KR102540330B1 - Nanodisc with phosphatidylethanolamine phospholipid - Google Patents
Nanodisc with phosphatidylethanolamine phospholipid Download PDFInfo
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- KR102540330B1 KR102540330B1 KR1020220135212A KR20220135212A KR102540330B1 KR 102540330 B1 KR102540330 B1 KR 102540330B1 KR 1020220135212 A KR1020220135212 A KR 1020220135212A KR 20220135212 A KR20220135212 A KR 20220135212A KR 102540330 B1 KR102540330 B1 KR 102540330B1
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- 239000002107 nanodisc Substances 0.000 title claims abstract description 64
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- -1 phosphatidylethanolamine phospholipid Chemical class 0.000 title claims abstract description 8
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Abstract
본 발명은 '막구조화 단백질 (membrane scaffold protein) 또는 양친매성 폴리머 (amphipathic polymer)' 및 인지질인 포스파티딜에탄올아민(phosphatidylethanolamine)을 포함하는 것을 특징으로 하는 나노디스크 및 이의 항바이러스 용도에 관한 것이다. 본 발명의 나노디스크는 인지질 중에서도 포스파티딜에탄올아민(phosphatidylethanolamine) 인지질을 포함하여 항바이러스 효능이 우수하다.The present invention relates to a nanodisc comprising a 'membrane scaffold protein or an amphipathic polymer' and phosphatidylethanolamine as a phospholipid and an antiviral use thereof. The nanodisc of the present invention contains phosphatidylethanolamine phospholipids among phospholipids, and has excellent antiviral efficacy.
Description
본 발명은 '막구조화 단백질 (membrane scaffold protein) 또는 양친매성 폴리머 (amphipathic polymer)' 및 인지질인 포스파티딜에탄올아민(phosphatidylethanolamine)을 포함하는 것을 특징으로 하는 나노디스크 및 이의 항바이러스 용도에 관한 것이다.The present invention relates to a nanodisc comprising a 'membrane scaffold protein or an amphipathic polymer' and phosphatidylethanolamine as a phospholipid and an antiviral use thereof.
인플루엔자 바이러스(Influenza virus)는 오르소믹소 계통(Family Orthomyxoviridae)에 속하는 RNA 바이러스로서 혈청형은 A형, B형, C형 등 3가지로 구분된다. 그 중 B형과 C형은 사람에서만 감염이 확인되고 있으며, A형은 사람, 말, 돼지, 기타 포유류 그리고 다양한 종류의 가금과 야생조류에서 감염이 확인되고 있다. A형 인플루엔자 바이러스의 혈청형은 바이러스 표면의 두 가지 단백질인 헤마글루티닌(Hemagglutinin, HA)과 뉴라미니다제(Neuraminidase, NA)의 종류에 따라 구분되는데, 지금까지 144종류(HA 단백질 16종과 NA 단백질 9종)가 알려져있다. HA는 바이러스가 체세포에 부착하는 역할을 하며, NA는 바이러스가 세포 내로 침투할 수 있도록 한다.Influenza virus is an RNA virus belonging to the Orthomyxoviridae family, and its serotypes are classified into three types: A, B, and C. Among them, types B and C have been confirmed to be infected only in humans, and type A has been confirmed to be infected in humans, horses, pigs, other mammals, and various types of poultry and wild birds. Serotypes of influenza A virus are classified according to the types of two proteins on the surface of the virus, hemagglutinin (HA) and neuraminidase (NA). So far, 144 types (16 HA proteins) and 9 NA proteins) are known. HA serves to attach viruses to somatic cells, and NA enables viruses to penetrate cells.
지금까지 개발된 바이러스 감염증 치료제로는 아만타딘(amantadine) 또는 리만타딘(rimantadine)계열의 M2 이온채널 억제제(M2 ion channel inhibitor)와 오셀타미비르(oseltamivir, 상품명 타미플루) 또는 자나미비르(zanamivir, 상품명 리렌자) 계열의 뉴라미니데이즈(neuraminidase) 억제제가 알려져 있으나, 이들 치료제는 그의 효과가 제한된다는 문제점이 있었다. 즉, 아만타딘 또는 리만타딘 계열의 유도체 화합물은 이에 대한 저항성 변종바이러스가 빠르게 생성되고, 일부 지역에서 검출된 H5N1 타입의 인플루엔자 바이러스는 아만타딘 또는 리만타딘 계열의 화합물에 대하여 내성을 나타내며, 인플루엔자 B 바이러스는 아만타딘 유도체에 민감하지 않다고 알려져 있다. 또한, 오셀타미비르 또는 자나미비르 계열의 유도체 화합물 역시 이에 대한 저항성 바이러스가 증가하고, 이러한 저항성 바이러스는 어린이에게서 빈번히 발생하고 있다고 알려져 있다.The drugs for viral infections developed so far include amantadine or rimantadine-type M2 ion channel inhibitors and oseltamivir (tamiflu) or zanamivir (trade name Relenza). ) series of neuraminidase inhibitors are known, but these treatments have a problem in that their effects are limited. That is, amantadine or rimantadine-based derivative compounds quickly generate resistant mutant viruses, H5N1 type influenza viruses detected in some regions show resistance to amantadine or rimantadine-based compounds, and influenza B virus is amantadine. It is known to be insensitive to derivatives. In addition, it is known that oseltamivir or zanamivir-based derivative compounds also increase resistant viruses, and these resistant viruses frequently occur in children.
한편, 2020년부터 현재 진행형인 코로나 바이러스에 의한 COVID-19 등 바이러스에 의한 질병의 유행이 근래 다수 발생하고 있다. On the other hand, since 2020, a number of epidemics of diseases caused by viruses such as COVID-19 caused by the ongoing corona virus have recently occurred.
코로나바이러스는 코로나바이러스과(Coronaviridae)의 코로나바이러스아과(Coronavirinae)에 속하는 RNA 바이러스로, 사람과 동물의 호흡기와 소화기계 감염을 유발한다. 주로 점막전염, 비말전파로 쉽게 감염되며, 사람에게는 일반적으로 경미한 호흡기 감염을 일으키지만 치명적인 감염을 유발하기도 하며, 소와 돼지는 설사, 닭은 호흡기 질환이 발생하기도 한다. 코로나바이러스는 현대 문명에서 치명적인 감염병을 일으키는 대표적인 바이러스다. 2003년 4월에는 중화인민공화국발 중증급성호흡기증후군, 일명 사스가 유행해 사망률 9.6%를 기록하며 많은 사람이 사망했다. 2015년에는 중동호흡기증후군, 일명 메르스가 중동에서 전 세계로 퍼지면서 사망률 약 36%로써 사망자가 다수 발생하였다. 또한 2019년 12월부터 중국 우한발 신종 코로나바이러스 감염증(코로나19,COVID-19)이 전 세계로 확진되면서 감염자가 늘어나고 있으며, 치사율은 2020년 2월까지 집계된 자료에 따르면 2.6%로 그나마 낮은 편이지만 전세계에서 확진자가 폭증하는 중이며 예방 또는 치료 목적으로 승인된 백신이나 항바이러스제는 없었다. 현재, 카모스타트, 렘데시비르, 하이도록시클로로퀸 등의 타 질환치료제로 이용되던 약물들이 코로나바이러스 치료제로의 가능성에 대해서 검토되고, 부작용과 미미한 효능으로 인하여 임상이 실패하거나 정지되어 있다.Coronaviruses are RNA viruses belonging to the Coronavirinae family of the family Coronaviridae and cause respiratory and digestive tract infections in humans and animals. It is easily infected mainly by mucosal transmission and droplet transmission, and generally causes mild respiratory infections in humans, but sometimes fatal infections, diarrhea in cattle and pigs, and respiratory diseases in chickens. Coronavirus is a representative virus that causes fatal infectious diseases in modern civilization. In April 2003, Severe Acute Respiratory Syndrome, also known as SARS, was prevalent in the People's Republic of China, with a mortality rate of 9.6% and many people dying. In 2015, Middle East Respiratory Syndrome, also known as MERS, spread from the Middle East to the world, resulting in a large number of deaths with a mortality rate of about 36%. In addition, as the new coronavirus infection (Corona 19, COVID-19) from Wuhan, China has been confirmed worldwide since December 2019, the number of infected is increasing, and the fatality rate is low at 2.6% according to data compiled until February 2020. However, the number of confirmed cases is exploding around the world, and there have been no approved vaccines or antivirals for preventive or therapeutic purposes. Currently, drugs used for the treatment of other diseases, such as camostat, remdesivir, and hydroxychloroquine, are being reviewed for their potential as coronavirus treatments, and clinical trials are failing or suspended due to side effects and insignificant efficacy.
상기한 바이러스들은 모두 지질이중막으로 구성된 외피를 가지고 있다는 공통점이 있다. 상기를 포함한 많은 동물 바이러스들(예, HIV, 간염 바이러스, MERS, 헤르페스 바이러스, 에볼라 바이러스 등)이 막단백질과 지질이중막 외피를 가지고 있다. 막단백질 혹은 표면 항원은 바이러스의 외피에 존재하며, 숙주세포의 수용체에 결합하는 특징을 갖고 있어 바이러스가 세포 내로 침투하는 것을 유도한다. 또한 외피는 바이러스의 감염 경로에서 매우 중요한 역할을 수행할 뿐만 아니라 스스로를 인체의 면역 체계로부터 보호하는 역할을 수행한다. 따라서 위와 같이 바이러스의 막단백질과 수용체 간의 결합을 방해하거나 외피를 손상시킴으로써 바이러스의 감염을 억제할 수 있다. 바이러스의 막단백질을 방해하여 바이러스 증식을 억제(virustatic)하는 약물 중 대표적으로 오셀타미비르(oseltamivir)가 알려져 있으나 이와 같은 항바이러스제의 경우 효과가 일시적이거나, 재발한다거나, 약효가 약하다거나 하는 것은 그 약의 작용양식이 바이러스의 증식을 억제하는 방식이기 때문에 그 약물의 농도가 낮아지면 다시 바이러스가 증식할 수 있다. 또한, 약물의 농도의 변화에 따라서 증식과 중지가 반복되면 당연히 바이러스는 "adaptive evolution"에 의한 결과로서 내성바이러스가 발생할 수 있다. 오셀타미비르(타미플루) 또한 내성바이러스들이 점점 많이 보고되고 있다. 중화 항체의 경우에도 단순히 바이러스 감염 사이클을 저해하는 것만으로는 충분하지 않기 때문에 치료제로 개발하기가 여의치 않은 상황이다. 외피 바이러스의 감염력을 감소시키는 약물은 살바이러스제(virucide)로 표현을 하며, 일상 생활에서는 비누와 소독제 같은 것들이 대표적인 살바이러스제로 사용되고 있다. 하지만, 상기와 같은 살바이러스제는 안전하지 않기 때문에 치료제로 사용하기에 제한적이다. All of the above viruses have in common that they have an envelope composed of a lipid bilayer. Many animal viruses including the above (eg, HIV, hepatitis virus, MERS, herpes virus, Ebola virus, etc.) have membrane proteins and lipid bilayer envelopes. Membrane proteins or surface antigens exist on the envelope of the virus and have a characteristic of binding to the host cell's receptor, leading the virus to penetrate into the cell. In addition, the envelope not only plays a very important role in the infection route of the virus, but also serves to protect itself from the body's immune system. Therefore, as described above, the infection of the virus can be inhibited by interfering with the binding between the membrane protein and the receptor of the virus or by damaging the envelope. Oseltamivir is known as a representative among drugs that inhibit virus proliferation by interfering with membrane proteins of viruses (virustatic). Since the mode of action of the drug is to inhibit the proliferation of viruses, if the concentration of the drug is lowered, the virus can proliferate again. In addition, if the proliferation and suspension are repeated according to the change in the concentration of the drug, naturally, the virus may develop a resistant virus as a result of "adaptive evolution". Oseltamivir (Tamiflu) also resistant viruses are increasingly being reported. Even in the case of neutralizing antibodies, it is not easy to develop them as therapeutic agents because simply inhibiting the viral infection cycle is not enough. Drugs that reduce the infectivity of enveloped viruses are expressed as virucides, and in everyday life, soaps and disinfectants are used as typical virucides. However, since these virucidal agents are not safe, their use as therapeutic agents is limited.
따라서 강한 약효를 기대하기 위해서는 바이러스 외피를 손상시키는 살바이러스제의 작용 방식이 유리하며, 이에 더하여 안전하고 광범위한 외피바이러스에 대한 적용이 가능한 살바이러스제가 필요하다. 이를 위하여 바이러스 외피를 파고들어 손상시키는 특성을 가진 나노디스크 구조체를 만들었는데, 상기 나노디스크 구조체의 항바이러스 효능을 더욱 향상시킬 수 있는 방법을 개발하고자 했다.Therefore, in order to expect strong drug efficacy, the method of action of a viricide that damages the viral envelope is advantageous, and in addition, a viricide that is safe and can be applied to a wide range of enveloped viruses is needed. To this end, a nanodisc structure having a property of penetrating and damaging the viral envelope was made, and a method for further improving the antiviral efficacy of the nanodisc structure was sought.
본 발명에서는 나노디스크의 항바이러스 효능을 더욱 향상시킬 수 있는 기술을 개발하고자 했다.In the present invention, it was intended to develop a technology capable of further improving the antiviral efficacy of the nanodisc.
본 발명은 인지질인 포스파티딜에탄올아민(phosphatidylethanolamine)을 사용하여 형성된 납작한 원반 형태의 이중층 구조로서, 친수성기는 외부로 배향되고, 소수성기는 내부로 배향되어 있는 지질 이중층 (lipid bilayer); 및 상기 지질 이중층의, '소수성기가 외부로 노출되어 있는 측면'을 소수성 결합으로 둘러싸는 '막구조화 단백질 (membrane scaffold protein) 또는 양친매성 폴리머 (amphipathic polymer)'를 포함하는 것을 특징으로 하는 나노디스크(nanodisc)를 제공한다.The present invention is a flat disk-shaped bilayer structure formed using phospholipid phosphatidylethanolamine, wherein the hydrophilic group is oriented to the outside and the hydrophobic group is oriented to the inside of the lipid bilayer; and a 'membrane scaffold protein or an amphipathic polymer' surrounding the 'side of the lipid bilayer where the hydrophobic group is exposed to the outside' with hydrophobic bonds ( nanodisc).
본 발명의 나노디스크에 있어서, 상기 포스파티딜에탄올아민(phosphatidylethanolamine)은 바람직하게 1-팔미토일-2-올레오일-sn-글리세로-3-포스포콜린(1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, POPE)인 것이 좋다. In the nanodisc of the present invention, the phosphatidylethanolamine is preferably 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (1-Palmitoyl-2-oleoyl-sn-glycero -3-phosphoethanolamine, POPE) is preferred.
본 발명의 나노디스크에 있어서, 상기 막구조화 단백질 (membrane scaffold protein)은 바람직하게 헬릭스(helix) 구조를 갖는 양친매성 단백질인 것이 좋다. 이때, 상기 막구조화 단백질 (membrane scaffold protein)은 더욱 바람직하게, 아포리포단백질(apolipoprotein) 또는 아포리포단백질의 '헬릭스(helix) 구조 및 양친매성 특성'이 유지된 아포리포단백질의 절편인 것이 좋다.In the nanodisc of the present invention, the membrane scaffold protein is preferably an amphiphilic protein having a helix structure. In this case, the membrane scaffold protein is more preferably an apolipoprotein or a fragment of an apolipoprotein that retains the 'helix structure and amphipathic properties' of the apolipoprotein.
본 발명의 나노디스크에 있어서, 상기 양친매성 폴리머 (amphipathic polymer)로 스티렌-말레산(Styrene-Maleic Acid, SMA), 디-이소부틸렌-말레산(Di-IsoButylene-Maleic Acid, DIBMA), 스티렌-말레이미드(Styrene-Maleimide, SMI), 폴리메타크릴레이트(Polymethyl Methacrylate, PMA) 중 선택되는 어느 하나 이상인 것을 사용할 수 있다.In the nanodisc of the present invention, as the amphipathic polymer, styrene-maleic acid (SMA), di-isobutylene-maleic acid (DIMBA), styrene -Any one or more selected from maleimide (Styrene-Maleimide, SMI) and polymethyl methacrylate (PMA) can be used.
본 발명의 나노디스크는 바람직하게 상기 지질 이중층 내부와 소수성 결합되는 '바이러스의 표면 항원에 대한 수용체(receptor)'을 더욱 포함하는 것이 좋다. 이때, 상기 '바이러스의 표면 항원에 대한 수용체(receptor)'는 바람직하게 안지오텐진 전환효소 2 또는 '일측단에 시알산(sialic acid)을 포함하는 화합물'인 것이 좋다.The nanodisc of the present invention preferably further includes a 'receptor for a surface antigen of a virus' hydrophobically bound to the inside of the lipid bilayer. In this case, the 'receptor for the surface antigen of the virus' is preferably
한편, 본 발명은 상기 본 발명의 나노디스크(nanodisc)를 포함하는 것을 특징으로 하는 바이러스 감염증 예방 또는 치료용 약학 조성물을 제공한다.Meanwhile, the present invention provides a pharmaceutical composition for preventing or treating a viral infection comprising the nanodisc of the present invention.
본 발명의 나노디스크는 지질로써, 포스파티딜에탄올아민 인지질을 포함하는 것을 특징으로 하는데, 다른 종류의 인지질을 포함하는 나노디스크 보다 항바이러스 효능이 우수하다.The nanodiscs of the present invention are characterized in that they contain phosphatidylethanolamine phospholipids as lipids, and are superior in antiviral efficacy to nanodiscs containing other types of phospholipids.
도 1은 본 발명 나노디스크의 구조를 개략적으로 보여준다.
도 2는 나노디스크에 포함된 인지질 종류에 따른 세포독성 차이를 보여준다.
도 3은 나노디스크에 포함된 인지질 종류에 따른 항바이러스 효능 차이를 보여준다. 한편, 도 3을 보면, 인지질 중 포스파티딜에탄올아민을 포함하는 나노디스크가 항바이러스 효능이 더욱 우수한 것을 확인할 수 있다.
도 4는 나노디스크에 포함된 인지질 종류에 따른 항바이러스 효능 차이를 보여준다. 한편, 도 4를 보면, 포스파티딜에탄올아민 중에서도, 1-팔미토일-2-올레오일-sn-글리세로-3-포스포콜린(1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, POPE)을 포함하는 나노디스크의 항바이러스 효능이 더욱 우수한 것을 확인할 수 있다. 1 schematically shows the structure of the nanodisc of the present invention.
Figure 2 shows the difference in cytotoxicity according to the type of phospholipids included in the nanodiscs.
Figure 3 shows the difference in antiviral efficacy according to the type of phospholipids included in the nanodiscs. Meanwhile, referring to FIG. 3 , it can be seen that nanodiscs containing phosphatidylethanolamine among phospholipids have more excellent antiviral efficacy.
Figure 4 shows the difference in antiviral efficacy according to the type of phospholipids included in the nanodiscs. 4, among phosphatidylethanolamines, 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, POPE ) It can be seen that the antiviral efficacy of the nanodisc containing the is more excellent.
본 발명은 인지질인 포스파티딜에탄올아민(phosphatidylethanolamine)을 사용하여 형성된 납작한 원반 형태의 이중층 구조로서, 친수성기는 외부로 배향되고, 소수성기는 내부로 배향되어 있는 지질 이중층 (lipid bilayer); 및 상기 지질 이중층의, '소수성기가 외부로 노출되어 있는 측면'을 소수성 결합으로 둘러싸는 '막구조화 단백질 (membrane scaffold protein) 또는 양친매성 폴리머 (amphipathic polymer)'를 포함하는 것을 특징으로 하는 나노디스크(nanodisc)를 제공한다.The present invention is a flat disk-shaped bilayer structure formed using phospholipid phosphatidylethanolamine, wherein the hydrophilic group is oriented to the outside and the hydrophobic group is oriented to the inside of the lipid bilayer; and a 'membrane scaffold protein or an amphipathic polymer' surrounding the 'side of the lipid bilayer where the hydrophobic group is exposed to the outside' with hydrophobic bonds ( nanodisc).
본 발명자는 대한민국 등록특허 제10-2181991호, 제10-2438720호 등을 통해 항바이러스 효능이 우수한 나노디스크를 개발하였다. 이후, 상기 항바이러스 효능을 더욱 향상시킬 수 있는 기술을 개발하던 중, 나노디스크에 포함되는 인지질의 종류에 따라 나노디스크의 항바이러스 효능이 달라지는 것을 확인하였고, 인지질 중 포스파티딜에탄올아민(phosphatidylethanolamine)을 사용한 경우가 항바이러스 효능이 특히 우수한 것을 확인할 수 있었다.The inventors of the present invention developed nanodiscs with excellent antiviral efficacy through Korean Patent Registration Nos. 10-2181991 and 10-2438720. Then, while developing a technology capable of further improving the antiviral efficacy, it was confirmed that the antiviral efficacy of the nanodisc varies depending on the type of phospholipid included in the nanodisk, and among the phospholipids, phosphatidylethanolamine was used. It was confirmed that the case had particularly excellent antiviral efficacy.
따라서, 본 발명의 나노디스크는 인지질인 포스파티딜에탄올아민(phosphatidylethanolamine)을 포함하는 것을 특징으로 한다. Accordingly, the nanodisc of the present invention is characterized by including phosphatidylethanolamine, which is a phospholipid.
한편, 상기 포스파티딜에탄올아민(phosphatidylethanolamine)은 일예로, DMPE(1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine), DPPE(1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine), DSPE(1,2-Distearoyl-sn-glycero-3-phosphoethanolamine), DOPE(1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine), DEPE(1,2-Dierucoyl-sn-glycero-3-phosphoethanolamine), DLPE(1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine) 또는 POPE(1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine)을 들 수 있는데, 하기 실시예에 따르면, 그 중에서도 POPE(1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine)를 사용한 나노디스크의 항바이러스 효능이 가장 우수하였다.On the other hand, the phosphatidylethanolamine (phosphatidylethanolamine) is, for example, DMPE (1,2-Dimyristoyl-sn-glycero-3-phosphoethanolamine), DPPE (1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine), DSPE (1 ,2-Distearoyl-sn-glycero-3-phosphoethanolamine), DOPE (1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine), DEPE (1,2-Dierucoyl-sn-glycero-3-phosphoethanolamine), DLPE ( 1,2-Dilauroyl-sn-glycero-3-phosphoethanolamine) or POPE (1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine) may be mentioned. Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine) showed the best antiviral efficacy of nanodisks.
한편, 본 발명의 지질 이중층 (lipid bilayer)은 '친수성 부위와 소수성 부위를 갖는 양친매성 지질 분자'의 일종인, 포스파티딜에탄올아민(phosphatidylethanolamine)으로부터 형성된 납작한 원반 형태의 이중층 구조로서, 친수성기는 외부로 배향되고, 소수성기는 내부로 배향되어 있는 것에 특징이 있다. 즉, 본 발명의 지질 이중층 (lipid bilayer)은 양친매성 지질 (amphipahtic lipid)의 친수성 부위뿐만 아니라, 소수성 부위도 측면을 통해 외부에 노출된 부분을 갖는 원반 형태 (도 1 참조)라는 점에서, 친수성 또는 소수성 부위 중 선택적으로 하나만이 외부에 노출되는 구(sphere) 형태의 리포좀(liposome)과 구조상 차이가 있다.On the other hand, the lipid bilayer of the present invention is a bilayer structure in the form of a flat disc formed from phosphatidylethanolamine, which is a kind of 'amphiphilic lipid molecule having a hydrophilic part and a hydrophobic part', and the hydrophilic group is oriented to the outside. and the hydrophobic group is characterized by being oriented inwardly. That is, the lipid bilayer of the present invention is a disk shape having not only the hydrophilic portion of the amphiphatic lipid but also the hydrophobic portion exposed to the outside through the side surface (see FIG. 1), which is hydrophilic. Alternatively, it is structurally different from a sphere-shaped liposome in which only one of the hydrophobic regions is selectively exposed to the outside.
또한, 본 발명에서는 '막구조화 단백질 (membrane scaffold protein) 또는 양친매성 폴리머 (amphipathic polymer)'을 사용하는데, 본 발명에서 '막구조화 단백질 (membrane scaffold protein) 또는 양친매성 폴리머 (amphipathic polymer)'는 본 발명 지질 이중층의 '소수성기가 외부로 노출되어 있는 측면'을 둘러싸는 역할을 수행하여, 본 발명 나노디스크가 안정한 형태로 유지될 수 있게 한다. In addition, in the present invention, 'membrane scaffold protein or amphipathic polymer' is used. In the present invention, 'membrane scaffold protein or amphipathic polymer' is used in the present invention. It serves to surround the 'side where the hydrophobic group is exposed to the outside' of the lipid bilayer of the invention, so that the nanodisc of the invention can be maintained in a stable form.
한편, 본 발명에서 상기 막구조화 단백질은 바람직하게 헬릭스(helix) 구조를 갖는 양친매성 단백질인 것이 좋다. 헬릭스(helix) 구조를 갖는 양친매성 막구조화 단백질(membrane scaffold protein)의 예로는 아포리포단백질(apolipoprotein)이 있다. 아포리포단백질(apolipoprotein)은 혈장지방단백질에 특이적으로 존재하는 단백질로, 지방단백질의 구조를 안정시키고, 지방단백질대사에 관여하는 효소를 활성화하며, 세포표면에 존재하는 지방단백질 수용체에 대한 배위자로서 기능을 수행하는 것으로 알려져 있다. 상기 아포리포단백질(apolipoprotein)은 일 예로서, 아포리포단백질 A1(ApoA-I), 아포리포단백질 A2(ApoA-2), 아포리포단백질 B(ApoB), 아포리포단백질 C(ApoC) 및 아포리포단백질 E(ApoE), MSP1(Membrane scaffold protein1), MSP1D1, MSP1D2, MSP1E1, MSP1E2, MSP1E3, MSP1E3D1, MSP2, MSP2N1, MSP2N2, MSP2N3, 등이 있다.Meanwhile, in the present invention, the membrane-structured protein is preferably an amphiphilic protein having a helix structure. An example of an amphiphilic membrane scaffold protein having a helix structure is apolipoprotein. Apolipoprotein is a protein that exists specifically in plasma lipoproteins, stabilizes the structure of lipoproteins, activates enzymes involved in lipoprotein metabolism, and acts as a ligand for lipoprotein receptors on the cell surface. known to perform the function. The apolipoprotein (apolipoprotein) is an example, apolipoprotein A1 (ApoA-I), apolipoprotein A2 (ApoA-2), apolipoprotein B (ApoB), apolipoprotein C (ApoC) and apolipoprotein Protein E (ApoE), MSP1 (Membrane scaffold protein 1), MSP1D1, MSP1D2, MSP1E1, MSP1E2, MSP1E3, MSP1E3D1, MSP2, MSP2N1, MSP2N2, MSP2N3, and the like.
상기에서 일 예로 언급한 ApoA-I은 주로 주변조직으로부터 콜레스테롤을 제거하여 간 또는 다른 리포단백질로 운반하는 직접적인 역할을 수행하는 고밀도 리포단백질(HDL)의 구성요소인 것으로 알려져 있다. Apo-A1은 분자량 28kDa의 243개의 아미노산으로 이루어진 단일 폴리펩타이드로 구성된다. 11개의 아미노산 혹은 22개의 아미노산으로 이루어진 8개의 반복 단위 도메인을 가지며, HDL을 이루는 2차 구조의 알파-헬릭스의 비율이 60 내지 75%인 단백질이다. 또한, ApoE는 ApoA1과 마찬가지로 콜레스테롤의 운반에 관여하는 것으로 알려져 있는데, 33kDa의 299개의 아미노산으로 이루어진 단일 폴리펩타이드로 구성된 단백질이다.ApoA-I mentioned above as an example is known to be a component of high-density lipoprotein (HDL) that plays a direct role in removing cholesterol from surrounding tissues and transporting it to the liver or other lipoproteins. Apo-A1 consists of a single polypeptide consisting of 243 amino acids with a molecular weight of 28 kDa. It is a protein having 8 repeating unit domains of 11 amino acids or 22 amino acids, and the ratio of alpha-helices in the secondary structure constituting HDL is 60 to 75%. In addition, ApoE, like ApoA1, is known to be involved in transporting cholesterol, and is a protein composed of a single polypeptide consisting of 299 amino acids of 33 kDa.
또한, 본 발명에서는 아포리포단백질의 헬릭스 구조 및 양친매성 특성'이 유지된 아포리포단백질의 절편을 사용할 수도 있다. 즉, 상기 아포리포단백질(apolipoprotein)의 '헬릭스 구조 및 양친매성 특성'을 소실하지 않는 범위 내에서, 아포리포단백질 전체가 아닌 그 일부(절편)를 사용할 수도 있는 것이다. In addition, in the present invention, fragments of apolipoprotein in which the helical structure and amphipathic characteristics of apolipoprotein are maintained may be used. That is, within the range of not losing the 'helix structure and amphipathic characteristics' of the apolipoprotein, a part (fragment) of the apolipoprotein may be used instead of the entire apolipoprotein.
한편, 본 발명에서 양친매성 폴리머는 양친매성을 가지고 있어서 소수성 꼬리 (acyl tail)가 물에 직접 노출되는 것을 방지하는 역할을 하는데, 이 양친매성 폴리머가 일정 비율로 존재하게 되면 지질이중막을 형성한 인지질의 중간층(2겹의 소수성 아실 꼬리(acyl tail))을 폴리머가 감싸게 된다. 결과적으로 디스크 형태의 지질이중막 모양이 수용액상에서 안정화된다. 이때, 인지질:폴리머 비율에 따라서 지질이중막 디스크의 크기가 결정된다.On the other hand, in the present invention, the amphiphilic polymer has amphiphilicity and serves to prevent the hydrophobic tail (acyl tail) from being directly exposed to water. The middle layer (two-layered hydrophobic acyl tail) of the polymer is wrapped. As a result, the disk-shaped lipid bilayer is stabilized in an aqueous solution. At this time, the size of the lipid bilayer disk is determined according to the phospholipid:polymer ratio.
본 발명에서 양친매성 폴리머는 바람직하게 스티렌-말레산(Styrene-Maleic Acid, SMA), 디-이소부틸렌-말레산(Di-IsoButylene-Maleic Acid, DIBMA), 스티렌-말레이미드(Styrene-Maleimide, SMI), 폴리메타크릴레이트(Polymethyl Methacrylate, PMA) 중 선택되는 어느 하나 이상을 사용하는 것이 좋다. 하기 실시예에서는 스티렌-말레산(Styrene-Maleic Acid, SMA)를 사용하였다.In the present invention, the amphiphilic polymer is preferably styrene-maleic acid (SMA), di-isobutylene-maleic acid (DIBMA), styrene-maleimide (Styrene-Maleimide, It is good to use at least one selected from SMI) and polymethyl methacrylate (PMA). In the following examples, styrene-maleic acid (SMA) was used.
한편, 본 발명의 나노디스크는 직경이 2 내지 50 nm에 해당하는 것일 수 있으나 이에 제한되는 것은 아니다. Meanwhile, the nanodisc of the present invention may have a diameter of 2 to 50 nm, but is not limited thereto.
한편, 본 발명의 나노디스크는 바람직하게 상기 지질 이중층 내부와 소수성 결합되는 '바이러스의 표면 항원에 대한 수용체(receptor)'를 더욱 포함하는 것이 좋다. 본 발명에서 표면 항원에 대한 수용체란, 표명 항원과 결합할 수 있는 수용체로써, 상기 표면 항원에 대한 항체, 상기 표면 항원이 결합할 수 있는 다른 세포막 결합 단백질, 상기 표면 항원이 결합할 수 있는 화합물 등이 될 수 있다. 즉, '바이러스의 표면 항원에 대한 수용체(receptor)'는 나노디스크가 바이러스와의 부착능을 더욱 향상시켜주는 역할을 한다. Meanwhile, the nanodisc of the present invention preferably further includes a 'receptor for a surface antigen of a virus' that is hydrophobically bound to the inside of the lipid bilayer. In the present invention, a receptor for a surface antigen is a receptor capable of binding to a labeled antigen, such as antibodies to the surface antigen, other cell membrane binding proteins capable of binding the surface antigen, compounds capable of binding the surface antigen, etc. This can be. In other words, the 'receptor for the surface antigen of the virus' plays a role in further enhancing the ability of the nanodisc to attach to the virus.
한편, 하기 실시예에서는 '바이러스의 표면 항원에 대한 수용체(receptor)'가 없는 나노디스크만을 사용해 항바이러스 실험을 진행해 하였다. 다만, 본 발명의 나노디스크에 '바이러스의 표면 항원에 대한 수용체(receptor)'를 포함함으로써, 바이러스와의 부착능을 더욱 향상시킬 수 있는데, 이에 나노디스크의 항바이러스 효능이 향상되는 것은 자명하다 할 것이다.Meanwhile, in the following examples, antiviral experiments were conducted using only nanodiscs having no 'receptors for surface antigens of viruses'. However, by including a 'receptor for the surface antigen of a virus' in the nanodisc of the present invention, the ability to attach to the virus can be further improved, and it is obvious that the antiviral efficacy of the nanodisc is improved. will be.
한편, 본 발명에서 '바이러스의 표면 항원에 대한 수용체(receptor)'는 바람직하게 안지오텐진 전환효소 2 또는 '일측단에 시알산(sialic acid)을 포함하는 화합물'인 것이 좋다.Meanwhile, in the present invention, the 'receptor for the surface antigen of the virus' is preferably
상기 안지오텐진 전환효소 2는 SARS-CoV와 SARS-CoV-2를 포함한 여러 코로나바이러스는 인체 세포로 침투하기 위해서 안지오텐신 전환효소 2 (Angiotensin converting enzyme 2, ACE2)를 수용체로 이용하는 것으로 알려졌다. 즉, 안지오텐진 전환효소 2를 포함시킴으로써 SARS-CoV와 SARS-CoV-2 등을 포함한 코로나바이러스와의 부착능을 향상시켜, 항바이러스 효능을 향상시킬 수 있는 것이다.The
상기 '일측단에 시알산(sialic acid)을 포함하는 화합물'은 일측단에 시알산(sialic acid)을 포함하여 바이러스와의 부착능이 있는 화합물 또는 시알산(sialic acid) 복합체를 말하는데, 일예로, 시알릴락토오스(sialyllactose), 강글리오시드(ganglioside) 등이 있다. 시알릴락토오스(sialyllactose) 및 강글리오시드(ganglioside)는 일측단에 시알산(sialic acid)을 포함하여 인플루엔자 바이러스와의 부착능이 있는 것으로 알려져 있다. The 'compound containing sialic acid at one end' refers to a compound or sialic acid complex containing sialic acid at one end and capable of attaching to a virus. For example, sialyllactose and ganglioside. It is known that sialyllactose and ganglioside contain sialic acid at one end and have an ability to attach to influenza virus.
한편, 본 발명은 상기 본 발명의 나노디스크(nanodisc)를 포함하는 것을 특징으로 하는 바이러스 감염증 예방 또는 치료용 약학 조성물을 제공한다.Meanwhile, the present invention provides a pharmaceutical composition for preventing or treating a viral infection comprising the nanodisc of the present invention.
본 발명에서 "바이러스 감염증"이란, 일 예로서, 버니아비리데 과의 바이러스의 감염에 의하여 발병되는 신증후근성출혈열(유행성출혈열); 코로나비리데 과의 바이러스의 감염에 의하여 발병되는 코감기 등 호흡기 질환 또는 코로나바이러스 감염증; 플라비비리데 과의 바이러스의 감염에 의하여 발병되는 C형 간염; 헤파드나비리데 과의 바이러스의 감염에 의하여 발병되는 B형 간염; 헤르페스비리데 과의 바이러스의 감염에 의하여 발병되는 대상포진; 오르소믹소비리데 과의 바이러스의 감염에 의하여 발병되는 독감 또는 인플루엔자 바이러스 감염증; 폭스비리데 과의 바이러스의 감염에 의하여 발병되는 천연두; 랍도비리데 과의 바이러스의 감염에 의하여 발병되는 광견병 또는 수포성 구내염; 레트로비리데과의 바이러스의 감염에 의하여 발병되는 후천성 면역결핍증 등이 될 수 있고, 다른 예로서, 오르소믹소비리데과에 속하는 인플루엔자 바이러스의 감염에 의하여 발병되는 독감 또는 인플루엔자 바이러스 감염증이 될 수 있다.In the present invention, "viral infection" is, as an example, nephropathy caused by infection with a virus of the family Vernia viride (epidemic hemorrhagic fever); Respiratory diseases such as a cold or a coronavirus infection caused by infection with a virus of the Coronaviridae family; hepatitis C caused by infection with a virus of the flaviviride family; hepatitis B caused by infection with a virus of the Hepadnaviridae family; herpes zoster caused by infection with a virus of the herpesviride family; Influenza or influenza virus infection caused by infection with a virus of the Orthomyxoviridae family; smallpox caused by infection with a poxviride family virus; rabies or vesicular stomatitis caused by infection with a virus of the Rhabdoviridae family; It may be acquired immunodeficiency syndrome caused by infection with a retroviridae family virus, and as another example, it may be influenza or influenza virus infection caused by infection with an influenza virus belonging to the orthomyxoviridae family.
본 발명의 약학 조성물은 유효성분인 상기 조성물 외에 약학으로 허용되는 담체를 포함할 수 있다.The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier in addition to the composition as an active ingredient.
본 발명의 약학 조성물에 포함되는 약학으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, calcium silicate , microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, but are not limited thereto no.
본 발명의 약학 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. The pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, and the like, in addition to the above components.
본 발명의 약학 조성물은 경구 또는 비경구로 투여할 수 있고, 예컨대 척추강 내 투여, 정맥내 투여, 피하 투여, 피내 투여, 근육내 투여, 복강내 투여, 흉골 내 투여, 종양 내 투여, 비내 투여, 뇌내 투여, 두개골 내 투여, 폐내 투여 및 직장내 투여 등으로 투여할 수 있으나 이에 한정되는 것은 아니다.The pharmaceutical composition of the present invention can be administered orally or parenterally, such as intrathecal administration, intravenous administration, subcutaneous administration, intradermal administration, intramuscular administration, intraperitoneal administration, intrasternal administration, intratumoral administration, intranasal administration, It may be administered by intracerebral administration, intracranial administration, intrapulmonary administration, intrarectal administration, etc., but is not limited thereto.
본 발명의 약학 조성물의 적합한 투여량은 제제화 방법, 투여방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하며, 보통으로 숙련된 의사는 소망하는 치료 또는 예방에 효과적인 투여량(약학 유효량)을 용이하게 결정 및 처방할 수 있다. 본 발명의 바람직한 구현예에 따르면, 본 발명의 약학 조성물의 1일 투여량은 0.0001-100 ㎎/㎏이다. The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, patient's age, weight, sex, disease condition, food, administration time, administration route, excretion rate and reaction sensitivity, usually As a result, a skilled physician can easily determine and prescribe an effective dosage (pharmaceutically effective amount) for the desired treatment or prevention. According to a preferred embodiment of the present invention, the daily dosage of the pharmaceutical composition of the present invention is 0.0001-100 mg/kg.
본 발명의 용어 "약학 유효량"은 상술한 질환을 예방 또는 치료하는 데 충분한 양을 의미한다.The term "pharmaceutically effective amount" of the present invention means an amount sufficient to prevent or treat the above-mentioned diseases.
본 발명의 용어 "예방"은 질환 또는 질환 상태의 방지 또는 보호적인 치료를 의미한다. 본 발명의 용어 "치료"는 질환 상태의 감소, 억제, 진정 또는 근절을 의미한다.The term “prophylaxis” as used herein refers to preventive or protective treatment of a disease or disease state. The term “treatment” as used herein refers to reduction, suppression, sedation or eradication of a disease state.
본 발명의 약학 조성물은 당해 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약학으로 허용되는 담체 및/또는 부형제를 이용하여 제제화 함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 이때 제형은 내복약, 주사제 등 다양하게 제조될 수 있고, 오일 또는 수성 매질중의 용액, 현탁액 또는 유화액 형태이거나 엑스제, 산제, 좌제, 분말제, 과립제, 정제 또는 캅셀제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다.The pharmaceutical composition of the present invention is prepared in unit dosage form by formulation using pharmaceutically acceptable carriers and/or excipients according to a method that can be easily performed by those skilled in the art, or It can be prepared by placing it in a multi-dose container. At this time, the dosage form may be prepared in various ways such as oral medicine and injection, and may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or in the form of an extract, powder, suppository, powder, granule, tablet, or capsule, and may be in the form of a dispersant or stabilizer. Additional topics may be included.
이하, 본 발명의 내용을 하기 실시예를 통하여 보다 상세하게 설명하고자 한다. 다만, 본 발명의 권리범위가 하기 실시예에만 한정되는 것은 아니고 그와 등가의 기술적 사상의 변형까지를 포함한다.Hereinafter, the contents of the present invention will be described in more detail through the following examples. However, the scope of the present invention is not limited only to the following examples, and includes modifications of equivalent technical ideas.
[실시예 1: 나노디스크의 제조][Example 1: Preparation of nanodisc]
1-1) MSP 나노디스크 제조1-1) Manufacture of MSP nanodisks
MSP 나노디스크를 제작하기 위해 막구조화 단백질(MSP, membrane scaffold protein)의 일종인 N말단에 his-tag가 MSP1E3D1(서열번호 1, 분자량 32.6 kDa)를 사용하고, 지질로는 POPC(1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine)를 사용하였다.To fabricate MSP nanodiscs, MSP1E3D1 (SEQ ID NO: 1, molecular weight: 32.6 kDa) was used as his-tag at the N-terminus, a type of membrane scaffold protein (MSP), and POPC (1-palmitoyl- 2-oleoyl-sn-glycero-3-phosphocholine) was used.
MSP 나노 디스크의 제조방법은 구체적으로 다음과 같다.The manufacturing method of the MSP nano-disc is specifically as follows.
POPC(1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine)를 클로로포름에 용해시켜 25 mg/ml 농도의 지질 용액을 준비하였다. 이후 0.5 ml의 소듐 콜레이트가 첨가된 PBS로 녹였을 때 지질의 농도가 10 mM이 되도록 25 mg/ml POPC 지질 용액의 152.02 μl를 유리 튜브에 옮겼다. 이후 질소가스를 가하고, 진공 상태에서 4시간동안 방치하여 용매를 제거하고, 리피드 필름 (lipid film)을 수득하였다. 상기 수득한 리피드 필름에 소듐 콜레이트(sodium cholate)가 첨가된 PBS의 0.5 ml을 이용하여, 상기 리피드 필름을 수화시키고, 초음파를 55℃에서 15 분간 처리하여, 리피드 필름이 분쇄된 리피드 필름 함유 현탁액을 수득하였다. 상기 수득한 현탁액에 막구조화 단백질로 MSP1E3D1(분자량 32.6 kDa)을 250μM 농도로 160 μl를 가하고, 전체 혼합액과 동일한 양(660 μl)의 바이오비드(bio-beads)를 처리(4℃, 12시간)함으로써, 자가조립과정을 통해 MPS 나노디스크를 제조할 수 있었다.A lipid solution having a concentration of 25 mg/ml was prepared by dissolving POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) in chloroform. Thereafter, 152.02 μl of the 25 mg/ml POPC lipid solution was transferred to a glass tube so that the lipid concentration was 10 mM when dissolved in PBS supplemented with 0.5 ml sodium cholate. Thereafter, nitrogen gas was added, and the mixture was left in a vacuum for 4 hours to remove the solvent, and a lipid film was obtained. Using 0.5 ml of PBS to which sodium cholate was added to the obtained lipid film, the lipid film was hydrated, and ultrasonic treatment was performed at 55 ° C. for 15 minutes to obtain a lipid film-containing suspension in which the lipid film was pulverized. obtained. To the obtained suspension, 160 μl of MSP1E3D1 (molecular weight 32.6 kDa) at a concentration of 250 μM was added as a membrane structure protein, and the same amount (660 μl) of the entire mixture was treated with bio-beads (4 ° C, 12 hours) By doing so, it was possible to fabricate MPS nanodisks through a self-assembly process.
1-2) 폴리머 나노디스크의 제조1-2) Preparation of polymer nanodiscs
폴리머 나노디스크를 제작하기 위해 폴리머로써 SMA(Styrene-Maleic Acid) 중 Styrene : Maleic acid가 2:1 또는 3:1의 몰비율로 중합된 두 종류의 폴리머를 사용하였고, 폴리머와 지질은 1:2의 중량비율로 사용하였다.To fabricate the polymer nanodisc, two types of polymers, in which styrene:maleic acid in SMA (Styrene-Maleic Acid) were polymerized at a molar ratio of 2:1 or 3:1, were used, and the polymer and lipid were 1:2 It was used in a weight ratio of
한편, 구성 지질은 POPC(1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 이하 약어로 기재), DPPE(1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine, 이하 약어로 기재), POPG(1-Palmitoyl-2-oleoyl-sn-glycero-3[Phospho-rac-(1-glycerol)], 이하 약어로 기재), 또는 POPE(1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, 이하 약어로 기재)을 사용하여 제조하였다 (표 1).On the other hand, constituent lipids are POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, hereinafter abbreviated), DPPE (1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine, hereinafter abbreviated) , POPG (1-Palmitoyl-2-oleoyl-sn-glycero-3 [Phospho-rac-(1-glycerol)], hereinafter abbreviated), or POPE (1-palmitoyl-2-oleoyl-sn-glycero-3 -phosphoethanolamine, described below as abbreviations) (Table 1).
폴리머 나노 디스크의 제조방법은 구체적으로 다음과 같다.The manufacturing method of the polymer nanodisc is specifically as follows.
먼저 고체 가루 형태의 지질을 유기용매에 녹인 후, 지질을 유리관에서 혼합한 뒤 질소 가스와 진공관을 이용해 유기용매를 완전히 기화시켜 필름 형태의 지질만 남겼다. 수화 버퍼(10 mM HEPES, 150 mM NaCl; pH 7.4)를 이용해 필름 형태의 지질을 5분 이상 충분히 녹인 후, 액체 질소와 50℃의 물을 이용해 동결과 융해과정을 5회 이상 수행하여 리포좀을 생성하였다. 이후 폴리머를 0.5-2%(w/v)의 양으로 첨가한 뒤 액체 질소와 50℃의 소니케이터를 이용해 동결 및 음파처리 과정을 5회 이상 수행하였다. 이를 통해 폴리머가 리포좀을 파고들면서 물 분자가 끼어들어 지질층 사이에 구멍(pore)을 형성하게 되고 최종적으로 나노디스크 구조가 형성되었다. 나노디스크를 형성하지 못한 폴리머와 직경이 큰 리포좀을 제거하기 위해 초고속 원심분리 여과(Ultracentrifugal filtration)를 수행하여 폴리머 나노디스크 농축액을 얻을 수 있었다.First, solid powdery lipids were dissolved in an organic solvent, then the lipids were mixed in a glass tube, and the organic solvent was completely vaporized using nitrogen gas and a vacuum tube, leaving only film-like lipids. After sufficiently dissolving film-type lipids for at least 5 minutes using a hydration buffer (10 mM HEPES, 150 mM NaCl; pH 7.4), liposomes are formed by performing freezing and thawing at least 5 times using liquid nitrogen and water at 50 ° C. did Thereafter, the polymer was added in an amount of 0.5-2% (w/v), and freezing and sonication were performed five or more times using liquid nitrogen and a sonicator at 50 ° C. Through this, as the polymer penetrates the liposome, water molecules are intervened to form pores between the lipid layers, and finally the nanodisc structure is formed. In order to remove polymers that did not form nanodiscs and liposomes having a large diameter, ultracentrifugal filtration was performed to obtain a polymer nanodisc concentrate.
한편, 폴리머 나노디스크 농축액의 지질 농도를 lipid quantification assay kit를 이용하여 측정하였고, 이렇게 얻은 지질 농도를 이하 실험에 활용하였다.Meanwhile, the lipid concentration of the polymer nanodisc concentrate was measured using a lipid quantification assay kit, and the obtained lipid concentration was used in the following experiments.
[실시예 2: 인지질 종류 변화에 따른 나노디스크의 세포독성 비교 실험][Example 2: Cytotoxicity comparison test of nanodisks according to phospholipid type changes]
본 실시예에서는 나노디스크에 포함된 인지질 종류 변화에 따라 세포독성이 나타나는지 확인하고자 했다.In this Example, it was attempted to confirm whether cytotoxicity appears according to the change in the type of phospholipids included in the nanodiscs.
세포독성은 세포 생존도 (Cell viability assay)를 정량화 할 수 있는 CCK-8 kit를 이용해 확인할 수 있었다. 구체적으로, 폴리머 기준 최고 농도 20 μM, 인지질 기준 200 μM 농도의 샘플을 희석하며 다양한 농도의 샘플을 준비하였다. 전날 96-웰 플레이트에 2x104 cells/100 μl로 시딩하여 90% 밀집도(confluency) 상태인 MDCK 세포에 순수 폴리머 또는 각종 인지질로 구성된 폴리머 나노디스크를 100 μl/well로 처리하였다. 24 시간 후 CCK-8 용액을 10 μl/well 로 처리하여 4시간 동안 37 ℃ 온도 조건에서 1시간동안 반응시켰다. 이후, 540 nm 파장에서 흡광도를 측정하여 세포 생존도를 정량화하고, 이를 통해 세포독성을 확인할 수 있었다 (도 5).Cytotoxicity was confirmed using a CCK-8 kit capable of quantifying cell viability assay. Specifically, samples of various concentrations were prepared by diluting samples with a maximum concentration of 20 μM based on polymer and 200 μM based on phospholipid. MDCK cells seeded at 2x10 4 cells/100 μl in a 96-well plate the day before and at 90% confluency were treated with 100 μl/well of polymer nanodisks composed of pure polymers or various phospholipids. After 24 hours, the CCK-8 solution was treated with 10 μl/well and reacted for 1 hour at 37 °C for 4 hours. Subsequently, cell viability was quantified by measuring absorbance at a wavelength of 540 nm, and cytotoxicity was confirmed through this (FIG. 5).
이를 통해, 폴리머 기준 20 μM, 인지질 기준 200 μM 농도에서 순수 폴리머와 인지질 POPG 구성의 나노디스크 실험군에서 세포 생존도가 대략 50%로 하락하는 것을 확인할 수 있었다. 반면, 인지질 POPC와 POPE 실험군의 경우 해당 최고 농도에서도 세포 생존도에 감소를 보이지 않고 100% 이상을 유지하며 무독성을 나타내는 것을 확인할 수 있었다.Through this, it was confirmed that cell viability decreased to approximately 50% in the nanodisc experimental group composed of pure polymer and phospholipid POPG at a concentration of 20 μM for polymer and 200 μM for phospholipid. On the other hand, in the case of the phospholipids POPC and POPE experimental groups, it was confirmed that cell viability did not decrease even at the highest concentration and maintained at 100% or more and exhibited non-toxicity.
[실시예 3: 인지질 종류 변화에 따른 나노디스크의 항바이러스 효능 비교 실험][Example 3: Comparison of antiviral efficacy of nanodiscs according to phospholipid types]
본 실시예에서는 어떤 종류의 지질을 포함하는 나노디스크가 더욱 우수한 항바이러스 효능을 가지지 확인하고자 했다. In this example, it was attempted to confirm whether nanodiscs containing any kind of lipids have better antiviral efficacy.
이를 위해 상기 실시예 1에서 제조한 나노디스크를 사용하여 항바이러스 효능 검증 실험을 진행하였다. To this end, an antiviral efficacy verification experiment was conducted using the nanodisc prepared in Example 1 above.
한편, 실험은 폴리머 나노디스크를 사용하여 실험을 진행하였는데, 폴리머 나노디스크는 일반적으로 알려진 막구조화 단백질(MSP, membrane scaffold protein)을 사용한 나노디스크의 막구조화 단백질을 양친매성 폴리머 성분으로 대체함으로써 나노디스크의 구조 안정성 향상, 항바이러스 활성 향상, 제조공정 개선 등을 위해 개발한 것으로, 양친매성 폴리머 성분을 사용한 나노디스크에서 나타나는 항바이러스 특징은 막구조화 단백질(MSP)을 사용한 나노디스크에서도 동일하게 적용할 수 있다. On the other hand, the experiment was conducted using a polymer nanodisk, which replaces the membrane scaffold protein of a nanodisc using a generally known membrane scaffold protein (MSP) with an amphiphilic polymer component. It was developed to improve structural stability, improve antiviral activity, and improve manufacturing process, and the antiviral characteristics shown in nanodiscs using amphipathic polymer components can be equally applied to nanodiscs using membrane structured protein (MSP). there is.
나노디스크의 항바이러스 효능은 지질 또는 상기 지질에 부착된 물질이 바이러스와 부착하는 것을 통해 나타내는데, 막구조화 단백질은 나노디스크에서 지질 조성을 원반형태로 붙잡아주는 역할을 하고 (도 1 참조), 상기와 막구조화 단백질을 폴리머 성분으로 대체하여도, 별다른 사정이 없는 이상 지질 또는 상기 지질에 부착된 물질을 방해하지 않기 때문이다.The antiviral efficacy of nanodiscs is shown through the attachment of lipids or substances attached to the lipids to viruses. Membrane-structured proteins play a role in holding the lipid composition in the nanodiscs in a disc shape (see FIG. 1), This is because, even if the structural protein is replaced with a polymer component, abnormal lipids or substances attached to the lipids are not disturbed without any special circumstances.
3-1) 나노디스크의 항바이러스 효능 도출 실험 방법3-1) Test method for deriving antiviral efficacy of nanodiscs
나노디스크의 항바이러스 효능은 37℃ 온도 조건에서 Microneutralization assay를 수행하여 도출하였다. 구체적으로, 폴리머 나노디스크 농축액을 인지질 기준 최고 농도 30 μM부터 희석하여 다양한 농도로 인플루엔자 바이러스(A/Puerto Rico/8/34)와 1시간 동안 처리하였다. 96웰 플레이트에 2x104 cells/100 μl로 시딩하여 90% 밀집도(confluency) 상태인 MDCK(Madin-Darby canine kidney cells) 세포에, 1시간 동안 반응시킨 '처리 물질 + 바이러스 혼합액'을 처리하였다. 24시간 후, 0.5 μM의 농도로 4-MUNANA(4-Methylumbelliferyl-N-acetyl-α-D-Neuraminic Acid)를 처리하고 37℃ 온도 조건에서 1시간 동안 반응시켰다. 이후 355/460 nm의 형광 값을 측정하여 바이러스의 NA(neuraminidase) 활성을 정량 분석해 항바이러스 효능을 도출하였다.The antiviral efficacy of the nanodisc was derived by performing a Microneutralization assay at 37°C. Specifically, the polymer nanodisc concentrate was diluted from a maximum concentration of 30 μM based on phospholipids and treated with influenza virus (A/Puerto Rico/8/34) at various concentrations for 1 hour. Madin-Darby canine kidney cells (MDCK) cells at 90% confluency by seeding at 2x10 4 cells/100 μl in a 96-well plate were treated with 'processing material + virus mixture' reacted for 1 hour. After 24 hours, 4-MUNANA (4-Methylumbelliferyl-N-acetyl-α-D-Neuraminic Acid) was treated at a concentration of 0.5 μM and reacted at 37° C. for 1 hour. Thereafter, by measuring fluorescence values at 355/460 nm, NA (neuraminidase) activity of the virus was quantitatively analyzed to derive antiviral efficacy.
3-2) 인지질 종류 변화에 따른 나노디스크의 항바이러스 효능 비교3-2) Comparison of antiviral efficacy of nanodiscs according to changes in phospholipid types
상기 실시예 1에서 제조한 SMA (2:1)-ND (POPC), SMA (2:1)-ND (POPE), SMA (3:1)-ND (POPC), SMA (3:1)-ND (POPE)의 항바이러스 효능을 비교하였다 (도 4).SMA (2: 1) -ND (POPC), SMA (2: 1) -ND (POPE), SMA (3: 1) -ND (POPC), SMA (3: 1) - prepared in Example 1 The antiviral efficacy of ND (POPE) was compared (FIG. 4).
이를 통해, SMA 2:1 기반 나노디스크의 경우 POPE를 사용한 경우, POPC를 사용한 실험군과 비교하여 EC50(해당 약물이 나타낼 수 있는 최대 효과의 절반 정도를 나타내는 약물의 최대 농도)이 대략 15 μM에서 4 μM로 감소하였고, SMA 3:1 기반 나노디스크의 경우, EC50이 대략 10 μM에서 0.5 μM로 20배 감소한 것을 확인할 수 있었다. 즉, 폴리머 성분의 조성변화에 상관없이 중성 인지질인 POPC 나노디스크에 비해 PE(phosphatidylethanolamine)가 포함된 나노디스크 실험군의 항바이러스 효능이 더욱 뛰어난 것을 확인할 수 있었다.As a result, in the case of SMA 2:1-based nanodiscs, when POPE was used, compared to the experimental group using POPC, the EC50 (maximum concentration of a drug that exhibits about half of the maximum effect that the drug can exhibit) was approximately 4 at 15 μM. μM, and in the case of the SMA 3:1 based nanodisc, it was confirmed that the EC50 decreased 20 times from approximately 10 μM to 0.5 μM. That is, it was confirmed that the antiviral efficacy of the nanodisk experimental group containing PE (phosphatidylethanolamine) was more excellent than that of the POPC nanodisc, which is a neutral phospholipid, regardless of the compositional change of the polymer component.
또한, 상기 실시예 1에서 제조한 SMA (3:1)-ND (POPE), SMA (3:1)-ND (DPPE), SMA (3:1)-ND (POPC)의 항바이러스 효능을 비교하였다 (도 5).In addition, the antiviral efficacy of SMA (3: 1) -ND (POPE), SMA (3: 1) -ND (DPPE), and SMA (3: 1) -ND (POPC) prepared in Example 1 was compared. (FIG. 5).
이를 통해, POPC 실험군의 경우 EC50이 대략 5 μM인데 반해, POPE 실험군의 경우 0.02 μM (POPC에 비해 250배 감소), DPPE의 경우 대략 0.1 μM (POPC에 비해50배 감소)가 나타나는 것을 확인할 수 있었다. 즉, 중성 인지질인 POPC 실험군에 비해 PE(phosphatidylethanolamine)가 포함된 POPE와 DPPE 실험군에서 우수한 항바이러스 효능이 나타냄을 확인할 수 있었고, 특히, PE(phosphatidylethanolamine) 인지질 중에서도 특히, POPE를 사용한 나노디스크가 더욱 우수한 항바이러스 효능이 나타냄을 확인할 수 있었다. Through this, in the case of the POPC experimental group, the EC50 was approximately 5 μM, whereas in the case of the POPE experimental group, it was 0.02 μM (250-fold decrease compared to POPC) and about 0.1 μM (50-fold decrease compared to POPC) in the case of DPPE. . That is, it was confirmed that the POPE and DPPE test groups containing PE (phosphatidylethanolamine) showed superior antiviral efficacy compared to the POPC test group, which is a neutral phospholipid. It was confirmed that the antiviral effect was shown.
Claims (8)
상기 지질 이중층의, '소수성기가 외부로 노출되어 있는 측면'을 소수성 결합으로 둘러싸는 '막구조화 단백질 (membrane scaffold protein) 또는 양친매성 폴리머 (amphipathic polymer)'를 포함하는 나노디스크(nanodisc)를 함유하는 바이러스 감염증 예방 또는 치료용 약학 조성물.
A lipid bilayer structure in the form of a flat disk formed using phospholipid phosphatidylethanolamine, wherein the hydrophilic group is oriented outwardly and the hydrophobic group is oriented inwardly; and
Containing a nanodisc containing a 'membrane scaffold protein or an amphipathic polymer' surrounding the 'side of the lipid bilayer where the hydrophobic group is exposed to the outside' with hydrophobic bonds A pharmaceutical composition for preventing or treating a viral infection.
상기 포스파티딜에탄올아민(phosphatidylethanolamine)은,
1-팔미토일-2-올레오일-sn-글리세로-3-포스포콜린(1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, POPE)인 것을 특징으로 하는 바이러스 감염증 예방 또는 치료용 약학 조성물.
According to claim 1,
The phosphatidylethanolamine (phosphatidylethanolamine),
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine, POPE) for preventing or treating viral infections pharmaceutical composition.
상기 막구조화 단백질 (membrane scaffold protein)은,
헬릭스(helix) 구조를 갖는 양친매성 단백질인 것을 특징으로 하는 바이러스 감염증 예방 또는 치료용 약학 조성물.
According to claim 1,
The membrane structure protein (membrane scaffold protein),
A pharmaceutical composition for preventing or treating viral infection, characterized in that it is an amphiphilic protein having a helix structure.
상기 막구조화 단백질 (membrane scaffold protein)은,
아포리포단백질(apolipoprotein) 또는 아포리포단백질의 '헬릭스(helix) 구조 및 양친매성 특성'이 유지된 아포리포단백질의 절편인 것을 특징으로 하는 바이러스 감염증 예방 또는 치료용 약학 조성물.
According to claim 3,
The membrane structure protein (membrane scaffold protein),
A pharmaceutical composition for preventing or treating viral infections, characterized in that it is a fragment of apolipoprotein or an apolipoprotein in which the 'helix structure and amphipathic characteristics' of the apolipoprotein are maintained.
상기 양친매성 폴리머 (amphipathic polymer)는,
스티렌-말레산(Styrene-Maleic Acid, SMA), 디-이소부틸렌-말레산(Di-IsoButylene-Maleic Acid, DIBMA), 스티렌-말레이미드(Styrene-Maleimide, SMI), 폴리메타크릴레이트(Polymethyl Methacrylate, PMA) 중 선택되는 어느 하나 이상인 것을 특징으로 하는 바이러스 감염증 예방 또는 치료용 약학 조성물.
According to claim 1,
The amphipathic polymer,
Styrene-Maleic Acid (SMA), Di-Isobutylene-Maleic Acid (DIBMA), Styrene-Maleimide (SMI), Polymethyl A pharmaceutical composition for preventing or treating viral infections, characterized in that at least one selected from methacrylate, PMA).
상기 나노디스크(nanodisc)는,
상기 지질 이중층 내부와 소수성 결합되는 '바이러스 리셉터 (virus receptor)'를 더욱 포함하는 것을 특징으로 하는 바이러스 감염증 예방 또는 치료용 약학 조성물.
According to claim 1,
The nanodisc,
A pharmaceutical composition for preventing or treating viral infection, characterized in that it further comprises a 'virus receptor' that is hydrophobically bound to the inside of the lipid bilayer.
상기 '바이러스 리셉터 (virus receptor)'는,
안지오텐진 전환효소 2 또는 '시알산(sialic acid)이 말단에 결합된 화합물'인 것을 특징으로 하는 바이러스 감염증 예방 또는 치료용 약학 조성물.
According to claim 6,
The 'virus receptor',
A pharmaceutical composition for preventing or treating a viral infection, characterized in that it is angiotensin converting enzyme 2 or 'a compound having sialic acid bound to its terminal'.
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