A botulinum toxin preparation with improved stability

Abstract

The present invention belongs to the field of botulinum toxin preparations, and specifically relates to a botulinum toxin preparation with improved stability. The raw materials of the botulinum toxin preparation include: botulinum toxin, buffer, zinc acetate, and one or more of sucrose, Tween 20, poloxamer 188, human albumin, glycerol, sodium chloride, mannitol, trehalose, methionine and sodium hyaluronate; the buffer is selected from any one of Tris-HCl buffer, sodium lactate buffer and histidine-glycine buffer. The botulinum toxin preparation in the present invention can significantly improve the activity of botulinum toxin and the stability of the preparation through a specific combination of ingredients.

Description

Technical Field

[0001] The present invention belongs to the field of botulinum toxin preparations, and in particular relates to a botulinum toxin preparation with improved stability. Background Art

[0002] Botulinum toxin, also known as botulinum toxin, is a bacterial endotoxin secreted by the deadly botulinum bacteria. It is a toxic protein secreted by botulinum bacteria during its reproduction process and has strong neurotoxicity. Botulinum toxin acts on the endings of cholinergic motor nerves, antagonizing the action of calcium ions in a certain way, interfering with the release of acetylcholine from the motor nerve endings, making the muscle fibers unable to contract and causing muscle relaxation to achieve the purpose of wrinkle removal and beauty. The greater the toxicity, the better the wrinkle removal and beauty effect.

[0003] The relative molecular mass of botulinum toxin is 150,000, and it is composed of a light chain with a relative molecular mass of 50,000 and a heavy chain with a relative molecular mass of 100,000. The heavy chain recognizes and binds to specific receptors on the presynaptic membrane of nerve endings; the light chain acts as a zinc-titanium endonuclease to hydrolyze the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, thereby affecting the fusion of synaptic vesicles with the presynaptic membrane, blocking the release of neurotransmitters such as acetylcholine, and causing chemical denervation effects such as muscle relaxation and glandular secretion disorders.

[0004] As a protein preparation, botulinum toxin has the problem that it is not easy to formulate into a pharmaceutical composition, nor is it easy to store, distribute and manage. This is due to the instability of the protein, and the problem is particularly serious when protein preparations such as botulinum toxin are formulated as pharmaceutical ingredients at extremely low concentrations. Botulinum toxin protein has the property of adhering to solid surfaces, so when the protein is injected into a container, a portion of the protein may adhere to the inner wall of the container, resulting in the loss of the active ingredient, and the protein may be easily oxidized or degraded into small fragments.

[0005] In order to prevent the denaturation of botulinum toxin to the greatest extent possible, the purified botulinum toxin during its production process is formulated as a lyophilized powder, which is diluted in saline immediately before clinical application and administered to patients in the form of a liquid. However, in this case, there is also a problem that medical accidents are likely to occur due to human errors (such as dilution factor errors caused by users or contamination of dilution saline). Therefore, there is an urgent need to develop stabilizers that can prevent protein denaturation even during the production and distribution of botulinum toxin liquid preparations.

[0006] Chinese invention patent CN116916894A discloses a long-term storable lyophilized botulinum toxin formulation composition, which comprises botulinum toxin, protamine sulfate, an isotonic agent and a buffer. The lyophilized botulinum toxin formulation according to the invention reduces the inactivation rate of botulinum toxin during the lyophilization process, can maintain the stability of botulinum toxin for a longer time than conventional lyophilized botulinum toxin formulations, forms a uniform particle size, makes the effects shown at the administration site the same, and can prevent the side effect of excessive toxin administration effects only at some sites.

[0007] Because there are individual differences in the required dosage from patient to patient, the dosage required for any individual patient may vary considerably. In addition, for some indications, clinicians must administer to patients only a small portion of the contents of a vial that has been prepared over a long period of time, which may be several hours. Although liquid botulinum toxin formulations can be refrozen and thawed with substantial activity retained, there is a loss of potency, resulting in significant waste of drug and patient expense.

[0008] Therefore, it is necessary to provide a botulinum toxin preparation that is easy to use and has significantly improved stability. Summary of the invention

[0009] In view of the deficiencies of the prior art, the present invention provides a botulinum toxin preparation with improved stability. The botulinum toxin preparation of the present invention has significantly improved stability and is easy to use.

[0010] In order to achieve the above-mentioned purpose of the present invention, the specific technical solution adopted by the present invention is:

[0011] A botulinum toxin preparation with improved stability, wherein the raw materials of the botulinum toxin preparation include: botulinum toxin, buffer, zinc acetate, and one or more of sucrose, Tween 20, poloxamer 188, human albumin, glycerol, sodium chloride, mannitol, trehalose, methionine and sodium hyaluronate;

[0012] The buffer is selected from any one of Tris-HCl buffer, sodium lactate buffer and histidine-glycine buffer.

[0013] Further preferably, the buffer is selected from any one of Tris-HCl buffer and sodium lactate buffer.

[0014] Preferably, the buffer is sodium lactate buffer.

[0015] Preferably, the raw materials of the botulinum toxin preparation include: botulinum toxin, sodium lactate buffer, zinc acetate, and one or more of sucrose, Tween 20, poloxamer 188, human serum albumin, glycerol and sodium chloride.

[0016] Further preferably, the raw materials of the botulinum toxin preparation include: botulinum toxin, sodium lactate buffer, zinc acetate and sucrose.

[0017] Further preferably, the raw materials of the botulinum toxin preparation include: botulinum toxin, sodium lactate buffer, zinc acetate, and two or three of sucrose, Tween 20, poloxamer 188, glycerol and human albumin.

[0018] Preferably, the raw materials of the botulinum toxin preparation include: botulinum toxin, sodium lactate buffer, zinc acetate, Tween 20 and human serum albumin.

[0019] Preferably, the raw materials of the botulinum toxin preparation include: botulinum toxin, sodium lactate buffer, zinc acetate, sucrose and human serum albumin.

[0020] Preferably, the raw materials of the botulinum toxin preparation include: botulinum toxin, sodium lactate buffer, zinc acetate, sucrose, human serum albumin and Tween 20.

[0021] Further preferably, the raw materials of the botulinum toxin preparation include: botulinum toxin, sodium lactate buffer, zinc acetate, and one or more of sucrose, Tween 20, human serum albumin, mannitol, trehalose, methionine and sodium hyaluronate.

[0022] Preferably, the raw materials of the botulinum toxin preparation include: botulinum toxin, sodium lactate buffer, zinc acetate, sucrose, Tween 20 and mannitol.

[0023] Preferably, the raw materials of the botulinum toxin preparation include: botulinum toxin, sodium lactate buffer, zinc acetate, sucrose, Tween 20, mannitol and methionine.

[0024] Preferably, the raw materials of the botulinum toxin preparation include: botulinum toxin, sodium lactate buffer, zinc acetate, Tween 20, human serum albumin, mannitol, trehalose and methionine.

[0025] Preferably, the raw materials of the botulinum toxin preparation include: botulinum toxin, sodium lactate buffer, zinc acetate, sucrose, Tween 20, human serum albumin and sodium hyaluronate.

[0026] Preferably, the raw materials of the botulinum toxin preparation include: botulinum toxin, sodium lactate buffer, zinc acetate, sucrose, Tween 20, human serum albumin and methionine.

[0027] Preferably, the concentration of sodium lactate in the botulinum toxin preparation is 3.1-6.2 mg / mL, and the concentration of botulinum toxin is 0.02 ng / mL-10 μg / mL.

[0028] Preferably, the concentration of zinc acetate in the botulinum toxin preparation is 45-55 μM, the concentration of sucrose is 2-100 mg / mL, the concentration of Tween 20 is 0.5-2 mg / mL, the concentration of human albumin is 0.5-1 mg / mL, the concentration of poloxamer 188 is 1.5-2.5 mg / mL, the concentration of glycerol is 15-25 mg / mL, the concentration of sodium chloride is 8-10 mg / mL, the concentration of mannitol is 50-75 mg / mL, the concentration of trehalose is 15-25 mg / mL, the concentration of methionine is 3-6 mg / mL, and the concentration of sodium hyaluronate is 0.8-1.2 mg / mL.

[0029] Preferably, the botulinum toxin preparation is in the form of a liquid preparation or a lyophilized powder preparation.

[0030] Preferably, the type of the botulinum toxin is selected from one or more of type A, type B, type C, type D, type E, type F, type G, type FA and type X.

[0031] More preferably, the botulinum toxin is type A.

[0032] The present invention also relates to the use of the above-mentioned botulinum toxin preparation in pharmaceutical manufacturing, and the obtained medicament is used to treat diseases characterized by excessive muscle activity or inappropriate contraction, including: local dystonia, spasticity, achalasia syndrome, anal spasm, nystagmus, hyperhidrosis, frown lines, crow's feet, forehead lines, myofascial pain syndrome, tension headache, migraine, face slimming, leg slimming, strabismus, depression and Parkinson's disease.

[0033] Preferably, the medicament has a therapeutic or cosmetic effect on the patient.

[0034] Compared with the prior art, the present invention has the following beneficial effects:

[0035] (1) The botulinum toxin preparation of the present invention can significantly improve the activity of botulinum toxin and the stability of the preparation through a specific combination of ingredients;

[0036] (2) The present invention prepares two dosage forms of botulinum toxin, namely, liquid preparation and lyophilized preparation, both of which have good stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a stability effect diagram at 5°C when the botulinum toxin concentration is 1 ng / mL in Example 7;

[0038] Figure 2 is a graph showing the stability effect of botulinum toxin at 25° C. when the concentration is 1 ng / mL in Example 7;

[0039] Figure 3This is a stability effect diagram at 40°C when the botulinum toxin concentration is 1 ng / mL in Example 7;

[0040] Figure 4 is a graph showing changes in the content of the target protein (botulinum toxin) at 25° C. when the botulinum toxin concentration is 10 μg / mL in Example 7;

[0041] Figure 5 This is a graph showing the purity change of the target protein (botulinum toxin) at 25° C. when the botulinum toxin concentration is 10 μg / mL in Example 7;

[0042] Figure 6 This is a graph showing the change in the content of the target protein (botulinum toxin) at 40° C. when the botulinum toxin concentration is 10 μg / mL in Example 7;

[0043] Figure 7 This is a graph showing the purity change of the target protein (botulinum toxin) at 40° C. when the botulinum toxin concentration is 10 μg / mL in Example 7;

[0044] Figure 8 is a graph showing changes in the target protein (botulinum toxin) content of formulation 18 in Example 8 at 25°C;

[0045] Fig. 9 This is a graph showing the purity change of the target protein (botulinum toxin) of formulation 18 in Example 8 at 25°C;

[0046] Fig.10 is a graph showing changes in the target protein (botulinum toxin) content of formulation 18 in Example 8 at 40°C;

[0047] Fig.11 This is a graph showing the purity change of the target protein (botulinum toxin) of formulation 18 in Example 8 at 40°C;

[0048] Fig.12 is a display diagram of the lyophilized samples in Example 10, wherein a is a 1 ng / mL lyophilized sample of Formulation 20, and b is a 1 ng / mL lyophilized sample of Formulation 21;

[0049] Fig.13 is a display diagram of a liquid preparation sample of formula 34 in Example 14;

[0050] Figure 12-13 The unclear contents do not affect the understanding of the present invention by those skilled in the art. DETAILED DESCRIPTION

[0051] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0052] The target protein (botulinum toxin type A protein) is of pharmaceutical grade and is a 150KDa core neurotoxin produced by Hebei Fengzhang Biotechnology Co., Ltd. through recombinant expression in E. coli and high purification;

[0053] Human albumin, 10g (20%, 50mL) / bottle, injection grade, manufacturer: Octapharma AB;

[0054] Tween 20, 500g / bottle, pharmaceutical grade, manufacturer: Nanjing Well Pharmaceutical Group Co., Ltd.;

[0055] Poloxamer 188, 500g / bottle, pharmaceutical grade, manufacturer: BASF (China) Co., Ltd. Shanghai Branch;

[0056] Sodium hyaluronate, 5mL / tube, injection grade, manufacturer: Shanghai Qisheng Biological Products Co., Ltd.;

[0057] Mice, KM mice, weight 14-20 g, manufacturer: Jinan Pengyue Experimental Animal Breeding Co., Ltd.

[0058] Example 1

[0059] The target protein (botulinum toxin) was prepared with four buffers at 0.2 ng / mL (as shown in Table 1) and 0.1 ng / mL (as shown in Table 3), and then injected intraperitoneally into mice (KM mice). The mice weighed 14-16 g and each was injected with 500 μL. The experimental results are shown in Tables 2 and 4.

[0060] Table 1 Effects of different buffers on the activity of 0.2 ng / mL target protein (botulinum toxin)

[0061]

[0062] Table 2 Experimental results of different buffers on the activity of 0.2 ng / mL target protein (botulinum toxin) in mice

[0063]

[0064] Table 3 Effects of different buffers on the activity of 0.1 ng / mL target protein (botulinum toxin)

[0065]

[0066] Table 4 Experimental results of different buffers on the activity of 0.1 ng / mL target protein (botulinum toxin) in mice

[0067]

[0068] From the results in Table 2 and Table 4, it can be seen that the activity of the target protein (botulinum toxin) is relatively high in the Tris-HCl and sodium lactate buffer systems, followed by the histidine system, while no mice died in the sodium citrate system. This shows that the target protein (botulinum toxin) can exert greater activity in the Tris-HCl or sodium lactate buffer system.

[0069] Example 2

[0070] In order to compare the effects of Tris-HCl and sodium lactate buffer systems on the activity of the target protein (botulinum toxin), the target protein (botulinum toxin) was prepared into 0.04 ng / mL and 0.02 ng / mL as shown in Table 5, and injected intraperitoneally into mice, the weight of the mice was in the range of 14-16 g, and each mouse was injected with 500 μL. The experimental results are shown in Table 6.

[0071] Table 5 Effects of Tris-HCl and sodium lactate on the activity of target protein (botulinum toxin)

[0072]

[0073] Table 6 Experimental results of Tris-HCl and sodium lactate on the activity of target protein (botulinum toxin) in mice

[0074]

[0075] From the results in Table 6, it can be seen that at the two target protein (botulinum toxin) concentrations, the mice in the sodium lactate buffer group all died, while the mice in the Tris-HCl group did not die. This indicates that when the target protein (botulinum toxin) concentration is lower, the sodium lactate system can still maintain the activity of the protein compared to the Tris-HC system.

[0076] Example 3

[0077] Based on the fact that the sodium lactate buffer can ensure the activity of the protein (botulinum toxin) in Example 2, the effect of adding human serum albumin on the activity of the target protein (botulinum toxin) was explored. The target protein (botulinum toxin) formula is shown in Table 7, and mice were intraperitoneally injected. The weight of the mice was in the range of 14-16g, and each mouse was injected with 500 μL. The experimental results are shown in Table 8.

[0078] Table 7 Effect of human serum albumin on the activity of target protein (botulinum toxin)

[0079]

[0080] Table 8 Results of mouse experiments on the effect of human serum albumin on the activity of target protein (botulinum toxin)

[0081]

[0082] From the results in Table 8, it can be seen that when the concentration of the target protein (botulinum toxin) was 0.04 ng / mL, the mice in the group with human albumin added died earlier than those without. When the concentration of the target protein (botulinum toxin) was 0.02 ng / mL, the mice in the group with human albumin added died, while the mice in the group without human albumin added did not die. This shows that the addition of human albumin can not only improve the activity of the target protein (botulinum toxin), but also maintain the activity of the target protein at a lower concentration of the target protein (botulinum toxin).

[0083] Example 4

[0084] The effect of zinc acetate on the activity of the target protein (botulinum toxin) was investigated. The formula of the target protein (botulinum toxin) is shown in Table 9. After incubation at room temperature for one night, the mice were intraperitoneally injected. The weight of the mice was in the range of 14-16 g, and each mouse was injected with 500 μL. The experimental results are shown in Table 10.

[0085] Table 9 Effect of zinc acetate on the activity of target protein (botulinum toxin)

[0086]

[0087] Table 10 Results of the mouse experiment on the effect of zinc acetate on the activity of the target protein (botulinum toxin)

[0088]

[0089] From the results in Table 10, it can be seen that when the concentration of the target protein (botulinum toxin) was 75 pg / mL and 40 pg / mL, the mice in the group with zinc acetate added died first. This shows that adding zinc acetate to the formula helps to improve the activity of the target protein.

[0090] Example 5

[0091] The target protein (botulinum toxin) (0.26 mg / mL) eluted with 200PG molecular sieve was diluted 10,000 times according to the formulation in Table 11 and then injected intraperitoneally into mice. Each mouse was injected with about 13 ng of the target protein (botulinum toxin), and the mouse death time was shown in Table 11. It can be seen that the mice in formulations 4, 5, 8, and 10 died earlier than those in the other 6 groups, and the activity of the target protein (botulinum toxin) was relatively high under these 4 formulations, indicating that adding sucrose, Tween 20, poloxamer 188, and human albumin, two or three of the four components, on the basis of sodium lactate and zinc acetate, is more conducive to exerting the activity of the target protein.

[0092] Table 11 Use different formulations to dilute the target protein (botulinum toxin) and inject it into mice

[0093]

[0094] Example 6

[0095] Based on the results of Example 5, the formulation composition was adjusted, and the formulation was prepared according to Table 12. The target protein (botulinum toxin) (0.26 mg / mL) eluted from the 200PG molecular sieve was diluted 10,000 times and then injected into the mouse intraperitoneally. Each mouse was injected with about 13 ng of the target protein (botulinum toxin), and the mouse death time was shown in Table 12. By comparing formulations 11 and 15, and formulations 12 and 17, it can be seen that the addition of Tween 20 is beneficial to improving the activity of the target protein (botulinum toxin); by comparing formulations 15 and 16, it can be seen that adding Tween 20 is better than glycerol on the basis of the same 4 components; by comparing formulations 15 and 17, on the basis of the same 4 components, adding human serum albumin is better than poloxamer 188; at the same time, the mouse death time of formulation 17 is the shortest, indicating that the activity of the target protein (botulinum toxin) under this formulation is higher than that of other formulations.

[0096] Table 12: Different formulations were used to dilute the target protein (botulinum toxin) and inject it into mice

[0097]

[0098] Example 7

[0099] The target protein (botulinum toxin) was diluted to 1 ng / mL and 10 μg / mL using the excipients of formula 17 in Example 6. The liquid preparation was directly placed in a stability test chamber (set at 5°C, 25°C, and 40°C) for long-term and accelerated experiments. The mice were injected intraperitoneally by regular sampling, and the death time of the mice was recorded to calculate the results. The stability test results are as follows Figure 1 , Figure 2 and Figure 3 As shown, Figure 1 , Figure 2 and Figure 3 The stability effects of formula 17 at 1 ng / mL at 5°C, 25°C and 40°C are shown respectively. The stability was investigated by detecting the content and purity of the target protein (botulinum toxin) by HPLC molecular exclusion. The results are as follows Figure 4 , Figure 5 , Figure 6 and Figure 7 As shown, Figure 4 and Figure 6 These are the changes in the target protein (botulinum toxin) content at 25°C and 40°C when the formula 17 is 10 μg / mL. Figure 5 and Figure 7These are the purity change graphs of the target protein (botulinum toxin) at 25°C and 40°C when the value of formula 17 is 10 μg / mL.

[0100] Depend on Figure 1 , Figure 2 , Figure 3 It can be seen that, up to 229 days, from the trend of the time of death of mice, the activity of the target protein (botulinum toxin) did not change much when stored at 5°C. The fluctuation of the time of death in the middle was analyzed to be caused by individual differences of mice; the time of death of mice at 25°C from 75 days to 199 days also showed a fluctuating trend. Analysis showed that it might also be caused by individual differences of mice or injection errors. The activity of the target protein remained stable; while under 40°C storage, the activity of the target protein (botulinum toxin) did not change significantly within 30 days, and it could not kill mice at 50 days and 75 days, but the mice all had mild symptoms of poisoning and were accompanied by weight loss, indicating that most of the target protein (botulinum toxin) had been inactivated at this time, retaining only extremely low activity. Liquid phase detection showed that the content of the sample at 5°C on the 70th day was 7.04μg / mL, the content on the 80th day was 9.77μg / mL, and the content on the 90th day was 10.31μg / mL. Figure 4 and Figure 6 From the results, the protein (botulinum toxin) content at 25℃ on the 90th day tended to be within the normal range, and the purity did not change except for the 40th day (the fluctuation was caused by the liquid equivalent condition); while at 40℃, the protein (botulinum toxin) content was severely degraded on the 50th day, and the purity did not change; it can be seen that the trend of the high concentration 10μg / mL and the low concentration 1ng / mL inspection results are the same. Based on the current stability results, if the target protein (botulinum toxin) is stored at 2-8℃, 25℃ is its accelerated temperature, the target protein (botulinum toxin) can be stored at low temperature (2-8℃) for 22.9-30.5 months, and if the target protein is stored at 25℃, it can be stored for at least 7-8 months.

[0101] Example 8

[0102] Based on the formula 17 of Example 6, the sucrose concentration was changed to 2 mg / mL, and the other components and contents remained unchanged, which was recorded as formula 18. The components and contents are shown in Table 13. The target protein was diluted to 1 ng / mL and 10 μg / mL. The prepared liquid preparation was directly placed in a stability test chamber (set at 5°C, 25°C, and 40°C) for long-term and accelerated experiments. The sample with a concentration of 10 μg / mL was tested for the content and purity of the target protein by high-performance liquid chromatography molecular exclusion to examine the stability. The results are as follows Figure 8 , Fig. 9 , Fig.10 and Fig.11 As shown; the sample with a concentration of 1 ng / mL was injected intraperitoneally into mice and the mortality was recorded to investigate the stability. The results are shown in Table 14.

[0103] Table 13 Ingredients and contents of formula 18

[0104]

[0105] Table 14 Results of stability study on formula 18 in mice

[0106]

[0107] Depend on Figure 8 It can be seen that the protein content of the target protein at a high concentration (10 μg / mL) at 25°C began to decrease from the 25th day, and the protein had been degraded by nearly 70% by the 60th day, and the protein purity did not change; while at 40°C, the protein content of the target protein at 10 μg / mL began to decrease from the 25th day, and the protein had been degraded by nearly 70% by the 60th day, Fig.10 It can be seen that the protein showed a rapid degradation trend from 0d to 5d, and could not be detected in the liquid phase on the 10th day; the protein concentration was 1ng / mL for a long-term observation at 5℃, with a low sampling frequency, and a high sampling frequency at 25℃ and 40℃. It can be seen from the results in Table 14 that the death time of mice at 25℃ was still normal at 180d, which is inconsistent with the result that the content of the protein at 10μg / mL at 25℃ showed a significant decrease at 25d; and from the results in the table at 40℃, it can be seen that the mouse could no longer be killed from the 50th day, indicating that the protein had been severely degraded on the 50th day, which is inconsistent with the phenomenon that the 10μg / mL sample was completely degraded at 40℃ on the 10th day.

[0108] In summary, the analysis shows that Formula 18 has a good effect on maintaining the activity of low-concentration (1ng / mL) target protein when the sucrose concentration is lower than that of Formula 17. It can be inferred that Formula 18 can maintain the activity of the target protein for at least 18-24 months at 5°C, while Formula 18 may not be able to maintain long-term activity for high-concentration (10μg / mL) protein.

[0109] Example 9

[0110] Based on the formulation 17 of Example 6, the components and contents were adjusted to be recorded as formulation 19. The components and contents are shown in Table 15. The target protein was diluted to 1 ng / mL and the liquid preparation was directly placed in a stability test chamber (set at 5°C, 25°C, and 40°C) for long-term and accelerated experiments. The stability was investigated by intraperitoneal injection of mice and recording the death situation (the sampling frequency was based on the actual experiment), and the results are shown in Table 16.

[0111] Table 15 Ingredients and contents of formula 19

[0112]

[0113] Table 16 Results of stability study on formula 19 in mice

[0114]

[0115] As shown in Table 16, the protein of Formula 19 could not kill mice at 15 days under 40℃, indicating that the protein of Formula 19 was unstable when stored at 40℃; the lethal time of mice was relatively stable at 90 days under 5℃, and the protein was still relatively stable after the 90th day under 25℃. The fact that mice did not die at 27 days and the death time of mice at 60 days was relatively long may be caused by individual differences of mice. Taking 25℃ as the accelerated temperature of 5℃, it is inferred that the target protein can be stored at 5℃ for at least 9-12 months based on Formula 19.

[0116] Example 10

[0117] On the basis of Formula 17 in Example 6, the concentrations of sucrose and Tween 20 were adjusted to investigate the effects of different sucrose concentrations on the activity of the target protein. The specific information of the formula is shown in Table 17. The target protein was diluted to 1 ng / mL and 50 μg / mL with the same formula liquid, and liquid preparations and lyophilized powders were prepared (the lyophilization of Formula 22 was not investigated at 1 ng / mL) and directly placed in a stability test chamber (set at 5°C, 25°C, and 40°C) for long-term and accelerated experiments. The 1 ng / mL sample was sampled regularly for intraperitoneal injection into mice, and the time of death of the mice was recorded to statistically analyze the results, as shown in Tables 18 and 19; the 50 μg / mL sample was regularly tested for content by liquid phase molecular exclusion, and the results are shown in Tables 20 and 21.

[0118] Table 17 Composition and content of auxiliary materials in formula 20-22

[0119]

[0120] Table 18 Results of mouse experiments on the stability of 1 ng / mL liquid preparations of formulas 20-22

[0121]

[0122] Table 19 Results of mouse experiments on the stability of 1 ng / mL lyophilized powder of formula 20 and 21

[0123]

[0124] Table 20 Liquid phase stability test results of 50 μg / mL liquid preparations of formula 20-22

[0125]

[0126] Table 21 Liquid phase stability test results of 50 μg / mL lyophilized powder of formula 20-22

[0127]

[0128] From the results in Table 18, it can be seen that for the low-concentration target protein (1 ng / mL) liquid preparation, increasing the concentration of sucrose on the basis of formula 17 is more conducive to maintaining the stability of the protein. It can be inferred that based on formula 22, if the acceleration temperature is 40°C, the protein can be maintained at 25°C for at least 8-10 months; if the acceleration temperature is 25°C, it can also be maintained stable for at least more than 18 months at 5°C; from the results in Table 19, it can be seen that the activity of the target protein of 1 ng / mL freeze-dried formulations 20 and 21 is still stable when observed at 5°C and 25°C for 154 days, and the fluctuations in the middle are caused by the differences in the freeze-drying process. It can be seen that the 1 ng / mL freeze-dried formulations 20 and 21 can be maintained stable for at least 5 months at 25°C, and if the acceleration temperature is 25°C, it can be maintained stable for at least 15-20 months at 5°C. Fig.12 a is the 1 ng / mL lyophilized sample of formulation 20, and b is the 1 ng / mL lyophilized sample of formulation 21; from the results in Table 20, it can be seen that the 50 μg / mL liquid preparations of formulations 20, 21 and 22 can maintain stability for at least 6 months at 5°C, can only maintain for about 20 days at 25°C, and are extremely unstable at 40°C; from the results in Table 21, it can be seen that the 50 μg / mL lyophilized powders of formulations 20, 21 and 22 are extremely unstable at 40°C, can be maintained for at least 6 months at 25°C, and from the trend from 90 days to 180 days, formulations 22 and 21 are better than formulation 20 at 25°C, which also shows that increasing the sucrose content is more conducive to maintaining the stability of the target protein; if 25°C is used as the acceleration temperature, the target protein can be stored at 5°C for at least 18-24 months based on formulations 20, 21 and 22.

[0129] Embodiment 11

[0130] Formula 23 and Formula 24 (excipient composition and content are shown in Table 22) were prepared to dilute the target protein to 1 ng / mL and 50 μg / mL, and the liquid preparations were directly placed in the stability test chamber (set at 5°C, 25°C, and 40°C) for long-term and accelerated experiments. The 1 ng / mL sample was sampled regularly for intraperitoneal injection into mice, and the time of mouse death was recorded to statistically calculate the results, as shown in Table 23; the 50 μg / mL sample was tested for stability by molecular exclusion detection of the target protein content by high performance liquid chromatography, and the results are shown in Table 24.

[0131] Table 22 Composition and content of auxiliary materials in formula 23 and 24

[0132]

[0133] Table 23 Results of mouse experiments on the stability of 1 ng / mL liquid preparations of formulations 23 and 24

[0134]

[0135] Table 24 Liquid phase test results of 50 μg / mL liquid preparation stability study of formulations 23 and 24

[0136]

[0137] As can be seen from Table 23, the lethal time of mice for the sample of liquid preparation 1 ng / mL formula 23 was basically stable when observed at 25℃ until the 90th day, while the lethal time of mice observed at 40℃ gradually increased and fluctuated, indicating that the protein will gradually degrade and inactivate at this temperature; the lethal time of mice for the sample of formula 24 was stable when observed at 5℃ until the 90th day, while the lethal time of mice observed at 25℃ increased significantly, indicating that from the 30th day, the protein began to degrade seriously, and the test at 40℃ could not kill mice at all; In summary, liquid preparation formula 23 is much better than formula 24. If 25℃ is used as the acceleration temperature, it can be inferred that the target protein 1 ng / mL liquid preparation made with formula 23 can maintain stability for at least 9-12 months at 5℃.

[0138] As shown in Table 24, the stability trend of the liquid preparation 50μg / mL based on formula 23 and 24 is basically the same as that of the low concentration 1ng / mL. The slight difference is that the results of the liquid phase test of formula 23 and 24 at 25℃ for 90 days are both stable. Analysis shows that the poisoning and death of mice may be greatly affected by the change of protein content. From the results of the liquid phase test at 40℃, it can be seen that formula 23 is still much better than formula 24, and formula 23 is more suitable for liquid preparations.

[0139] Example 12

[0140] On the basis of formula 17, the components were increased or decreased and the content was adjusted, and formula 25 and formula 26 (formula components and content are shown in Table 25) were prepared to dilute the target protein to 1 ng / mL, and the liquid preparation was placed in a stability test chamber (set at 5°C, 25°C, and 40°C) for long-term and accelerated experiments. The mice were injected intraperitoneally by regular sampling, and the death time of the mice was recorded to statistically calculate the results, as shown in Table 26.

[0141] Table 25 Ingredients and contents of formula 25 and formula 26

[0142]

[0143] Table 26 Results of mouse experiments on the stability of liquid preparations of Formulation 25 and Formulation 26

[0144]

[0145] As can be seen from Table 26, the lethal time of mice for Formulation 25 at 5°C and 25°C tended to be stable at 181 days, and the lethal time of mice at 40°C was slightly prolonged at 80 days. It can be inferred that if the target protein 1 ng / mL is made into a liquid preparation using Formulation 25 and stored at 25°C at an accelerated temperature of 40°C, it can be maintained stable for at least 8-12 months, and if stored at 5°C at an accelerated temperature of 25°C, it can be maintained stable for at least 18-24 months; while the lethal time of mice for Formulation 26 at 5°C tended to be stable at 80 days, and the lethal time of mice at 25°C showed an increasing trend, and mice could not be lethal at 40°C on the 10th day; in summary, Formulation 25 is superior to Formulation 26; combined with the previous examples, it can be analyzed that the effect of adding human serum albumin should be higher than zinc acetate and mannitol.

[0146] Example 13

[0147] On the basis of formula 17, the components were increased or decreased and the content was adjusted, and formula 27 and formula 28 (formula components and content are shown in Table 27) were prepared to dilute the target protein to 1 ng / mL, and the liquid preparation was placed in a stability test chamber (set at 5°C, 25°C, and 40°C) for long-term and accelerated experiments. The mice were injected intraperitoneally by regular sampling, and the death time of the mice was recorded to statistically calculate the results, as shown in Table 28.

[0148] Table 27 Ingredients and contents of formula 27 and formula 28

[0149]

[0150] Table 28 Results of mouse experiments on the stability of liquid preparations of Formulation 27 and Formulation 28

[0151]

[0152] As can be seen from Table 28, the lethal time of mice for Formulation 27 and Formulation 28 at 5°C and 25°C tended to be stable at 180 days, and the lethal time of mice at 40°C was longer at 120 days, which suggests that the target protein may have been severely degraded; while Formulation 28 at 40°C did not kill mice at 10 days and 53 days, indicating that the protein degradation was very serious at this time; from the current results, Formulation 27 is better than Formulation 28, which once again verifies that the addition of human serum albumin and zinc acetate can better maintain the stability of the target protein, and based on Formulation 27, the target protein can be kept stable for at least 6 months at 25°C. If 25°C is used as the accelerated temperature, the target protein can be kept stable for at least 18-24 months at 5°C.

[0153] Embodiment 14

[0154] On the basis of formula 17, components were increased or decreased and the contents were adjusted to prepare formulas 29-36 (formula ingredients and contents are shown in Table 29), and the target protein was added to 10 μg / mL. The liquid preparations were made and placed in a stability test chamber at 40°C for 2 weeks to examine the stability by detecting the target protein content by molecular exclusion using HPLC. The results are shown in Tables 30-31.

[0155] Table 29 Ingredients and contents of formula 29-36

[0156]

[0157] Table 30 Stability of formula 29-36 at 40℃ for 2 weeks liquid phase test results

[0158]

[0159] Table 31 Degradation rate results of formulations 29-36 at different observation times relative to the 4th day

[0160]

[0161] Due to problems with the liquid chromatography column during the measurement on the 0th day, the protein content result measured on the 0th day was unreliable. After replacing the chromatographic column, the protein content measured on the 4th day was used as a reference benchmark. As can be seen from Table 30, the test results from the 4th day to the 15th day fluctuated, but the protein content basically showed a downward trend, indicating that under the condition of 40°C, formulas 29-36 could not maintain protein stability well; from the results of the target protein content detected on the 15th day, formula 35 had the highest target protein content and was relatively optimal. As can be seen from Table 31, the degradation rate of the target protein content detected on the 15th day was the lowest for formula 34 relative to the 4th day. From the comparison of degradation rates, formula 34 was relatively optimal. For example, the liquid preparation sample of formula 34 Fig.13 shown.

[0162] The above detailed description is a specific description of one feasible embodiment of the present invention. The embodiment is not intended to limit the patent scope of the present invention. Any equivalent implementation or modification that does not deviate from the present invention should be included in the scope of the technical solution of the present invention.

Claims

1. A botulinum toxin preparation with improved stability, characterized in that: The concentration of sodium lactate in the botulinum toxin preparation is 3.1-6.2 mg / mL, the concentration of botulinum toxin is 0.02 ng / mL-10 μg / mL, the concentration of zinc acetate is 45-55 μM, the concentration of sucrose is 2-100 mg / mL, the concentration of Tween 20 is 0.5-2 mg / mL, the concentration of human albumin is 0.5-1 mg / mL, the concentration of poloxamer 188 is 1.5-2.5 mg / mL, the concentration of glycerol is 15-25 mg / mL, the concentration of sodium chloride is 8-10 mg / mL, the concentration of mannitol is 50-75 mg / mL, the concentration of trehalose is 15-25 mg / mL, the concentration of methionine is 3-6 mg / mL, and the concentration of sodium hyaluronate is 0.8-1.2 mg / mL.

2. The botulinum toxin preparation according to claim 1, characterized in that The dosage form of the botulinum toxin preparation is a liquid preparation or a lyophilized powder preparation.

3. The botulinum toxin preparation according to any one of claims 1 to 2, characterized in that: The type of the botulinum toxin is selected from one or more of type A, type B, type C, type D, type E, type F, type G, type FA and type X.

4. The botulinum toxin preparation according to claim 3, characterized in that The type of botulinum toxin is type A.

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