CN101160133B - Preparation and composition of inter-alpha inhibitor proteins from human plasma for therapeutic use - Google Patents

Abstract

The present invention relates to an inner-alpha inhibitor protein (I alpha Ip). The invention also relates to a method for purifying the I alpha Ip composition and the usage for treating the human diseases such as pyaemia, septicemic shock, atrophic arthritis, cancer and infectious disease.

Description

Therapeutic use from human plasma interior-preparation and the compositions of alpha inhibitor protein

Related application

The denomination of invention that the application comprises on November 8th, 2003 application for " therapeutic use from human plasma interior-preparation and the compositions of alpha inhibitor protein " temporary patent application series No.60/_____ in disclosed related subject, the disclosure of this application is incorporated herein by reference with its integral body at this.

Government supports

A part of the present invention is supported by the appropriation R01 GM053008 of National Institute of Health, R01GM057468 and R43 GM065667.

Background of invention

In-alpha inhibitor protein (I α Ip) family is the serpin of one group of blood plasma association.The member of this family is made of heavy chain and light chain polypeptide subunit, and is covalently bound by sugared aminoglycan.Light chain is also referred to as bikunin, and the serpin of undertaking molecule is active.Name " bikunin " reflects the protease repression domain that has 2 Kunitz types.In the normal blood plasma, find that most of bikunin is complex form, such as interior-alpha inhibitor (I α I), it has the molecular weight of 225kDa, and front-alpha inhibitor (P α I), and it has the molecular weight of 120kDa.Among the I α I, bikunin connects 2 polypeptide heavy chains, H1 and H2, and, among the P α I, only have single heavy chain (H3) to connect bikunin.In these complex forms, bikunin keeps passivation until discharge by the Partial Protein hydrolytic degradation, and this is as the mechanism of regulating active mode.After the division, by renal glomerular filtration the bikunin that activates is removed fast from blood plasma from complex, this is the process that promotes by low-molecular-weight and receptor-mediated absorption.US patent No.6,489,128 and 6,660,482 relate to cancer diagnosis and pyemic purposes separately.Also disclose and suppressed to shift and treat pyemic method, yet compositions not pure and has stable problem, i.e. short-decayed problem in fact.

Although introduced antibiotic before more than 50 years, it has seen that really mortality rate that sepsis induces is reduced to 35% from 55%, and medical science does not significantly reduce and is benefited from sepsis individual death rate.In fact, sepsis continues that to be one of intensive care unit's cause of death die from subsequently septic shock and multiple organ failure, MOF with a large amount of sepsis individualities.Sepsis is to infecting for example systemic reaction of antibacterial infection.It is normally caused by the endotoxin of gram negative bacteria or the extracellular toxin of gram-positive bacterium (it can cause the reaction of endotoxin sample).Systemic reaction can cause septic shock, it is characterized in that blood pressure drops, cardiovascular collapse and/or multiple organ failure, MOF.The mortality rate that is diagnosed as in the septic shock individuality can be up to 35-45%.Be difficult to treat fast and reliably sepsis with conventional medicine.

Sepsis is relevant with the activation of innate immunity and blood coagulation system with septic shock.Sepsis and septic shock clinically be characterized as systemic inflammatorome, coagulopathy, hypotension and many organ dysfunction (J.-L.Vincent etc., Annuals of Medicine 34(2002) 606-613).In several sepsis processes, the net of specific proteases activates thrombin, the fibrinolysis factor and complement factor.These protease can also cause tissue and organ injury and improve non-specific proteolysis (J.Wite etc., the Intensive CareMedicine of thrombin and complementary factor in the blood plasma 8(1982) 215-222; S.J.Weiss, New England Journal ofMedicine 320(1989) 365-376).

The invention summary

The present invention relates to Purification of Human blood plasma interior-method of alpha inhibitor protein (I α Ip) and uses thereof, be used for the treatment of the human disease, such as sepsis, acute inflammation disease, serious shock, septic shock, rheumatoid arthritis, cancer, cancerometastasis, infectious disease and preterm delivery; Or for reducing sepsis, acute inflammation disease, serious shock, septic shock, rheumatoid arthritis, cancer, cancerometastasis, mortality risk that infectious disease is relevant with preterm delivery.

According on the one hand, produce the method for the I α Ip compositions that is derived from blood plasma, wherein I α I and P α I are present in the mixture with the physiology ratio, comprise the blood plasma fraction that contains I α I and P α I from separating plasma, and wherein I α I and P α I exist with the physiology ratio; Assigning to obtain I α Ip purity with purification blood plasma level is approximately 85% to about 100% pure I α Ip compositions.

According on the other hand, in I α Ip compositions comprises-mixture of alpha inhibitor protein (I α I) and front-alpha protein (P α I), wherein I α I and P α I are present in the described mixture with the physiology ratio, and approximately 85% to approximately 100% pure.

In the related fields, I α Ip compositions comprises the mixture of interior-alpha inhibitor protein (I α I) and front-alpha protein (P α I), and wherein I α I and P α I are present in the described mixture with the physiology ratio and have high trypsin rejection ratio activity.

In another related fields, I α Ip compositions comprises that the half-life is higher than one hour I α I and P α I.

In the related fields, I α Ip compositions comprises I α I and P α I again, wherein I α I and P α I by with three heavy chain H1, H2 and H3 at least one combination interior-light chain of alpha inhibitor protein forms.

In another related fields, the compositions of I α Ip comprises the mixture of interior-alpha inhibitor protein (I α I) and front-alpha protein (P α I), wherein I α I and P α I are present in the mixture with the physiology ratio, comprise with four heavy chain H1, H2, H3 and H4 at least one combination interior-light chain of alpha inhibitor protein.

In the related fields, make I α Ip according to following methods again, the method comprises isolates the blood plasma fraction that contains I α I and P α I from blood plasma, and wherein I α I and P α I exist with the physiology ratio; Assigning to obtain I α Ip purity with purification blood plasma level is approximately 85% to about 100% pure I α Ip compositions.

On the other hand, pharmaceutical composition according to the present invention comprises acceptable carrier on the I α Ip compositions described herein for the treatment of effective dose and the materia medica.

Relate on the other hand the method for the treatment of individual inflammation related disease, cancer or infectious disease, it comprises the I α Ip that any method of the following stated produces that passes through of drug treatment effective dose.

In another related fields, the individual method for the treatment of comprises the front level of treatments one or more among I α I, P α I, I α Ip, H3, H4, H1, H2 and the LC of measuring; And the I α Ip that will treat effective dose delivers medicine to individuality.

In the related fields, the method for prediction to I α Ip therapeutic response described.The method comprises that test detects one or more level I α I, P α I, I α Ip, H3, H4, H1, H2 and the LC from the sample that individuality obtains; The level that wherein detects identify can sound response I α Ip treatment individuality.

In another related fields, describe the method for the individual progress for the treatment of of monitoring I α Ip treatment, and comprised the front level of treatments one or more among I α I, P α I, I α Ip, H3, H4, H1, H2 and the LC of measuring; The I α Ip for the treatment of effective dose is delivered medicine to individuality; And I α Ip treatment just after date measure individual one or more levels, wherein the raising of individual level represents that individuality may have good clinical response to I α Ip treatment after the I α Ip treatment.

On the other hand, describe the medicine box that is used for I α Ip treatment, comprised one or more among I α I, P α I, I α Ip, H3, H4, H1, H2 and the LC; With the description that is used for the treatment of use.

In the related fields, described the compositions that comprises container, comprised I α Ip in the container and be inserted with relevant label or the packing that I α Ip is delivered medicine to individual explanation.

In the related fields, described medicine box again, it comprises aforesaid compositions and the description that is used for the treatment of use.

Following discloses other embodiments of the present invention.

The accompanying drawing summary

Fig. 1 has described the histopathology of spleen.In the control animal (A), the protection white pulp loses the red myelocyte solvent on a large scale around broom shape tremulous pulse, in I α Ip treatment animal (B), has the white pulp and slight supracellular red pulp (the H ﹠amp that normally center on; E dyeing, amplification 20 *).

Fig. 2 has described the aminoacid sequence of H4.

Fig. 3 has described caecum ligation and puncture (CLP) or sham-operation (Sham) change of bikunin mrna expression in the liver in the time of rear 5 and 20 hours.Data are expressed as on average ± SE (n=8/ group) and relatively next by a tail analysis (ANOVA) and the Tukey test of variance: ^*P＜0.05 pair sham-operation.Fig. 3 a has shown amplification and the mRNA by size separation on the gel, and Fig. 3 b with chart drawing be normalized to the result of the bikunin level of G3PDH expression.

Fig. 4 has described the t of caecum ligation and puncture (CLP) or sham-operation (Sham) 125I-I α I in the time of rear 5 and 20 hours _1/2Change.Data are expressed as on average ± SE (n=5/ group) and relatively next by a tail analysis (ANOVA) and the Tukey test of variance:

Fig. 5 has described the change of the ligation of vehicle treatment caecum and puncture and caecum excision (CLP+ carrier) and the treatment caecum ligation of interior alpha inhibitor and puncture (CLP+I α Ip) survival rate after 10 days.Every group of 12 animals.Calculate survival rate and relatively next with the test of logarithm level by the Kaplan-Meier method. ^*P＜0.05 pair CLP+ carrier.

Fig. 6 has described the change of the ligation of vehicle treatment caecum and puncture and caecum excision (CLP+ carrier) and the treatment caecum ligation of interior alpha inhibitor and puncture (CLP+I α Ip) survival rate after 10 days.Every group of 11 to 12 animals.Calculate survival rate and relatively next with the test of logarithm level by the Kaplan-Meier method. ^*P＜0.05 pair CLP+ carrier.

Fig. 7 has described the change of the ligation of vehicle treatment caecum and puncture and caecum excision (CLP+ carrier) and the treatment caecum ligation of interior alpha inhibitor and puncture (CLP+I α Ip) survival rate after 10 days.Every group of 16 animals.Calculate survival rate and relatively next with the test of logarithm level by the Kaplan-Meier method. ^*P＜0.05 pair CLP+ carrier.

Fig. 8 a and 8b have described according to the present invention from the sketch map of human plasma purification I α Ip.

Fig. 9 beneficial effect of highly purified I α Ip in the sepsis zooscopy that presented in diagrammatic form.

Detailed Description Of The Invention

Disclosed herein is new method from blood plasma purification I α Ip.Further disclosed at this is the therapeutic combination of purification I α Ip, is used for delivering medicine to individuality and treats acute inflammation disease, sepsis, serious shock, septic shock, rheumatoid arthritis, cancer, cancerometastasis, infectious disease and preterm delivery.

Known purification contains the pollutant of factor X (FX) before, detects by western blot analysis to be the 80kDa band.In the blood coagulation test, also detect FX.It is important removing FX, if because think that FX is thrombosed and delivers medicine to the people and be harmful to.The FX pollution problem of I α Ip compositions before method described herein has solved.

In-alpha inhibitor protein (I α Ip) is serpin family relevant on the structure, finds to be relatively high concentration (400-800mg/L) in human plasma.I α Ip is large, multicomponent complex, as insulin-type protease inhibitor.Different with other inhibitor molecules, the inhibitor of this family by the compositions of polypeptide chain (light chain and heavy chain) uniquely covalently bound chondroitin sulfate chain form.The heavy chain (H1, H2 and H3) of interior-alpha protein is also referred to as hyaluronic acid (HA) in conjunction with albumen.The principal mode of finding in the human plasma is interior-α-inhibitor (I α I), and it is by two heavy chain (H1 ﹠amp; H2) and single light chain (L) form, and front-α-inhibitor (P α I), it is comprised of a heavy chain (H3) and a light chain (L).Known light chain (is also referred to as bikunin (two-the kunitz inhibitor, as to have two Kunitz domains) and extensively suppresses the blood plasma serine protease.Complex has demonstrated the importance in the protease inhibition array, and the protease array comprises neutrophilia elastoser, plasmin, trypsin, cathepsin G and acrosin.

Have been found that I α I and P α I and H4 are compound, H4 is another heavy chain of I α Ip protein.The I α Ip compositions of particular comprises the H4 compound with P α I, I α I or P α I and I α I according to the present invention.

Do not wish to be subject to the restriction of any scientific theory, it is rear in conjunction with HA that we infer that the heavy chain of I α Ip discharges from complex, to prevent that HA is in conjunction with its receptor CD44.When the heavy chain of I α Ip did not exist, HA was in connection with CD44 and cause the secretion of the proinflammatory factor.For example, TNF-α, and cause inflammation.Therebetween, the light chain of I α Ip just presents antiprotease activity in case discharged from complex.

" I α Ip compositions " refers to the preparation of I α Ip protein, comprises I α I and the P α I of physiology ratio.As used in this physiology ratio refer to comprise do not infected or the human or animal of disease in the ratio found, and/or the ratio of the I α I of natural appearance in the human plasma and P α I.The physiology ratio is generally approximately 60% to about 80%I α I and approximately 40% to about 20%P α I.Because the normal variation that genes of individuals consists of, the physiology ratio can be different from these scopes.

As used in this, " mixture of interior-alpha inhibitor protein (I α I) and front-alpha protein (P α I) " refers to the compositions that contains I α I and P α I complex.Mixture can also contain buffer agent, salt or for separating of other compositions of I α Ip complex.In the particular aspects, I α I and P α I are present in the mixture with the physiology ratio.

The I α I that exists in the blood plasma fraction and P α I have approximately 60,000 to approximately 280, the apparent molecular weight of 000kDa.Come determining molecular weight by SDS-PAGE.

The time quantum of half when " half-life " I α Ip activity of referring to administration is administration as used in this.The half-life of I α Ip compositions is according to the present invention, for example, and greater than approximately 1,1.5,2,2.5,3,3.5,4,4.5,5,7.5 or 10 hour.In the preferred embodiment, the half-life of I α Ip compositions is higher than approximately 5 hours.In the particularly preferred embodiment, the half-life of I α Ip compositions is higher than approximately 10 hours.Preferred long half-life, for example, because need to deliver medicine to individual dosage lower along with the time.

I α Ip compositions of the present invention has high trypsin inhibition activity.Be approximately 1000 to about 2000IU/mg according to the trypsin rejection ratio activity of I α Ip compositions of the present invention.Preferred trypsin rejection ratio activity is approximately 1200IU/mg, more preferably from about 1500IU/mg.Suppress test by trypsin and measure trypsin rejection ratio activity with L-BAPA as substrate.Referring to, HU Bergmeyer, editor: vol 5, the 3rd edition, 119 (1984) VerlagChemie, Weinheim:Chromogenic substrate for the assay of trypsin (testing tryptic chromophoric substrate): R.Geiger, H.Fritz, Methods of EnzymaticAnaIysis.

The compositions of I α Ip is the mixture of interior-alpha inhibitor protein (I α I) and front-alpha protein (P α I), wherein I α I and P α I are present in the described mixture with the physiology ratio, in comprising-and the light chain of alpha inhibitor protein is in conjunction with among three heavy chain H1, H2 and the H3 at least one.In also having according to compositions of the present invention-light chain of alpha inhibitor protein is in conjunction with among four heavy chain H1, H2, H3 and the H4 at least one.The example of each albumen is as follows in the I α Ip complex: Bikunin Genbank accession number: AAB84031, P02760; H1GenBank accession number: P19827, NP_002206; H2GenBank accession number: NP_002207, P19823; H3 GenBank accession number: NP_002208; H4 GenBank accession number: Q14624, NP_002209, each is incorporated herein by reference with its integral body at this.

" be derived from blood plasma " as used in this and refer at first from separating plasma or purification.That is, the natural environment of compositions is blood plasma.

" blood plasma fraction " is that original source is from blood plasma from the isolated or purified step fraction of chromatograph for example as used in this.Blood plasma fraction according to the present invention can be, for example, from the by-product of purifying blood coagulation factors IX acquisition, from the by-product of the compound concentrate acquisition of purifying blood coagulation proenzyme, the cold supernatant that obtains from CPP (is described in Hoffer etc., Journal ofChromatography B 669(1995) 187-196), or cold barren blood plasma (poorplasma), cold barren blood plasma is used alternatingly at this and cold supernatant.Cold barren blood plasma is the supernatant that obtains from cryoprecipitate.

By-product example according to the present invention obtains from the purifying blood coagulation factors IX.Shown that I α I/P α I mixture is present in the by-product of factors IX (FIX) purge process generation.The method that obtains by-product from the purifying blood coagulation factors IX is described in Hoffer etc., Journal ofChromatography B 669(1995) 187-196, it is incorporated herein by reference with its integral body at this.Other examples of by-product comprise from the by-product of purification FIX or from the by-product of the compound concentrate of purifying blood coagulation proenzyme, as are described in D.Josic etc., Thrombosis Research 100(2000) 433-441, it is incorporated herein by reference with its integral body at this; And the by-product that obtains cold supernatant separation as the blood plasma cryoprecipitate.(for example, suitable cryoprecipitate method is described in Hoffer etc., Journal of Chromatography B 669(1995) 187-196).Be used for comprising reinforcing YIN-essence ion exchange fraction and monolithic chromatogram fraction from other blood plasma fraction of blood purification I α Ip complex, will in following embodiment, describe.

According to the present invention, the blood plasma fraction can be from people, primates, cattle, pig, cat or dog source.

As used in this, term " acquisition " comprises purchase, synthesizes, separates or obtains in addition one or more used in the invention process materials.

Therefore, as used in this " acquisition blood " for example comprises obtaining blood from people, primates, cattle, pig, cat or dog source.For example, can originate to obtain and/or buy blood from blood bank, hospital, asylum, private company, WARF or any other blood.

As used in this " acquisition blood plasma ", for example comprise from people, primates, cattle, pig,, cat or dog source obtain blood plasma.For example, can originate to obtain and/or buy blood plasma from blood bank, hospital, asylum, private company, WARF or any other blood.Perhaps, in case obtain blood, also can be from blood separation blood plasma.The appropriate method of separated plasma comprises gravity and centrifugal.

As used in this, " obtaining from the by-product fraction of purifying blood coagulation factors IX acquisition " comprises for example from the company of daily purification of factor IX or the by-product fraction that hospital obtains and/or purchase obtains from the purifying blood coagulation factors IX.

As used in this, " obtain to obtain from the compound concentrate of purifying blood coagulation proenzyme by-product fraction " for example comprises from company, research organization or the hospital of purifying blood coagulation proenzyme complex obtaining and/or buy the by-product fraction.

As used in this, " obtain from the cold supernatant of blood plasma cryoprecipitate acquisition " to comprise for example from obtaining and/or buy cold supernatant with hospital, research organization or the company that is applicable to mode CPP of the present invention.

" solid carrier " refers to the solid material that can derive or connect in addition with capture agent capture agent.The example solid carrier comprises probe, microtitration plate and chromatography resin.

" absorption " refers to the non-covalent combination that detects of analyte and adsorbent or capture agent.Adsorbent surface refers to the surface in conjunction with adsorbent (being also referred to as " capture agent " or " affinity reagent ").Adsorbent be can bound analyte (for example, target polypeptides or nucleic acid) any material.

Chromatographic adsorbent refers to common used material in the chromatograph.Chromatographic adsorbent comprises, for example, and ion exchange material, metal-chelator (for example, nitrilo-acetic acid or iminodiacetic acid), the fixing metal chelate, the hydrophobicity exchange adsorbing substance, the hydrophilic exchange adsorbing substance, dyestuff, simple biomolecules is (for example, nucleotide, aminoacid, monosaccharide and fatty acid) and the adsorbent (for example, hydrophobic adsorption/electrostatic repulsion adsorbent) of mixed model.

The biologic specificity adsorbent refers to the adsorbent that comprises biomolecule, biomolecule for example, nucleic acid molecules is (for example, aptamer), polypeptide, polysaccharide, lipid, steroid or these conjugate are (for example, glycoprotein, lipoprotein, glycolipid, nucleic acid (for example, DNA)-protein conjugate).In the specific embodiment, the biologic specificity adsorbent is macromolecular structure such as polyprotein complex, biomembrane or virus.The example of biologic specificity adsorbent is antibody, receptor protein and nucleic acid.The biologic specificity adsorbent is usually high than chromatograph adsorbent to the specificity of target analytes.

" eluant " or " wash solution " refers to for impact or changes analyte and the absorption of adsorbent surface and/or remove the not reagent of bond material, normally solution from the surface.For example the eluting characteristic of eluant depends on pH, ionic strength, hydrophobicity, chaotic tropism's degree, detergent intensity and temperature.

" analyte " refers to any composition that expectation obtains detecting in the sample.Term can refer to single composition or the Multiple components of planting in the sample.

Term " polypeptide ", " peptide " and " protein " can be used alternatingly at this, are used for representing the polymer of amino acid residue.Term is applicable to the amino acid polymer that wherein one or more amino acid residues are the corresponding analog of natural generation aminoacid or analogies, and the amino acid polymer that is applicable to natural generation.Polypeptide can be modified, and for example, forms glycoprotein by adding carbohydrate.Term " polypeptide ", " peptide " and " protein " comprise glycoprotein and non-glycoprotein.

" immunoassay " is the test of using the antibody of specific binding antigen (for example, I α Ip complex).Immunoassay is characterised in that the specific binding characteristic of using specific antibodies and separator, target, and/or quantitative antigen.

" antibody " refers in fact the polypeptide ligand by an immunoglobulin gene or a plurality of immunoglobulin gene or its fragment coding, its specifically combination and identification epitope (for example, antigen).Known immunoglobulin gene comprises κ and the constant fragment gene of γ light chain, α, γ, δ, ε and the constant fragment gene of μ heavy chain, and countless immunoglobulin variable fragment genes.For example a plurality of fragments of the abundant sign that produces as complete immunoglobulin or as various peptide enzymic digestions of antibody exist.This comprises, for example, and Fab ' and F (ab) ' ₂Fragment.Antibody comprises polyclonal antibody and monoclonal antibody, chimera, and strand and humanized antibodies, and Fab fragment comprise the product in Fab or other immunoglobulin expression libraries.

Phrase " specificity (or selectivity) in conjunction with " antibody or " specificity (or selectivity) immunoreation " when relating to protein or peptide, refer in the multiple colony of protein and other biological goods and determine the association reaction that protein exists.Therefore, under the immunoassay condition of appointment, specific antibodies specific protein doubles at least background and there is no that other protein that exist are combined with significant quantity in sample.Need to select antibody to the specificity of specific protein with the specific binding of antibody under the condition like this.For example, the polyclonal antibody that can select to cause specific species such as rat, mice or people's I α Ip complex only obtain with I α Ip complex specific immune response and not with those polyclonal antibodies of other proteins reacts, except the allele of multiform variant and I α Ip complex.Obtain this selection by the antibody of getting rid of with the I α Ip complex molecule cross reaction of other species.Can select antibody with the specified protein specific immune response with various immunoassay forms.For example, usually with solid phase ELISA immunoassay select with the immunoreactive antibody of protein specific (referring to, for example, Harlow; Lane, Antibodies, A Laboratory Manual (1988) has described the immunoassay form and the condition that are used for measuring specific immune response).Usually specificity or selective reaction double background signal or noise at least, more generally are higher than 10 to 100 times of backgrounds.

" function equivalent " refers to present and substantially goes up in the similar body to said I α Ip protein or any albumen of external activity as used in this, for example, affects pyemic reduction.

For example, confirm structure and the purity of I α Ip product by HPLC or other chromatographic processes well known by persons skilled in the art.

" I α Ip complex " is used for comprising the biological activity variant of all natural generations of I α Ip protein as used in this, comprises the protein that contains deletion, insertion, adds and replace." the natural variant " of I α Ip protein is defined as the peptide with one or more amino acid change sequences that obtains from blood plasma.Variant can have " conservative " and change, and the aminoacid that wherein replaces has similar structure or chemical characteristic, and for example, isoleucine substitutes leucine.In another embodiment, variant can have " non-conservation " and change, and for example tryptophan substitutes glycine.Similar variant can also comprise aminoacid deletion or insertion, or both.Use computer program well known in the art, for example, which and how many amino acid residues DNASTAR software can be found to measure and be substituted, insert or detect and elimination biology or immunocompetent guidance.

" deletion " is defined as wherein one or more aminoacid or separately non-existent aminoacid or nucleotide sequence change of nucleotide residue.

" insertion " or " interpolation " be with the I α Ip composite bulk phase of natural generation relatively, separately so that the aminoacid that one or more aminoacid or nucleotide residue add or nucleotide sequence change.

Separately by different aminoacid or the one or more aminoacid of nucleotide substitution or nucleotide formation " replacement ".

Term " biological activity " refers to structure, adjusting or the biochemical function with natural generation I α Ip complex.Equally, " immunocompetence " orientates the ability that natural, restructuring or synthetic I α Ip complex or its any oligopeptide are induced specific immune response in suitable animal or the cell and be combined with specific antibodies as.

Term " derivant " refers to the chemical modification of the I α Ip complex of the nucleic acid of coding I α Ip complex or coding as used in this.Such modification illustrates by alkyl, acyl group or amino instead of hydrogen.Nucleic acid derivative will be encoded and be kept the polypeptide of the substantive biological nature of natural I α Ip complex.

" purification " refers to from I α Ip and removes the albumen of unwanted or pollution or the step that one-tenth assigns to produce purification I α Ip complex as used in this.For example, can be by the blood plasma level that the continuous chromatography step process contains the I α I of physiology ratio and the P α I purification I α Ip compositions of assigning to.

As used in this " separation " refer to from blood plasma and produce the blood plasma fraction, it contains I α I and the P α I of physiology ratio.For example, by being carried out chromatograph, blood plasma obtains the separated plasma fraction according to the present invention.Separate refer to from its primal environment (for example, if natural generation be natural environment) in the material that takes out, and therefore " pass through the people " from its native state and change.For example, the polypeptide of separation or protein can be the compositions of blood plasma, maybe can be contained in the cell and think " separation ", because blood plasma or specific cells can not be the primal environments of polypeptide.

Chromatograph can comprise anion-exchange chromatography as used in this.Anion-exchange chromatography can be based on granule, for example, and DEAE Sepharose, DEAE Sephadex A50, Toyopearl DEAE, TMAE Fractogel, DEAE Fractogel or Q-Sepharose.Anion-exchange chromatography can also pass through integral carrier, for example, and with CIM such as DEAE-CIM or the Q-CIM of immobilization anion exchange part.SEPHAROSE is the Pharmacia of New Jersey, and Inc. is to the trade name of high molecular weight material, and this material is used for the separation of macromole gel filtration.Anion-exchange column has two parts, substrate and parts.Substrate can be for example cellulose, glucosan, agarose or polystyrene.Part can be diethylamino ethyl (DEAE), polymine (PEI) or quaternary ammonium functional group.The intensity of anion-exchange column refers to the ionization state of part.Reinforcing YIN-essence ion exchange column, as have those of quaternary ammonium part, in wide pH scope with permanent positive charge.In the weak anionic exchange column, such as DEAE and PEI, the pH of pillar is depended in the existence of positive charge.Preferred reinforcing YIN-essence ion exchange column such as Q Sepharose FF or metal-chelating Sepharose (for example, Cu2+-chelating Sepharose).Anion-exchange column loads the low salt buffer agent that pH is higher than alpha-Glucosidase pI usually.

I α Ip compositions of the present invention is preferably approximately 85% to approximately 100% pure.As used in this, term " pure " refers to the I α Ip compositions of taking out from its natural environment, separation or separate, and at least about 85% to about 100% nothing, preferred 90% nothing, more preferably 95% without and its natural other relevant compositions.In the preferred embodiment, the protein of purification will consist of and be higher than 85%, 87.5%, 90%, 92.5%, 95%, 99% or even higher protein in the compositions basically.

That peptide, polypeptide or protein have wherein peptide, polypeptide or protein and substantially goes up purity level without other protein and biotic component as using peptide, polypeptide or the protein meaning to " being purified to homogeneous " of the present invention.One or more separating steps that any suitable materials and methods can be used for carrying out blood plasma obtain the I α Ip of purification.

Usually, the preparation of the I α Ip separation and the collection that relate to sample is used for measuring the fraction that contains target protein.The method of separating comprises, for example, solid phase extractions, chromatograph, for example, and anion-exchange chromatography, the size exclusion chromatograph, ion exchange chromatography, the heparin chromatograph, affinity chromatography is extracted gel electrophoresis and liquid chromatograph continuously.Preparation can also comprise purification, and it comprises chromatographic step, for example, ion exchange chromatography, the heparin chromatograph, affinity chromatography is extracted gel electrophoresis and liquid chromatograph continuously.

In the embodiment of the present invention, by anion-exchange chromatography with twice of Sample Purification on Single.Anion-exchange chromatography according to their charge characteristic with the purification roughly of protein in the sample.For example, can use Q anion exchange resin (for example, Q HyperD F, Biosepra), and with the continuous elution sample of the eluant of different pH.Anion-exchange chromatography so that in the sample biomolecule of more negative charges from the biomolecule of other types, separate.Albumen with the eluent elution of high pH may be weak negative charge, may be strong negative charge with the fraction of the eluent elution of low pH.Therefore, except reducing the complexity of sample, anion-exchange chromatography comes isolated protein according to their binding characteristic.

In the embodiment, by the heparin chromatograph sample is further purified again.The heparin chromatograph also is based on and the interactional affinity of heparin and charge characteristic so that the I α Ip complex in the sample is further purified.Heparin, the sulphation mucopolysaccharide, in connection with the I α Ip complex with positive charge part, and with the continuous elution sample of the eluent of different pH or salinity.I α Ip complex with the eluent elution of hanging down pH more may be weak positive charge.I α Ip complex with the eluent elution of high pH more may be strong positive charge.Therefore, the heparin chromatograph has also reduced the complexity of sample and has separated I α Ip complex according to their binding characteristic.

Can catch I α Ip complex with the capture agent that is fixed in carrier, carrier such as any biochip, porous microtitration plate, resin or have subsequently the NC Nitroncellulose film of probe as albumen.Especially, can catch I α Ip complex of the present invention at laser desorption/ionizing (SEIDI) protein bio-chip of surface-raising.Can catch at chromatographic surface or biologic specificity surface.Any SELDI protein-biochips that comprises reactive surfaces can be used for catching and detecting I α Ip complex of the present invention.These biological new films can derive with the antibody that specificity is caught I α Ip complex, maybe can derive with capture agent the protein A of capture agent such as binding domain-immunoglobulin or Protein G.Then use specific antibody that I α Ip complex is trapped in the solution neutralization by the I α Ip complex of capture agent separating trap on chip.

The whole bag of tricks of the quantitative albumen of disclosure according to the present invention, polypeptide or peptide purification degree is well known to a person skilled in the art.These comprise, for example, measure the specified protein activity of fraction, or measure the quantity of polypeptide in the fraction by gel electrophoresis.

Except those technology of following detailed description, the various other technologies that are applicable to protein purification are well known to a person skilled in the art.These comprise, and are for example, with the precipitation such as ammonium sulfate, PEG, antibody or by thermal denaturation, then centrifugal; Chromatographic step such as ion-exchange step, gel filtration step, anti-phase step, hydroxyapatite step, the affine step of lecithin, immune affinity chromatographic and other affinity chromatography steps; Isoelectric focusing; Gel electrophoresis, HPLC; Combination with these and other technology.In addition, if think needs, can carry out other purification step with the method for this area standard.These methods can fully give the pure preparation of height of protein.

In other embodiments, can use gel chromatography or molecular sieve chromatography.Gel chromatography is based on the particular type partition chromatography of molecular size.Principle after the gel chromatography is pillar, makes with containing foraminate fine grained inert substance, separates from than micromolecule according to the young pathbreaker is more macromolecular greatly when passing or center on the hole.As long as the material of granulation does not adsorb molecule, the unique factor that determines flow velocity is size.Therefore, molecule with size elution from pillar of successively decreasing, as long as shape is relatively constant.Gel chromatography is incompetent for the molecules that separate different sizes, because separate with every other factor such as pH, ionic strength, temperature etc. irrelevant.In fact also do not have absorption, the area distribution of minimizing is the simple factors relevant with molecular weight with elution volume.

In another embodiment, can use affinity chromatography.Affinity chromatography is the chromatographic process that relies on specificity affinity between material to be separated and the specific binding molecules.That is, for example, the receptor-ligand type interacts.Can be by synthesizing the pillar material in connection with the insoluble substrate of one of gametophyte covalent bond.Then the pillar material can be from solution the specific adsorption material.For example, carry out elution (for example, changing pH, ionic strength and temperature) by condition being changed over those that combination can not occur.

Method described herein is suitable for large-scale production, and forms protein, comprises the I α Ip complex and the H4 that are suitable for treating form of medication.

Any isolated or purified step that if necessary, can repeat said method obtains higher purity.Chromatographic step can be in batch or the pillar form.

Produce the method for optimizing that is derived from the I α Ip compositions of blood plasma of the present invention and comprise isolate the blood plasma fraction that contains I α I and P α I from blood plasma, wherein I α I and P α I exist with the physiology ratio; And purification blood plasma level assigns to obtain purity approximately 85% to about 100% pure I α Ip compositions.

The method according to this invention can also comprise being further purified of blood plasma fraction, for example, passes through the effluent of (not combination) fraction by heparin affinity column and collection.

Useful method can also be included in before the purification and separation step and/or afterwards with blood plasma fraction and/or purification I α Ip inactivation of virus among enforcement the present invention.Usual method comprises by solvent/detergent treatment or hot deactivation inactivation of virus.Hot deactivation or the pasteurize of the present composition can be for example approximately 55 xeothermic to approximately 65 ℃ or 70 to 120 ℃.

For the solvent detergent-treatment, the particular combinations of solvent and detergent, as, 0.3% 3-n-butyl phosphoric acid salt (TnBP) is in conjunction with 1% tween 80,24 ℃ 6 hours, effectively the deactivation enveloped virus (Horowitz etc. (1985) Transfusion 25, pp.516-522).Perhaps, any virus that in the presence of stabilizing agent, the I α Ip of fraction or purification is enough to the deactivation existence 55-65 ℃ of pasteurize.

In the process of purification or separating step, add stabilizing agent.The final composition of I α Ip can also contain stabilizing agent.For example, suitable stabilizing agent comprises albumin, Polyethylene Glycol, and α, α-trehalose, aminoacid, salt, glycerol, omega amino acid such as lysine, polylysine, arginine, EACA and tranexamic acid, sugar, such as sucrose, or its combination.

I α Ip protein of the present invention and compositions can be used for the treatment of the human disease.Such disease comprises, for example, acute inflammation disease, septicopyemia just, serious shock, septic shock, rheumatoid arthritis, cancer, cancerometastasis, preterm delivery and infectious disease.Assign to make the I α Ip compositions that is used for the treatment of disease by isolate the blood plasma level that contains I α I and P α I from blood plasma, wherein I α I and P α I exist with the physiology ratio; Assigning to obtain I α Ip purity with purification blood plasma level is approximately 85% to about 100% pure I α Ip compositions.

The present invention also comprises the pharmaceutical composition of I α Ip.The pharmaceutical composition of I α Ip can be acceptable carrier on treatment described herein any I α Ip compositions of effective dose and the materia medica.

For example, according to pharmaceutical composition of the present invention can be the treatment effective dose I α Ip compositions, it is interior-mixture of alpha inhibitor protein (I α I) and front-alpha protein (P α I), wherein I α I and P α I are present in the described mixture with the physiology ratio, for approximately 85% to approximately 100% pure.Pharmaceutical composition can also be the I α Ip compositions for the treatment of effective dose, it is interior-mixture of alpha inhibitor protein (I α I) and front-alpha protein (P α I), wherein I α I and P α I are present in the described mixture with the physiology ratio, comprise with three heavy chain H1, H2 and H3 or H1, H2, H3 and H4 at least one combination interior-light chain of alpha inhibitor protein.Pharmaceutical composition can also be the I α Ip compositions for the treatment of effective dose, it is interior-mixture of alpha inhibitor protein (I α I) and front-alpha protein (P α I), wherein I α I and P α I are present in the described mixture with the physiology ratio, and have high trypsin inhibitor specific activity.Pharmaceutical composition can also be the I α Ip compositions for the treatment of effective dose, it is interior-mixture of alpha inhibitor protein (I α I) and front-alpha protein (P α I), wherein I α I and P α I are present in the described mixture with the physiology ratio, and have the half-life that is higher than one hour, five hours or ten hours.

Term " acceptable carrier or adjuvant on the materia medica " refers to compositions of the present invention and delivers medicine to individuality and do not destroy its pharmaceutically active during with enough transmission therapeutic dose compositions administrations and be atoxic carrier or adjuvant.

Acceptable carrier on the used materia medica in the pharmaceutical composition of the present invention, adjuvant and excipient include, but not limited to ion-exchanger, aluminum, aluminium stearate, lecithin, self-emulsifying drug transfer system (SEDDS) is such as d-alpha-tocopherol cetomacrogol 1000 succinate, and used surfactant such as tween or other similar polymerizations transmit substrate, serum albumin in the pharmaceutical dosage form, such as the human serum albumin, buffer substance such as phosphate, glycerol, sorbic acid, potassium sorbate, the partial glycerol ester admixture of saturated vegetable fatty acid, water, salt or electrolyte, such as protamine sulfate, sodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salt, cabosil, magnesium trisilicate, polyvinylpyrrolidone, based on cellulosic material, Polyethylene Glycol, sodium carboxymethyl cellulose, polyacrylate, wax, polyethylene-polyoxypropylene block polymer, Polyethylene Glycol and lanoline.Cyclodextrin such as α-, β-, gamma-cyclodextrin, or the derivant of chemical modification such as hydroxyalkyl cyclodextrin comprise 2-and 3-HP-β-CD, or the derivant of other dissolvings also can be advantageously used in the transmission that improves said compositions.

Pharmaceutical composition of the present invention can oral, non-intestinal, suck spraying, part, rectum, nose, cheek, vagina or come administration by the implantation storage, preferably by oral administration or pass through drug administration by injection.

Pharmaceutical composition of the present invention can contain acceptable carrier, adjuvant or excipient on the atoxic materia medica of any routine.In the certain situation, improve the stability of compositions formulated or its delivery form with the pH of acceptable acid on the materia medica, alkali or buffer agent adjusting preparation.Within the non-intestinal of this used term comprises subcutaneous, Intradermal, intravenous, intramuscular, intraarticular, intra-arterial, synovial fluid, in the web, in the film, intracavity and intracranial injection or inculcate technology.Suitable medication can be tablet, capsule, or by intravenous injection.The administration of especially preferred injection form.

Pharmaceutical composition can be the form of aseptic injection preparation, for example, and as aseptic injection water or oleagenous suspension.Can prepare this suspension according to technology known in the art, use suitable dispersion or wetting agent (such as, for example, Tween 80) and suspending agent.Aseptic injection preparation can also be aseptic injectable solution or the suspension in the acceptable diluent of nontoxic non-intestinal or the solvent, for example, and as 1,3 butylene glycol solution.What can accept to use in carrier and the solvent is mannitol, water, Ringer's solution and isotonic sodium chlorrde solution.In addition, aseptic, nonvolatile oil is typically used as solvent or suspension media.For this purpose, can use the nonvolatile oil of any gentleness, comprise synthetic list-or two glyceride.Fatty acid can be used in the ejection preparation such as oleic acid and glyceride ester derivatives thereof, and acceptable oil on the natural materia medica, such as olive oil or Oleum Ricini, and their polyoxy ethylization form especially.These oil solutions or suspension can also contain long-chain alcohol diluent or dispersant, or carboxymethyl cellulose or be generally used for materia medica can accept dosage form such as emulsion with or suspension preparation in similar dispersant.For the purpose of preparing, can accept other conventional surfactants commonly used in the manufacturing of solid, liquid or other dosage forms such as tween or span and/or other similar emulsifying agents or bioavailability improving agent on the materia medica and also can use.

Pharmaceutical composition of the present invention can come oral administration with any oral acceptable dosage form, includes, but not limited to capsule, tablet, emulsion and aqueous suspension, dispersion and solution.In the situation of the tablet that orally uses, used carrier comprises lactose and corn starch usually.Usually also add lubricant, such as magnesium stearate.For the oral administration of capsule form, useful diluent comprises lactose and dry corn starch.When oral administration aqueous suspension and/or emulsion, will suspend or be dissolved in active component in the oil phase in conjunction with emulsifying agent and/or suspending agent.If necessary, can add specific sweeting agent and/or flavoring agent and/or pigment.

Pharmaceutical composition of the present invention can also be used for rectally with the form administration of suppository.By compositions of the present invention is mixed to prepare these compositionss with suitable non-irritating excipient, this excipient room temperature be solid but be liquid and therefore in rectum, melt and come release of active ingredients at rectal temperature.Such material includes, but not limited to cocoa butter, Apis cerana Fabricius and Polyethylene Glycol.

When required treatment related to topical application and holds accessible position or organ, the topical of pharmaceutical composition of the present invention was useful.For the topical application of skin, pharmaceutical composition should be prepared with containing the suitable ointment that suspends or be dissolved in the active component in the carrier.The carrier that is used for present composition topical includes, but not limited to mineral oil, liquid petroleum, white oil, polypropylene glycol, polyoxyethylene, polyoxypropylene compositions, emulsifing wax and water.Perhaps, pharmaceutical preparation can be prepared with suitable washing liquid or Emulsion and suitable emulsifying agent, and washing liquid or Emulsion contain and suspends or be dissolved in active compound in the carrier.Suitable carrier includes, but not limited to mineral oil, sorbitan stearate monoesters, polysorbate 60, cetyl esters wax, whale aryl alcohol, 2-octyldodecanol, benzyl alcohol and water.Pharmaceutical composition of the present invention also uses to lower intestinal tract by rectal suppository or suitable enema agent part.The present invention also comprises the sticking patch of local percutaneous.

Pharmaceutical composition of the present invention can or suck administration by the nose aerosol.Prepare such compositions and can make saline solution according to the known technology of field of pharmaceutical preparations, use benzyl alcohol or other suitable antiseptic, improve absorption enhancer, fluorocarbon and/or other stabilizing agents known in the art or the dispersant of bioavailability.

Treat the method for individual disease according to the present invention, comprise that administration is according to the I α Ip compositions of the treatment effective dose of the inventive method generation, these diseases comprise that inflammation related disease for example, rheumatoid arthritis, sepsis or septic shock, head trauma/damage and meningitis, inflammatory bowel (Crohn disease), chronic obstructive pulmonary disease, rhinitis; Cancer, preterm delivery or infectious disease.

Be about 1 to 50mg/kg body weight at the dosage of this compositions, preferred dose is 500mg to 1000mg/ agent, per 4 to 120 hours, or according to the needs of specific medication.Method at this comprises that the compositions of effective dosage obtains required or described effect.Usually, pharmaceutical composition every day of the present invention or every other day with continuous infusion administration approximately 1 to approximately 6 times.Such administration can be used as chronic or acute treatment.The active component content that produces the single dose form in conjunction with carrier mass changes according to host to be treated and specific administering mode.Exemplary formulations will contain has an appointment 5% to about 95% active compound (w/w).Perhaps, such preparation contains and has an appointment 20% to about 80% active compound.

It also is favourable being higher or lower than above-mentioned those dosage.Given dose and therapeutic scheme for particular individual will depend on many factors, comprise the activity of used particular composition, the age, body weight, general health situation, sex, meals, administration time, excretion rate, drug regimen, the order of severity of disease and process, disease or symptom, individual disposal to disease, disease or symptom, and treatment doctor's judgement.

If necessary, when disease is improved, the present composition or mixture that can the administration maintenance dose.Subsequently, as the function of symptom, the dosage of administration or frequency or both can be reduced to the level of improving disease of keeping, alleviate to desired level when symptom, treatment should stop.Yet, based on any recurrence of disease symptoms, the intermittent therapy that individual need is long-term.Can also judge based on the I α Ip level in the liver improvement of disease.If the I α Ip in the discovery liver is the normal physiological level and judges that by patient and treatment doctor patient's symptom is improved, and is individual with maintenance dose treatment.

When compositions of the present invention comprises the mixture of I α Ip compositions and one or more other treatment agent or preventive, the dosage level that compositions and other medicaments exist be in common single therapeutic scheme administration approximately 1 to 100%, 5 to 95% dosage more preferably from about.Other medicament can separate administration with compositions of the present invention, as the part of multiple dose scheme.Perhaps, those medicaments can be the parts of single dose form, are mixed in the single compositions with compositions of the present invention.

Treat individual acute inflammation disease, sepsis, serious shock, septic shock, rheumatoid arthritis, cancer, cancerometastasis, infectious disease, and/or the method for preterm delivery may further comprise the steps: measure I α I, P α I, I α Ip, H3, H4, H1, level before the one or more treatment among H2 and the LC; And the I α Ip that will treat effective dose delivers medicine to individuality.I α I, P α I, I α Ip, H3, H4, H1, level is the individual proteins level before administration I α Ip or any I α Ip compound protein for the first time before the treatment of H2 and LC.Level after the treatment is the I α Ip level of measuring behind the administration I α Ip.Method of the present invention comprises measures just after date I α I of I α Ip treatment, P α I, I α Ip, H3, H4, H1, level after the one or more treatment among H2 and the LC.The adjusting of I α Ip level represents to treat and is producing good clinical response.The initial stage for the treatment of is the time that the I α Ip plasma concentration of acquisition steady statue needs.

For example measure I α I by immunization method, P α I, I α Ip, H3, H4, H1, the level of H2 and LC.For example, can detect and/or measure I α Ip complex by various detection methods, comprise, for example, the gas phase ion spectrometry method, optical means, electrochemical method, atomic force microscopy, radio frequency method, surface plasma body resonant vibration, oval symmetrical immunity and Atomic Force Microscopy.

In another embodiment, can come I α Ip complex in the determination and analysis sample with immunoassay.The method comprises: the specific binding I α Ip antibody of complex (a) is provided; (b) sample is contacted with antibody; (c) detection is in conjunction with the existence of the antibody complex of I α Ip complex in the sample.The suitable antibodies that is used for the inventive method comprises MAb69.31, MAb69.26, anti-I α Ip polyclonal antibody (R16 or R20), and anti-bikunin monoclonal or polyclonal antibody.

Immunoassay is the test of using the antibody of specific binding antigen (for example, I α Ip complex).Immunoassay is characterised in that the specific binding characteristic of using specific antibodies and separator, target, and/or quantitative antigen.Phrase " specificity (or selectivity) in conjunction with " antibody or " specificity (or selectivity) immunoreation " when relating to protein or peptide, refer in the multiple colony of protein and other biological goods and determine the association reaction that protein exists.Therefore, under the immunoassay condition of appointment, specific antibodies specific protein doubles at least background and there is no that other protein that exist are combined with significant quantity in sample.Need to select antibody to the specificity of specific protein with the specific binding of antibody under the condition like this.For example, the polyclonal antibody that can select to cause specific species such as rat, mice or people's I α Ip complex only obtain with I α Ip complex specific immune response and not with those polyclonal antibodies of other proteins reacts, except the allele of multiform variant and I α Ip complex.Obtain this selection by the antibody of getting rid of with the I α Ip complex molecule cross reaction of other species.

Be suitable for individuality with I α Ip treatment can be accredited as suffer from inflammation, wound/damage, tumor invasion, neoplasm metastasis, sepsis, septic shock or infectious disease.Individuality can be the oneself identify or by doctor diagnosed for suffering from inflammation, tumor invasion, neoplasm metastasis, sepsis, septic shock or infectious disease.Individuality can be primates, people or other animals.

Method also comprises the co-administered with the other treatment agent.For example, other therapeutic agent can be anticarcinogen, antiinflammatory, anticoagulant or immunomodulator.For example; di-deoxynucleoside; for example; zidovudine (AZT); 2 '; 3 '-dideoxyinosine (ddI) and 2 '; 3 '-zalcitabine (ddC); lamivudine (3TC); stavudine (d4T) and TRIZIVIR (Abacavir+zidovudine+lamivudine); non-nucleoside; for example; efavirenz (DMP-266; DuPont Pharmaceuticals/Bristol Myers Squibb); nevirapine (Boehringer Ingleheim) and delaviridine (Pharmacia-Upjohn); TAT antagonist breast Ro 3-3335 and Ro 24-7429; protease inhibitor; for example; indinavir (Merck); ritonavir (Abbott); Saquinavir (Hoffmann LaRochw), viracept see nelfinaivr (Agouron Pharmaceuticals), 141W94 (Galxo-Wellcome); atazanavir (Bristol Myers Squibb); amprenavir (GlaxoSmithKline); fosamprenavir (GlaxoSmithKline), for Pune's Wei (BoehringerIngleheim), KALETRA (Lopinavir+ritonavir; Abbott); with other medicament breasts 9-(2-hydroxy ethoxy methyl) guanine (acycloguanosine), interferon, for example; alpha-interferon; the inhibitor of interleukin II and phosphonoformic acid salt (Foscarnet) or registration, for example T20 (enfuvirtide, Roche/Trimeris) or UK-427; 857 (Pfizer); levamisole or thymosin, cisplatin, NSC-241240; Docetaxel; paclitaxel, fluorouracil, capecitabine; gemcitabine; irinotecan, topotecan, etoposide; mitomycin; gefitinib, vincristine, vinblastine; amycin; cyclophosphamide, celecoxib, rofecoxib; Valdecoxib; ibuprofen, naproxen, ketoprofen; dexamethasone; prednisone, meticortelone, hydrocortisone; acetaminophen; misonidazole, amifostine, Tamsulosin; phenazopyridine; ondansetron, Ge Feisiqiong, A Luosi; palonosetron; promethazine, trimethobenzamide, aprepitant; diphenoxylate and atropine, and/or loperamide.Anticoagulant such as Antithrombin III, APC and protease inhibitor such as furin inhibitor.

Method also comprises prediction to the reaction of I α Ip treatment, measures the sample that obtains from individuality and detects I α I, P α I, I α Ip, H3, H4, H1, one or more level among H2 and the LC; The level that wherein detects represents that individuality can react I α Ip treatment well.For example, but the reduction of I α I and/or P α I detection level represents individual administration from I α Ip benefits.

Method also comprises the individual process of monitoring I α Ip treatment, measures I α I, P α I, I α Ip, H3, H4, H1, level before the one or more treatment among H2 and the LC; The I α Ip for the treatment of effective dose is delivered medicine to individuality; And measure one or more level in the first after date individuality of I α Ip treatment, wherein the raising of individual level represents that individuality may have good clinical response to I α Ip treatment after the I α Ip treatment.

The medicine box that is used for I α Ip treatment comprises I α I, P α I, I α Ip, H3, H4, H1, one or more among H2 and the LC; With the description that is used for the treatment of use.Medicine box also comprises I α Ip compositions described herein and operation instructions.For example, medicine box can contain I α I, P α I and the description that related dosage information, form of medication and preservation condition are provided.

Also relate to container, container comprises I α Ip and is inserted with label or the packing that I α Ip is delivered medicine to individual description.Description provides the explanation of dosage, form of medication and preservation condition.

Embodiment

To recognize and not will be understood that the present invention is subject to present described embodiment; On the contrary, will be understood that all equivalent variations that present invention resides in this any and all application and those skilled in the art's technical scope that provides.

Embodiment 1

Feature as the H4 of an I α Ip complex part

The SELDI-TOF mass spectral analysis shows that heavy chain 4 (H4) also is present in 125kDa band (P α I) and the 250kDa band (I α I) (data do not show).H4 in the 250kDa band exists more obvious.This expression I α I (H1+H2+LC) and P α I (H3+LC) another kind of complex protein in addition may be present in the some compositions of the present invention.The H4 of complex form was not also described up to now.Free H4 is less than 125 or 250kDa.Because the mass spectrum result, we think that H4 may with bikunin (light chain) or other be compound.

Embodiment 2

Pyemic animal model

Male Sprague-Dawley rat (275-325g) being housed in the room of control temperature, 12-h is bright/dark circulation and the solid type feedstuff of feeding standard P urina rat.Before inducing sepsis, with the rat overnight fasting, but can arbitrarily drink water.With isoflurane inhalation anesthesia rat, abdomen cervical region, abdominal part and groin are shaved the washing of Mao Bingyong 10% povidone iodine.Carry out 2-cm center line laparotomy.Caecum is exposed, and to avoid intestinal obstruction, with the pin puncture twice of 18-specification, slight extruding is flowed out a small amount of fecal matter from the hole, and gets back to the abdominal cavity in the ligation of ileocecal valve tip; Then sew up from level to level abdominal incision.Sham-operation is moved animal (that is, control animal) and is stood identical program, except caecum had not both had the not puncture of ligation liquid.With subcutaneous 3ml/100gBW normal saline animal is revived immediately after the operation.Then collection organization's sample is come with Animal Anesthesia in the different time interval after caecum ligation and puncture (CLP) or sham-operation.The laboratory animal usage criteria of all experimental basis National Inst. of Health (National Institutes of Health) carries out.This project obtains the approval of the Institutional Animal Care and Use Committee of North Shore Long Island Jewish Research Institute.

Preparation and the administration of people I α Ip

By-product as the program that designs from human plasma purifying blood coagulation Factor IX separates people I α Ip (I α I and P α I).This program relates to ion exchange and the size exclusion chromatograph of cryoprecipitate.Obtain approximately 70% purity after the chromatographic isolation.This preparation that contains I α Ip has very little side effect with regard to toxicity, thrombosis or hypotension.When sepsis outbreak rear 1,5,10 or 20h, under isoflurane anesthesia, use polyethylene 50-cannula in left femoral vein.By people I α Ip concentrate or the isopyknic carrier (normal saline, 1.5ml/ rat) of thigh conduit with the constant rate of inculcating intravenous administration 30mg/kg BW dosage.In experiment before, the administration of people I α Ip is in infusion procedure or after this do not change mean arterial pressure or heart rate (data do not show).

The mensuration of RNA extraction and liver bikunin gene

Liver is the main source of bikunin.Therefore, we measure the bikunin mrna expression in the liver.Extract total RNA by Tri-Reagent (Molecular Research Center, Cincinnati, OH) from liver.With the 100mg hepatic tissue homogenizing among the 1.5mlTri-Reagent, be separated into water and organic facies by adding chloroform, and centrifugal.Isolate RNA by adding isopropyl alcohol from aqueous phase, and use washing with alcohol.In, the deionized distilled water that 0.1%DEPC-processes with being precipitated and dissolved in.By 260 and 280nm measure concentration and the purity that absorptance is measured RNA.With 5 μ gRNA reverse transcription in 20 μ l reaction volumes of each tissue, reaction solution contains 50mM KCl, 10mM Tris-HCl, 5mM MgCl2,1mMdNTP, 20U RNA enzyme inhibitor, few d (T) 16 primers of 2.5mM and 50U reverse transcriptase.Reverse transcription reaction solution is hatched 1h at 42 ℃, then 95 ℃ of heating 5 minutes.With the cDNA of 3 ' and 5 ' primer amplification, the 1 μ l of 0.15 μ M separately, primer pair rat bikunin specificity (633bp) (5 ' TGA GGA ATA TGC CAT TTT CC 3 ', 5 ' CCACAG TAC TCC TTG CAC TCC 3 ') (accession number No.S87544), rat Glyceraldehyde-3-phosphate-dehydrogenase 7 (G3PDH) (24) is (5 ' TGA AGG TCGGTG TCA ACG GAT TTG GC 3 ' (983bp), 5 ' CAT GTA GGC CAT GAG GTCCAC CAC 3 '), in 25 μ l PCR mixture, contain 50mM KCl, 10mMTris-HCl, 2mM MgCl2,0.2mM dNTP and 0.7U AmpliTaq archaeal dna polymerase.At the enterprising performing PCR of Bio-Rad thermal cycler.Behind the RT-PCR, with reactant mixture electrophoresis in containing the 1.2%TBE-agarose of 0.22g/ml ethidium bromide of 5 μ l.Then produce gel and use Bio-Rad PS (Hercules, CA) by G3PDH with the band intensity standardization.

The mensuration of the radioiodination of protein and I α Ip half-life

Use Na ¹²⁵The I α Ip of I (Amersham, Arlington Heights, IL) radioiodination purification uses 1,3,4,6-tetrachloro-3a-6a-diphenylglycoluril (IODO-GEN iodating agent; Pierce, Rockford, IL) will.With reactant mixture use remove to the Excellulose GF-5 desalting column (Pierce) uncorporated ¹²⁵I.In gamma counter (Pharmacia-LKB, Piscataway, NJ), measure radioactivity.12 hours the time, suck Animal Anesthesia with isoflurane after CLP and the sham-operation.Intravenous injection pentobarbital sodium (～30mg/kg BW) is kept calm steady statue subsequently.Polyethylene-50 conduit is put into right jugular vein and left femoral artery, and pass through the I α Ip bolus injection (～500,000cpm/ rat) of neck sleeve pipe administration 125I-labelling.Measure remaining radioactivity in the syringe with gamma counter, and counting deducts the radioactivity that clean injection is measured in radiocounting before the initial injection.Collect immediately blood sample after the injection, then measured in the circulation in per 2 hours ₁₂₅Half-life (the t of I-I α Ip _1/2) 8 hours.Use gamma counter to measure the radioactivity (cmp) of each sample.According to Wu R, Zhou M, Cui X etc.: t is calculated in the removing (Chrelin clearance is reduced at the late stage of polymicrobialsepsis) that has reduced ghrelin in many microorganisms pyemic late period _1/2Int J Mol Med.2003；12：777-782。

Survival research

Carry out as mentioned above CLP.Behind CLP 1,5,10 or 10 and 20 hour the time, intravenous is inculcated people I α Ip concentrate (30mg/kg BW) or carrier (normal saline, the 1.5ml/ rat is behind the CLP 1 hour the time or behind the CLP 10 and 20 hours the time).20 hours the time, the downright bad caecum of excision also uses the warm physiological saline solution solution of 40ml with the abdominal cavity washed twice behind the CLP.Then abdominal incision is sewed up layer by layer.Simulation is excised program with regard to the caecum that the clinical setting of removing the septicopyemia focus carries out in the CLP animal whenever possible.Then allow animal arbitrarily feed and drinking-water and monitor over 10 days and record survival.

Statistical analysis

The result is expressed as on average ± SE.Use variance one tail analysis (ANOVA) and Tukey to test more on the same group laboratory animal.Calculate survival rate and relatively next by the test of logarithm level by the Kaplan-Meier method.If the difference of P＜0.05 value of thinking is significant.The change of bikunin mrna expression behind the CLP

Bikunin is the active part of I α Ip.Liver is the main source of bikunin.Therefore, we select bikunin mrna expression in the liver to react the generation of I α Ip.As shown in Figure 3, during 5h, the mrna expression of bikunin does not change in the liver behind CLP, yet, behind CLP, compare in the animal of the water of doing evil through another person during 20h, find to reduce by 32% (P＜0.05).

Behind the CLP ¹²⁵The t of I-I α Ip _1/2Change

The half-life that the blood content of the radioactive label I α Ip that injects during by the rear 12h of measurement sepsis outbreak changes to calculate I α Ip.As shown in Figure 4, behind the CLP ¹²⁵The t of I-I α Ip _1/2Significantly improve to 11.8 ± 2.7h (P＜0.05) from 5.6 ± 0.3h

I α Ip is to the effect of survival rate

The survival rate of single carrier administration after the excision of CLP and caecum when 1h (behind the CLP) was 75% at the 2nd day and was reduced to 50% (Fig. 5) at 5-10 days.Yet, behind the CPL during 1h administration people I α Ip in the whole 10 days observation stage, survival rate is increased to 92% (P＜0.05; Fig. 5).Although behind CLP 5 or during 10h administration people I α Ip survival rate is increased to 64% and 73% separately, these raisings do not have significance,statistical (Fig. 6).The survival rate of twice carrier administration after the excision of CLP and caecum when 20h (behind the CLP 10 and) was 56% at the 2nd day and was reduced to 44% (Fig. 7) at 5-10 days, and comparing with a carrier administration (Fig. 5) does not have significant difference.Yet, behind the CLP 10 and during 20h administration people I α Ip in the whole 10 days observation stage, survival rate is increased to 81%, comparing with vehicle group is (P＜0.05 of significant difference; Fig. 7).

Sepsis is the clinical symptoms that is characterised in that systemic inflammatorome, coagulopathy, respiratory failure, myocardial dysfunction, renal insufficiency and neuro-cognitive defective.It has been generally acknowledged that this symptom is that endogenous inflammatory mediator by the excessive initiation of invading micro-organism causes.These media comprise material such as cytokine, reactive oxygen species and the protease that mononuclear cell, macrophage, endotheliocyte and the neutrophil of activation discharge.In the serious inflammatory reaction, various blood and histiocyte comprise multinuclear type granulocyte, and born of the same parents discharge bacteriolyze protease outward and enter in the circulation.The normal born of the same parents' inner oxide matter that produces can cause tissue and organ injury and improve the clotting of plasma and the non-specific hydrolysis of complement factor in such protease and the phagocytosis.The release of neutrophil cell protease, especially human leukocyte elastase have related to the progress of the individual complication of sepsis.The order of severity of their blood plasma level and infection induced inflammation is closely related with the organ failure's who is about to occur high predicted.

In the process of people's septic shock, except the proteinase activity that improves, also reported the I α Ip level that reduces.The serious individuality that reduces of I α Ip concentration has higher mortality rate.Our result shows that the gene expression of bikunin in the CLP animal livers significantly is lower than in the sham-operation animal.In primates, pig and rodentine various tissues, detected and interior-mrna expression that the alpha inhibitor family protein is relevant.These genes that studies show that all member sources of I α Ip family are mainly at the liver transcription.Our result also shown with sham-operation and compared, and 5 or 20 hours the time, the bikunin gene expression in other organs (that is, intestinal and kidney) does not significantly change (data do not show) behind CLP.

Only observing remarkable downward modulation in liver shows that this organ is the important sources of I α Ip 20 hours the time behind the CLP, and in addition, in pyemic late period, the bikunin gene expression in the liver significantly reduces.In treatment, reported that bikunin has beneficial effect to the people as the prophylactic treatment of the organ injury after preventing the pancreatitis behind the gastrectomy or alleviating operation on heart.Measure bikunin in the bacteremic acute canine model of pathogenic escherichia coli effect studies show that similar result, have the improvement of hematodinamics variable and kinemic standardization and mean arterial pressure.Because the plasma half-life of bikunin very short (approximately 10 minutes), the half-life that therefore prolongs bikumin keeps the beneficial effect that this medicament continues and shows importance.Our result shows that the I α Ip half-life in the sham-operation animal is 5.6h.When we injected radiolabeled I α Ip when sepsis shows effect rear 12h, we found that the Increased Plasma Half-life of I α Ip is to 11.8h.These results show that I α Ip clearance rate significantly reduces in the sepsis process.Yet even have the clearance rate of reduction, the blood plasma level of I α Ip is still significantly lower in the sepsis individuality.That research before us has shown is early stage after the sepsis outbreak (that is, behind the CLP 1 hour), and administration low-purity I α Ip keeps whole body oxygen consumption and the whole body oxygen extraction ratio of cardiac output and whole body oxygen transmission and raising.In addition, the generation of I α Ip downward modulation TNF-α and reduce hepatocyte injury and lactic acidosis during 20h behind the CLP.Administration I α Ip had improved the survival of sepsis animal when in addition, sepsis showed effect rear 1h.

By-product as the classification of commercial scale blood plasma separates I α Ip albumen.Separation method height, Sync enrichment contain the main blood plasma form of bikunin albumen (I α I and P α I).Therefore, in this preparation, obtained the physiology compositions of blood plasma I α Ip.Survival rate when administration I α Ip excised rear 10 days with CLP and caecum when the mortality rate result of study of carrying out showed behind CLP 1 hour is increased to 92% from 50%.Although administration people I α Ip is increased to survival rate 64% and 73% separately 5 or 10 hours the time behind CLP, these raisings do not have significance,statistical.

Yet, behind CLP 10 and during 20h administration people I α Ip significantly survival rate is increased to 81% from 44%.Therefore, to appear be that useful additive comes for the survival rate that improves many microorganisms sepsis progression to I α Ip.In a word, in pyemic process, the bikunin gene expression in the liver reduces and the half-life of I α Ip was increased to 11.8 ± 2.7 hours from 5.6 ± 0.3 hours, has reduced the bikunin in the sepsis although show the clearance rate reduction.Behind the CLP 1 hour the time administration I α Ip survival rate is increased to 92% from 50%, and when behind the CLP 5 or during 10h administration I α Ip do not significantly improve.Yet 10 and 20 hours double injection I α Ip are increased to 81% with survival rate from 44% behind the CLP.The administration people I α Ip that postpones but repeat has improved the survival rate behind the CLP.

Embodiment 3

The purification of I α Ip

After eluate after application dialysis or the ultrafiltration/diafiltration is used DEAE Sephadex A50 solid phase extractions, with the 0.005M citric acid that pH6.0 contains 0.28M sodium chloride receive/0.0055M sodium phosphate buffer composition of combination a little less than the elution of DEAE-Sepharose FF post (is described in Hoffer etc., Journal of Chromatography B 669(1995) 187-196).In the step before, the 0.005M citric acid that contains 0.20M sodium chloride with pH6.0 is received/0.0055M sodium phosphate buffer washing pillar.

After the dialysis of 0.005M sodium phosphate buffer or ultrafiltration/diafiltration (UF/DF) to pH7.0, eluate is used to hydroxyapatite column.I α Ip does not collect in conjunction with pillar and as flowing out fraction.Can come elution impurity protein with the sodium phosphate buffer gradient of progressive concentration, mainly be FII, FVII and FX.I α I/P α I contains and is higher than 90% target protein, mainly is I α I and P α I.

Embodiment 4

Flow out purification I α Ip the fraction from factors IX

The unbinding protein (L.Hoffer etc., J.ofChromatography B) of heparin-agarose affinity chromatography is used to DEAE-Sepharose FF anion-exchange resin column.Behind the 0.005M phosphate buffer washing pillar with three column volume pH7.0 of minimum, come elution to contain the fraction of I α Ip/P α I with the 0.005M phosphate buffer that contains 0.55M sodium chloride (elution buffer) of pH7.0.Eluate contains the I α I/P α I of the 30-40% that has an appointment.After the dialysis of 0.005M sodium phosphate buffer or ultrafiltration/diafiltration (UF/DF) to pH7.0, the eluate of DEAESepharose FF is used to hydroxyapatite column.I α I/P α I does not collect in conjunction with pillar and as flowing out fraction.Can come elution impurity protein with the sodium phosphate buffer gradient of progressive concentration, mainly be FII and FX.The I α I/P α I of purification contains and is higher than 90% target protein.

Embodiment 5

With cold barren blood plasma at DEAE-Sephadex A50 (L.Hoffer etc., J.ofChromatography B) or Q-Sephadex A50 (D.Josic etc., ThrombosisResearch, with reference to above) on the eluant of solid phase extractions use (reference to the DEAC-CIM pipe monolith with 80mL column volume, K.Branovic etc., J.ofChromatography A 903(2000) 21-32).Collect unconjugated fraction (effluent).Use subsequently 0.02M Tris-HCl (level pad) the washing pillar of the pH7.4 of three column volumes.With the unconjugated protein of 0.02M Tris-HCl elution (eluate 1) of the pH7.4 that contains 0.35Mol/L sodium chloride, use the 0.02M Tris-HCl (elution 2) of the pH7.4 that contains 0.55M sodium chloride in the second step in the first step.In flowing out fraction and eluate 1, found I α I/P α I.The outflow fraction contains the I α I/P α I of the 35-45% that has an appointment.The amount of these target proteins is 20-30% in the eluate 1.The fraction that will contain I α I/P α I is accepted dialysis or the ultrafiltration/diafiltration (UF/DF) of the 0.005M sodium phosphate buffer of pH7 and is used to hydroxyapatite column.I α Ip does not collect in conjunction with pillar and as flowing out fraction.The outflow fraction contains and is higher than 90% I α I/P α I.

Fig. 8 a and 8b have described the exemplary purification scheme with the I α Ip of flowcharting that presents such as embodiment 3-5.

Embodiment 6

The beneficial effect of highly purified I α Ip in zooscopy

In male Sprague-Dawley rat, induce many microorganisms sepsis by caecum ligation and puncture (CLP).Before carrying out CLP, animal feed is spent the night but can arbitrarily drink water.During experiment, suck rat anesthesia by the methyl halothane, and carry out the 2-cm center line and cut open the belly.Caecum is exposed, only avoid intestinal obstruction in the ligation of ileocecal valve distally, with the pin puncture twice of 18-specification, leniently extrude a small amount of feces, then return the abdominal cavity.Abdominal incision is sewed up layer by layer, behind the CLP with animal immediately the subcutaneous 30mL/kg of acceptance body weight normal saline solution revive as fluid.Use two groups of rats, every group of n=12 in this experiment.Treatment group is accepted the highly purified I α Ip of 30mg/kg body weight 10 and 20 hours the time behind CLP.Matched group is accepted saline.Behind the CLP 20 hours the time, the caecum that excision is downright bad and with the warm physiological saline solution solution of 40mL with the abdominal cavity washed twice.Then abdominal incision is sewed up layer by layer.The caecum excision program of carrying out in the CLP animal is simulated the clinical setting of wherein removing the septicopyemia focus.The permission laboratory animal is arbitrarily taken food and is monitored the death of recording non-viable person over 10 days.Test the mortality rate between the more different treated animals and measure the p value by the Kaplan-Meier method with the logarithm level.Compare with the saline control group, observe significantly improve (81.3% surviving animals is to 44% survival (p value=0.0293) in the matched group in the treatment group) of surviving in the treatment group, show that highly purified I α Ip is bioactive and effective in the sepsis associated death that reduces the sepsis animal.The results are shown among Fig. 9.

The comparison of table 1.I α Ip rejection ratio activity

I α Ip preparation purified form	Protein concentration [mg/mL]	I α Ip concentration [mg/mL]	I α Ip purity [%]	Rejection ratio active [TIU/mg I α Ip]
					Cryoprecipitate	7.30	5.10	69.86％	1412±47.52
Cold barren blood plasma	17.30	17.00	98.27％	1409±42.50

TIU=TIU; On average ± SD from single independently the experiment

The rejection ratio of the I α Ip of computed altitude purification (come self cooling barren blood plasma) is active and compare with the I α Ip of cryoprecipitate purification, and cryoprecipitate is the by-product that the FVIII factor is produced.Suppress to use in the test product color substrate L-BAPA (N (α)-benzoyl-L-arginine-4-p-nitroanilide hydrochlorate) (Fluka Chemicals) to measure the biological activity of I α Ip at trypsin.This test is based on the ability that I α Ip suppresses the L-BAPA hydrolysis.δ absorptance by the 410nm place/minute rate reduction monitor inhibition.Test the quantitative measurement protein concentration by BioRad protein, and measure I α Ip concentration by competitive ELISA with MAb69.31, such as Lim etc., J.of Infectious Disease, described in 2003.Do not have significant difference (p value=0.939) from the specificity trypsin inhibition activity between the I α Ip preparation of cold barren blood plasma and cryoprecipitate purification, show that two kinds of I α Ip in the fraction have suitable biological activity.

Embodiment 7

By-product from the FIX purification

" washing fraction " is from thrombin FIX chromatogram purification on the DEAE-Sepharose FF (Josic etc., Journal of Chromatography, above drawing).Come this fraction of elution with the 0.01-0.1M sodium citrate that contains 0.25M sodium chloride of pH6.0/0.005-0.1M sodium phosphate buffer.I α I and P α I are the main components in this fraction.In next step, contain the fraction of FIX from the elution of DEAE-Sepharose FF post with the 0.01-0.1M sodium citrate that contains 0.3-0.6M sodium chloride of pH6.0/0.005-0.1M sodium phosphate buffer.Also contain the proconvertin (FVII) of other vitamin K-dependent clotting factors such as prothrombin (FII), Stuart factor (FX), lower content and the I α Ip of residual quantity in this fraction.After reducing the dialysis of osmotic pressure and salinity, by the affinity chromatography on the fixing heparin and increase progressively salinity and osmotic buffering agent in the elution step collect remaining I α Ip in the FIX fraction.Fraction in the lavation buffer solution of early stage elution step contains and surpasses 80% I α Ip and low-down FIX impurity.In higher salt concentrations after the elution of FIX occurs in and the osmotic pressure buffer step.Also exist not in conjunction with the outflow fraction of pillar, be without FIX's and contain I α Ip (30-40%), vitamin K-dependent clotting factor and be used for the mixture of the solvent/detergent (S/D) of inactivation of virus.Can in other DEAE-Sepharose FF chromatographic step, remove S/D.

Collect the fraction contain I α Ip from DEAE-Sepharose, the effluent that is fixed in heparin or heparin can be further purified or individually as the set of hydroxyapatite.Use any method, final preparation contains and is higher than 90% ITI.

Use the concentrate of these program purification to contain and be higher than 90% I α I/P α I.By solvent/detergent treatment with inactivation of virus.Can introduce with or without the final heating more than 30 minutes in the final container of stabilizing agent or in the presence of stabilizing agent 55-65 ℃ of pasteurize be used as second inactivation of virus step and activity is not significantly lost.

Process or as second inactivation step with S/D, with or without stabilizing agent with the final heating of the protein of purification 30 minutes, perhaps, 55-65 ℃ of pasteurize is with the resulting concentrate inactivation of viruses that is higher than 90%I α I/P α I that contains in the presence of stabilizing agent.

Reinforcing YIN-essence ion exchange fraction

Substitute the eluate after the weak anionic exchange DEAE Sephadex A50 solid phase extractions, can use the eluate after the reinforcing YIN-essence ion exchange Q Sephadex A50 solid phase extractions.The condition of elution is described among the German patent DE 4342131C1.The mixture of I α I and P α I not in conjunction with or just faintly in conjunction with whole anion-exchange support DEAE-CIM.Elution goes out other protein such as thrombin FII, FVII, FIX and FX in the fraction that separates, Coagulative inhibitors agent PC, PS and PZ, adhesion protein vitronectin and Protease F SAP.In next step, use hydroxyapatite chromatography can obtain the further separation of residual impurity.

The monolithic chromatogram fraction

The chromatographic isolation that DEAE CIM whole (film) can be used for the FIX purification substitutes DEAE Sepharose FF or other anion exchangers based on granule (referring to DE4342132C1).Surprisingly, I α I and P α I not in conjunction with or just faintly in conjunction with integral carrier.Other protein obtain elution such as FIX, vitronectin and FII, FVII (low amount) and FX as the fraction that separates.In this purification step, mainly from proteolytic activity (J.Roemisch, the Biological Chemistry of factor VII activator protein enzyme (FASP) 383(2002) 1119-1124) also obtained separating fully.In next step, obtain to contain the further separation of the residual impurity of I α I/P α I fraction with hydroxyapatite chromatography, i.e. trace vitamin K-dependent clotting factor FII, FVII and FX.

All publications of quoting in this application and patent document are incorporated herein by reference for all purposes to same degree with its integral body, and independently publication and patent document are separately expressions as each.Unless otherwise noted, has the meaning that those skilled in the art in the invention understand usually in these all used technology and scientific terminology.Following list of references provides the General Definition of used many terms: Singleton etc. among the present invention to the technical staff, microorganism and molecular biology dictionary (Dictionary of Microbiology and Molecular Biology) (the 2nd edition, 1994); Cambridge technical dictionary (The Cambridge Dictionary of Science andTechnology) (Walker edits, 1988); Hereditism's term (The Glossary ofGenetics), the 5th edition, R.Rieger etc. (editor), Spring Verlag (1991); And Hale﹠amp; Marham, Harper Collins dictionary biology (The Harper CollinsDictionary of Biology) (1991).

Introduce list of references

The content that runs through all lists of references (comprising list of references, disclosed patent, disclosed patent application and co-pending patent application) that the application quotes specially is incorporated herein by reference with its integral body at this.

Equivalent

Those skilled in the art use not transnormal experiment will recognize the many equivalents that maybe can determine particular of the present invention described herein.Determine that such equivalent is included by following claim.

Claims (98)

1. produce the method for the I α Ip compositions that is derived from blood plasma, said composition contains the mixture of interior-alpha inhibitor protein and front-alpha protein, wherein interior-alpha inhibitor protein and front-alpha protein are present in the described mixture with the physiology ratio, and the method comprises:

Isolate the blood plasma fraction that contains interior-alpha inhibitor protein and front-alpha protein from blood plasma, wherein interior-alpha inhibitor protein and front-alpha protein exist with the physiology ratio, wherein separate to comprise solid phase extractions or blood plasma is carried out chromatographic isolation; With

It is 85% to 100% pure I α Ip compositions that the described blood plasma level of purification assigns to obtain I α Ip purity, and wherein purification comprises the effluent of anion chromatographic and acquisition hydroxyapatite chromatography.

2. the process of claim 1 wherein and come solid phase extractions by DEAE Sephadex.

3. the process of claim 1 wherein that chromatograph comprises anion-exchange chromatography.

4. the method for claim 3, wherein anion-exchange chromatography is based on granule.

5. the method for claim 4, wherein granule contains immobilized anion exchange groups, and it is selected from DEAE Sephadex, DEAE Sephadex A50, Toyopearl DEAE, TMAE Fractogel, DEAE Fractogel or Q-Sepharose.

6. the process of claim 1 wherein and by integral carrier blood plasma is carried out chromatographic isolation.

7. the method for claim 6, wherein integral carrier is the CIM with immobilization anion exchange part, it is selected from DEAE-CIM or Q-CIM.

8. each method of claim 1-7, wherein the blood plasma fraction comprises the by-product fraction that the purifying blood coagulation factors IX obtains.

9. each method of claim 1-7, wherein the blood plasma fraction comprises the by-product fraction of purifying blood coagulation proenzyme complex concentrate.

10. each method of claim 1-7 is wherein come the separated plasma fraction as the cold supernatant that the blood plasma cryoprecipitate obtains.

11. each method of claim 1-7, wherein the blood plasma fraction is cold barren blood plasma.

12. each method of claim 1-7, wherein the blood plasma fraction is the blood plasma fraction of people, primates, cattle, pig, cat or dog.

13. each method of claim 1-7 further comprises acquisition blood.

14. each method of claim 1-7 further comprises acquisition blood plasma.

15. each method of claim 1-7 further comprises obtaining the by-product fraction that the purifying blood coagulation factors IX obtains.

16. each method of claim 1-7 further comprises the by-product fraction that obtains the compound concentrate of purifying blood coagulation proenzyme.

17. each method of claim 1-7 further comprises obtaining the cold supernatant that the blood plasma cryoprecipitate obtains.

18. each method of claim 1-7 further obtains cold barren blood plasma.

19. each method of claim 1-7 is wherein come purification by affinity chromatography.

20. the method for claim 19, wherein affinity chromatography is heparin.

21. the method for claim 18 is wherein come purification by ion exchange chromatography and hydroxyapatite chromatography.

22. each method of claim 1-7, wherein be present in the blood plasma fraction interior-inhibitor protein matter and front-alpha protein have approximately 60,000 to approximately 280, the apparent molecular weight of 000kDa.

23. the method for claim 22 is wherein come determining molecular weight by SDS-PAGE.

24. each method of claim 1-7 further comprises being further purified the blood plasma fraction.

25. the method for claim 24 wherein flows through (not combination) level by heparin affinity column and collection and assigns to be further purified.

26. each method of claim 1-7 comprises that further the I α Ip with blood plasma fraction and/or purification carries out inactivation of virus.

27. the method for claim 26 is wherein carried out inactivation of viruses by solvent/detergent treatment or hot deactivation.

28. the method for claim 27, wherein hot deactivation is approximately 55 to approximately 65 ℃ temperature or xeothermic at 70 to 120 ℃.

29. the method for claim 26 further comprises the adding stabilizing agent.

30. the method for claim 26, further comprise with the I α Ip pasteurize of purification or approximately 70 to approximately 120 ℃ xeothermic.

31. each method of claim 1-7 comprises that further the I α Ip with purification carries out anion-exchange chromatography.

32. the method for claim 31, wherein anion-exchange chromatography is DEAE Sepharose.

33. each method of claim 1-7, wherein I α Ip compositions has and is higher than 1 hour half-life.

34. each method of claim 1-7, wherein I α Ip compositions has and is higher than 5 hours half-life.

35. each method of claim 1-7, wherein to have high trypsin rejection ratio active for I α Ip compositions.

36. the method for claim 35, wherein trypsin rejection ratio activity is approximately 1000 to about 2000IU/mg.

37. in containing-the I α Ip compositions of the mixture of alpha inhibitor protein and front-alpha protein, wherein in-alpha inhibitor protein and front-alpha protein be present in the described mixture with the physiology ratio, for approximately 85% to approximately 100% pure.

38. the compositions of claim 37, wherein I α Ip comprise approximately 60% to approximately 80% interior-alpha inhibitor protein and approximately 40% to approximately 20% front-alpha protein.

39. the compositions of claim 37, wherein the physiology ratio be in the human plasma natural present interior-ratio of alpha inhibitor protein and front-alpha protein.

40. the compositions of claim 37, wherein protein is used for the treatment of the human disease.

41. the compositions of claim 40, wherein the human disease is acute inflammation disease, sepsis, serious shock, septic shock, rheumatoid arthritis, cancer, cancerometastasis, wound/damage, infectious disease and preterm delivery.

42. the compositions of claim 37 further comprises stabilizing agent.

43. the compositions of claim 42, wherein stabilizing agent is albumin, Polyethylene Glycol, α, α-trehalose, aminoacid, salt, glycerol, omega-amino acid, sugar or its combination.

44. contain the I α Ip compositions of the mixture of interior-alpha inhibitor protein and front-alpha protein, wherein interior-alpha inhibitor protein and front-alpha protein are present in the described mixture with the physiology ratio and have high trypsin rejection ratio activity.

45. the compositions of claim 44, wherein I α Ip comprise approximately 60% to approximately 80% interior-alpha inhibitor protein and approximately 40% to approximately 20% front-alpha protein.

46. the compositions of claim 44, wherein the physiology ratio is the natural ratio that presents in the human plasma.

47. the compositions of claim 44, wherein protein is used for the treatment of the human disease.

48. the compositions of claim 47, wherein the human disease is acute inflammation disease, sepsis, serious shock, septic shock, rheumatoid arthritis, cancer, cancerometastasis, infectious disease and preterm delivery.

49. the compositions of claim 44 further comprises stabilizing agent.

50. the compositions of claim 49, wherein stabilizing agent is albumin, Polyethylene Glycol, α, α-trehalose, aminoacid, salt, glycerol, omega-amino acid, sugar or its combination.

51. in containing-the I α Ip compositions of the mixture of alpha inhibitor protein and front-alpha protein, wherein in-alpha inhibitor protein and front-alpha protein be present in the described mixture with the physiology ratio and have and be higher than one hour half-life.

52. the compositions of claim 51, wherein I α Ip comprise approximately 60% to approximately 80% interior-alpha inhibitor protein and approximately 40% to approximately 20% front-alpha protein.

53. the compositions of claim 51, wherein I α Ip compositions has at least 5 hours half-life.

54. the compositions of claim 51, wherein I α Ip compositions has at least 10 hours half-life.

55. the compositions of claim 51, wherein the physiology ratio is the natural ratio that presents in the human plasma.

56. the compositions of claim 51, wherein protein is used for the treatment of the human disease.

57. the compositions of claim 56, wherein the human disease is acute inflammation disease, sepsis, serious shock, septic shock, rheumatoid arthritis, cancer, cancerometastasis, infectious disease and preterm delivery.

58. the compositions of claim 51 further comprises stabilizing agent.

59. the compositions of claim 58, wherein stabilizing agent is albumin, Polyethylene Glycol, α, α-trehalose, aminoacid, salt, glycerol, omega-amino acid, sugar or its combination.

60. contain the I α Ip compositions of the mixture of interior-alpha inhibitor protein and front-alpha protein, wherein-alpha inhibitor protein and front-alpha protein be present in the described mixture with the physiology ratio, comprise with three heavy chain H1, H2 and H3 at least one combination interior-light chain of alpha inhibitor protein.

61. the compositions of claim 60, wherein I α Ip comprise approximately 60% to approximately 80% interior-alpha inhibitor protein and approximately 40% to approximately 20% front-alpha protein.

62. the compositions of claim 60, wherein the physiology ratio be in the human plasma natural present interior-ratio of alpha inhibitor protein and front-alpha protein.

63. the compositions of claim 60, wherein protein is used for the treatment of the human disease.

64. the compositions of claim 63, wherein the human disease is acute inflammation disease, sepsis, serious shock, septic shock, rheumatoid arthritis, cancer, cancerometastasis and infectious disease.

65. the compositions of claim 60 further comprises stabilizing agent.

66. the compositions of claim 60, wherein stabilizing agent is albumin, Polyethylene Glycol, α, α-trehalose, aminoacid, salt, glycerol, omega-amino acid, sugar or its combination.

67. contain the I α Ip compositions of the mixture of interior-alpha inhibitor protein and front-alpha protein, wherein-alpha inhibitor protein and front-alpha protein be present in the described mixture with the physiology ratio, comprise with four heavy chain H1, H2, H3 and H4 at least one combination interior-light chain of alpha inhibitor protein.

68. the compositions of claim 67, wherein I α Ip comprise approximately 60% to approximately 80% interior-alpha inhibitor protein and approximately 40% to approximately 20% front-alpha protein.

69. the compositions of claim 67, wherein the physiology ratio be in the human plasma natural present interior-ratio of alpha inhibitor protein and front-alpha protein.

70. the compositions of claim 67, wherein protein is used for the treatment of the human disease.

71. the compositions of claim 70, wherein the human disease is acute inflammation disease, sepsis, serious shock, septic shock, rheumatoid arthritis, cancer, cancerometastasis, infectious disease and preterm delivery.

72. the compositions of claim 67 further comprises stabilizing agent.

73. the compositions of claim 72, wherein stabilizing agent is albumin, Polyethylene Glycol, α, α-trehalose, aminoacid, salt, glycerol, omega-amino acid, sugar or its combination.

74. the I α Ip compositions of the mixture that contains interior-alpha inhibitor protein and front-alpha protein that each makes according to claim 1-36, wherein interior-alpha inhibitor protein and front-alpha protein are present in the described mixture with the physiology ratio.

75. each compositions of claim 37-74 further comprises other therapeutic agent.

76. the compositions of claim 75, wherein other therapeutic agent is anticarcinogen, antiinflammatory, anticoagulant or immunomodulator.

77. pharmaceutical composition, comprise in the claim 37,44,51,60,67 or 74 each the treatment compositions useful and materia medica on acceptable carrier.

78. the I α Ip that each method of claim 1-36 produces is for the preparation of the purposes in the medicament for the treatment of individual inflammation related disease, cancer or infectious disease.

79. the purposes of claim 78, wherein, described I α Ip separates I α Ip from individuality.

80. the purposes of claim 79, wherein individuality is people, cattle, pig, goat or primate.

81. the purposes of claim 78 is wherein with tablet, capsule or injection preparation I α Ip.

82. the purposes of claim 78, wherein I α Ip is at least 85% pure.

83. the purposes of claim 78, wherein I α Ip is approximately 85% to approximately 100% pure.

84. the purposes of claim 78, wherein I α Ip comprise approximately 60% to approximately 80% interior-alpha inhibitor protein and approximately 40% to approximately 20% front-alpha protein.

85.I α Ip is preparing with the purposes in the reagent of following method, described method is the method for the individual acute inflammation disease for the treatment of, sepsis, serious shock, septic shock, rheumatoid arthritis, cancer, cancerometastasis, infectious disease or preterm delivery, and the method comprises:

(a) measure in the individuality level before the treatments one or more in the following level:

(i) level of interior-alpha inhibitor protein;

(ii) level of front-alpha protein;

(iii) level of I α Ip;

(iv) level of H3;

(v) level of H4;

(vi) level of H1;

(vii) level of H2; With

(viii) level of LC; With

The I α Ip that (b) will treat effective dose delivers medicine to individuality,

(c) measure level after the just treatment of the one or more levels of after date of I α Ip treatment, the wherein adjusting of the I α Ip level treatment that represents to produce good clinical response.

86. the purposes of claim 85, wherein said method further comprises:

(c) level after the treatment of the first one or more levels of after date of mensuration I α Ip treatment,

The wherein adjusting of the I α Ip level treatment that represents to produce good clinical response.

87. the purposes of claim 86, wherein regulating is the raising of I α Ip level.

88. the purposes of claim 85, in wherein measuring by immunization method-level of alpha inhibitor protein, front-alpha protein, I α Ip, H3, H4, H1, H2 and LC.

89. the purposes of claim 85, wherein inflammation, tumor invasion, neoplasm metastasis, septicopyemia are whole in order to suffer from, septic shock, rheumatoid arthritis, preterm delivery, cancer or infectious disease for Individual identification.

90. the purposes of claim 85, wherein the treatment initial stage is the I α Ip needed time of plasma concentration that obtains steady statue.

91. the purposes of claim 85 further comprises the therapeutic agent that administration is other.

92. the purposes of claim 91, wherein other therapeutic agent is anticarcinogen, antiinflammatory, anticoagulant or immunomodulator.

93.I α Ip is for the preparation of the purposes in the reagent of following method, described method is the method for prediction I α Ip therapeutic response, and the method comprises:

Mensuration detects with lower one or more level from the sample that individuality obtains:

(i) in-alpha inhibitor protein;

(ii) front-alpha protein;

(iii)IαIp；

(iv)H3；

(v)H4；

(vi)H1；

(vii) H2; With

(viii)LC；

The level that wherein detects represents the individuality of sound response I α Ip treatment.

94. the purposes of claim 93, the level that wherein detects is the reduction of interior-alpha inhibitor protein and front-alpha protein level.

95.I α Ip is for the preparation of the purposes in the reagent of following method, described method is the method for the individual progress of monitoring I α Ip treatment, and the method comprises:

(a) measure the front level of treatments one or more in the individual following level:

(i) level of interior-alpha inhibitor protein;

(ii) level of front-alpha protein;

(iii) level of I α Ip;

(iv) level of H3;

(v) level of H4;

(vi) level of H1;

(vii) level of H2; With

(viii) level of LC;

The I α Ip that (b) will treat effective dose delivers medicine to individuality; With

(c) level after the treatment of the first one or more levels of after date of mensuration I α Ip treatment,

Wherein the raising of individual level represents that individual I α Ip is treated has good clinical response after the I α Ip treatment.

96. be used for the medicine box of I α Ip treatment, comprise following one or more:

(i) in-alpha inhibitor protein;

(ii) front-alpha protein;

(iii)IαIp；

(iv)H3；

(v)H4；

(vi)H1；

(vii) H2; With

(viii) LC; With

The description that treatment is used.

97. comprise the compositions of container, container comprises I α Ip and is inserted with relevant label or the packing that I α Ip is delivered medicine to individual description.

98. medicine box comprises claim 37,44,51,60,67 or 74 each compositions and the description used for the treatment of.

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