Rapid efficacy expression effect of non-complexed botulinum toxin composition and method related thereto

Abstract

According to one embodiment of the present invention, provided is a botulinum toxin composition for use in a method for treating a disease in a subject in need of treatment, the botulinum toxin composition comprising a non-complexed botulinum toxin as an active ingredient and not containing any animal proteins, wherein the method comprises a step for topically administering a therapeutically effective amount of the composition to the subject in need of treatment, and the efficacy of the composition is expressed faster than the efficacy of a composition containing an animal protein and containing 19S complexed botulinum toxin as an active ingredient.

Description

Rapid onset effect of non-complex botulinum toxin composition and related methods

[0001] The present disclosure relates to a botulinum toxin preparation and a method related thereto, and more particularly, to a botulinum toxin preparation exhibiting a rapid onset of effect and a cosmetic or therapeutic method using the same.

[0002] Botulinum neurotoxin (BoNT) is a polypeptide product of the anaerobic bacterium Clostridium botulinum. It acts to paralyze muscles by preventing the release of neurotransmitters at the junction of nerves and muscles. Initially, botulinum toxin was a lethal toxic substance, but its use has since expanded to treat conditions such as strabismus, which are caused by excessive muscle contraction. Its use has since expanded to include a variety of other conditions. It is primarily used to treat dystonia, hemifacial spasm, tremor, and spasticity, as well as other non-dystonic movement disorders. It is also used for a variety of other indications, including ocular disease, pain-related conditions such as migraine, urological diseases, and the treatment of wrinkles for cosmetic purposes.

[0003] Meanwhile, type A botulinum toxin has the problem of not being able to expect immediate effects, as it takes several days or more for the effects to be manifested after administration of an effective dose. Korean Publication No. KR 10-2019-0126894 discloses information related to the properties and actions of type E botulinum toxin. However, type E botulinum toxin has a short duration of action, which increases the number of administrations, which hinders patient compliance.

[0004] One object of the present invention is to provide a composition for preventing and treating a disease comprising a non-complexed botulinum toxin and having a fast-acting effect, or a method for preventing or treating a disease by administering the composition to a subject in need of treatment for the disease.

[0005] According to one embodiment of the present invention, a botulinum toxin composition is provided, which comprises a non-complex botulinum toxin as an active ingredient and does not contain an animal protein for use in a method for treating a disease in a subject in need of treatment, the method comprising the step of topically administering a therapeutically effective amount of the composition to the subject in need of treatment, wherein the effect of the composition is developed more quickly than the effect of a composition containing an animal protein and containing a 19S complex botulinum toxin as an active ingredient.

[0006] In one embodiment, the time to reach maximum DAS after administration of an effective amount of the composition may be shorter than the time to reach maximum DAS after administration of an effective amount of a 19S complex botulinum toxin composition containing animal proteins.

[0007] The time to reach the maximum DAS after administering the 19S complex botulinum toxin composition containing the above animal protein to a mouse at a dose of 12 U / kg may be at least 40% longer than the time to reach the maximum DAS after administering the composition to a mouse at a dose of 12 U / kg.

[0008] After administering the above composition to mice at a dose of 12 U / kg, the time to reach maximum DAS may be 20 to 30 hours.

[0009] In one embodiment, the time taken to reach 75% of the pre-administration CMAP value after administering an effective amount of the composition may be shorter than the time taken to reach 75% of the pre-administration CMAP value after administering an effective amount of the 19S complex botulinum toxin composition containing animal protein.

[0010] In one embodiment, the time required to reach 75% of the pre-administration CMAP value after administering the composition to rats at a dose of 4 U / kg may be less than 8 hours.

[0011] In one embodiment, the area under the curve of the CMAP graph for 72 hours after administration of an effective amount of the composition may be less than the area under the curve of the CMAP graph for 72 hours after administration of an effective amount of the 19S complex botulinum toxin composition containing animal protein. The area under the curve of the CMAP graph for 72 hours after administration of the composition to rats at a dose of 4 U / kg may be 1300 mV*hours or less.

[0012] In one embodiment, the composition may comprise one or more components selected from the group consisting of botulinum toxin, a surfactant, an amino acid, and an ionic compound. The surfactant may comprise at least one component selected from the group consisting of polysorbate and poloxamer, and the amino acid may comprise methionine.

[0013] In one embodiment, the composition may be for improving skin wrinkles, square jaw, pointy jaw, wounds, skin softening, scars, acne, pores, elasticity or keloid symptoms, or for treating at least one condition selected from the group consisting of facial spasm, blepharospasm, torticollis, blepharospasm, cervical dystonia, mid-pharyngeal dystonia, spasmodic dysphonia, migraine, pruritus ani and hyperhidrosis.

[0014] The above botulinum toxin may include type A toxin.

[0015] In one embodiment of the present invention, a method for preventing or treating a disease by administering the aforementioned composition to a subject in need of treatment is provided.

[0016] According to one embodiment of the present invention, a fast-acting disease prevention and treatment composition comprising a non-complexed botulinum toxin is provided.

[0017] According to one embodiment of the present invention, a method for rapidly preventing or treating a disease by administering the composition to a subject in need thereof is provided.

[0018] Figure 1 is a graph showing temporal changes in DAS values ​​after administration of three different formulations of botulinum toxin type A to the right calf muscle in mice. DAS values ​​at each measurement time were analyzed using a t-test. *p<0.05, **p<0.01, vs. albumin-free BoNT / A(7S).

[0019] Figure 2 is a graph showing the peak DAS score (A) and the time to peak DAS score (B) after administration of three different formulations of botulinum toxin type A to the right calf muscle in mice. Analysis was performed using a t-test. *p<0.05 vs. albumin-free BoNT / A(7S). ns = not significant.

[0020] Figure 3 is a graph showing temporal changes in DAS values ​​after administration of two different formulations of botulinum toxin type A to the right calf muscle in rats. DAS values ​​at each measurement time were analyzed using a t-test. †p = 0.06 vs. albumin-free BoNT / A(7S).

[0021] Figure 4 is a graph showing temporal changes in CMAP values ​​after administration of two different formulations of botulinum toxin type A to the right calf muscle in rats. CMAP values ​​at each measurement time were analyzed using a t-test. #p = 0.08, †p = 0.05 vs. albumin-free BoNT / A(7S).

[0022] Figure 5 is a graph showing the lowest CMAP value (A), the time to reach 75% of the pre-administration CMAP value (B), and the CMAPAUC (C) after administration of two different formulations of botulinum toxin type A to the right calf muscle in rats. Analysis was performed using a t-test. *p<0.05, **p<0.01 vs. albumin-free BoNT / A(7S). ns = not significant.

[0023] Unless otherwise defined, technical and scientific terms used herein have the same meanings as commonly used in the art. To facilitate understanding of the present disclosure, the following definitions will apply, and singular expressions include plural expressions, and vice versa, unless the context clearly dictates otherwise.

[0024] The term "and / or" includes all combinations that the associated constructs may define.

[0025] When interpreting components, even if not explicitly stated otherwise, they are interpreted as including a tolerance. In this specification, the term "about" is used to encompass a typical tolerance in the relevant technical field. For example, the term "about" may mean that a tolerance of up to 10% exists in a given value or range of values.

[0026] As used herein, the term 'botulinum toxin' means a botulinum toxin protein molecule produced by bacteria or recombinantly, including all serotypes and variants or fusion proteins thereof.

[0027] In one embodiment, the botulinum toxin is not limited to serotype and may be selected from the group consisting of types A (BoNT / A), B (BoNT / B), C (BoNT / C), D (BoNT / D), E (BoNT / E), F (BoNT / F), G (BoNT / G), H (BoNT / H), X (BoNT / X), Enterococcus sp. botulinum toxin J (eBoNT / J) and mosaic botulinum toxins and / or variants thereof. Examples of mosaic toxins include BoNT / DC, BoNT / CD and BoNT / FA. For example, type A may be used.

[0028] In one embodiment, the botulinum toxin is derived from various BoNT / A subtypes, such as A1, A2, A3, A4, A5, A6, A7, A9, A10; BoNT / B subtypes, such as B1, B2, B3, B4, B5, B6, B7, B8, Bnp, and Bbv; BoNT / C subtypes, such as C and CD; BoNT / D subtypes, such as D and DC; BoNT / E subtypes, such as E1, E2, E3, E4, E5, E6, E7, E8, E9; BoNT / F subtypes, such as F1, F2, F3, F4, F5, F6, F7; and BoNT / G subtypes, such as subtype G. BoNT subtypes include chimeric BoNTs, such as BoNT / DC, BoNT / CD, BoNT / FA, etc.

[0029] Botulinum toxin includes botulinum toxin derivatives. The botulinum toxin derivatives may be derivatives of native botulinum toxin or recombinant native botulinum toxin having botulinum toxin activity, which contain one or more chemical or functional modifications on a portion or a portion of the chain. For example, the botulinum toxin derivatives may be modified toxins having one or more amino acids deleted, modified, or substituted. The botulinum toxin may be a recombinant peptide, a fusion protein, or a hybrid neurotoxin prepared, for example, from subunits or domains of different toxin serotypes. The botulinum toxin may also be part of a whole molecule having toxic activity, or may be used as part of a combination or conjugated molecule, for example, a fusion protein.

[0030] The term 'complexing protein' refers to hemagglutinin and a non-toxic component that does not have hemagglutinin activity (nontoxic nonhemagglutinin; NTNH). Clostridium botulinum strains produce an immunologically distinct neurotoxin (type AG). The molecular mass of type AG neurotoxin (NTX) is approximately 150 kDa (7S). In culture media and foods with acidic conditions, NTX combines with the non-toxic component to form larger complexes called progenitor toxins (PTX). Here, 'S' refers to the Svedberg sedimentation coefficient (Svedberg unit: 1S=10^-13 s). PTX can have molecular masses of 900 kDa (19S), 500 kDa (16S), and 300 kDa (12S). The 12S toxin consists of the neurotoxin NTX and a non-toxic component (NTNH, non-toxic non-hemagglutinin) that does not have hemagglutinin activity, while the 19S and 16S toxins consist of NTX, NTNH, and hemagglutinin. Type A strains produce three forms of toxins (19S, 16S, and 12S), while types B, C, and D strains produce 16S and 12S toxins. Under alkaline conditions, PTX dissociates into NTX and the non-toxic component, the 19S and 16S toxins dissociate into NTX, NTNH, and the non-toxic component of hemagglutinin, and the 12S toxin dissociates into NTX and NTNH.

[0031] The botulinum toxin composition of the present disclosure may be a complex botulinum toxin composition containing a complex protein. For example, the complex botulinum toxin may contain NTNH or hemagglutinin, in which case the complex botulinum toxin may have a molecular weight of about 900 kDa or 500 kDa.

[0032] The botulinum toxin composition of the present disclosure may be a non-complexed botulinum toxin composition that does not contain complexing proteins. For example, the non-complexed botulinum toxin may not contain NTNH or hemagglutinin, in which case the non-complexed botulinum toxin may have a molecular weight of about 150 kDa. As used herein, the non-complexed botulinum toxin may be referred to as 7S botulinum toxin or a botulinum toxin having a molecular weight of 150 kDa.

[0033] The term 'animal protein' refers to blood-derived, blood-infused or other animal-derived products.

[0034] The botulinum toxin composition of the present disclosure may be a botulinum toxin composition that does not contain animal proteins. For example, the botulinum toxin composition of the present disclosure may not contain a protein stabilizer derived from an animal. In one embodiment, the botulinum toxin composition of the present disclosure may not contain albumin, such as human serum albumin or recombinant human albumin.

[0035] As used herein, the term 'unit', 'unit(s)', or 'U' refers to the LD, which is defined as the amount of botulinum toxin that kills 50% of mice injected with botulinum toxin. 50 Refers to capacity and is used interchangeably within a single product.

[0036] A "pharmaceutical composition" means a preparation containing an active ingredient. The term "preparation" means that, in addition to the active ingredient (e.g., botulinum toxin), at least one additional ingredient, such as albumin (e.g., human serum albumin or recombinant human albumin) and / or sodium chloride, is present in the pharmaceutical composition. A pharmaceutical composition is a preparation suitable for administration to a subject, such as a human patient. The pharmaceutical composition may be in a lyophilized form, for example, a solution formed after reconstitution of a lyophilized pharmaceutical composition with saline or water, or in the form of a solution that does not require reconstitution. The pharmaceutical composition may be liquid or solid. The pharmaceutical composition may be free of animal-derived proteins and / or albumin.

[0037] The term "active ingredient" refers to a biologically or physiologically effective substance in a formulation or composition. Specifically, in the context of a pharmaceutical formulation or composition, the term "active ingredient" refers to a constituent substance that produces the desired pharmaceutical effect. For example, the active ingredient of the present invention may be uncomplexed botulinum toxin, free of complexing proteins.

[0038] "Administration" or "administering" refers to the step of providing a pharmaceutical composition or active ingredient to a subject. The pharmaceutical composition may be administered via various suitable routes.

[0039] In one embodiment, the pharmaceutical composition may be administered transdermally, subcutaneously, or intramuscularly. In a specific embodiment, the composition may be administered locally to a muscle or group of muscles. For example, reduction of forehead wrinkles or skin wrinkles may be achieved by administering the composition transdermally or subcutaneously to the wrinkle. For example, the composition of one embodiment may be administered by subcutaneous or intramuscular injection. The composition according to one embodiment may be administered by subcutaneous or intramuscular injection via a prefilled syringe. The composition of one embodiment may be administered transdermally using a transdermal patch or microneedles.

[0040] "Pharmaceutical composition" refers to a preparation containing an active ingredient. In addition to the active ingredient (e.g., botulinum toxin), the "pharmaceutical composition" may include at least one additional ingredient, such as albumin (human serum albumin or recombinant human albumin) and / or sodium chloride. The pharmaceutical composition is a preparation suitable for administration to a subject, such as a mammal including a human. The subject to which the composition of the present invention is administered may include, without limitation, a human or an animal, such as a human, pig, dog, cat, cow, horse, or rat.

[0041] The pharmaceutical composition may be in liquid or lyophilized form. If the pharmaceutical composition is a lyophilized formulation, the lyophilized pharmaceutical composition may be reconstituted using, for example, saline solution or water. The pharmaceutical composition may be in solution form. The pharmaceutical composition may be liquid or solid. The pharmaceutical composition may be free of animal-derived proteins and / or albumin.

[0042] "Treat," "treating," or "treatment" means the alleviation or reduction (including partial reduction, substantial reduction, almost complete reduction, and complete reduction), resolution, or prevention (whether temporary or permanent) of a disease, disorder, or condition, so as to achieve a desired therapeutic result, for example, by healing injured or damaged tissue, or by altering, changing, strengthening, improving, ameliorating, and / or beautifying an existing or recognized disease, disorder, or condition. As used herein, "treatment" includes prevention. "Prevention" means delaying the onset of a disease, disorder, or condition. Prevention may be considered complete if the onset of the disease, disorder, or condition is delayed for a predetermined period of time.

[0043] In one embodiment, 'treatment' means treating a disease, disorder, or medical condition in a patient, such as a mammal (particularly a human), comprising one or more of the following:

[0044] (a) preventing the occurrence of said disease, disorder, or medical condition, i.e. preventing the recurrence of said disease or medical condition, or prophylactic treatment of a patient pre-disposed to said disease or medical condition;

[0045] (b) ameliorating the disease, disorder, or medical condition, including antagonizing the effect of other treatments, i.e. eliminating or causing regression of the disease, disorder, or medical condition in a patient;

[0046] (c) suppressing said disease, disorder, or medical condition, i.e. slowing or arresting the development of said disease, disorder, or medical condition in the patient; or

[0047] (d) Alleviation of symptoms of the disease, disorder, or medical condition in the patient.

[0048] As used herein, the terms 'unit', 'unit(s)', or 'U' refer to the LD50 dose, defined as the amount of botulinum toxin that kills 50% of mice injected with botulinum toxin, and are used interchangeably within a product.

[0049] In one embodiment, a therapeutically effective amount of botulinum toxin is from about 0.01 U / kg to about 100 U / kg, from about 0.1 U / kg to about 100 U / kg, from about 0.2 U / kg to about 100 U / kg, from about 0.2 U / kg to about 50 U / kg, from about 0.2 U / kg to about 30 U / kg, from about 0.2 U / kg to about 10 U / kg, from about 0.2 U / kg to about 1 U / kg, from about 1.2 U / kg, from about 4 U / kg, or from about 12 U / kg. In another embodiment, the dose may be about 1 U to about 10,000 U, about 1 U to about 5,000 U, about 1 U to about 2,500 U, about 1 U to about 1,000 U, about 1 U to about 500 U, about 1 U to about 300 U, about 1 U to about 200 U, about 10 U to about 200 U, about 10 U to about 100 U, about 10 U to about 50 U, or about 10 U to 30 U for a 60 kg adult.

[0050] According to one embodiment of the present invention, a botulinum toxin composition is provided, which comprises a non-complex botulinum toxin as an active ingredient and does not contain an animal protein for use in a method for treating a disease in a subject in need of treatment, the method comprising the step of topically administering a therapeutically effective amount of the composition to the subject in need of treatment, wherein the effect of the composition is developed more quickly than the effect of a composition containing an animal protein and containing a 19S complex botulinum toxin as an active ingredient.

[0051] In the present invention, “does not contain animal protein” means that it does not substantially contain protein derived from animals.

[0052] The composition of one embodiment may be administered in a therapeutically effective amount, and the term "effective amount" or "therapeutically effective amount" as used herein refers to an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to any medical treatment or prevention. The effective dosage level may be determined according to the severity of the disease, the activity of the drug, the patient's age, weight, health, and sex, sensitivity to the drug, the timing of administration, the route of administration, and the rate of excretion of the composition of the present disclosure, the duration of treatment, drugs used concurrently or in combination with the composition of the present disclosure, and other factors known in the medical arts.

[0053] In one embodiment, the botulinum toxin may be administered in single or multiple treatment sessions. The dosage may be administered as a single or divided injection at the injection site. In multiple treatment sessions, the botulinum toxin may be administered at intervals of no more than six months, four months, or three months. In multiple treatment sessions, the interval between botulinum toxin administrations includes the first treatment and the second treatment, and the dosage of the second treatment may be less than, more than, or the same as the dosage of the first treatment.

[0054] The time to reach the maximum DAS (digit abduction score) after administration of an effective amount of the above composition may be shorter than the time to reach the maximum DAS after administration of an effective amount of a 19S complex botulinum toxin composition containing animal protein.

[0055] In one embodiment, the time to reach maximum DAS after administering a 19S complex botulinum toxin composition containing animal proteins to a mouse at a dose of 12 U / kg may be at least about 40% longer than the time to reach maximum DAS after administering the composition to a mouse at a dose of 12 U / kg. For example, the time to reach maximum DAS after administering a 19S complex botulinum toxin composition containing animal proteins to a mouse at a dose of 12 U / kg may be at least about 20%, at least about 30%, at least about 40%, at least about 50%, from about 30% to about 60%, from about 30% to about 55%, from about 35% to about 55%, or from about 40% to 50% longer than the time to reach maximum DAS after administering the composition to a mouse at a dose of 12 U / kg.

[0056] In this specification, when referring to a data value derived from a group consisting of two or more individuals, the value may mean an average value for the individuals. For example, the statement that the data value derived from group A is “higher”, “lower”, “longer”, “shorter”, “larger”, “smaller”, “faster” or “slower” than the data value derived from group B may mean that the average value derived from the individuals included in group A is “higher”, “lower”, “longer”, “shorter”, “larger”, “smaller”, “faster” or “slower” than the average value derived from the individuals included in group B. However, if the value refers to a value other than the average value, it is obvious that the usual interpretation shall be followed.

[0057] In this specification, DAS may be measured by visually evaluating the degree of mouse muscle paralysis according to the criteria described below by administering an effective amount of a composition to the calf muscle of a mouse.

[0058] In one embodiment, the 19S complex botulinum toxin composition containing an animal protein may include albumin (e.g., human serum albumin) as the animal protein. The 19S complex botulinum toxin composition containing an animal protein may include an animal protein (e.g., albumin) and sodium chloride as excipients.

[0059] In one embodiment, the time to reach maximum DAS after administering the composition to a mouse at a dose of 12 U / kg may be 20 to 30 hours. For example, the time to reach maximum DAS after administering the composition to a mouse at a dose of 12 U / kg may be about 18 to about 36 hours, about 20 to about 35 hours, about 20 to about 30 hours, about 20 to about 28 hours, about 22 to about 28 hours, about 23 to about 27 hours, about 24 to about 26 hours, or about 25 hours.

[0060] In one embodiment, the time taken to reach 75% of the pre-administration CMAP (compound muscle action potential) value after administering an effective amount of the composition of the present invention may be shorter than the time taken to reach 75% of the pre-administration CMAP value after administering an effective amount of the 19S complex botulinum toxin composition containing animal protein.

[0061] In one embodiment, the time required to reach 75% of the pre-administration CMAP value after administering the composition of the present invention to rats at a dose of 4 U / kg (CMAP T75% ) may be less than 8 hours. For example, CMAP after administering the composition of the present invention to rats at a dose of 4 U / kg T75%may be less than about 9 hours, less than about 8 hours, less than about 7.5 hours, from about 7 hours to about 9 hours, from about 7 hours to about 8.5 hours, from about 7.5 hours to about 9 hours, from about 7.5 hours to about 8.5 hours, or from about 7.5 hours to about 8 hours, for example, about 7.8 hours.

[0062] In the present specification, CMAP may be measured at the site of the administered muscle using a nerve and electromyography tester (e.g., model name UltraPro S100) after administering an effective amount of the composition to the calf muscle of a rat. For example, the anode may be placed on the right psoas muscle of the rat at the 4th to 5th lumbar vertebrae, the cathode may be placed on the right sciatic nerve based on the 5th to 6th lumbar vertebrae, the distance between the anode and the cathode may be maintained at about 1 cm, the recording electrode may be placed on the gastrocnemius muscle, the reference electrode may be placed on the Achilles tendon area, the ground electrode may be placed on the rectus femoris area, the stimulation level and duration may be 20 to 30 mA, 0.2 ms, and the filter range of the amplifier may be set to 2-10 K at 60 Hz.

[0063] In one embodiment, the area under the curve of the CMAP graph for 72 hours after administration of an effective amount of the composition (CMAP AUC ) may be smaller than the area under the curve of the CMAP graph for 72 hours after administration of an effective amount of a 19S complex botulinum toxin composition containing animal protein.

[0064] In one embodiment, the area under the curve of the CMAP graph for 72 hours after administration of an effective amount of the composition (CMAP AUC ) may be about 5% to about 15%, about 5% to about 12%, or about 7% to about 12%, for example, about 10% less than the area under the curve of the CMAP graph for 72 hours after administration of an effective amount of a 19S complex botulinum toxin composition containing animal protein.

[0065] In one embodiment, the area under the curve of the CMAP graph for 72 hours after administering the composition to rats at a dose of 4 U / kg may be 1300 mV*hours or less. For example, the area under the curve of the CMAP graph for 72 hours after administering the composition to rats at a dose of 4 U / kg may be 1350 mV*hours or less, 1300 mV*hours or less, 1250 mV*hours or less, 1150 mV*hours to 1350 mV*hours, 1200 mV*hours to 1300 mV*hours, 1250 mV*hours to 1300 mV*hours, or 1200 mV*hours to 1250 mV*hours.

[0066] In the past, in the case of 19S complex toxins, it took several days or more for the therapeutic effect to be maximized, which caused low patient satisfaction with the treatment. However, according to one embodiment of the present invention, a subject administered a therapeutically effective amount of the present composition containing non-complex botulinum toxin and containing no animal protein can experience faster improvement or treatment of the disease or symptom than when the same amount of 19S complex toxin composition was administered to the same area, thereby increasing patient satisfaction.

[0067] Meanwhile, in order to ship a botulinum toxin composition, it must undergo a potency test, and the only potency test method currently used is the mouse IP (intraperitoneal, intraperitoneal) LD50 measurement method through a mouse potency assay (MPA). According to one embodiment, when the composition of the present invention is administered to a mouse, a faster onset of effect is achieved compared to a non-complex botulinum toxin composition, so the time required for the potency test is shortened, and the efficiency of the botulinum toxin manufacturing process can be improved.

[0068] In one embodiment, the composition can include a pharmaceutically acceptable excipient or additive. A pharmaceutically acceptable excipient or additive can be a stabilizer, an ionic compound, a surfactant, a buffer, a lyoprotectant, or a combination thereof, such as an amino acid (e.g., methionine), a salt (e.g., NaCl), a buffer, a non-ionic surfactant (e.g., a polysorbate, e.g., polysorbate 20), a sugar (e.g., a disaccharide and / or a monosaccharide, e.g., glucose, fructose, galactose, trehalose, sucrose, and / or maltose), a polyol (e.g., a sugar alcohol such as mannitol, inositol, lactitol, isomalt, xylitol, erythritol, sorbitol, and / or glycerol), or a combination thereof.

[0069] In one embodiment, the nonionic surfactant may include, for example, polyoxyethylene compounds, ethoxylated alcohols, ethoxylated esters, ethoxylated amides, polyoxypropylene compounds, propoxylated alcohols, ethoxylated / propoxylated block polymers and propoxylated esters, alkanolamides, amine oxides, fatty acid esters of polyhydric alcohols, ethylene glycol esters, diethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl fatty acid esters, sorbitan esters, sucrose esters, and / or glucose (dextrose) esters. For example, it may include polyoxyethylene fatty alcohol esters, sorbitan fatty acid esters [Span], polyoxyethylene sorbitan fatty acid esters [e.g., polyoxyethylene (20) sorbitan monooleate (Tween 80 or polysorbate 80), polyoxyethylene (20) sorbitan monostearate (Tween 60 or polysorbate 60), polyoxyethylene (20) sorbitan monolaurate (Tween 20 or polysorbate 20) and other Tweens, sorbitan esters, glycerol esters.

[0070] The botulinum toxin composition of the present invention can be formulated in any form, such as a solid or liquid preparation, for example, a lyophilized powder, a vacuum-dried powder, a liquid, or a pre-filled syringe preparation.

[0071] The composition of one embodiment may be a vacuum-dried composition, a lyophilized composition, or a liquid composition.

[0072] In one embodiment, the botulinum toxin composition comprises non-complexed botulinum toxin, a surfactant, and / or an amino acid; and may include one or more ingredients selected from the group consisting of sugars, sugar alcohols, and ionic compounds.

[0073] The surfactant may be a nonionic surfactant, for example, an alkylene oxide surfactant such as an ethylene oxide surfactant or a propylene oxide surfactant. For example, it may be a polyoxyethylene sorbitan fatty acid ester and / or a polyoxyethylene-polyoxypropylene block copolymer, for example, a poloxamer and / or a polysorbate.

[0074] In one embodiment, the polysorbate can include, for example, polysorbate 20, 40, 60, 80, or 100, and can include, for example, polysorbate 20. In one embodiment, the amino acid can be at least one selected from the group consisting of methionine, arginine, alanine, glutamic acid, aspartic acid, arginine, glycine, and cysteine.

[0075] In one embodiment, the amino acid can comprise a natural amino acid (one or more of the 20 known natural amino acids or one or more of the 22 known natural amino acids including selenocysteine ​​and pyrrolysine). For example, the amino acid can comprise at least one selected from the group consisting of methionine, isoleucine, alanine, tryptophan, tyrosine, glutamic acid, aspartic acid, arginine, glycine, and cysteine, for example, methionine.

[0076] For example, the composition of one embodiment may include a polysorbate (e.g., polysorbate 20) and methionine as stabilizers.

[0077] In one embodiment, the sugar may include trehalose, sucrose, maltose, fructose, raffinose, lactose, or glucose. The sugar alcohol may include cyclodextrin, mannitol, sorbitol, glycerol, xylitol, or inositol. For example, the freeze-dried composition of one embodiment may include sucrose.

[0078] In one embodiment, the ionic compound may include sodium chloride, sodium phosphate, ammonium phosphate, magnesium sulfate, sodium acetate, sodium lactate, sodium succinate, sodium propionate, or potassium phosphate.

[0079] In one embodiment, the lyophilized composition comprises non-complexed botulinum toxin, a surfactant, and / or an amino acid; and may comprise one or more components selected from the group consisting of sugars, sugar alcohols, and ionic compounds. For example, the lyophilized composition may comprise non-complexed botulinum toxin, a non-ionic surfactant, an amino acid, a sugar or sugar alcohol, and an ionic compound. For example, the lyophilized composition may comprise non-complexed botulinum toxin; a poloxamer or polysorbate; at least one component selected from the group consisting of methionine, arginine, alanine, glutamic acid, aspartic acid, arginine, glycine, and cysteine; a sugar or sugar alcohol, and an ionic compound.

[0080] In one embodiment, the liquid composition may include uncomplexed botulinum toxin, a surfactant, an amino acid, and / or an ionic compound. For example, the liquid composition may include uncomplexed botulinum toxin; a poloxamer or polysorbate; at least one selected from the group consisting of methionine, arginine, alanine, glutamic acid, aspartic acid, arginine, glycine, and cysteine; and an ionic compound. The pH of the liquid composition in one embodiment may be from 5.5 to 7.0.

[0081] In one embodiment, the composition may be for improving skin wrinkles, square jaw, pointy jaw, wounds, skin softening, scars, acne, pores, elasticity or keloid symptoms, or for treating at least one condition selected from the group consisting of facial spasm, blepharospasm, torticollis, blepharospasm, cervical dystonia, mid-pharyngeal dystonia, spasmodic dysphonia, migraine, pruritus ani and hyperhidrosis.

[0082] According to one embodiment of the present invention, the botulinum toxin described above may comprise a type A botulinum toxin. For example, it may comprise a type A botulinum toxin subtype, such as an A1, A2, A3, A4, A5, A6, A7, A9, or A10 toxin. The type A botulinum toxin may comprise a chimeric toxin. For example, the light chain and / or heavy chain of the type A botulinum toxin may comprise a combination of the first to fourth domains of botulinum toxins, such as A1, A2, A3, A4, A5, A6, A7, A9, and / or A10 toxins. For example, one of the light chain and / or heavy chain of the type A botulinum toxin may comprise a combination of domains 1 to 4 of the A1, A2, A3, A4, A5, A6, A7, A9 and / or A10 toxins, and the other may comprise a light chain or heavy chain of a domain of a type B to H toxin.

[0083] According to one embodiment of the present invention, the botulinum toxin composition described above is administered to a mouse to obtain an LD of the composition. 50 comprising a step of measuring the LD 50 The time required to measure LD was calculated by administering a 19S complex botulinum toxin composition containing animal proteins to mice. 50 A method for shortening the time for measuring the potency of botulinum toxin is provided, which is shorter than the time for measuring the potency of botulinum toxin.

[0084] According to one embodiment of the present invention, there is provided a use of the above-described botulinum toxin composition in a method for shortening the time for measuring the titer of botulinum toxin for a mouse.

[0085] According to one embodiment of the present invention, a method of treating a disease in a subject is provided, comprising the step of locally administering a therapeutically effective amount of the aforementioned botulinum toxin composition to a subject in need of treatment.

[0086] According to one embodiment of the present invention, there is provided a use of an effective amount of the botulinum toxin preparation in the manufacture of a medicament for treating a disease.

[0087] According to one embodiment of the present invention, a method for rapidly improving or treating a disease or symptom of a subject is provided, comprising the step of locally administering a therapeutically effective amount of the aforementioned botulinum toxin composition to a subject in need of treatment.

[0088]

[0089] Hereinafter, the present invention will be described in more detail with examples, but these are only for the purpose of explaining the present invention and are not intended to limit its scope in any way.

[0090]

[0091] Example 1: Nonclinical Efficacy Test of Uncomplexed and Complexed Botulinum Toxin 1

[0092] Animals: Female CD1 (ICR) mice, 5 weeks old, were purchased from Orient Bio Co., Ltd., and 6-week-old mice were used in the experiment after 1 week of acclimatization and quarantine. Sterilized solid food for laboratory animals (R40-10, SAFE, France) was fed ad libitum, and tap water was sterilized at high temperature and high pressure and provided ad libitum. During the acclimatization, quarantine, and experimental periods, mice were raised under specific pathogen-free conditions set at a temperature of 23 ± 3°C, a relative humidity of 55 ± 15%, a lighting schedule of 12 h (8:00 AM to 8:00 PM), ventilation rate of 15 times / h, and illumination intensity of 150–300 Lux. This study was conducted after review and approval (A-2022-007) by the Animal Experiment Ethics Committee of Medytox Co., Ltd.

[0093] Test materials: Coretox (Lot No.: E623001, Medytox), MBA-P01 (Lot No.: TFXA21004, Medytox), and NXA20001 (Medytox) listed in Table 1 were used.

[0094] Test groupProduct name or code nameManufacturerBotulinum toxin additiveAlbumin-free BoNT / A(7S)CoretoxMedytoxApproximately 150 kDa, non-complexed botulinum toxin type Amethionine, polysorbate 20, sucrose, sodium chlorideAlbumin BoNT / A(19S)MBA-P01MedytoxApproximately 900 kDa, complexed botulinum toxin type Ahuman serum albumin, sodium chlorideAlbumin-free BoNT / A(19S)NXA20001MedytoxApproximately 900 kDa, complexed botulinum toxin type Amethionine, polysorbate 20, sucrose, sodium chloride

[0095] Each test substance was diluted to a concentration of 60 U / mL using sterile saline solution (Daehan Pharmaceutical Industry).

[0096] The day on which the test substance was administered was set as day 0, and the mice were anesthetized using an injection anesthetic (100 mg / kg ketamine hydrochloride + 10 mg / kg xylazine). Then, each test substance was administered to the right calf muscle of the mice at 0.2 mL / kg using a Hamilton syringe according to the group composition described in Table 2.

[0097] Military animal test group dose (U / kg) administration route evaluation index 16 Albumin-free BoNT / A (7S) 12 Right calf muscle DAS 2 16 Albumin BoNT / A (19S) 12 Right calf muscle DAS 3 10 Albumin-free BoNT / A (19S) 12 Right calf muscle DAS

[0098] The evaluation was conducted using the digit abduction score (DAS), which visually assesses the degree of muscle paralysis in mice. The DAS scores shown in Table 3 indicate the degree of muscle paralysis based on the shape of the toe on the side where the drug was administered.

[0099] Score Criteria 0 Normal, no different from the shape of the foot on the non-injected side 1 Narrowing of the space between the toes, or two toes close together and the rest fully extended 2 Significant narrowing of the space between all toes, or three toes close together 3 Curved foot with four toes close together 4 Curved foot with all toes close together

[0100] Statistics: GraphPad Prism 7.05 (GraphPad Software Inc., CA, USA) was used for graphical 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 are expressed as the mean ± standard deviation. Statistical analysis between the two groups was performed using a two-tailed t-test, and a p-value less than 0.05 was considered statistically significant.

[0101] result

[0102] Referring to Fig. 1, three different botulinum toxin formulations were administered at a dose of 12 U / kg to the right calf muscle of the mouse, and then the DAS was evaluated at different time points. As a result of the evaluation, the DAS score tended to increase in the BoNT / A(7S) non-albumin formulation administration group compared to the BoNT / A(19S) albumin formulation and non-albumin formulation administration groups until approximately 25 hours after administration, which is the time when the BoNT / A(7S) non-albumin formulation reached the maximum DAS score. In particular, at 8 hours after administration, the DAS score was significantly increased in the BoNT / A(7S) non-albumin formulation administration group compared to the BoNT / A(19S) albumin formulation and non-albumin formulation administration groups, confirming that the BoNT / A(7S) non-albumin formulation onset was faster.

[0103] To further confirm the rapid onset effect confirmed above, the time to reach maximum DAS (T DASmax) was analyzed and shown in Fig. 2. As a result, in the BoNT / A(7S) non-albumin formulation administration group, a significant increase of about 50% or more was observed compared to the BoNT / A(7S) non-albumin formulation administration group in the case of the BoNT / A(19S) albumin formulation (average 37.9 hours) and the BoNT / A(19S) non-albumin formulation (38.4 hours) at an average of 25 hours. From these results, the fast-onset effect was confirmed in the non-animal and non-complex botulinum toxin type A formulation compared to the animal and non-animal complex botulinum toxin type A formulations.

[0104] Example 2: Nonclinical Efficacy Test of Uncomplexed and Complexed Botulinum Toxin 2

[0105] The rapid onset effect of non-complex and non-albumin botulinum toxin type A formulation was confirmed in the rat model through the following experiment. Female 6-week-old SD rat mice were purchased from Orient Bio Co., Ltd., and 7-week-old rats were used in the experiment after one week of acclimatization and quarantine. Sterilized solid food for laboratory animals (R40-10, SAFE, France) was fed ad libitum, and tap water was sterilized at high temperature and high pressure and provided ad libitum. During the acclimatization, quarantine, and experimental periods, mice were housed under specific pathogen-free conditions set at a temperature of 23 ± 3℃, a relative humidity of 55 ± 15%, a lighting period of 12 h (8:00 AM to 8:00 PM), a ventilation rate of 15 times / h, and an illuminance of 150–300 Lux. This study was conducted after review and approval (A-2023-001) by the Animal Experiment Ethics Committee of Meditox Co., Ltd.

[0106] The test substances were prepared as follows. Coretox (Meditox), a 100-unit non-complexed and non-albumin botulinum toxin type A finished product, and MBA-P01 (Meditox), a complexed albumin botulinum toxin type A finished product, were used [Table 1]. Each test substance was diluted to a concentration of 40 U / mL using sterile saline (Daehan Pharmaceutical Industry).

[0107] The day on which the test substance was administered was set as day 0, and the rats were anesthetized using an injection anesthetic (60 mg / kg ketamine hydrochloride + 10 mg / kg xylazine). Then, each test substance was administered to the rats' right calf muscle at 0.1 mL / kg using a Hamilton syringe according to Table 4 below.

[0108] Military animal test group dose (U / kg) administration route evaluation index 18 Albumin-free BoNT / A (7S) 4 Right calf muscle DAS, CMAP 28 Albumin BoNT / A (19S) 4 Right calf muscle DAS, CMAP

[0109] As in Example 1, the digit abduction score (DAS) evaluation method (Table 3) was used to visually evaluate the degree of muscle paralysis in rats. The compound muscle action potential (CMAP) test was used for the evaluation. CMAP was measured in the right gastrocnemius muscle (injected muscle) of each rat at different time points after administration of the test substance using an UltraPro S100 (Natus neurology Inc.) device. After anesthetizing with an intravenous anesthetic, the hair in the measurement area was shaved and the rats were placed in a prone position. The anode was placed in the right psoas muscle at the 4th to 5th lumbar vertebrae of the rat, and the cathode was placed on the right sciatic nerve based on the 5th to 6th lumbar vertebrae. The distance between the anode and cathode was maintained at approximately 1 cm. The recording electrode was placed in the gastrocnemius muscle, the reference electrode was placed in the Achilles tendon area, and the grounding electrode was placed in the rectus femoris area. The stimulation level and duration were 20 to 30 mA, 0.2 ms, and the amplifier filter range was set to 2-10 K at 60 Hz. When measured under the conditions, the height from the baseline to the peak after the latency of the waveform was converted into data as the CMAP measurement value. The obtained data were statistically processed in the same way as in Example 1, and the AUC (area under the curve) of the CMAP graph was calculated using Graphpad Prism 7.05 (GraphPad Software Inc. CA, USA).

[0110] result

[0111] Referring to Fig. 3, when two different formulations of botulinum toxin type A were administered to the right calf muscle of rats at a dose of 4 U / kg, the DAS evaluation results by time zone showed that 24 hours after administration, the DAS score was 1 point in all 8 animals (100%) in the BoNT / A (7S) non-albumin formulation administration group, whereas 1 point was observed in only 5 out of 8 animals (62.5%) in the BoNT / A (19S) albumin formulation administration group, showing low expression. The average score at 24 hours also tended to increase in the BoNT / A (7S) non-albumin formulation administration group.

[0112] Referring to Figure 4, the CMAP score measured by time period after administration showed a tendency for the CMAP score to decrease in the BoNT / A(7S) non-albumin formulation administration group compared to the BoNT / A(19S) albumin formulation. In order to more clearly confirm the rapid onset effect confirmed above, the time until reaching 75% of the CMAP value before administration (CAMP) was measured. T75% ) and AUC of CMAP graph for 72 hours (CMAP AUC ) were calculated and analyzed. Referring to Figure 5, it was confirmed that the time to reach 75% of the CMAP value before administration was significantly shorter in the BoNT / A(7S) non-albumin formulation administration group compared to the BoNT / A(19S) albumin formulation. In addition, the CMAP was significantly reduced in the BoNT / A(7S) non-albumin formulation administration group compared to the BoNT / A(19S) albumin formulation. AUC The values ​​were observed. From these results, the rapid onset effect of the non-animal and non-complex botulinum toxin type A formulation was confirmed in a rat model.

Claims

1. A composition containing non-complex botulinum toxin as an active ingredient and containing no animal protein for use in a method for treating a disease of a subject requiring treatment, The method comprises the step of topically administering a therapeutically effective amount of the composition to a subject in need of treatment, A botulinum toxin composition, wherein the efficacy of the composition is expressed more quickly than that of a composition containing animal protein and comprising 19S complex botulinum toxin as an active ingredient.

2. In paragraph 1, A composition, wherein the time to reach maximum DAS after administration of an effective amount of the composition is shorter than the time to reach maximum DAS after administration of an effective amount of a 19S complex botulinum toxin composition containing animal protein.

3. In paragraph 2, A composition, wherein the time to reach the maximum DAS after administering a 19S complex botulinum toxin composition containing animal protein to a mouse at a dose of 12 U / kg is at least 40% longer than the time to reach the maximum DAS after administering the composition to a mouse at a dose of 12 U / kg.

4. In paragraph 2, A composition, wherein the time to reach maximum DAS is 20 to 30 hours after administering the composition to mice at a dosage of 12 U / kg.

5. In paragraph 1, A composition, wherein the time taken to reach 75% of the CMAP value before administration after administering an effective amount of the composition is shorter than the time taken to reach 75% of the CMAP value before administration after administering an effective amount of a 19S complex botulinum toxin composition containing animal protein.

6. In paragraph 5, A composition, wherein the time required to reach 75% of the CMAP value before administration is less than 8 hours after administering the composition to rats at a dose of 4 U / kg.

7. In paragraph 1, A composition, wherein the area under the curve of the CMAP graph for 72 hours after administration of an effective amount of the composition is smaller than the area under the curve of the CMAP graph for 72 hours after administration of an effective amount of the 19S complex botulinum toxin composition containing animal protein.

8. In paragraph 7, A composition, wherein the area under the curve of the CMAP graph for 72 hours after administering the composition to rats at a dosage of 4 U / kg is 1300 mV*hours or less.

9. In any one of paragraphs 1 to 8, A composition comprising at least one component selected from the group consisting of botulinum toxin, a surfactant, an amino acid, and an ionic compound.

10. In paragraph 9, A composition wherein the surfactant is at least one selected from the group consisting of polysorbate and poloxamer, and the amino acid is at least one selected from the group consisting of methionine, arginine, alanine, glutamic acid, aspartic acid, arginine, glycine, and cysteine.

11. A composition according to any one of claims 1 to 8, wherein the composition is for improving symptoms of skin wrinkles, square jaw, pointy jaw, wounds, skin softening, scars, acne, pores, elasticity or keloid, or for treating at least one disease selected from the group consisting of facial spasm, blepharospasm, torticollis, blepharospasm, cervical dystonia, central pharyngeal dystonia, spasmodic dysphonia, migraine, pruritus ani and hyperhidrosis.

12. A composition according to any one of claims 1 to 8, wherein the botulinum toxin comprises type A toxin.

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