TWI739368B - Type A botulinum toxin complex, its formulation and method of use - Google Patents

Abstract

The present disclosure relates to a type A botulinum toxin complex, and a formulation and used thereof. The botulinum toxin type A complex includes HA70, HA17, HA33, NTNH and BoNT / A1. The molecular weight of the botulinum toxin complex is 740 ~ 790 kDa. Comparing to the prior botulinum toxin complex, the botulinum toxin complex of the present disclosure has a smaller molecular weight, higher safety, and a considerable therapeutic effect.

Description

Type A botulinum toxin complex, its formulation and method of use

The present invention relates to the field of protein cosmetics or medicines, in particular to a botulinum toxin complex.

Wrinkles are an indicator of aging and can be caused by biochemical, histological, and physiological changes that accumulate environmental damage to the skin. In addition, other secondary factors can also cause the characteristic folds, grooves and wrinkles of facial wrinkles (Stegman et al., The Skin of the Aging Face Cosmetic Dermatological Surgery, 2nd ed., St. Louis, Mo.: Mosby Year Book : 5-15 (1990)). These secondary factors include: constant gravity pull, frequent and constant positional pressure on the skin (for example: during sleep) and repetitive facial movements caused by facial muscle contraction (Stegman et al., The Skin of the Aging Face Cosmetic Dermatological Surgery, 2nd ed., St. Louis, Mo.: Mosby Year Book: 5-15 (1990)).

Different technologies have been used to potentially relieve some signs of aging. These technologies range from facial moisturizers containing alpha-hydroxy acids and retinol to surgery and neurotoxin injections. For example: In 1986, Jean et al. developed a method of using botulinum toxin type A to treat movement-related wrinkles in the brow area (Schantz and Scott, In Lewis GE (Ed) Biomedical Aspects of Botulinum, New York: Academic Press, 143 -150 (1981)). This use of botulinum toxin type A to treat wrinkles led to the publication of this method in 1992 (Schantz and Scott, In Lewis G. E. (Ed) Biomedical Aspects of Botulinum, New York: Academic Press, 143-150 (1981)). Until 1994, the treatment of other sports-related wrinkles on the face was reported (Scott, Opthalmol, 87:1044-1049 (1980)). This led to the use of type A botulinum toxin for aesthetic medicine.

It is reported that botulinum toxin type A is the most lethal natural biological agent known to man. Spores of botulinum are present in the soil and can grow in improperly sterilized and sealed food containers. Botulism caused by the ingestion of bacteria can be fatal. Botulinum toxin prevents synaptic transmission by inhibiting the release of acetylcholine through neuromuscular junctions, thereby producing muscle paralysis, and it is believed that botulinum toxin also acts in other ways. Its effect essentially blocks the signals that usually cause muscle spasm or contraction, which leads to paralysis. In the past ten years, the muscle paralytic activity of botulinum toxin has been used to achieve various therapeutic effects. Controlled administration of botulinum toxin has been used to provide muscle paralysis to treat various medical conditions, such as neuromuscular conditions characterized by overactive skeletal muscles. Conditions treated with botulinum toxin include: hemifacial spasm, adult-onset spastic torticollis, anal fissure, blepharospasm, cerebral palsy, cervical dystonia, migraine, strabismus, temporomandibular joint disorders, and various types of muscle cramps and Convulsions. Recently, the muscle paralysis effect of botulinum toxin has been applied to the treatment and beauty of facial applications, such as the treatment of wrinkles and facial muscle spasms or contractions.

In addition to the type A form of botulinum toxin, there are seven serologically different botulinum toxin types, which are also produced by the gram-positive bacterium botulinum toxin. Among the eight serologically different types of botulinum toxin, the seven serotypes that can cause paralysis are named botulinum toxin serotypes A, B, C, D, E, F, and G. Among the above Each is distinguished by neutralization with serotype-specific antibodies. The molecular weight of each botulinum toxin protein is approximately 150kD. Due to the molecular size and molecular structure of botulinum toxin, it cannot penetrate the stratum corneum and the lower layers of the skin structure. The different serotypes of botulinum toxin differ in the effect, severity and duration of paralysis caused in different animal species. For example, in a rat experiment, by measuring the rate of paralysis, it has been determined that type A botulinum toxin is 500 times more potent than type B botulinum toxin. In addition, it has been determined that botulinum toxin type B is non-toxic at a dose of 480 U/kg in primates.

The botulinum toxin released by botulinum toxin is a component of a toxin complex containing approximately 150kD botulinum toxin protein molecules and related non-toxin proteins. It is believed that these endogenous non-toxin proteins include hemagglutinin protein (HA) family and non-hemagglutinin protein (NTNH). It is believed that the non-toxin protein stabilizes the botulinum toxin molecules in the toxin complex and protects it from digestive acid denaturation when the toxin complex is ingested. Therefore, when the toxin complex is administered through the gastrointestinal tract, the non-toxin protein of the toxin complex protects the activity of the botulinum toxin, thereby enhancing systemic penetration. In addition, it is believed that some non-toxin proteins specifically stabilize the botulinum toxin molecules in the blood.

The presence of non-toxin proteins in the toxin complex usually causes the molecular weight of the toxin complex to be greater than the molecular weight of the naked botulinum toxin molecule, which is about 150 kD as mentioned above. For example: Clostridium botulinum can produce a type A botulinum toxin complex with a molecular weight of about 900kD, 500kD or 300kD. Botulinum toxin types B and C are produced in the form of complexes with a molecular weight of about 700 kD or about 500 kD. Botulinum toxin type D is produced in the form of a complex with a molecular weight of approximately 300kD or 500kD. Botulinum toxin types E and F are only produced as complexes with a molecular weight of approximately 300 kD.

In order to provide additional stability to botulinum toxin, the toxin complex is usually stabilized by binding the complex to albumin during the manufacturing process. For example, Botox ^® (Allergan, Inc, Irvine, CA) is a preparation containing botulinum toxin, which contains 100 U of type A botulinum toxin and auxiliary proteins, 0.5 mg of human albumin, and 0.9 mg of sodium chloride. Albumin is used to bind and stabilize toxin complexes in different environments, including those related to manufacturing, transportation, storage, and administration.

However, the number of botulinum toxin type A complexes currently available is limited. There is still a need in the art for new type A botulinum toxin complexes and their formulations.

In view of the need for more type A botulinum toxin complexes in the art, the inventors provide a new type A botulinum toxin complex. Compared with existing products (for example: Botox ^® ), the botulinum toxin type A complex of the present invention has a smaller molecular weight, so the production cost is lower; and the stability and safety are higher.

In one aspect, the present invention provides a type A botulinum toxin complex, wherein the components include: HA70, HA17, HA33, NTNH, BoNT/A1, wherein the botulinum toxin composition has a 740~790 kDa Molecular weight.

In a specific embodiment, the botulinum toxin type A complex is composed of HA70, HA17, HA33, NTNH, and BoNT/A1, and has a molecular weight of about 760 kDa.

In a specific embodiment, HA70 comprises any one of the following: (1) the amino acid sequence of SEQ ID NO. 1; or (2) having one or more additions, deletions, substitutions or insertions in the sequence of SEQ ID NO. 1 Amino acid sequence derived from an amino acid, and retains the function of HA70.

In a specific embodiment, HA17 includes any one of the following: (1) the amino acid sequence of SEQ ID NO. 2; or (2) having one or more additions, deletions, substitutions or insertions in the sequence of SEQ ID NO. 2 The resulting amino acid sequence of an amino acid, and retains the function of HA17.

In a specific embodiment, HA33 includes any one of the following: (1) the amino acid sequence of SEQ ID NO. 3; or (2) having one or more additions, deletions, substitutions or insertions in the sequence of SEQ ID NO. 3 The resulting amino acid sequence of an amino acid, and retains the function of HA33.

In a specific embodiment, NTNH includes any one of the following: (1) the amino acid sequence of SEQ ID NO. 4; or (2) having one or more additions, deletions, substitutions or insertions in the sequence of SEQ ID NO. 4 Amino acid sequence derived from an amino acid, and retains the function of NTNH.

In a specific embodiment, BoNT/A1 includes any one of the following: (1) the amino acid sequence of SEQ ID NO. 5; or (2) having an addition, deletion, substitution or insertion in the sequence of SEQ ID NO. 5 Or more amino acid sequence derived from amino acid, and retain the function of BoNT/A1.

In another aspect, the present invention provides a nucleic acid for synthesizing the botulinum toxin type A complex of the present invention.

In another aspect, the present invention provides a pharmaceutical composition comprising the botulinum toxin type A complex of the present invention and a pharmaceutically or dermatologically acceptable carrier.

In yet another aspect, the present invention provides a liquid formulation comprising the type A botulinum toxin complex according to any one of claims 1 to 6, the composition of which comprises: human serum albumin, methionine , Arginine, sodium chloride, Tween 80, citrate buffer, and/or phosphate buffer.

In yet another aspect, the present invention provides a cosmetic method comprising administering to a subject in need an effective amount of the botulinum toxin type A complex, pharmaceutical composition, or liquid formulation of the present invention.

In a specific embodiment, the cosmetic method includes: reducing the appearance of fine lines and/or wrinkles, especially on the face, or expanding the eyes, lifting the corners of the mouth, or smoothing the lines that appear on the upper lip, or generally relieving muscle tension.

In yet another aspect, the present invention provides a method for treating or preventing diseases caused by synaptic transmission or release of acetylcholine in a subject in need thereof, which comprises administering to the subject an effective amount of the present invention Botulinum toxin type A complex, pharmaceutical composition, or liquid formulation.

In a specific embodiment, the disease is selected from neuromuscular symptoms characterized by overactive skeletal muscle, nerve pain, migraine, overactive bladder, rhinitis, sinusitis, acne, dystonia, dystonic contraction, hyperhidrosis Symptoms, hypersecretion of one or more glands controlled by the cholinergic nervous system, hemifacial spasm, adult-onset spastic torticollis, anal fissure, blepharospasm, cerebral palsy, cervical dystonia, strabismus, temporomandibular joint disorders And various types of muscle cramps and cramps.

In yet another aspect, the present invention provides the use of the botulinum toxin type A complex, pharmaceutical composition or liquid formulation of the present invention in the preparation of cosmetics or pharmaceutical compositions, which are used to reduce fine lines and / Or the appearance of wrinkles, especially on the face, or enlarge the eyes, lift the corners of the mouth, or smooth the lines that appear on the upper lip, or generally relieve muscle tone; the drug is used to treat or prevent synaptic transmission by acetylcholine Or release the disease caused by it.

In a specific embodiment, the disease is selected from neuromuscular disorders characterized by skeletal muscle hyperactivity, nerve pain, migraine, overactive bladder, rhinitis, sinusitis, acne, dystonia, dystonic contractions, hyperhidrosis , Hypersecretion of one or more glands controlled by the cholinergic nervous system, hemifacial spasm, adult-onset spastic torticollis, anal fissure, blepharospasm, cerebral palsy, cervical dystonia, strabismus, temporomandibular joint disorders, and Various types of muscle cramps and cramps.

In yet another aspect, the present invention provides a botulinum toxin type A complex, pharmaceutical composition or liquid formulation for reducing the appearance of fine lines and/or wrinkles, especially on the face, or enlarging the eyes and lifting the corners of the mouth , Or smooth the lines appearing on the upper lip, or generally relieve muscle tension; the drug is used to treat or prevent diseases caused by the synaptic transmission or release of acetylcholine.

In a specific embodiment, the disease is selected from neuromuscular disorders characterized by skeletal muscle hyperactivity, nerve pain, migraine, overactive bladder, rhinitis, sinusitis, acne, dystonia, dystonic contractions, hyperhidrosis , Hypersecretion of one or more glands controlled by the cholinergic nervous system, hemifacial spasm, adult-onset spastic torticollis, anal fissure, blepharospasm, cerebral palsy, cervical dystonia, strabismus, temporomandibular joint disorders, and Various types of muscle cramps and cramps.

The botulinum toxin type A complex of the present invention (also referred to as "OBI-858" in this article) has the following advantages compared ^{with the commercially available Botox ®:}

^{(1) Its therapeutic effect is equivalent to that of Botox ®} in mouse animal experiments (its effective dose: Effective Dose: ED50), but its lethal dose (Lethal Dose: LD50) is more lethal ^{than Botox ® in mouse animal experiments.} The dose is higher, and its safety margin (MOS) is also higher than Botox ^® in mouse animal experiments, which means that the present invention can produce Botox that is safer ^{than the commercial product Botox ®} Toxin preparations.

(2) The total protein molecular weight of the type A botulinum toxin complex of the present invention is 760 kDa, ^{which is different from Botox ® with a} molecular weight of 900 kDa, and is a new type A botulinum toxin complex.

(3) The formulation of the botulinum toxin type A complex of the present invention has improved stability.

The present invention relates to a type A botulinum toxin complex and its liquid formulation. As described herein, the composition of the present invention can be applied to the application of providing an injectable botulinum toxin to a subject, which can be used for various therapeutic, cosmetic and/or cosmetic purposes. Compared with the existing type A botulinum toxin complex product, the type A botulinum toxin complex of the present invention has better safety and enhanced or equivalent efficacy. Regarding the type A botulinum toxin complex of the present invention, the present invention provides a liquid formulation for the type A botulinum toxin complex to provide excellent stability.

As used herein, the term "type A botulinum toxin complex" refers to a type A botulinum toxin protein molecule of approximately 150 kD and related endogenous non-toxin proteins, namely hemagglutinin protein (HA) The complex formed with non-toxin non-hemagglutinin protein (NTNH). The hemagglutinin protein is composed of 70 kDa, 33 kDa, and 17 kDa subcomponents (ie, HA70, HA33, HA17). The botulinum toxin complex does not need to be derived from botulinum rod as a single toxin. For example, botulinum toxin or modified botulinum toxin can be prepared recombinantly and then combined with non-toxin proteins. You can also buy recombinant botulinum toxin and combine it with non-toxic proteins.

Here, the botulinum toxin type A complex may also contain HA70, HA33, HA17, NTNH or BoNT/A1 as described herein, or a variant thereof. The variant described herein is an amino acid sequence obtained by adding, deleting, substituting or inserting one or more amino acids in the sequence (for example: any one of SEQ ID No. 1 to 5).

The term "one or several" refers to the number of regions that do not damage the protein activity to a large extent. In addition, the number represented by the term "one or several" is, for example, 1 to 100, preferably 1 to 80, more preferably 1 to 50, still more preferably 1 to 40, and particularly preferably It is 1 to 30, 1 to 20, 1 to 10, or 1 to 5 (for example: 1, 2, 3, 4, or 5).

The following text provides the sequence of each component of the type A botulinum toxin complex described in the specific examples.

HA70 (SEQ ID NO 1.): MNSSIKKIYNDIQEKVINYSDTIDLADGNYVVRRGDGWILSRQNQILGGSVISNGSTGIVGDLRVNDNAIPYYYPTPSFNEEYIKNNIQTVFTNFTEANQIPIGFEFSKTAPSNKNLYMYLQYTYIRYEIIKVLQHEIIERAVLYVPSLGYVKSIEFNPGEKINKDFYFLTNDKCILNEQFLYKKILETTKNIPTNNIFNSKVSSTQRVLPYSNGLYVINKGDGYIRTNDKDLIGTLLIEAGSSGSIIQPRLRNTTRPLFTTSNDTKFSQQYTEERLKDAFNVQLFNTSTSLFKFVEEAPSDKNICIKAYNTYEKYELIDYQNGSIVNKAEYYLPSLGYCEVTNAPSPESEVVKMQVAEDGFIQNGPEEEIVVGVIDPSENIQEINTAISDNYTYNIPGIVNNNPFYILFTVNTTGIYKINAQNNLPSLKIYEAIGSGNRNFQSGNLCDDDIKAINYITGFDSPNAKSYLVVLLNKDKNYYIRVPQTSSNIENQIQFKREEGDLRNLMNSSVNIIDNLNSTGAHYYTRQSPDVHDYISYEFTIPGNFNNKDTSNIRLYTSYNQGIGTLFRVTETIDGYNLINIQQNLHLLNNTNSIRLLNGAIYILKVEVTELNNYNIRLHIDITN

HA17 (SEQ ID NO. 2): MSVERTFLPNGNYNIKSIFSGSLYLNPVSKSLTFSNESSANNQKWNVEYMAENRCFKISNVAEPNKYLSYDNFGFISLDSLSNRCYWFPIKIAVNTYIMLSLNKVNELDYAWDIYDTNENILSQPLLLLPNFDIYNSNQMFKLEKI

HA33 (SEQ ID NO 3.): MEHYSVIQNSLNDKIVTISCKADTNLFFYQVAGNVSLFQQTRNYLERWRLIYDSNKAAYKIKSMDIHNTNLVLTWNAPTHNISTQQDSNADNQYWLLLKDIGNNSFIIASYKNPNLVLYADTVARNLKLSTLNNSNYIKFIIEDYIISDLNNFTCKISPILDLNKVVQQVDVTNLNVNLYTWDYGRNQKWTIRYNEEKAAYQFFNTILSNGVLTWIFSNGNTVRVSSSNDQNNDAQYWLINPVSDTDETYTITNLRDTTKALDLYGGQTANGTAIQVFNYHGDDNQKWNIRNP

NTNH (SEQ ID NO 4.): MNINDNLSINSPVDNKNVVVVRARKTDTVFKAFKVAPNIWVAPERYYGESLSIDEEYKVDGGIYDSNFLSQDSEKDKFLQAIITLLKRINSTNAGEKLLSLISTAIPFPYGYIGGGYYAPNMITFGSAPKSNKKLNSLISSTIPFPYAGYRETNYLSSEDNKSFYASNIVIFGPGANIVENNTVFYKKEDAENGMGTMTEIWFQPFLTYKYDEFYIDPAIELIKCLIKSLYFLYGIKPSDDLVIPYRLRSELENIEYSQLNIVDLLVSGGIDPKFINTDPYWFTDNYFSNAKKVFEDHRNIYETEIEGNNAIGNDIKLRLKQKFRININDIWELNLNYFSKEFSIMMPDRFNNALKHFYRKQYYKIDYPENYSINGFVNGQINAQLSLSDRNQDIINKPEEIINLLNGNNVSLMRSNIYGDGLKSTVDDFYSNYKIPYNRAYEYHFNNSNDSSLDNVNIGVIDNIPEIIDVNPYKENCDKFSPVQKITSTREINTNIPWPINYLQAQNTNNEKFSLSSDFVEVVSSKDKSLVYSFLSNVMFYLDSIKDNSPIDTDKKYYLWLREIFRNYSFDITATQEINTNCGINKVVTWFGKALNILNTSDSFVEEFQNLGAISLINKKENLSMPIIESYEIPNDMLGLPLNDLNEKLFNIYSKNTAYFKKIYYNFLDQWWTQYYSQYFDLICMAKRSVLAQETLIKRIIQKKLSYLIGNSNISSDNLALMNLTTTNTLRDISNESQIAMNNVDSFLNNAAICVFESNIYPKFISFMEQCINNINIKTKEFIQKCTNINEDEKLQLINQNVFNSLDFEFLNIQNMKSLFSSETALLIKEETWPYELVLYAFKEPGNNVIGDASGKNTSIEYSKDIGLVYGINSDALYLNGSNQSISFSNDFFENGLTNSFSIYFWLRNLGKDTIKSKLIGSKEDNCGWEIYFQDTGLVFNMIDSNGNEKNIYLSDVSNNSWHYITISVDRLKEQLLIFI DDNLVANESIKEILNIYSSNIISLLSENNPSYIEGLTILNKPTTSQEVLSNYFEVLNNSYIRDSNEERLEYNKTYQLYNYVFSDKPICEVKQNNNIYLTINNTNNLNLQASKFKLLSINPNKQYVQKLDEVIISVLDNMEKYIDISEDNRLQLIDNKICKMKYIDISEDNRLQLIDNKICKMKYIDSNDIYCNNFKLSKMSKYIDSNDIYNSKMSKYD

BoNT / A1 (SEQ ID NO 5.): MPFVNKQFNYKDPVNGVDIAYIKIPNAGQMQPVKAFKIHNKIWVIPERDTFTNPEEGDLNPPPEAKQVPVSYYDSTYLSTDNEKDNYLKGVTKLFERIYSTDLGRMLLTSIVRGIPFWGGSTIDTELKVIDTNCINVIQPDGSYRSEELNLVIIGPSADIIQFECKSFGHEVLNLTRNGYGSTQYIRFSPDFTFGFEESLEVDTNPLLGAGKFATDPAVTLAHELIHAGHRLYGIAINPNRVFKVNTNAYYEMSGLEVSFEELRTFGGHDAKFIDSLQENEFRLYYYNKFKDIASTLNKAKSIVGTTASLQYMKNVFKEKYLLSEDTSGKFSVDKLKFDKLYKMLTEIYTEDNFVKFFKVLNRKTYLNFDKAVFKINIVPKVNYTIYDGFNLRNTNLAANFNGQNTEINNMNFTKLKNFTGLFEFYKLLCVRGIITSKTKSLDKGYNKALNDLCIKVNNWDLFFSPSEDNFTNDLNKGEEITSDTNIEAAEENISLDLIQQYYLTFNFDNEPENISIENLSSDIIGQLELMPNIERFPNGKKYELDKYTMFHYLRAQEFEHGKSRIALTNSVNEALLNPSRVYTFFSSDYVKKVNKATEAAMFLGWVEQLVYDFTDETSEVSTTDKIADITIIIPYIGPALNIGNMLYKDDFVGALIFSGAVILLEFIPEIAIPVLGTFALVSYIANKVLTVQTIDNALSKRNEKWDEVYKYIVTNWLAKVNTQIDLIRKKMKEALENQAEATKAIINYQYNQYTEEEKNNINFNIDDLSSKLNESINKAMININKFLNQCSVSYLMNSMIPYGVKRLEDFDASLKDALLKYIYDNRGTLIGQVDRLKDKVNNTLSTDIPFQLSKYVDNQRLLSTFTEYIKNIINTSILNLRYESNHLIDLSRYASKINIGSKVNFDPIDKNQIQLFNLESSKIEVILKNAIVYNSMYENFSTSFWIRIPKYFNSISLNNEYTIINCMENNSGWKVSL NYGEIIWTLQDTQEIKQRVVFKYSQMINISDYINRWIFVTITNNRLNNSKIYINGRLIDQKPISNLGNIHASNNIMFKLDGCRDTHRYIWIKYFNLFDKELNEKEIKDLYDNQSNSGILKDFWGDYLQYDKPYYMLNLYDPNKYVDVNNVGIRGYMYLKGPRGSVMTTNIYLNSSLYRGTKFIIKKYASGNKDNIVRNNDRVYINVVVKNKEYRLATNASQAGVEKILSALEIPDVGNLSQVVVMKSKNDQGITNKCKMNLQDNNGNDIGFIGFHQFNNIAKLVASNWYNRQIERSSRTLGCSWEFIPVDDGWGERPL

The present invention also relates to a method for producing a biological effect by injecting an effective amount of the composition of the present invention into a subject or patient in need. Among them, biological effects can include, for example, muscle paralysis, reduction of excessive secretion or sweating, treatment of neuropathic pain or migraine, control of rhinitis or sinusitis, treatment of overactive bladder, reduction of muscle spasm, prevention or reduction of acne, reduction or enhancement of immune response , Reduce wrinkles, or prevent or treat various other diseases.

The composition of the present invention can be injected into the skin or epithelium of a subject or patient (ie, a human or other mammal in need of specific treatment). The term "need" as used herein refers to needs related to drugs or health (for example, treatment of abnormal facial muscle spasms related to conditions), as well as cosmetic and subjective needs (for example: changing or improving the appearance of facial tissues). In the simplest form of the compositions of the present invention, they may contain an aqueous pharmaceutically acceptable diluent, such as buffered saline (eg, phosphate buffered saline). However, the composition may contain other ingredients commonly found in injectable pharmaceutical or cosmetic compositions, including a dermatologically or pharmaceutically acceptable carrier that is compatible with the tissue to be applied. As described herein, the term "dermatologically or pharmaceutically acceptable" means that the composition or its constituents described herein are suitable for contact with tissues or for use in general patients without toxicity, incompatibility, Instability, allergic reactions, etc. Where appropriate, the composition of the present invention may contain any components conventionally used in related fields, especially cosmetics and dermatology.

The botulinum toxin complex disclosed according to the present invention can be delivered to the muscles under the skin by injection (usually using a syringe), or to produce paralysis, produce relaxation, relieve contraction, prevent or relieve spasm, reduce glandular output or other needs The effective amount of action acts on the glandular structure in the skin. Local delivery of botulinum toxin in this manner can provide dose reduction, reduce toxicity, and allow more precise dose optimization relative to injectable or implantable materials to achieve the desired effect.

The botulinum toxin complex disclosed in the present invention is administered to deliver an effective amount of botulinum toxin. As used herein, the term "effective amount" refers to an amount that is sufficient to produce the desired muscle paralysis or other biological or aesthetic effects, but it implicitly also represents a safe amount, that is, it is low enough to avoid serious side effects . The desired effect includes relaxing certain muscles, for example, reducing the appearance of fine lines and/or wrinkles, especially on the face, or adjusting the appearance of the face in other ways, such as expanding the eyes, raising the corners of the mouth, or relieving muscle tension. Muscle tension can be relieved on the face or elsewhere. The botulinum toxin complex of the present invention can be administered to patients by injection for the treatment of wrinkles, undesirable facial muscles or other muscle spasms, hyperhidrosis, acne or other diseases of the body that require relief of muscle pain or spasms. The botulinum toxin complex is administered to muscles or other skin-related or other target tissue structures by injection. For example, for legs, shoulders, back (including lower back), armpits, palms, feet, neck, face, groin, back of hands or feet, elbows, upper arms, knees, upper legs, Administer the botulinum toxin complex to the buttocks, torso, pelvis, or any other part of the body in need.

The botulinum toxin complex disclosed in the present invention can also be used to treat other diseases, including any diseases where prevention of synaptic transmission or release of acetylcholine would bring therapeutic benefits. For example: the conditions treatable by the present invention include but are not limited to nerve pain, migraine or other headache pain, overactive bladder, rhinitis, sinusitis, acne, dystonia, dystonic contraction, hyperhidrosis, and cholinergic nerve A neuromuscular disorder characterized by hypersecretion of one or more glands controlled by the system, characterized by overactive skeletal muscles. Conditions that can be treated with botulinum toxin include: half-face cramps, hemifacial spasm, adult-onset spastic torticollis, anal fissure, blepharospasm, cerebral palsy, cervical dystonia, strabismus, temporomandibular joint disorders and various types of muscles Cramps and cramps. The disclosure of the present invention can also be used to reduce or enhance immune response, or to treat other diseases for which injection of botulinum toxin complex has been suggested or administered. The botulinum toxin complex disclosed in the present invention can be prepared in solid form, such as freeze-dried powder, tablet, etc., and re-dissolved with water for injection for use before use.

The botulinum toxin complex, composition or formulation disclosed in the present invention is administered by a physician or other health care professional or under the guidance of a physician or other health care professional. They can be administered in one treatment or over time in a series of treatments. In a preferred embodiment, the botulinum toxin complex, composition or formulation disclosed in the present invention is injected at one or more locations that require botulinum toxin-related effects. Due to the nature of the botulinum toxin complex, the botulinum toxin is preferably in an amount, application rate, and frequency of application that produces the desired result without producing any undesirable or undesirable results.

In the disclosure herein, the formulation includes human serum albumin, methionine, arginine, sodium chloride, Tween 80, citrate buffer, and/or phosphate buffer. Preferably, the formulation comprises human serum albumin, sodium chloride and Tween 80 dissolved in 50 mM citrate buffer. In the formulation, the concentration of human serum albumin may be 0.1 to 1 mg/mL, for example 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8 or 0.9 mg/mL. The concentration of sodium chloride can be 5-20 mg/mL, for example: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 mg/mL, more preferably The ground is 11.25 mg/mL. The concentration of Tween 80 dissolved in 50 mM citrate buffer may be 0.1 to 0.3, for example, 0.2 mg/mL, and optimally 0.188 mg/mL. The formulation has a certain pH value, for example: pH 4-8, preferably pH 5.6.

In the following disclosure, the inventive concept is described in more detail by referring to the following specific embodiments. However, these specific embodiments are for exemplary purposes only, and are not intended to limit the scope of the inventive concept.

Specific embodiment

Example 1: Preparation process of OBI-858

(1) Preparation process of OBI-858 API

Resuscitate the OBI-858 strain in the freezing tube of the cell bank, and use the medium to gradually expand it, and then inoculate it into the fermentation tank. After the cell culture is completed, the cells and cell debris are removed by filtration. The purification steps include: two-step precipitation, buffer exchange and chromatography steps. After the chromatographic step, the product is filtered through a 0.22 µm filter to the bulk drug container, and then lyophilized to obtain the OBI-858 bulk drug.

(2) Preparation process of pharmaceutical products (preparations):

The lyophilized OBI-858 bulk drug powder was re-dissolved with the preparation buffer. The re-dissolved solution is sterilized through a 0.22 µm filter, and transferred to a sterile isolator, and then lyophilized to obtain OBI-858 drug product.

Example 2: Identification of OBI-858

(1) Genomic sequence alignment

The OBI-858 production strain is a type A botulinum strain developed by self-selection. It was confirmed by whole-genome sequencing that 98.28% is similar to type A botulinum ATCC 3502 strain, and 89.69% is similar to type A botulinum Hall strain (Shown in Table 1 below), so it is different from the products currently on the market in terms of strain gene analysis.

Table 1: Genomic sequence alignment of Clostridium botulinum strains Clostridium botulinum strain Genome sequence similarity (%) Clostridium botulinum A str. ATCC 3502 98.28 Clostridium botulinum A str. ATCC 19397 90.29 Clostridium botulinum A str. Hall 89.69 Clostridium botulinum H04402 065(A5) 68.98 Clostridium botulinum A2 str. Kyoto 68.39 Clostridium botulinum A3 str. Loch Maree 48.84

(2) Analysis of protein composition

Experimental materials/processes:

In order to achieve the highest sequence coverage, different enzymes were used to decompose the OBI-858 protein samples and then analyzed by LC-MS/MS. OBI-858 protein was first denatured with 6 M urea, reduced with 10 mM DTT at 37 °C for 1 hour, and alkylated with 50 mM IAM in the dark at room temperature for 30 minutes. The resulting protein was decomposed in 50 mM ammonium bicarbonate under the following conditions:

(A) Decompose with trypsin at 37 °C for 18 hours;

(B) Decompose with Glu-C protease at 37 °C for 18 hours;

(C) Decompose with trypsin at 37 °C, and decompose with Glu-C protease for 18 hours;

(D) Decompose with Lys-C at 37 °C for 18 hours;

(E) Decompose with chymotrypsin at 37 °C for 18 hours;

(F) Decompose with thermolysin at 37 °C for 18 hours.

After decomposition, the protein sample was diluted and acidified with 0.1% FA for MS analysis.

The samples were analyzed with a Q Exactive mass spectrometer (Thermo Scientific) coupled to the Ultimate 3000 RSLC system (Dionex). Use a C18 column (Acclaim PepMap RSLC, 75 μm x 150 mm, 2 μm, 100 Å) to perform LC separation through the gradients disclosed below.

Table 2: Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis procedures Time (minutes) Mobile phase A (%) Mobile phase B (%) Flow rate (μL/min) 0 99 1 0.25 5.5 99 1 0.25 45 75 25 0.25 48 40 60 0.25 50 20 80 0.25 60 20 80 0.25 65 99 1 0.25 70 99 1 0.25 Mobile phase A: 0.1% formic acid Mobile phase B: 99% acetonitrile + 0.1% formic acid

A full MS scan was performed in the range of m/z 300~2000, and the 10 most intense ions in the MS scan were fragmented to obtain MS/MS spectra. Search the Mascot database through Proteome Discoverer 1.4 to process the original data into a peak list.

Experimental results:

The results of amino acid sequencing of OBI-858 botulinum toxin type A by liquid chromatography tandem mass spectrometry (LC-MS/MS) showed that the toxin protein is mainly composed of 71 kDa, 17 kDa, 34 kDa, 138 kDa and 149 kDa. The composition of protein molecules of different sizes (disclosed in Table 3 below). The five different proteins were sequenced to obtain the amino acid sequences of SEQ ID NO. 1~5.

Table 3: Theoretical values calculated according to the sequence sequence Protein sequence ID Theoretical molecular weight (Da) Protein name Number of amino acids Molecular formula A5HZZ4 71,391.01 HA-70 626 C ₃₂₀₀ H ₄₉₅₃ N ₈₃₇ O ₉₉₉ S ₈ A5HZZ5 17034.31 HA-17 146 C ₇₇₅ H ₁₁₆₉ N ₁₉₁ O ₂₃₀ S ₆ A5HZZ6 33872.73 HA-33 293 C ₁₅₁₅ H ₂₃₁₃ N ₄₀₉ O ₄₆₈ S ₄ A5HZZ8 138092.85 NTNH 1193 C ₆₂₄₀ H ₉₅₅₅ N ₁₅₆₉ O ₁₉₀₅ S ₃₃ A5HZZ9 149,425.84 BoNT/A1 1296 C ₆₇₆₃ H ₁₀₄₅₂ N ₁₇₄₄ O ₂₀₁₁ S ₃₃

(3) Molecular exclusion high performance liquid chromatography (SEC-HPLC) detects product purity and connection refractive index and molecular exclusion multi-angle laser scattering (SEC-MALS) detects product molecular weight

Experimental materials/processes:

SEC-MALS analysis is a combination of size exclusion chromatography and high performance liquid chromatography (SEC-HPLC) and static laser light scattering analysis, using Agilent 1260 HPLC system, and online connection to containing SRT SEC-300 (5 µm, 7.8×300mm ) WYATT_DAWN HELEOS static laser scattering detector of the column. Chemstation software (Agilent Technologies) was used to control the Agilent 1260 HPLC system. Use ASTRA V software (Wyatt Technology) to control and analyze the light scattering detector. Dissolve 50 μg OBI-858 bulk drug in 100 μL 0.15 M sulfate buffer (pH 6.0), and then inject the sample. Use mobile phase: 0.15 M phosphate buffer + 0.1 M NaCl and 200 ppm NaN3; pH 6.0 is used for isocratic extraction at a flow rate of 0.5 mL/min. The OBI-858 complex concentration during the light scattering experiment was monitored by UV absorbance at 280 nm. The specific refractive index increment (dn/dc) value of 0.185 mL/g and the UV extinction coefficient of 1.49 mL/(mg×cm) were used to calculate the molecular weight of the OBI-858 complex from the light scattering data.

Experimental results:

Figure 1 shows that the purity of the product is detected by size exclusion chromatography and high performance liquid chromatography (SEC-HPLC). The purity of the product reaches 99.36%. The refractive index of the connection and the molecular weight of the product detected by the multi-angle laser scattering (MALS) are close to that shown in Figure 2 760 kDa protein.

(4) Electron microscopy measurement

Experimental materials/processes:

Use 25 μg of OBI-858 bulk drug (test product) and L-PTC/A (reference product) dissolved in 10 μL of deionized distilled water (DDW) for sample preparation for negative staining. First, 4 μL of OBI-858 sample solution droplets were adsorbed onto a glow-discharged carbon-coated copper grid, washed dropwise with deionized water, stained with 2% uranyl acetate and air-dried. Sample images were recorded using a JEM-2100F transmission electron microscope (TEM), which was equipped with a field emission electron source and operated at an accelerating voltage of 200 kV. Record images on DE12 camera.

Experimental results: Figure 3 (a) ~ (b) show the determination of the total protein molecular weight of the product by electron microscopy, Figure 3 (a) shows the appearance of OBI-858 toxin particles (pointed by the arrow), Figure 3 (b) The display is compared with the reference product of L-PTC/A with a molecular weight of 760 kDa, and the arrows indicate four different directions. The above test results all show that OBI-858 is a toxin protein close to 760 kDa, so the total protein molecular weight of this product is also different from the 900 kDa botulinum toxin product on the market.

Example 3: OBI-858 Efficacy Study (Efficacy Study)

The purpose of this study was to evaluate the single-dose acute intramuscular (IM) efficacy of the OBI-858 test product in female ICR mice. The efficacy of the OBI-858 test product was determined by the Digit Abduction Scoring assay (DAS assay) to calculate the IM median effective dose (ED50). The safety margin was 72-hour IM LD50 and 48-hour IM The ratio of ED50 is determined.

Materials and Method

Test article, reference article, vehicle and control

Test product Name OBI-858 Supplier OBI Pharma, Inc. Lot number 14004-25, 14004-30, 14004-35, 14004-45 Storage conditions -15 – -25 °C Other features One bottle of sterile freeze-dried powder contains 1,350,440 units.

Reference product name BOTOX ^® supplier Allergan Inc. Lot number C3461 C2 Storage conditions -15 – -25 °C Other features 50 units of botulinum toxin type A [per vial]

Vehicle and control

Dilution buffer (30 mM phosphate buffered saline, 0.2% w/v gelatin, pH 6.8) was used as a vehicle for formulation and a negative control during administration.

Preparation and disposal of dosage formulations

All preparations were freshly prepared on the scheduled day of administration for use on the same day.

Test product re-dissolution and dosage formulation

The preparation procedures of all dosage formulations are carried out in a Class II biological safety cabinet located in the testing facility. The preparation of the dosage formulation is carried out on the day of administration before the administration program.

The test product is packaged as a lyophilized material in a sterile sealed vial. In order to re-dissolve the lyophilized test article, 1 mL of dilution buffer was injected into the vial containing the test article. Shake the vial vigorously to dissolve the test product until the particulate matter in the re-dissolved raw material is not visible to the naked eye. The stock concentration of the re-dissolved test product is 1,350440 LD50 units (U) per milliliter. Then, perform serial dilutions with the re-dissolved test product as disclosed in Table 4 below:

Table 4: Dilution ratios of test products used in the preparation of dosage formulations Raw material ID 0(Remelting) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Group N/A N/A N/A N/A N/A 12 11 10 9 8 7 6 5 4 3 2 Dilution buffer volume (μL) 1000 700 600 600 2000 2445 402 396 406 394 508 488 509 781 778 800 Raw material ID N/A 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 Raw material volume (μL) N/A 100 100 100 500 1000 2000 2000 2000 2000 1000 1000 1000 1000 1000 1000 Total volume (μL) 1000 800 700 700 2500 3445 2402 2396 2406 2394 1508 1488 1509 1781 1778 1800 Dosing volume (μL) N/A N/A N/A N/A N/A 1445 402 396 406 1394 508 488 509 781 778 1800 Dilution factor 1 8 56 392 1960 6752.2 8,109.4 9715.1 11687.2 13989.6 21096.3 31391.3 47369.5 84365 150001 270001.8 Multiple dilution 1 8 7 7 5 3.445 1.201 1.198 1.203 1.197 1.508 1.488 1.509 1.781 1.778 1.8 Dose concentration (U/mL) 1350440 168805 24115 3445 689 200 166.528 139.005 115.549 96.532 64.013 43.020 28.509 16.007 9.003 5.002

To prepare each serial dilution, transfer the required volume of dilution buffer through a micropipette into a fresh glass vial with an upper lid. Then transfer the required volume of the stock solution to be diluted to the vial containing the dilution buffer via a micropipette. Mix by repeatedly gently transferring the solution several times, then gently invert it repeatedly, and tighten the cap on the vial several times. At the completion of each transfer step, discard the used micropipette tips. The raw material 5 to raw material 15 obtained when the dosage formulation preparation is completed are used as the test product dosage formulation.

Reference product re-dissolution and dosage formulation

The reference product is packaged as a lyophilized material in a sterile sealed vial. To re-thaw the lyophilized reference product, 0.2 mL of dilution buffer was injected into the vial containing the test product. Then shake the vial vigorously to dissolve the test product until the particles in the re-dissolved raw material are not visible to the naked eye. The stock concentration of the reconstructed test product is 250 LD50 units (U) per milliliter. Then, perform serial dilutions with the re-dissolved reference product according to Table 5 below:

Table 5: Reference dilution ratios used in the preparation of dosage formulations Raw material ID 0 (rebuild) 1 2 3 4 5 6 7 8 9 10 11 Group N/A 12 11 10 9 8 7 6 5 4 3 2 Dilution buffer volume (μL) 1600 360 281 253 229 187 366 327 305 391 373 360 Raw material ID N/A 0 1 2 3 4 5 6 7 8 9 10 Raw material volume (μL) 0 1440 1400 1280 1130 950 720 670 600 500 480 450 Total volume (μL) 1600 1800 1681 1533 1359 1137 1086 997 905 891 853 810 Dosing volume (μL) 0 400 401 403 409 417 416 397 405 411 403 810 Dilution factor 1 1.25 1.501 1.798 2.162 2.587 3.903 5.807 8.759 15.609 27.739 49.930 Multiple dilution 1 1.25 1.201 1.198 1.203 1.197 1.508 1.488 1.508 1.782 1.777 1.8 Dose concentration (U/mL) 250 200 166.568 139.078 115.642 96.623 64.059 43.049 28.541 16.016 9.013 5.007

The dosage formulation was prepared by re-dissolving the reference product through the serial dilution scheme disclosed above. To prepare each serial dilution, transfer the required volume of dilution buffer via a micropipette to a fresh glass vial with a top cap. Then transfer the required volume of the stock solution to be diluted to the vial containing the dilution buffer via a micropipette. Mix by repeatedly gently pipetting the liquid several times, then gently invert it repeatedly, and tighten the cap on the vial several times. At the completion of each transfer step, discard the used micropipette tips. When the preparation of the dosage formulation is completed, raw materials 1 to 11 are used as reference dosage preparations.

Research animals

The study animals are described below. Breed ICR mice Source BioLASCO Taiwan Co., Ltd (Taipei, Taiwan) Group distribution of animal data 236 females per batch; 4 batches in total Spare animals 14 females per batch; 4 batches in total Weight on the day of administration 18-22 grams (the weight range is within plus or minus 10% of the average weight) Identify the cage card Ear engraving, dye marking the body or tail, and cage card

Reasons for selection: Using mice as the test system, a determination procedure was designed to determine the efficacy and toxicity of the drug categories of the test and reference products in this study. Female animals were used for the development of this program, and previous studies also showed that female animals are more susceptible to the pharmacological effects and toxicity of the drug category of the test article and reference article. According to established procedures and previous research results, female mice were selected as the test system for this study.

Environmental conditions

The feeding conditions of the research animals are as follows: the animals are individually raised in polycarbonate cages with straw bedding in animal houses approved by the Association for Assessment and Accreditation of Laboratory Animal Care International. Temperature 20.7~23.1 °C, relative humidity 51.2~67.1%, photoperiod: 12 hours of light / 12 hours of darkness; feed: high-temperature sterilized Rodent Diet 5010 (PMI ^® Nutrition International, Inc., MO, USA), arbitrarily provided. Water: provided freely. Analyze food and water regularly. Errors and water analysis results are kept in the archives of the testing facility. It is expected that no contaminants will be present in food and water.

experimental design

Animal adaptation and selection: before the administration, the animals are quarantined at the testing facility for 2 days and allowed to adapt for 3 to 4 days. At least one day before the administration, the animals were randomly divided into study groups in the study design (groups 1-12, each group has two subgroups A and B). Each assigned animal is given a four-digit serial number. There was no statistically significant difference between the average weights of the study group or subgroups of the study group (p <0.05), which was determined by one-way analysis of variance (ANOVA). Animals that are not assigned are designated as spare animals. If health problems (including weight exceeding the acceptable limit (24 grams)) and/or suspected dose errors occur on or before day 0 of the corresponding batch, the assigned animal will be replaced with a spare animal. After the first day, spare animals were excluded from the study.

Study design: dose group, nominal dose concentration, dose volume, nominal dose level and the number of animals in each dose group are as follows:

Table 6: Study Design Group Nominal dose concentration (U/mL) ^a Dose volume (mL/animal) Nominal dose (U/animal) ^b Total number of animals (F: female) OBI-858 BOTOX ^® 1 0 ^c 0.02 0 10F Subgroup A Subgroup B 2 5 0.02 0.1 10F 10F 3 9 0.02 0.18 10F 10F 4 16 0.02 0.32 10F 10F 5 28.5 0.02 0.57 10F 10F 6 43 0.02 0.86 10F 10F 7 64 0.02 1.28 10F 10F 8 96.5 0.02 1.93 10F 10F 9 115.5 0.02 2.31 10F 10F 10 139 0.02 2.78 10F 10F 11 166.5 0.02 3.33 10F 10F 12 200 0.02 4 10F 10F 13 844,025 0.1 84,402.5 3F+ 3F ^d 0 a: The calculated dose concentration is shown above. b: Except for group 13, the actual dose level is multiplied by the dose volume. c: The dose of the dilution buffer used in the first group of animals (negative control group). d: Group 13 is used to verify the integrity of the test product. The 13th group of animals was selected from the spare animals on the dosing day.

Verification of the integrity of the test product

On the administration day of each batch (day 0), 3 animals were randomly selected from the spare animal library. Separately, re-dissolve a vial of the test product (raw material 0), and dilute a portion of the raw material 0 1.6 times. Then, 0.1 mL of the re-dissolved and diluted test substance was injected intravenously into the selected test animals through the tail vein. When the integrity of the test article is verified by the mortality of at least two test animals administered intravenously within 1 hour after administration, the administration of the main study animal will continue. If at least 2 animals fail to die within 1 hour, the specific vial of test material 0 will be discarded. In this case, prepare a fresh vial of raw material 0 from the second test vial, and repeat the verification test with the second vial of raw material. If the second vial also fails the verification test, the specific batch of doses will be discontinued due to the unqualified test product efficacy. Since all the test product vials passed the test, the above follow-up operations were not performed after the initial test.

The route of administration of the test article: intramuscular injection into the right gastrocnemius muscle.

Reasons for choosing the dose level and route of administration

By selecting the nominal dose level, determine the expected intramuscular median effective dose (IM ED50) and intramuscular median lethal dose (IM LD50) with an order of magnitude difference.

Frequency of administration: single dose.

Negative control administration

As a negative control, 20 μL of dilution buffer was injected intramuscularly into the right gastrocnemius muscle of the first dose animals. The control injection procedure is the same as the test article injection procedure.

Mortality Observation

The mortality of the animals was observed at least 6 hours on day 0 after administration. Thereafter, the mortality of the animals was observed every day during the entire study period, and the DAS assay was performed at the same time (until the 3rd day). The mortality rate was scored separately and counted based on each group to calculate LD50.

Toe Abduction Score (DAS) measurement

For each batch, the muscle weakness of the study animals was observed 24 to 26, 48 to 50, and 72 to 74 hours after the first animal was administered on the 0th day of each batch. The muscle weakness was scored by DAS analysis. For each animal, two personnel independently recorded its muscle weakness score. Average the scores of two separate observations for analysis. The DAS measurement method is performed according to the content disclosed in the US Patent Application Publication No. US 2010/0168023 A1. Muscle paralysis was measured using the toe abduction score (DAS) measurement, for example: Aoki, KR “A comparison of the safety margins of botulinum neurotoxin serotypes A, B, and F in mice”, Toxicon 2001; 39(12): 1815 -As described in the 1820 article. In the DAS assay, the tail of the mouse is suspended briefly, causing a characteristic startle response in which the mouse stretches its hind limbs and abducts its hind toes. The extent to which mice can show this startle response is scored on a five-point scale (from 0 to 4), where 0 represents a normal startle response, and four represents the greatest reduction in toe abduction and leg extension. The scoring of toe abduction is based on the following instructions and criteria:

Table 7: Scale of the Toe Abduction Score (DAS) measurement method Toe abduction score 4 The legs are bent, with 5 toes touching. die 3 The legs are flat, with 5 toes touching. Bend legs, 4 toes touching 2 The legs are flat, with slight open space at the top of all toes. Flat legs, 3 toes touching 1 The legs are flat, and the toe abduction width is different compared to the uninjected leg Legs are flat, 2 toes are touching and the remaining toes are completely spread out 0 The appearance is completely normal (no change) For the purpose of DAS analysis, animals that died during the DAS study were assigned a score of "4".

Body weight: measure body weight at least once before grouping and on day 0 before administration.

Statistical analysis: Probability analysis is used to fit probability and logarithmic sigmoid toxin unit/DAS response curves, and calculate IM ED50 and IM LD50 values. Use Minitab® statistical software for analysis. The safety margin of each batch was calculated based on the IM ED50 (DAS) at 48 hours after administration and the IM LD50 at 72 hours after administration. The acceptance criterion for regression analysis is R ² ＞ 0.9.

Computer system: An online data collection system (version 6.3.2; Xybion Medical Systems Corporation) is used to capture and analyze weight data in the testing facility.

Results and discussion

OBI-858 batch processing integrity test

Table 8 below lists an overview of the OBI-858 integrity test.

Table 8: OBI-858 integrity test batch batch number Average survival time (minutes after administration) 1 14004-25 34.0 ± 2.6 2 14004-30 33.3 ± 1.5 3 14004-35 34.7 ± 3.1 4 14004-45 31.7 ± 2.3 All vials of the test product passed the integrity test, and the average survival time after administration was less than 1 hour.

Weight: For each batch, the weight range of a single dose group is within ±10% of their average weight. Animals weighing more than 24 grams were replaced.

DAS measurement and ED50 calculation

OBI-858

As early as 24 hours after administration, the animals showed signs of toe abduction. The severity of toe abduction is related to the dose increase of OBI-858 before reaching the plateau at the highest severity (including death) at higher doses. In the 48-hour IM ED50, all samples except batch 1 passed the acceptance criteria of regression analysis (R ² > 0.9). According to the table below, for batches 2, 3 and 4, the ED50 at 48 hours after administration was 0.33, 0.32 and 0.36 U/animal, respectively.

Table 9: 48-hour ED50 of test product OBI858 (U/animal) batch ED50 95% lower 95% higher R ² value 1 0.21 0.13 0.35 0.89 2 0.33 0.27 0.41 0.97 3 0.32 0.23 0.46 0.92 4 0.36 0.27 0.47 0.95

BOTOX ^®

As early as 24 hours after administration, the animals showed signs of toe abduction. The severity of toe abduction is related to ^{the increase in the dose of BOTOX ®} , but for the plateau of maximum severity (including death) obtained, ^{the dose of BOTOX ®} is smaller than the dose of OBI-858. In the 48-hour IM ED50, all samples except batch 1 passed the acceptance criteria of regression analysis (R ² > 0.9). According to the following table, for batches 2, 3 and 4, the ED50 at 48 hours after administration was 0.22, 0.24 and 0.23 U/animal, respectively. Overall, the 48-hour IM ED50 of ^{BOTOX ® is lower than OBI-858.}

Table 10: 48-hour ED50 of ^{BOTOX ® (U/animal)} batch ED50 95% lower 95% higher R ² value 1 0.36 0.21 0.60 0.82 2 0.22 0.19 0.25 0.99 3 0.24 0.22 0.26 1.00 4 0.23 0.19 0.29 0.98

Mortality and LD50 calculation

OBI-858

Most deaths occurred 72 hours after dosing, although deaths were observed as early as 48 hours after dosing for groups 11 and 12. According to the table below, 72 hours after administration, the LD50 of all four batches were 3.00, 3.42, 3.33, and 3.35 U/animal, respectively. However, at 72 hours after dosing, only batches 2, 3, and 4 had sufficient dose groups within the dose-related linear range for regression analysis, and the death percentage was between 0% and 100%. (At least three dose groups are required for each analysis). Among the three batches, only the second and third batches passed the acceptance criteria of regression analysis.

Table 11: 72-hour LD50 of test product OBI858 (U/animal) batch LD50 95% lower 95% higher R ² value 1 3.00 2 3.42 2.70 4.32 0.99 3 3.33 3.31 3.36 1.00 4 3.35 1.66 6.75 0.86

BOTOX ^®

In animals administered with BOTOX ^® , death occurred earlier (at the earliest 24 hours after administration), and the dose was lower than that observed in animals administered with OBI-858 (72 hours after administration) Group 6 to Group 12). 72 hours after administration, the LD50 of all four batches were 2.21, 1.63, 2.14, and 1.57 U/animal, respectively (as shown in Table 13 below). However, none of the four batches of mortality results passed the acceptance criteria of regression analysis. According to the table below, for batches 2, 3, and 4, 72 hours after dosing, there is no dose group within the dose-related linear range. Therefore, it is impossible to perform regression analysis on the calculated LD50 of these batches. For batch 1, the R ² value is 0.74, which is lower than the acceptance standard.

Table 12: 72-hour LD50 of ^{BOTOX ® (U/animal)} batch LD50 95% lower 95% higher R ² value 1 2.21 0.80 6.11 0.74 2 1.63 3 2.14 4 1.57

Assess the effect of weight deviation on ED50 results

For OBI-858 and BOTOX ^® , there is no consistent change trend between the calculated ED50 between animals within the required weight range (18-22 grams) and overweight (greater than 22 grams) animals at the time of administration . Compared with the total ED50 value, excluding overweight animals from the ED50 calculation does not result in a consistent change in the ED50 value, as shown in the table below.

Table 13: Comparison of IM 48-hour ED50 of ^{OBI858 and BOTOX ® by body weight range (U/animal)} batch OBI-858 BOTOX ^® 48 hours IM ED50 48 hours IM ED50 (weight 18-22 g) 48 hours IM ED50 (weight> 22 g) 48 hours IM ED50 48 hours IM ED50 (weight 18-22 g) 48 hours IM ED50 (weight> 22 g) 1 0.21 0.19 0.23 0.36 0.36 0.41 2 0.33 0.33 0.40 0.22 0.21 0.22 3 0.32 0.37 0.32 0.24 0.25 0.25 4 0.36 0.38 0.36 0.23 0.24 0.25

In addition, it was found that an animal weighed less than 17.7 grams (below the 18-gram threshold) at the time of administration. Underweight animals (such as ID 0193; Group 9, Subset B (Botox ^® ); Batch 2) achieved similar toe abduction scores in all scores to other animals in the same group. Therefore, the event had no effect on the results of the study (Table 14: Test product OBI858; Table 15: BOTOX ^® ).

Estimation of the margin of safety (MOS): The calculation method of the margin of safety is: the ratio of 72-hour IM LD50 to 48-hour IM ED50.

For the four batches of tests, the MOS values of OBI-858 were 14.07, 10.32, 10.33, and 9.29. For the four batches of tests, ^{the MOS values of BOTOX ®} are 6.20, 7.58, 8.98 and 6.73 respectively. The MOS of OBI-858 is always higher than BOTOX ^® (as revealed in Table 16).

in conclusion

All OBI-858 vials tested in this study passed the integrity test. The increase in body weight (22 to 24 grams) of the study animals at the time of certain dosing has no significant effect on the results of the ED50 calculation. For OBI-858, the 72-hour IM LD50 and 48-hour IM ED50 values were 3.42 and 0.33 U/animal (batch 2) and 3.33 and 0.32 U/animal (batch 3), respectively. The corresponding MOS values of batch 2 and batch 3 are 10.32 and 10.33, respectively. For BOTOX ^® , the 72-hour IM LD50 and 48-hour IM ED50 values were 1.63 and 0.22 U/animal (batch 2) and 2.14 and 0.24 U/animal (batch 3), respectively. The corresponding MOS values of batch 2 and batch 3 are 7.58 and 8.98, respectively. In short, the effectiveness of OBI-858 is comparable to BOTOX ^® , but it has a higher 72-hour IM LD50 and margin of safety value ^{than BOTOX ®.}

Table 14: 48-hour mouse DAS score of test product OBI858 Batch 4 0.00±0.00 (3) 0.90±0.99 (5) 1.70±1.06 (5) 1.83±0.98 (3) 2.80±0.42 (5) 2.63±0.92 (4) 3.17±0.75 (3) 2.83±0.75 (3) 3.50±0.52 (6) 3.75±0.46 (4) 4.00±0.00 (2) 4.00±0.00 (3) 0.21±0.43 (7) 0.80±0.42 (5) 1.00±0.67 (5) 1.17±0.83 (7) 2.40±0.52 (5) 3.08±0.79 (6) 3.14±0.53 (7) 3.57±0.51 (7) 3.75±0.46 (4) 4.00±0.00 (6) 4.00±0.00 (8) 4.00±0.00 (7) Batch 3 0.00±0.00 (1) 1.00±0.68 (7) 1.00±0.00 (1) 2.13±1.13 (4) 2.90±1.10 (5) 3.63±0.52 (4) 3.25±0.45 (6) 3.38±0.52 (4) 3.75±0.46 (4) 3.80±0.42 (5) 4.00±0.00 (5) 4.00±0.00 (2) 0.00±0.00 (9) 0.33±0.52 (3) 1.00±0.69 (6) 2.92±0.67 (6) 2.80±0.79 (5) 3.42±0.51 (6) 3.25±0.46 (4) 3.58±0.51 (6) 3.50±0.52 (6) 3.90±0.32 (5) 3.90±0.32 (5) 4.00±0.00 (8) Batch 2 0.00±0.00 (3) 0.40±0.52 (5) 1.50±0.58 (2) 1.88±1.25 (4) 2.90±0.57 (5) 3.17±0.41 (3) 3.29±0.47 (7) 4.00±0.00 (4) 3.00±0.00 (1) 4.00±0.00 (2) 4.00±0.00 (2) 4.00±0.00 (3) 0.00±0.00 (7) 0.90±0.32 (5) 0.94±0.44 (8) 2.17±0.83 (6) 2.50±0.85 (5) 3.43±0.65 (7) 3.17±0.75 (3) 3.75±0.45 (6) 4.00±0.00 (9) 4.00±0.00 (8) 4.00±0.00 (8) 4.00±0.00 (7) Batch 1 0.00±0.00 (4) 1.00±0.00 (1) 2.00±0.76 (4) 2.00±0.00 (1) 3.50±0.71 (1) 3.25±0.50 (2) 3.88±0.35 (4) 3.75±0.50 (2) 4.00±0.00 (1) 4.00±0.00 (4) 4.00±0.00 (4) 4.00±0.00 (2) 0.00±0.00 (6) 1.39±0.50 (9) 2.33±0.78 (6) 2.44±1.10 (9) 2.88±1.31* (8) 3.44±0.51 (8) 4.00±0.00 (6) 4.00±0.00 (8) 3.78±0.43 (9) 3.92±0.29 (6) 4.00±0.00 (6) 4.00±0.00 (8) Group (Dose level) (U/animal) 1 (0.00) 2 (0.10) 3 (0.18) 4 (0.32) 5 (0.57) 6 (0.86) 7 (1.28) 8 (1.93) 9 (2.31) 10 (2.78) 11 (3.33) 12 (4.00) *One mouse died during the experiment. This animal has been excluded from the 48-hour DAS analysis

Table 15: 48-hour mouse DAS score of ^{BOTOX ®} Batch 4 (n) 0.00±0.00 (3) 0.90±0.99 (5) 1.70±1.06 (5) 1.83±0.98 (3) 2.80±0.42 (5) 2.63±0.92 (4) 3.17±0.75 (3) 2.83±0.75 (3) 4.00±0.00 (2) 4.00±0.00 (5) 4.00±0.00 (2) 4.00±0.00 (1) (n) 0.21±0.43 (7) 0.80±0.42 (5) 1.00±0.67 (5) 1.71±0.83 (7) 2.40±0.52 (5) 3.08±0.79 (6) 3.14±0.53 (7) 3.57±0.51 (7) 4.00±0.00 (8) 4.00±0.00 (5) 4.00±0.00 (8) 4.00±0.00 (9) Batch 3 (n) 0.00±0.00 (1) 1.00±0.68 (7) 1.00±0.00 (1) 2.13±1.13 (4) 2.90±1.10 (5) 3.63±0.52 (4) 3.25±0.45 (6) 3.38±0.52 (4) 4.00±0.00 (6) 4.00±0.00 (5) 4.00±0.00 (3) 4.00±0.00 (5) (n) 0.00±0.00 (9) 0.33±0.52 (3) 1.00±0.69 (9) 2.92±0.67 (6) 2.80±0.79 (5) 3.42±0.51 (6) 3.25±0.46 (4) 3.58±0.51 (6) 4.00±0.00 (4) 4.00±0.00 (5) 4.00±0.00 (7) 4.00±0.00 (5) Batch 2 (n) 0.00±0.00 (3) 0.40±0.52 (5) 1.50±0.58 (2) 1.88±1.25 (4) 2.90±0.57 (5) 3.17±0.41 (3) 3.29±0.47 (7) 4.00±0.00 (4) 4.00±0.00 (6) 4.00±0.00 (5) 4.00±0.00 (3) 4.00±0.00 (3) (n) 0.00±0.00 (7) 0.90±0.32 (5) 0.94±0.44 (8) 2.17±0.83 (6) 2.50±0.85 (5) 3.43±0.65 (7) 3.17±0.75 (3) 3.75±0.45 (6) 4.00±0.00 (4) 4.00±0.00 (5) 4.00±0.00 (7) 4.00±0.00 (7) Batch 1 (n) 0.00±0.00 (4) 1.00±0.00 (1) 2.00±0.76 (4) 2.00±0.00 (1) 3.50±0.71 (1) 3.25±0.50 (2) 3.88±0.35 (4) 3.75±0.50 (2) 3.75±0.50 (2) 3.00±0.00 (1) 4.00±0.00 (2) 4.00±0.00 (4) (n) 0.00±0.00 (6) 1.39±0.50 (9) 2.33±0.78 (6) 2.44±1.10 (9) 2.88±1.31* (8) 3.44±0.51 (8) 4.00±0.00 (6) 4.00±0.00 (8) 3.69±0.48 (8) 3.61±0.50 (9) 4.00±0.00 (8) 4.00±0.00 (6) Group (Dose level) (U/animal) 1 (0.00) 2 (0.10) 3 (0.18) 4 (0.32) 5 (0.57) 6 (0.86) 7 (1.28) 8 (1.93) 9 (2.31) 10 (2.78) 11 (3.33) 12 (4.00)

Table 16: Margin of safety of ^{OBI858 and BOTOX ®} batch OBI858 BOTOX ^® 1 14.07 6.20 2 10.32 7.58 3 10.33 8.98 4 9.29 6.73

Example 4: Stability test of OBI-858 formulation

Experimental materials/processes:

(1) LF08 preparation: 0.5 mg/mL human serum albumin, 11.25 mg/mL sodium chloride and 0.188 mg/mL Tween 80 dissolved in 50 mM phosphate buffer (pH 6.3).

(2) LF09 preparation: 0.5 mg/mL human serum albumin, 11.25 mg/mL sodium chloride and 0.188 mg/mL Tween 80 are dissolved in 50 mM citrate (sodium citrate) buffer (pH 5.6).

(3) LF10 preparation: 0.5 mg/mL methionine, 11.25 mg/mL sodium chloride, 0.188 mg/mL Tween 80 and 150 mM arginine dissolved in 50 mM citrate buffer (pH 6.0).

(4) LF11 preparation: 11.25 mg/mL sodium chloride and 0.188 mg/mL Tween are dissolved in 50 mM phosphate buffer (pH 6.3).

(5) Experimental animals: Eight female ICR mice (body weight 18-22 grams) were injected with 0.1 mL OBI-858 intravenously for lethal dose (LD50/mL) test.

Experimental results:

(1) Stability test of different preparations:

The lyophilized OBI-858 crude drug was dissolved in the four preparations, stored at 4 ℃ for one day, and then stored at 45 ℃ for 45 days. After standing at room temperature for 1 hour, the mice were injected intravenously. . The following table shows the lethal dose (LD50/mL) of the four preparations, and the result is that the LF09 preparation has the best stability effect.

Table 17: Stability test of different formulations Formulation name LF08 LF09 LF10 LF11 Lethal dose before the test (LD50/mL) 6.15E+05 8.12E+05 6.75E+05 1.63E+05 Stability test temperature 45 ℃ Stability test time 14 days Lethal dose after the test (LD50/mL) 0 2.58E+05 0 0

(2) Accelerated stability test of LF09 formulation at 4-60 ℃:

The lyophilized OBI-858 bulk drugs were dissolved in LF09 preparations, stored at 4°C for one day, and then replaced at four temperatures (4°C, 25°C, 45°C and 60°C) and stored for seven days at room temperature. Let it stand for 1 hour and then give mice intravenous injection. The following table shows the lethal dose (LD50/mL) of the LF09 preparation. The results show that the LF09 preparation is still stable at 45°C.

Table 18: Accelerated Stability Test of LF09 Formulation Stability test temperature 4 ℃ 25 ℃ 45 ℃ 60 ℃ Lethal dose before the test (LD50/mL) 5.07E+05 5.07E+05 5.07E+05 5.07E+05 Stability test time 7 days Lethal dose after the test (LD50/mL) 5.07E+05 5.07E+05 4.06E+05 0

It should be understood that the illustrative specific embodiments described herein should be considered in a descriptive sense only and not for the purpose of limitation. It should generally be considered that the description of features or aspects within each exemplary embodiment can be used for other similar features or aspects in other exemplary embodiments.

Although one or more exemplary specific embodiments have been described with reference to the accompanying drawings, those of ordinary skill in the art should understand that they can be used without departing from the spirit and scope of the inventive concept as defined by the appended claims. Make various changes in form and details.

without

Figure 1 shows the use of size exclusion chromatography and high performance liquid chromatography (SEC-HPLC) to detect product purity.

Figure 2 shows the use of connection refractive index and multi-angle laser light scattering (MALS) to detect product molecular weight.

Figure 3 (a) ~ (b) is the use of electron microscopy (Electron microscopy) to determine the total protein molecular weight of the product. In Figure 3(a) OBI-858, the arrows indicate toxin particles; and Figure 3(b) the reference study of 760 kDa L-PTC/A, the arrows indicate four different directions.

Figure 4 is a scoring chart using the DAS measurement method.

                                  SEQUENCE LISTING
          <![CDATA[<110> Taiwan Hao Ding Biotechnology Co., Ltd.]]>
          <![CDATA[<120> Type A botulinum toxin complex, its formulation and method of use]]>
          <![CDATA[<130> OBIP-2TWPA]]>
          <![CDATA[<160> 5 ]]>
          <![CDATA[<170> PatentIn version 3.5]]>
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          Ile Pro Thr Asn Asn Ile Phe Asn Ser Lys Val Ser Ser Thr Gln Arg
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          Val Leu Pro Tyr Ser Asn Gly Leu Tyr Val Ile Asn Lys Gly Asp Gly
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          Tyr Ile Arg Thr Asn Asp Lys Asp Leu Ile Gly Thr Leu Leu Ile Glu
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          Ala Gly Ser Ser Gly Ser Ile Ile Gln Pro Arg Leu Arg Asn Thr Thr
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                  275 280 285
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          Tyr Gln Asn Gly Ser Ile Val Asn Lys Ala Glu Tyr Tyr Leu Pro Ser
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                      340 345 350
          Val Lys Met Gln Val Ala Glu Asp Gly Phe Ile Gln Asn Gly Pro Glu
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          Ile Asn Thr Ala Ile Ser Asp Asn Tyr Thr Tyr Asn Ile Pro Gly Ile
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          Val Asn Asn Asn Pro Phe Tyr Ile Leu Phe Thr Val Asn Thr Thr Gly
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          Ile Tyr Lys Ile Asn Ala Gln Asn Asn Leu Pro Ser Leu Lys Ile Tyr
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          Asp Asp Asp Ile Lys Ala Ile Asn Tyr Ile Thr Gly Phe Asp Ser Pro
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          Asn Ala Lys Ser Tyr Leu Val Val Leu Leu Asn Lys Asp Lys Asn Tyr
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          Tyr Ile Arg Val Pro Gln Thr Ser Ser Ser Asn Ile Glu Asn Gln Ile Gln
                          485 490 495
          Phe Lys Arg Glu Glu Gly Asp Leu Arg Asn Leu Met Asn Ser Ser Val
                      500 505 510
          Asn Ile Ile Asp Asn Leu Asn Ser Thr Gly Ala His Tyr Tyr Thr Arg
                  515 520 525
          Gln Ser Pro Asp Val His Asp Tyr Ile Ser Tyr Glu Phe Thr Ile Pro
              530 535 540
          Gly Asn Phe Asn Asn Lys Asp Thr Ser Asn Ile Arg Leu Tyr Thr Ser
          545 550 555 560
          Tyr Asn Gln Gly Ile Gly Thr Leu Phe Arg Val Thr Glu Thr Ile Asp
                          565 570 575
          Gly Tyr Asn Leu Ile Asn Ile Gln Gln Asn Leu His Leu Leu Asn Asn
                      580 585 590
          Thr Asn Ser Ile Arg Leu Leu Asn Gly Ala Ile Tyr Ile Leu Lys Val
                  595 600 605
          Glu Val Thr Glu Leu Asn Asn Tyr Asn Ile Arg Leu His Ile Asp Ile
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          Met Ser Val Glu Arg Thr Phe Leu Pro Asn Gly Asn Tyr Asn Ile Lys
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          Ser Ile Phe Ser Gly Ser Leu Tyr Leu Asn Pro Val Ser Lys Ser Leu
                      20 25 30
          Thr Phe Ser Asn Glu Ser Ser Ser Ala Asn Asn Gln Lys Trp Asn Val Glu
                  35 40 45
          Tyr Met Ala Glu Asn Arg Cys Phe Lys Ile Ser Asn Val Ala Glu Pro
              50 55 60
          Asn Lys Tyr Leu Ser Tyr Asp Asn Phe Gly Phe Ile Ser Leu Asp Ser
          65 70 75 80
          Leu Ser Asn Arg Cys Tyr Trp Phe Pro Ile Lys Ile Ala Val Asn Thr
                          85 90 95
          Tyr Ile Met Leu Ser Leu Asn Lys Val Asn Glu Leu Asp Tyr Ala Trp
                      100 105 110
          Asp Ile Tyr Asp Thr Asn Glu Asn Ile Leu Ser Gln Pro Leu Leu Leu
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          Leu Pro Asn Phe Asp Ile Tyr Asn Ser Asn Gln Met Phe Lys Leu Glu
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          Met Glu His Tyr Ser Val Ile Gln Asn Ser Leu Asn Asp Lys Ile Val
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          Thr Ile Ser Cys Lys Ala Asp Thr Asn Leu Phe Phe Tyr Gln Val Ala
                      20 25 30
          Gly Asn Val Ser Leu Phe Gln Gln Thr Arg Asn Tyr Leu Glu Arg Trp
                  35 40 45
          Arg Leu Ile Tyr Asp Ser Asn Lys Ala Ala Tyr Lys Ile Lys Ser Met
              50 55 60
          Asp Ile His Asn Thr Asn Leu Val Leu Thr Trp Asn Ala Pro Thr His
          65 70 75 80
          Asn Ile Ser Thr Gln Gln Asp Ser Asn Ala Asp Asn Gln Tyr Trp Leu
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          Leu Leu Lys Asp Ile Gly Asn Asn Ser Phe Ile Ile Ala Ser Tyr Lys
                      100 105 110
          Asn Pro Asn Leu Val Leu Tyr Ala Asp Thr Val Ala Arg Asn Leu Lys
                  115 120 125
          Leu Ser Thr Leu Asn Asn Ser Asn Tyr Ile Lys Phe Ile Ile Glu Asp
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          Leu Asp Leu Asn Lys Val Val Gln Gln Val Asp Val Thr Asn Leu Asn
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          Val Asn Leu Tyr Thr Trp Asp Tyr Gly Arg Asn Gln Lys Trp Thr Ile
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          Arg Tyr Asn Glu Glu Lys Ala Ala Tyr Gln Phe Phe Asn Thr Ile Leu
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          Ser Asn Gly Val Leu Thr Trp Ile Phe Ser Asn Gly Asn Thr Val Arg
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          Val Ser Ser Ser Asn Asp Gln Asn Asn Asp Ala Gln Tyr Trp Leu Ile
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          Asn Pro Val Ser Asp Thr Asp Glu Thr Tyr Thr Ile Thr Asn Leu Arg
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          Asp Thr Thr Lys Ala Leu Asp Leu Tyr Gly Gly Gln Thr Ala Asn Gly
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          Phe Lys Val Ala Pro Asn Ile Trp Val Ala Pro Glu Arg Tyr Tyr Gly
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          Asp Ser Asn Phe Leu Ser Gln Asp Ser Glu Lys Asp Lys Phe Leu Gln
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          Lys Leu Leu Ser Leu Ile Ser Thr Ala Ile Pro Phe Pro Tyr Gly Tyr
                      100 105 110
          Ile Gly Gly Gly Tyr Tyr Ala Pro Asn Met Ile Thr Phe Gly Ser Ala
                  115 120 125
          Pro Lys Ser Asn Lys Lys Leu Asn Ser Leu Ile Ser Ser Thr Ile Pro
              130 135 140
          Phe Pro Tyr Ala Gly Tyr Arg Glu Thr Asn Tyr Leu Ser Ser Glu Asp
          145 150 155 160
          Asn Lys Ser Phe Tyr Ala Ser Asn Ile Val Ile Phe Gly Pro Gly Ala
                          165 170 175
          Asn Ile Val Glu Asn Asn Thr Val Phe Tyr Lys Lys Glu Asp Ala Glu
                      180 185 190
          Asn Gly Met Gly Thr Met Thr Glu Ile Trp Phe Gln Pro Phe Leu Thr
                  195 200 205
          Tyr Lys Tyr Asp Glu Phe Tyr Ile Asp Pro Ala Ile Glu Leu Ile Lys
              210 215 220
          Cys Leu Ile Lys Ser Leu Tyr Phe Leu Tyr Gly Ile Lys Pro Ser Asp
          225 230 235 240
          Asp Leu Val Ile Pro Tyr Arg Leu Arg Ser Glu Leu Glu Asn Ile Glu
                          245 250 255
          Tyr Ser Gln Leu Asn Ile Val Asp Leu Leu Val Ser Gly Gly Ile Asp
                      260 265 270
          Pro Lys Phe Ile Asn Thr Asp Pro Tyr Trp Phe Thr Asp Asn Tyr Phe
                  275 280 285
          Ser Asn Ala Lys Lys Val Phe Glu Asp His Arg Asn Ile Tyr Glu Thr
              290 295 300
          Glu Ile Glu Gly Asn Asn Ala Ile Gly Asn Asp Ile Lys Leu Arg Leu
          305 310 315 320
          Lys Gln Lys Phe Arg Ile Asn Ile Asn Asp Ile Trp Glu Leu Asn Leu
                          325 330 335
          Asn Tyr Phe Ser Lys Glu Phe Ser Ile Met Met Pro Asp Arg Phe Asn
                      340 345 350
          Asn Ala Leu Lys His Phe Tyr Arg Lys Gln Tyr Tyr Lys Ile Asp Tyr
                  355 360 365
          Pro Glu Asn Tyr Ser Ile Asn Gly Phe Val Asn Gly Gln Ile Asn Ala
              370 375 380
          Gln Leu Ser Leu Ser Asp Arg Asn Gln Asp Ile Ile Asn Lys Pro Glu
          385 390 395 400
          Glu Ile Ile Asn Leu Leu Asn Gly Asn Asn Val Ser Leu Met Arg Ser
                          405 410 415
          Asn Ile Tyr Gly Asp Gly Leu Lys Ser Thr Val Asp Asp Phe Tyr Ser
                      420 425 430
          Asn Tyr Lys Ile Pro Tyr Asn Arg Ala Tyr Glu Tyr His Phe Asn Asn
                  435 440 445
          Ser Asn Asp Ser Ser Leu Asp Asn Val Asn Ile Gly Val Ile Asp Asn
              450 455 460
          Ile Pro Glu Ile Ile Asp Val Asn Pro Tyr Lys Glu Asn Cys Asp Lys
          465 470 475 480
          Phe Ser Pro Val Gln Lys Ile Thr Ser Thr Arg Glu Ile Asn Thr Asn
                          485 490 495
          Ile Pro Trp Pro Ile Asn Tyr Leu Gln Ala Gln Asn Thr Asn Asn Glu
                      500 505 510
          Lys Phe Ser Leu Ser Ser Asp Phe Val Glu Val Val Ser Ser Lys Asp
                  515 520 525
          Lys Ser Leu Val Tyr Ser Phe Leu Ser Asn Val Met Phe Tyr Leu Asp
              530 535 540
          Ser Ile Lys Asp Asn Ser Pro Ile Asp Thr Asp Lys Lys Tyr Tyr Leu
          545 550 555 560
          Trp Leu Arg Glu Ile Phe Arg Asn Tyr Ser Phe Asp Ile Thr Ala Thr
                          565 570 575
          Gln Glu Ile Asn Thr Asn Cys Gly Ile Asn Lys Val Val Thr Trp Phe
                      580 585 590
          Gly Lys Ala Leu Asn Ile Leu Asn Thr Ser Asp Ser Phe Val Glu Glu
                  595 600 605
          Phe Gln Asn Leu Gly Ala Ile Ser Leu Ile Asn Lys Lys Glu Asn Leu
              610 615 620
          Ser Met Pro Ile Ile Glu Ser Tyr Glu Ile Pro Asn Asp Met Leu Gly
          625 630 635 640
          Leu Pro Leu Asn Asp Leu Asn Glu Lys Leu Phe Asn Ile Tyr Ser Lys
                          645 650 655
          Asn Thr Ala Tyr Phe Lys Lys Ile Tyr Tyr Asn Phe Leu Asp Gln Trp
                      660 665 670
          Trp Thr Gln Tyr Tyr Ser Gln Tyr Phe Asp Leu Ile Cys Met Ala Lys
                  675 680 685
          Arg Ser Val Leu Ala Gln Glu Thr Leu Ile Lys Arg Ile Ile Gln Lys
              690 695 700
          Lys Leu Ser Tyr Leu Ile Gly Asn Ser Asn Ile Ser Ser Asp Asn Leu
          705 710 715 720
          Ala Leu Met Asn Leu Thr Thr Thr Asn Thr Leu Arg Asp Ile Ser Asn
                          725 730 735
          Glu Ser Gln Ile Ala Met Asn Asn Val Asp Ser Phe Leu Asn Asn Ala
                      740 745 750
          Ala Ile Cys Val Phe Glu Ser Asn Ile Tyr Pro Lys Phe Ile Ser Phe
                  755 760 765
          Met Glu Gln Cys Ile Asn Asn Ile Asn Ile Lys Thr Lys Glu Phe Ile
              770 775 780
          Gln Lys Cys Thr Asn Ile Asn Glu Asp Glu Lys Leu Gln Leu Ile Asn
          785 790 795 800
          Gln Asn Val Phe Asn Ser Leu Asp Phe Glu Phe Leu Asn Ile Gln Asn
                          805 810 815
          Met Lys Ser Leu Phe Ser Ser Glu Thr Ala Leu Leu Ile Lys Glu Glu
                      820 825 830
          Thr Trp Pro Tyr Glu Leu Val Leu Tyr Ala Phe Lys Glu Pro Gly Asn
                  835 840 845
          Asn Val Ile Gly Asp Ala Ser Gly Lys Asn Thr Ser Ile Glu Tyr Ser
              850 855 860
          Lys Asp Ile Gly Leu Val Tyr Gly Ile Asn Ser Asp Ala Leu Tyr Leu
          865 870 875 880
          Asn Gly Ser Asn Gln Ser Ile Ser Phe Ser Asn Asp Phe Phe Glu Asn
                          885 890 895
          Gly Leu Thr Asn Ser Phe Ser Ile Tyr Phe Trp Leu Arg Asn Leu Gly
                      900 905 910
          Lys Asp Thr Ile Lys Ser Lys Leu Ile Gly Ser Lys Glu Asp Asn Cys
                  915 920 925
          Gly Trp Glu Ile Tyr Phe Gln Asp Thr Gly Leu Val Phe Asn Met Ile
              930 935 940
          Asp Ser Asn Gly Asn Glu Lys Asn Ile Tyr Leu Ser Asp Val Ser Asn
          945 950 955 960
          Asn Ser Trp His Tyr Ile Thr Ile Ser Val Asp Arg Leu Lys Glu Gln
                          965 970 975
          Leu Leu Ile Phe Ile Asp Asp Asn Leu Val Ala Asn Glu Ser Ile Lys
                      980 985 990
          Glu Ile Leu Asn Ile Tyr Ser Ser Asn Ile Ile Ser Leu Leu Ser Glu
                  995 1000 1005
          Asn Asn Pro Ser Tyr Ile Glu Gly Leu Thr Ile Leu Asn Lys Pro
              1010 1015 1020
          Thr Thr Ser Gln Glu Val Leu Ser Asn Tyr Phe Glu Val Leu Asn
              1025 1030 1035
          Asn Ser Tyr Ile Arg Asp Ser Asn Glu Glu Arg Leu Glu Tyr Asn
              1040 1045 1050
          Lys Thr Tyr Gln Leu Tyr Asn Tyr Val Phe Ser Asp Lys Pro Ile
              1055 1060 1065
          Cys Glu Val Lys Gln Asn Asn Asn Ile Tyr Leu Thr Ile Asn Asn
              1070 1075 1080
          Thr Asn Asn Leu Asn Leu Gln Ala Ser Lys Phe Lys Leu Leu Ser
              1085 1090 1095
          Ile Asn Pro Asn Lys Gln Tyr Val Gln Lys Leu Asp Glu Val Ile
              1100 1105 1110
          Ile Ser Val Leu Asp Asn Met Glu Lys Tyr Ile Asp Ile Ser Glu
              1115 1120 1125
          Asp Asn Arg Leu Gln Leu Ile Asp Asn Lys Asn Asn Ala Lys Lys
              1130 1135 1140
          Met Ile Ile Ser Asn Asp Ile Phe Ile Ser Asn Cys Leu Thr Leu
              1145 1150 1155
          Ser Tyr Asn Gly Lys Tyr Ile Cys Leu Ser Met Lys Asp Glu Asn
              1160 1165 1170
          His Asn Trp Met Ile Cys Asn Asn Asp Met Ser Lys Tyr Leu Tyr
              1175 1180 1185
          Leu Trp Ser Phe Lys
              1190
          <![CDATA[<210> 5]]>
          <![CDATA[<211> 1296]]>
          <![CDATA[<212> PRT]]>
          <![CDATA[<213> Botulinum (Clostr]]>idium botulinum)
          <![CDATA[<400> 5]]>
          Met Pro Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val Asn Gly
          1 5 10 15
          Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Ala Gly Gln Met Gln Pro
                      20 25 30
          Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile Pro Glu Arg
                  35 40 45
          Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu
              50 55 60
          Ala Lys Gln Val Pro Val Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr
          65 70 75 80
          Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu Phe Glu
                          85 90 95
          Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser Ile Val
                      100 105 110
          Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys
                  115 120 125
          Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp Gly Ser Tyr
              130 135 140
          Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala Asp Ile
          145 150 155 160
          Ile Gln Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn Leu Thr
                          165 170 175
          Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro Asp Phe
                      180 185 190
          Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu Leu
                  195 200 205
          Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala His Glu
              210 215 220
          Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn Pro Asn
          225 230 235 240
          Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu
                          245 250 255
          Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala Lys
                      260 265 270
          Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr Asn
                  275 280 285
          Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile Val
              290 295 300
          Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys Glu Lys
          305 310 315 320
          Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val Asp Lys Leu
                          325 330 335
          Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr Glu Asp
                      340 345 350
          Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn
                  355 360 365
          Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val Asn Tyr
              370 375 380
          Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn
          385 390 395 400
          Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr Lys Leu
                          405 410 415
          Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Arg
                      420 425 430
          Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Asp Lys Gly Tyr Asn Lys
                  435 440 445
          Ala Leu Asn Asp Leu Cys Ile Lys Val Asn Asn Trp Asp Leu Phe Phe
              450 455 460
          Ser Pro Ser Glu Asp Asn Phe Thr Asn Asp Leu Asn Lys Gly Glu Glu
          465 470 475 480
          Ile Thr Ser Asp Thr Asn Ile Glu Ala Ala Glu Glu Asn Ile Ser Leu
                          485 490 495
          Asp Leu Ile Gln Gln Tyr Tyr Leu Thr Phe Asn Phe Asp Asn Glu Pro
                      500 505 510
          Glu Asn Ile Ser Ile Glu Asn Leu Ser Ser Asp Ile Ile Gly Gln Leu
                  515 520 525
          Glu Leu Met Pro Asn Ile Glu Arg Phe Pro Asn Gly Lys Lys Tyr Glu
              530 535 540
          Leu Asp Lys Tyr Thr Met Phe His Tyr Leu Arg Ala Gln Glu Phe Glu
          545 550 555 560
          His Gly Lys Ser Arg Ile Ala Leu Thr Asn Ser Val Asn Glu Ala Leu
                          565 570 575
          Leu Asn Pro Ser Arg Val Tyr Thr Phe Phe Ser Ser Asp Tyr Val Lys
                      580 585 590
          Lys Val Asn Lys Ala Thr Glu Ala Ala Met Phe Leu Gly Trp Val Glu
                  595 600 605
          Gln Leu Val Tyr Asp Phe Thr Asp Glu Thr Ser Glu Val Ser Thr Thr
              610 615 620
          Asp Lys Ile Ala Asp Ile Thr Ile Ile Ile Pro Tyr Ile Gly Pro Ala
          625 630 635 640
          Leu Asn Ile Gly Asn Met Leu Tyr Lys Asp Asp Phe Val Gly Ala Leu
                          645 650 655
          Ile Phe Ser Gly Ala Val Ile Leu Leu Glu Phe Ile Pro Glu Ile Ala
                      660 665 670
          Ile Pro Val Leu Gly Thr Phe Ala Leu Val Ser Tyr Ile Ala Asn Lys
                  675 680 685
          Val Leu Thr Val Gln Thr Ile Asp Asn Ala Leu Ser Lys Arg Asn Glu
              690 695 700
          Lys Trp Asp Glu Val Tyr Lys Tyr Ile Val Thr Asn Trp Leu Ala Lys
          705 710 715 720
          Val Asn Thr Gln Ile Asp Leu Ile Arg Lys Lys Met Lys Glu Ala Leu
                          725 730 735
          Glu Asn Gln Ala Glu Ala Thr Lys Ala Ile Ile Asn Tyr Gln Tyr Asn
                      740 745 750
          Gln Tyr Thr Glu Glu Glu Lys Asn Asn Ile Asn Phe Asn Ile Asp Asp
                  755 760 765
          Leu Ser Ser Lys Leu Asn Glu Ser Ile Asn Lys Ala Met Ile Asn Ile
              770 775 780
          Asn Lys Phe Leu Asn Gln Cys Ser Val Ser Tyr Leu Met Asn Ser Met
          785 790 795 800
          Ile Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp Ala Ser Leu Lys
                          805 810 815
          Asp Ala Leu Leu Lys Tyr Ile Tyr Asp Asn Arg Gly Thr Leu Ile Gly
                      820 825 830
          Gln Val Asp Arg Leu Lys Asp Lys Val Asn Asn Thr Leu Ser Thr Asp
                  835 840 845
          Ile Pro Phe Gln Leu Ser Lys Tyr Val Asp Asn Gln Arg Leu Leu Ser
              850 855 860
          Thr Phe Thr Glu Tyr Ile Lys Asn Ile Ile Asn Thr Ser Ile Leu Asn
          865 870 875 880
          Leu Arg Tyr Glu Ser Asn His Leu Ile Asp Leu Ser Arg Tyr Ala Ser
                          885 890 895
          Lys Ile Asn Ile Gly Ser Lys Val Asn Phe Asp Pro Ile Asp Lys Asn
                      900 905 910
          Gln Ile Gln Leu Phe Asn Leu Glu Ser Ser Lys Ile Glu Val Ile Leu
                  915 920 925
          Lys Asn Ala Ile Val Tyr Asn Ser Met Tyr Glu Asn Phe Ser Thr Ser
              930 935 940
          Phe Trp Ile Arg Ile Pro Lys Tyr Phe Asn Ser Ile Ser Leu Asn Asn
          945 950 955 960
          Glu Tyr Thr Ile Ile Asn Cys Met Glu Asn Asn Ser Gly Trp Lys Val
                          965 970 975
          Ser Leu Asn Tyr Gly Glu Ile Ile Trp Thr Leu Gln Asp Thr Gln Glu
                      980 985 990
          Ile Lys Gln Arg Val Val Phe Lys Tyr Ser Gln Met Ile Asn Ile Ser
                  995 1000 1005
          Asp Tyr Ile Asn Arg Trp Ile Phe Val Thr Ile Thr Asn Asn Arg
              1010 1015 1020
          Leu Asn Asn Ser Lys Ile Tyr Ile Asn Gly Arg Leu Ile Asp Gln
              1025 1030 1035
          Lys Pro Ile Ser Asn Leu Gly Asn Ile His Ala Ser Asn Asn Ile
              1040 1045 1050
          Met Phe Lys Leu Asp Gly Cys Arg Asp Thr His Arg Tyr Ile Trp
              1055 1060 1065
          Ile Lys Tyr Phe Asn Leu Phe Asp Lys Glu Leu Asn Glu Lys Glu
              1070 1075 1080
          Ile Lys Asp Leu Tyr Asp Asn Gln Ser Asn Ser Gly Ile Leu Lys
              1085 1090 1095
          Asp Phe Trp Gly Asp Tyr Leu Gln Tyr Asp Lys Pro Tyr Tyr Met
              1100 1105 1110
          Leu Asn Leu Tyr Asp Pro Asn Lys Tyr Val Asp Val Asn Asn Val
              1115 1120 1125
          Gly Ile Arg Gly Tyr Met Tyr Leu Lys Gly Pro Arg Gly Ser Val
              1130 1135 1140
          Met Thr Thr Asn Ile Tyr Leu Asn Ser Ser Leu Tyr Arg Gly Thr
              1145 1150 1155
          Lys Phe Ile Ile Lys Lys Tyr Ala Ser Gly Asn Lys Asp Asn Ile
              1160 1165 1170
          Val Arg Asn Asn Asp Arg Val Tyr Ile Asn Val Val Val Lys Asn
              1175 1180 1185
          Lys Glu Tyr Arg Leu Ala Thr Asn Ala Ser Gln Ala Gly Val Glu
              1190 1195 1200
          Lys Ile Leu Ser Ala Leu Glu Ile Pro Asp Val Gly Asn Leu Ser
              1205 1210 1215
          Gln Val Val Val Met Lys Ser Lys Asn Asp Gln Gly Ile Thr Asn
              1220 1225 1230
          Lys Cys Lys Met Asn Leu Gln Asp Asn Asn Gly Asn Asp Ile Gly
              1235 1240 1245
          Phe Ile Gly Phe His Gln Phe Asn Asn Ile Ala Lys Leu Val Ala
              1250 1255 1260
          Ser Asn Trp Tyr Asn Arg Gln Ile Glu Arg Ser Ser Arg Thr Leu
              1265 1270 1275
          Gly Cys Ser Trp Glu Phe Ile Pro Val Asp Asp Gly Trp Gly Glu
              1280 1285 1290
          Arg Pro Leu
              1295
          
      

Claims (16)

A type A botulinum toxin complex comprising: HA70, HA17, HA33, NTNH and BoNT/A1, wherein the botulinum toxin complex has a molecular weight of 740-790 kDa. The botulinum toxin type A complex according to claim 1, wherein the HA70 comprises any one of the following: (1) the amino acid sequence of SEQ ID NO. 1, or (2) the amino acid sequence of SEQ ID NO. 1. The amino acid sequence obtained by adding, deleting, substituting or inserting one or more amino acids in the amino acid sequence, and which retains the function of the HA70. The botulinum toxin type A complex according to claim 1, wherein the HA17 comprises any one of the following: (1) the amino acid sequence of SEQ ID NO. 2 or (2) the amino acid sequence of SEQ ID NO. 2. The amino acid sequence obtained by adding, deleting, substituting or inserting one or more amino acids in the amino acid sequence, and which retains the function of the HA17. The botulinum toxin type A complex according to claim 1, wherein the HA33 comprises any one of the following: (1) the amino acid sequence of SEQ ID NO. 3; or (2) the amino acid sequence of SEQ ID NO. 3. An amino acid sequence obtained by adding, deleting, substituting or inserting one or more amino acids into the amino acid sequence, and it retains the function of the HA33. The botulinum toxin type A complex according to claim 1, wherein NTNH comprises any one of the following: (1) the amino acid sequence of SEQ ID NO. 4, or (2) in the SEQ ID NO. 4 An amino acid sequence obtained by adding, deleting, substituting or inserting one or more amino acids into the amino acid sequence, and it retains the function of the NTNH. The botulinum toxin type A complex according to claim 1, wherein the BoNT/A1 comprises any one of the following: (1) the amino acid sequence of SEQ ID NO. 5, or (2) in the SEQ ID NO. ID NO. 5 The amino acid sequence obtained by adding, deleting, substituting or inserting one or more amino acids in the amino acid sequence, and it retains the function of BoNT/A1. The type A botulinum toxin complex according to any one of claims 1 to 6, wherein in a mouse/animal experiment, the therapeutically effective dose (ED50) of the type A botulinum toxin complex is 0.1 ~0.5U. The type A botulinum toxin complex according to any one of claims 1 to 6, wherein in a mouse/animal experiment, the lethal dose (LD50) of the type A botulinum toxin complex is 1 to 5 U. The type A botulinum toxin complex according to any one of claims 1 to 6, in a mouse/animal experiment, the safety margin (MOS) of the type A botulinum toxin complex is 2~ 50. A nucleic acid for encoding the type A botulinum toxin according to any one of claims 1 to 6. A pharmaceutical composition comprising: the botulinum toxin type A complex according to any one of claims 1 to 6 and a pharmaceutically or dermatologically acceptable carrier, wherein the pharmaceutical composition is freeze-dried form. A liquid formulation comprising: the type A botulinum toxin complex according to any one of claims 1 to 6. The liquid formulation according to claim 12, wherein the liquid formulation further comprises: human serum albumin, methionine, arginine, sodium chloride, Tween 80, citrate buffer, and/or Phosphate buffer. A type A botulinum toxin complex as described in any one of claims 1 to 6, a pharmaceutical composition as described in claim 11, or a liquid as described in any one of claims 12 and 13 The formulation is used to prepare cosmetic products, wherein the cosmetic products are used to reduce the appearance of fine lines and/or wrinkles on the face, eyes, and corners of the mouth, or generally relieve muscle tension. A type A botulinum toxin complex as described in any one of claims 1 to 6, a pharmaceutical composition as described in claim 11, or a liquid formulation as described in any one of claims 12 and 13 The preparation is used to prepare medicines for treating or preventing diseases caused by synaptic transmission or release of acetylcholine. The use of the medicament according to claim 15, wherein the disease is selected from neuromuscular disorders characterized by skeletal muscle hyperactivity, nerve pain, migraine, overactive bladder, rhinitis, sinusitis, acne, dystonia, tension Obstructive contraction, hyperhidrosis, excessive secretion of one or more glands controlled by the cholinergic nervous system, hemifacial spasm, adult-onset spastic torticollis, anal fissure, blepharospasm, cerebral palsy, cervical dystonia, Strabismus, temporomandibular joint disorders and various types of muscle cramps and spasms.

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