A human nerve growth factor composition

Abstract

This invention belongs to the pharmaceutical field and relates to a human nerve growth factor composition. This invention provides a human nerve growth factor composition that can be administered via multiple routes, maintains good stability without cryopreservation, and does not require fixed accessories, simplifying operation and improving the convenience of medication.

Description

Technical Field

[0001] This invention belongs to the field of medicine and relates to a human nerve growth factor composition. Background Technology

[0002] Nerve growth factor (NGF) is the earliest discovered and most typical neurotrophic factor, and also one of the most important bioactive molecules affecting the nervous system. Human nerve growth factor (hNGF) is a bioactive factor found in the human body that has a nutritional effect on normal nerve cells and regulates the repair function of damaged nerves. NGF not only maintains the survival and function of sympathetic and sensory nerves, promotes nerve cell differentiation, and determines the direction of axonal extension, but also plays a decisive role in promoting brain development, nervous system growth, and the regeneration and functional recovery of damaged nerves.

[0003] Diabetic skin ulcers are a serious complication of diabetes. Persistent hyperglycemia and the resulting cellular damage and senescence are the main pathophysiological changes associated with diabetes. The pathogenesis of diabetic skin ulcers is exceptionally complex, including skin barrier disruption and infection, persistent oxidative stress, neuropathy, microvascular complications, and chronic inflammation. NGF can promote the proliferation and / or differentiation of immature cells in the ulcer area, as well as angiogenesis and neurite growth, leading to sustained improvement in skin healing up to complete healing.

[0004] Sensorineural hearing loss is a type of hearing loss caused by damage to the cochlea, auditory nerve, auditory pathway, or auditory center, resulting in impaired sound perception or nerve impulse transmission. NGF can promote neuronal differentiation and improve hearing loss.

[0005] Neurotrophic keratitis (NK), also known as neurotrophic keratopathy, is a rare degenerative corneal disease caused by damage to the trigeminal nerve. It is characterized by decreased or absent corneal sensation, dry eye, corneal epithelial defects, and corneal ulcers, ultimately leading to corneal stromal melting and perforation. Nerve growth factor (NGF) has a certain therapeutic effect on neurotrophic keratitis.

[0006] Currently, the active ingredient in the market-available product for neurotrophic keratitis, xenegemin eye drops, is recombinant human nerve growth factor. Its formulation is complex, and the product is packaged in vials, requiring frozen storage. Once removed from the freezer, it must be used within 12 hours and requires specific accessories, making its operation relatively complicated and inconvenient. It can only be used for the treatment of neurotrophic keratitis and cannot be used for other routes of administration.

[0007] Therefore, there is an urgent need for a product that is stable in formulation and easy to use. Summary of the Invention

[0008] This invention provides a topical solution of human nerve growth factor that can be administered via multiple routes, such as ophthalmic, otomic, and dermal routes. It maintains good stability without the need for cryopreservation and is packaged in a simple, single-dose sterile packaging material, eliminating the need for fixed accessories and simplifying operation, thus improving the convenience of medication.

[0009] On one hand, the present invention provides a human nerve growth factor (NGF) composition comprising human nerve growth factor as an active ingredient and a stabilizer, wherein the stabilizer is selected from at least one of stevia, sorbitol or dodecyl-β-D-maltodextrin (DDM).

[0010] In some embodiments, the stabilizer is dodecyl-β-D-maltodextrin.

[0011] In some embodiments, the composition further comprises a solvent.

[0012] In some implementations, the solvent is water.

[0013] In some embodiments, the composition does not contain antibacterial agents.

[0014] In some embodiments, the human nerve growth factor compositions described herein exhibit excellent stability, such as physical stability, chemical stability, and / or biological stability.

[0015] The human nerve growth factor composition described herein is considered to "maintain its physical stability" in formulations if it shows substantially no signs of instability, such as aggregation, precipitation, and / or denaturation, upon visual inspection of color and / or transparency or by size exclusion chromatography. Aggregation is the process by which individual protein molecules or complexes covalently or nonvalently bind to form aggregates. Aggregation can proceed to the extent that visible precipitation is formed; for example, in some embodiments, the composition exhibits a slight opalescence; in other embodiments, the composition exhibits visible particles.

[0016] The human nerve growth factor composition described herein is considered "chemically stable" in formulations if its chemical stability over a given time period (e.g., 5 or 10 days) allows the protein to retain its biological activity (e.g., as described in the "Biological Activity" section below). Chemical stability can be assessed, for example, by detecting and quantifying the forms of chemical changes in the protein. Chemical changes may involve size alterations (e.g., shearing) and can be assessed using size exclusion chromatography, SDS-PAGE, and / or matrix-assisted laser desorption / ionization / time-of-flight mass spectrometry (MALDI / TOF MS). Other types of chemical changes include charge changes (e.g., changes due to deamidation or oxidation), which can be assessed, for example, by ion exchange chromatography.

[0017] The human nerve growth factor compositions described herein retain their biological activity in the formulation if the protein in the pharmaceutical preparation is biologically active for its intended purpose. In bioassays, the activity of the sample is tested on a sensitive cell line (e.g., human erythroblastic leukemia cells), and the results of this activity (e.g., cell proliferation) are compared with NGF standards. Biological activity can be assessed, for example, by the TF-1 cell / MTS colorimetric assay, see Example 2 below. Any suitable assay protocol known in the art is suitable for testing the biological activity of the human NGF compositions described herein.

[0018] The stability of proteins (such as human nerve growth factor as described herein) in protein formulations can be determined by a variety of methods. In some embodiments, protein stability is determined by size exclusion chromatography (SEC).

[0019] In some embodiments, the composition further comprises a viscosity modifier selected from at least one of hydroxypropyl methylcellulose, polyvinyl alcohol, or sodium hyaluronate.

[0020] In some embodiments, the viscosity modifier is polyvinyl alcohol.

[0021] In some embodiments, the stabilizer is dodecyl-β-D-maltodextrin and the viscosity modifier is polyvinyl alcohol.

[0022] In some embodiments, the human nerve growth factor comprises at least one of the amino acid sequences shown in SEQ ID NO:1-3.

[0023] The sequences are shown below:

[0024] SEQ ID NO:1: 117 amino acids;

[0025] SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINSVFKQYFFETKCRDPNPV DSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAV

[0026] SEQ ID NO:2: 118 amino acids;

[0027] SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINSVFKQYFFETKCRDPNPV DSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVR

[0028] SEQ ID NO:3: 120 amino acids;

[0029] SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINSVFKQYFFETKCRDPNPV DSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA

[0030] In some implementations, two or three amino acids are truncated relative to SEQ ID NO:3, SEQ ID NO:2, and SEQ ID NO:1.

[0031] In some embodiments, the human nerve growth factor comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with at least one of the amino acid sequences shown in SEQ ID NO:1-3.

[0032] In some embodiments, the stabilizer content is 0.001%-0.4% (w / v), for example, it can be 0.001%-0.05% (w / v), 0.001-0.1% (w / v), 0.1%-0.4% (w / v), 0.05%-0.4% (w / v), 0.05%-0.1% (w / v), 0.001%-0.2% (w / v), or 0.1%-0.2% (w / v), and more preferably 0.001%, 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, or 0.0 5%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, or any value between the two, preferably 0.05%-0.1% (w / v); in some embodiments, the stabilizer content is 0.001%, 0.05%, 0.1%, or 0.4%; in some preferred embodiments, the stabilizer content is 0.05% or 0.1% (w / v); in some preferred embodiments, the dodecyl-β-D-maltose content is 0.05% or 0.1% (w / v).

[0033] In some embodiments, the viscosity modifier content is 0-4% (w / v), for example, it can be 0-2% (w / v), 0-1% (w / v), 0-0.1% (w / v), 0-0.05% (w / v), 0.05%-4% (w / v), 0.05%-2%, 0.1%-2% (w / v) or 1%-2% (w / v), and more preferably 0%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 2%, 3%, 4% (w / v) or any value between the two; preferably 0.05%-4% (w / v), more preferably 1% (w / v). In some embodiments, the viscosity modifier content is 0%, 0.05%, 0.1%, 1%, 2%, or 4% (w / v); in some preferred embodiments, the viscosity modifier content is 0.05%, 0.1%, 1%, 2%, or 4% (w / v); in some more preferred embodiments, the viscosity modifier content is 1% (w / v); and in some even more preferred embodiments, the polyvinyl alcohol content is 1% (w / v).

[0034] The actual dose level of the active ingredient in the composition can be varied to obtain an amount of active ingredient that effectively achieves the desired therapeutic response for a specific patient, composition, and route of administration without being toxic to the patient.

[0035] In some embodiments, the content of the human nerve growth factor is any concentration to achieve the therapeutic effect, for example, it can be 0.001%-0.06% (w / v), and more specifically, it can be 0.001%, 0.002%, 0.003%, 0.004%, 0.005%, 0.006%, 0.007%, 0.008%, 0.009%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, or 0.06% (w / v); in some embodiments, the content of the human nerve growth factor is 0.001%, 0.005%, 0.01%, 0.02%, 0.04%, or 0.06% (w / v).

[0036] In some preferred embodiments, the content of the human nerve growth factor is 0.001%-0.02% (w / v).

[0037] In some embodiments, the composition includes a pH adjuster that adjusts the composition to a target pH. The pH adjuster can be any pH adjuster, such as hydrochloric acid and / or sodium hydroxide.

[0038] In some preferred embodiments, the target pH is 5.0-7.5, for example, it can be 5.0, 5.5, 6.0, 6.5, 7.0 or 7.5.

[0039] In some more preferred embodiments, the target pH is 6.0-6.5, for example 6.0 or 6.5.

[0040] In some embodiments, the composition further comprises an osmotic pressure regulator.

[0041] In some embodiments, the osmotic pressure regulator is selected from at least one of glycerol, glucose, citrate, phosphate, potassium chloride, sorbitol, sodium chloride, or mannitol.

[0042] In some preferred embodiments, the osmotic pressure regulator is selected from at least one of sodium chloride, glycerol, glucose, or mannitol.

[0043] In some embodiments, the osmotic pressure range of the composition is 260-320 mOsmol / kg, for example, it can be 260-310 mOsmol / kg, 260-300 mOsmol / kg, 260-290 mOsmol / kg, 260-280 mOsmol / kg, 260-270 mOsmol / kg, 270-320 mOsmol / kg, 270-310 mOsmol / kg, 270-300 mOsmol / kg, 270-290 mOsmol / kg, 270-280 mOsmol / kg, 280-300 mOsmol / kg, and more preferably 260, 270, 280, 290, 300, 310 or 320 mOsmol / kg, with 270-310 mOsmol / kg being the most preferred.

[0044] In some embodiments, the composition comprises the following components: 0.001%-0.06% (w / v) human nerve growth factor, 0.001%-0.4% (w / v) stabilizer, 0-4% (w / v) viscosity modifier, pH modifier to adjust the target pH to 5.0-7.5, and optionally an osmotic pressure modifier such that the osmotic pressure ranges from 260-320 mOsmol / kg, with the remainder being water.

[0045] On the other hand, the present invention provides a method for preparing the above-mentioned composition, wherein the preparation method comprises the following steps:

[0046] Dissolve or disperse the stabilizer and the osmotic pressure regulator in the appropriate amount of solvent, adjust to the target pH, add the human nerve growth factor stock solution, and stir until homogeneous to obtain the final product.

[0047] In some implementations, the solvent is water.

[0048] In some embodiments, the composition is formulated such that after being stored at 25°C for at least 10 days, the purity or activity of the active ingredient decreases by less than 5%, for example less than 4%, less than 3%, less than 2%, or less than 1%; preferably less than 4%, more preferably less than 2%, and even more preferably less than 1%.

[0049] In some embodiments, the composition is formulated such that after being stored at 40°C for at least 10 days, the purity or activity of the active ingredient decreases by less than 10%, for example, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%; preferably less than 8%, more preferably less than 5%, more preferably less than 4%, more preferably less than 3%, and more preferably less than 2%.

[0050] In some preferred embodiments, the preparation method includes the following steps: dissolving or dispersing the stabilizer and osmotic pressure regulator with an appropriate amount of water for injection, adjusting to the target pH, adding human nerve growth factor stock solution, and stirring evenly to obtain the final product.

[0051] In some embodiments, the composition is a solution.

[0052] In some embodiments, the composition is applied through the eyes, ears, or skin.

[0053] In some embodiments, the composition is an eye drop or an ophthalmic injection.

[0054] On the other hand, the present invention provides a pharmaceutical composition for treating neurotrophic keratitis, diabetic skin ulcers, or sensorineural hearing loss, the pharmaceutical composition comprising the above-mentioned composition.

[0055] In some embodiments, the pharmaceutical composition is administered via the eyes, ears, or skin.

[0056] In some embodiments, the pharmaceutical composition is formulated as a solution.

[0057] In some embodiments, the pharmaceutical composition is stored at room temperature or under refrigeration.

[0058] In some implementations, the pharmaceutical composition does not require a fixing device.

[0059] In some preferred embodiments, the composition is an eye drop or an ophthalmic injection.

[0060] On the other hand, the present invention provides the use of the above-described composition or the composition prepared by the above-described method in the preparation of a medicament or medicament for treating neurotrophic keratitis, diabetic skin ulcers or sensorineural hearing loss.

[0061] The present invention further relates to a medicament or kit comprising the human nerve growth factor composition described herein, and preferably provides instructions for use, such as for treating the diseases described herein.

[0062] The pillboxes described in this invention comprise one or more containers containing the human nerve growth factor composition described herein, and may further include descriptions of individuals (e.g., humans) selected for appropriate treatment based on identification of whether an individual has a disease and its stage. Instructions associated with the human nerve growth factor composition typically include information regarding the intended dosage, dosing schedule, and route of administration. Instructions are typically written instructions on a label or package insert (e.g., paper included in the pillbox), but machine-readable instructions (e.g., instructions stored on a disk or optical disc) are also acceptable. The containers may be single-dose vials, may be made of various materials such as glass or plastic, and are packaged using sterile packaging materials. The drug or pillbox may further contain a second active ingredient. The drug or pillbox may further contain a second container and other materials that may be further required. The quantity of the drug or pillbox packaged is sufficient for storage and use in a pharmacy, such as a hospital pharmacy or a multi-dose pharmacy.

[0063] In some implementations, the drug or medicament is administered via the eye, ear, or skin, for example, through topical application (such as eye drops).

[0064] In some implementations, the drug or medicament is formulated as a solution or an ophthalmic injection.

[0065] In some implementations, the drug or medicine box is stored at room temperature or under refrigeration, for example, at a temperature not exceeding 40°C.

[0066] In some implementations, the drug or medicine box does not need to be paired with fixed accessories, such as adapters, pipettes, disinfectant wipes, filters, needles, syringes, etc.

[0067] In some preferred embodiments, the drug or cassette is an eye drop or an ophthalmic injection.

[0068] Advantages of this invention

[0069] This invention provides a novel human nerve growth factor composition comprising human nerve growth factor as the active ingredient and a stabilizer, and may further comprise a viscosity modifier. The stabilizer is selected from at least one of stevia, sorbitol, or dodecyl-β-D-maltodextrin (DDM), and the viscosity modifier is selected from at least one of hydroxypropyl methylcellulose, polyvinyl alcohol, or sodium hyaluronate. The combined use of DDM and polyvinyl alcohol has been verified to have a good synergistic effect, maintaining the stability of the composition. The topical human nerve growth factor solution of this invention has a wider range of applications, including the eyes, ears, and skin surfaces. The formulation is simple, maintains stability without the addition of antibacterial agents, exhibits good safety, and can be stored at room temperature or under refrigeration without freezing. Using a common single-dose vial as the packaging, it eliminates the need for fixed accessories; one vial is used per dose, ensuring accurate dosage and convenient administration. Detailed Implementation

[0070] For the purpose of clarity and concise description, features are described herein as part of a number of identical or separate embodiments. However, it will be understood that the scope of this disclosure may include embodiments having combinations of all or some of the described features. The technical solutions of the present invention will now be described clearly and completely. Obviously, based on the specific embodiments of the present invention, all other embodiments obtained by those skilled in the art without inventive effort are within the scope of protection of the present invention.

[0071] I. Definition

[0072] Unless otherwise specified, the following terms used in the specification and claims shall have the following meanings:

[0073] The numerical ranges used in this article should be understood as including all numbers within that range. For example, the range 1 to 20 should be understood to include any number, combination of numbers, or subrange from the following group: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20.

[0074] As used herein, the terms “comprises” or “comprising” mean “including, but not limited to”. This term is intended to be open-ended to specify the presence of any of the stated features, elements, integers, steps, or components, but does not exclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Therefore, the term “comprising” includes the more restrictive terms “consisting of” and “substantially consisting of”. In one embodiment, the term “comprising” as used throughout the application, particularly in the claims, may be replaced by the term “consisting of”.

[0075] As used herein, the terms “optional,” “any,” “arbitrary,” or “any one” mean that the event or situation described below may, but does not have to, occur, including the circumstances in which the event or situation occurs or does not occur. As used herein, “an” and “a” refer to one or more grammatical objects.

[0076] As used in this article, “and / or” should be understood as meaning any one of the options or any combination of two or more of the options.

[0077] As used herein, nerve growth factor (NGF) is a neurotrophic factor that regulates neuronal survival and differentiation, playing a crucial role in nervous system development, neuronal protection, post-injury repair, and pain perception. During development and maturation, it is endogenously produced by various cell types, including neurons, Schwann cells, oligodendrocytes, lymphocytes, mast cells, macrophages, keratinocytes, and fibroblasts. These cells produce proNGF precursors, which are converted into biologically active NGF in the endoplasmic reticulum through proteolytic cleavage. NGF is present in various species, with abundant amounts found in the submandibular glands of male mice, bovine seminal plasma, snake venom, guinea pig prostate tissue, and human placental tissue. In some embodiments, the nerve growth factor is human nerve growth factor. In some embodiments, the human nerve growth factor comprises at least one of the amino acid sequences shown in SEQ ID NO:1-3.

[0078] As used in this article, N-Dodecyl-β-D-maltoside (DDM) is a pyrene (Py) derivative and a nonionic detergent commonly used to extract and dissolve proteins for studies of membranes and membrane-bound proteins. DDM has a hydrophilic maltose head and a hydrophobic long-chain alkyl tail, and compared to other maltoside-based detergents, it exhibits a relatively low critical micelle temperature.

[0079] The amino acid sequence “identity percentage (%)” is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in a specific polypeptide or polypeptide sequence. This percentage is maximized after sequence alignment and the introduction of gaps (if necessary), without considering any conserved substitutions as part of the sequence identity. To determine the amino acid sequence identity percentage, various alignment methods within the scope of the art can be used, such as publicly available computer software like BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine suitable parameters for measuring the alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared.

[0080] As used herein, a "stabilizer," when used with large charged biomolecules (such as proteins), can interact with charged groups on the side chains of amino acids, thereby reducing the likelihood of intermolecular and intramolecular interactions. In some embodiments, the stabilizer is present in an amount of 0.001%-0.4% (w / v); in some embodiments, the stabilizer is present in an amount of 0.05%-0.1% (w / v). In some embodiments, the stabilizer is selected from at least one of stevia, sorbitol, or dodecyl-β-D-maltose; in some embodiments, the stabilizer is dodecyl-β-D-maltose.

[0081] As used herein, the term "viscosity modifier" refers to a class of chemical agents that adjust the rheological properties of aqueous solutions or aqueous dispersions, changing their viscosity; these can be viscosity reducers or viscosity increasers (or thickeners). In some embodiments, the viscosity modifier is selected from at least one of hydroxypropyl methylcellulose (HPMC), polyvinyl alcohol, or sodium hyaluronate; in some embodiments, the viscosity modifier is polyvinyl alcohol. In some preferred embodiments, the viscosity modifier is present in a concentration of 0.05%-4% (w / v); in some more preferred embodiments, the viscosity modifier is present in a concentration of 1% (w / v).

[0082] As used herein, the term "pH adjuster" refers to a class of excipients used to control pH or adjust acidity / alkalinity. Based on their properties, they can be classified into three categories: acids, bases, and buffer solutions, and are mainly used in injections, eye drops, solutions, etc. In some embodiments, the pH adjuster is selected from at least one of hydrochloric acid or sodium hydroxide.

[0083] As used herein, the term "active ingredient" refers to one or more biologically active substances in a drug, and is a specific component that produces the desired effect on an individual given the drug. In some embodiments, the active ingredient is human nerve growth factor.

[0084] As used herein, the term "osmolarity regulator" refers to a substance used to regulate the osmotic pressure of the internal and external environments of an organism. It helps maintain the isotonicity of drug solutions, ensuring their safety and efficacy in vivo, and is commonly used in injections, eye drops, and other non-gastrointestinal formulations. In some embodiments, the osmolarity regulator is selected from at least one of sodium chloride or mannitol. In some embodiments, the osmolarity regulator adjusts the osmotic pressure to 260-320 mOsmol / kg, preferably 270-310 mOsmol / kg.

[0085] As used in this article, the term "antimicrobial agent" is also known as "preservative" and refers to chemical substances used to inhibit the growth of microorganisms. They are common excipients in non-solid dosage forms and can be broadly classified into acids, alcohols, quaternary ammonium salts, parabens, and organomercuries, such as sorbic acid, potassium sorbate, benzoic acid, sodium benzoate, benzyl alcohol, methylparaben, ethylparaben, propylparaben, and chlorobutanol.

[0086] II. Examples

[0087] The present invention will be described in detail below through specific embodiments. It should be understood that the following embodiments are for explanation and illustration only and do not limit the scope of the present invention in any way.

[0088] In the following embodiments, unless otherwise specified, all biochemical reagents are conventional reagents in the art, which can be prepared according to conventional methods in the art or obtained commercially, and the specification is laboratory grade.

[0089] The instruments and equipment used in the embodiments are shown in Table 1.

[0090] Table 1. Instruments and Equipment

[0091] Equipment Name Equipment Model Equipment manufacturers Electronic balance AR2140 Ohaus Electronic balance PX4202ZH1E Ohaus pH meter FE28 Mettler Toledo Magnetic stirrer MS-H-ProT Dalong Xingchuang Experimental Instruments (Beijing) Co., Ltd. Viscometer DV2TLVTJO Brookfield High Performance Liquid Chromatography e2695 waters Particle detector GWJ-16 Tianjin Tianda Tianfa Technology Co., Ltd.

[0092] Example 1: Preparation of recombinant human nerve growth factor polypeptide

[0093] Using the wild-type human nerve growth factor (NGF) sequence SEO ID NO:3 as the target protein sequence, upstream and downstream primer sequences were synthesized and cloned into a plasmid. This plasmid was then introduced into *E. coli* DH5α competent cells (source: American Type Culture Collection Center (ATCC)). After cultivation, the cells were lysed by sonication, and cell debris and lysates were separated by centrifugation. The cell lysate was purified by chromatography to remove impurities and other proteins, and the target protein sample was collected. The final purified product was validated by SDS-gel electrophoresis to ensure the correct structure and biological activity of the recombinant protein. The recombinant human NGF sequence aligned well with the wild-type NGF sequence, as shown in SEO ID NO:1 and SEO ID NO:2, containing 117 and 118 amino acids respectively, representing a 3- or 2-amino acid truncation compared to the wild-type sequence.

[0094] Example 2: Detection method for human nerve growth factor composition

[0095] Recombinant human neurotrophic factor SEQ ID NO:2 was used as the active ingredient. Different formulations were prepared according to the component ratios. A portion of the freshly prepared solution was used as the baseline value (0°C) for testing. The remaining solutions were placed in incubators at 25°C and 40°C, respectively. Samples were taken on days 5 and 10 for testing. The test indicators included appearance, purity, and biological activity. Evaluation was based on the percentage change in the test values. The percentage change formula is as follows:

[0096] Percentage change in difference = (Base value - Detected value) / Base value (%)

[0097] 1. Purity testing

[0098] This invention uses size exclusion chromatography to detect the purity of formulations.

[0099] Size exclusion chromatography (SUC) is a liquid chromatography technique that separates analytes based on their molecular size. The separation principle of SUC is based on the molecular sieving mechanism of gel chromatography columns. These columns typically use hydrophilic silica gel, gels, or modified gels such as Sephadex and Sepharose as packing materials. The surface of these packing materials has pores of varying sizes. After drug molecules enter the column, different components enter the corresponding pores according to their molecular size. Molecules larger than all pore sizes cannot enter the packing particles and are not retained during chromatography, being the first to be eluted by the mobile phase, resulting in shorter retention times. Molecules smaller than all pore sizes can freely enter all pores on the packing surface, resulting in longer retention times in the column. The remaining molecules are eluted sequentially according to their molecular size. Aggregates, compared to monomers, have larger molecules and shorter retention times during detection.

[0100] Operating Procedure: Use a chromatographic gel suitable for separating proteins with a molecular weight of 5–100 kDa as the stationary phase, and 0.25 mol / L phosphate buffer (containing 0.15 mol / L disodium hydrogen phosphate solution and 0.1 mol / L sodium dihydrogen phosphate solution)-acetonitrile (90:10) as the mobile phase; the detection wavelength is 214 nm. Take an appropriate amount of the test sample, dilute it with the mobile phase to prepare solutions of a certain concentration, accurately measure 20 μL and inject it into the liquid chromatograph, record for 30 minutes, record the chromatogram and calculate the peak area. Calculate the main peak area using the area normalization method.

[0101] 2. Assay for biological activity

[0102] This invention uses the TF-1 cell / MTS colorimetric method to determine biological activity.

[0103] This method is an arbitration method, which is based on the fact that the growth status of human erythroblastic leukemia cells (TF-1 cells) varies due to the different biological activities of human nerve growth factor (NGF), and uses this to detect the biological activity of NGF.

[0104] Reagents:

[0105] (1) RPMI 1640 culture medium: Take commercially available RPMI 1640 culture medium and add penicillin and streptomycin to a final concentration of 100 IU / ml.

[0106] (2) Basic culture medium: Measure 100 ml of fetal bovine serum (FBS) and add it to 900 ml of RPMI 1640 culture medium. Store at 4℃.

[0107] (3) Complete culture medium: Add NGF to the basic culture medium until the final concentration is 12U per 1ml.

[0108] (4) MTS solution: Take commercially available MTS and melt it at 4℃. Dispense 1.2ml / vial into EP tubes and store in the dark at -20℃.

[0109] (5) TF-1 cells: TF-1 cell line (source: American Type Culture Collection Center (ATCC)) was cultured in a complete culture medium at 37°C in a 5% CO2 incubator, with the cell concentration controlled at 1.0 × 10⁻⁶ cells per ml. 5 ~5.0×10 5 Cells were passaged and used for NGF biological activity assay 24 hours later.

[0110] Preparation of standard solutions: Take the national standard of NGF (source: National Institute for Biological Standards and Control, UK) and reconstitute it according to the instructions. Dilute with basal culture medium to a concentration of 100 U / ml or an appropriate concentration (each dilution should not exceed 10-fold). Perform a 3-fold serial dilution in a 96-well cell culture plate, for a total of 8 dilutions. Perform 2 wells for each dilution, retaining 100 μl of standard solution in each well and discarding any excess solution. All operations should be performed under clean conditions.

[0111] Preparation of the test solution: Reconstitute the test sample to the labeled amount, then dilute with basal culture medium to approximately 100 U per ml (each dilution should not exceed 10-fold). Perform a 3-fold serial dilution in a 96-well cell culture plate, for a total of 8 dilutions. Perform 2 wells for each dilution, retaining 100 μl of the standard solution in each well and discarding any excess solution. All procedures should be performed under clean conditions.

[0112] Assay Procedure: TF-1 cell lines were cultured in complete culture medium at 37°C and 5% carbon dioxide, with the cell concentration controlled at 1.0 × 10⁻⁶ cells per ml. 5 ~5.0×10 5 100 cells were passaged and used for biological activity assays 24 hours later. The experimental solution was preheated to 37°C. A sufficient volume of TF-1 cell culture was collected by centrifugation, and the cells were washed three times with basal culture medium, then resuspended in basal culture medium to prepare a solution containing 6.0 × 10⁶ cells per ml. 4 Cell suspensions of individual cells were prepared and stored at 37°C under 5% carbon dioxide conditions. 100 μl of cell suspension was added to each well of a 96-well cell culture plate containing standard and test solutions, and incubated at 37°C under 5% carbon dioxide for 66–72 hours. 20 μl of MTS solution was added to each well, and the plate was incubated at 37°C under 5% carbon dioxide for 3 hours. All procedures were performed under aseptic conditions. The samples were then placed in a microplate reader, and absorbance was measured at 490 nm using 550 nm as the reference wavelength. The results were recorded. Experimental data were processed using a computer program or a four-parameter regression method, and the results were calculated using the following formula:

[0113]

[0114] In the formula, Pr represents the biological activity of the standard, U / ml;

[0115] Ds is the pre-dilution factor of the test sample;

[0116] Dr represents the pre-dilution factor of the standard;

[0117] Es is the dilution factor of the test sample relative to half the effective amount of the standard;

[0118] Er is the dilution factor of the half-effective dose of the standard.

[0119] Example 3-1: Stabilizer Test

[0120] The sample formulations in this embodiment are shown in Table 2. Eight different samples were compared, with samples 1-1 to 1-5 serving as control samples. Sample 1-1 contained only recombinant nerve growth factor, sample 1-2 contained no stabilizer, samples 1-3 to 1-5 contained nonoxynol ether, PEG400, and polysorbate 20 as stabilizers, respectively, and samples 1-6 to 1-8 contained equal amounts of stevia, sorbitol, and dodecyl-β-D-maltose (DDM) as stabilizers, respectively. The results of appearance, purity, and biological activity tests are shown in Table 3, and the percentage changes in their differences are shown in Table 4.

[0121] The results showed that, compared with nonoxynol ether, PEG 400, and polysorbate 20, the addition of stevia, sorbitol, and DDM significantly slowed down the rate of purity reduction, i.e., slowed down the degradation of the active ingredient, and maintained the activity at a high level. The percentage decrease in purity and activity after 10 days at 25℃ was less than 4%, and the percentage decrease after 10 days at 40℃ was less than 8%. Among these, DDM showed the best effect as a stabilizer, achieving a percentage decrease in purity and activity of less than 1% after 10 days at 25℃ and less than 5% after 10 days at 40℃.

[0122] Table 2. Sample ratios in stabilizer testing

[0123]

[0124] Table 3. Sample test results in stabilizer testing

[0125]

[0126]

[0127] Table 4. Percentage Change in Sample Detection Results in Stabilizer Testing

[0128]

[0129] Example 3-2: Viscosity regulator test

[0130] The sample formulations in this embodiment are shown in Table 5. Eight different samples were compared. Sample 2-1 (same as sample 1-2) and sample 2-2 (same as sample 1-8) served as control samples. Samples 2-3 to 2-8 were compared by adding the same amounts of polyvinyl alcohol, sodium hyaluronate, and hydroxypropyl methylcellulose (HPMC) as viscosity modifiers, respectively, to samples containing and without DDM. The results of appearance, purity, and biological activity tests are shown in Table 6, and the percentage changes in their differences are shown in Table 7.

[0131] The results showed that samples 2-3, 2-5, and 2-7, which contained the same amounts of polyvinyl alcohol, sodium hyaluronate, and HPMC, exhibited improved formulation stability, purity, and activity reduction compared to control sample 2-1 without viscosity modifiers. Furthermore, samples 2-6, 2-8, and 2-4, which contained the same amounts of DDM and sodium hyaluronate, HPMC, and polyvinyl alcohol, showed improved formulation stability, purity, and activity reduction compared to sample 2-2 containing only DDM. This indicates that the combined use of DDM and viscosity modifiers slows down the rate of purity and activity reduction, especially the combination of DDM and polyvinyl alcohol, which has the most significant effect. The addition of polyvinyl alcohol further slows down the rate of purity and activity reduction, suggesting a synergistic effect between polyvinyl alcohol and DDM, which significantly improves formulation stability.

[0132] Table 5. Sample proportions in viscosity modifier testing

[0133]

[0134] Table 6. Sample test results in viscosity modifier testing

[0135]

[0136] Table 7. Percentage Change in Sample Detection Results in Viscosity Conditioner Testing

[0137]

[0138]

[0139] Example 3-3: Stabilizer Dosage Test

[0140] The sample formulations in this embodiment are shown in Table 8. Six different samples were compared. Sample 3-1 (same as sample 1-2) served as the control sample, while samples 3-2 to 3-6 contained DDM at concentrations ranging from 0.001% to 0.6%. Sample 3-4 was identical to samples 1-8. The results of appearance, purity, and biological activity tests are shown in Table 9, and the percentage changes in their differences are shown in Table 10.

[0141] The results showed that, compared with sample 3-1 without DDM and samples 3-6 with 0.6% DDM, a DDM concentration of 0.001%-0.4% significantly improved the stability of the formulation, with a purity and activity decrease of less than 4% after 10 days at 25℃ and less than 8% after 10 days at 40℃. The stability was even better when the DDM concentration was 0.05%-0.1%, with a purity and activity decrease of less than 1% after 10 days at 25℃ and less than 5% after 10 days at 40℃.

[0142] Table 8. Sample ratios in stabilizer dosage test

[0143]

[0144] Table 9. Sample test results in stabilizer dosage test

[0145]

[0146]

[0147] Table 10. Percentage Change in Sample Detection Results During Stabilizer Dosage Testing

[0148]

[0149] Examples 3-4: Viscosity modifier dosage test

[0150] The sample formulations in this embodiment are shown in Table 11. Seven different samples were compared. Sample 4-1 (same as samples 1-8) served as the control sample, while samples 4-2 to 4-7 contained polyvinyl alcohol at concentrations ranging from 0.05% to 5%. Sample 4-3 was identical to samples 2-4. The results of appearance, purity, and biological activity tests are shown in Table 12, and the percentage changes in their differences are shown in Table 13.

[0151] The results showed that the purity or activity of the active ingredient in samples 4-1 to 4-6, with concentrations ranging from 0-4%, decreased by less than 1% at 25℃ for 10 days and by less than 5% at 40℃ for 10 days, exhibiting good stability. Among these, the viscosity modifier concentrations in the range of 0.05%-4% showed even better stability. Although the addition of polyvinyl alcohol (PVA) improved the stability of the formulation, the viscosity increased significantly with increasing PVA content. Excessively high viscosity can affect the accuracy of eye drop administration, patient comfort, and is detrimental to sterile filtration.

[0152] Table 11. Sample proportions in the viscosity modifier dosage test

[0153]

[0154] Table 12. Sample test results in viscosity modifier dosage test

[0155]

[0156]

[0157] Table 13. Percentage Change in Sample Detection Results During Viscosity Modifier Dosage Test

[0158]

[0159] Examples 3-5: pH Range Testing

[0160] The sample formulations in this embodiment are shown in Table 14. Eight different samples were compared. Samples 5-1 to 5-8 were adjusted to different pH values ​​using hydrochloric acid or sodium hydroxide, ranging from 4.5 to 8.0. Sample 5-5 was identical to sample 4-4. The results of appearance, purity, and biological activity tests are shown in Table 15, and the percentage changes in their differences are shown in Table 16.

[0161] The results showed that, compared with samples 5-1 and 5-8 with pH values ​​of 4.5 and 8.0 respectively, samples 5-2 to 5-7 with pH values ​​in the range of 5.0-7.5 had better stability, with the purity or activity of the active ingredient decreasing by less than 4% at 25℃ for 10 days and less than 8% at 40℃ for 10 days.

[0162] Table 14. Sample ratios in pH range testing

[0163]

[0164]

[0165] Table 15. Sample detection results in pH range test

[0166]

[0167] Table 16. Percentage Change in Sample Detection Results in pH Range Test

[0168]

[0169] Examples 3-6: Synergistic Effect Test

[0170] Based on the aforementioned experiments, it was observed that DDM and polyvinyl alcohol may have a synergistic effect. Therefore, 0.05%-4% concentrations of polyvinyl alcohol and 0.001%-0.4% concentrations of DDM were selected for the experiments. Synergistic effect refers to comparing a parts of A and b parts of B with (a+b) parts of A and (a+b) parts of B, respectively. If the stability results of a parts of A and b parts of B are superior to the latter two, it is considered that a parts of A and b parts of B can synergistically enhance each other. The sample formulations in this embodiment are shown in Table 17. Nine different samples were compared. Sample 6-1 (same as sample 4-4) contained 0.1% DDM and 1% polyvinyl alcohol, while control samples 6-2 and 6-3 contained 1.1% polyvinyl alcohol and 1.1% DDM, respectively. Sample 6-6 contained 0.001% DDM and 4% polyvinyl alcohol, while control samples 6-4 and 6-5 contained 4.001% DDM and 4.001% polyvinyl alcohol, respectively. Sample 6-7 contained 0.4% DDM and 0.05% polyvinyl alcohol, while control samples 6-8 and 6-9 contained 0.45% DDM and 0.45% polyvinyl alcohol, respectively. The results of appearance, purity, and biological activity tests are shown in Table 18, and the percentage changes in their differences are shown in Table 19.

[0171] The results showed that, compared with the control sample containing only one reagent, the combined use of DDM and polyvinyl alcohol exhibited a better effect in reducing the degradation of active ingredients, with a smaller rate of decrease in the purity and activity of the formulation. DDM and polyvinyl alcohol had a synergistic effect in improving the stability of recombinant human nerve growth factor formulations.

[0172] Table 17. Sample ratios in the synergistic effect test

[0173]

[0174] Table 18. Sample detection results in synergistic effect test

[0175]

[0176] Table 19. Percentage Change in Difference of Sample Detection Results in Synergistic Effect Test

[0177]

[0178] Examples 3-7: Active ingredient concentration test

[0179] The sample formulations in this embodiment are shown in Table 20. Six different samples were compared, with sample 7-1 being identical to sample 4-4. Samples 7-1 to 7-6 contained 0.001%–0.06% of recombinant human nerve growth factor, respectively. The results of appearance, purity, and biological activity tests are shown in Table 21, and the percentage changes in their differences are shown in Table 22.

[0180] The results showed that the purity or activity of the active ingredients in samples 7-1 to 7-6 decreased by less than 1% at 25℃ for 10 days and by less than 2% at 40℃ for 10 days, indicating good stability. Therefore, the concentration range of the active ingredients from 0.001% to 0.06% is suitable for use. Since the activity increases with the concentration of the active ingredient, any concentration can be used to achieve the desired therapeutic effect.

[0181] Table 20. Sample ratios in the active ingredient concentration test

[0182]

[0183] Table 21. Sample Detection Results in Active Ingredient Concentration Test

[0184]

[0185]

[0186] Table 22. Percentage Change in Sample Detection Results in Active Ingredient Concentration Testing

[0187]

[0188] Examples 3-8: Osmotic Pressure Regulator Test

[0189] The sample formulations in this embodiment are shown in Table 23. Six different samples were compared, with samples 8-5 and 8-6 serving as controls. Sample 8-1 was identical to sample 4-4. Samples 8-1 through 8-5 were prepared with sodium chloride, glycerol, glucose, and mannitol as osmotic pressure regulators, respectively. Sample 8-5 was prepared without a viscosity regulator, and sample 8-6 was prepared without an osmotic pressure regulator. The results of appearance, purity, and biological activity tests are shown in Table 24, and the percentage changes in their differences are shown in Table 25.

[0190] The results showed that the purity or activity of the active ingredients in samples 8-1 to 8-6 decreased by less than 1% at 25℃ for 10 days and by less than 4% at 40℃ for 10 days, indicating that using different osmotic pressure regulators or not using any osmotic pressure regulator could achieve essentially equivalent stability. Furthermore, compared to sample 8-5, samples 8-1 to 8-4 and sample 8-6, which used both DDM and polyvinyl alcohol, exhibited better stability, with the purity or activity of the active ingredients decreasing by less than 1% at 25℃ for 10 days and by less than 3% at 40℃ for 10 days. Therefore, as long as the target osmotic pressure range of 260-320 mOsmol / kg can be achieved, the type of osmotic pressure regulator is not limited, and the selection of an osmotic pressure regulator is not mandatory.

[0191] Table 23. Sample ratios in osmotic pressure regulator testing

[0192]

[0193]

[0194] Table 24. Sample test results in the osmotic pressure regulator test

[0195]

[0196] Table 25. Percentage Change in Sample Detection Results in Osmotic Pressure Regulator Testing

[0197]

[0198] The above results demonstrate that this NGF composition does not require freezing and maintains good stability with minimal changes in activity and purity under storage conditions of 25°C and 40°C. Those skilled in the art can expect that under refrigeration conditions significantly lower than 25°C and 40°C, the rate of decrease in activity and purity will be even smaller, resulting in even better stability. In particular, the addition of DDM as a stabilizer and polyvinyl alcohol as a modifier fully leverages their synergistic effect.

[0199] In addition, the reagents in each experimental example of this application can be stored in conventional packaging containers under various storage conditions, and do not require the use of fixed accessories such as adapters, pipettes, disinfectant wipes, filters, needles, syringes, etc., making operation and use simple.

Claims

1. A storage-stable human nerve growth factor (NGF) composition, said composition comprising human nerve growth factor as an active ingredient and a stabilizer, wherein, The stabilizer is selected from at least one of stevia, sorbitol, or dodecyl-β-D-maltodextrin (DDM); Preferably, the stabilizer is dodecyl-β-D-maltodextrin; Preferably, the pH of the composition is 5.0-7.5, more preferably 6.0-6.

5.

2. The composition according to claim 1, wherein, The composition further comprises a viscosity modifier selected from at least one of hydroxypropyl methylcellulose, polyvinyl alcohol, or sodium hyaluronate; Preferably, the viscosity modifier is polyvinyl alcohol; Preferably, the viscosity modifier content is 0-4% (w / v); More preferably, the viscosity modifier content is 0.05%-4% (w / v); More preferably, the viscosity modifier content is 1% (w / v).

3. The composition according to claim 1 or 2, wherein, The human nerve growth factor contains at least one of the amino acid sequences shown in SEQ ID NO:1-3; Preferably, the human nerve growth factor comprises an amino acid sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with at least one of the amino acid sequences shown in SEQ ID NO:1-3; Preferably, the content of the human nerve growth factor is 0.001%-0.06% (w / v).

4. The composition according to any one of claims 1-3, wherein, The composition further comprises an osmotic pressure regulator; Preferably, the osmotic pressure regulator is selected from at least one of glycerol, glucose, citrate, phosphate, potassium chloride, sorbitol, sodium chloride, or mannitol; Preferably, the amount of osmotic pressure regulator used adjusts the osmotic pressure of the composition to a range of 260-320 mOsmol / kg, more preferably 270-310 mOsmol / kg.

5. The composition according to any one of claims 1-4, wherein, The stabilizer content is 0.001%-0.4% (w / v); Preferably, the content of the stabilizer is 0.05%-0.1% (w / v).

6. The composition according to any one of claims 1-5, wherein, The composition is formulated such that after storage at 25°C for at least 10 days, the purity or activity of the active ingredient decreases by less than 5%, preferably less than 4%, more preferably less than 2%, and more preferably less than 1%; and / or The composition is formulated such that after being stored at 40°C for at least 10 days, the purity or activity of the active ingredient decreases by less than 10%, preferably less than 8%, more preferably less than 5%, more preferably less than 4%, more preferably less than 3%, and more preferably less than 2%.

7. The composition according to any one of claims 1-6, wherein, The composition comprises the following components: 0.001%-0.06% (w / v) human nerve growth factor, 0.001%-0.4% (w / v) stabilizer, 0-4% (w / v) viscosity modifier, pH adjuster to adjust pH to 5.0-7.5, and optional osmotic pressure modifier such that the osmotic pressure ranges from 260-320 mOsmol / kg, with the remainder being water.

8. The composition according to any one of claims 1-7, wherein, The composition is a solution; Preferably, the composition is applied through the eyes, ears, or skin; Preferably, the composition is an eye drop or an ophthalmic injection.

9. The use of the composition according to any one of claims 1-8 in the preparation of a medicament or medicament for treating neurotrophic keratitis, diabetic skin ulcers, or sensorineural hearing loss; Preferably, the drug or medicament is administered through the eyes, ears, or skin; Preferably, the drug or medicine box is in the form of a solution; Preferably, the drug or medicine box is stored at room temperature or under refrigeration; Preferably, the medicine or medicine box does not need to be matched with fixed accessories; More preferably, the drug or medicine box is an eye drop or an ophthalmic injection.

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