WO2021195968A1 - A型肉毒杆菌毒素复合物、其配制剂和使用方法 - Google Patents

A型肉毒杆菌毒素复合物、其配制剂和使用方法 Download PDF

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Publication number
WO2021195968A1
WO2021195968A1 PCT/CN2020/082450 CN2020082450W WO2021195968A1 WO 2021195968 A1 WO2021195968 A1 WO 2021195968A1 CN 2020082450 W CN2020082450 W CN 2020082450W WO 2021195968 A1 WO2021195968 A1 WO 2021195968A1
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Prior art keywords
botulinum toxin
complex
component
seq
type
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PCT/CN2020/082450
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English (en)
French (fr)
Inventor
曹子文
王永凯
沈冠成
吴岳缙
游丞德
曾毓俊
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台湾浩鼎生技股份有限公司
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Priority to KR1020217039098A priority Critical patent/KR20220003061A/ko
Priority to MX2021014486A priority patent/MX2021014486A/es
Priority to PCT/CN2020/082450 priority patent/WO2021195968A1/zh
Priority to CN202310089032.2A priority patent/CN116041452A/zh
Application filed by 台湾浩鼎生技股份有限公司 filed Critical 台湾浩鼎生技股份有限公司
Priority to EP20926377.1A priority patent/EP3932938A4/en
Priority to NZ780855A priority patent/NZ780855A/en
Priority to KR1020237033930A priority patent/KR20230146125A/ko
Priority to CN202080028618.2A priority patent/CN114222754A/zh
Priority to SG11202111282SA priority patent/SG11202111282SA/en
Priority to JP2021571357A priority patent/JP7328714B2/ja
Priority to BR112021024058A priority patent/BR112021024058A2/pt
Priority to AU2020440362A priority patent/AU2020440362B2/en
Priority to US17/623,072 priority patent/US20220257730A1/en
Publication of WO2021195968A1 publication Critical patent/WO2021195968A1/zh
Priority to SA521430958A priority patent/SA521430958B1/ar

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    • 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)
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    • C07K14/33—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Clostridium (G)
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    • 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
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Definitions

  • 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.
  • 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, 2nd ed., St. Louis, Mo.: Mosby Year Book :5-15 (1990)).
  • These secondary factors include constant gravitational pull, frequent and constant positional pressure on the skin (for example, during sleep), and repetitive facial movements (Stegman et al., The Skin of the Aging Face Cosmetic Dermatological) caused by facial muscle contraction. Surgery, 2nd ed., St. Louis, Mo.: Mosby Year Book: 5-15 (1990)).
  • Botulinum toxin type A is reported to be the most lethal natural biological agent known to man. Spores of Clostridium 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 causing muscle paralysis, and it is believed that botulinum toxin also works in other ways. Its effect essentially blocks the signals that normally cause muscle spasms or contractions, leading 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 spasms .
  • the muscle paralysis effect of botulinum toxin has been applied to the treatment and cosmetic facial applications, such as the treatment of wrinkles and facial muscle spasms or contractions.
  • botulinum toxin serotypes A, B, C, D, E, F, and G are distinguished by neutralization with type-specific antibodies.
  • the molecular weight of each botulinum toxin protein is about 150kD. Due to the molecular size and structure of botulinum toxin, it cannot penetrate the stratum corneum and the lower layers of the skin structure.
  • botulinum toxin type A has been determined to be 500 times more potent than botulinum toxin type B, as measured by the rate of paralysis.
  • botulinum toxin type B is non-toxic at a dose of 480 U/kg in primates.
  • the botulinum toxin released by Clostridium botulinum bacteria 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 digestion and acid denaturation when the toxin complex is ingested.
  • HA hemagglutinin protein
  • NTNH non-hemagglutinin protein
  • the non-toxin protein of the toxin complex protects the activity of botulinum toxin, thereby enhancing systemic penetration.
  • some non-toxin proteins specifically stabilize the botulinum toxin molecules in the blood.
  • Clostridium botulinum bacteria can produce a botulinum toxin type A 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 produced only as complexes with a molecular weight of approximately 300 kD.
  • the toxin complex is usually stabilized by binding the complex to albumin during the manufacturing process.
  • albumin E.g, (Allergan, Inc, Irvine, CA) is a preparation containing botulinum toxin, which contains 100U type A botulinum toxin and auxiliary protein, 0.5mg human albumin and 0.9mg sodium chloride.
  • Albumin is used to bind and stabilize toxin complexes in different environments, including those related to manufacturing, transportation, storage, and administration.
  • 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.
  • the inventors provide a new type A botulinum toxin complex.
  • the type A botulinum toxin complex of the present invention has a smaller molecular weight, so the production cost is smaller; and the stability and safety are higher.
  • the present invention provides a type A botulinum toxin complex comprising HA70 component, HA17 component, HA33 component, NTNH component and BoNT/A1 component, wherein the botulinum toxin
  • the composition has a molecular weight of 740-790kDa.
  • the botulinum toxin type A complex is composed of HA70 component, HA17 component, HA33 component, NTNH component, and BoNT/A1 component, and has a molecular weight of about 760 kDa.
  • the HA70 component contains any of the following:
  • the HA17 component includes any of the following:
  • the HA33 component contains any of the following:
  • the NTNH component includes any of the following:
  • the BoNT/A1 component contains any of the following:
  • the present invention provides a nucleic acid encoding the botulinum toxin type A complex of the present invention.
  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the botulinum toxin type A complex of the present invention and a pharmaceutically or dermatologically acceptable carrier.
  • the present invention provides a liquid formulation comprising the type A botulinum toxin complex of any one of claims 1-6, the composition of which comprises human serum albumin, methionine, and Acid, sodium chloride, Tween 80, citrate buffer, and/or phosphate buffer.
  • 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.
  • the cosmetic method includes reducing the appearance of fine lines and/or wrinkles, particularly 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 tone.
  • 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 Type A of the present invention Botulinum toxin complex, pharmaceutical composition or liquid formulation.
  • the disease is selected from neuromuscular disorders characterized by skeletal muscle hyperactivity, nerve pain, migraine, overactive bladder, rhinitis, sinusitis, acne, dystonia, dystonic contractions, 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 cramps.
  • neuromuscular disorders characterized by skeletal muscle hyperactivity, nerve pain, migraine, overactive bladder, rhinitis, sinusitis, acne, dystonia, dystonic contractions, hyperhidrosis, Excessive secretion of one or more glands controlled by the cholinergic nervous system, hemifacial spasm, adult-onset spastic torticollis, anal fissure, blepharospasm
  • 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 medicines, 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 tension; the drug is used to treat or prevent diseases caused by the synaptic transmission or release of acetylcholine .
  • the disease is selected from neuromuscular disorders characterized by skeletal muscle hyperactivity, nerve pain, migraine, overactive bladder, rhinitis, sinusitis, acne, dystonia, dystonic contractions, 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 cramps.
  • neuromuscular disorders characterized by skeletal muscle hyperactivity, nerve pain, migraine, overactive bladder, rhinitis, sinusitis, acne, dystonia, dystonic contractions, hyperhidrosis, Excessive secretion of one or more glands controlled by the cholinergic nervous system, hemifacial spasm, adult-onset spastic torticollis, anal fissure, blepharospasm
  • 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.
  • the disease is selected from neuromuscular disorders characterized by skeletal muscle hyperactivity, nerve pain, migraine, overactive bladder, rhinitis, sinusitis, acne, dystonia, dystonic contractions, 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 cramps.
  • neuromuscular disorders characterized by skeletal muscle hyperactivity, nerve pain, migraine, overactive bladder, rhinitis, sinusitis, acne, dystonia, dystonic contractions, hyperhidrosis, Excessive secretion of one or more glands controlled by the cholinergic nervous system, hemifacial spasm, adult-onset spastic torticollis, anal fissure, blepharospasm
  • the botulinum toxin type A complex of the present invention (also referred to herein as "OBI-858") is compatible with commercially available products Compared with the following advantages:
  • the total protein molecular weight of the type A botulinum toxin complex of the present invention is 760kDa, and With a molecular weight of 900kDa, it is a new type A botulinum toxin complex.
  • Figure 1 Size exclusion high performance liquid chromatography (SEC-HPLC) to detect product purity.
  • Figure 2 Connecting refractive index and multi-angle laser light scattering (MALS) to detect product molecular weight.
  • MALS multi-angle laser light scattering
  • Figure 3 Electron microscopy to determine the molecular weight of the total protein of the product.
  • OBI-858 arrows indicate toxin particles; and
  • 760kDa L-PTC/A reference study arrows indicate four different directions.
  • Figure 4 Scoring chart of the DAS assay.
  • the present invention relates to a type A botulinum toxin complex and its liquid formulation.
  • the composition of the present invention can be used as an injectable application to provide botulinum toxin to a subject for various therapeutic, cosmetic and/or cosmetic purposes.
  • the type A botulinum toxin complex of the present invention has better safety and enhanced or equivalent efficacy.
  • the present invention provides a liquid formulation for the type A botulinum toxin complex, which provides excellent stability.
  • botulinum toxin type A complex refers to a botulinum toxin type A protein molecule of approximately 150 kD and related endogenous non-toxin proteins, namely hemagglutinin protein (HA) and non-toxin non-toxin proteins.
  • HA hemagglutinin protein
  • NTNH hemagglutinin protein
  • the hemagglutinin protein is composed of 70, 33 and 17kDa subcomponents, namely HA70, 33 and 17.
  • the botulinum toxin complex does not need to be derived from the Clostridium botulinum bacteria as a single toxin.
  • 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.
  • the botulinum toxin type A complex may also contain HA70, 33, 17, NTNH or BoNT/A1 as described herein, or a variant thereof.
  • a variant is an amino acid sequence obtained by adding, deleting, substituting or inserting one or more amino acids in the sequence described herein (for example, any one of SEQ ID No. 1-5).
  • one or several refers to the number of regions that do not impair protein activity to a large extent.
  • the number referred to 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 1 to 30, 1 to 20 , 1 to 10 or 1 to 5 (e.g. 1, 2, 3, 4, or 5).
  • BoNT/A1 (SEQ ID NO. 5)
  • 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.
  • 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 spasms, 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 is in a form that allows injection into the skin or epithelium of a subject or patient (ie, a human or other mammal in need of specific treatment).
  • the term “need” is meant to include needs related to drugs or health (for example, to treat conditions involving undesirable facial muscle spasms), as well as cosmetic and subjective needs (for example, to change or improve the appearance of facial tissues).
  • they may contain an aqueous pharmaceutically acceptable diluent, such as buffered saline (e.g., phosphate buffered saline).
  • 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.
  • the term "dermatologically or pharmaceutically acceptable” means that the composition or its components described herein are suitable for contact with tissues or for use in general patients without undue toxicity, incompatibility, Instability, allergic reactions, etc.
  • the composition of the present invention may contain any components conventionally used in the field under consideration, especially in cosmetics and dermatology.
  • the botulinum toxin complex 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 spasticity, reduce glandular output or other desired effects.
  • An effective amount 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 of the present invention is administered to deliver an effective amount of botulinum toxin.
  • effective amount refers to an amount that is sufficient to produce the desired muscle paralysis or other biological or aesthetic effects, but it is implicitly 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 muscle 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 administration of the botulinum toxin complex of the present invention can also be used to treat other conditions, including any conditions where prevention of synaptic transmission or release of acetylcholine would bring therapeutic benefits.
  • conditions that can be treated 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 nerves
  • a neuromuscular disorder characterized by hypersecretion of one or more glands controlled by the system, characterized by overactive skeletal muscles.
  • Conditions treated with botulinum toxin include hemifacial spasm, 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.
  • the present invention can also be used to reduce or enhance immune response, or to treat other conditions for which injection of botulinum toxin complex has been suggested or administered.
  • the botulinum toxin complex of the present invention can be prepared in solid form, such as freeze-dried powder, tablets, etc., and reconstituted in water for injection before use for use.
  • the botulinum toxin complex, composition or formulation of 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.
  • the botulinum toxin complex, composition or formulation according to the present invention is injected at one or more locations where botulinum toxin-related effects are required. Due to the nature of the botulinum toxin complex, the botulinum toxin is preferably administered in an amount, rate, and frequency that produces the desired result without producing any undesirable or undesirable results.
  • the formulation includes human serum albumin, methionine, arginine, sodium chloride, Tween 80, citrate buffer, and/or phosphate buffer.
  • the formulation comprises human serum albumin, sodium chloride, and Tween 80 dissolved in 50 mM citrate buffer.
  • the concentration of human serum albumin may be 0.1-1, 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, such as 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 mg/mL, more preferably 11.25 mg/mL .
  • the concentration of Tween 80 dissolved in 50 mM citrate buffer may be 0.1-0.3, for example, 0.2 mg/mL, more preferably 0.188 mg/mL.
  • the formulation has a certain pH value, for example 4-8, preferably pH 5.6.
  • the OBI-858 bacteria in the freezing tube of the cell bank were revived, and gradually expanded with the medium, and then inoculated into the production fermentor. After the cell culture is completed, the cells and cell debris are removed by filtration.
  • the purification step includes two steps of precipitation, buffer exchange and chromatography. After the chromatographic step, the product was filtered through a 0.22 ⁇ m filter to the bulk drug container, and then lyophilized to obtain the OBI-858 bulk drug.
  • the reconstituted solution was sterilized through a 0.22 ⁇ m filter, and transferred to a sterile isolator, and then lyophilized to obtain an OBI-858 drug product.
  • the strain used for OBI-858 production is a type A Clostridium botulinum strain developed by self-selection. It was confirmed by whole genome sequencing that 98.28% were similar to the 3502 Clostridium botulinum type A strain, and 89.69% were similar to the Hall clostridium botulinum type A strain. Strain [Table 1], so it is different from the products currently on the market in terms of strain gene analysis.
  • 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
  • OBI-858 protein samples were first denatured with 6M urea, reduced with 10mM DTT at 37°C for 1 hour, and alkylated with 50mM IAM for 30 minutes at room temperature in the dark.
  • the resulting protein was decomposed in 50 mM ammonium bicarbonate under the following conditions:
  • thermolysin (f) Decompose with thermolysin at 37°C for 18 hours.
  • the protein sample was diluted and acidified with 0.1% FA for MS analysis.
  • the full MS scan is performed in the range of m/z 300-2000, and the 10 most intense ions in the MS scan are fragmented to obtain MS/MS spectra.
  • the toxin protein is mainly composed of five different size protein molecules: 71kDa, 17kDa, 34kDa, 138kDa and 149kDa[ table 3].
  • LC-MS/MS liquid chromatography tandem mass spectrometry
  • SEC-MALS analysis is a combination of size exclusion high performance liquid chromatography (SEC-HPLC) and static light scattering analysis, using Agilent 1260HPLC system, and online connected to WYATT_DAWN containing SRT SEC-300 (5 ⁇ m, 7.8 ⁇ 300mm) column HELEOS static light scattering detector.
  • the Agilent 1260 HPLC system was controlled using Chemstation software (Agilent Technologies). Use ASTRA V software (Wyatt Technology) to control and analyze the light scattering detector. 50 ⁇ g of OBI-858 crude drug was dissolved in 100 ⁇ L of 0.15M sulfate buffer (pH 6.0), and then injected into the sample.
  • Figure 1 shows the purity of the product detected by SEC-HPLC. The purity of the product reaches 99.36%.
  • Figure 2 connects the refractive index and multi-angle laser light scattering (MALS) to detect the product with a molecular weight close to 760kDa.
  • MALS multi-angle laser light scattering
  • OBI-858 bulk drug (test product) and L-PTC/A (reference product) dissolved in 10 ⁇ L of deionized distilled water (DDW) to prepare negatively stained samples.
  • DSW deionized distilled water
  • 4 ⁇ L 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.
  • TEM transmission electron microscope
  • Figure 3 shows the measurement of the total protein molecular weight of the product by electron microscopy.
  • Figure 3a shows the appearance of OBI-858 toxin particles (pointed by the arrow), and
  • Figure 3b shows the comparison with the reference product of L-PTC/A with a molecular weight of 760kDa.
  • the arrows indicate four different directions.
  • the above test results all show that OBI-858 is a toxin protein close to 760kDa, so the total protein molecular weight of this product is also different from the 900kDa botulinum toxin product on the market.
  • 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), and the safety margin was 72 hours IM LD50 and 48 hours IM
  • DAS assay Digit Abduction Scoring assay
  • Test article Reference article, vehicle and control
  • Dilution buffer (30 mM phosphate buffered saline, containing 0.2% w/v gelatin, pH 6.8) was used as a vehicle for formulation and a negative control during administration.
  • 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.
  • test product is packaged as a lyophilized material in a sterile sealed vial.
  • 1 mL of dilution buffer was injected into the vial containing the test article. Shake the vial vigorously to dissolve the test article until the particulate matter in the reconstituted material is invisible to the naked eye.
  • the reserve concentration of the reconstituted test article is 1,350440 LD50 units (U) per milliliter. Then, perform serial dilutions with the reconstituted test product according to Table 4 below:
  • Table 4 Test product dilution scheme used for dosage formulation preparation
  • the reference product is packaged as a lyophilized material in a sterile sealed vial.
  • 0.2 mL of dilution buffer was injected into the vial containing the test product.
  • the vial is then shaken vigorously to dissolve the test article until the particles in the reconstituted material are invisible 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 reconstructed reference product according to the following table:
  • the dosage formulation was prepared from the reconstituted reference product by the above serial dilution scheme.
  • To prepare each serial dilution transfer the required volume of dilution buffer via a micropipette to a fresh glass vial with a 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 repeated gentle pipetting several times, then repeatedly gently invert, tighten the cap on the vial several times. At the completion of each transfer step, discard the used micropipette tips.
  • raw materials 1 to 11 are used as reference dosage preparations.
  • mice were used 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 the 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 and reference products. According to established procedures and previous research results, female mice were selected as the test system for this study.
  • 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 (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 light/12 hours dark; feed: high-temperature sterilized Rodent Diet 5010( Nutrition International, Inc., MO, USA), freely available. Water: provided freely. Analyze food and water regularly. The results of the food and water analysis are kept in the archives of the testing facility. It is expected that no contaminants will be present in food and water.
  • Animal adaptation and selection Before dosing, the animals are quarantined at the testing facility for 2 days and allowed to acclimatize for 3-4 days. At least one day before dosing, the animals were randomly divided into study groups in the study design (groups 1-12, each with 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). Unassigned animals 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 allocated animal will be replaced with a spare animal. After the first day, spare animals were excluded from the study.
  • c The dose of the dilution buffer used in the first group of animals (negative control group).
  • 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.
  • test article 0 On the administration day of each batch (day 0), 3 animals were randomly selected from the spare animal library. Separately, a single vial of the test article (material 0) was reconstituted, and a part of the material 0 was diluted 1.6 times. Then 0.1 mL of the reconstituted and diluted test article was injected intravenously into the selected test animal through the tail vein. When the integrity of the test article is confirmed by the death of at least two test animals administered intravenously within 1 hour after the 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.
  • the route of administration of the test article intramuscular injection into the right gastrocnemius muscle.
  • IM ED50 intramuscular median effective dose
  • IM LD50 intramuscular median lethal dose
  • Frequency of administration single dose.
  • control injection procedure is the same as the test article injection procedure.
  • 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.
  • DAS Toe Abduction Score
  • the muscle weakness of the study animals was observed 24-26, 48-50, and 72-74 hours after the first animal was administered on day 0 of each batch.
  • the muscle weakness was scored by DAS analysis.
  • the DAS measurement method was performed according to the following US Patent Application Publication No. US 2010/0168023 A1.
  • Muscle paralysis was measured using the toe abduction score (DAS) measurement method, such as Aoki, KR "A comparison of the safety margins of botulinum neurotoxin serotypes A, B, and F in mice", Toxicon 2001; 39(12): 1815- Described in 1820.
  • DAS toe abduction score
  • mice 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 0-4), 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:
  • 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 Statistical software for analysis. The safety margin of each batch was calculated based on the IM ED50 (DAS) at 48 hours after the administration and the IM LD50 at 72 hours after the administration. The acceptance criterion for regression analysis is R 2 >0.9.
  • Computer system Online data collection system (version 6.3.2; Xybion Medical Systems Corporation) is used to capture and analyze weight data in test facilities.
  • batch batch number Average survival time (number of 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
  • Body weight For each batch, the body weight of a single dose group is within ⁇ 10% of their average body weight. Animals weighing more than 24 grams were replaced.
  • the calculation method of the margin of safety is: the ratio of 72-hour IM LD50 to 48-hour IM ED50.
  • MOS values of OBI-858 were 14.07, 10.32, 10.33, and 9.29.
  • the MOS values are 6.20, 7.58, 8.98 and 6.73 respectively.
  • OBI-858's MOS is always higher than [Table 16].
  • mice Eight female ICR mice (body weight 18-22 grams) were injected with 0.1 mL OBI-858 intravenously for lethal dose (LD50/mL) test.
  • the lyophilized OBI-858 crude drug was dissolved in four preparations, stored at 4°C for one day, then stored at 45°C for 45 days, and allowed to stand at room temperature for 1 hour before intravenous injection in mice.
  • 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.
  • 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) for seven days, and kept at room temperature. Leave it for 1 hour and then implement intravenous injection in mice.
  • 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.

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Abstract

本发明涉及A型肉毒杆菌毒素复合物、其配制剂和使用方法。本发明提供了一种A型肉毒杆菌毒素复合物，其包含HA70组分、HA17组分、HA33组分、NTNH组分和BoNT/A1组分，其中所述肉毒杆菌毒素复合物具有740-790kDa的分子量。与现有的肉毒杆菌毒素复合物相比，本发明的肉毒杆菌毒素复合物的分子量更小，安全性更高，且具有相当的治疗效果。

Description

A型肉毒杆菌毒素复合物、其配制剂和使用方法 技术领域
本发明涉及蛋白质美容用品或药物领域，具体涉及肉毒杆菌毒素复合物。
背景技术
皱纹是衰老的一个指示性标志，可以由对皮肤的环境损害积累的生物化学、组织学和生理学变化引起。另外，其它次要因素也可以引起脸部皱纹的特征性褶、沟和皱(Stegman et al.,The Skin of the Aging Face Cosmetic Dermatological Surgery,2nd ed.,St.Louis,Mo.:Mosby Year Book:5-15(1990))。这些次要因素包括恒定的重力拉力、皮肤上的频繁且恒定的位置压力(例如，在睡眠期间)和由面部肌肉收缩引起的重复面部运动(Stegman et al.,The Skin of the Aging Face Cosmetic Dermatological Surgery,2nd ed.,St.Louis,Mo.:Mosby Year Book:5-15(1990))。
已经使用了不同的技术以潜在缓解一些衰老的体征。这些技术的范围包括从含有α-羟基酸和视黄醇的面部保湿剂到外科手术和神经毒素的注射。例如，在1986年，Jean等人开发了一种使用A型肉毒毒素治疗眉间区域运动相关皱纹的方法(Schantz and Scott,In Lewis G.E.(Ed)Biomedical Aspects of Botulinum,New York:Academic Press,143-150(1981))。A型肉毒毒素处理皱纹的这种用途导致1992年该方法的发表(Schantz and Scott,In Lewis G.E.(Ed)Biomedical Aspects of Botulinum,New York:Academic Press,143-150(1981))。直到1994年，又报道了脸上的其它与运动相关的皱纹的处理(Scott,Opthalmol,87:1044-1049(1980))。这导致了使用A型肉毒毒素进行美容医疗时代的到来。
据报道A型肉毒毒素是人类已知的最致死的天然生物制剂。肉毒梭菌的孢子存在于土壤中并可在不适当消毒且密封的食品容器中生长。由细菌的摄食引起的肉毒中毒可能是致命的。肉毒毒素通过抑制乙酰胆碱穿过神经肌肉连接处的释放来阻止突触传递，从而起到产生肌肉瘫痪的作用，并且认为肉毒毒素也以其它方式起作用。其作用实质上阻断了通常 会引起肌肉痉挛或收缩，导致瘫痪的信号。在过去的十年中，肉毒毒素的肌肉麻痹活性被用来达到各种治疗效果。肉毒毒素的控制施用已经用于提供肌肉麻痹以治疗各种医学病症，例如以骨骼肌过度活跃为特征的神经肌肉病症。用肉毒毒素治疗的病症包括偏侧面肌痉挛、成人发作痉挛性斜颈、肛裂、睑痉挛、脑瘫、颈椎张力障碍、偏头痛、斜视、颞颌关节病症和各种类型的肌肉抽筋和痉挛。最近，肉毒毒素的肌肉麻痹作用已经应用于治疗和美容的面部应用，例如皱纹和面部肌肉痉挛或收缩后果的治疗。
除了A型肉毒毒素形式外，还有7种血清学上不同的肉毒毒素形式，它们也由革兰氏阳性菌肉毒梭菌产生。在这八种血清学上不同类型的肉毒毒素中，可导致瘫痪的七种血清型被命名为肉毒毒素血清型A、B、C、D、E、F和G，这些中的每一种通过用类型特异性抗体中和来区分。每种肉毒毒素蛋白的分子量约为150kD。由于肉毒毒素的分子大小和分子结构，它不能穿过角质层和皮肤结构下部的多层。肉毒毒素的不同血清型在不同动物物种中引起的瘫痪的作用和严重程度和持续时间方面不同。例如，在大鼠中，已经确定A型肉毒毒素的效力是B型肉毒毒素的500倍，如通过瘫痪率测量的。另外，已经确定B型肉毒毒素在灵长类动物中在480U/kg剂量下是无毒的。
由肉毒梭菌细菌释放的肉毒毒素是含有约150kD肉毒毒素蛋白分子和相关非毒素蛋白的毒素复合物的组分。认为这些内源性非毒素蛋白包括血凝素蛋白(HA)家族，以及非血凝素蛋白(NTNH)。认为非毒素蛋白稳定毒素复合物中的肉毒毒素分子并当毒素复合物被摄取时保护其不被消化酸变性。因此，当毒素复合物经胃肠道给药时，毒素复合物的非毒素蛋白保护肉毒毒素的活性，从而增强全身渗透。另外，认为一些非毒素蛋白特异性稳定血液中的肉毒毒素分子。
毒素复合物中非毒素蛋白的存在通常导致毒素复合物的分子量大于裸肉毒毒素分子的分子量，如前所述，其约为150kD。例如，肉毒梭菌细菌可产生分子量约900kD、500kD或300kD的A型肉毒毒素复合物。B型和C型肉毒毒素以分子量为约700kD或约500kD的复合物的形式产生。D型肉毒毒素以分子量约为300kD或500kD的复合物的形式产生。E型和F型肉毒 毒素仅作为分子量约300kD的复合物产生。
为了对肉毒毒素提供额外的稳定性，毒素复合物通常在制造过程中通过将复合物与白蛋白结合来稳定。例如，
Figure PCTCN2020082450-appb-000001
(Allergan,Inc，Irvine，CA)是一种含有肉毒毒素的制剂，其含有100U A型肉毒杆菌毒素和辅助蛋白、0.5mg人白蛋白和0.9mg氯化钠。白蛋白用于在不同的环境中结合和稳定毒素复合物，包括与制造、运输、储存和给药相关的环境。
然而，目前可用的A型毒肉毒杆菌素复合物数目有限。本领域中仍然需要新的A型肉毒杆菌毒素复合物及其配制剂。
发明内容
鉴于本领域中需要更多的A型毒素复合物，发明人提供了一种新的A型肉毒杆菌毒素复合物。与现有的产品(例如
Figure PCTCN2020082450-appb-000002
)相比，本发明的A型肉毒杆菌毒素复合物分子量更小，因此生产成本更小；并且稳定性和安全性更高。
在一方面，本发明提供了一种A型肉毒杆菌毒素复合物，其包含HA70组分、HA17组分、HA33组分、NTNH组分和BoNT/A1组分，其中所述肉毒杆菌毒素组合物具有740-790kDa的分子量。
在一个实施方案中，A型肉毒杆菌毒素复合物由HA70组分、HA17组分、HA33组分、NTNH组分和BoNT/A1组分组成，并且具有约760kDa的分子量。
在一个实施方案中，HA70组分包含下列任一项：
(1)SEQ ID NO.1的氨基酸序列；或
(2)具有SEQ ID NO.1中添加、缺失、取代或插入一个或多个氨基酸得到的氨基酸序列，且保留HA70组分的功能。
在一个实施方案中，HA17组分包含下列任一项：
(1)SEQ ID NO.2的氨基酸序列；或
(2)具有SEQ ID NO.2中添加、缺失、取代或插入一个或多个氨基酸得到的氨基酸序列，且保留HA17组分的功能。
在一个实施方案中，HA33组分包含下列任一项：
(1)SEQ ID NO.3的氨基酸序列；或
(2)具有SEQ ID NO.3中添加、缺失、取代或插入一个或多个氨基酸得到的氨基酸序列，且保留HA33组分的功能。
在一个实施方案中，NTNH组分包含下列任一项：
(1)SEQ ID NO.4的氨基酸序列；或
(2)具有SEQ ID NO.4中添加、缺失、取代或插入一个或多个氨基酸得到的氨基酸序列，且保留NTNH组分的功能。
在一个实施方案中，BoNT/A1组分包含下列任一项：
(1)SEQ ID NO.5的氨基酸序列；或
(2)具有SEQ ID NO.5中添加、缺失、取代或插入一个或多个氨基酸得到的氨基酸序列，且保留BoNT/A1组分的功能。
在另一个方面，本发明提供了核酸，其编码本发明的A型肉毒杆菌毒素复合物。
在另一个方面，本发明提供了一种药物组合物，其包含本发明的A型肉毒杆菌毒素复合物和药学上或皮肤学上可接受的载体。
在又一个方面，本发明提供了一种液体配制剂，其包含权利要求1-6中任一项的A型肉毒杆菌毒素复合物，其组成包含人类血清白蛋白、甲硫氨酸、精氨酸、氯化钠、Tween 80、柠檬酸盐缓冲液、及/或磷酸盐缓冲液。
在又一个方面，本发明提供了一种美容方法，其包括对有需要的受试者施用有效量的本发明的A型肉毒杆菌毒素复合物、药物组合物或液体配制剂。
在一个实施方案中，美容方法包括减少细纹和/或皱纹的外观，特别是在脸部，或者扩大眼睛、提升嘴角、或者使上唇出现的纹路平滑，或者一般性缓解肌肉张力。
在又一个方面，本发明提供了一种治疗或预防有需要的受试者中的由乙酰胆碱突触传递或释放引起的疾病的方法，其包括对受试者施用有效量的本发明的A型肉毒杆菌毒素复合物、药物组合物或液体配制剂。
在一个实施方案中，疾病选自以骨骼肌过度活跃为特征的神经肌肉病症、神经疼痛、偏头痛、膀胱活动过度、鼻炎、鼻窦炎、痤疮、张力障碍、张力障碍性收缩、多汗症、胆碱能神经系统控制的一个或多个腺 体的分泌过多、偏侧面肌痉挛、成人发作痉挛性斜颈，肛裂，睑痉挛，脑瘫，颈肌张力障碍、斜视，颞颌关节病症和各种类型的肌肉抽筋和痉挛。
在又一个方面，本发明提供了本发明的A型肉毒杆菌毒素复合物、药物组合物或液体配制剂在制备美容用品或药物中的用途，所述美容用品用于减少细纹和/或皱纹的外观，特别是在脸部，或者扩大眼睛、提升嘴角、或者使上唇出现的纹路平滑，或者一般性缓解肌肉张力；所述药物用于治疗或预防由乙酰胆碱突触传递或释放引起的疾病。
在一个实施方案中，疾病选自以骨骼肌过度活跃为特征的神经肌肉病症、神经疼痛、偏头痛、膀胱活动过度、鼻炎、鼻窦炎、痤疮、张力障碍、张力障碍性收缩、多汗症、胆碱能神经系统控制的一个或多个腺体的分泌过多、偏侧面肌痉挛、成人发作痉挛性斜颈，肛裂，睑痉挛，脑瘫，颈肌张力障碍、斜视，颞颌关节病症和各种类型的肌肉抽筋和痉挛。
在又一个方面，本发明提供了A型肉毒杆菌毒素复合物、药物组合物或液体配制剂，用于减少细纹和/或皱纹的外观，特别是在脸部，或者扩大眼睛、提升嘴角、或者使上唇出现的纹路平滑，或者一般性缓解肌肉张力；所述药物用于治疗或预防由乙酰胆碱突触传递或释放引起的疾病。
在一个实施方案中，疾病选自以骨骼肌过度活跃为特征的神经肌肉病症、神经疼痛、偏头痛、膀胱活动过度、鼻炎、鼻窦炎、痤疮、张力障碍、张力障碍性收缩、多汗症、胆碱能神经系统控制的一个或多个腺体的分泌过多、偏侧面肌痉挛、成人发作痉挛性斜颈，肛裂，睑痉挛，脑瘫，颈肌张力障碍、斜视，颞颌关节病症和各种类型的肌肉抽筋和痉挛。
本发明的A型肉毒杆菌毒素复合物(在本文中也称为“OBI-858”)，与市售产品
Figure PCTCN2020082450-appb-000003
相比较具有以下优点：
(1)其治疗效果在小鼠动物实验中(有效剂量Effective Dose:ED50)与
Figure PCTCN2020082450-appb-000004
的治疗效果相当，但是在小鼠动物实验中其致死剂量(Lethal Dose:LD50)比
Figure PCTCN2020082450-appb-000005
的致死剂量更高，并且在小鼠动物实验中其安 全边际量(safety margin)(MOS)亦比
Figure PCTCN2020082450-appb-000006
更高，表明本发明可生产出比市售产品
Figure PCTCN2020082450-appb-000007
安全性更高的肉毒杆菌毒素制剂。
(2)本发明的A型肉毒杆菌毒素复合物的总蛋白质分子量为760kDa，与
Figure PCTCN2020082450-appb-000008
分子量900kDa不同，是一种新的A型肉毒杆菌毒素复合物。
(3)本发明的A型肉毒杆菌毒素复合物的配制剂具有改善的稳定性。
附图说明
图1：分子排阻高效液相色谱(SEC-HPLC)检测制品纯度。
图2：连接折射率和多角度激光散射(MALS)检测制品分子量。
图3：电子显微镜(Electron microscopy)测定产品总蛋白质分子量。(a)OBI-858，箭头指示毒素颗粒；和(b)760kDa L-PTC/A的参考品研究，箭头指示四种不同的方向。
图4：DAS测定法的评分图。
具体实施方式
本发明涉及A型肉毒杆菌毒素复合物以及其液体配制剂。如本文所述，本发明的组合物可用作向受试者提供肉毒毒素的可注射应用，用于各种治疗，美容和/或化妆目的。与现有A型肉毒杆菌毒素复合物产品相比，本发明的A型肉毒杆菌毒素复合物具有更好的安全性且增强或相当的效力。针对本发明的A型肉毒杆菌毒素复合物，本发明提供了用于该A型肉毒杆菌毒素复合物的液体配制剂，提供优异的稳定性。
如本文中使用，术语“A型肉毒杆菌毒素复合物”指约150kD的A型肉毒杆菌毒素蛋白分子以及相关的内源性非毒素蛋白，即血凝素蛋白(HA)和非毒素非血凝素蛋白(NTNH)形成的复合物。血凝素蛋白由70、33和17kDa的子组分，即HA70、33和17组成。肉毒杆菌毒素复合物无需作为一个单一毒素源自肉毒梭菌细菌。例如，肉毒杆菌毒素或经过修饰的肉毒杆菌毒素可以重组制备，然后与非毒素蛋白组合。也可以购买重组肉毒杆菌毒素，然后与非毒性蛋白质组合。
在本文中，A型肉毒杆菌毒素复合物也可以含有如本文中描述的HA70、33、17、NTNH或BoNT/A1，或者其变体。变体是在本文中描述 的序列(例如SEQ ID No.1-5中的任一种)中添加、缺失、取代或插入一个或多个氨基酸得到的氨基酸序列。
术语“一个或几个”是指很大程度上不损害蛋白质性活性的区域的数量。由术语“一个或几个”指代的数量是例如1至100，优选地1至80，更优选地1至50，仍更优选地1至40，并且特别优选地1至30、1至20、1至10或1至5(例如1、2、3、4或5)。
下文中提供了实施例中的A型肉毒杆菌毒素复合物的各组分的序列。HA70(SEQ ID NO.1)
Figure PCTCN2020082450-appb-000009
HA17(SEQ ID NO.2)
Figure PCTCN2020082450-appb-000010
HA33(SEQ ID NO.3)
Figure PCTCN2020082450-appb-000011
NTNH(SEQ ID NO.4)
Figure PCTCN2020082450-appb-000012
BoNT/A1 (SEQ ID NO. 5)
Figure PCTCN2020082450-appb-000013
Figure PCTCN2020082450-appb-000014
本发明还涉及一种通过将有效量的本发明组合物注射到有需要的受试者或患者上来产生生物效应的方法。生物效应可以包括例如肌肉麻痹，减少分泌过多或出汗，治疗神经性疼痛或偏头痛，控制鼻炎或鼻窦炎，治疗膀胱活动过度，减少肌肉痉挛，预防或减少痤疮，减少或增强免疫反应，减少皱纹，或预防或治疗各种其它病症。
本发明的组合物为允许注射到受试者或患者(即，需要特定治疗的人或其它哺乳动物)的皮肤或上皮中的形式。术语“需要”是指包括与药物或健康相关的需要(例如，治疗涉及不希望的面部肌肉痉挛的病症)，以及化妆和主观需要(例如，改变或改善面部组织的外观)。在它们的最简单形式中，它们可以包含水性药学上可接受的稀释剂，例如缓冲盐水(例如磷酸盐缓冲盐水)。然而，组合物可包含通常在可注射药物或化妆品组合物中发现的其它成分，包括与将被施用到的组织相容的皮肤学上或药学上可接受的载体。如本文所用，术语“皮肤学上或药学上可接受的”是指本文描述的组合物或其组分适于与组织接触或用于一般患者而没有不应有的毒性、不相容性、不稳定性、过敏反应等。适当时，本发明的组合物可包含常规用于所考虑的领域，特别是化妆品和皮肤病学中的任何组分。
根据本发明的肉毒杆菌毒素复合物可以通过注射(通常使用注射器) 递送到皮肤下面的肌肉，或以产生瘫痪，产生松弛，缓解收缩，预防或缓解痉挛，减少腺输出或其它所需作用的有效量作用于皮肤内的腺结构。以这种方式局部递送肉毒毒素可以提供剂量减少，降低毒性，并且允许相对于可注射或可植入材料更精确的剂量优化以获得所需的效果。
施用本发明的肉毒杆菌毒素复合物以递送有效量的肉毒毒素。本文所用术语“有效量”是指如下的量，其足以产生所需的肌肉麻痹或其它生物学或美学效果，但其隐含地是安全量，即其足够低以避免严重副作用。所需的效果包括放松某些肌肉，例如，减少细纹和/或皱纹的外观，特别是在脸部，或者以其它方式调节脸部外观，例如扩大眼睛，抬高嘴角，或者缓解肌肉张力。肌肉张力的缓解可以在脸部或其它地方进行。本发明的肉毒杆菌毒素复合物可通过注射给药于患者，用于治疗诸如皱纹，不希望的面部肌肉或其它肌肉痉挛，多汗症，痤疮或身体其它需要缓解肌肉疼痛或痉挛的疾病。肉毒杆菌毒素复合物通过注射施用于肌肉或其它皮肤相关或其它靶组织结构。可以例如，对腿部、肩部、背部(包括下背部)、腋部、手掌、足部、颈部、脸部、腹股沟、手或足的背部、肘部、上臂、膝部、上腿、臀部、躯干、骨盆或有需要的身体的任何其它部分施用肉毒杆菌毒素复合物。
本发明的肉毒杆菌毒素复合物的给药也可用于治疗其它病症，包括任何预防乙酰胆碱突触传递或释放会带来治疗益处的病症。例如，可由本发明治疗的病况包括但不限于神经疼痛、偏头痛或其它头痛疼痛、膀胱活动过度、鼻炎、鼻窦炎、痤疮、张力障碍、张力障碍性收缩、多汗症、以及胆碱能神经系统控制的一个或多个腺体的分泌过多、以骨骼肌过度活跃为特征的神经肌肉病症。用肉毒毒素治疗的病症包括半脸痉挛，偏侧面肌痉挛、成人发作痉挛性斜颈，肛裂，睑痉挛，脑瘫，颈肌张力障碍、斜视，颞颌关节病症和各种类型的肌肉抽筋和痉挛。本发明还可用于降低或增强免疫应答，或治疗已经建议或已经进行注射肉毒杆菌毒素复合物给药的其它病症。本发明的肉毒杆菌毒素复合物可以制备以固体形式制备，例如冻干末、片剂等，并且在使用前在注射用水中重建以供使用。
本发明的肉毒杆菌毒素复合物、组合物或配制剂由医师或其它保健 专业人员给药或在医师或其它保健专业人员指导下给药。它们可以在一次治疗中或在一系列治疗中随时间施用。在优选的实施方案中，在需要与肉毒毒素相关的作用的一个或多个位置注射根据本发明的肉毒杆菌毒素复合物、组合物或配制剂。由于肉毒杆菌毒素复合物的性质，肉毒毒素优选以产生所需结果而不产生任何不利或不希望的结果的量，施用速率和频率施用。
在本文中，配制剂包含人类血清白蛋白、甲硫氨酸、精氨酸、氯化钠、Tween 80、柠檬酸盐缓冲液、及/或磷酸盐缓冲液。优选地，配制剂包含人类血清白蛋白、氯化钠以及溶解于50mM柠檬酸盐缓冲液的Tween 80。在配制剂中，人类血清白蛋白的浓度可以为0.1-1，例如0.2、0.3、0.4、0.5、0.6、0.7、0.8或0.9mg/mL。氯化钠的浓度可以为5-20，例如6、7、8、9、10、11、12、13、14、15、16、17、18、或19mg/mL，更优选为11.25mg/mL。溶解于50mM柠檬酸盐缓冲液的Tween 80的浓度可以为0.1-0.3，例如0.2mg/mL，更优选为0.188mg/mL。配制剂具有一定的pH值，例如4-8，优选地pH 5.6。
在下文中，通过参考下述实施例来更为详细描述本发明构思。然而，这些实施例仅用于例示性目的，而不意图限制本发明构思的范围。
实施例
实施例1：OBI-858的制备过程
(1)OBI-858原料药制备过程:
将细胞库的冷冻管中的OBI-858细菌复苏，并且使用培养基逐步扩充，然后接种到生产发酵罐。完成细胞培养后，通过过滤除去细胞和细胞碎片。纯化步骤包括两步沉淀、缓冲液交换和色谱步骤。色谱步骤后，通过0.22μm滤器将产物过滤到原料药容器，然后冻干以得到OBI-858原料药。
(2)药物产品(制剂)制备过程：
用配制缓冲液重建冻干的OBI-858原料药粉末。重建后的溶液通过0.22μm滤器进行灭菌，并且转移到无菌隔离器中，然后冻干以获得OBI-858药物产品。
实施例2：OBI-858的鉴定
(1)基因组序列比对
OBI-858生产用菌种为自主筛选发展之A型肉毒梭菌株，经全基因组定序确认为98.28％相近于A型肉毒梭菌3502株，89.69％相近于A型肉毒梭菌Hall株[表1]，因此在菌种基因分析上有别于目前市面上发售之产品。
表1：肉毒杆菌菌株基因组序列比对
肉毒杆菌菌株 基因组序列似度(％)
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)蛋白组成分析
实验材料/流程：
为了实现最高的序列覆盖，用不同的酶分解OBI-858蛋白样品，然后进行LC-MS/MS分析。OBI-858蛋白首先用6M尿素变性，用10mM DTT于37℃还原1小时，在黑暗中于室温用50mM IAM烷基化30分钟。得到的蛋白质在50mM碳酸氢铵中在以下条件下进行分解：
(a)于37℃用胰蛋白酶分解18小时；
(b)于37℃用Glu-C蛋白酶分解18小时；
(c)于37℃用胰蛋白酶分解+于37℃用Glu-C蛋白酶分解18小时；
(d)于37℃用Lys-C分解18小时；
(e)于37℃用胰凝乳蛋白酶分解18小时；
(f)于37℃用嗜热菌蛋白酶分解18小时。
分解后，稀释蛋白质样品，并且用0.1％FA酸化以进行MS分析。
用与Ultimate 3000RSLC system(Dionex)偶联的Q Exactive质谱仪(Thermo Scientific)分析样品。使用C18柱(Acclaim PepMap RSLC,75μm x150mm,2μm,
Figure PCTCN2020082450-appb-000015
)通过以下所示的梯度进行LC分离。
表2：液相色谱串联质谱法(LC-MS/MS)分析程式
Figure PCTCN2020082450-appb-000016
流动相A:0.1％甲酸
流动相B:99％乙腈+0.1％甲酸
以m/z 300-2000的范围进行全MS扫描，并且MS扫描中10个最强烈的离子进行碎裂处理以得到MS/MS谱。通过Proteome Discoverer 1.4进行Mascot数据库搜索将原始数据加工成峰列表。
实验结果：
在液相色谱串联质谱法(LC-MS/MS)分析OBI-858A型肉毒毒素氨基酸测序结果显示，本毒素蛋白主要是由71kDa、17kDa、34kDa、138kDa与149kDa五种不同大小蛋白质分子组成[表3]。对这五种不同的蛋白质进行商业测序，分别得到SEQ ID NO.1-5的氨基酸序列。具体地，HA70组分的氨基酸序列为SEQ ID NO.1，HA17组分的氨基酸序列为SEQ ID NO.2，HA33组分的氨基酸序列为SEQ ID NO.3，NTNH组分的氨基酸序列为SEQ ID NO.4，以及BoNT/A1组分的氨基酸序列为SEQ ID NO.5。
表3：根据测序序列计算的理论数值
Figure PCTCN2020082450-appb-000017
(3)分子排阻高效液相色谱(SEC-HPLC)检测制品纯度与连接折射率和分子排阻多角度激光散射(SEC-MALS)检测制品分子量
实验材料/流程：
SEC-MALS分析是组合的大小排阻高效液相色谱(SEC-HPLC)和静态光散射分析，使用Agilent 1260HPLC系统进行，并且在线连接到含有SRT SEC-300(5μm,7.8×300mm)柱的WYATT_DAWN HELEOS静态光散射检测器。使用Chemstation软件(Agilent Technologies)控制Agilent 1260HPLC系统。使用ASTRA V软件(Wyatt Technology)控制和分析光散射检测器。将50μg OBI-858原料药溶解于100μL 0.15M硫酸盐缓冲液(pH 6.0)，然后注入样品。使用流动相：0.15M磷酸盐缓冲液+0.1M NaCl和200ppm NaN 3；pH 6.0以0.5mL/min的流速进行等度洗脱。以280nm的UV吸光度监测光散射实验期间的OBI-858复合物浓度。使用比折光指数增量(specific refractive index increment)(dn/dc)值0.185mL/g和UV消光系数1.49mL/(mg×cm)从光散射数据计算OBI-858复合物的分子量。
实验结果：
图1显示以分子排阻高效液相色谱(SEC-HPLC)检测制品纯度，本制品的纯度达到99.36％,图2连接折射率和多角度激光散射(MALS)检测制品分子量为接近760kDa的蛋白。
(4)电子显微镜(Electron microscopy)测定
实验材料/流程：
使用10μL去离子蒸馏水(DDW)中溶解的25μg OBI-858原料药(测试品)和L-PTC/A(参考品)进行负染色的样品制备。首先，将4μL OBI-858样品溶液液滴吸附到辉光放电的碳涂覆的铜栅格，用去离子水逐滴清洗,用2％醋酸铀酰染色，并且风干。使用JEM-2100F透射电子显微镜(TEM)记录样品图像，该透射电子显微镜装备有场发射电子源并且以200kV的加速电压操作。在DE12照相机上记录图像。
实验结果：
图3显示以电子显微镜(Electron microscopy)测定产品总蛋白质分子量，图3a显示OBI-858毒素颗粒外型(箭头指示处),图3b显示和分子量760kDa的L-PTC/A的参考品做比对，箭头指示分别朝向四种不同的方向。以上试验结果皆显示OBI-858为接近760kDa的毒素蛋白，因此本制品在总蛋白质分子量也有别于市面上900kDa的A型肉毒毒素产品。
实施例3：OBI-858功效性研究(Efficacy Study)
这项研究的目的是在雌性ICR小鼠中评估OBI-858测试品的单剂量急性肌内(IM)功效。通过后趾外展评分测定法(Digit Abduction Scoring assay，DAS测定法)确定OBI-858测试品的功效，以计算IM中值有效剂量(ED50)，安全边际量以72小时IM LD50与48小时IM ED50之比值来决定。
材料和方法
测试品、参考品、媒介物和对照
测试品
Figure PCTCN2020082450-appb-000018
参考品
Figure PCTCN2020082450-appb-000019
其它特征50个单位的A型肉毒杆菌毒素[每小瓶]
媒介物和对照
稀释缓冲液(30mM磷酸盐缓冲盐水，含0.2％w/v明胶，pH 6.8)用作配制制剂的媒介物和给药时的阴性对照。
剂量制剂的制备和处置
所有制剂均在预定的给药日新鲜制备以用于当日使用。
测试品重建和剂量配方
所有剂量制剂的制备程序均在位于测试设施中的II级生物安全柜中进行。在给药程序之前的给药日进行剂量制剂的制备。
将测试品作为冻干材料包装在无菌密封小瓶中。为了重建冻干的测试品，将1mL稀释缓冲液注入装有测试品的小瓶中。剧烈摇动小瓶以溶解测试品，直到肉眼看不到重建原料中的颗粒物质为止。重建的测试品的储备浓度为每毫升1350440LD50单位(U)。然后，按照下表4用重建的测试品进行系列稀释：
表4：用于剂量制剂制备的测试品稀释方案
Figure PCTCN2020082450-appb-000020
Figure PCTCN2020082450-appb-000021
为了制备每种系列稀释液，将所需体积的稀释缓冲液通过微量移液器转移至带盖的新鲜玻璃小瓶中。然后通过微量移液器将所需体积的待稀释原液转移到装有稀释缓冲液的小瓶中。通过反复轻柔移液几次进行混合，然后反复轻柔倒置，将小瓶上瓶盖拧紧几次。在每个转移步骤完成时，将用过的微量移液器吸头丢弃。剂量制剂制备完成时所得的原料5至原料15用作测试品剂量制剂。
参考品重建和剂量配方
将参考制品作为冻干材料包装在无菌密封小瓶中。为了重建冻干的参考制品，将0.2mL稀释缓冲液注入装有测试品的小瓶中。然后将小瓶剧烈摇动以溶解测试品，直到肉眼看不到重建原料中的颗粒物为止。重建的测试品的库存浓度为每毫升250LD50单位(U)。然后，按照下表用重建的参考品进行系列稀释：
表5：用于剂量制剂制备的参考品稀释方案
Figure PCTCN2020082450-appb-000022
Figure PCTCN2020082450-appb-000023
通过如上系列稀释方案由重建的参考品制备剂量制剂。为了制备每个系列稀释液，将所需体积的稀释缓冲液通过微量移液器转移至带盖的新鲜玻璃小瓶中。然后通过微量移液器将所需体积的待稀释原液转移到装有稀释缓冲液的小瓶中。通过反复轻柔移液几次进行混合，然后反复轻柔倒置，将小瓶上瓶盖拧紧几次。在每个转移步骤完成时，将用过的微量移液器吸头丢弃。剂量制剂制备完成时原料1至原料11用作参考品剂量制剂。
研究动物
以下描述了研究动物。
Figure PCTCN2020082450-appb-000024
选择理由：以小鼠为测试系统，设计了本研究中测试品和参考品的药物类别的功效和毒性确定的测定程序。雌性动物被用于该程序的开发，并且在先前的研究中还显示，雌性动物更容易受到该测试品和参考品的药物类别的药理作用和毒性的影响。根据既定程序和先前的研究结果，选择雌性小鼠作为本研究的测试系统。
环境条件
研究动物的饲养条件如下：动物在国际实验动物管理评鉴及认证协会(Association for Assessment and Accreditation of Laboratory Animal Care International)认可的动物房中在具有草垫的聚碳酸酯笼子中个别饲养。温度20.7–23.1℃，相对湿度51.2–67.1％，光周期：12小时光/12小时黑暗；饲料：高温灭菌的Rodent Diet 5010(
Figure PCTCN2020082450-appb-000025
Nutrition International,Inc.,MO,USA)，任意提供。水：任意提供。定期分析食物和水。食物和水的 分析结果保存在测试设施的档案中。预计食品和水中不会存在任何污染物。
实验设计
动物适应和选择：在给药前，将动物在测试设施处隔离2天，并使其适应3-4天。至少在给药前一天，在研究设计中将动物随机分为研究组(第1-12组，每组分别具有两个子组A和B)。每个分配的动物给四码序列号。研究组或研究组子组的平均体重之间无统计学显著性差异(p<0.05)，由单因素方差分析(ANOVA)确定。未分配的动物被指定为备用动物。如果在相应批次的第0天或之前出现健康问题[包括体重超过可接受的限制(24克)]和/或怀疑的剂量错误，则将分配的动物替换为备用动物。第1天后将备用动物排除在研究范围之外。
研究设计：剂量组、标称剂量浓度、剂量体积、标称剂量水平以及每个剂量组的动物数如下表：
表6：研究设计
Figure PCTCN2020082450-appb-000026
Figure PCTCN2020082450-appb-000027
a：有关计算出的剂量浓度的信息见上文。
b：除第13组外，实际剂量水平乘以剂量体积。
c：将稀释缓冲液用于第1组动物的剂量(阴性对照组)。
d：第13组用于验证测试品的完整性。在给药日从备用动物中选择第13组动物。
测试品完整性的验证
在每批的给药日(第0天)，从备用动物库中随机选择3只动物。单独地，重建单个小瓶的测试品(原料0)，并且将原料0的一部分稀释1.6倍。然后通过尾静脉向选择的测试动物静脉内注射0.1mL重建和稀释的测试品。当通过在给药后1小时内至少两只静脉内给药的试验动物的死亡证实了试验物品的完整性，将继续进行主要研究动物的给药。如果至少2只动物在1小时内未能死亡，则将丢弃特定小瓶的测试品原料0。在这种情况下，要从第二个测试品小瓶中制备新鲜的小瓶原料0，并用第二个小瓶的原料重复进行验证测试。如果第二个小瓶也未通过验证测试，则由于不合格的测试品效力，特定批次的剂量将被停止。由于所有测试品小瓶均通过了测试，因此在初始测试后没有进行上述后续操作。
测试品的施用途径：肌内注射右腓肠肌。
选择剂量水平和给药途径的理由
通过选择标称剂量水平，以确定预期有1个数量级差异的肌肉内中值有效剂量(IM ED50)和肌肉内中值致死剂量(IM LD50)。
施用频率：单剂量。
阴性对照施用
作为阴性对照，将20μL稀释缓冲液肌肉内注射到第1组剂量动物的右腓肠肌中。对照注射的程序与测试品注射的程序相同。
死亡率观察
在给药后第0天至少6小时观察一次动物的死亡率。此后，在整个研究期间每天观察动物的死亡率，同时进行DAS测定法(直至第3天)。对死亡率分别进行评分，并以每组为基础计数以计算LD50。
趾外展评分(DAS)测定法
对于每批，在各批次的第0天给药第一只动物后24-26、48-50和72-74小时观察研究动物的肌无力。通过DAS分析对肌无力进行评分。对于每只动物，各别由两位人员独立记录其肌无力得分。将两个单独观察的分数取平均值进行分析。DAS测定法依据以下美国专利申请公开号US 2010/0168023 A1进行。使用趾外展评分(DAS)测定法测量了肌肉麻痹，如Aoki,K.R.“A comparison of the safety margins of botulinum neurotoxin serotypes A,B,and F in mice”,Toxicon 2001；39(12):1815-1820所述。在DAS测定法中，小鼠的尾巴短暂地悬吊起来，引起特征性的惊吓反应，其中小鼠伸展其后肢并外展其后趾。小鼠能够表现出此惊吓反应的程度以五分制(从0-4)评分，其中0表正常的惊吓反应，四代表趾外展和腿伸展的最大减少。趾外展的评分基于以下说明和标准：
表7：趾外展评分(DAS)测定法的评分量表
Figure PCTCN2020082450-appb-000028
为了DAS分析的目的，在DAS研究时死亡的动物被赋予分数“4”。
体重：至少在分组前和给药前第0天测量体重一次。
统计分析：概率分析用于拟合概率和分对数S形毒素单位/DAS响应曲线，并计算IM ED50和IM LD50值。使用
Figure PCTCN2020082450-appb-000029
统计软件进行分析。根据给药后48小时的IM ED50(DAS)和给药后72小时的IM LD50计算各批次的安全边际量。回归分析的接受标准为R 2>0.9。
计算机系统：在线数据收集系统(版本6.3.2；Xybion Medical Systems Corporation)用于在测试设施中捕获和分析体重数据。
结果与讨论
OBI-858批次处理完整性的测试
下表列出了OBI-858完整性测试的概述。
表8：OBI-858完整性测试
批次 批次号 平均存活时间(给药后的分钟数)
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
测试品的所有小瓶均通过完整性测试，平均给药后存活时间少于1小时。
体重：对于每批，单个剂量组的体重范围在各自平均体重的±10％范围内。体重超过24克的动物被替换。
DAS测定法和ED50计算
OBI-858
早在给药后24小时，动物就表现出趾外展的体征。趾外展的严重程度与在更高剂量下最大严重程度(包括死亡)时达到平台期之前的OBI-858剂量增加有关。在48小时IM ED50中，除批次1外的所有样品均通过了回归分析的接受标准(R 2>0.9)。依據下表所示，对于第2、3和4批次，给药后48小时的ED50分别为0.33、0.32和0.36U/动物。
表9：测试品OBI858的48小时的ED50(U/动物)
批次 ED50 低95％ 高95％ R 2值
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
Figure PCTCN2020082450-appb-000030
早在给药后24小时，动物就表现出趾外展的体征。趾外展的严重程度与
Figure PCTCN2020082450-appb-000031
剂量的增加有关，但是对于获得的最大严重性(包括死亡)的平台期，
Figure PCTCN2020082450-appb-000032
的剂量比OBI-858的剂量小。在48小时的IM ED50中，除批次1外的所有样品均通过了回归分析的接受标准(R 2>0.9)。依據下表所示，对于第2、3和4批次，给药后48小时的ED50分别为0.22、0.24和0.23U/动物。总体而言，
Figure PCTCN2020082450-appb-000033
的48小时IM ED50低于OBI-858。
表10：
Figure PCTCN2020082450-appb-000034
的48小时的ED50(U/动物)
批次 ED50 低95％ 高95％ R 2值
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
死亡率和LD50计算
OBI-858
大多数死亡发生在给药后72小时，尽管对于第11组和第12组早在给药后48小时就观察到死亡。依據下表所示，给药后72小时，所有四个批次的LD50分别为3.00、3.42、3.33和3.35U/动物。然而，在给药后72小时，仅批次2、3和4具有在剂量相关线性范围内的足够剂量组用于回归分析，死亡百分率在0％至100％之间。(每个分析至少需要三个剂量组)。在这三个批次中，只有第2批次和第3批次通过了回归分析的接受标准。
表11：测试品OBI858的72小时的LD50(U/动物)
批次 LD50 低95％ 高95％ R 2值
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
Figure PCTCN2020082450-appb-000035
在以
Figure PCTCN2020082450-appb-000036
给药的动物中，死亡发生的时间更早(最早在给药后24小时)，且剂量低于在以OBI-858给药的动物中观察到的剂量(给药后72小时的第6组至第12组)。给药后72小时，所有四个批次的LD50分别为2.21、1.63、2.14和1.57U/动物[表13]。但是，四批死亡率结果均未通过回归分析的接受标准。依據下表所示，对于批次2、3和4，在给药后72小时，在剂量相关线性范围内没有剂量组。因此，无法对这些批次的计算得出的LD50进行回归分析。对于批次1，R 2值为0.74，低于接受标准。
表12：
Figure PCTCN2020082450-appb-000037
的72小时的LD50(U/动物)
批次 LD50 低95％ 高95％ R 2值
1 2.21 0.80 6.11 0.74
2 1.63      
3 2.14      
4 1.57      
评估体重偏差对ED50结果的影响
对于OBI-858和
Figure PCTCN2020082450-appb-000038
在给药时，在所需体重范围内(18-22克)的动物与超重(大于22克)的动物之间，在计算的ED50之间没有一致的变化趋势。与总ED50值相比，从ED50计算中排除超重动物不会导致ED50值出现一致变化，如下表所示。
表13：按体重范围比较OBI858与
Figure PCTCN2020082450-appb-000039
的IM 48小时ED50(U/动物)
Figure PCTCN2020082450-appb-000040
Figure PCTCN2020082450-appb-000041
此外，发现一只动物在给药时体重不足17.7克(低于18克阈值)。体重不足的动物[ID 0193；组9，子集
Figure PCTCN2020082450-appb-000042
第2批]在所有评分中都获得了与同一组其他动物相似的趾外展得分。因此，该事件对研究结果没有影响[表14：测试品OBI858；表15：
Figure PCTCN2020082450-appb-000043
]。
安全边际量(MOS)的估计：安全边际量的计算方法为：72小时IM LD50与48小时IM ED50的比率。
对于四批测试，OBI-858的MOS值分别为14.07、10.32、10.33和9.29。对于四批测试，
Figure PCTCN2020082450-appb-000044
的MOS值分别为6.20、7.58、8.98和6.73。OBI-858的MOS始终高于
Figure PCTCN2020082450-appb-000045
[表16]。
结论
本研究中测试的所有OBI-858小瓶均通过了完整性测试。某些给药时研究动物的体重升高(22至24克)对ED50计算的结果没有显著影响。对于OBI-858，72小时IM LD50和48小时IM ED50值分别为3.42和0.33U/动物(批次2)以及3.33和0.32U/动物(批次3)。批次2和批次3的相应MOS值分别为10.32和10.33。对于
Figure PCTCN2020082450-appb-000046
72小时IM LD50和48小时IM ED50值分别为1.63和0.22U/动物(批次2)以及2.14和0.24U/动物(批次3)。批次2和批次3的相应MOS值分别为7.58和8.98。简而言之，OBI-858的效力可与
Figure PCTCN2020082450-appb-000047
相当，但是它具有比
Figure PCTCN2020082450-appb-000048
高的72小时IM LD50和安全边际量值。
Figure PCTCN2020082450-appb-000049
Figure PCTCN2020082450-appb-000050
表16：OBI858与
Figure PCTCN2020082450-appb-000051
的安全边际值
Figure PCTCN2020082450-appb-000052
实施例4：OBI-858配制剂的稳定性测试
实验材料/流程：
(1)LF08制剂:
0.5mg/mL人类血清白蛋白,11.25mg/mL氯化钠以及0.188mg/mL Tween 80溶解于50mM磷酸盐缓冲液(pH 6.3)。
(2)LF09制剂:
0.5mg/mL人类血清白蛋白,11.25mg/mL氯化钠以及0.188mg/mL Tween 80溶解于50mM柠檬酸盐(柠檬酸钠)缓冲液(pH 5.6)。
(3)LF10制剂:
0.5mg/mL甲硫氨酸,11.25mg/mL氯化钠,0.188mg/mL Tween 80以及150mM精氨酸溶解于50mM柠檬酸盐缓冲液(pH 6.0)。
(4)LF11制剂:
11.25mg/mL氯化钠以及0.188mg/mL Tween溶解于50mM磷酸盐缓冲液(pH 6.3)。
(5)实验动物:
使用八只雌性ICR小鼠(体重18至22克)经由静脉注射0.1mL OBI-858以进行致死剂量(LD50/mL)测试。
实验结果：
(1)不同制剂稳定性试验:
将冻干后的OBI-858原料药分别于四种制剂中溶解，于4℃储放一天后置换于45℃储放四十五天，于室温下静置1小时再实施小鼠静脉注射。下表显示四种制剂的致死剂量(LD50/mL)，结果以LF09制剂的稳定 性效果最好。
表17：不同制剂稳定性试验
Figure PCTCN2020082450-appb-000053
(2)LF09制剂4-60℃稳定性加速试验:
将冻干后的OBI-858原料药分别于LF09制剂中溶解，于4℃储放一天后置换于四种温度(4℃、25℃、45℃与60℃)储放七天，于室温下静置1小时再实施小鼠静脉注射。下表显示LF09制剂的致死剂量(LD50/mL),结果显示LF09制剂在45℃环境下仍具有稳定性。
表18：LF09制剂稳定性加速试验
Figure PCTCN2020082450-appb-000054
应当理解，本文中描述的例示性的实施方案应当仅以描述意义而不是为了限制目的考虑。通常应当认为每个例示性的实施方案内的特征或方面的描述可用于其它例示性的实施方案中的其它类似的特征或方面。虽然已经参考附图描述了一个或多个例示性的实施方案，但是本领域普通技术人员应当理解可以在不背离如由所附权利要求书限定的发明构思的精神和范围的前提下对其进行形式和细节的各种变化。

Claims (21)

  1. 一种A型肉毒杆菌毒素复合物，其包含HA70组分、HA17组分、HA33组分、NTNH组分和BoNT/A1组分，其中所述肉毒杆菌毒素组合物具有740-790kDa的分子量。
  2. 根据权利要求1的A型肉毒杆菌毒素复合物，其中HA70组分包含下列任一项：
    (1)SEQ ID NO.1的氨基酸序列；或
    (2)具有SEQ ID NO.1中添加、缺失、取代或插入一个或多个氨基酸得到的氨基酸序列，且保留HA70组分的功能。
  3. 根据权利要求1的A型肉毒杆菌毒素复合物，其中HA17组分包含下列任一项：
    (1)SEQ ID NO.2的氨基酸序列；或
    (2)具有SEQ ID NO.2中添加、缺失、取代或插入一个或多个氨基酸得到的氨基酸序列，且保留HA17组分的功能。
  4. 根据权利要求1的A型肉毒杆菌毒素复合物，其中HA33组分包含下列任一项：
    (1)SEQ ID NO.3的氨基酸序列；或
    (2)具有SEQ ID NO.3中添加、缺失、取代或插入一个或多个氨基酸得到的氨基酸序列，且保留HA33组分的功能。
  5. 根据权利要求1的A型肉毒杆菌毒素复合物，其中NTNH组分包含下列任一项：
    (1)SEQ ID NO.4的氨基酸序列；或
    (2)具有SEQ ID NO.4中添加、缺失、取代或插入一个或多个氨基酸得到的氨基酸序列，且保留NTNH组分的功能。
  6. 根据权利要求1的A型肉毒杆菌毒素复合物，其中BoNT/A1组分包含下列任一项：
    (1)SEQ ID NO.5的氨基酸序列；或
    (2)具有SEQ ID NO.5中添加、缺失、取代或插入一个或多个氨基酸得到的氨基酸序列，且保留BoNT/A1组分的功能。
  7. 根据权利要求1-6中任一项的A型肉毒杆菌毒素复合物，在小鼠/动物实验中，其治疗有效剂量(ED50)包括0.1-0.5U。
  8. 根据权利要求1-6中任一项的A型肉毒杆菌毒素复合物，在小鼠/动物实验中，其致死剂量(LD50)包括1-5U。
  9. 根据权利要求1-6中任一项的A型肉毒杆菌毒素复合物，在小鼠/动物实验中，其安全边际量(MOS)包括2-50。
  10. 一种核酸，其编码根据权利要求1-6中任一项的A型肉毒杆菌毒素。
  11. 一种药物组合物，其包含权利要求1-6中任一项的A型肉毒杆菌毒素复合物和药学上或皮肤学上可接受的载体，优选地为冻干形式。
  12. 一种液体配制剂，其包含权利要求1-6中任一项的A型肉毒杆菌毒素复合物。
  13. 根据权利要求12所述的液体配制剂，其包含人类血清白蛋白、甲硫氨酸、精氨酸、氯化钠、Tween 80、柠檬酸盐缓冲液、及/或磷酸盐缓冲液。
  14. 一种美容方法，其包括对有需要的受试者施用有效量的权利要求1-6中任一项的A型肉毒杆菌毒素复合物、权利要求11的药物组合物或权利要求12或13的液体配制剂。
  15. 权利要求14的美容方法，其包括减少细纹和/或皱纹的外观，特别是在脸部，或者扩大眼睛、提升嘴角、或者使上唇出现的纹路平滑，或者一般性缓解肌肉张力。
  16. 一种治疗或预防有需要的受试者中的由乙酰胆碱突触传递或释放引起的疾病的方法，其包括对受试者施用有效量的权利要求1-6中任一项的A型肉毒杆菌毒素复合物、权利要求11的药物组合物或权利要求12或13的液体配制剂。
  17. 权利要求16的方法，其中所述疾病选自以骨骼肌过度活跃为特征的神经肌肉病症、神经疼痛、偏头痛、膀胱活动过度、鼻炎、鼻窦炎、痤疮、张力障碍、张力障碍性收缩、多汗症、胆碱能神经系统控制的一个或多个腺体的分泌过多、偏侧面肌痉挛、成人发作痉挛性斜颈，肛裂，睑痉挛，脑瘫，颈肌张力障碍、斜视，颞颌关节病症和各种类型的肌肉 抽筋和痉挛。
  18. 权利要求1-6中任一项的A型肉毒杆菌毒素复合物、权利要求11的药物组合物或权利要求12或13的液体配制剂在制备美容用品或药物中的用途，所述美容用品用于减少细纹和/或皱纹的外观，特别是在脸部，或者扩大眼睛、提升嘴角、或者使上唇出现的纹路平滑，或者一般性缓解肌肉张力；所述药物用于治疗或预防由乙酰胆碱突触传递或释放引起的疾病。
  19. 权利要求18的用途，其中所述疾病选自以骨骼肌过度活跃为特征的神经肌肉病症、神经疼痛、偏头痛、膀胱活动过度、鼻炎、鼻窦炎、痤疮、张力障碍、张力障碍性收缩、多汗症、胆碱能神经系统控制的一个或多个腺体的分泌过多、偏侧面肌痉挛、成人发作痉挛性斜颈，肛裂，睑痉挛，脑瘫，颈肌张力障碍、斜视，颞颌关节病症和各种类型的肌肉抽筋和痉挛。
  20. 权利要求1-6中任一项的A型肉毒杆菌毒素复合物、权利要求11的药物组合物或权利要求12或13的液体配制剂，用于减少细纹和/或皱纹的外观，特别是在脸部，或者扩大眼睛、提升嘴角、或者使上唇出现的纹路平滑，或者一般性缓解肌肉张力；所述药物用于治疗或预防由乙酰胆碱突触传递或释放引起的疾病。
  21. 权利要求20的A型肉毒杆菌毒素复合物、药物组合物或液体配制剂，其中所述疾病选自以骨骼肌过度活跃为特征的神经肌肉病症、神经疼痛、偏头痛、膀胱活动过度、鼻炎、鼻窦炎、痤疮、张力障碍、张力障碍性收缩、多汗症、胆碱能神经系统控制的一个或多个腺体的分泌过多、偏侧面肌痉挛、成人发作痉挛性斜颈，肛裂，睑痉挛，脑瘫，颈肌张力障碍、斜视，颞颌关节病症和各种类型的肌肉抽筋和痉挛。
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DATABASE PROTEIN 1 December 2020 (2020-12-01), ANONYMOUS: "Chain A, HA-33", XP055854952, retrieved from NCBI Database accession no. 4LO0_A *
DATABASE PROTEIN 18 July 2013 (2013-07-18), ANONYMOUS: "hemagglutinin [Clostridium botulinum]", XP055854955, retrieved from NCBI Database accession no. WP_003356711 *
DATABASE PROTEIN 19 April 2020 (2020-04-19), ANONYMOUS: "botulinum neurotoxin type A [Clostridium botulinum]", XP055854943, retrieved from NCBI Database accession no. WP_011948511 *
DATABASE PROTEIN 23 July 2021 (2021-07-23), ANONYMOUS: "botulinum neurotoxin hemagglutinin HA70 subunit [Clostridium botulinum]", XP055854960, retrieved from NCBI Database accession no. WP_011948507 *
DATABASE PROTEIN 29 August 2019 (2019-08-29), ANONYMOUS: "non-toxic nonhemagglutinin NTNH [Clostridium botulinum]", XP055854948, retrieved from NCBI Database accession no. WP_011948510 *
SCOTT, OPTHALMOL, vol. 87, 1980, pages 1044 - 1049

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023130875A1 (zh) * 2022-01-07 2023-07-13 重庆誉颜制药有限公司 一种肉毒毒素蛋白组合物
WO2023156385A1 (en) * 2022-02-15 2023-08-24 Merz Pharma Gmbh & Co. Kgaa Liquid botulinum toxin formulation and use thereof

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