WO2012171429A1 - 一种聚乙二醇干扰素偶联物 - Google Patents

一种聚乙二醇干扰素偶联物 Download PDF

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WO2012171429A1
WO2012171429A1 PCT/CN2012/075935 CN2012075935W WO2012171429A1 WO 2012171429 A1 WO2012171429 A1 WO 2012171429A1 CN 2012075935 W CN2012075935 W CN 2012075935W WO 2012171429 A1 WO2012171429 A1 WO 2012171429A1
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interferon
integer
formula
conjugate
conjugate according
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王亚里
吕爱锋
孙长安
王瑞军
李蕴波
房雷
徐刚
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Jiangsu Hengrui Medicine Co Ltd
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Jiangsu Hengrui Medicine Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • 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]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
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    • A61K38/19—Cytokines; Lymphokines; Interferons
    • A61K38/21—Interferons [IFN]
    • A—HUMAN NECESSITIES
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    • 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
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • 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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    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L71/00—Compositions of polyethers obtained by reactions forming an ether link in the main chain; Compositions of derivatives of such polymers
    • C08L71/02—Polyalkylene oxides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to a pegylated interferon conjugate synthesized by a novel bio-protein molecule polyethylene glycol modification technology, which has anti-viral infection, anti-tumor, immunomodulation and other biological activities.
  • the field of application of the invention relates to the treatment of biochemistry, medicinal chemistry and human diseases. Background technique
  • Interferon is a group of active proteins with multiple functions. It is an important family of cytokines with broad-spectrum antiviral, anti-cell proliferation, and immune regulation. Mammalian interferons can be divided into several types, such as ⁇ , ⁇ , ⁇ , and ⁇ . Among them, ⁇ interferon can be divided into more than ten subtypes. A large number of clinical studies have proved that ⁇ -interferon is an important antiviral and Anti-tumor treatment drugs. At present, the most widely used in China is recombinant human interferon alb, a2b and a2a.
  • Amgen a new protein engineering drug based on the homology of 13 alpha interferons, designed a new protein engineering drug, INFERGEN® (IFN-Con-1), which was approved by the FDA for marketing in 1997.
  • INFERGEN® IFN-Con-1
  • biomacromolecular drug products for parenteral administration usually have the following problems: (1) Biomacromolecules often have sensitizing reactions, and antibodies produced in the organism can cause serious damage to the organism and affect it. The treatment is carried out; (2) The biological macromolecular drug itself is affected by the antibody or the metabolism caused by the proteolytic enzyme, which causes the biological half-life to be greatly shortened; (3) The stability of the biological macromolecule is poor and the preservation is difficult.
  • P is an interferon, including all types of interferons, and all subtypes of these types, as well as complex interferons composed of different types and/or subclasses of interferon;
  • P can be of any origin, including natural sources, tissues An interferon cultured or obtained by genetic recombination technology;
  • P is preferably a natural or artificially recombinant interferon protein, more preferably recombinant human interferon, more preferably recombinant human interferon alpha, beta, gamma or ⁇ , most preferably a2b; It can be made by the technology disclosed in the art, or can be purchased on the market;
  • X is -(CH 2 ) k - or - CH 2 (OCH 2 CH 2 ) k -, k is selected from an integer of 2 to 10, preferably 1 to 4, further preferably 2;
  • Q is a methoxypolyethylene glycol having an average molecular weight of preferably 5,000 to 40,000 Daltons, more preferably 20,000 Daltons;
  • X is -(CH 2 ) k - or -CH 2 (OCH 2 CH 2 ) k -, the number of k is 1 to 10, and the number of m, n is selected from 2 to 10, selected from 100
  • the integer X between -2000 is preferably -(CH 2 ) k -, and the number of k may further preferably be from 1 to 4, and most preferably 2.
  • the present invention also provides a most preferred embodiment, the corresponding structural formula is:
  • m i is selected from an integer of 450 to 600, wherein P means interferon, further preferably recombinant human interferon, more preferably recombinant human interferon alpha, most preferably recombinant human interferon (x2b).
  • the invention also has a conjugate of the formula (III):
  • P refers to recombinant human interferon
  • X is -(CH 2 ) k - or - CH 2 (OCH 2 CH 2 ) k -
  • the number of k is selected from 1 to 10
  • the number of m is selected from 2 to 10, 13 ⁇ 4
  • P means recombinant human interferon a
  • X is -(C3 ⁇ 4) k -, wherein k is 2; the number of m is selected from 2; mi is selected from an integer of 450 to 600.
  • the invention also provides a conjugate of the formula (IV): Wherein P refers to recombinant human interferon, X is -(CH 2 ) k - or - CH 2 (OCH 2 CH 2 ) k -, the number of k is selected from 1 to 10, and the number of m is selected from 2 to 10, 13 ⁇ 4 An integer selected from 100 to 2000.
  • P means recombinant human interferon a;
  • X is -(C3 ⁇ 4) k -, wherein k is 2; the number of m is selected from 2 ; in is selected from an integer from 450 to 600.
  • the invention also provides a conjugate of the formula (V): Wherein P refers to recombinant human interferon, X is -(CH 2 ) k - or - CH 2 (OCH 2 CH 2 ) k -, the number of k is selected from 1 to 10, and 13 ⁇ 4 is selected from an integer between 100 and 2000. .
  • P means recombinant human interferon a
  • X is -(CH 2 :) k -, where k It is 2; 13 ⁇ 4 is an integer selected from 450 to 600.
  • P refers to recombinant human interferon
  • X is -(CH 2 ) k - or - CH 2 (OCH 2 CH 2 ) k -, the number of k is selected from 1 to 10, and the number of m is selected from 2 to 10, mi An integer selected from 100 to 2000.
  • P means recombinant human interferon a
  • X is -(CH 2 :) k -, where k is 2; Since 2, mi is selected from an integer of 450 to 600.
  • the invention also provides a conjugate of the formula (VII): H
  • P refers to recombinant human interferon
  • X is -(CH 2 ) k - or - CH 2 (OCH 2 CH 2 ) k -
  • the number of k is selected from 1 to 10
  • the number of m and n are each independently selected from An integer of 2 to 10
  • 13 ⁇ 4 is selected from an integer between 100 and 2000.
  • P means recombinant human interferon a
  • X is -(CH 2 ) k -, wherein k is 2; the number of m, n is selected from 2 ON; an integer selected from 450 to 600.
  • the invention also provides a conjugate of the formula:
  • P refers to recombinant human interferon
  • X is -(CH 2 ) k - or - CH 2 (OCH 2 CH 2 ) k -
  • the number of k is selected from 1 to 10
  • the number of m is selected from 2 to 10, 13 ⁇ 4
  • P means recombinant human interferon a
  • X is -(C3 ⁇ 4) k -, wherein k is 2; the number of m is selected from 2; mi is selected from an integer of 450 to 600.
  • the invention also provides a conjugate of the formula IX):
  • P refers to recombinant human interferon
  • X is -(CH 2 ) k - or - CH 2 (OCH 2 CH 2 ) k -, the number of k is selected from 1 to 10, and the number of n is selected from 2 to 10 , selected from an integer between 100 and 2000.
  • P means that the recombinant human interferon ⁇ X is -(C3 ⁇ 4 , wherein k is 2; m, the number of n is selected from 2; an integer selected from 450 to 600.
  • the invention also provides a conjugate of the formula (X):
  • P refers to recombinant human interferon
  • X is -(CH 2 ) k - or - CH 2 (OCH 2 CH 2 ) k -, the number of k is selected from 1 to 10, and the number of m is selected from 2 to 10, 13 ⁇ 4 It is further preferred to select a conjugate of the formula (X) between 100 and 2000, and the corresponding most preferred embodiment needs to satisfy the following conditions simultaneously:
  • P means recombinant human interferon a;
  • X is -(CH 2 k -, where k is 2; the number of m is selected from 2, 13 ⁇ 4 is an integer selected from 450 to 600.
  • the invention also provides a conjugate of the formula (XI): Wherein P refers to recombinant human interferon, X is -(CH 2 ) k - or - CH 2 (OCH 2 CH 2 ) k -, the number of k is selected from 1 to 10, and the number of m is selected from 2 to 10, mi further preferably the structure of formula (XI) is selected between 100 ⁇ 2000, the most preferred embodiment the corresponding need to meet the following criteria: P refers to a recombinant human interferon a; X is - (CH 2) k -, wherein k Is 2; the number of m is selected from 2, mi is selected from an integer of 450 to 600.
  • the invention also provides a conjugate of the formula ( ⁇ ):
  • ⁇ means recombinant human interferon
  • X is -(CH 2 ) k - or - CH 2 (OCH 2 CH 2 ) k .
  • the number is selected from 1 to 10, and the number of m and n are each independently selected from 2 ⁇ An integer of 10, selected from integers between 100 and 2000.
  • ⁇ means recombinant human interferon a;
  • X is -(CH 2 ) k -, wherein k is 2; the number of m, n is selected from 2; an integer selected from 450 to 600.
  • the invention also provides a conjugate of the formula (XIII):
  • P means that the recombinant human interferon X is -(CH 2 ) k - or - CH 2 (OCH 2 CH 2 ) k -, the number of k is selected from 1 to 10, and the number of m, n is independently selected from 2 An integer of ⁇ 10, 13 ⁇ 4 is selected from an integer between 100 and 2000.
  • conjugate of the formula (XIII) is required to simultaneously satisfy the following conditions: P means recombinant human interferon alpha; X is -(CH 2 :) k -, where k Is 2; the number of m, n is selected from 2; an integer selected from 450 to 600.
  • the steps thereof include:
  • the number of k is selected from 2 to 10, preferably 2;
  • a deprotecting agent is added to a buffer containing activated interferon to remove an acetyl protecting group of an activated interferon of the formula (XV) by a technique known in the art, followed by a structural formula (XVI).
  • Activated methoxypolyethylene glycol for pegylation is added to a buffer containing activated interferon to remove an acetyl protecting group of an activated interferon of the formula (XV) by a technique known in the art, followed by a structural formula (XVI).
  • the pH of the buffer system is selected from 5.0 to 7.0, preferably pH 6.2; the AG is selected from the group consisting of:
  • n is selected from 2 to 10, preferably 2;
  • the method for purifying the peginterferon conjugate is carried out by a technique known to those skilled in the art, such as ion exchange chromatography or gel chromatography.
  • a small molecule aldehyde compound of the formula (X IV) can be prepared by a known method disclosed in the literature, such as U.S. Patent 4,338,435.
  • It is still another object of the present invention to provide a pharmaceutical composition comprising the modified interferon conjugate comprising: (1) a therapeutic amount of a pegylated interferon conjugate provided by the present invention; An acceptable pharmaceutical carrier.
  • the interferon conjugate provided by the present invention can be formulated into a pharmaceutical composition suitable for injection by a method known in the art using a pharmaceutically acceptable carrier or excipient, and the pharmaceutical composition to be used can be formulated into a preparation and, as appropriate, Medical practices are administered in a consistent manner.
  • the compounds of the invention may be formulated in 10 mM sodium phosphate/potassium buffer pH 7 containing 132 mM sodium chloride.
  • the pharmaceutical composition may contain a preservative.
  • the pharmaceutical compositions may contain varying amounts of interferon, for example from 10 to 1000 micrograms per milliliter, such as 50 micrograms or 400 micrograms. Further, it is an object of the present invention to provide use of the interferon conjugate and a pharmaceutical composition containing the same for the preparation of an antiviral infection, antitumor, immunomodulatory drug.
  • the above diseases specifically include: chronic hepatitis B, hepatitis C and hepatitis E; viral diseases such as herpes zoster, flat and condyloma acuminata, papillomavirus infection, epidemic hemorrhagic fever and pediatric respiratory syncytial virus pneumonia; Malignant tumors such as hairy cell leukemia, chronic myeloid leukemia, melanoma, lymphoma, multiple myeloma, renal cell carcinoma, ovarian cancer, rectal cancer, liver cancer, lung cancer, and the like.
  • viral diseases such as herpes zoster, flat and condyloma acuminata, papillomavirus infection, epidemic hemorrhagic fever and pediatric respiratory syncytial virus pneumonia
  • Malignant tumors such as hairy cell leukemia, chronic myeloid leukemia, melanoma, lymphoma, multiple myeloma, renal cell carcinoma, ovarian cancer, rectal cancer
  • the biological activity of the interferon or the pegylated interferon conjugate provided by the present invention can be determined by the color reaction of the host cell pathogenic effect test. Host cell pathogenic effect
  • the (CytoPathic Effect, or CPE) test method was proposed by Foti (Methods in Enzymology, 119, 533, 198).
  • the method is a simple and quick color absorption reaction test method which is designed by the principle that the human host cell is treated by interferon to produce anti-viral infection and can prevent cytopathic death. Briefly, host cells are resistant to viral invasion after interferon treatment and thus prevent host cell death.
  • the antiviral biological activity of the interferon can be directly quantified by the dye absorbed by the viable cells.
  • the in vitro antiviral activity of interferon by the WISH-VSV system using the cytopathic inhibition method is a well-known method in the art, and specifically refers to the 2005 edition of the Pharmacopoeia of the People's Republic of China, the three appendix XC "interferon Biological activity assay".
  • test data of the pegylated interferon conjugate provided by the present invention indicates that the pegylated interferon conjugate provided by the invention can achieve the purpose of modifying the surface amino acid of the interferon a2b molecule and retain more interferon.
  • the biological activity of a2b provides good pharmacokinetic properties.
  • the interferon linker to -NH-CH 2 - linkages form, thereby enhancing the specificity of PEG-modified reaction.
  • Figure 1 is a comparison of serum antigen concentration-time changes after a single subcutaneous administration of the same dose of HH-IFN-003 and PEG-IFNa-2b (Peglntron, Schering-Plough) in cynomolgus monkeys.
  • Figure 2 is a graph comparing serum antiviral activity-time changes after single subcutaneous administration of 10 g.kg-1 HH-IFN-003 and Peglntron in cynomolgus monkeys. detailed description
  • the dialysis bag (molecular weight cutoff 3500) was dialyzed against 0.1 M sodium phosphate buffer solution containing 2 mM EDTA, pH 6.25 to remove excess small molecule aldehyde, and then hydroxylamine was added to remove the acetyl group to activate the interferon.
  • Example 2 The following examples (3 to 8) are the procedures in which the activated interferon obtained in Example 2 was modified with an active polyethylene glycol to obtain pegylated interferon.
  • the code and structure of the target are shown in the table below, where IFN is activating interferon a2b:
  • reaction solution was purified by ion exchange chromatography (SP Sepharose H.P.) to obtain poly(ethylene glycolated interferon) (HH-IFN-001), about 17.5 mg, and the molecular weight was 24.6 KD by MALDI-TOF-MS.
  • the reaction solution was purified by ion exchange chromatography (SP Sepharose H.P.) to obtain a polyethylene glycol interferon (HH-IFN-002), which was about 15.3 mg, and the molecular weight was 29.9 KD by MALDI-TOF-MS.
  • activated interferon a2b 1.0 mg/ml, 0.1 M sodium phosphate buffer, containing 2 mM EDTA, pH 6.25) containing free thiol obtained in Example 2, added 1.0 g mPEG-maleamide (20 kD from SU BIO), stir the reaction for 60 minutes; add N-methylmaleimide to a concentration of 5 mM, react at room temperature for 30 minutes to react the remaining sulfhydryl groups on the protein; The reaction was dialyzed into a 20 mM acetate buffer system.
  • reaction solution was purified by ion exchange chromatography (SP Sepharose H.P.) to obtain polyethylene glycol interferon (HH-IFN-003), about 16.0 mg, and the molecular weight was 40.3 KD by MALDI-TOF-MS.
  • activated interferon a2b 100 mg of activated interferon a2b (1.0 mg/ml, 0.1 M sodium phosphate buffer containing 2 mM EDTA, pH 6.25) containing free thiol obtained in Example 2, and 2.0 g of mPEG-maleamide (40 kD) was added.
  • mix reaction for 60 minutes add N-methyl maleimide to a concentration of 5 mM, react at room temperature for 30 minutes to react the remaining sulfhydryl groups on the protein; then dialyze the reaction solution to 20 mM Acetic acid buffer system.
  • the reaction solution was purified by ion exchange chromatography (SP Sepharose H.P.) to obtain a polyethylene glycol interferon (HH-IFN-004) of about 14.5 mg, which was detected by MALDI-TOF-MS to have a molecular weight of 60.1 KD.
  • activated interferon a2b 1.0 mg/ml, 0.1 M sodium phosphate buffer, containing 2 mM EDTA, pH 6.25) containing free sulfhydryl groups obtained in Example 2, and 1.0 g of gPEG-o-pyridyl-disulfide was added. (20KD, from SU BIO), stir the reaction for 60 minutes, then add N-methylmaleimide to a concentration of 5 mM, react at room temperature for 30 minutes to react the remaining sulfhydryl groups on the protein; then dialyze the reaction solution Up to 20 mM acetate buffer system.
  • reaction solution was purified by ion exchange chromatography (SP Sepharose H.P.) to obtain polyethylene glycol interferon (HH-IFN-005), about 16.0 mg, and the molecular weight was 41.0 KD by MALDI-TOF-MS.
  • activated interferon a2b 1.0 mg/ml, 0.1 M sodium phosphate buffer, containing 2 mM EDTA, pH 6.25) containing free thiol obtained in Example 2, and 1.0 g of iodoacetamide-mPEG (20 KD) was added. , from NOF CORPORATION), stir the reaction for 60 minutes, then add N-methyl maleimide to a concentration of 5 mM, react at room temperature for 30 minutes, The remaining sulfhydryl groups on the protein were reacted; the reaction solution was then dialyzed into a 20 mM acetate buffer system.
  • the reaction solution was purified by ion exchange chromatography (SP Sepharose H.P.) to obtain a polyethylene glycol interferon (HH-IFN-006), which was about 16.5 mg, and the molecular weight was 39.8 KD by MALDI-TOF-MS.
  • Example 9 In vitro antiviral activity test of interferon a2b and 6 pegylated interferon conjugates
  • the in vitro antiviral activity of interferon was determined by WISH-VSV system using cytopathic inhibition.
  • This example measures the in vitro antiviral activity of seven interferons (or derivatives), specifically including: IFNa2b (provided by Shanghai Hengrui Pharmaceutical Co., Ltd.), HH-IFN-001 (prepared by Example 3 of the present invention), HH-IFN-002 (prepared in Example 4 of the present invention), HH-IFN-003 (prepared in Example 5 of the present invention), HH-IFN-004 (prepared in Example 6 of the present invention), HH-IFN-005 (; Inventive Example 7 prepared:), HH-IFN-006 (Prepared in Example 8 of the present invention:).
  • the results of in vitro antiviral activity assay are shown in Table 1:
  • Example 10 Comparison of serum antigen concentration and pharmacokinetics of a single subcutaneous administration of a test article of cynomolgus monkey HH-IFN-003 and a reference product Peglntron
  • the concentration of the test article HH-IFN-003 and the reference Peglntron in the serum can be determined using a Hu-IFN-a ELISA kit.
  • Hu-IFN-a Immunoassay is an amplified double-sandwich antibody immunoassay. The test procedure is in accordance with the instructions of the kit.
  • the monoclonal antibody of IFN- ⁇ was coated, and the test substance HH-IFN-003 and the reference product Peglntron were separately added, incubated for a certain period of time, washed, plated with antibody dilution, sealed, incubated at room temperature, then washed, and added HRP (horseradish peroxidase) coupling, sealing plate, incubate at room temperature, wash plate thoroughly, finally add chromogenic substrate and incubate at room temperature in the dark until color appears; stop reaction by adding stop solution, measure 450 nm with microplate reader The value of light absorption at the wavelength.
  • HRP horseradish peroxidase
  • the logarithm of the light absorption value is positively correlated with the logarithm of the concentration of HH-IFN-003 and Peglntron in the sample.
  • the logarithm of the logarithm of the concentration and the logarithm of the light absorption value are subjected to a log-log linear regression, and a standard curve is drawn to calculate the unknown sample concentration.
  • EXPERIMENTAL RESULTS Compared with the single subcutaneous administration of Peglntron, 10 ⁇ - ⁇ , the serum antigen concentration of the HH-IFN-003 test group was greater than that of the Peglntron group. 1), AUC (0-96h), and AUC (O-inf) were higher than the Pegrntron group, and the pharmacokinetic parameters are shown in Table 2. After a single subcutaneous administration of the surface, the in vivo exposure of HH-IFN-003 was higher than that of PegIntron.
  • the method for measuring the activity of PEG-IFNa-2b and IFNa-2b adopts the cytopathic inhibition assay (CPE method), and the WISH cells are used as the host cells of the virus (susceptible cells), and the cells are cytopathic (CPE) after being attacked by the virus infection (CPE). ), the cells develop lesions, die, and fall off the bottom of the dish. Under the intervention of antiviral active drugs, the cells without lesions are attached to the culture dish.
  • the crystal violet dye can be used to dye the living cells, and the dye is removed by the decolorizing solution. The OD value is measured to reflect the cell adhesion in the culture. The state of survival on the dish.
  • the WISH cells in the logarithmic growth phase were digested, and the cell concentration was adjusted with a MEM medium containing 10% FBS, seeded in a 96-well culture plate, and incubated at 37 ° C, 5 % CO 2 for 24 hours. After washing with PBS, serum samples containing the test substance HH-IFN-003 or the reference product Peglntron diluted in 2% FBS MEM medium were added, and each concentration was set to three replicate wells, and no drug control wells were set. Continue to train for 24 hours. The virus was diluted with 100% TCID50 (50% cell infection amount) in 2% FBS MEM medium, and the virus-infected normal cell well and virus-infected control group were added, and culture was continued for 24 hours.
  • TCID50 50% cell infection amount

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Abstract

本发明公开了一种聚乙二醇干扰素偶联物，其制备方法，以及含有治疗有效量的该干扰素偶联物的药物组合物，及其在制备抗病毒、抗肿瘤、免疫调节药物中的用途。

Description

一种聚乙二醇干扰素偶联物
技术领域
本发明涉及一种聚乙二醇干扰素偶联物，该偶联物通过一种新颖 的生物蛋白分子的聚乙二醇修饰技术合成，具有抗病毒感染、抗肿瘤、 免疫调节等生物学活性， 本发明的应用领域涉及生物化学、药物化学 以及人类疾病的治疗。 背景技术
干扰素 （Interferon:) 是一组具有多种功能的活性蛋白质， 是一类 重要的家族性细胞因子， 具有广谱的抗病毒、 抗细胞增殖、 和免疫调 节作用。 哺乳动物的干扰素可以分为 α、 β、 γ、 ω等几种类型， 其中 α干扰素又可分十余种亚型， 经大量临床研究证明 α型干扰素是一种 重要的抗病毒和抗肿瘤治疗药物。目前我国在临床使用最广泛的主要 是重组人干扰素 alb、 a2b和 a2a。 另外， 美国 Amgen公司根据 13 种 α型干扰素的基因序列同源性，设计出的一种全新的蛋白质工程药 物干复津 （INFERGEN® , IFN-Con-1) 于 1997年经 FDA批准上市用 于治疗丙型肝炎， 其病毒活性是 a2b干扰素的 5〜10倍。
但是，一般来说，许多有生物技术 (如基因重组:)表达所得的产品， 包括干扰素，都是通过非肠道给药途径而进入生物体内发挥其药效作 用。此类由非肠道给药的生物大分子药物产品通常存在下面的一些问 题： （1) 生物大分子药物往往具有致敏性反应， 生物体内产生的抗体 会对生物体造成严重的伤害并影响治疗的进行； （2) 生物大分子药物 本身因受到抗体的影响或因蛋白分解酶而引起的代谢作用，致使其生 物半衰期大为缩短； （3) 生物大分子的稳定性差， 保存困难。 所以， 无论是干扰素 alb、 a2b和 a2a还是干复津， 作为蛋白质药物， 由于 稳定性差， 血桨清除率高， 体内半衰期短， 易产生抗原抗体反应等， 在临床治疗中受到很大的限制， 这些缺点造成的结果是： 需频繁注射 干扰素才能达到有效的血桨治疗浓度； 而且， 每次注射后均会导致血 液浓度的较大波动， 形成药物浓度的峰值与谷值。这样就可能增加了 治疗费用以及给药不便和不良反应的风险。 因此， 人们试图采用各种 药物传递技术 （Drug Delivery Technology), 来提高蛋白质药物的疗 效。 而目前在药物传递技术中研究最为广泛的是聚乙二醇修饰技术 (PEGNOLOGY)。
1980年， Davis等人在美国专利 4,179,337中公开了利用单一分 子不同聚合度的聚乙二醇对蛋白药物进行的化学修饰，在保持药物的 生物活性同时， 药物的抗原性降低， Verones等人在 Applied Biochem and Biotech 11, 142(1985)上公开发表了用氯甲酸苯酯活化的聚乙二醇 修饰核糖核酸酶和超氧化物歧化酶， 增加了蛋白的生物半衰期。上述 现有技术文献报导已表明聚乙二醇修饰技术的确可以解决非肠道给 药生物大分子药物存在的一些问题。
目前，市场上已经有两种聚乙二醇化干扰素产品，分别是 Schering Plough公司的 PEG-IFNa2b (PEG-INTRON) 与 Hoffman-la Roche 公 司的 PEG-IFNa2a (PEGASYS)， 提高了干扰素在体内的半衰期， 达到 每周注射一次， 使得干扰素的生物利用度得到了一定程度的提升。这 两个产品已占有国外干扰素市场 60%, 获得了巨大的经济效益。 发明内容
本发明的目的在于提供一种生物学活性更好、生物利用度更高的 的聚乙二醇修饰的干扰素偶联物， 其结构如式 （I )所示：
P-NH-CH2-X-S-Y-Q
( I )
其中， P是干扰素， 包括所有类型的干扰素， 以及这些类型所有 的亚类， 以及不同类型和 /或亚类干扰素组成的复合干扰素； P可以是 任何来源的， 包括天然来源、组织培养或由基因重组技术得到的干扰 素； P优选天然的或人工重组的干扰素蛋白， 更优选重组人干扰素， 更优选重组人干扰素 α、 β、 γ或 ω， 最优选 a2b; P既可通过本领域 公开的技术制得， 也可在市场上购得；
X是 -(CH2)k-或- CH2(OCH2CH2)k-， k选自 2〜10的整数，优选 1〜 4， 进一步优选为 2;
Q为甲氧基聚乙二醇，其平均分子量优选 5,000〜40,000道尔顿， 更优选 20,000道尔顿；
Figure imgf000004_0001
Figure imgf000004_0002
Figure imgf000004_0003
Figure imgf000004_0004
Figure imgf000004_0005
Y5 〇 工
Figure imgf000005_0001
一种结构通式为 （Π ) 的偶联
Figure imgf000006_0001
其中 P是指干扰素， X是 -(CH2)k-或 -CH2(OCH2CH2)k-， k的数目 1〜10， m、 n 的数目选自 2〜10， 选自 100〜2000之间的整 其中 X优选为 -(CH2)k-， k的数目可进一步优选自 1〜4， 最优选 的是 2。
对上述优选实施方案进一步优选，本发明还提供了最优选的一个 实施方案， 对应的结构式为：
Figure imgf000006_0002
mi选自 450〜600的整数， 其中 P是指干扰素， 可进一步优选重 组人干扰素， 再优选重组人干扰素 α， 最优选重组人干扰素 (x2b。
本发明还 一种结构通式为 （III) 的偶联物：
Figure imgf000006_0003
其中 P是指重组人干扰素， X是 -(CH2)k-或- CH2(OCH2CH2)k-， k 的数目选自 1〜10， m的数目选自 2〜10， 1¾选自 100〜2000之间的 对结构式 （ΠΙ) 进一步优选， 所对应的最优选实施方案需要同时 满足以下条件： P是指重组人干扰素 a; X是 -(C¾)k-， 其中 k是 2; m的数目选自 2; mi选自 450〜600的整数。
本发明还提供一种结构通式为 （IV) 的偶联物： 其中 P是指重组人干扰素， X是 -(CH2)k-或- CH2(OCH2CH2)k-， k 的数目选自 1〜10， m的数目选自 2〜10， 1¾选自 100〜2000之间的 整数。
对结构式 （IV) 进一步优选， 所对应的最优选实施方案需要同时 满足以下条件： P是指重组人干扰素 a; X是 -(C¾)k-， 其中 k是 2; m的数目选自 2; in选自 450〜600的整数。
本发明还提供一种结构通式为 （V) 的偶联物：
Figure imgf000007_0001
其中 P是指重组人干扰素， X是 -(CH2)k-或- CH2(OCH2CH2)k-， k 的数目选自 1〜10， 1¾选自 100〜2000之间的整数。
对结构通式为 （V) 的偶联物进一步优选， 所对应的最优选实施 方案需要同时满足以下条件： P是指重组人干扰素 a; X是 -(CH2：) k-， 其中 k是 2; 1¾选自 450〜600的整数。
本 一种结构通式为 （VI) 的偶联物：
Figure imgf000007_0002
其中 P是指重组人干扰素， X是 -(CH2)k-或- CH2(OCH2CH2)k-， k 的数目选自 1〜10， m的数目选自 2〜10， mi选自 100〜2000之间的 整数。
对结构式 （VI) 进一步优选， 所对应的最优选实施方案需要同时 满足以下条件： P是指重组人干扰素 a; X是 -(CH2：) k-， 其中 k是 2; m的数目选自 2， mi选自 450〜600的整数。
本发明还提供一种结构通式为 （VII) 的偶联物： H
P-NH— C—— X—— S-SH-CHp -C-NH-f-CH OCH2CH2 ~ OCH;
m m
(VII)
其中 P是指重组人干扰素， X是 -(CH2)k-或- CH2(OCH2CH2)k-， k 的数目选自 1〜10， m、 n的数目各自独立地选自 2〜10的整数， 1¾ 选自 100〜2000之间的整数。
对结构通式为 （VII) 的偶联物进一步优选， 所对应的最优选实施 方案需要同时满足以下条件： P是指重组人干扰素 a; X是 -(CH2)k-， 其中 k是 2; m、 n的数目选自 2 ON; 选自 450〜600的整数。
本发明还提供一种结构通式为 (覆)的偶联物：
Figure imgf000008_0001
其中 P是指重组人干扰素， X是 -(CH2)k-或- CH2(OCH2CH2)k-， k 的数目选自 1〜10， m的数目选自 2〜10， 1¾选自 100〜2000之间的 对结构式 （覆）进一步优选， 所对应的最优选实施方案需要同时 满足以下条件： P是指重组人干扰素 a; X是 -(C¾)k-， 其中 k是 2; m的数目选自 2; mi选自 450〜600的整数。
本发明还提供一种结构通式为 IX) 的偶联物：
P— NH— CH2— X— S— CH2CH2-— -S. CH2
Figure imgf000008_0002
(IX)
其中 P是指重组人干扰素， X是 -(CH2)k-或- CH2(OCH2CH2)k-， k 的数目选自 1〜10， m， n的数目选自 2〜10， 选自 100〜2000之 间的整数。 满足以下条件： P是指重组人干扰素^ X是 -(C¾ ， 其中 k是 2; m， n的数目选自 2; 选自 450〜600的整数。
本发明还提供一种结构通式为 （X) 的偶联物：
Figure imgf000009_0001
其中 P是指重组人干扰素， X是 -(CH2)k-或- CH2(OCH2CH2)k-， k 的数目选自 1〜10， m的数目选自 2〜10， 1¾选自 100〜2000之间 对结构通式为 （X) 的偶联物进一步优选， 所对应的最优选实施 方案需要同时满足以下条件： P是指重组人干扰素 a; X是 -(CH2)k-， 其中 k是 2; m的数目选自 2， 1¾选自 450〜600的整数。
本发明还提供一种结构通式为 （XI) 的偶联物：
Figure imgf000009_0002
其中 P是指重组人干扰素， X是 -(CH2)k-或- CH2(OCH2CH2)k-， k 的数目选自 1〜10， m的数目选自 2〜10， mi选自 100〜2000之间的 对结构式 （XI) 进一步优选， 所对应的最优选实施方案需要同时 满足以下条件： P是指重组人干扰素 a; X是 -(CH2)k-， 其中 k是 2; m的数目选自 2， mi选自 450〜600的整数。
本发明还提供一种结构通式为 （ΧΠ) 的偶联物：
-NH- -CH, ■ CHy -NH- CH2 -NH' CH' -NH- CH2CH2十 0CH3
(ΧΠ)
其中 Ρ是指重组人干扰素， X是 -(CH2)k-或- CH2(OCH2CH2)k. 的数目选自 1〜10， m、 n的数目各自独立地选自 2〜10的整数， 选自 100〜2000之间的整数。 对结构通式为 （ΧΠ) 的偶联物进一步优选， 所对应的最优选实施 方案需要同时满足以下条件： Ρ是指重组人干扰素 a; X是 -(CH2)k-， 其中 k是 2; m、 n的数目选自 2; 选自 450〜600的整数。
发明还提供一种结构通式为 （XIII) 的偶联物：
-ΝΗ- -CH2一 S- -CH7 -NH- CH2 - NH—— CH2CH2— fOCH2CH2i— OCH;
(XIII)
其中 P是指重组人干扰素 X是 -(CH2)k-或- CH2(OCH2CH2)k-， k 的数目选自 1〜10， m、 n的数目 自独立地选自 2〜10的整数， 1¾ 选自 100〜2000之间的整数。
对结构通式为 （XIII) 的偶联物进一步优选， 所对应的最优选实 施方案需要同时满足以下条件： P是指重组人干扰素 α; X是 -(CH2：) k-， 其中 k是 2; m、 n的数目选自 2; 选自 450〜600的整数。
本发明的又一目的在于提供一种制备所述干扰素偶联物的方法， 该方法主要包括两个阶段：首先是干扰素和含有已保护巯基的醛类物 质反应， 形成通过 -NH-CH2-键连接的活化干扰素蛋白； 然后， 所述活 化的干扰素脱保护， 与活性甲氧基聚乙二醇衍生物偶联， 通过分离而 得到。 具体而言， 其步骤包括：
(1) 制备结构通式为 （ 物：
Figure imgf000010_0001
(X IV)
其中 k的数目选自 2〜10， 优选 2;
(2) 将结构通式为 （X R 的小分子醛化合物与干扰素在缓冲液 里反应， 并加入还原剂得到结构通式为 （X V) 的活化干扰素；
Figure imgf000010_0002
其中 P是指干扰素， 优选重组人干扰素 (x2b， 缓冲液的 pH选自 4.0 7.0, 优选 4.5， 还原剂可以是硼氰化钠、 氰基硼氰化钠， 优选氰基 ί 氰化钠；
(3) 利用本领域公知的技术在含有活化干扰素的缓冲液里加入脱 保护剂脱去结构通式为 （X V) 的活化干扰素的乙酰基保护基， 随后 加入结构通式为 （XVI) 的活化甲氧基聚乙二醇进行聚乙二醇化反 应，
Figure imgf000011_0001
(XVI)
缓冲体系的 pH选自 5.0〜7.0， 优选 pH6.2; AG选自：
Figure imgf000011_0002
Figure imgf000012_0001
O
■CH -NH- CH2 -NH- CHp -NH-
O O
O
" CH, -NH- CH2 -NH—— CH?一 CH;
m
O
其中 m、 n的数目选自 2〜10， 优选 2;
(4) 聚乙二醇干扰素偶联物的纯化方法采用本领域技术人员公知 的技术， 如离子交换层析、 凝胶层析。
本发明制备所述干扰素偶联物的方法中， 结构通式为 （X IV) 的 小分子醛化合物可按文献公开的已知方法制备， 如 US4338435。
本发明的又一目的在于提供一种含有该被修饰的干扰素偶联物 的药物组合物， 其包含： （1) 本发明所提供的治疗量的聚乙二醇化干 扰素偶联物； 药学可接受的药物载体。 本发明提供的干扰素偶联 物可以通过本领域公知的方法用药学可接受载体或赋形剂制成适合 注射的药物组合物， 欲使用的药物组合物可制成制剂， 并按与合适的 医疗惯例一致的方式给药。本发明的化合物可以在含有 132 mM氯化 钠的 10 mM磷酸钠 /钾缓冲液 pH7中配制。 任选地， 药物组合物可 以含有防腐剂。 药物组合物可以含有不同量的干扰素， 例如 10-1000 微克 /毫升， 例如 50微克或 400微克。 进一步，本发明的目的在于提供所述干扰素偶联物以及含有它们 的药物组合物在制备抗病毒感染、 抗肿瘤、 免疫调节药物中的用途。 上述疾病具体包括： 慢性乙型肝炎、 丙型肝炎和戊型肝炎等； 病毒性 疾病如带状疱疹、 扁平和尖锐湿疣、 乳头瘤病毒感染、 流行性出血热 和小儿呼吸道合胞病毒肺炎等； 恶性肿瘤如毛细胞性白血病、慢性粒 细胞白血病、黑色素瘤、淋巴瘤、 多发性骨髓瘤、 肾细胞癌、卵巢癌、 直肠癌、 肝癌、 肺癌等。
干扰素或本发明提供的聚乙二醇干扰素偶联物的生物活性可由 宿主细胞致病效应测试法的颜色反应而确定。 宿主细胞致病效应
(CytoPathic Effect,or CPE)测试法系由 Foti 提出（Methods in Enzymology,119,533,198)。 本法利用人类宿主细胞经干扰素处理后产 生抵抗病毒感染的活性而能防止细胞病变死亡的原理设计而成的一 种简易快捷的颜色吸收反应测试方法。简单而言， 宿主细胞在经干扰 素处理后可抵抗病毒入侵因而可防止宿主细胞死亡。干扰素的抗病毒 生物活性即可经由存活细胞所吸收的染料而直接定量测量。由此而来 的采用细胞病变抑制法通过 WISH-VSV系统测定干扰素的体外抗病 毒活性， 是本技术领域公知的方法， 具体参照 2005版 《中华人民共 和国药典》 三部附录 X C"干扰素的生物活性测定"。
通过对本发明提供的聚乙二醇干扰素偶联物的测试数据表明：本 发明提供的聚乙二醇干扰素偶联物可以达到修饰干扰素 a2b 分子表 面氨基酸的目的且保留较多的干扰素 a2b 的生物活性并提供良好的 药代特性。
在本发明所提供的化合物中，干扰素与连接体以 -NH-CH2-键形式 连接， 由此增强了 PEG修饰反应的专一性。 附图说明
图 1 是食蟹猴单次皮下给药相同剂量 HH-IFN-003 与 PEG-IFNa-2b (Peglntron, 先灵葆雅） 后的血清抗原浓度 -时间变化比较图。
图 2是食蟹猴单次皮下给药 10 g.kg-l HH-IFN-003及 Peglntron后血 清抗病毒活性-时间变化的比较图。 具体实施方式
实施例 h 乙酰基巯基丙醛的制备
Figure imgf000014_0001
将 11.2 g (0.2mmol) 丙烯醛与干燥的 100 ml THF加入到反应瓶 中，冷却至 0°C，然后缓慢滴加 1.52 g (0.02 mol)硫代乙酸 /20 ml THF 的混合溶液。滴加完毕保温反应 2小时后。 35°C减压浓缩除去过量的 丙烯醛。 然后快速上柱 （洗脱液纯正己垸→正己垸 /乙酸乙酯 =50/1)， 合并收集产物点， 减压浓缩至干得油状液体 0.6 g。
实施例 2: 活化干扰素的制备
取 600 mg干扰素 a2b (来自上海恒瑞医药有限公司:)，蛋白浓度为 1.0 mg/ml, 共 600 ml， 体系为 0.1 M乙酸缓冲液， pH 4.5; 取 39.6 mg 乙酰基巯基丙醛溶于 400 ul乙腈后加入以上蛋白溶液；再称取 756 mg 氰基硼氢化钠加入上述反应液， 并慢速搅拌反应， 冰浴控制反应温度 在 10°C， 反应 4小时； 然后将反应液转入透析袋 （截留分子量 3500), 对 0.1 M磷酸钠盐缓冲液， 含 2 mM EDTA， pH 6.25进行透析， 以除 掉过量的小分子醛， 然后再加入羟胺脱去乙酰基， 得活化干扰素。
说明： 以下实施例 （3~8) 为将实施例 2得到的活化干扰素用活 性聚乙二醇修饰， 并得到聚乙二醇干扰素的过程。 目标物的代号及结 构示意图见下表， 其中 IFN是活化干扰素 a2b:
Figure imgf000014_0002
Figure imgf000015_0001
实施例 3: HH-IFN-001的制备
通过实施例 2得到的 100 mg活化干扰素 a2b (1.0 mg/ml, 0.1 M磷 酸钠盐缓冲液， 含 2mMEDTA， pH 6.25), 加入 0.25 g mPEG-马来酰 胺 （5kD， 来自 SU BIO), 搅拌反应 60分钟； 再加入 N-甲基马来酰 亚胺至浓度为 5mM，室温反应 30分钟， 以反应掉蛋白上剩余的巯基； 然后将反应液透析至 20 mM乙酸缓冲液体系。
反应液使用离子交换层析 （SP Sepharose H.P) 纯化， 得到聚乙二 醇化干扰素 （HH-IFN-001), 约 17.5mg， 经 MALDI-TOF-MS检测分子 量为 24.6KD。
实施例 4: HH-IFN-002的制备
向实施例 2得到的含有游离巯基的 100 mg活化干扰素 a2b (1.0 mg/ml, 0.1M磷酸钠盐缓冲液， 含 2mMEDTA， pH 6.25), 加入 0.5 g mPEG-马来酰胺（10kD， 来自 SUNBIO), 搅拌反应 60分钟； 再加入 N-甲基马来酰亚胺至浓度为 5 mM， 室温反应 30分钟， 以反应掉蛋白 上剩余的巯基； 然后将反应液透析至 20 mM乙酸缓冲液体系。
反应液使用离子交换层析 （SP Sepharose H.P) 纯化， 得到聚乙二 醇化干扰素 （HH-IFN-002),约 15.3mg，经 MALDI-TOF-MS检测分子 量为 29.9KD。
实施例 5: HH-IFN-003的制备
向实施例 2得到的含有游离巯基的 100 mg活化干扰素 a2b (1.0 mg/ml, 0.1M磷酸钠盐缓冲液， 含 2 mMEDTA， pH6.25), 加入 1.0 g mPEG-马来酰胺（20kD， 来自 SU BIO), 搅拌反应 60 分钟； 再加入 N-甲基马来酰亚胺至浓度为 5 mM， 室温反应 30分钟， 以反应掉蛋白 上剩余的巯基； 然后将反应液透析至 20 mM乙酸缓冲液体系。
反应液使用离子交换层析 （SP Sepharose H.P) 纯化， 得到聚乙二 醇化干扰素 （HH-IFN-003),约 16.0 mg，经 MALDI-TOF-MS检测分子 量为 40.3KD。
实施例 6: HH-IFN-004的制备
向实施例 2得到的含有游离巯基的 100 mg活化干扰素 a2b (1.0 mg/ml, 0.1 M磷酸钠盐缓冲液， 含 2 mM EDTA， pH6.25), 加入 2.0 g mPEG-马来酰胺 （40kD， 来自 SU BIO), 拌反应 60分钟， 再加入 N- 甲基马来酰亚胺至浓度为 5 mM， 室温反应 30分钟， 以反应掉蛋白上 剩余的巯基； 然后将反应液透析至 20 mM乙酸缓冲液体系。
反应液使用离子交换层析 （SP Sepharose H.P) 纯化， 得到聚乙二 醇化干扰素 （HH-IFN-004),约 14.5 mg，经 MALDI-TOF-MS检测分子 量为 60.1KD。
实施例 7: HH-IFN-005的制备
向实施例 2得到的含有游离巯基的 100 mg活化干扰素 a2b (1.0 mg/ml, 0.1M磷酸钠盐缓冲液， 含 2mM EDTA， pH 6.25), 加入 l.Og mPEG-邻吡啶基-二硫化物 （20KD, 来自 SU BIO), 搅拌反应 60分 钟， 再加入 N-甲基马来酰亚胺至浓度为 5 mM， 室温反应 30分钟， 以反应掉蛋白上剩余的巯基； 然后将反应液透析至 20 mM乙酸缓冲 液体系。
反应液使用离子交换层析 （SP Sepharose H.P) 纯化， 得到聚乙二 醇化干扰素 （HH-IFN-005),约 16.0 mg，经 MALDI-TOF-MS检测分子 量为 41.0KD。
实施例 8: HH-IFN-006的制备
向实施例 2得到的含有游离巯基的 100 mg活化干扰素 a2b (1.0 mg/ml, 0.1 M磷酸钠盐缓冲液， 含 2 mM EDTA， pH 6.25), 加入 l.Og 碘乙酰胺 -mPEG (20KD, 来自 NOF CORPORATION), 搅拌反应 60分 钟， 再加入 N-甲基马来酰亚胺至浓度为 5 mM， 室温反应 30分钟， 以 反应掉蛋白上剩余的巯基； 然后将反应液透析至 20 mM乙酸缓冲液体 系。
反应液使用离子交换层析 （SP Sepharose H.P) 纯化， 得到聚乙二 醇化干扰素 （HH-IFN-006),约 16.5 mg，经 MALDI-TOF-MS检测分子 量为 39.8KD。
实施例 9: 干扰素 a2b及 6种聚乙二醇干扰素偶联物的体外抗病 毒活性测试
采用细胞病变抑制法通过 WISH-VSV系统测定干扰素体外抗病毒 活性。
本实施例测定了 7种干扰素 （或衍生物） 的体外抗病毒活性，具体 包括： IFNa2b (上海恒瑞医药股份有限公司提供：)、 HH-IFN-001 (本发 明实施例 3制备)、 HH-IFN-002 (本发明实施例 4制备)、 HH-IFN-003 (本 发明实施例 5制备)、 HH-IFN-004 (；本发明实施例 6制备)、 HH-IFN-005 (；本发明实施例 7制备：)、 HH-IFN-006 (；本发明实施例 8制备：)。 体外抗 病毒活性测定结果见表 1 :
表 1 体外抗病毒活性测定结果
Figure imgf000017_0001
表 1中数据显示， 本发明所提供的聚乙二醇干扰素 a2b偶联物与 修饰前的干扰素 a2b相比， 仍保留有约 7-15%的抗病毒活性。
实施例 10: 食蟹猴单次皮下给药受试品 HH-IFN-003 和参比品 Peglntron后的血清抗原浓度和药代动力学比较实验
用 Hu-IFN-a ELISA药盒可以测定血清中受试品 HH-IFN-003和 参比品 Peglntron的浓度。 Hu-IFN-a Immunoassay是一种放大的双夹 心抗体免疫分析法， 试验操作步骤遵照药盒说明书， 96 孔板预先用 IFN- α的单抗包被，分别加入受试品 HH-IFN-003和参比品 Peglntron, 孵育一定时间， 洗板， 加抗体稀释液， 封板， 室温孵育， 再洗板， 另 加入携有 HRP (辣根过氧化物酶） 耦合物， 封板， 室温孵育， 彻底 洗板， 最后加入显色底物室温避光孵育直到出现颜色； 加入终止液终 止反应， 用酶标仪测定 450 nm波长处的光吸收值。 光吸收值的对数 值与样品中 HH-IFN-003和 Peglntron的浓度对数值呈正相关，对浓度 对数和光吸收值对数进行双对数线性回归， 绘制标准曲线， 计算未知 样品浓度。
实验结果：与单次皮下给药先灵葆雅公司参比品组 Peglntron, 10 μ^- Ι相比， HH-IFN-003受试品组各时间点血清抗原浓度在数值上 均大于 Peglntron组 （见图 1 )， AUC(0-96h)， 和 AUC(O-inf)均高于 Peglntron 组， 药代动力学参数见表 2。 表面单次皮下给药后， HH-IFN-003的体内暴露量要高于 PegIntron。
表 2 食蟹猴单次皮下注射受试品 HH-IFN-003与 Peglntron药代动力 学参数比较 受试品 HH-IFN-003 Peglntron 参 数 单位
lO g-kg-1 10 g-kg-1
AUC(0-96h) ng-h-mL"1 1167.0 + 146.5 782.9 + 304.2
AUC(O-inf) ng-h-mL"1 1187.3 ± 146.1 803.2 + 304.4 实施例 11 : 食蟹猴单次皮下给药 10 g'kg— 1 HH-IFN-003 及 Peglntron后血清抗病毒活性比较实验
PEG-IFNa-2b与 IFNa-2b活性测定方法采用细胞病变抑制试法 ( CPE法）， 以 WISH细胞作为病毒的宿主细胞 （易感细胞）， 细胞受 到病毒感染攻击后会产生细胞病变效应 （CPE ) , 细胞发生病变， 死 亡， 并从培养皿底部脱落。 在抗病毒活性药物的干预下， 未发生病变 的细胞附着在培养皿上， 利用结晶紫染料， 可使活细胞染色， 再用脱 色液将染料脱出， 通过测定其 OD值来反映细胞附着在培养皿上的存 活状态。 将处于对数生长期的 WISH细胞消化， 用含 10%FBS的 MEM培养 液调整细胞浓度， 接种于 96孔培养板中， 37°C， 5 %C02, 孵育 24小 时。 以 PBS清洗后， 分别加入以 2 %FBS的 MEM培养液稀释调配的含 有受试品 HH-IFN-003或参比品 Peglntron的血清样本，每个浓度设三复 孔， 并设无药物对照孔， 继续培养 24小时。 加入以 2%FBS的 MEM培 养液稀释调配的 100倍 TCID50 (50%细胞感染量） 的病毒， 并设无病 毒感染正常细胞孔和病毒感染对照组， 继续培养 24小时， 中性红染色 后甲醛固定， 洗涤后用脱色液溶解， 在 540nm处得到吸光度 (OD)值。 根据不同稀释度的待测样品抵抗病毒感染发生的细胞病变 (CPE)的保 护能力， 计算出干扰素抵抗病毒感染抑制率 （％)， 并得到半数有效 保护浓度 （EC50)。
实验结果： 食蟹猴单次皮下给药 lO kg- 1 HH-IFN-003后各时间 点血清抗病毒活性的绝对值高于同剂量的 PegIntron。 见表 3、 图 2。 这 个结果表明， 由于 HH-IFN-003血药浓度要高于同剂量的 Peglntron, 因 此其体内的抗病毒药效也优于 PegIntron。
表 3 食蟹猴皮下给药单剂量 Peglntron 以及 HH-IFN-003后血清抗病 毒活性-时间变化的比较
抗病毒活性 （IU.mL-1)
时
间 10 g.kg-1 10 g.kg-1
点 HH-IFN-003 Peglntron
0 ND ND
4 184.7± 89.7 164.7±36.9
12 223.0± 132.4 210.7±30.9
24 180.7± 113.4 122.7± 17.9
48 24.0± 12.0 19.7±7.4
96 5.0± 1.0 3.3 ±3.2

Claims

权利要求书
1、 一种结构如式 所示的干扰素偶联物，
P-NH-CH2-X-S-Y-Q
(I)
其中， P是干扰素； X是 -(CH2)k-或- CH2(OCH2CH2)k-， k选自 1〜10 的整数； Q是甲氧基聚乙二醇； Y选自 Y1〜Y12:
Figure imgf000020_0001
Figure imgf000020_0002
Υ2
Figure imgf000020_0003
Y3
Figure imgf000020_0004
Y4
Figure imgf000020_0005
Y5
Figure imgf000021_0001
其中 m、 n各自独立地选自 2〜10之间的整数。
2、 根据权利要求 1所述的干扰素偶联物， 其特征在于， 所述偶 联物选自式 （Π：)〜 (XIII)所示化合物：
CH:
Figure imgf000022_0001
( Π)
Figure imgf000022_0002
(ΠΙ)
Figure imgf000022_0003
(IV)
Figure imgf000022_0004
Figure imgf000022_0005
Figure imgf000023_0001
(XIII)
其中， P是干扰素， X是 -(CH2)k-或- CH2(OCH2CH2)k -， k选自 1〜 10， m、 n各自独立地选自 2〜10之间的整数， 1¾选自 100〜2000之 间的整数。
3、 根据权利要求 1或 2所述的干扰素偶联物， 其特征在于， 所 述干扰素选自所有类型的干扰素及其亚类中的任意一种，或者不同类 型和 /或亚类干扰素组成的复合干扰素。
4、 根据权利要求 3所述的干扰素偶联物， 其特征在于， 所述干 扰素是重组人干扰素 0 β、 γ或 ω， 优选重组人干扰素 α， 更优选重 组人干扰素 a2b。
5、 根据权利要求 4所述的干扰素偶联物， 其特征在于， m=2， n=2 o
6、 根据权利要求 5 所述的干扰素偶联物， 其特征在于， X 是 -(CH2)k-, k为 1〜4之间的整数， 优选 k=2。
7、 根据权利要求 6所述的干扰素偶联物， 其特征在于， mi选自 450-600之间的整数。
8、 根据权利要求 7所述的干扰素偶联物， 其特征在于， 每个甲 氧基聚乙二醇基团的平均分子量为 5,000-40,000 道尔顿， 优选为 20,000道尔顿。
9、 一种制备权利要求 1-8任意一项所述干扰素偶联物的方法， 其包括以下步骤：
1) 干扰素和含有已保护巯基的醛类物质反应， 形成通过 -NH-CH2-键连接的活化干扰素蛋白；
2) 所述活化的干扰素蛋白脱保护， 与活性甲氧基聚乙二醇衍生 物偶联。
10、根据权利要求 9所述的方法， 其特征在于所述醛类物质是式 (X IV) 的小分子醛化合
Figure imgf000024_0001
(XIV)
其中 k的数目选自 2〜10之间的整数， 优选 2(
11、根据权利要求 9或 10所述的方法， 其特征在于所述活性甲: 聚乙二醇衍生物是 （XVI) 的活化甲氧基聚乙二醇，
Figure imgf000025_0001
(XVI)
AG选自：
Figure imgf000025_0002
Figure imgf000025_0003
Figure imgf000026_0001
CH- -NH—— CH2
m
O
〇
•CH. -NH CH2 -NH ~ fCH -NH- m
〇 〇
〇
•CH -NH—— CH2 -NH—— CH?一 CH?- m
〇
其中 m、 n的数目选自 2〜10之间的整数， 优选 2(
12、 根据权利要求 9所述的方法， 包括以下步骤:
(1) 制备结构通式为 物，
Figure imgf000026_0002
(X IV)
其中 k的数目选自 2〜10之间的整数， 优选 2;
(2) 将结构通式为 （X R 的小分子醛化合物与干扰素在缓冲 里反应， 并加入还原剂得到结构通式为 （X V) 的活化干扰素，
Figure imgf000026_0003
其中 P是指干扰素， 优选重组人干扰素 (x2b; (3)在含有活化干扰素的缓冲液里加入脱保护剂脱去结构通式为 (X V) 的活化干扰素的乙酰基保护基， 随后加入结构通式为 （X VI) 的活化甲氧基聚乙二醇进行聚乙二醇化反应，得到聚乙二醇干扰素偶 联物，
Figure imgf000027_0001
(xvi)
AG选自
Figure imgf000027_0002
0
、 , H2 0 H H2 H2
-N— C — C -
Figure imgf000027_0003
Figure imgf000028_0001
O
■CH -NH—— CH2 -NH—— CH?一 CH?- m
O
其中 m、 n的数目选自 2〜10之间的整数， 优选 2;
(4) 纯化聚乙二醇干扰素偶联物。
13、 一种药物组合物， 其包含：
1) 如权利要求 1-8 任意一项所述的治疗有效量的聚乙二醇化干 扰素偶联物；
2) 药学可接受的药物载体。
14、权利要求 1-8所述的聚乙二醇化干扰素偶联物，权利要求 10 所述的药物组合物，在制备抗病毒、抗肿瘤、免疫调节药物中的用途; 优选在制备治疗慢性乙型肝炎、 丙型肝炎和戊型肝炎、 带状疱疹、 扁 平和尖锐湿疣、 乳头瘤病毒感染、 流行性出血热、 小儿呼吸道合胞病 毒肺炎、 毛细胞性白血病、 慢性粒细胞白血病、 黑色素瘤、 淋巴瘤、 多发性骨髓瘤、 肾细胞癌、 卵巢癌、 直肠癌、 肝癌、 肺癌的药物中的 用途。
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