NGF-containing ophthalmic composition and use thereof

Abstract

The present invention relates to the technical field of pharmaceuticals and provides an NGF-containing ophthalmic composition and use thereof. The ophthalmic composition comprises an NGF as an active substance, an ophthalmic excipient, and water. The ophthalmic excipient comprises a pH buffer and an osmotic pressure regulator. The content of the active substance NGF in the ophthalmic composition is 0.0001 wt% to 0.1 wt%, and the content of the pH buffer in the ophthalmic composition is 0.001 wt% to 2.5 wt%. The active substance NGF is rhNGF. The provided ophthalmic composition has relatively good stability at both 2-8 °C and room temperature, so that the activity of the NGF can be well maintained. Also provided is use of the ophthalmic composition in the preparation of a drug for preventing and / or treating eye diseases.

Description

An ophthalmic composition containing NGF and its uses Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to an ophthalmic composition containing NGF and its uses. Background Technology

[0002] Nerve growth factor (NGF) is a neurotrophic factor that nourishes neuronal cell bodies and promotes axonal growth. It is essential for the development and survival of specific neurons, including sympathetic and sensory neurons. It plays a crucial role in regulating neuronal growth, development, differentiation, determining the direction of axonal growth, and repairing damaged nerves. Numerous preclinical animal experiments and clinical studies have demonstrated that NGF has physiological functions in regulating the development, differentiation, and growth of central and peripheral neurons. Exogenous NGF can promote the repair and regeneration of damaged nerves. NGF is a complex protein containing α, β, and γ subunits, with the β subunit being the biologically active portion, known as β-NGF. The term NGF usually refers to β-NGF. NGF exerts its neuronal maintenance and differentiation functions through a specific high-affinity receptor (type I neurotrophic tyrosine receptor kinase, TrkA) and a non-specific low-affinity (p75NTR) nerve growth factor receptor signaling pathway. NGF can bind to either TrkA or p75NTR alone, or simultaneously to both. Both receptors are distributed in neuronal tissue, as well as other normal and pathological tissues, including epithelial, endothelial, and lymphoid tissues. The TrkA receptor promotes the growth and survival of neural synapses through the PI3K-Akt and Ras-MAP kinase pathways. Therefore, nerve growth factor (NGF) can regulate the development of peripheral and central neurons and maintain neuronal survival. Furthermore, NGF is also involved in the regulation of the immune and endocrine systems.

[0003] Clinical indications for NGF include peripheral sensory nerve diseases, Alzheimer's disease, and corneal ulcers. Currently, only one recombinant human nerve growth factor product has been approved for marketing: recombinant human nerve growth factor eye drops (Xenegiline eye drops / Oxervate®), indicated for neurotrophic keratitis. Neurotrophic keratitis (NK) is a degenerative corneal disease caused by damage to the corneal nerve function of the trigeminal nerve, leading to impaired corneal sensation and nutrition, resulting in decreased or absent corneal sensation, corneal epithelial defects, and corneal ulcers. The most common causes of corneal nerve injury include herpetic keratitis, chemical burns, physical injury, corneal surgery, prolonged use of contact lenses, and prolonged use of topical medications. Intracranial tumors (such as neuromas, meningiomas, and aneurysms) may compress the trigeminal nerve or ganglia, leading to impaired corneal sensitivity. Systemic diseases such as diabetes and multiple sclerosis may damage sensory nerves, resulting in decreased corneal sensation. Recent studies have also found that dry eye can cause corneal nerve damage, as well as corneal degenerative diseases such as keratoconus and malnutrition.

[0004] Nerve growth factor receptors TrkA and p75NTR are expressed in the anterior segment of the eye (cornea, conjunctiva, iris, ciliary body, and lens), lacrimal gland, and posterior segment intraocular tissues. The mechanism of action of NGF on NK cells can be summarized in four main points: participation in the maintenance of normal nerve function and stimulation of sensory nerve regeneration and survival; stimulation of corneal epithelial cell proliferation, migration, and differentiation; and direct promotion of tear secretion by binding to receptors on the lacrimal gland, restoring sensory-mediated reflexive tear secretion. Furthermore, NGF can inhibit inflammatory responses and modulate ocular immune responses. Therefore, NGF is essential for immunomodulation, maintaining corneal sensitivity, corneal nutrition, retinal neuroprotection, optic nerve protection, and ocular surface integrity. Technical issues

[0005] NGF has been developed into liquid pharmaceutical compositions for both ophthalmic and non-ophthalmic applications. Due to its dimer structure, the stability of NGF is more complex than typical chemical and physical degradation pathways. Combined with compatibility issues in container-sealed systems and the complexity of protein drug manufacturing processes, maintaining the stability of NGF is both complex and necessary. There is a need for NGF-containing formulations that can maintain NGF activity and stability for safer and more effective treatment, especially for the treatment of human diseases. Technical solutions

[0006] The purpose of this invention is to provide an ophthalmic composition containing NGF and its uses, thereby providing a drug with good stability and the ability to maintain NGF activity. The specific technical solution is as follows:

[0007] The first aspect of the present invention provides an ophthalmic composition containing NGF, comprising the active substance NGF, an ophthalmic excipient and water, wherein the ophthalmic excipient comprises a pH buffer and an osmotic pressure regulator;

[0008] The content of the active substance NGF in the ophthalmic composition is 0.0001wt%-0.1wt%, preferably 0.001wt%-0.01wt%, and more preferably 0.001wt%-0.005wt%.

[0009] The pH buffer in the ophthalmic composition is present in an amount of 0.001 wt%-2.5 wt%, preferably 0.01 wt%-1.5 wt%, and more preferably 0.05 wt%-0.5 wt%.

[0010] The active substance NGF is recombinant human nerve growth factor (rhNGF).

[0011] In some embodiments, the pH of the ophthalmic composition is 4.0-8.0, preferably 5.0-7.0, more preferably 5.5-6.5; and the osmotic pressure is 200-400 mOsmol / kg, preferably 240-380 mOsmol / kg, more preferably 280-320 mOsmol / kg.

[0012] In some embodiments, the pH buffer is selected from at least one of boric acid-borate, citrate-citrate, acetic acid-acetate, tris(hydroxymethyl)aminomethane hydrochloride, histidine, histidine-histidine hydrochloride, and phosphate; preferably, the pH buffer is citrate-citrate.

[0013] The borate is selected from at least one of sodium borate, potassium borate, and their hydrates;

[0014] The citrate is selected from at least one of potassium citrate, sodium citrate, disodium hydrogen citrate, sodium dihydrogen citrate, dipotassium hydrogen citrate, potassium dihydrogen citrate, and their hydrates;

[0015] The phosphate is selected from at least one of disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and their hydrates.

[0016] In some embodiments, the osmotic pressure regulator is selected from inorganic osmotic pressure regulators and / or organic osmotic pressure regulators;

[0017] The inorganic osmotic pressure regulator is selected from at least one of sodium chloride and potassium chloride;

[0018] The organic osmotic pressure regulator is selected from at least one of glycerol, mannitol, sorbitol, trehalose, L-carnitine, proline, ectoine, hyaluronic acid, erythritol, sucrose, and glucose.

[0019] In some embodiments, the inorganic osmotic pressure regulator is present in the ophthalmic composition at a content of 0.001 wt%-0.9 wt%, preferably 0.005 wt%-0.2 wt%, and more preferably 0.01 wt%-0.05 wt%.

[0020] The organic osmotic pressure regulator is present in the ophthalmic composition at a content of 0.01wt%-20wt%, preferably 0.1wt%-10wt%, and more preferably 0.5wt%-8wt%.

[0021] In some embodiments, the ophthalmic excipient further comprises a nonionic surfactant; the nonionic surfactant is selected from at least one of polysorbate, poloxamine, and poloxamer, preferably polysorbate;

[0022] The nonionic surfactant in the ophthalmic composition is present in an amount of 0.0001wt%-3wt%, preferably 0.001wt%-1wt%, and more preferably 0.01wt%-0.5wt%.

[0023] In some embodiments, the ophthalmic excipient further comprises a protein stabilizer; the protein stabilizer is selected from at least one of L-methionine and sodium thiosulfate;

[0024] Preferably, the protein stabilizer in the ophthalmic composition is 0.0001wt%-1wt%, more preferably 0.0001wt%-0.15wt%, and even more preferably 0.0005wt%-0.002wt%.

[0025] In some embodiments, the ophthalmic excipient further comprises a mucosal adhesive; the mucosal adhesive is selected from at least one of polyvinylpyrrolidone (PVP), hydroxypropyl methylcellulose (HPMC), carboxymethyl cellulose (CMC), and polyethylene glycol (PEG), preferably hydroxypropyl methylcellulose (HPMC).

[0026] Preferably, the content of the mucosal adhesive in the ophthalmic composition is 0.01wt%-0.5wt%, more preferably 0.02wt%-0.2wt%.

[0027] In some embodiments, the ophthalmic excipient further comprises a chelating agent; the chelating agent is selected from at least one of nitrotriacetic acid, ethylenediaminedisuccinic acid, iminodisuccinic acid, methylglycine diacetic acid, L-glutamic acid N,N-diacetic acid, ethylenediamine-N,N'-diglutamic acid, ethylenediamine-N,N'-dimalonic acid, 3-hydroxy-2,2-iminodisuccinic acid, 2-hydroxyethyliminodiacetic acid, pyridine-2,6-dicarboxylic acid, diethylenetriaminepentaacetic acid, hydroxyethyldiaminetriacetic acid, 1,2-diaminocyclohexanetetraacetic acid, hydroxyethylaminodiacetic acid, polyphosphate, citric acid and citrate, tartaric acid and tartrate, ethylenediaminetetraacetic acid and disodium ethylenediaminetetraacetic acid, and alkali metal salts of hexametaphosphate;

[0028] Preferably, the chelating agent is present in the ophthalmic composition at a content of 0.001wt%-1wt%, more preferably 0.1wt%-0.25wt%.

[0029] In some embodiments, the ophthalmic excipient is selected from any combination of the following:

[0030] Combination 1: pH buffer and osmotic pressure regulator; that is, ophthalmic excipients consist of two components: pH buffer and osmotic pressure regulator;

[0031] Or combination 2: pH buffer, osmotic pressure regulator, and at least two of the following: nonionic surfactant, mucosal adhesive, and protein stabilizer; that is: the ophthalmic excipient is a four-component or five-component composition consisting of at least two of the following: pH buffer, osmotic pressure regulator, nonionic surfactant, mucosal adhesive, and protein stabilizer; for example: a four-component composition consisting of pH buffer, osmotic pressure regulator, nonionic surfactant, and mucosal adhesive; another example: a four-component composition consisting of pH buffer, osmotic pressure regulator, nonionic surfactant, and protein stabilizer; yet another example: a five-component composition consisting of pH buffer, osmotic pressure regulator, nonionic surfactant, mucosal adhesive, and protein stabilizer.

[0032] Preferably, in the above combinations of the present invention, the pH buffer is citric acid-citrate; and / or the mucosal adhesive is at least one of hydroxypropyl methylcellulose and polyethylene glycol.

[0033] A second aspect of the present invention provides the use of the ophthalmic composition provided in the first aspect of the present invention in the preparation of a medicament for the prevention and / or treatment of eye diseases.

[0034] In some embodiments, the eye disease is an eye disease for which NGF treatment is applicable.

[0035] In some embodiments, the eye disease is selected from at least one of neurotrophic keratitis, corneal ulcers and injuries, dry eye disease, glaucoma, retinitis pigmentosa, and macular degeneration.

[0036] The ophthalmic composition containing the ophthalmic excipients of the present invention can be stored for at least 3-6 weeks at 2-8°C and at least 10 days at room temperature, exhibiting good stability and being able to stably maintain the activity of NGF; the ophthalmic composition containing at least one of nonionic surfactants, mucosal adhesives, protein stabilizers and chelating agents also exhibits better stability at 2-8°C and room temperature. Beneficial effects

[0037] Providing better storage conditions: Compared with commercially available products, the NGF in the ophthalmic composition of this invention exhibits significantly improved stability at 2-8°C. Commercially available Oxyvit only allows for patient storage at 2-8°C for one week under refrigeration and at 25°C for 24 hours. The ophthalmic composition of this invention, due to its superior stability at 2-8°C (allowing for storage for at least 3-6 weeks), offers greater convenience to patients.

[0038] Reducing the risk to patients using partially denatured or less effective rhNGF products: The ophthalmic composition of this invention can also be stored at room temperature for at least 10 days, or even a month. When patients fail to promptly return existing rhNGF-containing eye drops to the refrigerator (2-8°C) after medication use, rhNGF may become completely or partially denatured. Denatured protein drug formulations may be unable to treat eye diseases and endanger the patient's eye health. The ophthalmic composition of this invention can be stored at room temperature for 10 days or more, which can reduce the risk to patients using denatured drugs or rhNGF with reduced potency.

[0039] Of course, implementing any product or method of the present invention does not necessarily require achieving all of the advantages described above at the same time. Attached Figure Description

[0040] Figure 1 shows the proliferation curve of TF-1 cells under the action of rhNGF in the ophthalmic composition of Example 1 of the present invention;

[0041] Figure 2 shows the morphology of PC-12 cells cultured for 9 days at different concentrations of rhNGF in the ophthalmic composition of Example 1 of the present invention;

[0042] Figure 3 shows the rhNGF content of the ophthalmic compositions of Examples 1-2 after storage at different temperatures for different days, as determined by ELISA.

[0043] Figure 4 shows the rhNGF content of the ophthalmic compositions of Examples 3-5 after 20 days of storage at different temperatures, as determined by RP-HPLC.

[0044] Figure 5 shows the rhNGF content of the ophthalmic compositions of Examples 6-16 after 30 days of storage at different temperatures, as determined by RP-HPLC.

[0045] Figure 6 shows the rhNGF content of the ophthalmic composition of Example 17 after storage at different temperatures for different days, as determined by RP-HPLC.

[0046] Figure 7 shows the rhNGF content of the ophthalmic compositions of Examples 25-28 after 20 days of storage at different temperatures, as determined by RP-HPLC.

[0047] Figure 8 shows the rhNGF content of the ophthalmic compositions of Examples 29-34 after 30 days of storage at different temperatures, as determined by RP-HPLC.

[0048] Figure 9 shows the rhNGF content of the ophthalmic compositions of Examples 35-37 after 38 days of storage at different temperatures, as determined by RP-HPLC.

[0049] Figure 10 shows the rhNGF content of the ophthalmic compositions of Examples 38-40 after 43 days of storage at different temperatures, as determined by RP-HPLC.

[0050] Figure 11 shows the rhNGF content of the ophthalmic compositions of Examples 41-45 after 30 days of storage at different temperatures, as determined by RP-HPLC.

[0051] Figure 12 shows the rhNGF content of the ophthalmic compositions of Examples 46-48 after 17 days of storage at different temperatures, as determined by RP-HPLC.

[0052] Figure 13 shows the results of ELISA analysis of the ophthalmic compositions of Examples 25-34, with the rhNGF concentration of the ophthalmic compositions stored at -20°C for 3 days being 100% and the rhNGF concentration of the ophthalmic compositions stored at 40°C for 3 days being 100%. Embodiments of the present invention

[0053] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art based on the present invention are within the scope of protection of the present invention.

[0054] The first aspect of the present invention provides an ophthalmic composition containing NGF, comprising the active substance NGF, an ophthalmic excipient and water, wherein the ophthalmic excipient comprises a pH buffer and an osmotic pressure regulator (or osmotic pressure protectant).

[0055] The content of the active substance NGF in the ophthalmic composition is 0.0001wt%-0.1wt%, preferably 0.001wt%-0.01wt%, more preferably 0.001wt%-0.005wt%; for example, it can be 0.0001wt%, 0.0002wt%, 0.0004wt%, 0.0005wt%, 0.0008wt%, 0.001wt%, 0.002wt%, 0.003wt%, 0.005wt%, 0.007wt%, 0.01wt%, 0.02wt%, 0.05wt%, 0.08wt%, 0.1wt%, or a range of any two of these values;

[0056] The pH buffer in the ophthalmic composition is present in an amount of 0.001 wt%-2.5 wt%, preferably 0.01 wt%-1.5 wt%, more preferably 0.05 wt%-0.5 wt%; for example, it can be 0.001 wt%, 0.002 wt%, 0.003 wt%, 0.005 wt%, 0.007 wt%, 0.01 wt%, 0.02 wt%, 0.05 wt%, 0.08 wt%, 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.5 wt%, 0.8 wt%, 1 wt%, 1.2 wt%, 1.5 wt%, 1.8 wt%, 2 wt%, 2.3 wt%, 2.5 wt%, or a range of any two of these values;

[0057] The active substance NGF is rhNGF.

[0058] This invention provides an ophthalmic composition that offers improved stability and user convenience for nerve growth factor (NGF), particularly recombinant human nerve growth factor (rhNGF). The ophthalmic composition exhibits good stability while maintaining NGF activity. The ophthalmic composition provided by this invention can be stored at 2 to 8°C for at least 3 weeks and at room temperature (20°C-30°C) for at least 10 days, demonstrating good stability and remaining stable even after multiple freeze-thaw cycles.

[0059] The active pharmaceutical ingredient (API) in this invention is NGF, more specifically rhNGF. This invention provides an ophthalmic composition containing NGF that can promote nerve cell growth, repair, survival, proliferation, differentiation, and maturation, as well as promote tear secretion. When applied to the human eye, the final concentration or frequency of administration of the ophthalmic composition provided by this invention can be determined or adjusted according to the patient's condition, if necessary.

[0060] The rhNGF in this invention may contain 117-120 amino acids, but is not limited thereto. By way of example, the amino acid sequence of the rhNGF is selected from at least one of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, as follows:

[0061] SEQ ID NO:1:

[0062] SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAV,

[0063] SEQ ID NO:2:

[0064] SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVR,

[0065] SEQ ID NO:3:

[0066] SSSHPIFHRGEFSVCDSVSVWVGDKTTATDIKGKEVMVLGEVNINSVFKQYFFETKCRDPNPVDSGCRGIDSKHWNSYCTTTHTFVKALTMDGKQAAWRFIRIDTACVCVLSRKAVRRA.

[0067] The endogenous expression and activity of rhNGF in vivo, as described in this invention, requires multiple modification steps, including the cleavage of the leader peptide, dimer formation, and enzymatic hydrolysis of the terminal amino acids. For example, its monomeric sequence, due to post-translational modifications and C-terminal enzymatic cleavage in eukaryotic cells, typically exists in different forms of 117-120 amino acids (all formed from the 120-amino acid form through multiple enzymatic cleavage steps). Mature rhNGF exists in dimer form, containing six cysteine ​​residues within the chain, which can generate three pairs of intrachain disulfide bonds. These three pairs of disulfide bonds are crucial for maintaining the biological activity of rhNGF. Therefore, the maintenance of disulfide bonds and the stable existence of the dimer are important considerations for maintaining the activity of rhNGF. Its physicochemical properties and various degradation processes in vivo, such as protein aggregation, terminal truncation, and oxidation, can all affect the activity of rhNGF. Therefore, it is necessary to maintain the stability of rhNGF to better maintain the pharmacodynamic stability of ophthalmic compositions containing it.

[0068] The present invention does not particularly limit the water in the ophthalmic composition, as long as it can achieve the purpose of the present invention. For example, purified water or water for injection can be used.

[0069] In some embodiments, the pH of the ophthalmic composition is 4.0-8.0, preferably 5.0-7.0, more preferably 5.5-6.5; for example, it can be 4.0, 4.2, 4.5, 4.7, 5.0, 5.2, 5.5, 5.8, 6.0, 6.3, 6.5, 6.7, 7.0, 7.2, 7.5, 7.8, 8.0, or a range of any two of these values; the osmotic pressure is 200-400 mOsmol / kg, preferably 240-380 mOsmol / kg, more preferably 280-320 mOsmol / kg; for example, it can be 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400 mOsmol / kg, or a range of any two of these values. By adjusting the pH of the ophthalmic composition within the range of 4.0-8.0, this invention enables rhNGF to maintain good stability, making it easy to store and improving ease of use; when the pH of the ophthalmic composition is adjusted within the range of 5.0-7.0, the stability of rhNGF can be further improved.

[0070] The ophthalmic composition of the present invention may contain an ophthalmologically friendly and biocompatible buffering system to adjust the ophthalmic composition to approximate human tears. In some embodiments, the pH buffer is selected from at least one of boric acid-borate, citrate, acetate-acetate, Tris-Tris-HCl (tris(hydroxymethyl)aminomethane) hydrochloride, histidine, histidine-histidine hydrochloride, and phosphate.

[0071] The borate is selected from at least one of sodium borate, potassium borate, and their hydrates;

[0072] The citrate is selected from at least one of potassium citrate, sodium citrate, disodium hydrogen citrate, sodium dihydrogen citrate, dipotassium hydrogen citrate, potassium dihydrogen citrate, and their hydrates;

[0073] The acetate is selected from at least one of sodium acetate and potassium acetate;

[0074] The phosphate is selected from at least one of disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and their hydrates.

[0075] Preferably, the pH buffer is citric acid-citrate.

[0076] In some embodiments, the ophthalmic excipient comprises citric acid-citrate and an osmotic pressure regulator. The present invention further provides an ophthalmic composition containing rhNGF and citric acid-citrate, which exhibits improved stability; wherein the citric acid-citrate may simultaneously possess chelating and pH buffering capabilities.

[0077] The inventors have discovered that the ophthalmic composition of this invention, containing rhNGF and citrate, exhibits significantly better thermal stability than currently commercially available NGF products. The use of a pH buffering citrate system not only provides stable pH protection for the drug but also acts as a chelating agent, chelating metals and mitigating the negative effects of metal-induced rhNGF hydrolysis, thereby improving the stability of the rhNGF in the ophthalmic composition.

[0078] In some embodiments, the ophthalmic excipient comprises citric acid-citrate and an osmotic pressure regulator; the pH of the ophthalmic composition is 5.0-7.0. The present invention can further provide an ophthalmic composition containing rhNGF and citric acid-citrate, and adjust the pH of the ophthalmic composition to 5.0-7.0, which can further improve the stability of rhNGF in the ophthalmic composition.

[0079] In some embodiments, the osmotic pressure regulator is selected from inorganic osmotic pressure regulators and / or organic osmotic pressure regulators;

[0080] The inorganic osmotic pressure regulator is selected from at least one of sodium chloride and potassium chloride;

[0081] The organic osmotic pressure regulator is selected from at least one of glycerol, mannitol, sorbitol, trehalose, L-carnitine, proline, ectoine, hyaluronic acid, erythritol, sucrose, and glucose.

[0082] In some embodiments, the inorganic osmotic pressure regulator in the ophthalmic composition is present in an amount of 0.001-0.9 wt%, preferably 0.005 wt%-0.2 wt%, more preferably 0.01 wt%-0.05 wt%; for example, it can be 0.001 wt%, 0.002 wt%, 0.003 wt%, 0.005 wt%, 0.007 wt%, 0.01 wt%, 0.02 wt%, 0.05 wt%, 0.08 wt%, 0.1 wt%, 0.15 wt%, 0.2 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.8 wt%, 0.9 wt%, or a range of any two of these values.

[0083] In some embodiments, the organic osmotic pressure regulator is present in the ophthalmic composition at a content of 0.01wt%-20wt%, preferably 0.1wt%-10wt%, more preferably 0.5wt%-8wt%; for example, it may be 0.01wt%, 0.02wt%, 0.05wt%, 0.08wt%, 0.1wt%, 0.2wt%, 0.3wt%, 0.5wt%, 0.8wt%, 1wt%, 1.2wt%, 1.5wt%, 1.8wt%, 2wt%, 2.3wt%, 2.5wt%, 3wt%, 3.5wt%, 4wt%, 4.5wt%, 5wt%, 6wt%, 7wt%, 8wt%, 9wt%, 10wt%, 11wt%, 13wt%, 15wt%, 16wt%, 18wt%, 20wt%, or a range of any two of these values.

[0084] The inventors have discovered that inorganic osmotic pressure regulators, by providing effective osmotic pressure, may affect cell size and shape, and cause pain and / or irritation to injured ocular surfaces or tissues. When the content of the inorganic osmotic pressure regulator is controlled within the scope of this invention, an ophthalmic composition that is comfortable for the ocular surface and compatible with ocular tissues can be obtained. When the organic osmotic pressure regulator described in this invention is used, and its content is controlled within the scope of this invention, NGF, particularly rhNGF, can be further protected from denaturation, improving its stability. When used in the eye, it can improve the metabolic activity of related cells and the homeostasis of normal and / or injured ocular cells and tissues.

[0085] In some embodiments, the ophthalmic excipient further comprises a nonionic surfactant; the nonionic surfactant is selected from at least one of polysorbate, poloxamer, and poloxamer; the polysorbate is such as polysorbate 20 or polysorbate 80; the poloxamer is such as poloxamer 407 or poloxamer 188, etc., triblock copolymers.

[0086] In some embodiments, the nonionic surfactant in the ophthalmic composition is present in an amount of 0.0001 wt%-3 wt%, preferably 0.001 wt%-1 wt%, more preferably 0.01 wt%-0.5 wt%; for example, it can be 0.0001 wt%, 0.0002 wt%, 0.0004 wt%, 0.0005 wt%, 0.0008 wt%, 0.001 wt%, 0.002 wt%, 0.003 wt%, 0.005 wt%, 0.007 wt%, 0.01 wt%, 0.02 wt%, 0.05 wt%, 0.08 wt%, 0.1 wt%, 0.2 wt%, 0.3 wt%, 0.5 wt%, 0.8 wt%, 1 wt%, 1.2 wt%, 1.5 wt%, 1.8 wt%, 2 wt%, 2.3 wt%, 2.5 wt%, 2.8 wt%, 3 wt%, or a range of any two of these values.

[0087] In this invention, the addition of the nonionic surfactant minimizes the surface tension difference between the ophthalmic composition and the precorneal tear film. Simultaneously, it prevents NGF, such as rhNGF, from denaturing or becoming inactive under certain harsh storage conditions, thus improving the stability of NGF in the ophthalmic composition. Preferably, the use of polysorbate, such as polysorbate 80, can reduce the aggregation of peptides or proteins in the formulation, further enhancing the stability of the ophthalmic composition.

[0088] In some embodiments, the ophthalmic excipient further comprises a protein stabilizer; the protein stabilizer is selected from at least one of L-methionine (also known as L-methylthionine) and sodium thiosulfate.

[0089] In some embodiments, the protein stabilizer in the ophthalmic composition is present in an amount of 0.0001 wt%-0.15 wt%, preferably 0.0005 wt%-0.002 wt%; for example, it can be 0.0001 wt%, 0.0002 wt%, 0.0004 wt%, 0.0005 wt%, 0.0008 wt%, 0.001 wt%, 0.002 wt%, 0.003 wt%, 0.005 wt%, 0.007 wt%, 0.01 wt%, 0.02 wt%, 0.05 wt%, 0.08 wt%, 0.1 wt%, 0.12 wt%, 0.15 wt%, or a range of any two of these values. In this invention, adding the protein stabilizer and controlling its content within the range specified in this invention can further improve the stability of the ophthalmic composition and maintain the activity of NGF.

[0090] In polypeptide sequences, certain amino acids such as methionine, cysteine, histidine, and tyrosine are easily oxidized under certain conditions during manufacturing, storage, and transportation. The protein stabilizers described in this invention, such as L-methionine and sodium thiosulfate, can be used to protect the chemical and physical stability of NGF.

[0091] In some embodiments, the ophthalmic excipient further comprises a mucosal adhesive; the mucosal adhesive is selected from at least one of polyvinylpyrrolidone (PVP), hydroxypropyl methylcellulose (HPMC), carboxymethyl cellulose (CMC), and polyethylene glycol (PEG).

[0092] In some embodiments, the content of the mucosal adhesive in the ophthalmic composition is 0.01wt%-0.5wt%, preferably 0.02wt%-0.2wt%; for example, it can be 0.01wt%, 0.02wt%, 0.05wt%, 0.08wt%, 0.1wt%, 0.12wt%, 0.15wt%, 0.18wt%, 0.2wt%, 0.25wt%, 0.3wt%, 0.35wt%, 0.4wt%, 0.45wt%, 0.5wt%, or a range of any two of these values.

[0093] In this invention, the addition of the mucosal adhesive and the control of its content within the range of this invention can improve the stability of NGF in the ophthalmic composition. In some embodiments, the ophthalmic excipient also comprises a nonionic surfactant and a mucosal adhesive. The inventors have found that ophthalmic compositions containing both a nonionic surfactant and a mucosal adhesive have better stability and can be stored for at least 30 days at 2-8°C and room temperature.

[0094] In some embodiments, the ophthalmic excipient further comprises a chelating agent; the chelating agent is selected from at least one of nitrotriacetic acid, ethylenediaminedisuccinic acid, iminodisuccinic acid, methylglycine diacetic acid, L-glutamic acid N,N-diacetic acid, ethylenediamine-N,N'-diglutamic acid, ethylenediamine-N,N'-dimalonic acid, 3-hydroxy-2,2-iminodisuccinic acid, 2-hydroxyethyliminodiacetic acid, pyridine-2,6-dicarboxylic acid, diethylenetriaminepentaacetic acid, hydroxyethyldiaminetriacetic acid, 1,2-diaminocyclohexanetetraacetic acid, hydroxyethylaminodiacetic acid, polyphosphate, citric acid and citrate, tartaric acid and tartrate, ethylenediaminetetraacetic acid and disodium ethylenediaminetetraacetic acid, and alkali metal salts of hexametaphosphate.

[0095] In some embodiments, the chelating agent in the ophthalmic composition is present in an amount of 0.001 wt%-1 wt%, preferably 0.1 wt%-0.25 wt%; for example, it can be 0.001 wt%, 0.002 wt%, 0.003 wt%, 0.005 wt%, 0.007 wt%, 0.01 wt%, 0.02 wt%, 0.05 wt%, 0.08 wt%, 0.1 wt%, 0.12 wt%, 0.15 wt%, 0.18 wt%, 0.2 wt%, 0.23 wt%, 0.25 wt%, 0.3 wt%, 0.4 wt%, 0.5 wt%, 0.6 wt%, 0.8 wt%, 0.9 wt%, 1 wt%, or a range of any two of these values. In this invention, adding the chelating agent and controlling its content within the range specified in this invention can improve the stability of NGF in the ophthalmic composition.

[0096] The inventors discovered that the simultaneous use of protein stabilizers such as L-methionine and chelating agents such as citrate can produce a synergistic or additive effect: amino acids such as methionine, histidine, tyrosine, and cysteine ​​in NGF can be oxidized under certain conditions encountered during drug development. The addition of protein stabilizers and chelating agents can exert a synergistic effect, which can reduce the oxidation of NGF in ophthalmic compositions and thus improve the stability of ophthalmic compositions.

[0097] In this invention, the nonionic surfactants, protein stabilizers, mucosal adhesives, and chelating agents within the above-mentioned range can be used in any combination, as long as the purpose of this invention can be achieved.

[0098] In this invention, there are no particular limitations on the type, degree of polymerization, or molecular weight of each polymer, as long as the purpose of this invention can be achieved; for example, the polyvinylpyrrolidone can be PVP K30, the hydroxypropyl methylcellulose can be HPMC K4M, HPMC E4M or HPMC E15, the polyethylene glycol can be PEG6000 or PEG400, the poloxamer can be poloxamer 407 or poloxamer 188, the carboxymethyl cellulose can have a weight-average molecular weight of 400,000 to 600,000 Daltons, and the polysorbate can be polysorbate 80.

[0099] The present invention also provides a method for preparing the ophthalmic composition of the present invention, comprising: adding an ophthalmic excipient, preparing a diluent with water, adding rhNGF stock solution, mixing evenly, filtering, and obtaining the ophthalmic composition; wherein, the ophthalmic excipient may be a solid ophthalmic excipient and / or a stock solution of an ophthalmic excipient, and the stock solution of the excipient and the NGF stock solution may be selected and prepared according to the prescription amount. The present invention does not have any particular limitation on them, as long as the purpose of the present invention can be achieved.

[0100] The ophthalmic composition of the present invention has good stability and can be stored in different container closure systems to prepare single-dose or multi-dose ophthalmic compositions. Container closure systems include, but are not limited to, polypropylene-polyethylene tubes (PP-EP tubes), low-density polyethylene blow-fill-seal (LDPE-BFS), borosilicate vials, LDPE unit dose packs, LDPE multi-dose eye drop bottles, and glass containers with or without coatings.

[0101] A second aspect of the present invention provides the use of the ophthalmic composition provided in the first aspect of the present invention in the preparation of a medicament for the prevention and / or treatment of eye diseases.

[0102] In some embodiments, the eye disease is an eye disease for which NGF treatment is applicable.

[0103] In some embodiments, the eye disease is selected from at least one of neurotrophic keratitis, corneal ulcers and injuries, dry eye disease, glaucoma, retinitis pigmentosa, and macular degeneration.

[0104] A third aspect of the present invention provides a method for treating an eye disease sensitive to NGF therapy, the method comprising administering a therapeutically effective amount of the ophthalmic composition provided in the first aspect of the present invention to a patient in need of such treatment.

[0105] Example

[0106] The following examples illustrate the implementation of the present invention in more detail. Various tests and evaluations were performed according to the methods described below. Furthermore, unless otherwise specified, "parts" and "%" refer to quality standards.

[0107] I. Reagents, materials, and instruments:

[0108] Reference products: Recombinant human β-nerve growth factor (RhNGF Protein-QC), manufacturer: Beijing Yiqiao Shenzhou Technology Co., Ltd., product number: 11050-HNAC, concentration: 1 mg / mL, SEQ ID NO: 1; Xennegamine eye drops (Oxavir). ®Manufacturer: Dompe Pharmaceutical, Batch No.: 21D6580; Acetonitrile: Sigma-Aldrich, Catalog No.: 34851-4L; Trifluoroacetic acid: Merck, Catalog No.: 91707-250; Isopropanol: Sigma-Aldrich, Catalog No.: 34863; Column: YMC-Triart Bio C4, 4.6×250mm, S-5μm, 30nm, Manufacturer: YMC, Catalog No.: TB30S05-2546PTH; High performance liquid chromatograph: Agilent / 1260; Microplate reader: BioTek-Synergy H1.

[0109] II. Test methods and equipment:

[0110] This invention uses different analytical methods to analyze the content and purity of rhNGF in ophthalmic compositions to evaluate the stability of rhNGF.

[0111] 2.1 Enzyme-linked immunosorbent assay (ELISA) rapidly detects the rhNGF content in a sample through antigen-antibody reaction:

[0112] A human NGF enzyme-linked immunosorbent assay (ELISA) kit (Sino Biological, model: KIT11050) was used to quantitatively determine the content of human NGF (rhNGF) protein in samples. Specific experimental procedures were performed according to the kit's instructions. rhNGF-specific monoclonal antibodies were pre-coated onto well strips. A standard solution containing rhNGF (concentration identical to the standard solution in the kit, 40000 pg / mL) or the ophthalmic composition sample solution obtained in the examples was added to the wells. The rhNGF in the sample was captured by the immobilized antibody. After incubation, the wells were washed, and horseradish peroxidase-labeled anti-rhNGF antibody was added to generate an antibody-antigen-antibody complex. After washing to remove unbound antibody, a 3,3',5,5'-tetramethylbenzidine (TMB) substrate solution was added. The liquid color was proportional to the amount of rhNGF bound. Then, a stop solution was added to stop the reaction. The absorbance (OD) was measured at 450 nm using a microplate reader. 450 (value), OD 450 The value reflects the intensity of the liquid color. This ELISA kit uses a four-parameter fitting method for standard curve fitting. The OD value of the sample is then used to... 450 Substitute the values ​​into the standard curve to obtain the final rhNGF concentration of the sample.

[0113] 2.2 Quantitative detection of rhNGF bioactivity EC using TF-1 cell proliferation assay (MTS kit) 50 :

[0114] The cell proliferation assay is based on the fact that rhNGF protein drugs specifically bind to receptors on the surface of TF-1 cells and promote cell proliferation. The amount of rhNGF protein drug added is positively correlated with the number of proliferating cells. Therefore, the bioactivity of rhNGF protein drugs in promoting TF-1 cell proliferation can be determined by detecting the effect of rhNGF protein drugs on TF-1 cell proliferation using an MTS kit. Specific experimental procedures should be performed according to the kit instructions. TF-1 cells are derived from human leukemia cells. TF-1 cells have a high-affinity receptor (TrKA) on their surface. After NGF binds to TrKA, it induces TrkA autophosphorylation, thereby causing TF-1 cell proliferation in a concentration-dependent manner. Using cell culture medium (90% RPMI 1640 basal medium + 10% fetal bovine serum (FBS)), the rhNGF-containing sample (the rhNGF-containing ophthalmic composition solution of Example 1) was serially diluted 4-fold from an initial concentration of 200 ng / mL to a total of 10 concentrations. These concentrations were then added to TF-1 cells (Ningbo Mingzhou Biotechnology Co., Ltd.) and co-cultured for 2-4 days. MTS reagent (Promega) was added, and the cells were incubated for 1-4 hours. OD values ​​were measured at 490 nm using a microplate reader. Using Prism software, the proliferation curves of TF-1 cells under the action of rhNGF were obtained, as shown in Figure 1, and the corresponding half-maximal effective concentration (EC50) of rhNGF was determined. 50 The concentration of rhNGF in the EC ophthalmic composition of the present invention is 0.18 ng / mL. 50 All concentrations were between 0.1 and 2 ng / mL, and all exhibited similar effects in promoting neural differentiation. Among them, MTS is a new generation of tetrazolium blue salt compound, which is reduced to colored formazan by various dehydrogenases in the mitochondria of living cells, and the color intensity is highly correlated with the living cells.

[0115] 2.3 The PC-12 cell differentiation assay was used to confirm the role of rhNGF in promoting neural differentiation:

[0116] PC-12 cells are derived from a pheochromocytoma cell line in the adrenal medulla of adult rats. These cells exhibit a reversible neuronal phenotypic response to nerve growth factor (NGF). When PC-12 cells are exposed to NGF, they differentiate into neuron-like cells, promoting synaptic growth. PC-12 cells were induced by incubating them with rhNGF-containing samples (the rhNGF-containing ophthalmic composition solution from Example 1) at concentrations of 0, 1, 5, and 25 ng / mL using cell culture medium. The changes in synaptic length were observed using an inverted microscope (Olympus, model: CKX53), and the results are shown in Figure 2 (magnification 10X). Figure 2 shows that without rhNGF induction, PC-12 cells showed no synaptic growth and remained round. Different concentrations of rhNGF-containing samples resulted in different numbers of synapses, with the number of synapses showing a concentration-dependent relationship with the rhNGF concentration. Similar results were observed in other examples. Therefore, in the ophthalmic composition of the present invention, rhNGF has the effect of promoting neural differentiation.

[0117] 2.4 Determination of rhNFG content and purity using reversed-phase liquid chromatography (RP-HPLC) based on protein polarity separation:

[0118] Solution preparation: Mobile phase A (0.05% TFA in H2O): Accurately measure 1000 mL of ultrapure water, add 0.5 mL of trifluoroacetic acid (TFA), mix well, sonicate for 15 min, and store at 2-8℃. Mobile phase B (0.05% TFA in ACN): Accurately measure 1000 mL of acetonitrile (ACN), add 0.5 mL of TFA, mix well, sonicate for 15 min, and store at 2-8℃. 10% isopropanol aqueous solution: Measure 100 mL of isopropanol, pour it into a 1000 mL graduated cylinder, add purified water to 1000 mL, pour into a reagent bottle, mix well, sonicate for 15 min, and store at room temperature.

[0119] Sample preparation:

[0120] Quality control solution: Take an appropriate amount of reference standard and dilute it with mobile phase A to 100 μg / mL, then transfer it to the inner liner tube for later use.

[0121] Reference standard solutions (STD1-STD5): Dilute the reference standard with mobile phase A to 100 μg / mL, vortex to mix, centrifuge for 30 s, and collect the supernatant into the inner liner tube for later use. Based on the measured results, establish a standard curve with the injection volume (unit: μg) as the x-axis and the peak area of ​​the main peak as the y-axis. The injection volumes of the standards are shown in Table 1.

[0122] Table 1

[0123]

[0124] Test solution: Take an appropriate amount of test sample (i.e., the ophthalmic composition sample prepared in the example) and dilute it with mobile phase A to an NGF concentration of 100 μg / mL.

[0125] Blank control solution: Use mobile phase A as a blank control and place it in the inner liner tube for later use.

[0126] Vortex the treated solutions together, centrifuge for 30 seconds, and fill the supernatant into the sample vial.

[0127] Chromatographic conditions: Column: YMC-Triart Bio C4, 4.6×250mm column; Detection wavelength: 280nm; Run time: 40min; Column temperature: 40℃; Sample chamber temperature: 6℃; Injection volume: 20μL; Flow rate: 1.0mL / min; Needle wash solution: 10% isopropanol aqueous solution; Pump lubricating solution: 10% isopropanol aqueous solution; Pump head seal gasket cleaning solution: 10% isopropanol aqueous solution; Mobile phase A: 0.05% TFA in H2O; Mobile phase B: 0.05% TFA in ACN; Gradient elution program is shown in Table 2.

[0128] Table 2

[0129]

[0130] Sample injection sequence: The sample detection sequence is shown in Table 3.

[0131] Table 3

[0132]

[0133] Before injection, flush the column with mobile phase B at a flow rate of 1.0 mL / min for at least 30 min; then equilibrate the system and column with initial chromatographic conditions until the baseline is stable. Finally, inject and detect according to the above sequence.

[0134] III. Data Analysis:

[0135] The area normalization method was used to calculate the peak area and purity of the main peak of the test sample and the quality control sample, and the content (concentration) and purity of NGF were obtained.

[0136] The following embodiments include methods for preparing the rhNGF stock solution and the stock solutions of various ophthalmic excipients required in the ophthalmic compositions:

[0137] rhNGF stock solution (1 mg / mL): rhNGF lyophilized powder was directly diluted with distilled water to 1 mg / mL.

[0138] Trehalose (47wt%): Weigh 11.75g ​​of trehalose and dissolve in 25mL of water in a 25mL sterile centrifuge tube; Mannitol (12.2wt%): Weigh 3.05g of mannitol and dissolve in 25mL of water in a sterile centrifuge tube; Ectoin (20wt%): Weigh 1.0g of ectoin and dissolve in 5mL of water in a sterile centrifuge tube; Hyaluronic acid (5wt%): Weigh 1.25g of hyaluronic acid and dissolve in 25mL of water in a sterile centrifuge tube.

[0139] Disodium hydrogen phosphate (5.8wt%): Weigh 1.45g of Na2HPO4 and dissolve in 25mL of water in a sterile centrifuge tube; Sodium dihydrogen phosphate (1.84wt%): Weigh 0.46g of NaH2PO4 and dissolve in 25mL of water in a sterile centrifuge tube; Sodium citrate (16wt%): Weigh 4.0g of sodium citrate and dissolve in 25mL of water in a sterile centrifuge tube; Citric acid (4wt%): Weigh 1.0g of citric acid and dissolve in 25mL of water in a sterile centrifuge tube; Histidine (3.0wt%): Weigh 0.75g of histidine and dissolve in 25mL of water in a sterile centrifuge tube.

[0140] Polysorbate 80 (10wt%): Weigh 2.5g of polysorbate 80 and dissolve it in 25mL of water in a sterile centrifuge tube; Poloxamer 407 (0.5wt%): Weigh 0.125g of poloxamer 407 and dissolve it in 25mL of water in a sterile centrifuge tube.

[0141] Hydroxypropyl methylcellulose (1wt%): Weigh 0.5g of hydroxypropyl methylcellulose and slowly add it to 50mL of water, stirring until dissolved; PEG6000 (2wt%): Weigh 0.5g of PEG6000 and dissolve it in 25mL of water in a sterile centrifuge tube.

[0142] L-Methionine (0.2wt%): Weigh 0.05g of methionine into a sterile centrifuge tube and dissolve in 25mL of water.

[0143] Other ophthalmic excipient stock solutions of the present invention can be prepared according to the above-described method for preparing ophthalmic excipient stock solutions. The present invention does not have any particular limitations on this, as long as the purpose of the present invention can be achieved.

[0144] Example 1

[0145] According to the composition shown in Table 4, the prepared stock solutions of each ophthalmic excipient were added to sterile centrifuge tubes, and diluted with purified water. 10 mL of the diluted solution was taken and the required amount of rhNGF stock solution (1 mg / mL) was added. The mixture was mixed evenly, filtered, and the ophthalmic composition was obtained. The composition was placed in PP-EP tubes. The contents of rhNGF and ophthalmic excipients in the ophthalmic composition are shown in Table 4. The remainder was water. The pH and osmotic pressure of the ophthalmic composition were measured.

[0146] Examples 2-16

[0147] Except for adjusting the corresponding preparation parameters as shown in Table 4, everything else is the same as in Example 1.

[0148] Example 17

[0149] According to the composition shown in Table 4, the prepared stock solutions of each ophthalmic excipient were added to sterile centrifuge tubes, and diluted with purified water. 10 mL of the diluted solution was taken and the required amount of rhNGF stock solution (1 mg / mL) was added. The mixture was mixed evenly, filtered, and the ophthalmic composition was obtained. The composition was placed in a borosilicate glass vial. The contents of rhNGF and ophthalmic excipients in the ophthalmic composition are shown in Table 4. The remainder was water. The pH and osmotic pressure of the ophthalmic composition were measured.

[0150] Examples 18-24

[0151] Except for adjusting the corresponding preparation parameters as shown in Table 4, the rest is the same as in Example 17.

[0152] Example 25

[0153] According to the composition shown in Table 5, the prepared stock solutions of each ophthalmic excipient were added to sterile centrifuge tubes, and diluted with purified water. 10 mL of the diluted solution was taken and the required amount of rhNGF stock solution (1 mg / mL) was added. The mixture was mixed evenly, filtered, and the ophthalmic composition was obtained. The composition was placed in PP-EP tubes. The contents of rhNGF and ophthalmic excipients in the ophthalmic composition are shown in Table 5. The remainder is water.

[0154] Examples 26-45

[0155] Except for adjusting the corresponding preparation parameters as shown in Table 5, the rest is the same as in Example 25.

[0156] Example 46

[0157] According to the composition shown in Table 5, the prepared stock solutions of each ophthalmic excipient were added to sterile centrifuge tubes, and diluted with purified water. 10 mL of the diluted solution was taken and the required amount of rhNGF stock solution (1 mg / mL) was added. The mixture was mixed evenly, filtered, and the ophthalmic composition was obtained. The composition was placed in LDPE-BFS tubes. The contents of rhNGF and ophthalmic excipients in the ophthalmic composition are shown in Table 5, and the remainder is water.

[0158] Examples 47-48

[0159] Except for adjusting the corresponding preparation parameters as shown in Table 5, the rest is the same as in Example 46.

[0160] Example 49

[0161] According to the composition shown in Table 8, the prepared stock solutions of each ophthalmic excipient were added to sterile centrifuge tubes, and diluted with purified water. 10 mL of the diluted solution was taken and the required amount of rhNGF stock solution (1 mg / mL) was added. The mixture was mixed evenly, filtered, and the ophthalmic composition was obtained. The composition was placed in PP-EP tubes. The contents of rhNGF and ophthalmic excipients in the ophthalmic composition are shown in Table 8. The remainder is water.

[0162] Examples 50-58

[0163] Except for adjusting the corresponding preparation parameters as shown in Table 8, the rest is the same as in Example 50.

[0164] The pH, osmotic pressure, and stability of the ophthalmic compositions of Examples 1-17 and Examples 25-48 stored at different temperatures and for different numbers of days are shown in Table 6. The stability of the ophthalmic compositions of Examples 18-24 stored at 25°C and 40°C for 5 days is shown in Table 7. The stability of the ophthalmic compositions of Examples 49-58 stored at -20°C and 40°C for 3 days is shown in Table 8.

[0165] Table 4

[0166]

[0167] Note: In Table 4, " / " indicates that there are no corresponding preparation parameters.

[0168] Table 5

[0169]

[0170] Note: In Table 5, " / " indicates that there are no corresponding preparation parameters.

[0171] Table 6

[0172]

[0173] Note: "-" in Table 6 indicates that there are no corresponding test results.

[0174] Table 7

[0175]

[0176] Note: "-" in Table 7 indicates that there are no corresponding test results.

[0177] Table 8

[0178]

[0179] Note: In Table 8, " / " indicates that there are no corresponding preparation parameters.

[0180] The ophthalmic composition samples from Examples 1-2 were placed in environments of 4°C, 25°C, and 40°C, respectively, and ELISA was performed for 0, 5, 10, 30, and 67 days. For stability under different temperature conditions, the rhNGF content (concentration) at day 0 was taken as 100%. The results of the rhNGF content in each sample are shown in Figure 3. Taking the ophthalmic composition of Example 1 at 4°C on day 5 as an example, the rhNGF content in the ophthalmic composition on day 5 = the rhNGF concentration on day 5 / the rhNGF concentration on day 0 × 100%. The results of the rhNGF content in other ophthalmic compositions of the present invention at different temperatures and for different days are calculated similarly. According to the results in Figure 3, rhNGF exhibits better stability in the ophthalmic composition using a citrate buffer system, such as in Example 2, compared to the ophthalmic composition using a phosphate buffer system, such as in Example 1. As shown in Figure 3, the ophthalmic composition of Example 1 of the present invention can be stably stored for at least 30 days at 4°C and at least 10 days at 25°C; the ophthalmic composition of Example 2 of the present invention can be stably stored for at least 30 days at 4°C and at least 30 days at 25°C.

[0181] The ophthalmic composition samples of Examples 3-5 were placed in environments of -20℃, 4℃, and 25℃, respectively, and RP-HPLC was performed at 0 and 20 days. For the stability under different temperature environments, the rhNGF content at 0 days was taken as 100%, and the results of the rhNGF content in each sample at 20 days are shown in Figure 4. It can be seen that the ophthalmic compositions of Examples 3-5 of the present invention can be stably stored for at least 20 days in environments of -20℃, 4℃, and 25℃, and the lower the temperature, the better the stability.

[0182] The ophthalmic composition samples of Examples 6-16 were placed in environments of 4°C, 25°C, and 40°C, respectively, and RP-HPLC analysis was performed at 0 and 30 days. For stability under different temperature environments, the NGF content at day 0 was taken as 100%, and the results of rhNGF content in each sample at different temperatures after 30 days are shown in Figure 5. According to Figure 5, it can be seen that after storage at 4°C for 30 days, the rhNGF content in each ophthalmic composition remained above 95%, indicating that the ophthalmic compositions provided by the present invention, which also contain nonionic surfactants and mucosal adhesives, can be stably stored at 4°C for at least 30 days. It can also be seen that at 25°C, the rhNGF content in the ophthalmic compositions of Examples 9 and 14, which also contain protein stabilizers such as L-methionine, is higher than 90%, indicating that the ophthalmic compositions provided by the present invention, which also contain protein stabilizers, can be stably stored at 25°C for at least 30 days.

[0183] The ophthalmic composition samples from Example 17 were placed in environments of 4°C, -20°C, 25°C, and 30°C, respectively, and RP-HPLC analysis was performed at 0, 15, and 30 days. The rhNGF content at day 0 was taken as 100%, and the NGF content in each sample was obtained as shown in Figure 6. As can be seen from Figure 6, when the ophthalmic composition of the present invention is used as a container in PP-EP tubes, it can be stably stored for at least 30 days at -20°C and 4°C. At 25-30°C, the NGF content in the ophthalmic composition of the present invention is higher than 80% after 15 days. This indicates that the ophthalmic composition of the present invention can be stably stored at room temperature (25°C) for at least 15 days, and its stability is even better at low temperatures.

[0184] The ophthalmic composition samples from Examples 18-24 were placed in environments of 25°C and 40°C, respectively, and RP-HPLC analysis was performed on day 5. With the NGF content at day 0 as 100%, the results of the rhNGF content in each sample are shown in Table 7. Table 7 shows that a suitable salt concentration helps improve the stability of rhNGF. The addition of HPMC resulted in excellent stability in Examples 18, 22, and 23 during the 5-day observation at 25°C, with the rhNGF content decreasing by only about 5%. In particular, in Example 22, the rhNGF content decreased by less than 1% after HPMC was combined with a nonionic surfactant. In Example 24, increasing the amount of nonionic surfactant also showed good stability without the use of HPMC.

[0185] The ophthalmic composition samples from Examples 25-28 were placed in environments of 4°C and 25°C, respectively, and RP-HPLC analysis was performed at 0 and 20 days. For stability under different temperature conditions, the rhNGF content at day 0 was taken as 100%. The results of rhNGF content in each sample at different temperatures after 20 days are shown in Figure 7. According to Table 6 and Figure 7, it can be seen that after storage at 4°C and 25°C for 20 days, the rhNGF content in each ophthalmic composition remained above 90%. This indicates that the ophthalmic composition provided by this invention, containing at least two of the following: pH buffer, osmotic pressure regulator, nonionic surfactant, mucosal adhesive, and protein stabilizer, can be stably stored at 4°C and 25°C for at least 20 days.

[0186] The ophthalmic composition samples from Examples 29-34 were placed in environments of 4°C and 25°C, respectively, and RP-HPLC analysis was performed at 0 and 30 days. For stability under different temperature conditions, the rhNGF content at day 0 was taken as 100%. The results of rhNGF content in each sample at different temperatures after 30 days are shown in Figure 8. According to Table 6 and Figure 8, it can be seen that after 30 days of storage at 4°C, the rhNGF content in each ophthalmic composition remained above 90%, and after 30 days of storage at 25°C, the rhNGF content in each ophthalmic composition remained above 80%. This indicates that the ophthalmic composition provided by this invention, containing at least one of a pH buffer, an osmotic pressure regulator, a nonionic surfactant, and a mucosal adhesive, can be stably stored for at least 30 days at both 4°C and 25°C.

[0187] The ophthalmic composition samples from Examples 35-37 were placed in environments of 4°C and 25°C, respectively, and RP-HPLC analysis was performed at 0 and 38 days. For stability under different temperature conditions, the NGF content at day 0 was taken as 100%. The results of rhNGF content in each sample at different temperatures after 38 days are shown in Figure 9. According to Table 6 and Figure 9, it can be seen that after storage at 4°C for 38 days, the rhNGF content in each ophthalmic composition remained above 95%, and after storage at 25°C for 38 days, the rhNGF content in each ophthalmic composition remained above 80%. This indicates that the ophthalmic composition provided by this invention, containing at least one of a pH buffer, an osmotic pressure regulator, a mucosal adhesive, and a protein stabilizer, can be stably stored at 4°C and 25°C for at least 38 days.

[0188] The ophthalmic composition samples from Examples 38-40 were placed in environments of 4°C and 25°C, respectively, and RP-HPLC analysis was performed at 0 and 43 days. For stability under different temperature conditions, the NGF content at day 0 was taken as 100%. The results of rhNGF content in each sample at different temperatures after 43 days are shown in Figure 10. According to Table 6 and Figure 10, it can be seen that after 43 days of storage at 4°C, the rhNGF content in each ophthalmic composition remained above 95%, and after 43 days of storage at 25°C, the rhNGF content in each ophthalmic composition remained above 80%. This indicates that the ophthalmic composition provided by this invention, containing at least one of a pH buffer, an osmotic pressure regulator, a nonionic surfactant, and a protein stabilizer, can be stably stored for at least 43 days at both 4°C and 25°C.

[0189] The ophthalmic composition samples from Examples 41-45 were placed in environments of 4°C and 25°C, respectively, and RP-HPLC analysis was performed at 0 and 30 days. For stability under different temperature conditions, the NGF content at day 0 was taken as 100%. The results of rhNGF content in each sample at different temperatures after 30 days are shown in Figure 11. According to Table 6 and Figure 11, it can be seen that after 30 days of storage at 4°C, the rhNGF content in each ophthalmic composition remained above 95%, and after 30 days of storage at 25°C, the rhNGF content in each ophthalmic composition remained above 90%. This indicates that the ophthalmic composition provided by this invention, containing at least two of the following: pH buffer, osmotic pressure regulator, nonionic surfactant, mucosal adhesive, and protein stabilizer, can be stably stored for at least 30 days at both 4°C and 25°C.

[0190] The LDPE-BFS samples of the ophthalmic compositions from Examples 46-48 were placed in environments of 4°C and 25°C, respectively, and RP-HPLC analysis was performed at 0 and 17 days. For stability under different temperature conditions, the NGF content at day 0 was taken as 100%, and the results of rhNGF content in each sample at different temperatures after 17 days are shown in Figure 12. According to Table 6 and Figure 12, it can be seen that after 17 days of storage at 4°C, the rhNGF content in each ophthalmic composition remained above 90%, and after 17 days of storage at 25°C, the rhNGF content in each ophthalmic composition remained above 80%. This indicates that the ophthalmic composition provided by this invention, containing at least one of a pH buffer, osmotic pressure regulator, nonionic surfactant, mucosal adhesive, and protein stabilizer, can be stably stored for at least 17 days at both 4°C and 25°C when packaged in LDPE containers.

[0191] The ophthalmic compositions of Examples 49-58 were stored at -20°C and 40°C for 3 days respectively, and the concentration of rhNGF was detected using an ELISA kit. The results are shown in Table 8 and Figure 13. As can be seen from Table 8 and Figure 13, the concentration decrease in each example after three days of storage at 40°C was within 15%, indicating relatively stable results.

[0192] Based on the results of Examples 1-58, Tables 5-6, and Figures 1-13, the ophthalmic composition containing excipients of pH buffer and osmotic pressure regulator provided by the present invention can be stored for at least 3-6 weeks at 2-8°C, maintains good stability after 10 days at room temperature, and also exhibits good stability at 40°C. The present invention further includes ophthalmic compositions containing at least one of nonionic surfactants, mucosal adhesives, and protein stabilizers, which exhibit even better stability at 2-8°C and room temperature. Ophthalmic compositions containing at least two of nonionic surfactants, mucosal adhesives, and protein stabilizers show further improved stability. In contrast, commercially available ophthalmic compositions only support storage for one week at 2-8°C and 24 hours at 25°C. Therefore, the ophthalmic composition of the present invention exhibits superior low-temperature and room-temperature stability compared to ophthalmic compositions.

[0193] It should be noted that, in this document, the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, or article that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, or article.

[0194] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention are included within the scope of protection of the present invention.

Claims

1. An ophthalmic composition containing NGF, characterized in that, The ophthalmic composition comprises the active substance NGF, an ophthalmic excipient, and water, wherein the ophthalmic excipient comprises a pH buffer and an osmotic pressure regulator; The content of the active substance NGF in the ophthalmic composition is 0.0001wt%-0.1wt%, preferably 0.001wt%-0.01wt%, and more preferably 0.001wt%-0.005wt%. The pH buffer in the ophthalmic composition is present in an amount of 0.001 wt%-2.5 wt%, preferably 0.01 wt%-1.5 wt%, and more preferably 0.05 wt%-0.5 wt%. The active substance NGF is rhNGF.

2. The ophthalmic composition according to claim 1, characterized in that, The pH of the ophthalmic composition is 4.0-8.0, preferably 5.0-7.0, more preferably 5.5-6.5; the osmotic pressure is 200-400 mOsmol / kg, preferably 240-380 mOsmol / kg, more preferably 280-320 mOsmol / kg.

3. The ophthalmic composition according to claim 1, characterized in that, The pH buffer is selected from at least one of boric acid-borate, citrate-citrate, acetic acid-acetate, tris(hydroxymethyl)aminomethane hydrochloride, histidine, histidine-histidine hydrochloride, and phosphate. Preferably, the pH buffer is citrate-citrate. The borate is selected from at least one of sodium borate, potassium borate, and their hydrates; The citrate is selected from at least one of potassium citrate, sodium citrate, disodium hydrogen citrate, sodium dihydrogen citrate, dipotassium hydrogen citrate, potassium dihydrogen citrate, and their hydrates; The phosphate is selected from at least one of disodium hydrogen phosphate, sodium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and their hydrates.

4. The ophthalmic composition according to claim 1, characterized in that, The osmotic pressure regulator is selected from inorganic osmotic pressure regulators and / or organic osmotic pressure regulators, preferably organic osmotic pressure regulators; The inorganic osmotic pressure regulator is selected from at least one of sodium chloride and potassium chloride; The organic osmotic pressure regulator is selected from at least one of glycerol, mannitol, sorbitol, trehalose, L-carnitine, proline, ectoine, hyaluronic acid, erythritol, sucrose, and glucose, preferably at least one of mannitol, trehalose, ectoine, and hyaluronic acid.

5. The ophthalmic composition according to claim 4, characterized in that, The inorganic osmotic pressure regulator is present in the ophthalmic composition at a content of 0.001 wt%-0.9 wt%, preferably 0.005 wt%-0.2 wt%, and more preferably 0.01 wt%-0.05 wt%. The organic osmotic pressure regulator is present in the ophthalmic composition at a content of 0.01wt%-20wt%, preferably 0.1wt%-10wt%, and more preferably 0.5wt%-8wt%.

6. The ophthalmic composition according to claim 1, characterized in that, The ophthalmic excipient further comprises a nonionic surfactant; the nonionic surfactant is selected from at least one of polysorbate, poloxamine, and poloxamer, preferably polysorbate; Preferably, the nonionic surfactant in the ophthalmic composition is 0.0001wt%-3wt%, more preferably 0.001wt%-1wt%, and even more preferably 0.01wt%-0.5wt%.

7. The ophthalmic composition according to claim 1, characterized in that, The ophthalmic excipient further comprises a protein stabilizer; the protein stabilizer is selected from at least one of L-methionine and sodium thiosulfate. Preferably, the protein stabilizer in the ophthalmic composition is 0.0001wt%-1wt%, more preferably 0.0001wt%-0.15wt%, and even more preferably 0.0005wt%-0.002wt%.

8. The ophthalmic composition according to claim 1, characterized in that, The ophthalmic excipient further comprises a mucosal adhesive; the mucosal adhesive is selected from at least one of polyvinylpyrrolidone, hydroxypropyl methylcellulose, carboxymethyl cellulose, and polyethylene glycol, preferably at least one of hydroxypropyl methylcellulose and polyethylene glycol; Preferably, the content of the mucosal adhesive in the ophthalmic composition is 0.01wt%-0.5wt%, more preferably 0.02wt%-0.2wt%.

9. The ophthalmic composition according to claim 1, characterized in that, The ophthalmic excipient further comprises a chelating agent; the chelating agent is selected from at least one of the following: nitrotriacetic acid, ethylenediaminedisuccinic acid, iminodisuccinic acid, methylglycine diacetic acid, L-glutamic acid N,N-diacetic acid, ethylenediamine-N,N'-diglutamic acid, ethylenediamine-N,N'-dimalonic acid, 3-hydroxy-2,2-iminodisuccinic acid, 2-hydroxyethyliminodiacetic acid, pyridine-2,6-dicarboxylic acid, diethylenetriaminepentaacetic acid, hydroxyethyldiaminetriacetic acid, 1,2-diaminocyclohexanetetraacetic acid, hydroxyethylaminodiacetic acid, polyphosphate, citric acid and citrate, tartaric acid and tartrate, ethylenediaminetetraacetic acid and disodium ethylenediaminetetraacetic acid, and alkali metal salts of hexametaphosphate; The chelating agent is present in the ophthalmic composition at a content of 0.001wt%-1wt%, preferably 0.1wt%-0.25wt%.

10. The ophthalmic composition according to claim 1, characterized in that, The ophthalmic excipient is selected from any combination of the following: pH buffers and osmotic pressure regulators; Or at least two of the following: pH buffer, osmotic pressure regulator, and nonionic surfactant, mucosal adhesive, and protein stabilizer.

11. The ophthalmic composition according to claim 10, characterized in that, The pH buffer is citric acid-citrate; and / or the mucosal adhesive is at least one of hydroxypropyl methylcellulose and polyethylene glycol.

12. Use of the ophthalmic composition according to any one of claims 1-11 in the preparation of a medicament for the prevention and / or treatment of eye diseases.

13. The use according to claim 12, characterized in that, The eye disease is selected from at least one of neurotrophic keratitis, corneal ulcer and injury, dry eye disease, glaucoma, retinitis pigmentosa, and macular degeneration.

Images