Recombinant botulinum toxin light chain protein composition with improved stability

Abstract

The present disclosure relates to a composition comprising a recombinant botulinum toxin protein with improved stability. The composition according to the present disclosure comprises a sugar alcohol as a stabilizer. More specifically, the present disclosure provides a composition comprising a botulinum toxin type A light chain protein or a functional fragment thereof, and comprising, as a stabilizer, a sugar alcohol having six or fewer carbon atoms and / or a solubility in water of 150 mg / mL or more at 20°C. The composition of the present invention can achieve the effect of maintaining the biological activity of the recombinant botulinum toxin light chain protein at a usable level while sufficiently ensuring the stability of the botulinum toxin protein through the stabilizer.

Description

Technical Field

[0007]

[0001] This application claims priority based on Korean Patent Application No. 10-2020-0127249 filed on September 29, 2020, and all the contents disclosed in the specification and drawings of that application are incorporated herein by reference.

[0002] The present disclosure relates to a composition having improved stability of a recombinant botulinum toxin light chain protein.

Background Art

[0003] Botulinum toxin is a polypeptide product of the anaerobic bacterium Clostridium botulinum and is a type of neurotoxin. This toxin causes muscle paralysis by blocking the presynaptic release of the neurotransmitter acetylcholine at the neuromuscular junction of mammals.

[0004] Using the principle that only the muscles at the injection site are partially paralyzed when injected at a dose without side effects on the human body, botulinum toxin is therapeutically used in the treatment of involuntary muscle movement disorders. Involuntary muscle movement disorders include strabismus, idiopathic blepharospasm, hemifacial spasm, torticollis, masseter muscle dystonia, spastic dysphonia, and various types of muscle spasms and seizures.

[0005] Recently, botulinum toxin has been used to non-surgically reduce facial wrinkles by paralyzing the muscles that cause wrinkles around the eyes, between the eyebrows, and on the forehead, and is also used in aesthetic and cosmetological fields such as pore reduction, acne, elasticity enhancement, and relaxation of sagging skin.

[0006] <​​​​Botulinum toxin is approximately 150 kDa in size and consists of a complex of botulinum toxin protein and non-toxin proteins. The size of each complex can reach up to 900 kDa depending on the type of toxin. The mode of action, target, and duration of activity vary depending on the type of botulinum toxin, but botulinum toxin type A is known as one of the most lethal biological agents.

[0008] Regardless of the toxin type, the molecular mechanisms of botulinum toxin appear to be similar. Botulinum toxin is expressed in a single polypeptide and, through a post-expression reconstitution process, is divided into a heavy chain (H chain) of approximately 100 kDa and a light chain (L chain) of approximately 50 kDa. The H and L chains are then relinked by disulfide bonds. Although different botulinum toxin types have different targets, commonly, the H chain binds to receptors in nerve cells, forming toxin-containing endosomes via endocytosis. The botulinum toxin can then be taken into the cell through these endosomes. After being taken into the cell, the L chain escapes from the endosome and cleaves SNARE proteins in the cytoplasm, thereby inhibiting acetylcholine secretion and causing muscle paralysis.

[0009] Generally, protein preparations are prone to instability degradation due to protein denaturation, aggregation, and other factors. This instability of protein preparations increases with storage time. The main objective in formulating protein preparations is to maintain the solubility, stability, and viability of the protein. In particular, the viability of enzymes in protein preparations varies greatly depending on their three-dimensional structure. When purified botulinum toxin crystals are diluted in physiological saline or water, their biological activity and pharmacological properties are lost. Furthermore, they are known to denature easily at temperatures above 40°C. That is, it is well known that without appropriate stabilizers, the toxicity of botulinum toxin is rapidly eliminated.

[0010] Most botulinum toxins distributed for pharmaceutical purposes are in the form of a toxin complex containing a non-toxin protein and a botulinum toxin protein molecule. The non-toxin protein plays a role in stabilizing the botulinum toxin molecule in the toxin complex, protecting it from denaturation, and thus preserving the activity of the botulinum toxin. For botulinum toxin to be used in cosmetics, it must undergo a toxicity attenuation process and possess skin permeability. Therefore, there is a need to develop compositions that can maintain the activity of botulinum toxin using only the L chain, rather than in the form of a toxin complex with a non-toxin protein. [Overview of the Initiative] [Problems that the invention aims to solve]

[0011] The light chain of botulinum toxin is an endopeptidase capable of hydrolyzing peptide bonds. In the case of botulinum toxin type A, it cleaves SNAP-25, one of the SNARE proteins. Therefore, it can be said that the suppression of acetylcholine secretion in nerve cells is possible with the light chain alone. Focusing on this point, we attempted to develop a composition containing only the botulinum toxin light chain that exerts a muscle paralysis effect. Preferably, we aimed to develop it in a form that can be transmitted transdermally.

[0012] Therefore, the problem that this disclosure seeks to solve is to provide a stable composition containing recombinant botulinum toxin light chain protein.

[0013] Furthermore, this disclosure aims to provide a composition that maintains the endopeptidase activity of botulinum toxin not only under refrigerated conditions but also when stored at room temperature for extended periods. More preferably, this disclosure aims to provide a composition in liquid form rather than lyophilized form as a formulation to eliminate inconveniences in use. [Means for solving the problem]

[0014] The inventors have discovered a remarkable effect of maintaining the biological activity of recombinant botulinum toxin light chain protein at a usable level while ensuring sufficient stability of the recombinant botulinum toxin light chain protein through a specific stabilizer, leading to the completion of the present invention.

[0015] Specifically, the inventors attempted to produce a composition with enhanced protein stability at room temperature by including one or more stabilizers for protein stabilization, such as sugar alcohols, polyols, polymer compounds, surfactants, and antioxidants, in a composition containing recombinant botulinum toxin light chain protein. However, they confirmed that the stability of the recombinant botulinum toxin light chain protein varied greatly depending on the type of stabilizer used.

[0016] Among various protein stabilizers, only sugar alcohols significantly enhanced the stability of botulinum toxin type A light chain protein or its sections (sections with substantially the same efficacy). The inventors confirmed these unexpected experimental results and completed the present invention.

[0017] The sugar alcohols according to the present invention are typically organic compounds derived from sugars, and are substances in which one hydroxyl group (-OH) is attached to each carbon atom. The chain length varies depending on the number of carbon atoms, and various isomers exist depending on the orientation of the hydroxyl group. When a sugar alcohol contains multiple hydroxyl groups, it can be classified as a polyol, but in this specification, polyols and sugar alcohols are distinguished, and polyols are defined as substances having hydroxyl groups excluding sugar alcohols.

[0018] One aspect of the present disclosure provides a composition comprising botulinum toxin type A light chain protein or a section thereof, and a stabilizer, wherein the stabilizer is a sugar alcohol.

[0019] Preferably, the botulinum toxin type A light chain protein according to one aspect of the present disclosure is a botulinum toxin type A light chain protein comprising the 448 amino acid sequence of SEQ ID NO: 1. In a preferred aspect of the present disclosure, the botulinum toxin type A light chain protein according to the present invention comprises SEQ ID NO: 1.

[0020] In one aspect of this disclosure, the botulinum toxin type A light chain protein may further include additional amino acid sequences in addition to the light chain protein amino acid sequence for purposes such as protein production and protein purification. For example, the botulinum toxin type A light chain protein of the present invention may further include additional amino acid sequences, such as the 21-amino acid sequence of sequence number 3, in addition to the amino acid sequence of sequence number 1.

[0021] Furthermore, in the present invention, a section having the same efficacy as the botulinum toxin type A light chain protein according to the present invention may be used instead. For example, such a section may be a section of botulinum toxin type A light chain protein containing the 425 amino acid sequence of SEQ ID NO: 2. In one embodiment of the present disclosure, the section of botulinum toxin type A light chain protein according to the present invention consists of the sequence of SEQ ID NO: 2.

[0022] In one embodiment of this disclosure, a section of botulinum toxin type A light chain protein may further include additional amino acid sequences in addition to the amino acid sequence of the section, for purposes such as protein production and protein purification. For example, a section of botulinum toxin type A light chain protein according to one embodiment of the present invention may further include additional amino acid sequences, such as the 21-amino acid sequence of sequence number 3, in addition to the amino acid sequence of sequence number 2.

[0023] However, the inclusion of additional amino acid sequences does not alter the function of botulinum toxin type A light chain protein or its sections.

[0024] In one aspect of this disclosure, botulinum toxin type A light chain protein is a recombinant protein that uses the amino acid sequence of the N-terminal portion of the amino acid sequence of botulinum toxin type A protein that has enzymatic activity.

[0025] As used herein, the term "recombinant" protein is a protein encoded by a heterologous (e.g., recombinant) nucleic acid introduced into a host cell, such as a bacterial or eukaryotic cell. That is, it refers to a protein that has been genetically engineered for mass production.

[0026] The protein or a fragment thereof according to the present invention can be produced by conventional methods known in the art. For example, the methods disclosed in U.S. Patent No. 10,300,118 etc. can be used.

[0027] In a preferred form of the present disclosure, the composition is a liquid composition. The composition in liquid form can improve the inconvenience in use compared to the lyophilized form. Furthermore, the liquid form is advantageous in terms of economy as it does not require a nitrogen filling process in the manufacturing process of cosmetics. When using a protein in lyophilized form during the manufacture of cosmetics, a nitrogen filling process is required to ensure stability. Although stability usually decreases in liquid form, the composition of the present disclosure has a great advantage in that it can maintain stability even in such a liquid form.

[0028] In one aspect of the present disclosure, the composition according to the present invention is preferably a cosmetic composition, more preferably a cosmetic composition for improving skin wrinkles.

[0029] In the present invention, the term "wrinkle improvement" means suppressing or delaying the formation of wrinkles on the skin or alleviating existing wrinkles.

[0030] In one aspect of this disclosure, the cosmetic composition may be formulated in the form of solutions, suspensions, emulsions, lotions, oils, toners, softening waters, etc., which are commonly manufactured in the industry, but are not limited thereto. More specifically, it may be formulated in the form of softening lotions, nourishing lotions, skin, skin softeners, skin toners, astringents, milk lotions, moisturizing lotions, nourishing lotions, emulsions, lotions, essences, nourishing essences, etc., but are not limited thereto.

[0031] In one embodiment of this disclosure, the cosmetic composition of the present invention may further contain one or more cosmetically acceptable carriers. In one embodiment of this disclosure, the cosmetically acceptable carriers that can be contained in the cosmetic composition are diverse depending on the dosage form.

[0032] In one aspect of the present disclosure, when the dosage form of the composition is a solution or emulsion, a solvent, solubilizer, or emulsifier may be used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol, or fatty acid esters of sorbitan, either alone or in combination.

[0033] In one embodiment of the present disclosure, if the dosage form of the composition is a suspension, the carrier component may be a liquid diluent such as water, ethanol, or propylene glycol; a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, or polyoxyethylene sorbitan ester; microcrystalline cellulose; aluminum methhydroxyl; bentonite; agar; tracant, etc., either alone or in combination.

[0034] In one aspect of this disclosure, the cosmetic composition may be manufactured using a conventional cosmetic manufacturing method, and the cosmetic composition of the present invention may be manufactured in the form of a cosmetic. Such a cosmetic may be appropriately formulated with additional ingredients of any choice. For example, it may contain conventional ingredients such as thickeners, surfactants, humectants, abrasives, sweeteners, pH adjusters, preservatives, binders, wetting agents, foaming agents, solubilizers, pigments, or fragrances.

[0035] The stabilizer according to the present invention is a sugar alcohol. Among a variety of substances including sugar alcohols, polyols, and polymer compounds, sugar alcohols proved to be very effective in stabilizing botulinum toxin type A light chain protein or its sections according to the present invention.

[0036] In one embodiment of this disclosure, the stabilizer according to the present invention is preferably a sugar alcohol having 6 or fewer carbon atoms, and more preferably 2 to 6 carbon atoms.

[0037] In other aspects of this disclosure, the stabilizer according to the present invention is preferably a sugar alcohol having a solubility in water of 150 mg / mL or more at 20°C, and more preferably 150 mg / mL to 10,000 mg / mL at 20°C.

[0038] In other aspects of this disclosure, the stabilizer according to the present invention is a sugar alcohol having 6 or fewer carbon atoms and having a solubility in water of 150 mg / mL or more at 20°C. Preferably, the stabilizer is a sugar alcohol having 2 to 6 carbon atoms and having a solubility in water of 150 mg / mL to 10,000 mg / mL at 20°C.

[0039] In other aspects of this disclosure, the stabilizer according to the present invention is ethylene glycol, glycerol, erythritol, treitol, arabitol, xylitol, ribitol, sorbitol, iditol, or a mixture thereof. Preferably, the stabilizer of the present invention is glycerol, xylitol, sorbitol, or a mixture thereof, and more preferably, glycerol. Glycerol proved to be significantly more effective than the other stabilizers.

[0040] In one aspect of this disclosure, the present invention provides a composition in which the stabilizer is 50% by weight or less, preferably 20-50% by weight, relative to the total weight of the composition. In order for the stabilizer of the present invention to have an effective protein stabilization effect, it must be composed of 20% by weight or more relative to the total weight of the composition.

[0041] As used herein, the terms “stable,” “stabilize,” “to stabilize,” or “to stabilize” mean that recombinant botulinum toxin light chain protein is stored at 25°C or below and, when reconstituted with a specific composition, maintains enzyme activity at or above one-third of its original enzyme activity. In this specification, “enzyme activity at or above one-third of its original activity” is as defined in U.S. Patent No. 8,617,568. In U.S. Patent No. 8,617,568, when evaluating existing botulinum toxin protein, if it shows activity of 1 unit or more after being treated with 3 units, that activity is considered significant, and the stabilization described herein means showing activity at such a level.

[0042] Preferably, all components described in this invention do not exceed the maximum usage values ​​specified in relevant laws and regulations in countries such as South Korea, China, the United States, Europe, and Japan (e.g., regulations concerning cosmetic safety standards (South Korea), cosmetic safety technical standards (China), hygiene standards (China)). In other words, preferably, the cosmetic or personal care composition according to the present invention contains the components according to the present invention at content limits permitted by the relevant laws and regulations of each country. [Effects of the Invention]

[0043] This disclosure provides compositions comprising botulinum toxin type A light chain protein or a section thereof, which have improved stability, i.e., can maintain their effects over a long period of time. In particular, the compositions of the present invention can ensure stability even in liquid dosage forms. [Brief explanation of the drawing]

[0044] [Figure 1]This figure shows the stability period of recombinant botulinum toxin light chain protein in compositions containing various stabilizers. [Figure 2] This is the result of evaluating the botulinum toxin enzyme activity of a composition containing 0.2% by weight of polysorbate 20. [Figure 3] This is the result of evaluating the botulinum toxin enzyme activity of a composition containing 0.2% by weight of polysorbate 20 and 0.3% by weight of methionine. [Figure 4] This is the result of evaluating the botulinum toxin enzyme activity of a composition containing 1% by weight of poloxamer 188. [Figure 5] This is the result of evaluating the botulinum toxin enzyme activity of a composition containing 3% by weight of troxerutin. [Figure 6] This is the result of evaluating the botulinum toxin enzyme activity of compositions containing 20% ​​or 50% by weight of glycerol. [Figure 7] This is the result of evaluating the botulinum toxin enzyme activity of compositions containing 20% ​​or 50% by weight of xylitol. [Figure 8] This is the result of evaluating the botulinum toxin enzyme activity of compositions containing 20% ​​or 50% by weight of sorbitol. [Figure 9] This is the result of evaluating the botulinum toxin enzyme activity of a composition containing 20% ​​by weight of 1,3-butylene glycol (1,3-BG). [Figure 10] This is the result of evaluating the botulinum toxin enzyme activity of a composition containing 20% ​​by weight of dipropylene glycol (DPG). [Modes for carrying out the invention]

[0045] The present invention will be described in detail below with reference to examples to aid in understanding the present invention. However, the examples of the present invention are for illustrative purposes only and should not be construed as limiting the scope of the present invention to the following examples. The examples of the present invention are provided to give a more complete explanation of the present invention to a person with average skill in the art to which the present invention belongs. [Examples]

[0046] The endopeptidase activity capable of cleaving SNARE proteins was confirmed through a FRET (Fluorescence Resonance Energy Transfer)-based method for evaluating botulinum toxin type A enzyme activity (SNAPtide® 521, List Biological Laboratories, Inc.).

[0047] Compositions containing specific stabilizers for protein stabilization and recombinant botulinum toxin type A light chain protein (hereinafter referred to as "recombinant botulinum toxin light chain protein") were stored at 25°C, and their stability was evaluated using the SNAPtide evaluation method. The results showed that recombinant botulinum toxin protein remained stable for 10 weeks or more when specific sugar alcohols, such as glycerol, sorbitol, or xylitol, were used as stabilizers. Of the three sugar alcohols presented, it was confirmed that the shorter the carbon chain length, the greater the effect on stabilizing the recombinant botulinum toxin light chain protein.

[0048] In the experiments described later, the recombinant botulinum toxin type A light chain protein used was a protein obtained by adding the sequence of sequence number 3 to the protein of sequence number 2. Here, the sequence of sequence number 3 was added only to facilitate evaluation in the experiments described later, and is substantially identical to the effect of sequence number 2. The protein of sequence number 2 is obtained by removing some functionally irrelevant proteins from the protein of sequence number 1, and it is readily apparent to those skilled in the art that the performance of the protein of sequence number 2 is equivalent to that of sequence number 1, or can be easily confirmed experimentally.

[0049] 1. Combination Configuration The combination of recombinant botulinum toxin light chain protein with various stabilizers was evaluated. Through this, the stabilizing effect of the stabilizers on recombinant botulinum toxin light chain protein was confirmed. The weight excluding the stabilizer and protein combination consisted of water and a small amount of pH adjuster. In the examples of this invention, potassium phosphate (KH2PO4) and disodium phosphate (Na2HPO4) were used as pH adjusters. The control group and combinations 1-17 were completed by filling the weight excluding the stabilizer and protein with a phosphate buffer consisting of 0.14 wt% potassium phosphate, 0.8 wt% disodium phosphate, 9 wt% sodium chloride (NaCl), and 90.06 wt% water.

[0050] To confirm the protein stabilization effect of combining recombinant botulinum toxin light chain protein with sugar alcohol-based stabilizers, the following combinations 1 to 9 were constructed as specific examples. [Table 1]

[0051] To confirm the protein stabilization effect of combining recombinant botulinum toxin light chain protein with polyol-based stabilizers other than sugar alcohols, the following combinations 10 to 13 were constructed as specific examples. [Table 2]

[0052] To confirm the protein stabilization effect of recombinant botulinum toxin light chain protein combined with stabilizers other than polyols, the following combinations 14 to 17 were constructed as specific examples. [Table 3]

[0053] Experimental example: Evaluation of endopeptidase activity The protein stability of the recombinant botulinum toxin light chain protein used in this invention was evaluated by examining the changes in its endopeptidase activity, which is a biological function of the protein.

[0054] Compositions containing recombinant botulinum toxin light chain protein and stabilizers were prepared as shown in Tables 1 to 3, stored at 25°C, and the enzymatic activity of the recombinant botulinum toxin light chain protein was tracked using the SNAPtide evaluation method. Through this, the effect of improving protein stability was evaluated.

[0055] (1) We tracked the botulinum toxin enzyme activity of surfactants used as non-protein stabilizers for botulinum toxin distributed for pharmaceutical purposes, and of mixed compositions of surfactants and amino acids (combinations 14 and 15). The experimental results are shown in Figures 2 and 3.

[0056] The experimental results showed that when polysorbate 20, a surfactant, was mixed at 0.2% by weight, the stability period of recombinant botulinum toxin light chain protein increased compared to a composition with no added stabilizer (control group). However, the improvement was so slight that it was unusable.

[0057] Furthermore, when 0.3% by weight of methionine was mixed with 0.2% by weight of polysorbate 20, the protein stability period was similar to that of the control group. In other words, it was confirmed that a new stabilizer is needed to stabilize recombinant botulinum toxin light chain protein, rather than the conventional stabilizers used to stabilize botulinum toxin (Figures 2 and 3).

[0058] (2) To search for stabilizers of recombinant botulinum toxin light chain protein, the botulinum toxin enzyme activity of compositions (combinations 16 and 17) containing other types of compounds, namely high molecular weight polymers and antioxidants, was tracked. The experimental results are shown in Figures 4 and 5.

[0059] The experimental results showed that when the high molecular weight polymer poloxamer 188 or the antioxidant troxerutin was mixed with the protein, the period of protein stability was increased compared to the control group, but it was not at a practical level (Figures 4 and 5).

[0060] (3) The experimental results for compositions containing sugar alcohols (combinations 1 to 9) are shown in Figures 6 to 8.

[0061] Experimental results showed that when tracking the activity of recombinant botulinum toxin light chain protein in compositions containing xylitol and sorbitol, xylitol, with its shorter carbon chain length, exhibited superior protein stabilization compared to sorbitol, which has an even longer carbon chain length. Mannitol is an isomer of sorbitol and has a similar structure, so it was expected to have a similar level of protein stabilization effect as sorbitol. However, due to its low solubility, a significant protein stabilization effect could not be obtained.

[0062] We confirmed that the three sugar alcohols that exhibit protein stabilization effects—glycerol, xylitol, and sorbitol—all show a proportional relationship between their concentration and stabilization effect, and that the smaller the compound size, the greater the stabilization effect (Figures 6-8).

[0063] (4) Among polyols excluding sugar alcohols, the experimental results for compositions (combinations 10 to 13) containing polyols that have a short linear structure similar to glycerol and are widely used as cosmetics are shown in Figures 9 and 10.

[0064] The experimental results showed that while the combination of the two proposed polyols (1,3-butylene glycol (1,3-BG) and dipropylene glycol (DPG) at 20% by weight each) increased the protein stability period compared to the control group, it was not at a practical level. Furthermore, it was confirmed that, unlike sugar alcohols, the stabilizing effect of these two polyols was not proportional to their concentration (Figures 9 and 10).

[0065] Although preferred embodiments of the present invention have been described above, such embodiments are merely examples of how the technical idea of ​​the present invention can be realized, and other modifications or alterations that can realize the technical idea of ​​the present invention must also be interpreted as falling within the scope of the present invention.

[0066]

Claims

1. The composition contains botulinum toxin type A light chain protein or a fragment thereof as an active ingredient, and further contains a stabilizer. The stabilizer is glycerol, and the botulinum toxin type A light chain protein or a fragment thereof consists of the amino acid sequence described in SEQ ID NO: 1 or SEQ ID NO: 2, or the amino acid sequence obtained by adding SEQ ID NO: 3 to SEQ ID NO: 1 or 2. composition.

2. The composition according to claim 1, wherein the stabilizer is present in an amount of 20 to 50% by weight relative to the total weight of the composition.

3. The composition according to claim 1, wherein the composition is liquid.

4. The composition according to claim 1, wherein the composition is a cosmetic.

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