KR101478520B1 - Capsaicin-Peptide Having Improved Safety for Skin and Cosmetic External Preparation Composition for Skin Using the Same - Google Patents

Abstract

Disclosed in the present invention are a capsaicin-peptide having the improved safety for the skin, and a composition using the same for external application to the skin. Specifically, disclosed in the present invention are a capsaicin-peptide having the improved safety for the skin obtained by applying Arg-Gly-Asp(RGD), Glu-Cys-Gly(ECG) or Gly-His-Lys(GHK) in capsaicin, and a composition using the same for external application to the skin.

Description

[0001] The present invention relates to a capsaicin-peptide having improved skin safety and a composition for external application using the same. [0002] Capsaicin-

The present invention relates to a capsaicin-peptide having improved skin safety and a composition for external application to skin using the same.

Capsaicin is a type of alkaloid, and its related compounds are known as capsaicinoids and are known as spicy flavors. It is the most abundant in seeds of red pepper plants and has long been used as a spice, food additive or medicine.

As the effects of capsaicin on the nervous system, especially the pain transmission system, began to be known in the 1950's, research on neurophysiological effects was actively conducted and activation of the vanilloid receptor (VR1) was shown to act on sensory nerves (Caterina et al. , Nature 1997, 389 (6653): 816-24, Szallasi AND Blumberg, Pharmacol Rev 1999, 51, 159-211). VR1 is activated in noxious heat and acidic environment (proton, H +). It is presumed that the feeling of pepper in man is due to the nature of VR1. Activation of VR1 is caused by the nerves involved in pain and heat sensation 2001, Sep; 22 (9): 1439-46. Nathan et al., Am J Physiol Gastrointest Liver Physiol 2001 Nov; 281 (5): G1322 -8.). Persistent stimulation by capsaicin inhibits the transmission of pain and releases endorphins in the cerebrum, thereby exhibiting an analgesic effect. Accordingly, researches for the development of painkillers using capsaicin and capsaicin derivatives are actively conducted . It is currently applied as a topical application in the treatment of rheumatoid arthritis, osteoarthritis, diabetic neuropathy, postherpetic neuralgia, and cluster headache. In addition, it has been reported that the treatment effect of skin diseases such as psoriasis due to the improvement of pruritus and anti-inflammatory effect (Arnold & Kerkhof, J Am Acad Dermatol 1994 Jun; 31 (1): 135) .

In recent years, the anticancer effect of capsaicin on cell death (Mori et al., Cancer Res. 2006 Mar 15; 66 (6): 3222-9. Chow et al., Biochim Biophys Acta. 2007 Apr; : 565-76.) And anti-obesity effect through metabolism promotion and lipolysis promotion function (Lejeune et al., Br J Nutr 2003 Sep; 90 (3): 651-59 Yoneshiro et al., Am J Clin Nutr 2012 Apr; 95 (4): 845-50).

Capsaicin has the advantage of an effective therapeutic agent, but it is used within a limited range because it has strong skin irritation accompanied by a burning sensation and gives unpleasant irritation to the skin. Prior art techniques for reducing capsaicin skin irritation include U.S. Patent No. 5869533, which utilizes plant extracts and a method of mixing, a water-soluble strontium cation known to block neurotransmitters that act on epithelial nerve cells involved in depolarization and repolarization U.S. Patent No. 5,716,625, and Korean Patent No. 100395247 which promotes skin permeation by mixing a hydrophilic nonionic surfactant.

The present invention discloses novel capsaicin-peptides with improved skin safety.

It is an object of the present invention to provide a novel capsaicin-peptide having improved skin safety.

It is another object of the present invention to provide a composition for external application for skin using the capsaicin-peptide.

In one aspect, the present invention relates to a capsaicin-peptide of Formula 1:

[Chemical Formula 1]

Wherein L is absent or a linking group,

A is Arg-Gly-Asp (RGD), Glu-Cys-Gly (ECG) or Gly-His-Lys (GHK).

The capsaicin-peptide of the present invention can be prepared by synthesizing a peptide and then reacting the peptide with a capsaicin precursor (capsaicin having a carboxyl group introduced to the amino terminal of the peptide through an amide reaction). Specifically, the capsaicin-peptide of the present invention can be obtained by (i) first obtaining NH ₂ -protected peptide-resin by solid phase peptide synthesis known in the art, as shown in the following Reaction Scheme 1 (ii) Reacting the resulting NH ₂ -protected peptide-resin with a capsaicin precursor, and (iii) removing the resin. When a functional group is present on the side chain of the amino acid residue constituting the target peptide, a peptide can be synthesized using the amino acid protected with the functional group in the step (i), and the protecting group bonded to the functional group can be synthesized iii). For the more specific production process, reaction conditions and the like, the following examples can be referred to.

[Reaction Scheme 1]

In another aspect, the present invention relates to a composition for external application to skin comprising a capsaicin-peptide.

The capsaicin-peptide of the present invention showed no specific toxicity to keratinocytes as shown in the following experimental examples, and showed significantly lower skin irritation than capsaicin in human application tests.

Such experimental results may include various pharmacological effects of capsaicin or therapeutic or prophylactic effects of skin function (such as rheumatoid arthritis, osteoarthritis, diabetic neuropathy, postherpetic neuralgia, cluster headache, psoriasis, etc.) ), It is preferable to use the capsaicin-peptide of the present invention in place of capsaicin in the case of developing an external preparation for skin such as topical application medicines or cosmetics based on capsaicin.

The composition for external application for skin of the present invention may contain any amount (effective amount) of the capsaicin-peptide, which is an effective ingredient thereof, depending on the purpose of use, formulation, compounding purpose and the like, which can exhibit an intended pharmacological activity or skin function. Will be determined within the range of 0.001 wt% to 20.000 wt% based on the total weight of the composition. The term "effective amount" as used herein refers to the amount of the active ingredient that can exhibit the intended pharmacological activity or skin function. Such effective amounts can be determined experimentally within the ordinary skill of those skilled in the art.

The composition of the present invention can be identified as a cosmetic composition in a specific embodiment.

When the composition of the present invention is recognized as a cosmetic composition, the cosmetic composition may be prepared in various forms, for example, emulsion, lotion, cream (oil-in-water type, oil water type, multiphase), solution, suspension (Water and water), anhydrous products (oil and glycol), gel, mask, pack, powder and the like.

The composition of the present invention may contain an acceptable carrier in cosmetic preparations other than those containing the active ingredient.

As used herein, the term " acceptable carrier for cosmetic preparation "refers to a compound or composition which is already known and used in cosmetics, or which is a compound or composition to be developed in the future, and which has no toxicity that the human body can adapt to when contacted with skin.

The carrier may be included in the composition of the present invention in an amount of from about 1% by weight to about 99.99% by weight, preferably from about 50% by weight to about 99% by weight of the composition, based on the total weight thereof.

However, since the ratio depends on the above-mentioned formulation of the cosmetic product and its specific application site (face or hands) or the desired amount of application thereof, the ratio is to limit the scope of the present invention in any aspect It should not be.

Examples of the carrier include alcohols, oils, surfactants, fatty acids, silicone oils, humectants, moisturizers, viscosifiers, emulsifiers, stabilizers, sunscreens, coloring agents and perfumes.

The compounds / compositions which can be used as the carrier and which can be used as alcohols, oils, surfactants, fatty acids, silicone oils, wetting agents, moisturizers, viscosifiers, emulsions, stabilizers, sunscreens, A person skilled in the art can select and use appropriate substances / compositions.

The composition of the present invention may be identified as a pharmaceutical composition in another specific embodiment.

The pharmaceutical composition of the present invention may be formulated into a wide variety of dosage forms, including pharmaceutically acceptable carriers, excipients and the like, in addition to the active ingredient, and may be formulated into a wide variety of dosage forms such as a localized preparation such as cream, lotion, ointment (semi-solid external drug), micro loo emulsion, (TTS) (e.g., patches, bandages, etc.), and the like.

The term "pharmaceutically acceptable" as used herein means that the application (prescribing) subject does not have the above-mentioned toxicity that is adaptable without inhibiting the activity of the active ingredient.

Examples of pharmaceutically acceptable carriers include lactose, glucose, sucrose, starch (e.g., corn starch, potato starch and the like), cellulose, derivatives thereof (e.g. sodium carboxymethylcellulose, ethylcellulose, etc.), malt, gelatin, talc, (E.g., peanut oil, cottonseed oil, sesame oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerin, etc.), alginic acid, emulsifiers (e.g., TWEENS), wetting agents (For example, sodium lauryl sulfate), a coloring agent, a flavoring agent, a stabilizer, an antioxidant, a preservative, water, a saline solution, and a phosphate buffer solution. The carrier may be selected from one or more of suitable pharmaceutical formulations according to the formulation of the pharmaceutical composition of the present invention.

The excipient may be selected according to the formulation of the pharmaceutical composition of the present invention. For example, suspending agents such as sodium carboxymethyl cellulose, methyl cellulose, hydropropyl methyl cellulose, sodium alginate, polyvinyl pyrrolidone, .

The pharmaceutical compositions of the present invention may be administered orally or parenterally, and preferably topically.

The daily dose of the pharmaceutical composition of the present invention is usually 0.001 to 150 mg / kg body weight, and may be administered once or several times. However, since the dosage of the pharmaceutical composition of the present invention is determined in view of various related factors such as route of administration, age, sex, weight, and patient's severity of the patient, the dose is limited in any aspect to the scope of the present invention Should not be understood to be.

As described above, according to the present invention, a novel capsaicin-peptide having improved skin safety can be provided.

The capsaicin-peptide of the present invention can be used as a substitute for capsaicin in the development of external preparations for skin such as drugs and cosmetics based on the pharmacological effect of capsaicin or skin function.

1 is a synthesis diagram of a capsaicin precursor.
2 is a synthesis diagram of capsaicin-RGD (Capsavax).
3 is a synthesis diagram of capsaicin-ECG (Capsa Calm).
4 is a synthesis diagram of capsaicin-GHK (Capsanoin).
5 shows MALDI-TOF MS analysis results for confirming the molecular weight of capsaicin-RGD.
6 shows MALDI-TOF MS analysis results for confirming the molecular weight of capsaicin-ECG.
7 shows MALDI-TOF MS analysis results for confirming the molecular weight of capsaicin-GHK.

Hereinafter, the present invention will be described with reference to Examples and Experimental Examples. However, the scope of the present invention is not limited to these examples and experimental examples.

< Example > Capsaicin - Preparation of tripeptide

<Example 1> capsaicin - Preparation of RGD

&Lt; 1-1 > Preparation of capsaicin precursor having carboxyl group

In order to chemically bond capsaicin via an amide reaction to the amino terminal of the peptide, capsaicin without a carboxyl group was reacted with 1,1'-Carbonyldiimidazole (CDI) to prepare a capsaicin precursor having a carboxyl group introduced therein (see FIG. Specifically, CDI was added to the capsaicin solution dissolved in anhydrous THF (Tetrahydrofuran), refluxed for 7 hours, confirmed by TLC, and the reaction was terminated. The reaction product was poured into water and extracted with ether. Then, the organic layer was collected, washed with water, dried with anhydrous magnesium sulfate to remove a small amount of water, filtered, and the filtrate was concentrated under reduced pressure to prepare a gap precursor.

<1-2> Synthesis of NH ₂ -protected peptide-resin

The peptide was synthesized by a conventional solid phase peptide synthesis (SPPS) using 9-fluorenylmethoxycarbonyl (Fmoc) as a protecting group of an amino acid, and N-hydroxybenzotriazole N-hydroxybenzotriazole (HOBt) and N, N'-dicyclohexylcarbodiimide (DCC) were used as activating agents to extend the amino acid residues (Wang C. Chan, Perter D. White, 'Fmoc solid phase peptide synthesis', Oxford].

Specifically, 0.04 g of resin (Nova Biochem, Inc.) was swollen in 3 ml of N-methyl-2-pyrrolidone (NMP) for 20 minutes in a glass reactor, Aspartic acid (D), the first amino acid protected by Fmoc, was added to the resin and reacted at room temperature for 3 hours. The next solvent was removed and Fmoc was removed by treatment with 2 ml of 20% piperidine (3 ml). Next, the reaction was carried out twice with dichloromethane (DCM) and twice with NMP. Glycine (G), which is a second amino acid activated by HOBt-DCC, was added to the reactor and reacted at room temperature for about 3 hours to bind the second amino acid. The resulting NH ₂ -protected peptide (arginine-glycine-aspartic acid) -resin was washed twice with DCM, twice with NMP and twice with DCM, followed by coupling of the third amino acid, arginine (R) And then completely dried.

<1-3> Synthesis of capsaicin-RGD

After 20% piperidine / NMP solution was added to the synthesized NH ₂ -protected peptide (arginine-glycine-aspartic acid) -resin, the Fmoc bound to the amino group was removed, washed with NMP and DCM, With 5 equivalents of capsaicin precusor overnight at room temperature. After the reaction was completed, the reaction was washed several times with NMP and DCM, and then dried.

The dried capsaicin-peptide-resin was reacted with a mixed solution of trifluoroacetic acid: phenol: thioanisole: water: ethanedithiol (82.5: 5: 5: Butyloxycarbonyl (Boc) and trityl (triphenylmethyl) which are protecting groups of functional groups present in the side chain of the amino acid residues constituting the peptide are removed and the capsaicin-peptide is separated from the resin, Followed by precipitation to prepare capsaicin-RGD.

The synthesis of capsaicin-RGD (Capsavax) is shown in Fig.

Example 2 : Synthesis of capsaicin - ECG

&Lt; 2-1 > Preparation of capsaicin precursor having carboxyl group

A capsaicin precursor having a carboxyl group was prepared in the same manner as in <Example 1-1>.

<2-2> Synthesis of NH ₂ -protected peptide-resin

The <Example 1-2> By the same method as NH ₂ - protected peptide (ECG) - resin was synthesized.

<2-3> Synthesis of capsaicin-ECG

The 20% piperidine / NMP solution was added to the NH ₂ -protected peptide (glutamine-cysteine-glycine) -resin synthesized above to remove Fmoc bound to the amino group, washed with NMP and DCM, and capsaicin And 5 equivalents of a precursor (capsaicin precusor) were allowed to react overnight at room temperature. After the reaction was completed, the reaction was washed several times with NMP and DCM, and then dried.

The dried capsaicin-peptide-resin was reacted with a mixed solution of trifluoroacetic acid: phenol: thioanisole: water: ethanedithiol (82.5: 5: 5: Butyloxycarbonyl (Boc) and trityl (triphenylmethyl) which are protecting groups of functional groups present in the side chain of the amino acid residues constituting the peptide are removed and the capsaicin-peptide is separated from the resin, And precipitated to prepare capsaicin-ECG.

The synthesis of capsaicin-ECG (Capsa Calm) is shown in Fig.

&Lt; Example 3 & gt ; Synthesis of capsaicin - GHK

&Lt; 3-1 > Preparation of capsaicin precursor having carboxyl group

A capsaicin precursor having a carboxyl group was prepared in the same manner as in <Example 1-1>.

<3-2> Synthesis of NH ₂ -protected peptide-resin

In the same manner as <Example 1-2> NH ₂ - The synthesis was the resin-protected peptide (GHK).

<3-3> Synthesis of capsaicin-ECG

The 20% piperidine / NMP solution was added to the NH ₂ -protected peptide (glycine-histamine-lysine) -resin synthesized above to remove Fmoc bound to the amino group, washed with NMP and DCM, and then capsaicin And 5 equivalents of a precursor (capsaicin precusor) were allowed to react overnight at room temperature. After the reaction was completed, the reaction was washed several times with NMP and DCM, and then dried.

The dried capsaicin-peptide-resin was reacted with a mixed solution of trifluoroacetic acid: phenol: thioanisole: water: ethanedithiol (82.5: 5: 5: Butyloxycarbonyl (Boc) and trityl (triphenylmethyl) which are protecting groups of functional groups present in the side chain of the amino acid residues constituting the peptide are removed and the capsaicin-peptide is separated from the resin, And precipitated to prepare capsaicin-GHK.

The synthesis of capsaicin-GHK (Capsanoin) is shown in FIG.

Example 2: Preparation of capsaicin - peptide C18 Prep Column purification

For high - purity purification of synthesized capsaicin - peptides, HPLC analysis purification method widely used for peptide purification was used. In order to increase the solubility, gradient conditions of solvent analysis A and solvent B were changed and the basic purification conditions were as follows. When purifying by using these purification conditions, 60-70% of the reaction was observed immediately after the synthesis, but the purity was increased up to 95%.

HPLC instrument type: Waters 650E Advanced Protein Purification System

Column Type: Gemini 5u C18 110 A (Phenomenex sk)

Column Specification: 250 * 4.60 mm, 5 micron

Solvent conditions: Solvent A: Water (containing 0.1% TFA (Trifluoro acetic acid))

          Solvent B: Acetonitrile (containing 0.1% TFA (Trifluoro acetic acid))

Wavelength: 230 nm (UV lamp)

Flow rate: 1 ml / min

Solvent slope conditions (Gradient): [Table 1]

Solvent slope conditions Time (min) Solvent A (%) Solvent B (%) 0 100 0 30 35 65 31 35 65 36 100 0 41 100 0

&Lt; Example 3 > MALDI - TOF Mass Spectrometry analysis

To confirm the molecular weight of the capsaicin-peptide, MALDI-TOF MS analysis was performed under the following analysis conditions, and the results are shown in FIG. 1 to FIG. 3. From these results, it was confirmed that the capsaicin-peptide synthesized exactly matches the predicted molecular weight.

- Device name: Kratos PC AximaCFR V2.3.3

- Mode: Linear (Positive)

- Laser power: 63

- Matrix: alpha-Cyano-4-hydroxy-cinnamic acid (CHCA)

< Experimental Example > Capsaicin - Of peptide  Skin cell safety experiment and human skin Irritability  Reduction effect experiment

< Experimental Example  1> Skin cell safety experiment

The MTT assay was used to examine the cell safety of the HaCaT cell line, which is a keratinocyte cell line of capsaicin-peptides. To this end, human keratinocyte cell line HaCaT cells Count the cells in a 24-well plate using 5 × 10 ³ cells / well and 5 × 10 ⁴ cells / well using a heamacytometer. When cultured in DMEM containing 10% FBS for 48 hours and cultured to 50% of the culture vessel surface area, the sample was treated for each concentration and cultured for another 48 hours. After incubation, 50 μl of a solution of 2.5 mg / ml 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide (MTT, Sigma M5655, USA) was added and cultured for additional 3 hours. 100 μl aliquots were transferred into 96 wells and eluted with Enzyme-Linked Immunosorbent Assay (ELISA) at 570 cells / well. The cell lysates were discarded and 200 μl of dimethyl sulfoxide (DMSO, Sigma D2650, USA) nm absorbance was measured. The extent of promoting toxicity or proliferation to cells was expressed as a percentage based on the absorbance intensity of the control using pure water.

The results are shown in Table 2 below. Referring to [Table 2], it can be seen that both capsaicin-peptides do not exhibit any particular toxicity to keratinocytes.

Skin cell safety test results Treatment concentration
Cell proliferation (%) Example 1 Example 2 Example 2 Negative control group 100% 100% 100% 1 ppm 107 ± 4% 102 ± 5% 104 ± 3% 5 ppm 99 ± 1% 101 ± 2% 102 ± 7% 10 ppm 91 ± 15% 97 ± 8% 99 ± 4% 50 ppm 96 ± 11% 95 ± 11% 96 ± 7% 100 ppm 98 ± 4% 96 ± 7% 97 ± 6% Comparative Example 68 ± 6% * Comparative Example 1 treated capsaicin at 1 ppm

< Experimental Example  2> Human skin Irritability  Reduction effect experiment

A human patch test was performed on capsaicin-GHK (Example 3) among the capsaicin-tripeptides in order to determine the degree of reduction of the stimulation sensation on the human skin of the capsaicin-peptide.

To determine the degree of skin irritation, 0.1 μg / ml (0.1 ppm) of capsaicin dissolved in water and 0.1 μg / ml (0.1 ppm) of capsaicin-GHK (Capsinoin) test. A patch containing 100 samples of each sample was attached to a clean area on the inside of the subject's arm. After 48 hours, the subject was rinsed with flowing water, and the degree of stimulation after 30 minutes was determined. The negative control was pure water and the positive control was 5% SLS (Sodium Laureth Sulfate).

The evaluation of the skin reaction was divided into 5 steps (1 (less than 10, minimum stimulus), 2 (10-20, mild stimulation) 3 (20-30, moderate stimulation), 4 And the average degree of skin irritation (%) was calculated according to the following formula.

The average number of the skin irritation (%) = [(number of 4 stimuli corresponding to the number of subjects) + (number of 3 stimulants corresponding number of subjects) + (number of 2 stimulants corresponding number of subjects) + Number of subjects per subject)] / (highest stimulation value (4) total number of subjects) × 100

The results are shown in Table 3 below.

Skin irritation test result division Number of stimulation Skin irritation
Average (%) Irritability judgment 4 3 2 One 0 Negative control group 0 0 0 2 9 5 One Positive control group 0 One 5 4 One 39 4 Capsaicin 0.1 ug / ml concentration sample 7 3 One 0 0 89 4 Capsinoin 0.1 μg / ml concentration Sample 2 2 6 One 0 62 4

As shown in Table 3, the skin irritancy of capsaicin 0.1 ug / ml was 89% (irritation 4). Seven out of 11 subjects showed the strongest irritation and showed very strong skin irritation. 4.5% in the negative control group (irritative judgment 1) and 38.6% in the positive control group (irritative judgment 4). Although the stimuli were the same, the sensitivity of capsaicin-GHK (Capsinoin) was 62% (stimuli 4). Only 2 of 11 subjects showed the highest stimulation, and the majority of 6 people had relatively decreased stimulation And a mild stimulation of the second stage. As a result of the test for human stimulation, it was confirmed that the skin irritation of capsaicin-GHK (Capsinoin), which was developed by binding a peptide to cap ipsin, was significantly reduced.

Claims (5)

A capsaicin-peptide having the formula:

Wherein L is absent or a linking group,
A is Arg-Gly-Asp (RGD), Glu-Cys-Gly (ECG) or Gly-His-Lys (GHK).
A composition for external application for skin comprising a capsaicin-peptide having the following formula:

Wherein L is absent or a linking group,
A is Arg-Gly-Asp (RGD), Glu-Cys-Gly (ECG) or Gly-His-Lys (GHK). 3. The method of claim 2,
Wherein the composition is a cosmetic composition.
3. The method of claim 2,
Wherein the composition is a pharmaceutical composition.
(a) reacting capsaicin with 1,1'-carbonyldiimidazole (CDI) to prepare a capsaicin precursor having a carboxyl group introduced therein;
(b) reacting the capsaicin precursor with an NH ₂ -protected peptide-resin; And
(c) removing the resin.
&Lt; / RTI &gt;

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