WO2016169454A1 - 一种神经生长因子组合物及注射粉剂 - Google Patents

一种神经生长因子组合物及注射粉剂 Download PDF

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WO2016169454A1
WO2016169454A1 PCT/CN2016/079571 CN2016079571W WO2016169454A1 WO 2016169454 A1 WO2016169454 A1 WO 2016169454A1 CN 2016079571 W CN2016079571 W CN 2016079571W WO 2016169454 A1 WO2016169454 A1 WO 2016169454A1
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Prior art keywords
growth factor
nerve growth
factor composition
stabilizer
composition
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PCT/CN2016/079571
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English (en)
French (fr)
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谭剑平
王炳璋
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舒泰神（北京）生物制药股份有限公司
北京舒泰神新药研究有限公司
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Application filed by 舒泰神（北京）生物制药股份有限公司, 北京舒泰神新药研究有限公司 filed Critical 舒泰神（北京）生物制药股份有限公司
Priority to EP16782601.5A priority Critical patent/EP3287140B1/en
Priority to CN201680023115.XA priority patent/CN107660149B/zh
Priority to ES16782601T priority patent/ES2882106T3/es
Priority to US15/568,455 priority patent/US11077193B2/en
Priority to DK16782601.5T priority patent/DK3287140T3/da
Publication of WO2016169454A1 publication Critical patent/WO2016169454A1/zh

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    • 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
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3
    • 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
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system

Definitions

  • the invention relates to a nerve growth factor composition and an injectable powder, belonging to the field of medical biology.
  • Nerve growth factor is a neuronal growth regulator with neuronal nutrition and promoting the biological function of neurite outgrowth. It expresses the development, differentiation, growth, regeneration and functional properties of central and peripheral neurons. Both have important regulatory effects. NGF contains three subunits of ⁇ , ⁇ , and ⁇ . The ⁇ subunit is an active region, and a single chain composed of two 118 amino acids is formed by a combination of non-covalent bonds. In 1953, the Italian scientist Levi-Montalcini discovered the NGF and won the Nobel Prize. At present, a number of NGF products have been marketed at home and abroad, and are mainly used for the treatment of neurological dysplasia, including amblyopia, neuroma, various nerve injuries and neurological diseases.
  • NGF is similar to other protein drugs. Due to the short half-life of the protein, when exposed to extreme temperature and humidity conditions, or affected by physical and chemical factors, the spatial conformation of the protein is liable to change and the denatured protein is lost. In addition, because proteins often adhere to solid surfaces, some proteins adhere to the inner wall of the container during the filling process, resulting in loss of active ingredients. In order to ensure its biological activity, a stabilizer is added to prevent its loss of biological activity.
  • albumin is widely used as an excellent stabilizer and a cake forming agent in various biological products.
  • NGF composition is a non-sterilized sterile preparation, and the blood carries some unknown components that are difficult to detect, which is easy to cause pollution to the preparation; meanwhile, albumin
  • the long-term, large-scale application is also susceptible to the limitations of blood supply and production costs; again, albumin may interfere with relatively small amounts of NGF and affect product quality management when determining the content of intermediates and finished products. Therefore, in order to avoid the above problems, a stable albumin-removing NGF composition is sought.
  • the object of the present invention is to provide a nerve growth factor composition and an injectable powder, which not only protects the mouse-derived nerve growth factor (mNGF), but also ensures the human nerve.
  • mNGF mouse-derived nerve growth factor
  • rhNGF recombinant human nerve growth factor
  • the invention provides a nerve growth factor composition comprising the following components:
  • a nerve growth factor having a mass volume concentration of 10 ⁇ g/mL to 100 ⁇ g/mL;
  • a disaccharide stabilizer having a mass volume concentration of 10 mg/mL to 80 mg/mL;
  • An amino acid stabilizer having a mass volume concentration of 0 mg/mL to 30 mg/mL;
  • a surfactant having a mass volume concentration of from 0.01 mg/mL to 1 mg/mL;
  • a support agent having a mass volume concentration of 10 mg/mL to 50 mg/mL;
  • the solvent is water.
  • a stabilizer in the above nerve growth factor composition, the addition of a stabilizer can avoid or reduce protein aggregation and depolymerization caused by preparation or storage, and the term “stabilizer” refers to a method for preventing aggregation or depolymerization of the active ingredient in an aqueous solution.
  • stabilizers are often used as support agents to improve the formability of the product, and other effects are not excluded;
  • the disaccharide stabilizer has a mass volume concentration of 30 mg/mL to 70 mg/mL, and more preferably, the disaccharide stabilizer has a mass volume concentration of 30 mg/mL. ;
  • the disaccharide stabilizer may be at least one of maltose, trehalose, sucrose, and lactose;
  • the amino acid type stabilizer has a mass volume concentration of 2 mg/mL to 10 mg/mL, and more preferably, the amino acid type stabilizer has a mass volume concentration of 3 mg/mL to 10 mg. /mL, still more preferably, the amino acid type stabilizer has a mass volume concentration of 3 mg/mL and 10 mg/mL.
  • the amino acid stabilizer may be at least one of alanine, glycine, arginine, glutamic acid, histidine, and isoleucine.
  • the disaccharide stabilizer and the amino acid stabilizer may specifically be any one of the following 1) to 8):
  • sucrose and histidine 1) sucrose and histidine, 2) sucrose and glycine, 3) sucrose, glycine and histidine, 4) lactose and glycine, 5) lactose, sucrose and isoleucine, 6) trehalose, arginine and Histidine, 7) maltose, glycine and histidine, 8) maltose, alanine, arginine and glutamine acid.
  • the mass concentration of the surfactant is from 0.1 mg/mL to 0.5 mg/mL, and more preferably, the mass concentration of the surfactant is 0.2 mg/mL. 0.5 mg / mL, and more preferably, the mass concentration of the surfactant is 0.2 mg / mL, 0.5 mg / mL;
  • the surfactant may be a nonionic surfactant, and preferably, the nonionic surfactant may be poloxamer, polysorbate, and 15-hydroxystearic acid. At least one of a glycol ester (abbreviated as HS 15, the same applies hereinafter) may specifically be any one of poloxamer, polysorbate, and HS 15.
  • a glycol ester abbreviated as HS 15, the same applies hereinafter
  • the addition of the support agent can improve the appearance of the finished product, and ensure that the product has good moldability after lyophilization;
  • the mass concentration of the support agent is 20 to 50 mg / mL, and more preferably, the mass concentration of the support agent is 20 mg / mL, 50 mg / mL;
  • the support agent may be any one of mannitol, dextrin, and dextran.
  • the composition in order to ensure the maximum biological activity of the composition, it is usually necessary to control an optimum pH range, and the optimum pH range of the stability needs to be determined at the time of prescription screening, and the influence factor test is usually used ( Determination of light, high temperature, high humidity), acceleration and long-term stability tests, etc.
  • the influence factor test is usually used ( Determination of light, high temperature, high humidity), acceleration and long-term stability tests, etc.
  • the composition must be produced and stored during the process. Maintaining it in its optimum pH range, because the pH buffer has good buffering capacity, it can keep the pH of the product relatively stable within a certain range. Therefore, a buffer is often added to the prescription to control the pH value;
  • the molar concentration of the pH buffer is 10 mM to 50 mM, preferably, the molar concentration of the pH buffer is 20 mM to 25 mM, and more preferably, the molar concentration of the pH buffer is 20 mM, 25 mM;
  • the pH of the nerve growth factor composition is maintained at 6.80 to 7.00, and more preferably, the pH of the nerve growth factor composition is maintained at 6.86 to 6.91, and more preferably The pH of the nerve growth factor composition is maintained at 6.86, specifically 6.80, 6.82, 6.83, 6.85, 6.86, 6.87, 6.91, 6.92 or 7.00;
  • the pH buffering agent is at least one of phosphate, citrate, acetate, histidine hydrochloride and arginine hydrochloride, specifically 1) phosphoric acid Salt, 2) citrate, 3) acetate, 4) histidine hydrochloride, or 5) histidine hydrochloride and arginine hydrochloride, 5) histidine hydrochloride and The molar ratio of arginine hydrochloride can be 12:13.
  • the water is water for injection.
  • the nerve growth factor is a neuronal growth regulator having neuronal nutrition and promoting the biological function of neurite outgrowth, which develops, differentiates, and grows central and peripheral neurons. , regeneration and expression of functional properties have important regulatory effects;
  • the nerve growth factor has a mass volume concentration of 40 ⁇ g/mL to 60 ⁇ g/mL, and more preferably, the nerve growth factor has a mass volume concentration of 50 ⁇ g/mL;
  • the nerve growth factor is a mouse-derived nerve growth factor, a human nerve growth factor or a recombinant human nerve growth factor.
  • the concentration of each component may specifically be any one of the following 1) to 17):
  • nerve growth factor 40 ⁇ g/mL ⁇ 80 ⁇ g/mL
  • disaccharide stabilizer 10mg/mL ⁇ 40mg/mL
  • amino acid stabilizer 0mg/mL ⁇ 6mg/mL
  • surfactant 0.01mg/mL ⁇ 0.2mg/mL
  • support agent 10mg/mL ⁇ 30mg/mL
  • pH buffer 10 mM to 25 mM;
  • nerve growth factor 60 ⁇ g/mL ⁇ 100 ⁇ g/mL
  • disaccharide stabilizer 40mg/mL ⁇ 80mg/mL
  • amino acid stabilizer 6mg/mL ⁇ 30mg/mL
  • surfactant 0.2mg/mL ⁇ 1 mg / mL
  • support agent 30 mg / mL ⁇ 50 mg / mL
  • pH buffer 25 mM ⁇ 50 mM;
  • nerve growth factor 10 ⁇ g/mL ⁇ 50 ⁇ g/mL
  • disaccharide stabilizer 30mg/mL ⁇ 50mg/mL
  • amino acid stabilizer 2mg/mL ⁇ 10mg/mL
  • surfactant 0.05mg/mL ⁇ 0.4mg/mL
  • support agent 20mg/mL ⁇ 40mg/mL
  • pH buffer 20mM ⁇ 30mM
  • nerve growth factor 10 ⁇ g/mL ⁇ 50 ⁇ g/mL
  • disaccharide stabilizer 35mg/mL ⁇ 70mg/mL
  • amino acid stabilizer 3mg/mL ⁇ 13mg/mL
  • surfactant 0.1mg/mL ⁇ 0.5mg/mL
  • support agent 20mg/mL ⁇ 40mg/mL
  • pH buffer 20mM ⁇ 30mM
  • nerve growth factor 10 ⁇ g/mL ⁇ 50 ⁇ g/mL
  • disaccharide stabilizer 35mg/mL ⁇ 70mg/mL
  • amino acid stabilizer 4mg/mL ⁇ 14mg/mL
  • surfactant 0.1mg/mL ⁇ 0.5mg/mL
  • support agent 20mg/mL ⁇ 40mg/mL
  • pH buffer 20mM ⁇ 30mM
  • nerve growth factor 40 ⁇ g/mL ⁇ 60 ⁇ g/mL
  • disaccharide stabilizer 30mg/mL ⁇ 70mg/mL
  • amino acid stabilizer 0mg/mL ⁇ 10mg/mL
  • surfactant 0.2mg/mL ⁇ 0.5mg/mL
  • support agent 20mg/mL ⁇ 50mg/mL
  • pH buffer 20mM ⁇ 25mM
  • nerve growth factor 40 ⁇ g/mL ⁇ 60 ⁇ g/mL
  • disaccharide stabilizer 30mg/mL ⁇ 70mg/mL
  • amino acid stabilizer 3mg/mL ⁇ 10mg/mL
  • surfactant 0.2mg/mL ⁇ 0.5mg/mL
  • support agent 20mg/mL ⁇ 50mg/mL
  • pH buffer 20mM ⁇ 25mM
  • nerve growth factor 10 ⁇ g / mL
  • disaccharide stabilizer 10 mg / mL
  • amino acid stabilizer 0 mg / mL
  • surfactant 0.01 mg / mL
  • support agent 10 mg / mL
  • pH buffer 10 mM ;
  • nerve growth factor 40 ⁇ g / mL
  • disaccharide stabilizer 30 mg / mL
  • amino acid stabilizer 2 mg / mL
  • surfactant 0.05 mg / mL
  • support agent 20 mg / mL
  • pH buffer Agent 20mM
  • nerve growth factor 50 ⁇ g / mL
  • disaccharide stabilizer 35 mg / mL
  • amino acid stabilizer 3 mg / mL
  • surfactant 0.1 mg / mL
  • support agent 30 mg / mL
  • pH buffer 25 mM ;
  • nerve growth factor 60 ⁇ g / mL
  • disaccharide stabilizer 40 mg / mL
  • amino acid stabilizer 4 mg / mL
  • surfactant 0.2 mg / mL
  • support agent 40 mg / mL
  • pH buffer 30 mM ;
  • nerve growth factor 80 ⁇ g / mL
  • disaccharide stabilizer 50 mg / mL
  • amino acid stabilizer 6 mg / mL
  • surfactant 0.4 mg / mL
  • support agent 50 mg / mL
  • pH buffer 50 mM ;
  • nerve growth factor 100 ⁇ g / mL
  • disaccharide stabilizer 70 mg / mL
  • amino acid stabilizer 10 mg / mL
  • surfactant 0.5 mg / mL
  • support agent 50 mg / mL
  • pH buffer 50 mM ;
  • nerve growth factor 100 ⁇ g / mL
  • disaccharide stabilizer 80 mg / mL
  • amino acid stabilizer 13 mg / mL
  • surfactant 1 mg / mL
  • support agent 50 mg / mL
  • pH buffer 50 mM
  • nerve growth factor 100 ⁇ g / mL
  • disaccharide stabilizer 80 mg / mL
  • amino acid stabilizer 14 mg / mL
  • surfactant 1 ⁇ g / mL
  • support agent 50 mg / mL
  • pH buffer 50 mM
  • nerve growth factor 100 ⁇ g / mL
  • disaccharide stabilizer 80 mg / mL
  • amino acid stabilizer 30 mg / mL
  • surfactant 1 mg / mL
  • support agent 50 mg / mL
  • pH buffer 50 mM
  • nerve growth factor 50 ⁇ g / mL
  • disaccharide stabilizer 30 mg / mL
  • amino acid stabilizer 3 mg / mL
  • surfactant 0.2 mg / mL
  • support agent 50 mg / mL
  • pH buffer 25 mM ;
  • nerve growth factor 50 ⁇ g / mL
  • disaccharide stabilizer 30 mg / mL
  • amino acid stabilizer 10 mg / mL
  • surfactant 0.5 mg / mL
  • support agent 20 mg / mL
  • pH buffer 20 mM .
  • the present invention further provides a method for preparing the above-mentioned nerve growth factor composition injection powder, which can be obtained by freeze-drying the above nerve growth factor composition to obtain the nerve growth factor group.
  • Compound injection powder obtained by freeze-drying the above nerve growth factor composition to obtain the nerve growth factor group.
  • Another object of the present invention is to provide a nerve growth factor composition injection powder prepared by the above preparation method for administration by injection.
  • the water content of the injection powder may be 1.2% to 3.0%, specifically 1.3%, 1.7%, 1.9%, 2.2%, 2.3%, 2.4%, 2.5%, 2.7. Or 2.8%; the pH may be 6.80 to 7.20, specifically 6.80, 6.82, 6.83, 6.85, 6.92, 6.93, 6.95, 7.05, 7.10 or 7.20.
  • the nerve growth factor composition may specifically be a nerve growth factor composition injection powder.
  • the nerve injury may be an optic nerve injury, and the cause of the injury may specifically be a foot injury, a car accident, an object bruise or an eye blast injury.
  • the invention further provides a method of treating a nerve injury comprising the step of administering to a patient with a nerve injury an effective amount of a nerve growth factor composition.
  • the nerve growth factor composition may specifically be a nerve growth factor composition injection powder.
  • the administration may specifically be intramuscular injection.
  • the nerve injury may be an optic nerve injury, and the cause of the injury may specifically be a foot injury, a car accident, an object bruise or an eye blast injury.
  • Figure 1 is a graph showing the average content of NGF in the NGF composition injectable powder and the reference preparation prepared in the examples under accelerated conditions (25 ° C, RH 60 ⁇ 10%).
  • Figure 2 is a graph showing the average activity over time of the NGF composition injectable powder and the reference preparation prepared in the examples under accelerated conditions (25 ° C, RH 60 ⁇ 10%).
  • Figure 3 is a graph showing the average content of NGF in the NGF composition injectable powder and the reference preparation prepared in the examples under long-term stability conditions (6 ⁇ 2 ° C).
  • Figure 4 is a graph showing the average activity of the NGF composition injected powder and the reference preparation prepared in the examples under long-term stability conditions (6 ⁇ 2 ° C) as a function of time.
  • the mNGF and hNGF stock solutions used in the following examples were provided by Shutai Shen (Beijing) Bio-Pharmaceutical Co., Ltd., and the rhNGF stock solution was supplied by Beijing Yiqiao Shenzhou Biotechnology Co., Ltd.
  • the other excipients were injection grade unless otherwise specified.
  • the units of the concentration of the surfactant in the following examples are expressed in %, for example, 0.1% means 1 mg/mL.
  • the present invention has carried out a large number of exploratory experiments, using maltose, trehalose, sucrose, lactose, alanine, glycine, arginine, and valley as stabilizers for this product.
  • TW-20 polysorbate-20
  • TW-80 polysorbate-80
  • Polo Polo used as surfactants.
  • Various NGF compositions and injection powders were prepared by sam 188 (referred to as F68, the same below) and HS 15, and the moisture, pH, osmotic pressure, content and activity of the finished product were determined.
  • the NGF composition and the injected powder were prepared as follows:
  • the prescription composition in Table 1 weigh the prescribed amount of citrate, add appropriate amount of water for injection and stir until completely dissolved, then add the prescribed amount of trehalose, mannitol and F68 to completely dissolve, add hNGF stock solution, use NaOH or citric acid
  • the pH was adjusted to pH 6.85 as shown in Table 1, and the volume was adjusted to the mark with water for injection. After mixing uniformly, it was filtered through a 0.22 ⁇ m microporous membrane filter into a sterile container to prepare a composition having an NGF concentration of about 80 ⁇ g/mL. .
  • the above composition was dispensed into a borosilicate glass injection bottle at 0.63 ⁇ 0.03 mL/bottle, and lyophilized (a hNGF injection powder for hNGF injection) was prepared.
  • the NGF composition and the injected powder were prepared as follows:
  • TW-20 is in the water for injection. In the state of spherical aggregation, it is difficult to mix and evenly.
  • TW-20 is first dissolved in hot water for injection (40-80 ° C) to prepare a 1% aqueous solution, and after being cooled to room temperature, the solution is added after being converted according to the prescription amount.
  • the above composition was dispensed into a borosilicate glass injection bottle at 0.63 ⁇ 0.03 mL/bottle, and freeze-dried to obtain a rhNGF injection powder for rhNGF injection.
  • the NGF composition and the injected powder were prepared as follows:
  • HS 15 is in a spherical aggregation state in the water for injection, and it is difficult to mix uniformly.
  • HS 15 is first dissolved in hot water for injection (40-80 ° C) to prepare a 1% aqueous solution, and after cooling to room temperature, the amount is prescribed. After conversion, add the above solution and mix well.
  • hNGF stock solution adjust pH with NaOH or acetic acid to pH 6.92 shown in Table 1, add volume of water to the mark with water for injection, mix well, pass 0.22 ⁇ m microporous membrane The mixture was filtered into a sterile container to prepare a composition having an NGF concentration of about 40 ⁇ g/mL.
  • the above composition was dispensed into a borosilicate glass injection bottle at 0.63 ⁇ 0.03 mL/bottle, and freeze-dried to prepare a hNGF injection sterile powder, hNGF injection powder.
  • the NGF composition and the injected powder were prepared as follows:
  • TW-20 is first dissolved in hot water for injection (40-80 ° C) to prepare a 1% aqueous solution, and after being cooled to room temperature, the amount is converted according to the prescription amount. Add the above solution and mix well.
  • hNGF stock solution adjust the pH to pH 6.87 shown in Table 1 with HCl or L-histidine, add the volume of water to the mark with water for injection, mix well, and filter through 0.22 ⁇ m microporous.
  • the membrane was filtered into a sterile container to prepare a composition having an NGF concentration of about 60 ⁇ g/mL.
  • the above composition was dispensed into a borosilicate glass injection bottle at 0.63 ⁇ 0.03 mL/bottle, and freeze-dried to prepare a hNGF injection sterile powder, hNGF injection powder.
  • the NGF composition and the injected powder were prepared as follows:
  • TW-80 is in the water for injection.
  • the spherical aggregation state is not easy to be mixed uniformly.
  • TW-80 is first dissolved in hot water for injection (40-80 ° C) to prepare a 1% aqueous solution, and after being cooled to room temperature, it is added to the above solution after being converted according to the prescription amount.
  • mNGF stock solution adjust the pH to pH 6.82 shown in Table 1 with NaOH or citric acid, add the volume of water to the mark with the water for injection, mix well, and then filter into a sterile container through a 0.22 ⁇ m microporous membrane.
  • a composition having an NGF concentration of about 60 ⁇ g/mL was prepared.
  • the above composition was dispensed into a borosilicate glass injection bottle at 0.63 ⁇ 0.03 mL/bottle, and freeze-dried to obtain a sterile powder for mNGF injection, that is, an mNGF injection powder.
  • the NGF composition and the injected powder were prepared as follows:
  • TW-80 is spherical in water for injection. In the state of aggregation, it is not easy to mix and evenly.
  • TW-80 is first dissolved in hot water for injection (40-80 ° C) to prepare a 1% aqueous solution, and after being cooled to room temperature, it is added to the above solution after being converted according to the prescription amount.
  • the above composition was dispensed into a borosilicate glass injection bottle at 0.63 ⁇ 0.03 mL/bottle, and freeze-dried to prepare a hNGF injection sterile powder, hNGF injection powder.
  • the NGF composition and the injected powder were prepared as follows:
  • TW-80 is in a spherical aggregation state in water for injection, and it is not easy to mix and evenly.
  • TW-80 is first dissolved in hot water for injection (40-80 ° C) to prepare a 1% aqueous solution, and after cooling to room temperature, according to the prescription amount After the conversion, add the above solution and mix well.
  • the above composition was dispensed into a borosilicate glass injection bottle at 0.63 ⁇ 0.03 mL/bottle, and freeze-dried to obtain a rhNGF injection powder for rhNGF injection.
  • the NGF composition and the injected powder were prepared as follows:
  • HS 15 is first dissolved in hot water for injection (40-80 ° C) to prepare a 1% aqueous solution, and after being cooled to room temperature, it is added to the above solution after being converted according to the prescription amount, and finally mixed.
  • NGF NGF nitrate
  • the above composition was dispensed into a borosilicate glass injection bottle at 0.63 ⁇ 0.03 mL/bottle, and freeze-dried to prepare a hNGF injection sterile powder, hNGF injection powder.
  • the NGF composition and the injected powder were prepared as follows:
  • TW-20 is spherical in water for injection. In the state of aggregation, it is not easy to mix and evenly.
  • TW-20 is first dissolved in hot water for injection (40-80 ° C) to prepare a 1% aqueous solution, and after being cooled to room temperature, it is added to the above solution after being converted according to the prescription amount.
  • hNGF stock solution adjust the pH to pH 6.86 shown in Table 1 with NaOH or phosphoric acid, add the volume of water to the mark with the water for injection, mix well, and then filter into a sterile container through a 0.22 ⁇ m microporous membrane to prepare A composition having a NGF concentration of about 50 ⁇ g/mL.
  • the above composition was dispensed into a borosilicate glass injection bottle at 0.63 ⁇ 0.03 mL/bottle, and freeze-dried to prepare a hNGF injection sterile powder, hNGF injection powder.
  • the NGF composition and the injected powder were prepared as follows:
  • the prescription composition of Table 1 weigh the prescribed amount of histidine hydrochloride and arginine hydrochloride, add appropriate amount of water for injection and stir until completely dissolved, and then add the prescribed amount of trehalose, arginine, histidine and mannose. The alcohol is stirred until completely dissolved. Since the prescription TW-20 is in a spherical aggregation state in the water for injection, it is difficult to mix uniformly.
  • the present invention first dissolves TW-20 in hot water for injection (40-80 ° C) to prepare a 1% aqueous solution. After cooling to room temperature, the solution is added to the above solution and mixed uniformly.
  • the rhNGF stock solution is added, and the pH is adjusted to pH 6.83 shown in Table 1 with HCl or L-histidine or L-arginine.
  • the mixture was adjusted to volume with water for injection, and after mixing uniformly, it was filtered through a 0.22 ⁇ m microporous membrane filter into a sterile container to prepare a composition having an NGF concentration of about 60 ⁇ g/mL.
  • the above composition was dispensed into a borosilicate glass injection bottle at 0.63 ⁇ 0.03 mL/bottle, and freeze-dried to obtain a rhNGF injection powder for rhNGF injection.
  • the NGF composition and the injected powder were prepared as follows:
  • the prescription composition in Table 1 weigh the prescribed amount of phosphate, add appropriate amount of water for injection and stir until completely dissolved, then add the prescribed amount of lactose, sucrose, isoleucine, mannitol and F68 to dissolve completely dissolved, add mNGF stock solution, Adjust the pH to pH 6.85 shown in Table 1 with NaOH or phosphoric acid, and add to volume with water for injection. Mix well and filter through a 0.22 ⁇ m microporous membrane filter into a sterile container to prepare an NGF concentration of about 10 ⁇ g. /mL composition.
  • the above composition was dispensed into a borosilicate glass injection bottle at 0.63 ⁇ 0.03 mL/bottle, and freeze-dried to obtain a sterile powder for mNGF injection, that is, an mNGF injection powder.
  • NGF composition injection powder prepared in Examples 1-11 and the reference preparation (rat nerve growth factor for injection, trade name: threonide, supplied by Shutai Shen (Beijing) Biopharmaceutical Co., Ltd.) were observed. Appearance, measuring its moisture, osmotic pressure, pH, content and activity. Among them, the method for measuring the NGF content is carried out according to the method described in Example 1 in Patent No. 200510130348.3, entitled "Method for Measuring the Content of Nerve Growth Factor".
  • the activity test method was carried out by a cell method, and the detailed operation method was carried out in accordance with the method in Example 1 in which the patent publication number is CN103376248A, and the patent name is "Quantitative Method for Measuring Nerve Growth Factor Activity". The experimental results are shown in Table 2.
  • Example 2 According to the prescription composition of Example 2, 3 parts of the NGF composition was prepared, and the total amount of the phosphate was kept unchanged, and the amounts of NaH 2 PO 4 ⁇ H 2 O and Na 2 HPO 4 were adjusted to make the pH of the NGF composition 6.0 and 6.8, respectively. 7.4, respectively, filled in a vial and lyophilized to make a sterile powder for injection. Sampling, adding appropriate amount of sterile water for injection to prepare a solution, and at room temperature, observe the appearance, pH, content and activity changes at 0, 4, 8, 12, 24 h, respectively. The results are shown in the following table.
  • the NGF composition injectable powders of Examples 1 to 11 had a reduction rate of 7.8%, 6.1%, 8.0%, 3.2%, 5.5%, 3.6%, 7.9%, 3.0%, 2.4%, 5.2%, and 8.1%, respectively.
  • the stability of the examples of the invention under accelerated conditions is illustrated by the reference formulation. Among them, the content reduction rates of Examples 4, 6, 8, and 9 were 3.2%, 3.6%, 3.0%, and 2.4%, respectively, which were much lower than the reference preparation, indicating the stability of Examples 4, 6, 8, and 9 under accelerated conditions. Significantly better than the reference preparation.
  • Example 4 The stability of the inventive examples is due to the reference formulation. Among them, under accelerated conditions, the activity reduction rates of Example 4, Example 6, Example 8, and Example 9 were 8.9%, 9.2%, 6.3%, and 5.9%, respectively, which were much lower than the reference preparation, indicating that the accelerated conditions were implemented. The stability of Example 4, Example 6, Example 8 and Example 9 was significantly better than the reference formulation.
  • Example 4 The sampled NGF injection powder sample prepared in Example 4, Example 6, Example 8, and Example 9 was placed in accordance with the "Technical Guidelines for Biological Product Stability Research". The long-term stability of 0 ⁇ 12 months was investigated at 6 ⁇ 2°C. The appearance of the sample was observed and the water, osmotic pressure, pH, content and activity were measured. The results are shown in Table 5 and Figure 3 (average content versus time curve), Figure 4 (Average activity versus time curve).
  • the content reduction rates were 6.3%, 5.1%, 6.2%, 2.8%, 5.1%, 3.0%, 6.1%, 2.6%, 2.0%, 4.5%, and 6.0%, respectively.
  • the stability of the examples of the present invention under conditions (6 ⁇ 2 ° C) is superior to the reference formulation.
  • the content reduction rates of Example 4, Example 6, Example 8, and Example 9 were 2.8%, 3.0%, 2.6%, and 2.0%, respectively, which were much lower than the reference preparation, indicating that the long-term condition, Example 4,
  • the stability of Example 6, Example 8, and Example 9 was significantly better than that of the reference formulation.
  • Subject conditions 14 years of age or older.
  • Gender Male or female.
  • Subject source 409 patients with optic nerve injury caused by various reasons in various ophthalmology research units.
  • Treatment method The treatment group was intramuscularly injected (water for injection) using the reference preparation and the NGF composition injection powder of the present invention, once a day, 30 ⁇ g each time, and all cases were continuously administered for 12 weeks.
  • the placebo group used a negative control (without active ingredients, and the remaining excipients were combined with NGF) ()).
  • the treatment group consisted of 271 patients (271 eyes) with an average age of 34.2 years (range, 18-63 years); 189 males (189 eyes) and 82 females (82 eyes); 147 eyes in the right eye and 124 eyes in the left eye.
  • the main adverse reactions of the NGF composition prepared by the present invention are local pain, urticaria and dizziness compared with the placebo group; the NGF composition prepared by the present invention can be significantly reduced compared with the reference preparation.
  • Adverse reactions such as local pain, urticaria, and dizziness.
  • the NGF composition of the present invention and the powder thereof can effectively treat optic nerve damage caused by various reasons, and the comprehensive efficiency of treatment for optic nerve injury is better than that of the reference preparation. Compared with the preparation group, the incidence of adverse reactions can be significantly reduced, and the clinical treatment effect is good.
  • the nerve growth factor composition injection powder prepared by the invention maintains good stability during preparation, transportation and storage: (1) during preparation: the nerve growth factor composition prepared by the invention is injected at room temperature After 24 hours of standing, there was no significant change in the content and activity of NGF; (2) Injecting powder of NGF composition of the present invention during transportation and storage in a conventional (6 ⁇ 2 ° C) ratio compared with the reference preparation After 12 months, its effective content is only reduced by 2.0% to 6.3%, preferably by 2.0 to 3.0%, and its activity is only reduced by 3.4% to 10.9%, and has excellent stability.
  • the nerve growth factor composition injection powder prepared by the invention can significantly reduce the incidence of adverse reactions in clinical trials, has good clinical treatment effect, and has better clinical drug safety than the existing reference preparations. Quality controllability.
  • the nerve growth factor composition and the injectable powder of the present invention can avoid the potential risk of carrying a virus or other unknown component in albumin by using a combination of a disaccharide or a disaccharide and an amino acid in place of albumin as a stabilizer. .
  • the nerve growth factor composition injection powder of the invention not only protects the mouse-derived nerve growth factor (mNGF), but also ensures the preparation of human nerve growth factor (hNGF) and recombinant human nerve growth factor (rhNGF). Good stability, better clinical drug safety and quality controllability during transportation and storage.
  • the nerve growth factor composition and the injection powder of the invention have clear components, are easy to be qualitative and quantitative, and the stabilizers used have high purity, wide source, easy to carry out long-term mass production, and are convenient for controlling costs and improving product quality.

Abstract

一种神经生长因子组合物及注射粉剂。该神经生长因子组合物包括以下组分：10μg/mL～100μg/mL的神经生长因子；10mg/mL～80mg/mL的二糖类稳定剂；0mg/mL～30mg/mL的氨基酸类稳定剂；0.01mg/mL～1mg/mL的表面活性剂；10mg/mL～50mg/mL的支持剂；维持所述神经生长因子组合物的pH值为6.0～7.4的pH缓冲剂；溶剂为水。所述神经生长因子组合物及注射粉剂，通过使用二糖类或二糖类与氨基酸组合代替白蛋白作为稳定剂，能够避免因白蛋白中携带病毒或其他未知成分带来的潜在风险；不仅对mNGF具有保护作用，同时能够保证hNGF以及rhNGF在制备、运输及储存过程中保持良好的稳定性、更好的临床用药安全性及质量可控性；成分明确，易于定性及定量，所用稳定剂纯度高，来源广，容易进行长期大量生产，便于控制成本及提高产品质量。

Description

一种神经生长因子组合物及注射粉剂 技术领域
本发明涉及一种神经生长因子组合物及注射粉剂，属于医药生物领域。
背景技术
神经生长因子(NGF)是一种具有神经元营养和促进突起生长双重生物学功能的一种神经细胞生长调节因子，它对中枢及周围神经元的发育、分化、生长、再生和功能特性的表达均具有重要的调控作用。NGF包含α、β、γ三个亚单位，β亚单位是活性区，由两个118个氨基酸组成的单链通过非共价键结合而成。1953年，意大利科学家Levi-Montalcini发现了NGF并获得了诺贝尔奖。目前，国内外已有多个NGF产品上市，临床上主要用于治疗神经系统发育不良，包括弱视、神经瘤、各种神经损伤及神经系统病变等疾病。
NGF与其他蛋白质药物相似，由于蛋白质半衰期较短，当暴露于极端温度、湿度条件下，或受物理、化学因素的影响，蛋白质的空间构象容易发生改变而引起变性，变性的蛋白质丧失了原有的生物活性；此外，因蛋白质常易粘附于固体表面，在灌装过程中，部分蛋白质会粘附于容器内壁上而造成活性成分的损失。为保证其生物活性，需加入稳定剂以防止其生物活性丧失。
通常，白蛋白作为一种优良的稳定剂以及块状(cake)成型剂被广泛应用于各种生物制品中。然而，由于白蛋白主要来源于人血、胎盘血，NGF组合物作为一种非经过灭菌的无菌制剂，血液中携带着一些不易检测的未知成分容易给制剂带来污染；同时，白蛋白的长期、大量应用也容易受到血源供应的限制以及生产成本的影响；再次，在中间体及制剂成品含量测定时，白蛋白可能会对相对少量的NGF产生干扰从而影响产品的品质管理。因此，为避免上述问题，需寻求一种稳定的去白蛋白NGF组合物。
发明公开
本发明的目的是提供一种神经生长因子组合物及注射粉剂，不仅对鼠源性神经生长因子(mNGF)具有保护作用，同时还能够保证人源性神经 生长因子(hNGF)以及重组人神经生长因子(rhNGF)在制备、运输及储存过程中保持良好的稳定性、具有更好的临床用药安全性及质量可控性。
本发明提供的一种神经生长因子组合物，其包括以下组分：
质量体积浓度为10μg/mL～100μg/mL的神经生长因子；
质量体积浓度为10mg/mL～80mg/mL的二糖类稳定剂；
质量体积浓度为0mg/mL～30mg/mL的氨基酸类稳定剂；
质量体积浓度为0.01mg/mL～1mg/mL的表面活性剂；
质量体积浓度为10mg/mL～50mg/mL的支持剂；
维持所述神经生长因子组合物的pH值为6.0～7.4的pH缓冲剂；
溶剂为水。
上述神经生长因子组合物中，稳定剂的添加可避免或降低由制备或储存过程中引起的蛋白质聚集和解聚，术语“稳定剂”是指一种能防止活性成分在水溶液中聚集或解聚的物质，除了稳定作用之外，稳定剂通常也可作为支持剂以改善产品的成型性，且不排除还有其它作用；
上述神经生长因子组合物中，优选地，所述二糖类稳定剂的质量体积浓度为30mg/mL～70mg/mL，更优选地，所述二糖类稳定剂的质量体积浓度为30mg/mL；
上述神经生长因子组合物中，所述二糖类稳定剂可为麦芽糖、海藻糖、蔗糖和乳糖中的至少一种；
上述神经生长因子组合物中，优选地，所述氨基酸类稳定剂的质量体积浓度为2mg/mL～10mg/mL，更优选地，所述氨基酸类稳定剂的质量体积浓度为3mg/mL～10mg/mL，再优选地，所述氨基酸类稳定剂的质量体积浓度为3mg/mL、10mg/mL。
上述神经生长因子组合物中，所述氨基酸类稳定剂可为丙氨酸、甘氨酸、精氨酸、谷氨酸、组氨酸和异亮氨酸中的至少一种。
上述神经生长因子组合物中，所述二糖类稳定剂和所述氨基酸类稳定剂具体可为下述1)-8)中任一种：
1)蔗糖和组氨酸、2)蔗糖和甘氨酸、3)蔗糖、甘氨酸和组氨酸、4)乳糖和甘氨酸、5)乳糖、蔗糖和异亮氨酸、6)海藻糖、精氨酸和组氨酸、7)麦芽糖、甘氨酸和组氨酸、8)麦芽糖、丙氨酸、精氨酸和谷氨 酸。
上述神经生长因子组合物中，由于蛋白质具有较高的与表面相互作用的倾向，这使它们在气-液、瓶-液界面容易经受吸附和/或变性，这种相互作用与蛋白浓度成反比，并导致形成可溶性的和不可溶的蛋白质聚集体、或者溶液中的蛋白质通过吸附到界面而损失，表面活性剂的加入可防止溶液表面相互作用引发的吸附和/或变性，使得由表面引发的吸附和/或变性被减至最低；这是因为表面活性剂为两性分子，对于界面位置能竞争过蛋白质，表面活性剂分子中的疏水部分占据界面位置(如气-液)，而分子中的亲水部分保持指向溶剂主体，在足够的浓度下(通常在表面活性剂的临界胶束浓度左右)，表面活性剂分子的表面层起到防止蛋白质分子在界面吸附的作用；
上述神经生长因子组合物中，优选地，所述表面活性剂的质量体积浓度为0.1mg/mL～0.5mg/mL，更优选地，所述表面活性剂的质量体积浓度为0.2mg/mL～0.5mg/mL，再优选地，所述表面活性剂的质量体积浓度为0.2mg/mL、0.5mg/mL；
上述神经生长因子组合物中，所述表面活性剂可为非离子表面活性剂，优选地，所述非离子表面活性剂可为泊洛沙姆、聚山梨酯和15-羟基硬脂酸聚乙二醇酯(简称HS 15，下同)中的至少一种，具体可为泊洛沙姆、聚山梨酯和HS 15中的任一种。
上述神经生长因子组合物中，所述支持剂的加入可改善成品的外观，保证本品冻干后具有较好的成型性；
上述神经生长因子组合物中，优选地，所述支持剂的质量体积浓度为20～50mg/mL，更优选地，所述支持剂的质量体积浓度为20mg/mL、50mg/mL；
上述神经生长因子组合物中，所述支持剂可为甘露醇、糊精和右旋糖酐中的任一种。
上述神经生长因子组合物中，为保证组合物具有最大的生物活性，通常需要控制一个最适的pH范围，这种稳定性的最适pH范围需要在处方筛选时确定，通常采用影响因素试验(光照、高温、高湿)、加速及长期稳定性试验等方法进行确定，确定处方后，组合物在生产及贮存过程中必须 保持在其最适pH范围，由于pH缓冲剂具有良好的缓冲能力，在一定范围内能保持产品pH的相对稳定，因此，处方中经常添加缓冲剂用于控制pH值；
上述神经生长因子组合物中，所述pH缓冲剂的摩尔浓度为10mM～50mM，优选地，所述pH缓冲剂的摩尔浓度为20mM～25mM，更优选地，所述pH缓冲剂的摩尔浓度为20mM、25mM；
上述神经生长因子组合物中，优选地，维持所述神经生长因子组合物的pH值为6.80～7.00，更优选地，维持所述神经生长因子组合物的pH值为6.86～6.91，再优选地，维持所述神经生长因子组合物的pH值为6.86，具体可为6.80、6.82、6.83、6.85、6.86、6.87、6.91、6.92或7.00；
上述神经生长因子组合物中，所述pH缓冲剂为磷酸盐、柠檬酸盐、醋酸盐、组氨酸盐酸盐和精氨酸盐酸盐中的至少一种，具体可为1)磷酸盐、2)柠檬酸盐、3)醋酸盐、4)组氨酸盐酸盐、或5)组氨酸盐酸盐和精氨酸盐酸盐，5)中组氨酸盐酸盐和精氨酸盐酸盐的摩尔比可为12：13。
上述神经生长因子组合物中，所述水为注射用水。
上述神经生长因子组合物中，所述神经生长因子是一种具有神经元营养和促进突起生长双重生物学功能的一种神经细胞生长调节因子，它对中枢及周围神经元的发育、分化、生长、再生和功能特性的表达均具有重要的调控作用；
上述神经生长因子组合物中，优选地，所述神经生长因子的质量体积浓度为40μg/mL～60μg/mL，更优选地，所述神经生长因子的质量体积浓度为50μg/mL；
上述神经生长因子组合物中，所述神经生长因子为鼠源性神经生长因子、人源性神经生长因子或重组人神经生长因子。
上述神经生长因子组合物中，各组分的浓度具体可为下述1)-17)中的任一种：
1)神经生长因子：40μg/mL～80μg/mL、二糖类稳定剂：10mg/mL～40mg/mL、氨基酸类稳定剂：0mg/mL～6mg/mL、表面活性剂：0.01mg/mL～0.2mg/mL、支持剂：10mg/mL～30mg/mL、pH缓冲剂： 10mM～25mM；
2)神经生长因子：60μg/mL～100μg/mL、二糖类稳定剂：40mg/mL～80mg/mL、氨基酸类稳定剂：6mg/mL～30mg/mL、表面活性剂：0.2mg/mL～1mg/mL、支持剂：30mg/mL～50mg/mL、pH缓冲剂：25mM～50mM；
3)神经生长因子：10μg/mL～50μg/mL、二糖类稳定剂：30mg/mL～50mg/mL、氨基酸类稳定剂：2mg/mL～10mg/mL、表面活性剂：0.05mg/mL～0.4mg/mL、支持剂：20mg/mL～40mg/mL、pH缓冲剂：20mM～30mM；
4)神经生长因子：10μg/mL～50μg/mL、二糖类稳定剂：35mg/mL～70mg/mL、氨基酸类稳定剂：3mg/mL～13mg/mL、表面活性剂：0.1mg/mL～0.5mg/mL、支持剂：20mg/mL～40mg/mL、pH缓冲剂：20mM～30mM；
5)神经生长因子：10μg/mL～50μg/mL、二糖类稳定剂：35mg/mL～70mg/mL、氨基酸类稳定剂：4mg/mL～14mg/mL、表面活性剂：0.1mg/mL～0.5mg/mL、支持剂：20mg/mL～40mg/mL、pH缓冲剂：20mM～30mM；
6)神经生长因子：40μg/mL～60μg/mL、二糖类稳定剂：30mg/mL～70mg/mL、氨基酸类稳定剂：0mg/mL～10mg/mL、表面活性剂：0.2mg/mL～0.5mg/mL、支持剂：20mg/mL～50mg/mL、pH缓冲剂：20mM～25mM；
7)神经生长因子：40μg/mL～60μg/mL、二糖类稳定剂：30mg/mL～70mg/mL、氨基酸类稳定剂：3mg/mL～10mg/mL、表面活性剂：0.2mg/mL～0.5mg/mL、支持剂：20mg/mL～50mg/mL、pH缓冲剂：20mM～25mM；
8)神经生长因子：10μg/mL、二糖类稳定剂：10mg/mL、氨基酸类稳定剂：0mg/mL、表面活性剂：0.01mg/mL、支持剂：10mg/mL、pH缓冲剂：10mM；
9)神经生长因子：40μg/mL、二糖类稳定剂：30mg/mL、氨基酸类稳定剂：2mg/mL、表面活性剂：0.05mg/mL、支持剂：20mg/mL、pH缓冲 剂：20mM；
10)神经生长因子：50μg/mL、二糖类稳定剂：35mg/mL、氨基酸类稳定剂：3mg/mL、表面活性剂：0.1mg/mL、支持剂：30mg/mL、pH缓冲剂：25mM；
11)神经生长因子：60μg/mL、二糖类稳定剂：40mg/mL、氨基酸类稳定剂：4mg/mL、表面活性剂：0.2mg/mL、支持剂：40mg/mL、pH缓冲剂：30mM；
12)神经生长因子：80μg/mL、二糖类稳定剂：50mg/mL、氨基酸类稳定剂：6mg/mL、表面活性剂：0.4mg/mL、支持剂：50mg/mL、pH缓冲剂：50mM；
13)神经生长因子：100μg/mL、二糖类稳定剂：70mg/mL、氨基酸类稳定剂：10mg/mL、表面活性剂：0.5mg/mL、支持剂：50mg/mL、pH缓冲剂：50mM；
14)神经生长因子：100μg/mL、二糖类稳定剂：80mg/mL、氨基酸类稳定剂：13mg/mL、表面活性剂：1mg/mL、支持剂：50mg/mL、pH缓冲剂：50mM；
15)神经生长因子：100μg/mL、二糖类稳定剂：80mg/mL、氨基酸类稳定剂：14mg/mL、表面活性剂：1μg/mL、支持剂：50mg/mL、pH缓冲剂：50mM；
16)神经生长因子：100μg/mL、二糖类稳定剂：80mg/mL、氨基酸类稳定剂：30mg/mL、表面活性剂：1mg/mL、支持剂：50mg/mL、pH缓冲剂：50mM；
17)神经生长因子：50μg/mL、二糖类稳定剂：30mg/mL、氨基酸类稳定剂：3mg/mL、表面活性剂：0.2mg/mL、支持剂：50mg/mL、pH缓冲剂：25mM；
18)神经生长因子：50μg/mL、二糖类稳定剂：30mg/mL、氨基酸类稳定剂：10mg/mL、表面活性剂：0.5mg/mL、支持剂：20mg/mL、pH缓冲剂：20mM。
本发明进一步提供了一种上述神经生长因子组合物注射粉剂的制备方法，将上述的神经生长因子组合物冷冻干燥即可得到所述神经生长因子组 合物注射粉剂。
本发明的另一个目的是提供一种上述制备方法制备得到的神经生长因子组合物注射粉剂，为注射给药。
上述神经生长因子组合物注射粉剂中，所述注射粉剂的含水率可为1.2％～3.0％，具体可为1.3％、1.7％、1.9％、2.2％、2.3％、2.4％、2.5％、2.7或2.8％；pH值可为6.80～7.20，具体可为6.80、6.82、6.83、6.85、6.92、6.93、6.95、7.05、7.10或7.20。
上述神经生长因子组合物在治疗神经损伤或制备治疗神经损伤的药物中的应用，也在本发明的保护范围内。所述神经生长因子组合物具体可为神经生长因子组合物注射粉剂。所述神经损伤可以是视神经损伤，其致伤原因具体可为拳脚伤、车祸、物体崩伤或眼部爆炸伤。
本发明进一步提供了一种治疗神经损伤的方法，它包括如下步骤：给神经损伤患者施用有效剂量的神经生长因子组合物。所述神经生长因子组合物具体可为神经生长因子组合物注射粉剂。所述施用具体可为肌肉注射。所述神经损伤可以是视神经损伤，其致伤原因具体可为拳脚伤、车祸、物体崩伤或眼部爆炸伤。
附图说明
图1为在加速条件下(25℃，RH 60±10％)，实施例中制备得到的NGF组合物注射粉剂及参比制剂中NGF的平均含量随时间的变化曲线。
图2为在加速条件下(25℃，RH 60±10％)，实施例中制备得到的NGF组合物注射粉剂及参比制剂的平均活性随时间的变化曲线。
图3为在长期稳定性条件下(6±2℃)，实施例中制备得到的NGF组合物注射粉剂及参比制剂中NGF的平均含量随时间的变化曲线。
图4为在长期稳定性条件下(6±2℃)，实施例中制备得到的NGF组合物注射粉剂及参比制剂的平均活性随时间的变化曲线。
实施发明的最佳方式
为使本领域的技术人员更好地理解本发明的技术方案，下面，结合实施例及试验例对本发明的具体实施方式做进一步阐述，但是这些实施例不能被解释为限制本发明。
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
下述实施例中所使用的mNGF及hNGF原液由舒泰神(北京)生物制药股份有限公司提供，rhNGF原液由北京义翘神州生物技术有限公司提供，其余辅料除非特别说明，均为注射级别。
下述实施例中表面活性剂的浓度的单位均采用％表示，如0.1％表示1mg/mL。
为了寻找替代白蛋白的物质作为NGF的稳定剂，本发明进行了大量探索性实验，作为本品的稳定剂使用的麦芽糖、海藻糖、蔗糖、乳糖、丙氨酸、甘氨酸、精氨酸、谷氨酸、组氨酸、异亮氨酸以及作为表面活性剂使用的聚山梨酯-20(简称TW-20，下同)、聚山梨酯-80(简称TW-80，下同)、泊洛沙姆188(简称F68，下同)、HS 15制备了各种NGF组合物及注射粉剂，并测定了成品的水分、pH、渗透压、含量及活性。
实施例1、NGF组合物及注射粉剂的制备
按下述步骤制备NGF组合物及注射粉剂：
按照表1中处方组成称取处方量柠檬酸盐，加入适量注射用水并搅拌至完全溶解，再加入处方量的海藻糖、甘露醇及F68搅拌至完全溶解，加入hNGF原液，用NaOH或柠檬酸调pH至表1中所示pH 6.85，并加注射用水定容至刻度，混合均匀后，经0.22μm微孔滤膜过滤至无菌容器中，制备成NGF浓度约为80μg/mL的组合物。
将上述组合物，按0.63±0.03mL/瓶分装至硼硅玻璃注射瓶中，冷冻干燥(制得hNGF注射用无菌粉末即hNGF注射粉剂。
实施例2、NGF组合物及注射粉剂的制备
按下述步骤制备NGF组合物及注射粉剂：
按照表1处方组成称取处方量磷酸盐，加入适量注射用水并搅拌至完全溶解，再加入处方量的蔗糖、甘氨酸、组氨酸和右旋糖酐搅拌至完全溶解，由于处方中TW-20在注射用水中呈球形聚集状态，不易混合均匀，本发明先将TW-20溶解于热的注射用水(40～80℃)中配制成1％水溶液，待冷却至室温后按处方量经折算后加入上述溶液中混合均匀，最后加入rhNGF原液，用NaOH或磷酸调pH至表1中所示pH 7.00，并加注射用水 定容至刻度，混合均匀后，经0.22μm微孔滤膜过滤至无菌容器中，制备成NGF浓度约为50μg/mL的组合物。
将上述组合物，按0.63±0.03mL/瓶分装至硼硅玻璃注射瓶中，冷冻干燥制得rhNGF注射用无菌粉末即rhNGF注射粉剂。
实施例3、NGF组合物及注射粉剂的制备
按下述步骤制备NGF组合物及注射粉剂：
按照表1处方组成称取处方量醋酸盐，加入适量注射用水并搅拌至完全溶解，再加入处方量的麦芽糖、丙氨酸、精氨酸、谷氨酸和甘露醇搅拌至完全溶解，由于处方中HS 15在注射用水中呈球形聚集状态，不易混合均匀，本发明先将HS 15溶解于热的注射用水(40～80℃)中配制成1％水溶液，待冷却至室温后按处方量经折算后加入上述溶液中混合均匀，最后加入hNGF原液，用NaOH或醋酸调pH至表1中所示pH 6.92，并加注射用水定容至刻度，混合均匀后，经0.22μm微孔滤膜过滤至无菌容器中，制备成NGF浓度约为40μg/mL的组合物。
将上述组合物，按0.63±0.03mL/瓶分装至硼硅玻璃注射瓶中，冷冻干燥制得hNGF注射用无菌粉末即hNGF注射粉剂。
实施例4、NGF组合物及注射粉剂的制备
按下述步骤制备NGF组合物及注射粉剂：
按照表1处方组成称取处方量组氨酸盐酸盐，加入适量注射用水并搅拌至完全溶解，再加入处方量的蔗糖、组氨酸和甘露醇搅拌至完全溶解，由于处方中TW-20在注射用水中呈球形聚集状态，不易混合均匀，本发明先将TW-20溶解于热的注射用水(40～80℃)中配制成1％水溶液，待冷却至室温后按处方量经折算后加入上述溶液中混合均匀，最后加入hNGF原液，用HCl或L-组氨酸调pH至表1中所示pH 6.87，并加注射用水定容至刻度，混合均匀后，经0.22μm微孔滤膜过滤至无菌容器中，制备成NGF浓度约为60μg/mL的组合物。
将上述组合物，按0.63±0.03mL/瓶分装至硼硅玻璃注射瓶中，冷冻干燥制得hNGF注射用无菌粉末即hNGF注射粉剂。
实施例5、NGF组合物及注射粉剂的制备
按下述步骤制备NGF组合物及注射粉剂：
按照表1处方组成称取处方量柠檬酸盐，加入适量注射用水并搅拌至完全溶解，再加入处方量的海藻糖、蔗糖甘露醇搅拌至完全溶解，由于处方中TW-80在注射用水中呈球形聚集状态，不易混合均匀，本发明先将TW-80溶解于热的注射用水(40～80℃)中配制成1％水溶液，待冷却至室温后按处方量经折算后加入上述溶液中混合均匀，最后加入mNGF原液，用NaOH或柠檬酸调pH至表1中所示pH 6.82，并加注射用水定容至刻度，混合均匀后，经0.22μm微孔滤膜过滤至无菌容器中，制备成NGF浓度约为60μg/mL的组合物。
将上述组合物，按0.63±0.03mL/瓶分装至硼硅玻璃注射瓶中，冷冻干燥制得mNGF注射用无菌粉末即mNGF注射粉剂。
实施例6、NGF组合物及注射粉剂的制备
按下述步骤制备NGF组合物及注射粉剂：
按照表1处方组成称取处方量磷酸盐，加入适量注射用水并搅拌至完全溶解，再加入处方量的蔗糖、甘氨酸和甘露醇搅拌至完全溶解，由于处方中TW-80在注射用水中呈球形聚集状态，不易混合均匀，本发明先将TW-80溶解于热的注射用水(40～80℃)中配制成1％水溶液，待冷却至室温后按处方量经折算后加入上述溶液中混合均匀，最后加入hNGF原液，用NaOH或磷酸调pH至表1中所示pH 6.91，并加注射用水定容至刻度，混合均匀后，经0.22μm微孔滤膜过滤至无菌容器中，制备成NGF浓度约为50μg/mL的组合物。
将上述组合物，按0.63±0.03mL/瓶分装至硼硅玻璃注射瓶中，冷冻干燥制得hNGF注射用无菌粉末即hNGF注射粉剂。
实施例7、NGF组合物及注射粉剂的制备
按下述步骤制备NGF组合物及注射粉剂：
按照表1处方组成称取处方量组氨酸盐酸盐，加入适量注射用水并搅拌至完全溶解，再加入处方量的麦芽糖、甘氨酸、组氨酸和糊精搅拌至完全溶解，由于处方中TW-80在注射用水中呈球形聚集状态，不易混合均匀，本发明先将TW-80溶解于热的注射用水(40～80℃)中配制成1％水溶液，待冷却至室温后按处方量经折算后加入上述溶液中混合均匀，最后加入rhNGF原液，用HCl或L-组氨酸调pH至表1中所示pH 6.80，并加 注射用水定容至刻度，混合均匀后，经0.22μm微孔滤膜过滤至无菌容器中，制备成NGF浓度约为100μg/mL的组合物。
将上述组合物，按0.63±0.03mL/瓶分装至硼硅玻璃注射瓶中，冷冻干燥制得rhNGF注射用无菌粉末即rhNGF注射粉剂。
实施例8、NGF组合物及注射粉剂的制备
按下述步骤制备NGF组合物及注射粉剂：
按照表1处方组成称取处方量柠檬酸盐，加入适量注射用水并搅拌至完全溶解，再加入处方量的海藻糖和甘露醇搅拌至完全溶解，由于处方中HS 15在注射用水中呈球形聚集状态，不易混合均匀，本发明先将HS 15溶解于热的注射用水(40～80℃)中配制成1％水溶液，待冷却至室温后按处方量经折算后加入上述溶液中混合均匀，最后加入hNGF原液，用NaOH或柠檬酸调pH至表1中所示pH 6.86，并加注射用水定容至刻度，混合均匀后，经0.22μm微孔滤膜过滤至无菌容器中，制备成NGF浓度约为40μg/mL的组合物。
将上述组合物，按0.63±0.03mL/瓶分装至硼硅玻璃注射瓶中，冷冻干燥制得hNGF注射用无菌粉末即hNGF注射粉剂。
实施例9、NGF组合物及注射粉剂的制备
按下述步骤制备NGF组合物及注射粉剂：
按照表1处方组成称取处方量磷酸盐，加入适量注射用水并搅拌至完全溶解，再加入处方量的乳糖、甘氨酸和甘露醇搅拌至完全溶解，由于处方中TW-20在注射用水中呈球形聚集状态，不易混合均匀，本发明先将TW-20溶解于热的注射用水(40～80℃)中配制成1％水溶液，待冷却至室温后按处方量经折算后加入上述溶液中混合均匀，最后加入hNGF原液，用NaOH或磷酸调pH至表1中所示pH 6.86，并加注射用水定容至刻度，混合均匀后，经0.22μm微孔滤膜过滤至无菌容器中，制备成NGF浓度约为50μg/mL的组合物。
将上述组合物，按0.63±0.03mL/瓶分装至硼硅玻璃注射瓶中，冷冻干燥制得hNGF注射用无菌粉末即hNGF注射粉剂。
实施例10、NGF组合物及注射粉剂的制备
按下述步骤制备NGF组合物及注射粉剂：
按照表1处方组成称取处方量组氨酸盐酸盐和精氨酸盐酸盐，加入适量注射用水并搅拌至完全溶解，再加入处方量的海藻糖、精氨酸、组氨酸和甘露醇搅拌至完全溶解，由于处方中TW-20在注射用水中呈球形聚集状态，不易混合均匀，本发明先将TW-20溶解于热的注射用水(40～80℃)中配制成1％水溶液，待冷却至室温后按处方量经折算后加入上述溶液中混合均匀，最后加入rhNGF原液，用HCl或L-组氨酸或L-精氨酸调pH至表1中所示pH 6.83，并加注射用水定容至刻度，混合均匀后，经0.22μm微孔滤膜过滤至无菌容器中，制备成NGF浓度约为60μg/mL的组合物。
将上述组合物，按0.63±0.03mL/瓶分装至硼硅玻璃注射瓶中，冷冻干燥制得rhNGF注射用无菌粉末即rhNGF注射粉剂。
实施例11、NGF组合物及注射粉剂的制备
按下述步骤制备NGF组合物及注射粉剂：
按照表1中处方组成称取处方量磷酸盐，加入适量注射用水并搅拌至完全溶解，再加入处方量的乳糖、蔗糖、异亮氨酸、甘露醇及F68搅拌至完全溶解，加入mNGF原液，用NaOH或磷酸调pH至表1中所示pH6.85，并加注射用水定容至刻度，混合均匀后，经0.22μm微孔滤膜过滤至无菌容器中，制备成NGF浓度约为10μg/mL的组合物。
将上述组合物，按0.63±0.03mL/瓶分装至硼硅玻璃注射瓶中，冷冻干燥制得mNGF注射用无菌粉末即mNGF注射粉剂。
表1、实施例1-11中各组分的用量
Figure PCTCN2016079571-appb-000001
Figure PCTCN2016079571-appb-000002
Figure PCTCN2016079571-appb-000003
实施例12、实施例1-11中制备得到的NGF组合物注射粉剂性能测试
(1)外观、水分、渗透压、pH、含量及活性测试
分别观察实施例1-11中制备得到的NGF组合物注射粉剂及参比制剂(注射用鼠神经生长因子，商品名：苏肽生，由舒泰神(北京)生物制药股份有限公司提供)的外观，测定其水分、渗透压、pH、含量及活性。其中，NGF含量的测定方法为按照专利号200510130348.3，专利名称为“测定神经生长因子含量的方法”中实施例1所述方法进行操作。活性测试方法采用细胞法进行测定，详细操作方法按照专利公开号为CN103376248A，专利名称为“神经生长因子活性定量测定方法”中实施例1中的方法进行操作。实验结果如表2所示。
表2各实施例检查项目检测结果
Figure PCTCN2016079571-appb-000004
由上述表2可知，本发明制备得到的NGF组合物注射粉剂各项指标均符合质量标准要求。
(2)稳定pH范围筛选
根据实施例2中处方组成配制3份NGF组合物，保持磷酸盐总量不 变，调节NaH2PO4·H2O与Na2HPO4用量，使NGF组合物pH值分别为6.0、6.8及7.4，分别灌装于西林瓶中，冷冻干燥制成注射用无菌粉末。取样，加入适量灭菌注射用水配制成溶液，置于室温条件下，分别于第0、4、8、12、24h观察外观、测定pH、含量及活性变化情况，测定结果如下表所示。
表3 NGF组合物稳定pH范围筛选结果
Figure PCTCN2016079571-appb-000005
由上表数据可知，三种不同pH的NGF组合物在室温下放置24h后，各项指标较0h无明显变化，均符合质量标准要求，说明室温条件下样品溶液pH在6.0～7.4范围内24h内能保持稳定，优选pH为6.8。
(3)稳定性测试
A、加速稳定性试验
根据《生物制品稳定性研究技术指导原则》要求，将参比制剂(苏肽生)与实施例制备得到的分装的NGF组合物注射粉剂样品置于25℃，RH 60±10％条件下进行0～6月加速稳定性考察，观察样品外观，进行水分、渗 透压、pH、含量及活性的测试，结果见表4及图1(平均含量随时间的变化曲线)、图2(平均活性随时间的变化曲线)。
表4 NGF组合物注射粉剂及参比制剂加速稳定性试验结果
Figure PCTCN2016079571-appb-000006
Figure PCTCN2016079571-appb-000007
Figure PCTCN2016079571-appb-000008
Figure PCTCN2016079571-appb-000009
由图1可以看出，在加速条件下(25℃，RH 60±10％)下放置，随着时间的变化，参比制剂、实施例制备得到的NGF组合物注射粉剂中NGF的平均含量呈下降趋势，其中，参比制剂中NGF的平均含量的下降速度明显大于本发明实施例。由表4结果可知，在加速条件(25℃，RH 60±10％)下放置6个月，各样品外观、水分、pH及渗透压无明显变化，而参比制剂含量降低约10.5％，实施例1～11的NGF组合物注射粉剂含量降低率分别为7.8％、6.1％、8.0％、3.2％、5.5％、3.6％、7.9％、3.0％、2.4％、5.2％及8.1％。说明加速条件下本发明实施例的稳定性由于参比制剂。其中，实施例4、6、8、9含量降低率分别为3.2％、3.6％、3.0％、2.4％，远低于参比制剂，说明加速条件下实施例4、6、8、9稳定性明显优于参比制剂。
由图2可以看出，在加速条件下(25℃，RH 60±10％)下放置，随着时间的变化，参比制剂、实施例制备得到的分装的NGF组合物注射粉剂中NGF的平均活性整体呈下降趋势，其中，参比制剂的活性在加速两个月后迅速下降。由表4结果可知，在加速条件(25℃，RH 60±10％)下放置6个月，各样品外观、水分、pH及渗透压无明显变化，参比制剂的活性下降17.7％，实施例1～11的NGF组合物注射粉剂活性分别下降14.5％、12.5％、15.3％、8.9％、10.6％、9.2％、12.9％、6.3％、5.9％、11.1％及13.8％，说明加速条件下本发明实施例的稳定性由于参比制剂。其中，加速条件下，实施例4、实施例6、实施例8和实施例9活性降低率分别为8.9％、9.2％、6.3％、5.9％，远低于参比制剂，说明加速条件下实施例4、实施例6、实施例8和实施例9稳定性明显优于参比制剂。
B、长期稳定性试验
根据《生物制品稳定性研究技术指导原则》要求，将参比制剂(苏肽生)与实施例4、实施例6、实施例8、实施例9制备得到的分装的NGF注射粉剂样品置于6±2℃条件下进行0～12月长期稳定性考察，观察样品外观，测定水分、渗透压、pH、含量及活性，结果见表5及图3(平均含量随时间变化曲线)、图4(平均活性随时间变化曲线)。
表5NGF组合物注射粉剂及参比制剂长期稳定性试验结果
Figure PCTCN2016079571-appb-000010
Figure PCTCN2016079571-appb-000011
Figure PCTCN2016079571-appb-000012
Figure PCTCN2016079571-appb-000013
Figure PCTCN2016079571-appb-000014
由图3可以看出，在长期稳定性条件下(6±2℃)下放置，随着时间的变化，参比制剂、实施例制备得到的分装的NGF组合物注射粉剂中NGF的平均含量均呈下降趋势，其中，参比制剂中NGF的平均含量的下降速度明显大于本发明实施例。由表5结果可知，在长期条件下放置12个月，各样品外观、水分、pH及渗透压无明显变化，而参比制剂含量降低约6.7％，实施例1～11的NGF组合物注射粉剂含量降低率分别为6.3％、5.1％、6.2％、2.8％、5.1％、3.0％、6.1％、2.6％、2.0％、4.5％及6.0％。说明在长期稳定性 条件下(6±2℃)下本发明实施例的稳定性优于参比制剂。其中，实施例4、实施例6、实施例8、实施例9含量降低率分别为2.8％、3.0％、2.6％、2.0％，远低于参比制剂，说明长期条件下，实施例4、实施例6、实施例8、实施例9稳定性明显优于参比制剂。
由图4可以看出，在长期稳定性条件下(6±2℃)下放置，随着时间的变化，参比制剂、实施例中制备得到的分装的NGF组合物注射粉剂中NGF的平均活性整体均呈下降趋势，其中，参比制剂的活性的下降速度大于本发明实施例。由表4结果可知，在长期条件下放置12个月，各样品外观、水分、pH及渗透压无明显变化，参比制剂的活性下降12.8％，实施例1～11的NGF组合物注射粉剂的活性分别下降10.9％、9.6％、10.3％、4.8％、8.7％、5.4％、9.2％、3.8％、3.4％、8.1％及10.2％，说明说明在长期稳定性条件下(6±2℃)下本发明实施例的稳定性优于参比制剂。其中，实施例4、实施例6、实施例8、实施例9的活性分别下降4.8％、5.4％、3.8％、3.4％，远低于参比制剂，说明长期条件下，实施例4、实施例6、实施例8、实施例9稳定性明显优于参比制剂。
(4)临床评价
采用多中心非随机对照研究设计，271名视神经损伤患者进行为期12周的临床试验，受试者年龄为14周岁以上，所有受试者均能够与研究者进行良好沟通，理解并服从本临床试验要求，能够理解本临床试验并签署知情同意书。
受试者条件：14周岁以上。
性别：男性或女性。
受试者来源：在以各大眼科研究单位纳入各种原因所致的视神经损伤患者409例。
致伤原因：拳脚伤136例(136眼)，车祸152例(152眼)，物体崩伤73例(73眼)，眼部爆炸伤49例(49眼)。所有病例均不伴眼球破裂伤及眶壁骨折压迫视神经所致损伤。
治疗方法：治疗组分别采用采用参比制剂及本发明NGF组合物注射粉剂进行肌肉注射(注射用水)，每日1次，每次30μg，所有病例连续给药12周。安慰剂组采用阴性对照(不含活性成分，其余辅料同NGF组合 物)。
治疗组271例(271眼)，年龄18～63岁，平均34.2岁；男189例(189眼)，女82例(82眼)；右眼147眼，左眼124眼。安慰剂组138例(138眼)，年龄13～55岁，平均33.9岁，男96例(96眼)，女42例(42眼)；右眼45眼，左眼93眼。
分组设计，按下表所示进行分组：
表6临床试验分组及受试者数量
Figure PCTCN2016079571-appb-000015
注：采用实施例9制得的神经生长因子组合物，下同。
临床常见不良反应评价：按安慰剂组、参比制剂组及NGF组合物组分别给药，观察各组不良反应症状及发生率，结果如下表所示。
表7 NGF临床常见不良反应
Figure PCTCN2016079571-appb-000016
注：*为与安慰剂组相比，有显著差异(P<0.05)；#为与参比制剂组相比，有显著差异(P<0.05)。
由上表结果可知，与安慰剂组相比，本发明制备的NGF组合物主要的不良反应为局部疼痛、荨麻疹和头晕；与参比制剂相比，本发明制备的NGF组合物能显著减轻局部疼痛、荨麻疹、头晕等不良反应。
临床疗效：按安慰剂组、参比制剂组及NGF组合物组分别给药，观察各组的临床疗效，结果如下表所示。
表8 NGF临床疗效对比
Figure PCTCN2016079571-appb-000017
上述表格中的临床疗效采用如下临床疗效评价标准进行评价：
①痊愈：视力恢复到1.0以上，视野中心暗点消失；
②显效：视力提高4行上，视野中心暗点缩小或绝对暗点变为相对暗点；
③好转：视力提高2行以上，视野无变化；
④无效：视力和视野与治疗前相同或有退步。
由上表的结果可知，与安慰剂组相比，本发明的NGF组合物及其注射粉剂能够有效治疗各种原因所致的视神经损伤，对视神经损伤治疗的综合有效率优于参比制剂参比制剂组，能够明显降低不良反应发生率，临床治疗效果好。
虽然，上文中已经用一般性说明、具体实施方案及实施例对本发明做了详尽的描述，但在本发明的基础上，所属技术领域的技术人员可以在不偏离本发明主旨的范围内对之进行修改和改进，这些修改或改进均属于本发明要求保护的范围。
工业应用
本发明制备得到的神经生长因子组合物注射粉剂，在制备、运输及储存过程中均保持良好的稳定性：(1)在制备过程中：本发明制备得到的神经生长因子组合物注射粉剂在室温条件下放置24小时后，其中NGF的含量和活性均无明显变化；(2)与参比制剂相比，在常规(6±2℃)的运输及储存过程中，本发明NGF组合物注射粉剂在12月后，其有效含量仅降低2.0％～6.3％，优选降低2.0～3.0％，其活性仅降低3.4％～10.9％，具有优异的稳定性。
本发明制备得到的神经生长因子组合物注射粉剂，在临床试验中，能够明显降低不良反应发生率，临床治疗效果好，与现有的参比制剂相比，具有更好的临床用药安全性及质量可控性。
本发明具有如下有益效果：
(1)本发明神经生长因子组合物及注射粉剂，通过使用二糖类或二糖类与氨基酸组合代替白蛋白作为稳定剂，能够避免因白蛋白中携带病毒或其他未知成分带来的潜在风险。
(2)本发明神经生长因子组合物注射粉剂，不仅对鼠源性神经生长因子(mNGF)具有保护作用，同时能够保证人源性神经生长因子(hNGF)以及重组人神经生长因子(rhNGF)在制备、运输及储存过程中保持良好的稳定性、更好的临床用药安全性及质量可控性。
(3)本发明神经生长因子组合物及注射粉剂中，成分明确，易于定性及定量，所用稳定剂纯度高，来源广，容易进行长期大量生产，便于控制成本及提高产品质量。

Claims (17)

  1. 一种神经生长因子组合物，其特征在于：所述神经生长因子组合物包括以下组分：
    质量体积浓度为10μg/mL～100μg/mL的神经生长因子；
    质量体积浓度为10mg/mL～80mg/mL的二糖类稳定剂；
    质量体积浓度为0mg/mL～30mg/mL的氨基酸类稳定剂；
    质量体积浓度为0.01mg/mL～1mg/mL的表面活性剂；
    质量体积浓度为10mg/mL～50mg/mL的支持剂；
    维持所述神经生长因子组合物的pH值为6.0～7.4的pH缓冲剂；
    溶剂为水。
  2. 根据权利要求1所述的神经生长因子组合物，其特征在于：所述二糖类稳定剂的质量体积浓度为30mg/mL～70mg/mL。
  3. 根据权利要求1或2所述的神经生长因子组合物，其特征在于：所述二糖类稳定剂为麦芽糖、海藻糖、蔗糖和乳糖中的至少一种。
  4. 根据权利要求1-3任一项所述的神经生长因子组合物，其特征在于：所述氨基酸类稳定剂的质量体积浓度为2mg/mL～10mg/mL，优选地，所述氨基酸类稳定剂的质量体积浓度为3mg/mL～10mg/mL。
  5. 根据权利要求1-4任一项所述的神经生长因子组合物，其特征在于：所述氨基酸类稳定剂为丙氨酸、甘氨酸、精氨酸、谷氨酸、组氨酸和异亮氨酸中的至少一种。
  6. 根据权利要求1-5任一项所述的神经生长因子组合物，其特征在于：所述氨基酸类稳定剂为丙氨酸、甘氨酸、精氨酸、谷氨酸、组氨酸和异亮氨酸中的至少一种；优选地，所述二糖类稳定剂和氨基酸类稳定剂为下述1)-8)中任一种：
    1)蔗糖和组氨酸、2)蔗糖和甘氨酸、3)蔗糖、甘氨酸和组氨酸、4)乳糖和甘氨酸、5)乳糖、蔗糖和异亮氨酸、6)海藻糖、精氨酸和组氨酸、7)麦芽糖、甘氨酸和组氨酸、8)麦芽糖、丙氨酸、精氨酸和谷氨酸。
  7. 根据权利要求1-6任一项所述的神经生长因子组合物，其特征在于：所述表面活性剂的质量体积浓度为0.1mg/mL～0.5mg/mL。
  8. 根据权利要求1-7任一项所述的神经生长因子组合物，其特征在 于：所述表面活性剂为非离子表面活性剂，优选地，所述非离子表面活性剂为泊洛沙姆、聚山梨酯和15-羟基硬脂酸聚乙二醇酯中的至少一种。
  9. 根据权利要求1-8任一项所述的神经生长因子组合物，其特征在于：所述支持剂的质量体积浓度为20～50mg/mL。
  10. 根据权利要求1-9任一项所述的神经生长因子组合物，其特征在于：所述支持剂为甘露醇、糊精和右旋糖酐中的任一种。
  11. 根据权利要求1-10任一项所述的神经生长因子组合物，其特征在于：所述pH缓冲剂的摩尔浓度为10mM～50mM的，优选地，所述pH缓冲剂的摩尔浓度为20mM～25mM；所述pH缓冲剂为磷酸盐、柠檬酸盐、醋酸盐、组氨酸盐酸盐和精氨酸盐酸盐中的至少一种；
    所述水为注射用水。
  12. 根据权利要求1-11中任一项所述的神经生长因子组合物，其特征在于：所述神经生长因子的质量体积浓度为40μg/mL～60μg/mL；
    优选地，所述神经生长因子为鼠源性神经生长因子、人源性神经生长因子或重组人神经生长因子。
  13. 根据权利要求1-12中任一项所述的神经生长因子组合物，其特征在于：所述神经生长因子组合物中：
    所述神经生长因子的质量体积浓度为50μg/mL；
    所述二糖类稳定剂的质量体积浓度为30mg/mL，所述氨基酸类稳定剂的质量体积浓度为10mg/mL；
    所述表面活性剂的质量体积浓度为0.5mg/mL；
    所述支持剂的质量体积浓度为20mg/mL；
    所述pH缓冲剂的摩尔浓度为20mM。
  14. 一种神经生长因子组合物注射粉剂的制备方法，其特征在于：将权利要求1-13中任一项所述的神经生长因子组合物冷冻干燥即可得到所述神经生长因子组合物注射粉剂。
  15. 权利要求14所述的制备方法制备得到的神经生长因子组合物注射粉剂。
  16. 权利要求1-13中任一项所述的神经生长因子组合物在治疗神经损伤或制备治疗神经损伤的药物中的应用，其中神经损伤可以是视神经损 伤。
  17. 一种治疗神经损伤的方法，它包括如下步骤：给神经损伤患者施用有效剂量的权利要求1-13中任一项所述的神经生长因子组合物，其中神经损伤可以是视神经损伤。
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EP3287140B1 (en) 2021-06-02
CN107660149A (zh) 2018-02-02
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