Liquid composition for stabilizing botulinum toxin

Abstract

The present invention relates to a liquid composition for stabilizing botulinum toxin, and it was confirmed that a stabilizer comprising a non-ionic surfactant, an amino acid, and disaccharides as active ingredients can significantly increase the stability of botulinum toxin. Specifically, in the present invention, polysorbate 20, histidine, and disaccharides are combined, and the concentration conditions of each ingredient were established to prepare the optimal stabilizer through the combination. Thus, it is possible to prepare a botulinum toxin liquid formulation with low cytotoxicity and increased stability without containing animal-derived substances, and therefore the present invention can be used in various fields using botulinum toxin.

Description

Botulinum toxin stabilization liquid composition

[0001] The present invention relates to a liquid composition for stabilizing botulinum toxin.

[0002] The present application claims priority based on Korean Patent Application No. 10-2024-0132104 filed on September 27, 2024, and all contents disclosed in the specification and drawings of said application are incorporated into the present application.

[0003] Various strains of the genus Clostridium that secrete toxins with neurotoxic effects have been discovered since the 1890s, and the characteristics of the toxins secreted by these strains have been identified over the past 70 years. Among them, botulinum toxin is known to cause generalized weakness by inhibiting the exocytosis of acetylcholine at the cholinergic presynapse of the neuromuscular junction in animals with neurological function.

[0004] Research and development are being conducted in various fields to utilize the properties of botulinum toxin for cosmetic or therapeutic purposes. For example, technologies have been attempted to use botulinum toxin in the treatment of ophthalmic diseases, pain, various autonomic nervous system dysfunctions including hyperhidrosis, migraine pain, post-surgical pain and visceral pain, psoriasis and dermatitis, various cancers, and neuroinflammation.

[0005] However, as botulinum toxin is a protein-based product, there have been issues regarding its storage, distribution, and management, which involve the pharmaceuticalization process. This is attributed to the instability of proteins, and the problem is particularly severe for protein-based products like botulinum toxin that are pharmaceuticalized at extremely low concentrations. Since botulinum toxin proteins have the property of adhering to solid surfaces, when injected into containers, parts of the protein adhere to the inner walls, causing a loss of the active ingredient; furthermore, the protein can easily oxidize or break down into small fragments.

[0006] To minimize the denaturation of botulinum toxin, purified botulinum toxin is distributed in the form of freeze-dried powder after the manufacturing process and administered to patients in liquid form by diluting it with a solvent immediately before clinical use. However, this method also presents a problem with a high risk of medical accidents due to human error, such as mistakes in dilution ratios or contamination of the dilution solvent. Therefore, there is an urgent need to develop stabilizers capable of preventing protein denaturation during the manufacturing and distribution of liquid botulinum toxin formulations. Furthermore, while albumin has traditionally been widely used as a stabilizer to maintain the activity of botulinum toxin, the development of non-animal-derived preparations is currently required due to the risks of cross-infection and side effects associated with animal-derived components.

[0007] Nevertheless, there has not been much research to date on formulations using stabilizers not derived from animal sources in the pharmaceuticalization process of botulinum toxin proteins, and on liquid compositions that do not require a rehydration process prior to use.

[0008] The object of the present invention is to provide a botulinum toxin-stabilizing liquid composition comprising polysorbate 20, histidine, and a disaccharide as active ingredients.

[0009] Another objective of the present invention is to provide the above-mentioned botulinum toxin stabilizing liquid composition and a botulinum toxin liquid composition comprising botulinum toxin as an active ingredient.

[0010]

[0011] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art to which the present invention belongs from the description below.

[0012] The present invention provides a botulinum toxin-stabilizing liquid composition comprising polysorbate 20, histidine, and a disaccharide as active ingredients.

[0013] In one embodiment of the present invention, the disaccharide may be sucrose or lactose, but is not limited thereto.

[0014] In one embodiment of the present invention, the concentration ratio of polysorbate 20 : histidine : disaccharide in the composition may be (0.001 to 0.1) : (0.01 to 10) : (0.1 to 10), but is not limited thereto.

[0015] In one embodiment of the present invention, the composition is,

[0016] (1 to 20) parts by weight of polysorbate 20;

[0017] (1 to 2000) parts by weight of histidine; and

[0018] It may include (5 to 5000) parts by weight of disaccharides, but is not limited thereto.

[0019] In one embodiment of the present invention, the composition may additionally include an isotonic agent, but is not limited thereto.

[0020] In one embodiment of the present invention, the isotonic agent may be one or more selected from the group consisting of sodium chloride, glycerin, potassium chloride, and dextrose, but is not limited thereto.

[0021] In one embodiment of the present invention, in the above composition,

[0022] The concentration ratio of polysorbate 20 : histidine : disaccharide : isotonic agent may be (0.001 to 0.1) : (0.01 to 10) : (0.1 to 10) : (0.1 to 10), but is not limited thereto.

[0023] In one embodiment of the present invention, the composition is,

[0024] (1 to 20) parts by weight of polysorbate 20;

[0025] (1 to 2000) parts by weight of histidine;

[0026] (5 to 5000) parts by weight of disaccharide; and

[0027] It may include (1 to 9000) parts by weight of an isotonic agent, but is not limited thereto.

[0028] In one embodiment of the present invention, the composition is,

[0029] Polysorbate 20 of 0.005 mg / mL to 0.02 mg / mL;

[0030] 0.05 mg / mL to 4 mg / mL of histidine;

[0031] 0.2 mg / mL to 10 mg / mL of disaccharide; and

[0032] It may contain an isotonic agent in an amount of 1 mg / mL to 20 mg / mL, but is not limited thereto.

[0033] In one embodiment of the present invention, the botulinum toxin may be one or more selected from the group consisting of botulinum toxins types A, B, C, D, E, F, and G, but is not limited thereto.

[0034] The present invention provides the above-described botulinum toxin stabilizing liquid composition and a botulinum toxin liquid composition comprising botulinum toxin as an active ingredient.

[0035] In one embodiment of the present invention, the botulinum toxin may be one or more selected from the group consisting of botulinum toxins types A, B, C, D, E, F, and G, but is not limited thereto.

[0036] In one embodiment of the present invention, the composition may contain 1 to 500 Unit / mL of botulinum toxin based on the total amount of the composition, but is not limited thereto.

[0037] In addition, the present invention provides a botulinum toxin stabilizing liquid composition comprising, as active ingredients, a nonionic surfactant comprising polysorbate 20, an amino acid comprising histidine, and a disaccharide comprising sucrose; or a botulinum toxin stabilizing liquid composition comprising, as active ingredients, a nonionic surfactant comprising polysorbate 20, an amino acid comprising histidine, a disaccharide comprising sucrose, and an isotonic agent comprising sodium chloride.

[0038] In addition, the present invention provides a use for stabilizing botulinum toxin using the above-described botulinum toxin stabilization liquid composition.

[0039] In addition, the present invention provides a method for stabilizing botulinum toxin using the above-described botulinum toxin stabilizing liquid composition.

[0040] In addition, the present invention provides a use for manufacturing a botulinum toxin liquid stabilization preparation of the above-described botulinum toxin stabilization liquid composition.

[0041]

[0042] In addition, the present invention provides the above-mentioned botulinum toxin stabilizing liquid composition and a botulinum toxin liquid pharmaceutical composition comprising botulinum toxin as an active ingredient.

[0043] In addition, the present invention provides the above-mentioned botulinum toxin stabilizing liquid composition and a botulinum toxin liquid pharmaceutical composition for treating neurological diseases or cosmetic treatment, comprising botulinum toxin as an active ingredient.

[0044] In addition, the present invention provides a method for treating neurological diseases or cosmetic treatments, comprising the step of administering the above-described botulinum toxin stabilizing liquid composition and a composition containing botulinum toxin as an active ingredient, i.e., the above-described botulinum toxin liquid pharmaceutical composition, to an individual in need of such treatment in a pharmaceutically effective amount.

[0045] In addition, the present invention provides the above-described botulinum toxin stabilizing liquid composition and a composition comprising botulinum toxin as an active ingredient, i.e., the above-described botulinum toxin liquid pharmaceutical composition, for use in treating neurological diseases or cosmetic treatments.

[0046] In addition, the present invention provides a use for preparing a formulation for the treatment of neurological diseases or cosmetic treatment of the above-described botulinum toxin stabilization liquid composition and a composition containing botulinum toxin as an active ingredient, namely, the above-described botulinum toxin liquid pharmaceutical composition.

[0047] In one embodiment of the present invention, the composition may be characterized as a cosmetic treatment being a wrinkle cosmetic treatment, but is not limited thereto.

[0048] In one embodiment of the present invention, the wrinkle may be a facial wrinkle, but is not limited thereto.

[0049] In one embodiment of the present invention, the neurological disease may be one or more selected from the group consisting of torticollis, blepharospasm, muscle tone such as in the hands and feet, unilateral facial spasm, migraine, lower back pain, cervical spine disorder, and hyperstimulation of salivary glands, but is not limited thereto.

[0050] According to the botulinum toxin stabilization liquid composition, it was confirmed that a stabilizer comprising a nonionic surfactant, an amino acid, and a disaccharide as active ingredients can significantly increase the stability of botulinum toxin. Specifically, the present invention combines polysorbate 20, histidine, and a disaccharide, and establishes concentration conditions for each component to produce the best stabilizer through the above composition. As such, it is possible to produce a botulinum toxin liquid formulation with low cytotoxicity and increased stability without containing animal-derived materials, and thus it can be utilized in various fields utilizing botulinum toxin.

[0051] The present invention provides a liquid composition for stabilizing botulinum toxin comprising polysorbate 20, histidine, and a disaccharide as active ingredients, and may additionally provide a liquid composition for stabilizing botulinum toxin comprising an isotonic agent. In the present invention, the combination of polysorbate 20, histidine, and a disaccharide, which are essential components for significantly increasing the stabilization activity of botulinum toxin, and the concentration conditions thereof have been established.

[0052] In Example 2-2 of the present invention, among the types of polysorbates that are nonionic surfactants, polysorbate 20 and polysorbate 80 were combined with histidine, a disaccharide (sucrose), and botulinum toxin to investigate their stability. As a result, it was confirmed that the stability of polysorbate 20 was significantly superior, and the concentration conditions at that time were established.

[0053] In addition, in Examples 2-3 of the present invention, histidine and a disaccharide (sucrose) were combined with polysorbate 20 and botulinum toxin to investigate stability, and concentration conditions that exhibit the best stability effect were established.

[0054] Furthermore, in Example 3, the experimental results from Example 2 were reconfirmed.

[0055] That is, the present invention was completed by confirming that the botulinum toxin stabilization effect is significantly superior when polysorbate 20, histidine, and a disaccharide (sucrose) are combined under conditions of specific concentrations.

[0056] Accordingly, the botulinum toxin stabilization liquid composition and the botulinum toxin liquid composition of the present invention can be free from the potential risk of infection by blood-derived pathogens or infectious microorganisms that can be induced by animal-derived proteins, in that they produce an excellent botulinum toxin stabilization effect using only the minimum essential components of polysorbate 20, histidine, and disaccharide (sucrose) without containing animal-derived proteins. Furthermore, there is an advantage in that safety for the human body can be increased in that it stabilizes botulinum toxin while having significantly low cytotoxicity.

[0057] Accordingly, the present invention provides a botulinum toxin-stabilizing liquid composition comprising polysorbate 20, histidine, and a disaccharide as active ingredients.

[0058] In the present invention, “botulinum toxin” refers to a neurotoxic protein produced by the bacterium Clostridium botulinum. The genus Clostridium comprises more than 127 species and is classified according to morphology and function. The anaerobic, Gram-positive bacterium Clostridium botulinum produces botulinum toxin, a potent polypeptide neurotoxin that causes a neuroparalytic disease called botulism in humans and animals. Many factors contribute to botulism, such as the fact that spores of Clostridium botulinum are found in soil and can be cultured in sealed canned food containers that are not properly sterilized.

[0059] Symptoms of botulism typically appear 18 to 36 hours after consuming food contaminated with Clostridium botulinum cultures or spores. Botulinum toxin appears capable of penetrating the intestinal lining without reduced toxicity and exhibits a high affinity for cholinergic motor neurons. In cases of botulinum toxin poisoning, symptoms can progress to gait disturbances, dysphagia and speech disorders, paralysis of respiratory muscles, and death.

[0060] This toxin causes muscle paralysis in mammals by blocking the presynaptic release of the neurotransmitter acetylcholine at the neuromuscular junction. Recently, utilizing this effect, botulinum toxin has been used therapeutically to treat strabismus, idiopathic blepharospasm, and hemifacial spasm, as well as torticollis, ortonia, or mandibular dystonia, spastic dysphonia, and any involuntary muscle movement disorder including dystonia. Additionally, it is used in various cosmetic fields, such as the non-surgical reduction of facial wrinkles, and to treat hyperhidrosis.

[0061] Currently, serotypes A and B of botulinum toxin proteins are being formulated for the treatment of blepharospasm, torticollis, and hyperhidrosis, as well as for facial wrinkle removal; however, many problems arise during the formulation process for protein preparations such as these botulinum toxins.

[0062] As mentioned above, this is due to the instability of the protein, the loss of active ingredients caused by the botulinum toxin protein's tendency to adhere to solid surfaces, and its high oxidative and degradable properties; therefore, it is necessary to add a stabilizer as a substance capable of preventing these disadvantages.

[0063] In order to resolve the instability of such botulinum toxin, the present invention has invented a botulinum toxin stabilization liquid composition comprising polysorbate 20, histidine, and a disaccharide as active ingredients. In the present invention, the “stabilizing agent” is a “botulinum toxin stabilizing agent” and can be used interchangeably with the “botulinum toxin stabilization (liquid) composition.”

[0064] In the present invention, the term “stabilizer” refers to any additive added to increase the stability of the active ingredient and to prevent the active ingredient from being arbitrarily altered, oxidized, crystallized, or denatured into a related substance; it is not significantly limited as long as it is pharmaceutically acceptable. The evaluation of the stabilizing efficacy of the stabilizer can be performed without temperature limitations, but it is generally understood that the stabilizing effect is maintained for a longer period at lower temperatures than at higher temperatures. In the present invention, the effect was confirmed by performing a stability test under harsh conditions (35°C).

[0065] In the liquid composition for stabilizing botulinum toxin according to the present invention, polysorbate 20 was used as a nonionic surfactant. In the present invention, “nonionic surfactant” is used to lower the surface tension of a protein solution to prevent the adsorption or aggregation of proteins on hydrophobic surfaces, and can collectively refer to nonionic substances that can be used for the above purpose. Representative specific examples of nonionic surfactants may include polysorbates. For example, polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate), polysorbate 40 (polyoxyethylene (20) sorbitan monopalmitate), polysorbate 60 (polyoxyethylene (20) sorbitan monostearate), and polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). The number (20) following polyoxyethylene above may represent the total number of oxyethylene groups (- (CH2CH2O)-), but is not limited thereto. Also, poloxamer (PEO-PPO-PEO copolymer; PEO: poly (ethylene oxide), PPO: poly (propylene oxide)), polyethylene-polypropylene glycol, polyoxyethylene compounds (e.g., polyoxyethylene stearate, polyoxyethylene alkyl ether (alkyl: C1-C 30 ), polyoxyethylene monoallyl ether, alkylphenyl polyoxyethylene copolymer (alkyl: C1-C 30 It may be sodium dodecyl sulfate (SDS), etc., or polysorbate or poloxamer, and may also be included in the form of one or more combinations of these.

[0066] In the industry, among nonionic surfactants, polysorbates are sometimes understood to be interchangeable with other types of compounds within a similar range. However, as a result of a comparative analysis of the stability effects of polysorbate 20 and polysorbate 80 in one embodiment of the present invention, polysorbate 80 was found to be effective as a stabilizer, while polysorbate 20 was found to be about 10 to 20% more effective in stabilizing the botulinum toxin than polysorbate 80. Furthermore, it was confirmed that such a botulinum toxin stabilizing effect appeared over a wider range than polysorbate 80 when used under the same composition and concentration conditions.

[0067] That is, the combination of histidine, an isotonic agent, and a disaccharide as a composition for stabilizing botulinum toxin showed superior stability in the same concentration range when polysorbate 20 was used compared to polysorbate 80, and this may be derived from the total number of oxyethylene groups, since the number (20) after polyoxyethylene refers to the total number of oxyethylene groups (-(CH2CH2O)-), but is not limited thereto.

[0068] Accordingly, the botulinum toxin stabilizing liquid composition of the present invention comprises polysorbate 20, histidine, and sucrose as active ingredients, and the excellent stabilizing effect thereof can occur when all concentration conditions of polysorbate 20, histidine, and sucrose in the present invention are satisfied.

[0069] In the present invention, “histidine (symbol, His, H)” is an essential amino acid used in protein biosynthesis and may be represented by the following structural formula, but is not limited thereto, and may include a pharmaceutically or chemically effective salt thereof. Histidine contains an α-amino group, a carboxylic acid group, and an imidazole side chain, and can be classified as an amino acid that carries a positive charge at physiological pH.

[0070]

[0071] In the present invention, “disaccharide” may be a sugar formed when two monosaccharides are connected by a glycosidic bond. Like monosaccharides, disaccharides are soluble in water, and representative types of disaccharides may be sucrose (sugar) or lactose (milk sugar) in one embodiment of the present invention, preferably sucrose, but are not limited thereto.

[0072] In one embodiment of the present invention, the concentration ratio of polysorbate 20 : histidine : disaccharide in the composition may be (0.001 to 0.1) : (0.01 to 10) : (0.1 to 10), but is not limited thereto. For example, the concentration ratio of polysorbate 20 : histidine : disaccharide is (0.001 to 0.08) : (0.01 to 8) : (0.1 to 10), (0.001 to 0.06) : (0.01 to 6) : (0.1 to 10), (0.001 to 0.04) : (0.01 to 5) : (0.1 to 10), (0.001 to 0.02) : (0.01 to 4) : (0.1 to 10), (0.003 to 0.1) : (0.03 to 10) : (0.15 to 10), (0.003 to 0.08) : (0.03 to 8) : (0.15 to 10), (0.003 to 0.06) : (0.03 to 6) : (0.15 to 10), (0.003 to 0.04) : (0.03 to 5) : (0.15 to 10), (0.003 to 0.02) : (0.03 to 4) : (0.15 to 10), (0.005 to 0.1) : (0.05 to 10) : (0.2 to 10), (0.005 to 0.08) : (0.05 to 8) : (0.2 to 10), (0.005 to 0.06) : (0.05 to 6) : (0.2 to 10), (0.005 to 0.04) : (0.05 to 5) : (0.2 to 10), or (0.005 to 0.02) : (0.05 to 4) : (0.2 to 10). However, it is not limited thereto. Alternatively, in one embodiment of the present invention, the concentration ratio of polysorbate 20 : histidine : disaccharide in the composition may be (0.01 to 0.02) : (0.1 to 2) : (0.5 to 5), but is not limited thereto. In this case, the unit of concentration may be mg / mL, but is not limited thereto.

[0073] In one embodiment of the present invention, the composition is,

[0074] (1 to 20) parts by weight of polysorbate 20;

[0075] (1 to 2000) parts by weight of histidine; and

[0076] It may include (5 to 5000) parts by weight of disaccharides, but is not limited thereto. For example, in the composition of the present invention, the parts by weight of polysorbate 20, histidine, and disaccharides may be (1 to 10), (1 to 1500), and (20 to 4000) parts by weight; (1 to 8), (5 to 1000), and (25 to 3500) parts by weight; (1 to 6), (7 to 900), and (30 to 3000) parts by weight; (1 to 5), (9 to 850), and (35 to 2500) parts by weight; or (1 to 4), (10 to 800), and (40 to 2000) parts by weight.

[0077] In addition, for example, (1 to 4), (10 to 600), and (40 to 1000) parts by weight; (1 to 4), (10 to 400), and (40 to 800) parts by weight; (1 to 4), (10 to 300), and (40 to 600) parts by weight; (1 to 4), (10 to 200), and (40 to 500) parts by weight; (1 to 3), (10 to 600), and (50 to 1000) parts by weight; (1 to 3), (10 to 400), and (50 to 800) parts by weight; (1 to 3), (10 to 300), and (50 to 600) parts by weight; (1 to 3), (10 to 200), and (50 to 500) parts by weight; (1 to 2), (10 to 600), and (50 to 1000) parts by weight; (1 to 2), (10 to 400), and (50 to 800) parts by weight; (1 to 2), (10 to 300), and (50 to 600) parts by weight; or (1 to 2), (10 to 200), and (50 to 500) parts by weight; but is not limited thereto. In the present invention, the parts by weight may be based on polysorbate 20.

[0078] The above-described concentration ratio and weight ratio were established based on the concentration range confirmed to have an excellent botulinum toxin stabilization effect when polysorbate 20, histidine, and disaccharide were combined as confirmed in one embodiment of the present invention. For example, polysorbate 20, histidine, and disaccharide may each be 0.005 to 0.02 mg / mL; 0.05 to 4 mg / mL; and 0.2 to 10 mg / mL; or 0.01 to 0.02 mg / mL; 0.1 to 2 mg / mL; and 0.5 to 5 mg / mL.

[0079] Accordingly, in one embodiment of the present invention, the composition is,

[0080] Polysorbate 20 of 0.005 mg / mL to 0.02 mg / mL;

[0081] 0.05 mg / mL to 4 mg / mL of histidine; and

[0082] It may include 0.2 mg / mL to 10 mg / mL of disaccharide; but is not limited thereto.

[0083] In addition, in one embodiment of the present invention, the composition is,

[0084] Polysorbate 20 of 0.01 mg / mL to 0.02 mg / mL;

[0085] 0.1 mg / mL to 10 mg / mL of histidine; and

[0086] It may include 0.5 mg / mL to 5 mg / mL of disaccharide; but is not limited thereto.

[0087] In one embodiment of the present invention, the composition may additionally include an isotonic agent, but is not limited thereto.

[0088] In the present invention, the “isotonic agent” is a substance added to adjust the osmotic pressure of a liquid formulation, and may be included in the liquid formulation of the present invention without limitation as long as it contributes to stabilizing botulinum toxin while maintaining an appropriate osmotic pressure. The isotonic agent may include a water-soluble inorganic salt, and specifically, the liquid formulation of the present invention may be one or more selected from the group consisting of sodium chloride, glycerin, potassium chloride, and dextrose, but is not limited thereto.

[0089] In one embodiment of the present invention, in the above composition,

[0090] The concentration ratio of polysorbate 20 : histidine : disaccharide : isotonic agent may be (0.001 to 0.1) : (0.01 to 10) : (0.1 to 10) : (0.1 to 10), but is not limited thereto. For example, the ratio of polysorbate 20 : histidine : disaccharide may be applied in the same way as the example described above, and the concentration ratio of the isotonic agent may be (1 to 10), (3 to 10), (5 to 10), (6 to 10), (7 to 10), (8 to 10), (1 to 9), (3 to 9), (5 to 9), (6 to 9), (7 to 9), (8 to 9), or 9. That is, in one embodiment of the present invention, the concentration ratio of polysorbate 20 : histidine : disaccharide : isotonic agent in the composition may be (0.005 to 0.02) : (0.05 to 4) : (0.2 to 10) : 9, and may also be (0.01 to 0.02) : (0.1 to 2) : (0.5 to 5) : 9, but is not limited thereto.

[0091] In one embodiment of the present invention, the composition is,

[0092] (1 to 20) parts by weight of polysorbate 20;

[0093] (1 to 2000) parts by weight of histidine;

[0094] (5 to 5000) parts by weight of disaccharide; and

[0095] It may include (1 to 9000) parts by weight of an isotonic agent, but is not limited thereto. For example, the parts by weight of polysorbate 20, histidine, and disaccharide may be applied in the same manner as in the example described above. In this case, the part by weight of the isotonic agent may be (1 to 5000) parts by weight, (1 to 4000) parts by weight, (1 to 3000) parts by weight, (1 to 2000) parts by weight, (1 to 1900) parts by weight, or (1 to 1800) parts by weight. That is, in one embodiment of the present invention, the composition comprises (1 to 4) parts by weight of polysorbate 20, (10 to 800) parts by weight of histidine, (40 to 2000) parts by weight of disaccharide, and (1 to 1800) parts by weight of an isotonic agent; or may include (1 to 2) parts by weight of polysorbate 20, (10 to 200) parts by weight of histidine, (50 to 500) parts by weight of disaccharide, and (1 to 1800) parts by weight of isotonic agent; but is not limited thereto.

[0096] In one embodiment of the present invention, the composition is,

[0097] Polysorbate 20 of 0.005 mg / mL to 0.02 mg / mL;

[0098] 0.05 mg / mL to 4 mg / mL of histidine;

[0099] 0.2 mg / mL to 10 mg / mL of disaccharide; and

[0100] It may contain an isotonic agent in an amount of 1 mg / mL to 20 mg / mL, but is not limited thereto.

[0101] For example, in the present invention, polysorbate 20 may be 0.005 mg / mL to 0.02 mg / mL, 0.006 mg / mL to 0.02 mg / mL, 0.007 mg / mL to 0.02 mg / mL, 0.008 mg / mL to 0.02 mg / mL, 0.009 mg / mL to 0.02 mg / mL, or 0.01 mg / mL to 0.02 mg / mL, but is not limited thereto.

[0102] In addition, in the present invention, histidine is 0.05 mg / mL to 4 mg / mL, 0.06 mg / mL to 4 mg / mL, 0.07 mg / mL to 4 mg / mL, 0.08 mg / mL to 4 mg / mL, 0.09 mg / mL to 4 mg / mL, 0.1 mg / mL to 4 mg / mL, 0.05 mg / mL to 3 mg / mL, 0.06 mg / mL to 3 mg / mL, 0.07 mg / mL to 3 mg / mL, 0.08 mg / mL to 3 mg / mL, 0.09 mg / mL to 3 mg / mL, 0.1 mg / mL to 3 mg / mL, 0.05 mg / mL to 2 mg / mL, 0.06 mg / mL to 2 mg / mL, 0.07 mg / mL to 2 mg / mL, 0.08 mg / mL to 2 It may be mg / mL, 0.09 mg / mL to 2 mg / mL, or 0.1 mg / mL to 2 mg / mL, but is not limited thereto.

[0103] In addition, in the present invention, the disaccharide sucrose may be 0.2 mg / mL to 10 mg / mL, 0.2 mg / mL to 8 mg / mL, 0.2 mg / mL to 6 mg / mL, 0.2 mg / mL to 5 mg / mL, 0.3 mg / mL to 10 mg / mL, 0.3 mg / mL to 8 mg / mL, 0.3 mg / mL to 6 mg / mL, 0.3 mg / mL to 5 mg / mL, 0.4 mg / mL to 10 mg / mL, 0.4 mg / mL to 8 mg / mL, 0.4 mg / mL to 6 mg / mL, 0.4 mg / mL to 5 mg / mL, 0.5 mg / mL to 10 mg / mL, 0.5 mg / mL to 8 mg / mL, 0.5 mg / mL to 6 mg / mL, or 0.5 mg / mL to 5 mg / mL, but It is not limited to this.

[0104] In addition, in the present invention, the isotonic agent is 1 mg / mL to 20 mg / mL, 1 mg / mL to 15 mg / mL, 1 mg / mL to 14 mg / mL, 1 mg / mL to 13 mg / mL, 1 mg / mL to 12 mg / mL, 1 mg / mL to 11 mg / mL, 1 mg / mL to 10 mg / mL, 1 mg / mL to 9 mg / mL, 3 mg / mL to 20 mg / mL, 3 mg / mL to 15 mg / mL, 3 mg / mL to 14 mg / mL, 3 mg / mL to 13 mg / mL, 3 mg / mL to 12 mg / mL, 3 mg / mL to 11 mg / mL, 3 mg / mL to 10 mg / mL, 3 mg / mL to 9 mg / mL, 6 mg / mL to 20 mg / mL, 6 mg / mL to 15 mg / mL, 6 mg / mL to It may be 14 mg / mL, 6 mg / mL to 13 mg / mL, 6 mg / mL to 12 mg / mL, 6 mg / mL to 11 mg / mL, 6 mg / mL to 10 mg / mL, 6 mg / mL to 9 mg / mL, 8 mg / mL to 20 mg / mL, 8 mg / mL to 15 mg / mL, 8 mg / mL to 14 mg / mL, 8 mg / mL to 13 mg / mL, 8 mg / mL to 12 mg / mL, 8 mg / mL to 11 mg / mL, 8 mg / mL to 10 mg / mL, 8 mg / mL to 9 mg / mL, or 9 mg / mL.

[0105] Accordingly, in the present invention, the weight parts of polysorbate 20, histidine, disaccharide, and isotonic agent may be (1 to 4), (10 to 800), (40 to 2000), and 1800, respectively, and the polysorbate 20, histidine, disaccharide, and isotonic agent may be 0.005 to 0.02 mg / mL, 0.05 to 4 mg / mL, 0.2 to 10 mg / mL, and 9 mg / mL, respectively, but are not limited thereto.

[0106] In the present invention, the weight parts of polysorbate 20, histidine, disaccharide, and isotonic agent may be (1 to 2), (1 to 200), (5 to 500), and 1800, respectively, and the polysorbate 20, histidine, disaccharide, and isotonic agent may be 0.01 to 0.02 mg / mL, 0.1 to 2 mg / mL, 0.5 to 5 mg / mL, and 9 mg / mL, respectively, but are not limited thereto.

[0107] In particular, in one embodiment of the present invention, 0.02 mg / mL polysorbate 20, 1 mg / mL histidine, and 1 mg / mL disaccharide (test group 4);

[0108] 0.02 mg / mL polysorbate 20, 2 mg / mL histidine, and 1 mg / mL disaccharide (test group 9);

[0109] It was confirmed that the botulinum toxin stabilization effect was best with 0.02 mg / mL polysorbate 20, 0.1 mg / mL histidine, and 1 mg / mL disaccharide (test group 13), but is not limited thereto. In addition, the sodium chloride in all cases may be 9 mg / mL.

[0110] The present invention provides the above-described botulinum toxin stabilizing liquid composition and a botulinum toxin liquid composition comprising botulinum toxin as an active ingredient.

[0111] In one embodiment of the present invention, the botulinum toxin may be one or more selected from the group consisting of botulinum toxins types A, B, C, D, E, F, and G, and in the present invention, conditions for stabilizing the toxin using type A botulinum toxin were established, but are not limited thereto.

[0112] Botulinum toxin type A is the most lethal natural biological agent known to humans. LD50 of commercially available botulinum toxin type A (purified neurotoxin complex) 50It is approximately 50 picograms (i.e., 1 unit). On a molar basis (M), botulinum toxin type A is 1.8 billion times more lethal than diphtheria, 600 million times more lethal than sodium cyanide, 30 million times more lethal than cobra toxin, and 12 million times more lethal than cholera. 1 unit (U) of botulinum toxin is administered via intraperitoneal injection to female Swiss Webster mice or similar mice weighing 18–20 g each (mLD). 50 It can be defined as. Generally, the seven immunologically distinct botulinum neurotoxins are characterized by neurotoxin serotypes A, B, C1, D, E, F, and G, respectively, which are distinguished by neutralization with type-specific antibodies. Different serotypes of botulinum toxins differ depending on the animal species acted upon and the degree and duration of paralysis caused. For example, when measured by the paralysis rate in rats, botulinum toxin type A was measured to be 500 times more potent than botulinum toxin type B. Furthermore, botulinum toxin type B is botulinum toxin type A primate LD50 50 It was measured to be non-toxic even when administered to primates at a dose of 480 U / kg, which is about 12 times the amount. Botulinum toxin is believed to bind with a strong affinity to cholinergic motor neurons, enter the neurons, and inhibit the release of acetylcholine. It can be absorbed not only through phagocytosis and pinocytosis, but also through low-affinity receptors.

[0113] The molecular weight of botulinum toxin molecules is approximately 150 kD in all seven known botulinum toxin serotypes. Meanwhile, botulinum toxin is released by Clostridium bacteria as a complex containing a 150 kD botulinum toxin protein molecule along with associated non-toxin proteins. Therefore, botulinum toxin type A complex can be produced by Clostridium bacteria in 900 kD, 500 kD, and 300 kD forms. Botulinum toxin types B and C can be produced as 500 kD complexes, and botulinum toxin type D can be produced as 300 kD and 500 kD complexes. Botulinum toxin types E and F can be produced as approximately 300 kD complexes. These complexes (i.e., those with a molecular weight greater than approximately 150 kD) are believed to contain non-toxic hemagglutinin proteins and non-toxin and non-toxic non-hemagglutinin proteins.

[0114] Botulinum toxin comprises a pure neurotoxin component of about 150 kD along with a high molecular weight complex form containing non-toxin proteins. Thus, the complexed form may comprise botulinum neurotoxin proteins and one or more non-toxin hemagglutinin proteins and / or one or more non-toxin non-hemagglutinin proteins. Specifically, the botulinum toxin complex protein may be a complex of botulinum neurotoxin (BoNT), nontoxic nonhemagglutinin (NTNH), and hemagglutinin (HA) proteins. The molecular weight of the complex may be greater than about 150 kD. For example, the complexed form of botulinum toxin type A may have a molecular weight of about 900 kD, about 500 kD, or about 300 kD.

[0115] The botulinum toxin complex protein of the present invention may have a molecular weight greater than 150 kD. Specifically, it may have a molecular weight of about 250 kD to 1400 kD, and more specifically, it may have a molecular weight of 280 kD to 1300 kD, 300 kD to 1200 kD, 700 kD to 1100 kD, 800 kD to 1000 kD, or about 900 kD, but is not limited thereto.

[0116] In the present invention, the botulinum toxin may include natural or recombinant botulinum toxin, but is not limited thereto.

[0117] The botulinum toxin of the present invention may include both that obtained from a culture medium of Clostridium botulinum and that obtained commercially, but is not limited thereto.

[0118] In one embodiment of the present invention, the composition may contain 1 to 500 Unit / mL of botulinum toxin based on the total amount of the composition, but is not limited thereto.

[0119] The liquid formulation of the present invention comprises the botulinum toxin in an amount of 1 unit / mL to 400 unit / mL, 1 unit / mL to 300 unit / mL, 1 unit / mL to 200 unit / mL, 1 unit / mL to 100 unit / mL, 1 unit / mL to 80 unit / mL, 1 unit / mL to 60 unit / mL, 1 unit / mL to 50 unit / mL, 1 unit / mL to 40 unit / mL, 10 unit / mL to 400 unit / mL, 10 unit / mL to 300 unit / mL, 10 unit / mL to 200 unit / mL, 10 unit / mL to 100 unit / mL, 10 unit / mL to 80 unit / mL, 10 unit / mL to 60 unit / mL, 10 unit / mL to 50 unit / mL, 10 unit / mL to 40 unit / mL, and 20 unit / mL. 400 unit / mL, 20 unit / mL to 300 unit / mL, 20 unit / mL to 200 unit / mL, 20 unit / mL to 100 unit / mL, 20 unit / mL to 80 unit / mL, 20 unit / mL to 60 unit / mL, 20 unit / mL to 50 unit / mL, 20 unit / mL to 40 unit / mL, 30 unit / mL to 400 unit / mL, 30 unit / mL to 300 unit / mL, 30 unit / mL to 200 unit / mL, 30 unit / mL to 100 unit / mL, 30 unit / mL to 80 unit / mL, 30 unit / mL to 60 unit / mL, 30 unit / mL to 50 unit / mL, 30 unit / mL to 40 unit / mL, or 100 unit / mL, 90 unit / mL, It may be included at a concentration of 80 unit / mL, 70 unit / mL, 60 unit / mL, 50 unit / mL, or 40 unit / mL, but is not limited thereto.

[0120] The botulinum toxin liquid composition of the present invention may include a pharmaceutically acceptable salt form. Accordingly, the present invention can provide a botulinum toxin liquid pharmaceutical composition.

[0121] Botulinum toxin is widely used for both cosmetic and pharmaceutical purposes, and it is evident in the industry that stabilizing botulinum toxin increases its use in cosmetic and pharmaceutical applications for neurological diseases. In this invention, a stabilizing liquid composition capable of enhancing the stabilization of botulinum toxin has been developed, and a composition comprising both the botulinum toxin stabilizing liquid composition and botulinum toxin is provided, which is defined as the botulinum toxin liquid composition.

[0122] As described above, botulinum toxin liquid compositions can be used pharmaceutically in accordance with general practices in the art. Accordingly, the present invention provides a botulinum toxin stabilizing liquid composition and a botulinum toxin liquid pharmaceutical composition comprising botulinum toxin as an active ingredient.

[0123] In addition, the present invention provides a botulinum toxin stabilization liquid composition and a botulinum toxin liquid pharmaceutical composition for treating neurological diseases or cosmetic treatment, comprising botulinum toxin as an active ingredient.

[0124] In one embodiment of the present invention, the neurological disease may be one or more selected from the group consisting of torticollis, blepharospasm, muscle tone such as in the hands and feet, unilateral facial spasm, migraine, lower back pain, cervical spine disorder, and hyperstimulation of salivary glands, but is not limited thereto.

[0125] In one embodiment of the present invention, the composition may be characterized as a cosmetic treatment being a wrinkle cosmetic treatment, but is not limited thereto.

[0126] In one embodiment of the present invention, the wrinkle may be a facial wrinkle, but is not limited thereto.

[0127] As described above, it is common in the art for pharmaceutical compositions containing botulinum toxin as an active ingredient to be used for cosmetic treatments, particularly for wrinkle treatment, and for neurological diseases. Since the botulinum toxin stabilization composition of the present invention significantly increases the stability of botulinum toxin, it can be easily deduced that it will enhance the therapeutic activity of botulinum toxin for cosmetic and neurological disease treatment; thus, the therapeutic activity of the present invention for cosmetic and neurological diseases can be obviously inferred.

[0128] In the present invention, “pharmaceuticalally acceptable” means a substance that can be effectively used for a desired purpose without causing excessive toxicity, irritation, or allergic reactions within the scope of pharmaceutical judgment. The term “pharmaceuticalally acceptable salt” in the present invention includes a salt derived from a pharmaceutically acceptable inorganic acid, organic acid, or base. Examples of suitable acids include hydrochloric acid, bromic acid, sulfuric acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, toluene-p-sulfonic acid, tartaric acid, acetic acid, citric acid, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, benzenesulfonic acid, etc. Salts derived from suitable bases may include alkali metals such as sodium and potassium, alkaline earth metals such as magnesium, and ammonium, etc.

[0129] In addition, the liquid composition according to the present invention may further include pharmaceutically acceptable carriers, excipients, etc., and such carriers and excipients may be non-naturally present. Specifically, “pharmaceutically acceptable carrier” refers to a carrier or diluent that does not irritate living organisms and does not impair the properties of the liquid formulation of the present invention. As pharmaceutical carriers acceptable for a composition formulated as a liquid solution, they may be sterile and biocompatible, such as saline solution, sterile water, Ringer’s solution, buffered saline solution, albumin injection solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these components, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents may be added as needed.

[0130] More specifically, carriers, excipients, and diluents that may be included in the above pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, polycaprolactone, polylactic acid, poly-L-lactic acid, mineral oil, etc.

[0131] The liquid formulation of the present invention can be used for the treatment or prevention of diseases for which the effect of prevention, treatment, or improvement can be obtained by the administration of botulinum toxin, for example, neuromuscular disorders characterized by hyperactive skeletal muscle. In addition, headache, migraine, tension headache, sinus headache, cervical headache, sweating disorders, axillary hyperhidrosis, palmar hyperhidrosis, plantar hyperhidrosis, Frey's syndrome, hyperkinetic skin line, facial wrinkles, glabellar wrinkles, crow's feet, perioral wrinkles, nose-lip wrinkles, skin disorders, achalasia, strabismus, chronic dentition, blepharospasm, musculoskeletal pain, fibromyalgia, pancreatitis, tachycardia, prostatic hypertrophy, prostatitis, urinary retention, urinary incontinence, overactive bladder, hemifacial spasm, tremor, muscle spasm, gastrointestinal disorders, diabetes mellitus, hypersalivation, detrusor-spyin coordination disorder, post-stroke spasticity, wound healing, pediatric cerebral palsy, smooth muscle spasm, restenosis, focal dystonia, epilepsy, cervical dystonia, thyroid disorders, hypercalcemia, obsessive-compulsive disorder, arthritis pain, Raynaud's syndrome, stretch marks, peritoneal adhesions, It can be used for various purposes, such as treating conditions like vasospasm, runny nose, muscle spasms, laryngeal dystonia, writing spasms, and carpal tunnel syndrome, as well as for cosmetic purposes, but is not limited thereto.

[0132] The “pharmaceutical composition” according to the present invention may further include a suitable carrier, excipient, and diluent commonly used in the manufacture of pharmaceutical compositions. The excipient may be one or more selected from the group consisting of, for example, diluents, binders, disintegrants, lubricants, adsorbents, humectants, film-coating materials, and controlled-release additives.

[0133] The pharmaceutical composition according to the present invention may be formulated and used in the form of external preparations such as powders, granules, sustained-release granules, enteric granules, liquids, eye drops, ellipsoids, emulsions, suspensions, ethanol tablets, troches, fragrances, limonades, tablets, sustained-release tablets, enteric tablets, sublingual tablets, hard capsules, soft capsules, sustained-release capsules, enteric capsules, pills, tinctures, soft extracts, dry extracts, fluid extracts, injections, capsules, irrigation solutions, warning agents, lotions, pastes, sprays, inhalants, patches, sterile injectable solutions, or aerosols, according to conventional methods, and the external preparations may have formulations such as creams, gels, patches, sprays, ointments, warning agents, lotions, liniments, pastes, or cataplasms.

[0134] Carriers, excipients, and diluents that may be included in the pharmaceutical composition according to the present invention include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0135] When formulating, it is prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrants, and surfactants.

[0136] Excipients such as corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium monohydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, refined lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropylmethylcellulose (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, primogel, etc., as additives to tablets, powders, granules, capsules, pills, and lozenges according to the present invention; Gelatin, gum arabic, ethanol, agar powder, cellulose phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium casein, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch paste, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, polyvinylpyrrolidone, etc. may be used as binders, and hydroxypropylmethylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, Calcium carboxymethylcellulose, calcium citrate, sodium lauryl sulfate, anhydrous silica, 1-hydroxypropylcellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethylcellulose, sucrose, magnesium aluminum silicate, D-sorbitol solution, hard anhydrous silica, etc. disintegrants;Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium pods, kaolin, petroleum jelly, sodium stearate, cocoa paste, sodium salicylate, magnesium salicylate, polyethylene glycol 4000, PEG 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, anhydrous silica, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and hard anhydrous silica may be used.

[0137] As additives to the liquid formulation according to the present invention, water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, monostearic acid sucroses, polyoxyethylene sorbitol fatty acid esters (tween esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, water ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethylcellulose, sodium carboxymethylcellulose, etc. may be used.

[0138] In the syrup preparation according to the present invention, a solution of white sugar, other sugars or sweeteners, etc. may be used, and if necessary, flavorings, coloring agents, preservatives, stabilizers, suspending agents, emulsifiers, viscosity enhancers, etc. may be used.

[0139] Purified water may be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

[0140] In the suspension agent according to the present invention, suspending agents such as acacia, tragacanthus, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose, HPMC 1828, HPMC 2906, and HPMC 2910 may be used, and surfactants, preservatives, stabilizers, coloring agents, and fragrances may be used as needed.

[0141] The injectable preparation according to the present invention comprises solvents such as distilled water for injection, 0.9% sodium chloride injection solution, Ringer's injection solution, dextrose injection solution, dextrose + sodium chloride injection solution, PEG, lactated Ringer's injection solution, ethanol, propylene glycol, non-volatile oils—sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; solubilizing agents such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, tween, nijungtinamide, hexamine, and dimethylacetamide; and buffers such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptone, and gums. It may include isotonic agents such as sodium chloride; stabilizers such as sodium bisulfite (NaHSO3), carbon dioxide gas, sodium metabisulfite (Na2S2O5), sodium sulfite (Na2SO3), nitrogen gas (N2), and ethylenediaminetetraacetic acid; sulfating agents such as sodium bisulfide 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetic acid, and sodium bisulfite acetone; non-analging agents such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium CMC, sodium alginate, Tween 80, and aluminum monostearate.

[0142] The suppository according to the present invention contains cocoa dough, lanolin, Witepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, a mixture of stearic acid and oleic acid, Subanal, cottonseed oil, peanut oil, palm oil, cocoa butter + cholesterol, lecithin, lanette wax, glycerol monostearate, Tween or Spandex, Imhausen, monollene (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, Cebes Pharma 16, hexalide base 95, Cotomar, Hydrocote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydrocote Bases such as (Hydrokote) 25, Hydrokote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Masa-MF, Masupol, Masupol-15, Neosupostal-N, Paramount-B, Suposiro (OSI, OSIX, A, B, C, D, H, L), suppository base type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Supostal (N, Es), Wekovi (W, R, S, M, Fs), and Tegestor triglyceride base (TG-95, MA, 57) may be used.

[0143] Solid dosage forms for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid dosage forms are prepared by mixing at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc., with the extract. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used.

[0144] Liquid preparations for oral administration include suspensions, oral liquids, emulsions, and syrups; in addition to commonly used simple diluents such as water and liquid paraffin, they may contain various excipients, such as humectants, sweeteners, flavorings, and preservatives. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0145] The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, a “pharmaceutically effective amount” means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined based on factors including the type and severity of the patient’s disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.

[0146] The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects by considering all the above-mentioned factors, and this can be easily determined by a person skilled in the art to which the present invention belongs.

[0147] The pharmaceutical composition of the present invention may be administered to an individual by various routes. All modes of administration are expected, for example, oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal (intradural) injection, sublingual administration, buccal mucosal administration, rectal insertion, vaginal insertion, ocular administration, ear administration, nasal administration, inhalation, spray through the mouth or nose, skin administration, transdermal administration, etc.

[0148] The pharmaceutical composition of the present invention is determined by the type of active ingredient drug, along with various relevant factors such as the disease to be treated, the route of administration, the patient's age, gender, weight, and the severity of the disease. Specifically, the effective dose of the composition according to the present invention may vary depending on the patient's age, gender, and weight, and generally, 0.001 to 150 mg, preferably 0.01 to 100 mg per kg of body weight, may be administered daily or every other day, or divided into 1 to 3 doses per day. However, since the dose may be increased or decreased depending on the route of administration, the severity of the disease, gender, weight, age, etc., the above dosage does not limit the scope of the present invention in any way.

[0149] In the present invention, the term “individual” refers to a subject requiring treatment for a disease, and more specifically, to mammals such as humans or non-human primates, mice, rats, dogs, cats, horses, and cattle.

[0150] In the present invention, “administration” means providing a specific composition of the present invention to an individual by any appropriate method. In the present invention, “prevention” means any act of suppressing or delaying the onset of a target disease; “treatment” means any act of improving or beneficially altering the target disease and the associated metabolic abnormality symptoms by administering a pharmaceutical composition according to the present invention; and “improvement” means any act of reducing parameters related to the target disease, such as the severity of symptoms, by administering a composition according to the present invention.

[0151] Preferred embodiments are presented below to aid in understanding the present invention. However, the following embodiments are provided merely to facilitate a better understanding of the invention, and the scope of the invention is not limited by the following embodiments.

[0152]

[0153] [Example]

[0154]

[0155] Example 1. Design of Botulinum Toxin Stabilization Liquid Composition

[0156] To prepare a botulinum toxin-stabilized liquid composition with excellent stability, compositions were designed as shown in Tables 1 to 3. Table 1 shows the composition of each composition designed to compare the stability of polysorbate 20 and polysorbate 80. Tables 2 and 3 show the compositions designed to evaluate stability at different concentrations of sucrose and histidine, respectively. In this case, botulinum toxin type A was used.

[0157]

[0158]

[0159]

[0160]

[0161]

[0162]

[0163]

[0164] Example 2. Confirmation of stability according to the composition of a botulinum toxin-stabilizing liquid composition

[0165] Example 2-1. Stability analysis method

[0166] The stability of the 18 types of botulinum toxin-stabilizing liquid compositions designed in Example 1 was analyzed. Stability was evaluated using potency (mLD). 50 Stability was evaluated by potency per vial using the test method, identical to the evaluation method used for actual commercially sold products. Specifically, a liquid botulinum toxin type A preparation was prepared at 40 units / mL, and 3.0 mL of the composition (Table 1) was filled into each vial. 600 μL of physiological saline solution was added to 5 mL of the liquid preparation to prepare a solution of 200 U / 5.6 mL. Next, 4.4 mL of the above-prepared test solution was added to a 15 mL tube containing 1.45 mL of diluent (physiological saline solution). Subsequently, test solutions 1 through 7 were prepared as shown in Table 2 below. During stepwise dilution, the lids of the 15 mL tubes containing the test solution and diluent were closed to ensure thorough mixing. Subsequently, 0.1 mL of the solution was intraperitoneally injected into the lower abdominal cavity of 7 groups of 10 ICR mice each. After the injection, the mortality rate of the mice was observed for 3 days, and the titer (mLD) was calculated using the statistical program (Combistats). 50 ) was calculated (condition 35℃).

[0167]

[0168]

[0169]

[0170] Example 2-2. Results of stability analysis according to polysorbate type

[0171] The results of the stability analysis for compositions #1 to #6 in Table 1 were confirmed as shown in Table 5 below.

[0172]

[0173]

[0174]

[0175] As a result, polysorbate 20 showed a stable effect in the concentration range of 0.005 to 0.02 mg / mL, and in particular, the stability was found to be superior in the range of 0.001 to 0.02 mg / mL. On the other hand, polysorbate 80 showed an appropriate effect as a stabilizer in the range of 0.001 to 0.02 mg / mL, but was found to have poor stability at 0.005 mg / mL.

[0176]

[0177] Example 2-3. Results of stability analysis according to sucrose concentration

[0178] The results of the stability analysis for compositions #7 to #12 in Table 2 were confirmed as shown in Table 6 below.

[0179]

[0180]

[0181]

[0182] As a result, it was found that sucrose had a sufficiently excellent stability effect in the range of 0.2 mg / mL to 10 mg / mL, and in particular, it was confirmed that it further increased the stability of the botulinum toxin stabilizing liquid composition when combined with other components and concentrations in the range of 0.5 mg / mL to 5 mg / mL.

[0183]

[0184] Example 2-4. Results of stability analysis according to histidine concentration

[0185] The results of the stability analysis for compositions #13 to #18 in Table 3 were confirmed as shown in Table 7 below.

[0186]

[0187]

[0188]

[0189] As a result, histidine was found to have a sufficiently excellent stability effect in the range of 0.05 mg / mL to 4 mg / mL, and in particular, it was confirmed to further increase the stability of the botulinum toxin liquid composition when combined with other compositions and concentration conditions in the range of 0.1 mg / mL to 2 mg / mL.

[0190]

[0191] According to Examples 2-2 to 2-4, the botulinum toxin stabilization liquid composition was found to have the best stability under conditions of 0.01 mg / mL to 0.02 mg / mL of polysorbate 20, 0.5 mg / mL to 5 mg / mL of sucrose, and 0.1 mg / mL to 2 mg / mL of histidine.

[0192]

[0193] Example 3. Stability evaluation of DoE-based liquid formulation candidates

[0194] A group of liquid formulation candidates was extracted based on DoE, and a stability evaluation was performed on them. The stability evaluation method was carried out in the same manner as in Example 2-1.

[0195]

[0196] As a result, the results shown in Table 8 below were confirmed.

[0197]

[0198]

[0199]

[0200] According to these results, the liquid compositions of test groups 4, 9, and 13 were confirmed to have the best stability. Since test groups 4, 9, and 13 satisfy all the concentration ranges and weight parts conditions of polysorbate 20, histidine, and sucrose identified in Examples 2-2 to 2-4, the experimental results of Example 3 were found to correspond to the experimental results of Example 2.

[0201]

[0202] As a result, it was confirmed that a composition containing 0.01 mg / mL to 0.02 mg / mL of polysorbate 20, 0.1 mg / mL to 2 mg / mL of histidine, and 0.5 mg / mL to 5 mg / mL of sucrose increases the stability of the botulinum toxin stabilization liquid composition. Furthermore, it was confirmed that a composition containing 0.02 mg / mL of polysorbate 20, 0.1 mg / mL to 2 mg / mL of histidine, and 1 mg / mL of sucrose exhibits the best stabilization effect based on 40 Unit / mL of botulinum toxin.

[0203]

[0204] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0205] The present invention relates to a liquid composition for stabilizing botulinum toxin, and it has been confirmed that a stabilizer comprising a nonionic surfactant, an amino acid, and a disaccharide as active ingredients can significantly increase the stability of botulinum toxin. Specifically, the present invention combines polysorbate 20, histidine, and a disaccharide, and establishes concentration conditions for each component to produce the best stabilizer through the above composition. As such, it is possible to produce a liquid botulinum toxin preparation with low cytotoxicity and increased stability without containing animal-derived materials, and thus it can be utilized in various fields using botulinum toxin, and thus its industrial applicability is recognized.

Claims

1. A liquid composition for stabilizing botulinum toxin, comprising polysorbate 20, histidine, and a disaccharide as active ingredients.

2. In Paragraph 1, A botulinum toxin stabilizing liquid composition in which the above disaccharide is sucrose or lactose.

3. In claim 1 or 2, in the above composition, A botulinum toxin-stabilizing liquid composition having a concentration ratio of polysorbate 20 : histidine : disaccharide of (0.001 to 0.1) : (0.01 to 10) : (0.1 to 10).

4. In claim 1 or 2, the composition is, (1 to 20) parts by weight of polysorbate 20; (1 to 2000) parts by weight of histidine; and A botulinum toxin stabilizing liquid composition comprising (5 to 5000) parts by weight of a disaccharide.

5. In Paragraph 1 or 2, The above composition is a botulinum toxin stabilizing liquid composition that additionally comprises an isotonic agent.

6. In Paragraph 5, A botulinum toxin stabilizing liquid composition wherein the isotonic agent is one or more selected from the group consisting of sodium chloride, glycerin, potassium chloride, and dextrose.

7. In paragraph 5, in the above composition, A liquid composition for stabilizing botulinum toxin, wherein the concentration ratio of polysorbate 20 : histidine : disaccharide : isotonic agent is (0.001 to 0.1) : (0.01 to 10) : (0.1 to 10) : (0.1 to 10).

8. In paragraph 5, the above composition is, (1 to 20) parts by weight of polysorbate 20; (1 to 2000) parts by weight of histidine; (5 to 5000) parts by weight of disaccharide; and A botulinum toxin stabilizing liquid composition comprising (1 to 9000) parts by weight of an isotonic agent.

9. In paragraph 5, the above composition is, Polysorbate 20 of 0.005 mg / mL to 0.02 mg / mL; 0.05 mg / mL to 4 mg / mL of histidine; 0.2 mg / mL to 10 mg / mL of disaccharide; and A botulinum toxin stabilizing liquid composition comprising an isotonic agent in an amount of 1 mg / mL to 20 mg / mL.

10. In Paragraph 1 or 2, A botulinum toxin stabilization liquid composition wherein the botulinum toxin is one or more selected from the group consisting of botulinum toxins A, B, C, D, E, F, and G.

11. A botulinum toxin stabilizing liquid composition of claim 1 or 2, and a botulinum toxin liquid composition comprising botulinum toxin as an active ingredient.

12. In Paragraph 11, A liquid botulinum toxin composition wherein the above botulinum toxin is one or more selected from the group consisting of botulinum toxins A, B, C, D, E, F, and G.

13. In Paragraph 11 or 12, A botulinum toxin liquid composition comprising 1 to 500 Unit / mL of botulinum toxin based on the total amount of the composition.

14. Use of the liquid composition for stabilizing botulinum toxin according to claim 1 for stabilizing botulinum toxin.

15. Use of the botulinum toxin stabilizing liquid composition of claim 1 for manufacturing a botulinum toxin liquid stabilizing preparation.

16. The botulinum toxin stabilizing liquid composition of claim 1, and a botulinum toxin liquid pharmaceutical composition for the treatment of neurological diseases or cosmetic treatment, comprising botulinum toxin as an active ingredient.

17. A method for treating neurological diseases or cosmetic treatments comprising the step of administering the botulinum toxin-stabilizing liquid composition of claim 1 and a composition containing botulinum toxin as an active ingredient to an individual in need of the same in a pharmaceutically effective amount.

18. Use of the botulinum toxin-stabilizing liquid composition of claim 1, and the composition comprising botulinum toxin as an active ingredient for the treatment of neurological diseases or cosmetic treatment.

19. Use for manufacturing a preparation for the treatment of neurological diseases or cosmetic treatment of the botulinum toxin-stabilizing liquid composition of claim 1 and a composition containing botulinum toxin as an active ingredient.

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