WO2006116948A1 - Conjugue de polyethylene glycol et d’interleukine-6 et son procede de preparation et son utilisation - Google Patents

Conjugue de polyethylene glycol et d’interleukine-6 et son procede de preparation et son utilisation 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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    • 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

La présente invention concerne un conjugué de polyéthylène glycol et d’interleukine-6 et son procédé de préparation et ses compositions médicales comprenant le conjugué et des excipients acceptables sur le plan pharmaceutique. Le conjugué en accord avec l’invention est utilisé pour produire des médicaments traitant la thrombocytopénie, des adjuvants de chimiothérapie ou des médicaments améliorant l’immunité. L’IL-6 modifiée par le mono-PEG améliore la biostabilité de façon marquée, a une plus longue demi-vie in vivo et une clairance plasmatique plus faible comparée avec l’IL-6 non modifiée, ce qui résulte en une grande diminution de la fréquence et des doses d’administration de même que des effets secondaires. L’IL-6 modifiée par le mono-PEG en accord avec la présente invention peut atteindre des normes médicinales du fait de sa bonne uniformité.
PCT/CN2006/000891 2005-04-30 2006-04-30 Conjugue de polyethylene glycol et d’interleukine-6 et son procede de preparation et son utilisation WO2006116948A1 (fr)

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EP2217264A4 (fr) * 2007-11-28 2011-11-30 Hadasit Med Res Service Procédés de traitement de lésion tissulaire induite par un rayonnement ou une chimiothérapie
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CN114668852A (zh) * 2017-09-30 2022-06-28 天津键凯科技有限公司 一种聚乙二醇-多肽和蛋白类药物的结合物
CN112110982A (zh) * 2020-09-24 2020-12-22 科兴生物制药股份有限公司 一种蛋白质定点聚乙二醇化修饰的制备方法
CN112110982B (zh) * 2020-09-24 2021-12-07 科兴生物制药股份有限公司 一种蛋白质定点聚乙二醇化修饰的制备方法

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