Botulinum toxin composition for long-term sustained release

Abstract

The present invention relates to: a botulinum toxin composition comprising a botulinum toxin, a poloxamer, and methyl cellulose; and a method for preparing same. In addition, the botulinum toxin composition according to the present invention comprises a cell-penetrating peptide. The composition according to the present invention exhibits an increase in potency and a decrease in erosion rate, thereby enabling long-term controlled release of the toxin, and thus can exhibit long-acting properties.

Description

Long-lasting release botulinum toxin composition

[0001] The present invention relates to a botulinum toxin composition comprising botulinum toxin, poloxamer, and methylcellulose, and a method for preparing the same. In addition, the botulinum toxin composition of the present invention comprises a cell-penetrating peptide.

[0002] Botulinum toxin is a type of protein produced by the bacterium Clostridium botulinum. It works by irreversibly attaching to presynaptic nerve endings and inhibiting the secretion of acetylcholine at the synapse, thereby blocking muscle contraction and secondarily producing a muscle relaxant effect. Consequently, botulinum toxin is currently being used to treat various involuntary muscle movement disorders, including strabismus, idiopathic blepharospasm, blepharospasm, and hemifacial spasm, as well as torticollis, spastic dysphonia, and dystonia. Furthermore, it is utilized in various cosmetic applications, such as the non-surgical reduction of facial wrinkles, as well as for the treatment of hyperhidrosis.

[0003] Since such botulinum toxins can cause fatal toxicity and side effects even in minute amounts, there is a need to develop a botulinum toxin composition that reduces the frequency of administration, increases convenience, and improves safety and stability by extending the duration of activity with a single administration.

[0004] The object of the present invention is to provide a botulinum toxin composition comprising botulinum toxin; poloxamer; and methylcellulose. The botulinum toxin composition may exhibit a long sustained-release ability.

[0005] Another objective of the present invention is to provide a method for preparing the above-mentioned botulinum toxin composition.

[0006] Another object of the present invention is to provide a method for improving skin or a method for treating a disease, comprising the step of administering a botulinum toxin composition comprising botulinum toxin; poloxamer; and methylcellulose to an individual.

[0007] Another objective of the present invention is to provide a botulinum toxin composition comprising botulinum toxin; poloxamer; and methylcellulose for use in improving skin or treating diseases.

[0008] Another objective of the present invention is to provide a composition for manufacturing a botulinum toxin pharmaceutical preparation comprising botulinum toxin; poloxamer; and methylcellulose.

[0009] One aspect of the present invention for achieving the above objectives relates to a botulinum toxin composition comprising botulinum toxin; poloxamer; and methylcellulose.

[0010] In the present invention, "Botulinum Toxin" refers to a neurotoxin produced by the Gram-positive bacterium Clostridium botulinum. Botulinum toxin acts to cause muscle paralysis by blocking the synaptic transmission or release of acetylcholine through the neuromuscular junction, and may also act through other mechanisms. Such action can result in paralysis by essentially blocking the signals that normally induce muscle spasms or contractions. The botulinum toxin includes all serotypes produced by bacteria or recombinant production, as well as their derivatives or fusion proteins.

[0011] The above "botulinum toxin derivative" may be a derivative comprising one or more chemical or functional modifications on a part or part of the chain of a natural botulinum toxin or recombinant circular botulinum toxin having botulinum toxin activity. For example, the botulinum toxin derivative may be a modified toxin having one or more deleted, modified, or substituted amino acids.

[0012] Specifically, the botulinum toxin may be one or more selected from the group consisting of botulinum toxins A, B, C, D, E, F, G, and H.

[0013] The above-mentioned botulinum toxin is serologically related and is classified into eight neurotoxins. Among these, eight types, namely botulinum neurotoxin serotypes A, B, C, D, E, F, G, and H, can cause paralysis. Each serotype is distinguished by neutralization by serotype-specific antibodies. Depending on the serotype, the animal species affected, the degree of paralysis caused, and the duration differ, and an appropriate serotype can be selected and applied according to the purpose.

[0014] More specifically, the botulinum toxin may be botulinum toxin type A.

[0015] In the present invention, "botulinum toxin type A" is one of the most lethal natural biological agents known to humans. The LD50 of commercially available botulinum toxin type A is about 50 picograms (i.e., 1 unit), which is known to be 1.8 billion times more lethal than diphtheria, 600 million times more lethal than sodium cyanide, 30 million times more lethal than cobra venom, and 12 million times more lethal than cholera on a molar (M) basis. Thus, 1 unit (U) of botulinum toxin, which serves as the standard for lethality, can be defined as the LD50 via intraperitoneal injection into female Swiss Webster mice weighing 18-20 g each.

[0016] Specifically, the poloxamer of the composition of the present invention may be included in an amount of 10 to 30 weight% based on the total weight of the composition, but is not limited thereto. More specifically, the poloxamer may be included in an amount of 10 to 25 weight%, 10 to 20 weight%, 15 to 30 weight%, 20 to 30 weight%, or 25 to 30 weight%, but is not limited thereto. Additionally, specifically, the poloxamer may be poloxamer 407 or poloxamer 124, but is not limited thereto.

[0017] In the present invention, "Poloxamer" is a tripolymer composed of the hydrophilic polymer PEO (Polyethylene oxide) and the hydrophobic polymer PPO (Polypropylene oxide). In particular, Poloxamer 407 is composed of approximately 70% PEO and 30% PPO by weight and is widely used in the fields of intravenous injection and biomaterials due to its low toxicity and drug delivery efficiency. Poloxamer 407 gels by forming micelles in response to temperature changes above a certain concentration, and this gelling characteristic is controlled by the composition and environment. This is advantageous for the controlled release and delivery of drugs.

[0018] Additionally, specifically, the methylcellulose of the composition of the present invention may be included in an amount of 0.5 to 10 weight% based on the total weight of the composition, but is not limited thereto. More specifically, it may be included in an amount of 1 to 10 weight%, 1 to 9 weight%, 1 to 8 weight%, 1 to 7 weight%, 1 to 6 weight%, or 1 to 5 weight%, but is not limited thereto.

[0019] In addition, the methylcellulose may be 1 to 500 centipoise (cP), but is not limited thereto. More specifically, it may be 2 to 500 cP, 3 to 500 cP, 4 to 500 cP, 1 to 400 cP, 2 to 400 cP, 3 to 400 cP, 4 to 400 cP, or 5 to 400 cP, but is not limited thereto.

[0020] Since the release of botulinum toxin occurs more as the erosion rate increases, a composition containing methylcellulose that significantly reduces the erosion rate can exhibit a long-term sustained effect of the toxin along with a long-term delayed release.

[0021] Additionally, specifically, the composition may further include glycerol. More specifically, the glycerol may be included in an amount of 0.5 to 5 weight% based on the total weight of the composition, but is not limited thereto. More specifically, the glycerol may be included in an amount of 0.5 to 4 weight%, 0.5 to 3 weight%, or 0.5 to 2 weight%, but is not limited thereto.

[0022] Additionally, specifically, the above composition may further include a cell-penetrating peptide. More specifically, the cell-penetrating peptide may be any one selected from the group consisting of SEQ ID NOs 1 to 6, but is not limited thereto.

[0023] Another aspect of the present invention relates to a botulinum toxin composition comprising botulinum toxin and a cell-permeable peptide.

[0024] Specifically, the cell-permeating peptide may be composed of any one selected from the group consisting of SEQ ID NOs 1 to 6, but is not limited thereto.

[0025] The botulinum toxin composition of the present invention may include excipients in the manufacturing process to facilitate convenient and accurate administration as a carrier for active action, to increase the volume of a formulation containing a potent active ingredient, or to optimize the handling of the active substance. For example, it includes antioxidants, solubilization enhancers, stabilizers, anti-aggregation / precipitation agents, anti-adsorption agents, buffers, preservatives, osmotic pressure regulators, carriers, isotonic agents, surfactants, etc. Although not limited thereto, the composition may further include antioxidants and stabilizers.

[0026] The botulinum toxin composition of the present invention can be used for the treatment of neuromuscular disorders characterized by hyperactive skeletal muscle. In addition, headache, migraine, tension headache, sinus headache, cervical headache, sweating disorders, axillary hyperhidrosis, palmar hyperhidrosis, plantar hyperhidrosis, Frey's syndrome, hyperkinetic skin line, facial wrinkles, glabellar wrinkles, crow's feet, perioral wrinkles, nose-lip wrinkles, skin disorders, achalasia, strabismus, chronic dentition, blepharospasm, musculoskeletal pain, fibromyalgia, pancreatitis, tachycardia, prostatic hypertrophy, prostatitis, urinary retention, urinary incontinence, overactive bladder, hemifacial spasm, tremor, muscle spasm, gastrointestinal disorders, diabetes mellitus, hypersalivation, detrusor-spyin coordination disorder, post-stroke spasticity, wound healing, pediatric cerebral palsy, smooth muscle spasm, restenosis, focal dystonia, epilepsy, cervical dystonia, thyroid disorders, hypercalcemia, obsessive-compulsive disorder, arthritis pain, Raynaud's syndrome, stretch marks, peritoneal adhesions, It can be used for various purposes, such as treating conditions like vasospasm, runny nose, muscle spasms, laryngeal dystonia, writing spasms, and carpal tunnel syndrome, as well as for cosmetic purposes.

[0027] The composition of the present invention exists in the form of a product to be applied to the skin, epithelium, intradermal (ID), subcutaneous (SC), or intramuscular (IM) of humans or other mammals requiring administration of botulinum toxin, but is not limited thereto.

[0028] In the present invention, "needing" is intended to include both pharmaceutical or health needs, e.g., treatment of the said disease, and cosmetic or subjective needs, e.g., the need to modify or improve the appearance of facial tissues.

[0029] In addition, the botulinum toxin composition of the present invention may be an injectable in the form of a ready-to-use (RTU). The injectable may further comprise a sugar alcohol selected from the group consisting of mannitol, erythritol, sorbitol, isomalt, trehalose, maltitol, xylitol, and mixtures thereof, but is not limited thereto and may be used without limitation as needed.

[0030] Another aspect of the present invention comprises the step of administering the composition to an individual who requires it for the treatment of a disease or for cosmetic purposes, for neuromuscular disorders, headache, migraine, tension headache, sinus headache, cervical headache, sweating disorders, axillary hyperhidrosis, palmar hyperhidrosis, plantar hyperhidrosis, Frey syndrome, hyperkinetic skin line, facial wrinkles, glabellar wrinkles, crow's feet, perioral wrinkles, nose-lip wrinkles, skin disorders, achalasia, strabismus, chronic dentition, blepharospasm, musculoskeletal pain, fibromyalgia, pancreatitis, tachycardia, prostatic hypertrophy, prostatitis, urinary retention, urinary incontinence, overactive bladder, hemifacial spasm, tremor, muscle spasm, gastrointestinal disorders, diabetes mellitus, hypersalivation, detrusor-spyintosis, spasticity after stroke, wound healing, pediatric cerebral palsy, smooth muscle spasm, restenosis, focal dystonia, epilepsy, cervical dystonia, thyroid disorders, This relates to a method for treating one or more of the following conditions: hypercalcemia, obsessive-compulsive disorder, arthritis pain, Raynaud's syndrome, stretch marks, peritoneal adhesions, vasospasm, runny nose, muscle spasms, laryngeal dystonia, writing spasm, and carpal tunnel syndrome.

[0031] The above composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. Additionally, the composition of the present invention may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects, taking all of the above factors into consideration, and this amount may be determined by modification as necessary.

[0032] The term "individual" in the present invention includes animals or humans whose symptoms of the disease may be improved by the administration of the composition according to the present invention. By administering the composition according to the present invention to an individual, neuromuscular disorders, etc., can be effectively prevented and treated, and cosmetic improvement effects can be exhibited.

[0033] The term "administration" in the present invention means introducing a specific substance into a human or animal by any appropriate method, and the administration route of the composition according to the present invention may be parenteral through any general route as long as it can reach the target tissue.

[0034] The preferred dosage of the composition according to the present invention varies depending on the patient's condition and weight, the degree of disease, the required dosage for cosmetic improvement, the route of administration, and the duration, and can be appropriately selected as needed.

[0035] Another aspect of the present invention relates to a method for preparing a botulinum toxin composition comprising the step of mixing botulinum toxin; poloxamer; and methylcellulose.

[0036] Specifically, the poloxamer may be mixed in an amount of 10 to 30 weight% based on the total weight of the composition; and the methylcellulose may be mixed in an amount of 0.5 to 10 weight% based on the total weight of the composition, but is not limited thereto.

[0037] Specifically, the poloxamer may be mixed in an amount of 10 to 30 weight% based on the total weight of the composition, but is not limited thereto. More specifically, the poloxamer may be mixed in an amount of 10 to 25 weight%, 10 to 20 weight%, 15 to 30 weight%, 20 to 30 weight%, or 25 to 30 weight%, but is not limited thereto. Additionally, specifically, the poloxamer may be poloxamer 407 or poloxamer 124, but is not limited thereto.

[0038] Additionally, specifically, the methylcellulose may be mixed in an amount of 0.5 to 10 weight% based on the total weight of the composition, but is not limited thereto. More specifically, it may be mixed in an amount of 1 to 10 weight%, 1 to 9 weight%, 1 to 8 weight%, 1 to 7 weight%, 1 to 6 weight%, or 1 to 5 weight%, but is not limited thereto.

[0039] Additionally, specifically, the composition may include a step of additionally mixing glycerol. More specifically, the glycerol may be included in an amount of 0.5 to 5 weight% based on the total weight of the composition, but is not limited thereto. More specifically, the glycerol may be included in an amount of 0.5 to 4 weight%, 0.5 to 3 weight%, or 0.5 to 2 weight%, but is not limited thereto.

[0040] Additionally, specifically, the composition may further include the step of mixing a cell-penetrating peptide. More specifically, the cell-penetrating peptide may be any one selected from the group consisting of SEQ ID NOs 1 to 6, but is not limited thereto.

[0041] Another aspect of the present invention relates to a method for preparing a botulinum toxin composition, comprising the step of mixing botulinum toxin and a cell-permeable peptide.

[0042] Specifically, the cell-penetrating peptide may be composed of any one selected from the group consisting of SEQ ID NOs 1 to 6, but is not limited thereto. The description of the cell-penetrating peptide is as described above.

[0043] Another aspect of the present invention relates to a method for cosmetic improvement of wrinkles, square jaw, skin softening, acne, pores, elasticity, or keloid symptoms, comprising the step of administering a botulinum toxin composition comprising botulinum toxin; poloxamer; and methylcellulose to an individual.

[0044] Botulinum toxin relieves hyperexcitation of skeletal muscles and autonomic innervation targets by selectively inhibiting acetylcholine release through the cleavage of SNARE proteins, such as SNAP-25, at target nerve endings. As the contractile force of facial muscles is reduced, the formation of dynamic wrinkles is inhibited, and the deepening of skin creases caused by repetitive contractions can be alleviated. Additionally, hypertrophy or hypertonicity of masticatory muscles is reduced, making the shape of the mandibular angle more flexible, and the skin surface can be maintained more smoothly through the continuous relaxation of skin tension and the reduction of microcontraction. Furthermore, the administration of the composition of the present invention can suppress excessive sebum and sweat secretion by regulating sympathetic cholinergic secretion, and can contribute to the alleviation of inflammatory skin conditions by influencing local cytokine secretion.

[0045] The botulinum toxin composition of the present invention is a formulation comprising poloxamer and methylcellulose, which exhibits long-term release control and a reduced erosion rate effect, thereby imparting persistence and spatial localization of the botulinum toxin, allowing the acetylcholine inhibitory effect to be expressed more uniformly and continuously, and can improve the cosmetic improvement method.

[0046] Another aspect of the present invention relates to a method for treating neuromuscular disorders, facial spasms, facial spasms, blepharospasm, strabismus, dystonia, hyperhidrosis, migraine, or pain, comprising the step of administering to an individual a botulinum toxin composition comprising botulinum toxin; poloxamer; and methylcellulose.

[0047] Botulinum toxin can inhibit the fusion of synaptic vesicles and selectively block the release of acetylcholine by cleaving SNARE complex components such as SNAP-25 at cholinergic nerve endings, and accordingly, for neuromuscular disorders, facial spasms, blepharospasm, strabismus, and dystonia, hyperexcitation and sustained muscle contraction of the terminal motor plate are relieved, pathophysiological muscle tone is reduced, and involuntary movement symptoms can be improved or treated by blocking the chain of abnormal co-contraction and pathological reflex activity.

[0048] In addition, botulinum toxin can inhibit the release of pain and inflammation-related neurotransmitters such as glutamate, substance P, and CGRP from sensory nerve endings, thereby inhibiting the vicious cycle of peripheral sensitization and central sensitization mediated by it in migraines and various pain conditions.

[0049] The botulinum toxin composition of the present invention includes poloxamer and methylcellulose to induce long-term sustained release at the site of administration and significantly reduce the erosion rate, thereby ensuring stable local concentration and sustained effect. When administered via intradermal, subcutaneous, or intramuscular injection, the action of botulinum toxin at the neuromuscular junction and cholinergic terminals is maintained for a certain period, thereby enabling the continuation of therapeutic effects and the extension of the administration interval for the said neuromuscular disorders, facial spasms, blepharospasm, strabismus, dystonia, hyperhidrosis, migraine, or pain.

[0050] Another aspect of the present invention relates to the use of a botulinum toxin composition comprising botulinum toxin; poloxamer; and methylcellulose for improving symptoms of wrinkles, square jaw, skin softening, acne, pores, elasticity, or keloids. The improvement of symptoms of wrinkles, square jaw, skin softening, acne, pores, elasticity, or keloids is as described above.

[0051] Another aspect of the present invention relates to the use of a botulinum toxin composition comprising botulinum toxin; poloxamer; and methylcellulose for the treatment of neuromuscular disorders, facial spasms, blepharospasm, strabismus, dystonia, hyperhidrosis, migraines, or pain. The treatment of neuromuscular disorders, facial spasms, blepharospasm, strabismus, dystonia, hyperhidrosis, migraines, or pain is as described above.

[0052] Another aspect of the present invention relates to a composition for manufacturing a botulinum toxin pharmaceutical preparation comprising botulinum toxin; poloxamer; and methylcellulose.

[0053] The above composition for manufacturing a botulinum toxin pharmaceutical preparation comprises botulinum toxin as an active ingredient and is a composition intended for manufacturing into a finished product formulation. It may be in the form of an aqueous solution, suspension, concentrate, or lyophilized material, and may additionally include additives to impart structural or functional stability, homogeneity, etc., of the botulinum toxin during the dilution, filtration, filling, and packaging stages of the manufacturing process, and, if necessary, may add buffers, isotonic agents, chelating agents, antioxidants, cryoprotectants, surfactants, etc.

[0054] The composition for manufacturing a botulinum toxin pharmaceutical preparation according to the present invention can be converted into a final botulinum toxin preparation having various concentrations or viscoelasticity depending on the addition of a diluent solvent, a stabilizer, a buffer, etc., and by including the poloxamer and methylcellulose, the dispersibility and biological activity of the botulinum toxin can be stably maintained.

[0055] The botulinum toxin composition of the present invention exhibits long-term release control and reduced erosion rate effects by including poloxamer and methylcellulose in the botulinum toxin. In addition, the botulinum toxin composition containing a cell-penetrating peptide can exhibit a long-term sustained effect of the toxin by allowing long-term release to continue along with increased potency.

[0056] The effects of the present invention are not limited to the effects described above, and should be understood to include all effects that can be inferred from the configuration of the invention described in the detailed description or claims of the present invention.

[0057] Figures 1 and 2 show the results of the emission control evaluation of the composition of the present invention.

[0058] Figure 3 shows the results confirming the toxin release delay effect of the composition of the present invention.

[0059] Figure 4 shows the results confirming the erosion inhibition effect of the composition of the present invention.

[0060] Figures 5 and 6 show the results of the emission control evaluation according to the inclusion of methylcellulose in the composition of the present invention.

[0061] Figure 7 shows the results of evaluating the compatibility of cell-penetrating peptides.

[0062] Figure 8 shows the results of confirming the long-term persistence of a composition with added cell-permeable peptides.

[0063] The present invention will be explained in detail below by way of examples. However, the following examples are merely illustrative of the present invention, and the present invention is not limited by the following examples.

[0064] Preparation Example 1. Preparation of a Botulinum Toxin Composition I

[0065] Each example composition was prepared by mixing according to the compositions in Tables 1 and 2 below. Type A botulinum toxin was used.

[0066] Ingredients Example 1 Example 2 Example 3 Botulinum toxin (unit) 20,000 20,000 20,000 Poloxamer 407 (wg%) 20 20 20 Sodium chloride (wg%) - 0.9-Methylcellulose (wg%) - 2.0 Poloxamer 124 (wg%) 2.0--Phosphoric acid (mM) 50 50 50

[0067] Ingredients Example 4 Example 5 Example 6 Example 7 Example 8 Botulinum toxin (unit) 20,000 20,000 20,000 20,000 20,000 20,000 Poloxamer 407 (wg%) 20 20 20 20 20 Methylcellulose (wg%) 2.0 2.0 2.0 2.0 2.0 Arg-HCl 4.2 ---- Glycerol 1.0 --- Poloxamer 124 (wg%) -- 2.0 -- α-Cyclodextrin --- 1.0 -Hydroxypropyl -β-Cyclodextrin 1.0 Phosphate (mM) 50 50 50 50 50

[0068] Furthermore, compositions were prepared with different content and viscosity (cp) of the polymer methylcellulose (MC) according to Table 3 below. The content of botulinum toxin was kept the same at 20,000 units. As a comparative example, a composition was prepared using hydroxypropyl cellulose (HPC) instead of methylcellulose.

[0069] Formulation medium / dissolution medium P407 (wt%) MC (wt%) MC Viscosity (cp) HPC (wt%) HPC Molecular Weight Example 3 - 150 mM PhosphatepH 6.0 / 50 mM Phosphate 150 mM MSodium chloride pH 6.0 201.0 400 -- Example 3 - 2 202.0 400 -- Example 3 - 3 203.0 400 -- Example 3 - 4 204.05 -- Example 3 - 5 206.0 15 -- Comparative Example 20-- 4.0 Low MW

[0070] Preparation Example 2. Preparation of a Botulinum Toxin Composition II

[0071] 2,000 units / mL of botulinum toxin type A was mixed with 300 mM trehalose, 50 mM phosphate buffer (pH 6.0), 0.5% HSA, and 0.9% sodium chloride. Then, the cell-penetrating peptides of Table 4 below were added at a concentration of 5.0 mg / mL to prepare the compositions of Examples 9 to 17.

[0072] Peptide Amino Acid Sequence Sequence Number Example 9 Protamine sulfate DHYNC VSSGG QCLYSACPIFTKIQGTCYRGKAKCCK1 Example 10 TAT-PTD GRKKRRQRRR2 Example 11 PenetraMax KWFKIQ MQIRRWKNKR3 Example 12 Penetratin RQIKWFQNRRMKWKK4 Example 13 MPGGALFLGFLGAAGSTMGAWS QPKKKRKV5 Example 14 poly-Arginine RRRRRRRRR6 Example 15 TCTP-PTDM IIYRDLISH7 Example 16 TCTP-PTD 13M2MIIFRLLAYKKK8 Example 17 TCTP-PTD 13M3MIIFRLLAYHKK9

[0073] Experimental Example 1. Evaluation of Emission Control

[0074] Under the assumption that the botulinum toxin of the composition of the present invention dissociates in the body and exhibits activity, the amount of toxin released into the release medium per unit time was analyzed by ELISA.

[0075] Specifically, the compositions of each of Examples 1 to 8 prepared in Preparation Example 1 were cultured in an incubator at 37°C for 1 hour, and then introduced into an eluent at 37°C.

[0076] The eluent was composed of 50 mM phosphate buffer (pH 6.0), 150 mM sodium chloride (NaCl), 0.02% sodium azide (NaN3), and 1 mg / mL human serum albumin. After introducing the composition into the eluent, it was mixed using an orbital shaker at 30 rpm.

[0077] Subsequently, 100 μL of each sample was dispensed into each well of a 96-well plate. The ELISA analysis was performed using a solid-phase sandwich ELISA method.

[0078] 0.05% Tween in the washing step 　 PBS buffer containing 20 was used, and PBS containing 1% BSA was used as the diluent. To stop the reaction, 2 N sulfuric acid (H2SO4) solution was added to terminate the enzymatic reaction.

[0079] As a result, as shown in Figure 1, after 14 days, Example 3 exhibited a release pattern that showed a significant difference (p<0.01) from Example 2 and showed the highest toxin residue. In addition, unlike the other examples, Example 3 did not show a rapid release pattern. From this, it was confirmed that when methylcellulose is included, long-term release can be controlled and long-term persistence can be observed.

[0080] In addition, as shown in Figure 2, the composition of Example 5 showed the highest long-term sustainability after 14 days and exhibited a release pattern that showed a significant difference (p<0.01) from Example 2. Through this, it was confirmed that adding glycerol together with methylcellulose resulted in longer sustainability along with more distinct release control.

[0081] Additionally, residual toxins were measured after 14 days for the compositions of Examples 2, 4 to 8.

[0082] Composition Remaining toxin Example 2 15.13% Example 4 15.38% Example 5 25.41% Example 6 18.74% Example 7 17.45% Example 8 13.36%

[0083] As a result, it was confirmed that a large amount of toxin remained in the composition of Example 5, enabling longer-term release. It was confirmed that the higher residual toxin content, resulting from improved release control by poloxamer and glycerol, allows for sustained toxin release over a longer period. Additionally, toxin release was measured for 14 days for the compositions of Examples 2, 3, and 5. A composition mixed with phosphate buffer and botulinum toxin was used as a control. As a result, as shown in Figure 3, Example 3, which contained methylcellulose, showed delayed toxin release compared to Example 2, which did not contain methylcellulose. Furthermore, it was confirmed that Example 5, which additionally contained glycerol, showed even further delayed toxin release, demonstrating long-term persistence.

[0084] Experimental Example 2. Evaluation of Swelling / Erosion

[0085] 2-1. Scanning Electron Microscope Observation

[0086] To confirm the erosion effect, the compositions of Example 2 and Example 3 were exposed to a buffer solution for 3 days, then freeze-dried and observed using a scanning electron microscope (SEM).

[0087] As a result, as shown in Figure 4, it was confirmed that while Example 2, composed solely of poloxamer, easily eroded after 3 days, Example 3, with the addition of methylcellulose, inhibited erosion. In particular, it was confirmed that there was a significant difference in erosion rates, with the erosion rate of Example 2 being 47% and the erosion rate of Example 3 being 10%.

[0088] 2-2. Confirmation of Erosion Rate According to Methylcellulose Content and Molecular Weight

[0089] For each example composition according to the above preparation example, the weight was measured after exposure to a buffer solution for 3 days, and the erosion rate was calculated according to the following [Formula]. Specifically, erosion was induced by adding 2 mL of release solution to 2 mL of the example composition, and the remaining weight was measured after removing the release solution. The swelling / erosion ratio was calculated according to the following formula after weight measurement.

[0090] [ceremony]

[0091]

[0092] As a result, as shown in Table 7 below, the erosion rate during 3 days of exposure in the buffer solution was observed at 3 hours, 6 hours, 1 day, and 3 days to determine the erosion rate over time. In the case without methylcellulose, a high erosion rate was observed from the beginning, whereas in the case of 3-2 and 3-5 containing methylcellulose, it was confirmed that the initial erosion rate was very low and remained very low even up to the 3rd day.

[0093] Erosion Rate (%) Composition 3 Hour 6 Hour 1 Day 3 Day Example 2 10.12 ± 0.24 25.34 ± 1.85 49.51 ± 2.39 84.08 ± 2.02 Example 3 - 25.47 ± 0.53 11.62 ± 1.77 15.28 ± 0.95 20.10 ± 1.18 Example 3 - 50.96 ± 0.25 1.53 ± 0.33 1.63 ± 0.21 -0.09 ± 0.75

[0094] In addition, as shown in FIGS. 5 and 6, the compositions of Examples 3-4 and 3-5, which have a low erosion rate, showed a gradual release compared to Example 2, which has a high erosion rate. In contrast, the comparative example containing HPC instead of methylcellulose showed an unstable release pattern, exhibiting two rapid releases along with a high erosion rate compared to Examples 3-2, 3-4, and 3-5, which contain methylcellulose. After 14 days, Example 2 showed no residual toxin, whereas Example 3-4 showed 12% and Example 3-5 showed 15% residual toxin, confirming that there is long-term persistence capable of releasing toxins even after 14 days.

[0095] Experimental Example 3. Measurement of potency activity following the addition of cell-penetrating peptides

[0096] For protamine sulfate, TAT-PTD, PenetraMax, Penetratin, MPG, TCTP-PTD, TCTP-PTD 13M2, and TCTP-PTD 13M3, each peptide was diluted and mixed with 5.0 mg / mL of botulinum toxin and 0.5 mg / mL of botulinum toxin, and the compatibility of the toxin and peptide was evaluated using Dynamic Light Scattering (DLS) analysis. Approximately 100 μL of the sample was placed in a quartz cell and analyzed under the conditions shown in Table 8 below.

[0097] Cuvette: Low volume - quartz (PN: ZEN2112) Temperature: Initial at 20 ℃, 20–60 ℃, Step 2 ℃ / step Concentration: 0.5 mg / mL Equilibrium time: 2 min

[0098] As a result, as shown in Fig. 7(a), PenetraMax, MPG, Protamine sulfate, TAT-PTD, and Penetratin peptide did not exhibit a rapid change in Z-average size immediately after addition, confirming that they are more suitable for addition to botulinum toxin. Among these, Protamine sulfate at approximately 46 ℃ T onset, PenetraMax has a high T of about 50°C onset It was confirmed that it exhibits [this]. On the other hand, as shown in Fig. 7(b), in the case of TCTP-PTD, TCTP-PTD 13M2, and TCTP-PTD 13M3, the Z-average size increased rapidly immediately after addition, indicating that the suitability was somewhat reduced.

[0099] Experimental Example 4. Confirmation of potency and long-term persistence following the addition of cell-penetrating peptides

[0100] The electromyography of the gastrocnemius muscle of mice was evaluated after injecting the compositions of Examples 10, 11, and 14. Specifically, 3-week-old mice were acquired and acclimatized for 3 days, and at 4 weeks of age, the composition was injected into the left lateral gastrocnemius muscle, after which the compound muscle action potential (CMAP) was measured using an electromyography device. The relationship of duration was evaluated through electromyography monitoring.

[0101] Composition Objective Potency Test Potency Example 102.7 x 10 8 3.9 ~ 4.2 x 10 8 (143 ~ 154%) Example 113.5 ~ 3.7 x 10 8 (130 ~ 137%) Example 143.2 ~ 3.3 x 10 8 (117 ~ 123 %)

[0102] As a result, as shown in Table 9 above, it was confirmed that adding cell-penetrating peptides to botulinum toxin resulted in an activity more than 120% higher than the target potency. In addition, as shown in Figure 8, it was confirmed that the toxin can exhibit a long-term sustained effect by showing long-term release for more than 16 weeks.

[0103] The foregoing description of the present invention is for illustrative purposes only, and those skilled in the art will understand that other specific forms can be easily modified without altering the technical spirit or essential features of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. For example, each component described as a single unit may be implemented in a distributed manner, and components described as distributed may likewise be implemented in a combined form.

Claims

1. Botulinum toxin; A botulinum toxin composition comprising poloxamer; and methylcellulose.

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

3. In Paragraph 1, A botulinum toxin composition in which the above-mentioned poloxamer is included in an amount of 10 to 30 weight% based on the total weight of the composition.

4. In Paragraph 1, A botulinum toxin composition in which the above-mentioned methylcellulose is included in an amount of 0.5 to 10 weight% based on the total weight of the composition.

5. In Paragraph 1, A botulinum toxin composition in which the methylcellulose is 1 to 500 centipoise (cP).

6. In Paragraph 1, The above composition is a botulinum toxin composition further comprising glycerol.

7. In Paragraph 6, A botulinum toxin composition in which the above glycerol is included in an amount of 0.5 to 5 weight% based on the total weight of the composition.

8. In Paragraph 1, The above composition is a botulinum toxin composition further comprising a cell-penetrating peptide.

9. In Paragraph 8, A botulinum toxin composition wherein the cell-penetrating peptide is composed of any one selected from the group consisting of SEQ ID NOs 1 to 6.

10. A botulinum toxin composition comprising botulinum toxin and a cell-penetrating peptide.

11. In Paragraph 10, A botulinum toxin composition wherein the cell-penetrating peptide is composed of any one selected from the group consisting of SEQ ID NOs 1 to 6.

12. Botulinum toxin; A method for preparing a botulinum toxin composition comprising the step of mixing poloxamer and methylcellulose.

13. In Paragraph 12, The above poloxamer is 10 to 30 weight percent based on the total weight of the composition; A method for preparing a botulinum toxin composition, wherein the above-mentioned methylcellulose is mixed in an amount of 0.5 to 10 weight percent based on the total weight of the composition.

14. A method for preparing a botulinum toxin composition according to claim 12, comprising the step of additionally mixing glycerol.

15. A method for preparing a botulinum toxin composition according to claim 12, comprising the step of additionally mixing a cell-penetrating peptide.

16. A method for preparing a botulinum toxin composition comprising the step of mixing botulinum toxin and a cell-penetrating peptide.

17. A method for preparing a botulinum toxin composition according to claim 16, wherein the cell-penetrating peptide is composed of any one selected from the group consisting of SEQ ID NOs 1 to 6.

18. A method for cosmetic improvement of wrinkles, square jaw, skin softening, acne, pores, elasticity, or keloid symptoms, comprising the step of administering a botulinum toxin composition comprising botulinum toxin; poloxamer; and methylcellulose to an individual.

19. A method for treating neuromuscular disorders, facial spasms, facial spasms, blepharospasm, strabismus, dystonia, hyperhidrosis, migraine, or pain, comprising the step of administering a botulinum toxin composition comprising botulinum toxin; poloxamer; and methylcellulose to an individual.

20. Use of a botulinum toxin composition comprising botulinum toxin; poloxamer; and methylcellulose for improving wrinkles, square jaw, skin softening, acne, pores, elasticity, or keloid symptoms.

21. Use of a botulinum toxin composition comprising botulinum toxin; poloxamer; and methylcellulose for the treatment of neuromuscular disorders, facial spasms, blepharospasm, strabismus, dystonia, hyperhidrosis, migraine, or pain.

22. A composition for manufacturing a botulinum toxin pharmaceutical preparation comprising botulinum toxin; poloxamer; and methylcellulose.

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