WO2022095973A1 - 尿酸氧化酶制剂及其应用 - Google Patents

尿酸氧化酶制剂及其应用 Download PDF

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WO2022095973A1
WO2022095973A1 PCT/CN2021/129071 CN2021129071W WO2022095973A1 WO 2022095973 A1 WO2022095973 A1 WO 2022095973A1 CN 2021129071 W CN2021129071 W CN 2021129071W WO 2022095973 A1 WO2022095973 A1 WO 2022095973A1
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urate oxidase
polyethylene glycol
preparation according
modified
urate
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PCT/CN2021/129071
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English (en)
French (fr)
Inventor
何云凤
王志明
付志成
王彧
文海燕
闫天文
胡春兰
苏国威
刘日勇
丁旭朋
范开
王宏英
汪倩
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杭州远大生物制药有限公司
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Priority claimed from CN202011224613.5A external-priority patent/CN114438048B/zh
Application filed by 杭州远大生物制药有限公司 filed Critical 杭州远大生物制药有限公司
Priority to AU2021376596A priority Critical patent/AU2021376596A1/en
Priority to JP2023527119A priority patent/JP2023548207A/ja
Priority to EP21888665.3A priority patent/EP4335454A1/en
Priority to CA3197424A priority patent/CA3197424A1/en
Priority to KR1020237018857A priority patent/KR20230104666A/ko
Publication of WO2022095973A1 publication Critical patent/WO2022095973A1/zh
Priority to US18/312,292 priority patent/US20230346959A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0012—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7)
    • C12N9/0044—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on other nitrogen compounds as donors (1.7)
    • C12N9/0046—Oxidoreductases (1.) acting on nitrogen containing compounds as donors (1.4, 1.5, 1.6, 1.7) acting on other nitrogen compounds as donors (1.7) with oxygen as acceptor (1.7.3)
    • C12N9/0048—Uricase (1.7.3.3)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • 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
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y107/00—Oxidoreductases acting on other nitrogenous compounds as donors (1.7)
    • C12Y107/03—Oxidoreductases acting on other nitrogenous compounds as donors (1.7) with oxygen as acceptor (1.7.3)
    • C12Y107/03003—Factor-independent urate hydroxylase (1.7.3.3), i.e. uricase
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • 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
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner

Definitions

  • the present invention relates to the field of biomedicine, in particular to urate oxidase preparations and pharmaceutical compositions.
  • Gout is a disease caused by long-term purine metabolism disorder or decreased uric acid excretion. Its clinical characteristics are hyperuricemia, and due to poor solubility of uric acid, crystal deposits accumulate in the subcutaneous, joints, and kidneys to form tophi, resulting in repeated attacks. of acute arthritis and involvement of the kidneys causing uric acid urolithiasis and interstitial nephritis. Purines in the human body are converted into uric acid, the final product by enzymes. Under normal circumstances, the blood uric acid content of men is 149-416 mmol/L, and the uric acid content of women's blood is 89-357 mol/L. day, in a state of balance.
  • uric acid nephropathy The deposition of urate in renal tubules and renal interstitium causes inflammation and can lead to chronic urate nephropathy; in patients with severe hyperuricemia (such as patients with malignant tumors such as leukemia and lymphoma), a large amount of uric acid deposition in the short term can cause urinary tract Obstruction leads to acute renal failure, also known as uric acid nephropathy.
  • hyperuricemia is related to the mutation inactivation of the uricase gene during human evolution, so humans cannot synthesize active uricase by themselves.
  • One of the current treatments for hyperuricemia is to use uricase to lower the level of uric acid in a patient's body.
  • Uric acid oxidase (EC 1.7.3.3) is widely found in microorganisms (Bacillus reticulum, Candida monocytogenes, Aspergillus flavus), plants (soybeans, chickpeas), animals (pigs, cattle, dogs, baboons) (Suzuki K, Sakasegawa S, Misaki H, Sugiyama M.J Biosci Bioeng. 2004. 98: 153-158), it can catalyze the oxidation of allantoin by uric acid and release carbon dioxide in the presence of oxygen (Retailleau P, Colloc'h, Denis V, Francoise B. Acta Cryst D. 2004. 60: 453-462.).
  • Active uricase is a tetrameric protein composed of identical subunits, each with a molecular weight of about 34kD and consisting of 301-304 amino acids.
  • the pH value of the highest enzymatic activity of uricase in each solution was 8.0 (Bayol A et al. Biophys Chem. 1995. 54: 229-235.).
  • Active uricase is a tetrameric protein composed of identical subunits, each with a molecular weight of about 34kD and consisting of 301-304 amino acids.
  • the pH value of the highest enzymatic activity of uricase in each solution was 8.0 (Bayol A et al. Biophys Chem. 1995. 54: 229-235.).
  • the highest activity is derived from Aspergillus flavus, reaching 27IU/mg; the second is from Bacillus fastidious, its activity remains at 13IU/mg (HuangSH, Wu T K. Eur J Biochem. 2004. 271: 517-523.).
  • the activity of uricase derived from legumes is only 2-6IU/mg; the uricase derived from mammals, after recombinant expression, the uricase activity of pigs can reach 5IU/mg, and the uricase activity of baboons can reach 5IU/mg. Only 1 IU/mg (Michael H, Susan J.K. 2006. US7056713B1), and human uricase is inactive.
  • urate oxidase needs to ensure its enzymatic activity, stability and storage time when it is prepared into pharmaceutical preparations, and different preparation methods, dosage forms, buffers, stabilizers, etc. will affect the storage time and storage time of urate oxidase.
  • Activity, and at the same time, chemical modification on the protein structure can also affect the stability of urate oxidase preparations.
  • Active urate oxidase is a homotetrameric protein, of which one-third of the amino acids are strongly hydrophobic amino acids, and the tetrameric proteins are easily aggregated to form octamers and larger aggregates.
  • Molecules with a molecular weight of more than 100kDa can effectively induce the body to produce an immune response, while the molecular weight of the unmodified polymer uricase protein has reached 140kDa, and the polymer uricase with a larger molecular weight will have higher immunogenicity.
  • the human body is prone to produce anti-uricase antibodies, which rapidly weaken its efficacy and cause severe allergic reactions, which cannot be used for long-term treatment.
  • Covalent modification of proteins with PEG polyethylene glycol has been shown to reduce protein immunogenicity, increase protein solubility, and prolong protein half-life.
  • a chimeric uricase study of pig origin and baboon origin was performed by Duke University and Sagent (Michael H, Susan J.K. 2006. US7056713B1).
  • the method of this research is to ensure that the activity of the enzyme is not significantly reduced, and the ⁇ - of lysine residues of porcine uricase-like uricase is treated by a 10KDa methoxy-containing polyethylene glycol (10KDa-mPEG-NPC) with a molecular weight of 10KDa.
  • Amino group is modified (the modified product obtained is Pegloticase), and the goal of treating intractable gout in humans has been initially achieved.
  • pegloticase is not suitable for injection, it is suitable for intravenous injection, which reduces the long-term use compliance of subjects, thus severely limiting its clinical application. So far, there are no longer-acting urate oxidase drugs that are less immunogenic and that can be administered subcutaneously.
  • the present invention aims to solve one of the technical problems in the related art at least to a certain extent.
  • the present invention provides a urate oxidase preparation.
  • the urate oxidase preparation includes: an active ingredient, the active ingredient is selected from polyethylene glycol-modified urate oxidase; and an adjuvant, the adjuvant is selected from a buffer reagent, wherein the buffer reagent Include at least one of the following: phosphate, hydrochloride, and carbonate.
  • the formulation of the formulation according to the embodiment of the present invention has a simple composition, the urate oxidase has high stability under the formulation formulation, and the preparation of the formulation can save production costs and has high production efficiency.
  • the urate oxidase preparation according to the above-mentioned embodiment of the present invention also has the following additional technical features:
  • At least 11 of the following amino acid sites in the uricase have PEG modifications: T 1 , K 3 , K 4 , K 30 , K 35 , K 76 , K 79 , K 97 , K 112 , K 116 , K 120 , K 152 , K 179 , K 222 , K 231 , K 266 , K 272 , K 285 , K 291 , K 293 .
  • it further includes ultrafiltration and/or purification of the coupling reaction product. Further, unmodified polyethylene glycol and by-products such as NHS can be effectively removed, and the purity of the obtained polyethylene glycol-modified uric acid oxidase can be effectively improved.
  • At least one of the following 4 amino acid positions has PEG modification, K 30 , K 35 , K 222 and K 231 .
  • the amino acid site positioning is based on the amino acid sequence shown in SEQ ID NO: 1.
  • the urate oxidase has the amino acid sequences shown in SEQ ID NOs: 1-7.
  • the amino acid sequence shown in SEQ ID NO:1 is the amino acid sequence of pig-derived and baboon-derived chimeric uricase (pig baboon);
  • the amino acid sequence shown in SEQ ID NO:2 is the amino acid sequence of pig-derived uricase;
  • the amino acid sequence shown in SEQ ID NO:3 is the amino acid sequence of canine-derived and baboon-derived (canine baboon) chimeric uric acid oxidase;
  • the amino acid sequence shown in SEQ ID NO:4 is the amino acid sequence of canine-derived uric acid oxidase;
  • SEQ ID NO:4 The amino acid sequence shown in ID NO: 5 is the amino acid sequence of bovine urate oxidase;
  • the amino acid sequence shown in SEQ ID NO: 6 is the amino acid sequence of monkey urate oxidase;
  • the amino acid sequence shown in SEQ ID NO: 7 is the baboon amino acid sequence The amino acid sequence of urate oxid
  • K 4 refers to the lysine located at position 4 based on the amino acid sequence shown in SEQ ID NO: 1. amino acid.
  • Uricases having the amino acid sequences shown in SEQ ID NOs: 1-7 or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% compared to SEQ ID NOs: 1-7 , polypeptides with at least 99% identity; or polypeptides with one or more amino acid substitutions, deletions and/or additions compared with SEQ ID NOs: 1-7 have homology in structure, and those skilled in the art can process Sequence alignment to determine SEQ ID NOs: 2-7 or at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% compared to SEQ ID NOs: 1-7 % identical to T 1 , K 3 , K 4 , K 30 , K 35 , on polypeptides having one or more amino acid substitutions, deletions and/or additions compared to SEQ ID NOs: 1-7.
  • the corresponding sites include M 1 , K 9 , K 10 , K 36 , K 41 , K 82 , K 85 , K 103 , K 118 , K 122 , K 126 , K 158 , K 185 , K 228 , K 237 , K 272 , K 278 , K 297 , K 299
  • the corresponding sites of the sequence shown in SEQ ID NO:3 and the corresponding site of the sequence shown in SEQ ID NO:1 include M 1 , K
  • the inventors have found through experiments that after at least 11 positions of the corresponding positions of the amino acid sequences shown in the above SEQ ID NOs: 2 to 7 are modified by PEG, the obtained PEG-modified urate oxidase has low immunogenicity and high in vivo stability. Sex, suitable for the advantages of intramuscular injection.
  • the peptide map of the polyethylene glycol-modified uric acid oxidase has at least 11 peaks of predetermined peptide segments compared with the peptide map of the uric acid oxidase not modified with polyethylene glycol.
  • the relative ratio of the area reduction is not lower than 75%, preferably not lower than 80%, more preferably not lower than 90%.
  • the polyethylene glycol-modified urate oxidase according to the embodiments of the present invention has the advantages of low immunogenicity, high in vivo stability, and suitability for intramuscular injection.
  • the molecular weight of the polyethylene glycol for PEG modification does not exceed 6KD.
  • the inventors found that by using polyethylene glycol with a molecular weight of no more than 6KD for modification, the long-term effect of the obtained urate oxidase in the body is further enhanced, and serious anti-PEG antibodies will not be produced due to excessive molecular weight, that is, the immunogenicity is further reduced. .
  • the polyethylene glycol has a monomethoxy group or a hydroxyl group.
  • the polyethylene glycol has a linear or branched chain structure.
  • the polyethylene glycol is coupled with urate oxidase through an amide bond.
  • the polyethylene glycol is a modified polyethylene glycol
  • the modified group of the modified polyethylene glycol includes at least one selected from the following: N hydroxysuccinimide, N Hydroxysuccinimide Carbonate, N-Hydroxysuccinimide Acetate, N-Hydroxysuccinimide Propionate, N-Hydroxysuccinimide Butyrate, N-Hydroxysuccinimide Succinate, and p-Nitrogen phenyl carbonate.
  • the modified group of the modified polyethylene glycol is N-hydroxysuccinimide propionate.
  • the immunogenicity of urate oxidase can be effectively reduced, and the obtained urate oxidase has higher in vivo safety and longer effect.
  • the buffer reagent includes at least one of the following: disodium hydrogen phosphate, sodium dihydrogen phosphate monohydrate and sodium chloride.
  • disodium hydrogen phosphate, sodium dihydrogen phosphate monohydrate and sodium chloride as auxiliary materials can ensure the enzyme specific activity of urate oxidase, and it can be stored for 30 minutes under the conditions of low temperature, normal temperature and high temperature. The enzyme specific activity, protein degradation and aggregation degree were all in line with expectations.
  • the method of the present invention has a simple prescription and high stability of the preparation.
  • the buffering agent refers to a buffering agent that resists pH changes through the action of its acid-base conjugated components.
  • Buffers may be present in the liquid or solid formulations of the present invention, the buffers of the present invention adjust the pH of the formulation to 7-9, and the buffers that control the pH within the range of 7-9, alone or in combination, include acetic acid Salt, succinate, gluconate, histidine, citrate, phosphate, maleate, dimethylarsinate, 2-[N-morpholinyl]ethanesulfonic acid (MES), Bis(2-hydroxyethyl)iminotris[hydroxymethyl]methane (Bis-Tris), N-[2-acetamido]-2-iminodiacetic acid (ADA), Glycyl Glycine and other organic acid buffers.
  • acetic acid Salt succinate, gluconate, histidine, citrate, phosphate, maleate, dimethylarsinate
  • the pH of the urate oxidase preparation is 7-9, preferably 7.4-8.2.
  • the inventors found that the polyethylene glycol-modified urate oxidase preparation has high stability under the above pH conditions, the urate oxidase is not easy to aggregate and degrade, and the enzyme specific activity is high.
  • the pH of the uricase preparation is: 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2.
  • the mass ratio of the polyethylene glycol-modified urate oxidase to the buffer reagent is (5-6):(6-37), preferably 6:10.
  • the above ratio is used to prepare the preparation, so that the pH of the preparation can be guaranteed to be between 7.4 and 8.2, and the stability of the urate oxidase can be ensured.
  • the mass ratio of the polyethylene glycol-modified urate oxidase to the buffer reagent is 6:10, 6:11, 5:10, 5:11, 5:9, 6:9.
  • the mass ratio of the polyethylene glycol-modified urate oxidase to the phosphate is (5-6): (1-7).
  • the mass ratio of the polyethylene glycol-modified urate oxidase to the sodium chloride is (5-6):(5-30).
  • the mass ratio of the phosphate to the sodium chloride is (1-7): (5-30), wherein the phosphate is disodium hydrogen phosphate and/or sodium dihydrogen phosphate .
  • the urate oxidase preparation provided by the present invention does not need to add stabilizers such as glycerol, glucose, mannitol, Tween-80, etc., and only needs to use a buffer to ensure the urate oxidase provided by the present invention. stability, and obtain a urate oxidase preparation with high stability.
  • stabilizers such as glycerol, glucose, mannitol, Tween-80, etc.
  • the addition of mannitol and/or glycerol to the urate oxidase according to the embodiment of the present invention increases the particle size of the urate oxidase, while the addition of Tween-80 has no significant effect on the stability of the urate oxidase in the embodiment of the present invention.
  • the dosage form of the urate oxidase preparation includes at least one of the following: liquid, semi-solid, and solid.
  • the urate oxidase preparation according to the embodiment of the present invention may be a liquid preparation or a freeze-dried dosage form, which is dissolved into a liquid when used.
  • the urate oxidase preparation is an injection preparation, which can be injected into the body of a patient by intravenous injection, intramuscular injection, etc. .
  • the preparation is in the form of a single dosage form, and each preparation contains 6 mg of urate oxidase.
  • the single-dose urate oxidase preparation of the embodiment of the present invention the single-dose form of urate oxidase is administered in a simple manner, does not require repeated administration every day, is convenient for patients to use, achieves maximum efficacy, and is easy to store.
  • the present invention proposes the use of the urate oxidase preparation proposed in the first aspect of the present invention in preparing a medicament for treating or preventing hyperuricemia and related diseases.
  • the hyperuric acid-related diseases include chronic hyperuricemia, gout, kidney disease, hyperuricemic arthritis, kidney stones, gout nodules, hypertension, diabetes, high triglycerides Hyperuricemia caused by hyperuricemia, metabolic syndrome, coronary heart disease, atherosclerosis, cancer chemotherapy.
  • the present invention provides a pharmaceutical composition.
  • the pharmaceutical composition includes the urate oxidase preparation proposed in the first aspect of the present invention.
  • the pharmaceutical composition further includes at least one of the following additional technical features:
  • the above-mentioned pharmaceutical composition further includes other drugs for treating or preventing hyperuricemia and related diseases.
  • the polyethylene glycol-modified urate oxidase or pharmaceutical compositions of the present invention when administered in combination therapy with other drugs, they may be administered to an individual sequentially or simultaneously.
  • the pharmaceutical composition of the present invention may comprise the polyethylene glycol-modified urate oxidase of the present invention, a pharmaceutically acceptable carrier or a pharmaceutically acceptable excipient, and other therapeutic or preventive drugs known in the art The combination.
  • the pharmaceutical composition has the advantages of low immunogenicity, high in vivo stability, and suitability for intramuscular injection, and can be used for the treatment or prevention of hyperuric acid-related diseases.
  • the pharmaceutical composition further includes a pharmaceutically acceptable adjuvant.
  • the adjuvants include any solvents, solid excipients, diluents, binders, disintegrating agents, or other liquid excipients, dispersing agents, flavoring or suspending agents, surfactants, isotonic agents, enhancers. Thickening agents, emulsifiers, preservatives, solid binders, glidants or lubricants, etc., are suitable for the particular target dosage form.
  • Fig. 1 is the PHC physical and chemical reference substance-SEC-HPLC-UV detection figure according to the embodiment of the present invention
  • Fig. 2 is the PHC physical and chemical reference substance-SEC-HPLC-RI detection figure according to the embodiment of the present invention
  • Fig. 3 is according to the PEG reference substance-SEC-HPLC-RI detection figure of the embodiment of the present invention.
  • Fig. 4 is the PU5 modified product-SEC-HPLC-UV detection figure according to the embodiment of the present invention.
  • Fig. 5 is the PU5 modified product-SEC-HPLC-RI detection chart according to the embodiment of the present invention.
  • Fig. 6 is according to the embodiment of the present invention PHC and PU5 adopt Lys-c and trypsin double enzyme digestion comparison diagram respectively;
  • Fig. 7 is a PU5 Lys-C enzyme cleavage diagram according to an embodiment of the present invention.
  • Fig. 9 is the SEC graph of prescription 5 at 37°C for 30 days according to an embodiment of the present invention.
  • 11 is an SEC graph of formulation 7 at 37°C for 30 days according to an embodiment of the present invention.
  • Figure 12 is the serum uric acid level after intramuscular administration of different doses of model rats according to an embodiment of the present invention.
  • Figure 13 is a score chart of renal injury, necrosis and inflammation according to an embodiment of the present invention.
  • Figure 14 is the average blood concentration-time curve diagram of each group after single intravenous injection of the same dose (1.0mg/kg) of Pegloticase and PEGylated uricase injection in SD rats according to the embodiment of the present invention
  • Figure 15 is the mean blood concentration-time curve diagram of each group after single intramuscular injection of Pegloticase and different doses of PEGylated uricase injection in SD rats according to the embodiment of the present invention
  • Figure 16 is the average blood concentration-time curve diagram of each group after single intramuscular injection of different doses of pegylated uricase injection in SD rats according to the embodiment of the present invention
  • Fig. 17 is the mean value-time curve diagram of blood uric acid in each group at different times after single intramuscular/intravenous injection of different doses of Pegloticase and PEGylated uricase injection in SD rats according to the embodiment of the present invention;
  • Figure 18 is a graph of male and female average blood drug concentration-time curves after the first (Day1) intravenous injection of the same dose (1.0 mg/kg) of Pegloticase and pegylated uricase injection in SD rats according to the embodiment of the present invention;
  • Figure 19 is a graph of the average blood concentration-time curve of male and female after the last (Day22) intravenous injection of the same dose (1.0 mg/kg) of Pegloticase and PEGylated uricase injection in SD rats according to an embodiment of the present invention
  • Figure 20 is a male and female average blood concentration-time curve diagram after intramuscular injection of the same dose (1.0mg/kg) of Pegloticase and pegylated uricase in SD rats for the first time (Day1) according to the embodiment of the present invention;
  • Figure 21 is a male and female average blood concentration-time curve diagram after the last (Day22) intramuscular injection of the same dose (1.0 mg/kg) of Pegloticase and pegylated uricase injection in SD rats according to the embodiment of the present invention;
  • Fig. 22 is the mean value-time curve of blood uric acid at different times after multiple intravenous injections of Pegloticase and pegylated uricase injection in SD rats according to an embodiment of the present invention.
  • Figure 23 is a graph of the mean value-time curve of blood uric acid at different times after multiple intramuscular injections of Pegloticase and pegylated uricase injection in SD rats according to an embodiment of the present invention.
  • the purpose of the present invention is to provide a novel polyethylene glycol-modified urate oxidase preparation.
  • Another object of the present invention is to provide the application of the above polyethylene glycol-modified urate oxidase preparation, which can achieve a long-acting and significantly reducing blood uric acid level in vivo, and can be used for the treatment of hyperuricemia and ventilation.
  • a polyethylene glycol-modified urate oxidase preparation is provided.
  • the urate oxidase is not particularly limited, and can be any source of urate oxidase and urate oxidase analogs thereof, and representative examples include but are not limited to mammalian sources, microorganisms, plants, and the like.
  • the urate oxidase from different species of the present invention can be obtained through various ways, including but not limited to natural extraction, chemical synthesis, recombinant expression by genetic engineering, and the like.
  • the recombinant expression strain is prepared by using Escherichia coli or yeast as the host, and more preferably Escherichia coli is used as the host strain for recombinant expression.
  • the prepared preparation can maximize the enzyme activity and improve the preparation storage stability.
  • the polyethylene glycol urate oxidase preparation does not need to add other stabilizers, such as mannitol, glycerol, Tween-80, etc., to have high storage stability, thus, the The urate oxidase preparation has simple formula, simple preparation process and low cost.
  • the application of the above polyethylene glycol-modified urate oxidase preparation is provided.
  • the polyethylene glycol uric acid oxidase preparation is more suitable as a medicine for treating chronic hyperuricemia or ventilation and its composition.
  • the main symptoms of hyperuricemia and ventilation include, but are not limited to, uric acid nephropathy and ventilation arthritis.
  • the administration route of the polyethylene glycol urate oxidase includes but is not limited to intravenous injection, subcutaneous injection, intramuscular injection and intraperitoneal injection, etc., preferably intravenous injection, intramuscular injection, more preferably intramuscular injection.
  • the polyethylene glycol uricase oxidase has lower in vivo immunogenicity.
  • the low immunogenicity of the PEG oxidase means that after intramuscular injection of PEG oxidase in humans or animals, the body does not produce antibodies against polyethylene glycol molecules or produce low titers. anti-PEG molecule antibody. Antibodies against urate oxidase are not produced.
  • the polyethylene glycol uric acid oxidase After intramuscular injection, the polyethylene glycol uric acid oxidase has a longer half-life in the body and the effect of reducing the level of uric acid in the body.
  • first and second are only used for descriptive purposes, and should not be construed as indicating or implying relative importance or implying the number of indicated technical features. Thus, a feature delimited with “first”, “second” may expressly or implicitly include at least one of that feature.
  • plurality means at least two, such as two, three, etc., unless otherwise expressly and specifically defined.
  • the cDNA sequence of uricase protein (code: PHC) (SEQ ID NO: 1) was designed, and the whole gene was synthesized and named as pUC-57-PHC plasmid. Nde I and BamHI were used as the target gene insertion sites, and the pET-30a plasmid was used as the expression vector (pET-30a-PHC).
  • the expression vector pET-30a-PHC was introduced into Escherichia coli BL21 (DE3) by the CaCl 2 method, and kanamycin was used for resistance screening to screen out high-expressing clones, and save the original seed bank strain (E3B). Methods commonly used in the field of biology are implemented.
  • Frozen cells were taken and suspended in 25mmol/L Tris, 5mmol/L EDTA buffer, and the suspension ratio was 1:10 (W/V). After breaking the bacterial cells by high pressure, the urate oxidase precipitate was collected by centrifugation. After the precipitate was washed once with 50mmol/L NaHCO 3 , the enriched uricase precipitate was suspended in 100mmol/L Na 2 HCO 3 (pH9.7 ⁇ 10.3) buffer In the liquid, the suspension ratio was 1:50 (W/V), and the mixture was stirred overnight at room temperature to dissolve, and then the supernatant was collected by centrifugation.
  • Uric acid oxidase was further purified by several chromatographic steps. The purity was more than 95% detected by SDS-PAGE, and the purity was more than 95% detected by Superdex 200 column. There was no aggregate form. Uric acid oxidase activity was measured by meter, and 1 unit (U) of enzyme activity was defined as the amount of enzyme required to convert 1 ⁇ mol of uric acid per minute under the optimal reaction temperature of 37° C. and the optimal pH of 9.0 buffer.
  • the concentration of urate oxidase in the coupling reaction is 10 mg/ml, and the coupling reaction needs to be stirred at 5 to 30 °C for 60 minutes. minutes or more until the degree of PEG coupling no longer changes with time.
  • unmodified PEG and by-products are removed from the reaction by ultrafiltration and/or chromatography. Appropriate molecular sieve chromatography media can be selected for the separation and removal of modified by-products. Finally, sterile filtration to obtain 5K-modified PEGylated urate oxidase (codenamed PU5).
  • the protein concentration was determined by Lowry method, and the activity of polyethylene glycol uricase was determined by spectrophotometer.
  • the maximum UV absorption wavelength of uricase substrate uric acid is 293 nm, while the maximum UV absorption wavelength of product allantoin is 224 nm. Within a certain concentration range, the absorption value of uric acid at 293 nm is proportional to its concentration. Quantitative determination. The specific process is as follows: turn on the UV-Vis spectrophotometer, adjust the wavelength to 293 nm, and turn on the water bath circulation system of the instrument to keep the temperature at 37 °C.
  • Degradation concentration and calculation of enzyme activity definition of enzyme activity: when the optimum reaction temperature is 37°C and the optimum reaction pH is 9.5, the amount of enzyme required to convert 1 ⁇ mol of uric acid into allantoin per minute is defined as one activity unit (U) .
  • the average modification degree of PEG uricase was detected by SEC-HPLC in tandem with UV/RI (a combination of UV and refractive index detectors).
  • UV/RI a combination of UV and refractive index detectors.
  • the protein has the maximum absorption peak at ultraviolet 280nm, and PEG has no absorption at this wavelength, and the absorption value of the differential refraction detector for protein and PEG in a certain range is proportional to its various concentrations. Therefore, the respective contents of PEG and protein moieties in PEGylated urate oxidase can be obtained by the external standard method of PEG reference substance and PHC physical and chemical reference substance, and then the number of PEG molecules on each urate oxidase monomer can be obtained by the following calculation method, That is, the average modification degree.
  • Average degree of modification of PEG urate oxidase (relative molecular weight of urate oxidase subunit ⁇ amount of PEG in sample)/(relative molecular weight of PEG ⁇ amount of protein in sample).
  • Example 2 Under different feeding ratios, the enzymatic activity and average modification degree of the obtained polyethylene glycol oxidase uricase are shown in Table 1.
  • Protein:5K-PEG feeding molar ratio Enzyme activity Enzyme activity retention average modification unmodified uricase 11.4U/mg 100% 0 1:48 10.71U/mg 94% 10.3 1:56 11.17U/mg 103.4% 11.4 1:68 12.2U/mg 107.1% 11.9 1:82 12.02U/mg 105.4% 12.3 1:94 11.75U/mg 103.1% 12.1 1:110 10.83U/mg 95% 11.5 1:150 10.03U/mg 88% 10.1
  • the average modification degree indicates the number of PEG molecules bound to each uricase monomer.
  • the average modification degree of the polyethylene glycol urate oxidase of the present application in the range of protein:5K-PEG feeding ratio 1:56-1:110 is stable at more than 11, and the enzyme activity is compared to Unmodified urate oxidase has a high retention rate of enzymatic activity, the enzymatic activity does not decrease, but increases, and the enzymatic activity is relatively stable.
  • the polyethylene glycol urate oxidase of the present application has a higher average modification degree of polyethylene glycol than the marketed drugs, and has also achieved unexpected technical effects in terms of enzyme activity retention.
  • the inventors speculate that it may be caused by the difference in the degree of PEG modification or modification site of PEG uricase.
  • Example 2 the inventors detected the modification site of the urate oxidase obtained in Example 2.
  • the PEG-modified site of PEG-modified urate oxidase can be digested with one or more enzymes for non-PEGylated and PEGylated urate oxidase, and then subjected to chromatographic detection to obtain a chromatogram, That is, peptide map confirmation.
  • Non-pegylated and pegylated urate oxidase can be digested by single enzyme (Lys-C or Trypsin) and or double enzyme (Lys-C and Trypsin combined).
  • Reversed-phase column was used to separate the digested fragments, and the modification site of PEG uricase was determined by the internal reference peptide correction and comparison of the disappearance or reduction ratio of the peptide.
  • Lys-C can specifically cut the C-terminus of lysine (K), and trypsin uses the basic amino acid-arginine (R ) and lysine (K) were used as restriction sites to specifically cut the C-terminal peptide bond. Comparing the changes of the corresponding peptides before and after enzyme cleavage in PHC and PU5, and combining with the internal standard peptides, the relative proportion of PEG-modified peptides reduced or disappeared can be analyzed and confirmed.
  • the modification ratio can be determined by the relative ratio of the reduction or disappearance of the peptide. It should be pointed out that PEG modification is a non-uniform modification, and a site with a high modification ratio can be considered to be modified.
  • Sample treatment Take urate oxidase and PEGylated urate oxidase and dissolve them in enzyme digestion buffer (25mmol/L Tris-HCl, 20% acetonitrile, pH 9.0) to dilute to 1mg/ml, respectively, take 100 ⁇ l of each , add 2 ⁇ l Lys-C, and digest at 37°C for 4 hours. The solution was transferred to a pancreatin reaction tube (1:100 ratio) at 37°C for 2 hours, and 4 ⁇ l of TCEP reducing solution was used to continue the reaction for 30 minutes, and then 10 ⁇ l of 1mol/L hydrochloric acid solution was added to stop the reaction.
  • enzyme digestion buffer 25mmol/L Tris-HCl, 20% acetonitrile, pH 9.0
  • liquid A water solution containing 0.1% TFA
  • liquid B acetonitrile solution containing 0.1% TFA
  • Ion source ESI
  • Ion type positive ion
  • the post-column split was about 0.3 ml/min.
  • the sample volume was injected 100 ⁇ l, and the chromatogram was recorded.
  • the peak area of PU5 peptide corresponding to the same concentration of PU5 and PHC can be calculated by the following formula:
  • a 1 is the peak area of the PU5 peptide after conversion of the two internal reference peptides
  • a 0 is the measured peak area of the PU5 peptide map
  • t is the average ratio of the peak area of the PHC peptide map to the PU5 peptide map in the internal reference peptides T30 and T31. value, which is 0.588.
  • the relative percentage reduction of the peak area of a peptide in the PU5 peptide map can be calculated by using the following formula to calculate the peak area of the peptide segment converted from the internal reference and the peak area of the PHC peptide map:
  • a 2 is the peak area of a peptide in the PHC peptide map
  • a 1 is the peak area of the peptide in PU5 after conversion by the internal reference.
  • Table 5 Summary results of peptides with reduced peak area in the peptide map after double digestion of PU5
  • the potential sites for urate oxidase modification include T 1 , K 3 , K 4 , K 17 , K 21 , K 30 , K 35 , K 48 , K 49 , K 66 , K 74 , K 76 , K 79 , K 97 , K 112 , K 116 , K 120 , K 152 , K 155 , K 158 , K 169 , K 179 , K 190 , K 215 , K 222 , K 231 , K 266 , K 272 , K 285 , K 291 , K 293 and other 31 sites.
  • the inventors found that the modification sites of the polyethylene glycol-modified urate oxidase of the present application are more than those of the marketed drugs, and there are significant differences.
  • the disappearance ratio of ethylene glycol-modified urate oxidase at the four sites of K 30 , K 35 , K 222 and K 231 is higher than 80%, while the similar drug Krystexx (pegloticase), which has been listed on the market, is analyzed by the method.
  • the peptide segments where these four sites are located have hardly disappeared, that is, the modification rate of the similar drugs on the market at K 30 , K 35 , K 222 and K 231 is much lower than that of the polyethylene glycol-modified urate oxidase of this application .
  • the polyethylene glycol-modified urate oxidase of the present application has significantly lower immunogenicity than the marketed drugs, and the inventor speculates that it may be related to the number of modification sites and the difference in modification sites. Due to the different modification sites and degrees of modification, the protection of the enzyme's immunogenic site in vivo and the exposure of the enzyme's active center are different. The above differences result in different biological properties of different modified enzymes in vivo.
  • urate oxidase is a protein preparation, different chemical modifications will have different effects on its stability. Therefore, when preparing a preparation, it is necessary to select different excipients, pH, etc. for specific urate oxidase active ingredients to prepare urate oxidase preparations , the following will describe the preparation of uric acid oxidase preparations with high stability and high biological activity for the polyethylene glycol-modified uric acid oxidase of the present invention.
  • the components of the polyethylene glycol-modified uricase preparation were screened to obtain polyglycolase with high uricase activity and high stability. Glycol uricase preparation.
  • the screening includes: excipient formula screening and stabilizer screening.
  • Prescription excipients include: carbonate, phosphate, hydrochloride, citrate.
  • the excipients are in the same concentration range of 10-50 mmol/L and under the same pH (pH7-9) conditions, polyethylene glycol-modified uric acid oxidase preparations
  • the stability is basically the same, but the concentration ratio of different excipients and the difficulty of controlling the osmotic pressure of the preparation are different in the preparation process. Among them, phosphate and hydrochloride are easier to control osmotic pressure and pH than carbonate and citrate.
  • Mannitol, glycerin and Tween-80 are added to the basic formulation, and it is necessary to add corresponding stabilizers to the formulation of this product through stability inspection.
  • the basic formulation was used as a control.
  • Stabilizers such as 4% mannitol, 2% glycerol, and 0.04% Tween-80 were added to the basic formulation.
  • the sample numbers were recorded as sample 1 to sample 4, and samples 1-4 were placed in 45 °C was investigated for 7 days, the particle size was detected by sampling, and the effect of stabilizer on the stability of PU5 preparation was studied.
  • Control means placed under the condition of 2 ⁇ 8°C.
  • the pH of the formulation formulation solution was adjusted with buffer salts to be 7.8, 7.0, 7.4, 8.2, and 8.6, respectively.
  • the samples were placed at 2-8°C, 25 ⁇ 2°C and 37°C for stability study, respectively, and the content of high and low molecular weight proteins and enzyme activities were measured in 30 days.
  • the polymerization or degradation of PU5 protein and changes in enzyme activity in each formulation were compared.
  • the polyethylene glycol-modified urate oxidase of the present application has significantly lower immunogenicity than the marketed drugs, and the inventor speculates that it may be related to the number of modification sites and the difference in modification sites. Due to the different modification sites and degrees of modification, the protection of the enzyme's immunogenic site and the exposure of the enzyme's active center in vivo are different. The above differences may cause different biological properties of different modified enzymes in vivo.
  • the in vivo evaluation of the polyethylene glycol-modified urate oxidase preparation (PU5 preparation) of the present application will be described in detail below.
  • the pegloticase used in the experiment refers to a similar drug already on the market, and the batch number is 5085B.
  • the rat model of chronic hyperuricemia was induced by potassium oxonate drinking water combined with high uric acid diet, and the therapeutic effect of polyethylene glycol uric acid oxidase (PU5 preparation) on chronic hyperuricemia in rats was evaluated.
  • PU5 preparation polyethylene glycol uric acid oxidase
  • model rats were selected and randomly divided into 4 groups, namely model group, low-dose pegylated uricase administration group (0.3 mg/kg), and pegylated uricase medium-dose administration group (1.0 mg/kg). kg), pegylated uricase high-dose administration group (3.0 mg/kg), 10 rats in each group, and 10 normal SD rats were selected as blank control group.
  • the experiment was continuously established for 5 weeks, and intramuscular administration was started after 1 week of modeling, once a week for 4 consecutive weeks, and the serum uric acid and serum urea of the rats before administration and 7 days after each administration were detected respectively. Nitrogen, serum creatinine levels, and histological changes in rat kidneys were observed after the experiment.
  • each administration group of PEGylated uricase significantly improved renal tubular dilatation, renal necrosis and inflammation.
  • Get 36 SD rats, half male and half are randomly divided into 6 groups (see Table 9), namely the marketed drug Pegloticase intravenous injection group, intramuscular injection group, polyethylene glycol uric acid oxidase intravenous injection group and polyethylene glycol uric acid oxidation group.
  • Enzyme low, medium and high (0.5, 1.0, 2.0 mg/kg) dose intramuscular injection groups, the specific dosing schedule and dose are shown in Table 9. Jugular vein blood was taken to detect PK and PD.
  • the serum drug concentration levels of all individuals in SD rats were lower than the lower limit of quantification (LLOQ: 312.500ng/mL) before administration, a single intramuscular injection of 0.5, 1.0, 2.0 mg/kg, at 0-168h (0-7 days) Within the range, the serum drug concentration after pegloticase injection (PU5 preparation) was dose-related, and the overall level increased with the increase of the administered dose.
  • LLOQ lower limit of quantification
  • the AUClast of the SD rats given the same dose (1.0 mg/kg) of the marketed drug Pegloticase intravenously was 426.48 ⁇ 65.34, the AUClast of the intramuscular injection group was 264.19 ⁇ 78.22; the AUClast of the PU5 formulation injection intravenously administered group was 565.61 ⁇ 161.60, the AUClast of the intramuscular injection group was 337.86 ⁇ 227.34.
  • the AUClast of the PU5 preparation was higher than that of the marketed drug Pegloticase.
  • SD rats were given the same dose (1.0mg/kg) of the marketed drug Pegloticase intravenously, and the t1/2(h) of the group was 49.51 ⁇ 8.12, and the t1/2(h) of the intramuscular administration group was 55.21 ⁇ 13.50.
  • the t1/2(h) of the intravenous administration group of PU5 preparation injection was 86.12 ⁇ 33.82, and the t1/2(h) of the intramuscular administration group was 60.45 ⁇ 21.37.
  • the t1/2(h) of PU5 preparation injection was longer than that of the marketed drug Pegloticase.
  • Table 10 Individual blood concentration data and statistical analysis data of single intravenous injection of 1.0 mg/kg Pegloticase in SD rats (unit: ⁇ g/mL)
  • Table 11 Individual blood drug concentration data and statistical analysis data of SD rats with a single intravenous injection of 1.0 mg/kg pegylated uricase injection (unit: ⁇ g/mL)
  • Table 12 Individual blood concentration data and statistical analysis data of single intramuscular injection of 1.0 mg/kg Pegloticase in SD rats (unit: ⁇ g/mL)
  • Table 13 Individual blood concentration data and statistical analysis data of SD rats with a single intramuscular injection of 1.0 mg/kg PEGylated uricase injection (unit: ⁇ g/mL)
  • Table 14 Mean pharmacokinetic parameters after a single intravenous injection of Pegloticase and PEGylated uricase in SD rats
  • the uric acid concentration was maintained at a low level 1 day and 3 days after administration, and the uric acid level of each dose group began to start 7 days after administration. recovery, and the higher the dose, the longer the uric acid remains low in the body.
  • the time of maintaining a low level of serum uric acid in the intravenous injection group of PU5 preparations was longer than that in the intravenous injection group of pegloticase.
  • the time of maintaining a low level of serum uric acid in the intramuscular injection group of PU5 preparations was longer than that in the intramuscular injection group of pegloticase.
  • the time of maintaining the low level of serum uric acid in the intravenous injection or intramuscular injection group of PU5 preparation was longer than that in the intravenous injection group or intramuscular injection group of pegloticase, that is, the time for PU5 preparation to maintain the low concentration level in vivo was longer than that of pegloticase. longer.
  • the results are shown in Figure 17.
  • Pegloticase intravenous injection group There are 4 groups in this experiment, namely the marketed drug Pegloticase intravenous injection group, intramuscular injection group, PEGylated uricase injection (PU5) intravenous injection group, and intramuscular injection group, with 8 animals in each group, half male and half male, with a total of 32 animals. SD rats.
  • the intravenous injection group of Pegloticase and PEGylated uricase injection was administered intravenously; the intramuscular injection of Pegloticase and PEGylated uricase injection group was administered intramuscularly.
  • the administered doses were all 1.0 mg/kg. Administered once a week for 4 consecutive doses.
  • the antibodies produced by the PU5 preparation and pegloticase are mainly antibodies against the PEG moiety, not against the urate oxidase moiety, indicating that both can effectively shield the immunogenic site of urate oxidase.
  • the production of PEG antibodies can lead to some side effects in vivo.
  • the immunogenicity of the PU5 preparation of the present application is lower than that of the commercial product pgeloticase.
  • both the PU5 preparation and pegloticase were better in the intramuscular administration group than in the intravenous administration group, and the anti-PEG antibody produced in the intravenous administration group, PU5 preparation was better than pegloticase; intramuscular administration group The anti-PEG antibody produced, the PU5 formulation was superior to pegloticase.
  • SD rats were given the same dose (1.0mg/kg) of the marketed drug Pegloticase by intravenous/muscular injection. After the first administration, the absolute bioavailability in rats was 51.35%; The absolute bioavailability in vivo was 45.98%, respectively. SD rats were given the same dose (1.0 mg/kg) of pegylated uricase injection by multiple intravenous/muscular injections. After the first dose, the absolute bioavailability in rats was 58.29%; the last dose was 58.29%. After that, the absolute bioavailability in rats was 52.60%.
  • SD rats were injected intravenously and intramuscularly 4 times (1 time/week) with 1.0 mg/kg Pegloticase and PEGylated uricase injection.
  • Serum uric acid concentration was maintained at a low level after each administration. 14 days after intramuscular injection of Pegloticase, recovery occurred, and the other groups recovered 18 days after the last administration.
  • the maintenance time of the intravenous injection groups of the two drugs was relatively consistent, while the maintenance time of the intramuscular injection group of PEGylated uricase injection was longer than that of the marketed drug, that is, the PU5 preparation was administered intramuscularly. Efficacy is better than Pegloticase.

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Abstract

提供了一种尿酸氧化酶制剂，该尿酸氧化酶制剂包括：活性成分聚乙二醇修饰的尿酸氧化酶，以及辅料缓冲试剂。

Description

尿酸氧化酶制剂及其应用 技术领域
本发明涉及生物医药领域，具体地，涉及尿酸氧化酶制剂、药物组合物。
背景技术
痛风是长期嘌呤代谢紊乱或尿酸排泄减少所引起的疾病，其临床特点为高尿酸血症，并因尿酸盐溶解性较差，结晶沉淀累积在皮下、关节、肾脏形成痛风石，导致反复发作的急性关节炎，并累及肾脏引起尿酸性尿路结石和间质性肾炎。人体内嘌呤经酶作用转化终产物尿酸，正常情况下，男性血液中尿酸含量149～416mmol/L,女性血液中尿酸含量89～357mol/L，体内尿酸量约1200mg，生成与排泄量约600mg/天，处于平衡状态。但机体摄入尿酸过多或排泄机制出现障碍，造成尿酸在血液中累积达到70mg/L以上，则会引起高尿酸血症。尿酸钠在血液或滑膜液中达到饱和状态结晶析出，或长时间高尿酸血症在关节、软组织周围结晶沉淀，可导致痛风性急性关节炎或痛风性慢性关节炎以及关节畸形。尿酸盐在肾小管、肾间质内的沉积引发炎症可导致慢性尿酸盐肾病；严重的高尿酸血症患者(如白血病和淋巴瘤等恶性肿瘤患者)，短期内大量尿酸沉积造成尿路阻塞引发急性肾功能衰竭，又称尿酸肾病。
高尿酸血症的成因，与人在进化过程中尿酸酶基因的突变失活有关，因此人类不能自身合成具有活性的尿酸酶。目前治疗高尿酸血症的方法之一是使用尿酸酶降低患者体内的尿酸含量。
尿酸氧化酶(E C 1.7.3.3)广泛存在于微生物(苛求芽孢杆菌、单假丝酵母、黄曲霉)、植物(大豆、鹰嘴豆)、动物(猪、牛、狗、狒狒)中(Suzuki K，Sakasegawa S，Misaki H，SugiyamaM.J Biosci Bioeng.2004.98：153-158)，在氧气存在的情况下，它能催化尿酸氧化尿囊素并放出二氧化碳(Retailleau P，Colloc’h，Denis V，Francoise B.Acta Cryst D.2004.60：453-462.)。具有活性的尿酸酶是一四聚体蛋白，由相同的亚基组成，每个亚基分子量在34kD左右，由301-304个氨基酸组成。每个溶液中尿酸酶的酶活力最高的pH值为8.0(Bayol A etal.Biophys Chem.1995.54：229-235.)。
具有活性的尿酸酶是一四聚体蛋白，由相同的亚基组成，每个亚基分子量在34kD左右，由301-304个氨基酸组成。每个溶液中尿酸酶的酶活力最高的pH值为8.0(Bayol A etal.Biophys Chem.1995.54：229-235.)。在目前所知的所有来源的尿酸酶中，活性最高的来源于黄曲霉，达到27IU/mg；其次是来源于苛求芽胞杆菌，它的活性保持在13IU/mg(HuangS H，Wu T K.Eur J Biochem.2004.271：517-523.)。另外，来源于豆类植物的尿酸酶，其活性只有2～6IU/mg；来源于哺乳动物的尿酸酶，经过重组表达以后，猪的尿酸酶活性可以达到5IU/mg，狒狒的尿酸酶酶活性只有1IU/mg(Michael H，Susan J.K.2006.US7056713B1)，而人源尿酸酶无活性。
作为人体应用，微生物尿酸酶的高活性和哺乳动物尿酸酶的低免疫原性，使得这两大来源的尿酸酶成了目前开发应用的重组尿酸酶的研究热点。但黄曲霉来源的尿酸酶与推测出的人源尿酸酶的同源性不足40％(Lee C C，Wu X，Gibbs R A，Cook R G，Muzny D M，CaskeyC T.Science.1988.239：1288-1291.)，人体易产生抗尿酸酶抗体，黄曲霉尿酸酶的功效迅速减弱，同时引发严重过敏性反应，无法用于长期治疗。
尿酸氧化酶作为一种蛋白质，在制备成药物制剂时需保证其酶活、稳定性及存放时间，而不同的制备方法、剂型、缓冲剂、稳定剂等均会影响尿酸氧化酶的存放时间及活性，同时，在蛋白结构上进行化学修饰也会影响尿酸氧化酶制剂的稳定性。
因此，研发一种能够稳定存放，保证尿酸氧化酶活性的尿酸氧化酶制剂很有必要。
发明内容
本申请是基于发明人对以下事实和问题的发现和认识作出的：
活性尿酸氧化酶是同源四聚体蛋白，其中三分之一的氨基酸为强疏水性氨基酸，四聚体蛋白间容易聚集形成八聚体及更大的聚合体。分子量超过100kDa的分子即可有效诱导机体产生免疫反应，而未修饰聚体尿酸氧化酶蛋白的分子量已经达到140kDa，更大分子量的多聚体尿酸酶将具有更高的免疫原性。人体易产生抗尿酸酶抗体，使其功效迅速减弱，同时引发严重过敏性反应，无法用于长期治疗。用PEG(聚乙二醇)对蛋白进行共价修饰，已证明可降低蛋白免疫原性、增加蛋白溶解度并延长蛋白的半衰期。
杜克大学(Duke University)和山景公司(Savient)进行了猪来源和狒狒来源嵌合尿酸酶研究(Michael H，Susan J.K.2006.US7056713B1)。该研究的方法是在保证酶活性不明显减少的情况下，通过分子量为10KDa含甲氧基的聚乙二醇(10KDa-mPEG-NPC)对类猪源尿酸酶赖氨酸残基的ε-氨基进行修饰(所获得的修饰产品为Pegloticase)，初步实现了在人体中用于治疗顽固性痛风的目标。发明人发现，上述研究成果并不能彻底解决药物引起的免疫原性问题，临床受试者多次注射后出现了尿酸酶疗效消失的现象，发明人推测这可能与Pegloticase蛋白分子量过大有关(采用10kd的PEG，导致Pegloticase分子量为540kDa)，同时由于pegloticase不适于注射，适于静脉推注，降低了受试者长期使用的依从性，进而严重限制了其临床应用。到目前为止，还没有免疫原性更低的以及可以采用皮下注射的长效尿酸氧化酶药物。
使用聚乙二醇对尿酸氧化酶进行修饰，依据修饰数量及修饰位点的不同，会改变尿酸氧化酶的性质，进而尿酸氧化酶制剂的配方需要改变，以保证不同尿酸氧化酶在制剂中的酶活、稳定性及保存时间符合标准。
本发明旨在至少在一定程度上解决相关技术中的技术问题之一。
在本发明的第一方面，本发明提出了一种尿酸氧化酶制剂。根据本发明的实施例，所述尿酸氧化酶制剂包括：活性成分，所述活性成分选自聚乙二醇修饰的尿酸氧化酶；以及辅料，所述辅料选自缓冲试剂，其中所述缓冲试剂包括下列至少之一：磷酸盐、盐酸盐和碳酸盐。根据本发明实施例的制剂的处方构成简单，尿酸氧化酶在制剂处方下稳定性较高，该制剂的制备能够节约生产成本，生产效率高。
另外，根据本发明上述实施例的尿酸氧化酶制剂，还具有如下附加的技术特征：
根据本发明的实施例，所述尿酸氧化酶中的下列氨基酸位点中的至少11个具有PEG修饰：T 1、K 3、K 4、K 30、K 35、K 76、K 79、K 97、K 112、K 116、K 120、K 152、K 179、K 222、K 231、K 266、K 272、K 285、K 291、K 293。
根据本发明的实施例，进一步包括将偶联反应产物进行超滤和/或纯化处理。进而可有效去除未修饰的聚乙二醇及副产物，如NHS，有效提高所获得的聚乙二醇修饰的尿酸氧化酶的纯度。
根据本发明的实施例，下列4个氨基酸位点的至少之一具有PEG修饰，K 30、K 35、K 222和K 231。
根据本发明的实施例，所述氨基酸位点定位是以SEQ ID NO:1所示的氨基酸序列定位的。
Figure PCTCN2021129071-appb-000001
根据本发明的实施例，所述尿酸氧化酶具有SEQ ID NO:1～7所示的氨基酸序列。
Figure PCTCN2021129071-appb-000002
Figure PCTCN2021129071-appb-000003
其中，SEQ ID NO:1所示的氨基酸序列是猪来源和狒狒来源嵌合尿酸酶(猪狒狒)的氨基酸序列；SEQ ID NO:2所示的氨基酸序列是猪源尿酸氧化酶的氨基酸序列；SEQ ID NO:3所示的氨基酸序列是犬来源和狒狒来源(犬狒狒)嵌合尿酸氧化酶的氨基酸序列；SEQ ID NO:4所示的氨基酸序列是犬源尿酸氧化酶的氨基酸序列；SEQ ID NO:5所示的氨基酸序列是牛源尿酸氧化酶的氨基酸序列；SEQ ID NO:6所示的氨基酸序列是猴子的尿酸氧化酶氨基酸序列；SEQ ID NO:7所示的氨基酸序列是狒狒的尿酸氧化酶氨基酸序列。
需要说明的是，本申请的赖氨酸的定位是依据SEQ ID NO:1所示的氨基酸序列进行的，如K 4是指基于SEQ ID NO:1所示氨基酸序列，位于第4位的赖氨酸。具有SEQ ID NO:1～7所示氨基酸序列的尿酸酶或与SEQ ID NO:1～7相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少99％同一性的多肽；或与SEQ ID NO:1～7相比具有一个或者多个氨基酸的取代、缺失和/或添加的多肽在结构上具有同源性，本领域技术人员可经过序列比对，确定SEQ ID NO:2～7或与SEQ ID NO:1～7相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少99％同一性的多肽或与SEQ ID NO:1～7相比具有一个或者多个氨基酸的取代、缺失和/或添加的多肽上对应于T 1、K 3、K 4、K 30、K 35、K 76、K 79、K 97、K 112、K 116、K 120、K 152、K 179、K 222、K 231、K 266、K 272、K 285、K 291、K 293位点的对应位点，进而在上述多肽所比对上的对应位点发生PEG修饰，实现本申请的聚乙二醇修饰的尿酸氧化酶的低免疫原性、高体内稳定性、适于肌肉注射的优势。
例如，根据本发明的实施例，SEQ ID NO:2所示序列与SEQ ID NO:1所示序列的T 1、K 3、K 4、K 30、K 35、K 76、K 79、K 97、K 112、K 116、K 120、K 152、K 179、K 222、K 231、K 266、K 272、K 285、K 291、K 293位点的对应的位点包括M 1、K 9、K 10、K 36、K 41、K 82、K 85、K 103、K 118、K 122、K 126、K 158、K 185、K 228、K 237、K 272、K 278、K 297、K 299；SEQ ID NO:3所示序列与SEQ ID NO:1所示序列相应位点的对应位点包括M 1、K 3、K 29、K 34、K 75、K 78、K 111、K 115、K 119、K 151、K 178、K 221、K 230、K 265、K 271、K 284、K 290、K 292；SEQ ID NO:4所示序列与SEQ ID NO:1所示序列相应位点的对应位点包括M 1、K 9、K 10、K 36、K 41、K 82、K 85、K 118、K 122、K 126、K 158、K 185、K 228、K 237、K 272、K 278、K 297、K 299；SEQ ID NO:5所示序列与SEQ ID NO:1所示序列相应位点的对应位点包括M 1、K 10、K 36、K 41、K 82、K 85、K 118、K 122、K 126、K 158、K 185、K 228、K 237、K 272、K 278、K 297、K 299；SEQ ID NO:6所示序列与SEQ ID NO:1所示序列相应位点的对应位点包括M 1、K 9、K 10、K 36、K 41、K 82、K 85、K 118、K 122、K 126、K 158、K 185、K 228、K 237、K 272、K 278、K 291、K 297、K 299；SEQ ID NO:7所示序列与SEQ ID NO:1所示序列相应位点的对应位点包括M 1、K 9、K 10、K 36、K 41、K 82、K 85、K 118、K 122、K 126、K 158、K 185、K 228、K 237、K 272、K 278、K 291、K 297、K 299。发明人通过实验发现，上述SEQ ID NO:2～7所示氨基酸序列的相应位点的至少11个位点发生PEG修饰后，所获得PEG修饰的尿酸氧化酶具有低免疫原性、高体内稳定性、适于肌肉注射的优势。
根据本发明的实施例，所述聚乙二醇修饰的尿酸氧化酶的肽图与未被聚乙二醇修饰的所述尿酸氧化酶的肽图相比，具有至少11个预定肽段的峰面积减少的相对比例不低于75％，优选不低于80％，更优选不低于90％。根据本发明的实施例的聚乙二醇修饰的尿酸氧化酶具有低免疫原性、高体内稳定性、适 于肌肉注射的优势。
根据本发明的实施例，所述PEG修饰用聚乙二醇的分子量不超过6KD。发明人发现，采用分子量不超过6KD聚乙二醇进行修饰，所获得的尿酸氧化酶在体内的长效性进一步增强，且不会因分子量过大产生严重抗PEG抗体，即免疫原性进一步降低。
根据本发明的实施例，所述聚乙二醇具有单甲氧基或羟基。
根据本发明的实施例，所述聚乙二醇为直链或支链结构。
根据本发明的实施例，所述聚乙二醇与尿酸氧化酶通过酰胺键偶联。
根据本发明的实施例，所述聚乙二醇为修饰性聚乙二醇，所述修饰性聚乙二醇的修饰基团包括选自下列的至少之一：N羟基琥珀酰亚胺、N羟基琥珀酰亚胺碳酸酯、N羟基琥珀酰亚胺乙酸酯、N羟基琥珀酰亚胺丙酸酯、N羟基琥珀酰亚胺丁酸酯、N羟基琥珀酰亚胺琥珀酸酯和对硝基苯碳酸酯。
根据本发明的实施例，所述修饰性聚乙二醇的修饰基团为N羟基琥珀酰亚胺丙酸酯。
根据本发明实施例的方法，可有效降低尿酸氧化酶的免疫原性，所得到的尿酸氧化酶的体内安全性更高，作用更加长效。
可以理解的是，前面所述的制备聚乙二醇修饰的尿酸氧化酶方法的附加技术特征和附加技术特征所具有的技术效果适用于根据本发明实施例的上述降低尿酸氧化酶免疫原性的方法的附加技术特征，根据本发明实施例的上述降低尿酸氧化酶免疫原性的方法的附加技术特征在此不再赘述。
根据本发明的实施例，所述缓冲试剂包括下列至少之一：磷酸氢二钠、一水合磷酸二氢钠和氯化钠。根据本发明实施例的尿酸氧化酶制剂，采用磷酸氢二钠、一水合磷酸二氢钠和氯化钠作为辅料可以保证尿酸氧化酶的酶比活性，在低温，常温，高温的条件下保存30天的酶比活性、蛋白质降解与聚集程度等指标均符合预期。并且，相对于常规的尿酸氧化酶制剂需要添加甘氨酸、蔗糖等，本发明的方法处方简单，且制剂的稳定性高。
根据本发明的实施例，所述缓冲试剂是指通过其酸碱共轭组分的作用抵抗pH变化的缓冲剂。缓冲剂可存在于本发明的液体或固体制剂中，本发明的缓冲剂将制剂的pH调节至7～9，将控制pH在7～9范围内的呈单独或组合形式的缓冲剂包括乙酸盐、丁二酸盐、葡糖酸盐、组氨酸、柠檬酸盐、磷酸盐、顺丁烯二酸盐、二甲基胂酸盐、2-[N-吗啉基]乙烷磺酸(MES)、双(2-羟乙基)亚氨基三[羟甲基]甲烷(Bis-Tris)、N-[2-乙酰胺基]-2-亚氨基二乙酸(ADA)、甘氨酰甘氨酸和其它有机酸缓冲剂。
根据本发明的实施例，所述尿酸氧化酶制剂的pH为7～9，优选7.4～8.2。发明人经过反复试验及分析，发现聚乙二醇修饰的尿酸氧化酶制剂在上述pH的条件下的稳定性高，尿酸氧化酶不易聚集、降解，酶比活性高。
根据本发明的实施例，所述尿酸酶制剂的pH为：7.4、7.5、7.6、7.7、7.8、7.9、8.0、8.1、8.2。
根据本发明的实施例，所述聚乙二醇修饰的尿酸氧化酶与缓冲试剂的质量比为(5～6):(6～37)，优选为6:10。根据本发明实施例的尿酸氧化酶制剂，采用上述比例配制制剂，可以保证制剂的pH在7.4～8.2，保证所述尿酸氧化酶的稳定性。
根据本发明的实施例，所述聚乙二醇修饰的尿酸氧化酶与缓冲试剂的质量比为6:10、6:11、5:10、5:11、5:9、6:9。
根据本发明的实施例，所述聚乙二醇修饰的尿酸氧化酶与所述磷酸盐的质量比为(5～6)：(1～7)。
根据本发明的实施例，所述聚乙二醇修饰的尿酸氧化酶与所述氯化钠的质量比为(5～6)：(5～30)。
根据本发明的实施例，所述磷酸盐与所述氯化钠的质量比为(1～7)：(5～30)，其中所述磷酸盐为磷酸氢二钠和/或磷酸二氢钠。
根据本发明的实施例，本发明所提供的尿酸氧化酶制剂不需加入甘油、葡萄糖、甘露醇、Tween-80等稳定剂，只需使用缓冲剂即可保证本发明所提供的尿酸氧化酶的稳定性，得到稳定性高的尿酸氧化酶制剂。根据本发明实施例的尿酸氧化酶加入甘露醇和/或甘油，反而使尿酸氧化酶粒径增大，而加入Tween-80对本发明实施例的尿酸氧化酶稳定性无明显影响。根据本发明的实施例，所述尿酸氧化酶制剂的剂型包括下列至少之一：液体、半固体、固体。根据本发明实施例的尿酸氧化酶制剂可以为液体制剂或冻干剂型，在使用时溶解为液体，此外，所述尿酸氧化酶制剂为注射制剂，可以静脉注射、肌肉注射等方式注射入患者体内。
根据本发明的实施例，所述制剂为单剂型形式，每份制剂含有尿酸氧化酶6mg。根据本发明实施例的单剂量形式的尿酸氧化酶制剂，所述单剂量形式的尿酸氧化酶给药方式简单，每天无需重复给药，便于病人使用，从而达到最大药效，并且易于保存。
在本发明的第二方面，本发明提出了在本发明第一方面所提出的尿酸氧化酶制剂在制备药物中的用途，所述药物用于治疗或预防高尿酸血症及其相关性疾病。根据本发明的实施例，所述高尿酸相关性疾病包括选自慢性高尿酸血症、痛风、肾脏疾病、高尿酸性关节炎、肾结石、痛风结节、高血压、糖尿病、高甘油三酯血症、代谢综合征、冠心病、动脉粥样硬化、癌症化疗引起的高尿酸血症。
在本发明的第三方面，本发明提出了一种药物组合物。根据本发明的实施例，所述药物组合物包括在本发明第一方面所提出的尿酸氧化酶制剂。
根据本发明的实施例，所述药物组合物进一步包括如下附加技术特征至少之一：
根据本发明的实施例，上述药物组合物进一步包括其它用于治疗或预防高尿酸血症及相关性疾病的药物。
根据本发明的实施例，当本发明的聚乙二醇修饰的尿酸氧化酶或药物组合物在采用与其它药物的联合疗法中给药时，它们可序贯地或同时地给予个体。或者，本发明的药物组合物可包含本发明的聚乙二醇修饰的尿酸氧化酶、药学上可接受的载体或药学上可接受的赋形剂以及本领域已知的其它治疗药或预防药的组合。
根据本发明的实施例，所述药物组合物具有低免疫原性、高体内稳定性、适于肌肉注射的优势，可用于高尿酸相关性疾病的治疗或预防。
根据本发明的实施例，所述药物组合物进一步包括药学上可接受的辅剂。所述辅剂包括任何溶剂、固体赋形剂、稀释剂、粘合剂、崩解剂、或其他液体赋形剂、分散剂、矫味剂或悬浮剂、表面活性剂、等渗剂、增稠剂、乳化剂、防腐剂、固体粘合剂、助流剂或润滑剂，等等，适合于特有的目标剂型。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1是根据本发明实施例的PHC理化对照品-SEC-HPLC-UV检测图；
图2是根据本发明实施例的PHC理化对照品-SEC-HPLC-RI检测图；
图3是根据本发明实施例的PEG参照品-SEC-HPLC-RI检测图；
图4是根据本发明实施例的PU5修饰产物-SEC-HPLC-UV检测图；
图5是根据本发明实施例的PU5修饰产物-SEC-HPLC-RI检测图；
图6是根据本发明实施例的PHC和PU5分别采用Lys-c和胰蛋白酶双酶切比对图；
图7是根据本发明实施例的PU5 Lys-C酶切图；
图8是根据本发明实施例的处方5在0天时的SEC图；
图9是根据本发明实施例的处方5在37℃30天的SEC图；
图10是根据本发明实施例的处方6在37℃30天的SEC图；
图11是根据本发明实施例的处方7在37℃30天的SEC图。
图12是根据本发明实施例的模型大鼠肌肉给药不同给药剂量后血清尿酸水平；
图13是根据本发明实施例的肾损伤坏死及炎症评分图；
图14是根据本发明实施例的SD大鼠单次静脉注射相同剂量(1.0mg/kg)Pegloticase和聚乙二醇化尿酸酶注射液后各组平均血药浓度-时间曲线图；
图15是根据本发明实施例的SD大鼠单次肌肉注射Pegloticase和不同剂量聚乙二醇化尿酸酶注射液后各组平均血药浓度-时间曲线图；
图16是根据本发明实施例的SD大鼠单次肌肉注射不同剂量聚乙二醇化尿酸酶注射液后各组平均血药浓度-时间曲线图；
图17是根据本发明实施例的SD大鼠单次肌肉/静脉注射不同剂量Pegloticase和聚乙二醇化尿酸酶注射液后各组不同时间血尿酸平均值-时间曲线图；
图18是根据本发明实施例的SD大鼠首次(Day1)静脉注射相同剂量(1.0mg/kg)Pegloticase和聚乙二醇化尿酸酶注射液后雄性与雌性平均血药浓度-时间曲线图；
图19是根据本发明实施例的SD大鼠末次(Day22)静脉注射相同剂量(1.0mg/kg)Pegloticase和聚乙二醇化尿酸酶注射液后雄性与雌性平均血药浓度-时间曲线图；
图20是根据本发明实施例的SD大鼠首次(Day1)肌肉注射相同剂量(1.0mg/kg)Pegloticase和聚乙二醇化尿酸酶注射液后雄性与雌性平均血药浓度-时间曲线图；
图21是根据本发明实施例的SD大鼠末次(Day22)肌肉注射相同剂量(1.0mg/kg)Pegloticase和聚乙二醇化尿酸酶注射液后雄性与雌性平均血药浓度-时间曲线图；
图22是根据本发明实施例的SD大鼠多次静脉注射Pegloticase和聚乙二醇化尿酸酶注射液后不同时间血尿酸平均值-时间曲线图；以及
图23是根据本发明实施例的SD大鼠多次肌肉注射Pegloticase和聚乙二醇化尿酸酶注射液后不同时间血尿酸平均值-时间曲线图。
具体实施方式
下面详细描述本发明的实施例，所述实施例的示例在附图中示出，其中自始至终相同或类似的标号表示相同或类似的元件或具有相同或类似功能的元件。下面通过参考附图描述的实施例是示例性的，旨在用于解释本发明，而不能理解为对本发明的限制。
本发明的目的是提供一种新型的聚乙二醇修饰的尿酸氧化酶制剂。
本发明的另一目的是提供上述聚乙二醇修饰的尿酸氧化酶制剂的应用，可以实现体内长效且显著降低血液尿酸水平的功效，可用于高尿酸血症及通风的治疗。
在本发明的一方面提供了一种聚乙二醇修饰的尿酸氧化酶制剂。
尿酸氧化酶没有特别的限制，可以是任何来源的尿酸氧化酶及其尿酸氧化酶类似物，代表的例子包括但并不限于哺乳动物来源、微生物、植物等。
本发明所述的不同种属来源尿酸氧化酶，可通过多种途径获得，包括但不限于天然提取、化学合成、基因工程重组表达等。
在另一优选例中，以大肠杆菌或酵母作为宿主，构建重组表达菌株的方法制备而得，更优选大肠杆菌作为宿主菌进行重组表达。
在另一优选例中，以所述聚乙二醇尿酸氧化酶为活性成分，添加磷酸二氢钠、磷酸氢二钠和氯化钠，所制备的制剂能够最大限度保证酶活，并且提高制剂的存放稳定性。
在另一优选例中，所述聚乙二醇尿酸氧化酶制剂不需要再添加其他稳定剂，如甘露醇、甘油和Tween-80等，即可具有较高的存放稳定性，由此，所述尿酸氧化酶制剂配方简单，制备工艺简便，成本低廉。
在本发明的一方面提供了上述聚乙二醇修饰的尿酸氧化酶制剂的应用。
所述的聚乙二醇尿酸氧化酶制剂更适用于作为一种治疗慢性高尿酸血症或通风的药物及其组合物。所述高尿酸血症及通风的主要症状包括但不限于尿酸性肾病和通风性关节炎。
所述的聚乙二醇尿酸氧化酶的给药途径包括但不局限于静脉注射、皮下注射、肌肉注射和腹腔注射等，优选静脉注射、肌肉注射，更优选肌肉注射。
所述的聚乙二醇尿酸氧化酶具有更低的体内免疫原性。
所述的聚乙二醇尿酸氧化酶具有低免疫原性是指在人或动物体内经肌肉注射聚乙二醇尿酸氧化酶后，机体不产生抗聚乙二醇分子的抗体或产生低滴度的抗聚乙二醇分子抗体。不产生针对尿酸氧化酶的抗体。
所述的聚乙二醇尿酸氧化酶经过经肌肉注射后体内具有更长的半衰期和降低体内尿酸水平的功效。
此外，术语“第一”、“第二”仅用于描述目的，而不能理解为指示或暗示相对重要性或者隐含指明所 指示的技术特征的数量。由此，限定有“第一”、“第二”的特征可以明示或者隐含地包括至少一个该特征。在本发明的描述中，“多个”的含义是至少两个，例如两个，三个等，除非另有明确具体的限定。
下面参考具体实施例，对本发明进行描述，需要说明的是，这些实施例仅仅是描述性的，而不以任何方式限制本发明。
实施例1重组尿酸氧化酶的制备
1.1用于尿酸酶表达的基因和表达质粒的构建
根据E.coli密码子使用偏好性数据，结合密码子偏爱性及GC含量等因素，设计尿酸酶蛋白(代号：PHC)(SEQ ID NO：1)的cDNA序列，进行全基因合成，并命名为pUC-57-PHC质粒。以Nde I和BamH I作为目的基因插入位点，采用pET-30a质粒作为表达载体(pET-30a-PHC)。
1.2表达质粒向细菌宿主细胞中的转化
采用CaCl 2法将表达载体pET-30a-PHC引导入到大肠杆菌BL21(DE3)中，Kanamycin进行抗性筛选，筛选出高表达克隆，并保存原始种子库菌种(E3B)，这些步骤按照分子生物学领域常用方法实施。
1.3重组尿酸氧化酶的制备
将转化的工程菌种采用发酵罐进行发酵表达，控制条件为先30℃、pH约7.2培养至OD 600＝30以上，添加IPTG至0.5mmol/L，继续诱导3h以上，以让尿酸氧化酶积累。离心收集细胞，然后放置在-15℃以下保存。
取冻存菌体，悬浮于25mmol/L Tris，5mmol/L EDTA缓冲中，悬浮比例1:10(W/V)。采用高压破开细菌细胞后，离心收集尿酸氧化酶沉淀，沉淀用50mmol/L NaHCO 3洗涤一次后，将富集的尿酸酶沉淀悬浮于100mmol/L Na 2HCO 3(pH9.7～10.3)缓冲液中，悬浮比例1:50(W/V)，室温搅拌过夜溶解，随后离心收集上清。
尿酸氧化酶通过几个色谱步骤作进一步的纯化，SDS-PAGE检测纯度大于95％以上，用Superdex 200柱检测出纯度大于95％以上，无聚集体形式，采用Lowry法测定蛋白浓度，用分光光度计测定尿酸氧化酶活性，1单位(U)酶活定义为，在最适反应温度37℃、最适pH9.0缓冲液条件下，每分钟转化1μmol尿酸所需酶的量。
实施例2聚乙二醇化氧化尿酸酶的制备
将不同分子量的(500～20000Da)单甲氧基PEG衍生物，如5K分子量的N-琥珀酰亚胺丙酸酯PEG(5K-PEG-SPA)用1～5mmol/L酸溶液溶解成100～300mmol/L的PEG溶液，溶解后以1：45～1：150摩尔比(尿酸氧化酶：5K-PEG-SPA)，加入溶有尿酸氧化酶的碳酸盐浓度为0.1～0.3mol/L pH10.0的碳酸盐缓冲液中，使得PEG与尿酸氧化酶发生偶联反应，偶联反应的尿酸氧化酶的浓度为10mg/ml，偶联反应需在5～30℃的条件下搅拌反应60分钟以上，直到PEG偶联程度不再随时间而变化。反应结束后，通过超滤和/或色谱法将未修饰的PEG及副产物从反应中去除。可以选用合适的分子筛层析介质，进行修饰副产物的分离去除。最后无菌过滤即得5K修饰的聚乙二醇化尿酸氧化酶(代号为PU5)所示。
实施例3聚乙二醇化氧化尿酸酶的特性分析
3.1平均修饰度及酶活检测
采用Lowry法测定蛋白浓度，聚乙二醇尿酸氧化酶活性测定在分光光度计下进行。尿酸酶底物尿酸最大紫外吸收波长为293nm，而产物尿囊素的最大紫外吸收波长为224nm，在一定浓度范围内，尿酸在293nm的吸收值与其浓度成正比，可用分光光度计法进行尿酸的定量测定。具体过程如下：打开紫外可见分光光度计，将波长调至293nm，并打开该仪器水浴循环系统使温度保持为37℃。以四硼酸钠缓冲液作空白对照，校零点；取2.95ml底物反应液(0.1mol/L四硼酸钠、100μmol/L尿酸，pH9.5，37℃预热)置石英比色杯中，然后加入50μl的供试品迅速混匀后于293nm处测定吸收值。连续测定293nm处吸收度变化；根据C＝A/εL(其中A为特定浓度尿酸在293nm处吸光值，ε为尿酸摩尔消光系数，L为比色杯光程，C为尿酸摩尔浓度)计算尿酸降解浓度，并计算酶活；酶活定义：在最适反应温度37℃，最适反应pH9.5时，每分钟转化1μmol尿酸为尿囊素所需的酶量定义为一个活性单位(U)。
采用SEC-HPLC串联UV/RI(紫外和折光指数检测器联合)进行聚乙二醇尿酸氧化酶的平均修饰度检测。根据蛋白在紫外280nm处有最大吸收峰，而该波长下PEG没有吸收，同时示差折光检测器对蛋白和PEG在一定范围内吸收值与其各种浓度成正比。因此可通过PEG参照品和PHC理化对照品外标方式得到聚乙二醇化尿酸氧化酶中PEG和蛋白部分各自的含量，进而可通过以下计算方式获得每一个尿酸氧化酶单体上PEG分子数，即平均修饰度。
PEG尿酸氧化酶平均修饰度＝(尿酸氧化酶亚基相对分子量×样品中PEG的量)/(PEG相对分子量×样品中蛋白质的量)。
其中，PHC理化对照品、PEG参照品、PU5修饰产物SEC-HPLC-UV/RI检测图见图1至图5。
实施例2不同投料比下，所获得的聚乙二醇氧化尿酸酶的酶活和平均修饰度如表1所示。
表1：5K-PEG下不同投料比下的酶活和平均修饰度
蛋白：5K-PEG投料摩尔比 酶活 酶活保留 平均修饰度
未修饰尿酸氧化酶 11.4U/mg 100％ 0
1:48 10.71U/mg 94％ 10.3
1:56 11.17U/mg 103.4％ 11.4
1:68 12.2U/mg 107.1％ 11.9
1:82 12.02U/mg 105.4％ 12.3
1:94 11.75U/mg 103.1％ 12.1
1:110 10.83U/mg 95％ 11.5
1:150 10.03U/mg 88％ 10.1
备注：平均修饰度表示每一个尿酸氧化酶单体上结合的PEG分子数。
由表1可以看出，本申请的聚乙二醇尿酸氧化酶在蛋白：5K-PEG投料比1:56-1:110范围内的平均修饰度稳定在11个以上，且酶活相比于未修饰的尿酸氧化酶，酶活保留率高，酶活性并没有降低，反而升高，且酶活相对稳定。这与已上市的药物Krystexxa(pegloticase)完全不同，根据山景公司专利中所披露的内容(CN1264575C，图2A-图3B)和本领域技术人员的一般认识，随着5kD PEG修饰度的提高，酶活性显著下降。然而出乎意料的是，本申请获得的聚乙二醇尿酸酶在超过11的平均修饰度下，酶活性和未修饰时相比无明显变化。因此本申请的聚乙二醇尿酸氧化酶与已上市药相比，聚乙二醇平均修饰度更高，在酶活保留方面也取得了预料不到的技术效果。发明人推测可能是由于聚乙二醇尿酸氧化酶的PEG修饰度或修饰位点不同导致的。
3.2聚乙二醇修饰位点检测
在以下步骤中，发明人对实施例2所获得的尿酸氧化酶进行修饰位点的检测。
聚乙二醇修饰的尿酸氧化酶的PEG修饰位点可通过对非聚乙二醇化和聚乙二醇化尿酸氧化酶采用一种或多种酶进行酶切，然后经过色谱检测，获得色谱图，即肽图确认。非聚乙二醇化和聚乙二醇化尿酸氧化酶可以采用单酶切(Lys-C或Trypsin)和或双酶切(Lys-C和Trypsin联用)进行酶切。反相柱分离酶切片段，通过内参肽段校正比较肽段消失或降低比例判断聚乙二醇尿酸氧化酶的修饰位点。
胰蛋白酶和Lys-C双酶切质量肽图中修饰位点分析原理：Lys-C可对赖氨酸(K)C端进行特异性酶切，胰蛋白酶以碱性氨基酸-精氨酸(R)和赖氨酸(K)作为酶切位点，对其C端肽键进行特异性酶切。比较PHC和PU5中酶切前后对应各肽段变化情况，并结合内标肽段可分析确认PEG修饰肽段减小或消失的相对比例。通过肽段的减小或消失相对比例，可判定肽段上该赖氨酸位点是否被PEG修饰以及修饰的比例。需要指出的是，PEG修饰是一种非均一的修饰，某位点修饰比例高则可以认为该位点被修饰。
具体如下：
(1)样品处理：取尿酸氧化酶和聚乙二醇化尿酸氧化酶分别用酶切缓冲液(25mmol/L Tris-HCl，20％乙腈，pH9.0)溶解稀释为1mg/ml，各取100μl，加入2μl Lys-C，于37℃酶切4小时。即将该溶液转移至胰酶反应管中(1：100的比例)于37度继续酶切2小时，用4μl TCEP还原溶液继续反应30分钟，再加入10μl 1mol/L盐酸溶液终止反应。
(2)分析条件：
仪器：Thermo Ultimate 3000 HPLC和MSQ Plus；
色谱柱：月旭Welch Materials
Figure PCTCN2021129071-appb-000004
(4.6mm×250mm，5μm)；
分析条件：A液(含0.1％TFA的水溶液)，B液(含0.1％TFA的乙腈溶液)；
梯度：0-70min，B从3-70％；
LC检测波长：214nm。
离子源：ESI；
离子类型：正离子；
锥孔电压：50V；
扫描范围：300-2000Da；
扫描时间：1S；
柱后分流约0.3ml/min。
样品量进样100μl，记录色谱图。
(3)结果处理：
比较尿酸氧化酶和聚乙二醇化尿酸氧化酶的色谱图(肽图)，计算差异肽段的面积减少的相对比例。
(4)实验结果见表2至表5，及图6～图7。
表2：PHC经Lys-C酶切肽段表
Figure PCTCN2021129071-appb-000005
Figure PCTCN2021129071-appb-000006
表3：PHC经Lys-C和胰蛋白酶双酶切后的肽段表
Figure PCTCN2021129071-appb-000007
Figure PCTCN2021129071-appb-000008
Figure PCTCN2021129071-appb-000009
PU5肽段峰面积的降低百分比计算方法：
利用以下公式可计算PU5与PHC相同浓度下对应的PU5肽段峰面积：
A 1＝A 0×t
其中A 1为经内参两肽段换算后PU5肽段峰面积，A 0为PU5肽图肽段实测峰面积，t为T30、T31号内参肽段中PHC肽图与PU5肽图峰面积比值平均值，即0.588。
表4：PHC和PU5内参肽段比对
Figure PCTCN2021129071-appb-000010
将内参换算后肽段峰面积与PHC肽图峰面积利用如下公式，即可计算出PU5肽图中某肽段峰面积减少的相对百分比例：
P(％)＝(A 2-A 1)/A 2×100％
其中，A 2为某肽段在PHC肽图中肽段峰面积，A 1为经内参换算后该肽段在PU5肽段峰面积。
表5：PU5经双酶切后肽图中峰面积降低肽段总结结果
Figure PCTCN2021129071-appb-000011
根据本实施例的蛋白序列(SEQ ID NO:1)分析可知，尿酸氧化酶被修饰的潜在位点有T 1、K 3、K 4、K 17、K 21、K 30、K 35、K 48、K 49、K 66、K 74、K 76、K 79、K 97、K 112、K 116、K 120、K 152、K 155、K 158、K 169、K 179、K 190、K 215、K 222、K 231、K 266、K 272、K 285、K 291、K 293等31个位点。
通过对实施例2中所获得聚乙二醇修饰的尿酸氧化酶修饰位点的分析可知，如表2、表3、表4、表5和图6分析所示，PU5酶切后肽段消失在90％以上的位点有K 3、K 4、K 35、K 97、K 112、K 116、K 120、K 152、K 222、K 266、K 285，PU5酶切后肽段消失在80％～90％范围内的位点有K 76、K 231，在PU5中，这些位点均被修饰。
同时发明人发现，本申请的聚乙二醇修饰的尿酸氧化酶的修饰位点相比于已上市药物，修饰位点更多，具有显著差异，如通过对单酶切发现，本申请的聚乙二醇修饰的尿酸氧化酶在K 30、K 35、K 222和K 231四个位点所在的肽段消失比例高于80％以上，而通过方法分析已上市类似药Krystexx(pegloticase)，在这四个位点所在的肽段几乎未消失，即上市类似药在K 30、K 35、K 222和K 231四个位点修饰率远低于本申请的聚乙二醇修饰的尿酸氧化酶。另外，本申请的聚乙二醇修饰的尿酸氧化酶，相比于已上市药物，免疫原性显著更低，发明人推测可能与修饰位点的多少和修饰位点的不同有关。由于修饰位点的不同以及修饰度的不同，导致酶在体内免疫原性位点的保护和酶活性中心的暴露均不同，上述不同造成了不同修饰酶在体内生物性质的不同。
由于尿酸氧化酶为蛋白制剂，不同的化学修饰会对其稳定性造成不同的影响，因此在制备成制剂时，需要针对特定的尿酸氧化酶活性成分选用不同的辅料、pH等制备尿酸氧化酶制剂，下面将描述针对本发明的聚乙二醇修饰的尿酸氧化酶制备稳定性高、生物活性高的尿酸氧化酶制剂。
实施例5：处方筛选
以上述实施例中的到的聚乙二醇修饰的尿酸氧化酶PU5为活性成分，对聚乙二醇修饰的尿酸酶制剂的组分进行筛选，以获得尿酸酶活性高、稳定性高的聚乙二醇尿酸酶制剂。所述筛选包括：辅料配方筛选和稳定剂筛选。
1、辅料筛选
制剂处方辅料包括：碳酸盐、磷酸盐、盐酸盐、枸橼酸盐。
使用上述缓冲液辅料与活性成分制备聚乙二醇修饰的尿酸氧化酶制剂，辅料在相同浓度范围10～50mmol/L，相同pH(pH7～9)条件下，聚乙二醇修饰的尿酸氧化酶稳定性基本一致，但制剂过程中不同辅料浓度比例和制剂渗透压控制难易程度不同，其中，磷酸盐、盐酸盐比碳酸盐和枸橼酸盐更容易控制渗透压和pH。通过缓冲液筛选最终发现，采用磷酸盐、盐酸盐作为辅料，磷酸 盐浓度范围10～50mmol/L，盐酸盐浓度范围100～200mmol/L均可满足制剂工艺需求，其中，15mmol/L磷酸二氢钠/磷酸氢二钠、0.136mol/L氯化钠，pH为7.4的制剂具有相对较好的稳定性。发明人将其作为基础配方，并进行更进一步的筛选。
2、稳定剂筛选实验
基础制剂处方添加甘露醇、甘油和Tween-80，通过稳定性考察，来确定本品制剂中是否有必要添加相应稳定剂。
2.1稳定剂初步筛选实验
基础制剂处方作为对照，在基础制剂处方基础上分别添加4％甘露醇、2％甘油和0.04％Tween-80等稳定剂，样品编号分别记录为样品1至样品4，样品1-4放置于45℃考察7天，取样检测粒径，研究稳定剂对PU5制剂稳定性的影响。
通过实验结果可以看出，加入甘油和甘露醇的配方，其粒径(分别为19.44，20.51)比基础制剂配方的粒径(18.57)大，加入Tween-80的制剂配方其粒径(18.10)略小于基础制剂配方，考虑在制剂配方中加入Tween-80可防止PU5粒径增加。
2.2稳定剂再次筛选实验
由稳定剂初步筛选实验可知，在制剂配方中加入Tween-80可防止PU5粒径增加，通过稳定性进一步确认。将初步筛选的45℃考察7天的2个样品，样品1、样品4重新放入45℃考察箱，继续放置15天后，取样检测粒径，研究Tween-80稳定剂对PU5制剂稳定性的影响。
(1)粒径检测结果见表6
表6：稳定剂再次筛选粒径结果统计
Figure PCTCN2021129071-appb-000012
注：对照表示在2～8℃条件下放置。
(2)结论
从45℃考察7天和22天与对照组的数据分析：加入0.04％Tween-80，在45℃考察7天内样品的PDI明显较小，样品较均一，45℃考察22天检测时，是否加Tween-80对PDI没有影响，说明在高温时加入吐温-80对阻止或延缓PU5粒径增加没有意义。
2.3第三次稳定剂筛选实验
为最终确定是否在制剂配方中添加Tween-80，将添加Tween-80和不添加Tween-80的两个处方，分别置于25℃考察1个月和37℃考察1个月，取样检测粒径、高低分子蛋白含量、pH和酶比活性，结果如表7所示。
表7：加速稳定性考察结果
Figure PCTCN2021129071-appb-000013
Figure PCTCN2021129071-appb-000014
由表7可知，PU5制剂中加入Tween-80与不加Tween-80的制剂，进行加速稳定性考察，其高低分子、酶活性和粒径变化行为基本一致。
经过多次研究，添加Tween-80前后其高、低分子蛋白含量和粒径均无明显差别，故确定在制剂处方中不添加稳定剂。
实施例6：制剂pH筛选
1、pH范围优化
(1)研究方法
用缓冲盐调节制剂处方溶液pH分别为7.8、7.0、7.4、8.2、8.6。将样品分别放置于2～8℃、25±2℃和37℃进行稳定性考察，分别于30天测定高、低分子蛋白含量和酶活性。比较各处方中PU5蛋白的聚合或降解情况以及酶活性的变化。
(2)测定结果
测定结果见表8和图8～11。
表8：高、低分子蛋白含量及酶活性测定结果统计表(％)
Figure PCTCN2021129071-appb-000015
注：“长期”表示2～8℃，“加速”表示25±2℃，“高温”表示37℃。
由图8～11和表8可知：在2～8℃和25±2℃考察30天，处方5(pH 7.8)、处方6(pH 8.2)、处方7(pH 7.4)的高分子蛋白含量呈降低的趋势；低分子蛋白含量呈略微增加的趋势，其中处方7＞处方5＞处方6，三个处方中酶活性均未见明显变化。由此确定PU5制剂pH控制在7.4～8.2。37℃高温考察30天，处方5、6、7的高分子蛋白含量三者间没有明显差异，但低分子蛋白含量增加，处方7＞处方5＞处方6。由此确定PU5制剂的pH中点为7.8。
综上所述，确定本品PU5注射液制剂pH范围在7.4～8.2。
本申请的聚乙二醇修饰的尿酸氧化酶，相比于已上市药物，免疫原性显著更低，发明人推测可能与修饰位点的多少和修饰位点的不同有关。由于修饰位点的不同以及修饰度的不同，导致酶在体内免疫原性位点的保护和酶活性中心的暴露均不同，上述不同可能造成了不同修饰酶在体内生物性质的不同。
下面将详细描述本申请的聚乙二醇修饰的尿酸氧化酶制剂(PU5制剂)的药物动物体内评价，其中，实验中所用到的pegloticase是指已上市类似药，批号为5085B。
实施例7聚乙二醇尿酸氧化酶制剂体内药效学研究
7.1、聚乙二醇尿酸氧化酶在模型大鼠体内药效评价
采用氧嗪酸钾饮水联合高尿酸饲料诱导大鼠慢性高尿酸血症模型，评价聚乙二醇尿酸氧化酶(PU5 制剂)对大鼠慢性高尿酸血症的治疗作用。
选取40只模型大鼠，随机分为4组，即模型组、聚乙二醇化尿酸酶低剂量给药组(0.3mg/kg)、聚乙二醇化尿酸酶中剂量给药组(1.0mg/kg)、聚乙二醇化尿酸酶高剂量给药组(3.0mg/kg)，每组10只，另选10只正常SD大鼠为空白对照组。试验连续造模5周，造模1周后开始肌肉给药，每周给药一次，连续给药4周，分别检测给药前及每次给药后7天的大鼠血清尿酸、血清尿素氮、血清肌酐水平，试验结束后观察大鼠肾脏组织学变化。
图12结果显示，在造模后第7、14、21、28和35天，与空白对照组相比，模型对照组血尿酸水平均显著增加，造模7天后模型组大鼠血清尿素氮、肌酐及尿酸分别为空白组大鼠的2.73倍、2.40倍和7.83倍。从肾脏病理来看(如图13所示)，模型对照组的肾小管扩张、坏死、炎症及纤维化评分均显著增加，同时尿酸盐结晶数量也显著增加。受试物聚乙二醇化尿酸酶中高剂量均显著降低血清尿酸水平，且呈剂量相关性，在14天～35天期间，中剂量组血尿酸水平均值维持在303.80-660.60μmol/L，高剂量组血尿酸水平均值维持在153.70-403.40μmol/L，与模型组相比，中剂量组血尿酸降低的幅度为34.46-67.94％；高剂量组血尿酸降低的幅度为65.67-83.78％。与模型对照组相比，聚乙二醇化尿酸酶各给药组对肾小管扩张、肾脏坏死及炎症均有显著改善作用。
7.2、聚乙二醇尿酸氧化酶大鼠体内单次给药评价
取36只SD大鼠，雌雄各半随机分为6组(见表9)，即上市药Pegloticase静脉注射组、肌肉注射组，聚乙二醇尿酸氧化酶静脉注射组及聚乙二醇尿酸氧化酶低、中、高(0.5、1.0、2.0mg/kg)剂量肌肉注射组，给药具体方案和剂量见表9。颈静脉取血检测PK和PD。
表6：动物分组及剂量设计
Figure PCTCN2021129071-appb-000016
7.2.1、药代动力学对比
SD大鼠给药前所有个体的血清药物浓度水平均低于定量下限(LLOQ：312.500ng/mL)，单次肌肉注射0.5、1.0、2.0mg/kg，在0～168h(0～7天)范围内，聚乙二醇化尿酸酶注射液(PU5制剂)后血清药物浓度具有剂量相关性，整体水平随给药剂量增加而增加，当超过168h后，pegloticase肌肉给药组的血药浓度低于定量下限，而PU5制剂肌肉给药组可继续维持至240h以上。
给药后，1.0mg/kg Pegloticase静脉注射和肌肉注射组，1.0mg/kg聚乙二醇化尿酸酶注射液静脉注射及0.5、1.0、2.0mg/kg聚乙二醇化尿酸酶注射液肌肉注射组各组雌性与雄性SD大鼠体内Cmax(C5min)比值在0.75～0.99范围内，AUClast比值在0.54～0.94范围内，AUC0-∞比值在0.58～0.97范围内。由此可见，Pegloticase和聚乙二醇化尿酸酶(PU5制剂)注射液在SD大鼠体内暴露水平无明显性别差异。
然而，SD大鼠给予相同剂量(1.0mg/kg)的上市药Pegloticase静脉给药组的AUClast为426.48±65.34，肌肉注射组的AUClast为264.19±78.22；PU5制剂注射液静脉给药组的AUClast为565.61±161.60，肌肉注射组的AUClast为337.86±227.34。在相同剂量，相同给药方式条件下，PU5制剂的AUClast高于上市药Pegloticase。
SD大鼠给予相同剂量(1.0mg/kg)的上市药Pegloticase静脉给药组t1/2(h)为49.51±8.12，肌肉给药组t1/2(h)为55.21±13.50。PU5制剂注射液静脉给药组t1/2(h)为86.12±33.82，肌肉给药组t1/2(h)为 60.45±21.37。在相同剂量，相同给药方式条件下，PU5制剂注射液的t1/2(h)长于上市药Pegloticase。
上述药代动力学结果如表10～表15，图14～图16所示。
表10：SD大鼠单次静脉注射1.0mg/kg Pegloticase的个体血药浓度数据及统计分析数据(单位：μg/mL)
Figure PCTCN2021129071-appb-000017
备注：“/”表示无相关信息。
表11：SD大鼠单次静脉注射1.0mg/kg聚乙二醇化尿酸酶注射液的个体血药浓度数据及统计分析数据(单位：μg/mL)
Figure PCTCN2021129071-appb-000018
备注：“/”表示无相关信息。
表12：SD大鼠单次肌肉注射1.0mg/kg Pegloticase的个体血药浓度数据及统计分析数据(单位：μg/mL)
Figure PCTCN2021129071-appb-000019
备注：“/”表示无相关信息。
表13：SD大鼠单次肌肉注射1.0mg/kg聚乙二醇化尿酸酶注射液的个体血药浓度数据及统计分析数据(单位：μg/mL)
Figure PCTCN2021129071-appb-000020
备注：“/”表示无相关信息。
表14：SD大鼠单次静脉注射Pegloticase和聚乙二醇化尿酸酶注射液后平均药代动力学参数
Figure PCTCN2021129071-appb-000021
表15：SD大鼠单次肌肉注射Peglocticase和聚乙二醇化尿酸酶注射液后平均药代动力学参数
Figure PCTCN2021129071-appb-000022
Figure PCTCN2021129071-appb-000023
7.2.2、体内药效对比(尿酸)
单次肌肉注射0.5、1.0、2.0mg/kg聚乙二醇化尿酸酶注射液后，给药后1天、3天尿酸浓度均维持在较低水平，给药后7天各剂量组尿酸水平开始恢复，而给药剂量越高，尿酸在体内维持低水平的时间越长。与相同剂量静脉注射组的相比，PU5制剂静脉注射组血清尿酸维持低浓度水平的时间均较pegloticase静脉注射组长。与相同剂量肌肉注射组的相比，PU5制剂肌肉注射组血清尿酸维持低浓度水平的时间均较pegloticase肌肉注射组长。与相同剂量组相比，PU5制剂静脉注射或肌肉注射组血清尿酸维持低浓度水平的时间均较pegloticase静脉注射组或肌肉注射组长，即PU5制剂在体内维持维持低浓度水平的时间均较pegloticase更长。结果如图17所示。
7.3、聚乙二醇尿酸氧化酶大鼠体内多次给药评价
本试验设4个组，分别为上市药Pegloticase静脉注射组、肌肉注射组，聚乙二醇化尿酸酶注射液(PU5)静脉注射组、肌肉注射组，每组8只，雌雄各半，共32只SD大鼠。Pegloticase及聚乙二醇化尿酸酶注射液静脉注射组采用静脉注射；Pegloticase和聚乙二醇化尿酸酶注射液肌肉注射组采用肌肉注射。给药剂量均为1.0mg/kg。每周给药1次，连续给药4次。
结果分析可知：
SD大鼠多次静脉/肌肉注射1.0mg/kg Pegloticase和聚乙二醇化尿酸酶注射液。大鼠一般状况未见与药物相关异常改变。
7.3.1抗PEG抗体检测
SD大鼠连续4次给药后，首次给药前，所有个体动物均未检出抗PEG抗体和抗PHC抗体；给药结束后，所有动物未检出抗PHC抗体，Pegloticase静脉、肌肉注射组，聚乙二醇化尿酸酶注射液静脉、肌肉注射组各组检出抗PEG抗体，阳性结果的比例分别为：3/8、1/8、1/8、1/8。通过PEG免疫组织化学检查发现：pegloticase静脉注射组和肌肉注射组的脾脏、肝脏和肾脏可见PEG较弱阳性表达。聚乙二醇化尿酸酶注射液静脉注射组和肌肉注射组未见PEG阳性表达，结果见表13。
从以上分析可知，PU5制剂和pegloticase产生的抗体主要是针对PEG部分的抗体，而非针对尿酸氧化酶部分的抗体，说明二者均能有效遮蔽尿酸氧化酶的免疫原性位点。PEG抗体的产生在体内会导致部分副作用的发生，根据表16的结果显示，本申请PU5制剂免疫原性低于市售产品pgeloticase。
针对PEG抗体以及PEG免疫组化的结果可知，PU5制剂和pegloticase均是肌肉给药组优于静脉给药组，其中静脉给药组产生的抗PEG抗体，PU5制剂优于pegloticase；肌肉给药组产生的抗PEG抗体， PU5制剂优于pegloticase。
表16：PEG免疫组织化学阳性表达结果
Figure PCTCN2021129071-appb-000024
阳性分级：1＝较弱阳性，2＝弱阳性，3＝中度阳性，4＝强阳性。
4.3.2药代动力学检测
SD大鼠多次静脉、肌肉注射给予Pegloticase和聚乙二醇化尿酸酶注射液后，各组动物主要药代动力学参数无明显性别差异。连续4次给药后，两种药物在大鼠体内有轻微蓄积。
SD大鼠多次静脉/肌肉注射给予相同剂量(1.0mg/kg)的上市药Pegloticase,首次给药后，在大鼠体内的绝对生物利用度分别为51.35％；末次给药后，在大鼠体内的绝对生物利用度分别为45.98％。SD大鼠多次静脉/肌肉注射给予相同剂量(1.0mg/kg)的聚乙二醇化尿酸酶注射液，首次给药后，在大鼠体内的绝对生物利用度分别为58.29％；末次给药后，在大鼠体内的绝对生物利用度分别为52.60％。
4.3.3体内药效对比(尿酸)
SD大鼠连续4次(1次/周)静脉、肌肉注射1.0mg/kg Pegloticase和聚乙二醇化尿酸酶注射液，每次给药后血清尿酸浓度均维持在较低水平，末次给药后14天Pegloticase肌肉注射出现恢复，其余各组于末次给药后18天出现恢复。与相同剂量的上市药Pegloticase相比，两种药物静脉注射组的维持时间比较一致，而聚乙二醇化尿酸酶注射液肌肉注射组的维持时间较上市药长，即PU5制剂在肌肉给药的疗效优于Pegloticase。
上述结果如表17～表19，图18～图22所示。
表17：SD大鼠连续静脉注射Pegloticase和聚乙二醇化尿酸酶注射液后平均药代动力学参数结果
Figure PCTCN2021129071-appb-000025
Figure PCTCN2021129071-appb-000026
表18：SD大鼠连续肌肉注射Pegloticase和聚乙二醇化尿酸酶注射液后平均药代动力学参数结果
Figure PCTCN2021129071-appb-000027
Figure PCTCN2021129071-appb-000028
表19：SD大鼠多次肌肉/静脉注射Pegloticase和聚乙二醇化尿酸酶注射液后各剂量组尿酸统计结果(Mean+SD)
Figure PCTCN2021129071-appb-000029
Figure PCTCN2021129071-appb-000030
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (24)

  1. 一种尿酸氧化酶制剂，其特征在于，包括：
    活性成分，所述活性成分选自聚乙二醇修饰的尿酸氧化酶；以及
    辅料，所述辅料选自缓冲试剂，其中所述缓冲试剂包括下列至少之一：磷酸盐和盐酸盐。
  2. 根据权利要求1所述的尿酸氧化酶制剂，其特征在于，所述尿酸氧化酶中的下列氨基酸位点中的至少11个具有PEG修饰：T 1、K 3、K 4、K 30、K 35、K 76、K 79、K 97、K 112、K 116、K 120、K 152、K 179、K 222、K 231、K 266、K 272、K 285、K 291和K 293，所述氨基酸位点定位是以SEQ ID NO:1所示的氨基酸序列定位的。
  3. 根据权利要求2所述的尿酸氧化酶制剂，所述尿酸氧化酶中的下列4个氨基酸位点中的至少1个、至少两个、至少三个或四个具有PEG修饰：K 30、K 35、K 222和K 231。
  4. 根据权利要求1所述的尿酸氧化酶制剂，其特征在于，所述聚乙二醇具有单甲氧基或羟基。
  5. 根据权利要求1所述的尿酸氧化酶制剂，其特征在于，所述聚乙二醇为直链或支链结构。
  6. 根据权利要求1所述的尿酸氧化酶制剂，其特征在于，所述聚乙二醇与尿酸氧化酶通过酰胺键偶联。
  7. 根据权利要求1-6任一项所述的尿酸氧化酶制剂，其特征在于，所述聚乙二醇为修饰性聚乙二醇，所述修饰性聚乙二醇的修饰基团选自下列的至少一种：
    N羟基琥珀酰亚胺、N羟基琥珀酰亚胺碳酸酯、N羟基琥珀酰亚胺乙酸酯、N羟基琥珀酰亚胺丙酸酯、N羟基琥珀酰亚胺丁酸酯、N羟基琥珀酰亚胺琥珀酸酯和对硝基苯碳酸酯。
  8. 根据权利要求7所述的尿酸氧化酶制剂，其特征在于，所述修饰性聚乙二醇的修饰基团为N羟基琥珀酰亚胺丙酸酯。
  9. 根据权利要求1所述的尿酸氧化酶制剂，其特征在于，所述尿酸氧化酶具有SEQ ID NO:1～7所示的氨基酸序列；或
    与SEQ ID NO:1～7相比具有至少70％、至少75％、至少80％、至少85％、至少90％、至少95％、至少99％同一性的多肽；或
    与SEQ ID NO:1～7相比具有一个或者多个氨基酸的取代、缺失和/或添加的多肽。
  10. 根据权利要求1所述的尿酸氧化酶制剂，其特征在于，所述尿酸氧化酶具有SEQ ID NO:1～4所示的氨基酸序列。
  11. 根据权利要求1所述的尿酸氧化酶制剂，其特征在于，所述缓冲试剂包括选自下列至少之一：磷酸氢二钠、磷酸二氢钠和氯化钠。
  12. 根据权利要求1所述尿酸氧化酶制剂，其特征在于，所述尿酸氧化酶制剂的pH为7～9。
  13. 根据权利要求12所述的尿酸氧化酶制剂，其特征在于，所述尿酸氧化酶制剂的pH为7.4～8.2。
  14. 根据权利要求9或10所述的尿酸氧化酶制剂，其特征在于，所述聚乙二醇修饰的尿酸氧化酶与缓冲试剂的质量比为(5～6):(6～37)。
  15. 根据权利要求14所述的尿酸氧化酶制剂，其特征在于，所述聚乙二醇修饰的尿酸氧化酶与缓冲试剂的质量比为6:10。
  16. 根据权利要求9或10所述的尿酸氧化酶制剂，其特征在于，所述聚乙二醇修饰的尿酸氧化酶与所述磷酸盐的质量比为(5～6)：(1～7)，其中，所述磷酸盐为磷酸氢二钠、磷酸二氢钠。
  17. 根据权利要求9或10所述的尿酸氧化酶制剂，其特征在于，所述聚乙二醇修饰的尿酸氧化酶与所述氯化钠的质量比为(5～6)：(5～30)。
  18. 根据权利要求9或10所述的尿酸氧化酶制剂，其特征在于，所述磷酸盐与所述氯化钠的质量比为(1～7)：(5～30)，其中所述磷酸盐为磷酸氢二钠和/或磷酸二氢钠。
  19. 根据权利要求1所述的尿酸氧化酶制剂，其特征在于，所述尿酸氧化酶制剂的剂型包括下列至少之一：液体、半固体、固体。
  20. 根据权利要求1所述的尿酸氧化酶制剂，其特征在于，所述制剂为单剂型形式，每份制剂含有尿酸氧化酶6mg。
  21. 权利要求1～20任一项所述的尿酸氧化酶制剂在制备药物中的用途，所述药物用于治疗或预防高尿酸血症及其相关性疾病。
  22. 一种药物组合物，其特征在于，包含权利要求1～20任一项所述尿酸氧化酶制剂。
  23. 根据权利要求22所述的药物组合物，其特征在于，进一步包括其它用于治疗或预防高尿酸血症及相关性疾病的药物。
  24. 一种治疗或预防高尿酸血症及相关性疾病的方法，其特征在于，包含：
    向受试者施用药学上可接受量的权利要求1～20所述的尿酸氧化酶制剂或权利要求22～23任一项所述的组合物。
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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7056713B1 (en) 1998-08-06 2006-06-06 Duke University Urate oxidase
CN101302501A (zh) * 2007-05-10 2008-11-12 刘国安 聚乙二醇化尿酸氧化酶化合物及其制备方法和其制剂及应用
CN101928704A (zh) * 2010-03-04 2010-12-29 杭州北斗生物技术有限公司 黄曲霉尿酸氧化酶的聚乙二醇修饰物及其制备方法
CN102634492A (zh) * 2011-02-14 2012-08-15 重庆富进生物医药有限公司 聚乙二醇化犬源尿酸氧化酶类似物及其制备方法和应用
CN105087530A (zh) * 2015-08-14 2015-11-25 扬州艾迪生物科技有限公司 聚乙二醇修饰的低分子量尿激酶及其制备方法和应用

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7056713B1 (en) 1998-08-06 2006-06-06 Duke University Urate oxidase
CN101302501A (zh) * 2007-05-10 2008-11-12 刘国安 聚乙二醇化尿酸氧化酶化合物及其制备方法和其制剂及应用
CN101928704A (zh) * 2010-03-04 2010-12-29 杭州北斗生物技术有限公司 黄曲霉尿酸氧化酶的聚乙二醇修饰物及其制备方法
CN102634492A (zh) * 2011-02-14 2012-08-15 重庆富进生物医药有限公司 聚乙二醇化犬源尿酸氧化酶类似物及其制备方法和应用
CN105087530A (zh) * 2015-08-14 2015-11-25 扬州艾迪生物科技有限公司 聚乙二醇修饰的低分子量尿激酶及其制备方法和应用

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
BAYOL A ET AL., BIOPHYS CHEM, vol. 54, 1995, pages 229 - 235
BAYOL A ET AL., BIOPHYS CHEM., vol. 54, 1995, pages 229 - 235
DATABASE Proetin GenPept; ANONYMOUS : "uricase precursor [Sus scrofa] - Protein - NCBI", XP055929016, retrieved from NCBI *
HUANGS HWU T K, EUR J BIOCHEM., vol. 271, 2004, pages 517 - 523
LEE C CWU XGIBBS R ACOOK R GMUZNY D MCASKEYC T, SCIENCE, vol. 239, 1988, pages 1288 - 1291
RETAILLEAU PCOLLOC'HDENIS VFRANCOISE B, ACTA CRYST, vol. 60, 2004, pages 453 - 462
SUZUKI KSAKASEGAWA SMISAKI HSUGIYAMA M, J BIOSCI BIOENG., vol. 98, 2004, pages 153 - 158

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