Human nerve growth factor pharmaceutical composition and use thereof

Abstract

Disclosed is a human nerve growth factor pharmaceutical composition. In particular, the present invention relates to a human nerve growth factor eye drop with the human nerve growth factor as an active ingredient, wherein a pH buffer comprises or is histidine, and a stabilizer comprises or is albumin.

Description

Human nerve growth factor pharmaceutical compositions and their uses

[0001] Cross-references to related applications

[0002] This application claims priority to Chinese Patent Application No. 202411758261.X, filed on December 2, 2024, the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention relates to pharmaceutical compositions for treating nerve damage diseases, and more specifically, to human nerve growth factor eye drops, their preparation methods, and their uses. Background Technology

[0004] Nerve growth factor (NGF) is a neuronal growth regulator with dual biological functions: nourishing neurons and promoting neurite growth. It promotes the growth, development, differentiation, and maturation of central and peripheral neurons, maintains normal nervous system function, and accelerates the repair of nervous system injuries. NGF is widely distributed in various tissues and organs of the body (including the brain). NGF contains three subunits: α, β, and γ. The β subunit is the active region, composed of two single chains of 118 amino acids linked by non-covalent bonds. In 1953, Italian scientist Levi-Montalcini discovered NGF and was awarded the Nobel Prize.

[0005] Currently, several NGF products are available on the market both domestically and internationally. Clinically, they are mainly used to treat developmental disorders of the nervous system, including amblyopia, neuroma, various nerve injuries, and other neurological diseases. Among them, neurotrophic keratitis (NK), also known as neurotrophic keratopathy (NK), is a rare corneal degenerative disease caused by damage to the trigeminal nerve. The disease is characterized by decreased or absent corneal sensation, dry eye, corneal epithelial defects, and corneal ulcers, eventually leading to corneal stromal melting and perforation. NGF is believed to maintain corneal integrity through three mechanisms: (1) tear secretion: NGF binds to lacrimal gland receptors, promoting sensory nerve-mediated reflexive tear secretion; (2) corneal nerve innervation: NGF plays a role in the recovery of nerve function, promoting the regeneration and survival of sensory nerves; (3) cell proliferation and differentiation: NGF stimulates the proliferation, differentiation, and survival of corneal epithelial cells. In clinical applications, NGF liquid preparations (such as eye drops) can be used for topical application to the patient's eye, allowing NGF to promote the proliferation of corneal epithelial cells and increase the density of subbasal nerve fibers in the cornea, thereby playing a therapeutic role in persistent corneal epithelial defects and corneal ulcers.

[0006] Similar to other protein drugs, NGF is prone to denaturation due to its short half-life. Exposure to extreme temperatures and humidity, or physical and chemical factors, can easily alter its spatial conformation, leading to denaturation and loss of its original biological activity. Furthermore, because proteins readily adhere to solid surfaces, some protein may adhere to the container walls during filling, resulting in the loss of active ingredients. Therefore, to ensure its biological activity, a stable NGF-containing liquid formulation (e.g., eye drops) is needed, which can be safely and effectively used in mammals, especially humans. However, currently available NGF eye drops can only be frozen and must be used within 12 hours of removal from the freezer. They also require specific accessories, making the process relatively complex and inconvenient.

[0007] There is a need to develop a growth factor eye drop that is highly stable, easy to store, convenient to administer, and has a high safety profile. It should be able to be stored under different temperature conditions, and the appearance, concentration, and purity of the formulation should not change significantly during long-term storage. At the same time, it is desirable that the formulation can be packaged using the single-dose packaging material commonly used for eye drops, which is simple and easy to use.

[0008] Invention Overview

[0009] To address the aforementioned issues, this invention has successfully developed a human nerve growth factor eye drop that is highly stable, can be stored at room temperature or under refrigeration, is convenient to administer, and has high safety through formulation optimization. It is administered to patients via eye drops, ensuring accurate dosage and ease of use. By rationally selecting and determining the dosage of excipients, the stability of the formulation is improved; the product shows no significant changes in appearance, concentration, or purity after being stored at 2–8°C for 12 months and at 25°C for 6 months. The eye drops maintain good stability without freezing, and are packaged in simple, single-dose aseptic packaging, avoiding the use of antibacterial agents in the formulation. It also eliminates the need for fixed accessories, simplifying operation and thus improving the convenience of product use.

[0010] The present invention provides a pharmaceutical composition comprising an active ingredient, a pH buffer, an osmotic pressure regulator, and a stabilizer, wherein the active ingredient is human nerve growth factor, the pH buffer comprises or is histidine, and the stabilizer comprises or is albumin.

[0011] In some embodiments, the pharmaceutical composition is a human nerve growth factor eye drop or eye preparation.

[0012] In some embodiments, the pharmaceutical composition does not contain an antibacterial agent.

[0013] In some preferred embodiments, the albumin is human serum albumin.

[0014] In some embodiments, the osmotic pressure regulator is selected from one or more of sodium chloride, boric acid, borax, and glucose, preferably sodium chloride.

[0015] In some embodiments, the human nerve growth factor comprises any amino acid sequence selected from those shown in SEQ ID NO: 1-3.

[0016] The exemplary amino acid sequence of human nerve growth factor is as follows:

[0017] Among them, SEQ ID NO:3 is wild-type human nerve growth factor with a length of 120aa; SEQ ID NO:1 and SEQ ID NO:2 are recombinant human nerve growth factor with sequences of 117aa and 118aa, respectively.

[0018] The present invention also provides a component-optimized pharmaceutical composition comprising an active ingredient, a pH buffer, an osmotic pressure regulator, and a stabilizer, wherein the active ingredient is human nerve growth factor, the pH buffer is histidine, and the stabilizer is albumin. In some embodiments, the osmotic pressure regulator is sodium chloride. In some preferred embodiments, the stabilizer is human serum albumin.

[0019] In some embodiments, the concentration of the human nerve growth factor is 0.0001%-0.2%, for example 0.0002%-0.1%, preferably 0.0005%-0.05%, and most preferably 0.001%-0.03%.

[0020] In some embodiments, the concentration of histidine is 0.1%-1%, preferably 0.17%-0.5%, more preferably 0.24%-0.38%, and most preferably 0.31%.

[0021] In some embodiments, the concentration of the human serum albumin is 0.005%-1%, preferably 0.01%-0.06%, more preferably 0.01-0.04%, and most preferably 0.02%.

[0022] In some embodiments, the pH of the pharmaceutical composition is 4.5-7.5, preferably 5.0-7.0, more preferably 5.5-6.5 or 5.5-6.0.

[0023] In some embodiments, the concentration of sodium chloride is 0.1%-5%, preferably 0.25%-1.75%, more preferably 0.55%-1.45%, and most preferably 0.85%.

[0024] In some embodiments, the pharmaceutical composition comprises: human nerve growth factor at a concentration of 0.0005%-0.05%, preferably 0.001%-0.03%; histidine at a concentration of 0.24%-0.38%, preferably 0.31%; human serum albumin at a concentration of 0.01-0.04%, preferably 0.02%; and a pH of 5.5-6.0 for the pharmaceutical composition. Preferably, the pharmaceutical composition further comprises sodium chloride at a concentration of 0.55%-1.45%, preferably 0.85%.

[0025] The pharmaceutical compositions of the present invention are tested for their appearance, related substances, and SEC properties using methods commonly used in the art. In some embodiments, the pharmaceutical compositions include:

[0026] a) When placed at 45°C for 5 days, the change in the content of its active ingredient does not exceed 6%; the change in the oxidation peak of related substances does not exceed 4%, or the change in the denaturation peak of related substances does not exceed 3%; and / or

[0027] b) When placed at 25°C for 10 days, the change in the content of its active ingredient does not exceed 6%; the change in the oxidation peak of related substances does not exceed 4%, or the change in the denaturation peak of related substances does not exceed 3%; and / or

[0028] c) When stored at 25°C for 6 months, the content of its active ingredient changes by no more than 9%; the oxidation peak of related substances changes by no more than 7%, or the denaturation peak of related substances changes by no more than 7%; and / or

[0029] d) When stored at 2-8℃ for 12 months, the content of its active ingredients changes by no more than 5%; the oxidation peak of related substances changes by no more than 4%; or the denaturation peak of related substances changes by no more than 4%.

[0030] Preferably, a) when placed at 45°C for 5 days, the change in the content of its active ingredient does not exceed 5.6%; the change in the oxidation peak of related substances does not exceed 4.7%, or the change in the denaturation peak of related substances does not exceed 2.6%; and / or

[0031] b) When placed at 25°C for 10 days, the change in the content of its active ingredient does not exceed 4.9%; the change in the oxidation peak of related substances does not exceed 3.2%, or the change in the denaturation peak of related substances does not exceed 2.5%; and / or

[0032] c) When stored at 25°C for 6 months, the change in the content of its active ingredient does not exceed 8.8%; the change in the oxidation peak of related substances does not exceed 6.7%, or the change in the denaturation peak of related substances does not exceed 6.8%; and / or

[0033] d) When stored at 2-8℃ for 12 months, the content of its active ingredients changes by no more than 4.1%; the oxidation peak of related substances changes by no more than 3.1%; or the denaturation peak of related substances changes by no more than 3.5%.

[0034] A method for preparing the pharmaceutical composition is also provided, comprising the following steps:

[0035] 1) Dissolve or disperse the pH buffer and osmotic pressure regulator with an appropriate amount of water for injection, and adjust to the target pH; and

[0036] 2) Add stabilizer and human nerve growth factor stock solution, and stir well.

[0037] The use of the pharmaceutical composition described herein in the preparation of a medicament for treating diseases caused by nerve damage is also provided. Preferably, the diseases caused by nerve damage are selected from, for example, neurotoxicity, peripheral nerve injury, diabetic peripheral neuropathy, Alzheimer's disease, Parkinson's disease, facial neuritis, neuroma, amblyopia, corneal epithelial defects, corneal ulcers, neurotrophic keratitis, dry eye syndrome, optic nerve injury, glaucoma, and retinal degeneration. Summary of the Invention

[0038] Unless otherwise specified, the terms used herein have the common meaning as understood by one of ordinary skill in the art to which they pertain.

[0039] In addition, the concentration ranges of each component (e.g., active ingredient, pH buffer, osmotic pressure regulator and stabilizer) in the pharmaceutical compositions disclosed herein, all intermediate values ​​in the numerical ranges, i.e. all values ​​between the endpoint values, and the numerical ranges defined by any two listed endpoint values ​​as endpoints, or any subranges within such numerical ranges, have also been disclosed and are claimed.

[0040] I. Active ingredients and their dosage in pharmaceutical compositions

[0041] The term "active ingredient" refers to a substance, compound, or molecule that has biological or other activity and induces a biological or physiological effect on a subject to which it is administered. In other words, "active ingredient" refers to one or more components of a composition attributable to all or part of the effects of the composition.

[0042] In particular, the active ingredient in the pharmaceutical composition of the present invention is nerve growth factor (NGF), specifically, the nerve growth factor is human nerve growth factor expressed in a eukaryotic system.

[0043] A. Nerve growth factor / NGF

[0044] As used herein, the term “nerve growth factor” or “NGF” refers to a group of neurotrophic factors and neuropeptides that are primarily involved in the regulation of the growth, maintenance, proliferation, and survival of certain target neurons, particularly those that transmit pain, temperature, and touch (sensory neurons). Unless otherwise stated, the term “NGF” as described herein includes wild-type NGF from any species described herein and any NGF with mutations, substitutions, and / or modifications to the β chain.

[0045] The human nerve growth factor described herein comprises a bioactive fragment of a full-length NGF polypeptide (NGF precursor polypeptide or mature NGF polypeptide). In some embodiments, the human nerve growth factor comprises the amino acid sequence shown in SEQ ID NO: 1. The term "bioactive fragment" refers to a portion of a wild-type NGF polypeptide of any species, including any possible substitutions, mutations, deletions, insertions, fusions, and / or other modifications, while maintaining at least one biological function.

[0046] This article provides a human nerve growth factor, which includes, but is not limited to, the following amino acid sequence:

[0047] Among them, SEQ ID NO:3 is wild-type human nerve growth factor with a length of 120aa; SEQ ID NO:1 and SEQ ID NO:2 are recombinant human nerve growth factor with sequences of 117aa and 118aa, respectively.

[0048] In the preparation of pharmaceutical compositions containing human nerve growth factor and their eye drops, the concentration range of human nerve growth factor can be 0.0001%-0.2% w / v, or 0.0002%-0.1% w / v, or 0.0005%-0.05% w / v, or 0.001%-0.03% w / v; specifically, the concentration of human nerve growth factor can be 0.0001% w / v, 0.0005% w / v, 0.001% w / v, 0.005% w / v, 0.008% w / v, 0 .01% w / v, 0.02% w / v, 0.03% w / v, 0.04% w / v, 0.05% w / v, 0.06% w / v, 0.07% w / v, 0.08% w / v, 0.09% w / v, 0.10% w / v, 0 .12% w / v, 0.14% w / v, 0.16% w / v, 0.18% w / v, 0.20% w / v, 0.22% w / v, 0.24% w / v, 0.26% w / v, 0.28% w / v, 0.030% w / v. Preferred are 0.001% w / v, 0.005% w / v, 0.008% w / v, 0.010% w / v, 0.020% w / v, and 0.030% w / v. In some implementations, the concentration range of human nerve growth factor also includes a numerical range defined by any two endpoint values ​​listed above, or any subrange within that numerical range.

[0049] II. Auxiliary materials and their dosage

[0050] As used herein, the term "excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient, and includes, but is not limited to: pH buffers, osmolarity regulators, stabilizers, surfactants, adjuvants, and ionic strength enhancers. For example, pH buffers include, but are not limited to, one or more of histidine, disodium hydrogen phosphate, sodium acetate, and sodium citrate, and may also be selected from other buffers well known in the art; stabilizers include, but are not limited to, one or more of polysorbate 20, polysorbate 80, and albumin, and may also be selected from other stabilizers well known in the art, wherein albumin is preferably human serum albumin; osmolarity regulators include, but are not limited to, sodium chloride, and may also be selected from other osmolarity regulators well known in the art; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants, such as Tween-80.

[0051] A. pH buffer

[0052] The term "pH buffer" refers to a compound or composition that can be used to maintain the pH of a composition. In some embodiments, the pH buffer includes histidine.

[0053] In some embodiments, the pH buffer in the pharmaceutical compositions disclosed herein is histidine. In some embodiments, the pharmaceutical compositions disclosed herein include histidine. In some embodiments, the concentration of histidine is 0.1%-1% w / v. In some embodiments, the concentration of histidine is 0.17%-0.5% w / v. In some embodiments, the concentration of histidine is 0.24%-0.38% w / v. In some embodiments, the concentration of histidine is 0.1% w / v, 0.17% w / v, 0.24% w / v, 0.31% w / v, 0.38% w / v, 0.50% w / v, 0.57% w / v, 0.64% w / v, 0.71% w / v, 0.78% w / v, 0.85% w / v, 0.92% w / v, 0.99% w / v, or 1% w / v. In some preferred embodiments, the concentration of histidine is 0.17% w / v, 0.24% w / v, 0.31% w / v, 0.38% w / v, or 0.50% w / v. In some most preferred embodiments, the concentration of histidine is 0.31% w / v. In some embodiments, the concentration range of histidine also includes a numerical range defined by any two of the endpoints listed above, or any subrange within that numerical range.

[0054] B. Stabilizers

[0055] The term "stabilizer" refers to a substance that prevents active ingredients from agglomerating or depolymerizing in aqueous solutions. Besides its stabilizing effect, stabilizers often act as support agents to improve product moldability, and may also have other functions. The addition of stabilizers can prevent or reduce protein aggregation and depolymerization caused during preparation or storage. Stabilizers include albumin, with human serum albumin being preferred.

[0056] In some embodiments, the pharmaceutical compositions disclosed herein include human serum albumin. In some embodiments, the concentration of human serum albumin is 0.005%-1% w / v. In some embodiments, the concentration of human serum albumin is 0.01%-0.06% w / v. In some embodiments, the concentration of human serum albumin is 0.01-0.04% w / v. In some implementations, the concentration of human serum albumin is 0.005% w / v, 0.01% w / v, 0.015% w / v, 0.02% w / v, 0.025% w / v, 0.03% w / v, 0.035% w / v, 0.04% w / v, 0.045% w / v, 0.05% w / v, 0.055% w / v, 0.06% w / v, 0.065% w / v, 0.07% w / v, 0.075% w / v, 0.08% w / v, 0.085% w / v, 0.09% w / v, 0.095% w / v, or 0.1% w / v. In some preferred embodiments, the concentration of human serum albumin is 0.01% w / v, 0.02% w / v, 0.04% w / v, or 0.06% w / v. In some optimal embodiments, the concentration of human serum albumin is 0.02% w / v. In some embodiments, the concentration range of human serum albumin also includes a numerical range defined by any two of the endpoints listed above, or any subrange within that numerical range.

[0057] C. Osmotic pressure regulator

[0058] The term "osmolarity regulator" primarily functions to regulate the osmotic pressure of the ophthalmic solution. It is well known in the art that a solution with the same osmotic pressure as plasma or tears is called an isotonic solution. The osmotic pressure of ophthalmic solutions should ideally be isotonic with that of tears, which have an osmotic pressure of 296-308 mOsm / L. Hypertonic solutions can cause the cornea to lose moisture, leading to dryness and discomfort; hypotonic solutions can cause corneal cell swelling and irritation. Furthermore, excessively high or low osmotic pressure can stimulate the eye, increasing tear production and causing rapid dilution or rinsing away of the medication. Therefore, it is generally recommended that ophthalmic solutions have their osmotic pressure regulated. Osmolarity regulators include, but are not limited to, one or more of sodium chloride, boric acid, borax, and glucose, and may also be selected from other osmolarity regulators well known in the art.

[0059] In some embodiments, the pharmaceutical compositions disclosed herein use sodium chloride as an osmotic pressure regulator, and the pharmaceutical compositions comprise sodium chloride. In some embodiments, the concentration of sodium chloride is 0.1%-5% w / v. In some embodiments, the concentration of sodium chloride is 0.25%-1.75% w / v. In some embodiments, the concentration of sodium chloride is 0.55%-1.45% w / v. In some embodiments, the concentration of sodium chloride is 0.1% w / v, 0.15% w / v, 0.25% w / v, 0.35% w / v, 0.45% w / v, 0.55% w / v, 0.65% w / v, 0.75% w / v, 0.85% w / v, 0.95% w / v, 1.05% w / v, 1.15% w / v, 1.25% w / v, 1.35% w / v, 1.45% w / v, 1.55% w / v, 1.65% w / v, or 1.75% w / v. In some preferred embodiments, the concentration of sodium chloride is 0.25% w / v, 0.55% w / v, 0.85% w / v, 1.15% w / v, 1.45% w / v, or 1.75% w / v; in some optimal embodiments, the concentration of sodium chloride is 0.85% w / v. In some embodiments, the concentration range of sodium chloride also includes a numerical range defined by any two of the endpoints listed above, or any subrange within that numerical range.

[0060] II. Formulation and Administration

[0061] A. Eye drops or eye ointments

[0062] As used herein, the terms "eye drops" or "eye solutions" are interchangeable. Eye drops or eye solutions refer to sterile liquid preparations made from active pharmaceutical ingredients and suitable excipients for instillation into the eye. They can be classified as solutions, suspensions, or emulsions, and are commonly used for sterilization, anti-inflammation, astringency, miosis, anesthesia, or diagnosis. Some can also be used as lubricants or as a substitute for tears. Although eye drops are for external use, their quality requirements are similar to those of injectable drugs, with specific requirements for pH, osmotic pressure, sterility, and visible foreign matter.

[0063] pH has a significant impact on eye drops. Irritation caused by improper pH settings can increase tear secretion, leading to rapid drug loss and even corneal damage. The pH adjustment of eye drops should consider the requirements of drug solubility, stability, and irritation, while also taking into account the effect of pH on drug absorption and efficacy. In some embodiments, the pH of the composition is maintained below 7.5. In some embodiments, the pH of the composition is maintained above 4.5. In some embodiments, the pH of the composition is maintained in the range of about 4.5 to 7.5. In some embodiments, the pH of the composition is maintained in the range of 5.0 to 7.0. In some embodiments, the pH of the composition is maintained in the range of about 5.5 to about 6.6. In some embodiments, the pH of the composition is maintained in the range of about 5.5 to about 6.0. In some embodiments, the pH of the composition is maintained at 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, or 7.5. In some preferred embodiments, the pH of the composition is maintained at 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, or 7.5. In some embodiments, the pH range also includes a numerical range defined by any two of the endpoints listed above, or any subrange within that numerical range.

[0064] Unless otherwise specified, eye drops should be isotonic with tears. General eye drops (i.e., eye drops used without ocular trauma) must be free of pathogenic bacteria (Pseudomonas aeruginosa and Staphylococcus aureus must not be detected). Eye drops should be tested according to the visible foreign matter examination method in Part IV, Chapter 0903 of the Chinese Pharmacopoeia (2020 edition), and should comply with the regulations. Suspension eye drops should also be tested according to the method under "Particle Size" in Part IV, Chapter 0105 of the Chinese Pharmacopoeia (2020 edition), and should comply with the regulations.

[0065] The pharmaceutical compositions disclosed herein include ophthalmic drop formulations containing a concentration of human nerve growth factor albumin as the active ingredient and other pharmaceutically acceptable excipients. These pharmaceutically acceptable excipients include, but are not limited to, pH buffers, stabilizers, and osmotic pressure regulators. In some embodiments, the pharmaceutical compositions disclosed herein comprise human nerve growth factor, histidine, albumin, and sodium chloride; wherein human nerve growth factor is the active ingredient, histidine is the pH buffer, and albumin is the stabilizer; in some preferred embodiments, albumin is human serum albumin. In some embodiments, the pharmaceutical composition comprises human nerve growth factor at a concentration of 0.0005%-0.05% w / v, preferably 0.001%-0.03% w / v; histidine at a concentration of 0.24%-0.38% w / v, preferably 0.31% w / v; human serum albumin at a concentration of 0.01-0.04% w / v, preferably 0.02% w / v; and sodium chloride at a concentration of 0.55%-1.45% w / v, preferably 0.85% w / v, wherein the pH of the pharmaceutical composition is 5.5-6.0.

[0066] B. Preparation of eye drops

[0067] The general manufacturing process for ophthalmic liquid preparations includes: raw materials and excipients → preparation and filtration → filtrate → sterilization / aseptic dispensing → quality inspection → printing and packaging. This process is suitable for drugs with stable properties. For heat-sensitive active pharmaceutical ingredients, aseptic processing is required. Preparations intended for ophthalmic surgery or trauma can be packaged in single-dose vials using glass tubular / molded injection bottles, manufactured according to glass tubular / molded injection bottle production processes to ensure complete sterility. Eye wash solutions are packaged in infusion bottles and processed according to infusion procedures.

[0068] Eye drops require sterility. Small-scale preparation can be carried out in a sterile operating cabinet, while large-scale production should be carried out according to the standards and requirements for injection manufacturing processes. The drug and excipients are dissolved in an appropriate amount of solvent. If necessary, activated charcoal (0.05%–0.3%) is added for treatment. The solution is then filtered through a filter rod, sintered glass filter ball, or microporous membrane until clear. Solvent is added to the required volume, and after sterilization, a semi-finished product inspection is performed. When preparing ophthalmic suspensions, the micronized drug is first sterilized. A surfactant and suspending agent are separately prepared with a small amount of sterile distilled water to form a viscous solution. This solution is then mixed with the active pharmaceutical ingredient using a homogenizer. Sterile distilled water is added to the final volume to complete the preparation.

[0069] The quality evaluation of eye drops includes items such as active pharmaceutical ingredient content, visible foreign matter, fill volume, osmolality, and sterility. For specific methods, please refer to Part IV, 0105 of the Chinese Pharmacopoeia (2020 edition).

[0070] III. Uses of the formulation

[0071] In some embodiments, the therapeutic composition comprising the human nerve growth factor described herein is used to treat a disease or condition in a subject. Such a disease or condition may include, for example, any condition related to a deficiency in the amount, level, and / or activity of endogenous NGF genes, mRNA, and / or proteins, such as neurological disorders. In some embodiments, the therapeutic composition described herein also comprises another agent capable of treating the disease or condition described herein.

[0072] In some implementations, the subjects described herein suffer from neurological disorders. The human NGF described herein can be used to promote the development, maintenance, or regeneration of neurons (including central neurons) in vitro and in vivo. In this invention, the human nerve growth factor described herein can be administered to patients whose nervous systems have been damaged by trauma, surgery, stroke, local ischemia, infection, metabolic diseases, nutritional deficiencies, malignancies, or toxic agents to promote survival or growth.

[0073] Therefore, the human nerve growth factor (rhNGF) described herein can be used in methods for treating various neurological disorders and conditions. In a preferred embodiment, the formulation of the present invention is administered to a patient to treat one or more neurological disorders or conditions. "Neurological disorders" as used herein refers to diseases of the central and / or peripheral nervous systems caused by nerve injury. Specific examples of neurological disorders include, but are not limited to, neurotoxicity, neuroma, facial neuritis, peripheral nerve injury (diabetic peripheral neuropathy), and neurodegenerative diseases (Alzheimer's disease, Parkinson's disease). Furthermore, diseases caused by nerve injury may also include ophthalmic diseases, including but not limited to: amblyopia, corneal epithelial defects, corneal ulcers, neurotrophic keratitis, dry eye syndrome, optic nerve injury, glaucoma, and retinal degeneration.

[0074] The human nerve growth factor described in this article can be used to treat neurotoxicity, neuroma, and facial neuritis. Neurotoxicity refers to the symptoms resulting from a series of central nervous system dysfunctions caused by external toxins invading the body and affecting the central nervous system. Neuroma is a benign tumor that usually occurs in nerve tissue. It can be neurofibroma, schwannoma, neurofibromatosis, etc. Facial neuritis, also known as facial nerve palsy or facial paralysis, is a neurological deficit symptom caused by damage to the facial nerve, leading to facial muscle paralysis. Depending on the location of the facial nerve injury, facial nerve palsy can be divided into central facial nerve palsy and peripheral facial nerve palsy. Common causes include stroke, tumors, intracranial infection, Hunter syndrome, Lyme disease, encephalitis, otitis media, etc., with about 70% of patients having idiopathic facial nerve palsy. The onset is related to cold weather and seasonal changes.

[0075] The human nerve growth factor described in this article can also be used to treat peripheral neuropathy. "Peripheral neuropathy" refers to diseases affecting the peripheral nervous system, most commonly manifesting as one or a combination of motor, sensory, sensorimotor, or autonomic dysfunction. The diverse forms of peripheral neuropathy can each be uniquely attributed to a wide range of causes. For example, peripheral neuropathy can be hereditary, caused by systemic diseases, or induced by toxic agents. Examples include, but are not limited to, diabetic peripheral neuropathy, distal sensorimotor neuropathy, or autonomic neuropathy such as decreased gastrointestinal motility or bladder weakness.

[0076] The human nerve growth factor described in this article can be used to treat human neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, epilepsy, multiple sclerosis, Huntington's disease, Down syndrome, sensorineural hearing loss, and Meniere's disease. The human nerve growth factor described in this article can also be used as a cognitive enhancer, for example, to enhance learning, including in patients with dementia or trauma.

[0077] The human nerve growth factor described in this article can also be used to treat ophthalmic diseases caused by nerve damage, including but not limited to amblyopia, corneal epithelial defects, corneal ulcers, neurotrophic keratitis, dry eye, optic nerve injury, glaucoma, and retinal degeneration. NGF is naturally present in the eye, produced by retinal neurons and glial cells or by paracrine secretion, and circulates back to the retina from the superior colliculus. It is crucial for the survival and growth of sympathetic and sensory neurons, as well as the differentiation of neurons in the central nervous system (CNS). NGF receptors (including the high-affinity TrkA receptor and the low-affinity receptor p75NTR) are present in NGF-responsive neurons and glial cells, respectively. The downstream PI3K / Akt pathway activated by NGF / TrkA is an important intracellular signaling pathway involved in a series of cellular responses in retinal microvascular endothelial cells and pericytes, such as cell proliferation, survival, apoptosis, cytoskeleton construction, vesicle transport, glucose transport, and platelet function. NGF is not only an important regulator of the survival and death of neurons and glial cells in the eye, but also mediates the development, survival, and regeneration of microvascular cells. Supplementation with exogenous NGF can promote corneal epithelial cell proliferation and increase the density of subbasal corneal nerve fibers, thereby playing a therapeutic role in persistent corneal epithelial defects, corneal ulcers, and neurotrophic keratitis. NGF can promote the survival of retinal ganglion cells, prevent permanent nerve cell loss, and maintain healthy neuronal function, thus playing a therapeutic role in glaucoma. Ocular application of rhNGF can restore lost RGCs and stimulate cell growth and / or axonal regeneration, demonstrating a therapeutic effect on optic nerve injury. Detailed Implementation

[0078] The following describes embodiments of the present invention, but the present invention is not limited thereto. The present invention is not limited to the various configurations described below, and various modifications can be made within the scope of the claims. Embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples are also included in the technical scope of the present invention.

[0079] Unless otherwise stated herein, the ranges of values ​​listed herein are intended only as a convenient way to refer to each individual value falling within the range, and each individual value is incorporated into the specification as if it were listed separately herein.

[0080] The following embodiments are used to further describe this disclosure, but these embodiments are not intended to limit the scope of this disclosure.

[0081] To prepare eye drops with high stability, easy storage, convenient administration, and high safety, this invention selects an amino acid sequence containing recombinant human nerve growth factor as shown in SEQ ID NO: 1, based on the structural characteristics of nerve growth factor. Furthermore, extensive exploratory experiments were conducted by screening and optimizing excipients and their dosages, including pH buffers, stabilizers, and osmotic pressure regulators. Finally, various human nerve growth factor drug compositions and their eye drops were prepared using different concentrations of histidine, human serum albumin, and sodium chloride. The appearance, active ingredient content, related substances, and SEC of various eye drops were measured at different temperatures. The instruments and equipment used in the experiments of this invention are shown in Table 1 below.

[0082] Table 1. Names of Instruments and Equipment

[0083] This invention employs size exclusion chromatography to detect monomers and aggregates in the formulation; and high performance liquid chromatography to detect the content of human nerve growth factor and related substances (oxidation peak, denaturation peak) in the formulation.

[0084] Size exclusion chromatography

[0085] 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.

[0086] Size exclusion chromatography 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.

[0087] High performance liquid chromatography

[0088] The chromatographic stationary phase was a gel suitable for separating proteins with a molecular weight of 5–60 kD. The mobile phase was 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). The detection wavelength was 214 nm. Appropriate amounts of the test sample and standard were diluted with the mobile phase to prepare solutions of specific concentrations. 20 μL of each solution was accurately injected into the liquid chromatograph, and the chromatogram was recorded after 30 minutes. The peak area was calculated. The content of human nerve growth factor in the test sample was calculated by peak area using the external standard method. The oxidation and denaturation peaks were calculated by normalizing the peak area.

[0089] Example 1: Histidine and human serum albumin have a synergistic effect in the formulation.

[0090] The inventors unexpectedly discovered that the addition of histidine and human serum albumin to human nerve growth factor formulations produces a synergistic effect, effectively stabilizing the active ingredients and extending shelf life. To further confirm the synergistic effect of histidine and human serum albumin, and whether this synergistic effect exists in other buffer solutions and stabilizers, human nerve growth factor formulations were prepared using different types of buffer solutions and stabilizers. Changes in the content of active ingredients, appearance, oxidation peaks, and denaturation peaks were examined. The results showed that the synergistic effect only exists when histidine and human serum albumin are combined.

[0091] 1.1. Verification of the synergistic effect of histidine and human serum albumin

[0092] Dissolve or disperse different types of buffer solutions (disodium hydrogen phosphate, sodium acetate, sodium citrate, or histidine) in an appropriate amount of water for injection, adjust to the target pH, add different types of stabilizers (polysorbate 20, polysorbate 80, or human serum albumin) and human nerve growth factor stock solution, stir evenly to obtain the final product, as shown in Table 1-1 below.

[0093] 1.2. The appearance, content, and related substances were tested, and the results are shown in Tables 1-2 and 1-3 below:

[0094] As shown in Table 1-2, regarding the changes in the content of active ingredients, compared with the baseline group (Examples 1-4), under the condition of 40°C for 5 days, the change in human nerve growth factor content from day 0 to day 5 was reduced by more than 4 times when histidine alone (Examples 1-2) or human serum albumin alone (Examples 1-3) was used, while when histidine was combined with human serum albumin (Example 1-1), the change was reduced by about 26 times. However, when human serum albumin was combined with other types of buffer solutions (Examples 1-5, Examples 1-7), and histidine was combined with other types of stabilizers (Examples 1-11, Examples 1-13), the change in human nerve growth factor content was not significantly improved compared with the corresponding buffer solution groups (Examples 1-8, Examples 1-10) and stabilizer groups (Examples 1-12, Examples 1-14), and no synergistic effect was observed.

[0095] Furthermore, by calculating the degradation rate constant of the active ingredient in each group of formulations (lnC0 / Cn=kt, where C0 and Cn are the content values ​​of the active ingredient on day 0 and day n, respectively, k is the degradation rate constant, and t is time), as shown in Table 1-2, the histidine and human serum albumin group (Example 1-1) had the lowest degradation rate constant, and the degradation rate was reduced by about 1-2 orders of magnitude compared to the other groups of formulations. This indicates that the histidine and human serum albumin group (Example 1-1) can effectively stabilize the content of human nerve growth factor and play a synergistic role in improving the stability of the formulation.

[0096] As shown in Table 1-3, based on the results of appearance, oxidation peak, and denaturation peak, the formulations containing histidine and human serum albumin maintained a consistently colorless and clear appearance, with no SEC aggregates and relatively low levels of related impurities, indicating good product stability. In contrast, some formulations in other groups exhibited slight opalescence, visible particles, or even turbidity, and the levels of related substances and aggregates showed a significant increasing trend, indicating poor product stability. This further illustrates the synergistic effect of histidine and human serum albumin in stabilizing the formulations.

[0097] In summary, compared to human serum albumin and other buffer solutions, and histidine and other stabilizers, only the combination of histidine and human serum albumin exhibits a synergistic effect. When both are present, the oxidation and denaturation peaks of formulation-related substances show the lowest growth rates; the absence of either one causes significant fluctuations in these peaks. In conclusion, both must be added simultaneously to the formulation to achieve a higher quality product.

[0098] 2.1 Screening of histidine dosage

[0099] To determine the optimal concentration of the buffer, further screening of buffer concentrations was conducted. Histidine was dissolved or dispersed in an appropriate amount of water for injection, adjusted to the target pH, and then human serum albumin and human nerve growth factor stock solution were added and stirred until homogeneous. The formulation is shown in Table 2-1 below:

[0100] The appearance, content, and related substances were tested, and the results are shown in Table 2-2 below:

[0101] The test results showed that the appearance of the preparation did not change with different concentrations of histidine, and the content and related substances changed only slightly.

[0102] 2.2. Screening of Human Serum Albumin Dosage

[0103] To determine the optimal dosage of human serum albumin, further screening of human serum albumin dosage was conducted. Histidine was dissolved or dispersed in an appropriate amount of water for injection, adjusted to the target pH, and then human serum albumin and human nerve growth factor stock solution were added and stirred until homogeneous. The solution was then aseptically filtered through a 0.22 μm filter membrane before filling. The formulation is shown in Table 2-3 below:

[0104] The appearance, content, and related substances were tested, and the results are shown in Table 2-4 below:

[0105] The test results showed that the appearance of the preparation of human serum albumin did not change at a dosage of 0.005-0.06%, and the content and related substances changed little. Among them, the content did not change much at a dosage of 0.02%, so 0.02% human serum albumin is the preferred dosage.

[0106] 2.3. Screening of Osmotic Pressure Regulators

[0107] Sodium chloride is used as an osmotic pressure regulator. Histidine and sodium chloride are dissolved or dispersed in an appropriate amount of water for injection, and the pH is adjusted to the target level. Human serum albumin and human nerve growth factor stock solution are added and stirred until homogeneous. The solution is then aseptically filtered through a 0.22 μm membrane before filling. The formulation is shown in Table 2-5 below:

[0108] The appearance, osmotic pressure, content, and related substances were tested, and the results are shown in Table 2-6 below:

[0109] The results showed that the addition or absence of the osmotic pressure regulator sodium chloride, and its different amounts, only affected the osmotic pressure, without affecting the appearance, content, or related substances of the formulation. When eye drops are formed, excessively high or low osmotic pressure can cause irritation after instillation, increasing tear secretion, diluting or rinsing away the medication, and reducing its efficacy. The eye has a relatively high tolerance to osmotic pressure, approximately equivalent to an osmotic pressure range of 200–500 mOsm / kg, which is tolerable for most people. Therefore, the amount of osmotic pressure regulator sodium chloride added is 0.55%–1.45%, with 0.85% being closer to isotonicity; therefore, the preferred amount of osmotic pressure regulator is 0.85%.

[0110] 2.4. pH screening

[0111] Dissolve or disperse histidine in an appropriate amount of water for injection, adjust to the target pH, add human serum albumin and human nerve growth factor stock solution, and stir until homogeneous. After aseptic filtration through a 0.22 μm filter membrane, fill into vials. The formulation is shown in Table 2-7 below:

[0112] The appearance, content, and related substances of the formulation were tested, and the test results are shown in Table 2-8 below:

[0113] The test results showed that the higher the pH, the faster the denaturation peak of the formulation-related substances grew; conversely, the lower the pH, the faster the oxidation peak grew. The growth rates of both the oxidation and denaturation peaks were lowest at a pH of 5.5–6.0. ​​Furthermore, excessively low pH (Comparative Example 2-17) or excessively high pH (Comparative Example 2-18) caused changes in the appearance of the formulation under high-temperature conditions, indicating a decrease in formulation stability. In summary, the optimal pH range is 4.5–7.5, with 5.5–6.0 being preferred.

[0114] It is worth noting that, in order to further explore the synergistic effect of histidine and human serum albumin, the applicant also prepared histidine and human serum albumin combination formulations at other concentrations (e.g., the combination of minimum histidine concentration and maximum albumin concentration in Examples 2-26, and the combination of minimum albumin concentration and maximum histidine concentration in Examples 2-29) to verify their synergistic effect, as shown in Table 2-9 below:

[0115] The appearance, content, and related substances of the preparation were tested, as shown in Table 2-10.

[0116] The results, as shown in Table 2-10, indicate that, compared with the human serum albumin group or the histidine group alone, the combined preparations of histidine and human serum albumin at other concentrations (Examples 2-26, Examples 2-29) still had a degradation rate constant that was reduced by 1-2 orders of magnitude, indicating a synergistic effect.

[0117] Based on the results of appearance, oxidation peak, and denaturation peak, the combined preparations of histidine and human serum albumin at other concentrations in Examples 2-26 and 2-29 still maintained a colorless and clear appearance, with no SEC aggregates appearing, and the content of related impurities remained relatively low, indicating good product stability.

[0118] Example 3: Changes in physicochemical properties and other key test items of formulations at different concentrations

[0119] 3.1. Preparation process

[0120] The preparation method of the human nerve growth factor drug composition or eye drops is as follows:

[0121] Step 1: Prepare excipient solution: Weigh the prescribed amount of buffer and osmotic pressure regulator, add water for injection to dissolve, and adjust the pH value using hydrochloric acid or sodium hydroxide;

[0122] Step 2: Add the prescribed amount of stabilizer and human nerve growth factor stock solution to the above-prepared excipient solution, and then add water for injection to bring the volume to a final volume. After aseptic filtration through a 0.22μm filter membrane, the solution is filled into vials.

[0123] 3.2. Changes in the physicochemical properties and other key test items of formulations at different concentrations

[0124] Dissolve or disperse histidine and sodium chloride in an appropriate amount of water for injection, adjust to the target pH, add human serum albumin and human nerve growth factor stock solution, and stir until homogeneous. After aseptic filtration through a 0.22 μm filter membrane, fill into vials to obtain the formulation shown in Table 3-1 below.

[0125] The physicochemical properties (appearance, osmotic pressure, insoluble particles), content, and related substances were tested, and the results are shown in Table 3-2 below:

[0126] The results showed that the formulations of each concentration could be stored at 25℃ (room temperature) for 6M and at 2-8℃ (refrigeration) for 12M. During the storage process, the physicochemical properties, content, and related substances of the formulations did not change significantly, and the formulations had good stability.

[0127] Although the present invention has been described in detail above with general descriptions, specific implementation schemes and embodiments, those skilled in the art can make modifications and improvements to it without departing from the spirit of the present invention, and such modifications or improvements are all within the scope of protection claimed by the present invention.

[0128] Industrial applications

[0129] The nerve growth factor drug composition and eye drops prepared by this invention are administered to patients via eye drops, ensuring accurate dosage and ease of use. Furthermore, they maintain good stability without the need for cryopreservation; they contain no antibacterial agents and are packaged in simple, single-dose sterile packaging materials, eliminating the need for fixed accessories and simplifying operation, thus improving the convenience of medication use.

[0130] The beneficial effects of this invention are as follows:

[0131] (1) The pharmaceutical composition of human nerve growth factor and its eye drops of the present invention have the active ingredient being human nerve growth factor expressed by eukaryotes, which has a low mismatch rate and high activity. At the same time, histidine is used as a pH buffer and albumin is used as a stabilizer. The two work synergistically. When they are both present, the growth rate of the oxidation peak and denaturation peak of the formulation-related substances is the lowest. The absence of one of them will cause large fluctuations in the oxidation peak and denaturation peak of the formulation.

[0132] (2) The pharmaceutical composition of human nerve growth factor and its eye drops of the present invention are human nerve growth factor eye drops that can be stored at room temperature or refrigerated. The single-dose packaging material commonly used for eye drops is used as packaging, which avoids the use of antibacterial agents in the preparation. The packaging form is convenient for administration and the safety is also improved, thus improving the convenience of medication.

[0133] (3) The present invention relates to a pharmaceutical composition of nerve growth factor and its eye drops. By rationally selecting and determining the dosage of excipients, the stability of the formulation is improved. The product shows no significant changes in appearance, concentration, and purity after being stored at 2–8°C for 12 months and at 25°C for 6 months. This formulation effectively solves the problem of long-term storage stability and has high drug safety.

[0134] (4) The present invention has a pharmaceutical composition of nerve growth factor and its eye drops. The composition has clear components, is easy to identify and quantify, and uses high-purity excipients with wide availability. It is easy to carry out long-term mass production, which facilitates cost control and product quality improvement.

Claims

1. A pharmaceutical composition comprising an active ingredient, a pH buffer, and a stabilizer; wherein the active ingredient is human nerve growth factor, the pH buffer comprises or is histidine, and the stabilizer comprises or is albumin.

2. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition is human nerve growth factor eye drops.

3. The pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical composition does not contain an antibacterial agent.

4. The pharmaceutical composition according to any one of claims 1-3, wherein the human nerve growth factor comprises an amino acid sequence selected from any one of SEQ ID NO: 1-3.

5. The pharmaceutical composition according to any one of claims 1-4, wherein the concentration of the human nerve growth factor is 0.0001%-0.2% w / v, for example 0.0002%-0.1% w / v, preferably 0.0005%-0.05% w / v, and most preferably 0.001%-0.03% w / v.

6. The pharmaceutical composition according to any one of claims 1-5, wherein the concentration of histidine is 0.1%-1% w / v, preferably 0.17%-0.5% w / v, more preferably 0.24%-0.38% w / v, and most preferably 0.31% w / v.

7. The pharmaceutical composition according to any one of claims 1-6, wherein the albumin is human serum albumin.

8. The pharmaceutical composition according to any one of claims 7, wherein the concentration of human serum albumin is 0.005%-1% w / v, preferably 0.01%-0.06% w / v, more preferably 0.01-0.04% w / v, and most preferably 0.02% w / v.

9. The pharmaceutical composition according to any one of claims 1-8, wherein the pharmaceutical composition further comprises an osmotic pressure regulator.

10. The pharmaceutical composition according to claim 9, wherein the osmotic pressure regulator comprises one or more selected from sodium chloride, boric acid, borax, and glucose, preferably sodium chloride.

11. The pharmaceutical composition according to claim 10, wherein the concentration of sodium chloride is 0.1%-5% w / v, preferably 0.25%-1.75% w / v, more preferably 0.55%-1.45% w / v, and most preferably 0.85% w / v.

12. The pharmaceutical composition according to any one of claims 1-11, wherein the pH of the pharmaceutical composition is 4.5-7.5, preferably 5.0-7.0, more preferably 5.5-6.5 or 5.5-6.

0.

13. The pharmaceutical composition according to any one of claims 1-12, wherein the pharmaceutical composition comprises: Human nerve growth factor, at a concentration of 0.0005%-0.05% w / v, preferably 0.001%-0.03% w / v; Histidine, at a concentration of 0.24%-0.38% w / v, preferably 0.31% w / v; Human serum albumin, with a concentration of 0.01-0.04% w / v, preferably 0.02% w / v; and The pH of the pharmaceutical composition is 5.5-6.

0.

14. The pharmaceutical composition according to claim 13, further comprising sodium chloride at a concentration of 0.55%-1.45% w / v, preferably 0.85% w / v.

15. The pharmaceutical composition according to any one of claims 1-14, wherein the pharmaceutical composition: a) When placed at 45°C for 5 days, the change in the content of its active ingredient does not exceed 6%; the change in the oxidation peak of related substances does not exceed 4%, or the change in the denaturation peak of related substances does not exceed 3%; and / or b) When placed at 25°C for 10 days, the change in the content of its active ingredient does not exceed 6%; the change in the oxidation peak of related substances does not exceed 4%, or the change in the denaturation peak of related substances does not exceed 3%; and / or c) When stored at 25°C for 6 months, the content of its active ingredient changes by no more than 9%; the oxidation peak of related substances changes by no more than 7%, or the denaturation peak of related substances changes by no more than 7%; and / or d) When stored at 2-8℃ for 12 months, the content of its active ingredients changes by no more than 5%; the oxidation peak of related substances changes by no more than 4%; or the denaturation peak of related substances changes by no more than 4%.

16. A method for preparing the pharmaceutical composition according to any one of claims 1-15, comprising the following steps: 1) Dissolve or disperse the pH buffer and osmotic pressure regulator with an appropriate amount of water for injection, and adjust to the target pH; and 2) Add stabilizer and human nerve growth factor stock solution, and stir well.

17. Use of the pharmaceutical composition of any one of claims 1-16 in the preparation of a medicament for treating diseases caused by nerve damage.

18. The use according to claim 17, wherein the disease caused by nerve injury is selected from, for example, nerve poisoning, peripheral nerve injury, diabetic peripheral neuropathy, Alzheimer's disease, Parkinson's disease, facial nerve inflammation, neuroma, amblyopia, corneal epithelial defects, corneal ulcers, neurotrophic keratitis, dry eye syndrome, optic nerve injury, glaucoma, and retinal degeneration.

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