Botulinum toxin stabilization composition, botulinum toxin preparation containing the same, and polypeptide for use therein

Abstract

Provided are a botulinum toxin stabilization composition, a botulinum toxin preparation containing the same, and a polypeptide for use therein.

Description

Technical Field

[0001] The present disclosure relates to a botulinum toxin stabilization composition, a botulinum toxin preparation containing the same, and a polypeptide for use therein.

Background Art

[0002] Botulinum neurotoxin (BoNT) is a polypeptide product of the anaerobic bacterium Clostridium botulinum and is a toxic substance that acts specifically on nerve cells. Botulinum toxin is originally a lethal toxic substance, but it is used for the treatment of cervical dystonia (CD), blepharospasm, hyperhidrosis, strabismus, achalasia, neurogenic bladder, urologic disease, migraine, and the like.

[0003] International Publication No. WO2008 / 082889 discloses a method and composition for stabilizing botulinum toxin using a polypeptide that is an HIV-TAT fragment or a derivative of an HIV-TAT fragment.

[0004] International Publication No. WO2010 / 078242 discloses an injectable composition in which a positively charged carrier containing a specific amino acid sequence is non-covalently bound to botulinum toxin.

[0005] Alternative stabilization methods for preventing the diffusion of botulinum toxin are required.

Summary of the Invention

Problems to be Solved by the Invention

[0006] The problem to be solved by the present invention is to provide a botulinum toxin-stabilizing composition containing a polypeptide that inhibits the diffusion of botulinum toxin in vivo.

[0007] Another problem to be solved by the present invention is to provide a botulinum toxin preparation containing a botulinum toxin and the botulinum toxin-stabilizing composition.

[0008] Yet another problem to be solved by the present invention is to provide a polypeptide for use in the composition or preparation.

Means for Solving the Problems

[0009] According to one aspect, the present invention relates to a botulinum toxin-stabilizing composition containing a polypeptide of the following Chemical Formula 1 that inhibits the diffusion of botulinum toxin in vivo.

[0010] [Chemical Formula 1] (C) n -V In the above formula, C represents a moiety that interacts with the cell membrane of nerve cells, V represents a moiety that interacts with botulinum toxin while interacting with the cell membrane of nerve cells, and n is an integer of 0 or 1.

[0011] According to another aspect, the present invention relates to a botulinum toxin preparation containing a botulinum toxin and the botulinum toxin-stabilizing composition.

[0012] According to still another aspect, the present invention relates to a polypeptide consisting of the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, or SEQ ID NO: 20.

Effects of the Invention

[0013] According to the present disclosure, there are provided a botulinum toxin-stabilizing composition that prevents the diffusion of botulinum toxin in vivo and improves stability, a botulinum toxin preparation containing the same, and a polypeptide that can be used therefor.

[0014] The botulinum toxin preparation according to the present disclosure can exhibit one or more advantages compared to a normal botulinum toxin preparation, including a decrease in undesired diffusion, an increase in the duration of clinical efficacy, an improvement in stability, and a decrease in antigenicity after injection.

Brief Description of the Drawings

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Mode for Carrying Out the Invention

[0016] As used herein, "Botulinum toxin (BoNT)" is also any polypeptide or fragment of botulinum toxin. In a specific example, botulinum toxin means a botulinum toxin derivative, that is, a compound having botulinum toxin activity and containing one or more chemical modifications or functional modifications on any part or any chain with respect to native botulinum toxin or recombinant native botulinum toxin. For example, botulinum toxin is a modified neurotoxin having one or more amino acids that are deleted, modified, or substituted compared to the prototype, or the modified neurotoxin is also a neurotoxin produced by recombinant techniques or a derivative thereof, or a fragment thereof.

[0017] The botulinum toxin is also a botulinum toxin (a botulinum toxin protein molecule of about 150 kD belonging to one of botulinum toxin serotypes A-G) that is either bound to or not bound to endogenous non-toxin proteins (hemagglutinin protein and non-toxin non-hemagglutinin protein produced by Clostridium botulinum).

[0018] The botulinum toxin is divided into seven serotypes (botulinum toxin type A, botulinum toxin type B, botulinum toxin type C, botulinum toxin type D, botulinum toxin type E, botulinum toxin type F, and botulinum toxin type G), and they are further subdivided into subtypes based on mutations in the amino acid sequence. Botulinum toxins of each serotype vary in the animal species they affect and the severity and duration of the paralysis they induce.

[0019] The term "amino acid" refers to not only natural and unnatural amino acids, but also amino acids such as proline, amino acid analogs, and amino acid mimetics that function in a manner similar to natural amino acids. That is, the amino acid mimetics have a structure different from the general chemical structure of natural amino acids, but mean that they function in a manner similar to natural amino acids.

[0020] The terms "polypeptide" and "peptide" are used interchangeably herein and refer to polymers composed of amino acid residues.

[0021] The term "identity" indicates the relationship between the sequences of two or more polypeptides or polynucleotides determined by comparing the sequences. The identity indicates the degree of sequence relatedness determined by the number of matches between strings of two or more amino acid residues or nucleotide residues. The identity of related polypeptides or polynucleotides can be calculated by known methods. The "% identity" applied to a polypeptide or polynucleotide is defined as the percentage of residues in a candidate amino acid sequence or nucleotide sequence that are the same as the residues of a second sequence after aligning the candidate sequence with the second sequence to obtain maximum percent identity and introducing gaps if necessary. Methods and programs for such alignment are known. The programs are, for example, BLAST, Smith-Waterman algorithm, or Needleman-Wunsch algorithm.

[0022] One aspect provides a botulinum toxin stabilization composition comprising a polypeptide of the following Chemical Formula 1 that inhibits the diffusion of botulinum toxin in vivo.

[0023] [Chemical Formula 1] (C) n -V In the above formula, C represents a moiety that interacts with the cell membrane of a nerve cell, V represents a moiety that interacts with botulinum toxin while interacting with the cell membrane of a nerve cell, and n is an integer of 0 or 1.

[0024] In one aspect, V is also VAMP (vesicle associated membrane protein), BDNF (brain-derived neurotrophic factor), fragments thereof or variants thereof.

[0025] In one aspect, V is also VpN of VAMP, fragments thereof or variants thereof.

[0026] In one aspect, VAMP is also VAMP1, VAMP2 or VAMP3, for example, VAMP2.

[0027] For example, V is also VAMP2, a fragment thereof or a variant thereof. For example, V is also VpN of VAMP2, a fragment thereof or a variant thereof.

[0028] In one aspect, V also comprises the amino acid sequence of SEQ ID NO: 1.

[0029] In one aspect, V consists of the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10; or a sequence in which one amino acid is substituted in the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8. The amino acid of SEQ ID NO: 8 is VpN, the amino acids of SEQ ID NO: 1 to 7 are fragments of VpN, and SEQ ID NO: 9 and 10 are variants of VpN.

[0030] V can be selected from the amino acid sequences of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 and 8; and amino acid sequences in which one amino acid is substituted in the amino acid sequences of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 and 8.

[0031] A sequence in which one amino acid among the amino acids of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7 or 8 is substituted is also one in which the hydrophobic interaction is reduced compared to the sequence before substitution. For example, it is also one in which methionine and / or threonine is substituted with alanine in the amino acid sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, or 8. For example, it is also the M47A or T36A mutation of the amino acid sequence of SEQ ID NO: 8. In one aspect, C is also a cell penetrating peptide or a cationic peptide.

[0032] For example, the cationic amino acid can consist of lysine, arginine, and histidine. For example, the cationic amino acid is also arginine.

[0033] In a specific example, C is also an arginine-rich peptide. For example, it is also one in which 3 / 5 or more or 2 / 3 or more of the total amino acids of C are composed of arginine.

[0034] In one aspect, C can further contain aromatic amino acids.

[0035] For example, the aromatic amino acid can be selected from tryptophan, phenylalanine, and tyrosine.

[0036] In a specific example, the aromatic amino acid is also tryptophan.

[0037] In a specific example, C can contain 6 to 9 arginines and 2 to 3 tryptophans. For example, C can be composed of 6 to 9 arginines and 2 to 3 tryptophans.

[0038] For example, when C is composed of 6 to 9 arginines and 2 to 3 tryptophans, the amino acids of the same sequence can be repeated up to 3 times at most.

[0039] In one aspect, C also has a three-dimensional structure in which arginine and tryptophan are biased to one side.

[0040] In one aspect, C can consist of the amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.

[0041] In one aspect, the polypeptide can consist of 10 to 50, 10 to 40, 15 to 40, or 15 to 35 amino acids, for example, 17 to 32 or 25 to 32 amino acids, for example, 17, 25, 26, 31, or 32 amino acids.

[0042] In one aspect, the polypeptide can consist of the amino acid sequence of SEQ ID NO: 8 (TSP19), SEQ ID NO: 14 (TSP11), SEQ ID NO: 15 (TSP28), SEQ ID NO: 16 (TSP17), SEQ ID NO: 17 (TSP35), SEQ ID NO: 18 (TSP23), SEQ ID NO: 19 (TSP2), SEQ ID NO: 20 (TSP56), or SEQ ID NO: 21 (TSP61).

[0043] The polypeptide is also a variant of the disclosed amino acid sequence. The variant is also a polypeptide having at least 80% sequence identity with the disclosed amino acid sequence. For example, it can have at least 82.5%, 85%, 90%, 95%, or 98% sequence identity with the disclosed amino acid sequence.

[0044] In the polypeptide, the N-terminus is also protected by a protecting group. The N-terminus is bound to an acetyl group, a fluoreonylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, a butoxycarbonyl group, an allyloxycarbonyl group, or polyethylene glycol (PEG) as the protecting group.

[0045] The C-terminus of the polypeptide is also protected by a protecting group. The C-terminus is bound to an amino group (-NH2), a tertiary alkyl group, or an azide (-NHNH2) as the protecting group. In the tertiary alkyl group, the alkyl group has 4 to 20, 4 to 16, 4 to 12, 4 to 8, 5 to 20, 5 to 16, 5 to 12, or 5 to 8 carbon atoms.

[0046] Another aspect provides a method of inhibiting the in vivo diffusion of botulinum toxin by providing the polypeptide of Chemical Formula 1 to botulinum toxin or a preparation containing the same.

[0047] Another aspect provides a method of stabilizing botulinum toxin by providing the polypeptide of Chemical Formula 1 to botulinum toxin or a preparation containing the same.

[0048] Another aspect provides the use of the polypeptide of Chemical Formula 1 for inhibiting the in vivo diffusion of botulinum toxin.

[0049] Another aspect provides the use of the polypeptide of Chemical Formula 1 for stabilizing botulinum toxin.

[0050] Another aspect provides a use for the polypeptide of Chemical Formula 1 as a botulinum toxin excipient.

[0051] Another aspect provides a botulinum toxin preparation comprising a botulinum toxin and the botulinum toxin stabilization composition.

[0052] Another aspect provides the polypeptide of Chemical Formula 1 for inhibiting the diffusion of botulinum toxin in vivo.

[0053] Another aspect provides the polypeptide of Chemical Formula 1 for stabilizing botulinum toxin.

[0054] Another aspect provides the polypeptide of Chemical Formula 1 for use as a botulinum toxin excipient.

[0055] In one aspect, the botulinum toxin is also of type A, B, C, D, E, F or G.

[0056] In one aspect, the botulinum toxin is also a natural or recombinant toxin.

[0057] In a specific example, the recombinant toxin is also the one disclosed in Korean Application No. 10-2021-0083930 (the disclosure content of which is incorporated herein by reference in its entirety), and the recombinant toxin is not a type A4 botulinum toxin, but contains a recombinant type A botulinum toxin light chain in which the sequence of the third domain among the first, second, third, and fourth domains of the type A botulinum toxin light chain is replaced with the sequence of the third domain of the type A4 botulinum toxin or its variant. For example, it may also be one in which the sequence of the fourth domain among the first, second, third, and fourth domains of the recombinant type A botulinum toxin light chain is further replaced with the sequence of the fourth domain of the type A4 botulinum toxin or its variant. For example, the type A botulinum toxin light chain may also be a type A1, A2, A3, A5, A6, A7, or A8 botulinum toxin, for example, a type A1 botulinum toxin. For example, the recombinant type A botulinum toxin light chain may also contain A1-A1-A4-A4 as the first, second, third, and fourth domains. For example, the recombinant type A botulinum toxin light chain may consist of the amino acid sequence of SEQ ID NO: 23.

[0058] In a specific example, the recombinant toxin is also the one disclosed in Korean Application No. 10-2021-0083950 (the disclosure content of which is incorporated herein by reference in its entirety), and the recombinant toxin is not a type A1 botulinum toxin, but may contain a recombinant type A botulinum toxin light chain in which the sequence of the second domain among the first, second, third, and fourth domains of the type A botulinum toxin light chain is replaced with the sequence of the second domain of the type A1 botulinum toxin or its variant. For example, it may also be one in which the sequence of the fourth domain among the first, second, third, and fourth domains of the recombinant type A botulinum toxin light chain is further replaced with the sequence of the fourth domain of the type A1 botulinum toxin or its variant. For example, the type A botulinum toxin light chain may also be a type A2, A3, A4, A5, A6, A7, or A8 botulinum toxin, for example, a type A4 botulinum toxin. For example, the recombinant type A botulinum toxin light chain may also contain A4-A1-A4-A1 as the first, second, third, and fourth domains.

[0059] In one aspect, the botulinum toxin is also in the form of 7S (150 kD) or 19S (900 kD).

[0060] In one aspect, the amount of the polypeptide used is not particularly limited as long as it exhibits the activity of inhibiting the diffusion of botulinum toxin and does not inhibit the desired clinical efficacy of botulinum toxin. For example, it can be 10 to 10 10 times, 10 2 to 10 9 times, 10 3 to 10 8 times, 10 3 to 10 7 times, 10 3 to 10 6 times, or an amount of 3×10 3 to 10 6 times.

[0061] The botulinum toxin preparation may further contain a pharmaceutically acceptable excipient or carrier.

[0062] The term "pharmaceutically acceptable" means that the botulinum toxin stabilization composition or component is suitable for use in contact with tissues or for use in patients without excessive toxicity, instability, or allergic reaction. When pharmaceutically or cosmetically typically acceptable, the botulinum toxin preparation of the present disclosure may contain any component commonly used in the fields of pharmacy and dermatology.

[0063] The botulinum toxin formulation of the present disclosure can be manufactured by mixing with one or more additional pharmaceutically acceptable excipients or additives. For example, it can contain merely aqueous pharmaceutically acceptable excipients or additives such as buffered saline. In that case, the botulinum toxin formulation of the present disclosure can be applied by injection. Pharmaceutically acceptable excipients or additives are stabilizers, ionic compounds, surfactants, buffers, cryoprotectants, or combinations thereof, for example, amino acids (e.g., methionine), salts (e.g., NaCl), buffers, nonionic surfactants (e.g., polysorbates, e.g., polysorbate 20), sugars (e.g., disaccharides such as sucrose), sugar alcohols (e.g., sorbitol), or combinations thereof.

[0064] The botulinum toxin formulation also does not contain albumin or components or polysaccharides derived from animals.

[0065] The botulinum toxin formulation can be formulated into any form, such as a solid or liquid formulation, for example, a lyophilized powder, a liquid, or a pre-filled syringe formulation.

[0066] The botulinum toxin formulation of the present disclosure can include a solution, an emulsion (including a microemulsion), a suspension, a cream, a lotion, a gel, a powder, or other typical solid or liquid compositions for use in applying the botulinum toxin formulation of the present disclosure to the skin or other tissues to which it is applied. Such compositions can include, in addition to the botulinum toxin and the stabilizing peptide, other commonly used ingredients, such as antimicrobial agents, humectants, and hydrating agents, penetration agents, preservatives, emulsifiers, natural or synthetic oils, solvents, surfactants, detergents, gelling agents, emollients, antioxidants, fragrances, fillers, thickeners, waxes, odor absorbers, dyes, colorants, powders, viscosity modifiers, and water, and optionally, anesthetic agents, anti-itch actives, plant extracts, conditioning agents, darkening agents or lightening agents, glitter, humectants, mica, minerals, polyphenols, silicon or its derivatives, sunblocks, vitamins, and phytomedicinals.

[0067] Other aspects provide a polypeptide consisting of the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, or SEQ ID NO: 18 for use in the botulinum toxin stabilizing compositions and formulations.

[0068] Other aspects provide for the use of the botulinum toxin formulation for administering to an individual to ameliorate or treat a disease.

[0069] "Administer" or "administering" means the step of providing a pharmaceutical composition or an active ingredient to a subject. The pharmaceutical composition can be administered through a variety of suitable routes.

[0070] "Pharmaceutical composition" means a formulation in which the active ingredient is also botulinum toxin. The term "formulation" means that there is at least one additional ingredient in the pharmaceutical composition in addition to the botulinum neurotoxin active ingredient, for example, albumin (human serum albumin or recombinant human albumin) and / or sodium chloride. The pharmaceutical composition is a formulation suitable for administration to a subject such as a human patient. The pharmaceutical composition is also in a lyophilized form, for example, a solution formed after reconstitution of a lyophilized pharmaceutical composition using saline or water, or in a solution form that does not require reconstitution. The pharmaceutical composition can be liquid or solid.

[0071] The present disclosure is applied such that an effective amount of the botulinum toxin formulation is administered. As used herein, the term "effective amount" means an amount of the botulinum toxin formulation that is sufficient to produce the desired effect but is safe, i.e., can prevent serious side effects. The desired effect includes, for example, reducing fine wrinkles, particularly facial fine wrinkles, or adjusting the facial contour, or relaxing specific muscles for the purpose of overall relaxation of muscle tension. Overall relaxation of muscle tension can occur in the face or other areas.

[0072] "Treat", "treating" or "treatment" means, for example, achieving a desired therapeutic result by wounding, healing damaged tissue, or altering, changing, enhancing, improving, ameliorating and / or beautifying an existing or recognized disease, disorder or abnormality, including reduction or decrease (including partial reduction, substantial reduction, near complete reduction and complete reduction), resolution or prevention (temporary or permanent) of a disease, disorder or abnormality. As used herein, "treatment" is a concept that includes prevention. "Prevention" means delaying the onset of a disease, disorder or illness. Prevention is considered complete if the onset of the disease, disorder or illness is delayed for the expected period.

[0073] Another aspect is to provide the use of the botulinum toxin formulation for administering to an individual to improve or treat a disease.

[0074] The "individual" means a subject in need of treatment for a disease. The individual is also a mammal such as, for example, a human, mouse, cat, horse, and cow.

[0075] The present disclosure is for administering the botulinum toxin preparation to a patient to improve or treat a disease. In a specific example, the stabilized botulinum toxin stabilized composition is applied in an effective amount to an individual or patient in need of treatment for a disease, for example, treatment of muscle paralysis, reduction of hypersecretion or sweating, treatment of nervous system pain or migraine, reduction of muscle spasm, prevention or reduction of acne, reduction or enhancement of immune response, reduction of wrinkles, or prevention or treatment of various other diseases. In one specific example, the botulinum toxin preparation is administered by parenteral injection such as, for example, subcutaneous injection. Administration is, for example, to the leg, shoulder, waist, palm of the hand, foot, neck, elbow, back of the hand, foot, wrist, upper arm, knee, upper limb, buttocks, torso, pelvis, or a body part where administration of the botulinum toxin composition is desired.

[0076] In addition, the present disclosure includes a transdermal delivery device for delivering the botulinum toxin stabilized composition described herein through the skin. Such a device can also be a simple structure such as a skin patch, or a device including means for dispensing the composition, monitoring the dispensing of the composition, and means for monitoring the individual's response to the dispensed pharmaceutical composition. / / The botulinum toxin preparation of the present disclosure may have a pH suitable for use in a physiological environment having a pH in the range of about 4.5 to 6.3. The botulinum toxin preparation according to the present disclosure can be stored at room temperature or under refrigeration conditions.

[0077] The therapeutically effective amount of botulinum toxin is about 0.01 U / kg to 100 U / kg, about 0.1 U / kg to 100 U / kg, about 0.2 U / kg to 100 U / kg, about 0.2 U / kg to 50 U / kg, about 0.2 U / kg to 30 U / kg, about 0.2 U / kg to 10 U / kg, about 0.2 U / kg to 1 U / kg. In other embodiments, based on an adult of 60 kg body weight, it is also about 1 U to 10,000 U, about 1 U to 5,000 U, about 1 U to 2,500 U, about 1 U to 1,000 U, about 1 U to 500 U, about 1 U to 300 U, about 1 U to 200 U, about 10 U to 200 U, about 10 U to 100 U, about 10 U to 50 U.

[0078] As used herein, the terms "unit", "unit(s)", or "U" are defined as the amount of botulinum toxin that kills 50% of the mice that have received a botulinum toxin injection and are used interchangeably to refer to the dose. 50

[0079] Yet another embodiment is to provide a method of stabilizing botulinum toxin by administering the botulinum toxin preparation to an individual.

[0080] The term "stabilization" minimizes the formation of aggregates (insoluble and / or soluble) or chemical decomposition during the storage or production of a pharmaceutical composition, and means maintaining the pH and native structure of a protein to maintain substantial retention of biological activity and protein stability. It also means preventing the diffusion of botulinum toxin to other tissues and reducing the risk of side effects.

[0081] When botulinum toxin is applied topically to the body for use in improving or treating a disease, it diffuses over time, which can cause unwanted muscle paralysis in other parts of the body. For example, if botulinum toxin is administered around the eyes for wrinkle improvement and diffuses to adjacent tissues, "Ptosis" may occur and interfere with normal vision.

[0082] The botulinum toxin formulation of the present disclosure can reduce diffusion after administration to the human body. Thereby, it can be accurately transmitted to the target tissue of the botulinum toxin and reduce the unwanted side effects associated with the diffusion of the botulinum toxin.

[0083] The botulinum toxin formulation of the present disclosure can reduce the unwanted side effects associated with the diffusion of the botulinum toxin, and thus can contain an amount of botulinum toxin increased compared to the amount normally used (used in formulations without polypeptides) in the formulation. In that case, it can show an increased clinical efficacy duration of the botulinum toxin compared to a normal botulinum toxin formulation. Thereby, the administration cycle can be extended and the administration frequency can be reduced. In a specific example, the botulinum toxin formulation can contain 1.5 times, 2 times, 3 times, 4 times, 5 times or more the amount of botulinum toxin normally used. In a specific example, the botulinum toxin formulation can be administered at an administration cycle 1.5 times, 2 times, 3 times, 4 times or more the normal administration cycle, for example, a cycle exceeding 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months or 12 months or more.

[0084] Hereinafter, the present disclosure will be described in detail with examples, which are merely illustrative for explaining the present disclosure and are not intended to limit its scope.

[0085] In this experiment, the polypeptides shown in Table 1 below were synthesized (company name: AnyGen) and used. In the structures of Table 1 below, in addition to the amino acid structure by the sequence number, the presence or absence of an amino group bond as a protecting group was also indicated.

[0086]

Table 1

[0087] Example 1: Non-clinical efficacy test of a mixture type of botulinum toxin type A and TSP11 in mice Five-week-old female CD1 (ICR) mice were purchased from Orient Bio Inc. After one week of acclimation and quarantine, six-week-old mice were used in the experiment. The mice were fed with sterilized solid feed for experimental animals (R40-10, SAFE, France) ad libitum, and the drinking water was autoclaved tap water and given ad libitum. During the acclimation, quarantine, and experimental periods, the mice were housed under specific pathogen-free conditions with a temperature of 23 ± 3°C, relative humidity of 55 ± 15%, a lighting period of 12 hours (8:00 am to 8:00 pm), a ventilation rate of 15 times / hour, and an illuminance of 150 - 300 Lux. This study was conducted after review and approval by the Animal Experiment Ethics Committee of Meditox Inc. (A-2020-016).

[0088] The test substance used was Coretox injection (Meditox), and the placebo (sham-administered group) used was sterilized physiological saline (Korea Pharmaceutical Industry). The toxin-stabilizing peptide (TSP11) was mixed with the peptide at a ratio of 3x10 4 ~1x10 5 and used in the experiment (Table 2). The mixture of botulinum toxin and toxin-stabilizing peptide was diluted to a concentration of 120 U / mL and used in the experiment.

[0089] The day when the test substance was administered was designated as day 0. After anesthetizing the mice with an injection anesthetic (100 mg / kg ketamine hydrochloride + 10 mg / kg xylazine), each test substance was administered at 0.2 mL / kg to the right gastrocnemius muscle of the mice using a Hamilton syringe according to the group composition in Table 2.

[0090]

Table 2

[0091] The evaluation used a compound muscle action potential (CMAP) test method that measures the action potential of muscles in response to external electrical stimulation. CMAP was measured from the right gastrocnemius muscle site (administration site) and the left gastrocnemius muscle (non-administration site) of each mouse using Nicolet Viking Quest (Viasys Healthcare, Inc.) or UltraPro S100 (Natus Neurology Inc.) equipment. After anesthesia with an injection anesthetic, the measurement site was depilated and the mouse was positioned in a prone position. The negative electrode was positioned at the sciatic nerve site of the leg to be measured, and the positive electrode was positioned at a site approximately 1 cm away from that site with the spine as a reference. The recording electrode and the reference electrode were positioned at the gastrocnemius muscle site and the Achilles tendon site, respectively, and the ground electrode was positioned at the rectus femoris muscle site. The stimulation level and duration were set at 7 - 8 mA and 0.1 ms, and the filter range of the amplifier was set at 60 Hz and 2 - 10 K. When measured under these conditions, the height from the base to the highest point of the waveform was converted into data as the CMAP measurement value. After administration of the test substance, measurements were taken on the gastrocnemius muscle at the administration site and the gastrocnemius muscle of the contralateral leg on days 7 and 14.

[0092] Graphpad Prism 7.05 (GraphPad Software Inc., CA, USA) was used for graph presentation, and SPSS software 25.0 (SPSS Inc., IL, USA) and Excel (2013, MS, USA) were used for statistical analysis. The results of the experiment were expressed as mean ± standard deviation. A normality test was performed using the Kolmogorov - Smirnov test. Non-parametric data were statistically analyzed through the Mann-Whitney test, and in the case of parametric data, a two-tailed t-test was performed. When the p-value was less than 0.05, it was judged to be statistically significant.

[0093] After administering the BoNT / A+TSP11 test substance, which was a mixture of botulinum toxin type A (BoNT / A) and a toxin-stabilizing peptide, at a dose of 24 U / kg to the right gastrocnemius muscle of a mouse, CMAP measurements were performed on the administered-site muscle and the non-administered-site muscle on the opposite side on days 7 and 14. As a result, no significant difference in the CMAP value of the administered-site muscle was observed between the BoNT / A single administration group and the BoNT / A+TSP11 administration group. However, the CMAP value of the contralateral muscle was significantly higher in the BoNT / A+15.5 ng / U TSP11 administration group and the BoNT / A+4.65 ng / U TSP11 administration group compared to the BoNT / A single administration group (Figure 1). From such results, a reduced diffusion effect to the contralateral leg was confirmed through the mixing of botulinum toxin and TSP11.

[0094] Example 2: Non-clinical efficacy test of a recombinant botulinum toxin type A and TSP11 mixture in mice Similar to Example 1, 5-week-old CD1 (ICR) female mice were purchased from Orient Bio Inc. After 1 week of acclimation and quarantine, 6-week-old mice were used in the experiment. The feed, drinking water, and breeding conditions were the same as those in Example 1. This study was conducted after review and approval by the Medytox Animal Experiment Ethics Committee (A-2020-016).

[0095] For the placebo (sham administration group), sterilized physiological saline (Korea Pharmaceutical Industry) was used. The recombinant botulinum toxin type A (eBoNT / A) was a recombinant botulinum toxin in which the light chain of the botulinum toxin consisted of a combination of BoNT / A1 light chain and BoNT / A4 light chain. The recombinant full-length botulinum toxin gene (SEQ ID NO: 24) was cloned by the infusion method (infusion-HD, Takara) using the pMTL80000 vector system, and a detoxified hall A-hyper strain in which the toxin gene was inactivated was used for production and purified into a form of approximately 150 kDa without complex components. All production of the recombinant botulinum toxin was carried out in a facility where toxin production was permitted by government regulations. The toxin-stabilizing peptide (TSP11) was used in the experiment by mixing the peptide at a ratio of 4.65 to 46.5 ng / U (Table 3).

[0096] The administration doses of each test substance were all administered identically based on unit (U: a value indicating the biological activity of botulinum toxin, where 1 U is the median lethal dose for mice by intraperitoneal administration to mice). The mixture of botulinum toxin and toxin-stabilizing peptide was diluted to a concentration of 60 U / mL and used in the experiment.

[0097] The day when the test substance was administered was set as day 0. After anesthetizing the mice using an injection anesthetic (100 mg / kg ketamine hydrochloride + 10 mg / kg xylazine), each test substance was administered at 0.2 mL / kg to the right gastrocnemius muscle of the mice using a Hamilton syringe according to the group composition in Table 3. Six mice were used per test substance for each test, and a total of 2 to 3 repeated tests were performed for each test substance. For the evaluation, the compound muscle action potential CMAP test method, which measures the action potential of the muscle that responds to external electrical stimulation, was used. The specific evaluation method and statistical analysis were carried out in the same manner as in Example 1.

[0098]

Table 3

[0099] After administering recombinant botulinum toxin type A (eBoNT / A) and the eBoNT / A+TSP11 test substance, which is a mixture of recombinant botulinum toxin type A and a toxin-stabilizing peptide, to the right gastrocnemius muscle of mice at a dose of 12 U / kg, CMAP measurements were performed on the muscle at the administration site and the non-administration site muscle on the opposite side on days 7 and 14. As a result, for the CMAP value of the muscle at the administration site, a significant increase in CMAP was observed in the eBoNT / A+46.5 ng / U TSP11 administration group on day 7 after administration compared to the eBoNT / A alone administration group, but on day 14 after administration, it was not observed in the test group mixed with the toxin-stabilizing peptide compared to the eBoNT / A alone administration group (Figure 2). For the CMAP value in the muscle on the opposite side, a significant increase was observed in the eBoNT / A+TSP11 administration group compared to the eBoNT / A alone administration group on both days 7 and 14 after administration (Figure 2). From such results, it was confirmed that the diffusion of recombinant botulinum toxin type A to the opposite leg is reduced through mixing with the toxin-stabilizing peptide (TSP11).

[0100] Example 3: IM LD for botulinum toxin type A and TSP1, TSP11, TSP28 mixture types in mice 50 Test Similar to Example 1, 5-week-old CD1 (ICR) female mice were purchased from Orient Bio Inc., and after 1 week of acclimation and quarantine, 6-week-old mice were used in the experiment. The feed, drinking water, and breeding conditions were the same as in Example 1. This study was conducted after review and approval by the Medytox Animal Experiment Ethics Committee (A-2020-015).

[0101] The test substance used was Coaxin Injection (Medytox), and the toxin-stabilizing peptides (TSP1, TSP11, TSP28) were mixed with the peptide at a ratio of 3×10 5 to 1 mole of botulinum toxin and used in the experiment (Table 4). The mixture of botulinum toxin and toxin-stabilizing peptide was diluted to concentrations of 300, 450, 675, and 1012.5 U / mL and used in the experiment.

[0102] The day when the test substance was administered was designated as day 0, and the anesthesia of the experimental animals and the administration of the test substance were carried out in the same manner as in Example 1. Ten mice were used per test group for each test, and a total of two repeated tests were performed. After administering the test substance to the right gastrocnemius muscle, the number of dead individuals was confirmed for 7 days. For the dead individuals in each administration dose, the IM LD 50 value was calculated through probit analysis using SPSS software 25.0 (SPSS Inc., IL, USA).

[0103]

Table 4

[0104]

Table 5

[0105] As shown in Table 5, after administering the test substances of BoNT / A + TSP1, BoNT / A + TSP28, and BoNT / A + TSP11, in which botulinum toxin type A (BoNT / A) and a toxin-stabilizing peptide were mixed, to the right gastrocnemius muscle of mice, the occurrence of dead individuals was observed for 7 days, and the median lethal dose (IM LD 50 ) was calculated. As a result, the BoNT / A alone administration group was observed to have an average of 91.1 U / kg, the BoNT / A + TSP1 administration group had an average of 76.6 U / kg, the BoNT / A + TSP28 administration group had an average of 125.3 U / kg, and the BoNT / A + TSP11 administration group had an average of 147.5 U / kg. An approximately 1.4-fold increase was observed in the BoNT / A + TSP28 administration group and an approximately 1.6-fold increase in the BoNT / A + TSP11 administration group compared to the BoNT / A alone administration group (Table 5). From such results, the effect of improving the stability through the mixing of botulinum toxin and toxin-stabilizing peptides (TSP11, TSP28) was confirmed.

[0106] Example 4: IM LD for a recombinant botulinum toxin type A and TSP11 mixture in mice 50 Test Similar to Example 1, 5-week-old CD1 (ICR) female mice were purchased from Orient Bio Inc., and after 1 week of acclimation and quarantine, 6-week-old mice were used in the experiment. The feed, drinking water, and breeding conditions were the same as those in Example 1. This study was conducted after examination and approval (A-2020-015) by the Meditox Animal Experiment Ethics Committee, Inc.

[0107] As the test substance, the same recombinant botulinum toxin type A as in Example 2 was used. The toxin-stabilizing peptide (TSP11) was used in the experiment at a ratio of 46.5 ng / U (Table 6). The mixture of recombinant botulinum toxin type A and the toxin-stabilizing peptide was diluted at concentrations of 300, 450, and 675 U / mL and used in the experiment. The day when the test substance was administered was designated as Day 0, and the anesthesia of the experimental animals and the administration of the test substance were carried out in the same manner as in Example 1. After administering the test substance to the right gastrocnemius muscle, the number of dead individuals was confirmed for 14 days. For the dead individuals at each administration dose, the IM LD 50 value was calculated through probit analysis using SPSS software 25.0 (SPSS Inc., IL, USA).

[0108]

Table 6

[0109]

Table 7

[0110] As shown in Table 7, after administering the eBoNT / A+TSP11 test substance, which is a mixture of recombinant botulinum toxin type A (eBoNT / A) and a toxin-stabilizing peptide, to the right gastrocnemius muscle of mice, the occurrence of dead individuals was observed for 14 days to calculate the median lethal dose. As a result, the eBoNT / A alone administration group was observed at 63.8 U / kg, and the eBoNT / A+TSP11 administration group was observed at 81.2 U / kg, showing an approximately 1.3-fold increase compared to the eBoNT / A alone administration group (Table 7). From such results, the effect of improving the stability of recombinant botulinum toxin through the mixing of recombinant botulinum toxin and a toxin-stabilizing peptide (TSP11) was confirmed.

[0111] Example 5: Non-clinical Efficacy Test for a Mixture of Botulinum Toxin Type A and TSP11, TSP17, and TSP19 in Mice Similar to Example 1, 5-week-old CD1 (ICR) female mice were purchased from Orient Bio Inc., and after 1 week of acclimation and quarantine, 6-week-old mice were used in the experiment. The feed, drinking water, and breeding conditions were the same as those in Example 1.

[0112] Coax toxin injection (Meditox) was used as the test substance, and placebo (sham drug administration group) used sterilized physiological saline (Korea Pharmaceutical Industry). Toxin-stabilizing peptides (TSP11, TSP17, TSP19) were synthesized (company name: AnyGen) and used, and the peptides were mixed at a ratio of 3×10 5 with respect to 1 mole of botulinum toxin and used in the experiment (Table 8). The mixture of botulinum toxin and toxin-stabilizing peptide was diluted to a concentration of 120 U / mL and used in the experiment.

[0113] The day when the test substance was administered was set as day 0, and the anesthesia of the experimental animals and the administration of the test substance were carried out in the same manner as in Example 1.

[0114] For the evaluation, the compound muscle action potential CMAP test method, which measures the action potential of muscles that respond to external electrical stimulation, was used. The specific evaluation method and statistical analysis were carried out in the same manner as in Example 1.

[0115]

Table 8

[0116] After administering test substances BoNT / A+TSP17, BoNT / A+TSP19, and BoNT / A+TSP11, in which botulinum toxin type A (BoNT / A) and a toxin-stabilizing peptide were mixed, to the right gastrocnemius muscle of a mouse at a dose of 24 U / kg, CMAP measurements were performed on the administered-site muscle and the non-administered-site muscle on the opposite side on days 7 and 14. As a result, no significant difference in the CMAP value of the administered-site muscle was observed between the group administered BoNT / A alone and the groups administered BoNT / A+TSP17, BoNT / A+TSP19, and BoNT / A+TSP11. However, the CMAP value of the muscle on the opposite side was significantly higher in the groups administered BoNT / A+TSP19 and BoNT / A+TSP11 on day 7 after administration and in the groups administered BoNT / A+TSP17 and BoNT / A+TSP11 on day 14 after administration, as compared with the group administered BoNT / A alone (Figure 3). From such results, a reduced diffusion effect to the opposite leg was confirmed through the mixing of botulinum toxin and toxin-stabilizing peptides (TSP11, TSP17, TSP19).

[0117] Example 6: Non-clinical efficacy test for a mixture of botulinum toxin type A and TSP11 and TSP28 in mice Similar to Example 1, 5-week-old CD1 (ICR) female mice were purchased from Orient Bio Inc., and after 1 week of acclimation and quarantine, 6-week-old mice were used in the experiment. The feed, drinking water, and breeding conditions were the same as those in Example 1.

[0118] Coax toxin injection (Meditox) was used as the test substance, and placebo (sham-dosing group) used sterile physiological saline (Korea Pharmaceutical Industry). Toxin-stabilizing peptides (TSP11, TSP28) were mixed at a ratio of 3×10 5 to 1 mole of botulinum toxin and used in the experiment (Table 9). The mixture of botulinum toxin and toxin-stabilizing peptide was diluted to a concentration of 120 U / mL and used in the experiment.

[0119] The day when the test substance was administered was defined as day 0, and the anesthesia of the experimental animals and the administration of the test substance were carried out in the same manner as in Example 1.

[0120] For the evaluation, the compound muscle action potential CMAP test method, which measures the muscle action potential in response to external electrical stimulation, was used. The specific evaluation method and statistical analysis were carried out in the same manner as in Example 1.

[0121]

Table 9

[0122] After administering the test substances BoNT / A+TSP11 and BoNT / A+TSP24, in which botulinum toxin type A (BoNT / A) and a toxin-stabilizing peptide were mixed, to the right gastrocnemius muscle of mice at a dose of 24 U / kg, CMAP measurements were performed on the muscle at the administration site and the non-administration site muscle on the opposite side on days 7 and 14. As a result, no significant difference was observed in the CMAP values of the muscle at the administration site among the BoNT / A alone administration group, the BoNT / A+TSP11 group, and the BoNT / A+TSP28 group. However, the CMAP values in the muscle on the opposite side were significantly higher in both the BoNT / A+TSP11 and BoNT / A+TSP28 administration groups on days 7 and 14 after administration compared to the BoNT / A alone administration group (Figure 4). From such results, a reduced diffusion effect to the opposite leg was confirmed through the mixing of botulinum toxin and toxin-stabilizing peptides (TSP11, TSP28).

[0123] Example 7: Non-clinical efficacy test of a mixture of botulinum toxin type A 19S and TSP11 in mice Similar to Example 1, 5-week-old CD1 (ICR) female mice were purchased from Orient Bio Inc., and after 1 week of acclimation and quarantine, 6-week-old mice were used in the experiment. The feed, drinking water, and breeding conditions were the same as in Example 1.

[0124] As the test substance, a 19S non-albumin formulation (Meditox; a formulation containing 19S toxin instead of the 7S toxin of Coax) was used, and for the placebo (sham administration group), sterilized physiological saline (Korea Pharmaceutical Industry) was used. The toxin-stabilizing peptide (TSP11) was 3×10 with respect to 1 mole of botulinum toxin 5The peptides were mixed at the ratio shown in (Table 10) and used in the experiment. The mixture of botulinum toxin and the toxin-stabilizing peptide was diluted to a concentration of 120 U / mL and used in the experiment.

[0125] The day when the test substance was administered was defined as day 0, and the anesthesia of the experimental animals and the administration of the test substance were carried out in the same manner as in Example 1. For the evaluation, the compound muscle action potential (CMAP) test method, which measures the action potential of the muscle that responds to external electrical stimulation, was used. The specific evaluation method and statistical analysis were carried out in the same manner as in Example 1.

[0126]

Table 10

[0127] After administering the BoNT / A19S+TSP11 test substance, which is a mixture of botulinum toxin type A 19S (BoNT / A) and the toxin-stabilizing peptide, to the right gastrocnemius muscle of mice at a dose of 24 U / kg, CMAP measurements were performed on the muscle at the administration site and the non-administration site on the opposite side on days 7 and 14. As a result, no significant difference was observed in the CMAP values of the muscle at the administration site between the group administered BoNT / A19S alone and the group administered BoNT / A19S+TSP11. However, the CMAP value in the muscle on the opposite side was significantly higher in the BoNT / A19S+TSP11 administration group than in the BoNT / A19S alone administration group on day 14 after administration (Figure 5). From such results, a reduced diffusion effect to the opposite leg was confirmed through the mixing of botulinum toxin 19S and the toxin-stabilizing peptide (TSP11).

[0128] Example 8: Non-clinical efficacy test of a mixture of botulinum toxin type A and TSP1 and TSP2 in mice In the same manner as in Example 1, 5-week-old CD1 (ICR) female mice were purchased from Orient Bio Inc., and after 1 week of acclimation and quarantine, 6-week-old mice were used in the experiment. The feed, drinking water, and breeding conditions were the same as in Example 1.

[0129] Coretox injection (Meditox) was used as the test substance, and placebo (sham drug administration group) used sterile physiological saline (Korea Pharmaceutical Industry). Toxin-stabilizing peptides (TSP1, TSP2) were mixed with 1 mol of botulinum toxin at a ratio of 1×10 6 and used in the experiment (Table 11). The mixture of botulinum toxin and toxin-stabilizing peptide was diluted to a concentration of 60 U / mL and used in the experiment.

[0130] The day when the test substance was administered was designated as day 0, and anesthesia of the experimental animals and administration of the test substance were carried out in the same manner as in Example 1.

[0131] For the evaluation, the compound muscle action potential CMAP test method, which measures the action potential of muscles that respond to external electrical stimulation, was used. The specific evaluation method and statistical analysis were carried out in the same manner as in Example 1.

[0132]

Table 11

[0133] After administering the test substances BoNT / A+TSP1 and BoNT / A+TSP2, which are mixtures of botulinum toxin type A (BoNT / A) and toxin-stabilizing peptides, to the right gastrocnemius muscle of mice at a dose of 12 U / kg, CMAP measurements were performed on the muscle at the administration site and the non-administration site muscle on the opposite side on days 7 and 14. As a result, no significant difference was observed in the CMAP values of the muscle at the administration site among the BoNT / A alone administration group, the BoNT / A+TSP1 administration group, and the BoNT / A+TSP2 administration group. However, the CMAP value in the muscle on the opposite side was significantly higher in the BoNT / A+TSP2 administration group compared to the BoNT / A alone administration group (Figure 6). On the other hand, in the case of the BoNT / A+TSP1 administration group, no significant difference was observed in the CMAP value in the muscle on the opposite side compared to the BoNT / A alone administration group. From such results, a reduced diffusion effect to the opposite leg was confirmed through the mixing of botulinum toxin and TSP2.

[0134] Example 9: Non-clinical efficacy test of a mixture of botulinum toxin type A and TSP11, TSP23 in mice Similar to Example 1, 5-week-old CD1 (ICR) female mice were purchased from Orient Bio Inc. After 1 week of acclimation and quarantine, 6-week-old mice were used in the experiment. The feed, drinking water, and breeding conditions were the same as those in Example 1.

[0135] Coretox injection (Meditox) was used as the test substance, and placebo (sham drug administration group) used sterile physiological saline (Korea Pharmaceutical Industry). Toxin-stabilizing peptides (TSP11, TSP23) were mixed with botulinum toxin at a ratio of 3×10 5 and used in the experiment (Table 12). The mixture of botulinum toxin and toxin-stabilizing peptide was diluted to a concentration of 120 U / mL and used in the experiment.

[0136] The day when the test substance was administered was set as day 0, and the anesthesia of the experimental animals and the administration of the test substance were carried out in the same manner as in Example 1.

[0137] For the evaluation, the compound muscle action potential CMAP test method, which measures the action potential of muscles that respond to external electrical stimulation, was used. The specific evaluation method and statistical analysis were carried out in the same manner as in Example 1.

[0138]

Table 12

[0139] After administering the test substances BoNT / A+TSP11 and BoNT / A+TSP23, which are mixtures of botulinum toxin type A (BoNT / A) and toxin-stabilizing peptide, to the right gastrocnemius muscle of mice at a dose of 24 U / kg, CMAP measurements were performed on the muscle at the administration site and the non-administration site muscle on the opposite side on days 7 and 14. As a result, no significant difference was observed in the CMAP values of the muscle at the administration site between the BoNT / A alone administration group and the BoNT / A+TSP11 administration group, but a significant difference was confirmed between the BoNT / A alone administration group and the BoNT / A+TSP23 administration group. The CMAP values in the opposite muscle were significantly higher in the BoNT / A+TSP11 and BoNT / A+TSP23 administration groups compared to the BoNT / A alone administration group (Figure 7).

[0140] Example 10: Non-clinical Efficacy Test of Botulinum Toxin Type A and TSP11, TSP35 Mixture in Mice Similar to Example 1, 5-week-old CD1 (ICR) female mice were purchased from Orient Bio Inc. After 1 week of acclimation and quarantine, 6-week-old mice were used in the experiment. The feed, drinking water, and breeding conditions were the same as those in Example 1.

[0141] Coax toxin injection (Meditox) was used as the test substance, and placebo (sham drug administration group) used sterile physiological saline (Korea Pharmaceutical Industry). The toxin-stabilizing peptides (TSP11, TSP35) were mixed with the peptide at a ratio of 3×10 5 to 1 mole of botulinum toxin and used in the experiment (Table 13). The mixture of botulinum toxin and toxin-stabilizing peptide was diluted to a concentration of 120 U / mL and used in the experiment.

[0142] The day when the test substance was administered was set as day 0, and the anesthesia of the experimental animals and the administration of the test substance were carried out in the same manner as in Example 1.

[0143] For the evaluation, the compound muscle action potential CMAP test method, which measures the action potential of the muscle that responds to external electrical stimulation, was used. The specific evaluation method and statistical analysis were carried out in the same manner as in Example 1.

[0144]

Table 13

[0145] After administering test substances BoNT / A+TSP11 and BoNT / A+TSP35, which were mixtures of botulinum toxin type A (BoNT / A) and a toxin-stabilizing peptide, to the right gastrocnemius muscle of mice at a dose of 24 U / kg, CMAP measurements were performed on the administered-site muscle and the non-administered-site muscle on the opposite side on days 7 and 14. As a result, significant differences in CMAP values of the administered-site muscle on day 7 were observed between the BoNT / A-alone administration group and the BoNT / A+TSP11 administration group, and between the BoNT / A-alone administration group and the BoNT / A+TSP35 administration group. However, on day 14, a significant difference was observed only between the BoNT / A-alone group and the BoNT / A+TSP35 group. On the other hand, significant differences in CMAP values of the non-administered-site muscle on the opposite side were observed between the BoNT / A-alone group and the BoNT / A+TSP11 group, and between the BoNT / A-alone group and the BoNT / A+TSP35 group on both days 7 and 14 (Figure 8).

[0146] Example 11: Non-clinical Efficacy Test of a Mixture of Botulinum Toxin Type A and TSP11 in Mice Similar to Example 1, 5-week-old CD1 (ICR) female mice were purchased from Orient Bio Inc., and after 1 week of acclimation and quarantine, 6-week-old mice were used in the experiment. The feed, drinking water, and breeding conditions were the same as those in Example 1.

[0147] Coax toxin injection (Meditox) was used as the test substance, and placebo (sham drug administration group) used sterile physiological saline (Korea Pharmaceutical Industry). The toxin-stabilizing peptide (TSP11) was mixed at a ratio of 3×10 5 to 1 mole of botulinum toxin and used in the experiment (Table 14). The mixture of botulinum toxin and toxin-stabilizing peptide was diluted to concentrations of 30 and 60 U / mL and used in the experiment.

[0148] The day when the test substance was administered was designated as day 0, and the anesthesia of the experimental animals and the administration of the test substance were carried out in the same manner as in Example 1.

[0149] Evaluation was performed using the DAS (digit abduction score), which macroscopically evaluates the degree of muscle paralysis in mice. The DAS numerical values shown in Table 15 indicate the degree of muscle paralysis based on the toe morphology on the side administered to the mice. The DAS recovery period (botulinum toxin action period) indicates the period until the DAS score returns to 0 after administration of the test substance. Body weight was measured once a week.

[0150]

Table 14

[0151]

Table 15

[0152]

Table 16

[0153] After administering the BoNT / A+TSP11 test substance, which is a mixture of 6 U / kg botulinum toxin type A (BoNT / A) and 12 U / kg toxin-stabilizing peptide, to the right gastrocnemius muscle of mice, DAS and body weight were measured for 7 weeks. As a result of measuring the change in body weight for each week based on the body weight at the time of administration of the test substance (0th week), no significant difference was observed between the 6 U / kg BoNT / A administration group and the 12 U / kg BoNT / A+TSP11 administration group (Table 16). In the 6 U / kg BoNT / A administration group, DAS recovery was observed on average on the 23.8th day, and in the 12 U / kg BoNT / A+TSP11 administration group, DAS recovery was observed on average on the 35.6th day, and an action period approximately 1.5 times longer was observed compared to the BoNT / A alone administration group (Figure 9). From such results, it was confirmed that when the dose of botulinum toxin was increased in a mixture of botulinum toxin and toxin-stabilizing peptide (TSP11), the action period of botulinum toxin could be increased without any change in stability.

[0154] Example 12: Non-clinical efficacy test of botulinum toxin type A and a mixture of TSP47 and TSP48 in mice Similar to Example 1, 5-week-old CD1 (ICR) female mice were purchased from Orient Bio Inc. After 1 week of acclimation and quarantine, 6-week-old mice were used in the experiment. The feed, drinking water, and breeding conditions were the same as those in Example 1. This study was conducted after review and approval by the Meditox Animal Experiment Ethics Committee (A-2022-007).

[0155] For the test substance, Coretox Injection (Meditox) was used, and for the placebo (sham dosing group), sterilized physiological saline (Korea Pharmaceutical Industry) was used. The toxin-stabilizing peptides (TSP47, TSP48) were mixed with the botulinum toxin at a ratio of 3x10 5 and used in the experiment (Table 17). The mixture of botulinum toxin and toxin-stabilizing peptide was diluted to a concentration of 120 U / mL and used in the experiment.

[0156] The day on which the test substance was administered was designated as Day 0, and the anesthesia of the experimental animals and the administration of the test substance were carried out in the same manner as in Example 1.

[0157] For the evaluation, the compound muscle action potential CMAP test method, which measures the action potential of the muscle that responds to external electrical stimulation, was used. The specific evaluation method and statistical analysis were carried out in the same manner as in Example 1.

[0158]

Table 17

[0159] After administering the BoNT / A+TSP47 test substance and the BoNT / A+TSP48 test substance, which were mixtures of botulinum toxin type A (BoNT / A) and a toxin-stabilizing peptide, to the right gastrocnemius muscle of a mouse at a dose of 24 U / kg each, CMAP measurements were performed on the administered-site muscle and the non-administered-site muscle on the opposite side on days 7 and 14. As a result, the CMAP value of the administered-site muscle showed a significant increase in the BoNT / A+TSP47 administration group compared to the BoNT / A-alone administration group on day 7 after administration, but no significant difference was observed on day 14 after administration compared to the BoNT / A-alone administration group. On the other hand, the CMAP values of the BoNT / A+TSP47 administration group and the BoNT / A+TSP48 administration group in the opposite-side muscle were both significantly higher than those of the BoNT / A-alone administration group on days 7 and 14 after administration. From such results, a reduced diffusion effect to the opposite leg was confirmed through the mixing of botulinum toxin and TSP47 or TSP48 (Figure 10).

[0160] Example 13: Non-clinical efficacy test of botulinum toxin type A and TSP56, TSP61 mixture type in mice Similar to Example 1, 5-week-old CD1 (ICR) female mice were purchased from Orient Bio Inc., and after 1 week of acclimation and quarantine, 6-week-old mice were used in the experiment. The feed, drinking water, and breeding conditions were the same as those in Example 1. This study was conducted after review and approval by the Meditox Animal Experiment Ethics Committee (A-2022-007).

[0161] The test substance used was Coretox Injection (Meditox), and the placebo (sham administration group) used was sterile physiological saline (Korea Pharmaceutical Industry). The toxin-stabilizing peptides (TSP56, TSP61) were mixed with the peptide at a ratio of 3x10 5 to 1 mole of botulinum toxin and used in the experiment (Table 18). The mixture of botulinum toxin and the toxin-stabilizing peptide was diluted to a concentration of 120 U / mL and used in the experiment.

[0162] The day when the test substance was administered was designated as day 0, and the anesthesia of the experimental animals and the administration of the test substance were carried out in the same manner as in Example 1.

[0163] For the evaluation, the compound muscle action potential CMAP test method, which measures the muscle action potential in response to external electrical stimulation, was used. The specific evaluation method and statistical analysis were carried out in the same manner as in Example 1.

[0164]

Table 18

[0165] After administering the test substances BoNT / A+TSP56 and BoNT / A+TSP61, in which botulinum toxin type A (BoNT / A) and a toxin-stabilizing peptide were mixed, to the right gastrocnemius muscle of mice at a dose of 24 U / kg each, CMAP measurements were performed on the muscle at the administration site and the non-administration site muscle on the opposite side on days 7 and 14. As a result, significant CMAP increases were observed in the BoNT / A+TSP56 administration group on days 7 and 14 after administration compared to the BoNT / A alone administration group in the CMAP values of the muscle at the administration site. However, the CMAP values of the BoNT / A+TSP56 administration group in the contralateral muscle were significantly higher on days 7 and 14 after administration compared to the BoNT / A alone administration group. When mixing botulinum toxin and TSP56, although the CMAP in the muscle at the administration site slightly increased compared to the administration of botulinum toxin alone, the CMAP value in the contralateral leg further increased, indicating a confirmed effect of reducing diffusion through the mixing of TSP56. In the case of the BoNT / A+TSP61 administration group, no significant difference was observed in the CMAP values of the muscle at the administration site compared to the BoNT / A alone administration group, but the CMAP value in the contralateral muscle was significantly higher compared to the BoNT / A alone administration group, indicating a confirmed effect of reducing diffusion through the mixing of TSP61. Sequence Listing Free Text

[0166] SEQ ID NOs: 1 to 10 are exemplary sequences related to moieties that interact with botulinum toxin while interacting with the cell membrane of nerve cells. SEQ ID NOs: 11 to 13 are exemplary sequences related to moieties that interact with the cell membrane of nerve cells. SEQ ID NOs: 14 to 21 are exemplary sequences of polypeptides included in the botulinum toxin stabilization composition. SEQ ID NO: 22 is a substance used as a control for the polypeptide included in the toxin stabilization composition according to one embodiment. SEQ ID NO: 23 is the sequence of the recombinant botulinum neurotoxin serotype A light chain. SEQ ID NO: 24 is the sequence of a recombinant botulinum neurotoxin serotype A in which the light chain of the botulinum toxin consists of a combination of BoNT / A1 light chain and BoNT / A4 light chain.

Claims

**Claim 1** A botulinum toxin stabilization composition comprising a polypeptide of the following Chemical Formula 1 that inhibits the diffusion of botulinum toxin in vivo: [Chemical Formula 1] (C) ｎ -V In the above formula, C represents a moiety that interacts with the cell membrane of nerve cells, V represents a moiety that interacts with botulinum toxin while interacting with the cell membrane of nerve cells, and n is an integer of 0 or 1. **Claim 2** The botulinum toxin stabilization composition according to claim 1, wherein V is VAMP (vesicle associated membrane protein), BDNF (brain-derived neurotrophic factor), a fragment thereof, or a variant thereof. **Claim 3** The botulinum toxin stabilization composition according to claim 1 or 2, wherein V is VpN of VAMP, a fragment thereof, or a variant thereof. **Claim 4** The botulinum toxin stabilization composition according to claim 2 or 3, wherein VAMP is VAMP1, VAMP2, VAMP3, a fragment thereof, or a variant thereof. **Claim 5** The botulinum toxin stabilization composition according to any one of claims 1 to 4, wherein V comprises the amino acid sequence of SEQ ID NO:

1. **Claim 6** The botulinum toxin stabilization composition according to claim 5, wherein V is selected from the amino acid sequences of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, and 8; and amino acid sequences in which one amino acid is substituted in the amino acid sequences of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, and 8. **Claim 7** The botulinum toxin stabilization composition according to any one of claims 1 to 6, wherein C is a cell-penetrating peptide or a cationic peptide. **Claim 8** The botulinum toxin stabilization composition according to claim 7, wherein C is an arginine-rich peptide. **Claim 9** The botulinum toxin stabilization composition according to claim 7 or 8, wherein C further comprises an aromatic amino acid. **Claim 10** The botulinum toxin stabilization composition according to claim 9, wherein the aromatic amino acid is tryptophan. **Claim 11** The botulinum toxin stabilization composition according to any one of claims 7 to 10, wherein C comprises 6 to 9 arginines and 2 to 3 tryptophans. **Claim 12** The botulinum toxin stabilization composition according to any one of claims 7 or 11, wherein C has a three-dimensional structure in which arginine and tryptophan are biased to one side. **Claim 13** C is the botulinum toxin stabilization composition according to any one of claims 7 to 12, which consists of the amino acid sequence of SEQ ID NO: 11, SEQ ID NO: 12 or SEQ ID NO:

13.

14. The polypeptide is the botulinum toxin stabilization composition according to any one of claims 1 to 13, which consists of the amino acid sequence of SEQ ID NO: 8, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20 or SEQ ID NO:

21.

15. A botulinum toxin preparation comprising a botulinum toxin and the botulinum toxin stabilization composition according to any one of claims 1 to 14.

16. The botulinum toxin is of type A, B, C, D, E, F or G, and the botulinum toxin preparation according to claim 15.

17. The botulinum toxin is a natural or recombinant toxin, and the botulinum toxin preparation according to claim 15 or 16.

18. The botulinum toxin is in the 7S or 19S form, and the botulinum toxin preparation according to any one of claims 15 to 17.

19. The botulinum toxin preparation according to any one of claims 16 to 19, which contains an increased amount of botulinum toxin compared to the case where the botulinum toxin stabilization composition is not included.

20. A polypeptide consisting of the amino acid sequence of SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO: 20.

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