Preparation method and application of recombinant porcine-human urate oxidase enteric-coated capsules and enteric-coated tablets

Abstract

The present invention relates to a preparation method and application of a recombinant porcine-human urate oxidase enteric-coated capsule and enteric-coated tablet. The method comprises: using a modifier monomethoxy polyethylene glycol succinimidyl propionate or monomethoxy polyethylene glycol succinimide to perform PEGylation modification on the purified recombinant porcine-human urate oxidase, performing molecular sieve chromatography on the modified product to obtain PEGylated recombinant porcine-human urate oxidase, and then preparing it into a recombinant porcine-human urate oxidase enteric-coated capsule or enteric-coated tablet. The molecular weight of mPEG-SPA is 5kDa, and the end group substitution rate is 95%; the molecular weight of mPEG-SC is 10kDa, and the end group substitution rate is 99%. The molar ratio of the modifier to the recombinant porcine-human urate oxidase is 3, 6 or 12. The recombinant porcine-human urate oxidase enteric-coated preparation prepared by the method of the present invention overcomes the defects of the recombinant porcine-human urate oxidase injection in terms of convenience, safety and repeated use effectiveness, significantly improves the convenience and safety of drug use, and has a good market promotion prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of biopharmaceuticals, and in particular relates to a preparation method and application of recombinant porcine-human urate oxidase oral enteric-coated capsules and enteric-coated tablets for reducing the uric acid content in human blood. Background Art

[0002] Uricase (Urate Oxidase, EC 1.7.3.3, UOX or UO) can catalyze the oxidation of uric acid to produce allantoin, CO2 and H2O2. This enzyme is found in the liver, kidney and other tissues of most vertebrates, but birds, humans and some primates with similar genes to humans have lost the ability to synthesize active urate oxidase due to gene mutations.

[0003] Uric acid is the final product of purine metabolism in the human body. Uric acid exists mainly in the form of sodium salt in the body. The solubility of free uric acid and its sodium salt in water is very low. If the body's purine metabolism is disturbed and excessive uric acid is produced, or uric acid excretion is blocked, the uric acid concentration in the blood will increase, causing diseases such as hyperuricemia and gout. Malignant blood system diseases and tumor chemotherapy can also cause a sharp increase in blood uric acid concentration.

[0004] Under normal physiological conditions, uric acid is mainly excreted by the kidneys, accounting for about 2 / 3 of its excretion (about 400 mg / day), and the other 1 / 3 of uric acid (about 200 mg / day) is eliminated through intestinal secretion. In patients with chronic kidney disease, uric acid excreted through intestinal secretion can increase to 50%-70% of the total. Most of the uric acid secreted by the kidneys and intestines will be reabsorbed into the blood circulation, affecting the effective excretion of uric acid. Modern pharmacological studies have confirmed that oral administration of different dosage forms of urate oxidase can quickly decompose uric acid secreted by the intestines, greatly reducing the reabsorption of uric acid in the intestines, thereby reducing the uric acid level in the blood.

[0005] In June 2011, our research team successfully applied for the national invention patent of "A method for preparing and using a PEGylated recombinant porcine-human urate oxidase fusion protein", and obtained the authorization from the State Intellectual Property Office on April 3, 2013, with the patent number: ZL 201110181447.X. This invention uses recombinant porcine-human urate oxidase to prepare an injection that can quickly reduce the uric acid content in human blood after PEGylation. However, this invention still has some imperfections in terms of convenience of use, effectiveness of repeated use, and safety of use. Based on this invention, our research team conducted further in-depth research and successfully developed a method for preparing oral preparations using the recombinant porcine-human urate oxidase and expanded its application scenarios. Summary of the invention

[0006] In order to overcome the defects of recombinant porcine-human urate oxidase injection in terms of convenience, safety and repeated use effectiveness, the present invention provides a new method for preparing recombinant porcine-human urate oxidase enteric-coated capsules or enteric-coated tablets. The present invention proposes a method for preparing oral enteric-coated capsules or enteric-coated tablets using PEGylated or non-PEGylated recombinant porcine-human urate oxidase. The present technology can also be derived or expanded to prepare other dosage forms. The present invention also relates to the use of enteric-coated capsules and enteric-coated tablets prepared by the present method in reducing the uric acid content in human blood.

[0007] Specifically, the present invention provides a method for preparing recombinant porcine-human urate oxidase enteric-coated capsules or enteric-coated tablets, the method comprising: using a modifier to perform PEGylation modification on purified recombinant porcine-human urate oxidase (rPHUOX), performing molecular sieve chromatography on the modified product to obtain PEGylated recombinant porcine-human urate oxidase, and then preparing the PEGylated recombinant porcine-human urate oxidase into recombinant porcine-human urate oxidase enteric-coated capsules or enteric-coated tablets; The modifying agent includes, but is not limited to, monomethoxy polyethylene glycol succinimidyl propionate (mPEG-SPA) and monomethoxy polyethylene glycol succinimide (mPEG-SC).

[0008] Furthermore, in the method of the present invention, the recombinant porcine-human uricase is a tetramer composed of a recombinant porcine-human uricase fusion protein, the molecular weight of the recombinant porcine-human uricase fusion protein is 33 kDa, and its amino acid sequence refers to sequence 2 in the sequence table of the specification of the invention patent "A method for preparing and applying a PEGylated recombinant porcine-human uricase fusion protein (patent number: ZL201110181447.X)".

[0009] Further, in the method of the present invention, the molecular weight of the monomethoxy polyethylene glycol succinimidyl propionate is 5 kDa, and the molecular weight of the monomethoxy polyethylene glycol succinimide is 10 kDa; The monomethoxy polyethylene glycol succinimide propionate and monomethoxy polyethylene glycol succinimide are linear or branched.

[0010] Furthermore, in the method of the present invention, the terminal group substitution rate of the monomethoxy polyethylene glycol succinimide propionate is 95%; the terminal group substitution rate of the monomethoxy polyethylene glycol succinimide is 99%.

[0011] Furthermore, in the method of the present invention, the molar ratio of monomethoxy polyethylene glycol succinimidyl propionate and monomethoxy polyethylene glycol succinimide to recombinant porcine-human urate oxidase is 3, 6, or 12.

[0012] Furthermore, in the method of the present invention, the purified recombinant porcine-human uricase is subjected to PEGylation modification treatment using a modifying agent, comprising: The amount of the modifier is calculated according to the molar ratio of the modifier to the recombinant porcine-human uricase, and the modifier is weighed according to the amount of the modifier. First, 1 / 2 of the total amount of the modifier is slowly added to the recombinant porcine-human uricase solution. The recombinant porcine-human uricase solution is continuously stirred during the addition process. After the addition is completed, stirring is continued for 30 minutes at 15-25°C; then 1 / 4 of the total amount of the modifier is taken and added to the recombinant porcine-human uricase solution in the same way. After stirring for 30 minutes, the remaining modifier is added to the recombinant porcine-human uricase solution and stirred for 30 minutes; then, modification is carried out at 2-8°C for 1 hour to obtain PEGylated recombinant porcine-human uricase.

[0013] Furthermore, in the method of the present invention, the raw material formula of the recombinant porcine-human urate oxidase enteric-coated capsules comprises: PEGylated recombinant porcine-human urate oxidase, 7500 U; Microcrystalline cellulose or starch, 240 g; Trehalose or mannitol, 10g.

[0014] Furthermore, in the method of the present invention, the urate oxidase activity of the recombinant porcine-human urate oxidase enteric-coated capsules or enteric-coated tablets is 7.5 U / capsule or 7.5 U / tablet.

[0015] On the other hand, the present invention also provides a recombinant porcine-human urate oxidase enteric-coated capsule or enteric-coated tablet, the preparation method of which includes the above-mentioned preparation method.

[0016] The present invention also provides a recombinant porcine-human uricase enteric-coated capsule or enteric-coated tablet, comprising: PEGylated recombinant porcine-human uricase, a matrix and a stabilizer.

[0017] Furthermore, in the recombinant porcine-human uricase enteric-coated capsules or enteric-coated tablets provided by the present invention, the modifier of the PEGylated recombinant porcine-human uricase includes: monomethoxypolyethylene glycol succinimidyl propionate and / or monomethoxypolyethylene glycol succinimide.

[0018] Furthermore, in the recombinant porcine-human urate oxidase enteric-coated capsules or enteric-coated tablets provided by the present invention, the matrix is ​​microcrystalline cellulose or starch.

[0019] Furthermore, in the recombinant porcine-human urate oxidase enteric-coated capsules or enteric-coated tablets provided by the present invention, the stabilizer is trehalose or mannitol.

[0020] In addition, the present invention also relates to the use of the above-mentioned recombinant porcine-human urate oxidase enteric-coated capsules or enteric-coated tablets in the treatment of hyperuricemia and gout.

[0021] Finally, the present invention also provides a method for evaluating the efficacy of the above-mentioned recombinant porcine-human urate oxidase enteric-coated capsules or enteric-coated tablets, comprising: 1. One-time drug administration experiment Experimental animals: pigeons, chickens, quails; Experimental grouping and administration method: The experimental animals were divided into 8 groups, with 5-10 experimental animals in each group. They were fasted for 12 hours before taking the medicine and began to eat freely 30 minutes after taking the medicine. Serum uric acid detection and statistical analysis: Sampling time for blood uric acid test in experimental animals: before administration, 1, 2, 4, 6, 8, 10, and 12 hours after administration; Statistical methods: SPSS statistical analysis software, statistical items include: descriptive statistics mean and standard deviation, one-way analysis of variance and SNK method analysis of differences between groups; Statistical analysis: The test results of blood samples collected at each sampling time were analyzed, and the blood uric acid levels among the groups were analyzed by one-way variance (ANOVA). Sig>0.05 indicated that there was no significant difference in the blood uric acid levels among the groups. SNK analysis was performed on the blood uric acid levels among the groups to determine whether there was a significant difference between the experimental group and the blank control group; to determine whether there was a significant difference between the experimental groups; Result calculation: Calculate the amplitude and time of the decrease in blood uric acid content in each group, and select the experimental group with the most time points of decrease of more than 10% and the longest effective effect time as the experimental group with the best drug efficacy; 2. Repeated dosing experiment Experimental animals: pigeons, chickens, quails; Experimental grouping and administration method: The experimental animals were divided into 2 groups, with 5-10 experimental animals in each group. Group 1 was the blank control group, which took drugs without rPHUOX, and Group 2 was the experimental group, which took drugs containing rPHUOX. The animals were fasted for 12 hours before taking the drugs, and began to eat freely 30 minutes after taking the drugs. Serum uric acid detection and statistical analysis: Sampling time for blood uric acid test in experimental animals: 4 hours after administration, peripheral blood was collected to determine the blood uric acid content; after the first medication test, the next medication was given 2 weeks apart, and the medication was repeated 4 times in total; Statistical methods: SPSS statistical analysis software, statistical items include: descriptive statistics mean and standard deviation, one-way analysis of variance and SNK method analysis of differences between groups; Statistical analysis: The test results of blood samples collected at each sampling time were analyzed, and one-way analysis of variance (ANOVA) was performed on the blood uric acid content between the blank control group and the experimental group to determine whether there was a significant difference between the two; SNK analysis was performed on the blood uric acid content of the experimental group at each sampling time to determine whether there was a significant difference in blood uric acid content between the experimental groups at different dosing times.

[0022] In summary, the recombinant porcine-human uricase enteric-coated capsules or enteric-coated tablets prepared by the method of the present invention overcome the defects of the recombinant porcine-human uricase injection in terms of convenience, safety and repeated use effectiveness, significantly improve the convenience and safety of drug use, and have good market promotion prospects. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments, and the present invention can also be implemented or applied through other different specific implementation methods. The details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention.

[0024] At the same time, it should be understood that the protection scope of the present invention is not limited to the specific embodiments described below; it should also be understood that the terms used in the embodiments of the present invention are for describing specific embodiments rather than for limiting the protection scope of the present invention.

[0025] When the embodiments give numerical ranges, it should be understood that, unless otherwise specified in the present invention, the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, according to the grasp of the prior art by those skilled in the art and the record of the present invention, any method, equipment, and material of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0026] In the present invention, unless otherwise specified, all parts and percentages are by weight, and unless otherwise specified, all equipment and raw materials are commercially available or commonly used in the industry. The methods in the following embodiments are conventional methods in the art unless otherwise specified.

[0027] The applicant once again specifically states that the present invention is a further improved invention based on the invention patent ZL 201110181447.X, and the patent ZL 201110181447.X is a part of the present invention. The entire text of the aforementioned patent is cited here. For the sake of brevity, the contents disclosed in the aforementioned patent will not be repeated here.

[0028] Example: Preparation method and application of recombinant porcine-human urate oxidase enteric-coated capsules 1. Preparation of recombinant porcine-human urate oxidase enteric-coated capsules 1. Preparation of recombinant porcine-human urate oxidase (rPHUOX) (1) According to the method disclosed in Examples 1-4 of the specification of the invention patent "A method for preparing and using a PEGylated recombinant porcine-human urate oxidase fusion protein (patent number: ZL 201110181447.X)", the recombinant porcine-human urate oxidase protein was expressed, synthesized, separated and purified to obtain purified recombinant porcine-human urate oxidase. The concentration of the purified rPHUOX was 33.8 mg / ml and the specific activity was 9.6 U / mg.

[0029] The nucleotide sequence of the recombinant porcine-human uricase may be SEQ ID NO.1: accatggact acaaaaagaa tgatgaggta gagtttgtcc gaactggcta tgggaaggat atgataaaag ttctccatat tcagcgagat ggaaaatatc acagcattaa agaggtggca acttcagtgc aactgacttt gagctccaaa aaagattacc tgcatggaga caattcagat gtcatcccta cagacaccat caagaacaca gttaatgtcc tggcgaagtt caacggcatc aaaagcatag aaacttttgc tgtgactatc tgtgagcatt tcctttcttc cttcaagcat gtcatcagag ctcaagtcta tgtgaagaa gttccttgga agcgttttga aaagaatgga gttaagcatg tccatgcatt tatttatact cctactggaa cgcacttctg tgaggttgaa cagataaga atggacctcc agttattcat tctggaatca aagacctaaa agtcttgaaa acaaccagt ctggctttga aggattcatc aaggaccagt tcaccaccct ccctgaggtg aaggaccggt gctttgccac ccaagtgac tgcaaatggc gctaccacca gggcagagat gtggactttg aggccacctg ggacactgtt aggagcattg tcctgcagaa atttgctggg ccctatgaca aaggtgaata cttgacctct gtgcagaaga ccctctgtga tatccaggtg ctctccctga gccgagttcc tgcgatagaa gatatggaaa tcagcctgcc aaacattcac tacttcaaca tagacatgtc caaaatgggt ctgatcaaca aggaagaggt attgctgcca ttagacaatc catatggaaa aattactggt acagtcaaga ggaagttgtc ttcaagactg tgactcgag The amino acid sequence of the recombinant porcine-human urate oxidase described above may be SEQ ID NO.2: MDYKKNDEVE FVRTGYGKDM IKVLHIQRDG KYHSIKEVAT SVQLTLSSKK DYLHGDNSDVIPTDTIKNTV NVLAKFNGIK SIETFAVTIC EHFLSSFKHV IRAQVYVEEV PWKRFEKNGV KHVHAFIYTPTGTHFCEVEQ IRNGPPVIHS GIKDLKVLKT TQSGFEGFIK DQFITLPEVK DRCFATQVYC KWRYHQGRDVDFEATWDTVR SIVLQKFAGP YDKGEYSPSV QKTLYDIQVL SLSRVPAIED MEISLPNIHY FNIDMSKMGLINKEEVLLPL DNPYGKITGT VKRKLSSRL (2) Preparation of PEGylated recombinant porcine-human uricase: According to the preparation method of PEGylated recombinant porcine-human uricase conjugate disclosed in Example 5 of the specification of the invention patent "Preparation and application method of a PEGylated recombinant porcine-human uricase fusion protein (patent number: ZL 201110181447.X)", the purified recombinant porcine-human uricase obtained in the previous step was prepared into PEGylated recombinant porcine-human uricase.

[0030] In the present invention, the PEGylation preparation method is optimized and expanded, as follows: The modifiers used were activated PEG products produced by Beijing Kaizheng Bioengineering Development Co., Ltd., including: (a) monomethoxy polyethylene glycol succinimidyl propionate (mPEG-SPA, 5 kDa), (b) monomethoxy polyethylene glycol succinimide (mPEG-SC, 10 kDa).

[0031] The PEGylation method is selected from any one of the following: 1) The modifier is monomethoxy polyethylene glycol succinimidyl propionate (mPEG-SPA, 5 kDa), and the molar ratio of mPEG-SPA to rPHUOX is 2.5-3.8, preferably 3.0.

[0032] The molecular weight of mPEG-SPA is 5,000; the molecular weight of rPHUOX is 33,000; the terminal group substitution rate of the modifier mPEG-SPA (5kDa) is preferably 0.95 or 95%.

[0033] Calculation: The isomeric molar ratio of monomethoxypolyethylene glycol succinimidyl propionate (mPEG-SPA, 5 kDa) to rPHUOX = (1 / 5000) / (1 / 33000) = 6.6.

[0034] Calculation: The weight of mPEG-SPA (5 kDa) required for PEGylation of 10 ml of rPHUOX at a concentration of 33.8 mg / ml is W （mPEG-SPA，5kDa） , W （mPEG-SPA，5kDa） = (target molar ratio × W （rPHUOX） ) / (modifier end group substitution rate × equal mass molar ratio), Taking the target molar ratio as 3.0 as an example: W （mPEG-SPA，5kDa） = (3×33.8mg / ml×10ml) / (0.95×6.6)=162mg.

[0035] 162 mg of the modifier mPEG-SPA (5 kDa) was weighed and added in batches to 10 ml of 33.8 mg / ml rPHUOX solution (first, 1 / 2 of the total amount of the modifier was slowly added to the rPHUOX solution, and the rPHUOX solution was continuously stirred during the addition process to avoid excessive local concentration; after the addition, stirring was continued for 30 minutes at 15-25°C; then 1 / 4 of the total amount of the modifier was added to the rPHUOX solution in the same way, and after stirring for 30 minutes, the remaining modifier was added to the rPHUOX solution and stirred for 30 minutes), and then modified at 2-8°C for 1 hour to obtain a modified product referred to as: 3×5kDa PEG-rPHUOX.

[0036] The inventor needs to specially point out that the above-mentioned proportions, methods and steps are the result of the inventor's intensive research and creative experimental summaries, and are technical solutions with obvious advantages in terms of efficiency, yield, experimental success rate, etc.

[0037] 2) The molar ratio of the modifier to rPHUOX is 5.5-6.3, preferably 6.0.

[0038] Calculation: The weight of mPEG-SPA (5 kDa) required for PEGylation of 10 ml of rPHUOX at a concentration of 33.8 mg / ml is W （mPEG-SPA，5kDa） , W （mPEG-SPA，5kDa） = (target molar ratio × W （rPHUOX） ) / (modifier end group substitution rate × equal mass molar ratio), Take the target molar ratio of 6.0 as an example: W （mPEG-SPA，5kDa） = (6×33.8mg / ml×10ml) / (0.95×6.6)=323.4mg.

[0039] Weigh 323 mg of the modifying agent mPEG-SPA (5 kDa) and add it in batches to 10 ml of 33.8 mg / ml rPHUOX solution (addition method and technical effect are the same as before). Modify at 2-8°C for 1 hour to obtain a modified product referred to as: 6×5kDaPEG-rPHUOX.

[0040] 3) The modifier is monomethoxy polyethylene glycol succinimidyl propionate (mPEG-SPA, 5 kDa), and the molar ratio of mPEG-SPA to rPHUO is 11.5-12.6, preferably 12.0.

[0041] Calculation: The weight of mPEG-SPA (5 kDa) required for PEGylation of 10 ml of rPHUOX at a concentration of 33.8 mg / ml is W （mPEG-SPA，5kDa） , W （mPEG-SPA，5kDa） = (target molar ratio × W （rPHUOX） ) / (modifier end group substitution rate × equal mass molar ratio), Taking the target molar ratio of 12.0 as an example: W （mPEG-SPA，5kDa） = (12×33.8mg / ml×10ml) / (0.95×6.6)=646.9mg.

[0042] Weigh 647 mg of the modifying agent mPEG-SPA (5 kDa) and add it in batches to 10 ml of 33.8 mg / ml rPHUOX solution (addition method and technical effect are the same as before). Modify at 2-8°C for 1 hour to obtain a modified product referred to as: 12×5kDa PEG-rPHUOX.

[0043] 4) The modifier is monomethoxypolyethylene glycol succinimide (mPEG-SC, 10 kDa), and the molar ratio of mPEG-SC to rPHUOX is 2.5-3.8, preferably 3.0.

[0044] The molecular weight of mPEG-SC is 10,000; the molecular weight of rPHUOX is 33,000; the terminal group substitution rate of the modifier mPEG-SC (10kDa) is 0.99 or 99%.

[0045] Calculation: The molar ratio of monomethoxypolyethylene glycol succinimide (mPEG-SC, 10 kDa) to rPHUOX = (1 / 10000) / (1 / 33000) = 3.3.

[0046] Calculation: The weight of mPEG-SC (10 kDa) required for PEGylation of 10 ml of rPHUOX at a concentration of 33.8 mg / ml is W （mPEG-SC，10kDa） , Take the target molar ratio of 3.0 as an example: W （mPEG-SC，10kDa） = (target molar ratio × W （rPHUOX） ) / (modifier end group substitution rate × equal mass molar ratio), W （mPEG-SC，10kDa） = (3×33.8mg / ml×10ml) / (0.99×3.3)=310mg.

[0047] Weigh 310 mg of the modifier mPEG-SC (10 kDa) and add it in batches to 10 ml of 33.8 mg / ml rPHUOX solution (addition method and technical effect are the same as before). Modify at 2-8°C for 1 hour to obtain a modified product referred to as: 3×10 kDa PEG-rPHUOX.

[0048] 5) The modifier is monomethoxypolyethylene glycol succinimide (mPEG-SC, 10 kDa), and the molar ratio of mPEG-SC to rPHUOX is 5.5-6.3, preferably 6.

[0049] Calculation: The weight of mPEG-SC (10 kDa) required for PEGylation of 10 ml of rPHUOX at a concentration of 33.8 mg / ml is W （mPEG-SC，10kDa） , W （mPEG-SC，10kDa） = (target molar ratio × W （rPHUOX） ) / (modifier end group substitution rate × equal mass molar ratio), W （mPEG-SC，10kDa） = (6×33.8mg / ml×10ml) / (0.99×3.3)=621mg.

[0050] Weigh 621 mg of the modifier mPEG-SC (10 kDa) and add it in batches to 10 ml of 33.8 mg / ml rPHUOX solution (addition method and technical effect are the same as before). Modify at 2-8°C for 1 hour to obtain a modified product referred to as: 6×10 kDa PEG-rPHUOX.

[0051] 6) The modifier is monomethoxypolyethylene glycol succinimide (mPEG-SC, 10 kDa), and the molar ratio of mPEG-SC to rPHUOX is 11.5-12.6, preferably 12.0.

[0052] Calculation: The weight of mPEG-SC (10 kDa) required for PEGylation of 10 ml of rPHUOX at a concentration of 33.8 mg / ml is W （mPEG-SC，10kDa） , W （mPEG-SC，10kDa） = (target molar ratio × W （rPHUOX） ) / (modifier end group substitution rate × equal mass molar ratio), Take the target molar ratio of 12.0 as an example: W （mPEG-SC，10kDa） =(12×33.8mg / ml×10ml) / (0.99×3.3)=1241.5mg.

[0053] An equal volume of pH 10.14, 0.02 M carbonate buffer was added to 10 ml of 33.8 mg / ml rPHUOX solution, 1241.5 mg of the modifier mPEG-SC (10 kDa) was weighed and added in batches to 20 ml of 16.9 mg / ml rPHUOX solution (addition method, technical effect is the same as before), modified at 2-8°C for 1 hour, and the modified product was abbreviated as: 12×10 kDa PEG-rPHUOX.

[0054] (3) Purification of PEGylated recombinant porcine-human urate oxidase: The above-mentioned modified products are subjected to molecular sieve chromatography respectively to separate the modified enzyme protein molecules and the remaining part of the modifying agent.

[0055] The molecular sieve chromatography column was filled with TA-GF200 filler produced by Chutian Microsphere Biotechnology Co., Ltd., and then balanced with 0.1M, pH 7.4 phosphate buffer. The above PEGylated recombinant porcine-human urate oxidase was loaded onto the column, eluted with 0.02M, pH 7.4 phosphate buffer, and the protein peak containing enzyme activity was collected, freeze-dried, and stored at 4°C.

[0056] 2. Preparation of recombinant porcine-human urate oxidase enteric-coated capsules Basic formula (based on 1000 capsules, each capsule contains 250mg of filler): Microcrystalline cellulose, Anhui Shanhe Pharmaceutical Excipients Co., Ltd.; No. 1 white plant enteric-coated capsules, Qingdao PolyOcean Algae Industry Group Co., Ltd.; Table 1. Formula and composition of enteric-coated capsules (per 1000 capsules)

[0057] * According to the activity test results of rPHUOX in groups 2, 3, 4, 5, 6, 7, and 8, 7500 U of enzyme preparation was weighed and microcrystalline cellulose was added to 10 g; 10 g of microcrystalline cellulose was taken in group 1.

[0058] 2. Animal Experiments 1. One-time drug administration experiment Experimental animals: Avian animals, such as pigeons, chickens, quails, etc. As a preferred embodiment of the present invention, 80 American king pigeons were selected, with an average weight of 439g / pigeon and a standard deviation of 45.44g; divided into 8 groups, each with 5-10 experimental animals, fasted for 12 hours before taking the capsules, and began to eat freely 30 minutes after taking the medicine. The dosing methods of each experimental group are shown in Table 2.

[0059] Table 2. Drug administration methods in each experimental group

[0060] Serum uric acid test results and statistical analysis: The sampling time for blood uric acid determination of experimental pigeons was before administration, and 1, 2, 4, 6, 8, 10, and 12 hours after administration.

[0061] Statistical methods: SPSS statistical analysis software, statistical items include: descriptive statistics mean and standard deviation (mean ± Std.), one-way analysis of variance (ANOVA) and SNK method for inter-group difference analysis. The analysis results are shown in Table 3.

[0062] Table 3. Blood uric acid test values ​​and statistical results at different time periods after administration

[0063] Note: * There are significant differences between the data with different suffixes (a, b, c, d) after Mean±Std. in the same row.

[0064] ** Mean change = (the current group's immediate mean - the mean of peer group 1) / the mean of peer group 1 × 100%.

[0065] According to the data in Table 3, the blood uric acid content of the experimental pigeons is explained and analyzed as follows: 1. Results and analysis of blood uric acid test before administration Before administration, the blood uric acid levels among the groups were analyzed by ANOVA, F=1.224, Sig=0.301>0.05, and there was no significant difference in blood uric acid levels. SNK analysis showed that there was no significant difference in blood uric acid levels among the groups.

[0066] 2. Results and analysis of blood uric acid test 1 hour after administration One hour after taking the medicine, the blood uric acid test data of the experimental pigeons were analyzed by ANOVA, F=4.676, Sig<0.001, and there was a significant difference in the blood uric acid content among the experimental pigeon groups. SNK analysis showed that the 12×5kDa PEG-rPHUOX group (Group 3), the unPEGylated rPHUOX group (Group 2) and the 12×10kDa PEG-rPHUOX group (Group 6) were significantly different from the blank control group (Group 1). However, there was no significant difference between the 6×5kDa PEG-rPHUOX group (Group 4), the 3×5kDa PEG-rPHUOX group (Group 5), the 3×10kDa PEG-rPHUOX group (Group 8), and the 6×10kDa PEG-rPHUOX group (Group 7) and the blank control group (Group 1). There was no significant difference in the blood uric acid content among all rPHUOX capsule groups, whether taking non-PEGylated rPHUOX or taking various forms of PEGylated rPHUOX capsules.

[0067] 3. Results and analysis of blood uric acid test 2 hours after administration Two hours after taking the medicine, the blood uric acid test data of the experimental pigeons were analyzed by ANOVA, F=7.977, Sig<0.001, and there was a significant difference in the blood uric acid content between the groups. SNK showed that the blood uric acid content of all experimental pigeons taking rPHUOX capsules was significantly lower than that of the blank control group. There was no significant difference between the experimental groups taking rPHUOX capsules.

[0068] 4. Results and analysis of blood uric acid test 4 hours after administration Four hours after taking the medicine, the blood uric acid test data of the experimental pigeons were analyzed by ANOVA, F=34.174, Sig<0.001, and there were significant differences in the blood uric acid content among the groups. SNK showed that 1) the blood uric acid content of all experimental pigeons taking rPHUOX capsules was significantly lower than that of the blank control group; 2) among the groups taking rPHUOX, the blood uric acid content of the PEGylated rPHUOX capsule group was lower than that of the non-PEGylated rPHUOX capsule group, and the effect of PEGylated rPHUOX in promoting the decrease of blood uric acid was more obvious; 3) among all PEGylated rPHUOX groups, the blood uric acid content of the experimental pigeons taking the 12×10kDa PEG-rPHUOX group was the lowest; 4) among the other various PEGylated rPHUOX capsules, although the measured values ​​were different, they were not significant.

[0069] 5. Results and analysis of blood uric acid test 6 hours after administration Six hours after taking the medicine, the blood uric acid test data of the experimental pigeons were analyzed by ANOVA, F=12.044, Sig<0.001, and there were significant differences in the blood uric acid content among the groups. SNK showed that 1) the blood uric acid content of all experimental pigeons taking rPHUOX capsules was significantly lower than that of the blank control group; 2) the blood uric acid decrease in the 12×10kDa PEG-rPHUOX group and the 12×5kDa PEG-rPHUOX group was greater than that in the non-PEGylated rPHUOX group; 3) the blood uric acid decrease in the 12×10kDa PEG-rPHUOX group was significantly greater than that in the 3×10kDa PEG-rPHUOX group.

[0070] 6. Results and analysis of blood uric acid test 8 hours after administration Eight hours after taking the medicine, the blood uric acid test data of the experimental pigeons were analyzed by ANOVA, F=3.845, Sig=0.001<0.05, and there was a significant difference in the blood uric acid content between the groups. SNK showed that 1) the blood uric acid content of the 12×10kDa PEG-rPHUOX group and the 12×5kDa PEG-rPHUOX group was significantly different from that of the blank control group; 2) there was no significant difference between the other experimental groups and the blank control group.

[0071] 7. Results and analysis of blood uric acid test 10 hours after administration Ten hours after taking the medicine, the blood uric acid test data of the experimental pigeons were analyzed by ANOVA, F=2.908, Sig=0.010<0.05, and there was a significant difference in blood uric acid content among the groups. SNK analysis showed that 1) there was a significant difference between the 12×10kDa PEG-rPHUOX group and the blank control group; 2) there was no significant difference between the other groups that took rPHUOX capsules and the blank control group.

[0072] 8. Results and analysis of blood uric acid test 12 hours after administration 12 hours after taking the medicine, the blood uric acid test data of the experimental pigeons were analyzed by ANOVA, F=1.972, Sig=0.071>0.05, and there was no significant difference in the blood uric acid content among the groups. SNK analysis showed that there was no significant difference between the groups taking rPHUOX capsules and the blank control group.

[0073] 9. Table 4 is summarized based on the magnitude and time of the decrease in blood uric acid in each group: Table 4. The extent and time of serum uric acid decrease in each group

[0074] It can be seen from Table 4 that: 1) PEG-rPHUOX capsules (5Ka or 10Ka) with a 12-fold molar ratio have a better effect in lowering blood uric acid; 2) The maximum decrease in blood uric acid in the non-PEGylated rPHUOX group occurred 2 hours after medication, while the maximum decrease in other PEG-rPHUOX groups occurred 4 hours after medication; 3) The PEG-rPHUOX capsules (5Ka or 10Ka) with a 12-fold molar ratio had more time points at which the decrease was more than 10%, and the effective action time was relatively longer.

[0075] 10. Conclusions of the one-time administration animal experiment: (1) rPHUOX can significantly reduce the level of uric acid in the blood; (2) The medicinal efficacy of PEGylated rPHUOX is significantly higher than that of non-PEGylated rPHUOX; (3) The overall difference in the medicinal efficacy of the two modifiers, monomethoxypolyethylene glycol succinimidyl propionate (mPEG-SPA, 5kDa) and monomethoxypolyethylene glycol succinimide propionate (mPEG-SC, 10kDa), is not significant; (4) Different modification degrees are preferred, and the 12-fold molar ratio is better than the 6-fold molar ratio and the 3-fold molar ratio; (5) The 12×10kDa PEG-rPHUOX group has the best efficacy, which is mainly manifested in a relatively longer effective action time.

[0076] 2. Repeated dosing animal experiments Experimental animals: 20 American king pigeons, with an average body weight of 442.3 g / pigeon and a standard deviation of 68.0 g; they were randomly divided into 2 groups, group 1 was a blank control group, taking 1 capsule without rPHUOX, and group 2 was an experimental group, with 5-10 experimental animals in each group, taking 1 capsule of 12×5kDa PEG-rPHUOX, containing 7.5 U / capsule.

[0077] Fast for 12 hours before taking the capsules, and start eating freely 30 minutes after taking the capsules. Peripheral blood was collected 4 hours after taking the capsules to measure the blood uric acid content. After the first medication test, the next medication was given at an interval of 2 weeks, and the medication was repeated 4 times in total. The experimental grouping method is shown in Table 5.

[0078] Table 5. Grouping of repeated dosing animal experiments

[0079] The results of blood uric acid test of experimental pigeons after administration are shown in Table 6.

[0080] Table 6. Results of blood uric acid test after repeated administration

[0081] *SNK analysis showed that there were significant differences between the data in the same column with different suffixes (a, b) after Mean±Std, Sig<0.05.

[0082] **Mean change = (experimental group - control group) / control group × 100%.

[0083] Conclusion of repeated administration animal experiment: (1) Four hours after repeated administration at an interval of 2 weeks, there was a significant difference in the blood uric acid level between the blank control group and the experimental group each time. The blood uric acid level of the experimental pigeons taking 12×5kDa PEG-rPHUOX was significantly lower than that of the blank control group that did not take rPHUOX.

[0084] (2) After four repeated administrations, there was no significant difference in the blood uric acid levels of the experimental pigeons in the 12×5kDa PEG-rPHUOX group.

[0085] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Any technician familiar with the profession can make some changes or modifications to the technical contents disclosed above without departing from the scope of the technical solution of the present invention to obtain equivalent embodiments of equivalent changes. However, any simple modification, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention should be included in the scope of protection of the claims of the present invention.

Claims

1. A method for preparing recombinant porcine-human urate oxidase enteric-coated capsules or enteric-coated tablets, characterized in that: The method comprises: using a modifying agent to perform PEGylation modification on purified recombinant porcine-human uricase, performing molecular sieve chromatography on the modified product to obtain PEGylated recombinant porcine-human uricase, and then preparing the PEGylated recombinant porcine-human uricase into recombinant porcine-human uricase enteric-coated capsules or enteric-coated tablets; The modifying agent includes: monomethoxy polyethylene glycol succinimide propionate and / or monomethoxy polyethylene glycol succinimide.

2. The method according to claim 1, characterized in that: The recombinant porcine-human uricase is a tetramer composed of a recombinant porcine-human uricase fusion protein, and the molecular weight of the recombinant porcine-human uricase fusion protein is 33 kDa.

3. The method according to claim 1, characterized in that: The molecular weight of the monomethoxy polyethylene glycol succinimidyl propionate is 5 kDa, and the molecular weight of the monomethoxy polyethylene glycol succinimide is 10 kDa; The monomethoxy polyethylene glycol succinimide propionate and monomethoxy polyethylene glycol succinimide are linear or branched.

4. The method according to claim 3, characterized in that The molar ratio of the monomethoxy polyethylene glycol succinimidyl propionate and / or monomethoxy polyethylene glycol succinimide to the recombinant porcine-human urate oxidase is 3-12.

5. The method according to claim 1, characterized in that The raw material formula of the recombinant porcine-human urate oxidase enteric-coated capsule or enteric-coated tablet comprises: PEGylated recombinant porcine-human urate oxidase, 7500 U; The matrix is ​​microcrystalline cellulose or starch, 240 g or other matrix; The stabilizer is trehalose or mannitol, 10 g or other stabilizers.

6. A recombinant porcine-human urate oxidase enteric-coated capsule or enteric-coated tablet, characterized in that: include: PEGylated recombinant porcine-human urate oxidase, matrix and stabilizer.

7. The recombinant porcine-human urate oxidase enteric-coated capsule or enteric-coated tablet according to claim 6, characterized in that: The modifying agents of PEGylated recombinant porcine-human uricase include: monomethoxypolyethylene glycol succinimidyl propionate and / or monomethoxypolyethylene glycol succinimide.

8. The recombinant porcine-human urate oxidase enteric-coated capsule or enteric-coated tablet according to claim 6 or 7, wherein the preparation method thereof comprises the method according to any one of claims 1 to 5.

9. Use of the recombinant porcine-human urate oxidase enteric-coated capsules or enteric-coated tablets according to any one of claims 6 to 8 in the treatment of hyperuricemia or gout.

10. A method for evaluating the efficacy of the recombinant porcine-human urate oxidase enteric-coated capsule or enteric-coated tablet according to any one of claims 6 to 8, comprising:

1. One-time drug administration experiment Experimental animals: pigeons, chickens, quails; Experimental grouping and administration method: The experimental animals were divided into 8 groups, with 5-10 experimental animals in each group. They were fasted for 12 hours before taking the medicine and began to eat freely 30 minutes after taking the medicine. Serum uric acid detection and statistical analysis: Sampling time for blood uric acid test in experimental animals: before administration, 1, 2, 4, 6, 8, 10, and 12 hours after administration; Statistical methods: SPSS statistical analysis software, statistical items include: descriptive statistics mean and standard deviation, one-way analysis of variance and SNK method analysis of differences between groups; Statistical analysis: The test results of blood samples collected at each sampling time were analyzed, and the blood uric acid levels among the groups were subjected to one-way analysis of variance. Sig>0.05 indicated that there was no significant difference in the blood uric acid levels among the groups; SNK analysis was performed on the blood uric acid levels among the groups to determine whether there was a significant difference between the experimental group and the blank control group; to determine whether there was a significant difference between the experimental groups; Result calculation: Calculate the amplitude and time of the decrease in blood uric acid content in each group, and select the experimental group with the most time points of decrease of more than 10% and the longest effective effect time as the experimental group with the best drug efficacy; 2. Repeated dosing experiment Experimental animals: pigeons, chickens, quails; Experimental grouping and administration method: The experimental animals were divided into 2 groups, with 5-10 experimental animals in each group. Group 1 was the blank control group, which took drugs without rPHUOX, and Group 2 was the experimental group, which took drugs containing rPHUOX. The animals were fasted for 12 hours before taking the drugs, and began to eat freely 30 minutes after taking the drugs. Serum uric acid detection and statistical analysis: Sampling time for blood uric acid test in experimental animals: 4 hours after administration, peripheral blood was collected to determine the blood uric acid content; after the first medication test, the next medication was given 2 weeks apart, and the medication was repeated 4 times in total; Statistical methods: SPSS statistical analysis software, statistical items include: descriptive statistics mean and standard deviation, one-way analysis of variance and SNK method analysis of differences between groups; Statistical analysis: The test results of blood samples collected at each sampling time were analyzed, and one-way analysis of variance was performed on the blood uric acid content between the blank control group and the experimental group to determine whether there was a significant difference between the two; SNK analysis was performed on the blood uric acid content of the experimental group at each sampling time to determine whether there was a significant difference in blood uric acid content between the experimental groups at different dosing times.

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