KR101732124B1 - Novel transdermal delivery peptides and use thereof - Google Patents

Abstract

The present invention relates to novel peptides and derivatives thereof having excellent skin permeability, and a method of delivering a novel peptide or a derivative thereof to a specific site in vivo by fusing a biological or pharmaceutical active substance.
Accordingly, it is possible to deliver a substance having biological or pharmaceutical activity through various routes including oral, oral, intranasal, subcutaneous, intradermal, mucosal, and inhalation, thereby maximizing the therapeutic effect and minimizing side effects. System can be developed.

Description

NOVEL TRANSDERMAL DELIVERY PEPTIDES AND USE THEREOF FIELD OF THE INVENTION The present invention relates to novel skin permeable peptides,

TECHNICAL FIELD The present invention relates to a novel skin permeable peptide, and a method for delivering a biologically or pharmaceutically active substance into skin by permeation through the skin using the same.

Skin is the largest surface organ in the human body and is the route through which drugs can be effectively delivered when appropriate methods are used. However, due to the extrinsic skin permeability of foreign substances due to the stratum corneum, which is composed of dead cells at the outer most part of the skin, it is difficult to transfer large molecular weight and hydrophilic substances. Various skin permeation promotion studies have been attempted to overcome the skin barrier of drug delivery. However, it is almost impossible to transfer the drug only to the chemical without stimulating the skin physically, and development of a chemical promoter that can overcome this is required.

As a result of the research on this demand, the membrane permeation protein domain including Tat (47-57; YGRKKRRQRRR) derived from the transcription factor of human immunodeficiency virus-1 (HIV-1) et al., 2001; Tyagi et al., 2001; < RTI ID = 0.0 > Rusnati et al., 1997; Chen et al., 2006). PTD is composed mostly of electrically positively charged arginine and lysine amino acids and these sequences are thought to act on the cell lipid membrane with negative charge to induce the permeation of the peptide. However, since cell membrane permeability is inherently distinct from skin permeation, further studies are required. Rather, it is expected that effective intradermal drug delivery may be difficult due to penetration of epidermal cells. Nevertheless, the study of skin penetration of cell permeable peptides is a mere reality.

Therefore, there is a continuing need to develop a method for analyzing the membrane permeability of various peptides, thereby effectively overcoming the limitation of skin permeability using peptides and peptides that pass through the skin, and delivering biological or pharmaceutical active substances.

The present invention provides novel peptides and derivatives thereof having excellent skin permeability.

It is another object of the present invention to provide a skin permeable fused product in which a biological or pharmaceutical active substance is fused to the peptide and its derivative.

Another object of the present invention is to provide a pharmaceutical composition for transdermal delivery comprising the fusion substance.

It is another object of the present invention to provide a method for delivering a biological or pharmaceutical active substance to a specific site in vivo using the fusant.

As one embodiment of the present invention, there is provided a skin permeable peptide or a derivative thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1 to 3.

In another aspect of the present invention, there is provided a skin-permeable fusion body in which a biological or pharmaceutical active substance is bound to the peptide or a derivative thereof.

In another aspect of the present invention, there is provided a composition for transdermal delivery of a biologically or pharmaceutically active substance containing the above-mentioned skin permeable fusant as an active ingredient.

Also, as another embodiment of the present invention, there is provided a percutaneous permeation delivery method for binding a peptide with a target substance of biological or pharmacological activity to prepare a delivery complex and injecting the peptide into a living body or a cell of a mammal.

The present invention provides a method for discovering a novel skin permeable peptide which binds to a biological or pharmaceutical active substance and enhances skin permeation of the substance, and transmits a biologically or pharmaceutically active substance through the skin to the living body do. Accordingly, it is possible to deliver a substance having biological or pharmaceutical activity through various routes including oral, oral, intranasal, subcutaneous, intradermal, mucosal, and inhalation, thereby maximizing the therapeutic effect and minimizing side effects. System can be developed.

In FIG. 1, a and b are photographs of SKH-1 mouse skin coated with the skin permeable peptide of the present invention and observed with a confocal microscope, and quantified therefrom.
FIG. 2 is a graph showing skin permeation of skin permeable peptides of the present invention using a Franz diffusion cell apparatus. FIG.
FIG. 3 is a graph showing the skin permeability of FK506-peptide compound prepared by synthesizing the skin permeable peptide of the present invention together with FK506 using a Franz diffusion cell instrument.
4 is a photograph showing the cell membrane permeability of the skin permeable peptide of the present invention using a confocal microscope.
5 is a graph showing the safety of the skin permeation peptide.

Hereinafter, the present invention will be described in detail.

In general, membrane permeable peptides have been suggested that amino acids with positivity such as arginine or lysine may be important for peptide membrane permeation, but skin permeation-promoting peptides such as TD-1 have only one lysine, Is not applicable to skin permeation, and it is disproved that cell membrane permeation and skin permeation are completely different. Accordingly, the present inventors conducted a skin permeation screening with a known cell permeable peptide, and developed a new skin permeable peptide by analyzing the amino acid sequence and structure constituting peptides capable of transmitting skin. Thus, the peptide provided by the present invention means a peptide having a novel function that permeates through the skin without functional similarity to PTD known to pass through the cell membrane. Based on these results, we hypothesized that amino acids with positively charged amino acids such as arginine or lysine may be important in cell permeation, and that repeated permeation of hydrophilic amino acids and hydrophobic amino acids in skin permeation is important for skin permeation. The present invention has been completed by developing a peptide sequence having superior skin permeability than previously known peptides.

The amino acid character codes used in the present specification are character codes commonly used in this technical field, and the contents thereof are shown in Table 1 below.

[Table 1]

As used herein, the term " peptide " refers to a polymer typically composed of 10 or less, preferably 3 to 10 amino acid residues linked by an amide bond (or peptide bond) Means an amino acid. The peptide molecule may be one obtained by extracting a protein in vivo and treating it with proteolytic enzymes to lower molecular weight or synthesized using a gene recombination and protein expression system and preferably synthesized in vitro through a peptide synthesizer or the like . In the present invention, the peptide molecule may be a derivative in which a specific atom or atomic group is substituted with a hydroxyl group or the like, as desired. To the COOH terminus of the peptide molecule C- terminal, NH ₂ terminal is referred to as N- terminus.

The present invention provides, as a specific aspect, a skin permeable peptide or a derivative thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1 to 3. SEQ ID NOS: 1 to 3 are as set forth in Table 2 below:

[Table 2]

The peptide or derivative may have a substance-transferring function, and since it is a very small peptide, biological interference with a substance to be delivered can be minimized.

The derivative may be modified such that the amino acid fragment or a part of the peptide is substituted or deleted, a part of the amino acid sequence is modified to a structure capable of increasing stability in vivo, a part of the amino acid sequence is modified to increase the hydrophilicity, Or all of which may be substituted with L- or D-amino acids, or a part of the amino acid may be modified. The derivative will be modified to retain cellular permeability and intracellular delivery function.

As a preferred derivative of the present invention, the C-terminal of the peptide may be modified with a hydroxy group (-OH), an amino group (-NH 2), an azide (-NHNH 2), or the like.

As a preferred derivative of the present invention, the N-terminal of the peptide is selected from the group consisting of an acetyl group, a fluorenylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group and a polyethylene glycol (PEG) A protecting group may be bonded.

According to another aspect of the present invention, there is provided a skin-permeable fusion substance in which a biological or pharmaceutical active substance is fused to the peptide or a derivative thereof.

In the present specification, the term 'skin permeable fusion substance' means a substance having a substance-transferring activity of a skin permeation peptide linked chemically and physically or non-covalently with a biological or pharmaceutical active substance. Amino acid selected from the group consisting of SEQ ID NOS: 1 to 3, or a peptide consisting of a mutant sequence in which some of the amino acid sequences selected from the group consisting of SEQ ID NOS: 1 to 3 are substituted with structurally and functionally similar amino acids.

As used herein, a substance having biological or pharmacological activity capable of binding to a cell-penetrating peptide to be a fused substance means a substance capable of controlling all physiological phenomena in living body, and includes DNA, RNA, proteins, peptides, fats , A carbohydrate and a chemical compound, or a fluorescent labeling substance, and examples thereof include an anti-cancer agent, an immunological disease agent, an antiviral agent, an antibiotic, an organism growth, development or differentiation factor, It is not. Since the cell permeable peptide having the mass transfer function according to the present invention is a very small peptide, it is possible to minimize the biological interference with the active substance that may be generated. When the fusion substance is to be delivered to a specific cell, tissue or organ, the substance having biological activity may be an extracellular partial protein of a ligand capable of selectively binding to a receptor specifically expressed in a specific cell, tissue or organ, or Monoclonal antibodies (mAbs) that can specifically bind to these receptors or ligands and fused forms in combination with modified forms. Binding of the peptide to a substance having biological activity can be achieved by indirect coupling by cloning techniques using an expression vector at the nucleotide level or by direct or indirect coupling by chemical or physical covalent or noncovalent coupling of a substance having biological activity with the peptide can do.

According to a preferred embodiment of the present invention, the biological or pharmaceutical active substance is FITC or FK-506.

According to another aspect of the present invention, there is provided a transdermal delivery composition comprising a skin permeable fused product in which a biological or pharmaceutical active substance is fused to a peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOS: 1 to 3.

The transdermal delivery composition according to the present invention includes a pharmaceutically acceptable carrier in addition to the active ingredient. The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, It is not. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc., in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

A suitable dose of the pharmaceutical composition of the present invention may be variously prescribed by factors such as the formulation method, the administration method, the age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient .

The pharmaceutical composition of the present invention may be prepared in a unit dosage form by formulating it with a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs Or into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

In yet another embodiment of the present invention, there is provided a method for preparing a delivery complex comprising the steps of: preparing a delivery complex by binding a skin permeable peptide comprising any one of SEQ ID NOS: 1 to 3 with a target substance of biological or pharmaceutical activity; And administering the delivery complex in vivo to the mammal. The present invention also provides a method for transdermal delivery of a biological or pharmacologically active target substance comprising the delivery complex.

The binding of step 1 may be achieved by indirect coupling by cloning techniques using an expression vector at the nucleotide level or by chemical or physical covalent or noncovalent coupling of the peptide or derivative with a substance having biological or pharmacological activity .

Hereinafter, the constitution and effects of the present invention will be described in more detail with reference to Examples, Comparative Examples and Experimental Examples. However, the following examples, comparative examples, and experimental examples are provided for illustrative purposes only in order to facilitate understanding of the present invention, but the scope and scope of the present invention are not limited thereto.

[ Example  One]

The Peptides  making

In this example, the synthesis was carried out according to the general peptide solid phase synthesis method (Wang C. Chan, Perter D. White, "Fmoc Solid Phase Peptide Synthesis", Oxford) in order to synthesize various cell permeation peptide derivatives. More specifically, coupling was performed one by one from the C-terminus using an automatic synthesizer (ASP48S, Peptron, Inc.) using Fmoc-SPPS (9-Fluorenyl methyl oxycarbonyl solid phase peptide synthesis) method. All monomeric materials used in the synthesis of peptide derivatives are protected with Fmoc at the N-terminus and amino acids protected with trityl (Trt), t-butyloxycarbonyl (Boc), t-butyl Respectively. As the coupling reagent, HBTU (2- (1H-Benzotirazloe-1-yl) -1,1,3,3-tetramethyluronium hexafluorophophate / HOBt (Hydroxybenzo triazloe) / NMM (N-methylmorpholine) was used.

(1) Protected amino acid (8 eq.) And coupling agent HBTU (8 eq.) / NMM (16 eq.) Were dissolved in DMF (dimethyl formamide) and reacted at room temperature for 2 hours. (2) To remove Fmoc, 20% (v / v) Piperidine / DMF was added and reacted twice at room temperature for 5 minutes. The peptide derivatives were produced by repeating the reactions (1) and (2). Then, to confirm the skin permeability, 6-aminohexanoic acid and fluorescein isothiocyanate (FITC) were bonded to the N-terminal portion of the peptide as a linker.

Peptide separation at Resin uses TFA (Trifluoroacetic acid) / EDT (1,2-ethanedithiol) / Thioanisole / TIS (Triisopropylsilane) / H ₂ O (mixing ratio (by weight) = 90 / 2.5 / 2.5 / 2.5 / 2.5) Respectively. The thus obtained mixed solution is subjected to an excess treatment of refrigerated diethyl ether solvent to produce a precipitate. The resulting precipitate was centrifuged to complete precipitation, excess trifluoroacetic acid, thianyzole, and ethanedithiol were firstly removed, and then the same procedure was repeated twice to obtain a solidified precipitate.

The resulting precipitate was dissolved in a water-containing solution containing 0.1% (v / v) Trifluoroacetic acid using a high performance liquid chromatographic apparatus (Shimadzu Prominence HPLC, Japan) using a C18 column (250 mm × 22 mm, 10 μm, Vydac Everest, USA) acetonitrile liner gradient (Acetonitrile concentration: 10 ~ 75% (v / v)). The molecular weights of the purified peptide derivatives were confirmed using LC / MS (HP 1100 series, Agilent Technologies, Germany) and the pure purified fractions were lyophilized to give the following peptides as TFA (Trifluoroacetic acid) salt in the form of a white powder .

Specifically, the sequences and molecular weights of the synthesized peptide derivatives are shown below.

Derivatives A: QYRISH-NH ₂ : Rt = 5.89 min (from v / v) to 100% (v / v) acetonitrile / water containing 0.01% Concentration gradient); MS (ESI) 801.4 m / e, [M + H] < ⁺ > = 802.5

Derivative _{B: QIYRISH-NH 2: Rt} = 7.21 bun (0.01% (v / v) 5% containing TFA (v / v) to 100% (v / v) acetonitrile / water, various over a period of 30 minutes from the Concentration gradient); MS (ESI) 914.5 m / e, [M + H] < ⁺ > = 915.5

Derivative _{C: QIIYRPLG-NH 2: Rt} = 6.23 bun (0.01% (v / v) 5% containing TFA (v / v) to 100% (v / v) acetonitrile / water, various over a period of 30 minutes from the Concentration gradient); MS (ESI) 957.6 m / e, [M + H] < ⁺ > = 958.5

Fusions A: FITC-linker-QYRISH- NH 2: Rt = 7.83 bun (0.01% (v / v) 5% containing TFA (v / v) to 100% (v / v) acetonitrile / from the water 30 in the 0.0 > minutes) < / RTI > MS (ESI) 1303.9 m / e, [M + H] < ⁺ > = 1304.0

Fusions B: FITC-linker-QIYRISH- NH 2: Rt = 6.33 bun (0.01% (v / v) 5% containing TFA (v / v) to 100% (v / v) acetonitrile / from the water 30 in the 0.0 > minutes) < / RTI > MS (ESI) 1417.1 m / e, [M + H] < ⁺ > = 1418.4

Fusions C: FITC-linker-QIIYRPLG- NH 2: Rt = 5.83 bun (0.01% (v / v) 5% containing TFA (v / v) to 100% (v / v) acetonitrile / 30 from water of 0.0 > minutes) < / RTI > MS (ESI) 1460.1 m / e, [M + H] < ⁺ > = 1461.5

[ Example  2]

Skin permeation Of peptide  When a derivative and a substance having a biological activity are fused Fused  Synthesis and separation

In this example, FK506 (Tacrolimus, fujimycin), which is used as an immunosuppressant to inhibit rejection after various organ transplantation of a human, is ligated to the N-terminal of the skin permeable peptide to synthesize a fusion. For this purpose, the hydroxy moiety of the cyclohexyl of FK506 was activated with 4-nitrophenyl chloroformate to facilitate connection to the N-terminal of the skin permeable peptide, and then ligated to the skin permeable peptide.

Specifically, the sequence and molecular weight of the synthesized peptide derivatives are shown in Table 3 below.

Fusions _{D: FK506-QYRISH-NH 2} : Rt = 15.71 bun (0.01% (v / v) for 30 minutes of acetonitrile / water in 5% (v / v) to 100% (v / v) containing TFA Various concentration gradients throughout); MS (ESI) 1631.9 m / e, [M + H] < ⁺ > = 1632.8

Fusions _{E: FK506-QIYRISH-NH 2} : Rt = 15.74 bun (0.01% (v / v) for 30 minutes of acetonitrile / water in 5% (v / v) to 100% (v / v) containing TFA Various concentration gradients throughout); MS (ESI) 1745.1 m / e, [M + H] < ⁺ > = 1745.7

[Table 3]

Each peptide was labeled with FITC labeling at the amino terminus and protected at the carboxy terminus for the detection of fluorescence with a purity of 95% or more.

[ Experimental Example  One]

Of peptide  Skin permeation screening

Skin of 7-week-old male SKH-1 mice was harvested and the peptides permeated into the skin were observed using a confocal microscope. The peptide conjugated with FITC conjugate (fucoid AC) was applied onto mouse skin and fluorescence intensities of each skin depth were analyzed through a confocal microscope (FV1000-IX81, Olympus Co., Japan) to select peptides whose skin permeability was higher than PTD of TAT Respectively. (Figs. 1A and 1B)

[ Experimental Example  2]

Franz Diffusion  Cell-based Of peptide  Skin penetration test

Skin of 7 week old male SKH-1 mice was harvested and measured using a Franz diffusion cell (FCDV-15, Labfine, Korea) at 37 +/- 0.5 DEG C with stirring at 600 rpm. At this time, the area of the skin to which the skin permeable peptide was administered was 1.77 cm ² , and the receptor phase was filled with phosphate buffer (pH 7.4) and the volume was 12.5 ml. FITC-conjugated peptides (fused AC) were applied to the skin surface. After 24 hours, 1 ml of the receptor phase was collected and analyzed by using Nanodrop 2000 (Thermo scientific, USA). (Fig. 2)

[ Experimental Example  3]

Franz Diffusion  Cell-based Peptides - physiologically active substance Fused  Skin penetration test

Skin of 7 week old male SKH-1 mice was harvested and measured using a Franz diffusion cell at 37 +/- 0.5 DEG C with stirring at 600 rpm. At this time, the area of the skin to which the skin permeable peptide was administered was 1.77 cm ² , and the receptor phase was filled with phosphate buffer (pH 7.4) and the volume was 12.5 ml. After 24 hours, 1 ml of the receptor phase was collected and analyzed using a 4000 Q Trap LC-MS / MS system (AB Sciex, USA). (Fig. 3)

[ Experimental Example  4]

Skin permeation Of peptide  Cell membrane permeation screening

B16F10 (mouse melanoma) cells were seeded on plates to grow about 70-80%, and then the experiment was carried out when stabilized. After washing twice with DMEM medium at 37 ° C, FITC conjugated peptide (fucoid AC) was treated with 100 μM of the culture medium, incubated for 1 hour at 37 ° C in a CO ₂ incubator, and the cells were washed 3 times with PBS And fluorescence of FITC, which is an indicator of the protein entering the cell, was measured at 525 nm of excitation and 490 nm of emission using an immediate confocal microscope (FV1000-IX81, Olympus Co., Japan). (Figure 4)

[ Experimental Example  5]

Skin permeation Of peptide  Safety check

MCF 7 cells were treated with a skin permeation peptide (derivative A-C) at a concentration of 100 μM and their safety was confirmed by MTT assay. The peptide was treated in the same manner as in [Experimental Example 4] and treated as nothing but a control. After the test sample was added, it was cultured for 72 hours, treated with MTT and cultured for 2 hours. After that, the culture solution was removed, and the cell viability was calculated by measuring the absorbance at 540 nm by adding DMSO. (Fig. 5)

<110> PEPTRON &Lt; 120 > NOVEL TRANSDERMAL DELIVERY PEPTIDES AND USE THEREOF} <130> 5489 <160> 3 <170> Kopatentin 2.0 <210> 1 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> TRANSDERMAL PEPTIDE <400> 1 Gln Tyr Arg Ile Ser His   1 5 <210> 2 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> TRANSDERMAL PEPTIDE <400> 2 Gln Ile Tyr Arg Ile Ser His   1 5 <210> 3 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> TRANSDERMAL PEPTIDE <400> 3 Gln Ile Ile Tyr Arg Pro Leu Gly   1 5

Claims (8)

A skin permeable peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOS: 1 to 3; Or a derivative of said skin permeable peptide selected from the group consisting of A, B, and C:
Derivative A: QYRISH-NH _2;
Derivative B: QIYRISH-NH _2; And
Derivative C: QIIYRPLG-NH _2. delete A fused product selected from the group consisting of A, B, C, D and E, wherein the skin permeable peptide of claim 1 or a derivative of said skin permeable peptide is linked to fluorescein isothiocyanate (FITC) or immunosuppressant FK506 :
Fusions A: FITC-linker-QYRISH- NH 2;
Fusions B: FITC-linker-QIYRISH- NH 2;
Fusions C: FITC-linker-QIIYRPLG- NH 2;
Fusions _{D: FK506-QYRISH-NH 2} ; And
Fusions _{E: FK506-QIYRISH-NH 2} :
However, the linker in the above sequence is 6-aminohexanoic acid.

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