Stabilizer of urate oxidase and polyethylene glycol conjugate thereof and pharmaceutical application of stabilizer

Abstract

The invention provides a method for improving the stability of urate oxidase. The inventor finds that the active ingredients, namely urate oxidase and chemically modified urate oxidase, are combined with a stabilizer, namely xanthine in a non-covalent bond form, so that the tetramer stability of the urate oxidase and the chemically modified urate oxidase inside and outside an organism can be remarkably improved, and the stability of the urate oxidase is effectively improved.

Description

Technical Field

[0001] The present invention relates to the biological field, and in particular to a stabilizer for uricase and a polyethylene glycol conjugate thereof and a pharmaceutical use thereof. Background Art

[0002] Gout is a disease caused by long-term purine metabolism disorder or reduced uric acid excretion. Its clinical characteristics are hyperuricemia. Due to the poor solubility of urate, crystals precipitate and accumulate in the subcutaneous tissue, joints, and kidneys to form tophi, leading to recurrent acute arthritis and involving the kidneys to cause uric acid urinary stones and interstitial nephritis. Purine in the human body is converted into the final product uric acid by enzymes. Under normal circumstances, the uric acid content in male blood is 149-416mmol / L, and the uric acid content in female blood is 89-357mol / L. The amount of uric acid in the body is about 1200mg, and the production and excretion are about 600mg / day, which is in a state of balance. However, if the body takes in too much uric acid or the excretion mechanism is impaired, causing uric acid to accumulate in the blood to more than 70mg / L, it will cause hyperuricemia. When sodium urate reaches saturation in the blood or synovial fluid and crystallizes, or when hyperuricemia lasts for a long time and crystallizes around joints and soft tissues, it can lead to acute gouty arthritis or chronic gouty arthritis and joint deformities. Urate deposition in the renal tubules and renal interstitium can cause inflammation and lead to chronic urate nephropathy. In patients with severe hyperuricemia (such as patients with malignant tumors such as leukemia and lymphoma), a large amount of uric acid deposition in a short period of time causes urinary tract obstruction and acute renal failure, also known as uric acid nephropathy.

[0003] The cause of hyperuricemia is related to the inactivation of the uricase gene during human evolution, so humans cannot synthesize active uricase by themselves. The active uricase in non-human primates or other mammals can convert uric acid produced by purine metabolism into more soluble allantoin to improve the excretion efficiency of the kidney (Wortmann RL, Kelley W N. Kelley's textbook of rheumatology (6th). 2001: 1339-1376). Hyperuricemia may be caused by excessive uric acid production or insufficient uric acid excretion (Hershfield, Seegmiller, 1976; Roessler, 1995). In recent years, people's quality of life has improved and their living and eating habits have changed, and they have consumed more high-protein and high-purine foods. On the other hand, drugs during certain tumor chemotherapy and drugs such as azathioprine to prevent organ transplant rejection can cause significant hyperuricemia and may also cause severe gout or renal damage. Mild hyperuricemia before clinical symptoms appear can be controlled by restricting diet; when clinical symptoms appear, appropriate drug treatment is required. Conventional treatments currently used in clinical practice include: analgesic and anti-inflammatory drugs, such as colchicine, ibuprofen, naproxen, etc., which are mainly used to control the acute symptoms of gouty arthritis and eliminate local joint pain, swelling and inflammation; uricosuric agents that promote uric acid excretion (ineffective if renal function is reduced), such as probenicid, sulfinpyrazone, benzbromarone, etc.; drugs that inhibit uric acid synthesis, such as allopurinol, can inhibit xanthine oxidase from converting hypoxanthine or xanthine into uric acid, and instead slowly oxidize it to form isoxanthine that is easily soluble in water. However, allopurinol therapy may cause hypersensitivity syndromes such as acute renal and liver failure, skin damage, and is unlikely to be effective in patients with severe chronic hyperuricemia who have already developed tophi.

[0004] Uricase (EC 1.7.3.3) is widely present in microorganisms (Bacillus fastidious, Candida monocytogenes, Aspergillus flavus), plants (soybeans, chickpeas), and animals (pigs, cattle, dogs, baboons) (Suzuki K, Sakasegawa S, Misaki H, Sugiyama M. J Biosci Bioeng. 2004. 98: 153-158). In the presence of oxygen, it can catalyze uric acid to oxidize allantoin and release carbon dioxide (Retailleau P, Colloc'h, Denis V, Francoise B. Acta Cryst D. 2004. 60: 453-462.). Active uricase is a tetrameric protein composed of identical subunits, each of which has a molecular weight of about 34 kD and is composed of 301-304 amino acids. The pH value at which the uricase activity in each solution reaches its maximum is 8.0 (Bayol A et al. Biophys Chem. 1995. 54: 229-235).

[0005] In early studies, non-recombinant natural uricase was obtained by using microorganisms. That is, there were reports of using uric acid or its structural analogs such as adenine, guanine, xanthine and hypoxanthine as culture medium inducers to increase the expression of uricase oxidase (Liu Jianguo et al., Conditions for the formation of uricase in Candida, Acta Microbiologica Sinica 29(1):45-50, 1989). The results showed that the enzyme activity was the highest when uric acid was used as an inducer, and xanthine and guanine also had an induction effect. In this induction mechanism, substrate analogs such as purine enter cells and are converted into uric acid under the action of corresponding enzymes, which induces cells to form uricase oxidase (Machida, Y.: Agric. Boil. Chem, 44(12), 1980).

[0006] Uricase prepared from Aspergillus flavus has been used to treat hyperuricemia caused by leukemia-related chemotherapy for more than 20 years (London and Hudson, 1957; Kissel et al., 1968; Masera et al., 1982; Duke University, CN 101280293A). Uricase has better efficacy than allopurinol. For gout treatment, infusion of uricase can block acute attacks of hyperuricemia and reduce the volume of tophi (Brogard et al., 1978). In 2002, the recombinant Aspergillus flavus uricase produced by brewer's yeast, ELITEK, developed by Sanofi in France, passed FDA certification and was used for short-term treatment of hyperuricemia caused by tumor chemotherapy. However, this uricase infusion therapy is used to treat recurrent gout. Due to the heterogeneity of the microbial source, the anti-uricase antibodies in the patient's body clear the uricase after repeated infusions, resulting in a rapid decrease in the efficacy of the drug after administration (Donadio et al., 1981; Pui et al., 1997).

[0007] Therefore, how to effectively improve the stability of urate oxidase is a key issue that scientific researchers are waiting to solve. Summary of the invention

[0008] The present invention aims to solve at least one of the technical problems existing in the prior art to at least a certain extent.

[0009] During the research and development process, the inventors of the present application unexpectedly and pleasantly discovered that xanthine can effectively improve the stability of urate oxidase.

[0010] To this end, in one aspect of the present invention, the present invention proposes a uricase oxidase composition. According to an embodiment of the present invention, the uricase oxidase composition comprises an active ingredient and a stabilizer, the active ingredient comprises uricase oxidase, and the stabilizer comprises xanthine. The inventor unexpectedly discovered during the research and development process that xanthine can effectively improve the stability of uricase oxidase. The uricase oxidase composition according to an embodiment of the present invention contains xanthine as a stabilizer, and its stability is greatly improved.

[0011] According to an embodiment of the present invention, the uricase composition may further include at least one of the following additional technical features:

[0012] According to an embodiment of the present invention, the molar ratio of xanthine to uricase tetramer is not less than 0.5: 1. According to a specific embodiment of the present invention, when the uricase concentration is lower, the amount of xanthine that can be added to the uricase composition system is more, and the uricase composition is more stable.

[0013] According to an embodiment of the present invention, the uricase is naturally extracted uricase, recombinantly expressed uricase or modified uricase.

[0014] According to a specific embodiment of the present invention, the naturally extracted uricase is derived from Aspergillus flavus, wild-type Micrococcus or mammals.

[0015] According to a specific embodiment of the present invention, the modified uricase comprises at least one selected from polyethylene glycol-modified uricase, polysaccharide side chain-modified uricase or polymer-oligopeptide-modified uricase.

[0016] It should be noted that xanthine can greatly reduce the immunogenicity of enzymes from different sources while maximizing enzyme activity, and further improve the in vivo stability of urate oxidase.

[0017] According to an embodiment of the present invention, the uricase oxidase is combined with xanthine in the form of a non-covalent bond, which can significantly improve the tetramer stability of the uricase oxidase and the chemically modified uricase in vitro and in vivo.

[0018] According to a specific embodiment of the present invention, the uricase binds to xanthine via hydrogen bonds, salt bonds, hydrophobic interactions or van der Waals interactions.

[0019] According to an embodiment of the present invention, the concentration of the uricase in the composition is 1-12 mg / ml, and the molar ratio of xanthine to uricase tetramer is (0.5-200):1.

[0020] According to an embodiment of the present invention, the concentration of the uricase in the composition is 1-12 mg / ml, and the molar ratio of xanthine to the uricase tetramer is (1-156): 1. Within this range, the stability of uricase is higher.

[0021] According to an embodiment of the present invention, the concentration of the uricase in the composition is 1 mg / ml, and the molar ratio of xanthine to uricase tetramer is (0.5-200):1.

[0022] According to an embodiment of the present invention, the concentration of the uricase in the composition is 1 mg / ml, and the molar ratio of xanthine to uricase tetramer is (1-156):1.

[0023] According to an embodiment of the present invention, the concentration of the uricase in the composition is 6 mg / ml, and the molar ratio of xanthine to uricase tetramer is (0.5-34):1.

[0024] According to an embodiment of the present invention, the concentration of the uricase in the composition is 6 mg / ml, and the molar ratio of xanthine to uricase tetramer is (0.5-33.3):1.

[0025] According to an embodiment of the present invention, the concentration of the uricase in the composition is 6 mg / ml, and the molar ratio of xanthine to the uricase tetramer is (1-26):1.

[0026] According to an embodiment of the present invention, the concentration of the uricase in the composition is 8 mg / ml, and the molar ratio of xanthine to the uricase tetramer is (0.5-25):1.

[0027] According to an embodiment of the present invention, the concentration of the uricase in the composition is 8 mg / ml, and the molar ratio of xanthine to the uricase tetramer is (1-20):1.

[0028] According to an embodiment of the present invention, the concentration of the uricase in the composition is 8 mg / ml, and the molar ratio of xanthine to uricase tetramer is (1-19.5):1.

[0029] According to an embodiment of the present invention, the concentration of the uricase in the composition is 12 mg / ml, and the molar ratio of xanthine to uricase tetramer is (0.5-17):1.

[0030] According to an embodiment of the present invention, the concentration of the uricase in the composition is 12 mg / ml, and the molar ratio of xanthine to uricase tetramer is (0.5-16.7):1.

[0031] According to an embodiment of the present invention, the concentration of the uricase in the composition is 12 mg / ml, and the molar ratio of xanthine to uricase tetramer is (1-13):1.

[0032] It should be noted that the uricase or polyethylene glycol-modified uricase involved in the present invention exists in the form of a tetramer. The molar ratio of xanthine to uricase involved in the whole text refers to the molar ratio of xanthine to uricase tetramer. In addition, when it comes to the molar ratio of xanthine to uricase, the uricase oxidase can be a uricase oxidase tetramer or a polyethylene glycol uricase tetramer.

[0033] According to an embodiment of the present invention, the uricase composition further includes a pharmaceutically acceptable excipient or carrier, and the excipient or carrier is selected from at least one of the following: hydroxypropyl methylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, mannitol, sucrose, fructose, methyl cellulose, vinyl acetate copolymer, chitosan, sodium alginate, xanthan gum, sodium carboxymethyl cellulose, polyethylene oxide, methacrylic acid copolymer, maleic anhydride-methyl vinyl ether copolymer, carbomer, polyvinyl pyrrolidone, ethyl cellulose, cellulose acetate, methacrylate, polyoxyethylene, polyvinyl alcohol, behenyl glyceryl, monostearate glyceryl, cellulose acetate, cellulose acetate phthalate, ethyl cellulose, PLA, PLGA and polyethylene glycol.

[0034] According to an embodiment of the present invention, the uricase composition is an injection, so that the uricase composition can be distributed in the body faster, thereby achieving a better therapeutic effect.

[0035] In another aspect of the present invention, the present invention proposes the use of xanthine in improving the stability of uricase oxidase. The inventors have found that xanthine can significantly improve the tetramer stability of uricase oxidase and modified uricase in vivo and in vitro by specifically binding to uricase oxidase and chemically modified uricase oxidase in the form of non-covalent bonds. According to an embodiment of the present invention, xanthine is introduced during the preparation of uricase oxidase and modified uricase, and xanthine binds to uricase oxidase, effectively improving the stability of uricase oxidase.

[0036] In another aspect of the present invention, a method for improving the stability of uricase is provided. According to an embodiment of the present invention, the method comprises contacting uricase with xanthine. According to the method of an embodiment of the present invention, uricase can be specifically bound to xanthine, thereby effectively improving the stability of uricase.

[0037] In another aspect of the present invention, a method for preparing uricase is provided. According to an embodiment of the present invention, the method comprises contacting uricase with xanthine. According to the method of an embodiment of the present invention, uricase oxidase can be specifically bound to xanthine, thereby effectively improving the stability of uricase oxidase.

[0038] According to an embodiment of the present invention, the uricase is naturally extracted uricase, recombinantly expressed uricase or modified uricase.

[0039] According to a specific embodiment of the present invention, the naturally extracted uricase is derived from Aspergillus flavus, wild-type Micrococcus or mammals.

[0040] The sequence information of urate oxidase from Aspergillus flavus is as follows:

[0041] SAVKAARYGKDNVRVYKVHKDEKTGVQTVYEMTVCVLLEGEIETSYTKADNSVIVATDSIKNTIYITAKQNPVTPPELFGSILGTHFIEKYNHIHAAHVNIVCHRWTRMDIDGKPHPHSFIRDSEEKRNVQVDVVEGKGIDIKSSLSGLT VLKSTNSQFWGFLRDEYTTLKETWDRILSTDVDATWQWKNFSGLQEVRSHVPKFDATWATAREVTLKTFAEDNSASVQATMYKMAEQILARQQLIETVEYSLPNKHYFEIDLSWHKGLQNTGKNAEVFAPQSDPNGLIKCTVGRSSLKSKL

[0042] The sequence information of wild-type Micrococcus-derived urate oxidase is as follows:

[0043] MTNIILGKNQYGKAEVRLVKVTRNTCRHEIQDLNVTSQLRGDFEAAAHTAGDNAHVVATDTQKNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWDRINDHDHAFSRNKSEVRTAVLEISGSEQAIVAGIEGL TVLKSTGSEFHGFPRDKYTTLPETEDRILATDVSARWRYNTVEVDFDAVYADVRGLLLKAFAETHSLALQQTMYEMGRAVIETHPEIDEIKMSLPNKHHFLVDLSPFGQDNPNEVFYAADRPYGLIEATIQREGCRADHPIWSN

[0044] According to a specific embodiment of the present invention, the modified uricase comprises at least one selected from polyethylene glycol-modified uricase, polysaccharide side chain-modified uricase or polymer-oligopeptide-modified uricase.

[0045] It should be noted that xanthine can greatly reduce the immunogenicity of enzymes from different sources while maximizing enzyme activity, and further improve the in vivo stability of urate oxidase.

[0046] According to an embodiment of the present invention, the contact between the uricase oxidase and xanthine is carried out in the following manner: the engineered bacteria carrying the nucleic acid molecule expressing the uricase oxidase are fermented in a fermentation liquid containing xanthine to obtain the uricase oxidase. Therefore, the xanthine can be more fully combined with the uricase oxidase to improve the stability of the uricase oxidase.

[0047] According to an embodiment of the present invention, the amount of xanthine in the xanthine-containing fermentation broth is not less than 0.1 g / L.

[0048] According to an embodiment of the present invention, the contact between uricase oxidase and xanthine is carried out in the following manner: fermenting an engineered bacterium carrying a nucleic acid molecule expressing the uricase oxidase in a fermentation broth containing xanthine to obtain the uricase oxidase; and modifying the uricase oxidase with polyethylene glycol and purifying the uricase oxidase to obtain polyethylene glycol-modified uricase oxidase. Therefore, the stability of the uricase oxidase can be improved.

[0049] According to an embodiment of the present invention, the contact between uricase oxidase and xanthine is carried out in the following manner: fermenting an engineered bacterium carrying a nucleic acid molecule expressing the uricase oxidase to obtain the uricase oxidase, wherein the fermentation system contains or does not contain xanthine; the uricase oxidase is subjected to polyethylene glycol modification and purification treatment to obtain polyethylene glycol-modified uricase oxidase, and xanthine is added to the polyethylene glycol modification or purification system, that is, the polyethylene glycol-modified uricase oxidase is contacted with xanthine. When the pegylated uricase oxidase tetramer is added with xanthine, it shows a more stable trend, and the addition of xanthine does not affect the activity of the pegylated uricase oxidase.

[0050] In another aspect of the present invention, the present invention provides a uricase. According to an embodiment of the present invention, the uricase is prepared according to the method described above. Thus, the obtained uricase has high stability.

[0051] In another aspect of the present invention, the present invention provides the use of the aforementioned uricase composition or the aforementioned uricase in the preparation of a medicament for preventing or treating a hyperuricemia-related disease.

[0052] According to an embodiment of the present invention, the hyperuric acid-related diseases include chronic hyperuricemia, gout, kidney disease, hyperuricemia arthritis, kidney stones, gouty nodules, hypertension, diabetes, hypertriglyceridemia, metabolic syndrome, coronary heart disease, atherosclerosis, and hyperuricemia caused by cancer chemotherapy.

[0053] In yet another aspect of the present invention, the present invention provides use of the aforementioned uricase composition or the aforementioned uricase for reducing the uric acid level in a biological fluid.

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

[0055] It can be understood that the technical effects of the additional technical features of the uricase oxidase composition described above are applicable to the additional technical features of the method for improving the stability of uricase oxidase and the method for preparing uricase according to an embodiment of the present invention, and the additional technical features of the method for improving the stability of uricase oxidase and the method for preparing uricase according to an embodiment of the present invention are not described in detail herein.

[0056] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0058] Figure 1 The RP-HPLC diagram of urate oxidase obtained after xanthine is introduced into the fermentation process according to one embodiment of the present invention is shown;

[0059] Figure 2 The SEC-HPLC diagram of urate oxidase obtained after the introduction of xanthine during the fermentation process according to one embodiment of the present invention is shown;

[0060] Figure 3 shows a chromatogram of urate oxidase obtained without adding xanthine during the purification process according to one embodiment of the present invention;

[0061] Figure 4 A chromatogram of urate oxidase obtained by adding xanthine during purification according to one embodiment of the present invention is shown;

[0062] Figure 5 A blood drug concentration variation curve according to an embodiment of the present invention is shown;

[0063] Figure 6 A graph showing changes in serum uric acid levels according to an embodiment of the present invention is shown. DETAILED DESCRIPTION

[0064] Embodiments of the present invention are described in detail below, examples of which are shown in the accompanying drawings, wherein the same or similar reference numerals throughout represent the same or similar elements or elements having the same or similar functions. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, and should not be construed as limiting the present invention.

[0065] In addition, the terms "first" and "second" are used for descriptive purposes only and should not be understood as indicating or implying relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of the features. In the description of the present invention, the meaning of "plurality" is at least two, such as two, three, etc., unless otherwise clearly and specifically defined.

[0066] As used herein, the terms "uricase" and "uricase" are used interchangeably and refer to a class of enzymes described in the present invention that can catalyze the oxidation of uric acid to produce allantoin and hydrogen peroxide. The terms "uricase analogs", "uricase analogs", and "uricase derivatives" are used interchangeably and refer to the protein structure sequence of uricase oxidase, which can be subjected to structural modification such as partial amino acid substitution, deletion or addition, or xanthine according to an embodiment of the present invention can be added in the preparation process on the basis of retaining the activity of uricase oxidase in specifically catalyzing the conversion of uric acid into allantoin and hydrogen peroxide, thereby achieving the effects of this example, including but not limited to increasing the stability of the protein, etc.

[0067] There is no particular limitation on the uricase, and the uricase may be from any source and its analogs. Representative examples include but are not limited to mammalian sources, microorganisms, plants, and the like.

[0068] In another preferred embodiment, the uricase and uricase analogs thereof are derived from mammals, preferably the amino acid sequences shown in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 4, and more preferably SEQ ID NO: 1.

[0069] TYKKNDEVEFVRTGYGKDMIKVLHIQRDGKYHSIKEVATTVQLTLSSKKDYLHGDNSDVIPTDTIKNTVNVLAKFKGIKSIETFAVTICEHFLSSFKHVIRAQVYVEEVPWKRFEKNGVKHVHAFIYTPTGTHFCEVEQIRNGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATQVYCKWRYHQGRDVDFEATWDTVRSIVLQKFAGPYDKGEYSPSVQKTLYDIQVLTLGQVPEIEDMEISLPNIHYLNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRL(SEQ ID NO:1)。

[0070] MAHYRNDYKKNDEVEFVRTGYGKDMIKVLHIQRDGKYHSIKEVATSVQLTLSSKKDYLHGDNSDVIPTDTIKNTVNVLAKFKGIKSIETFAVTICEHFLSSFKHVIRAQVYVEEVPWKRFEKNGVKHVHAFIYTPTGTHFCEVEQIRNGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATQVYCKWRYHQGRDVDFEATWDTVRSIVLQKFAGPYDKGEYSPSVQKTLYDIQVLTLGQVPEIEDMEISLPNIHYLNIDMSKMGLINKEEVLLPLDNPYGRITGTVKRKLTSRL(SEQ ID NO:2)。

[0071] MYKNDEVEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSVQLTLSSKKDYVYGDNSDIIPTDTIKNTVHVLAKFKGIKSIETFAMNICEHFLSSFNHVIRAQVYVEEVPWKRFEKNGVKHVHAFIHNPTGTHFCEVEQMRSGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATKVYCKWRYHQGRDVDFEATWDTVRDIVLEKFAGPYDKGEYSPSVQKTLYDIQVHSLSRVPEMEDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRL(SEQ ID NO:3)。

[0072] MAHYHNDYKKNDEVEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSVQLTLSSKKDYVYGDNSDIIPTDTIKNTVHVLAKFKGIKSIETFAMNICEHFLSSFNHVIRAQVYVEEVPWKRFEKNGVKHVHAFIHNPTGTHFCEVEQMRSGPPVIHSGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATKVYCKWRYHQGRDVDFEATWDTVRDIVLEKFAGPYDKGEYSPSVQKTLYDIQVHSLSRVPEMEDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGRITGTAKRKLASKL(SEQ ID NO:4)。

[0073] MAHYHNDYQKNDEVEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSVQLTLNSRREYLHGDNSDIIPTDTIKNTVQVLAKFKGIKSIETFAMNICEHFLSSFNHVIRVQVYVEEVPWKRFEKNGVKHVHAFIHTPTGTHFCEVEQLRSGPPVIHSGIKDLKVLKTTQSGFEGFLKDQFTTLPEVKDRCFATQVYCKWRYHQGRDVDFEATWEAVRGIVLKKFAGPYDKGEYSPSVQKTLYDIQVLSLSQLPEIEDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGRITGTVKRKLTSRL(SEQ ID NO:5)。

[0074] MAHYHNDYKKNDEVEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSVQLTLSSKKDYLHGDNSDIIPTDTIKNTVHALAKFKGIKSIEAFAVNICQHFLSSFNHVIRTQVYVEEIPWKRLEKNGVKHVHAFIHTPTGTHFCEVEQLRSGPPVIH SGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFAAQVYCKWRYHQCRDVDFEATWDTIRDVVLEKFAGPYDKGEYSPSVQKTLYDIQVVSLSQVPEIDDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRL(SEQ ID NO:6).

[0075] MADYHNNYKKNDELEFVRTGYGKDMVKVLHIQRDGKYHSIKEVATSVQLTLSSKKDYLHGDNSDIIPTDTIKNTVHVLAKFKGIKSIEAFGVNICEYFLSSFNHVIRAQVYVEEIPWKRLEKNGVKHVHAFIHTPTGTHFCEVEQLRSGPPVIH SGIKDLKVLKTTQSGFEGFIKDQFTTLPEVKDRCFATQVYCKWRYHQCRDVDFEATWGTIRDLVLEKFAGPYDKGEYSPSVQKTLYDIQVLSLSRVPEIEDMEISLPNIHYFNIDMSKMGLINKEEVLLPLDNPYGKITGTVKRKLSSRL(SEQ ID NO:7).

[0076] Among them, the amino acid sequence shown in SEQ ID NO:1 is the amino acid sequence of a chimeric uricase from pig and baboon (pig-baboon); the amino acid sequence shown in SEQ ID NO:2 is the amino acid sequence of pig uricase; the amino acid sequence shown in SEQ ID NO:3 is the amino acid sequence of a chimeric uricase from dog and baboon (dog baboon); the amino acid sequence shown in SEQ ID NO:4 is the amino acid sequence of dog uricase; the amino acid sequence shown in SEQ ID NO:5 is the amino acid sequence of bovine uricase; the amino acid sequence shown in SEQ ID NO:6 is the amino acid sequence of monkey uricase; and the amino acid sequence shown in SEQ ID NO:7 is the amino acid sequence of baboon uricase.

[0077] The uricase having the 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%, or at least 99% identity with SEQ ID NO: 1-7; or a polypeptide having structural homology with one or more amino acid substitutions, deletions, and / or additions compared to SEQ ID NO: 1-7, are all within the scope of the uricase of the present application.

[0078] The urate oxidases from different species described in the present invention can be obtained through various ways, including but not limited to natural extraction, chemical synthesis, genetic engineering recombinant expression, etc.

[0079] In another preferred embodiment, the uricase protein sequence (SEQ ID NO: 1) is recombinantly expressed in a host cell by recombinant technology.

[0080] In another preferred embodiment, the recombinant expression strain is prepared by using Escherichia coli or yeast as a host, and more preferably, Escherichia coli is used as a host strain for recombinant expression.

[0081] S1 (from Aspergillus flavus) urate oxidase sequence information:

[0082] SAVKAARYGKDNVRVYKVHKDEKTGVQTVYEMTVCVLLEGEIETSYTKADNSVIVATDSIKNTIYITAKQNPVTPPELFGSILGTHFIEKYNHIHAAHVNIVCHRWTRMDIDGKPHPHSFIRDSEEKRNVQVDVVEGKGIDIKSSLSGLT VLKSTNSQFWGFLRDEYTTLKETWDRILSTDVDATWQWKNFSGLQEVRSHVPKFDATWATAREVTLKTFAEDNSASVQATMYKMAEQILARQQLIETVEYSLPNKHYFEIDLSWHKGLQNTGKNAEVFAPQSDPNGLIKCTVGRSSLKSKL

[0083] S3 (Micrococcus) urate oxidase sequence information:

[0084] MTNIILGKNQYGKAEVRLVKVTRNTCRHEIQDLNVTSQLRGDFEAAAHTAGDNAHVVATDTQKNTVYAFARDGFATTEEFLLRLGKHFTEGFDWVTGGRWAAQQFFWDRINDHDHAFSRNKSEVRTAVLEISGSEQAIVAGIEGL TVLKSTGSEFHGFPRDKYTTLPETEDRILATDVSARWRYNTVEVDFDAVYADVRGLLLKAFAETHSLALQQTMYEMGRAVIETHPEIDEIKMSLPNKHHFLVDLSPFGQDNPNEVFYAADRPYGLIEATIQREGCRADHPIWSN

[0085] As used herein, the polyethylene glycol uricase of the present invention refers to uricase obtained by covalently modifying uricase with polyethylene glycol. The polyethylene glycol (PEG) refers to a mixture of ethylene oxide condensation product and water, represented by the general formula H(OCH2CH2)nOH, which is a pH neutral, non-toxic, highly water-soluble hydrophilic polymer with a linear or branched structure.

[0086] The object of the present invention is to provide a method for preparing uricase non-covalently bound to xanthine and a method for chemically modified uricase, which can be introduced into the preparation process of uricase or chemically modified uricase.

[0087] Another object of the present invention is to provide a stabilizer for uricase and chemically modified uricase. Specifically, the stabilizer is xanthine, which can significantly improve the in vivo and in vitro stability of uricase and chemically modified uricase.

[0088] Xanthine is widely distributed in human organs and body fluids. It is often used as a mild stimulant and bronchodilator and can be used to treat asthma symptoms. The mechanism of action of xanthine drugs is mainly to inhibit phosphodiesterase that can decompose and destroy cAMP, increase the intracellular cAMP content, and thus produce a wide range of physiological activities. According to its pharmacological activity, this type of drug is mainly used in central nervous system excitation, cardiovascular and cerebrovascular dilation, increasing glomerular filtration rate and increasing primary urine volume to exert diuretic and antiasthmatic effects (Ren Junguo, New Progress in Xanthine Drugs; Foreign Medicine. Synthetic Drugs. Biochemical Drugs. Preparations, 1989). Therefore, as a stabilizer for drug preparation, it has no safety issues itself, and can improve the in vivo and in vitro stability of urate oxidase and its pegylated urate oxidase, which improves and improves the safety and effectiveness of the drug.

[0089] According to the embodiments of the present invention, the uricase combined with xanthine of the present invention can greatly improve the in vivo stability of uricase while ensuring the enzyme activity to the maximum extent. Therefore, the polyethylene glycol-modified uricase combined with xanthine of the present invention and the pharmaceutical composition comprising the polyethylene glycol-modified uricase can be administered for the treatment or prevention of hyperuricemia-related diseases.

[0090] As used herein, the term "administration" refers to the introduction of a predetermined amount of a substance into a patient by some suitable means. The polyethylene glycol-modified uricase of the present invention can be administered by any common route, as long as it can reach the desired tissue. Various modes of administration are contemplated, including peritoneal, intravenous, intramuscular, subcutaneous, cortical, oral, topical, nasal, pulmonary and rectal administration, but the present invention is not limited to these exemplified modes of administration. 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.

[0091] The administration frequency and dosage of the pharmaceutical composition of the present invention can be determined by a number of relevant factors, including the type of disease to be treated, the administration route, the patient's age, sex, weight and severity of the disease, and the type of drug as the active ingredient.

[0092] The term "therapeutically effective amount" refers to an amount of a compound sufficient to significantly improve certain symptoms associated with a disease or condition, that is, an amount that provides a therapeutic effect for a given condition and dosing regimen. For example, in the treatment of chronic hyperuricemia or gout, a drug or compound that reduces, prevents, delays, inhibits or blocks any symptom of a disease or condition should be therapeutically effective. A therapeutically effective amount of a drug or compound does not need to cure a disease or condition, but will provide treatment for the disease or condition so that the onset of the individual's disease or condition is delayed, stopped or prevented, or the symptoms of the disease or condition are alleviated, or the duration of the disease or condition is changed, or, for example, the disease or condition becomes less severe, or recovery is accelerated.

[0093] The term "treatment" is used to refer to obtaining a desired pharmacological and / or physiological effect. The effect may be preventive in terms of completely or partially preventing a disease or its symptoms, and / or may be therapeutic in terms of partially or completely curing a disease and / or the adverse effects caused by the disease. "Treatment" as used herein covers the treatment of mammals, especially human diseases (mainly hyperuricemia-related diseases), including: (a) preventing the disease in individuals who are susceptible to the disease but have not yet been diagnosed with the disease; (b) inhibiting the disease, such as blocking the progression of the disease; or (c) alleviating the disease, such as alleviating symptoms associated with the disease. "Treatment" as used herein covers any medication that administers a drug or compound to an individual to treat, cure, alleviate, improve, reduce or inhibit an individual's disease, including but not limited to administering a polyethylene glycol-modified uricase as described herein to an individual in need.

[0094] According to an embodiment of the present invention, the uricase or pharmaceutical composition combined with xanthine of the present invention can be used in combination with conventional treatment methods and / or therapies, or can be used separately from conventional treatment methods and / or therapies. When the uricase or pharmaceutical composition combined with xanthine of the present invention is administered in a combination therapy with other drugs, they can be administered to an individual sequentially or simultaneously. Alternatively, the pharmaceutical composition of the present invention can include a combination of the uricase of the present invention combined with xanthine, a pharmaceutically acceptable carrier or a pharmaceutically acceptable excipient, and other therapeutic or preventive drugs known in the art.

[0095] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the various technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution, which can be more clearly understood by referring to the following examples. Due to space limitations, the examples are only for illustrative purposes and are not intended to limit the present invention.

[0096] Embodiments of the present invention will be described in further detail below, examples of which are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be construed as limiting the present invention.

[0097] Example 1 Preparation of uricase from different sources

[0098] Uricase from three different sources (Micrococcus, Aspergillus flavus, and pig-baboon) was prepared. The specific sample information is shown in Table 1.

[0099] Table 1 Information on urate oxidase from different sources and modified urate oxidase

[0100]

[0101] 1. S2 enzyme preparation process:

[0102] 1.1 Construction of genes and expression plasmids for uricase expression

[0103] According to the E. coli codon usage preference data, combined with codon preference and GC content, the cDNA sequence of uricase protein (code: 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 used as the target gene insertion sites, and pET-30a plasmid was used as the expression vector (pET-30a-PHC).

[0104] 1.2 Transformation of expression plasmids into bacterial host cells

[0105] The expression vector pET-30a-PHC was introduced into Escherichia coli BL21 (DE3) using the CaCl2 method, and Kanamycin was used for resistance screening to select high-expression clones, and the original seed bank strain (E3B) was preserved. These steps were implemented according to common methods in the field of molecular biology.

[0106] 1.3 Preparation of recombinant uricase

[0107] The transformed engineered strains were fermented and expressed in a fermenter under the following conditions: first cultured at 30°C and pH about 7.2 until OD 600 When the cell culture temperature is above 30, add IPTG to 0.5mmol / L and continue induction for more than 3h to allow urate oxidase to accumulate. Collect the cells by centrifugation and store them below -15℃.

[0108] Take the frozen bacterial cells and suspend them in 25mmol / L Tris, 5mmol / L EDTA buffer at a suspension ratio of 1:10 (W / V). After breaking the bacterial cells with high pressure, collect the urate oxidase precipitate by centrifugation, wash the precipitate once with 50mmol / L NaHCO3, and suspend the enriched urate oxidase precipitate in 100mmol / L Na2HCO3 (pH9.7-10.3) buffer at a suspension ratio of 1:50 (W / V), stir at room temperature overnight to dissolve, and then collect the supernatant by centrifugation.

[0109] Urate oxidase was further purified through several chromatographic steps. The purity was greater than 95% as detected by SDS-PAGE and greater than 95% as detected by Superdex 200 column. The protein concentration was determined by Lowry method and the activity of uricase was determined by spectrophotometer. One unit (U) of enzyme activity was defined as the amount of enzyme required to convert 1 μmol of uric acid per minute under the optimum reaction temperature of 37°C and the optimum pH buffer of 9.0.

[0110] 2. S5 enzyme preparation process:

[0111] 5KD molecular weight N-succinimidyl propionate PEG (5K-PEG-SPA) is dissolved with 2mmol / L HCl to form a 200mmol / L PEG solution, and after dissolution, the solution is added to a carbonate buffer solution with a carbonate concentration of 0.1-0.3mol / L and a pH of 10.0 containing urate oxidase at a molar ratio of 1:55-1:95 (uricase: 5K-PEG-SPA), wherein the molar ratio of uricase oxidase to 5K-PEG-SPA is calculated in monomer form, i.e., the molar ratio of 1:55-1:95 refers to the molar ratio of uricase oxidase in monomer form to 5K-PEG-SPA, so that PEG and uricase undergo coupling reaction, and the coupling reaction needs to be stirred at 5-30°C for more than 60 minutes until the degree of PEG coupling no longer changes with time. After the reaction is completed, the unmodified PEG and by-products are removed from the reaction by ultrafiltration and / or chromatography. A suitable molecular sieve chromatography medium can be selected to separate and remove the modified by-products. Finally, the 5K PEG-modified urate oxidase (code-named PU5) was obtained by sterile filtration.

[0112] 3. S3 enzyme preparation process:

[0113] Using E. coli BL21 (DE3) host bacteria, PET28a was used as an expression vector, and the nucleic acid sequence expressing urate oxidase was introduced between the NcoI and XhoI double restriction sites to construct an expression bacterium. Through transformation experiments, the recombinant plasmid containing the S3 enzyme expression gene was introduced into the E. coli BL21 (DE3) host bacteria, and the target strain containing the expression gene was screened in a medium containing 50 μg / L kanamycin. The screened strain was cultured overnight at 30°C in 100 ml LB medium containing glucose (containing 50 μg / L kanamycin) to expand the strain, and the OD 600 The expanded bacterial suspension was inoculated into 5L of fresh LB medium and cultured at 37℃ for 4h. 600 The temperature was about 2.5, and the temperature was lowered to 25°C. IPTG was added to make the final concentration 0.5mmol / L to induce the expression of S3 enzyme. The induction lasted for about 3.5h. After the induction, OD 600About 3.5. After the culture was completed, the bacterial suspension was centrifuged to collect the bacterial cells to obtain the bacterial cells expressing the S3 enzyme. The fermented bacterial cells were resuspended in a lysis buffer (200mM NaH2PO4-Na2HPO4, 50mM EDTA) at a ratio of 1:10, and after high-pressure homogenization and crushing, the supernatant was loaded into the Xanthine Agrose filler, eluted and purified by eluent (100mM Na2CO3-NaHCO3, 0.5mM Uricase), and then the SEC solution was changed to the equilibrium solution (200mM NaH2PO4-Na2HPO4), and finally ultrafiltration and concentration were performed to obtain the S3 enzyme.

[0114] 4. S4 enzyme preparation process:

[0115] The 5KD molecular weight methoxy PEG maleimide (M-PEG-MAL) was dissolved with 1mmol / L HCl to form a 200mmol / L PEG solution, which was added to the S1 enzyme solution in a certain proportion for modification for 1h. Finally, the solution was replaced by ultrafiltration and concentrated to obtain the S4 enzyme solution.

[0116] 5. S6 enzyme preparation process:

[0117] The 5KD molecular weight methoxy PEG maleimide (M-PEG-MAL) was dissolved with 1mmol / L HCl to form a 200mmol / L PEG solution, which was added to the S3 enzyme solution in a certain proportion for modification for 1h. Finally, the solution was replaced by ultrafiltration and concentrated to obtain the S6 enzyme solution.

[0118] Example 2 Effect of xanthine on the stability of urate oxidase from different sources

[0119] 1. Detection method of high and low molecular weight proteins of urate oxidase

[0120] Use SEC, 7.8×300mm chromatographic column or equivalent column; use 0.05mol / L Na2HPO4-NaH2PO4, 0.3mol / LNaCl, pH 7.0 as mobile phase; set the flow rate of HPLC to 0.5ml / min, the detection wavelength to 280nm; isocratic elution for 35min; injection volume is 10μl.

[0121] 2. Enzyme activity detection method

[0122] Method for determining the activity of urate oxidase and pegylated urate oxidase: Using an ultraviolet spectrophotometer, taking advantage of the characteristic that the substrate uric acid has a maximum ultraviolet absorption wavelength of 293nm, within a certain concentration range, the absorption value of uric acid at 293nm is proportional to its concentration. Therefore, the ultraviolet spectrophotometer detects the wavelength of 293nm, uses the buffer as a blank control, and calibrates the absorption zero point; in the quartz cuvette, add 2.95ml of substrate reaction solution, then add 50μl of the test solution and mix quickly, and continuously measure the 293nm light absorption value. Calculate the uric acid degradation concentration according to C=A / εL (where A is the absorbance value of a specific concentration of uric acid at 293nm, ε is the molar extinction coefficient of uric acid, L is the optical path of the cuvette, and C is the molar concentration of uric acid), and calculate the enzyme activity.

[0123] Definition of uricase oxidase and pegylated uricase activity: At the optimal reaction temperature of 37°C and the optimal reaction pH of 9.5, the amount of enzyme required to convert 1 μmol uric acid into allantoin per minute is defined as one activity unit (U).

[0124] 3. Effect of xanthine on the stability of urate oxidase from different sources

[0125] (1) Comparison of the effect of xanthine on the stability of enzymes derived from Aspergillus flavus and mammals

[0126] In the embodiment of the present invention, xanthine was added to the S1 and S2 samples at a molar ratio of 26 times (xanthine: uricase); the samples were packaged and placed at 37°C for stability investigation, and the investigation time points were day 0, 1.5 days, 6 days, and 16 days. The investigation indicators included high and low molecular weight protein content and enzyme specific activity. The quantitative results of small molecule content and enzyme activity in each group of samples are shown in Table 2.

[0127] Table 2 Stability of xanthine to urate oxidase from Aspergillus flavus and mammals

[0128]

[0129]

[0130] By comparing the stability of xanthine on enzymes from different sources, it can be seen that xanthine can not only significantly improve the product stability of commercial products and PHC, but also reduce the molecular protein content. By comparing the test results at 0d and different days, it can be seen that the test method results are stable.

[0131] (2) Comparison of the effect of xanthine on the stability of enzymes from Micrococcus

[0132] Table 3 Xanthine stability of urate oxidase from Micrococcus

[0133]

[0134] From the SEC results of the stability test at 2-8°C in Table 3, it can be seen that the total peak area of ​​the group with small molecules added on day 15 is larger and the main peak ratio is higher. Xanthine can improve the stability of the enzyme derived from Micrococcus to a certain extent.

[0135] Example 3 Preparation of uricase oxidase or polyethylene glycol-modified uricase containing xanthine during fermentation

[0136] Urate oxidase was prepared by referring to the method described for enzyme S2 in Example 1, and the fermentation broth contained no less than 0.1 g / L of xanthine. The fermentation product was further purified by breaking bacteria, ion exchange or hydrophobic chromatography to obtain uricase that non-covalently bound xanthine. RP-HPLC and SEC-HPLC tests proved that the uricase prepared by recombinant expression contained non-covalently bound xanthine. The RP-HPLC and SEC-HPLC test diagrams are shown in FIG. Figure 1 and Figure 2 shown.

[0137] According to the method described in Example 1, the recombinantly expressed uricase was modified with polyethylene glycol.

[0138] Example 4 Purification and preparation process of urate oxidase or polyethylene glycol-modified urate oxidase containing xanthine

[0139] Urate oxidase was prepared by referring to the method described in Example 1. A uricase protein solution was obtained by breaking the bacteria, ion exchange or hydrophobic chromatography, polyethylene glycol modification and purification. In the uricase protein solution containing a certain amount of xanthine, uricase non-covalently bound to xanthine was prepared.

[0140] The polyethylene glycol modified uricase was prepared by referring to the method described in Example 1 for the enzyme S2. The polyethylene glycol modified uricase protein solution was obtained by breaking the bacteria, ion exchange or hydrophobic chromatography, polyethylene glycol modification and purification. A certain amount of xanthine was added before the polyethylene glycol modified uricase protein or before the polyethylene glycol modified uricase protein solution, so that the polyethylene glycol uricase non-covalently bound to xanthine could be prepared. The chromatograms detected by SEC-HPLC before and after the addition of xanthine are shown in FIG. Figure 3 and Figure 4 shown.

[0141] Example 5 Effects of different xanthine contents on the activity of uricase and pegylated uricase

[0142] Referring to Example 2, the enzyme activity detection method is used. Taking pegylated uricase as an example, pegylated uricase is taken and dissolved in ultrapure water to prepare a solution. The pegylated uricase solution is mixed with different amounts of xanthine according to the molar ratio of 0.05 mg / ml pegylated uricase to xanthine of 1:0, 1:3, 1:11, and 1:44, respectively, and then the urate oxidase and pegylated uricase enzyme activity are measured according to the above-mentioned urate oxidase and pegylated uricase enzyme activity determination method. The measurement results are shown in Table 4.

[0143] Table 4 Effect of xanthine on the activity of PEGylated urate oxidase

[0144]

[0145] As shown in Table 4 above, the addition of xanthine had no effect on the activity of pegylated uricase.

[0146] Example 6 Effects of different xanthine contents on the stability of urate oxidase and pegylated urate oxidase

[0147] The results of the study showed that xanthine has good solubility under alkaline conditions. As the amount of xanthine added increases, the pH of the solution needs to be further increased to dissolve xanthine. However, under high pH conditions, it will cause protein denaturation. After screening the concentration of xanthine, the molar concentration ratio of xanthine to uricase was selected in the range of (1.6-26):1 to study the effect of xanthine on the stability of uricase. Taking the modified pegylated uricase from mammals as an example, the final concentration of pegylated uricase protein was 6 mg / ml. Pegylated uricase samples containing different concentrations of xanthine of 2.56 μg / ml, 42.24 μg / ml, and 168.96 μg / ml were prepared and placed at 37°C for accelerated observation for 0, 1 month, and 2 months. The stability change trend of pegylated uricase tetramer was detected by the pegylated uricase high and low molecular weight protein detection method. The test results are shown in Table 5:

[0148] Table 5 Effects of different xanthine contents on the stability of PEGylated urate oxidase (37°C)

[0149]

[0150]

[0151] The results of the effect of different xanthine contents on the stability of pegylated uricase show that when the molar ratio of xanthine to pegylated uricase is in the range of 1.6:1 to 25.3:1, the stability of pegylated uricase tetramer tends to be more stable with the increase of xanthine concentration. The results of the study combined with Example 2 show that xanthine has a stabilizing effect on the stability of uricase from different sources within a 26-fold molar ratio (xanthine: uricase).

[0152] During the research process, the inventors found that when the xanthine content reached 168.96 μg / ml, it was basically saturated. The molar ratio of xanthine to pegylated uricase was studied to be higher than 26:1. It was necessary to continue to increase the pH of the solution to increase the solubility of xanthine, because the high pH range will cause the uricase protein to denature, thereby affecting the effect of xanthine in improving stability. Therefore, at a concentration of 6 mg / ml pegylated uricase, the optimal molar ratio concentration range of xanthine to the stability of uricase and pegylated uricase is selected within the range of (1 to 26):1.

[0153] The researchers of the present application found that the amount of xanthine added is related to the concentration of uricase oxidase or polyethylene glycol uricase, and the amount of xanthine added may be reduced as the concentration of uricase oxidase or polyethylene glycol uricase in the uricase oxidase solution increases.

[0154] Based on the above research, the inventors referred to the method described above and studied the optimal amount of xanthine added to 1 mg / ml, 8 mg / ml, and 12 mg / ml uricase solutions or polyethylene glycol uricase solutions, and found that when the final concentration of uricase protein was 1 mg / ml, the molar ratio of xanthine to uricase tetramer was (0.5-200):1, preferably (1-156):1, which had the best effect on improving the stability of uricase or polyethylene glycol uricase; for uricase oxidase, the optimal amount of xanthine added to uricase tetramer was (0.5-200):1, preferably (1-156):1, which had the best effect on improving the stability of uricase or polyethylene glycol uricase; for uricase When the final protein concentration is 8 mg / ml, the molar ratio of xanthine to urate oxidase tetramer is (0.5-25):1, preferably (1-20):1, which has the best effect on improving the stability of urate oxidase or polyethylene glycol urate oxidase; when the final urate oxidase protein concentration is 12 mg / ml, the molar ratio of xanthine to urate oxidase tetramer is (0.5-17):1, preferably (1-13):1, which has the best effect on improving the stability of urate oxidase or polyethylene glycol urate oxidase.

[0155] In summary, within the range of 1 to 12 mg / ml, the amount of xanthine added is in the range of (0.5 to 200):1, preferably in the range of (1 to 156):1 (molar ratio of xanthine to urate oxidase tetramer), which has the best effect on improving the stability of urate oxidase or polyethylene glycol-containing urate oxidase.

[0156] Example 7 Effects of xanthine on the pharmacokinetics and pharmacodynamics of pegylated urate oxidase injection in rats

[0157] Taking the modified polyethylene glycol-modified urate oxidase from mammals as an example, the pharmacokinetic and pharmacodynamic indicators of xanthine-containing and non-xanthine-containing pegylated urate oxidase in rats were evaluated in vivo.

[0158] Principle of pharmacokinetic (PK) test of pegylated uricase oxidase: The concentration of pegylated uricase injection in SD rat serum samples was determined by a methodologically validated analytical method (fluorescence method). Uricase catalyzes the conversion of uric acid into allantoin, H2O2 and CO2. H2O2 reacts with Amplex Red Reagent (10-acetyl-3,7-dihydroxyphenazine) in the presence of horseradish peroxidase (HRP) in a 1:1 ratio to generate a red fluorescent oxidation product, resorufin. The amount of pegylated uricase injection is correlated with the depth of color produced by the final reaction. For fluorescence detection, the excitation light is set to 485nm and the emission light is set to 630nm.

[0159] Principle of Detection of Uric Acid (PD) in Serum: The concentration of uric acid in SD rat serum samples is directly detected by biochemical analyzer (Enzymatic Colorimetric Test). Uric acid in the sample generates allantoin and hydrogen peroxide (H2O2) under the action of uricase, and H2O2 oxidizes 4-aminoaminopyrine (4-AAP) under the action of peroxidase (POD) to generate red quinone imine. The absorbance change is measured at 520nm, and the absorbance change is proportional to the uric acid concentration in the sample.

[0160]

[0161]

[0162] The reaction formula is:

[0163] Test samples: xanthine-free PEGylated uricase, code: PEG-Uricase(-X); xanthine-containing PEGylated uricase, code: PEG-Uricase(+X), each 1mg PEGylated uricase protein contains 1.5μg xanthine.

[0164] Animal grouping: Twelve SD rats were randomly divided into two groups, with 6 rats in each group (half male and half female). The dosage of the rats was 1 mg / kg according to the urate oxidase protein content, and the rats were given intravenously as a single dose, namely, the PEG-Uricase (-X)-iv group and the PEG-Uricase (+X)-iv group.

[0165] Blood collection time: before administration, and 1 day, 3 days, 5 days, 7 days, 9 days, 11 days, and 13 days after administration to measure blood drug concentration and serum uric acid level.

[0166] The blood drug concentration measurement statistics are shown in Table 6 and the blood drug concentration change curve is shown in Figure 5 As shown, it can be seen that on the 5th day, the concentration of xanthine-containing PEGylated uricase in the blood is much greater than that of xanthine-free PEGylated uricase, and on the 9th day, the concentration of xanthine-free PEGylated uricase in the blood is completely below the detection limit, while the average concentration of xanthine-containing PEGylated uricase in the blood is 0.081 μg / ml. This shows that the added xanthine can improve the stability of PEGylated uricase in the blood and slow down the clearance of PEGylated uricase by the system.

[0167] The statistics of serum uric acid content are shown in Table 7 and the changes of serum uric acid level are shown in Table 7. Figure 6 As shown in the figure, it can be seen that within the first 5 days, the uric acid content of both xanthine-containing PEGylated uricase and non-xanthine PEGylated uricase in serum was below the detection limit. On the 7th day, the uric acid content of non-xanthine PEGylated uricase in serum began to exceed the detection limit, while the uric acid content of xanthine PEGylated uricase in serum was still below the detection limit. On the 9th day, the uric acid content of non-xanthine PEGylated uricase in serum was much higher than the detection limit. In the following days, the uric acid content in the PEGylated uricase containing xanthine was always greater than that in the PEGylated uricase containing xanthine. This shows that the stability of PEGylated uricase containing xanthine in serum is significantly higher than that of PEGylated uricase not containing xanthine, and it can inhibit the formation of uric acid in serum and reduce the content of uric acid in serum for a longer time.

[0168] Table 6 Statistical table of blood drug concentration determination

[0169]

[0170] * Note: ND means the content is below the detection limit, the same below.

[0171] Table 7 Statistics of uric acid content in serum

[0172]

[0173]

[0174] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0175] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A uricase composition, characterized in that: The invention comprises an active ingredient and a stabilizer, wherein the active ingredient comprises urate oxidase, and the stabilizer comprises xanthine.

2. The uricase composition according to claim 1, characterized in that: The molar ratio of xanthine to urate oxidase tetramer is not less than 0.5:

1.

3. The uricase composition according to claim 1, characterized in that: The uricase is naturally extracted uricase, recombinantly expressed uricase or modified uricase; Optionally, the naturally extracted urate oxidase is derived from Aspergillus flavus, wild-type Micrococcus or mammals; Optionally, the modified uricase comprises at least one selected from polyethylene glycol-modified uricase, polysaccharide side chain-modified uricase or poly-oligopeptide-modified uricase.

4. The uricase composition according to claim 1, characterized in that: The urate oxidase is bound to xanthine via a non-covalent bond; Optionally, the uricase binds to xanthine via hydrogen bonding, salt bonding, hydrophobic interaction or van der Waals force.

5. The uricase composition according to claim 1, characterized in that: The concentration of the uricase in the composition is 1-12 mg / ml, and the molar ratio of xanthine to uricase tetramer is (0.5-200):1, preferably (1-156):1; Optionally, the concentration of the uricase in the composition is 1 mg / ml, and the molar ratio of xanthine to uricase tetramer is (0.5-200):1, preferably (1-156):1; Optionally, the concentration of the uricase in the composition is 6 mg / ml, and the molar ratio of xanthine to uricase tetramer is (0.5-34):1, preferably (1-26):1; Optionally, the concentration of the uricase in the composition is 8 mg / ml, and the molar ratio of xanthine to uricase tetramer is (0.5-25):1, preferably (1-20):1; Optionally, the concentration of uricase in the composition is 12 mg / ml, and the molar ratio of xanthine to uricase tetramer is (0.5-17):1, preferably (1-13):

1.

6. The uricase composition according to claim 1, characterized in that: Further comprising a pharmaceutically acceptable excipient or carrier; Optionally, the excipient or carrier is selected from at least one of the following: hydroxypropyl methylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, mannitol, sucrose, fructose, methyl cellulose, vinyl acetate copolymer, chitosan, sodium alginate, xanthan gum, sodium carboxymethyl cellulose, polyethylene oxide, methacrylic acid copolymer, maleic anhydride-methyl vinyl ether copolymer, carbomer, polyvinyl pyrrolidone, ethyl cellulose, cellulose acetate, methacrylate, polyoxyethylene, polyvinyl alcohol, behenyl glyceryl, monostearate, cellulose acetate, cellulose acetate phthalate, ethyl cellulose, PLA, PLGA and polyethylene glycol.

7. The uricase composition according to claim 6, characterized in that: The composition is an injection.

8. Use of xanthine in improving the stability of urate oxidase.

9. A method for improving the stability of urate oxidase, characterized in that: Urate oxidase is contacted with xanthine.

10. A method for preparing urate oxidase, characterized in that: Urate oxidase is contacted with xanthine.

11. The method according to claim 9 or 10, characterized in that: The uricase is naturally extracted uricase, recombinantly expressed uricase or modified uricase; Optionally, the naturally extracted urate oxidase is derived from Aspergillus flavus, wild-type Micrococcus or mammals; Optionally, the modified uricase comprises at least one selected from polyethylene glycol-modified uricase, polysaccharide side chain-modified uricase or poly-oligopeptide-modified uricase.

12. The method according to claim 9 or 10, characterized in that: Urate oxidase contacts xanthine in the following way: The engineered bacteria carrying the nucleic acid molecule expressing the uricase oxidase are fermented in a fermentation liquid containing xanthine to obtain the uricase oxidase.

13. The method according to claim 12, characterized in that The amount of xanthine in the xanthine-containing fermentation liquid is not less than 0.1 g / L.

14. The method according to claim 12, characterized in that Further including: The uricase is subjected to polyethylene glycol modification and purification treatment to obtain polyethylene glycol-modified uricase.

15. The method according to any one of claims 9 to 11, characterized in that: The contacting of uricase oxidase with xanthine is performed as follows: uricase oxidase modified with polyethylene glycol is contacted with xanthine.

16. A urate oxidase, characterized in that: It is obtained according to the method according to any one of claims 10 to 15.

17. Use of the uricase composition according to any one of claims 1 to 7 or the uricase according to claim 16 in the preparation of a medicament for preventing or treating a hyperuricemia-related disease; Optionally, the hyperuric acid-related diseases include chronic hyperuricemia, gout, kidney disease, hyperuricemia arthritis, kidney stones, gouty nodules, hypertension, diabetes, hypertriglyceridemia, metabolic syndrome, coronary heart disease, atherosclerosis, and hyperuricemia caused by cancer chemotherapy.

18. Use of the uricase composition according to any one of claims 1 to 7 or the uricase according to claim 16 for reducing the level of uric acid in a biological fluid; Optionally, the biological fluid is urine or blood.

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