Freeze-dried powder and nasal preparation containing neurotoxin

Abstract

The present invention relates to the field of biomedicine, and specifically provides a lyophilized powder and nasal preparation containing neurotoxin, comprising neurotoxin, stabilizer and excipient; the excipient comprises one or more of acidic gelatin, alkaline gelatin and sucrose, and the stabilizer comprises one or more of block polyether F-68, block polyether F-127 and Tween 80; by using 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, a synergistic effect is produced, so that the stability of the neurotoxin at room temperature is significantly improved, the degradation of the neurotoxin is slowed down, and the time for maintaining the biological activity of the neurotoxin at more than 95% is significantly prolonged, and at the same time, the preparation can be administered nasally, which increases the penetration of the neurotoxin through the blood-brain barrier and greatly reduces the toxicity.

Description

Technical Field

[0001] The invention relates to the field of biomedicine, and in particular to a freeze-dried powder and a nasal preparation containing neurotoxin. Background Art

[0002] Snake venom is a natural protein secreted by the poison glands of snakes. It contains a variety of proteins, peptides, enzymes and other small molecules. It has strong biological activity and is one of the most studied and used animal toxins by humans. As people conduct in-depth research on snake venom neurotoxins, they have found that neurotoxins can be divided into three categories: presynaptic neurotoxins (β-neurotoxins), postsynaptic neurotoxins (α-neurotoxins) and neurotoxin-like toxins. The former two mainly inhibit neuromuscular conduction at the motor end plate, causing muscle paralysis and respiratory failure, while the latter can block smooth muscle contraction and ion channels, showing neurotoxin-like activity.

[0003] There are two kinds of cobras in my country. One is the Chinese cobra (Naja atra), also known as the Zhoushan cobra, which is distributed in most provinces of China. The other is the Thai cobra (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 snake venom. Since the first isolation of neurotoxins from cobra venom in 1965, the biochemistry and biological activity of cobra neurotoxins have been widely studied. Cobra neurotoxins belong to postsynaptic neurotoxins (α-neurotoxins) and are members of the three-finger protein superfamily. α-neurotoxins isolated from cobra venom can specifically and almost irreversibly bind to acetylcholine receptors (AChR). Therefore, since the 1960s, it has been an ideal tool for studying the structure and function of AChR. At the same time, because α-neurotoxins have high stability, contain multiple pairs of disulfide bonds, and have a large number of natural homologous proteins, they also become a good model for studying protein folding and structure-function relationships.

[0004] The venom of Chinese cobra contains two short-chain neurotoxins, of which the short-chain cobra neurotoxin (cobrotoxin, trade name cobrotoxin) consists of 62 amino acids. It is an alkaline protein with a molecular weight of about 7 kD, an isoelectric point of pH 8.8, and four disulfide bonds in the molecule. The venom of Thai cobra contains long-chain cobra neurotoxin (cobratoxin), which consists of 71 amino acids. Thai cobras also contain several short-chain neurotoxins. However, due to geographical separation, the Thai cobras distributed in China only contain three short-chain neurotoxins and do not contain long-chain cobra neurotoxin (cobratoxin).

[0005] The neurotoxin extracted from snake venom is highly hygroscopic and hydrophilic, easily soluble in water, and has poor stability in aqueous solutions. The storage conditions are harsh, and neither water injections nor powder injections can be left at room temperature. Commercially available water injections (such as cobotin injection) need to be stored at 0~10°C, and powder injections (such as cobotin for injection) need to be stored at 2~10°C. They require cold chain transportation, have high transportation costs, and a short effective storage period, which is very unfavorable for drug promotion and use. Therefore, how to improve the stability of neurotoxins is a technical problem that technicians in this field urgently need to solve. Summary of the invention

[0006] Therefore, the first technical problem to be solved by the present invention is to overcome the problem of low stability of existing preparations containing neurotoxins, and to provide a freeze-dried powder containing neurotoxins, in which the stability of the neurotoxins is significantly improved, and the preparations can be stored for a long time at room temperature without the need for cold chain transportation, which greatly reduces the cost of drug transportation and storage, reduces the risk of deterioration and degradation during storage or use, and ensures the safety and effectiveness of the medication. In order to change the current method of neurotoxins (such as cobotin injection) that can only be administered by injection, the present invention improves the stability of neurotoxins at room temperature while taking into account that the preparations are suitable for nasal administration after being reduced to a solution, so that the neurotoxins can pass through the blood-brain barrier and greatly reduce toxicity.

[0007] To this end, the present invention provides a lyophilized powder containing neurotoxin, including neurotoxin, stabilizer and excipient, wherein the excipient comprises one or more of acidic gelatin, alkaline gelatin and sucrose, and the stabilizer comprises one or more of block polyether F-68, block polyether F-127 and Tween 80.

[0008] In certain preferred embodiments, the excipient comprises acidic gelatin, and the stabilizer comprises block polyether F-68 and Tween 80.

[0009] Gelatin produced by alkaline hydrolysis is called alkaline gelatin. The isoelectric point of alkaline gelatin is 4.7-5.2.

[0010] Gelatin produced by acid hydrolysis is called acid gelatin. The isoelectric point of acid gelatin is 7.0-9.0.

[0011] Furthermore, the neurotoxin is cobra neurotoxin.

[0012] Furthermore, the cobra neurotoxin is a long-chain neurotoxin or a short-chain neurotoxin, such as a long-chain cobra neurotoxin (cobratoxin) or a short-chain cobra neurotoxin (cobrotoxin).

[0013] Among them, long-chain neurotoxins refer to neurotoxins with more than 63 amino acids (e.g., 63-74 amino acids), and short-chain neurotoxins refer to neurotoxins with less than 63 amino acids (e.g., 60-62 amino acids).

[0014] Furthermore, the neurotoxin is cobra neurotoxin cobrotoxin or cobotin.

[0015] Furthermore, the lyophilized powder satisfies at least one of the following AC:

[0016] A. The freeze-dried powder contains or does not contain a penetration enhancer;

[0017] B. The freeze-dried powder contains or does not contain a preservative;

[0018] C. In terms of weight, the lyophilized powder includes 3.5 parts of neurotoxin, 200-5000 parts of excipients and 10-1300 parts of stabilizers.

[0019] Furthermore, the penetration enhancer comprises borneol and / or menthol.

[0020] Furthermore, the preservative is a conventional preservative, such as benzalkonium chloride.

[0021] Furthermore, the mass ratio of the penetration enhancer to the neurotoxin is 3.5:20-400.

[0022] Further, the lyophilized powder does not contain a penetration enhancer (such as borneol and / or menthol). For example, the lyophilized powder is composed of the following components in parts by weight: neurotoxin: 3.5 parts, acid gelatin or alkaline gelatin: 200-300 parts, block polyether F-68: 375-1250 parts; or,

[0023] The freeze-dried powder is composed of the following components in parts by weight: neurotoxin: 3.5 parts, acid gelatin or alkaline gelatin: 200-300 parts, block polyether F-68: 375-1250 parts, and Tween 80: 10-12 parts.

[0024] In certain preferred 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.

[0025] In certain more preferred 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.

[0026] Further, the lyophilized powder contains 20-400 parts by weight of a penetration enhancer. That is, in terms of weight, the lyophilized powder includes 3.5 parts of neurotoxin, 200-5000 parts of excipients, 20-400 parts of penetration enhancers and 10-1300 parts of stabilizers. For example, the lyophilized powder is composed of the following components in weight: neurotoxin: 3.5 parts, acid gelatin or alkaline 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 weight: neurotoxin: 3.5 parts; acid gelatin or alkaline gelatin: 200-300 parts; menthol: 350-400 parts; block polyether F-68: 375-1250 parts; Tween 80: 10-12 parts.

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

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

[0029] The present invention also provides a method for preparing any of the above-mentioned lyophilized powders, wherein the lyophilized powder is prepared by freeze-drying a mixed solution containing a neurotoxin, a stabilizer and an excipient.

[0030] Furthermore, 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 is also mixed with a permeation enhancer.

[0031] Furthermore, the concentration of the penetration enhancer in the mixed solution is 0.5-7.5 mg / mL.

[0032] Furthermore, the mixed solution contains 1-1000 µg / ml of neurotoxin, 0.4-10% (w / v, g / 100 mL) of excipient and 0.75-2.5% (w / v, g / 100 mL) of stabilizer.

[0033] Furthermore, the mixed solution contains 1-1000 µg / ml of neurotoxin, 0.4-10% (w / v, g / 100 mL) of excipient, 0.05-0.75% (w / v, g / 100 mL) of permeation enhancer and 0.75-2.5% (w / v, g / 100 mL) of stabilizer.

[0034] Furthermore, the freeze-drying includes: 1) a pre-freezing stage: running at -45°C to -35 for 15 to 25 minutes, and running at -55 to -45°C for 1 to 2 hours; 2) a drying stage: vacuuming, maintaining the vacuum degree at 4-8 pa, running at -10 to -5°C for 4 to 6 hours, running at -3 to -1°C for 4 to 6 hours, running at -2 to 0°C for 10 to 14 hours, running at 0 to 2°C for 17 to 19 hours, and running at 18 to 22°C for 4 to 6 hours.

[0035] In certain preferred embodiments, the mixed solution contains 70 µg / ml of neurotoxin, 0.5% (w / v, g / 100 mL) of acidic gelatin, 0.75% (w / v, g / 100 mL) of menthol and 0.75% (w / v, g / 100 mL) of block polyether F-68.

[0036] In certain preferred embodiments, the mixed solution contains 70 µg / ml of neurotoxin, 0.5% (w / v, g / 100 mL) of acidic gelatin, 0.75% (w / v, g / 100 mL) of menthol, 0.75% (w / v, g / 100 mL) of block polyether F-68, and 0.02% (v / v, ml / 100 ml) of Tween 80.

[0037] Furthermore, the solvent of the mixed solution is water.

[0038] Furthermore, the method for preparing the mixed solution comprises the following steps: dissolving the neurotoxin in water to obtain liquid 1;

[0039] Dissolving the excipient in water to prepare liquid 2;

[0040] The stabilizer is dissolved in water to obtain liquid 3, and the liquid 1, liquid 2 and liquid 3 are mixed to obtain a mixed solution; or the stabilizer is mixed with liquid 1 and liquid 2 to obtain a mixed solution.

[0041] In certain embodiments, the process of mixing the excipient and water further includes the step of adding a penetration enhancer.

[0042] If necessary, conventional heating methods can be used to accelerate the dissolution of the remaining ingredients except the neurotoxin in water, for example, the heating temperature is 30-50°C.

[0043] In certain preferred embodiments, the method for preparing the lyophilized powder comprises the following steps:

[0044] Weigh 250 parts by weight of acidic gelatin and 375 parts by weight of menthol, add 30 parts by volume of water, and heat to dissolve at 40° C. to prepare liquid 1;

[0045] Weigh 375 parts by weight of block polyether F-68, add 10 parts by volume of water, and heat to dissolve at 40° C. to obtain liquid 2;

[0046] 5 parts by volume of a 0.70 mg / ml aqueous solution of neurotoxin, liquid 1 and liquid 2, and 0.01 parts by volume of Tween 80 are mixed, and water is added or not added to obtain a mixed solution, and the mixed solution is freeze-dried to obtain a freeze-dried powder; when the unit of the weight part is mg, the unit of the volume part is mL.

[0047] The second technical problem to be solved by the present invention is to overcome the problem of small safety range of water injection or powder injection treatment in the prior art, and provide a nasal preparation. The use of nasal spray greatly improves the safety range of drug treatment compared with the existing injection.

[0048] The present invention provides a nasal preparation, comprising any of the above-mentioned lyophilized powders or the lyophilized powder prepared by any of the above-mentioned preparation methods, and also comprising a solvent, wherein the lyophilized powder and the solvent are packaged independently.

[0049] The solvent may be a conventional solvent that can be used in pharmacy, such as water, phosphoric acid aqueous solution or physiological saline, etc. The solvent may contain a preservative or may not contain a preservative.

[0050] Furthermore, before use, the lyophilized powder is dissolved in a solvent to obtain a drug solution, wherein the concentration of the neurotoxin in the drug solution is 1-1000 µg / mL.

[0051] Preferably, the concentration of the neurotoxin in the drug solution is 30-100 µg / mL.

[0052] The nasal preparation may be a conventional nasal preparation such as a spray, an aerosol or nasal drops.

[0053] The technical solution of the present invention has the following advantages:

[0054] 1. The freeze-dried powder containing neurotoxin provided by the present invention includes neurotoxin, stabilizer and excipient; the excipient includes one or more of acidic gelatin, alkaline gelatin and sucrose, and the stabilizer includes one or more of block polyether F-68, block polyether F-127 and Tween 80; by using a stabilizer including one or more of block polyether F-68, block polyether F-127 and Tween 80 and an excipient including one or more of acidic gelatin, alkaline gelatin and sucrose, a synergistic effect is produced, so that the stability of the neurotoxin is significantly improved, the degradation of the neurotoxin is greatly alleviated, and the time for maintaining the biological activity of the neurotoxin at more than 95% is significantly prolonged.

[0055] In particular, the effect is better when acid gelatin is used as an excipient. Among them, the neurotoxin has better stability when block polyether F-68 and acid gelatin groups and block polyether F-68, Tween 80 and acid gelatin groups are added; the neurotoxin freeze-dried powder can be stable for at least one year at 25°C (HPLC purity ≥ 95%), which greatly reduces the risk of neurotoxin degradation or failure during storage, transportation and use; after the freeze-dried powder is dissolved with a solvent before use, the solution can be stable at room temperature (25°C) for at least 10 days, and the solution can be used for one week, so patients will not encounter the problem of degradation of the active ingredient in daily use. Therefore, the present invention is of great significance to improving the safety and effectiveness of clinical use of cobotide.

[0056] In addition, the study found that compared with using block polyether F-68, block polyether F-127, and Tween 80 alone as stabilizers, the combined use of block polyether F-68 and Tween 80 as stabilizers can significantly improve the stability of neurotoxins and significantly prolong the time that the neurotoxins are more than 95% biologically active.

[0057] 2. The preparation method of the freeze-dried powder containing neurotoxin provided by the present invention is simple to operate, easy to control quality, and convenient for large-scale production. In particular, the freeze-dried powder obtained by the freeze-drying procedure shown below has better stability. The freeze-drying includes: 1) pre-freezing stage: running at -45℃~-35 for 15~25 minutes, and running at -55~-45℃ for 1~2 hours; 2) drying stage: vacuuming, maintaining the vacuum degree at 4-8pa, running at -10~-5℃ for 4~6 hours, running at -3~-1℃ for 4~6 hours, running at -2~0℃ for 10~14 hours, running at 0~2℃ for 17~19 hours, and running at 18~22℃ for 4~6 hours.

[0058] 3. The nasal preparation provided by the present invention includes a lyophilized powder and a solvent, and the lyophilized powder and the solvent are packaged independently. Compared with the existing injection, the use of the nasal preparation greatly reduces the toxicity of the drug, greatly improves the safety range of drug treatment, and improves the safety of medication. The animal experiment of this study showed that the minimum lethal dose of the nasal preparation of the present invention for ICR mice was 1 mg / kg, compared with the minimum lethal dose of the traditional injection of 95 µg / kg. The minimum lethal doses of the two preparations differ by more than ten times. The therapeutic dose of the nasal preparation is 6 µg / kg-12 µg / kg, and the neurotoxin has no toxic and side effects at this dose. The implementation of the technology of the present invention can greatly reduce the toxicity of neurotoxin administration and improve the safety of neurotoxin use while ensuring the therapeutic effect.

[0059] 4. In the past, most neurotoxin drugs were injected and then entered the blood circulation to take effect. The main indication of commercially available neurotoxin drugs is analgesia. Due to the existence of the blood-brain barrier, the utilization of peripheral administration is low. The blood-brain barrier refers to the barrier between the plasma and brain cells formed by the walls of brain capillaries and glial cells, and the barrier between the plasma and cerebrospinal fluid formed by the choroid plexus. These barriers can prevent certain substances from entering the brain tissue from the blood. It is difficult and easy for a variety of solutes in the blood to enter the brain tissue from the brain capillaries; some pass quickly, some slowly, and some cannot pass at all. However, in order to exert a central analgesic effect or treat central nervous system diseases such as Parkinson's disease, drugs need to enter the central nervous system. However, peripheral administration of neurotoxins is believed to be unable to enter the central nervous system or only a very small amount can enter the central nervous system. Therefore, exploring new preparations of neurotoxins will be of great help in improving the utilization of neurotoxin drugs.

[0060] The nasal preparation provided by the present invention can also penetrate the blood-brain barrier and reach the drug action site. When menthol and / or borneol are used as penetration enhancers, the penetration rate of neurotoxins can be increased. Among them, it is preferred to select menthol as the penetration enhancer, which will not affect the stability of neurotoxins.

[0061] 5. Currently, all neurotoxin products on the market are injected intramuscularly and require professional medical staff to operate. Since most neurotoxin products need to be injected daily, and the treatment of their indications does not require hospitalization, patients need to go to the hospital for medication every day, which is extremely inconvenient. In addition, daily injections are prone to cause pain at the injection site, which further reduces the patient's compliance and makes it easy for them to give up treatment. The nasal preparation of the present invention reduces the pain of injection, and patients can take the medication themselves according to the doctor's instructions after dispensing, which improves the patient's compliance and greatly facilitates the use of patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0063] Figure 1 These are photos of each group of freeze-dried powder in Experimental Example 3 before being placed in a constant temperature and humidity chamber, from left to right: Sample 3-1, Sample 3-4, Sample 3-3, the freeze-dried powder prepared in Comparative Example 1, and Sample 3-2;

[0064] Figure 2 This is a photo of sample 3-1 in Experimental Example 3 placed in a constant temperature and humidity chamber for 150 days;

[0065] Figure 3 This is the analgesic result of the nasal preparation in Experimental Example 9;

[0066] Figure 4 This is the analgesic result of the injection in Experimental Example 9;

[0067] Figure 5 This is the brain tissue imaging result after drug administration in Experimental Example 10;

[0068] Figure 6 These are the staining results of different brain tissues in Experimental Example 10. DETAILED DESCRIPTION

[0069] The following examples are provided for a better understanding of the present invention, but are not intended to limit the best mode of implementation, nor to limit the content and protection scope of the present invention. Any product identical or similar to the present invention obtained by anyone under the inspiration of the present invention or by combining the features of the present invention with other prior arts shall fall within the protection scope of the present invention.

[0070] If no specific experimental steps or conditions are specified in the examples, the conventional experimental steps or conditions described in the literature in the field can be used. If no manufacturer is specified for the reagents or instruments used, they are all conventional reagent products that can be obtained commercially.

[0071] In the following neurotoxin stability test, the purity of the neurotoxin was determined by high performance liquid chromatography, and the chromatographic conditions were as follows:

[0072] Chromatographic column: model Sepax Bio-C18 4.6×150 mm; detection wavelength: 280 nm; mobile phase A: acetonitrile; mobile phase B: 0.1% trifluoroacetic acid; flow rate: 1 mL / min; injection volume: 20 μL; column temperature: 40°C. Gradient elution according to Table 1:

[0073] Table 1 Gradient elution table

[0074]

[0075] Injection sequence: Inject blank solvent and test solution into the liquid chromatograph in the following order and record the chromatogram. Blank ≥ 1 injection, test solution 1 injection.

[0076] In the sample chromatogram, the percentage of the area of ​​the neurotoxin chromatographic peak to the area of ​​all chromatographic peaks is the purity of the neurotoxin calculated by the area normalization method.

[0077] The raw material neurotoxin powder used in each embodiment, comparative example and experimental example of the present invention is Chinese cobra neurotoxin separated and purified by Suzhou Renben Pharmaceutical. After sequencing, the neurotoxin is composed of 62 amino acids with a molecular weight of 6944 Daltons. The amino acid sequence obtained by protein full coverage amino acid sequencing is lechnqqssq tptttgcsggetncykkrwr dhrgyrterg cgcpsvkngi einccttdrc nn (see SEQ ID No: 1), which is consistent with the amino acid sequence of cobrotoxin in the gene library.

[0078] Acidic gelatin was purchased from Yuanye Biotechnology, model: M241S210427, specification: S25197-100g; alkaline gelatin was purchased from Yuanye Biotechnology, model: J29HS184618, specification: S30925-100g.

[0079] Example 1

[0080] This embodiment provides a freeze-dried powder, the product specifications are: in terms of neurotoxin, the content of each bottle is 0.07 mg, and the prescription of 50 bottles is: neurotoxin 3.5 mg, acid gelatin 250 mg, menthol 375 mg, block polyether F-68 375 mg. The preparation method is as follows:

[0081] (1) Weigh 35 mg of neurotoxin and dissolve it in pure water in a 50 ml volumetric flask to make a neurotoxin stock solution with a concentration of 0.70 mg / ml.

[0082] (2) Weigh 250 mg of acid gelatin and 375 mg of menthol into another 50 ml volumetric flask, add 30 ml of pure water, and heat to dissolve in a 40°C water bath. Weigh 375 mg of block polyether F-68 and 10 ml of pure water, heat to dissolve in a 40°C water bath, and transfer to the above volumetric flask. Transfer 5 ml of the neurotoxin stock solution into the above volumetric flask, dilute to 50 ml with pure water, and shake well to obtain a mixed solution.

[0083] (3) Pipette 1 ml of the mixed solution into 15 4 ml vials and freeze-dry to obtain 15 vials of freeze-dried powder. The freeze-drying procedure is as follows: 1) Pre-freezing stage: run at -40°C for 20 minutes, run 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, run at -2°C for 5 hours, run at -2°C for 12 hours, run at 0°C for 18 hours, and run at 20°C for 5 hours.

[0084] This embodiment also provides a nasal preparation, including a lyophilized powder prepared according to the method of this embodiment and a solvent. The solvent is normal saline, and the preparation method is: weigh 0.9 g of sodium chloride to a 100 ml volumetric flask, dilute to 100 ml with pure water, and sterilize to obtain the nasal preparation.

[0085] The lyophilized powder and solvent are packaged separately. When used, 1 mL of solvent is injected into each bottle (containing 0.07 mg of neurotoxin) of lyophilized powder and shaken, and the lyophilized powder is quickly reconstituted. After reconstitution, the concentration of neurotoxin in the drug solution is 70 g / 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.

[0086] Example 2

[0087] This embodiment provides a freeze-dried powder, the product specifications are: in terms of neurotoxin, the content of each bottle is 0.07 mg, and the prescription of 50 bottles is: neurotoxin 3.5 mg, acid gelatin 250 mg, menthol 375 mg, block polyether F-68 625 mg. The preparation method is as follows:

[0088] Weigh 250 mg of acid gelatin and 375 mg of menthol into another 50 ml volumetric flask, add 30 ml of pure water, and heat to dissolve in a 40°C water bath. Weigh 625 mg of block polyether F-68 and 10 ml of pure water, heat to dissolve in a 40°C water bath, and transfer to the above volumetric flask. Transfer 5 ml of the neurotoxin mother solution prepared in Example 1 into the above volumetric flask, dilute to 50 ml with pure water, and shake well to obtain a mixed solution. Transfer 1 ml of the mixed solution to 15 4 ml vials for freeze drying to obtain 15 bottles of freeze-dried powder. The freeze-drying procedure is the same as in Example 1.

[0089] This embodiment also provides a nasal preparation, which is basically the same as Example 1, except that the lyophilized powder prepared by the method of this embodiment is used instead of the lyophilized powder prepared by 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.

[0090] Example 3

[0091] This embodiment provides a freeze-dried powder, the product specifications are: in terms of neurotoxin, the content of each bottle is 0.07 mg, and the prescription of 50 bottles is: neurotoxin 3.5 mg, acid gelatin 250 mg, menthol 375 mg, block polyether F-68 1000 mg. The preparation method is as follows:

[0092] Weigh 250 mg of acid gelatin and 375 mg of menthol into another 50 ml volumetric flask, add 30 ml of pure water, and heat in a 40°C water bath to dissolve. Weigh 1 g of block polyether F-68 and 10 ml of pure water, heat in a 40°C water bath to dissolve, and transfer to the above volumetric flask. Transfer 5 ml of the neurotoxin mother solution prepared in Example 1 into the above volumetric flask, dilute to 50 ml with pure water and shake well to obtain a mixed solution. Transfer 1 ml of the mixed solution to 15 4 ml vials for freeze drying to obtain 15 bottles of freeze-dried powder. The freeze-drying procedure is the same as in Example 1.

[0093] This embodiment also provides a nasal preparation, which is basically the same as Example 1, except that the lyophilized powder prepared by the method of this embodiment is used instead of the lyophilized powder prepared by 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.

[0094] Example 4

[0095] This embodiment provides a freeze-dried powder, the product specifications are: in terms of neurotoxin, the content of each bottle is 0.07 mg, and the prescription of 50 bottles is: neurotoxin 3.5 mg, acid gelatin 250 mg, menthol 375 mg, block polyether F-68 1250 mg. The preparation method is as follows:

[0096] Weigh 250 mg of acid gelatin and 375 mg of menthol into another 50 ml volumetric flask, add 30 ml of pure water, and heat to dissolve in a 40°C water bath. Weigh 1.25 g of block polyether F-68 and 10 ml of pure water, heat to dissolve in a 40°C water bath, and transfer to the above volumetric flask. Transfer 5 ml of the neurotoxin mother solution prepared in Example 1 into the above volumetric flask, dilute to 50 ml with pure water, and shake well to obtain a mixed solution. Transfer 1 ml of the mixed solution to 15 4 ml vials for freeze drying to obtain 15 bottles of freeze-dried powder. The freeze-drying procedure is the same as in Example 1.

[0097] This embodiment also provides a nasal preparation, which is basically the same as Example 1, except that the lyophilized powder prepared by the method of this embodiment is used instead of the lyophilized powder prepared by 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.

[0098] Comparative Example 1

[0099] This comparative example provides a freeze-dried powder, the product specification is: in terms of neurotoxin, the content of each bottle is 0.07 mg, and the prescription of 50 bottles is: neurotoxin 3.5 mg, acid gelatin 250 mg. The preparation method is as follows:

[0100] (1) Weigh 35 mg of neurotoxin and dissolve it in pure water in a 50 ml volumetric flask to make a neurotoxin stock solution with a concentration of 0.70 mg / ml.

[0101] (2) Weigh 250 mg of acid gelatin into another 50 ml volumetric flask, add 30 ml of pure water, and heat in a 40°C water bath to dissolve. Pipette 5 ml of the neurotoxin mother solution into the above volumetric flask and dilute to 50 ml with pure water, shaking well to obtain a mixed solution. Pipette 1 ml of the mixed solution into 15 4 ml vials for freeze drying to obtain 15 bottles of freeze-dried powder. The freeze drying procedure is the same as in Example 1.

[0102] This comparative example also provides a nasal preparation, which is basically the same as Example 1, except that the lyophilized powder prepared by the method of this comparative example is used instead of the lyophilized powder prepared by the method of Example 1, and the lyophilized powder is reconstituted with physiological saline to obtain a drug solution without menthol and block polyether F-68.

[0103] Comparative Example 2

[0104] This comparative example provides a freeze-dried powder, the product specification is: in terms of neurotoxin, the content of each bottle is 0.07 mg, and the prescription of 50 bottles is: neurotoxin 3.5 mg, acid gelatin 250 mg, menthol 375 mg. The preparation method is as follows:

[0105] (1) Weigh 35 mg of neurotoxin and dissolve it in pure water in a 50 ml volumetric flask to make a neurotoxin stock solution with a concentration of 0.70 mg / ml.

[0106] (2) Weigh 250 mg of acid gelatin and 375 mg of menthol into another 50 ml volumetric flask, add 30 ml of pure water, and heat in a 40°C water bath to dissolve. Pipette 5 ml of the neurotoxin mother solution into the above volumetric flask and dilute to 50 ml with pure water, shaking well to obtain a mixed solution. Pipette 1 ml of the mixed solution into 15 4 ml vials for freeze drying to obtain 15 bottles of freeze-dried powder. The freeze drying procedure is the same as in Example 1.

[0107] This comparative example also provides a nasal preparation, which is basically the same as Example 1, except that the lyophilized powder prepared by the method of this comparative example is used instead of the lyophilized powder prepared by the method of Example 1, and the lyophilized powder is reconstituted with physiological saline to obtain a drug solution without block polyether F-68.

[0108] Experimental Example 1

[0109] The vials loaded with the lyophilized powders of Examples 1-4 and Comparative Examples 1-2 were sealed with caps and placed in a constant temperature and humidity chamber at a temperature of 40°C and a humidity of 50%±5%RH. Before placement (recorded as 0 month), and at the same time of 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 after placement, 1 vial of lyophilized powder was reconstituted with 1 ml of normal saline, and the purity of the neurotoxin in the reconstituted solution was tested. The results are shown in Table 2.

[0110] Table 2 Stability and degradation time of neurotoxins

[0111]

[0112] The experiment ended when the purity dropped below 95%.

[0113] It can be seen from the results in Table 2 that compared with the freeze-dried powder prepared in Comparative Examples 1 and 2, the stability of the neurotoxin in the freeze-dried powder comprising neurotoxin, stabilizer and excipient in Examples 1-4 of the present invention is significantly improved, the degradation of the neurotoxin is greatly alleviated, and the time for maintaining the biological activity of the neurotoxin at more than 95% is significantly prolonged, from 8 months to 9-11 months.

[0114] In addition, compared with Examples 1-4, it can be seen that Examples 1-3 (especially Example 1) can further improve the stability of the neurotoxin by limiting the weight ratio or concentration of the block polyether F-68 within a preferred range.

[0115] Experimental Example 2

[0116] 1 mg of neurotoxin powder was weighed and dispensed into 22 vials, which were sealed with caps and evenly divided into two groups. One group was placed in a constant temperature and humidity chamber at 25°C and 50%±5%RH, and the other group was placed in a constant temperature and humidity chamber at 40°C and 50%±5%RH. At the same time every 3 months, 1 bottle of neurotoxin powder was reconstituted with 1 mL of normal saline, and the purity of the neurotoxin in the reconstituted solution was tested. The results are shown in Table 3.

[0117] Table 3 Stability and degradation time of neurotoxins

[0118]

[0119] From the results in Table 3, it can be seen that the raw material neurotoxin powder can maintain more than 95% biological activity of the neurotoxin in the 40°C accelerated test for only 3 months, while the neurotoxin can maintain more than 95% biological activity at room temperature for 9 months, which is less than 12 months.

[0120] Experimental Example 3 Excipient Selection

[0121] The purpose of this experimental example is to investigate the effects of different excipients on lyophilized powder.

[0122] Sample 3-1: Weigh 5 g of sucrose into a 50 ml volumetric flask, add 30 ml of pure water, and heat in a 40°C water bath to dissolve. Pipette 5 ml of the neurotoxin mother solution prepared in Comparative Example 1 into the above volumetric flask, dilute to 50 ml with pure water, and shake well to obtain a mixed solution. Pipette 1 ml of the mixed solution into 15 4 ml vials for freeze drying. The freeze drying procedure is the same as in Example 1, and 15 bottles of freeze-dried powder are obtained.

[0123] Sample 3-2: Weigh 250 mg of alkaline gelatin into a 50 ml volumetric flask, add 30 ml of pure water, and heat in a 40°C water bath to dissolve. Pipette 5 ml of the neurotoxin mother solution prepared in Comparative Example 1 into the above volumetric flask and dilute to 50 ml with pure water and shake well to obtain a mixed solution. Pipette 1 ml of the mixed solution into 15 4 ml vials for freeze drying. The freeze drying procedure is the same as in Example 1 to obtain 15 bottles of freeze-dried powder.

[0124] Sample 3-3: Weigh 200 mg of glycine into a 50 ml volumetric flask, add 30 ml of pure water, and heat in a 40°C water bath to dissolve. Pipette 5 ml of the neurotoxin mother solution prepared in Comparative Example 1 into the above volumetric flask and dilute to 50 ml with pure water, shaking well to obtain a mixed solution. Pipette 1 ml of the mixed solution into 15 4 ml vials for freeze drying, and the freeze drying procedure is the same as in Example 1 to obtain 15 bottles of freeze-dried powder.

[0125] Sample 3-4: Weigh 5 g of mannitol into a 50 ml volumetric flask, add 30 ml of pure water, and heat in a 40°C water bath to dissolve. Pipette 5 ml of the neurotoxin mother solution prepared in Comparative Example 1 into the above volumetric flask, dilute to 50 ml with pure water, and shake well to obtain a mixed solution. Pipette 1 ml of the mixed solution into 15 4 ml vials for freeze drying. The freeze drying procedure is the same as in Example 1, and 15 bottles of freeze-dried powder are obtained.

[0126] The vials loaded with samples 3-1 to 3-4 were sealed with caps and placed in a constant temperature and humidity chamber at 40°C and 50%±5%RH. At the same time every month, 1 bottle of freeze-dried powder was reconstituted with 1 ml of saline, and the purity of the neurotoxin in the reconstituted solution was tested. The purity results of the comparison with those of comparative example 1 in experimental example 1 are shown in Table 4.

[0127] Table 4 Stability and degradation time of neurotoxins

[0128]

[0129] The experiment ended when the purity dropped below 95%.

[0130] From the results in Tables 3 and 4, it can be seen that when glycine and mannitol are used as excipients, the time for maintaining the biological activity of neurotoxins at more than 95% in the 40°C accelerated experiment is only 2-3 months, which cannot effectively improve the stability of neurotoxins. However, when acid gelatin and alkaline gelatin are used as excipients, the time for maintaining the biological activity of neurotoxins at more than 95% in the 40°C accelerated experiment is extended to 7-8 months, which significantly improves the stability of neurotoxins, especially acid gelatin.

[0131] Among them, sucrose as an excipient dissolved after liquefaction at 150 days and did not form, while the other groups had good formability within 9 months of placement, such as Figure 1-2 .

[0132] Experimental Example 4 Investigation of penetration enhancers

[0133] The purpose of this experimental example is to investigate the effects of different penetration enhancers on lyophilized powder.

[0134] Sample 4-1: Weigh 250 mg of acid gelatin and 25 mg of borneol into a 50 ml volumetric flask, add 30 ml of pure water, and heat in a 40°C water bath to dissolve. Pipette 5 ml of the neurotoxin mother solution prepared in Comparative Example 2 into the above volumetric flask and dilute to 50 ml with pure water and shake well to obtain a mixed solution. Pipette 1 ml of the mixed solution into 15 4 ml vials for freeze drying. The freeze drying procedure is the same as in Example 1 to obtain 15 bottles of freeze-dried powder.

[0135] Sample 4-2: Weigh 250 mg of alkaline gelatin and 375 mg of menthol into a 50 ml volumetric flask, add 30 ml of pure water, and heat in a 40°C water bath to dissolve. Pipette 5 ml of the neurotoxin mother solution prepared in Comparative Example 2 into the above volumetric flask, dilute to 50 ml with pure water and shake well to obtain a mixed solution. Pipette 1 ml of the mixed solution into 15 4 ml vials for freeze drying. The freeze drying procedure is the same as in Example 1, and 15 bottles of freeze-dried powder are obtained.

[0136] Sample 4-3: Weigh 250 mg of alkaline gelatin and 50 mg of borneol into a 50 ml volumetric flask, add 30 ml of pure water, and heat in a 40°C water bath to dissolve. Pipette 5 ml of the neurotoxin mother solution prepared in Comparative Example 2 into the above volumetric flask, dilute to 50 ml with pure water and shake well to obtain a mixed solution. Pipette 1 ml of the mixed solution into 15 4 ml vials for freeze drying. The freeze drying procedure is the same as in Example 1, and 15 bottles of freeze-dried powder are obtained.

[0137] The vials loaded with samples 4-1 to 4-3 were sealed with caps and placed in a constant temperature and humidity chamber at 40°C and 50%±5%RH. At the same time every month, 1 bottle of freeze-dried powder was reconstituted with 1 ml of saline, and the purity of the neurotoxin in the reconstituted solution was tested. The purity results of the comparison with those of comparative example 2 in experimental example 1 are shown in Table 5.

[0138] Table 5 Stability and degradation time of neurotoxins

[0139]

[0140] The experiment ended when the purity dropped below 95%.

[0141] From the results in Table 5, it can be seen that when menthol is used as a penetration enhancer, the stability of the neurotoxin is better than that of borneol. When acid gelatin and menthol are used in combination, the biological activity of the neurotoxin can be maintained at more than 95% and extended to 8 months in the 40°C accelerated experiment, which significantly improves the stability of the neurotoxin.

[0142] It can be seen from the results in Tables 4 and 5 that the use of borneol will affect the stability of neurotoxins and weaken the degree to which the excipient improves the stability of neurotoxins, while menthol will not, so menthol is preferred.

[0143] Experimental Example 5 Investigation of Stabilizers

[0144] The purpose of this experimental example is to investigate the effects of different stabilizers on freeze-dried powder.

[0145] Weigh 35 mg of neurotoxin and dissolve it in Watson's water in a 50 ml volumetric flask to make up to volume to prepare the neurotoxin stock solution with a concentration of 0.70 mg / ml.

[0146] Sample 5-1: Weigh 250 mg of acid gelatin and 375 mg of menthol into another 50 ml volumetric flask, add 30 ml of pure water, and heat to dissolve in a 40°C water bath. Weigh 375 mg of block polyether F-68 and 10 ml of pure water, heat to dissolve in a 40°C water bath, and transfer to the above volumetric flask. Transfer 5 ml of neurotoxin mother solution to the above volumetric flask, dilute to 50 ml with pure water and shake well to obtain a mixed solution. Transfer 1 ml of the mixed solution to 15 4 ml vials for freeze drying. The freeze drying procedure is the same as in Example 1, and 15 bottles of freeze-dried powder are obtained. The freeze drying procedure is the same as in Example 1.

[0147] Sample 5-2: The preparation method is 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 is used instead of 375 mg of block polyether F-68.

[0148] Sample 5-3: The preparation method is basically 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 basically the same as that of sample 5-1, except that 0.01 ml Tween 80 is added. Specifically, 250 mg of acid gelatin and 375 mg of menthol are weighed and added to another 50 ml volumetric flask. 30 ml of pure water are added and heated in a 40°C water bath to dissolve. 375 mg of block polyether F-68 and 10 ml of pure water are weighed and heated in a 40°C water bath to dissolve, and then transferred to the above volumetric flask. 0.01 ml of Tween 80 is transferred to the above volumetric flask. 5 ml of the mother solution of neurotoxin is transferred to the above volumetric flask. Pure water is adjusted to 50 ml and shaken to obtain a mixed solution. 1 ml of the mixed solution is transferred to 15 4 ml vials for freeze drying. The freeze drying procedure is the same as in Example 1, and 15 bottles of freeze-dried powder are obtained.

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

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

[0152] Table 6 Stability and degradation time of neurotoxins

[0153]

[0154] The experiment ended when the purity dropped below 95%.

[0155] From the results of Table 6 and Table 2, it can be seen that compared with Comparative Examples 1 and 2, the stability of neurotoxin in freeze-dried powder can be further improved by adding stabilizers in samples 5-1 to 5-5. The time for maintaining more than 95% biological activity of neurotoxin in the 40°C accelerated test was extended from 8 months to 9-12 months. Among them, the combined use of Tween 80 and F-68 had the best effect.

[0156] Experimental Example 6 Investigation of preservatives

[0157] Weigh 35 mg of neurotoxin and dissolve it in pure water in a 50 ml volumetric flask to make up to volume to prepare the neurotoxin stock solution with a concentration of 0.70 mg / ml.

[0158] Sample 6-1: Weigh 250 mg of acid gelatin and 375 mg of menthol into another 50 ml volumetric flask, add 30 ml of pure water, and heat to dissolve in a 40°C water bath. Weigh 375 mg of block polyether F-68 and 10 ml of pure water, heat to dissolve in a 40°C water bath, transfer to the above volumetric flask, transfer 5 ml of neurotoxin mother solution into the above volumetric flask, dilute to 50 ml with pure water, and shake well to obtain a mixed solution. Transfer 1 ml of the mixed solution into 15 4 ml vials for freeze drying to obtain 15 bottles of freeze-dried powder, seal with a cap, and place in a constant temperature and humidity chamber at a temperature of 40°C and a humidity of 50%±5%RH. Weigh 0.9 g of sodium chloride into a 100 ml volumetric flask, dilute to 100 ml with pure water, and sterilize and dispense. Redissolve the freeze-dried powder with 1 ml of normal saline at the same time every month to test the purity of the neurotoxin.

[0159] Sample 6-2: Weigh 250 mg of acid gelatin and 375 mg of menthol into another 50 ml volumetric flask, add 25 ml of pure water, and heat to dissolve in a 40°C water bath. Weigh 375 mg of block polyether F-68 and 10 ml of pure water, heat to dissolve in a 40°C water bath, and transfer to the above volumetric flask. Weigh 5 mg of benzalkonium chloride, dissolve in pure water, and transfer to the above volumetric flask. Transfer 5 ml of neurotoxin mother solution to the above volumetric flask, dilute to 50 ml with pure water, and shake well to obtain a mixed solution. Transfer 1 ml of the mixed solution to 15 4 ml vials for freeze drying, and obtain 15 bottles of freeze-dried powder. After sealing with a cap, place in a constant temperature and humidity chamber at a temperature of 40°C and a humidity of 50%±5%RH. Weigh 0.9 g of sodium chloride into a 100 ml volumetric flask, dilute to 100 ml with pure water, and dispense after sterilization. At the same time every month, reconstitute the lyophilized powder with 1 ml of normal saline to test the purity of the neurotoxin.

[0160] Sample 6-3: Weigh 250 mg of acid gelatin and 375 mg of menthol into another 50 ml volumetric flask, add 30 ml of pure water, and heat to dissolve in a 40°C water bath. Weigh 375 mg of block polyether F-68 and 10 ml of pure water, heat to dissolve in a 40°C water bath, and transfer to the above volumetric flask. Transfer 5 ml of the neurotoxin mother solution to the above volumetric flask and dilute to 50 ml with pure water and shake well to obtain a mixed solution. Transfer 1 ml of the mixed solution to 15 4 ml vials for freeze drying to obtain 15 bottles of freeze-dried powder, seal them with a cap, and place them in a constant temperature and humidity chamber at 40°C and 50%±5%RH. Weigh 0.9 g of sodium chloride and 0.01 g of benzalkonium chloride into a 100 ml volumetric flask, dilute to 100 ml with pure water, and sterilize and package. Redissolve the freeze-dried powder with 1 ml of normal saline at the same time every month to test the purity of the neurotoxin.

[0161] Table 7 Stability and degradation time of neurotoxins

[0162]

[0163] The data showed that there was no significant difference among the three groups, indicating that the scheme of the present invention can ensure the stability of the purity of neurotoxins in the absence of preservatives.

[0164] Experimental Example 7 Room-temperature stability of the solution obtained after reconstitution

[0165] The purpose of this experimental example is to investigate the synergistic effect of stabilizers and excipients on the stability of lyophilized powder.

[0166] 1.1 Weigh 35 mg of neurotoxin and dissolve it in a 50 ml volumetric flask with Watson's water to make up to volume to prepare the neurotoxin stock solution with a concentration of 0.70 mg / ml.

[0167] 1.2 Sample 7-1: Weigh 250 mg of acid gelatin and 375 mg of menthol into a 50 ml volumetric flask, add 30 ml of pure water, heat in a 40°C water bath to dissolve, transfer 5 ml of the neurotoxin stock solution into a volumetric flask, dilute to 50 ml with pure water, shake well, and obtain a mixed solution. Transfer 1 ml of the mixed solution into 8 4 ml vials (8 bottles);

[0168] Sample 7-2: Weigh 375 mg of menthol and add 30 ml of pure water, heat in a 40°C water bath to dissolve, weigh 375 mg of block polyether F-68 and add 10 ml of pure water to dissolve in a 40°C water bath, transfer the two solutions to a 50 ml volumetric flask, add 0.01 ml of Tween 80, transfer 5 ml of the neurotoxin mother solution to a volumetric flask and dilute to 50 ml with pure water, shake well, and obtain a mixed solution. Transfer 1 ml of the mixed solution to 8 4 ml vials (8 vials);

[0169] Sample 7-3: Weigh 250 mg of acid gelatin, 375 mg of menthol, add 30 ml of pure water, heat in a 40℃ water bath to dissolve, weigh 375 mg of block polyether F-68, 10 ml of pure water, heat in a 40℃ water bath to dissolve, transfer the above two solutions to a 50 ml volumetric flask, mix, add 0.01 ml of Tween 80, transfer 5 ml of neurotoxin mother solution to a volumetric flask, dilute to 50 ml with pure water, shake well, and obtain a mixed solution. Transfer 1 ml of the mixed solution to 8 4 ml vials (8 vials).

[0170] 1.3 The above three groups of samples (samples 7-1, 7-2 and 7-3) were packaged and placed in a freeze dryer for freeze drying. The freeze drying procedure was the same as in Example 1.

[0171] 1.4 After freeze-drying, press the lid, seal it and place it in a constant temperature and humidity chamber at 40°C and 50%±5%RH. At the same time every month, reconstitute it with 1 ml of 0.9% sodium chloride solution and test the purity for stability assessment. Tukey's multiple comparison test was used to analyze the test results and calculate the p value between the two groups. The results are shown in Tables 8 and 9.

[0172] Table 8 Stability and degradation time of neurotoxins

[0173]

[0174] Table 9 Tukey's multiple comparison test

[0175]

[0176] The lower the relative standard deviation, the better the stability. Comparative data show that the stability of sample 7-3 mixed with excipients and stabilizers is better than that of a single added component, and the difference is significant, indicating that the excipients and stabilizers play a synergistic role.

[0177] After placing sample 7-3 in a constant temperature and humidity chamber at 40°C and 50%±5%RH for 6 months, it was taken out, re-dissolved with 1ml of 0.9% sodium chloride solution, and placed in a constant temperature and humidity chamber at 25°C and 50%±5%RH to measure the purity stability at the same time every two days. The results are shown in Table 10.

[0178] Table 10 Purity of neurotoxins (%)

[0179]

[0180] The data showed that after dissolving the freeze-dried powder with a solvent, the solution could remain stable at room temperature (25°C) for at least 10 days, meeting the requirement that the preparation be used for one week.

[0181] Experimental Example 8 Safety

[0182] (1) 40 ICR mice (22.0-25.0 g) were randomly divided into 4 groups, with 10 mice in each group, half male and half female. Mice were anesthetized with cotton balls soaked in anhydrous ether before intranasal administration. Neurotoxin freeze-dried powder was prepared according to the method of Example 1, dissolved in physiological saline, and prepared into solutions (nasal preparations) with concentrations of 9.2 mg / mL, 18.4 mg / mL, 0.92 mg / mL, and 1.38 mg / mL. The solutions were administered intranasally (see Experimental Example 10 for intranasal or transnasal administration) at 2.5 μL / mouse and 5 μL / mouse. The dosages are shown in Table 11. The symptoms and deaths of mice were observed and recorded.

[0183] Table 11 Experimental results

[0184]

[0185] The data show that after nasal administration of 200µg / kg and 300µg / kg of neurotoxin nasal preparations, all experimental mice survived; after nasal administration of 1 mg / kg of neurotoxin nasal preparations, two of 10 experimental mice died; after nasal administration of 2 mg / kg of neurotoxin nasal preparations, four of 10 experimental mice died. The above data show that the minimum lethal dose of this batch of neurotoxin nasal preparations is 1 mg / kg.

[0186] (2) Eighteen ICR mice (18.0-22.0 g) were randomly divided into three groups, with six mice in each group. 771.4 µL, 814.2 µL, and 857.1 µL of the commercially available 35 µg / mL cobotin injection (Tonghua Wellcome Biopharmaceutical Co., Ltd.) were taken and placed in 5 mL EP tubes, respectively, and the volume was adjusted to 3 mL with saline to prepare cobotin solutions of different concentrations. The groups and dosages were 90 µg / kg, 95 µg / kg, and 100 µg / kg of cobotin, and subcutaneous injection was performed at a volume of 0.1 mL / 10 g to observe the status of the mice and record whether they survived. The experimental results are shown in Table 12.

[0187] Table 12 Minimum lethal dose

[0188]

[0189] The data show that after subcutaneous injection of 90µg / kg of cobotide at a volume of 0.1 mL / 10 g, all 6 mice survived; after subcutaneous injection of 95µg / kg of cobotide at a volume of 0.1 mL / 10 g, only one male mouse died among the 6 mice; after subcutaneous injection of 100µg / kg of cobotide at a volume of 0.1 mL / 10 g, all 6 mice died. The above data show that the minimum lethal dose of this batch of cobotide injection is 95µg / kg.

[0190] This experiment shows that the minimum lethal dose of the neurotoxin nasal preparation of the present invention is 1 mg / kg, and the minimum lethal dose of cobotin is 95µg / kg, which is more than ten times the difference. Therefore, compared with cobotin, the nasal preparation greatly reduces the toxicity of the neurotoxin and improves the safety.

[0191] Experimental Example 9 Analgesia Experiment

[0192] Animal screening was carried out the day before the experiment. Mice were placed on a 55°C hot plate. Thermal stimulation of the mice's feet produced a painful licking response. The time when the mice first licked their hind paws was recorded, i.e., the pain threshold. Mice with a pain threshold of more than 30 seconds were eliminated.

[0193] (1) Experimental Animals Female ICR mice were selected, and the basal pain threshold of the mice was measured. The mice were placed on a hot plate at 55°C, and the licking of the feet by the mice was used as an observation index. 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. Neurotoxin freeze-dried powder was prepared according to the method in Example 1, and 70 μg / ml neurotoxin solution was obtained after dissolving it with physiological saline. The test drug solution of different concentrations was obtained by diluting with physiological saline. After the basal pain threshold of each mouse in each group was measured and recorded before administration (0 hours), the two treatment groups were given the test drug solution intranasally. The dosage of the neurotoxin was 6 and 12 μg / kg animal body weight, respectively, and the administration volume for each mouse was 0.8 μL / 10g (mouse body weight). The control group was given the same volume of physiological saline intranasally. The intranasal administration operation is shown in 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 s, the mouse was removed and calculated as 60 s) to observe the analgesic effect of different doses of neurotoxin.

[0194] Results Figure 3 As shown, after using the nasal preparation of the neurotoxin provided in this study, the mice had a significant analgesic effect at a dose of 6µg / kg and 12µg / kg compared with the control group (p<0.01 or p<0.001), and showed a good dose relationship.

[0195] (2) Take 35 µg / mL of cobotin injection and dilute it with sodium chloride injection to a solution of 0.6 µg / ml (6 µg / kg) or 0.9 µg / ml (9 µg / kg) for experimental use. Prepare it for immediate use.

[0196] Twenty-four mice with qualified pain threshold were selected and randomly divided into three groups, each with 8 mice, namely, the 6 µg / kg group of cobotide injection, the 9 µg / kg group of cobotide injection and the control group. After the basal pain threshold of each mouse in each group was recorded before administration (0 hour), the corresponding concentration of cobotide injection was subcutaneously injected in the drug-treated group, and the same volume of sodium chloride injection was subcutaneously injected in the normal control group. After administration, the pain threshold of each mouse in each group was measured and recorded at 0.5 h, 1 h, 2 h, 3 h, 4 h, and 5 h. The time the mice were placed on the hot plate was limited to 60 seconds, and the pain threshold greater than 60 seconds was counted as 60 seconds.

[0197] Results Figure 4 As shown, after using the neurotoxin injection, compared with the control group, the mice had no significant analgesic effect at 6 µg / kg (p>0.05), but had a significant analgesic effect at a dose of 9 µg / kg (p<0.001). This shows that compared with the commercially available injection, the nasal preparation of the present invention can achieve a good therapeutic effect while reducing the dosage.

[0198] Experimental Example 10

[0199] 1. Experimental Materials

[0200] Lyophilized powder of neurotoxin labeled with FITC (fluorescein isothiocyanate): Lyophilized powder was prepared in accordance with the method of Example 1, except that in step (1), FITC-labeled neurotoxin (Beijing Zhongke Chenyu Co., Ltd. was commissioned to label the neurotoxin with FITC) was used instead of the neurotoxin in Example 1.

[0201] Before use, the FITC-labeled neurotoxin freeze-dried powder was dissolved and diluted with physiological saline to prepare neurotoxin solutions of different concentrations.

[0202] 2. Experimental methods

[0203] Eight 9-month-old WT mice were randomly divided into two groups, with 4 mice in each group. One group was given an equal amount of saline and 3, 6, and 12 µg / kg of animal body weight of FITC-labeled neurotoxins by subcutaneous administration on the back; the other group was given an equal amount of saline and 3, 6, and 12 µg / kg of animal body weight of FITC-labeled neurotoxins by nasal administration. The administration volume for each mouse in both groups was 0.8 µL / 10g.

[0204] The nasal administration method is: select a 10-microliter liquid injection needle, put a very thin hose on the needle tip as a tool for nasal administration. After the mouse is anesthetized with isoflurane, pinch the skin of its head and neck to fix the head, hold the injection needle and carefully insert the hose about 7 mm into the mouse's nose, slowly push the drug solution in, and carefully pull out the hose after completion. The whole process should be as gentle as possible to avoid causing harm to the experimental animals.

[0205] Both groups of animals were given the drug once a day for three consecutive days. Three hours after the end of the drug administration on the last day, the mice were perfused by heart, and the brain tissues were removed for organ imaging using an IVIS small animal imager.

[0206] Brain tissue sectioning method:

[0207] 1) Anesthetize the mouse with 4% chloral hydrate, fix it on a foam board, cut open the chest cavity and expose the heart;

[0208] 2) Make a small cut at the left auricle, insert an intravenous needle from the apex of the heart, and slowly perfuse 40 mL of pre-cooled PBS solution (phosphate buffered saline). After the blood is completely perfused, slowly perfuse 20 mL of pre-cooled 4% paraformaldehyde solution.

[0209] 3) Remove the mouse brain tissue and fix it in 4% paraformaldehyde at 4°C for 24 h;

[0210] 4) Soak the brain tissue in a 20% sucrose solution, wait for it to sink to the bottom, and then soak it in a 30% sucrose solution for 24 hours;

[0211] 5) Frozen sections: Use filter paper to absorb the surface moisture of the brain tissue and freeze it in a -80 ℃ refrigerator. When slicing, wrap the brain tissue in an embedding agent. The slice thickness is 20 μm. Select brain slices from the olfactory bulb, cerebral cortex, striatum, hippocampus, and cerebellum and stick them on adhesive slides. Dry them in the dark and store them at 4 ℃.

[0212] 6) Select brain slices from each part of the brain tissue, take them out in advance and restore them to room temperature;

[0213] 7) Wash with PBS 3 times, 5 min each time;

[0214] 8) Seal the slides with a sealing solution containing DAPI (4',6-diamidino-2-phenylindole), let them dry, and take pictures under a confocal microscope.

[0215] 3. Experimental results

[0216] Table 13 Fluorescence intensity results of each group (average value)

[0217]

[0218] from Figure 5 ,6 As shown in Table 13, FITC-labeled nasal preparations of neurotoxins can be absorbed through the nasal mucosa, pass through the blood-brain barrier into the brain, and are distributed in various areas of the brain. Compared with injections, the content of neurotoxins in brain tissue was significantly increased using the nasal preparations of neurotoxins provided in this study. This result lays a good foundation for subsequent research on the therapeutic effect of nasal preparations of neurotoxins on neurodegenerative diseases.

[0219] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A freeze-dried powder containing neurotoxin, characterized in that: The invention comprises a neurotoxin, a stabilizer and an excipient, wherein the excipient comprises one or more of acidic gelatin and alkaline gelatin, the stabilizer comprises one or more of block polyether F-68 and block polyether F-127, or a composition comprising one or more of block polyether F-68, block polyether F-127 and Tween 80; the neurotoxin is cobra neurotoxin cobrotoxin.

2. The freeze-dried powder according to claim 1, characterized in that The lyophilized powder satisfies at least one of the following AC: A. The freeze-dried powder contains or does not contain a penetration enhancer; B. The freeze-dried powder contains or does not contain a preservative; C. In terms of weight, the lyophilized powder includes 3.5 parts of neurotoxin, 200-5000 parts of excipients and 10-1300 parts of stabilizers.

3. A method for preparing the freeze-dried powder according to claim 1 or 2, characterized in that: The freeze-dried powder is prepared by freeze-drying a mixed solution containing neurotoxin, a stabilizer and an excipient.

4. The method for preparing the freeze-dried powder according to claim 3, 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 permeation enhancer; and / or, the mixed solution contains or does not contain a preservative.

5. The method for preparing the freeze-dried powder according to claim 3, characterized in that: The preparation method of the freeze-dried 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 heat to dissolve at 40° C. to prepare liquid 1; Weigh 375 parts by weight of block polyether F-68, add 10 parts by volume of water, and heat to dissolve at 40° C. to obtain liquid 2; 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 are pipetted, mixed with or without water to obtain a mixed solution, and the mixed solution is freeze-dried to obtain a freeze-dried powder; when the unit of the weight part is mg, the unit of the volume part is mL.

6. A nasal preparation, characterized in that: The invention comprises the freeze-dried powder according to claim 1 or 2 or the freeze-dried powder prepared by the preparation method according to any one of claims 3 to 5, and also comprises a solvent, wherein the freeze-dried powder and the solvent are packaged independently.

7. The nasal preparation according to claim 6, characterized in that Before use, the lyophilized powder is dissolved in a solvent to obtain a drug solution, wherein the concentration of the neurotoxin in the drug solution is 1-1000 µg / mL.

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