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EP-4736866-A1 - STABILIZER FOR URATE OXIDASE AND PEGYLATED CONJUGATE THEREOF, AND PHARMACEUTICAL USE OF STABILIZER

EP4736866A1EP 4736866 A1EP4736866 A1EP 4736866A1EP-4736866-A1

Abstract

Provided is a method for improving the stability of urate oxidase. The inventors have discovered that combining an active ingredient urate oxidase or chemically modified urate oxidase with a stabilizer xanthine through a non-covalent bond can significantly improve the in vivo and in vitro stability of urate oxidase and chemically modified urate oxidase in the form of a tetramer, thereby effectively improving the stability of urate oxidase.

Inventors

  • WANG, QIAN
  • FU, Zhicheng
  • DING, Xupeng
  • XU, YING
  • CHEN, ZEYU
  • ZHANG, HUI
  • ZHOU, Yatong
  • FAN, KAI
  • WANG, YU
  • LIU, Riyong
  • WANG, HONGYING
  • HU, CHUNLAN
  • YAN, Tianwen
  • HE, YUNFENG
  • SU, Guowei

Assignees

  • Hangzhou Grand Biologic Pharmaceutical Inc.
  • Peg-Bio Biopharm Co., Ltd. (Chongqing)

Dates

Publication Date
20260506
Application Date
20240628

Claims (20)

  1. A urate oxidase composition, comprising: an active ingredient, comprising urate oxidase; and a stabilizer, comprising xanthine.
  2. The urate oxidase composition according to claim 1, wherein a molar ratio of xanthine to urate oxidase tetramer is not less than 0.5:1.
  3. The urate oxidase composition according to claim 1, wherein the urate oxidase is extracted natural urate oxidase, expressed recombinant urate oxidase, or modified urate oxidase.
  4. The urate oxidase composition according to claim 3, wherein: the extracted natural urate oxidase is derived from Aspergillus flavus, wild-type Micrococcus, or mammals; and the modified urate oxidase is selected from the group consisting of polyethylene glycol-modified urate oxidase, polysaccharide side chain-modified urate oxidase, polyoligopeptide-modified urate oxidase, and a combination thereof.
  5. The urate oxidase composition according to claim 1, wherein the urate oxidase is bound to xanthine through a non-covalent bond.
  6. The urate oxidase composition according to claim 5, wherein the urate oxidase is bound to xanthine through hydrogen bonding, salt bonding, hydrophobic interaction, or van der Waals force.
  7. The urate oxidase composition according to claim 1, wherein: a concentration of the urate oxidase in the composition ranges from 1 mg/ml to 12 mg/ml; and a molar ratio of xanthine to urate oxidase tetramer is (0.5 to 200): 1, and preferably (1 to 156): 1.
  8. The urate oxidase composition according to claim 1, wherein: a concentration of the urate oxidase in the composition is 1 mg/ml; and a molar ratio of xanthine to urate oxidase tetramer is (0.5 to 200): 1, and preferably (1 to 156): 1.
  9. The urate oxidase composition according to claim 1, wherein: a concentration of the urate oxidase in the composition is 6 mg/ml; and a molar ratio of xanthine to urate oxidase tetramer is (0.5 to 34): 1, and preferably (1 to 26): 1.
  10. The urate oxidase composition according to claim 1, wherein: a concentration of the urate oxidase in the composition is 8 mg/ml; and a molar ratio of xanthine to urate oxidase tetramer is (0.5 to 25): 1, and preferably (1 to 20): 1.
  11. The urate oxidase composition according to claim 1, wherein: a concentration of the urate oxidase in the composition is 12 mg/ml; and a molar ratio of xanthine to urate oxidase tetramer is (0.5 to 17): 1, and preferably (1 to 13): 1.
  12. The urate oxidase composition according to claim 1, further comprising a pharmaceutically acceptable excipient or carrier, wherein: the excipient or carrier is selected from the group consisting of hydroxypropyl methylcellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, mannitol, sucrose, fructose, methyl cellulose, vinyl acetate copolymer, chitosan, sodium alginate, xanthan gum, sodium carboxymethyl cellulose, polyethylene oxide, methacrylic acid copolymer, maleic anhydride-methyl vinyl ether copolymer, carbomer, polyvinyl pyrrolidone, ethyl cellulose, cellulose acetate, methacrylate, polyoxyethylene, polyvinyl alcohol, glyceryl behenate, glycerol monostearate, cellulose acetate, cellulose acetate phthalate, ethyl cellulose, PLA, PLGA, polyethylene glycol, and combinations thereof.
  13. The urate oxidase composition according to claim 12, wherein the composition is an injection.
  14. Use of xanthine in improving the stability of urate oxidase.
  15. A method for improving the stability of urate oxidase, comprising contacting the urate oxidase with xanthine.
  16. A method for preparing urate oxidase, comprising: contacting urate oxidase with xanthine.
  17. The method according to claim 15 or 16, wherein the urate oxidase is extracted natural urate oxidase, expressed recombinant urate oxidase, or modified urate oxidase.
  18. The method according to claim 17, wherein: the extracted natural urate oxidase is derived from Aspergillus flavus, wild-type Micrococcus, or mammals; and the modified urate oxidase is selected from the group consisting of polyethylene glycol-modified urate oxidase, polysaccharide side chain-modified urate oxidase, polyoligopeptide-modified urate oxidase, and a combination thereof.
  19. The method according to claim 15 or 16, wherein said contacting urate oxidase with xanthine is performed by: fermenting engineered bacteria carrying a nucleic acid molecule expressing the urate oxidase in a xanthine-containing fermentation broth, to obtain the urate oxidase.
  20. The method according to claim 19, wherein an amount of xanthine in the xanthine-containing fermentation broth is not less than 0.1 g/L.

Description

FIELD The present disclosure relates to the field of biology, and more particularly, to a urate oxidase, a stabilizer for PEGylated conjugate thereof, and pharmaceutical use thereof. BACKGROUND Gout is a disease caused by a long-term disorder of purine metabolism or a decrease in uric acid excretion. Its clinical feature is hyperuricemia. Due to the poor solubility of urate, crystals deposit and accumulate subcutaneously and in joints and kidney to form tophi, leading to recurrent acute arthritis and involving the kidneys to cause uric acid stones and interstitial nephritis. In human body, purine is converted into end-product uric acid through enzyme action. Under normal conditions, the uric acid content is 149 to 416 mmol/L in male blood and 89 to 357 mol/L in female blood. The amount of uric acid in the body is about 1,200 mg, and the amount of production and excretion is about 600 mg/day, which is in the equilibrium state. However, hyperuricemia may be caused by excessive intake of uric acid or dysfunction of the excretion mechanism, resulting in the accumulation of uric acid above 70 mg/L in the blood. When sodium urate reaches saturation in the blood or synovial fluid and crystallizes, or when hyperuricemia lasts for a long time and crystallizes around joints and soft tissues, it can lead to acute gouty arthritis or chronic gouty arthritis and joint deformities. The deposition of urate in the renal tubules and interstitium may induce inflammation that leads to chronic urate nephropathy. In patients with severe hyperuricemia (such as patients with malignant tumors such as leukemia and lymphoma), massive uric acid deposition in a short period of time causes urinary tract obstruction and acute renal failure, also known as uric acid nephropathy. The cause of hyperuricemia is related to the mutation and inactivation of the uricase gene during human evolution, so humans cannot synthesize active uricase by themselves. The active uricase in non-human primates or other mammals can convert uric acid produced by purine metabolism into more soluble allantoin to improve the excretion efficiency of the kidney (Wortmann R L, Kelley W N. Kelley's textbook of rheumatology (6th). 2001: 1339-1376). Hyperuricemia may be caused by excessive production of uric acid or insufficient excretion of uric acid (Hershfield, Seegmiller, 1976; Roessler, 1995). In recent years, people's quality of life has improved and their living and eating habits have changed, and they consume more high-protein and high-purine foods. In addition, some drugs used during chemotherapy for tumors and drugs such as azathioprine used to prevent organ transplant rejection can cause significant hyperuricemia and may also cause severe gout or renal damage. Mild hyperuricemia before the onset of clinical symptoms can be controlled by restricting diet; however, once clinical symptoms appear, appropriate drug treatment is required. Conventional clinical treatments currently include: analgesic and anti-inflammatory drugs, such as colchicine, ibuprofen, naproxen, etc., which are mainly used to control the symptoms of acute attacks of gouty arthritis, eliminating local pain, swelling and inflammation in joints; uricosuric agents that promote the excretion of uric acid (ineffective if the kidney function is reduced), such as probenicid, sulfinpyrazone, benzbromarone, etc.; drugs that inhibit the synthesis of uric acid, such as allopurinol, which can inhibit xanthine oxidase from converting hypoxanthine or xanthine into uric acid, and instead slowly oxidize it to produce isoxanthine that is easily soluble in water. However, allopurinol therapy may cause hypersensitivity syndromes such as acute renal and liver failure, skin damage, and is unlikely to be effective in patients with severe chronic hyperuricemia who have already developed tophi. Uricase (EC 1.7.3.3) is widely present in microorganisms (Bacillus fastidious, Candida monocytogenes, Aspergillus flavus), plants (soybeans, chickpeas), animals (pigs, cattle, dogs, baboons) (Suzuki K, Sakasegawa S, Misaki H, Sugiyama M. J Biosci Bioeng. 2004. 98: 153-158). In the presence of oxygen, it can catalyze uric acid to oxidize allantoin to release carbon dioxide (Retailleau P, Colloc'h, Denis V, Francoise B. Acta Cryst D.2004.60: 453-462.). Active uricase is a tetrameric protein composed of identical subunits, each of which has a molecular weight of about 34 kD and is composed of 301 to 304 amino acids. The pH at the highest enzyme activity of uricase in each solution is 8.0 (Bayol A et al. Biophys Chem.1995.54: 229-235.). In early studies, microorganisms were used to obtain non-recombinant natural uricase. Uric acid or its structural analogs such as adenine, guanine, xanthine, and hypoxanthine were reported as culture medium inducers to increase the expression of urate oxidase (Liu Jianguo et al., Culture conditions for uricase formation of Candida utilis, Wei Sheng Wu Xue Bao, 29(1):45-50, 1989). The results showed t