WO2015101033A1 - 多臂聚乙二醇修饰剂的新用途及其在修饰门冬酰胺酶中的应用 - Google Patents

多臂聚乙二醇修饰剂的新用途及其在修饰门冬酰胺酶中的应用 Download PDF

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WO2015101033A1
WO2015101033A1 PCT/CN2014/083143 CN2014083143W WO2015101033A1 WO 2015101033 A1 WO2015101033 A1 WO 2015101033A1 CN 2014083143 W CN2014083143 W CN 2014083143W WO 2015101033 A1 WO2015101033 A1 WO 2015101033A1
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peg
polyethylene glycol
arm
modified
protein
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PCT/CN2014/083143
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French (fr)
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马永
王俊
吴鼎龙
徐春林
王耀方
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江苏众红生物工程创药研究院有限公司
常州京森生物医药研究所有限公司
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Priority to US15/109,304 priority Critical patent/US10406235B2/en
Publication of WO2015101033A1 publication Critical patent/WO2015101033A1/zh

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    • A—HUMAN NECESSITIES
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    • A61K38/44—Oxidoreductases (1)
    • A—HUMAN NECESSITIES
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    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/50—Hydrolases (3) acting on carbon-nitrogen bonds, other than peptide bonds (3.5), e.g. asparaginase
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
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    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • 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
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0014—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4)
    • C12N9/0016—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on the CH-NH2 group of donors (1.4) with NAD or NADP as acceptor (1.4.1)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • C12N9/80—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
    • C12N9/82—Asparaginase (3.5.1.1)
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
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    • C12Y104/00—Oxidoreductases acting on the CH-NH2 group of donors (1.4)
    • C12Y104/01—Oxidoreductases acting on the CH-NH2 group of donors (1.4) with NAD+ or NADP+ as acceptor (1.4.1)
    • C12Y104/01002—Glutamate dehydrogenase (1.4.1.2)
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    • C12Y301/00—Hydrolases acting on ester bonds (3.1)
    • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
    • C12Y301/03001—Alkaline phosphatase (3.1.3.1)
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    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
    • C12Y305/01001—Asparaginase (3.5.1.1)
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    • C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
    • C12Y305/01—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in linear amides (3.5.1)
    • C12Y305/01005—Urease (3.5.1.5)

Definitions

  • the present invention relates to novel uses of multi-arm polyethylene glycol modifiers and their use in modifying asparaginase. Background technique
  • Polyethylene glycol is a linear, free-curing, uncharged polymer that is non-toxic, weakly antigenic, and biocompatible. Covalent modification of proteins can increase the in vivo circulating half-life of the protein and reduce its antigenicity, increase the solubility of the protein and alter the biological distribution of the protein in the human body. Since 1977, Abuchowski, Davis (J. Biol. Chem. 1977, 252: 3578-3581.) et al. first reported the use of PEG to modify proteins, PEG modification technology has been widely used in biomedical and biotechnology fields, PEG has It is widely used in the modification of protein and peptide drugs. Protein PEGylation technology has become one of the most effective methods for reducing the immunogenicity of protein biopharmaceuticals and improving their pharmacokinetic/pharmacodynamic properties, and has been approved by the FDA for use in pharmaceuticals, foods and cosmetics.
  • the activity of the pegylated protein drug is reduced compared to the unmodified native protein, and the generally modified protein activity is only 30% to 40% or even lower of the original protein.
  • Schering-Plough's PEG-Intron which uses PEG with a molecular weight of 5000, modifies interferon, and its activity is only 8% of the original protein.
  • the activity of the modified protein decreases more significantly.
  • erythropoietin EPO
  • PEG having a molecular weight of 20 kDa and 30 KDa 40 KDa
  • the activity is significantly reduced as the molecular weight of PEG increases (Yin-jue Wang, journal of controlled release, 2010 (145): 306-313).
  • Bailon et al. modified Interferon-a-2a with a branched 40 kDa PEG, and the resulting single modification achieved a longer circulating half-life, but retained only 7% of in vitro activity (Bailon P, Bioconjugate Chem., 2001, 12: 195-202). . )
  • polyethylene glycol modification technology has undergone several decades of development and is currently mature. However, it is not possible to find a universal polyethylene glycol modifier and modification method to modify all protein drugs.
  • polyethylene glycol modifiers are the most important factors affecting the physical properties of modification, biological activity in vitro and in vivo, pharmacokinetics, pharmacodynamics and clinical manifestations of modified products. Therefore, the choice of modifier (type of modifier, molecular weight) and control of modification reactions play an important role in polyethylene glycol modification technology.
  • a multi-subunit protein refers to a polymer that is bound together by two or more independent subunits through interaction between molecules. Wherein, each subunit is generally composed of one peptide chain, but two or more peptide chains are also composed of disulfide bonds.
  • alkaline phosphatase consists of two subunits, each having a molecular weight of about 28 KDa and a molecular weight of about 56 KDa; human tumor necrosis factor is composed of three subunits, each having a molecular weight of about 17 kDa.
  • the molecular weight of the whole molecule is about 51KDa ; the active form of L-asparaginase is a homotetramer structure composed of 4 subunits, each subunit consists of 326 amino acids, and the molecular weight of the whole protein molecule is about 140.
  • KDa; Hp urease monomer consists of two subunits A and B, which are hexamers.
  • the molecular weights of A and B submonomers are about 30 KDa and 64 KDa, respectively, and the ratio is 1:1.
  • the biological activity of multi-subunit proteins is often related to the structure of their aggregates.
  • the human tumor necrosis factor T F- ⁇ trimer has the highest biological activity, which is 8 times the activity of each subunit alone.
  • L-asparaginase only has a homotetrameric structure composed of 4 subunits to have corresponding biological activities.
  • the focus of maintaining the biological activity of the multi-subunit protein is how to prevent depolymerization of the subunits.
  • L-asparaginase A protein with L-asparagine aminohydrolase activity (often referred to as L-asparaginase, L-asparaginase, or asparaginase) is effective in the treatment of acute lymphoblastic leukemia in children or adults. (ALL).
  • L-asparaginase-containing drugs have been used in combination with chemotherapy to treat K/T cell lymphoma, and have achieved good therapeutic results.
  • K/T cell lymphoma is a special type of non-Hodgkin's lymphoma, which is more common in Asia and Latin America, and its incidence is relatively high in China.
  • K/T cell lymphoma can be divided into nasal K/T cell lymphoma and non-nasal K/T cell lymphoma.
  • L-asparaginase is also used to treat Hodgkin's disease, acute myeloid leukemia, acute myeloid monocytic leukemia, chronic lymphocytic leukemia, lymphosarcoma, reticulum sarcoma and melanoma (Kotzia and labrou, J. Biotechnol. 127 (2007) 657-669).
  • the active form of L-asparaginase is a homotetrameric structure composed of 4 subunits, each subunit consisting of 326 amino acids.
  • L-asparaginase was originally purified from several organisms, including E. coli and Erwinia carotovora. In mammals, slightly higher levels of L-asparaginase were found only in guinea pigs (Cavioidea superfamily) and certain New World monkeys. However, since it is derived from a foreign organism, it is a foreign protein to humans, has strong immunogenicity, and is clinically common for progressive immune response and systemic allergic reaction, which limits its clinical application. (Zhang Lina, Gong Daohua. Jiangsu Medicine. L-asparaginase in the treatment of acute lymphoblastic leukemia in children. 2005, 31(5): 392; Wang Ningling, Liu Zhiyu et al. L-asparaginase in the treatment of childhood leukemia Toxic side effects and prevention. Chinese Pediatric Blood, 2005, 10(3): 133).
  • the PEG-modified L-asparaginase currently marketed in China is only the "Pendoorase” of Hengrui Company. It is a generic drug of Oncaspar, and there is also the problem that PEG is easily degraded and shed.
  • the polyethylene glycol modifier is preferably coupled to the amino group of the multi-subunit protein.
  • the polyethylene glycol modifier used in the present invention is an aldehyde-activated or ester-activated four to eight-arm polyethylene glycol modifier.
  • a four-arm polyethylene glycol modifier is preferred.
  • the structural formula of the multi-arm polyethylene glycol modifier used in the present invention is as shown in formula (I) or formula (II):
  • n is an integer value from 1 to 2000, preferably an integer value between 2 and 500, more preferably an integer between 25 and 100; k is 1 or 2, preferred 1; m is an integer between 2 and 16, preferred 4; p is an integer between 1-4, preferably 2; the molecular weight of the polyethylene glycol modifier is between 1 and 100 kDa, preferably between 1 and 40 kDa, more preferably between 5 and 10 kDa.
  • the above ester-activated polyethylene glycol modifier is four-arm polyethylene glycol succinimide acetate (4ARM-SCM), four-arm polyethylene glycol succinimide propionate (4ARM) -SPA) or four-arm polyethylene glycol succinimide carbonate (4ARM-SC). Most preferred is a four-arm polyethylene glycol succinimide acetate (4ARM-SCM).
  • the polyethylene glycol modifier molecule may be any molecule having a molecular weight of 2 KDa to 40 KDa depending on the degree of polymerization, and a polyethylene glycol molecule having a molecular weight of 5 KDa is preferred.
  • the multi-arm PEG used is preferred to the above structural formula, but is not limited to the above structural formula.
  • the above-described multi-subunit proteins include, but are not limited to, L-asparaginase, alkaline phosphatase, and urease and glutamic acid.
  • Another object of the present invention is to provide a PEGylated multi-subunit as described above.
  • the method of preparing protein includes the following steps:
  • the multi-subunit protein to be modified and the multi-arm polyethylene glycol modifier are mixed in a ratio of 1:5-1:200, and after mixing, the modification reaction is carried out in a buffer;
  • the multi-arm polyethylene glycol modifier which is not reacted with the protein in the modified product is removed by ion exchange chromatography;
  • the multi-arm polyethylene glycol modifier and the multi-substrate chalk used therein are as described above.
  • the pH of the buffer ranges from about 5.0 to 6.0.
  • the pH of the buffer ranges from about 7.0 to 8.0.
  • the modification reaction of the above step (1) is carried out at a protein concentration of from 3 mg/mL to 15 mg/mL.
  • Another object of the invention is to provide a multi-arm polyethylene glycol modified multi-subunit protein.
  • the multi-arm polyethylene glycol modifier is preferably coupled to the amino group of the multi-subunit protein.
  • the multi-arm polyethylene glycol modifier and multi-subunit protein used therein are as described above.
  • a fourth object of the present invention is to provide a pharmaceutical composition
  • a pharmaceutical composition comprising a multi-arm polyethylene glycol modified multi-subunit protein or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant.
  • the pharmaceutical composition described above is a lyophilized powder injection.
  • the excipients include pharmaceutically acceptable carriers and/or excipients and the like.
  • the multi-arm polyethylene glycol modified multi-subunit protein and the pharmaceutical composition thereof are administered by a muscle, intravenous or subcutaneous route.
  • compositions are non-toxic in the amounts and concentrations to which they are administered.
  • the preparation of such salts promotes pharmaceutical applications by altering the physical characteristics of the compound without impeding its physiologic effects.
  • Useful changes in physical properties include lowering the melting point to promote transmucosal administration, and increasing solubility to facilitate administration of higher concentrations of the drug.
  • the pharmaceutically acceptable salts described above include acid addition salts, for example, sulfates, hydrochlorides, fumarates, maleates, phosphates, acetates, citrates, lactates, tartrates. , methanesulfonate, besylate, and the like.
  • the pharmaceutically acceptable salts described above may be derived from acids including hydrochloric acid, maleic acid, sulfuric acid, phosphoric acid, acetic acid, citric acid, lactic acid, tartaric acid and the like.
  • Carriers suitable for the practice of the present invention include calcium carbonate, calcium phosphate, various sugars (lactose, glucose, sucrose), or various starches, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and physiologically compatible Solvents (including sterile aqueous solutions for injection, saline solutions and dextran, etc.).
  • a fifth object of the present invention is to provide a multi-arm polyethylene glycol modified asparaginase.
  • the polyethylene glycol is preferably coupled to the amino group of the multi-subunit protein.
  • the multi-arm polyethylene glycol used therein is as described above.
  • the invention also provides the use of a multi-arm polyethylene glycol modified L-asparaginase for the treatment of acute leukemia.
  • the depolymerization of the subunits of the multi-subunit protein is mainly due to the non-covalent bond between the protein subunits to maintain its high-order structure, which is not as stable as the covalent bond, so It is easily degraded in the body, and more epitopes of each subunit are exposed after depolymerization, resulting in stronger immunogenicity.
  • the solution to this problem is to increase the force between the subunits without affecting the activity of the protein, or the effect is small. Applicants therefore consider modifying with polyethylene glycol, but currently the commonly used monomethoxy PEG modifiers clearly do not enhance the force between the subunits.
  • the multi-arm PEG of the present invention Modifiers meet this requirement. These PEG modifiers are characterized by: A PEG molecule with multiple activating groups can react with the amino acid residues of the protein.
  • the multi-arm PEG modifiers used in the present invention are all currently available PEG modifiers, it has not been reported in the literature prior to the present invention that such multi-arm PEG modifiers are used to modify multi-subunit proteins to solve many problems. Problems such as reduced stability, inactivation, and enhanced immunogenicity caused by depolymerization of each subunit in the subunit protein.
  • the multi-arm PEG-modified multi-subunit protein provided by the present invention can well couple multi-subunits compared with the original protein or a general polyethylene glycol-modified multi-subunit protein. , more effectively prevent the problem of subunit depolymerization, and better stability.
  • the multi-arm polyethylene glycol modified L-asparaginase, alkaline phosphatase, urease, etc. provided by the present invention have higher stability and retention than the original protein or modified with a general PEG modifier.
  • the ability to biologically activate, and the structure is stable and uniform.
  • the PEG-modified asparaginase prepared in the present invention has the advantages of low immunogenicity, high stability, and PEG not easy to fall off, compared with the commercially available PEGylated asparaginase product "Pemenixase", and
  • the multi-arm PEG-modified asparaginase provided by the invention also retains high biological activity, and has the characteristics of prolonged half-life and stable structure.
  • Perenzyme is a product prepared by modifying PEG to Escherichia coli-derived L-asparaginase, and the polyethylene glycol-modified L-asparaginase provided in the present invention is also of Escherichia coli. Therefore, it should also have a therapeutic effect similar to that of Pembellase. That is, the multi-arm polyethylene glycol modified L-asparaginase provided in the present invention can be used in combination with other chemotherapeutic drugs to treat K/T cell lymphoma.
  • FIG. 1 Protein electrophoresis analysis of different PEG-ASP conjugates.
  • Electropherograms of proteins after different PEG modified ASP were protein Marker, Pemetrex, 4SCM5K-ASP, 8SCM10K-ASP, 4PALD5K-ASP, 4PALD10K-ASP. It can be seen from the protein results that the molecular weight of the enantiomerase is diffuse, and the molecular weight of the PEG-ASP conjugate prepared by the multi-arm PEG modifier is relatively simple. It can be seen that the conjugate prepared by the multi-arm PEG modifier has excellent uniformity. Modified with a monomethoxy PEG modifier
  • P Figure 2 Electropherograms of proteins after PEG modification of AKP, URE and GDH.
  • the samples in lanes 1-3 of Figure a are protein Marker, SCM5K-AKP, and 4SCM5K-AKP, respectively.
  • the samples in lanes 1-3 of Figure b are the proteins Marker, SCM5K-URE, 4SCM5K-URE.
  • the samples in lanes 1-3 of Figure c are the proteins Marker, SCM5K-GDH, 4SCM5K-GDH.
  • the PEG modifier stabilizes the active structure of the multi-subunit protein, and the multi-arm PEG-modified conjugate sample is more uniform.
  • the multi-arm PEG modifier modified multi-subunit protein has a higher molecular weight than the conventional monomethoxy PEG modifier.
  • the homogeneity of the conjugate is better.
  • Figure 3 Thermal stability study of PEG-AKP, PEG-URE, PEG-GDH conjugates.
  • Figure a is the test result of SCM5K-AKP and 4SCM5K-AKP.
  • the results show that the activity of SCM5K-AKP is significantly decreased within 2h, and the total activity is lost after 5h, while the activity of 4SCM5K-AKP is slower during the detection. After 60h, 60% of the original activity can still be retained.
  • Figure b is a test result of SCM5K-URE and 4SCM5K-URE.
  • FIG. 4 In vitro biological activity of different PEG-ASP conjugates.
  • Figure 5 Stability studies of different PEG-ASP conjugates.
  • Figure 6 Results of PEG protein electrophoresis iodine staining of PEG-ASP conjugate and aspartic enzyme.
  • Figure a is a protein electrophoresis pattern of Pemetrexase after being placed in a 37 ° C water bath for different periods of time.
  • Lane 1 is the protein Marker
  • Lane 2 is the monomethoxy PEG with a molecular weight of 5000
  • lanes 3-12 are respectively placed in the 37 ° C water bath for 10h, 12h, 24h, 36h, 48h, 60h, 72h.
  • Figure b shows the electropherogram of 4SCM5K-ASP after being placed in a 37 °C water bath for different time.
  • Lane 1 is protein Marker
  • lane 2 is 4SCM5K
  • lanes 3-12 are 4SCM5K-ASP for protein electrophoresis after being placed in a 37 °C water bath for 0h, 12h, 24h, 36h, 48h, 60h, 72h, 84h, 96h, 108h.
  • Figure 7 Comparison of circular dichroism spectra of different PEG-ASP conjugates and proproteins.
  • Figure a is a far-ultraviolet circular dichroism of the PEG-ASP conjugate
  • Figure b is a near-ultraviolet circular dichroic chromatogram of the PEG-ASP conjugate.
  • the circular dichroism chromatograms of different PEG-ASP conjugates and proproteins were compared and their structural differences were analyzed. It can be seen from the circular dichroic chromatogram that, similar to the aspartase, after the modification of ASP with different multi-arm PEG, the near-ultraviolet and far-ultraviolet circular dichroism diagrams have not changed substantially, indicating the secondary structure of the modified ASP and The tertiary structure has not changed.
  • Figure 8 Comparison of the efficacy of different PEG-ASP conjugates. The efficacy of different PEG-ASP conjugates was compared and compared with the proprotein and aspartic enzyme. The pharmacodynamic test showed that 4SCM10K-ASP and 4PALD10K-ASP inhibited tumor cells better than Pembellase.
  • PEG polyethylene glycol
  • PEG modifier polyethylene glycol modifier
  • multi-arm polyethylene glycol modifier polyethylene glycol molecule containing two or more activating groups.
  • Polyethylene glycol (PEG, HO-(CH 2 CH 2 0) n -CH 2 CH 2 OH;) is a linear polymer with hydroxyl groups at both ends, and polyethylene glycol is polymerized by ethylene oxide. Made of repeating oxyethylene, branched, linear and multi-armed. PEG is also known as poly(ethyleneoxide) (PEO), poly(oxy-ethylene) (POE), or polyoxirane. In general, a molecular weight of less than 20,000 is called PEG, and a larger molecular weight is called PEO. Ordinary polyethylene glycol has a hydroxyl group at each end. If one end is blocked with a methyl group, methoxypolyethylene glycol (mPEG) is obtained, which is the most commonly used in protein pegylation technology.
  • PEG poly(ethyleneoxide)
  • POE poly(oxy-ethylene)
  • PEO poly(oxy-ethylene)
  • PEO poly(oxy-ethylene)
  • Ordinary polyethylene glycol has a hydroxy
  • the polyethylene glycol modifier refers to a polyethylene glycol derivative with a functional group, and refers to an activated polyethylene glycol, which is mainly used for protein and polypeptide drug modification, and is also called modified polyethylene glycol. Modified PEG.
  • 4SCM5K four-arm polyethylene glycol succinimide acetate having a molecular weight of 5KDa;
  • 8SCM10K eight-arm polyethylene glycol succinimide acetate having a molecular weight of lOKDa;
  • 4PALD5K four-arm polyethylene glycol propionaldehyde with a molecular weight of 5KDa;
  • 4PALD10K four-arm polyethylene glycol propionaldehyde with a molecular weight of lOKDa;
  • AKP alkaline phosphatase
  • ASP asparaginase
  • URE urease
  • glutamate dehydrogenase GDH.
  • conjugate refers to a polysubunit protein such as polyethylene glycol modified asparaginase or urease. Modified product obtained afterwards;
  • modified products of polyethylene glycol modified asparaginase may be referred to herein as "4SCM5K-ASP, 8SCM10K-ASP, 4PALD5K-ASP, 4PALD10K-ASP", collectively referred to as PEG-ASP or PEG modified ASP coupling Things.
  • the modified product after modification of the alkaline phosphatase with polyethylene glycol can be referred to herein as "4SCM5K-AKP, SCM5K-AKP," a conjugate that is collectively referred to as PEG-AKP or PEG-modified AKP.
  • the modified product after modification of the urease with polyethylene glycol can be referred to herein as "4SCM5K-URE, SCM5K-URE,” a conjugate that is collectively referred to as PEG-URE or PEG modified URE.
  • the modified product after modification of the glutamate dehydrogenase by polyethylene glycol can be referred to herein as "4SCM5K-GDH, SCM5K-GDH,” a conjugate that is collectively referred to as PEG-GDH or PEG-modified GDH.
  • the polyethylene glycol modifier of the present invention preferably has a molecular weight in the range of from about 2 KDa to about 40 KDa. More specifically, the polyethylene glycol modifier has a molecular weight selected from the group consisting of 2 KDa, 5 KDa, 10 KDa. In a particular embodiment, the polyethylene glycol modifier has a molecular weight of 5 kDa, 10 kDa.
  • the polyethylene glycol modifier used in the present invention is preferably the following: aldehyde-activated and ester-activated polyethylene glycol, more specifically, the polyethylene glycol modifier is propionaldehyde-activated and succinimide acetate Ester activated polyethylene glycol.
  • the modified protein may be a multi-subunit protein of any origin.
  • the modified multi-subunit protein is an alkaline phosphatase and a urease.
  • the modified multi-subunit protein is an asparaginase, which can be derived, cloned or produced from any source, including from an animal or by genetic recombination techniques or a combination thereof.
  • asparaginase can be extracted from E. coli, including but not limited to E. coli.
  • the asparaginase has at least about 60% sequence identity to the protein comprising the sequence of SEQ ID ⁇ : 1. More particularly with at least about 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% of the protein comprising SEQ ID NO: , 97%, 98% or 100% sequence identity.
  • the protein is an E. coli-derived asparaginase having the sequence of SEQ ID NO: l.
  • a fragment of the protein of SEQ ID NO: 1 is also included in the definition of the protein used in the conjugate of the present invention.
  • the "fragment of the protein of SEQ ID NO : 1" means that the sequence of the polypeptide may comprise fewer amino acids than SEQ ID NO : 1.
  • a polypeptide can be modified by substitution, insertion, deletion and/or addition of one or more amino acids while retaining its enzymatic activity. For example, it is common to replace an amino acid with a chemically equivalent amino acid at a given position without affecting the functional properties of the protein. Thus, it is expected that the following changes will result in a functionally equivalent product: a change that results in a change in one negatively charged residue or another that produces a positively charged residue in place of the other.
  • the linking group used to covalently bind the PEG to the multi-subunit protein can be any biocompatible linking group.
  • Biocompatible means that the compound or group is non-toxic and can be used in vitro or in vivo without causing damage, vomiting, disease or death.
  • PEG can bind to a linking group, such as through an ester bond, a thiol bond, or an amide bond.
  • a common feature of the most preferred biocompatible linking groups in the present invention is that they are coupled to the amino group of the multi-subunit protein via a succinimide acetate group or a propionaldehyde group.
  • the protein can be directly coupled to the PEG via an amino group, a thiol group, a hydroxyl group or a carboxyl group.
  • the PEG is coupled to an amino group on an asparaginase, alkaline phosphatase, urease, glutamate dehydrogenase.
  • the invention relates to a method of preparing a conjugate comprising reacting a certain amount of a multi-arm PEG modifier with an amount of asparaginase, alkaline phosphatase or urease in a buffer solution for a sufficient amount of time So that PEG is covalently bound to it.
  • the asparaginase is derived from E. coli, and more particularly, the amino acid sequence of the asparaginase is set forth in SEQ ID NO: 1.
  • the PEG is 4SCM5K and 4ALD10K.
  • the pH of the buffer is in the range of from about 4.0 to about 9.0.
  • the most preferred pH range is from about 5.0 to 6.0, for example, a pH of about 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9 or 6.0.
  • the pH of the buffer is in the range of from about 6.0 to about 9.0.
  • the most preferred pH range is from about 7.0 to 8.0, for example, a pH of about 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0.
  • PEGylation of asparaginase, alkaline phosphatase, urease, glutamate dehydrogenase is carried out at the following protein concentrations: about 0.5-30 mg/mL, more specifically about 2 mg/mL-20 mg/ The mL, most specifically about 3 mg/mL to 15 mg/mL.
  • the asparaginase PEGylated at these protein concentrations is from E. coli, and more particularly, the asparaginase comprises the sequence of SEQ ID NO: 1.
  • the PEGylation reaction proceeds rapidly, in less than 3 hours.
  • the molar ratio of PEG applied to asparaginase, alkaline phosphatase, urease, and glutamate dehydrogenase is at most 200:1.
  • the molar ratio is 200: 1, 150: 1, 100: 1, 80: 1, 70: 1, 60: 1, 50: 1, 40: 1, 30: 1, 20: 1, 15: 1, 10 : 1, 5: 1.
  • the asparaginase (purchased from Chihong Pharmaceutical, having the sequence of SEQ ID NO: 1) was dissolved in 50 mM acetic acid-sodium acetate buffer (purchased from Sinopharm Group) at pH 5.0 to prepare a solution of 8 mg/mL, respectively.
  • 4PALD5K , 4PALD10K (purchased from Beijing Keykai Technology Co., Ltd.) modified as a polyethylene glycol modifier
  • asparaginase polyethylene glycol modifier: reducing agent (sodium cyanoborohydride, purchased from sigma)
  • the reaction was carried out at a molar ratio of 1:50:2500, and after reacting at 4 ° C for 12 hours, the reaction was terminated by adding 1 M glycine.
  • Chromatographic conditions Q ion exchange column (purchased from GE, HiTrap Q HP 5 mL), equilibration buffer: 20 mM Tris-Hcl (pH 8.0), (reagent purchased from Sinopharm Group). Elution buffer: 20 mM Tris-HCl (pH 8.0) containing 1 M NaCl (reagent purchased from Sinopharm Group). The flow rate was 2.5 mL/min and the detection wavelength was 280 nm.
  • Chromatographic conditions Hiload 16/60 Superdex 200 pg (available from GE) Semi-preparative gel filtration column with PBS at a flow rate of 1.5 mL/min and a detection wavelength of 280 nm.
  • the protein concentrate was 5% and the separation gel was 7%.
  • the concentrated gel buffer was 0.5 M Tris-HCl buffer (pH 6.8); the separation gel buffer was 1.5 mol/L Tris-HCl buffer (pH 8.8).
  • Tris-HCl buffer pH 8.8
  • Preparation Examples 2 to 4 were the same as Preparation Example 1 except that the pH value, reaction temperature, reaction time, protein concentration, and molar ratio were different, and the specific parameters and yields are shown in the following table:
  • Alkaline phosphatase is widely distributed in various organs of the human body.
  • Alkaline phosphatase is an enzyme that can dephosphorylate the corresponding substrate, that is, the phosphate group on the substrate molecule by hydrolyzing the phosphate monoester. The group is removed and produces phosphate ions and free hydroxyl groups.
  • substrates include nucleic acids, proteins, alkaloids and the like.
  • AKP is a homodimeric protein with a molecular weight of 28 kDa per subunit.
  • the Hp urease monomer consists of two subunits, A and B, which are hexamers.
  • the molecular weights of the two subunits A and B are 30 KDa and 64 KDa, respectively, and the ratio is 1:1.
  • the urease used is a 6-mer that cannot be formed, but is a monomeric protein formed by two subunits of hydrazine and hydrazine.
  • Glutamate dehydrogenase catalyzes the deamination of glutamate to form alpha-ketoglutarate and ammonia. And glutamate dehydrogenase is the only enzyme that can utilize both NAD+ and NADH+ as reducing equivalents. It is an aerobic dehydrogenase.
  • An important part of amino acid metabolism is an allosteric enzyme, which is composed of 6 identical subunits, each of which has a molecular weight of 56,000.
  • Alkaline phosphatase purchased from SIGMA
  • urease purchased from SIGMA
  • glutamate dehydrogenase purchased from SIGMA
  • SCM5K, 4SCM5K purchased from Beijing Keykai Technology Co., Ltd.
  • SCM5K, 4SCM5K was modified as a polyethylene glycol modifier, and the reaction was carried out according to the alkaline phosphatase or urease: polyethylene glycol modifier at a molar ratio of 1:50. , reacted at 4 ° C for 2 h.
  • the subunits of the protein can be coupled together by modifying the alkaline phosphatase, asparaginase, urease and glutamate dehydrogenase proteins using a multi-arm PEG modifier.
  • the uniformity of the modified product is significantly improved.
  • alkaline phosphatase and urease are di-subunit proteins
  • asparaginase is a tetra-subunit protein
  • glutamate dehydrogenase is a 6-subunit protein.
  • the multi-arm PEG modifier is suitable for the modification of multi-subunit proteins.
  • the subunits can be coupled to improve the homogeneity of the modified product. purpose.
  • Example 3 Thermal stability of PEG conjugates of alkaline phosphatase, urease and glutamate dehydrogenase
  • the specific method is as follows: A sample having a protein concentration of lmg/mL dissolved in PBS buffer is placed in a 60 ° C water bath, sampled at intervals and placed in a 4 ° C ice box for use. The activity was measured after the end of sampling, and its stability was evaluated based on the change in its activity. The result is shown in Figure 3 below.
  • 4SCM5K-URE retains 40% activity at 120min in 60°C water bath, while SCM5K-URE is almost complete. Inactivated. It is indicated that the coupling of the URE subunit with multi-arm PEG is more stable than the modified product modified with conventional monomethoxy PEG. Thus, the stability of the di-subunit protein after coupling with the multi-arm PEG is superior to that of the product coupled with the PEG with monomethoxy.
  • a similar method was used to compare the activities of 4SCM5K-GDH and SCM5K-GDH at 0.5 h, and similar results were obtained. Thus, the coupling of the 6-subunit protein with the multi-arm PEG is superior to the product after the PEG coupling with the monomethoxy group. Therefore, it can be reasonably assumed that the modification of the multi-subunit protein with the multi-arm PEG is superior to the modified product modified with conventional monomethoxy PEG.
  • Example 4 Detection of in vitro activity of PEG-ASP conjugates
  • Asparaginase can hydrolyze the amide group of asparagine, and according to this principle, the activity of asparaginase can be measured.
  • the specific measurement method is carried out by referring to the method described on page 31 of the second edition of the Pharmacopoeia 2005.
  • the reagents required for the measurement were purchased from Sinopharm Chemical Reagent Co., Ltd.
  • the samples tested were 4SCM5K-ASP, 8SCM10K-ASP, 4PALD5K- ASP, 4PALD10K-ASP and unmodified original protein and the same kind of product on the market, Pemetrexase (purchased from Jiangsu Hengrui Pharmaceutical Co., Ltd.). Their relative activity comparison results are shown in Figure 4.
  • this example detects 4SCM5K-ASP, 8SCM10K-ASP, 4PALD5K-ASP, 4PALD10K in Example 1, respectively.
  • the activity of the four modified products of -AS P and the original protein and the enzymatic enzyme were allowed to stand in a water bath at 37 ° C for a period of time.
  • the specific method is as follows: A sample having a protein concentration of lmg/mL dissolved in PBS buffer is placed in a 37 ° C water bath, sampled at intervals and placed in a refrigerator at 4 ° C for use. The biological activity was measured after the sampling was completed. The test results are shown in Figure 5 below.
  • the original protein showed a significant decrease in activity after 25 hours, and was completely inactivated after 48 hours.
  • the stability of the enantiomerase was higher than that of the original protein, but the activity also began to decrease significantly after 75 h, and the activity was completely lost by 120 h.
  • the multi-arm PEG modified products 4SCM5K-ASP, 8SCM10K-ASP, 4PALD5K-ASP and 4PALD10K-ASP of the present invention have higher stability and slower activity, and the activities of 4SCM5K-ASP and 8SCM10K-ASP are basically unchanged.
  • thermal stability of 4SCM5K-ASP, 8SCM10K-ASP, 4PALD5K-ASP, 4PALD10K-ASP prepared by the present invention is much higher than that of the enzyme.
  • the modification of the multi-subunit protein with the multi-arm PEG has a significantly better stability of the modified product than that obtained by modification with a conventional monomethoxy PEG. Modify the product.
  • the multi-arm PEG used in the present invention is suitable for the modification of all multi-subunit proteins, and by covalently coupling the subunits, the stability is greatly improved, and the dissociation of the subunits is prevented, thereby reducing The resulting loss of activity and the production of immunogenicity.
  • Example 6 Stability of Coupling Groups of PEG-ASP Conjugates
  • the polyethylene glycol modifier can enhance the stability of the original protein, but due to the difference of the coupling group, there is a phenomenon that the PEG is detached due to the degradation of the coupling group, thereby affecting the stability and biological activity of the drug.
  • the PEG shedding of 4SCM5K-ASP and Pemetrexase after a period of time in a 37 ° C water bath was examined. .
  • the specific method is as follows: Protein dissolved in PBS buffer Samples with a degree of lmg/mL were placed in a 37 ° C water bath, sampled at intervals and placed in a 4 ° C refrigerator for later use. After the sampling, protein electrophoresis was carried out, and after the electrophoresis, PEG was stained with iodine. The test results are shown in Figure 6.
  • the protein drug modified by the multi-arm PEG is highly stable, and the PE G molecule is not easily detached, thereby increasing the stability of the modified drug and reducing the probability of the drug eliciting an immune response.
  • Example 7 Circular dichroism analysis of PEG-ASP conjugates and proproteins
  • the secondary and tertiary structures of the modified and unmodified proteins can be characterized by a circular dichroism spectrometer.
  • the protein concentration range is 0.1 to 0.2 mg/mL.
  • the sample was added to a circular dichroic cuvette with a diameter of 1 mm to detect the circular dichroism spectrum in the far ultraviolet region (190 nm-250 nm) and the near ultraviolet region (253 nm-480 nm).
  • the scanning bandwidth was 1 nm and the scanning speed was 500 nm/min.
  • Each test was averaged three times with the corresponding buffer as the background. It can be seen from Fig.
  • THP-1 derived from human monocyte leukemia cell line
  • U937 derived from humans
  • Nuclear cell leukemia cell line Raji (human-derived lymphoma cell line)
  • Jurkat derived human acute T cell leukemia cell line
  • L1210 leukemia cell derived from mouse
  • L5178Y derived mouse lymph Tumor cells
  • the multi-arm PEG-modified asparaginase of the present invention is generally more toxic to the above six kinds of tumor cells than the unmodified asparaginase, and the anti-inhibition of the 4SCM5K-ASP and 4PALD10K-ASP of the present invention.
  • the tumor activity was significantly higher than that of the enzyme, and it showed good anti-tumor effect on various cell lines, especially 4PALD10K-ASP.
  • IC 5Q was the lowest in all cell lines, and the anti-tumor activity was the highest.
  • Example 9 Comparison of in vivo inhibition of tumors by different PEG-ASP conjugates
  • nude mice 4-6 weeks old nude mice (BALB/cA-nu) 32, 15-18g, female, divided into 5 groups, 8 in each group, BP: Pemetrexase group, ASP group, PBS group, 4PALD10K-ASP , 4SCM10K-ASP group.
  • BP Pemetrexase group
  • ASP group Pemetrexase group
  • PBS group PBS group
  • 4PALD10K-ASP 4SCM10K-ASP group.
  • liver function was measured.
  • each nude mouse was intraperitoneally injected with cyclophosphamide 100 mg/kg for 4 consecutive days. On the fifth day, about 5 ⁇ 10 6 tumor cells L1210 were intraperitoneally injected. Administered once a day, intramuscularly injected with parmozyme, ASP, PBS,
  • 4PALD10K-ASP, 4SCM10K-ASP was administered intramuscularly at 4.7 mg/kg, and the tumor inhibition rate was calculated on the third and sixth days after the injection.
  • the experimental results are shown in Figure 8 and Table 3.
  • PEG molecules can reduce or eliminate the ability to induce neutralizing antibodies and bind to antibodies, making them difficult to recognize and clear by the immune system, so PEG-modified proteins can reduce immunogenicity to some extent.
  • This example measures the relative immunogenicity of different PEG-ASP conjugates in mice and compares them with proprotein and Pemetrein.
  • the test was divided into 4PALD10K-ASP group, 4SCM5K-ASP group, parental enzyme group and proprotein group.
  • the tail vein was injected into the tail vein at a dose of 2 mg/kg every 8 weeks for 8 weeks (according to the amount of protein). Calculate the above protein.
  • Blood was taken from the eyelids 1 week after the end of the administration.
  • the level of anti-asparaginase antibody in serum was determined by indirect ELISA. (The secondary antibody used was purchased from SIGMA) The results are shown in Figure 9.
  • the steric hindrance of PEG greatly enhances the resistance of the modified protein to protease digestion, and the molecular size of the modified protein is significantly increased, resulting in a significant decrease in renal filtration clearance, thereby increasing the in vivo half-life of the original protein. .
  • the in vivo blood concentration of PEG-modified asparaginase was studied by 125 1 isotope labeling and tracer method.
  • the thyroid of the rat was saturated with 1% KI solution ImL intraperitoneally about 8 hours before the experiment.
  • Rats were randomly divided into 4SCM5K-ASP group and Pemetrexase group, 8 in each group, half male and half female.
  • the specific operation method is as follows:
  • Radioactive iodine replaces the hydroxyl group on the benzene ring of the amino acid residue in the protein molecule to obtain a 125 1 protein with radioactive tracer.
  • Rat plasma was centrifuged at 5000 rpm/3 min, and plasma activity was measured with a gamma counter (Anke Zhongjia, GC-911) for a measurement time of lmin.
  • the plasma was recovered after the measurement of the activity was completed, and the undegraded proteinogenic drug was separated by HPLC (Shimadzu LC-20AT).
  • the half-life of the 4SCM5K-ASP pharmacological parameters was significantly longer than that of the parmozyme, from 45h to 45h, and the area under the curve (AUC) was significantly improved from 459mg/L*h to 805 mg/L*h.
  • AUC area under the curve
  • the clearance rate of 4SCM5K-ASP in the blood was also lower than that of the enzyme. It can be seen that the asparaginase modified by the multi-arm PEG has a significantly enhanced stability in vivo compared with the aspartase, so the half-life is significantly improved, the metabolic rate in the blood is significantly reduced, and the drug is effectively prolonged. Pharmacological time.
  • the multi-arm PEG-modified asparaginase used in the present invention has improved stability due to its ability to enhance the interaction between protein subunits, and the results of pharmacokinetic experiments have also confirmed this. Since the 4SCM10K-ASP activating group is the same as 4SCM5K-ASP and the PEG chain is longer, it can be inferred from the data of the 4SCM5K-ASP group that the half-life of the 4SCM10K-ASP group is significantly greater than 45.90 h, and the AUC value is also higher. , its pharmacokinetic data should be significantly better than the 4SCM5K-ASP group.

Abstract

本发明公开了多臂聚乙二醇修饰剂的新用途及其在修饰门冬酰胺酶中的应用。本发明提供的多臂PEG修饰剂可用于加强多亚基蛋白的亚基间作用力，使多亚基蛋白保持聚合的形式，从而增加多聚体蛋白的稳定性，增强或保持多亚基蛋白的生物活性，同时减少各亚基解聚后抗原位点暴露的几率，降低免疫原性。

Description

多臂聚乙二醇修饰剂的新用途及其在修饰门冬酰胺酶中的应用
技术领域
本发明涉及多臂聚乙二醇修饰剂的新用途及其在修饰门冬酰胺酶中的应用。 背景技术
聚乙二醇 (PEG)是一种线性、在溶液中可自由卷曲的不带电荷的聚合物， 具有无毒、微弱 的抗原性和良好的生物相容性。 用它来共价修饰蛋白质， 可以增加蛋白质的体内循环半衰期 和减小其抗原性， 增加蛋白质的溶解性并会改变蛋白质在人体内的生物学分布。 自 1977 年 Abuchowski, Davis(J. Biol. Chem. 1977, 252: 3578-3581.)等人首次报道用 PEG修饰蛋白质以 来， PEG修饰技术在生物医学和生物技术领域得到了广泛的应用， PEG已经被广泛地应用于 蛋白质， 多肽类药物的修饰研究。 目前蛋白质 PEG化技术已经成为降低蛋白质生物药物的免 疫原性， 以及改善其药代动力学 /药效学性质最有效的方法之一， 且已通过 FDA认证可用于 药物、 食品和化妆品。
在大多数情况下， 与未修饰的原蛋白相比， 聚乙二醇化的蛋白药物的活性会降低， 一般 修饰后的蛋白活性仅有原蛋白的 30%-40%, 甚至更低。例如 Schering-Plough公司的 PEG-Intron, 是用分子量为 5000的 PEG修饰干扰素， 修饰后其活性只有原蛋白的 8%。 此外， 一般随着 PEG 分子量的增大， 修饰后的蛋白质活性降低更明显。 例如用分子量为 20KDa、 30 KDa 40 KDa 的 PEG修饰促红细胞生成素（EPO)后，活性随着 PEG分子量的增大而显著减小 (Yin-jue Wang, journal of controlled release, 2010(145):306-313 )。 Bailon 等采用分支型 40 kDa PEG 修饰 Interferon-a-2a,所得单修饰物获得较长的循环半衰期，但是仅仅保留了 7% 的体外活性 (Bailon P, Bioconjugate Chem., 2001, 12: 195-202. ) 。
聚乙二醇修饰技术经历了几十年的发展， 目前虽然已经比较成熟。 但并不能找出一种通 用的聚乙二醇修饰剂以及修饰方法来对所有的蛋白药物进行修饰。 蛋白的结构以及所用 PEG 的分子量、形状以及修饰的位点等对聚乙二醇化的蛋白质的生物活性以及药效有很大的影响。 对于特定药物的修饰， 聚乙二醇修饰剂是影响修饰产物理化性质、 体内外生物活性、 药代动 力学、 药效学以及修饰产物临床表现最重要的因素。 因此， 修饰剂的选择 (修饰剂种类、 分子 量大小)以及修饰反应的控制在聚乙二醇修饰技术中占有重要地位。蛋白质结构的解析并不能 准确预测天然蛋白质的药代动力学行为， 而在蛋白偶联上 PEG以后由于引入了许多新的变量 如分子量、 修饰剂种类等， 对于 PEG偶联物的药代动力学行为预测变得更加不可能。 为此， 针对不同的蛋白质药物， 需要通过选择不同种类以及不同分子量的修饰剂， 并通过理化性质 检测、 动物实验评价等确定最佳方案。 多亚基蛋白质 （寡聚或多聚亚基蛋白质） 指的是由两个以上彼此独立的亚基通过分子之 间相互作用结合在一起的聚合体。 其中， 每个亚基一般由一条肽链组成， 但也有两条以上肽 链经二硫键相连组成。 每个亚基自身折叠成一定的空间构型， 不同亚基之间依靠疏水作用、 氢键、 离子键与作用力聚集在一起， 形成蛋白质的四级结构， 亚基之间相互作用构成有生物 化学活性的整体。 在自然界中， 这种多亚基蛋白质在整个蛋白质家族中为数不少。 例如， 碱 性磷酸酶由二个亚基构成， 每个亚基分子量约为 28 KDa， 整个分子的分子量约为 56KDa; 人 肿瘤坏死因子就是三个亚基构成， 每个亚基分子量约为 17KDa， 整个分子的分子量约为 51KDa; L-门冬酰胺酶的活性形式为 4个亚基组成的同源四聚体结构， 每一亚基由 326个氨基 酸组成， 整个蛋白分子的分子量约为 140 KDa; Hp尿素酶单体由 A、 B两个亚单位组成， 呈六 聚体， A、 B两个亚单体的分子量分别为 30 KDa和 64 KDa左右， 其比例为 1： 1。 多亚基蛋白 质的生物学活性常常与其聚集体的结构有关。 人肿瘤坏死因子 T F-α三聚体的生物学活性最 高， 是每个亚基单独存在时活性的 8倍。 而 L-门冬酰胺酶只有在 4个亚基组成的同源四聚体结 构才能具有相应的生物学活性。 另一方面， 多亚基类蛋白解聚后不仅其活性一般会有显著降 低， 且作为药物在体内降解后， 容易暴露出更多抗原表位， 引起免疫反应， 从而降低药效， 引起副反应。 因此， 保持多亚基蛋白的生物学活性的重点在于如何防止亚基的解聚。
具有 L-天冬酰胺氨基水解酶活性的蛋白 （通常被称为 L-天冬酰胺酶， L-门冬酰胺酶， 或 门冬酰胺酶）可以有效的治疗儿童或成人中的急性淋巴细胞白血病 （ALL)。 最近几年， 含有 L-天冬酰胺酶的药物已经用于联合化疗方案来治疗 K/T细胞淋巴瘤， 并取得了较好的治疗 效果。 K/T细胞淋巴瘤是中特殊类型的非霍奇金淋巴瘤， 多见于亚洲和拉丁美洲， 我国发病 率相对较高。 根据肿瘤发生部门， K/T 细胞淋巴瘤可分为鼻型 K/T 细胞淋巴瘤和非鼻型 K/T细胞淋巴瘤。 另外 L-天冬酰胺酶还被用于治疗何杰金氏病， 急性骨髓白血病， 急性骨 髓单核细胞白血病， 慢性淋巴细胞白血病， 淋巴肉瘤， 网状细胞肉瘤和黑素肉瘤 （Kotzia and labrou, J. Biotechnol. 127(2007)657-669)。 L- 门冬酰胺酶的活性形式为 4个亚基组成的同源 四聚体结构， 每一亚基由 326 个氨基酸组成。 L-天冬酰胺酶最初从若干生物体中纯化， 包括 大肠杆菌 E.coli)和软腐欧文氏菌（Erwinia carotovora)。在哺乳动物中， 仅在豚鼠 (Cavioidea 超科)和某些阔鼻猴 (New World monkey)中发现略高于痕量的 L-天门冬酰胺酶。 但是， 由于它 来源于外源生物， 对人而言是一种外源蛋白， 有较强的免疫原性， 临床上常见进行性免疫反 应和全身性过敏反应， 而限制了其临床应用。 （ 张丽娜， 宫道华. 江苏医药. 左旋门冬酰胺酶 治疗小儿急性淋巴细胞性白血病的毒副反应.2005， 31(5) ： 392 ； 王宁玲， 刘芝璋等. 左旋 门冬酰胺酶治疗儿童白血病的毒副作用及防治. 中国小儿血液， 2005， 10(3) ： 133)。
目前针对 L-门冬酰胺酶的聚乙二醇修饰研究已有很多。国外利用聚乙二醇修饰的 L-门冬 酰胺酶的产品 Oncaspai<Enzon inc)早在 1994年就已上市， 并在 2006年起被批准作为儿童和 成人的 ALL的一线疗法。 但是 Oncaspar使用的 PEG是琥珀酰亚胺琥珀酸酯 （SS-PEG), 这 种 PEG含有对酶的水解敏感的、或者在微碱性 PH值时不稳定的酯键 (美国专利号 4670417)。 这些性质显著降低了体外和体内的稳定性并且副作用也较大。而国内目前上市的 PEG修饰的 L-门冬酰胺酶只有恒瑞公司的"培门冬酶"， 它是 Oncaspar的仿制药， 也存在着 PEG容易降解 脱落的问题。
国内相关的已授权的专利主要有两个，分别是连云港新阳医药有限公司的"聚乙二醇修饰 门冬酰胺酶的制备方法" （申请号 02149328.6 )， 和上海医药工业研究院的"聚乙二醇修饰的 L-门冬酰胺酶" （申请号 200610027026.0)。 其中连云港新阳医药有限公司的专利中所采用的 修饰方法是两步反应法， 首先用分子量较小的 PEG修饰剂进行修饰， 然后再用分子量较大的 PEG修饰剂进行二次修饰。 这种修饰方法步骤繁多， 而且修饰产物的均一性难以控制， 纯化 方法复杂。而上海医药工业研究院的专利中使用的 PEG修饰剂与门冬酰胺酶结合后不易水解， 但修饰后活性损失较大， 保留了原蛋白 60%的活性。 国外的阿利兹第二药物公司最近也申请 了关于 PEG修饰 L-门冬酰胺酶的专利。 这个专利中所使用的 PEG修饰剂也是较常用的随机 修饰氨基的修饰剂，修饰的 L-门冬酰胺酶是欧文氏菌来源的。另外国内外也有多篇 PEG修饰 L-门冬酰胺酶的论文发表， 用的都是常规的单甲氧基 PEG修饰剂。 所有的这些专利和文章中 PEG修饰后的 L-门冬酰胺酶和未修饰的原蛋白比较， 免疫原性有了降低， 但依然存在有免疫 原性的问题。 并且， 这些专利和文章也都没有涉及解决多亚基蛋白质的亚基解聚的问题。 发明内容
为了克服现有技术中的上述技术问题， 本发明的目的之一是提供多臂聚乙二醇修饰剂在 修饰多亚基蛋白中的应用。
所述聚乙二醇修饰剂优选与多亚基蛋白质的氨基偶联。
优选的， 本发明使用的聚乙二醇修饰剂为醛基活化的或酯活化的四至八臂聚乙二醇修饰 剂。 优选为四臂聚乙二醇修饰剂。
更优选的， 本发明使用的多臂聚乙二醇修饰剂的结构式分别如式 （I) 或式 （II) 所示：
Figure imgf000005_0001
(I) C CH2—— O - CH2CH -)— CH2— C— N H
Figure imgf000006_0001
(II)
n 为 1到 2000 整数值， 优选为 2-500 之间的整数值， 更优选为 25-100 之间的整数； k为 1或 2， 首选 1 ; m 为 2-16 之间的整数， 首选 4; p为 1-4之间的整数， 首选为 2; 聚乙二醇修饰剂的分 子量在 l~100kDa之间， 优选为 l~40kDa之间， 更优选为 5~10kDa。
更优选的， 上述酯活化的聚乙二醇修饰剂是， 四臂聚乙二醇琥珀酰亚胺乙酸酯 (4ARM-SCM) 、 四臂聚乙二醇琥珀酰亚胺丙酸酯 （4ARM-SPA) 或四臂聚乙二醇琥珀酰亚 胺碳酸酯 （4ARM-SC)。 最优选为四臂聚乙二醇琥珀酰亚胺乙酸酯 （4ARM-SCM)。
上述醛基活化的聚乙二醇修饰剂是， 四臂聚乙二醇丙醛 (4ARM-PALD)、 四臂聚乙二醇丁 醛 (4ARM-BALD；)、 四臂聚乙二醇乙醛 (4ARM-ALD)或四臂聚乙二醇戊醛 (4ARM-AALD；)。 最 优选为四臂聚乙二醇丙醛。 0c=
聚乙二醇修饰剂分子根据聚合度不同， 可以是分子量为 2KDa 〜 40KDa 的任一分子， 其中优选分子量为 5KDa 的聚乙二醇分子。 所使用的多臂 PEG首选上述结构式的， 但不局限 于上述结构式。
上述所述的多亚基蛋白质包括但不限于 L-门冬酰胺酶， 碱性磷酸酶以及尿素酶和谷氨酸 本发明的另一目的是提供一种上述的聚乙二醇化的多亚基蛋白质的制备方法， 包含以下 几个步骤：
( 1 ) 需要修饰的多亚基蛋白质和多臂聚乙二醇修饰剂按摩尔比 1 :5-1 :200的比例混合， 混合后在缓冲液中进行修饰反应；
(2)修饰反应结束后用离子交换层析去除掉修饰产物中未与蛋白发生反应的多臂聚乙二 醇修饰剂；
(3 ) 用凝胶过滤层析对修饰产物进行纯化， 收集目的修饰产物；
其中使用的多臂聚乙二醇修饰剂和多亚基蚩白质同上所述。
优选的， 当上述所述多臂聚乙二醇修饰剂为丙醛活化的多臂聚乙二醇时， 所述缓冲液的 pH值范围是大约 5.0至 6.0。
优选的， 当上述所述多臂聚乙二醇修饰剂为琥珀酰亚胺乙酸酯活化的多臂聚乙二醇时， 所述缓冲液的 pH值范围是大约 7.0至 8.0。
优选的， 上述步骤 （1 ) 的修饰反应在蛋白浓度为 3mg/mL-15mg/mL下进行。 本发明的另一个目的是提供一种多臂聚乙二醇修饰的多亚基蛋白质。 所述多臂聚乙二醇 修饰剂优选与多亚基蛋白质的氨基偶联。
其中使用的多臂聚乙二醇修饰剂和多亚基蛋白同上所述。
本发明的第四个目的是提供一种药物组合物， 所述药物组合物含有多臂聚乙二醇修饰的 多亚基蛋白质或其药学上可接受的盐， 和药学上可接受的辅料。
优选的， 上述所述药物组合物为冻干粉针剂。 所述的辅料包括药学上可接受的载体和 /或 赋形剂等。
优选的， 上述所述多臂聚乙二醇修饰的多亚基蛋白质及其药物组合物通过肌肉、 静脉或 皮下途径给药。
药学上可接受的盐在其被施用的量和浓度存在时是非毒性的。 制备此类盐可通过改变化 合物的物理特征而不妨碍其发挥生理学效果来促进药物应用。 在物理特性方面有用的改变包 括降低熔点以促进经粘膜施用， 以及增加溶解性以促进施用更高浓度的药物。
上述所述的药学上可接受的盐包括酸加成盐， 例如含有硫酸盐、 盐酸盐、 延胡索酸盐、 马来酸盐、 磷酸盐、 醋酸盐、 柠檬酸盐、 乳酸盐、 酒石酸盐、 甲磺酸盐、 苯磺酸盐等。 上述 所述的药学上可接受的盐可来自酸， 包括盐酸、 马来酸、 硫酸、 磷酸、 乙酸、 柠檬酸、 乳酸、 酒石酸等。
药学上可接受的载体和 /或赋形剂也可被掺入根据本发明的药物组合物中促进特定门冬 酰胺酶的施用。 适于实施本发明的载体包括碳酸钙， 磷酸钙， 各种糖类 （乳糖、 葡萄糖、 蔗 糖）， 或各种淀粉， 纤维素衍生物， 明胶， 植物油， 聚乙二醇以及生理学上相容的溶剂 （包括 用于注射的无菌水溶液， 盐溶液和右旋糖苷等）。
本发明的第五个目的是提供一种多臂聚乙二醇修饰的门冬酰胺酶。 所述聚乙二醇优选与 多亚基蛋白质的氨基偶联。 其中使用的多臂聚乙二醇同上所述。
本发明还提供了多臂聚乙二醇修饰的 L-门冬酰胺酶在治疗急性白血病的用途。
有益效果：
本发明中， 经过申请人研究发现多亚基蛋白质各亚基解聚主要是由于蛋白亚基之间靠非 共价键作用来维持其高级结构， 这种作用力不如共价键稳定， 因此在体内很容易降解， 并且 解聚后各亚基更多的抗原表位暴露， 导致免疫原性较强。 解决这一问题的方法就是要增强亚 基之间的作用力， 并且同时不影响蛋白的活性， 或者是影响较小。 因此申请人考虑用聚乙二 醇修饰， 但目前常用的单甲氧基 PEG修饰剂显然无法增强亚基之间的作用力。 因此需要一种 修饰剂， 可以与蛋白偶联的同时， 增强各亚基之间的作用力。 各亚基之间的作用力增强后， 在体内就不易降解，从而不会暴露出抗原表位而产生免疫原性的问题。而本发明中的多臂 PEG 修饰剂正符合这种要求， 这类 PEG修饰剂的特点是： 一个 PEG分子上具有多个活化基团可 以和蛋白的氨基酸残基进行反应。 虽然， 本发明中所采用的多臂 PEG修饰剂都是目前现有的 PEG修饰剂，但是在本发明之前并没有文献报道采用该类多臂 PEG修饰剂来修饰多亚基蛋白 质， 以解决多亚基蛋白质中各亚基解聚而引起的稳定性降低、 失活、 免疫原性增强等问题。
并且， 经实验验证， 本发明提供的多臂 PEG修饰的多亚基蛋白质， 和原蛋白或者一般的 聚乙二醇修饰的多亚基蛋白质相比， 能够很好的把多亚基进行偶联， 更有效的防止了亚基解 聚的问题， 稳定性更好。 例如， 本发明提供的多臂聚乙二醇修饰的 L-门冬酰胺酶， 碱性磷酸 酶以及尿素酶等， 比原蛋白或者用一般 PEG修饰剂修饰后， 具有更高的稳定性和保持生物活 性的能力， 且结构稳定均一。 而本发明中制备的 PEG修饰后的门冬酰胺酶和市场上的 PEG 化门冬酰胺酶产品"培门冬酶"比较， 具有低免疫原性， 高稳定性， PEG不易脱落等优点， 而 且， 本发明提供的多臂 PEG修饰的门冬酰胺酶还保留了较高的生物活性， 且具有半衰期明显 延长、 结构稳定均一的特点。
目前已有文献报道用培门冬酶联合化疗治疗 K/T细胞淋巴瘤， 并取得较好疗效。 培门 冬酶是 PEG对大肠杆菌来源的 L-门冬酰胺酶进行修饰后制备得到的产品，而本发明中提供的 聚乙二醇修饰的 L-门冬酰胺酶也是大肠杆菌来源的。 因此也应该具有和培门冬酶相似的治疗 效果。 也就是说本发明中提供的多臂聚乙二醇修饰的 L-门冬酰胺酶可以用于联合其它化疗药 物来治疗 K/T细胞淋巴瘤。 附图说明
图 1 : 不同 PEG-ASP偶联物的蛋白电泳分析。
不同 PEG修饰 ASP后的蛋白电泳图。 第 1 -6泳道的样品分别是蛋白 Marker、 培门冬酶、 4SCM5K-ASP、 8SCM10K-ASP、 4PALD5K-ASP、 4PALD10K-ASP。 通过蛋白结果可以看出 培门冬酶的分子量呈现弥散状，而多臂 PEG修饰剂制备的 PEG-ASP偶联物分子量较为单一， 可见用多臂 PEG修饰剂制备的偶联物均一性显著优于用单甲氧基 PEG修饰剂进行修饰的样
P 图 2: 不同 PEG修饰 AKP、 URE和 GDH后的蛋白电泳图。
其中， 图 a中第 1-3泳道的样品分别是蛋白 Marker、 SCM5K-AKP、 4SCM5K-AKP。 图 b 中第 1-3泳道的样品分别是蛋白 Marker、 SCM5K-URE、 4SCM5K-URE。 图 c中第 1-3泳道的 样品分别是蛋白 Marker、 SCM5K-GDH、 4SCM5K-GDH。 通过蛋白电泳结果可以看出用多臂
PEG修饰剂能够稳定多亚基蛋白的活性结构， 多臂 PEG修饰后的偶联物样品更均一。 由此可 见， 与传统的单甲氧基 PEG修饰剂相比， 多臂 PEG修饰剂修饰的多亚基蛋白的分子量较单 一， 偶联物的均一性较好。
图 3 : PEG-AKP, PEG-URE, PEG-GDH偶联物的热稳定性研究。
通过测定 PEG-AKP, PEG-URE, PEG-GDH偶联物在 60°C水浴条件下， 不同时间的活性 变化来评价其稳定性。其中， 图 a是 SCM5K-AKP、 4SCM5K-AKP的测试结果图， 结果显示， SCM5K-AKP在 2h内活性发生显著降低， 5h后基本丧失全部活性， 而 4SCM5K-AKP在检测 过程中活性降低较慢， 5h后依然可保留 60%的原有活性。图 b是 SCM5K-URE、4SCM5K-URE 的测试结果图。 结果显示， SCM5K-URE在 lh内基本丧失活性， 而 4SCM5K-URE在 2h后依 然可保存原有活性的 40%。同样，我们做了类似的比较实验。对 SCM5K-GDH、 4SCM5K-GDH 样品在 0.5h 取样检测其活性。 结果发现， SCM5K-GDH 在 0.5h 内基本丧失活性， 而 8SCM10K-GDH在 0.5h依然可保存原有活性的 65%。 由此可见， 与传统的单甲氧基 PEG修 饰剂相比， 多臂 PEG修饰剂可以显著提高多亚基蛋白的稳定性。
图 4: 不同 PEG-ASP偶联物的体外生物活性。
比较不同 PEG-ASP 偶联物的体外生物活性。 生物活性检测结果表明培门冬酶的活性较 低， 仅有原蛋白的 40%; 而 4SCM5K-ASP, 8SCM10K-ASP, 4PALD5K-ASP, 4PALD10K-ASP 这 4种修饰产物的活性都比较高， 保留了原蛋白 80%的活性。
图 5 : 不同 PEG-ASP偶联物的稳定性研究。
比较了不同 PEG-ASP偶联物在 37°C下的稳定性， 并和培门冬酶进行比较。 稳定性分析 结果表明 4SCM5K-ASP, 8SCM10K-ASP的稳定性较好，活性基本没有下降。 4PALD5K-ASP, 4PALD10K-ASP的活性虽有显著降低， 但仍优于培门冬酶。
图 6: PEG-ASP偶联物和培门冬酶的 PEG蛋白电泳碘染结果。
通过蛋白电泳后的碘染结果来分析偶联物的 PEG脱落情况。其中图 a为培门冬酶在 37°C 水浴中放置不同时间后的蛋白电泳图。 泳道 1为蛋白 Marker, 泳道 2为分子量为 5000的单 甲氧基 PEG, 泳道 3-12分别为培门冬酶在 37°C水浴中放置 0h、 12h、 24h、 36h、 48h、 60h、 72h、 84h、 96h、 108h后的蛋白电泳图； 图 b为 4SCM5K-ASP在 37°C水浴中放置不同时间后 的蛋白电泳图。 泳道 1为蛋白 Marker, 泳道 2为 4SCM5K, 泳道 3-12分别为 4SCM5K-ASP 在 37°C水浴中放置 0h、 12h、 24h、 36h、 48h、 60h、 72h、 84h、 96h、 108h后的蛋白电泳图。
根据上两图的比较结果显示培门冬酶的稳定性较差， 存在明显的 PEG脱落现象， 并且随 着时间的延长， PEG脱落进一步加剧。而 4SCM5K-ASP在 108小时未发生 PEG脱落的现象。
图 7: 不同 PEG-ASP偶联物和原蛋白的圆二色谱图比较。
其中图 a为 PEG-ASP偶联物的远紫外区圆二色谱图， 图 b为 PEG-ASP偶联物的近紫外 区圆二色谱图。 比较不同 PEG-ASP偶联物和原蛋白的圆二色谱图， 分析其结构上的差别。通过圆二色谱 图可以看出， 和培门冬酶相同， ASP用不同的多臂 PEG修饰以后， 其近紫外和远紫外的圆二 色谱图基本没有改变， 说明修饰后 ASP的二级结构和三级结构没有发生改变。
图 8: 不同 PEG-ASP偶联物的药效比较。 比较了不同 PEG-ASP偶联物的药效， 并和原 蛋白以及培门冬酶做比较。药效试验表明 4SCM10K-ASP和 4PALD10K-ASP对肿瘤细胞的抑 制作用好于培门冬酶。
图 9: 不同 PEG- ASP偶联物的免疫原性。
比较了不同 PEG-ASP偶联物的免疫原性， 并和原蛋白以及培门冬酶做比较。从结果可以 看出 4SCM5K-ASP和 4PALD10K-ASP产生的抗体滴度低于原蛋白和培门冬酶。 具体实施例
定义：
本发明使用的缩写含义如下：
PEG, 聚乙二醇； PEG修饰剂， 聚乙二醇修饰剂； 多臂聚乙二醇修饰剂， 含有两个及以 上活化基团的聚乙二醇分子。
聚乙二醇 (PEG, HO-(CH2CH20)n-CH2CH2OH；)是一种两端带羟基的线型聚合物， 聚乙二 醇是经环氧乙烷聚合而成的， 由重复的氧乙烯基组成， 有分支型， 直链型和多臂型。 PEG也 被称为 poly(ethyleneoxide) (PEO)， poly(oxy-ethylene) (POE), 或者 polyoxirane。 一般情况下， 分子量低于 20,000 的被称为 PEG, 分子量更大的被称为 PEO。 普通的聚乙二醇两端各有一 个羟基， 若一端以甲基封闭则得到甲氧基聚乙二醇 （mPEG)， 这种衍生物是蛋白质聚乙二醇 化技术中最常用到。
聚乙二醇修饰剂， 则是指带有官能团的聚乙二醇衍生物， 是指经过活化的聚乙二醇， 目 前主要用于蛋白质以及多肽药物修饰， 又叫修饰性聚乙二醇， 修饰性 PEG。
4SCM5K, 分子量为 5KDa的四臂聚乙二醇琥珀酰亚胺乙酸酯；
8SCM10K, 分子量为 lOKDa的八臂聚乙二醇琥珀酰亚胺乙酸酯；
4SCM10K, 分子量为 lOKDa的四臂聚乙二醇琥珀酰亚胺乙酸酯；
4PALD5K, 分子量为 5KDa的四臂聚乙二醇丙醛；
4PALD10K, 分子量为 lOKDa的四臂聚乙二醇丙醛；
SCM5K, 分子量为 5KDa的单甲氧基琥珀酰亚胺乙酸酯；
AKP, 碱性磷酸酶； ASP, 门冬酰胺酶； URE， 尿素酶； 谷氨酸脱氢酶, GDH。
本申请中所用， 术语"偶联物"， 是指聚乙二醇修饰门冬酰胺酶或尿素酶等多亚基蛋白质 后得到的修饰产物；
几种聚乙二醇修饰门冬酰胺酶的修饰产物可在本申请中称为 "4SCM5K-ASP , 8SCM10K-ASP, 4PALD5K-ASP, 4PALD10K-ASP"统称为 PEG-ASP或 PEG修饰 ASP的偶联 物。聚乙二醇修饰碱性磷酸酶后的修饰产物可在本申请中称为" 4SCM5K-AKP, SCM5K-AKP, " 统称为 PEG-AKP或 PEG修饰 AKP的偶联物。聚乙二醇修饰尿素酶后的修饰产物可在本申请 中称为 "4SCM5K-URE, SCM5K-URE, "统称为 PEG-URE或 PEG修饰 URE的偶联物。聚乙二 醇修饰谷氨酸脱氢酶后的修饰产物可在本申请中称为" 4SCM5K-GDH, SCM5K-GDH, "统称为 PEG-GDH或 PEG修饰 GDH的偶联物。
本发明的聚乙二醇修饰剂优选具有下列范围内的分子量： 大约 2KDa至大约 40KDa。 更 具体地， 聚乙二醇修饰剂具有选自下列的分子量： 2KDa， 5KDa， 10KDa。 在特定的实施方 式中， 聚乙二醇修饰剂的分子量为 5KDa， 10KDa。
本发明使用的聚乙二醇修饰剂优选下面几种： 醛基活化的和酯活化的聚乙二醇， 更具体 地， 聚乙二醇修饰剂为丙醛活化的和琥珀酰亚胺乙酸酯活化的聚乙二醇。
在本发明中， 所修饰的蛋白， 可以是任何来源的多亚基蛋白质。 在特定实施例中， 所修 饰的多亚基蛋白为碱性磷酸酶和尿素酶。 在特定实施例中， 所修饰的多亚基蛋白为门冬酰胺 酶， 可从任何来源衍生、 克隆或产生， 包括来自动物或通过基因重组技术或它们的组合。 例 如， 门冬酰胺酶可从大肠杆菌中提取， 包括但不限于大肠杆菌。 在本发明的偶联物的特定实 施方式中， 门冬酰胺酶与包含 SEQ ID ΝΟ: 1的序列的蛋白具有至少约 60%的序列一致性。 更 特别地与包含 SEQ ID NO: l的蛋白具有至少约 65%， 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%或 100%的序列一致性。
在特定的实施方式中， 所述蛋白为大肠杆菌来源的门冬酰胺酶， 其具有 SEQ ID NO: l的 序列。
SEQ ID ΝΟ: 1的蛋白的片段也被包含在本发明的偶联物中所使用的蛋白的定义中。 所述 的" SEQ ID NO: l的蛋白的片段 "是指多肽的序列可包括比 SEQ ID NO: l更少的氨基酸。
本领域熟知的是， 可通过置换、 插入、 缺失和 /或添加一个或多个氨基酸来修饰多肽但同 时保留其酶活性。 例如， 在给定的位置通过化学上等同的氨基酸来置换一个氨基酸而不影响 蛋白的功能特性是常见的。 因此， 可预期如下改变将产生功能上等同的产物： 产生以一个带 负电的残基取代另一个的改变或产生一个带正电的残基取代另一个的改变。
对氨基酸序列中氨基酸残基被修饰的位置以及经受修饰的氨基酸的数目没有特别的限 制。 技术人员能够识别可被引入而不影响蛋白活性的修饰。
用于制备偶联物的方法- 门冬酰胺酶、碱性磷酸酶或尿素酶可使用本领域已知的方法通过连接基团共价结合 PEG, 例如 《聚乙二醇化学： 生物技术和生物医学应用》， J.M. Harris编辑 （1992)， 所揭示的纳入 本文供参考。
用来使 PEG共价结合多亚基蛋白的连接基团可以是任何生物相容的连接基团。 "生物相 容"表示化合物或基团是非毒性且可在体外或体内使用而不引起损伤、呕吐、疾病或死亡。 PEG 可结合连接基团， 例如通过酯键、 硫醇键或酰胺键。
本发明中， 最优选的生物相容连接基团的共同特征是它们通过琥珀酰亚胺乙酸酯基团或 丙醛基团与多亚基蛋白的氨基偶联。 另外， 蛋白可通过氨基、 巯基、 羟基或羧基直接与 PEG 偶联。 在一个最优实施方案中， PEG偶联门冬酰胺酶、 碱性磷酸酶、 尿素酶、 谷氨酸脱氢酶 上的氨基。
一方面， 本发明涉及制备偶联物的方法， 所述方法包括使用一定量的多臂 PEG修饰剂与 一定量的门冬酰胺酶、碱性磷酸酶或尿素酶在缓溶液中反应足量时间至使 PEG与之共价结合。 在特定的实施方式中， 所述门冬酰胺酶来自大肠杆菌， 并且更特别的， 所述门冬酰胺酶的氨 基酸序列如 SEQ ID NO: 1所示。 在一个实施方式中， 所述 PEG为 4SCM5K和 4ALD10K。
在一个特定的实施方式中， 当使用的是丙醛活化的 PEG时， 缓冲液的 pH值在大约 4.0 至大约 9.0的范围内。 最优选的 pH值范围是大约 5.0至 6.0， 例如， pH值约为 5.0， 5.1， 5.2， 5.3 , 5.4， 5.5， 5.6， 5.7， 5.8， 5.9或 6.0。
当使用的是琥珀酰亚胺乙酸酯活化的 PEG时，缓冲液的 pH值在大约 6.0至大约 9.0的范 围内。 最优选的 pH值范围是大约 7.0至 8.0， 例如， pH值约为 7.0， 7.1， 7.2， 7.3， 7.4， 7.5， 7.6， 7.7， 7.8， 7.9或 8.0。
此外， 门冬酰胺酶、 碱性磷酸酶、 尿素酶、 谷氨酸脱氢酶的聚乙二醇化在下列蛋白浓度 下进行： 大约 0.5-30mg/mL， 更特别的大约 2mg/mL-20mg/mL， 最特别地大约 3mg/mL-15mg/mL。 在特定的实施方式中， 在这些蛋白浓度下聚乙二醇化的门冬酰胺酶来自 大肠杆菌， 并且更特别的， 所述门冬酰胺酶包含 SEQ ID NO: 1的序列。
在升高的蛋白浓度下， 聚乙二醇化反应快速进行， 在少于 3h之内。此外， 所应用的 PEG 与门冬酰胺酶、 碱性磷酸酶、 尿素酶、 谷氨酸脱氢酶的摩尔比至多为 200: 1。 例如， 摩尔比 为 200: 1， 150： 1， 100： 1， 80： 1， 70： 1， 60： 1， 50： 1， 40： 1， 30： 1， 20： 1， 15： 1， 10： 1， 5： 1。
本发明通过下述实施例进一步阐明， 但任何实施例或其组合不应当理解为对本发明的范 围或实施方式的限制。 本发明的范围由所附权利要求书限定， 结合本说明书和本领域一般常 识， 本领域普通技术人员可以清楚地明白权利要求书所限定的范围。 在不偏离本发明的精神 和范围的前提下， 本领域技术人员可以对本发明的技术方案进行任何修改或改变， 这种修改 和改变也包含在本发明的范围内。 实施例 1 : 门冬酰胺酶的 PEG偶联物的制备及分析
制备例 1， 本发明聚乙二醇化门冬酰胺酶通过如下方法制备、 纯化、 鉴定：
1 . PEG偶联物样品制备
用 50mM pH5.0的乙酸-乙酸钠缓冲液（购自国药集团）溶解门冬酰胺酶（购自千红制药， 具有 SEQ ID NO: l的序列） 以配制成为 8mg/mL的溶液， 分别用 4PALD5K ， 4PALD10K (购 自北京键凯科技有限公司） 作为聚乙二醇修饰剂进行修饰， 门冬酰胺酶： 聚乙二醇修饰剂:还 原剂 （氰基硼氢化钠， 购自 sigma公司） 为 1 :50:2500的摩尔比进行反应， 在 4°C下反应 12h 后， 加入 1M甘氨酸终止反应。
另外以 50mM pH7.5的 PB缓冲液（磷酸氢二钠和磷酸二氢钠配置而成，试剂购自国药集 团） 溶解门冬酰胺酶， 配制成为 8mg/mL的溶液， 分别用 4SCM5K, 8SCM10K (购自北京键凯 科技有限公司)作为聚乙二醇修饰剂进行修饰， 按门冬酰胺酶： 聚乙二醇修饰剂为 1 : 50的摩 尔比进行反应， 在 4°C下反应 2h。
2. PEG偶联物样品纯化
2.1 色谱法去除未反应 PEG
色谱法条件： Q离子交换柱（购自 GE公司， HiTrap Q HP 5mL)， 平衡缓冲液： 20mM的 Tris-Hcl (pH8.0)， （试剂购自国药集团）。 洗脱缓冲液： 含 1M NaCl 的 20mM 的 Tris-HCl (pH8.0)， （试剂购自国药集团）。 流速 2.5mL/min， 检测波长为 280nm。
上样： 上述修饰反应产物用 0.5M的 NaOH溶液调节至 pH 8.0， 结合至 Q离子交换柱。 平衡： 平衡缓冲液液冲洗 5个柱体积。
收集： 50%的洗脱缓冲液洗脱， 并收集洗脱峰样品。
2.2色谱法纯化单修饰 PEG偶联物
色谱法条件： Hiload 16/60 Superdex 200 pg (购自 GE公司） 半制备型凝胶过滤柱， 洗脱 液为 PBS， 流速 1.5mL/min， 检测波长为 280nm。
3. PEG偶联物样品的蛋白电泳检测
蛋白浓缩胶为 5%， 分离胶为 7%。浓缩胶缓冲液为 0.5 M Tris-HCl 缓冲液 (pH 6.8); 分离胶 缓冲液为 1.5 mol/L Tris-HCl缓冲液 (pH8.8)。 取 10ug蛋白样品， 与样品缓冲液等体积混合， 在 100 °C下煮沸 5 min后上样运行， 电泳结束后用考马斯亮蓝 R250(购自国药集团)染色。
由图 1 可以看出， 修饰产物 4SCM5K-ASP， 8SCM10K-ASP, 4PALD5K-ASP， 4PALD10K-ASP的电泳条件非常均一， 说明 ASP的 4个亚基得到了很好的偶联。而比较市场 上的同类产品——培门冬酶 （购自江苏恒瑞医药股份有限公司）， 均一性得到很大提高。
制备例 2至 4除了 pH值、 反应温度、 反应时间、 蛋白浓度、 摩尔比不同之外， 其他方法 与制备例 1相同， 具体参数与得率如下表所示：
表 1 制备例 2至 4相关参数
Figure imgf000014_0001
实施例 2: 碱性磷酸酶和尿素酶以及谷氨酸脱氢酶的 PEG偶联物的制备及分析
碱性磷酸酶 （AKP) 是广泛分布于人体各脏器器官中， 碱性磷酸酶是一种能够将对应底 物去磷酸化的酶， 即通过水解磷酸单酯将底物分子上的磷酸基团除去， 并生成磷酸根离子和 自由的羟基， 这类底物包括核酸、 蛋白、 生物碱等。 AKP为同源二聚体蛋白， 每个亚基的分 子量为 28KDa。
Hp尿素酶单体由 A、 B两个亚单位组成， 呈六聚体， A、 B两个亚单体的分子量分别为 30 KDa和 64 KDa左右，其比例为 1： 1，本实施例中所使用的尿素酶是不能形成的 6聚体的， 只是由 Α,Β两个亚基形成的单体蛋白。
谷氨酸脱氢酶可以催化谷氨酸脱氨生成 α-酮戊二酸和氨的反应。 且谷氨酸脱氢酶是唯一 既能利用 NAD+又能利用 NADH+作为还原当量的酶。 它是一种不需氧的脱氢酶。 在氨基酸 代谢中占有重要地位是一种变构酶，由 6个相同的亚基聚合而成，每个亚基的分子量为 56000.
用 50mM pH7.5 的 PB 缓冲液分别溶解碱性磷酸酶 （购自 SIGMA) 和尿素酶 （购自 SIGMA), 以及谷氨酸脱氢酶 （购自 SIGMA) 配制成为 8mg/mL 的溶液， 分别用 SCM5K, 4SCM5K, (购自北京键凯科技有限公司） 作为聚乙二醇修饰剂进行修饰， 按碱性磷酸酶或尿素 酶： 聚乙二醇修饰剂为 1 : 50的摩尔比分别进行反应， 在 4°C下反应 2h。
修饰产物的制备和蛋白电泳分析和实施例 1中的制备例 1相同。分析结果见图 2所示。如图 2可以看出， 4SCM5K-AKP、 4SCM5K-URE和 4SCM5K-GDH的均一性要显著高于 SCM5K-AKP、 SCM5K-URE和 SCM5K-GDH, 可见四臂 PEG修饰剂能够很好把 AKP、 URE或 GDH的亚基都偶联在一起，而用单甲氧基的 PEG修饰剂修饰后得到的修饰产物的均一性较差， 单甲氧基的 PEG修饰剂无法达到聚合各亚基的作用。
通过实施例 1和 2可以看出， 使用多臂 PEG修饰剂对碱性磷酸酶、 门冬酰胺酶、 尿素酶 以及谷氨酸脱氢酶蛋白进行修饰， 可以把蛋白的亚基偶联在一起， 使得修饰产物的均一性显 著提高。 其中碱性磷酸酶和尿素酶是二亚基蛋白， 门冬酰胺酶是四亚基蛋白， 谷氨酸脱氢酶 是 6亚基蛋白。 由此可见， 多臂 PEG修饰剂适用于多亚基类蛋白的修饰， 和单甲氧基的 PEG 修饰剂比较， 可以把亚基之间进行偶联， 从而起到提高修饰产物的均一性的目的。 实施例 3: 碱性磷酸酶、 尿素酶以及谷氨酸脱氢酶的 PEG偶联物的热稳定性研究
为了比较单甲氧基 PEG修饰剂修饰后的碱性磷酸酶、尿素酶和谷氨酸脱氢酶的稳定性和 多臂 PEG修饰剂修饰后的稳定性。本实施例分别检测了实施例 2中的 4SCM5K-AKP、 4SCM 5K-URE、 4SCM5K-GDH 、 SCM5K-AKP、 SCM5K-GDH和 SCM5K-URE这 6种修饰产物在 60°C水浴放置一段时间后的活性变化， 以此来研究其稳定性的差别。 具体方法如下： 把 PBS 缓冲液溶解的蛋白浓度为 lmg/mL的样品置于 60°C水浴中， 每隔一段时间取样并放入 4°C冰 箱备用。 取样结束后测定其活性， 根据其活性的变化评价其稳定性。 结果如下图 3所示。
由图 3a可以看出， 4SCM5K-AKP的稳定性显著好于 SCM5K-AKP, 在 60°C水浴条件下 120min时， 4SCM5K-AKP还保留了 70%的活性， 而 SCM5K-AKP的活性则下降到 40%左右， 在 400min时， 4SCM5K-AKP还保留了 60%左右的活性， 而 SCM5K-AKP则基本完全失活。 说明用多臂 PEG对 AKP的亚基进行偶联后， 其稳定性要高于用常规单甲氧基 PEG对其修饰 的修饰产物。 同样在图 3b中可以看出， 4SCM5K-URE的稳定性显著好于 SCM5K-URE, 在 60°C水浴条件下 120min时， 4SCM5K-URE还保留了 40%的活性， 而 SCM5K-URE则几乎完 全失活。 说明用多臂 PEG对 URE的亚基进行偶联后， 其稳定性要高于用常规单甲氧基 PEG 对其修饰的修饰产物。 由此可见， 用多臂 PEG对二亚基蛋白进行偶联后， 其稳定性优于比用 单甲氧基的 PEG偶联后的产物。用类似的方法比较了 4SCM5K-GDH和 SCM5K-GDH在 0.5h 的活性， 得到了相似的结果。 由此可见， 用多臂 PEG对 6亚基蛋白进行偶联， 其稳定性优于 用单甲氧基的 PEG偶联后的产物。 因此可以合理推测， 用多臂 PEG对多亚基蛋白进行修饰 后， 其稳定性要优于用常规单甲氧基 PEG对其修饰的修饰产物。 实施例 4: PEG-ASP偶联物的体外活性检测
门冬酰胺酶可以水解门冬酰胺的酰胺基， 根据这一原理， 可以对门冬酰胺酶的活性进行 测定。 具体测定方法参照 2005版药典第二部第 31页所述方法进行。 测活所需的试剂都购自 国药集团化学试剂有限公司。 检测的样品分别是 4SCM5K-ASP, 8SCM10K-ASP, 4PALD5K- ASP, 4PALD10K-ASP以及未修饰的原蛋白和市场上的同类产品培门冬酶 (购自江苏恒瑞医药 股份有限公司)。 它们的相对活性比较结果如图 4所示。
由图 4活性测定的结果可以看出，本发明的 PEG修饰门冬酰胺酶后的活性和市场上的同 类产品培门冬酶都有一定程度的降低，但本发明的 PEG修饰门冬酰胺酶都能保持原蛋白 80% 左右的活性， 而培门冬酶的活性显著下降， 只有原蛋白 50%左右的活性。
总体比较可以得出 4SCM5K-ASP, 8SCM10K-ASP, 4PALD5K-ASP, 4PALD10K-ASP这 4种修饰产物的比活大于培门冬酶。 实施例 5: PEG-ASP偶联物的热稳定性研究
为了验证 PEG修饰后可提高门冬酰胺酶的稳定性并和培门冬酶的稳定性做比较， 本实施 例分别检测了实施例 1中的 4SCM5K-ASP, 8SCM10K-ASP, 4PALD5K-ASP, 4PALD10K-AS P这 4种修饰产物及原蛋白和培门冬酶在 37°C水浴放置一段时间后的活性。 具体方法如下： 把 PBS缓冲液溶解的蛋白浓度为 lmg/mL的样品置于 37°C水浴中，每隔一段时间取样并放入 4°C冰箱备用。 取样结束后检测其生物活性。 检测结果如下图 5所示。
通过活性测定的结果可以看出， 原蛋白在 25h后活性显著下降， 48h后就已完全失活。 培门冬酶的稳定性高于原蛋白， 但是在 75h后活性也开始显著下降， 到 120h时活性已完全丧 失。 而本发明多臂 PEG 修饰产物 4SCM5K-ASP , 8SCM10K-ASP, 4PALD5K-ASP , 4PALD10K-ASP的稳定性较高， 活性降低较为缓慢， 其中 4SCM5K-ASP, 8SCM10K-ASP的 活性基本没有变化。
由此可见，本发明制备的 4SCM5K-ASP, 8SCM10K-ASP, 4PALD5K-ASP, 4PALD10K-ASP 热稳定性均远高于培门冬酶。
根据实施例 3、 4、 5的结果可以得知， 用多臂 PEG对多亚基类蛋白进行修饰后， 其修饰 产物的稳定性显著好于用常规的单甲氧基 PEG进行修饰而得到的修饰产物。本发明所使用的 多臂 PEG适用于对所有多亚基类蛋白的修饰， 通过对亚基之间进行共价键偶联后， 大幅提高 其稳定性， 防止亚基的解离， 降低由此带来的活性损失及免疫原性的产生。 实施例 6: PEG-ASP偶联物的偶联基团的稳定性研究
聚乙二醇修饰剂可增强原蛋白的稳定性， 但是由于偶联基团的不同， 会存在偶联基团降 解而引起的 PEG脱落的现象， 从而影响药物的稳定性及生物活性。 为了比较本发明的 PEG- ASP偶联物与市售培门冬酶的 PEG修饰的稳定性， 分别检测了 4SCM5K-ASP, 和培门冬酶 在 37°C水浴放置一段时间后的 PEG脱落情况。 具体方法如下： 把 PBS缓冲液溶解的蛋白浓 度为 lmg/mL的样品置于 37°C水浴中， 每隔一段时间取样并放入 4°C冰箱备用。 取样结束后 进行蛋白电泳， 电泳结束后用碘染色对 PEG进行染色。 检测结果如图 6所示。
由图 6a可以看出， 培门冬酶样品中含有游离 PEG, 说明在进行稳定性考察前， PEG已 开始脱落， 并且随着时间的延长， PEG脱落进一步加剧。 而从图 6b 中可以看出， 4SCM5K- ASP在整个研究时间内未发生 PEG脱落的现象。 说明偶联的 PEG与蛋白的氨基形成的共价 键非常稳定， 显著好于培门冬酶。 PEG修饰类的蛋白药物一般都是注射剂， 如果在体内发生 PEG脱落， 会使得蛋白的抗原决定簇暴露出来， 引起显著的免疫原反应， 从而导致药效降低 并带来副作用。 根据实验结果可知， 多臂 PEG修饰后的蛋白药物稳定性较高， 不易发生 PE G分子的脱落， 从而增加了修饰后药物的稳定性， 减少了药物引发机体免疫反应的几率。 实施例 7: PEG-ASP偶联物和原蛋白的圆二色谱图分析
用圆二色光谱仪可以表征修饰蛋白以及未修饰蛋白的二级结构及三级结构。 蛋白浓度范 围是 0.1~0.2 mg/mL。样品加入 1 mm光径的圆二色比色杯，检测其远紫外区（190 nm-250 nm ) 和近紫外区 （253nm-480nm) 的圆二色光谱， 扫描带宽为 1 nm， 扫描速度为 500 nm/min。 每 次检测以相应缓冲液为背景， 测三次取平均值。 由图 7可以看出， PEG-ASP偶联物的远紫外 区圆二色谱图和原蛋白相比， 偶联物的光谱几乎没有发生峰的位移， 但峰值有一定程度的变 化， 这是由于 PEG的修饰对其吸收值有一定的影响。 但 PEG-ASP偶联物和培门冬酶的图谱 基本重合， 说明用不同修饰剂修饰后的二级结构没有差别， 这个结果符合 PEG的特性， 由于 PEG在溶液中是一种柔性的两亲性大分子， 因此偶联到蛋白表面后， 不会对其结构有显著影 响。 说明 PEG修饰没有影响 KLK1 的二级结构。 同样的， PEG-ASP偶联物的近紫外区圆二 色谱图和原蛋白相比， 偶联物的光谱也几乎没有发生峰的位移， 虽然峰值有一定变化， 但和 培门冬酶的谱图几乎重合， 这说明 PEG修饰没有影响 ASP的三级结构。 总体看来， 通过不 同修饰剂制备的偶联物， ASP的高级结构基本没有发生改变。 由于偶联物经过 PEG修饰后， 其结构未变化， 因此其活性和原蛋白比较， 损失较少。 实施例 8: 不同 PEG^ASP偶联物对不同肿瘤细胞的抑制作用比较
为了评价 PEG-ASP 偶联物对肿瘤细胞的抑制率， 并和培门冬酶进行比较， 我们选择了 THP-1 (来源于人的单核细胞白血病细胞系）、 U937 (来源于人的单核细胞白血病细胞系、 Raji (来源于人的淋巴瘤细胞系）、 Jurkat (来源人的急性 T细胞白血病细胞系）、 L1210 (来源于 小鼠的白血病细胞）、 L5178Y (来源于的小鼠淋巴瘤细胞） 这六种细胞进行评价。 通过 MTT 法检测细胞的抑制率， 考察了不同给药浓度的抑制率， 并最终计算出 IC5Q值， 计算结果如表 2所示。
表 2 不同 PEG-ASP偶联物及培门冬酶对肿瘤细胞的 IC5o值
Figure imgf000018_0001
根据实验结果，本发明的多臂 PEG修饰后的门冬酰胺酶对上述 6种肿瘤细胞的杀伤性普 遍高于未修饰的门冬酰胺酶， 而本发明的 4SCM5K-ASP和 4PALD10K-ASP的抗肿瘤活性明 显高于培门冬酶， 对多种细胞株均表现出良好的抗肿瘤作用， 特别是 4PALD10K-ASP, 在多 种细胞株中 IC5Q均为最低， 抗肿瘤活性最高。 实施例 9: 不同 PEG-ASP偶联物对肿瘤的体内抑制作用比较
4-6周龄裸鼠 （BALB/cA-nu) 32只， 15-18g， 雌性， 分为 5组， 每组 8只， BP : 培门冬酶 组， ASP组， PBS组， 4PALD10K-ASP, 4SCM10K-ASP组。 实验前， 记录每只裸鼠的体重并 检测肝功能。 试验开始时， 每只裸鼠腹腔注射环磷酰胺 100mg/kg， 连续 4天。 第五天腹腔注 射约 5 X 106个肿瘤细胞 L1210。 第二天给药一次， 肌肉注射培门冬酶， ASP , PBS ,
4PALD10K-ASP, 4SCM10K-ASP, 按 4.7mg/kg肌肉注射给药， 并于注射完的第三天和第六 天计算抑瘤率。 实验结果如图 8和表 3所示。
表 3 PEG修饰物对肿瘤接种小鼠抑瘤率的影响
(个) d3 d6_
模型组 / Γ
ASP 8 7.1 32.9 培门冬酶 8 19.9 35.5
4PALD10K-ASP 8 36.3 40.0
4SCM10K-ASP 8 37.6 43.5 由图 8和表 3可以得出， PEG修饰后的门冬酰胺酶对肿瘤的抑制率显著高于修饰之前， 门冬酰胺酶组的第 3天和第 6天的抑制率都低于其它 PEG修饰组的。 另外， 4PALD10K-ASP 和 4SCM10K-ASP组对肿瘤的抑瘤率好于培门冬酶组， 特别是第 3天的抑瘤率。 说明用多臂 PEG修饰后的门冬酰胺酶的药效优于用传统的单甲氧基的 PEG修饰的门冬酰胺酶。这可能和 4PALD10K-ASP以及 4SCM10K-ASP的稳定性更高有关。 本实施例初步说明 4SCM10K-ASP 和 4PALD10K-ASP对肿瘤的抑制效果优于市场上的同类产品培门冬酶。 实施例 10: PEG-ASP偶联物的免疫原性
PEG分子的结构特异性可以降低或者消除诱导产生中和抗体和与抗体结合的能力， 使其 难以被免疫系统识别和清除， 因此 PEG修饰的蛋白可以一定程度上降低免疫原性。
本实施例测定了不同 PEG-ASP偶联物的对小鼠的相对免疫原性，并和原蛋白以及培门冬 酶进行了比较。 试验分 4PALD10K-ASP组、 4SCM5K-ASP组、 培门冬酶组及原蛋白组， 以 每 2周 1次的频率， 连续 8周的方法给小鼠尾静脉注射 2mg/kg (按蛋白的量计算） 的上述蛋 白。 在给药结束后 1周从眼眶取血。 通过间接 ELISA测定血清中抗门冬酰胺酶抗体的水平。 (所使用的二抗购自 SIGMA公司） 结果如图 9中所示。
从结果可以得出， 4PALD10K-ASP组、 4SCM5K-ASP组对小鼠的抗体滴度均远远低于原 蛋白以及培门冬酶组。 说明 PEG 修饰后能够明显降低蛋白的免疫原性。 并且由于 4PALD10K-ASP组、 4SCM5K-ASP组是用多臂 PEG进行修饰的， 亚基得到了充分的偶联。 因此在体内不容易降解， 从而不易发生免疫反应。 培门冬酶虽然也是用 PEG进行了修饰， 但 是由于其各亚基没有充分偶联， 分子稳定性较低， 容易在体内发生亚基的解离， 从而暴露了 抗原表位， 导致免疫反应。 实施例 11: PEG-ASP偶联物的药代动力学研究
PEG的空间阻碍作用使得被修饰蛋白对蛋白酶酶解的抵抗能力大为提高， 同时被修饰蛋 白的分子排阻体积明显增大， 使得其肾脏过滤清除率降低显著， 从而可以提高原蛋白的体内 半衰期。
本实施例采用 1251同位素标记示踪法对 PEG修饰后的门冬酰胺酶的体内血药浓度进行了 研究。其中， 为了减少大鼠甲状腺对标记药物的吸收， 实验前约 8h腹腔注射 1%KI溶液 ImL 饱和大鼠的甲状腺。 将大鼠随机分为 4SCM5K-ASP组、 培门冬酶组， 每组 8只， 雌雄各半。 具体操作方法如下：
1. 放射性碘取代蛋白质分子中氨基酸残基苯环上的羟基， 得到具有放射性示踪作用的 1251 蛋白。
2. 用苦味酸在其身上按照编号规则进行编号， 称重。 用大鼠固定器固定后， 由尾静脉注射给 药 （1.5U/kg)。 给药后立即放开大鼠， 自由活动， 自由饮水和喂食。 3. 分别于给药后 lmin, 5min、 10min、 20min、 30min、 60min、 3h、 6h、 12h、 24h、 48h、 84 h、 96h、 144h、 168h、 192h、 216h、 264h从眼眶取血约 0.2mL， 并加入 EDTA抗凝。
4. 5000rpm/3min离心分离大鼠血浆， 用伽马计数器（安可中佳， GC-911 )测定血浆放射性活 度， 测量时间为 lmin。
5. 放射性活度测量完毕后回收血浆， HPLC (岛津 LC-20AT) 分离未降解的蛋白原性药物。
动物实验结束后经过数据处理等，可以得到每个时间点的 SD大鼠的血药浓度。通过时间 和对应的血药浓度， 用药动学专用软件 DAS计算该药物的药动学参数。
实验结果表明， 培门冬酶以及 4SCM5K-ASP药物药动学模型属于三房室模型。各参数的 计算结果见表 4所示。
PEG-ASP偶联物和培门冬酶的药代动力学参数比较
Sample Τ1/2γ(1ι) AUC(mg/L*h) CL(L/h/kg) 培门冬酶 25.12 459 0.028
4SCM5K-ASP 45.90 805 0.015
4SCM5K-ASP的药代参数与培门冬酶的参数相比， 半衰期明显延长， 从 25h左右提高到 了 45h， 并且药时曲线下面积 （AUC) 有了显著提高， 从 459mg/L*h提高到 805 mg/L*h.其次， 4SCM5K-ASP在血液中的清除速率也低于培门冬酶。 由此可见， 用多臂 PEG修饰后的门冬酰胺 酶和培门冬酶相比， 在体内的稳定性显著增强， 因此其半衰期显著提高， 血液中代谢速度明 显降低， 有效的延长了药物的药效时间。 可以合理推测， 本发明中所使用的多臂 PEG修饰门 冬酰胺酶后， 由于其能够增强蛋白亚基间的作用力， 因此提高了稳定性， 药代实验的结果也 验证了这一点。 由于 4SCM10K-ASP活化基团和 4SCM5K-ASP相同， 并且 PEG的链更长， 因此可 以根据 4SCM5K-ASP组的数据推测 4SCM10K-ASP组的半衰期会显著大于 45. 90 h， 另外 AUC值 也更高， 其药代数据应显著优于 4SCM5K-ASP组。

Claims

权利要求书
1、 多臂 PEG在修饰多亚基蛋白中的应用。
2、根据权利要求 1所述的应用，其特征在于所述的多臂 PEG选自以下任意一种结构所示的 PEG:
C
Figure imgf000021_0001
o
(1)， — H
Figure imgf000021_0002
(11)，
其中 n选自 1到 2000 整数值， k为 1或 2， m 选自 2-16 之间的整数， p选自 1-4之间的整数， 多臂 PEG的分子量在 l~100kDa之间。
3、 根据权利要求 2所述的应用， 其特征在于所述的 n选自 2-500 之间的整数值， 优选为 25-100 之间的整数； 所述的 k为 l，m 为 4， p为 2; 多臂 PEG的分子量在 l~40kDa之间， 优选为 5~10kDa。
4、 根据权利要求 3所述的应用， 其特征在于所述的多臂 PEG为醛基活化的或酯活化的多臂聚乙 二醇衍生物； 其中酯活化的聚乙二醇衍生物选自 4臂聚乙二醇琥珀酰亚胺乙酸酯、 4臂聚乙二醇 琥珀酰亚胺丙酸酯、 4臂聚乙二醇琥珀酰亚胺碳酸酯中的任意一种； 醛基活化的聚乙二醇衍生 物选自 4臂聚乙二醇丙醛、 4臂聚乙二醇丁醛、 4臂聚乙二醇乙醛、 4臂聚乙二醇戊醛中的任意一 种,优选 4臂聚乙二醇丙醛。
5、根据权利要求广 4中任一项所述的应用， 其特征在于所述的多亚基蛋白选自碱性磷酸酶、 门 冬酰胺酶或尿素酶中的任意一种。
6、 一种利用多臂 PEG修饰多亚基蛋白的方法， 其特征在于包含以下步骤；
( 1 ) 需要修饰的蛋白和 PEG修饰剂按比例混合后在缓冲液中进行修饰反应；
(2) 修饰反应结束后用离子交换层析去除掉修饰产物中未与蛋白发生反应的 PEG修饰剂；
(3) 用凝胶过滤层析对修饰产物进行纯化， 收集目的修饰产物；
其中所述的 PEG修饰剂选自以下任意一种结构所示的 PEG: C
Figure imgf000022_0001
o
(I),
Figure imgf000022_0002
( 11)，
其中 n选自 1到 2000 整数值， k为 1或 2， m 选自 2-16 之间的整数， p选自 1-4之间的整数， 多臂 PEG的分子量在 l~100kDa之间。
7、 根据权利要求 6所述的方法， 其特征在于所述的 n选自 2-500 之间的整数值， 优选为 25-100 之间的整数； 所述的 k为 l，m 为 4， p为 2; 多臂 PEG的分子量在 l~40kDa之间， 优选为 5~10kDa。
8、 根据权利要求 7所述的方法， 其特征在于所述的多臂 PEG为醛基活化的或酯活化的多臂聚乙 二醇衍生物； 其中酯活化的聚乙二醇衍生物选自 4臂聚乙二醇琥珀酰亚胺乙酸酯、 4臂聚乙二醇 琥珀酰亚胺丙酸酯、 4臂聚乙二醇琥珀酰亚胺碳酸酯中的任意一种，优选 4臂聚乙二醇琥珀酰亚 胺乙酸酯； 醛基活化的聚乙二醇衍生物选自 4臂聚乙二醇丙醛、 4臂聚乙二醇丁醛、 4臂聚乙二 醇乙醛、 4臂聚乙二醇戊醛中的任意一种，优选 4臂聚乙二醇丙醛。
9、根据权利要求 8所述的方法， 其特征在于所述的多亚基蛋白选自碱性磷酸酶、 门冬酰胺酶或 尿素酶中的任意一种。
10、 多臂 PEG修饰的多亚基蛋白。
11、根据权利要求 10所述的多臂 PEG修饰的多亚基蛋白，其特征在于所述的多臂 PEG修饰的多亚 基蛋白按照权利要求 6〜9中任一项所述的方法获得。
12、根据权利要求 10所述的多臂 PEG修饰的多亚基蛋白，其特征在于所述的多臂 PEG修饰的多亚 基蛋白为多臂 PEG修饰的门冬酰胺酶。
13、 根据权利要求 10所述的多臂 PEG修饰的多亚基蛋白， 其特征在于所述的多臂 PEG修饰剂 选自以下任意一种结构所示的 PEG: C
Figure imgf000023_0001
o
(I),
Figure imgf000023_0002
( 11)，
其中 n选自 1到 2000 整数值， k为 1或 2， m 选自 2-16 之间的整数， p选自 1-4之间的整数， 多臂 PEG的分子量在 l~100kDa之间。
14、 一种药物组合物， 其特征在于所述药物组合物含有如权利要求 12所述的多臂 PEG修饰的门 冬酰胺酶和药学上可接受的辅料； 所述的药物组合物的剂型优选冻干粉针制剂。
15、 权利要求 12所述的多臂 PEG修饰的门冬酰胺酶或权利要求 14所述的药物组合物在制备治疗 肿瘤的药物中的应用。
16、 权利要求 12所述的多臂 PEG修饰的门冬酰胺酶或权利要求 14所述的药物组合物在制备治疗 急性白血病的药物中的应用。
PCT/CN2014/083143 2013-12-30 2014-07-28 多臂聚乙二醇修饰剂的新用途及其在修饰门冬酰胺酶中的应用 WO2015101033A1 (zh)

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