KR20160066281A - Novel cell permeable peptide with high stability in plasma and use thereof - Google Patents

Abstract

Provided are a novel peptide which has high cell-permeability and is stable in the body, and a derivative thereof. More specifically, the purpose of the present invention is to provide a use thereof for delivering a biologically active substance into the body. To this end, the cell-permeable peptide of the present invention is composed of amino acid sequences selected from the group including sequence number 1 to 12.

Description

TECHNICAL FIELD [0001] The present invention relates to a novel cell permeable peptide having increased stability in blood plasma and a use thereof. BACKGROUND OF THE INVENTION 1. Field of the Invention [0001]

The present invention relates to a novel cytotoxic peptide or derivative thereof with increased stability in plasma.

Cell Penetrating Peptides (CPP) are cell membrane permeable peptides composed of about 10-30 short peptides, most of which are derived from the protein-transduction domain or the membranetranslocating sequence Endoh and Ohtsuki, 2010; Joliot and Prochiantz, 2004; Mogi and Kondo, 2010). In addition, CPP has electrochemical and physicochemical properties that allow the cell membrane to permeate through an oligopeptide that has the property of reacting with endocytosis or directly through the cell membrane in response to the cell membrane (Jung et al., 2007; Kwon Et al., 2009; M et al., 2009). These CPPs have been studied in connection with applications to drug delivery systems and are well known in the field of medicine and have been used in a specific biological system and have been widely used as a tool for controlling gene expression. In addition, efforts to use in disease diagnosis have been racing (Dietz, 2010; Gao et al., 2010; Hao et al., 2010; Standley et al., 2010).

Of the peptides best known as CPP, TAT peptides are derived from Human Immuno-deficiency Virus (HIV) and are currently used in a variety of applications, such as cellular therapeutics and diagnostic reagents. It has been shown that the portion consisting of the 11 basic amino acids (YGRKKRRQRRR) of the protein transduction domain (PTD) present in the TAT protein plays an important role in passing through the cell membrane (Vives et al., 1997; Futaki et al., 2001, Suzuki et al., 2002, Hakansson et al., 2001, Tyagi et al., 2001, Rusnati et al., 1997). In addition, TAT peptides (YGRKKRRQRRR) have high affinity for amino acids with positive charge. Therefore, the permeation efficiency was compared and analyzed by varying the PTD amino acid sequence by various methods. As a result, TAT peptide (RRRRRRRRR) (Wender et al., 2000). In particular, it is possible to introduce full-length proteins into cells without affecting the size or function by preparing a TAT fusion protein by linking a vector using 11 amino acids that perform protein permeation among other TATs to another peptide or protein "He said. Therefore, it is considered that the application field of CPP is almost infinite due to the fact that there is almost no toxicity in transporting a biologically active substance into a desired cell, and the efficiency of transporting a carrier material to various organelles in a cell is very high.

Korean Patent No. 10-0935030 entitled " New cell permeable peptide and method for delivering biologically active substance using the same " discloses that a macromolecular substance can be transported to cells or intracellular organs without damage to cells and substances, Discloses a novel hydrophobic cell permeable peptide PLAC that can be usefully used in delivery systems and drug therapy technologies. Korean Patent No. 10-0811745 " A novel antimicrobial peptide Pep 1 designed from a cell permeable peptide Pep 1 K and its uses "not only show strong antibacterial activity in strains resistant to various strains and antibiotics, but also exhibit no hemolytic activity in human red blood cells, so that they can be used as non-toxic antibacterial agents, food preservatives and cosmetic additives, De Pep- a 1-K are provided. Also, Korean Patent No. 10-0951719 entitled " Composite of Cellular Permeable Peptide and Fluorescently labeled Magnetic Nanoparticle and Its Use " includes a method of chemically bonding an intracellular permeable peptide to a fluorescently labeled magnetic nanoparticle, There is disclosed a cell permeable peptide-fluorescently labeled magnetic nanoparticle complex capable of stably introducing labeled magnetic nanoparticles into a cell directly, and a composition for cell imaging containing the same. In addition, many CPPs have been reported and commercialized.

However, basically, many CPPs contain a high proportion of positively charged amino acids such as lysine (K) and arginine (R), and they are easily degraded in vivo by peptide degrading enzymes, The lowering disadvantages are shown. In the case of TAT, the half-life in the body was 3.5 minutes (Bioconjug Chem. 2009 August 19; 20 (8): 1531-1537). This short half-life limits the use in the pharmaceutical field to a very limited extent, and a method of modifying the sequence of a peptide to overcome it (WO 2008089491 A2), a method of linking and stabilizing a large protein (WO 2003078470 A1, Bioconjug Chem. 2009 August 19 ; 20 (8): 1531-1537) have been studied, and since stability in the body is one of the methods for increasing the bioavailability, it is urgently required in the field of peptide pharmaceuticals to produce peptide derivatives with increased stability in vivo.

In order to solve the above problems, the present invention provides a novel peptide having high cell permeability and stable in the body and a derivative thereof, and to provide a method of delivering a biologically active substance in vivo using the peptide.

As described above, the present invention provides peptides and derivatives which are stable in the body with high cell permeability.

For this purpose, the inventors of the present invention have conducted extensive studies to overcome the disadvantage that peptides are easily decomposed by enzymes in vivo, and found that amino acids having a positive charge such as lysine (K) and arginine (R) And then permeated into the cells through various methods. It was confirmed that the peptides exhibited excellent effects in the stability test using human plasma, and the peptides were found to have a biological activity And fused to exhibit more excellent activity.

Also, as an embodiment of the present invention, there is provided a cell permeable peptide or a derivative thereof comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1 to 12.

In another aspect of the present invention, there is provided a fusion substance in which a substance having biological or pharmacological activity is fused to a cell permeable peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 1 to 12.

In another aspect of the present invention, there is provided a composition for intracellular delivery of a biologically active substance containing the fusion substance as an active ingredient.

Further, as another embodiment of the present invention, there is provided a gene therapy composition comprising the fusion substance as an active ingredient.

The present invention relates to a method for delivering a target substance having biological activity, comprising the steps of: preparing a delivery complex by binding a stable peptide in vivo with a target substance having biological activity while having high cell permeability; And injecting the delivery complex into the organism or cells of the mammal.

The present invention also relates to a gene therapy method comprising the steps of: preparing a delivery complex by binding a stable peptide in vivo to a target gene with high cell permeability; And injecting said delivery complex into the cells of a mammal.

Since the fusions in which the cell permeable peptide according to the present invention is fused with the biologically active substance can permeate and transport the cell membrane while maintaining its function without damaging the biologically active substance, And can be usefully used in therapeutic techniques.

FIG. 1 is a view showing a cell permeation of a cell permeable peptide derivative through a confocal microscope.
FIG. 2 shows the results obtained by flow cytometry of the cell permeability of the cell permeable peptide derivative.
3 is a graph showing the stability in human plasma of a cell permeation peptide derivative.
FIG. 4 is a graph showing a toxicity analysis result using a fusion substance in which a cell permeation peptide derivative and a substance having biological activity are fused.
FIG. 5 is a graph showing cell death results using a fusion substance in which a cell permeation peptide derivative and a substance having biological activity are fused.

Hereinafter, the present invention will be described in detail.

The present invention provides a cell permeable peptide or a derivative thereof which is stable in the body with high cell permeability.

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]

In the present specification, 'peptide' refers to a polymer composed of 10 or less, preferably 3 to 10 amino acid residues connected by an amide bond (or a peptide bond). In the present invention, . 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.

As an embodiment of the present invention, the novel peptides according to the present invention may be selected from the group consisting of SEQ ID NOs: 1 to 12, and more preferably, those represented by SEQ ID NO: 12. SEQ ID NOS: 1 to 12 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.

Preferred derivatives include FITC or an amino acid fragment in which the acetyl group is substituted at the C-terminus or the N-terminus. For example, the derivative shown in Example 2 can be mentioned.

Also, as one embodiment of the present invention, there is provided a fusion substance in which a peptide or a derivative thereof is fused with a substance having biological or pharmacological activity.

In the present specification, a substance having biological or pharmacological activity, which can bind to a cell-penetrating peptide and become a fused substance, means a substance capable of controlling all physiological phenomena in living body, and includes DNA, RNA, protein, , 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. The fusion of the peptide or derivative with a substance having biological activity may be administered intravenously, intraperitoneally, intramuscularly, subcutaneous, intradermal, intranasal, mucosal, inhalation, and oral routes. 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 MPRFMDYWEGLN or MTX.

In another embodiment of the present invention, there is provided a composition for intracellular delivery using the fusion substance and a therapeutic composition.

The pharmaceutical compositions of the present invention can be administered to mammals, including humans, in a variety of routes. The mode of administration may be any conventionally used route and may be administered, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dermal or intracerebral injection. The pharmaceutical composition of the present invention can be administered orally or parenterally in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, transdermal preparations, suppositories and sterilized injection solutions Can be used.

The pharmaceutical compositions of the present invention may be administered to humans alone, but may be administered in admixture with a pharmaceutical carrier selected generally in consideration of the mode of administration and standard phamaceutical practice. For example, the pharmaceutical composition of the present invention may be in the form of tablets containing starch or lactose, in the form of capsules containing the active ingredient alone or as an excipient, or as an elixir or a suspending agent containing a flavoring or coloring agent Orally, orally or sublingually. Such liquid preparations may contain suspending agents (e. G., A mixture of a semisynthetic glyceride such as methylcellulose, witepsol or apricot kernel oil and a PEG-6 ester or a mixture of PEG-8 and caprylic / A glyceride mixture such as a mixture of glycerides, such as a mixture of glycerides).

The present invention relates to a method for delivering a target substance having biological or pharmacological activity, comprising the steps of: (1) preparing a delivery complex by binding a stable peptide in vivo with a target substance having biological or pharmaceutical activity while having high cell permeability; And (2) injecting the delivery complex into the organism or cells of the mammal.

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 . In vivo or intracellular infusion of a delivery complex of a substance having biological or pharmacological activity with the peptide or derivative may be administered intravenously, intraperitoneally, intramuscularly, subcutaneous, intradermal Or by injecting into a pathway such as a nasal cavity, a nasal cavity, a mucosal, an inhalation or an oral cavity. The delivery method of the present invention can be sufficiently extended not only to cultured cells but also to general in vivo delivery, that is, delivery to animal cells, animal tissues and animals.

The cytotoxicity of the peptides of the present invention prepared by the method of Example 1 was confirmed in order to confirm whether or not the peptides of the present invention can be used harmlessly in vivo polymer delivery. As a result, it was confirmed that the fusants showed no cytotoxicity even at a concentration of 100 μM as shown in FIG. 4

The present invention also relates to a gene therapy method, which comprises: (1) preparing a delivery complex by binding a stable peptide in vivo to a target gene with high cell permeability; And (2) injecting the delivery complex into a cell of a mammal.

The binding of step 1 can be by chemical or physical covalent linkage of the gene with the peptide or derivative and by direct linkage by noncovalent linkage. The in vivo or intracellular injection of the peptide or derivative delivery complex with the substance having biological or pharmaceutical activity can be administered by the same route as described above. The delivery method of the present invention can be sufficiently extended not only to cultured cells but also to general in vivo delivery, that is, delivery to animal cells, animal tissues and animals.

In addition, the gene delivery complex is non-immunogenic, noninfective, and is not limited by the size of the plasmid since the DNA is not packaged in vector organisms such as retroviruses or adenoviruses. And thus can be used in recombinant gene expression constructs of any practical size.

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 1] Synthesis and separation of cell-permeable peptide derivatives

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 cell permeability, 6-aminohexanoic acid and fluorescein isothiocyanate (FITC) were bound to the N-terminal portion of the peptide as a linker, To confirm stability and cytotoxicity, acetylation was performed at the N-terminal portion using acetic anhydride and diisopropylethylamine (DIEA).

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 weight of the purified peptide derivative was confirmed using LC / MS (Agilient HP 1100 series), and the pure purified fractions were lyophilized to obtain 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.

Derivative 1: FITC-linker-QIIYRITNSH- NH 2: Rt = 7.63 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) 1745.23 m / e, [M + H] < ⁺ > = 1745.44

Derivative 2: FITC-linker-QQIYRITASH-NH ₂ : Rt = 6.56 min (from v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / 0.0 > minutes) < / RTI > MS (ESI) 1017.20 m / e, [M + H] < ⁺ > = 1018.7

Derivative 3: FITC-linker-QAIYRITVSH-NH ₂ : Rt = 5.67 min. 30% (v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / 0.0 > minutes) < / RTI > MS (ESI) 1688.21 m / e, [M + H] < ⁺ > = 1689.3

Derivative 4: FITC-linker-QVIYRITASH-NH ₂ : Rt = 5.77 min (from v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / 0.0 > minutes) < / RTI > MS (ESI) 1688.21 m / e, [M + H] < ⁺ > = 1689.4

Derivative 5: FITC-linker-QAIYRITASH-NH ₂ : Rt = 6.55 min (from v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / 0.0 > minutes) < / RTI > MS (ESI) 1660.18 m / e, [M + H] < ⁺ > = 1661.6

Derivative 6: FITC-linker-QVIYRITVSH- NH 2: Rt = 5.78 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) 1716.24 m / e, [M + H] < ⁺ > = 1717.7

Derivative 7: FITC-linker-VAIYRITASH-NH ₂ : Rt = 7.01 min (from v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / 0.0 > minutes) < / RTI > MS (ESI) 1631.19 m / e, [M + H] < ⁺ > = 1632.6

Derivative 8: FITC-linker-EAIYRITASH-NH ₂ : Rt = 6.70 min. 30% (v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / 0.0 > minutes) < / RTI > MS (ESI) 1661.16 m / e, [M + H] < ⁺ > = 1662.4

Derivative 9: FITC-linker-QAIYTITASH-NH ₂ : Rt = 5.76 min. 30% (v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / 0.0 > minutes) < / RTI > MS (ESI) 1605.13 m / e, [M + H] < ⁺ > = 1606.6

Derivative 10: FITC-linker-QAIYSITASH- NH 2: Rt = 5.73 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) 1591.11 m / e, [M + H] < ⁺ > = 1592.5

Derivative 11: FITC-linker-QAIYYITASH- NH 2: Rt = 7.34 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) 1667.14 m / e, [M + H] < ⁺ > = 1668.6

Derivative 12: FITC-linker-QAIRYITASH-NH ₂ : Rt = 6.59 min (from v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / 0.0 > minutes) < / RTI > MS (ESI) 1660.18 m / e, [M + H] < ⁺ > = 1661.5

30 minutes from acetonitrile / water at Rt = 7.58 bun (0.01% (v / v) 5% containing TFA (v / v) to 100% (v / v): derivative 13: Ac-QIIYRITNSH-NH ₂ Various concentration gradients throughout); MS (ESI) 1284 m / e, [M + H] < ⁺ > = 1285.5

Derivative 14: Ac-QQIYRITASH-NH ₂ : Rt = 6.28 min (from v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / Various concentration gradients throughout); MS (ESI) 1256.66 m / e, [M + H] < ⁺ > = 1257.5

Derivative 15: Ac-QAIYRITVSH-NH ₂ : Rt = 6.98 min (from v / v) to 100% (v / v) acetonitrile / water containing 0.01% Various concentration gradients throughout); MS (ESI) 1227.67 m / e, [M + H] < ⁺ > = 1228.5

Derivative 16: Ac-QVIYRITASH-NH ₂ : Rt = 7.49 min (from v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / Various concentration gradients throughout); MS (ESI) 1227.67 m / e, [M + H] < ⁺ > = 1228.5

Derivative 17: Ac-QAIYRITASH-NH ₂ : Rt = 6.64 min (from v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / Various concentration gradients throughout); MS (ESI) 1199.64 m / e, [M + H] < ⁺ > = 1200.5

Derivative 18: Ac-QVIYRITVSH-NH ₂ : Rt = 7.42 min (from v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / Various concentration gradients throughout); MS (ESI) 1255.70 m / e, [M + H] < ⁺ > = 1256.6

The reaction was carried out in the presence of 5% (v / v) to 100% (v / v) acetonitrile / water containing 30% Ac-VAIYRITASH-NH ₂ : Rt = 7.56 min Various concentration gradients throughout); MS (ESI) 1170.65 m / e, [M + H] < ⁺ > = 1171.5

Derivative 20: Ac-EAIYRITASH-NH ₂ : Rt = 7.05 min (from v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / Various concentration gradients throughout); MS (ESI) 1200.63 m / e, [M + H] < ⁺ > = 1201.5

Derivative 21: Ac-QAIYTITASH-NH ₂ : Rt = 6.88 min (from v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / Various concentration gradients throughout); MS (ESI) 1144.59 m / e, [M + H] < ⁺ > = 1145.5

Derivative 22: Ac-QAIYSITASH-NH ₂ : Rt = 6.57 min (from v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / Various concentration gradients throughout); MS (ESI) 1130.57 m / e, [M + H] < ⁺ > = 1131.4

Derivative 23: Ac-QAIYYITASH-NH ₂ : Rt = 7.82 min (from v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / Various concentration gradients throughout); MS (ESI) 1206.60 m / e, [M + H] < ⁺ > = 1207.4

Derivative 24: Ac-QAIRYITASH-NH ₂ : Rt = 6.78 min (from v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / Various concentration gradients throughout); MS (ESI) 1199.64 m / e, [M + H] < ⁺ > = 1200.5

[Example 2]

Synthesis and Separation of Derivatives of Cell Permeable Peptides and Fused Fusions with Biologically Active Substances

In this example, MPRFMDYWEGLN (International Journal of Peptide Research and Therapeutics, Vol. 12, March 2006, 3-19), which is known as a sequence that inhibits the binding of HDM2 and HDMX to p53 and stabilizes p53 to inhibit cancer cell growth, MTX (Methotrexate), which is used as a therapeutic agent for choriocarcinoma, rheumatoid arthritis, and psoriasis, was ligated to the N-terminal of the cell permeable peptide using the method of Example 1 to synthesize a fusion.

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

Fusion 1: Ac-MPRFMDYWEGLNQIIYRITNSH-NH2 : Rt = 6.72 min (from v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / Various concentration gradients); MS (ESI) 2824.36 m / e, [M + H] < ⁺ > = 2825.82

Fusion 2: MTX-QIIYRITNSH-NH2 : Rt = 7.63 min (from v / v) to 100% (v / v) acetonitrile / water containing 0.01% (v / v) Various concentration gradients); MS (ESI) 1745.23 m / e, [M + H] < ⁺ > = 1745.44

[Experimental Example 1] Confirmation of cell permeation using a confocal microscope

As an animal cell for confirming the cell permeability of the cell permeable peptide, HL-60 ( human promyelocytic leukemia cells were cultured in RPMI 1640 (Welgene) supplemented with 10% (v / v) fetal bovine serum, 300 mg / L glutamine and 1% (v / v) , Korea) media, and cultured in a 37 ° C incubator with 5% CO ₂ . Cells were cultured in 12-well plates with coverslips coated with gamma-methacryloxypropyltrimethoxysilane for 1 day, and 10 μM of fluorescent-labeled cell-permeable peptides, respectively And reacted at 37 DEG C for 10 minutes. The cell permeability was observed with a confocal laser scanning microscope (FV10i-W, Olympus Co., Tokyo, Japan). To _{TAT (47-58, GRKKRRQRRR-NH 2} ) as a positive control was as a negative control using the FITC-linker-QIIYRIAASH-NH2.

The results are shown in Fig. It was confirmed that the cell permeable peptide derivative of the present invention showed a similar transmittance as that of the positive control peptide TAT.

[Experimental Example 2] Measurement of cell permeability using flow cytometry

To quantitatively determine the degree of cell permeability of the cell permeable peptide, HL-60 ( Human promyelocytic leukemia cells (Korean Cell Line Bank) were dispensed in a 6-well plate at a rate of 1 × 10 ⁵ cells / well, and then the cells were treated with 10% (v / v) fetal bovine serum, 300 mg / L glutamine and 1% (Welgene, Korea) media supplemented with 10% fetal bovine serum. After washing each well with phosphate buffered saline (PBS) for 30 minutes, the cells were separated from the wells and washed with phosphate buffered saline (PBS) , Followed by centrifugation to remove the supernatant. The procedure was repeated twice to remove the extracellular fluorescent-labeled cell-permeable peptides. The cells were then suspended by adding 300 ml of phosphate buffer solution (PBS) and then observed at FL-1 (488 nm) using FACSCalibur (BD, USA). TAT (47-58, GRKKRRQRRR-NH ₂ ) was used as a positive control.

The results are shown in Fig. The peptide derivatives of SEQ ID NOS: 1 and 12 of the present invention showed similar transmittance to that of the control peptide TAT.

[Experimental Example 3] The stability in human plasma

The reaction solution was prepared by adding 25% (v / v) human plasma (Sigma, Korea) to RPMI 1640 (Welgene, Korea) media and then incubated at 37 ° C for 15 minutes to activate the enzymes present in the plasma. Then, 1 mg of the peptide derivative to be measured was added thereto, and the mixture was treated at 37 ° C for each hour. To complete the reaction, 20% TFA (Trifluoro acetic acid) was added. Finally, only the supernatant was separated by centrifugation at 13,000 rpm for 10 minutes to precipitate suspended solids in solution. The separated supernatants were collected in vials, and the area was determined by 3 repeated measurements using high performance liquid chromatography (Shimadzu Prominence HPLC, Japan) and reversed phase column (Vydac, Everest C18 column, USA). TAT (47-58, GRKKRRQRRR-NH ₂ ) was used as a control.

The results are shown in Fig. As shown in FIG. 3, the peptide derivative of the derivative 24 of the present invention was found to have increased stability about 1,000 times as compared with the control peptide TAT (3.5-minute half-life in the body).

[Experimental Example 4] Cytotoxicity experiment

(V / v) fetal bovine serum, 300 mg / L glutamine and 10% (v / v) fetal bovine serum were obtained by dividing 1 × 10 ⁴ cells in MCF-7 (Michigan Cancer Foundation- (Overnight starvation) in RPMI 1640 (Welgene, Korea) media supplemented with 1% (v / v) antibiotics for 20 hours. The media was exchanged in 96-well RPMI 1640 (Welgene, Korea) media supplemented with 1% (v / v) antibiotic. After that, 100 uM of each cell permeable peptide was treated and cultured at 37 ° C for 72 hours, and cell viability and toxicity were analyzed using MTT assay method.

The results are shown in Fig. As shown in FIG. 4, all the cytotoxic peptide derivatives of the present invention did not kill normal cells at a concentration of 100 uM. Therefore, it was confirmed as a safe material.

[Experimental Example 5] Cell death using a fused fusion of a cell permeable peptide derivative and a substance having biological activity

(V / v) fetal bovine serum, 300 mg / L glutamine and 10% (v / v) fetal bovine serum were obtained by dividing 1 × 10 ⁴ cells in MCF-7 (Michigan Cancer Foundation- (Overnight starvation) in RPMI 1640 (Welgene, Korea) media supplemented with 1% (v / v) antibiotics for 20 hours. The media was exchanged in 96-well RPMI 1640 (Welgene, Korea) media supplemented with 1% (v / v) antibiotic. Then, 20 uM of each cell permeable peptide was treated and incubated at 37 ° C for 72 hours, and cell viability was analyzed using MTT assay method. Ac-MPRFMDYWEGLN-NH ₂ was used as a control and Nutlin-3a (Sigma-aldrich, USA) was used as a positive control.

The results are shown in Fig. As shown in FIG. 5, the fused product in which the derivative of a cell permeable peptide of the present invention is fused with a substance having biological activity was confirmed to be a substance capable of effectively exhibiting the existing activity with high permeability of the cell permeable peptide.

<110> PEPTRON <120> NOVEL CELL PERMEABLE PEPTIDE WITH HIGH STABILITY IN PLASMA AND          USE THEREOF <130> 5488 <160> 12 <170> Kopatentin 2.0 <210> 1 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CELL PERMEABLE ARTIFICIAL PEPTIDE <400> 1 Gln Ile Ile Tyr Arg Ile Thr Asn Ser His   1 5 10 <210> 2 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CELL PERMEABLE ARTIFICIAL PEPTIDE <400> 2 Gln Gln Ile Tyr Arg Ile Thr Ala Ser His   1 5 10 <210> 3 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CELL PERMEABLE ARTIFICIAL PEPTIDE <400> 3 Gln Ala Ile Tyr Arg Ile Thr Val Ser His   1 5 10 <210> 4 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CELL PERMEABLE ARTIFICIAL PEPTIDE <400> 4 Gln Val Ile Tyr Arg Ile Thr Ala Ser His   1 5 10 <210> 5 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CELL PERMEABLE ARTIFICIAL PEPTIDE <400> 5 Gln Ala Ile Tyr Arg Ile Thr Ala Ser His   1 5 10 <210> 6 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CELL PERMEABLE ARTIFICIAL PEPTIDE <400> 6 Gln Val Ile Tyr Arg Ile Thr Val Ser His   1 5 10 <210> 7 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CELL PERMEABLE ARTIFICIAL PEPTIDE <400> 7 Val Ala Ile Tyr Arg Ile Thr Ala Ser His   1 5 10 <210> 8 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CELL PERMEABLE ARTIFICIAL PEPTIDE <400> 8 Glu Ala Ile Tyr Arg Ile Thr Ala Ser His   1 5 10 <210> 9 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CELL PERMEABLE ARTIFICIAL PEPTIDE <400> 9 Gln Ala Ile Tyr Thr Ile Thr Ala Ser His   1 5 10 <210> 10 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CELL PERMEABLE ARTIFICIAL PEPTIDE <400> 10 Gln Ala Ile Tyr Ser Ile Thr Ala Ser His   1 5 10 <210> 11 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CELL PERMEABLE ARTIFICIAL PEPTIDE <400> 11 Gln Ala Ile Tyr Tyr Ile Thr Ala Ser His   1 5 10 <210> 12 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> CELL PERMEABLE ARTIFICIAL PEPTIDE <400> 12 Gln Ala Ile Arg Tyr Ile Thr Ala Ser His   1 5 10

Claims (10)

Wherein the peptide is an amino acid sequence selected from the group consisting of SEQ ID NOS: 1 to 12, or a derivative thereof.
The cell permeable peptide or derivative thereof according to claim 1, wherein the peptide or derivative thereof is improved in plasma stability.
10. A fusion substance comprising the cell-permeable peptide of claim 1 or a derivative thereof fused with a substance having biological or pharmacological activity.
4. The fusion substance according to claim 3, wherein the substance having biological activity is at least one selected from the group consisting of DNA, RNA, protein, fat, carbohydrate and compound.
[Claim 4] The method according to claim 3, wherein the substance having the pharmacological activity is any one or more selected from the group consisting of an anti-cancer agent, an immune disease therapeutic agent, an antiviral agent, an antibiotic or an agent for growth, .
4. The fused product of claim 3, wherein the fused product further comprises a ligand that selectively binds to a specific cell, tissue or organ receptor.
A composition for intracellular delivery of a biological or pharmaceutical active substance comprising the fusion substance of claim 3 as an active ingredient.
A composition for gene therapy comprising the fused product of claim 3 as an active ingredient.
(1) preparing a delivery complex by binding the peptide of claim 1 with a target substance having biological or pharmaceutical activity; And
(2) injecting the delivery complex into the organism or cells of the mammal
Wherein the target substance has a biological or pharmaceutical activity.
(1) preparing a delivery complex by binding the peptide of claim 1 to a target gene; And
(2) injecting the above delivery complex into the cells of a mammal
&Lt; / RTI &gt;

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