WO2023130875A1 - 一种肉毒毒素蛋白组合物 - Google Patents

一种肉毒毒素蛋白组合物 Download PDF

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WO2023130875A1
WO2023130875A1 PCT/CN2022/136597 CN2022136597W WO2023130875A1 WO 2023130875 A1 WO2023130875 A1 WO 2023130875A1 CN 2022136597 W CN2022136597 W CN 2022136597W WO 2023130875 A1 WO2023130875 A1 WO 2023130875A1
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botulinum toxin
protein
toxin protein
composition according
amino acid
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PCT/CN2022/136597
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English (en)
French (fr)
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杨武
巩慧
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重庆誉颜制药有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/183—Amino acids, e.g. glycine, EDTA or aspartame
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/4886—Metalloendopeptidases (3.4.24), e.g. collagenase
    • A61K38/4893—Botulinum neurotoxin (3.4.24.69)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/16—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing nitrogen, e.g. nitro-, nitroso-, azo-compounds, nitriles, cyanates
    • A61K47/18—Amines; Amides; Ureas; Quaternary ammonium compounds; Amino acids; Oligopeptides having up to five amino acids
    • A61K47/186—Quaternary ammonium compounds, e.g. benzalkonium chloride or cetrimide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/18—Antioxidants, e.g. antiradicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A61P21/02—Muscle relaxants, e.g. for tetanus or cramps
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P23/00—Anaesthetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/06—Antimigraine agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A61P29/02—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID] without antiinflammatory effect
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/24—Metalloendopeptidases (3.4.24)
    • C12Y304/24069—Bontoxilysin (3.4.24.69), i.e. botulinum neurotoxin
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the invention relates to the field of pharmaceutical preparations, in particular to a botulinum toxin protein composition and a preparation method thereof.
  • BoNTs Natural botulinum neurotoxins
  • Clostridium anaerobic botulinum which cause muscle paralysis in mammals by blocking the presynaptic release of the neurotransmitter acetylcholine at neuromuscular junctions.
  • Seven types of BoNTs have been discovered so far, named BoNT/A to BoNT/G. Due to the unique pharmacological properties of BoNTs, including high specificity to the nervous system, limited diffusion by local injection, and reversibility of action, BoNTs have rapidly developed into a class of drugs for the treatment of cholinergic hyperstimulation in the fields of medical and aesthetics. Has a wide range of applications.
  • therapeutically active proteins are usually formulated in liquid solutions, particularly for injection.
  • Therapeutically active proteins are usually prepared in compositions and sold commercially as ready-to-use solutions or in lyophilized form for reconstitution from liquid solutions.
  • BoNT/A products on the market are all vacuum-dried or freeze-dried solid preparations, which need to be reconstituted by medical staff before use, so the reconstitution volume needs to be precisely controlled.
  • the dosage required for each treatment varies greatly. A small portion of the content in the bottle is administered to the patient, and the rest is kept in the refrigerator for further use.
  • botulinum toxin due to the extremely low concentration of botulinum toxin, in order to maintain the stability of botulinum toxin protein at an extremely low concentration and avoid the adsorption of drugs on solid surfaces, high concentrations of exogenous proteins are generally added for preparation. Most of the botulinum toxin formulations use albumin or gelatin as a stabilizer, which will not only cause immune reactions, but also introduce the possibility of contamination.
  • the present invention provides a botulinum toxin protein composition and a preparation method thereof.
  • the invention provides a botulinum toxin protein composition, comprising the following components:
  • Botulinum toxin protein and protein stabilizer Botulinum toxin protein and protein stabilizer.
  • the protein stabilizer is selected from any one of amino acids or amino acid-like compounds;
  • the amino acid-like compound is L-carnitine (alias: vitamin Bt, L-carnitine).
  • the molar content of the L-carnitine is 5-20 mM, and in a specific embodiment, the molar content of the L-carnitine is 10 mM.
  • the molar content of the amino acid is 5-20 mM; in a specific embodiment, the molar content of the amino acid is 10 mM.
  • the amino acid is selected from one or more of L-histidine, L-glycine, L-arginine, L-methionine, L-histidine, L-lysine .
  • proteins are easily degraded and inactivated at low concentrations ( ⁇ 0.1mg/ml).
  • Some amino acids and amino acid-like compounds of the present application can increase the solubility of proteins at a specific pH and improve their stability.
  • Amino acids and amino acid-like structural compounds can not only reduce surface adsorption and protect protein conformation, but also prevent protein denaturation and aggregation.
  • L-carnitine makes the formula have the characteristics of low viscosity, and can reduce the aggregation and precipitation of botulinum toxin protein, which is beneficial to maintain the stability of botulinum toxin protein.
  • the botulinum toxin protein is selected from botulinum toxin protein type A, botulinum toxin protein type B, botulinum toxin protein type C1, botulinum toxin protein type C2, botulinum toxin protein type D, and botulinum toxin protein type E Any one of toxin protein, F-type botulinum toxin protein or G-type botulinum toxin protein; more preferably, the botulinum toxin protein is type A botulinum toxin protein.
  • the botulinum toxin is recombinant botulinum toxin.
  • the recombinant botulinum toxin protein consists of at least one dimer structure composed of single-chain polypeptides cleaved by proteases;
  • the single-chain polypeptide comprises:
  • a first polypeptide fragment comprising:
  • the second functional amino acid structural region which comprises a receptor binding domain capable of binding to target cell surface receptors and/or a translocation domain capable of mediating translocation of polypeptides across vesicle membranes.
  • tag protein refers to a class of protein molecules that can bind to a specific ligand.
  • the tagged protein is selected from tagged proteins known to those skilled in the art or tagged proteins designed by computer programming, and the tagged protein can specifically bind to known substrates.
  • the tag protein is selected from but not limited to the following proteins: glutathione S-transferase (GSTs), C-myc, chitin binding domain, maltose binding protein (MBP), SUMO heterologous Affinity Moiety, Monoclonal Antibody or Protein A, Streptavidin Binding Protein (SBP), Cellulose Binding Domain, Calmodulin Binding Peptide, S Tag, Strep Tag II, FLA, Protein A, Protein G, Group Amino acid affinity tag (HAT), polyhistidine.
  • GSTs glutathione S-transferase
  • C-myc C-myc
  • chitin binding domain chitin binding domain
  • MBP maltose binding protein
  • SBP Streptavidin Binding Protein
  • SBP Streptavidin Binding Protein
  • HAT Group Amino acid affinity tag
  • polyhistidine polyhistidine
  • the "tag protein” can increase the solubility of the toxin polypeptide precursor in the host.
  • the "tag protein” allows the precursor of the toxin polypeptide to exist in the host cell in a soluble manner.
  • the tag protein is located at the N-terminus of the single-chain polypeptide.
  • the tag protein is glutathione s-transferase (GSTs), more preferably, the amino acid sequence of the glutathione s-transferase is shown in SEQ ID NO: 2 .
  • neither the first protease cleavage site nor the second protease cleavage site can be cleaved by human protease or protease produced by the host cell expressing the single-chain polypeptide.
  • first protease cleavage site and the second protease cleavage site are the same or different.
  • the first protease cleavage site and the second protease cleavage site are not recognized and cleaved by the endogenous protease of the host cell when expressed or when used.
  • the specific protease is selected from but not limited to one or a combination of two of the following proteases: non-human enterokinase, tobacco etch virus protease, protease derived from Bacillus subtilis, derived from Bacillus amyloliquefaciens proteases derived from rhinoviruses, papain, homologues of insect papain or homologues of crustacean papain.
  • both the protease specifically recognizing the first protease and the protease specifically recognizing the cleavage site of the second protease are proteases derived from rhinovirus.
  • the second enzyme cleavage site is embedded, partially replaced or completely replaced by the naturally occurring loop region between the first functional peptide and the second functional peptide.
  • the embedding refers to inserting between certain two amino acids in the loop region;
  • the partial replacement refers to the replacement of part of the amino acid sequence of the loop region by the second restriction site ;
  • the complete replacement means that the amino acid sequence of the natural loop region is completely replaced by the second enzyme cutting site.
  • the structural region comprising the first protease cleavage site and the structural region comprising the second protease cleavage site are selected from, but not limited to, one or a combination of two of the following cleavage sites: DDDDK, EXXYXQS /G, HY, YH or LEVLFQGP.
  • the structural region containing the first protease cleavage site and the structural region containing the second protease cleavage site are both LEVLFQGP, and the cleavage site is between Q-G.
  • the connecting short peptide has no more than 5 amino acids.
  • the connecting short peptide can make the first protease cleavage site easier to be recognized or bound by its protease.
  • the connecting short peptide does not affect the function of the single-chain polypeptide.
  • the connecting short peptide remaining at the N-terminal of the second polypeptide fragment will not affect the function of the second polypeptide fragment.
  • the GS part of the connecting short peptide does not exceed 5 amino acid residues. More preferably, the connecting short peptide is selected from glycine-serine (Glycine-Serine, GS for short) short peptides, GGS, GGGS, GGGGS, GSGS, GGSGS, GSGGS, GGSGS, GGGSS and other linking short peptides.
  • Glycine-Serine Glycine-Serine, GS for short
  • amino acid sequence of the structural region comprising the first protease cleavage site and the connecting short peptide is LEVLFQGPLGS.
  • the metal ion-dependent protease activity domain is a Zn 2+ -dependent protease activity domain.
  • the first functional amino acid structural region and/or the second functional amino acid structural region are encoded by natural sequences and/or artificially synthesized sequences. More preferably, the first functional amino acid structural region of the single-chain polypeptide comprises the Zn 2+ protease active domain of the light chain of Clostridial toxin.
  • the target cell capable of binding the receptor binding domain of human cell surface antigen in the second functional amino acid structural region refers to the target cell with SNARE complex.
  • SNARE complex For example: nerve cells, pancreatic cells or other cells where SNARE complexes exist.
  • the target cells in the second functional amino acid structural region refer to human nerve cells or pancreatic cells
  • the receptor binding domain is a receptor binding domain capable of specifically binding to human nerve cells or pancreatic cells.
  • the receptor binding domain of the second functional amino acid structural region is the cell surface antigen binding domain of the heavy chain of Clostridial toxin, and the mediator polypeptide of the second functional amino acid structural region crosses the vesicle membrane
  • the transferred translocation domain is the domain that mediates the transfer of Clostridial toxins across the vesicle membrane.
  • Clostridial toxin is botulinum toxin or tetanus toxin.
  • the botulinum toxin is selected from any of the serotypes BoNT/A-BoNT/H and their derivatives known in the prior art.
  • Clostridial toxin is selected from any one of tetanus toxin or its derivatives.
  • the first functional amino acid structural region comprises BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, BoNT/G, BoNT/H or tetanus toxin Part or all of the light chain.
  • the second functional amino acid structural region comprises BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, BoNT/G, BoNT/H or tetanus toxin Part or all of the heavy chain.
  • the first functional amino acid structural region is the light chain of BoNT-/A
  • the second functional amino acid structural region is the heavy chain of BoNT-/A
  • the first functional amino acid structural region and the second functional amino acid structural region can be from different serotypes. It can be any combination of the above serotypes, for example, the first functional amino acid structural region is derived from the light chain of BoNT-/A, and the second functional amino acid structural region is derived from BoNT-/B, BoNT/C, The heavy chain of BoNT/D, BoNT/E, BoNT/F or BoNT/G, or, the first functional amino acid structural region is derived from BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/ F or the light chain of BoNT/G, and the second functional amino acid structural region is derived from the heavy chain of BoNT/A, etc.
  • amino acid sequence of the second polypeptide fragment comprises the fragment shown in SEQ ID NO:7.
  • the single-chain polypeptide comprises: glutathione S-transferase, LEVLFQGPLGS, light chain of BoNT/A, LEVLFQGP and heavy chain of BoNT/A sequentially from the N-terminus.
  • amino acid sequence of the single-chain polypeptide is shown in SEQ ID NO: 11.
  • the recombinant botulinum toxin type A protein is cleaved by the first protease to remove the tag protein of the single-chain polypeptide, and the first functional amino acid structural region and the second functional amino acid structural region are cleaved by the second protease to form at least one binary Polymers are prepared.
  • the first functional amino acid structural region and the second functional amino acid structural region are connected to form a dimer structure through a disulfide bond.
  • the recombinant type A botulinum toxin protein of the present invention is formed by a specific single-chain polypeptide, wherein the involved single-chain polypeptide has a specific structure, and the single-chain polypeptide of this structure has the characteristics of strong specificity and low toxicity.
  • the tag protein in the single-chain polypeptide can bind to a specific ligand, and then detach from the ligand, making it easier for molecules co-expressed with the tag protein to be extracted from different In the preparation process, the purity of the product can be significantly improved, resulting in higher product yield and higher purity; from the perspective of low toxicity, the tag protein in the single-chain polypeptide makes The protease domain of the first functional amino acid structural region in the polypeptide cannot function as a protease, and can only be recognized by a specific enzyme by replacing the natural enzyme cleavage site between the first functional amino acid structural region and the second functional amino acid structural region site, so that the molecular activity (neurotoxicity) of the single-chain polypeptide is reduced to less than one-ten-thousandth of the natural toxin, which effectively reduces the toxicity.
  • the single-chain polypeptide in the recombinant botulinum toxin protein involved in the present invention is easier to carry out industrial production and Purification operation.
  • the composition further includes a solubilizer and/or a salt compound.
  • the volume percentage concentration of the solubilizer is 0.05%-0.2%.
  • the mass percent concentration of the salts is 0.9%.
  • the solubilizer is selected from Tweens, Spans, sodium lauryl sulfate;
  • the Tweens are selected from one or more of Tween 20, Tween 40, Tween 60, or Tween 80;
  • the Spans are selected from one or more of Spans 20, Spans 40, Spans 60, Spans 80, or Spans 85;
  • the solubilizer is Tween 80.
  • the salts are selected from any one of inorganic salts or organic salts;
  • the inorganic salt is selected from one or more of sodium chloride, sodium phosphate, magnesium sulfate, sodium acetate, sodium propionate and potassium phosphate;
  • the organic salt is selected from one or more of sodium lactate and sodium succinate;
  • the inorganic salt is sodium chloride.
  • the composition includes botulinum toxin protein, L-carnitine, Tween 80, and sodium chloride;
  • the composition comprises botulinum toxin protein, amino acid, Tween 80 and sodium chloride.
  • the molar content of said L-carnitine is 5-20mM, preferably 10mM;
  • the molar content of the amino acid is 5-20 mM; preferably 10 mM.
  • the volume percentage concentration of the Tween 80 is 0.05%-0.2%.
  • the concentration of Tween 80 is 0.1% by volume.
  • the mass percent concentration of the sodium chloride is 0.9%.
  • the composition is a liquid formulation.
  • the present invention also provides a preparation method of the botulinum toxin protein composition, and the preparation method includes the step of mixing the components.
  • the steps of the above-mentioned preparation method include:
  • the water is water for injection.
  • the step (b) also includes adding solubilizers and/or salts.
  • botulinum toxin protein protein stabilizer, solubilizer and/or salt are as defined above.
  • the preparation method comprises:
  • the water is water for injection.
  • the present invention also provides the application of the above-mentioned botulinum toxin protein composition in the preparation of medicines for preventing and/or treating diseases related to cholinergic nerves innervating muscles or exocrine glands, and medical cosmetology.
  • Such diseases or conditions associated with cholinergic nerves innervating muscles or eccrine glands include: Exemplary diseases or conditions associated with hyperactivity of muscles or eccrine glands innervated by cholinergic nerves include, for example, Frey syndrome, crocodile tears syndrome, axillary hyperhidrosis, plantar hyperhidrosis, head and neck hyperhidrosis, body hyperhidrosis, rhinorrhea, Parkinson's disease, amyotrophic lateral sclerosis, hypersalivation, drooling, salivation, spastic conditions, palate Myoclonus, myoclonus, myofibrillar twitch, rigidity, benign muscle spasm, hereditary jaw tremor, paradoxical jaw muscle movement, hemimasticatory muscle spasm, hypertrophic gill myopathy, masseter hypertrophy, tibialis anterior hypertrophy, Nystagmus, vibratory hallucinations, supranuclear gaze palsy, status partial epile
  • the medical cosmetology includes smoothing or preventing wrinkles, thinning the face, and repairing scars.
  • the wrinkles can be seen as folds, ridges or creases in the skin. Wrinkles visible in the human face are preferred. Examples of wrinkles include tear troughs, glabellar lines, crow's feet, commissure lines, jaw lines, perioral wrinkles, cheek folds, marionette lines, lip lines, forehead lines, frown lines, hare lines, nasolabial folds, under-eye lines and jawline.
  • the present invention has the following beneficial effects:
  • the present invention provides a stable botulinum toxin protein composition.
  • amino acids or quasi-amino acids are used to replace human-derived proteins (such as human serum albumin), which eliminates the threat of human-derived viruses.
  • human-derived proteins such as human serum albumin
  • Tween 80 to prevent adsorption. It can be stored stably for 1 year or longer, and can be stored stably for 6 months or longer in liquid state at room temperature (25°C).
  • the present invention provides a recombinant botulinum toxin protein composition, which uses a high-purity recombinant botulinum toxin protein prepared by a specific single-chain polypeptide, which can improve the purity of the botulinum toxin protein in the preparation, avoiding the
  • the possible immune risk caused by inactive botulinum toxin protein contained in botulinum toxin type A extracted from toxin bacilli provides a new option for expanding the application range of recombinant botulinum toxin type A protein.
  • botulinum toxin protein composition of the present invention When used as a liquid preparation, it increases the convenience of clinical use, and improves the freeze-dried or vacuum products. Defects such as artificial contamination or inaccurate reconstitution.
  • Figure 1 SDS-PAGE results of the preliminary purification of GSTs-BoNT/A and the further removal of the GSTs tag, in which the first lane is the GSTs-BoNT/A obtained through the preliminary purification of the GSTs affinity chromatography column, and the second lane is the preliminary purified GSTs-BoNT/A
  • the protein is digested with Rinovirus 3C Protease to remove the GSTs tagged protein to obtain BoNT/A after GSTs removal.
  • Figure 2 SDS-PAGE results of highly pure BoNT/A protein obtained by further purification of the GSTs tag-cut product through an ion-exchange column.
  • Figure 3 Verification results of double-strand dissociation of BoNT/A under reducing conditions.
  • the term "U” refers to a potency unit, which is a variety of units used (expressing) biological abilities in pharmacy. Because the chemical composition of these drugs is not constant or their quality specifications cannot be verified by physical and chemical methods so far, biological experimental methods are often used to verify their potency by comparing them with standard products. Through this kind of biological test, the minimum potency unit with a certain biological efficacy is called “unit” (U); the standard unit stipulated by international negotiations is called “international unit” (IU);
  • BoNT/A refers to botulinum toxin type A protein
  • tagged protein refers to a polypeptide or protein that is fused and expressed with the target protein using DNA in vitro recombination technology, so as to facilitate the expression and detection of the target protein.
  • HSA Human serum albumin
  • body fluids HSA can transport fatty acids, bile pigments, amino acids, steroid hormones, metal ions and many therapeutic molecules, etc.: while maintaining Normal osmotic pressure of blood.
  • HSA can be used to treat shock and burns, to supplement blood loss caused by surgery, accidents or hemorrhage, and to be used as a plasma expander.
  • dimer structure refers to a polymer complex composed of two molecules, usually bonded by non-covalent bonds.
  • a nucleic acid molecule contains, in order from the 5' end:
  • nucleotide sequence encoding glutathione s-transferase is as shown in SEQ ID NO: 1, and the amino acid sequence of its encoded glutathione s-transferase is as shown in SEQ ID NO: 2;
  • nucleotide sequence encoding the GS-linked short peptide is shown in SEQ ID NO: 5, which is GGATCC;
  • the nucleotide sequence of coding second polypeptide fragment comprises the nucleotide sequence of BoNT/A light chain, second protease cleavage site and BoNT/A heavy chain, as shown in SEQ ID NO:6,
  • the encoded amino acid sequence is shown in SEQ ID NO:7; wherein, the nucleotide sequence encoding the second protease cleavage site is shown in SEQ ID NO:8, and the encoded amino acid sequence is shown in SEQ ID NO:9 ;
  • BoNT/A light chain and the BoNT/A heavy chain There may also be no natural loop region between the BoNT/A light chain and the BoNT/A heavy chain, such as removing the KTKSLDKGYNK linking sequence between the light chain and the heavy chain to reduce non-specific protease cleavage.
  • the nucleotide sequence encoding the single-chain polypeptide is shown in SEQ ID NO:10, and the amino acid sequence of the encoded single-chain polypeptide is shown in SEQ ID NO:11.
  • NdeI and NotI enzyme cutting sites were added to both ends of the synthesis, after NdeI and NotI digestion at 37 degrees Celsius (New England Biolabs), use QIquick gel extraction kit (Qiagen) to purify , using T4 DNA ligase (NEB) to insert the NdeI and NotI sites in the pET28a (Novagen) plasmid vector.
  • Qiagen QIquick gel extraction kit
  • NEB T4 DNA ligase
  • Components and proportions of the medium 11.8g/L tryptone, 23.6g/L yeast extract, 9.4g/L K 2 HPO 4 , 2.2g/L KH 2 PO 4 , 4ml/L glycerin.
  • the OD600 can be 0.2-1.5, the temperature can be 37°C to 10°C, and the expression time can be 5-16 hours.
  • the conventional method is as follows: use 20 times column volume of phosphate buffer to wash the chromatography column, and use 10 times column volume of freshly prepared 10mM glutathione elution buffer (0.154g reduced glutathione dissolved in 50ml 50mM Tris -HCl (pH 8.0)) for GSTs-BoNT/A elution, and the elution of the fusion protein was monitored using absorbance readings at 280 nm. The eluted protein was cut off GSTs tagged protein under the action of Rinovirus 3C Proteas.
  • GSTs-BoNT/A re-adsorbed on the glutathione purification resin chromatography column was treated with GenScript 3C enzyme, and the first enzyme cleavage site between GSTs and BoNT/A was broken under the action of 3C enzyme Open, the GSTs disengage, and at the same time the second enzyme cleavage site between the light chain and the heavy chain of BoNT/A is broken.
  • the glutathione purification resin column was treated with phosphate buffer, and the GSTs tagged protein remained on the column and was removed, while the light chain and heavy chain of BoNT/A were eluted by phosphate buffer.
  • the GSTs-removed product was taken for a conventional SDS-PAGE experiment. As shown in Figure 2, there was no GSTs-tagged protein in the obtained band, indicating that the GSTs-tagged protein had been completely removed. Use SDS-PAGE to scan the pictures, and calculate the BoNT/A obtained according to the gray density of the bands with a purity of more than 90%.
  • the effect is similar to GS, and the tagged protein can be well exposed, so that it can be completely excised.
  • step 6 The product of step 6 is carried out reduction experiment:
  • the sample was treated at 100 degrees Celsius for five minutes to reduce the sample, and the sample was separated by 4-12% SDS-PAGE (Biorad) electrophoresis at 200 volts to separate the heavy chain and light chain.
  • SDS-PAGE Biorad electrophoresis at 200 volts to separate the heavy chain and light chain.
  • the product obtained in step 6 was reduced under reducing conditions, and the obtained product was subjected to a conventional SDS-PAGE experiment to obtain two different bands with molecular weights of 100Kda and 50Kda respectively, proving that the product formed in step 6
  • the product is a dimer structure of two peptides linked by disulfide bonds.
  • the LD 50 of intraperitoneal injection of mice is between 45ng-450ng; considering the purity of the injected botulinum toxin protein, the converted LD 50 is between 22.5ng-225ng, the median value 123.75ng.
  • the LD 50 of the BoNT/A obtained in step 5 by intraperitoneal injection in mice was between 0.02ng–0.05ng. Considering the purity of the injected botulinum toxin protein, the converted LD 50 is between 0.006ng-0.015ng, and the median value is 0.0105ng (see Table 1).
  • GSTs-BoNT/A has the activity of botulinum toxin.
  • its median lethal dose (LD 50 ) is about 11786 times higher than that of BoNT/A protein LD 50 , indicating that GSTs-BoNT/A recombinant protein toxin polypeptide
  • the precursor molecule was about 11786 times less active than the final product, BoNT/A.
  • This experiment proves that the GSTs-BoNT/A recombinant protein toxin polypeptide precursor molecule has the activity of botulinum toxin. Due to the ultra-high toxicity of botulinum toxin, in the production process, a high level of safety operation protection is required before the precursor molecules are activated.
  • the activity of the botulinum toxin protein was evaluated by the median lethal dose of mice, and it was shown in mouse units (U), and one unit was defined as the intraperitoneal injection of recombinant type A required for killing 50% of the total number of mice Botulinum toxin protein.
  • mice of the CD-1 variety were selected, grouped, weighed, and numbered to ensure that the number of mice in each group was equal and the body weight was similar.
  • mice were given a single intraperitoneal injection of 0.5 mL of recombinant botulinum toxin protein preparations of different formulations.
  • the amount of recombinant botulinum toxin type A protein injected intraperitoneally into mice is 2X LD 50 (2U).
  • each animal was weighed once (before administration on the first day) and recorded. After the administration, the body weight was weighed every 24 hours. After administration, 24h ( ⁇ 1h), 48h ( ⁇ 1h), and 72h ( ⁇ 1h), 96h ( ⁇ 1h), check the mortality rate.
  • formula 1 can maintain the activity of recombinant botulinum toxin protein after being placed at 4°C for 211 days
  • formula 2 can maintain the activity of recombinant botulinum toxin protein after being placed at 4°C for 70 days.
  • the median lethal dose (LD 50 ) of recombinant botulinum toxin type A calculated through multiple dose-activity relationships has no significant change after being placed at 4°C for 44, 70 and 211 days, and the median lethal dose remains basically unchanged, which fully proves that The formulation of the invention can effectively maintain the long-term stability of the biological activity of the recombinant type A botulinum toxin.
  • the present invention adopts recombinant botulinum toxin, uses amino acids or amino acid-like compounds as protein stabilizers, and can be stored at low temperatures (such as at 4°C) for a long time (such as at 4°C for more than 70 days), and the formulation of the present invention can also be Maintain the activity of natural botulinum toxin proteins (botulinum toxin type A, botulinum toxin type B, botulinum toxin type C1, botulinum toxin type C2, botulinum toxin type D, and botulinum toxin type E proteins) .

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Abstract

一种肉毒毒素蛋白组合物，包含肉毒毒素蛋白和蛋白稳定剂，蛋白稳定剂选自氨基酸或类氨基酸化合物中任一种，氨基酸选自L-组氨酸、L-甘氨酸、L-精氨酸、L-甲硫氨酸、L-赖氨酸中的一种或多种，类氨基酸化合物包括左旋卡尼汀。可作为液体制剂用于医疗和美容领域，可保持肉毒毒素蛋白在液体状态下活性稳定。

Description

一种肉毒毒素蛋白组合物 技术领域
本发明涉及药物制剂领域，具体涉及一种肉毒毒素蛋白组合物及其制备方法。
背景技术
天然肉毒杆菌神经毒素(BoNTs)是由梭菌属厌氧肉毒杆菌产生的毒素蛋白，通过阻断神经肌肉接点神经递质乙酰胆碱的突触前释放引起哺乳类动物肌肉麻痹。目前已经发现了七种类型的BoNTs，分别命名为BoNT/A到BoNT/G。BoNTs由于独特的药理学特性，包括对神经系统高度特异性，局部注射有限的扩散，以及作用的可逆性，已经迅速发展成为了一类治疗胆碱能过度兴奋的药物，在医疗和美容领域中具有广泛的应用。
一般说来，治疗用药物活性蛋白通常在液体溶液中配制，特别是用于注射剂。治疗用活性蛋白通常制备成组合物，在商业上以即用溶液形式出售或以用液体溶液重构的冻干形式提供。例如市场上的BoNT/A产品均为真空干燥或冷冻干燥后的固体制剂，在使用时需要医护人员进行复溶，因此需要精确地控制复溶体积。此外，由于不同的治疗目的以及患者间在需用剂量上存在个体差异，每个治疗所需的剂量差别相当大，市场产品所含的药物量在按照厂家要求的体积复溶后，临床医生往往将瓶中的含量的一小部分给药于患者，其余放置冰箱再用。有研究估计，当复溶的BoNT/A在冰箱(约4℃)下贮藏12小时后，其效力损失至少44％，所以一般在实际操作中，建议复溶后4小时内使用，这会导致药品的浪费和患者承担了不必要的经济损失。
此外，由于肉毒毒素使用浓度极低，为了在极低浓度下保持肉毒毒素蛋白质的稳定性，同时避免药物在固态表面的吸附，一般会添加高浓度外源蛋白进行配制。在肉毒毒素配方中大多数使用白蛋白或明胶用作稳定剂，不但会引起免疫反应，并有引入污染源的可能。
发明内容
针对背景技术中提到的技术问题，本发明提供一种肉毒毒素蛋白组合物及其制备方法。
本发明的技术方案如下：
本发明提供一种肉毒毒素蛋白组合物，包括以下组分：
肉毒毒素蛋白和蛋白稳定剂。
所述蛋白稳定剂选自氨基酸或类氨基酸化合物中任一种；
优选的，所述类氨基酸化合物为左旋卡尼汀(别名：维生素Bt，左旋肉碱)。
更优选的，所述左旋卡尼汀的摩尔含量为5-20mM，在一个具体的实施方式中，所述左旋卡尼汀的摩尔含量为10mM。
优选的，所述氨基酸的摩尔含量为5-20mM；在一个具体的实施方式中，所述氨基酸的摩尔含量为10mM。
更优选的，所述氨基酸选自L-组氨酸、L-甘氨酸、L-精氨酸、L-甲硫氨酸、L-组氨酸、L-赖氨酸中的一种或多种。
一般说来，蛋白在低浓度时(<0.1mg/ml)极易降解而失活，本申请的一些氨基酸以及类氨基酸化合物，可以增加蛋白质在特定pH的溶解度，并可提高其稳定性，另外氨基酸及类氨基酸结构化合物除了可以降低表面吸附和保护蛋白质的构象之外，还可以防止蛋白质的变性与聚集，另外，当本发明的蛋白稳定剂为左旋卡尼汀时，与常用的辅料相比，左旋卡尼汀使配方具有低粘度的特点、并且能够减少肉毒毒素蛋白聚集和沉淀，有利于维持肉毒毒素蛋白稳定。
优选的，所述肉毒毒素蛋白选自A型肉毒毒素蛋白、B型肉毒毒素蛋白、C1型肉毒毒素蛋白、C2型肉毒毒素蛋白、D型肉毒毒素蛋白、E型肉毒毒素蛋白、F型肉毒毒素蛋白或G型肉毒毒素蛋白中的任一种；更优选的，所述肉毒毒素蛋白为A型肉毒毒素蛋白。
更优选的，所述肉毒毒素为重组肉毒毒素。
更为优选的，所述重组肉毒毒素蛋白由单链多肽经蛋白酶切割后形成多肽组成的至少一个二聚体结构；
所述的单链多肽包含：
(I)第一多肽片段，所述第一多肽片段包含：
(a)标签蛋白，
(b)包含第一蛋白酶切割位点的结构区；
(c)连接短肽，
(II)第二多肽片段，所述第二多肽片段包含：
(d)第一功能氨基酸结构区，其包含金属离子依赖的蛋白酶活性域；
(e)包含第二蛋白酶切割位点的结构区；
(f)第二功能氨基酸结构区，其包含可与靶细胞表面受体结合的受体结合域和/或可以介导多肽跨囊泡膜转移的易位域。
术语“标签蛋白”是指可以与特定的配基结合的一类蛋白分子。
优选的，所述的标签蛋白选自本领域技术人员已知的标签蛋白或者经过计算机程序设计的标签蛋白，所述的标签蛋白能够与已知底物特异结合。
更优选的，所述的标签蛋白选自但不限于以下蛋白：谷胱甘肽S－转移酶(GSTs)、C-myc、几丁质结合结构域、麦芽糖结合蛋白(MBP)、SUMO异源亲和部分、单克隆抗体或蛋白A、链霉亲和素结合蛋白(SBP)、纤维素结合结构域、钙调蛋白结合肽、S标签、Strep标签II、FLA、蛋白A、蛋白G、组氨酸亲和标签(HAT)、多聚组氨酸。
优选的，所述的“标签蛋白”可以增加毒素多肽前体的在宿主中的可溶性。
优选的，所述的“标签蛋白”使得毒素多肽前体以可溶的方式存在于宿主细胞中。
在一个具体实施例中，所述的标签蛋白位于单链多肽的N端。
在一个具体实施例中，所述的标签蛋白为谷胱甘肽s－转移酶(GSTs)，更优选的，所述谷胱甘肽s－转移酶的氨基酸序列如SEQ ID NO:2所示。
优选的，所述的第一蛋白酶切割位点和第二蛋白酶切割位点均不能被人蛋白酶或表达所述单链多肽的宿主细胞自身产生的蛋白酶切割。
更优选的，所述的第一蛋白酶切割位点和第二蛋白酶切割位点相同或不同。
所述的第一蛋白酶切割位点和第二蛋白酶切割位点不被表达时的宿主细胞或使用时机体内源性的蛋白酶识别和切割。
更优选的，所述的特定的蛋白酶选自但不限于以下蛋白酶的一种或两种的组合：非人肠激酶、烟草蚀刻病毒蛋白酶、来源于枯草芽孢杆菌的蛋白酶、来源于解淀粉芽孢杆菌的蛋白酶、来源于鼻病毒的蛋白酶、木瓜蛋白酶、昆虫木瓜蛋白酶的同源物或甲壳动物木瓜蛋白酶的同源物。
在一个具体实施例中，所述的特异地识别第一蛋白酶和所述的特异地识别第二蛋白酶切割位点的蛋白酶均为来源于鼻病毒的蛋白酶。
优选的，第二酶切位点嵌入、部分替换或全部替换第一功能肽段和第二功能 肽段之间的天然存在的环状区域。所述的嵌入是指插入在所述的环状区域的某两个氨基酸之间；所述的部分替换是指所述的第二酶切位点替换了所述的环状区域的部分氨基酸序列；所述的完全替换是指天然的环状区域的氨基酸序列完全被第二酶切位点所取代。
更优选的，所述的包含第一蛋白酶切割位点的结构区和包含第二蛋白酶切割位点的结构区选自但不限于以下酶切位点的一种或两种的组合：DDDDK、EXXYXQS/G、HY、YH或LEVLFQGP。
在一个具有实施例中，所述的包含第一蛋白酶切割位点的结构区和包含第二蛋白酶切割位点的结构区均为LEVLFQGP，切割位点在Q-G之间。
优选的，所述的连接短肽不超过5个氨基酸。
优选的，所述连接短肽能够使第一蛋白酶切割位点更容易被其蛋白酶识别或结合。
更优选的，所述的连接短肽不影响单链多肽的功能。
更优选的，所述的第一蛋白酶切割位点被切开后，保留在第二多肽片段N端的连接短肽不会影响第二多肽片段的功能。
更优选的，所述的连接短肽GS部分不超过5个氨基酸残基，更优选的，所述的连接短肽选自甘氨酸-丝氨酸(Glycine-Serine,简称GS)短肽，GGS,GGGS,GGGGS,GSGS,GGSGS,GSGGS,GGSGS,GGGSS等连接短肽。
在一个具体实施例中，所述的包含第一蛋白酶切割位点的结构区和连接短肽的氨基酸序列为LEVLFQGPLGS。
优选的，所述的金属离子依赖的蛋白酶活性域为Zn 2+依赖的蛋白酶活性域。
优选的，所述的第一功能氨基酸结构区和/或第二功能氨基酸结构区由天然序列和/或人工合成的序列编码。更优选的，所述的单链多肽的第一功能氨基酸结构区包含梭菌毒素轻链的Zn 2+蛋白酶活性域。
优选的，所述的第二功能氨基酸结构区中的能够结合人体细胞表面抗原的受体结合域的靶细胞是指存在SNARE复合体的靶细胞。例如：神经细胞、胰腺细胞或其他存在SNARE复合体的细胞。
更优选的，所述的第二功能氨基酸结构区中的靶细胞是指人神经细胞或胰腺细胞，所述的受体结合域为能够特定结合人神经细胞或胰腺细胞的受体结合域。
更优选的，所述的第二功能氨基酸结构区的受体结合域为梭菌毒素的重链的细胞表面抗原结合结构域，所述的第二功能氨基酸结构区的介导多肽跨囊泡膜转移的易位域为介导梭菌毒素跨囊泡膜转移的结构域。
更优选的，所述的梭菌毒素为肉毒杆菌毒素或破伤风毒素。
更优选的，所述的肉毒杆菌毒素选自现有技术已知的血清型BoNT/A-BoNT/H以及它们的衍生物中的任意一种。
更优选的，所述的梭菌毒素选自破伤风毒素或其衍生物中的任意一种。
更优选的，所述的第一功能氨基酸结构区包含BoNT/A、BoNT/B、BoNT/C、BoNT/D、BoNT/E、BoNT/F、BoNT/G、BoNT/H或破伤风毒素的轻链的部分或全部。
更优选的，所述的第二功能氨基酸结构区包含BoNT/A、BoNT/B、BoNT/C、BoNT/D、BoNT/E、BoNT/F、BoNT/G、BoNT/H或破伤风毒素的重链的部分或全部。
在一个具体实施例中，所述的第一功能氨基酸结构区为BoNT-/A的轻链，所述的第二功能氨基酸结构区为BoNT-/A的重链。
更优选的，所述的第一功能氨基酸结构区和第二功能氨基酸结构区可以来自不同的血清型。可以是上述血清型的任意组合，例如，所述的第一功能氨基酸结构区衍生自BoNT-/A的轻链，所述的第二功能氨基酸结构区衍生自BoNT-/B、BoNT/C、BoNT/D、BoNT/E、BoNT/F或BoNT/G的重链，或者，所述的第一功能氨基酸结构区衍生自BoNT/B、BoNT/C、BoNT/D、BoNT/E、BoNT/F或BoNT/G的轻链，所述的第二功能氨基酸结构区衍生自BoNT/A的重链等。
更优选的，所述的第二多肽片段的氨基酸序列包含SEQ ID NO:7所示的片段。
在一个具体实施例中，所述的单链多肽从N端开始依次包含：谷胱甘肽S-转移酶、LEVLFQGPLGS、BoNT/A的轻链、LEVLFQGP和BoNT/A的重链。
更优选的，所述的单链多肽的氨基酸序列如SEQ ID NO:11所示。
更优选的，重组A型肉毒毒素蛋白通过将单链多肽的经第一蛋白酶切割去除标签蛋白，和经第二蛋白酶切割后第一功能氨基酸结构区和第二功能氨基酸结构区形成至少一个二聚体来制备。
优选的，所述第一功能氨基酸结构区和第二功能氨基酸结构区经过二硫键连接成二聚体结构。
本发明的重组A型肉毒毒素蛋白，由特定的单链多肽形成，其中，所涉及的单链多肽具有特定的结构，该结构的单链多肽具有特异性强、毒性低的特点。具体的，从特异性强的角度来说，该单链多肽中的标签蛋白其能够与特异的配体结合，再经过与配体的脱离，使得与标签蛋白共表达的分子更易于从不同的多肽或蛋白产物中被选择出来，在制备过程中，可以显著的提升产品的纯度，使得产品得率更高、纯度更高；从毒性低的角度来说，该单链多肽中的标签蛋白使得多肽中第一功能氨基酸结构区的蛋白酶结构域不能发挥蛋白酶的功能，通过将第一功能氨基酸结构区和第二功能氨基酸结构区之间的天然酶切位点替换为仅特定的酶才可以识别的位点，从而使得该单链多肽的分子活性(神经毒性)降低至天然毒素的万分之一以下，有效的降低了毒性，由于其毒性被大幅降低，从而提高了生产过程中的安全性，使得整个工业化生产更易于进行。也就是说，本发明所涉及到重组肉毒毒素蛋白中的单链多肽，由于得率更高、纯度更高以及安全性也更高，相比于现有技术，更易于进行工业化的生产和纯化操作。
优选的，所述组合物还包括增溶剂和/或盐类化合物。
更优选的，所述增溶剂的体积百分比浓度为0.05％-0.2％。
更优选的，所述盐类的质量百分比浓度为0.9％。
更优选的，所述增溶剂选自吐温类、司盘类、十二烷基硫酸钠；
更优选的，所述吐温类选自吐温20、吐温40、吐温60、或吐温80中的一种或多种；
进一步优选的，所述司盘类选自司盘20、司盘40、司盘60、司盘80、或司盘85中的一种或多种；
在一个具体的实施方式中，所述增溶剂为吐温80。
优选的，所述盐类选自无机盐或有机盐中任一种；
更优选的，所述无机盐选自氯化钠、磷酸钠、硫酸镁、乙酸钠、丙酸钠和磷酸钾中的一种或多种；
进一步优选的，所述有机盐选自乳酸钠、琥珀酸钠中的一种或多种；
在一个具体的实施方式中，所述无机盐为氯化钠。
优选的，所述组合物包括肉毒毒素蛋白、左旋卡尼汀、吐温80、氯化钠；
或，
所述组合物包括肉毒毒素蛋白、氨基酸、吐温80、氯化钠。
更优选的，所述左旋卡尼汀的摩尔含量为5-20mM，优选为10mM；
或，所述氨基酸的摩尔含量为5-20mM；优选为10mM。
更优选的，所述吐温80的体积百分比浓度为0.05％-0.2％。
在一个具体的实施方式中，所述吐温80的体积百分比浓度为0.1％。
更优选的，所述氯化钠的质量百分比浓度为0.9％。
所述组合物为液体制剂。
本发明还提供肉毒毒素蛋白组合物的制备方法，所述制备方法包括将各组分混合的步骤。
优选的，上述制备方法的步骤包括：
(a)制备、纯化肉毒毒素蛋白；
(b)向所述肉毒毒素蛋白中加入蛋白稳定剂水。
所述水为注射用水。
更优选的，所述步骤(b)中还包括加入增溶剂和/或盐类。
其中，所述肉毒毒素蛋白、蛋白稳定剂、增溶剂和/或盐类如上所限定。
在一个具体的实施方式中，所述制备方法包括：
(a)制备、纯化重组A型肉毒毒素蛋白；
(b)向所述重组A型肉毒毒素蛋白中加入蛋白稳定剂水、吐温80和氯化钠、水。
所述水为注射用水。
本发明还提供了上述肉毒毒素蛋白组合物在制备用于预防和/或治疗与支配肌肉或外分泌腺的胆碱能神经相关的疾病、医学美容的药物中的应用。
所述与支配肌肉或外分泌腺的胆碱能神经相关的疾病或病症包括：与胆碱能神经支配相关的肌肉或外分泌腺过度活跃的示例性疾病或病症包括，例如弗雷综合征、鳄鱼泪综合征、腋下多汗症、足底多汗症、头颈多汗症、身体多汗症、鼻漏、帕金森氏病、肌萎缩侧索硬化、多涎、垂涎、流涎、痉挛状况、腭肌阵挛、肌阵挛、肌纤维颤搐、僵直、良性肌肉痉挛、遗传性下巴颤抖、反常性下颌肌肉 活动、半侧咀嚼肌痉挛、肥大型鳃肌病、嚼肌肥大、胫骨前肌肥大、眼球震颤、振动幻视、核上凝视麻痹、持续状态部分性癫痫、痉挛性斜颈手术计划、展肌声带麻痹、顽固性突变性发音障碍、上食道括约肌功能障碍、声带肉芽肿、口吃、抽动秽语综合征、中耳肌阵挛、保护性喉闭合、喉切除术后言语失败、保护性上睑下垂、睑内翻、奥狄氏括约肌功能障碍、假性食道弛缓不能、非失弛缓性食道运动障碍、阴道痉挛、术后卧床、震颤、膀胱功能障碍、半面痉挛、神经支配恢复术运动障碍、僵人综合征、破伤风、前列腺增生、肥胖治疗、婴儿大脑性麻痹、失弛缓症和肛裂。
所述医学美容包括平滑或预防皱纹、瘦脸、疤痕修复。
所述皱纹可被视为皮肤中的褶皱、嵴或褶痕。优选人类的面部中可见的皱纹。皱纹的例子包括泪沟、眉间纹、鱼尾纹、颊连合处纹、下颌纹、口周皱纹、颊褶皱、木偶纹、唇纹、前额皱纹、皱眉纹、兔纹、鼻唇沟、眼下皱纹和下巴褶皱。
与现有技术相比，本发明具有如下有益效果：
(1)本发明提供了一种稳定的肉毒毒素蛋白组合物。其组合物中使用氨基酸或类氨基酸取代了人源蛋白(例如人血白蛋白)，杜绝了人源病毒的威胁，同时配合吐温80防止吸附，能在标准冰箱(约4℃)内以液体状态稳定贮藏1年或更长，能在室温(25℃)以液体状态稳定贮藏6个月或更长。
(2)本发明提供一种重组肉毒毒素蛋白组合物，采用特定单链多肽制备的高纯度重组A型肉毒毒素蛋白，能够提升制剂中A型肉毒毒素蛋白的纯度，避免了从肉毒杆菌提取A型肉毒毒素所包含的非活性肉毒杆菌蛋白摄入人体可能产生的免疫风险，为扩大重组A型肉毒毒素蛋白的应用范围提供了新的选择。
(3)本发明的肉毒毒素蛋白组合物作为液体制剂使用时，增加了临床使用方便性，改善了冻干或真空产品复溶后通常保存时间较短，易造成使用浪费以及复溶过程中人为污染或复溶不准确等缺陷。
附图说明
图1:GSTs-BoNT/A初步纯化和进一步去除GSTs标签的SDS-PAGE结果，其中第1泳道为经过GSTs亲和层析柱初步纯化得到的GSTs-BoNT/A，第2泳道为初步纯化的蛋白经过Rinovirus 3C Protease的酶切去除GSTs标签蛋白得到切除GSTs后的BoNT/A。
图2：切除了GSTs标签的产物经过离子交换柱进一步纯化得到高纯度的BoNT/A蛋白的SDS-PAGE结果。
图3：BoNT/A在还原条件下双链解离的验证结果。
具体实施方式
除非另有定义，本发明中所使用的所有科学和技术术语具有与本发明涉及技术领域的技术人员通常理解的相同的含义。
本发明中，术语“U”是指效价单位，药剂学上使用的(表示)生物学能力的多样的单位。因为这些药物的化学成分不恒定或至今还不能用理化方法检定其质量规格，往往采用生物实验方法并与标准品加以比较来检定其效价。通过这种生物检定，具有一定生物效能的最小效价单元就叫“单位”(U)；经由国际协商规定出的标准单位，称为“国际单位”(IU)；
术语“BoNT/A”是指A型肉毒毒素蛋白；
术语“标签蛋白”是指利用DNA体外重组技术，与目的蛋白一起融合表达的一种多肽或者蛋白，以便于目的蛋白的表达、检测。
术语“人血清白蛋白”(Human serum albumin)，简称HSA，是指人血浆中的蛋白质，在体液中HSA可以运输脂肪酸、胆色素、氨基酸、类固醇激素、金属离子和许多治疗分子等：同时维持血液正常的渗透压。在临床上HSA可用于治疗休克与烧伤，用于补充因手术、意外事故或大出血所致的血液丢失，也可以作为血浆增容剂。
术语“二聚体结构”是指为由两个分子组合而成的高分子配合物，常以非共价键键结。
下面将结合本发明实施例，对本发明的技术方案进行清楚、完整地描述。显然，所描述的实施例仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实验例1重组A型肉毒毒素蛋白的制备
具体分为以下8个步骤：
1、设计和构建编码经修饰的肉毒毒素的单链多肽的核酸分子：
核酸分子从5’端起依次包含：
(a)编码谷胱甘肽s-转移酶的核苷酸序列如SEQ ID NO:1所示，其编码的谷胱甘肽s-转移酶的氨基酸序列如SEQ ID NO:2所示；
(b)编码第一蛋白酶切割位点的核苷酸序列如SEQ ID NO:3所示，其编码的氨基酸序列如SEQ ID NO:4所示；
(c)编码GS连接短肽的核苷酸序列如SEQ ID NO:5所示，为GGATCC；
(d)编码第二多肽片段的核苷酸序列，其包含BoNT/A轻链、第二蛋白酶切割位点和BoNT/A重链的核苷酸序列，如SEQ ID NO:6所示，其编码的氨基酸序列如SEQ ID NO:7所示；其中，编码第二蛋白酶切割位点的核苷酸序列如SEQ ID NO:8所示，其编码的氨基酸序列如SEQ ID NO:9所示；
BoNT/A轻链和BoNT/A重链之间也可以不存在天然的环状区域，例如去除轻链和重链之间的KTKSLDKGYNK连接序列，以减少非特异性蛋白酶切割。
编码单链多肽的核苷酸序列如SEQ ID NO:10所示，其编码的单链多肽的氨基酸序列如SEQ ID NO:11所示。
2、构建包含步骤1序列的核酸分子的质粒
在上述人工合成基因工程优化后的GSTs-BoNT/A，两端合成加入NdeI和NotI酶切位点，经过NdeI和NotI 37摄氏度酶切(New England Biolabs)，使用QIquick gel extraction kit(Qiagen)纯化，使用T4 DNA ligase(NEB)插入pET28a(Novagen)质粒载体中的NdeI和NotI的位点。
3、转染步骤2构建的质粒到宿主细胞
(a)感受态细胞的制备：取一管大肠杆菌细胞E.coli BL21 DE3(New England Biolabs)，接种至含有3ml LB培养液的试管中，37℃振荡培养过夜。次日取菌液0.5ml接种至含有50ml LB培养基的250ml烧瓶中，37℃剧烈震荡培养约3-5小时(250rpm)，当菌落600nm OD值达到0.3-0.4时，将烧瓶取出放置冰上10-15分钟。在无菌条件下把菌液倒入50ml离心管中。4℃，1000g离心10分钟。弃上清，收集加10ml 0.1M的CaCl 2到离心管中，振荡混匀，悬浮菌体，冰浴30分钟，4℃，1000g离心10分钟，弃上清，收集加4ml冰预冷的0.1M的CaCl 2，重悬浮菌体。每管0.2ml分装，至4℃保存备用，24小时内使用，其余保存于-70℃的低温冰箱中。
(b)转染：适量DNA(ng)和感受态大肠杆菌混合放置冰浴15分钟，42℃热击30秒，放置冰浴5分钟，放入SOC培养基250rpm摇晃1小时，涂抹在有抗生素的平板上，37℃隔夜培养。
4、培养宿主细胞和诱导表达单链多肽
培养基的组分和配比：11.8g/L胰蛋白胨、23.6g/L酵母提取物、9.4g/L K 2HPO 4、2.2g/L KH 2PO 4、4ml/L甘油。
培养条件：37℃隔夜250rpm震荡培养细胞。
诱导表达：当大肠杆菌生长到OD600＝1的时候，加入1mM IPTG诱导表达在25℃表达5个小时。OD600可以选用0.2-1.5，温度可以选用37℃到10℃，表达时间可以是5-16个小时。
收获细胞：3000rpm 30分钟离心收集大肠杆菌。
5、单链多肽表达水平的鉴定
使用金斯瑞谷胱甘肽纯化树脂制备一根直径1厘米，高1厘米的谷胱甘肽纯化树脂层析柱，缓慢地倾倒25毫升裂解液到层析柱上层，注意不要冲起树脂，经过一小时缓慢过柱吸附裂解液中的GSTs-BoNT/A。按照常规方法洗脱获得GSTs-BoNT/A。
常规方法如下：使用20倍柱体积的磷酸缓冲液洗涤层析柱，用10倍柱体积的新鲜制备的10mM谷胱甘肽洗脱缓冲液(0.154g还原的谷胱甘肽溶解在50ml 50mM Tris-HCl(pH 8.0))进行GSTs-BoNT/A洗脱，使用280nm处的吸光度读数监控融合蛋白的洗脱。洗脱后的蛋白在Rinovirus 3C Proteas作用下切除GSTs标签蛋白。
取去除GSTs标签蛋白之前的产物和之后的产物进行常规的SDS-PAGE实验，如图1所示，与没有标签蛋白去除的第1泳道相比，在Rinovirus 3C Protease的作用下，第1泳道的GSTs标签蛋白被切除，得到没有GSTs的BoNT/A分子。
GSTs-BoNT/A在总蛋白水平中所占的比例的测定：使用4-12％SDS-PAGE(Biorad)对纯化的GSTs-BoNT/A在200伏电压下电泳分离，175kd分子量主要条带是GSTs-BoNT/A。
6、标签蛋白的去除以及提纯
对将步骤5的产物进行GSTs标签蛋白的去除和有毒多肽的提纯：
用金斯瑞3C酶处理重新吸附在谷胱甘肽纯化树脂层析柱上的GSTs-BoNT/A，在3C酶的作用下，GSTs和BoNT/A之间的第一酶切位点被断开，GSTs脱离，同时BoNT/A的轻链和重链之间的第二酶切位点被断开。
以磷酸缓冲液处理谷胱甘肽纯化树脂层析柱，GSTs标签蛋白留在柱子上，被去除掉，而BoNT/A的轻链和重链被磷酸缓冲液洗脱下来。
取去除了GSTs的产物进行常规的SDS-PAGE实验，如图2所示，得到的条带中不再有GSTs标签蛋白，说明GSTs标签蛋白已经被完全去除。使用SDS-PAGE扫描图片，根据条带灰色密度来计算所得BoNT/A为90％纯度以上。
采用GSS、GSGS、GGSGS多肽代替链接短肽GS部分，其效果和GS相似，标签蛋白可以很好得暴露，从而能被完全切除。
7、二聚体BoNT/A的双链在还原条件下的解离
将步骤6的产物进行还原实验：
使用100mM的二硫苏糖醇，在100摄氏度下处理样品五分钟，使样品还原，样品经过4-12％SDS-PAGE(Biorad)200伏电压电泳分离，分离出重链和轻链。如图3所示，将步骤6得到的产物在还原条件下进行还原，得到的产物进行常规的SDS-PAGE实验，得到两条不同的条带，分子量分别在100Kda和50Kda，证明步骤6形成的产物是以二硫键链接两个肽段的二聚体结构。
8、GSTs-BoNT/A的毒性试验
用步骤5获得的GSTs-BoNT/A经小鼠腹腔注射的LD 50在45ng-450ng之间；考虑到所注射的肉毒毒素蛋白纯度，折算的LD 50在22.5ng-225ng之间，中间值为123.75ng。用步骤5获得的BoNT/A经小鼠腹腔注射的LD 50在0.02ng–0.05ng之间。考虑到所注射的肉毒毒素蛋白纯度，折算的LD 50在0.006ng-0.015ng之间，中间值为0.0105ng(见表1)。
表1：GSTs-BoNT/A和BoNT/A分子在小鼠生物毒性实验中的毒性比较
Figure PCTCN2022136597-appb-000001
GSTs-BoNT/A具有肉毒毒素的活性，经小鼠腹腔注射后，其半数致死量(LD 50)大约比BoNT/A的蛋白LD 50高11786倍，表明GSTs-BoNT/A重组蛋白毒素多肽前体分子活性弱于最终产品BoNT/A大约11786倍。这个实验证明，GSTs-BoNT/A重组蛋白毒素多肽前体分子具有肉毒毒素的活性。由于肉毒毒素的超高毒性，因此生产过程中，在未对前体分子作活化处理前就需要高度安全操作防护。
实施例2重组A型肉毒毒素蛋白组合物的优化
通过小鼠半数致死量评估肉毒毒素蛋白(实施例1制备)的活性，并以小鼠单位(U)显示，一个单位定义为杀死小鼠总体数量50％所需的腹腔注射重组A型肉毒毒素蛋白量。
一、动物实验过程：
1、选种分组：
选取CD-1品种的成年小鼠，分组，称重、编号、保证每组小鼠数量相等，体重相近。
2、给药：
表2中不同配方制剂给药，给药过程中，腹腔单次注射给予CD-1小鼠不同制剂配方重组A型肉毒毒素蛋白制剂均为0.5mL。经小鼠腹腔注射药的重组A型肉毒毒素蛋白量为2X LD 50(2U)。
3、观察：
试验开始前，每只动物称量一次体重(第一天给药前)并记录，给药后，每24h称量一次体重，在给药后24h(±1h)、48h(±1h)、72h(±1h)、96h(±1h)，检查死亡率。
二、实验结果
选取表2中所列的含有2U肉毒毒素的各组分配方(1-4)注射小鼠腹腔，观察各制剂储存在4℃条件下0至211天后2U肉毒毒素对每组小鼠的致死率，实验过程同上述动物实验，实验结果如表2所示。
表2.各组分配方制剂储存不同时间后的活性测定
Figure PCTCN2022136597-appb-000002
从表2的结果中可以看出，表中4个配方在4℃条件下放置0至211天后，重组A型肉毒毒素对小鼠的致死作用(药物活性)没有减弱，仍然保持重组A型肉毒毒素蛋白在放置4℃保存前的生物活性，证明本发明所选择的配方能够维持重组A型肉毒毒素的长期稳定性。
选取表2中两个制剂测定了重组A型肉毒毒素储存在4℃ 44，75和211天后的小鼠半数致死量(LD 50)。实验结果见表3.
表3.重组A型肉毒毒素在4℃保存44，75和211天后的小鼠LD 50测定值
Figure PCTCN2022136597-appb-000003
Figure PCTCN2022136597-appb-000004
从表3中可以看出，配方1在4℃放置211天后，能保持重组A型肉毒毒素蛋白的活性，配方2在4℃放置70天后，能保持重组A型肉毒毒素蛋白的活性，经过多剂量-活性关系计算的重组A型肉毒毒素的小鼠半数致死量(LD 50)经过4℃分别放置44，70和211天后没有明显变化，半数致死量仍然保持基本不变，充分证明本发明的制剂配方能够有效维持重组A型肉毒毒素的生物活性长期稳定。
本发明采用重组肉毒毒素，使用氨基酸或类氨基酸化合物作为蛋白稳定剂，可以在低温条件(如4℃下)长时间储存(如4℃下储存70天以上)，本发明的制剂配方也可保持天然肉毒素蛋白(A型肉毒毒素蛋白、B型肉毒毒素蛋白、C1型肉毒毒素蛋白、C2型肉毒毒素蛋白、D型肉毒毒素蛋白、E型肉毒毒素蛋白)的活性。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明。凡在本发明的精神和原则之内所作的任何修改、等同替换等，均应包含在本发明的保护范围之内。
本文描述的前述实施例和方法可以基于本领域技术人员的能力、经验和偏好而有所不同。
本发明中，仅按一定顺序列出方法的步骤并不构成对方法步骤顺序的任何限制。

Claims (17)

  1. 一种肉毒毒素蛋白组合物，其特征在于，包括以下组分：
    肉毒毒素蛋白和蛋白稳定剂；
    所述蛋白稳定剂选自氨基酸或类氨基酸化合物中任一种。
  2. 如权利要求1所述的组合物，其特征在于，所述类氨基酸化合物包括左旋卡尼汀(L-carnitine)。
  3. 如权利要求2所述的组合物，其特征在于，所述左旋卡尼汀的摩尔含量为5-20mM。
  4. 如权利要求1所述的组合物，其特征在于，所述氨基酸的摩尔含量为5-20mM。
  5. 如权利要求4所述的组合物，其特征在于，所述氨基酸选自L-组氨酸、L-甘氨酸、L-精氨酸、L-甲硫氨酸、L-组氨酸和L-赖氨酸中的一种或多种。
  6. 如权利要求1-5任一项所述的组合物，其特征在于，所述肉毒毒素蛋白选自A型肉毒毒素蛋白、B型肉毒毒素蛋白、C1型肉毒毒素蛋白、C2型肉毒毒素蛋白、D型肉毒毒素蛋白、E型肉毒毒素蛋白、F型肉毒毒素蛋白、G型肉毒毒素蛋白或H型肉毒毒素蛋白中的任一种；优选的，所述肉毒毒素蛋白为A型肉毒毒素蛋白，更优选的，所述肉毒毒素为重组肉毒毒素。
  7. 如权利要求1-6任一项所述的组合物，其特征在于，所述组合物还包括增溶剂和/或盐类。
  8. 如权利要求7所述的组合物，其特征在于，所述增溶剂的体积百分比浓度为0.01％-0.2％。
  9. 如权利要求7-8任一项所述的组合物，其特征在于，所述的盐类的质量百分比为0.9％。
  10. 如权利要求7-9任一项所述的组合物，其特征在于，所述增溶剂选自吐温类、司盘类、十二烷基硫酸钠；
    优选的，所述吐温类选自吐温20、吐温40、吐温60、或吐温80中的一种或多种；
    优选的，所述司盘类选自司盘20、司盘40、司盘60、司盘80、或司盘85中的一种或多种；
    更优选的，所述增溶剂为吐温80。
  11. 如权利要求7-10任一项所述的组合物，其特征在于，所述盐类选自无机盐或有机盐中任一种；
    优选的，所述无机盐选自氯化钠、磷酸钠、硫酸镁、乙酸钠、丙酸钠和磷酸钾中的一种或多种；
    优选的，所述有机盐选自乳酸钠、琥珀酸钠中的一种或多种；
    更优选的，所述无机盐类为氯化钠。
  12. 如权利要求1-11任一项所述的组合物，其特征在于，所述组合物包括肉毒毒素蛋白、左旋卡尼汀、吐温80、氯化钠；
    或，
    所述组合物包括肉毒毒素蛋白、氨基酸、吐温80、氯化钠。
  13. 如权利要求12所述的组合物，其特征在于，所述吐温80的体积百分比浓度为0.05％-0.2％；
    优选的，所述吐温80的体积百分比浓度为0.1％。
  14. 如权利要求12-13任一项所述的组合物，其特征在于，所述氯化钠的质量百分比浓度为0.9％。
  15. 如权利要求1-14任一项所述的组合物，其特征在于，所述组合物为液体制剂。
  16. 一种权利要求1-15任一项所述的组合物的制备方法，其特征在于，所述制备方法包括将各组分混合的步骤。
  17. 权利要求1-15任一项所述的组合物在制备用于预防和/或治疗支配肌肉或外分泌腺的胆碱能神经相关疾病的药物中的应用，
    或，
    在制备用于医学美容中药物的应用；
    优选的，所述医学美容包括平滑或预防皱纹、瘦脸、形体塑型、疤痕修复；
    优选的，所述的胆碱能神经相关疾病选自：
    弗雷综合征、鳄鱼泪综合征、腋下多汗症、足底多汗症、头颈多汗症、身体多汗症、鼻漏、帕金森氏病、肌萎缩侧索硬化、多涎、垂涎、流涎、痉挛状况、腭肌阵挛、肌阵挛、肌纤维颤搐、僵直、良性肌肉痉挛、遗传性下巴颤抖、反常性下颌肌肉活动、半侧咀嚼肌痉挛、肥大型鳃肌病、嚼肌肥大、胫骨前肌肥大、 眼球震颤、振动幻视、核上凝视麻痹、持续状态部分性癫痫、痉挛性斜颈手术计划、展肌声带麻痹、顽固性突变性发音障碍、上食道括约肌功能障碍、声带肉芽肿、口吃、抽动秽语综合征、中耳肌阵挛、保护性喉闭合、喉切除术后言语失败、保护性上睑下垂、睑内翻、奥狄氏括约肌功能障碍、假性食道弛缓不能、非失弛缓性食道运动障碍、阴道痉挛、术后卧床、震颤、膀胱功能障碍、半面痉挛、神经支配恢复术运动障碍、僵人综合征、破伤风、前列腺增生、肥胖治疗、婴儿大脑性麻痹、失弛缓症和肛裂中的一种或多种。
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CN106163545A (zh) * 2013-12-12 2016-11-23 株式会社美得拓石 长效的新肉毒杆菌毒素配制剂
WO2021195968A1 (zh) * 2020-03-31 2021-10-07 台湾浩鼎生技股份有限公司 A型肉毒杆菌毒素复合物、其配制剂和使用方法
CN113876936A (zh) * 2021-11-09 2022-01-04 达尔文新研(北京)生物科技有限公司 一种高稳定性的注射用肉毒毒素组合物及其制备方法和其应用

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