Method of maintaining hyaluronidase activity in composition

Abstract

The invention belongs to the technical field of pharmacy, and particularly relates to a method for maintaining the activity of hyaluronidase in a composition. The invention provides a method for maintaining stability of hyaluronidase. The method can eliminate potential safety hazards caused by instability of hyaluronidase in the process of assisting antibiotic administration with hyaluronidase, promote rapid absorption of antibiotics after administration, effectively improve anaphylactic reaction and remarkably improve patient compliance.

Description

[0001] Priority Statement This application claims priority to Chinese Patent Application No. 2025101276765, filed on January 27, 2025, the entire contents of which are incorporated herein by reference. Technical Field

[0002] This disclosure belongs to the field of pharmaceutical technology, and specifically relates to a method for maintaining hyaluronidase activity in a composition. Background Technology

[0003] Antibiotics are the most commonly used drugs for treating infections in clinical practice. After many years of development, there are now thousands of types, with hundreds commonly used in clinical practice. The main classifications include β-lactams, aminoglycosides, macrolides, lincomycins, polypeptides, quinolones, sulfonamides, anti-tuberculosis drugs, antifungal drugs, and other antibiotics.

[0004] Antibiotics can be administered in various ways, with oral, intramuscular, and intravenous administration being the most common. However, these methods of administration often have limitations, drawbacks, and side effects.

[0005] Intradermal or subcutaneous administration refers to injecting medication into the intradermal or subcutaneous tissue for rapid absorption. Subcutaneous administration is less expensive than intravenous administration, offers more site options, is simpler to insert, causes less pain or discomfort, and is easier to re-insert at different sites. The needle can be placed in relatively non-sensitive areas of the skin. Furthermore, intradermal or subcutaneous administration can be performed in almost any environment, making it more suitable than intravenous infusion in situations with limited nursing resources. However, intradermal or subcutaneous infusion volumes exceeding 2 mL can cause tissue deformation, increase interstitial pressure, and lead to pain and discomfort, thus limiting the infusion volume and rate at a single subcutaneous infusion site.

[0006] However, antibiotics are limited by infusion volume and the potential skin irritation caused by high concentrations. When antibiotics are injected subcutaneously or intramuscularly, the hyaluronic acid in the tissue can hinder drug diffusion and absorption. Hyaluronic acid is a major component of the extracellular matrix, forming a viscous gel-like structure in tissues, acting to fill and stabilize them. Adding hyaluronidase can disrupt this hyaluronic acid barrier, allowing antibiotics to diffuse more quickly within the tissues and shortening the onset of action. This is because hyaluronidase breaks down the hyaluronic acid in subcutaneous tissues, creating more unobstructed diffusion channels for antibiotics, enabling more antibiotics to rapidly enter the capillaries and bloodstream to exert their effects.

[0007] WO2023001264A1 provides a composition containing an antibiotic and hyaluronidase, utilizing the aforementioned function of hyaluronidase. Examples of this composition were used to investigate the compatibility of different antibiotics and hyaluronidase, demonstrating good compatibility and indicating that the composition containing both components can be used for drug development. However, through experimentation, the applicant unexpectedly discovered that extending the compatibility time, for example, to more than one hour, or even two to twelve hours, resulted in different antibiotics exhibiting varying effects on hyaluronidase activity. Direct mixing of these antibiotics into a drug presents a potential safety hazard due to the instability of hyaluronidase activity.

[0008] Therefore, when using hyaluronidase to enhance the efficacy of antibiotic administration, it is necessary to maintain the stability of hyaluronidase activity. Summary of the Invention

[0009] This disclosure provides a method for maintaining the stability of hyaluronidase, which can eliminate the safety risks caused by the instability of hyaluronidase during the administration of antibiotics with the assistance of hyaluronidase, promote the rapid absorption of antibiotics after administration, effectively improve allergic reactions, and significantly improve patient compliance.

[0010] The first aspect of this disclosure provides a method for maintaining the activity of hyaluronidase or recombinant hyaluronidase in a liquid formulation, wherein the liquid formulation contains an antibiotic and hyaluronidase or recombinant hyaluronidase; the method includes containing albumin in the liquid formulation.

[0011] In an optional embodiment, the concentration of the antibiotic is 1-500 mg / ml; alternatively, the concentration of the antibiotic is 20-300 mg / ml.

[0012] In an optional embodiment, the concentration of albumin is 0.01~10 g / L; optionally, the concentration of albumin is 0.02~1 g / L; optionally, the concentration of albumin is 0.05~1 g / L, for example 0.05 g / L, 0.1 g / L or 1 g / L.

[0013] In an optional embodiment, the mass ratio of albumin to antibiotic is 1:10 to 50000; it can be 1:10 to 100, for example 1:10, 1:20, 1:25, 1:50 or 1:100.

[0014] In an optional embodiment, the enzyme activity of the hyaluronidase or recombinant hyaluronidase is 45 IU / ml to 3,000,000 IU / ml; optionally, the enzyme activity of the hyaluronidase or recombinant hyaluronidase is 45 IU / ml to 1,500,000 IU / ml; for example, 45 IU / ml to 300,000 IU / ml.

[0015] In an optional embodiment, the antibiotic includes cephalosporin antibiotics; such as one or more of ceftazidime, cefuroxime sodium, ceftriaxone sodium, cefazolin sodium, cefoperazone / sulbactam sodium, cefazolin sodium for injection, cefazolin sodium, cefoperazone sodium, or cefoperazone.

[0016] In an optional embodiment, the liquid formulation further comprises a buffer, a stabilizer, and a nonionic surfactant; optionally, the buffer comprises phosphate buffer and / or Tris buffer, and the nonionic surfactant comprises one or more of polysorbate 20, polysorbate 80, or poloxamer 188; optionally, the stabilizer is selected from (i) or (ii): (i) methionine and sodium chloride; (ii) is composed of (i) and (iii), wherein (iii) is trehalose or sucrose.

[0017] In an optional embodiment, the concentration of the buffer is 2-50 mM; the stabilizer is selected from (i) or (ii): (i) Methionine and sodium chloride; the concentration of said methionine is 2-50 mM, for example 2 mM, 2.6 mM, 3.3 mM, 5 mM, 10 mM or 50 mM; the concentration of said sodium chloride is 25-200 mM, optionally 30 mM-200 mM, for example 30 mM, 130 mM, 145 mM, 154 mM or 180 mM; (ii) is composed of (i) and (iii), wherein (c) is trehalose or sucrose, and the concentration of the trehalose or sucrose is 25-200 mM, for example 25 mM, 53 mM or 200 mM; The concentration of the nonionic surfactant is 0.01% to 0.1% (w / v), for example, 0.02% (w / v).

[0018] In an optional embodiment, the concentration of methionine in the stabilizer (i) is 2-15 mM, and the concentration of sodium chloride is 50-180 mM.

[0019] In an optional embodiment, the concentration of the phosphate buffer or Tris buffer is 2 mM, 2.6 mM, 3.3 mM, 5 mM, 10 mM or 50 mM.

[0020] In an optional embodiment, the liquid formulation further comprises a buffer, a cryoprotectant, an excipient, an enzyme protectant, and a surfactant.

[0021] In an optional embodiment, the buffer comprises a phosphate buffer, such as sodium dihydrogen phosphate; the cryoprotectant comprises sucrose and / or trehalose; the excipient comprises mannitol; the enzyme activity protectant comprises a chelating agent and a polyvalent metal salt ion, such as disodium EDTA and calcium chloride; and the surfactant comprises polysorbate 20 and / or polysorbate 80.

[0022] In an optional embodiment, the concentration of the phosphate buffer is 0.1~50mM, for example, 0.1mM, 0.2mM, 0.5mM, 1mM, 2mM, 2.6mM, 3.3mM, 5mM, 10mM or 50mM; the concentration of the cryoprotectant is 1~60mM; the concentration of the excipient is 1~300mM; the concentration of the chelating agent is 0.01~10mM; the concentration of the polyvalent metal salt ion is 0.01~10mM; and the concentration of the surfactant is 0.001~0.1g / L.

[0023] In an optional embodiment, the composition of the liquid formulation is selected from any of the following: (1) 50 IU / ml~500 IU / ml hyaluronidase or recombinant hyaluronidase, about 116 mg / ml cefuroxime sodium, about 10 mM phosphate buffer, about 145 mM sodium chloride, about 10 mM methionine, about 0.02% (w / v) polysorbate 20, about 1 g / L albumin; (2) 50 IU / ml~500 IU / ml hyaluronidase or recombinant hyaluronidase, about 116 mg / ml cefuroxime sodium, about 10 mM phosphate buffer, about 145 mM sodium chloride, about 10 mM methionine, about 0.02% polysorbate 20, about 1 g / L albumin; (3) 50 IU / ml to 500 IU / ml hyaluronidase or recombinant hyaluronidase, approximately 116 mg / ml cefuroxime sodium, approximately 2.6 to 3.3 mM phosphate buffer, approximately 38 to 48 mM sodium chloride, approximately 2.6 to 3.3 mM methionine, approximately 0.005% (w / v) to 0.007% (w / v) polysorbate 20, approximately 0.26 to 0.33 g / L albumin; (4) 50 IU / ml~500 IU / ml hyaluronidase or recombinant hyaluronidase, about 260 mg / ml ceftazidime, about 10 mM phosphate buffer, about 145 mM sodium chloride, about 10 mM methionine, about 0.02% polysorbate 20, about 1 g / L albumin. (5) 50 IU / ml to 500 IU / ml of hyaluronidase or recombinant hyaluronidase, about 260 mg / ml of ceftazidime, about 2.6 to 3.3 mM of phosphate buffer, about 145 to 154 mM of sodium chloride, about 2.6 to 3.3 mM of methionine, about 0.005% (w / v) to 0.007% (w / v) of polysorbate 20, and about 0.26 to 0.33 g / L of albumin.

[0024] In an optional embodiment, the enzyme activity of hyaluronidase or recombinant hyaluronidase in the liquid formulation is maintained for more than 1 hour, for example, 1 to 24 hours, or 2 to 12 hours.

[0025] A second aspect of this disclosure provides the use of reagents containing hyaluronidase and / or recombinant hyaluronidase in the preparation of combination reagents, said combination reagents comprising individually packaged reagents (a) containing an antibiotic, and reagents (b) containing hyaluronidase and / or recombinant hyaluronidase.

[0026] In an optional embodiment, the antibiotic includes cephalosporin antibiotics; such as one or more of ceftazidime, cefuroxime sodium, ceftriaxone sodium, cefazolin sodium, cefoperazone / sulbactam sodium, cefazolin sodium, cefotaxime sodium, or cefoperazone.

[0027] In an optional embodiment, the enzyme activity of the hyaluronidase or recombinant hyaluronidase in reagent (b) is 45 IU / ml to 3,000,000 IU / ml; optionally, the enzyme activity of the hyaluronidase or recombinant hyaluronidase is 45 IU / ml to 1,500,000 IU / ml; for example, 45 IU / ml to 300,000 IU / ml.

[0028] In an optional embodiment, reagent (b) further comprises a buffer, a stabilizer, and a nonionic surfactant; optionally, the buffer comprises phosphate buffer and / or Tris buffer, and the nonionic surfactant comprises one or more of polysorbate 20, polysorbate 80, or poloxamer 188; optionally, the stabilizer is selected from (i) or (ii): (i) methionine and sodium chloride; (ii) is composed of (i) and (iii), wherein (iii) is trehalose or sucrose.

[0029] In an optional embodiment, the concentration of the buffer is 2-50 mM; the stabilizer is selected from (i) or (ii): (i) Methionine and sodium chloride; the concentration of said methionine is 2-50 mM, for example 2 mM, 2.6 mM, 3.3 mM, 5 mM, 10 mM or 50 mM; the concentration of said sodium chloride is 25-200 mM, optionally 30 mM-200 mM, for example 30 mM, 130 mM, 145 mM, 154 mM or 180 mM; (ii) is composed of (i) and (iii), wherein (c) is trehalose or sucrose, and the concentration of the trehalose or sucrose is 25-200 mM, for example 25 mM, 53 mM or 200 mM; The concentration of the nonionic surfactant is 0.01% to 0.1% (w / v), for example, 0.02% (w / v).

[0030] In an optional embodiment, the concentration of methionine in the stabilizer (i) is 2-15 mM, and the concentration of sodium chloride is 50-180 mM.

[0031] In an optional embodiment, the concentration of the phosphate buffer or Tris buffer is 0.1 to 50 mM, for example, 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 2 mM, 2.6 mM, 3.3 mM, 5 mM, 10 mM or 50 mM.

[0032] In another alternative embodiment, the reagent (b) contains hyaluronidase and / or recombinant hyaluronidase, buffer, cryoprotectant, excipient, enzyme activity protectant and surfactant.

[0033] In an optional embodiment, the buffer includes a phosphate buffer, such as sodium dihydrogen phosphate; the cryoprotectant includes sucrose and / or trehalose; the excipient includes mannitol; and the enzyme activity protectant includes a chelating agent and a polyvalent metal salt ion, such as disodium ethylenediaminetetraacetate and calcium chloride.

[0034] In an optional embodiment, the lyophilized formulation contains, prior to lyophilization, the following in the liquid formulation: The enzyme activity of hyaluronidase and / or recombinant hyaluronidase is 45 IU / ml to 3,000,000 IU / ml; the concentration of the buffer is 0.014 to 70 g / L, for example 0.14 to 14 g / L; the cryoprotectant is 0.2 to 400 g / L, for example 2 to 200 g / L; the excipient is 0.4 to 2000 g / L, for example 4 to 400 g / L; the concentration of the chelating agent is 0.009 to 45 g / L, for example 0.09 to 9 g / L; and the concentration of the polyvalent metal salt ion is 0.03 to 3 g / L.

[0035] In an optional embodiment, the lyophilized formulation contains, prior to lyophilization, the following in the liquid formulation: The enzyme contained hyaluronidase and / or recombinant hyaluronidase with an enzyme activity of 45 IU / ml to 3,000,000 IU / ml, 1.4 g / L sodium dihydrogen phosphate, 0.9 g / L disodium ethylenediaminetetraacetate, 0.3 g / L calcium chloride, 40 g / L mannitol and 20 g / L trehalose.

[0036] A third aspect of this disclosure provides a combination reagent comprising individually packaged reagent (a) containing an antibiotic, and reagent (b) containing hyaluronidase and / or recombinant hyaluronidase; said reagent (b) may or may not contain albumin.

[0037] In an optional embodiment, the antibiotic includes cephalosporin antibiotics; such as one or more of ceftazidime, cefuroxime sodium, ceftriaxone sodium, cefazolin sodium, cefoperazone / sulbactam sodium, cefazolin sodium, cefotaxime sodium, or cefoperazone.

[0038] In an optional embodiment, the antibiotic includes ceftazidime, cefuroxime sodium, ceftriaxone sodium, cefazolin sodium, and cefoperazone / sulbactam sodium, and the reagent (b) does not contain albumin.

[0039] In another alternative embodiment, the antibiotic includes ceftazidime, cefuroxime sodium, ceftriaxone sodium, cefazolin sodium, and cefoperazone / sulbactam sodium, and the reagent (b) contains albumin.

[0040] In an optional embodiment, reagent (b) is a liquid formulation containing hyaluronidase or recombinant hyaluronidase, an enzyme, a buffer, a stabilizer, and a nonionic surfactant; optionally, the buffer includes phosphate buffer and / or Tris buffer, and the nonionic surfactant includes one or more of polysorbate 20, polysorbate 80, or poloxamer 188; optionally, the stabilizer is selected from (i) or (ii): (i) methionine and sodium chloride; (ii) is composed of (i) and (iii), wherein (iii) is trehalose or sucrose.

[0041] In an optional embodiment, the concentration of the buffer is 2-50 mM; the stabilizer is selected from (i) or (ii): (i) Methionine and sodium chloride; the concentration of said methionine is 2-50 mM, for example 2 mM, 2.6 mM, 3.3 mM, 5 mM, 10 mM or 50 mM; the concentration of said sodium chloride is 25-200 mM, optionally 30 mM-200 mM, for example 30 mM, 130 mM, 145 mM, 154 mM or 180 mM; (ii) is composed of (i) and (iii), wherein (c) is trehalose or sucrose, and the concentration of the trehalose or sucrose is 25-200 mM, for example 25 mM, 53 mM or 200 mM; The concentration of the nonionic surfactant is 0.01% to 0.1% (w / v), for example, 0.02% (w / v).

[0042] In an optional embodiment, the concentration of methionine in the stabilizer (i) is 2-15 mM, and the concentration of sodium chloride is 50-180 mM.

[0043] In an optional embodiment, the concentration of the phosphate buffer or Tris buffer is 2 mM, 2.6 mM, 3.3 mM, 5 mM, 10 mM or 50 mM.

[0044] In an optional embodiment, reagent (b) is a lyophilized formulation containing hyaluronidase and / or recombinant hyaluronidase, buffer, cryoprotectant, excipient, enzyme activity protectant, and surfactant.

[0045] In an optional embodiment, the buffer includes a phosphate buffer, such as sodium dihydrogen phosphate; the cryoprotectant includes sucrose and / or trehalose; the excipient includes mannitol; and the enzyme activity protectant includes a chelating agent and a polyvalent metal salt ion, such as disodium ethylenediaminetetraacetate and calcium chloride.

[0046] In an optional embodiment, the lyophilized formulation contains, prior to lyophilization, the following in the liquid formulation: The enzyme activity of hyaluronidase and / or recombinant hyaluronidase is 45 IU / ml to 3,000,000 IU / ml; the concentration of the buffer is 0.014 to 70 g / L, for example 0.14 to 14 g / L; the cryoprotectant is 0.2 to 400 g / L, for example 2 to 200 g / L; the excipient is 0.4 to 2000 g / L, for example 4 to 400 g / L; the concentration of the chelating agent is 0.009 to 45 g / L, for example 0.09 to 9 g / L; and the concentration of the polyvalent metal salt ion is 0.03 to 3 g / L.

[0047] In an optional embodiment, the lyophilized formulation contains, prior to lyophilization, the following in the liquid formulation: The enzyme contained hyaluronidase and / or recombinant hyaluronidase with an enzyme activity of 45 IU / ml to 3,000,000 IU / ml, 1.4 g / L sodium dihydrogen phosphate, 0.9 g / L disodium ethylenediaminetetraacetate, 0.3 g / L calcium chloride, 40 g / L mannitol and 20 g / L trehalose.

[0048] In an optional implementation, the combined reagents are selected from any of the following: (A) The reagent (a) is ceftazidime; Reagent (b) is a liquid formulation comprising 250 IU / ml hyaluronidase, 10 mM disodium hydrogen phosphate, 10 mM methionine, 145 mM sodium chloride, 0.02% (w / v) polysorbate 20 and 1 g / L albumin. After dissolving reagent (a) with reagent (b), the concentration of ceftazidime is approximately 260 mg / ml; (B) The reagent (a) mentioned is ceftazidime; Reagent (b) is a liquid formulation comprising 150 IU / ml hyaluronidase, 10 mM disodium hydrogen phosphate, 10 mM methionine, 145 mM sodium chloride, 0.02% (w / v) polysorbate 20 and 1 g / L albumin. After dissolving reagent (a) with reagent (b), the concentration of ceftazidime is approximately 260 mg / ml; (C) The reagent (a) is ceftazidime; Reagent (b) is a liquid formulation comprising 150 IU / ml hyaluronidase, 10 mM disodium hydrogen phosphate, 10 mM methionine, 130 mM sodium chloride, 0.02% (w / v) polysorbate 20 and 1 g / L albumin. After dissolving reagent (a) with reagent (b), the concentration of ceftazidime is approximately 260 mg / ml; (D) The reagent (a) is cefuroxime sodium; Reagent (b) is a liquid formulation comprising 500 IU / ml hyaluronidase, 10 mM disodium hydrogen phosphate, 10 mM methionine, 145 mM sodium chloride, 0.02% (w / v) polysorbate 20 and 1 g / L albumin. After dissolving reagent (a) with reagent (b), the concentration of ceftazidime is approximately 116 mg / ml; (E) The reagent (a) is cefuroxime sodium; Reagent (b) is a liquid formulation comprising 150 IU / ml hyaluronidase, 10 mM disodium hydrogen phosphate, 10 mM methionine, 145 mM sodium chloride, 0.02% (w / v) polysorbate 20 and 1 g / L albumin. After dissolving reagent (a) with reagent (b), the concentration of ceftazidime is approximately 116 mg / ml; (F) The reagent (a) mentioned is cefuroxime sodium; Reagent (b) is a liquid formulation comprising 150 IU / ml hyaluronidase, 10 mM disodium hydrogen phosphate, 10 mM methionine, 130 mM sodium chloride, 0.02% (w / v) polysorbate 20 and 1 g / L albumin. After dissolving reagent (a) with reagent (b), the concentration of ceftazidime is approximately 116 mg / ml; (G) The reagent (a) mentioned is ceftazidime; Reagent (b) is a lyophilized formulation, which, before lyophilization, comprises hyaluronidase and / or recombinant hyaluronidase with an enzyme activity of 45 IU / ml to 3,000,000 IU / ml, 1.4 g / L sodium dihydrogen phosphate, 0.9 g / L disodium ethylenediaminetetraacetate, 0.3 g / L calcium chloride, 40 g / L mannitol, and 20 g / L trehalose; (H) The reagent (a) mentioned is cefuroxime sodium; Reagent (b) is a lyophilized formulation, which, before lyophilization, comprises hyaluronidase and / or recombinant hyaluronidase with an enzyme activity of 45 IU / ml to 3,000,000 IU / ml, 1.4 g / L sodium dihydrogen phosphate, 0.9 g / L disodium ethylenediaminetetraacetate, 0.3 g / L calcium chloride, 40 g / L mannitol, and 20 g / L trehalose.

[0049] Fourth aspect: This disclosure provides a method for preparing a liquid formulation, comprising mixing reagent (a) and reagent (b) in any of the preceding embodiments of the combined reagent, regardless of the order of addition. Optionally, the reagent (b) is a liquid preparation, and the preparation method includes mixing the reagent (a) and the reagent (b) in any order of addition, or mixing the reagent (a), the reagent (b), and the reconstituter in any order; the reconstituter may be physiological saline. Optionally, the reagent (b) is a lyophilized preparation, and the preparation method includes mixing the reagent (a), the reagent (b), and the reconstituter in any order; the reconstituter may be physiological saline.

[0050] In an optional embodiment, the liquid formulation is composed of the liquid formulation described in any of the aforementioned methods for maintaining hyaluronidase stability.

[0051] Fifthly, this disclosure provides the use of the combined reagents described in any of the foregoing embodiments, or the liquid preparations obtained by the preparation methods described in any of the foregoing embodiments, in the preparation of medicaments for treating diseases; the diseases include infections caused by bacteria, fungi, actinomycetes, mycoplasma, chlamydia, spirochetes, or amoebae, or diseases caused by infections caused by the aforementioned pathogens.

[0052] In an optional embodiment, the treatment for the disease includes administering to a subject an effective dose of a liquid formulation prepared using any of the foregoing embodiments.

[0053] In an optional embodiment, the liquid formulation is a liquid formulation prepared by any of the preparation methods described in the foregoing embodiments and left for more than 1 hour; for example, 2 to 12 hours.

[0054] In optional embodiments, the administration method includes subcutaneous administration, intramuscular administration, or intravenous administration.

[0055] In a sixth aspect, this disclosure provides a method for treating a disease, comprising administering to a subject an effective dose of a liquid preparation prepared by any of the foregoing embodiments; wherein the disease includes infection by bacteria, fungi, actinomycetes, mycoplasma, chlamydia, spirochetes, or amoebae, or a disease caused by infection with the aforementioned pathogens.

[0056] In an optional embodiment, the liquid formulation is a liquid formulation prepared by using the combination reagents described in any of the foregoing embodiments and the preparation method described in any of the foregoing embodiments.

[0057] In an optional embodiment, the liquid formulation is a liquid formulation that has been left to stand for more than 1 hour; for example, 2 to 12 hours.

[0058] In optional embodiments, the administration method includes subcutaneous administration, intramuscular administration, or intravenous administration.

[0059] In a seventh aspect, this disclosure provides a combination reagent or liquid preparation for treating diseases; said diseases include infections caused by bacteria, fungi, actinomycetes, mycoplasma, chlamydia, spirochetes, or amoebae, or diseases caused by infections of the aforementioned pathogens; said combination reagent includes the combination reagent described in any of the foregoing embodiments; said liquid preparation includes a liquid preparation prepared by mixing and formulating the combination reagent described in any of the foregoing embodiments, or a liquid preparation prepared by the preparation method described in any of the foregoing embodiments.

[0060] In an optional embodiment, the liquid formulation is a liquid formulation that has been left to stand for more than 1 hour; for example, 2 to 12 hours.

[0061] In an optional embodiment, the treatment for the disease includes administering to a subject an effective dose of a liquid formulation prepared using any of the foregoing embodiments.

[0062] In optional embodiments, the administration method includes subcutaneous administration, intramuscular administration, or intravenous administration.

[0063] In a seventh aspect, this disclosure provides a packaging body containing the combined reagents described in any of the foregoing embodiments, or a liquid formulation prepared by the preparation method described in any of the foregoing embodiments.

[0064] In an optional embodiment, the packaging body is used to store or transport the combined reagents described in any of the foregoing embodiments, or the liquid formulation prepared by any of the preparation methods described in any of the foregoing embodiments.

[0065] In an optional embodiment, the packaging is used to formulate or administer a liquid formulation prepared by any of the preparation methods described in the foregoing embodiments.

[0066] The method for maintaining hyaluronidase activity disclosed herein allows for the addition or omission of albumin depending on the antibiotic used. This method is simple and easy to implement, and the albumin used is readily available. Therefore, the preparation method disclosed herein is suitable for widespread production and has industrialization prospects.

[0067] The inventors of this disclosure have discovered for the first time that when certain antibiotics are mixed with hyaluronidase, prolonged storage can reduce the activity of the hyaluronidase, which is detrimental to long-term storage or transportation and also poses safety risks for drug administration. Therefore, this disclosure provides different combinations of reagents or liquid formulations for different antibiotics. Detailed Implementation

[0068] Based on the above content of this disclosure, and in accordance with common technical knowledge and practices in the field, various other modifications, substitutions, or alterations can be made without departing from the basic technical ideas of this disclosure.

[0069] I. Definition Unless otherwise expressly stated, throughout the specification and claims, the term "comprising" or its variations such as "including" or "comprises" shall be understood to include the stated elements or components without excluding other elements or other components.

[0070] In the description of this disclosure, "multiple" means two or more, unless otherwise expressly and specifically defined.

[0071] The term “optional” or “optionally” means that the event or situation described below may or may not occur, including both the occurrence and non-occurrence of the event or situation.

[0072] "Combined with" means to administer two or more therapeutic agents together, as a single agent, simultaneously or sequentially as a single agent to the patient in any order.

[0073] "Pharmaceutical composition" refers to a product obtained by mixing an antibiotic with hyaluronidase, and includes both fixed and non-fixed combinations. Pharmaceutical compositions typically contain pharmaceutically acceptable excipients. "Fixed combination" refers to a single pharmaceutical composition containing an antibiotic and hyaluronidase administered simultaneously as a single entity or in a single dose. "Non-fixed combination" refers to an independent pharmaceutical composition or IU dosage form of an antibiotic and hyaluronidase administered simultaneously, in parallel, or sequentially as independent entities without specific time interval restrictions, wherein such administration provides an effective level of both compounds in the subject.

[0074] In this patent, the term "recombinant" primarily refers to recombinant proteins. Recombinant proteins are proteins obtained using recombinant DNA or recombinant RNA technology. The methods for obtaining them can be divided into in vitro and in vivo methods. Both methods require the application of gene recombination technology to obtain a recombinant vector containing a gene fragment that can be translated into the target protein. This vector is then transferred into a host cell capable of expressing the target protein, thereby expressing the specific recombinant protein molecule.

[0075] The term "antibiotics" as used in this article includes cephalosporin antibiotics; such as one or more of ceftazidime, cefuroxime sodium, ceftriaxone sodium, cefazolin sodium, cefoperazone / sulbactam sodium, cefazolin sodium, cefotaxime sodium, or cefoperazone.

[0076] The term "hyaluronic acid" refers to hyaluronic acid (HA), also known as glucuronic acid, hyaluronic acid, or hyaluronic acid. It is a linear high-molecular-weight glycosaminoglycan composed of two repeating disaccharide units, D-glucuronic acid and N-acetylglucosamine. It is widely found in the connective tissue, mucous tissue, lens of the eye, and skin of vertebrates, and is particularly abundant in embryos, cartilage, synovial fluid, vitreous humor, umbilical cord, and rooster comb. Hyaluronic acid is the most widely distributed acidic mucopolysaccharide in the human tissue matrix, forming a network barrier between the collagen fiber backbone of the extracellular matrix. This hyaluronic acid barrier is also one of the main factors hindering the rapid absorption of subcutaneously injected drugs.

[0077] The term "hyaluronidase" refers to an endoglucosidase that degrades hyaluronic acid, producing low-molecular-weight hyaluronic acid. It increases tissue permeability and enhances the ability of fluids to permeate tissues by hydrolyzing the β-1,4 glycosidic bonds in the HA chain to yield small-molecule HA or oligosaccharides. In this invention, "hyaluronidase" can also be referred to as "hyaluronidase," such as PH20 or rPH20.

[0078] The term "hyaluronidase activity" refers to the ability of hyaluronidase to cleave hyaluronic acid. In vitro assays for determining the hyaluronidase activity of hyaluronidases (e.g., recombinant human hyaluronidase PH20) are known in the art and are described herein. Exemplary assays include microturbidity assays, which indirectly determine the cleavage of hyaluronidase by hyaluronidase by detecting the insoluble precipitate formed when uncleaved hyaluronic acid binds to serum albumin. Examples include methods for hyaluronidase activity detection provided in Chinese Pharmacopoeia 2015, Part IV, General Chapter 1207 - Hyaluronidase Assay; USP 20130011378A or USP 20130011378B; and Chinese Invention Patent CN2025100465702.

[0079] "Pharmaceuticalally acceptable excipients" refer to components in a pharmaceutical composition other than the active ingredient that are non-toxic to the subjects. Pharmaceutically acceptable excipients include, but are not limited to, buffer solutions, excipients, stabilizers, preservatives, pH adjusters, nonionic surfactants, and / or solubilizers.

[0080] Subcutaneous administration refers to injecting medication into the subcutaneous tissue for absorption. Subcutaneous infusion offers numerous advantages in terms of economy and ease of operation: 1. Lower cost; 2. More suitable sites, simpler needle insertion, less pain or discomfort compared to intravenous insertion, and easier re-insertion at different sites, as the needle can be placed in relatively non-sensitive areas of the skin; 3. Subcutaneous infusion can be performed in almost any environment, and is more suitable than intravenous infusion in situations with limited nursing resources. From a safety perspective, the presence of a structurally fixed fibrous framework in the subcutaneous tissue restricts the flow of molecules larger than 200 nm in diameter, avoiding the risk of introducing large, insoluble microparticles into the central circulation. Subcutaneous infusion has the following safety advantages: 1. No risk of thrombosis; 2. Less likely to cause pulmonary edema and fluid overload; 3. No risk of thrombophlebitis; 4. No risk of complications such as sepsis or systemic infection.

[0081] The term "about" refers to a range of acceptable error for a particular value as determined by those skilled in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. In the context of a particular measurement, result, or implementation, unless otherwise expressly stated in the embodiments or elsewhere in the specification, "about" means within one standard deviation, or up to 20% (whichever is greater), according to convention in the art.

[0082] The term "disease" as used herein includes infections caused by bacteria, fungi, actinomycetes, mycoplasma, chlamydia, spirochetes, or amoebae, or diseases caused by the aforementioned pathogens. The bacteria or fungi may be Haemophilus influenzae type B, Pseudomonas aeruginosa types A and B, Staphylococcus aureus, Group B Streptococcus, Streptococcus pneumoniae (types 1, 3, 4, 6, 7, 8, 9, 12, 14, 18, 19, and 23), Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus pyogenes, and others. Streptococcus viridans, Streptococcus agalactiae, Streptococcus pneumoniae, Bacillus anthracis, Corynebacterium diphtheriae, Bordetella pertussis, Clostridium tetani, Mycobacterium tuberculosis, Bacillus subtilis, Clostridium perfringens, Mycobacterium leprae, Neisseria gonorrhoeae, Neisseria meningitidis, Legionella pneumophila, Shigella dysenteriae, Pseudomonas aeruginosa, Proteus, Vibrio cholerae, Bacillus parahaemolyticus, Escherichia coli, Salmonella typhi, Salmonella fulvum, Haemophilus influenzae, Acinetobacter bacillus, Aspergillus, Candida albicans, Cryptococcus, Mucor, Fusarium. The diseases mentioned include: chronic osteomyelitis caused by Staphylococcus aureus, endocarditis caused by drug-resistant Staphylococcus aureus or Enterococcus, typhoid fever, paratyphoid fever, bacterial food poisoning, bacterial infectious diarrhea, cholera, bacterial dysentery, brucellosis, plague, anthrax, diphtheria, pertussis, scarlet fever, epidemic cerebrospinal meningitis, tuberculosis, bacterial bloodstream infection, bacterial upper respiratory tract infection, bacterial lower respiratory tract infection, bacterial urinary tract infection, bacterial abdominal infection, dermatophytosis, fungal stomatitis, candidal vaginitis, fungal pneumonia, fungal urinary tract infection, fungal bacteremia, cryptococcosis, candidiasis, aspergillosis, and pneumocystis infection.

[0083] The term "packaging" as used herein includes containers for storing and transporting the combined reagents or liquid formulations, containers for preparing liquid formulations using the combined reagents, and injection systems for administering the prepared liquid formulations. Materials of the packaging include, but are not limited to, individually packaged tubular vials, reagent kits or reagent boxes repackaged from multiple individually packaged vials, syringes, or test tubes. Injection systems include, but are not limited to, syringes, infusion pumps, injection pens, needle-free devices, or subcutaneous patch delivery devices. The components of the injection device may be conventional in the art, including, a container, a seal, and an injection needle. The container includes, but is not limited to, tubular vials, syringes, or test tubes. The material of the container may be conventional in the art, such as glass or plastic. The seal includes, but is not limited to, sealing plugs or sealing rings. The material of the seal may be conventional in the art, such as rubber, plastic, or polymer materials. The injection needle includes, but is not limited to, aqueous syringes, single needles, and microneedle assemblies. The material of the injection needle may be conventional in the art, such as metals, silicon, silica, glass, nickel, titanium, or biodegradable polymers. The aforementioned water injection includes, but is not limited to, vials, ampoules, or pre-filled injection systems. The ampoule-based water injection can be made of glass or plastic. The pre-filled injection system can be conventional in the art, such as a pre-filled syringe. Optionally, the container is a vial made of neutral borosilicate glass with a volume of 0.1~20ml. The sealing element is a sealing plug made of halogenated butyl rubber; the injection needle is a single needle or a microneedle assembly. Optionally, the single needle can be made of 304 or 316 stainless steel with a size of 30G, 24G, 27G, or 29G; the microneedle assembly can be made of 304 or 316 stainless steel or a biodegradable polymer with a nanometer-sized needle with a height of 10~2000μm and a width of 10~50μm.

[0084] II. Detailed Description of the Invention The first aspect of this disclosure provides a method for maintaining the activity of hyaluronidase or recombinant hyaluronidase in a liquid formulation, wherein the liquid formulation contains an antibiotic and hyaluronidase or recombinant hyaluronidase; the method includes containing albumin in the liquid formulation.

[0085] In optional embodiments, the concentration of the antibiotic is 1-500 mg / ml; optionally, the concentration of the antibiotic is 20-300 mg / ml; optionally, the concentration of the antibiotic is 50-260 mg / ml; optionally, the concentration of the antibiotic is 90-260 mg / ml; optionally, the concentration of the antibiotic is 90-116 mg / ml; optionally, the concentration of the antibiotic is 50-100 mg / ml; optionally, the concentration of the antibiotic is 100-150 mg / ml, including but not limited to 1 mg / ml, 5 mg / ml, 10 mg / ml, 15 mg / ml, 20 mg / ml, etc. ml, 25mg / ml, 30mg / ml, 35mg / ml, 40mg / ml, 45mg / ml, 50mg / ml, 55mg / ml, 60mg / ml, 65mg / ml, 70mg / ml, 75mg / ml, 80mg / ml, 85mg / ml, 90mg / ml, 95mg / ml, 100mg / ml, 116mg / ml, 150mg / ml, 200mg / ml, 250mg / ml, 260mg / ml, 300mg / ml, 350mg / ml, 400mg / ml, 450mg / ml or 500mg / ml.

[0086] In optional embodiments, the concentration of albumin is 0.01~10 g / L; optionally, the concentration of albumin is 0.01~5 g / L; optionally, the concentration of albumin is 0.02~1 g / L; optionally, the concentration of albumin is 0.05~1 g / L, for example 0.01 g / L, 0.02 g / L, 0.03 g / L, 0.04 g / L, 0.05 g / L, 0. 06g / L, 0.07g / L, 0.08g / L, 0.09g / L, 0.1g / L, 0.2g / L, 0.3g / L, 0.4g / L, 0.5g / L, 0.6g / L, 0.7g / L, 0.8g / L, 0.9g / L, 1g / L, 2g / L, 3g / L, 4g / L, 5g / L, 6g / L, 7g / L, 8g / L, 9g / L or 10g / L.

[0087] In optional embodiments, the mass ratio of albumin to antibiotic is 1:10~50000; optionally, it is 1:10~100, optionally, 1:50~10000, optionally, 1:116~446.2, optionally, 1:351.5~446.2, optionally, 1:260~1000, optionally, 1:787.8~1000, optionally, 1:50~100, for example, 1:10, 1:20, 1:25, 1:50, 1:100, 1:116, 1:260, 1:351.5, 1:446.2, 1:787.8, 1:1000, 1:2000, or 1:10000.

[0088] In an optional embodiment, the enzyme activity of the hyaluronidase or recombinant hyaluronidase is 45 IU / ml to 3,000,000 IU / ml; optionally, the enzyme activity of the hyaluronidase or recombinant hyaluronidase is 45 IU / ml to 1,500,000 IU / ml; for example, 45 IU / ml to 300,000 IU / ml, for example, 50 IU / ml to 500 IU / ml, including but not limited to 45 IU / ml, 50 IU / ml, 100 IU / ml, 150 IU / ml, 200 IU / ml, 250 IU / ml. U / ml, 300IU / ml, 350IU / ml, 400IU / ml, 450IU / ml, 500IU / ml, 600IU / ml, 800IU / ml, 1000IU / ml, 2000IU / ml, 4000IU / ml, 6000IU / ml, 8000IU / ml, 10000IU / ml, 50000IU / ml, 100000IU / ml, 500000IU / ml, 1000000IU / ml or 1500000IU / ml.

[0089] In optional embodiments, the antibiotics include cephalosporin antibiotics or β-lactam antibiotics; for example, the cephalosporin antibiotics include one or more of ceftazidime, cefuroxime sodium, ceftriaxone sodium, cefazolin sodium, cefoperazone / sulbactam sodium, cefazolin sodium for injection, cefazolin sodium, cefoperazone sodium, or cefoperazone; for example, the β-lactam antibiotics include meropenem.

[0090] In an optional embodiment, the liquid formulation further comprises a buffer, a stabilizer, and a nonionic surfactant; optionally, the buffer comprises a phosphate buffer and / or a Tris buffer, optionally the phosphate buffer comprises disodium hydrogen phosphate, and the nonionic surfactant comprises one or more of polysorbate 20, polysorbate 80, or poloxamer 188; optionally, the stabilizer is selected from (i) or (ii): (i) methionine and sodium chloride; (ii) is composed of (i) and (iii), wherein (iii) is trehalose or sucrose.

[0091] In an optional embodiment, the concentration of the buffer is 2-50 mM; the stabilizer is selected from (i) or (ii): (i) Methionine and sodium chloride; the concentration of said methionine is 2-50 mM, for example 2 mM, 2.6 mM, 3.3 mM, 5 mM, 10 mM or 50 mM; the concentration of said sodium chloride is 25-200 mM, optionally 30 mM-200 mM, for example 30 mM, 38 mM, 48 mM, 130 mM, 145 mM, 154 mM or 180 mM; (ii) is composed of (i) and (iii), wherein (c) is trehalose or sucrose, and the concentration of the trehalose or sucrose is 25-200 mM, for example 25 mM, 53 mM or 200 mM; The concentration of the nonionic surfactant is 0.01% to 0.1% (w / v), for example, 0.02% (w / v), and the concentration of the nonionic surfactant can also be 0.005% to 0.007% (w / v).

[0092] In an optional embodiment, the concentration of methionine in the stabilizer (i) is 2-15 mM, and the concentration of sodium chloride is 50-180 mM.

[0093] In an optional embodiment, the concentration of the phosphate buffer or Tris buffer is 2 mM, 2.6 mM, 3.3 mM, 5 mM, 10 mM or 50 mM.

[0094] In an optional embodiment, the liquid formulation further comprises a buffer, a cryoprotectant, an excipient, an enzyme protectant, and a surfactant.

[0095] In an optional embodiment, the liquid formulation further contains an enzyme activity protectant; optionally, the enzyme activity protectant includes a chelating agent and a polyvalent metal salt ion, such as disodium ethylenediaminetetraacetate and calcium chloride; optionally, the concentration of the chelating agent is 0.01~10 mM, and the concentration of the polyvalent metal salt ion is 0.01~10 mM.

[0096] In optional embodiments, the liquid formulation comprises hyaluronidase or recombinant hyaluronidase, albumin, antibiotics, buffers, and enzyme activity protectants and stabilizers. Optionally, the buffer comprises phosphate buffer and / or Tris buffer; optionally, the phosphate buffer comprises disodium hydrogen phosphate. Optionally, the concentration of the buffer is 2-50 mM. Optionally, the enzyme activity protectant comprises a chelating agent and a polyvalent metal salt ion, such as disodium ethylenediaminetetraacetate and calcium chloride; optionally, the concentration of the chelating agent is 0.01-10 mM, and the concentration of the polyvalent metal salt ion is 0.01-10 mM. Optionally, the enzyme activity of the hyaluronidase or recombinant hyaluronidase is 45 IU / ml to 3,000,000 IU / ml. Optionally, the concentration of the albumin is 0.01-10 g / L. Optionally, the antibiotic includes cephalosporin antibiotics or β-lactam antibiotics; optionally, the mass ratio of albumin to antibiotic is 1:10 to 50000.

[0097] In an optional embodiment, the buffer comprises a phosphate buffer, such as sodium dihydrogen phosphate; the cryoprotectant comprises sucrose and / or trehalose; the excipient comprises mannitol; the enzyme activity protectant comprises a chelating agent and a polyvalent metal salt ion, such as disodium EDTA and calcium chloride; and the surfactant comprises polysorbate 20 and / or polysorbate 80.

[0098] In an optional embodiment, the concentration of the phosphate buffer is 0.1~50mM, for example, 0.1mM, 0.2mM, 0.5mM, 1mM, 2mM, 2.6mM, 3.3mM, 5mM, 10mM or 50mM; the concentration of the cryoprotectant is 1~60mM; the concentration of the excipient is 1~300mM; the concentration of the chelating agent is 0.01~10mM; the concentration of the polyvalent metal salt ion is 0.01~10mM; and the concentration of the surfactant is 0.001~0.1g / L.

[0099] In an optional embodiment, the composition of the liquid formulation is selected from any of the following: (1) 50 IU / ml~500 IU / ml hyaluronidase or recombinant hyaluronidase, about 50~260 mg / ml cefuroxime sodium, about 10 mM phosphate buffer, about 145 mM sodium chloride, about 10 mM methionine, about 0.02% (w / v) polysorbate 20, about 1 g / L albumin; (2) 150 IU / ml~500 IU / ml hyaluronidase or recombinant hyaluronidase, about 50~260 mg / ml cefuroxime sodium, about 10 mM phosphate buffer, about 145 mM sodium chloride, about 10 mM methionine, about 0.02% (w / v) polysorbate 20, about 1 g / L albumin; (3) 50 IU / ml to 500 IU / ml hyaluronidase or recombinant hyaluronidase, approximately 50 to 260 mg / ml cefuroxime sodium, approximately 2.6 to 3.3 mM phosphate buffer, approximately 38 to 48 mM sodium chloride, approximately 2.6 to 3.3 mM methionine, approximately 0.005% (w / v) to 0.007% (w / v) polysorbate 20, approximately 0.26 to 0.33 g / L albumin; (4) 50 IU / ml~500 IU / ml hyaluronidase or recombinant hyaluronidase, about 50~260 mg / ml ceftazidime, about 10 mM phosphate buffer, about 145 mM sodium chloride, about 10 mM methionine, about 0.02% polysorbate 20, about 1 g / L albumin; (5) 50 IU / ml to 500 IU / ml hyaluronidase or recombinant hyaluronidase, approximately 50 to 260 mg / ml ceftazidime, approximately 2.6 to 3.3 mM phosphate buffer, approximately 145 to 154 mM sodium chloride, approximately 2.6 to 3.3 mM methionine, approximately 0.005% (w / v) to 0.007% (w / v) polysorbate 20, approximately 0.26 to 0.33 g / L albumin; (6) 200 IU / ml hyaluronidase or recombinant hyaluronidase, approximately 50~260 mg / ml ceftazidime, approximately 1.4 g / L disodium hydrogen phosphate, approximately 0.9 g / L disodium ethylenediaminetetraacetate, approximately 0.3 g / L calcium chloride buffer and approximately 0.01 g / L~5 g / L albumin; (7) 200 IU / ml hyaluronidase or recombinant hyaluronidase, approximately 50~260 mg / ml ceftriaxone, approximately 1.4 g / L disodium hydrogen phosphate, approximately 0.9 g / L disodium ethylenediaminetetraacetate, approximately 0.3 g / L calcium chloride buffer and approximately 0.01 g / L~5 g / L albumin; (8) 200 IU / ml hyaluronidase or recombinant hyaluronidase, approximately 50~260 mg / ml meropenem, approximately 1.4 g / L disodium hydrogen phosphate, approximately 0.9 g / L disodium ethylenediaminetetraacetate, approximately 0.3 g / L calcium chloride buffer and approximately 0.01 g / L~5 g / L albumin; (9) 200 IU / ml hyaluronidase or recombinant hyaluronidase, approximately 50-260 mg / ml cefazolin sodium, approximately 1.4 g / L disodium hydrogen phosphate, approximately 0.9 g / L disodium ethylenediaminetetraacetate, approximately 0.3 g / L calcium chloride buffer, and approximately 0.01 g / L-5 g / L albumin.

[0100] In an optional embodiment, the composition of the liquid formulation is selected from any of the following: (1) 50 IU / ml~500 IU / ml hyaluronidase or recombinant hyaluronidase, about 116 mg / ml cefuroxime sodium, about 10 mM phosphate buffer, about 145 mM sodium chloride, about 10 mM methionine, about 0.02% (w / v) polysorbate 20, about 1 g / L albumin; (2) 150 IU / ml~500 IU / ml hyaluronidase or recombinant hyaluronidase, approximately 116 mg / ml cefuroxime sodium, approximately 10 mM phosphate buffer, approximately 145 mM sodium chloride, approximately 10 mM methionine, approximately 0.02% (w / v) polysorbate 20, approximately 1 g / L albumin; (3) 50 IU / ml to 500 IU / ml hyaluronidase or recombinant hyaluronidase, approximately 116 mg / ml cefuroxime sodium, approximately 2.6 to 3.3 mM phosphate buffer, approximately 38 to 48 mM sodium chloride, approximately 2.6 to 3.3 mM methionine, approximately 0.005% (w / v) to 0.007% (w / v) polysorbate 20, approximately 0.26 to 0.33 g / L albumin; (4) 50 IU / ml~500 IU / ml hyaluronidase or recombinant hyaluronidase, about 260 mg / ml ceftazidime, about 10 mM phosphate buffer, about 145 mM sodium chloride, about 10 mM methionine, about 0.02% polysorbate 20, about 1 g / L albumin. (5) 50 IU / ml to 500 IU / ml hyaluronidase or recombinant hyaluronidase, approximately 260 mg / ml ceftazidime, approximately 2.6 to 3.3 mM phosphate buffer, approximately 145 to 154 mM sodium chloride, approximately 2.6 to 3.3 mM methionine, approximately 0.005% (w / v) to 0.007% (w / v) polysorbate 20, approximately 0.26 to 0.33 g / L albumin; (6) 200 IU / ml hyaluronidase or recombinant hyaluronidase, about 100 mg / ml ceftazidime, about 1.4 g / L disodium hydrogen phosphate, about 0.9 g / L disodium ethylenediaminetetraacetate, about 0.3 g / L calcium chloride buffer and about 0.01 g / L~5 g / L albumin; (7) 200 IU / ml hyaluronidase or recombinant hyaluronidase, approximately 100 mg / ml ceftriaxone, approximately 1.4 g / L disodium hydrogen phosphate, approximately 0.9 g / L disodium ethylenediaminetetraacetate, approximately 0.3 g / L calcium chloride buffer and approximately 0.01 g / L to 5 g / L albumin; (8) 200 IU / ml hyaluronidase or recombinant hyaluronidase, approximately 50 mg / ml meropenem, approximately 1.4 g / L disodium hydrogen phosphate, approximately 0.9 g / L disodium ethylenediaminetetraacetate, approximately 0.3 g / L calcium chloride buffer and approximately 0.01 g / L to 5 g / L albumin; (9) 200 IU / ml hyaluronidase or recombinant hyaluronidase, approximately 100 mg / ml cefazolin sodium, approximately 1.4 g / L disodium hydrogen phosphate, approximately 0.9 g / L disodium ethylenediaminetetraacetate, approximately 0.3 g / L calcium chloride buffer, and approximately 0.01 g / L to 5 g / L albumin.

[0101] In an optional embodiment, the enzyme activity of hyaluronidase or recombinant hyaluronidase in the liquid formulation is maintained for more than 1 hour, for example, 1 to 24 hours, or 2 to 12 hours.

[0102] A second aspect of this disclosure provides the use of reagents containing hyaluronidase and / or recombinant hyaluronidase in the preparation of combination reagents, said combination reagents comprising individually packaged reagent (a) containing an antibiotic, and reagent (b) containing hyaluronidase and / or recombinant hyaluronidase; said reagent (b) further containing albumin; said combination reagents being used in the method of maintaining hyaluronidase or recombinant hyaluronidase activity in a liquid formulation as described in any of the foregoing embodiments.

[0103] In an optional embodiment, the antibiotic includes cephalosporins or β-lactam antibiotics; the cephalosporins include one or more of, for example, ceftazidime, cefuroxime sodium, ceftriaxone sodium, cefazolin sodium, cefoperazone / sulbactam sodium, cefazolin sodium, cefotaxime sodium, or cefoperazone; and the β-lactam antibiotics include, for example, meropenem.

[0104] In an optional embodiment, the enzyme activity of the hyaluronidase or recombinant hyaluronidase in reagent (b) is 45 IU / ml to 3,000,000 IU / ml; optionally, the enzyme activity of the hyaluronidase or recombinant hyaluronidase is 45 IU / ml to 1,500,000 IU / ml; for example, 45 IU / ml to 300,000 IU / ml, for example, 50 IU / ml to 500 IU / ml.

[0105] In an optional embodiment, reagent (b) further comprises a buffer, a stabilizer, and a nonionic surfactant; optionally, the buffer comprises phosphate buffer and / or Tris buffer, optionally, the phosphate buffer comprises disodium hydrogen phosphate, and the nonionic surfactant comprises one or more of polysorbate 20, polysorbate 80, or poloxamer 188; optionally, the stabilizer is selected from (i) or (ii): (i) methionine and sodium chloride; (ii) is composed of (i) and (iii), wherein (iii) is trehalose or sucrose.

[0106] In an optional embodiment, the concentration of the buffer is 2-50 mM; the stabilizer is selected from (i) or (ii): (i) Methionine and sodium chloride; the concentration of said methionine is 2-50 mM, for example 2 mM, 2.6 mM, 3.3 mM, 5 mM, 10 mM or 50 mM; the concentration of said sodium chloride is 25-200 mM, optionally 30 mM-200 mM, for example 30 mM, 38 mM, 48 mM, 130 mM, 145 mM, 154 mM or 180 mM; (ii) is composed of (i) and (iii), wherein (c) is trehalose or sucrose, and the concentration of the trehalose or sucrose is 25-200 mM, for example 25 mM, 53 mM or 200 mM; The concentration of the nonionic surfactant is 0.01% to 0.1% (w / v), for example, 0.02% (w / v), and the concentration of the nonionic surfactant can also be 0.005% to 0.007% (w / v).

[0107] In an optional embodiment, the concentration of methionine in the stabilizer (i) is 2-15 mM, and the concentration of sodium chloride is 50-180 mM.

[0108] In an optional embodiment, the concentration of the phosphate buffer or Tris buffer is 0.1 to 50 mM, for example, 0.1 mM, 0.2 mM, 0.5 mM, 1 mM, 2 mM, 2.6 mM, 3.3 mM, 5 mM, 10 mM or 50 mM.

[0109] In another alternative embodiment, the reagent (b) contains hyaluronidase and / or recombinant hyaluronidase, buffer, cryoprotectant, excipient, enzyme activity protectant and surfactant.

[0110] In an optional embodiment, the liquid formulation further contains an enzyme activity protectant; optionally, the enzyme activity protectant includes a chelating agent and a polyvalent metal salt ion, such as disodium ethylenediaminetetraacetate and calcium chloride; optionally, the concentration of the chelating agent is 0.01~10 mM, and the concentration of the polyvalent metal salt ion is 0.01~10 mM.

[0111] In an optional embodiment, the buffer includes a phosphate buffer, such as sodium dihydrogen phosphate; the cryoprotectant includes sucrose and / or trehalose; the excipient includes mannitol; and the enzyme activity protectant includes a chelating agent and a polyvalent metal salt ion, such as disodium ethylenediaminetetraacetate and calcium chloride.

[0112] In an optional embodiment, the lyophilized formulation contains, prior to lyophilization, the following in the liquid formulation: The enzyme activity of hyaluronidase and / or recombinant hyaluronidase is 45 IU / ml to 3,000,000 IU / ml; the concentration of the buffer is 0.014 to 70 g / L, for example 0.14 to 14 g / L; the cryoprotectant is 0.2 to 400 g / L, for example 2 to 200 g / L; the excipient is 0.4 to 2000 g / L, for example 4 to 400 g / L; the concentration of the chelating agent is 0.009 to 45 g / L, for example 0.09 to 9 g / L; and the concentration of the polyvalent metal salt ion is 0.03 to 3 g / L.

[0113] In an optional embodiment, the lyophilized formulation contains, prior to lyophilization, the following in the liquid formulation: The enzyme contained hyaluronidase and / or recombinant hyaluronidase with an enzyme activity of 45 IU / ml to 3,000,000 IU / ml, 1.4 g / L sodium dihydrogen phosphate, 0.9 g / L disodium ethylenediaminetetraacetate, 0.3 g / L calcium chloride, 40 g / L mannitol and 20 g / L trehalose.

[0114] A third aspect of this disclosure provides a combination reagent comprising an individually packaged reagent (a) containing an antibiotic, and a reagent (b) containing hyaluronidase and / or recombinant hyaluronidase; said reagent (b) may or may not contain albumin; said combination reagent is used for the method of maintaining hyaluronidase or recombinant hyaluronidase activity in a liquid formulation as described in any of the preceding embodiments.

[0115] In an optional embodiment, the antibiotic includes cephalosporin antibiotics or β-lactam antibiotics; for example, one or more of ceftazidime, cefuroxime sodium, ceftriaxone sodium, cefazolin sodium, cefoperazone / sulbactam sodium, cefazolin sodium, cefotaxime sodium, or cefoperazone.

[0116] In an optional embodiment, the antibiotics include cephalosporins, penicillins, β-lactams, aminoglycosides, macrolides, lincomycins, polypeptides, quinolones, sulfonamides, antituberculosis drugs, antifungal drugs, and other antibiotics; cephalosporins include ceftazidime, cefuroxime sodium, ceftriaxone sodium, cefazolin sodium, and cefoperazone / sulbactam sodium, and the reagent (b) does not contain albumin.

[0117] In an optional embodiment, the β-lactam antibiotic includes carbapenem antibiotics, such as meropenem.

[0118] In another alternative embodiment, the antibiotic includes ceftazidime, cefuroxime sodium, ceftriaxone sodium, cefazolin sodium, cefoperazone / sulbactam sodium, or meropenem, and the reagent (b) contains albumin.

[0119] In an optional embodiment, reagent (b) is a liquid formulation containing hyaluronidase or recombinant hyaluronidase, an enzyme, a buffer, a stabilizer, and a nonionic surfactant; optionally, the buffer includes phosphate buffer and / or Tris buffer, optionally, the phosphate buffer includes disodium hydrogen phosphate, and the nonionic surfactant includes one or more of polysorbate 20, polysorbate 80, or poloxamer 188; optionally, the stabilizer is selected from (i) or (ii): (i) methionine and sodium chloride; (ii) is composed of (i) and (iii), wherein (iii) is trehalose or sucrose.

[0120] In an optional embodiment, the concentration of the buffer is 2-50 mM; the stabilizer is selected from (i) or (ii): (i) Methionine and sodium chloride; the concentration of said methionine is 2-50 mM, for example 2 mM, 2.6 mM, 3.3 mM, 5 mM, 10 mM or 50 mM; the concentration of said sodium chloride is 25-200 mM, optionally 30 mM-200 mM, for example 30 mM, 38 mM, 48 mM, 130 mM, 145 mM, 154 mM or 180 mM; (ii) is composed of (i) and (iii), wherein (c) is trehalose or sucrose, and the concentration of the trehalose or sucrose is 25-200 mM, for example 25 mM, 53 mM or 200 mM; The concentration of the nonionic surfactant is 0.01% to 0.1% (w / v), for example, 0.02% (w / v), and the concentration of the nonionic surfactant can also be 0.005% to 0.007% (w / v).

[0121] In an optional embodiment, the concentration of methionine in the stabilizer (i) is 2-15 mM, and the concentration of sodium chloride is 50-180 mM.

[0122] In an optional embodiment, the concentration of the phosphate buffer or Tris buffer is 2 mM, 2.6 mM, 3.3 mM, 5 mM, 10 mM or 50 mM.

[0123] In an optional embodiment, the reagent (b) further contains an enzyme activity protectant; optionally, the enzyme activity protectant includes a chelating agent and a polyvalent metal salt ion, such as disodium ethylenediaminetetraacetate and calcium chloride; optionally, the concentration of the chelating agent is 0.01~10 mM, and the concentration of the polyvalent metal salt ion is 0.01~10 mM.

[0124] In optional embodiments, the liquid formulation comprises hyaluronidase or recombinant hyaluronidase, albumin, antibiotics, buffers, and enzyme activity protectants and stabilizers. Optionally, the buffer comprises phosphate buffer and / or Tris buffer; optionally, the phosphate buffer comprises disodium hydrogen phosphate. Optionally, the concentration of the buffer is 2-50 mM. Optionally, the enzyme activity protectant comprises a chelating agent and a polyvalent metal salt ion, such as disodium ethylenediaminetetraacetate and calcium chloride; optionally, the concentration of the chelating agent is 0.01-10 mM, and the concentration of the polyvalent metal salt ion is 0.01-10 mM. Optionally, the enzyme activity of the hyaluronidase or recombinant hyaluronidase is 45 IU / ml to 3,000,000 IU / ml. Optionally, the concentration of the albumin is 0.01-10 g / L. Optionally, the antibiotic includes cephalosporin antibiotics or β-lactam antibiotics; optionally, the mass ratio of albumin to antibiotic is 1:10 to 50000.

[0125] In an optional embodiment, reagent (b) is a lyophilized formulation containing hyaluronidase and / or recombinant hyaluronidase, buffer, cryoprotectant, excipient, enzyme activity protectant, and surfactant.

[0126] In an optional embodiment, the buffer includes a phosphate buffer, such as sodium dihydrogen phosphate; the cryoprotectant includes sucrose and / or trehalose; the excipient includes mannitol; and the enzyme activity protectant includes a chelating agent and a polyvalent metal salt ion, such as disodium ethylenediaminetetraacetate and calcium chloride.

[0127] In an optional embodiment, the lyophilized formulation contains, prior to lyophilization, the following in the liquid formulation: The enzyme activity of hyaluronidase and / or recombinant hyaluronidase is 45 IU / ml to 3,000,000 IU / ml; the concentration of the buffer is 0.014 to 70 g / L, for example 0.14 to 14 g / L; the cryoprotectant is 0.2 to 400 g / L, for example 2 to 200 g / L; the excipient is 0.4 to 2000 g / L, for example 4 to 400 g / L; the concentration of the chelating agent is 0.009 to 45 g / L, for example 0.09 to 9 g / L; and the concentration of the polyvalent metal salt ion is 0.03 to 3 g / L.

[0128] In an optional embodiment, the lyophilized formulation contains, prior to lyophilization, the following in the liquid formulation: The enzyme contained hyaluronidase and / or recombinant hyaluronidase with an enzyme activity of 45 IU / ml to 3,000,000 IU / ml, 1.4 g / L sodium dihydrogen phosphate, 0.9 g / L disodium ethylenediaminetetraacetate, 0.3 g / L calcium chloride, 40 g / L mannitol and 20 g / L trehalose.

[0129] In an optional implementation, the combined reagents are selected from any of the following: (A) The reagent (a) is ceftazidime; Reagent (b) is a liquid formulation comprising 250 IU / ml hyaluronidase, 10 mM disodium hydrogen phosphate, 10 mM methionine, 145 mM sodium chloride, 0.02% (w / v) polysorbate 20 and 1 g / L albumin. After dissolving reagent (a) with reagent (b), the concentration of ceftazidime is approximately 260 mg / ml; (B) The reagent (a) mentioned is ceftazidime; Reagent (b) is a liquid formulation comprising 150 IU / ml hyaluronidase, 10 mM disodium hydrogen phosphate, 10 mM methionine, 145 mM sodium chloride, 0.02% (w / v) polysorbate 20 and 1 g / L albumin. After dissolving reagent (a) with reagent (b), the concentration of ceftazidime is approximately 260 mg / ml; (C) The reagent (a) is ceftazidime; Reagent (b) is a liquid formulation comprising 150 IU / ml hyaluronidase, 10 mM disodium hydrogen phosphate, 10 mM methionine, 130 mM sodium chloride, 0.02% (w / v) polysorbate 20 and 1 g / L albumin. After dissolving reagent (a) with reagent (b), the concentration of ceftazidime is approximately 260 mg / ml; (D) The reagent (a) is cefuroxime sodium; Reagent (b) is a liquid formulation comprising 500 IU / ml hyaluronidase, 10 mM disodium hydrogen phosphate, 10 mM methionine, 145 mM sodium chloride, 0.02% (w / v) polysorbate 20 and 1 g / L albumin. After dissolving reagent (a) with reagent (b), the concentration of ceftazidime is approximately 116 mg / ml; (E) The reagent (a) is cefuroxime sodium; Reagent (b) is a liquid formulation comprising 150 IU / ml hyaluronidase, 10 mM disodium hydrogen phosphate, 10 mM methionine, 145 mM sodium chloride, 0.02% (w / v) polysorbate 20 and 1 g / L albumin. After dissolving reagent (a) with reagent (b), the concentration of ceftazidime is approximately 116 mg / ml; (F) The reagent (a) mentioned is cefuroxime sodium; Reagent (b) is a liquid formulation comprising 150 IU / ml hyaluronidase, 10 mM disodium hydrogen phosphate, 10 mM methionine, 130 mM sodium chloride, 0.02% (w / v) polysorbate 20 and 1 g / L albumin. After dissolving reagent (a) with reagent (b), the concentration of ceftazidime is approximately 116 mg / ml; (G) The reagent (a) mentioned is ceftazidime; Reagent (b) includes hyaluronidase and / or recombinant hyaluronidase with an enzyme activity of 200 U / ml, sodium dihydrogen phosphate at 1.4 g / L, disodium ethylenediaminetetraacetate at 0.9 g / L, calcium chloride at 0.3 g / L, and albumin at 0.01 g / L to 5 g / L. After dissolving reagent (a) with reagent (b), the concentration of ceftazidime is 100 mg / ml; (H) The reagent (a) mentioned is ceftriaxone; Reagent (b) includes hyaluronidase and / or recombinant hyaluronidase with an enzyme activity of 200 U / ml, sodium dihydrogen phosphate at 1.4 g / L, disodium ethylenediaminetetraacetate at 0.9 g / L, calcium chloride at 0.3 g / L, and albumin at 0.01 g / L to 5 g / L. After dissolving reagent (a) with reagent (b), the concentration of ceftazidime is 100 mg / ml; (I) The reagent (a) is meropenem; Reagent (b) includes hyaluronidase and / or recombinant hyaluronidase with an enzyme activity of 200 U / ml, sodium dihydrogen phosphate at 1.4 g / L, disodium ethylenediaminetetraacetate at 0.9 g / L, calcium chloride at 0.3 g / L, and albumin at 0.01 g / L to 5 g / L. After dissolving reagent (a) with reagent (b), the concentration of ceftazidime is 100 mg / ml; (J) The reagent (a) mentioned is cefazolin sodium; Reagent (b) includes hyaluronidase and / or recombinant hyaluronidase with an enzyme activity of 200 U / ml, sodium dihydrogen phosphate at 1.4 g / L, disodium ethylenediaminetetraacetate at 0.9 g / L, calcium chloride at 0.3 g / L, and albumin at 0.01 g / L to 5 g / L. After dissolving reagent (a) with reagent (b), the concentration of ceftazidime is 100 mg / ml.

[0130] Fourth aspect: This disclosure provides a method for preparing a liquid formulation, comprising mixing reagent (a) and reagent (b) in any of the preceding embodiments of the combined reagent, regardless of the order of addition. Optionally, the reagent (b) is a liquid preparation, and the preparation method includes mixing the reagent (a) and the reagent (b) in any order of addition, or mixing the reagent (a), the reagent (b), and the reconstituter in any order; the reconstituter may be physiological saline. Optionally, reagent (b) is a lyophilized preparation, and the preparation method includes mixing reagent (a), reagent (b) and reconstituter in any order; the reconstituter may be physiological saline, glucose solution or water.

[0131] In an optional embodiment, the liquid formulation is composed of the liquid formulation described in any of the aforementioned methods for maintaining hyaluronidase stability.

[0132] Fifthly, this disclosure provides the use of the combined reagents described in any of the foregoing embodiments, or the liquid preparations obtained by the preparation methods described in any of the foregoing embodiments, in the preparation of medicaments for treating diseases; the diseases include infections caused by bacteria, fungi, actinomycetes, mycoplasma, chlamydia, spirochetes, or amoebae, or diseases caused by infections caused by the aforementioned pathogens.

[0133] In an optional embodiment, the treatment for the disease includes administering to a subject an effective dose of a liquid formulation prepared using any of the foregoing embodiments.

[0134] In an optional embodiment, the liquid formulation is a liquid formulation prepared by any of the preparation methods described in the foregoing embodiments and left for more than 1 hour; for example, 2 to 12 hours.

[0135] In optional embodiments, the administration method includes subcutaneous administration, intramuscular administration, or intravenous administration.

[0136] In a sixth aspect, this disclosure provides a method for treating a disease, comprising administering to a subject an effective dose of a liquid preparation prepared by any of the foregoing embodiments; wherein the disease includes infection by bacteria, fungi, actinomycetes, mycoplasma, chlamydia, spirochetes, or amoebae, or a disease caused by infection with the aforementioned pathogens.

[0137] In an optional embodiment, the liquid formulation is a liquid formulation prepared by using the combination reagents described in any of the foregoing embodiments and the preparation method described in any of the foregoing embodiments.

[0138] In an optional embodiment, the liquid formulation is a liquid formulation that has been left to stand for more than 1 hour; for example, 2 to 12 hours.

[0139] In optional embodiments, the administration method includes subcutaneous administration, intramuscular administration, or intravenous administration.

[0140] In a seventh aspect, this disclosure provides a combination reagent or liquid preparation for treating diseases; said diseases include infections caused by bacteria, fungi, actinomycetes, mycoplasma, chlamydia, spirochetes, or amoebae, or diseases caused by infections of the aforementioned pathogens; said combination reagent includes the combination reagent described in any of the foregoing embodiments; said liquid preparation includes a liquid preparation prepared by mixing and formulating the combination reagent described in any of the foregoing embodiments, or a liquid preparation prepared by the preparation method described in any of the foregoing embodiments.

[0141] In an optional embodiment, the liquid formulation is a liquid formulation that has been left to stand for more than 1 hour; for example, 2 to 12 hours.

[0142] In an optional embodiment, the treatment for the disease includes administering to a subject an effective dose of a liquid formulation prepared using any of the foregoing embodiments.

[0143] In optional embodiments, the administration method includes subcutaneous administration, intramuscular administration, or intravenous administration.

[0144] In a seventh aspect, this disclosure provides a packaging body containing the combined reagents described in any of the foregoing embodiments, or a liquid formulation prepared by the preparation method described in any of the foregoing embodiments.

[0145] In an optional embodiment, the packaging body is used to store or transport the combined reagents described in any of the foregoing embodiments, or the liquid formulation prepared by any of the preparation methods described in any of the foregoing embodiments.

[0146] In an optional embodiment, the packaging is used to formulate or administer a liquid formulation prepared by any of the preparation methods described in the foregoing embodiments.

[0147] In an optional embodiment, the packaging includes the combination reagent described in any of the foregoing embodiments and instructions for use of the combination reagent.

[0148] Eighth aspect: This disclosure provides an use of albumin in maintaining the activity of hyaluronidase or recombinant hyaluronidase in a liquid formulation, wherein the liquid formulation contains an antibiotic and hyaluronidase or recombinant hyaluronidase.

[0149] Optionally, the concentration of the antibiotic is 1~500 mg / ml; Optionally, the enzyme activity of the hyaluronidase or recombinant hyaluronidase is 45 IU / ml to 3,000,000 IU / ml; Optionally, the antibiotic includes cephalosporin antibiotics or β-lactam antibiotics; for example, one or more of ceftazidime, cefuroxime sodium, ceftriaxone sodium, cefazolin sodium, cefoperazone / sulbactam sodium, cefazolin sodium, cefotaxime sodium, or cefoperazone.

[0150] Example The present disclosure is further illustrated below with reference to embodiments. The description of specific exemplary embodiments of the present disclosure is for illustrative and explanatory purposes. These descriptions are not intended to limit the present disclosure to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the teachings of this specification. The exemplary embodiments were chosen and described in order to explain the specific principles of the present disclosure and their practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the present disclosure, as well as various different choices and variations.

[0151] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0152] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0153] Example 1. Hyaluronidase Production CHO cells were cultured in suspension in a self-developed serum-free medium and fed-batch culture in a self-developed serum-free feed medium. The culture was gradually scaled up to a 30L reactor through shake flask culture.

[0154] During the 3-4 day culture period, the amount of supplemental culture medium added to the bioreactor daily was 1%-6% of the actual culture volume. The culture temperature was controlled at 35℃-37℃, and the pH was maintained at 7.0 by supplementing with 10% (w / v) Na₂CO₃ and CO₂. The reactor aeration rate was controlled at 0.015-0.15 vvm; the rotation speed was controlled at 80-150 rpm; and the dissolved oxygen level was controlled at 20%-40%. During cell culture, samples were taken daily to monitor temperature, pH, glucose concentration, lactate concentration, molar osmolality, and protein expression levels. Culture was terminated when the CHO cell viability was below 80% or the culture period reached 14-20 days, yielding recombinant human hyaluronidase. The amino acid sequence of recombinant human hyaluronidase is shown in SEQ ID NO: 1. LNFRAPPVIPNVPFLWAWNAPSEFCLGKFDEPLDMSLFSFIGSPRINATGQGVTIFYVDRLGYYPYIDSITGVTVNGGIPQKISLQDHLDKAKKDITFYMPVDNLGMAVIDW EEWRPTWARNWKPKDVYKNRSIELVQQQNVQLSLTEATEKAKQEFEKAGKDFLVETIKLGKLLRPNHLWGYYLFPDCYNHHYKKPGYNGSCFNVEIKRNDDLSWLWNESTALY PSIYLNTQQSPVAATLYVRNRVREAIRVSKIPDAKSPLPVFAYTRIVFTDQVLKFLSQDELVYTFGETVALGASGIVIWGTLSIMRSMKSCLLLDNYMETILNPYIINVTLAA KMCSQVLCQEQGVCIRKNWNSSDYLHLNPDNFAIQLEKGGKFTVRGKPTLEDLEQFSEKFYCSCYSTLSCKEKADVKDTDAVDVCIADGVCIDAFLKPPMETEEPQIFY(SEQ ID NO: 1).

[0155] The obtained recombinant human hyaluronidase supernatant was sequentially purified by filtration chromatography to obtain recombinant human hyaluronidase stock solution with SEC purity greater than 95% and hyaluronidase with RP purity greater than 85%.

[0156] Example 2. Compatibility test of different antibiotics with hyaluronidase Antibiotics were weighed and dissolved separately using water for injection. A certain amount of hyaluronidase prepared in Example 1 was added, and the mixture was mixed thoroughly. The mixture was then divided into 3-4 tubes and placed in an incubator at 25°C. After 0, 2, 6, and 12 hours, the remaining hyaluronidase activity was measured, and the percentage relative to the theoretical enzyme activity was calculated. The results showed that the following antibiotics had different effects on the stability of the hyaluronidase after being mixed for more than 2 hours.

[0157] Table 1. Effects of different antibiotics on hyaluronidase activity Example 3. Albumin stability experiment against hyaluronidase Based on Example 2, this embodiment selects cefazolin sodium from Table 1 as an example. It is dissolved in water for injection containing 1 g / L albumin, and then a certain amount of hyaluronidase prepared in Example 1 is added. After mixing, a mixture is obtained, which is the experimental group. At the same time, a control group is set up in which no albumin is added and only water for injection is used to dissolve cefazolin sodium (Table 2). The mixtures of the experimental group and the control group are divided into two tubes and placed in an incubator at 25°C. After 0 h and 4 h, the remaining hyaluronidase activity is detected and the percentage of theoretical enzyme activity is calculated.

[0158] Table 2. Effect of albumin on hyaluronidase activity in different cefazolin sodium solutions. The above results demonstrate that human serum albumin can significantly increase the long-term enzyme activity of hyaluronidase in solutions containing cefazolin sodium.

[0159] Referring to the experimental method for hyaluronidase activity in cefazolin sodium solution, another cephalosporin antibiotic, ceftazidime, and a carbapenem antibiotic, meropenem, were selected to investigate the effect of albumin on hyaluronidase activity in the solutions of the two antibiotics. The results are shown in Table 3.

[0160] Table 3. Effects of albumin on hyaluronidase activity in different antibiotic solutions. The above results demonstrate that the long-term stabilizing effect of albumin on hyaluronidase activity is also applicable in solutions containing ceftazidime and meropenem.

[0161] Example 4. Stability experiment of albumin to hyaluronidase in buffer solution. Based on Example 3, this embodiment selects meropenem as an example to investigate the stabilizing effect of albumin on hyaluronidase activity in the buffer solution. The buffer solution used in this embodiment consists of: 1.4 g / L (9 mM) disodium hydrogen phosphate, 0.9 g / L (2.4 mM) EDTA, 0.3 g / L (8.7 mM) calcium chloride buffer, pH 7.0.

[0162] Meropenem, an antibiotic from Table 3, was selected and dissolved in a buffer solution containing 1 g / L albumin. A certain amount of hyaluronidase prepared in Example 1 was added, and the mixture was mixed to obtain the experimental group. At the same time, a control group was set up in which meropenem was dissolved using only the buffer solution without the addition of albumin (Table 4). The mixtures of the experimental group and the control group were divided into two equal tubes and placed in an incubator at 25°C. After 0 h and 4 h, the remaining hyaluronidase activity was detected, and the percentage of theoretical enzyme activity was calculated.

[0163] Table 4. Effect of albumin on hyaluronidase activity in meropenem buffer solution. The above results indicate that meropenem significantly reduces hyaluronidase activity in the buffer solution provided in this embodiment, while the long-term storage activity of hyaluronidase is significantly improved after the addition of albumin.

[0164] Referring to the experimental method for hyaluronidase activity in meropenem buffer solution mentioned above, two cephalosporin antibiotics, ceftriaxone and cefazolin sodium, were selected to investigate the effect of albumin on hyaluronidase activity in ceftriaxone buffer solution. The results are shown in Table 5.

[0165] Table 5. Effect of albumin on hyaluronidase activity in buffer solutions of ceftriaxone and cefazolin sodium. The above results demonstrate that the long-term stabilizing effect of albumin on hyaluronidase activity is also applicable in buffer solutions containing ceftriaxone and cefazolin sodium.

[0166] Example 5. Effect of different albumin concentrations on hyaluronidase stability This embodiment, based on Example 4, selects ceftazidime as the target antibiotic to investigate the effect of different concentrations of albumin on the long-term stability of hyaluronidase in the above-mentioned buffer solution system. The experimental method is the same as in Example 4, and the experimental results are shown in Table 6.

[0167] Table 6. Effects of different concentrations of albumin on hyaluronidase activity in ceftazidime buffer solution. The above results demonstrate that albumin's long-term stabilizing effect on hyaluronidase activity is also applicable in buffer solutions containing ceftazidime. Furthermore, as the albumin concentration increases, its stabilizing effect on hyaluronidase activity gradually strengthens. When the albumin concentration reaches 0.5 g / L or higher, the activity of hyaluronidase can still be maintained at around 95% after 4 hours of storage.

[0168] Example 6. Compatibility test between different antibiotics and different hyaluronidase preparations The compatibility of the two substances was studied by preparing solutions containing hyaluronidase and antibiotics. Quantitative amounts of both reconstitution solvents were transferred to antibiotic vials, the vials were sealed with rubber stoppers, the antibiotic samples were reconstituted, and the entire volume was transferred to a volumetric flask and brought to volume. Samples were collected at 25°C for 0 h, 1 h, and 2 h, and hyaluronidase activity was measured at each incubation period.

[0169] This embodiment investigated how albumin improves the compatibility of different antibiotics with hyaluronidase under different buffer systems.

[0170] Among them, G1, G4, G5 and blank-2 groups used disodium hydrogen phosphate solution as phosphate buffer, and added stabilizers methionine and sodium chloride, surfactant Tween 20, and albumin. This formulation can be used to prepare liquid preparations containing antibiotics and hyaluronidase.

[0171] Groups G2, G3, and Blank-1, based on the buffer solutions provided in Examples 4 and 5 above, further added the stabilizers sodium chloride and trehalose, as well as the excipient mannitol, without adding albumin. This formulation can be used to prepare lyophilized preparations containing antibiotics and hyaluronidase.

[0172] The specific dosage of each component and the results of the hyaluronidase stability test are shown in Table 7 below. It can be seen that the stability of hyaluronidase after mixing with reconstituted ceftazidime in each of the following groups is good, with no significant decrease.

[0173] Table 7. Stability of ceftazidime-hyaluronidase mixture Note: Due to volume changes after reconstitution, the formulation content in the table may fluctuate by 20%.

[0174] The same experiments were conducted on cefuroxime sodium using the same grouping method, as shown in Table 8. The cefuroxime sodium and hyaluronidase mixture was stable in the liquid formulation containing albumin, but showed a significant decrease in the albumin-free formulation. The hyaluronidase group, i.e., the blank formulation, remained stable throughout. Therefore, cefuroxime sodium interferes with the hyaluronidase activity in the system, leading to decreased stability, but the addition of albumin has no effect and can significantly improve enzyme stability. In addition, comparing the hyaluronidase stability results corresponding to group G2 in Tables 7 and 8, it can be seen that in the liquid formulation system of group G2 (G3 and blank-1), ceftazidime and cefuroxime sodium have different effects on hyaluronidase stability, while in the liquid formulation system of group G1 (G4, G5 and blank-2), neither ceftazidime nor cefuroxime sodium has a significant effect on hyaluronidase stability, proving that the presence of albumin can eliminate the differences in the effects of different antibiotics on hyaluronidase stability.

[0175] Table 8. Stability of cefuroxime sodium-hyaluronate mixture Note: Due to volume changes after reconstitution, the formulation content in the table may fluctuate by 20%.

[0176] While specific embodiments of this disclosure have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of this disclosure. Therefore, the scope of protection of this disclosure is defined by the appended claims.

Claims

1. A method for maintaining the activity of hyaluronidase or recombinant hyaluronidase in a liquid formulation, wherein, The liquid formulation contains antibiotics and hyaluronidase or recombinant hyaluronidase; the method includes making the liquid formulation contain albumin; Optionally, the concentration of the antibiotic is 1~500 mg / ml; Optionally, the concentration of the antibiotic is 20-300 mg / ml; Optionally, the concentration of albumin is 0.01~10 g / L.

2. The method as described in claim 1, wherein, The mass ratio of albumin to antibiotic is 1:10 to 50000; it can be selected as 1:10 to 100, for example 1:10, 1:20, 1:25, 1:50 or 1:

100.

3. The method as described in claim 1 or 2, wherein, The enzyme activity of the hyaluronidase or recombinant hyaluronidase is 45 IU / ml to 3,000,000 IU / ml; optionally, the enzyme activity of the hyaluronidase or recombinant hyaluronidase is 45 IU / ml to 1,500,000 IU / ml; for example, 45 IU / ml to 300,000 IU / ml.

4. The method according to any one of claims 1 to 3, wherein, The antibiotics include cephalosporin antibiotics or β-lactam antibiotics; for example, one or more of ceftazidime, cefuroxime sodium, ceftriaxone sodium, cefazolin sodium, cefoperazone / sulbactam sodium, cefazolin sodium, cefotaxime sodium, or cefoperazone.

5. The method according to any one of claims 1 to 4, wherein, The liquid formulation also contains buffers, stabilizers, and nonionic surfactants; Optionally, the buffer includes phosphate buffer and / or Tris buffer, and the nonionic surfactant includes one or more of polysorbate 20, polysorbate 80, or poloxamer 188. Optionally, the stabilizer is selected from (i) or (ii): (i) methionine and sodium chloride; (ii) is composed of (i) and (iii), wherein (iii) is trehalose or sucrose; Optionally, the concentration of the buffer is 2~50mM; Optionally, the stabilizer is selected from (i) or (ii): (i) Methionine and sodium chloride; the concentration of said methionine is 2-50 mM, for example 2 mM, 2.6 mM, 3.3 mM, 5 mM, 10 mM or 50 mM; the concentration of said sodium chloride is 25-200 mM, optionally 30 mM-200 mM, for example 30 mM, 130 mM, 145 mM, 154 mM or 180 mM; (ii) is composed of (i) and (iii), wherein (c) is trehalose or sucrose, and the concentration of the trehalose or sucrose is 25-200 mM, for example 25 mM, 53 mM or 200 mM; Optionally, the concentration of the nonionic surfactant is 0.01% to 0.1% (w / v), for example, 0.02% (w / v); Optionally, the concentration of methionine in the stabilizer (i) is 2-15 mM, and the concentration of sodium chloride is 50-180 mM; Optionally, the concentration of the phosphate buffer or Tris buffer is 0.1~50mM, for example, 0.1mM, 0.2mM, 0.5mM, 1mM, 2mM, 2.6mM, 3.3mM, 5mM, 10mM or 50mM.

6. The method according to any one of claims 1 to 5, wherein, The composition of the liquid formulation is selected from any of the following: (1) 50 IU / ml~500 IU / ml hyaluronidase or recombinant hyaluronidase, 116 mg / ml cefuroxime sodium, 10 mM phosphate buffer, 145 mM sodium chloride, 10 mM methionine, 0.02% (w / v) polysorbate 20, 1 g / L albumin. (2) 150 IU / ml~500 IU / ml hyaluronidase or recombinant hyaluronidase, 116 mg / ml cefuroxime sodium, 10 mM phosphate buffer, 145 mM sodium chloride, 10 mM methionine, 0.02% (w / v) polysorbate 20, 1 g / L albumin; (3) 50 IU / ml~500 IU / ml hyaluronidase or recombinant hyaluronidase, 116 mg / ml cefuroxime sodium, 2.6~3.3 mM phosphate buffer, 38~48 mM sodium chloride, 2.6~3.3 mM methionine, 0.005% (w / v)~0.007% (w / v) polysorbate 20, 0.26~0.33 g / L albumin; (4) 50 IU / ml~500 IU / ml hyaluronidase or recombinant hyaluronidase, 260 mg / ml ceftazidime, 10 mM phosphate buffer, 145 mM sodium chloride, 10 mM methionine, 0.02% polysorbate 20, 1 g / L albumin. (5) 50 IU / ml to 500 IU / ml hyaluronidase or recombinant hyaluronidase, 260 mg / ml ceftazidime, 2.6 to 3.3 mM phosphate buffer, 145 to 154 mM sodium chloride, 2.6 to 3.3 mM methionine, 0.005% (w / v) to 0.007% (w / v) polysorbate 20, and 0.26 to 0.33 g / L albumin.

7. The method according to any one of claims 1 to 6, wherein, The activity of hyaluronidase or recombinant hyaluronidase in the liquid formulation is maintained for more than 1 hour, for example, 1 to 24 hours or 2 to 12 hours.

8. A combination reagent comprising individually packaged reagent (a) containing an antibiotic, and reagent (b) containing hyaluronidase and / or recombinant hyaluronidase; said reagent (b) further containing albumin; said combination reagent being used to carry out the method according to any one of claims 1 to 7; Optionally, the antibiotic includes cephalosporin antibiotics or β-lactam antibiotics; for example, one or more of ceftazidime, cefuroxime sodium, ceftriaxone sodium, cefazolin sodium, cefoperazone / sulbactam sodium, cefazolin sodium, cefotaxime sodium, or cefoperazone. Optionally, the reagent (b) is a liquid preparation, and the reagent (b) contains hyaluronidase or recombinant hyaluronidase, an enzyme, a buffer, a stabilizer, and a nonionic surfactant; Optionally, the buffer includes phosphate buffer and / or Tris buffer, and the nonionic surfactant includes one or more of polysorbate 20, polysorbate 80, or poloxamer 188. Optionally, the phosphate buffer solution comprises disodium hydrogen phosphate; Optionally, the stabilizer is selected from (i) or (ii): (i) methionine and sodium chloride; (ii) is composed of (i) and (iii), wherein (iii) is trehalose or sucrose; Optionally, the concentration of the buffer is 2~50mM; Optionally, the stabilizer is selected from (i) or (ii): (i) Methionine and sodium chloride; the concentration of said methionine is 2-50 mM, for example 2 mM, 2.6 mM, 3.3 mM, 5 mM, 10 mM or 50 mM; the concentration of said sodium chloride is 25-200 mM, optionally 30 mM-200 mM, for example 30 mM, 130 mM, 145 mM, 154 mM or 180 mM; (ii) is composed of (i) and (iii), wherein (c) is trehalose or sucrose, and the concentration of the trehalose or sucrose is 25-200 mM, for example 25 mM, 53 mM or 200 mM; Optionally, the concentration of the nonionic surfactant is 0.01% to 0.1% (w / v), for example, 0.02% (w / v); Optionally, the concentration of methionine in the stabilizer (i) is 2-15 mM, and the concentration of sodium chloride is 50-180 mM; Optionally, the concentration of the phosphate buffer or Tris buffer is 2 mM, 2.6 mM, 3.3 mM, 5 mM, 10 mM or 50 mM.

9. The combination reagent as described in claim 8, wherein, The combined reagents are selected from any of the following: (A) The reagent (a) is ceftazidime; Reagent (b) is a liquid formulation comprising 250 IU / ml hyaluronidase, 10 mM disodium hydrogen phosphate, 10 mM methionine, 145 mM sodium chloride, 0.02% (w / v) polysorbate 20 and 1 g / L albumin. After dissolving reagent (a) with reagent (b), the concentration of ceftazidime is 260 mg / ml; (B) The reagent (a) mentioned is ceftazidime; Reagent (b) is a liquid formulation comprising 150 IU / ml hyaluronidase, 10 mM disodium hydrogen phosphate, 10 mM methionine, 145 mM sodium chloride, 0.02% (w / v) polysorbate 20 and 1 g / L albumin. After dissolving reagent (a) with reagent (b), the concentration of ceftazidime is 260 mg / ml; (C) The reagent (a) mentioned is ceftazidime; Reagent (b) is a liquid formulation comprising 150 IU / ml hyaluronidase, 10 mM disodium hydrogen phosphate, 10 mM methionine, 130 mM sodium chloride, 0.02% (w / v) polysorbate 20 and 1 g / L albumin. After dissolving reagent (a) with reagent (b), the concentration of ceftazidime is 260 mg / ml; (D) The reagent (a) is cefuroxime sodium; Reagent (b) is a liquid formulation comprising 500 U / ml hyaluronidase, 10 mM disodium hydrogen phosphate, 10 mM methionine, 145 mM sodium chloride, 0.02% (w / v) polysorbate 20 and 1 g / L albumin. After dissolving reagent (a) with reagent (b), the concentration of ceftazidime is 116 mg / ml; (E) The reagent (a) is cefuroxime sodium; Reagent (b) is a liquid formulation comprising 150 U / ml hyaluronidase, 10 mM disodium hydrogen phosphate, 10 mM methionine, 145 mM sodium chloride, 0.02% (w / v) polysorbate 20 and 1 g / L albumin. After dissolving reagent (a) with reagent (b), the concentration of ceftazidime is 116 mg / ml; (F) The reagent (a) mentioned is cefuroxime sodium; Reagent (b) is a liquid formulation comprising 150 U / ml hyaluronidase, 10 mM disodium hydrogen phosphate, 10 mM methionine, 130 mM sodium chloride, 0.02% (w / v) polysorbate 20 and 1 g / L albumin. After dissolving reagent (a) with reagent (b), the concentration of ceftazidime is 116 mg / ml.

10. A method for preparing a liquid formulation, comprising mixing reagent (a) and reagent (b) of the combined reagent of claim 8 or 9 in any order of addition. Optionally, the preparation method includes mixing reagent (a) and reagent (b) in any order of addition, or mixing reagent (a), reagent (b), and the reconstituter in any order; the reconstituter may be physiological saline, glucose solution, and water; Optionally, the composition of the liquid formulation is as described in the method of claim 5 or 6.

11. The use of the combination reagent according to claim 8 or 9, or the liquid formulation prepared by the preparation method according to claim 10, in the preparation of medicaments for treating diseases; The diseases mentioned include infections caused by bacteria, fungi, actinomycetes, mycoplasma, chlamydia, spirochetes, or amoebae, or diseases caused by infections with the aforementioned pathogens; Optionally, the treatment for the disease includes administering to a subject an effective dose of a liquid formulation prepared by the preparation method of claim 10. Optionally, the liquid formulation is a liquid formulation prepared by the preparation method according to claim 10 and left for more than 1 hour; for example, 2 to 12 hours. Optionally, the administration method includes subcutaneous administration, intramuscular administration, or intravenous administration.

12. A packaging containing the combined reagent of claim 8 or 9, or a liquid formulation prepared by the preparation method of claim 10; Optionally, the packaging is used to store or transport the combined reagent of claim 8 or 9, or the liquid formulation prepared by the preparation method of claim 10; Optionally, the packaging is used to formulate or administer a liquid formulation prepared by the preparation method of claim 10.

13. The application of albumin in maintaining the activity of hyaluronidase or recombinant hyaluronidase in liquid formulations, wherein, The liquid formulation contains antibiotics, and hyaluronidase or recombinant hyaluronidase; Optionally, the concentration of the antibiotic is 1~500 mg / ml; Optionally, the enzyme activity of the hyaluronidase or recombinant hyaluronidase is 45 IU / ml to 3,000,000 IU / ml; Optionally, the antibiotic includes cephalosporin antibiotics or β-lactam antibiotics; for example, one or more of ceftazidime, cefuroxime sodium, ceftriaxone sodium, cefazolin sodium, cefoperazone / sulbactam sodium, cefazolin sodium, cefotaxime sodium, or cefoperazone.

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