CN101343318A - Peptide-polymer conjugates - Google Patents

Peptide-polymer conjugates Download PDF

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CN101343318A
CN101343318A CNA200810130543XA CN200810130543A CN101343318A CN 101343318 A CN101343318 A CN 101343318A CN A200810130543X A CNA200810130543X A CN A200810130543XA CN 200810130543 A CN200810130543 A CN 200810130543A CN 101343318 A CN101343318 A CN 101343318A
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binding substances
described binding
interferon
peptide
rabbit
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林国钟
吴逸之
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PharmaEssentia Corp
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A61K38/212—IFN-alpha
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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Abstract

This invention relates to a peptide-polymer conjugate of the following formula: A-G1-L-G2-B, wherein A is a polymer moiety; each of G1 and G2, independently, is a bond or a linking functional group; L is an alkenylene or alkynylene moiety; and B is a peptide moiety. Also disclosed are a method for preparing a peptide-polymer conjugate and its use in treating hepatitis C virus infection or hepatitis B virus infection.

Description

The binding substances of peptide-polymer
Cross reference
The application advocates the right of priority of the U.S. Provisional Application 60/948,095 submitted on July 5th, 2007.The content of this provisional application is introduced by reference.
Background technology
Cytobiology and recombinant protein Progress in technique cause the generation of protein for treatment.
Yet, still have a main difficult problem.Proteolytic degradation takes place in most albumen easily, therefore has the short life-span.Other shortcoming comprises inducing of water-soluble low and neutralizing antibody.
(for example polyoxyethylene glycol (PEG) is bonded to albumen and has hindered proteolytic ferment near the albumen skeleton polymkeric substance, causes the enhanced protein stability.In addition, it also can improve water-soluble and minimize immunogenicity.Need conjugated polymer to proteic effective ways.
Summary of the invention
The part that the present invention is based on unexpected discovery peptide can be coupled to polymkeric substance to form the binding substances of peptide-polymer by unsaturated joint, is used as protein drug.
On the one hand, the invention is characterized in the polymer-peptide conjugates of following general formula:
A-G 1-L-G 2-B，
Wherein, A is the part of polymkeric substance; G 1And G 2Each is key independently or connects functional group; L is alkenylene (alkenylene) or alkynylene (alkynylene) part; With B be peptide moiety.
With reference to above-mentioned general formula, polymer-peptide conjugates can have one or more following characteristics: A and have molecular weight 2～100kD (preferred 12～30kD) polyalkylene oxides (polyalkylene oxide) part, G 1And G 2Each is a key, and B is that Interferon, rabbit part or modified interferon and the L that contains 1～4 other amino-acid residue are C 6Alkenylene.
Term " polyalkylene oxides part " refers to the monoradical derived from linearity, branch or star polyalkylene oxides.The molecular weight of polyalkylene oxides part can be 2～100kD.Polyalkylene oxides partly is saturated or undersaturated.The example of polyalkylene oxides part includes but not limited to polyethylene oxide, polyoxyethylene glycol, polyisobutylene oxide compound, polybutene oxide compound and multipolymer thereof.Other polymkeric substance is dextran, polyvinyl alcohol, poly-propionic acid amide or also can be used for replacing the polyalkylene oxides part based on the polymkeric substance of carbohydrate for example, as long as it is not antigenic, deleterious or causes immune response.Polyalkylene oxides partly is that replace or unsubstituted.For example, it can be the end capped polyoxyethylene glycol of methoxyl group (methoxy-capped polyethylene glycol (mPEG)).
Term " peptide moiety " refers to the singly-bound group from native peptides or modified peptides.Native peptides can be an interferon-' alpha ' 2b, interferon-beta, tethelin or antibody.Modified peptides can be for example to contain Interferon, rabbit and at interferon-' alpha ' 2bThe N-end peptide of 1～4 other amino-acid residue is arranged.The example of this modified interferon is
Figure A20081013054300061
IFN represents interferon-' alpha ' 2bPart, its N-end is bonded to carbonyl.
Term " connect functional group " refers to divalence functional group, and the one end is connected with polymer moieties and the other end is connected with peptide moiety.Example includes but not limited to-O-,-S-, carboxylicesters, carbonyl, carbonic ether, amine, carbaminate, urea, alkylsulfonyl, sulfinyl, amino, imino-, hydroxyl amino, phosphoric acid salt or bound phosphate groups.
Term " alkenylene " refers to contain the divalence straight chain or the ramose hydrocarbon polymer of 2～10 carbon atoms and one or more pair key.The example of alkenylene includes but not limited to vinyl (ethenylenyl), propylene and 2-butylene.Term " alkynylene " refers to contain 2～10 carbon atoms and one or more triple-linked divalence straight chain or ramose hydrocarbon polymer.The example of alkynylene includes but not limited to acetylene, 1-propine and 2-butyne.
Alkenylene that this paper mentions and alkynylene comprise replacement and do not replace part.Substituent example comprises C 1～C 10Alkyl, C 2～C 10Thiazolinyl, C 2～C 10Alkynyl, C 3～C 8Cycloalkyl, C 5～C 8Cycloalkenyl group, C 1～C 10Alkoxyl group, aryl, aryloxy, heteroaryl, heteroaryloxy, amino, C 1～C 10Alkylamino, C 1～C 20Dialkylamino, virtue amino, diarylamino, hydroxylamino, alkoxy amino, C 1～C 10Alkyl sulfonamide, aryl sulfonic acid amides, hydroxyl, halogen, sulfenyl, C 1～C 10Alkylthio, arylthio, nitrile, nitrogen, acyl group, acyloxy, carboxyl and carboxylicesters.
If of course, the binding substances of above-mentioned peptide-polymer can be the free form or the form of salt.For example, can between the negatively charged ion of peptide-polymer binding substances of the present invention and positive charge group (for example amino), form salt.Suitable negatively charged ion comprises chlorion, bromide anion, iodide ion, sulfate radical, nitrate radical, phosphate radical, citrate, methanesulfonates, trifluoro-acetate and acetic ester.Equally, can between the positively charged ion of polypeptide-polymer binding substances of the present invention and negative electricity group (for example carboxylicesters), form salt.Suitable positively charged ion comprises sodium ion, potassium ion, magnesium ion, calcium ion and ammonium ion, for example tetramethyl ammonium.
In addition, the peptide-polymer binding substances can have one or more pairs of keys, or one or more asymmetric center.This binding substances can be racemoid, racemic mixture, single enantiomorph, single diastereomer, non-enantiomer mixture and trans or cis or E-or Z-double-bond isomerism form.
Two examples of polymer-peptide conjugates of the present invention are as follows:
Figure A20081013054300071
Wherein, mPEG has molecular weight 20kD and IFN is an interferon-' alpha ' 2bPart.
On the other hand, the invention is characterized in a kind of method for preparing the peptide-polymer binding substances.This method comprises makes A-G 1-L '-CHO and H 2N-B ' coupling, and reduce this coupled product to form
A-G 1-L '-CH 2The peptide-polymer binding substances of NHB '; Wherein, A is a polymer moieties, G 1Be key or connection functional group, L ' is alkenylene or alkynylene, and H 2N-B ' is N-free end peptide, for example Interferon, rabbit of Interferon, rabbit or modification.
Interferon, rabbit is the immunoregulation druge that is used for the treatment of HCV or HBV infection.Referring to for example Journal ofVascular and Interventional Radiology 13 (2002): 191-196.Therefore, more on the one hand, the invention is characterized in that the Interferon, rabbit-polymer conjugates treatment hepatitis C virus (HCV) that utilizes following general formula infects or hepatitis B virus (HBV) infects:
A-G 1-L-G 2-B，
Wherein, A is the part of polymkeric substance, G 1And G 2Each is key independently or connects functional group; L is alkenylene or alkynylene part; With B be N-free end peptide Interferon, rabbit or the Interferon, rabbit that contains the modification of 1～4 other amino-acid residue at the N-end.
In addition, be to contain only to describe to be used for the treatment of polypeptide-polymer binding substances and this therepic use and the application of this binding substances in the medicine of making the above-mentioned infection of mentioning of treatment that HCV infects or HBV infects within the scope of the invention.
The details of one or more embodiments of invention will propose in following explanation.The further feature of invention, purpose and advantage will be apparent from specification sheets and claim.
Detailed description of the invention
The present invention relates to the peptide-polymer binding substances, wherein peptide moiety is coupled to polymkeric substance by unsaturated joint.
Can prepare peptide-polymer binding substances of the present invention by the well-known synthetic method of chemical field.For example, the linkers that contains leavings group (for example bromine) can connect by ether and at first be coupled to the mPEG that contains the hydroxyl terminal group.Subsequently, the peptide molecule that contains functional group (for example amino) can react to form peptide-polymer binding substances of the present invention with the functional group (for example aldehyde functional group) of joint.
Above-mentioned chemical reaction comprises use solvent, reagent, catalyzer, blocking group and removes the blocking group reagent, and some reaction conditions.They can comprise step in addition before the specifically described step of this paper or after the step, to allow synthetic peptide-polymer binding substances and add or remove suitable blocking group for final.In addition, the various synthesis steps order or the order that can change carries out to obtain desired polypeptides-polymer conjugates.Synthetic chemistry useful in the peptide-polymer binding substances that is being suitable for changes and the blocking group methodology is known in the art and for example comprises synthesizing, in following described R.Larock, ComprehensiveOrganic Transformations, VCH Publishers (1989); T.W.Greene and P.G.M.Wuts, Protective Groups in Organic Synthesis, 2d.Ed., John Wiley and Sons (1991); L.Fieser and M.Fieser, Fieser and Fieser ' s Reagents forOrganic Synthesis, John Wiley and Sons (1994); And L.Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995) and later release thereof.
Following route 1 shows the example of preparation peptide-polymer binding substances of the present invention.Chemical I has polymer moieties and aldehyde radical functional group.It can react with peptide II (having free ammonia functional group).Generate product III subsequently for example by hydrogenation or by NaBH 3The CN reduction is to provide peptide-polymer binding substances IV.
A is a polymer moieties
G 1Be key or connection functional group
L ' alkenylene or alkynylene
B is a peptide moiety
Route 1
Therefore synthetic peptide-polymer binding substances can be further by such as the method for column chromatography or high pressure liquid chromatography and purifying.
Peptide-polymer binding substances of the present invention can be with the form of binding substances but pharmaceutical active.Perhaps, it can discharge pharmaceutically active peptides (for example by hydrolysis) in vivo by the connection that enzymatic cuts between peptide moiety and polymer moieties.Relate to the example that cuts the enzyme that connects in the body and comprise oxydase (for example peroxidase, amine oxidase or desaturase), reductase enzyme (for example ketone reductase enzyme) and lytic enzyme (for example proteolytic enzyme, esterase, sulfatase or phosphoesterase).
Therefore, one aspect of the present invention relates to the method that one or more above-mentioned peptide-polymer binding substancess that give significant quantity are used for the treatment of disease (for example HCV or HBV infect).Particularly, can treat disease by one or more peptide-polymer binding substancess that give experimenter's significant quantity.This experimenter can be identified according to the result of any suitable diagnostic method by the health care expert.
As used herein, term " treatment " or " treatment " are defined as the Secondary cases illness of using or having illness, condition symptoms, disease or this illness, the experimenter (human or animal) who perhaps tends to this illness comprises the composition of peptide-polymer binding substances, its purpose is to cure, alleviate, alleviate, treat or improve the Secondary cases illness of this illness, condition symptoms, disease or this illness, the perhaps tendency of this illness." significant quantity " refers to give the amount by the peptide-polymer binding substances of treatment experimenter result of treatment.Result of treatment can be passive (promptly measurable by some detections or mark) or (being hint or the sensation that the experimenter provides this effect) initiatively.
The pharmaceutical composition that contains at least a above-mentioned peptide-polymer binding substances of significant quantity and pharmaceutical acceptable carrier also within the scope of the invention.In addition, the present invention includes the method for one or more peptide-polymer binding substancess of experimenter's significant quantity of suffering from one or more diseases.As is known to the person skilled in the art, effectively dosage changes according to the use of the hydrolysis rate of for example peptide-polymer binding substances, disease type to be treated, route of administration, vehicle and with the common possibility of using of treatment on other therapeutics.
In order to put into practice method of the present invention, have one or more above-mentioned mention compound compositions can parenteral, oral, nasal cavity, rectum, part or orally administering.That term used herein " parenteral " refers to is subcutaneous, in the intracutaneous, intravenously, intramuscular, intraarticular, intra-arterial, synovial membrane, in the breastbone, in the sheath, in the intralesional, intraperitoneal, tracheae or intracranial injection and any suitable implantttion technique.
The Injectable composition of sterilization can be solution or the suspension in atoxic parenteral acceptable diluent or the solvent, for example solution of 1,3 butylene glycol.Operable acceptable carrier and solvent are N.F,USP MANNITOL, water, Ringer ' s solution and isotonic sodium chlorrde solution.In addition, daily use fixedly carburetion as solvent or suspension medium (for example synthetic list-or two-glyceryl ester).It is useful with its polyoxyethylene form when preparing injection especially that lipid acid (for example oleic acid) and glyceride derivative thereof can be accepted oil (for example sweet oil or Viscotrol C) as natural drug.These oil solutions or suspension also can contain long-chain alcohol thinner or dispersion agent, perhaps methyl carboxylic acids Mierocrystalline cellulose or similarly dispersion agent.Other is generally used for making pharmacy can accept the purpose that the normally used tensio-active agent of solid, liquid or other formulation (for example Tweens or Spans or other similar emulsifying agent or bioavailability toughener) also can be used to prepare.
Liquid preparations for oral administration can be any oral acceptable forms, comprises capsule, tablet, emulsion and aqueous suspensions, dispersion agent and solution.Under the situation of tablet, normally used carrier comprises lactose and W-Gum.Usually also add lubricant (for example Magnesium Stearate).For the oral administration of capsule pattern, useful thinner comprises lactose and dried corn starch.When oral when giving aqueous suspensions or emulsion, activeconstituents can suspend or be dissolved into emulsifying agent or suspension agent bonded oil phase in.If desired, can add some sweeting agent, seasonings or tinting material.
Can prepare nose with aerosol or composition for inhalation according to medicine formulation art technique known.For example, said composition can use the suitable sanitas of phenylcarbinol or other, absorption enhancer to prepare with salts solution to strengthen bioavailability, fluorine carbon and/or other solubilizing agent known in the art or dispersion agent.Composition with one or more above-claimed cpds also can give with the suppository form that is used for rectal administration.
Pharmaceutical acceptable carrier uses with one or more above-mentioned compounds of mentioning usually.The carrier of pharmaceutical composition must be that " acceptable " condition is the activeconstituents compatible (and preferably can stablize this activeconstituents) of itself and composition and harmless to experimenter to be treated.One or more solubilizing agent can be as pharmaceutical excipient to send the above-mentioned compound of mentioning.The example of other carrier comprises colloid silica, Magnesium Stearate, Mierocrystalline cellulose, sodium lauryl sulphate and D﹠amp; The yellow #10 of C.
The following examples are regarded as merely illustrative and all the other contents of limit publicity text never in any form.Do not need further to elaborate, believe that those skilled in the art can be based on the explanation of this paper, utilize the present invention to its to greatest extent.All documents that this paper quotes are whole by reference to be introduced.
Preparation (Z)-six-4-thiazolinyl mPEG aldehyde
(i) preparation 2-[5-bromo-(Z)-five-3-thiazolinyl]-1,3-dioxolane (compound 1)
Figure A20081013054300101
At N 2Down in-40 ℃ in THF (120mL) (Z)-5-(1,3-dioxolane-2-yl) five-2-alkene-1-alcohol (3.0g, 19mmol) solution add triethylamine (5.8mL, 41.7mmol) and Methanesulfonyl chloride (2.2mL, 28.5mmol).After stirring 1h, add LiBr (16.5g, 190mmol) solution among the THF (20mL).Reaction mixture is warmed to room temperature and stirs 1h so that white precipitate to be provided.(MTBE is 60mL) with water saturation NH to add methyl t-butyl ether 4Cl (120mL).Remove organic layer and extract water layer with methyl t-butyl ether (100mL).At MgSO 4In dry bonded organic layer and concentrating to obtain thick residue, its mixture that advances-go on foot utilize to use 20: 1 hexane/EtOAc as the silica gel chromatography of eluent to obtain 2-[5-bromo-(Z)-five-3-thiazolinyl as yellowish liquid]-1,3-dioxolane (3.4g, 80%).
1H?NMR(400MHz，CDCl 3)：5.82-5.76(m，1H)，5.69-5.62(m，1H)，4.92(t，J＝4.6Hz，1H)，4.06(d，J＝8.4Hz，2H)，4.04-3.97(m，2H)，3.94-3.88(m，2H)，2.35-2.29(m，2H)，1.83-1.78(m，2H)。
(ii) prepare (Z)-six-4-thiazolinyl mPEG aldehyde Glycol Acetal (compound 2)
20kD mPEGOH (49.7g in 900mL exsiccant THF, 2.49mmol) (SunBio Inc., CA, USA) solution adds 60% sodium hydride (1.5g, 37.4mmol), add 2-[5-bromo-(Z)-five-3-thiazolinyl subsequently]-1, the 3-dioxolane (5.5g, 24.9mmol) and potassiumiodide (0.32g).The mixture backflow 20h that generates is cooled to room temperature, slowly quenches with cold water (600mL), and vacuum concentration is to remove THF then.(3 * 900mL) extract residue with methylene dichloride.At MgSO 4In dry bonded organic layer and be concentrated into about 400mL in a vacuum.This solution is added dropwise to MTBE (7.0L) then in 2h.The white precipitate that collect to generate and in vacuum-drying to produce compound 2 (45.0g, 91%) as white powder.
1H?NMR(400MHz，CDCl 3)δ5.55-5.52(m，2H)，4.82(t，J＝4.0Hz，1H)，4.06(d，J＝4.0Hz，2H)，3.95-3.89(m，2H)，3.34(s，3H)，2.19-2.14(m，2H)，1.71-1.66(m，2H)。
(iii) prepare (Z)-six-4-thiazolinyl mPEG aldehyde (compound 3)
(45.0g 2.25mmol) is dissolved in the damping fluid of 700mL pH 2 (in the citric acid-HCl-NaCl) to compound 2.This solution of stirring at room 48h also uses methylene dichloride (3 * 1.0L) extractions.Organic layer is combined, with normal saline washing, use anhydrous MgSO 4Drying is concentrated into about 350mL in a vacuum and is added dropwise to MTBE (6.0L) in 2h.The white precipitate that collection generates is also dry to produce white powder compound 3 (42.0g, 93%) under vacuum.
1H?NMR(400MHz，CDCl 3)δ9.74(s，1H)，5.60-5.55(m，1H)，5.53-5.48(m，1H)，4.06(d，J＝6.4Hz，2H)，3.34(s，3H)，2.49(t，J＝6.8Hz，2H)，2.39-2.35(m，2H)。
Preparation (E)-six-4-thiazolinyl mPEG aldehyde (compound 4)
Prepare compound 4 with the method identical, except using 2-[5-bromo-(Z)-five-3-thiazolinyl with compound 3]-1, the 3-dioxolane replaces 2-[5-bromo-(E)-five-3-thiazolinyl]-1, the 3-dioxolane.
1H?NMR(400MHz，CDCl 3)δ9.71(s，1H)，5.70-5.61(m，1H)，5.68-5.49(m，1H)，3.90(d，J＝5.6Hz，2H)，3.32(s，3H)，2.51-2.47(m，2H)，2.35-2.30(m，2H)。
Preparation precursor I FN
The recombined human interferon-alpha that end user's genomic dna utilizes the PCR method clone to modify as template 2b, i.e. precursor I FN.Based on human interferon-alpha 2bFlanking sequence (the GenBank number of landing #J00207) synthetic oligonucleotide.The PCR product subclone that obtains is to pGEM-T carrier (Promega).By pGEM-T clone once more pcr amplification IFN variant and utilize subsequently NdeI/BamHI as the cloning site subclone to protein expression vector pET-24a (Novagen), a kind of t7 rna polymerase promoters driven carrier.Then carrier pET-24a is transformed into E.coli BL21-CodonPlus (DE 3)-RIL (Stratagene) strain system.By existing E.coliBL21-CodonPlus (the DE 3)-RIL of maintenance conversion down to screen high-expression clone in kantlex (50 μ g/mL) and paraxin (50 μ g/mL).
(BD 200mL) breeds BL21-CodonPlus (DE 3)-RIL and precursor I FN gene at 1000mL in flask to use the superfine product broth culture.Flask sways 16h at 37 ℃ with 230rpm.(Bioflo 3000 at the 5L fermentor tank; New Brunswick Scientific Co., Edison carries out in batches and feed supplement-batch fermentation in NJ).Batch fermentation uses spend the night pre-incubated inoculum and 3L of 150mL to have kantlex (50 μ g/mL), paraxin (50ug/mL), 0.4% glycerol and 0.5% (v/v) trace elements (FeSO of 10g/L 47H 2O, the ZnSO of 2.25g/L 47H 2O, the CuSO of 1g/L 45H 2O, the MnSO of 0.5g/L 4H 2O, the H of 0.3g/L 3BO 3, the CaCl of 2g/L 22H 2O, (the NH of 0.1g/L 4) 6Mo 7O 24, 0.84g/L EDTA, 50ml/L HCl) the superfine product broth culture.The dissolved oxygen concentration is controlled at 35% and by the NaOH aqueous solution that adds 5N pH is remained on 7.2.Preparation contains the MgSO of 600g/L glucose and 20g/L 47H 2The feed solution of O.When pH is increased to the numerical value that is higher than the set-point, the feed solution that adds proper volume is to improve the glucose concn in the culture meat soup.Induce precursor I FN expression of gene and after cultivating 3h, collect culture meat soup to final concentration 1mM by adding IPTG.
The cell mass of collecting with TEN damping fluid (50mM Tris-HCl (pH 8.0), 1mM EDTA, the 100mM NaCl) resuspension of about ratio 1: 10 (weight in wet base g/mL), and utilize the microjet crusher machine, then in 10, the centrifugal 20min of 000rpm.Contain the granule 2 times of inclusion body (IB) and press above-mentioned centrifugal with the flushing of TEN damping fluid.Suspending in the 4M of 150mL Guanidinium hydrochloride (GuHCl) aqueous solution then contains the granule of IB and 20, the centrifugal 15min of 000rpm.In the 6M of 50mL GuHCl solution, dissolve IB then.IB by dilution sex change in freshly prepd refolding damping fluid (100mM Tris-HCl (pH 8.0), 0.5M L-arginine, 2mM EDTA) begins refolding, and this damping fluid is only adding fashionable stirring.Allow the refolding reaction mixture under not stirring, to mix 48h.(have 2mM EDTA and 0.1M urea, pH7.0) recombined human interferon-alpha of dialysis refolding with 20mM Tris damping fluid 2b(that is precursor I FN) is used for the Q-Sepharose column chromatography and is further purified.
With the recombinant human protein's precursor I FN after the refolding go up sample to the Q-Sepharose post (GE AmershamPharmacia, Pittsburgh, PA).Post washes by pre-equilibration and with 20mM Tris-HCl damping fluid (pH 7.0).The product mixture wash-out of 20mM Tris-HCl damping fluid (pH 7.0) and 200mM NaCl.The flow point that contains precursor I FN is absorbed in 280nm based on it and collects.(Pierce, Rockford IL) determine the concentration of precursor I FN to utilize the Bradford method by protein detection kit.
In conjunction with mPEG aldehyde and Pro-IFN
The precursor I FN (1mg) of the Q-Sepharose purifying of preparation and compound 3 reactions in the foregoing description 3.Final reaction product contains 50mM sodium phosphate (pH 6.0), (Aldrich, Milwaukee is WI) with 10mg compound 3 for 5mM hydroboration cyanogen sodium.Mixture is cultivated 20h to form as main in room temperature then
Figure A20081013054300141
The mono-pegylated precursor I FN of product (compound 5), it uses SP XL Sepharose chromatogram (GEAmersham Pharmacia, Pittsburgh, PA) purifying then.Particularly, the SP post washes by pre-equilibration and with 20mM sodium acetate (pH 5.4).Then with the buffer solution elution compound 5 that contains 20mM sodium acetate (pH 5.4) and 60mM NaCl.Unreacted IFN (being precursor I FN) is by containing the buffer solution elution of 20mM sodium acetate (pH 5.4) and 100mM NaCl.The gel electrophoresis analysis of the sodium laurylsulfonate by 12%-policapram gel is dyed detection by wash-out flow point and this signal by coomassie brilliant blue staining R-250 and silver.Collect the flow point that contains compound 5 based on its retention time with in the absorption of 280nm.(Pierce, Rockford IL) determine the concentration of compound 5 to utilize the Bradford method by protein detection kit.The isolated yield of compound 5 is 30%～40%.
Compound 4 and precursor I FN are reacted so that the binding substances with E form to be provided.
Other embodiment
Disclosed all features can make up with any combination in the specification sheets.Disclosed every kind of feature can be used to identical, equivalence or the alternative features of similar purpose replaces in this specification sheets.Therefore, except as otherwise noted, disclosed every kind of feature only is the general equivalence or the example of similar characteristics series.
From above-mentioned explanation, those skilled in the art can determine essential feature of the present invention easily, and can carry out various changes and modification so that it is applicable to various uses and condition and does not deviate from its spirit and scope to invention.Therefore, other embodiment also within the scope of the following claims.

Claims (27)

1, a kind of binding substances of polymkeric substance-peptide has following general formula:
A-G 1-L-G 2-B，
Wherein, A is the part of polymkeric substance, G 1And G 2Each is key independently or connects functional group; L is alkenylene or alkynylene part; With B be peptide moiety.
2, the described binding substances of claim 1, wherein said A is the mPEG part, its molecular weight is 2～100kD.
3, the described binding substances of claim 2, wherein said G 1And G 2Each is a key.
4, the described binding substances of claim 3, wherein said B is the Interferon, rabbit part.
5, the described binding substances of claim 3, wherein said B are the Interferon, rabbit parts that contains the modification of 1～4 other amino-acid residue.
6, the described binding substances of claim 5, wherein said B is IFN is an interferon-' alpha ' 2bPart, its N-end is bonded to carbonyl.
7, the described binding substances of claim 6, wherein said L is C 6Alkenylene.
8, the described binding substances of claim 7, wherein said A is the mPEG with molecular weight 12～30kD.
9, the described binding substances of claim 1, wherein said G 1And G 2Each is a key.
10, the described binding substances of claim 1, wherein said B is the Interferon, rabbit part.
11, the described binding substances of claim 1, wherein said B are the Interferon, rabbit parts that contains the modification of 1～4 other amino-acid residue.
12, the described binding substances of claim 1, wherein said B is
Figure A2008101305430002C2
IFN is an interferon-' alpha ' 2bPart, its N-end is bonded to carbonyl.
13, the described binding substances of claim 1, wherein said L is C 6Alkenylene.
14, the described binding substances of claim 1, wherein said A is the polyalkylene oxides part.
15, the described binding substances of claim 14, the molecular weight of wherein said A is 12～30kD.
16, the described binding substances of claim 1, wherein said binding substances is
Figure A2008101305430002C3
Or
Figure A2008101305430002C4
The molecular weight of wherein said mPEG is that 20kD and IFN are interferon-' alpha 's 2bPart, its N-end is bonded to carbonyl.
17, a kind of method for preparing the binding substances of peptide-polymer comprises: coupling A-G 1-L '-CHO and H 2N-B ' and
The reductive coupling product is to form general formula: A-G 1-L '-CH 2The peptide-polymer binding substances of NHB '; Wherein A is the part of polymkeric substance, G 1Be key or connection functional group, L ' is alkenylene or alkynylene part; And H 2N-B ' is a N-free end peptide.
18, the described binding substances of claim 17, wherein G 1It is key; With A be mPEG with molecular weight 12～30kD.
19, the described binding substances of claim 18, wherein H 2N-B ' is the Interferon, rabbit part.
20, the described binding substances of claim 18, wherein H 2N-B ' is the Interferon, rabbit part that contains the modification of 1～4 other amino-acid residue at N-terminal.
21, the described binding substances of claim 17, wherein said peptide-polymer binding substances is
Or
Figure A2008101305430003C2
The molecular weight of wherein said mPEG is that 20kD and IFN are interferon-' alpha 's 2bPart, its N-end is bonded to carbonyl.
22, a kind of hepatitis C virus or hepatitis b virus infected method for the treatment of comprises the peptide-polymer binding substances of the following general formula of the experimenter's significant quantity that needs:
A-G 1-L-G 2-B，
Wherein, A is the part of polymkeric substance; G 1And G 2Each is key independently or connects functional group; L is alkenylene or alkynylene part; With B be N-free end peptide Interferon, rabbit or the Interferon, rabbit that contains the modification of 1～4 other amino-acid residue at the N-end.
23, the described binding substances of claim 22, wherein said G 1And G 2Each is a key; With A be mPEG with molecular weight 12～30kD.
24, the described binding substances of claim 23, wherein said B is the Interferon, rabbit part.
25, the described binding substances of claim 23, wherein said B are the Interferon, rabbit parts that contains the modification of 1～4 other amino-acid residue.
26, the described binding substances of claim 25, wherein said B is
Figure A2008101305430004C1
IFN is an interferon-' alpha ' 2bPart, its N-end is bonded to carbonyl.
27, the described binding substances of claim 22, wherein said peptide-polymer binding substances is
Figure A2008101305430004C2
Or
Figure A2008101305430004C3
The molecular weight of wherein said mPEG is that 20kD and IFN are interferon-' alpha 's 2bPart, its N-end is bonded to carbonyl.
CNA200810130543XA 2007-07-05 2008-07-07 Peptide-polymer conjugates Pending CN101343318A (en)

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