KR20180049621A - Composition for treating antivirus containing micafungin or pharmaceutically acceptable salts thereof as an active ingredient - Google Patents
Composition for treating antivirus containing micafungin or pharmaceutically acceptable salts thereof as an active ingredient Download PDFInfo
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- KR20180049621A KR20180049621A KR1020160145770A KR20160145770A KR20180049621A KR 20180049621 A KR20180049621 A KR 20180049621A KR 1020160145770 A KR1020160145770 A KR 1020160145770A KR 20160145770 A KR20160145770 A KR 20160145770A KR 20180049621 A KR20180049621 A KR 20180049621A
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- South Korea
- Prior art keywords
- micafungin
- enterovirus
- antiviral
- cells
- cvb3
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Abstract
Description
본 발명은 미카펀진(micafungin) 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 항바이러스용 약학적 조성물에 관한 것이다.
The present invention relates to an antiviral pharmaceutical composition comprising micafungin or a pharmaceutically acceptable salt thereof as an active ingredient.
엔테로바이러스(enterovirus)는 인체나 포유류의 장(腸)에 감염을 일으키는 바이러스를 통칭하며, 새롭게 발견될 때마다 EV68, EV71, EV73 등으로 명명된다. 엔테로바이러스는 발병 시 손발과 입에 염증 및 발진 등이 일어나 수족구병(手足口病)을 일으키기도 하지만 일부 환자에게서는 무균성수막염, 바이러스성 폐렴, 뇌염 또는 급성이완성 마비현상 등을 동반한다. 면역체계가 아직 발달되지 못한 신생아의 경우 심하면 신경원성 폐부종으로 인해 사망할 수도 있다. 중국에서는 2008년 5월, EV71 감염병의 확산으로 큰 혼란을 겪은 바 있으며, 우리나라는 2010년 12월 30일부터 보건복지가족부 장관의 고시에 따라 엔테로바이러스 감염증과 수족구병을 감시활동을 필요로 하는 지정감염병으로 지정한 바 있다.Enteroviruses are viruses that cause infection in the intestines of humans and mammals, and are named EV68, EV71, and EV73 whenever they are found. Enteroviruses cause inflammation and rashes in the hands and mouth of the onset of the disease, which can cause hand and foot disease. However, in some patients, aseptic meningitis, viral pneumonia, encephalitis or acute complete paralysis is accompanied. Newborn infants whose immune system has not yet developed may die from neurotic pulmonary edema. In China, in May 2008, the spread of the EV71 infection caused great confusion. In accordance with the notification of Minister of Health, Welfare and Family Affairs from Dec. 30, 2010, It has been designated as an infectious disease.
엔테로바이러스는 주로 하절기에 발병하며, 감염자의 면역학적 특성에 따라 중증감염을 초래하기도 한다. 특히 위생이 나쁜 환경에서 흔하게 전파되는 전염성 병원체로써 감염경로는 분변 또는 구강 등이며, 오염된 물과 토양을 통한 경구적인 전파로도 이어진다. 감염자에서 배출된 분변 속의 바이러스가 오수나 폐수를 통해 지하수, 하천, 해수로 흘러들어가 다시 사람에게로 전염되는데, 드물게는 호흡기 분비물을 통해서도 감염된다. 엔테로바이러스는 소화기를 통해 감염된 후 인후두 부위나 소장의 림프절에서 일차적으로 증식한 후 신체의 각 장기로 이동한다. 임상증상은 감기 등의 가벼운 증상부터 심각한 마비까지 매우 다양하다. 소아인 경우 비폴리오성 엔테로바이러스(enterovirus) 감염은 50%정도 불현성 감염으로 나타나며, 상기도감염이나, 소화기증상, 결막염, 중이염, 피부발진, 무균성수막염, 포진성구협염, 수족구병 및 고환염 등의 증상을 보인다. 드물게 심근염, 뇌염, Guillian-Barre syndrome, 실조증, 말단신경염, 횡단성척수염, 사지마비, 당뇨병 및 유행성 출혈성 각결막염 등 치명적이거나 합병증을 남기는 경우도 보고되어 있다. 엔테로바이러스에 의한 대표적인 증상은 무균성 수막염이고 주로 하절기에 발생하는 것으로 알려져 있으나 봄이나 늦가을, 그리고 겨울에도 산발적으로 발생하는 경우가 있어 일 년 내내 감염될 위험이 존재한다. 또한 알려져 있는 주된 발생 연령층은 영ㆍ유아이나, 경우에 따라 소아 및 노령층에서도 발생할 수 있다. EV71(enterovirus 71)의 경우 다른 엔테로바이러스보다 더 심각한 임상경과를 보이며, 특히 어린 아이에서 높은 비율로 신경계 합병증을 일으켜, 뇌간 뇌염, 신경인성 폐부종, 폐출혈, 쇼크 및 급속한 사망에 이를 수 있다. Enteroviruses occur mainly during the summer season and may cause serious infections depending on the immunological characteristics of the infected person. It is an infectious pathogen that is commonly spread in hygienic environments, such as feces or oral cavity, and it also leads to oral transmission through contaminated water and soil. The fecal virus from the infected person flows into the groundwater, the river, and the seawater through sewage or wastewater, and then it is transmitted to humans again. In rare cases, it is also transmitted through the respiratory secretions. The enteroviruses are transmitted through the digestive tract and then propagate primarily in the larynx or in the lymph nodes of the small intestine and then move to the organs of the body. Clinical symptoms vary from mild symptoms such as colds to severe paralysis. Enterovirus infection is a 50% incidence of infectious infection in children. Symptoms such as upper respiratory infection, digestive symptoms, conjunctivitis, otitis media, skin rash, aseptic meningitis, herpes zoster, . Rarely, there are reports of fatal or complications such as myocarditis, encephalitis, Guillian-Barre syndrome, ataxia, terminal neuritis, transverse myelitis, limb paralysis, diabetes mellitus and epidemic keratoconjunctivitis. A typical symptom caused by enterovirus is aseptic meningitis, which is known to occur mainly during the summer, but sporadically occurs in spring, late autumn, and winter, so there is a risk of infection throughout the year. Also known are the main age groups that can occur in infants and young children, and in some cases children and elderly people. EV71 (enterovirus 71) has a more severe clinical course than other enteroviruses, causing a high rate of neurological complications, especially in young children, resulting in brain encephalitis, neurogenic pulmonary edema, pulmonary hemorrhage, shock, and rapid death.
엔테로바이러스는 분류학적으로 Picornaviridae과에 속하며, 현재까지 약 68여 종의 혈청형이 알려져 있다. 혈청형에 따라 크게 3가지 혈청형의 Poliovirus (PV: 1~3)와 23가지 혈청형의 Coxsackievirus A군(CVA: 1~22, 24), 6가지 혈청형의 Coxsackievirus B군(CVB: 1~6), 그리고 28가지 혈청형의 Echovirus군(ECV: 1~7, 9, 11~21, 24~27, 29~33) 및 기타 Human Enterovirus (EV: 68~116) 등으로 구성되어 있다.Enterobacteriaceae belong to the genus Picornaviridae, and about 68 serotypes have been known to date. There were three serotypes of poliovirus (PV: 1 ~ 3), 23 serotypes of Coxsackievirus A (CVA: 1 ~ 22, 24) and 6 serotypes of Coxsackievirus B (CVB: 6) and 28 serotypes of Echovirus (ECV: 1 to 7, 9, 11 to 21, 24 to 27, 29 to 33) and other human enteroviruses (EV: 68 to 116).
엔테로바이러스는 엔벨로프(envelope)를 갖지 않는 27 내지 30 nm 크기의 바이러스로서 정이십면체의 형태를 가지며, 약 7.2 내지 7.5 kb 크기의 single stranded positive sense RNA를 유전물질로 함유하고 있다. 하나의 open reading frame (ORF)과 5´ 및 3´말단에 단백질로 발현되지 않는 non-coding region (NCR)으로 구성되어 있는데, ORF는 전체 유전자의 약 90%를 차지하고 하나의 polyprotein으로 발현되는데, 약 2,185개의 아미노산으로 이루어져 있다. 이 polyprotein은 바이러스의 단백질 분해효소에 의해서 여러 개의 다른 단백질로 나누어진다. 하나의 polyprotein은 P1, P2, P3로 구분되어 있는데, P1부분은 바이러스의 캡시드 단백질의 구성요소인 VP4, VP2, VP3, VP1을 순서대로 암호화 하고 있고, 캡시드는 32 mer의 capsomer로 구성되어 있다. P2부분은 2Apro 단백질분해효소와 현재까지 기능이 정확하게 알려지지 않은 단백질들을 암호화하고 있으며, P3부분은 VPg 단백질과 3Cpro단백질분해효소 및 RNA dependent RNA polymerase (RdRp)가 암호화되어있다. VPg 단백질은 primer로써 3´쪽에 작용하여, negative sense strand의 게놈 형성에 관여 한다. 5´NCR은 700~800 bp로 구성되어 있고, 매우 잘 보존된 복잡한 RNA 2차 구조를 형성한다. 이러한 RNA 2차 구조는 두 가지 기능을 갖는데, 첫 번째는 RdRp에 의해서 positive sense RNA가 합성될 때 RNA 합성에 필요한 시작점을 제공한다. 두 번째는 cap-independent 단백질 합성과정에서 숙주의 ribosome이 최초로 결합하는 internal ribosomal entry site(IRES)를 제공함으로써 바이러스 단백질을 합성할 수 있도록 한다. 3´NCR은 100-150 bp로 구성되어 있으며, 이것의 2차 구조는 RNA 게놈의 주형인 negative sense RNA가 합성될 때 primer의 기능에 관여한다고 알려져 있다.The enteroviruses are 27 to 30 nm in size with no envelope. They are in the form of a regular dodecahedron and contain a single stranded positive sense RNA of about 7.2 to 7.5 kb in size. The ORF consists of a single open reading frame (ORF) and a non-coding region (NCR) that is not expressed at the 5'and 3'ends. The ORF occupies about 90% of the entire gene and is expressed as a polyprotein. It is made up of about 2,185 amino acids. This polyprotein is divided into several different proteins by the proteolytic enzyme of the virus. One polyprotein is divided into P1, P2, and P3. The P1 part encodes VP4, VP2, VP3, VP1, which are the constituents of the virus's capsid protein, and the capsid is composed of a 32 mer capsomer. The P2 part is 2A pro It encodes proteases and proteins that are not known to function until now. The P3 part encodes VPg protein, 3C pro protease and RNA dependent RNA polymerase (RdRp). The VPg protein acts as a primer on the 3 'side and is involved in the genome formation of a negative sense strand. The 5 'NCR consists of 700-800 bp and forms a very well conserved complex RNA secondary structure. These RNA secondary structures have two functions, the first of which provides a starting point for RNA synthesis when positive sense RNA is synthesized by RdRp. The second is to allow the synthesis of viral proteins by providing an internal ribosomal entry site (IRES) in which the host's ribosome first binds during cap-independent protein synthesis. 3'NCR consists of 100-150 bp, and its secondary structure is known to be involved in primer function when negative sense RNA, the template of the RNA genome, is synthesized.
항바이러스제(antiviral drug)의 개발은 내성 바이러스의 출현, 약제 부작용, 높은 생산비용 등을 극복하는 방식으로의 지속적인 요구를 가지고 있다. 장바이러스의 감염 증상은 EV71(enterovirus 71)을 제외하고는 대부분 치명적이지 않지만, 면역력이 약한 영유아에게 문제를 일으키고 돌연변이율이 높아 독성이 강한 신,변종의 출현이 예상되므로 이들에 대한 치료제나 예방 백신의 개발이 필요하다. 그럼에도 현재까지의 예방백신에 대한 연구는 EV71을 제외하고는 거의 없는 실정이고, 현재까지 장바이러스의 유일한 치료제로 개발되어 있는 플레코나릴(Pleconaril)은 치료효과가 비교적 뛰어난 편이나, 부작용이 심각한 수준이어서 FDA의 승인을 받지 못하고 위급한 상황에만 제한적으로 사용되고 있다. 또한 점차 치료물질에 내성을 보이는 바이러스가 계속 보고되고 있어, 새로운 작용기전으로 다양한 장바이러스에 폭넓게 작용할 수 있는 치료용 약물의 개발이 시급하게 요구되고 있다.
The development of antiviral drugs has a continuing need in the form of overcoming the emergence of resistant viruses, drug side effects, and high production costs. Infectious symptoms of intestinal virus are mostly not fatal except for EV71 (enterovirus 71). However, it is expected that the emergence of new and mutant strains with high immunity and high mutation rate, Development is needed. However, studies on preventive vaccines to date have been rare except for EV71. Pleconaril, which has been developed as the sole treatment for enteroviruses, has a relatively high therapeutic efficacy, but serious side effects It is not approved by the FDA and is used only in emergency situations. In addition, viruses that are gradually resistant to therapeutic agents are continuously being reported, and it is urgently required to develop therapeutic drugs that can act widely against various enteroviruses with a new mechanism of action.
한편, 미카펀진(micafungin)은 정맥에 투여하는 에치노칸딘(echinocandin) 계열의 항진균제(antifungal drug)로서, 진균 세포벽의 필수 구성물질인 베타(beta)-1,3-글루칸(glucan)의 생성을 억제하는 메커니즘을 갖는, 부작용이 적고 선택적(selective) 독성이 높은 약물로 잘 알려져 있다. 그러나 미카펀진이 항바이러스 효과를 나타낸다는 연구결과는 아직까지 보고된 바가 없다.
On the other hand, micafungin is an antifungal drug of the echinocandin system administered intravenously. It produces beta-1,3-glucan, an essential constituent of the fungal cell wall. Is known to be a drug with low side effects and high selective toxicity. However, no study has yet been reported on the antiviral effect of microfunzine.
이에, 본 발명자들은 항바이러스 활성을 가진 신규한 조성물을 찾기 위해 노력하던 중, 미카펀진(micafungin)이 항바이러스 효능을 가짐을 확인하여 항바이러스용 약학적 조성물로써 유용하게 이용될 수 있음을 밝힘으로써, 본 발명을 완성하였다.
Accordingly, the present inventors have found that micafungin has an antiviral effect while trying to find a novel composition having antiviral activity, and thus it can be used as a pharmaceutical composition for antiviral use , Thereby completing the present invention.
본 발명의 목적은 미카펀진(micafungin) 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 항바이러스용 약학적 조성물을 제공하는 것이다.
It is an object of the present invention to provide a pharmaceutical composition for antiviral containing micafungin or a pharmaceutically acceptable salt thereof as an active ingredient.
상기 목적을 달성하기 위하여, 본 발명은 미카펀진(micafungin) 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 항바이러스용 약학적 조성물을 제공한다.In order to achieve the above object, the present invention provides a pharmaceutical composition for antiviral comprising micafungin or a pharmaceutically acceptable salt thereof as an active ingredient.
또한, 본 발명은 미카펀진(micafungin) 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 바이러스 질환의 개선용 건강기능식품을 제공한다.
The present invention also provides a health functional food for improving viral diseases containing micafungin or a pharmaceutically acceptable salt thereof as an active ingredient.
본 발명의 미카펀진(micafungin) 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 조성물은 EV71(enterovirus 71)(BrCr 타입, H 타입 및 1095 타입), CVB3(coxsackievirus B3)및 HRV(human rhinovirus)(HRV-14, HRV-21, HRV-71)로 감염시킨 세포의 생존률을 증가시키고, CVB3가 감염된 마우스의 몸무게 저하 및 췌장 병리를 개선하는 것을 확인하여, 항바이러스용 약학적 조성물로 유용하게 이용될 수 있다.
The composition comprising the micafungin of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient is selected from the group consisting of EV71 (Enterovirus 71) (BrCr type, H type and 1095 type), CVB3 (coxsackievirus B3) and HRV (human rhinovirus (HRV-14, HRV-21, and HRV-71), and decreased CVB3-infected mice and improved pancreatic pathology. Thus, they are useful as antiviral pharmaceutical compositions Can be used.
도 1은 스크리닝 과정을 거쳐 EV71(enterovirus 71)에 항바이러스 효능을 나
나타내는 미카펀진(micafungin)을 발굴하는 과정을 나타낸 도이다:
A: 바이러스의 복제 활성을 루시퍼라아제(luciferase) 발현으로 측정할 수 있는 EV71 리플리콘(replicon) 시스템의 모식도;
B: Vero 세포에 EV71 리플리콘(replicon)을 도입한 뒤 FDA 승인 약물들로 스크리닝(screening)하여, DMSO 처리 대비 60% 이상 luciferase 활성을 감소시키는 것으로 나타난 21개의 약물들: Gem은 gemcitabine, Mica는 micafungin, Rup는 rupintrivir를 나타냄;
C: EV71(enterovirus 71)에 감염된 LLC-MK2 유도세포에 21개의 약물들을 처리한 후 MTT 어세이(assay)로 세포 생존율을 측정한 결과; 및
D: 미카펀진(micafungin)의 화학 구조.
도 2는 Vero 세포에 EV71(enterovirus 71) 리플리콘(replicon) RNA를 형질주
입(transfection)시킨 후, 루시퍼라아제(luciferase)의 활성을 시간 의존적으로(time-dependent) 측정한 결과를 나타낸 도이다.
도 3은 미카펀진(micafungin)의 EV71(enterovirus 71) 리플리콘(replicon)의 복제 억제효과를 나타낸 도이다:
A: Vero 세포에 EV71(enterovirus 71) 리플리콘(replicon) RNA를 형질주입(transfection)시킨 후, 다양한 농도의 미카펀진(micafungin)을 처리하여 루시퍼라아제(luciferase)의 활성(activity)을 측정한 결과; 및
B : 다양한 농도의 미카펀진(micafungin)에서 세포의 독성을 평가한 결과.
도 4는 미카펀진(micafungin)의 CVB3(coxsackievirus B3) 리플리콘(replicon)의 복제 억제효과를 나타낸 도이다:
A: Vero 세포에 CVB3(coxsackievirus B3) 리플리콘(replicon) RNA를 형질주입(transfection)시킨 후, 다양한 농도의 미카펀진(micafungin)을 처리하여 루시퍼라아제(luciferase)의 활성(activity)을 측정한 결과; 및
B: 다양한 농도의 미카펀진(micafungin)에서 세포의 독성을 평가한 결과.
도 5는 미카펀진(micafungin)에 장시간 노출시 세포의 독성을 평가한 결과를 나타낸 도이다.
A: 24시간 동안 각 농도별 미카펀진(micafungin)의 처리 결과; 및
B: 48시간 동안 각 농도별 미카펀진(micafungin)의 처리 결과.
도 6은 EV71(enterovirus 71)를 감염시킨 LLC-MK2 유도세포에서 미카펀진(micafungin)의 항바이러스 효과를 확인한 도이다:
A: EV71을 감염시킨 LLC-MK2 유도세포에서 미카펀진(micafungin)을 농도별로 4일 동안 처리 후, MTT 어세이(assay)를 통해 항바이러스 효과를 측정한 결과;
B: 동일 조건의 세포에서 미카펀진(micafungin)에 의한 세포의 독성을 측정한 결과;
C: 미카펀진(micafungin)의 처리 농도에 따른 3C 단백질의 발현 양상;
D: C의 세포로부터 분리한 바이러스 RNA(3BC 부위)의 양을 RT-PCR한 결과;
및
E: EV71(enterovirus 71)의 dsRNA를 항체로 염색(녹색)하고, 핵의 DNA는 DAPI로 염색한 사진(파란색).
도 7은 EV71(enterovirus 71)(H 및 1095 strain)에 대한 미카펀진(micafungin)의 항바이러스 효과를 MTT 어세이(assay)를 통해 측정한 도이다.
도 8은 상기 도 6E의 EV71(enterovirus 71)이 감염된 LLC-MK2 유도세포에서 나타나는 dsRNA 형광신호를 정량적으로 분석하여 미카펀진(micafungin)의 항바이러스 효과를 나타낸 도이다.
도 9는 CVB3(coxsackievirus B3)에 대한 미카펀진(micafungin)의 항바이러스 효과를 나타낸 도이다:
A: HeLa 세포에 CVB3(coxsackievirus B3)를 감염시켜 농도별로 미카펀진 (micafungin)을 처리한 후, 항바이러스 효과를 MTT 어세이(assay)로 측정한 결과; 및
B: 동일 조건의 세포에서 세포의 독성을 측정한 결과.
도 10은 세 종류의 HRV(human rhinovirus)(HRV-14, HRV-21 및 HRV-71)에 대한 미카펀진(micafungin)의 항바이러스 효과를 MTT 어세이(assay)로 측정한 결과:
A: HRV14;
B: HRV 21; 및
C: HRV 71.
도 11은 EV71(enterovirus 71)의 감염 전후 미카펀진(micafungin)의 처리 시간에 따른 항바이러스 효과를 나타낸 도이다.
도 12는 미카펀진(micafungin)의 항바이러스 효과의 작용점을 추적하기 위해
수행한 일련의 실험들을 나타낸 도이다:
A: 미카펀진(micafungin)이 IRES(internal ribosomal entry site)의 활성을 억제하는지 여부를 확인한 실험결과;
B: 미카펀진(micafungin)이 3C 프로테아제(protease)를 억제하는지의 여부를 확인한 실험결과;
C: 미카펀진(micafungin)이 2C 또는 3A를 통해 항바이러스의 효과를 갖는지의 여부를 확인한 실험결과.
도 13은 CVB3(coxsackievirus B3)를 감염시킨 마우스 모델에서 미카펀진(micafungin)의 체중저하 억제 효과를 나타낸 도이다:
A: Body weight(g); 및
B: Body weight(%).
도 14는 CVB3(coxsackievirus B3)를 감염시킨 마우스의 췌장 조직에서 분리한 RNA를 이용하여 Real time-PCR을 수행한 결과를 나타낸 도이다.
도 15는 CVB3(coxsackievirus B3)를 감염시킨 마우스의 췌장 조직에서 미카펀진(micafungin)에 의한 췌장 선포세포(acinar cell)의 손상 억제 효과를 H&E 염색(staining)으로 나타낸 도이다.Figure 1 shows the antiviral efficacy of EV71 (Enterovirus 71) after screening.
This figure shows the process of excavating a micafungin representing:
A: A schematic diagram of an EV71 replicon system capable of measuring the replication activity of a virus by expression of luciferase;
B: Twenty-one drugs that have been shown to reduce the luciferase activity by more than 60% compared to DMSO treatment by introducing EV71 replicon into Vero cells and then screening with FDA-approved drugs: Gem is gemcitabine, Mica is micafungin, Rup denotes rupintrivir;
C: Cell viability of LLC-MK2-inducible cells infected with EV71 (enterovirus 71) was measured by MTT assays after treatment with 21 drugs. And
D: Chemical structure of micafungin .
Fig. 2 shows the expression of EV71 (enterovirus 71) replicon RNA in Vero cells
FIG. 5 shows the result of time-dependent measurement of the activity of luciferase after transfection.
3 is a diagram showing the replication inhibition effect of EV71 (enterovirus 71) replicon of micafungin:
A: Vero cells were transfected with EV71 (enterovirus 71) replicon RNA and treated with various concentrations of micafungin to measure the activity of luciferase result; And
B: Results of cell toxicity evaluation at various concentrations of micafungin.
Figure 4 is a diagram showing the effect of inhibiting the replication of CVB3 (coxsackievirus B3) replicon of micafungin:
A: Vero cells were transfected with CVB3 (coxsackievirus B3) replicon RNA and treated with various concentrations of micafungin to measure the activity of luciferase result; And
B: Results of cell toxicity evaluation at various concentrations of micafungin.
FIG. 5 is a graph showing the results of evaluating the toxicity of cells upon prolonged exposure to micafungin. FIG.
A: Results of treatment with micafungin for each concentration for 24 hours; And
B: Treatment results of micafungin at each concentration for 48 hours.
6 shows the antiviral effect of micafungin in LLC-MK2 inducing cells infected with EV71 (enterovirus 71)
A: The antiviral effect was measured by MTT assay after 4 days treatment with micafungin in LLC-MK2 inducing cells infected with EV71.
B: Measurement of cell toxicity by micafungin in cells of the same condition;
C: Expression pattern of 3C protein according to treatment concentration of micafungin;
RT-PCR of the amount of viral RNA (3BC region) isolated from D: C cells;
And
E: A dsRNA of EV71 (enterovirus 71) is stained with antibody (green), and DNA of the nucleus is stained with DAPI (blue).
FIG. 7 shows the antiviral effect of micafungin on EV71 (enterovirus 71) (H and 1095 strain) measured by MTT assays.
FIG. 8 is a graph showing the antiviral effect of micafungin by quantitatively analyzing dsRNA fluorescence signals appearing in LLC-MK2 inducing cells infected with EV71 (enterovirus 71) of FIG. 6E.
Figure 9 shows the antiviral effect of micafungin on CVB3 (coxsackievirus B3): < RTI ID = 0.0 >
A: The anti-viral effect of HeLa cells treated with micafungin (CVB3, coxsackievirus B3) was measured by MTT assays. And
B: The result of measurement of cell toxicity in cells of the same condition.
FIG. 10 shows the antiviral effect of micafungin on three types of human rhinovirus (HRV-14, HRV-21 and HRV-71) measured by an MTT assay:
A: HRV14;
B:
C:
Fig. 11 shows the antiviral effect according to the treatment time of micafungin before and after infection of EV71 (enterovirus 71). Fig.
Figure 12 shows the effect of the antiviral effect of < RTI ID = 0.0 > micafungin < / RTI &
A series of experiments performed:
A: Experimental results confirming whether micafungin inhibits IRES (internal ribosomal entry site) activity;
B: Experimental results confirming whether or not micafungin inhibits 3C protease;
C: Experimental results confirming whether micafungin has an antiviral effect through 2C or 3A.
13 is a graph showing the effect of micafungin on weight loss in a mouse model infected with CVB3 (coxsackievirus B3)
A: Body weight (g); And
B: Body weight (%).
FIG. 14 is a graph showing the result of Real time-PCR using RNA isolated from pancreatic tissue of mice infected with CVB3 (coxsackievirus B3). FIG.
FIG. 15 shows H & E staining of the effect of micafungin on the damage of pancreatic acinar cells in pancreatic tissues of mice infected with CVB3 (coxsackievirus B3).
이하 본 발명을 상세히 설명한다.
Hereinafter, the present invention will be described in detail.
본 발명은 미카펀진(micafungin) 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 항바이러스용 약학적 조성물을 제공한다.The present invention provides an antiviral pharmaceutical composition comprising micafungin or a pharmaceutically acceptable salt thereof as an active ingredient.
상기 바이러스는 엔테로바이러스(enterovirus)로 이루어진 군으로부터 선택되는 어느 하나 이상인 것이 바람직하나, 이에 한정되지 않는다.The virus is preferably one or more selected from the group consisting of enterovirus, but is not limited thereto.
상기 바이러스는 라이노바이러스(rhinovirus)로 이루어진 군으로부터 선택되는 어느 하나 이상인 것이 바람직하나, 이에 한정되지 않는다.The virus is preferably at least one selected from the group consisting of rhinovirus, but is not limited thereto.
상기 미카펀진은 EV71(enterovirus 71)의 3C 단백질의 발현과 바이러스 RNA의 발현을 억제하는 것이 바람직하나, 이에 한정되지는 않는다.It is preferable, but not limited, to inhibit the expression of 3C protein of EV71 (enterovirus 71) and viral RNA expression.
또한, 상기 미카펀진은 바이러스 감염에 의한 체중의 저하를 억제하는 것이 바람직하며, 췌장조직을 구성하고 있는 췌장 선포세포(acinar cell)의 손상을 억제하는 것이 바람직하나, 이에 한정되지는 않는다.
In addition, it is preferable that the mycophenazine inhibits the decrease of body weight due to viral infection, and it is preferable to inhibit the damage of pancreatic acinar cells constituting the pancreatic tissue, but the present invention is not limited thereto.
본 발명의 구체적인 실시예 및 실험예에서, FDA 승인 약물들 중 EV71(enterovirus 71)에 항바이러스 효과를 나타내는 유효물질을 발굴하기 위한 EV71(enterovirus 71) 리플리콘(replicon) 시스템을 활용하기 위해, 바이러스의 구조 유전자인 P1(VP4-VP1) 부분에 반딧불이 루시퍼라아제(firefly luciferase) 유전자를 대체하여 삽입함으로써 세포 내에서 바이러스 복제를 쉽고 정량적으로 측정해 낼 수 있는 시스템을 제작한 뒤(도 1), 968개의 FDA 승인 약물들을 EV71 리플리콘(replicon) RNA 형질주입(transfection)과 동시에 처리하여 8시간이 지난 후에 루시퍼라아제(luciferase)의 활성을 측정하여(도 2), 21개의 유효 약물을 도출하였고, 이들 중 미카펀진(micafungin)이 EV71 및 CVB3 리플리콘(replicon) 모두에서 미카펀진 농도 의존적으로 루시퍼라아제(luciferase)의 활성을 감소시키고(도 3 및 도 4), 세포에 독성이 없는 신규한 용도의 물질임을 확인하였다(도 5). 또한, 실제 EV71(enterovirus 71)(BrCr 타입)으로 감염시킨 세포에서 미카펀진이 상기와 유사한 항바이러스 효과가 있음을 MTT 어세이(assay), 웨스턴 블롯팅(western blotting), RT-PCR 및 dsRNA 항체 형광 실험 등을 통해 확인하였고(도 6 및 8), 다른 종류의 EV71(enterovirus 71)(H 타입 및 1095 타입)로 감염시킨 세포에서도 미카펀진이 항바이러스 활성이 있음을 확인하였으며(도 7), CVB3(coxsackievirus B3)를 감염시킨 세포 및 HRV(human rhinovirus)(HRV-14, HRV-21, HRV-71)로 감염시킨 세포에서도 미카펀진에 의한 항바이러스 활성이 있음을 확인하였다(도 9 및 10). 또한, 미카펀진(micafungin)의 항바이러스 효과의 작용점과 관련해서, 바이러스 감염 후 1시간 시점에 미카펀진을 처리한 경우에도 바이러스 감염 전(-1시간) 및 감염과 동시에 미카펀진의 처리가 이뤄진 경우와 비슷하게 강력한 항바이러스 효과가 나타남을 확인하였고(도 11), 보다 세부적으로는 바이러스의 세포내 프로세스 중IRES 의존적 번역, 3C 프로테아제(protease)에 의한 폴리단백질 프로세싱(polyprotein processing) 및 2C 또는 3A 등과는 상관없이 다른 방식으로 항바이러스 효과를 가짐을 확인하였다(도 12). 또한, CVB3(coxsackievirus B3)를 감염시킨 마우스 그룹에서 나타나는 체중저하가 미카펀진에 의해 억제되며(도 13), CVB3의 RNA 발현량이 미카펀진에 의해서 감소하였음을 확인함과 동시에, CVB3(coxsackievirus B3)를 감염시킨 마우스의 췌장조직 내의 선포세포(acinar cell)의 손상이 미카펀진에 의해 억제됨을 최종적으로 확인하였다(도 15).
In order to utilize the EV71 (enterovirus 71) replicon system for identifying an effective substance showing an antiviral effect on EV71 (enterovirus 71) among the FDA-approved drugs in the specific examples and experimental examples of the present invention, A system capable of easily and quantitatively measuring viral replication in the cell by inserting a firefly luciferase gene into the P1 (VP4-VP1) structural gene of the present invention (FIG. 1) After 968 FDA-approved drugs were treated concurrently with EV71 replicon RNA transfection, the activity of luciferase was measured 8 hours later (FIG. 2), resulting in 21 active drugs , Of which micafungin reduced the activity of luciferase in both EV71 and CVB3 replicon in a dependent manner on the mycophenazine concentration (Figures 3 and 4) It was confirmed that the toxicity of this substance is not novel use (Fig. 5). In addition, MTT assays, western blotting, RT-PCR, and dsRNA antibodies (hereinafter referred to as " anti-viral " Neon (FIG. 6 and FIG. 8), it was confirmed that the microorganisms were also infected with other types of EV71 (enterovirus 71) (H type and 1095 type) cells were infected with coxsackievirus B3 and cells infected with human rhinovirus (HRV-14, HRV-21, HRV-71) were also confirmed to have antiviral activity by mikaphengin (FIGS. 9 and 10) . In addition, with regard to the point of action of the antiviral effect of micafungin, even when the microfunzine was treated at 1 hour after the virus infection, the microfunzine treatment was performed before the virus infection (-1 hour) (FIG. 11). More specifically, IRES-dependent translation among intracellular processes of viruses, polyprotein processing by 3C protease, and 2C or 3A It was confirmed that it had an antiviral effect in different ways irrespective of the presence or absence (FIG. 12). In addition, it was confirmed that the weight decrease in the mouse group infected with CVB3 (coxsackievirus B3) was inhibited by mikaphengin (Fig. 13), and that the amount of CVB3 RNA expression was decreased by the microcapyrin and CVB3 (coxsackievirus B3) (Fig. 15). It was confirmed that the damage of the acinar cells in the pancreatic tissues of the mice infected with < RTI ID = 0.0 >
따라서, 본 발명의 미카펀진(micafungin) 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 약학적 조성물은 항바이러스의 용도로 유용하게 사용될 수 있다.
Therefore, the pharmaceutical composition containing the micafungin of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient can be usefully used for antiviral use.
또한, 본 발명은 상기 미카펀진(micafungin) 뿐만 아니라, 이의 약학적으로 허용되는 염, 이로부터 제조될 수 있는 가능한 용매화물, 수화물, 라세이체 또는 입체이성질체를 모두 포함한다.In addition, the present invention encompasses not only the above-mentioned micafungins but also pharmaceutically acceptable salts thereof, possible solvates, hydrates, racemates or stereoisomers thereof which can be prepared therefrom.
본 발명은 상기 미카펀진(micafungin) 또는 이의 약학적으로 허용되는 염의 형태로 사용할 수 있으며, 염으로는 약학적으로 허용 가능한 유리산(free acid)에 의해 형성된 산 부가염이 유용하다. 산 부가염은 염산, 질산, 인산, 황산, 브롬화수소산, 요오드화수소산, 아질산 또는 아인산과 같은 무기산류와 지방족 모노 및 디카르복실레이트, 페닐-치환된 알카노에이트, 하이드록시 알카노에이트 및 알칸이도에이트, 방향족 산류, 지방족 및 방향족 설폰산류와 같은 무독성 유기산으로부터 얻는다. 이러한 약학적으로 무독한 염류로는 설페이트, 피로설페이트, 바이설페이트, 설파이트, 바이설파이트,니트레이트, 포스페이트, 모노하이드로겐 포스페이트, 디하이드로겐 포스페이트, 메타포스페이트, 피로포스페이트 클로라이드, 브로마이드, 아이오다이드, 플루오라이드, 아세테이트, 프로피오네이트, 데카노에이트, 카프릴레이트, 아크릴레이트, 포메이트, 이소부티레이트, 카프레이트, 헵타노에이트, 프로피올레이트, 옥살레이트, 말로네이트, 숙시네이트, 수베레이트, 세바케이트, 푸마레이트, 말리에이트, 부틴-1,4-디오에이트, 헥산-1,6-디오에이트, 벤조에이트, 클로로벤조에이트, 메틸벤조에이트, 디니트로 벤조에이트, 하이드록시벤조에이트, 메톡시벤조에이트, 프탈레이트, 테레프탈레이트, 벤젠설포네이트, 톨루엔설포네이트, 클로로벤젠설포네이트, 크실렌설포네이트, 페닐아세테이트, 페닐프로피오네이트, 페닐부티레이트, 시트레이트, 락테이트, 하이드록시부티레이트, 글리콜레이트, 말레이트, 타트레이트, 메탄설포네이트, 프로판설포네이트, 나프탈렌-1-설포네이트, 나프탈렌-2-설포네이트 또는 만델레이트를 포함한다.
The present invention can be used in the form of micafungin or a pharmaceutically acceptable salt thereof. As salts, acid addition salts formed by pharmaceutically acceptable free acids are useful. Acid addition salts include those derived from inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, nitrous acid or phosphorous acid, and aliphatic mono- and dicarboxylates, phenyl-substituted alkanoates, hydroxyalkanoates, Derived from non-toxic organic acids, such as, for example, diesters, aromatic acids, aliphatic and aromatic sulfonic acids. Such pharmaceutically innocuous salts include, but are not limited to, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogenphosphate, dihydrogenphosphate, metaphosphate, pyrophosphate chloride, bromide, Butyrate, caprate, heptanoate, propiolate, oxalate, malonate, succinate, succinic acid, succinic acid, succinic acid, succinic acid, , Sebacate, fumarate, maleate, butyne-1,4-dioate, hexane-1,6-dioate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate, Methoxybenzoate, phthalate, terephthalate, benzenesulfonate, toluene sulfonate, chlorobenzene sulfoxide Sulfonate, methanesulfonate, propanesulfonate, naphthalene-1-sulphonate, naphthalene-1-sulphonate, , Naphthalene-2-sulfonate or mandelate.
본 발명에 따른 산 부가염은 통상의 방법, 예를 들면 상기 미카펀진(micafungin) 화합물을 과량의 산 수용액 중에 용해시키고, 이 염을 수혼화성 유기 용매, 예를 들면 메탄올, 에탄올, 아세톤 또는 아세토니트릴을 사용하여 침전시켜서 제조할 수 있다. 또한, 동량의 상기 미카펀진 화합물, 및 산 수용액 또는 알코올을 가열하고, 이어서 이 혼합물을 증발시켜서 건조하거나 또는 석출된 염을 흡입 여과시켜 제조할 수도 있다.The acid addition salt according to the present invention can be prepared by a conventional method, for example, by dissolving the above micafungin compound in an excess amount of an acid aqueous solution, and mixing the salt with a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile ≪ / RTI > Alternatively, the same amount of the above-mentioned mycophenazine compound and acidic aqueous solution or alcohol may be heated, followed by evaporation of the mixture, followed by drying or precipitation of the precipitated salt by suction filtration.
또한, 염기를 사용하여 약학적으로 허용가능 한 금속염을 만들 수 있다. 알칼리 금속 또는 알칼리 토금속 염은, 예를 들면 화합물을 과량의 알칼리 금속 수산화물 또는 알칼리 토금속 수산화물 용액 중에 용해하고, 비용해 화합물 염을 여과하고, 여액을 증발, 건조시켜 얻는다. 이때, 금속염으로는 나트륨, 칼륨 또는 칼슘염을 제조하는 것이 제약 상 적합하다. 또한, 이에 대응하는 은 염은 알칼리 금속 또는 알칼리 토금속 염을 적당한 음염(예, 질산은)과 반응시켜 얻는다.
In addition, bases can be used to make pharmaceutically acceptable metal salts. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and evaporating and drying the filtrate. At this time, it is preferable for the metal salt to produce sodium, potassium or calcium salt. The corresponding silver salt is also obtained by reacting an alkali metal or alkaline earth metal salt with a suitable salt (such as silver nitrate).
상기 조성물을 제제화할 경우, 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제 등의 희석제 또는 부형제를 사용하여 제조된다.When the composition is formulated, it is prepared using diluents or excipients such as fillers, extenders, binders, humectants, disintegrants, surfactants and the like which are usually used.
경구 투여를 위한 고형제에는 정제, 환제, 산제, 과립제, 캡슐제, 트로키제 등이 포함되며, 이러한 고형 제제는 하나 이상의 본 발명의 미카펀진 화합물에 적어도 하나 이상의 부형제 예를 들면, 전분, 탄산칼슘, 수크로스(sucrose) 또는 락토오스(lactose) 또는 젤라틴 등을 섞어 조제된다. 또한, 단순한 부형제 외에 마그네슘 스티레이트 탈크 같은 윤활제들도 사용된다. 경구 투여를 위한 액상 제제로는 현탁제, 내용액제, 유제 또는 시럽제 등이 해당되는데, 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제 등이 포함될 수 있다.Solid formulations for oral administration include tablets, pills, powders, granules, capsules, troches and the like, which may contain one or more excipients such as starch, calcium carbonate Sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Liquid preparations for oral administration include suspensions, solutions, emulsions or syrups. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances and preservatives may be included. have.
비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁용제, 유제, 동결건조제제, 좌제 등이 포함된다.Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, suppositories, and the like.
비수성용제, 현탁용제로는 프로필렌글리콜, 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 81, 카카오지, 타우린지, 글리세롤, 젤라틴 등이 사용될 수 있다.
Examples of the non-aqueous solvent and suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 81, cacao butter, taurine, glycerol, gelatin and the like.
본 발명에 따른 조성물은 약제학적으로 유효한 양으로 투여한다. 본 발명에 있어서, "약제학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효용량 수준은 환자의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기한 요소들을 모두 고려하여 부작용없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, " pharmaceutically effective amount " means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level will depend on the type of disease, severity, , Sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition of the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.
구체적으로, 본 발명에 따른 조성물의 유효량은 환자의 나이, 성별, 체중에 따라 달라질 수 있으며, 일반적으로는 체중 1 kg 당 0.1 mg 내지 100 mg, 바람직하게는 0.5 mg 내지 10 mg을 매일 또는 격일 투여하거나, 1일 1 내지 3회로 나누어 투여할 수 있다. 그러나 투여 경로, 질환의 중증도, 성별, 체중, 연령 등에 따라서 증감될 수 있으므로 상기 투여량이 어떠한 방법으로도 본 발명의 범위를 한정하는 것은 아니다.Specifically, the effective amount of the composition according to the present invention may vary depending on the age, sex, and body weight of the patient. In general, 0.1 mg to 100 mg, preferably 0.5 mg to 10 mg per kg of body weight is administered daily or every other day Or one to three times a day. However, the dosage may be varied depending on the route of administration, the severity of the disease, sex, weight, age, and the like, and therefore the dose is not limited to the scope of the present invention by any means.
본 발명의 조성물은 단독으로, 또는 수술, 방사선 치료, 호르몬 치료, 화학치료 및 생물학적 반응 조절제를 사용하는 방법들과 병용하여 사용될 수 있다.
The composition of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers.
또한, 본 발명은 미카펀진(micafungin) 또는 이의 약학적으로 허용 가능한 염을 유효성분으로 함유하는 바이러스 질환의 개선용 건강기능식품을 제공한다.The present invention also provides a health functional food for improving viral diseases containing micafungin or a pharmaceutically acceptable salt thereof as an active ingredient.
상기 바이러스는 엔테로바이러스(enterovirus)로 이루어진 군으로부터 선택되는 어느 하나 이상인 것이 바람직하며, 라이노바이러스(rhinovirus)로 이루어진 군으로부터 선택되는 어느 하나 이상인 것이 바람직하나, 이에 한정되는 것은 아니다.The virus is preferably at least one selected from the group consisting of enterovirus, and is preferably at least one selected from the group consisting of rhinovirus, but is not limited thereto.
본 명세서의 "건강기능식품"이란 일상 식사에서 결핍되기 쉬운 영양소나 인체에 유용한 기능을 가진 원료나 성분 (기능성 원료)을 사용하여 제조한 것으로, 인체의 정상적인 기능을 유지하거나 생리기능 활성화를 통하여 건강을 유지하고 개선하는 식품으로 식품의약품안전처장이 정한 것을 의미하나, 이에 한정되지 않으며 통상적인 의미의 건강식품을 배제하는 의미로 사용된 것이 아니다.As used herein, the term " health functional food " is produced by using raw materials or ingredients (functional raw materials) having functions useful for nutrients or human body that are likely to be deficient in daily eating, and is intended to maintain the normal function of the human body, But is not limited to, and is not meant to exclude health food in the usual sense.
본 발명의 조성물은 식품에 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효 성분의 혼합량은 그의 사용 목적(예방 또는 개선용)에 따라 적합하게 결정될 수 있다. 일반적으로, 건강기능식품 중의 상기 조성물의 양은 전체 식품 중량의 0.01 내지 90 중량부로 가할 수 있다. 그러나 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취 시에는 상기 양은 상기 범위 이하일 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로도 사용될 수 있다.The composition of the present invention can be added directly to food or used together with other food or food ingredients, and can be suitably used according to conventional methods. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (for prevention or improvement). Generally, the amount of the composition in the health functional food may be from 0.01 to 90 parts by weight of the total food. However, when consumed for a long period of time for the purpose of health and hygiene or for health control purposes, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.
본 발명의 건강 기능성 음료 조성물은 지시된 비율로 필수 성분으로서 상기 조성물을 함유하는 외에는 다른 성분에는 특별한 제한이 없으며 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물의 예는 단당류, 예를 들어, 포도당, 과당 등; 이당류, 예를 들어 말토스, 수크로스 등; 및 다당류, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당, 및 자일리톨, 소르비톨, 에리트리톨 등의 당알콜이다. 상술한 것 이외의 향미제로서 천연 향미제{타우마틴, 스테비아 추출물(예를 들어 레바우디오시드 A, 글리시르히진 등)} 및 합성 향미제(사카린, 아스파르탐 등)를 유리하게 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 조성물 100 g 당 일반적으로 약 1 내지 20 g, 바람직하게는 약 5 내지 12 g이다.The health functional beverage composition of the present invention is not particularly limited to other components other than those containing the above-mentioned composition as an essential ingredient at the indicated ratio, and may contain various flavors or natural carbohydrates as an additional ingredient such as ordinary beverages. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; Disaccharides such as maltose, sucrose, and the like; And polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin and the like, and sugar alcohols such as xylitol, sorbitol and erythritol. As a flavor other than the above, a natural flavoring agent {tau martin, stevia extract (for example, rebaudioside A, glycyrrhizin etc.)} and synthetic flavorings (saccharin, aspartame, etc.) have. The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 g of the composition of the present invention.
상기 외에 본 발명의 조성물은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 그 밖에 본 발명의 조성물은 천연 과일 쥬스 및 과일쥬스 음료 및 야채 음료의 제조를 위한 과육을 함유할 수 있다.In addition to the above-mentioned composition, the composition of the present invention can be used as a flavoring agent such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, coloring agents and intermediates (cheese, chocolate etc.), pectic acid and its salts, Salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonating agents used in carbonated beverages and the like. In addition, the composition of the present invention may contain natural fruit juice and pulp for the production of fruit juice drinks and vegetable drinks.
이러한 성분은 독립적으로 또는 조합하여 사용할 수 있다. 이러한 첨가제의 비율은 그렇게 중요하진 않지만 본 발명의 조성물 100 중량부 당 0.1 내지 약 20 중량부의 범위에서 선택되는 것이 일반적이다.
These components may be used independently or in combination. The ratio of such additives is not so important, but is generally selected in the range of 0.1 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.
이하, 본 발명을 실시예 및 실험예에 의해서 상세히 설명한다Hereinafter, the present invention will be described in detail with reference to examples and experimental examples
단, 하기 실시예 및 실험예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예 및 실험예에 의해서 한정되는 것은 아니다.
However, the following Examples and Experimental Examples are merely illustrative of the present invention, and the content of the present invention is not limited by the following Examples and Experimental Examples.
바이러스 복제 정량시스템을 이용한 스크리닝Screening using virus replication quantitative system
<1-1> 바이러스 복제 정량시스템의 도입<1-1> Introduction of virus replication quantitative system
우선, 본 발명자들은 안정성 및 임상 적용 가능성이 높은 FDA 승인 약물들 중에서 EV71(enterovirus 71)에 항바이러스 효과를 나타내는 유효물질을 발굴하기 위한 EV71(enterovirus 71) 리플리콘(replicon) 시스템을 활용하기 위해, 바이러스의 구조 유전자인 P1(VP4-VP1) 부분에 반딧불이 루시퍼라아제(firefly luciferase) 유전자를 대체하여 삽입함으로써 세포 내에서 바이러스 복제를 쉽고 정량적으로 측정해 낼 수 있는 시스템을 Dr. Van Kuppeveld(University of Utrecht, 네덜란드)로부터 제공 받아 사용하였다(도 1의 A)In order to utilize the EV71 (enterovirus 71) replicon system for finding an effective substance exhibiting an antiviral effect on EV71 (enterovirus 71) among the FDA-approved drugs having high stability and clinical applicability, A system that can easily and quantitatively measure viral replication in cells by inserting the firefly luciferase gene into the P1 (VP4-VP1) structural gene of the virus. Van Kuppeveld (University of Utrecht, The Netherlands) (Figure 1 A)
실험에 앞서서, EV71 리플리콘(replicon) RNA를 Vero 세포에 형질주입(transfection)시킨 후, 반딧불이 루시퍼라아제(firefly luciferase)의 활성을 시간 의존적으로(time-dependent) 측정하여 10시간째에 가장 높은 루시퍼라아제(luciferase) 활성을 보이는 것을 확인하여, 이후의 FDA 승인 약물의 실험은 EV71 리플리콘(replicon) RNA 형질주입(transfection)과 동시에 처리하여 8시간이 지난 후에 루시퍼라아제(luciferase)의 활성을 측정하기로 최종 결정하였다(도 2).
Prior to the experiment, EV71 replicon RNA was transfected into Vero cells, and then the activity of firefly luciferase was measured in a time-dependent manner to determine the highest After confirming that luciferase activity was shown, subsequent FDA-approved drug experiments were performed simultaneously with EV71 replicon RNA transfection, and after 8 hours, the activity of luciferase (luciferase) (Fig. 2).
<1-2> 약물 스크리닝<1-2> Drug Screening
다음으로, 본 발명자들은 EV71(enterovirus 71) 리플리콘(replicon) RNA를 Vero 세포에 형질주입(transfection)시킨 후, 동시에 968개의 각 FDA 승인 약물들을 10 μM 농도로 8시간 동안 처리하고 루시퍼라아제(luciferase)의 활성을 측정한 결과, 21개의 약물에서 DMSO 처리군 대비 60% 이상 루시퍼라아제(luciferase) 활성이 감소되는 것을 확인하였다(도 1의 B). 이때, 대조군의 약물로는 강력한 3C 프로테아제 억제제로 알려져 있는 rupintrivir를 사용하였다. Next, the inventors transfected Vero cells with EV71 (enterovirus 71) replicon RNA, and at the same time, 968 respective FDA-approved drugs were treated at a concentration of 10 [mu] M for 8 hours and luciferase luciferase activity was measured. As a result, it was confirmed that luciferase activity was reduced by 60% or more in 21 drugs compared with the DMSO-treated group (Fig. 1B). At this time, rupintrivir, which is known as a potent 3C protease inhibitor, was used as a control.
또한, 상기의 21개 약물이 EV71 리플리콘(replicon)뿐만 아니라 EV71(enterovirus 71)의 증식을 억제하는지 여부를 확인하고자, EV71를 감염시킨 LLC-MK2 유도세포를 이용하여 세포 생존율을 측정하는 방식으로 항바이러스(antivirus)의 효과를 측정하였다. EV71이 감염된 세포는 세포독성(cytotoxicity)이 나타나게 되는데 이때 약물에 의해 EV71의 증식이 억제된다면 세포 생존율이 증가할 것이라고 예상하였으므로 LLC-MK2 유도세포에 EV71을 감염시키고, 동시에 약물들을 2 및 10 μM 의 농도로 96 시간 동안 처리한 후 MTT 어세이(assay)를 통해 항바이러스 효과를 평가하였다. In addition, in order to confirm whether the 21 drugs inhibited the proliferation of EV71 (enterovirus 71) as well as EV71 replicon, a method of measuring cell viability using LLC-MK2 inducing cells infected with EV71 The effect of antivirus was measured. Since EV71-infected cells are expected to show cytotoxicity when cell proliferation of EV71 is inhibited by the drug, LLC-MK2 induction cells are infected with EV71, and drugs are added at a concentration of 2 and 10 μM And the antiviral effect was evaluated by MTT assays.
그 결과, 21개의 약물들 중 미카펀진(Micafungin sodium, Selleckchem(S4287))이 가장 높은 항바이러스(antivirus) 효과를 보였고, DMSO 처리군 대비 60% 이상 향상된 세포 생존율을 확인하였다. 또한, 효능이 이미 검증되어 있는 젬시타빈(gemcitabine) 대비 우수한 효과를 확인하였다(도 1의 C).
Among the 21 drugs, Micafungin sodium (Selleckchem (S4287)) showed the highest antiviral effect, and the cell survival rate was improved by more than 60% compared with the DMSO treatment group. In addition, excellent efficacy was confirmed as compared to gemcitabine, whose efficacy has already been verified (Fig. 1C).
<< 실험예Experimental Example 1> 1> 미카펀진Mikafunjin (( micafunginmicafungin )의 항바이러스 효과 확인) To confirm the antiviral effect
<1-1> <1-1> EV71EV71 (( enterovirusenterovirus 71) 및 71) and CVB3CVB3 (( coxsackieviruscoxsackievirus B3)에 대한 항바이러스 효과 확인 B3) against the antiviral effect
본 발명자들은 상기 <실시예 1>에서 EV71(enterovirus 71)의 억제능을 확인한 미카펀진(micafungin)의 항바이러스 효과를 검증하고, 또한 다른 엔테로바이러스(enterovirus) 종류인 CVB3(coxsackievirus B3)에서의 항바이러스 효과를 검증하기 위해 다음과 같이 실험하였다.The present inventors have verified the antiviral effect of micafungin confirming the inhibitory effect of EV71 (enterovirus 71) in the above <Example 1>, and also confirmed the antiviral effect of the enterovirus type CVB3 (coxsackievirus B3) To verify the effect, the following experiment was conducted.
EV71과 CVB3 리플리콘(replicon) RNA를 Vero 세포에 각각 형질주입(transcfection)한 후 동시에 미카펀진(Micafungin sodium, Selleckchem(S4287))을 0.08 μM 부터 50 μM까지 다양한 농도로 8시간 동안 처리한 후 결과를 평가하였다.EV71 and CVB3 replicon RNAs were transfected into Vero cells and treated with various concentrations of micafungin (Micafungin sodium, Selleckchem (S4287)) from 0.08 μM to 50 μM for 8 hours. .
그 결과, 도 3 및 도 4에 나타내 바와 같이, EV71 및 CVB3 리플리콘(replicon) 모두에서 미카펀진(micafungin) 농도 의존적으로 루시퍼라아제(luciferase)의 활성이 감소하는 것을 확인하였다(도 3의 A 및 도 4의 A). 이때, EV71와 CVB3에 대한 IC50은 5 내지 8 μM로 비슷하게 나타났으며, 리플리콘(replicon) 형질주입(transcfection)을 하지 않은 세포에 동일한 농도 조건으로 미커펀진(micafungin)을 처리한 후 CellTiter-GloTM 기법을 사용하여 세포의 독성을 측정했을 때, 거의 독성이 나타나지 않음을 확인하였다(도 3의 B 및 도 4의 B).
As a result, as shown in FIG. 3 and FIG. 4, it was confirmed that the activity of luciferase was decreased in all of the EV71 and CVB3 replicons in a micafungin concentration-dependent manner (FIG. 3A And FIG. 4A). At this time, the IC 50 for EV71 and CVB3 was similar to 5 to 8 μM. Cells without replicon transfection were treated with micafungin under the same concentration conditions, and CellTiter- Glo TM (Fig. 3B and Fig. 4B). The results are shown in Fig.
<1-2> 장시간 처리시 세포 독성(cell <1-2> Cytotoxicity during prolonged treatment (cell cytotoxicitycytotoxicity ) 여부 확인) Check whether
본 발명자들은 미카펀진(micafungin)을 장시간 처리하였을 때 세포독성(cell cytotoxicity)의 여부를 알아보기 위해, Vero 세포에 미카펀진을 상기 실험예 <1-1>에서와 같은 농도조건으로 24시간 및 48시간 동안 처리하여 세포의 독성 여부를 측정하였다.In order to examine the cytotoxicity when micafungin was treated for a long time, the inventors of the present invention found that when Verapamil is administered at 24 hours and 48 hours under the same conditions as in Experimental Example 1-1, For a period of time to determine the toxicity of the cells.
그 결과, 도 5에 나타낸 바와 같이, 미카펀진이 세포 독성을 거의 보이지 않음을 확인하였다(도 5의 A 및 B).
As a result, as shown in Fig. 5, it was confirmed that mikapanzin showed almost no cytotoxicity (Figs. 5A and 5B).
<< 실험예Experimental Example 2> 바이러스 감염 세포에서의 2 > virus in infected cells 미카펀진Mikafunjin (( micafunginmicafungin ) 항바이러스 효과 확인) Identification of antiviral effect
<2-1> <2-1> EV71EV71 (( BrCrBrCr 타입) 감염 세포에서의 Type) in infected cells 미카펀진Mikafunjin 항바이러스 효과 확인 Identification of antiviral effect
본 발명자들은 미카펀진(micafungin)의 항바이러스 효과를 앞선 <실험예 1>에서와 같은 리플리콘(replicon)에서뿐만 아니라, 바이러스가 직접 감염된 세포 조건에서도 확인하기 위해 다음과 같이 실험하였다.The present inventors conducted the following experiment to confirm the antiviral effect of micafungin not only in the replicon as in Experimental Example 1 but also in the cell condition directly infected with virus as follows.
먼저, LLC-MK2 유도세포에 EV71(enterovirus 71)(BrCr 타입)을 감염시키고 동시에 미카펀진(micafungin)을 다양한 농도로 4일 동안 처리 후, MTT 어세이(assay)로 세포 생존율을 측정하여 항바이러스의 효과를 평가하였다.First, LLC-MK2-inducible cells were infected with EV71 (enterovirus 71) (BrCr type) and at the same time treated with micafungin at various concentrations for 4 days. Cell viability was measured by MTT assays, Were evaluated.
그 결과, 도 6에 나타낸 바와 같이, EC50가 5 μM 정도로 측정되어 리플리콘(replicon)에서 확인했던 항바이러스 효과와 비슷한 수준의 효과를 보임을 확인하였으며(도 6의 A), 앞서 미카펀진(micafungin)을 Vero 세포에서 24 및 48시간 동안 처리하였을 때 세포 독성을 거의 보이지 않았던 상기 실험예 <1-2>에서의 결과와는 달리(도 5), 미카펀진 50μM의 농도에서 96시간 동안 처리한 조건에서는 약간의 세포 독성을 확인하였다(도 6의 B).
As a result, as shown in FIG. 6, it was confirmed that the EC 50 was measured to be about 5 μM and showed an effect similar to that of the antiviral effect confirmed by replicon (FIG. 6A) micafungin) was treated for 96 hours at a concentration of 50 μM of mycophenol (Fig. 5), unlike the results in Experimental Example <1-2>, which showed almost no cytotoxicity when treated with Vero cells for 24 and 48 hours (Fig. 6B). ≪ tb >< TABLE >
<2-2> <2-2> EV71EV71 (H 타입 및 1095 타입) 감염 세포에서의 (H type and 1095 type) in infected cells 미카펀진Mikafunjin 항바이러스 효과 확인 Identification of antiviral effect
다음으로, 본 발명자들은 미카펀진(micafungin)이 EV71(BrCr 타입)외에 다른 타입의 바이러스에서도 유사한 항바이러스 효과를 보이는지 확인하기 위해, EV71(H 타입)과 EV71(1095 타입)이 감염된 LLC-MK2 유도세포에서 상기 <실험예 2-1>와 같은 조건에서 실험을 수행하였다.Next, the inventors of the present invention found that LLC-MK2 induction with EV71 (H type) and EV71 (1095 type) induces similar antiviral effects to other types of viruses other than EV71 (BrCr type) Cells were tested under the same conditions as those of <Experimental Example 2-1>.
그 결과, 도 7에 나타낸 바와 같이, 미카펀진이 EV71(H 타입) 또는 EV71(1095 타입)이 감염된 LLC-MK2 유도세포에서도 기존 타입(BrCr 타입) 대비 유사한 항바이러스 효과를 보이지만, EV71(BrCr 타입)에서의 최대 효능보다는 낮은 수준으로 나타나는 것을 확인하였다(도 7의 A 및 B).
As a result, as shown in Fig. 7, even in the LLC-MK2 inducing cells in which Mikapentin was infected with EV71 (H type) or EV71 (1095 type) showed similar antiviral effects as the conventional type (BrCr type) (Fig. 7A and Fig. 7B). ≪ tb >< TABLE >
<2-3> <2-3> 웨스턴Western 블롯팅Blotting (western blotting)(western blotting)
본 발명자들은 미카펀진(micafungin)에 의한 항바이러스 효과를 확인해보기 위해 다음과 같이 웨스턴 블롯팅(western blotting) 실험을 수행하였다.The present inventors conducted western blotting experiments as follows to confirm the antiviral effect of micafungin.
EV71(enterovirus 71)을 20시간 동안 감염시킨 LLC-MK2 유도세포에 미카펀진을 다양한 농도로 처리한 후 세포를 수확 및 용해(lysate)하여 얻은 단백질을 SDS-PAGE로 분리한 다음, PVDF 막(membrane)으로 옮긴 후 (transfer), PVDF 막 위의 비특이적(non-specific) 단백질을 차단하기 위하여 1시간 동안 블로킹(blocking)처리하였고, 워싱(washing)처리하여 EV71 3C 일차 항체와 4℃에서 15시간 동안 반응시켰다. 다시 워싱(washing)처리 후, 일차 항체에 대한 이차 항체와 1 시간 동안 상온에서 반응시킨 다음, 각 반응 사이 TBST 솔루션(solution)으로 5분씩 3번 반복 세척한 후, 항체에 대한 단백질 밴드(band)를 확인하고자 ECL용액으로 반응시킨 후 감광하여 EV71 3C의 단백질 밴드를 확인하였다. 그리고 동일한 양의 단백질을 넣었는지 확인하기 위해 베타-액틴(beta actin) 항체를 이용하여 추가로 단백질 밴드를 확인하였다. MK-induced cells were treated with various concentrations of LLC-MK2-inducible cells infected with EV71 (enterovirus 71) for 20 hours. After harvesting and lysing the cells, the protein was separated by SDS-PAGE, and the PVDF membrane ), Blocked for 1 hour to block non-specific proteins on the PVDF membrane, washed and treated with
그 결과, 도 6에 나타낸 바와 같이, EV71(enterovirus 71)의 3C 단백질이 미카펀진(micafungin) 10 μM의 농도부터 거의 나타나지 않음으로써, 미카펀진에 의한 항바이러스 효과를 확인하였다(도 6의 C).
As a result, as shown in FIG. 6, the 3C protein of EV71 (enterovirus 71) hardly appeared from the concentration of 10 μM of micafungin, confirming the antiviral effect of mikaphengin (FIG. 6C) .
<2-4> RT-<2-4> RT- PCRPCR
또한, 본 발명자들은 미카펀진(micafungin)에 의한 항바이러스 효과를 확인해보기 위해 다음과 같이 RT-PCR 실험을 수행하였다.In addition, the present inventors conducted RT-PCR experiments as follows to confirm the antiviral effect of micafungin.
EV71(enterovirus 71)을 20시간 동안 감염시킨 LLC-MK2 유도세포에 미카펀진을 다양한 농도로 처리한 후 RNeasy mini kit를 사용하여 세포의 RNA를 추출하였으며, random hexamer와 Superscript cDNA reverse transcription kit를 사용하여 cDNA를 만들고, 이를 주형(template)으로 하여 각각의 유전자를 증폭시킬 수 있는 3B-3C 부분에 해당되는 프라이머(primer)(표 1)와 함께 reverse transcriptase PCR 장비를 사용하여 cDNA의 증폭을 수행하였다.
Cells were harvested using LLC-MK2 inducible cells (EV71, enterovirus 71) at various concentrations and then RNA was extracted using RNeasy mini kit. Using a random hexamer and Superscript cDNA reverse transcription kit cDNA was amplified using a reverse transcriptase PCR apparatus together with a primer (Table 1) corresponding to the 3B-3C portion in which each gene was amplified using a template.
그 결과, 도 6에 나타낸 바와 같이, 미카펀진(micafungin) 10 μM의 농도부터 EV71(enterovirus 71)의 RNA 양(3BC)이 급격히 감소하는 것을 확인하였다(도 6의 D). 이러한 결과는 앞선 <실험예 1>의 EV71 리플리콘(replicon) 및 상기의 세포 감염 실험에서 확인된 항바이러스 효과와 매우 유사한 수준을 보여준다.
As a result, as shown in Fig. 6, it was confirmed that the RNA amount (3BC) of EV71 (enterovirus 71) rapidly decreased from a concentration of 10 μM of micafungin (Fig. 6D). These results show a level very similar to the antiviral effect confirmed in the EV71 replicon of the above <Experimental Example 1> and the above cell infection experiment.
<2-5> <2-5> dsRNAdsRNA 항체 Antibody 형광법Fluorescence method
다음으로, 본 발명자들은 미카펀진(micafungin)에 의한 항바이러스 효과를 확인해보기 위해, 상기의 실험예 <2-4>와 동일한 조건으로 처리 후 LLC-MK2 유도세포에서 dsRNA(double strand RNA)를 항체로 염색하여 분석하였다. Next, in order to confirm the antiviral effect by micafungin, the inventors treated dsRNA (double strand RNA) in LLC-MK2 inducing cells with the same conditions as Experimental Example <2-4> And analyzed.
EV71(enterovirus 71)을 20시간 동안 감염시킨 LLC-MK2 유도세포에 미카펀진을 다양한 농도로 처리한 후 세포를 고정(fixing)하고 dsRNA에 대한 항체를 처리하여 1시간 동안 반응시켰다. 다음으로, 녹색 형광을 나타내는 이차 항체를 1시간 동안 반응시킨 후, 세포핵은 DAPI 용액을 이용하여 따로 염색하였다. 바이러스가 감염되었는지의 여부는 Operetta 형광 현미경을 사용하여 관찰하였고, 현미경에 설치된 프로그램을 이용하여 감염된 정도를 계산하였다.LLC-MK2-induced cells infected with EV71 (enterovirus 71) for 20 hours were treated with various concentrations of mitochondria, fixed with cells, treated with antibodies against dsRNA, and reacted for 1 hour. Next, the secondary antibody expressing green fluorescence was reacted for 1 hour, and the nucleus was separately stained with DAPI solution. Whether or not the virus was infected was observed using an Operetta fluorescence microscope and the degree of infection was calculated using a microscope program.
그 결과, 도 6에 나타낸 바와 같이, 바이러스가 감염되지 않은 세포에서는 염색이 거의 되지 않다가 EV71(enterovirus 71)을 감염시킨 세포에서 뚜렷한 녹색 형광의 신호가 나타나는 것을 확인하였다. 또한, 미카펀진(micafungin)의 농도 의존적으로 녹색 형광의 신호가 감소함을 확인하여, 미카펀진에 의한 항바이러스 효과를 확인하였다(도 6의 E 및 도 8).
As a result, as shown in Fig. 6, it was confirmed that there was almost no staining in the virus-uninfected cells, but a clear green fluorescence signal appeared in cells infected with EV71 (enterovirus 71). Further, it was confirmed that the signal of green fluorescence decreased in a concentration-dependent manner of micafungin, confirming antiviral effect by mikafugine (FIG. 6E and FIG. 8).
<< 실험예Experimental Example 3> 3> CVB3CVB3 (( coxsackieviruscoxsackievirus B3) 및 B3) and HRVHRV (human rhinovirus)에서의 미카펀진((human rhinovirus) micafunginmicafungin ) 항바이러스 효과 확인) Identification of antiviral effect
본 발명자들은 미카펀진(micafungin)이 광범위한 항바이러스의 효능을 보이는지의 여부를 확인해 보기 위해 EV71과 매우 유사한 엔테로바이러스(enterovirus) 종류인 CVB3(coxsackievirus B3)와 HRV(human rhinovirus)를 사용하여 다음과 같이 실험을 수행하였다. CVB3(coxsackievirus B3)는 장바이러스들 중 가장 많은 연구가 이루어진 바이러스이며 바이러스성 뇌수막염, 심근염 및 췌장염의 주요 원인이 되는 것으로 잘 알려져 있다. 또한 HRV(human rhinovirus)는 감기의 주요 원인 바이러스 중 하나이다. In order to confirm whether or not the micafungin exhibits a broad spectrum of antiviral efficacy, the present inventors used CVV3 (coxsackievirus B3) and HRV (human rhinovirus), which are very similar enterovirus types to EV71, Experiments were performed. CVB3 (coxsackievirus B3) is the most studied virus among the longest viruses and is well known to be a major cause of viral meningitis, myocarditis and pancreatitis. HRV (human rhinovirus) is also one of the major causative viruses of the cold.
먼저, HeLa 세포에는 CVB3(coxsackievirus B3)를 감염시키고, H1HeLa 세포에는 HRV(human rhinovirus)의 세 종류 바이러스(HRV 14, HRV 21 및 HRV 71)를 각각 감염시킨 후, 미카펀진을 0.08 μM 부터 50 μM까지 다양한 농도로 48시간 동안 처리 후 결과를 평가하였다.First, HeLa cells were infected with CVB3 (coxsackievirus B3) and H1HeLa cells were infected with three types of HRV (human rhinovirus) viruses (
그 결과, 도 9에 나타낸 바와 같이, CVB3(coxsackievirus B3)를 감염시킨 HeLa 세포에서 미카펀진(micafungin)은 항바이러스 효과를 보이지만, EV71(enterovirus 71)을 감염시킨 LLC-MK2 유도세포에서의 효과(도 6의 A) 대비 낮은 항바이러스 효과를 확인하였으며(도 9의 A), 미카펀진 50μM의 농도에서 48시간 동안 처리한 조건에서는 약간의 세포 독성을 확인하였다(도 9의 B). As a result, as shown in FIG. 9, micafungin showed an antiviral effect in HeLa cells infected with CVB3 (coxsackievirus B3), but the effect on LLC-MK2 induced cells infected with EV71 (enterovirus 71) (Fig. 9A), and slight cytotoxicity was observed under conditions of 48 hours treatment with a concentration of 50 mu M of mycophenol (Fig. 9B).
또한, 도 10에 나타낸 바와 같이, 세 종류의 다른 HRV(human rhinovirus)(HRV-14, HRV-21 및 HRV-71)를 각각 감염시킨 H1HeLa 세포에서 미카펀진(micafungin)이 비교적 낮은 수준의 항바이러스 효과를 갖는 것을 확인하였다(도10의 A, B 및 C).
In addition, as shown in Fig. 10, in H1HeLa cells infected with three different human rhinoviruses (HRV-14, HRV-21 and HRV-71), micafungin has a relatively low level of antiviral (Figs. 10A, 10B and 10C).
<< 실험예Experimental Example 4> 4> 미카펀진Mikafunjin (( micafunginmicafungin ) 항바이러스 효과의 작용점 추적) Track the action point of the antiviral effect
<4-1> time of addition 실험<4-1> time of addition experiment
본 발명자들은 미카펀진(micafungin)이 바이러스 감염의 주기(cycle) 중 어떤 과정에서 항바이러스 효과를 나타내는지 확인하기 위해 우선, 바이러스 감염 전후의 다양한 시간(-1, 0, 1, 3, 5, 7, 9 및 20 시간)들에 미카펀진을 처리한 후 viral dsRNA의 항체 형광을 정량화하였다. 그 결과, 도 11에 나타낸 바와 같이, 바이러스 감염 후 1시간 시점에서도 바이러스 감염 전(-1시간) 및 감염과 미카펀진의 처리가 동시에 이뤄진 시점(0시간)과 동일하게 강력한 항바이러스 효과가 나타남을 확인하였다(도 11). 감염 후 9 시간째 까지도 미카펀진의 항바이러스 효과가 지속되는 것을 확인하였으며, 이러한 효과는 기존에 잘 알려진 3C 프로테아제 억제제인 rupintrivir와 유사한 경향성을 보여주는 것으로, 미카펀진이 바이러스 감염 초기의 엔트리(entry) 과정 이후 특정 과정을 표적화(targeting)하여 EV71(enterovirus 71)의 증식을 억제하는 것을 의미한다. 아울러, 상기의 실험예 <1-1>의 EV71(enterovirus 71) 리플리콘(replicon)을 이용한 실험에서의 항바이러스 효능(도 3)은, 미카펀진이 바이러스의 입자와는 상관없이 세포 내 과정(intracellular process)만을 통해 항바이러스 효능을 가짐을 나타낸다.
The inventors of the present invention conducted a study to determine whether the micafungin exhibits an antiviral effect during the course of the viral infection at various times (-1, 0, 1, 3, 5, 7 , 9 and 20 hours) were quantified by immunofluorescence of viral dsRNA. As a result, as shown in FIG. 11, a strong antiviral effect was observed at 1 hour after the virus infection, just before the virus infection (-1 hour) and at the same time as the infection and the treatment with the mycophenazine (Fig. 11). It was confirmed that the antiviral effect of mikafunzine persisted up to 9 hours after infection. This effect showed a tendency similar to that of rupintrivir, a well known 3C protease inhibitor, And then targeting a specific process to inhibit the proliferation of EV71 (enterovirus 71). In addition, the antiviral efficacy (FIG. 3) in the experiment using the EV71 (enterovirus 71) replicon of the above Experimental Example <1-1> shows that the mitochondrial activity intracellular < / RTI > process).
<4-2> 이중 <4-2> Double 루시퍼라아제Luciferase 리포터 실험 Reporter experiment
다음으로, 본 발명자들은 미카펀진(micafungin) 항바이러스 효능의 작용 단계를 구체적으로 알아보기 위한 실험을 다음과 같이 수행하였다.Next, the present inventors carried out an experiment to specifically examine the action stage of the micafungin antiviral effect as follows.
먼저, 미카펀진이 EV71(enterovirus 71) RNA의 5'NCR에서 번역 개시를 일으키는 internal ribosomal entry site(IRES)의 활성을 억제하는지의 여부를 확인하기 위해 이중 루시퍼라아제 리포터 시스템(dual luciferase reporter system)을 제작하였다. 이 리포터(reporter) DNA는 레닐라 루시퍼라아제(renilla luciferase) 유전자와 반딧불이 루시퍼라아제(firefly luciferase) 유전자 사이에 EV71 IRES 서열을 넣은 것으로, 레닐라 루시퍼라아제(renilla luciferase) 발현은 cap 의존적 번역을, 반딧불이 루시퍼라아제(firefly luciferase)의 발현은 EV71 IRES의존적 단백질 발현을 나타내는 분석법(assay)이다. 이 이중 루시퍼라아제 리포터 DNA(dual luciferase reporter DNA)를 293T 세포에 발현시킨 후, 미카펀진을 다양한 농도로 24시간 동안 처리하여 각각의 루시퍼라아제(luciferase)의 활성을 측정하였다.First, a dual luciferase reporter system was used to confirm whether the microfunzine inhibited the activity of the internal ribosomal entry site (IRES) causing translation initiation in 5 'NCR of EV71 (enterovirus 71) RNA. Respectively. This reporter DNA is the EV71 IRES sequence between the renilla luciferase gene and the firefly luciferase gene. The renilla luciferase expression is cap-dependent translation , And the expression of firefly luciferase is an assay showing EV71 IRES-dependent protein expression. After expressing the dual luciferase reporter DNA in 293T cells, the activity of each luciferase was measured by treating the mycophenazine with various concentrations for 24 hours.
그 결과, 도 12의 A에 나타낸 바와 같이, 미카펀진(micafungin)의 항(anti) EV71(enterovirus 71) 효과를 설명할 만한 억제효과는 나타나지 않았다(도 12의 A).
As a result, as shown in Fig. 12A, there was no inhibitory effect to explain the anti-EV71 (enterovirus 71) effect of micafungin (Fig. 12A).
<4-3> <4-3> 웨스턴Western 블롯팅Blotting (western blotting)(western blotting)
다음으로, 미카펀진(micafungin)이 3C 프로테아제(protease)를 억제하는지의 여부를 확인하기 위해, 다음과 같이 웨스턴 블롯팅(western blotting) 실험을 수행하였다.Next, in order to confirm whether or not micafungin inhibits 3C protease, a western blotting experiment was carried out as follows.
EV71(enterovirus 71)의 3C-3D 앞에 플래그 꼬리붙임(flag tagged) 형태로 293T 세포에 발현시키고, 9시간 동안 미카펀진을 처리한 후, 웨스턴 블롯팅의 결과를 통해 3C의 억제효과를 평가하고자 하였다. 3C가 억제될 경우에는 3CD precursor 형태의 단백질이 축적되어 잘려진 3C의 양이 감소하게 된다. Expression was made in 293T cells in a flag tagged form in front of 3C-3D of EV71 (enterovirus 71), and the inhibitory effect of 3C was evaluated through Western blotting after 9 hours of treatment with mikaphengin . When 3C is inhibited, 3CD precursor proteins accumulate and the amount of truncated 3C decreases.
우선, 상기의 조건으로 미카펀진을 처리한 293T 세포의 추출물을 수확하여 SDS-PAGE로 분리한 다음, PVDF 막(membrane)으로 옮긴 후(transfer), PVDF 막 위의 비특이적(non-specific) 단백질을 차단하기 위하여 2시간 동안 블로킹(blocking)처리하였고, 워싱(washing)처리하여 플래그(flag)-3C 및 플래그(flag)-3CD에 대한 일차 항체인 항-플래그(anti-flag)와 2시간 동안 반응시켰다. 다시 워싱(washing)처리 후, 일차 항체에 대한 이차 항체와 1시간 동안 상온에서 반응시킨 다음, 각 반응 사이 TBST 솔루션(solution)으로 5분씩 4번 반복 세척한 후, 항체에 대한 단백질 밴드(band)를 확인하고자 ECL용액으로 반응시킨 후 감광하여 각각의 단백질 밴드를 확인하였다.First, the extract of 293T cells treated with mycophenazine under the above conditions was harvested, separated by SDS-PAGE, transferred to a PVDF membrane, and transferred to a non-specific protein on the PVDF membrane Blocking for 2 hours to block and then washing for 2 hours with an anti-flag as the primary antibody against flag-3C and flag-3CD. . After washing again, the cells were reacted with the secondary antibody against the primary antibody for 1 hour at room temperature. Then, the cells were washed 4 times for 5 minutes each time with a TBST solution between each reaction, Were reacted with ECL solution to confirm their respective protein bands.
그 결과, 도 12의 B에 나타낸 바와 같이, 강력한 3C 프로테아제(protease) 저해제로 잘 알려진 rupintrivir를 처리한 경우에는 3C를 억제하여 플래그(flag)-3CD가 축적되고 플래그(flag)-3C가 감소하는 반면, 미카펀진(micafungin)을 처리한 경우에는 처리하지 않은 경우와 큰 차이를 나타내지 않음을 확인하였다(도 12의 B). 이는 미카펀진이 3C 프로테아제를 억제하지 않음을 의미한다.
As a result, when rupintrivir, which is well known as a powerful 3C protease inhibitor, was treated as shown in Fig. 12B, 3C was suppressed and flag-3CD was accumulated and flag-3C was decreased On the other hand, it was confirmed that when micafungin was treated, it did not show a significant difference from the case without treatment (FIG. 12B). This means that mycophenazine does not inhibit 3C protease.
<4-4> 돌연변이 <4-4> Mutation 리플리콘Reply (( repliconreplicon ) RNA의 형질주입() Transfection of RNA ( transcfection전사 ) 실험) Experiment
아울러, 기존 항바이러스 효과를 갖는 약물들 중에서 2C나 3A를 표적화(targeting)하는 것으로 알려져 있는 약물들이 존재하므로, 본 발명자들은 다음의 실험을 통해 미카펀진(micafungin)도 2C나 3A를 통해 항바이러스의 효능을 나타내는지의 여부를 확인해 보았다.In addition, among the drugs having existing antiviral effects, there are drugs known to target 2C or 3A. Therefore, the inventors of the present invention found that micafungin also inhibited the antiviral activity of 2C or 3A And whether or not it represents efficacy.
미카펀진(micafungin)이 2C나 3A를 통해 항바이러스의 효능을 나타낸다면, 2C 또는 3A 돌연변이를 포함하는 리플리콘(replicon)에서 항바이러스의 효과를 보이지 않을 것이므로, 본 발명자들은 2C 또는 3A 돌연변이 리플리콘(replicon) RNA를 Vero 세포에 각각 형질주입(transcfection)한 후, 미카펀진을 농도별 처리하여 그 결과를 분석하였다.Since micafungin exhibits antiviral efficacy via 2C or 3A, it will not show the antiviral effect in replicons including 2C or 3A mutants, and therefore, the inventors believe that 2C or 3A mutant replicons The replicon RNAs were transfected into Vero cells, respectively, and the results were analyzed by treatment with mycophenazine.
그 결과, 미카펀진(micafungin)이 2C 또는 3A 돌연변이 리플리콘(replicon)에서도 정상적인 리플리콘(replicon)에서의 실험결과 대비 유사한 수준의 항바이러스 효과를 보임을 확인하였다(도 12의 C). 이는 미카펀진이 2C 또는 3A를 통해 항바이러스 효과를 갖는 것이 아님을 의미한다.As a result, it was confirmed that the micafungin exhibited a similar level of antiviral effect to that of the normal replicon in the 2C or 3A mutant replicon (FIG. 12C). This means that the mycophanase does not have an antiviral effect through 2C or 3A.
이러한 상기의 결과들은 미카펀진이 IRES 의존적 번역, 3C 프로테아제(protease)에 의한 폴리단백질 프로세싱(polyprotein processing) 및 2C 또는 3A 단백질의 세포 내 초기 처리 과정(intracellular process)등과는 상관없이 다른 방식으로 항바이러스 효과를 가짐을 보여준다.
These results suggest that the mycotoxin may be expressed in other ways, irrespective of IRES dependent translation, polyprotein processing by 3C protease, and intracellular process of 2C or 3A protein, Effect.
<< 실험예Experimental Example 5> 바이러스 감염 동물모델에서의 5> In viral infected animal models 미카펀진Mikafunjin (( micafunginmicafungin ) 항바이러스 효과 확인) Identification of antiviral effect
<5-1> <5-1> 엔테로바이러스Enterovirus (( enterovirusenterovirus ) 감염 동물 모델의 제작) Production of infected animal models
본 발명자들은 마우스의 in vivo 실험을 위해, CVB3(coxsackievirus B3)를 1 x 107 TCID 50/100 ㎕의 농도로 복강에 투여(injection)하여 CVB3 감염 마우스 모델을 유도하였다.
For in vivo experiments of mice, we injected CVB3 (coxsackievirus B3) at a concentration of 1 x 10 7
<< 5-2> 바이러스 감염 동물모델의 체중변화 확인5-2> Confirmation of weight change in animal model of virus infection
본 발명자들은, 미카펀진(micafungin)의 항바이러스 효과를 in vivo 에서 검증하기 위해, 상기 <실험예 5-1>에서 제작한 엔테로바이러스(enterovirus) 감염 마우스 모델에서 다음과 같이 실험하였다.The inventors have found that the antiviral effect of micafungin in vivo , The following experiment was conducted in an enterovirus-infected mouse model prepared in the above <Experimental Example 5-1>.
다양한 엔테로바이러스(enterovirus) 중에서도 마우스 감염에 매우 효과적인 것으로 알려져 있으면서, 미카펀진(micafungin)에 의한 항바이러스 효능을 이미 확인했었던 CVB3(coxsackievirus B3)를 감염시켜 만든 마우스 동물 모델에 미카펀진을 2 mg/kg 및 10 mg/kg의 농도로 5일 동안 매일 복강에 투여(injection)하면서, 5일간의 마우스 체중(body weight)의 변화를 측정하였다.Among the various enteroviruses, a mouse animal model infected with CVB3 (coxsackievirus B3), which has been known to be highly effective in mouse infection and has already confirmed its antiviral efficacy by micafungin, And 10 mg / kg for 5 days, the change in body weight for 5 days was measured.
그 결과, 도 13에 나타낸 바와 같이, CVB3(coxsackievirus B3)를 감염시킨 마우스 그룹에서 약 4% 정도의 체중저하가 나타나는 것을 확인하였으나, 2 mg/kg 의 미카펀진(micafungin)을 투여한 마우스 그룹에서는 CVB 감염에 의한 체중저하가 전혀 관찰되지 않았으며, 10 mg/kg의 미카펀진을 투여한 마우스 그룹에서는 약 1% 정도의 체중저하가 관찰되었다(도 13). 이는 미카펀진(micafungin)이 CVB3 감염에 의한 마우스의 체중저하를 억제함을 나타낸다(도 13의 A 및 B).
As a result, as shown in FIG. 13, it was confirmed that weight loss of about 4% was observed in a mouse group infected with CVB3 (coxsackievirus B3), but in a group of mice administered with 2 mg / kg of micafungin No weight loss due to CVB infection was observed, and a weight loss of about 1% was observed in a mouse group to which 10 mg / kg of mycophenazine was administered (FIG. 13). This indicates that micafungin inhibits weight loss in mice due to CVB3 infection (FIGS. 13A and 13B).
<< 5-3> RT-5-3> RT- PCRPCR
본 발명자들은 상기 <실험예 5-1>에서 바이러스 감염에 따른 마우스의 체중저하가 미카펀진(micafungin)에 의해 억제되는 현상이, 미카펀진의 CVB3(coxsackievirus B3)의 억제 효과에 의한 것인지의 여부를 확인하기 위해서CVB3의 주요 감염 조직인 췌장(pancreas)에서의 CVB3 RNA의 양을 Real time-PCR을 이용하여 다음과 같이 측정하였다. The inventors of the present invention conducted a study on whether or not the decrease in weight of mice caused by viral infection by micafungin is due to the inhibitory effect of CVB3 (coxsackievirus B3) of mycophenazine in <Experimental Example 5-1> For confirmation, the amount of CVB3 RNA in the pancreas, the main infectious tissue of CVB3, was measured using Real time-PCR as follows.
상기 <실험예 5-2>에서의 마우스 동물 모델을 CO2 처리하여 췌장을 각각 분리한 후, TRI 용액을 사용하여 세포의 RNA를 추출하였으며, cDNA reverse transcription kit를 사용하여 cDNA를 만들고, 이를 주형(template)으로 하는 프라이머(primer)(표 2)와 함께 reverse transcriptase PCR 장비를 사용하여 cDNA의 증폭을 수행하였다. 그리고 그 결과를 GAPDH RNA 양으로 정규화(nomalization)하여 비교하였다.
The mouse animal model in Experimental Example 5-2 was subjected to CO 2 treatment to isolate pancreas. RNA was extracted from the cells using a TRI solution. CDNA was prepared using cDNA reverse transcription kit, amplification of the cDNA was carried out using a reverse transcriptase PCR apparatus together with a primer (template 2) (Table 2). And the results were compared by normalization with the amount of GAPDH RNA.
그 결과, 도 14에 나타낸 바와 같이, 10 mg/kg의 미카펀진(micafungin)을 투여한 마우스 그룹에서는 CVB3(coxsackievirus B3)의 RNA 발현량이 CVB3 감염 마우스 그룹(대조군)과 비교하여 크게 감소함을 확인하였다(도 14).
As a result, as shown in Fig. 14, it was confirmed that the expression amount of CVB3 (coxsackievirus B3) RNA significantly decreased in the mouse group administered with 10 mg / kg of micafungin compared to the CVB3-infected mouse group (control group) (Fig. 14).
<< 5-4> 조직병리학적 변화 확인5-4> Confirmation of histopathological change
다음으로, 본 발명자들은 마우스 동물 모델에서 미카펀진(micafungin)에 의한 췌장의 조직병리학적 변화를 육안으로 관찰하기 위해, 다음과 같이 실험하였다.Next, in order to visually observe the histopathological changes of the pancreas by micafungin in a mouse animal model, the present inventors conducted the following experiment.
상기 <실험예 5-2>에서의 마우스 동물 모델들의 췌장조직 일부를 얻어, 4% 파라포름 알데히드(paraformaldehyde)로 상온에서 1시간 동안 고정한 뒤, 파라핀 블록을 제작하여 표준 H/E 염색(staining)을 수행하였다. 염색한 조직의 사진은 디지털 사진기가 장착된 현미경 장비를 이용하여 얻었다.Partial pancreatic tissues of mouse animal models in the above Experimental Example 5-2 were obtained and fixed with 4% paraformaldehyde at room temperature for 1 hour. Paraffin blocks were prepared and stained with standard H / E, Respectively. Photographs of the stained tissue were obtained using a microscope equipped with a digital camera.
그 결과, 도 15에 나타낸 바와 같이, CVB3(coxsackievirus B3) 감염 마우스그룹(대조군)에서는 췌장조직을 구성하고 있는 선포세포(acinar cell)가 손상되어 있는 것을 뚜렷하게 확인할 수 있었으나, 2 mg/kg 및 10 mg/kg의 미카펀진( micafungin)을 투여한 마우스 그룹에서는 췌장 선포세포(acinar cell)의 손상이 현저하게 완화되어 있는 것을 확인하였다(도 15). As a result, as shown in FIG. 15, it was clearly confirmed that the acinar cell constituting the pancreatic tissue was damaged in the CVB3 (coxsackievirus B3) -infected mouse group (control group), but 2 mg / kg and 10 In the mouse group administered with mg / kg of micafungin, it was confirmed that damage of pancreatic acinar cells was remarkably alleviated (Fig. 15).
이러한 결과들은 미카펀진(micafungin)이 CVB3(coxsackievirus B3)감염 마우스 모델에서도 항바이러스 효과를 보이고, 그 결과, 췌장조직의 괴사 또한 억제함을 보여준다.These results show that micafungin also exhibits antiviral effects in CVB3 (coxsackievirus B3) -infected mouse models and as a result inhibits necrosis of pancreatic tissue.
<110> Korea research institute of bioscience and biotechnology
Korea research institute of chemical technology
Kangwon national university university -industry cooperation foundation
<120> Composition for treating antivirus containing micafungin or
pharmaceutically acceptable salts thereof as an active ingredient
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<110> Korea research institute of bioscience and biotechnology
Korea research institute of chemical technology
Kangwon national university university -industry cooperation foundation
<120> Composition for treating antivirus containing micafungin or
pharmaceutically acceptable salts thereof as an active ingredient
<130> 2016P-09-051
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Claims (10)
An antiviral pharmaceutical composition comprising, as an active ingredient, micafungin or a pharmaceutically acceptable salt thereof
2. The antiviral pharmaceutical composition according to claim 1, wherein the virus is an enterovirus or a rhinovirus.
3. The antiviral pharmaceutical composition according to claim 2, wherein the enterovirus is any one selected from the group consisting of poliovirus, coxsackievirus and echovirus. .
The antiviral pharmaceutical composition according to claim 1, wherein the microcavity inhibits the expression of 3C gene or protein of EV71 (enterovirus 71).
The antiviral pharmaceutical composition according to claim 1, wherein the microcapsule inhibits the expression of viral RNA.
2. The antiviral pharmaceutical composition according to claim 1, wherein the microcavity inhibits the decrease in body weight due to viral infection.
The antiviral pharmaceutical composition according to claim 1, wherein the microcavity inhibits damage to pancreatic acinar cells caused by viral infection.
An antiviral health functional food containing, as an active ingredient, micafungin or a pharmaceutically acceptable salt thereof.
The antifungal health food according to claim 8, wherein the virus is an enterovirus or a rhinovirus.
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