WO2015183044A1 - 신규한 세포투과성 펩타이드 및 이와 보툴리눔 독소 결합체 및 이들의 용도 - Google Patents
신규한 세포투과성 펩타이드 및 이와 보툴리눔 독소 결합체 및 이들의 용도 Download PDFInfo
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- WO2015183044A1 WO2015183044A1 PCT/KR2015/005434 KR2015005434W WO2015183044A1 WO 2015183044 A1 WO2015183044 A1 WO 2015183044A1 KR 2015005434 W KR2015005434 W KR 2015005434W WO 2015183044 A1 WO2015183044 A1 WO 2015183044A1
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- botulinum toxin
- cell
- recombinant protein
- peptide
- protein
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/55—Fusion polypeptide containing a fusion with a toxin, e.g. diphteria toxin
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Definitions
- the present invention relates to a novel cell permeable peptide and a cell permeable botulinum toxin recombinant protein fused to one end of the cell permeable peptide and the botulinum toxin light chain and their use.
- Botulinum toxin is a neurotoxin produced by Clostridium botulinum , a gram-positive anaerobic bacterium that grows in rotten canned or spoiled meat. It is classified into eight neurotoxins, of which seven (A, B, C, D, E, F, G) can cause nerve paralysis. It is about 150 kDa in size, and consists of a complex of non-toxins in addition to the botulinum toxin protein. The size of each complex is generated up to 900 kDa depending on the type of neurotoxin. Depending on the botulinum toxin type, the type of action, the subject, and the duration of activity vary. Among them, botulinum toxin type A is known as one of the deadly biological agents.
- Botulinum toxin acts as a paralytic by blocking signals that cause muscle spasms or contractions, which has been used for therapeutic or cosmetic purposes since the US FDA approval in 1989.
- neuromuscular diseases such as strabismus, torticollis or blepharospasm
- cosmetic purposes for wrinkles, frown removal and square jaw treatment, hyperhidrosis or migraine It is used as an injection.
- Adverse events such as dysphagia, voice change, dry mouth and blurred vision have been reported, but there are no direct deaths due to botulinum toxin, but if used properly It is considered a safe drug.
- the application is limited in pregnant or lactating women.
- botulinum toxin In the current application of botulinum toxin, the duration of botulinum toxin injected into the skin tissue is within 3-6 months, and when the signal between nerve and muscle is blocked by the botulinum toxin, a new nerve branch is created, which is caused by botulinum toxin. Regular treatment is necessary because it reduces the paralysis effect of nerves. In addition, when the botulinum toxin is repeatedly administered, an antibody to the botulinum toxin in vivo is formed, and thus the effect is reduced.
- the skin which is a body tissue that is always in contact with the external environment, plays a major role as a protective barrier that prevents fluid leakage, infection, and water loss, and is composed of epidermis, dermis and subcutaneous tissue.
- the stratum corneum of the epidermis is in the outermost part of the skin and prevents the skin from drying out by inhibiting the loss of moisture and electrolytes out of the skin and provides an environment for normal biochemical metabolism of the skin.
- the stratum corneum of the skin protects the body from external physical damage and chemicals, and plays an important role in preventing bacteria, fungi, viruses, etc. from invading the skin.
- the stratum corneum of the skin is a natural constituent of the keratinocyte (keratinocyte) is a natural death and forms a dense structure in the outermost layer of the skin, due to the sweat and various lipid components show an acidity of pH around 5.
- keratinocyte keratinocyte
- the molecular weight should be as small as 1,000 or less, and that it should have lipophilic properties.
- Low molecular synthetic compounds or natural compounds which are frequently used as cosmetic and medical raw materials, are known to be easily transferred into cells.
- macromolecules such as proteins, peptides, and nucleic acids have a double lipid membrane structure due to their molecular weight and hydrophilic properties. Since it is difficult to penetrate into the cell membrane, the permeation efficiency of low molecular weight materials is extremely low due to the intrinsic properties of the stratum corneum which actually constitutes the skin barrier, and the permeation efficiency of high molecular weight materials is known to be lower.
- PTDs protein transduction domains
- HIV-Tat and antennapedia are short-charged peptides that are positively charged and contain DNA, RNA, fat, carbohydrates, compounds, or viruses as well as proteins. Is known to be able to deliver intracellularly, and is receptor-independent and has been reported to penetrate the cell membrane as a mechanism of endocytosis or phagocytosis.
- MTD macromolecular transfer domain
- the cell membrane permeation of these peptides can increase the value of development as a new therapeutic drug by transferring a nucleic acid material such as therapeutic protein, DNA or siRNA, which was difficult to use as a drug due to rapid in vivo half-life or cell membrane permeation.
- MTD is known to be highly useful when developing an external preparation of botulinum toxin because the delivery efficiency of compounds, peptides, and proteins, such as cargo materials, is higher than that of HIV-Tat-derived peptides.
- the light or light chain derivative of skin permeation and neuronal cell permeation botulinum toxin to be pursued in the present invention should be limited to the concentration of 1 ⁇ 10ppm in order to ensure safety even after attenuation (toxicity attenuation) process
- PTD toxicity attenuation
- MTD can simultaneously penetrate the skin barrier and penetrate nerve cells and penetrate the skin barrier in a concentration-dependent manner even at low concentrations. The development of an MTD having such characteristics or using a new one was required.
- the present invention is designed to efficiently penetrate the botulinum toxin protein that is difficult to deliver through the skin due to the size of the molecular weight and the intrinsic properties of the stratum corneum as described above, and then to the nerve cells present in the skin tissue, botulinum toxin It is an object of the present invention to provide a novel cell-penetrating peptide capable of mediating intracellular transport of biologically active molecules derived from the translocation domain of the heavy chain.
- the present invention provides a composition comprising the botulinum toxin recombinant protein as an active ingredient, more specifically, it enables the transdermal delivery of cell permeable botulinum toxin recombinant protein, and for various dermatological and cosmetic purposes It is yet another object to provide a composition which can be used topically for this purpose.
- the present invention provides a peptide capable of mediating the delivery of a biologically active molecule into a cell, wherein the peptide provides a cell permeable peptide consisting of the amino acid sequence of SEQ ID NO: 1.
- the present invention provides a polynucleotide encoding the peptide.
- the polynucleotide may be composed of the nucleotide sequence of SEQ ID NO: 2.
- the present invention provides a cell-permeable botulinum toxin recombinant protein in which a cell-permeable peptide consisting of the amino acid sequence of SEQ ID NO: 1 is fused to one side or sock end of a botulinum toxin light chain.
- the botulinum toxin recombinant protein may be composed of an amino acid sequence selected from the group consisting of SEQ ID NO: 31 to SEQ ID NO: 58.
- the botulinum toxin light chain may consist of an amino acid sequence selected from the group consisting of SEQ ID NO: 3 to SEQ ID NO: 9.
- the botulinum toxin light chain may further include a hexahistidine tag at one end.
- the botulinum toxin light chain may be selected from the group consisting of botulinum toxin serotypes A, B, C, D, E, F and G.
- the fusion may be a cell-penetrating peptide fused to the carboxy terminal, amino terminal or both of the botulinum toxin light chain.
- the fusion may be made by peptide bond or covalent bond.
- the present invention provides a polynucleotide encoding the cell permeable botulinum toxin recombinant protein.
- the polynucleotide may comprise a nucleotide sequence selected from the group consisting of SEQ ID NO: 59 to SEQ ID NO: 86.
- the present invention provides a recombinant expression vector comprising the polynucleotide.
- the recombinant expression vector is an affinity label selected from the group consisting of His, HAT, FLAG, c-myc, SBP, Chitin-binding domain, Glutathione-S transferase and Maltose-binding protein tag).
- the present invention provides a bacterium transformed with the recombinant expression vector.
- the present invention provides a pharmaceutical composition for treating a disease selected from the group consisting of migraine, anal pruritus and hyperhidrosis.
- the pharmaceutical composition may be for transdermal administration.
- the present invention provides an external composition for skin comprising the cell permeable botulinum toxin recombinant protein as an active ingredient.
- the present invention provides a cosmetic composition comprising the cell-permeable botulinum toxin recombinant protein as an active ingredient.
- the composition may be applied to improve wrinkles, square jaws and pointed jaws, wounds, softening, scars, acne, pores, elasticity or keloids.
- the present invention facial spasm, eyelid spasm, quadrilateral, blepharospasm, cervical myotonic dystrophy, pharyngeal central myotonic dystrophy, convulsive dyskinesia, migraine headaches comprising transdermal administration of the cell permeable botulinum toxin recombinant protein to the subject It provides a method for treating a disease selected from the group consisting of anal pruritus and hyperhidrosis.
- the present invention provides a method of improving wrinkles, softening, scars, acne, pores, elasticity or keloids, comprising the step of transdermally administering the cell permeable botulinum toxin recombinant protein to an individual.
- the present invention is directed from the group consisting of facial convulsions, eyelid convulsions, quadriceps, blepharospasm, cervical dystonia, central pharyngeal dystonia, convulsive dysphonia, migraine, anal pruritus and hyperhidrosis of the cell-permeable botulinum toxin recombinant protein. Provides use for treating the disease of choice.
- the present invention provides a use of the cell permeable botulinum toxin recombinant protein for use in ameliorating wrinkles, square and pointed jaws, wounds, softening, scars, acne, pores, elasticity or keloid symptoms.
- the present invention provides a cell permeable botulinum toxin recombinant protein production method comprising culturing the transformed bacteria.
- Botulinum toxin works by causing paralysis by blocking signals that cause muscle spasms or contractions.
- the muscle paralysis effect of botulinum toxin is currently used in the treatment of facial spasms, dystonia, migraine, disorder of the jaw joint, hyperhidrosis, and aesthetic and cosmetic fields such as wrinkle improvement, pore reduction, acne, elasticity and square jaw relief.
- a topical application of botulinum toxin that does not require injection would be a safer and more desirable therapeutic alternative.
- the cell-penetrating peptide-botulinum toxin recombinant protein of the present invention can exhibit activity by cleaving a snare protein of nerve cells through the skin complex layer and nerve cells, the molecular weight of the botulinum toxin Significantly small, the possibility of antibody production can be significantly reduced, thereby reducing the efficacy of neutralizing antibody formation.
- the cell-penetrating peptide-botulinum toxin recombinant protein of the present invention can be delivered through the percutaneous, retaining the inherent efficacy of botulinum toxin, and at the same time, the ease of use is expanded, and thus, various diseases can be treated, aesthetic and / or cosmetic purposes using the same. It can be effectively used as a topical agent.
- botulinum toxin type A expresses severe toxicity with only a few picograms (pg), but the cell-permeable botulinum toxin according to the present invention has been attenuated to express toxicity at the microgram ( ⁇ g) level. Full safety from toxicity can be ensured.
- Figure 1 shows the characteristics of the cell permeable peptide TD1 in a table.
- Figure 2 is a graphical illustration of the structure of the cell permeable peptide TD1.
- 3A and 3B confirm the in vitro permeability of the cell permeable peptide TD1 to the keratinocytes (HaCaT cells) using flow cytometry.
- 3c shows the in vitro permeability of the cell permeable peptide TD1 to neurons (SiMa cells) using flow cytometry.
- 3d shows the in vitro permeability of cell permeable peptide TD1 to neurons (U-87MG cells) using flow cytometry.
- 3e shows the in vitro permeability of the cell permeable peptide TD1 to HeLa cells using flow cytometry.
- Figure 4a is confirmed by the confocal microscopy (Confocal microscopy) of the cell permeable peptide TD1 in vitro transmission in keratinocytes (HaCaT cells).
- 4b shows the in vitro transmission ability of the cell-permeable peptide TD1 in neurons (SiMa cell) using confocal microscopy.
- Figure 4c is confirmed by the confocal microscopy (confocal microscopy) of the cell permeable peptide TD1 in vitro transmission in neurons (U-87MG cells).
- 4d shows the in vitro permeability of the cell permeable peptide TD1 in HeLa cells using confocal microscopy.
- FIG. 5 is a schematic diagram showing the purification of the botulinum toxin recombinant protein TD1-Lc bound to the cell permeable peptide TD1.
- Figure 6 shows the purity and molecular weight of the purified cell permeable botulinum toxin recombinant protein TD1-Lc through SDS-PAGE.
- FIG. 7a shows the in vitro permeability of the cell-permeable botulinum toxin recombinant protein TD1-Lc to nerve cells (SiMa cells) using flow cytometry.
- 7b shows the in vitro permeability of the cell-permeable botulinum toxin recombinant protein TD1-Lc in neurons (SiMa cell) using confocal microscopy.
- FIG. 7C shows the in vitro permeability of the cell-permeable botulinum toxin recombinant protein TD1-Lc in keratinocytes (HaCaT cells) using confocal microscopy.
- Figure 8 confirms the permeability of the cell-permeable botulinum toxin recombinant protein TD1-Lc in artificial skin mimetics.
- Figure 9 shows the in vitro SNAP25 cleavage activity of the purified cell permeable botulinum toxin recombinant protein TD1-Lc by SDS-PAGE.
- Figure 10a confirms the in vitro SNAP25 cleavage activity of the cell permeable botulinum toxin recombinant protein TD1-Lc in keratinocytes (HaCaT cells).
- Figure 10b confirms the in vitro SNAP25 cleavage activity of the cell-permeable botulinum toxin recombinant protein TD1-Lc in neurons (SiMa cell).
- FIG. 11A evaluates the cytotoxicity of the cell permeable botulinum toxin recombinant protein TD1-Lc in keratinocytes (HaCaT cells).
- FIG. 11B shows the cytotoxicity of the cell-permeable botulinum toxin recombinant protein TD1-Lc in neuronal cells (SiMa cells).
- Figure 12 confirms the stability of the purified cell permeable botulinum toxin recombinant protein TD1-Lc according to the storage period by SDS-PAGE.
- 13 is a result of evaluating the safety and skin irritation test of the cell-permeable botulinum toxin recombinant protein TD1-Lc prepared in a cosmetic preparation through a clinical laboratory.
- 14a, 14b and 14c are the results of evaluating the clinical efficacy of the cell-permeable botulinum toxin recombinant protein TD1-Lc prepared in a cosmetic preparation through a clinical laboratory.
- the present invention provides a novel cell permeable peptide and a transdermal delivery composition and method of a botulinum toxin light chain using the same.
- the novel cell-penetrating peptide TD1 developed has been found to be suitable as a delivery system that allows botulinum toxin light chains to be administered transdermally by topical application of an appropriate agent.
- the botulinum toxin is expressed as a polypeptide, but is divided into a heavy chain (H chain) of about 100 kDa and a light chain (L chain) of about 50 kDa by the reconstitution process after expression.
- the chains are connected by disulfide bonds.
- the H chain binds to receptors on nerve cells, allowing botulinum toxin to enter through endocytosis.
- the L chain of botulinum toxin enters the cell, then exits the endosomes, enters the cytoplasm, and cleaves the SNARE protein in the cytoplasm to inhibit acetylcholine secretion, thereby exhibiting muscle paralysis effects.
- the isolated botulinum toxin light chain having a molecular weight of 50 kDa cannot penetrate the cell membrane and thus cannot function by itself.
- the help of a botulinum toxin heavy chain of about 100 kDa is necessary for the botulinum toxin light chain to be delivered to the cytoplasm of neurons and thus exhibit botulinum toxin-specific activity.
- the botulinum toxin heavy chain is composed of two sites: a receptor-binding domain that binds to receptors of the neuronal membrane and a translocation domain that is internal to the cell membrane to facilitate the translocation of the light chain.
- the botulinum toxin As a result of research on a method for efficiently delivering the botulinum toxin, more specifically, the botulinum toxin light chain to the skin and nerve cells, through the structural analysis of the botulinum toxin heavy chain, a novel Cell permeable peptides were developed.
- the three-dimensional structure of the translocation domain of the botulinum toxin heavy chain was analyzed in silico to extract and select a sequence of a protein binding site that may be developed as a cell permeable peptide.
- the MTD which is a signal protein or a viral protein-derived peptide involved in the secretion of various proteins, penetrates the cell membrane and mediates the introduction of macromolecules such as proteins into the cell.
- Macromolecule Transduction Domain Karl Patent No. 10-1258279
- the peptide is amphiphilic, and the arrangement of polar amino acids increases cell membrane accessibility, improves physical properties and solubility, and adds a non-polar amino acid to give hydrophobicity suitable for permeation of the cell membrane to develop a novel cell-permeable peptide,
- the novel cell permeable peptide has been confirmed to possess permeability to human skin keratinocytes and neurons at the same time, and completed the present invention based thereon.
- the present invention provides a novel cell permeable peptide, and more specifically, as a peptide capable of mediating the intracellular transport of a biologically active molecule, provides a cell permeable peptide consisting of the amino acid sequence of SEQ ID NO: 1.
- the novel cell permeable peptide is a peptide capable of mediating the delivery of biologically active molecules into cells, and was named "TD1".
- Theoretical pi can be analyzed as 9.31;
- amphiphilic peptides having at least 60% hydrophobic amino acid composition of the fragments
- GRAVY Gram Average of Hydropathicity
- the SVM value is a sequence with a property of -0.15.
- the cell permeable peptide itself preferably does not have a defined enzyme or therapeutic biological activity, but acts as a carrier to enable intracellular transmission through the cell membrane. It may be attached to the N-terminus or C-terminus of the cargo to be transferred into the cell and to the sock end, and may be attached forward or reverse at each end.
- the peptide according to the present invention is preferably applied as a monomer, but is not limited thereto, and may be used in the form of a dimer or a polymer.
- the peptide according to the present invention may be a peptide containing the amino acid sequence of SEQ ID NO: 1 in minimum units.
- one or more amino acids may be added to one or the sock end to change cell membrane accessibility, permeability and physical properties.
- the present invention provides a polynucleotide encoding the peptide. That is, it encodes a cell-penetrating peptide consisting of the amino acid sequence of SEQ ID NO: 1, but may consist of the nucleotide sequence of SEQ ID NO: 2, but is not limited thereto.
- the polynucleotides according to the invention may be in the form of RNA or DNA, wherein the DNA comprises cDNA and synthetic DNA.
- DNA can be single stranded or double stranded. If single stranded, it may be a coding strand or a non-coding (antisense) strand.
- the coding sequence may be identical to the nucleotide sequence of SEQ ID NO: 2, or may be another coding sequence, wherein the coding sequence may contain the same polypeptide as a result of the degeneracy or redundancy of the genetic code. Can be encoded.
- the present invention provides a cell-permeable botulinum toxin recombinant protein in which a cell-permeable peptide consisting of the amino acid sequence of SEQ ID NO: 1 is fused to one side or sock end of the botulinum toxin light chain.
- cell-permeable botulinum toxin recombinant protein includes a novel cell-permeable peptide TD1 and a botulinum toxin light chain, and means a binder formed by chemical bonds such as peptide bonds or covalent bonds. That is, the cell-permeable botulinum toxin recombinant protein delivers botulinum toxin light chain into the cell efficiently by fusion of a specific cell-permeable peptide to the botulinum toxin light chain, which is a large molecule that is not easily introduced into the cell.
- the cell permeable peptide may be fused to a carboxy terminal, an amino terminal, or both of the botulinum toxin light chain.
- botulinum toxin means any known kind of botulinum toxin, whether or not it can be subsequently found, including variants or fusion proteins produced or engineered by bacteria or by recombinant technology. do.
- the botulinum toxin light chain may be selected from the group consisting of botulinum toxin serotypes A, B, C, D, E, F and G, wherein the botulinum toxin light chain is SEQ ID NO: 3 to SEQ ID NO: It may consist of an amino acid sequence selected from the group consisting of nine.
- one end may further include a hexahistidine tag.
- the botulinum toxin light chain may alternatively be a botulinum toxin derivative, ie, a compound having botulinum toxin activity but optionally one or more modifications in part or sequence.
- a botulinum toxin derivative ie, a compound having botulinum toxin activity but optionally one or more modifications in part or sequence.
- light chain endopeptidase may be applied by methods such as deletion, modification, replacement, or chimeric fusion on the amino acid sequence.
- the modified form may be modified in such a manner as to maintain the activity of the compound and enhance the properties or reduce the side effects.
- a portion of a botulinum toxin light chain or a botulinum toxin light chain prepared by recombinant or chemical synthesis can be used.
- the cell permeable botulinum toxin recombinant protein may be composed of an amino acid sequence selected from the group consisting of SEQ ID NO: 31 to SEQ ID NO: 58, the polynucleotide encoding them is selected from the group consisting of SEQ ID NO: 59 to SEQ ID NO: 86
- the base sequence may be formed, but is not limited thereto.
- the skin keratinocytes HaCaT cell
- nerve cells SiMa cell, U-87 MG cell
- skin-derived artificial membrane It was confirmed that the cell permeability was significantly superior to (Start-M) (see Examples 6 and 7).
- the present invention provides a recombinant expression vector comprising a polynucleotide encoding the cell permeable botulinum toxin recombinant protein.
- a "recombinant expression vector” refers to a gene construct that is capable of expressing a protein of interest or RNA of interest in a suitable host cell, and includes a gene construct comprising essential regulatory elements operably linked to express the gene insert.
- operably linked means that the nucleic acid expression control sequence and the nucleic acid sequence encoding the protein or RNA of interest are functionally linked to perform a general function.
- a promoter and a nucleic acid sequence encoding a protein or RNA may be operably linked to affect the expression of the encoding nucleic acid sequence.
- Operative linkage with recombinant expression vectors can be prepared using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation uses enzymes commonly known in the art.
- Expression vectors usable in the present invention include, but are not limited to, plasmid vectors, cosmid vectors, bacteriophage vectors, viral vectors, and the like. Suitable expression vectors include membrane targeting or in addition to expression control sequences such as promoters, operators, initiation codons, termination codons, polyadenylation signals, and enhancers. It may be prepared in various ways according to the purpose, including a signal sequence (leader sequence) or a signal sequence for secretion. The promoter of the expression vector may be constitutive or inducible.
- the expression vector may include a selection marker for selecting a host cell containing the vector, and may include the origin of replication when the expression vector capable of replication. Also, from the group consisting of His, HAT, FLAG, c-myc, SBP, chitin-binding domain, glutathione-S transferase and maltose-binding protein The affinity tag selected may also be included.
- the present invention provides a transformed bacterium transformed with the recombinant expression vector.
- the present invention provides a method for producing a cell-permeable botulinum toxin recombinant protein comprising the step of culturing the transforming bacteria.
- the production method is carried out by culturing the transformed bacteria under appropriate media and conditions such that the polynucleotide encoding the cell permeable botulinum toxin recombinant protein of the present invention is expressed in the recombinant expression vector introduced into the transformed bacterium of the present invention.
- Methods for culturing the transformed bacteria to express the recombinant protein are known in the art, for example, inoculated in a suitable medium in which the transformed bacteria can grow, followed by seed culture, and then inoculated in the medium for culture and suitable Expression of the protein can be induced by culturing in the presence of conditions such as isopropyl- ⁇ -D-thiogalactoside (IPTG), which is a gene expression inducer.
- IPTG isopropyl- ⁇ -D-thiogalactoside
- substantially pure recombinant protein Upon completion of the culture, substantially pure recombinant protein can be recovered from the culture.
- substantially pure in the present invention means that the sequence of the recombinant protein of the present invention and the polynucleotide encoding it is substantially free of other proteins derived from host cells.
- Recovery of the recombinant protein expressed in the transgenic bacteria can be carried out through various separation and purification methods known in the art, and typically centrifuged cell lysate to remove cell debris, culture impurities, etc.
- precipitation for example, salting out (ammonium sulfate precipitation and sodium phosphate precipitation), solvent precipitation (protein fraction precipitation using acetone, ethanol, isopropyl alcohol, etc.) and the like can be carried out, and dialysis, electrophoresis and Various column chromatography can be performed.
- chromatography techniques such as ion exchange chromatography, gel-filtration chromatography, HPLC, reverse phase-HPLC, adsorption chromatography, affinity column chromatography, and ultrafiltration may be used alone or in combination.
- the recombinant protein expressed in bacteria transformed with the recombinant expression vector may be classified into a soluble fraction and an insoluble fraction depending on the characteristics of the protein when the protein is separated. If most of the expressed protein is in the soluble fraction, the protein can be easily isolated and purified according to the method described above, but most of the expressed protein is in the form of an insoluble fraction, i.e. an inclusion body. In the case of dissolving the protein as much as possible with a solution containing a protein denaturant such as urea, surfactant, etc., it can be purified by centrifugation to carry out dialysis, electrophoresis and various column chromatography filled with various kinds of resin.
- a protein denaturant such as urea, surfactant, etc.
- the desalting and reconstituting may be performed by dialysis and dilution using a solution containing no protein denaturant, or centrifugation using a filter.
- the salt concentration in the solution used for purification is high, such a desalting and reconstitution step can be performed.
- the activity of the botulinum toxin is maintained even in the cell-permeable botulinum toxin recombinant protein (TD1-Lc) It was confirmed that the same function as the botulinum toxin (see Examples 8 and 9). In addition, it was confirmed that not only cytotoxicity was observed in human keratinocytes (HaCaT cells) and neurons (SiMa cells) (see Example 10), but also excellent stability (see Example 11). Therefore, the cell permeable botulinum toxin recombinant protein (TD1-Lc) according to the present invention can be effectively used as a topical agent for various disease treatment, aesthetic and / or cosmetic purposes.
- the present invention comprises a cell-permeable botulinum toxin recombinant protein, facial spasm, eyelid spasm, quadrilateral, eyelid spasm, cervical dystonia, pharyngeal dystonia,
- a pharmaceutical composition for treating a disease selected from the group consisting of convulsive dysphonia, migraine, anal pruritus and hyperhidrosis.
- the pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier in addition to the cell permeable botulinum toxin recombinant protein as an active ingredient, wherein the pharmaceutically acceptable carrier included in the pharmaceutical composition of the present invention includes saline, Buffered saline, water, glycerol and ethanol, and the like, but are not limited to this. Any suitable agent known in the art may be used.
- the present invention provides a skin external composition or cosmetic composition comprising a botulinum toxin recombinant protein as an active ingredient.
- the composition may be applied to alleviate or ameliorate wrinkles, square jaw and point jaw correction, wounds, softening, scars, acne, pore reduction, elasticity, lifting or keloid symptoms, but is not limited thereto.
- the compositions according to the invention can be used to induce paralysis in muscles or glandular structures beneath the skin to relieve contraction or cause relaxation or to deliver an effective amount for other cosmetically desired effects.
- the cosmetic composition of the present invention may be prepared in any formulation commonly prepared in the art, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansing , Oils, powder foundations, emulsion foundations, wax foundations and the like can be formulated, but is not limited thereto. More specifically, it may be prepared in the form of a flexible lotion, nutrition lotion, nutrition cream, massage cream, essence, eye cream, cleansing cream, cleansing foam, cleansing water, pack, or powder.
- the cosmetically effective carrier contained in the cosmetic composition of the present invention a carrier commonly used in the art may be used depending on the dosage form.
- a carrier commonly used in the art may be used depending on the dosage form.
- the formulation of the present invention is a paste, cream or gel, animal oils, vegetable oils, waxes, paraffins, starches, trachants, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicas, talc or zinc oxide may be used as carrier components.
- a carrier commonly used in the art may be used depending on the dosage form.
- a solvent, solubilizer or emulsifier is used as the carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 Fatty acid esters of, 3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan.
- liquid carrier diluents such as water, ethanol or propylene glycol
- suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystals Soluble cellulose, aluminum metahydroxy, bentonite, agar or tracant and the like can be used.
- the carrier component is an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, an isethionate, an imidazolinium derivative, a methyltaurate, a sarcosinate, a fatty acid amide.
- Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.
- the components included in the cosmetic composition of the present invention may include components conventionally used in cosmetic compositions, for example, moisturizers, antioxidants, fragrances, fillers, thickeners, dyes, colorants, surfactants. , Natural or synthetic oils, preservatives, penetrants, hydrating agents, antifungal agents, emulsifier solvents, emollients, deodorants, waxes, and the like, optionally with plant extracts, conditioning agents, pigmented or whitening agents, sunscreens, wetting agents, vitamins and Other components conventionally used in such products, including derivatives and the like.
- components conventionally used in cosmetic compositions for example, moisturizers, antioxidants, fragrances, fillers, thickeners, dyes, colorants, surfactants.
- Natural or synthetic oils preservatives, penetrants, hydrating agents, antifungal agents, emulsifier solvents, emollients, deodorants, waxes, and the like, optionally with plant extracts, conditioning agents, pigmente
- the present invention provides facial spasms, eyelid spasms, quadriceps, blepharospasm, cervical dystonia, comprising the steps of local administration of the cell-permeable botulinum toxin recombinant protein to a subject.
- Treatment of diseases selected from the group consisting of central pharyngeal dystonia, convulsive dysphonia, migraine, anal pruritus and hyperhidrosis or improvement of wrinkles, square jaw and pointed jaw, wound, skin softening, scar, acne, pores, elasticity or keloid Provide a method.
- "individual” means a subject in need of treatment of a disease or improvement of skin, and more specifically human or non-human primates, mice, rats, dogs, cats, horses And mammals such as cows.
- Topical administration means the direct administration of a drug to or near an area on or in an animal body in need of the biological effect of the medicament.
- Topical administration excludes systemic routes of administration such as intravenous or oral administration.
- Topical administration is included as a form of topical administration in which the pharmaceutical formulation is applied to the skin of a human.
- the composition of the present invention is preferably administered transdermally for the dermatological and cosmetic desired effect.
- the total effective amount of the recombinant protein of the present invention may be administered to a patient in a single dose, and the fractionated treatment protocol in which multiple doses are administered for a long time. It may be administered by, and the content of the active ingredient may vary depending on the degree of symptoms. This means an amount that is sufficient but intrinsically safe to produce the desired muscle paralysis or biological or aesthetic effect.
- the effective dose of the recombinant protein may be determined in consideration of various factors such as the age, weight, health condition, sex, severity of the disease, diet, and excretion rate, as well as the route and frequency of treatment of the drug. .
- a novel dermal and cell permeable peptide has been developed that enables transdermal delivery of botulinum toxin light chains.
- the structure and function of the botulinum toxin heavy and light chains were analyzed, and the sequence was selected based on the fact that the heavy chain plays an important role in neuronal permeation of botulinum toxin type A.
- the amphiphilic arrangement of polar amino acids increases cell membrane accessibility, improves physical properties and solubility, and adds nonpolar amino acids to hydrophobicity suitable for cell membrane permeation.
- TD1 The cell permeable peptide designed as above was named TD1, and its characteristics and structure were analyzed by the ProtParam program ( http://web.expacy.org/protparam), and the results are shown in FIGS. 1 and 2.
- HaCaT cells were cultured using DMEM complete media (10% FBS, 1% penicillin / streptomycin). For flow cytometry, transfer from 12 well plates and incubate for another 16 to 24 hours, each sample for 1 hour (treatment concentration 5 ⁇ M, 10 ⁇ M) and 3 hours (serum free medium) without FBS. Concentrations of 2.5 ⁇ M, 5 ⁇ M, 10 ⁇ M). After the reaction time is over, the sample is washed twice with DPBS to remove the residual sample, treated with 0.05% trypsin-EDTA, and reacted for 10 minutes with light blocking, and then the trypsin-EDTA is burned using complete media.
- SiMa cell has a weak cell adhesion to the culture plate, and uses a culture plate coated with gelatin (sigma, G2500), coated with 0.1% gelatin solution on the culture plate, removes the solution after 1 hour at room temperature, and dried. Used as. Complete media were passaged at 80% or more confluency using RPMI1640 (10% FBS, 1% penicillin / streptomycin). Cells were stabilized by repeated passage and inoculated with 5 ⁇ 10 5 / well per 100 mm dish, and then cultured overnight in an incubator at 37 ° C. with 5% CO 2 .
- Each sample (control: cell only, FITC only, comparative: HIV-Tat & kFGF4-derived peptide, test substance: TD1) was treated at 5 ⁇ M concentration for 1 and 6 hours in non-serum medium without FBS. . After the reaction time was completed, the sample was washed twice with DPBS to remove the residual sample, treated with 0.05% trypsin-EDTA, and reacted for 10 minutes while blocking the light, followed by inactivation of trypsin-EDTA using complete media. Cells were then collected in the prepared tube, 3 mL of phosphate buffer solution was added, and centrifuged at 2,000 rpm for 3 minutes.
- Trypsin-EDTA was inactivated using complete media. Cells were then collected in the prepared tube, 3 mL of phosphate buffer solution was added, and centrifuged at 2,000 rpm for 3 minutes. After removing the supernatant, 200 ⁇ L of phosphate buffer solution was added to each FACS tube, and the cells were fully resuspended to perform flow cytometry. FL-1 wavelength was used to measure the level of FITC transmitted into the cells, and the transmission capacity was determined based on the scramble peptide value to correct the fluorescence value of the sample at the measured geo.mean value of Fl-1. As a result, as shown in Figure 3d, it was confirmed that TD1 exhibits excellent cell permeability in neurons (U-87 MG cell) compared to the peptide known from the cell-penetrating peptide, kFGF4.
- HeLa cells Human cervix adenocarcinoma cells
- MEM complete media 10% FBS, 1% penicillin / streptomycin
- the cells were transferred from 12 well plates and incubated for 16 to 24 hours, and each sample was reacted with each time and concentration in a serum free medium without FBS. After completion of the reaction time, the sample was washed twice with DPBS to remove the residual sample, treated with 0.05% trypsin-EDTA, and reacted for 10 minutes while blocking the light. Trypsin-EDTA using complete media was inactivated.
- phosphate buffer solution was added, and centrifuged at 2,000 rpm for 3 minutes. After removing the supernatant, 200 ⁇ L of phosphate buffer solution was added to each FACS tube, and the cells were fully resuspended to perform flow cytometry.
- TD1, HIV-Tat and kFGF4-derived peptides were used as the experimental group, and the transmission capacity of each time and concentration was determined by the measured geo.mean value of Fl-1.
- TD1 flows into the cells in a concentration-dependent manner within 12 hours in HeLa cells, HIV-Tat and kFGF4-derived peptides are mostly introduced into the cells at a concentration of 5 ⁇ M or more, It can be seen that it is quite weak compared to TD1. As described above, it was confirmed that TD1 showed very excellent cell permeability in HeLa cells.
- DMEM complete media (10% FBS, 1% penicillin / streptomycin) to check the cell permeability in HaCaT cells, skin keratinocytes.
- 12 mm cover glass was flame sterilized and then placed in each well of each 24 well plate, inoculated with HaCaT cells and incubated for 16 to 24 hours.
- Samples (control: Vehicle, comparative: HIV-Tat, peptide derived from kFGF4, test substance: TD1) were treated at 3 ⁇ M and 5 ⁇ M concentrations in serum-free medium without FBS for 1 and 3 hours.
- the medium was completely removed by suction, and then phosphate buffered solution was added thereto, shaken lightly and washed twice. Then, 200 ⁇ L of 10% formalin solution was added to each well, and the mixture was lightly stirred for 10 minutes. Cells were fixed. After cell fixation, fixative was removed and washed twice with phosphate buffer for 10 minutes. Thereafter, a counterstain was performed at room temperature for 10 minutes in a shaded state using Hoechst and DAPI staining solution. After the reaction, the dyeing solution was removed and washed twice with phosphate buffer. After that, the cover glass was recovered, and the cover glass was slowly lowered and mounted so that air bubbles did not enter the slide glass in which the mounting solution was deposited.
- SiMa cells complete media were cultured at 80% confluency using RPMI1640 (10% FBS, 1% penicillin / streptomycin). After stabilization of the cells by repeated passage, flame sterilization of 12 mm cover glass for microscopic analysis, and then inoculated SiMa cells into each well of each 24 well plate and inoculated with SiMa cells for 16 to 24 hours. Each sample (HIV-Tat, kFGF4-derived peptide, TD1) was treated at 5 ⁇ M concentration for 6 hours in non-serum medium without FBS. After the reaction was completed, the medium was completely removed by inhalation, and then phosphate buffered solution was added and gently shaken twice.
- RPMI1640 % FBS, 1% penicillin / streptomycin
- the solution was washed twice with 200 ⁇ L of 10% formalin solution in each well and lightly stirred for 10 minutes. Cells were fixed. After cell fixation, fixative was removed and washed twice with phosphate buffer for 10 minutes. Thereafter, a counterstain was performed at room temperature for 10 minutes in a shaded state. After the reaction, the dyeing solution was removed and washed twice with phosphate buffer. After that, the cover glass was recovered, and the cover glass was slowly lowered and mounted so that air bubbles did not enter the slide glass in which the mounting solution was deposited. After drying sufficiently in a shaded state, cells were observed using a confocal microscope (Zeiss LSM700). As a result, as shown in Fig. 4b, compared with the kFGF4-derived peptide, excellent cell permeability of TD1 was also confirmed in neurons.
- U-87 MG cells were cultured using DMEM complete media (10% FBS, 1% penicillin / streptomycin) to confirm cell permeability in U-87 MG cells.
- DMEM complete media 10% FBS, 1% penicillin / streptomycin
- 12 mm cover glass was flame sterilized, and then one was put into each well of each 24 well plate, and the cells were inoculated and incubated for 16 to 24 hours.
- Each sample (kFGF4-derived peptide, TD1) was treated at 5 ⁇ M concentration for 6 hours in serum-free medium without FBS. After the reaction was completed, the treated samples were removed and washed twice with PBS. Then, 200 ⁇ L of 10% formalin solution was added to each well, and the cells were fixed for 10 minutes.
- the fixation solution was removed and washed twice with PBS for 10 minutes, and then stained with the Hoechst and DAPI staining solutions for 10 minutes at room temperature in a shaded state. After staining, the solution was removed, washed twice with PBS, and the cover glass was recovered to mount the bubbles in the slide glass in which the mounting solution was deposited. After drying in a shaded state, the cells were observed using a confocal microscope (Zeiss LSM700). As a result, as shown in Fig. 4C, neurons In U-87 MG cells, TD1 was visually confirmed to show excellent cell permeability compared to the kFGF4-derived peptide.
- HeLa cells Human cervix adenocarcinoma cells
- MEM complete media 10% FBS, 1% penicillin / streptomycin.
- 12 mm cover glass was flame sterilized, and then one in each well of each 24 well plate was inoculated with the cells and incubated for 16 to 24 hours.
- Each sample (HIV-Tat, kFGF4-derived peptide, TD1) was reacted for 6 hours and 24 hours at 5 ⁇ M concentration in a serum free medium without FBS. After the reaction was completed, the treated samples were removed and washed twice with PBS.
- botulinum toxin type A light chain protein (Lc) and recombinant protein combining MTD (TD1) and botulinum toxin light chain protein (Lc) was prepared.
- the codon-optimized botulinum neurotoxin type A light chain sequence synthesized by Bioneer was polymerized with primer pairs specifically designed for each. Enzyme chain reaction (PCR) was performed. At this time, the sequence information of each primer is shown in Table 1 below.
- the PCR reaction was carried out with 50 ng of codon optimized Lc template, a final concentration of 0.4 mM each dNTP mixture, 1 ⁇ M of each primer, 5 ⁇ l of 10 ⁇ EX taq buffer, and 0.25 ⁇ l of EX taq polymerase (Takara). It carried out as a reaction liquid. PCR reaction conditions were first denatured at 95 ° C. for 5 minutes, and then repeated 30 times at 95 ° C., 1 minute at 58 ° C. and 1 minute at 72 ° C. for 30 minutes, and finally amplified at 72 ° C. for 8 minutes. It was. After the reaction, electrophoresis was performed on 1% agarose gel to confirm the amplified product.
- the amplified recombinant fragment was recovered from the agarose gel, and then used as a commercial gel extraction kit (Intron, Korea) extracted and purified. Each purified PCR product was treated with NdeI and XhoI enzymes at 37 ° C. for 2 hours, followed by electrophoresis on an agarose gel to digest each recombinant fragment with a gel extraction kit (Intron, Korea). Was purified. Meanwhile, the recombinant vector pET-21b (+) vector (Novagen, USA) having histidine-tag and T7 promoter was cut under the same conditions using restriction enzymes NdeI and XhoI to purify each recombinant.
- the fragment and the cleaved pET-21b (+) vector were mixed, followed by ligation at 16 ° C. for 16 hours after the addition of T4 DNA ligase (Intron, Korea). This was transformed into E. coli DH5 ⁇ -sensitized cells to finally obtain a recombinant protein expression vector.
- the expression vector was treated with the same NdeI and XhoI restriction enzymes and 1% agarose gel electrophoresis. +) It was confirmed that it was correctly inserted into the vector.
- the resulting recombinant protein expression vectors were named pET21b (+)-Lc and pET21b (+)-TD1-Lc, respectively.
- IPTG which is an inducer of protein expression
- Example 5-1 The soluble fraction obtained in Example 5-1 was purified using Fast Protein Liquid Chromatography (FPLC, Bio-rad). The soluble fraction was bound to an affinity chromatography column while flowing in FPLC, and washed by flowing a washing buffer. Thereafter, the imidazole concentration was gradually increased to obtain a purified sample, and dialyzed while stirring at 4 ° C. for 16-20 hours using a dialysis membrane in phosphate buffer or PBS.
- FPLC Fast Protein Liquid Chromatography
- FITC-labeled protein was prepared. In the shaded state, 50 mM Boric acid, 0.1 ng / mL FITC and 0.5 ⁇ g / mL of protein were mixed to make 10 mL of protein suspension and reacted at 4 ° C. for 8 hours. After the reaction was completed, the protein suspension was placed in a dialysis tube, and the dialysis proceeded by replacing with DPBS at intervals of 4 hours-4 hours-16 hours for 3 days at 4 ° C in a shaded state.
- the FITC labeled protein was filtered using a 0.2 ⁇ m syringe filter, and the obtained protein was quantified by Bradford assay and selectively concentrated according to the required concentration.
- the fluorescence intensity (RFU) was measured by diluting to the lowest concentration of the measured protein.
- the fluorescence intensity of the FITC fusion protein used for verification was compared based on the measured RFU.
- SiMa cell The cell permeability of the cell permeable botulinum toxin recombinant protein (TD1-Lc) on the neuronal cell line, SiMa cell, was evaluated.
- SiMa cell has a weak cell adhesion to the culture plate, and uses a culture plate coated with gelatin (sigma, G2500), coated with 0.1% gelatin solution on the culture plate, removes the solution after 1 hour at room temperature, and dried. Used as. Complete media were passaged at 80% or more confluency using RPMI1640 (10% FBS, 1% penicillin / streptomycin). Cells were stabilized by repeated passage and inoculated with 5 ⁇ 10 5 / well per 100 mm dish, incubated for 16-20 hours in an incubator at 37 ° C, 5% CO 2 , and used for experiments. .
- Each sample (vehicle, FITC only, Lc-FITC, TD1-Lc-FITC) was treated at concentrations of 1.5 ⁇ g / ml and 7.5 ⁇ g / ml for 6 hours in non-serum medium without FBS. After the reaction time was completed, the sample was washed twice with DPBS to remove the residual sample, treated with 0.05% trypsin-EDTA, and reacted for 10 minutes while blocking the light, followed by inactivation of trypsin-EDTA using complete media. Cells were then collected in the prepared tube, 3 mL of phosphate buffer solution was added, and centrifuged at 2,000 rpm for 3 minutes.
- TD1-Lc cell-permeable botulinum toxin recombinant protein
- SiMa cells complete media was used at 80% or more confluency using RPMI1640 (10% FBS, 1% penicillin / streptomycin). Cells were cultured. After stabilizing the cells by repeated passage, flame sterilized the 12 mm cover glass for microscopic analysis, and then put one cover glass into each well of each 24 well plate and inoculating SiMa cells and incubating for 16 to 24 hours. Each sample (vehicle, Lc, TD1-Lc) was treated at a concentration of 5 ⁇ g / ml for 3 hours in serum-free medium without FBS.
- the medium was completely removed through an inhaler, washed twice with phosphate buffer solution, 200 ⁇ L of 10% formalin solution was added to each well, and the cells were fixed for 10 minutes.
- the fixative was removed and washed twice with phosphate buffer for 10 minutes.
- the dye solution was removed and washed twice with phosphate buffer.
- the cover glass was recovered for cell observation, and the cover glass was slowly lowered and mounted so that no bubbles entered the slide glass in which the mounting solution was deposited. After drying in a shaded state, the cells were observed using a confocal microscope (Zeiss LSM700).
- DMEM complete media (10% FBS, 1% penicillin / streptomycin) to confirm the cell permeability of the cell permeable botulinum toxin recombinant protein (TD1-Lc) against HaCaT cells, skin keratinocytes.
- TD1-Lc botulinum toxin recombinant protein
- 12 mm cover glass was flame sterilized and then placed in each well of each 24 well plate, inoculated with HaCaT cells and incubated for 16 to 24 hours.
- Each sample (Vehicle, Lc, TD1-Lc) was treated at 5 ⁇ M concentration for 1 hour, 3 hours and 6 hours in serum-free medium without FBS.
- the medium of each well was removed and washed twice with phosphate buffer solution, and then 200 ⁇ L of 10% formalin solution was added to each well, and the cells were fixed for 10 minutes.
- the fixer was removed, washed twice with phosphate buffer for 10 minutes, and counterstained at room temperature for 10 minutes using a Hoechst and DAPI staining solution in a shaded state.
- the staining solution was removed, washed twice with phosphate buffer, and the cover glass was recovered for cell observation.
- the mounting solution was slowly lowered to prevent air bubbles from entering the slide glass. After drying in a shaded state, the cells were observed using a confocal microscope (Zeiss LSM700).
- the TD1-Lc recombinant protein to which the cell-penetrating peptide was bound showed a remarkably excellent cell permeability even in keratinocytes as compared with the Lc protein.
- the automatic skin permeator (MicroettePlus) on the skin-simulated artificial membrane (Start-M) was confirmed.
- Skin simulation artificial membrane is composed of PES (polyether sulfone) which inhibits absorption and polyolefin of lower layer which can differentiate absorption by creating porous structure. It is easy to store and can be directly applied to the system without pretreatment. There is this. In addition, the amount of penetration that is difficult to measure in actual skin can be quantified under conditions similar to that of skin, and thus it is widely used.
- SNAP25 protein is a kind of SNARE protein that is cut by the light chain of botulinum toxin type A.
- the SNAP25 cleavage assay is used in vitro to examine the activity of botulinum toxin.
- BoNT / A Light chain (Lc) cleavage assay was performed.
- cleavage assay buffer (10 mM DTT, 10 mM HEPES, 10 mM NaCl & 20 uM ZnCl 2 ) was added to 2 ⁇ g of GST-SNAP25-EGFP fusion protein, and the concentration of recombinant protein TD1-Lc was 10, 30, 90, 270, respectively. After addition at a concentration of 810 ng, the reaction was carried out at 37 °C for 4 hours. As a positive control, 270 ng of botulinum toxin mixture (BoNT / A complex) was added, and the total volume was adjusted to 20 ⁇ l with tertiary distilled water and reacted under the same conditions.
- BoNT / A complex botulinum toxin mixture
- a SNAP25 cleavage assay was performed by western blot method to confirm the efficacy through cleavage of SNAP25 protein.
- HaCaT cells were incubated for 24 hours at a cell number of 1 ⁇ 10 4 / well in a 24 well plate, transfected with pcDNA3.1-SNAP25 plasmid, and cotransfection with pcDNA3.1-Lc plasmid as a positive control. After overexpressing SNAP25 through 16 hours of incubation, the medium was replaced with FBS-free medium.
- SiMa cells which are human neuroblastoma cells
- a SNAP25 cleavage assay was performed by western blot method to confirm the efficacy through cleavage of SNAP25 protein.
- differentiation medium (10% FBS, RPMI, Glutamax, 1X NEAA, 1X B27, 1X N2, 5uM RA, 2.5uM PUR) was prepared by inducing differentiation.
- Neurons (SiMa cells) were incubated in a cell number of 5 ⁇ 10 5 / well in a 24well plate and then differentiated according to neuronal differentiation method. The cells were exchanged for the last differentiation medium and treated with recombinant protein TD1-Lc after 4 hours. After 48 hours of protein treatment, the medium was removed, washed with PBS, and 200 ⁇ l of RIPA buffer (intron) was added to each well to lyse the cells, and centrifuged at 4 ° C.
- RIPA buffer intron
- the second antibody (Millipore, AP192P) was diluted 1: 5500 in 5% BSA and reacted at room temperature for 1 hour. .
- membranes were washed three times or more at 10-minute intervals with PBST, treated with ECL solution for further reactions, transferred to cassettes, and confirmed by photosensitive X-ray film in the dark.
- Figure 10b it was confirmed that only the TD1-Lc protein can effectively penetrate the neurons. This confirmed that TD1-Lc protein can effectively penetrate not only skin cells but also nerve cells.
- MTT assay was performed to measure cell viability.
- keratinocytes HaCaT cells
- HaCaT cells keratinocytes
- MTT assay was performed to measure cell viability.
- Human neurons (SiMa cells) were cultured in a 24 well plate at a cell number of 5 ⁇ 10 5 / well, and differentiation was induced according to neuronal differentiation methods.
- the recombinant protein TD1-Lc was treated. The protein was treated from 0.625 ⁇ g / ml to a concentration of 40 ⁇ g / ml, and reacted for 48 hours. After 5 mg / ml of MTT (sigma) was added, 10 ⁇ l of each was further reacted for 4 hours.
- each of the recombinant proteins TD1-Lc was dissolved and subjected to electrophoresis on a 12% SDS-PAGE gel according to the course of each period. Check for changes. As a result, as shown in Figure 12, even after six months it was confirmed that the recombinant protein is maintained stable without a change in the pattern of the protein.
- Example 12 Cell Permeability Peptide Botulinum Toxin Recombinant Protein TD1-bound with TD1 Lc Preparation of cosmetic composition and evaluation of skin irritation safety
- liposomes were entrusted to H & A pharmachem and processed and then processed into cosmetic raw materials to produce cosmetic compositions.
- the test was submitted to ICI Korea Co., Ltd. (Korea), a specialized clinical trial agency (CRO).
- Kirea a specialized clinical trial agency
- 31 healthy men and women were placed in the IQ chamber and placed on the back skin.
- the dermatologist judged the safety of human skin and evaluated and analyzed the degree of stimulation.
- the patch method was performed by a single and closed patch test, and the stimulus degree was evaluated and analyzed by Flosch & Kligman's designed measurement method by applying the CTFA guideline commonly used for skin irritation evaluation.
- the volunteers selected 32 healthy adult men and women who met the criteria of selection and exclusion of subjects through medical history examination, questionnaire and screening, and promotion if necessary.
- the investigator and the investigator shall do their best to the safety of the subject, and in the event of any skin abnormality, take prompt and appropriate measures to minimize the reaction.
- the investigator and the investigator take appropriate measures together with the dermatological evaluation and record the case and the situation in detail.
- the measurement of the result was made by the subject visiting the laboratory and waiting at least 15 minutes in a constant temperature and humidity room (22 ⁇ 2 ° C and 50 ⁇ 5%) to stabilize the skin.
- the skin roughness parameter was analyzed using a PRIMOS system after scanning the nasolabial folds before and after 4 weeks of use of the sample.
- Parameters expressing skin roughness are as follows.
- Ra arithmetic average
- Skin elasticity was measured by using a custometer to measure the elasticity (elastic resilience) of the pores. The procedure of inhalation for 2 seconds and reduction for 2 seconds at 400 mb was repeated three times. The pretension time was set to 0.1 seconds to increase the reproducibility of the measurement results.
- the meaning of each parameter value obtained as a measured value when the skin is inhaled and relaxed is as follows.
- nasolabial fold roughness was verified before and after the use of the sample. The results were interpreted before and after using the sample, roughness of the nasolabial folds, skin elasticity parameters, and dermatologist's nasal wrinkle evaluation When there was a significant change in, it was interpreted that the nasolabial folds or elasticity were improved.
- SPSS 14.0 was used, and the normality of the data of the instrument measurement was verified by Shapiro-Wilk test. All 22 subjects were selected as suitable subjects, and all subjects completed the test normally until the last visit, and the last 22 subjects (average 46.1 years old) obtained valid data.
- the cell-penetrating peptide-botulinum toxin recombinant protein of the present invention can be delivered through the transdermal and retains the inherent efficacy of botulinum toxin, and at the same time, ease of use, and can be a safer and preferred therapeutic alternative. It can be effectively used as a topical agent for the treatment of a variety of diseases, aesthetic and / or cosmetic purposes.
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Abstract
Description
Lc Forward primer | GGAATTCCATATGCCCTTTGTCAACAAACAGTTC (서열번호 87) |
Lc Reverse primer | CCGCTCGAGCTTGTTGTAGCCTTTGTCAAG (서열번호 88) |
TD1-LcForward primer | GGAATTCCATATGAAGGCCATGATCAATATTAACAAGTTCTTAAATCAATGTCCCTTTGTCAACAAACAGTTC (서열번호 89) |
TD1-LcReverse primer | CTTGACAAAGGCTACAACAAGCACCACCACCACAGCGGCGGTGGTATGTGACTCGAGCGG (서열번호 90) |
Claims (23)
- 생물학적 활성 분자의 세포 내로의 운반을 매개할 수 있는 펩타이드로서, 상기펩타이드가 서열번호 1의 아미노산 서열로 이루어진, 세포투과성 펩타이드.
- 제1항의 펩타이드를 인코딩하는 폴리뉴클레오티드.
- 제2항에 있어서,상기 폴리뉴클레오티드가 서열번호 2의 염기서열로 이루어진 것을 특징으로 하는, 폴리뉴클레오티드.
- 보툴리눔 독소 경쇄의 일측 또는 양말단에 서열번호 1의 아미노산 서열로 이루어진 세포투과성 펩타이드가 융합된, 세포투과성 보툴리눔 독소 재조합 단백질.
- 제4항에 있어서,상기 보툴리눔 독소 재조합 단백질은 서열번호 31 내지 서열번호 58로 이루어진 군으로부터 선택되는 아미노산 서열로 이루어진 것을 특징으로 하는, 세포투과성 보툴리눔 독소 재조합 단백질.
- 제4항에 있어서,상기 보툴리눔 독소 경쇄는 서열번호 3 내지 서열번호 9으로 이루어진 군으로부터 선택되는 아미노산 서열로 이루어진 것을 특징으로 하는, 세포투과성 보툴리눔 독소 재조합 단백질.
- 제4항에 있어서,상기 보툴리눔 독소 경쇄는 일측 말단에 헥사히스티딘(hexahistidine) 태그(tag)를 더 포함하는 것을 특징으로 하는, 세포투과성 보툴리눔 독소 재조합 단백질.
- 제4항에 있어서,상기 보툴리눔 독소 경쇄는 보툴리눔 독소 혈청형(serotype) A, B, C, D, E, F 및 G로 이루어진 군으로부터 선택되는 것을 특징으로 하는, 세포투과성 보툴리눔 독소 재조합 단백질.
- 제4항에 있어서,상기 융합은 상기 보툴리눔 독소 경쇄의 카복시 말단, 아미노 말단 또는 이들 모두에 상기 세포투과성 펩타이드가 융합되는 것을 특징으로 하는, 세포 투과성 보툴리눔 독소 재조합 단백질.
- 제4항에 있어서,상기 융합은 펩타이드 결합 또는 공유결합에 의해 이루어지는 것을 특징으로 하는, 세포투과성 보툴리눔 독소 재조합 단백질.
- 제4항의 세포투과성 보툴리눔 독소 재조합 단백질을 인코딩하는 폴리뉴클레오티드.
- 제11항에 있어서,상기 폴리뉴클레오티드가 서열번호 59 내지 서열번호 86으로 구성된 군으로부터 선택되는 염기서열로 이루어진 것을 특징으로 하는, 폴리뉴클레오티드.
- 제11항의 폴리뉴클레오티드를 포함하는 재조합 발현벡터.
- 제13항에 있어서,상기 재조합 발현벡터는 His, HAT, FLAG, c-myc, SBP, Chitin-binding domain, Glutathione-S transferase 및 Maltose-binding protein으로 이루어진 군으로부터 선택되는 친화성 표지(affinity tag)를 포함하는 것을 특징으로 하는, 재조합 발현벡터.
- 제13항의 재조합 발현벡터로 형질전환된 세균.
- 제4항의 세포투과성 보툴리눔 독소 재조합 단백질을 유효성분으로 포함하는, 안면경련, 눈꺼풀 경련, 사경(斜頸), 안검경련, 경부 근긴장 이상증, 인두 중앙부 근긴장 이상증, 경련성 발성 장애, 편두통, 항문 소양증 및 다한증으로 구성된 군으로부터 선택되는 질환 치료용 약제학적 조성물.
- 제16항에 있어서,상기 약제학적 조성물은 경피 투여용인 것을 특징으로 하는, 조성물.
- 제4항의 세포투과성 보툴리눔 독소 재조합 단백질을 유효성분으로 포함하는, 피부 외용제 조성물.
- 제4항의 세포투과성 보툴리눔 독소 재조합 단백질을 유효성분으로 포함하는, 화장료 조성물.
- 제18항 또는 제19항에 있어서,상기 조성물은 주름살, 사각턱 및 뾰쪽턱, 상처, 피부연화, 흉터, 여드름, 모공, 탄력 또는 켈로이드 증상을 개선시키는데 적용되는 것을 특징으로 하는, 조성물.
- 제4항의 세포투과성 보툴리눔 독소 재조합 단백질을 개체에 경피 투여하는 단계를 포함하는, 안면경련, 눈꺼풀 경련, 사경(斜頸), 안검경련, 경부 근긴장 이상증, 인두 중앙부 근긴장 이상증, 경련성 발성 장애, 편두통, 항문 소양증 및 다한증으로 구성된 군으로부터 선택되는 질환 치료방법.
- 제4항의 세포투과성 보툴리눔 독소 재조합 단백질을 개체에 경피 투여하는 단계를 포함하는, 주름살, 사각턱 및 뾰쪽턱, 상처, 피부연화, 흉터, 여드름, 모공, 탄력 또는 켈로이드 개선방법.
- 제15항의 형질전환 세균을 배양하는 단계를 포함하는, 세포투과성 보툴리눔 독소 재조합 단백질 생산방법.
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CN201580028681.5A CN106459155B (zh) | 2014-05-29 | 2015-05-29 | 新颖细胞穿透肽、其与肉毒杆菌毒素的缀合物以及其用途 |
EP15800126.3A EP3156412B1 (en) | 2014-05-29 | 2015-05-29 | Novel cell penetrating peptide, conjugate thereof with botulinum toxin, and use thereof |
KR1020167032746A KR101882461B1 (ko) | 2014-05-29 | 2015-05-29 | 신규한 세포투과성 펩타이드 및 이와 보툴리눔 독소 결합체 및 이들의 용도 |
JP2017515651A JP6243577B2 (ja) | 2014-05-29 | 2015-05-29 | 新規な細胞透過性ペプチド及びこれとボツリヌストキシン結合体、およびこれらの用途 |
US15/313,259 US10300118B2 (en) | 2014-05-29 | 2015-05-29 | Cell penetrating peptide, conjugate thereof with botulinum toxin, and use thereof |
CA2949653A CA2949653C (en) | 2014-05-29 | 2015-05-29 | Novel cell penetrating peptide, conjugate thereof with botulinum toxin, and use thereof |
RU2016146659A RU2670135C2 (ru) | 2014-05-29 | 2015-05-29 | Новый проникающий в клетки пептид, его конъюгат с ботулотоксином и их применение |
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106117352A (zh) * | 2016-07-05 | 2016-11-16 | 中国人民解放军军事医学科学院军事兽医研究所 | 抗A型肉毒毒素的人源单链抗体E3‑scFv及其应用 |
WO2017180587A2 (en) | 2016-04-11 | 2017-10-19 | Obsidian Therapeutics, Inc. | Regulated biocircuit systems |
US20180169182A1 (en) * | 2015-06-11 | 2018-06-21 | Merz Pharma Gmbh & Co. Kgaa | Novel recombinant clostridial neurotoxins with increased duration of effect |
US10300118B2 (en) * | 2014-05-29 | 2019-05-28 | Procell Therepautics Inc. | Cell penetrating peptide, conjugate thereof with botulinum toxin, and use thereof |
WO2019241315A1 (en) | 2018-06-12 | 2019-12-19 | Obsidian Therapeutics, Inc. | Pde5 derived regulatory constructs and methods of use in immunotherapy |
WO2020086742A1 (en) | 2018-10-24 | 2020-04-30 | Obsidian Therapeutics, Inc. | Er tunable protein regulation |
KR102192191B1 (ko) * | 2019-12-06 | 2020-12-17 | 주식회사 칸젠 | 신규한 세포 투과성 펩타이드 및 이의 용도 |
KR102192192B1 (ko) * | 2019-12-06 | 2020-12-17 | 주식회사 칸젠 | 보툴리눔 독소 및 세포 투과성 펩타이드를 포함하는 재조합 단백질과 이를 포함하는 화장료 조성물 |
WO2021046451A1 (en) | 2019-09-06 | 2021-03-11 | Obsidian Therapeutics, Inc. | Compositions and methods for dhfr tunable protein regulation |
US11155802B2 (en) | 2017-07-06 | 2021-10-26 | Merz Pharma Gmbh & Co. Kgaa | Recombinant botulinum neurotoxins with increased duration of effect |
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US11952601B2 (en) | 2017-06-20 | 2024-04-09 | Merz Pharma Gmbh & Co. Kgaa | Recombinant botulinum toxin with increased duration of effect |
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Families Citing this family (15)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR101636846B1 (ko) * | 2016-06-08 | 2016-07-07 | (주)넥스젠바이오텍 | 피부 세포 증식 및 항산화 효과가 증가한 보툴리눔 톡신-인간상피세포성장인자 융합단백질 및 이를 유효성분으로 함유하는 피부 재생 및 주름 개선용 화장료 조성물 |
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US11547649B2 (en) | 2018-06-14 | 2023-01-10 | Avixgen Inc. | Fusion protein bound to cell-permeable peptide, and composition comprising fusion protein or cell-permeable peptide and epithelial cell growth factor as active ingredients |
EP3808842A4 (en) * | 2018-06-14 | 2022-11-23 | Avixgen Inc. | PHARMACEUTICAL COMPOSITION FOR THE TREATMENT OF SEVERE COMPLEX IMMUNE DEFICIENCY WITH A CELL-PERFORMING PEPTIDE AND ADENOSINDEAMINASE FUSION PROTEIN |
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Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20040209797A1 (en) * | 2003-03-04 | 2004-10-21 | Michael Karas | Intracellular delivery of small molecules, proteins, and nucleic acids |
US7192596B2 (en) * | 1996-08-23 | 2007-03-20 | The Health Protection Agency Ipsen Limited | Recombinant toxin fragments |
KR20090103957A (ko) * | 2007-01-29 | 2009-10-01 | 주식회사 프로셀제약 | 신규한 거대분자 전달 도메인 및 이의 동정 방법 및 용도 |
US8568740B2 (en) * | 2005-11-17 | 2013-10-29 | Revance Therapeutics, Inc. | Compositions and methods of topical application and transdermal delivery of botulinum toxins with reduced non-toxin proteins |
Family Cites Families (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20040009180A1 (en) * | 2002-07-11 | 2004-01-15 | Allergan, Inc. | Transdermal botulinum toxin compositions |
WO2005030119A2 (en) * | 2003-04-11 | 2005-04-07 | Allergan, Inc. | Botulinum toxin a peptides and methods of predicting and reducing immunoresistance to botulinum toxin therapy |
US20100266638A1 (en) * | 2004-02-26 | 2010-10-21 | Allergan, Inc. | Headache treatment method |
KR100612673B1 (ko) * | 2004-08-20 | 2006-08-14 | (주)바이오버드 | 세포도입성 보톡신 융합단백질 |
US8273865B2 (en) * | 2006-03-15 | 2012-09-25 | Allergan, Inc. | Multivalent clostridial toxins |
KR101258279B1 (ko) | 2011-11-23 | 2013-04-25 | 주식회사 프로셀제약 | 세포 투과능을 개선한 개량형 신규 거대 분자 전달 도메인 개발 및 이의 이용방법 |
ES2621337T3 (es) * | 2011-11-23 | 2017-07-03 | Procell Therapeutics Inc. | Desarrollo de nuevo dominio de transducción macromolecular con mejor permeabilidad celular y método de uso del mismo |
US8420106B1 (en) * | 2012-03-12 | 2013-04-16 | William J. Binder | Extramuscular treatment of traumatic-induced migraine headache |
ES2759478T3 (es) * | 2012-03-12 | 2020-05-11 | William J Binder | Tratamiento de las cefaleas por migraña con neurotoxina presináptica |
CA2880897C (en) * | 2012-11-21 | 2020-01-14 | Syntaxin Limited | Methods for the manufacture of proteolytically processed polypeptides |
CA2949653C (en) * | 2014-05-29 | 2019-12-17 | Procell Therapeutics Inc. | Novel cell penetrating peptide, conjugate thereof with botulinum toxin, and use thereof |
-
2015
- 2015-05-29 CA CA2949653A patent/CA2949653C/en active Active
- 2015-05-29 CN CN201580028681.5A patent/CN106459155B/zh active Active
- 2015-05-29 RU RU2016146659A patent/RU2670135C2/ru active
- 2015-05-29 WO PCT/KR2015/005434 patent/WO2015183044A1/ko active Application Filing
- 2015-05-29 KR KR1020167032746A patent/KR101882461B1/ko active IP Right Grant
- 2015-05-29 BR BR112016027773-2A patent/BR112016027773B1/pt active IP Right Grant
- 2015-05-29 EP EP15800126.3A patent/EP3156412B1/en active Active
- 2015-05-29 JP JP2017515651A patent/JP6243577B2/ja active Active
- 2015-05-29 US US15/313,259 patent/US10300118B2/en active Active
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7192596B2 (en) * | 1996-08-23 | 2007-03-20 | The Health Protection Agency Ipsen Limited | Recombinant toxin fragments |
US20040209797A1 (en) * | 2003-03-04 | 2004-10-21 | Michael Karas | Intracellular delivery of small molecules, proteins, and nucleic acids |
US8568740B2 (en) * | 2005-11-17 | 2013-10-29 | Revance Therapeutics, Inc. | Compositions and methods of topical application and transdermal delivery of botulinum toxins with reduced non-toxin proteins |
KR20090103957A (ko) * | 2007-01-29 | 2009-10-01 | 주식회사 프로셀제약 | 신규한 거대분자 전달 도메인 및 이의 동정 방법 및 용도 |
Non-Patent Citations (1)
Title |
---|
BRUNGER, A. T. ET AL.: "Botulinum neurotoxin heavy chain belt as an intramolecular chaperone for the light chain", PLOS PATHOGENS, vol. 1, no. 10, 2006, pages 1191 - 1194, XP055240720, ISSN: 0021-9258 * |
Cited By (18)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10300118B2 (en) * | 2014-05-29 | 2019-05-28 | Procell Therepautics Inc. | Cell penetrating peptide, conjugate thereof with botulinum toxin, and use thereof |
US20180169182A1 (en) * | 2015-06-11 | 2018-06-21 | Merz Pharma Gmbh & Co. Kgaa | Novel recombinant clostridial neurotoxins with increased duration of effect |
US10603353B2 (en) * | 2015-06-11 | 2020-03-31 | Merz Pharma Gmbh & Co. Kgaa | Recombinant clostridial neurotoxins with increased duration of effect |
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US11969461B2 (en) | 2016-03-02 | 2024-04-30 | Merz Pharma Gmbh & Co. Kgaa | Composition comprising botulinum toxin |
WO2017180587A2 (en) | 2016-04-11 | 2017-10-19 | Obsidian Therapeutics, Inc. | Regulated biocircuit systems |
CN106117352A (zh) * | 2016-07-05 | 2016-11-16 | 中国人民解放军军事医学科学院军事兽医研究所 | 抗A型肉毒毒素的人源单链抗体E3‑scFv及其应用 |
CN106117352B (zh) * | 2016-07-05 | 2020-10-09 | 中国人民解放军军事医学科学院军事兽医研究所 | 抗A型肉毒毒素的人源单链抗体E3-scFv及其应用 |
US11952601B2 (en) | 2017-06-20 | 2024-04-09 | Merz Pharma Gmbh & Co. Kgaa | Recombinant botulinum toxin with increased duration of effect |
US11155802B2 (en) | 2017-07-06 | 2021-10-26 | Merz Pharma Gmbh & Co. Kgaa | Recombinant botulinum neurotoxins with increased duration of effect |
WO2019241315A1 (en) | 2018-06-12 | 2019-12-19 | Obsidian Therapeutics, Inc. | Pde5 derived regulatory constructs and methods of use in immunotherapy |
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KR102192192B1 (ko) * | 2019-12-06 | 2020-12-17 | 주식회사 칸젠 | 보툴리눔 독소 및 세포 투과성 펩타이드를 포함하는 재조합 단백질과 이를 포함하는 화장료 조성물 |
KR102192191B1 (ko) * | 2019-12-06 | 2020-12-17 | 주식회사 칸젠 | 신규한 세포 투과성 펩타이드 및 이의 용도 |
WO2022228443A1 (en) * | 2021-04-26 | 2022-11-03 | Shanghaitech University | Intramuscular compositions of botulinum neurotoxins |
KR102666595B1 (ko) | 2023-06-16 | 2024-05-16 | ㈜에스에이치랩 | 박테리오파지 유래 펩타이드를 포함하는 피부 주름 개선용 생분해성 고분자 필러 및 이의 제조방법 |
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JP6243577B2 (ja) | 2017-12-06 |
CN106459155A (zh) | 2017-02-22 |
RU2670135C2 (ru) | 2018-10-18 |
CN106459155B (zh) | 2019-10-25 |
CA2949653C (en) | 2019-12-17 |
EP3156412A4 (en) | 2017-11-29 |
RU2016146659A (ru) | 2018-05-29 |
JP2017527300A (ja) | 2017-09-21 |
KR101882461B1 (ko) | 2018-07-27 |
BR112016027773A2 (pt) | 2017-10-31 |
US20170246266A1 (en) | 2017-08-31 |
EP3156412A1 (en) | 2017-04-19 |
CA2949653A1 (en) | 2015-12-03 |
BR112016027773B1 (pt) | 2023-12-26 |
RU2016146659A3 (ko) | 2018-05-29 |
EP3156412B1 (en) | 2020-01-08 |
KR20170002475A (ko) | 2017-01-06 |
US10300118B2 (en) | 2019-05-28 |
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