WO2006116948A1 - Interleukin-6 polyethylene glycol conjugate and its preparing method and use - Google Patents

Interleukin-6 polyethylene glycol conjugate and its preparing method and use Download PDF

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WO2006116948A1
WO2006116948A1 PCT/CN2006/000891 CN2006000891W WO2006116948A1 WO 2006116948 A1 WO2006116948 A1 WO 2006116948A1 CN 2006000891 W CN2006000891 W CN 2006000891W WO 2006116948 A1 WO2006116948 A1 WO 2006116948A1
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interleukin
polyethylene glycol
conjugate
peg
modified
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PCT/CN2006/000891
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Chinese (zh)
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Xuemei Zhang
Tao Yuan
Ke Zhang
Hailin Rao
Jie Deng
Zhijie Wang
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Chengdu Institute Of Biological Products
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Publication of WO2006116948A1 publication Critical patent/WO2006116948A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • 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
    • C07K14/54—Interleukins [IL]
    • C07K14/5412—IL-6

Definitions

  • Interleukin-6 polyethylene glycol conjugate and preparation method and application thereof
  • the present invention relates to an interleukin-6 polyethylene glycol conjugate
  • the invention also relates to methods and uses for the preparation of interleukin-6 polyethylene glycol conjugates. Background technique
  • Interleukin 6 also known as interferon beta-6, is a multifunctional cytokine.
  • IL-6 mainly acts on the immune system, promotes the differentiation of sputum cells and nerve cells, stimulates the growth of sputum cells and stem cells, induces the differentiation of multinucleated cells, and promotes platelet production.
  • IL-6 There are three main aspects of the use of IL-6: (1) It has a certain effect on the reduction of platelets caused by chemotherapy and radiotherapy. It was once used as a substitute for platelet preparations, and it is used in the treatment of hematopoietic insufficiency caused by anticancer agents.
  • IL-6 can enhance humoral and cellular immunity, Some patients with immunodeficiency have a therapeutic effect.
  • the development of IL-6 in the treatment of thrombocytopenia has progressed to the phase II clinical trial stage, but because the half-life of IL-6 in plasma is too short and the dosage is large, it is easy to cause adverse reactions (such as fever, Headache, serum albumin decreased), failed to achieve practical application.
  • Polyethylene glycol can form a combination with proteins and peptide drugs.
  • PEG-modified proteins and peptides can alter the properties of protein drugs, such as increasing the solubility and stability of such drugs, attenuating or eliminating immunogenicity, antigenicity and toxicity, increasing the therapeutic index of drugs, and expanding the scope of clinical use. , as well as improving the in vivo pharmacokinetic properties of the drug, prolonging the half-life of the drug in the body.
  • the pharmacokinetic properties of polyethylene glycol-based finishes vary depending on the nature of the protein they are decorated with, the relative molecular weight of the modified product, and the mode of administration.
  • the modification pathways of polyethylene glycol to proteins and peptides mainly include amino modification (including acylation modification of quinone amino group, acylation modification of lysine side chain amino group, alkylation modification of quinone amino group), carboxyl modification, hydrazine Base modification, etc. Because of the presence of multiple amino groups in the structure of a protein or polypeptide, the structure is complex and diverse, so controlling and determining the degree of modification and the site of modification has been a difficulty in the modification of polyethylene glycols of proteins and polypeptides.
  • U.S. Patent No. 5,264,209 discloses a PEG-IL-6.
  • Human IL-6 was modified using PEG with molecular weights of 4500, 10000 and 12000, of which PEG4500 and PEG12000 were linear PEG and bis-PEG5000 (total molecular weight 10000) was branched PEG.
  • the modified products obtained in this patent are mixed products with different degrees of modification (two to nine PEG modifications). Animal experiments show that the proliferation of platelets is higher than that of the same dose (4 g ⁇ 10 mg/kg).
  • Modified IL-6 It is also believed that at least 2, and preferably more than 5, PEG modifications are required to ensure the in vivo activity of the modified product.
  • Tsunoda S, Tsutsumi Y and others compared the in vivo platelet proliferative activity of PEG ( PEG-5000 ) modified IL-6 ( MPEG- IL-6 ) and unmodified IL-6 ( IL-6 ) ( PEGylation of interleukin-6 Effectively increases Its Thrombopoietic Potency.
  • the residence time in the body is about 25 hours, which is not much different from the IL-6, and its activity in the body is also significantly lower than that of the highly modified component, and its activity in vivo is lower than 50 times the dose of IL-6.
  • the single-modified DmPEG-IL-6 prepared by using DMMAn as an amino-protecting agent has not only a significant difference in the pharmacokinetic parameters and in vivo activity of the single-modified PEG-IL-6, but also requires a 3-step reaction to obtain The preparation process is complicated.
  • the PEG modified products of the above-mentioned 16 are mostly mixtures of products with different modification sites and degree of modification. Due to the heterogeneity of the products, effective quality control cannot be performed, and it is difficult to ensure safety, effectiveness, quality control, and it is difficult to put into practice. Application; In rare cases, the single modified IL-6 polyethylene glycol conjugate has shortcomings such as short half-life, low activity, poor efficacy, and low yield. At present, there is an urgent need in the art to develop a single modified PEGylation which can overcome the shortcomings of existing products, has good homogeneity, is safe, effective, quality controllable, low cost, and can be mass-produced as a drug. Recombinant human interleukin-6. Summary of the invention
  • a first object of the present invention is to provide an interleukin-6 polyethylene glycol conjugate (hereinafter referred to as IL-6 polyethylene glycol conjugate or PEG-IL-6).
  • the IL-6 polyethylene glycol conjugate is obtained by covalently modifying the IL-6 by polyethylene glycol, wherein each interleukin-6 molecule is covalently bound to a polyethylene glycol molecule, polyethylene.
  • the molecular weight of the diol molecule is from 15,000 to 30,000. .
  • interleukin-6 polyethylene glycol conjugate of the present invention the preferred poly(diethylenediamine) modification of interleukin-6 at the side chain amino group or IL- of the Lys residue of the IL-6 molecule. 6-molecular peptide chain N-terminal gas group.
  • the polyethylene glycol molecule used for the modification may be a linear polyethylene glycol molecule, that is, on the side chain amino group of the Lys residue of the interleukin-6 or the N-terminal amino group of the peptide chain of the interleukin-6 molecule.
  • Connecting a PEG chain, which may also be a branched polyethylene glycol molecule, branching polyethylene glycol means connecting two or more PEG chains on the activating group, and the molecular weight is the sum of the molecular weights of the two chains.
  • the selectivity of the two polyethylene glycol molecules to the binding site in the modification of the interleukin-6 molecule may be different due to their different spatial structures.
  • m represents a methyl group
  • i, j is an integer of 100-1000, and the sum of i and j causes the molecular weight of the conjugated polyethylene glycol molecule to be 15,000 to 30000.
  • the PEG molecule which can be used as a modifier is, for example, a branched structure of mPEG2-NHS, and a linear structure of mPEG-aldehyde.
  • the IL-6 polyethylene glycol conjugate of the present invention is prepared by reacting an IL-6 molecule with a polyethylene glycol molecule at a certain temperature, pH and reaction time, and thus the present invention also provides the preparation of the above IL-6.
  • a polyethylene glycol conjugate method comprising the steps of:
  • Interleukin-6 is prepared as a solution having a protein concentration between 0.05 and 20 mg/ml and a pH of 6.5 to 10.0;
  • the SDS-PAGE purity of IL-6 in step 1) is greater than 95%, and the solution has a protein concentration of 0.5 ⁇ lmg/ml and a pH of 8.7 ⁇ 9.3.
  • the purification step in the step 3) of the above method comprises: desalting the reaction solution obtained in the step 2) through a G-25 gel filtration column, and then performing preliminary separation using a cation exchange chromatography column, and finally condensing with Superdex 200.
  • the gel filtration column was purified to obtain a single modified product.
  • the present invention also provides a pharmaceutical composition prepared by adding the IL-6 polyethylene glycol conjugate of the present invention to a pharmaceutically acceptable adjuvant.
  • the present invention also provides a polyethylene glycol conjugate of the above IL-6 for preparing a medicament for treating thrombocytopenia
  • the IL-6 of the present invention can be of various origins, and the homologous peptide chain having a peptide chain structure similar to that of natural human IL-6, whether synthetic or expressed by prokaryotic and eukaryotic systems, or even
  • the modified polyethylene glycol modified IL-6 of the present invention can be obtained as a raw material for the PEG modification of the present invention.
  • the modifier used in the present invention may be an activated PEG ester, and other methods may be used to covalently couple the PEG of the present invention to the Lys residue of IL-6. It is also within the scope of the invention to include the use of PEG having other types of activating groups, or to activate the Lys residue site of the IL-6 peptide chain.
  • the single modified PEG-IL-6 (monoPEG-IL-6) content of the product obtained by the method of the present invention can be greater than 85%.
  • the polyethylene glycol conjugate of IL-6 of the present invention may be prepared by adding a pharmaceutically acceptable auxiliary component such as an injection solution, a lyophilized preparation or the like.
  • the polyethylene glycol mono-modified interleukin-6 of the invention not only has the physiological activity of interleukin-6, but also has greatly improved stability due to the selection of polyethylene glycol of a suitable molecular weight; its long half-life in vivo, The serum clearance rate is low, the dosage and frequency of use are greatly reduced, and the side effects are greatly reduced.
  • the properties of each aspect are obviously superior to the currently disclosed interleukin-6 and single modified interleukin-6, which are convenient for patients to use. Reduce the cost of use, and also improve the safety of use, which can greatly reduce the suffering of patients.
  • the polyethylene glycol single modified interleukin-6 provided by the invention has good homogeneity, is convenient for quality control, can meet the requirements of safe, effective and quality control of clinical medication, and can be mass-produced, better than poly
  • the multi-modified interleukin-6 of ethylene glycol makes the industrial production and practical application of PEGylated recombinant human interleukin-6 possible, and has an excellent market prospect.
  • Figure 1 SDS-PAGE of the modified product of PEG modified IL-6 and its purified product; wherein: 1 and 9 are protein molecular weight standards; 10 and 13 are PEG modified products; 2 ⁇ 8, 11 ⁇ 12 and 15 are Each eluted group purified by SP Sepharose High Performance cation exchange chromatography 15 is a single modified product; 14 is a single modified product isolated and purified by Superdex 200 gel filtration chromatography.
  • the main band of the modified product (MW60, 000) is greater than 85%. Mass spectrometry indicates that the molecular weight of the modified protein is about 46,000, indicating that only one PEG 20,000 molecules have been modified;
  • mPEG-aldehyde 1.0 ug/head
  • PEG-SPA l.Oug/only
  • mPEG-SPA 2.5 ug/;
  • Figure 3 is a flow chart showing the preparation process of the PEG-IL-6 lyophilized preparation.
  • the sub-quantity is higher selectivity of lOkDa, the multi-molecular weight is smaller, and the air-bearing characteristic group, a PEG fraction
  • Child can have the same two bits
  • the purified rhIL-6 pure product was modified with these four PEG modification reagents, and the modified product was purified to remove unmodified rhIL-6 and modified by-products.
  • the molecular weight distribution, in vitro activity retention and drug efficacy in mice were determined.
  • the molecular weight distribution of PEG-modified rhIL-6 was determined by SDS-PAGE. After electrophoresis, the molecular weight and content of each band were calculated by gel scanning imaging system. Since the PEG long chain is a linear macromolecule, the apparent molecular weight in SDS-PAGE is usually 2-4 times its true molecular weight, so the molecular weight of the modified rhIL-6 cannot be accurately calculated, as listed in Table 2. Molecular weights are estimated apparent molecular weights. Apparent molecular weight distribution after four PEG modified rhIL-6
  • the in vitro cell viability determined by the MTT assay can indirectly reflect the in vivo activity of rhIL-6 and PEG-modified rhIL-6, but its platelet proliferative activity still needs to be confirmed by animal experiments.
  • the specific test protocol is as follows: On the 1-5th day, each mouse is injected subcutaneously with 0.5 ml of the test sample per day; from the third day of the injection test-risk sample, each mouse is intraperitoneally injected with 2 mg of cyclophosphamide per day for 3 consecutive days; Before the first day of injection of the test sample and ⁇ 8 17 days, every day from each small The tail of the rat was collected lOul, diluted 6 times, and the number of platelets was measured by Cell-DYN1600 instrument. Since the four PEG-modified rhLL-6 are successive animal tests, we used relative ratios to reflect the number of platelets.
  • the number of platelets before the test sample was injected on the first day was determined as 100%, and the relative percentage of platelets after the first day was calculated for statistical analysis.
  • mPEG2-NHS is significantly better than the other three.
  • mPEG2-NHS with a molecular weight of 20 kDa can only be modified to a modified amino group on the surface of rhIL-6 due to the steric hindrance effect of larger molecular weight and branched structure.
  • other PEG molecules are also difficult to access the rhIL-6 molecule due to steric hindrance, so that a single modified isomer can be obtained (monoPEG-rhIL- 6)
  • the main modified product is significantly better than the other three.
  • mPEG2-NHS binds to two lOkDa PEG long chains on the surface of the protein, which blocks the binding of rhIL-6 to the cell surface receptor, making the activity only 5-15% of rhIL-6.
  • PEG-aldehyde has high selectivity for the N-terminal amino acid of the protein, and the modified product is mostly N-terminal single modification, and the activity in vitro is also better, and the activity in vivo is lower than that of mPEG2-NHS.
  • PEG is a linear linear molecule, its apparent molecular weight is smaller than that of a branched type, and it is easily cleared by the kidneys in the body.
  • PEG-SPA The molecular weight of PEG in PEG-SPA and mPEG-SPA is small, and the plasma clearance rate is relatively fast, so a larger dose is required to obtain significant platelet proliferation activity.
  • the amount of small modified products (such as diPEG-rh-6, triPEG-rML-6, polyPEG-rhIL-6) is more, and the molecular weight is continuously distributed, which is unfavorable for subsequent purification and identification and quality control.
  • PEG-SPA is a bifunctional modifier that also causes coupling between protein molecules.
  • mPEG2-NHS As the PEG modification reagent, the conditions of the modification reaction need to be optimized to obtain the optimal yield and the optimal physical and chemical properties and biological activity of the modified product.
  • the reaction of mPEG2-NHS with protein molecules can be briefly expressed as:
  • the mPEG2-NHS molecule has a faster hydrolysis rate in aqueous solution. At pH 8.0, the half-life is only 4.9 minutes at 25 °C, so the modification reaction is almost completed in 45 minutes, and the residual mPEG2-NHS molecule is less than 0.1%.
  • the modifier reacts with the amino group, it also reacts ineffectively with the water molecule. Therefore, it is necessary to optimize the reaction conditions, reduce the ineffective reaction, and increase the modification yield. The following optimizations were carried out on three important conditions: the reaction system ' ⁇ , reaction time, molar ratio of modifier to protein.
  • the rhIL-6 samples with the required purity were divided into five groups.
  • the pH of the buffer was adjusted to 7.5, 8.0, 8.5, 9.0, and 9.5, respectively, and the same amount of mPEG2-NHS was added, mixed, and reacted in a 25-inch water bath for 45 minutes. Sampling was performed by SDS-PAGE electrophoresis, scanning imaging, and the ratio of various products was calculated. See Table 4 for the balance. Table 4. Effect of mPEG2-NHS modification of rhIL-6 at different pH conditions
  • reaction system can obtain higher yield of single modified PEG-rh-6 at pH above 9.0.
  • the rhIL-6 samples with the required purity were divided into 5 groups, the pH was adjusted to 9.0, and the protein concentration was adjusted to 0.8 mg/ml.
  • Each of the mPEG2-NHS was added so that the molar ratio of rhIL-6 to the modifier was 1:1, 1:3, 1:5, 1:10, 1:20, and the mixture was mixed and reacted in a 25 ° C water bath for 45 minutes.
  • SDS-PAGE detection, scanning imaging, and calculation of the ratio of products of different degrees of modification to the total modified product The results are shown in Table 5.
  • the molar ratio of rhIL-6 to the modifier is between 1:1 and 1:3, and the main component in the modified product obtained is a single modified product, ie monoPEG- rhIL-6, the product is higher, which is conducive to subsequent purification.
  • Sample pure rhIL-6, SDS-PAGE purity greater than 95%, protein concentration between 0.5 ⁇ 1mg/ml, pH 9.0, buffer PB, no other amino-containing compound decoration reagent: mPEG2-NHS MW20kDa, -20 °C low temperature dry preservation
  • hIL-6 water bath heated to 25 ° C weigh 1 to 2 times the total amount of rhIL-6 mPEG2-NHS, loaded: into a dry clean sterile pyrogen-free container;
  • hIL-6 is poured into a container containing mPEG2-NHS, rapidly mixed to completely dissolve mPEG2-NHS, and reacted in a water bath at 25 ° C for 45 minutes; add glycine to 0.45 M to terminate the reaction; c, after the reaction is completed, Store at 4 ° C, sample and test, and other samples to be purified by column chromatography.
  • i and j are integers of from 100 to 1000, and the sum of i and j is such that the molecular weight of the mPEG moiety of the conjugate is from 15,000 to 30,000, preferably 20,000, and the amino group of -NH-IL-6 in the reaction structure is a Lys residue. Side chain amino group.
  • the finishing process is as follows:
  • Modification reagent mPEG2-NHS MW20kDa, -20 °C dry storage
  • IL-6 is poured into a container containing mPEG2-NHS, and rapidly mixed to completely dissolve mPEG2-NHS, and reacted in a water bath at 25 ° C for 45 minutes;
  • the three batches of the product obtained by the method of the first method were uniformly mixed, and desalted and exchanged with a G-25 gel column equilibrated to H5.0 with 10 mM acetate buffer, and the sample buffer (buffer formulation '. Na2HPO 12H2O, 15.04 g/L; NaH2P04 2H20, 1.25 g L; NaCl 8.77 g/L.
  • the target sample separated by Superdex 200 gel filtration chromatography column was tested for purity by SDS-PAGE.
  • the results showed that the single modified PEG-IL-6 (monoPEG-IL-6) contained in the sample after this purification contained More than 85% (see lane 1 of Figure 1), the total amount of PEG-IL-6 with various degrees of modification is greater than 95%, meeting or exceeding the quality requirements of other PEG-modified protein peptides at home and abroad.
  • the above purification steps should be carried out under aseptic and pyrogen-free conditions to ensure that the products meet the relevant requirements of the national biochemical drugs.
  • PEG-rhIL-6 was prepared as described above
  • mice were randomly divided into 10 groups of 5 each. On day 1.-5, each mouse was injected subcutaneously into the test sample (PEG-IL-6 prepared according to the method of the foregoing examples) 0.5 ml; On the third day of injection of the test sample, each mouse was intraperitoneally injected with 2 mg of cyclophosphamide per day for 3 consecutive days; ⁇ before the first day of injection of the test sample and on days 8-17, every day from the tail of each mouse was licked. () ul, diluted 6 times with • Cell-DYN1600 instrument to measure the number of platelets.
  • test sample IL-6 (0.20 ug/ml) 0.5 ml per day; O.lug/only
  • test sample IL-6 (2ug/ml) 0.5ml per day; l.Oug/only
  • A4 subcutaneous injection test-risk sample IL-6 (5ug/ml) 0.5ml per day; 2.5ug/only
  • the drug group was compared with the control group (the previous data was analyzed):
  • the effective dose range of (PEG-IL-6) is in the range of 0.01 ug to 0.5 ug per mouse.
  • Bl (PEG-IL-6) is much less than A4 (IL-6), and can be reduced by up to 250 times, and the amount of the drug used is greatly reduced.
  • [Test Example 2] Pharmacodynamic test of PEG-IL-6 on cyclophosphamide-induced thrombocytopenia in mice and Beagle dogs
  • mice and Beagle dogs Two experimental animal models of hematopoietic damage caused by cyclophosphamide were used: mice and Beagle dogs, respectively, with three doses of high, medium, and four doses.
  • the positive control substance was a commercially available IL-11 drug (Jijufen, Yixing et al, 3 mg/branch).
  • the results showed that the duration of thrombocytopenia in each dose group was shorter than that of the model group, and the recovery was faster, and the degree of thrombocytopenia was higher than that of the model.
  • the light weight of the group indicates that the product can significantly increase the number of platelets in two animal models, reduce the degree of thrombocytopenia, shorten the duration of thrombocytopenia, and accelerate the recovery rate.
  • the dose used is much lower than that of the positive drug.
  • this product can also temporarily increase canine leukocytes and lymphocytes, and has no obvious effect on red blood cells, hemoglobin and reticulocytes.
  • mice The subcutaneous, tail vein and intraperitoneal injection of PEG-IL-6 prepared according to the above examples is equivalent to 1000 doses of general clinical dose (15 g/d, 0.3 g/kg). Times. After continuous observation for 14 days, no poisoning reaction was observed. The LD 50 >400 ( ⁇ g /kg) in the subcutaneous, tail vein and intraperitoneal injection of mice.
  • Beagle dog chronic toxicity test The three doses of PEG-IL-6 of the invention are large, medium and small (30.0, 12.0, 6.0 ⁇ ⁇ .1 - 1 ). The Beagle dog is injected subcutaneously every day for 32 consecutive days. Observed for 15 days. The results showed that subcutaneous injection of this product is less than 12.0 ⁇ ⁇ /] 3 ⁇ 4 is a safe dose for Beagle dogs.
  • Rats were injected subcutaneously with 125 PEG-rhIL-6. Its metabolism accords with the one-compartment distribution model, the distribution phase half-life is 1.4 ⁇ 5.1h, the elimination phase half-life is 58.3 ⁇ 236.5h, the peak time is 7.9 ⁇ 13.3h, and the body retention time is 42 ⁇ 52h.
  • Rats were injected intravenously with 125 I-PEG-rhIL-6. Its metabolism accords with the two-compartment distribution model, the distribution phase half-life $ ⁇ t 1/2 ( ⁇ ) is about ll ⁇ 2.5h, and the elimination phase half-life t 1/2 ( ⁇ ) is 13 ⁇ 18h.
  • Beagle dogs were injected subcutaneously with 125 I-PEG-rhIL-6.
  • Three dose groups were 2C ⁇ g/kg, 10 g/kg, 5 g/kg, and blood was collected from the forelimb vein after administration.
  • the results showed that the metabolism accorded with the one-compartment distribution model.
  • the half-life of the distribution phase of subcutaneous injection was 0.1 ⁇ 2.2h
  • the elimination phase half-life was 70.8 ⁇ 247.lh
  • the peak time was 0.9 ⁇ 10.4h
  • the body retention time was 69.7 ⁇ 91.7h.
  • interleukin-6 modified by PEG alone is greatly improved, its in vivo half-life is long, serum clearance rate is low, the dosage and frequency of use are greatly reduced, and side effects are greatly reduced. It can not only be convenient for patients to use, reduce the cost of use, but also improve the safety of use and greatly reduce the suffering of patients.
  • the single modified human interleukin-6 polyethylene glycol conjugate of the invention has good homogeneity, can meet the requirements of safe, effective and quality control of clinical drugs, and can be produced on a large scale with good application prospect.

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Abstract

A interleukin-6 polyethylene glycol conjugate and its preparing method and medical compositions comprising the conjugate and pharmaceutically acceptable excipients. The conjugate according to the invention is used for producing medicines treating thrombocytopenia, chemotherapy adjuvants or medicines enhancing immunity. The mono-PEG-modified IL-6 enhances markedly biostability, has longer in vivo half life and lower plasma clearance compared with unmodified IL-6, resulting in a great decrease in frequency and dose of administration as well as side effects. The mono-PEG-modified IL-6 according to the present invention can reach medicinal standards due to its good uniformity.

Description

白细胞介素 -6聚乙二醇结合物及其制备方法和应用 扶术领域  Interleukin-6 polyethylene glycol conjugate and preparation method and application thereof
本发明涉及一种白细胞介素- 6聚乙二醇结合物；  The present invention relates to an interleukin-6 polyethylene glycol conjugate;
本发明还涉及白细胞介素 -6聚乙二醇结合物的制备方法和应用。 背景技术  The invention also relates to methods and uses for the preparation of interleukin-6 polyethylene glycol conjugates. Background technique
白细胞介素 -6 ( Interleukin 6, 筒称 IL-6 )又名干扰素 β2, 是一种多功能的 细胞因子。 IL-6主要作用于免疫***， 可以促进 Τ细胞和神经细胞的分化， 激发 Τ细胞、 干细胞的生长， 诱导多核细胞的分化， 从而促进血小板的生产。 IL-6的医药用途主要有 3个方面： （1 )对因化疗、放疗引起的血小板降低有一 定的疗效， 曾一度作为血小板制剂的代用品，在用于抗癌剂导致的造血功能不 全方面寄予了很大的期望； （2 )可增强癌症患者的免疫力， 清除癌症病人手术 治疗后体内残留的瘤细胞，预防恶性肿瘤的复发； ( 3 ) IL-6可以增强体液与细 胞免疫， 对一部分免疫功能缺陷病人有治疗作用。 过去对 IL-6在治疗血小板 减少症方面的研制开发已进行到了 II期临床实验阶段， 但由于 IL-6在血浆中 的半衰期太短、 使用剂量较大， 易引起不良反应.（如发热、 头疼、 血清白蛋白 下降）， 未能实现实际应用。  Interleukin 6, also known as interferon beta-6, is a multifunctional cytokine. IL-6 mainly acts on the immune system, promotes the differentiation of sputum cells and nerve cells, stimulates the growth of sputum cells and stem cells, induces the differentiation of multinucleated cells, and promotes platelet production. There are three main aspects of the use of IL-6: (1) It has a certain effect on the reduction of platelets caused by chemotherapy and radiotherapy. It was once used as a substitute for platelet preparations, and it is used in the treatment of hematopoietic insufficiency caused by anticancer agents. It has great expectations; (2) It can enhance the immunity of cancer patients, eliminate the residual tumor cells in the cancer patients after surgery, and prevent the recurrence of malignant tumors; (3) IL-6 can enhance humoral and cellular immunity, Some patients with immunodeficiency have a therapeutic effect. In the past, the development of IL-6 in the treatment of thrombocytopenia has progressed to the phase II clinical trial stage, but because the half-life of IL-6 in plasma is too short and the dosage is large, it is easy to cause adverse reactions (such as fever, Headache, serum albumin decreased), failed to achieve practical application.
聚乙二醇（ polyethylene glycol, 简称 PEG )可以与蛋白质和多肽类药物形 成结合物。 经过 PEG修饰的蛋白质和多肽类药物， 可改变蛋白质药物的性质， 如增加此类药物的溶解度和稳定性， 减弱或消除免疫原性、抗原性和毒性，提 高药物的治疗指数， 扩大临床用药范围， 以及改善药物的体内药动学性质， 延 长药物在体内的半衰期等。聚乙二醇类务饰剂的药代动力学性质因它们所 饰 的蛋白质的性质、修饰产物的相对分子量和给药方式而不同。聚乙二醇对蛋白 和多肽的修饰途径主要有氨基修饰 （包括 Ν端氨基的酰化修饰、 赖氨酸侧链 氨基的酰化修饰、 Ν端氨基的烷基化修饰）、 羧基修饰、 琉基修飾等。 因为蛋 白或多肽结构中存在多个氨基，结构复杂多样，所以控制和确定修饰程度及修 饰位点一直是蛋白和多肽的聚乙二醇修饰中的难点。在多肽的聚乙二醇化过程 中还发现很多其他问题： 比如（1 )蛋白盾的 tyrosine残基的酰氨化还导致修 饰产物的生物活性降低； （2 )有一些聚乙二醇化蛋白极其不稳定， 完全没有药 用价值； （3 )修饰过程的试剂也很难选择， 有一些在反应过程中用的试剂不能 合适地完成反应， 从而花费很长时间， 以致于在修饰前、 后的蛋白质变性， 或 者根本不进行反应； ( 4 )在生理 PH条件时水溶液中蛋白质的水解作用会倾向 于阻止蛋白质的 PEG化， 由此产生很多修饰困难。 Polyethylene glycol (PEG) can form a combination with proteins and peptide drugs. PEG-modified proteins and peptides can alter the properties of protein drugs, such as increasing the solubility and stability of such drugs, attenuating or eliminating immunogenicity, antigenicity and toxicity, increasing the therapeutic index of drugs, and expanding the scope of clinical use. , as well as improving the in vivo pharmacokinetic properties of the drug, prolonging the half-life of the drug in the body. The pharmacokinetic properties of polyethylene glycol-based finishes vary depending on the nature of the protein they are decorated with, the relative molecular weight of the modified product, and the mode of administration. The modification pathways of polyethylene glycol to proteins and peptides mainly include amino modification (including acylation modification of quinone amino group, acylation modification of lysine side chain amino group, alkylation modification of quinone amino group), carboxyl modification, hydrazine Base modification, etc. Because of the presence of multiple amino groups in the structure of a protein or polypeptide, the structure is complex and diverse, so controlling and determining the degree of modification and the site of modification has been a difficulty in the modification of polyethylene glycols of proteins and polypeptides. Many other problems have been discovered during the pegylation of peptides: for example, (1) the amidation of the tyrosine residue of the protein shield also leads to repair The biological activity of the decorative product is reduced; (2) some PEGylated proteins are extremely unstable and have no medicinal value at all; (3) the reagents for the modification process are also difficult to select, and some reagents used in the reaction process are not suitable. The reaction is completed to take a long time, so that the protein is denatured before or after the modification, or does not react at all; (4) The hydrolysis of the protein in the aqueous solution tends to prevent the PEGylation of the protein under physiological pH conditions. This creates a lot of modification difficulties.
为了克服这些困难， 研究者对蛋白药物的聚乙二醇化方面进行了大量的研 究工作 , 在对 IL-6的 PEG修饰研究上也取得了一些进展。  In order to overcome these difficulties, the researchers have done a lot of research on the PEGylation of protein drugs, and some progress has been made in the study of PEG modification of IL-6.
美国专利 US 5,264,209公开了一种 PEG-IL-6。使用了分子量为 4500、 10000 和 12000的 PEG对人 IL-6进行修饰，其中 PEG4500和 PEG12000为直链 PEG , bis-PEG5000 (总分子量 10000 )为分枝结构 PEG。 该专利中得到的修饰产物 为不同修饰度（二到九个 PEG修饰）的混合产物， 动物实验结果表明， 在相 同使用剂量的情况下（4 g~10mg/kg )其增生血小板活性高于未修饰的 IL- 6。 并且认为至少 2个， 最好超过 5个 PEG修饰才能保证修饰产物的体内活性。  U.S. Patent No. 5,264,209 discloses a PEG-IL-6. Human IL-6 was modified using PEG with molecular weights of 4500, 10000 and 12000, of which PEG4500 and PEG12000 were linear PEG and bis-PEG5000 (total molecular weight 10000) was branched PEG. The modified products obtained in this patent are mixed products with different degrees of modification (two to nine PEG modifications). Animal experiments show that the proliferation of platelets is higher than that of the same dose (4 g~10 mg/kg). Modified IL-6. It is also believed that at least 2, and preferably more than 5, PEG modifications are required to ensure the in vivo activity of the modified product.
Tsunoda S, Tsutsumi Y 等比较了 PEG ( PEG-5000 ) 修饰的 IL-6 ( MPEG- IL- 6 )和未修饰 IL-6 ( IL-6 )的体内血小板增生活性（ PEGylation of interleukin-6 Effectively increases Its Thrombopoietic Potency. Tsunoda S, Tsutsumi Y .Thrombopoietic and heamostasis; 77(1)168-173,1997 )„其以 PEG5000与 IL-6 反应而获得 MPEG-IL-6,其中 IL-6的 14个 Lys残基有 54%与 PEG发生偶联， 但却只表现出约 51%的 IL-6生物学活性。 药效学试验表明， 从笫 2天到第 7 天， 分别给小鼠皮下注射 IL-6和 MPEG-IL-6。 IL-6不仅增加了外周血小板计 数， 也增加了血浆 IgGl的水平。 与 IL-6相比， MPEG-IL-6在增生血小板时没 有增加 IgGl的水平。 MPEG-IL-6可显著的刺激 5-氟尿嘧啶处理小鼠的血小板 恢复，而 IL-6的作用可以忽略。 MPEG-IL-6的血浆半衰期比 IL- 6长约 100倍。  Tsunoda S, Tsutsumi Y and others compared the in vivo platelet proliferative activity of PEG ( PEG-5000 ) modified IL-6 ( MPEG- IL-6 ) and unmodified IL-6 ( IL-6 ) ( PEGylation of interleukin-6 Effectively increases Its Thrombopoietic Potency. Tsunoda S, Tsutsumi Y .Thrombopoietic and heamostasis; 77(1)168-173,1997 ) „It reacts with PEG5000 to IL-6 to obtain MPEG-IL-6, of which 14 Lys residues of IL-6 54% of the base was coupled to PEG, but only showed about 51% of IL-6 biological activity. Pharmacodynamic tests showed that mice were injected subcutaneously with IL-6 from day 2 to day 7. And MPEG-IL-6. IL-6 not only increased the peripheral platelet count, but also increased the level of plasma IgGl. Compared with IL-6, MPEG-IL-6 did not increase the level of IgGl in the proliferation of platelets. MPEG-IL -6 can significantly stimulate platelet recovery in 5-fluorouracil-treated mice, while the effect of IL-6 is negligible. MPEG-IL-6 has a plasma half-life of about 100-fold longer than IL-6.
Tsunoda S等后来又将一种可逆的氨基保护试剂 DMMAn用在了 IL-6的 PEG修饰中 ( Selective enhancement of thrombopoietic activity of PEGylated interle kin 6 by a simple procedure using a reversible amino- protective reagent. Br J Haematol. 2001 Jan;112(l):181-8 )。 依然以 PEG- 5000对 IL- 6进行修饰 , 该研 究中得到了分子量为 26，500的单修饰产物组分 Fr3 ,该組分尽管保留了较多的 体外活性， 但用其皮下注射小鼠发现其体内半衰期极短（消除相半衰期约 12 小时）， 体内滞留时间约 25小时， 与未^ ίι爹饰 IL-6差别不大， 其体内活性也明 显不及修饰度高的组分，并且其体内活性低于 50倍剂量的 IL-6。而用 DMMAn 作为氨基保护剂制备的单修饰 DmPEG-IL-6不但与单修饰 PEG-IL-6在药代动 力学参数和体内活性方面没有极显著的差异， 更是需要进行 3 步反应才能得 到， 制备工艺复杂。 Tsunoda S et al later used a reversible amino-protection reagent DMMAn in PEGylated interle kin 6 by a simple procedure using a reversible amino-protective reagent. Br J Haematol 2001 Jan;112(l):181-8). IL-6 was still modified with PEG-5000. In this study, a single modified product fraction, Fr3, with a molecular weight of 26,500 was obtained. This component, although retaining more in vitro activity, was found by subcutaneous injection of mice. Its half-life in vivo is extremely short (eliminating phase half-life is about 12 Hours), the residence time in the body is about 25 hours, which is not much different from the IL-6, and its activity in the body is also significantly lower than that of the highly modified component, and its activity in vivo is lower than 50 times the dose of IL-6. However, the single-modified DmPEG-IL-6 prepared by using DMMAn as an amino-protecting agent has not only a significant difference in the pharmacokinetic parameters and in vivo activity of the single-modified PEG-IL-6, but also requires a 3-step reaction to obtain The preparation process is complicated.
以上公开的 1 6的 PEG修饰产物大多是不同修饰位点和修饰度的产物的 混合物， 由于产物的不均一，无法进行有效的质量控制，难以保证安全、有效、 质量可控， 很难投入实际应用； 而极少情况下得到的单修饰 IL- 6的聚乙二醇 结合物却存在半衰期太短， 活性低， 疗效差， 产量低等缺陷。 目前本领域急需 开发出一种能克服现有产品缺点的， 均一性好、 符合临床用药安全、有效、 质 量可控要求、成本低廉、并能大规模生产作为药物使用的单修饰聚乙二醇化重 组人白细胞介素 -6。 发明内容  The PEG modified products of the above-mentioned 16 are mostly mixtures of products with different modification sites and degree of modification. Due to the heterogeneity of the products, effective quality control cannot be performed, and it is difficult to ensure safety, effectiveness, quality control, and it is difficult to put into practice. Application; In rare cases, the single modified IL-6 polyethylene glycol conjugate has shortcomings such as short half-life, low activity, poor efficacy, and low yield. At present, there is an urgent need in the art to develop a single modified PEGylation which can overcome the shortcomings of existing products, has good homogeneity, is safe, effective, quality controllable, low cost, and can be mass-produced as a drug. Recombinant human interleukin-6. Summary of the invention
本发明的第一个目的是提供一种白细胞介素 -6聚乙二醇结合物（下称 IL-6 聚乙二醇结合物或 PEG-IL-6 )。 该 IL- 6聚乙二醇结合物是由聚乙二醇对 IL- 6 进行共价单修饰得到的，其中每个白细胞介素 -6分子上共价结合一个聚乙二醇 分子， 聚乙二醇分子的分子量为 15000 ~ 30000。 .  A first object of the present invention is to provide an interleukin-6 polyethylene glycol conjugate (hereinafter referred to as IL-6 polyethylene glycol conjugate or PEG-IL-6). The IL-6 polyethylene glycol conjugate is obtained by covalently modifying the IL-6 by polyethylene glycol, wherein each interleukin-6 molecule is covalently bound to a polyethylene glycol molecule, polyethylene. The molecular weight of the diol molecule is from 15,000 to 30,000. .
本发明的白细胞介素 -6聚乙二醇结合物中，优选的聚乙二'醇对白细胞介素 -6进行修饰的位点在 IL-6分子的 Lys残基的侧链氨基或 IL-6分子的肽链 N末 端氣基。  In the interleukin-6 polyethylene glycol conjugate of the present invention, the preferred poly(diethylenediamine) modification of interleukin-6 at the side chain amino group or IL- of the Lys residue of the IL-6 molecule. 6-molecular peptide chain N-terminal gas group.
用于修饰的聚乙二醇分子可以为线型聚乙二醇分子，即在所述白细胞介素 -6的 Lys残基的侧链氨基或白细胞介素 -6分子的肽链 N末端氨基上连接一条 PEG链， 也可以为分枝聚乙二醇分子， 分枝聚乙二醇是指在活化基团上连接 两条或以上的 PEG链， 其分子量为两条链的分子量之和， 这两种聚乙二醇分 子因其空间结构不同而在对白细胞介素 -6分子的修饰中对结合位点的选择性 可能有所差异。  The polyethylene glycol molecule used for the modification may be a linear polyethylene glycol molecule, that is, on the side chain amino group of the Lys residue of the interleukin-6 or the N-terminal amino group of the peptide chain of the interleukin-6 molecule. Connecting a PEG chain, which may also be a branched polyethylene glycol molecule, branching polyethylene glycol means connecting two or more PEG chains on the activating group, and the molecular weight is the sum of the molecular weights of the two chains. The selectivity of the two polyethylene glycol molecules to the binding site in the modification of the interleukin-6 molecule may be different due to their different spatial structures.
对于选择分枝聚乙二醇修饰的 IL-6聚乙二醇结合物， 其具有式（I )所述 的结构： m-(-ocH2cH2)j- c - I For the selection of a branched polyethylene glycol modified IL-6 polyethylene glycol conjugate having the structure of formula (I): M-(-ocH 2 cH 2 )j- c - I
(CHj 4 (CHj 4
0 CH  0 CH
II /\  II /\
m~( CH2CH2j~ C— NH C— NH— IL-6 m~( CH 2 CH 2 j~ C— NH C— NH— IL-6
Ί l |  Ί l |
O  O
(I)  (I)
其中 m代表甲基， i、 j为 100_1000的整数， i、 j之和使结合物的聚乙二 醇分子的分子量为 15000 ~ 30000。  Wherein m represents a methyl group, i, j is an integer of 100-1000, and the sum of i and j causes the molecular weight of the conjugated polyethylene glycol molecule to be 15,000 to 30000.
作为本发明的优选实施方案， 可以作为修饰剂使用的 PEG分子为例如， 分枝结构的 mPEG2-NHS、 以及线型结构的 mPEG-aldehyde。  As a preferred embodiment of the present invention, the PEG molecule which can be used as a modifier is, for example, a branched structure of mPEG2-NHS, and a linear structure of mPEG-aldehyde.
通过在一定的温度、 PH和反应时间下，将 IL-6分子与聚乙二醇分子反应， 制备本发明的 IL- 6聚乙二醇结合物， 因此本发明也提供制备上述 IL-6的聚乙 二醇结合物方法， 该方法包括以下步驟：  The IL-6 polyethylene glycol conjugate of the present invention is prepared by reacting an IL-6 molecule with a polyethylene glycol molecule at a certain temperature, pH and reaction time, and thus the present invention also provides the preparation of the above IL-6. A polyethylene glycol conjugate method, the method comprising the steps of:
1 ) 白细胞介素 -6制备成蛋白浓度在 0.05 - 20mg/ml之间， pH为 6.5 ~ 10.0 的溶液；  1) Interleukin-6 is prepared as a solution having a protein concentration between 0.05 and 20 mg/ml and a pH of 6.5 to 10.0;
2 ) 将制备的白细胞介素 -6溶液与活化聚乙二醇反应，所述聚乙二醇的量 为白细胞介素 -6重量的 1― 100倍， 反应温度为 15 ~ 35°C , 时间为 5 ~ 100分 钟， 得到白细胞介素 -6聚乙二醇结合物；  2) reacting the prepared interleukin-6 solution with activated polyethylene glycol, the amount of the polyethylene glycol being 1-100 times the weight of the interleukin-6, the reaction temperature being 15 ~ 35 ° C, time For 5 to 100 minutes, an interleukin-6 polyethylene glycol conjugate is obtained;
3) 分离纯化获得的白细胞介素 -6聚乙二醇结合物，得到聚乙二纯单修饰 的白细胞介素- 6聚乙二醇结合物。  3) The obtained interleukin-6 polyethylene glycol conjugate was isolated and purified to obtain a polyethylene-pure-modified interleukin-6 polyglycol conjugate.
其中， 上迷方法步骤 1 ) 中的 IL-6的 SDS-PAGE纯度大于 95%, 并配成 蛋白浓度在 0.5 ~ lmg/ml之间， pH为 8.7 ~ 9.3的溶液。  Among them, the SDS-PAGE purity of IL-6 in step 1) is greater than 95%, and the solution has a protein concentration of 0.5 ~ lmg/ml and a pH of 8.7 ~ 9.3.
其中， 上述方法步骤 3 ) 中的纯化步 包括： 将步骤 2 )所得到的反应液 经 G-25凝胶过滤柱脱盐处理后， 再用阳离子交换层析柱进行初步分离， 最后 用 Superdex 200凝胶过滤柱纯化得单修饰产物。  The purification step in the step 3) of the above method comprises: desalting the reaction solution obtained in the step 2) through a G-25 gel filtration column, and then performing preliminary separation using a cation exchange chromatography column, and finally condensing with Superdex 200. The gel filtration column was purified to obtain a single modified product.
本发明也提供一种药物组合物， 将本发明的 IL-6聚乙二醇结合物添加药 学上可接受的辅料制备而成。  The present invention also provides a pharmaceutical composition prepared by adding the IL-6 polyethylene glycol conjugate of the present invention to a pharmaceutically acceptable adjuvant.
本发明还提供上述 IL- 6的聚乙二醇结合物在制备治疗血小板减少症的药 物、 放化疗辅助用药物、 免疫增强药物的应用。 The present invention also provides a polyethylene glycol conjugate of the above IL-6 for preparing a medicament for treating thrombocytopenia The application of drugs, radiotherapy and chemotherapy, and immunopotentiating drugs.
本发明所迷的 IL-6可有多种来源， 凡肽链结构与天然人 IL-6相近的同源 肽链， 无论是人工合成的， 还是通过原核和真核***表达的， 甚至是经过改造 的，都可以作为本发明 PEG修饰的原料,从而获得本发明的聚乙二醇修饰 IL-6。  The IL-6 of the present invention can be of various origins, and the homologous peptide chain having a peptide chain structure similar to that of natural human IL-6, whether synthetic or expressed by prokaryotic and eukaryotic systems, or even The modified polyethylene glycol modified IL-6 of the present invention can be obtained as a raw material for the PEG modification of the present invention.
本发明使用的修饰剂可为活化 PEG酯， 也可采用其它的方法将本发明所 述的 PEG与 IL-6的 Lys残基共价偶联。 包括采用具有其他种类活化基团的 PEG, 或对 IL-6肽链的 Lys残 目应位点也进行活化， 同样在本发明的范围 之内。  The modifier used in the present invention may be an activated PEG ester, and other methods may be used to covalently couple the PEG of the present invention to the Lys residue of IL-6. It is also within the scope of the invention to include the use of PEG having other types of activating groups, or to activate the Lys residue site of the IL-6 peptide chain.
由于每种蛋白的性盾和体内药代性质不同， PEG试剂分子量和性质各异， 蛋白的 PEG化方式是多种多样的，因此使蛋白药物的 PEG修饰变得异常复杂。 同时 PEG修饰反应需要高度特异并温和的反应条件。 根本无法预料一种蛋白 如何能进行成功的 PEG化而得到高产率的、 均一的目的修饰产物。 本发明方 法制得的产物中的单修饰 PEG-IL-6 ( monoPEG-IL-6 )含量能大于 85%。 而本 发明 IL-6的聚乙二醇结合物可以添加药学上可以接受的辅助性成分制备各种 制剂， 比如注射液， 冻干制剂等。  Due to the different nature of the sexual shield and in vivo pharmacokinetics of each protein, the molecular weight and properties of PEG reagents vary, and the PEGylation of proteins is varied, thus making the PEG modification of protein drugs extremely complicated. At the same time, PEG modification requires highly specific and mild reaction conditions. It is simply impossible to predict how a protein can be successfully PEGylated to yield a high yield, uniform target modified product. The single modified PEG-IL-6 (monoPEG-IL-6) content of the product obtained by the method of the present invention can be greater than 85%. Further, the polyethylene glycol conjugate of IL-6 of the present invention may be prepared by adding a pharmaceutically acceptable auxiliary component such as an injection solution, a lyophilized preparation or the like.
本发明的聚乙二醇单修饰的白细胞介素 -6, 由于选择了合适分子量的聚乙 二醇， 不但具有白细胞介素 -6的生理活性， 并且稳定性大大提高； 它的体内半 衰期长， 血清清除率低， 使用剂量和使用频率大为降低， 副作用也大大減小，. 各方面性质明显优于目前公开的白细胞介素 -6和单修饰白细胞介素 -6,既能方 便患者使用， 降低使用成本， 也提高了使用安全性， 能大大减轻患者的痛苦。 同时本发明提供的聚乙二醇单修饰的白细胞介素 -6的均一性好,便于进行质量 控制， 能达到临床用药安全、有效、质量可控的要求， 并能大规模生产， 优于 聚乙二醇多修饰的白细胞介素 -6，使得聚乙二醇化的重组人白细胞介素 -6的工 业化生产和实际应用成为可能， 具有极好的市场前景。 附图的简要说明  The polyethylene glycol mono-modified interleukin-6 of the invention not only has the physiological activity of interleukin-6, but also has greatly improved stability due to the selection of polyethylene glycol of a suitable molecular weight; its long half-life in vivo, The serum clearance rate is low, the dosage and frequency of use are greatly reduced, and the side effects are greatly reduced. The properties of each aspect are obviously superior to the currently disclosed interleukin-6 and single modified interleukin-6, which are convenient for patients to use. Reduce the cost of use, and also improve the safety of use, which can greatly reduce the suffering of patients. At the same time, the polyethylene glycol single modified interleukin-6 provided by the invention has good homogeneity, is convenient for quality control, can meet the requirements of safe, effective and quality control of clinical medication, and can be mass-produced, better than poly The multi-modified interleukin-6 of ethylene glycol makes the industrial production and practical application of PEGylated recombinant human interleukin-6 possible, and has an excellent market prospect. BRIEF DESCRIPTION OF THE DRAWINGS
图 1 PEG修饰 IL-6后的修饰产物及其纯化产物的 SDS-PAGE图谱； 其 中： 1和 9为蛋白分子量标准； 10和 13为 PEG修饰产物； 2 ~ 8、 11 ~ 12 及 15为经 SP Sepharose High Performance阳离子交换层析純化后的各洗脱组 分，其中 15为单修饰产物； 14为 Superdex 200凝胶过滤层析分离纯化后的单 修饰产物， 修饰产物的主带（MW60, 000 )大于 85%, 质谱分析表明该修饰 蛋白的分子量约为 46, 000, 说明只修饰上了一个 PEG20, 000分子； Figure 1 SDS-PAGE of the modified product of PEG modified IL-6 and its purified product; wherein: 1 and 9 are protein molecular weight standards; 10 and 13 are PEG modified products; 2 ~ 8, 11 ~ 12 and 15 are Each eluted group purified by SP Sepharose High Performance cation exchange chromatography 15 is a single modified product; 14 is a single modified product isolated and purified by Superdex 200 gel filtration chromatography. The main band of the modified product (MW60, 000) is greater than 85%. Mass spectrometry indicates that the molecular weight of the modified protein is about 46,000, indicating that only one PEG 20,000 molecules have been modified;
图 2 四种 PEG修饰的 rhIL-6在小鼠体内的生物学活性比较； 其中 PBS: 阴性对照； rhIL-6: 2.5ug/只； mPEG2-NHS: 0.05ug/只；  Figure 2 Comparison of the biological activities of four PEG-modified rhIL-6 in mice; PBS: negative control; rhIL-6: 2.5ug/mo; mPEG2-NHS: 0.05ug/only;
mPEG-aldehyde:1.0ug/只； PEG-SPA: l.Oug/只； mPEG-SPA:2.5ug/ ； mPEG-aldehyde: 1.0 ug/head; PEG-SPA: l.Oug/only; mPEG-SPA: 2.5 ug/;
图 3为 PEG-IL-6冻干制剂的制剂工艺流程图。 发明的具体实施方式  Figure 3 is a flow chart showing the preparation process of the PEG-IL-6 lyophilized preparation. DETAILED DESCRIPTION OF THE INVENTION
下面结合附图，通过对本发明较佳实施方式的详细描述，说明但不限制本 发明。  The invention will be described, but not limited, by the following detailed description of the preferred embodiments of the invention.
下面的实施例中所使用的材料如无特别说明， 均为市售购买。  The materials used in the following examples are commercially available unless otherwise stated.
PEG修饰 rhIL-6的工艺条件选择  Selection of process conditions for PEG modification of rhIL-6
1. 修饰试剂 （PEG )的筛选 1. Screening of modification reagents (PEG)
1.1 四种修饰试剂 （PEG )结构特点的比较  1.1 Comparison of structural characteristics of four modified reagents (PEG)
在进行正式动物试验前的研究阶段， 陆续试验了四种 PEG修饰试剂， 这 四种修饰试剂的各种特性见表 1。  Four PEG modification reagents were tested in the research stage prior to the formal animal test. The various characteristics of these four modification reagents are shown in Table 1.
表 1. 四种 PEG修饰剂的比较  Table 1. Comparison of four PEG modifiers
简称 mPEG2-NHS mPEG-aldehyde PEG-SPA mPEG-SPA 分子量（kDa ) 20 20 10 5  Abbreviation mPEG2-NHS mPEG-aldehyde PEG-SPA mPEG-SPA Molecular Weight (kDa) 20 20 10 5
北京凯正生物工 北京凯正生物工 生产厂商 美国 Nektar公司 韩国 SunBio公司  Beijing Kaizheng Biotech Beijing Kaizheng Biotech Manufacturer US Nektar Corporation Korea SunBio Corporation
程公司 程公司  Company
分子结构 分枝 线型 线型 线型 活化官能团 N-羟基琥珀酸 醛基 丙酸玻珀酰亚胺 丙酸琥珀酰亚胺 蛋白修饰位点 α或 ε氨基 ct或 ε氣基 ct或 ε氛基 α或 ε氣基 Molecular structure, branched line, linear linear activation functional group, N-hydroxysuccinic acid, aldehyde, propionate, glassylide, propionic acid, succinimide, protein modification site, α or ε amino ct or ε gas group, ct or ε Alpha or ε gas base
PEG的两个末端 Both ends of PEG
分枝结构，有两条分 对 Ν-末端氨基有  Branched structure, there are two pairs of Ν-terminal amino groups
都有活化官能  Activated function
子量均为 lOkDa的 较高的选择性，多 分子量较小，受空 特点 团， 一个 PEG分  The sub-quantity is higher selectivity of lOkDa, the multi-molecular weight is smaller, and the air-bearing characteristic group, a PEG fraction
PEG长链，表观分子 形成 Ν-末端修- 间位阻效应最小。  PEG long chain, apparent molecular formation Ν-terminal repair - steric effect is minimal.
子可以同两个位  Child can have the same two bits
量很大。 饰。  A lot. Decoration.
点反应。 四种 饰剂的分子式如下： Point reaction. The molecular formulas of the four fragrances are as follows:
Figure imgf000008_0001
Figure imgf000008_0001
Figure imgf000008_0002
Figure imgf000008_0002
1.2 四种 PEG修饰 rhIL-6后的分子量分布，体外活性及小鼠体内药效比较  1.2 Molecular weight distribution, in vitro activity and drug efficacy of four PEG modified rhIL-6
分别用这四种 PEG修饰试剂修饰纯化后的 rhIL-6纯品， 并对修饰后产物 进行纯化，去除未被修饰的 rhIL-6和修饰副产物。分别测定了分子量分布情况， 体外活性保留和小鼠体内药效。  The purified rhIL-6 pure product was modified with these four PEG modification reagents, and the modified product was purified to remove unmodified rhIL-6 and modified by-products. The molecular weight distribution, in vitro activity retention and drug efficacy in mice were determined.
1.2.1四种 PEG修饰 rhIL-6后的分子量分布 1.2.1 Molecular Weight Distribution of Four PEG Modified rhIL-6
用 SDS- PAGE测定 PEG修饰 rhIL-6的分子量分布， 电泳后用凝胶扫描成 像***计算各条带的分子量和含量。 由于 PEG长链是一种线型大分子， 在 SDS-PAGE 中的表观分子量通常是其真实分子量的 2-4倍， 因此修饰后的 rhIL-6的分子量不能准确计算， 表 2中所列分子量都是估计的表观分子量。 四种 PEG修饰 rhIL- 6后的表观分子量分布 The molecular weight distribution of PEG-modified rhIL-6 was determined by SDS-PAGE. After electrophoresis, the molecular weight and content of each band were calculated by gel scanning imaging system. Since the PEG long chain is a linear macromolecule, the apparent molecular weight in SDS-PAGE is usually 2-4 times its true molecular weight, so the molecular weight of the modified rhIL-6 cannot be accurately calculated, as listed in Table 2. Molecular weights are estimated apparent molecular weights. Apparent molecular weight distribution after four PEG modified rhIL-6
Figure imgf000009_0001
Figure imgf000009_0001
1.2.2 四种 PEG修饰 rhIL-6后的体外活性比较 1.2.2 Comparison of in vitro activities of four PEG modified rhIL-6
与 rhIL-6相似， PEG修饰后的 rhIL-6的体外活性同样用 7TO1细胞 MTT 法测定。 将阳性对照 rhIL-6的比活性定为 100%, 各种 PEG修饰后 rhIL-6的 比活性与^ }目比， 计算百分含量。 各种文献报道和我们的试验都表明， 蛋白在 PEG修饰之后，由于 PEG长链对蛋白表面的活性位点产生了遮蔽和阻挡作用， 限制了蛋白与受体、 辅酶、 辅基等的结合， 因此酶活性或生物学活性都有不同 程度的降低。 修饰后的 rhIL-6的比活性都低于修饰前。 结果见表 3。 四种 PEG修饰 rhIL-6后的体外活性比较  Similar to rhIL-6, the in vitro activity of PEG-modified rhIL-6 was also determined by the 7TO1 cell MTT assay. The specific activity of the positive control rhIL-6 was determined to be 100%, and the specific activity of rhIL-6 after various PEG modification was compared with the ratio of ^}, and the percentage was calculated. Various literature reports and our experiments have shown that after PEG modification, the long chain of PEG blocks and blocks the active site on the surface of the protein, which limits the binding of proteins to receptors, coenzymes, prosthetic groups, etc. Therefore, the enzyme activity or biological activity has a different degree of reduction. The specific activity of the modified rhIL-6 was lower than that before the modification. The results are shown in Table 3. Comparison of in vitro activities of four PEG modified rhIL-6
Figure imgf000009_0002
Figure imgf000009_0002
1.2.3 四种 PEG修饰 rML-6后的小鼠体内生物学活性比较 1.2.3 Comparison of biological activities of four PEG-modified mice after rML-6
由 MTT法测定的体外细胞活性可以间接反映 rhIL-6和 PEG修饰 rhIL-6的 体内活性，但是其血小板增生活性仍需要用动物试验来证实。我们用注射了环 磷酰胺的小鼠模拟放化疗后血小板减少， 注射 rML-6和 PEG修饰 rhIL-6, 监 测血小板的恢复情况。 具体试验方案如下： 第 1-5天每天每只小鼠皮下注射试 验样品 0.5ml; 从注射试-险样品的第三天起每天每只小鼠腹腔注射环磷酰胺 2mg, 连续注射 3天； 于第一天注射试脸样品前和笫 8 17天， 每天自每只小 鼠尾部采血 lOul，稀释 6倍， 用 Cell-DYN1600仪测血小板数。 由于四种 PEG 修饰的 rhlL- 6是先后进行的动物试验，因此我们用相对比值来反应血小板的数 量。将第一天注射试臉样品前的血小板数定为 100%, 计算其后的血小板数与 第一天的相对百分比， 进行统计学分析。 为了便于比较， 我们取每种 PEG修 饰 rhIL-6中有显著性差异的剂量组中的最低剂量和 rML-6中有显著性差异的 最低剂量列表比较。 结果见图 2。 The in vitro cell viability determined by the MTT assay can indirectly reflect the in vivo activity of rhIL-6 and PEG-modified rhIL-6, but its platelet proliferative activity still needs to be confirmed by animal experiments. We used cyclophosphamide-injected mice to simulate thrombocytopenia after chemoradiotherapy, and injected rML-6 and PEG to modify rhIL-6 to monitor platelet recovery. The specific test protocol is as follows: On the 1-5th day, each mouse is injected subcutaneously with 0.5 ml of the test sample per day; from the third day of the injection test-risk sample, each mouse is intraperitoneally injected with 2 mg of cyclophosphamide per day for 3 consecutive days; Before the first day of injection of the test sample and 笫8 17 days, every day from each small The tail of the rat was collected lOul, diluted 6 times, and the number of platelets was measured by Cell-DYN1600 instrument. Since the four PEG-modified rhLL-6 are successive animal tests, we used relative ratios to reflect the number of platelets. The number of platelets before the test sample was injected on the first day was determined as 100%, and the relative percentage of platelets after the first day was calculated for statistical analysis. For ease of comparison, we compared the lowest dose in the dose group with a significant difference in each PEG-modified rhIL-6 and the lowest dose list in the rML-6 with a significant difference. The results are shown in Figure 2.
1.3 结论 1.3 Conclusion
综合分析以上结果， 我们得出以下结论：  Based on a comprehensive analysis of the above results, we draw the following conclusions:
( 1 ) rhIL-6经过各种 PEG剂修饰后， 生物学活性都得到了不同程度的保 留。 但是体外活性和体内活性并不相符， mPEG2-NHS体外活性只有 5~15% , 但只需要 0.05 g/只的剂量就可以达到 2.5μβ/只小鼠 rhIL-6的血小板增生活性， 可以相应的认为修饰后的活性（体内）较修饰前提高了 50倍。 这可能是 PEG 修饰使得药物在血浆内的清除率降低，保留时间延长， 同时生物利用度得以提 高， 因此药物可以在体内作用更长的时间。 同时由于 PEG修饰还可以延緩蛋 白酶的降解， .在另一方面保持了 rML- 6在体内的活性。 (1) After the modification of rhIL-6 by various PEG agents, the biological activities were preserved to varying degrees. Activity in vitro and in vivo activity but does not match, mPEG2-NHS in vitro activity of only 5 to 15%, but only 0.05 g / dose can be achieved only 2.5μ β / rhIL-6 mice platelet proliferative activity may be appropriate It is considered that the modified activity (in vivo) is 50 times higher than that before the modification. This may be because the PEG modification reduces the clearance of the drug in the plasma, prolongs the retention time, and increases the bioavailability, so the drug can act in the body for a longer period of time. At the same time, PEG modification can also delay the degradation of proteases, and on the other hand maintain the activity of rML-6 in vivo.
( 2 )几种 PEG修饰试剂从体内活性来看， mPEG2-NHS明显优于其他三 种。 参考各种文献和专利资料， 我们认为， 分子量为 20kDa的 mPEG2-NHS 由于较大的分子量和分枝状结构产生的空间位阻效应 ,在修饰反应中只能接近 rhIL-6表面的可修饰氨基位点； 并且， 在结合了一个这样的分子后， 其他的 PEG分子也因为空间位阻的关系， 很难再接近 rhIL-6分子， 因此能够获得主 要以单修饰异构体 ( monoPEG-rhIL-6 )为主的修饰产物。 虽然在体外活性测定 中， mPEG2-NHS结合在蛋白表面两条 lOkDa的 PEG长链阻碍了 rhIL-6和细 胞表面受体的结合， 使得活性只有 rhIL-6的 5~15%。 但是其在体内延长保留 时间， 延緩蛋白酶水解的作用要优于其他三种 PEG。  (2) From the in vivo activity of several PEG modification reagents, mPEG2-NHS is significantly better than the other three. With reference to various literatures and patents, we believe that mPEG2-NHS with a molecular weight of 20 kDa can only be modified to a modified amino group on the surface of rhIL-6 due to the steric hindrance effect of larger molecular weight and branched structure. And; after combining one such molecule, other PEG molecules are also difficult to access the rhIL-6 molecule due to steric hindrance, so that a single modified isomer can be obtained (monoPEG-rhIL- 6) The main modified product. Although in the in vitro activity assay, mPEG2-NHS binds to two lOkDa PEG long chains on the surface of the protein, which blocks the binding of rhIL-6 to the cell surface receptor, making the activity only 5-15% of rhIL-6. However, it prolongs the retention time in vivo and delays the hydrolysis of proteases over the other three PEGs.
( 3 ) PEG-aldehyde对于蛋白的 N-末端氨基酸有较高的选择性， 修饰的产 物多为 N-末端单修饰，体外活性也较好，体内活性要低于 mPEG2-NHS。但由 于其 PEG为直链线型分子， 表观分子量较分枝型小， 在体内易被肾脏清除。  (3) PEG-aldehyde has high selectivity for the N-terminal amino acid of the protein, and the modified product is mostly N-terminal single modification, and the activity in vitro is also better, and the activity in vivo is lower than that of mPEG2-NHS. However, because its PEG is a linear linear molecule, its apparent molecular weight is smaller than that of a branched type, and it is easily cleared by the kidneys in the body.
( 4 ) PEG-SPA和 mPEG-SPA中 PEG的分子量较小， 血浆清除速率都比 较快， 因此需要较大的剂量才能获得显著的血小板增生活性。 同样， 因为分子 量较小， 多修饰的产物（如 diPEG- rhIL-6、 triPEG- rML- 6、 polyPEG-rhIL-6 ) 较多， 分子量呈连续分布， 对于后续纯化鉴定以及质量控制都不利。 PEG-SPA 是双官能团的修饰剂， 还会引起蛋白分子之间的偶联。 (4) The molecular weight of PEG in PEG-SPA and mPEG-SPA is small, and the plasma clearance rate is relatively fast, so a larger dose is required to obtain significant platelet proliferation activity. Again, because of the molecule The amount of small modified products (such as diPEG-rh-6, triPEG-rML-6, polyPEG-rhIL-6) is more, and the molecular weight is continuously distributed, which is unfavorable for subsequent purification and identification and quality control. PEG-SPA is a bifunctional modifier that also causes coupling between protein molecules.
综上所述， 我们认为选择 mPEG2-NHS ( 20kDa )作为 rhIL-6的 PEG修饰 试剂较为理想，不仅能达到我们预想的修饰后对 rhIL-6药效和药代动力学的改 变， 同时修饰产物的质量易于控制， 可以保证最终制品的品质和效果。  In summary, we believe that the choice of mPEG2-NHS (20kDa) as a PEG modification reagent for rhIL-6 is ideal, not only to achieve the desired modification of rhIL-6 pharmacodynamics and pharmacokinetics, but also to modify the product. The quality is easy to control and guarantees the quality and effectiveness of the final product.
2. rhIL-6的 PEG化学修饰工艺的优化 2. Optimization of PEG chemical modification process of rhIL-6
在选定了 mPEG2- NHS作为 PEG修饰试剂后，还需要对修饰反应的条件进 行优化， 以获得最优的收率和修饰产物的最优理化特性和生物活性。 mPEG2-NHS与蛋白质分子的反应可以简要的表示为：  After selecting mPEG2-NHS as the PEG modification reagent, the conditions of the modification reaction need to be optimized to obtain the optimal yield and the optimal physical and chemical properties and biological activity of the modified product. The reaction of mPEG2-NHS with protein molecules can be briefly expressed as:
Figure imgf000011_0001
Figure imgf000011_0001
mPEG2-NHS分子在水溶液中的水解速率较快， pH8.0, 25°C时半衰期只 有 4.9分钟， 因此修饰反应在 45分钟内就基本完成， 残余的 mPEG2- NHS分 子少于 0.1%。 修饰剂在与氨基反应的同时， 也会和水分子发生无效反应， 因 此需要优化反应条件， 减少无效反应， 提高修饰收率。 下面从反应体系 ' ρΗ、 反应时间、 修饰剂与蛋白的摩尔比三个重要的条件上进行筛选优化。  The mPEG2-NHS molecule has a faster hydrolysis rate in aqueous solution. At pH 8.0, the half-life is only 4.9 minutes at 25 °C, so the modification reaction is almost completed in 45 minutes, and the residual mPEG2-NHS molecule is less than 0.1%. When the modifier reacts with the amino group, it also reacts ineffectively with the water molecule. Therefore, it is necessary to optimize the reaction conditions, reduce the ineffective reaction, and increase the modification yield. The following optimizations were carried out on three important conditions: the reaction system 'ρΗ, reaction time, molar ratio of modifier to protein.
2.1反应体系 ρΗ的优化 2.1 Reaction System Optimization of ρΗ
取纯度符合要求的 rhIL-6样品， 分为五组， 分别将緩冲液 pH调整为 7.5、 8.0、 8.5、 9.0、 9.5, 各加入等量 mPEG2-NHS, 混匀， 25Ό水浴反应 45min。 取样做 SDS-PAGE电泳， 扫描成像， 计算各种产物的比率。 结杲见表 4。 表 4. mPEG2-NHS在不同 pH条件下修饰 rhIL-6的效果 The rhIL-6 samples with the required purity were divided into five groups. The pH of the buffer was adjusted to 7.5, 8.0, 8.5, 9.0, and 9.5, respectively, and the same amount of mPEG2-NHS was added, mixed, and reacted in a 25-inch water bath for 45 minutes. Sampling was performed by SDS-PAGE electrophoresis, scanning imaging, and the ratio of various products was calculated. See Table 4 for the balance. Table 4. Effect of mPEG2-NHS modification of rhIL-6 at different pH conditions
pH 〜20kDa 〜80 ~100kDa  pH ~20kDa ~80 ~100kDa
(rhIL-6) (monoPEG-rhIL-6 ) (diPEG-rhIL-6)  (rhIL-6) (monoPEG-rhIL-6 ) (diPEG-rhIL-6)
7.5 88% 12%  7.5 88% 12%
8.0 65% 30% 5%  8.0 65% 30% 5%
8.5 71% 28% 1%  8.5 71% 28% 1%
9.0 42% 44% 12%  9.0 42% 44% 12%
9.5 40% 46% 13% 反应体系在 pH 大于 9.0 都能获得较高收率的单修饰 PEG- rhIL-6 9.5 40% 46% 13% The reaction system can obtain higher yield of single modified PEG-rh-6 at pH above 9.0.
( monoPEG-rhIL-6 ), 因为较高的 pH使氨基更具有亲核性， 易于与亲电性的 修饰剂结合。 但过高的 pH可能使 rhIL-6不稳定， 因此没有使用更高的 pH。 考虑到 pH9.0和 9.5下收率相似，我们选取 H9.0为修饰反应的最佳 pH条件。 2.2反应时间的优化 (monoPEG-rhIL-6), because the higher pH makes the amino group more nucleophilic and easily binds to the electrophilic modifier. However, too high a pH may make rhIL-6 unstable, so no higher pH is used. Considering the similar yields at pH 9.0 and 9.5, we chose H9.0 as the optimum pH condition for the modification reaction. 2.2 Optimization of reaction time
取纯度符合要求的 rhIL-6样品，将 pH调整为 9.0,加入 mPEG2-NHS, 混 匀， 25。C水浴反应。 在加入修饰剂后的 25min、 lh、 2h、 4h、 7h取样作 FPLC 检测，积分计算被修饰的 rhIL-6和未被修饰的 rhIL-6的比率。 由于随 pH升高 反应速率加快， 因此反应开始 25分钟后 98%以上的 mPEG2-NHS就已反应或 7 解，被修饰 rhIL-6的比率就保持不变了。我们将反应时间控制在 30-45分钟 就足以完成整个反应过程。  Take the rhIL-6 sample with the required purity, adjust the pH to 9.0, add mPEG2-NHS, and mix well, 25. C water bath reaction. Samples were taken at 25 min, 1 h, 2 h, 4 h, and 7 h after addition of the modifier for FPLC detection, and the ratio of modified rhIL-6 to unmodified rhIL-6 was calculated by integration. Since the reaction rate increases with increasing pH, more than 98% of mPEG2-NHS reacts or resolves after 25 minutes from the start of the reaction, and the ratio of modified rhIL-6 remains unchanged. We can control the reaction time to 30-45 minutes to complete the entire reaction process.
2.3蛋白与修饰剂比例的优化  2.3 Optimization of protein to modifier ratio
取純度符合要求的 rhIL-6样品， 样品分为 5組， 将 pH调整为 9.0， 蛋白 浓度调整到 0.8mg/ml。各加入 mPEG2-NHS,使得 rhIL-6与修饰剂的摩尔比分 別为 1:1、 1:3、 1:5、 1:10、 1:20, 混匀， 25°C水浴反应 45min。 SDS-PAGE检 测， 扫描成像， 计算不同修饰度的产物所占总修饰产物的比率。 结果见表 5。  The rhIL-6 samples with the required purity were divided into 5 groups, the pH was adjusted to 9.0, and the protein concentration was adjusted to 0.8 mg/ml. Each of the mPEG2-NHS was added so that the molar ratio of rhIL-6 to the modifier was 1:1, 1:3, 1:5, 1:10, 1:20, and the mixture was mixed and reacted in a 25 ° C water bath for 45 minutes. SDS-PAGE detection, scanning imaging, and calculation of the ratio of products of different degrees of modification to the total modified product. The results are shown in Table 5.
表 5不同比例的蛋白与修饰剂修饰 rhIL-6的效果  Table 5 Effect of different ratios of protein and modifier on rhIL-6
比例 monoPEG-rML-6 diPEG-rhIL-6 triPEG-rhIL-6 1:1 89% 11% Proportional monoPEG-rML-6 diPEG-rhIL-6 triPEG-rhIL-6 1:1 89% 11%
1:3 67% 32% 1%  1:3 67% 32% 1%
1:5 52% 43% 5%  1:5 52% 43% 5%
1:10 23% 56% 21%  1:10 23% 56% 21%
1:20 5% 33% 62% 从表 5可见, rhIL-6与修饰剂的摩尔比在 1:1到 1:3之间， 获得的修饰产 物中的主要成分为单修饰产物， 即 monoPEG- rhIL- 6, 产物均一度较高， 有利 于后续纯化。  1:20 5% 33% 62% As seen from Table 5, the molar ratio of rhIL-6 to the modifier is between 1:1 and 1:3, and the main component in the modified product obtained is a single modified product, ie monoPEG- rhIL-6, the product is higher, which is conducive to subsequent purification.
2.4 PEG修饰工艺的硝定  2.4 Nitration of PEG modification process
经过对反应条件的优化筛选， 确立了以下稳定高效的修饰工艺：  After optimizing the screening of the reaction conditions, the following stable and efficient modification process was established:
样品： rhIL-6纯品， SDS-PAGE纯度大于 95%, 蛋白浓度在 0.5~lmg/ml 之间， pH为 9.0, 緩冲液为 PB, 不能有其他含有氨基的化合物 饰试剂： mPEG2-NHS MW20kDa， -20 °C低温干燥保存  Sample: pure rhIL-6, SDS-PAGE purity greater than 95%, protein concentration between 0.5~1mg/ml, pH 9.0, buffer PB, no other amino-containing compound decoration reagent: mPEG2-NHS MW20kDa, -20 °C low temperature dry preservation
修饰程序：  Modification program:
a、 hIL-6水浴加热至 25°C , 称取 rhIL-6总量 1~2倍的 mPEG2-NHS, 装: 入干燥清洁无菌无热原的容器中；  a, hIL-6 water bath heated to 25 ° C, weigh 1 to 2 times the total amount of rhIL-6 mPEG2-NHS, loaded: into a dry clean sterile pyrogen-free container;
b、 hIL-6倒入盛有 mPEG2-NHS的容器中， 迅速混匀使 mPEG2-NHS完 全溶解， 25°C水浴反应 45分钟； 加入甘氨酸至 0.45M以终止反应； c、 反应完成后， 于 4°C保存， 取样检测， 其他样品留待柱层析纯化。 白细胞介素 -6聚乙二醇结合物的制备  b, hIL-6 is poured into a container containing mPEG2-NHS, rapidly mixed to completely dissolve mPEG2-NHS, and reacted in a water bath at 25 ° C for 45 minutes; add glycine to 0.45 M to terminate the reaction; c, after the reaction is completed, Store at 4 ° C, sample and test, and other samples to be purified by column chromatography. Preparation of interleukin-6 polyethylene glycol conjugate
【实施例 1】 白细胞介素 -6聚乙二醇结合物（ PEG-n^6 )的制备 [Example 1] Preparation of interleukin-6 polyethylene glycol conjugate (PEG-n^6)
制备 IL-6聚乙二醇结合物的化学修饰反应式如下： ρ The chemical modification reaction formula for preparing IL-6 polyethylene glycol conjugate is as follows: ρ
nV-(-OCH2CH2^- C -nV-(-OCH 2 CH 2 ^- C -
(CH^) (CH^)
H H
Figure imgf000014_0001
Figure imgf000014_0001
o  o
其中 i、 j为 100-1000的整数， i、 j之和使结合物的 mPEG部分的分子量 为 15000-30000， 优选为 20000, 反应式结构中的 -NH-IL- 6的氨基为 Lys残基 的侧链氨基。  Wherein i and j are integers of from 100 to 1000, and the sum of i and j is such that the molecular weight of the mPEG moiety of the conjugate is from 15,000 to 30,000, preferably 20,000, and the amino group of -NH-IL-6 in the reaction structure is a Lys residue. Side chain amino group.
修饰工艺如下：  The finishing process is as follows:
样品： 人 IL-6纯品（ Sigma公司）， SDS-PAGE纯度大于 95%, 蛋白浓度 在 0.5 - lmg/ml之间， pH为 9.0, 緩冲液为 PB, 不能含有其他含氨基的化合 物  Sample: Human IL-6 pure product (Sigma), SDS-PAGE purity greater than 95%, protein concentration between 0.5 - lmg/ml, pH 9.0, buffer PB, can not contain other amino-containing compounds
修饰试剂： mPEG2-NHS MW20kDa, -20°C氐温干燥保存  Modification reagent: mPEG2-NHS MW20kDa, -20 °C dry storage
修饰过程：  Modification process:
a、 IL-6水浴加热至 25 °C , 称取 IL-6总量 1 ~ 2倍的 mPEG2-NHS, 装入干 燥清洁无菌无热原的容器中；  a, IL-6 water bath heated to 25 °C, weighed 1 ~ 2 times the total amount of IL-6 mPEG2-NHS, into a dry clean sterile pyrogen-free container;
b、 IL-6倒入盛有 mPEG2-NHS的容器中， 迅速混匀使 mPEG2-NHS完全溶 解， 25 °C水浴反应 45分钟；  b. IL-6 is poured into a container containing mPEG2-NHS, and rapidly mixed to completely dissolve mPEG2-NHS, and reacted in a water bath at 25 ° C for 45 minutes;
c、 反应完成后， 于 4°C保存， 取样检测， 其他样品留待柱层析纯化。  c. After the reaction is completed, store at 4 ° C, sample and test, and other samples to be purified by column chromatography.
FPLC检测显示被修饰产物（包括各种修饰度的 PEG-IL-6 ) 占 40 ~ 60%, FPLC detection showed that the modified product (including various degrees of modification of PEG-IL-6) accounted for 40 ~ 60%,
SDS-PAGE显示单修饰产物（ monoPEG-IL- 6 ) 占被修饰物 60%以上 (结果见附 图 1中的泳道 10和泳道 13)。 【实施例 2】 PEG-IL-6的純化 SDS-PAGE showed that the single modified product (monoPEG-IL-6) accounted for more than 60% of the modified product (results see lanes 10 and 13 in Figure 1). [Example 2] Purification of PEG-IL-6
首先将采用实施 1方法所得的三批产物均匀混合， 用经 10mM醋酸緩冲 液平衡至 H5.0的 G-25凝胶柱进行脱盐换液， 把样品緩冲液 (緩冲液配方'. Na2HPO 12H2O , 15.04g/L; NaH2P04 2H20, 1.25g L; NaCl 8.77g/L。 pH 9.0 ) pH从 9.0调整为 5.0;再用一步 SP Sepharose High Performance阳离子 交换层析对修饰反应的混合物进行分离（洗脱液配方： A液醋酸钠 0.82 g/L , 醋酸调 pH至 5.0; B液醋酸钠 0.82 g/L,氯化钠 29.25 g/L,醋酸调 pH至 5.0 )。 在此 ^牛下， 水解的 mPEG分子因为不带电荷或带负电荷不能吸附在柱上而 穿出， 然后在逐渐增加的盐离子梯度洗脱下， 多修饰的 mPEG- IL-6先被洗脱 下来， 其次是单修饰的 mPEG-IL-6, 最后是未修饰的 IL-6。  First, the three batches of the product obtained by the method of the first method were uniformly mixed, and desalted and exchanged with a G-25 gel column equilibrated to H5.0 with 10 mM acetate buffer, and the sample buffer (buffer formulation '. Na2HPO 12H2O, 15.04 g/L; NaH2P04 2H20, 1.25 g L; NaCl 8.77 g/L. pH 9.0) pH was adjusted from 9.0 to 5.0; and the modified reaction mixture was separated by one step SP Sepharose High Performance cation exchange chromatography ( Eluent formulation: A solution of sodium acetate 0.82 g / L, acetic acid adjusted to pH 5.0; B solution of sodium acetate 0.82 g / L, sodium chloride 29.25 g / L, acetic acid adjusted to pH 5.0). Under this calf, the hydrolyzed mPEG molecule can be eluted because it is uncharged or negatively charged and cannot be adsorbed on the column. Then, with the increasing gradient of salt ion elution, the multi-modified mPEG-IL-6 is washed first. Take off, followed by a single modified mPEG-IL-6, and finally an unmodified IL-6.
分别收集各洗脱组分做 SDS-PAGE检测，结果见附图 1 lane2-8和 lanell-12 所^。 经过阳离子交换层析的单修饰 mPEG-IL-6 中还残留了少量多务饰的 mPEG-IL-6和未修饰的 IL- 6, 由于这三者在分子量上也有较大的差别 （相差 20kDa以上）， 用 Superdex 200凝胶过滤层析凝胶过滤柱进行分离。  Each eluted fraction was collected for SDS-PAGE detection. The results are shown in Figure 1 lane 2-8 and lanell-12. A small amount of mPEG-IL-6 and unmodified IL-6 remained in the mono-modified mPEG-IL-6 after cation exchange chromatography. Because of the large difference in molecular weight (the difference is 20kDa). Above), separation was performed using a Superdex 200 gel filtration chromatography gel filtration column.
用 Superdex 200凝胶过滤层析柱分离后的目标样品经 SDS-PAGE检测纯· 度， 结果表明经过这步纯化后样品中的单修饰 PEG-IL-6 ( monoPEG-IL-6 )含' 量大于 85% (见附图 1 lanel4 )， 各种修饰度的 PEG-IL-6的总舍量大于 95%, 达到或超过了国内外其他的 PEG修饰蛋白多肽的质量要求。 同时， 上述纯化 步骤需在无菌、无热原条件下进行，保证产品的符合国家对生化药物的相关要 求。  The target sample separated by Superdex 200 gel filtration chromatography column was tested for purity by SDS-PAGE. The results showed that the single modified PEG-IL-6 (monoPEG-IL-6) contained in the sample after this purification contained More than 85% (see lane 1 of Figure 1), the total amount of PEG-IL-6 with various degrees of modification is greater than 95%, meeting or exceeding the quality requirements of other PEG-modified protein peptides at home and abroad. At the same time, the above purification steps should be carried out under aseptic and pyrogen-free conditions to ensure that the products meet the relevant requirements of the national biochemical drugs.
【实施例 3】 制剂处方和工艺流程  [Example 3] Formulation and process flow
1 制剂处方  1 preparation prescription
1.1 制剂处方 （以 1000瓶计 )  1.1 Formulation prescription (in 1000 bottles)
： PEG-rhIL-6按照前述方法制备 : PEG-rhIL-6 was prepared as described above
主药 PEG-rML-6 15mg Main drug PEG-rML-6 15mg
k白蛋白 10g  k albumin 10g
甘氨酸 25g  Glycine 25g
Na2HP04 12H20 0.619g KH2PO4 0.103g Na2HP04 12H20 0.619g KH2PO4 0.103g
NaCl 3.440g  NaCl 3.440g
KC1 0.086g  KC1 0.086g
1.2 制剂类型 1.2 Type of preparation
属于 2005年版《中华人民共和国药典》三部附录 I "制剂通则" 规定的注 射用冻干制剂。  It is a lyophilized preparation for injection prescribed in the 2005 edition of the Pharmacopoeia of the People's Republic of China, Appendix III, "General Provisions for Preparation".
1.3制剂规格 1.3 preparation specifications
15 μ g/0.5ml/瓶 (20万活性单位）  15 μg/0.5ml/bottle (200,000 active units)
2 制剂工艺流程参见图 3。 药效学试验、 安全性试验和药代动力学试验 2 See Figure 3 for the formulation process. Pharmacodynamic test, safety test and pharmacokinetic test
【试验例 1】 PEG-IL-6对环磷酰胺致小鼠血小板减少症的作用剂量筛选 实验动物： 18-20g雌性 Balb/c小鼠。 [Test Example 1] Effect of PEG-IL-6 on cyclophosphamide-induced thrombocytopenia in mice Dose screening Experimental animals: 18-20 g female Balb/c mice.
实验方案： 将小鼠随机分为 10组， 每组 5只. 第 1.-5天每天每只小鼠皮 下注射试验样品（依前述实施例方法制备的 PEG- IL- 6 ) 0.5ml; 从注射试验样 品的第三天起每天每只小鼠腹腔注射环磷酰胺 2mg, 连续注射 3天； 亍第一天 注射试验样品前和第 8-17天， 每天自每只小鼠尾部釆血 l()ul,稀释 6倍 用 •Cell-DYN1600仪测血小板数。  Experimental protocol: The mice were randomly divided into 10 groups of 5 each. On day 1.-5, each mouse was injected subcutaneously into the test sample (PEG-IL-6 prepared according to the method of the foregoing examples) 0.5 ml; On the third day of injection of the test sample, each mouse was intraperitoneally injected with 2 mg of cyclophosphamide per day for 3 consecutive days; 亍 before the first day of injection of the test sample and on days 8-17, every day from the tail of each mouse was licked. () ul, diluted 6 times with • Cell-DYN1600 instrument to measure the number of platelets.
实验分组方案：  Experimental grouping plan:
. AO 每天皮下注射 PBS 0.5ml/只  AO daily subcutaneous injection of PBS 0.5ml / only
A1 每天皮下注射试验样品 IL-6 (0.20ug/ml)0.5ml; O.lug/只  A1 subcutaneous injection of test sample IL-6 (0.20 ug/ml) 0.5 ml per day; O.lug/only
A2 每天皮下注射试-险样品 IL-6 (lug/ml)0.5ml; 0.5ug/只  A2 subcutaneous injection test-risk sample IL-6 (lug/ml) 0.5ml per day; 0.5ug/only
A3 每天皮下注射试验样品 IL-6 (2ug/ml)0.5ml; l.Oug/只  A3 subcutaneous injection of test sample IL-6 (2ug/ml) 0.5ml per day; l.Oug/only
A4 每天皮下注射试-险样品 IL-6 (5ug/ml)0.5ml; 2.5ug/只  A4 subcutaneous injection test-risk sample IL-6 (5ug/ml) 0.5ml per day; 2.5ug/only
B1 每天皮下注射试 ¾r样品 PEG-IL-6 (0.1ug/ml)0.5ml; 0.05ug/只  B1 daily subcutaneous injection test 3⁄4r sample PEG-IL-6 (0.1ug/ml) 0.5ml; 0.05ug/only
B2 每天皮下注射试验样品 PEG-IL-6 (0.2ug/ml)0.5ml; O.lug/只  B2 subcutaneous injection test sample per day PEG-IL-6 (0.2ug/ml) 0.5ml; O.lug/only
B3 每天皮下注射试验样品 PEG-IL-6 (lug/ml)0.5ml; 0.5ug/只  B3 subcutaneous injection test sample per day PEG-IL-6 (lug/ml) 0.5ml; 0.5ug/only
B4 每天皮下注射试验样品 PEG-IL-6 (2ug/ml)0.5ml; 1.Oug/只  B4 subcutaneous injection test sample PEG-IL-6 (2ug/ml) 0.5ml per day; 1.Oug/only
B5 每天皮下注射试脸样品 PEG- IL- 6 (5ug/ml)0.5ml. 2.5ug/只 实验结果见表 6: 表 6.对皮下注射 IL-6和 PEG-IL-6的化疗小鼠血小板计数（X 109/L) 分組 AO A1 A2 A3 A4 B1 B2 B3 B4 B5 B5 daily subcutaneous injection test sample PEG- IL-6 (5ug/ml) 0.5ml. 2.5ug / only The experimental results are shown in Table 6: Table 6. Platelet counts of chemotherapy mice injected subcutaneously with IL-6 and PEG-IL-6 ( X 10 9 /L) Group AO A1 A2 A3 A4 B1 B2 B3 B4 B5
天数 Number of days
给药前 1186 1026 1049 1134 1030 1133 1006 1090 1200 956 Before administration 1186 1026 1049 1134 1030 1133 1006 1090 1200 956
+109 +214 土 224 +182 土 118 +271 +307 ±115 ±61 ±105 给药 8天 570 590 580 572 559 762 592 569 676 684  +109 +214 soil 224 +182 soil 118 +271 +307 ±115 ±61 ±105 administration 8 days 570 590 580 572 559 762 592 569 676 684
+197 +90 ±140 +112 +82 ±275 ±80 ±111 ±81 ±106 给药 9天 499 532 479 579 500 602 490 439 503 550  +197 +90 ±140 +112 +82 ±275 ±80 ±111 ±81 ±106 Dosing 9 days 499 532 479 579 500 602 490 439 503 550
±58 ±122 土 191 +182 +133 +223 ±74 ±45 ±121 +77 给药 10天 472 488 553 634 601 637 522 515 619 712  ±58 ±122 soil 191 +182 +133 +223 ±74 ±45 ±121 +77 administration 10 days 472 488 553 634 601 637 522 515 619 712
±77 ±169 ±142 +292 ±186 ±199 ±54 ±80 ±80 ±24 给药 11天 713 796 721 881 744 935 788 721 737 •899  ±77 ±169 ±142 +292 ±186 ±199 ±54 ±80 ±80 ±24 Dosing 11 days 713 796 721 881 744 935 788 721 737 •899
±141 ±127 ±129 ±240 ±165 ±279 ±91 ±115 ±92 ±55 给药 12天 841 953 968 1054 943 1040 1006 962 1037 1144  ±141 ± 127 ± 129 ± 240 ± 165 ± 279 ± 91 ± 115 ± 92 ± 55 for 12 days 841 953 968 1054 943 1040 1006 962 1037 1144
±95' ±145 土 164 ±137 ±145 +197 土 177 土 163 +115 ±109 给药 13天 883 1009 1072 900 940 937 972 895 1104 1110  ±95' ±145 soil 164 ±137 ±145 +197 soil 177 soil 163 +115 ±109 administration 13 days 883 1009 1072 900 940 937 972 895 1104 1110
±130 土 201 +166 +223 土 134 ±142 +129 +135 ±159 ±86 给药 15天 889 1062 1454 1259 978 1247 1174 1047 1236 1405  ±130 soil 201 +166 +223 soil 134 ±142 +129 +135 ±159 ±86 administration 15 days 889 1062 1454 1259 978 1247 1174 1047 1236 1405
+230 +214 土 440 +588 +196 +236 +165 +284 +274 ±192 给药 16天 894 958 1189 1033 1116 1176 1141 1169 1092 1386  +230 +214 soil 440 +588 +196 +236 +165 +284 +274 ±192 administration 16 days 894 958 1189 1033 1116 1176 1141 1169 1092 1386
±188 +280 +254 +336 +282 ±186 ±170 ±89 +304 ±261 给药 17天 1212 1088 1324 1317 1230 1416 1330 1277 1130 1273  ±188 +280 +254 +336 +282 ±186 ±170 ±89 +304 ±261 administration 17 days 1212 1088 1324 1317 1230 1416 1330 1277 1130 1273
±207 ±229 ±407 ±117 ±288 ±118 +89 ±219 ±271 ±222 按所有小鼠的平均血小板数 1092xl09/L计算起始值（正常值）， 超过的扣 去， 不足的补充， 以消除误差。 以各组起始值为 100%， 计算各组别各天平均 值占起始值的百分数， 以表明血小板数目的恢复率， 结果见表 7。 表 7.各组小鼠血小板计数的恢复率 ( % ) 分组 AO A1 A2 A3 A4 B1 B2 B3 B4 B5 天数 ±207 ± 229 ± 407 ± 117 ± 288 ± 118 +89 ± 219 ± 271 ± 222 Calculate the starting value (normal value) according to the average platelet count of all mice, 1092xl0 9 /L, excess deduction, insufficient supplement, To eliminate the error. The starting value of each group was 100%, and the percentage of the mean value of each group in each group was calculated to indicate the recovery rate of the number of platelets. The results are shown in Table 7. Table 7. Recovery rate of platelet counts in each group of mice (%) Group AO A1 A2 A3 A4 B1 B2 B3 B4 B5 days
给药前 100 100 100 100 100 100 100 100 100 100 给药 8天 43.6 63.8 60.4 51.5 60.3 70.1 65.8 55.4 55.2 77.1 给药 9天 39.4 58.1 50.7 51.8 54.6 54.5 56 42.9 38.4 64 给药 10天 36.7 53.8 57.9 57.5 64.4 57.9 59.1 50.2 49.7 79.8 给药 11天 60.1 83.7 74.2 81.5 78.3 86.8 84.9 70.3 61.1 97.9 给药 12天 72.6 99 98.3 98.3 97.7 97.1 106.1 93.6 90.3 126.7 给药 13天 76.7 104.5 108.3 83.4 97.3 87.1 102.8 87.2 96.8 118.3 给药 15天 77.3 109.2 145.5 106.6 101.1 117.2 122.4 101.7 109.6 147.1 给药 16天 68.2 73.1 90.8 63.3 86.1 89.7 87.1 89.2 93.3 105.8 给药 17天 70.5 83.1 101 100.5 93.9 108.1 101.5 97.4 86.3 97.2 统计分析： 按均值的百分数计算整体值： t0.。5=2.179 (n=7> Before administration 100 100 100 100 100 100 100 100 100 100 Administration 8 days 43.6 63.8 60.4 51.5 60.3 70.1 65.8 55.4 55.2 77.1 Administration 9 days 39.4 58.1 50.7 51.8 54.6 54.5 56 42.9 38.4 64 Administration 10 days 36.7 53.8 57.9 57.5 64.4 57.9 59.1 50.2 49.7 79.8 Administration 11 days 60.1 83.7 74.2 81.5 78.3 86.8 84.9 70.3 61.1 97.9 Administration 12 days 72.6 99 98.3 98.3 97.7 97.1 106.1 93.6 90.3 126.7 Administration 13 days 76.7 104.5 108.3 83.4 97.3 87.1 102.8 87.2 96.8 118.3 Administration 15 Day 77.3 109.2 145.5 106.6 101.1 117.2 122.4 101.7 109.6 147.1 Administration 16 days 68.2 73.1 90.8 63.3 86.1 89.7 87.1 89.2 93.3 105.8 Administration 17 days 70.5 83.1 101 100.5 93.9 108.1 101.5 97.4 86.3 97.2 Statistical analysis: Calculate the overall value as a percentage of the mean: t 0 . 5 = 2.179 (n=7>
用药组与对照组相比较 (分析了前 Ί个数据)：  The drug group was compared with the control group (the previous data was analyzed):
A1-A0 t=2.13  A1-A0 t=2.13
A2-A0 t=1.85 无显著差异  A2-A0 t=1.85 No significant difference
A3-A0 t=1.63 无显著差异  A3-A0 t=1.63 no significant difference
A4-A0 t=2.17  A4-A0 t=2.17
B1-A0 t=2.3 有显箸差异  B1-A0 t=2.3 has a significant difference
B2-A0 t=2.29 有显著差异  B2-A0 t=2.29 has significant difference
B3-A0 t=1.24 无显箸差异  B3-A0 t=1.24 No significant difference
B4-A0 t=l.ll 无显著差异  B4-A0 t=l.ll No significant difference
据实猃结果可得, 修饰前（IL-6 ) 的有效剂量为 2.5ug/只小鼠。 修饰后 According to the results, the effective dose before modification (IL-6) was 2.5 ug/mouse. Modified
( PEG-IL-6 )的有效剂量范围在 0.01ug-0. 5ug /只小鼠的范围内。 在有效用药 剂量中， Bl ( PEG-IL-6 ) 比 A4 ( IL-6 )减少了很多， 最高可减少 250倍的用 量， 使用药剂量大为降低。 【试验例 2】 PEG-IL-6对环磷酰胺致小鼠和 Beagle犬血小板减少症的药效 试验 The effective dose range of (PEG-IL-6) is in the range of 0.01 ug to 0.5 ug per mouse. Among the effective doses, Bl (PEG-IL-6) is much less than A4 (IL-6), and can be reduced by up to 250 times, and the amount of the drug used is greatly reduced. [Test Example 2] Pharmacodynamic test of PEG-IL-6 on cyclophosphamide-induced thrombocytopenia in mice and Beagle dogs
采用环磷酰胺所致的造血***受损的两种实验动物模型： 小鼠和 Beagle 犬， 分别试 -臉了高、 中、 4氐三个剂量。 阳性对照品为市售的 IL-11药品（吉巨 芬， 依星等， 3mg/支）， 结果表明， 各剂量组血小板减少持续时间较模型组短， 恢复较快，且血小板减少程度较模型组轻，表明本品对两种动物模型可明显增 加血小板数， 减轻血小板减少程度， 缩短血小板减少持续时间， 加快恢复速。 在与阳性药达到相同效果的情况下，其使用剂量比阳性药低得多。 同时本品还 可以短暂增加犬白细胞和淋巴细胞，对红细胞、血红蛋白和网织红细胞均无明 显影响。  Two experimental animal models of hematopoietic damage caused by cyclophosphamide were used: mice and Beagle dogs, respectively, with three doses of high, medium, and four doses. The positive control substance was a commercially available IL-11 drug (Jijufen, Yixing et al, 3 mg/branch). The results showed that the duration of thrombocytopenia in each dose group was shorter than that of the model group, and the recovery was faster, and the degree of thrombocytopenia was higher than that of the model. The light weight of the group indicates that the product can significantly increase the number of platelets in two animal models, reduce the degree of thrombocytopenia, shorten the duration of thrombocytopenia, and accelerate the recovery rate. In the case of achieving the same effect as the positive drug, the dose used is much lower than that of the positive drug. At the same time, this product can also temporarily increase canine leukocytes and lymphocytes, and has no obvious effect on red blood cells, hemoglobin and reticulocytes.
【试臉例 3】 PEG-IL-6体外生物学活性试验结果  [Test face example 3] PEG-IL-6 in vitro biological activity test results
使用 7TD1细胞/ MTT比色法检测： PEG-IL-6体外生物学活性， 结果见表 8。 Using 7TD1 cells/MTT colorimetric assay: PEG-IL-6 in vitro biological activity, the results are shown in Table 8.
表 8体外生物学活性的检测结果  Table 8 Test results of in vitro biological activity
检测样品 体外生物学活性 Test sample in vitro biological activity
IL-6原料 (修饰前） 1.82xl07AU/ml IL-6 raw material (before modification) 1.82xl0 7 AU/ml
PEG-IL-6原液 (修饰后） 1.59xl06AU/ml PEG-IL-6 stock solution (after modification) 1.59xl0 6 AU/ml
PEG-IL-6冻干成品 (药品） 3.35xl05 AU/瓶 PEG-IL-6 freeze-dried finished product (drug) 3.35xl0 5 AU/bottle
活性实验结果表明本发明 PEG-IL-6的体外活性低于未修饰 IL-6, 与现有研究 一致。 The results of the activity experiments showed that the in vitro activity of the PEG-IL-6 of the present invention was lower than that of the unmodified IL-6, which was consistent with the existing studies.
【试验例 4】 PEG-IL-6安全性试验  [Test Example 4] PEG-IL-6 safety test
1、 小鼠急性毒性试猃： 小鼠皮下、 尾静脉及腹腔注射依前述实施例制备 的 PEG-IL-6, 剂量相当于一般临床用剂量（15 g/支 ， 0.3 g/kg )的 1000倍。 连续观察 14 天， 未观察到中毒反应。 小鼠皮下、 尾静脉、 腹腔注射的 LD50>400(^g/kg。 1. Acute toxicity test in mice: The subcutaneous, tail vein and intraperitoneal injection of PEG-IL-6 prepared according to the above examples is equivalent to 1000 doses of general clinical dose (15 g/d, 0.3 g/kg). Times. After continuous observation for 14 days, no poisoning reaction was observed. The LD 50 >400 (^ g /kg) in the subcutaneous, tail vein and intraperitoneal injection of mice.
2、 Beagle犬慢性毒性试验： 本发明 PEG-IL-6大、 中、 小三个剂量（30.0、 12.0、 6.0μ§.1 -1)给 Beagle犬每日皮下注射， 连续 32天， 停药恢复期观察 15 天。 结果表明， 本品皮下注射低于 12.0μβ/]¾对 Beagle犬是安全剂量。 2. Beagle dog chronic toxicity test: The three doses of PEG-IL-6 of the invention are large, medium and small (30.0, 12.0, 6.0μ § .1 - 1 ). The Beagle dog is injected subcutaneously every day for 32 consecutive days. Observed for 15 days. The results showed that subcutaneous injection of this product is less than 12.0μ β /] 3⁄4 is a safe dose for Beagle dogs.
本实验结果表明本发明 PEG-IL-6的安全性好。 【试验例 5】 PEG-IL-6药代动力学试验 The results of this experiment show that the PEG-IL-6 of the present invention is safe. [Test Example 5] PEG-IL-6 pharmacokinetic test
用 1251标记 PEG-rhIL-6 (依照前述实施例制备）， 获得了符合常规药代动 力学试验要求的 125I-PEG- rhIL-6。 按常规药代动力学试验要求进行以下实验。 With 1251 labeled PEG-rhIL-6 (prepared in accordance with the aforementioned Example), 125 I-PEG- obtained consistent with conventional pharmacokinetic studies required rhIL-6. The following experiments were carried out in accordance with the requirements of conventional pharmacokinetic tests.
大鼠皮下注射 125 PEG-rhIL-6。 其代谢符合一房室分布模型， 分布相半衰 期为 1.4~5.1h, 消除相半衰期为 58.3~236.5h, 达峰时间 7.9~13.3h, 体内滞留 时间 42~52h。 Rats were injected subcutaneously with 125 PEG-rhIL-6. Its metabolism accords with the one-compartment distribution model, the distribution phase half-life is 1.4~5.1h, the elimination phase half-life is 58.3~236.5h, the peak time is 7.9~13.3h, and the body retention time is 42~52h.
大鼠静脉注射 125I-PEG-rhIL-6。 其代谢符合二房室分布模型， 分布相半衰 $\ t1/2 ( α )约为 l.l~2.5h, 消除相半衰期 t1/2 (β ) 为 13~18h。 Rats were injected intravenously with 125 I-PEG-rhIL-6. Its metabolism accords with the two-compartment distribution model, the distribution phase half-life $\ t 1/2 (α) is about ll~2.5h, and the elimination phase half-life t 1/2 (β) is 13~18h.
Beagle 犬皮下注射 125I-PEG-rhIL-6。 分三个剂量组 2C^g/kg、 10 g/kg、 5 g/kg, 给药后于前肢静脉采血。 结果显示其代谢符合一房室分布模型， 皮下 注射给药分布相半衰期为 0.1~2.2h, 消除相半衰期为 70.8~247.lh， 达峰时间 0.9~10.4h, 体内滞留时间 69.7~91.7h。 Beagle dogs were injected subcutaneously with 125 I-PEG-rhIL-6. Three dose groups were 2C^g/kg, 10 g/kg, 5 g/kg, and blood was collected from the forelimb vein after administration. The results showed that the metabolism accorded with the one-compartment distribution model. The half-life of the distribution phase of subcutaneous injection was 0.1~2.2h, the elimination phase half-life was 70.8~247.lh, the peak time was 0.9~10.4h, and the body retention time was 69.7~91.7h.
药代动力学实验结果表明， 本发明聚乙二醇单修饰的人白细胞介素 -6 的 体内滞留时间、达峰时间、半衰期等指标均比现有文献报道的单修饰人白细胞 介素 -6有了极大的延长 (参见文献 Selective enhancement of thrombopoietic activity of PEGylated interleukin 6 by a simple procedure using a reversible amino-protective reagent. Br J Haematol. 2001 Jan;l 12(1) :181 -8 )。 工业应用性  The results of pharmacokinetic experiments show that the in vivo retention time, peak time and half-life of the human polyethylene interleukin-6 modified by the polyethylene glycol of the present invention are more than the single modified human interleukin-6 reported in the prior literature. There is a great extension (see Selectedive enhancement of thrombopoietic activity of PEGylated interleukin 6 by a simple procedure using a reversible amino-protective reagent. Br J Haematol. 2001 Jan; l 12(1): 181 -8 ). Industrial applicability
经 PEG单修饰后的白细胞介素 -6的稳定性大大提高，它的体内半衰期长， 血清清除率低，使用剂量和频率大能为减少， 副作用也大为降低。 既能方便患 者使用， 降低使用成本， 也能提高使用安全性， 大大减轻患者的痛苦。 同时， 本发明的单修饰的人白细胞介素 -6聚乙二醇结合物的均一性好，能达到临床用 药安全、 有效、 质量可控的要求， 并能大规模生产， 应用前景好。 以上对本发明的详细描述并不限制本发明，本领域技术人员可以根据本发 明作出各种改变或变形，只要不脱离本发明的精神， 均属于本发明所附权利要 求所定义的范围。  The stability of interleukin-6 modified by PEG alone is greatly improved, its in vivo half-life is long, serum clearance rate is low, the dosage and frequency of use are greatly reduced, and side effects are greatly reduced. It can not only be convenient for patients to use, reduce the cost of use, but also improve the safety of use and greatly reduce the suffering of patients. At the same time, the single modified human interleukin-6 polyethylene glycol conjugate of the invention has good homogeneity, can meet the requirements of safe, effective and quality control of clinical drugs, and can be produced on a large scale with good application prospect. The above description of the present invention is not intended to limit the invention, and various modifications and changes may be made by those skilled in the art without departing from the spirit and scope of the invention.

Claims

权利要求书 Claim
1、 一种白细胞介素 -6聚乙二醇结合物，其特征在于：所述的白细胞介素 -6聚乙二醇结合物是由聚乙二醇对白细胞介素- 6进行共价单修饰得到的， 其 中每个白细胞介素 -6分子上共价结合一个聚乙二醇分子， 聚乙二醇分子的分 子量为 15000 ~ 30000Da。 What is claimed is: 1. An interleukin-6 polyethylene glycol conjugate, characterized in that the interleukin-6 polyethylene glycol conjugate is covalently bound to interleukin-6 by polyethylene glycol. Modified, wherein each interleukin-6 molecule is covalently bound to a polyethylene glycol molecule, and the molecular weight of the polyethylene glycol molecule is 15000 ~ 30000Da.
2、 权利要求 1所述的白细胞介素 -6聚乙二醇结合物，其特征在于： 所述 聚乙二醇分子与白细胞介素 -6分子的 Lys残基的侧链氨基或白细胞介素 -6分 子的肽链 N末端氨基结合。  The interleukin-6 polyethylene glycol conjugate according to claim 1, wherein: the polyethylene glycol molecule and the side chain amino group or interleukin of the Lys residue of the interleukin-6 molecule The N-terminal amino group of the 6-molecular peptide chain is bound.
3、 权利要求 2所述的白细胞介素- 6聚乙二醇结合物，其特征在于： 所述 聚乙二醇为分枝聚乙二醇， 所述分枝聚乙二醇为在所迷白细胞介素 -6的 Lys 残基的侧链氨基或白细胞介素- 6分子的肽链 N末端氨基上连接两条或两条以 上的 PEG链。  The interleukin-6 polyglycol conjugate according to claim 2, wherein the polyethylene glycol is branched polyethylene glycol, and the branched polyethylene glycol is in the fascination. The side chain amino group of the Lys residue of interleukin-6 or the N-terminal amino group of the peptide chain of the interleukin-6 molecule is linked to two or more PEG chains.
4、 权利要求 3所述的白细胞介素 -6聚乙二醇结合物， 其特征在于： 具有 式（I )所迷的结构：
Figure imgf000021_0001
The interleukin-6 polyethylene glycol conjugate according to claim 3, which has the structure of the formula (I):
Figure imgf000021_0001
p CH  p CH
/ \  / \
m-(-OCH2CH2t- C - NH C-NH - iL.6 M-(-OCH 2 CH 2 t- C - NH C-NH - i L . 6
o  o
(I)  (I)
其中 m代表甲基， i、 j为 100 ~ 1000的整数， i、 j之和使结合物的聚乙 二醇分子的分子量为 15000 ~ 30000Da。  Wherein m represents a methyl group, and i and j are integers of from 100 to 1000, and the sum of i and j causes the molecular weight of the polyethylene glycol molecule of the combination to be 15,000 to 30000 Da.
5、 权利要求 4所述的白细胞介素 -6聚乙二醇结合物， 其中用于对白细 胞介素 -6进行共价修饰的聚乙二醇分子为 mPEG2-NHS。  The interleukin-6 polyethylene glycol conjugate according to claim 4, wherein the polyethylene glycol molecule for covalently modifying the interleukin-6 is mPEG2-NHS.
6、 权利要求 2所述的白细胞介素 -6聚乙二醇结合物， 其特征在于： 所 述聚乙二醇为线型聚乙二醇， 所述线型聚乙二醇为在所述白细胞介素 -6 的 Lys残基的侧链氨基或白细胞介素 -6分子的肽链 N末端氨基上连接一条 PEG 链。 6. The interleukin-6 polyethylene glycol conjugate according to claim 2, wherein: the polyethylene glycol is a linear polyethylene glycol, and the linear polyethylene glycol is in the A side chain amino group of the Lys residue of interleukin-6 or a N-terminal amino group of the peptide chain of the interleukin-6 molecule is linked to a PEG chain.
7、 权利要求 6所述的白细胞介素- 6聚乙二醇结合物， 其中用于对白细 胞介素 -6进行共价修饰的聚乙二醇分子为 mPEG- aldehyde。 The interleukin-6 polyglycol conjugate according to claim 6, wherein the polyethylene glycol molecule for covalently modifying the interleukin-6 is mPEG-aldehyde.
8、 权利要求 1所述的白细胞介素- 6聚乙二醇结合物， 其中所述白细胞 介素 -6聚乙二醇结合物通过下述方法制备：  The interleukin-6 polyethylene glycol conjugate according to claim 1, wherein the interleukin-6 polyethylene glycol conjugate is prepared by the following method:
1 )将白细胞介素 -6制备成蛋白浓度在 0.05 - 20mg/ml之间， pH为 6.5 ~ 1) The interleukin-6 is prepared to a protein concentration of 0.05 - 20 mg/ml, and the pH is 6.5 ~
10.0的溶液； a solution of 10.0;
2 )将制备的白细胞介素 -6溶液与聚乙二醇反应， 所述聚乙二醇的量为 白细胞介素- 6重量的 1 ~ 100倍，反应温度为 15 ~ 35°C ,时间为 5 ~ 100分钟， 得到白细胞介素 -6聚乙二醇结合物；  2) reacting the prepared interleukin-6 solution with polyethylene glycol, the amount of the polyethylene glycol is 1 to 100 times the weight of the interleukin-6, and the reaction temperature is 15 to 35 ° C for a time of 5 ~ 100 minutes, get interleukin-6 polyethylene glycol conjugate;
3 )分离纯化获得的白细胞介素 -6聚乙二醇结合物， 得到聚乙二醇单修 饰的白细胞介素 -6聚乙二醇结合物。  3) The obtained interleukin-6 polyethylene glycol conjugate was isolated and purified to obtain a polyethylene glycol single-modified interleukin-6 polyethylene glycol conjugate.
9、 杈利要求 8所述的白细胞介素 -6聚乙二醇结合物， 其中所述白细胞 介素 -6聚乙二醇结合物通过下述方法制备：  9. The interleukin-6 polyethylene glycol conjugate of claim 8, wherein the interleukin-6 polyethylene glycol conjugate is prepared by the following method:
① 将 SDS-PAGE纯度大于 95%的白细胞介素 -6用磷酸緩冲液制备成蛋白 浓度在 0.5~lmg/ml之间， pH为 9.0的溶液；  1 Prepare a solution with a protein concentration of 0.5 to 1 mg/ml and a pH of 9.0 using an interleukin-6 with a SDS-PAGE purity greater than 95%;
② 将制备好的溶液水浴加热至 25°C， 与 mPEG2- NHS 反应， 所述 mPEG2-NHS的量为白细胞介素 -6总量的 1~2倍， 25°C水浴反应 45分钟； 加 入甘氨酸至 0.45M以终止反应， 获得白细胞介素- 6聚乙二醇结合物；  2 The prepared solution water bath is heated to 25 ° C, and reacts with mPEG2-NHS, the amount of the mPEG2-NHS is 1 to 2 times of the total amount of interleukin-6, and the reaction at 25 ° C for 45 minutes in water bath; To 0.45 M to terminate the reaction, obtaining an interleukin-6 polyethylene glycol conjugate;
③ 纯化分离获得的白细胞介素 -6 聚乙二醇结合物得到聚乙二醇单修饰 的白细胞介素 -6聚乙二醇结合物。  3 Purification of the isolated interleukin-6 polyethylene glycol conjugate to obtain a polyethylene glycol mono-modified interleukin-6 polyethylene glycol conjugate.
10、 制备权利要求 1所述的白细胞介素 -6聚乙二醇结合物的方法， 包括 以下步 ：  10. A method of preparing the interleukin-6 polyethylene glycol conjugate of claim 1, comprising the steps of:
1 ) 将白细胞介素 -6制备成蛋白浓度在 0.05 - 20mg/ml之间， pH为 6.5 10.0的溶液；  1) preparing interleukin-6 into a solution having a protein concentration of 0.05 - 20 mg/ml and a pH of 6.5 10.0;
2 ) 将制备的白细胞介素 _6溶液与聚乙二醇反应， 所述聚乙二醇的量 为白细胞介素 -6重量的 1 ~ 100倍，反应温度为 15 ~ 35'C , 时间为 5 ~ 100分 钟， 得到白细胞介素 -6聚乙二醇结合物；  2) reacting the prepared interleukin-6 solution with polyethylene glycol, the amount of the polyethylene glycol being 1 to 100 times the weight of the interleukin-6, and the reaction temperature is 15 to 35'C, the time is 5 ~ 100 minutes, get interleukin-6 polyethylene glycol conjugate;
3)分离纯化获得的白细胞介素 -6聚乙二醇结合物，得到聚乙二醇单修 饰的白细胞介素 -6聚乙二醇结合物。 3) The obtained interleukin-6 polyethylene glycol conjugate was isolated and purified to obtain a polyethylene glycol mono-modified interleukin-6 polyethylene glycol conjugate.
11、 权利要求 10迷的方法， 其中步驟 1 ) 中所述的白细胞介素 -6 的 SDS-PAGE纯度大于 95%,制备成蛋白浓度在 0.5 ~ lmg/ml之间， pH为 8.7 ~ 9.3的溶液。 11. The method of claim 10, wherein the interleukin-6 described in step 1) has a SDS-PAGE purity greater than 95%, a protein concentration of between 0.5 and 1 mg/ml, and a pH of 8.7 to 9.3. Solution.
12、 权利要求 10所述的方法， 其中步骤 3 )所述的纯化包括下述步骤： 将步骤 2 )获得的白细胞介素 -6聚乙二醇结合物经 G-25凝胶过滤-柱脱盐 处理后，再用阳离子交换层析柱进行初步分离， 最后用 Superdex 200凝胶过 滤柱纯化得到聚乙二醇单修饰的白细胞介素 -6聚乙二醇结合物。  12. The method of claim 10, wherein the purifying of step 3) comprises the step of: desalting the interleukin-6 polyethylene glycol conjugate obtained in step 2) by G-25 gel filtration-column dehydration After the treatment, preliminary separation was carried out by using a cation exchange chromatography column, and finally purified by a Superdex 200 gel filtration column to obtain a polyethylene glycol mono-modified interleukin-6 polyethylene glycol conjugate.
13、 权利要求 10所述的方法， 其中包括下述步骤：  13. The method of claim 10, comprising the steps of:
① 将 SDS-PAGE纯度大于 95%的白细胞介素 -6用磷酸緩冲液制备 成蛋白浓度在 0.5-lmg/ml之间， pH为 9.0的溶液；  1 A solution having a SDS-PAGE purity greater than 95% and a solution having a protein concentration of 0.5-l mg/ml and a pH of 9.0 is prepared using a phosphate buffer;
② 将制备好的溶液水浴加热至 25 °C , 与 mPEG2-NHS反应， 所述 mPEG2-NHS的量为白细胞介素- 6总量的 1~2倍， 25°C水浴反应 45分钟； 加 入甘氨酸至 0.45M以终止反应， 获得白细胞介素 -6聚乙二醇结合物；  2 The prepared solution water bath is heated to 25 ° C, and reacts with mPEG2-NHS, the amount of the mPEG2-NHS is 1 to 2 times of the total amount of interleukin-6, and the reaction at 25 ° C for 45 minutes in water bath; To 0.45 M to terminate the reaction, obtaining an interleukin-6 polyethylene glycol conjugate;
③ 纯化分离获得的白细胞介素 -6 聚乙二醇结合物得到聚乙二醇单 修饰的白细胞介素 -6聚乙二醇结合物。  3 Purification of the isolated interleukin-6 polyethylene glycol conjugate to obtain a polyethylene glycol mono-modified interleukin-6 polyethylene glycol conjugate.
14、 一种药物组合物， 含有权利要求 1所述的白细胞介素 -6聚乙二醇结 合物以及药学上可接受的辅料。  A pharmaceutical composition comprising the interleukin-6 polyethylene glycol composition of claim 1 and a pharmaceutically acceptable adjuvant.
15、 权利要求 1所述的白细胞介素 -6聚乙二醇结合物在制备治疗血小板 减少症的药物、 放化疗辅助用药物或免疫增强药物中的应用。  The use of the interleukin-6 polyethylene glycol conjugate according to claim 1 for the preparation of a medicament for treating thrombocytopenia, a chemotherapeutic adjuvant or an immunopotentiating medicament.
PCT/CN2006/000891 2005-04-30 2006-04-30 Interleukin-6 polyethylene glycol conjugate and its preparing method and use WO2006116948A1 (en)

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