WO2016169455A1 - 神经生长因子组合物和注射粉剂 - Google Patents

神经生长因子组合物和注射粉剂 Download PDF

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WO2016169455A1
WO2016169455A1 PCT/CN2016/079572 CN2016079572W WO2016169455A1 WO 2016169455 A1 WO2016169455 A1 WO 2016169455A1 CN 2016079572 W CN2016079572 W CN 2016079572W WO 2016169455 A1 WO2016169455 A1 WO 2016169455A1
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growth factor
nerve growth
composition
nerve
factor composition
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PCT/CN2016/079572
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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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Priority to DK16782602.3T priority Critical patent/DK3287141T3/da
Priority to CN201680023059.XA priority patent/CN107708723B/zh
Priority to US15/568,450 priority patent/US20180110833A1/en
Priority to ES16782602T priority patent/ES2893952T3/es
Priority to EP16782602.3A priority patent/EP3287141B1/en
Publication of WO2016169455A1 publication Critical patent/WO2016169455A1/zh

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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
    • A61K38/16—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
    • 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
    • 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
    • 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
    • A61K47/06—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
    • 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, and belongs 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 human nerve growth factor (hNGF) and recombinant human nerve growth factor (rhNGF). ) in preparation, transportation and storage The process maintains good stability, better clinical drug safety and quality controllability.
  • mNGF mouse-derived nerve growth factor
  • hNGF human nerve growth factor
  • rhNGF recombinant human nerve growth factor
  • the invention provides a nerve growth factor composition, which comprises a nerve growth factor, a stabilizer, a support agent, a pH buffering agent and water;
  • the stabilizers are arginine, glutamic acid, glycine and isoleucine.
  • the mass concentration of the nerve growth factor is from 10 ⁇ g / mL to 100 ⁇ g / mL;
  • the mass concentration of the stabilizer is 6 mg / mL ⁇ 30 mg / mL;
  • the mass concentration of the support agent is from 20 mg/mL to 50 mg/mL;
  • the pH buffer maintains the pH of the nerve growth factor composition from 6.80 to 7.00;
  • 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 stabilizer has a mass volume concentration of 10 mg/mL to 20 mg/mL, and more preferably, the stabilizer has a mass volume concentration of 13 mg/mL;
  • the mass ratio of the components of arginine, glutamic acid, glycine and isoleucine in the stabilizer is 1: (1 to 3): (1 to 3): (1 to 3.6), specifically 1: (1 to 1.3): (1 to 1.8): (1 to 1.3), 1: (1.3 to 3): (1.8 to 3.0): (1.3 to 3.6), 1: (1.2) ⁇ 2): (1.5 to 2.0): (1.2 to 1.5), 1: (1.25 to 2.8): (1.6 to 2.8): (1.25 to 3.0), 1:1:1:1, 1:1.2:3: 1.3, 1:2:3:1, 1:1.25:1.5:1.25, 1:1:2.8:1.5, 1:2:2:3.6, 1:1.3:3:1.3, 1:3:3:3, 1:1.2:1.6:1.2 or 1:2.8:1.8:1.5, preferably, the mass ratio of each component of arginine, glutamic acid, glycine and isoleucine in the stabilizer is 1:1.25:1.5: 1.25
  • 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 from 30 mg/mL to 50 mg/mL, and more preferably, the mass concentration of the support agent is 50. Mg/mL;
  • the support agent is any one of mannitol, dextrin, and sorbitol.
  • 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 ( The method of light, high temperature, high humidity), acceleration and long-term stability test is determined.
  • the composition After the prescription is determined, the composition must be kept at its optimum pH range during production and storage. Because the buffer has a good buffering capacity, the pH of the product can be kept relatively stable within a certain range. Therefore, a buffer is often added to the prescription for controlling 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 25 mM.
  • the pH of the nerve growth factor composition is maintained at 6.81 to 6.91, and more preferably, the pH of the nerve growth factor composition is maintained at 6.82.
  • the pH buffering agent is selected from one or more of a phosphate, a citrate, an acetate, a histidine hydrochloride, and an arginine hydrochloride, and specifically Any of the following 1)-5): 1) arginine hydrochloride, 2) phosphate, 3) citrate, 4) acetate, 5) histidine hydrochloride and arginine Hydrochloride.
  • 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 mass concentration of the nerve growth factor is from 40 ⁇ g/mL to 80 ⁇ g/mL, and more preferably, the mass concentration of the nerve growth factor is 50 ⁇ g/mL.
  • the nerve growth factor is selected from the group consisting of a murine nerve growth factor, a human nerve growth factor, or a recombinant human nerve growth factor.
  • the concentration of each component may specifically be the following 1) -15) Any of them:
  • nerve growth factor 10 ⁇ 50 ⁇ g / mL
  • stabilizer 6 ⁇ 13mg / mL
  • support agent 20mg / mL ⁇ 40mg / mL
  • pH buffer 10mM ⁇ 25mM
  • nerve growth factor 60 ⁇ 100 ⁇ g / mL
  • stabilizer 6.5 ⁇ 14mg / mL
  • support agent 30mg / mL ⁇ 50mg / mL
  • pH buffer 25mM ⁇ 50mM
  • nerve growth factor 40 ⁇ 80 ⁇ g / mL, stabilizer: 8.9 ⁇ 15mg / mL, support agent: 30mg / mL ⁇ 50mg / mL, pH buffer: 20mM ⁇ 30mM;
  • nerve growth factor 40 ⁇ 80 ⁇ g / mL
  • stabilizer 10 ⁇ 20mg / mL
  • support agent 30mg / mL ⁇ 50mg / mL
  • pH buffer 20mM ⁇ 25mM;
  • nerve growth factor 50 ⁇ 80 ⁇ g / mL
  • stabilizer 10 ⁇ 20mg / mL
  • support agent 40mg / mL ⁇ 50mg / mL
  • pH buffer 20mM ⁇ 25mM;
  • nerve growth factor 10 ⁇ g / mL
  • stabilizer 6 mg / mL
  • support agent 20 mg / mL
  • pH buffer 10 mM
  • nerve growth factor 40 ⁇ g / mL
  • stabilizer 6.5 mg / mL
  • support agent 30 mg / mL
  • pH buffer 20 mM
  • nerve growth factor 50 ⁇ g / mL, stabilizer: 8.9 mg / mL, support agent: 40 mg / mL, pH buffer: 25 mM;
  • nerve growth factor 60 ⁇ g / mL
  • stabilizer 10 mg / mL
  • support agent 50 mg / mL
  • pH buffer 30 mM
  • nerve growth factor 80 ⁇ g / mL, stabilizer: 13 mg / mL, support agent: 50 mg / mL, pH buffer: 30 mM;
  • nerve growth factor 100 ⁇ g / mL, stabilizer: 14 mg / mL, support agent: 50 mg / mL, pH buffer: 30 mM;
  • nerve growth factor 100 ⁇ g / mL, stabilizer: 15 mg / mL, support agent: 50 mg / mL, pH buffer: 30 mM;
  • nerve growth factor 100 ⁇ g / mL, stabilizer: 20 mg / mL, support agent: 50 mg / mL, pH buffer: 30 mM;
  • nerve growth factor 100 ⁇ g / mL, stabilizer: 30 mg / mL, support agent: 50 mg / mL, pH buffer: 30 mM;
  • nerve growth factor 50 ⁇ g/mL
  • stabilizer 10mg/mL
  • support agent 40 Mg/mL
  • pH buffer 25 mM
  • the pH of the nerve growth factor composition may be 6.80 to 7.00, specifically 6.80, 6.81, 6.83, 6.85, 6.91 or 7.00.
  • proteins tend to undergo adsorption and/or denaturation at the gas-liquid, bottle-liquid interface due to their high tendency to interact with the surface. This interaction is inversely proportional to the protein concentration and results in the formation of soluble and insoluble protein aggregates, or proteins in solution that are lost by adsorption to the interface.
  • Surfactants are commonly used in protein formulations to prevent adsorption and/or denaturation caused by surface interactions.
  • Surfactants are amphiphilic molecules that compete for proteins at the interfacial position.
  • the hydrophobic portion of the surfactant molecule occupies the interfacial position (eg, gas-liquid) while the hydrophilic portion of the molecule remains directed toward the solvent body.
  • the surface layer of the surfactant molecule acts to prevent adsorption of protein molecules at the interface.
  • the surfactant can reduce the freezing and dehydration denaturation caused by the interfacial tension of ice water during the freezing and dehydration process, and can also moisturize the active components during the rehydration process.
  • the above nerve growth factor composition may further include a surfactant.
  • the mass concentration of the surfactant is from 0 mg/mL to 1.0 mg/mL, but not 0; preferably, the mass concentration of the surfactant is from 0.2 mg/mL to 1.0 mg/mL; preferably, The surfactant has a mass-volume concentration of 0.2 to 0.5 mg/ml; more preferably, the surfactant has a mass-volume concentration of 0.2 mg/mL.
  • the surfactant is any one of poloxamer, polysorbate, and polyethylene glycol 15-hydroxystearate (referred to as HS 15, the same below), preferably,
  • the surfactant is poloxamer 188 (abbreviated as F68), polysorbate 20 (abbreviated as TW-20) polysorbate 80 (abbreviated as TW-80) and HS 15.
  • the present invention further provides a method for preparing a nerve growth factor composition injection powder, which is obtained by freeze-drying the above nerve growth factor composition to obtain the nerve growth factor composition injection powder.
  • the present invention also provides a nerve growth factor composition injection powder for administration by injection.
  • the nerve growth factor composition injection powder may have a water content of 1.0 to 3.0%, preferably 1.0% to 2.0%, specifically 1.1% to 1.9%, 1.1%, 1.3%, 1.5%, 1.6%, 1.7%. Or 1.9%.
  • 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 present invention conducts a large number of exploratory experiments by screening and optimizing the types and amounts of amino acids, and finally adopts different concentrations and ratios of glycine, Preparation of sterile powder for injection of each NGF composition by arginine, glutamic acid and isoleucine, and measuring the water after lyophilization Fraction, pH, osmotic pressure, content and activity.
  • the NGF composition and the injected powder were prepared as follows:
  • the prescription composition in Table 1 weighed the prescription arginine hydrochloride, added appropriate amount of water for injection and stirred until completely dissolved, and then added the prescribed amount of glutamic acid, arginine, isoleucine, glycine, mannitol, F68 stir until completely dissolved, add rhNGF stock solution, adjust pH with arginine or HCl to pH 6.85 shown in Table 1, and add volume of water to the mark with water for injection. After mixing evenly, filter through 0.22 ⁇ m microporous membrane to no In the bacterial container, a composition having an NGF concentration of about 10 ⁇ g/mL was prepared.
  • the above rhNGF composition was dispensed into a neutral borosilicate glass controlled injection bottle at 0.63 ⁇ 0.03 mL/bottle, and lyophilized to prepare a sterile powder for injection, that is, a rhNGF composition injection powder.
  • the NGF composition and the injected powder were prepared as follows:
  • 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 glutamic acid, arginine, isoleucine, glycine and mannitol to stir until completely dissolved.
  • hNGF stock solution adjust the pH to pH 6.83 shown in Table 1 with NaOH or H 3 PO 4 , add the volume of water to the mark with water for injection, mix well, and then filter into a sterile container through 0.22 ⁇ m microporous membrane to prepare.
  • the above hNGF composition was dispensed into a neutral borosilicate glass controlled injection bottle at 0.63 ⁇ 0.03 mL/bottle, and lyophilized to prepare a sterile powder for injection, that is, a hNGF composition injection powder.
  • the NGF composition and the injected powder were prepared as follows:
  • 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 glutamic acid, arginine, isoleucine, glycine and mannitol to stir until completely dissolved.
  • hNGF stock solution adjust the pH to pH 6.80 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 to prepare A composition having a NGF concentration of about 40 ⁇ g/mL.
  • the above hNGF composition was dispensed into a neutral borosilicate glass controlled injection bottle at 0.63 ⁇ 0.03 mL/bottle, and lyophilized to prepare a sterile powder for injection, that is, a hNGF composition injection powder.
  • the NGF composition and the injected powder were prepared as follows:
  • 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 glutamic acid, arginine, isoleucine, glycine and mannitol to stir until completely dissolved.
  • hNGF stock solution adjust the pH to pH 6.91 shown in Table 1 with NaOH or H 3 PO 4 , 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 an NGF concentration of about 80 ⁇ g/mL was formed.
  • the above hNGF composition was dispensed into a neutral borosilicate glass controlled injection bottle at 0.63 ⁇ 0.03 mL/bottle, and lyophilized to prepare a sterile powder for injection, that is, a hNGF composition injection powder.
  • the NGF composition and the injected powder were prepared as follows:
  • composition in Table 1 weigh the prescribed amount of acetate, add appropriate amount of water for injection and stir until completely dissolved, then add the prescribed amount of glutamic acid, arginine, isoleucine, glycine and mannitol to stir until completely dissolved.
  • Add rhHIF stock solution adjust the pH to pH 6.82 shown in Table 1 with NaOH or HAc, add the volume of water to the mark with the water for injection, mix well, filter into a sterile container through a 0.22 ⁇ m microporous membrane to prepare NGF.
  • the above rhNGF composition was dispensed into a neutral borosilicate glass controlled injection bottle at 0.63 ⁇ 0.03 mL/bottle, and lyophilized to prepare a sterile powder for injection, that is, a rhNGF composition injection powder.
  • the NGF composition and the injected powder were prepared as follows:
  • 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 glutamic acid, arginine, isoleucine, glycine and sorbitol to stir until completely dissolved.
  • hNGF 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 to prepare A composition having a NGF concentration of about 50 ⁇ g/mL.
  • the above hNGF composition was dispensed into a neutral borosilicate glass controlled injection bottle at 0.63 ⁇ 0.03 mL/bottle, and lyophilized to prepare a sterile powder for injection, that is, a hNGF composition injection powder.
  • the NGF composition and the injected powder were prepared as follows:
  • TW-80 is in a spherical aggregation state in the water for injection, it is difficult to mix uniformly.
  • TW-80 is first dissolved 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.
  • hNGF stock solution adjust the pH to pH 6.81 shown in Table 1 with NaOH or HAc, and add to volume with water for injection.
  • the composition was filtered into a sterile container through a 0.22 ⁇ m microporous membrane to prepare a composition having an NGF concentration of about 60 ⁇ g/mL.
  • the above hNGF composition was dispensed into a neutral borosilicate glass controlled injection bottle at 0.63 ⁇ 0.03 mL/bottle, and lyophilized to prepare a sterile powder for injection, that is, a hNGF composition injection powder.
  • the NGF composition and the injected powder were prepared as follows:
  • the prescription composition in Table 1 weigh the prescription 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 glutamic acid, arginine, isoleucine The acid, glycine and dextrin were stirred until completely dissolved.
  • the mNGF stock solution was added, and the pH was adjusted to pH 6.85 as shown in Table 1 with arginine or HCl, and the volume was adjusted to the mark with water for injection. After mixing uniformly, the micropores were passed through 0.22 ⁇ m. The filter was filtered into a sterile container to prepare a composition having an NGF concentration of about 60 ⁇ g/mL.
  • the above mNGF composition was dispensed into a neutral borosilicate glass controlled injection bottle at 0.63 ⁇ 0.03 mL/bottle, and lyophilized to prepare a sterile powder for injection.
  • the NGF composition and the injected powder were prepared as follows:
  • the prescription composition in Table 1 weigh the prescription 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 glutamic acid, arginine, isoleucine The acid, glycine and mannitol were stirred until completely dissolved.
  • the hNGF stock solution was added, and the pH was adjusted to pH 6.83 as shown in Table 1 with arginine or HCl, and the volume was adjusted to the mark with water for injection. After mixing uniformly, the micropores were passed through 0.22 ⁇ m. The filter was filtered into a sterile container to prepare a composition having an NGF concentration of about 50 ⁇ g/mL.
  • the above hNGF composition was dispensed into a neutral borosilicate glass controlled injection bottle at 0.63 ⁇ 0.03 mL/bottle, and lyophilized to prepare a sterile powder for injection, that is, a hNGF composition injection powder.
  • the NGF composition and the injected powder were prepared as follows:
  • 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 glutamic acid, arginine, isoleucine, glycine and mannitol to stir until completely dissolved.
  • mNGF stock solution adjust the pH to pH 7.00 shown in Table 1 with NaOH or H 3 PO 4 , 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.
  • the above mNGF composition was dispensed into a neutral borosilicate glass controlled injection bottle at 0.63 ⁇ 0.03 mL/bottle, and lyophilized to prepare a sterile powder for injection, that is, an injection powder of mNGF composition.
  • the NGF composition and the injected powder were prepared as follows:
  • composition in Table 1 weigh the prescribed amount of acetate, add appropriate amount of water for injection and stir until completely dissolved, then add the prescribed amount of glutamic acid, arginine, isoleucine, glycine and mannitol to stir until completely dissolved.
  • hNGF stock solution adjust the pH to pH 6.83 shown in Table 1 with NaOH or HAc, add the volume of water to the mark with the water for injection, mix well, filter into a sterile container through a 0.22 ⁇ m microporous membrane to prepare NGF.
  • the above hNGF composition was dispensed into a neutral borosilicate glass controlled injection bottle at 0.63 ⁇ 0.03 mL/bottle, and lyophilized to prepare a sterile powder for injection, that is, a hNGF composition injection powder.
  • 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 glutamic acid, arginine, isoleucine, glycine and sorbitol to stir to completely dissolve. Since HS 15 is a bulk solid under normal temperature conditions, it is difficult to mix uniformly.
  • the present invention first dissolves HS 15 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 conversion, add the above solution and mix well.
  • the above hNGF composition was dispensed into a neutral borosilicate glass controlled injection bottle at 0.63 ⁇ 0.03 mL/bottle, and lyophilized to prepare a sterile powder for injection, that is, a hNGF composition injection powder.
  • 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 glutamic acid, arginine, isoleucine, glycine and mannitol to stir 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, and after cooling to room temperature. After the conversion of the prescription amount, add the above solution and mix well.
  • the above hNGF composition was dispensed into a neutral borosilicate glass controlled injection bottle at 0.63 ⁇ 0.03 mL/bottle, and lyophilized to prepare a sterile powder for injection, that is, a hNGF composition injection powder.
  • NGF composition injection powder and the reference preparation prepared in Examples 1-13 were observed, and the water, osmotic pressure, pH, content and activity were measured.
  • the NGF content was measured in accordance with the method described in Example 1 of Patent Application 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.
  • the osmotic pressure is related to the ion concentration in the composition, the osmotic pressure differs in each of the above embodiments, and the concentration of each embodiment of the present invention is slightly lower than the isotonic concentration, but does not cause significant discomfort to the patient. It can be seen from the above results that the remaining indexes of the NGF composition injection powder prepared by the invention meet the requirements of the quality standard.
  • NGF composition 3 parts was prepared, the total amount of citrate was kept unchanged, and the relative ratio of citric acid to sodium citrate was adjusted, so that the pH values of the NGF composition were 6.0, 6.8 and 7.4, respectively.
  • a vial 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 reference preparation and the NGF composition injection powder sample prepared in Example 1-13 were placed at 25 ° C under RH 60 ⁇ 10% for 0 to 6 months according to the "Guidelines for Technical Studies of Biological Product Stability". Accelerate stability investigation, observe the appearance of the sample, measure moisture, osmotic pressure, pH, content and activity. The results are shown in Table 4 and Figure 1 (average content versus time), Figure 2 (average activity versus time curve, activity unit: U/bottle, per bottle: 0.63 ⁇ 0.03 mL).
  • Examples 1 to 13 were 7.1%, 7.0%, 4.9%, 3.8%, 6.9%, 2.7%, 3.6%, 6.4%, 4.5%, 5.5%, 3.8%, 4.8%, and 4.9%, respectively.
  • the stability of Examples 1 to 13 under accelerated conditions was better than that of the reference preparation, wherein the stability of Example 3, Example 4, Example 6, Example 7 and Example 11 was significantly better than that of the reference preparation, especially It is Example 4, Example 6, Example 7, and Example 11.
  • Example 1 ⁇ 13 decreased by 9.3%, 8.7%, 8.1%, 5.8%, 9.5%, 4.8%, 5.4%, 7.8%, 7.0%, 6.1%, 5.6%, 7.5% and 7.8, respectively, therefore, under accelerated conditions, examples
  • the stability of 1 to 13 is better than that of the reference preparation, wherein the stability of Example 4, Example 6, Example 7 and Example 11 is obviously better than that of the reference preparation, especially Example 4, Example 6, implementation Example 7 and Example 11.
  • the NGF composition injection powder of the present invention uses four amino acids of arginine, glutamic acid, glycine and isoleucine as stabilizers, and alanine and fine relative to the reference preparation.
  • amino acid and glycine have more excellent stability, especially Example 4, Example 6, and Example 7.
  • Subject conditions 14 years of age or older.
  • Gender Male or female.
  • Subject source Inclusion of optic nerve damage caused by various causes in major ophthalmology research units 409 patients.
  • Treatment method The treatment group was intramuscularly injected with 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 ingredient, the remaining excipients were combined with NGF composition).
  • 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.
  • NGF composition group in the above table uses the nerve growth factor composition prepared in Example 6, the same below.
  • the clinical efficacy of the drug the patients were given a placebo, a reference preparation and an NGF composition injection powder for intramuscular injection, and the therapeutic effect was evaluated according to recovery, marked effect, improvement and ineffectiveness, and the results were as follows:
  • the NGF composition of the present invention can effectively treat optic nerve damage caused by various reasons compared with the placebo group, and the treatment efficiency of the optic nerve injury is better than that of the reference preparation group, and the clinical treatment effect is good. .
  • the nerve growth factor composition prepared by the invention and the injection powder maintain good stability during preparation, transportation and storage: (1) the nerve growth factor composition prepared by the invention is injected into the powder during preparation. After standing for 24 hours at room temperature, there was no significant change in the content and activity of NGF; (2) The NGF composition of the present invention was transported and stored during conventional (6 ⁇ 2 ° C) comparison with the reference preparation. After 12 months, the effective amount of the injected powder was only reduced by 2.4% to 3.4%, and the activity was also reduced little, and the stability was excellent.
  • 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 an amino acid instead of albumin as a stabilizer.
  • the nerve growth factor composition injection powder of the invention not only has protective effect on mouse nerve growth factor (mNGF), but also ensures preparation, transportation and storage of human nerve growth factor (hNGF) and recombinant human nerve growth factor (rhNGF). Maintain good stability, better clinical drug safety and quality controllability during the process.
  • mNGF mouse nerve growth factor
  • hNGF human nerve growth factor
  • rhNGF recombinant human nerve growth factor
  • the nerve growth factor composition and the injection powder of the invention have clear components, are easy to be qualitative and quantitative, and the stabilizer used has high purity, wide source, easy to carry out long-term mass production, and is convenient for controlling cost and improving product quality.
  • the nerve growth factor composition injection powder of the invention can well solve the problem of long-term storage stability of the product, and has higher clinical effectiveness.

Abstract

一种神经生长因子组合物和注射粉剂。所述神经生长因子组合物包括神经生长因子、稳定剂、支持剂、pH缓冲剂和水；所述稳定剂为精氨酸、谷氨酸、甘氨酸和异亮氨酸。所述神经生长因子组合物还包括表面活性剂。通过使用氨基酸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，同时可保证hNGF以及rhNGF在制备、运输及储存过程中保持良好的稳定性、临床用药安全性及质量可控性；组合物中成分明确，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。

Description

神经生长因子组合物和注射粉剂 技术领域
本发明涉及神经生长因子组合物和注射粉剂，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神经生长因子组合物和注射粉剂，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，它包括神经生长因子、稳定剂、支持剂、pH缓冲剂和水；
所述稳定剂为精氨酸、谷氨酸、甘氨酸和异亮氨酸。
上述神经生长因子的组合物中：
所述神经生长因子的质量体积浓度为10μg/mL～100μg/mL；
所述稳定剂的质量体积浓度为6mg/mL～30mg/mL；
所述支持剂的质量体积浓度为20mg/mL～50mg/mL；
所述pH缓冲剂维持所述神经生长因子组合物的pH值为6.80～7.00；
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，所述稳定剂的质量体积浓度为10mg/mL～20mg/mL，更优选地，所述稳定剂的质量体积浓度为13mg/mL；
上述神经生长因子组合物中，所述稳定剂中精氨酸、谷氨酸、甘氨酸和异亮氨酸各组分质量比为1：(1～3)：(1～3)：(1～3.6)，具体可为1：(1～1.3)：(1～1.8)：(1～1.3)、1：(1.3～3)：(1.8～3.0)：(1.3～3.6)、1：(1.2～2)：(1.5～2.0)：(1.2～1.5)、1：(1.25～2.8)：(1.6～2.8)：(1.25～3.0)、1：1：1：1、1：1.2：3：1.3、1：2：3：1、1：1.25：1.5：1.25、1：1：2.8：1.5、1：2：2：3.6、1：1.3：3：1.3、1：3：3：3、1：1.2：1.6：1.2或1：2.8：1.8：1.5，优选地，所述稳定剂中精氨酸、谷氨酸、甘氨酸和异亮氨酸各组分质量比为1：1.25：1.5：1.25，。
上述神经生长因子组合物中，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30mg/mL～50mg/mL，更优选地，所述支持剂的质量体积浓度为50 mg/mL；
所述支持剂为甘露醇、糊精和山梨醇中任一种。
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6.81～6.91，更优选地，维持所述神经生长因子组合物的pH值为6.82。
上述神经生长因子组合物中，所述pH缓冲剂选自磷酸盐、柠檬酸盐、醋酸盐、组氨酸盐酸盐和精氨酸盐酸盐中的一种或几种，具体可为下述1)-5)中任一种：1)精氨酸盐酸盐、2)磷酸盐、3)柠檬酸盐、4)醋酸盐、5)组氨酸盐酸盐和精氨酸盐酸盐。
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～80μg/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，所述神经生长因子选自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。
上述神经生长因子的组合物中，各组分的浓度具体可为下述1)-15) 中的任一种：
1)神经生长因子：10～50μg/mL、稳定剂：6～13mg/mL、支持剂：20mg/mL～40mg/mL、pH缓冲剂：10mM～25mM；
2)神经生长因子：60～100μg/mL、稳定剂：6.5～14mg/mL、支持剂：30mg/mL～50mg/mL、pH缓冲剂：25mM～50mM；
3)神经生长因子：40～80μg/mL、稳定剂：8.9～15mg/mL、支持剂：30mg/mL～50mg/mL、pH缓冲剂：20mM～30mM；
4)神经生长因子：40～80μg/mL、稳定剂：10～20mg/mL、支持剂：30mg/mL～50mg/mL、pH缓冲剂：20mM～25mM；
5)神经生长因子：50～80μg/mL、稳定剂：10～20mg/mL、支持剂：40mg/mL～50mg/mL、pH缓冲剂：20mM～25mM；
6)神经生长因子：10μg/mL、稳定剂：6mg/mL、支持剂：20mg/mL、pH缓冲剂：10mM；
7)神经生长因子：40μg/mL、稳定剂：6.5mg/mL、支持剂：30mg/mL、pH缓冲剂：20mM；
8)神经生长因子：50μg/mL、稳定剂：8.9mg/mL、支持剂：40mg/mL、pH缓冲剂：25mM；
9)神经生长因子：60μg/mL、稳定剂：10mg/mL、支持剂：50mg/mL、pH缓冲剂：30mM；
10)神经生长因子：80μg/mL、稳定剂：13mg/mL、支持剂：50mg/mL、pH缓冲剂：30mM；
11)神经生长因子：100μg/mL、稳定剂：14mg/mL、支持剂：50mg/mL、pH缓冲剂：30mM；
12)神经生长因子：100μg/mL、稳定剂：15mg/mL、支持剂：50mg/mL、pH缓冲剂：30mM；
13)神经生长因子：100μg/mL、稳定剂：20mg/mL、支持剂：50mg/mL、pH缓冲剂：30mM；
14)神经生长因子：100μg/mL、稳定剂：30mg/mL、支持剂：50mg/mL、pH缓冲剂：30mM；
15)神经生长因子：50μg/mL、稳定剂：10mg/mL、支持剂：40 mg/mL、pH缓冲剂：25mM。
上述神经生长因子的组合物中，所述神经生长因子组合物的pH值可为6.80～7.00，具体可为6.80、6.81、6.83、6.85、6.91或7.00。
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。
表面活性剂通常用于蛋白质配方中以防止表面相互作用引发的吸附和/或变性。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。此外，在生物制品的冷冻干燥全过程中，表面活性剂既能在冻结和脱水过程中降低冰水界面张力所引起的冻结和脱水变性；又能在复水过程中对活性组分起到润湿剂和重褶皱剂的作用。
上述神经生长因子组合物还可以包括表面活性剂。所述表面活性剂的质量体积浓度为0mg/mL～1.0mg/mL，但不为0；优选地，所述表面活性剂的质量体积浓度为0.2mg/mL～1.0mg/mL；优选地，所述表面活性剂的质量体积浓度为0.2～0.5mg/ml；更优选地，所述表面活性剂的质量体积浓度为0.2mg/mL。
上述神经生长因子组合物中，所述表面活性剂为泊洛沙姆、聚山梨酯、15-羟基硬脂酸聚乙二醇酯(简称HS 15，下同)中任一种，优选地，所述表面活性剂为泊洛沙姆188(简称F68)，聚山梨酯20(简称TW-20)聚山梨酯80(简称TW-80)及HS 15。
本发明进一步提供了一种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。
本发明也提供了一种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，其含水率可为1.0～3.0％，优选为1.0％～2.0％，具体可为1.1％～1.9％、1.1％、1.3％、1.5％、1.6％、1.7％或 1.9％。
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
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。
为了寻找具有更佳稳定性的物质作为NGF的稳定剂，本发明根据NGF的结构特性，通过对氨基酸种类及用量进行筛选及优化，进行了大量探索性实验，最终采用不同浓度和比例的甘氨酸、精氨酸、谷氨酸、异亮氨酸制备了各NGF组合物注射用无菌粉末，并测定了冷冻干燥后成品的水 分、pH、渗透压、含量及活性。
实施例1、NGF组合物和注射粉剂的制备
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，再加入处方量的谷氨酸、精氨酸、异亮氨酸、甘氨酸、甘露醇、F68搅拌至完全溶解，加入rhNGF原液，用精氨酸或HCl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10μg/mL的组合物。
将上述rhNGF组合物，按0.63±0.03mL/瓶分装至中性硼硅玻璃管制注射剂瓶中，冷冻干燥，制备成注射用无菌粉末即rhNGF组合物注射粉剂。
实施例2、NGF组合物和注射粉剂的制备
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，再加入处方量的谷氨酸、精氨酸、异亮氨酸、甘氨酸和甘露醇搅拌至完全溶解，加入hNGF原液，用NaOH或H3PO4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40μg/mL的组合物。
将上述hNGF组合物，按0.63±0.03mL/瓶分装至中性硼硅玻璃管制注射剂瓶中，冷冻干燥，制备成注射用无菌粉末即hNGF组合物注射粉剂。
实施例3、NGF组合物和注射粉剂的制备
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，再加入处方量的谷氨酸、精氨酸、异亮氨酸、甘氨酸和甘露醇搅拌至完全溶解，加入hNGF原液，用NaOH或柠檬酸调pH至表1中所示pH 6.80，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40μg/mL的组合物。
将上述hNGF组合物，按0.63±0.03mL/瓶分装至中性硼硅玻璃管制注射剂瓶中，冷冻干燥，制备成注射用无菌粉末即hNGF组合物注射粉剂。
实施例4、NGF组合物和注射粉剂的制备
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，再加入处方量的谷氨酸、精氨酸、异亮氨酸、甘氨酸和甘露醇搅拌至完全溶解，加入hNGF原液，用NaOH或H3PO4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80μg/mL的组合物。
将上述hNGF组合物，按0.63±0.03mL/瓶分装至中性硼硅玻璃管制注射剂瓶中，冷冻干燥，制备成注射用无菌粉末即hNGF组合物注射粉剂。
实施例5、NGF组合物和注射粉剂的制备
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，再加入处方量的谷氨酸、精氨酸、异亮氨酸、甘氨酸和甘露醇搅拌至完全溶解，加入rhNGF原液，用NaOH或HAc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80μg/mL的组合物。
将上述rhNGF组合物，按0.63±0.03mL/瓶分装至中性硼硅玻璃管制注射剂瓶中，冷冻干燥，制备成注射用无菌粉末即rhNGF组合物注射粉剂。
实施例6、NGF组合物和注射粉剂的制备
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，再加入处方量的谷氨酸、精氨酸、异亮氨酸、甘氨酸和山梨醇搅拌至完全溶解，加入hNGF原液，用NaOH或柠檬酸调pH至表1中所示pH 6.82，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50μg/mL的组合物。
将上述hNGF组合物，按0.63±0.03mL/瓶分装至中性硼硅玻璃管制注射剂瓶中，冷冻干燥，制备成注射用无菌粉末即hNGF组合物注射粉剂。
实施例7、NGF组合物和注射粉剂的制备
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，再加入处方量的谷氨酸、精氨酸、异亮氨酸、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，加入hNGF原液，用NaOH或HAc调pH至表1中所示pH 6.81，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hNGF组合物，按0.63±0.03mL/瓶分装至中性硼硅玻璃管制注射剂瓶中，冷冻干燥，制备成注射用无菌粉末即hNGF组合物注射粉剂。
实施例8、NGF组合物和注射粉剂的制备
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，再加入处方量的谷氨酸、精氨酸、异亮氨酸、甘氨酸和糊精搅拌至完全溶解，加入mNGF原液，用精氨酸或HCl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60μg/mL的组合物。
将上述mNGF组合物，按0.63±0.03mL/瓶分装至中性硼硅玻璃管制注射剂瓶中，冷冻干燥，制备成注射用无菌粉末。
实施例9、NGF组合物和注射粉剂的制备
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，再加入处方量的谷氨酸、精氨酸、异亮氨酸、甘氨酸和甘露醇搅拌至完全溶解，加入hNGF原液，用精氨酸或HCl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50μg/mL的组合物。
将上述hNGF组合物，按0.63±0.03mL/瓶分装至中性硼硅玻璃管制注射剂瓶中，冷冻干燥，制备成注射用无菌粉末即hNGF组合物注射粉剂。
实施例10、NGF组合物和注射粉剂的制备
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，再加入处方量的谷氨酸、精氨酸、异亮氨酸、甘氨酸和甘露醇搅拌至完全溶解，加入mNGF原液，用NaOH或H3PO4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100μg/mL的组合物。
将上述mNGF组合物，按0.63±0.03mL/瓶分装至中性硼硅玻璃管制注射剂瓶中，冷冻干燥，制备成注射用无菌粉末即mNGF组合物注射粉剂。
实施例11、NGF组合物和注射粉剂的制备
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，再加入处方量的谷氨酸、精氨酸、异亮氨酸、甘氨酸和甘露醇搅拌至完全溶解，加入hNGF原液，用NaOH或HAc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hNGF组合物，按0.63±0.03mL/瓶分装至中性硼硅玻璃管制注射剂瓶中，冷冻干燥，制备成注射用无菌粉末即hNGF组合物注射粉剂。
实施例12、NGF组合物和注射粉剂的制备
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，再加入处方量的谷氨酸、精氨酸、异亮氨酸、甘氨酸和山梨醇搅拌至完全溶解，由于HS 15在常温条件下为块状固体，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，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50μg/mL的组合物。
将上述hNGF组合物，按0.63±0.03mL/瓶分装至中性硼硅玻璃管制注射剂瓶中，冷冻干燥，制备成注射用无菌粉末即hNGF组合物注射粉剂。
实施例13、NGF组合物和注射粉剂的制备
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，再加入处方量的谷氨酸、精氨酸、异亮氨酸、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，加入hNGF原液，用NaOH或HAc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50μg/mL的组合物。
将上述hNGF组合物，按0.63±0.03mL/瓶分装至中性硼硅玻璃管制注射剂瓶中，冷冻干燥，制备成注射用无菌粉末即hNGF组合物注射粉剂。
表1、实施例1～13中各组分用量
Figure PCTCN2016079572-appb-000001
Figure PCTCN2016079572-appb-000002
实施例14、实施例1-13中制备得到的NGF组合物注射粉剂性能测试
下述性能测试中的参比制剂均采用发明名称为“去白蛋白神经生长因子制剂”，公开号为CN 1318087C的专利中实施例14中的去白蛋白神经生长因子冻干粉针制剂。
(1)外观、水分、渗透压、pH、含量及活性测试
分别观察实施例1-13中制备得到的NGF组合物注射粉剂及参比制剂的外观，测定其水分、渗透压、pH、含量及活性。其中，NGF含量测定按照专利申请号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 PCTCN2016079572-appb-000003
Figure PCTCN2016079572-appb-000004
因渗透压与组合物中的离子浓度有关，上述各实施例中渗透压有所差异，本发明各实施例浓度略低于等渗浓度，但不会引起患者明显不适。由上表结果可知，本发明制备得到的NGF组合物注射粉剂的其余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3中处方组成配制3份NGF组合物，保持柠檬酸盐总量不变，调节柠檬酸与柠檬酸钠相对比例，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pH范围筛选
Figure PCTCN2016079572-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。
(3)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，将参比制剂与实施例1-13中制备得到的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，测定水分、渗透压、 pH、含量及活性。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，活性单位：U/瓶，每瓶装量：0.63±0.03mL)。
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 PCTCN2016079572-appb-000006
Figure PCTCN2016079572-appb-000007
Figure PCTCN2016079572-appb-000008
Figure PCTCN2016079572-appb-000009
Figure PCTCN2016079572-appb-000010
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，参比制剂及各实施例制备得到的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大于本发明实施例。由表4结果可知，在加速条件下(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，但参比制剂含量降低约8.2％，实施例1～13含量降低率分别为7.1％、7.0％、4.9％、3.8％、6.9％、2.7％、3.6％、6.4％、4.5％、5.5％、3.8％、4.8％及4.9％，因此，加速条件下实施例1～13稳定性均优于参比制剂，其中，实施例3、实施例4、实施例6、实施例7和实施例11的稳定性明显优于参比制剂，尤其是实施例4、实施例6、实施例7和实施例11。
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，参比制剂及各实施例制备得到的NGF组合物注射粉剂中 NGF的平均活性呈下降趋势，但由图可以看出参比制剂的平均活性下降速度大于本发明制备得到的NGF组合物注射粉剂。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，参比制剂活性降低10.2％，实施例1～13分别降低9.3％、8.7％、8.1％、5.8％、9.5％、4.8％、5.4％、7.8％、7.0％、6.1％、5.6％、7.5％及7.8，因此，加速条件下，实施例1～13稳定性均优于参比制剂，其中，实施例4、实施例6、实施例7和实施例11的稳定明明显优于参比制剂，尤其是实施例4、实施例6、实施例7和实施例11。
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，将参比制剂与实施例1-13中制备得到的NGF注射粉剂样品均置于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，活性单位：U/瓶，每瓶装量：0.63±0.03mL)。
表5 NGF组合物注射粉剂及参比制剂长期稳定性试验结果
Figure PCTCN2016079572-appb-000011
Figure PCTCN2016079572-appb-000012
Figure PCTCN2016079572-appb-000013
Figure PCTCN2016079572-appb-000014
Figure PCTCN2016079572-appb-000015
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，参比制剂及各实施例制备得到的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，参比制剂含量降低约5.9％，实施例1～13含量降低率分别为4.8％、4.9％、3.6％、3.3％、5.4％、2.2％、2.9％、5.0％、4.0％、4.7％。3.7％、3.8％及3.9％，因此，长期条件下实施例1～13稳定性均优于参比制剂，其中，实施例3、实施例4、实施例6、实施例7和实施例11的稳定性明显优于参比制剂，尤其是实施例4、实施例6、实施例7和实施例11。
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，参比制剂及各实施例制备得到的NGF组合物注射粉剂中NGF的平均活性整体均呈下降趋势，然而，由图可以看出，参比制剂的活性下降速度大于本发明实施例。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，参比制剂活性降低8.4％，实施例1～13活性分别降低7.7％、7.8％、5.4％、4.8％、7.9％、4.1％、4.5％、7.7％、5.5％、5.9％、4.8％、5.6％及5.8％，因此，长期条件下实施例1～13稳定性均优于参比制剂，其中，实施例3、实施例4、实施例6、实施例7和实施例11的稳定性明显优于参比制剂，尤其是实施例4、实施例6、实施例7和实施例11。
由上述稳定性测试结果可以看出，本发明NGF组合物注射粉剂采用精氨酸、谷氨酸、甘氨酸和异亮氨酸四种氨基酸作为稳定剂，相对于参比制剂采用丙氨酸、精氨酸和甘氨酸作为稳定剂，具有更加优异的稳定性，尤其是实施例4、实施例6、实施例7和。
(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：治疗组分别采用参比制剂及本发明NGF组合物注射粉剂进行肌肉注射，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，按照下表所示进行分组：
表6 临床试验分组及受试者数量
Figure PCTCN2016079572-appb-000016
注：上表中的NGF组合物组采用实施例6制得的神经生长因子组合物，下同。
药物临床疗效：分别给予患者安慰剂、参比制剂及NGF组合物注射粉剂进行肌肉注射，治疗效果按痊愈、显效、好转及无效等进行评价，结果如下：
表7 NGF临床疗效对比
Figure PCTCN2016079572-appb-000017
Figure PCTCN2016079572-appb-000018
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有效率优于参比制剂组，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及注射粉剂，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，将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℃)在运输及储存过程中，本发明NGF组合物注射粉剂在12月后，其有效含量仅降低2.4％～3.4％，其活性降低也很少，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。
本发明的有益效果在于：
(1)本发明神经生长因子组合物及注射粉剂，通过使用氨基酸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，不仅对鼠神经生长因子(mNGF)具有保护作用，同时能够保证人神经生长因子(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，组合物中成分明确，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。
(4)本发明神经生长因子组合物注射粉剂能够很好地解决产品长期贮存稳定性问题，并且临床有效性更高。

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神经生长因子、稳定剂、支持剂、pH缓冲剂和水；
    所述稳定剂为精氨酸、谷氨酸、甘氨酸和异亮氨酸。
  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：所述神经生长因子的组合物中：
    所述神经生长因子的质量体积浓度为10μg/mL～100μg/mL；
    所述稳定剂的质量体积浓度为6mg/mL～30mg/mL；
    所述支持剂的质量体积浓度为20mg/mL～50mg/mL；
    所述pH缓冲剂维持所述神经生长因子组合物的pH值为6.80～7.00；
    溶剂为水。
  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：所述稳定剂的质量体积浓度为10mg/mL～20mg/mL。
  4. 根据权利要求1-3中任一项所述的神经生长因子组合物，其特征在于：所述稳定剂中精氨酸、谷氨酸、甘氨酸和异亮氨酸各组分质量比为1：(1～3)：(1～3)：(1～3.6)，优选地，所述稳定剂中精氨酸、谷氨酸、甘氨酸和异亮氨酸各组分质量比为1：1.25：1.5：1.25。
  5. 根据权利要求1-4中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、糊精和山梨醇中任一种。
  6. 根据权利要求1-5中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，优选为20mM～25mM。
  7. 根据权利要求1-6中任一项所述的神经生长因子组合物，其特征在于：所述pH缓冲剂选自磷酸盐、柠檬酸盐、醋酸盐、组氨酸盐酸盐和精氨酸盐酸盐中的一种或几种。
  8. 根据权利要求1-7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～80μg/mL。
  9. 根据权利要求1-8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还包括表面活性剂。
  10. 根据权利要求1-9中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0mg/mL～1.0mg/mL，但不为0； 优选地，所述表面活性剂的质量体积浓度为0.2mg/mL～0.5mg/mL。
  11. 根据权利要求1-10中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。
  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组合物中：
    所述神经生长因子的质量体积浓度为50μg/mL；
    所述稳定剂的质量体积浓度为10mg/mL；
    所述支持剂的质量体积浓度为40mg/mL；
    所述pH缓冲剂的摩尔浓度为25mM。
  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：所述神经生长因子选自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。
  14. 一种神经生长因子组合物注射粉剂制备方法，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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