KR101968873B1 - Cosmetic composition comprising peptide derived from Botulinum toxin having improved cell penetrating ability - Google Patents

Abstract

The present invention relates to a recombinant protein in which a translocation region of heavy chains and light chains of botulinum toxin and a protein transporter are fused, and a composition comprising the same, in order to enhance an effect of botulinum toxin and cell penetration. More specifically, the present invention relates to a cosmetic composition comprising a transdermal absorption enhancer and the fusion protein.

Description

[0001] The present invention relates to a cosmetic composition comprising a botulinum-derived peptide having excellent cell penetration effect,

The present invention relates to a novel skin permeable fusion protein exhibiting excellent skin permeability and cell permeability and having improved botulinum toxin activity, and a cosmetic composition containing the same.

Specifically, the present invention relates to a cosmetic composition comprising a protein transporter, a light chain of a botulinum toxin, and a botulinum toxin fusion protein comprising a translocation domain of a heavy chain of a botulinum toxin and a transdermal absorption enhancer.

The botulinum toxin is a neurotoxin protein produced by Clostridium botulinum and has been reported to inhibit the secretion of neurotransmitter acetol and catecholamines. There are eight kinds of botulinum toxin types A, B, C, D, E, F, G and H, and types A and B are commercially available.

In order to secrete neurotransmitters, synaptobrevin (Vesicle-associate membrane protein, VAMP), present on the surface of the vesicle carrying neurotransmitter, and pre-synaptic plasma membrane SNAP-25 (synaptosomal associated protein 25), and Syntaxin 3 proteins form a SNARE complex and secreted extracellularly by stimulation of Ca ²⁺ .

Subtypes of botulinum toxin inhibit the secretion of neurotransmitters by interrupting the formation of SNARE complexes by cleaving specific positions of VAMP-2, SNAP-25 and Syntaxin that form the SNARE complex (Schiavo et < RTI ID = 0.0 > al., Nature 359, 832-835, 1992).

The botulinum toxin protein is approximately 150 kDa in size, the light chain in the N-terminal is 50 kDa as Zn ^{2 +} metalloprotease, the heavy chain is 100 kDa, Are connected by a disulfide bond. The heavy chain consists of the N-terminal translocation domain and the C-terminal receptor-binding domain.

The botulinum toxin is a neurotoxin protein. Human lethal dose is 1.3 ~ 2.1 ng / kg for intravenous or intramuscular injection and 10 ~ 13 ng / kg for inhalation. However, It can be used as a medicine for treating diseases, hyperhidrosis, quadriceps, etc., and is most widely used for cosmetic purposes such as wrinkles and calf muscle reduction. Domestic botulinum toxins are reported to be Botox (Allergan), Botolex, Meditoxin, Dysport and BTXA.

These botulinum toxins have been used as medicines administered to the body through intramuscular injections. Recently, various researches and developments have been actively carried out for use as cosmetics directly delivered to the skin.

However, because the botulinum toxin is a very large molecule with a light chain and a heavy chain at 150 kDa, it is difficult to penetrate the skin, and the transfer of the botulinum toxin into the nerve cell is possible through the endocytosis of the heavy chain of the botulinum toxin. In recent years, protein transporters and fusion proteins have been produced using only light chains having enzymatic activity to induce skin permeation and intracellular delivery.

The protein transduction domain (PTD) is a small peptide composed of 10 to 16 amino acids called a cell permeable peptide (CPP) or a membrane-translocating sequence (MTS). Protein transporters accumulate in cells through plasma membranes with substances that bind themselves or with themselves, without the aid of special receptors. They can bind to DNA, RNA, heparin and sialic acid, as well as proteins, peptides, anti-sense sequences, plasmids, microbeads, liposomes, You can move freely.

The protein with the first protein transduction phenomenon is the Tat protein of HIV-1 (Human Immunodeficiency Virus type 1). The Tat protein is a transacting transcription factor of HIV-1 consisting of 86 amino acids that not only activates transcription of HIV-1 but also affects the induction of viral replication in chronically infected cells. It is also known to induce reactivation of infected cells in the resting state.

In addition, VP22, a skin protein existing between the envelope protein and capsid of HSV-1 (Herpes Simplex Virus type 1), and the transcription factor involved in the development of the fruit fly and neuron formation, Have been reported to induce protein transduction of various species such as the antennapedia protein, which is a type of homeoprotein.

The human protein transporter has been found in home proteins such as Hox-A5 and plays an important role in inducing intracellular entry. Recently, a protein transporter having a property of passing through a cell membrane through synthesis has been developed.

Intracellular delivery by the protein transporter is a completely different mechanism than conventional protein delivery, such as endocytosis. In the case of a positively charged protein transporter containing a basic amino acid arginine, it is transferred into the cell by macropinocytosis. In the case of the hydrophobic protein transporter, the inverted micelle is formed by binding with the phospholipid, It is known.

Recently, an attempt has been made to use a fusion protein composed of a protein carrier and a light chain of a botulinum toxin as a cosmetic product. However, when such a fusion protein is applied to skin, not only a problem of having excellent skin permeability and cell permeability but also a sufficient effect of a botulinum toxin in a cell is not sufficiently verified.

In order to improve the activity of the botulinum toxin in the cell even when applied directly to the skin, the inventors of the present invention have found that the translocation domain of the N-terminal, which plays an important role in the translocation of the target protein in the cytoplasm of the heavy chain of the botulinum toxin ) Was fused to the light chain of the botulinum toxin to complete the present invention.

In order to improve the skin permeability and cell permeability of the fusion protein, the present inventors identified and fused the protein carrier most suitable for the fusion protein.

That is, the present invention relates to a fusion protein comprising a protein transporter, a light chain of a botulinum toxin, and a translocation domain at the N-terminal of the heavy chain of the botulinum toxin.

The present invention also provides a cosmetic composition comprising the botulinum toxin fusion protein as an active ingredient.

Specifically, the present invention provides a transdermal absorption enhancer which comprises a botulinum toxin fusion protein capable of transdermal delivery, exhibiting excellent activity of a botulinum toxin, exhibiting no skin irritation, as an active ingredient, and enhancing cell penetration of the fusion protein Which further comprises a cosmetic composition.

The present invention also provides a cosmetic composition having an effect of improving or preventing skin wrinkles comprising a botulinum toxin fusion protein and a transdermal absorption promoter.

The present invention utilizes a protein transporter consisting of an amino acid sequence of any one of SEQ ID NOS: 1 to 10 as a carrier of a light chain peptide of a botulinum toxin, which comprises a cation transporter and a hydrophobic protein transporter.

The present invention relates to a fusion protein comprising a protein carrier consisting of an amino acid sequence of any one of SEQ ID NOS: 1 to 10 and a light chain peptide of a botulinum toxin.

The present invention provides a fusion protein comprising a protein carrier comprising an amino acid sequence of any one of SEQ ID NOS: 1 to 10, a light chain peptide of a botulinum toxin, and a translocation domain peptide of a botulinum toxin heavy chain.

Specifically, the botulinum toxin light chain peptide may be composed of the amino acid sequence of SEQ ID NO: 21.

Specifically, the translocation domain peptide of the botulinum toxin heavy chain may be composed of the amino acid sequence of SEQ ID NO: 22.

Specifically, a fusion protein consisting of the protein transporter, the light chain peptide of the botulinum toxin, and the translocation domain peptide of the botulinum toxin heavy chain, wherein the protein carrier and the botulinum toxin light chain peptide and / or the botulinum toxin light chain peptide and the botulinum toxin heavy chain Lt; RTI ID = 0.0 > a < / RTI >

Specifically, the linker may be composed of (G) _N , wherein N is an integer of 1 to 20, and G is an amino acid sequence of glycine. Preferably, the linker may be GGGGG.

The present invention also provides a polynucleotide encoding a fusion protein comprising a protein transporter consisting of an amino acid sequence of any one of SEQ ID NOS: 1 to 10, a light chain peptide of a botulinum toxin, and a translocation domain peptide of a heavy chain of a botulinum toxin.

The present invention also provides a recombinant expression vector comprising the polynucleotide.

The present invention also provides a bacterium transformed with said recombinant expression vector.

The polynucleotide of the protein transporter may be any one of the nucleotide sequences of SEQ ID NOS: 11 to 20.

The present invention also provides a composition comprising a protein carrier comprising an amino acid sequence of any one of SEQ ID NOS: 1 to 10, a light chain peptide of a botulinum toxin, and a fusion protein comprising a heavy chain translocation domain peptide of a botulinum toxin.

Specifically, the present invention relates to a cosmetic composition comprising a protein carrier consisting of an amino acid sequence of any one of SEQ ID NOS: 1 to 10, a light chain peptide of a botulinum toxin, and a fusion protein comprising a translocation domain peptide of a heavy chain of a botulinum toxin and a transdermal absorption enhancer .

The " transdermal absorption enhancer " is an ingredient that affects skin permeation among emulsifiers and is usually used in transdermal patches. In the present invention, the fusion protein has a role of enhancing the penetration of the skin and penetration of cells. Examples of the protein include lecithin, lauryl pyrrolidone, glycerol monooleate, glycerol monolaurate ), Propylene glycol monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monolaurate (polyoxyethylene sorbitan monolaurate), polyoxyethylene sorbitan monolaurate But are not limited to, polyoxyethylene sorbitan monolaurate, sorbitan monooleate, sorbitan monostearate, sorbitan monolaurate, and the like.

The transdermal absorption enhancer of the present invention may preferably be lecithin, lauryl pyrrolidone, polyoxyethylene sorbitan monooleate or sorbitan monostearate, more preferably lecithin.

The present invention also relates to a cosmetic composition comprising the fusion protein, transdermal absorption promoter and medium chain glyceride of the present invention.

The fusion proteins, transdermal absorption enhancers and heavy chain glycerides may form micelles in cosmetic compositions. Micelle is a microparticle formed as aggregate when surfactant is above a certain concentration. In the present invention, it is difficult to form micelles only with an active ingredient and a surfactant, and it is preferable to add an oil component such as an heavy chain glyceride to form micelles.

Usually, the transdermal absorption enhancer imparts fluidity to the horny phospholipid layer to help to transmit relatively small molecular weight (less than 500 Da) compounds. However, since the molecular weight of the fusion protein of the present invention is as high as 100 kDa or more, Skin permeation of proteins and penetration of cells are difficult to form micelles, so that they can be intracellularly transferred into micelles, thereby increasing skin permeation and cell penetration.

The heavy chain glyceride of the present invention can be, but is not limited to, caprylic / capric triglyceride.

In the present invention, lecithin acting as a transdermal absorption enhancer rapidly increases the skin permeability at a concentration of 0.5% by weight or more based on the total weight of the composition, which corresponds to a critical micelle concentration (CMC) forming micelles.

In addition, the composition of the present invention may further comprise sugar alcohol to stabilize the fusion protein, and the sugar alcohol may be mannitol, erythritol, xylitol, sorbitol, etc., and preferably mannitol.

The composition of the present invention relates to a cosmetic composition for improving or preventing skin wrinkles.

The term " skin permeability " in the present invention means the ability or property to permeate through the skin to penetrate into the skin, and the fusion protein of the present invention and the composition containing the same have significantly superior skin permeability compared to the conventional botulinum toxin. .

In the present invention, "fusion protein" is a protein artificially synthesized so that the skin permeable peptide (protein transporter) binds to another protein or peptide. The structure of the fusion protein according to the present invention is shown in Fig.

The fusion protein of the present invention or the polypeptide constituting the fusion protein may be produced by a chemical peptide synthesis method known in the art, or may be prepared by amplifying the gene encoding the fusion protein by PCR (polymerase chain reaction) Followed by expression and cloning into an expression vector.

As used herein, the term "expression vector" refers to a recombinant vector capable of expressing a target peptide in a desired host cell, and a gene construct containing an essential regulatory element operatively linked to the expression of the 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 part of the nucleotide sequence encoding the polypeptide, and when the gene product is administered, And must be in coding sequence and in frame. The promoter of the vector may be constitutive or inducible.

In the present invention, the expression vector may include a nucleotide sequence encoding a skin permeable peptide (protein transporter) of the present invention and a fusion protein comprising the light chain of the botulinum toxin and the N-terminus of the heavy chain. Is not particularly limited as long as it is capable of producing a fusion protein comprising the light-permeable peptide of the present invention and the N-terminal of the light chain and the heavy chain of the botulinum toxin.

The "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 derivative that helps expression of a target protein. Ubiquitin is cleaved and removed by Ubiquitin Specific Protease (USP) during the purification process.

The " linker " of the present invention is a peptide used for physicochemical distances or binding between domains and domains in a fusion protein. The fusion protein of the present invention may comprise 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 proteins of the present invention and compositions comprising them have the advantage of improving skin permeability and cell permeability and improving the activity of botulinum toxin.

Figure 1 shows the domain structure of the fusion protein according to the present invention.
Fig. 2 shows the results of treatment of a pATP vector into which a DNA encoding a fusion protein is inserted, with a restriction enzyme.
Fig. 3 shows the result of expressing a fusion protein in an E. coli strain.
Fig. 4 shows the result of purifying the peptide of the botulinum toxin fusion protein.
FIG. 5 shows experimental results of transfer of botulinum toxin to the fusion protein.
Figure 6 is a schematic diagram of a Franz Diffusion Cell used for skin permeability analysis.
FIG. 7 shows the result of skin permeation assay according to the fusion protein.
Figure 8 shows the average of the composite muscle activity potentials recorded by stimulation of the sciatic nerve at 50 Hz at 2 Hz in the complex muscle activity potential test.
FIG. 9 shows the result of measuring the complex muscle activity potential with time after injection of the fusion protein.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

[Examples 1 to 3]

Expression and purification of fusion proteins

 (1) the light chain (LC) of the botulinum toxin; (2) an amino acid (S + LC fusion protein) consisting of a protein carrier represented by SEQ ID NO: 2, a linker (GGGGG) and a light chain (LC) of a botulinum toxin; And (3) an amino acid (S + LC + HC fusion protein) in the order of the protein transducer, the linker (GGGGG), the LC, the linker (GGGGG) and the translocation domain of the heavy chain of the botulism toxin, (Fig. 1). ≪ tb > < TABLE >

The synthesized polynucleotide was inserted into the expression vector pAPT to prepare expression vectors for the three fusion proteins. H6Ub protein was used as a fusion partner to induce expression.

BL21 (DE3) starpLysS E. coli strain (Invitrogen, NY, USA) transformed with the plasmid encoding the fusion protein was cultured in LB (Luria-Bertani) medium containing kanamycin (50 mg / nm until absorbance at 0.6 was obtained. The expression results of the fusion protein are shown in Fig.

Protein expression was induced with 0.1 M IPTG (isopropyl- beta -D-thiogalactopyranoside) and cells were further cultured for 16 hours. Collect cells by centrifugation, resuspended in buffer and Lai sheath _{(50 mM NaH 2 PO 4,} 100 mM NaCl, 5mM imidazole, protease inhibitor cocktail tablet, pH 8.0), was crushed by ultrasonic waves. Then, the mixture was centrifuged at 13,000 rpm at 4 ° C for 15 minutes, and the supernatant was subjected to SDS-PAGE to confirm the expression level and the degree of water solubility of the fusion protein. The results of restriction enzyme treatment are shown in Fig.

Purification of the fusion protein was carried out by purifying the supernatant using IMAC 6FF (GE Healthcare Life Sciences) and Q FF (GE Healthcare Life Sciences), and then using a Sephadex G-25 (GE Healthcare Life Science) Respectively.

Purification of the fusion protein was carried out by purifying the supernatant using HisPrep FF (GE Healthcare Life Sciences) and HighTrep Desalting (GE Healthcare Life Science). The purified fusion protein is shown in Fig.

(1) the light chain (LC) of the botulinum toxin; (2) an amino acid (S + LC fusion protein) consisting of a protein carrier represented by SEQ ID NO: 2, a linker (GGGGG) and a light chain (LC) of a botulinum toxin; And (3) an amino acid (S + LC + HC fusion protein) in the order of the protein transducer, the linker (GGGGG), the LC, the linker (GGGGG) and the translocation domain of the heavy chain of the botulism toxin, . The polypeptides synthesized from the polynucleotides of Examples 1 to 3 were as follows.

[Production Examples 1 to 12]

Composition comprising a botulinum-derived fusion protein

A composition comprising the fusion protein of Example 3 and a transdermal absorption enhancer was prepared by the following method.

A mixture of glycerin, mannitol, arginine, phenoxyethanol and purified water was dissolved at 70 ° C, and the mixture was mixed and homogenized in a homomixer at 3000 rpm for 5 minutes Lt; / RTI > Then, the fusion protein and the carboxy polymer solution were added and mixed for 5 minutes to prepare Preparative Example 1

Further, a solution prepared by mixing a mixture of cetearyl alcohol, caprylic / capric triglyceride and lecithin, glycerin, mannitol, arginine, phenoxyethanol and purified water was dissolved at 70 ° C, And emulsified at 3000 rpm for 5 minutes. Then, the fusion protein and the carboxy polymer solution were added and mixed for 5 minutes to prepare Preparative Example 2.

The components and contents of Production Examples 1 and 2 are shown in Table 1 below.

Manufacturing example 1 (% by weight) 2 (% by weight) The fusion protein (Example 3) 0.1 0.1 lecithin - 0.5 Cetearyl Alcohol 1.0 1.0 Caprylic / capric triglyceride 5.0 5.0 glycerin 3.0 3.0 Mannitol 1.0 1.0 Carboxy polymer (carbomer) 0.2 0.2 Arginine 0.2 0.2 Phenoxyethanol Suitable amount Suitable amount Purified water Balance Balance Sum 100 100

In place of lecithin, which is a transdermal absorption enhancer of Production Example 2, lauryl pyrrolidone, glycerol monooleate, glycerol monolaurate, propylene glycol monolaurate ( Propylene glycol monolaurate, polyoxyethylene sorbitan monooleate, polyoxyethylene sorbitan monostearate, polyoxyethylene sorbitan monolaurate, sorbitan monooleate, Preparation Examples 3-12 were prepared by adding Sorbitan Monooleate, Sorbitan Monostearate, and Sorbitan Monolaurate.

[Test Example 1]

Intracellular delivery effect of fusion protein

To analyze the effect of the intracellular botulinum toxin, FITC (Sigma, Fluorescein-5-isothiocyanate, 27072-45-3), which is a fluorescent substance, was labeled on the polypeptides of Examples 1 to 3 and labeled with FACs (FACSCalibur, Becton Dickinson). PC 12 cells (Pheochromocytoma, ATCC CRL-1721) were reacted at the same reaction time for 1 hour.

The results of the experiment are shown in Fig. As shown in FIG. 5, Examples 2 and 3 showed high intracellular delivery effect, and Example 3 showed better intracellular delivery effect than Example 2.

[Test Example 2]

Skin permeation effect using Franz Diffusion cell

Flux was analyzed using the Franz Diffusing Cell, which is the most widely used for the analysis of skin permeability (Fig. 6).

Guinea pig (Hartley Guinea Pig) was used for the test. The temperature was maintained at 32 ° C using a constant-temperature water bath, and the medium of the receptor was PBS at 600 rpm. The skin permeation rate (Flux) was calculated by sampling at intervals of 2 hours for 10 hours.

Cy-5 was conjugated to the polypeptides of Examples 1 and 3 using the Cy5 Fast conjugation kit (ab188288), and analyzed using a Waters 2475 Fluorescence Detector (excitation 630 nm, emission 660 nm).

The results of the skin permeation analysis in the Franz Diffusion Cell according to the protein carrier are shown in FIG.

As shown in Fig. 7, the skin permeation of Examples 1 and 3 was analyzed. As a result, it was found that the skin permeability of the fusion protein of Example 3 was significantly higher than that of the botulinum toxin of Example 1 Excellent.

[Test Example 3]

Catecholamine secretion inhibition assay

Catecholamines are an important indicator of sympathetic nervous system functioning as a sympathetic nerve stimulator. Because botulinum toxin paralyzes muscles by inhibiting the secretion of neurotransmitters from neurons, we examined the inhibitory effects of dopamine and norepinephrine on the secretion of catecholamines.

PC 12 cells (Pheochromocytoma, ATCC CRL-1721) were cultured in 15 ml of F-12K medium (Gibco) containing 2 mM L-glutamine, 1.5 g / L sodium bicarbonate and 10% FBS. And cultured for 2 days in a cell culture flask T75 (Nunc) to a cell density of 625,000 cells / cm2.

Then, the cultured cells were centrifuged to remove the supernatant, and the same volume of F-12K medium and the fusion proteins of Examples 1 to 3 were added, respectively, and cultured for one day. The amount of dopamine and norepinephrine secreted by stimulation with Ca ²⁺ at a concentration of 10 uM was analyzed by liquid chromatography-mass spectrometry (LC / Mass).

The fusion proteins of Examples 1 to 3 were treated at a concentration of 10 ^-5 M and 10 ^-6 M, and the results are shown in Table 2 below.

As shown in Table 2, the light chain of the botulinum toxin of Example 1 had no difference in secretion amount of neurotransmitter compared to the positive control, and it was confirmed that there was no inhibitory effect on catecholamine secretion.

The fusion proteins of Examples 2 and 3 showed the effect of suppressing the secretion of neurotransmitters, and Example 3 showed more excellent inhibitory effect than that of Example 2.

[Test Example 4]

Complex muscle activity potential test

In order to confirm the performance of the botulinum toxin, electrical stimulation of the nerve should be performed to record the compound muscle action potentials (CMAP) of the muscle in question, because the botulinum toxin paralyzes the muscles by inhibiting the secretion of neurotransmitters Evaluation is a general performance evaluation test.

The complex muscle activity potentials of the fusion proteins of Examples 1 to 3 were analyzed as follows.

After rats were anesthetized, they were placed in a prone position (upright posture), and the hip, knee, and ankle joints were bent and fixed to 90 °. BOTOX cosmetics (onabotulinum toxin A, Allergan) 1 to 3 fusion proteins were injected.

The injection method of BOTOX Cosmetics was performed by dissolving 100 units of the manufacturer in 2.5 mL of 0.9% physiological saline and injecting 5 uL once. In addition, the fusion proteins of Examples 1 to 3 were each injected at a concentration of 100 ng / 5 uL and compared.

When 0.2 unit of BOTOX Cosmetics was injected, muscle was paralyzed completely by 3 weeks, and this was compared.

Electrical stimulation of the sciatic nerve was used to measure the amplitude of the complex muscle activity potential in the tibialis anterior. Fig. 8 shows the mean of the complex muscle activity potentials recorded by stimulating the sciatic nerve 50 times at 2 Hz.

Stimulation of 150% of the stimulus size causing the maximum amplitude was repeated 50 times at 2 Hz and 20 Hz to induce repeated complex muscle activity potentials. After 1, 2, 3, and 4 weeks of injection, The results of measuring the amplitude of the muscle activity potential are shown in Fig.

As shown in Fig. 9, in the case of the light chain (LC) of the botulinum toxin of Example 1, no effect was given to the cells and no effect of muscle paralysis was observed. On the other hand, the fusion proteins of Examples 2 and 3 gradually increased the muscle paralysis effect. The fusion protein of Example 3 showed more excellent muscle paralysis than the fusion protein of Example 2, And showed almost the same muscle paralysis effect.

[Test Example 5]

Skin irritation experiment

A skin irritation test was conducted on the fusion protein of Example 3.

Three male New Zealand white rabbits were used as experimental animals. 24 hours before application of the test substance, an electric epilator was used to make a healthy skin that was epilated so as not to injure the skin of the rabbit. The epilated skin was divided into left and right, and the left was applied to the compartment and the right was made to the contrast compartment.

As a method of application, 0.5 ml (concentration of 1 mg / ml) of the test substance was applied onto the skin of application area of 2.5 cm × 2.5 cm, and the gauze was covered so that the test substance and the skin were in good contact with each other. The control compartment covered only gauze. On the gauze, a non-permeable, non-irritating tape was wrapped around to prevent evaporation of the test material.

The application of the test substance was applied once after 24 hours, and was gently rinsed with physiological saline so that the test substance did not remain. Changes in erythema, edema, hemorrhage, scar formation and the like at the applied local sites were visually observed at 24 hours, 48 hours, and 72 hours after application of the test substance. Table 3 and Table 4 show skin reaction evaluation criteria.

Formation reaction of erythema and scar Degree No erythema 0 Very light erythema (barely visible to the naked eye) One A clear erythema 2 Some severe erythema 3 Severe erythema (blushing redness) and mild degree of scar formation (deep injury) 4

Edema formation reaction Degree No edema at all 0 Very light edema (only visible to the naked eye) One Mild edema (if clearly distinct and swollen marginally distinct) 2 Severe swelling (about 1mm swelling) 3 Severe edema (swelling greater than 1 mm and extending beyond the exposed area) 4

Using the scoring of the skin reaction, the mean value was calculated by adding the erythema rating and edema score at 24 hours and 72 hours, and the skin irritation was evaluated by this value. The primary skin irritation index was used for comparison of skin irritation. The primary irritation index (PII) is the average sum of skin response scores for each individual divided by four. Table 5 shows the degree of irritation according to the primary skin irritation index.

Primary skin irritation index division 0.0 - 0.5 Non-magnetic 0.6 - 2.0 Weak irritation 2.1 - 5.0 Moderate irritancy 5.1 - 8.0 Strong irritant

According to the application of the fusion protein of Example 3, mild erythema was observed in all three rabbits at 24 hours and mild erythema was observed in only one rabbit at 48 hours and 72 hours. Thus, it was confirmed that the fusion protein of Example 3 was a non-magnetic substance having a primary skin irritation index (PII) value of 0.25.

[Test Example 6]

Wrinkle improvement effect

To evaluate the degree of skin wrinkle improvement of the cosmetic composition containing the fusion protein of Example 3, the clinical efficacy of 15 healthy women over 30 years old was evaluated.

The effect of wrinkle improvement according to product use was analyzed using a skin image analyzer (Visiometer). For this purpose, a specimen of eye area was prepared at 0, 4, and 8 weeks. The simulated plate was made of silicon material under constant temperature and humidity (22 ± 2 ° C, 40% ~ 60% humidity) conditions.

The simulated plate 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 emitted from the light emitted from the artificial light source through the silicone material was analyzed by the Lambert & Beer's law, and the degree of improvement of the skin wrinkles was measured.

The measurement variable by the skin image analyzer is represented by R2, which represents the maximum roughness. The smaller the value of the variable is, the more the wrinkles of the skin are improved and the depth of the wrinkles is lowered. 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.

 Wrinkle improvement effect Improvement rate (%) p-value After 4 weeks 5.67 0.11248 After 8 weeks 9.28 0.00097

The simulated plate was analyzed using software of a skin image analyzer (Skin-Visiometer SV 600, Courage & Khazaha, Gemany).

From the results of the above experiment, it was confirmed that the cosmetic containing the fusion protein of Example 3 according to the present invention exhibited excellent wrinkle-reducing effect.

[Test Example 7]

Skin permeation test of a composition comprising a botulinum-derived fusion protein

Skin permeation tests were conducted in the same manner as in Test Example 2 to measure the degree of skin permeation of the compositions of Production Examples 1 to 12, wherein the cumulative skin permeation amount for 10 hours was measured, Respectively.

As shown in Table 7, it was confirmed that Production Example 2-12 to which a transdermal absorption enhancer was added showed a skin permeability about 2-3 times better than that of Production Example 1 to which no transdermal absorption promoter was added.

Production Example 2 including lecithin exhibited the best skin permeability, and Production Example 3 containing lauryl pyrrolidone, Production Example 8 containing polyoxyethylene sorbitan monostearate, and sorbitan monostearate the Preparation 11 containing rate was found to exhibit superior skin permeability than 15㎍ / cm ^2.

[Test Example 8]

Skin permeation test of a composition comprising botulinum-derived fusion protein and lecithin

Preparation Examples 13 to 15 were prepared in the same manner as in Preparation Example 2, in order to carry out a test for confirming the skin permeability according to the content of lecithin in a composition containing botulinum toxin and lecithin.

The skin permeability test was carried out in the same manner as in Test Example 7, and the results are shown in Table 8 below.

As shown in Table 8, when the content of the lecithin is 0.1 and 0.3 wt% of the skin permeation amount of 8.2 and were similar to 9.7㎍ / cm ^2, when the content of the lecithin is 0.5% by weight drastically by 20.3 ㎍ / cm ² Respectively.

It is known that transdermal absorption enhancers impart fluidity to the keratinous phospholipid layer to help transmit relatively small molecular weight (less than 500 Da) compounds. In the case of the fusion protein of the present invention, since the molecular weight is as large as 100 kDa or more, permeation of the fusion protein to the skin is difficult only by the fluidization of the keratin.

As a result of the experiment, Caprylic / Capric Triglyceride, which is one kind of transdermal absorption promoter and Medium Chain Glyceride, forms a micelle together with the fusion protein , Endocytosis in the form of micelles, and increased skin permeation.

From the results of experiments in which the skin permeability increases sharply at a content of lecithin as a transdermal absorption promoter of 0.5 wt% or more, it can be seen that the above content is a critical micelle concentration (CMC) forming micelles.

<110> Iphama CO., LTD.          ICURE Pharmaceutical Inc. <120> Cosmetic composition comprising peptide derived from Botulinum          toxin having improved cell penetrating ability <130> P170021 <160> 22 <170> KoPatentin 3.0 <210> 1 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> penetratin <400> 1 Arg Gln Ile Lys Ile Trp Phe Gln Asn Arg Arg Met Lys Trp Lys Lys   1 5 10 15 <210> 2 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> TAT <400> 2 Arg Lys Lys Lys Arg Arg Gln Arg Arg Arg   1 5 10 <210> 3 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> VP22 <400> 3 Asn Ala Lys Thr Arg Arg His Glu Arg Arg Arg Lys Leu Ala Ile Glu   1 5 10 15 Arg     <210> 4 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> Pep-1 <400> 4 Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys   1 5 10 15 Lys Lys Arg Lys Val              20 <210> 5 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> kFGF <400> 5 Ala Ala Val Ala Leu Ala Leu Pro Ala Val Leu Leu Ala Leu   1 5 10 15 <210> 6 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> Polyarginine (R9) <400> 6 Arg Arg Arg Arg Arg Arg Arg Arg Arg   1 5 <210> 7 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> MPG <400> 7 Gly Ala Leu Phe Leu Gly Phe Leu Gly Ala Ala Gly Ser Thr Met Gly   1 5 10 15 Ala     <210> 8 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> YTA2 <400> 8 Tyr Thr Ala Ile Ala Trp Val Lys Ala Phe Ile Arg Lys Leu Arg Lys   1 5 10 15 <210> 9 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> YTA4 <400> 9 Ile Ala Trp Val Lys Ala Phe Ile Arg Lys Leu Arg Lys Gly Pro Leu   1 5 10 15 Gly     <210> 10 <211> 18 <212> PRT <213> Artificial Sequence <220> <223> pVEC <400> 10 Leu Leu Ile Ile Leu Arg Arg Arg Ile Arg Lys Gln Ala His Ala His   1 5 10 15 Ser Lys         <210> 11 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide of protein transduction domain <400> 11 aggcagatca agatctggtt ccagaacagg aggatgaagt ggaagaag 48 <210> 12 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide of protein transduction domain <400> 12 aggaagaaga agaggaggca gaggaggagg 30 <210> 13 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide of protein transduction domain <400> 13 aacgccaaga ccaggaggca cgagaggagg aggaagctgg ccatcgagag g 51 <210> 14 <211> 63 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide of protein transduction domain <400> 14 aaggagacct ggtgggagac ctggtggacc gagtggagcc agcccaagaa gaagaggaag 60 gtg 63 <210> 15 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide of protein transduction domain <400> 15 gccgccgtgg ccctgctgcc cgccgtgctg ctggccctgc tggccccc 48 <210> 16 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide of protein transduction domain <400> 16 aggaggagga ggaggaggag gaggagg 27 <210> 17 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide of protein transduction domain <400> 17 ggcgccctgt tcctgggctt cctgggcgcc gccggcagca ccatgggcgc c 51 <210> 18 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide of protein transduction domain <400> 18 tacaccgcca tcgcctgggt gaaggccttc atcaggaagc tgaggaag 48 <210> 19 <211> 51 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide of protein transduction domain <400> 19 atcgcctggg tgaaggcctt catcaggaag ctgaggaagg gccccctggg c 51 <210> 20 <211> 54 <212> DNA <213> Artificial Sequence <220> <223> polynucleotide of protein transduction domain <400> 20 ctgctgatca tcctgaggag gaggatcagg aagcaggccc acgcccacag caag 54 <210> 21 <211> 448 <212> PRT <213> Artificial Sequence <220> <223> Botulinum toxin light chain peptide <400> 21 Met Pro Phe Val Asn Lys Gln Phe Asn Tyr Lys Asp Pro Val Asn Gly   1 5 10 15 Val Asp Ile Ala Tyr Ile Lys Ile Pro Asn Val Gly Gln Met Gln Pro              20 25 30 Val Lys Ala Phe Lys Ile His Asn Lys Ile Trp Val Ile Pro Glu Arg          35 40 45 Asp Thr Phe Thr Asn Pro Glu Glu Gly Asp Leu Asn Pro Pro Pro Glu      50 55 60 Ala Lys Gln Val Val Ser Ser Tyr Tyr Asp Ser Thr Tyr Leu Ser Thr  65 70 75 80 Asp Asn Glu Lys Asp Asn Tyr Leu Lys Gly Val Thr Lys Leu Phe Glu                  85 90 95 Arg Ile Tyr Ser Thr Asp Leu Gly Arg Met Leu Leu Thr Ser Ile Val             100 105 110 Arg Gly Ile Pro Phe Trp Gly Gly Ser Thr Ile Asp Thr Glu Leu Lys         115 120 125 Val Ile Asp Thr Asn Cys Ile Asn Val Ile Gln Pro Asp Gly Ser Tyr     130 135 140 Arg Ser Glu Glu Leu Asn Leu Val Ile Ile Gly Pro Ser Ala Asp Ile 145 150 155 160 Ile Gln Phe Glu Cys Lys Ser Phe Gly His Glu Val Leu Asn Leu Thr                 165 170 175 Arg Asn Gly Tyr Gly Ser Thr Gln Tyr Ile Arg Phe Ser Pro Asp Phe             180 185 190 Thr Phe Gly Phe Glu Glu Ser Leu Glu Val Asp Thr Asn Pro Leu Leu         195 200 205 Gly Ala Gly Lys Phe Ala Thr Asp Pro Ala Val Thr Leu Ala His Glu     210 215 220 Leu Ile His Ala Gly His Arg Leu Tyr Gly Ile Ala Ile Asn Pro Asn 225 230 235 240 Arg Val Phe Lys Val Asn Thr Asn Ala Tyr Tyr Glu Met Ser Gly Leu                 245 250 255 Glu Val Ser Phe Glu Glu Leu Arg Thr Phe Gly Gly His Asp Ala Lys             260 265 270 Phe Ile Asp Ser Leu Gln Glu Asn Glu Phe Arg Leu Tyr Tyr Tyr Asn         275 280 285 Lys Phe Lys Asp Ile Ala Ser Thr Leu Asn Lys Ala Lys Ser Ile Val     290 295 300 Gly Thr Thr Ala Ser Leu Gln Tyr Met Lys Asn Val Phe Lys Glu Lys 305 310 315 320 Tyr Leu Leu Ser Glu Asp Thr Ser Gly Lys Phe Ser Val Asp Lys Leu                 325 330 335 Lys Phe Asp Lys Leu Tyr Lys Met Leu Thr Glu Ile Tyr Thr Glu Asp             340 345 350 Asn Phe Val Lys Phe Phe Lys Val Leu Asn Arg Lys Thr Tyr Leu Asn         355 360 365 Phe Asp Lys Ala Val Phe Lys Ile Asn Ile Val Pro Lys Val Asn Tyr     370 375 380 Thr Ile Tyr Asp Gly Phe Asn Leu Arg Asn Thr Asn Leu Ala Ala Asn 385 390 395 400 Phe Asn Gly Gln Asn Thr Glu Ile Asn Asn Met Asn Phe Thr Lys Leu                 405 410 415 Lys Asn Phe Thr Gly Leu Phe Glu Phe Tyr Lys Leu Leu Cys Val Arg             420 425 430 Gly Ile Ile Thr Ser Lys Thr Lys Ser Leu Asp Lys Gly Tyr Asn Lys         435 440 445 <210> 22 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> The translocation domain of the Botulinum toxin heavy          chain <400> 22 Ser Met Ile Pro Tyr Gly Val Lys Arg Leu Glu Asp Phe Asp Ala Ser   1 5 10 15 Leu Lys Asp            

Claims (14)

A botulinum toxin fusion protein and a transdermal absorption promoter,
The botulinum toxin fusion protein comprises a protein carrier comprising the amino acid sequence of SEQ ID NO: 2, a botulinum toxin light chain peptide comprising the amino acid sequence of SEQ ID NO: 21, and a translocation domain peptide of the botulinum toxin heavy chain comprising the amino acid sequence of SEQ ID NO: Lt; / RTI &
The fusion protein is fused in the order of a protein transporter, a botulinum toxin light chain peptide, and a translocation domain peptide of a botulinum toxin heavy chain,
The transdermal absorption enhancer may be at least one selected from the group consisting of lecithin, lauryl pyrrolidone, polyoxyethylene sorbitan monooleate and sorbitan monostearate. Wherein the cosmetic composition is a cosmetic composition.
delete delete The cosmetic composition according to claim 1, wherein the cosmetic composition comprises a linker between at least one of the protein carrier and the botulinum toxin light chain peptide, and between the botulinum toxin light chain peptide and the translocation domain peptide of the botulinum toxin heavy chain.
5. The cosmetic composition according to claim 4, wherein the linker is represented by (G) _N , wherein N is an integer of 1 to 20, and G is glycine.
The cosmetic composition according to claim 5, wherein the linker is GGGGG.
The cosmetic composition according to claim 1, wherein the transdermal absorption enhancer is lecithin.
The cosmetic composition according to claim 7, wherein the lecithin is contained in an amount of 0.5% by weight or more based on the total weight of the composition.
The cosmetic composition according to claim 7, further comprising a medium chain glyceride.
The cosmetic composition according to claim 9, wherein the heavy chain glyceride is caprylic / capric triglyceride
11. The cosmetic composition according to claim 10, wherein the fusion protein, lecithin and caprylic / capric triglyceride form micelles.
The cosmetic composition according to claim 1, further comprising a sugar alcohol for stabilizing the fusion protein
13. The cosmetic composition according to claim 12, wherein the sugar alcohol is mannitol
The cosmetic composition according to claim 1, wherein the composition is used for improving or preventing skin wrinkles.

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