CN101160133A - 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

The proteinic preparation of interior-alpha inhibitor and the compositions from human plasma of therapeutic use

Related application

The denomination of invention that the application comprises application on November 8th, 2003 is disclosed related subject among the temporary patent application series No.60/__ of " the proteinic preparation of interior-alpha inhibitor and the compositions from human plasma of therapeutic use ", and 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, undertakes the serpin activity of molecule.Name " bikunin " reflects that the protease that has 2 Kunitz types suppresses domain.In the normal blood plasma, find that most of bikunin is a complex form, as interior-alpha inhibitor (I α I), it has molecular weight and the preceding-alpha inhibitor (P α I) of 225kDa, 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, have only single heavy chain (H3) to connect bikunin.In these complex forms, bikunin keeps passivation until discharging by the Partial Protein hydrolytic degradation, and this is as the mechanism of regulating active mode.After the division, by renal glomerular filtration activated bikunin 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, promptly short-decayed problem in fact.

Although introduced antibiotic before more than 50 years, it has seen that really the inductive mortality rate of sepsis is reduced to 35% from 55%, and medical science does not significantly reduce and is benefited from sepsis individual death rate.In fact, to continue be one of intensive care unit's cause of death and a large amount of sepsis individualities is died from subsequently septic shock and multiple organ failure, MOF to sepsis.Sepsis is to infecting for example systemic reaction of bacterial infection.It is normally caused by 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 use conventional medicine to treat sepsis fast and reliably.

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 the method and uses thereof of the interior-alpha inhibitor protein (I α Ip) of purification human plasma, be used for the treatment of the human disease, as sepsis, acute inflammation disease, serious shock, septic shock, rheumatoid arthritis, cancer, cancerometastasis, infectious disease and premature labor childbirth; Or be used to reduce sepsis, acute inflammation disease, serious shock, septic shock, rheumatoid arthritis, cancer, cancerometastasis, mortality rate risk that infectious disease is relevant with the premature labor childbirth.

According on the one hand, produce the I α Ip method for 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 about 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 preceding-alpha protein (P α I), wherein I α I and P α I are present in the described mixture with the physiology ratio, and about 85% to about 100% is pure.

In the related fields, I α Ip compositions comprises the mixture of interior-alpha inhibitor protein (I α I) and preceding-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 bonded in-the proteinic light chain of alpha inhibitor forms.

In another related fields, the compositions of I α Ip comprises the mixture of interior-alpha inhibitor protein (I α I) and preceding-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 bonded in-the proteinic light chain of alpha inhibitor.

In the related fields, make I α Ip according to following method again, this 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 about 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 to the method for the treatment of individual inflammation related disease, cancer or infectious disease on the other hand, 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 of treatment comprises the preceding 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.This 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 detected identify can sound response I α Ip treatment individuality.

In another related fields, describe the method for the individual progress of treatment of monitoring I α Ip treatment, and comprised the preceding 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 of treatment 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 I α Ip treatment back individual level represents that individuality may have good clinical response to 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 the slight supracellular red pulp (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 the change that bikunin mRNA expresses in the liver in the time of 5 and 20 hours of sham-operation (Sham) back.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 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) back 125I-I α I in the time of 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 use the test of logarithm level to come relatively by the KapIan-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 use the test of logarithm level to come relatively by the KapIan-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 use the test of logarithm level to come relatively by the KapIan-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 purifying I α Ip. Further disclosed at this is the therapeutic combination of purifying I α Ip, is used for delivering medicine to individuality and treats acute inflammation disease, pyemia, serious shock, septic shock, rheumatoid arthritis, cancer, metastasis of cancer, infectious disease and preterm delivery.

Known purifying 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 FX be form thrombus and deliver medicine to the people and be harmful to. The FX pollution problem of I α Ip composition before method described herein has solved.

In-alpha inhibitor protein (I α Ip) is serine protease inhibitors family relevant on the structure, finds to be relatively high concentration (400-800mg/L) in people's blood plasma. I α Ip is large, multicomponent compound, as insulin-type protease inhibitors. Different with other inhibitor molecules, the inhibitor of this family by the composition of polypeptide chain (light chain and heavy chain) uniquely covalently bound sulfuric acid chondroitin chain form. The heavy chain (H1, H2 and H3) of interior-alpha protein is also referred to as hyalomitome acid (HA) in conjunction with albumen. The main form of finding in people's blood plasma is interior-α-inhibitor (I α I), it is comprised of two heavy chains (H1﹠H2) and single light chain (L), with 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 structure territories) and extensively suppresses blood plasma serine protease. Compound has demonstrated the importance in suppressing the protease array, and the protease array comprises neutrophilia elastoser, plasmin, trypsase, organizes protease G and sperm enzyme.

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 composition 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 compound, to prevent that HA is in conjunction with its acceptor 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 compound.

" I α Ip composition " 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 illness in the ratio found, and/or the ratio of the I α I of natural appearance in people's blood plasma and P α I. The physiology ratio is generally about 60% to about 80%I α I and about 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 contain the composition of I α I and P α I complex. Mixture can also contain buffer, salt or for separating of other compositions of I α Ip compound. 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 about 60,000 to about 280, the apparent molecular weight of 000kDa. Come determining molecular weight by sodium dodecyl sulfate polyacrylamide gel electrophoresis.

The time area of a room of half when " half-life " refers to that the I α Ip activity of administration is administration as used in this. The half-life of I α Ip composition is according to the present invention, for example, and greater than about 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 composition is higher than about 5 hours. In the particularly preferred embodiment, the half-life of I α Ip composition is higher than about 10 hours. Preferred long half-life, for example, because need to deliver medicine to individual dosage lower along with the time.

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

The composition 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 composition 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 plasma separation or purifying. That is, the natural environment of composition is blood plasma.

" blood plasma fraction " is that original source is from blood plasma from the isolated or purified step fraction of chromatogram for example as used in this. Blood plasma fraction according to the present invention can be, for example, from the accessory substance of purifying blood coagulation factors IX acquisition, from the accessory substance of the compound concentrate acquisition of purifying blood coagulation proenzyme, the cold supernatant liquid that obtains from cryoprecipitate blood plasma (is described in Hoffer etc., Journal of Chromatography B669(1995) 187-196), or cold barren blood plasma (poor plasma), cold barren blood plasma is used alternatingly at this and cold supernatant liquid. Cold barren blood plasma is the supernatant liquid that obtains from cryoprecipitate.

Accessory substance 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 accessory substance of factors IX (FIX) purge process generation. The method that obtains accessory substance from the purifying blood coagulation factors IX is described in Hoffer etc., Journal of Chromatography B669(1995) 187-196, it is incorporated herein by reference with its integral body at this. Other examples of accessory substance comprise from the accessory substance of purifying FIX or from the accessory substance of the compound concentrate of purifying blood coagulation proenzyme, as be 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 accessory substance that obtains cold supernatant liquid separation as the blood plasma cryoprecipitate. (for example, suitable cryoprecipitate method is described in Hoffer etc., Journal of Chromatography B669(1995) 187-196). Be used for comprising strong anion exchange fraction and monolithic chromatogram fraction from other blood plasma fractions of blood purifying I α Ip complex, will in following embodiment, describe.

According to the present invention, the blood plasma fraction can be from people, primate, ox, 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, primate, ox, pig, cat or dog source. For example, can originate to obtain and/or buy blood from blood bank, hospital, collecting post, private company, research foundation meeting or any other blood.

" acquisition blood plasma " as used in this, for example comprise from people, primates, cattle, pig,, cat or dog source obtain blood plasma.For example, can originate from blood bank, hospital, asylum, private company, WARF or any other blood and obtain and/or buy blood plasma.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 " 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 from the cold supernatant of blood plasma cryoprecipitate acquisition.

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

" absorption " refers to analyte and combines with detecting of adsorbent or capture agent is non-covalent.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, hydrophobicity absorption/electrostatic repulsion adsorbent) of mixed model.

The biologic specificity adsorbent refers to the adsorbent that comprises biomolecule, biomolecule for example, nucleic acid molecules (for example, adaptive son), polypeptide, polysaccharide, lipid, steroid or these conjugate (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 to the specificity of target analytes usually than chromatograph adsorbent height.

" eluant " or " wash solution " refers to and is used to influence or changes the absorption of analyte and 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 composition 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 that wherein one or more amino acid residues are amino acid polymers of natural generation aminoacid respective analogs 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 to use the test of the antibody of specificity conjugated antigen (for example, I α Ip complex).Immunoassay is characterised in that the specificity binding characteristic that uses 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 combination specifically 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 to the association reaction that decision protein exists in the multiple colony of protein and other biological goods.Therefore, under specified immunoassay condition, specific antibodies specific protein double at least background and basically not with sample in other protein of existing combine with significant quantity.Combine needs with the specificity of antibody under the condition like this and select the specificity of antibody specific protein.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 use various immunoassay forms to select antibody with the specified protein specific immune response.For example, use usually solid phase ELISA immunoassay select with the immunoreactive antibody of protein specific (referring to, for example, Harlow﹠amp; Lane, Antibodies, ALaboratory Manual (1988) has described the immunoassay form and the condition that are used to measure 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 goes up in the similar body substantially to said I α Ip protein or any albumen of external activity as used in this, for example, influences pyemic reduction.

For example, confirm the 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 proteinic all the natural generations of I α Ip as used in this, comprises the protein that contains deletion, insertion, interpolation and replace.I α Ip proteinic " natural variant " 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 wherein the aminoacid of Qu Daiing 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 the biological or immunocompetent guidance of elimination.

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

" insertion " or " interpolation " is to compare with the I α Ip complex of natural generation, makes aminoacid or nucleotide sequence that one or more aminoacid or nucleotide residue add change separately.

Separately by different aminoacid or 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 " orientate as natural, reorganization or synthetic I α Ip complex or its any oligopeptide induce in suitable animal or the cell specific immune response and with the bonded ability of specific antibodies.

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 the 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.Isolating refer to from its primal environment (for example, if natural generation for natural environment) in the material that takes out, and therefore " pass through the people " and change from its native state.For example, isolating polypeptide or protein can be the compositions of blood plasma, maybe can be contained in the cell and think " isolating ", 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 particulate, 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 carriers, for example, has CIM such as the 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.The reinforcing YIN-essence ion exchange column, as have those of quaternary ammonium part, in wide pH scope, have permanent positive charge.In the weak anionic exchange column, 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 preferably about 85% to about 100% is pure.As used in this, term " pure " refers to the I α Ip compositions of taking out from its natural environment, and is isolating or separate, and do not have at least about 85% to about 100%, and preferred 90% does not have, and more preferably 95% does not have and its natural other relevant compositions.In the preferred embodiment, the protein of purification will constitute and be higher than 85%, 87.5%, 90%, 92.5%, 95%, 99% or even higher protein in the compositions basically.

As utilization to the peptide of " being purified to homogeneous " of the present invention, polypeptide or the protein meaning is that peptide, polypeptide or protein have the purity level that wherein peptide, polypeptide or protein are gone up other protein of nothing and biotic component substantially.One or more separating steps that any suitable material and method 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.Isolating method 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.Anion-exchange chromatography according to their charge characteristic with the purification roughly of protein in the sample.For example, can use the 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 makes that the biomolecule of more negative charges is separated in the sample from the biomolecule of other types.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, sample is further purified again by the heparin chromatograph.The heparin chromatograph makes the I α Ip complex in the sample be further purified, and also is based on and interactional affinity of heparin and charge characteristic.Heparin, the sulphation mucopolysaccharide will be in conjunction 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 the NC Nitroncellulose film of probe subsequently as albumen.Especially, can on laser desorption/ionizing (SEIDI) protein bio-chip of surface-raising, catch I α Ip complex of the present invention.Can on chromatographic surface or biologic specificity surface, catch.Any SELDI protein-biochips that comprises reactive surfaces can be used to catch and detect I α Ip complex of the present invention.These biological new films can be derived with the antibody that specificity is caught I α Ip complex, maybe can derive the protein A of capture agent such as binding domain-immunoglobulin or Protein G with capture agent.Use specific antibody that I α Ip complex is trapped in the solution neutralization then and on chip, separate the I α Ip complex of catching by capture agent.

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, be applicable to that the various other technologies of protein purification are well known to a person skilled in the art.These comprise that for example, usefulness ammonium sulfate, PEG, antibody etc. precipitate or passes through thermal denaturation, and is 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 use the method for this area standard to carry out other purification step.These methods can fully give proteinic height pure preparation.

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 a pillar, makes with the fine grained inert substance that contains aperture, separates from than micromolecule according to the young pathbreaker is more macromolecular greatly when passing or center on the hole.Do not have absorbing molecules as long as make particulate material, unique factor of decision flow velocity is a size.Therefore, molecule, as long as shape is constant relatively with size elution from pillar of successively decreasing.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 the insoluble substrate of one of binding partners covalent bond be synthesized the pillar material.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 changing Cheng Buhui is taken place bonded those.

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.

If desired, any isolated or purified step that 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 and from blood plasma, isolate the blood plasma fraction that contains I α I and P α I that wherein I α I and P α I exist with the physiology ratio; And purification blood plasma level assigns to obtain the pure I α Ip compositions of purity about 85% to about 100%.

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 purification and the separating 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.The hot deactivation or the pasteurize of the present composition can be for example about 55 xeothermic to about 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 stabilizers comprises albumin, Polyethylene Glycol, and α, α-trehalose, aminoacid, salt, glycerol, omega amino acid such as lysine, polylysine, arginine, EACA and tranexamic acid, sugar, 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, premature labor childbirth 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 about 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 an 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 I α Ip compositions of treatment effective dose, it is interior-mixture of alpha inhibitor protein (I α I) and preceding-alpha protein (P α I), and wherein I α I and P α I are present in the described mixture with the physiology ratio, for about 85% to about 100% pure.Pharmaceutical composition can also be the I α Ip compositions of treatment effective dose, it is interior-mixture of alpha inhibitor protein (I α I) and preceding-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 bonded in-the proteinic light chain of alpha inhibitor.Pharmaceutical composition can also be the I α Ip compositions of treatment effective dose, it is interior-mixture of alpha inhibitor protein (I α I) and preceding-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 of treatment effective dose, it is interior-mixture of alpha inhibitor protein (I α I) and preceding-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 as d-alpha-tocopherol cetomacrogol 1000 succinate, and used surfactant such as tween or other similar polymerization transmit substrate, serum albumin in the pharmaceutical dosage form, 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, 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-, or other dissolved derivants 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.In 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 (as, for example, Tween 80) and suspending agent.Aseptic injection preparation can also be aseptic injectable solution or the suspension in nontoxic non-intestinal acceptable diluent 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 used as solvent or suspension media usually.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 as oleic acid and glyceride ester derivatives thereof, and acceptable oil on the natural materia medica, 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 forms, 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, as magnesium stearate.For the oral administration of capsule form, useful diluent comprises lactose and exsiccant 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 desired, 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.Prepare these compositionss by compositions of the present invention is mixed with suitable non-irritating excipient, this excipient is solid in room temperature but is liquid and therefore melts in rectum 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 approaching easily position of topical application 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 carriers 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 field of pharmaceutical preparations technique known, 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 the I α Ip compositions of administration according to 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, premature labor childbirth or infectious disease.

Dosage in this compositions is about 1 to 50mg/kg body weight, and preferred dose is 500mg to 1000mg/ agent, per 4 to 120 hours, or according to the needs of certain drug.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 about 1 to about 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 5% to about 95% the active compound (w/w) of having an appointment.Perhaps, such preparation contains 20% to about 80% the active compound of having an appointment.

It also is favourable being higher or lower than above-mentioned those dosage.Given dose and therapeutic scheme for particular individual will depend on multiple factor, 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 to the disposal of disease, disease or symptom and treatment doctor's judgement.

If desired, 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 secular intermittent therapy of individual need.Can also judge the improvement of disease based on the I α Ip level in the liver.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 comprised 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 was the about 1 to 100% of administration in common single therapeutic scheme, more preferably from about 5 to 95% dosage.Other medicament can with compositions separate administration 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 premature labor childbirth 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 I α Ip treatment after date I α I just, 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 is represented to treat and is being produced good clinical response.The initial stage of treatment is the time that the I α Ip plasma concentration of acquisition steady statue needs.

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

In another embodiment, can use immunoassay detect with analytic sample in I α Ip complex.This method comprises: the antibody of specificity in conjunction with I α Ip 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 to use the test of the antibody of specificity conjugated antigen (for example, I α Ip complex).Immunoassay is characterised in that the specificity binding characteristic that uses 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 to the association reaction that decision protein exists in the multiple colony of protein and other biological goods.Therefore, under specified immunoassay condition, specific antibodies specific protein double at least background and basically not with sample in other protein of existing combine with significant quantity.Combine needs with the specificity of antibody under the condition like this and select the specificity of antibody specific protein.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 the doctor be diagnosed as suffer 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-Upj ohn); 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; cut down ground former times cloth; 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, reactive protein C and protease inhibitor such as furin inhibitor.

Method also comprises the reaction of prediction to 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; Wherein the level of Jian Ceing 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 represent individual administration and benefit from I α Ip.

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 of treatment 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 I α Ip treatment back individual level represents that individuality may have good clinical response to 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 not shown).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,, but can arbitrarily drink water the rat overnight fasting.Suck anesthetized rat with isoflurane, 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 spot of fecal matter from the hole, and gets back to the abdominal cavity in the ligation of ileocecal valve tip; Sew up abdominal incision then from level to level.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.Different time after caecum ligation and puncture (CLP) or sham-operation comes collection organization's sample with Animal Anesthesia at interval then.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 comes separation of human I α Ip (I α I and P α I).This program relates to the ion exchange and the size exclusion chromatograph of cryoprecipitate.Obtain about 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 back 1,5,10 or 20h, under isoflurane anesthesia, use polyethylene 50-cannula in left femoral vein.By the thigh conduit with constant people I α Ip concentrate or the isopyknic carrier (normal saline, 1.5ml/ rat) of inculcating rate 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 not shown).

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.(Molecular Research Center, Cincinnati OH) extracts total RNA from liver by Tri-Reagent.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.Resolution of precipitate is handled in 0.1%DEPC-, in the deionized distilled water.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 is to 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.Produce gel then and use the Bio-Rad PS (Hercules, CA) by G3PDH with the band intensity standardization.

Proteinic radioiodination and I α Ip half life determination

Use Na ¹²⁵(IL) the I α Ip of radioiodination purification uses 1,3,4,6-tetrachloro-3a-6a-diphenyl glycoluril (IODO-GEN iodating agent to I for Amersham, Arlington Heights; 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) the middle radioactivity of measuring.12 hours the time, suck Animal Anesthesia after CLP and the sham-operation with isoflurane.Intravenous injection pentobarbital sodium (～30mg/kg BW) is kept the abirritative 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, the 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 blood sample after the injection immediately, measured in the circulation in per then 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 CLP as mentioned above.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.Allow animal ad libitum access and drinking-water then and monitor over 10 days and write down survival.

Statistical analysis

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

The change that bikunin mRNA expresses behind the CLP

Bikunin is the active part of I α Ip.Liver is the main source of bikunin.Therefore, we select the bikunin mRNA in the liver to express the generation of reacting I α Ip.As shown in Figure 3, during 5h, the mRNA of bikunin does not express and changes 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

Change the half-life of calculating I α Ip by the blood content of measuring the radioactive label I α Ip that injects when sepsis shows effect back 12h.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 CLP and caecum excision back single carrier administration 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 the twice carrier administration in CLP and caecum excision back when 20h (behind the CLP 10 and) was 56% at the 2nd day and was reduced to 44% (Fig. 7) at 5-10 days that 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 activated mononuclear cell, macrophage, endotheliocyte and neutrophil 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,, also reported the I α Ip level that reduces except the proteinase activity that improves.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.Having detected the mRNA relevant with interior-alpha inhibitor family protein in primates, pig and rodentine various tissues expresses.These genes that studies show that all member sources of I α Ip family are mainly transcribed in liver.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 not shown) behind CLP.

Only observing remarkable downward modulation in liver shows that this organ is the important source 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 Research on effect in the bacteremic acute canine model of pathogenic escherichia coli and shown similar result, have the improvement of hematodinamics variable and kinemic standardization and mean arterial pressure.Because the plasma half-life of bikunin is lacked (about 10 minutes) very much, 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 during at sepsis outbreak back 12h, we found that the half-life of I α Ip extends 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.Research before us has shown that sepsis outbreak back early stage (that is, behind the CLP 1 hour) administration low-purity I α Ip keeps the 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 back 1h.

Separate I α Ip albumen as the fractionated by-product of commercial scale blood plasma.Separation method height, enrichment simultaneously 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 back 10 days with CLP and caecum when the mortality rate result of study of being carried 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 the survival rate that useful additive is used for improving 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.Postpone but multiple administration people I α Ip 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 bonded composition 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 use the sodium phosphate buffer gradient of progressive concentration to come elution impurity protein, 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 use the sodium phosphate buffer gradient of progressive concentration to come elution impurity protein, 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 eluant utilization (reference to the DEAC-CIM pipe monolith of solid phase extractions with 80mL column volume, K.Branovic etc., J.ofChromatography A, 903 (2000) 21-32).Collect unconjugated fraction (effluent).Use 0.02M Tris-HCl (level pad) the washing pillar of the pH7.4 of three column volumes subsequently.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 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 the 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 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,, leniently extrude a small amount of feces, return the abdominal cavity then with the pin puncture twice of 18-specification in the ligation of ileocecal valve distally.Abdominal incision is sewed up layer by layer, behind the CLP the subcutaneous immediately 30mL/kg of acceptance body weight of animal normal saline solution is revived as fluid.Use two groups of rats, every group of n=12 in this experiment.Time treatment winding in 10 and 20 hours is subjected to the highly purified I α Ip of 30mg/kg body weight 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.Allow the laboratory animal ad libitum access and monitor the death of writing down non-survivor over 10 days.Use the logarithm level to test the mortality rate between the more different treated animals and measure the p value by the Kaplan-Meier method.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 active comparison of table 1.I α Ip rejection ratio

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

The 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 speed reduce and monitor inhibition.Test the quantitative measurement protein concentration by BioRad protein, and use MAb69.31 to measure I α Ip concentration by competitive ELISA, 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 " from thrombin FIX chromatogram purification on the DEAE-Sepharose FF (Josic etc., Journal of Chromatography, more than drawn).The 0.01-0.1M sodium citrate/0.005-0.1M sodium phosphate buffer that contains 0.25M sodium chloride with pH6.0 comes this fraction of elution.I α I and P α I are the main components in this fraction.The fraction that in next step, contains FIX from the elution of DEAE-Sepharose FF post with the 0.01-0.1M sodium citrate/0.005-0.1M sodium phosphate buffer that contains 0.3-0.6M sodium chloride of pH6.0.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 the higher salt concentrations and osmotic pressure buffer step after the elution of FIX occurs in.Also exist not in conjunction with the outflow fraction of pillar, be no 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 in the final container of stabilizing agent surpass 30 minutes 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.

Handle 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, blood coagulation inhibitor 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 integral body (film) can be used for the FIX purification substitutes DEAE Sepharose FF or other are based on particulate anion exchanger (referring to DE4342132C1).Surprisingly, I α I and P α I not in conjunction with or just faintly in conjunction with integral carriers.Other protein obtain elution 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, use hydroxyapatite chromatography to obtain to contain the further separation of the residual impurity of I α I/P α I fraction, i.e. trace vitamin K-dependent clotting factor FII, FVII and FX.

All publications quoted in this application and patent document are incorporated herein by reference with its integral body and are used for all purposes to same degree, and independently publication and patent document are expressions separately as each.Unless otherwise noted, the meaning that has those skilled in the art in the invention's common sense at 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 (101)

1. produce the I α Ip method for compositions that is derived from blood plasma, said composition contains the mixture of interior-alpha inhibitor protein (I α I) and preceding-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 this method comprises:

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; With

It is about 85% to about 100% pure I α Ip compositions that the described blood plasma level of purification assigns to obtain I α Ip purity.

2. the process of claim 1 wherein and separate by solid phase extractions.

3. the method for claim 2 is wherein come solid phase extractions by DEAE Sephadex.

4. the process of claim 1 wherein to separate and comprise blood plasma is carried out chromatographic isolation.

5. the method for claim 4, wherein chromatograph comprises anion-exchange chromatography.

6. the method for claim 5, wherein anion-exchange chromatography is based on particulate.

7. the method for claim 6, wherein granule contains immobilized anion exchange groups such as DEAE Sephadex, DEAE Sephadex A50, Toyopearl DEAE, TMAEFractogel, DEAE Fractogel or Q-Sepharose.

8. the method for claim 4 is wherein carried out chromatographic isolation by integral carriers with blood plasma.

9. the method for claim 8, wherein integral carriers is CIM such as DEAE-CIM or the Q-CIM with immobilization anion exchange part.

10. the method for each claim before, wherein the blood plasma fraction comprises the by-product fraction that the purifying blood coagulation factors IX obtains.

11. the method for each claim before, wherein the blood plasma fraction comprises the by-product fraction of purifying blood coagulation proenzyme complex concentrate.

12. the method for each claim is wherein come the separated plasma fraction as the cold supernatant that the blood plasma cryoprecipitate obtains before.

13. the method for each claim before, wherein the blood plasma fraction is cold barren blood plasma.

14. the method for each claim before, wherein the blood plasma fraction is the blood plasma fraction of people, primates, cattle, pig, cat or dog.

15. the method for each claim further comprises acquisition blood before.

16. the method for each claim further comprises acquisition blood plasma before.

17. the method for each claim further comprises obtaining the by-product fraction that the purifying blood coagulation factors IX obtains before.

18. the method for each claim further comprises the by-product fraction that obtains the compound concentrate of purifying blood coagulation proenzyme before.

19. the method for each claim further comprises obtaining the cold supernatant that the blood plasma cryoprecipitate obtains before.

20. the method for each claim further obtains cold barren blood plasma before.

21. the method for each claim is wherein come purification by hydroxyapatite chromatography before.

22. each method of claim 1-21 is wherein come purification by affinity chromatography.

23. the method for claim 22, wherein affinity chromatography is a heparin.

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

25. the method for each claim before wherein is present in the I α I of blood plasma fraction and P α I and has about 60,000 to about 280, the apparent molecular weight of 000kDa.

26. the method for claim 25 is wherein come determining molecular weight by SDS-PAGE.

27. the method for each claim further comprises being further purified the blood plasma fraction before.

28. the method for claim 27 wherein flows through (not combination) level by heparin affinity column and collection and assigns to be further purified.

29. the method for each claim comprises that further the I α Ip with blood plasma fraction and/or purification carries out inactivation of virus before.

30. the method for claim 29 is wherein carried out inactivation of viruses by solvent/detergent treatment or hot deactivation.

31. the method for claim 30, wherein hot deactivation are about 55 to about 65 ℃ temperature or xeothermic at 70 to 120 ℃.

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

33. the method for claim 29, further comprise with the I α Ip pasteurize of purification or about 70 to about 120 ℃ xeothermic.

34. the method for each claim comprises that further the I α Ip with purification carries out anion-exchange chromatography before.

35. the method for claim 34, wherein anion-exchange chromatography is DEAESepharose.

36. the method for each claim before, wherein I α Ip compositions has and is higher than 1 hour half-life.

37. each method of claim 1-36, wherein I α Ip compositions has and is higher than 5 hours half-life.

38. the method for each claim before, wherein I α Ip compositions has high trypsin rejection ratio activity.

39. the method for claim 38, wherein trypsin rejection ratio activity is about 1000 to about 2000IU/mg.

40. in containing-the I α Ip compositions of the mixture of alpha inhibitor protein (I α I) and preceding-alpha protein (P α I), wherein I α I and P α I are present in the described mixture with the physiology ratio, for about 85% to about 100% pure.

41. the compositions of claim 40, wherein I α Ip comprises about 60% to about 80% I α I and about 40% to about 20% P α I.

42. the compositions of claim 40, wherein the physiology ratio is the ratio of the natural I α I that presents and P α I in the human plasma.

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

44. the compositions of claim 43, wherein the human disease is acute inflammation disease, sepsis, serious shock, septic shock, rheumatoid arthritis, cancer, cancerometastasis, wound/damage, infectious disease and premature labor childbirth.

45. the compositions of claim 40 further comprises stabilizing agent.

46. the compositions of claim 45, wherein stabilizing agent is albumin, Polyethylene Glycol, α, α-trehalose, aminoacid, salt, glycerol, omega-amino acid, sugar or its combination.

47. contain the I α Ip compositions of the mixture of interior-alpha inhibitor protein (I α I) and preceding-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 rejection ratio activity.

48. the compositions of claim 47, wherein I α Ip comprises about 60% to about 80% I α I and about 40% to about 20% P α I.

49. the compositions of claim 47, wherein the physiology ratio is the natural ratio that presents in the human plasma.

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

51. the compositions of claim 50, wherein the human disease is acute inflammation disease, sepsis, serious shock, septic shock, rheumatoid arthritis, cancer, cancerometastasis, infectious disease and premature labor childbirth.

52. the compositions of claim 47 further comprises stabilizing agent.

53. the compositions of claim 52, wherein stabilizing agent is albumin, Polyethylene Glycol, α, α-trehalose, aminoacid, salt, glycerol, omega-amino acid, sugar or its combination.

54. in containing-the I α Ip compositions of the mixture of alpha inhibitor protein (I α I) and preceding-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 and be higher than one hour half-life.

55. the compositions of claim 54, wherein I α Ip comprises about 60% to about 80% I α I and about 40% to about 20% P α I.

56. the compositions of claim 54, wherein I α Ip compositions has at least 5 hours half-life.

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

58. the compositions of claim 54, wherein the physiology ratio is the natural ratio that presents in the human plasma.

59. the compositions of claim 54, wherein protein is used for the treatment of the human disease.

60. the compositions of claim 59, wherein the human disease is acute inflammation disease, sepsis, serious shock, septic shock, rheumatoid arthritis, cancer, cancerometastasis, infectious disease and premature labor childbirth.

61. the compositions of claim 54 further comprises stabilizing agent.

62. the compositions of claim 61, wherein stabilizing agent is albumin, Polyethylene Glycol, α, α-trehalose, aminoacid, salt, glycerol, omega-amino acid, sugar or its combination.

63. contain the I α Ip compositions of the mixture of interior-alpha inhibitor protein (I α I) and preceding-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 at least one bonded in-the proteinic light chain of alpha inhibitor.

64. the compositions of claim 63, wherein I α Ip comprises about 60% to about 80% I α I and about 40% to about 20% P α I.

65. the compositions of claim 63, wherein the physiology ratio is the ratio of the natural I α I that presents and P α I in the human plasma.

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

67. the compositions of claim 66, wherein the human disease is acute inflammation disease, sepsis, serious shock, septic shock, rheumatoid arthritis, cancer, cancerometastasis and infectious disease.

68. the compositions of claim 63 further comprises stabilizing agent.

69. the compositions of claim 63, wherein stabilizing agent is albumin, Polyethylene Glycol, α, α-trehalose, aminoacid, salt, glycerol, omega-amino acid, sugar or its combination.

70. contain the I α Ip compositions of the mixture of interior-alpha inhibitor protein (I α I) and preceding-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 four heavy chain H1, H2, H3 and H4 at least one bonded in-the proteinic light chain of alpha inhibitor.

71. the compositions of claim 70, wherein I α Ip comprises about 60% to about 80% I α I and about 40% to about 20% P α I.

72. the compositions of claim 70, wherein the physiology ratio is the ratio of the natural I α I that presents and P α I in the human plasma.

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

74. the compositions of claim 73, wherein the human disease is acute inflammation disease, sepsis, serious shock, septic shock, rheumatoid arthritis, cancer, cancerometastasis, infectious disease and premature labor childbirth.

75. the compositions of claim 70 further comprises stabilizing agent.

76. the compositions of claim 75, wherein stabilizing agent is albumin, Polyethylene Glycol, α, α-trehalose, aminoacid, salt, glycerol, omega-amino acid, sugar or its combination.

77. the I α Ip compositions of the mixture that contains interior-alpha inhibitor protein (I α I) and preceding-alpha protein (P α I) that each makes according to claim 1-39, wherein I α I and P α I are present in the described mixture with the physiology ratio.

78. each compositions of claim 44-77 further comprises other therapeutic agent.

79. the compositions of claim 78, wherein other therapeutic agent are anticarcinogen, antiinflammatory, anticoagulant or immunomodulator.

80. pharmaceutical composition, comprise in the claim 40,47,54,63,70 or 77 each the treatment compositions useful and materia medica on acceptable carrier.

81. treat the method for individual inflammation related disease, cancer or infectious disease, comprise the I α Ip of each method generation of claim 1-39 of drug treatment effective dose.

82. the method for claim 81 is wherein separated I α Ip from individuality.

83. the method for claim 82, wherein individuality is people, cattle, pig, goat or primate.

84. the method for claim 81 is wherein with tablet, capsule or injection administration I α Ip.

85. the method for claim 81, wherein I α Ip is at least 85% pure.

86. the method for claim 81, wherein I α Ip is about 85% to about 100% pure.

87. the method for claim 81, wherein I α Ip comprises about 60% to about 80% I α I and about 40% to about 20% P α I.

88. treat the method for individual acute inflammation disease, sepsis, serious shock, septic shock, rheumatoid arthritis, cancer, cancerometastasis, infectious disease or premature labor childbirth, comprising:

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

(i) level of I α I;

The (ii) level of P α I;

The (iii) level of I α Ip;

The (iv) level of H3;

(the v) level of H4;

(the vi) level of H1;

(the vii) level of H2; With

(the viii) level of LC; With

(b) the I α Ip that will treat effective dose delivers medicine to individuality.

89. the method for claim 88 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 of representing to produce good clinical response.

90. the method for claim 89, wherein regulating is the raising of I α Ip level.

91. the method for claim 88 is wherein measured the level of I α I, P α I, I α Ip, H3, H4, H1, H2 and LC by immunization method.

92. the method for claim 88, wherein individuality be accredited as suffer from inflammation, tumor invasion, neoplasm metastasis, septicopyemia are whole, septic shock, rheumatoid arthritis, premature labor childbirth, cancer or infectious disease.

93. the method for claim 88, wherein the treatment initial stage is the I α Ip needed time of plasma concentration that obtains steady statue.

94. the method for claim 88 further comprises the therapeutic agent that administration is other.

95. the method for claim 94, wherein other therapeutic agent are anticarcinogen, antiinflammatory, anticoagulant or immunomodulator.

96. the method for prediction I α Ip therapeutic response comprises:

The sample that mensuration obtains from individuality detect following one or more levels:

(i)IαI；

(ii)PαI；

(iii)IαIp；

(iv)H3；

(v)H4；

(vi)H1；

(vii) H2; With

(viii)LC；

Wherein the level of Jian Ceing is represented the individuality of sound response I α Ip treatment.

97. the method for claim 96, wherein the level of Jian Ceing is the reduction of I α I and P α I level.

98. the method for the individual progress of monitoring I α Ip treatment comprises:

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

(i) level of I α I;

The (ii) level of P α I;

The (iii) level of I α Ip;

The (iv) level of H3;

(the v) level of H4;

(the vi) level of H1;

(the vii) level of H2; With

(the viii) level of LC;

(b) the I α Ip that 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 I α Ip treatment back individual level represents that individual I α Ip is treated has good clinical response.

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

(i)IαI；

(ii)PαI；

(iii)IαIp；

(iv)H3；

(v)H4；

(vi)H1；

(vii) H2; With

(viii) LC; With

The description that treatment is used.

100. 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.

101. medicine box comprises claim 40,47,54,63,70 or 77 each compositions and the description used of treatment.

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