Botulinum toxin freeze-dried preparation dosage form capable of being stored for long time

Abstract

The present invention relates to a composition of a botulinum toxin lyophilized dosage form that can be stored for a long period of time, comprising botulinum toxin, protamine sulfate, an isotonic agent and a buffer, and the botulinum toxin lyophilized dosage form according to the present invention reduces the rate of inactivation of botulinum toxin during lyophilization, according to the present invention, the stability of the botulinum toxin can be maintained for a longer time than the conventional botulinum toxin lyophilized dosage form, uniform particle size is formed, the same effect is exhibited at the administration site, and the side effect that the excessive toxin administration effect is exhibited only at some sites can be prevented.

Description

[0001] This application is a divisional application of the invention patent application with the application date of February 7, 2022, application number 2022800137666, and invention name "Botulinum toxin freeze-dried preparation dosage form that can be stored for a long time". Technical Field

[0002] The present invention relates to a lyophilized botulinum toxin composition capable of long-term storage, and more particularly to a lyophilized botulinum toxin composition capable of long-term storage comprising a complex of botulinum toxin and protamine sulfate, an isotonicity agent and a buffer. Background Art

[0003] Various Clostridium strains secreting neurotoxic toxins have been discovered since the 1890s, and the characterization of the toxins secreted by these strains has been achieved over the past 70 years.

[0004] The neurotoxic toxin derived from Clostridium strains, i.e., botulinum toxin, is divided into seven types, A to G, according to its serological characteristics. Each toxin has a toxin protein of about 150 kDa, and is naturally composed of a complex into which various non-toxic proteins are incorporated. The medium complex (300 kDa) is composed of toxin protein and non-toxic non-hemagglutinin protein, and the large complex (450 kDa) and the giant complex (900 kDa) are configured so that the medium complex is bound to hemagglutinin (Sugiyama, H, Microbiol Rev., 44: 419, 1980). These non-toxic non-hemagglutinin proteins are known to have the function of protecting the toxin from the low pH in the intestine and the effects of various proteolytic enzymes.

[0005] The toxin is synthesized in the cell as a single polypeptide with a molecular weight of about 150kDa, and then cut at a position 1 / 3 away from the N-terminus by the action of intracellular proteolytic enzymes or artificial treatment with enzymes such as trypsin, and is therefore divided into two units, i.e., a light chain (L: molecular weight: 50kDa) and a heavy chain (H: molecular weight: 100kDa). The toxicity of the toxin so divided is significantly increased than that of a single polypeptide. The two units are interconnected by a disulfide bond, and each has a different function. The heavy chain binds to the receptor of the target cell and reacts with the biofilm at low pH (pH 4) to form a channel (Mantecucco, C et al, TIBS, 18: 324, 1993), and the light chain has pharmacological activity, so detergents are used to confer cell penetration activity or interfere with the secretion of neurotransmitters when injected into cells by electroporation, etc.

[0006] The toxin inhibits the exocytosis of acetylcholine at the pre-cholinergic synapses of the neuromuscular junction, causing general weakness. It is believed that the toxin has no enzymatic activity, as toxicity occurs even in very small amounts.

[0007] Recently, it was discovered that the toxin has metallopeptidase activity and its substrates are synaptobrevin, syntaxin, and synaptosome-associated protein of 25 kDa (SNAP25), which are unit proteins of the exocytosis machinery complex. Each type of toxin uses one of these three proteins as a substrate. Types B, D, F, and G are known to cleave synaptobrevin at a specific site, types A and E cleave SNAP25 at a specific site, and type C cleaves syntaxin at a specific site.

[0008] In particular, botulinum toxin type A is known to be soluble in dilute aqueous solutions at pH 4.0 to 6.8. It is known that at a pH of about 7 or higher, the non-toxin proteins that play a stabilizing role separate from the neurotoxin, resulting in a gradual loss of toxicity, particularly with increasing pH and temperature.

[0009] Since a small amount of botulinum toxin is lethal to humans and is easy to produce on a large scale, it is a toxin that can be used in four major bioterrorist weapons, along with Bacillus anthracis, Yersinia pestis, and smallpox virus. However, in particular, it has been found that when injected at a dose equal to or less than that which does not affect the human body systemically, botulinum toxin type A can paralyze local muscles at the site of injection. Utilizing these characteristics, botulinum toxin type A can be used as a wrinkle remover and a therapeutic agent for spastic hemiplegia and cerebral palsy, and therefore the demand for it is rapidly increasing, and methods for producing botulinum toxin are being thoroughly studied to meet this demand.

[0010] Currently, the representative commercially available product is Allergan's (a purified neurotoxin complex of botulinum toxin type A), and 100-unit vials It contains about 5 ng of purified botulinum toxin type A complex, 0.5 mg of human serum albumin and 0.9 mg of sodium chloride and is provided in a vacuum-dried form and reconstituted with sterile saline (0.9% sodium chloride injection) without any preservatives. Other commercially available products include (a botulinum toxin type A hemagglutinin complex containing lactose and human serum albumin in a botulinum toxin pharmaceutical composition, reconstituted with 0.9% sodium chloride prior to use) and Solstice Neurosciences (an injectable solution containing botulinum toxin type B, human serum albumin, sodium succinate and sodium chloride, with a pH of approximately 5.6).

[0011] Botulinum toxin is mainly distributed in the form of lyophilized preparations, and many studies have been conducted to keep the lyophilized preparations stable for a long time. For example, US 7,744,904 B1 attempts to improve the stability of botulinum toxin by simply mixing α, β and γ cyclodextrins with botulinum toxin in phosphate buffered saline to form a cyclodextrin-botulinum toxin complex. However, in the case of α, β and γ cyclodextrins, oral administration is allowed, but the problem is that due to their limited solubility and nephrotoxicity, they are not suitable as excipients for botulinum toxins that are mainly used as injections. In addition, when botulinum toxin with uneven particle size is injected into the body, it cannot function normally in the body, so that the desired treatment or cosmetic effect may not be achieved, resulting in various side effects.

[0012] Therefore, the present inventors have endeavored to develop a lyophilized preparation in which botulinum toxin is uniformly granulated and stability is also enhanced, and thus determined that when a botulinum toxin lyophilized preparation containing protamine sulfate as an excipient is prepared, protamine sulfate and botulinum toxin form a complex so that the efficacy of botulinum toxin can be sustained for 6 months or longer compared to the previous 3 months, and botulinum toxin particles having a PDI value of 0.3 or less can be uniformly prepared, thereby achieving the present invention.

[0013] Citation List

[0014] Patent Literature

[0015] (Patent Document 1) US 7,744,904 B1 Summary of the invention

[0016] An object of the present invention is to provide a novel lyophilized botulinum toxin preparation composition having uniform botulinum toxin particle size and improved long-term storage stability, and a method for preparing the same.

[0017] In order to achieve the above object, the present invention provides a lyophilized botulinum toxin preparation composition comprising a complex of botulinum toxin and protamine sulfate, an isotonic agent and a buffer.

[0018] In addition, the present invention provides a method for preparing a lyophilized botulinum toxin preparation, comprising:

[0019] (a) forming a complex of botulinum toxin and protamine sulfate by mixing botulinum toxin and protamine sulfate;

[0020] (b) preparing a botulinum toxin stock solution by adding at least one excipient selected from the group consisting of polysorbate 20, hydroxypropyl-β-cyclodextrin, human serum albumin and mannitol, an isotonicity agent and a buffer to a complex of botulinum toxin and protamine sulfate; and

[0021] (c) Lyophilizing the botulinum toxin stock solution. DETAILED DESCRIPTION

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Generally, the terms used herein are well known in the art and are typical.

[0023] In the present invention, it was demonstrated that when a complex of botulinum toxin and protamine sulfate is prepared and formulated in a lyophilized form, the long-term storage stability of the lyophilized preparation can be improved while maintaining the uniformity of its size.

[0024] Therefore, aspects of the present invention relate to a lyophilized botulinum toxin formulation composition comprising a complex of botulinum toxin and protamine sulfate, an isotonicity agent, and a buffer.

[0025] In the present invention, negatively charged botulinum toxin and positively charged protamine sulfate form an ion complex when reacting in a solution, and the ion complex is specifically configured so that the protamine sulfate binds to the high molecular weight botulinum toxin protein by ionic bonding. Therefore, the amount of protamine sulfate bound to the botulinum toxin can be varied depending on the concentration of the protamine sulfate added, and the surface of the botulinum toxin bound to the protamine sulfate can be positively charged, thereby forming an additional complex with the botulinum toxin.

[0026] Therefore, in order to form a complex with improved long-term storage stability of the lyophilized preparation according to the present invention, the concentration of protamine sulfate in the present invention may be 1.72 nM to 0.22 mM, preferably 1.72 nM to 17.2 nM, more preferably 1.72 nM to 6.98 nM, and even more preferably 1.72 nM to 3.44 nM, but is not limited thereto.

[0027] Furthermore, in the present invention, botulinum toxin and protamine sulfate may form a complex in a mass ratio of 1:3 to 1:20, preferably 1:5 to 1:20, more preferably 1:5 to 1:10, most preferably 1:5, but the present invention is not limited thereto.

[0028] In the present invention, the composition may further include at least one excipient selected from the group consisting of polysorbate 20, hydroxypropyl-β-cyclodextrin, human serum albumin and mannitol, and preferably includes hydroxypropyl-β-cyclodextrin, human serum albumin and mannitol, but the present invention is not limited thereto.

[0029] In the present invention, the concentration of hydroxypropyl-β-cyclodextrin can be 1 to 30 w / v%, preferably 2 to 25 w / v%, more preferably 5 to 20 w / v%, for example, 5 to 15 w / v%, 5 to 10 w / v%, 8 to 15 w / v%, 8 to 10 w / v%, 10 to 20 w / v% or 15 to 20 w / v%, but is not limited thereto.

[0030] In the present invention, the concentration of human serum albumin may be 0.1 to 1.0 w / v%, preferably 0.2 to 0.7 w / v%, more preferably 0.2 to 0.5 w / v%, and most preferably 0.35 to 0.5 w / v%, but is not limited thereto.

[0031] In the present invention, mannitol acts as a cryoprotectant, and sucrose, glycerol, trehalose or lactose may be used instead, but the present invention is not limited thereto. The amount of the cryoprotectant may be 0.1 to 8 w / v % based on the total amount of the composition, but is not limited thereto.

[0032] In the present invention, the cryoprotectant is preferably mannitol, and the concentration of mannitol may be 0.1 to 10 w / v%, preferably 0.1 to 8 w / v%, more preferably 4 to 8 w / v%, but is not limited thereto.

[0033] In the present invention, the isotonic agent used may be sodium chloride, glycerol, mannitol, sucrose, potassium chloride or dextrose, but is not limited thereto. The amount of the isotonic agent may be 0.7 to 0.95 w / v % based on the total amount of the composition, but is not limited thereto.

[0034] In the present invention, the isotonic agent is preferably sodium chloride, and the concentration of sodium chloride may be 0.7 to 1.0 w / v%, preferably 0.7 to 0.95 w / v%, more preferably 0.8 to 0.95 w / v%, and most preferably 0.9 w / v%, but is not limited thereto.

[0035] The composition according to the invention may comprise a physiologically compatible buffer showing a pH equal to or above the isoelectric point, thereby ensuring long-term stability.

[0036] In the present invention, the pH of the physiologically compatible buffer must be maintained in the range of 6.0 to 7.0, preferably in the range of 6.4 to 6.6, and it is most preferred to use a buffer such that the pH is about 6.5.

[0037] In the present invention, the physiologically compatible buffer used may be sodium citrate, succinic acid, phosphoric acid, potassium dihydrogen phosphate, sodium acetate or sodium chloride, but is not limited thereto.

[0038] In the present invention, the concentration of the buffer suitable for lyophilization may be 10 to 35 mM, preferably 10 to 30 mM, and most preferably 15 to 25 mM, based on the total concentration of the composition.

[0039] Preferably, the buffer has a pH of 6.0 to 7.0 and a concentration of 10 to 35 mM, but the present invention is not limited thereto.

[0040] In an embodiment, phosphoric acid at a pH of 6.5 may be included as a physiologically compatible buffer at a concentration of 20 mM based on the total concentration of the composition.

[0041] The lyophilized preparation composition according to the present invention is imparted with hydrophobicity by forming a complex of botulinum toxin and protamine sulfate and then adding hydroxypropyl-β-cyclodextrin and human serum albumin thereto. In addition, the solubility of botulinum toxin can be increased by using an isotonic agent, preferably sodium chloride.

[0042] In the present invention, protamine sulfate is a molecule containing a large number of positively charged amino acids and forms a complex with botulinum toxin, thereby contributing to the stability of botulinum toxin.

[0043] In the present invention, hydroxypropyl-β-cyclodextrin (which may be used interchangeably with "Betadex") has a sugar chain with a ring structure and has amphiphilic properties similar to polysorbate 20 or polysorbate 80, thereby contributing to the stability of botulinum toxin.

[0044] In the present invention, "botulinum toxin" may include not only neurotoxins (NTX) produced by Clostridium botulinum strains or variants thereof, but also modified, recombinant, hybrid and chimeric botulinum toxins. In the present invention, the recombinant botulinum toxin may have a light chain and / or a heavy chain recombinantly produced by a non-Clostridium species. In the present invention, botulinum toxin includes pure botulinum toxin (i.e., a neurotoxic molecule of about 150 kDa), and botulinum toxin complexes (i.e., complexes of 300, 600 and 900 kDa).

[0045] In the present invention, the botulinum toxin may be selected from the group consisting of serotypes A, B, C, D, E, F and G, but is not limited thereto.

[0046] Preferably, the botulinum toxin contained in the freeze-dried composition of the present invention is botulinum toxin type A. The composition according to the present invention may contain about 10 to 200 units / ml, preferably about 20 to 150 units / ml, more preferably about 40 to 100 units / ml of botulinum toxin, but the present invention is not limited thereto.

[0047] In the present invention, the freeze-dried composition may further contain an antioxidant, and the antioxidant may be α-tocopherol, but is not limited thereto.

[0048] Another aspect of the present invention relates to a method for preparing a lyophilized botulinum toxin formulation, comprising:

[0049] (a) forming a complex of botulinum toxin and protamine sulfate by mixing botulinum toxin and protamine sulfate;

[0050] (b) preparing a botulinum toxin stock solution by adding at least one excipient selected from the group consisting of polysorbate 20, hydroxypropyl-β-cyclodextrin, human serum albumin and mannitol, an isotonicity agent and a buffer to a complex of botulinum toxin and protamine sulfate; and

[0051] (c) Lyophilizing the botulinum toxin stock solution.

[0052] The present invention can be better understood by the following examples. It is obvious to those skilled in the art that these examples are only for illustrating the present invention, rather than for limiting the scope of the present invention.

[0053] Experimental Example 1. Preparation of Botulinum Toxin / Protamine Sulfate Complex and Measurement of Its Size

[0054] In order to maintain the stability of botulinum toxin, positively charged protamine sulfate is used to prepare botulinum toxin / protamine sulfate complex. Relatively negatively charged botulinum toxin uses a buffer solution of pH 6 or higher to form a complex with protamine sulfate having a pH of about 6. 20mM phosphate buffered saline (pH 6.5) is used as a buffer solution. 0.1mg / ml of botulinum toxin type A (900kDa, Daewoong Co., Ltd.) is prepared, and protamine sulfate is dissolved in 20mM phosphate buffered saline (pH 6.5) at 0.5, 1.0 and 2.0mg / ml, then mixed at 1:1 (v / v), and then a complex is formed at room temperature for about 30 minutes.

[0055] In order to confirm whether the complex is formed, the particle size of botulinum toxin or botulinum toxin / protamine sulfate complex is measured using a nanoparticle size analyzer (ZSP zeta sizer, Malvern).Nanoparticle size analyzer is a device for measuring particle size by applying light to a sample and measuring the refractive index when the light is reflected from the sample.Based on the test results, as shown in Table 1 below, it is determined that the size of type A botulinum toxin is about 19nm, and the size of the botulinum toxin / protamine sulfate complex determined to be 1:5 is about 182nm.At 1:10 and 1:20, its respective size is determined to be about 335 and 481nm.In addition, based on the measurement result of polydispersity index (PDI), when forming a complex with a ratio of 1:5 of botulinum toxin and protamine sulfate, its PDI value is 0.3 or less, which shows that the complex of the corresponding ratio has a uniform particle size.Therefore, when injected into the body, it is most advantageous to find that the above-mentioned composition ratio plays the same effect for a single complex.

[0056] [Table 1]

[0057] Measurement of the size of the botulinum toxin / protamine sulfate complex

[0058] sample Size(nm) PDI Botulinum toxin type A 18.58±5.12 0.258 Botulinum toxin type A / protamine sulfate 1:5 complex 182±51.9 0.269 Botulinum toxin type A / protamine sulfate 1:10 complex 334.7±74.8 0.391 Botulinum toxin type A / protamine sulfate 1:20 complex 481.4±79.1 0.488

[0059] Experimental Example 2. Screening of excipients in a lyophilized preparation composition comprising botulinum toxin as a main ingredient and evaluation of stability under harsh conditions.

[0060] For a botulinum toxin lyophilized preparation including a botulinum toxin / protamine sulfate 1:5 complex as a basic composition, an excipient composition having stability was screened. In a lyophilized preparation composition including a botulinum toxin / protamine sulfate complex using 20 mM phosphate buffered saline (pH 6.5) as a basic solvent, various excipients such as stabilizers, antioxidants, cryoprotectants, and isotonic agents were screened, and the stability of the botulinum toxin was confirmed.

[0061] Therefore, the botulinum toxin of 40U / ml was used for the experiment, and the final stock solution with the excipient composition shown in Table 2 below was prepared, packaged in 50u / bottle, and lyophilized. After lyophilization, stability tests were performed under harsh conditions to indirectly confirm long-term stability. The samples were stored in a small chamber under harsh conditions of 40°C for 12 weeks, and the toxin storage stability was confirmed by measuring the change in titer. In particular, the stability of botulinum toxin was evaluated by performing animal titer tests within a specified specific time using 4-week-old ICR mice (Koatech, Korea), and the test materials were rehydrated with saline, and then diluted to seven concentrations, and the diluted solution was administered with 0.1ml / mouse, and the death of the animals was observed for 3 days, and the LD50 value was determined to calculate the titer value. The experiment was performed with 10 mice at each concentration, and the results were expressed as their average values.

[0062] Here, the titer of each preparation was set to 100% at the time of preparation. However, since botulinum toxin is a protein and is affected by the composition of the excipients, the external environment, freeze-drying parameters, etc., the titer was converted to 100% again on the first day under strict conditions, and then the change in titer was calculated. In this way, based on the initial titer value of 100%, the titer was judged to be appropriate up to ±20%.

[0063] [Table 2]

[0064]

[0065] Therefore, when human serum albumin (HSA) was used as a stabilizer and mannitol was used as a cryoprotectant (Comparative Example 1), and when Betadex and human serum albumin were used as stabilizers and sodium chloride was used as an isotonic agent (Comparative Example 2), based on the measurement results of the botulinum toxin titer after 12 weeks under harsh conditions, Comparative Examples 1 and 2 were both judged to be inappropriate. However, when Betadex and human serum albumin were used as stabilizers, sodium chloride was used as an isotonic agent, and mannitol was used as a cryoprotectant (Example 1), it was confirmed that the titer of botulinum toxin after 12 weeks under strict conditions was the best. In addition, when polysorbate, human serum albumin and Betadex were used as stabilizers, sodium chloride was used as an isotonic agent, and mannitol was used as a cryoprotectant (Example 2), and when α-tocopherol was further added thereto as an antioxidant (Example 3), the titer after 12 weeks under strict conditions was judged to be appropriate. However, considering the above experimental results and the economic cost of excipients, Example 1 was selected as the basic excipient composition and further studies were performed.

[0066] Experimental Example 3. Optimization of excipient composition and evaluation of stability under harsh conditions

[0067] Botulinum toxin / protamine sulfate 1:5 complex was used as the basic composition, and 0.9% sodium chloride and 20mM phosphate buffered saline (pH 6.5) were fixed, and in order to confirm the effect in the concentration range containing 0.35-0.5 (w / v)% human serum albumin, 5-20 (w / v)% Betadex and 4-8 (w / v)% mannitol, three additional compositions were prepared as shown in Table 3 below. All compositions were prepared in the same manner except for the concentration of the excipients. After preparation, the composition was dispensed into 10 ml vials and lyophilized, and the stability of the botulinum toxin was evaluated under harsh conditions for 12 weeks.

[0068] [Table 3]

[0069]

[0070] Therefore, it has been experimentally confirmed that within a concentration range containing 0.35-0.5% human serum albumin, 5-20% Betadex and 4-8% mannitol, the stability of botulinum toxin is maintained for 8 weeks or more, especially 12 weeks under harsh conditions. Based on the results of conventional studies, the present inventors have confirmed that when stability for 2 weeks is ensured under harsh conditions, stability can be maintained for up to about 6 months under refrigeration, and when stability for 8 weeks is ensured under harsh conditions, stability can be maintained for about 2 years or more under refrigeration. Therefore, it was found that the efficacy of a botulinum toxin lyophilized preparation containing 0.35-0.5% human serum albumin, 10-20% Betadex and 4-8% mannitol as an optimal composition with stability for up to 12 weeks in a titer range of 80 to 120% under harsh conditions is maintained during distribution for at least 6 months, preferably 12 months or more, and most preferably 24 months or more.

[0071] Experimental Example 4. Evaluation of the Effect of Cyclodextrin-Based Excipients on the Stability of Botulinum Toxin

[0072] Betadex is a derivative of β-cyclodextrin and is a material with improved hydrophilicity by introducing hydroxyl functional groups and propyl functional groups. Betadex is currently an excipient approved by FDA, EU, etc. as a pharmaceutical excipient and a main ingredient stabilizer for use as a drug. Cyclodextrin is divided into three types according to its structure: alpha (α), beta (β), and gamma (γ), and the internal structure with relatively hydrophobic properties is stabilized by the inclusion of this main ingredient. Therefore, the effect of cyclodextrin-based excipients similar to Betadex on the stability of botulinum toxin was evaluated.

[0073] Here, since α, β, and γ cyclodextrins are nephrotoxic and evaluated by FDA and KFDA as unsuitable excipients for drug injections, they were excluded from this experiment, and it was tested whether methyl-β-cyclodextrin, β-cyclodextrin sulfate, sulfobutyl ether β-cyclodextrin sodium, hydroxyethyl-β-cyclodextrin, hydroxypropyl-γ-cyclodextrin, or hydroxypropyl-α-cyclodextrin could be used instead of Betadex (hydroxypropyl-β-cyclodextrin).

[0074] To this end, a botulinum toxin / protamine sulfate 1:5 complex was used as a basic composition, and 0.9% sodium chloride, 20 mM phosphate buffered saline (pH 6.5), 0.5% human serum albumin, and 4% mannitol were fixed, and only the cyclodextrin-based excipient was changed, and the experiment was performed. Here, the concentration of the cyclodextrin-based excipient was set to 5%.

[0075] [Table 4]

[0076]

[0077] Therefore, as shown in Table 4, when other cyclodextrin-based excipients other than Betadex (hydroxypropyl-β-cyclodextrin, Example 7) according to the present invention were used, the storage stability of botulinum toxin for 4 weeks under harsh conditions was determined to be inappropriate, and the titer stability could not be maintained for 6 weeks or more under harsh conditions. Therefore, it was found that in the preparation for cryopreservation of botulinum toxin according to the present invention, Betadex in cyclodextrin was very effective.

[0078] Experimental Example 5. Evaluation of the effect of protamine sulfate on the stability of botulinum toxin

[0079] Protamine sulfate is a positively charged peptide that can form a complex with the relatively negatively charged botulinum toxin through ionic bonds. The formation of a complex with botulinum toxin can serve as a factor for stably maintaining the botulinum toxin protein. Therefore, a comparative test was conducted to confirm the effect of the presence / absence of protamine sulfate on the stability of botulinum toxin.

[0080] For this purpose, 0.9% sodium chloride, 20 mM phosphate buffered saline (pH 6.5), 0.5% human serum albumin, 4% mannitol and 10% Betadex were fixed and compared with botulinum toxin alone or a botulinum toxin / protamine sulfate 1:5 complex.

[0081] [Table 5]

[0082]

[0083] Therefore, as shown in Table 5, it was confirmed that the stability of the lyophilized botulinum toxin preparations was maintained only in the preparations containing protamine sulfate.

[0084] The specific parts of the present invention have been described in detail above, and it is obvious to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. Therefore, the essential scope of the present invention is defined by the appended claims and their equivalents.

[0085] Industrial Applicability

[0086] According to the present invention, the lyophilized botulinum toxin preparation can reduce the inactivation rate of botulinum toxin during the lyophilization process, and produce an effective and stable lyophilized preparation. In addition, compared with conventional lyophilized botulinum toxin preparations, the lyophilized botulinum toxin preparation according to the present invention can maintain the stability of botulinum toxin for a long time, and due to the formation of uniform particle size, it also has a consistent effect at the administration site, and can prevent the side effect of excessive toxin administration effect only in some sites.

Claims

1. A lyophilized botulinum toxin preparation composition, comprising a complex of botulinum toxin and protamine sulfate, an isotonic agent and a buffer.

2. The lyophilized botulinum toxin preparation composition according to claim 1, wherein The concentration of the protamine sulfate is 1.72 nM to 0.22 mM.

3. The lyophilized botulinum toxin preparation composition according to claim 1, wherein The botulinum toxin and the protamine sulfate form a complex at a mass ratio of 1:5 to 1:

20.

4. The lyophilized botulinum toxin preparation composition according to claim 1, wherein The composition further comprises at least one excipient selected from the group consisting of polysorbate 20, hydroxypropyl-β-cyclodextrin, human serum albumin and mannitol.

5. The lyophilized botulinum toxin preparation composition according to claim 4, wherein: The composition further comprises hydroxypropyl-β-cyclodextrin, human serum albumin and mannitol.

6. The lyophilized botulinum toxin preparation composition according to claim 5, wherein: The concentration of the hydroxypropyl-β-cyclodextrin is 5 to 20 w / v%.

7. The lyophilized botulinum toxin preparation composition according to claim 5, wherein: The concentration of human serum albumin is 0.35 to 0.5 w / v %.

8. The lyophilized botulinum toxin preparation composition according to claim 5, wherein: The concentration of mannitol is 4 to 8 w / v%.

9. The lyophilized botulinum toxin preparation composition according to claim 1, wherein The concentration of the isotonic agent is 0.7 to 1.0 w / v %.

10. The lyophilized botulinum toxin preparation composition according to claim 1, wherein The buffer has a pH of 6.0 to 7.0 and a concentration of 10 to 35 mM.

11. The lyophilized botulinum toxin preparation composition according to claim 1, wherein The botulinum toxin is selected from the group consisting of serotypes A, B, C, D, E, F and G.

12. A method for preparing a lyophilized botulinum toxin preparation, comprising: (a) forming a complex of botulinum toxin and protamine sulfate by mixing botulinum toxin and protamine sulfate; (b) preparing a botulinum toxin stock solution by adding at least one excipient selected from the group consisting of polysorbate 20, hydroxypropyl-β-cyclodextrin, human serum albumin and mannitol, an isotonicity agent and a buffer to the complex of botulinum toxin and protamine sulfate; and (c) Lyophilizing the botulinum toxin stock solution.

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