Botulinum toxin formulations with reduced tolerance expression and methods related thereto

Abstract

Provided are a botulinum toxin preparation containing a botulinum toxin in an amount corresponding to a desired potency, a method for producing the same, a cosmetic method or therapeutic method using the same, and a method for reducing the immunogenicity of the botulinum toxin preparation. Thus, the formulation according to the present invention has low immunogenicity and can exhibit low tolerance expression when administered to a subject.

Description

Technical Field

[0001] The present disclosure relates to a botulinum toxin preparation and related methods, and more particularly, to a botulinum toxin preparation, a method for producing the same, a cosmetic method or a therapeutic method using the same, and a method for reducing the immunogenicity of a botulinum toxin preparation using the same, wherein the botulinum toxin preparation contains an amount of botulinum toxin corresponding to a desired efficacy. Background Art

[0002] Generally, it is not preferred to compensate for decomposition by overdosing the drug substance during the manufacturing process or product storage. Whether or not it is included in the final formulated product, for the finished drug product manufacturing process, the safety and efficacy of the product need to be considered to justify the overdosing. Information should be provided to justify the amount of overdosing, the reason for overdosing (e.g., to compensate for expected and recorded manufacturing losses), and the rationality of overdosing. Overdosing should be included in the amount of the drug substance listed in the batch formula.

[0003] If overdosing occurs during the manufacturing process, the finished product will contain more protein than the final target amount. This means that it contains more protein than the amount required for the final product. In this case, in addition to the active protein contained in the finished product administered to the patient, inactive protein is also administered together. Generally, exogenous proteins act as immunogens when administered to the body, thereby causing an immune response and producing neutralizing antibodies, thereby removing the protein from the body. In this regard, the protein that is overdosed due to overdosing acts as an immunogen in the body and produces neutralizing antibodies, resulting in a situation where the appropriate efficacy cannot be demonstrated. In particular, most botulinum toxins require regular repeated administration, so the subjects who are administered botulinum toxin are continuously exposed to botulinum toxin. Therefore, in order to reduce the role of botulinum toxin preparations as immunogens, it is important to minimize overdosing when manufacturing the finished product for formulation.

[0004] Botulinum neurotoxin (BoNT) is a polypeptide product of the anaerobic bacterium Clostridium botulinum, which paralyzes muscles by blocking the secretion of neurotransmitters at the junction of nerves and muscles. Botulinum toxin was originally a deadly toxic substance. It was initially used to treat diseases caused by excessive muscle contraction, such as strabismus, and its scope of use has been continuously expanded. It is currently used for a variety of purposes. Botulinum toxin is mainly used to treat dystonia or other dystonia movement diseases such as hemifacial spasm, tremor, spasticity, etc. In addition, it is also used for various indications such as eye diseases, pain-related diseases such as headaches, urinary system diseases, and wrinkle treatment for cosmetic purposes.

[0005] The manufacturing (formulation) process of a botulinum toxin pharmaceutical composition generally requires an overdose of 50% or more. An overdose of 50% of botulinum toxin in the manufacturing process of a botulinum toxin pharmaceutical composition means that a manufacturing process starting with 1.5 times the amount (i.e., 150 units) of botulinum toxin produces a botulinum pharmaceutical composition having 1 times the amount (e.g., 100 units) of botulinum toxin. The manufacturing process or formulation process is to dilute, mix, and process botulinum toxin obtained by bacterial fermentation (referred to as bulk or raw toxin) to make a botulinum toxin pharmaceutical composition suitable for administration to humans for treatment and / or beauty. Therefore, it is known that 50% or more of the potency of botulinum toxin is lost in the formulation process for unknown reasons, and an overdose of 50% or more is required due to the denaturation and or loss of botulinum toxin in the formulation process.

[0006] International Publication No. WO2015-089452 relates to the use of a botulinum toxin composition that does not contain animal protein in the preparation of a medicament for treating a disease, disorder, or a patient in need of the above treatment, and specifically discloses that the effect of botulinum toxin is prolonged by comparing a botulinum toxin composition that does not contain animal protein with a botulinum toxin composition that contains animal protein. However, it does not disclose or suggest overdosing of botulinum toxin.

[0007] International Publication No. WO2007-016018 provides a pharmaceutical composition that does not require excessive dosage of botulinum toxin by optimizing the ratio of excipients and performing a vacuum drying process under appropriate conditions. Specifically, the disclosed pharmaceutical composition includes botulinum toxin, a first excipient (albumin) and a second excipient (e.g., sodium chloride), and the ratio of the first excipient to the second excipient in the pharmaceutical composition is greater than 0.6 and less than about 100. In particular, The HSA (human serum albumin):NaCl weight ratio of 0.6 increased 47-fold to 28. However, this only applies to preparations containing the animal protein albumin. Summary of the invention

[0008] Technical issues

[0009] An object of the present disclosure is to provide a botulinum toxin preparation that is free of albumin and contains an amount of botulinum toxin corresponding to a desired potency.

[0010] One object of the present disclosure is to provide a method for producing the botulinum toxin preparation.

[0011] An object of the present disclosure is to provide a method for reducing the potency loss of botulinum toxin during the manufacturing process using the botulinum toxin preparation.

[0012] An object of the present disclosure is to provide a method for treating various diseases or improving wrinkles, etc. by administering the botulinum toxin preparation to a subject.

[0013] An object of the present disclosure is to provide a method for reducing the immunogenicity of a botulinum toxin preparation by administering the botulinum toxin preparation to a subject.

[0014] Technical Solution

[0015] One aspect of the present disclosure relates to a botulinum toxin preparation,

[0016] It contains no animal protein.

[0017] It contains an amount of botulinum toxin corresponding to the desired potency.

[0018] Among them, the amount corresponding to the desired efficacy includes

[0019] Less than 50% overdosage.

[0020] In one embodiment, the formulation includes a botulinum toxin overdose that is less than the botulinum toxin overdose of botulinum toxin formulations containing animal protein.

[0021] In one embodiment, the botulinum toxin formulation can be a lyophilized formulation or a liquid formulation.

[0022] In one embodiment, the lyophilized formulation may include botulinum toxin, a surfactant, an amino acid, and one or more components selected from the group consisting of sugars, sugar alcohols, and ionic compounds. For example, the amino acid may include methionine. For example, the surfactant may include one or more of polysorbates and poloxamers.

[0023] In one embodiment, the liquid formulation may include botulinum toxin, a surfactant, and an amino acid. For example, the amino acid may include methionine. For example, the surfactant may include one or more of polysorbate and poloxamer.

[0024] In one embodiment, the liquid formulation may include 0.05 mg / ml to 50 mg / ml of a surfactant and 0.05 mg / ml to 8 mg / ml of an amino acid.

[0025] In one embodiment, the liquid formulation may include botulinum toxin, a surfactant, and an amino acid.

[0026] In one embodiment, the surfactant may include one or more of polysorbates and poloxamers.

[0027] In one embodiment, the botulinum toxin is selected from the group consisting of botulinum toxin serotypes A, B, C, D, E, F, and G. For example, the botulinum toxin can be botulinum toxin serotype A.

[0028] In one embodiment, a botulinum toxin formulation can be used to improve wrinkles, square chins, pointy chins, wounds, dermatomas, scars, acne, pores, elasticity, or keloids, or to treat facial spasms, blepharospasm, torticollis, blepharospasm, cervical dystonia, oropharyngeal dystonia, spasmodic dysphonia, migraines, anal pruritus, or hyperhidrosis.

[0029] Another aspect of the present disclosure relates to a method for producing a botulinum toxin preparation, comprising obtaining the botulinum toxin preparation by mixing a botulinum toxin and a pharmaceutically acceptable excipient,

[0030] Wherein, the pharmaceutically acceptable excipient does not contain albumin,

[0031] Botulinum toxin is used in an amount corresponding to the desired efficacy.

[0032] Among them, the amount corresponding to the desired efficacy includes

[0033] Less than 50% overdosage.

[0034] In one embodiment, the step of mixing the excipients may include mixing 0.05 mg / ml to 50 mg / ml of a surfactant and 0.05 mg / ml to 8 mg / ml of an amino acid.

[0035] Another aspect of the present disclosure relates to a method for reducing the potency loss of a botulinum toxin during a manufacturing process, comprising obtaining the botulinum toxin preparation.

[0036] Another aspect of the present disclosure relates to a method of reducing the immunogenicity of a botulinum toxin formulation comprising administering a botulinum toxin formulation according to the present invention to a subject in need thereof.

[0037] Another aspect of the present disclosure relates to a method for improving wrinkles, square chin, pointed chin, wounds, skin softening, scars, acne, pores, elasticity or keloids or for treating facial spasm, blepharospasm, torticollis, blepharospasm, cervical dystonia, oropharyngeal dystonia, spasmodic dysphonia, migraine, anal pruritus or hyperhidrosis, comprising administering a botulinum toxin formulation according to the present invention to a subject in need thereof.

[0038] Another aspect of the present disclosure relates to use of an effective amount of a botulinum toxin preparation according to the present invention in the manufacture of a medicament for improving wrinkles, square chin, pointed chin, wounds, skin softening, scars, acne, pores, elasticity or keloids, or for treating facial spasm, blepharospasm, torticollis, blepharospasm, cervical dystonia, oropharyngeal dystonia, spasmodic dysphonia, migraine, anal pruritus or hyperhidrosis.

[0039] Beneficial Effects

[0040] The present disclosure provides a botulinum toxin preparation, a method for producing the same, a method for reducing the loss of potency of botulinum toxin during the production process, a cosmetic or therapeutic method using the same, and a method for reducing the immunogenicity of a botulinum toxin preparation using the same, wherein the botulinum toxin preparation contains an amount of botulinum toxin corresponding to a desired potency. DETAILED DESCRIPTION

[0041] The meanings of technical and scientific terms used in this specification are the same as those commonly used in the art, unless otherwise defined. To help understand the present disclosure, the following definitions apply, and terms expressed in the singular include plural expressions, and vice versa.

[0042] The terms "first", "second" and the like may be used to describe various components, but the above components should not be limited by the above terms. The above terms are only used to distinguish one component from another. For example, without departing from the scope of the present invention, the first component may be named as the second component, and similarly, the second component may also be named as the first component.

[0043] The term "and / or" includes all definable combinations of the associated constituents.

[0044] As used herein, the term "botulinum toxin" refers to a botulinum toxin protein molecule produced bacterially or recombinantly, including all serotypes and variants or fusion proteins thereof.

[0045] In one embodiment, the use of botulinum toxin may not be limited by serotype, and may be selected from the group consisting of type A (BoNT / A), type B (BoNT / B), type C (BoNT / C), type D (BoNT / D), type E (BoNT / E), type F (BoNT / F), type G (BoNT / G), type H (BoNT / H), type X (BoNT / X), enterococcal botulinum toxin J (eBoNT / J), and mosaic botulinum toxin (Mosaic Botulinum Toxin) and / or variants thereof. Examples of mosaic botulinum toxins include BoNT / DC, BoNT / CD, and BoNT / FA. For example, type A may be used.

[0046] In one approach, the botulinum toxin can be derived from various BoNT / A subtypes, such as A1, A2, A3, A4, A5, A6, A7, A9, A10; BoNT / B subtypes, such as B1, B2, B3, B4, B5, B6, B7, B8, Bnp, and Bbv; BoNT / C subtypes, such as C and CD; BoNT / D subtypes, such as D and DC; BoNT / E subtypes, such as E1, E2, E3, E4, E5, E6, E7, E8, E9; BoNT / F subtypes, such as F1, F2, F3, F4, F5, F6, F7; and BoNT / G subtypes, such as G subtype. BoNT subtypes include chimeric BoNTs, such as BoNT / DC, BoNT / CD, BoNT / FA, etc.

[0047] Botulinum toxin includes botulinum toxin derivatives. Botulinum toxin derivatives can include at least one chemical or functional modification on a portion or partial chain of a natural botulinum toxin or a recombinant prototype (recombinant native) botulinum toxin with botulinum toxin activity. For example, a botulinum toxin derivative can be a modified toxin with at least one amino acid that is missing, modified or substituted. Botulinum toxin can be a recombinant peptide, a fusion protein, or a hybrid neurotoxin (e.g., made of subunits or domains of different toxin serotypes). Botulinum toxin can also be part of an integral molecule with toxin activity, and can be used as a combined molecule or conjugated molecule, for example, a part of a fusion protein.

[0048] The term "complex protein" refers to hemagglutinin and a nontoxic component (nontoxic nonhemagglutinin; NTNH) that does not have hemagglutinin activity. Clostridium botulinum strains produce immunologically different neurotoxins (AG type). The molecular weight of AG type neurotoxin (NTX) is about 150kDa (7S). In culture media and foods under specific acidic conditions, NTX forms a larger complex called PTX (progenitor toxins) by combining with nontoxic components. PTX can have molecular weights of 900kDa (19S), 500kDa (16S) and 300kDa (12S). 12S toxin consists of NTX and a nontoxic component (NTNH) that does not have hemagglutinin activity, and 19S and 16S toxins consist of NTX, NTNH and hemagglutinin. Type A strains produce three forms of toxins (19S, 16S and 12S), and type B, type C and type D strains produce 16S and 12S toxins. Under alkaline conditions, PTX dissociates into NTX and non-toxic components, 19S and 16S toxins dissociate into NTX and NTNH and the non-toxic component of hemagglutinin, and 12S toxin dissociates into NTX and NTNH.

[0049] The botulinum toxin composition of the present disclosure may be a complex botulinum toxin composition containing a complex protein. For example, the complex botulinum toxin may contain NTNH or hemagglutinin, and the molecular weight of the complex botulinum toxin may be about 900 kDa or 500 kDa.

[0050] The botulinum toxin composition of the present disclosure may be a non-complexed botulinum toxin composition that does not contain complexing proteins. For example, the non-complexed botulinum toxin may not contain NTNH or hemagglutinin, and the molecular weight of the non-complexed botulinum toxin may be about 150 kDa.

[0051] The term "animal protein" refers to products derived from, mixed with blood or derived from other animals.

[0052] The botulinum toxin composition of the present disclosure may be a botulinum toxin composition that does not contain animal protein. For example, the botulinum toxin composition of the present disclosure may not contain an animal-derived protein stabilizer. In one embodiment, the botulinum toxin composition of the present disclosure may not include albumin, such as human serum albumin or recombinant human albumin.

[0053] The term "overdosing" refers to the excessive amount of toxins added to compensate for the loss of active ingredients in the finished drug, as well as the excessive amount of toxins added to compensate for the loss of active ingredients during the manufacturing process or product storage. Therefore, "overdosing" is when the initial toxin input amount is calculated based on the target toxin potency and the final potency is confirmed by the potency determination method after production. If the final potency is lower than the target potency due to the inactivation of toxin proteins produced in the process, the initial toxin input amount is overdosed to compensate for the inactivation of toxin proteins in the process. Therefore, the overdosing can be calculated as follows.

[0054] Excess dosing (%) = ((100 / efficiency recovery rate)-1) × 100)

[0055] In the present specification, potency can be measured by a mouse potency assay or a potency assay by a method that shows substantially equivalent results thereto (eg, a cell-based potency assay).

[0056] As used herein, the term "unit", "unit(s)" or "U" refers to LD 50 dose, which can be used interchangeably in one product, LD 50 The dose was defined as the amount of botulinum toxin that killed 50% of the mice injected with botulinum toxin.

[0057] In the present specification, the term "an amount corresponding to the desired efficacy" may be meant to include an overdosage significantly lower than the amount conventionally required for the manufacturing process, for example, an overdosage of less than 50%.

[0058] In the present specification, "excluding overdosage" means minimizing the overdosage routinely required in the manufacturing process of a botulinum toxin preparation, for example, less than 50%, less than or equal to 45%, less than or equal to 40%, less than or equal to 35%, less than or equal to 30%, less than or equal to 25%, less than or equal to 20%, less than or equal to 10%, less than or equal to 5%, less than or equal to 3%, less than or equal to 1%, or less than or equal to 0.1%.

[0059] In the present specification, the difference between the botulinum toxin overdose (%) of the botulinum toxin preparation containing animal protein and the botulinum toxin overdose (%) of the preparation in one embodiment is the value of subtracting the botulinum toxin overdose (%) of the preparation in one embodiment from the botulinum toxin overdose (%) of the botulinum toxin preparation containing animal protein, and can be, for example, greater than or equal to 10, greater than or equal to 15, greater than or equal to 20, greater than or equal to 25, greater than or equal to 30, greater than or equal to 35, greater than or equal to 40, greater than or equal to 45, greater than or equal to 50, greater than or equal to 55, greater than or equal to 60, greater than or equal to 65, greater than or equal to 70, greater than or equal to 75, greater than or equal to 80, greater than or equal to 85, or greater than or equal to 90.

[0060] "Pharmaceutical composition" refers to a preparation containing an active ingredient. The term "preparation" refers to the presence of at least one additional ingredient in the pharmaceutical composition in addition to the active ingredient (e.g., botulinum toxin), such as albumin (e.g., human serum albumin or recombinant human albumin) and / or sodium chloride. A pharmaceutical composition is a preparation suitable for administration to a subject, such as a human patient. The pharmaceutical composition may be in a lyophilized form, such as a solution formed after reconstitution of a lyophilized pharmaceutical composition using saline or water, or in the form of a solution without reconstitution. The pharmaceutical composition may be a liquid or a solid. The pharmaceutical composition may be free of animal-derived protein and / or albumin.

[0061] The term "active ingredient" refers to a substance in a biologically or physiologically effective preparation or composition. In particular, in the context of a pharmaceutical preparation or composition, the term "active ingredient" refers to a component substance that exhibits the target pharmaceutical effect. For example, the active ingredient of the present disclosure can be a botulinum toxin without a complexing protein.

[0062] "Administering" or "administered" refers to the step of providing a pharmaceutical composition or active ingredient to a subject. The pharmaceutical composition can be administered by various appropriate routes.

[0063] In one embodiment, the pharmaceutical composition can be administered transdermally, subcutaneously or intramuscularly. In one embodiment, the composition can be applied topically to a muscle or muscle group. For example, forehead wrinkles and skin wrinkles can be reduced by applying the composition transdermally or subcutaneously to the wrinkles.

[0064] "Treatment", "therapeutic" or "treatment" means to alleviate or reduce (including partial reduction, significant reduction, almost complete reduction, and complete reduction), resolve or prevent (temporarily or permanently) a disease, disorder or abnormality, such as by healing wounds or damaged tissues or changing, altering, strengthening, improving, modifying and / or beautifying an existing or known disease, disorder or abnormality, to achieve the desired therapeutic result. In this specification, "treatment" is a concept that includes prevention. "Prevention" means delaying the occurrence of a disease, disorder or condition. If the occurrence of a disease, disorder or condition is delayed within the expected time, prevention can be considered to have been completed.

[0065] In one embodiment, "treatment" refers to the treatment of a disease, disorder or medical condition in a mammal (especially a human) patient, including one or more of the following:

[0066] (a) preventing the onset of the disease, disorder or medical condition, i.e. preventing the recurrence of the disease or medical condition, or prophylactic treatment of a patient susceptible to the disease or medical condition;

[0067] (b) ameliorating the disease, disorder or medical condition, i.e., eliminating or regressing the disease, disorder or medical condition in a patient, including antagonizing the effects of other therapeutic agents;

[0068] (c) inhibiting the disease, disorder or medical condition, i.e., slowing or delaying the development of the disease, disorder or medical condition in a patient; or

[0069] (d) alleviating the symptoms of the disease, disorder or medical condition in a patient.

[0070] In one embodiment, the therapeutically effective amount of botulinum toxin is about 0.01 U / kg to about 100 U / kg, about 0.1 U / kg to about 100 U / kg, about 0.2 U / kg to about 100 U / kg, about 0.2 U / kg to about 50 U / kg, about 0.2 U / kg to about 30 U / kg, about 0.2 U / kg to about 10 U / kg, about 0.2 U / kg to about 1 U / kg. In another embodiment, based on an adult weighing 60 kg, the therapeutically effective amount of botulinum toxin is about 1 U to about 10000 U, about 1 U to about 5000 U, about 1 U to about 2500 U, about 1 U to about 1000 U, about 1 U to about 500 U, about 1 U to about 300 U, about 1 U to about 200 U, about 10 U to about 200 U, about 10 U to about 100 U, about 10 U to about 50 U.

[0071] According to one aspect of the present disclosure, a botulinum toxin preparation is provided.

[0072] It contains no animal protein.

[0073] It contains an amount of botulinum toxin corresponding to the desired potency.

[0074] Among them, the amount corresponding to the desired efficacy includes

[0075] Less than 50% overdosage.

[0076] In one embodiment, the overdose of the botulinum toxin formulation can be less than 50%, less than or equal to 45%, less than or equal to 40%, less than or equal to 35%, less than or equal to 30%, less than or equal to 25%, less than or equal to 20%, less than or equal to 15%, less than or equal to 10%, less than or equal to 5%, less than or equal to 3%, less than or equal to 1%, or less than or equal to 0.1%.

[0077] In one embodiment, the botulinum toxin formulation does not include overdosing.

[0078] In one embodiment, the difference between the botulinum toxin overdose (%) of the botulinum toxin formulation containing animal protein and the botulinum toxin overdose (%) in the formulation according to one embodiment can be greater than or equal to 40, greater than or equal to 45, greater than or equal to 50, greater than or equal to 55, greater than or equal to 60, greater than or equal to 65, greater than or equal to 70, greater than or equal to 75, greater than or equal to 80, greater than or equal to 85, or greater than or equal to 90.

[0079] In one embodiment, the amount of botulinum toxin included in the botulinum toxin formulation can be 50% to 90%, 50% to 85%, 50% to 80%, 50% to 75%, 50% to 70%, 50% to 65%, 50% to 60%, 50% to 55%, 60% to 90%, 60% to 85%, 60% to 80%, 60% to 75%, 60% to 70%, or 60% to 65% of the amount of botulinum toxin included in the botulinum toxin formulation containing animal source protein.

[0080] That is, the preparation according to an embodiment may include a smaller amount of botulinum toxin than a botulinum toxin preparation containing an animal-derived protein.

[0081] Compared to existing preparations, the overdosage of toxins in the preparation according to one embodiment is reduced, and thus includes less toxins (active toxins and inactive toxins) in absolute amounts. Therefore, the overdosage of proteins that become immunogens when the preparation according to one embodiment is administered to a subject can be reduced by 50% or more, so that immunogenicity is reduced, and the generation of neutralizing antibodies can be suppressed or reduced by suppressing the immune response in the subject. Therefore, in a subject administered with a preparation according to one embodiment, the expression of resistance to botulinum toxin is reduced.

[0082] In one embodiment, the botulinum toxin formulation may contain a pharmaceutically acceptable excipient or additive, and the pharmaceutically acceptable excipient or additive may include a stabilizer (eg, amino acid), an ionic compound, a surfactant, a buffer, a lyoprotectant, or a combination thereof.

[0083] The surfactant can be a nonionic surfactant, such as an ethylene oxide surfactant, an alkylene oxide surfactant (e.g., a propylene oxide surfactant), for example, polyoxyethylene sorbitan fatty acid esters and / or polyoxyethylene-polyoxypropylene block copolymers, such as poloxamer and / or polysorbate.

[0084] For example, it can be an amino acid (e.g., methionine, arginine, alanine, glutamic acid, aspartic acid, arginine, glycine and / or cysteine, such as methionine), a salt (e.g., NaCl), a buffer, a non-ionic surfactant (e.g., a poloxamer and / or a polysorbate, such as polysorbate 20 and / or polysorbate 80), a sugar (e.g., a disaccharide such as sucrose), a sugar alcohol (e.g., sorbitol), or a combination thereof.

[0085] For example, pharmaceutically acceptable excipients or additives may be surfactants (eg, nonionic surfactants) and amino acids.

[0086] In one embodiment, the lyophilized preparation may include botulinum toxin, a surfactant, an amino acid, and one or more components selected from the group consisting of sugars, sugar alcohols, and ionic compounds. For example, the surfactant may include polysorbate and / or poloxamer, and the amino acid may include methionine.

[0087] In one embodiment, the lyophilized formulation may include a predetermined amount of a pharmaceutically acceptable excipient or additive.

[0088] In the present specification, the content of excipients or additives included in the lyophilized preparation is based on the content in the liquid phase before the lyophilization process.

[0089] In one embodiment, the lyophilized preparation may include 0.01 mg / ml to 100 mg / ml, 0.01 mg / ml to 80 mg / ml, 0.01 mg / ml to 50 mg / ml, 0.03 mg / ml to 100 mg / ml, 0.03 mg / ml to 80 mg / ml, 0.03 mg / ml to 80 mg / ml, 0.05 mg / ml to 100 mg / ml, 0.05 mg / ml to 80 mg / ml or 0.05 mg / ml to 50 mg / ml of a surfactant (a non-ionic surfactant, for example, an ethylene oxide surfactant or a propylene oxide surfactant, such as an alkylene oxide surfactant), for example, polyoxyethylene sorbitan fatty acid esters and / or polyoxyethylene-polyoxypropylene block copolymers, for example, poloxamers and / or polysorbates). In one embodiment, the lyophilized formulation may include 0.01 mg / ml to 30 mg / ml, 0.01 mg / ml to 20 mg / ml, 0.01 mg / ml to 15 mg / ml, 0.01 mg / ml to 10 mg / ml, 0.03 mg / ml to 30 mg / ml, 0.03 mg / ml to 20 mg / ml, 0.03 mg / ml to 15 mg / ml, 0.03 mg / ml to 10 mg / ml, 0.05 mg / ml to 30 mg / ml, 0.05 mg / ml to 20 mg / ml, 0.05 mg / ml to 15 mg / ml, 0.05 mg / ml to 10 mg / ml, 0.05 mg / ml to 8 mg / ml, 0.05 mg / ml to 3 mg / ml of amino acids (e.g., methionine, arginine, alanine, glutamic acid, aspartic acid, arginine, glycine and / or cysteine, such as methionine). In one embodiment, the lyophilized formulation may include 0.05 mg / ml to 50 mg / ml of a surfactant and 0.05 mg / ml to 8 mg / ml of an amino acid. In one embodiment, the lyophilized formulation may include 0.05 mg / ml to 50 mg / ml of a polysorbate or poloxamer and 0.05 mg / ml to 8 mg / ml of an amino acid.

[0090] In one embodiment, the lyophilized formulation may include 0.1 mg / ml to 50 mg / ml, 0.5 mg / ml to 50 mg / ml, 1 mg / ml to 50 mg / ml, 1 mg / ml to 40 mg / ml, 0.1 mg / ml to 30 mg / ml, 0.1 mg / ml to 20 mg / ml, 0.1 mg / ml to 15 mg / ml, 0.1 mg / ml to 10 mg / ml, 0.5 mg / ml to 30 mg / ml, 0.5 mg / ml to 20 mg / ml, 3 mg / ml to 15 mg / ml, 3 mg / ml to 20 mg / ml, or 5 mg / ml to 15 mg / ml of sodium chloride.

[0091] In one embodiment, the lyophilized formulation may include sucrose at 1 mg / ml to 100 mg / ml, 1 mg / ml to 90 mg / ml, 1 mg / ml to 80 mg / ml, 1 mg / ml to 70 mg / ml, 1 mg / ml to 60 mg / ml, 1 mg / ml to 50 mg / ml, 3 mg / ml to 50 mg / ml, 5 mg / ml to 50 mg / ml, 7 mg / ml to 50 mg / ml, 10 mg / ml to 50 mg / ml, 10 mg / ml to 40 mg / ml or 20 mg / ml to 40 mg / ml.

[0092] In one embodiment, the liquid formulation may include botulinum toxin, polysorbate, and amino acid. For example, the surfactant may include polysorbate and / or poloxamer, and the amino acid may include methionine.

[0093] In one embodiment, the liquid preparation may include 0.01 mg / ml to 100 mg / ml, 0.01 mg / ml to 80 mg / ml, 0.01 mg / ml to 50 mg / ml, 0.03 mg / ml to 100 mg / ml, 0.03 mg / ml to 80 mg / ml, 0.03 mg / ml to 80 mg / ml, 0.05 mg / ml to 100 mg / ml, 0.05 mg / ml to 80 mg / ml or 0.05 mg / ml to 50 mg / ml of a surfactant (e.g., a nonionic surfactant, for example, an ethylene oxide surfactant or a propylene oxide surfactant, such as an alkylene oxide surfactant), such as a polyoxyethylene sorbitan fatty acid ester and / or a polyoxyethylene-polyoxypropylene block copolymer, for example, a poloxamer and / or a polysorbate).

[0094] In one embodiment, the liquid formulation may include 0.01 mg / ml to 30 mg / ml, 0.01 mg / ml to 20 mg / ml, 0.01 mg / ml to 15 mg / ml, 0.01 mg / ml to 10 mg / ml, 0.03 mg / ml to 30 mg / ml, 0.03 mg / ml to 20 mg / ml, 0.03 mg / ml to 15 mg / ml, 0.03 mg / ml to 10 mg / ml, 0.05 mg / ml to 30 mg / ml, 0.05 mg / ml to 20 mg / ml, 0.05 mg / ml to 15 mg / ml, 0.05 mg / ml to 10 mg / ml, 0.05 mg / ml to 8 mg / ml, 0.05 mg / ml to 3 mg / ml of an amino acid (e.g., methionine, arginine, alanine, glutamic acid, aspartic acid, arginine, glycine and / or cysteine, such as methionine). In one embodiment, the liquid preparation may include 0.05mg / ml to 50mg / ml of a surfactant and 0.05mg / ml to 8mg / ml of an amino acid. In one embodiment, the liquid preparation may include 0.05mg / ml to 50mg / ml of a polysorbate or poloxamer and 0.05mg / ml to 8mg / ml of an amino acid.

[0095] In one embodiment, the liquid formulation may include 0.1 mg / ml to 50 mg / ml, 0.5 mg / ml to 50 mg / ml, 1 mg / ml to 50 mg / ml, 1 mg / ml to 40 mg / ml, 0.1 mg / ml to 30 mg / ml, 0.1 mg / ml to 20 mg / ml, 0.1 mg / ml to 15 mg / ml, 0.1 mg / ml to 10 mg / ml, 0.5 mg / ml to 30 mg / ml, 0.5 mg / ml to 20 mg / ml, 3 mg / ml to 15 mg / ml, 3 mg / ml to 20 mg / ml or 5 mg / ml to 15 mg / ml of sodium chloride.

[0096] The botulinum toxin preparations of the present disclosure may be formulated into any form, such as a solid preparation or a liquid preparation, such as a lyophilized powder, a liquid, or a pre-filled syringe preparation, etc.

[0097] In one embodiment, the botulinum toxin formulation is a lyophilized formulation or a liquid formulation.

[0098] In one embodiment, the botulinum toxin is selected from the group consisting of botulinum toxin serotypes A, B, C, D, E, F, and G. For example, the botulinum toxin is botulinum toxin serotype A.

[0099] In one embodiment, a botulinum toxin formulation is used to improve wrinkles, square chin, pointed chin, wounds, dermatomas, scars, acne, pores, elasticity, or keloids, or to treat facial spasm, blepharospasm, torticollis, blepharospasm, cervical dystonia, oropharyngeal dystonia, spasmodic dysphonia, migraine, anal pruritus, or hyperhidrosis.

[0100] The following is a description of a method related to a preparation of an embodiment. The above description of the preparation is also applicable to the following description of the method related to the preparation, as long as they are not contradictory.

[0101] According to one embodiment, a method for producing a botulinum toxin preparation is provided, comprising:

[0102] The botulinum toxin preparation is obtained by mixing botulinum toxin and a pharmaceutically acceptable excipient,

[0103] Wherein, the pharmaceutically acceptable excipient does not contain albumin,

[0104] Botulinum toxin is used in an amount corresponding to the desired efficacy.

[0105] Among them, the amount corresponding to the desired efficacy includes

[0106] Less than 50% overdosage.

[0107] In one embodiment of the method, the difference between the botulinum toxin overdose (%) of the botulinum toxin preparation containing animal protein and the botulinum toxin overdose (%) in the preparation can be greater than or equal to 40.

[0108] In the method of one embodiment, the pharmaceutically acceptable excipients may include a surfactant and an amino acid.

[0109] In the method of one embodiment, the step of mixing the excipients may include mixing 0.05 mg / ml to 50 mg / ml of a surfactant and 0.05 mg / ml to 8 mg / ml of an amino acid.

[0110] Different from this, a method for reducing the potency loss of botulinum toxin during a manufacturing process is provided, which comprises obtaining the botulinum toxin preparation.

[0111] According to another aspect, a method for improving or treating a disease is provided, comprising administering an effective amount of the botulinum toxin preparation to a subject in need thereof.

[0112] In contrast, a method for reducing the immunogenicity of a botulinum toxin preparation is provided, comprising administering an effective amount of the botulinum toxin preparation to a subject in need thereof.

[0113] In another aspect, there is provided a use of an effective amount of the botulinum toxin preparation in the manufacture of a medicament for treatment.

[0114] In one embodiment, botulinum toxin can be administered in a single or multiple treatment courses. Taking dosage as an example, it can be administered to the injection site in a single or divided injection. In multiple treatment courses, botulinum toxin can be administered at intervals of 6 months, 4 months, or 3 months or shorter. In multiple treatment courses, the administration interval of botulinum toxin includes the first treatment and the second treatment, and the administration amount of the second treatment can be less than, more than, or the same as the administration amount of the first treatment.

[0115] The dosage, administration time, administration method, administration period or interval of botulinum toxin can be appropriately selected by a person skilled in the art according to the patient's weight, age, gender, health condition, disease severity, etc.

[0116] The subject to which the botulinum toxin or the composition containing the botulinum toxin is administered may include, without limitation, humans or animals, such as humans, pigs, dogs, cats, cows, horses, rats, and the like.

[0117] The present disclosure is described in detail below through examples, but the examples are only used to describe the present disclosure and are not intended to limit the scope thereof in any form.

[0118] Example: Manufacture of lyophilized and liquid formulations of botulinum toxin with minimal potency loss during manufacturing

[0119] Potency assay, mouse LD 50

[0120] The efficacy was determined as follows. For freeze-dried preparations, 2.8 mL of saline was added to two bottles of samples and dissolved (5.6 mL in total). 0.6 mL of saline was added thereto to make an initial dilution of 5.6 mL. Afterwards, the dilution method was as follows. 4.4 mL was taken from each initial dilution and 1.45 mL of saline was added, and the liquid was used as test solution 1. 4.4 mL of test solution 1 was added to 1.45 mL of saline, and the liquid was used as test solution 2. The same method was further diluted 8 times and used as each test solution. For mice (CD1, female) weighing 17 g to 22 g, 0.1 mL of each test solution of test solution 3 to test solution 6 was injected intraperitoneally for every 10 mice, and the mortality rate was measured three days later, and the efficacy was calculated after statistics using the Combistats method.

[0121] Control group preparation: botulinum toxin A complex with sodium chloride (NaCl, 0.9%), human serum albumin (HSA, 0.5%) lyophilized preparation

[0122] 100 units of botulinum toxin A complex (1000 units / mL), to make 100 units of toxin preparation, 191 units (91% overdose) of toxin were added and made into a pharmaceutical composition containing sodium chloride (9.0 mg / mL) and human serum albumin (5.0 mg / mL) as an excipient of animal-derived protein, and after being formulated through commercial manufacturing procedures, 0.1 mL (100 units) / vial was filled and lyophilized into powder. Then, physiological saline was re-dissolved and the potency assay (mouse LD 50 The potency recovery (%) was calculated by comparing the potency measured after lyophilization to the initial input potency, and the overdose required for the manufacturing process was determined based on this (((100 / potency recovery)-1)×100).

[0123] Preparation 1

[0124] A pharmaceutical composition containing 100 units of botulinum toxin A complex (1000 units / mL) and excipients of amino acids (0.05 mg / mL to 8.0 mg / mL), surfactants (0.05 mg / mL to 50 mg / mL), and sodium chloride (9.0 mg / mL) was prepared and formulated through commercial manufacturing procedures and filled into 0.1 mL / vial to form a liquid. The amino acid used in formulation 1 is methionine, and the surfactant used is polysorbate 20. Then, the potency assay (mouse LD 50 ) The potency was measured. The potency recovery (%) was calculated by comparing the potency measured after charging the liquid to the potency of the initial input, and based on this, the excess feed required for the manufacturing process was determined (((100 / potency recovery)-1)×100).

[0125] Preparation 2

[0126] A pharmaceutical composition containing 100 units of botulinum toxin A complex (1000 units / mL) and excipients of amino acids (0.05 mg / mL to 8.0 mg / mL), surfactants (0.05 mg / mL to 50 mg / mL), and sodium chloride (9.0 mg / mL) was prepared and formulated through commercial manufacturing procedures and filled into 0.1 mL / vial to form a liquid. The amino acid used in formulation 1 is methionine, and the surfactant used is polysorbate 80. Then, the potency assay (mouse LD 50 ) The potency was measured. The potency recovery (%) was calculated by comparing the potency measured after charging the liquid to the potency of the initial input, and based on this, the excess feed required for the manufacturing process was determined (((100 / potency recovery)-1)×100).

[0127] Preparation 3

[0128] A pharmaceutical composition containing 100 units of botulinum toxin A complex (1000 units / mL) and excipients of amino acids (0.05 mg / mL to 8.0 mg / mL), surfactants (0.01 mg / mL to 50 mg / mL), and sodium chloride (9.0 mg / mL) was prepared and formulated through commercial manufacturing procedures and filled into 0.1 mL / vial to form a liquid. The amino acid used in formulation 1 was methionine, and the surfactant used was poloxamer 188. Then, the potency assay (mouse LD 50 ) The potency was measured. The potency recovery (%) was calculated by comparing the potency measured after charging the liquid to the potency of the initial input, and based on this, the excess feed required for the manufacturing process was determined (((100 / potency recovery)-1)×100).

[0129] result

[0130] The results are shown in Table 1.

[0131]

Table 1

[0132]

[0133]

[0134] *: Efficacy recovery rate = (semi-finished product efficacy / initial efficacy) × 100

[0135] **: Excess dosing (%) = ((100 / efficiency recovery rate)-1) × 100

[0136] As shown in Table 1, it was confirmed that 91% overdosage was required to compensate for the potency loss in the manufacturing process of the control preparation (lyophilized preparation of botulinum toxin A complex and sodium chloride (NaCl, 0.9%) and human serum albumin (HSA, 0.5%)). In contrast, it was confirmed that Formulation 1 (liquid preparation containing botulinum toxin A complex and methionine (0.05 mg / mL to 8.0 mg / mL), polysorbate 20 (0.05 mg / mL to 50 mg / mL), and sodium chloride (9.0 mg / mL)) required 0% to 20.9% overdosage depending on the concentration combination, which was improved by a minimum of 77% overdosage compared to the control group. It was confirmed that formulation 2 (a liquid formulation containing botulinum toxin A complex and methionine (0.05 mg / mL to 8.0 mg / mL), polysorbate 80 (0.05 mg / mL to 50 mg / mL), and sodium chloride (9.0 mg / mL)) required an overdose of 0% to 17.4% depending on the concentration combination, which was an 81% improvement over the overdose in the control group. It was confirmed that formulation 3 (a liquid formulation containing botulinum toxin A complex and methionine (0.05 mg / mL to 8.0 mg / mL), poloxamer 188 (0.05 mg / mL to 50 mg / mL), and sodium chloride (9.0 mg / mL)) required an overdose of 0% to 14.8% depending on the concentration combination. Thus, it was confirmed that an 84% improvement was achieved over the control group.

[0137] Thus, it was confirmed that in the preparation containing amino acids and nonionic surfactants during the manufacturing process, liquid preparations with improved overdosing were provided due to reduced inactivation of toxin proteins compared to the control preparation (animal-derived protein, NaCl).

[0138] Preparation 4

[0139] A pharmaceutical composition containing 100 units of botulinum toxin A complex (1000 units / mL) and excipients of amino acids (0.05 mg / mL to 8.0 mg / mL), surfactants (0.05 mg / mL to 50 mg / mL), sodium chloride (9.0 mg / mL), and sucrose (30 mg / mL) was prepared and formulated by commercial manufacturing procedures, filled into 0.1 mL / vial, and lyophilized into powder. The amino acid used in formulation 4 was methionine, and the surfactant used was polysorbate 20. Then, physiological saline was re-dissolved and the potency was determined by a potency assay (mouse LD 50 The potency recovery (%) was calculated by comparing the potency measured after lyophilization to the initial input potency, and the overdose required for the manufacturing process was determined based on this (((100 / potency recovery)-1)×100).

[0140] Preparation 5

[0141] A freeze-dried powder formulation was prepared by the same method as formulation 4, except that the type of surfactant used was changed to polysorbate 80. Then, physiological saline was reconstituted and the efficacy was measured by a potency assay (mouse LD 50 The potency recovery (%) was calculated by comparing the potency measured after lyophilization to the initial input potency, and the overdose required for the manufacturing process was determined based on this (((100 / potency recovery)-1)×100).

[0142] Preparation 6

[0143] A freeze-dried powder formulation was prepared by the same method as formulation 4, except that the type of surfactant used was changed to poloxamer 188. Then, physiological saline was reconstituted and the formulation was tested by potency assay (mouse LD 50 The potency recovery (%) was calculated by comparing the potency measured after lyophilization to the initial input potency, and the overdose required for the manufacturing process was determined based on this (((100 / potency recovery)-1)×100).

[0144] result

[0145] The results are shown in Table 2.

[0146]

Table 2

[0147]

[0148]

[0149] *: Efficacy recovery rate = (semi-finished product efficacy / initial efficacy) × 100

[0150] **: Excess dosing (%) = ((100 / efficiency recovery rate)-1) × 100

[0151] As shown in Table 2, it was confirmed that 91% overdosage was required to compensate for the potency loss during the manufacturing process of the control formulation (lyophilized formulation of botulinum toxin A complex with sodium chloride (NaCl, 0.9%) and human serum albumin (HSA, 0.5%)). In contrast, it was confirmed that formulation 4 (lyophilized formulation containing botulinum toxin A complex and methionine (0.05 mg / mL to 8.0 mg / mL), polysorbate 20 (0.05 mg / mL to 50 mg / mL), sodium chloride (9.0 mg / mL), and sucrose (30 mg / mL)) required 0% to 26.6% overdosage. As for formulations 5 and 6, which included the same formulation as the second formulation of formulation 4 but changed the type of surfactant, it was confirmed that no overdosage of botulinum toxin was required. Thus, it was confirmed that a lyophilized formulation with improved overdosage of toxin was provided due to reduced inactivation of toxin protein during the manufacturing process.

[0152] According to an embodiment, the botulinum toxin preparation containing no animal protein contains an amount of botulinum toxin corresponding to the desired potency, wherein the amount corresponding to the desired potency includes less than 50% overdose. Therefore, compared with preparations containing animal-derived proteins, the immune response when administered to a subject is suppressed, and the production of neutralizing antibodies is suppressed or reduced. Therefore, the preparation of an embodiment reduces the expression of tolerance in the body, thereby enabling continuous and repeated use without reducing the potency.

Claims

1. A botulinum toxin preparation which contains no animal protein and contains an amount of botulinum toxin corresponding to the desired potency, in, Amounts corresponding to the desired potency include less than 50% overdosage.

2. The botulinum toxin preparation according to claim 1, wherein The formulation includes a botulinum toxin overdose that is less than the botulinum toxin overdose of botulinum toxin formulations containing animal protein.

3. The botulinum toxin preparation according to any one of claims 1 and 2, wherein The overfeed is less than 30%.

4. The botulinum toxin preparation according to any one of claims 1 to 3, wherein It is a lyophilized preparation or a liquid preparation.

5. The botulinum toxin preparation according to claim 4, wherein The lyophilized preparation includes botulinum toxin, a surfactant, an amino acid, and one or more components selected from the group consisting of sugars, sugar alcohols, and ionic compounds.

6. The botulinum toxin preparation according to claim 5, wherein The surfactant includes one or more of polysorbates and poloxamers.

7. The botulinum toxin preparation according to claim 5, wherein Amino acids include methionine.

8. The botulinum toxin preparation according to claim 5, wherein The lyophilized formulation includes 0.05 mg / ml to 50 mg / ml of a surfactant and 0.05 mg / ml to 8 mg / ml of an amino acid.

9. The botulinum toxin preparation according to claim 4, wherein The liquid formulation includes botulinum toxin, a surfactant, and an amino acid.

10. The botulinum toxin preparation according to claim 9, wherein The surfactant includes one or more of polysorbates and poloxamers.

11. The botulinum toxin preparation according to claim 9, wherein Amino acids include methionine.

12. The botulinum toxin preparation according to claim 9, wherein The liquid formulation includes 0.05 mg / ml to 50 mg / ml of a surfactant and 0.05 mg / ml to 8 mg / ml of an amino acid.

13. The botulinum toxin preparation according to any one of claims 1 to 12, wherein The botulinum toxin is selected from the group consisting of botulinum toxin serotypes A, B, C, D, E, F and G.

14. The botulinum toxin preparation according to any one of claims 1 to 13, wherein It is used to improve wrinkles, square chins, pointed chins, wounds, skin softening, scars, acne, pores, elasticity, or keloids, or to treat facial spasms, eyelid spasms, torticollis, blepharospasm, cervical dystonia, oropharyngeal dystonia, spasmodic dysphonia, migraines, anal pruritus, or hyperhidrosis.

15. The botulinum toxin preparation according to any one of claims 1 to 14, wherein Its immunogenicity is reduced.

16. A method for producing a botulinum toxin preparation, comprising: A botulinum toxin preparation is obtained by mixing botulinum toxin and a pharmaceutically acceptable excipient, Wherein, the pharmaceutically acceptable excipient does not contain albumin, Botulinum toxin is used in an amount corresponding to the desired efficacy. The amount corresponding to the desired efficacy includes an overdose of less than 50%.

17. The method for producing a botulinum toxin preparation according to claim 16, wherein: The botulinum toxin preparation includes a botulinum toxin overdose that is less than the botulinum toxin overdose of a botulinum toxin preparation containing animal protein.

18. The method for producing a botulinum toxin preparation according to any one of claims 16 and 17, wherein Pharmaceutically acceptable excipients include surfactants and amino acids.

19. The method for producing a botulinum toxin preparation according to any one of claims 16 to 18, wherein: The step of mixing the excipients comprises, 0.05 mg / ml to 50 mg / ml of a surfactant is mixed, and 0.05 mg / ml to 8 mg / ml of an amino acid is mixed.

20. A method for reducing the potency loss of botulinum toxin during a manufacturing process, comprising: The step of obtaining a botulinum toxin preparation according to any one of claims 1 to 15.

21. A method for reducing the immunogenicity of a botulinum toxin preparation, comprising: An effective amount of the botulinum toxin formulation of any one of claims 1 to 15 is administered to a subject in need thereof.

22. A method for improving wrinkles, square chin, pointed chin, wounds, skin softening, scars, acne, pores, elasticity or keloids or for treating facial spasm, eyelid spasm, torticollis, blepharospasm, cervical dystonia, oropharyngeal dystonia, spasmodic dysphonia, migraine, anal pruritus or hyperhidrosis, comprising: An effective amount of the botulinum toxin formulation of any one of claims 1 to 13 is administered to a subject in need thereof.

23. Use of an effective amount of the botulinum toxin preparation according to any one of claims 1 to 13 in the manufacture of a medicament for improving wrinkles, square chin, pointed chin, wounds, skin softening, scars, acne, pores, elasticity or keloids, or for treating facial spasm, blepharospasm, torticollis, blepharospasm, cervical dystonia, oropharyngeal dystonia, spasmodic dysphonia, migraine, anal pruritus or hyperhidrosis.

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