KR102129178B1 - Composition for myocardial infarction treatment or prevention containing recombinant protein for myocardial infarction treatment or prevention - Google Patents
Composition for myocardial infarction treatment or prevention containing recombinant protein for myocardial infarction treatment or prevention Download PDFInfo
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- KR102129178B1 KR102129178B1 KR1020180128033A KR20180128033A KR102129178B1 KR 102129178 B1 KR102129178 B1 KR 102129178B1 KR 1020180128033 A KR1020180128033 A KR 1020180128033A KR 20180128033 A KR20180128033 A KR 20180128033A KR 102129178 B1 KR102129178 B1 KR 102129178B1
- Authority
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- South Korea
- Prior art keywords
- myocardial infarction
- recombinant protein
- preventing
- prevention
- protein
- Prior art date
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Abstract
본 발명은 Milk fat globule-EGF factor 8(MFG-E8) 단백질을 기반으로 재조합된 심근경색 예방 또는 치료용 재조합 단백질로서, 서열번호1의 아미노산 서열로 이루어져 심근경색에 의해 섬유화를 통한 리모델링이 진행된 치료대상의 심장조직에 우수한 항섬유화 효과를 제공하여 심장 크기 및 박동 기능을 정상 수준으로 회복시킬 수 있다.
아울러, 본 발명은 서열번호1의 아미노산 서열로 이루도록 Milk fat globule-EGF factor 8(MFG-E8) 단백질을 기반으로 재조합된 심근경색 예방 또는 치료용 재조합 단백질을 유효성분으로 포함하는 약학 조성물에 관한 것이기도 하다.The present invention is a recombinant protein for the prevention or treatment of myocardial infarction based on the milk fat globule-EGF factor 8 (MFG-E8) protein. It provides an excellent antifibrotic effect to the heart tissue of the subject, thereby restoring heart size and heartbeat to normal levels.
In addition, the present invention relates to a pharmaceutical composition comprising a recombinant protein for prevention or treatment of recombinant myocardial infarction based on the milk fat globule-EGF factor 8 (MFG-E8) protein to form the amino acid sequence of SEQ ID NO: 1 Do
Description
본 발명은 심근경색 예방 또는 치료용 재조합 단백질 및 이를 포함하는 심근경색 예방 또는 치료용 조성물에 관한 것이다.The present invention relates to a recombinant protein for preventing or treating myocardial infarction and a composition for preventing or treating myocardial infarction comprising the same.
심근경색증(Myocardial infarction)은 심장의 관상동맥이 혈전에 의해 완전히 막혀 심장 근육의 일부가 파괴 또는 괴사되어 혈관 내 혈액의 흐름이 원활하지 못하게 되어 통증을 유발하는 허혈성 심질환의 하나이다.Myocardial infarction is one of the ischemic heart diseases that causes pain due to the fact that the coronary arteries of the heart are completely blocked by blood clots, and part of the heart muscle is destroyed or necrotic, preventing the flow of blood in the blood vessels.
심근경색과 같은 심장 질환은 전 세계 사망률 1위의 질병으로, 특히 심근경색의 경우 치료제가 없고 다른 장기와 같이 이식이 용이하지 않아 현재 심장 이식 외에 주로 약물 치료에 의존하고 있는 실정이다.Heart disease, such as myocardial infarction, is the number one mortality rate in the world. In particular, myocardial infarction has no treatment and is not easily transplanted like other organs.
주로 심근경색 치료에 이용되는 약물에는 현재 아스피린, 클로피도그렐, 베타차단제, 에이스 억제제, 안지오텐신 차단제 등이 이용되는데, 이들은 손상된 심근세포의 회복을 통한 근본적 치료가 이루어지는 것이 아니라 심장의 기능을 보완하거나 기능 악화를 늦춰주는 수준에 그치고 있다. Drugs used mainly for the treatment of myocardial infarction currently use aspirin, clopidogrel, beta blockers, ace inhibitors, angiotensin blockers, etc., which do not provide basic treatment through the recovery of damaged myocardial cells, but complement the function of the heart or worsen its function. It's just slowing down.
최근 성체 줄기세포의 이식이나 줄기세포를 이용한 심근세포 이식 등의 형태로 마련된 심근경색 치료제가 개발되고 있으나 효과가 미비한 상태이다.Recently, a therapeutic agent for myocardial infarction provided in the form of transplantation of adult stem cells or transplantation of myocardial cells using stem cells has been developed, but the effect is insufficient.
아울러, 심근경색 치료용으로 마련된 세포형 치료제와 관련된 종래기술에 대한 선행문헌에는 대한민국 등록특허공보 제10-1628453호의"심근경색 치료용 면역관용 수지상 세포 및 그 제조방법"(이하, '종래기술'이라고 함)이 있다.In addition, prior literature on prior art related to a cell-type therapeutic agent provided for the treatment of myocardial infarction includes "Registration of immune dendritic cells for myocardial infarction and its manufacturing method" of Korean Patent Registration No. 10-1628453 (hereinafter referred to as'prior art') Is called).
이러한 종래기술을 비롯한 기존의 심근경색의 치료를 위해 마련된 약학 조성물의 경우, 손상된 심근세포의 치료를 통한 근본적 심근경색 치료가 이루어지지 못해 결국에는 심근경색으로 괴사된 심근세포 조직이 박동 기능성을 상실한 섬유화 조직으로 대체되고, 이에 따라 심장이 비대해지면서 정상적 심장 기능을 잃고 환자는 결국 사망에 이르게 되는 문제가 있었다.In the case of the conventional pharmaceutical composition prepared for the treatment of myocardial infarction, including the prior art, the myocardial infarction tissue that has been necrotic with myocardial infarction has failed to perform the basic myocardial infarction treatment through the treatment of damaged myocardial cells. There was a problem that the tissue was replaced, and as a result, the heart became enlarged, the normal cardiac function was lost, and the patient eventually died.
또한, 종래기술을 비롯한 기존의 심근경색의 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, in the case of a pharmaceutical composition prepared for the treatment of myocardial infarction, including the prior art, the biocompatibility of the composition based on the chemical component is low, the incidence of side effects is high, and it suffers from mass production and quality control considering productivity. have.
본 발명은 상기 문제점을 해결하기 위해 창작된 것으로써, 본 발명의 목적은 심근경색에 의해 괴사되어 섬유화가 진행된 심근세포 조직을 근본적으로 치료하여 박동 기능성을 정상 수준으로 회복시키고, 더 나아가 심장의 비대화를 막을 수 있는 심근경색 예방 또는 치료용 재조합 단백질 및 이를 포함하는 조성물을 제공하는데 있다.The present invention was created to solve the above problems, and the object of the present invention is to fundamentally treat myocardial cell tissues that have undergone fibrosis and have undergone fibrosis, thereby restoring pulsatile functionality to normal levels, and furthermore, enlargement of the heart. It is to provide a composition containing a recombinant protein and the same for preventing or treating myocardial infarction that can be prevented.
상기 목적을 달성하기 위하여 본 발명에 따른 심근경색 예방 또는 치료용 재조합 단백질은, Milk fat globule-EGF factor 8(MFG-E8) 단백질 기반의 재조합 단백질로서, 서열번호1의 아미노산 서열로 이루어진다.To achieve the above object, the recombinant protein for preventing or treating myocardial infarction according to the present invention is a milk fat globule-EGF factor 8 (MFG-E8) protein-based recombinant protein, and is composed 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 심근경색 예방 또는 치료용 재조합 단백질은 예방 또는 치료 대상 심장의 좌심실 박출률(EF, Ejection Fraction)을 현저히 높일 수 있다.Here, the milk fat globule-EGF factor 8 (MFG-E8) protein-based recombinant protein for the prevention or treatment of myocardial infarction consisting of the amino acid sequence of SEQ ID NO: 1 is the left ventricular ejection fraction (EF, Ejection Fraction) of the heart to be prevented or treated ) Can be significantly increased.
또한, Milk fat globule-EGF factor 8(MFG-E8) 단백질 기반의 재조합되어 서열번호1의 아미노산 서열로 이루는 심근경색 예방 또는 치료용 재조합 단백질은 예방 또는 치료 대상 심장의 좌심실 분획단축률(FS, Fractional shortening)을 현저히 높일 수 있다.In addition, the recombinant protein for the prevention or treatment of myocardial infarction 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 a fraction of the left ventricular fraction of the heart to be prevented or treated (FS, Fractional shortening) can be significantly increased.
또한, Milk fat globule-EGF factor 8(MFG-E8) 단백질 기반의 재조합되어 서열번호1의 아미노산 서열로 이루는 심근경색 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 the prevention or treatment of myocardial infarction consisting of the amino acid sequence of SEQ ID NO: 1, which is recombinant based on the milk fat globule-EGF factor 8 (MFG-E8) protein, is administered intralesionally, intravascularly, subcutaneously, or intranasally. Or it can be formulated for intraperitoneal administration.
한편, 상기 목적을 달성하기 위하여 본 발명에 따른 심근경색 예방 또는 치료용 조성물은, Milk fat globule-EGF factor 8(MFG-E8) 단백질 기반의 재조합되어 서열번호1의 아미노산 서열로 이루는 심근경색 예방 또는 치료용 재조합 단백질을 유효성분으로 포함한다.On the other hand, in order to achieve the above object, the composition for preventing or treating myocardial infarction according to the present invention is a milk fat globule-EGF factor 8 (MFG-E8) protein-based recombination to prevent myocardial infarction consisting of the amino acid sequence of SEQ ID NO: 1 The therapeutic recombinant protein 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 심근경색 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 myocardial infarction consisting of an amino acid sequence of SEQ ID NO: 1 that is recombined based on 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 심근경색 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 includes a gene encoding a recombinant protein for preventing or treating myocardial infarction consisting of an amino acid sequence of SEQ ID NO: 1 by recombination based on Milk fat globule-EGF factor 8 (MFG-E8) protein. The recombinant vector is provided as another aspect.
본 발명에 의하면 다음과 같은 효과가 있다.According to the present invention has the following effects.
첫째, 심근경색 예방 또는 치료용 재조합 단백질을 통해 심근경색에 의해 섬유화를 통한 리모델링이 진행된 치료대상의 심장조직에 우수한 항섬유화 효과를 제공하여 심장 크기 및 박동 기능을 정상 수준으로 회복시킬 수 있다.First, through the recombinant protein for preventing or treating myocardial infarction, the heart size and heartbeat function can be restored to a normal level by providing an excellent anti-fibrotic effect to the heart tissue of a treatment target undergoing remodeling through fibrosis through myocardial infarction.
둘째, 심근경색 예방 또는 치료용 재조합 단백질을 통해 심근경색에 의해 섬유화를 통한 리모델링이 진행된 치료대상 심장의 좌심실 박출률(EF, Ejection Fraction)을 현저히 높일 수 있다.Second, the left ventricular ejection fraction (EF) of the target heart, which has been remodeled through fibrosis due to myocardial infarction, can be significantly increased through a recombinant protein for preventing or treating myocardial infarction.
셋째, 심근경색 예방 또는 치료용 재조합 단백질을 통해 심근경색에 의해 섬유화를 통한 리모델링이 진행된 치료대상 심장의 분획단축률(FS, Fractional shortening)을 현저히 높일 수 있다.Third, by using a recombinant protein for preventing or treating myocardial infarction, fractional shortening (FS) of the target heart, which has undergone remodeling through fibrosis due to myocardial infarction, can be significantly increased.
넷째, 심근경색 예방 또는 치료용 재조합 단백질의 투여로 인해 심근경색에 의해 섬유화를 통한 리모델링이 진행된 치료대상의 별도 심각한 병리적 증상이 유발되지 않는다.Fourth, due to the administration of a recombinant protein for the prevention or treatment of myocardial infarction, no serious serious pathological symptoms of the treatment target undergoing remodeling through fibrosis due to myocardial infarction are caused.
다섯째, 생체 친화적이고, 부작용이 적은 심근경색 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 myocardial infarction, which is bio-friendly and has fewer side effects, and a composition comprising the same.
여섯째, 대량 생산 및 품질관리가 용이한 심근경색 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 myocardial infarction, which is easy to mass-produce and quality control, and a composition containing the same.
도1은 본 발명의 심근경색 예방 또는 치료용 재조합 단백질(NP-011)을 코딩하는 유전자가 삽입된 Backbone Vector의 구조를 도시하고 있다.
도2는 도1에 도시된 Backbone Vector의 구조 내 삽입될 수 있으며, 본 발명의 심근경색 예방 또는 치료용 재조합 단백질(NP-011)을 코딩하는 유전자의 DNA 염기서열을 도시하고 있다.
도3은 본 발명의 심근경색 예방 또는 치료용 재조합 단백질(NP-011)의 아미노산 서열을 도시하고 있다.
도4는 본 발명의 심근경색 예방 또는 치료용 재조합 단백질(NP-011)의 효과 검증 시험의 설계를 나타낸 모식도이다.
도5는 본 발명의 심근경색 예방 또는 치료용 재조합 단백질(NP-011)의 효과 검증을 위해 설계된 동물실험 모델들의 2주차 좌심실 박출률(EF)과 분획 단축률(FS)의 측정 결과를 나타낸 그래프이다.
도6은 본 발명의 심근경색 예방 또는 치료용 재조합 단백질(NP-011)의 효과 검증을 위해 설계된 동물실험 모델들의 4주차 좌심실 박출률(EF)과 분획 단축률(FS)의 측정 결과를 나타낸 그래프이다.
도7은 본 발명의 심근경색 예방 또는 치료용 재조합 단백질(NP-011)의 효과 검증을 위해 설계된 동물실험 모델들의 8주차 좌심실 박출률(EF)과 분획 단축률(FS)의 측정 결과를 나타낸 그래프이다.
도8은 본 발명의 심근경색 예방 또는 치료용 재조합 단백질(NP-011)의 효과 검증을 위해 설계된 동물실험 모델들의 진행시간에 따른 좌심실 박출률(EF) 변화 측정 결과를 나타낸 그래프이다.
도9는 본 발명의 심근경색 예방 또는 치료용 재조합 단백질(NP-011)의 효과 검증을 위해 설계된 동물실험 모델들의 진행시간에 따른 좌심실 분획 단축률(FS) 변화 측정 결과를 나타낸 그래프이다.
도10은 본 발명의 심근경색 예방 또는 치료용 재조합 단백질(NP-011)의 효과 검증을 위해 설계된 동물실험 모델들의 치료 진행에 따른 체중변화를 비교한 결과를 나타낸 그래프이다.
도11은 본 발명의 심근경색 예방 또는 치료용 재조합 단백질(NP-011)의 효과 검증을 위해 설계된 동물실험 모델들의 심장조직 MT 및 H&E 염색 결과를 나타낸 사진이다.
도12는 본 발명의 심근경색 예방 또는 치료용 재조합 단백질(NP-011)의 효과 검증을 위해 설계된 동물실험 모델들의 도11을 기준으로 한 좌심실 전체 면적 대비 지엽적으로 섬유화된 부분의 면적 값의 계산 결과를 나타낸 그래프이다.1 shows the structure of a backbone vector in which a gene encoding a recombinant protein (NP-011) for preventing or treating myocardial infarction of the present invention is inserted.
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 the prevention or treatment of myocardial infarction of the present invention.
Figure 3 shows the amino acid sequence of a recombinant protein (NP-011) for the prevention or treatment of myocardial infarction of the present invention.
Figure 4 is a schematic diagram showing the design of the effect verification test for a recombinant protein for preventing or treating myocardial infarction (NP-011) of the present invention.
Figure 5 is a graph showing the measurement results of the second week left ventricular ejection rate (EF) and fractional shortening rate (FS) of animal experimental models designed to verify the effect of the recombinant protein for preventing or treating myocardial infarction (NP-011) of the present invention to be.
Figure 6 is a graph showing the results of measuring the left ventricular ejection fraction (EF) and fractional shortening rate (FS) at
7 is a graph showing the results of measurement of the left ventricular ejection fraction (EF) and fractional shortening rate (FS) at
8 is a graph showing the results of measuring changes in left ventricular ejection fraction (EF) according to the progression time of animal experimental models designed to verify the effect of a recombinant protein for preventing or treating myocardial infarction (NP-011) of the present invention.
9 is a graph showing the results of measuring changes in left ventricular fraction shortening rate (FS) according to the progression time of animal experimental models designed to verify the effect of recombinant protein for preventing or treating myocardial infarction (NP-011) of the present invention.
Figure 10 is a graph showing the results of comparing the changes in body weight according to the treatment progress of animal experimental models designed to verify the effect of the recombinant protein for preventing or treating myocardial infarction (NP-011) of the present invention.
FIG. 11 is a photograph showing the results of staining of MT and H&E of cardiac tissues of animal experimental models designed to verify the effect of recombinant protein for preventing or treating myocardial infarction of the present invention (NP-011).
12 is a result of calculating the area value of the locally fibrous portion compared to the total area of the left ventricle based on FIG. 11 of animal experimental models designed to verify the effect of the recombinant protein for preventing or treating myocardial infarction (NP-011) of the present invention. It is a graph showing.
본 발명의 바람직한 실시예에 대하여 첨부된 도면을 참조하여 더 구체적으로 설명하되, 이미 주지된 기술적 부분에 대해서는 설명의 간결함을 위해 생략하거나 압축하기로 한다.With reference to the accompanying drawings, a preferred embodiment of the present invention will be described in more detail, but for the brevity of description, the already known technical parts will be omitted or compressed.
1. 심근경색 예방 또는 치료용 재조합 단백질(NP-011)에 관한 설명1. Description of recombinant protein for preventing or treating myocardial infarction (NP-011)
본 발명에 따른 심근경색 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 myocardial infarction 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) 심근경색 예방 또는 치료용 재조합 단백질(NP-011)의 수득(1) Obtaining recombinant protein for preventing or treating myocardial infarction (NP-011)
먼저 심근경색 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)를 획득(서열번호4 참고)한다.First, the process of expression of the recombinant protein for preventing or treating myocardial infarction (NP-011) will be described.For the improvement of the protein structure of Milk fat globule-EGF factor 8 (MFG-E8), Origene's MFG-E8 (NM_005928) Human cDNA Clone ( Cat. No. RG217163), which is 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 having 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 심근경색 예방 또는 치료용 재조합 단백질(NP-011)에 해당하는 재조합 단백질의 발현을 확인한다.Thereafter, plasmid DNA is extracted and transfected into HEK293 cells. After 2 days, the culture solution is collected, and immunoprecipitation reaction (IP, Immunoprecipitation) is performed with FLAG resin to prevent or treat myocardial infarction by Western Blotting. The expression of the recombinant protein corresponding to the protein (NP-011) is confirmed.
다음으로 심근경색 예방 또는 치료용 재조합 단백질(NP-011)의 대량 생산 과정을 설명하면, 발현의 확인을 거친 plasmid DNA를 Maxi prep을 통해 대랑 확보한 후, HEK293 세포를 준비하여 대량의 plasmid DNA를 HEK293 세포에 형질도입하여 심근경색 예방 또는 치료용 재조합 단백질(NP-011)에 해당하는 재조합 단백질의 대량 생산을 수행할 수 있다.Next, the process of mass production of a recombinant protein for preventing or treating myocardial infarction (NP-011) will be described. After confirming expression, the plasmid DNA that has been confirmed is secured through Maxi prep, and HEK293 cells are prepared to prepare a large amount of plasmid DNA. It can be transduced into HEK293 cells to perform mass production of a recombinant protein corresponding to a recombinant protein for preventing or treating myocardial infarction (NP-011).
다음으로 심근경색 예방 또는 치료용 재조합 단백질(NP-011)의 품질 관리를 위한 정제 과정을 설명하면, Cornin사의 CellSTACK Cell Culture Chamer 10개를 준비하여 HEK293 세포를 도포한 뒤, 1600μg의 plasmid DNA와 3200μl의 Transfection Reagents를 상온에서 15분간 혼합한 후, 형질도입을 수행한다. Next, to describe the purification process for quality control of recombinant protein for preventing or treating myocardial infarction (NP-011), 10 CornST CellSTACK Cell Culture Chamers were prepared, coated with HEK293 cells, and 1600 μg of plasmid DNA and 3200 μl After mixing the Transfection Reagents of at room temperature for 15 minutes, transfection is 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 transfection, the culture medium was replaced and further cultured for 6 days, and the culture medium was collected three 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(서열번호6 참고)되어 있음으로 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 (refer to SEQ ID NO: 6), only the target protein is bound using the FLAG affinity resin, and then the target protein is washed by washing buffer. Excluded 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 Coomassie Blue staining and Western Blotting using 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 심근경색 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)의 구축을 위해 당업계의 기술자들에게 공지되고 자명한 수준의 기술을 이용한 다양한 방식으로 구현 가능하다.Based on such milk fat globule-EGF factor 8 (MFG-E8) protein, it is recommended for cloning, mass production and purification for expression of recombinant protein (NP-011) for prevention or treatment of myocardial infarction prepared by recombination. The series of specific processes described herein are not limited to this, and are known to those skilled in the art for the construction of specific amino acid sequence structures (SEQ ID NOs: 1 and 3) of the recombinant protein to be described below. It can be implemented in various ways.
예를 들어, Milk fat globule-EGF factor 8(MFG-E8) 단백질에 기반을 두고 재조합되어 마련되는 심근경색 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 transformation of the expression vector used for cloning of recombinant protein (NP-011) for prevention or treatment of myocardial infarction prepared based on milk fat globule-EGF factor 8 (MFG-E8) protein. Techniques such as performance targets, production conditions and morphology, purification conditions and morphology can be variously implemented.
아울러, Milk fat globule-EGF factor 8(MFG-E8) 단백질에 기반을 두고 재조합되어 마련되는 심근경색 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 실시될 수도 있다.In addition, DNA fragments that are cloned into an expression vector in relation to expression of a recombinant protein (NP-011) for prevention or treatment of myocardial infarction prepared based on milk fat globule-EGF factor 8 (MFG-E8) protein are fragmented (fragments) The structure of) is also based on the structure of FIG. 2, but a specific DNA nucleotide sequence encoding a specific Signal Peptide may be additionally linked to DNA fragments according to the implementation.
(2) 심근경색 예방 또는 치료용 재조합 단백질(NP-011)의 구조(2) Structure of recombinant protein (NP-011) for prevention or treatment of myocardial infarction
앞 서 설명한, 재조합 단백질의 클로닝, 분리, 생산, 정제 등의 과정을 거쳐 최종적으로 마련되는 Milk fat globule-EGF factor 8(MFG-E8) 단백질 기반의 심근경색 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 myocardial infarction 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 (NP-011) ) Has the amino acid sequence structure as shown in SEQ ID NO: 1 or FIG. 3 in the sequence listing below.
특징적으로, Milk fat globule-EGF factor 8(MFG-E8) 단백질 기반의 심근경색 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 myocardial infarction 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 Domain. Heavy EGF-like Domain (see SEQ ID NO: 2) and C1 Domain (see SEQ ID NO: 3) are recombined into structures.
다시 말해, 본 발명의 Milk fat globule-EGF factor 8(MFG-E8) 단백질 기반의 심근경색 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 prevention or treatment of myocardial infarction 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 심근경색 예방 또는 치료용 재조합 단백질(NP-011)은 심근경색 예방 또는 치료용으로 이용되는 약학적 조성물의 주요 유효성분으로 이용될 수 있다.The milk fat globule-EGF factor 8 (MFG-E8) protein-based recombinant protein for preventing or treating myocardial infarction (NP-011) of the present invention is the main active ingredient of a pharmaceutical composition used for preventing or treating myocardial infarction Can be used as
아울러, 본 발명의 Milk fat globule-EGF factor 8(MFG-E8) 단백질 기반의 심근경색 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(좌심실 조직)내 투여용으로 제형화할 수 있다.In addition, the milk fat globule-EGF factor 8 (MFG-E8) protein-based recombinant protein for preventing or treating myocardial infarction (NP-011) of the present invention and a pharmaceutical composition using the same as the main active ingredient are administered in a lesion, blood vessel ( It can be formulated for intrathecal administration, subcutaneous administration, intranasal administration, intraperitoneal administration, or administration within a specific tissue (left ventricular tissue).
한편, 본 발명의 Milk fat globule-EGF factor 8(MFG-E8) 단백질 기반의 심근경색 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.On the other hand, the milk fat globule-EGF factor 8 (MFG-E8) protein-based recombinant protein for preventing or treating myocardial infarction (NP-011) of the present invention is DNA cloned into an expression vector in relation to the expression of the recombinant protein described above Corresponding to the scope of the structural embodiments of the fragments (fragments) may be made according to the implementation of additional linkage of a specific Signal Peptide structure.
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 (see SEQ ID NO: 5) such as "MPRPRLLAALCGALLCAPSLLVA", but is not limited thereto.
2. 심근경색 예방 또는 치료용 재조합 단백질(NP-011)의 효과 검증 시험 결과에 관한 설명2. Description of the test results of the verification of the effect of recombinant protein for preventing or treating myocardial infarction (NP-011)
본 발명의 Milk fat globule-EGF factor 8(MFG-E8) 단백질 기반의 심근경색 예방 또는 치료용 재조합 단백질(NP-011)과 관련하여 해당 재조합 단백질에 기인한 심근경색 예방 또는 치료의 효과 수준 검증 및 MFG-E8 단백질과의 효과 비교를 시험을 통해 확인하였으며, 해당 시험은 당업계의 기술자들에게 자명한 수단에 의한 성질 등을 정의하기 위한 목적으로 하기 실험 방법들을 이용하였다. In relation to the milk fat globule-EGF factor 8 (MFG-E8) protein-based myocardial infarction prevention or treatment recombinant protein (NP-011) of the present invention, verification of the effect level of myocardial infarction prevention or treatment due to the recombinant protein and The effect comparison with the MFG-E8 protein was confirmed through a test, and the following test methods were used for the purpose of defining properties by means obvious to those skilled in the art.
(1) 심근경색 동물모델의 제작 및 실험설계(1) Production and experimental design of myocardial infarction animal model
우선, 무게가 180~200g 정도 되는 8~10주령 수컷 랫트(Rat)을 이용하여 급성심근경색(AMI, Acute myocardial infarction) 유도 동물모델을 제작한다.First, an acute myocardial infarction (AMI)-induced animal model is prepared using an 8-10 week old male rat weighing 180-200 g.
다음으로, 실험군은 도4에 도시된 바와 같이 정상(Normal)의 비교군 랫트 동물모델(제1실험군), 급성심근경색(AMI)이 유도된 랫트 동물모델(제2실험군), 급성심근경색(AMI)이 유도된 랫트에 식염수(PBS, Phosphate Buffered Saline)를 주입한 동물모델(제3실험군), 급성심근경색(AMI)이 유도된 랫트에 MFG-E8(Milk fat globule-EGF factor 8)을 주입한 동물모델(제4실험군) 및 급성심근경색(AMI)이 유도된 랫트에 본 발명의 심근경색 예방 또는 치료용 재조합 단백질(NP-011)을 주입한 동물모델(제5실험군) 군으로 나누어 각 군당 5마리, 총 30마리의 랫트을 이용하여 실험을 진행하는 것이 바람직하다. Next, the experimental group, as shown in Figure 4, the normal (Normal) rat animal model (first experimental group), acute myocardial infarction (AMI) induced rat animal model (second experimental group), acute myocardial infarction ( AMI)-induced rats infused with saline (PBS, Phosphate Buffered Saline) in an animal model (3rd experimental group) and MFG-E8 (Milk fat globule-EGF factor 8) in acute myocardial infarction (AMI)-induced rats The injected animal model (4th experimental group) and the acute myocardial infarction (AMI)-induced rat myocardial infarction prevention or treatment recombinant protein (NP-011) injected into animal model (5th experimental group) group It is desirable to conduct experiments using 5 rats per group, a total of 30 rats.
여기서, 급성심근경색(AMI)이 유도된 랫트 동물모델(제2실험군)은 케타민(Ketamine) 60mg/kg과 하이드로클로라이드(Hydrochloride) 7.5mg/kg을 혼합하여 랫트에 복강 주사한 후 완전히 마취되면 수술을 통해서 흉부를 개방한 후 6-0 silk suture를 이용하여 왼쪽 관상동맥을 묶어서 심근경색을 유도였다.Here, acute myocardial infarction (AMI) induced rat animal model (2nd experimental group) is mixed with 60 mg/kg of ketamine (Ketamine) and 7.5 mg/kg of hydrochloride (Hydrochloride) and injected into the rat intraperitoneally. After opening the chest through, the left coronary artery was tied using 6-0 silk suture to induce myocardial infarction.
급성심근경색(AMI)이 유도된 랫트 동물모델(제2실험군)은 심근경색 유도 직후 바로 봉합 수술을 진행했으며, 식염수(PBS), MFG-E8 혹은 심근경색 예방 또는 치료용 재조합 단백질(NP-011) 주입이 이루어진 동물모델(제3실험군 내지 제5실험군)은 심근경색 유도 직후 도4에 도시된 바와 같이 좌심실 부위 3 곳에 식염수(PBS), 심근경색 예방 또는 치료용 재조합 단백질(NP-011) 혹은 MFG-E8을 160μg/kg 투여하고 봉합 수술을 진행하여 8주 내지 10주간 사육하면서 기능 검증 검사를 수행하고 각각의 결과를 검증하였다.Acute myocardial infarction (AMI) induced rat animal model (2nd experimental group) immediately underwent suture surgery immediately after myocardial infarction, saline (PBS), MFG-E8 or recombinant protein for prevention or treatment of myocardial infarction (NP-011) ) Injected animal model (3rd experimental group to 5th experimental group) immediately after induction of myocardial infarction, as shown in FIG. 4, saline (PBS), a recombinant protein for prevention or treatment of myocardial infarction (NP-011) or 3 MFG-E8 was administered at 160 μg/kg, and suture surgery was performed to breed for 8 to 10 weeks, and functional verification tests were performed and each result was verified.
(2) 심근경색 유도 동물모델의 심초음파 검사(Echocardiography)를 이용한 심장 박동기능 개선 효과 검증(2) Verifying the effect of improving heart rate using echocardiography in an animal model of myocardial infarction
본 발명의 Milk fat globule-EGF factor 8(MFG-E8) 단백질 기반의 심근경색 예방 또는 치료용 재조합 단백질(NP-011)의 심장기능 향상성을 확인하기 위하여, 심전도를 측정을 기반으로 좌심실의 박출률(EF)과 분획 단축률(FS)을 확인하였다. Milk fat globule-EGF factor 8 (MFG-E8) protein-based myocardial infarction prevention or treatment of the present invention in order to confirm the cardiac function improvement of the recombinant protein (NP-011), based on the electrocardiogram measurement of the left ventricle The yield rate (EF) and fractional shortening rate (FS) were confirmed.
우선, 앞서 제작 설정한 제1실험군 내지 제5실험군 각각을 8주 동안 0일차(Day 0), 1일차(Day1), 14일차(Day 14), 28일차(Day28), 56일차(Day56)에 총 5번에 걸처 Vivid 7(GE medical systems) system과 10MHz small linear array transducer (i13L, GE medical systems)을 이용하여 심전도를 측정한다.First, each of the first experimental group and the fifth experimental group, which were previously set, were set to Day 0 (Day 0), Day 1 (Day1), Day 14 (Day 14), Day 28 (Day28), and Day 56 (Day56) for 8 weeks. The ECG was measured using a Vivid 7 (GE medical systems) system and a 10 MHz small linear array transducer (i13L, GE medical systems) for a total of five times.
여기서, 각각의 실험군에 해당되는 랫트들은 케타민(Ketamine) 60mg/kg과 하이드로콜라이드(Hydrochloride) 7.5mg/kg을 혼합하여 복강 주사한 후 완전히 마취된 상태에서 연이은 두 번의 심장 싸이클 동안의 심전도를 측정하여 평균값을 이용하는 것이 바람직하다. Here, rats corresponding to each experimental group were injected intraperitoneally by mixing ketamine 60mg/kg and hydrochloride 7.5mg/kg, and then electrocardiogram was measured during two consecutive cardiac cycles under complete anesthesia. Therefore, it is preferable to use the average value.
다음으로, 제1실험군 내지 제5실험군의 심초음파 검사(Echocardiography)를 이용해 측정된 심전도 결과를 기반으로 좌심실의 박출률(EF)과 분획 단축률(FS)을 계산하여 상호 결과를 비교 분석하였다.Next, the ejection rate (EF) and fractional shortening rate (FS) of the left ventricle were calculated based on the ECG results measured using echocardiography of the first to fifth experimental groups to compare and analyze the mutual results.
여기서, 좌심실의 분획 단축률(FS, Fractional Shortening)은 대표적인 심장 수축 값으로 측정하며, 좌심실의 박출률(EF, Ejection Fraction)은 확장기말용적(end-diastolic volume)과 수축기말용적(end-systolic volume)을 이용해 계산한다.Here, the fractional shortening (FS) of the left ventricle is measured by a representative cardiac contraction value, and the ejection fraction (EF) of the left ventricle is the end-diastolic volume and the end-systolic volume. volume).
이에 따라, 제1실험군 내지 제5실험군의 심초음파 검사(Echocardiography)를 이용해 측정된 심전도 결과를 기반으로 각각 도출된 좌심실의 박출률(EF)과 분획 단축률(FS)의 결과는 도5 내지 도9에 도시된 바와 같다.Accordingly, the results of the ejection rate (EF) and fractional shortening rate (FS) of the left ventricle respectively derived based on the results of ECG measured using echocardiography of the first to fifth experimental groups are shown in FIGS. 5 to 5 As shown in 9.
도5에 도시된 바와 같이 제1실험군 내지 제5실험군의 동물모델들의 2주차(14일차) 심전도측정에 따른 좌심실의 박출률(EF)과 분획 단축률(FS)의 결과를 살펴보면, 급성심근경색(AMI)이 유도된 랫트에 본 발명의 심근경색 예방 또는 치료용 재조합 단백질(NP-011)을 주입하더라도 좌심실의 박출률(EF)과 분획 단축률(FS)의 수준이 제2실험군 내지 제4실험군과 큰 차이 없이 유사하고, 제1실험군의 정상 수준과 비교해 유의한 개선이 이루어지지 않았음을 알 수 있다.As shown in FIG. 5, when looking at the results of the ejection rate (EF) and fractional shortening rate (FS) of the left ventricle according to the electrocardiogram measurement of the second week (day 14) of the animal models of the first experimental group to the fifth experimental group, acute myocardial infarction Even when the recombinant protein for preventing or treating myocardial infarction (NP-011) of the present invention is injected into the (AMI)-induced rat, the levels of ejection rate (EF) and fractional shortening rate (FS) of the left ventricle are
그 후, 도6에 도시된 바와 같이 제1실험군 내지 제5실험군의 동물모델들의 4주차(28일차) 심전도측정에 따른 좌심실의 박출률(EF)과 분획 단축률(FS)의 결과를 살펴보면, 급성심근경색(AMI)이 유도된 랫트에 본 발명의 심근경색 예방 또는 치료용 재조합 단백질(NP-011)을 주입하더라도 좌심실의 박출률(EF)과 분획 단축률(FS)의 수준이 제2실험군 내지 제4실험군과 비교해 뚜렷한 차이를 보이며 향상되고 있음이 나타나고, 제1실험군의 정상 수준과 비교해 유의한 개선이 이루어지고 있음을 알 수 있다. Thereafter, as shown in FIG. 6, the results of the ejection rate (EF) and fractional shortening rate (FS) of the left ventricle according to the 4th week (Day 28) ECG measurement of the animal models of the first experimental group to the fifth experimental group, Acute myocardial infarction (AMI) induced rat myocardial infarction prevention or treatment of recombinant protein (NP-011) of the present invention, the level of left ventricular ejection (EF) and fractional shortening rate (FS) in the second experimental group It can be seen that the improvement is shown by showing a distinct difference compared to the fourth to fourth experimental groups, and a significant improvement is made compared to the normal level of the first experimental group.
마지막으로, 도7에 도시된 바와 같이 제1실험군 내지 제5실험군의 동물모델들의 8주차(56일차) 심전도측정에 따른 좌심실의 박출률(EF)과 분획 단축률(FS)의 결과를 살펴보면, 급성심근경색(AMI)이 유도된 랫트에 본 발명의 심근경색 예방 또는 치료용 재조합 단백질(NP-011)을 주입하더라도 좌심실의 박출률(EF)과 분획 단축률(FS)의 수준이 제2실험군 내지 제4실험군과 비교해 더 큰 차이를 보이며 확연한 향상을 나타내고 있고, 제1실험군의 정상 수준과 비교해 상호 더욱 가까워지며 유의한 의미를 이루어 심장 기능이 정상 수준으로 회복됨을 알 수 있다.Finally, as shown in Figure 7, looking at the results of the ejection rate (EF) and fractional shortening rate (FS) of the left ventricle according to the 8th week (day 56) ECG measurement of the animal models of the first experimental group to the fifth experimental group, Acute myocardial infarction (AMI) induced rat myocardial infarction prevention or treatment of recombinant protein (NP-011) of the present invention, the level of left ventricular ejection (EF) and fractional shortening rate (FS) in the second experimental group It can be seen that the heart function is restored to a normal level by showing a significant difference and showing a marked improvement compared to the fourth to fourth experimental groups, closer to each other than the normal level of the first experimental group, and having a significant meaning.
앞 서 설명한 결과를 더욱 확실히 확인하기 위해 도8 및 도9에 도시된 바와 같이 시간의 흐름에 따른 각 실험군 별 좌심실의 박출률(EF)과 분획 단축률(FS)의 변화 양상을 살펴보면, 급성심근경색(AMI)이 유도된 랫트에 본 발명의 심근경색 예방 또는 치료용 재조합 단백질(NP-011)을 주입하면, MFG-E8를 주입한 제4실험군에 비해 좌심실의 박출률(EF)과 분획 단축률(FS)의 수준이 더 확실하고 큰 증가폭을 보이며 증가하여, 심장 박동 기능의 꾸준하며 빠른 회복률과 정상 수준에 가까운 고도한 회복 수준을 제공할 수 있다. In order to confirm the above-described results more clearly, as shown in FIGS. 8 and 9, when examining the changes in the ejection rate (EF) and fractional shortening rate (FS) of the left ventricle for each experimental group over time, acute myocardium When the recombinant protein for preventing or treating myocardial infarction of the present invention (NP-011) is injected into an infarct (AMI)-induced rat, the ejection rate (EF) and fraction of the left ventricle are shortened compared to the fourth experimental group injected with MFG-E8. The level of the rate (FS) can be increased with greater certainty and with a large increase, providing a steady and rapid recovery rate of the heart rate function and a high level of recovery close to the normal level.
(3) 심근경색 유도 동물모델의 체중 변화 검사(3) Examination of weight change in animal models of myocardial infarction
본 발명의 Milk fat globule-EGF factor 8(MFG-E8) 단백질 기반의 심근경색 예방 또는 치료용 재조합 단백질(NP-011)의 심장기능 향상과정에서의 체중 변화를 확인하였다.The milk fat globule-EGF factor 8 (MFG-E8) protein-based recombinant protein for preventing or treating myocardial infarction (NP-011) of the present invention was examined for weight changes during cardiac function improvement.
우선, 앞서 제작 설정한 제2실험군과 제5실험군 각각을 8주 동안의 체중변화 양상을 1일차(Day1), 28일차(Day28) 및 56일차(Day56)에 총 3번에 걸처 측정하여 그래프화하였다.First, each of the 2nd experimental group and the 5th experimental group, which were previously set up, is measured and graphed over a total of 3 times on the 1st day (Day1), 28th day (Day28), and 56th day (Day56). Did.
그 결과, 도10에 도시된 바와 같이 급성심근경색(AMI)이 유도된 랫트와 비교해 급성심근경색(AMI)이 유도된 랫트에 본 발명의 심근경색 예방 또는 치료용 재조합 단백질(NP-011)을 주입 경우, 8주간 체중의 큰 변화가 보이지 않아 체중변화에 큰 영향을 주지 않음을 알 수 있다.As a result, as shown in FIG. 10, the recombinant protein for preventing or treating myocardial infarction (NP-011) of the present invention was compared to a rat with acute myocardial infarction (AMI) induced as compared to a rat with acute myocardial infarction (AMI). In the case of infusion, it can be seen that no significant change in body weight was observed for 8 weeks, and thus there was no significant effect on body weight change.
이는 급성심근경색(AMI)이 유도된 랫트에 본 발명의 심근경색 예방 또는 치료용 재조합 단백질(NP-011)을 주입하더라도 심장 박동 기능의 회복이 진행되는 과정에서 별도의 병리적으로 심각한 부작용은 나타나지 않는 것을 확인하였다.This is acute myocardial infarction (AMI) induced rat myocardial infarction prevention or treatment recombinant protein (NP-011) of the present invention in the process of recovery of cardiac pulsation function in the process of progressing a separate pathologically serious side effects appear It was confirmed that it does not.
(4) 심근경색 유도 동물모델의 MT 및 H&E 염색을 통한 항섬유화 효과 검증(4) Verification of antifibrotic effect through MT and H&E staining of myocardial infarction induced animal model
본 발명의 Milk fat globule-EGF factor 8(MFG-E8) 단백질 기반의 심근경색 예방 또는 치료용 재조합 단백질(NP-011)의 심장기능 향상성을 확인하기 위하여, MT염색 및 H&E염색을 통한 심장 조직 섬유화 수준을 측정하였다.Cardiac tissue through MT staining and H&E staining to confirm cardiac function improvement of the recombinant protein (NP-011) for prevention or treatment of myocardial infarction based on the milk fat globule-EGF factor 8 (MFG-E8) protein of the present invention Fibrosis level was measured.
우선, 앞서 제작 설정한 제1실험군 내지 제5실험군 각각의 동물 모델들 심장 조직 섬유화 정도를 분석하기 위해 8주차 진행을 거친 각각의 랫트에서 적출한 심장을 4% 파라포름알데히드(Paraformaldehyde)로 고정 후, 파라핀 블록을 제조하여 4μm 두께로 절제하여 H&E staining 및 Masson's trichrome(MT) staining을 통하여 심장 조직의 섬유화 정도를 분석한다. First, after analyzing the animal tissues of each of the first experimental group and the fifth experimental group, which were previously set, the heart extracted from each rat, which has undergone the 8th week, is fixed with 4% Paraformaldehyde. , Paraffin block is prepared and excised to a thickness of 4 μm to analyze the degree of fibrosis of the heart tissue through H&E staining and Masson's trichrome (MT) staining.
여기서, H&E염색을 위해서는 Sigma에서 구입한 헤마톡실린(Hematoxylin) 및 에오신(Eosin)을 사용하였으며, MT염색을 위해서는 Sigma에서 구입한 Trichrome Stain Kit(HT15, Sigma-Aldrich)를 사용하였다. Here, Hematoxylin and Eosin purchased from Sigma were used for H&E staining, and Trichrome Stain Kit (HT15, Sigma-Aldrich) purchased from Sigma was used for MT staining.
또한, MT 및 H&E 염색을 통해 각 실험군 별 심장 조직 샘플의 사진을 도11과 같이 수득하여 좌심실 전체 면적 대비 지엽적으로 섬유화된 부분의 면적 값을 계산하였다.In addition, MT and H&E staining to obtain a picture of a heart tissue sample for each experimental group as shown in FIG. 11 to calculate the area value of the locally fibrous portion compared to the total area of the left ventricle.
아울러, 각 실험군 별 좌심실 전체 면적 대비 지엽적으로 섬유화된 부분의 면적 값의 계산은 Mathworks 사 (社)의 Matlab (R2018b) software를 이용했으며, 상대적 넓이의 단위는 arbitrary unit (a.u.)으로 나타냈다. In addition, the calculation of the area value of the locally fibrous portion compared to the total area of the left ventricle for each experimental group was performed using Mathworks' Matlab (R2018b) software, and the unit of the relative width was expressed as an arbitrary unit (a.u.).
이와 같이 도11의 제1실험군 내지 제5실험군 8주차 심장 조직 샘플 사진을 기준으로 측정된 섬유화 면적 비율에 대한 결과는 도12에 도시된 바와 같다.As described above, the results of the ratio of the fibrous area measured based on the 8th week heart tissue sample photograph of the first experimental group to the fifth experimental group of FIG. 11 are as shown in FIG. 12.
도11 및 도12를 통해 각 실험군 별 심장 조직의 섬유화 정도 및 심근경색을 통한 리모델링이 진행된 심장 조직의 크기 증대의 정도를 확인할 수 있다.11 and 12, the degree of fibrosis of the heart tissue for each experimental group and the degree of increase in the size of the heart tissue undergoing remodeling through myocardial infarction can be confirmed.
구체적으로, 급성심근경색(AMI)이 유도된 랫트에 본 발명의 심근경색 예방 또는 치료용 재조합 단백질(NP-011)을 주입한 제5실험군의 경우, 급성심근경색(AMI)이 유도된 랫트인 제2실험군과 비교해 약 80%의 항섬유화 효과를 나타내고 있다.Specifically, in the case of the fifth experimental group in which the recombinant protein for preventing or treating myocardial infarction (NP-011) of the present invention was injected into a rat in which acute myocardial infarction (AMI) was induced, acute myocardial infarction (AMI) was induced. Compared with the 2nd experimental group, it shows an anti-fibrotic effect of about 80%.
아울러, 급성심근경색(AMI)이 유도된 랫트에 본 발명의 심근경색 예방 또는 치료용 재조합 단백질(NP-011)을 주입한 제5실험군의 경우, 급성심근경색(AMI)이 유도된 랫트에 MFG-E8을 주입한 제4실험군과 비교해 약 34% 높은 항섬유화 효과를 보인다.In addition, in the case of the fifth experimental group in which the recombinant protein for preventing or treating myocardial infarction (NP-011) of the present invention was injected into a rat in which acute myocardial infarction (AMI) was induced, MFG in acute myocardial infarction (AMI) induced rats -Compared to the 4th experimental group injected with E8, it shows about 34% higher antifibrosis effect.
즉, 본 발명의 심근경색 예방 또는 치료용 재조합 단백질(NP-011)은 압도적으로 우수한 항섬유화능의 제공을 통해 심근경색에 의해 괴사되어 섬유화가 진행된 심근 세포 조직을 비롯한 각종 심장조직의 섬유화를 방지하여, 조직 섬유화로 인한 심장 박동 기능의 비정상화 및 심장 비대화 현상을 해소하고 심근경색의 본질적 치료가 이루어질 수 있도록 한다.That is, the recombinant protein for preventing or treating myocardial infarction of the present invention (NP-011) prevents fibrosis of various cardiac tissues, including myocardial cell tissue that has undergone fibrosis by necrosis by myocardial infarction through providing an overwhelmingly excellent antifibrotic ability. Thus, the abnormality of the heartbeat function due to tissue fibrosis and the cardiac hypertrophy phenomenon are solved and essential treatment of myocardial infarction can be achieved.
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, 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.
<110> NEXEL CO.,LTD. <120> RECOMBINANT PROTEIN FOR MYOCARDIAL INFARCTION TREATMENT OR PREVENTION AND COMPOSITION FOR MYOCARDIAL INFARCTION TREATMENT OR PREVENTION CONTAINING THEREOF <130> LP18-108 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 202 <212> PRT <213> Artificial Sequence <220> <223> RECOMBINANT PROTEIN(NP-011) FOR MYOCARDIAL INFARCTION TREATMENT OR PREVENTION <400> 1 Leu Asp Ile Cys Ser Lys Asn Pro Cys His Asn Gly Gly Leu Cys Glu 1 5 10 15 Glu Ile Ser Gln Glu Val Arg Gly Asp Val Phe Pro Ser Tyr Thr Cys 20 25 30 Thr Cys Leu Lys Gly Tyr Ala Gly Asn His Cys Glu Thr Lys Cys Val 35 40 45 Glu Pro Leu Gly Met Glu Asn Gly Asn Ile Ala Asn Ser Gln Ile Ala 50 55 60 Ala Ser Ser Val Arg Val Thr Phe Leu Gly Leu Gln His Trp Val Pro 65 70 75 80 Glu Leu Ala Arg Leu Asn Arg Ala Gly Met Val Asn Ala Trp Thr Pro 85 90 95 Ser Ser Asn Asp Asp Asn Pro Trp Ile Gln Val Asn Leu Leu Arg Arg 100 105 110 Met Trp Val Thr Gly Val Val Thr Gln Gly Ala Ser Arg Leu Ala Ser 115 120 125 His Glu Tyr Leu Lys Ala Phe Lys Val Ala Tyr Ser Leu Asn Gly His 130 135 140 Glu Phe Asp Phe Ile His Asp Val Asn Lys Lys His Lys Glu Phe Val 145 150 155 160 Gly Asn Trp Asn Lys Asn Ala Val His Val Asn Leu Phe Glu Thr Pro 165 170 175 Val Glu Ala Gln Tyr Val Arg Leu Tyr Pro Thr Ser Cys His Thr Ala 180 185 190 Cys Thr Leu Arg Phe Glu Leu Leu Gly Cys 195 200 <210> 2 <211> 44 <212> PRT <213> Artificial Sequence <220> <223> EGF-like domain part in Recombinant Protein(NP-011) <400> 2 Leu Asp Ile Cys Ser Lys Asn Pro Cys His Asn Gly Gly Leu Cys Glu 1 5 10 15 Glu Ile Ser Gln Glu Val Arg Gly Asp Val Phe Pro Ser Tyr Thr Cys 20 25 30 Thr Cys Leu Lys Gly Tyr Ala Gly Asn His Cys Glu 35 40 <210> 3 <211> 158 <212> PRT <213> Artificial Sequence <220> <223> C1 domain part in Recombinant Protein(NP-011) <400> 3 Thr Lys Cys Val Glu Pro Leu Gly Met Glu Asn Gly Asn Ile Ala Asn 1 5 10 15 Ser Gln Ile Ala Ala Ser Ser Val Arg Val Thr Phe Leu Gly Leu Gln 20 25 30 His Trp Val Pro Glu Leu Ala Arg Leu Asn Arg Ala Gly Met Val Asn 35 40 45 Ala Trp Thr Pro Ser Ser Asn Asp Asp Asn Pro Trp Ile Gln Val Asn 50 55 60 Leu Leu Arg Arg Met Trp Val Thr Gly Val Val Thr Gln Gly Ala Ser 65 70 75 80 Arg Leu Ala Ser His Glu Tyr Leu Lys Ala Phe Lys Val Ala Tyr Ser 85 90 95 Leu Asn Gly His Glu Phe Asp Phe Ile His Asp Val Asn Lys Lys His 100 105 110 Lys Glu Phe Val Gly Asn Trp Asn Lys Asn Ala Val His Val Asn Leu 115 120 125 Phe Glu Thr Pro Val Glu Ala Gln Tyr Val Arg Leu Tyr Pro Thr Ser 130 135 140 Cys His Thr Ala Cys Thr Leu Arg Phe Glu Leu Leu Gly Cys 145 150 155 <210> 4 <211> 609 <212> DNA <213> Artificial Sequence <220> <223> DNA fragment encoding Recombinant Protein(NP-011) <400> 4 ctggatatct gcagcaaaaa cccgtgccac aacggtggcc tgtgcgagga aattagccaa 60 gaggtgcgtg gtgacgtttt cccgagctac acctgcacct gcctgaaggg ctatgcgggt 120 aaccactgcg agaccaaatg cgtggaaccg ctgggcatgg agaacggtaa catcgcgaac 180 agccagattg cggcgagcag cgtgcgtgtt acctttctgg gcctgcaaca ctgggttccg 240 gaactggcgc gtctgaaccg tgcgggtatg gttaacgcgt ggaccccgag cagcaacgac 300 gataacccgt ggatccaggt gaacctgctg cgtcgtatgt gggttaccgg cgtggttacc 360 cagggtgcga gccgtctggc gagccacgag tacctgaagg cgttcaaagt tgcgtatagc 420 ctgaacggcc acgaattcga ctttattcac gatgtgaaca agaaacacaa ggagttcgtt 480 ggtaactgga acaaaaacgc ggtgcacgtt aacctgtttg agaccccggt ggaagcgcag 540 tacgttcgtc tgtatccgac cagctgccac accgcgtgca ccctgcgttt tgaactgctg 600 ggttgctaa 609 <210> 5 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> Signal peptide Example for NP-011 <400> 5 Met Pro Arg Pro Arg Leu Leu Ala Ala Leu Cys Gly Ala Leu Leu Cys 1 5 10 15 Ala Pro Ser Leu Leu Val Ala 20 <210> 6 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> FLAG tag in pLFCF Vector <400> 6 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 <110> NEXEL CO.,LTD. <120> RECOMBINANT PROTEIN FOR MYOCARDIAL INFARCTION TREATMENT OR PREVENTION AND COMPOSITION FOR MYOCARDIAL INFARCTION TREATMENT OR PREVENTION CONTAINING THEREOF <130> LP18-108 <160> 6 <170> KoPatentIn 3.0 <210> 1 <211> 202 <212> PRT <213> Artificial Sequence <220> <223> RECOMBINANT PROTEIN(NP-011) FOR MYOCARDIAL INFARCTION TREATMENT OR PREVENTION <400> 1 Leu Asp Ile Cys Ser Lys Asn Pro Cys His Asn Gly Gly Leu Cys Glu 1 5 10 15 Glu Ile Ser Gln Glu Val Arg Gly Asp Val Phe Pro Ser Tyr Thr Cys 20 25 30 Thr Cys Leu Lys Gly Tyr Ala Gly Asn His Cys Glu Thr Lys Cys Val 35 40 45 Glu Pro Leu Gly Met Glu Asn Gly Asn Ile Ala Asn Ser Gln Ile Ala 50 55 60 Ala Ser Ser Val Arg Val Thr Phe Leu Gly Leu Gln His Trp Val Pro 65 70 75 80 Glu Leu Ala Arg Leu Asn Arg Ala Gly Met Val Asn Ala Trp Thr Pro 85 90 95 Ser Ser Asn Asp Asp Asn Pro Trp Ile Gln Val Asn Leu Leu Arg Arg 100 105 110 Met Trp Val Thr Gly Val Val Thr Gln Gly Ala Ser Arg Leu Ala Ser 115 120 125 His Glu Tyr Leu Lys Ala Phe Lys Val Ala Tyr Ser Leu Asn Gly His 130 135 140 Glu Phe Asp Phe Ile His Asp Val Asn Lys Lys His Lys Glu Phe Val 145 150 155 160 Gly Asn Trp Asn Lys Asn Ala Val His Val Asn Leu Phe Glu Thr Pro 165 170 175 Val Glu Ala Gln Tyr Val Arg Leu Tyr Pro Thr Ser Cys His Thr Ala 180 185 190 Cys Thr Leu Arg Phe Glu Leu Leu Gly Cys 195 200 <210> 2 <211> 44 <212> PRT <213> Artificial Sequence <220> <223> EGF-like domain part in Recombinant Protein (NP-011) <400> 2 Leu Asp Ile Cys Ser Lys Asn Pro Cys His Asn Gly Gly Leu Cys Glu 1 5 10 15 Glu Ile Ser Gln Glu Val Arg Gly Asp Val Phe Pro Ser Tyr Thr Cys 20 25 30 Thr Cys Leu Lys Gly Tyr Ala Gly Asn His Cys Glu 35 40 <210> 3 <211> 158 <212> PRT <213> Artificial Sequence <220> <223> C1 domain part in Recombinant Protein (NP-011) <400> 3 Thr Lys Cys Val Glu Pro Leu Gly Met Glu Asn Gly Asn Ile Ala Asn 1 5 10 15 Ser Gln Ile Ala Ala Ser Ser Val Arg Val Thr Phe Leu Gly Leu Gln 20 25 30 His Trp Val Pro Glu Leu Ala Arg Leu Asn Arg Ala Gly Met Val Asn 35 40 45 Ala Trp Thr Pro Ser Ser Asn Asp Asp Asn Pro Trp Ile Gln Val Asn 50 55 60 Leu Leu Arg Arg Met Trp Val Thr Gly Val Val Thr Gln Gly Ala Ser 65 70 75 80 Arg Leu Ala Ser His Glu Tyr Leu Lys Ala Phe Lys Val Ala Tyr Ser 85 90 95 Leu Asn Gly His Glu Phe Asp Phe Ile His Asp Val Asn Lys Lys His 100 105 110 Lys Glu Phe Val Gly Asn Trp Asn Lys Asn Ala Val His Val Asn Leu 115 120 125 Phe Glu Thr Pro Val Glu Ala Gln Tyr Val Arg Leu Tyr Pro Thr Ser 130 135 140 Cys His Thr Ala Cys Thr Leu Arg Phe Glu Leu Leu Gly Cys 145 150 155 <210> 4 <211> 609 <212> DNA <213> Artificial Sequence <220> <223> DNA fragment encoding Recombinant Protein (NP-011) <400> 4 ctggatatct gcagcaaaaa cccgtgccac aacggtggcc tgtgcgagga aattagccaa 60 gaggtgcgtg gtgacgtttt cccgagctac acctgcacct gcctgaaggg ctatgcgggt 120 aaccactgcg agaccaaatg cgtggaaccg ctgggcatgg agaacggtaa catcgcgaac 180 agccagattg cggcgagcag cgtgcgtgtt acctttctgg gcctgcaaca ctgggttccg 240 gaactggcgc gtctgaaccg tgcgggtatg gttaacgcgt ggaccccgag cagcaacgac 300 gataacccgt ggatccaggt gaacctgctg cgtcgtatgt gggttaccgg cgtggttacc 360 cagggtgcga gccgtctggc gagccacgag tacctgaagg cgttcaaagt tgcgtatagc 420 ctgaacggcc acgaattcga ctttattcac gatgtgaaca agaaacacaa ggagttcgtt 480 ggtaactgga acaaaaacgc ggtgcacgtt aacctgtttg agaccccggt ggaagcgcag 540 tacgttcgtc tgtatccgac cagctgccac accgcgtgca ccctgcgttt tgaactgctg 600 ggttgctaa 609 <210> 5 <211> 23 <212> PRT <213> Artificial Sequence <220> <223> Signal peptide Example for NP-011 <400> 5 Met Pro Arg Pro Arg Leu Leu Ala Ala Leu Cys Gly Ala Leu Leu Cys 1 5 10 15 Ala Pro Ser Leu Leu Val Ala 20 <210> 6 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> FLAG tag in pLFCF Vector <400> 6 Asp Tyr Lys Asp Asp Asp Asp Lys 1 5
Claims (7)
심근경색 예방 또는 치료용 조성물.
Characterized in that it comprises a milk fat globule-EGF factor 8 (MFG-E8) protein-based recombinant protein consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient
A composition for preventing or treating myocardial infarction.
상기 재조합 단백질은 예방 또는 치료 대상 심장의 좌심실 박출률(EF, Ejection Fraction)을 높이는 것을 특징으로 하는
심근경색 예방 또는 치료용 조성물.
According to claim 1,
The recombinant protein is characterized by increasing the left ventricular ejection fraction (EF, Ejection Fraction) of the heart to be prevented or treated
A composition for preventing or treating myocardial infarction.
상기 재조합 단백질은 예방 또는 치료 대상 심장의 좌심실 분획단축률(FS, Fractional shortening)을 높이는 것을 특징으로 하는
심근경색 예방 또는 치료용 조성물.
According to claim 1,
The recombinant protein is characterized by increasing the left ventricular fractional shortening (FS) of the heart to be prevented or treated
A composition for preventing or treating myocardial infarction.
상기 심근경색 예방 또는 치료용 조성물은 병변내 투여, 혈관내 투여, 피하 투여, 비강내 투여 또는 복강내 투여용으로 제형화되는 것을 특징으로 하는
심근경색 예방 또는 치료용 조성물.According to claim 1,
The composition for preventing or treating myocardial infarction is formulated for intralesional administration, intravascular administration, subcutaneous administration, intranasal administration, or intraperitoneal administration.
A composition for preventing or treating myocardial infarction.
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| PCT/KR2018/012780 WO2020085545A1 (en) | 2018-10-25 | 2018-10-26 | Recombinant protein for prevention or treatment of myocardial infarction and composition comprising same for prevention or treatment of myocardial infarction |
| EP19875681.9A EP3870210A4 (en) | 2018-10-25 | 2019-10-24 | Compositions and methods for treating or preventing fibrosis |
| PCT/IB2019/001136 WO2020084344A2 (en) | 2018-10-25 | 2019-10-24 | Compositions and methods for treating or preventing fibrosis |
| JP2021547960A JP2022509445A (en) | 2018-10-25 | 2019-10-24 | Compositions and Methods for Treating or Preventing Fibrosis |
| CN201980086450.8A CN113301914A (en) | 2018-10-25 | 2019-10-24 | Compositions and methods for treating and preventing fibrosis |
| US17/285,040 US20210388041A1 (en) | 2018-10-25 | 2019-10-24 | Compositions and methods for treating or preventing fibrosis |
| JP2024092192A JP2024133483A (en) | 2018-10-25 | 2024-06-06 | Compositions and methods for treating or preventing fibrosis |
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| Barallobre-Barreiro J et al., Circulation. 2012, 125(6), pp.789-802 (2012.02.14.) |
| Deng et al., Hypertension, 2017,10, pp.770-779 (2017.10.) |
| Genebank, AAP36434.1, Homo sapiens milk fat globule-EGF factor 8 protein, partial [synthetic construct], (2016.07.25.) |
| Genebank, XP_016877695.1, lactadherin isoform X2 [Homo sapiens], (2018.05.26.) |
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