CN104151421A - Pegylated interferon conjugate - Google Patents
Pegylated interferon conjugate Download PDFInfo
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- CN104151421A CN104151421A CN201410394786.XA CN201410394786A CN104151421A CN 104151421 A CN104151421 A CN 104151421A CN 201410394786 A CN201410394786 A CN 201410394786A CN 104151421 A CN104151421 A CN 104151421A
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/555—Interferons [IFN]
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- 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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- C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
- C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
- C08G65/32—Polymers modified by chemical after-treatment
- C08G65/329—Polymers modified by chemical after-treatment with organic compounds
- C08G65/333—Polymers modified by chemical after-treatment with organic compounds containing nitrogen
- C08G65/33331—Polymers modified by chemical after-treatment with organic compounds containing nitrogen containing imide group
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Abstract
The invention discloses a pegylated interferon conjugate, a preparation method thereof, a pharmaceutical composition comprising a therapeutically effective amount of the interferon conjugate, and an application thereof in preparing anti-viral, anti-tumor, and immunoregulatory medicines.
Description
The application is that application number is 201280002760.5, and the applying date is on May 23rd, 2012, and what denomination of invention was the Chinese patent application of " a kind of polyoxyethylene glycol interferon conjugate " divides an application.
Technical field
The present invention relates to a kind of polyoxyethylene glycol interferon conjugate, this conjugate is synthetic by a kind of polyethyleneglycol modified technology of bioprotein molecule of novelty, have the biologic activity such as anti-virus infection, antitumor, immunomodulatory, Application Areas of the present invention relates to the treatment of biological chemistry, pharmaceutical chemistry and human diseases.
Background technology
Interferon, rabbit (Interferon) is one group of active protein with several functions, is the important familial cytokine of a class, has antiviral, inhibition of cell proliferation and the immunoregulation effect of wide spectrum.Mammiferous Interferon, rabbit can be divided into the several types such as α, β, γ, ω, and wherein interferon-alpha can divide again more than ten to plant hypotype, through a large amount of clinical study proof alpha-interferons, is a kind of important antiviral and antineoplaston medicines.At present China clinical most popular be mainly recombinant human interferon alpha 1 b, α 2b and α 2a.In addition, U.S. Amgen company is according to the gene order homology of 13 kinds of alpha-interferons, a kind of brand-new protein engineering drugs Infergen of designing (
iFN-Con-1) in 1997, through FDA approval listing, be used for the treatment of hepatitis C, its virus activity is 5～10 times of α 2b Interferon, rabbit.
But in general, many products that have biotechnology (as gene recombination) to express gained, comprise Interferon, rabbit, be all to enter and in organism, bring into play its drug action by parenterai administration approach.Conventionally there are some problems below in this type of biopharmaceutical macromolecular drug product by parenterai administration: (1) biopharmaceutical macromolecular drug often has sensitization reaction, and the antibody producing in organism can cause serious injury and affect on organism the carrying out for the treatment of; (2) metabolism of biopharmaceutical macromolecular drug because being subject to the impact of antibody or causing because of protease itself, causes shorten dramatically its biological half-life; (3) poor stability of biomacromolecule, preserves difficulty.So, no matter be Interferon α1 b, α 2b and α 2a or Infergen, as pharmaceutical grade protein, due to poor stability, plasma clearance is high, and Half-life in vivo is short, easily produces antigen antibody reaction etc., in clinical treatment, be very restricted, the result that these shortcomings cause is: need frequent injection of interferon just can reach effective plasma treatment concentration; And, after per injection, all can cause the larger fluctuation of haemoconcentration, form peak value and the valley of drug level.So just may increase the risk of medical expense and administration inconvenience and untoward reaction.Therefore, people attempt to adopt various useful for drug delivery technology (Drug Delivery Technology), improve the curative effect of pharmaceutical grade protein.And study in useful for drug delivery technology at present, be polyethyleneglycol modified technology (PEGNOLOGY) the most widely.
1980, the people such as Davis are at United States Patent (USP) 4,179, the chemically modified that utilizes the polyoxyethylene glycol of single molecule different polymerization degree to carry out protein drug is disclosed in 337, in the biological activity while that keeps medicine, the antigenicity of medicine reduces, and the people such as Verones are at Applied Biochem and Biotech 11, on 142 (1985), publish polyethyleneglycol modified rnase and superoxide-dismutase with phenyl chloroformate activation, increased the biological half-life of albumen.Above-mentioned prior art reported in literature has shown that polyethyleneglycol modified technology can solve some problems that parenterai administration biopharmaceutical macromolecular drug exists really.
At present, on market, there have been two kinds of Peg-Intron products, respectively the PEG-IFN α 2b (PEG-INTRON) of Schering Plough company and the PEG-IFN α 2a (PEGASYS) of Hoffman-la Roche company, improved the Interferon, rabbit transformation period in vivo, reaching injection weekly once, makes the bioavailability of Interferon, rabbit obtain lifting to a certain degree.These two products have occupied external Interferon, rabbit market 60%, have obtained huge economic benefit.
Summary of the invention
The object of the present invention is to provide that a kind of biologic activity is better, bioavailability is higher polyethyleneglycol modified interferon conjugate, its structure is as shown in formula I:
P-NH-CH
2-X-S-Y-Q
(Ⅰ)
Wherein, P is Interferon, rabbit, comprises all types of Interferon, rabbit, and all subclass of these types, and Interferon alfacon-1 dissimilar and/or that subclass Interferon, rabbit forms; P can be any source, comprises natural origin, tissue culture or the Interferon, rabbit being obtained by gene recombination technology; P is interferon protein natural or artificial recombination, more preferably recombinant human interferon alpha 2, more preferably recombinant human interferon-alpha, β, γ or ω, most preferably α 2b preferably; P both can make by the disclosed technology in this area, also can on market, buy;
X is-(CH
2)
k-or-CH
2(OCH
2cH
2)
k-, k is selected from 2～10 integer, and preferably 1～4, more preferably 2;
Q is methoxy poly (ethylene glycol), and its molecular-weight average is 5,000～40,000 dalton preferably, more preferably 20,000 dalton;
Y is selected from Y1～Y12, and wherein, m, n are selected from 2～10 integer independently of one another, are preferably 2,
Particularly, the invention provides the conjugate that a kind of general structure is (II):
Wherein P refers to Interferon, rabbit, and X is-(CH
2)
k-or-CH
2(OCH
2cH
2)
k-, the number that the number of k is selected from 1～10, m, n is selected from 2～10, m
1be selected from the integer between 100～2000.
Be preferably-(CH of X wherein
2)
k-, the number of k can be further preferably from 1～4, and most preferably 2.
Further preferred to above-mentioned preferred embodiment, the present invention also provides a most preferred embodiment, and corresponding structural formula is:
M
1be selected from 450～600 integer, wherein P refers to Interferon, rabbit, can be further preferred recombinant human interferon alpha 2, more preferably recombinant human interferon-alpha, most preferably recombinant human interferon alpha 2 b.
The present invention also provides the conjugate that a kind of general structure is (III):
Wherein P refers to recombinant human interferon alpha 2, and X is-(CH
2)
k-or-CH
2(OCH
2cH
2)
k-, the number that the number of k is selected from 1～10, m is selected from 2～10, m
1be selected from the integer between 100～2000.
Further preferred to structure formula III, corresponding most preferred embodiment need to meet the following conditions simultaneously: P refers to recombinant human interferon-alpha; X is-(CH
2)
k-, wherein k is 2; The number of m is selected from 2; m
1be selected from 450～600 integer.
The present invention also provides the conjugate that a kind of general structure is (IV):
Wherein P refers to recombinant human interferon alpha 2, and X is-(CH
2)
k-or-CH
2(OCH
2cH
2)
k-, the number that the number of k is selected from 1～10, m is selected from 2～10, m
1be selected from the integer between 100～2000.
Further preferred to structure formula IV, corresponding most preferred embodiment need to meet the following conditions simultaneously: P refers to recombinant human interferon-alpha; X is-(CH
2)
k-, wherein k is 2; The number of m is selected from 2; m
1be selected from 450～600 integer.
The present invention also provides the conjugate that a kind of general structure is (V):
Wherein P refers to recombinant human interferon alpha 2, and X is-(CH
2)
k-or-CH
2(OCH
2cH
2)
k-, the number of k is selected from 1～10, m
1be selected from the integer between 100～2000.
To general structure, be that the conjugate of (V) is further preferred, corresponding most preferred embodiment need to meet the following conditions simultaneously: P refers to recombinant human interferon-alpha; X is-(CH
2)
k-, wherein k is 2; m
1be selected from 450～600 integer.
The present invention also provides the conjugate that a kind of general structure is (VI):
Wherein P refers to recombinant human interferon alpha 2, and X is-(CH
2)
k-or-CH
2(OCH
2cH
2)
k-, the number that the number of k is selected from 1～10, m is selected from 2～10, m
1be selected from the integer between 100～2000.
Further preferred to structure formula VI, corresponding most preferred embodiment need to meet the following conditions simultaneously: P refers to recombinant human interferon-alpha; X is-(CH
2)
k-, wherein k is 2; The number of m is selected from 2, m
1be selected from 450～600 integer.
The present invention also provides the conjugate that a kind of general structure is (VII):
Wherein P refers to recombinant human interferon alpha 2, and X is-(CH
2)
k-or-CH
2(OCH
2cH
2)
k-, the number that the number of k is selected from 1～10, m, n is selected from 2～10 integer, m independently of one another
1be selected from the integer between 100～2000.
To general structure, be that the conjugate of (VII) is further preferred, corresponding most preferred embodiment need to meet the following conditions simultaneously: P refers to recombinant human interferon-alpha; X is-(CH
2)
k-, wherein k is 2; The number of m, n is selected from 2; m
1be selected from 450～600 integer.
The present invention also provides the conjugate that a kind of general structure is (VIII):
Wherein P refers to recombinant human interferon alpha 2, and X is-(CH
2)
k-or-CH
2(OCH
2cH
2)
k-, the number that the number of k is selected from 1～10, m is selected from 2～10, m
1be selected from the integer between 100～2000.
Further preferred to structural formula (VIII), corresponding most preferred embodiment need to meet the following conditions simultaneously: P refers to recombinant human interferon-alpha; X is-(CH
2)
k-, wherein k is 2; The number of m is selected from 2; m
1be selected from 450～600 integer.
The present invention also provides the conjugate that a kind of general structure is (IX):
Wherein P refers to recombinant human interferon alpha 2, and X is-(CH
2)
k-or-CH
2(OCH
2cH
2)
k-, the number of k is selected from 1～10, m, and the number of n is selected from 2～10, m
1be selected from the integer between 100～2000.
Further preferred to structural formula (IX), corresponding most preferred embodiment need to meet the following conditions simultaneously: P refers to recombinant human interferon-alpha; X is-(CH
2)
k-, wherein k is 2; M, the number of n is selected from 2; m
1be selected from 450～600 integer.
The present invention also provides the conjugate that a kind of general structure is (X):
Wherein P refers to recombinant human interferon alpha 2, and X is-(CH
2)
k-or-CH
2(OCH
2cH
2)
k-, the number that the number of k is selected from 1～10, m is selected from 2～10, m
1be selected from the integer between 100～2000.
To general structure, be that the conjugate of (X) is further preferred, corresponding most preferred embodiment need to meet the following conditions simultaneously: P refers to recombinant human interferon-alpha; X is-(CH
2)
k-, wherein k is 2; The number of m is selected from 2, m
1be selected from 450～600 integer.
The present invention also provides the conjugate that a kind of general structure is (XI):
Wherein P refers to recombinant human interferon alpha 2, and X is-(CH
2)
k-or-CH
2(OCH
2cH
2)
k-, the number that the number of k is selected from 1～10, m is selected from 2～10, m
1be selected from the integer between 100～2000.
Further preferred to structural formula (XI), corresponding most preferred embodiment need to meet the following conditions simultaneously: P refers to recombinant human interferon-alpha; X is-(CH
2)
k-, wherein k is 2; The number of m is selected from 2, m
1be selected from 450～600 integer.
The present invention also provides the conjugate that a kind of general structure is (XII):
Wherein P refers to recombinant human interferon alpha 2, and X is-(CH
2)
k-or-CH
2(OCH
2cH
2)
k-, the number that the number of k is selected from 1～10, m, n is selected from 2～10 integer, m independently of one another
1be selected from the integer between 100～2000.
To general structure, be that the conjugate of (XII) is further preferred, corresponding most preferred embodiment need to meet the following conditions simultaneously: P refers to recombinant human interferon-alpha; X is-(CH
2)
k-, wherein k is 2; The number of m, n is selected from 2; m
1be selected from 450～600 integer.
Invention also provides the conjugate of a kind of general structure for (X III):
Wherein P refers to recombinant human interferon alpha 2, and X is-(CH
2)
k-or-CH
2(OCH
2cH
2)
k-, the number that the number of k is selected from 1～10, m, n is selected from 2～10 integer, m independently of one another
1be selected from the integer between 100～2000.
To general structure, be that the conjugate of (X III) is further preferred, corresponding most preferred embodiment need to meet the following conditions simultaneously: P refers to recombinant human interferon-alpha; X is-(CH
2)
k-, wherein k is 2; The number of m, n is selected from 2; m
1be selected from 450～600 integer.
Another object of the present invention is to provide a kind of method of preparing described interferon conjugate, and the method mainly comprises two stages: be first that Interferon, rabbit protects the aldehyde material of sulfydryl to react with containing, form pass through-NH-CH
2the activation interferon protein that-key connects; Then, the Interferon, rabbit deprotection of described activation, with the coupling of active methoxy poly (ethylene glycol) derivative, by being isolated.Particularly, its step comprises:
(1) prepare the small molecules aldehyde cpd that general structure is (X IV):
Wherein the number of k is selected from 2～10, and preferably 2;
(2) the small molecules aldehyde cpd that is (X IV) by general structure reacts in damping fluid with Interferon, rabbit, and adds reductive agent to obtain general structure for the activation Interferon, rabbit of (X V);
Wherein P refers to Interferon, rabbit, preferred recombinant human interferon alpha 2 b, and the pH of damping fluid is selected from 4.0～7.0, and preferably 4.5, reductive agent can be boron sodium cyanide, sodium cyanoborohydride, preferably sodium cyanoborohydride;
(3) utilize technology well known in the art containing the ethanoyl protecting group that adds deprotection agent to slough the activation Interferon, rabbit that general structure is (X V) in the damping fluid that activates Interferon, rabbit; adding subsequently general structure is that the activation methoxy poly (ethylene glycol) of (X VI) carries out pegylation reaction
The preferred azanol of deprotection agent wherein adding; The pH of buffer system is selected from 5.0～7.0, preferably pH6.2; AG is selected from:
Wherein the number of m, n is selected from 2～10, and preferably 2;
(4) purification process of polyoxyethylene glycol interferon conjugate adopts and well known to a person skilled in the art technology, as ion exchange chromatography, gel chromatography.
The present invention is prepared in the method for described interferon conjugate, and general structure is that the small molecules aldehyde cpd of (X IV) can be by the disclosed currently known methods preparation of document, as US4338435.
Another object of the present invention is to provide a kind of pharmaceutical composition that contains this adorned interferon conjugate, and it comprises: the Peg-Intron conjugate of (1) therapeutic dose provided by the present invention; (2) the acceptable pharmaceutical carrier of pharmacy.Interferon conjugate provided by the invention can make with pharmaceutical acceptable carrier or vehicle the pharmaceutical composition that is applicable to injection by means commonly known in the art, the pharmaceutical composition that wish is used can be made into preparation, and by the mode administration consistent with suitable medical practice.Compound of the present invention can be prepared in the l0mM sodium phosphate/potassium pH of buffer 7 that contains 132mM sodium-chlor.Optionally, pharmaceutical composition can contain sanitas.Pharmaceutical composition can contain the Interferon, rabbit of different amounts, for example 10-1000 mcg/ml, for example 50 micrograms or 400 micrograms.
Further, the purposes of the pharmaceutical composition that the object of the present invention is to provide described interferon conjugate and contain them in preparing anti-virus infection, antitumor, immunoregulation druge.Above-mentioned disease specifically comprises: chronic hepatitis B, hepatitis C and hepatitis E etc.; Virus disease is as zoster, flat and pointed condyloma, parillomarvirus infections, epidemic hemorrhagic fever and children RSV pneumonia etc.; Malignant tumour is as hairy cell, chronic myelocytic leukemia, melanoma, lymphoma, multiple myeloma, renal cell carcinoma, ovarian cancer, the rectum cancer, liver cancer, lung cancer etc.
The biological activity of Interferon, rabbit or polyoxyethylene glycol interferon conjugate provided by the invention can be determined by the color reaction of the pathogenic effect method of testing of host cell.Host cell causes a disease effect (CytoPathic Effect, or CPE) the test genealogy of law by Foti proposition (Methods in Enzymology, 119,533,198).This law is utilized human host cell after Interferon, rabbit is processed, to produce the activity of opposing virus infection and can be prevented a kind of simple and easy reaction test of the color absorption efficiently method that the principle design of cytopathy death forms.Briefly, host cell can resisted poisoning intrusion thereby can prevent that host cell is dead after Interferon, rabbit is processed.The dyestuff that the antiviral biological activity of Interferon, rabbit can absorb via survivaling cell and direct quantitative are measured.Resultant employing cytopathic-effect inhibition assay is active by the extracorporeal antivirus effect of WISH-VSV systems measurement Interferon, rabbit, method well-known in the art, concrete with reference to 2005 editions < < Pharmacopoeia of People's Republic of China > tri-appendix X С of > " biological activity determination of Interferon, rabbit ".
By the test data of polyoxyethylene glycol interferon conjugate provided by the invention is shown: polyoxyethylene glycol interferon conjugate provided by the invention can reach the amino acid whose object of modified interferon α 2b molecular surface and retain the biological activity of more interferon alpha 2 b and provide good medicine for characteristic.
In compound provided by the present invention, Interferon, rabbit and linker are with-NH-CH
2-key form connects, and has strengthened thus the specificity of PEG modification reaction.
Accompanying drawing explanation
Fig. 1 changes comparison diagram the serum antigen concentration-time after cynomolgus monkey single subcutaneous administration same dose HH-IFN-003 and PEG-IFN α-2b (PegIntron, Schering Plough).
Fig. 2 is the comparison diagram changing serum antiviral activity after cynomolgus monkey single subcutaneous administration 10 μ gkg-1HH-IFN-003 and PegIntron-time.
Embodiment
Embodiment 1: the preparation of ethanoyl sulfydryl propionic aldehyde
11.2g (0.2mmol) propenal and dry 100ml THF are joined in reaction flask, be cooled to 0 ℃, then slowly drip the mixing solutions of 1.52g (0.02mol) thioacetic acid/20 ml THF.Dropwise insulation reaction after 2 hours.35 ℃ of concentrating under reduced pressure are removed excessive propenal.Then quick upper prop (pure hexane → n-hexane/ethyl acetate=50/1 of elutriant), merges and collects product point, is evaporated to dry oily liquids 0.6g.
Embodiment 2: the preparation of activation Interferon, rabbit
Get 600mg interferon alpha 2 b (from Hengrui Pharmaceutical Co., Ltd., Shanghai), protein concentration is 1.0mg/ml, is total to 600ml, and system is 0.1M acetate buffer, pH4.5; Get after 39.6mg ethanoyl sulfydryl propionic aldehyde is dissolved in 400ul acetonitrile and add above protein solution; Take again 756mg sodium cyanoborohydride and add above-mentioned reaction solution, and low rate mixing reaction, ice bath is controlled temperature of reaction at 10 ℃, reacts 4 hours; Then reaction solution is proceeded to dialysis tubing (molecular weight cut-off 3500), to 0.1M sodium phosphate salt damping fluid, containing 2mM EDTA, pH6.25 dialyses, and to remove excessive small molecules aldehyde, and then adds azanol deacetylate, obtains activation Interferon, rabbit.
Illustrate: following examples (3～8) are that the activation Interferon, rabbit that embodiment 2 is obtained is polyethyleneglycol modified by activity, and obtain the process of polyoxyethylene glycol Interferon, rabbit.Code name and the structural representation of target compound see the following form, and wherein IFN is activation interferon alpha 2 b:
The preparation of embodiment 3:HH-IFN-001
The 100mg activation interferon alpha 2 b obtaining by embodiment 2 (1.0mg/ml, 0.1M sodium phosphate salt damping fluid, containing 2mM EDTA, pH6.25), adds 0.25g mPEG-maleinamide (5kD, from SUNBIO), stirring reaction 60 minutes; Adding N-methyl maleimide to concentration is 5mM again, and room temperature reaction 30 minutes, to react away remaining sulfydryl on albumen; Then reaction solution is dialysed to 20mM acetate buffer system.
Reaction solution is used ion exchange chromatography (SP Sepharose H.P) purifying, obtains Peg-Intron (HH-IFN-001), and about 17.5mg, is 24.6KD through MALDI-TOF-MS detection molecules amount.
The preparation of embodiment 4:HH-IFN-002
The 100mg that the contains free sulfhydryl groups activation interferon alpha 2 b obtaining to embodiment 2 (1.0mg/ml, 0.1M sodium phosphate salt damping fluid, containing 2mM EDTA, pH6.25), add 0.5g mPEG-maleinamide (10kD, from SUNBIO), stirring reaction 60 minutes; Adding N-methyl maleimide to concentration is 5mM again, and room temperature reaction 30 minutes, to react away remaining sulfydryl on albumen; Then reaction solution is dialysed to 20mM acetate buffer system.
Reaction solution is used ion exchange chromatography (SP Sepharose H.P) purifying, obtains Peg-Intron (HH-IFN-002), and about 15.3mg, is 29.9KD through MALDI-TOF-MS detection molecules amount.
The preparation of embodiment 5:HH-IFN-003
The 100mg that the contains free sulfhydryl groups activation interferon alpha 2 b obtaining to embodiment 2 (1.0mg/ml, 0.1M sodium phosphate salt damping fluid, containing 2mM EDTA, pH6.25), add 1.0g mPEG-maleinamide (20kD, from SUNBIO), stirring reaction 60 minutes; Adding N-methyl maleimide to concentration is 5mM again, and room temperature reaction 30 minutes, to react away remaining sulfydryl on albumen; Then reaction solution is dialysed to 20mM acetate buffer system.
Reaction solution is used ion exchange chromatography (SP Sepharose H.P) purifying, obtains Peg-Intron (HH-IFN-003), and about 16.0mg, is 40.3KD through MALDI-TOF-MS detection molecules amount.
The preparation of embodiment 6:HH-IFN-004
The 100mg that the contains free sulfhydryl groups activation interferon alpha 2 b (1.0mg/ml obtaining to embodiment 2,0.1M sodium phosphate salt damping fluid, containing 2mM EDTA, pH6.25), add 2.0g mPEG-maleinamide (40kD, from SUNBIO), mix reaction 60 minutes, adding N-methyl maleimide to concentration is 5mM again, and room temperature reaction 30 minutes, to react away remaining sulfydryl on albumen; Then reaction solution is dialysed to 20mM acetate buffer system.
Reaction solution is used ion exchange chromatography (SP Sepharose H.P) purifying, obtains Peg-Intron (HH-IFN-004), and about 14.5mg, is 60.1KD through MALDI-TOF-MS detection molecules amount.
The preparation of embodiment 7:HH-IFN-005
The 100mg that the contains free sulfhydryl groups activation interferon alpha 2 b (1.0mg/ml obtaining to embodiment 2,0.1M sodium phosphate salt damping fluid, containing 2mM EDTA, pH6.25), add the adjacent pyridyl-disulphide (20KD, from SUNBIO) of 1.0g mPEG-, stirring reaction 60 minutes, adding N-methyl maleimide to concentration is 5mM again, and room temperature reaction 30 minutes, to react away remaining sulfydryl on albumen; Then reaction solution is dialysed to 20mM acetate buffer system.
Reaction solution is used ion exchange chromatography (SP Sepharose H.P) purifying, obtains Peg-Intron (HH-IFN-005), and about 16.0mg, is 41.0KD through MALDI-TOF-MS detection molecules amount.
The preparation of embodiment 8:HH-IFN-006
The 100mg that the contains free sulfhydryl groups activation interferon alpha 2 b (1.0mg/ml obtaining to embodiment 2,0.1M sodium phosphate salt damping fluid, containing 2mM EDTA, pH6.25), add 1.0g iodo-acid amide-mPEG (20KD, from NOF CORPORATION), stirring reaction 60 minutes, adding N-methyl maleimide to concentration is 5mM again, and room temperature reaction 30 minutes, to react away remaining sulfydryl on albumen; Then reaction solution is dialysed to 20mM acetate buffer system.
Reaction solution is used ion exchange chromatography (SP Sepharose H.P) purifying, obtains Peg-Intron (HH-IFN-006), and about 16.5mg, is 39.8KD through MALDI-TOF-MS detection molecules amount.
Embodiment 9: the extracorporeal antivirus effect active testing of interferon alpha 2 b and 6 kinds of polyoxyethylene glycol interferon conjugates
Adopt cytopathic-effect inhibition assay active by WISH-VSV systems measurement Interferon, rabbit extracorporeal antivirus effect.
The present embodiment has been measured the extracorporeal antivirus effect activity of 7 kinds of Interferon, rabbit (or derivative), specifically comprises: IFN α 2b (the permanent auspicious medical limited-liability company in Shanghai provides), HH-IFN-001 (embodiment of the present invention 3 preparations), HH-IFN-002 (embodiment of the present invention 4 preparations), HH-IFN-003 (embodiment of the present invention 5 preparations), HH-IFN-004 (prepared by the embodiment of the present invention 6), HH-IFN-005 (embodiment of the present invention 7 preparations), HH-IFN-006 (embodiment of the present invention 8 preparations).Extracorporeal antivirus effect determination of activity the results are shown in Table 1:
Table 1 extracorporeal antivirus effect determination of activity result
| Title | Specific activity (* 10 8IU/mg) | Active retain (%) |
| IFNα2b | 1.18 | / |
| HH-IFN-001 | 0.18 | 15％ |
| HH-IFN-002 | 0.15 | 13％ |
| HH-IFN-003 | 0.13 | 11％ |
| HH-IFN-004 | 0.08 | 7％ |
| HH-IFN-005 | 0.12 | 10％ |
| HH-IFN-006 | 0.11 | 9％ |
Data presentation in table 1, polyoxyethylene glycol interferon alpha 2 b conjugate provided by the present invention is compared with the interferon alpha 2 b before modification, still retains the antiviral activity of the 7-15% that has an appointment.
Embodiment 10: cynomolgus monkey single subcutaneous administration is subject to serum antigen concentration and the pharmacokinetics comparative experiments after test product HH-IFN-003 and reference product PegIntron
With Hu-IFN-α ELISA medicine box, can measure the concentration that is subject to test product HH-IFN-003 and reference product PegIntron in serum.Hu-IFN-α Immunoassay is a kind of double fastener heart antibody immunoassay method of amplification, test operation step is in accordance with medicine box specification sheets, 96 orifice plates are coated with the monoclonal antibody of IFN-α in advance, add respectively and be subject to test product HH-IFN-003 and reference product PegIntron, hatch certain hour, wash plate, add antibody diluent, shrouding, incubated at room, wash again plate, separately add and carry HRP (horseradish peroxidase) couplings, shrouding, incubated at room, thoroughly wash plate, finally add chromogenic substrate room temperature lucifuge to hatch until there is color; Add stop buffer termination reaction, by microplate reader, measure the absorbance value at 450nm wavelength place.In the logarithmic value of absorbance value and sample, the concentration logarithmic value of HH-IFN-003 and PegIntron is proportionate, and concentration logarithm and absorbance value logarithm are carried out to double-log linear regression, and drawing standard curve calculates unknown sample concentration.
Experimental result: with the reference product group PegIntron of single subcutaneous administration Schering Plough company, 10 μ gkg-1 compare, HH-IFN-003 is subject to each time point serum antigen concentration of test product group to be numerically all greater than PegIntron group (seeing Fig. 1), AUC (0-96h), with AUC (0-inf) is all higher than PegIntron group, pharmacokinetic parameter is in Table 2.After the single subcutaneous administration of surface, in the body of HH-IFN-003, exposed amount will be higher than PegIntron.
Table 2 cynomolgus monkey single subcutaneous injection is subject to test product HH-IFN-003 and the comparison of PegIntron pharmacokinetic parameter
Embodiment 11: cynomolgus monkey single subcutaneous administration 10 μ gkg
-1serum antiviral activity comparative experiments after HH-IFN-003 and PegIntron
PEG-IFN α-2b and IFN α-2b activity determination method adopt cytopathic effect inhibition examination method (CPE method), using WISH cell as viral host cell (permissive cell), cell is subject to can producing cytopathic effect (CPE) after virus infection attack, cell generation pathology, death, and come off from culture dish bottom.Under the intervention of antiviral activity medicine, there is not the cell attachment of pathology on culture dish, utilize Crystal Violet Dye, can make viable cell dyeing, then with destainer, dyestuff be deviate from, by measuring its OD value, reflect the existing state of cell attachment on culture dish.
By the WISH cell digestion in logarithmic phase, with the MEM nutrient solution containing 10%FBS, adjust cell concn, be inoculated in 96 well culture plates, 37 ℃, 5%CO2, hatches 24 hours.After cleaning with PBS, add respectively the serum sample that is subject to test product HH-IFN-003 or reference product PegIntron that contains of allocating with the MEM nutrient solution dilution of 2%FBS, each concentration is established three multiple holes, and establishes without medicine control wells, continues to cultivate 24 hours.The virus that adds the 100 times of TCID50 (50% cell infection amount) that allocate with the MEM nutrient solution dilution of 2%FBS, and establish virus-free infection normal cell hole and virus infection control group, continue to cultivate 24 hours, after neutral red staining, formaldehyde is fixed, after washing, with destainer, dissolve, at 540nm place, obtain absorbancy (OD) value.The protective capability of the cytopathy (CPE) occurring according to the dilution testing sample of difference opposing virus infection, calculates interferon resistance Gene therapy rate (%), and obtains half and effectively protect concentration (EC50).
Experimental result: cynomolgus monkey single subcutaneous administration 10 μ gkg
-1after HH-IFN-003, the absolute value of each time point serum antiviral activity is higher than the PegIntron of same dosage.In Table 3, Fig. 2.This result shows, because HH-IFN-003 Plasma Concentration will be higher than the PegIntron of same dosage, so the antiviral drug effect in its body is also better than PegIntron.
The comparison that serum antiviral activity after table 3 cynomolgus monkey subcutaneous administration single dose PegIntron and HH-IFN-003-time changes
Claims (14)
1. structure is suc as formula the interferon conjugate shown in (I),
P-NH-CH
2-X-S-Y-Q
(I)
Wherein, P is Interferon, rabbit; X is-(CH
2)
k-or-CH
2(OCH
2cH
2)
k-, k is selected from 1～10 integer; Q is methoxy poly (ethylene glycol); Y is selected from Y1～Y12:
Wherein m, n are selected from the integer between 2～10 independently of one another.
2. interferon conjugate according to claim 1, is characterized in that, described conjugate is selected from compound shown in formula II～(X III):
Wherein, P is Interferon, rabbit, and X is-(CH
2)
k-or-CH
2(OCH
2cH
2)
k-, k is selected from 1～10, m, n and is selected from independently of one another the integer between 2～10, m
1be selected from the integer between 100～2000.
3. interferon conjugate according to claim 1 and 2, is characterized in that, described Interferon, rabbit is selected from any one in all types of Interferon, rabbit and subclass thereof, or Interferon alfacon-1 dissimilar and/or that subclass Interferon, rabbit forms.
4. interferon conjugate according to claim 3, is characterized in that, described Interferon, rabbit is recombinant human interferon-alpha, β, γ or ω, preferably recombinant human interferon-alpha, more preferably recombinant human interferon alpha 2 b.
5. interferon conjugate according to claim 4, is characterized in that, m=2, n=2.
6. interferon conjugate according to claim 5, is characterized in that, X is-(CH
2)
k-, k is the integer between 1～4, preferably k=2.
7. interferon conjugate according to claim 6, is characterized in that, m
1be selected from the integer between 450-600.
8. interferon conjugate according to claim 7, is characterized in that, the molecular-weight average of each methoxy poly (ethylene glycol) group is 5,000-40, and 000 dalton is preferably 20,000 dalton.
9. a method of preparing interferon conjugate described in claim 1-8 any one, it comprises the following steps:
1) Interferon, rabbit protects the aldehyde material of sulfydryl to react with containing, forms pass through-NH-CH
2the activation interferon protein that-key connects;
2) the interferon protein deprotection of described activation, with the coupling of active methoxy poly (ethylene glycol) derivative.
10. method according to claim 9, is characterized in that described aldehyde material is the small molecules aldehyde cpd of formula (X IV),
Wherein the number of k is selected from the integer between 2～10, and preferably 2.
11. according to the method described in claim 9 or 10, it is characterized in that described active methoxy poly (ethylene glycol) derivative is that general formula is the activation methoxy poly (ethylene glycol) of (X VI),
AG is selected from:
Wherein the number of m, n is selected from the integer between 2～10, and preferably 2.
12. methods according to claim 9, comprise the following steps:
1) prepare the small molecules aldehyde cpd that general structure is (X IV),
Wherein the number of k is selected from the integer between 2～10, and preferably 2;
2) the small molecules aldehyde cpd that is (X IV) by general structure reacts in damping fluid with Interferon, rabbit, and adds reductive agent to obtain general structure for the activation Interferon, rabbit of (X V),
Wherein P refers to Interferon, rabbit, preferably recombinant human interferon alpha 2 b;
3) containing the ethanoyl protecting group that adds deprotection agent to slough the activation Interferon, rabbit that general structure is (X V) in the damping fluid that activates Interferon, rabbit; adding subsequently general structure is that the activation methoxy poly (ethylene glycol) of (X VI) carries out pegylation reaction; obtain polyoxyethylene glycol interferon conjugate
AG is selected from:
Wherein the number of m, n is selected from the integer between 2～10, and preferably 2;
4) purifying polyoxyethylene glycol interferon conjugate.
13. a pharmaceutical composition, it comprises:
1) the Peg-Intron conjugate of the treatment significant quantity as described in claim 1-8 any one;
2) the acceptable pharmaceutical carrier of pharmacy.
Peg-Intron conjugate described in 14. claim 1-8, pharmaceutical composition claimed in claim 10, the purposes in antiviral, antitumor, the immunoregulation druge of preparation; The preferred purposes in the medicine of preparation treatment chronic hepatitis B, hepatitis C and hepatitis E, zoster, flat and pointed condyloma, parillomarvirus infections, epidemic hemorrhagic fever, children RSV pneumonia, hairy cell, chronic myelocytic leukemia, melanoma, lymphoma, multiple myeloma, renal cell carcinoma, ovarian cancer, the rectum cancer, liver cancer, lung cancer.
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|---|---|---|---|
| CN201410394786.XA CN104151421A (en) | 2011-06-14 | 2012-05-23 | Pegylated interferon conjugate |
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| CN2011101586331A CN102824646A (en) | 2011-06-14 | 2011-06-14 | Conjugate of polyethylene glycol interferon |
| CN201410394786.XA CN104151421A (en) | 2011-06-14 | 2012-05-23 | Pegylated interferon conjugate |
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| CN201280002760.5A Division CN103097406B (en) | 2011-06-14 | 2012-05-23 | Polyethylene glycol-interferon conjugate |
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| CN201410394786.XA Pending CN104151421A (en) | 2011-06-14 | 2012-05-23 | Pegylated interferon conjugate |
| CN201280002760.5A Active CN103097406B (en) | 2011-06-14 | 2012-05-23 | Polyethylene glycol-interferon conjugate |
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| US5252714A (en) * | 1990-11-28 | 1993-10-12 | The University Of Alabama In Huntsville | Preparation and use of polyethylene glycol propionaldehyde |
| KR100507796B1 (en) * | 2003-04-03 | 2005-08-17 | 한미약품 주식회사 | Peg-biologically active polypeptide homodimer conjugate having enhanced half life in blood and process for the preparation thereof |
| CN100355784C (en) * | 2004-02-12 | 2007-12-19 | 江苏恒瑞医药股份有限公司 | Method for preparing polyethylene glycol-modified alpha-interferon 1b |
| CN1754889A (en) * | 2004-06-30 | 2006-04-05 | 深圳科兴生物工程有限公司 | Pegylated interferon alpha-1b |
| TW200643029A (en) * | 2005-06-09 | 2006-12-16 | Egen Corp | Pegylated interferon alpha-1B |
-
2011
- 2011-06-14 CN CN2011101586331A patent/CN102824646A/en not_active Withdrawn
-
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- 2012-05-23 WO PCT/CN2012/075935 patent/WO2012171429A1/en active Application Filing
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| CN103097406A (en) | 2013-05-08 |
| CN102824646A (en) | 2012-12-19 |
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