Botulinum toxin liquid formulation comprising botulinum toxin and liquid composition for botulinum toxin stabilization, and method for preparing same

Abstract

The present invention relates to a botulinum toxin liquid formulation comprising botulinum toxin and a liquid composition for botulinum toxin stabilization and a method for preparing same and, more specifically, provides a liquid composition for botulinum toxin stabilization, which is capable of maintaining the activity and stability of botulinum toxin during preparation, storage, and distribution processes even when the botulinum toxin is stabilized in a liquid form, a liquid formulation and an injectable agent each comprising botulinum toxin and the liquid composition for botulinum toxin stabilization, and a method for preparing a botulinum toxin liquid formulation.

Description

Botulinum toxin liquid preparation comprising botulinum toxin and a liquid composition for stabilizing botulinum toxin, and a method for producing the same

[0001] This application claims priority to Republic of Korea Patent Application No. 10-2024-0029757, filed February 29, 2024, the entire disclosure of which is incorporated herein by reference.

[0002] The present invention relates to a botulinum toxin liquid formulation comprising botulinum toxin and a liquid composition for stabilizing botulinum toxin, and a method for producing the same. More specifically, the present invention provides a liquid composition for stabilizing botulinum toxin capable of maintaining the activity and stability of botulinum toxin during the manufacturing, storage, and distribution processes even when botulinum toxin is formulated in a liquid form, a liquid formulation comprising botulinum toxin and the liquid composition for stabilizing botulinum toxin, an injection, and a method for producing the liquid botulinum toxin formulation.

[0003] Botulinum toxin is a neurotoxin produced by the anaerobic bacterium Clostridium botulinum and is classified into seven serotypes, labeled A through G. Botulinum toxin types A and B are widely used to treat dystonia and astonic movement disorders, as well as axillary hyperhidrosis, spasticity, tremors, and several other medical conditions, including pain management.

[0004] However, during the manufacturing, storage, and distribution of botulinum toxin, problems arise where botulinum toxin may be adsorbed onto the surface of the container due to the instability of the protein preparation, or may be decomposed by protein oxidation, resulting in loss of activity. These problems may be particularly evident when formulated at low concentrations. In addition, since existing powdered botulinum toxin must go through a dilution process, there is a high risk of medical accidents due to management errors such as errors in the dilution ratio during administration or contamination of the dilution solvent during preparation.

[0005] As a prior art for minimizing denaturation of botulinum toxin, Patent Document 1 (Korean Patent Publication No. 10-2023-0107137) discloses a botulinum toxin composition containing albumin. However, albumin is an animal-derived ingredient, which raises concerns about adverse effects such as immune system abnormalities and cross-infection. Furthermore, its unstable supply and relatively high price hinder its industrial application.

[0006] As a prior art for stabilizing a liquid preparation of botulinum toxin, according to Patent Document 2 (Korean Patent Publication No. 10-2022-0088623), a liquid composition of botulinum toxin containing hyaluronic acid is disclosed, and according to Patent Document 3 (Korean Patent Publication No. 10-2013-0106557), a liquid product of botulinum toxin containing a dextrose solution is disclosed. However, there is a lack of research on a liquid preparation of botulinum toxin that utilizes a natural substance, amino acid, and has proven stability under body temperature and pH conditions.

[0007] Accordingly, the inventors of the present invention have completed the present invention by confirming that when botulinum toxin includes at least one selected from the group consisting of phenylalanine, threonine, and serine as a stabilizer, the botulinum toxin is stable at room temperature and can be stably provided under the temperature and pH conditions of the human body.

[0008] [Prior Art Literature]

[0009] [Patent Document]

[0010] (Patent Document 0001) Republic of Korea Publication Patent Document No. 10-2023-0107137 (July 14, 2023)

[0011] (Patent Document 0002) Republic of Korea Publication Patent Document No. 10-2022-0088623 (June 28, 2022)

[0012] (Patent Document 0003) Republic of Korea Publication Patent Document No. 10-2013-0106557 (September 30, 2013)

[0013] The problem to be solved by the present invention is to provide a liquid composition of botulinum toxin that does not undergo a dilution process, thus having no side effects such as cross-contamination, reducing distribution costs, and improving stability.

[0014] Accordingly, an object of the present invention is to provide a liquid composition for stabilizing botulinum toxin, comprising a stabilizer including a specific type of amino acid, a nonionic surfactant and an isotonic agent.

[0015] Another object of the present invention is to provide a liquid composition for stabilizing botulinum toxin and a liquid botulinum toxin preparation containing botulinum toxin.

[0016] Another object of the present invention is to provide an injection comprising the above botulinum toxin liquid preparation.

[0017] Another object of the present invention is to provide a use of a liquid composition comprising a stabilizer comprising a specific type of amino acid, a nonionic surfactant and a tonicity agent for the preparation of a stabilized liquid botulinum toxin preparation.

[0018] Another object of the present invention is to provide a method for producing the above botulinum toxin liquid preparation.

[0019] In order to solve the above-described problem, the present invention provides a liquid composition for stabilizing botulinum toxin, comprising as active ingredients at least one stabilizer selected from the group consisting of phenylalanine, threonine and serine, a nonionic surfactant and an isotonic agent.

[0020] In addition, the present invention provides the use of a liquid composition comprising at least one stabilizer selected from the group consisting of phenylalanine, threonine and serine, a nonionic surfactant and an isotonic agent for the preparation of a stabilized liquid botulinum toxin preparation.

[0021] In the present invention, the stabilizer may be included at a concentration of 0.001 to 2.0% (w / v) based on the total volume of the liquid composition.

[0022] In the present invention, the nonionic surfactant may be at least one selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80 with respect to the total volume of the liquid composition.

[0023] In the present invention, the nonionic surfactant may be included at a concentration of 0.001 to 1.0% (w / v) based on the total volume of the liquid composition.

[0024] In the present invention, the isotonic agent may be at least one selected from the group consisting of sodium chloride, mannitol, glycerol, sucrose, potassium chloride, sorbitol, glucose, and dextrose.

[0025] In the present invention, the isotropic agent may be included at a concentration of 0.01 to 1.0% (w / v) based on the total volume of the liquid composition.

[0026] In the present invention, the pH of the liquid composition may be 5 to 7.

[0027] In addition, the present invention provides a liquid composition for stabilizing the aforementioned botulinum toxin and a liquid botulinum toxin preparation containing the botulinum toxin.

[0028] In the present invention, the botulinum toxin may be selected from the group consisting of type A, type B, type C, type D, type E, type F, type G, or derivatives thereof.

[0029] In the present invention, the botulinum toxin liquid preparation may additionally contain a local anesthetic.

[0030] In the present invention, the local anesthetic may be selected from the group consisting of lidocaine, bupivacaine, and mepivacaine.

[0031] In the present invention, the local anesthetic may be included in a concentration of 0.01 to 1.0% (w / v) based on the total volume of the botulinum toxin liquid preparation.

[0032] In the present invention, the botulinum toxin liquid preparation can be prepared through the following steps (a) to (d):

[0033] (a) a step of preparing a first mixture by mixing water and an isotonic agent;

[0034] (b) a step of preparing a second mixture by adding a stabilizer to the first mixture;

[0035] (c) a step of preparing a liquid composition by adding a nonionic surfactant to the second mixed solution; and

[0036] (d) A step of preparing a liquid botulinum toxin preparation by adding botulinum toxin to the above liquid composition.

[0037] In the present invention, when the botulinum toxin liquid preparation contains a local anesthetic, the local anesthetic may be added in any one of steps (a) to (c).

[0038] Additionally, the present invention provides an injection comprising the aforementioned botulinum toxin liquid preparation.

[0039] In addition, the present invention provides a method for preparing a liquid botulinum toxin preparation, comprising the following steps (a) to (d):

[0040] (a) a step of preparing a first mixture by mixing water and an isotonic agent;

[0041] (b) a step of preparing a second mixture by adding at least one stabilizer selected from the group consisting of phenylalanine, threonine, and serine to the first mixture;

[0042] (c) a step of preparing a liquid composition by adding a nonionic surfactant to the second mixed solution; and

[0043] (d) A step of preparing a liquid botulinum toxin preparation by adding botulinum toxin to the above liquid composition.

[0044] In the present invention, the botulinum toxin can be selected from the group consisting of botulinum toxin types A, B, C, D, E, F, and G.

[0045] In the present invention, the isotonic agent of step (a) may be at least one selected from the group consisting of sodium chloride, mannitol, glycerol, sucrose, potassium chloride, sorbitol, glucose, and dextrose.

[0046] In the present invention, the nonionic surfactant of step (c) may be at least one selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80.

[0047] In the present invention, when the botulinum toxin liquid preparation contains a local anesthetic, the local anesthetic may be added in any one of steps (a) to (c).

[0048] In the present invention, the local anesthetic may be at least one selected from the group consisting of lidocaine, bupivacaine, and mepivacaine.

[0049] The present invention relates to a liquid botulinum toxin preparation comprising botulinum toxin and a stabilizer, a nonionic surfactant and an isotonic agent as active ingredients, and a method for preparing the same. By adding at least one selected from the group consisting of phenylalanine, threonine and serine as a stabilizer, it has been confirmed that long-term stability and effectiveness are maintained even under harsh conditions such as 40°C, so that stability can be maintained when botulinum toxin is formulated, stored and distributed as a liquid composition, the original activity of botulinum toxin can be maintained under human body temperature and pH conditions, and convenience is improved because a dilution process is not required.

[0050] Figure 1 shows the results of testing stabilizer candidates using enzyme-linked immunosorbent assay to select stabilizers for implementing a liquid composition containing botulinum toxin.

[0051] Figure 2 shows the results of testing the effect on stability when the concentration of a stabilizer is changed in a liquid composition containing botulinum toxin, or when two or more stabilizers are used.

[0052] Figure 3 shows the results of an animal titer test conducted on a selected stabilizer using enzyme-linked immunosorbent assay, confirming that the stabilizer actually works.

[0053] Figure 4 shows the results of a long-term preservation test confirming the stability of a prescription based on the results of enzyme immunoassay and animal titer tests.

[0054] Hereinafter, the present invention will be described in more detail.

[0055] All technical terms used in this invention, unless otherwise defined, have the same meaning as commonly understood by those skilled in the art. While preferred methods and samples are described herein, similar or equivalent methods are also included within the scope of this invention.

[0056] A first aspect of the present invention relates to a liquid composition for stabilizing botulinum toxin, comprising, consisting essentially of, or consisting of at least one stabilizer, a nonionic surfactant, and an isotonic agent selected from the group consisting of phenylalanine, threonine, and serine as an active ingredient.

[0057] In relation to the first aspect, the present invention relates to a liquid composition comprising, consisting essentially of, or consisting of at least one stabilizer, a nonionic surfactant, and an isotonic agent selected from the group consisting of phenylalanine, threonine, and serine, as an active ingredient for use in a stabilized botulinum toxin liquid formulation.

[0058] In connection with the first aspect, the present invention further relates to the use of a liquid composition comprising at least one stabilizer selected from the group consisting of phenylalanine, threonine and serine, a nonionic surfactant and an isotonic agent for the preparation of a stabilized liquid botulinum toxin preparation.

[0059] In the present invention, the term "stabilizer" means a pharmaceutically acceptable additive added to prevent an active ingredient from being decomposed, crystallized, or adsorbed on the surface of a storage container due to external stimuli (stress) such as temperature, pH, or light.

[0060] In the liquid composition of the present invention, the stabilizer may play a role in preventing the decomposition or adsorption of the botulinum toxin to the container, thereby preserving and maintaining the biological activity of the botulinum toxin without a decrease in potency even under harsh conditions such as 40°C, but is not limited thereto.

[0061] The above "phenylalanine" has a molecular formula of C9H 11 It is an α-amino acid with a molecular weight of 165.19, is water-soluble, and is used in protein biosynthesis. The abbreviation for the residue is 'Phe', and when further simplified, it is written as 'F'. Phenylalanine is an α-amino group (protonated under biological conditions -NH3 + It contains an α-carboxyl group (in the form of -COO-, which is deprotonated under biological conditions) and a side chain benzyl group. Phenylalanine is an aromatic amino acid classified as neutral and nonpolar due to the inertness and hydrophobicity of the side chain benzyl group.

[0062] The above "threonine" is a water-soluble amino acid with a molecular formula of C4H9NO3 and a molecular weight of 119.12, and is an α-amino acid used in protein biosynthesis. The abbreviation for the residue is 'Thr', and when further simplified, it is expressed as 'T'. Threonine is an α-amino group (-NH3, which is protonated under biological conditions). + It is an amino acid classified as a polar uncharged amino acid containing an α-carboxyl group (in the form of -COO-, which is deprotonated under biological conditions) and a side chain hydroxyl group, with the side chain group forming a hydrogen bond.

[0063] The above "serine" is a water-soluble amino acid with a molecular formula of C3H7NO3 and a molecular weight of 105.09, and is an α-amino acid used in protein biosynthesis. The abbreviation for the residue is 'Ser', and when further simplified, it is expressed as 'S'. Serine is an α-amino group (-NH3, which is protonated under biological conditions). + Serine is a non-essential amino acid that can be synthesized in the human body. Serine is an amino acid classified as a polar uncharged amino acid.

[0064] In a specific embodiment of the present invention, 12 amino acids and mannitol, as shown in Table 1, were used as stabilizers for a liquid botulinum toxin preparation, respectively, to prepare a liquid composition containing botulinum toxin, and after culturing under harsh conditions for 0 to 8 weeks, the recovery rate (%) of botulinum toxin was measured using enzyme-linked immunosorbent assay to select an effective botulinum toxin stabilizer. As a result, as confirmed in Table 1 and Figure 1, when phenylalanine, threonine, and serine were each used as stabilizers compared to other amino acids, the recovery rate (%) of botulinum toxin in the liquid preparation was found to be significantly higher during the evaluation period of 8 weeks.

[0065] In another specific embodiment of the present invention, in order to confirm the optimal concentration of the stabilizer, the recovery rate (%) of botulinum toxin according to the concentration change of the selected stabilizer was measured by enzyme-linked immunosorbent assay to derive the optimal concentration range. Referring to the results in Table 2 and Fig. 2, when phenylalanine and threonine were each used in a concentration range of 0.2 to 1.2% (w / v) with respect to the total volume of the liquid composition, there was no significant change in the recovery rate (%) of botulinum toxin from week 0 to week 8 under harsh conditions, confirming that the structural stability of the botulinum toxin was well maintained. In addition, it was confirmed that the botulinum toxin was maintained in a stable state until week 8 even when phenylalanine and threonine were used in combination.

[0066] Therefore, in the liquid composition of the present invention, the stabilizer may be included in a concentration range of 0.001 to 2.0% (w / v) based on the total volume of the liquid composition, preferably 0.01 to 1.7% (w / v), and more preferably 0.1 to 1.5% (w / v).

[0067] In the liquid composition of the present invention, if the stabilizer is included in an amount of less than 0.001% (w / v), the ability to prevent botulinum toxin from being adsorbed to a container may be reduced, and if it is included in an amount of more than 2.0% (w / v), the protein three-dimensional structure of the botulinum toxin may be deformed, which may cause a decrease in stability.

[0068] In the liquid composition of the present invention, when the stabilizer is used as a combination of two or more amino acids, the stabilizer may be included in a concentration range of 0.001 to 1.0% (w / v) based on the total volume of the liquid composition, preferably 0.01 to 0.9% (w / v), more preferably 0.1 to 0.8% (w / v), but is not limited thereto.

[0069] In the liquid composition of the present invention, when the stabilizer is used as a combination of two or more amino acids, it is preferable that the stabilizer is a combination of phenylalanine and threonine, and phenylalanine and threonine may be included in different or identical contents.

[0070] In the present invention, the term "non-ionic surfactant" refers to a surfactant having a chemical structure with hydrophilicity and hydrophobicity within a single molecule so as to relax the boundary of the interface where gas and liquid, liquid and liquid, or liquid and solid come into contact with each other and weaken the surface tension of the interface, and the hydrophilic part has a non-electrolyte, that is, a hydrophilic part that has not been ionized, and there exist low-molecular-weight series such as alkyl glycol or high-molecular-weight non-ionic surfactants such as polyethylene glycol and polyvinyl alcohol. These have the advantage of having little foaming properties, performing surfactant action at low concentrations, and having little toxicity, so they are widely used as additives in pharmaceuticals, especially as additives for injections.

[0071] In the liquid composition of the present invention, the nonionic surfactant may be at least one selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80, but is not limited thereto.

[0072] Polysorbate, a surfactant of the polyethylene glycol series, can play a role in preventing botulinum toxin from adsorbing to the surface of a storage container or syringe.

[0073] "Polysorbate 20" has the chemical name polyoxyethylene(20) sorbitan monolaurate and the molecular formula is C58 H 114 O 26 It is a nonionic surfactant with an HLB value of 16.7.

[0074] "Polysorbate 40" is a chemical compound whose molecular formula is C and whose chemical name is polyoxyethylene(20) sorbitan monopalmitate. 62 H 122 O 26 , and is a nonionic surfactant with an HLB value of 15.6.

[0075] "Polysorbate 60" is a chemical compound whose molecular formula is C and whose chemical name is polyoxyethylene(20) sorbitan monostearate. 64 H 126 O 26 , and is a nonionic surfactant with an HLB value of 14.9.

[0076] "Polysorbate 80" has the chemical name polyoxyethylene(20) sorbitan monooleate and the molecular formula is C 64 H 124 O 26 , and is a nonionic surfactant with an HLB value of 15.0.

[0077] In the liquid composition of the present invention, the nonionic surfactant may be included in a concentration of 0.001 to 1.0% (w / v) based on the total volume of the liquid composition, preferably 0.005 to 0.5% (w / v), more preferably 0.005 to 0.05% (w / v), but is not limited thereto.

[0078] In the liquid composition of the present invention, if the nonionic surfactant is less than 0.001% (w / v), the ability to prevent botulinum toxin from being adsorbed on a container may be reduced, and if it is more than 1.0% (w / v), many bubbles may be generated in the liquid composition, causing a problem of decomposition of botulinum toxin at the interface, and further, a process for removing bubbles may be added, causing a problem of reducing the productivity of the liquid preparation.

[0079] In the present invention, the term "isotonic agent" refers to an additive that alleviates pain or discomfort when administering injections, eye drops, nasal drops, etc., and prevents red blood cell crenation or hemolysis. Maintaining isotonicity is crucial in storage or hypertonic solutions, as cell destruction or protein stability may occur.

[0080] In the liquid composition of the present invention, the isotonic agent may be at least one selected from the group consisting of sodium chloride, mannitol, glycerol, sucrose, potassium chloride, sorbitol, glucose, and dextrose, and is preferably sodium chloride or mannitol, but is not limited thereto.

[0081] The isotonic agent can prevent the stability of botulinum toxin, the protein in a liquid botulinum toxin formulation, from deteriorating, or can alleviate pain caused by cell shrinkage or hemolysis during injection. Furthermore, it maintains the osmotic pressure of the liquid composition at 300±50 mOsm / kg, thereby preventing or alleviating pain at the injection site during injection.

[0082] Sodium chloride (NaCl) has a molecular weight of 58.44 and a pH of 6.7 to 7.3 in saturated solutions. It is a widely used additive as an isotonic agent in pharmaceuticals such as injections. It also has a freezing point depression effect, preventing liquid formulations from freezing when exposed to low temperatures, thereby deteriorating product quality.

[0083] "Mannitol" has a molecular formula of C6H 14 It is O6, has a molecular weight of 181.2, and is a type of sugar alcohol. Mannitol is non-hygroscopic and is used as an additive in the formulation of moisture-sensitive drugs, helps lyophilized powders form a homogeneous cake, and is used as an osmotic pressure regulator in injections.

[0084] In the liquid composition of the present invention, the isotonic agent may be included in a concentration of 0.01 to 1.0% (w / v) based on the total volume of the liquid composition, preferably 0.1 to 1.0% (w / v), more preferably 0.3 to 0.9% (w / v), but is not limited thereto.

[0085] In the liquid composition of the present invention, if the isotonic agent is included in an amount of less than 0.01% (w / v) or more than 1.0% (w / v), the osmotic pressure may exceed 300±50 mOsm / kg, and when the botulinum toxin liquid composition is administered, pain, swelling, etc. may occur at the injection site, which may lower patient compliance.

[0086] The liquid composition of the present invention may exhibit a pH of 5 to 7, preferably a pH of 5.5 to 6.5, and the stability and activity of the botulinum toxin may be maintained under the above pH conditions.

[0087] A second aspect of the present invention relates to a liquid botulinum toxin preparation and an injection containing the same, comprising the liquid composition for stabilizing the botulinum toxin and the botulinum toxin described above, consisting essentially of the liquid composition for stabilizing the botulinum toxin and the botulinum toxin, or consisting of the liquid composition for stabilizing the botulinum toxin and the botulinum toxin.

[0088] In the liquid preparation of the present invention, the description of the liquid composition for stabilizing botulinum toxin is the same as described above, and therefore, its description is omitted.

[0089] Botulinum toxin is a neurotoxic protein produced by the bacterium Clostridium botulinum. When a therapeutic botulinum toxin preparation is injected into a target tissue, it enters peripheral cholinergic nerve terminals and acts as a metalloproteinase to cleave proteins, a key component of the neuronal exocytosis machinery, causing sustained and reversible inhibition of neurotransmitter release. Although the precise molecular mechanism of botulinum toxin action is not yet fully understood, existing experimental evidence suggests that botulinum toxin intoxication occurs through a multistep process involving each functional domain of the toxin. These steps include neurotoxin binding to specific receptors on presynaptic nerve terminals, toxin internalization into neurons and translocation across the endosomal membrane, and intracellular endoprotease activity on proteins crucial for neurotransmitter release (Roshan K., et al., Res. Reports Biochem. 2015, 5, 173-183).

[0090] The above "botulinum toxin" refers to a neurotoxin produced by a strain of Clostridium botulinum or a variant thereof. Furthermore, "botulinum toxin" includes botulinum toxin serotypes A, B, C, D, E, F, and G, and may include pure botulinum toxin (150 kDa) as well as botulinum toxin complexes (i.e., 300, 600, and 900 kDa).

[0091] In the liquid formulation of the present invention, the botulinum toxin may be included in an amount of 10 to 500 Units, preferably 40 to 300 Units, and more preferably 40 to 250 Units per mL of the liquid formulation.

[0092] The above botulinum toxin 1 Unit (U) is the LD that causes 50% mortality through intraperitoneal injection in female Swiss Webster mice weighing 17 to 22 g. 50 can be defined as

[0093] The liquid formulation of the present invention may additionally contain a local anesthetic.

[0094] In the present invention, the term "local anesthetic" refers to a drug that chemically and reversibly blocks the conduction of nerves controlling a part of the body, and a method of obtaining an anesthetic effect using this drug is called local anesthesia or regional anesthesia.

[0095] In the liquid formulation of the present invention, the local anesthetic may be at least one selected from the group consisting of lidocaine, bupivacaine, and mepivacaine in the amide form, but is not limited thereto. The local anesthetic is used for the purpose of blocking pain when administering botulinum toxin subcutaneously.

[0096] "Lidocaine" has a molecular formula of C14 H 22 N 2O It has a molecular weight of 234.3, is water-soluble, has a relatively short half-life in the blood of 1.5 to 2 hours, has a fast onset of action, does not cause irritation to local tissues, has a higher strength and longer duration of action than procaine, and has a surface anesthetic effect and does not cause allergic reactions.

[0097] "Bupivacaine" has a molecular formula of C 18 H 28 N 2O It has a molecular weight of 288.4 and a relatively long half-life in blood of 3.5 hours in adults and 8.1 hours in newborns. The onset of anesthetic effect is within 4 to 10 minutes, and it is used for peripheral nerve block, infiltration, sympathetic nerve block, caudal and epidural block, and block of the back of the eye.

[0098] "Mepivacaine" has a molecular formula of C 15 H 22 N 2O It has a molecular weight of 246.4, a relatively short half-life in blood of 1.9 hours, and an onset of action of 7 to 15 minutes. It is used for nerve blockade, caudal blockade, infiltration, and therapeutic blockade.

[0099] In a specific embodiment of the present invention, when phenylalanine or threonine was used as a stabilizer in the manufacture of a liquid botulinum toxin composition containing lidocaine as a local anesthetic as shown in Table 3, the recovery rate (%) of botulinum toxin was measured by enzyme-linked immunosorbent assay for 0 to 8 weeks to confirm the change in stability of botulinum toxin according to the addition of the local anesthetic. At this time, the local anesthetic was added at 0.24 or 0.25% (w / v) based on the total volume of the liquid botulinum toxin composition, and the stabilizer was added at 0.4 to 0.8% (w / v) based on the total volume of the liquid botulinum toxin composition. Referring to the results in Table 3, when phenylalanine and threonine were each used as stabilizers, there was no significant change in the recovery rate (%) of botulinum toxin from 0 to 8 weeks under harsh conditions, confirming that the structural stability of botulinum toxin was still well maintained.

[0100] In the liquid preparation of the present invention, the local anesthetic may be included in a concentration of 0.01 to 1.0% (w / v), preferably 0.05 to 0.7% (w / v), and more preferably 0.1 to 0.5% (w / v) based on the total volume of the botulinum toxin liquid preparation.

[0101] In the present invention, the liquid formulation can be prepared in the following order of steps (a) to (d):

[0102] (a) a step of preparing a first mixture by mixing water and an isotonic agent;

[0103] (b) a step of preparing a second mixture by adding a stabilizer to the first mixture;

[0104] (c) a step of preparing a liquid composition by adding a nonionic surfactant to the second mixed solution; and

[0105] (d) A step of preparing a liquid botulinum toxin preparation by adding botulinum toxin to the above liquid composition.

[0106] In the present invention, when the botulinum toxin liquid preparation contains a local anesthetic, the local anesthetic may be added in any one of steps (a) to (c).

[0107] The method for manufacturing the above liquid preparation will be described in more detail in the method for manufacturing the botulinum toxin liquid preparation of the third aspect described below.

[0108] In the present invention, the term "formulation" may also be referred to as a dosage form, and refers to a product made into a certain form by mixing and processing a pharmaceutical product for a therapeutic purpose.

[0109] In a specific embodiment of the present invention, in order to evaluate the degree of toxicity of botulinum toxin depending on whether phenylalanine or threonine, which is a stabilizer, is included, a botulinum toxin liquid composition was prepared as shown in Table 4, cultured under harsh conditions for 0 to 8 weeks, and then the botulinum toxin liquid composition was administered to mice, and the titer (LD) was determined based on the number of deaths over 3 days. 50 ) was evaluated to observe whether there was a change in toxicity. Referring to the results in Table 4 and Fig. 3, when phenylalanine or threonine, which are stabilizers, were not included, the titer was not confirmed at week 8, whereas when phenylalanine or threonine, respectively, was included as stabilizers, the titer was stably maintained until week 8, confirming that the activity of botulinum toxin was excellently maintained.

[0110] In another specific embodiment of the present invention, in order to evaluate the long-term stability under harsh conditions of a liquid botulinum toxin composition containing phenylalanine or threonine as a stabilizer and to evaluate the long-term stability according to the additional inclusion of a local anesthetic, a liquid botulinum toxin composition was prepared as shown in Table 5, stored at 4°C as a distribution condition for 0 to 10 months, and then the liquid botulinum toxin composition was administered to mice and the titer (LD) was determined based on the number of deaths for 3 days. 50) was evaluated to observe whether there was any change in toxicity. Referring to the results in Table 5 and Figure 4, when phenylalanine or threonine, which are stabilizers, were included as stabilizers, the potency was maintained stably for up to 10 months regardless of whether a local anesthetic was included, confirming that the activity of botulinum toxin was maintained excellently.

[0111] In the present invention, the liquid composition and / or liquid preparation may further comprise, in addition to the aforementioned components, suitable carriers, excipients, and diluents commonly used in the manufacture of pharmaceutical compositions. The excipients may be, for example, one or more selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, moisturizers, film-coating materials, and controlled-release additives.

[0112] In the present invention, the liquid preparation may be formulated and used in the form of a powder, granule, sustained-release granule, enteric-coated granule, solution, eye drop, ellipsoid, emulsion, suspension, alcohol, troche, nasal spray, limonade, tablet, sustained-release tablet, enteric-coated tablet, sublingual tablet, hard capsule, soft capsule, sustained-release capsule, enteric capsule, pill, tincture, soft extract, dry extract, fluid extract, injection, capsule, irrigation solution, ointment, lotion, paste, spray, inhalant, patch, sterile injection solution, or external preparation such as an aerosol, according to a conventional method, and the external preparation may have a formulation such as a cream, gel, patch, spray, ointment, ointment, lotion, liniment, paste, or cataplasma.

[0113] Carriers, excipients and diluents that may be included in the liquid composition and / or liquid preparation of the present invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

[0114] When formulating, it is usually prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0115] The additives of the tablets, powders, granules, capsules, pills, and troches according to the present invention include excipients such as corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium hydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropyl methylcellulose (HPMC), HPMC 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, and Primogel; Gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethylcellulose, calcium carboxymethylcellulose, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, refined shellac, starch starch, hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl alcohol, polyvinyl pyrrolidone, and binders such as hydroxypropyl methylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, Disintegrants such as carboxymethylcellulose calcium, calcium citrate, sodium lauryl sulfate, anhydrous silicic acid, 1-hydroxypropyl cellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum, baking soda, polyvinylpyrrolidone, calcium phosphate, gelled starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethyl cellulose, sucrose, magnesium aluminum silicate, di-sorbitol solution, and light anhydrous silicic acid;Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium dentata, kaolin, petrolatum, sodium stearate, cocoa butter, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, anhydrous silicic acid, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light anhydrous silicic acid can be used.

[0116] As additives of the liquid formulation according to the present invention, water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, monostearate sucrose, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, ammonia water, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethyl cellulose, sodium carboxymethyl cellulose, etc. can be used.

[0117] The syrup according to the present invention may include a solution of white sugar, other sugars, or sweeteners, and may also include a fragrance, a coloring agent, a preservative, a stabilizer, a suspending agent, an emulsifier, a viscosity modifier, and the like, as needed.

[0118] Purified water may be used in the emulsion according to the present invention, and emulsifiers, preservatives, stabilizers, fragrances, etc. may be used as needed.

[0119] The suspension according to the present invention may include suspending agents such as acacia, tragacanth, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, and HPMC 2910, and, if necessary, surfactants, preservatives, stabilizers, colorants, and fragrances may be used.

[0120] The injection according to the present invention includes a solvent such as distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated Ringer's injection, ethanol, propylene glycol, non-volatile oils - sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, and benzene benzoate; a solubilizing agent such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, tween, nitrile acid amide, hexamine, and dimethylacetamide; a buffer such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumins, peptones, 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; oxidizing agents such as sodium bisulfite 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetic acid, and acetone sodium bisulfite; analgesics such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium cis-methylenediamine, sodium alginate, Tween 80, and aluminum monostearate.

[0121] The liquid formulation and / or injection according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level may be determined based on factors including the type and severity of the patient's disease, drug activity, drug sensitivity, administration time, administration route and excretion rate, treatment period, concurrently used drugs, and other factors well known in the medical field.

[0122] The liquid formulation and / or injection according to the present invention can be administered as an individual treatment or in combination with other treatments, sequentially or simultaneously with conventional treatments, or in single or multiple doses. Considering all of the above factors, it is important to administer the amount that achieves maximum effect with the minimum amount possible without adverse effects. This can be readily determined by those skilled in the art. The formulations administered in combination are not limited.

[0123] The liquid formulation and / or injection of the present invention can be administered to a subject by various routes. All modes of administration are conceivable, including oral ingestion, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, intrathecal injection, sublingual administration, buccal mucosa administration, rectal insertion, vaginal insertion, ocular administration, otic administration, nasal administration, inhalation, spraying through the mouth or nose, dermal administration, and transdermal administration.

[0124] The liquid formulation and / or injection of the present invention is determined according to the type of drug as an active ingredient along with various related factors such as the disease to be treated, the route of administration, the patient's age, sex, weight, and the severity of the disease.

[0125] In the present invention, the term "subject" means a subject requiring administration of botulinum toxin, and more specifically, may mean a mammal such as a human or non-human primate, mouse, dog, cat, horse, or cow.

[0126] The liquid formulation and / or injection of the present invention can be used for the treatment of neuromuscular disorders characterized by hyperactive skeletal muscles. Also, headache, migraine, tension headache, sinus headache, cervicogenic headache, sweating disorder, axillary hyperhidrosis, palmar hyperhidrosis, foot hyperhidrosis, Frey's syndrome, hyperkinetic skin folds, facial wrinkles, glabellar wrinkles, crow's feet wrinkles, mouth wrinkles, nasolabial folds, skin disorders, achalasia, strabismus, chronic fissures, blepharospasm, musculoskeletal pain, fibromyalgia, pancreatitis, tachycardia, benign prostatic hyperplasia, prostatitis, urinary retention, urinary incontinence, overactive bladder, hemifacial spasm, tremor, muscle spasms, gastrointestinal disorders, diabetes, hypersalivation, detrusor-sphincteric dyssynergia, post-stroke spasticity, wound healing, pediatric cerebral palsy, smooth muscle spasm, restenosis, focal dystonia, epilepsy, cervical dystonia, thyroid disorders, hypercalcemia, obsessive-compulsive disorder, arthritic pain, Raynaud's syndrome, stretch marks, peritoneal adhesions, It can be used for various purposes, including but not limited to, the treatment of conditions such as vasospasm, runny nose, muscle spasm, laryngeal dystonia, writer's spasm, and carpal tunnel syndrome, as well as for cosmetic purposes.

[0127] A third aspect of the present invention relates to a method for producing the aforementioned liquid botulinum toxin preparation and a method for stabilizing botulinum toxin in the liquid preparation.

[0128] Specifically, the method for manufacturing a liquid botulinum toxin preparation according to the present invention may include the following steps (a) to (d):

[0129] (a) a step of preparing a first mixture by mixing water and an isotonic agent;

[0130] (b) a step of preparing a second mixture by adding at least one stabilizer selected from the group consisting of phenylalanine, threonine, and serine to the first mixture;

[0131] (c) a step of preparing a liquid composition by adding a nonionic surfactant to the second mixed solution; and

[0132] (d) A step of preparing a liquid botulinum toxin preparation by adding botulinum toxin to the above liquid composition.

[0133] In the present invention, when the botulinum toxin liquid preparation includes a local anesthetic, the local anesthetic may be added in any one of steps (a) to (c), and a person skilled in the art can appropriately select the step of adding the local anesthetic before adding the botulinum toxin.

[0134] In the method for manufacturing the liquid formulation of the present invention, the description of the role, type, content, etc. of the isotonic agent, stabilizer, nonionic surfactant, local anesthetic, and botulinum toxin used in steps (a) to (d) is the same as described above, and therefore, description thereof is omitted.

[0135] In the method for manufacturing a liquid formulation of the present invention, the water in step (a) may be distilled water, sterilized water, sterile distilled water, physiological saline solution, purified water, or water for injection (WFI), but is not limited thereto.

[0136] In the method for manufacturing the liquid formulation of the present invention, step (a) can be performed at 30 to 60°C for 10 to 40 minutes.

[0137] When the first mixture is prepared in which the stabilizer is dissolved in water, step (b) of adding the stabilizer is performed.

[0138] In the method for manufacturing a liquid formulation of the present invention, step (b) may be performed at a temperature of 30 to 60°C until the stabilizer is completely dissolved in the first mixture. At this time, stirring may be performed to increase dissolution efficiency.

[0139] When a second mixture is prepared in which the stabilizer is completely dissolved in the first mixture, step (c) of adding a nonionic surfactant is performed.

[0140] In the method for manufacturing a liquid formulation of the present invention, step (c) may be performed at a temperature of 30 to 60°C until the nonionic surfactant is completely dissolved in the second mixture. At this time, stirring may be performed to increase dissolution efficiency.

[0141] The method for producing a liquid formulation of the present invention may optionally include a step (c') of cooling the liquid composition between steps (c) and (d). For example, if the temperature range of the liquid composition obtained after all nonionic surfactants are dissolved in step (c) is a high temperature (e.g., 60°C or higher) that may negatively affect the stability and activity of the botulinum toxin, it is preferable that the cooling step be performed before step (d) of adding the botulinum toxin to the obtained liquid composition. The final temperature of the liquid composition after cooling may be a temperature suitable for adding the botulinum toxin, for example, a temperature that does not negatively affect the stability and activity of the botulinum toxin. The temperature of the liquid composition after cooling may be preferably 15 to 30°C, but is not limited thereto, and a person skilled in the art can appropriately select a temperature range that does not negatively affect the stability and activity of the botulinum toxin and perform the cooling step.

[0142] Once the dissolution of the nonionic surfactant is complete, step (d) of adding botulinum toxin or local anesthetic and botulinum toxin is performed.

[0143] In the method for manufacturing a liquid formulation of the present invention, step (d) may be performed at 15 to 30°C until both the botulinum toxin or local anesthetic and the botulinum toxin are dissolved. At this time, stirring may be performed to increase the dissolution efficiency, but it is preferable to perform stirring so as to prevent the formation of air bubbles.

[0144] It is important that the method for manufacturing the liquid formulation of the present invention is performed sequentially in steps (a) to (d), and if the order of addition of each component is changed, the structural stability and activity of the botulinum toxin may not be maintained as intended.

[0145] The method for stabilizing botulinum toxin in the liquid preparation according to the present invention is substantially the same as the method for manufacturing the liquid preparation, and therefore, its description is omitted.

[0146] In the present invention, when the term "comprising" is used, it means that other components can be included rather than excluding other components unless specifically stated otherwise. As used throughout the present invention, the terms "step of ~" or "step of ~" do not mean "step for ~."

[0147] Hereinafter, the present invention will be described in more detail through examples. These examples are intended to further illustrate the present invention, and the scope of the present invention is not limited by the following examples, and unless otherwise defined, it is intended that those skilled in the art understand the scope of the present invention.

[0148] [Manufacturing Example 1]

[0149] Preparation of a liquid composition containing botulinum toxin

[0150] Sodium chloride (NaCl) was mixed in water for injection (WFI) at 60°C for more than 20 minutes, a stabilizer was added, and the mixture was stirred at 300 rpm at 60°C for more than 30 minutes to completely dissolve. After adding a certain amount of polysorbate 20, the mixture was stirred at 50 to 100 rpm at 60°C for more than 30 minutes to dissolve, and then slowly cooled until the temperature of the mixed solution reached 20°C. A botulinum toxin solution was added to this, and the mixture was stirred at 10 to 50 rpm at 20°C for more than 20 minutes to prevent bubbles from forming during mixing.

[0151] [Manufacturing Example 2]

[0152] Preparation of a liquid composition containing botulinum toxin and a local anesthetic

[0153] Sodium chloride (NaCl) was mixed in water for injection (WFI) at 60°C for more than 20 minutes, a stabilizer was added, and the mixture was stirred at 300 rpm at 60°C for more than 30 minutes to completely dissolve. After adding a certain amount of polysorbate 20, the mixture was stirred at 50 to 100 rpm at 60°C for more than 30 minutes to dissolve, and then slowly cooled until the temperature of the mixed solution reached 20°C. Lidocaine, a local anesthetic, was added and dissolved at any of the above steps. The botulinum toxin solution was added at the last step after all components were dissolved, and the mixture was prepared by stirring at 10 to 50 rpm at 20°C for more than 20 minutes to prevent bubbles from forming during mixing.

[0154] [Example 1]

[0155] Stabilizer screening using enzyme-linked immunosorbent assay

[0156] Botulinum toxin type A produced by Jetema (Korea) was used, and a liquid composition containing botulinum toxin was prepared in the same manner as in Manufacturing Example 1 to select a new stabilizer rather than an animal-derived stabilizer such as albumin. At this time, botulinum toxin was added to be 100 U / mL, polysorbate 20 was added in an amount of 0.01% (w / v) based on the total volume of the liquid composition, sodium chloride was added in an amount of 0.8% (w / v) based on the total volume of the liquid composition, and the stabilizer was added in an amount of 0.4% (w / v) based on the total volume of the liquid composition. For the control group, a liquid composition was prepared under the same conditions without adding any additives used as stabilizers. Afterwards, each experimental group was cultured under harsh conditions at 40℃ for 0 to 8 weeks, and the recovery rate (% recovery) of botulinum toxin was measured using enzyme-linked immunosorbent assay (ELISA).

[0157] Toxin amount measurement was performed according to the test method of Botulinum Neurotoxin Type A DuoSet ELISA (R&D system). Botulinum Antitoxin (NIBSC, 59 / 021) was diluted in PBS, 100 μL was dispensed into each well of a 96-well microplate, and coated for more than 16 hours. Then, each well was washed with washing buffer. This process was repeated three times in total. Each well was blocked with reagent diluent and washed with washing buffer.

[0158] 100 μL of sample was added to each well, reacted for 2 hours, and each well was washed with wash buffer. This process was repeated three times in total. 100 μL of detection antibody dilution was added to each well, reacted for 2 hours, and each well was washed with wash buffer. This process was repeated three times in total. 100 μL of streptavidin-horseradish peroxidase dilution was added to each well, reacted for 20 minutes, and each well was washed with wash buffer. This process was repeated three times in total. 100 μL of substrate solution was added to each well, reacted for 20 minutes, and 50 μL of stop solution was added to each well and mixed appropriately to stop the enzyme reaction. The absorbance at 450 nm in each well was measured using a microplate reader (Table 1).

[0159] Classification Concentration (%, w / v) Recovery % Week 0 Week 1 Week 2 Week 4 Week 8 Control -123.279.191.1453.35ND Valine 0.4120.571.553.835.9ND Leucine 0.4117.695.454.1ND Phenylalanine 0.4116.9120.0107.1112.72106.5 Serine 0.4115.199.794.287.677.9 Threonine 0.4117.7113.1105.5122.4105.9 Arginine )0.4ND----Alanine0.4169.494.568.3ND-Lysine0.4111.586.375.3ND-Histidine0.4102.443.3ND--Mannitol0.499.093.3ND--Isoleucine0.498.678.2ND--Glycine0.499.977.339.0ND-Tryptophan0.4100.164.243.2ND-ND: not detected

[0160] *Recovery% represents the percentage of botulinum toxin recovered compared to the reference concentration.

[0161] As a result of the experiment, it was confirmed that the liquid botulinum toxin composition using phenylalanine, serine and threonine as stabilizers was stable up to 8 weeks when evaluated by enzyme-linked immunosorbent assay (Fig. 1).

[0162] [Example 2]

[0163] Stability evaluation according to stabilizer concentration

[0164] Botulinum toxin type A produced by Jetema (Korea) was used as the botulinum toxin, and a liquid composition containing botulinum toxin was prepared in the same manner as in Manufacturing Example 1 to evaluate the stability when the concentration of the stabilizer and the inclusion of complex components in the botulinum toxin liquid composition are varied. At this time, botulinum toxin was added to be 100 U / mL, and phenylalanine, threonine, and sodium chloride were prepared in various concentration conditions (% (w / v) with respect to the total volume of the liquid composition) as shown in Table 2. At this time, polysorbate 20 was used as a nonionic surfactant at 0.01% (w / v) with respect to the total volume of the liquid composition. Afterwards, each experimental group was cultured under harsh conditions of 40℃ for 0 to 8 weeks, and the recovery rate (% recovery) of botulinum toxin was measured using enzyme-linked immunosorbent assay.

[0165] Toxin amount measurement was performed according to the test method of Botulinum Neurotoxin Type A DuoSet ELISA (R&D system). Botulinum Antitoxin (NIBSC, 59 / 021) was diluted in PBS, prepared, and 100 μL was dispensed into each well of a 96-well microplate. After coating for more than 16 hours, each well was washed with washing buffer. This process was repeated three times in total. Each well was blocked with reagent diluent and washed with washing buffer.

[0166] 100 μL of sample was added to each well, reacted for 2 hours, and each well was washed with a wash buffer. This process was repeated three times in total. 100 μL of detection antibody dilution was added to each well, reacted for 2 hours, and each well was washed with a wash buffer. This process was repeated three times in total. 100 μL of streptavidin-HRP (Streptavidin-horseradish peroxidase) dilution was added to each well, reacted for 20 minutes, and each well was washed with a wash buffer. 100 μL of substrate solution was added to each well, reacted for 20 minutes, and 50 μL of stop solution was added to each well and mixed appropriately to stop the enzyme reaction. The absorbance at 450 nm in each well was measured using a microplate reader (Table 2).

[0167] Concentration (%, w / v) Recovery % Phenylalanine Threonine Sodium chloride Week 0 Week 2 Week 4 Week 8 0.2-0.85 15 7.78 0.68 6.5 10 1.5 0.4-0.80 116.9 10 7.11 12.7 10 6.5 0.8-0.75 142.9 13 1.5 9 6.2 119.7 1.2-0.65 17 0.11 2 9.69 2. 9107.6-0.20.85153.6146.899.1101.8-0.40.80117.7105.5122.4105.9-1.20.70157.2143.194.2109.20.20.20.80140.5132.098.8115.120.40.40.75146.2101.280.288.0

[0168] As a result of the experiment, the recovery rate (% recovery) of botulinum toxin did not change significantly depending on the concentration change of phenylalanine and threonine (0.2 to 1.2% w / v), and it was confirmed that the botulinum toxin liquid composition was stable up to 8 weeks when evaluated by enzyme-linked immunosorbent assay even when used in combination (Fig. 2).

[0169] [Example 3]

[0170] Safety assessment when local anesthetics are included

[0171] Botulinum toxin type A produced by Jetema (Korea) was used as the botulinum toxin, and in order to evaluate the stability of the botulinum toxin liquid composition when containing the local anesthetic lidocaine, a liquid composition containing botulinum toxin was prepared in the same manner as in Manufacturing Example 2, but the liquid compositions were prepared with phenylalanine, threonine, and lidocaine at various concentration conditions as shown in Table 3. At this time, the botulinum toxin was added to be 200 U / mL, and polysorbate 20 was used as a nonionic surfactant at 0.01% (w / v) based on the total volume of the liquid composition, and sodium chloride was used as an isotonic agent at 0.7% (w / v) based on the total volume of the liquid composition. Afterwards, each experimental group was cultured under harsh conditions of 40℃ for 0 to 8 weeks, and the recovery rate (% recovery) of the botulinum toxin was measured using enzyme-linked immunosorbent assay.

[0172] Toxin amount measurement was performed according to the test method of Botulinum Neurotoxin Type A DuoSet ELISA (R&D system). Botulinum Antitoxin (NIBSC, 59 / 021) was diluted in PBS, prepared, and 100 μL was dispensed into each well of a 96-well microplate. After coating for more than 16 hours, each well was washed with washing buffer. This process was repeated three times in total. Each well was blocked with reagent diluent and washed with washing buffer.

[0173] 100 μL of sample was added to each well, reacted for 2 hours, and each well was washed with a wash buffer. This process was repeated three times in total. 100 μL of detection antibody dilution was added to each well, reacted for 2 hours, and each well was washed with a wash buffer. This process was repeated three times in total. 100 μL of streptavidin-HRP (Streptavidin-horseradish peroxidase) dilution was added to each well, reacted for 20 minutes, and each well was washed with a wash buffer. This process was repeated three times in total. 100 μL of substrate solution was added to each well, reacted for 20 minutes, and 50 μL of stop solution was added to each well and mixed appropriately to stop the enzyme reaction. The absorbance at 450 nm in each well was measured using a microplate reader (Table 3).

[0174] Concentration (%, w / v) Recovery % Phenylalanine Threonine Lidocaine Week 0 Week 2 Week 4 Week 8 0.4-0.24 137.21 30.6 104.3 145.10.8-0.24 130.8 133.4 118.6 138.4-0.4 0.25 142.8 127.11 10.69 5.4-0.8 0.25 145.5 128.8 126.4 101.0

[0175] Experimental results showed that when phenylalanine or threonine was used as a stabilizer and lidocaine was added, the botulinum toxin liquid composition was stable for up to 8 weeks as evaluated by enzyme-linked immunosorbent assay.

[0176] [Example 4]

[0177] Potency evaluation of a liquid botulinum toxin composition containing a stabilizer

[0178] Botulinum toxin type A produced by Jetema (Korea) was used as the botulinum toxin, and in order to evaluate the potency of the liquid composition containing botulinum toxin, the botulinum toxin liquid composition was prepared in the same manner as in Manufacturing Example 1, but the concentration range of the isotonic agent, stabilizer, and nonionic surfactant (% (w / v) based on the total volume of the liquid composition) was set to the conditions shown in Table 4 to prepare the liquid composition. At this time, the botulinum toxin was added to be 100 U / mL (Table 4). Afterwards, each experimental group was cultured for 0 to 8 weeks under harsh conditions of 40°C, diluted to a concentration of 3 to 30 U / mL, and administered once (0.1 mL / mouse) to the peritoneal cavity of 10 mice for each concentration, and the result of the number of deaths for 3 days was applied to CombiStats and statistically processed using the Probit method to obtain the potency (LD 50 ) was evaluated (Fig. 3). LD 50 (%) is 100%, which means that the mortality rate was 50% when 1U was administered to each mouse. Therefore, LD 50 (%) exceeding 100% corresponds to a case where the mortality rate exceeds 50%, which can be interpreted as indicating that the toxicity of botulinum toxin is high.

[0179] Concentration (%, w / v)LD 50 (%) Phenylalanine Threonine Polysorbate 20 Sodium Chloride Week 0 Week 4 Week 8 --0.010.8137.860.000.4-0.010.8137.7126.8100.9-0.40.010.8136.5100.980.4

[0180] The experimental results showed that the activity of the botulinum toxin in the composition was maintained well, as the composition contained phenylalanine or threonine, compared to the composition not containing phenylalanine or threonine, maintained the potency stably for up to 8 weeks.

[0181] [Example 5]

[0182] Evaluation of the potency and stability of liquid botulinum toxin compositions with and without local anesthetics

[0183] Botulinum toxin type A produced by Jetema (Korea) was used as the botulinum toxin, and in order to evaluate the stability of the liquid composition containing botulinum toxin when containing the local anesthetic lidocaine, a liquid composition containing botulinum toxin and a liquid composition containing botulinum toxin and a local anesthetic were prepared in the same manner as in Manufacturing Examples 1 and 2, respectively. At this time, the botulinum toxin was added to be 100 to 200 U / mL. The concentration of the components of each liquid composition (% (w / v) with respect to the total volume of the liquid composition) is as shown in Table 5. Afterwards, each experimental group was stored at 4℃, which is the distribution condition, and a stability evaluation was conducted for 10 months. At the 0M, 1M, 3M, 6M, and 10M time points, the samples were diluted to concentrations of 3 to 30 U / mL, and each concentration was administered once (0.1 mL / mouse) to the peritoneum of 10 mice. Based on the results of the number of deaths over 3 days, CombiStats was applied and statistically processed using the Probit method to obtain the titer (LD). 50 ) was evaluated (Fig. 4).

[0184] Concentration (%, w / v)LD 50 (%)0 M1M3M6M10MPhe(0.40%)+Polysorbate 20(0.01%)+NaCl(0.80%)136.7143.9138.1134.1145.2Thr(0.40%)+Polysorbate 20(0.01%)+NaCl(0.80%)133.6145.2133.1122.9137.8Thr(0.80%)+Polysorbate 20(0.01%)+NaCl(0.75%)+Lido(0.30%)135.2149.4137.8145.0143.9

[0185] The experimental results showed that both liquid compositions, regardless of whether they contained a local anesthetic, maintained their potency stably for up to 10 months, indicating that the activity of the botulinum toxin in the compositions was excellently maintained.

Claims

1. At least one stabilizer selected from the group consisting of phenylalanine, threonine and serine; nonionic surfactants; and A liquid composition for stabilizing botulinum toxin, comprising a topical agent as an active ingredient.

2. A liquid composition for stabilizing botulinum toxin, wherein the stabilizer is included in a concentration of 0.001 to 2.0% (w / v) based on the total volume of the liquid composition in the first paragraph.

3. A liquid composition for stabilizing botulinum toxin, wherein the nonionic surfactant in the first paragraph is at least one selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80, based on the total volume of the liquid composition.

4. A liquid composition for stabilizing botulinum toxin, wherein the nonionic surfactant is included in a concentration of 0.001 to 1.0% (w / v) based on the total volume of the liquid composition.

5. A liquid composition for stabilizing botulinum toxin, wherein the isotonic agent in paragraph 1 is at least one selected from the group consisting of sodium chloride, mannitol, glycerol, sucrose, potassium chloride, sorbitol, glucose, and dextrose.

6. A liquid composition for stabilizing botulinum toxin, wherein the isostatic agent is included in a concentration of 0.01 to 1.0% (w / v) based on the total volume of the liquid composition.

7. A liquid composition for stabilizing botulinum toxin, wherein the pH of the liquid composition is 5 to 7 in the first paragraph.

8. A liquid composition for stabilizing botulinum toxin according to any one of claims 1 to 7; and A liquid botulinum toxin preparation comprising botulinum toxin.

9. A liquid botulinum toxin preparation according to claim 8, wherein the botulinum toxin is selected from the group consisting of type A, type B, type C, type D, type E, type F, type G, or derivatives thereof.

10. A liquid botulinum toxin preparation according to claim 8, further comprising a local anesthetic.

11. A liquid botulinum toxin preparation in claim 10, wherein the local anesthetic is at least one selected from the group consisting of lidocaine, bupivacaine, and mepivacaine.

12. A botulinum toxin liquid formulation in claim 10, wherein the local anesthetic is contained in a concentration of 0.01 to 1.0% (w / v) based on the total volume of the botulinum toxin liquid formulation.

13. A liquid botulinum toxin preparation manufactured in the following steps (a) to (d) in paragraph 8: (a) a step of preparing a first mixture by mixing water and an isotonic agent; (b) a step of preparing a second mixture by adding a stabilizer to the first mixture; (c) a step of preparing a liquid composition by adding a nonionic surfactant to the second mixed solution; and (d) A step of preparing a liquid botulinum toxin preparation by adding botulinum toxin to the above liquid composition.

14. In the 13th paragraph, if the botulinum toxin liquid preparation contains a local anesthetic, the local anesthetic is added in any one of the steps (a) to (c).

15. An injection comprising a liquid botulinum toxin preparation according to any one of claims 8 to 14. 16.(a) A step of preparing a first mixture by mixing water and an isotonic agent; (b) a step of preparing a second mixture by adding at least one stabilizer selected from the group consisting of phenylalanine, threonine, and serine to the first mixture; (c) a step of preparing a liquid composition by adding a nonionic surfactant to the second mixed solution; and (d) A method for producing a liquid botulinum toxin preparation, comprising the step of adding botulinum toxin to the liquid composition.

17. A method for producing a liquid botulinum toxin preparation, wherein the botulinum toxin in paragraph 16 is selected from the group consisting of botulinum toxin types A, B, C, D, E, F, and G.

18. A method for producing a liquid botulinum toxin formulation in claim 16, wherein the isotonic agent of step (a) is at least one selected from the group consisting of sodium chloride, mannitol, glycerol, sucrose, potassium chloride, sorbitol, glucose, and dextrose.

19. A method for producing a liquid botulinum toxin preparation, wherein the nonionic surfactant of step (c) is at least one selected from the group consisting of polysorbate 20, polysorbate 40, polysorbate 60, and polysorbate 80.

20. A method for producing a liquid botulinum toxin preparation, wherein, in the 16th paragraph, the liquid botulinum toxin preparation includes a local anesthetic, the local anesthetic is added in any one of steps (a) to (c).

21. A method for producing a liquid botulinum toxin preparation in claim 20, wherein the local anesthetic is at least one selected from the group consisting of lidocaine, bupivacaine, and mepivacaine.

22. At least one stabilizer selected from the group consisting of phenylalanine, threonine and serine; nonionic surfactants; and A liquid composition for use in a stabilized botulinum toxin liquid formulation, comprising a topical agent as an active ingredient.

23. Use of a liquid composition comprising, as active ingredients, at least one stabilizer selected from the group consisting of phenylalanine, threonine and serine; a nonionic surfactant; and an isotonic agent for the manufacture of a stabilized liquid botulinum toxin preparation.

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