Composition for preventing or treating keloid or hypertrophic scar

Abstract

The present invention relates to a pharmaceutical composition for the prevention and treatment of hypertrophic scars. The inventors have shown that the expression inhibition of thioredoxin 5, PRRC1, calcytin, galectin 1, serine A, eukaryotic initiation factor-5A, annexin A2 and fatty acid binding protein-5 can become a new target for the improvement and treatment of hypertrophic scars. In the present invention, thioredoxin 5, PRRC1, calcytin, galectin 1, serine A, eukaryotic initiation factor-5A, annexin A2 and fatty acid binding protein-5-specific siRNA are manufactured, and the therapeutic possibility of hypertrophic scars is confirmed. Therefore, the inhibition of the protein or the gene encoding it induces cell elimination and reduces the expression of collagen in hypertrophic scars, so that it can be very useful in scar treatment.

Description

Technical Field

[0001] The present invention relates to a composition for preventing or treating keloid or hypertrophic scar. Background Art

[0002] When the deep dermis is damaged due to surgery or trauma, the collagen in the dermis that maintains the tension of the skin will overproliferate, so that the thinned skin will show through after the wound heals, leaving traces of skin recovery, which is called "ordinary scars". This is the result of wound healing, or when the function of properly regulating and inhibiting the wound healing process is impaired, the fibrous tissue grows abnormally densely, thereby generating hypertrophic scars (Hypertrophic scars) or keloids (Keloid). Hypertrophic scars do not extend beyond the scope of the wound and will gradually disappear over time, but keloids have the difference that they will grow wider than the damaged area over time and invade into normal skin. In order to treat hypertrophic scars or keloids, attempts have been made including surgical treatment, radiotherapy, steroid treatment, silicone blocking dressings, and laser treatment, but they only have limited effects and there is no established method yet.

[0003] The cause of the formation of hypertrophic scars or keloids is known to be excessive activation of cell migration during the wound healing stage, or excessive formation of tissue cells and capillaries due to abnormal proliferation, or excessive accumulation of collagen due to failure to decompose normally. Registered Patent No. 10-1505294 discloses a starfish incubation extract that can be used to hydrolyze collagen to reduce scar formation, and Registered Patent No. 10-1697396 discloses a method for treating hypertrophic scars using a compound that targets connective tissue growth factor (CTGF) associated with fibrosis.

[0004] There is little information on biomarkers that are involved in the formation of collagen that accumulates above the necessary level during the healing process of scars and in cell proliferation. By selecting such biomarkers, abnormal scars such as hypertrophic scars can be accurately diagnosed, and substances that target the biomarkers can be screened to inhibit and treat the formation of hypertrophic scars or keloids. Summary of the invention

[0005] Problems to be solved by the invention

[0006] The object of the present invention is to provide a pharmaceutical composition for preventing or treating keloid or hypertrophic scars.

[0007] Another object of the present invention is to provide a method for preventing or treating keloid or hypertrophic scars.

[0008] Another object of the present invention is to provide a method for screening substances for preventing or treating keloid or hypertrophic scars.

[0009] Another object of the present invention is to provide a method for diagnosing keloid or hypertrophic scars.

[0010] Solutions for solving problems

[0011] To achieve the above purpose, the inventors compared the expression differences of multiple proteins shown in hypertrophic scar tissue and normal tissue, thereby assuming that protein markers showing abnormal expression states are the cause of hypertrophic scars, and selected proteins showing different expression states from normal tissue.

[0012] Thus, the present invention provides a composition for diagnosing keloid or hypertrophic scar, the diagnostic composition comprising a substance for measuring the expression level of one or more genes selected from the group consisting of thioredoxin 5 (TXNDC5), PRRC1, calcein (S100A11), galectin 1 (Galectin1), serine A (Filamin A), eukaryotic initiation factor-5A (eIF-5A), annexin A2 (Annexin A2) and fatty acid binding protein-5 (FABP5), or one or more proteins selected from the group consisting of thioredoxin 5, PRRC1, calcein, galectin 1, serine A, eukaryotic initiation factor-5A, annexin A2 and fatty acid binding protein-5.

[0013] Furthermore, the present invention provides a method for diagnosing keloid or hypertrophic scar, the method comprising the following steps: measuring, in keloid or hypertrophic scar tissue, the expression level of one or more genes selected from the group formed by thioredoxin 5, PRRC1, calcein, galectin 1, serine A, eukaryotic initiation factor-5A, annexin A2 and fatty acid binding protein-5, or the expression level of one or more proteins selected from the group formed by thioredoxin 5, PRRC1, calcein, galectin 1, serine A, eukaryotic initiation factor-5A, annexin A2 and fatty acid binding protein-5; and comparing the measurement result with the expression level of the gene and protein in normal tissue.

[0014] The measurement of the gene or protein expression amount can be implemented by various methods known in the art. For example, it can be implemented using RT-PCR (Sambrook et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)), Northern blotting (Peter B. Kaufma et al., Molecular and Cellular Methods in Biology and Medicine, 102-108, CRC press), hybridization reaction using cDNA microarray (Sambrook et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)), immunoblotting or in situ hybridization reaction (Sambrook et al., Molecular Cloning. A Laboratory Manual, 3rd ed. Cold Spring Harbor Press (2001)).

[0015] In addition, the inventors confirmed the expression of the selected proteins in keloids or hypertrophic scars and normal tissues by immunoblotting and immunohistochemical staining. When the respective genes encoding the proteins were knocked down in keloids or hypertrophic scar tissues, it was confirmed that the expression of type I collagen, α-SMA and PCNA proteins was reduced. These results support that the inhibition of the proteins or the reduction of the genes encoding them is very useful for the treatment of keloids or hypertrophic scars.

[0016] Thus, the present invention provides a pharmaceutical composition for preventing or treating keloid or hypertrophic scars, the pharmaceutical composition comprising as an active ingredient a substance that inhibits the expression of one or more genes selected from the group consisting of thioredoxin 5, PRRC1, calcein, galectin 1, serine A, eukaryotic initiation factor-5A, annexin A2 and fatty acid binding protein-5, or inhibits the activity of one or more proteins selected from the group consisting of thioredoxin 5, PRRC1, calcein, galectin 1, serine A, eukaryotic initiation factor-5A, annexin A2 and fatty acid binding protein-5.

[0017] In addition, the present invention provides a method for treating an individual having a keloid disease or a hypertrophic scar, the method comprising administering a medical composition comprising, as an active ingredient, a substance that inhibits the expression of one or more genes selected from the group consisting of thioredoxin 5, PRRC1, calcein, galectin 1, serine A, eukaryotic initiation factor-5A, annexin A2, and fatty acid binding protein-5, or inhibits the activity of one or more proteins selected from the group consisting of thioredoxin 5, PRRC1, calcein, galectin 1, serine A, eukaryotic initiation factor-5A, annexin A2, and fatty acid binding protein-5 to an individual having a hypertrophic scar or a keloid wound.

[0018] In the present invention, the substance that inhibits the expression of one or more genes selected from the group formed by thioredoxin 5, PRRC1, calcein, galectin 1, serine A, eukaryotic initiation factor-5A, annexin A2 and fatty acid binding protein-5 may include shRNA (short hairpin RNA), siRNA (small interference RNA), miRNA (microRNA), Crispr-cas9, Crispr-cpf1, ribozyme, DNAzyme, PNA (peptide nucleic acids), antisense oligonucleotide, and the substance that inhibits the activity of the protein expressed by the gene may include antibodies, aptamers, natural extracts or chemical substances, and any substance that inhibits the expression of the gene and the activity of the protein may be included without limitation.

[0019] The term "shRNA (short hairpin RNA)" used in this specification refers to a single-strand RNA with a length of 45 to 70 nucleotides. An oligonucleotide DNA with a 3-10 base linker peptide is synthesized between the sense and complementary nonsense sequences of the target gene siRNA base sequence and then cloned into a plasmid vector, or the shRNA is inserted into a retrovirus such as a lentivirus or adenovirus and expressed, thereby producing an shRNA with a circular hairpin structure, which is converted into siRNA by Dicer in the cell to exhibit an RNAi effect.

[0020] The term "siRNA" in the present invention means a nucleic acid molecule that interferes with RNA or can mediate gene silencing (see WO00 / 44895, WO01 / 36646, WO99 / 32619, WO01 / 29058, WO99 / 07409 and WO00 / 44914). siRNA can inhibit the expression of target genes and is therefore provided as an effective gene reduction method or gene therapy method. siRNA was first discovered in plants, worms, fruit flies and parasites, and recently developed / utilized siRNA for use in mammalian cell research (Degot S, et al. 2002; Degot S, et al. 2004; Ballut L, et al. 2005).

[0021] The siRNA molecule of the present invention may have a double-stranded structure formed by sense lines (corresponding sequences in the mRNA sequences of each gene) and antisense lines (sequences complementary to the mRNA sequences of each gene) located on opposite sides of each other. In addition, the siRNA molecule of the present invention may have a single-stranded structure with self-complementary sense and antisense lines. siRNA is not limited to the complete pairing of the double-stranded RNA portion formed by pairing only between RNAs, and may include portions that are not paired due to mispairing (corresponding bases are not complementary), protrusions (no bases corresponding to one side of the chain), etc. The entire length is 10 to 100 bases, preferably 15 to 8 bases, more preferably 20 to 70 bases, and most preferably 20-30 bases.

[0022] The term "miRNA (microRNA)" used in the present invention is a single-line RNA molecule of 21-25 nucleotides, which is a regulatory substance that controls the gene expression of eukaryotic organisms by inhibiting the destruction or detoxification step of the target mRNA. These miRNAs are formed by a two-step process. The miRNA primary transcript (primary miRNA) is made into a stem-loop structure of 70-90 bases, i.e., premiRNA, by the RNase III type enzyme of Drosha in the nucleus, and then moves to the cytoplasm and is cut by the Dicer enzyme to make a mature miRNA of 21-25 bases. The miRNA formed in this way complementarily binds to the mRNA and acts as a post-transcriptional gene suppressor, and induces translation inhibition and mRNA instability. miRNA is involved in a variety of physiological phenomena and diseases.

[0023] The term "antisense oligonucleotide" used in this specification means a DNA or RNA containing a complementary nucleic acid sequence in a specific mRNA sequence, or these derivatives, which bind to the complementary sequence in the mRNA and act to hinder the translation of the mRNA as a protein. The antisense sequence of the present invention means a DNA or RNA sequence that is complementary to each gene and can be bound to the mRNA of each gene, which can hinder the translation of the mRNA of the gene, the translocation (translocation) in the cytoplasm, the maturation (maturation) or other necessary activities of all other overall biological functions. The length of the antisense nucleic acid can be 6 to 100 bases, preferably 8 to 60 bases, and more preferably 10 to 40 bases.

[0024] According to a specific example of the present invention, the substance can be siRNA. More specifically, the siRNA that reduces thioredoxin-5 can be composed of sense 5'-GGCCCUAACUAGAGUUCUAtt-3' (SEQ ID NO.1) and antisense 5'-UAGAACUCUAGUUAGGGCCtt-3' (SEQ ID NO.2); the two siRNAs that reduce PRRC1 are composed of sense 5'-CAAGAAGACCCUAGAAUUAtt-3' (SEQ ID NO.3) and antisense 5'-UAAUUCUAGGGUCUUCUUGtt-3' (SEQ ID NO.4) and sense 5'-UAUCAAAUCUGGUGAAtt-3' (SEQ ID NO.5) and antisense siRNAUUCACCUCCAGAUUUGAUAtt-3' (SEQ ID NO.6); and the siRNA that reduces calcein is composed of senseGAACUAGCUGCCACAAtt-3' (SEQ ID NO.7) and the antisense base sequence 5'-UUGUGAAGGCAGCUAGUUCtt-3' (SEQ ID NO.8).

[0025] The term "treatment" in this specification means (i) prevention of keloid or hypertrophic scar; (ii) inhibition or improvement of the formation of keloid or hypertrophic scar; and (iii) alleviation of related diseases or diseases according to inhibition or improvement of the formation of keloid or hypertrophic scar. The term "therapeutically effective amount" in this specification means a sufficient amount to achieve the pharmacological effect.

[0026] According to another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating keloid disease or hypertrophic scar, the pharmaceutical composition comprising (a) as an active ingredient, a therapeutically effective amount of a substance that inhibits the expression of one or more genes selected from the group consisting of thioredoxin 5, PRRC1, calcein, galectin 1, serine A, eukaryotic initiation factor-5A, annexin A2 and fatty acid binding protein-5, or inhibits the activity of one or more proteins selected from the group consisting of thioredoxin 5, PRRC1, calcein, galectin 1, serine A, eukaryotic initiation factor-5A, annexin A2 and fatty acid binding protein-5; and (b) pharmaceutically acceptable carriers.

[0027] The pharmaceutically acceptable carriers included in the composition of the present invention are generally used in preparations, including: lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinyl pyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, etc., but are not limited thereto. In addition to the above ingredients, the pharmaceutical composition of the present invention may also include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

[0028] The pharmaceutical composition of the present invention is preferably administered parenterally, for example, by intravenous administration, intraperitoneal administration, intratumoral administration, intramuscular administration, subcutaneous administration, portal vein administration, hepatic artery administration or local perfusion administration.

[0029] The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, patient's age, weight, sex, degree of disease symptoms, diet, administration time, administration route, excretion rate and reaction sensitivity, and a generally skilled physician can easily determine and prescribe an effective dosage for the target treatment.

[0030] According to the method that is convenient for those skilled in the art to implement, the pharmaceutical composition of the present invention is formulated using pharmaceutically acceptable carriers and / or excipients, so as to be manufactured in the form of unit volume or loaded in a multi-volume container. In this case, the dosage form can be a solution, suspension or emulsion in oil or aqueous medium or an extract, powder, granule, tablet or capsule form, and may also include a dispersant or stabilizer.

[0031] The pharmaceutical composition of the present invention can be used for improving or treating keloid or hypertrophic scar with skin topical preparation. In this case, according to the body part, its dosage form is not particularly limited. Specifically, for example, it can be a cosmetic composition with the dosage form of a softening lotion, a nourishing lotion, a massage cream, a nourishing cream, a facial mask, a gel or a skin adhesive type cosmetic, and can be a transdermal dosage form such as an emulsion, an ointment, a gel, a cream, a patch or a spray. In addition, in the topical preparation composition of each dosage form, in addition to the above-mentioned pharmaceutical composition of the present invention, other ingredients, according to the dosage form or the purpose of use of other skin topical preparations, can be conveniently selected and combined by those skilled in the art, and when used simultaneously with other raw materials at this time, an ascending effect can be produced.

[0032] In addition, the present invention may provide a method for screening a preventive or therapeutic substance for keloid disease or hypertrophic scar, comprising the following steps: (a) treating keloid or hypertrophic scar cells with a test substance; and (b) analyzing the activity of one or more proteins selected from the group consisting of thioredoxin 5, PRRC1, calcein, galectin 1, serinamide A, eukaryotic initiation factor-5A, annexin A2, and fatty acid binding protein-5, or the expression level of a gene encoding the protein, from tissues or cells treated with the test substance, and when the activity of the protein or the expression state of the gene encoding the protein in normal tissue is different, the test substance may be judged to be a preventive or therapeutic substance for keloid or hypertrophic scar.

[0033] According to the method of the present invention, the keloid or hypertrophic scar tissue is first contacted with the test substance to be analyzed. The term "test substance" used in the screening method of the present invention means an unknown substance used for screening in order to check the expression amount of the gene or the amount of the protein or whether it affects the activity. The test substance includes chemical substances, nucleotides, antisense RNA, shRNA, miRNA, siRNA (small interference RNA) and natural extracts, but is not limited thereto.

[0034] Next, the expression levels of the genes and proteins in the cells are analyzed from the tissues or cells treated with the test substance. When the measurement result shows that the expression of the genes in the cells is reduced or the activity or expression of the proteins is reduced, the test substance can be determined as a preventive or therapeutic substance for keloid or hypertrophic scar.

[0035] Effects of the Invention

[0036] The present invention clarifies proteins involved in abnormal collagen formation at wound sites that may be the cause of keloids or hypertrophic scars, and can accurately diagnose hypertrophic scars using the corresponding proteins or genes encoding them. In addition, a substance that inhibits the expression and activity of the protein or gene is provided, which can be effectively used in the improvement or treatment of keloids or hypertrophic scars. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The figure shows the result of immunohistochemical staining for the complementary protein of human hypertrophic scar tissue.

[0038] Figure 2 The results show the results of confirming the protein knockdown efficiency when siRNAs targeting the three candidate proteins were processed by immunoblotting.

[0039] Figure 3 The results show that the expression of collagen was confirmed by immunoblotting in hypertrophic scar tissue in which the expression of post-protein was suppressed. DETAILED DESCRIPTION

[0040] The present invention is described in more detail below by way of examples. These examples are only intended to more specifically describe the present invention, and those skilled in the art should understand that the scope of the present invention is not limited to these examples according to the key points of the present invention.

[0041] Example

[0042] Preparation of human skin fibroblasts, normal tissue, and hypertrophic scar tissue

[0043] Skin tissues used for the experiment were obtained from 3 patients, and IHC was performed using normal and scar tissues of both sexes, and the effects of siRNA transformation were observed using primary fibroblasts derived from each tissue.

[0044] After washing the tissue with 70% ethanol, the tissue for IHC was isolated by trimming to remove fat and chopping. After further trimming of the remaining tissue, it was minced and placed in a solution mixed with collagenase, trypsin, and EDTA, and the cells were isolated at 37°C and 100 rpm. The isolated cells were cultured in F12 medium supplemented with 10% fetal bovine serum (FBS) and gentamycin.

[0045] Comparison of protein expression in hypertrophic scar tissue by immunohistochemistry (IHC)

[0046] The tissue obtained from biopsy was placed in OCT compound (Cell Poth, KMA-0100-00A) and frozen on dry ice. The frozen tissue was fixed with a 1:1 buffer of acetone and methanol. After removing the buffer, 0.5% Triton X-100 was treated for 10 minutes at room temperature and permeabilized, and then the activity of peptidase was inhibited using Ultravision hydrogen peroxide (Thermo kit / Ultravision LPdetection system). After reacting with Ultravision block buffer at room temperature for 10 minutes, the corresponding primary antibody was treated and treated at 4°C overnight. The primary antibodies used were thioredoxin 5 (Abcam, Ab155684), PRRC1 (Abcam / ab12544), calcein (Abcam / ab97329), galectin 1 (Abcam, Ab108389), serine A (Millipore / MAB1680), eIF5-A (Abcam / ab32014), annexin A2 (Cell singnaling / 8235), and fatty acid binding protein-5 (Abcam / ab37267). After treating the primary antibody enhancer at room temperature for 10 minutes, the enzyme-labeled secondary antibody (HRP polymer) was reacted at room temperature for 15 minutes in the light-blocking state. After removing the HRP polymer, all tissues except PRRC1 were treated with AEC (Spring, ASS-125), and PRRC1 was treated with DAB (Thermo, TA-125-HDX) for 1 minute. The tissue was stained with Mayer's shematoxylin at room temperature and dehydrated, and then mounted with Canada balsam mixed xylene and observed under a microscope.

[0047] IHC results showed that thioredoxin 5, PRRC1, calcein, galectin 1, serine A, eukaryotic initiation factor-5A, annexin A2, and fatty acid binding protein-5 showed increased expression in hypertrophic scar tissue compared to normal tissue. The increased expression showed differences depending on the location of the dermis and epidermis, and the expression difference results of the supplementary proteins are shown in Tables 1 and Figure 1 Displayed.

[0048]

Table 1

[0049] Comparison of protein expression and location in normal and scar human fibroblasts

[0050] Candidate Expression difference results Thioredoxin 5 Scar epidermis, increased expression in dermis PRRC1 Scar epidermis, increased expression in dermis Calcein Scar epidermis, increased expression in dermis Galectin-1 Increased expression in scar dermis Serinamide A Scar epidermis, increased expression in dermis Eukaryotic initiation factor-5A Scar epidermis, increased expression in dermis Annexin A2 Scar epidermis, increased expression in dermis Fatty acid binding protein-5 Increased expression in scar epidermis

[0051] Changes in the expression of collagen type I, a-SMA and PCNA in fibroblasts of hypertrophic scar tissue after the transformation of protein-specific siRNA

[0052] Among the proteins selected by the ICH results, three (thioredoxin 5, PRRC1, and calcein) were knocked down using siRNA, and then the expression of collagen and fibroblast proliferation-related proteins overexpressed in scars were analyzed.

[0053] Specifically, the siRNA used to reduce thioredoxin-5 can be composed of sense 5'-GGCCCUAACUAGAGUUCUAtt-3' (SEQ ID NO.1) and antisense 5'-UAGAACUCUAGUUAGGGCCtt-3' (SEQ ID NO.2); the two siRNAs used to reduce PRRC1 are composed of sense 5'-CAAGAAGACCCUAGAAUUAtt-3' (SEQ ID NO.3) and antisense 5'-UAAUUCUAGGGUCUUCUUGtt-3' (SEQ ID NO.4) and sense 5'-UAUCAAAUCUGGUGAAtt-3' (SEQ ID NO.5) and antisense siRNA UUCACCUCCAGAUUUGAUAtt-3' (SEQ ID NO.6); and the siRNA used to reduce calcein is composed of sense GAACUAGCUGCCACAAtt-3' (SEQ ID NO.7), antisense 5'-UUGUGAAGGCAGCUAGUUCtt-3' (SEQ ID NO.8).

[0054] Specifically, fibroblasts isolated from hypertrophic scar tissue were cultured in F12 medium supplemented with 10% FBS, and then transfected with the siRNA that reduces the expression of each protein. 48 hours after siRNA transfection, the lysate was extracted and immunoblot was performed to observe the expression changes of collagen and the like. As a result, it was confirmed that the target protein transfected by siRNA was reduced ( Figure 2), the effect of the reduction of the target protein on the function and growth of fibroblasts was confirmed by changes in the expression of collagen-1, α-SMA and PCNA proteins. The results of the observation showed that when the expression of all the three genes was blocked, the expression of collagen-1, α-SMA and PCNA proteins was inhibited ( Figure 3 ).

[0055] In conclusion, inhibition of each of the proteins effectively inhibited the synthesis of collagen overproduced in hypertrophic scars and the proliferation of fibroblasts and induced cell elimination, and therefore, it was confirmed that the supplementary proteins could be targets for improving or treating hypertrophic scars.

[0056] The above describes in detail the specific parts of the present invention, and those skilled in the art should understand that these specific technologies are only preferred embodiments and cannot limit the scope of the present invention. Therefore, the essential scope of the present invention can be defined by the claims and their equivalents.

Claims

1. A pharmaceutical composition for treating keloid or hypertrophic scar, wherein: The pharmaceutical composition includes siRNA that inhibits the expression of a gene encoding thioredoxin 5 as an active ingredient, and the siRNA is formed by SEQ ID NO.1 and SEQ ID NO.

2.

2. A method for screening a substance for treating keloid or hypertrophic scar, wherein: The screening method is as follows: (a) a step of treating tissue obtained from a patient with keloid or hypertrophic scar with a test substance; as well as (b) analyzing the expression level of thioredoxin 5 in the tissue treated with the test substance, When the expression level of the protein is lower than that of the tissue before treatment with the test substance, the test substance is judged to be a therapeutic substance for keloid or hypertrophic scar.

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