Method for preparing polyethylene glycol-modified uric acid oxidase

Abstract

A method for producing a polyethylene glycol-modified uric acid oxidase is provided, wherein at least 11 of the following amino acid sites of the polyethylene glycol-modified uric acid oxidase have PEG modification: T1, K3, K4, K30, K35, K76, K79, K97, K112, K116, K120, K152, K179, K222, K231, K266, K272, K285, K291, K293. The method comprises the step of conjugating the uric acid oxidase with polyethylene glycol, wherein the polyethylene glycol is provided in the form of an acidic solution, and the molar ratio of the uric acid oxidase to the polyethylene glycol is 1:(56 to 94) to obtain a polyethylene glycol-modified uric acid oxidase.

Description

Technology Field

[0001] The present invention relates to the field of biopharmaceuticals, specifically to a method for producing uric acid oxidase modified with polyethylene glycol, and more specifically to a method for producing uric acid oxidase modified with polyethylene glycol, a method for reducing the immunogenicity of uric acid oxidase, uric acid oxidase modified with polyethylene glycol, a pharmaceutical composition, and a pharmaceutical use of uric acid oxidase modified with polyethylene glycol. Background Technology

[0002] Gout is a disease caused by a disorder of purine metabolism, characterized by hyperuricemia; urate deposits accumulate in the subcutaneous tissue, joints, and kidneys, forming tofus. Purines in the body undergo a series of transformations to ultimately form uric acid; blood uric acid levels exceeding 70 mg / L can induce hyperuricemia, and 5% to 12% of patients with hyperuricemia may develop gout. When the concentration of sodium urate in the blood or synovial fluid reaches saturation, microcrystals of sodium urate form, which can cause gouty arthritis. Over time, chronic hyperuricemia can also deposit destructive crystalline uric acid deposits around the joints, soft tissues, and certain organs, leading to conditions such as acute gouty arthritis, chronic tofusal arthritis, and joint deformities. Kidney damage is considered the second most common clinical manifestation of gout. Due to the progressive nature of chronic hyperuricemia, uric acid is deposited in the medulla, renal tubules, and renal interstitium, irritating local areas and causing an inflammatory response, which is referred to as chronic uric acid nephropathy; in patients with severe hyperuricemia (e.g., some malignancies, particularly leukemia and lymphoma), large amounts of uric acid are deposited in the renal collecting ducts, renal pelvis, renal calyces, and ureters in a short period, causing lumen obstruction and urinary obstruction, leading to acute renal failure (also referred to as uric acid nephropathy).

[0003] Over the past few decades, as people's quality of life has improved and dietary and lifestyle habits have changed, the consumption of high-protein and high-purine foods has increased, leading to a yearly upward trend in the number of gout patients. In Europe, the number of gout patients has approximately doubled over the past 20 years, and currently in China, the incidence of hyperuricemia and gout has risen to about 2–3%. For hyperuricemia, dietary control is used in the absence of clinical symptoms, while drug treatment is necessary when clinical symptoms appear. The common treatment methods currently used in clinical practice are as follows: Analgesics and anti-inflammatory drugs such as colchicine, ibuprofen, and naproxen are primarily used to control the symptoms of acute attacks of gouty arthritis and to relieve localized joint pain, swelling, and inflammation; uric acid medications such as probenicid, sulfinpyrazone, and benzbromarone (ineffective in cases of impaired renal function) promote uric acid excretion; Uric acid synthesis inhibitors such as allopurinol are major therapeutic drugs for patients with tophus gout, renal failure, leukemia, and some genetic diseases. They work by inhibiting xanthine oxidase, which prevents hypoxanthine and xanthine from being converted into uric acid. This oxidizes in the body to produce oxypurinol, which is easily soluble in water and excreted through urine. However, it is difficult to treat patients with chronic gout that has developed tophus with all existing treatments. Furthermore, if the aforementioned drugs are taken for a long period, patients inevitably develop complications such as leukopenia, cardiac dysfunction, liver and kidney dysfunction, gastrointestinal irritation, aplastic anemia leading to diabetes, and gout.

[0004] Human hyperuricemia is associated with mutations and inactivation of the uricase gene during human evolution, and mutations introduce an early stop codon (Wu X, Lee CC, Muzny DM, Caskey C T.Proc Natl Acad SciUSA.1989.86: 9412-9416.) into the coding sequence of the human uricase gene. Consequently, since humans cannot synthesize active uricase on their own, human purine degradation metabolism terminates at uric acid (Wu X, Muzny DM, Lee CC, Caskey C TJ Mol Evol.1992.34: 78-84.). Active uricases in the hepatic peroxisomes of non-human primates and other mammals convert less soluble urate (~11 mg / 100 ml water) into more soluble allantoin (~147 mg / 100 ml water), which can be more effectively excreted by the kidneys (Wortmann RL, Kelley W N. Kelley's textbook of rheumatology (6th).2001:1339-1376). In Europe and the United States, uricases (Uricozymes) prepared from Aspergillus flavus have been used for over 10 years to treat severe hyperuricemia associated with tumor chemotherapy (Zittoun R, Dauchy F, Teilaud C, Barthelemy M, Bouchard P.Ann Med Interne.1978.127: 479-482.). ELITEK, a recombinant Aspergillus flavus uricase drug developed by Sanofi of France and produced by fermenting brewer's yeast, received FDA approval in 2002 and is used for the short-term treatment of severe hyperuricemia caused by tumor chemotherapy (Pui CH, Relling MV, Lascombes F, HarrisonP L, Struxiano A et al. Leukemia.1997.11:1813-1816.).In addition, it was demonstrated that the infusion of ELITEK can also reduce the volume of tofus (Potaux L, Aparicio M, Maurel C, Ruedas ME, Mart in C L. Nouv PresseMed. 1975.4: 1109-1112.). In September 2010, the FDA approved PEG-modified recombinant porcine uricase (Pegloticase) produced by the publicly traded U.S. company Savient as a treatment for intractable gout, but due to unresolved immunogenicity issues, about 50% of patients do not see results in clinical application.

[0005] Uricase (EC 1.7.3.3) 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 by catalyzing uric acid in the presence of oxygen and release carbon dioxide (Retailleau P, Colloc'h, Denis V, Francoise B.Acta Cryst D.2004.60: 453-462.).

[0006] 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.

[0007] In terms of 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 readily produces antibodies against uricases, the efficacy of Aspergillus flavus uricases rapidly weakens, simultaneously inducing severe allergic reactions, making them unsuitable for long-term treatment. Human uricase genes mutate, lose their activity, and become pseudogenes.

[0008] Therefore, uric acid oxidase-based treatment technology for hyperuricemia still requires further development and improvement.

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

[0013] In a first aspect of the present invention, the present invention proposes a method for preparing a uric acid oxidase modified with polyethylene glycol. According to an embodiment of the present invention, at least 11 of the following amino acid sites of the uric acid oxidase modified with polyethylene glycol have PEG modification: T 1 , K 3 , K 4 , K 30 , K 35 , K 76 , K 79 , K 97 , K112 , K 116 , K 120 , K 152 , K 179 , K 222 , K 231 , K 266 , K 272 , K 285 , K 291 , K 293 The above method comprises the step of conjugating uric acid oxidase with polyethylene glycol, wherein the polyethylene glycol is provided in the form of an acidic solution, and obtaining uric acid oxidase modified with polyethylene glycol by setting the molar ratio of uric acid oxidase to polyethylene glycol to 1:(56–94). Modification site T of the polyethylene glycol modified uric acid oxidase prepared according to the method of the embodiment of the present invention. 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 293At least 11 of the sites have PEG modifications. It should be noted that the term "uric acid oxidase" prepared in the present invention should be understood in a broad sense and refers to a collective term for mixtures of uric acid oxidases produced in the same batch in actual production practices. The polyethylene glycol-modified uric acid oxidase obtained according to the method of the embodiments of the present invention significantly improves the stability of the uric acid oxidase in vivo, reduces immunogenicity, and, in addition, the in vivo efficacy after intramuscular injection can reach an in vivo efficacy equivalent to that after intravenous injection of a similar commercially available drug, provided that the enzyme activity is guaranteed to the maximum.

[0014] According to an embodiment of the present invention, the method may further include at least one of the following additional technical features.

[0015] According to an embodiment of the present invention, the polyethylene glycol is provided in the form of an acidic solution. By dissolving PEG in an acidic solution, the inventor prevents the active group of the polyethylene glycol from being hydrolyzed before it reacts with the protein, thereby effectively securing the effective activation efficiency of the polyethylene glycol. At the same time, when the polyethylene glycol solid is directly dissolved in a conjugation reaction buffer, defects such as high local concentration, non-uniform dissolution, non-uniform contact between the polyethylene glycol and the conjugated protein, and the generation of bubbles that hinder the conjugation reaction can be resolved, thereby effectively improving the conjugation reaction efficiency between uric acid oxidase and polyethylene glycol.

[0016] According to an embodiment of the present invention, the acidic solution is an acidic solution containing at least one selected from organic acids and inorganic acids.

[0017] According to an embodiment of the present invention, the organic acid is,

[0018] Selected from acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, malonic acid, adipic acid, ascorbic acid, benzenesulfonic acid, benzoic acid, butyric acid, cyclopentylpropionic acid, digluconic acid, dodecyl sulfonic acid, ethylsulfonic acid, formic acid, fumaric acid, glucoheptonic acid, glycerophosphate, gluconic acid, heptanoic acid, caproic acid, 2-hydroxyethanesulfonic acid, lacturonic acid, lactic acid, lauric acid, lauryl sulfic acid, malic acid, malonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, niacin, oleic acid, palmitic acid, pectinic acid, 3-phenylpropionate, picrate, pivalic acid, propionic acid, stearic acid, p-toluenesulfonic acid, undecanic acid, and valeric acid;

[0019] According to an embodiment of the present invention, the inorganic acid is,

[0020] It is selected from hydrochloric acid, hydrobromide, phosphoric acid, sulfuric acid, perchloric acid, hydroiodic acid, nitric acid, persulfuric acid, boric acid, dichromate, silicic acid, chromate, and thiocyanate.

[0021] According to an embodiment of the present invention, the concentration of hydrogen ions in the acidic solution is 1 to 5 mmol / L.

[0022] According to an embodiment of the present invention, the acidic solution contains hydrochloric acid, sulfuric acid, or glacial acetic acid.

[0023] According to an embodiment of the present invention, the concentration of the acid in the acidic solution is 1 to 5 mmol / L.

[0024] According to an embodiment of the present invention, the molar ratio of uric acid oxidase to polyethylene glycol is 1:(45 to 110).

[0025] According to an embodiment of the present invention, the molar ratio of uric acid oxidase to polyethylene glycol is 1:(56 to 94).

[0026] According to an embodiment of the present invention, the concentration of polyethylene glycol in the acidic solution is 100 to 300 mmol / L. The inventors discovered that when the concentration of polyethylene glycol in the acid is within the above concentration range, the viscosity of the reaction solution is appropriate, which is advantageous for improving reaction efficiency and expanding industrial production.

[0027] According to an embodiment of the present invention, the molecular weight of the polyethylene glycol is 6 kD or less. The inventors discovered that polyethylene glycol modified uric acid oxidase obtained by using polyethylene glycol with a molecular weight of 6 kD or less in a conjugation reaction with uric acid oxidase further enhances in vivo long-term effects, does not produce anti-uricase antibodies, and hardly produces anti-PEG antibodies, that is, immunogenicity is further reduced.

[0028] According to an embodiment of the present invention, the polyethylene glycol has a monomethoxyl group or a hydroxyl group.

[0029] According to an embodiment of the present invention, the polyethylene glycol has a linear or branched structure.

[0030] According to an embodiment of the present invention, the polyethylene glycol and uric acid oxidase are joined through an amide bond.

[0031] According to an embodiment of the present invention, the polyethylene glycol is modified polyethylene glycol, and the modifier of the modified polyethylene glycol is,

[0032] It is selected from N-hydroxysuccinimide, N-hydroxysuccinimidyl carbonate, N-hydroxysuccinimidyl acetate, N-hydroxysuccinimidylpropionate, N-hydroxysuccinimidylbutyrate, N-hydroxysuccinylsuccinate and bis(p-nitrophenyl)carbonate.

[0033] According to an embodiment of the present invention, the modifying group of the modified polyethylene glycol is N-hydroxysuccinimide.

[0034] According to an embodiment of the present invention, the conjugation reaction is performed in a carbonate buffer solution.

[0035] According to an embodiment of the present invention, the pH of the carbonate buffer solution is 9 to 11. The inventors discovered that if the buffer pH is less than 9.0, it has a severe effect on the solubility of uricase and prevents the subsequent modification reaction from being performed, and if the buffer pH is 11 or higher, it has a severe effect on the activity of uricase and simultaneously reduces the conjugation efficiency of polyethylene glycol and uric acid oxidase.

[0036] According to an embodiment of the present invention, the concentration of the uric acid oxidase in the conjugation reaction system is 10 mg / ml. The inventors discovered that when the concentration of the uric acid oxidase in the conjugation reaction system is within the above concentration range, the viscosity of the reaction solution is appropriate, which is advantageous for improving reaction efficiency and expanding industrial production. In addition, the inventors discovered that the protein concentration of the uric acid oxidase affects the average strain of the uric acid oxidase, and that when the obtained uric acid oxidase has the same average PEG strain, the PEG supply ratio required for 10 mg / ml of uric acid oxidase is low, thereby reducing production costs.

[0037] According to an embodiment of the present invention, the conjugation reaction is performed for at least 60 minutes under conditions of 5 to 30°C. By performing the conjugation reaction for at least 60 minutes under the above temperature conditions, the T of uric acid oxidase 1 , K 3 , K 4 , K 30 , K 35 , K 76 , K 79 , K 97 , K 112 , K 116 , K 120 , K 152 , K 179 , K 222 , K 231 , K 266 , K 272 , K 285, K 291 , K 293 It is possible to effectively implement PEG modification in at least 11 of the sites.

[0038] 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.

[0039] 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 .

[0040] 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.

[0041] TYKKNDEVEFVRTGYGKDMIKVLHIQRDGKYHSIKEVATTVQLTLSSKKDYLHGDNSDVIPTDTIKNTVNVLAKFKGIKSIETFAVTICEHFLSSFKHVIRAQVYVEEVPWKRFEKNGVKHVHAFIYTPTGTHFCEVEQIRNGPPVIHSGI KDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATQVYCKWRYHQGRDVDFEATWDTVRSIVLQKFAGPYDKGEYSPSVQKTLYDIQVLTLGQVPEIEDMEISLPNIHYLNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRL(SEQ ID NO:1).

[0042] According to an embodiment of the present invention, the uric acid oxidase has an amino acid sequence represented by SEQ ID NO:1~7.

[0043] MAHYRNDYKKNDEVEFVRTGYGKDMIKVLHIQRDGKYHSIKEVATSVQLTLSSKKDYLHGDNSDVIPTDTIKNTVNVLAKFKGIKSIETFAVTICEHFLSSFKHVIRAQVYVEEVPWKRFEKNGVKHVHAFIYTPTGTHFCEVEQIRNGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATQVYCKWRYHQGRDVDFEATWDTVRSIVLQKFAGPYDKGEYSPSVQKTLYDIQVLTLGQVPEIEDMEISLPNIHYLNIDMSKMGLINKEEVLLPLDNPYGRITGTVKRKLTSRL(SEQ ID NO:2).

[0044] MYKNDEVEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSVQLTLSSKKDYVYGDNSDIIPTDTIKNTVHVLAKFKGIKSIETFAMNICEHFLSSFNHVIRAQVYVEEVPWKRFEKNGVKHVHAFIHNPTGTHFCEVEQMRSGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATKVYCKWRYHQGRDVDFEATWDTVRDIVLEKFAGPYDKGEYSPSVQKTLYDIQVHSLSRVPEMEDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRL(SEQ ID NO:3).

[0045] MAHYHNDYKKNDEVEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSVQLTLSSKKDYVYGDNSDIIPTDTIKNTVHVLAKFKGIKSIETFAMNICEHFLSSFNHVIRAQVYVEEVPWKRFEKNGVKHVHAFIHNPTGTHFCEVEQMRSGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATKVYCKWRYHQGRDVDFEATWDTVRDIVLEKFAGPYDKGEYSPSVQKTLYDIQVHSLSRVPEMEDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGRITGTAKRKLASKL(SEQ ID NO:4).

[0046] MAHYHNDYQKNDEVEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSVQLTLNSRREYLHGDNSDIIPTDTIKNTVQVLAKFKGIKSIETFAMNICEHFLSSFNHVIRVQVYVEEVPWKRFEKNGVKHVHAFIHTPTGTHFCEVEQLRSGPPVIHSGIKDLKVLKTTQSGFEGFLKDQFTTLPEVKDRCFATQVYCKWRYHQGRDVDFEATWEAVRGIVLKKFAGPYDKGEYSPSVQKTLYDIQVLSLSQLPEIEDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGRITGTVKRKLTSRL(SEQ ID NO:5).

[0047] MAHYHNDYKKNDEVEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSVQLTLSSKKDYLHGDNSDIIPTDTIKNTVHALAKFKGIKSIEAFAVNICQHFLSSFNHVIRTQVYVEEIPWKRLEKNGVKHVHAFIHTPTGTHFCEVEQLRSGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFAAQVYCKWRYHQCRDVDFEATWDTIRDVVLEKFAGPYDKGEYSPSVQKTLYDIQVVSLSQVPEIDDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRL(SEQ ID NO:6).

[0048] MADYHNNYKKNDELEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSVQLTLSSKKDYLHGDNSDIIPTDTIKNTVHVLAKFKGIKSIEAFGVNICEYFLSSFNHVIRAQVYVEEIPWKRLEKNGVKHVHAFIHTPTGTHFCEVEQLRSGPPVIH SGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATQVYCKWRYHQCRDVDFEATWGTIRDLVLEKFAGPYDKGEYSPSVQKTLYDIQVLSLSRVPEIEDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRL(SEQ ID NO:7).

[0049] 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.

[0050] 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 , 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 293By 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.

[0051] 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 , 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: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 , 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: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 299 It 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.

[0052] 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.

[0053] According to an embodiment of the present invention, the peptide map of the polyethylene glycol-modified uric acid oxidase has a peak area reduced peptide fragment as shown in Table 8.

[0054] According to an embodiment of the present invention, the peptide map of the uric acid oxidase modified with polyethylene glycol is as shown in FIG. 6 or FIG. 7.

[0055] In a second aspect of the present invention, the present invention proposes a method for reducing the immunogenicity of uric acid oxidase. According to an embodiment of the present invention, PEG modification occurs at least 11 of the following amino acid sites of the polyethylene glycol-modified uric acid oxidase, and: 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 293The above method comprises the step of conjugating uric acid oxidase with polyethylene glycol, wherein the polyethylene glycol is provided in the form of an acidic solution and the molar ratio of uric acid oxidase to polyethylene glycol is 1:(56 to 94); 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.

[0056] 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.

[0057] In a third aspect of the present invention, the present invention proposes a uric acid oxidase modified with polyethylene glycol. According to an embodiment of the present invention, the uric acid oxidase is obtained by the method described above. According to the polyethylene glycol modified uric acid oxidase of an embodiment of the present invention, the stability of the uric acid oxidase in vivo is greatly improved and immunogenicity is reduced under the premise of ensuring maximum enzyme activity, and furthermore, the in vivo efficacy after intramuscular injection can reach an in vivo efficacy equivalent to that after intravenous injection of a similar commercially available drug.

[0058] In a fourth 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 aforementioned uric acid oxidase. The pharmaceutical composition according to an embodiment of the present invention has the advantages of low immunogenicity, high in vivo stability, and suitability for intramuscular injection, and can be used for the treatment or prevention of hyperuric acid-related diseases.

[0059] According to an embodiment of the present invention, the pharmaceutical composition further comprises at least one of the following additional technical features.

[0060] According to an embodiment of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable adjuvant.

[0061] According to an embodiment of the present invention, the pharmaceutical composition further comprises other drugs that treat or prevent hyperacidity-related diseases.

[0062] In a fifth aspect of the present invention, the present invention proposes the use of the aforementioned uric acid oxidase or the aforementioned pharmaceutical composition in the preparation of a drug for treating hyperuric acid-related diseases and reducing uric acid levels in the biological fluid of a subject in need. The uric acid oxidase according to an embodiment of the present invention has the advantages of low immunogenicity, high in vivo stability, and suitability for intramuscular injection, and has significant advantages for the treatment of hyperuric acid-related diseases.

[0063] According to an embodiment of the present invention, the use may further include at least one of the following additional technical features.

[0064] According to an embodiment of the present invention, the hyperuricemia-related disease includes a disease selected from chronic hyperuricemia, gout, kidney disease, hyperuricemia arthritis, kidney stones, gouty nodules, hypertension, diabetes mellitus, hypertriglyceridemia, metabolic syndrome, and coronary heart disease.

[0065] According to an embodiment of the present invention, the biological fluid is urine or blood. Brief explanation of the drawing

[0066] 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. FIG. 8 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. 9 is a diagram showing kidney damage, necrosis, and inflammation scores according to an embodiment of the present invention. FIG. 10 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. 11 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. 12 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. 13 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 to SD rats according to an embodiment of the present invention. FIG. 14 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. 15 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. 16 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. 17 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. 18 is a figure showing the average blood uric acid level-time curve over time after several intravenous injections of pegloticase and pegylated uricase injection solutions into SD rats according to an embodiment of the present invention. FIG. 19 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

[0067] Hereinafter, embodiments of the present invention will be described in detail and illustrated in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are illustrative and are intended only to interpret the present invention and should not be understood as limiting the present invention.

[0068] The object of the present invention is to provide a method for producing uric acid oxidase modified with polyethylene glycol.

[0069] Another objective of the present invention is to provide a new uric acid oxidase modified with polyethylene glycol.

[0070] Another objective of the present invention is to provide a method for effectively reducing the immunogenicity of uric acid oxidase, and the technology can effectively reduce the immunogenicity of uric acid oxidase and improve the safety and stability of uric acid oxidase in vivo.

[0071] Another objective of the present invention is to provide an application of the polyethylene glycol oxyuricase conjugate obtained above, which can achieve in vivo persistence, significantly reduce blood uric acid levels, and be used for the treatment of hyperuricemia and gout.

[0072] As used herein, the terms "uric acid oxidase" and "uricase" are interchangeable and both refer to a type of enzyme capable of catalyzing the oxidation of uric acid described in the present invention to produce allantoin and hydrogen peroxide. The terms "uric acid oxidase analog," "uricase analog," and "uricase derivative" are interchangeable and, based on maintaining the activity of uric acid oxidase that specifically catalyzes the conversion of uric acid into allantoin and hydrogen peroxide, the protein structural sequence of uric acid oxidase can be structurally improved by partial amino acid substitution, deletion, or addition, and further achieve the advantages of the present embodiment, including but not limited to, reducing immunogenicity, increasing protein stability, and being favorable for further modification of polyethylene glycol.

[0073] Uric acid oxidase is not particularly limited and may be uric acid oxidase derived from any source and its uric acid oxidase analogs, representative examples include, but are not limited to, mammalian-derived, microorganisms, and plants.

[0074] In another preferred example, the uric acid oxidase and its uric acid oxidase analog are derived from mammals. Preferably, they are derived from the amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, more preferably from the amino acid sequence represented by SEQ ID NO:1.

[0075] 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.

[0076] In another preferred example, uric acid oxidase is recombinantly expressed in a host cell through recombinant technology, with the coding sequence of the uric acid oxidase protein sequence (SEQ ID NO:1) being recombinantly expressed.

[0077] 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.

[0078] As used in the specification, the polyethylene glycol uric acid oxidase described in the present invention is obtained by covalently modifying uric acid oxidase with polyethylene glycol. The polyethylene glycol (PEG) refers to a mixture of an ethylene oxide condensation polymer and water, represented by the general formula H(OCH2CH2)nOH. It is a hydrophilic polymer with a neutral pH, no toxicity, and excellent water solubility, and has a linear or branched structure. Due to the non-toxicity and good biocompatibility of PEG, the FDA has currently approved the release of various PEG-modified recombinant protein drugs, which demonstrates that PEG can be used to reduce the immunogenicity of proteins, increase protein solubility, and extend the half-life of proteins. For PEG to bind to a protein, one or multiple groups of PEG must be activated; depending on the modified target protein, such as amino groups, sulfhydryl groups, carboxyl groups, or hydroxyl groups, the corresponding modification group can be selected and activated.

[0079] In another preferred example, the site used for PEG modification of oxyuricase and uricase analogs in the present invention is the ε-amino group of a lysine residue, but a small amount of the α-amino group of an N-terminal lysine residue is also modified. Uric acid oxidase is covalently linked to the modification group of PEG through an amino-lipid bond, a secondary amino bond, or an amide bond, and preferably, a polyethylene glycol molecule and uric acid oxidase are conjugated to form an amide bond, and the modification group of polyethylene glycol includes but is not limited to the N-hydroxysuccinimide system, including but not limited to N-hydroxysuccinimide (NHS), N-hydroxysuccinimideyl carbonate (SC), N-hydroxysuccinimideyl acetate (SCM), N-hydroxysuccinimideyl propionate (SPA), N-hydroxysuccinimideyl butyrate (SBA), N-hydroxysuccinylsuccinate (SS), etc. Here, the blocking group of polyethylene glycol includes, but is not limited to, a monomethoxyl group, an ethoxyl group, glucose or galactose, preferably a monomethoxyl group.

[0080] In another preferred example, polyethylene glycol may be linear or linear.

[0081] In another preferred example, the relative molecular weight of polyethylene glycol applied to polyethylene glycol uric acid oxidase is 6 KD or less, preferably 1 KD to 5 KD, more preferably 2 KD, 5 KD, and most preferably 5 KD. It should be noted that the "relative molecular weight of polyethylene glycol" mentioned in the present invention refers to the relative molecular weight of polyethylene glycol without modifier groups, which has a general meaning in the art. After PEG is activated by an active group, the total relative molecular weight is slightly greater than 5 KD, such as in the range of 5 KD + 10%.

[0082] In another preferred example, the uric acid oxidase modified with the polyethylene glycol has the following characteristics.

[0083] (1) At least 11 of the following amino acid sites of uric acid oxidase have PEG modifications:

[0084] 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 .

[0085] (2) One uric acid oxidase monomer molecule is conjugated with an average of 11 to 13 polyethylene glycol molecules.

[0086] (3) Seq ID NO:1 K among the uric acid oxidase sequences 30 and / or K 35 Includes, and K 222 and / or K 231 Polyethylene glycol bonding deformation occurs.

[0087] (4) Polyethylene glycol uric acid oxidase has lower in vivo immunogenicity.

[0088] In another aspect of the present invention, a method is provided for effectively reducing the immunogenicity of uric acid oxidase. The technique can effectively reduce the immunogenicity of uric acid oxidase and improve the stability of uric acid oxidase in vivo.

[0089] As used herein, uric acid oxidase modified with polyethylene glycol has the following characteristics. Uric acid oxidase is not particularly limited and may be uric acid oxidase derived from any source and uric acid oxidase analogs thereof, and representative examples include, but are not limited to, mammalian-derived, microorganisms, plants, etc.

[0090] In another preferred example, the uric acid oxidase and its uric acid oxidase analog are derived from mammals. Preferably, they are derived from the amino acid sequence represented by SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, and SEQ ID NO:4, more preferably from the amino acid sequence represented by SEQ ID NO:1.

[0091] 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.

[0092] 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.

[0093] The uric acid oxidase described in the present invention can be recombinantly expressed in Escherichia coli to obtain a large amount of uric acid oxidase, and the expressed uric acid oxidase may be expressed within the cell or on the cell membrane, or secreted outside the cell. If necessary, high-purity uric acid oxidase can be obtained using methods well known to those skilled in the art. Examples of such methods include, but are not limited to, centrifugation, sterilization, salting out, ultrafiltration, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, and combinations of various other techniques.

[0094] The uric acid oxidase obtained above can be covalently bonded to polyethylene glycol through a linker using a method known in the art.

[0095] In another preferred example, polyethylene glycol directionally modifies lysine residues on the surface of the uric acid oxidase space structure. The uric acid oxidase is covalently linked to a modifier group (also referred to as an active group) of PEG via an amide bond, and the modifier group (also referred to as an active group) of polyethylene glycol comprises, but is not limited to, N-hydroxysuccinimide (NHS), N-hydroxysuccinimidylcarbonate (SC), N-hydroxysuccinimidylacetate (SCM), N-hydroxysuccinimidylpropionate (SPA), N-hydroxysuccinimidylbutyrate (SBA), and N-hydroxysuccinylsuccinate (SS), wherein the blocker group of polyethylene glycol comprises, but is not limited to, a monomethoxyl group, an ethoxyl group, glucose or galactose, preferably a monomethoxyl group.

[0096] In another preferred example, polyethylene glycol may be linear or branched.

[0097] In another preferred example, the relative molecular weight of polyethylene glycol is 6 KD or less, preferably 1 KD to 5 KD, most preferably 5 KD.

[0098] In another preferred example, the present invention provides a method for producing uric acid oxidase modified with polyethylene glycol having one or more of the following features.

[0099] (1) The modified feed molar ratio of uric acid oxidase to polyethylene glycol is 1:45 to 1:150 (uric acid oxidase: polyethylene glycol), preferably modified to a feed molar ratio of 1:45 to 1:110, and more preferably modified to a feed molar ratio of 1:56 to 1:94.

[0100] (2) The conjugation reaction system is a carbonate buffer with a modified pH range of 9 to 11.

[0101] (3) In the conjugation reaction system, the concentration of uric acid oxidase protein is 10 mg / ml.

[0102] The above method for producing polyethylene glycol-modified uric acid oxidase obtains high-purity polyethylene glycol-modified uric acid oxidase by applying various purification means.

[0103] In another preferred example, the purification of the modified sample includes, but is not limited to, molecular sieve chromatography, ion exchange chromatography, hydrophobic chromatography, tangential flow ultrafiltration, or a combination thereof, and more preferably molecular sieve chromatography and tangential flow ultrafiltration.

[0104] In another aspect of the present invention, the polyethylene glycol-modified uric acid oxidase and its applications are provided. The conjugate achieves in vivo persistence and can significantly reduce blood uric acid levels and can be used to treat hyperuricemia and gout.

[0105] The above polyethylene glycol uric acid oxidase 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.

[0106] 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.

[0107] The above polyethylene glycol uric acid oxidase has lower in vivo immunogenicity.

[0108] 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.

[0109] 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.

[0110] According to some embodiments of the present invention, a pharmaceutical composition comprising polyethylene glycol-modified uric acid oxidase of the present invention may further comprise a pharmaceutically acceptable carrier, and the formulation and mode of administration of the pharmaceutical composition are not particularly limited. In the case of an injectable formulation, the pharmaceutically acceptable carrier may comprise a buffer, a preservative, an analgesic, a solubilizer, an isotonic agent, and a stabilizer. In the case of a topical formulation, the pharmaceutically acceptable carrier may comprise an alkali, an excipient, a lubricant, and a preservative. The pharmaceutical composition of the present invention may be prepared in various formulations by combining with the above pharmaceutically acceptable carrier.

[0111] Herein, according to some specific examples of the present invention, excipients and diluents in a carrier suitable for drug formulation may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylparaben, propylparaben, talc, magnesium stearate, and mineral oil.

[0112] According to other embodiments of the present invention, the pharmaceutical composition of the present invention may further include a filler, an anticoagulant, a lubricant, a buffer, an osmotic pressure regulator, a humectant, a fragrance, and a preservative.

[0113] According to an embodiment of the present invention, the polyethylene glycol-modified uric acid oxidase and the pharmaceutical composition of the present invention significantly improve the stability of the in vivo uric acid oxidase and reduce immunogenicity under the premise of ensuring maximum enzyme activity, and furthermore, the in vivo pharmacological effect after intramuscular injection can reach an in vivo pharmacological effect equivalent to that after intravenous injection of a commercially available drug. Accordingly, the polyethylene glycol-modified uric acid oxidase of the present invention and the pharmaceutical composition comprising the polyethylene glycol-modified uric acid oxidase can be administered for the treatment or prevention of hyperuricemia-related diseases.

[0114] As used herein, the term "administration" means introducing a predetermined amount of a substance into a patient in an appropriate manner. The polyethylene glycol-modified uric acid oxidase of the present invention may be administered by any general route as long as it can reach the desired tissue. Various methods including peritoneum, vein, muscle, subcutaneous, cortical, oral, local site, nasal cavity, lung, and rectum are considered, but the present invention is not limited to these exemplified methods of administration. However, in the case of oral administration, the active ingredient of the composition for oral administration must be coated or formulated so as not to be degraded in the stomach. Preferably, the composition of the present invention is administered in the form of an injectable formulation. In addition, the pharmaceutical composition of the present invention may be administered using a specific device that delivers the active ingredient to target cells.

[0115] The frequency and dosage of administration of the pharmaceutical composition of the present invention may be determined by a plurality of relevant factors, including the type of disease to be treated, the route of administration, the patient's age, gender, weight, and severity of the disease, and the type of drug that is the active ingredient.

[0116] The term "therapeutic effective dose" refers to an amount of a compound sufficient to significantly improve some symptoms associated with a disease or pathology, that is, an amount that provides a therapeutic effect for a given pathology and dosage. For example, in the treatment of chronic hyperuricemia or gout, a drug or compound that reduces, prevents, delays, suppresses, or blocks any symptom of the disease or pathology must be therapeutically effective. A therapeutically effective dose of a drug or compound does not need to cure the disease or pathology, but provides treatment for the disease or pathology to delay, inhibit, or prevent the onset of the disease or pathology in the individual, or to alleviate symptoms of the disease or pathology, change the duration of the disease or pathology, or, for example, make the disease or pathology less severe or accelerate recovery.

[0117] The term “treatment” is used to refer to obtaining a desired pharmacological and / or physiological effect. Such effect may be preventive in that it completely or partially prevents a disease or its symptoms, or therapeutic in that it partially or completely cures the disease and / or adverse effects caused by the disease. As used herein, “treatment” includes the treatment of a disease in mammals, particularly humans (meaning a disease related to hyperuricemia), including (a) prevention of the disease in individuals susceptible to the disease but not yet diagnosed; (b) suppression of the disease, such as blocking the progression of the disease; or (c) alleviation of the disease, such as reducing disease-related symptoms. As used herein, “treatment” includes any drug or compound administered to an individual to treat, cure, alleviate, improve, reduce, or suppress a disease of gas. It includes, but is not limited to, the step of administering the polyethylene glycol-modified uric acid oxidase described herein to an individual in need.

[0118] According to embodiments of the present invention, the polyethylene glycol-modified uric acid oxidase or pharmaceutical composition of the present invention may be used in combination with general treatment methods and / or therapies, or separately from general treatment methods and / or therapies. When the polyethylene glycol-modified uric acid oxidase or pharmaceutical composition of the present invention is administered as a combination therapy with 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 pharmaceutically acceptable excipient, and other therapeutic or prophylactic drugs known in the art.

[0119] The term "average strain" refers to the number of PEGs bound to each uricase monomer.

[0120] In this specification, unless otherwise specified, the expression “the amino acid region has PEG modification” means that in the three-dimensional structure of the polypeptide, PEG molecules cover said amino acid region so that at least a portion of said amino acid region is not exposed. Those skilled in the art will understand that whether a specific amino acid region is modified by PEG molecules can be determined by conventional technical means, and can be verified, for example, by referring to the method listed in the “detection of a region modified by polyethylene glycol” section of Example 3 of the present invention. In summary, the method comprises: 1) digesting non-pegylated and pegylated uric acid oxidases by applying one or more enzymes, such as single enzymatic digestion with Lys-C or Trypsin, or double enzymatic digestion with Lys-C and Trypsin; 2) separating the digested fragments by a high-performance liquid chromatography method to generate a chromatogram of the non-pegylated and pegylated uric acid oxidases, i.e., a peptide map; 3) The method includes the step of comparing the difference between the peptide maps of non-pegylated and pegylated uric acid oxidases, determining the relative ratio of the reduction or disappearance of the peptide fragment peak where a specific amino acid site is located in the pegylated uric acid oxidase by binding with a predetermined internal standard peptide fragment, and further determining whether the specific amino acid site in the peptide fragment has been modified by PEG. Specifically, in Example 3 of the present invention, the relative ratio of the reduction or disappearance of the peak area of ​​the peptide fragment where a specific amino acid site is located can be calculated through the following formula.

[0121] P(%)=(A2-A1) / A2×100%,

[0122] Here, A1 = A0 × t, and

[0123] A0 is the actual measured peak area of ​​the peptide fragment where a specific amino acid site of the modified protein to be measured is located, and t is the average value of the ratio of the peak areas of the internal reference peptide fragment in the PHC peptide map and the modified protein to be measured peptide map;

[0124] P(%) represents the relative ratio of the decrease or loss of the peak area of ​​the peptide fragment where the specific amino acid site is located, A2 is the peptide fragment peak area where the specific amino acid site is located in the PHC peptide map, and A1 is the peak area of ​​the peptide fragment where the specific amino acid site is located converted to an internal reference in the modified protein peptide map to be measured.

[0125] It should be understood that within the scope of the present invention, each of the technical features of the present invention and the various technical features specifically described below (e.g., in the embodiments) may be combined with one another to form a novel or desirable technical solution, which will be more clearly understood by referring to the following embodiments. Due to limitations of scope, the above embodiments are for illustrative purposes only and are not intended to limit the present invention.

[0126] Hereinafter, embodiments of the present invention are described in more detail, and examples of such embodiments are illustrated in the drawings. 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.

[0127] Example 1: Preparation of recombinant uric acid oxidase

[0128] 1.1 Construction of Gene and Expression Plasmid for Uricase Expression

[0129] E.coliBased 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).

[0130] 1.2 Transformation of Expression Plasmids into Bacterial Host Cells

[0131] 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.

[0132] 1.3 Preparation of Recombinant Uric Acid Oxidase

[0133] 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.

[0134] 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.

[0135] 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.

[0136] Example 2: Preparation of Pegylated Oxyuricase

[0137] 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.

[0138] Example 3: Characterization of Pegylated Oxyuricase

[0139] 3.1 Detection of Average Strain and Enzyme Activity

[0140] 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).

[0141] 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 determined by a differential refractive index detector, 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 a PEG reference material and a 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.

[0142] 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)

[0143] 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.

[0144] The enzymatic activity and average strain of polyethylene glycol oxyuricase obtained under different feed ratios in Example 2 are shown in Table 1.

[0145] Table 1: Enzyme activity and mean strain of 5K-PEG at different feed ratios

[0146] 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

[0147] The enzymatic activity and average strain of polyethylene glycol oxyuricase obtained under a protein-to-PEG feed molar ratio of 1:68 with different PEG molecular weights from Example 2 are shown in Table 2.

[0148] Table 2: Enzyme activity and average modification under PEG modifications with different molecular weights

[0149] PEG MW(KD) Average strain Enzyme activity (U / mg) Enzyme activity retention rate 0 - 11.3 100% 2 11.7 11.6 102.7% 3.5 11.5 11.4 100.9% 5 11.8 12.1 107.1% 10 11.7 11.8 104.4%

[0150] Note: Average strain represents the number of PEG molecules in each uric acid oxidase monomer.

[0151] From Tables 1 and 2, it can be seen that 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. This is inconsistent with the teaching of the original study drug that enzyme activity would decrease due to low molecular weight PEG modification. The average degree of modification of the polyethylene glycol-modified uric acid oxidase obtained in the present invention is higher, and unexpected technical effects were achieved in terms of maintaining enzyme activity.

[0152] In addition, the applicant measured the immunogenicity of uric acid oxidase obtained under PEG modifications having different molecular weights, and the experimental results are shown in Table 3.

[0153] Table 3: In vivo antibody results of PEGylated uric acid oxidase with different molecular weights in mice

[0154] PEG MW(KD) Average strain Anti-PEG antibody Anti-uricase protein antibody Antibody positivity rate Antibody titer range Antibody positivity rate Antibody titer range 0 (non-deformed) - 0 / 8 - 8 / 8 1:51200~1:204800 2 11.7 1 / 8 1:100~1:400 2 / 8 1:100~1:300 3.5 11.5 1 / 8 1:100~1:600 2 / 8 1:50~1:100 5 11.8 2 / 8 1:100~1:700 1 / 8 1:20~1:50 10 11.7 4 / 8 1:1600~1:12800 2 / 8 1:50~1:100

[0155] In the experiment, mice were grouped into groups of eight and intravenously injected with 1 mg / kg once a week per animal. Blood samples were collected after four consecutive administrations to evaluate the immunogenicity of anti-PEG and anti-uric acid oxidase. From the results in Table 3, it can be observed that when the average strain was consistent, the positive rate of the generated anti-PEG antibody increased with increasing molecular weight of PEG, and when the molecular weight of PEG exceeded 5 KD, the positive rate and antibody titer of the anti-PEG antibody significantly increased. From the analysis of the anti-uric acid oxidase results, it can be seen that PEG modification can significantly reduce the positive rate and antibody titer of the anti-uric acid oxidase antibody, and when the average strain was consistent, within the range of 2–5 KD, the positive rate and antibody titer of the generated anti-uric acid oxidase antibody decreased as the molecular weight of PEG increased, and when the molecular weight of PEG was greater than 5 KD, the risk of anti-uric acid oxidase antibodies also appeared. In conclusion, 2~5K PEG-modified uric acid oxidases are superior to the 10K PEG-modified uric acid oxidase group, and 5KD is more desirable.

[0156] Finally, the inventors measured the enzymatic activity and average degree of deformation of uric acid oxidase obtained by modifying PEG dissolved in different acidic solutions, and the acidic solutions used were acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, malonic acid, adipic acid, ascorbic acid, benzenesulfonic acid, benzoic acid, butyric acid, cyclopentylpropionic acid, digluconic acid, dodecyl sulfonic acid, ethylsulfonic acid, formic acid, fumaric acid, glucoheptonic acid, glycerophosphate, gluconic acid, heptanoic acid, caproic acid, 2-hydroxyethanesulfonic acid, lacturonic acid, lactic acid, lauric acid, lauryl sulfonic acid, malic acid, malonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, niacin, oleic acid, palmitic acid, pectinic acid, 3-phenylpropionate, picrate, pivalic acid, It may be selected from organic acids such as propionic acid, stearic acid, p-toluenesulfonic acid, undecanic acid, and valeric acid; or it may be selected from inorganic acids such as hydrochloric acid, hydrobromide, phosphoric acid, sulfuric acid, perchloric acid, hydroiodide, nitric acid, persulfuric acid, boric acid, dichromate, silicic acid, chromic acid, and thiocyanate. Various types of acidic solutions can dissolve PEG and modify uricase. The feeding methods and feeding ratios of different PEGs are shown in Table 4 below.

[0157] Table 4: Effects of Different Feeding Methods and Feeding Costs on PEG Modification Results

[0158] PEG supply method Supply Ratio (Molar Supply Ratio) Protein: 5K-PEG Average strain Enzyme activity Dry powder supply 1:48 8.8 8.96 U / mg 1:56 9.3 9.18 U / mg 1:94 9.8 9.26 U / mg 1:110 9.2 9.16 U / mg hydrochloric acid dissolution 1:48 10.8 10.96 U / mg 1:56 11.4 11.21 U / mg 1:94 12.4 11.35 U / mg 1:110 12.2 10.76 U / mg sulfuric acid dissolution 1:94 11.9 11.16 U / mg Dissolution of glacial acetic acid 1:94 11.7 11.03 U / mg

[0159] The inventors discovered that the method of dissolving PEG in acid before supplying results in a higher modification rate of the obtained protein, higher enzyme activity, and effectively reduced PEG usage compared to the method of supplying in the form of a directly dried powder.

[0160] 3.2 Detection of areas modified by polyethylene glycol

[0161] In the following steps, the inventors performed detection of modified sites on existing commercial products and uric acid oxidase obtained in the examples.

[0162] 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 in combination). Digestion fragments were separated using a reverse-phase column, and the modification site of polyethylene glycol uric acid oxidase and the polyethylene glycol modification ratio of the corresponding site were determined and calculated through internal reference peptide fragment correction and comparison of the loss or reduction ratio of the peptide fragments.

[0163] 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 combining with an internal standard peptide fragment, the relative ratio of reduction or loss of PEG-modified peptide fragments can be analyzed and confirmed. 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 relative ratio of modification; that is, it is possible to obtain whether a specific amino acid (e.g., lysine) of the peptide fragment has been modified by PEG.

[0164] To explain in detail, it is as follows.

[0165] (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.

[0166] (2) Analysis conditions:

[0167] Instruments: Thermo Ultimate 3000 HPLC and MSQ Plus;

[0168] Chromatics Column: Welch Materials μltimate ® XB-C18(4.6mm Υ250mm, 5μm);

[0169] Analysis conditions: Solution A (aqueous solution containing 0.1% TFA), Solution B (acetonitrile solution containing 0.1% TFA);

[0170] Gradient: 0~70 min, B is 3~70%;

[0171] LC detection wavelength: 214nm.

[0172] Ion source: ESI;

[0173] Ion type: Cation;

[0174] Cone voltage: 50V;

[0175] Scanning range: 300~2000Da;

[0176] Scanning time: 1S;

[0177] Post-column flow switching: approx. 0.3 ml / min.

[0178] 100 μl of sample was injected and a chromatogram was recorded.

[0179] (3) Result processing:

[0180] 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.

[0181] (4) The experimental results are shown in Tables 5 to 8 and Figures 6 to 7.

[0182] Table 5: List of peptide fragments of PHC after digestion with Lys-C

[0183] 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

[0184] Table 6: List of peptide fragments of PHC after dual enzyme digestion with Lys-C and trypsin

[0185] 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 /

[0186] Calculation method for the percentage reduction in peak area of ​​the PU5 peptide fragment:

[0187] 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.

[0188] A1 = A0 × t

[0189] 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.

[0190] Table 7: Comparison of PHC and PU5 internal reference peptide fragments

[0191] 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

[0192] The relative percentage of the 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.

[0193] P(%)=(A2-A1) / A2×100%

[0194] 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.

[0195] Table 8: Summary results of peptide fragments with reduced peak area in the peptide map after diubal enzyme digestion of PU5

[0196] 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%

[0197] 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 parts.

[0198] As shown in the analysis of Tables 5, 6, 7, 8 and Figure 6, from the analysis of the polyethylene glycol-modified uric acid oxidase modified site obtained in Example 2, the site where more than 90% of the peptide fragment was lost after PU5 digestion is 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 It can be seen that.

[0199] 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 231The strain occurring in 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 modified sites and differences in modified sites.

[0200] The in vivo drug evaluation of the polyethylene glycol-modified uric acid oxidase (PU5) of the present invention in animals will be described in detail below. Here, the pegloticase used in the experiment refers to a similar drug commercially available under batch number 5085B.

[0201] Example 4: In vivo pharmacodynamic study of polyethylene glycol uric acid oxidase

[0202] 4.1 In vivo evaluation of polyethylene glycol uric acid oxidase in model rats

[0203] The therapeutic effect of polyethylene glycol uric acid oxidase (PU5) on chronic hyperuricemia in rats was evaluated by inducing chronic hyperuricemia model rats using a combination of oxaginate drinking water and a hyperuricemia diet.

[0204] Forty model rats were selected and randomly divided into four groups: a model group, a low-dose pegylated uricase group (0.3 mg / kg), a medium-dose pegylated uricase group (1.0 mg / kg), and a high-dose pegylated uricase group (3.0 mg / kg). Ten rats were selected per group, and an additional 10 normal SD rats were selected as a blank control group. The model construction study was conducted for five consecutive weeks, and intramuscular administration began one week after the start of the model construction. Serum uric acid, serum urea nitrogen, and serum creatinine levels were measured before administration and on the seventh day after administration, and histological changes in the rat kidneys were observed after the end of the study.

[0205] The results in Figure 8 indicate that compared to the blank control group, blood uric acid levels in the model control group significantly increased on days 7, 14, 21, 28, and 35 after model establishment. Seven days after model establishment, serum urea nitrogen, creatinine, and uric acid in the model group rats were 2.73 times, 2.40 times, and 7.83 times higher, respectively, than those in the blank group rats. From a renal pathological perspective (as shown in Figure 9), the scores for tubular dilation, necrosis, inflammation, and fibrosis in the model control group all significantly increased, along with a significant increase in the number of uric acid crystals. Pegylated uricase, the test substance at intermediate and high doses, significantly reduced serum uric acid levels, exhibiting a dose-dependent relationship. During the period from day 14 to day 35, the average blood uric acid levels of the medium-dose group were maintained at 303.80–660.60 μmol / L, and the average blood uric acid levels of the high-dose group were maintained at 153.70–403.40 μmol / L. Compared to the model group, the reduction in blood uric acid levels in the medium-dose group was 34.46–67.94%; and in the high-dose group, the reduction was 65.67–83.78%. Compared to the model control group, each administration group of pegylated uricase showed significant improvement in tubular dilation, renal necrosis, and inflammation.

[0206] 4.2 Evaluation of a Single Dose of Polyethylene Glycol Uric Acid Oxidase in Rats

[0207] Thirty-six SD rats (half female, half male) were randomly divided into six groups (see Table 6): 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 6. Blood was collected from the jugular vein to detect PK and PD.

[0208] Table 9: Animal Grouping and Dosage Design

[0209] number By group Route of administration 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 Intravenous Injection Group Intravenous injection 1 time 1.0 0.1 10.0 3 3 4 PU5 low-dose intramuscular injection group Intramuscular injection 1 time 0.5 0.5 1.0 3 3 5 PU5 Medium-dose Intramuscular Injection Group Intramuscular injection 1 time 1.0 1.0 1.0 3 3 6 PU5 High-dose Intramuscular Injection Group Intramuscular injection 1 time 2.0 2.0 1.0 3 3

[0210] 4.2.1, Pharmacokinetic Comparison

[0211] In SD rats, the serum drug concentration levels of all individuals prior to administration were lower than the lower limit of quantification (LLOQ: 312.500 ng / mL), and upon a single intramuscular injection of 0.5, 1.0, and 2.0 mg / kg, the serum drug concentration of pegloticase injection solution (PU5) was dose-dependent during the period of 0 to 168 h (0 to 7 days), with the overall level increasing as the dose increased. After 168 h, the blood drug concentration of the pegloticase intramuscular administration group was lower than the lower limit of quantification, whereas the PU5 intramuscular administration group continued to maintain it for more than 240 h.

[0212] 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.

[0213] 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 lastIt 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.

[0214] 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.

[0215] The above pharmacokinetic results are shown in Tables 10 to 15 and Figures 10 to 12.

[0216] 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)

[0217] 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 / /

[0218] Note: " / " means there is no relevant information.

[0219] 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)

[0220] 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

[0221] Note: " / " means there is no relevant information.

[0222] 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)

[0223] 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 / /

[0224] Note: " / " means there is no relevant information.

[0225] Table 13: Blood drug concentration data and statistical analysis data for SD rats administered a single intramuscular injection of 1.0 mg / kg pegylated uricase solution (Unit: μg / mL)

[0226] 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

[0227] Table 14: Mean pharmacokinetic parameters after a single intravenous injection of pegloticase and pegylated uricase solution in SD rats

[0228] 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

[0229] Table 15: Mean pharmacokinetic parameters after a single intramuscular injection of Peglocticase and pegylated uricase solution in SD rats

[0230] 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

[0231] 4.2.2, Comparison of In vivo efficacy (Uric acid)

[0232] When 0.5, 1.0, and 2.0 mg / kg pegylated uricase injections 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 compared to the pegloticase intravenous injection group, and the PU5 intramuscular injection group maintained low serum uric acid concentrations for a longer period compared to the pegloticase intramuscular injection group. When comparing the same dose groups, the PU5 intravenous or intramuscular injection groups maintained low serum uric acid concentrations for a longer period compared to the pegloticase intravenous or intramuscular injection groups; that is, PU5 maintained low in vivo uric acid concentrations for a longer period compared to pegloticase in each case, and the results are shown in Figure 13.

[0233] 4.3 Evaluation of Multiple Administrations of Polyethylene Glycol Uric Acid Oxidase in Rats

[0234] 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.

[0235] From the analysis of results,

[0236] 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.

[0237] 4.3.1 Detection of Anti-PEG Antibodies

[0238] 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 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 intravenous and intramuscular injection groups, and the results are shown in Table 16.

[0239] 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 mainly antibodies against the PEG portion.

[0240] From the results of PEG antibody and PEG immunohistochemical tests, both PU5 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 compared to pegloticase; and the anti-PEG antibody produced in the intramuscular administration group was superior in PU5 compared to pegloticase.

[0241] Table 16: PEG Immunohistochemical Positive Expression Results

[0242] 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

[0243] Positive grade: 1=Very weak positive, 2=Weak positive, 3=Moderately positive, 4=Strong positive.

[0244] 4.3.2 Pharmacokinetic Detection

[0245] 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.

[0246] 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.

[0247] 4.3.3 Comparison of In vivo Efficacy (Uric Acid)

[0248] 1.0 mg / kg of Pegloticase and pegylated uricase injections were administered intravenously and intramuscularly to SD rats four consecutive times (once per week). Serum uric acid levels remained low after each administration, and the intramuscular Pegloticase 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 administration groups of the two drugs were relatively consistent; however, the retention time of the intramuscular administration group of pegylated uricase was longer than that of the commercially available drug, indicating that the efficacy of PU5 was superior to that of Pegloticase upon intramuscular administration.

[0249] The above results are shown in Tables 17–20 and Figures 14–19.

[0250] Table 17: Mean pharmacokinetic parameters after serial intravenous injection of pegloticase and pegylated uricase injection solutions in SD rats

[0251] 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

[0252] Table 18: Results of mean pharmacokinetic parameters after serial intramuscular injection of pegloticase and pegylated uricase injection solutions in SD rats

[0253] 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

[0254] 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)

[0255] 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

[0256] 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 connection with said embodiment or example are included in at least one embodiment or example of the present invention. A general expression of said 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.

Claims

Claim 1 A method for preparing a polyethylene glycol-modified uric acid oxidase, characterized by conjugating a uric acid oxidase with polyethylene glycol, wherein the polyethylene glycol is provided in the form of an acidic solution, and the molar ratio of the uric acid oxidase to the polyethylene glycol is 1:(56 to 94) to obtain a polyethylene glycol-modified uric acid oxidase. Claim 2 A method according to claim 1, characterized in that the acidic solution comprises one or more selected from organic acids and / or inorganic acids. Claim 3 In paragraph 2, the organic acid is derived from acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid, malonic acid, adipic acid, ascorbic acid, benzenesulfonic acid, benzoic acid, butyric acid, cyclopentylpropionic acid, digluconic acid, dodecyl sulfonic acid, ethylsulfonic acid, formic acid, fumaric acid, glucoheptonic acid, glycerophosphate, gluconic acid, heptanoic acid, caproic acid, 2-hydroxyethanesulfonic acid, lacturonic acid, lactic acid, lauric acid, lauryl sulfonic acid, malic acid, malonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, niacin, oleic acid, palmitic acid, pectinic acid, 3-phenylpropionate, picrate, pivalic acid, propionic acid, stearic acid, p-toluenesulfonic acid, undecanic acid, and valeric acid. A method characterized by being selected; wherein the inorganic acid is selected from hydrochloric acid, hydrobromide, phosphoric acid, sulfuric acid, perchloric acid, hydroiodic acid, nitric acid, persulfuric acid, boric acid, dichromate, silicic acid, chromic acid, and thiocyanate, or wherein the concentration of hydrogen ions in the acidic solution is 1 to 5 mmol / L, or wherein the acidic solution contains hydrochloric acid, sulfuric acid, or glacial acetic acid. Claim 4 A method according to claim 3, characterized in that the concentration of the acid in the acidic solution is 1 to 5 mmol / L. Claim 5 A method according to claim 1, characterized in that the concentration of the polyethylene glycol in the acidic solution is 100 to 300 mmol / L. Claim 6 A method according to claim 1, wherein the molecular weight of the polyethylene glycol is 6 KD or less, or the polyethylene glycol has a monomethoxyl group or a hydroxyl group, or the polyethylene glycol has a linear or branched structure, or the polyethylene glycol and uric acid oxidase are conjugated via an amide bond, or the polyethylene glycol is a modified polyethylene glycol, and the modified group of the modified polyethylene glycol is selected from at least one of N-hydroxysuccinimide, N-hydroxysuccinimidylcarbonate, N-hydroxysuccinimidylacetate, N-hydroxysuccinimidylpropionate, N-hydroxysuccinimidylbutyrate, N-hydroxysuccinylsuccinate, and bis(p-nitrophenyl)carbonate. Claim 7 A method according to claim 6, characterized in that the modifier group of the modified polyethylene glycol is N-hydroxysuccinimidylpropionate. Claim 8 A method according to claim 1, characterized in that the conjugation reaction is performed in a carbonate buffer solution. Claim 9 A method according to claim 8, characterized in that the pH of the carbonate buffer solution is 9 to 11. Claim 10 A method according to claim 1, characterized in that the concentration of the uric acid oxidase in the conjugation reaction system is 10 mg / ml. Claim 11 A method according to claim 1, characterized in that the conjugation reaction is performed for at least 60 minutes under conditions of 5 to 30°C. Claim 12 In claim 1, the following amino acid site of the uric acid oxidase: 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 A method characterized in that at least 11 of them have PEG modifications, and the amino acid sites are positioned by an amino acid sequence represented by SEQ ID NO:

1. Claim 13 A method according to claim 1, characterized in that the uric acid oxidase has an amino acid sequence represented by SEQ ID NO:1~7. Claim 14 A method according to claim 12, characterized in that the uric acid oxidase has an amino acid sequence represented by SEQ ID NO:1~4. Claim 15 In any one of claims 1 to 14, the following four amino acid sites of the polyethylene glycol-modified uric acid oxidase: K 30 , K 35 , K 222 and K 231 A method characterized in that at least one of them has a PEG modification, and the amino acid site is positioned by an amino acid sequence represented by SEQ ID NO:

1. Claim 16 A method according to any one of claims 1 to 14, wherein, compared with 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 peak area reduction of at least 11 specific peptide fragments of 80% or more, wherein at least 11 specific peptide fragments are selected from the following peptide fragments: . Claim 17 A method according to claim 16, characterized in that, 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 peak area reduction of at least 11 predetermined peptide fragments of 90% or more. Claim 18 A method according to claim 16, wherein the peptide map of the polyethylene glycol-modified uric acid oxidase has a peptide fragment with reduced peak area, and the relative ratio of the reduced peak area of ​​the peptide fragment is as listed below: . Claim 19 delete Claim 20 delete Claim 21 delete Claim 22 delete Claim 23 delete Claim 24 delete Claim 25 delete Claim 26 delete Claim 27 delete Claim 28 delete Claim 29 delete Claim 30 delete Claim 31 delete Claim 32 delete Claim 33 delete Claim 34 delete Claim 35 delete Claim 36 delete Claim 37 delete Claim 38 delete Claim 39 delete Claim 40 delete Claim 41 delete Claim 42 delete Claim 43 delete Claim 44 delete Claim 45 delete Claim 46 delete Claim 47 delete Claim 48 delete Claim 49 delete Claim 50 delete Claim 51 delete

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