Recombinant botulinum toxin and pharmaceutical composition containing same
Patent Information
- Authority / Receiving Office
- WO · WO
- Patent Type
- Applications
- Current Assignee / Owner
- MVRIX CO LTD
- Filing Date
- 2025-11-13
- Publication Date
- 2026-07-30
Smart Images
Figure KR2025018776_30072026_PF_FP_ABST
Abstract
Description
Recombinant botulinum toxin and pharmaceutical composition containing the same
[0001] The present invention relates to a recombinant botulinum toxin and a pharmaceutical composition containing the same, and more specifically, to a recombinant botulinum toxin and a pharmaceutical composition containing the same, characterized in that the light chain is an LC derived from A6 type botulinum toxin, the Hn domain is an Hn domain derived from A1 type botulinum toxin, and the Hc domain is an Hc domain derived from A2 type botulinum toxin.
[0002]
[0003] Botulinum toxin is a protein toxin produced by the strain *Clostridium botulinum* that penetrates nerve cells and inhibits the release of neurotransmitters. For nerve cells to release neurotransmitters, SNARE (Soluble N-ethylmaleimide-sensitive factor attachment protein receptors) protein complexes must fuse vesicles storing neurotransmitters to the nerve cell membrane; however, botulinum toxin inhibits this fusion between the vesicle and the nerve cell membrane by cleaving SNAP-25, syntaxin, or VAMP, which are components of the SNARE protein. If the release of neurotransmitters is inhibited, it causes neuroparalysis, leading to various behavioral disorders and paralysis of the respiratory muscles, which ultimately results in death.
[0004] Botulinum toxin exists in serotypes A, B, C, D, E, F, and G, and approximately 40 subtypes have been discovered based on them. It is known that there is a sequence variation rate of about 2.6–31.6% between serotypes and subtypes, and accordingly, substrate activity and specificity, duration of toxicity, and receptor specificity differ. For example, type A exhibits toxicity more slowly than type E but lasts longer, and types A, C, and E have the characteristic of cleaving SNAP-25, while types B, D, F, and G have the characteristic of cleaving VAMP.
[0005] Botulinum toxin consists of three domains: the light chain domain (LC), which is responsible for matrix cleavage; the heavy chain translocation domain (Hn), which delivers the LC into the cytoplasm; and the heavy chain retraction domain (Hc), which binds to receptors on the surface of nerve cells. The reason each subtype has different characteristics is that the domains of the respective subtype possess unique characteristics depending on their sequences. Therefore, it is theoretically possible to create a botulinum toxin with new characteristics by introducing and fusing LC, Hn, and Hc from different subtypes.
[0006] The persistence of botulinum toxin is influenced by the intracellular stability of LC. For example, type A, which has a deubiquitinase binding site in LC, has a longer persistence than other serotypes, and even within type A, type A3, which has relatively many sequence variations including deletions in the deubiquitinase binding site, has a shorter duration of toxicity compared to other types A.
[0007] Meanwhile, the onset of action and diffusivity of botulinum toxin are primarily influenced by the binding affinity between Hc and neuronal receptors. The higher the binding affinity, the better the botulinum toxin is delivered to neurons, and the lower the likelihood of diffusion to other sites. It is known that A2, which has a higher binding affinity to receptors than A1, moves into cells more rapidly and exhibits lower diffusivity than A1.
[0008]
[0009] In this invention, sequences related to characteristic differences of each domain constituting botulinum toxin were analyzed, and based on this, mutations were introduced to realize a safe botulinum toxin with a rapid onset of action, a long duration, and low diffusivity using recombinant production technology.
[0010]
[0011] The present invention provides a recombinant botulinum toxin formed by combining a light chain (LC) of botulinum toxin and a heavy chain (HC) of botulinum toxin composed of an Hn domain (Translocation Domain) and an Hc domain (Receptor Binding Domain), wherein the light chain is an LC derived from A6 type botulinum toxin, the Hn domain is an Hn domain derived from A1 type botulinum toxin, and the Hc domain is an Hc domain derived from A2 type botulinum toxin.
[0012] In addition, the present invention provides a pharmaceutical composition comprising the recombinant botulinum toxin of the present invention and a pharmaceutically acceptable excipient or additive.
[0013] At this time, the above pharmaceutical composition may preferably be intended to improve wrinkles, square jaw, pointed jaw, wounds, skin softening, scars, acne, pores, elasticity, or keloid symptoms.
[0014] At this time, the above pharmaceutical composition may preferably be intended to treat facial spasms, eyelid spasms, torticollis, blepharospasm, cervical dystonia, central pharyngeal dystonia, spasmodic dysphonia, migraine, anal pruritus, or hyperhidrosis.
[0015] At this time, the above pharmaceutical composition may preferably be for transdermal, subcutaneous, or intramuscular administration.
[0016]
[0017] The recombinant botulinum toxin developed in this invention has safe characteristics, including a rapid onset of action, a long duration, and low diffusivity.
[0018]
[0019] Figure 1 shows the results of the Digit Abduction Score (DAS) assay in mice, comparing the daily paralysis levels of recombinant botulinum toxin. In Figure 1, the line with a DAS score of 2 represents the EC50 line, which indicates that after the DAS score rises to the highest score of 4, the paralysis effect drops to half of that level.
[0020] Figure 2 shows the results of a mouse Digit Abduction Score (DAS) assay that more precisely analyzed the duration of muscle paralysis by administering the MBT-002 of the present invention in the same unit as the existing wild-type BoNT / A1 (900 kDa). In Figure 2, the line with a DAS score of 2 represents the EC50 line, which indicates that the paralysis effect dropped to half of the maximum score of 4 after the DAS score had risen.
[0021] Figure 3 shows the mortality results of mice induced by botulinum toxin, with the LD50 of botulinum toxin 50 The toxicity of MBT-001 (rBoNT / A1, 150kDa) and the MBT-002 of the present invention was compared through calculation.
[0022] Figure 4 shows the toxicity development rate of botulinum toxin, and is the result of comparing the toxicity development rates of BoNT / A1 (900 kDa) and MBT-002 of the present invention. In Figure 4, the line with a DAS score of 2 represents half of the highest DAS score of 4, i.e., the EC50 line.
[0023] Figure 5 shows the change in body weight of mice caused by botulinum toxin.
[0024]
[0025] Existing botulinum toxins are extracted from Clostridium botulinum, making it very difficult to improve their performance through engineering. Therefore, to develop a product with new performance, it is necessary to find a new serotype or subtype of strain, but it is not easy to find a strain of toxin with the desired performance. In this invention, we aim to develop and provide a recombinant botulinum toxin with improved performance and safety by utilizing recombinant technology.
[0026] Botulinum toxin consists of a light chain (LC) that cleaves the matrix within nerve cells, a translocation domain (Hn) of the heavy chain that moves the light chain into the cytoplasm, and a retraction domain (Hc) of the heavy chain that binds to receptors; the characteristics of the entire botulinum toxin vary depending on the performance of each domain. For example, if the stability of the LC is low, the duration of the muscle paralysis effect is short, and if the activity of Hn is slow, the onset of the drug effect may be delayed. Additionally, if the receptor binding ability of Hc is low, the diffusivity of the toxin increases, which may compromise safety.
[0027] In the present invention, by comparing the characteristics of various serotypes or subtypes and introducing LC, Hn, and Hc domains by substitution, a recombinant botulinum toxin (so-called 'MBT-002') was developed that has a fast onset of action, a long duration, low diffusivity, and is safe.
[0028] Based on this, the present invention provides a recombinant botulinum toxin formed by combining a light chain (LC) of botulinum toxin and a heavy chain (HC) of botulinum toxin composed of an Hn domain (Translocation Domain) and an Hc domain (Receptor Binding Domain), wherein the light chain is an LC derived from A6 type botulinum toxin, the Hn domain is an Hn domain derived from A1 type botulinum toxin, and the Hc domain is an Hc domain derived from A2 type botulinum toxin.
[0029] As used in the present invention, the terms 'protein' and 'polypeptide' refer to polymers of amino acid residues, variants thereof, and synthetic analogs thereof, and are to be used interchangeably in the present invention. These terms are interpreted to include polymers of naturally occurring amino acids, including, for example, chemical analogs of related naturally occurring amino acids, which are synthesized from one or more amino acid residues. These terms are also interpreted to include post-translational modifications of polypeptides, for example, glycosylation, phosphorylation, and acetylation.
[0030] In the present invention, the term 'Botulinum toxin (BoNT)' encompasses any polypeptide or fragment of botulinum toxin. In specific embodiments, botulinum toxin is interpreted as comprising full-length botulinum toxin or botulinum toxin-derived fragments. Botulinum toxin can enter neurons and execute an entire cellular mechanism that inhibits the release of neurotransmitters.
[0031] In the present invention, the term 'domain' refers to a conserved portion of a given protein sequence that is a tertiary structure capable of evolving, functioning, and existing independently of the rest of the protein chain. Because the domain is independently stable, domain swaps can be performed between one protein and another through genetic engineering to produce a recombinant protein.
[0032] In this invention, the term "recombination" refers to the process in which elements constituting genes or proteins, such as DNA (Deoxyribonucleic Acid) or RNA (Ribonucleic Acid), are altered from their original sequences during the process of disassembly and reassembly. DNA fragments can be artificially recombined through molecular biology experiments. DNA that has been artificially recombined in this way is called recombinant DNA, and the protein produced by interpreting it is called recombinant protein.
[0033] Meanwhile, the present invention provides a pharmaceutical composition comprising the recombinant botulinum toxin of the present invention and a pharmaceutically acceptable excipient or additive.
[0034] In one embodiment, pharmaceutically acceptable excipients or additives may be stabilizers, ionic compounds, surfactants, buffers, lyophilizing protective agents, or combinations thereof, for example, amino acids (e.g., methionine), salts (e.g., NaCl), buffers, nonionic surfactants (e.g., polysorbates, e.g., polysorbate 20), sugars (e.g., disaccharides such as sucrose), sugar alcohols (e.g., sorbitol), or combinations thereof.
[0035] In one embodiment, the composition may be formulated in any form, such as a solid or liquid formulation, for example, a freeze-dried powder, a liquid, or a pre-filled syringe formulation.
[0036] According to another aspect of the present invention, the present invention provides a use for administering a composition comprising the recombinant botulinum toxin and a pharmaceutically acceptable excipient or additive to an individual to improve or treat a disease.
[0037] In one embodiment, a composition comprising the recombinant botulinum toxin, pharmaceutically acceptable excipients or additives may be administered to an individual for use in improving wrinkles, square jaw, pointed jaw, wounds, skin softening, scars, acne, pores, elasticity, or keloids.
[0038] In addition, in one embodiment, the composition comprising the recombinant botulinum toxin, pharmaceutically acceptable excipients or additives may be administered to an individual for use in treating facial spasms, blepharospasm, torticollis, blepharospasm, cervical dystonia, central pharyngeal dystonia, spasmodic dysphonia, migraine, anal pruritus, or hyperhidrosis.
[0039] The pharmaceutical composition of the present invention may be administered to an individual by various routes. For example, it may be administered by oral administration, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, injection into the paraspinal space (intradural space), sublingual administration, administration into the cheek mucosa, rectal insertion, vaginal insertion, ocular administration, ear administration, nasal administration, inhalation, spray through the mouth or nose, skin administration, transdermal administration, etc. In a specific embodiment, the composition may be administered locally to a muscle or group of muscles. More specifically, it may be administered transdermally or subcutaneously to a fold.
[0040] The pharmaceutical composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means a reasonable amount applicable to medical treatment, and the effective amount level may be determined by the judgment of a physician based on factors including the type and severity of the patient's disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field.
[0041] The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects by considering all of the above-mentioned factors, and this can be easily determined by a person skilled in the art to which the present invention pertains.
[0042] The pharmaceutical composition of the present invention is determined by the type of active ingredient drug, along with various relevant factors such as the disease to be treated, the route of administration, the patient's age, gender, weight, and the severity of the disease.
[0043]
[0044] Hereinafter, the contents of the present invention will be explained in more detail through the following examples and experimental examples. However, the scope of the present invention is not limited to the following examples and experimental examples, but includes variations of equivalent technical concepts.
[0045]
[0046] [Example 1: Preparation of Recombinant Botulinum Toxin of the Present Invention]
[0047] The recombinant botulinum toxin produced in the present invention was produced in Escherichia coli based on a three-fragment production method (LC fragment, Hn fragment, Hc fragment) (Republic of Korea Registered Patent 10-2597062 (2023.10.27)). The specific production method is to be replaced with the method described in the above patent document.
[0048] In this embodiment, LC and Hc derived from A2 and A6, which have a long duration of action and high toxicity of botulinum toxin, were recombined with Hn derived from A1 to create new variants of LC, Hn, and Hc.
[0049] The origins of the sequences introduced into each variant are summarized in Table 1.
[0050] Botulinum toxin variants LC (sequence origin)Hn (sequence origin)Hc (sequence origin)Variant 1(A112)A1A1A2Variant 2(A116)A1A1A6Variant 3(A212)A2A1A2Variant 4(A611)A6A1A1Variant 5(A612,MBT-002)A6A1A2Variant 6(A616)A6A1A6
[0051]
[0052] LC derived from A1 has the nucleic acid sequence of SEQ ID NO. 1 and the amino acid sequence of SEQ ID NO. 2.
[0053] LC derived from A2 has the nucleic acid sequence of SEQ ID NO. 3 and the amino acid sequence of SEQ ID NO. 4.
[0054] LC derived from A6 has the nucleic acid sequence of SEQ ID NO. 5 and the amino acid sequence of SEQ ID NO. 6.
[0055] Hn derived from A1 has the nucleic acid sequence of SEQ ID NO. 7 and the amino acid sequence of SEQ ID NO. 8.
[0056] Hc derived from A1 has the nucleic acid sequence of SEQ ID NO. 9 and the amino acid sequence of SEQ ID NO. 10.
[0057] Hc derived from A2 has the nucleic acid sequence of SEQ ID NO. 11 and the amino acid sequence of SEQ ID NO. 12.
[0058] Hc derived from A6 has the nucleic acid sequence of SEQ ID NO. 13 and the amino acid sequence of SEQ ID NO. 14.
[0059]
[0060] [Experimental Example 1: Comparison of Durability of the Recombinant Botulinum Toxin of the Present Invention]
[0061] In this experimental example, we intended to confirm the persistence of the wild-type BoNT / A1 (900 kDa, a complex of core toxin and non-toxic non-hemaglutinin protein), rBoNT / A1 (150 kDa core toxin excluding non-toxic non-hemaglutinin protein, hereinafter referred to as "MBT-001" in the present invention), variant 1, variant 5 (A612, MBT-002), and variant 6 variants among the above samples through a Digit abduction score (DAS) assay.
[0062] For the DAS assay, variants diluted to 10 μL were injected into the gastrocnemius muscle of mice acclimatized for more than 3 days, and the feet were observed to record the score of the degree of paralysis. Figure 1 shows the results of the Digit Abduction Score (DAS) assay in mice, comparing the daily degree of paralysis of recombinant botulinum toxin. As shown in Figure 1, Variant 5 (A612, MBT-002) was confirmed to have the longest duration of action. In Figure 1, "BoNT / A1 (900 kDa)" is a complete wild-type form of botulinum A1 type toxin with a protective protein attached, and "rBoNT / A1" is a recombinant form of A1 toxin (approx. 150 kDa) (MBT-001) created by recombining LC, Hn, and Hc of botulinum A1 type, excluding the non-toxic non-hemaglutinin protein.
[0063] Meanwhile, Figure 2 shows the results of a mouse Digit Abduction Score (DAS) assay in which the duration of muscle paralysis was analyzed more precisely by administering the MBT-002 of the present invention in the same unit (0.8 unit) as the existing wild-type BoNT / A1 (900 kDa), and it was confirmed that the MBT-002 of the present invention showed approximately twice the duration of efficacy.
[0064]
[0065] [Experimental Example 2: Comparison of Toxicity of the Recombinant Botulinum Toxin of the Present Invention]
[0066] In this example, to compare the toxicity of MBT-002 and MBT-001 (rBoNT / A1, 150 kDa), 100 μL of MBT-001 and MBT-002 prepared at different concentrations were administered into the peritoneal cavity of mice, and the mortality rate was observed for 3 days. As a result of the observation, MBT-002 had an LD50 compared to MBT-001. 50 It was confirmed that this was improved (Fig. 3). MBT-001 is LD 50 While this is about 40 pg, MBT-002 is LD 50 This was approximately 20 pg. Figure 3 shows the mortality results of mice induced by botulinum toxin, with the LD50 of botulinum toxin. 50 The toxicity of MBT-001 and MBT-002 was compared through calculations.
[0067]
[0068] [Experimental Example 3: Comparison of Maximum Duration of Effect and Rate of Onset of Effect of the Recombinant Botulinum Toxin of the Present Invention]
[0069] In this example, a DAS assay was performed to compare the maximum duration of efficacy and expression rate of MBT-002 and BoNT / A1 (900 kDa).
[0070] BoNT / A1 (900 kDa) reached a maximum DAS score of 4, then became 0 after 26 days, and the muscle paralysis effect (EC50) dropped to half (2) after 11 days. In contrast, MBT-002 took 41 days to decrease from 4 to 0, and it took about 20 days to decrease to EC50 (DAS score value of 2). Through this, it was confirmed that the duration of muscle paralysis increased by approximately 1.5 to 2 times (not shown).
[0071] Meanwhile, the degree of paralysis onset was observed from immediately after administration to within 1 day. It was confirmed that the rate of paralysis onset was improved with MBT-002 compared to BoNT / A1 (900 kDa) (Fig. 4). Fig. 4 shows the toxicity onset rate of botulinum toxin, and is the result of comparing the toxicity onset rates of BoNT / A1 (900 kDa) and MBT-002.
[0072]
[0073] [Experimental Example 4: Observation of changes in mouse body weight following injection of the recombinant botulinum toxin of the present invention (Verification of the diffusion of the toxin in the body)]
[0074] In this experiment, changes in mouse body weight were observed following the administration of MBT-002 and BoNT / A1 (900 kDa). Initially, both BoNT / A1 (900 kDa) and MBT-002 showed a decrease in body weight; however, MBT-002 began to recover its body weight faster than BoNT / A1 (900 kDa), and this recovery continued steadily, narrowing the gap with the saline administration group. This result indicates that MBT-002 has fewer effects other than paralysis of the administered muscle compared to BoNT / A1 (900 kDa). Figure 5 shows the changes in mouse body weight caused by botulinum toxin.
Claims
1. Light chain (Light Chain, LC) of botulinum toxin; and, In a botulinum toxin formed by the combination of a heavy chain (HC) of botulinum toxin composed of an Hn domain (Translocation Domain) and an Hc domain (Receptor Binding Domain), The above light chain is an LC derived from A6 type botulinum toxin, and The above Hn domain is an Hn domain derived from A1 type botulinum toxin, and A recombinant botulinum toxin characterized in that the above Hc domain is an Hc domain derived from an A2 type botulinum toxin.
2. A pharmaceutical composition comprising the recombinant botulinum toxin of claim 1 and pharmaceutically acceptable excipients or additives.
3. In Paragraph 2, The above pharmaceutical composition is, A pharmaceutical composition for improving wrinkles, square jaw, pointed jaw, wounds, skin softening, scars, acne, pores, elasticity, or keloid symptoms.
4. In Paragraph 2, The above pharmaceutical composition is, A pharmaceutical composition for treating facial spasms, blepharospasm, torticollis, blepharospasm, cervical dystonia, central pharyngeal dystonia, spasmodic dysphonia, migraine, anal pruritus, or hyperhidrosis.
5. In Paragraph 2, The above pharmaceutical composition is, A pharmaceutical composition for transdermal, subcutaneous, or intramuscular administration.