Uric acid oxidase preparations and their applications

Abstract

A uric acid oxidase preparation is provided, wherein the uric acid oxidase preparation comprises an active ingredient selected from uric acid oxidase modified with polyethylene glycol; and an auxiliary ingredient selected from buffer reagents.

Description

Technology Field

[0001] The present invention relates to the field of biopharmaceuticals, specifically to uric acid oxidase preparations and pharmaceutical compositions. Background Technology

[0002] Gout is a disease caused by long-term purine metabolic disorders or reduced uric acid excretion, characterized by hyperuricemia. Due to the decreased solubility of urate, crystals accumulate in the subcutaneous tissue, joints, and kidneys, forming gouty nodules that lead to recurrent acute arthritis. Furthermore, it affects the kidneys, causing urolithiasis and interstitial nephritis. Purines in the human body are converted into uric acid, the final product, through enzymatic action. Under normal conditions, uric acid levels in the blood range from 149 to 416 mmol / L in men and 89 to 357 mol / L in women; the total body uric acid is approximately 1,200 mg, while production and excretion are in a balanced state at about 600 mg / day. However, hyperuricemia occurs when uric acid accumulates in the blood at 70 mg / L or more due to excessive intake or a dysfunction in the excretion mechanism. Sodium urate can crystallize and precipitate when it reaches saturation in the blood or synovial fluid, or crystallize and precipitate around joints and soft tissues due to prolonged hyperuricemia, potentially causing acute gouty arthritis, chronic gouty arthritis, and joint deformities. Urate can be deposited in the renal tubules and interstitium, causing chronic uric acid nephropathy; in patients with severe hyperuricemia (e.g., patients with malignant tumors such as leukemia and lymphoma), the deposition of large amounts of uric acid in a short period causes urinary tract obstruction and acute renal failure, also known as uric acid nephropathy.

[0003] The cause of hyperuricemia is related to mutations and inactivation of the uricase gene during the process of human evolution, so humans cannot synthesize uricase on their own. Currently, one of the methods to treat hyperuricemia is to use uricase to reduce the uric acid content in the patient's body.

[0004] Uric acid oxidase (EC1.7.3.3) is widely present in microorganisms (Bacillus fastidious, Candida monocytogenes, Aspergillus flavus), plants (soybeans, chickpeas), and animals (pigs, cattle, dogs, baboons) (Suzuki K, Sakasegawa S, Misaki H, Sugiyama M.J Biosci Bioeng.2004.98: 153-158). It can oxidize allantoin and release carbon dioxide by catalyzing uric acid 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 subunit having a molecular weight of approximately 34 kD and consisting of 301 to 304 amino acids. The pH value at which the enzymatic activity of uricase is highest in each solution is 8.0 (Bayol A et al. Biophys Chem. 1995.54: 229-235.).

[0005] Active uricases are tetrameric proteins composed of identical subunits, each with a molecular weight of approximately 34 kD and consisting of 301 to 304 amino acids. The pH value at which uricase enzymatic activity is highest in each solution is 8.0 (Bayol A et al. Biophys Chem. 1995.54: 229-235.). Among all currently known sources of uricases, the one with the highest activity is derived from Aspergillus flavus, reaching 27 IU / mg; the second is derived from Bacillus fastidious, maintaining an activity of 13 IU / mg (Huang S H, Wu T K. Eur J Biochem. 2004.271:517-523.). In addition, uricases derived from legumes possess only 2–6 IU / mg of activity; After recombinant expression of mammalian-derived uricases, porcine-derived uricase activity can reach 5 IU / mg, baboon-derived uricase activity is only 1 IU / mg (Michael H, Susan JK2006.US7056713B1), and human-derived uricase is inactivated.

[0006] In human applications, due to the high activity of microbial uricases and the low immunogenicity of mammalian uricases, uricases derived from them are currently the focus of research in the development and application of recombinant uricases. However, the homology between uricases derived from Aspergillus flavus and speculated human uricases is less than 40% (Lee CC, Wu X, Gibbs RA, Cook RG, Muzny DM, Caskey C T. Science. 1988. 239: 1288-1291.). Since the human body is prone to producing antibodies against uricases, the efficacy of Aspergillus flavus uricases rapidly weakens, and they cannot be used for long-term treatment as they induce severe allergic reactions.

[0007] As a protein, uric acid oxidase must ensure enzyme activity, stability, and shelf life when manufactured into pharmaceutical preparations; however, different manufacturing methods, formulations, buffers, and stabilizers all affect the shelf life and activity of uric acid oxidase, and furthermore, chemical modifications of the protein structure also affect the stability of the uric acid oxidase preparation.

[0008] Therefore, there is a need to develop a uric acid oxidase preparation that can be stably preserved and ensure the activity of uric acid oxidase.

[0009] The present invention is based on the inventor's findings and understanding of the following facts and problems.

[0010] Active uric acid oxidase is a tetrameric protein in which one-third of its amino acids are strongly hydrophobic, and the tetrameric proteins readily aggregate to form octamers and larger aggregates. While molecules with a molecular weight of 100 kDa or more can effectively induce an immune response in the body, the molecular weight of unmodified polymeric uric acid oxidase protein already reaches 140 kDa, and polymeric uricases with larger molecular weights possess higher immunogenicity. Because the human body readily produces antibodies against uricase, it rapidly weakens its efficacy and causes severe allergic reactions, making it unsuitable for long-term treatment. It has been demonstrated that covalently modifying the protein with PEG can reduce protein immunogenicity, increase protein solubility, and extend the protein's half-life.

[0011] Duke University and Savient conducted research on chimeric uricases derived from pigs and baboons (Michael H, Susan JK2006.US7056713B1). This research method essentially achieved the goal of treating intractable gout in humans by modifying the ε-amino group of the lysine residue of porcine uricase using methoxy-containing polyethylene glycol (10KDa-mPEG-NPC) with a molecular weight of 10KDa without significantly reducing enzyme activity (the resulting modified product is pegloticase). The inventors discovered that the results of this research could not completely resolve drug-induced immunogenicity; clinical subjects exhibited a loss of uricase efficacy after multiple injections, which the inventors speculated might be related to the excessively large molecular weight of the pegloticase protein (applying 10KDa PEG results in a pegloticase molecular weight of 540 kDa). At the same time, since pegloticase is suitable for intravenous blue injection but not for injection, it reduces long-term patient compliance and further severely limits its clinical application. To date, there are no sustained-release uric acid oxidase drugs available that are immunogenic and suitable for subcutaneous injection.

[0012] When uric acid oxidase is modified using polyethylene glycol, the properties of the uric acid oxidase are altered depending on the different number of modifications and modification sites, and furthermore, the composition of the uric acid oxidase preparation must be modified so that the enzyme activity, stability, and retention time of the different uric acid oxidases in the preparation meet the standards.

[0013] The present invention aims to solve at least one of the technical problems of the related technology to some extent.

[0014] In a first aspect of the present invention, the present invention proposes a uric acid oxidase preparation. According to an embodiment of the present invention, the uric acid oxidase preparation comprises an active ingredient selected from a uric acid oxidase modified with polyethylene glycol; and an auxiliary ingredient selected from a buffer reagent comprising at least one of a phosphate, a hydrochloride, and a carbonate. The formulation of the preparation according to an embodiment of the present invention has a simple composition, the uric acid oxidase has high stability under the formulation, and the manufacture of the preparation can reduce production costs and has high production efficiency.

[0015] In addition, the uric acid oxidase preparation according to the above embodiment of the present invention further comprises the following additional technical features.

[0016] According to an embodiment of the present invention, at least 11 of the following amino acid regions of the uric acid oxidase have a PEG modification: 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 .

[0017] According to an embodiment of the present invention, the step of ultrafiltration and / or purification of the conjugation reaction product is further included. Accordingly, by-products such as unmodified polyethylene glycol and NHS can be effectively removed, and the purity of the polyethylene glycol-modified uric acid oxidase obtained can be effectively improved.

[0018] According to an embodiment of the present invention, at least one of the following four amino acid sites has a PEG modification: K 30 , K 35 , K 222 and K 231 .

[0019] According to an embodiment of the present invention, the amino acid site is located by an amino acid sequence represented by SEQ ID NO:1.

[0020] TYKKNDEVEFVRTGYGKDMIKVLHIQRDGKYHSIKEVATTVQLTLSSKKDYLHGDNSDVIPTDTIKNTVNVLAKFKGIKSIETFAVTICEHFLSSFKHVIRAQVYVEEVPWKRFEKNGVKHVHAFIYTPTGTHFCEVEQIRNGPPVIHSGI KDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATQVYCKWRYHQGRDVDFEATWDTVRSIVLQKFAGPYDKGEYSPSVQKTLYDIQVLTLGQVPEIEDMEISLPNIHYLNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRL(SEQ ID NO:1).

[0021] According to an embodiment of the present invention, the uric acid oxidase has an amino acid sequence represented by SEQ ID NO:1~7.

[0022] MAHYRNDYKKNDEVEFVRTGYGKDMIKVLHIQRDGKYHSIKEVATSVQLTLSSKKDYLHGDNSDVIPTDTIKNTVNVLAKFKGIKSIETFAVTICEHFLSSFKHVIRAQVYVEEVPWKRFEKNGVKHVHAFIYTPTGTHFCEVEQIRNGPPVIH SGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATQVYCKWRYHQGRDVDFEATWDTVRSIVLQKFAGPYDKGEYSPSVQKTLYDIQVLTLGQVPEIEDMEISLPNIHYLNIDMSKMGLINKEEVLLPLDNPYGRITGTVKRKLTSRL(SEQ ID NO:2).

[0023] MYKNDEVEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSVQLTLSSKKDYVYGDNSDIIPTDTIKNTVHVLAKFKGIKSIETFAMNICEHFLSSFNHVIRAQVYVEEVPWKRFEKNGVKHVHAFIHNPTGTHFCEVEQMRSGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATKVYCKWRYHQGRDVDFEATWDTVRDIVLEKFAGPYDKGEYSPSVQKTLYDIQVHSLSRVPEMEDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRL(SEQ ID NO:3).

[0024] MAHYHNDYKKNDEVEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSVQLTLSSKKDYVYGDNSDIIPTDTIKNTVHVLAKFKGIKSIETFAMNICEHFLSSFNHVIRAQVYVEEVPWKRFEKNGVKHVHAFIHNPTGTHFCEVEQMRSGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATKVYCKWRYHQGRDVDFEATWDTVRDIVLEKFAGPYDKGEYSPSVQKTLYDIQVHSLSRVPEMEDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGRITGTAKRKLASKL(SEQ ID NO:4).

[0025] MAHYHNDYQKNDEVEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSVQLTLNSRREYLHGDNSDIIPTDTIKNTVQVLAKFKGIKSIETFAMNICEHFLSSFNHVIRVQVYVEEVPWKRFEKNGVKHVHAFIHTPTGTHFCEVEQLRSGPPVIHSGIKDLKVLKTTQSGFEGFLKDQFTTLPEVKDRCFATQVYCKWRYHQGRDVDFEATWEAVRGIVLKKFAGPYDKGEYSPSVQKTLYDIQVLSLSQLPEIEDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGRITGTVKRKLTSRL(SEQ ID NO:5).

[0026] MAHYHNDYKKNDEVEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSVQLTLSSKKDYLHGDNSDIIPTDTIKNTVHALAKFKGIKSIEAFAVNICQHFLSSFNHVIRTQVYVEEIPWKRLEKNGVKHVHAFIHTPTGTHFCEVEQLRSGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFAAQVYCKWRYHQCRDVDFEATWDTIRDVVLEKFAGPYDKGEYSPSVQKTLYDIQVVSLSQVPEIDDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRL(SEQ ID NO:6).

[0027] MADYHNNYKKNDELEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSVQLTLSSKKDYLHGDNSDIIPTDTIKNTVHVLAKFKGIKSIEAFGVNICEYFLSSFNHVIRAQVYVEEIPWKRLEKNGVKHVHAFIHTPTGTHFCEVEQLRSGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATQVYCKWRYHQCRDVDFEATWGTIRDLVLEKFAGPYDKGEYSPSVQKTLYDIQVLSLSRVPEIEDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRL(SEQ ID NO:7).

[0028] Here, the amino acid sequence indicated by SEQ ID NO:1 is the amino acid sequence of a pig-derived and baboon-derived chimeric uricase (pig-baboon); the amino acid sequence indicated by SEQ ID NO:2 is the amino acid sequence of a pig-derived uric acid oxidase; the amino acid sequence indicated by SEQ ID NO:3 is the amino acid sequence of a dog-derived and baboon-derived (dog-baboon) chimeric uric acid oxidase; the amino acid sequence indicated by SEQ ID NO:4 is the amino acid sequence of a dog-derived uric acid oxidase; the amino acid sequence indicated by SEQ ID NO:5 is the amino acid sequence of a bovine-derived uric acid oxidase; the amino acid sequence indicated by SEQ ID NO:6 is the amino acid sequence of a monkey uric acid oxidase; and the amino acid sequence indicated by SEQ ID NO:7 is the amino acid sequence of a baboon uric acid oxidase.

[0029] It should be noted that the lysine of the present invention is positioned by the amino acid sequence represented by SEQ ID NO:1, for example, K4 means lysine located at the fourth position based on the amino acid sequence represented by SEQ ID NO:1. Uricase having amino acid sequences represented by SEQ ID NO:1 to 7, or a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity compared to SEQ ID NO:1 to 7; Or, compared to SEQ ID NO:1–7, a polypeptide having one or more amino acids substituted, deleted, and / or added has structural homology, and a person skilled in the art, through sequence comparison, compares the polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with SEQ ID NO:2–7 or SEQ ID NO:1–7, or T in the polypeptide having one or more amino acids substituted, deleted, and / or added compared to SEQ ID NO:1–7. 1 , K3 , 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 By determining a corresponding site corresponding to the site and further determining the PEG modification occurring at the corresponding site in the comparison of the polypeptide, the advantages of the polyethylene glycol-modified uric acid oxidase of the present invention, such as low immunogenicity, high in vivo stability, and suitability for intramuscular injection, can be realized.

[0030] For example, according to an embodiment of the present invention, T of the sequence represented by SEQ ID NO:2 and the sequence represented by SEQ ID NO:1 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 The corresponding part of the area is M 1 , K 9 , K 10 , K 36 , K 41 , K 82 , K 85 , K 103 , K118 , K 122 , K 126 , K 158 , K 185 , K 228 , K 237 , K 272 , K 278 , K 297 , K 299 Includes; the corresponding region of the sequence indicated by SEQ ID NO:3 and the sequence indicated by SEQ ID NO:1 is 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 Includes; the corresponding region of the sequence indicated by SEQ ID NO:4 and the sequence indicated by SEQ ID NO:1 is 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 Includes; the corresponding region of the sequence indicated by SEQ ID NO:5 and the sequence indicated by SEQ ID NO:1 is M 1 , K 10 , K 36 , K41 , 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 Includes; the corresponding region of the sequence indicated by SEQ ID NO:6 and the sequence indicated by SEQ ID NO:1 is 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 Includes; the corresponding region of the sequence indicated by SEQ ID NO:7 and the sequence indicated by SEQ ID NO:1 is 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 299It includes. The inventors discovered through experiments that after PEG-modifying at least 11 of the corresponding sites of the amino acid sequences represented by SEQ ID NO:2~7, the PEG-modified uric acid oxidase obtained has the advantages of low immunogenicity, high in vivo stability, and suitability for intramuscular injection.

[0031] According to an embodiment of the present invention, compared to a peptide map of the uric acid oxidase not modified with polyethylene glycol, the peptide map of the uric acid oxidase modified with polyethylene glycol has a relative ratio of reduced peak area having at least 11 predetermined peptide fragments of 75% or more, preferably 80% or more, and more preferably 90% or more. The uric acid oxidase modified with polyethylene glycol according to an embodiment of the present invention has the advantages of low immunogenicity, high in vivo stability, and suitability for intramuscular injection.

[0032] According to an embodiment of the present invention, the molecular weight of the polyethylene glycol for PEG modification is 6 kD or less. The inventors discovered that uric acid oxidase obtained by modification with polyethylene glycol having a molecular weight of 6 kD or less enhances in vivo organ effects and does not produce severe anti-PEG antibodies due to excessive molecular weight, that is, immunogenicity is further reduced.

[0033] According to an embodiment of the present invention, the polyethylene glycol has a monomethoxyl group or a hydroxyl group.

[0034] According to an embodiment of the present invention, the polyethylene glycol has a linear or branched structure.

[0035] According to an embodiment of the present invention, the polyethylene glycol and uric acid oxidase are joined through an amide bond.

[0036] According to an embodiment of the present invention, the polyethylene glycol is a modified polyethylene glycol, and the modified group of the modified polyethylene glycol comprises at least one selected from N-hydroxysuccinimide, N-hydroxysuccinimidyl carbonate, N-hydroxysuccinimidyl acetate, N-hydroxysuccinimidylpropionate, N-hydroxysuccinimidylbutyrate, N-hydroxysuccinylsuccinate, and bis(p-nitrophenyl)carbonate.

[0037] According to an embodiment of the present invention, the modifier of the modified polyethylene glycol is N-hydroxysuccinimidylpropionate.

[0038] According to the method of the embodiment of the present invention, the immunogenicity of uric acid oxidase can be effectively reduced, the stability of the obtained in vivo uric acid oxidase is higher, and the action lasts longer.

[0039] It can be understood that the technical effect of the additional technical feature of the method for preparing a polyethylene glycol-modified uric acid oxidase described above may be applied to the additional technical feature of the method for reducing the immunogenicity of the uric acid oxidase according to an embodiment of the present invention. The additional technical feature of the method for reducing the immunogenicity of the uric acid oxidase according to an embodiment of the present invention is not described further herein.

[0040] According to an embodiment of the present invention, the buffer reagent comprises at least one of disodium hydrogen phosphate, sodium dihydrogen phosphate monohydrate, and sodium chloride. The uric acid oxidase preparation according to an embodiment of the present invention can ensure the enzymatic inactivity of uric acid oxidase by using disodium hydrogen phosphate, sodium dihydrogen phosphate monohydrate, and sodium chloride as auxiliary ingredients, and indicators such as enzymatic inactivity, protein degradation, and degree of aggregation preserved for 30 days under low, room, and high temperature conditions all met expectations. Furthermore, compared to conventional uric acid oxidase preparations that require the addition of glycine, sucrose, etc., the method of the present invention is simpler to formulate and has high stability of the preparation.

[0041] According to an embodiment of the present invention, the buffer reagent refers to a buffer that resists pH changes through the action of an acid-base conjugated component. The buffer may be present in the liquid or solid formulation of the present invention, and the buffer of the present invention controls the pH of the formulation to 7 to 9. Buffers in a single or combined form that control the pH within the range of 7 to 9 include acetate, succinate, gluconate, histidine, citrate, phosphate, maleate, dimethylarcinate, 2-[N-morpholino]ethanesulfonic acid (MES), bis(2-hydroxyethyl)iminotris[hydroxymethyl]methane (Bis-Tris), N-[2-acetamido]-2-iminodiacetic acid (ADA), glycerin aminoacylglycine, and other organic acid buffers.

[0042] According to an embodiment of the present invention, the pH of the uric acid oxidase preparation is 7 to 9, preferably 7.4 to 8.2. As a result of repeated testing and analysis, the inventors discovered that the uric acid oxidase preparation modified with polyethylene glycol has high stability under the above pH conditions, the uric acid oxidase does not easily aggregate or decompose, and has high enzyme inactivity.

[0043] According to an embodiment of the present invention, the pH of the uricase preparation is 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, and 8.2.

[0044] According to an embodiment of the present invention, the mass ratio of the polyethylene glycol-modified uric acid oxidase to the buffer reagent is (5 to 6):(6 to 37), preferably 6:10. According to the uric acid oxidase preparation of an embodiment of the present invention, by preparing the preparation at the above ratio, the pH of the preparation can be ensured to be 7.4 to 8.2, and the stability of the uric acid oxidase can be ensured.

[0045] According to an embodiment of the present invention, the mass ratio of the polyethylene glycol-modified uric acid oxidase to the buffer reagent is 6:10, 6:11, 5:10, 5:11, 5:9, and 6:9.

[0046] According to an embodiment of the present invention, the mass ratio of the polyethylene glycol-modified uric acid oxidase to the phosphate is (5-6):(1-7).

[0047] According to an embodiment of the present invention, the mass ratio of the polyethylene glycol-modified uric acid oxidase to the sodium chloride is (5-6):(5-30).

[0048] According to an embodiment of the present invention, the mass ratio of the phosphate to the sodium chloride is (1 to 7):(5 to 30), wherein the phosphate is disodium hydrogen phosphate and / or sodium dihydrogen phosphate.

[0049] According to an embodiment of the present invention, the uric acid oxidase preparation provided by the present invention ensures the stability of the uric acid oxidase provided by the present invention by using only a buffer without the need to add stabilizers such as glycerin, glucose, mannitol, or Tween-80, thereby obtaining a highly stable uric acid oxidase preparation. When mannitol and / or glycerol are added to the uric acid oxidase according to an embodiment of the present invention, the particle size of the uric acid oxidase increases, but the addition of Tween-80 does not significantly affect the stability of the uric acid oxidase of the embodiment of the present invention. According to an embodiment of the present invention, the formulation of the uric acid oxidase preparation comprises at least one of liquid, semi-solid, and solid. The uric acid oxidase preparation according to an embodiment of the present invention may be a liquid formulation or a freeze-dried formulation, and dissolves into a liquid upon use. Additionally, the uric acid oxidase preparation may be an injectable formulation and can be injected into a patient's body by means such as intravenous injection or intramuscular injection.

[0050] According to an embodiment of the present invention, the preparation is in a single formulation form, and each preparation contains 6 mg of uric acid oxidase. According to the single-dose uric acid oxidase preparation of the embodiment of the present invention, the single-dose uric acid oxidase has a simple administration method and does not require repeated daily administration, making it convenient for patients to use, thereby achieving maximum efficacy and easy storage.

[0051] In a second aspect of the present invention, the present invention proposes the use of the uric acid oxidase preparation proposed in the first aspect of the present invention in the manufacture of a drug for treating or preventing hyperuricemia and related diseases. According to an embodiment of the present invention, the hyperuricemia-related diseases include chronic hyperuricemia, gout, kidney disease, hyperuric arthritis, kidney stones, gouty nodules, hypertension, diabetes mellitus, hypertriglyceridemia, metabolic syndrome, coronary heart disease, arteriosclerosis, and hyperuricemia caused by anticancer chemotherapy.

[0052] In a third aspect of the present invention, the present invention proposes a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises the uric acid oxidase preparation proposed in the first aspect of the present invention.

[0053] According to an embodiment of the present invention, the pharmaceutical composition further comprises at least one of the following additional technical features.

[0054] According to an embodiment of the present invention, the pharmaceutical composition further comprises other drugs for treating or preventing hyperuricemia and related diseases.

[0055] According to an embodiment of the present invention, when the polyethylene glycol-modified uric acid oxidase or pharmaceutical composition of the present invention is administered in combination therapy using other drugs, it may be administered to an individual sequentially or simultaneously. Alternatively, the pharmaceutical composition of the present invention may comprise a combination of the polyethylene glycol-modified uric acid oxidase of the present invention, a pharmaceutically acceptable carrier or a pharmaceutically acceptable excipient, and other therapeutic or prophylactic drugs known in the art.

[0056] According to an embodiment of the present invention, the pharmaceutical composition has the advantages of low immunogenicity, high in vivo stability, and suitability for intramuscular injection, so it can be used for the treatment or prevention of hyperuricemia-related diseases.

[0057] According to an embodiment of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant. The adjuvant comprises any solvent, solid excipient, diluent, binder, disintegrant or other liquid excipient, dispersant, deodorant or suspending agent, surfactant, isotonic agent, enhancer, thickener, emulsifier, preservative, solid binder, fluidizing agent or lubricant, etc., and is suitable for a specific target formulation.

[0058] Additional aspects and advantages of the present invention will be described in part below, some of which will become obvious from the following description or will be understood through the practice of the invention. Brief explanation of the drawing

[0059] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments together with the following drawings. Herein, Figure 1 is a PHC physicochemical reference material-SEC-HPLC-UV detection spectrum according to an embodiment of the present invention. Figure 2 is a PHC physicochemical reference material-SEC-HPLC-RI detection spectrum according to an embodiment of the present invention. Figure 3 is a PEG reference substance-SEC-HPLC-RI detection spectrum according to an embodiment of the present invention. Figure 4 is a PU5 modified product-SEC-HPLC-UV detection spectrum according to an embodiment of the present invention. Figure 5 is a PU5 modified product-SEC-HPLC-RI detection spectrum according to an embodiment of the present invention. Figure 6 is a comparative figure of PHC and PU5 to which Lys-c and trypsin dual enzyme digestion according to an embodiment of the present invention were applied, respectively. Figure 7 is a digestion diagram of PU5 Lys-C according to an embodiment of the present invention. Figure 8 is a SEC graph of Day 0 prescription 5 according to an embodiment of the present invention. Figure 9 is a SEC graph of prescription 5 on day 30 at 37℃ according to an embodiment of the present invention. Figure 10 is a SEC graph of prescription 6 on day 30 at 37°C according to an embodiment of the present invention. Figure 11 is a SEC graph of prescription 7 on day 30 at 37°C according to an embodiment of the present invention. FIG. 12 is a diagram showing serum uric acid levels after intramuscular administration of different doses to model rats according to an embodiment of the present invention. FIG. 13 is a diagram showing kidney damage, necrosis, and inflammation scores according to an embodiment of the present invention. FIG. 14 is a figure showing the average blood concentration-time curves of each group after a single intravenous injection of the same dose (1.0 mg / kg) of pegloticase and pegylated uricase solution into SD rats according to an embodiment of the present invention. FIG. 15 is a figure showing the average blood concentration-time curves of each group after a single intramuscular injection of pegloticase and different doses of pegylated uricase injection solution into SD rats according to an embodiment of the present invention. FIG. 16 is a figure showing the average blood concentration-time curves of each group after a single intramuscular injection of pegylated uricase solution at different doses into SD rats according to an embodiment of the present invention. FIG. 17 is a figure showing the average blood uric acid level-time curves of each group after a single intramuscular / intravenous injection of pegloticase and pegylated uricase solutions at different doses in SD rats according to an embodiment of the present invention. FIG. 18 is a figure showing the average blood concentration-time curves of males and females after intravenously injecting pegloticase and pegylated uricase solution at the same dose (1.0 mg / kg) into SD rats according to an embodiment of the present invention first (Day 1). FIG. 19 is a figure showing the average blood concentration-time curves of males and females after intravenously injecting pegloticase and pegylated uricase solution at the same dose (1.0 mg / kg) into SD rats according to an embodiment of the present invention on Day 22. FIG. 20 is a figure showing the average blood concentration-time curves of males and females after intramuscular injection of pegloticase and pegylated uricase solution at the same dose (1.0 mg / kg) into SD rats according to an embodiment of the present invention on Day 1. FIG. 21 is a figure showing the average blood concentration-time curves of males and females after intramuscular injection of pegloticase and pegylated uricase solution at the same dose (1.0 mg / kg) in SD rats according to an embodiment of the present invention on Day 22. FIG. 22 is a figure showing the average blood uric acid level-time curve over time after several intravenous injections of pegloticase and pegylated uricase solutions into SD rats according to an embodiment of the present invention. FIG. 23 is a time-average blood uric acid level-time curve after several intramuscular injections of pegloticase and pegylated uricase injection solutions into SD rats according to an embodiment of the present invention. Specific details for implementing the invention

[0060] Hereinafter, embodiments of the present invention are described in detail, and examples of said embodiments are illustrated in the drawings. Throughout, identical or similar reference numerals indicate identical or similar components or components having identical or similar functions. The embodiments described below with reference to the attached drawings are illustrative and are intended only to interpret the present invention and should not be understood as limiting the present invention.

[0061] The object of the present invention is to provide a new uric acid oxidase preparation modified with polyethylene glycol.

[0062] Another objective of the present invention is to provide an application of the polyethylene glycol modified uric acid oxidase preparation, which can achieve in vivo persistence, significantly reduce blood uric acid levels, and be used for the treatment of hyperuricemia and gout.

[0063] In one embodiment of the present invention, a uric acid oxidase preparation modified with polyethylene glycol is provided.

[0064] Uric acid oxidase is not particularly limited and may be uric acid oxidase derived from any source and its uric acid oxidase analogs, and representative examples include, but are not limited to, mammalian-derived, microorganisms, and plants.

[0065] The uric acid oxidase derived from heterologous sources described in the present invention can be obtained through various routes including, but not limited to, natural extraction, chemical synthesis, and genetic engineering recombinant expression.

[0066] In another preferred example, a recombinant expression strain is prepared by a method of constructing a recombinant expression strain using E. coli or yeast as a host, and more preferably, recombinant expression is performed using E. coli as the host strain.

[0067] In another preferred example, a formulation prepared by using the polyethylene glycol uric acid oxidase as an active ingredient and adding sodium dihydrogen phosphate, disodium hydrogen phosphate, and sodium chloride can ensure maximum enzyme activity and improve the storage stability of the formulation.

[0068] In another preferred example, the polyethylene glycol uric acid oxidase preparation has high storage stability without the need to add other stabilizers such as mannitol, glycerin, or Tween-80, so the uric acid oxidase preparation has a simple composition, a simple manufacturing process, and a low cost.

[0069] In one embodiment of the present invention, an application of the uric acid oxidase preparation modified with polyethylene glycol is provided.

[0070] The above polyethylene glycol uric acid oxidase preparation is more suitable as a drug and combination thereof for treating chronic hyperuricemia or gout. The main symptoms of the above hyperuricemia and gout include, but are not limited to, uric acid nephropathy and gouty arthritis.

[0071] The administration route of the above polyethylene glycol uric acid oxidase includes, but is not limited to, intravenous injection, subcutaneous injection, intramuscular injection, and intraperitoneal injection, preferably intravenous injection, intramuscular injection, and more preferably intramuscular injection.

[0072] The above polyethylene glycol uric acid oxidase has lower in vivo immunogenicity.

[0073] The fact that the above polyethylene glycol uric acid oxidase has low immunogenicity means that after intramuscular injection of polyethylene glycol uric acid oxidase into the body of a human or animal, the body does not produce anti-polyethylene glycol molecular antibodies or produces anti-polyethylene glycol molecular antibodies of low titer and does not produce antibodies against uric acid oxidase.

[0074] The above polyethylene glycol uric acid oxidase has a longer half-life in vivo after intramuscular injection and has the effect of lowering uric acid levels in vivo.

[0075] Furthermore, the terms "first" and "second" are used solely for descriptive purposes and should not be understood as indicating or implying relative importance, or implicitly specifying the number of indicated technical features. Accordingly, the features limited to "first" and "second" may explicitly or implicitly include at least one of these features. In the description of the present invention, "plural" means at least two, such as two or three, unless otherwise clearly and specifically limited.

[0076] The present invention will be described below with reference to specific embodiments, but these embodiments are for illustrative purposes only and do not limit the invention in any way.

[0077] Example 1: Preparation of recombinant uric acid oxidase

[0078] 1.1 Construction of Gene and Expression Plasmid for Uricase Expression

[0079] E.coli Based on codon usage preference data, the cDNA sequence of the uricase protein (code: PHC) (SEQ ID NO: 1) was designed in conjunction with factors such as codon preference and GC content, and the entire gene was synthesized and named the pUC-57-PHC plasmid. Nde I and BamH I were used as target gene insertion sites, and the pET-30a plasmid was used as the expression vector (pET-30a-PHC).

[0080] 1.2 Transformation of Expression Plasmids into Bacterial Host Cells

[0081] The expression vector pET-30a-PHC was introduced into E. coli BL21 (DE3) via the CaCl2 method, high-expression clones were screened through resistance screening using Kanamycin, and the original seed bank strain (E3B) was preserved. These steps were performed according to methods commonly used in the field of molecular biology.

[0082] 1.3 Preparation of Recombinant Uric Acid Oxidase

[0083] The transformed and engineered strains were fermented and expressed in a fermenter under the following control conditions. First, at 30°C and pH approximately 7.2, OD 600 The cells were cultured at a concentration of 30 or higher, and IPTG was added at 0.5 mmol / L. The cells were then continuously induced for at least 3 hours to allow for the accumulation of uric acid oxidase. After centrifugation, the cells were collected and stored at -15℃ or below.

[0084] Cryopreserved bacteria were taken and suspended in 25 mmol / L Tris and 5 mmol / L EDTA buffer at a suspension ratio of 1:10 (W / V). After rupturing the bacterial cells under high pressure, the uric acid oxidase precipitate was collected by centrifugation. The precipitate was washed once with 50 mmol / L NaHCO3. The concentrated uricase precipitate was then suspended in 100 mmol / L Na2HCO3 (pH 9.7–10.3) buffer at a suspension ratio of 1:50 (W / V). The solution was dissolved by stirring overnight at room temperature, and the supernatant was collected by centrifugation.

[0085] Uric acid oxidase was further purified through several chromatographic steps, and the purity detected by SDS-PAGE was over 95%, and the purity detected by Superdex 200 column was over 95%, with no aggregates. Protein concentration was measured by the Lowry method, and the activity of uric acid oxidase was measured by a spectrophotometer. Here, 1 unit (U) of enzyme activity is defined as the amount of enzyme required to convert 1 μmol of uric acid per minute under optimal pH 9.0 buffer conditions at an optimal reaction temperature of 37°C.

[0086] Example 2: Preparation of Pegylated Oxyuricase

[0087] Monomethoxy PEG derivatives of different molecular weights (500–20000 Da), such as N-succinimidylpropionate PEG (5K-PEG-SPA) with a molecular weight of 5K, were dissolved in a 100–300 mmol / L PEG solution in an acidic solution of 1–5 mmol / L. After dissolution, the mixture was added to a carbonate buffer solution containing dissolved uric acid oxidase at a molar ratio of 1:45–1:150 (uric acid oxidase: 5K-PEG-SPA) with a carbonate concentration of 0.1–0.3 mol / L and a pH of 10.0 to induce a conjugation reaction between PEG and uric acid oxidase. The concentration of uric acid oxidase for the conjugation reaction was 10 mg / ml, and the reaction was stirred for at least 60 minutes under a temperature of 5–30°C until the degree of PEG conjugation no longer changed over time. After the reaction was completed, unmodified PEG and byproducts were removed from the reaction by ultrafiltration and / or chromatography. Modified byproducts could be separated and removed by selecting an appropriate molecular sieve chromatography medium, and finally, 5K modified pegylated uric acid oxidase (code: PU5) was obtained by sterile filtration.

[0088] Example 3: Characterization of Pegylated Oxyuricase

[0089] 3.1 Detection of Average Strain and Enzyme Activity

[0090] Protein concentration was measured using the Lowry method, and the activity of polyethylene glycol uric acid oxidase was measured using a spectrophotometer. The maximum UV absorption wavelength of uric acid, the substrate of uricase, is 293 nm, and the maximum UV absorption wavelength of the product allantoin is 224 nm. Since the absorption value of uric acid at 293 nm is proportional to concentration within a certain concentration range, and the quantitative measurement of uric acid can be performed using the spectrophotometer, the specific procedure is as follows. The UV-Vis spectrophotometer was turned on and the wavelength was adjusted to 293 nm, and then the water bath circulation system of the instrument was turned on to maintain the temperature at 37°C. Sodium tetraborate buffer was used as a blank control and zero-point calibration was performed; 2.95 ml of substrate reaction solution (0.1 mol / L sodium tetraborate, 100 μmol / L uric acid, pH 9.5, preheated to 37°C) was taken and placed in a quartz cuvette, 50 μl of the test sample was added and mixed rapidly to measure the absorption value at 293 nm. The change in absorption at 293 nm was measured continuously; the uric acid degradation concentration and enzyme activity were calculated according to C = A / εL (where A is the absorption value of uric acid at a specific concentration at 293 nm, ε is the molar extinction coefficient of uric acid, L is the optical path of the cuvette, and C is the molar concentration of uric acid); enzyme activity was defined as the amount of enzyme required to transform 1 μmol of uric acid into allantoin per minute at an optimal reaction temperature of 37°C and an optimal reaction pH of 9.5, with 1 active unit (U).

[0091] The average strain of polyethylene glycol uric acid oxidase was detected using SEC-HPLC connected in series with UV / RI (combined ultraviolet and refractive index detectors). Proteins have a maximum absorption peak at 280 nm ultraviolet, whereas PEG does not absorb at the above wavelength, and within a certain range by differential refractive index detectors, the absorption values ​​of protein and PEG are proportional to various concentrations. Therefore, the content of the PEG portion and the protein portion of pegylated uric acid oxidase can be obtained using an external standard method with PEG reference material and PHC physicochemical reference material, and furthermore, the number of PEG molecules in each uric acid oxidase monomer, i.e., the average strain, can be calculated using the following calculation method.

[0092] PEG Uric Acid Oxidase Average Strain = (Relative Molecular Weight of Uric Acid Oxidase Subunit × Amount of PEG in Sample) / (Relative Molecular Weight of PEG × Amount of Protein in Sample)

[0093] Here, the SEC-HPLC-UV / RI detection spectra of the PHC physicochemical reference material, the PEG reference material, and the PU5 modified product are shown in Figures 1 to 5.

[0094] The enzymatic activity and average strain of polyethylene glycol oxyuricase obtained under different feed ratios in Example 2 are shown in Table 1.

[0095] Table 1: Enzyme activity and mean strain of 5K-PEG at different feed ratios

[0096] Protein: 5K-PEG feed molar ratio Enzyme activity Enzyme activity retention rate Average strain Non-modified uric acid oxidase 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

[0097] Note: The average degree of modification represents the number of PEG molecules bound to each uric acid oxidase monomer. From Table 1, it can be seen that within the protein:5K-PEG feed ratio range of 1:56 to 1:110, the average degree of modification of the polyethylene glycol uric acid oxidase of the present invention stabilizes at 11 or higher, and the enzyme activity retains a higher rate than that of the unmodified uric acid oxidase. Furthermore, the enzyme activity does not decrease but rather increases and remains relatively stable, which is completely different from the commercially available drug Krystexx (pegloticase). According to the contents disclosed in Savient’s patent (CN1264575C, FIG. 2A–3B) and the general knowledge of those skilled in the art, enzyme activity decreases significantly as the degree of modification of 5kD PEG increases. However, unexpectedly, the polyethylene glycol uricase obtained in the present invention did not show a significant change in enzyme activity at an average degree of modification of 11 or higher compared to when it was unmodified. Therefore, the polyethylene glycol uric acid oxidase of the present invention has a higher average polyethylene glycol modification degree compared to commercially available drugs, and has obtained unexpected technical effects in terms of retaining enzyme activity. According to the inventor's conjecture, this may be due to differences in the PEG modification degree or modification sites of the polyethylene glycol uric acid oxidase.

[0098] 3.2 Detection of areas modified by polyethylene glycol

[0099] In the following steps, the inventor performed detection of modified sites on the uric acid oxidase obtained in Example 2.

[0100] The PEG modification site of polyethylene glycol-modified uric acid oxidase can be identified by first digesting non-pegylated and pegylated uric acid oxidases with one or more enzymes, and then obtaining a chromatogram, i.e., a peptide map, through chromatographic detection. Non-pegylated and pegylated uric acid oxidases can be digested via single enzymatic digestion (Lys-C or Trypsin) and / or double enzymatic digestion (Lys-C and Trypsin combined). Digestion fragments were separated using a reverse-phase column, and the modification site of polyethylene glycol uric acid oxidase was determined by internal reference peptide fragment correction and comparison of the loss or reduction rate of the peptide fragments.

[0101] Principle of analysis of modified sites in the Trypsin and Lys-C dual enzyme digestion quality peptide map: Lys-C can specifically digest the C-terminus of lysine (K), and trypsin specifically digests C-terminal peptides using the basic amino acids arginine (R) and lysine (K) as digestion sites. By comparing the changes in each corresponding peptide fragment before and after digestion by PHC and PU5, and by binding them to an internal standard peptide fragment, the relative ratio of reduction or loss of PEG-modified peptide fragments can be analyzed and verified. Through the relative ratio of reduction or loss of peptide fragments, it is possible to determine whether the lysine region of the peptide fragment has been modified by PEG and the ratio of such modification. It should be noted that PEG modification is non-uniform, and a high modification ratio in a particular region may be considered as modification of that region.

[0102] To explain in detail, it is as follows.

[0103] (1) Sample treatment: Uric acid oxidase and pegylated uric acid oxidase were each taken and dissolved in digestion buffer (25 mmol / L Tris-HCl, 20% acetonitrile, pH 9.0) to dilute to 1 mg / ml. 100 μl of each diluted solution was taken, 2 μl of Lys-C was added, and the solution was digested at 37°C for 4 hours. Then, the solution was transferred to a pancreatin reaction tube (ratio 1:100) and digestion was continued at 37°C for 2 hours. Afterward, 4 μl of TCEP reduction solution was added and the reaction was continued for 30 minutes, and then 10 μl of 1 mol / L hydrochloric acid solution was added to terminate the reaction.

[0104] (2) Analysis conditions:

[0105] Instruments: Thermo Ultimate 3000 HPLC and MSQ Plus;

[0106] Chromatography column: Welch Materials μltimate®XB-C18(4.6mm Υ250mm, 5μm);

[0107] Analysis conditions: Solution A (aqueous solution containing 0.1% TFA), Solution B (acetonitrile solution containing 0.1% TFA);

[0108] Gradient: 0-70 min, B is 3-70%;

[0109] LC detection wavelength: 214nm.

[0110] Ion source: ESI;

[0111] Ion type: Cation;

[0112] Cone voltage: 50V;

[0113] Scanning range: 300-2000Da;

[0114] Scanning time: 1S;

[0115] Post-column flow switching: approx. 0.3 ml / min.

[0116] 100 μl of sample was injected and a chromatogram was recorded.

[0117] (3) Result processing:

[0118] The chromatograms (peptide maps) of uric acid oxidase and pegylated uric acid oxidase were compared, and the relative ratio of the reduction in the area of ​​differential peptide fragments was calculated.

[0119] (4) The experimental results are shown in Tables 2 to 5 and Figures 6 to 7.

[0120] Table 2: List of peptide fragments of PHC after digestion with Lys-C

[0121] peptide fragment Digestive area order Theoretical molecular weight (Da) Actual measured molecular weight T1 3 TYK 410.47 410.2 T2 4 K 146.189 / T3 17 NDEVEFVRTGYGK 1513.627 / T2+T3 KNDEVEFVRTGYGK 1641.089 1642.2 T4 21 DMIK 505.63 505.4 T5 30 VLHIQRDGK 1065.241 1065 T6 35 YHSIK 646.744 646.5 T7 48 EVATTVQLTLSSK 1376.57 / T8 49 K 146.189 / T9 66 DYLHGDNSDVIPTDTIK 1903.032 1903 T10 74 NTVNVLAK 858.005 857.7 T11 76 FK 293.366 293.1 T12 79 GIK 316.401 316.2 T13 97 SIETFAVTICEHFLSSFK 2059.364 2059 T14 112 HVIRAQVYVEEVPWK 1853.154 1852.8 T15 116 RFEK 578.669 578.4 T16 120 NGVK 416.478 417.2 T17 152 HVHAFIYTPTGTHFCEVEQIRNGPPVIHSGIK 3586.088 3586.2 T18 155 DLK 374.437 374.1 T19 158 VLK 358.481 358.2 T20 169 TTQSGFEGFIK 1214.34 1213.8 T21 179 DQFTTLPEVK 1177.32 1176.8 T22 190 DRCFATQVYCK 1333.543 1333.2 T23 215 WRYHQGRDVDFEATWDTVRSIVLQK 3106.45 3106.5 T24 222 FAGPYDK 796.878 796.5 T25 231 GEYSPSVQK 994.069 993.7 T26 266 TLYDIQVLTLGQVPEIEDMEISLPNIHYLNIDMSK 4046.66 4046.1 T27 272 MGLINK 674.856 / T28 285 EEVLLPLDNPYGK 1486.685 1486.6 T27+ T28 MGLINK EEVLLPLDNPYGK 2143.54 2143.2 T29 291 ITGTVK 617.743 617.4 T30 293 RK 302.377 / T31 298 LSSRL 574.678 574.4

[0122] Table 3: List of peptide fragments of PHC after dual enzyme digestion with Lys-C and trypsin

[0123] peptide fragment Sequence position order Theoretical relative molecular weight [Da] Actual measured molecular weight T1 1-3 TYK 410.470 410.3 T2 4 K 146.189 / T3 5-12 NDEVEFVR 1007.068 / T2+3 4-12 KNDEVEFVR 1135.4 T4 13-17 TGYGK 524.574 524.5 T5 18-21 DMIK 505.630 505.5 T6 22-27 VLHIQR 764.926 764.8 T7 28-30 DGK 318.330 / T8 31-35 YHSIK 646.744 646.7 T9 36-48 EVATTVQLTLSSK 1376.570 / T10 49 K 146.189 / T11 50-66 DYLHGDNSDVIPTDTIK 1903.032 1903.4 T12 67-74 NTVNVLAK 858.005 857.9 T13 75-76 FK 293.366 293.1 T14 77-79 GIK 316.401 / T15 80-97 SIETFAVTICEHFLSSFK 2059.364 2059.6 T16 98-101 HVIR 523.636 523.6 T17 102-112 AQVYVEEVPWK 1347.534 1347.4 T18 113 R 174.203 / T19 114-116 FEK 422.481 / T18+19 113-116 RFEK 578.684 578.6 T20 117-120 NGVK 416.478 417.1 T21 121-141 HVHAFIYTPTGTHFCEVEQIR 2485.802 2486.8 T22 142-152 NGPPVIHSGIK 1118.301 1118.8 T21+22 121-152 3586.103 3587.7 T23 153-155 DLK 374.437 / T24 156-158 VLK 358.481 358.3 T25 159-169 TTQSGFEGFIK 1214.340 1214.2 T26 170-179 DQFTTLPEVK 1177.320 1177.2 T27 181-181 DR 289.291 / T28 182-190 CFATQVYCK 1062.267 / T27+28 181-190 1333.558 1333.6 T29 191-192 WR 360.416 360.1 T30 193-197 YHQGR 659.702 659.6 T31 198-209 DVDFEATWDTVR 1453.528 1453.6 T32 210-215 SIVLQK 686.850 686.8 T33 216-222 FAGPYDK 796.878 796.8 T34 223-231 GEYSPSVQK 994.069 994.1 T35 262-266 TLYDIQVLTLGQVPEIEDMEISLPNIHYLNIDMSK 4046.660 4047 T36 267-272 MGLINK 674.856 674.7 T37 273-285 EEVLLPLDNPYGK 1486.685 1486.7 T36+37 2143.541 2143.6 T38 286-291 ITGTVK 617.743 617.7 T39 292 R 174.203 / T40 293 K 146.189 / T41 294-297 LSSR 461.519 461.5 T42 298 L 131.175 /

[0124] Method for calculating the percentage reduction in the peak area of ​​a PU5 peptide fragment: Using the following formula, the peak area of ​​the corresponding PU5 peptide fragment at a PU5 concentration equal to the concentration of PHC can be calculated.

[0125] A1 = A0 × t

[0126] Here, A1 is the peak area of ​​the PU5 peptide fragment after the conversion of two internal reference peptide fragments, A0 is the actual measured peak area of ​​the PU5 peptide map peptide fragment, and t is the average value of the ratio of the peak areas of the PHC peptide map and the PU5 peptide map in the T30 and T31 internal reference peptide fragments, i.e., 0.588.

[0127] Table 4: Comparison of PHC and PU5 internal reference peptide fragments

[0128] Peptide fragment number order PHC Peptide Map PU5 Peptide Map PHC and PU5 peak area ratio Retention time Peak area Retention time Peak area Each value medium T30 YHQGR 7.5 13.4 7.467 22.9 0.585 0.588 T31 DVDFEATWDTVR 28.31 35.5 28.28 60.1 0.591

[0129] The relative percentage reduction in the peak area of ​​a specific peptide fragment in the PU5 peptide map can be calculated using the following formula with the peak area of ​​the peptide fragment converted to an internal reference and the peak area of ​​the PHC peptide map: P(%)=(A2-A1) / A2×100%

[0130] Here, A2 is the peak area of ​​a specific peptide fragment in the PHC peptide map, and A1 is the peak area of ​​the peptide fragment in PU5 after conversion to an internal reference.

[0131] Table 5: Summary results of peptide fragments with reduced peak area in the peptide map after diubal enzyme digestion of PU5

[0132] Peptide fragment location Peptide fragment sequence Relative ratio of reduced peak area of ​​peptide fragments 1-3 TYK 100.00% 4-12 KNDEVEFVR 94.07% 31-35 YHSIK 100.00% 75-76 FK 82.27% 80-97 SIETFAVTICEHFLSSFK 100.00% 102-112 AQVYVEEVPWK 100.00% 113-116 RFEK 100.00% 117-120 NGVK 100.00% 121-152 HVHAFIYTPTGTHFCEVEQIRNGPPVIHSGIK 100.00% 193-197 YHQGR Internal reference peptide fragment 198-209 DVDFEATWDTVR Internal reference peptide fragment 216-222 FAGPYDK 91.37% 223-231 GEYSPSVQK 86.40% 232-266 TLYDIQVLTLGQVPEIEDMEISLPNIHYLNIDMSK 100.00% 273-285 EEVLLPLDNPYGK 100.00%

[0133] From the analysis of the protein sequence (SEQ ID NO:1) of this example, the potential site for modification of uric acid oxidase is 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 It can be seen that there are 31 sites. As shown in the analysis of Tables 2, 3, 4, 5 and Figure 6, from the analysis of the polyethylene glycol-modified uric acid oxidase sites obtained in Example 2, the sites where more than 90% of the peptide fragment was lost after PU5 digestion are K 3 , K 4 , K 35 , K 97, K 112 , K 116 , K 120 , K 152 , K 222 , K 266 , K 285 And, the site where peptide fragments were lost within the 80%–90% range after PU5 digestion is K 76 , K 231 And, it can be seen that all these parts are deformed in PU5.

[0134] Furthermore, the inventors discovered that the polyethylene glycol-modified uric acid oxidase of the present invention has more modification sites and exhibits significant differences compared to commercially available drugs. For example, through single-stage digestion of the enzyme, K in the polyethylene glycol-modified uric acid oxidase of the present invention 30 , K 35 , K 222 and K 231 The loss rate of peptide fragments located in these four sites is over 80%, but analysis of the commercially available analog drug Krystexx (pegloticase) using this method showed that peptide fragments located in these four sites were hardly lost. In other words, the commercially available analog drug K 30 , K 35 , K 222 and K 231 The strain occurring at these four sites was much lower than that of the polyethylene glycol-modified uric acid oxidase of the present invention. In addition, the polyethylene glycol-modified uric acid oxidase of the present invention has significantly lower immunogenicity compared to commercially available drugs, and the inventor speculates that this may be related to the number of modification sites and differences in modification sites. Due to different modification sites and degrees of modification, the protection of the enzyme's immunogenic sites and the exposure of the enzyme's active center in vivo are both different. These differences may cause differences in the biological properties of different modified enzymes in vivo.

[0135] Since uric acid oxidase is a protein preparation, the effect on stability varies depending on chemical modification; therefore, when manufacturing the preparation, it is necessary to select different auxiliary materials, pH, etc., according to the specific active ingredient of the uric acid oxidase to produce the uric acid oxidase preparation. Below, the preparation of a uric acid oxidase preparation having high stability and high biological activity for the polyethylene glycol-modified uric acid oxidase of the present invention is described.

[0136] Example 5: Prescription Screening

[0137] Polyethylene glycol modified uric acid oxidase PU5 obtained in the above example was used as the active ingredient, and the components of polyethylene glycol modified uricase preparations were screened to obtain a polyethylene glycol uricase preparation having high uricase activity and high stability. The screening includes screening of auxiliary ingredient compositions and screening of stabilizers.

[0138] 1. Screening of auxiliary materials

[0139] The auxiliary ingredients of the formulation include carbonates, phosphates, hydrochlorides, and citrates.

[0140] The above buffer auxiliary raw materials and the uric acid oxidase preparation modified with polyethylene glycol for the preparation of the active ingredient are used. Under conditions where the auxiliary raw materials have the same concentration range of 10–50 mmol / L and the same pH (pH 7–9), the stability of the uric acid oxidase modified with polyethylene glycol is basically consistent, but the ratio of different auxiliary raw material concentrations and the difficulty of controlling the osmotic pressure of the preparation differ during the preparation process. Among them, phosphate and hydrochloride are easier to control in terms of osmotic pressure and pH than carbonate and citrate. When phosphate and hydrochloride are finally used as auxiliary raw materials through buffer screening, the phosphate concentration range is 10–50 mmol / L and the hydrochloride concentration range is 100–200 mmol / L, both of which can satisfy the requirements of the preparation process. Among them, the preparation with 15 mmol / L sodium dihydrogen phosphate / disodium hydrogen phosphate, 0.136 mol / L sodium chloride, and a pH of 7.4 has relatively superior stability. The inventor used this as the basic composition to perform additional screening.

[0141] 2. Stabilizer screening experiment

[0142] Mannitol, glycerin, and Tween-80 were added to the basic formulation, and through a stability review, it was determined whether it was necessary to add the corresponding stabilizers to the formulation.

[0143] 2.1 Stabilizer Initial Screening Experiment

[0144] Stabilizers such as 4% mannitol, 2% glycerin, and 0.04% Tween-80 were added to the basic formulation using the basic formulation as a control, and sample numbers were recorded as Samples 1 to 4, respectively. Samples 1 to 4 were examined at 45°C for 7 days, and the effect of the stabilizers on the stability of the PU5 formulation was studied by detecting and sampling the particle size.

[0145] From the experimental results, it can be seen that the particle size of the composition with added glycerin and mannitol (19.44 and 20.51, respectively) is larger than the particle size (18.57) of the basic formulation composition, and the particle size (18.10) of the formulation composition with added Tween-80 is slightly smaller than the basic formulation composition. Therefore, it may be considered to prevent an increase in the PU5 particle size by adding Tween-80 to the formulation composition.

[0146] 2.2 Stabilizer Re-screening Experiment

[0147] From the initial screening experiment of the stabilizer, it was found that adding Tween-80 to the formulation composition can prevent an increase in PU5 particle size, which is further confirmed through stability. Samples 1 and 4, which were initially screened and examined at 45°C for 7 days, were placed back into a 45°C examination box and left for 15 days, after which samples were taken to detect particle sizes, and the effect of the Tween-80 stabilizer on the stability of the PU5 formulation was investigated.

[0148] (1) The particle size detection results are shown in Table 6.

[0149] Table 6: Stabilizer Re-screening Particle Size Statistics

[0150] Sample Name Processing method Average particle size (nm) Maximum particle size (nm) PDI (Polyvariance Index) Percentage of particles with a diameter of 20 nm (%) Number 1 control group 18.46 20.51 0.108 4.0 45℃ 7 days 18.57 20.81 0.123 3.2 45℃ 22 days 18.14 20.59 0.111 2.4 Number 4 control group 17.51 18.98 0.073 3.7 45℃ 7 days 18.10 19.91 0.089 3.7 45℃ 22 days 18.47 20.87 0.119 2.9

[0151] Note: The control group is indicated by being left under conditions of 2–8°C.

[0152] (2) Conclusion

[0153] Data analysis of the control group and the group examined for 7 and 22 days at 45°C: When 0.04% Tween-80 was added, the PDI of the sample examined within 7 days at 45°C was much smaller and the sample was uniform, and when examined for 22 days at 45°C, the addition of Tween-80 did not affect the PDI, indicating that the addition of Tween-80 at high temperatures is not meaningful in preventing or delaying the increase in PU5 particle size.

[0154] 2.3 Third Stabilizer Screening Experiment

[0155] To finally determine whether to add Tween-80 to the formulation composition, formulations with and without Tween-80 were placed at 25°C and 37°C, respectively, and observed for one month. Samples were taken to detect particle size, high and low molecular weight protein content, pH, and enzyme activity, and the results are shown in Table 7.

[0156] Table 7: Results of Acceleration Stability Analysis

[0157] Consideration Indicators Content (mg / ml) high molecular weight protein content low molecular weight protein content Enzyme inactivity (U / mg) Average particle size (nm) Tween-80 not added Before the discussion 6.15 0.31% Undetected 10.45 / 25℃ 1 month 5.98 0.17% Undetected 9.60 17.93 37℃ 1 month 5.78 0.14% 9.20% 9.60 17.78 Tween-80 added Before the discussion 6.46 0.37% Undetected 9.07 / 25℃ 1 month 6.04 0.18% Undetected 8.95 17.62 37℃ 1 month 6.00 0.20% 9.90% 8.75 17.32

[0158] From Table 7, the accelerated stability analysis of the PU5 formulation with and without Tween-80 revealed that the behavior regarding changes in high and low molecular weight proteins, enzyme activity, and particle size was essentially consistent. After extensive research, it was decided not to add a stabilizer to the formulation, as there were no significant differences in high and low molecular weight protein content or particle size before and after the addition of Tween-80.

[0159] Example 6: Formulation pH Screening

[0160] 1. pH range optimization

[0161] (1) Research method

[0162] The pH of the formulation solutions was adjusted to 7.8, 7.0, 7.4, 8.2, and 8.6, respectively, using buffer salts. Stability was assessed by placing the samples at 2–8°C, 25±2°C, and 37°C, respectively, and the high and low molecular weight protein content and enzyme activity were measured on the 30th day for each. The polymerization or degradation status of PU5 protein and changes in enzyme activity were compared for each formulation.

[0163] (2) Measurement results

[0164] The measurement results are shown in Table 8 and Figures 8–11.

[0165] Table 8: Statistical table of measurement results for high and low molecular weight protein content and enzyme activity (%)

[0166] / Prescription 5 Prescription 6 Prescription 7 Prescription 8 Prescription 9 7.8 8.2 7.4 7.0 8.6 polymer content 0 days 0.56% 0.82% 0.67% 0.33% 0.22% 2~8℃ 30 days 0.19% 0.16% 0.19% 0.31% 0.26% 25±2℃ 30 days 0.14% 0.12% 0.15% 0.21% 0.13% 37℃ 30 days 0.04% 0.03% Undetected 0.17% 0.05% low molecular weight content 0 days ＜1.0% ＜1.0% ＜1.0% 1.3% ＜1.0% 2~8℃ 30 days ＜1.0% ＜1.0% ＜1.0% 2.3% 1.4% 25±2℃ 30 days ＜1.0% ＜1.0% ＜1.0% 4.8% 1.7% 37℃ 30 days 14.7% 10.5% 23.4% 27.1% 19.6% Enzyme inactivity (U / mg) 0 days 9.77 10.10 9.98 9.76 8.13 25±2℃ 30 days 9.71 9.58 9.36 8.40 9.09 37℃ 30 days 8.95 9.95 8.20 8.11 8.50

[0167] Note: "Long-term" refers to 2–8°C, "Accelerated" refers to 25±2°C, and "High temperature" refers to 37°C. From Figures 8–11 and Table 8, the high molecular weight protein content of Prescription 5 (pH 7.8), Prescription 6 (pH 8.2), and Prescription 7 (pH 7.4), examined for 30 days at 2–8°C and 25±2°C, showed a decreasing trend; the low molecular weight protein content showed a slight increasing trend, with Prescription 7 > Prescription 5 > Prescription 6, and it can be seen that the enzyme activity of all three prescriptions did not show significant changes. From this, it was determined that the pH of the PU5 preparation was controlled to 7.4–8.2. As a result of high-temperature testing at 37°C for 30 days, there was no significant difference in the high molecular weight protein content of Prescriptions 5, 6, and 7, but the low molecular weight protein content increased, with Prescription 7 > Prescription 5 > Prescription 6. From this, it was determined that the midpoint of the PU5 formulation is 7.8.

[0168] In summary, the pH range of the PU5 injection formulation of this product is determined to be 7.4 to 8.2.

[0169] The polyethylene glycol-modified uric acid oxidase of the present invention exhibits significantly lower immunogenicity compared to commercially available drugs, which the inventors speculate is related to the number and difference of modification sites. Due to differences in modification sites and degrees of modification, both the protection of the enzyme's immunogenic sites and the exposure of the enzyme's active center differ in vivo, and these differences may lead to differences in the biological properties of the different modified enzymes in vivo.

[0170] Hereinafter, the in vivo evaluation of the polyethylene glycol-modified uric acid oxidase preparation (PU5 preparation) of the present invention will be described in detail, wherein the pegloticase used in the experiment refers to a commercially available similar drug, and batch number is 5085B.

[0171] Example 7: In vivo pharmacodynamic study of a polyethylene glycol uric acid oxidase preparation

[0172] 7.1 Evaluation of the efficacy of polyethylene glycol uric acid oxidase in model rats

[0173] A chronic hyperuricemia model in rats was induced by combining a high uric acid diet with potassium oxoate drinking water, and the therapeutic effect of polyethylene glycol uric acid oxidase (PU5 preparation) on chronic hyperuricemia in rats was evaluated.

[0174] Forty model rats were selected and randomly divided into four groups of 10 rats each: a model group, a pegylated uricase low-dose group (0.3 mg / kg), a pegylated uricase medium-dose group (1.0 mg / kg), and a pegylated uricase high-dose group (3.0 mg / kg). Another 10 normal SD rats were selected as a blank control group. In the study, the model was established continuously for 5 weeks, and intramuscular administration was started 1 week after modeling. The rats were administered once a week for 4 consecutive weeks, and serum uric acid, serum enzyme nitrogen, and serum creatinine levels were detected before administration and 7 days after each administration. Histological changes in the rat kidneys were observed after the end of the study.

[0175] The results in Figure 12 show that blood uric acid levels in the model control group were all significantly increased compared to the blank control group on days 7, 14, 21, 28, and 35 after modeling, and that serum urea nitrogen, creatinine, and uric acid in the model group rats 7 days after modeling were 2.73 times, 2.40 times, and 7.83 times higher, respectively, than those in the blank group rats. In terms of renal pathology (shown in Figure 13), the scores for renal tubular dilation, necrosis, inflammation, and fibrosis in the model control group were all significantly increased, and the number of uric acid crystals also significantly increased. Both medium and high doses of the test substance, pegylated uricase, significantly reduced serum uric acid levels and showed dose-relatedness. Over 14 to 35 days, the average blood uric acid levels in the medium dose group remained between 303.80 and 660.60 μmol / L, while the average blood uric acid levels in the high dose group remained between 153.70 and 403.40 μmol / L. Compared to the model group, the reduction in blood uric acid in the medium dose group was 34.46–67.94%, and in the high dose group, it was 65.67–83.78%. Compared to the model control group, each administration group of pegylated uricase showed significant improvement in renal tubular dilation, renal necrosis, and inflammation.

[0176] 7.2 Evaluation of a Single Dose of Polyethylene Glycol Uric Acid Oxidase in Rats

[0177] Thirty-six SD rats (half female, half male) were randomly divided into six groups (see Table 9): an intravenous group for the commercial drug Pegloticase, an intramuscular group, an intravenous group for polyethylene glycol uric acid oxidase, and intramuscular groups for low, medium, and high doses (0.5, 1.0, 2.0 mg / kg) of polyethylene glycol uric acid oxidase. Specific administration regimens and dosages are shown in Table 9. Blood was collected from the jugular vein to detect PK and PD.

[0178] Table 9: Animal Grouping and Dosage Design

[0179] number By group Administration route Frequency of administration Dosage (mg / kg) Administered concentration (mg / ml) Administered volume (ml / kg) Number of animals cock female 1 pegloticase intravenous injection group Intravenous injection 1 time 1.0 0.1 10.0 3 3 2 pegloticase intramuscular injection group Intramuscular injection 1 time 1.0 1.0 1.0 3 3 3 PU5 preparation intravenous injection group Intravenous injection 1 time 1.0 0.1 10.0 3 3 4 PU5 formulation low-dose intramuscular injection group Intramuscular injection 1 time 0.5 0.5 1.0 3 3 5 PU5 formulation medium-dose intramuscular injection group Intramuscular injection 1 time 1.0 1.0 1.0 3 3 6 PU5 formulation high-dose intramuscular injection group Intramuscular injection 1 time 2.0 2.0 1.0 3 3

[0180] 7.2.1, Pharmacokinetic Comparison. In SD rats, serum drug concentration levels in all individuals were lower than the lower limit of quantification (LLOQ: 312.500 ng / mL) prior to administration. When 0.5, 1.0, and 2.0 mg / kg were administered as a single intramuscular injection, the serum drug concentration of pegloticase injection (PU5) was dose-dependent during the period of 0 to 168 h (0 to 7 days), and the overall level increased as the dose increased. After 168 h, the blood drug concentration in the pegloticase intramuscular administration group was lower than the lower limit of quantification, whereas in the PU5 intramuscular administration group, it was maintained until 240 h or later.

[0181] After administration, in vivo C levels in female and male SD rats of the 1.0 mg / kg pegloticase intravenous and intramuscular injection groups, the 1.0 mg / kg pegylated uricase injection intravenous group, and the 0.5, 1.0, and 2.0 mg / kg pegloticase injection intramuscular groups, respectively. max (C 5min The ratio is within the range of 0.75 to 0.99, and the AUC last The ratio is within the range of 0.54 to 0.94, and the AUC 0-Δ The ratio is within the range of 0.58 to 0.97. From this, it can be seen that there is no significant difference in the levels of exposure to pegloticase and pegylated uricase (PU5) injection in SD rats according to sex.

[0182] However, the AUC of the intravenous administration group of the commercially available drug Pegloticase administered to SD rats at the same dose (1.0 mg / kg) last is 426.48±65.34, and the AUC of the intramuscular injection group last is 264.19±78.22; and the AUC of the PU5 injection intravenous administration group last is 565.61±161.60, and the AUC of the intramuscular injection group last It was 337.86±227.34. The AUC of PU5 under the same dose and administration method conditions.last It is higher than the commercially available drug Pegloticase.

[0183] T of the intravenous administration group of SD rats administered the same dose (1.0 mg / kg) of the commercially available drug Pegloticase 1 / 2 (h) is 49.51±8.12, and T of the intramuscular administration group 1 / 2 (h) is 55.21±13.50, and T of the PU5 injection intravenous administration group 1 / 2 (h) is 86.12±33.82, and T of the intramuscular administration group 1 / 2 (h) was 60.45±21.37. T of PU5 injection solution under the same dose and administration method conditions 1 / 2 (h) is longer than the commercial drug Pegloticase.

[0184] The above pharmacokinetic results are shown in Tables 10–15 and Figures 14–16.

[0185] Table 10: Blood drug concentration data and statistical analysis data for SD rats administered a single intravenous injection of 1.0 mg / kg Pegloticase (Unit: μg / mL)

[0186] Collection time (h) cock female Female + Male 1M001 1M002 1M003 N Mean SD 1F001 1F002 1F003 N Mean SD N Mean SD 0 BLQ BLQ BLQ 0 / / BLQ BLQ BLQ 0 / / 0 / / 0.08333 8.03 7.466 8.078 3 7.858 0.340 6.495 6.402 7.828 3 6.908 0.798 6 7.383 0.756 0.5 8.042 7.352 7.926 3 7.773 0.369 6.257 6.141 7.618 3 6.672 0.821 6 7.223 0.830 2 5.917 7.235 6.914 3 6.689 0.687 6.056 5.875 6.836 3 6.256 0.511 6 6.472 0.591 4 7.598 7.047 6.757 3 7.134 0.427 5.595 4.922 7.164 3 5.894 1.150 6 6.514 1.031 8 7.144 5.852 6.492 3 6.496 0.646 5.005 4.121 5.748 3 4.958 0.815 6 5.727 1.069 24 4.992 3.923 4.469 3 4.461 0.535 3.764 3.341 4.862 3 3.989 0.785 6 4.225 0.654 48 3.552 2.934 3.304 3 3.263 0.311 2.988 2.415 3.836 3 3.080 0.715 6 3.172 0.503 72 3.009 2.271 2.422 3 2.567 0.390 2.223 1.994 3.103 3 2.440 0.585 6 2.504 0.450 120 1.522 1.483 1.246 3 1.417 0.149 0.985 1.098 1.734 3 1.272 0.404 6 1.345 0.284 168 0.652 0.629 0.316 3 0.532 0.188 0.497 0.672 0.726 3 0.632 0.120 6 0.582 0.151 240 BLQ BLQ BLQ 0 / / BLQ BLQ BLQ 0 / / 0 / / 336 BLQ BLQ BLQ 0 / / BLQ BLQ BLQ 0 / / 0 / /

[0187] Table 11: Blood drug concentration data and statistical analysis data for SD rats administered a single intravenous injection of 1.0 mg / kg pegylated uricase solution (Unit: μg / mL)

[0188] Collection time cock female Female + Male (h) 3M001 3M002 3M003 N Mean SD 3F001 3F002 3F003 N Mean SD N Mean SD 0 BLQ BLQ BLQ 0 / / BLQ BLQ BLQ 0 / / 0 / / 0.08333 7.364 9.941 7.74 3 8.348 1.392 7.236 5.991 6.657 3 6.628 0.623 6 7.488 1.348 0.5 7.316 9.469 7.693 3 8.159 1.150 7.051 5.513 6.36 3 6.308 0.770 6 7.234 1.340 2 7.742 9.084 7.338 3 8.055 0.914 6.063 5.522 6.44 3 6.008 0.461 6 7.032 1.294 4 7 8.837 6.997 3 7.611 1.061 6.508 5.735 6.288 3 6.177 0.398 6 6.894 1.064 8 6.628 7.43 6.61 3 6.889 0.468 5.387 4.85 5.52 3 5.252 0.355 6 6.071 0.971 24 4.672 5.628 4.746 3 5.015 0.532 4.291 3.919 4.129 3 4.113 0.187 6 4.564 0.609 48 3.307 4.264 3.497 3 3.689 0.507 3.406 3.042 3.014 3 3.154 0.219 6 3.422 0.456 72 2.933 3.762 3.124 3 3.273 0.434 2.859 2.596 2.319 3 2.591 0.270 6 2.932 0.494 120 1.986 2.279 1.989 3 2.085 0.168 1.604 1.617 1.454 3 1.558 0.091 6 1.822 0.313 168 1.268 1.742 1.391 3 1.467 0.246 1.187 1.031 0.699 3 0.972 0.249 6 1.220 0.350 240 0.67 1.19 0.734 3 0.865 0.284 BLQ BLQ BLQ 0 / / 3 0.865 0.284 336 BLQ 0.853 0.368 2 0.611 0.343 BLQ BLQ BLQ 0 / / 2 0.611 0.343

[0189] Note: " / " means there is no relevant information.

[0190] Table 12: Blood drug concentration data and statistical analysis data for SD rats administered a single intramuscular injection of 1.0 mg / kg Pegloticase (Unit: μg / mL)

[0191] Collection time cock female Female + Male (h) 2M001 2M002 2M003 N Mean SD 2F001 2F002 2F003 N Mean SD N Mean SD 0 BLQ BLQ BLQ 0 / / BLQ BLQ BLQ 0 / / 0 / / 0.5 0.652 BLQ 0.581 2 0.617 0.050 0.388 BLQ BLQ 1 0.388 / 3 0.540 0.137 2 1.337 1.249 1.62 3 1.402 0.194 1.172 1.135 BLQ 2 1.154 0.026 5 1.303 0.194 4 2.298 1.699 2.348 3 2.115 0.361 1.812 1.371 0.773 3 1.319 0.521 6 1.717 0.593 8 2.56 2.058 2.396 3 2.338 0.256 1.915 1.657 1.273 3 1.615 0.323 6 1.977 0.474 24 3.808 3.235 3.309 3 3.451 0.312 2.947 2.808 2.493 3 2.749 0.233 6 3.100 0.456 48 3.188 2.618 2.749 3 2.852 0.299 2.317 2.279 1.729 3 2.108 0.329 6 2.480 0.495 72 2.694 2.263 2.211 3 2.389 0.265 1.984 2.016 1.261 3 1.754 0.427 6 2.072 0.471 120 1.56 1.169 1.332 3 1.354 0.196 0.884 1.111 0.174 3 0.723 0.489 6 1.038 0.480 168 BLQ 0.341 0.869 2 0.605 0.373 BLQ 0.635 BLQ 1 0.635 / 3 0.615 0.265 240 BLQ BLQ BLQ 0 / / BLQ BLQ BLQ 0 / / 0 / / 336 BLQ BLQ BLQ 0 / / BLQ BLQ BLQ 0 / / 0 / /

[0192] Note: " / " means there is no relevant information.

[0193] Table 13: Blood drug concentration data and statistical analysis of SD rats administered a single intramuscular injection of 1.0 mg / kg pegylated uricase solution

[0194] Collection time cock female Female + Male (h) 5M001 5M002 5M003 N Mean SD 5F001 5F002 5F003 N Mean SD N Mean SD 0 BLQ BLQ BLQ 0 / / BLQ BLQ BLQ 0 / / 0 / / 0.5 BLQ 1.421 0.328 2 0.875 0.773 BLQ BLQ BLQ 0 / / 2 0.875 0.773 2 BLQ 2.295 0.923 2 1.609 0.970 0.593 0.905 0.674 3 0.724 0.162 5 1.078 0.695 4 0.729 2.897 1.648 3 1.758 1.088 1.356 1.222 0.762 3 1.113 0.312 6 1.436 0.798 8 1.305 3.628 2.054 3 2.329 1.186 1.559 1.249 1.266 3 1.358 0.174 6 1.844 0.926 24 2.408 4.617 3.069 3 3.365 1.134 3.01 2.339 2.216 3 2.522 0.427 6 2.943 0.895 48 2.068 3.877 2.4 3 2.782 0.963 2.739 2.298 2.189 3 2.409 0.291 6 2.595 0.668 72 1.76 3.606 2.027 3 2.464 0.998 2.385 1.761 1.863 3 2.003 0.335 6 2.234 0.712 120 1.042 2.9 1.107 3 1.683 1.054 1.169 0.811 0.926 3 0.969 0.183 6 1.326 0.782 168 0.479 2.419 0.631 3 1.176 1.079 0.595 BLQ BLQ 1 0.595 / 4 1.031 0.928 240 BLQ 1.303 BLQ 1 1.303 / BLQ BLQ BLQ 0 / / 1 1.303 / 336 BLQ 0.719 BLQ 1 0.719 / BLQ BLQ BLQ 0 / / 1 0.719 /

[0195] Note: " / " means there is no relevant information.

[0196] Table 14: Mean pharmacokinetic parameters after a single intravenous injection of pegloticase and pegylated uricase solution in SD rats

[0197] volume gender parameters t 1 / 2 C 5min AUC last AUC 0-Δ Vz Cl MRT last (mg / kg) (h) (μg / mL) (h*μg / mL) (h*μg / mL) (mL / kg) (mL / h / kg) (h) 1.0(Pegloticase) cock N 3 3 3 3 3 3 3 Mean 45.70 7.86 448.57 484.58 136.90 2.08 52.36 SD 7.57 0.34 42.16 46.96 26.57 0.19 2.58 female N 3 3 3 3 3 3 3 Mean 53.32 6.91 404.38 453.63 173.91 2.26 54.64 SD 7.98 0.80 86.21 90.21 43.64 0.40 2.62 Female + Male N 6 6 6 6 6 6 6 Mean 49.51 7.39 426.48 469.11 155.40 2.17 53.50 SD 8.12 0.76 65.34 66.52 38.14 0.30 2.64 1.0(PU5) cock N 3 3 3 3 3 3 3 Mean 105.16 8.35 692.29 794.77 186.76 1.31 90.87 SD 41.08 1.39 128.22 197.50 24.94 0.29 14.06 female N 3 3 3 3 3 3 3 Mean 67.09 6.63 438.93 535.17 180.63 1.88 58.58 SD 9.24 0.62 26.51 59.13 11.64 0.21 2.76 Female + Male N 6 6 6 6 6 6 6 Mean 86.12 7.49 565.61 664.97 183.70 1.59 74.73 SD 33.82 1.35 161.60 192.92 17.73 0.39 19.87

[0198] Table 15: Mean pharmacokinetic parameters after a single intramuscular injection of Peglocticase and pegylated uricase solution in SD rats

[0199] volume gender parameters t 1 / 2 T max C max AUC last AUC 0-Δ Vz_F Cl_F MRT last (mg / kg) (h) (h) (μg / mL) (h*μg / mL) (h*μg / mL) (mL / kg) (mL / h / kg) (h) 1.0(Pegloticase) cock N 3 3 3 3 3 3 3 3 Mean 58.31 24.00 3.45 318.23 405.13 203.75 2.54 60.57 SD 20.10 0.00 0.31 15.37 80.13 42.90 0.54 6.54 female N 3 3 3 3 3 3 3 3 Mean 52.12 24.00 2.75 210.14 278.56 276.83 3.72 51.27 SD 4.78 0.00 0.23 79.35 60.90 50.57 0.91 15.28 Female + Male N 6 6 6 6 6 6 6 6 Mean 55.21 24.00 3.10 264.19 341.85 240.29 3.13 55.92 SD 13.50 0.00 0.46 78.22 94.12 57.97 0.93 11.68 0.5(PU5) cock N 3 3 3 3 3 3 3 3 Mean 63.57 24.00 1.93 181.10 233.11 199.06 2.21 60.26 SD 18.68 0.00 0.26 79.19 48.71 56.50 0.45 23.89 female N 3 3 3 3 3 3 3 3 Mean 48.20 24.00 1.91 170.63 205.87 167.09 2.56 55.67 SD 17.38 0.00 0.14 41.99 61.41 21.47 0.67 11.48 Female + Male N 6 6 6 6 6 6 6 6 Mean 55.88 24.00 1.92 175.87 219.49 183.07 2.38 57.97 SD 18.20 0.00 0.19 56.98 51.77 42.05 0.54 16.95 1.0(PU5) cock N 3 3 3 3 3 3 3 3 Mean 70.47 24.00 3.36 439.83 504.61 225.86 2.57 84.20 SD 28.55 0.00 1.13 307.66 344.91 54.21 1.32 31.10 female N 3 3 3 3 3 3 3 3 Mean 50.44 24.00 2.52 235.90 293.04 252.46 3.46 58.28 SD 5.05 0.00 0.43 58.01 45.24 46.82 0.50 6.96 Female + Male N 6 6 6 6 6 6 6 6 Mean 60.45 24.00 2.94 337.86 398.83 239.16 3.02 71.24 SD 21.37 0.00 0.89 227.34 248.66 47.59 1.02 24.65 2.0(PU5) cock N 3 3 3 3 3 3 3 3 Mean 66.65 24.00 4.84 590.58 649.31 292.61 3.10 85.42 SD 20.11 0.00 0.46 59.68 55.26 64.39 0.27 19.91 female N 3 3 3 3 3 3 3 3 Mean 72.51 32.00 4.55 537.05 628.72 339.98 3.22 79.26 SD 15.56 13.86 0.91 124.85 78.17 100.19 0.42 9.60 Female + Male N 6 6 6 6 6 6 6 6 Mean 69.58 28.00 4.70 563.81 639.01 316.30 3.16 82.34 SD 16.40 9.80 0.66 92.30 61.59 79.67 0.32 14.38

[0200] 7.2.2, Comparison of In vivo Efficacy (Uric Acid) When 0.5, 1.0, and 2.0 mg / kg pegloticase injection solutions were administered once intramuscularly, uric acid concentrations were maintained at low levels 1 and 3 days after administration, and uric acid levels in each dose group began to recover 7 days after administration. The higher the dose, the longer the time that uric acid remained at low levels in vivo. When comparing the intravenous injection groups of the same dose, the PU5 intravenous injection group maintained low serum uric acid concentrations for a longer period than the pegloticase intravenous injection group, and the PU5 intramuscular injection group maintained low serum uric acid concentrations for a longer period than the pegloticase intramuscular injection group. When comparing the same dosage groups, the PU5 intravenous or intramuscular injection group maintained lower serum uric acid concentrations for a longer period compared to the pegloticase intravenous or intramuscular injection group; that is, PU5 maintained lower in vivo uric acid concentrations for a longer period compared to pegloticase in each case, and the results are shown in Figure 17.

[0201] 7.3 Evaluation of Multiple Administrations of Polyethylene Glycol Uric Acid Oxidase in Rats

[0202] For this study, four groups were established: an intravenous injection group for the commercial drug Pegloticase, an intramuscular injection group, an intravenous injection group for pegylated uricase injection (PU5), and an intramuscular injection group. The study consisted of a total of 32 SD rats, with 8 rats per group (half male, half female). The intravenous injection groups for Pegloticase and pegylated uricase injection were administered intravenously; the intramuscular injection groups for Pegloticase and pegylated uricase injection were administered intramuscularly. The dosage for all groups was 1.0 mg / kg, administered once a week for four consecutive weeks.

[0203] From the analysis of results,

[0204] It can be seen that there were no abnormal drug-related changes in the general situation of SD rats that were injected intravenously or intramuscularly with 1.0 mg / kg Pegloticase and pegylated uricase solution multiple times.

[0205] 7.3.1 Detection of Anti-PEG Antibodies

[0206] The drug was administered to SD rats four times in succession. Before the first administration, neither anti-PEG nor anti-PHC antibodies were detected in any of the animals; after the administration was completed, anti-PHC antibodies were not detected in any of the animals, while anti-PEG antibodies were detected in the pegloticase intravenous and intramuscular injection groups, and the pegylated uricase injection intravenous and intramuscular injection groups, respectively, with positive result ratios of 3 / 8, 1 / 8, 1 / 8, and 1 / 8, respectively. As a result of PEG immunohistochemical examination, weak PEG-positive expression was observed in the spleen, liver, and kidney of the pegloticase intravenous and intramuscular injection groups, while no PEG-positive expression was observed in the pegloticase injection intravenous and intramuscular injection groups, and the results are shown in Table 13.

[0207] From the analysis, it can be seen that the antibodies produced by PU5 and pegloticase are not antibodies against the uric acid oxidase portion but are primarily antibodies against the PEG portion, which means that both can effectively mask the immunogenic site of uric acid oxidase. The production of PEG antibodies can cause some side effects in vivo, and according to the results in Table 16, the immunogenicity of PU5 of the present invention is lower than that of commercially available pgeloticase.

[0208] From the results of PEG antibody and PEG immunohistochemical tests, both PU5 agent and pegloticase were superior in intramuscular administration compared to the intravenous administration group, and among them, the anti-PEG antibody produced in the intravenous administration group was superior in PU5 agent compared to pegloticase; and the anti-PEG antibody produced in the intramuscular administration group was superior in PU5 agent compared to pegloticase.

[0209] Table 16: PEG Immunohistochemical Positive Expression Results

[0210] Incidence pegloticase intravenous injection group pegloticase intramuscular injection group PU5 Intravenous Injection Group PU5 Intramuscular Injection Group Microscopic observation cock female cock female cock female cock female spleen Total number of checks: 4 4 4 4 4 4 4 4 --PEG, white pulp 1 0 1 1 2 0 0 0 0 Total number of occurrences: 0 1 1 2 0 0 0 0 --PEG, red pulp 1 0 1 0 0 0 0 0 0 Total number of occurrences: 0 1 0 0 0 0 0 0 liver Total number of checks: 4 4 4 4 4 4 4 4 --PEG, vascular endothelial cells / Kupffer cells 1 4 4 1 3 0 0 0 0 Total number of occurrences: 4 4 1 3 0 0 0 0 height Total number of checks: 4 4 4 4 4 4 4 4 --PEG, renal tubule 1 3 1 1 1 0 0 0 0 Total number of occurrences: 3 1 1 1 0 0 0 0 -- PEG, vascular endothelial cells 1 0 1 0 0 0 0 0 0 Total number of occurrences: 0 1 0 0 0 0 0 0

[0211] Positive grade: 1=Very weak positive, 2=Weak positive, 3=Intermediate positive, 4=Strong positive. 4.3.2 Pharmacokinetic Detection

[0212] After multiple intravenous and intramuscular injections of pegloticase and pegylated uricase solutions into SD rats, the major pharmacokinetic parameters of the animals in each group did not show significant sex differences. After four consecutive administrations, the two drugs accumulated slightly in the rats' bodies.

[0213] When the same dose (1.0 mg / kg) of the commercially available drug Pegloticase was administered to SD rats via intravenous / intramuscular injection multiple times, the absolute bioavailability in the rats after the first administration was 51.35%, respectively; and after the last administration, the absolute bioavailability in the rats was 45.98%, respectively. When the same dose (1.0 mg / kg) of pegylated uricase injection solution was administered to SD rats via intravenous / intramuscular injection multiple times, the absolute bioavailability in the rats after the first administration was 58.29%, respectively; and after the last administration, the absolute bioavailability in the rats was 52.60%, respectively.

[0214] 4.3.3 Comparison of In vivo Efficacy (Uric Acid)

[0215] SD rats were injected intravenously and intramuscularly with 1.0 mg / kg of pegloticase and pegylated uricase solution four consecutive times (once a week). Serum uric acid levels remained low after each administration, and the pegloticase intramuscular injection group recovered 14 days after the last administration, while the other groups recovered 18 days after the last administration. Compared to the commercially available drug pegloticase at the same dose, the retention times of the intravenous injection groups of the two drugs were relatively consistent, but the retention time of the pegylated uricase intramuscular injection group was longer than that of the commercially available drug; that is, the efficacy of the PU5 agent was superior to that of pegloticase upon intramuscular administration.

[0216] The above results are shown in Tables 17–19 and Figures 18–22.

[0217] Table 17: Mean pharmacokinetic parameters after serial intravenous injection of pegloticase and pegylated uricase injection solutions in SD rats

[0218] Exam time volume gender parameters t 1 / 2 T max C max AUC last AUC 0-Δ Vz Cl MRT last Drug names (h) (h) (μg / mL) (h*μg / mL) (h*μg / mL) (mL / kg) (mL / h / kg) (h) Day 1 1.0 mg / kg Pegloticase cock N 4 4 4 4 4 4 4 4 Mean 72.17 2.13 11.12 632.58 778.65 133.74 1.29 56.91 SD 4.53 1.44 0.28 25.32 45.00 4.49 0.07 1.17 female N 4 4 4 4 4 4 4 4 Mean 66.24 0.50 8.84 514.21 633.80 149.07 1.59 54.93 SD 17.91 0.00 1.07 50.98 64.84 27.23 0.16 7.58 Female + Male N 8 8 8 8 8 8 8 8 Mean 69.21 1.31 9.98 573.40 706.22 141.41 1.44 55.92 SD 12.51 1.28 1.42 73.43 93.08 19.84 0.20 5.13 1.0 mg / kgPU5 cock N 4 4 4 4 4 4 4 4 Mean 67.98 1.75 9.47 527.73 634.48 158.66 1.60 55.50 SD 8.87 1.66 0.91 91.07 91.12 39.53 0.22 0.89 female N 4 4 4 4 4 4 4 4 Mean 79.29 1.38 6.42 369.05 481.09 238.12 2.16 59.68 SD 17.07 1.75 0.61 49.11 98.31 15.34 0.53 2.84 Female + Male N 8 8 8 8 8 8 8 8 Mean 73.63 1.56 7.94 448.39 557.79 198.39 1.88 57.59 SD 13.97 1.59 1.78 108.54 120.10 50.74 0.48 2.96 Day 22 1.0 mg / kg Pegloticase cock N 4 4 4 4 4 4 4 4 Mean 135.63 1.75 12.33 1159.18 1300.99 149.28 0.78 118.29 SD 40.45 1.66 0.94 134.20 208.65 28.13 0.13 9.91 female N 4 4 4 4 4 4 4 4 Mean 96.09 0.88 8.02 672.95 747.61 186.89 1.35 92.46 SD 7.69 0.75 0.87 78.50 78.66 24.21 0.14 9.52 Female + Male N 8 8 8 8 8 8 8 8 Mean 115.86 1.31 10.17 916.07 1024.30 168.09 1.07 105.37 SD 34.25 1.28 2.45 279.12 329.86 31.53 0.33 16.48 1.0 mg / kgPU5 cock N 4 4 4 4 4 4 4 4 Mean 149.80 2.25 9.00 840.78 947.08 229.16 1.07 117.99 SD 24.68 2.02 0.73 91.96 104.82 36.69 0.11 6.88 female N 4 4 4 4 4 4 4 4 Mean 103.90 1.25 6.63 576.81 636.52 236.33 1.63 101.66 SD 21.25 0.87 0.72 128.81 128.02 17.98 0.39 20.03 Female + Male N 8 8 8 8 8 8 8 8 Mean 126.85 1.75 7.82 708.79 791.80 232.75 1.35 109.82 SD 32.51 1.54 1.44 175.05 198.21 27.02 0.40 16.38

[0219] Table 18: Results of mean pharmacokinetic parameters after serial intramuscular injection of pegloticase and pegylated uricase injection solutions in SD rats

[0220] Exam time volume gender parameters t 1 / 2 T max C max AUC last AUC 0-Δ Vz_F Cl_F MRT last Drug names (h) (h) (μg / mL) (h*μg / mL) (h*μg / mL) (mL / kg) (mL / h / kg) (h) Day 1 1.0 mg / kg Pegloticase cock N 4 4 4 4 4 4 4 4 Mean 59.68 24.00 3.16 318.22 395.66 217.53 2.54 62.05 SD 13.70 0.00 0.38 29.92 29.26 49.49 0.19 6.59 female N 4 4 4 4 4 4 4 4 Mean 80.53 24.00 2.80 270.69 415.60 282.07 2.48 57.80 SD 16.48 0.00 0.36 66.91 84.31 37.74 0.52 6.21 Female + Male N 8 8 8 8 8 8 8 8 Mean 70.11 24.00 2.98 294.46 405.63 249.80 2.51 59.92 SD 17.91 0.00 0.39 54.29 59.39 53.39 0.36 6.35 1.0 mg / kgPU5 cock N 4 4 4 4 4 4 4 4 Mean 82.25 6.00 3.06 290.64 403.89 294.92 2.50 61.83 SD 9.79 2.31 0.25 34.67 48.73 29.13 0.31 6.88 female N 4 4 4 4 4 4 4 4 Mean 70.44 48.00 2.21 232.11 306.59 340.32 3.50 66.28 SD 13.41 19.60 0.26 59.53 90.62 46.97 1.09 10.28 Female + Male N 8 8 8 8 8 8 8 8 Mean 76.34 27.00 2.63 261.38 355.24 317.62 3.00 64.06 SD 12.57 25.90 0.51 54.89 85.10 43.56 0.91 8.44 Day 22 1.0 mg / kg Pegloticase cock N 3 4 4 4 3 3 3 4 Mean 198.20 25.00 3.18 486.70 799.90 353.52 1.35 112.01 SD 83.85 18.00 0.85 298.21 293.71 23.56 0.42 60.30 female N 4 4 4 4 4 4 4 4 Mean 97.92 20.00 2.69 355.77 427.75 344.97 2.65 94.51 SD 33.98 8.00 0.71 134.18 155.69 88.41 1.19 30.45 Female + Male N 7 8 8 8 7 7 7 8 Mean 140.90 22.50 2.93 421.23 587.25 348.64 2.09 103.26 SD 76.12 13.17 0.77 225.23 283.63 64.14 1.12 45.20 1.0 mg / kgPU5 cock N 4 4 4 4 4 4 4 4 Mean 140.04 15.00 2.52 395.82 478.83 610.95 9.64 102.76 SD 90.61 10.52 1.15 255.45 300.26 419.13 16.09 61.50 female N 4 4 4 4 4 4 4 4 Mean 122.51 30.00 2.41 349.84 428.61 416.41 2.82 103.39 SD 59.33 12.00 0.50 178.08 204.30 72.32 1.36 44.91 Female + Male N 8 8 8 8 8 8 8 8 Mean 131.27 22.50 2.46 372.83 453.72 513.68 6.23 103.08 SD 71.52 13.17 0.82 205.33 239.26 297.23 11.18 49.85

[0221] Table 19: Statistical results of uric acid in each dose group after multiple intramuscular / intravenous injections of pegloticase and pegylated uricase solutions in SD rats (Mean+SD)

[0222] Gender measurement time 1.0 mg / kg Pegloticase Intravenous Injection Group 1.0 mg / kg Pegloticas intramuscular injection group 1.0 mg / kg PU5 intravenous injection group 1.0 mg / kg PU5 intramuscular injection group n n n n cock Before the first administration 4 71.250± 19.103 4 62.250± 5.315 4 58.750± 7.632 4 50.750± 6.850 3 days after the first administration 4 0.250± 0.500 4 0.500± 0.577 4 0± 0 4 0± 0 Before the second administration 4 0.500± 0.577 4 69.750± 46.133 4 0.500± 0.577 4 0.250± 0.500 3 days after the second administration 4 1.000± 0 4 20.750± 40.178 4 0.500± 0.577 4 17.500± 33.670 Before the 3rd administration 4 1.000± 0 4 26.500± 30.116 4 1.250± 0.957 4 18.000± 32.680 3 days after the 3rd administration 4 1.000± 0.816 4 19.000± 37.336 4 0.500± 0.577 4 20.500± 38.336 Before the 4th administration 4 0± 0 4 15.500± 31.000 4 0.250± 0.500 4 19.250± 37.170 1 day after the 4th administration 4 0.750± 0.500 4 1.750± 1.500 4 0.750± 0.500 4 9.750± 16.840 3 days after the 4th administration 4 0.500± 0.577 4 10.500± 21.000 4 0± 0 4 12.750± 24.838 7 days after the 4th administration 4 0.250± 0.500 4 22.250± 44.500 4 0± 0 4 16.250± 31.837 10 days after the 4th administration 4 0± 0 4 20.000± 38.670 4 0.250± 0.500 4 16.000± 30.681 14 days after the 4th administration 4 1.250± 0.500 4 29.250± 28.123 4 2.000± 0.816 4 19.250± 30.015 18 days after the 4th administration 4 25.000± 10.296 4 53.500± 14.933 4 24.000± 13.614 4 50.000± 29.833 female Before the first administration 4 61.250± 8.057 4 63.000± 15.470 4 50.250± 7.500 4 43.250± 9.743 3 days after the first administration 4 0± 0 4 0± 0 4 0.250± 0.500 4 0± 0 Before the second administration 4 42.000± 48.132 4 38.250± 44.507 4 0.500± 0.577 4 4.000± 7.348 3 days after the second administration 4 0.250± 0.500 4 40.250± 41.080 4 0.250± 0.500 4 0.500± 0.577 Before the 3rd administration 4 19.500± 35.01 4 46.750± 28.547 4 1.000± 0.816 4 13.500± 25.000 3 days after the 3rd administration 4 1.250± 0.500 4 0.500± 0.577 4 0.750± 0.500 4 0.750± 0.500 Before the 4th administration 4 0.250± 0.500 4 33.750± 40.285 4 0.500± 0.577 4 3.750± 6.850 1 day after the 4th administration 4 0.750± 0.500 4 3.000± 2.708 4 0.500± 0.577 4 1.250± 0.500 3 days after the 4th administration 4 0.250± 0.500 4 0± 0 4 0± 0 4 0± 0 7 days after the 4th administration 4 0.250± 0.500 4 6.750± 12.842 4 1.000± 0 4 1.750± 1.258 10 days after the 4th administration 4 0± 0 4 8.500± 16.340 4 0.250± 0.500 4 2.000± 2.828 14 days after the 4th administration 4 6.750± 7.089 4 33.500± 19.689 4 1.500± 0.577 4 9.500± 8.699 18 days after the 4th administration 4 48.000± 24.993 4 54.750± 4.031 4 14.000± 6.00. 4 32.000± 13.638

[0223] In the description of this specification, any description referring to terms such as “one embodiment,” “some embodiment,” “example,” “specific example,” or “some example” implies that specific features, structures, materials, or properties described in conjunction with such embodiment or example are included in at least one embodiment or example of the present invention. A general expression of the above terms in this specification does not necessarily refer to the same embodiment or example. Furthermore, the described specific features, structures, materials, or properties may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples and features of different embodiments or examples described in this specification, provided that such combinations are not contradictory. Although embodiments of the present invention have been illustrated and described above, it should be understood that such embodiments are illustrative and should not be interpreted as limiting the present invention. Those skilled in the art may change, modify, substitute, and alter the above embodiments within the scope of the present invention.

Claims

Claim 1 A pharmaceutical composition for the treatment or prevention of hyperuricemia comprising: a uric acid oxidase modified with polyethylene glycol; and a buffer reagent comprising at least one of a phosphate and a hydrochloride; wherein the phosphate is disodium hydrogen phosphate or sodium dihydrogen phosphate, and the hydrochloride is sodium chloride; wherein the uric acid oxidase has an amino acid sequence represented by SEQ ID NO:1; and at least 11 of the following amino acid sites of the uric acid oxidase have a PEG modification: 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 and K 293 A pharmaceutical composition characterized in that the amino acid site is positioned by an amino acid sequence indicated by SEQ ID NO:1; and the polyethylene glycol is N-succinimidylpropionate PEG having a molecular weight of 5K. Claim 2 A pharmaceutical composition according to claim 1, wherein at least one, at least two, at least three, or four of the following four amino acid sites of the uric acid oxidase are PEG modified: K 30 , K 35 , K 222 and K 231 . Claim 3 A pharmaceutical composition according to claim 1, characterized in that the pH of the composition is 7 to 9. Claim 4 A pharmaceutical composition according to claim 3, characterized in that the pH of the composition is 7.4 to 8.

2. Claim 5 A pharmaceutical composition according to claim 1, characterized in that the mass ratio of the polyethylene glycol-modified uric acid oxidase to the buffer reagent is (5-6):(6-37). Claim 6 A pharmaceutical composition according to claim 5, characterized in that the mass ratio of the polyethylene glycol-modified uric acid oxidase to the buffer reagent is 6:

10. Claim 7 A pharmaceutical composition according to claim 1, wherein the mass ratio of the polyethylene glycol-modified uric acid oxidase to the phosphate is (5-6):(1-7), and the phosphate is disodium hydrogen phosphate or sodium dihydrogen phosphate. Claim 8 A pharmaceutical composition according to claim 1, characterized in that the mass ratio of the polyethylene glycol-modified uric acid oxidase to the sodium chloride is (5-6):(5-30). Claim 9 A pharmaceutical composition according to claim 1, wherein the mass ratio of the phosphate to the sodium chloride is (1 to 7): (5 to 30), and the phosphate is disodium hydrogen phosphate or sodium dihydrogen phosphate. Claim 10 A pharmaceutical composition according to claim 1, characterized in that the formulation of the composition is one of liquid, semi-solid, and solid. Claim 11 A pharmaceutical composition according to claim 1, wherein the composition is administered as a single formulation, and the single formulation contains 6 mg of polyethylene glycol-modified uric acid oxidase. Claim 12 delete Claim 13 delete Claim 14 delete Claim 15 delete Claim 16 delete Claim 17 delete Claim 18 delete Claim 19 delete Claim 20 delete Claim 21 delete Claim 22 delete Claim 23 delete Claim 24 delete

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