WO2020085548A1 - Recombinant protein for prevention or treatment of idiopathic pulmonary fibrosis and composition comprising same for prevention or treatment of idiopathic pulmonary fibrosis - Google Patents
Recombinant protein for prevention or treatment of idiopathic pulmonary fibrosis and composition comprising same for prevention or treatment of idiopathic pulmonary fibrosis Download PDFInfo
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- WO2020085548A1 WO2020085548A1 PCT/KR2018/012801 KR2018012801W WO2020085548A1 WO 2020085548 A1 WO2020085548 A1 WO 2020085548A1 KR 2018012801 W KR2018012801 W KR 2018012801W WO 2020085548 A1 WO2020085548 A1 WO 2020085548A1
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- idiopathic pulmonary
- pulmonary fibrosis
- recombinant protein
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- fibrosis
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- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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Definitions
- the present invention relates to a recombinant protein for preventing or treating idiopathic pulmonary fibrosis and a composition for preventing or treating idiopathic pulmonary fibrosis comprising the same.
- Idiopathic pulmonary fibrosis is an unknown cause of pulmonary interstitial tissue, and inflammation occurs repeatedly, resulting in permanent scarring and tissue fibrosis, resulting in structural changes in lung tissue, leading to deterioration of lung function and death. It is a fatal disease.
- idiopathic pulmonary fibrosis is difficult to treat because the exact cause of the disease is unknown, and anticoagulants, tyrosine kinase inhibitors, ER-A endothelial receptor antagonists, anti-fibrotic drugs, antacids, and immunosuppressive drugs are used in combination therapy. It is proposed as a treatment.
- the idiopathic pulmonary fibrosis guidelines conditionally recommends only some treatments that have a high incidence of adverse events but have no statistical significance or improve mortality based on clinical trial results, and others recommend strong non-recommendations or recommendations. Delayed.
- the partial treatment effect is provided or the level of providing the effect is insignificant and thus it is not meaningful to delay or treat the onset or progression of pulmonary fibrosis. There was a problem that could not provide a fundamental effect.
- the pharmaceutical composition prepared for the treatment of idiopathic pulmonary fibrosis including the prior art, has a low biocompatibility of the composition based on chemical components, a high incidence of side effects, and difficulties in mass production and quality control considering productivity. Going through.
- the present invention was created to solve the above problems, and an object of the present invention is to fundamentally treat pulmonary tissue in which fibrosis has progressed by idiopathic pulmonary fibrosis to restore pulmonary function to a normal level, and further develop idiopathic pulmonary fibrosis. It is to provide a composition containing a recombinant protein and the same for preventing or treating idiopathic pulmonary fibrosis that can delay or stop the.
- the recombinant protein for preventing or treating idiopathic pulmonary fibrosis is a recombinant protein based on the Milk fat globule-EGF factor 8 (MFG-E8) protein, and consists of the amino acid sequence of SEQ ID NO: 1.
- MFG-E8 Milk fat globule-EGF factor 8
- the recombinant protein for preventing or treating idiopathic pulmonary fibrosis consisting of the amino acid sequence of SEQ ID NO: 1 by recombination based on milk fat globule-EGF factor 8 (MFG-E8) protein is collagen, ⁇ -SMA ( ⁇ -Smooth Muscle) in lung tissue. Actin) and phosphorylated Extracellular Signal-Regulated Kinase (ERK) can significantly reduce expression.
- the recombinant protein for preventing or treating idiopathic pulmonary fibrosis consisting of the amino acid sequence of SEQ ID NO: 1 by recombination based on the milk fat globule-EGF factor 8 (MFG-E8) protein is Col1a1, MMP-2 in lung tissue to be prevented or treated.
- MMP-12 and TIMP1 can be significantly reduced.
- the recombinant protein for preventing or treating idiopathic pulmonary fibrosis consisting of the amino acid sequence of SEQ ID NO: 1 that is recombinant based on Milk fat globule-EGF factor 8 (MFG-E8) protein is administered intralesionally, intravascularly, subcutaneously, intranasally It can be formulated for administration or intraperitoneal administration.
- MFG-E8 Milk fat globule-EGF factor 8
- the composition for preventing or treating idiopathic pulmonary fibrosis according to the present invention is idiopathic pulmonary fibrosis consisting of the milk fat globule-EGF factor 8 (MFG-E8) protein-based recombinant amino acid sequence of SEQ ID NO: 1
- the recombinant protein for prevention or treatment is included as an active ingredient.
- the present invention is a gene encoding a recombinant protein for preventing or treating idiopathic pulmonary fibrosis consisting of an amino acid sequence of SEQ ID NO: 1 that is recombined based on the milk fat globule-EGF factor 8 (MFG-E8) protein. It provides as another aspect.
- the present invention is a gene encoding a recombinant protein for preventing or treating idiopathic pulmonary fibrosis consisting of an amino acid sequence of SEQ ID NO: 1 that is recombined based on the milk fat globule-EGF factor 8 (MFG-E8) protein.
- a recombinant vector comprising the same is provided as another aspect.
- ERK extracellular signal-regulated kinase
- SMAD2 phosphorylated extracellular signal-regulated kinase
- FIG. 1 shows the structure of a backbone vector in which a gene encoding a recombinant protein (NP-011) for preventing or treating idiopathic pulmonary fibrosis of the present invention is inserted.
- NP-011 a recombinant protein
- Figure 2 can be inserted into the structure of the Backbone Vector shown in Figure 1, shows the DNA sequence of the gene encoding a recombinant protein (NP-011) for preventing or treating idiopathic pulmonary fibrosis of the present invention.
- NP-011 a recombinant protein
- Figure 3 shows the amino acid sequence of the recombinant protein (NP-011) for preventing or treating idiopathic pulmonary fibrosis of the present invention.
- Figure 4 is a schematic diagram showing the production process of an animal experimental model designed for verifying the effect of the recombinant protein for preventing or treating idiopathic pulmonary fibrosis of the present invention (NP-011).
- Figure 5 shows the staining results showing the distribution of collagen and ⁇ -SMA in the lung tissue of animal experimental models designed to verify the effect of the recombinant protein for preventing or treating idiopathic pulmonary fibrosis of the present invention (NP-011).
- Figure 6 is a graph showing the expression patterns of lung tissue fibrosis-related biomarkers in lung tissue of animal experimental models designed to verify the effect of recombinant protein (NP-011) for preventing or treating idiopathic pulmonary fibrosis of the present invention.
- Figure 7 shows the expression pattern of the sub-factors of the signaling system related to inducing fibrosis of lung tissue in lung tissue of animal experimental models designed to verify the effect of recombinant protein (NP-011) for preventing or treating idiopathic pulmonary fibrosis of the present invention. It is the graph shown.
- FIG. 8 is a schematic diagram showing the production process of two test groups with different injection timings of NP-011 among animal experimental models designed for verifying the effect of recombinant protein (NP-011) for preventing or treating idiopathic pulmonary fibrosis of the present invention.
- Figure 10 is collagen in the lung tissue of the animal experimental model test group injected with NP-011 at day 5 of the animal experimental model designed for verifying the effect of the recombinant protein for preventing or treating idiopathic pulmonary fibrosis of the present invention (NP-011) And staining results comparing the distribution of ⁇ -SMA distribution.
- Figure 11 is a lung in the lung tissue of the animal experimental model test group injected with NP-011 at day 3 of the animal experimental model designed for verifying the effect of the recombinant protein for preventing or treating idiopathic pulmonary fibrosis of the present invention (NP-011)
- NP-011 idiopathic pulmonary fibrosis of the present invention
- Figure 12 is a lung in the lung tissue of the animal experimental model test group injected with NP-011 at the time of day 5 of the animal experimental model designed for verifying the effect of the recombinant protein for preventing or treating idiopathic pulmonary fibrosis of the present invention (NP-011)
- NP-011 idiopathic pulmonary fibrosis of the present invention
- Figure 13 is a lung in the lung tissue of the animal experimental model test group injected with NP-011 at day 3 of the animal experimental model designed for verifying the effect of the recombinant protein (NP-011) for preventing or treating idiopathic pulmonary fibrosis of the present invention
- NP-011 the recombinant protein
- NP-011 idiopathic pulmonary fibrosis
- NP-011 idiopathic pulmonary fibrosis according to the present invention
- NP-011 recombinant protein for preventing or treating idiopathic pulmonary fibrosis
- NP-011 idiopathic pulmonary fibrosis
- MFG-E8 Origene's MFG-E8 (NM_005928) Human cDNA Clone (Cat. No. RG217163) was purchased, used as a template and subjected to PCR to obtain DNA fragments as shown in FIG. 2 (see SEQ ID NO: 4).
- FIG. 2 obtained through PCR amplification at the HindIII and SalI restriction sites (A portion of FIG. 1) of the pLFCF Vector, which is a Mammalian expression Vector of the structure as shown in FIG. 1 (see SEQ ID NO: 4). Proceed with cloning to insert.
- plasmid DNA is extracted and transfected into HEK293 cells, and after 2 days, the culture is collected, and immunoprecipitation reaction (IP, Immunoprecipitation) is performed with FLAG resin to prevent or treat idiopathic pulmonary fibrosis by Western Blotting.
- IP immunoprecipitation reaction
- NP-011 The expression of the recombinant protein corresponding to the recombinant protein (NP-011) is confirmed.
- NP-011 idiopathic pulmonary fibrosis
- NP-011 idiopathic pulmonary fibrosis
- the culture medium was replaced and further cultured for 6 days, and the culture medium was collected 3 times every 2 days to perform Affinity-type protein purification from the collected culture medium.
- NP-011 for prevention or treatment of idiopathic pulmonary fibrosis, which is prepared based on the milk fat globule-EGF factor 8 (MFG-E8) protein.
- MFG-E8 milk fat globule-EGF factor 8
- NP-011 recombinant protein
- MFG-E8 milk fat globule-EGF factor 8
- a DNA fragment that is cloned into an expression vector in relation to the expression of a recombinant protein for preventing or treating idiopathic pulmonary fibrosis (NP-011), which is prepared based on milk fat globule-EGF factor 8 (MFG-E8) protein
- NP-011 idiopathic pulmonary fibrosis
- MFG-E8 milk fat globule-EGF factor 8
- the structure of fragments is also based on the structure of FIG. 2, but according to an embodiment, a specific DNA sequence encoding a specific signal peptide may be additionally linked to DNA fragments.
- Recombinant protein for prevention or treatment of idiopathic pulmonary fibrosis based on milk fat globule-EGF factor 8 (MFG-E8) protein which is finally prepared through the processes of cloning, separation, production, and purification of the recombinant protein described above.
- 011 has the amino acid sequence structure as shown in SEQ ID NO: 1 or 3 in the following sequence listing.
- the recombinant protein for preventing or treating idiopathic pulmonary fibrosis based on the milk fat globule-EGF factor 8 (MFG-E8) protein (NP-011) is a structural component of the MFG-E8 protein, EGF-like Domain, C1 Domain, and C2.
- EGF-like domain (refer to SEQ ID NO: 2)
- C1 domain (refer to SEQ ID NO: 3) are recombined into structures.
- the recombinant protein for preventing or treating idiopathic pulmonary fibrosis based on the milk fat globule-EGF factor 8 (MFG-E8) protein of the present invention forms an amino acid sequence of "EGF-like Domain + C1 Domain" It is used as a basic structure, and is characterized in that the C2 domain is excluded from the structure.
- the milk fat globule-EGF factor 8 (MFG-E8) protein-based idiopathic pulmonary fibrosis prevention or treatment recombinant protein (NP-011) of the present invention is a major pharmaceutical composition used for the prevention or treatment of idiopathic pulmonary fibrosis. It can be used as an active ingredient.
- milk fat globule-EGF factor 8 (MFG-E8) protein-based idiopathic pulmonary fibrosis prevention or treatment recombinant protein (NP-011) of the present invention and a pharmaceutical composition using it as a main active ingredient are administered in a lesion, blood vessels It can be formulated for intrathecal administration (artery, vein, etc.), subcutaneous administration, intranasal administration, intraperitoneal administration, or administration in a specific tissue (lung tissue).
- the recombinant protein for preventing or treating idiopathic pulmonary fibrosis based on the milk fat globule-EGF factor 8 (MFG-E8) protein of the present invention (NP-011) is cloned into an expression vector in relation to the expression of the recombinant protein described above.
- additional linkage of a specific signal peptide structure may be made.
- a specific Signal Peptide structure may be implemented in a form that is connected to the EGF-like domain tip by having an amino acid sequence such as "MPRPRLLAALCGALLCAPSLLVA” (see SEQ ID NO: 5), but is not limited thereto.
- an animal model for inducing acute idiopathic pulmonary fibrosis is prepared by instilling 3 mg / kg of bleomycin into the C57BL / 6 mice.
- the experimental group for verifying the effect of the recombinant protein for preventing or treating idiopathic pulmonary fibrosis of the present invention is acute idiopathic pulmonary fibrosis inducing animal of the 'BLM' model group prepared as shown in FIG.
- the model mice were prepared by administering 160 ⁇ g / kg NP-011 in the vein of the tail of the mouse on a daily basis, and are designated as 'BLM + NP011' based on the drawing.
- the experimental group of 'BLM + NP011' prepared as described above was changed for 3 days after injection of bleomycin by varying the administration time of the recombinant protein for preventing or treating idiopathic pulmonary fibrosis (NP-011). It was classified into the first experimental group (CASE 1) in which NP-011 was injected once at the time point and the second experimental group (CASE 2) in which NP-011 was injected once at 5 days after the injection of bleomycin.
- CASE 1 idiopathic pulmonary fibrosis
- 'BLM' model group which is made of animal model mice induced with Idiopathic Pulmonary Fibrosis, and thereby changes in lung tissue. Lung tissues of animal model mice were extracted for 4 times over 3, 5, 7, and 14 days, respectively, to verify tissue staining analysis and expression of specific components related to lung tissue fibrosis.
- mice of the 'BLM' model group were injected with bleomycin, followed by anesthesia after 3 days, 5 days, 7 days, and 14 days, followed by cardiac perfusion through PBS. Lung tissue was removed after injection.
- the extracted lung tissue is refrigerated for 4 days in a 4% PFA solution, and the lung tissue embedded with paraffin is cut to a thickness of 4 ⁇ m using a sectioning machine (CM3050S, Leica), and a slide (silane coated slide) ).
- a sectioning machine C3050S, Leica
- a slide silane coated slide
- the lung tissue sections are first defatted in xylene, hydrated in a 100% alcohol solution, and the slides washed with tap water are stained with Hematoxylin for 5 minutes, Biebrich Scarlet-Acid Fuchsin The dye was stained for 15 minutes, phosphomolybdic / phosphotungstic acid for 10 minutes, aniline blue for 5 minutes, and 1% acetic acid for 3 minutes, then dehydrated and sealed with a cover slip.
- the acute idiopathic pulmonary fibrosis-inducing animal model mice of the 'BLM' model group have an expression distribution of collagen, which is a major fibrosis index in lung tissue from day 3 after injection of bleomycin. Increases little by little, shows a remarkable increase after 5 days, shows the highest increase level on the 7th day, and shows a similar level to the 7th day on the 14th.
- lung tissue of the lung tissue extracted from the 3rd, 5th, 7th and 14th days after injection of bleomycin into the acute idiopathic pulmonary fibrosis-induced animal model mice of the 'BLM' model group, respectively.
- the expression pattern of biomarkers related to fibrosis was verified.
- PCR polymerase chain reaction
- each extracted lung tissue section was extracted with total RNA using an RNeasy mini kit, and cDNA was synthesized through reverse transcriptase PCR (RT-PCR), and the synthesized cDNA was expressed through quantitative RT-PCR.
- RT-PCR reverse transcriptase PCR
- sequence structure (refer to SEQ ID NO: 6 to SEQ ID NO: 9) for the primer used therein is shown in Table 1 below.
- FIG. 6 the results of expression patterns for each extraction time of biomarkers related to lung tissue fibrosis of the extracted lung tissue are as shown in FIG. 6.
- FIG. 6 (refer to the drawing- * P ⁇ 0.05, ** As shown in P ⁇ 0.01, *** P ⁇ 0.001), the acute idiopathic pulmonary fibrosis-inducing animal model mice of the 'BLM' model group were injected with bleomycin, followed by the 'CON' normal control (Wild Type Control). From day 3 compared to normal mice, it can be seen that the expression of Col1a1, MMP-2, MMP-12 and TIMP1, which are biomarkers related to fibrosis in the lung tissue, maintains a high level.
- lung tissue of the lung tissue extracted from the 3rd, 5th, 7th and 14th days after injection of bleomycin into the acute idiopathic pulmonary fibrosis-induced animal model mice of the 'BLM' model group, respectively.
- the expression pattern of sub-factors of the fibrosis-related signaling system was verified.
- each lung tissue extracted was pulverized to obtain a protein, and then proteins were separated by size through SDS-PAGE, transferred to a PVDF membrane, and immunized at 4 ° C as a primary antibody.
- Primary antibodies used for this are ⁇ -SMA (SC-53015), pERK (# 4370), tERK (# 4695), pSMAD2 (3104S), tSMAD2 (3102S) and Actin (SC-47778).
- the immune antibody was reacted with a secondary antibody combined with HRP (Horse Radish Peroxidase) to develop and quantify the band, thereby verifying the expression pattern of the sub-factors of the signal transduction system related to lung tissue fibrosis of the extracted lung tissue.
- HRP Hase Radish Peroxidase
- the acute idiopathic pulmonary fibrosis-induced animal model mice of the 'BLM' model group compared to normal mice of the 'CON' normal control (Wild Type Control) after injection of bleomycin
- the expression of the phosphorylated form of extracellular signal-regulated kinase (ERK), a sub-factor of the MARK signal of the fibrosis-related signaling system increased from the 5th day, and was significantly increased on the 14th day to have the highest level.
- ERK extracellular signal-regulated kinase
- Milk fat globule-EGF factor 8 of the present invention (MFG-E8) protein-based idiopathic pulmonary fibrosis prevention or treatment of the recombinant protein (NP-011) in order to confirm the lung tissue fibrosis preventing function, the first experimental group and the second experimental group
- the expression patterns of collagen and ⁇ -SMA in the lung tissue of the corresponding experimental animal mice were examined.
- NP-011 was injected once on the 3rd day, and then anesthetized at the 6th day. After infusion of PBS through perfusion, lung tissue was removed.
- Trichrome staining and Sirius red staining process for evaluating the expression distribution of collagen and ⁇ -SMA in lung tissue using lung tissue of the first and second experimental groups extracted as described above and the process of deriving the results are described above. It is omitted because it is the same as the method for acute idiopathic pulmonary fibrosis-induced animal model mice in the 'BLM' model group.
- the lung tissue slides of each animal model mice of the first experimental group after Trichrome staining and Sirius red staining were obtained by fluorescence microscopy (BX61; Olympus), as shown in FIG. 9.
- the lung tissue slides of each animal model mice of the second experimental group after Trichrome staining and Sirius red staining were obtained by fluorescence microscopy (BX61; Olympus), as shown in FIG. 10.
- the animal model mice of the first experimental group and the second experimental group of 'BLM + NP011' are acute idiopathic pulmonary fibrosis-induced animal model mice of the 'BLM' model group after injection of NP-011. It can be seen that the expression distribution of collagen, which is a major fibrosis index in lung tissue, is significantly reduced compared to those of the above.
- the animal model mice of the first experimental group and the second experimental group of 'BLM + NP011' are acute idiopathic pulmonary fibrosis-induced animal model mice of the 'BLM' model group after injection of NP-011.
- ⁇ -SMA a major fibrosis index in lung tissue
- NP-011 when NP-011 is administered to the acute idiopathic pulmonary fibrosis-induced animal model mice of the 'BLM' model group at the time of day 3 or 5 after injection of bleomycin, collagen, which is a major fibrosis index in lung tissue ( Collagen) and ⁇ -SMA are significantly reduced in expression distribution, thereby providing an excellent level of antifibrosis effect for preventing lung tissue fibrosis.
- Milk fat globule-EGF factor 8 of the present invention (MFG-E8) protein-based idiopathic pulmonary fibrosis prevention or treatment of the recombinant protein (NP-011) in order to confirm the lung tissue fibrosis preventing function, the first experimental group and the second experimental group
- the expression patterns of the subfactors of the MARK signal and the TGF ⁇ signal in the lung tissue fibrosis-related signaling system in the lung tissue of experimental animal mice were examined.
- NP-011 was injected once on the 3rd day, and then anesthetized at the 6th day. After infusion of PBS through perfusion, lung tissue was removed.
- the animal model mice of the first experimental group of 'BLM + NP011' compared to those of the animal model mice inducing acute idiopathic pulmonary fibrosis of the 'BLM' model group after injection of NP-011
- ERK extracellular signal-regulated kinase
- the animal model mice of the 'BLM + NP011' first experimental group compared to the acute idiopathic pulmonary fibrosis-induced animal model mice of the 'BLM' model group after injection of NP-011 lung tissue in the lung tissue. It can be seen that the expression of SMAD2 in the phosphorylated form, which is a subfactor of the TGF ⁇ signal of the fibrosis-related signaling system, is also significantly reduced.
- the animal model mice of the 'BLM + NP011' second experimental group compared to the acute idiopathic pulmonary fibrosis-induced animal model mice of the 'BLM' model group after injection of NP-011 lung tissue in the lung tissue. It can be confirmed that the expression of the extracellular signal-regulated kinase (ERK) in the phosphorylated form, which is a sub-factor of the MARK signal of the fibrosis-related signaling system, is significantly reduced, which can be confirmed to be similar to that of normal mice.
- ERK extracellular signal-regulated kinase
- the animal model mice of the 'BLM + NP011' second experimental group were in lung tissue compared to the acute idiopathic pulmonary fibrosis-induced animal model mice of the 'BLM' model group after injection of NP-011. It can be seen that the expression of ⁇ -SMA, a major pulmonary tissue fibrosis indicator, is significantly reduced, which is similar to that of normal mice.
- NP-011 when NP-011 is administered to the acute idiopathic pulmonary fibrosis-induced animal model mice of the 'BLM' model group at the time of day 3 or 5 after injection of bleomycin, lung tissue fibrosis-related signaling in lung tissue System MARK signal sub-factor phosphorylated form of ERK and TGF ⁇ signal sub-factor phosphorylated form of SMAD2 rapidly decreased, and further, the expression of ⁇ -SMA, a major lung tissue fibrosis index, was also significantly reduced, resulting in the expression of normal mice. Similar to the level, it can provide an excellent level of antifibrosis effect for preventing lung tissue fibrosis.
- NP-011 was injected once on the 3rd day, and then anesthetized at the 6th day. After infusion of PBS through perfusion, lung tissue was removed.
- PCR Polymerase chain reaction
- the animal model mice of the first experimental group 'BLM + NP011' showed lung in lung tissue compared to the acute idiopathic pulmonary fibrosis-induced animal model mice of the 'BLM' model group after injection of NP-011. It can be confirmed that the expression of tissue fibrosis-related biomarkers Col1a1, MMP-2, MMP-12, and TIMP1 is markedly reduced, thereby approaching the expression level of normal mice.
- NP-011 when NP-011 is administered to the acute idiopathic pulmonary fibrosis-induced animal model mice of the 'BLM' model group at the third day after injection of bleomycin, Col1a1, a biomarker related to lung tissue fibrosis in the lung tissue,
- MMP-2, MMP-12 and TIMP1 are significantly reduced, which is similar to that of normal mice, and thus can provide an excellent level of antifibrosis effect for preventing lung tissue fibrosis.
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Abstract
The present invention relates to a recombinant protein for prevention or treatment of idiopathic pulmonary fibrosis, which is recombined on the basis of milk fat globule-EGF factor 8(MFG-E8) and has the amino acid sequence of SEQ ID NO: 1. The recombinant protein can fundamentally treat a lung tissue that has undergone fibrosis due to idiopathic pulmonary fibrosis and as such, can recover lung function to a normal level. In addition, the present invention relates to a pharmaceutical composition comprising as an active ingredient a recombinant protein for prevention or treatment of idiopathic pulmonary fibrosis, which is recombined on the basis of milk fat globule-EGF factor 8(MFG-E8) and has the amino acid sequence of SEQ ID NO: 1.
Description
본 발명은 특발성 폐섬유증 예방 또는 치료용 재조합 단백질 및 이를 포함하는 특발성 폐섬유증 예방 또는 치료용 조성물에 관한 것이다.The present invention relates to a recombinant protein for preventing or treating idiopathic pulmonary fibrosis and a composition for preventing or treating idiopathic pulmonary fibrosis comprising the same.
특발성 폐섬유증(Idiopathic Pulmonary Fibrosis)은 폐 간질 조직에 알 수 없는 원인으로 염증이 반복 발생하여 영구적인 상흔 및 조직 섬유화를 초래하여 폐 조직의 구조적 변화를 유발함으로써, 폐 기능이 저하되어 사망에 이르게 되는 치명적인 질환이다.Idiopathic pulmonary fibrosis is an unknown cause of pulmonary interstitial tissue, and inflammation occurs repeatedly, resulting in permanent scarring and tissue fibrosis, resulting in structural changes in lung tissue, leading to deterioration of lung function and death. It is a fatal disease.
전 세계 약 5백만 명의 발병자가 있으며, 국내에서는 간질성 폐 질환 중 50%가 넘게 조사될 정도로 가장 흔히 발병하는 질환이다. 이러한 대부분의 특발성 폐섬유증은 만성적으로 1년 내지 2년에 걸쳐 천천히 발병하여 병이 진행될수록 호흡곤란이 심해져 일상적인 생활도 힘들어지게 된다.There are about 5 million people worldwide, and it is the most common disease in Korea, with over 50% of interstitial lung diseases being investigated. Most of these idiopathic pulmonary fibrosis chronically develops slowly over a period of 1 to 2 years, and as the disease progresses, respiratory distress increases and daily life becomes difficult.
현재 특발성 폐섬유증은 정확한 발병원인이 밝혀지지 않아 치료에 어려움을 겪고 있고, 항응고제, 티로신인산화효소(Tyrosine kinase) 억제제, ER-A 내피 수용체 길항체, 항섬유화 약물, 제산제 및 면역억제제의 병용 요법 등이 치료법으로 제시되고 있다.Currently, idiopathic pulmonary fibrosis is difficult to treat because the exact cause of the disease is unknown, and anticoagulants, tyrosine kinase inhibitors, ER-A endothelial receptor antagonists, anti-fibrotic drugs, antacids, and immunosuppressive drugs are used in combination therapy. It is proposed as a treatment.
이와 관련하여 특발성 폐섬유증 치료용 약학 조성물의 종래기술에 대한 선행문헌에는 대한민국 등록특허공보 제10-1845862호의"특발성 폐섬유증의 치료 또는 예방을 위한 약학적 조성물"(이하, '종래기술'이라고 함)이 있다.In this regard, prior literature on the prior art of the pharmaceutical composition for the treatment of idiopathic pulmonary fibrosis described in Korean Patent Publication No. 10-1845862 "Pharmaceutical composition for the treatment or prevention of idiopathic pulmonary fibrosis" (hereinafter referred to as 'prior art') ).
특히, 2015년 특발성 폐섬유증 가이드라인의 업데이트 내용을 살펴보면 "특정 치료 전략의 우위 또는 단독 병용요법을 권고하고 있지 않다"라고 강조하며 치료 대상에 따라 적절한 치료 전략의 선택이 필요함을 강조 했다.In particular, looking at the updated contents of the Idiopathic Pulmonary Fibrosis Guidelines in 2015, he emphasized that "the superiority of a specific treatment strategy or a single combination therapy is not recommended," and emphasized that it is necessary to select an appropriate treatment strategy according to the treatment target.
여기서, 특발성 폐섬유증 가이드라인은 임상 시험 결과를 바탕으로 앞 서 설명한 치료법 중 유해사건 발생률은 높지만 통계적 유의성이 없거나, 사망률 개선을 보인 일부의 치료법만을 조건부 권고하고 나머지는 강한 비권고 또는 권고 사항 제시를 지연하였다.Here, the idiopathic pulmonary fibrosis guidelines conditionally recommends only some treatments that have a high incidence of adverse events but have no statistical significance or improve mortality based on clinical trial results, and others recommend strong non-recommendations or recommendations. Delayed.
아울러, 이러한 종래기술을 비롯한 기존의 특발성 폐섬유증의 치료를 위해 마련된 약학 조성물의 경우, 부분적 치료 효과가 제공되거나 효과의 제공 수준이 미미하여 유의성을 갖추지 못하여 폐섬유증의 발병 혹은 진행을 지연하거나 치료할 수 있는 근본적 효과를 제공하지 못하는 문제점이 있었다.In addition, in the case of the pharmaceutical composition prepared for the treatment of idiopathic pulmonary fibrosis, including the prior art, the partial treatment effect is provided or the level of providing the effect is insignificant and thus it is not meaningful to delay or treat the onset or progression of pulmonary fibrosis. There was a problem that could not provide a fundamental effect.
또한, 종래기술을 비롯한 기존의 특발성 폐섬유증의 치료를 위해 마련된 약학 조성물의 경우, 화학 성분에 기초한 조성의 생체 친화성이 낮고, 부작용 발생률이 높았으며, 생산성을 고려한 대량 생산 및 품질 관리에서 어려움을 겪고 있다.In addition, the pharmaceutical composition prepared for the treatment of idiopathic pulmonary fibrosis, including the prior art, has a low biocompatibility of the composition based on chemical components, a high incidence of side effects, and difficulties in mass production and quality control considering productivity. Going through.
본 발명은 상기 문제점을 해결하기 위해 창작된 것으로써, 본 발명의 목적은 특발성 폐섬유증에 의해 섬유화가 진행된 폐 조직을 근본적으로 치료하여 폐 기능성을 정상 수준으로 회복시키고, 더 나아가 특발성 폐섬유증의 발병을 지연하거나 막을 수 있는 특발성 폐섬유증 예방 또는 치료용 재조합 단백질 및 이를 포함하는 조성물을 제공하는데 있다.The present invention was created to solve the above problems, and an object of the present invention is to fundamentally treat pulmonary tissue in which fibrosis has progressed by idiopathic pulmonary fibrosis to restore pulmonary function to a normal level, and further develop idiopathic pulmonary fibrosis. It is to provide a composition containing a recombinant protein and the same for preventing or treating idiopathic pulmonary fibrosis that can delay or stop the.
상기 목적을 달성하기 위하여 본 발명에 따른 특발성 폐섬유증 예방 또는 치료용 재조합 단백질은, Milk fat globule-EGF factor 8(MFG-E8) 단백질 기반의 재조합 단백질로서, 서열번호1의 아미노산 서열로 이루어진다.In order to achieve the above object, the recombinant protein for preventing or treating idiopathic pulmonary fibrosis according to the present invention is a recombinant protein based on the Milk fat globule-EGF factor 8 (MFG-E8) protein, and consists of the amino acid sequence of SEQ ID NO: 1.
여기서, Milk fat globule-EGF factor 8(MFG-E8) 단백질 기반의 재조합되어 서열번호1의 아미노산 서열로 이루는 특발성 폐섬유증 예방 또는 치료용 재조합 단백질은 폐 조직 내 Collagen, α-SMA(α-Smooth Muscle Actin) 및 인산화된 ERK(Extracellular signal-Regulated Kinase) 중 적어도 하나 이상의 발현을 현저히 감소시킬 수 있다.Here, the recombinant protein for preventing or treating idiopathic pulmonary fibrosis consisting of the amino acid sequence of SEQ ID NO: 1 by recombination based on milk fat globule-EGF factor 8 (MFG-E8) protein is collagen, α-SMA (α-Smooth Muscle) in lung tissue. Actin) and phosphorylated Extracellular Signal-Regulated Kinase (ERK) can significantly reduce expression.
또한, Milk fat globule-EGF factor 8(MFG-E8) 단백질 기반의 재조합되어 서열번호1의 아미노산 서열로 이루는 특발성 폐섬유증 예방 또는 치료용 재조합 단백질은 예방 또는 치료 대상 폐 조직 내 Col1a1, MMP-2, MMP-12 및 TIMP1 중 적어도 하나 이상의 발현을 현저히 감소시킬 수 있다.In addition, the recombinant protein for preventing or treating idiopathic pulmonary fibrosis consisting of the amino acid sequence of SEQ ID NO: 1 by recombination based on the milk fat globule-EGF factor 8 (MFG-E8) protein is Col1a1, MMP-2 in lung tissue to be prevented or treated. The expression of at least one of MMP-12 and TIMP1 can be significantly reduced.
또한, Milk fat globule-EGF factor 8(MFG-E8) 단백질 기반의 재조합되어 서열번호1의 아미노산 서열로 이루는 특발성 폐섬유증 예방 또는 치료용 재조합 단백질은 병변내 투여, 혈관내 투여, 피하 투여, 비강내 투여 또는 복강내 투여용으로 제형화될 수 있다.In addition, the recombinant protein for preventing or treating idiopathic pulmonary fibrosis consisting of the amino acid sequence of SEQ ID NO: 1 that is recombinant based on Milk fat globule-EGF factor 8 (MFG-E8) protein is administered intralesionally, intravascularly, subcutaneously, intranasally It can be formulated for administration or intraperitoneal administration.
한편, 상기 목적을 달성하기 위하여 본 발명에 따른 특발성 폐섬유증 예방 또는 치료용 조성물은, Milk fat globule-EGF factor 8(MFG-E8) 단백질 기반의 재조합되어 서열번호1의 아미노산 서열로 이루는 특발성 폐섬유증 예방 또는 치료용 재조합 단백질을 유효성분으로 포함한다.On the other hand, in order to achieve the above object, the composition for preventing or treating idiopathic pulmonary fibrosis according to the present invention is idiopathic pulmonary fibrosis consisting of the milk fat globule-EGF factor 8 (MFG-E8) protein-based recombinant amino acid sequence of SEQ ID NO: 1 The recombinant protein for prevention or treatment is included as an active ingredient.
또한, 상기 목적을 달성하기 위하여 본 발명은 Milk fat globule-EGF factor 8(MFG-E8) 단백질 기반의 재조합되어 서열번호1의 아미노산 서열로 이루는 특발성 폐섬유증 예방 또는 치료용 재조합 단백질을 코딩하는 유전자를 또 다른 하나의 양태로서 제공한다.In addition, in order to achieve the above object, the present invention is a gene encoding a recombinant protein for preventing or treating idiopathic pulmonary fibrosis consisting of an amino acid sequence of SEQ ID NO: 1 that is recombined based on the milk fat globule-EGF factor 8 (MFG-E8) protein. It provides as another aspect.
또한, 상기 목적을 달성하기 위하여 본 발명은 Milk fat globule-EGF factor 8(MFG-E8) 단백질 기반의 재조합되어 서열번호1의 아미노산 서열로 이루는 특발성 폐섬유증 예방 또는 치료용 재조합 단백질을 코딩하는 유전자를 포함하는 재조합 벡터를 또 다른 하나의 양태로서 제공한다.In addition, in order to achieve the above object, the present invention is a gene encoding a recombinant protein for preventing or treating idiopathic pulmonary fibrosis consisting of an amino acid sequence of SEQ ID NO: 1 that is recombined based on the milk fat globule-EGF factor 8 (MFG-E8) protein. A recombinant vector comprising the same is provided as another aspect.
본 발명에 의하면 다음과 같은 효과가 있다.According to the present invention has the following effects.
첫째, 특발성 폐섬유증 예방 또는 치료용 재조합 단백질을 통해 특발성 폐섬유증에 의해 치료대상의 폐 조직에 우수한 항섬유화 효과를 제공하여 폐 구조 및 기능을 정상 수준으로 회복시킬 수 있다.First, it is possible to restore the lung structure and function to a normal level by providing excellent antifibrosis effect to the lung tissue of the treatment target by idiopathic pulmonary fibrosis through recombinant protein for prevention or treatment of idiopathic pulmonary fibrosis.
둘째, 특발성 폐섬유증 예방 또는 치료용 재조합 단백질을 통해 특발성 폐섬유증에 의해 치료대상 폐 조직의 섬유화 지표인 Collagen 및 α-SMA(α-Smooth Muscle Actin)의 발현을 현저히 낮출 수 있다.Second, through the use of a recombinant protein for preventing or treating idiopathic pulmonary fibrosis, expression of collagen and α-SMA (α-Smooth Muscle Actin), which are fibrosis indicators of lung tissue to be treated, can be significantly lowered by idiopathic pulmonary fibrosis.
셋째, 특발성 폐섬유증 예방 또는 치료용 재조합 단백질을 통해 특발성 폐섬유증에 의해 치료대상 폐 조직의 섬유화 지표인 Col1a1, MMP-2, MMP-12 및 TIMP1의 발현을 현저히 낮출 수 있다.Third, through the use of a recombinant protein for preventing or treating idiopathic pulmonary fibrosis, expression of Col1a1, MMP-2, MMP-12, and TIMP1, which are fibrosis indicators of lung tissue to be treated, can be significantly lowered by idiopathic pulmonary fibrosis.
넷째, 특발성 폐섬유증 예방 또는 치료용 재조합 단백질을 통해 특발성 폐섬유증에 의해 치료대상 폐 조직의 섬유화 유발과 관련된 신호전달체계의 하위인자인 인산화된 ERK(Extracellular signal-Regulated Kinase) 및 인산화된 SMAD2의 발현을 현저히 낮출 수 있다.Fourth, expression of phosphorylated extracellular signal-regulated kinase (ERK) and phosphorylated SMAD2, which are sub-factors of the signaling system associated with inducing fibrosis of lung tissue to be treated by idiopathic pulmonary fibrosis through recombinant proteins for prevention or treatment of idiopathic pulmonary fibrosis. Can be significantly lowered.
다섯째, 생체 친화적이고, 부작용이 적은 특발성 폐섬유증 예방 또는 치료용 재조합 단백질 및 이를 포함하는 조성물을 제공할 수 있다.Fifth, it is possible to provide a recombinant protein for preventing or treating idiopathic pulmonary fibrosis, which is bio-friendly and has fewer side effects, and a composition comprising the same.
여섯째, 대량 생산 및 품질관리가 용이한 특발성 폐섬유증 예방 또는 치료용 재조합 단백질 및 이를 포함하는 조성물을 제공할 수 있다.Sixth, it is possible to provide a recombinant protein for preventing or treating idiopathic pulmonary fibrosis that is easy to mass production and quality control and a composition containing the same.
도1은 본 발명의 특발성 폐섬유증 예방 또는 치료용 재조합 단백질(NP-011)을 코딩하는 유전자가 삽입된 Backbone Vector의 구조를 도시하고 있다.1 shows the structure of a backbone vector in which a gene encoding a recombinant protein (NP-011) for preventing or treating idiopathic pulmonary fibrosis of the present invention is inserted.
도2는 도1에 도시된 Backbone Vector의 구조 내 삽입될 수 있으며, 본 발명의 특발성 폐섬유증 예방 또는 치료용 재조합 단백질(NP-011)을 코딩하는 유전자의 DNA 염기서열을 도시하고 있다.Figure 2 can be inserted into the structure of the Backbone Vector shown in Figure 1, shows the DNA sequence of the gene encoding a recombinant protein (NP-011) for preventing or treating idiopathic pulmonary fibrosis of the present invention.
도3은 본 발명의 특발성 폐섬유증 예방 또는 치료용 재조합 단백질(NP-011)의 아미노산 서열을 도시하고 있다.Figure 3 shows the amino acid sequence of the recombinant protein (NP-011) for preventing or treating idiopathic pulmonary fibrosis of the present invention.
도4는 본 발명의 특발성 폐섬유증 예방 또는 치료용 재조합 단백질(NP-011)의 효과 검증을 위해 설계된 동물실험 모델의 제작 과정을 나타낸 모식도이다.Figure 4 is a schematic diagram showing the production process of an animal experimental model designed for verifying the effect of the recombinant protein for preventing or treating idiopathic pulmonary fibrosis of the present invention (NP-011).
도5는 본 발명의 특발성 폐섬유증 예방 또는 치료용 재조합 단백질(NP-011)의 효과 검증을 위해 설계된 동물실험 모델들의 폐 조직 내 Collagen 및 α-SMA의 분포를 보여주는 염색 결과를 도시하고 있다.Figure 5 shows the staining results showing the distribution of collagen and α-SMA in the lung tissue of animal experimental models designed to verify the effect of the recombinant protein for preventing or treating idiopathic pulmonary fibrosis of the present invention (NP-011).
도6은 본 발명의 특발성 폐섬유증 예방 또는 치료용 재조합 단백질(NP-011)의 효과 검증을 위해 설계된 동물실험 모델들의 폐 조직 내 폐 조직 섬유화 관련 바이오마커들의 발현양상을 나타낸 그래프이다. Figure 6 is a graph showing the expression patterns of lung tissue fibrosis-related biomarkers in lung tissue of animal experimental models designed to verify the effect of recombinant protein (NP-011) for preventing or treating idiopathic pulmonary fibrosis of the present invention.
도7은 본 발명의 특발성 폐섬유증 예방 또는 치료용 재조합 단백질(NP-011)의 효과 검증을 위해 설계된 동물실험 모델들의 폐 조직 내 폐 조직의 섬유화 유발과 관련된 신호전달체계의 하위인자들의 발현양상을 나타낸 그래프이다. Figure 7 shows the expression pattern of the sub-factors of the signaling system related to inducing fibrosis of lung tissue in lung tissue of animal experimental models designed to verify the effect of recombinant protein (NP-011) for preventing or treating idiopathic pulmonary fibrosis of the present invention. It is the graph shown.
도8은 본 발명의 특발성 폐섬유증 예방 또는 치료용 재조합 단백질(NP-011)의 효과 검증을 위해 설계된 동물실험 모델 중 NP-011을 주입 시점을 달리한 두 시험군의 제작 과정을 나타낸 모식도이다.8 is a schematic diagram showing the production process of two test groups with different injection timings of NP-011 among animal experimental models designed for verifying the effect of recombinant protein (NP-011) for preventing or treating idiopathic pulmonary fibrosis of the present invention.
도9는 본 발명의 특발성 폐섬유증 예방 또는 치료용 재조합 단백질(NP-011)의 효과 검증을 위해 설계된 동물실험 모델 중 3일차 시점에 NP-011을 주입한 동물실험 모델 시험군의 폐 조직 내 Collagen 및 α-SMA의 분포 변화를 비교한 염색 결과를 도시하고 있다.9 is collagen in lung tissue of the animal experimental model test group injected with NP-011 at day 3 of the animal experimental model designed for verifying the effect of the recombinant protein for preventing or treating idiopathic pulmonary fibrosis of the present invention (NP-011) And staining results comparing the distribution of α-SMA distribution.
도10은 본 발명의 특발성 폐섬유증 예방 또는 치료용 재조합 단백질(NP-011)의 효과 검증을 위해 설계된 동물실험 모델 중 5일차 시점에 NP-011을 주입한 동물실험 모델 시험군의 폐 조직 내 Collagen 및 α-SMA의 분포 변화를 비교한 염색 결과를 도시하고 있다.Figure 10 is collagen in the lung tissue of the animal experimental model test group injected with NP-011 at day 5 of the animal experimental model designed for verifying the effect of the recombinant protein for preventing or treating idiopathic pulmonary fibrosis of the present invention (NP-011) And staining results comparing the distribution of α-SMA distribution.
도11은 본 발명의 특발성 폐섬유증 예방 또는 치료용 재조합 단백질(NP-011)의 효과 검증을 위해 설계된 동물실험 모델 중 3일차 시점에 NP-011을 주입한 동물실험 모델 시험군의 폐 조직 내 폐 조직의 섬유화 유발과 관련된 신호전달체계의 하위인자들의 발현양상 변화 및 차이를 비교한 그래프이다. Figure 11 is a lung in the lung tissue of the animal experimental model test group injected with NP-011 at day 3 of the animal experimental model designed for verifying the effect of the recombinant protein for preventing or treating idiopathic pulmonary fibrosis of the present invention (NP-011) This graph compares the changes in expression patterns and differences in the sub-factors of the signaling system related to tissue fibrosis.
도12는 본 발명의 특발성 폐섬유증 예방 또는 치료용 재조합 단백질(NP-011)의 효과 검증을 위해 설계된 동물실험 모델 중 5일차 시점에 NP-011을 주입한 동물실험 모델 시험군의 폐 조직 내 폐 조직의 섬유화 유발과 관련된 신호전달체계의 하위인자들의 발현양상 변화 및 차이를 비교한 그래프이다. Figure 12 is a lung in the lung tissue of the animal experimental model test group injected with NP-011 at the time of day 5 of the animal experimental model designed for verifying the effect of the recombinant protein for preventing or treating idiopathic pulmonary fibrosis of the present invention (NP-011) This graph compares the changes in expression patterns and differences in the sub-factors of the signaling system related to tissue fibrosis.
도13은 본 발명의 특발성 폐섬유증 예방 또는 치료용 재조합 단백질(NP-011)의 효과 검증을 위해 설계된 동물실험 모델 중 3일차 시점에 NP-011을 주입한 동물실험 모델 시험군의 폐 조직 내 폐 조직 섬유화 관련 바이오마커들의 발현양상 변화 및 차이를 비교한 그래프이다. Figure 13 is a lung in the lung tissue of the animal experimental model test group injected with NP-011 at day 3 of the animal experimental model designed for verifying the effect of the recombinant protein (NP-011) for preventing or treating idiopathic pulmonary fibrosis of the present invention It is a graph comparing the changes in expression patterns and differences of biomarkers related to tissue fibrosis.
본 발명의 바람직한 실시예에 대하여 첨부된 도면을 참조하여 더 구체적으로 설명하되, 이미 주지된 기술적 부분에 대해서는 설명의 간결함을 위해 생략하거나 압축하기로 한다.Preferred embodiments of the present invention will be described in more detail with reference to the accompanying drawings, but for the brevity of description, the already-known technical parts will be omitted or compressed.
1. 특발성 폐섬유증 예방 또는 치료용 재조합 단백질(NP-011)에 관한 설명1. Description of recombinant protein for preventing or treating idiopathic pulmonary fibrosis (NP-011)
본 발명에 따른 특발성 폐섬유증 예방 또는 치료용 재조합 단백질(NP-011)의 수득이 이루어지는 과정 및 수득된 단백질의 구조적 특징에 대해 이하에서 도1 내지 도3을 참조하여 상세하게 설명한다.The process of obtaining the recombinant protein for preventing or treating idiopathic pulmonary fibrosis according to the present invention (NP-011) and the structural characteristics of the obtained protein will be described in detail with reference to FIGS. 1 to 3 below.
(1) 특발성 폐섬유증 예방 또는 치료용 재조합 단백질(NP-011)의 수득(1) Obtaining recombinant protein (NP-011) for preventing or treating idiopathic pulmonary fibrosis
먼저 특발성 폐섬유증 예방 또는 치료용 재조합 단백질(NP-011)의 발현 과정을 설명하면, Milk fat globule-EGF factor 8(MFG-E8) 단백질 구조 개량을 위해 Origene사의 MFG-E8(NM_005928) Human cDNA Clone (Cat. No. RG217163)을 구입하여, 이를 주형(Template)으로 사용하고 PCR을 진행하여 도2에 도시된 바와 같은 DNA 단편(fragments)을 획득(서열번호4 참고)한다.First, the process of expression of the recombinant protein for preventing or treating idiopathic pulmonary fibrosis (NP-011) is described. To improve the protein structure of milk fat globule-EGF factor 8 (MFG-E8), Origene's MFG-E8 (NM_005928) Human cDNA Clone (Cat. No. RG217163) was purchased, used as a template and subjected to PCR to obtain DNA fragments as shown in FIG. 2 (see SEQ ID NO: 4).
그 후, 도1에 도시된 바와 같은 구조의 Mammalian expression Vector인 pLFCF Vector의 HindⅢ 및 SalⅠ 제한효소 부위(도1의 A부위)에 PCR 증폭을 통해 수득한 도2의 DNA 단편(서열번호4 참고)을 삽입하는 클로닝을 진행한다.Thereafter, the DNA fragment of FIG. 2 obtained through PCR amplification at the HindIII and SalI restriction sites (A portion of FIG. 1) of the pLFCF Vector, which is a Mammalian expression Vector of the structure as shown in FIG. 1 (see SEQ ID NO: 4). Proceed with cloning to insert.
이 후, plasmid DNA를 추출하여 HEK293 세포로 형질도입(Transfection)하여 2일 후 배양액을 모아 FLAG resin으로 면역침강반응(IP, Immunoprecipitation)하여 웨스턴 블로팅(Western Blotting)으로 특발성 폐섬유증 예방 또는 치료용 재조합 단백질(NP-011)에 해당하는 재조합 단백질의 발현을 확인한다.Thereafter, plasmid DNA is extracted and transfected into HEK293 cells, and after 2 days, the culture is collected, and immunoprecipitation reaction (IP, Immunoprecipitation) is performed with FLAG resin to prevent or treat idiopathic pulmonary fibrosis by Western Blotting. The expression of the recombinant protein corresponding to the recombinant protein (NP-011) is confirmed.
다음으로 특발성 폐섬유증 예방 또는 치료용 재조합 단백질(NP-011)의 대량 생산 과정을 설명하면, 발현의 확인을 거친 plasmid DNA를 Maxi prep을 통해 다량 확보한 후, HEK293 세포를 준비하여 대량의 plasmid DNA를 HEK293 세포에 형질도입하여 특발성 폐섬유증 예방 또는 치료용 재조합 단백질(NP-011)에 해당하는 재조합 단백질의 대량 생산을 수행할 수 있다.Next, when the mass production process of the recombinant protein for preventing or treating idiopathic pulmonary fibrosis (NP-011) is described, a large amount of plasmid DNA that has been confirmed through expression is secured through Maxi prep, and then HEK293 cells are prepared to prepare a large amount of plasmid DNA. And transduced into HEK293 cells to perform mass production of a recombinant protein corresponding to the recombinant protein for preventing or treating idiopathic pulmonary fibrosis (NP-011).
다음으로 특발성 폐섬유증 예방 또는 치료용 재조합 단백질(NP-011)의 품질 관리를 위한 정제 과정을 설명하면, Corning사의 CellSTACK Cell Culture Chamer 10개를 준비하여 HEK293 세포를 도포한 뒤, 1600μg의 plasmid DNA와 3200μl의 Transfection Reagents를 상온에서 15분간 혼합한 후, 형질도입을 수행한다. Next, the purification process for quality control of the recombinant protein for preventing or treating idiopathic pulmonary fibrosis (NP-011) is described. After preparing 10 CellSTACK Cell Culture Chamers of Corning and applying HEK293 cells, 1600μg of plasmid DNA and After 3200 μl of Transfection Reagents were mixed at room temperature for 15 minutes, transduction was performed.
형질 도입 수행 4시간 뒤, 배양액을 교체해주고 6일간 추가 배양을 진행하였으며, 배양액은 2일마다 총 3번 수거하여 수거된 배양액에서 Affinity식 단백질 정제를 진행한다.After 4 hours of transduction, the culture medium was replaced and further cultured for 6 days, and the culture medium was collected 3 times every 2 days to perform Affinity-type protein purification from the collected culture medium.
이와 같은 정제 과정은 각각의 단백질의 C-terminal에 FLAG 유전자가 발현(서열번호10 참고)되어 있으므로 FLAG affinity resin을 이용해 표적 단백질만 결합시킨 뒤, 세척 완충액(Washing Buffer)으로 세척하여 표적 단백질을 제외한 단백질들을 제거할 수 있다.In this purification process, since the FLAG gene is expressed in the C-terminal of each protein (see SEQ ID NO: 10), only the target protein is bound using FLAG affinity resin, and then washed with washing buffer to exclude the target protein. Proteins can be removed.
이 후, 용출 완충액(Elution Buffer)을 이용해 순수한 표적 단백질만을 추출한 뒤, 최종적으로 획득한 단백질 확인을 위해 SDS-PAGE를 진행하여 Coomassie Blue 염색 및 Anti-FLAG 항체를 이용한 웨스턴 블로팅(Western Blotting)을 통해 표적단백질의 생산 및 순도를 확인할 수 있다.Thereafter, only pure target protein was extracted using Elution Buffer, and then SDS-PAGE was performed to confirm the finally obtained protein, followed by Western blotting using Coomassie Blue staining and Anti-FLAG antibody. Through this, the production and purity of the target protein can be confirmed.
이와 같은 Milk fat globule-EGF factor 8(MFG-E8) 단백질에 기반을 두고 재조합되어 마련되는 특발성 폐섬유증 예방 또는 치료용 재조합 단백질(NP-011)의 발현을 위한 클로닝, 대량생산 및 정제 등을 위해 앞 서 설명된 일련의 구체적 과정은 이에 한정되는 것이 아니라, 아래 설명되어질 재조합 단백질의 구체적인 아미노산 서열 구조(서열번호1 및 도3 참고)의 구축을 위해 당업계의 기술자들에게 공지되고 자명한 수준의 기술을 이용한 다양한 방식으로 구현 가능하다.For cloning, mass production and purification for the expression of recombinant protein (NP-011) for prevention or treatment of idiopathic pulmonary fibrosis, which is prepared based on the milk fat globule-EGF factor 8 (MFG-E8) protein. The series of specific processes described above is not limited to this, and it is known and obvious to those skilled in the art for the construction of specific amino acid sequence structures (see SEQ ID NOs: 1 and 3) of the recombinant protein to be described below. It can be implemented in various ways using technology.
예를 들어, Milk fat globule-EGF factor 8(MFG-E8) 단백질에 기반을 두고 재조합되어 마련되는 특발성 폐섬유증 예방 또는 치료용 재조합 단백질(NP-011)의 클로닝에 이용되는 발현 벡터의 구조, 형질전환 수행 대상, 생산 조건 및 형태, 정제 조건 및 형태 등의 기술은 다양하게 실시 가능하다.For example, the structure and traits of expression vectors used for cloning of recombinant protein (NP-011) for prevention or treatment of idiopathic pulmonary fibrosis, which is prepared based on milk fat globule-EGF factor 8 (MFG-E8) protein, and recombined. Techniques such as conversion targets, production conditions and forms, purification conditions and forms can be carried out in various ways.
아울러, Milk fat globule-EGF factor 8(MFG-E8) 단백질에 기반을 두고 재조합되어 마련되는 특발성 폐섬유증 예방 또는 치료용 재조합 단백질(NP-011)의 발현과 관련하여 발현 벡터에 클로닝되는 DNA 단편(fragments)의 구조 역시 도2의 구조를 기본으로 하되, 실시에 따라 특정 Signal Peptide를 코딩하는 특정 DNA 염기서열이 추가로 DNA 단편(fragments)에 연결되는 형태로 실시 될 수도 있다.In addition, a DNA fragment that is cloned into an expression vector in relation to the expression of a recombinant protein for preventing or treating idiopathic pulmonary fibrosis (NP-011), which is prepared based on milk fat globule-EGF factor 8 (MFG-E8) protein ( The structure of fragments) is also based on the structure of FIG. 2, but according to an embodiment, a specific DNA sequence encoding a specific signal peptide may be additionally linked to DNA fragments.
(2) 특발성 폐섬유증 예방 또는 치료용 재조합 단백질(NP-011)의 구조(2) Structure of recombinant protein (NP-011) for preventing or treating idiopathic pulmonary fibrosis
앞 서 설명한, 재조합 단백질의 클로닝, 분리, 생산, 정제 등의 과정을 거쳐 최종적으로 마련되는 Milk fat globule-EGF factor 8(MFG-E8) 단백질 기반의 특발성 폐섬유증 예방 또는 치료용 재조합 단백질(NP-011)은 하기 서열목록 내 서열번호1 또는 도3에 도시된 바와 같은 아미노산 서열 구조를 갖춘다.Recombinant protein for prevention or treatment of idiopathic pulmonary fibrosis based on milk fat globule-EGF factor 8 (MFG-E8) protein, which is finally prepared through the processes of cloning, separation, production, and purification of the recombinant protein described above. 011) has the amino acid sequence structure as shown in SEQ ID NO: 1 or 3 in the following sequence listing.
특징적으로, Milk fat globule-EGF factor 8(MFG-E8) 단백질 기반의 특발성 폐섬유증 예방 또는 치료용 재조합 단백질(NP-011)은 MFG-E8 단백질의 구조적 구성인 EGF-like Domain, C1 Domain 및 C2 Domain 중 EGF-like Domain(서열번호2 참고) 및 C1 Domain(서열번호3 참고)을 포함하는 구조로 재조합된다.Characteristically, the recombinant protein for preventing or treating idiopathic pulmonary fibrosis based on the milk fat globule-EGF factor 8 (MFG-E8) protein (NP-011) is a structural component of the MFG-E8 protein, EGF-like Domain, C1 Domain, and C2. Among domains, EGF-like domain (refer to SEQ ID NO: 2) and C1 domain (refer to SEQ ID NO: 3) are recombined into structures.
다시 말해, 본 발명의 Milk fat globule-EGF factor 8(MFG-E8) 단백질 기반의 특발성 폐섬유증 예방 또는 치료용 재조합 단백질(NP-011)은"EGF-like Domain+C1 Domain"의 아미노산 서열을 이루는 것을 기본 구조로 삼고 있고, C2 Domain이 구조 상 배제되어짐을 특징으로 한다.In other words, the recombinant protein for preventing or treating idiopathic pulmonary fibrosis based on the milk fat globule-EGF factor 8 (MFG-E8) protein of the present invention (NP-011) forms an amino acid sequence of "EGF-like Domain + C1 Domain" It is used as a basic structure, and is characterized in that the C2 domain is excluded from the structure.
이와 같은 본 발명의 Milk fat globule-EGF factor 8(MFG-E8) 단백질 기반의 특발성 폐섬유증 예방 또는 치료용 재조합 단백질(NP-011)은 특발성 폐섬유증 예방 또는 치료용으로 이용되는 약학적 조성물의 주요 유효성분으로 이용될 수 있다.The milk fat globule-EGF factor 8 (MFG-E8) protein-based idiopathic pulmonary fibrosis prevention or treatment recombinant protein (NP-011) of the present invention is a major pharmaceutical composition used for the prevention or treatment of idiopathic pulmonary fibrosis. It can be used as an active ingredient.
아울러, 본 발명의 Milk fat globule-EGF factor 8(MFG-E8) 단백질 기반의 특발성 폐섬유증 예방 또는 치료용 재조합 단백질(NP-011) 및 이를 주요 유효성분으로 하는 약학적 조성물은 병변내 투여, 혈관(동맥, 정맥 등)내 투여, 피하 투여, 비강내 투여, 복강내 투여 또는 특정 조직(폐 조직)내 투여용으로 제형화할 수 있다.In addition, the milk fat globule-EGF factor 8 (MFG-E8) protein-based idiopathic pulmonary fibrosis prevention or treatment recombinant protein (NP-011) of the present invention and a pharmaceutical composition using it as a main active ingredient are administered in a lesion, blood vessels It can be formulated for intrathecal administration (artery, vein, etc.), subcutaneous administration, intranasal administration, intraperitoneal administration, or administration in a specific tissue (lung tissue).
한편, 본 발명의 Milk fat globule-EGF factor 8(MFG-E8) 단백질 기반의 특발성 폐섬유증 예방 또는 치료용 재조합 단백질(NP-011)은 앞 서 설명한 재조합 단백질의 발현과 관련하여 발현 벡터에 클로닝되는 DNA 단편(fragments)의 구조적 실시 형태의 범위와 대응되어 실시에 따라 특정 Signal Peptide 구조의 추가 연결이 이루어질 수도 있다.Meanwhile, the recombinant protein for preventing or treating idiopathic pulmonary fibrosis based on the milk fat globule-EGF factor 8 (MFG-E8) protein of the present invention (NP-011) is cloned into an expression vector in relation to the expression of the recombinant protein described above. Corresponding to the scope of the structural embodiment of the DNA fragments (fragments), depending on the implementation, additional linkage of a specific signal peptide structure may be made.
예를 들어, 특정 Signal Peptide 구조는"MPRPRLLAALCGALLCAPSLLVA"와 같은 아미노산 서열(서열번호5 참고)을 갖추어 EGF-like Domain 선단에 연결되는 형태로 실시 가능하나 이에 한정되지는 아니한다.For example, a specific Signal Peptide structure may be implemented in a form that is connected to the EGF-like domain tip by having an amino acid sequence such as "MPRPRLLAALCGALLCAPSLLVA" (see SEQ ID NO: 5), but is not limited thereto.
2. 특발성 폐섬유증 예방 또는 치료용 재조합 단백질(NP-011)의 효과 검증 시험 결과에 관한 설명2. Description of the test results for the verification of the effect of the recombinant protein for preventing or treating idiopathic pulmonary fibrosis (NP-011)
본 발명의 Milk fat globule-EGF factor 8(MFG-E8) 단백질 기반의 특발성 폐섬유증 예방 또는 치료용 재조합 단백질(NP-011)과 관련하여 해당 재조합 단백질에 기인한 특발성 폐섬유증 예방 또는 치료의 효과 수준 검증을 시험을 통해 확인하였으며, 해당 시험은 당업계의 기술자들에게 자명한 수단에 의한 성질 등을 정의하기 위한 목적으로 하기 실험 방법들을 이용하였다. Effect level of the prevention or treatment of idiopathic pulmonary fibrosis caused by the recombinant protein in relation to the recombinant protein (NP-011) for preventing or treating idiopathic pulmonary fibrosis based on the milk fat globule-EGF factor 8 (MFG-E8) protein of the present invention Verification was confirmed through tests, and the following test methods were used for the purpose of defining properties by means obvious to those skilled in the art.
(1) 특발성 폐섬유증 동물모델의 제작 및 실험설계(1) Idiopathic pulmonary fibrosis animal model production and experimental design
우선, C57BL/6 생쥐에 3mg/kg의 블레오마이신(Bleomycin)을 기관 내 점적 주입하여 급성특발성 폐섬유증(Idiopathic Pulmonary Fibrosis) 유도 동물모델을 제작한다.First, an animal model for inducing acute idiopathic pulmonary fibrosis is prepared by instilling 3 mg / kg of bleomycin into the C57BL / 6 mice.
이를 통해 제작된 급성특발성 폐섬유증(Idiopathic Pulmonary Fibrosis) 유도 동물모델 생쥐(n=7)는 도면 기준'BLM'로 표시되는 모델군으로 분류하였으며, 급성특발성 폐섬유증 유발이 진행되지 않은 10주령의 C57BL/6 생쥐(n=5)인 동물모델은 정상적 대조군(Wild Type Control)인'CON'으로 분류하였다.The resulting acute idiopathic pulmonary fibrosis induced animal model mice (n = 7) were classified into the model group indicated by the drawing 'BLM', and the 10-week-old C57BL with no acute idiopathic pulmonary fibrosis induced progression Animal models of / 6 mice (n = 5) were classified as 'CON', a normal control (Wild Type Control).
이 후, 제작 설계된 'BLM'모델군은 도4에 도시된 바와 같이 급성특발성 폐섬유증의 유도를 위한 블레오마이신(Bleomycin)의 주입 후 각각 3일째, 5일째, 7일째 및 14일째에 걸친 4회에 동안 동물모델 생쥐의 폐 조직을 추출하여 분석을 진행하였다. Thereafter, the design and design of the 'BLM' model group, as shown in FIG. 4, 4 times over 3, 5, 7, and 14 days after injection of bleomycin for induction of acute idiopathic pulmonary fibrosis, respectively In the meantime, the lung tissues of animal model mice were extracted and analyzed.
다음으로, 본 발명의 특발성 폐섬유증 예방 또는 치료용 재조합 단백질(NP-011)의 효과 검증을 위한 실험군은 도8에 도시된 바와 같이 앞 서 제작된'BLM'모델군의 급성특발성 폐섬유증 유도 동물모델 생쥐에 1일 기준 160μg/kg의 NP-011을 생쥐 꼬리의 정맥 내에 투여하여 마련하였으며, 도면 기준'BLM+NP011'로 표시된다. Next, the experimental group for verifying the effect of the recombinant protein for preventing or treating idiopathic pulmonary fibrosis of the present invention (NP-011) is acute idiopathic pulmonary fibrosis inducing animal of the 'BLM' model group prepared as shown in FIG. The model mice were prepared by administering 160 μg / kg NP-011 in the vein of the tail of the mouse on a daily basis, and are designated as 'BLM + NP011' based on the drawing.
이와 같이 마련되는 'BLM+NP011'의 실험군은 도8에 도시된 바와 같이 특발성 폐섬유증 예방 또는 치료용 재조합 단백질(NP-011)의 투여시기를 달리하여 블레오마이신(Bleomycin)의 주입 후 3일 경과시점에 NP-011를 1회 주입한 제1실험군(CASE 1)과 블레오마이신(Bleomycin)의 주입 후 5일 경과시점에 NP-011를 1회 주입한 제2실험군(CASE 2)으로 분류하였다.As shown in FIG. 8, the experimental group of 'BLM + NP011' prepared as described above was changed for 3 days after injection of bleomycin by varying the administration time of the recombinant protein for preventing or treating idiopathic pulmonary fibrosis (NP-011). It was classified into the first experimental group (CASE 1) in which NP-011 was injected once at the time point and the second experimental group (CASE 2) in which NP-011 was injected once at 5 days after the injection of bleomycin.
이를 통해 제작되는 제1실험군의 급성특발성 폐섬유증 유도 동물모델 생쥐(n=7)는 폐 조직의 추출을 통한 효과 분석의 진행을 블레오마이신(Bleomycin)의 주입 후 6일 경과시점에 수행했으며, 제2실험군의 급성특발성 폐섬유증 유도 동물모델 생쥐(n=7)는 폐 조직의 추출을 통한 효과 분석의 진행을 블레오마이신(Bleomycin)의 주입 후 8일 경과시점에 수행했다.The acute idiopathic pulmonary fibrosis-induced animal model mice (n = 7) of the first experimental group produced through this were performed at 6 days after injection of bleomycin through the effect analysis through extraction of lung tissue. The acute idiopathic pulmonary fibrosis-induced animal model mice (n = 7) in the experimental group 2 were performed 8 days after the injection of bleomycin through the effect analysis through extraction of lung tissue.
(2) 특발성 폐섬유증 동물모델의 발병 여부 및 수준 검증(2) Verification and development of idiopathic pulmonary fibrosis animal model
우선적으로, 급성특발성 폐섬유증(Idiopathic Pulmonary Fibrosis) 유도 동물모델 생쥐로 제작된 'BLM'모델군의 급성특발성 폐섬유증 발병 여부 및 이로 인한 폐 조직의 변화를 관찰하기 위해 블레오마이신(Bleomycin)의 주입 후 각각 3일째, 5일째, 7일째 및 14일째에 걸친 4회에 동안 동물모델 생쥐의 폐 조직을 추출하여 조직 염색 분석 및 폐 조직 섬유화 관련 특정 성분의 발현 양상을 검증하였다.First of all, after the injection of bleomycin to observe whether acute idiopathic pulmonary fibrosis develops in the 'BLM' model group, which is made of animal model mice induced with Idiopathic Pulmonary Fibrosis, and thereby changes in lung tissue. Lung tissues of animal model mice were extracted for 4 times over 3, 5, 7, and 14 days, respectively, to verify tissue staining analysis and expression of specific components related to lung tissue fibrosis.
먼저, 'BLM'모델군의 급성특발성 폐섬유증 유도 동물모델 생쥐들을 블레오마이신(Bleomycin)의 주입 후 각각 3일째, 5일째, 7일째 및 14일째가 경과된 시점마다 마취 후 심장관류를 통해 PBS를 주입 후 폐 조직을 적출하였다. First, acute idiopathic pulmonary fibrosis-inducing animal model mice of the 'BLM' model group were injected with bleomycin, followed by anesthesia after 3 days, 5 days, 7 days, and 14 days, followed by cardiac perfusion through PBS. Lung tissue was removed after injection.
이와 같이 적출된 폐 조직은 4% PFA 용액에서 4일간 냉장 보관한 후, 파라핀(Paraffin)으로 포매한 폐 조직은 절편기(CM3050S, Leica)를 사용하여 4㎛의 두께로 잘라 슬라이드(silane coated slide)에 부착시켰다. The extracted lung tissue is refrigerated for 4 days in a 4% PFA solution, and the lung tissue embedded with paraffin is cut to a thickness of 4 μm using a sectioning machine (CM3050S, Leica), and a slide (silane coated slide) ).
그 후, 각각의 폐 조직 내 콜라겐(Collagen) 및 α-SMA의 발현 분포를 평가하기 위해 Trichrome 염색 및 Sirius red 염색을 수행하였다. Then, Trichrome staining and Sirius red staining were performed to evaluate the expression distribution of collagen and α-SMA in each lung tissue.
여기서, Trichrome 염색의 경우, 먼저 폐 조직 절편을 자일렌에서 지방질이 제거되고, 100% 알코올 용액에서 수화하고, 수돗물로 세척된 슬라이드는 헤마톡실린(Hematoxylin)에서 5분간 염색, Biebrich Scarlet-Acid Fuchsin에서 15분간 염색, Phosphomolybdic/phosphotungstic acid에서 10분간, 아닐린블루(Aniline blue)에서 5분간 염색, 1% 아세트산(Acetic acid)서 3분간 잠갔다가 탈수과정을 거쳐 커버슬립으로 봉입하여 진행하였다. Here, in the case of Trichrome staining, the lung tissue sections are first defatted in xylene, hydrated in a 100% alcohol solution, and the slides washed with tap water are stained with Hematoxylin for 5 minutes, Biebrich Scarlet-Acid Fuchsin The dye was stained for 15 minutes, phosphomolybdic / phosphotungstic acid for 10 minutes, aniline blue for 5 minutes, and 1% acetic acid for 3 minutes, then dehydrated and sealed with a cover slip.
아울러, Sirius red 염색의 경우, 먼저 폐 조직 절편을 자일렌에서 지방질이 제거되도록 하고, 연속적으로 알코올 용액 (100%, 95%, 80% 및 70%)을 거쳐 수화하고, 이 후 Sirius red에 7분간 염색하고 세척하여 탈수과정을 거친 뒤 커버슬립으로 봉입하여 진행하였다. In addition, in the case of Sirius red staining, first, lung tissue sections are defatted from xylene, and subsequently hydrated through an alcohol solution (100%, 95%, 80% and 70%), after which Sirius red 7 After dyeing and washing for a minute, it was dehydrated and then sealed with a cover slip to proceed.
이와 같은 Trichrome 염색 및 Sirius red 염색을 마친 'BLM'모델군의 급성특발성 폐섬유증 유도 동물모델 생쥐들의 시기별 폐 조직 슬라이드는 형광현미경 (BX61; Olympus)하에서 이미지를 얻어 도5에 도시된 바와 같다.Acute idiopathic pulmonary fibrosis-inducing animal model mice of the 'BLM' model group after the Trichrome staining and Sirius red staining, as shown in FIG. 5, obtained by pulmonary tissue slides by fluorescence microscope (BX61; Olympus).
구체적으로, 도5에 도시된 바와 같이 'BLM'모델군의 급성특발성 폐섬유증 유도 동물모델 생쥐들은 블레오마이신(Bleomycin)의 주입 후 3일째부터 폐 조직 내 주요 섬유화 지표인 콜라겐(Collagen)의 발현 분포가 조금 씩 증가하여 5일 이후로는 현저한 증가 추세를 보이고, 7일 째에는 가장 많은 증가 수준을 보이며, 14일 째에는 7일째와 유사한 수준을 보인다.Specifically, as shown in FIG. 5, the acute idiopathic pulmonary fibrosis-inducing animal model mice of the 'BLM' model group have an expression distribution of collagen, which is a major fibrosis index in lung tissue from day 3 after injection of bleomycin. Increases little by little, shows a remarkable increase after 5 days, shows the highest increase level on the 7th day, and shows a similar level to the 7th day on the 14th.
아울러, 구체적으로, 도5에 도시된 바와 같이 BLM'모델군의 급성특발성 폐섬유증 유도 동물모델 생쥐들은 블레오마이신(Bleomycin)의 주입 후 3일째부터 폐 조직 내 주요 섬유화 지표인 α-SMA(α-Smooth Muscle Actin)의 발현 분포가 혈관(Vessesl) 주변 및 세(細)기관지(Bronchiole) 주변에서 조금 씩 증가하여 시간이 지남에 따라 더욱 크게 증가하는 양상을 보인다.In addition, specifically, as shown in Figure 5, the acute idiopathic pulmonary fibrosis-inducing animal model mice of the BLM 'model group, α-SMA (α-), a major fibrosis index in lung tissue from day 3 after injection of bleomycin, The expression distribution of Smooth Muscle Actin increases slightly over time around the vessel (Vessesl) and around the bronchial (Bronchiole).
다음으로, 'BLM'모델군의 급성특발성 폐섬유증 유도 동물모델 생쥐들을 블레오마이신(Bleomycin)의 주입 후 각각 3일째, 5일째, 7일째 및 14일째가 경과된 시점에서 적출된 폐 조직의 폐 조직 섬유화 관련 바이오 마커(Biomarker)들의 발현양상을 검증하였다.Next, lung tissue of the lung tissue extracted from the 3rd, 5th, 7th and 14th days after injection of bleomycin into the acute idiopathic pulmonary fibrosis-induced animal model mice of the 'BLM' model group, respectively. The expression pattern of biomarkers related to fibrosis was verified.
구체적으로, 폐 조직에서의 섬유화 관련 바이오 마커들의 발현양상을 평가하기 위해서 프라이머를 이용한 중합효소연쇄반응(PCR, Polymerase chain reaction) 분석을 수행하였다. Specifically, polymerase chain reaction (PCR) analysis using primers was performed to evaluate the expression pattern of biomarkers related to fibrosis in lung tissue.
여기서, 적출된 각각의 폐 조직 절편은 RNeasy mini kit를 사용하여 total RNA를 추출하고 reverse transcriptase PCR (RT-PCR)을 통하여 cDNA를 합성하였으며, 이와 같이 합성된 cDNA는 quantitative RT-PCR을 통하여 발현양상을 확인하였다. Here, each extracted lung tissue section was extracted with total RNA using an RNeasy mini kit, and cDNA was synthesized through reverse transcriptase PCR (RT-PCR), and the synthesized cDNA was expressed through quantitative RT-PCR. Was confirmed.
또한, 이에 사용된 프라이머에 대한 서열구조(서열번호6 내지 서열번호9 참고)는 하기 표1과 같다.In addition, the sequence structure (refer to SEQ ID NO: 6 to SEQ ID NO: 9) for the primer used therein is shown in Table 1 below.
| 섬유화 바이오마커Fiberized biomarker | 정방향(5'→3')Forward (5 '→ 3') |
| COL1A1COL1A1 | CTGGCGGTTCAGGTCCAATTTCCAGGCAATCCACGAGCCTGGCGGTTCAGGTCCAATTTCCAGGCAATCCACGAGC |
| MMP-2MMP-2 | GCGATGTCGCCCCTAAAACAGCTGTATGTGATCTGGTTCTTGTCCGCGATGTCGCCCCTAAAACAGCTGTATGTGATCTGGTTCTTGTCC |
| MMP-12MMP-12 | TGGTATTCAAGGAGATGCGGTTTGTGCCTTGAAAACTGGTATTCAAGGAGATGCGGTTTGTGCCTTGAAAAC |
| TIMP1TIMP1 | GGGTTCCCCAGAAATCAACGAGACAGAGGCTTTCCATGACTGGGGTGGGGTTCCCCAGAAATCAACGAGACAGAGGCTTTCCATGACTGGGGTG |
그 결과, 적출된 폐 조직의 폐 조직 섬유화 관련 바이오 마커(Biomarker)들의 적출 시점별 발현양상의 결과는 도6에 도시된 바와 같다.구체적으로, 도6(도면참고- *P<0.05, **P<0.01, ***P<0.001)에 도시된 바와 같이 'BLM'모델군의 급성특발성 폐섬유증 유도 동물모델 생쥐들은 블레오마이신(Bleomycin)의 주입 후 'CON'정상적 대조군(Wild Type Control)의 정상 생쥐와 비교해 3일차부터 모두 폐 조직 내 섬유화 관련 바이오 마커들인 Col1a1, MMP-2, MMP-12 및 TIMP1의 발현이 큰 폭으로 높은 수준을 유지함을 확인할 수 있다.As a result, the results of expression patterns for each extraction time of biomarkers related to lung tissue fibrosis of the extracted lung tissue are as shown in FIG. 6. Specifically, FIG. 6 (refer to the drawing- * P <0.05, ** As shown in P <0.01, *** P <0.001), the acute idiopathic pulmonary fibrosis-inducing animal model mice of the 'BLM' model group were injected with bleomycin, followed by the 'CON' normal control (Wild Type Control). From day 3 compared to normal mice, it can be seen that the expression of Col1a1, MMP-2, MMP-12 and TIMP1, which are biomarkers related to fibrosis in the lung tissue, maintains a high level.
다음으로, 'BLM'모델군의 급성특발성 폐섬유증 유도 동물모델 생쥐들을 블레오마이신(Bleomycin)의 주입 후 각각 3일째, 5일째, 7일째 및 14일째가 경과된 시점에서 적출된 폐 조직의 폐 조직 섬유화 관련 신호전달체계의 하위인자들의 발현양상을 검증하였다.Next, lung tissue of the lung tissue extracted from the 3rd, 5th, 7th and 14th days after injection of bleomycin into the acute idiopathic pulmonary fibrosis-induced animal model mice of the 'BLM' model group, respectively. The expression pattern of sub-factors of the fibrosis-related signaling system was verified.
이를 위해, 적출된 각각의 폐 조직에서 MARK신호 및 TGFβ신호와 관련된 신호전달체계의 하위인자 발현양상을 확인하기 위해서는 웨스턴 블럿(Western blotting)을 수행하였다.To this end, Western blotting was performed to confirm the expression pattern of the sub-factor of the signaling system related to the MARK signal and the TGFβ signal in each of the extracted lung tissues.
먼저, 적출된 각각의 폐 조직을 분쇄하여 단백질을 얻은 뒤 SDS-PAGE를 통하여 사이즈 별로 단백질을 분리하고 PVDF membrane에 옮겨 일차항체로 4℃C에서 면역반응시켰다. 이에 사용된 일차항체는 α-SMA(SC-53015), pERK(#4370), tERK(#4695), pSMAD2(3104S), tSMAD2(3102S) 및 Actin(SC-47778)와 같다. First, each lung tissue extracted was pulverized to obtain a protein, and then proteins were separated by size through SDS-PAGE, transferred to a PVDF membrane, and immunized at 4 ° C as a primary antibody. Primary antibodies used for this are α-SMA (SC-53015), pERK (# 4370), tERK (# 4695), pSMAD2 (3104S), tSMAD2 (3102S) and Actin (SC-47778).
그 후, HRP(Horse Radish Peroxidase)가 결합된 이차항체로 면역반응 시킨 뒤 현상하여 band를 얻고 정량화하였으며, 이에 따른 적출된 폐 조직의 폐 조직 섬유화 관련 신호전달체계의 하위인자들의 발현양상을 검증한 결과는 도7에 도시된 바와 같다.Subsequently, the immune antibody was reacted with a secondary antibody combined with HRP (Horse Radish Peroxidase) to develop and quantify the band, thereby verifying the expression pattern of the sub-factors of the signal transduction system related to lung tissue fibrosis of the extracted lung tissue. The results are as shown in FIG. 7.
구체적으로, 도7에 도시된 바와 같이 'BLM'모델군의 급성특발성 폐섬유증 유도 동물모델 생쥐들은 블레오마이신(Bleomycin)의 주입 후 'CON'정상적 대조군(Wild Type Control)의 정상 생쥐와 비교해 폐 조직 섬유화 관련 신호전달체계의 MARK신호 하위인자인 인산화된 형태의 ERK(Extracellular signal-Regulated Kinase) 발현이 5일째부터 증가를 보여 14일 째에는 큰 폭으로 증가되어 가장 높은 수준을 갖춘다.Specifically, as shown in Figure 7, the acute idiopathic pulmonary fibrosis-induced animal model mice of the 'BLM' model group compared to normal mice of the 'CON' normal control (Wild Type Control) after injection of bleomycin The expression of the phosphorylated form of extracellular signal-regulated kinase (ERK), a sub-factor of the MARK signal of the fibrosis-related signaling system, increased from the 5th day, and was significantly increased on the 14th day to have the highest level.
또한, 도7에 도시된 바와 같이 'BLM'모델군의 급성특발성 폐섬유증 유도 동물모델 생쥐들은 블레오마이신(Bleomycin)의 주입 후 'CON'정상적 대조군(Wild Type Control)의 정상 생쥐와 비교해 폐 조직 섬유화 관련 신호전달체계의 TGFβ신호 하위인자인 인산화된 형태의 SMAD2 발현이 5일째부터 큰 폭으로 증가해 높은 수준을 갖추어 정상 수준과 큰 차이를 보인다.In addition, as shown in Figure 7, the acute idiopathic pulmonary fibrosis-induced animal model mice of the 'BLM' model group compared to normal mice of the 'CON' normal control (Wild Type Control) after injection of bleomycin, lung tissue fibrosis The expression of SMAD2 in the phosphorylated form of the TGFβ signal sub-factor of the relevant signaling system increased significantly from the 5th day, showing a large difference from the normal level.
아울러, 도7에 도시된 바와 같이 'BLM'모델군의 급성특발성 폐섬유증 유도 동물모델 생쥐들은 블레오마이신(Bleomycin)의 주입 후 'CON'정상적 대조군(Wild Type Control)의 정상 생쥐와 비교해 폐 조직 섬유화 관련 지표인 α-SMA 발현이 5일째부터 큰 폭으로 증가해 높은 수준을 갖추어 정상 수준과 큰 차이를 보인다.In addition, as shown in Figure 7, the acute idiopathic pulmonary fibrosis-inducing animal model mice of the 'BLM' model group compared to normal mice of the 'CON' normal control (Wild Type Control) after injection of bleomycin, lung tissue fibrosis The expression of α-SMA, a related indicator, has increased significantly from the 5th day and has a high level, showing a large difference from the normal level.
즉, 'BLM'모델군의 급성특발성 폐섬유증 유도 동물모델 생쥐들은 블레오마이신(Bleomycin)의 주입을 통해 급성특발성 폐섬유증이 발병하였음을 알 수 있고, 구체적으로 폐 조직 내 폐 섬유화 주요 지표인 콜라겐(Collagen) 및 α-SMA의 발현, 폐 조직 섬유화 관련 바이오 마커인 Col1a1, MMP-2, MMP-12 및 TIMP1의 발현, 폐 조직 섬유화 관련 신호전달체계 MARK신호 및 TGFβ신호의 하위인자인 인산화된 형태의 ERK 및 인산화된 형태의 SMAD2 발현이 모두 정상 생쥐의 폐 조직에 비해 증가하여 높은 수준으로 차이를 나타낸다.That is, in the animal model mice inducing acute idiopathic pulmonary fibrosis of the 'BLM' model group, it can be seen that acute idiopathic pulmonary fibrosis has occurred through injection of bleomycin, and specifically collagen (a major indicator of lung fibrosis in lung tissue) Collagen) and the expression of α-SMA, the expression of the biomarkers Col1a1, MMP-2, MMP-12 and TIMP1 related to lung tissue fibrosis, and the phosphorylated form of the signaling factor MARK signal and the TGFβ signal of the lung tissue fibrosis Both ERK and phosphorylated SMAD2 expression increased compared to the lung tissue of normal mice, showing a high level of difference.
(3) 특발성 폐섬유증 동물모델의 Collagen 및 α-SMA 발현 검사(3) Collagen and α-SMA expression test in idiopathic pulmonary fibrosis animal model
본 발명의 Milk fat globule-EGF factor 8(MFG-E8) 단백질 기반의 특발성 폐섬유증 예방 또는 치료용 재조합 단백질(NP-011)의 폐 조직 섬유화 방지 기능을 확인하기 위하여, 제1실험군 및 제2실험군에 해당하는 실험동물 생쥐의 폐 조직 내 콜라겐(Collagen) 및 α-SMA의 발현 양상을 검사하였다.Milk fat globule-EGF factor 8 of the present invention (MFG-E8) protein-based idiopathic pulmonary fibrosis prevention or treatment of the recombinant protein (NP-011) in order to confirm the lung tissue fibrosis preventing function, the first experimental group and the second experimental group The expression patterns of collagen and α-SMA in the lung tissue of the corresponding experimental animal mice were examined.
먼저, 'BLM+NP011'제1실험군의 급성특발성 폐섬유증 유도 동물모델 생쥐들을 블레오마이신(Bleomycin)의 주입 후 각각 3일째에 NP-011를 1회 주입 후 6일째가 경과된 시점에 마취 후 심장관류를 통해 PBS를 주입 후 폐 조직을 적출하였다.First, after injecting bleomycin into the acute idiopathic pulmonary fibrosis-induced animal model mice of the first experimental group of 'BLM + NP011', NP-011 was injected once on the 3rd day, and then anesthetized at the 6th day. After infusion of PBS through perfusion, lung tissue was removed.
아울러, BLM+NP011'제2실험군의 급성특발성 폐섬유증 유도 동물모델 생쥐들을 블레오마이신(Bleomycin)의 주입 후 각각 5일째에 NP-011를 1회 주입 후 8일째가 경과된 시점에 마취 후 심장관류를 통해 PBS를 주입 후 폐 조직을 적출하였다.In addition, after injecting NP-011 once on the 5th day after injecting bleomycin into the acute idiopathic pulmonary fibrosis-inducing animal model mice of the BLM + NP011 '2nd experimental group, after anesthesia, cardiac perfusion after 8 days After injecting PBS through, lung tissue was extracted.
이와 같이 적출된 제1실험군과 제2실험군의 폐 조직을 이용한 폐 조직 내 콜라겐(Collagen) 및 α-SMA의 발현 분포 평가를 위한 Trichrome 염색 및 Sirius red 염색의 진행 방법 및 결과 도출 과정은 앞 서 설명한 'BLM'모델군의 급성특발성 폐섬유증 유도 동물모델 생쥐를 대상으로 한 방법과 동일하므로 생략한다.Trichrome staining and Sirius red staining process for evaluating the expression distribution of collagen and α-SMA in lung tissue using lung tissue of the first and second experimental groups extracted as described above and the process of deriving the results are described above. It is omitted because it is the same as the method for acute idiopathic pulmonary fibrosis-induced animal model mice in the 'BLM' model group.
이에 따라, Trichrome 염색 및 Sirius red 염색을 마친 제1실험군의 동물모델 생쥐들의 시기별 폐 조직 슬라이드는 형광현미경 (BX61; Olympus)하에서 이미지를 얻어 도9에 도시된 바와 같다.Accordingly, the lung tissue slides of each animal model mice of the first experimental group after Trichrome staining and Sirius red staining were obtained by fluorescence microscopy (BX61; Olympus), as shown in FIG. 9.
이에 따라, Trichrome 염색 및 Sirius red 염색을 마친 제2실험군의 동물모델 생쥐들의 시기별 폐 조직 슬라이드는 형광현미경 (BX61; Olympus)하에서 이미지를 얻어 도10에 도시된 바와 같다.Accordingly, the lung tissue slides of each animal model mice of the second experimental group after Trichrome staining and Sirius red staining were obtained by fluorescence microscopy (BX61; Olympus), as shown in FIG. 10.
구체적으로, 도9 및 도10에 도시된 바와 같이 'BLM+NP011'제1실험군 및 제2실험군의 동물모델 생쥐들은 NP-011의 주입 후 ' BLM'모델군의 급성특발성 폐섬유증 유도 동물모델 생쥐들에 비해 폐 조직 내 주요 섬유화 지표인 콜라겐(Collagen)의 발현 분포가 현저히 감소함을 알 수 있다.Specifically, as shown in FIGS. 9 and 10, the animal model mice of the first experimental group and the second experimental group of 'BLM + NP011' are acute idiopathic pulmonary fibrosis-induced animal model mice of the 'BLM' model group after injection of NP-011. It can be seen that the expression distribution of collagen, which is a major fibrosis index in lung tissue, is significantly reduced compared to those of the above.
아울러, 도9 및 도10에 도시된 바와 같이 'BLM+NP011'제1실험군 및 제2실험군의 동물모델 생쥐들은 NP-011의 주입 후 ' BLM'모델군의 급성특발성 폐섬유증 유도 동물모델 생쥐들에 비해 폐 조직 내 주요 섬유화 지표인 α-SMA의 발현 분포가 현저히 감소함을 알 수 있다.In addition, as shown in FIGS. 9 and 10, the animal model mice of the first experimental group and the second experimental group of 'BLM + NP011' are acute idiopathic pulmonary fibrosis-induced animal model mice of the 'BLM' model group after injection of NP-011. In comparison, it can be seen that the expression distribution of α-SMA, a major fibrosis index in lung tissue, is significantly reduced.
다시 말해, 'BLM'모델군의 급성특발성 폐섬유증 유도 동물모델 생쥐들에 NP-011을 블레오마이신(Bleomycin)의 주입 후 3일째 또는 5일째의 시점에 투여하면 폐 조직 내 주요 섬유화 지표인 콜라겐(Collagen) 및 α-SMA의 발현 분포가 현저히 감소하여 폐 조직 섬유화 방지를 위한 우수한 수준의 항섬유화 효과를 제공할 수 있다.In other words, when NP-011 is administered to the acute idiopathic pulmonary fibrosis-induced animal model mice of the 'BLM' model group at the time of day 3 or 5 after injection of bleomycin, collagen, which is a major fibrosis index in lung tissue ( Collagen) and α-SMA are significantly reduced in expression distribution, thereby providing an excellent level of antifibrosis effect for preventing lung tissue fibrosis.
(4) 특발성 폐섬유증 동물모델의 섬유화 관련 신호전달체계 변화 검사(4) Examination of changes in signaling system related to fibrosis in idiopathic pulmonary fibrosis animal model
본 발명의 Milk fat globule-EGF factor 8(MFG-E8) 단백질 기반의 특발성 폐섬유증 예방 또는 치료용 재조합 단백질(NP-011)의 폐 조직 섬유화 방지 기능을 확인하기 위하여, 제1실험군 및 제2실험군에 해당하는 실험동물 생쥐의 폐 조직 내 폐 조직 섬유화 관련 신호전달체계 MARK신호 및 TGFβ신호의 하위인자 발현 양상을 검사하였다.Milk fat globule-EGF factor 8 of the present invention (MFG-E8) protein-based idiopathic pulmonary fibrosis prevention or treatment of the recombinant protein (NP-011) in order to confirm the lung tissue fibrosis preventing function, the first experimental group and the second experimental group The expression patterns of the subfactors of the MARK signal and the TGFβ signal in the lung tissue fibrosis-related signaling system in the lung tissue of experimental animal mice were examined.
먼저, 'BLM+NP011'제1실험군의 급성특발성 폐섬유증 유도 동물모델 생쥐들을 블레오마이신(Bleomycin)의 주입 후 각각 3일째에 NP-011를 1회 주입 후 6일째가 경과된 시점에 마취 후 심장관류를 통해 PBS를 주입 후 폐 조직을 적출하였다.First, after injecting bleomycin into the acute idiopathic pulmonary fibrosis-induced animal model mice of the first experimental group of 'BLM + NP011', NP-011 was injected once on the 3rd day, and then anesthetized at the 6th day. After infusion of PBS through perfusion, lung tissue was removed.
아울러, 'BLM+NP011'제2실험군의 급성특발성 폐섬유증 유도 동물모델 생쥐들을 블레오마이신(Bleomycin)의 주입 후 각각 5일째에 NP-011를 1회 주입 후 8일째가 경과된 시점에 마취 후 심장관류를 통해 PBS를 주입 후 폐 조직을 적출하였다.In addition, after injecting NP-011 once on the 5th day after injecting bleomycin into the acute idiopathic pulmonary fibrosis-induced animal model mice of the 'BLM + NP011' 2nd experimental group, after anesthesia, the heart after anesthesia After infusion of PBS through perfusion, lung tissue was removed.
이와 같이 적출된 제1실험군과 제2실험군의 폐 조직을 이용한 폐 조직 내 폐 조직 섬유화 관련 신호전달체계 MARK신호 및 TGFβ신호의 하위인자 발현 양상 검사를 위한 웨스턴 블럿(Western blotting) 기반의 검사 진행 방법 및 정량화 결과 도출 방법은 앞 서 설명한 'BLM'모델군의 급성특발성 폐섬유증 유도 동물모델 생쥐를 대상으로 한 방법과 동일하므로 생략한다.Western blotting-based testing method for examining the expression of subfactors of the MARK signal and TGFβ signal in the lung tissue fibrosis-related signaling system in lung tissue using lung tissue of the first and second experimental groups thus extracted And the method of deriving the quantification results is the same as the method for the acute idiopathic pulmonary fibrosis-induced animal model mice of the 'BLM' model group described above, and thus is omitted.
이에 따라, 제1실험군의 동물모델 생쥐들의 적출된 폐 조직 내 폐 조직 섬유화 관련 신호전달체계의 하위인자들의 발현양상을 검증한 결과는 도11에 도시된 바와 같다.Accordingly, the results of verifying the expression patterns of sub-factors of the signal transduction system related to pulmonary tissue fibrosis in the extracted lung tissue of the animal model mice of the first experimental group are shown in FIG. 11.
또한, 제2실험군의 동물모델 생쥐들의 적출된 폐 조직 내 폐 조직 섬유화 관련 신호전달체계의 하위인자들의 발현양상을 검증한 결과는 도12에 도시된 바와 같다.In addition, the results of verifying the expression patterns of sub-factors of the signaling system related to lung tissue fibrosis in the extracted lung tissue of the animal model mice of the second experimental group are shown in FIG. 12.
구체적으로, 도11에 도시된 바와 같이 'BLM+NP011'제1실험군의 동물모델 생쥐들은 NP-011의 주입 후 ' BLM'모델군의 급성특발성 폐섬유증 유도 동물모델 생쥐들에 비해 폐 조직 내 폐 조직 섬유화 관련 신호전달체계의 MARK신호 하위인자인 인산화된 형태의 ERK(Extracellular signal-Regulated Kinase) 발현이 현저히 감소함을 확인할 수 있다.Specifically, as shown in FIG. 11, the animal model mice of the first experimental group of 'BLM + NP011' compared to those of the animal model mice inducing acute idiopathic pulmonary fibrosis of the 'BLM' model group after injection of NP-011 It can be seen that the expression of the extracellular signal-regulated kinase (ERK) in phosphorylated form, which is a sub-factor of the MARK signal of the tissue fibrosis-related signaling system, is significantly reduced.
아울러, 도11에 도시된 바와 같이 'BLM+NP011'제1실험군의 동물모델 생쥐들은 NP-011의 주입 후 ' BLM'모델군의 급성특발성 폐섬유증 유도 동물모델 생쥐들에 비해 폐 조직 내 폐 조직 섬유화 관련 신호전달체계의 TGFβ신호 하위인자인 인산화된 형태의 SMAD2 발현이 역시 현저히 감소함을 확인할 수 있다.In addition, as shown in Figure 11, the animal model mice of the 'BLM + NP011' first experimental group compared to the acute idiopathic pulmonary fibrosis-induced animal model mice of the 'BLM' model group after injection of NP-011 lung tissue in the lung tissue. It can be seen that the expression of SMAD2 in the phosphorylated form, which is a subfactor of the TGFβ signal of the fibrosis-related signaling system, is also significantly reduced.
또한, 도12에 도시된 바와 같이 'BLM+NP011'제2실험군의 동물모델 생쥐들은 NP-011의 주입 후 ' BLM'모델군의 급성특발성 폐섬유증 유도 동물모델 생쥐들에 비해 폐 조직 내 폐 조직 섬유화 관련 신호전달체계의 MARK신호 하위인자인 인산화된 형태의 ERK(Extracellular signal-Regulated Kinase) 발현이 현저히 감소하여 정상 생쥐의 발현 수준과 유사해짐을 확인할 수 있다.In addition, as shown in Figure 12, the animal model mice of the 'BLM + NP011' second experimental group compared to the acute idiopathic pulmonary fibrosis-induced animal model mice of the 'BLM' model group after injection of NP-011 lung tissue in the lung tissue. It can be confirmed that the expression of the extracellular signal-regulated kinase (ERK) in the phosphorylated form, which is a sub-factor of the MARK signal of the fibrosis-related signaling system, is significantly reduced, which can be confirmed to be similar to that of normal mice.
아울러, 또한, 도12에 도시된 바와 같이 'BLM+NP011'제2실험군의 동물모델 생쥐들은 NP-011의 주입 후 ' BLM'모델군의 급성특발성 폐섬유증 유도 동물모델 생쥐들에 비해 폐 조직 내 주요 폐 조직 섬유화 지표인 α-SMA의 발현이 현저히 감소하여 정상 생쥐의 발현 수준과 유사해짐을 확인할 수 있다.In addition, as shown in FIG. 12, the animal model mice of the 'BLM + NP011' second experimental group were in lung tissue compared to the acute idiopathic pulmonary fibrosis-induced animal model mice of the 'BLM' model group after injection of NP-011. It can be seen that the expression of α-SMA, a major pulmonary tissue fibrosis indicator, is significantly reduced, which is similar to that of normal mice.
다시 말해, ' BLM'모델군의 급성특발성 폐섬유증 유도 동물모델 생쥐들에 NP-011을 블레오마이신(Bleomycin)의 주입 후 3일째 또는 5일째의 시점에 투여하면 폐 조직 내 폐 조직 섬유화 관련 신호전달체계 MARK신호 하위인자인 인산화된 형태의 ERK 및 TGFβ신호 하위인자인 인산화된 형태의 SMAD2의 발현이 급감하게 되고, 더 나아가 주요 폐 조직 섬유화 지표인 α-SMA의 발현 역시 현저히 감소하여 정상 생쥐의 발현 수준과 유사해져 폐 조직 섬유화 방지를 위한 우수한 수준의 항섬유화 효과를 제공할 수 있다.In other words, when NP-011 is administered to the acute idiopathic pulmonary fibrosis-induced animal model mice of the 'BLM' model group at the time of day 3 or 5 after injection of bleomycin, lung tissue fibrosis-related signaling in lung tissue System MARK signal sub-factor phosphorylated form of ERK and TGFβ signal sub-factor phosphorylated form of SMAD2 rapidly decreased, and further, the expression of α-SMA, a major lung tissue fibrosis index, was also significantly reduced, resulting in the expression of normal mice. Similar to the level, it can provide an excellent level of antifibrosis effect for preventing lung tissue fibrosis.
(5) 특발성 폐섬유증 동물모델의 폐섬유화 관련 바이오마커 발현 검사(5) Lung fibrosis-related biomarker expression test in idiopathic pulmonary fibrosis animal model
본 발명의 Milk fat globule-EGF factor 8(MFG-E8) 단백질 기반의 특발성 폐섬유증 예방 또는 치료용 재조합 단백질(NP-011)의 폐 조직 섬유화 방지 기능을 확인하기 위하여, 제1실험군에 해당하는 실험동물 생쥐의 폐 조직 내 폐 조직 섬유화 관련 바이오 마커인 Col1a1, MMP-2, MMP-12 및 TIMP1의 발현 양상을 검사하였다.In order to confirm the lung tissue fibrosis preventing function of the recombinant protein (NP-011) for preventing or treating idiopathic pulmonary fibrosis based on the milk fat globule-EGF factor 8 (MFG-E8) protein of the present invention, an experiment corresponding to the first experimental group The expression patterns of Col1a1, MMP-2, MMP-12 and TIMP1, biomarkers related to lung tissue fibrosis in the lung tissue of animal mice, were examined.
먼저, 'BLM+NP011'제1실험군의 급성특발성 폐섬유증 유도 동물모델 생쥐들을 블레오마이신(Bleomycin)의 주입 후 각각 3일째에 NP-011를 1회 주입 후 6일째가 경과된 시점에 마취 후 심장관류를 통해 PBS를 주입 후 폐 조직을 적출하였다.First, after injecting bleomycin into the acute idiopathic pulmonary fibrosis-induced animal model mice of the first experimental group of 'BLM + NP011', NP-011 was injected once on the 3rd day, and then anesthetized at the 6th day. After infusion of PBS through perfusion, lung tissue was removed.
이와 같이 적출된 제1실험군의 폐 조직을 이용한 폐 조직 내 폐 조직 섬유화 관련 바이오 마커들의 발현 분포 평가를 위한 프라이머를 이용한 중합효소연쇄반응(PCR, Polymerase chain reaction) 분석 방법, 이용되는 프라이머의 서열 및 결과 도출 과정은 앞 서 설명한 'BLM'모델군의 급성특발성 폐섬유증 유도 동물모델 생쥐를 대상으로 한 방법과 동일하므로 생략한다.Polymerase chain reaction (PCR) analysis method using primers for evaluating expression distribution of lung tissue fibrosis-related biomarkers in lung tissue using lung tissue of the first experimental group thus extracted, the sequence of the primer used, and The process of deriving the results is the same as the method for the acute idiopathic pulmonary fibrosis-induced animal model mice of the 'BLM' model group described above, and thus is omitted.
이에 따라, 제1실험군의 동물모델 생쥐들의 적출된 폐 조직 내 폐 조직 섬유화 관련 바이오 마커인 Col1a1, MMP-2, MMP-12 및 TIMP1의 발현 양상을 검증한 결과는 도13에 도시된 바와 같다.Accordingly, the results of verifying the expression patterns of the biomarkers Col1a1, MMP-2, MMP-12 and TIMP1 in the lung tissue fibrosis in the extracted lung tissue of the animal model mice of the first experimental group are shown in FIG. 13.
구체적으로, 도13에 도시된 바와 같이 'BLM+NP011'제1실험군의 동물모델 생쥐들은 NP-011의 주입 후 'BLM'모델군의 급성특발성 폐섬유증 유도 동물모델 생쥐들에 비해 폐 조직 내 폐 조직 섬유화 관련 바이오 마커인 Col1a1, MMP-2, MMP-12 및 TIMP1의 발현이 현저히 감소하여 정상 생쥐의 발현 수준에 가까워짐을 확인할 수 있다.Specifically, as shown in FIG. 13, the animal model mice of the first experimental group 'BLM + NP011' showed lung in lung tissue compared to the acute idiopathic pulmonary fibrosis-induced animal model mice of the 'BLM' model group after injection of NP-011. It can be confirmed that the expression of tissue fibrosis-related biomarkers Col1a1, MMP-2, MMP-12, and TIMP1 is markedly reduced, thereby approaching the expression level of normal mice.
다시 말해, 'BLM'모델군의 급성특발성 폐섬유증 유도 동물모델 생쥐들에 NP-011을 블레오마이신(Bleomycin)의 주입 후 3일째의 시점에 투여하면 폐 조직 내 폐 조직 섬유화 관련 바이오 마커인 Col1a1, MMP-2, MMP-12 및 TIMP1의 발현이 현저히 감소하여 정상 생쥐의 발현 수준과 유사해져 폐 조직 섬유화 방지를 위한 우수한 수준의 항섬유화 효과를 제공할 수 있다.In other words, when NP-011 is administered to the acute idiopathic pulmonary fibrosis-induced animal model mice of the 'BLM' model group at the third day after injection of bleomycin, Col1a1, a biomarker related to lung tissue fibrosis in the lung tissue, The expression of MMP-2, MMP-12 and TIMP1 is significantly reduced, which is similar to that of normal mice, and thus can provide an excellent level of antifibrosis effect for preventing lung tissue fibrosis.
본 발명에 개시된 실시예는 본 발명의 기술 사상을 한정하기 위한 것이 아니라 설명하기 위한 것이고, 이러한 실시예에 의해서 본 발명의 기술 사상의 범위가 한정되는 것은 아니다. 보호범위는 아래 청구범위에 의하여 해석되어야 하며, 그와 동등한 범위 내에 있는 모든 기술 사상은 본 발명의 권리 범위에 포함되는 것으로 해석되어야 할 것이다.The embodiments disclosed in the present invention are not intended to limit the technical spirit of the present invention, but to explain them, and the scope of the technical spirit of the present invention is not limited by these embodiments. The scope of protection should be interpreted by the claims below, and all technical spirits within the scope equivalent thereto should be interpreted as being included in the scope of the present invention.
Claims (7)
- Milk fat globule-EGF factor 8(MFG-E8) 단백질 기반의 재조합 단백질로서, 서열번호1의 아미노산 서열로 이루어진 것을 특징으로 하는Milk fat globule-EGF factor 8 (MFG-E8) protein-based recombinant protein, characterized by consisting of the amino acid sequence of SEQ ID NO: 1특발성 폐섬유증 예방 또는 치료용 재조합 단백질.Recombinant protein for prevention or treatment of idiopathic pulmonary fibrosis.
- 제1항에 있어서,According to claim 1,상기 재조합 단백질은 예방 또는 치료 대상 폐 조직 내 Collagen, α-SMA(α-Smooth Muscle Actin) 및 인산화된 ERK(Extracellular signal-Regulated Kinase) 중 적어도 하나 이상의 발현을 감소시키는 것을 특징으로 하는The recombinant protein is characterized by reducing the expression of at least one of collagen, α-SMA (α-Smooth Muscle Actin) and phosphorylated extracellular signal-regulated kinase (ERK) in lung tissue to be prevented or treated.특발성 폐섬유증 예방 또는 치료용 재조합 단백질.Recombinant protein for prevention or treatment of idiopathic pulmonary fibrosis.
- 제1항에 있어서,According to claim 1,상기 재조합 단백질은 예방 또는 치료 대상 폐 조직 내 Col1a1, MMP-2, MMP-12 및 TIMP1 중 적어도 하나 이상의 발현을 감소시키는 것을 특징으로 하는The recombinant protein is characterized by reducing the expression of at least one of Col1a1, MMP-2, MMP-12 and TIMP1 in the lung tissue to be prevented or treated.특발성 폐섬유증 예방 또는 치료용 재조합 단백질.Recombinant protein for prevention or treatment of idiopathic pulmonary fibrosis.
- 제1항에 있어서,According to claim 1,상기 재조합 단백질은 병변내 투여, 혈관내 투여, 피하 투여, 비강내 투여 또는 복강내 투여용으로 제형화되는 것을 특징으로 하는The recombinant protein is formulated for intralesional administration, intravascular administration, subcutaneous administration, intranasal administration, or intraperitoneal administration.특발성 폐섬유증 예방 또는 치료용 재조합 단백질.Recombinant protein for prevention or treatment of idiopathic pulmonary fibrosis.
- 제1항 내지 제4항 중 어느 한 항에 따른 특발성 폐섬유증 예방 또는 치료용 재조합 단백질을 유효성분으로 포함하는 특발성 폐섬유증 예방 또는 치료용 조성물.A composition for preventing or treating idiopathic pulmonary fibrosis comprising the recombinant protein for preventing or treating idiopathic pulmonary fibrosis according to any one of claims 1 to 4 as an active ingredient.
- 제1항 내지 제4항 중 어느 한 항에 따른 특발성 폐섬유증 예방 또는 치료용 재조합 단백질을 코딩하는 유전자.A gene encoding a recombinant protein for preventing or treating idiopathic pulmonary fibrosis according to any one of claims 1 to 4.
- 제1항 내지 제4항 중 어느 한 항에 따른 특발성 폐섬유증 예방 또는 치료용 재조합 단백질을 코딩하는 유전자를 포함하는 재조합 벡터.Recombinant vector comprising a gene encoding a recombinant protein for preventing or treating idiopathic pulmonary fibrosis according to any one of claims 1 to 4.
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| KR10-2018-0128625 | 2018-10-26 | ||
| KR1020180128625A KR102161892B1 (en) | 2018-10-26 | 2018-10-26 | Composition for idiopathic pulmonary fibrosis treatment or prevention containing recombinant protein for idiopathic pulmonary fibrosis treatment or prevention |
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| KR20230066169A (en) * | 2021-11-05 | 2023-05-15 | (주) 넥셀 | Recombinant protein for ulcerative colitis treatment or prevention and composition for ulcerative colitis treatment or prevention containing thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20130060670A (en) * | 2011-11-30 | 2013-06-10 | 고려대학교 산학협력단 | Use of liver regeneration and improvement of liver diseases of using milk fat globule-egf factor 8 |
| KR20170013621A (en) * | 2015-07-28 | 2017-02-07 | (주) 넥셀 | Composition for preventing or treating tissue fibrosis by using milk fat globule-EGF factor 8 |
| KR20170055626A (en) * | 2015-11-11 | 2017-05-22 | (주)나디안바이오 | Pharmaceutical compositions for treatment or prevention of idiopathic pulmonary fibrosis |
-
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20130060670A (en) * | 2011-11-30 | 2013-06-10 | 고려대학교 산학협력단 | Use of liver regeneration and improvement of liver diseases of using milk fat globule-egf factor 8 |
| KR20170013621A (en) * | 2015-07-28 | 2017-02-07 | (주) 넥셀 | Composition for preventing or treating tissue fibrosis by using milk fat globule-EGF factor 8 |
| KR20170055626A (en) * | 2015-11-11 | 2017-05-22 | (주)나디안바이오 | Pharmaceutical compositions for treatment or prevention of idiopathic pulmonary fibrosis |
Non-Patent Citations (3)
| Title |
|---|
| A N, S. Y. ET AL.: "Milk fat globule-EGF factor 8, secreted by mesenchymal stem cells , protects against liver fibrosis in mice", GASTROENTEROLOGY, vol. 152, 2017, pages 1174 - 1186, XP029955901, DOI: 10.1053/j.gastro.2016.12.003 * |
| ATABAI, KAMRAN ET AL.: "Mfge8 diminishes the severity of tissue fibrosis in mice by binding and targeting collagen for uptake by macrophages", THE JOURNAL OF CLINICAL INVESTIGATION, vol. 119, no. 12, 2009, pages 3713 - 3722, XP055350117, DOI: 10.1172/JCI40053 * |
| DATABASE GenBank 25 July 2016 (2016-07-25), "Homo sapiens milk fat globule-EGF factor 8 protein, partial [synthetic construct", XP055710669, Database accession no. AAP36434.1 * |
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