WO2020052457A1 - 一种尿酸酶外用凝胶制剂、其制备方法及用途 - Google Patents

一种尿酸酶外用凝胶制剂、其制备方法及用途 Download PDF

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WO2020052457A1
WO2020052457A1 PCT/CN2019/103968 CN2019103968W WO2020052457A1 WO 2020052457 A1 WO2020052457 A1 WO 2020052457A1 CN 2019103968 W CN2019103968 W CN 2019103968W WO 2020052457 A1 WO2020052457 A1 WO 2020052457A1
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uric acid
urase
gel preparation
preparation
carbomer
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French (fr)
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陈建华
黄秀华
王玉霞
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China Pharmaceutical University
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China Pharmaceutical University
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/44—Oxidoreductases (1)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/32—Macromolecular compounds obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. carbomers, poly(meth)acrylates, or polyvinyl pyrrolidone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/38—Cellulose; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y107/00—Oxidoreductases acting on other nitrogenous compounds as donors (1.7)
    • C12Y107/03—Oxidoreductases acting on other nitrogenous compounds as donors (1.7) with oxygen as acceptor (1.7.3)
    • C12Y107/03003—Factor-independent urate hydroxylase (1.7.3.3), i.e. uricase

Definitions

  • the invention relates to a uric acid external gel preparation, which can be used as a pharmaceutical preparation for treating hyperuricemia, gout and diseases caused by hyperuricemia, and also relates to a preparation method and use of the gel preparation, and belongs to biological medicine. Technical field of preparations.
  • uric acid (UAric acid, UA), as the final product of human purine metabolism, is mainly produced by metabolism in the liver, gastrointestinal tract, etc., enters the blood and forms a uric acid metabolism pool.
  • Hyperuricemia is formed when the uric acid metabolism of the human body is disturbed, resulting in excessive uric acid production or reduced excretion.
  • the diagnostic index of hyperuricemia is usually that the female blood uric acid level is greater than 6 mg / dl (360 ⁇ M (M is mol / L, the same below)), and the male blood uric acid level is greater than 7 mg / dl (420 ⁇ M).
  • uric acid forms monosodium urate crystals and deposits in the joint cavity or soft tissues to form tophi.
  • the main clinical features of gout are hyperuricemia, recurrent acute and chronic attacks of gout caused by hyperuricemia, arthritis, joint deformities, uric acid urinary stones, which cause damage to the kidneys, cause interstitial nephritis, and renal failure. Wait.
  • serum and / or intracellular urate can stimulate the renin-angiotensin-aldosterone system and induce hypertension (Johnson RJ, Bakris GL, Borghi C, et al.
  • uric acid has important significance in diabetic nephropathy, calcineurin inhibitor nephrotoxicity and acute kidney injury (Johnson RJ, Bakris GL, Borghi C, et al. American Journal of Kidd Diseases , 2018: 851-865). Gout is often accompanied by clinical manifestations such as abdominal obesity, hyperlipidemia, hypertension, type II diabetes, and cardiovascular disease.
  • Serum uric acid is mainly excreted by the sweat glands of the kidneys, intestines, and skin.
  • the kidney is the main organ of uric acid excretion.
  • Uric acid can pass freely through the glomeruli, and is reabsorbed and secreted in the proximal tubule. Most of the secreted uric acid is reabsorbed, and less than 10% is excreted.
  • the process of reabsorption and secretion depends on ion channels and related urate transporters.
  • uric acid in the intestine accounts for about one-third of the daily excretion.
  • the mechanism of uric acid transport in the intestine is not clear, but GLUT9 and ABCG2 transport uric acid to the intestine.
  • GLUT9 gene knockout can cause hyperuricemia.
  • ABCG2 gene knockout can cause hyperuricemia and "overload" uric acid.
  • Urine disease Johnson RJ, Bakris G L, Borghi C, et al. American Journal of Kidney Diseases, 2018: 851-865).
  • Human skin contains about 2-5 million sweat glands. Under the control of sympathetic nerves (autonomic nerves), sweat is secreted to the surface of the skin to avoid excessive body temperature and subsequent body dysfunction caused by the increase in ambient temperature. Provide a steady state for the balance of the human body's internal and external environment.
  • the skin is the main excretory organ following the lungs, kidneys, and intestines. Through the sweating effect of the sweat glands, uric acid and urea can be excreted from the body. It plays a role of assisting or replacing the kidney. It can be regarded as a special form Kidney (Hanafusa N, Lodebo B, T, Shah A, et al.
  • Gout is a kind of "wealthy disease”. With the improvement of living standards, the prevalence of hyperuricemia and gout in the Chinese population has gradually increased. According to statistics, the prevalence of gout in China in 2014 was 1.4%, while the prevalence of gout in 2000-2005 was only 0.9% (Liu R, Han C, Wu D, et al. Biomed Research International, 2015, 2015 (15, supplement): 1-12.).
  • Gout treatment is a long-term process. In addition to reasonable weight loss, avoid alcohol (especially beer and spirits), sugary drinks, overeating, and excessive intake of meat and seafood. Encourage low-fat dairy products.
  • the treatment of gout attacks with drugs has a significant short-term effect.
  • the first-line drug colchicine for the treatment of acute gout attacks and other drugs such as non-steroidal anti-inflammatory drugs, glucocorticoids, allopurinol, etc. ( Benn, C, L, Dua, P, Gurrell, R, et al. Frontiers in medicine, 2018, 5: 160-188.).
  • uric acid enzymes can be used as patient drugs.
  • the drug of choice for treatment (Richette P, Doherty M, Pascual E, et al. Annals of the Rheumatic Diseases, 2017, 76 (1): 29-42.).
  • the European League against Rheumatism (EULAR) in 2016 recommended that patients receiving uric acid lowering therapy should monitor serum uric acid values and should continue to be below 360 ⁇ M; for severe (gout, chronic joint disease, regular Onset of gout patients, the serum uric acid level should be lower than 300 ⁇ M, which promotes the rapid dissolution of uric acid crystals until the uric acid crystals are completely dissolved and the gout disappears; during long-term treatment, the uric acid level should not be lower than 180 ⁇ M.
  • EULAR European League against Rheumatism
  • urase drugs have been developed internationally at present: (1) Uricozyme derived from Aspergillus flavus (Uricozyme) approved for marketing in France and Italy in the 1970s; (2) in the United States in 2000 And EU-approved recombinant urase (Rasburicase) expressed by yeast genetically engineered uric acid gene (derived from Aspergillus flavus); (3) recombination of polyethylene glycol modified pigs and baboons approved for marketing by the US FDA in 2010 Chimeric urase (Pegloticase).
  • the first two urase drugs are derived from microorganisms and have strong immunogenicity.
  • Pegloticase is chimeric and recombinantly expressed from two mammalian urase genes, pig and baboon. After modified with polyethylene glycol, the half-life of intravenous administration of Pegloticase was significantly increased. However, 41% of patients developed high titers against Pegloticase. Antibodies, in which 40% of patients produced high titer antibodies against PEG (Lipsky, E, Calabrese, H, Kavanaugh A, et al.
  • nanoparticle combination treatment method degrades urate crystals, and the PLGA emulsification solvent is evaporated by a double emulsification solvent evaporation method to prepare nanoparticles containing urase and diclofenac; due to the small particle size, large specific surface area, and other drug delivery systems
  • the penetration rate of the drug through the skin is improved, and the further prepared nanoparticle gel delivers the drug to the synovial fluid percutaneously to exert a therapeutic effect.
  • the preparation process of the dosage form is complicated and the stability is poor.
  • the half-life of the nanoparticle gelling agent under refrigeration is only 45.22 days.
  • the inventor's research group is committed to the research of urase and its pharmaceutical preparations, and has applied for several Chinese invention patents, such as Chinese invention patent with patent number CN201410048071.9, authorization announcement number CN103834623B, application number CN201510066745.2, application publication number CN104630168A Chinese invention patent application, application number CN201610316709.1, application publication number CN105838686A Chinese invention patent application.
  • the research group of the inventor has made further research results on uric acid preparations.
  • the main purpose of the present invention is to overcome the problems existing in the prior art and provide a uric acid external gel preparation, which is applied on the skin surface.
  • the preparation is easy to prepare, has good stability, is convenient to use, and has a stable drug effect. It is easy to control the uric acid level autonomously during the medication, and the efficacy of the preparation does not depend on the urase entering the body through the skin barrier.
  • the present invention also provides a method for preparing the preparation, and uses of the preparation.
  • the pH of the formulation is adjusted to 5-10 by a pH adjuster.
  • the uric acid enzyme includes natural uric acid enzyme, recombinant uric acid enzyme, chimeric uric acid enzyme, and fusion uric acid enzyme;
  • the natural uric acid enzyme is derived from prokaryote or eukaryote;
  • the recombinant uric acid enzyme and chimeric uric acid enzyme The fusion urase is prepared by bioengineering technology, respectively;
  • the natural urase includes urase derived from microorganisms and urase derived from mammals, and the microorganisms include Candida utilis and Aspergillus flavus, and Mammals include pigs and dogs;
  • the recombinant uric acid enzyme includes recombinant uric acid expressed by a natural uric acid gene prepared by using bioengineering technology; and
  • the chimeric uric acid enzyme includes a nucleotide sequence embedded between different natural uric acid genes.
  • the fusion uric acid enzyme comprises a natural uric acid gene fused to a human serum albumin gene, a recombinantly expressed human serum albumin-uric acid fusion protein, and a natural uric acid gene and human Fc fragment urinase antibody fusion protein, recombinantly expressed Fc-urase fusion protein.
  • the uric acid enzyme has a chemical modification including a PEG modification.
  • the aqueous gel matrix is selected from natural polymers or semi-synthetic polymers or synthetic polymers;
  • the natural polymers include starch, alginate, gum arabic, tragacanth, agar, and gelatin;
  • the The semi-synthetic polymer includes modified cellulose and modified starch.
  • the modified cellulose includes carboxymethyl cellulose and methyl cellulose.
  • the synthetic polymer includes carbomer and sodium polyacrylate.
  • the aqueous gel matrix is carbomer 934, carbomer 940, carbomer 941, carbomer 942, carbomer 971, carbomer 974, carbomer 980, carbomer 981 One or a combination.
  • the protein protecting agent is selected from one of bovine serum albumin (BSA), mannitol, sucrose, sodium citrate, sorbitol, or a combination thereof;
  • BSA bovine serum albumin
  • mannitol sucrose
  • sodium citrate sodium citrate
  • sorbitol or a combination thereof;
  • the humectant is selected from one of glycerin, propylene glycol, ethanol, hyaluronic acid, or a combination thereof;
  • the chelating agent is selected from one of the salts of ethylenediaminetetraacetic acid or a combination thereof.
  • the salts of ethylenediaminetetraacetic acid include ethylenediaminetetraacetic acid disodium salt (EDTA-2Na), ethylenediaminetetraacetic acid. Dipotassium salt (EDTA-2K);
  • the transdermal enhancer is selected from one of Azone, Propylene Glycol, Hyaluronic Acid, Cholate, Deoxycholate, Urea, Cyclodextrin, Tween-80 or a combination thereof;
  • the preservative is selected from one of benzoic acid, sodium benzoate, methyl paraben, ethyl paraben, phenol, sorbic acid, or a combination thereof;
  • the pH adjusting agent is selected from one of triethanolamine, NaOH, and NaHCO 3 .
  • the content of the urase is 0.001-0.1%; the pH of the preparation is 6-9.
  • the present invention also provides a method for preparing the uric acid external gel preparation described above, which is characterized in that it includes the following steps:
  • the first step is to take an appropriate amount of water for injection, add a transdermal enhancer, humectant, and chelating agent, and dissolve and stir well; then add an aqueous gel matrix, and swell and / or dissolve and stir well. Sterilize to obtain spares. ;
  • the second step is to add the protein protectant, preservative, and the main drug urase to the appropriate amount of water for injection to dissolve, and filter the bacteria through a microporous filter for later use;
  • the preservative obtained in the second step is added to the standby material obtained in the first step, and the pH value is adjusted to 5-10 with a pH adjuster, and then the protein protective agent, the main drug urase and the balance are added in the second step. Water for injection, stir evenly and dispense it to obtain finished product of uric acid external gel preparation.
  • the time for swelling is overnight, and the conditions of the wet heat sterilization are 115 ° C and 30 minutes; in the second step, the pore size of the microporous film is 0.22 ⁇ m; in the third step, the pH after adjustment is 6 -9; at the time of dispensing, it is dispensed into a pharmaceutically acceptable container, which includes a pharmaceutical aluminum tube, a plastic tube, an aluminum-plastic tube, a polyethylene composite hose, or, when dispensing, coating A gel patch is made from hygienic materials; after dispensing, the contents are stored in a refrigerator at 4 ° C.
  • the invention also provides: the use of the uric acid external gel preparation described above for preparing a medicine or a pharmaceutical composition, the role of the medicine or the pharmaceutical composition is to lower uric acid, or to treat hyperuricemia, or to treat gout, or Treatment of diseases caused by hyperuricemia.
  • the administration route of the gel preparation of the present invention is to apply to the skin surface for external use, degrade uric acid secreted from the skin surface through sweat glands, generate allantoin, and then form a concentration gradient of uric acid inside and outside the skin, and promote the continuous secretion of uric acid and other metabolism by the sweat glands in the form of microfluids.
  • the product can play a significant role in lowering uric acid in the body; the effectiveness of the preparation does not depend on urase entering the body through the skin barrier, unlike lyophilized powder injection, which needs to be administered intravenously into the body, avoiding allergic reactions and infusion reactions of urase And antibodies; the preparation has the effect of degrading uric acid, in addition to being used to treat hyperuricemia and gout, it can also be used to treat complications caused by increased uric acid concentration in the blood, such as diabetes, hypertension, and interstitial nephritis , Renal failure and other cardiovascular system diseases.
  • the preparation method of the invention can not only maintain the urase activity, but also can maintain the stability of the urase gel preparation for a long time.
  • the preparation process of the invention is simple, the preparation has good stability, is convenient to use, and has a stable drug effect. It is easy to control the uric acid level autonomously during the medication process.
  • FIG. 1 is a uric acid standard curve diagram of Example 9 of the present invention.
  • FIG. 2 is a uric acid standard curve measured by high performance liquid chromatography in Example 11 of the present invention.
  • FIG. 3 is a diagram illustrating an example of detecting a uric acid solution by high performance liquid chromatography according to Example 11 of the present invention.
  • FIG. 4 is a graph showing measurement results of uric acid content on the skin surface of each experimental group in Example 11 of the present invention.
  • FIG. 5 is a graph showing measurement results of uric acid content in skin tissue of each experimental group in Example 11 of the present invention.
  • FIG. 6 is an allantoin standard curve determined by high performance liquid chromatography in Example 12 of the present invention.
  • FIG. 7 is a graph showing the results of allantoin on the skin surface of each experimental group in Example 12 of the present invention
  • FIG. 8 is a diagram showing the results of Example 13 of the present invention.
  • test methods in the following examples are conventional methods; reagents and materials can be obtained through commercial means unless otherwise specified.
  • the uric acid external gel preparation of this embodiment is composed of the following components (based on 100 g of gel):
  • a 15% triethanolamine solution (pH adjuster) was used to adjust the pH of the formulation to 7.4.
  • the uric acid external gel preparation of this embodiment is composed of the following components (based on 100 g of gel):
  • a 15% NaOH solution (pH adjuster) was used to adjust the formulation pH to 7.5.
  • the uric acid external gel preparation of this embodiment is composed of the following components (based on 100 g of gel):
  • a 15% NaOH solution (pH adjuster) was used to adjust the formulation pH to 8.5.
  • the uric acid external gel preparation of this embodiment is composed of the following components (based on 100 g of gel):
  • a 10% NaHCO 3 solution (pH adjuster) was used to adjust the formulation pH to 8.0.
  • the uric acid external gel preparation of this embodiment is composed of the following components (based on 100 g of gel):
  • a 10% NaHCO 3 solution (pH adjuster) was used to adjust the formulation pH to 8.0.
  • the uric acid external gel preparation of this embodiment is composed of the following components (based on 100 g of gel):
  • a 15% triethanolamine solution (pH adjuster) was used to adjust the pH of the formulation to 7.4.
  • the uric acid external gel preparation of this embodiment is composed of the following components (based on 100 g of gel):
  • a 15% NaOH solution (pH adjuster) was used to adjust the formulation pH to 7.5.
  • the uric acid external gel preparation of this embodiment is composed of the following components (based on 100 g of gel):
  • a 15% NaOH solution (pH adjuster) was used to adjust the formulation pH to 8.5.
  • Example 1 According to the method for preparing the urase external gel preparation in Example 1, three batches of samples were prepared, with the production batch numbers of 20170912, 20170913, and 20170914, respectively.
  • the stability of the gel preparation was studied by the following methods.
  • the gel preparation urase external gel preparation was placed in a closed sterile aluminum tube, and stored at 37 ° C, room temperature and 4 ° C, respectively, and the appearance, dehydration, pH, and mildew of the gel preparation were checked regularly. Then, take a sample to determine the retention rate of urase activity in the gel preparation, and test the stability of the urase activity in the gel preparation urase external gel preparation.
  • uric acid standard curve The prepared 600 ⁇ M uric acid mother liquor was diluted to 60 ⁇ M, 54 ⁇ M, 48 ⁇ M, 42 ⁇ M, 36 ⁇ M, 30 ⁇ M, 24 ⁇ M, 18 ⁇ M, 12 ⁇ M, and 0 ⁇ M with 0.1M borax-boric acid solution (pH 8.5). The OD 293 of the solution is shown in Table 1; a standard curve was drawn based on the measured data, and the results are shown in Figure 1.
  • the recombinant human-pig chimeric gel preparation urase external gel preparation was placed at 4 ° C and 37 ° C, and samples were taken at different time points to test the recombinant human-pig chimera urase activity. The results are shown in the table below:
  • the gel preparation urase external gel preparation maintains an enzyme activity retention rate of more than 90% for 24 hours at 37 ° C., 85% for 48 hours, and 72 hours, and the enzyme activity retention rate can still reach 81%.
  • the gel preparation urase external gel preparation has an enzyme activity retention rate of 85% or more at 4 ° C for 6 months.
  • the uric acid external gel preparation of the present invention can protect the uric acid enzyme from various external factors, and is beneficial to stabilize the uric acid enzyme and protect the uric acid activity.
  • Example 10 Local irritation test of urase external gel preparation
  • Example 11 Determination of skin uric acid content
  • mice in each group had a normal diet and were not given uric acid topical gel preparation or aqueous gel matrix.
  • the mice in the model group were injected intraperitoneally with sodium urate suspension, and the yeast was mixed with food. Powdery feed was used to prepare a mouse hyperuricemia model; the negative control group was modeled in the same way as the model group.
  • the aqueous gel matrix ie, blank gel
  • the urase gel was administered.
  • the modeling method was the same as that of the model group. After the successful modeling, the urase external gel preparation of Example 1 was administered.
  • the exposed skin on the back of each group of mice was disinfected and cleaned with a cotton ball. After wiping, the mice were returned to their cages.
  • the gel administration group was given urase external gel and the negative control group was given blank gel. After the gel is allowed to air dry naturally, the mice are returned to their cages, drinking and drinking freely.
  • use a cotton swab to soak a suitable amount of normal saline to wipe the skin on the back of each group of mice. Then squeeze the water from the cotton swab into the EP tube. , Marked as "Skin Wipe Sample". After the obtained sample was centrifuged, a certain percentage of a protein precipitant was added, and the supernatant was taken for HPLC measurement.
  • the integration method was used to obtain the uric acid peak area, and the uric acid concentration was calculated according to the standard curve.
  • the uric acid concentration on the skin surface of the mice in the blank group and the model group gradually reaches the upper limit after 8 hours, and the uric acid content on the skin surface of the model group is 2.4 times that of the blank group.
  • the negative control group is the same as the model group. Since the urase in the gel can continuously degrade the uric acid secreted from the sweat glands to the skin surface, the uric acid content on the skin surface is lower than that of the blank group at 4-8h, but exceeds the blank group after 12h, and the uric acid concentration on the skin surface after 24h Exceeding the model group, because the uric acid concentration on the skin surface is proportional to the serum uric acid concentration.
  • the skin uric acid concentration relative to blood uric acid that is, the uric acid concentration in the skin of the model group, and the uric acid excretion on the skin surface of the urase gel administration group significantly exceeds that of the model group, so it can be explained that the urase gel can not only degrade the skin surface Uric acid can also promote skin uric acid excretion.
  • mice were sacrificed by cervical dislocation.
  • the skin tissue of each group of mice was removed and homogenized. After centrifugation, the supernatant was pipetted into an EP tube. A protein precipitant was added at a certain ratio. After centrifugation, the supernatant was used for HPLC. Measurement, integration method to obtain the uric acid peak area, and then calculate the uric acid concentration according to the standard curve, and then obtain the skin uric acid content per unit weight according to the following formula:
  • the uric acid level in the skin tissue of the hyperuricemia mice (model group) is twice as high as the uric acid level in the skin tissue of normal mice (the blank group); It has the effect of decomposing uric acid, so it cannot prevent the increase of uric acid level in the skin of mice.
  • the uric acid level in the skin tissue is close to that in the model group, which is still 2 times the uric acid level in the blank group. Because urase can play a role in degrading uric acid, it can continuously degrade the uric acid secreted from the skin, thereby reducing the level of uric acid in the skin tissue.
  • the uric acid level in the skin tissue of the mice in the administration group and the skin in the blank group Tissue uric acid levels are similar, which indicates that one week of uric acid topical gel preparation can significantly reduce the uric acid in the skin tissue of mice, and the blood uric acid level of hyperuricemia mice is directly proportional to the skin uric acid level.
  • Decreased tissue uric acid means that the blood uric acid level of the mice in the administration group will also decrease, which will reduce the blood uric acid level. Effect.
  • Analytical balance was used to accurately weigh 0.02g of allantoin standard in 100ml of water. Stir and dissolve thoroughly. Drain with a glass rod into a 250ml clean volumetric flask. Continue to add water to the mark. After mixing, 500 ⁇ M allantoin is obtained. ⁇ standard solution.
  • the "skin wipe samples" of the uric acid gel administration group were collected, centrifuged to take the supernatant and added a certain proportion of protein precipitant, and the supernatant was centrifuged for HPLC detection.
  • the integration method was used to obtain the allantoin peak area, and then according to the standard The curve calculates the allantoin concentration.
  • the uric acid in the gel has been fully reacted or inactivated by proteases on the skin surface and licked and inactivated by the mouse, but uric acid is still continuously excreted from the body to the body at this time.
  • uric acid excretion in the final administration group for 24 hours exceeded the skin surface uric acid content in the model group for 24 hours. Therefore, it can be explained that the uric acid gel preparation can convert uric acid on the skin surface to allantoin with higher water solubility, and 12 hours after administration, the uric acid gel preparation on the skin surface still has activity, which makes the blood uric acid keep flowing. It is excreted in the body, and also increases the excretion of uric acid in the skin, thereby reducing the blood uric acid level rapidly and continuously.
  • the blank group was fed with ordinary feed only; the model group, the negative control group, and the administration group were injected intraperitoneally with sodium urate suspension once a day, and the yeast powder-containing feed was given at the same time.
  • UOX 83 gel the recombinant human of Example 1- Pig chimeric urase external gel preparation, specification 25mg / 100g, batch: 20180512
  • administration group 2 UOX P
  • Blank group no modeling, no medicine
  • model group daily modeling, no medicine
  • negative control group continuous modeling, back hair removal, apply blank gel (using the aqueous gel matrix of Example 1); administration Group: continuous modeling. After successful modeling, the back hair was applied daily with the corresponding urase external gel preparation. Except for the blank group, no other mice were used for modeling.
  • orbital blood was taken from each group of mice. Let stand at 4 ° C for a period of time, centrifuge the supernatant to obtain 20 ⁇ L by HPLC, record the uric acid peak area, and calculate the blood uric acid content according to the uric acid standard curve.
  • the serum uric acid level of the mice in the model group, the negative control group, the administration group 1, the administration group 2, the administration group 3 and the administration group 4 after being modeled by hyperuricemia is The serum uric acid level of the mice in the blank group was more than twice.
  • the administration group 1, administration group 2, administration group 3 and administration group 4 could all reduce the blood uric acid level of the mice to be similar to that of the administration group. .
  • the uric acid external gel preparation of the present invention not only has significant degradation of uric acid on the skin surface to be converted into allantoin, but also promotes continuous excretion of blood uric acid to the skin surface, thereby reducing the serum uric acid level.

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Abstract

一种尿酸酶外用凝胶制剂由以下组分按重量百分比组成：尿酸酶大于0且小于或等于1.0％，水性凝胶基质0.05-20％，蛋白保护剂0.15-20％，保湿剂2-25％，螯合剂0.01-0.20％，透皮促进剂0.001-3.0％，防腐剂0.005-0.5％，余量为注射用水，所述制剂的pH经pH调节剂调整为5-10。所述凝胶制剂的制备方法包括：以部分组分制得备用物，分别以注射用水配制各剩余组分，将防腐剂加入备用物，调节pH，加入蛋白保护剂、尿酸酶以及余量注射用水，搅拌均匀后分装即得。

Description

一种尿酸酶外用凝胶制剂、其制备方法及用途 技术领域
本发明涉及一种尿酸酶外用凝胶制剂，可作为治疗高尿酸血症、痛风以及由高尿酸血症引起的疾病的药物制剂，同时还涉及该凝胶制剂的制备方法及用途，属于生物医药制剂技术领域。
背景技术
据发明人了解，尿酸(Uric acid，UA)作为人体嘌呤代谢的最终产物，主要由肝脏、胃肠道等代谢产生，进入血液并形成尿酸代谢池。当人体内尿酸代谢发生障碍，使尿酸产生过多或排泄减少时，即形成高尿酸血症(hyperuricemia)。高尿酸血症的诊断指标通常为女性血尿酸水平大于6mg/dl(360μM(M即mol/L，下同))，男性血尿酸水平大于7mg/dl(420μM)。长期过饱和尿酸形成单钠尿酸盐结晶并沉积于关节腔内或软组织中即形成痛风石。痛风的主要临床特征为高尿酸血症，以及高尿酸血症引起的痛风反复急、慢性发作，关节炎症、关节畸形、尿酸性尿路结石，对肾脏造成损害，引发间质性肾炎、肾衰竭等。此外，血清和/或细胞内的尿酸盐可以刺激肾素-血管紧张素-醛固酮系统，诱发高血压(Johnson R J,Bakris G L,Borghi C,et al.American Journal of Kidney Diseases,2018:851-865)；研究亦发现尿酸在糖尿病肾病、钙调神经磷酸酶抑制剂肾毒性和急性肾损伤中具有重要意义(Johnson R J,Bakris G L,Borghi C,et al.American Journal of Kidney Diseases,2018:851-865)。痛风常伴腹型肥胖、高脂血症、高血压、Ⅱ型糖尿病及心血管病等临床表现。
血尿酸主要由肾脏、肠道、皮肤汗腺排出体外。肾脏是尿酸排泄的主要器官，尿酸能够自由透过肾小球，在近端小管重吸收和分泌，分泌的尿酸绝大部分被重吸收，只有不到10％被排泄，在近端肾小管的重吸收和分泌过程依赖于离子通道及相关的尿酸盐转运体。
尿酸在肠道的排泄约占日排泄量的三分之一。肠道中尿酸的转运机制尚不明确，但GLUT9、ABCG2将尿酸运输到肠道，肠道GLUT9基因敲除可引发高尿酸血症，ABCG2基因敲除可导致高尿酸血症和“超负荷”尿酸尿症(Johnson R J,Bakris G L,Borghi C,et al.American Journal of Kidney Diseases,2018:851-865)。
人皮肤中约含有200-500万个汗腺，在交感神经(自主神经)的控制下将汗液分泌到皮肤表面，避免因周围环境温度上升而导致机体温度过高以及随之而来的机体功能紊乱，为人体内外环境的平衡提供稳态。皮肤是继肺脏、肾脏、肠道之外的最主要的排泄器官，通过汗腺的排汗作用，可以将尿酸、尿素等排泄出体外，发挥着辅助或替代肾脏的功能，可被视为特殊形式的肾脏(Hanafusa N,Lodebo B T,Shah A,et al.Journal of Renal Nutrition the Official Journal of the Council on Renal Nutrition of the National  Kidney Foundation,2017,27(5):295-302.)。汗腺与肾脏的排泄作用配合非常紧密，当肾脏的排泄功能损害时，汗液中的尿素等氮代谢物含量增加，可在一定程度上补偿肾脏功能的不足。研究表明，汗液中的尿酸约24.5μM，约占血尿酸(serum uric acid,SUA)水平的6.3％(Huang C T,Chen M L,Huang L L,et al.Chinese Journal of Physiology,2002,45(3):109-115.)，尿酸通过汗腺透过皮肤屏障到达皮肤表面，为降解尿酸药物的局部给药制剂的研发提供了理论基础。
痛风是一种“富贵病”。随着生活水平的提高，中国人人群中高尿酸血症及痛风的患病率逐渐增高。据统计在中国大陆，2014年痛风的患病率为1.4％，而2000-2005年痛风的患病率仅为0.9％(Liu R,Han C,Wu D,et al.Biomed Research International,2015,2015(15,supplement):1-12.)。流行病学研究发现，从1948年国内首次发现的痛风患者开始，到2016年我国国内高尿酸血症患者的发病率为10％，患者数目高达1.35亿，痛风患者约在1700万左右，已成为我国第二大代谢疾病，是继高血糖、高血脂之后的第三类富贵病(中国人民解放军总医院.中国药物应用与监测[M].)。
痛风治疗是一个长期的过程，除了合理减重，避免摄入酒精(尤其是啤酒以及烈酒)、含糖饮料、暴饮暴食以及过多摄入肉类、海鲜，鼓励摄入低脂奶制品及规律运动等外，以药物治疗痛风发作在短期内具有显著的疗效，例如治疗急性痛风发作的一线药物秋水仙碱以及其它药物如非甾体抗炎药、糖皮质激素、别嘌呤醇等(Benn C L,Dua P,Gurrell R,et al.Frontiers in medicine,2018,5:160-188.)。但对于体内证实有尿酸盐晶体、严重慢性痛风石以及累及生活质量的痛风患者，即使采用最大剂量降尿酸药物(包括联合用药)而血尿酸浓度仍然无法达标的患者，尿酸酶可以作为患者药物治疗的首选药物(Richette P,Doherty M,Pascual E,et al.Annals of the Rheumatic Diseases,2017,76(1):29-42.)。此外，2016年欧洲抗风湿病联盟(European League Against Rheumatism，EULAR)建议，接受降尿酸治疗的患者，应监测血清尿酸值，并应持续低于360μM；对于严重(痛风石、慢性关节病、经常发作)的痛风患者，血清尿酸水平应低于300μM，促使尿酸结晶更快溶解，直到尿酸结晶全部溶解，痛风消失为止；在长期治疗过程中，尿酸水平不应低于180μM。
经检索发现，目前国际上开发了三种尿酸酶类上市药物，分别为：(1)20世纪70年代在法国和意大利批准上市的黄曲霉来源尿酸酶(Uricozyme)；(2)2000年在美国和欧盟批准上市的尿酸酶基因(来源于黄曲霉)的酵母基因工程菌表达的重组尿酸酶(Rasburicase)；(3)2010年由美国FDA批准上市的聚乙二醇修饰的猪和狒狒的重组嵌合尿酸酶(Pegloticase)。前两种尿酸酶类药物均来源于微生物，具有较强的免疫原性。Pegloticase由猪和狒狒这两种哺乳动物尿酸酶基因嵌合重组表达，经聚乙二醇化学修饰后，Pegloticase静脉给药的半衰期显著增加，然而，41％的病人产生了针对Pegloticase的高滴度抗体，这其中40％的病人产生了抗PEG的高滴度抗体(Lipsky P E,Calabrese L H,Kavanaugh A,et al.Arthritis Research&Therapy,2014, 16(2):(R60)1-8.)；临床研究发现，静脉滴注后45％的病人产生输液反应，包括胸闷(15％)，潮红(12％)，呼吸困难(11％)等(Baraf H S,Yood R A,Ottery F D,et al.Journal of Clinical Rheumatology Practical Reports on Rheumatic & Musculoskeletal Diseases,2014,20(8):427-432.)。上述尿酸酶类药物均为冻干粉针剂，经静脉滴注应用于人体，虽降解尿酸的作用发挥迅速，但可引起77％患者的痛风急性发作(Lyseng-Williamson K A.Drugs,2011,71(16):2179-2192.)。在药物开发过程中，哺乳动物尿酸酶的低免疫原性和微生物尿酸酶的高比活性，使这两大来源的重组尿酸酶的开发及应用成为热点。
另有一种纳米颗粒组合治疗方法降解尿酸盐结晶，采用复乳化溶剂蒸发法蒸发PLGA乳化溶剂制备包含尿酸酶与双氯芬酸的纳米颗粒；由于纳米颗粒粒径小，比表面积大，和其它药物递送系统相比，提高了药物通过皮肤的渗透率，进一步制备的纳米颗粒凝胶经皮递送药物至关节滑液发挥治疗作用。该剂型制备工艺复杂，稳定性较差，在冷藏条件下纳米颗粒凝胶剂半衰期仅为45.22天，由于大分子药物难以透过皮肤屏障，难以发挥药效(Tiwari S,Dwivedi H,Kymonil K M,et al.Drug Deliv Transl Res,2015,5(3):219-230.)。
发明人所在课题组致力于尿酸酶及其药物制剂的研究，并已申请若干项中国发明专利，如专利号CN201410048071.9、授权公告号CN103834623B的中国发明专利，申请号CN201510066745.2、申请公布号CN104630168A的中国发明专利申请，申请号CN201610316709.1、申请公布号CN105838686A的中国发明专利申请。目前，发明人所在课题组在尿酸酶制剂方面有了进一步的研究成果。
发明内容
本发明的主要目的是：克服现有技术存在的问题，提供一种尿酸酶外用凝胶制剂，使用方式为涂抹于皮肤表面，该制剂易于制备，稳定性好，使用方便，药效发挥平稳，用药过程中易于自主控制尿酸水平，制剂疗效的发挥不依赖于尿酸酶透过皮肤屏障进入机体。此外，本发明还提供该制剂的制备方法，以及该制剂的用途。
本发明解决其技术问题的技术方案如下：
一种尿酸酶外用凝胶制剂，其特征是：所述制剂由以下组分按重量百分比组成：
Figure PCTCN2019103968-appb-000001
所述制剂的pH经pH调节剂调整为5-10。
优选地，所述尿酸酶包括天然尿酸酶、重组尿酸酶、嵌合尿酸酶、以及融合尿酸酶；所述天然尿酸酶来源于原核生物或真核生物；所述重组尿酸酶、嵌合尿酸酶、融合尿酸酶分别经生物工程技术制备而得；所述天然尿酸酶包括来源于微生物的尿酸酶、以及来源于哺乳动物的尿酸酶，所述微生物包括产朊假丝酵母、黄曲霉，所述哺乳动物包括猪、犬；所述重组尿酸酶包括采用生物工程技术制备的天然尿酸酶基因表达的重组尿酸酶；所述嵌合尿酸酶包括由不同天然尿酸酶基因之间的核苷酸序列嵌合、重组表达的尿酸酶嵌合蛋白；所述融合尿酸酶包括由天然尿酸酶基因与人血清白蛋白基因融合、重组表达的人血清白蛋白-尿酸酶融合蛋白，由天然尿酸酶基因与人源化抗体Fc片段核苷酸序列融合、重组表达的Fc-尿酸酶融合蛋白。
更优选地，所述尿酸酶具有化学修饰，所述化学修饰包括PEG修饰。
优选地，所述水性凝胶基质选自天然高分子或半合成高分子或合成高分子；所述天然高分子包括淀粉、海藻酸盐、阿拉伯胶、西黄蓍胶、琼脂和明胶；所述半合成高分子包括改性纤维素和改性淀粉，所述改性纤维素包括羧甲基纤维素、甲基纤维素；所述合成高分子包括卡波姆、聚丙烯酸钠。
更优选地，所述水性凝胶基质为卡波姆934、卡波姆940、卡波姆941、卡波姆942、卡波姆971、卡波姆974、卡波姆980、卡波姆981之一或其组合。
优选地，所述蛋白保护剂选自牛血清白蛋白(BSA)、甘露醇、蔗糖、柠檬酸钠、山梨醇之一或其组合；
所述保湿剂选自甘油、丙二醇、乙醇、透明质酸之一或其组合；
所述螯合剂选自乙二胺四乙酸的盐类之一或其组合，所述乙二胺四乙酸的盐类包括乙二胺四乙酸二钠盐(EDTA-2Na)、乙二胺四乙酸二钾盐(EDTA-2K)；
所述透皮促进剂选自氮酮、丙二醇、透明质酸、胆酸盐、脱氧胆酸盐、尿素、环状糊精、吐温-80之一或其组合；
所述防腐剂选自苯甲酸、苯甲酸钠、对羟基苯甲酸甲酯、对羟基苯甲酸乙酯、苯酚、山梨酸之一或其组合；
所述pH调节剂选自三乙醇胺、NaOH、NaHCO 3之一。
优选地，所述尿酸酶的含量为0.001-0.1％；所述制剂的pH为6-9。
本发明还提供：前文所述尿酸酶外用凝胶制剂的制备方法，其特征是，包括以下步骤：
第一步、取适量的注射用水，加入透皮促进剂、保湿剂、以及螯合剂并溶解搅拌均匀；然后加入水性凝胶基质，经溶胀和/或溶解后搅拌均匀，湿热灭菌得备用物；
第二步、将蛋白保护剂、防腐剂、主药尿酸酶分别加入适量注射用水溶解，并经微孔滤膜过滤除菌备用；
第三步、将第二步所得防腐剂加入第一步所得备用物中，并以pH调节剂调节pH值至5-10，然后加入第二步所得蛋白保护剂、主药尿酸酶以及余量注 射用水，搅拌均匀后分装，即得尿酸酶外用凝胶制剂成品。
优选地，第一步中，溶胀所用时间为过夜，湿热灭菌的条件为115℃30min；第二步中，所述微孔薄膜的孔径为0.22μm；第三步中，调节后pH为6-9；在分装时，分装至药学上可接受的容器中，所述容器包括药用铝管、塑胶管、铝塑管、聚乙烯复合软管，或者，在分装时，涂布于卫生材料制成凝胶贴剂；分装后，将所得分装物保存于4℃冰箱内。
本发明还提供：前文所述尿酸酶外用凝胶制剂用于制备药物或药物组合物的用途，所述药物或药物组合物的作用为降尿酸、或治疗高尿酸血症、或治疗痛风、或治疗高尿酸血症引起的疾病。
与现有技术相比，本发明的有益效果如下：
本发明凝胶制剂的给药途径为外用涂抹于皮肤表面，降解通过汗腺分泌皮肤表面的尿酸，生成尿囊素，进而形成皮肤内外尿酸的浓度梯度，促进汗腺以微流形式持续分泌尿酸等代谢产物，发挥显著的降体内尿酸作用；该制剂疗效的发挥不依赖于尿酸酶透过皮肤屏障进入机体，不同于冻干粉针剂需静脉给药进入机体，避免了尿酸酶的过敏反应、输液反应和抗体产生；该制剂具有降解尿酸作用，除可以用于治疗高尿酸血症、痛风外，还可以用于治疗由于血液中尿酸浓度增高引起的并发症，如糖尿病、高血压、间质性肾炎、肾衰竭及其它心血管系统疾病等辅助治疗。
本发明制备方法既能够保持尿酸酶活性又能够使尿酸酶凝胶制剂长期保持稳定性。本发明制备工艺简单，制剂稳定性好，使用方便，药效发挥平稳，用药过程中易于自主控制尿酸水平。
附图说明
图1为本发明实施例9的尿酸标准曲线图。
图2为本发明实施例11的高效液相色谱法测定的尿酸标准曲线图。
图3为本发明实施例11的高效液相色谱检测尿酸溶液的示例图。
图4为本发明实施例11的各实验组皮肤表面尿酸含量测定结果图。
图5为本发明实施例11的各实验组皮肤组织尿酸含量测定结果图。
图6为本发明实施例12的高效液相色谱法测定的尿囊素标准曲线图。
图7为本发明实施例12的各实验组皮肤表面尿囊素含量测定结果图
图8为本发明实施例13的结果示意图。
具体实施方式
下面参照附图并结合实施例对本发明作进一步详细描述。但是本发明不限于所给出的例子。下述实施例中的试验方法，如无特殊说明，均为常规方法；试剂和材料，如无特殊说明，均可通过商业途径获得。
实施例1、尿酸酶外用凝胶制剂制备方案一
本实施例的尿酸酶外用凝胶制剂由以下组分组成(按100g凝胶计)：
Figure PCTCN2019103968-appb-000002
Figure PCTCN2019103968-appb-000003
采用15％三乙醇胺溶液(pH调节剂)调节制剂的pH至7.4。
本实施例尿酸酶外用凝胶制剂的制备方法包括：
(1)主药重组人猪尿酸酶的制备：按照中国专利104630168A构建重组E.coli工程菌，将工程菌接种于LB固体培养基上37℃培养进行活化，12h后挑取单菌落于LB液体培养基中，37℃继续培养12h，再按体积比为1％的接种量加入发酵培养基，200rpm，37℃培养4h后，加入IPTG诱导尿酸酶蛋白表达，继续培养6h后离心收集湿菌体，加入Tris-Hcl缓冲液重悬沉淀，超声破壁，破壁条件为：功率为60％，超3s隔3s，30min。破壁菌液离心后收集沉淀，按m:V＝1g:10ml比例加入Tris-Hcl缓冲液重悬沉淀，离心后收集上清，采用硫酸铵分级沉淀对目的蛋白进行盐析，盐析后沉淀用蒸馏水洗3遍，按m:V＝1g:10ml加入tris-Hcl缓冲液复溶，得主药人猪嵌合尿酸酶粗酶液。
(2)取占注射用水总量60％重量的注射用水，加入透明质酸、甘油、EDTA-2K并溶解搅拌均匀；然后加入羧甲基纤维素，经溶解后搅拌均匀，115℃湿热灭菌30min得备用物；
(3)将苯甲酸钠、BSA、主药重组人-猪嵌合尿酸酶分别加入适量注射用水溶解，经0.22μm微孔滤膜过滤除菌备用；
(4)将(3)所得苯甲酸钠加入(2)所得备用物中，并以15％三乙醇胺溶液调节pH值至7.4，然后加入(3)所得BSA、主药重组人-猪嵌合尿酸酶以及余量注射用水，搅拌均匀，分装到无菌铝管中，即得尿酸酶外用凝胶制剂成品，保存于4℃冰箱中备用。
实施例2、尿酸酶外用凝胶制剂制备方案二
本实施例的尿酸酶外用凝胶制剂由以下组分组成(按100g凝胶计)：
Figure PCTCN2019103968-appb-000004
采用15％的NaOH溶液(pH调节剂)调节制剂pH至7.5。
本实施例尿酸酶外用凝胶制剂的制备方法包括：
(1)取占注射用水总量60％重量的注射用水，加入透明质酸、甘油、EDTA-2Na并溶解搅拌均匀；然后加入海藻酸钠，经过夜溶胀溶解后，115℃湿热灭菌30min得备用物；
(2)将山梨酸、BSA、主药产朊假丝酵母尿酸酶分别加入适量注射用水溶解，经0.22μm微孔滤膜过滤除菌备用；
(3)将(2)所得山梨酸加入(1)所得备用物中，并15％的NaOH溶液调节pH值至7.5，然后加入(2)所得BSA、主药产朊假丝酵母尿酸酶以及余量注射用水，搅拌均匀，分装到无菌的聚乙烯复合软管中，即得尿酸酶外用凝胶制剂成品，保存于4℃冰箱中备用。
实施例3、尿酸酶外用凝胶制剂制备方案三
本实施例的尿酸酶外用凝胶制剂由以下组分组成(按100g凝胶计)：
Figure PCTCN2019103968-appb-000005
采用15％的NaOH溶液(pH调节剂)调节制剂pH至8.5。
本实施例尿酸酶外用凝胶制剂的制备方法包括：
(1)取占注射用水总量60％重量的注射用水，加入甘油、丙二醇、EDTA-2K并溶解搅拌均匀；然后加入卡波姆941，经过夜溶胀溶解后，115℃湿热灭菌30min得备用物；
(2)将对羟基苯甲酸甲酯、甘露醇、主药黄曲霉来源尿酸酶分别加入适量注射用水溶解，经0.22μm微孔滤膜过滤除菌备用；
(3)将(2)所得对羟基苯甲酸甲酯加入(1)所得备用物中，并以15％的NaOH溶液调节pH值至8.5，然后加入(2)所得甘露醇、主药黄曲霉来源尿酸酶以及余量注射用水，搅拌均匀，分装到药用铝塑管中，即得尿酸酶外用凝胶制剂成品，保存于4℃冰箱中备用。
实施例4、尿酸酶外用凝胶制剂制备方案四
本实施例的尿酸酶外用凝胶制剂由以下组分组成(按100g凝胶计)：
Figure PCTCN2019103968-appb-000006
采用10％NaHCO 3溶液(pH调节剂)调节制剂pH至8.0。
本实施例尿酸酶外用凝胶制剂的制备方法包括：
(1)聚乙二醇修饰尿酸酶的制备方法:按文献(张纯.尿酸酶的聚乙二醇 修饰及其成药性的初步评价[D].重庆医科大学,2011.)方法对产朊假丝酵母尿酸酶进行聚乙二醇修饰，修饰后分离纯化，去除反应多余的聚乙二醇，制备足够多的聚乙二醇修饰尿酸酶蛋白样品。
(2)取占注射用水总量60％重量的注射用水，加入甘油、丙二醇、EDTA-2Na并溶解搅拌均匀；然后加入甲基纤维素，经溶解后搅拌均匀，115℃湿热灭菌30min得备用物；
(3)将苯甲酸、甘露醇、主药聚乙二醇修饰尿酸酶分别加入适量注射用水溶解，经0.22μm微孔滤膜过滤除菌备用；
(4)将(3)所得苯甲酸加入(2)所得备用物中，并以10％NaHCO 3溶液调节pH值至8.0，然后加入(3)所得甘露醇、主药聚乙二醇修饰尿酸酶，搅拌均匀，分装到塑胶管中，即得尿酸酶外用凝胶制剂成品，保存于4℃冰箱中备用。
实施例5、尿酸酶外用凝胶制剂制备方案五
本实施例的尿酸酶外用凝胶制剂由以下组分组成(按100g凝胶计)：
Figure PCTCN2019103968-appb-000007
采用10％NaHCO 3溶液(pH调节剂)调节制剂pH至8.0。
本实施例尿酸酶外用凝胶制剂的制备方法包括：
(1)取占注射用水总量60％重量的注射用水，加入丙二醇、无水乙醇、EDTA-2Na并溶解搅拌均匀；然后加入卡波姆942，经过夜溶胀溶解后，115℃湿热灭菌30min得备用物；
(2)将苯甲酸、甘露醇、主药Fc-尿酸酶融合蛋白分别加入适量注射用水溶解，经0.22μm微孔滤膜过滤除菌备用；
(3)将(2)所得苯甲酸加入(1)所得备用物中，并以10％NaHCO 3溶液调节pH值至8.0，然后加入(2)所得甘露醇、主药Fc-尿酸酶融合蛋白以及余量注射用水，搅拌均匀，分装到塑胶管中，即得尿酸酶外用凝胶制剂成品，保存于4℃冰箱中备用。
实施例6、尿酸酶外用凝胶制剂制备方案六
本实施例的尿酸酶外用凝胶制剂由以下组分组成(按100g凝胶计)：
Figure PCTCN2019103968-appb-000008
Figure PCTCN2019103968-appb-000009
采用15％三乙醇胺溶液(pH调节剂)调节制剂的pH至7.4。
本实施例尿酸酶外用凝胶制剂的制备方法包括：
(1)取占注射用水总量60％重量的注射用水，加入氮酮、甘油、EDTA-2K并溶解搅拌均匀；然后加入阿拉伯胶，过夜溶胀，115℃湿热灭菌30min得备用物；
(2)将苯甲酸钠、蔗糖、甘露醇、主药重组人-猪嵌合尿酸酶分别加入适量注射用水溶解，经0.22μm微孔滤膜过滤除菌备用；
(3)将(2)所得苯甲酸钠加入(1)所得备用物中，并以15％三乙醇胺溶液调节pH值至7.4，然后加入(2)所得蔗糖、甘露醇、主药重组人-猪嵌合尿酸酶以及余量注射用水，搅拌均匀，分装到无菌铝管中，即得尿酸酶外用凝胶制剂成品，保存于4℃冰箱中备用。
实施例7、尿酸酶外用凝胶制剂制备方案七
本实施例的尿酸酶外用凝胶制剂由以下组分组成(按100g凝胶计)：
Figure PCTCN2019103968-appb-000010
采用15％的NaOH溶液(pH调节剂)调节制剂pH至7.5。
本实施例尿酸酶外用凝胶制剂的制备方法包括：
(1)取占注射用水总量60％重量的注射用水，加入丙二醇、甘油、苯甲酸钠并溶解搅拌均匀；然后加入甲基纤维素，经过夜溶胀溶解后，115℃湿热灭菌30min得备用物；
(2)将EDTA-2Na、甘露醇、主药产朊假丝酵母尿酸酶分别加入适量注射用水溶解，经0.22μm微孔滤膜过滤除菌备用；
(3)将(2)所得苯甲酸钠加入(1)所得备用物中，并15％的NaOH溶液调节pH值至7.5，然后加入(2)所得甘露醇、主药产朊假丝酵母尿酸酶以及余量注射用水，搅拌均匀，分装到无菌的聚乙烯复合软管中，即得尿酸酶外用凝胶制剂成品，保存于4℃冰箱中备用。
实施例8、尿酸酶外用凝胶制剂制备方案三
本实施例的尿酸酶外用凝胶制剂由以下组分组成(按100g凝胶计)：
Figure PCTCN2019103968-appb-000011
Figure PCTCN2019103968-appb-000012
采用15％的NaOH溶液(pH调节剂)调节制剂pH至8.5。
本实施例尿酸酶外用凝胶制剂的制备方法包括：
(1)取占注射用水总量60％重量的注射用水，加入甘油、透明质酸、EDTA-2K并溶解搅拌均匀；然后加入卡波姆934，经过夜溶胀溶解后，115℃湿热灭菌30min得备用物；
(2)将对羟基苯甲酸甲酯、BSA、主药黄曲霉来源尿酸酶分别加入适量注射用水溶解，经0.22μm微孔滤膜过滤除菌备用；
(3)将(2)所得对羟基苯甲酸甲酯加入(1)所得备用物中，并以15％的NaOH溶液调节pH值至8.5，然后加入(2)所得BSA、主药黄曲霉来源尿酸酶以及余量注射用水，搅拌均匀，分装到药用铝塑管中，即得尿酸酶外用凝胶制剂成品，保存于4℃冰箱中备用。
实施例9、尿酸酶外用凝胶制剂稳定性及酶活稳定性研究
A.尿酸酶外用凝胶制剂稳定性研究
按实施例1中尿酸酶外用凝胶制剂的制备方法，共制备三批样品，生产批号分别为20170912、20170913、20170914，通过下列方法进行凝胶制剂稳定性研究。
将上述凝胶制剂尿酸酶外用凝胶制剂置于密闭无菌铝管中，分别于37℃、室温及4℃条件下保存，定时检查凝胶制剂的外观、失水、酸碱度及霉败等现象，并取样测定凝胶制剂中尿酸酶的酶活保有率，检测凝胶制剂尿酸酶外用凝胶制剂中尿酸酶的酶活稳定性。
结果表明，三批凝胶制剂尿酸酶外用凝胶制剂37℃保存3个月以及室温和4℃保存6个月后，凝胶制剂无失水、也无霉败、外观、pH均无变化。(注：此处受篇幅限制，未将具体实验数据列于此处)
B.尿酸酶活性测定的标准曲线测定
尿酸标准曲线的测定：用0.1M硼砂-硼酸溶液(pH8.5)将配制好的600μM尿酸母液分别稀释为60μM、54μM、48μM、42μM、36μM、30μM、24μM、18μM、12μM、0μM，测定各溶液的OD 293，结果如表1所示；根据测定的数据绘制标准曲线，结果见图1。
表1 尿酸梯度浓度溶液配制
Figure PCTCN2019103968-appb-000013
Figure PCTCN2019103968-appb-000014
C.尿酸酶活性检测：
根据测定的尿酸标准曲线的线性范围，向比色皿中加入尿酸溶液，根据酶液的浓度向尿酸溶液中加入适量酶液以确保底物过量，将两者快速混匀后测定3min内293nm处的吸光值，根据尿酸标准曲线计算尿酸的消耗量，计算酶活。公式如下，其中U＝尿酸酶活力单位；(A 0﹣A)/3表示在反应的3min内尿酸溶液在293nm处吸光值下降的斜率；Vt＝反应液总体积(mL)；12.078为尿酸在293nm处的微摩尔消光系数；Ve＝酶液体积(mL)。
Figure PCTCN2019103968-appb-000015
D.人-猪嵌合凝胶制剂尿酸酶外用凝胶制剂的酶活稳定性研究
将重组人-猪嵌合凝胶制剂尿酸酶外用凝胶制剂放置在4℃、37℃条件下，于不同时间点取样，检测重组人-猪嵌合尿酸酶活性，结果见下表：
表2：37℃凝胶制剂酶活稳定性
Figure PCTCN2019103968-appb-000016
由表2可知，凝胶制剂尿酸酶外用凝胶制剂在37℃条件下24小时酶活保有率保持在90％以上，48小时85％，72小时，酶活保有率仍可达到81％。
表3：4℃凝胶制剂尿酸酶外用凝胶制剂酶活稳定性
Figure PCTCN2019103968-appb-000017
由表3可知，凝胶制剂尿酸酶外用凝胶制剂在4℃条件下6个月酶活保有率保持在85％以上。
综上可知，本发明的尿酸酶外用凝胶制剂，可以保护尿酸酶免于外界多种因素的影响，有利于稳定尿酸酶，保护尿酸酶活性。
实施例10、尿酸酶外用凝胶制剂局部刺激性实验
取健康家兔6只，体重1.8-2.2kg,于背部脊柱两侧对称剃毛，每侧约4×4cm 2，休息24h，正常饲养。一侧涂适量实施例1尿酸酶外用凝胶制剂，另一侧涂适量实施例1采用的水性凝胶基质，24h洗掉残留物，连续观察20天，家兔皮肤并未出现红斑、丘疹、水泡等现象。
实施例11、皮肤尿酸含量测定
A.尿酸标准曲线绘制
(1)检测尿酸方法
色谱条件：Waters C18柱(250mm×4.6mm，5μm)流动相：磷酸3mL，甲醇30mL，加超纯水定容；流速：1.0mL/min；检测波长：293nm；柱温：30℃，进样量20μL。
(2)尿酸母液(5000μM)配制：
称取84mg尿酸于100mL烧杯中，加入适量硼砂-硼酸缓冲溶液，磁力搅 拌器搅拌至其完全溶解，定容至100ml即得。
(3)0-500μM尿酸标曲绘制：
取5mM尿酸，依次用硼砂-硼酸缓冲溶液稀释至500，250，100，50，20，10μM，吸取20μL上述溶液，按体积比1:10比例加入蛋白沉淀剂，冰水浴5min，离心后吸取上清进样后以尿酸浓度和峰面积绘制标准曲线，如图2所示；检测尿酸溶液的色谱图示例如图3所示。
B.高效液相色谱测定皮肤表面的尿酸含量
取5-6周龄雄性昆明小鼠24只，按体重随机法分成以下4组：(1)空白组，(2)模型组，(3)阴性对照组，(4)尿酸酶凝胶给药组。每组6只小鼠，空白组小鼠正常饮食，不给予尿酸酶外用凝胶制剂或水性凝胶基质；模型组小鼠采用每日腹腔注射尿酸钠混悬液，同时拌食法给予含酵母粉饲料，制备小鼠高尿酸血症模型；阴性对照组造模方法同模型组，造模成功后，给予实施例1采用的水性凝胶基质(即空白凝胶)；尿酸酶凝胶给药组：造模方法同模型组，造模成功后，给予实施例1的尿酸酶外用凝胶制剂。
第一次给药前，先将各组小鼠背部裸露皮肤消毒并用棉球清洗，擦拭干净后将小鼠放回笼中，凝胶给药组给予尿酸酶外用凝胶，阴性对照组给予空白凝胶，待凝胶自然风干后，小鼠放回笼中，自由饮食饮水，分别在不同时段，用棉签沾取适量生理盐水擦拭各组小鼠背部皮肤，再将棉签上的水分挤入EP管中，标记为“皮肤擦拭样品”。所得样品离心后加入一定比例蛋白沉淀剂，离心后取上清用于HPLC测定，积分法得到尿酸峰面积，再根据标准曲线算出尿酸浓度。
由图4可以看出，空白组和模型组小鼠皮肤表面尿酸在8h后尿酸浓度逐渐达上限，且模型组小鼠皮肤表面尿酸含量是空白组的2.4倍，阴性对照组与模型组相同，给药组由于凝胶中的尿酸酶可不断降解由汗腺向皮肤表面分泌的尿酸，因此4-8h皮肤表面尿酸含量均比空白组低，但12h后超过空白组，24h后，皮肤表面尿酸浓度超过模型组，因为皮肤表面尿酸与血清尿酸浓度之间呈正比关系，由于皮肤表面尿酸浓度的降低，致使皮肤内外尿酸形成了浓度梯度，使体内尿酸通过汗腺自发不断地排到皮肤表面，以到达与血液尿酸相对于的皮肤尿酸浓度，也就是模型组皮肤尿酸浓度，而尿酸酶凝胶给药组的皮肤表面尿酸排泄量明显超过了模型组，因此可以说明尿酸酶凝胶不仅可以降解皮肤表面尿酸，同时也能起到促进皮肤尿酸排泄的作用。
C.高效液相色谱测定皮肤组织的尿酸含量
给药一周后，颈椎脱臼处死小鼠，将各组小鼠皮肤组织取下并匀浆，离心后吸取上清至EP管中，按一定比例加入蛋白沉淀剂，离心后吸取上清用于HPLC测定，积分法得到尿酸峰面积，再根据标准曲线算出尿酸浓度，再按下述公式得到单位重量的皮肤尿酸含量：
单位皮肤尿酸含量＝皮肤尿酸含量(μM)/皮肤重量(g)
由图5可以看出，高尿酸血症的小鼠(模型组)的皮肤组织尿酸水平为正常小鼠(空白组)的皮肤组织尿酸水平的2倍；阴性对照组由于给予空白凝胶，不具有分解尿酸的作用，因而不能阻止小鼠皮肤尿酸水平的升高，其 皮肤组织尿酸水平与模型组接近，仍然为空白组皮肤尿酸水平的2倍；给药组给予尿酸酶外用凝胶制剂，由于尿酸酶能够发挥降解尿酸的作用，可以将不断分泌出皮肤的尿酸降解，从而使皮肤组织尿酸水平降低，且给药一周后，给药组小鼠的皮肤组织尿酸水平与空白组小鼠皮肤组织尿酸水平相近，说明给予一周的尿酸酶外用凝胶制剂能起到非常显著的降低小鼠皮肤组织尿酸的效果，且高尿酸血症小鼠的血尿酸水平与皮肤尿酸水平呈正比关系，皮肤组织的尿酸水平降低，意味着给药组小鼠的血尿酸水平也会跟着降低，从而起到降低血尿酸水平的作用。
实施例12、皮肤尿囊素含量的检测
A.尿囊素标准曲线的绘制
(1)检测尿囊素的方法
色谱条件：Dubhe C18柱(250mm×4.6mm,5μm)，流动相：甲醇-水(10：90)；柱温：30℃；检测波长：210nm；流速：1ml×min -1；进样10μl。
(2)尿囊素母液(500μM)配制：
用分析天平精密称取0.02g尿囊素标准品于100ml水中，搅拌并溶解充分，用玻璃棒引流至250ml洁净的容量瓶中，继续加水定容至刻度线，混合均匀后即得500μM尿囊素标准溶液。
(3)0-500μM尿囊素标曲绘制：
取500μM尿囊素溶液，依次用蒸馏水稀释至250，100，50，20，10μM，吸取20μL上述溶液，按一定比例加入蛋白沉淀剂，冰水浴5min，离心后吸取上清进样后以尿囊素浓度和峰面积绘制标准曲线，如图6所示。
B.高效液相色谱测定给药后皮肤表面的尿囊素含量
将收集的尿酸酶凝胶给药组的“皮肤擦拭样品”，离心取上清后加入一定比例蛋白沉淀剂，离心后上清用于HPLC检测，积分法得到尿囊素峰面积，再根据标准曲线算出尿囊素浓度。
结果如图7所示，给药4h后，给药组由于皮肤表面涂抹了尿酸酶凝胶，可不断降解由体内排泄到皮肤表面的尿酸，生成尿囊素，因此皮肤表面的尿酸含量低于空白组，4-12h，尿囊素生成量不断上升，说明此时凝胶依然保有酶活，具有降解皮肤尿酸的能力，直至24h，此时尿囊素生成量相比于12h多出了0.3μM，二者相接近，推测此时凝胶中的尿酸酶已被充分反应，或是被皮肤表面的蛋白酶降解及小鼠舔舐而失活，但此时依然有尿酸不断从体内排到体外，在皮肤表面不断的积累，最终给药组24h的尿酸排泄量超过模型组24h的皮肤表面尿酸含量。因此可以说明，尿酸酶凝胶制剂可以将皮肤表面尿酸转化为水溶性更高的尿囊素，且给药12h后，皮肤表面的尿酸酶凝胶制剂依然还残有活性，使得血尿酸源源不断的排出体内，同时还增加了皮肤尿酸的排泄量，从而起到快速持续的降低血尿酸水平的作用。
实施例13、尿酸酶外用凝胶制剂药效学研究
A：小鼠高尿酸血症造模
空白组仅给予普通饲料饲养；模型组、阴性对照组、给药组每日腹腔注射尿酸钠混悬液1次，同时拌食法给予含酵母粉饲料。
B：动物分组、给药
取5-6周龄雄性昆明小鼠56只，按体重随机法分为以下7组：空白组、模型组、阴性对照组、给药组1(UOX 83凝胶：实施例1的重组人-猪嵌合尿酸酶外用凝胶制剂,规格25mg/100g,批次：20180512)、给药组2(UOX PEU凝胶：实施例2的产朊假丝酵母尿酸酶外用凝胶制剂，规格25mg/100g，批次：20180512)、给药组3(实施例4的聚乙二醇修饰尿酸酶外用凝胶制剂，规格25mg/100g，批次：20180513)、给药组4(实施例5的Fc-尿酸酶融合蛋白外用凝胶制剂，规格25mg/100g，批次：20180513)。
空白组：不造模、不用药；模型组：每日造模、不用药；阴性对照组：连续造模、背部去毛涂抹空白凝胶(采用实施例1的水性凝胶基质)；给药组：连续造模，造模成功后，背部去毛每日涂抹相应的尿酸酶外用凝胶制剂；除空白组不用造模外，其他小鼠给药期间正常造模。
C.血尿酸水平测定
连续给药一周后，对各组小鼠进行眼眶取血。4℃静置一段时间，离心取上清，HPLC进样20μL，记录尿酸峰面积，根据尿酸标准曲线计算血液尿酸含量。
表4：连续给药一周后各组小鼠血清尿酸水平(
Figure PCTCN2019103968-appb-000018
n＝8,μM)
Figure PCTCN2019103968-appb-000019
Figure PCTCN2019103968-appb-000020
注：*，**和***分别表示在P<0.05，P<0.01，P<0.001的水平上与模型组之间差异的显著性。
由上表及图8可知，经过高尿酸血症造模的模型组、阴性对照组、给药组1、给药组2、给药组3及给药组4的小鼠的血清尿酸水平为空白组小鼠血清尿酸水平的两倍以上，给药组1、给药组2、给药组3及给药组4在给药一周后均可以将小鼠血尿酸水平降至与空白组相近。
综上，本发明的尿酸酶外用凝胶制剂不仅具有显著降解皮肤表面的尿酸，以转化成尿囊素，还可促进血液尿酸不断向皮肤表面排泄，进而降低血清尿酸水平。
除上述实施例外，本发明还可以有其他实施方式。凡采用等同替换或等效变换形成的技术方案，均落在本发明要求的保护范围。

Claims (10)

  1. 一种尿酸酶外用凝胶制剂，其特征是：所述制剂由以下组分按重量百分比组成：
    Figure PCTCN2019103968-appb-100001
    所述制剂的pH经pH调节剂调整为5-10。
  2. 根据权利要求1所述的尿酸酶外用凝胶制剂，其特征是，所述尿酸酶包括天然尿酸酶、重组尿酸酶、嵌合尿酸酶、以及融合尿酸酶；所述天然尿酸酶来源于原核生物或真核生物；所述重组尿酸酶、嵌合尿酸酶、融合尿酸酶分别经生物工程技术制备而得；所述天然尿酸酶包括来源于微生物的尿酸酶、以及来源于哺乳动物的尿酸酶，所述微生物包括产朊假丝酵母、黄曲霉，所述哺乳动物包括猪、犬；所述重组尿酸酶包括采用生物工程技术制备的天然尿酸酶基因表达的重组尿酸酶；所述嵌合尿酸酶包括由不同天然尿酸酶基因之间的核苷酸序列嵌合、重组表达的尿酸酶嵌合蛋白；所述融合尿酸酶包括由天然尿酸酶基因与人血清白蛋白基因融合、重组表达的人血清白蛋白-尿酸酶融合蛋白，由天然尿酸酶基因与人源化抗体Fc片段核苷酸序列融 合、重组表达的Fc-尿酸酶融合蛋白。
  3. 根据权利要求1或2所述一种尿酸酶外用凝胶制剂，其特征是，所述尿酸酶具有化学修饰，所述化学修饰包括PEG修饰。
  4. 根据权利要求1或2所述一种尿酸酶外用凝胶制剂，其特征是，所述水性凝胶基质选自天然高分子或半合成高分子或合成高分子；所述天然高分子包括淀粉、海藻酸盐、阿拉伯胶、西黄蓍胶、琼脂和明胶；所述半合成高分子包括改性纤维素和改性淀粉，所述改性纤维素包括羧甲基纤维素、甲基纤维素；所述合成高分子包括卡波姆、聚丙烯酸钠。
  5. 根据权利要求4所述一种尿酸酶外用凝胶制剂，其特征是，所述水性凝胶基质为卡波姆934、卡波姆940、卡波姆941、卡波姆942、卡波姆971、卡波姆974、卡波姆980、卡波姆981之一或其组合。
  6. 根据权利要求1或2所述一种尿酸酶外用凝胶制剂，其特征是，所述蛋白保护剂选自牛血清白蛋白、甘露醇、蔗糖、柠檬酸钠、山梨醇之一或其组合；
    所述保湿剂选自甘油、丙二醇、乙醇、透明质酸之一或其组合；
    所述螯合剂选自乙二胺四乙酸的盐类之一或其组合，所述乙二胺四乙酸的盐类包括乙二胺四乙酸二钠盐、乙二胺四乙酸二钾盐；
    所述透皮促进剂选自氮酮、丙二醇、透明质酸、胆酸盐、脱氧胆酸盐、尿素、环状糊精、吐温-80之一或其组合；
    所述防腐剂选自苯甲酸、苯甲酸钠、对羟基苯甲酸甲酯、对羟基苯甲酸乙酯、苯酚、山梨酸之一或其组合；
    所述pH调节剂选自三乙醇胺、NaOH、NaHCO 3之一。
  7. 根据权利要求1或2所述尿酸酶外用凝胶制剂，其特征是，所述尿酸酶的含量为0.001-0.1％；所述制剂的pH为6-9。
  8. 根据权利要求1至7任一项所述尿酸酶外用凝胶制剂的制备方法，其特征是，包括以下步骤：
    第一步、取适量的注射用水，加入透皮促进剂、保湿剂、以及螯合剂并溶解搅拌均匀；然后加入水性凝胶基质，经溶胀和/或溶解后搅拌均匀，湿热灭菌得备用物；
    第二步、将蛋白保护剂、防腐剂、主药尿酸酶分别加入适量注射用水溶解，并经微孔滤膜过滤除菌备用；
    第三步、将第二步所得防腐剂加入第一步所得备用物中，并以pH调节剂调节pH值至5-10，然后加入第二步所得蛋白保护剂、主药尿酸酶以及余量注射用水，搅拌均匀后分装，即得尿酸酶外用凝胶制剂成品。
  9. 根据权利要求8所述的制备方法，其特征是，第一步中，溶胀所用时间为过夜，湿热灭菌的条件为115℃30min；第二步中，所述微孔薄膜的孔径为0.22μm；第三步中，调节后pH为6-9；在分装时，分装至药学上可接受的容器中，所述容器包括药用铝管、塑胶管、铝塑管、聚乙烯复合软管，或者，在分装时，涂布于卫生材料制成凝胶贴剂；分装后，将所得分装物保存于4℃冰箱内。
  10. 根据权利要求1至7任一项所述尿酸酶外用凝胶制剂用于制备药物或药物组合物的用途，所述药物或药物组合物的作用为降尿 酸、或治疗高尿酸血症、或治疗痛风、或治疗高尿酸血症引起的疾病。
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10815461B2 (en) 2017-07-07 2020-10-27 Allena Pharmaceuticals, Inc. Recombinant uricase enzyme

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EP3681479B1 (en) 2017-09-15 2024-01-31 Dyve Biosciences, Inc. Sodium bicarbonate for use in the treatment of gout and related disorders
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CN115531346B (zh) * 2022-09-24 2024-01-26 重庆医科大学 一种仿生融合膜包裹尿酸酶、铂纳米粒和白藜芦醇脂质纳米粒及其制备方法

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101302501A (zh) * 2007-05-10 2008-11-12 刘国安 聚乙二醇化尿酸氧化酶化合物及其制备方法和其制剂及应用
CN104066324A (zh) * 2011-11-04 2014-09-24 西玛贝医药公司 治疗痛风急性发作的方法
CN106902349A (zh) * 2017-03-07 2017-06-30 云南中医学院 一种口服降尿酸药物的制备与应用
CN109223707A (zh) * 2018-09-13 2019-01-18 中国药科大学 一种尿酸酶外用凝胶制剂、其制备方法及用途

Family Cites Families (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NZ234453A (en) * 1989-07-13 1993-01-27 Sanofi Sa Recombinant dna encoding urate oxidase, and vector, host, protein and pharmaceutical compositions associated therewith
CA2506839A1 (en) * 2002-11-20 2004-06-03 Prometic Biosciences, Inc. Composition and method for treating inflammatory diseases using protease inhibitors
CN1864744B (zh) * 2005-05-18 2010-09-29 杭州北斗生物技术有限公司 含有尿酸氧化酶的药用制剂
CN101168052A (zh) * 2007-10-26 2008-04-30 西安交通大学 一种防治高尿酸血症及痛风的肠溶制剂
CN101390969A (zh) * 2008-11-03 2009-03-25 中国人民解放军军事医学科学院生物工程研究所 降低血液中尿酸浓度的药物
CN101816630B (zh) * 2009-02-26 2012-07-04 重庆医科大学 尿酸酶脂质纳米粒及其制备方法
CN102357242B (zh) * 2011-11-02 2012-12-19 珠海亿胜生物制药有限公司 重组牛碱性成纤维细胞生长因子凝胶
CN103083226B (zh) * 2012-10-26 2015-11-25 温州医学院 成纤维细胞生长因子-1改构体凝胶剂制备及在糖尿病足治疗中的应用
US8778365B1 (en) * 2013-01-31 2014-07-15 Merz Pharmaceuticals, Llc Topical compositions and methods for making and using same
CN103977396A (zh) * 2014-05-26 2014-08-13 重庆医科大学 一种尿酸酶复合过氧化氢酶脂质体及其制备方法
CN104645313A (zh) * 2015-01-28 2015-05-27 南京航空航天大学 一种修复止痒贻贝粘蛋白凝胶剂及其制备方法
CN104630168A (zh) * 2015-02-04 2015-05-20 中国药科大学 一种重组人猪嵌合尿酸酶的构建和表达
CN105062987B (zh) * 2015-09-01 2018-09-25 山西锦波生物医药股份有限公司 重组人/狒狒嵌合尿酸酶蛋白及其制备方法
CN106554948B (zh) * 2015-09-29 2019-06-25 上海生物制品研究所有限责任公司 突变型尿酸酶、peg修饰的突变型尿酸酶及其应用
CN105838686B (zh) * 2016-05-13 2019-10-08 中国药科大学 具有催化活性的人猪嵌合尿酸氧化酶
CN108379561B (zh) * 2018-03-01 2021-11-26 修正生物医药（杭州）研究院有限公司 一种聚乙二醇化尿酸氧化酶冻干粉剂及其制备方法

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101302501A (zh) * 2007-05-10 2008-11-12 刘国安 聚乙二醇化尿酸氧化酶化合物及其制备方法和其制剂及应用
CN104066324A (zh) * 2011-11-04 2014-09-24 西玛贝医药公司 治疗痛风急性发作的方法
CN106902349A (zh) * 2017-03-07 2017-06-30 云南中医学院 一种口服降尿酸药物的制备与应用
CN109223707A (zh) * 2018-09-13 2019-01-18 中国药科大学 一种尿酸酶外用凝胶制剂、其制备方法及用途

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10815461B2 (en) 2017-07-07 2020-10-27 Allena Pharmaceuticals, Inc. Recombinant uricase enzyme

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