CN101125206A - Diol polymer-interleukin-1 receptor antagonist conjugate, preparation method and use - Google Patents
Diol polymer-interleukin-1 receptor antagonist conjugate, preparation method and useDownload PDFInfo
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- CN101125206A CN101125206ACNA200610089288XACN200610089288ACN101125206ACN 101125206 ACN101125206 ACN 101125206ACN A200610089288X ACNA200610089288X ACN A200610089288XACN 200610089288 ACN200610089288 ACN 200610089288ACN 101125206 ACN101125206 ACN 101125206A
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Abstract
Translated fromChineseDescription
Technical field
The invention belongs to the protein chemistry field, relate to the diol polymer modification, preparation method of interleukin-1 receptor antagonist and as the application of depot drug product.
Background technology
Il-1 (Interleukin-1, IL-1) be the important pro-inflammatory cytokine of a class, when organism is subjected to wound or inflammation, the mononuclear phagocyte of inflamed sites can produce a large amount of IL-1, with the receptors bind that is positioned at cell surfaces such as fibroblast and T cell, stimulus signal conduction and cell activation.This mutual effect has caused reacting between many cells, discharges neutrophilic granulocyte as promoting bone marrow, induces mononuclear cell and multinuclear granulocyte chemotactic to soak into the inflammation part, discharges lysosomal enzyme in the part.IL-1 also causes basophilic granulocyte and mast cell degranulation, discharges inflammatory mediator.In the articular cavity of rheumatoid arthritis, IL-l impels synovial cell and chondrocyte proliferation and produces PGE2, collagenase and phospholipase A etc., cause arthritis.Interleukin-1 receptor antagonist (IL-1Ra) is a kind of endogenic anti-inflammatory cytokine, and it is the natural specific antagonists of pro-inflammatory cytokine IL-1 α and IL-1 β.The specific receptor that combines cell surface with IL-1 competition of IL-1Ra, it with receptors bind after, stimulus signal conducts, thereby can not cause a series of cell effect.By blocking effect (D.Boraschi, P.Boss ù, G.Macchia, P.Ruggiero, A.Tagliabue.Frontiers in bioscience 1, d270-308,1995 that IL-1 and receptors bind are brought into play its anti-inflammatory; C.Dinarello.FASEB J.8,1314-1325,1994; A.Kluczyk, M.Cebrat, R.
, M.Lisowski, P.Stefanowicz, Z.Wieczorek, I.Z.siemion.Acta Biochimica Polonica 5l, 57-66,2004).
The Antril (Synergen) (Anakinra) that (Amgen) company produces is pacified into by the U.S., a kind of IL-1Ra of gene recombinaton, in calendar year 2001 JIUYUE 14 days be used to improve the sings and symptoms of moderate and severe activeness patient with rheumatoid arthritis, the progress of the arthritis that slows down infringement by American National drug and food management board (FDA) approval.This medicine is suitable for the patient with rheumatoid arthritis that the crowd fails to respond to any medical treatment for age 〉=18 year old, through one or more resisting rheumatoid disease medicines.This recombinant interleukin at expression in escherichia coli-1 receptor antagonist has the biological function identical with natural IL-1Ra.Comparing major different with natural IL-1Ra is: it is non-glycosylated protein for (1); (2) its nitrogen end is Duoed methionine (H.A.Schreuder, J.M.Rondeau, C.Tardif, an A.Soffientini than natural IL-1Ra, E.Sarubbi, A.Akeson, T.L.Bowlin, S.Yanofsky, R.W.Barrett.Eur.J.Biochem.227,838-847,1995).The clinical using dosage of Anakinra is 100mg/day, and patient needs injection every day once, and follows the injection site reaction sometimes.Prepare a kind of low (nothing) immunogenicity, the half life that circulates, is long, and the derivant with IL-1Ra biologic activity can effectively reduce the number of times of injection, and can weaken the immunoreation that natural IL-1Ra causes, thereby, be a job highly significant.
Diol polymer such as Polyethylene Glycol are a kind of good biocompatibilities, and the inertia macromole of nontoxic, no antigen and non-immunogenicity shows as the absorption of low-protein and low platelet in vivo and hangs down cell adhesion.With itself and protein, polypeptide compounds and micromolecule covalent coupling, the outstanding character that can give these reactive proteins, polypeptide class and micromolecular compound diol polymer is effectively improved these materials bio distribution in vivo.It is very extensive that these character make diol polymer use at biomedical sector, obtained the additive that the FDA approval is used to enter human body.This diol polymer such as Polyethylene Glycol have been applied to modify on circulation half life is short, immunogenicity the is strong medical protein by success, as alpha-interferon, granulocyte colony-stimulating factor, (S.P.Monkarsh, Y.Ma such as arginase, A.Aglione, P.Bailon, D.Ciolek, B.DeBarbieri, M.C.Graves, K.Hollfelder, H.Michel, A.Palleroni, J.E.Porter, E.Russonman, S.Roy, and Y.-C.E.Pan.Anal.Biochem.247,434-440,1997; R.Clark, K.Olson, G.Fuh, M.Marian, D.Mortensen, G.Teshima, S.Chang, H.Chu, V.Mukku, E.C.Davis, T.Somers, M.Cronin, M.Winkler, and J.A.Wells.J.Biol.Chem.271,21969-21977,1996; Y.Ashihara, T.Kono, S.Yamazaki, Y.Inada.Biochem.Biophys.Res.Commun.83,385-391,1978).
Interleukin-1 receptor antagonist is brought into play its anti-inflammatory effect by the interleukin I receptor that combines cell surface with the il-1 competition.W16, Q20, Y34, Q36 and Y147 in the one grade amino acid sequence is accredited as the binding site (B.J.Stockman of interleukin-1 receptor antagonist and interleukin I receptor, T.A.Scahill, N.A.Strakalaitis, D.P.Brunner, A.W.Yem, M.R.Deibel Jr.FEBS Lett.349,79-83,1994; R.J.Evans, J.Bray, J.D.Childs, G.P.A.Vigers, B.J.Brandhuber, J.J.Skalicky, R.C.Thompson, S.P.Eisenberg.J.Biol.Chem.270,11477-11483,1995).This five important amino acid residues have surrounded a land with the effect of interleukin I receptor.So when considering the modification of interleukin-1 receptor antagonist, the target decorating site of selection should have great importance to the biologic activity that keeps conjugate like this away from this land.But interleukin-1 receptor antagonist has 38 decorating sites, comprising nitrogen end, 9 lysines, 4 free cysteine, 2 histidine, 11 serines, 3 tyrosine and 8 threonine.Our modification generally all is to carry out at the higher lysine of selectivity, nitrogen end, cysteine and histidine.The nitrogen end of interleukin-1 receptor antagonist and histidine residues are all away from its receptor binding domain, and its 116th and 122 two free cysteine and its receptor binding domain also have one section moderate distance.It all is more satisfactory decorating site.9 lysine residues of interleukin-1 receptor antagonist distribute and disperse, wherein the 21st and 145 lysine residue is very near apart from the receptor binding domain distance of interleukin-1 receptor antagonist, if modify and occur in this two sites, the biologic activity of possible conjugate can be subjected to bigger influence.And the lysine residue of other position, as the 6th, 9,64,93,96,71 all away from the receptor binding domain of interleukin-1 receptor antagonist.Also belong to more satisfactory decorating site.
Usefulness ends such as Thompson are that the succinimide activated polyglycol carried out modifying research (R.C.Thompson, C.H.Hannum, S.P.Eisenberg to the il-1 receptor antagonist, W.P.Arend, F.G.Joslin, A.Sommer.US Pat No.6858409,2005).On specific the 116th free sulfhydryl groups that is connected interleukin-1 receptor antagonist of the Polyethylene Glycol after the succinimide activation, this Polyethylene Glycol-interleukin-1 receptor antagonist the conjugate that generates is through subcutaneous administration, compare with unmodified protein, it obtains significant prolongation at the intravital blood plasma residence time of rat, as unmodified protein is 2.7 hours, and be that its average residence time of the polyethyleneglycol modified conjugate of 5000 dalton is extended for 6.5 hours with mean molecule quantity, be that its average residence time of the polyethyleneglycol modified conjugate of 12000 dalton is extended for 9.0 hours with mean molecule quantity.And its blood plasma residence time of intravenously administrable is extended for 0.37 hour (mean molecule quantity of Polyethylene Glycol is 5000) and 0.76 hour (mean molecule quantity of Polyethylene Glycol is 12000) by 0.25 hour of unmodified protein.Hakimi etc. have also protected a kind of structure to be by the form of patentConjugate (J.Hakimi, P.Kilian, P.Rosen.US Pat No.457057,1995).As in embodiment 6, using α-[(1,2-dihydroxy-2-oxygen-1-pyridine radicals) thiocarbonyl-ω-mono methoxy polyethylene glycol (mean molecule quantity 10000 dalton) after 24 hours, can also detect interleukin-1 receptor antagonist to the administration of C57BF/6 mice with the conjugate of interleukin-1 receptor antagonist reaction generation is subcutaneous in the blood plasma of mice; And unmodified protein behind 8 hours of administration just by the complete metabolism of mice.These examples show, diol polymer is modified and can effectively be prolonged interleukin-1 receptor antagonist circulation half life in vivo, thereby realizes its purpose as the long-effect active compound medicine.
The invention provides class Diolpolymer-interleukin-1 receptor antagonists conjugate and preparation method thereof.Our invention is the difference of Diolpolymer-interleukin-1 receptor antagonists conjugate on connection site and uses the different of diol polymer with the difference of above disclosed patent or document.The amino of selecting interleukin-1 receptor antagonist still is that the imidazole radicals of histidine depends on the activation method of diol polymer and the adjustment of the condition of modification.When the end of diol polymer was activated to aldehyde, diol polymer can specificly be modified on the nitrogen end of interleukin-1 receptor antagonist.When the end of diol polymer is activated to N-hydroxy-succinamide, can be modified at diol polymer under the slant acidity condition on the imidazole radicals and lysine amino of interleukin-1 receptor antagonist histidine away from its receptor binding domain.These conjugates with ad hoc structure obtain electrophoretically pure Diolpolymer-interleukin-1 receptor antagonists conjugate by the liquid chromatograph separation.This conjugate has kept the biologic activity of unmodified protein 45%-75%.Zoopery shows that its circulating half-life in vivo brings up to 18 hours from 6 hours of unmodified protein.In the rheumatoid arthritis rat model test of bovine collagen albumen moulding, conjugate also significantly improves the disease of animal ankle joint and hind paw (p＜0.05), illustrates that this conjugate can be used as the long-effect active compound medicine and is used for the treatment of rheumatoid arthritis effectively.
Summary of the invention
The objective of the invention is to overcome in the prior art interleukin-1 receptor antagonist in being used for the treatment of the process of rheumatoid arthritis, the deficiency of its circulation half life short (4-6 hour), administration number of times frequent (administration every day), and the long lasting Diolpolymer-interleukin-1 receptor antagonists conjugate of a class and preparation method and purposes are provided; Diol polymer covalently bound at interleukin-1 receptor antagonist amino or the imidazole radicals of histidine on the conjugate that forms, can effectively keep under the bioactive condition of interleukin-1 receptor antagonist (biologic activity that has kept unmodified protein 45%-75%), prolong its circulating half-life (bringing up to 18 hours) in vivo significantly from 6 hours of unmodified protein.As everyone knows, the half life of this significant prolongation, provide theoretical support for reducing the medication number of times clinically.The more important thing is that conjugate significantly improves the disease of animal ankle joint and hind paw in the rheumatoid arthritis rat model test of bovine collagen albumen moulding (p＜0.05), shown its effectiveness in the treatment rheumatoid arthritis.
Description to the diol polymer molecular weight is meant its mean molecule quantity.Interleukin-1 receptor antagonist mainly refers to secreting type human interleukin-1 receptor antagonist and gene recombinaton type human interleukin-1 receptor antagonist.
Technical scheme of the present invention is as follows:
Diolpolymer-interleukin-1 receptor antagonists conjugate provided by the invention, this conjugate are the conjugate on the imidazole radicals of diol polymer amino or its histidine of being covalently attached to interleukin-1 receptor antagonist;
Described diol polymer is covalently attached to the general structure of conjugate of the amino of interleukin-1 receptor antagonist
Be .[R-(O-CH2-CH2-)n-(O-CH2-CH2-CH2-)m]k-W-IL-1Ra
Wherein R is the alkyl that contains 1～6 carbon atom; N and m are 0～900 integer, and n+m is not less than 45, and k is 1～4 integer, the aminoacid that W is hydroxyl, be connected with diol polymer with amido link or the primary alconol of 1～6 carbon atom, and IL-1Ra is an interleukin-1 receptor antagonist;
The general structure of the conjugate on the imidazole radicals of the described histidine that diol polymer is covalently attached to interleukin-1 receptor antagonist is:
Wherein R is the alkyl that contains 1～6 carbon atom; N and m are 0～900 integer, and n+m is not less than 45, and k is 1～4 integer, the aminoacid that W is hydroxyl, be connected with diol polymer with amido link or the primary alconol of 1～6 carbon atom, and IL-1Ra is an interleukin-1 receptor antagonist;
The mean molecule quantity of described diol polymer is 2000～40000 dalton.
The amino of the covalently bound interleukin-1 receptor antagonist of diol polymer is the amino of interleukin-1 receptor antagonist lysine amino or nitrogen end in the conjugate.
Described interleukin-1 receptor antagonist is that natural secreting type human interleukin-1 receptor antagonist or gene recombinaton type human interleukin-1 receptor are picked up anti-agent; Preferred gene recombinant type human interleukin-1 receptor antagonist (recombinanthuman interleukin-1 receptor antagonist, RhIL-1Ra).
The preparation method of Diolpolymer-interleukin-1 receptor antagonists conjugate provided by the invention, its step is as follows:
1) the diol polymer activation is terminal reactive intermediate with aldehyde radical or N-hydroxy-succinamide functional group;
2) reactive intermediate that then step 1) is obtained and interleukin-1 receptor antagonist reaction makes the conjugate of the covalently bound imidazole radicals at interleukin-1 receptor antagonist amino or histidine of diol polymer;
It is as follows that described diol polymer is connected the general structure of conjugate of interleukin-1 receptor antagonist amino:
[R-(O-CH2-CH2-)n-(O-CH2-CH2-CH2-)m]k-W-IL-1Ra
Wherein R is the alkyl that contains 1～6 carbon atom; N and m are 0～900 integer, and n+m is not less than 45, and k is 1～4 integer, the aminoacid that W is hydroxyl, be connected with diol polymer with amido link or the primary alconol of 1～6 carbon atom, and IL-1Ra is an interleukin-1 receptor antagonist;
The general structure of the covalently bound conjugate at the interleukin-1 receptor antagonist histidine of described diol polymer is as follows:
Wherein R is the alkyl that contains 1～6 carbon atom; N and m are 0～900 integer, and n+m is not less than 45, and k is 1～4 integer, the aminoacid that W is hydroxyl, be connected with diol polymer with amido link or the primary alconol of 1～6 carbon atom, and IL-1Ra is an interleukin-1 receptor antagonist;
The mean molecule quantity of described diol polymer is 2000～40000 dalton.
The amino of the covalently bound interleukin-1 receptor antagonist of diol polymer is the amino of interleukin-1 receptor antagonist lysine amino or nitrogen end in the conjugate.
Described interleukin-1 receptor antagonist is natural secreting type human interleukin-1 receptor antagonist or gene recombinaton type human interleukin-1 receptor antagonist; Preferred gene recombinant type human interleukin-1 receptor antagonist (recombinanthuman interleukin-1 receptor antagonist, RhIL-1Ra).
The purposes of Diolpolymer-interleukin-1 receptor antagonists conjugate provided by the invention is: the application in preparation treatment medicine for treating rheumatoid arthritis.
The invention has the advantages that: the type by selecting different diol polymers and its activation method make diol polymer optionally be covalently attached on the imidazole radicals of the amino of interleukin-1 receptor antagonist or histidine.This Diolpolymer-interleukin-1 receptor antagonists conjugate that generates has kept the biologic activity of unmodified protein 45%-75% effectively; But its circulating half-life in vivo is significantly improved, and brings up to 18 hours from 6 hours of unmodified protein.The more important thing is that in the rheumatoid arthritis rat model test of bovine collagen albumen moulding, conjugate significantly improves (p＜0.05) because the animal ankle joint that rheumatoid arthritis causes and the swelling of hind paw.Studies show that Diolpolymer-interleukin-1 receptor antagonists conjugate in this patent protection is as the effectiveness of a kind of long lasting interleukin-1 receptor antagonist in the treatment rheumatoid arthritis.
Description of drawings:
Fig. 1 is the SDS-PAGE testing result of the activatory diol polymer modified interleukin-1 of terminal propionic aldehyde receptor antagonist among the embodiment 2, wherein
A band: standard molecular weight albumen;
B band: the gene recombinant human IL-1Ra of unmodified;
C band: the gene recombinant human IL-1Ra of the N-terminal coupling diol polymer of purification (mean molecule quantity is 5000 dalton);
Fig. 2 is the SDS-PAGE testing result of the activatory diol polymer modified interleukin-1 of terminal N-hydroxy-succinamide receptor antagonist among the embodiment 6, wherein
A band: the gene recombinant human IL-1Ra of unmodified;
B band: the gene recombinant human IL-1Ra of the terminal coupling diol polymer of the histidine of purification (mean molecule quantity is 10000 dalton)
C band: standard molecular weight albumen;
Fig. 3 is the MALDI-TOF spectrum of the activatory mono methoxy ethylene glycol polymer of propionic aldehyde-interleukin-1 receptor antagonist conjugate behind tryptic digestion among the embodiment 2;
Fig. 4 determines the coupling site of interleukin-1 receptor antagonist conjugate among the embodiment 6 for the oxammonium hydrochloride method.4a is for before adding oxammonium hydrochloride, and 4b is for after adding oxammonium hydrochloride.
The specific embodiment
Embodiment 1
Preparation has the reactive intermediate of the mono methoxy ethylene glycol polymer of propionic aldehyde end
Take by weighing mono methoxy ethylene glycol polymer (mean molecule quantity 5000 dalton) 5 gram, be dissolved in fill 20 milliliters exsiccant 1, in the round-bottomed flask of 4-dioxane, stirring and dissolving; The 3-chlorine propionyl Anaesthetie Ether and the 2 gram sodium hydroxide that in it, add 1 milliliter, closed system refluxed 24 hours; The reactant liquor rotation is concentrated into about 5 milliliters the cold diethyl ether precipitation.Will be after filtration, exsiccant diol polymer water 30 ml waters dissolvings, dilute hydrochloric acid is regulated its pH value to 2, hydrolysis atroom temperature 4 hours.Dichloromethane extraction with 10 * 3; Cold diethyl ether precipitation, filtration, drying behind combining extraction liquid, the concentrating under reduced pressure.
The proton nmr spectra of mono methoxy ethylene glycol polymer reactive intermediate with propionic aldehyde end is as follows: 3.24 (3H ,-OCH3), 3.56 (4nH ,-O-CH2-CH2), 9.71 (1H ,-CHO), 2.57 (2H ,-CH2-CHO), 3.70 (2H ,-O-CH2-CH2-CHO)
Embodiment 2
Reactive intermediate with embodiment 1 preparation prepares the conjugate that diol polymer is coupled at the interleukin-1 receptor antagonist N-terminal
The gene recombinaton type people IL-1Ra (RhIL-1Ra) that with concentration is 20mg/ml is diluted to 1mg/ml with the sodium phosphate of 40mM, and regulating its pH is 5.0.The activatory mono methoxy ethylene glycol polymer of the propionic aldehyde of 20 times of protein moles (molecular weight 5000) is joined in the protein solution, and adding sodium cyanoborohydride to concentration is 1mM, hatches under 4 ℃ 12 hours.Mixture gel chromatography separation with reaction.(1.0 * 30cm), mobile phase is the sodium phosphate (pH7.0) that contains the 50mM of 0.15M sodium chloride to chromatographic condition: Superdex 75 posts, flow velocity 0.5ml/min.Receive the protein peak of modifying, SDS-PAGE electrophoresis detection (Fig. 1).The biological activity assay result shows that the biological activity of this conjugate has kept 75% of unmodified protein.
Embodiment 3
Preparation has the single isopropyl ethylene glycol of propionic aldehyde end and the reactive intermediate of propylene glycol copolymers
Take by weighing single isopropyl ethylene glycol and glycol polymers (mean molecule quantity 40000 dalton) 10 gram, be dissolved in fill 50 milliliters exsiccant 1, at the bottom of three hole circles of 4-dioxane in the flask, stirring and dissolving.Add fresh activatory manganese dioxide (placing 4 hours in 250 degrees centigrade of following muffle furnaces) 20 grams in it, under the condition that is connected with oxygen (0.2 atmospheric pressure), stirring reaction spends the night.Remove manganese dioxide powder with buchner funnel, filtrate decompression is concentrated, the cold diethyl ether precipitation, getting terminal is activatory single isopropyl ethylene glycol of propionic aldehyde and glycol polymers.Its proton nmr spectra is as follows: 1.16 (6H, (CH3)2-CH-), 3.19 (1H, (CH3)2-CH-), 3.54 (4nH ,-O-CH2-CH2), 3.37 (4mH ,-O-CH2-CH2-CH2), 1.63 (2mH ,-O-CH2-CH2-CH2), 3.70 (2H ,-O-CH2-CH2-CHO) 9.71 (1H ,-CHO)
Reactive intermediate with embodiment 3 preparations prepares the conjugate that diol polymer is coupled at the interleukin-1 receptor antagonist N-terminal
The secreting type people IL-1Ra that with concentration is 20mg/ml is diluted to 1mg/ml with acetic acid-sodium acetate of 40mM, and regulating its pH is 6.0.Activatory single isopropyl ethylene glycol of the propionic aldehyde of 50 times of protein moles and glycol polymers (molecular weight 40000) are joined in the protein solution, and adding sodium cyanoborohydride to concentration is 10mM, hatches under 1 ℃ 36 hours.Mixture gel chromatography separation with reaction.(1.0 * 30cm), mobile phase is the sodium phosphate (pH7.0) that contains the 50mM of 0.15M sodium chloride to chromatographic condition: Superdex 75 posts, flow velocity 0.5ml/min.Receive the protein peak of modifying, the SDS-PAGE electrophoresis detection.The biological activity assay result shows that the biological activity of this conjugate has kept 61% of unmodified protein.
The preparation end is the activatory propionyloxy mono methoxy of a N-hydroxy-succinamide ethylene glycol polymer
Take by weighing mono methoxy ethylene glycol polymer (mean molecule quantity 5000 dalton) 10 gram, be dissolved in 20 milliliters of ultra-pure waters, add 0.2 gram potassium hydroxide, the abundant cooling solution to 0 of dissolving back ice degree centigrade.2.8 the gram acetonitrile is divided 3 addings, ice bath reacted 4 hours down.Regulating its pH value with 0.5 mole disodium phosphate soln is 7.With dichloromethane extraction, concentrated, cold diethyl ether precipitation (precipitation 1).
Add 50 milliliters of concentrated hydrochloric acid in above-mentioned precipitation 1, stirring reaction diluted with 2 liters ultra-pure waters after 48 hours under the room temperature, dichloromethane extraction, concentrated, cold diethyl ether precipitation (precipitation 2).To precipitate 2 and be dissolved in 1 liter of ultra-pure water, add the potassium hydroxide of 68 grams, and stir under the room temperature and spend the night, dichloromethane extraction, concentrated, cold diethyl ether precipitation (precipitation 3).Get 2 gram precipitations 3, be dissolved in 10 milliliters the anhydrous methylene chloride, adds 100 milligrams N-hydroxy-succinamide and 200 milligrams dicyclohexylcarbodiimide, sealed reaction system reacted 24 hours under the room temperature.Elimination precipitation, filtrate decompression concentrates, the cold diethyl ether precipitation, dry terminal be the activatory propionyloxy mono methoxy of N-hydroxy-succinamide ethylene glycol polymer, its proton nmr spectra is as follows: 3.24 (3H ,-O-CH3), 3.54 (4nH ,-O-CH2-CH2), 3.65 (2H ,-O-CH2-CH2-CO), 2.35 (2H ,-O-CH2-CH2-CO), 2.80 (4H, 4 H on the succinimide)
Embodiment 6
Reactive intermediate withembodiment 5 preparations prepares the conjugate that diol polymer is coupled at the imidazole radicals of interleukin-1 receptor antagonist histidine
The gene recombinaton type people IL-1Ra of 20mg/ml is diluted to 5mg/ml with the citric acid-sodium citrate of 40mM, and regulating its pH is 5.5.The activatory propionyloxy mono methoxy of the N-hydroxy-succinamide of 20 times of protein moles ethylene glycol polymer (SPA, molecular weight 5000) is joined in the protein solution, under 37 ℃, hatched 24 hours.Mixture gel chromatography separation with reaction.(1.0 * 30cm), mobile phase is to contain the O.15M sodium phosphate of the 50mM of sodium chloride (pH7.0), flow velocity 0.5ml/min to chromatographic condition: Superdex 75 posts.Receive the protein peak that PEG modifies, SDS-PAGE electrophoresis detection (accompanying drawing 2).The biological activity assay result shows that the biological activity of this conjugate has kept 67% of unmodified protein.
Embodiment 7
Preparation is terminal to be restrained for the activatory ramiform diol polymer of N-hydroxy-succinamide takes by weighing mono methoxy ethylene glycol polymer (mean molecule quantity 1000 dalton) 20, be dissolved in 40 milliliters of ultra-pure waters, in it, add 2 of 8mg, 2,6,6-tetramethyl-piperidines-1-oxygen base (TEMPO) and 48mg potassium bromide, stirring and dissolving under the ice-water bath.Regulating its pH of liquor natrii hypochloritis's (concentration is 8%) with 4N HCl is 10, in ice-water bath cooling down, adds 15 milliliters in reaction system, regulates with 0.5 mole sodium hydroxide, and the pH that keeps reaction system is about 10.React after 6 hours, add 1 milliliter of ethanol cessation reaction.With dichloromethane extraction aqueous solution, concentrated, cold diethyl ether precipitation.Precipitation is dissolved in 50 milliliters the anhydrous methylene chloride, adds the ethyl ester of lysine hydrochlorate of 0.8 gram and 0.5 milliliter triethylamine, back flow reaction 72 hours.Filtering solution, concentrated, dry.Get solid 16.3 grams.It is dissolved in 20 milliliters the anhydrous methylene chloride, adds the N-hydroxy-succinamide of 0.4 gram and the dicyclohexylcarbodiimide of 0.8 gram, sealed reaction system, reaction is 24 hours under the room temperature.Elimination precipitation, filtrate decompression concentrates, the cold diethyl ether precipitation, dry terminal be the activatory ramiform diol polymer of N-hydroxy-succinamide solid, this polymer has two polyglycol chains, its molecular weight is 2000 dalton.Its proton nmr spectra is as follows: 3.24 (6H ,-O-CH3), 3.54 (8nH ,-O-CH2-CH2), 4.26 (4H ,-O-CH2-NH), 8.05 (2H ,-CO-NH), 1.78 (2H, the β H of lysine), 1.55 (2H, the γ H of lysine), 3.20 (2H, the ω H of lysine), 2.80 (4H, 4 H on the succinimide)
Embodiment 8
Reactive intermediate with embodiment 7 preparations prepares the conjugate that diol polymer is coupled at interleukin-1 receptor antagonist histidine imidazole radicals
The secreting type people IL-1Ra (hIL-1Ra) of 20mg/ml is diluted to 3mg/ml with the citric acid-sodium citrate of 40mM, and regulating its pH is 5.5.The activatory ramiform diol polymer of the N-hydroxy-succinamide of 40 times of protein moles (molecular weight 2000) is joined in the protein solution, under room temperature, hatched 15 hours.Mixture gel chromatography separation with reaction.(1.0 * 30cm), mobile phase is the sodium phosphate (pH7.0) that contains the 50mM of 0.15M sodium chloride to chromatographic condition: Superdex 75 posts, flow velocity 0.5ml/min.Receive the protein peak that PEG modifies, the SDS-PAGE electrophoresis detection.The biological activity assay result shows that the biological activity of this conjugate has kept 45% of unmodified protein.
Embodiment 9
Reactive intermediate withembodiment 5 preparations prepares the conjugate that diol polymer is coupled at the interleukin-1 receptor antagonist lysine amino
The gene recombinant human IL-1Ra of 20mg/ml is diluted to 5mg/ml with the citric acid-sodium citrate of 40mM, and regulating its pH is 6.8.The activatory propionyloxy mono methoxy of the N-hydroxy-succinamide of 12 times of protein moles ethylene glycol polymer (SPA, molecular weight 5000) is joined in the protein solution, under 37 ℃, hatched 24 hours.Mixture gel chromatography separation with reaction.(1.0 * 30cm), mobile phase is the sodium phosphate (pH7.0) that contains the 50mM of 0.15M sodium chloride to chromatographic condition: Superdex 75 posts, flow velocity 0.5ml/min.Receive the protein peak that PEG modifies, the biological activity assay result shows that the biological activity of this conjugate has kept 58% of unmodified protein.
Determining of Diolpolymer-interleukin-1 receptor antagonists conjugate nitrogen terminal amino group decorating site
The conjugate of embodiment 2 purification is dissolved with 0.1 mole tris-Hcl (pH8.0) buffer that contains 2.0 mole of urea, according to albumen: the trypsin mass ratio is that 80: 1 amount adds trypsin, in 37 degrees centigrade of followingenzyme action 20 hours, MALDI-TOF composed detection.In the interscan of 5000 dalton-15000 dalton scope, only scan a diol polymer modified polypeptides peak.The peak is bell-shaped distribution, is the characteristic peak of Polyethylene Glycol, and peak point molecular weight is 6062.83kDa, adds molecular weight (5036kDa) the sum (see figure 3) of a diol polymer for (1041kDa) molecular weight of recombination human interleukin N-terminal MRPSGRKSS.
Embodiment 11
Determining of Diolpolymer-interleukin-1 receptor antagonists conjugate histidine imidazole radicals decorating site
The coupled product that diol polymer generates when being modified at the histidine of interleukin-1 receptor antagonist is unstable under the condition that oxammonium hydrochloride exists, and diol polymer can be discharged free interleukin-1 receptor antagonist by the oxammonium hydrochloride aminolysis.Determine the coupling site of the interleukin-1 receptor antagonist conjugate among the embodiment 6 with the method.The pure conjugate of 2mg/ml concentration joined in 0.5 mole the oxyammonia (pH is 7.0), reaction is after 4 hours down in 37 degrees centigrade, and to the phosphate dialysis of 50mM, Superdex 75 posts detect.Chromatographic condition: 0.5ml/min, last sample 100 microlitres, 280nm detects.Can see, behind the oxammonium hydrochloride aminolysis, albumen on the gel chromatography go out the peak position after move to unreacted albumen place.Thus, can identify that diol polymer is modified on the histidine site of interleukin-1 receptor antagonist (seeing Fig. 4 a and 4b).
Embodiment 12
Determining of the amido modified site of Diolpolymer-interleukin-1 receptor antagonists conjugate lysine
The conjugate of purification among the embodiment 9 is dissolved with 0.1 mole tris-Hcl (pH8.0) buffer that contains 2.0 mole of urea, according to albumen: the trypsin mass ratio is that 100: 1 amount adds trypsin, in 37 degrees centigrade of following enzyme action 18 hours, MALDI-TOF composed detection.In the interscan of 5000 dalton-15000 dalton scope, scan 4 groups of polypeptide peak with Polyethylene Glycol feature, the peak is bell-shaped distribution.Molecular weight calculates and to show, diol polymer is connected on 93,6,71 and 64 of interleukin-1 receptor antagonist.
Embodiment 13 modifies the mensuration of each derivant cytoactive of back
The determination of activity of IL-1Ra then is that the survey live body at above-mentioned EL-4/CTLL-2IL-1 is on the basis, adds not commensurability IL-1Ra sample, is that index reflects its cytoactive with it to the active inhibitory action of IL-1, the results are shown in Table 1.
Embodiment 14 modifies each derivants circulation of back mensuration of half life
36 of Wistar rats, body weight restrains at 200-220, is divided into 3 groups (N-terminal diol polymer modification groups of the histidine diol polymer modification group of unmodified protein group, RhIL-1Ra and RhIL-1Ra) at random.Every group 12.Every Mus intravenously administrable, dosage 3mg/kg.Get blood from the rat tails vein, ELISA (R﹠amp; The test kit that D Systems provides) measures the concentration of RhIL-1Ra in the blood plasma and PEGization derivant thereof, determine its circulation half life in blood plasma in vivo, the results are shown in Table 2.
The effect of Diolpolymer-interleukin-1 receptor antagonists conjugate in treatment rat kind rheumatic arthritis
40 of Lewis rats, body weight restrains at 180-220.After 3 days, select 30 rats that it is carried out the rheumatoid arthritis moulding in normal raising at random, remain 10 as normal control group (A group).At the 0th and the 7th day, with II type bovine collagen albumen (2mg/ml is dissolved in the incomplete Freund) through the subcutaneous administration 250 μ l of animal root of the tail portion.The pathological characters of rheumatoid arthritis occurred the 12nd day 24 animal, and the pathological characters of rheumatoid arthritis all occurred the 14th day all animal, the rheumatoid arthritis model that shows animal is by successful moulding.Significant swelling has all been taken place by the ankle joint of moulding animal in all.These model mouses are divided into 3 groups (B, C, D groups), 10 every group at random.Wherein B organizes the interleukin-1 receptor antagonist of subcutaneous administration reorganization, 20mg/kg/day, successive administration 7 days.The interleukin-1 receptor antagonist (preparation process is seen embodiment 2) that C group administration N-terminal is modified, 35mg/kg, the 0th day and the twice subcutaneous injection administration in the 3rd day that begins in treatment respectively.The interleukin-1 receptor antagonist (preparation process is seen embodiment 6) that D group administration histidine imidazole radicals is modified, 35mg/kg, the 0th day and the twice subcutaneous injection administration in the 3rd day that begins in treatment respectively.Begin to end in administration, all measure body weight, ankle joint size, the hind paw thickness of each treated animal every day.The results are shown in Table 3
The cytoactive of table 1 interleukin-1 receptor antagonist and conjugate thereof
| Sample | Cytoactive (IU/mg) |
| RhIL-1Ra embodiment 2 | 9.17E4 6.88E4 5.60E4 6.14E4 4.12E4 5.32E4 |
The plasma half-life of table 2 interleukin-1 receptor antagonist and the administration of conjugate rat vein thereof
| Sample | Half-life (h) |
| RhIL-1Ra embodiment 2 samples (diol polymer mean molecule quantity 20000 dalton) embodiment 8 samples (diol polymer mean molecule quantity 40000 dalton) | 4.5～6 12～15 16～18.5 |
The effect of the rheumatoid arthritis that table 3 interleukin-1 receptor antagonist and conjugate treatment glue II type bovine collagen albumen thereof cause
| Group | Body weight change (g) | Ankle joint change in size (cm) | Hind paw thickness (mm) |
| A B C | 20.87±2.93* -8.99±2.08* -6.34±1.74* | 0.02±0.06 -0.97±0.21* -1.53±0.38* | 0.05±0.07 -0.63±0.14 -0.87±0.18 |
| D | -8.23±2.71* | -1.76±0.24* | -0.73±0.26 |
*p＜0.05，n＝10
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