KR102026138B1 - Composition for Inhibition or Treatment of Keloids and Hypertrophic Scar Comprising CRIF1 Antagonist - Google Patents

Abstract

The present invention relates to compositions that are effective in inhibiting or treating the formation of keloids and hypertrophic scars by inhibiting proliferation and neovascularization, cell migration and ECM production. More particularly, CRIF1 (CR6 interacting) Factor 1) relates to a pharmaceutical composition for inhibiting or treating keloid and hypertrophic scar formation comprising an inhibitor of gene expression or an inhibitor of activity of CRIF1 protein as an active ingredient.

Description

Composition for Inhibition or Treatment of Keloids and Hypertrophic Scar Comprising CRIF1 Antagonist

The present invention relates to compositions that are effective in inhibiting the formation of keloids and thickening scars or treating keloids and thickening scars later in the wound healing process by inhibiting proliferation of keloid cells, migration of cells and production of ECM.

A wound is a condition in which external or tissue continuity is destroyed by external pressure, or a defect occurs in some areas. Injuries can cause symptoms of pain, bleeding, dysfunction, and inflammation, and the living body responds to it quickly to restore its original condition. Wound healing can be defined as 'remaining the continuity that cells have lost due to regeneration, differentiation and proliferation'. The wound healing process is known to go through inflammatory, proliferative, and reconstructed stages.

Inflammation is a blood clot at the wound site where hemostasis occurs, while immune cells, including leukocytes and neutrophils, move to the site to remove harmful substances and damaged tissues from outside by an immune response. In the proliferation stage, vascular endothelial cells move to the wound site and new blood vessels are formed into the damaged tissue. Tissue-forming proteins such as collagen and elastin are generated, and fibroblasts move to the wound site to form granulation tissue. In addition, as the skin and tissue around the wound shrink and epithelial cells proliferate from the periphery of the wound, the size of the wound decreases. The reconstruction step is the final stage of the wound healing process in which the proliferated epithelial cells mature, and the adhesion and elasticity between newly synthesized tissue and the proliferated cells are increased by the formation of fibrous tissue. It takes up to three weeks for the new epithelial cells to proliferate and no exudates after the onset of the wound, but it takes months to years to complete the reconstruction of the wound. Poor form or shape leads to poor results.

If the wound is damaged to the deep layer of the dermis due to surgery or trauma, the collagen in the dermal layer, which maintains the tension of the skin, excessively proliferates, pushing the thinned skin even after the wound is healed, and leaving a trace of healing of the skin. It is called "normal scar". This may be the result of wound healing or abnormal growth of fibrous tissues resulting in hypertrophic scars or keloids in the event of impairment in the ability to properly control and inhibit the wound healing process. Hypertrophic scars do not extend beyond the wound and tend to disappear over time, but keloids grow wider than damaged areas over time and invade normal skin. Many attempts have been made to treat hypertrophic scars or keloids, including surgical therapies, radiation therapy, steroid therapy, closure dressing with silicone gel, and laser therapy.

The formation of hypertrophic scars or keloids is due to excessive accumulation of collagen due to over-activation of cell migration or abnormal proliferation of tissue cells and capillaries due to abnormal growth of cells and capillaries. Patent No. 10-1505294 discloses a hatching enzyme isolated from starfish hatching extract which can be used to reduce keloid or scar formation due to the hydrolytic effect on collagen, and Patent No. 10-1697396 discloses binding associated with fibrosis. A method for treating keloids or thickening scars using antisense compounds targeting tissue growth factor (CTGF) has been reported.

CRIF1 (CR6 interacting factor 1) is a protein present in mitochondria, and the lack of CRIF1 is known to induce mitochondrial dysfunction and stimulate ROS production. Korean Patent Publication No. 10-2016-0022628 reports that CRIF1 deficiency causes endothelial dysfunction and vascular inflammation, which can be effectively used for diagnosis and treatment of vascular diseases related to endothelial dysfunction and vascular inflammation. Korean Patent Publication No. 10-2009-0103763 reports that CRIF1 protein has a therapeutic effect on cancer by controlling abnormal replication of DNA through the regulation of geminin through cc-domains present at the C-terminus.

However, it is not known at all that CRIF1 affects cell migration or factors associated with the formation of keloids or thickening scars.

Patent Registration No. 10-1505294 Patent Registration No. 10-1697396

The present invention is to suppress the formation of keloids and thickening scars effective in the prevention and treatment of keloids or hypertrophic scars causing a wide range of involvement, such as causing cosmetic problems or obstructing the movement of joints when occurring in important areas such as joints or It is an object to provide a therapeutic composition.

The present invention for achieving the above object relates to a pharmaceutical composition for inhibiting or treating keloid and thickening scar formation comprising an inhibitor of the expression of the CR6 interacting factor 1 (CRIF1) gene or an active inhibitor of the CRIF1 protein as an active ingredient.

Cell proliferation, angiogenesis, and cell migration play an important role in wound healing, but depending on the stage of healing, they may play a positive role in promoting wound healing or negatively, leaving scars such as keloids and thickening scars. In other words, in the inflammatory and proliferative stages of wound healing, wound healing is accelerated as cell proliferation, angiogenesis, and cell migration become more efficient as the mechanism of removing damaged tissue and forming new tissue is activated. However, in the reconstruction stage, the final stage of wound healing, if cell proliferation, angiogenesis, and cell migration are overactivated, they cause keloids or thickening scars, and therefore, it is necessary to suppress them.

By adjusting the expression level of CRIF1 in cells by CRIF1 knockdown using siRNA, it was confirmed that Akt phosphorylation, which is known to affect vascular proliferation and fibroblast proliferation of conventional vascular endothelial cells, was inhibited according to the degree of inhibition of CRIF1 expression. there was. In addition, it was confirmed that CRIF1 regulates the expression of Rho GDI2, which is known to modulate conventional cell migration according to the regulation of expression level, and can inhibit cell mobility by inhibiting the expression or activity of CRIF1 even in wound recovery evaluation. Showed. This means that either the expression inhibitor of CRIF1 or the inhibitor of activity of CRIF1 can be used to form keloids and thickening scars by the effect of inhibiting angiogenesis, fibroblast proliferation, and cell migration in the reconstruction phase during wound healing.

CRIF1 deficiency in experiments with keloid fibroblasts effectively prevents keloid proliferation, migration to normal tissue, and excessive ECM production, which are the major causes of keloid and hypertrophic scar formation, preventing and treating keloid and hypertrophic scars. It was confirmed that it can be used effectively.

The expression inhibitor of the CRIF1 gene may be an antisense oligonucleotide or siRNA that specifically binds to the CRIF1 gene. As used herein, an "antisense nucleotide that specifically binds to the CRIF1 gene" refers to an oligonucleotide that specifically binds to the mRNA of CRIF1 by having a sequence complementary to a specific region of the mRNA of CRIF1 to inhibit the translation of the mRNA into a protein. Means. The antisense nucleotides may be composed of DNA, RNA, PNA or derivatives thereof. As used herein, "siRNA" refers to double-stranded RNA that inhibits the production of proteins by cleaving the mRNA of CRIF1. Antisense nucleotides and siRNAs that specifically bind to the CRIF1 gene may be readily designed by those skilled in the art with reference to the mRNA sequences of human CRIF1 known in the art. In the following examples, double-stranded RNA consisting of SEQ ID NO: 1 and SEQ ID NO: 2 was used as siRNA, but is not limited thereto.

Sense: 5'-UGGAGGCCGAAGAACGCGAAUGGUA-3 '(SEQ ID NO: 1)

Antisense: 5'-UACCAUUCGCGUUCUUCGGCCUCCA-3 '(SEQ ID NO: 2)

The inhibitor of activity of the protein may be an antibody specific for the CRIF1 protein. The antibody specifically binds to the CRIF1 protein to inhibit the activity of the CRIF1 protein, and may be a polyclonal antibody or a monoclonal antibody. Antibodies to the CRIF1 protein can be prepared by methods conventional in the art.

The wound care composition of the present invention may be prepared and used in various formulations by mixing with itself or a pharmaceutically acceptable carrier, excipient, forming agent, diluent, etc. by methods known in the pharmaceutical art. For example, it may be prepared and used in oral formulations such as powders, granules, tablets, capsules, syrups, emulsions, or formulations such as external preparations or injections. In particular, in consideration of the purpose of wound healing, the composition of the present invention is more preferably in the form of a topical or injectable skin for topical administration. Examples of the external preparation for skin include liquids, ointments, creams, lotions, sprays, patches, gels, aerosols, and the like. The pharmaceutical composition according to the invention may be administered in a pharmaceutically effective amount. In the present invention, the 'pharmaceutically effective amount' means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment. Effective dose levels are well known for factors including the type of wound, severity, age, sex, activity of the drug, sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, concurrently used drugs, and other medical fields. It can be determined according to known factors. The compositions of the present invention may be administered alone or in combination with other therapeutic agents and may be administered sequentially or simultaneously with conventional therapeutic agents. Considering all of the above factors, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, which can be easily determined by those skilled in the art.

As described above, the composition of the present invention has an effect of inhibiting the proliferation of keloids, the formation of neovascularization, the migration of cells, and the production of ECM, and thus can be usefully used as a pharmaceutical composition for inhibiting or treating keloids and hypertrophic scar formation.

1 is an electrophoretic image and quantitative graph showing Akt phosphorylation inhibition by CRIF1 knockdown.
2 is an electrophoretic image and quantitative graph showing increased Rho GDI2 expression by CRIF1 knockdown.
Figure 3 is an image and quantitative graph showing the results of wound recovery evaluation by CRIF1 knockdown.
4 is a Western blot image showing reduction of CRIF1 expression by CRIF1 knockdown in keloid fibroblasts.
Figure 5 is a graph and Western blot image showing the cell proliferation inhibitory effect of keloid fibroblasts by CRIF1 knockdown.
Figure 6 is an image and quantitative graph showing the cell migration inhibitory effect of keloid fibroblasts by CRIF1 knockdown.
7 is a graph showing the effect of CRIF1 knockdown on the expression of ECM related genes in keloid fibroblasts.

Hereinafter, the present invention will be described in more detail with reference to the accompanying examples. However, such an embodiment is only an example for easily describing the content and scope of the technical idea of the present invention, whereby the technical scope of the present invention is not limited or changed. It will be apparent to those skilled in the art that various modifications and variations are possible within the scope of the present invention based on these examples.

EXAMPLE

Example 1 Evaluation of the Effect of CRIF1 on the Cell Migration of Vascular Endothelial Cells

1) Knockdown of CRIF1 Gene by Cell Culture and siRNA Infection

Human umbilical vein endothelial cells (HUVECs) were purchased from Clonetics (USA) and incubated in Endothelial Growth Medium-2 (Lonza, USA) at 37 ° C., 5% CO ₂ conditions according to the manufacturer's instructions. Sub-confluent proliferative HUVECs were used at 2-8 passages. HUVECs were infected with siRNA against CRIF1 of SEQ ID NOs 1 and 2 as described above using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer's manual. Infected cells were incubated for 48 hours at 37 ° C., 5% CO ₂ for knockdown of the CRIF1 gene. The control group was infected with Stealth RNAi Negative Control Duplexes and Medium GC Duplex (Invitrogen) instead of siRNAs of SEQ ID NOs: 1 and 2.

2) Confirmation of Akt Phosphorylation Inhibition by CRIF1 Knockdown

In 1), siRNA-infected cells were cultured with RIPA (10 mM Tris-HCl at pH 8.0, 1 mM EDTA, 140 mM NaCl, 0.1% SDS, 0.1% 200 sodium deoxycholate, and 1% Triton X-100) and protease inhibitor cocktail ( Lysis using Roche). 30 μg of the lysate was dissolved in SDS-PAGE and then transferred to the PVDF membrane to quantify CRIF1, Akt and p-Akt proteins by conventional Western blot methods known in the art. As an antibody for Western blot, Anti-CRIF1, anti-phospho Akt antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA), and Akt antibody was purchased from Cell Signaling Technology (Beverly, MA, USA). Perxosidase conjugated secondary antibody (Santa Cruz) was treated for visualization of the separated proteins, and chemiluminescent signals were developed using Super Fisher West Pico or Femto Substrate from Thermo Fisher Scientific. The density of developed blots and bands was quantified with Gel Doc 2000 Chemi Doc system using Quantity One software (Bio-Rad, Hercules, CA).

Figure 1 is an electrophoretic image and quantitative graph showing the Western blot results show that Akt phosphorylation process is also inhibited as the expression of CRIF1 is reduced by the infection of siRNA.

3) Evaluation of expression level of Rho GDI2 by CRIF1 knockdown

Rho GDI2 is known to regulate cell migration. This was confirmed by Western blot whether the knockdown of CRIF1 influences the expression of Rho GDI2. Western blot was performed in the same manner as 2), and an anti-Rho GDI2 antibody (Santa Cruz Biotechnology) was used as the antibody.

2 is an electrophoretic photograph and a quantitative graph showing the Wessett blot results. 2 shows that CRIF1 can regulate cell mobility by regulating the expression of Rho GDI2.

4) Wound healing assasy

HUVECs were cultured according to the method described in 1) in 6-well plates and infected with siRNA for CRIF1. The cells were then further incubated for 24 hours, wound to a constant width using a 200 μl pipette tip sterilized in the fused cell monolayer, and the debris was washed using PBS. Thereafter, 48 hours of incubation was performed, and wound recovery ability was evaluated by measuring the moving distance of the cells at the wound site using Image J software from the photos before and after the culture.

Figure 3 is a graph quantifying the wound image and the microscope image of the wound recovery evaluation experiment. 3 shows that the wound recovery ability is greatly reduced when CRIF1 is knocked down compared to the control group. From this, when the expression of CRIF1 is suppressed, the mobility of cells is reduced. This is consistent with the increased expression of Rho GDI2, which inhibits cell mobility of 3), suggesting that CRIF1 affects cell mobility by regulating the expression of Rho GDI2.

Example 2 Evaluation of the Effect of CRIF1 on the Proliferation and Cell Migration of Keloid Fibroblasts

As shown in Example 1 that inhibition of CRIF1 expression affects vascular endothelial migration, it was determined that cell migration would be available for the prevention and treatment of keloid or hypertrophic scars associated with hyperactivation.

1) Knockdown of CRIF1 gene by culturing keloid fibroblasts and siRNA infection

Keloid fibroblasts were purchased from ATCC, 37 ° C., 5% using DMEM medium containing 4 mM L-glutamine, 100 μg / ml streptomycin, 100 U / ml penicillin and 10% fetal bovine serum. Incubated in CO ₂ conditions. The keloid fibroblasts cultured by this method were infected with siRNA against CRIF1 of SEQ ID NOs 1 and 2 described above using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer's manual. Infected cells were incubated for 48 hours at 37 ° C., 5% CO ₂ for knockdown of the CRIF1 gene. The control group was infected with Stealth RNAi Negative Control Duplexes and Medium GC Duplex (Invitrogen) instead of siRNAs of SEQ ID NOs: 1 and 2.

Expression of CRIF1 was analyzed by Western blot using the cells infected with siRNA according to the method of Example 1 2). 4 shows that the Western blot image showing the results showed that the CRIF1 gene was effectively knocked down by siRNA infection.

2) Confirmation of proliferation inhibitory effect of keloid fibroblasts by knockdown of CRIF1 gene

In 1), keloid fibroblasts knocked down the CRIF1 gene or control keloid fibroblasts were cultured by inoculating 6-well plates at a density of 5 × 10 ⁴ cells / well. After 24 hours of incubation, 50 μl of the reagent of CCK-8 kit (Dojindo, Japan) was treated, and 1 hour later, the cell proliferation rate was measured by measuring the OD value at 450 nm. Relative proliferation was obtained by setting the cell number of the control group to 100 and the results are shown in FIG. 5. Expression of PCNA protein, a cell proliferation marker, was measured separately by Western blot and the results are shown together in FIG. 5.

5, the proliferation of keloid fibroblasts was reduced by about 30% due to the lack of CRIF1, and the expression of PCNA protein was also reduced compared to the control group.

3) Inhibition of migration of keloid fibroblasts by knockdown of CRIF1 gene

In keloid fibroblasts, the effect of CRIF1 gene on the migration of cancer cells was evaluated by the same method as in Example 4).

6 is a graph quantifying the wound recovery capacity and the microscopic image of the wound recovery evaluation experiment showing the results. In FIG. 6, when knocking down CRIF1, the wound repair ability is greatly reduced compared to the control group. From this, when the expression of CRIF1 is suppressed, the mobility of keloid fibroblasts is also reduced.

4) Effect of knockdown of CRIF1 gene on expression of ECM-related genes in keloid fibroblasts

Keloids are lesions in which extra components of ECM (extracellular matrix) accumulate in excess and infiltrate into normal tissues, and CRIF1 deficiency is used to confirm the prevention and treatment of keloids and hypertrophic scars by the control of CRIF1. The effect on the expression of ECM related genes was confirmed.

Specifically, keloid fibroblasts were inoculated in 6well plates and cultured for 24 hours, followed by knockdown of the CRIF1 gene by the same method as 1). After 48 hours of incubation, the culture medium in which the cells were grown was suctioned and treated with 1 ml of Trizol reagent (Invitrogen, USA). The cells were scraped and collected, treated with 200 μl of chloroform, and centrifuged at 12000 g for 15 minutes. The supernatant was collected, treated with 500 μl of isopropanol, and centrifuged at 12000 g for 10 minutes to obtain an RNA precipitate. The precipitate was washed with 1 ml of 75% ethanol and dried at room temperature. 1 μg of the dried precipitate was dissolved in 15 μl of Rnase-Free water, followed by PCR at 45 ° C. 60 minutes and 95 ° C. 5 minutes using RT-preMix (Intron, KOREA) to obtain cDNA.

Real-time PCR was performed using SYBR green (Enzynomics, KOREA), a reagent that directly binds to the obtained cDNA and the primers and DNA shown in Table 1 below, to quantify the expression of ECM-related mRNA. The rt PCR denatured the cDNA at 95 ° C for 10 minutes, followed by 40 cycles of 95 ° C 10 seconds, annealing 60 ° C 20 seconds, and elongation 72 ° C 30 seconds. The obtained results were corrected using the house keeping gene GAPHDH as a control, and the relative results of the control cells that did not knock down CRIF1 were set to 1 as relative values.

FIG. 7 is a graph showing the results, although the expression of transforming growth factor-β (TGF-β) mRNA, which is known to be most importantly involved in ECM synthesis in FIG. And expression of ECM can be seen to be reduced.

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Claims (8)

delete A pharmaceutical composition for inhibiting or treating keloid and hypertrophic scar formation, comprising an antisense nucleotide or siRNA that specifically binds to a CR6 interacting factor 1 (CRIF1) gene.
The method of claim 2,
The siRNA is a pharmaceutical composition for inhibiting or treating keloid and thickening scar formation, characterized in that having the sequence of SEQ ID NO: 1 and 2.
A pharmaceutical composition for inhibiting or treating keloid and hypertrophic scar formation, comprising an antibody specific for CRIF1 (CR6 interacting factor 1) protein.
The method of claim 2 or 3,
The composition is a pharmaceutical composition for inhibiting or treating keloid and hypertrophic scar formation, characterized in that used in the reconstruction step of the wound healing process.
The method of claim 2 or 3,
The composition is a pharmaceutical composition for inhibiting or treating keloid and thickening scar formation, characterized in that it is provided in the form of an external preparation for skin or injection.
The method of claim 4, wherein
The composition is a pharmaceutical composition for inhibiting or treating keloid and hypertrophic scar formation, characterized in that used in the reconstruction step of the wound healing process.
The method of claim 4, wherein
The composition is a pharmaceutical composition for inhibiting or treating keloid and thickening scar formation, characterized in that it is provided in the form of an external preparation for skin or injection.

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