KR101315056B1 - PEGylated anti-wrinkle skin-restoring pentapeptide derivatives - Google Patents

Abstract

The present invention is a skin functional pentapeptide derivative in which polyethylene glycol is bound. More specifically, polyethylene glycol obtained by binding monodisperse polyethylene glycol (PEG) to an inactive site of pentapeptide (KTTKS) having a wrinkle improvement function This is a combined skin functional collagen peptide derivative, which is chemically stable, low toxicity and excellent physiological activity.

Description

PEGylated anti-wrinkle skin-restoring pentapeptide derivatives}

The present invention relates to a PEGylated (PEGylated, polyethylene glycol coupled) skin wrinkle functional pentapeptide derivative, and more particularly to a penta peptide (KTTKS) having a skin wrinkle improvement function, polyethylene glycol (PEG) The present invention relates to a PEGylated skin functional pentapeptide derivative and a skin wrinkle improving functional cosmetic material, which are chemically stable, low toxicity, and excellent in physiological activity.

The skin covering our body is composed of epidermis, dermis and subcutaneous fat. The epidermis is the thinnest layer and consists of organic combination of keratinocytes and melanocytes. The dermis accounts for about 95% of the skin and moisturizes the skin. It is a protective layer, and collagen and elastin, proteins that play an important role in skin elasticity, are woven like a net, and contain moisturizing factors such as mast cells and hyaluronic acid that are involved in allergic reactions. Subcutaneous fat serves as the epidermis and dermis to provide nutrition, body shape, body temperature, external shock absorption and subcutaneous fat cell protection.

As you age, your skin function is drastically deteriorated due to endogenous or exogenous causes. As aging progresses, the epidermis, dermis and subcutaneous tissue become thinner, collagen and elastin become thinner and the structure loosens, resulting in less elasticity and wrinkles. In addition, as the lipid composition and content of the lipid barrier, which functions as a skin barrier, are changed, the skin's moisture content decreases and the skin dries, resulting in physiological changes such as blemishes, freckles, pigmentation and various other skin lesions. Triggered. Recently, studies on various bioactive components and functional cosmetics containing the same for improving the skin aging phenomenon have been attracting attention.

Peptides are recently attracting attention as the material of functional cosmetics, because (1) peptides are biomaterials composed of amino acids, so they are less toxic and safe, (2) they have strong physiological activities, and (3) precise activity mechanisms. This is because it is widely used, it is (4) stable to light, and (5) has a strong affinity with water, so it has strong moisturizing power.

   Functional cosmetic peptide materials that have been widely used recently include penta peptide (pentapeptide), a collagen-derived peptide, acetyl hexapeptide-3, a botox peptide, and copper peptide (copper peptide) containing copper.

Collagen-derived pentapeptide (Lys-Thr-Thr-Lys-Ser) is a carboxyl terminal fragment of collagen type I that plays an important role in skin connective tissue and wrinkle formation and moisturizing. Cell culture of fibroblasts, cells that synthesize collagen It is known to promote the synthesis of collagen type I and collagen type III, fibronectin, and growth factor TGF-β, and to promote its transcription by binding to the porogen of the collagen gene (Katayama et al . (1993). J. Biol. Chem., 268, 9941-9944).

The collagen is present in a large amount in the skin, blood vessels, internal organs, bones, etc., and 70% of the dermis constituting the skin is composed of collagen. Collagen, which accounts for 30% of the total weight of a living protein, functions to induce mechanical toughness of the skin, resistance of connective tissues and binding of tissues, support of cell adhesion, cell division and differentiation during growth or wound healing of the organism. Collagen is biosynthesized in fibroblasts, the amount of which is reduced by natural and photoaging, and it is known that a 65% reduction occurs between the ages of 20 and 80 (Shuster S., (1975) British Journal of Dermatology, 93 , 639-643). As the synthesis of collagen in vivo becomes active, dermal matrix components are increased, and it is found that the effect of wound healing, elasticity, and wrinkle improvement can be expected, and is used in cosmetics, food, and medicine.

However, peptides exhibit a variety of physical and chemical instabilities, depending on the given environment. Peptides that exhibit physical instability such as adsorption, aggregation, and precipitation, and are relatively small in size, deamidation, oxidation, hydrolysis, racemization, and heterogeneity Activity is denatured or degraded by various chemical degradation pathways such as nitriding (isomerization). Therefore, there is a need for a technique for stabilizing peptide structures in formulations or in biological tissues.

As a method of stabilizing the peptide, a bioconjugation technique that chemically binds a biopolymer or a fatty acid is useful. The most notable technique is a PEGylation technique that chemically bonds polyethylene glycol (PEG) polymer. )to be. PEGylation technology prevents the breakdown of peptides by blocking the accessibility of proteases, increases the solubility of peptides by the solubility of polyethylene glycol (PEG), and increases their size when applied to proteins to prevent loss from the kidneys And significantly increase blood half-life (Harris JM and Chess RB (2003) Nat. Rev. Drug Discov., 2, 214-221).

However, since the PEGylated peptide used in the related art uses non-uniform polydisperse polyethylene glycol (PEG), there has been a problem of low productivity and low yield and low purity in the preparation of peptide derivatives.

The present invention has been invented to solve the conventional problems as described above has the following object.

The present invention is a PEGylation in which monodisperse polyethylene glycol (PEG) is selectively bound to N-terminal amine of pentapeptide (KTTKS) in place of polydisperse polyethylene glycol (PEG). The purpose is to provide a pentapeptide derivative.

The present invention provides a method for preparing pegylated dermal functional pentapeptide derivatives having a significantly improved production yield by selectively binding monodisperse polyethylene glycol (PEG) to N-terminal amine of pentapeptide (KTTKS). The purpose is to provide.

The present invention provides monodisperse monopegylated pentapeptide (mono-PEG-KTTKS) in which monodisperse polyethylene glycol (PEG) is selectively bound to N-terminal amine of collagen peptide (KTTKS) with high production yield. It is an object of the present invention to provide a method for preparing pegylated skin functional pentapeptide derivatives which can be prepared in high purity.

In order to achieve the above object, the present invention is implemented by an embodiment having the following configuration.

The first embodiment of the present invention is characterized in that the PEGylated skin wrinkle improvement functional pentapeptide derivative represented by the following formula.
[Chemical Formula]
CH ₃ O- (CH ₂ CH ₂ O) _m- (CH ₂ ) _n -CH ₂ -NH-Lys-Thr-Thr-Lys-Ser
Wherein m is an integer ranging from 2 to 48, n is an integer ranging from 1 to 3, and NH is an N-terminal amine group of Lys.

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The second embodiment of the present invention is characterized in that the monodisperse PEGylated skin wrinkle improving functional pentapeptide derivative wherein m of the first embodiment is an integer ranging from 2 to 24.

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The present invention has the following effects with the above-described configuration.

The present invention is a monodisperse that selectively binds monodisperse polyethylene glycol (PEG) to the N-terminal amine of pentapeptide (KTTKS) in place of polydisperse polyethylene glycol (PEG) conventionally used It is effective to provide PEGylated pentapeptide derivatives (PEG-KTTKS).

The present invention provides the production yield and purity of monodisperse PEGylated pentapeptide derivative (PEG-KTTKS) by selectively binding monodisperse polyethylene glycol (PEG) to N-terminal amine of pentapeptide (KTTKS). Has the effect of greatly improving.

1 is a view showing the advantages of PEG-peptide preparation using monodisperse polyethylene glycol (PEG)
2 is a view showing a process for preparing pegylated pentapeptides
Figure 3 is a purified purified HPLC chromatogram of Mono-PEG-2000-KTTKS (phase: chromatogram of the reaction of the pre-separation purification, bottom: chromatogram of mono-PEG-2000-KTTKS after separation and purification)
Figure 4 is a purified purified HPLC chromatogram of monodisperse mono-PEG-220-KTTKS (phase: chromatogram of reaction mixture, bottom: chromatogram of mono-PEG-220-KTTKS after separation purification).
Figure 5 is a purified purified HPLC chromatogram of monodisperse mono-PEG-572-KTTKS (phase: chromatogram of reaction mixture, bottom: chromatogram of mono-PEG-572-KTTKS after separation purification).
Figure 6 LC-MS mass spectrometry of pentapeptide (KTTKS).
7 is an LC-MS mass spectrometry spectrum of Mono-PEG-220-KTTKS.
8 is an LC-MS mass spectrometry spectrum of Mono-PEG-572-KTTKS.
9 is an LC-MS mass spectrometry spectrum of polydisperse mono-PEG-2000-KTTKS.
Figure 10 shows the results of comparing the stability of pentapeptide (KTTKS) and mono-PEG-220-KTTKS
FIG. 11 shows cytotoxicity (* p <0.05) of peptide (KTTKS), monodisperse mono-PEG-220-KTTKS, monodisperse mono-PEG-572-KTTKS, polydisperse mono-PEG-2000-KTTKS
12 shows collagen synthesis activity (* p <0.05) of peptide (KTTKS), monodisperse mono-PEG-220-KTTKS, monodisperse mono-PEG-572-KTTKS, polydisperse mono-PEG-2000-KTTKS

In general, PEGylation of the amine group (-NH ₂ ) of the peptide involves polyethylene glycol (PEG) on the amines of N-terminal amines and lysine residues. Is combined. This PEGylation is called non-specific PEGylation. In this case, polyethylene glycol (PEG) is bound to various sites, causing deactivation of peptides and producing products of various sizes. There is a problem of poor efficiency. As a complementary method, site-specific PEGylation, which selectively binds polyethylene glycol (PEG) to inactive sites, is used. However, even in this case, using polydisperse PEG, which is generally used, remains a problem that products of various sizes are produced.

The present invention provides a method for the production of PEGylated collagen peptides in a high production yield to bind monodisperse polyethylene glycol (monodisperse PEG) to the N- terminal amine (N-terminal amine) which is an inactive portion of the collagen peptide . The reason why N-terminal amine is selected as a PEGylation site is that of N-terminal amine in the study of palmitoyl pentapeptide (palmitoyl-KTTKS), which is currently developed as a functional cosmetic material. It is based on the fact that fatty acid is not bound to the activity.

Monodisperse polyethylene glycol is a single molecular weight polyethylene glycol (PEG), which is distinguished from polydisperse polyethylene glycol (PEG) having various molecular weight distributions. When monodisperse polyethylene glycol (PEG) is conjugated to the peptide by site-specific mono-PEGylation, a mono-polyethylene glycol conjugate of a uniform size can be obtained in high yield. Therefore, the PEGylated peptide production method is greatly improved the conventional PEGylation (PEGylation) method using the polydisperse polyethylene glycol (PEG). That is, it is possible to maximize the PEGylation (PEGylation) effect such as increased stability without affecting the activity of the peptide, it is possible to produce a polyethylene glycol conjugate (PEG conjugate) that can be obtained with a high production yield (see Fig. 1).

Hereinafter, the present invention will be described with reference to Examples. However, the embodiments according to the present invention may be modified in various forms, and the scope of the present invention should not be construed as being limited to the embodiments described below. The embodiments of the present invention are provided to more completely explain the present invention to those having ordinary skill in the art.

< Pentapeptide Pegillation >

In order to bind polyethylene glycol (PEG) to the N-terminus of pentapeptide (KTTKS), a monomethoxy PEG-aldehyde (mPEG-ALD) containing an aldehyde group was used. The three mPEG-ALDs used were polydisperse mPEG-ALD (hereinafter referred to as mPEG-ALD-2000) having a molecular weight of 2000, monodisperse mPEG-ALD having a molecular weight of 220 (hereinafter referred to as mPEG-ALD-220) and a molecular weight of 572. Monodisperse mPEG-ALD (hereinafter mPEG-ALD-572) was used.

PEG Molecular weight (Da) manufacturer Monodisperse PEG (mPEG-ALD-220) 220 Celares GmbH (Germany) Monodisperse PEG (mPEG-ALD-572) 572 Celares GmbH (Germany) Polydisperse PEG (mPEG-ALD-2000) 2,000 NEKTAR (USA)

<MPEG-ALD used for experiment>

N-terminal PEGylation reaction of collagen peptide (KTTKS) was performed in the presence of sodium cyanoborohydride in a weakly acidic buffer at pH 5.5 (see FIG. 2).

PEGylation (PEGylation) was reacted in various molar ratios at the most optimal condition 1: 1.5 to obtain each mono-pegylated pentapeptide (mono-PEGylated KTTKS). Reaction conditions were to dissolve pentapeptide (KTTKS) in deionized water at a concentration of 10 mg / mL, and then dissolve each polyethylene glycol (PEG) at 10 mg / mL in 0.1 M acetate buffer (pH 5.5). According to the molar ratio, an appropriate amount was added in the presence of 20 mM sodium cyanoborohydride and reacted for 4 to 24 hours. The resulting mono-pegylated pentapeptide (mono-PEGylated KTTKS) was collected under respective separation conditions using HPLC, and then the organic solvent was removed through a vacuum dryer (Speed Vac) and then lyophilized to mono-pegylated. Pentapeptide (mono-PEGylated KTTKS) was obtained.

< Pegylated Pentapeptide HPLC  Separation Purification>

Isolation and purification of mono-pegylated pentapeptide (mono-PEG-KTTKS) was performed using HPLC. A Dionex Ultimate 3000 HPLC system and a Phenomenex Gemini C-18 (4.6 x 250 mm, 5 micron particle size) column were used as mobile phase and 0.1% triloacetic acid (TFA) containing water (mobile phase A) and 0.1 Isolate mono-pegylated pentapeptides (mono-PEG-KTTKS) by isocratic elution or gradient elution using acetonitrile (mobile phase B) containing% TFA It was. Detection was carried out at UV 215 nm, column temperature maintained at 25 degrees and flow rate of mobile phase was 1 mL / min. Mono-pegylated pentapeptide with a molecular weight of 220 (Mono-PEG-220-KTTKS) was isolated by isocratic elution of mobile phase B 8%, mono-pegylated pentapeptide with a molecular weight of 572 (mono -PEG-572-KTTKS) was separated by isocratic elution of 16% of mobile phase B, and mono-pegylated pentapeptide (mono-PEG-2000-KTTKS) having molecular weight of 2000 was 10-50% of mobile phase B. Separated by gradient elution.

< LC - MS Using Pegylated Pentapeptide  Check>

The molecular weight and polyethylene glycol (PEG) binding site of the prepared mono-pegylated pentapeptide (mono-PEG-KTTKS) were confirmed using liquid chromatography-mass spectrometry (LC-MS). Liquid Chromatography-Mass Spectrometry (LC-MS) was performed using an Agilent 6410 triple quadrupole LC-MS system and Dionex Acclaim 120 C18 colume (2.1 x 150 mm, 3 micron particle size) and 0.1% formic acid as mobile phase. Water containing (mobile phase A) and acetonitrile (mobile phase B) containing 0.1% formic acid were used. The mobile phase was gradient elution of mobile phase B 20-60% for 20 minutes, the column temperature was maintained at 25 degrees, and the flow rate was 0.2 mL / min. The mass spectrometer used a scan mode with positive ion monitoring, a fragment voltage of 50 V, a drying gas (N2) flow of 10 L / min, Conditions of drying gas termperature 350 degrees, nebulizer pressure 278 kPa, and capillary voltage 3.5 kV were used.

< Pegylated Pentapeptide  Manufacturing Yield and Purity Measurements>

The production rate of the desired mono-pegylated pentapeptide (mono-PEG-KTTKS) in pegylated pentapeptide (KTTKS) was determined by analyzing the reaction mixture under the above HPLC conditions, and the final yield was separated and purified by HPLC. One mono-pegylated penta peptide (mono-PEG-KTTKS) was again analyzed by HPLC to determine its concentration and amount. The purity of the final product was determined as a percentage of the main peak area of the mono-pegylated pentapeptide (mono-PEG-KTTKS) divided by the total area of all peaks by measuring main peak and other analog peaks on the HPLC chromatogram.

In the present invention, a monodisperse monomethoxy polyethyleneglycol-aldehyde having a molecular weight of 220 as compared to a polydisperse monomethoxy polyethyleneglycol-aldehyde having a molecular weight of 2000 is commonly used (monomethoxy). PEG-aldehyde, mPEG-ALD-220) and a monodisperse monomethoxy polyethyleneglycol-aldehyde having a molecular weight of 572 (monomethoxy PEG-aldehyde, mPEG-ALD-572) were used. The productivity and production yield of mono-PEG-KTTKS) were measured.

     First, the reaction molar ratio of the optimal peptide (KTTKS): PEG was searched using HPLC to obtain the desired mono-pegylated pentapeptide (mono-PEG-KTTKS) in high yield. A typical example is the production of mono-pegylated pentapeptides (mono-PEG-KTTKS) with monodisperse mPEG-ALD-220 under conditions of reaction molar ratio (KTTKS: PEG) 1: 1, 1: 1.5, 1: 2. When the reaction molar ratio was increased, the amount of unreacted peptide (KTTKS) decreased and the amount of PEGylated peptide (PEGylated KTTKS) increased, and most peptides (KTTKS) reacted at a molar ratio of 1: 2. Although participated in, the amount of dipegylated peptide (di-PEGylated KTTKS) was found to increase. Therefore, it was confirmed that 1: 1.5 is the optimal condition for generating the most mono-pegylated peptide (mono-PEG-KTTKS). At the molar ratio 1: 1.5, mono-PEG-220-KTTKS was the most obtained with 82.8%. Monodisperse mPEG-ALD-220 had the most mono-pegylated pentapeptide (mono-PEG-KTTKS) at a molar ratio of 1: 1.5, but monodisperse mPEG-ALD-572 and polydisperse mPEG-ALD-2000 Most mono-pegylated pentapeptides (mono-PEG-KTTKS) were produced at a molar ratio of 1: 2 (Table 2).

Molar ratio (KTTKS: PEG) 1: 1 1: 1.5 1: 2 1: 3 1: 4 mono-PEG-220-KTTKS 79.3% 82.8% 75.6% - - mono-PEG-572-KTTKS 56.8% 70.2% 77.3% - - mono-PEG-2000-KTTKS 55.1% - 64.9% 61.1% 54.0%

<Production rate of mono-pegylated pentapeptide (mono-PEG-KTTKS) according to reaction molar ratio (KTTKS: PEG)>

HPLC conditions for the separation of mono-pegylated peptide (Mono-PEG-KTTKS) were carried out using deionized water (mobile phase A) containing 0.1% TFA and acetonitrile (mobile phase B) containing 0.1% TFA. It was established under isocratic or gradient elution conditions. The mono-pegylated peptide of interest (Mono-PEG-KTTKS) was isolated according to the established separation conditions. Mono-pegylated peptide (Mono-PEG-KTTKS) prepared using polydisperse mPEG-ALD-2000 was isolated under the gradient elution condition of mobile phase B 20-50% for 10 minutes (FIG. 3). Reference).

Mono-pegylated peptide (Mono-PEG-KTTKS) prepared using monodisperse mPEG-ALD-220 was isolated under isocratic elution conditions of mobile phase B 8% (see Figure 4), monodisperse Mono-pegylated peptide (Mono-PEG-KTTKS) prepared using mPEG-ALD-572 was isolated under isocratic elution conditions of mobile phase B 16% (see Figure 5).

Mono-pegylated peptide (Mono-PEG-KTTKS) (mono-PEG-220-KTTKS, mono-PEG-572-KTTKS, mono-PEG-2000-KTTKS) was isolated and confirmed by LC-MS. It was. First, KTTKS is measured as [M + H] ⁺ = m / z 564.4, mono-PEG-220-KTTKS is [M + H] ⁺ = m / z 768.5, and mono-PEG-572-KTTKS is [M + 2H ²⁺ = m / z 560.9 and the actual size was m / z 1121.8 (see Figures 6-8). Monodisperse mono-pegylated pentapeptide (mono-PEG-KTTKS) showed a single molecular weight peak as expected. However, the polydisperse mono-PEG-2000-KTTKS showed a cluster of peaks of various masses due to the polydispersity of polyethylene glycol (PEG) (see FIG. 9). This feature of polydisperse polyethylene glycol (PEG) not only makes it difficult to set the specification of the material but also adversely affects the polyethylene glycol (PEG) bonding reaction and purity. The monodisperse PEGylated peptide of the present invention (PEG-KTTKS) can overcome this problem.

Polyethylene glycol (PEG) binding sites of monodisperse mono-pegylated peptides (mono-PEG-220-KTTKS and mono-PEG-572-KTTKS) were confirmed by LC-MS analysis after trypsin treatment. When trypsin treatment of peptide (KTTKS), the positions of Lys ¹ and Lys ⁴ are cleaved into three segments, Lys, Thr-Thr-Lys and Ser. Trypsin-treated peptide (KTTKS) sample LC-MS showed Lys ( m / z 147.8) and Thr-Thr-Lys ( m / z 349.4) peaks. In addition, the mono-PEG-220-KTTKS sample was trypsin (trypsin) after LC-MS analysis showed that the peak m / z 351.0 corresponding to PEG-220-Lys ¹ was confirmed. On the other hand, To obtain specific polyethylene glycol (PEG) binding to the position of the Lys ⁴ m / z 639.3, which may receive and Lys ¹ Mass peaks corresponding to ( m / z 147.8) were not observed, indicating that polyethylene glycol (PEG) was bound to the N-terminus in mono-PEG-220-KTTKS. Trypsin-treated samples of mono-PEG-572-KTTKS showed two mass peaks of m / z 345.4 and 516.9, respectively, which were PEG-572-Lys ¹ and PEG-572-Lys-Thr-Thr-Lys. Polyethylene peaks corresponding to [M + 2H] ²⁺ , and no mass peaks corresponding to PEG-572-TTKS and Lys ¹ ( m / z 147.8) bound PEG at the position of Lys ⁴ were observed. Glycol (PEG) was confirmed to be bound to the N-terminus (N-terminus).

Three mono-pegylated peptides (mono-PEG-220-KTTKS, mono-PEG-572-KTTKS, mono-PEG-2000-KTTKS) were successfully isolated and lyophilized mono-pegylated peptides after separation. The purity of (mono-PEG-KTTKS) was determined by HPLC (Table 3). Mono-PEG-220-KTTKS and mono-PEG-572-KTTKS prepared from monodisperse polyethylene glycol (PEG) were confirmed to have high purity of 100% and 97.2%, respectively. Mono-PEG-2000-KTTKS prepared showed a purity of 91.7%.

product Purity (%) mono-PEG-220-KTTKS 100.0 mono-PEG-572-KTTKS 97.2 mono-PEG-2000-KTTKS 91.7

<Purity of mono-pegylated peptide (mono-PEG-KTTKS)>

The yield of each mono-pegylated peptide (mono-PEG-KTTKS) product was quantified by HPLC. As a result, the yield of mono-PEG-2000-KTTKS made of polydisperse polyethylene glycol (PEG) was only 47.0%. On the other hand, mono-PEG-220-KTTKS and mono-PEG-572-KTTKS made of monodispersible polyethylene glycol (PEG) were confirmed to be produced in high yield of 79.5% and 76.5%, respectively (Table 4).

product Final yield (%) mono-PEG-220-KTTKS (monodispersible) 79.5 mono-PEG-572-KTTKS (monodispersible) 76.5 mono-PEG-2000-KTTKS (polydispersity) 47.0

Final yield of mono-pegylated peptide (mono-PEG-KTTKS) isolated and purified>

The reason why the mono-pegylated peptide (mono-PEG-KTTKS) made of monodisperse polyethylene glycol (PEG) shows a high production yield is not only due to the high production rate in the PEGylation reaction but also to the monodisperse property. This is because the yield of the separation process is high because of the good separation ability with di-pegylated polyethylene glycol conjugate (di-PEG conjugate) and the like by HPLC.

In conclusion, the PEGylation method using monodisperse polyethylene glycol (PEG) of the present invention has a higher purity (more than 95%) of mono with higher yield (75% or more) than the conventional method using polydisperse polyethylene glycol (PEG). -PEG-KTTKS can be prepared.

<Pegylated Pentapeptide  Stability Test

The stability of monodisperse mono-PEG-220-KTTKS was measured in comparison with native peptide (KTTKS). The stability test in the aqueous solution of pentapeptide (KTTKS) and mono-pegylated pentapeptide (mono-PEG-KTTKS) prepared was carried out by preparing substances in concentrations of 1 mg / mL in four buffers. PH pH 0.1 M glycine buffer (pH 2.5), 0.1 M acetate buffer (pH 4.0), 0.1 M phosphate buffer (pH 7.4), 0.1 M borate buffer (pH 9.0) Each sample was dissolved in) and the change in main peak for 7 days at 55 ° C was analyzed by HPLC.

Peptides (KTTKS) were tested for 7 days at 55 ° C, resulting in 96% and 99% at pH 2.5 and pH 4.0, respectively, at 79% at pH 7.4 and 70% at pH 9.0. Mono-PEG-220-KTTKS showed more than 99% stability at pH 2.5, 4.0 and 7.4 except pH 9. However, at pH 9, it showed a slight instability of about 88% at 7 days, but the stability was superior to that of the native peptide (KTTKS) (see FIG. 10).

<Pegylated Pentapeptide  Cytotoxicity Test>

Monodisperse mono-pegylated pentapeptides (mono-PEG-KTTKS) and polydisperse mono-pegylated pentapeptides (mono-PEG-KTTKS) and pentapeptides (KTTKS), and palmitoyl, a commercially available functional cosmetic material Cytotoxicity of pentapeptide (palmitoyl pentapeptide, pal-KTTKS) was compared. First, human normal fibroblasts (Human CCD-986sk, human fibroblast cells) were dispensed in a 96-well plate by dispensing 1 × 0 ⁴ cells / well in 10% Fetal Bovine Serum (FBS). After 24 hours incubation in DMEM (Dulbecco's Modified Eagle Media, Gibco BRL), the sample was washed twice and gradually diluted with serum-free medium, followed by a carbon dioxide incubator for 24 hours. ₂ incubator). To this was added MTT solution (5 mg / mL) 20 followed by 37 to 5% CO ₂ After 4 hours incubation in incubator gSanyo, Japan, the formed formazan was dissolved in DMSO and absorbed at UV 570 nm with an enzyme-linked immunosorbent assay (ELISA) reader (EL _X 800, BioTek, USA). Was measured.

Cytotoxicity of monodisperse mono-PEG-220-KTTKS and mono-PEG-572-KTTKS, and polydisperse mono-PEG-2000-KTTKS were evaluated in comparison with native peptide (KTTKS) (see FIG. 11). . Peptides (KTTKS) showed cytotoxicity at concentrations of 500 μM, whereas monodisperse mono-PEG-220-KTTKS and mono-PEG-572-KTTKS and polydisperse mono-PEG-2000-KTTKS ranged from 1-500 μM It was determined not to show clear cytotoxicity. Therefore, it was confirmed that PEG binding has an effect of reducing the cytotoxicity of the peptide (KTTKS).

<Pegylated Pentapeptide  Collagen Synthesis Activity Test>

In order to measure the physiological activity of the prepared mono-pegylated pentapeptide (mono-PEG-KTTKS), a sample was added to human dermal fibroblasts to measure the effect of promoting intracellular collagen synthesis. Biosynthetic collagen was measured by an enzyme-linked immunosorbent assay (ELISA assay) and calculated as 100% of no addition of the sample as a control. First, human normal fibroblasts (Human CCD-986sk, human fibroblast cells) were dispensed in 48 well plates at 5 × 10 ⁴ cells / well, incubated in DMEM with 10% FBS for 24 hours, and then washed twice. (washing) and adding a sample diluted step by step with serum-free medium, followed by a carbon dioxide incubator (CO ₂₎ for 24 hours. incubator). At this time, the amount of collagen in the cell culture was measured using a procollagen type C-peptide EIA kit (Takara, Japan). First, 100 μL of the antibody-POD conjugate solution was added to each well, and 20 μL of the sample or standard was added. At this time, the cell culture medium, which was used, was diluted 5 times, and shaken to mix evenly in a well, and then sealed with a foil and reacted for 37 to 3 hours. After the reaction was completed, the contents in the well (well) were removed and washed four times with 400 μL of phosphate buffered saline (PBS). After washing, 100 μL of the substrate solution was added and reacted for 15 minutes at 20-30. Then, 100 μL of 1 MH ₂ SO ₄ was added to stop the reaction, followed by shaking. After absorbance was measured at 450 nm, a standard concentration curve was prepared to measure the amount of collagen.

 Collagen synthesis activity of monodisperse mono-PEG-220-KTTKS and mono-PEG-572-KTTKS and polydisperse mono-PEG-2000-KTTKS was evaluated in comparison with native pentapeptide (KTTKS) at 100 μM concentration (See FIG. 12). Polydisperse mono-PEG-2000-KTTKS showed lower activity than pentapeptide (KTTKS), while monodisperse mono-PEG-220-KTTKS and mono-PEG-572-KTTKS clearly showed better bioactivity than pentapeptide (KTTKS) Indicated.

Claims (3)

PEGylated skin wrinkle improvement functional pentapeptide derivative represented by the following formula.
[Chemical Formula]
CH ₃ O- (CH ₂ CH ₂ O) _m- (CH ₂ ) _n -CH ₂ -NH-Lys-Thr-Thr-Lys-Ser
Wherein m is an integer ranging from 2 to 48, n is an integer ranging from 1 to 3, and NH is an N-terminal amine group of Lys.
The monodisperse PEGylated skin wrinkle improving functional pentapeptide derivative according to claim 1, wherein m is an integer ranging from 2 to 24.




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