Neurotoxin-containing freeze-dried powder and nasal formulation

Abstract

The present application relates to the field of biopharmaceuticals and specifically provides a neurotoxin-containing freeze-dried powder and a nasal formulation. The freeze-dried powder comprises a neurotoxin, and further comprises a stabilizer and an excipient. The excipient comprises one or more of acidic gelatin, alkaline gelatin, and sucrose. The stabilizer comprises one or more of block polyether F-68, block polyether F-127, and Tween 80. The use of the stabilizer comprising one or more of block polyether F-68, block polyether F-127, and Tween 80 and the excipient comprising one or more of acidic gelatin, alkaline gelatin, and sucrose produces a synergistic effect, such that the stability of the neurotoxin at room temperature is significantly improved, thereby slowing down the degradation of the neurotoxin, and significantly extending the period during which the bioactivity of the neurotoxin is maintained at 95% or higher. Moreover, the formulation can be nasally administered to increase the delivery of the neurotoxin across the blood-brain barrier and greatly reduce the toxicity.

Description

A lyophilized powder and a nasal preparation containing neurotoxin

[0001] Cross-reference to Related Applications

[0002] This application claims priority to the Chinese patent application No. 202411027921.7, filed on July 30, 2024, and entitled "A lyophilized powder and a nasal preparation containing neurotoxin", the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present application relates to the field of biological medicine, in particular to a lyophilized powder and a nasal preparation containing neurotoxin. BACKGROUND

[0004] Snake venom is a natural protein secreted by the venom gland of snakes, containing various proteins, polypeptides, enzymes and other small molecule substances, and has strong biological activity. It is one of the animal toxins that humans have researched and utilized the most. With the in-depth study of snake venom neurotoxin, it is found that neurotoxin can be divided into three types: presynaptic neurotoxin (beta-neurotoxin), postsynaptic neurotoxin (alpha-neurotoxin) and neurotoxin-like. The former two mainly inhibit the neuromuscular transmission at the motor endplate, causing muscle paralysis and respiratory failure, while the latter can block smooth muscle contraction and ion channels, showing neurotoxin-like activity.

[0005] There are two species of cobra in China, one is Naja atra, also known as Zhoushan cobra, which is distributed in most provinces of China, and the other is Naja kaouthia, which is distributed in Guangxi Zhuang Autonomous Region and Yunnan Province of China. Neurotoxin is the main lethal component of cobra venom, accounting for about 4% to 8% of the total dry weight of the venom. Since the first neurotoxin was isolated from cobra venom in 1965, the biochemical and biological activity of cobra neurotoxin has been extensively studied. Cobra neurotoxin belongs to postsynaptic neurotoxin (alpha-neurotoxin) and is a member of the three-finger protein superfamily. The alpha-neurotoxin isolated from cobra venom can specifically and almost irreversibly bind to acetylcholine receptor (AChR). Therefore, since the 1960s, it has been an ideal tool for studying the structure and function of AChR. At the same time, because alpha-neurotoxin has high stability, contains multiple disulfide bonds, and has a large number of natural homologous proteins, it has also become a good model for studying protein folding and structure-function relationship.

[0006] The Chinese cobra venom contains two short-chain neurotoxins, among which the short-chain cobrotoxin (trade name Cobropeptide) consists of 62 amino acids, is a basic protein with a molecular weight of about 7 kD, an isoelectric point of pH 8.8, and four disulfide bonds in the molecule. The Thai cobra venom contains a long-chain cobrotoxin (cobratoxin) consisting of 71 amino acids, and the Thai cobra also contains several short-chain neurotoxins. However, due to geographical separation, the Thai cobra distributed in China contains only three short-chain neurotoxins and does not contain the long-chain cobrotoxin (cobratoxin).

[0007] However, the neurotoxin extracted from the venom has extremely strong hygroscopicity and hydrophilicity, is easily soluble in water, and the neurotoxin has poor stability in the aqueous solution. The storage conditions are harsh, and neither the water injection nor the powder injection can be placed at room temperature. The commercially available water injection (such as Cobropeptide Injection) needs to be stored at 0-10℃, and the powder injection (such as Cobropeptide for Injection) needs to be stored at 2-10℃, which requires cold chain transportation, has high transportation cost, short effective storage period, and is very unfavorable for drug promotion and use. Therefore, how to improve the stability of the neurotoxin is a technical problem to be solved by the technical personnel in the field at present.

[0008] SUMMARY

[0009] Therefore, the first technical problem to be solved by the present application is to overcome the low stability problem of the existing neurotoxin-containing preparation, and to provide a freeze-dried powder containing neurotoxin, the stability of the neurotoxin in the freeze-dried powder is significantly improved, the freeze-dried powder can be stored at room temperature for a long time, does not need cold chain transportation, greatly reduces the cost of drug transportation and storage, reduces the risk of deterioration and degradation during storage and use, and ensures the safety and effectiveness of drug use. In order to change the current mode of injecting neurotoxin (such as Cobropeptide Injection) for administration, the present application improves the stability of neurotoxin at room temperature, and also takes into account that the preparation is suitable for nasal administration after being reduced to a solution, so that the neurotoxin can penetrate the blood-brain barrier and greatly reduce the toxicity.

[0010] To this end, the present application provides a freeze-dried powder containing neurotoxin, which comprises neurotoxin, further comprises a stabilizer and an excipient, the excipient comprises one or more of acid gelatin, basic gelatin and sucrose, and the stabilizer comprises one or more of block polyether F-68, block polyether F-127 and Tween 80.

[0011] In some optional embodiments, the excipient comprises acid gelatin, and the stabilizer comprises block polyether F-68 and Tween 80.

[0012] The gelatin produced by alkaline hydrolysis is called basic gelatin. The isoelectric point of basic gelatin is 4.7-5.2.

[0013] Gelatin produced by acid hydrolysis is called acid gelatin. The isoelectric point of acid gelatin is 7.0-9.0.

[0014] Further, the neurotoxin is a cobra neurotoxin.

[0015] Further, the cobra neurotoxin is a long-chain neurotoxin or a short-chain neurotoxin. For example, a long-chain cobra neurotoxin (cobratoxin) or a short-chain cobra neurotoxin (cobrotoxin).

[0016] In this regard, the long-chain neurotoxin refers to a neurotoxin having more than 63 amino acids (e.g., 63-74 amino acids). The short-chain neurotoxin refers to a neurotoxin having less than 63 amino acids (e.g., 60-62 amino acids).

[0017] Further, the neurotoxin is a cobra neurotoxin cobrotoxin or cobropeptide.

[0018] Further, the freeze-dried powder satisfies at least one of the following A-C:

[0019] A. The freeze-dried powder contains or does not contain a penetration enhancer;

[0020] B. The freeze-dried powder contains or does not contain a preservative;

[0021] C. The freeze-dried powder comprises 3.5 parts of a neurotoxin, 200-5000 parts of an excipient, and 10-1300 parts of a stabilizer, in terms of weight fraction.

[0022] Further, the penetration enhancer comprises borneol and / or menthol.

[0023] Further, the preservative is a conventional preservative, such as benzalkonium chloride.

[0024] Further, the mass ratio of the penetration enhancer to the neurotoxin is 3.5:20-400.

[0025] Further, the freeze-dried powder does not contain a penetration enhancer (e.g., borneol and / or menthol). For example, the freeze-dried powder is composed of the following components in terms of weight fraction: a neurotoxin: 3.5 parts, acid gelatin or basic gelatin: 200-300 parts, and block polyether F-68: 375-1250 parts; or,

[0026] The freeze-dried powder is composed of the following components in terms of weight fraction: a neurotoxin: 3.5 parts, acid gelatin or basic gelatin: 200-300 parts, block polyether F-68: 375-1250 parts, and Tween 80: 10-12 parts.

[0027] In some alternative embodiments, the lyophilized powder is composed of the following components in parts by weight: neurotoxin: 3.5 parts, acid gelatin: 250 parts, block polyether F-68: 375-1000 parts.

[0028] In some alternative embodiments, the lyophilized powder is composed of the following components in parts by weight: neurotoxin: 3.5 parts, acid gelatin: 250 parts, block polyether F-68: 375-1000 parts, Tween 80: 11 parts.

[0029] Further, the lyophilized powder contains 20-400 parts by weight of a penetration enhancer. That is, the lyophilized powder includes 3.5 parts of neurotoxin, 200-5000 parts of excipient, 20-400 parts of penetration enhancer, and 10-1300 parts of stabilizer, in parts by weight. For example, the lyophilized powder is composed of the following components in parts by weight: neurotoxin: 3.5 parts, acid gelatin or basic gelatin: 200-300 parts, menthol: 350-400 parts, block polyether F-68: 375-1250 parts; or, the lyophilized powder is composed of the following components in parts by weight: neurotoxin: 3.5 parts; acid gelatin or basic gelatin: 200-300 parts; menthol: 350-400 parts; block polyether F-68: 375-1250 parts; Tween 80: 10-12 parts.

[0030] In some alternative embodiments, the lyophilized powder is composed of the following components in parts by weight: neurotoxin: 3.5 parts, acid gelatin: 250 parts, block polyether F-68: 375-1000 parts.

[0031] In some alternative embodiments, the lyophilized powder is composed of the following components in parts by weight: neurotoxin: 3.5 parts, acid gelatin: 250 parts, block polyether F-68: 375-1000 parts.

[0032] The present application also provides a method for preparing the lyophilized powder as described above, which is prepared by lyophilizing a mixed solution containing neurotoxin, stabilizer, and excipient.

[0033] Further, the concentration of the stabilizer in the mixed solution is 7.5-25 mg / mL; and / or, the concentration of the excipient in the mixed solution is 4-100 mg / mL; and / or, the concentration of the neurotoxin in the mixed solution is 1-1000 μg / mL (preferably 30-100 μg / mL, for example, 30 μg / mL, 50 μg / mL, 70 μg / mL, 100 μg / mL); and / or, the mixed solution further contains a penetration enhancer.

[0034] Further, the concentration of the penetration enhancer in the mixed solution is 0.5-7.5 mg / mL.

[0035] Further, the mixed solution contains 1-1000 μg / ml of the neurotoxin, 0.4-10% (w / v, g / 100 mL) of the excipient, and 0.75-2.5% (w / v, g / 100 mL) of the stabilizer.

[0036] Further, the mixed solution contains 1-1000 μg / ml of the neurotoxin, 0.4-10% (w / v, g / 100 mL) of the excipient, 0.05-0.75% (w / v, g / 100 mL) of the penetration enhancer, and 0.75-2.5% (w / v, g / 100 mL) of the stabilizer.

[0037] Further, the freeze-drying comprises: 1) a pre-freezing stage: running at -45℃ to -35℃ for 15-25 minutes and at -55℃ to -45℃ for 1-2 hours; and 2) a drying stage: vacuumizing, maintaining the vacuum degree at 4-8 pa, running at -10℃ to -5℃ for 4-6 hours, at -3℃ to -1℃ for 4-6 hours, at -2℃ to 0℃ for 10-14 hours, at 0℃ to 2℃ for 17-19 hours, and at 18℃ to 22℃ for 4-6 hours.

[0038] In some alternative embodiments, the mixed solution contains 70 μg / ml of the neurotoxin, 0.5% (w / v, g / 100 mL) of the acid gelatin, 0.75% (w / v, g / 100 mL) of the menthol, and 0.75% (w / v, g / 100 mL) of the block polyether F-68.

[0039] In some alternative embodiments, the mixed solution contains 70 μg / ml of the neurotoxin, 0.5% (w / v, g / 100 mL) of the acid gelatin, 0.75% (w / v, g / 100 mL) of the menthol, 0.75% (w / v, g / 100 mL) of the block polyether F-68, and 0.02% (v / v, ml / 100 ml) of the Tween 80.

[0040] Further, the solvent of the mixed solution is water.

[0041] Further, the method for preparing the mixed solution comprises the following steps: dissolving the neurotoxin in water to obtain liquid 1;

[0042] dissolving the excipient in water to obtain liquid 2;

[0043] dissolving the stabilizer in water to obtain liquid 3, mixing the liquid 1, the liquid 2, and the liquid 3 to obtain the mixed solution, or mixing the stabilizer with the liquid 1 and the liquid 2 to obtain the mixed solution.

[0044] In some embodiments, the step of adding a penetration enhancer is further included in the mixing of the excipient with water.

[0045] If necessary, the dissolution of the remaining ingredients in water, except for the neurotoxin, can be accelerated by conventional heating, for example, at a temperature of 30-50°C.

[0046] In some alternative embodiments, the method for preparing the freeze-dried powder comprises the following steps:

[0047] Take 250 parts by weight of acid gelatin and 375 parts by weight of menthol, add 30 parts by volume of water, and dissolve at 40°C to prepare liquid 1;

[0048] Take 375 parts by weight of block polyether F-68, add 10 parts by volume of water, and dissolve at 40°C to prepare liquid 2;

[0049] Take 5 parts by volume of 0.70 mg / ml neurotoxin aqueous solution, liquid 1 and liquid 2, and 0.01 parts by volume of Tween 80, mix with or without water to obtain a mixed solution, and freeze-dry the mixed solution to obtain a freeze-dried powder. When the parts by weight is mg, the parts by volume is mL.

[0050] The second technical problem to be solved by the present application is to overcome the small safety range of water injection or powder injection in the prior art, and to provide a nasal preparation. The use of nasal spray greatly improves the safety range of drug treatment compared with the existing injection.

[0051] The present application provides a nasal preparation, which comprises the freeze-dried powder of any of the above or the freeze-dried powder prepared by any of the preparation methods, and further comprises a solvent, wherein the freeze-dried powder and the solvent are packaged separately.

[0052] The solvent can be a conventional solvent used in pharmacy, such as water, aqueous phosphoric acid, or physiological saline, etc. The solvent can contain or not contain a preservative.

[0053] Further, before use, the freeze-dried powder is dissolved in the solvent to obtain a medicinal solution, and the concentration of the neurotoxin in the medicinal solution is 1-1000 μg / mL.

[0054] Alternatively, the concentration of the neurotoxin in the medicinal solution is 30-100 μg / mL.

[0055] The nasal preparation can be a conventional nasal preparation, such as a spray, an aerosol, or a nasal drop, etc.

[0056] The present application also relates to the use of the freeze-dried powder or the nasal preparation in the preparation of analgesic drugs.

[0057] The application also provides a method for treating central nervous system diseases, comprising administering the freeze-dried powder or the nasal preparation to a patient in need.

[0058] The application also relates to the use of the freeze-dried powder or the nasal preparation in the preparation of a medicament for treating central nervous system diseases.

[0059] 1. The freeze-dried powder containing neurotoxin provided by the application comprises neurotoxin, and further comprises stabilizers and excipients; the excipients comprise one or more of acidic gelatin, basic gelatin and sucrose, and the stabilizers comprise one or more of block polyether F-68, block polyether F-127 and Tween 80; the use of the stabilizers comprising one or more of block polyether F-68, block polyether F-127 and Tween 80 and the excipients comprising one or more of acidic gelatin, basic gelatin and sucrose produces a synergistic effect, so that the stability of the neurotoxin is significantly improved, the degradation of the neurotoxin is greatly alleviated, and the time for maintaining the neurotoxin to be more than 95% bioactive is significantly prolonged.

[0060] Especially, the effect is better when acidic gelatin is used as the excipient, wherein the neurotoxin has good stability in the group of block polyether F-68 and acidic gelatin and the group of block polyether F-68, Tween 80 and acidic gelatin; the freeze-dried neurotoxin can be stable at 25℃ for more than one year (HPLC purity ≥ 95%), which greatly reduces the risk of neurotoxin degradation or failure during storage and use; after the freeze-dried powder is dissolved with a solvent before use, the solution can be stable at room temperature (25℃) for more than 10 days, while the solution is used for one week, so that the patient will not encounter the problem of degradation of effective components in daily use. Therefore, the application is of great significance to improve the safety and effectiveness of clinical use of Cerebrolysin.

[0061] In addition, it is found that, compared with the use of block polyether F-68, block polyether F-127 and Tween 80 as stabilizers alone, the use of block polyether F-68 and Tween 80 as stabilizers in combination can significantly improve the stability of the neurotoxin and significantly prolong the time for maintaining the neurotoxin to be more than 95% bioactive.

[0062] 2. The method for preparing the lyophilized powder containing neurotoxin provided by the application, the preparation method is simple to operate, convenient for quality control, and convenient for mass production, especially the lyophilized powder obtained by using the lyophilization procedure shown below has better stability, the lyophilization comprises: 1) pre-freezing stage: running at -45℃ to -35℃ for 15 to 25 minutes, and running at -55℃ to -45℃ for 1 to 2 hours; 2) drying stage: vacuumizing, maintaining the vacuum degree at 4 to 8 pa, running at -10℃ to -5℃ for 4 to 6 hours, running at -3℃ to -1℃ for 4 to 6 hours, running at -2℃ to 0℃ for 10 to 14 hours, running at 0℃ to 2℃ for 17 to 19 hours, and running at 18℃ to 22℃ for 4 to 6 hours.

[0063] 3. The nasal preparation provided by the application comprises a lyophilized powder and a solvent, and the lyophilized powder and the solvent are independently packaged. Compared with the existing injection, the nasal preparation greatly reduces the toxicity of the drug and greatly improves the safety range of the drug treatment, thereby improving the safety of drug use. Animal experiments in the present study show that the minimum lethal dose of the nasal preparation of the application for ICR mice is 1 mg / kg, and the minimum lethal dose of the traditional injection is 95 μg / kg. The minimum lethal dose of the two preparations is more than ten times different. The therapeutic dose of the nasal preparation is 6 μg / kg to 12 μg / kg, and at this dose, the neurotoxin has no any toxic side effect. According to the technology of the application, the toxicity of neurotoxin administration can be greatly reduced while ensuring the therapeutic effect, and the safety of neurotoxin use can be improved.

[0064] 4. Most of the past neurotoxin drugs enter the blood circulation after being administered by injection to take effect. The main indication of the commercially available neurotoxin drugs is analgesia. Due to the existence of the blood-brain barrier, the peripheral administration has low utilization. The blood-brain barrier refers to the barrier between the plasma and the brain cells formed by the brain capillary wall and the glial cells and the barrier between the plasma and the cerebrospinal fluid formed by the choroid plexus, which can prevent certain substances from entering the brain tissue from the blood. Many solutes in the blood enter the brain tissue from the brain capillary, some quickly, some slowly, and some completely cannot. However, in order to exert the central analgesic effect or treat central nervous system diseases such as Parkinson's disease, the drug needs to enter the central nervous system. Peripheral administration of neurotoxin is considered to be unable to enter the central nervous system or only a very small amount can enter the central nervous system. Therefore, exploring a new preparation of neurotoxin can greatly help to improve the utilization of neurotoxin drugs.

[0065] The nasal preparation provided by the application can also penetrate the blood-brain barrier and reach the drug action site. When menthol and / or borneol are used as the penetration enhancer, the penetration rate of the neurotoxin can be improved. Among them, the use of menthol as the penetration enhancer is preferred, which does not affect the stability of the neurotoxin.

[0066] 5. The currently marketed neurotoxin products are all injected intramuscularly, which need professional medical staff to operate. Since most of the neurotoxin products need to be injected daily, and the treatment of the indications thereof does not require hospitalization, the patients need to go to the hospital for administration every day, which is extremely inconvenient. Moreover, the injection site is prone to pain after daily injection, which further reduces the compliance of the patients and makes them easily give up treatment. The nasal preparation of the present application reduces the injection pain, and the patients can self-administer according to the doctor's advice after dispensing, which improves the compliance of the patients and greatly facilitates the use of the patients. BRIEF DESCRIPTION OF DRAWINGS

[0067] In order to more clearly illustrate the specific embodiments of the present application or the technical solutions in the prior art, the drawings needed in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description are some embodiments of the present application, and those skilled in the art can also obtain other drawings according to these drawings without creative labor.

[0068] Fig. 1 is a photograph of the freeze-dried powders of each group before being placed in the constant-temperature and constant-humidity box in experimental example 3. From left to right, they are sample 3-1, sample 3-4, sample 3-3, and the freeze-dried powder prepared by comparative example 1, and sample 3-2;

[0069] Fig. 2 is a photograph of sample 3-1 placed in the constant-temperature and constant-humidity box for 150 days in experimental example 3;

[0070] Fig. 3 is the analgesic result of the nasal preparation in experimental example 9;

[0071] Fig. 4 is the analgesic result of the injection in experimental example 9;

[0072] Fig. 5 is the brain tissue imaging result after administration in experimental example 10;

[0073] Fig. 6 is the staining result of different brain tissues in experimental example 10. DETAILED DESCRIPTION

[0074] The following examples are provided in order to better further understand the present application, and do not limit the best embodiments described, and do not constitute a limitation on the content and protection scope of the present application. Any person who obtains any product identical or similar to the present application under the inspiration of the present application or by combining the present application with other prior art features falls within the protection scope of the present application.

[0075] The specific experimental steps or conditions not mentioned in the examples can be performed according to the conventional experimental steps described in the literature in the art or the operation or conditions. The reagents or instruments not mentioned by the manufacturer are all conventional reagent products that can be obtained by purchase.

[0076] In the following neurotoxin stability test, the purity of the neurotoxin is determined by high performance liquid chromatography, and the chromatographic conditions are as follows:

[0077] Chromatographic column: Sepax Bio-C18 4.6x150mm; detection wavelength: 280nm; mobile phase A: acetonitrile; mobile phase B: 0.1% trifluoroacetic acid; flow rate: 1mL / min; injection volume: 20μL; column temperature: 40℃. Gradient elution according to Table 1:

[0078] Table 1 Gradient elution table

[0079] Injection sequence: inject the blank solvent and the test sample solution into the liquid chromatograph in the following order, and record the chromatogram. Blank ≥ 1 needle, test sample solution 1 needle.

[0080] In the sample chromatogram, calculate the percentage of the area of the neurotoxin chromatographic peak in the total chromatographic peak area, which is the purity of the neurotoxin calculated by area normalization method.

[0081] The raw material neurotoxin powder used in the various embodiments, comparative examples and experimental examples of the present application is the Chinese cobra neurotoxin isolated and purified by Suzhou Renben Pharmaceutical Co., Ltd. After sequencing, the neurotoxin has 62 amino acids and a molecular weight of 6944 daltons. The amino acid sequence obtained by protein full coverage amino acid sequencing is lechnqqssq tptttgcsgg etncykkrwr dhrgyrterg cgcpsvkngi einccttdrc nn (see SEQ ID No: 1), which is consistent with the amino acid sequence of cobrotoxin in GenBank.

[0082] Acid gelatin is purchased from Gen-Leaf Biologics, model number M241S210427. Specification: S25197-100g; basic gelatin is purchased from Gen-Leaf Biologics, model number J29HS184618. Specification: S30925-100g.

[0083] Example 1

[0084] This example provides a lyophilized powder, the product specification is: 0.07mg of neurotoxin per bottle, the prescription for 50 bottles is: 3.5mg of neurotoxin, 250mg of acid gelatin, 375mg of menthol, and 375mg of block polyether F-68. The preparation method is as follows:

[0085] (1) Weigh 35mg of neurotoxin into a 50ml volumetric flask and dissolve in purified water to constant volume to prepare a neurotoxin stock solution. The stock solution concentration is 0.70mg / ml.

[0086] (2) Take 250 mg of acid gelatin and 375 mg of menthol in another 50 ml volumetric flask, add 30 ml of pure water, and dissolve in a 40°C water bath. Take 375 mg of block polyether F-68 and 10 ml of pure water, heat and dissolve in a 40°C water bath, and then move to the above-mentioned volumetric flask. Move 5 ml of the neurotoxin mother liquor prepared in Example 1 to the above-mentioned volumetric flask, and add pure water to make up to 50 ml. Shake well to obtain a mixed solution.

[0087] (3) Move 1 ml of the mixed solution to 15 4 ml vials respectively, and freeze-dry to obtain 15 bottles of freeze-dried powder. The freeze-drying procedure is as follows: 1) pre-freezing stage: run at -40°C for 20 minutes, and at -45°C for 1 hour and 30 minutes; 2) drying stage: vacuumize, maintain the vacuum degree at 4-8 pa, run at -6°C for 5 hours and 30 minutes, at -2°C for 5 hours, at -2°C for 12 hours, at 0°C for 18 hours, and at 20°C for 5 hours.

[0088] The present embodiment also provides a nasal preparation, which comprises the freeze-dried powder prepared according to the method of the present embodiment and a solvent. The solvent is physiological saline, which is prepared by taking 0.9 g of sodium chloride into a 100 ml volumetric flask, and adding pure water to make up to 100 ml. After sterilization, the physiological saline is obtained.

[0089] The freeze-dried powder and the solvent are packaged separately. When used, 1 mL of the solvent is added to each bottle (containing 0.07 mg of neurotoxin) of freeze-dried powder, and shaken to quickly reconstitute the freeze-dried powder. After reconstitution, the concentration of neurotoxin in the drug solution is 70 μg / ml, the concentration of acid gelatin is 5 mg / ml, the concentration of menthol is 7.5 mg / ml, and the concentration of block polyether F-68 is 7.5 mg / ml.

[0090] Example 2

[0091] The present embodiment provides a freeze-dried powder, which has a product specification of 0.07 mg of neurotoxin per bottle. The prescription for 50 bottles is as follows: 3.5 mg of neurotoxin, 250 mg of acid gelatin, 375 mg of menthol, and 625 mg of block polyether F-68. The preparation method is as follows:

[0092] Take 250 mg of acid gelatin and 375 mg of menthol in another 50 ml volumetric flask, add 30 ml of pure water, and dissolve in a 40°C water bath. Take 625 mg of block polyether F-68 and 10 ml of pure water, heat and dissolve in a 40°C water bath, and then move to the above-mentioned volumetric flask. Move 5 ml of the neurotoxin mother liquor prepared in Example 1 to the above-mentioned volumetric flask, and add pure water to make up to 50 ml. Shake well to obtain a mixed solution. Move 1 ml of the mixed solution to 15 4 ml vials respectively, and freeze-dry to obtain 15 bottles of freeze-dried powder. The freeze-drying procedure is the same as in Example 1.

[0093] The present example also provides a nasal preparation, which is basically the same as that of Example 1, except that the lyophilized powder prepared according to the method of the present example is used instead of the lyophilized powder prepared according to the method of Example 1, and the concentration of the block polyether F-68 in the drug solution obtained by reconstituting the lyophilized powder with physiological saline is 12.5 mg / ml.

[0094] Example 3

[0095] The present example provides a lyophilized powder, which has the following product specifications: 0.07 mg of neurotoxin per bottle, and the prescription for 50 bottles is: 3.5 mg of neurotoxin, 250 mg of acid gelatin, 375 mg of menthol, and 1000 mg of block polyether F-68. The preparation method is as follows:

[0096] Take 250 mg of acid gelatin and 375 mg of menthol, add 30 ml of pure water to another 50 ml volumetric flask, and dissolve in a 40°C water bath. Take 1 g of block polyether F-68, dissolve in 10 ml of pure water in a 40°C water bath, and then transfer to the above-mentioned volumetric flask. Take 5 ml of the neurotoxin stock solution prepared in Example 1 to the above-mentioned volumetric flask, and add pure water to 50 ml to obtain a mixed solution. Transfer 1 ml of the mixed solution to 15 4 ml vials, and freeze-dry to obtain 15 bottles of lyophilized powder. The freeze-drying procedure is the same as that of Example 1.

[0097] The present example also provides a nasal preparation, which is basically the same as that of Example 1, except that the lyophilized powder prepared according to the method of the present example is used instead of the lyophilized powder prepared according to the method of Example 1, and the concentration of the block polyether F-68 in the drug solution obtained by reconstituting the lyophilized powder with physiological saline is 20 mg / ml.

[0098] Example 4

[0099] The present example provides a lyophilized powder, which has the following product specifications: 0.07 mg of neurotoxin per bottle, and the prescription for 50 bottles is: 3.5 mg of neurotoxin, 250 mg of acid gelatin, 375 mg of menthol, and 1250 mg of block polyether F-68. The preparation method is as follows:

[0100] Take 250 mg of acid gelatin and 375 mg of menthol, add 30 ml of pure water to another 50 ml volumetric flask, and dissolve in a 40°C water bath. Take 1.25 g of block polyether F-68, dissolve in 10 ml of pure water in a 40°C water bath, and then transfer to the above-mentioned volumetric flask. Take 5 ml of the neurotoxin stock solution prepared in Example 1 to the above-mentioned volumetric flask, and add pure water to 50 ml to obtain a mixed solution. Transfer 1 ml of the mixed solution to 15 4 ml vials, and freeze-dry to obtain 15 bottles of lyophilized powder. The freeze-drying procedure is the same as that of Example 1.

[0101] The present example also provides a nasal preparation, which is substantially the same as that of Example 1, except that the lyophilized powder prepared by the method of the present example is used instead of the lyophilized powder prepared according to the method of Example 1, and the concentration of the block polyether F-68 in the drug solution obtained by reconstituting the lyophilized powder with physiological saline is 25 mg / ml.

[0102] Comparative Example 1

[0103] The present comparative example provides a lyophilized powder, which has the following product specifications: 0.07 mg of neurotoxin per bottle, and the prescription for 50 bottles is 3.5 mg of neurotoxin and 250 mg of acid gelatin. The preparation method is as follows:

[0104] (1) 35 mg of neurotoxin was weighed into a 50 ml volumetric flask and dissolved in pure water to prepare a neurotoxin stock solution, and the stock solution had a concentration of 0.70 mg / ml.

[0105] (2) 250 mg of acid gelatin and 375 mg of menthol were weighed into another 50 ml volumetric flask, 30 ml of pure water was added, and the mixture was heated and dissolved in a 40°C water bath. 5 ml of the neurotoxin stock solution was transferred to the above-mentioned volumetric flask, and the volume was adjusted to 50 ml with pure water to obtain a mixed solution. 1 ml of the mixed solution was transferred to 15 4 ml vials, respectively, and lyophilized to prepare 15 bottles of lyophilized powder. The lyophilization procedure was the same as that of Example 1.

[0106] The present comparative example also provides a nasal preparation, which is substantially the same as that of Example 1, except that the lyophilized powder prepared by the method of the present comparative example is used instead of the lyophilized powder prepared according to the method of Example 1, and the lyophilized powder is reconstituted with physiological saline to obtain a drug solution that does not contain menthol and block polyether F-68.

[0107] Comparative Example 2

[0108] The present comparative example provides a lyophilized powder, which has the following product specifications: 0.07 mg of neurotoxin per bottle, and the prescription for 50 bottles is 3.5 mg of neurotoxin and 250 mg of acid gelatin. The preparation method is as follows:

[0109] (1) 35 mg of neurotoxin was weighed into a 50 ml volumetric flask and dissolved in pure water to prepare a neurotoxin stock solution, and the stock solution had a concentration of 0.70 mg / ml.

[0110] (2) 250 mg of acid gelatin and 375 mg of menthol were weighed into another 50 ml volumetric flask, 30 ml of pure water was added, and the mixture was heated and dissolved in a 40°C water bath. 5 ml of the neurotoxin stock solution was transferred to the above-mentioned volumetric flask, and the volume was adjusted to 50 ml with pure water to obtain a mixed solution. 1 ml of the mixed solution was transferred to 15 4 ml vials, respectively, and lyophilized to prepare 15 bottles of lyophilized powder. The lyophilization procedure was the same as that of Example 1.

[0111] The comparative example also provides a nasal preparation which is substantially the same as Example 1, except that the lyophilized powder prepared according to the method of the comparative example is used instead of the lyophilized powder prepared according to the method of Example 1, and the physiological saline is used to reconstitute the lyophilized powder to obtain a drug solution, and the block polyether F-68 is not used.

[0112] Experimental Example 1

[0113] The vials loaded with the lyophilized powder of Examples 1-4 and Comparative Examples 1-2 were capped and sealed and then placed in a constant temperature and humidity chamber at a temperature of 40°C and a humidity of 50% ± 5% RH. The purity of the neurotoxin in the solution obtained by reconstituting 1 vial of the lyophilized powder with 1 mL of physiological saline was detected at the same time before storage (0 months) and after storage for 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months and 12 months, respectively. The results are shown in Table 2.

[0114] Table 2 Stability and degradation time of neurotoxin

[0115] The experiment was ended when the purity was reduced to less than 95%

[0116] As can be seen from the results in Table 2, the stability of the neurotoxin in the lyophilized powder containing the neurotoxin, the stabilizer and the excipient according to Examples 1-4 of the present application is significantly improved compared to the lyophilized powder prepared according to Comparative Examples 1 and 2, the degradation of the neurotoxin is greatly alleviated, and the time for maintaining the neurotoxin at a biological activity of more than 95% is significantly prolonged from 8 months to 9-11 months.

[0117] In addition, as can be seen from Examples 1-4, the stability of the neurotoxin can be further improved by limiting the weight ratio or concentration of the block polyether F-68 to the preferred range according to Examples 1-3 (especially Example 1).

[0118] Experimental Example 2

[0119] 1 mg of neurotoxin powder was weighed into 22 vials, which were capped and sealed and then divided into two groups, one group was placed in a constant temperature and humidity chamber at 25°C and a humidity of 50% ± 5% RH, and the other group was placed in a constant temperature and humidity chamber at 40°C and a humidity of 50% ± 5% RH. The purity of the neurotoxin in the solution obtained by reconstituting 1 vial of the neurotoxin powder with 1 mL of physiological saline was detected at the same time every 3 months. The results are shown in Table 3.

[0120] Table 3 Stability and degradation time of neurotoxin

[0121] From the results of Table 3, it can be seen that the raw drug neurotoxin powder can maintain neurotoxin with a biological activity of 95% or more for only 3 months in the 40°C accelerated experiment, and for 9 months at room temperature, less than 12 months.

[0122] Experimental Example 3: Excipient Selection

[0123] The purpose of this experimental example is to investigate the effects of different excipients on the freeze-dried powder.

[0124] Sample 3-1: 5 g of sucrose was weighed into a 50 ml volumetric flask, 30 ml of pure water was added, and the mixture was dissolved in a 40°C water bath. 5 ml of the neurotoxin stock solution prepared in Comparative Example 1 was transferred to the above-mentioned volumetric flask, and the volume was adjusted to 50 ml with pure water. The mixture was shaken to obtain a mixed solution. 1 ml of the mixed solution was transferred to 15 4 ml vials, and the freeze-drying procedure was the same as in Example 1 to obtain 15 bottles of freeze-dried powder.

[0125] Sample 3-2: 250 mg of basic gelatin was weighed into a 50 ml volumetric flask, 30 ml of pure water was added, and the mixture was dissolved in a 40°C water bath. 5 ml of the neurotoxin stock solution prepared in Comparative Example 1 was transferred to the above-mentioned volumetric flask, and the volume was adjusted to 50 ml with pure water. The mixture was shaken to obtain a mixed solution. 1 ml of the mixed solution was transferred to 15 4 ml vials, and the freeze-drying procedure was the same as in Example 1 to obtain 15 bottles of freeze-dried powder.

[0126] Sample 3-3: 200 mg of glycine was weighed into a 50 ml volumetric flask, 30 ml of pure water was added, and the mixture was dissolved in a 40°C water bath. 5 ml of the neurotoxin stock solution prepared in Comparative Example 1 was transferred to the above-mentioned volumetric flask, and the volume was adjusted to 50 ml with pure water. The mixture was shaken to obtain a mixed solution. 1 ml of the mixed solution was transferred to 15 4 ml vials, and the freeze-drying procedure was the same as in Example 1 to obtain 15 bottles of freeze-dried powder.

[0127] Sample 3-4: 5 g of mannitol was weighed into a 50 ml volumetric flask, 30 ml of pure water was added, and the mixture was dissolved in a 40°C water bath. 5 ml of the neurotoxin stock solution prepared in Comparative Example 1 was transferred to the above-mentioned volumetric flask, and the volume was adjusted to 50 ml with pure water. The mixture was shaken to obtain a mixed solution. 1 ml of the mixed solution was transferred to 15 4 ml vials, and the freeze-drying procedure was the same as in Example 1 to obtain 15 bottles of freeze-dried powder.

[0128] The vials loaded with samples 3-1 to 3-4 were sealed and placed in a constant temperature and humidity chamber at 40°C and 50% ± 5% RH. At the same time every month, 1 ml of physiological saline was used to reconstitute 1 bottle of freeze-dried powder, and the neurotoxin purity of the reconstituted solution was detected. The results are shown in Table 4.

[0129] Table 4: Stability and degradation time of neurotoxin

[0130] The experiment ends when the purity is reduced to less than 95%

[0131] As can be seen from the results of Table 3 and Table 4, using glycine and mannitol as excipients, the time for maintaining the neurotoxin at a biological activity of 95% or more in the 40°C accelerated experiment is only 2-3 months, which cannot effectively improve the stability of the neurotoxin, while using acid gelatin and basic gelatin as excipients, the time for maintaining the neurotoxin at a biological activity of 95% or more in the 40°C accelerated experiment is extended to 7-8 months, which significantly improves the stability of the neurotoxin, especially the acid gelatin.

[0132] Among them, sucrose as an excipient is dissolved after liquefaction for 150 days and does not form a shape, while the other groups have good formability within 9 months of placement, as shown in Figures 1-2.

[0133] Experimental Example 4 Investigation of Penetration Enhancers

[0134] The purpose of this experimental example is to investigate the effect of different penetration enhancers on the freeze-dried powder.

[0135] Sample 4-1: Take 250 mg of acid gelatin and 25 mg of borneol into a 50 ml volumetric flask, add 30 ml of pure water, and heat to dissolve in a 40°C water bath. Take 5 ml of the neurotoxin stock solution prepared in Comparative Example 2 into the above-mentioned volumetric flask, dilute to 50 ml with pure water, and shake to obtain a mixed solution. Take 1 ml of the mixed solution into 15 4 ml vials, and freeze-dry, with the freeze-drying procedure being the same as in Example 1, to obtain 15 bottles of freeze-dried powder.

[0136] Sample 4-2: Take 250 mg of acid gelatin and 25 mg of borneol into a 50 ml volumetric flask, add 30 ml of pure water, and heat to dissolve in a 40°C water bath. Take 5 ml of the neurotoxin stock solution prepared in Comparative Example 2 into the above-mentioned volumetric flask, dilute to 50 ml with pure water, and shake to obtain a mixed solution. Take 1 ml of the mixed solution into 15 4 ml vials, and freeze-dry, with the freeze-drying procedure being the same as in Example 1, to obtain 15 bottles of freeze-dried powder.

[0137] Sample 4-3: Take 250 mg of acid gelatin and 25 mg of borneol into a 50 ml volumetric flask, add 30 ml of pure water, and heat to dissolve in a 40°C water bath. Take 5 ml of the neurotoxin stock solution prepared in Comparative Example 2 into the above-mentioned volumetric flask, dilute to 50 ml with pure water, and shake to obtain a mixed solution. Take 1 ml of the mixed solution into 15 4 ml vials, and freeze-dry, with the freeze-drying procedure being the same as in Example 1, to obtain 15 bottles of freeze-dried powder.

[0138] The vials loaded with samples 4-1 to 4-3 were sealed and placed in a constant temperature and humidity chamber at 40°C and 50% RH ± 5%. At the same time each month, one vial of lyophilized powder was reconstituted with 1 ml of normal saline, and the purity of the neurotoxin in the reconstituted solution was determined. The results of the purity of the neurotoxin in the reconstituted solution were compared with the purity of the comparative example 2 in the experimental example 1, and are shown in Table 5.

[0139] Table 5 Stability and degradation time of neurotoxin

[0140] The experiment was ended when the purity was reduced to less than 95%

[0141] As shown in Table 5, the stability of the neurotoxin was better when the penetration enhancer was menthol than when the penetration enhancer was borneol. When the acidic gelatin and menthol were used in combination, the neurotoxin remained above 95% bioactivity for 8 months in the accelerated experiment at 40°C, and the stability of the neurotoxin was significantly improved.

[0142] As shown in Table 4 and Table 5, the use of borneol would affect the stability of the neurotoxin and weaken the improvement of the stability of the neurotoxin by the excipient, while the use of menthol would not. Therefore, menthol is preferred.

[0143] Experimental example 5 Investigation of stabilizers

[0144] The purpose of this experimental example is to investigate the effect of different stabilizers on the lyophilized powder.

[0145] 35 mg of neurotoxin was weighed into a 50 ml volumetric flask, dissolved in water, and diluted to volume. The neurotoxin mother liquor was prepared, and the concentration of the mother liquor was 0.70 mg / ml.

[0146] Sample 5-1: 250 mg of acidic gelatin and 375 mg of menthol were weighed into another 50 ml volumetric flask, and 30 ml of pure water was added. The mixture was dissolved in a 40°C water bath. 375 mg of block polyether F-68 and 10 ml of pure water were dissolved in a 40°C water bath, and then transferred to the above volumetric flask. 5 ml of the neurotoxin mother liquor was transferred to the above volumetric flask, and the volume was adjusted to 50 ml with pure water. The mixture was shaken to obtain a mixed solution. 1 ml of the mixed solution was transferred to 15 4 ml vials, and lyophilized according to the procedure of Example 1. The lyophilization procedure was the same as that of Example 1.

[0147] Sample 5-2: The preparation method was basically the same as that of sample 5-1, except that a mixture of 187.5 mg of block polyether F-68 and 187.5 mg of block polyether F-127 was used instead of 375 mg of block polyether F-68.

[0148] Sample 5-3: The preparation method is substantially the same as that of sample 5-1, except that 375 mg of the mixture of block polyether F-127 is used instead of 375 mg of block polyether F-68.

[0149] Sample 5-4: The preparation method is substantially the same as that of sample 5-1, except that 0.01 ml of Tween 80 is additionally added, specifically: 250 mg of acid gelatin and 375 mg of menthol are weighed in another 50 ml volumetric flask, and 30 ml of pure water is added, and the mixture is dissolved in a 40 °C water bath. 375 mg of block polyether F-68 and 10 ml of pure water are weighed and dissolved in a 40 °C water bath, and then transferred to the above-mentioned volumetric flask. 0.01 ml of Tween 80 is transferred to the above-mentioned volumetric flask. 5 ml of neurotoxin stock solution is transferred to the above-mentioned volumetric flask, and the volume is adjusted to 50 ml with pure water, and the mixture is shaken to obtain a mixed solution. 1 ml of the mixed solution is transferred to 15 4 ml vials, and the vials are freeze-dried according to the procedure of Example 1 to obtain 15 bottles of freeze-dried powder.

[0150] Sample 5-5: The preparation method is substantially the same as that of sample 5-3, except that 0.01 ml of Tween 80 is additionally added, specifically: 250 mg of acid gelatin and 375 mg of menthol are weighed in another 50 ml volumetric flask, and 30 ml of pure water is added, and the mixture is dissolved in a 40 °C water bath. 375 mg of block polyether F-127 and 10 ml of pure water are weighed and dissolved in a 40 °C water bath, and then transferred to the above-mentioned volumetric flask. 0.01 ml of Tween 80 is transferred to the above-mentioned volumetric flask. 5 ml of neurotoxin stock solution is transferred to the above-mentioned volumetric flask, and the volume is adjusted to 50 ml with pure water, and the mixture is shaken to obtain a mixed solution. 1 ml of the mixed solution is transferred to 15 4 ml vials, and the vials are freeze-dried according to the procedure of Example 1 to obtain 15 bottles of freeze-dried powder.

[0151] The vials loaded with samples 5-1 to 5-5 are sealed and placed in a constant temperature and humidity chamber at 40 °C and 50% ± 5% RH. At the same time every month, 1 ml of physiological saline is used to reconstitute 1 bottle of freeze-dried powder, and the purity of the neurotoxin in the reconstituted solution is detected. The results of the purity of the comparative example 2 in the experimental example 1 are shown in Table 6.

[0152] Table 6 Stability and degradation time of neurotoxin

[0153] The experiment is ended when the purity is reduced to below 95%

[0154] From the results of Table 6 and Table 2, compared with Comparative Example 1 and Comparative Example 2, the stability of the neurotoxin in the freeze-dried powder of Sample 5-1 to 5-5 can be further improved by adding the stabilizer. The time for maintaining the neurotoxin at more than 95% bioactivity in the accelerated experiment at 40°C is prolonged from 8 months to 9-12 months. The best effect is obtained by using Tween 80 and F-68 in combination.

[0155] Experimental Example 6 Investigation of Preservatives

[0156] Dissolve 35 mg of the neurotoxin in 50 ml of pure water in a volumetric flask to prepare a neurotoxin stock solution, and the concentration of the stock solution is 0.70 mg / ml.

[0157] Sample 6-1: Take 250 mg of acid gelatin and 375 mg of menthol into another 50 ml volumetric flask, and add 30 ml of pure water to dissolve at 40°C in a water bath. Take 375 mg of block polyether F-68 and 10 ml of pure water to dissolve at 40°C in a water bath, and then transfer to the above-mentioned volumetric flask. Take 5 ml of the neurotoxin stock solution to the above-mentioned volumetric flask, and add pure water to 50 ml to shake well to obtain a mixed solution. Take 1 ml of the mixed solution into 15 4 ml vials respectively to freeze-dry, and prepare 15 bottles of freeze-dried powder. Seal the vials with caps, and place in a constant temperature and humidity chamber at a temperature of 40°C and a humidity of 50% ± 5% RH. Take 0.9 g of sodium chloride into a 100 ml volumetric flask, and add pure water to 100 ml. Sterilize and then divide into portions. At the same time every month, dissolve the freeze-dried powder with 1 ml of normal saline to detect the purity of the neurotoxin.

[0158] Sample 6-2: Take 250 mg of acid gelatin and 375 mg of menthol into another 50 ml volumetric flask, and add 25 ml of pure water to dissolve at 40°C in a water bath. Take 375 mg of block polyether F-68 and 10 ml of pure water to dissolve at 40°C in a water bath, and then transfer to the above-mentioned volumetric flask. Take 5 mg of benzalkonium chloride to dissolve in pure water, and then transfer to the above-mentioned volumetric flask. Take 5 ml of the neurotoxin stock solution to the above-mentioned volumetric flask, and add pure water to 50 ml to shake well to obtain a mixed solution. Take 1 ml of the mixed solution into 15 4 ml vials respectively to freeze-dry, and prepare 15 bottles of freeze-dried powder. Seal the vials with caps, and place in a constant temperature and humidity chamber at a temperature of 40°C and a humidity of 50% ± 5% RH. Take 0.9 g of sodium chloride into a 100 ml volumetric flask, and add pure water to 100 ml. Sterilize and then divide into portions. At the same time every month, dissolve the freeze-dried powder with 1 ml of normal saline to detect the purity of the neurotoxin.

[0159] Sample 6-3: 250 mg of acid gelatin and 375 mg of menthol were weighed into another 50 ml volumetric flask, and 30 ml of pure water was added and dissolved in a 40 °C water bath. 375 mg of block polyether F-68 was weighed into 10 ml of pure water and dissolved in a 40 °C water bath, and then transferred to the above-mentioned volumetric flask. 5 ml of neurotoxin stock solution was transferred to the above-mentioned volumetric flask, and the volume was adjusted to 50 ml with pure water. The mixture was shaken to obtain a mixed solution. 1 ml of the mixed solution was transferred to 15 4 ml vials, respectively, and freeze-dried to obtain 15 bottles of freeze-dried powder. The vials were sealed and placed in a constant temperature and humidity box at 40 °C and 50% ± 5% RH. 0.9 g of sodium chloride and 0.01 g of benzalkonium chloride were weighed into a 100 ml volumetric flask, and the volume was adjusted to 100 ml with pure water. After sterilization, it was divided into portions. At the same time every month, 1 ml of physiological saline was used to reconstitute the freeze-dried powder to detect the purity of the neurotoxin.

[0160] Table 7 Stability and degradation time of neurotoxin

[0161] The data shows that there is no significant difference between the three groups, indicating that the scheme of the present application can ensure the stability of the neurotoxin without preservatives. Experimental Example 7 Stability of the solution obtained after reconstitution at room temperature

[0162] The purpose of this experimental example is to investigate the synergistic effect of stabilizers and excipients on the stability of freeze-dried powder.

[0163] 1.1 35 mg of neurotoxin was weighed into a 50 ml volumetric flask and dissolved with Aesop water to make a neurotoxin stock solution. The concentration of the stock solution was 0.70 mg / ml.

[0164] 1.2 Sample 7-1: 250 mg of acid gelatin and 375 mg of menthol were weighed into a 50 ml volumetric flask and dissolved in 30 ml of pure water in a 40 °C water bath. 5 ml of neurotoxin stock solution was transferred to the volumetric flask, and the volume was adjusted to 50 ml with pure water. The mixture was shaken to obtain a mixed solution. 1 ml of the mixed solution was transferred to 8 4 ml vials (8 vials) respectively;

[0165] Sample 7-2: 375 mg of menthol was weighed into 30 ml of pure water and dissolved in a 40 °C water bath. 375 mg of block polyether F-68 was weighed into 10 ml of pure water and dissolved in a 40 °C water bath. The above two solutions were transferred to a 50 ml volumetric flask and mixed. 0.01 ml of Tween 80 was added, and 5 ml of neurotoxin stock solution was transferred to the volumetric flask. The volume was adjusted to 50 ml with pure water, and the mixture was shaken to obtain a mixed solution. 1 ml of the mixed solution was transferred to 8 4 ml vials (8 vials) respectively;

[0166] Sample 7-3: 250 mg of acid gelatin and 375 mg of menthol were weighed into 30 ml of purified water, and heated to dissolve in a 40°C water bath. 375 mg of block polyether F-68 was weighed into 10 ml of purified water, and heated to dissolve in a 40°C water bath. The two solutions were mixed in a 50 ml volumetric flask, 0.01 ml of Tween 80 was added, 5 ml of neurotoxin stock solution was added, and the volume was made up to 50 ml with purified water. The mixture was shaken well, and 1 ml of the mixture was taken into 8 4 ml vials (8 vials).

[0167] 1.3 The above 3 groups of samples (samples 7-1, 7-2 and 7-3) were placed in a freeze dryer after being divided into portions. The freeze drying procedure was the same as in Example 1.

[0168] 1.4 After freeze drying, the samples were capped and sealed, and placed in a constant temperature and humidity chamber at 40°C and 50% ± 5% RH. At the same time each month, 1 ml of 0.9% sodium chloride solution was used to reconstitute the samples, and the purity was detected and the stability was investigated. Tukey's multiple comparison test was used to analyze the test results, and the p value between the two groups was calculated. The results are shown in Tables 8 and 9.

[0169] Table 8 Stability and degradation time of neurotoxin

[0170] Table 9 Tukey's multiple comparison test

[0171] The lower the relative standard deviation, the better the stability. The comparison data show that the stability of sample 7-3, which is mixed with excipients and stabilizers, is better than that of a single component, and there is a significant difference, indicating that the excipients and stabilizers have a synergistic effect.

[0172] After sample 7-3 was placed in a constant temperature and humidity chamber at 40°C and 50% ± 5% RH for 6 months, it was taken out, reconstituted with 1 ml of 0.9% sodium chloride solution, and placed in a constant temperature and humidity chamber at 25°C and 50% ± 5% RH. The purity stability was measured at the same time every two days. The results are shown in Table 10.

[0173] Table 10 Purity of neurotoxin (%)

[0174] The data show that after the freeze-dried powder is dissolved in a solvent, the solution can be stable at room temperature (25°C) for at least 10 days, meeting the requirement of one week of use time for the preparation.

[0175] Experimental Example 8 Safety

[0176] (1) ICR mice (22.0-25.0 g), 40, randomly divided into 4 groups, half male and half female, a total of 10. Before nasal administration, the mice were anesthetized with a cotton ball with anhydrous ether. The neurotoxin was prepared into a freeze-dried powder according to the method of Example 1, dissolved in normal saline, and prepared into a solution (nasal preparation) with a concentration of 9.2 mg / mL, 18.4 mg / mL, 0.92 mg / mL, and 1.38 mg / mL. Nasal administration (nasal administration or transnasal administration operation, see Experimental Example 10) was performed at 2.5 μL / mouse and 5 μL / mouse, and the administration doses are shown in Table 11. The symptoms and death of the mice were observed and recorded.

[0177] Table 11 Experimental results

[0178] From the data, it can be seen that after nasal administration of 200 μg / kg and 300 μg / kg doses of neurotoxin nasal preparation, all the experimental mice survived. After nasal administration of 1 mg / kg dose of neurotoxin nasal preparation, 2 of 10 experimental mice died. After nasal administration of 2 mg / kg dose of neurotoxin nasal preparation, 4 of 10 experimental mice died. The above data show that the minimum lethal dose of this batch of neurotoxin nasal preparation is 1 mg / kg.

[0179] (2) ICR mice (18.0-22.0 g), half male and half female, a total of 18, were randomly divided into 3 groups, 6 in each group. 771.4 μL, 814.2 μL, and 857.1 μL of commercially available cobotoxin injection solution (Tonghua Huikang Biopharmaceutical Co., Ltd.) with a concentration of 35 μg / mL were taken and placed in 5 mL EP tubes, and then diluted to 3 mL with normal saline to prepare cobotoxin solutions with different concentrations. The groups and administration doses were cobotoxin 90 μg / kg, cobotoxin 95 μg / kg, and cobotoxin 100 μg / kg, and subcutaneous injection was performed at a dose of 0.1 mL / 10 g to observe the state of the mice and record whether they survived. The experimental results are shown in Table 12.

[0180] Table 12 Minimum lethal dose

[0181] From the data, it can be seen that after subcutaneous injection of cobotoxin 90 μg / kg at a dose of 0.1 mL / 10 g, all 6 mice survived. After subcutaneous injection of cobotoxin 95 μg / kg at a dose of 0.1 mL / 10 g, only one male mouse died out of 6 mice. After subcutaneous injection of cobotoxin 100 μg / kg at a dose of 0.1 mL / 10 g, all 6 mice died. The above data show that the minimum lethal dose of this batch of cobotoxin injection solution is 95 μg / kg.

[0182] The experiment shows that the minimum lethal dose of the nerve toxin nasal preparation of the application is 1 mg / kg, the minimum lethal dose of cobropeptide is 95 μg / kg, which is more than ten times different, so the nasal preparation greatly reduces the toxicity of the nerve toxin compared with cobropeptide, and improves the safety.

[0183] Experimental Example 9 Analgesia Experiment

[0184] The day before the experiment, the animals were screened by placing the mice on a hot plate at 55°C, and the mice were stimulated by heat to produce a pain response to lick their feet. The time at which the mice first licked their feet was recorded as the pain threshold. Mice with a pain threshold of more than 30 seconds were excluded.

[0185] (1) Female ICR mice were selected as experimental animals, and the baseline pain threshold of the mice was determined. The mice were placed on a hot plate at 55°C, and the mice were observed for licking their feet as an indicator. The mice were divided into three groups according to the length of the pain threshold, two treatment groups and one control group, with 10 mice in each group. The nerve toxin was prepared into a freeze-dried powder according to the method of Example 1, and then dissolved in physiological saline to obtain a nerve toxin solution of 70 μg / ml. The nerve toxin solution was diluted with physiological saline to obtain different concentrations of the test drug solution. After recording the baseline pain threshold of each mouse in each group before administration (0 hours), the two treatment groups were given the test drug solution intranasally. The dose of the nerve toxin was 6 μg / kg and 12 μg / kg of animal body weight, respectively, and the volume of the drug given to each mouse was 0.8 μL / 10 g (mouse body weight). The control group was given the same volume of physiological saline intranasally. The nasal administration was performed according to the method of Experimental Example 10. The pain threshold was measured at 0.5, 1, 2, 3, and 4 hours after administration (if there was no obvious pain response within 60 seconds, the mouse was removed and the time was calculated as 60 seconds), and the analgesic effect of different doses of nerve toxin was observed.

[0186] The results are shown in Figure 3. Compared with the control group, the mice showed significant analgesic effect at doses of 6 μg / kg and 12 μg / kg (p<0.01 or p<0.001) after nasal administration of the nerve toxin nasal preparation provided in this study, and showed a good dose relationship.

[0187] (2) 35 μg / mL cobropeptide injection was diluted with sodium chloride injection to 0.6 μg / ml (6 μg / kg) and 0.9 μg / ml (9 μg / kg) solutions for experimental use, and the solutions were prepared and used immediately.

[0188] Select 24 mice with qualified pain threshold, randomly divide into 3 groups, 8 mice in each group, cobo peptide injection 6 μg / kg group, cobo peptide injection 9 μg / kg group and control group. Each mouse in each group is detected to record the basic pain threshold before administration (0 hour), then the mice in the administration groups are subcutaneously injected with cobo peptide injection of corresponding concentration, the normal control group is subcutaneously injected with sodium chloride injection of the same volume, and the pain threshold of each mouse in each group is measured and recorded at 0.5 h, 1 h, 2 h, 3 h, 4 h and 5 h after administration. The time limit for the mouse to be placed on the hot plate instrument is 60 seconds, and the pain threshold greater than 60 seconds is counted as 60 seconds.

[0189] The results are shown in Figure 4. After using the neurotoxin injection, compared with the control group, the mice had no obvious analgesic effect at 6 μg / kg (p>0.05), but had obvious analgesic effect at 9 μg / kg (p<0.001). It shows that compared with the commercially available injection, the nasal preparation of the present application can achieve good therapeutic effect while reducing the administration dose.

[0190] Experimental Example 10

[0191] 1. Experimental materials

[0192] FITC (fluorescein isothiocyanate) labeled neurotoxin freeze-dried powder: The freeze-dried powder was prepared according to the method of Example 1, except that FITC labeled neurotoxin (FITC labeled neurotoxin was entrusted to Beijing Zhongke Chenyu Technology Co., Ltd.) was used instead of neurotoxin in step (1) of Example 1.

[0193] Before use, the FITC labeled neurotoxin freeze-dried powder was dissolved and diluted with normal saline to prepare neurotoxin solutions of different concentrations.

[0194] 2. Experimental method

[0195] Select 8 WT mice of 9 months old, and randomly divide them into two groups, 4 mice in each group. One group is given the same amount of normal saline and 3, 6 and 12 μg / kg of FITC labeled neurotoxin per animal weight by subcutaneous administration in the back; the other group is given the same amount of normal saline and 3, 6 and 12 μg / kg of FITC labeled neurotoxin per animal weight by nasal administration. The administration volume of each mouse in the two groups is 0.8 μL / 10 g.

[0196] Nasal administration method: select a ten-microliter liquid sample injection needle, and cover a very thin hose at the needle tip as a tool for nasal administration. After the mouse is anesthetized with isoflurane, the head is fixed by pinching the skin of the head and neck, the injection needle is carefully inserted into the mouse's nose about 7 mm, and the drug solution is slowly pushed in. After completion, the hose is carefully pulled out, and the whole process is as gentle as possible to avoid harm to the experimental animals.

[0197] Both groups of animals were continuously dosed for three days, once a day, and 3 hours after the last dose, the mice were heart perfused and the brain tissue was removed for organ imaging using the IVIS small animal imaging instrument.

[0198] Brain tissue section method:

[0199] 1) Anesthetize the mouse with 4% chloral hydrate, fix it on a foam board, cut open the chest, and expose the heart;

[0200] 2) Cut a small opening at the left auricle, insert a intravenous injection needle from the apex of the heart, slowly perfuse 40 mL of pre-cooled PBS solution (phosphate buffered saline), and after the blood is perfused clean, slowly perfuse 20 mL of pre-cooled 4% paraformaldehyde solution;

[0201] 3) Remove the mouse brain tissue and soak it in 4% paraformaldehyde at 4°C for 24 hours;

[0202] 4) Soak the brain tissue in 20% sucrose solution, and after it sinks to the bottom, place it in 30% sucrose solution for 24 hours;

[0203] 5) Frozen section: filter paper to absorb the surface moisture of the brain tissue, freeze in -80°C refrigerator, wrap the brain tissue in embedding agent when sectioning, section thickness is 20 μm, select brain slices of olfactory bulb, cerebral cortex, striatum, hippocampus, and cerebellum respectively and adhere to the adhesive glass slide, avoid light, dry, and store at 4°C;

[0204] 6) Select brain slices of each part of the brain tissue and take them out in advance to room temperature;

[0205] 7) Wash with PBS for 3 times, 5 minutes each time;

[0206] 8) Seal with DAPI (4', 6-diamidino-2-phenylindole) containing mounting fluid, dry, and take pictures with confocal microscope.

[0207] 3. Experimental results

[0208] Table 13: Fluorescence intensity results (average value) of each group

[0209] As can be seen from FIGS. 5, 6 and Table 13, the FITC-labeled neurotoxin nasal preparation can be absorbed through the nasal mucosa, penetrate the blood-brain barrier into the brain, and be distributed in various regions of the brain. Compared with the injection, the use of the neurotoxin nasal preparation provided in this study significantly improves the content of neurotoxin in the brain tissue. This result lays a good foundation for subsequent exploration of the treatment effect of neurotoxin nasal preparation on neurodegenerative diseases.

[0210] Obviously, the above embodiments are merely example for clearly illustrating but not limitation to the embodiments. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. Here, all the embodiments need not and can not be enumerated. The obvious changes or variations derived from the above description are still within the protection scope of the present application.

Claims

1. A lyophilized powder containing a neurotoxin, characterized in that, The neurotoxin, a stabilizer and an excipient, wherein the excipient comprises one or more of acidic gelatin, basic gelatin and sucrose, and the stabilizer comprises one or more of Pluronic F-68, Pluronic F-127 and Tween 80.

2. The lyophilized powder according to claim 1, characterized in that, The neurotoxin is a cobra neurotoxin.

3. The lyophilized powder according to claim 2, characterized in that, The cobra neurotoxin is a long-chain neurotoxin or a short-chain neurotoxin.

4. The lyophilized powder of claim 2, wherein, The cobra neurotoxin is cobra neurotoxin cobrotoxin or cobropeptide.

5. The lyophilized powder according to any one of claims 1 to 4, characterized in that, The lyophilized powder satisfies at least one of the following A-C: A. The lyophilized powder contains or does not contain a penetration enhancer; B. The lyophilized powder contains or does not contain a preservative; C. The lyophilized powder comprises 3.5 parts of neurotoxin, 200-5000 parts of excipient and 10-1300 parts of stabilizer by weight.

6. A process for the preparation of a lyophilized powder according to any one of claims 1 to 5, characterized in that, The lyophilized powder is prepared by lyophilizing a mixed solution containing neurotoxin, stabilizer and excipient.

7. The method of preparing a lyophilized powder according to claim 6, characterized in that, The concentration of the stabilizer in the mixed solution is 7.5-25 mg / mL; and / or, the concentration of the excipient in the mixed solution is 4-100 mg / mL; and / or, the concentration of the neurotoxin in the mixed solution is 1-1000 μg / mL; and / or, the mixed solution contains or does not contain a penetration enhancer; and / or, the mixed solution contains or does not contain a preservative.

8. The method of preparing a lyophilized powder according to claim 6, characterized in that, The preparation method of the lyophilized powder comprises the following steps: Weigh 250 parts by weight of acidic gelatin and 375 parts by weight of menthol, add 30 parts by volume of water, and dissolve at 40°C to prepare liquid 1; Weigh 375 parts by weight of Pluronic F-68, add 10 parts by volume of water, and dissolve at 40°C to prepare liquid 2; Remove 5 parts by volume of 0.70 mg / ml neurotoxin aqueous solution, liquid 1 and liquid 2, and 0.01 parts by volume of Tween 80, mix with or without water to obtain a mixed solution, and lyophilize the mixed solution to obtain a lyophilized powder; when the parts by weight unit is mg, the parts by volume unit is mL.

9. A nasal preparation, characterized by comprising The lyophilized powder prepared by the preparation method of any one of claims 6-8, further comprises a solvent, and the lyophilized powder and the solvent are packaged separately.

10. The nasal preparation according to claim 9, characterized in that Before use, the lyophilized powder is dissolved in a solvent to obtain a drug solution, and the concentration of the neurotoxin in the drug solution is 1-1000 μg / mL.

11. Use of the lyophilized powder of any one of claims 1-5 or the nasal preparation of any one of claims 9-10 in the preparation of an analgesic drug.

12. A method for treating central nervous system diseases, comprising administering the lyophilized powder of any one of claims 1-5 or the nasal preparation of any one of claims 9-10 to a patient in need.

13. Use of the lyophilized powder of any one of claims 1-5 or the nasal preparation of any one of claims 9-10 in the preparation of a drug for treating central nervous system diseases.

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