Method for producing polyethylene glycol-modified urate oxidase

Abstract

The present invention provides a method for producing polyethylene glycol-modified urate oxidase, which comprises the steps of: 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 At least 11 of the above are PEG-modified, and the method for preparing the urate oxidase includes a step of coupling urate oxidase with polyethylene glycol, wherein the polyethylene glycol is provided in the form of an acidic solution, and the molar ratio of the urate oxidase to the polyethylene glycol is 1:(56-94), to obtain polyethylene glycol-modified urate oxidase.

Description

[Technical field]

[0001] The present invention relates to the field of biomedicine. In particular, the present invention relates to a method for producing polyethylene glycol-modified urate oxidase. More particularly, the present invention relates to a method for producing polyethylene glycol-modified urate oxidase, a method for reducing the immunogenicity of urate oxidase, polyethylene glycol-modified urate oxidase, a pharmaceutical composition, and pharmaceutical uses of polyethylene glycol-modified urate oxidase. [Background technology]

[0002] Gout is a disease caused by the disturbance of purine metabolism. Its clinical characteristic is hyperuricemia, and uric acid salts are deposited in the subcutaneous tissue, joints, and kidneys, resulting in the formation of gout stones. Purines in the human body undergo a series of changes, and the final product formed is uric acid. When the blood uric acid concentration exceeds 70 mg / L, it causes hyperuricemia. 5% to 12% of patients with hyperuricemia may develop gout. When the sodium urate concentration reaches saturation in the blood or synovial fluid, microcrystals of sodium urate salt are formed, causing gouty arthritis. Over time, chronic hyperuricemia also deposits around the joints, soft tissues, and some organs to produce destructive crystalline uric acid deposits, causing diseases such as gouty acute arthritis, gouty chronic arthritis, and joint deformity. Kidney damage is considered the second most common clinical manifestation of gout. Chronic hyperuricemia gradually progresses, causing urate deposition in the medulla, tubules and renal interstitium, irritating the local area and causing inflammatory reactions, which is called chronic urate nephropathy. In patients with severe hyperuricemia (e.g. some malignant tumors, especially leukemia and lymphoma patients), large amounts of uric acid are deposited in the renal collecting ducts, renal pelvis, renal calyx and ureters in a short period of time, causing luminal obstruction and urinary retention, which may lead to acute renal failure (also known as uric acid nephropathy).

[0003] In recent decades, with the improvement of people's quality of life and changes in eating and lifestyle habits, the intake of high-protein and high-purine foods has increased, and the number of gout patients has been increasing year by year. In Europe, the number of gout patients has nearly doubled in the past 20 years. The current incidence rate of hyperuricemia and gout in China has risen to about 2-3%. If hyperuricemia does not show clinical symptoms, it is sufficient to control the diet, but if clinical symptoms caused by hyperuricemia appear, drug treatment is required. The common treatment methods currently used in clinical practice include analgesics and anti-inflammatory drugs such as colchicine, ibuprofen, and naproxen, which are mainly used to control acute attacks of gouty arthritis and relieve local pain, swelling, and inflammation in the joints, uricosurics that promote uric acid excretion such as probenicid, sulfinpyrazone, and benzobromarone (ineffective if renal function is reduced), and drugs that inhibit uric acid synthesis such as allopurinol. For patients suffering from lithiasis, renal failure, leukemia, and some genetic diseases, allopurinol is the main treatment drug, which inhibits xanthine oxidase and prevents hypoxanthine and xanthine from being converted into uric acid, which is gradually oxidized in the human body to produce isoxanthine, which is easily soluble in water, and is excreted in urine. However, all conventional treatment methods are difficult to cure chronic gout patients who have formed gout stones. Moreover, after taking the above drugs for a long time, patients cannot avoid complications such as reduction in white blood cells, damage to heart function, damage to liver and kidney function, stimulation of gastrointestinal system, diabetes mellitus caused by regenerative anemia, gout, etc.

[0004] Human hyperuricemia is associated with the mutational inactivation of the uric acid enzyme gene during human evolution; the mutation introduced a premature stop codon into the coding sequence of the human uric acid enzyme gene (Wu X, Lee CC, Muzny DM, Caskey C T. Proc Natl Acad SciUSA. 1989.86:9412-9416.), which means that humans are unable to synthesize active uric acid enzyme themselves, and the degradation of purines in humans ends with uric acid (Wu X, Muzny DM, Lee CC, Caskey C TJ Mol Evol. 1992.34:78-84.). Active uric acid enzymes in the liver peroxidases of non-human primates and other mammals convert the less soluble urate (~11 mg / 100 ml water) to the more soluble allantoin (~147 mg / 100 ml water), which can be more effectively excreted by the kidney (Wortmann RL, Kelley W N.Kelley's textbook of rheumatology (6th).2001 :1339-1376). In Europe and the United States, uric acid enzymes (Uricozyme) produced from aflatoxins (Aspergillus flavus) have been used for more than 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 aflatoxin uric acid enzyme drug produced by brewer's yeast fermentation developed by Sanofi in France, received FDA approval in 2002 and is used for the short-term treatment of severe hyperuricemia caused by cancer chemotherapy (Pui CH, Relling MV, Lascombes F, Harrison P L, Struxiano A et al. Leukemia. 1997.11:1813-1816.), and at the same time, it has been proven that ELITEK infusion can also reduce the volume of gout stones (Potaux L, Aparicio M, Maurel C, Ruedas ME, Mart in C L. Nouv Presse Med. 1975.4:1109-1112.).PEG-modified recombinant porcine uric acid enzyme (Pegloticase), manufactured by Savient, a US company, was approved for marketing by the FDA in September 2010 and has been used to treat stubborn gout. However, because it did not resolve the issue of immunogenicity, it was ineffective in approximately 50% of patients in clinical use.

[0005] Uric acid enzymes (EC 1.7.3.3) are widely present in microorganisms (Bacillus fastidiosus, Monocandida, Aflatoxin), plants (soybean, chickpea) and animals (pig, cow, dog, baboon) (Suzuki K, Sakasegawa S, Misaki H, Sugiyama M. J Biosci Bioeng. 2004.98 :153-158) and can catalyze the oxidation of allantoin by uric acid in the presence of oxygen, releasing carbon dioxide (Retailleau P, Colloc'h, Denis V, Francoise B. Acta Cryst D. 2004.60 :453-462.).

[0006] The active uric acid enzyme is a tetrameric protein, composed of the same subunits, each of which has a molecular weight of about 34 kD and consists of 301-304 amino acids. The pH value at which the enzyme activity of the uric acid enzyme in each solution is highest is 8.0 (Bayol A et al. Biophys Chem. 1995.54 :229-235.). Among all currently known sources of uric acid enzyme, the highest activity is from aflatoxin, which reaches 27 IU / mg, followed by Bacillus fastidiosus, whose activity remains at 13 IU / mg (Huang S H, Wu T K. Eur J Biochem. 2004.271 :517-523.). In addition, the uric acid enzyme derived from legume plants has an activity of only 2-6 IU / mg, and the uric acid enzyme derived from mammals can reach 5 IU / mg in pigs after recombinant expression, and the enzyme activity of baboon uric acid enzyme is only 1 IU / mg (Michael H, Susan JK2006.US7056713B1), and the uric acid enzyme derived from humans is inactive.

[0007] For human application, due to the high activity of microbial uric acid enzyme and the low immunogenicity of mammalian uric acid enzyme, these two major origins of uric acid enzyme are the focus of research on recombinant uric acid enzymes currently being developed and applied. However, the homology between the aflatoxin-derived uric acid enzyme and the putative human-derived uric acid enzyme is less than 40% (Lee CC, Wu X, Gibbs RA, Cook RG, Muzny DM, Caskey C T. Science. 1988. 239: 1288-1291.), and the human body is prone to the development of anti-uric acid enzyme antibodies, which rapidly weakens the effect of aflatoxin uric acid enzyme and causes severe allergic reactions, making it unusable for long-term treatment. The human uric acid enzyme gene mutates and loses activity, becoming a pseudogene.

[0008] For this reason, urate oxidase-based treatment techniques for hyperuricemia still require further development and improvement. Summary of the Invention [Problem to be solved by the invention]

[0009] The present application has been prepared based on the inventor's discovery and recognition of the following facts and problems.

[0010] Active urate oxidase is a homologous tetrameric protein, of which one-third of the amino acids are strongly hydrophobic amino acids, and the tetrameric proteins are prone to aggregate with each other to form octamers and larger polymers. Molecules with a molecular weight of more than 100 kDa can effectively induce immune responses in the body, but the molecular weight of unmodified polyurate oxidase protein has already reached 140 kDa, and polymeric uric acid enzymes with larger molecular weights have higher immunogenicity. The human body is prone to producing anti-uric acid enzyme antibodies, which quickly weaken its effect and at the same time cause serious allergic reactions, making it unusable for long-term treatment. It has been proven that covalent modification of proteins with PEG can reduce the immunogenicity of proteins, increase the solubility of proteins, and extend the half-life of proteins.

[0011] Duke University and Savient conducted research on chimeric uric acid enzymes derived from pigs and baboons (Michael H, Susan JK2006.US7056713B1). The method of this research was to modify the ε-amino group of the lysine residue of uric acid enzyme derived from pigs with methoxy group-containing polyethylene glycol (10KDa-mPEG-NPC) with a molecular weight of 10KDa (the resulting modified product is Pegloticase), while ensuring that the enzyme activity is not significantly reduced, and thus the goal of treating stubborn gout in the human body was initially achieved. The inventor found that the above research results did not completely solve the problem of drug immunogenicity, and found that the clinical subjects showed a phenomenon in which the therapeutic effect of uric acid enzyme disappeared after repeated injections, and speculated that this may be related to the molecular weight of pegloticase protein being too large (the molecular weight of pegloticase is 540 kDa), and at the same time, pegloticase is not suitable for injection, but is suitable for intravenous injection, which reduces the compliance of subjects with long-term use and severely limits its clinical application. Until now, there has been no long-life uric acid oxidase drug with lower immunogenicity or that can be used for subcutaneous injection.

[0012] The present invention aims to solve, at least to a certain extent, one of the technical problems in the related art. [Means for solving the problem]

[0013] In a first aspect of the present invention, a method for producing a polyethylene glycol-modified urate oxidase is provided. According to an embodiment of the present invention, the polyethylene glycol-modified urate oxidase has an amino acid site selected from the group consisting of 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 , K222 , K 231 , K 266 , K 272 , K 285 , K 291 , K 293 At least 11 of the above-mentioned compounds have PEG modification, and the method includes a step of coupling reaction between urate oxidase and polyethylene glycol, the polyethylene glycol being provided in the form of an acidic solution, and the molar ratio of the urate oxidase to the polyethylene glycol is 1:(56-94), so as to obtain a polyethylene glycol-modified urate oxidase. The polyethylene glycol-modified urate oxidase produced by the method according to the embodiment of the present invention has its modification site 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 At least 11 sites of the urate oxidase are PEG-modified. Note that the term "urate oxidase" in the present application should be understood in a broad sense, and is a general term for a mixture of urate oxidases produced in the same batch in actual production practice. The polyethylene glycol-modified urate oxidase obtained by the method according to the embodiment of the present invention can greatly improve the stability of urate oxidase in the body and reduce immunogenicity while ensuring the maximum enzyme activity, and the internal efficacy after intramuscular injection can reach the internal efficacy after intravenous injection of the original drug.

[0014] According to an embodiment of the present invention, the above 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.The inventors have found that dissolving PEG in an acidic solution can prevent the hydrolysis of active groups before the polyethylene glycol reacts with protein, and can effectively ensure the effective activation efficiency of polyethylene glycol, while solving the defects that occur when the solid polyethylene glycol is directly dissolved in the coupling reaction buffer solution, such as the local concentration is too high, the dissolution is not uniform, the contact between polyethylene glycol and the coupling protein is not uniform, and bubbles are generated to interfere with the coupling reaction, and the coupling reaction efficiency of urate oxidase and polyethylene glycol can be effectively improved.

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

[0017] According to an embodiment of the present invention, the organic acid is The acid may be 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, lauryl sulfate, ethanesulfonic acid, formic acid, transbutenedioic acid, glucoheptonic acid, glyceryl phosphate, gluconic acid, heptanoic acid, caproic acid, 2-hydroxyethanesulfonic acid, lacturonic acid, lactic acid, lauric acid, lauryl sulfate, malic acid, malonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, nicotinic acid, oleic acid, palmitic acid, pectic acid, 3-phenylpropionate, picrate, pivalic acid, propionic acid, stearic acid, paratoluenesulfonic acid, undecanoic acid, and valeric acid. According to an embodiment of the present invention, the inorganic acid is The acid is selected from hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, perchloric acid, hydroiodic acid, nitric acid, persulfuric acid, boric acid, dichromic acid, silicic acid, chromic acid, and thiocyanic acid.

[0018] According to an embodiment of the present invention, the concentration of hydrogen ions in the acidic solution is 1-5 mmol / L.

[0019] According to an embodiment of the present invention, the acid solution includes hydrochloric acid or sulfuric acid or glacial acetic acid.

[0020] According to an embodiment of the present invention, the concentration of the acid in the acidic solution is 1-5 mmol / L.

[0021] According to an embodiment of the present invention, the molar ratio of urate oxidase to polyethylene glycol is 1:(45-110).

[0022] According to an embodiment of the present invention, the molar ratio of urate oxidase to polyethylene glycol is 1:(56-94).

[0023] According to an embodiment of the present invention, the concentration of the polyethylene glycol in the acidic solution is 100-300 mmol / L. The inventors have found that the concentration of the polyethylene glycol in the acid is within the above range, the viscosity of the reaction solution is appropriate, and it is favorable for improving the reaction efficiency and expanding industrial production.

[0024] According to the embodiment of the present invention, the molecular weight of said polyethylene glycol is less than 6KD. The inventors have found that by coupling polyethylene glycol with molecular weight less than 6KD with urate oxidase, the life span of the obtained polyethylene glycol-modified urate oxidase in the body is further improved, and anti-uric acid enzyme antibody is not produced, and anti-PEG antibody is hardly produced, that is, immunogenicity is further reduced.

[0025] According to an embodiment of the present invention, the polyethylene glycol has a monomethoxy or hydroxyl group.

[0026] According to an embodiment of the present invention, the polyethylene glycol is a linear or branched structure.

[0027] According to an embodiment of the present invention, the polyethylene glycol and the urate oxidase are coupled via an amide bond.

[0028] According to an embodiment of the present invention, the polyethylene glycol is a modified polyethylene glycol, and the modifying group of the modified polyethylene glycol is: It is selected from N-hydroxysuccinimide, N-hydroxysuccinimide carbonate, N-hydroxysuccinimide acetate, N-hydroxysuccinimide propionate, N-hydroxysuccinimide butyrate, N-hydroxysuccinimide succinate and p-nitrophenyl carbonate.

[0029] According to an embodiment of the present invention, the modifying group of the modifying polyethylene glycol is N-hydroxysuccinimide.

[0030] According to an embodiment of the present invention, the coupling reaction is carried out in a carbonate buffer solution.

[0031] According to an embodiment of the present invention, the pH of the carbonate buffer solution is 9 to 11. The inventors have found that if the buffer pH is below 9.0, it will seriously affect the solubility of uric acid enzyme, making it impossible to carry out the next modification reaction, and if the buffer pH is above 11, it will seriously affect the activity of uric acid enzyme, and at the same time, it will also reduce the coupling efficiency between polyethylene glycol and uric acid oxidase.

[0032] According to an embodiment of the present invention, the concentration of said urate oxidase in the coupling reaction system is 10mg / ml.The inventors found that the concentration of urate oxidase in the coupling reaction system is within the above range, the viscosity of the reaction solution is appropriate, which is favorable for improving the reaction efficiency and expanding industrial production.At the same time, the inventors found that the protein concentration of urate oxidase affects the average modification degree of urate oxidase, and under the condition that the obtained urate oxidase has the same average modification degree of PEG, 10mg / ml urate oxidase requires a lower PEG input ratio, which can save production costs.

[0033] According to an embodiment of the present invention, the coupling reaction is carried out at 5 to 30° C. for at least 60 minutes. By carrying out the coupling reaction at the above temperature condition for at least 60 minutes, the T 1 , K 3 , K 4 , K 30 , K 35 , K 76 , K 79 , K 97 , K 112 , K 116 , K 120 , K 152 , K 179 , K 222 , K 231 , K 266 , K 272 , K 285 , K 291 , K 293 It is effectively achieved that at least 11 of the sites are PEG-modified.

[0034] According to an embodiment of the present invention, the method further includes ultrafiltration and / or purification of the coupling reaction product, which can effectively remove unmodified polyethylene glycol and by-products such as NHS, and effectively improve the purity of the obtained polyethylene glycol-modified urate oxidase.

[0035] According to an embodiment of the present invention, K 30 , K 35 , K 222 and K 231 At least one of the amino acid sites of has a PEG modification.

[0036] According to an embodiment of the invention, the amino acid site is localized to the amino acid sequence shown in SEQ ID NO:1. TYKKNDEVEFVRTGYGKDMIKVLHIQRDGKYHSIKEVATTVQLTLSSKKDYLHGDNSDUVIPTDTIKNTVNVLAKFKGIKSIETFAVTICEHFLSSFKHVIRAQVYVEEVPWKRFEKNGVKHVHAFIYTPTGTHFCEVEQIRNGPPVIHSGI KDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATQVYCKWRYHQGRDVDFEATWDTVRSIVLQKFAGPYDKGEYSPSVQKTLYDIQVLTLGQVPEIEDMEISLPNIHYLNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRL(SEQ ID NO:1).

[0037] According to an embodiment of the present invention, the urate oxidase has an amino acid sequence shown in SEQ ID NO:1-7. MAHYRNDYKKNDEVEFVRTGYGKDMIKVLHIQRDGKYHSIKEVATSVQLTLSSKKDYLHGDNSDIVIPTDTIKNTVNVLAKFKGIKSIETFAVTICEHFLSSFKHVIRAQVYVEEVPWKRFEKNGVKHVHAFIYTPTGTHFCEVEQIRNGPPVIH SGIKDLKVLKTTQSGFEGFIKDQFTTLEVKDRCFATQVYCKWRYHQGRDVDFEATWDTVRSIVLQKFAGPYDKGEYSPSVQKTLYDIQVLTLGQVPEIEDMEISLPNIHYLNIDMSKMGLINKEEVLLPLDNPYGRITGTVKRKLTSRL(SEQ ID NO:2). MYKNDEVEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSVQLTLSSKKDYVYGDNSDIIPTDTIKNTVHVLAKFKGIKSIETFAMNICEHFLSSFNHVIRAQVYVEEVPWKRFEKNGVKHVHAFIHNPTGTHFCEVEQMRSGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATKVYCKWRYHQGRDVDFEATWDTVRDIVLEKFAGPYDKGEYSPSVQKTLYDIQVHSLSRVPEMEDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRL(SEQ ID NO:3)。 MAHYHNDYKKNDEVEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSVQLTLSSKKDYVYGDNSDIIPTDTIKNTVHVLAKFKGIKSIETFAMNICEHFLSSFNHVIRAQVYVEEVPWKRFEKNGVKHVHAFIHNPTGTHFCEVEQMRSGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATKVYCKWRYHQGRDVDFEATWDTVRDIVLEKFAGPYDKGEYSPSVQKTLYDIQVHSLSRVPEMEDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGRITGTAKRKLASKL(SEQ ID NO:4)。 MAHYHNDYQKNDEVEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSVQLTLNSRREYLHGDNSDIIPTDTIKNTVQVLAKFKGIKSIETFAMNICEHFLSSFNHVIRVQVYVEEVPWKRFEKNGVKHVHAFIHTPTGTHFCEVEQLRSGPPVIHSGIKDLKVLKTTQSGFEGFLKDQFTTLPEVKDRCFATQVYCKWRYHQGRDVDFEATWEAVRGIVLKKFAGPYDKGEYSPSVQKTLYDIQVLSLSQLPEIEDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGRITGTVKRKLTSRL(SEQ ID NO:5)。 MAHYHNDYKKNDEVEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSVQLTLSSKKDYLHGDNSDIIPTDTIKNTVHALAKFKGIKSIEAFAVNICQHFLSSFNHVIRTQVYVEEIPWKRLEKNGVKHVHAFIHTPTGTHFCEVEQLRSGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFAAQVYCKWRYHQCRDVDFEATWDTIRDVVLEKFAGPYDKGEYSPSVQKTLYDIQVVSLSQVPEIDDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRL (SEQ ID NO:6). MADYHNNYKKNDELEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSVQLTLSSKKDYLHGDNSDIIPTDTIKNTVHVLAKFKGIKSIEAFGVNICEYFLSSFNHVIRAQVYVEEIPWKRLEKNGVKHVHAFIHTPTGTHFCEVEQLRSGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATQVYCKWRYHQCRDVDFEATWGTIRDLVLEKFAGPYDKGEYSPSVQKTLYDIQVLSLSRVPEIEDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRL (SEQ ID NO:7).

[0038] The amino acid sequence represented by SEQ ID NO:1 is the amino acid sequence of a chimeric urate enzyme derived from pig and baboon (pig-baboon), the amino acid sequence represented by SEQ ID NO:2 is the amino acid sequence of a urate oxidase derived from pig, the amino acid sequence represented by SEQ ID NO:3 is the amino acid sequence of a chimeric urate oxidase derived from dog and baboon (dog-baboon), the amino acid sequence represented by SEQ ID NO:4 is the amino acid sequence of a urate oxidase derived from dog, the amino acid sequence represented by SEQ ID NO:5 is the amino acid sequence of a urate oxidase derived from cow, the amino acid sequence represented by SEQ ID NO:6 is the amino acid sequence of a monkey urate oxidase, and the amino acid sequence represented by SEQ ID NO:7 is the amino acid sequence of a urate oxidase from baboon.

[0039] In addition, the localization of lysine in the present application is performed based on the amino acid sequence shown in SEQ ID NO:1. 4 is a lysine located at position 4 based on the amino acid sequence shown in SEQ ID NO: 1. A uric acid enzyme having an amino acid sequence shown in SEQ ID NO: 1-7 or a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99% identity compared to SEQ ID NO: 1-7, or a polypeptide having one or more amino acid substitutions, deletions and / or additions compared to SEQ ID NO: 1-7, is structurally homologous, and a person skilled in the art can easily determine by sequence comparison that a uric acid enzyme having an amino acid sequence shown in SEQ ID NO: 2 .... 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 corresponding site corresponding to the site can be determined, and further, the above polypeptide undergoes PEG modification at the corresponding site in comparison, thereby realizing the advantages of the polyethylene glycol-modified urate oxidase of the present application, such as low immunogenicity, high stability in the body, and suitability for intramuscular injection.

[0040] For example, according to an embodiment of the present invention, the sequence shown in SEQ ID NO:2 and the sequence shown in 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 site's compatible sites are 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 299The corresponding sites of the sequence shown in SEQ ID NO:3 and the sequence shown in SEQ ID NO:1 are 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 The corresponding sites of the sequence shown in SEQ ID NO:4 and the sequence shown in SEQ ID NO:1 are 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 The corresponding sites of the sequence shown in SEQ ID NO:5 and the sequence shown in SEQ ID NO:1 are 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 299The corresponding sites of the sequence shown in SEQ ID NO:6 and the sequence shown in SEQ ID NO:1 are 1 , K 9 , K 10 , K 36 , K 41 , K 82 , K 85 , K 118 , K 122 , K 126 , K 158 , K 185 , K 228 , K 237 , K 272 , K 278 , K 291 , K 297 , K 299 The corresponding sites of the sequence shown in SEQ ID NO:7 and the sequence shown in SEQ ID NO:1 are 1 , K 9 , K 10 , K 36 , K 41 , K 82 , K 85 , K 118 , K 122 , K 126 , K 158 , K 185 , K 228 , K 237 , K 272 , K 278 , K 291 , K 297 , K 299 The inventors have found through testing that after PEG modification is performed on at least 11 sites corresponding to the amino acid sequences shown in SEQ ID NOs: 2 to 7, the resulting PEG-modified urate oxidase has the advantages of low immunogenicity, high stability in vivo, and suitable for intramuscular injection.

[0041] According to an embodiment of the present invention, the peptide map of the polyethylene glycol-modified urate oxidase is reduced by 75% or more, preferably 80% or more, more preferably 90% or more in peak area having at least 11 predetermined peptide segments compared with the peptide map of the urate oxidase not modified with polyethylene glycol. The polyethylene glycol-modified urate oxidase according to the embodiment of the present invention has the advantages of low immunogenicity, high stability in the body, and suitable for intramuscular injection.

[0042] According to an embodiment of the present invention, the peptide map of the polyethylene glycol-modified urate oxidase has the peak area-reducing peptide segments shown in Table 8.

[0043] According to an embodiment of the present invention, the peptide map of the polyethylene glycol-modified urate oxidase is shown in FIG. 6 or FIG.

[0044] In a second aspect of the present invention, the present invention provides a method for reducing the immunogenicity of urate oxidase. According to an embodiment of the present invention, the T of the polyethylene glycol-modified urate oxidase is 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 amino acid sites are PEG-modified, the method includes a step of coupling urate oxidase with polyethylene glycol, the polyethylene glycol is provided in the form of an acidic solution, and the molar ratio of the urate oxidase to the polyethylene glycol is 1:(56-94). According to the method of the embodiment of the present invention, the immunogenicity of urate oxidase can be effectively reduced, and the obtained urate oxidase has higher safety in the body and longer-lasting action.

[0045] It is to be understood that the additional technical features of the method for producing polyethylene glycol-modified urate oxidase and the technical effects of the additional technical features are applicable to the additional technical features of the method for reducing the immunogenicity of urate oxidase according to the embodiment of the present invention, and the additional technical features of the method for reducing the immunogenicity of urate oxidase according to the embodiment of the present invention will not be described repeatedly here.

[0046] In a third aspect of the present invention, the present invention provides a polyethylene glycol-modified urate oxidase. According to an embodiment of the present invention, the urate oxidase is obtained by the above-mentioned method. The polyethylene glycol-modified urate oxidase according to the embodiment of the present invention can greatly improve the stability of urate oxidase in the body and reduce immunogenicity while maximizing the enzyme activity, and the internal efficacy after intramuscular injection is equivalent to that after intravenous injection of a similar drug already on the market.

[0047] In a fourth aspect of the present invention, the present invention provides a pharmaceutical composition. According to an embodiment of the present invention, the pharmaceutical composition comprises the above-mentioned urate oxidase. The pharmaceutical composition according to the embodiment of the present invention has the advantages of low immunogenicity, high stability in the body, and suitable for intramuscular injection, and can be used for the treatment and prevention of hyperuric acid-related diseases.

[0048] According to an embodiment of the present invention, the pharmaceutical composition further comprises at least one of the following additional technical features:

[0049] According to an embodiment of the present invention, the pharmaceutical composition further comprises a pharma- ceutically acceptable adjuvant.

[0050] According to an embodiment of the present invention, the pharmaceutical composition further comprises other drugs for treating or preventing hyperuric acid-related diseases.

[0051] In a fifth aspect of the present invention, the present invention provides a use of the above urate oxidase or the above drug in the manufacture of a composition for treating hyperuric acid-related diseases and reducing the uric acid level in a biological fluid of a subject. The urate oxidase according to the embodiment of the present invention has the advantages of low immunogenicity, high stability in the body, and suitable for intramuscular injection, and has a significant advantage in treating hyperuric acid-related diseases.

[0052] According to an embodiment of the present invention, the above use may further include at least one of the following additional technical features:

[0053] According to an embodiment of the present invention, the hyperuric acid related disease includes a disease selected from chronic hyperuricemia, gout, kidney disease, hyperuricemia arthritis, kidney stones, gouty tophi, hypertension, diabetes, hypertriglyceridemia, metabolic syndrome, and coronary heart disease.

[0054] According to an embodiment of the present invention, the biological fluid is urine or blood. [Brief description of the drawings]

[0055] [Figure 1] FIG. 2 is a SEC-HPLC-UV detection diagram of a PHC physicochemical control sample according to an embodiment of the present invention. [Diagram 2] FIG. 2 is a SEC-HPLC-RI detection diagram of a PHC physicochemical control sample according to an embodiment of the present invention. [Diagram 3] FIG. 2 is a PEG reference sample-SEC-HPLC-RI detection diagram according to an embodiment of the present invention. [Figure 4] FIG. 2 shows SEC-HPLC-UV detection of PU5 modified products according to an embodiment of the present invention. [Diagram 5]FIG. 2 shows SEC-HPLC-RI detection of PU5 modified products according to an embodiment of the present invention. [Figure 6] FIG. 1 is a comparative diagram of double enzyme cleavage of PHC and PU5 according to an embodiment of the present invention using Lys-c and trypsin, respectively. [Figure 7] FIG. 2 is a diagram showing PU5 Lyc enzymatic cleavage according to an embodiment of the present invention. [Figure 8] 1 shows serum uric acid levels after intramuscular administration of different drug doses to model rats according to an embodiment of the present invention. [Figure 9] FIG. 2 is a kidney injury necrosis and inflammation score diagram according to an embodiment of the present invention. [Figure 10] FIG. 1 shows the average blood concentration-time curves for each group after a single intravenous injection of equal amounts (1.0 mg / kg) of Pegloticase and polyethylene glycolated uric acid enzyme injection solution into SD rats according to an embodiment of the present invention. [Figure 11] FIG. 2 is a graph showing the average blood concentration-time curves of each group after a single intramuscular injection of Pegloticase and different doses of polyethylene glycolated uric acid enzyme injection solution in SD rats according to an embodiment of the present invention. [Figure 12] FIG. 2 is a graph showing the average blood concentration-time curves of each group of SD rats after a single intramuscular injection of different doses of polyethylene glycol-modified uric acid enzyme injection solution according to an embodiment of the present invention. [Figure 13] FIG. 2 is a graph showing the average blood uric acid value-time curves at different times for each group after a single intramuscular / intravenous injection of different doses of Pegloticase and polyethylene glycolated uric acid enzyme injection solution according to an embodiment of the present invention into SD rats. [Figure 14] This is a graph showing the average blood concentration-time curves in males and females after the initial (Day 1) intravenous injection of the same amount (1.0 mg / kg) of Pegloticase and polyethylene glycolated uric acid enzyme injection solution into SD rats according to an embodiment of the present invention. [Figure 15] This is a graph showing the average blood concentration-time curves in males and females after the final (Day 22) intravenous injection of the same amount (1.0 mg / kg) of Pegloticase and polyethylene glycolated uric acid enzyme injection solution into SD rats according to an embodiment of the present invention. [Figure 16] This is a graph showing the average blood concentration-time curves in males and females after the first (Day 1) intramuscular injection of the same amount (1.0 mg / kg) of Pegloticase and polyethylene glycolated uric acid enzyme injection solution into SD rats according to an embodiment of the present invention. [Figure 17] This is a graph showing the average blood concentration-time curves in males and females after the final (Day 22) intravenous injection of the same amount (1.0 mg / kg) of Pegloticase and polyethylene glycolated uric acid enzyme injection solution into SD rats according to an embodiment of the present invention. [Figure 18] FIG. 2 is a graph showing the average blood uric acid value vs. time curves at different times after multiple intravenous injections of Pegloticase and polyethylene glycolated uric acid enzyme injections into SD rats according to an embodiment of the present invention. [Figure 19] FIG. 2 is a graph showing the average blood uric acid value-time curve at different times after multiple intramuscular injections of Pegloticase and polyethylene glycolated uric acid enzyme injection solution into SD rats according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0056] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, the embodiments of the present invention will be described in detail, and examples of the embodiments are shown in the drawings. The embodiments described below with reference to the drawings are illustrative and are intended to interpret the present invention, but should not be understood as limiting the present invention.

[0057] An object of the present invention is to provide a method for producing polyethylene glycol-modified urate oxidase.

[0058] Another object of the present invention is to provide a novel polyethylene glycol-modified urate oxidase.

[0059] Another object of the present invention is to provide a method for effectively reducing the immunogenicity of urate oxidase, which can effectively reduce the immunogenicity of urate oxidase and improve the safety and stability of urate oxidase in vivo.

[0060] Another object of the present invention is to provide an application of the polyethylene glycol urate oxidase coupling product obtained above, which can achieve a long-term and significant effect of lowering blood uric acid level in the body, and can be used for the treatment of hyperuricemia and gout.

[0061] As used herein, the terms "urate oxidase" and "uric acid enzyme" can be used interchangeably and refer to a type of enzyme that can catalyze and oxidize uric acid according to the present invention to produce allantoin and hydrogen peroxide.The terms "uric acid oxidase analog", "uric acid enzyme analog", and "uric acid enzyme derivative" can be used interchangeably, and all of them can carry out structural modifications such as substitution, deletion or addition of a part of amino acid in the protein structural sequence of uric acid oxidase while maintaining the activity of uric acid oxidase that specifically catalyzes and converts uric acid into allantoin and hydrogen peroxide, and further realize the following, including but not limited to the reduction of immunogenicity in this example, the improvement of protein stability, and the further promotion of polyethylene glycol modification.

[0062] The urate oxidase is not particularly limited and may be urate oxidase of any origin and its analogues, representative examples of which include, but are not limited to, those derived from mammals, microorganisms, plants, etc.

[0063] In another preferred embodiment, the urate oxidase and its analogues are derived from mammals, and preferably have the amino acid sequence shown in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, more preferably SEQ ID NO:1.

[0064] The heterologous urate oxidase according to the present invention can be obtained by various methods including, but not limited to, natural extraction, chemical synthesis, and recombinant expression by genetic engineering.

[0065] In another preferred embodiment, the urate oxidase is recombinantly expressed in a host cell by recombinant techniques using a coding sequence for the urate oxidase protein sequence (SEQ ID NO:1).

[0066] In another preferred example, the gene is produced by a method for constructing a recombinant expression strain using E. coli or yeast as a host, and it is more preferred to carry out recombinant expression using E. coli as a host.

[0067] As used herein, the polyethylene glycol urate oxidase according to the present invention is obtained by covalently modifying urate oxidase with polyethylene glycol. The polyethylene glycol (PEG) is a mixture of a polycondensate of ethylene oxide and water, and has the general formula H(OCH 2 CH 2 )nOH, it is a pH-neutral, non-toxic, highly water-soluble hydrophilic polymer, and can have a linear or branched structure. Due to its non-toxicity and good biocompatibility, FDA has approved the marketing of many kinds of PEG-modified recombinant protein drugs, proving that PEG can be used to reduce protein immunogenicity, increase protein solubility, and extend protein half-life. For PEG to bind to protein, one or both ends of PEG can be activated, and the corresponding modification groups, such as amino, mercapto, carboxyl, or hydroxyl groups, can be selected and activated according to the target protein to be modified.

[0068] In another preferred embodiment, the site used for PEG modification of the urate oxidase and urate enzyme analogs of the present invention is the ε-amino group of the lysine residue, but there is also a small amount of modification of the α-amino group of the N-terminal lysine residue. Urate oxidase is preferably covalently bonded to the modifying group of PEG via a urethane bond, a secondary ammonium bond or an amide bond, and the polyethylene glycol molecule is coupled with the urate oxidase to form an amide bond, and the modifying group of the polyethylene glycol includes, but is not limited to, N-hydroxysuccinimide based, including, but not limited to, N-hydroxysuccinimide (NHS), N-hydroxysuccinimide carbonate (SC), N-hydroxysuccinimide acetate (SCM), N-hydroxysuccinimide propionate (SPA), N-hydroxysuccinimide butyrate (SBA), N-hydroxysuccinimide succinate (SS) and the like, and the blocking group of the polyethylene glycol includes, but is not limited to, monomethoxy, ethoxy, glucose or galactose, and is preferably monomethoxy.

[0069] In another preferred embodiment, the polyethylene glycol is linear or straight chain.

[0070] In another preferred embodiment, the relative molecular weight of polyethylene glycol used in polyethylene glycol urate oxidase is 6 KD or less, preferably 1 KD to 5 KD, and most preferably 5 KD. Note that the "relative molecular weight of polyethylene glycol" described in the present application refers to the relative molecular weight of polyethylene glycol having no modifying group and has the general meaning in the art, and the total relative molecular weight after PEG is activated by an activating group is slightly larger than 5 KD, for example, within the range of 5 KD + 10%.

[0071] In another preferred embodiment, the polyethylene glycol-modified urate oxidase has the following characteristics: (1) At least eleven of the following amino acid sites in urate oxidase are PEG-modified: T 1 , K3 , K 4 , K 30 , K 35 , K 76 , K 79 , K 97 , K 112 , K 116 , K 120 , K 152 , K 179 , K 222 , K 231 , K 266 , K 272 , K 285 , K 291 , K 293 . (2) One urate oxidase molecule is coupled to an average of 11 to 13 polyethylene glycol molecules. (3) K located in Seg ID NO:1 urate oxidase sequence 30 and / or K. 35 Including K 222 and / or K. 231 generates a polyethylene glycol coupling modification. (4) Polyethylene glycol urate oxidase has lower immunogenicity in the body.

[0072] In another aspect of the present invention, a method for effectively reducing the immunogenicity of urate oxidase is provided, which can effectively reduce the immunogenicity of urate oxidase and improve the stability of urate oxidase in vivo.

[0073] As used herein, the polyethylene glycol-modified urate oxidase is characterized in that the urate oxidase is not particularly limited and may be urate oxidase of any origin and its urate oxidase analogues, representative examples of which include, but are not limited to, those derived from mammals, microorganisms, plants, etc.

[0074] In another preferred embodiment, the urate oxidase and its analogs are of mammalian origin, preferably having the amino acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, more preferably SEQ ID NO:1.

[0075] The heterologous urate oxidase according to the present invention can be obtained by various methods including, but not limited to, natural extraction, chemical synthesis, and recombinant expression by genetic engineering.

[0076] In another preferred example, the gene is produced by a method for constructing a recombinant expression strain using E. coli or yeast as a host, and it is more preferred to carry out recombinant expression using E. coli as a host.

[0077] The recombinant expression of urate oxidase in E. coli according to the present invention can obtain a large amount of urate oxidase, and the expressed urate oxidase can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, high purity urate oxidase can be obtained by methods well known to those skilled in the art. These methods include, for example, but are not limited to, centrifugation, lysis, salting out, ultrafiltration, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, and a combination of various other techniques.

[0078] The urate oxidase obtained above is covalently bound to polyethylene glycol via a linking group using methods known in the art.

[0079] In another preferred embodiment, polyethylene glycol orients and modifies lysine residues on the surface of the spatial structure of urate oxidase. Urate oxidase is covalently bound to a modifying group (also called an active group) of PEG via an amide bond, and the modifying group (also called an active group) of polyethylene glycol includes, but is not limited to, N-hydroxysuccinimide (NHS), N-hydroxysuccinimide carbonate (SC), N-hydroxysuccinimide acetate (SCM), N-hydroxysuccinimide propionate (SPA), N-hydroxysuccinimide butyrate (SBA), and N-hydroxysuccinimide succinate (SS), where the blocking group of polyethylene glycol includes, but is not limited to, monomethoxy, ethoxy, glucose, or galactose, preferably monomethoxy.

[0080] In other preferred embodiments, the polyethylene glycol may be linear or branched.

[0081] In another preferred embodiment, the relative molecular weight of the polyethylene glycol is 6 KD or less, preferably 1 KD to 5 KD, more preferably 2 KD or 5 KD, and most preferably 5 KD.

[0082] In another preferred embodiment, the present invention provides a method for producing a polyethylene glycol-modified urate oxidase, the method having one or more of the following characteristics: (1) The molar ratio of urate oxidase to polyethylene glycol used for modification is 1:45 to 1:150 (urate oxidase:polyethylene glycol), preferably 1:45 to 1:110, more preferably 1:56 to 1:94. (2) The coupling reaction system is a carbonate buffer solution, and its modified pH range is 9-11. (3) The urate oxidase protein concentration in the coupling reaction system was 10 mg / ml.

[0083] The method for producing the polyethylene glycol-modified urate oxidase described above uses a plurality of purification means to obtain a highly pure polyethylene glycol-modified urate oxidase.

[0084] In another preferred embodiment, the modified sample is purified by methods including, but not limited to, molecular sieve chromatography, ion exchange chromatography, hydrophobic chromatography, tangential flow ultrafiltration, or a combination thereof, more preferably molecular sieve chromatography, tangential flow ultrafiltration.

[0085] In another aspect of the present invention, the above polyethylene glycol-modified urate oxidase and its application are provided, which can achieve a long-term and significant effect of lowering blood uric acid level in the body, and can be used for the treatment of hyperuricemia and gout.

[0086] Said polyethylene glycol urate oxidase is more suitable as a drug and composition for treating chronic hyperuricemia or gout, the main symptoms of which include, but are not limited to, uric acid nephropathy and gouty arthritis.

[0087] The administration route of the polyethylene glycol urate oxidase includes, but is not limited to, intravenous injection, subcutaneous injection, intramuscular injection, and intraperitoneal injection, and is preferably intravenous injection or intramuscular injection, and more preferably intramuscular injection.

[0088] Said polyethylene glycol urate oxidase has lower immunogenicity in the body.

[0089] The above-mentioned polyethylene glycol urate oxidase has low immunogenicity, which means that after the polyethylene glycol urate oxidase is injected intramuscularly into the human or animal body, the body does not produce antibodies against the polyethylene glycol molecule, or produces a low level of anti-polyethylene glycol molecule antibodies, and does not produce antibodies against urate oxidase.

[0090] Said polyethylene glycol urate oxidase has a longer half-life in the body after intramuscular injection and the effect of lowering the uric acid level in the body.

[0091] According to some embodiments of the present invention, the pharmaceutical composition comprising the polyethylene glycol-modified urate oxidase of the present invention may include a pharmacopoeia acceptable carrier, and the dosage form and administration method of the pharmaceutical composition are not particularly limited. In the case of an injection preparation, the pharmacopoeia acceptable carrier may include a buffer, a preservative, an analgesic, a solubilizer, an isotonic agent, and a stabilizer. In the case of a topical preparation, the pharmacopoeia acceptable carrier may include an alkali, an excipient, a lubricant, and a preservative. The pharmaceutical composition of the present invention may be combined with the above pharmacopoeia acceptable carrier to prepare various dosage forms.

[0092] Thus, according to some embodiments of the present invention, excipients and diluents among the carriers suitable for drug formulations can include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltose alcohol, starch, gum arabic, alginate, gel, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, hydroxyphenylpropyl, talc, magnesium stearate, and mineral oil.

[0093] According to some 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 adjusting agent, a moisturizer, a fragrance, and a preservative.

[0094] According to the embodiment of the present invention, the polyethylene glycol-modified urate oxidase and pharmaceutical composition of the present invention can greatly improve the stability of urate oxidase in the body, reduce immunogenicity, and achieve the same efficacy after intramuscular injection as that after intravenous injection of the original drug.Therefore, the polyethylene glycol-modified urate oxidase of the present invention and pharmaceutical composition containing said polyethylene glycol-modified urate oxidase can be administered in the treatment or prevention of hyperuric acid-related diseases.

[0095] The term "administration" as used herein means that a predetermined amount of a substance is introduced into a patient in a suitable manner. The polyethylene glycol-modified urate oxidase of the present invention can be administered in any conventional manner as long as it can reach the desired tissue. Various modes of administration can be envisaged, including peritoneal, intravenous, muscular, subcutaneous, cortical, oral, topical, nasal, pulmonary and rectal, but the present invention is not limited to these exemplary modes of administration. However, in the case of oral administration, the active ingredient of the orally administered composition should be coated or prepared to prevent degradation in the stomach. Preferably, the composition of the present invention can be administered in an injection formulation. In addition, the pharmaceutical composition of the present invention can be administered using a specific device that delivers the active ingredient to the target cell.

[0096] The frequency and dosage of administration of the pharmaceutical composition of the present invention can be determined depending on several relevant factors including the type of disease to be treated, the administration route, the age, sex, weight and severity of the disease of the patient, and the type of drug as the active ingredient.

[0097] The term "therapeutically effective amount" refers to an amount of a compound sufficient to significantly improve some symptoms associated with a disease or condition, i.e., an amount that provides a therapeutic effect for a particular condition and means of administration. For example, in chronic hyperuricemia or gout treatment, a drug or compound must be therapeutically effective to reduce, prevent, delay, inhibit or inhibit any symptoms of the disease or condition. A therapeutically effective amount of a drug or compound need not cure the disease or condition, but may provide treatment for the disease or condition, such that an attack of the disease or condition in an individual is delayed, inhibited or prevented, the symptoms of the disease or condition are alleviated, the duration of the disease or condition is altered, e.g., the disease or condition is less severe or recovery is accelerated.

[0098] The term "treatment" means to obtain a desired pharmacological and / or physiological effect. The effect may be prophylactic, in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic, in terms of partially or completely curing a disease and / or adverse effects of the disease. As used herein, "treatment" covers the treatment of a mammalian, particularly a human, disease (mainly referring to hyperuric acid-related diseases), to (a) prevent the disease in an individual susceptible to the disease but not yet diagnosed with the disease, (b) inhibit the disease, e.g., inhibit the development of the disease, or (c) ameliorate the disease, e.g., reduce symptoms associated with the disease. As used herein, "treatment" covers any administration of a drug or compound to an individual to treat, cure, alleviate, ameliorate, reduce or inhibit a disease in an individual, including, but not limited to, administration of a polyethylene glycol-modified urate oxidase as described herein to an individual in need thereof.

[0099] According to an embodiment of the present invention, the polyethylene glycol-modified urate oxidase or pharmaceutical composition of the present invention may be used in combination with conventional treatment methods and / or therapies, or may be used separately from conventional treatment methods and / or therapies. When the polyethylene glycol-modified urate oxidase or pharmaceutical composition of the present invention is administered in a combination therapy with other drugs, they may be administered to an individual sequentially or simultaneously. Alternatively, the pharmaceutical composition of the present invention may include a combination of the polyethylene glycol-modified urate oxidase of the present invention, a pharma- ceutically acceptable carrier or a pharma-ceutically acceptable excipient, and other therapeutic or prophylactic agents known in the art.

[0100] The term "average degree of modification" refers to the number of PEGs bound to each uric acid enzyme unit.

[0101] In this specification, unless otherwise specified, the expression "having PEG modification at an amino acid site" means that in the three-dimensional structure of the corresponding polypeptide, the amino acid site is covered by a PEG molecule, and at least some groups at the amino acid site are not exposed. It can be understood that a person skilled in the art can determine whether a specific amino acid site is modified by a PEG molecule by ordinary technical means, for example, by referring to the exemplary method in the part of Example 3 "Detection of polyethylene glycol modified site" of the present application. Briefly, the method includes the steps of: 1) enzymatically cleaving non-polyethylene glycolated and polyethylene glycolated urate oxidase with one or more enzymes, for example, monoenzymatic cleavage with Lys-C or Trypsin, or bienzymatic cleavage with Lys-C and Trypsin; 2) separating the enzymatic cleavage fragments by high performance liquid chromatography to generate chromatograms, i.e., peptide maps, of non-polyethylene glycolated and polyethylene glycolated urate oxidase; and 3) comparing the differences between the peptide maps of non-polyethylene glycolated and polyethylene glycolated urate oxidase, and determining the relative percentage of the reduction or disappearance of the peak of the peptide segment in which a specific amino acid site exists in the polyethylene glycolated urate oxidase with reference to a predetermined internal standard peptide segment, and further determining whether the specific amino acid site on the peptide segment is modified by PEG. Specifically, in Example 3 of the present application, the relative percentage of the reduction or disappearance of the peak area of ​​the peptide segment in which a specific amino acid site exists can be calculated by the following formula: P(%)=(A 2 - A 1 ) / A 2 ×100%, However, A 1 =A 0 ×t. A 0 is the measured peak area of ​​the peptide segment in which a specific amino acid site of the modified protein to be measured is present, t is the average peak area ratio of the internal reference peptide segment in the PHC peptide map and the peptide map of the modified protein to be measured, P(%) is the relative percentage of the decrease or disappearance of the peak area of ​​the peptide segment in which a particular amino acid site exists, and A 2 is the peak area of ​​a peptide segment in the PHC peptide map where a specific amino acid site exists, and A 1 is the peak area in the peptide map of the modified protein to be measured of the peptide segment in which a specific amino acid site exists after conversion using an internal reference.

[0102] It should be understood that, within the scope of the present invention, the above technical features of the present invention and various technical features specifically described below (e.g., in the Examples) can be combined with each other to form new or preferred technical solutions, which can be more clearly understood by referring to the following examples. For the sake of illustration only, the examples are provided for illustrative purposes and are not intended to limit the present invention.

[0103] Hereinafter, the present invention will be described in more detail with reference to the embodiments, examples of which are shown in the drawings. The embodiments described below with reference to the drawings are illustrative and are used to interpret the present invention, but are not to be construed as limiting the present invention.

[0104] Example 1 Production of recombinant urate oxidase

[0105] 1.1 Construction of genes and expression plasmids for uric acid enzyme expression Based on the E. coli codon usage preference data, combining factors such as codon preference and GC content, cDNA sequence of uric acid enzyme protein (code name: PHC) (SEQ ID NO: 1) was designed, and the whole gene was synthesized and named pUC-57-PHC plasmid. Nde I and BamH I were inserted into the site as the target gene, and pET-30a plasmid was used as the expression carrier (pET-30a-PHC).

[0106] 1.2 Transformation of expression plasmids into bacterial host cells CaCl 2The expression carrier pET-30a-PHC was introduced into E. coli BL21(DE3) using the method, kanamycin resistance screening was performed, high-expression clones were screened, and the original seed bank strain (E3B) was preserved, and these steps were carried out according to the methods commonly used in the molecular biology field.

[0107] 1.3 Production of recombinant urate oxidase The engineered strain was fermented in a fermenter and expressed at OD at 30°C and pH 7.2. 600 Control conditions consist of culturing the cells at 37°C for 30 min or more, then raising the temperature to approximately 37°C, adding IPTG to 0.5 mmol / L, and inducing urate oxidase to accumulate for 3 h or more. Cells are harvested by centrifugation and then stored at -15°C or colder.

[0108] The frozen bacterial cells 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 urate oxidase precipitate was collected by centrifugation and the precipitate was diluted with 50 mmol / L NaHCO 3 After washing once with 100 mmol / L Na 2 HCO 3 The cells were then suspended in a pH 9.7-10.3 buffer at a suspension ratio of 1:50 (W / V), stirred at room temperature to dissolve overnight, and then centrifuged to collect the supernatant.

[0109] The urate oxidase was further purified through several chromatographic steps, with a purity of 95% or more detected by SDS-PAGE and 95% or more pure and free of aggregate form detected by Superdex 200 column, protein concentration was measured by Lowry method, and urate oxidase activity was measured by spectrophotometer, with 1 unit (U) enzyme activity defined as the amount of enzyme required to convert 1 μmol of uric acid per minute under the buffer conditions of reaction temperature of 37°C and optimal pH of 9.0.

[0110] Example 2: Preparation of polyethylene glycol-modified urate oxidase

[0111] Monomethoxy PEG derivatives of different molecular weights (500-20000Da), such as 5K molecular weight N-succinimide propionate PEG (5K-PEG-SPA), are dissolved in 100-300mmol / L PEG solution with 1-5mmol / L acid solution, and after dissolution, urate oxidase is dissolved in a carbonate buffer solution with a carbonate concentration of 0.1-0.3mol / L and pH of 10.0 at a molar ratio of 1:45-1:150 (urate oxidase:5K-PEG-SPA), and PEG and urate oxidase are subjected to a coupling reaction. The concentration of urate oxidase in the coupling reaction is 10mg / ml, and the coupling reaction is stirred for 60 minutes or more under conditions of 5-30°C until the degree of PEG coupling does not change with time. After the reaction is completed, unmodified PEG and by-products are removed from the reaction by ultrafiltration and / or chromatography. Separation and removal of the modified by-products can be achieved using suitable molecular sieve chromatography media, and finally, sterile filtration is performed to obtain the 5K modified PEGylated urate oxidase (codenamed PU5).

[0112] Example 3 Characterization of polyethylene glycol-modified urate oxidase

[0113] 3.1 Average modification degree and enzyme activity detection Protein concentration is measured by Lowry method, and polyethylene glycol urate oxidase activity is measured by spectrophotometer. The maximum ultraviolet absorption wavelength of uric acid enzyme substrate uric acid is 293nm, while the maximum ultraviolet absorption wavelength of the product allantoin is 224nm. Within a certain concentration range, the absorption value of uric acid at 293nm is proportional to its concentration, and the quantitative measurement of uric acid can be performed by spectrophotometer method. The specific process is as follows: open the UV-visible spectrophotometer, adjust the wavelength to 293nm, open the water bath circulation system of the device and keep the temperature at 37℃. Use sodium tetraborate buffer as blank control to calibrate the zero point, take 2.95ml of substrate reaction solution (0.1mol / L sodium tetraborate, 100μmol / L uric acid, pH 9.5, preheated at 37℃) and put it into a quartz cuvette, then add 50μl of sample and mix quickly and uniformly, then measure the absorption value at 293nm. The change in absorbance at 293 nm was continuously measured, and the uric acid decomposition concentration was calculated by C = A / εL (where A is the absorbance value at 293 nm for a specific concentration of uric acid, ε is the molar extinction coefficient of uric acid, L is the light path of the cuvette, and C is the molar concentration), and the enzyme activity was calculated. The enzyme activity is defined as one activity unit (U), which is the amount of enzyme required to convert 1 μmol of uric acid per minute at the optimal reaction temperature of 37°C and the optimal reaction pH of 9.5.

[0114] SEC-HPLC tandem UV / RI (combined ultraviolet and refractive index detector) is used to detect the average modification degree of polyethylene glycol urate oxidase. According to protein, there is a maximum absorption peak at 280 nm of ultraviolet light, and PEG is not absorbed at this wavelength, while the differential refractive index detector shows that the absorption value of protein and PEG is proportional to their various concentrations within a certain range. Therefore, the content of PEG and protein moieties in polyethylene glycol urate oxidase can be obtained by the external standard method of PEG reference sample and PHC physicochemical control sample, and the number of PEG molecules per unit urate oxidase, that is, the average modification degree, can be obtained by the following calculation method. Average degree of PEG urate oxidase modification = (relative molecular weight of urate oxidase subunit x amount of PEG in sample) / (relative molecular weight of PEG x amount of protein in sample).

[0115] Here, the SEC-HPLC-UV / RI detection patterns of the PHC physicochemical control sample, the PEG reference sample, and the PU5 modified product are shown in Figs. 1 to 5.

[0116] In Example 2, the enzyme activity and average modification degree of the polyethylene glycol urate oxidase obtained under different input ratios are shown in Table 1.

[0117] [Table 1]

[0118] In Example 2, the enzyme activity and average modification degree of the obtained polyethylene glycol urate oxidase with different PEG molecular weights and the molar ratio of protein to PEG input of 1:68 are shown in Table 2.

[0119] [Table 2]

[0120] As can be seen from Tables 1 and 2, the average modification degree of the polyethylene glycol urate oxidase of the present application is stable at 11 or more, and compared with the unmodified urate oxidase, the enzyme activity retention rate is higher, the enzyme activity does not decrease, but increases, and the enzyme activity is relatively stable. This is inconsistent with the view taught by Genken that low molecular weight PEG modification leads to decrease in enzyme activity, and the average modification degree of polyethylene glycol of the polyethylene glycol-modified urate oxidase obtained in the present application is higher, which has an unexpected technical effect on enzyme activity retention.

[0121] At the same time, the applicant also measured the immunogenicity of urate oxidase obtained by modifying with PEG of different molecular weights, and the experimental results are shown in Table 3.

[0122] [Table 3]

[0123] In the experiment, mice were divided into groups, with 8 mice in each group, and each animal was intravenously administered 1 mg / kg, once a week, and blood was collected after 4 consecutive doses to evaluate the immunogenicity of anti-PEG and anti-uric acid oxidase. As can be seen from the results in Table 3, when the average modification degree is consistent, the larger the PEG molecular weight, the higher the positive rate of the anti-PEG antibody produced, and when the PEG molecular weight exceeds 5KD, the positive rate and antibody titer of the anti-PEG antibody increase significantly. As can be seen from the analysis of the results of anti-uric acid oxidase, PEG modification can significantly reduce the antibody positive rate and antibody titer of uric acid oxidase, and when the average modification degree is consistent, within the range of 2-5K, the antibody positive rate and antibody titer of the anti-uric acid oxidase produced decreases with the increase in the PEG molecular weight, and when the PEG molecular weight is greater than 5K, there is also a risk of anti-uric acid enzyme antibodies. As described above, the 2-5K PEG-modified uric acid oxidase is superior to the 10K PEG-modified uric acid oxidase group, and 5KD is more preferable.

[0124] Finally, the inventors simultaneously measured the enzyme activity and average modification degree of the obtained urate oxidase modified with different acid-soluble PEGs, and the acid 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, lauryl sulfate, ethanesulfonic acid, formic acid, transbutenedioic acid, glucoheptonic acid, glycerol phosphate, gluconic acid, heptanoic acid, caproic acid, 2-hydroxy-ethanesulfonic acid. , lacturonic acid, lactic acid, lauric acid, lauryl sulfuric acid, malic acid, malonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, nicotinic acid, oleic acid, palmitic acid, pectic acid, 3-phenylpropionate, picrate, pivalic acid, propionic acid, stearic acid, paratoluenesulfonic acid, undecanoic acid, valeric acid and other organic acids, hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, perchloric acid, hydroiodic acid, nitric acid, persulfuric acid, boric acid, dichromate, silicic acid, chromic acid, thiocyanic acid and other inorganic acids. Different types of acid solutions can all realize the dissolution of PEG and modify uric acid enzyme. The dosing methods and dosing ratios of different PEGs are shown in Table 4.

[0125] [Table 4]

[0126] The inventors have discovered that by choosing the method of dissolving in acid before adding PEG rather than directly adding the dry powder, the obtained protein modification rate is higher, the enzyme activity is higher, and the dosage of PEG is effectively saved.

[0127] 3.2 Detection of polyethylene glycol modification sites

[0128] In the following steps, the inventors will detect modification sites in conventional commercial products and the urate oxidase obtained in the Examples.

[0129] The PEG modification sites of the polyethylene glycol-modified urate oxidase are confirmed by performing enzymatic cleavage of non-polyethylene glycol-modified and polyethylene glycol-modified urate oxidase with one or more enzymes, and then obtaining a chromatogram, i.e., a peptide map, by chromatography detection. Enzymatic cleavage of non-polyethylene glycol-modified and polyethylene glycol-modified urate oxidase can be performed with single enzyme cleavage (Lys-C or Trypsin) and / or double enzyme cleavage (combination of Lys-C and Trypsin). The enzymatic cleavage fragments are separated on a reversed-phase column, and the disappearance or reduction rate of the peptide segment is compared by calibration with an internal reference peptide segment, and the modification sites of the polyethylene glycol-modified urate oxidase and the percentage of the polyethylene glycol modification of the sites are determined and calculated.

[0130] Principle of analysis of modification sites in trypsin and Lys-C bienzyme cleavage mass peptide map: Lys-C can perform enzymatic cleavage specifically at the C-terminus of lysine (K), while trypsin uses basic amino acid-arginine (R) and lysine (K) as enzymatic cleavage sites and performs enzymatic cleavage specifically at the C-terminal peptide bond. The changes in the corresponding peptide segments before and after enzymatic cleavage in PHC and PU5 are compared, and the relative percentage of the decrease or disappearance of the PEG-modified peptide segments is analyzed and confirmed by referring to the internal standard peptide segment. The relative percentage of the decrease or disappearance of the peptide segment can be determined to determine the relative percentage of the lysine site on the peptide segment that is modified with PEG, and it can be known whether a specific amino acid (e.g., lysine) on a specific peptide segment is modified with PEG.

[0131] Specifically, the following applies: (1) Sample treatment: Urate oxidase and polyethylene glycol-modified urate oxidase are dissolved and diluted to 1 mg / mL with enzyme cleavage buffer (25 mmol / L Tris-HCl, 20% acetonitrile, pH 9.0), and 100 μL of each is taken, and 2 μL of Lys-C is added to perform enzyme cleavage at 37°C for 4 hours. That is, the solution is transferred to a pancreatic enzyme reaction tube (1:100 ratio), and enzyme cleavage is continued at 37°C for 2 hours, and the reaction is continued with 4 μL of TCEP reducing solution for 30 minutes, and the reaction is terminated by adding 10 μL of 1 mol / L hydrochloric acid solution. (2)Analysis conditions: Instrumentation: Thermo Ultimate 3000 HPLC, MSQ Plus; Chromatography column: Tsukiasahi Welch Materials μltimate (R) XB-C18 (4.6mm × 250mm, 5μm); Analysis conditions: Solution A (aqueous solution containing 0.1% TFA), Solution B (acetonitrile solution containing 0.1% TFA); Gradient: 0-70min, B 3-70%; LC detection wavelength: 214 nm. Ion source: ESI; Ion type: positive ion; Taper voltage: 50V; Scanning range: 300-2000Da; Scan time: 1s; The post-column flow rate is approximately 0.3 mL / min. A sample volume of 100 μL is injected and the chromatogram is recorded.

[0132] (3) Result processing: The chromatograms (peptide maps) of urate oxidase and polyethylene glycol-modified urate oxidase are compared, and the relative percentage reduction in the differential peptide segment area is calculated.

[0133] (4) The experimental results are shown in Tables 5 to 8 and Figures 6 to 7.

[0134] [Table 5-1] [Table 5-2]

[0135] [Table 6-1] [Table 6-2] [Table 6-3]

[0136] How to calculate the percent reduction in PU5 peptide segment peak area: The corresponding PU5 peptide segment peak area at the same concentration of PU5 and PHC can be calculated by the following formula: A 1 =A 0 ×t Here, A 1 is the PU5 peptide segment peak area after reduction using two internal reference peptide segments, and A 0 is the measured peak area of ​​the PU5 peptide map peptide segment, and t is the average value of the peak area ratio of the PHC peptide map to the PU5 peptide map in the internal reference peptide segments numbered T30 and T31, i.e., 0.588.

[0137] [Table 7]

[0138] The peptide segment peak area and the PHC peptide map peak area after conversion using the internal reference can be used to calculate the relative rate at which a certain peptide segment peak area in the PU5 peptide map is reduced by the following formula: P(%)=(A 2 - A 1 ) / A 2 ×100% However, A 2 is the peptide segment peak area in the PHC peptide map, and A 1 is the PU5 peptide segment peak area for that peptide segment after internal referencing reduction.

[0139] [Table 8]

[0140] As can be seen from the protein sequence analysis (SEQ ID NO:1) in this example, potential sites for modification of urate oxidase include T 1 , K 3 , K 4 , K 17 , K 21 , K 30 , K 35 , K 48 , K 49 , K 66 , K 74 , K 76 , K 79 , K 97 , K 112 , K 116 , K 120 , K 152 , K 155 , K 158 , K 169 , K 179 , K 190 , K 215 , K 222 , K 231 , K 266 , K 272 , K 285 , K 291 , K 293 There are 31 sites such as:

[0141] As can be seen from the analysis of the polyethylene glycol-modified urate oxidase-modified site obtained in Example 2, as shown in Tables 5, 6, 7, 8 and FIG. 6, the site in which 90% or more of the peptide segment was lost after PU5 enzyme cleavage was K 3 , K 4 , K 35 , K 97 , K112 , K 116 , K 120 , K 152 , K 222 , K 266 , K 285 The sites where the peptide segments were lost within 80% to 90% after PU5 enzyme cleavage were K 76 , K 231 There is.

[0142] At the same time, the inventors discovered that the modification sites of the polyethylene glycol-modified urate oxidase of the present invention are more numerous than those of commercially available drugs, and that there is a significant difference between them. By cleaving the single enzyme, the polyethylene glycol-modified urate oxidase of the present invention can inhibit K 30 , K 35 , K 222 and K 231 The disappearance rate of peptide segments containing the four sites is 80% or more, and similar drugs on the market are hardly disappeared in peptide segments containing these four sites, i.e., K 30 , K 35 , K 222 and K 231の The modification rates of the commercially available similar drugs at the four sites are much lower than that of the polyethylene glycol-modified urate oxidase of the present application.In addition, the immunogenicity of the polyethylene glycol-modified urate oxidase of the present application is significantly reduced compared to that of the commercially available drugs, and the inventors speculate that this may be related to the number and difference of the modification sites.

[0143] The following provides a detailed description of the in vivo animal evaluation of the polyethylene glycol-modified urate oxidase (PU5) of the present invention. The pegloticase used in the experiment is a commercially available similar drug with batch number 5085B.

[0144] Example 4: Polyethylene glycol urate oxidase in vivo drug efficacy study

[0145] 4.1. Evaluation of in vivo efficacy of polyethylene glycol urate oxidase in rat models A rat model of chronic hyperuricemia is induced by combining potassium oxazinate drinking water and high uric acid feed, and the therapeutic effect of polyethylene glycol urate oxidase (PU5) on chronic hyperuricemia in rats is evaluated.

[0146] 40 model rats were selected and randomly divided into 4 groups, namely model group, low dose polyethylene glycol uric acid enzyme group (0.3mg / kg), medium dose polyethylene glycol uric acid enzyme group (1.0mg / kg), high dose polyethylene glycol uric acid enzyme group (3.0mg / kg), 10 rats per group, and 10 normal SD rats were selected as blank control group. The test was performed for 5 weeks of continuous modeling, and after 1 week of modeling, intramuscular administration was started, and administration was performed once a week, for 4 weeks of continuous administration. The serum uric acid, serum urea nitrogen, and serum creatinine levels of rats were detected before administration and 7 days after each administration, and the rat kidney histological changes were observed after the end of the test.

[0147] As shown in the results of Figure 8, on the 7th, 14th, 21st, 28th and 35th days after modeling, the blood uric acid levels of the model control group were all significantly increased compared with the blank control group, and on the 7th day after modeling, the serum urea nitrogen, creatinine and uric acid of the rats in the model group were 2.73 times, 2.40 times and 7.83 times that of the rats in the blank control group, respectively. From the perspective of kidney pathology (as shown in Figure 9), the renal tubule dilation, necrosis, inflammation and fibrosis scores of the model control group were all significantly increased, and at the same time, the number of urate crystals was also significantly increased. All the polyethylene glycol uric acid enzymes of the test material at medium and high doses significantly reduce serum uric acid level, and show correlation with dose. Between 14 and 35 days, the mean value of the blood uric acid level of the medium dose group is maintained at 303.80-660.60μmol / L, and the mean value of the blood uric acid level of the high dose group is maintained at 153.70-403.40μmol / L. Compared with the model group, the reduction of blood uric acid of the medium dose group is 34.46-67.94%, and the reduction of blood uric acid of the high dose group is 65.67-83.78%. Compared with the model control group, each administration group of polyethylene glycol uric acid enzyme has a significant improvement effect on renal tubule dilatation, renal necrosis and inflammation.

[0148] 4.2. Evaluation of single-dose administration of polyethylene glycol urate oxidase in rats Thirty-six SD rats were taken, and half each of the males and females were randomly divided into six groups (see Table 9), namely, a commercially available drug Pegloticase intravenous injection group, an intramuscular injection group, a polyethylene glycol urate oxidase intravenous injection group, and a polyethylene glycol urate oxidase low, medium, and high (0.5, 1.0, 2.0 mg / kg) dose intramuscular injection group, the specific administration methods and doses are shown in Table 9. PK and PD were detected by blood sampling from the jugular vein.

[0149] [Table 9]

[0150] 4.2.1. Pharmacokinetic comparison Before administration to SD rats, the serum drug concentration levels of all individuals were below the lower limit of quantification (LLOQ: 312.500ng / mL). After intramuscular injection of 0.5, 1.0, and 2.0mg / kg once, within the range of 0-168h (0-7 days), the serum drug concentration after polyethylene glycolated uric acid enzyme injection (PU5) was correlated with the dose, and the overall level increased with increasing drug dose. After 168h, the blood concentration of the pegloticase intramuscular administration group was below the lower limit of quantification, and that of the PU5 intramuscular administration group could be maintained for more than 240h.

[0151] After administration, the internal C of female and male SD rats in each group was measured using 1.0 mg / kg Pegloticase intravenous and intramuscular injections, 1.0 mg / kg polyethylene glycolated uric acid enzyme injection intravenous injections, and 0.5, 1.0, and 2.0 mg / kg polyethylene glycolated uric acid enzyme injection intramuscular injections. max (C 5min ) ratio was in the range of 0.75 to 0.99, and the AUC last Ratios are in the range of 0.54 to 0.94, and AUC 0-∞The ratio was in the range of 0.58 to 0.97. As can be seen, there was no clear gender difference in the exposure levels of pegloticase and polyethylene glycol-modified uric acid enzyme (PU5) injection in SD rats.

[0152] However, when SD rats were administered the same amount (1.0 mg / kg) of the commercially available drug Pegloticase, the AUC last The AUC of the intramuscular injection group was 426.48 ± 65.34. last The AUC last was 565.61 ± 161.60, and the AUC last The AUC of PU5 was 337.86 ± 227.34 under the same dosage and administration conditions. last is higher than the over-the-counter drug Pegloticase.

[0153] The same amount (1.0mg / kg) of the commercially available drug Pegloticase was administered to SD rats. The t1 / 2(h) of the intravenous administration group was 49.51±8.12, and that of the intramuscular administration group was 55.21±13.50. The t1 / 2(h) of the intravenous administration group of PU5 injection was 86.12±33.82, and that of the intramuscular administration group was 60.45±21.37. Under the same dose and administration method conditions, the t1 / 2(h) of the PU5 injection was longer than that of the commercially available drug Pegloticase.

[0154] The pharmacokinetic results are shown in Tables 10 to 15 and Figures 10 to 12.

[0155] [Table 10]

[0156] [Table 11]

[0157] [Table 12]

[0158] [Table 13]

[0159] [Table 14]

[0160] [Table 15-1] [Table 15-2]

[0161] 4.2.2. Comparison of drug effects in the body (uric acid) After a single intramuscular injection of 0.5, 1.0, and 2.0 mg / kg polyethylene glycol uric acid enzyme injection, the uric acid concentration was maintained at a low level on days 1 and 3 after administration, and the uric acid level of each dosage group began to recover on day 7 after administration. The higher the drug dosage, the longer the time that uric acid maintained a low level in the body. Compared with the same amount of intravenous injection group, the time that serum uric acid maintained a low level in the PU5 intravenous injection group was longer than that of the pegloticase intravenous injection group. Compared with the same amount of intramuscular injection group, the time that serum uric acid maintained a low level in the PU5 intramuscular injection group was longer than that of the pegloticase intramuscular injection group. Compared with the same amount of intramuscular injection group, the time that serum uric acid maintained a low level in the PU5 intravenous or intramuscular injection group was longer than that of the pegloticase intravenous or intramuscular injection group, that is, the time that PU5 maintained a low level in the body was longer than that of the pegloticase. The results are shown in Figure 13.

[0162] 4.3. Evaluation of polyethylene glycol urate oxidase by multiple administration to rats In this study, there were four groups: a group receiving intravenous injections of the commercially available drug Pegloticase, a group receiving intramuscular injections, a group receiving intravenous injections of polyethylene glycolated uric acid enzyme injections (PU5), and a group receiving intramuscular injections. There were eight rats in each group, half male and half female, for a total of 32 SD rats. The group receiving intravenous injections of Pegloticase and polyethylene glycolated uric acid enzyme injections received intramuscular injections, while the group receiving intramuscular injections of Pegloticase and polyethylene glycolated uric acid enzyme injections received intramuscular injections. The drug dosage was 1.0mg / kg for both groups. The drug was administered once a week for four consecutive doses.

[0163] As can be seen from the analysis of the results, SD rats were given multiple intravenous / muscular injections of 1.0mg / kg Pegloticase and polyethylene glycol-glucose-containing uric acid enzyme injections. No drug-related abnormal changes were observed in the general condition of the rats.

[0164] 4.3.1 Anti-PEG antibody detection After four consecutive doses in SD rats, anti-PEG and anti-PHC antibodies were not detected in any individual animals before the first dose, and after the end of the dose, anti-PHC antibodies were not detected in any animals. Anti-PEG antibodies were detected in the pegloticase intravenous and intramuscular injection groups, and the polyethylene glycolated uric acid enzyme injection intravenous and intramuscular injection groups, with the percentages of positive results being 3 / 8, 1 / 8, 1 / 8, and 1 / 8, respectively. PEG immunohistochemistry showed weak positive expression of PEG in the spleen, liver, and kidney of the pegloticase intravenous and intramuscular injection groups. No positive expression of PEG was observed in the polyethylene glycolated uric acid enzyme injection intravenous and intramuscular injection groups. The results are shown in Table 16.

[0165] As can be seen from the above analysis, the antibodies produced by PU5 and pegloticase are not antibodies against the urate oxidase moiety, but are mainly antibodies against the PEG moiety.

[0166] As can be seen from the results of PEG antibody and PEG immunohistochemistry, both PU5 and pegloticase were superior in the intramuscular administration group compared to the intravenous administration group, with PU5 producing superior anti-PEG antibodies than pegloticase in the intravenous administration group, and PU5 producing superior anti-PEG antibodies than pegloticase in the intramuscular administration group.

[0167] [Table 16]

[0168] 4.3.2 Pharmacokinetic detection After multiple intravenous and intramuscular injections of Pegloticase and polyethylene glycolated uric acid enzyme injections into SD rats, there were no obvious sex differences in the main pharmacokinetic parameters of animals in each group. After four consecutive doses, the two drugs were slightly accumulated in the rats' bodies.

[0169] The same amount (1.0mg / kg) of the commercially available drug Pegloticase was administered to SD rats by intravenous / muscular injection multiple times. After the first administration, the absolute bioavailability in the rats was 51.35%, and after the last administration, the absolute bioavailability in the rats was 45.98%. The same amount (1.0mg / kg) of polyethylene glycol uric acid enzyme injection was administered to SD rats by intravenous / muscular injection multiple times. After the first administration, the absolute bioavailability in the rats was 58.29%, and after the last administration, the absolute bioavailability in the rats was 52.60%.

[0170] 4.3.3 Comparison of drug effects in the body (uric acid) SD rats were given 1.0mg / kg Pegloticase and polyethylene glycolated uric acid enzyme injections four times (once a week) intravenously and intramuscularly, and serum uric acid concentrations were maintained at low levels after each injection, with the Pegloticase intramuscular injection group beginning to recover 14 days after the last injection, and the other groups beginning to recover 18 days after the last injection. Compared with the same amount of commercially available Pegloticase, the retention times of the two drug intravenous injection groups were relatively consistent, and the retention time of the polyethylene glycolated uric acid enzyme injection intramuscular injection group was longer than that of the commercially available drug, that is, the therapeutic effect of intramuscular administration of PU5 is superior to that of Pegloticase.

[0171] The results are shown in Tables 17 to 20 and Figs. 14 to 19.

[0172] [Table 17-1] [Table 17-2]

[0173] [Table 18-1] [Table 18-2]

[0174] [Table 19]

[0175] In the present specification, the reference words such as "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" refer to specific features, structures, materials, or characteristics described in the embodiment or example in combination with each other, which are included in at least one embodiment or example of the present invention. In the present specification, the above-mentioned terms and exemplary descriptions do not necessarily refer to the same embodiment or example. In addition, specific features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, if not in conflict with each other, a person skilled in the art may combine different embodiments or examples described herein and features of different embodiments or examples.

Claims

1. A method for preparing polyethylene glycol-modified urate oxidase, comprising: coupling urate oxidase with polyethylene glycol, the polyethylene glycol being provided in the form of an acidic solution, and the molar ratio of the urate oxidase to the polyethylene glycol being 1:(56-94), to obtain the polyethylene glycol-modified urate oxidase; A method for producing polyethylene glycol-modified urate oxidase, wherein the concentration of the urate oxidase in the coupling reaction system is 10 mg / ml.

2. The method according to claim 1 , wherein the acidic solution contains at least one acid selected from the group consisting of organic acids and / or inorganic acids.

3. The organic acid is 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, lauryl sulfate, ethanesulfonic acid, formic acid, transbutenedioic acid, glucoheptonic acid, glyceryl phosphate, gluconic acid, heptanoic acid, caproic acid, 2-hydroxy-ethanesulfonic acid, lacturonic acid, lactic acid, lauric acid, lauryl sulfate, malic acid, malonic acid, methanesulfonic acid, 2-naphthalenesulfonic acid, nicotinic acid, oleic acid, palmitic acid, pectic acid, 3-phenylpropionate, picrate, pivalic acid, propionic acid, stearic acid, paratoluenesulfonic acid, undecanoic acid, valeric acid, The inorganic acid is selected from hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid, perchloric acid, hydroiodic acid, nitric acid, persulfuric acid, boric acid, dichromic acid, silicic acid, chromic acid, thiocyanic acid, or The concentration of hydrogen ions in the acidic solution is 1 to 5 mmol / L; or The acid solution comprises hydrochloric acid, sulfuric acid, or glacial acetic acid; The method according to claim 2, wherein the concentration of the acid in the acidic solution is preferably 1 to 5 mmol / L.

4. 2. The method according to claim 1, wherein the concentration of the polyethylene glycol in the acidic solution is 100 to 300 mmol / L.

5. The polyethylene glycol molecular weight is 6 KD or less; or The polyethylene glycol has a monomethoxy or hydroxyl group, or The polyethylene glycol is linear or branched; or The polyethylene glycol and urate oxidase are coupled via an amide bond; or The polyethylene glycol is a modified polyethylene glycol, and the modifying group of the modified polyethylene glycol is selected from at least one of N-hydroxysuccinimide, N-hydroxysuccinimide carbonate, N-hydroxysuccinimide acetate, N-hydroxysuccinimide propionate, N-hydroxysuccinimide butyrate, N-hydroxysuccinimide succinate, and p-nitrophenyl carbonate; The method according to claim 1, characterized in that the modifying group of the modifying polyethylene glycol is N-hydroxysuccinimide propionate.

6. The coupling reaction is carried out in a carbonate buffer solution; 2. The method of claim 1, wherein the carbonate buffer solution has a pH of 9 to 11.

7. The method of claim 1, wherein the coupling reaction is carried out at 5 to 30° C. for at least 60 minutes.

8. As the amino acid site of the urate 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 At least 11 of the above have PEG modifications, The method of claim 1, characterized in that the amino acid site is localized to the amino acid sequence shown in SEQ ID NO:

1.

9. The urate oxidase has an amino acid sequence as set forth in SEQ ID NO: 1-7; or having a polypeptide having at least 90% identity to SEQ ID NO: 1-7; or 2. The method of claim 1, comprising a polypeptide having one or several amino acid substitutions, deletions and / or additions compared to SEQ ID NOs: 1-7.

10. The method according to claim 8, wherein the urate oxidase has an amino acid sequence represented by SEQ ID NO: 1 to 4.

11. The polyethylene glycol-modified urate oxidase is K ３０ , K ３５ , K ２２２ and K. ２３１ At least one of the four amino acid sites is PEGylated, The method according to any one of claims 1 to 10, characterized in that the amino acid site is localized to the amino acid sequence shown in SEQ ID NO:

1.

12. the relative reduction in peak area having at least 11 predetermined peptide segments in the peptide map of the polyethylene glycol-modified urate oxidase compared with the peptide map of the urate oxidase not modified with polyethylene glycol is 80% or more; The method according to any one of claims 1 to 10, characterized in that the at least eleven predefined peptide segments are selected from the following table: 【Table 1】

13. The method according to claim 12, characterized in that the relative reduction in peak area having at least 11 specified peptide segments in the peptide map of the polyethylene glycol-modified urate oxidase compared to the peptide map of the urate oxidase not modified with polyethylene glycol is 90% or more.

14. The method of claim 12, characterized in that the peptide map of the polyethylene glycol-modified urate oxidase has peptide segments with reduced peak areas, and the relative percentages of the peak areas of the peptide segments are as shown in the following table: 【Table 2】

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