WO2020060128A1 - Composition cosmétique comprenant un peptide dérivé de la toxine botulique ayant une excellente capacité de pénétration cellulaire - Google Patents

Composition cosmétique comprenant un peptide dérivé de la toxine botulique ayant une excellente capacité de pénétration cellulaire Download PDF

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WO2020060128A1
WO2020060128A1 PCT/KR2019/011945 KR2019011945W WO2020060128A1 WO 2020060128 A1 WO2020060128 A1 WO 2020060128A1 KR 2019011945 W KR2019011945 W KR 2019011945W WO 2020060128 A1 WO2020060128 A1 WO 2020060128A1
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Prior art keywords
botulinum toxin
cosmetic composition
fusion protein
protein
composition according
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PCT/KR2019/011945
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English (en)
Korean (ko)
Inventor
최영권
한창규
정현식
장관영
조현영
소우섭
이문행
김성수
호환기
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아이큐어 주식회사
주식회사 아이큐어비앤피
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Publication of WO2020060128A1 publication Critical patent/WO2020060128A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/34—Alcohols
    • A61K8/345—Alcohols containing more than one hydroxy group
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/33—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds containing oxygen
    • A61K8/37—Esters of carboxylic acids
    • A61K8/375—Esters of carboxylic acids the alcohol moiety containing more than one hydroxy group
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/55—Phosphorus compounds
    • A61K8/553—Phospholipids, e.g. lecithin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations

Definitions

  • the present invention relates to a novel skin permeable fusion protein having excellent skin permeability and cell permeability and improved activity of botulinum toxin, and a cosmetic composition comprising the same.
  • the present invention relates to a cosmetic composition
  • a cosmetic composition comprising a botulinum toxin fusion protein and a transdermal absorption accelerator comprising a protein transporter, a light chain of botulinum toxin, and a translocation domain of a heavy chain of botulinum toxin.
  • Botulinum toxin is a neurotoxin protein produced by Clostridium botulinum and has been reported to inhibit the secretion of neurotransmitters acetcholine and catecholamine from neurons.
  • Botulinum toxins There are eight types of botulinum toxins of the A, B, C, D, E, F, G and H types, and the A and B types are commercially used.
  • Synaptobrevin (Vesicle-associate membrane protein, VAMP) present on the surface of vesicles carrying neurotransmitters for the secretion of neurotransmitters, pre-synaptic plasma membrane SNAP-25 (synaptosomal associated protein 25), and Syntaxin three proteins form a snare complex and are secreted extracellularly by stimulation of Ca 2+ .
  • Subtypes of botulinum toxin inhibit the secretion of neurotransmitters by disrupting the formation of snare complexes by cutting specific positions of VAMP-2, SNAP-25 and Syntaxin forming the snare complex (Schiavo et. al. Nature 359, 832-835, 1992).
  • the size of the botulinum toxin protein is about 150 kDa
  • the N-terminal light chain is Zn 2+ metalloprotease 50 kDa
  • the heavy chain is 100 kDa
  • the two proteins Are connected by disulfide bonds.
  • the heavy chain is composed of an N-terminal translocation domain and a C-terminal receptor-binding domain.
  • Botulinum toxin is a neurotoxin protein, and human mortality is very toxic with intravenous injection or intramuscular injection of 1.3 to 2.1 ng / kg and 10 to 13 ng / kg when inhaled. It can be used as a drug to treat diseases, hyperhidrosis, blind jaw, etc., and is most frequently used for cosmetic purposes such as wrinkles and calf muscle contraction.
  • Botulinium toxins on the market are reported to include Botox (Allergan), Botolex, Meditoxin, Disport and BTXA.
  • botulinum toxins have been used as medicines administered to the body through intramuscular injection, and recently, various researches and developments have been actively conducted for use as cosmetics directly delivered to the skin.
  • botulinum toxin is a very large molecule with a combined light and heavy chain of 150 kDa, it is difficult to penetrate the skin, and delivery into the neurons is possible through the endocytosis by binding the heavy chain of botulinum toxin to the receptor.
  • protein transducers and fusion proteins are produced using only light chains having enzymatic activity to induce skin permeation and intracellular delivery.
  • Protein transduction domain is a small peptide consisting of 10 to 16 amino acids called a cell penetration peptide (CPP) or membrane translocating sequence (MTS).
  • CPP cell penetration peptide
  • MTS membrane translocating sequence
  • Protein transporters accumulate in cells through the plasma membrane with themselves or substances that bind themselves without the aid of special receptors. They can bind DNA, RNA, heparin and sialic acid, as well as proteins, peptides, antisense sequences, plasmids, microbeads, liposomes, etc. You can move freely.
  • the protein with the first protein transduction was the HIV-1 (Human Immunodeficiency Virus type 1) Tat protein.
  • the Tat protein is a transacting transcription factor of HIV-1 composed of 86 amino acids, which not only activates transcription of HIV-1, but also influences induction of viral replication in chronically infected cells. It is also known to induce reactivation of infected cells in a resting state.
  • VP22 which is a protein between the envelope protein and the capsid of HSV-1 (Herpes Simplex Virus type 1), and the transcription factor involved in the development process and nerve formation of Drosophila. Sequences that induce protein transduction of various species have been reported, such as antennapedia protein, which is a type of homeoprotein.
  • Intracellular delivery by protein transporters acts as a completely different mechanism than conventional protein delivery, such as endocytosis.
  • a positively charged protein carrier that contains a large amount of the basic amino acid arginine
  • it is transferred into the cell by macropinocytosis
  • a hydrophobic protein carrier it is combined with phospholipids to form inverted micelles and transferred into the cell. Is known.
  • the present inventor In order to improve the activity of botulinum toxin in cells even when applied directly to the skin, the present inventor has an N-terminal translocation domain that plays an important role in the translocation of target proteins in the cytoplasm during heavy chain of botulinum toxin. ) Was fused to the light chain of botulinum toxin to complete the present invention.
  • the present inventors identified and fused the most suitable protein carrier for the fusion protein.
  • the present invention relates to a fusion protein comprising a protein transporter, a light chain of botulinum toxin, and an N-terminal translocation domain of a heavy chain of botulinum toxin.
  • the present invention provides a cosmetic composition comprising the botulinum toxin fusion protein as an active ingredient.
  • the present invention provides a transdermal absorption accelerator that enables delivery through the percutaneous layer, exhibits excellent activity of botulinum toxin, contains botulinum toxin fusion protein that does not exhibit skin irritation as an active ingredient, and improves cell penetration of the fusion protein.
  • a cosmetic composition further comprising.
  • the present invention provides a cosmetic composition having an improvement or prevention effect of skin wrinkles including a botulinum toxin fusion protein and a transdermal absorption accelerator.
  • the present invention uses a protein transporter consisting of an amino acid sequence of any one of SEQ ID NOs: 1 to 10, including a cationic transporter and a hydrophobic protein transporter, among the protein transporters as carriers for light chain peptides of botulinum toxin.
  • the present invention relates to a fusion protein comprising a protein carrier consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 10 and a light chain peptide of botulinum toxin.
  • the present invention provides a fusion protein comprising a protein delivery system consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 10, a light chain peptide of botulinum toxin, and a translocation region peptide of botulinum toxin heavy chain.
  • the botulinum toxin light chain peptide may consist of the amino acid sequence of SEQ ID NO: 21.
  • the translocation region peptide of the heavy chain of botulinum toxin may consist of the amino acid sequence of SEQ ID NO: 22.
  • the fusion protein consisting of the protein carrier, the light chain peptide of botulinum toxin, and the translocation region peptide of the botulinum toxin heavy chain, between the protein carrier and the botulinum toxin light chain peptide and / or the botulinum toxin light chain peptide and the botulinum toxin heavy chain It may include a linker between the translocation region of the peptide.
  • the linker is represented by (G) N , where N is an integer from 1 to 20, and G may be composed of an amino acid sequence that is glycine.
  • the linker may be GGGGG.
  • the present invention provides a polynucleotide encoding a fusion protein comprising a protein delivery system consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 10, a light chain peptide of botulinum toxin and a translocation region peptide of heavy chain of botulinum toxin.
  • the present invention provides a recombinant expression vector comprising the polynucleotide.
  • the present invention provides bacteria transformed with the recombinant expression vector.
  • the polynucleotide of the protein delivery system may be composed of any one of SEQ ID NOs: 11 to 20.
  • the present invention provides a composition comprising a fusion protein comprising a protein delivery system consisting of the amino acid sequence of any one of SEQ ID NOs: 1 to 10, a light chain peptide of botulinum toxin and a translocation region peptide of heavy chain of botulinum toxin.
  • the present invention is a cosmetic composition
  • a cosmetic composition comprising a fusion protein and a transdermal absorption accelerator comprising a protein carrier consisting of any one of the amino acid sequences of SEQ ID NOs: 1 to 10, a light chain peptide of botulinum toxin and a translocation region peptide of heavy chain of botulinum toxin.
  • the "percutaneous absorption accelerator” is a component that affects skin permeation among emulsifiers and is a component commonly used in transdermal patch. In the present invention, it serves to increase the skin penetration and cell penetration of the fusion protein, Lecithin, Lauryl Pyrrolidone, Glycerol Monooleate, Glycerol Monolaurate ), Propylene Glycol Monolaurate, Polyoxyethylene Sorbitan Monooleate, Polyoxyethylene Sorbitan Monostearate, Polyoxyethylene Sorbitan Monostearate, Polyoxyethylene Sorbitan Monolaurate, Sorbitan Monooleate, Sorbitan Monostearate, Sorbitan Monolaurate, but is not limited thereto.
  • the transdermal absorption accelerator of the present invention may preferably be lecithin, lauryl pyrrolidone, polyoxyethylene sorbitan monooleate or sorbitan monostearate, and more preferably lecithin.
  • the present invention relates to a cosmetic composition
  • a cosmetic composition comprising the fusion protein of the present invention, a transdermal absorption accelerator and a medium chain glyceride (Medium Chain Glyceride).
  • the fusion protein, transdermal absorption accelerator, and medium chain glyceride may form micelles in a cosmetic composition.
  • a micelle has a microsphere shape formed as an aggregate when the surfactant is at a certain concentration or more.
  • it is difficult to form micelles only with an active ingredient and a surfactant, and it is preferable to form an micelle by adding an oil component such as a medium chain glyceride.
  • the percutaneous absorption accelerator helps to permeate compounds of relatively small molecular weight (500 Da or less) by imparting fluidity to the stratum corneum phospholipid layer, but because the molecular weight of the fusion protein of the present invention is very large, 100 kDa or more, it is fused only by fluidization of keratin.
  • the cell permeation and cell permeation may be increased due to the intracellular induction (endocytosis) in the form of micelles.
  • the medium chain glyceride of the present invention may be caprylic / capric triglyceride, but is not limited thereto.
  • Lecithin acting as a transdermal absorption accelerator in the present invention rapidly increases skin permeability at 0.5% by weight or more based on the total weight of the composition, which corresponds to a critical concentration (CMC; Critical Micelle Concentration) to form micelles.
  • CMC Critical Micelle Concentration
  • composition of the present invention may further include a sugar alcohol that stabilizes the fusion protein, and the sugar alcohol may be mannitol, erythritol, xylitol, sorbitol, and the like, preferably mannitol.
  • the composition of the present invention relates to a cosmetic composition for improving or preventing skin wrinkles.
  • skin permeability means the ability or property to penetrate the skin and penetrate into the skin, and the fusion protein of the present invention and the composition comprising the same have remarkably excellent skin permeability compared to conventional botulinum toxin. Shows.
  • fusion protein is a protein artificially synthesized such that the skin-permeable peptide (protein transporter) is bound to another protein or peptide.
  • the composition of the fusion protein according to the present invention is shown in FIG. 1.
  • the fusion protein of the present invention or the polypeptide constituting the fusion protein is prepared by a chemical peptide synthesis method known in the art, or the gene encoding the fusion protein is amplified by polymerase chain reaction (PCR) or by a known method. After synthesis, it can be produced by cloning and expressing it in an expression vector.
  • PCR polymerase chain reaction
  • expression vector is a recombinant vector capable of expressing a target peptide in a desired host cell, and means a gene construct comprising essential regulatory elements operably linked to express a gene insert.
  • the expression vector includes expression control elements such as an initiation codon, a termination codon, a promoter, an operator, etc., the initiation codon and the termination codon are generally regarded as a part of a nucleotide sequence encoding a polypeptide, and when a gene construct is administered. It must represent an action in and must be in frame with the coding sequence.
  • the promoter of the vector can be constitutive or inducible.
  • the expression vector may include a nucleotide sequence encoding a fusion protein comprising the skin-permeable peptide (protein transporter) of the present invention and the N-terminus of the light and heavy chains of botulinum toxin. Is not particularly limited as long as it can produce a fusion protein comprising the skin-permeable peptide of the present invention and the N-terminus of the light and heavy chains of botulinum toxin.
  • H6Ub protein of the present invention is a protein in which six histidine proteins (H6) are bound to a ubiquitin protein.
  • Ubiquitin is a protein known as an expression inducer that helps the expression of a target protein.
  • Ubiquitin is cleaved and removed by Ubiquitin Specific Protease (USP) during the purification process.
  • USP Ubiquitin Specific Protease
  • the "linker” of the present invention is a peptide used for a physicochemical distance between domains or domains within a fusion protein, or for linkage.
  • the fusion protein of the present invention may include a linker between the skin permeable peptide and the botulinum toxin light chain peptide and / or between the botulinum toxin light chain peptide and the N-terminal peptide of the botulinum toxin heavy chain.
  • the fusion protein of the present invention and a composition comprising the same, has the advantage of improving skin permeability and cell permeability, and improving the activity of botulinum toxin.
  • Figure 1 shows the domain configuration of the fusion protein according to the present invention.
  • Figure 2 shows the results of processing a pATP vector inserted with DNA encoding a fusion protein with a restriction enzyme.
  • Figure 3 shows the results of expressing the fusion protein in the strain E. coli.
  • Figure 4 shows the results of purifying the peptide of the botulinum toxin fusion protein.
  • Figure 5 shows the results of the intracellular botulinum toxin delivery experiment according to the fusion protein.
  • FIG. 6 is a schematic diagram of Franz Diffusion Cell used for skin permeability analysis.
  • Figure 8 shows the average of the composite muscle action potential recorded by stimulating the sciatic nerve 50 times at 2 Hz in the complex muscle action potential test.
  • Figure 9 shows the results of measuring the complex muscle action potential over time after injection of the fusion protein.
  • LC light chain of botulinum toxin
  • S + LC fusion protein amino acid consisting of the sequence of the protein carrier represented by SEQ ID NO: 2, the linker (GGGGG) and the light chain (LC) of botulinum toxin
  • S + LC + HC fusion protein amino acid consisting of the protein transporter represented by SEQ ID NO: 2, linker (GGGGG), LC, linker (GGGGG), and the translocation region of the heavy chain of botulinum toxin, respectively.
  • the polynucleotide was synthesized (FIG. 1).
  • expression vectors for three fusion proteins were prepared.
  • H6Ub protein was used as a fusion partner to induce expression.
  • the BL21 (DE3) starpLysS E. coli strain (Invitrogen, NY, USA) transformed with the plasmid encoding the fusion protein was 600 in 37 LC (Luria-Bertani) medium containing kanamycin (50 mg / L). Incubation was performed until the absorbance at nm became 0.6. The expression results of the fusion protein are shown in FIG. 2.
  • Protein expression was induced with 0.1M of IPTG (isopropyl- ⁇ -D-thiogalactopyranoside), and the cells were further cultured for 16 hours.
  • Cells were collected by centrifugation and re-suspended in Lysis buffer (50 mM NaH 2 PO 4 , 100 mM NaCl, 5 mM imidazole, protease inhibitor cocktail tablets, pH 8.0) and crushed by sonication. Then, centrifugation was performed at 13,000 rpm and 4 ° C. for 15 minutes, and the supernatant was checked for SDS-PAGE to express the expression rate and acceptability of the fusion protein.
  • the restriction enzyme treatment results are shown in FIG. 3.
  • the supernatant was purified using IMAC 6FF (GE Healthcare Life Sciences) and Q FF (GE Healthcare Life Sciences), and then buffered with a stabilized buffer using Sephadex G-25 (GE Healthcare Life Science). Replaced.
  • fusion protein Purification of the fusion protein was purified using the HisPrep FF (GE Healthcare Life Sciences) and HighTrep Desalting (GE Healthcare Life Science). The purified fusion protein is shown in FIG. 4.
  • the light chain of botulinum toxin (LC); (2) an amino acid (S + LC fusion protein) consisting of the sequence of the protein carrier represented by SEQ ID NO: 2, the linker (GGGGG) and the light chain (LC) of botulinum toxin; And (3) the amino acid (S + LC + HC fusion protein) consisting of the protein transporter represented by SEQ ID NO: 2, linker (GGGGG), LC, linker (GGGGG), and the translocation region of the heavy chain of botulinum toxin, respectively.
  • Polypeptides synthesized from the polynucleotide described above were used as Examples 1 to 3.
  • composition comprising botulinum-derived fusion protein
  • a composition comprising the fusion protein of Example 3 and a percutaneous absorption accelerator was prepared by the following method.
  • a solution of a mixture of cetearyl alcohol and caprylic / capric triglyceride, and a solution of glycerin, mannitol, arginine, phenoxyethanol, and purified water are dissolved at 70 ° C, respectively, and mixed for 5 minutes at 3000 rpm in a homomixer. While emulsifying. Thereafter, a fusion protein and a carboxypolymer solution were added and mixed for 5 minutes to prepare Preparation Example 1.
  • a solution of a mixture of cetearyl alcohol, caprylic / capric triglyceride, and lecithin, and a solution of glycerin, mannitol, arginine, phenoxyethanol, and purified water are dissolved at 70 ° C, respectively, and mixed to obtain a homomixer. Emulsified at 3000 rpm for 5 minutes. Thereafter, a fusion protein and a carboxypolymer solution were added and mixed for 5 minutes to prepare Preparation Example 2.
  • the fluorescent substances FITC Sigma, Fluorescein-5-isothiocyanate, 27072-45-3) are labeled with FACs (FACSCalibur, Becton) for the polypeptides of Examples 1 to 3, respectively. Dickinson).
  • the reaction time was reacted to PC 12 cells (Pheochromocytoma, ATCC CRL-1721) for 1 hour.
  • Example 3 shows a better intracellular delivery effect than Example 2.
  • the skin used for the test was a Guinea Pig (Hartley Guinea Pig). 32 ° C was maintained using a constant temperature water bath, and the medium of the receptor was PBS, and was set at 600 rpm. Sampling was performed at intervals of 2 hours for 10 hours to calculate skin penetration rate (Flux).
  • Cy-5 was conjugated to the polypeptides of Examples 1 and 3 using a Cy5 Fast conjugation kit (ab188288), and analyzed using a Waters 2475 Fluorescence Detector (excitation 630 nm, emission 660 nm).
  • the skin permeability of the fusion protein of Example 3 was significantly 2-3 times greater than in the case of only the light chain of the botulinum toxin of Example 1 It was confirmed that it was excellent.
  • Catecholamine is an sympathetic nerve stimulator and is an important indicator to consider the function of the adrenal water-sympathetic nervous system. Since botulinum toxin paralyzes muscles by inhibiting the secretion of neurotransmitters from nerve cells, the effect of inhibiting the secretion of catecholamines, dopamine and norepinephrine, was analyzed to confirm the neurotransmitter secretion inhibiting function.
  • PC 12 cells (Pheochromocytoma, ATCC CRL-1721) were cultured in 15 ml of F-12K medium (Gibco) with 2 mM L-glutamine, 1.5 g / L sodium bicarbonate and 10% FBS. For 2 days, the cells were cultured in a cell culture flask T75 (Nunc) to have a cell density of 625,000 cells / cm 2.
  • the fusion proteins of Examples 1 to 3 were treated at concentrations of 10 -5 M and 10 -6 M, and the results are shown in Table 2 below.
  • the light chain of botulinum toxin of Example 1 had no difference in the amount of neurotransmitter secretion compared to the positive control group, and it was confirmed that there was no inhibitory effect of catecholamine secretion.
  • Example 2 The fusion proteins of Examples 2 and 3 showed the effect of suppressing the secretion of neurotransmitters, and Example 3 showed a better inhibitory effect than Example 2.
  • Botulinum toxin paralyzes muscles by inhibiting the secretion of neurotransmitters from nerve cells, so to confirm the performance of botulinum toxin, it stimulates nerves and records the compound muscle action potentials (CMAP) of the muscles. Evaluation is a general performance evaluation test.
  • BOTOX Cosmetics onabotulinum toxin A, Allergan
  • Example 1 to 3 fusion proteins were injected.
  • the injection method of BOTOX Cosmetics was to dissolve 100 units suggested by the manufacturer in 2.5 mL of 0.9% physiological saline and inject 5 uL once.
  • the fusion proteins of Examples 1 to 3 were compared by injection once at a concentration of 100 ng / 5 uL each.
  • the sciatic nerve was electrically stimulated to measure the amplitude of the compound muscle action potential in the anterior sciatic muscle.
  • the average of the composite muscle action potential recorded by stimulating the sciatic nerve 50 times at 2 Hz is shown in FIG. 8.
  • the stimulation around 150% of the size of the stimulus causing the maximum amplitude was repeated 50 times at 2 Hz and 20 Hz to induce repeated complex muscle action potentials, and was compounded at 1, 2, 3, and 4 weeks after injection, respectively. 9 shows the results of measuring the amplitude of the muscle action potential.
  • the gauze As a method of application, after applying 0.5 ml (concentration of 1 mg / ml) of test substance onto the skin of 2.5 cm X 2.5 cm, the gauze was applied so that the test substance and the skin were in good contact. The control section was covered only with gauze. On the gauze, a tape with low impermeability and low irritation was wound to prevent evaporation of the test material.
  • test substance was applied once, removed after 24 hours, and lightly washed with physiological saline so that the test substance did not remain. After application of the test substance, changes in erythema, edema, bleeding, peel formation, etc. of the topical site were observed visually at 24 hours, 48 hours, and 72 hours. Table 3 and Table 4 show the evaluation criteria for skin reactions.
  • the primary skin irritation index is the average sum of skin response scoring results for each individual divided by four. Table 5 below shows the degree of irritation according to the primary skin irritation index.
  • the fusion protein of Example 3 is a non-irritating substance having a primary skin irritation index (PII) value of 0.25.
  • the wrinkle improvement effect according to the use of the product was analyzed using a skin image analyzer (Visiometer). To this end, a phantom plate of the designated eye area was prepared at 0, 4, and 8 weeks. The simulated plate was manufactured using a silicone material under constant temperature and humidity (22 ⁇ 2 ° C, 40% to 60% humidity) conditions.
  • the simulation board was analyzed using software of a skin image analyzer (Skin-Visiometer SV 600, Courage & Khazaha, Gemany).
  • Analysis of the simulated plate was performed by spectroscopy. Specifically, the intensity of light generated by the light emitted from the artificial light source passing through the silicon material was analyzed by Lambert & Beer's law, and the degree of skin wrinkle improvement was measured.
  • the measured variable by the skin image analyzer is indicated by R2, which represents the maximum roughness.
  • R2 represents the maximum roughness.
  • the skin wrinkle improvement rate (%) in the skin wrinkle improvement evaluation test of the cosmetic composition according to the present invention is shown in Table 6 below.
  • the simulation board was analyzed using software of a skin image analyzer (Skin-Visiometer SV 600, Courage & Khazaha, Gemany).
  • Preparation Example 2 containing lecithin showed the best skin permeability
  • Preparation Example 3 containing lauryl pyrrolidone
  • Preparation Example 8 comprising polyoxyethylene sorbitan monostearate
  • sorbitan monostea It was confirmed that Preparation Example 11 including a rate showed excellent skin permeability of 15 ⁇ g / cm 2 or more.
  • Preparation Examples 13 to 15 were prepared in the same manner as in Preparation Example 2 to perform a test for confirming skin permeability according to the content of lecithin.
  • Percutaneous absorption accelerators are known to impart fluidity to the stratum corneum phospholipid layer and help to permeate compounds of relatively small molecular weight (below 500 Da).
  • molecular weight is significantly larger than 100 kDa, the skin permeation of the fusion protein is difficult only by fluidization of keratin.
  • capillary / capric triglyceride a type of transdermal absorption accelerator and medium chain glyceride, forms micelles together with a fusion protein.
  • skin penetration was increased by intracellular migration (endocytosis) in the form of micelles.

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Abstract

La présente invention concerne un support protéique, une protéine recombinante dans laquelle les régions de translocation de la chaîne légère et de la chaîne lourde de la toxine botulique sont fusionnées, et une composition comprenant ceux-ci, afin d'améliorer les effets et la pénétration cellulaire d'une toxine botulique. En particulier, la présente invention concerne une composition cosmétique comprenant la protéine de fusion et un accélérateur d'absorption transdermique.
PCT/KR2019/011945 2018-09-21 2019-09-16 Composition cosmétique comprenant un peptide dérivé de la toxine botulique ayant une excellente capacité de pénétration cellulaire WO2020060128A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11596673B2 (en) * 2013-12-12 2023-03-07 Medy-Tox Inc. Long lasting effect of new botulinum toxin formulations
WO2023075060A1 (fr) * 2021-10-25 2023-05-04 비피메드(주) Composition comprenant un peptide dérivé de la toxine botulinique pour soulager la douleur

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