Botulinum toxin protein composition and application thereof

Abstract

The invention discloses a botulinum toxin protein composition and application thereof, and particularly relates to a botulinum toxin protein composition containing L-carnitine or structural analogues thereof, which can be applied to the fields of medical treatment and beauty treatment as a liquid preparation. The composition has better stability, especially long-term stability, and is beneficial to storage of botulinum toxin protein.

Description

Technical Field

[0001] The present invention relates to the field of pharmaceutical preparations, and in particular to a botulinum toxin protein composition and application thereof, in particular to a botulinum toxin protein composition comprising L-carnitine or a structural analogue thereof. Background Art

[0002] Natural botulinum neurotoxins (BoNTs) are toxin proteins produced by anaerobic Clostridium botulinum, which cause muscle paralysis in mammals by blocking the presynaptic release of the neurotransmitter acetylcholine at the neuromuscular junction. Seven types of BoNTs have been discovered, named BoNT / A to BoNT / G. Due to their unique pharmacological properties, including high specificity for the nervous system, limited diffusion after local injection, and reversibility of action, BoNTs have rapidly developed into a class of drugs for the treatment of cholinergic hyperexcitability, with a wide range of applications in the medical and cosmetic fields.

[0003] Generally speaking, therapeutic active proteins are usually formulated in liquid solutions, especially for injections. Therapeutic active proteins are usually prepared into compositions and sold commercially as ready-to-use solutions or provided in lyophilized form reconstituted with liquid solutions. For example, BoNT / A products on the market are solid preparations that are vacuum dried or freeze-dried, and medical staff are required to reconstitute them when used, so the reconstitution volume needs to be precisely controlled. In addition, due to different therapeutic purposes and individual differences in the required doses between patients, the dose required for each treatment varies greatly. After the amount of drug contained in the market product is reconstituted according to the volume required by the manufacturer, clinicians often administer a small portion of the content in the bottle to the patient, and the rest is placed in the refrigerator for reuse. Studies have estimated that when the reconstituted BoNT / A is stored in a refrigerator (about 4°C) for 12 hours, its potency is lost by at least 44%, so in actual operation, it is generally recommended to use it within 4 hours after reconstitution, which will lead to waste of drugs and unnecessary economic losses for patients.

[0004] In addition, since botulinum toxin is used at extremely low concentrations, in order to maintain the stability of botulinum toxin protein at extremely low concentrations and avoid drug adsorption on solid surfaces, high concentrations of exogenous proteins are generally added for preparation. Most botulinum toxin formulas use albumin or gelatin as stabilizers, which not only cause immune responses, but also have the potential to introduce contamination sources. Summary of the invention

[0005] To overcome the deficiencies of the prior art, the present invention provides a botulinum toxin protein composition and a preparation method and application thereof.

[0006] In a first aspect of the present invention, a botulinum toxin protein composition is provided, comprising: a botulinum toxin protein, L-carnitine or a derivative thereof or a salt thereof.

[0007] In one embodiment of the present invention, the composition is a liquid composition, which further comprises a solvent, such as water (especially water for injection), sodium chloride injection (such as 0.9% sodium chloride injection, compound sodium chloride injection).

[0008] Specifically, the solvent is a sterile solvent, such as sterile water for injection and 0.9% sodium chloride injection.

[0009] Specifically, in the composition, the concentration of L-carnitine or its derivatives or salts thereof is 1-50 mM (e.g., 1, 2, 3, 4, 4.5, 4.8, 4.9, 5, 6, 8, 9, 10, 11, 12, 14, 15, 20, 20.1, 20.2, 20.5, 25, 30, 35, 40, 45, 50 mM), for example, 1-4.9 mM, 2-4.5 mM, 1-20 mM, 20.1-50 mM, 20.5-40 mM, 20.5-30 mM.

[0010] In one embodiment of the present invention, the composition comprises: botulinum toxin protein and L-carnitine.

[0011] In one embodiment of the present invention, the composition comprises: botulinum toxin protein and a salt of L-carnitine.

[0012] In one embodiment of the present invention, the composition comprises: botulinum toxin protein, a derivative of L-carnitine or a salt thereof.

[0013] In the composition, L-carnitine or its derivatives or their salts are used as protein stabilizers; in one embodiment, the protein stabilizer is L-carnitine; in another embodiment, the protein stabilizer is a salt of L-carnitine; in another embodiment, the protein stabilizer is a derivative of L-carnitine; in another embodiment, the protein stabilizer is a salt of an L-carnitine derivative.

[0014] In some embodiments of the present invention, the L-carnitine derivative is alkanoyl L-carnitine, whose structural formula is Wherein R is an alkanoyl group, which can be saturated or unsaturated, linear or branched, having 2-26 carbon atoms (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 14, 16, 18, 20, 22, 24, 26 carbon atoms), such as acetyl, propionyl, butyryl, isobutyryl, valeryl, isovaleryl. In one embodiment of the invention, the alkanoyl L-carnitine is O-acetyl-L-carnitine.

[0015] Specifically, the salt of L-carnitine or its derivative is preferably a pharmaceutically acceptable salt, which is any salt of L-carnitine or its derivative with an acid that does not produce toxic side effects, including, but not limited to, salts thereof with inorganic acids, for example, hydrochlorides, nitrates, phosphates, sulfates, hydrobromides, hydroiodides, salts thereof with inorganic acids such as aliphatic monocarboxylic acids and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, alkanedioic acids, aromatic acids and aliphatic and aromatic sulfonic acids, for example, sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogen phosphates, dihydrogen phosphates, metaphosphates, pyrophosphates, Phosphate, hydrochloride, hydrobromide, iodate, acetate, propionate, octanoate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, tartrate, methanesulfonate, gluconate, galacturonate, mucate, glucose phosphate, trichloroacetate, trifluoroacetate, aspartate, fumarate, glycerophosphate, methanesulfonate, choline tartrate, etc. In some embodiments of the present invention, the salt is tartrate, hydrochloride, citrate, succinate, fumarate, etc.

[0016] In one embodiment of the present invention, the salt is hydrochloride.

[0017] In one embodiment of the present invention, the salt is tartrate.

[0018] Generally speaking, proteins are easily degraded and inactivated at low concentrations (<0.1 mg / ml). The present invention finds that L-carnitine or its derivatives (such as alkanoyl L-carnitine) or its salts (such as tartrate, hydrochloride) can make the formula have the characteristics of low viscosity and can reduce the aggregation and precipitation of botulinum toxin protein, which is beneficial to maintaining the stability of botulinum toxin protein.

[0019] Specifically, the botulinum toxin protein can be selected from any one of: type A botulinum toxin protein, type B botulinum toxin protein, type C1 botulinum toxin protein, type C2 botulinum toxin protein, type D botulinum toxin protein, type E botulinum toxin protein, type F botulinum toxin protein or type G botulinum toxin protein, in particular type A botulinum toxin protein.

[0020] In one embodiment of the present invention, the botulinum toxin is a recombinant botulinum toxin, in particular a recombinant botulinum toxin type A, such as the toxin polypeptide described in WO2022 / 179552A1, in particular the dimer BoNT / A prepared in the examples thereof.

[0021] Specifically, in the composition, the concentration of botulinum toxin protein can be 1-1000 U / mL (e.g., 1, 10, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1000 U / mL), especially 50-200 U / mL.

[0022] In one embodiment of the present invention, the composition further comprises a solubilizer.

[0023] Specifically, in the composition, the volume percentage concentration of the solubilizer is 0-0.1% (eg, 0.01%, 0.02%, 0.05%, 0.1%), for example, 0.01-0.1%.

[0024] Specifically, the solubilizing agent is selected from one or more of Tweens, Spans, sodium lauryl sulfate, and poloxamers.

[0025] More specifically, the Tween solubilizing agent is selected from one or more of Tween 20, Tween 40, Tween 60, or Tween 80.

[0026] More specifically, the Span-type solubilizing agent is selected from one or more of Span 20, Span 40, Span 60, Span 80, or Span 85.

[0027] In some embodiments of the present invention, the solubilizing agent is Tween 80.

[0028] In one embodiment of the present invention, the composition is a liquid composition, which comprises water (especially water for injection) as a solvent, and the composition further comprises an osmotic pressure regulator.

[0029] Specifically, the osmotic pressure regulator is selected from: sodium chloride, glucose, glycerol, fructose, sorbitol, magnesium chloride, phosphate, sodium citrate, mannitol, especially sodium chloride.

[0030] Specifically, in the composition, the mass percentage concentration of the osmotic pressure regulator is 0.6%-0.9% (e.g. 0.6%, 0.7%, 0.8%, 0.9%); preferably, the osmotic pressure regulator is an isotonic regulator, and its dosage is based on adjusting the osmotic pressure of the composition to be equal to or close to the osmotic pressure of plasma.

[0031] In some embodiments of the present invention, the osmotic pressure regulator is sodium chloride, and its mass percentage concentration in the composition is 0.9%.

[0032] Specifically, the composition may comprise: botulinum toxin protein, a protein stabilizer (L-carnitine or its derivatives or their salts), a solubilizer, and an osmotic pressure regulator, wherein the concentration of L-carnitine or its derivatives or their salts is 1-50 mM, the volume percentage concentration of the solubilizer is 0-0.1%, and the osmotic pressure regulator is an isotonic regulator; wherein the selection of each component is as described above in the present invention.

[0033] More specifically, the composition comprises: botulinum toxin protein, 2-10 mM protein stabilizer (L-carnitine or its derivatives or salts thereof), 0-0.1% Tween 80, and 0.9% sodium chloride.

[0034] In some embodiments of the present invention, the composition comprises: botulinum toxin protein, 10 mM protein stabilizer (L-carnitine or its derivatives or salts thereof), 0.1% Tween 80, and 0.9% sodium chloride.

[0035] Specifically, the pH of the composition is 5.0-8.0 (e.g., 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0).

[0036] In one embodiment of the present invention, the composition further comprises a second (cosmetic) active ingredient, such as one or more of hyaluronic acid (HA) or its salts, polylactic acid, polycaprolactone, collagen, etc., to achieve a combined effect (e.g., shrinking pores, brightening skin tone).

[0037] In one embodiment of the present invention, the composition further comprises a local anesthetic, such as lidocaine, bupivacaine, ropivacaine, tetracaine, and the like.

[0038] Specifically, the composition may further include one or more auxiliary materials such as a co-solvent, a pH adjuster, an antioxidant, a buffer, etc.

[0039] In a second aspect of the present invention, a method for preparing the composition of the first aspect is provided, which comprises the step of mixing the components.

[0040] In one embodiment of the present invention, the composition is a liquid composition, which further comprises a solvent, and the preparation method comprises the following steps:

[0041] (1) mixing a solvent, L-carnitine or its derivatives or salts thereof (optionally, a solubilizer, and, if necessary, other components);

[0042] (2) diluting the botulinum toxin protein with the mixture obtained in step (1);

[0043] Optionally, (3) the mixture obtained in step (2) is divided into several packages and stored.

[0044] In some embodiments of the present invention, the solvent is 0.9% sodium chloride injection, and step (1) comprises: mixing the solvent, L-carnitine or its derivatives or their salts, and a solubilizing agent.

[0045] Specifically, the dilution in step (2) can be one or more consecutive dilutions to a desired concentration.

[0046] Specifically, the packaging in step (3) can be carried out using vials.

[0047] Specifically, the storage temperature in step (3) is low temperature, especially 2-8°C (e.g. 2, 4, 6, 8°C).

[0048] In the third aspect of the present invention, there is provided use of the composition of the first aspect in the preparation of a medicament for preventing and / or treating a disease.

[0049] In one embodiment of the invention, the disease is associated with overactive neurons or glands, such as neuromuscular disease, autonomic disorders, pain.

[0050] More specifically, the diseases include: spasmodic dystonia, spasmodic torticollis, laryngeal dystonia, oromandibular dystonia, tongue dystonia, cervical dystonia, focal dystonia, blepharospasm, strabismus, hemifacial spasm, eyelid disorder, cerebral palsy, focal spasm and other language disorders, spastic colitis, neurogenic bladder, pelvic floor dystonia, limb spasm, convulsions, tremors, bruxism, anal fissures, achalasia, dysphagia and other dystonias and other disorders characterized by involuntary movements of muscle groups, tearing, sweating, excessive salivation, excessive gastrointestinal secretion, secretory disorders, pain caused by muscle spasms, headaches, etc.

[0051] In one embodiment of the invention, the disease is not associated with unwanted neuronal activity, such as psoriasis, allergies, eosinophilic lymphohistiocytosis and alcoholic pancreatic disease.

[0052] In one embodiment of the invention, the disease is a thalamus-mediated disorder, such as epilepsy, chronic pain.

[0053] More specifically, the chronic pain is selected from the group consisting of central sensitized chronic pain, central pain after stroke, regional pain, phantom limb pain and demyelinating pain.

[0054] In one embodiment of the invention, the disease is a neuropsychiatric disorder, such as schizophrenia, delirium, Alzheimer's disease, depression, mania, attention deficit disorder, drug addiction, dementia, agitation, apathy, anxiety, psychosis, personality disorder, bipolar disorder, obsessive-compulsive disorder, eating disorder, post-traumatic stress disorder, irritability and disinhibition, in particular schizophrenia, Alzheimer's disease, mania, anxiety, depression.

[0055] Specifically, the medicine can be an injection.

[0056] In a fourth aspect of the present invention, there is provided use of the composition of the first aspect in preparing a cosmetic product.

[0057] Specifically, the beauty treatment includes: removing or preventing wrinkles, slimming the face, repairing scars, improving or removing blackheads, acne, changing eyebrow shape, lifting mouth corners, body shaping (such as slimming legs and shoulders), removing body odor, hair growth and hair retention, etc., especially removing wrinkles and slimming the face.

[0058] In particular, the wrinkles can be considered as folds, ridges or creases in the skin. Preferred are wrinkles visible in the human face. Wrinkles include, but are not limited to, tear troughs, frown lines, crow's feet, cheek commissure lines, chin lines, perioral wrinkles, cheek wrinkles, marionette lines, lip lines, forehead wrinkles, frown lines, rabbit lines, nasolabial folds, under-eye wrinkles, chin wrinkles, neck wrinkles, etc., especially frown lines, crow's feet, forehead wrinkles.

[0059] Specifically, the product can be an injection.

[0060] In the fifth aspect of the present invention, a method for preventing and / or treating a disease is provided, which comprises the step of administering an effective amount of the composition of the first aspect to a subject in need thereof.

[0061] Specifically, the disease is as described in the third aspect of the present invention.

[0062] Specifically, the subject is a mammal, especially a human.

[0063] Specifically, the administration method is injection, such as subcutaneous injection, intradermal injection, and intramuscular injection.

[0064] In a sixth aspect of the present invention, a cosmetic method is provided, comprising the step of administering an effective amount of the composition of the first aspect to a subject in need thereof.

[0065] Specifically, the beauty treatment includes: removing or preventing wrinkles, slimming the face, repairing scars, improving or removing blackheads, acne, changing eyebrow shape, lifting mouth corners, body shaping (such as slimming legs and shoulders), removing body odor, hair growth and hair retention, etc., especially removing wrinkles and slimming the face.

[0066] Specifically, the subject is a mammal, especially a human.

[0067] Specifically, the administration method is injection, such as subcutaneous injection, intradermal injection, and intramuscular injection.

[0068] Compared with the prior art, the present invention has the following beneficial effects:

[0069] The present invention provides a stable botulinum toxin protein composition, wherein L-carnitine or its derivatives or their salts are used as protein stabilizers to replace human proteins (such as human serum albumin), thereby eliminating the threat of human viruses. At the same time, Tween 80 is used to effectively prevent adsorption, and has good long-term stability, and can even maintain high protein activity for more than two years. In addition, the botulinum toxin protein composition is a liquid preparation, and the preparation method is simpler, which can effectively reduce costs and be used for industrial production.

[0070] When the botulinum toxin protein composition of the present invention is used as a liquid preparation, it increases the convenience of clinical use and improves the defects of freeze-dried or vacuum products, such as a short storage time after reconstitution, which easily causes waste of use, human contamination during the reconstitution process, or inaccurate reconstitution. DETAILED DESCRIPTION

[0071] Unless otherwise defined, all scientific and technical terms used in the present invention have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention relates.

[0072] It is reported that the use of botulinum toxin to treat various spastic muscle disorders can relieve depression and anxiety while relieving muscle spasms. Murry T., et al., Spasmodic dysphonia; emotional status and botulinum toxin treatment, Arch Otolaryngol 1994 Mar; 120 (3): 310-316; Jahanshahi M., et al., Psychological functioning before and after treatment of torticollis with botulinum toxin, J Neurol Neurosurg Psychiatry 1992; 55 (3): 229-231. In addition, German patent application DE10150415 discusses intramuscular injection of botulinum toxin to treat depression and related emotional disorders.

[0073] Botulinum toxin has also been proposed or used to treat skin lesions (U.S. Patent 6,447,787), various autonomic dysfunctions (U.S. Patent 5,766,605), tension headaches (U.S. Patent 6,458,365), migraines (U.S. Patent 5,714,468), sinus headaches (U.S. Patent No. 429069), postoperative pain and visceral pain (U.S. Patent 6,464,986), neuralgia (U.S. Patent No. 630,587), hair growth (Hair The invention relates to hair growth and hair retention (U.S. Patent 6,299,893), dental disease (U.S. Provisional Patent Application No. 60 / 418,789), fibromyalgia (U.S. Patent 6,623,742), various skin diseases (U.S. Patent Application No. 10 / 731973), motion sickness (U.S. Patent Application No. 752,869), psoriasis and dermatitis (U.S. Patent 5,670,484), muscle damage (U.S. Patent 6,423,319), various cancers (U.S. Patent 6,139,845), smooth muscle disorders (U.S. Patent 5,437,291), lip corner ptosis (U.S. Patent 6,358,917), nerve entrapment syndrome (U.S. Patent Application 20030224019), various impulse disorders (U.S. Patent Application No. 423,380), acne (WO03 / 011333), and neurogenic inflammation (U.S. Patent 6,063,768).

[0074] In the present invention, the term "U" refers to a potency unit, a variety of units used in pharmacy to express biological potency. Because the chemical composition of these drugs is not constant or their quality specifications cannot be tested by physical and chemical methods, biological experimental methods are often used to test their potency by comparing them with standard products. Through this biological test, the minimum potency unit with a certain biological potency is called a "unit" (U); the standard unit determined by international consultation is called an "international unit" (IU).

[0075] The term "tag protein" refers to a polypeptide or protein that is fused and expressed with a target protein using DNA in vitro recombination technology to facilitate the expression and detection of the target protein.

[0076] The term "dimer structure" refers to a polymer complex composed of two molecules, usually bonded by non-covalent bonds.

[0077] "L-Carnitine", "L-carnitine", "L-carnitine", "L-carnitine" represent the same substance and can be used interchangeably.

[0078]

[00136] Various publications, patents, and published patent specifications are cited herein, the disclosures of which are incorporated by reference in their entireties.

[0079] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0080] The method for detecting the amount of botulinum toxin protein used in the examples is as follows:

[0081] The activity of botulinum toxin protein was assessed by mouse median lethal dose and is shown in mouse units (U), where one unit is defined as the amount of botulinum toxin protein injected intraperitoneally required to kill 50% of the total number of mice.

[0082] Animal experiment process:

[0083] 1. Seed selection and grouping:

[0084] Adult mice of the CD-1 strain were selected, divided into groups, weighed, and numbered to ensure that the number of mice in each group was equal and the weight was similar.

[0085] 2. Drug administration:

[0086] CD-1 mice were given a single intraperitoneal injection of 0.5 mL of botulinum toxin protein preparations of different formulations (diluted if necessary).

[0087] 3. Observation:

[0088] Before the start of the experiment, each animal was weighed once (before administration on the first day) and recorded. After administration, the body weight was weighed every 24 hours. The mortality rate was checked at 24 hours (±1 hour), 48 hours (±1 hour), 72 hours (±1 hour), and 96 hours (±1 hour) after administration. The 95% confidence limit should be between 50% and 200% of the potency.

[0089] The recombinant botulinum toxin type A used in the examples was prepared according to the method described in Examples 1-6 of WO2022 / 179552A1, i.e., dimer BoNT / A.

[0090] Example 1

[0091] Each preparation was prepared according to the formula shown in Table 1, wherein the botulinum toxin was recombinant botulinum toxin type A, and the content of the recombinant botulinum toxin type A in the preparation was 100 U / ml.

[0092] Taking Formula 1 in Table 1 as an example, the preparation steps are as follows:

[0093] 1. Prepare solvent (100ml):

[0094] a) Pipette 99 ml of 0.9% sodium chloride injection into the solution bottle;

[0095] b) Measure 0.202 g of L-carnitine hydrochloride and 0.1 ml of Tween 80 into a solution bottle;

[0096] c) Mix at 100 rpm for 15 minutes and set aside.

[0097] 2. Dilute recombinant botulinum toxin type A preparation:

[0098] a) The first step is dilution, the dilution multiple is 100 times;

[0099] b) The second dilution step is 100 times;

[0100] c) The third step is dilution, diluting to 100 U / ml;

[0101] d) Dispense into vials, label them and store them at a predetermined temperature (2-8°C).

[0102] Samples were taken at different time points to detect the botulinum toxin activity of the obtained preparation. The results are shown in Table 1.

[0103] Table 1 Formulation and its long-term stability test results

[0104]

[0105] Note: The protein activity of Formulation 2 was greater than 100 U / ml after 60 days of preparation, which was a normal test fluctuation and met the requirements for accuracy and precision of the determination methods involving animal tests in the General Principles for Technical Review of Validation of Analytical Methods for Quality Control of Biological Products issued by the Center for Drug Evaluation. The same is true for other embodiments.

[0106] As shown in Table 1, the preparation containing L-carnitine salt has better stability and can effectively maintain protein activity within 60 days.

[0107] Example 2

[0108] The preparation was prepared according to the formula shown in Table 2 and the preparation steps of Example 1, wherein the botulinum toxin was recombinant botulinum toxin type A, and the content of the recombinant botulinum toxin type A in the preparation was 100 U / ml.

[0109] After the preparation was completed, the botulinum toxin activity of the obtained preparation was tested, and the results are shown in Table 2.

[0110] Table 2 Formulation and activity test results

[0111]

[0112] As shown in Table 2, alkanoyl L-carnitine can also act as a protein stabilizer, playing the same role as L-carnitine.

[0113] Example 3

[0114] Each preparation was prepared according to the formulation shown in Table 3 with reference to the preparation steps of Example 1, wherein the botulinum toxin was recombinant botulinum toxin type A, and the content of recombinant botulinum toxin type A in the preparation was 350 U / ml.

[0115] After preparation, the botulinum toxin activity of each preparation was tested, and the results are shown in Table 3.

[0116] Table 3 Formulation and activity test results

[0117] Recipe Number composition active 1 10mM L-Carnitine, 0.01% Tween 80, 0.9% NaCl 339U / ml 2 10mM L-Carnitine, 0.02% Tween 80, 0.9% NaCl 400U / ml 3 10mM L-Carnitine, 0.05% Tween 80, 0.9% NaCl 357U / ml 4 10mM L-Carnitine, 0.1% Tween 80, 0.9% NaCl 359U / ml

[0118] As shown in Table 3, 0.01-0.1% Tween can effectively maintain the activity of botulinum toxin.

[0119] Example 4

[0120] Each preparation was prepared according to the formulation shown in Table 4 and with reference to the preparation steps of Example 1, wherein the botulinum toxin was recombinant botulinum toxin type A, and the content of the recombinant botulinum toxin type A in the preparation was 200 U / ml.

[0121] After preparation, the botulinum toxin activity of each preparation was tested, and the results are shown in Table 4.

[0122] Table 4 Formulation and activity test results

[0123] Recipe Number composition active 1 2mM L-Carnitine, 0.02% Tween 80, 0.9% NaCl 299U / ml 2 2mM L-Carnitine, 0.05% Tween 80, 0.9% NaCl 236U / ml 3 2mM L-Carnitine, 0.1% Tween 80, 0.9% NaCl 228U / ml 4 10mM L-Carnitine, 0.02% Tween 80, 0.9% NaCl 277U / ml 5 10mM L-Carnitine, 0.05% Tween 80, 0.9% NaCl 235U / ml 6 10mM L-Carnitine, 0.1% Tween 80, 0.9% NaCl 222U / ml

[0124] As shown in Table 4, the preparations containing 2-10 mM L-carnitine can effectively maintain the activity of botulinum toxin.

[0125] Example 5

[0126] Each preparation was prepared according to the formulation shown in Table 5 and with reference to the preparation steps of Example 1, wherein the botulinum toxin was recombinant botulinum toxin type A, and the content of the recombinant botulinum toxin type A in the preparation was 100 U / ml.

[0127] Samples were taken at different time points to detect the botulinum toxin activity of the obtained preparation to investigate the long-term stability of the preparation. The results are shown in Table 5.

[0128] Table 5 Formulation and its long-term stability test results

[0129]

[0130] As shown in Table 5, the formulation containing L-carnitine has excellent long-term stability and can maintain high activity for more than two years.

[0131] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

[0132] The aforementioned embodiments and methods described in the present invention may be varied based on the ability, experience and preference of those skilled in the art.

[0133] In the present invention, merely listing the steps of the method in a certain order does not constitute any limitation on the order of the method steps.

Claims

1. A botulinum toxin protein composition, comprising: botulinum toxin protein, L-carnitine or its derivatives or salts thereof; Preferably, the composition is a liquid composition, and more preferably, the composition further comprises a solvent, such as water for injection or 0.9% sodium chloride injection.

2. The composition according to claim 1, characterized in that The concentration of the L-carnitine or its derivatives or salts thereof is 1-50 mM, preferably 1-20 mM.

3. The composition according to claim 1 or 2, characterized in that The derivative of L-carnitine is alkanoyl L-carnitine; Preferably, the alkanoyl group has 2 to 26 carbon atoms; More preferably, the alkanoyl group is acetyl, propionyl, butyryl, isobutyryl, valeryl or isovaleryl; More preferably, the L-carnitine derivative is O-acetyl-L-carnitine.

4. The composition according to claim 1 or 2, characterized in that The salt of L-carnitine or its derivative is a pharmaceutically acceptable salt, preferably selected from: tartrate, hydrochloride, citrate, succinate, fumarate, preferably tartrate or hydrochloride.

5. The composition according to claim 1, characterized in that The composition further comprises a solubilizer; Preferably, the volume percentage concentration of the solubilizer is 0-0.1%; Preferably, the solubilizing agent is selected from: one or more of Tweens, Spans, sodium lauryl sulfate, and poloxamer; More preferably, the solubilizing agent is Tween 80.

6. The composition according to claim 1, characterized in that The composition further comprises an osmotic pressure regulator; Preferably, the osmotic pressure regulator is selected from the group consisting of sodium chloride, glucose, glycerol, fructose, sorbitol, magnesium chloride, phosphate, sodium citrate, and mannitol; More preferably, the osmotic pressure regulator is sodium chloride, and its mass percentage concentration in the composition is 0.9%.

7. The composition according to any one of claims 1 to 6, characterized in that The botulinum toxin protein is selected from any one of type A botulinum toxin protein, type B botulinum toxin protein, type C1 botulinum toxin protein, type C2 botulinum toxin protein, type D botulinum toxin protein, type E botulinum toxin protein, type F botulinum toxin protein or type G botulinum toxin protein, preferably type A botulinum toxin protein; Preferably, the botulinum toxin is a recombinant botulinum toxin.

8. The composition according to claim 7, characterized in that The composition comprises: botulinum toxin protein, L-carnitine or its derivatives or salts thereof, a solubilizer, and an osmotic pressure regulator, wherein the concentration of L-carnitine or its derivatives or salts thereof is 1-50 mM, the volume percentage concentration of the solubilizer is 0-0.1%, and the osmotic pressure regulator is an isotonic regulator; Preferably, the composition comprises: botulinum toxin protein, 2-10 mM L-carnitine or its derivatives or salts thereof, 0-0.1% Tween 80, 0.9% sodium chloride; More preferably, the composition comprises: botulinum toxin protein, 10 mM L-carnitine or its derivatives or salts thereof, 0.1% Tween 80, and 0.9% sodium chloride.

9. Use of the composition according to any one of claims 1 to 8 in the preparation of medicines and cosmetic products for preventing and / or treating diseases.

10. The use according to claim 9, characterized in that The disease is selected from the group consisting of: neuromuscular disease, autonomic disorder, pain, neuropsychiatric disorder, skin disease, dental disease, cancer; Preferably, the disease is selected from the group consisting of: spasmodic dysphonia, spasmodic torticollis, laryngeal dystonia, oromandibular dysphonia, tongue dystonia, cervical dystonia, focal dystonia, blepharospasm, strabismus, hemifacial spasm, eyelid disorder, cerebral palsy, focal spasm and other language disorders, spastic colitis, neurogenic bladder, pelvic floor dystonia, limb spasm, convulsions, tremors, bruxism, anal fissures, achalasia, dysphagia and other dystonias and other disorders characterized by involuntary movements of muscle groups, lacrimation, hyperhidrosis, excessive salivation, excessive gastrointestinal secretions, secretory disorders, pain caused by muscle spasms, headaches; Psoriasis, allergies, eosinophilic lymphohistiocytosis and alcoholic pancreatic disease; epilepsy, central sensitized chronic pain, central pain after stroke, regional pain, phantom limb pain and demyelinating pain; schizophrenia, Alzheimer's disease, mania, anxiety, depression; tension headache, migraine, sinus headache, postoperative pain, visceral pain, neuralgia, fibromyalgia; The beauty treatment is selected from the group consisting of: removing or preventing wrinkles, slimming the face, repairing scars, improving or removing blackheads, acne, changing eyebrow shape, lifting mouth corners, body shaping, removing body odor, hair growth and hair maintenance.

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