WO2024001877A1 - Polypeptide drug solution preparation and method for preparing same - Google Patents

Polypeptide drug solution preparation and method for preparing same Download PDF

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WO2024001877A1
WO2024001877A1 PCT/CN2023/101441 CN2023101441W WO2024001877A1 WO 2024001877 A1 WO2024001877 A1 WO 2024001877A1 CN 2023101441 W CN2023101441 W CN 2023101441W WO 2024001877 A1 WO2024001877 A1 WO 2024001877A1
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polypeptide
pharmaceutical preparation
polypeptide pharmaceutical
preparation according
preparation
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PCT/CN2023/101441
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French (fr)
Chinese (zh)
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虞振静
余东梁
王红昀
刘雯君
陈健韬
张伟明
唐洋明
余品香
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深圳翰宇药业股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/02—Peptides of undefined number of amino acids; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/02—Inorganic compounds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/14—Esters of carboxylic acids, e.g. fatty acid monoglycerides, medium-chain triglycerides, parabens or PEG fatty acid esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/24—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing atoms other than carbon, hydrogen, oxygen, halogen, nitrogen or sulfur, e.g. cyclomethicone or phospholipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A61K47/40—Cyclodextrins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/44—Oils, fats or waxes according to two or more groups of A61K47/02-A61K47/42; Natural or modified natural oils, fats or waxes, e.g. castor oil, polyethoxylated castor oil, montan wax, lignite, shellac, rosin, beeswax or lanolin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses

Definitions

  • the invention relates to the technical field of pharmaceutical preparations and belongs to a polypeptide pharmaceutical liquid preparation and a preparation method thereof.
  • HY3000 peptide drugs can be used to treat and prevent new coronavirus pneumonia (Covid-19).
  • HY3000 is unstable in the liquid state and easily aggregates to form precipitates or gels. It can only be stored in solid form and must be prepared as a solution before use, which increases the risk to patients.
  • the polypeptide drug liquid preparation includes the following components in terms of mass percentage:
  • polypeptide pharmaceutical preparation includes the following components in terms of mass percentage:
  • the surfactant is selected from phospholipids, polysorbates, sorbitol fatty acid esters, poloxamers, One or more polyethylene glycol esters.
  • the solubilizing agent is selected from one or more of cyclodextrin, cyclodextrin derivatives, chitosan and chitosan derivatives.
  • the osmotic pressure regulator is selected from one or more of sodium chloride, phosphate, citrate, glycerol, mannitol, sorbitol, glucose, fructose, lactose and trehalose.
  • the pH adjuster is selected from one or more of citric acid and citrate, acetic acid and acetate, phosphoric acid and phosphate, sodium hydroxide, hydrochloric acid, lactic acid and maleic acid.
  • the solvent is an alcoholic organic solvent or water.
  • it also includes a preservative and/or bacteriostatic agent, the preservative or the bacteriostatic agent being selected from benzalkonium chloride, hydroxyphenyl esters, chlorobutanol, benzyl alcohol and phenethyl alcohol. one or more.
  • a preservative and/or bacteriostatic agent selected from benzalkonium chloride, hydroxyphenyl esters, chlorobutanol, benzyl alcohol and phenethyl alcohol. one or more.
  • the preservative is selected from one or more of chlorobutanol and benzyl alcohol
  • the bacteriostatic agent is selected from one or more of benzalkonium chloride and phenethyl alcohol.
  • Another object of the present invention is to provide a method for preparing a polypeptide pharmaceutical preparation.
  • the preparation method of the polypeptide pharmaceutical preparation includes dissolving HY3000 as described in any one of the above, the surfactant and/or solubilizer, the osmotic pressure regulator and the preservative in 10% to 95% of the solvent in, stir until dissolved, add the pH adjuster to make the pH value of the solution between 6 and 9, add the remaining solvents, mix, filter, and fill into a spray device to obtain the polypeptide pharmaceutical preparation.
  • the polypeptide pharmaceutical preparation provided by the invention improves the stability of HY3000 in liquid and reduces the risk of use for patients.
  • Figure 1 is a protein fibrillation test chart provided in Example 2;
  • Figure 2 is a protein fibrillation test chart provided in Example 3.
  • Figure 3 is a protein fibrillation test chart provided in Example 4.
  • Figure 4 shows the inhibition rate at different concentrations of each polypeptide added with the original strain pseudovirus provided in the application example
  • Figure 5 shows the inhibition rate at different concentrations of each polypeptide added with the mutant strain pseudovirus provided in the application example.
  • HY3000 involved in the present invention is a polypeptide or a derivative thereof of the sequence described in any one of the following (1)-(8),
  • a polypeptide comprising an amino acid sequence in which one or more amino acid residues are substituted, deleted, added or inserted in any one of the amino acid sequences of the polypeptides (1) to (6), and the polypeptide has a specificity against coronavirus inhibitory activity; or
  • a polypeptide comprising at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 93%, 94% of the same amino acid sequence as any one of the polypeptides (1) to (6). % or 95% identical amino acid sequence, and the polypeptide has inhibitory activity against coronavirus;
  • sequence of the polypeptide is SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.12, SEQ ID NO.13 or SEQ ID NO.14.
  • the polypeptide derivative is a derivative mentioned in the patent number "202210333422.5" previously applied by the applicant.
  • Polypeptide drug preparation prescription composition :
  • polypeptide pharmaceutical preparation prescription consists of:
  • Solubilizer and/or surfactant dodecylphosphocholine 0.1% ⁇ 20%;
  • Osmotic pressure regulator sorbitol 0.5 ⁇ 10%
  • Preservative benzalkonium chloride 0.001% ⁇ 0.5%
  • pH adjuster citric acid and/or sodium hydroxide 0.001% ⁇ 0.2%;
  • the new coronavirus pseudovirus used in this application was obtained in the following ways:
  • Cell preparation Plate HEK293T cells in a 10cm cell culture dish so that the cell confluence density reaches 80% the next day.
  • Transfection Take the expression plasmids pCAGGS-SARS-CoV-2-WT-S-del18 and pCAGGS-BF.7-S-del18 of the new coronavirus outer membrane S protein obtained in the above step 2) and press them with PEI respectively. Mix evenly at a ratio of 1:3 and transfect HEK293T cells.
  • the transfection specification is 30 ⁇ g plasmid/10cm cell culture dish. Change the culture medium (DMEM medium containing 10% FBS) every 4-6 hours and culture at 37°C for 24 hours.
  • Toxin addition Add the lentiviral vector G*VSV-delG (purchased from Wuhan Shumi Brain Science and Technology Co., Ltd.) to the above-transfected HEK293T cells, incubate at 37°C for 2 hours, and change the culture medium (DMEM containing 10% FBS) base), and add VSV-G antibody (hybridoma cells expressing this antibody were purchased from ATCC cell bank), and continue to culture in the incubator for 30 h.
  • G*VSV-delG purchased from Wuhan Shumi Brain Science and Technology Co., Ltd.
  • Collect the virus Collect the supernatant and centrifuge it at 3000 rpm for 10 minutes, filter it through a 0.45 ⁇ m sterile filter in a clean workbench, remove cell debris, and obtain the original SARS-CoV-2 strain (WT) and the mutant strain Omicron (BF.7). ) pseudoviruses were aliquoted separately and stored in a -80°C refrigerator.
  • Kolliphor HS15 polyethylene glycol-12-hydroxystearate
  • KolliphorELP polyoxyethylene (35) castor oil
  • Tween 80 polyoxyethylene sorbitan monooleate, referred to as polysorbate-80
  • Tween 20 polysorbate-20
  • poloxamer 188 ⁇ -hydrogen- ⁇ -hydroxy poly(oxygen)
  • hydroxypropyl ⁇ -cyclodextrin When hydroxypropyl ⁇ -cyclodextrin is used as a stabilizer, the clear and transparent liquid properties can be better maintained. However, hydroxypropyl ⁇ -cyclodextrin can significantly accelerate the degradation of the raw material and significantly increase the total impurities.
  • DPC dodecylphosphocholine
  • the polypeptide drug preparations with prescription numbers 21-27 were prepared as samples. Different samples contain different amounts of dodecylphosphocholine.
  • Amyloid fibrillation The amyloid fibrillation and aggregation of each prescription example was detected through thioflavin (ThT).
  • the total impurity growth of each sample is calculated based on the impurity content of the same sample on the first day.
  • the growth rate of total impurities in all products from prescriptions 21 to 27 during storage was less than 10%, and all products passed the test.
  • HY3000 maintains good stability in aqueous solution, especially when the mass fraction of dodecylphosphocholine in the reagent is 0.5%-1.5%, HY3000 has excellent stability in aqueous solution
  • the polypeptide pharmaceutical preparations with prescription numbers 28-34 were prepared as samples according to the prescription composition of the polypeptide pharmaceutical preparations.
  • the pH values of polypeptide pharmaceutical preparations with different prescription numbers are different.
  • Amyloid fibrillation The amyloid fibrillation and aggregation of each prescription sample was detected through ThT.
  • the total impurity growth of each sample is calculated based on the impurity content of the same sample on the first day. It can be seen from the table above that the growth rate of total impurities in all products from prescriptions 28 to 34 during storage was less than 10%, and the tests were all qualified. The higher the pH value, the greater the growth rate of total impurities.
  • the polypeptide drug preparations with prescription numbers 35-41 were prepared as samples. Different samples have different concentrations of raw materials.
  • Amyloid fibrillation Detect the amyloid fibrillation and aggregation of each prescription sample through ThT.
  • the total impurity growth of each sample is calculated based on the impurity content of the same sample on the first day. It can be seen from the above table that the growth rate of total impurities in all products from prescription 35 to prescription 42 during storage was less than 10%, and all products passed the test.
  • the polypeptide pharmaceutical preparation provided by the present invention improves the stability of HY3000 in liquid and reduces the risk of use for patients.
  • Preparation of polypeptide solution Use pure water, CZ430 excipient solution, CZ442 excipient solution, CZ443 excipient solution, CZ444 excipient solution and CZ445 excipient solution to dissolve HY3000 polypeptide to prepare HY3000 polypeptide solution and preparations CZ430, CZ442, CZ443, CZ444, CZ445 mother liquor, and then The stock solution was diluted to 20 ⁇ M with DMEM medium containing 10% FBS as the stock solution for further gradient dilution.
  • the concentration of the polypeptide in the preparation CZ430 mother solution is 5 mg/mL
  • the concentration of dodecylphosphocholine is 5 mg/mL
  • the concentration of sorbitol is 40 mg/mL
  • the concentration of phenethyl alcohol is 9 mg/mL.
  • the concentration of the polypeptide in the mother solution of preparation CZ442 is 5 mg/mL
  • the concentration of hydroxypropyl betacyclodextrin is 100 mg/mL
  • the concentration of sorbitol is 50 mg/mL
  • the concentration of benzalkonium chloride is 0.2 mg/mL.
  • the concentration of the polypeptide in the mother solution of preparation CZ443 is 5 mg/mL, the concentration of hydroxypropyl betacyclodextrin is 100 mg/mL, the concentration of sorbitol is 50 mg/mL, and the concentration of phenethyl alcohol is 9 mg/mL.
  • the concentration of the polypeptide in the mother solution of preparation CZ444 is 5 mg/mL, the concentration of dodecylphosphocholine is 5 mg/mL, the concentration of sorbitol is 50 mg/mL, and the concentration of benzalkonium chloride is 0.2 mg/mL.
  • the concentration of polypeptide, dodecylphosphocholine, and sorbitol in the mother solution of preparation CZ445 is 5 mg/mL, 5 mg/mL.
  • Preparation of polypeptide gradient dilutions Use DMEM medium containing 10% FBS to dilute the above 20 ⁇ M polypeptide stock solution by 2 times, and dilute a total of 9 gradients (respectively 10, 5, 2.5, 1.25, 0.625, 0.3125 , 0.156, 0.078, 0.039 ⁇ M), each gradient has 3 duplicate wells, 50 ⁇ L per well.
  • Determination of the dosage of pseudovirus Quantify the original SARS-CoV-2 strain or mutant strain Omicron (BF.7) pseudovirus stock solution and a series of dilutions on HEK293ThACE2 cells, and use the dilution when 1000 FFU appears as Dosage of virus when evaluating the inhibitory effect of polypeptides (dilution factor ranges from 6 to 20 times).
  • step c Carefully discard the supernatant of the 96-well cell culture plate in step a, add the polypeptide-pseudovirus mixture (100 ⁇ L/well) in step b, and continue culturing in the incubator for 15h-24h.
  • each prescription preparation has a good inhibitory effect on both the original strain and the mutated strain (110.15nM ⁇ EC 50 ⁇ 379.35nM).
  • the inhibitory effect of each prescription preparation on the SARS-CoV-2 virus has not changed significantly, but the addition of the excipient solution can improve the stability of HY3000, and has antibacterial efficacy and drug potential.
  • each prescription preparation has a good inhibitory effect on both the original strain and the mutated strain.

Abstract

The present application relates to the technical field of pharmaceutical preparations and provides a polypeptide drug liquid preparation and a method for preparing same. The polypeptide drug liquid preparation comprises the following components in percentage by mass: 0.01-5% of HY3000; 0.1-20% of a surfactant and/or a solubility enhancer; 0.5-10% of an osmotic pressure regulator; 0.001-0.2% of a pH regulator; and 64.8-99.4% of a solvent. The provided polypeptide pharmaceutical preparation enhances the stability of HY3000 in a liquid, and use risks borne by a patient are reduced.

Description

一种多肽药物溶液制剂及其制备方法A polypeptide drug solution preparation and its preparation method 技术领域Technical field
本发明涉及药物制剂技术领域，属于一种多肽药物液体制剂及其制备方法。The invention relates to the technical field of pharmaceutical preparations and belongs to a polypeptide pharmaceutical liquid preparation and a preparation method thereof.
背景技术Background technique
在新冠肺炎大流行的大背景下，非注射途径的预防和治疗药物显示出极大的优势和极度紧张的临床需求。鼻喷雾剂便于给药，患者顺应性高，安全风险小于注射给药，同时鼻腔给药接近新冠病毒入侵的人体部位，具有显著优势。In the context of the COVID-19 pandemic, non-injection preventive and therapeutic drugs have shown great advantages and extremely tight clinical needs. Nasal spray is easy to administer, has high patient compliance, and has lower safety risks than injection administration. At the same time, nasal administration is close to the part of the human body where the new coronavirus invades, which has significant advantages.
HY3000多肽药物可用于治疗和预防新冠病毒肺炎(Covid-19)。HY3000在液体状态下不稳定，易聚集形成沉淀或凝胶，只能以固体形式储存，在临用前配制为溶液形式给药，这增加了患者使用的风险。HY3000 peptide drugs can be used to treat and prevent new coronavirus pneumonia (Covid-19). HY3000 is unstable in the liquid state and easily aggregates to form precipitates or gels. It can only be stored in solid form and must be prepared as a solution before use, which increases the risk to patients.
因此，亟需一种多肽药物液体制剂及其制备方法，以降低上述使用风险。Therefore, there is an urgent need for a polypeptide drug liquid preparation and a preparation method thereof to reduce the above-mentioned use risks.
发明内容Contents of the invention
为了降低上述使用风险，本发明提供一种多肽药物液体制剂。该多肽药物液体制剂包括按百分质量计的以下组分：
In order to reduce the above-mentioned use risks, the present invention provides a polypeptide pharmaceutical liquid preparation. The polypeptide drug liquid preparation includes the following components in terms of mass percentage:
可选地，所述的多肽药物制剂包括按百分质量计的以下组分：
Optionally, the polypeptide pharmaceutical preparation includes the following components in terms of mass percentage:
可选地，所述表面活性剂选自磷脂类、聚山梨酯类、山梨醇脂肪酸酯类、泊洛沙姆类、 聚乙二醇酯类中的一种或多种。Optionally, the surfactant is selected from phospholipids, polysorbates, sorbitol fatty acid esters, poloxamers, One or more polyethylene glycol esters.
可选地，所述增溶剂选自环糊精、环糊精类衍生物、壳聚糖和壳聚糖衍生物中的一种或多种。Optionally, the solubilizing agent is selected from one or more of cyclodextrin, cyclodextrin derivatives, chitosan and chitosan derivatives.
可选地，所述渗透压调节剂选自氯化钠、磷酸盐、枸橼酸盐、甘油、甘露醇、山梨醇、葡萄糖、果糖、乳糖和海藻糖中的一种或多种。Optionally, the osmotic pressure regulator is selected from one or more of sodium chloride, phosphate, citrate, glycerol, mannitol, sorbitol, glucose, fructose, lactose and trehalose.
可选地，所述pH调节剂选自枸橼酸及枸橼酸盐、醋酸及醋酸盐、磷酸及磷酸盐、氢氧化钠、盐酸、乳酸和马来酸中的一种或多种。Optionally, the pH adjuster is selected from one or more of citric acid and citrate, acetic acid and acetate, phosphoric acid and phosphate, sodium hydroxide, hydrochloric acid, lactic acid and maleic acid.
可选地，所述溶剂为醇类有机溶剂或水。Optionally, the solvent is an alcoholic organic solvent or water.
可选地，还包括防腐剂和/或抑菌剂，所述防腐剂或所述抑菌剂选自苯扎氯铵、羟苯酯类、三氯叔丁醇、苯甲醇和苯乙醇中的一种或多种。Optionally, it also includes a preservative and/or bacteriostatic agent, the preservative or the bacteriostatic agent being selected from benzalkonium chloride, hydroxyphenyl esters, chlorobutanol, benzyl alcohol and phenethyl alcohol. one or more.
可选地，所述防腐剂选自三氯叔丁醇和苯甲醇中的一种或多种，所述抑菌剂选自苯扎氯铵和苯乙醇中一种或多种。Optionally, the preservative is selected from one or more of chlorobutanol and benzyl alcohol, and the bacteriostatic agent is selected from one or more of benzalkonium chloride and phenethyl alcohol.
本发明的另一目的在于提供一种多肽药物制剂的制备方法。该多肽药物制剂的制备方法包括将如上任一项所述HY3000、所述表面活性剂和/或增溶剂、所述渗透压调节剂和所述防腐剂溶于10％～95％所述的溶剂中，搅拌至溶解，加入所述pH调节剂使溶液的PH值位于6～9，加入其余所述溶剂，混匀，过滤，灌装至喷雾装置中，即得所述多肽药物制剂。Another object of the present invention is to provide a method for preparing a polypeptide pharmaceutical preparation. The preparation method of the polypeptide pharmaceutical preparation includes dissolving HY3000 as described in any one of the above, the surfactant and/or solubilizer, the osmotic pressure regulator and the preservative in 10% to 95% of the solvent in, stir until dissolved, add the pH adjuster to make the pH value of the solution between 6 and 9, add the remaining solvents, mix, filter, and fill into a spray device to obtain the polypeptide pharmaceutical preparation.
本发明所提供的多肽药物制剂提高了HY3000在液体中的稳定性，降低了患者的使用风险。The polypeptide pharmaceutical preparation provided by the invention improves the stability of HY3000 in liquid and reduces the risk of use for patients.
附图说明Description of drawings
图1为实施例2提供的蛋白纤维化测试图；Figure 1 is a protein fibrillation test chart provided in Example 2;
图2为实施例3提供的蛋白纤维化测试图；Figure 2 is a protein fibrillation test chart provided in Example 3;
图3为实施例4提供的蛋白纤维化测试图；Figure 3 is a protein fibrillation test chart provided in Example 4;
图4为应用例提供的添加原始毒株假病毒的各多肽在不同浓度下的抑制率；Figure 4 shows the inhibition rate at different concentrations of each polypeptide added with the original strain pseudovirus provided in the application example;
图5为应用例提供的添加变异毒株假病毒的各多肽在不同浓度下的抑制率。Figure 5 shows the inhibition rate at different concentrations of each polypeptide added with the mutant strain pseudovirus provided in the application example.
具体实施方式Detailed ways
为了使本发明的上述目的、特征和优点能够更加明显易懂，下面结合附图对本发明的具体实施方式做详细的说明，但不能理解为对本发明的可实施范围的限定。 In order to make the above objects, features and advantages of the present invention more obvious and understandable, the specific embodiments of the present invention are described in detail below with reference to the accompanying drawings, but this should not be understood as limiting the implementable scope of the present invention.
本发明中所涉及的HY3000为以下(1)-(8)任一所述序列的多肽或其衍生物，HY3000 involved in the present invention is a polypeptide or a derivative thereof of the sequence described in any one of the following (1)-(8),
(1)多肽，其包含以SEQ ID NO.1所示的氨基酸序列；(1) Polypeptide, which contains the amino acid sequence shown in SEQ ID NO.1;
(2)多肽，其包含以SEQ ID NO.2所示的氨基酸序列；(2) Polypeptide, which contains the amino acid sequence shown in SEQ ID NO.2;
(3)多肽，其包含以SEQ ID NO.3所示的氨基酸序列；(3) Polypeptide, which contains the amino acid sequence shown in SEQ ID NO.3;
(4)多肽，其包含以SEQ ID NO.12所示的氨基酸序列；(4) Polypeptide, which contains the amino acid sequence shown in SEQ ID NO. 12;
(5)多肽，其包含以SEQ ID NO.13所示的氨基酸序列；(5) Polypeptide, which contains the amino acid sequence shown in SEQ ID NO. 13;
(6)多肽，其包含以SEQ ID NO.14所示的氨基酸序列；(6) Polypeptide, which contains the amino acid sequence shown in SEQ ID NO. 14;
(7)多肽，其包含在所述多肽(1)-(6)任一的氨基酸序列中取代、缺失、添加或插入1个或多个氨基酸残基的氨基酸序列，且该多肽具有针对冠状病毒的抑制活性；或(7) A polypeptide comprising an amino acid sequence in which one or more amino acid residues are substituted, deleted, added or inserted in any one of the amino acid sequences of the polypeptides (1) to (6), and the polypeptide has a specificity against coronavirus inhibitory activity; or
(8)多肽，其包含与所述多肽(1)-(6)任一的氨基酸序列具有至少60％、65％、70％、75％、80％、85％、90％、93％、94％或95％同一性的氨基酸序列，且该多肽具有针对冠状病毒的抑制活性；(8) A polypeptide comprising at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 93%, 94% of the same amino acid sequence as any one of the polypeptides (1) to (6). % or 95% identical amino acid sequence, and the polypeptide has inhibitory activity against coronavirus;
优选地，所述多肽的序列为SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.12、SEQ ID NO.13或SEQ ID NO.14。Preferably, the sequence of the polypeptide is SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.12, SEQ ID NO.13 or SEQ ID NO.14.
优选的，所述多肽衍生物为本申请人在先申请的专利号为“202210333422.5”的专利中所提及的衍生物。Preferably, the polypeptide derivative is a derivative mentioned in the patent number "202210333422.5" previously applied by the applicant.
多肽药物制剂处方组成：
Polypeptide drug preparation prescription composition:
优选地，多肽药物制剂处方组成：Preferably, the polypeptide pharmaceutical preparation prescription consists of:
HY3000：0.01％～5％；HY3000: 0.01%～5%;
增溶剂和/或表面活性剂：十二烷基磷酸胆碱0.1％～20％；Solubilizer and/or surfactant: dodecylphosphocholine 0.1% ~ 20%;
渗透压调节剂：山梨醇0.5～10％；Osmotic pressure regulator: sorbitol 0.5~10%;
防腐剂：苯扎氯铵0.001％～0.5％；Preservative: benzalkonium chloride 0.001% ~ 0.5%;
pH调节剂：枸橼酸和/或氢氧化钠0.001％～0.2％；pH adjuster: citric acid and/or sodium hydroxide 0.001% ~ 0.2%;
溶剂：64.3％-99.4％。Solvent: 64.3%-99.4%.
多肽药物制剂制备方法： Preparation method of polypeptide drug preparation:
将HY3000、十二烷基磷酸胆碱、山梨醇、苯扎氯铵溶于处方量10％～95％的水中，搅拌至溶解，然后加入枸橼酸和/或氢氧化钠使溶液pH值至6～9，加水至100％，混匀，过滤，灌装至喷雾装置中，即得多肽药物制剂。Dissolve HY3000, dodecylphosphocholine, sorbitol, and benzalkonium chloride in water with 10% to 95% of the prescription amount, stir until dissolved, then add citric acid and/or sodium hydroxide to bring the solution pH to 6 to 9, add water to 100%, mix, filter, and fill into a spray device to obtain a polypeptide pharmaceutical preparation.
按照上述多肽药物制剂处方组成和多肽药物制剂制备方法配置实施例1-4的样品和空白样品。The samples and blank samples of Examples 1-4 were prepared according to the above polypeptide pharmaceutical preparation prescription composition and polypeptide pharmaceutical preparation preparation method.
本申请所用到的新型冠状病毒假病毒由如下方式获得：The new coronavirus pseudovirus used in this application was obtained in the following ways:
构建新型冠状病毒外膜S蛋白的质粒Plasmid for constructing novel coronavirus outer membrane S protein
1)将编码SARS-CoV-2原始毒株(WT)及变异毒株Omicron(BF.7)的S蛋白的后18位氨基酸的核苷酸去掉，分别得到核苷酸序列SARS-CoV-2-WT-S-del18和BF.7-S-del18，(其序列分别如SEQ ID NO:01-02所示)，由苏州金唯智公司进行合成。1) Remove the nucleotides encoding the last 18 amino acids of the S protein of the original strain (WT) and variant strain Omicron (BF.7) of SARS-CoV-2 to obtain the nucleotide sequences of SARS-CoV-2 respectively. -WT-S-del18 and BF.7-S-del18, (their sequences are shown in SEQ ID NO:01-02 respectively), were synthesized by Suzhou Jinweizhi Company.
2)分别将1)中获得的各核苷酸序列克隆到pCAGGS表达载体上，得到表达质粒pCAGGS-SARS-CoV-2-WT-S-del18、pCAGGS-BF.7-S-del18。2) Clone each nucleotide sequence obtained in 1) into the pCAGGS expression vector to obtain expression plasmids pCAGGS-SARS-CoV-2-WT-S-del18 and pCAGGS-BF.7-S-del18.
SARS-CoV-2原始毒株及变异毒株假病毒的包装Packaging of SARS-CoV-2 original strain and mutated strain pseudovirus
a.细胞准备：在10cm细胞培养皿中铺HEK293T细胞，使第二天细胞汇合密度至80％。a. Cell preparation: Plate HEK293T cells in a 10cm cell culture dish so that the cell confluence density reaches 80% the next day.
b.转染：取上述步骤2)中获得的新型冠状病毒外膜S蛋白的表达质粒pCAGGS-SARS-CoV-2-WT-S-del18、pCAGGS-BF.7-S-del18各自与PEI按1:3比例混匀后转染HEK293T细胞，转染规格为30μg质粒/10cm细胞培养皿，4-6h换一次培养液(含10％FBS的DMEM培养基)，37℃培养24h。b. Transfection: Take the expression plasmids pCAGGS-SARS-CoV-2-WT-S-del18 and pCAGGS-BF.7-S-del18 of the new coronavirus outer membrane S protein obtained in the above step 2) and press them with PEI respectively. Mix evenly at a ratio of 1:3 and transfect HEK293T cells. The transfection specification is 30 μg plasmid/10cm cell culture dish. Change the culture medium (DMEM medium containing 10% FBS) every 4-6 hours and culture at 37°C for 24 hours.
c.加毒：将慢病毒载体G*VSV-delG(购自武汉枢密脑科学技术有限公司)加入上述转染后的HEK293T细胞，37℃孵育2h，换培养液(含10％FBS的DMEM培养基)，并加入VSV-G抗体(表达该抗体杂交瘤细胞购自ATCC细胞库)，在培养箱中继续培养30h。c. Toxin addition: Add the lentiviral vector G*VSV-delG (purchased from Wuhan Shumi Brain Science and Technology Co., Ltd.) to the above-transfected HEK293T cells, incubate at 37°C for 2 hours, and change the culture medium (DMEM containing 10% FBS) base), and add VSV-G antibody (hybridoma cells expressing this antibody were purchased from ATCC cell bank), and continue to culture in the incubator for 30 h.
d.收毒：收上清3000rpm离心10min,在超净工作台中经0.45μm无菌滤器过滤，去除细胞碎片，得到SARS-CoV-2原始毒株(WT)及变异毒株Omicron(BF.7)的假病毒，各自进行分装，-80℃冰箱冻存。d. Collect the virus: Collect the supernatant and centrifuge it at 3000 rpm for 10 minutes, filter it through a 0.45 μm sterile filter in a clean workbench, remove cell debris, and obtain the original SARS-CoV-2 strain (WT) and the mutant strain Omicron (BF.7). ) pseudoviruses were aliquoted separately and stored in a -80°C refrigerator.
实施例1Example 1
1.1配置样品和空白样品1.1 Configure samples and blank samples
按照多肽药物制剂处方组成配置处方号为2-20的多肽药物制剂为样品，不同样品的增溶剂和/或稳定性剂的种类或用量不同，配置处方号为1的多肽药物制剂为空白样品，其中含增溶剂和/或稳定性剂的量为0。 Configure polypeptide pharmaceutical preparations with prescription numbers 2-20 as samples according to the prescription composition of the polypeptide pharmaceutical preparations. Different samples have different types or dosages of solubilizers and/or stabilizers. Configure the polypeptide pharmaceutical preparation with prescription number 1 as a blank sample. The amount of solubilizing agent and/or stabilizing agent is 0.
1.1.1性状：取储存温度为40℃和25℃第1、5、10、23、30天的样品和空白样品，观察其性状。1.1.1 Properties: Take samples and blank samples stored at 40°C and 25°C for the 1st, 5th, 10th, 23rd and 30th days and observe their properties.
1.1.2有关物质(纯度)：取储存温度为25℃第23天的样品和空白样品，采用高效液相色谱法HPLC检测杂质的生成长情况。1.1.2 Related substances (purity): Take the samples and blank samples stored at 25°C on the 23rd day, and use high-performance liquid chromatography (HPLC) to detect the formation and growth of impurities.
1.2测试结果如下表所示：
1.2 The test results are shown in the following table:
由上表可知：考察乙醇、丙二醇、甘油、聚乙二醇等常用溶剂作为增溶剂时，所制备的样品在40℃放置第5天后，样品溶液仍然出现聚集析出现象。It can be seen from the above table that when the commonly used solvents such as ethanol, propylene glycol, glycerin, and polyethylene glycol are used as solubilizers, the sample solution still exhibits aggregation and precipitation after the prepared sample is placed at 40°C for 5 days.
考察Kolliphor HS15(聚乙二醇-12-羟基硬脂酸酯)、KolliphorELP(聚氧乙烯(35)蓖麻油)、 吐温80(聚氧乙烯脱水山梨醇单油酸酯，简称聚山梨酯-80)、吐温20(聚山梨酯-20)、泊洛沙姆188(α-氢-ω-羟基聚(氧乙稀)a-聚(氧丙烯)b-聚(氧乙稀))等常用表面活性剂作为稳定剂时，原料药的聚集析出问题有所改善。Examine Kolliphor HS15 (polyethylene glycol-12-hydroxystearate), KolliphorELP (polyoxyethylene (35) castor oil), Tween 80 (polyoxyethylene sorbitan monooleate, referred to as polysorbate-80), Tween 20 (polysorbate-20), poloxamer 188 (α-hydrogen-ω-hydroxy poly(oxygen) When commonly used surfactants such as ethylene), a-poly(oxypropylene), b-poly(oxyethylene)) are used as stabilizers, the problem of aggregation and precipitation of raw materials has been improved.
采用羟丙基β环糊精作为稳定剂时，可较好的维持澄清透明的药液性状，但羟基倍他环糊精可显著加快原料药的降解，使总杂质显著增加。When hydroxypropyl β-cyclodextrin is used as a stabilizer, the clear and transparent liquid properties can be better maintained. However, hydroxypropyl β-cyclodextrin can significantly accelerate the degradation of the raw material and significantly increase the total impurities.
采用十二烷基磷酸胆碱(DPC)作为稳定剂时，可使药液的性状较好维持澄清透明，且在已考察的方案中，使用该物料作稳定剂的样品化学稳定性表现最好。When dodecylphosphocholine (DPC) is used as a stabilizer, the properties of the liquid can be better maintained to be clear and transparent. Among the solutions that have been investigated, the chemical stability of the sample using this material as a stabilizer is the best. .
实施例2Example 2
2.1配置样品2.1 Configuration sample
按照多肽药物制剂处方组成配置处方号为21-27的多肽药物制剂为样品，不同样品含有十二烷基磷酸胆碱量不同。According to the prescription composition of the polypeptide drug preparation, the polypeptide drug preparations with prescription numbers 21-27 were prepared as samples. Different samples contain different amounts of dodecylphosphocholine.
2.1.1实施例的成分如下表所示：
2.1.1 The ingredients of the embodiment are as shown in the following table:
2.1.2性状：取储存第1、5、10、20、30天的样品，观察其性状。2.1.2 Properties: Take samples stored for 1, 5, 10, 20 and 30 days and observe their properties.
2.1.3淀粉样蛋白纤维化：通过硫磺素(ThT)检测各处方实施例的淀粉样蛋白纤维化聚集情况。2.1.3 Amyloid fibrillation: The amyloid fibrillation and aggregation of each prescription example was detected through thioflavin (ThT).
2.1.4有关物质(总杂质增长量)：取储存第1、10、20、30天的样品，采用高效液相色谱法HPLC检测杂质增长量。2.1.4 Related substances (total impurity growth): Take samples stored for 1, 10, 20, and 30 days, and use high-performance liquid chromatography (HPLC) to detect the impurity growth.
2.2稳定性检测结果2.2 Stability test results
2.2.1性状

2.2.1 Characteristics

由上表可知，储存期间处方23～处方26所有产品均为无色澄清透明，性状稳定，但处方21、处方22、处方27出现可见异物或浑浊，说明十二烷基磷酸胆碱的用量对HY3000多肽的稳定性产生影响。As can be seen from the table above, during storage, all products from prescriptions 23 to 26 were colorless, clear and transparent, with stable properties. However, visible foreign matter or turbidity appeared in prescriptions 21, 22, and 27, indicating that the dosage of dodecylphosphocholine is Impact on the stability of HY3000 polypeptide.
2.2.2淀粉样蛋白纤维化2.2.2 Amyloid Fibrosis
参阅说明书图1可知，随十二烷基磷酸胆碱浓度的增大，实施例在ThT中的logtime(即样品在仪器上维持不发生淀粉样蛋白纤维化的时间，若时间越长，说明样品稳定性越好)越大，说明十二烷基磷酸胆碱对HY3000多肽的淀粉样蛋白纤维化有较明显的影响，可提高HY3000多肽的稳定性。Referring to Figure 1 of the specification, it can be seen that as the concentration of dodecylphosphocholine increases, the logtime of the example in ThT (that is, the time the sample maintains on the instrument without amyloid fibrillation), if the time is longer, it means that the sample (the better the stability), the larger it is, indicating that dodecylphosphocholine has a more obvious effect on the amyloid fibrillation of HY3000 polypeptide and can improve the stability of HY3000 polypeptide.
2.2.3有关物质(总杂质增长量)
2.2.3 Related substances (increased amount of total impurities)
上表中，各样品的总杂质增长量均是基于第1天相同样品的杂质含量计算得出。由上表可知，储存期间处方21～处方27所有产品的总杂质增长幅度均小于10％，检测均合格。In the above table, the total impurity growth of each sample is calculated based on the impurity content of the same sample on the first day. As can be seen from the table above, the growth rate of total impurities in all products from prescriptions 21 to 27 during storage was less than 10%, and all products passed the test.
综合2.2.1～2.2.3稳定性各项检测结果，发现十二烷基磷酸胆碱的用量对HY3000溶液稳定性至关重要，在本实施例提供的十二烷基磷酸胆碱用量下，HY3000在水溶液中保持良好的稳定性，尤其是十二烷基磷酸胆碱在试剂中的质量分数为0.5％-1.5％的情况下，HY3000在水溶液中具有优异的稳定性Based on the stability test results from 2.2.1 to 2.2.3, it was found that the dosage of dodecylphosphocholine is crucial to the stability of HY3000 solution. Under the dosage of dodecylphosphocholine provided in this example, HY3000 maintains good stability in aqueous solution, especially when the mass fraction of dodecylphosphocholine in the reagent is 0.5%-1.5%, HY3000 has excellent stability in aqueous solution
实施例3Example 3
3.1、配置样品3.1. Configure samples
按照多肽药物制剂处方组成配置处方号为28-34的多肽药物制剂为样品，不同处方号的多肽药物制剂PH值不同。The polypeptide pharmaceutical preparations with prescription numbers 28-34 were prepared as samples according to the prescription composition of the polypeptide pharmaceutical preparations. The pH values of polypeptide pharmaceutical preparations with different prescription numbers are different.
3.1.1制剂pH值考察设计如下表：

3.1.1 The pH value investigation design of the preparation is as follows:

3.1.2性状：取储存第1、5、10、20、30天的样品，观察其性状。3.1.2 Properties: Take samples stored for 1, 5, 10, 20 and 30 days and observe their properties.
3.1.3淀粉样蛋白纤维化：通过ThT检测各处方样品的淀粉样蛋白纤维化聚集情况。3.1.3 Amyloid fibrillation: The amyloid fibrillation and aggregation of each prescription sample was detected through ThT.
3.1.4有关物质(总杂质增长量)：取储存第1、10、20、30天的样品，采用高效液相色谱法HPLC检测有关物质。3.1.4 Related substances (increased amount of total impurities): Take samples stored on the 1st, 10th, 20th, and 30th day, and use high-performance liquid chromatography (HPLC) to detect related substances.
3.2稳定性检测结果3.2 Stability test results
3.2.1性状
3.2.1 Characteristics
由上表可知，储存期间处方30～处方34所有样品均为无色澄清透明，性状稳定，但处方28、处方29出现可见异物或析出沉淀，说明pH值对HY3000多肽的稳定性产生影响，在pH6～9范围内，pH值越高，药液性状越稳定。As can be seen from the above table, during storage, all samples from prescriptions 30 to 34 were colorless, clear and transparent, with stable properties. However, visible foreign matter or precipitates appeared in prescriptions 28 and 29, indicating that the pH value affects the stability of HY3000 polypeptide. Within the range of pH 6 to 9, the higher the pH value, the more stable the liquid properties will be.
3.2.2淀粉样蛋白纤维化3.2.2 Amyloid Fibrosis
参阅说明书图2可知，随pH升高，样品在ThT中的logtime越大，说明pH对HY3000多肽的淀粉样蛋白纤维化有较明显的影响。 Referring to Figure 2 of the instruction manual, it can be seen that as the pH increases, the logtime of the sample in ThT becomes larger, indicating that pH has a more obvious impact on the amyloid fibrillation of HY3000 polypeptide.
3.2.3有关物质(总杂质增长量)
3.2.3 Related substances (increased amount of total impurities)
上表中，各样品的总杂质增长量均是基于第1天相同样品的杂质含量计算得出。由上表可知，储存期间处方28～处方34所有产品的总杂质增长幅度均小于10％，检测均合格，且pH值越高，总杂质增长幅度越大。In the above table, the total impurity growth of each sample is calculated based on the impurity content of the same sample on the first day. It can be seen from the table above that the growth rate of total impurities in all products from prescriptions 28 to 34 during storage was less than 10%, and the tests were all qualified. The higher the pH value, the greater the growth rate of total impurities.
综合3.2.1～3.2.3稳定性各项检测结果，发现pH值对HY3000溶液稳定性至关重要，在pH值在6-9的范围内，HY3000在水溶液中保持良好的稳定性，尤其是PH值在7.2-8.5范围内，HY3000在水溶液中具有优异的稳定性。Based on the stability test results from 3.2.1 to 3.2.3, it was found that the pH value is crucial to the stability of HY3000 solution. Within the pH range of 6-9, HY3000 maintains good stability in aqueous solutions, especially With a pH value in the range of 7.2-8.5, HY3000 has excellent stability in aqueous solutions.
实施例4Example 4
4.1配置样品4.1 Configuration sample
按照多肽药物制剂处方组成配置处方号为35-41的多肽药物制剂为样品，不同样品原料药浓度不同。According to the prescription composition of the polypeptide drug preparation, the polypeptide drug preparations with prescription numbers 35-41 were prepared as samples. Different samples have different concentrations of raw materials.
4.1.1原料药浓度筛选设计如下表：
4.1.1 The concentration screening design of raw materials is as follows:
4.1.2性状：取储存第1、5、10、20、30天的供试品，观察其性状。4.1.2 Properties: Take the test samples stored for 1, 5, 10, 20 and 30 days and observe their properties.
4.1.3淀粉样蛋白纤维化：通过ThT检测各处方样品的淀粉样蛋白纤维化聚集情况。4.1.3 Amyloid fibrillation: Detect the amyloid fibrillation and aggregation of each prescription sample through ThT.
4.1.4有关物质(总杂质增长量)：取储存第1、10、20、30天的供试品，采用高效液相色谱法HPLC检测有关物质。4.1.4 Related substances (increased amount of total impurities): Take the test samples stored for 1, 10, 20, and 30 days, and use high-performance liquid chromatography (HPLC) to detect related substances.
4.2稳定性检测结果4.2 Stability test results
4.2.1性状
4.2.1 Characteristics
由上表可知，储存期间处方35～处方42所有产品均为无色澄清透明，性状稳定，说明HY3000多肽原料药的浓度在0.5～20mg/ml范围内，可维持制剂的性状。It can be seen from the above table that during storage, all products from Prescription 35 to Prescription 42 are colorless, clear and transparent, with stable properties, indicating that the concentration of HY3000 polypeptide API is in the range of 0.5 to 20 mg/ml, which can maintain the properties of the preparation.
4.2.2淀粉样蛋白纤维化4.2.2 Amyloid Fibrosis
参阅说明书图3可知，随HY3000多肽原料药浓度的增大，样品在ThT中的logtime越小，说明HY3000多肽原料药浓度对HY3000多肽的淀粉样蛋白纤维化有较明显的影响。Referring to Figure 3 of the instruction manual, it can be seen that as the concentration of HY3000 polypeptide API increases, the logtime of the sample in ThT becomes smaller, indicating that the concentration of HY3000 polypeptide API has a significant impact on the amyloid fibrillation of HY3000 polypeptide.
4.2.3有关物质(总杂质增长量)
4.2.3 Related substances (increased amount of total impurities)
上表中，各样品的总杂质增长量均是基于第1天相同样品的杂质含量计算得出。由上表可知，储存期间处方35～处方42所有产品的总杂质增长幅度均小于10％，检测均合格。In the above table, the total impurity growth of each sample is calculated based on the impurity content of the same sample on the first day. It can be seen from the above table that the growth rate of total impurities in all products from prescription 35 to prescription 42 during storage was less than 10%, and all products passed the test.
综合4.2.1～4.2.3稳定性各项检测结果，发现HY3000多肽原料药浓度对HY3000溶液稳定性有明显影响，在实施例4提供的HY3000多肽原料药浓度下，HY3000在水溶液中保持良好的稳定性。Based on the stability test results of 4.2.1 to 4.2.3, it was found that the concentration of HY3000 polypeptide API has a significant impact on the stability of HY3000 solution. Under the concentration of HY3000 polypeptide API provided in Example 4, HY3000 maintains good stability in the aqueous solution. stability.
综上所述，本发明所提供的多肽药物制剂提高了HY3000在液体中的稳定性，降低了患者的使用风险。To sum up, the polypeptide pharmaceutical preparation provided by the present invention improves the stability of HY3000 in liquid and reduces the risk of use for patients.
应用例Application examples
HY3000多肽溶液制剂CZ430、CZ442、CZ443、CZ444、CZ445对原始毒株(SARS-CoV-2WT)及变异毒株Omicron(BF.7)的假病毒的抑制效果：The inhibitory effect of HY3000 polypeptide solution preparations CZ430, CZ442, CZ443, CZ444, and CZ445 on pseudoviruses of the original strain (SARS-CoV-2WT) and the mutant strain Omicron (BF.7):
按照实验过程中动物菌株+新型冠状病毒假病毒+制剂组合的不同将实验分为以下几组：According to the different combinations of animal strains + new coronavirus pseudovirus + preparations during the experiment, the experiments are divided into the following groups:
HEK293ThACE2细胞+原始毒株或变异毒株假病毒+原料药HY3000溶液(HY3000处理组)；HEK293ThACE2 cells + original strain or mutant strain pseudovirus + API HY3000 solution (HY3000 treatment group);
HEK293ThACE2细胞+原始毒株或变异毒株假病毒+制剂CZ430(CZ430处理组)；HEK293ThACE2 cells + original strain or mutant strain pseudovirus + preparation CZ430 (CZ430 treatment group);
HEK293ThACE2细胞+原始毒株或变异毒株假病毒+制剂CZ442(CZ442处理组)； HEK293ThACE2 cells + original strain or mutant strain pseudovirus + preparation CZ442 (CZ442 treatment group);
HEK293T hACE2细胞+原始毒株或变异毒株假病毒+制剂CZ443(CZ443处理组)；HEK293T hACE2 cells + original strain or variant strain pseudovirus + preparation CZ443 (CZ443 treatment group);
HEK293T hACE2细胞+原始毒株或变异毒株假病毒+制剂CZ444(CZ444处理组)；HEK293T hACE2 cells + original strain or mutant strain pseudovirus + preparation CZ444 (CZ444 treatment group);
HEK293T hACE2细胞+原始毒株或变异毒株假病毒+制剂CZ445(CZ445处理组)。HEK293T hACE2 cells + original strain or mutant strain pseudovirus + preparation CZ445 (CZ445 treatment group).
辅料溶液的配制：Preparation of excipient solution:
取十二烷基磷酸胆碱、山梨醇和苯乙醇，用纯水配制得到CZ430辅料溶液，含十二烷基磷酸胆碱5mg/mL，山梨醇40mg/mL，苯乙醇9mg/mL；Take dodecylphosphocholine, sorbitol and phenethyl alcohol and prepare it with pure water to obtain a CZ430 excipient solution, containing dodecylphosphocholine 5mg/mL, sorbitol 40mg/mL and phenethyl alcohol 9mg/mL;
取羟丙基倍他环糊精、山梨醇和苯扎氯铵，用纯水配制得到CZ442辅料溶液，含羟丙基倍他环糊精100mg/mL，山梨醇50mg/mL，苯扎氯铵0.2mg/mL；Take hydroxypropyl beta-cyclodextrin, sorbitol and benzalkonium chloride and prepare it with pure water to obtain CZ442 excipient solution, containing hydroxypropyl beta-cyclodextrin 100 mg/mL, sorbitol 50 mg/mL, and benzalkonium chloride 0.2 mg/mL;
取羟丙基倍他环糊精、山梨醇和苯乙醇，用纯水配制得到CZ443辅料溶液，含羟丙基倍他环糊精100mg/mL，山梨醇50mg/mL，苯乙醇9mg/mL；Take hydroxypropyl beta-cyclodextrin, sorbitol and phenethyl alcohol and prepare it with pure water to obtain a CZ443 excipient solution, containing hydroxypropyl beta-cyclodextrin 100 mg/mL, sorbitol 50 mg/mL, and phenethyl alcohol 9 mg/mL;
取十二烷基磷酸胆碱、山梨醇和苯扎氯铵，用纯水配制得到CZ444辅料溶液，含十二烷基磷酸胆碱5mg/mL，山梨醇50mg/mL，苯扎氯铵0.2mg/mL；Take dodecylphosphocholine, sorbitol and benzalkonium chloride and prepare it with pure water to obtain CZ444 excipient solution, containing dodecylphosphocholine 5mg/mL, sorbitol 50mg/mL, benzalkonium chloride 0.2mg/ mL;
取十二烷基磷酸胆碱和山梨醇，用纯水配制得到CZ445辅料溶液，含十二烷基磷酸胆碱5mg/mL和山梨醇50mg/mL。Take dodecylphosphocholine and sorbitol and prepare it with pure water to obtain a CZ445 excipient solution, containing 5mg/mL dodecylphosphocholine and 50mg/mL sorbitol.
多肽溶液配制：分别使用纯水、CZ430辅料溶液、CZ442辅料溶液、CZ443辅料溶液、CZ444辅料溶液和CZ445辅料溶液溶解HY3000多肽制得HY3000多肽溶液和制剂CZ430、CZ442、CZ443、CZ444、CZ445母液，再用含10％FBS的DMEM培养基将母液稀释成20μM，作为进一步梯度稀释的原液。制剂CZ430母液中多肽的浓度为5mg/mL、十二烷基磷酸胆碱的浓度为5mg/mL，山梨醇的浓度为40mg/mL，苯乙醇的浓度为9mg/mL。制剂CZ442母液中多肽的浓度为5mg/mL、羟丙基倍他环糊精的浓度为100mg/mL，山梨醇的浓度为50mg/mL，苯扎氯铵的浓度为0.2mg/mL。制剂CZ443母液中多肽的浓度为5mg/mL、羟丙基倍他环糊精的浓度为100mg/mL，山梨醇的浓度为50mg/mL，苯乙醇的浓度为9mg/mL。制剂CZ444母液中多肽的浓度为5mg/mL、十二烷基磷酸胆碱的浓度为5mg/mL，山梨醇的浓度为50mg/mL，苯扎氯铵的浓度为0.2mg/mL。制剂CZ445母液中多肽的浓度为5mg/mL、十二烷基磷酸胆碱的浓度为5mg/mL、山梨醇的浓度为50mg/mL。Preparation of polypeptide solution: Use pure water, CZ430 excipient solution, CZ442 excipient solution, CZ443 excipient solution, CZ444 excipient solution and CZ445 excipient solution to dissolve HY3000 polypeptide to prepare HY3000 polypeptide solution and preparations CZ430, CZ442, CZ443, CZ444, CZ445 mother liquor, and then The stock solution was diluted to 20 μM with DMEM medium containing 10% FBS as the stock solution for further gradient dilution. The concentration of the polypeptide in the preparation CZ430 mother solution is 5 mg/mL, the concentration of dodecylphosphocholine is 5 mg/mL, the concentration of sorbitol is 40 mg/mL, and the concentration of phenethyl alcohol is 9 mg/mL. The concentration of the polypeptide in the mother solution of preparation CZ442 is 5 mg/mL, the concentration of hydroxypropyl betacyclodextrin is 100 mg/mL, the concentration of sorbitol is 50 mg/mL, and the concentration of benzalkonium chloride is 0.2 mg/mL. The concentration of the polypeptide in the mother solution of preparation CZ443 is 5 mg/mL, the concentration of hydroxypropyl betacyclodextrin is 100 mg/mL, the concentration of sorbitol is 50 mg/mL, and the concentration of phenethyl alcohol is 9 mg/mL. The concentration of the polypeptide in the mother solution of preparation CZ444 is 5 mg/mL, the concentration of dodecylphosphocholine is 5 mg/mL, the concentration of sorbitol is 50 mg/mL, and the concentration of benzalkonium chloride is 0.2 mg/mL. The concentration of polypeptide, dodecylphosphocholine, and sorbitol in the mother solution of preparation CZ445 is 5 mg/mL, 5 mg/mL.
多肽梯度稀释液的配制：用含10％FBS的DMEM培养基将上述20μM的各多肽原液以2倍的倍比稀释，共稀释9个梯度(分别为10、5、2.5、1.25、0.625、0.3125、0.156、0.078、0.039μM)，每个梯度3个复孔，每孔50μL。Preparation of polypeptide gradient dilutions: Use DMEM medium containing 10% FBS to dilute the above 20 μM polypeptide stock solution by 2 times, and dilute a total of 9 gradients (respectively 10, 5, 2.5, 1.25, 0.625, 0.3125 , 0.156, 0.078, 0.039μM), each gradient has 3 duplicate wells, 50μL per well.
假病毒用量的确定：将SARS-CoV-2原始毒株或变异毒株Omicron(BF.7)假病毒原液及一系列稀释液在HEK293ThACE2细胞上进行定量，将出现1000个FFU时的稀释度作为评价多肽抑制效果时的病毒用量(稀释倍数介于6～20倍之间)。 Determination of the dosage of pseudovirus: Quantify the original SARS-CoV-2 strain or mutant strain Omicron (BF.7) pseudovirus stock solution and a series of dilutions on HEK293ThACE2 cells, and use the dilution when 1000 FFU appears as Dosage of virus when evaluating the inhibitory effect of polypeptides (dilution factor ranges from 6 to 20 times).
病毒抑制效果的测定方法为：The method for measuring the virus inhibitory effect is:
a.在96孔细胞培养板中铺HEK293ThACE2细胞，培养使第二天细胞汇合密度达到80-90％。a. Plate HEK293ThACE2 cells in a 96-well cell culture plate and culture until the cell confluence density reaches 80-90% the next day.
b.根据上述实验分组，取上述各多肽梯度稀释液，加入等体积(50μL)的定量的假病毒稀释液，混匀后，在37℃孵育1h。b. According to the above experimental grouping, take the above-mentioned gradient dilution of each polypeptide, add an equal volume (50 μL) of quantitative pseudovirus dilution, mix well, and incubate at 37°C for 1 hour.
c.将步骤a中的96孔细胞培养板上清小心弃去，加入步骤b中的多肽-假病毒混合液(100μL/孔)，在培养箱中继续培养15h-24h。c. Carefully discard the supernatant of the 96-well cell culture plate in step a, add the polypeptide-pseudovirus mixture (100 μL/well) in step b, and continue culturing in the incubator for 15h-24h.
d.利用高内涵显微镜统计被感染的细胞数，计算各多肽在不同浓度下的抑制率，再利用GraphPad计算出各制剂溶液的EC50(如图1、图2所示)，结果见表1；并计算出制剂溶液相较于HY3000多肽溶液对SARS-CoV-2原始毒株及变异毒株Omicron(BF.7)假病毒抑制效果的提升倍数，结果见表2。d. Use high-content microscopy to count the number of infected cells, calculate the inhibition rate of each polypeptide at different concentrations, and then use GraphPad to calculate the EC 50 of each preparation solution (as shown in Figure 1 and Figure 2). The results are shown in Table 1 ; and calculated the improvement fold of the inhibitory effect of the preparation solution on the original strain of SARS-CoV-2 and the mutant strain Omicron (BF.7) pseudovirus compared with the HY3000 polypeptide solution. The results are shown in Table 2.
表1各制剂对SARS-CoV-2原始毒株及其变异毒株假病毒抑制效果(EC50)
Table 1 The inhibitory effect (EC 50 ) of each preparation on the original strain of SARS-CoV-2 and its mutant strain pseudovirus
表2制剂溶液相较于HY3000多肽溶液对SARS-CoV-2原始毒株及其变异毒株假病毒抑制效果的提升倍数
Table 2 Compared with the HY3000 polypeptide solution, the improvement factor of the inhibitory effect of the preparation solution on the original strain of SARS-CoV-2 and its mutant strain pseudovirus
从表1和表2可知，各处方制剂对原始毒株和变异毒株均具有很好的抑制效果(110.15nM≤EC50≤379.35nM)。相比于HY3000多肽溶液，各处方制剂对SARS-CoV-2病毒抑制效力没有显著变化，但辅料溶液的加入能够提高HY3000的稳定性，并具备抑菌效力，具备成药性。It can be seen from Table 1 and Table 2 that each prescription preparation has a good inhibitory effect on both the original strain and the mutated strain (110.15nM ≤ EC 50 ≤ 379.35nM). Compared with the HY3000 polypeptide solution, the inhibitory effect of each prescription preparation on the SARS-CoV-2 virus has not changed significantly, but the addition of the excipient solution can improve the stability of HY3000, and has antibacterial efficacy and drug potential.
从图4和图5中得知，各处方制剂对原始毒株和变异毒株均具有很好的抑制效果。



It can be seen from Figure 4 and Figure 5 that each prescription preparation has a good inhibitory effect on both the original strain and the mutated strain.



Claims (10)

  1. 一种多肽药物制剂，其特征在于，包括按百分质量计的以下组分：
    A polypeptide pharmaceutical preparation, characterized in that it includes the following components in terms of mass percentage:
  2. 如权利要求1所述的多肽药物制剂，其特征在于，包括按百分质量计的以下组分：
    The polypeptide pharmaceutical preparation according to claim 1, characterized in that it includes the following components in terms of mass percentage:
  3. 如权利要求1所述的多肽药物制剂，其特征在于，所述表面活性剂选自磷脂类、聚山梨酯类、山梨醇脂肪酸酯类、泊洛沙姆类、聚乙二醇酯类中的一种或多种。The polypeptide pharmaceutical preparation according to claim 1, wherein the surfactant is selected from the group consisting of phospholipids, polysorbates, sorbitol fatty acid esters, poloxamers, and polyethylene glycol esters. one or more.
  4. 如权利要求1所述的多肽药物制剂，其特征在于，所述增溶剂选自环糊精、环糊精类衍生物、壳聚糖和壳聚糖衍生物中的一种或多种。The polypeptide pharmaceutical preparation according to claim 1, wherein the solubilizing agent is selected from one or more of cyclodextrin, cyclodextrin derivatives, chitosan and chitosan derivatives.
  5. 如权利要求1所述的多肽药物制剂，其特征在于，所述渗透压调节剂选自氯化钠、磷酸盐、枸橼酸盐、甘油、甘露醇、山梨醇、葡萄糖、果糖、乳糖和海藻糖中的一种或多种。The polypeptide pharmaceutical preparation according to claim 1, wherein the osmotic pressure regulator is selected from the group consisting of sodium chloride, phosphate, citrate, glycerol, mannitol, sorbitol, glucose, fructose, lactose and seaweed One or more types of sugar.
  6. 如权利要求1所述的多肽药物制剂，其特征在于，所述pH调节剂选自枸橼酸及枸橼酸盐、醋酸及醋酸盐、磷酸及磷酸盐、氢氧化钠、盐酸、乳酸和马来酸中的一种或多种。The polypeptide pharmaceutical preparation according to claim 1, wherein the pH regulator is selected from the group consisting of citric acid and citrate, acetic acid and acetate, phosphoric acid and phosphate, sodium hydroxide, hydrochloric acid, lactic acid and One or more types of maleic acid.
  7. 如权利要求1所述的多肽药物制剂，其特征在于，所述溶剂为醇类有机溶剂或水。The polypeptide pharmaceutical preparation according to claim 1, wherein the solvent is an alcoholic organic solvent or water.
  8. 如权利要求1所述的多肽药物制剂，其特征在于，还包括防腐剂和/或抑菌剂,所述防腐剂或所述抑菌剂选自苯扎氯铵、羟苯酯类、三氯叔丁醇、苯甲醇和苯乙醇中的一种或多种。The polypeptide pharmaceutical preparation according to claim 1, further comprising a preservative and/or bacteriostatic agent, the preservative or the bacteriostatic agent being selected from benzalkonium chloride, hydroxyphenyl esters, trichloride One or more of tert-butyl alcohol, benzyl alcohol and phenethyl alcohol.
  9. 如权利要求8所述的多肽药物制剂，其特征在于，所述防腐剂选自三氯叔丁醇和苯甲醇中的一种或多种，所述抑菌剂选自苯扎氯铵和苯乙醇中一种或多种。The polypeptide pharmaceutical preparation according to claim 8, wherein the preservative is selected from one or more of chlorobutanol and benzyl alcohol, and the bacteriostatic agent is selected from benzalkonium chloride and phenylethyl alcohol. one or more of them.
  10. 一种多肽药物制剂的制备方法，其特征在于，将如权利要求1-9任一项所述的HY3000、所述表面活性剂和/或增溶剂、所述渗透压调节剂和所述防腐剂溶于10％～95％所述的溶剂中，搅拌至溶解，加入所述pH调节剂使溶液的PH值位于6～9，加入其余所述溶剂，混匀，过滤，灌装至喷雾装置中，即得所述多肽药物制剂。 A method for preparing a polypeptide pharmaceutical preparation, characterized in that the HY3000 according to any one of claims 1 to 9, the surfactant and/or solubilizer, the osmotic pressure regulator and the preservative Dissolve in 10% to 95% of the solvent, stir until dissolved, add the pH adjuster to make the pH value of the solution between 6 and 9, add the rest of the solvent, mix, filter, and fill into a spray device , to obtain the polypeptide pharmaceutical preparation.
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