KR20240083166A - Biodegradable lipid nanoparticles for targeted delivery to the lungs and their use thereof - Google Patents
Biodegradable lipid nanoparticles for targeted delivery to the lungs and their use thereof Download PDFInfo
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- KR20240083166A KR20240083166A KR1020230145077A KR20230145077A KR20240083166A KR 20240083166 A KR20240083166 A KR 20240083166A KR 1020230145077 A KR1020230145077 A KR 1020230145077A KR 20230145077 A KR20230145077 A KR 20230145077A KR 20240083166 A KR20240083166 A KR 20240083166A
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Abstract
본 발명은 약물의 경폐 전달용 조성물 및 이의 용도에 관한 것으로, 본 발명의 이온화 가능 지질 및 양이온성 지질을 포함하는 지질나노입자는 폐 조직 및 이의 특정 세포에 특이적으로 전달되고 생체 친화도가 우수하며, 고효율로 음이온성 약물을 전달할 수 있어 지질나노입자 매개 유전자 치료 등 관련 기술분야에 유용하게 활용될 수 있다.The present invention relates to a composition for transpulmonary delivery of drugs and its use. Lipid nanoparticles containing ionizable lipids and cationic lipids of the present invention are specifically delivered to lung tissue and its specific cells and have excellent biocompatibility. Since it can deliver anionic drugs with high efficiency, it can be usefully used in related technical fields such as lipid nanoparticle-mediated gene therapy.
Description
본 발명은 특정 구조의 이온화 가능 지질 및 양이온성 지질을 포함하는 지질나노입자를 포함하는, 약물의 경폐 전달용 조성물 및 이의 폐 질환의 예방 또는 치료 용도에 관한 것이다.The present invention relates to a composition for transpulmonary delivery of a drug, comprising lipid nanoparticles containing ionizable lipids and cationic lipids of a specific structure, and its use in the prevention or treatment of lung diseases.
Antisense RNA, mRNA 등의 핵산은 생체 내에서 특정 단백질의 발현을 억제할 수 있는 물질로, 암, 유전병, 감염질병, 자가면역 질환 등의 치료에 중요한 도구로 각광받고 있다 (Novina and Sharp, Nature, 430, 161-164, 2004). 그러나 핵산은 세포 내로 직접 전달이 어렵고, 혈액 내에서 효소에 의해 쉽게 분해되므로 이를 극복하기 위한 연구들이 많이 진행되고 있다.Nucleic acids such as antisense RNA and mRNA are substances that can inhibit the expression of specific proteins in vivo, and are attracting attention as important tools in the treatment of cancer, genetic diseases, infectious diseases, and autoimmune diseases (Novina and Sharp, Nature, 430, 161-164, 2004). However, since nucleic acids are difficult to transfer directly into cells and are easily decomposed by enzymes in the blood, many studies are being conducted to overcome this problem.
약물전달시스템 (DDS; Drug Delivery System)은 약물의 부작용을 줄이고 효능 및 효과를 극대화시켜 필요한 양의 약물을 효율적으로 전달할 수 있도록 설계한 기술이다. 특히, 유전자 치료에 있어서 약물 전달체로는 종래 바이러스 전달체가 효과적임이 입증되었으나, 면역원성 (immunogenicity), 주입된 DNA 크기의 한계 및 대량생산의 어려움과 같은 여러 결점으로 인해 유전자 전달시스템으로서 바이러스의 이용이 제한되고 있다.Drug Delivery System (DDS) is a technology designed to efficiently deliver the required amount of drugs by reducing the side effects of drugs and maximizing their efficacy and effectiveness. In particular, conventional viral carriers have proven to be effective as drug carriers in gene therapy, but the use of viruses as a gene delivery system has been discouraged due to several drawbacks such as immunogenicity, limitations in the size of the injected DNA, and difficulties in mass production. It is being restricted.
이에, 바이러스성 시스템의 대체 수단으로 핵산을 세포 내로 운반하는 방법으로는 현재까지 양전하 지질 또는 중합체와 섞어 운반하는 방법 (각각, 지질-DNA 접합체 (lipoplex) 및 폴리머-DNA 접합체 (polyplex)라 명명됨)이 주로 사용되고 있다 (Hirko et al., Curr, Med, Chem., 10, 1185-1193, 2003; Merdan et al., Adv. Drug. Deliv.Rev., 54, 715-758, 2002; Spagnou et al., Biochemistry, 43, 13348-13386, 2004). 특히, 지질-DNA 접합체는 핵산과 결합하여 세포 내로 핵산을 잘 전달시켜 세포수준에서 많이 사용되고 있으나, 생체 내에서는 국부적으로 주사 시 많은 경우 체내에서 염증을 유발시키며 (Filonand and Phillips, Biochim. Biophys/Acta, 1329, 345-356, 1997), 혈관 내 주사 시 주로 1차 통과기관들인 폐, 간, 비장 등과 같은 조직에 축적되는 단점이 있다 (Ren et al., Gene Therapy. 7, 764-768, 2000).Accordingly, as an alternative to the viral system, the method of transporting nucleic acids into cells is currently mixing them with positively charged lipids or polymers (named lipid-DNA conjugates (lipoplex) and polymer-DNA conjugates (polyplex), respectively). ) is mainly used (Hirko et al., Curr, Med, Chem., 10, 1185-1193, 2003; Merdan et al., Adv. Drug. Deliv.Rev., 54, 715-758, 2002; Spagnou et al. al., Biochemistry, 43, 13348-13386, 2004). In particular, lipid-DNA conjugates are widely used at the cellular level because they bind to nucleic acids and deliver nucleic acids well into cells, but when injected locally in vivo, they often cause inflammation in the body (Filonand and Phillips, Biochim. Biophys/Acta , 1329, 345-356, 1997), it has the disadvantage of accumulating in tissues such as the lung, liver, and spleen, which are the first passage organs during intravascular injection (Ren et al., Gene Therapy. 7, 764-768, 2000 ).
이러한 배경 하에서 본 발명자들은 폐 조직 특이적으로 핵산 등의 음이온성 약물을 효과적으로 전달할 수 있는 전달체를 개발하고자 예의 노력한 결과, 특정한 구조를 갖는 이온화 가능 지질에 추가로 양이온성 지질을 포함하는 지질나노입자 제형이 우수한 폐 특이적 약물 전달 효능을 가짐을 확인함으로써 본 발명을 완성하였다.Under this background, the present inventors have made diligent efforts to develop a carrier that can effectively deliver anionic drugs such as nucleic acids specifically to lung tissue. As a result, a lipid nanoparticle formulation containing cationic lipids in addition to ionizable lipids with a specific structure has been developed. The present invention was completed by confirming that it has excellent lung-specific drug delivery efficacy.
본 발명의 하나의 목적은, 특정 구조를 갖는 이온화 가능 지질 및 양이온성 지질을 포함하는 지질나노입자를 포함하는, 약물의 경폐 전달용 조성물을 제공하는 것이다.One object of the present invention is to provide a composition for transpulmonary delivery of a drug, comprising lipid nanoparticles containing ionizable lipids and cationic lipids having a specific structure.
본 발명의 다른 하나의 목적은, 상기 지질나노입자 및 음이온성 약물을 포함하는, 폐 질환의 예방 또는 치료용 약학적 조성물을 제공하는 것이다.Another object of the present invention is to provide a pharmaceutical composition for preventing or treating lung diseases, comprising the lipid nanoparticles and anionic drugs.
이를 구체적으로 설명하면 다음과 같다. 한편, 본 발명에서 개시된 각각의 설명 및 실시형태는 각각의 다른 설명 및 실시형태에도 적용될 수 있다. 즉, 본 발명에서 개시된 다양한 요소들의 모든 조합이 본 발명의 범주에 속한다. 또한, 하기 기술된 구체적인 서술에 의하여 본 발명의 범주가 제한된다고 볼 수 없다.This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention can also be applied to each other description and embodiment. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Additionally, the scope of the present invention cannot be considered limited by the specific description described below.
상기 목적을 달성하기 위한 본 발명의 하나의 양태는, 하기 화학식 1로 표시되는 이온화 가능 지질 (ionizable lipid) 및 양이온성 지질 (cationic lipid)을 포함하는 지질나노입자 (Lipid nanoparticle, LNP)를 포함하는, 약물의 경폐 전달용 조성물이다.One aspect of the present invention for achieving the above object includes lipid nanoparticles (LNPs) containing ionizable lipids and cationic lipids represented by the following formula (1): , a composition for transpulmonary delivery of drugs.
[화학식 1][Formula 1]
본 발명에서 "이온화 가능 지질 (ionizable lipid)"은 용이하게 양성자화될 수 있는 아민-함유 지질을 의미하며, 지질 유사체 (lipidoid)로도 명명된다. 상기 이온화 가능 지질은, 주변 pH에 따라 전하상태가 변할 수 있으므로, 음이온성 약물과 정전기적 상호작용을 통하여 상기 약물이 지질나노입자 내에 높은 효율로 봉입되도록 하는 역할을 수행하며, 지질나노입자의 구조를 형성하는데 기여한다. 본 발명의 지질나노입자는 상기 화학식 1의 이온화 가능 지질을 포함하는 것을 하나의 특징으로 한다.In the present invention, “ionizable lipid” refers to an amine-containing lipid that can be easily protonated, and is also called a lipid analog (lipidoid). Since the charge state of the ionizable lipid can change depending on the surrounding pH, it plays a role in ensuring that the drug is encapsulated within the lipid nanoparticle with high efficiency through electrostatic interaction with the anionic drug, and the structure of the lipid nanoparticle contributes to forming One feature of the lipid nanoparticles of the present invention is that they contain an ionizable lipid of the above formula (1).
상기 화학식 1의 이온화 가능 지질은 복수 개의 3차 아민을 갖는 헤드 그룹과 에스터 작용기 및 탄소 이중결합을 포함하는 사슬 꼬리를 갖는 물질로서, 본 발명에서 "244-cis"로 명명된다. 본 발명자들은 국제공개공보 WO2023/136689에서, 244-cis 또는 이와 유사한 구조를 갖는 이온화 가능 지질은 지질나노입자의 일 구성으로 사용될 수 있고, 상기 지질나노입자는 mRNA와 같은 음이온성 약물 전달에 매우 우수한 효과가 있음을 보고한 바 있다. 그러나 본 발명자들은 상기 이온화 가능 지질이 놀랍게도 이와 조합되는 헬퍼 지질 (helper lipid)의 종류에 따라 표적 조직이 달라지며, 특히 양이온성 지질을 헬퍼 지질로 사용하여 지질나노입자를 제조하였을 때 폐 특이적으로 전달되는 것을 확인하였다. 또한, 본 발명의 지질나노입자는 면역원성이 거의 없이 mRNA를 안정적으로 전달하여 표적 부위에서 높은 수준으로 발현되는 것을 확인하였다.The ionizable lipid of Formula 1 is a substance having a head group having a plurality of tertiary amines, an ester functional group, and a chain tail containing a carbon double bond, and is named “244-cis” in the present invention. In International Publication WO2023/136689, the present inventors found that ionizable lipids with 244-cis or similar structures can be used as a component of lipid nanoparticles, and that the lipid nanoparticles are excellent for delivering anionic drugs such as mRNA. It has been reported that it is effective. However, the present inventors found that the target tissue of the ionizable lipid surprisingly varies depending on the type of helper lipid with which it is combined, and in particular, when lipid nanoparticles are manufactured using cationic lipids as helper lipids, the target tissue is lung-specific. Delivery was confirmed. In addition, it was confirmed that the lipid nanoparticles of the present invention stably delivered mRNA with little immunogenicity and were expressed at a high level at the target site.
따라서, 본 발명은 화학식 1로 표시되는 이온화 가능 지질, 즉 244-cis 뿐만 아니라 이와 유사한 구조를 갖는 이온화 가능 지질에도 마찬가지로 적용될 수 있다. 당업자는 화학식 1의 이온화 가능 지질의 범위를 넘어, 예컨대 WO2023/136689에 기재된 다른 유사 구조의 이온화 가능 지질의 경우에도 본 발명에서 확인한 것과 마찬가지의 효과를 보여줄 수 있음을 충분히 기대할 수 있다. 그러므로, 상기 244-cis와 유사 구조의 이온화 가능 지질 역시 모두 균등 범위로서 본 발명의 범주 내로 포함될 수 있다.Therefore, the present invention can be applied not only to the ionizable lipid represented by Formula 1, that is, 244-cis, but also to the ionizable lipid having a similar structure. Those skilled in the art can fully expect that beyond the scope of the ionizable lipid of Formula 1, for example, other ionizable lipids with similar structures described in WO2023/136689 can show the same effects as those confirmed in the present invention. Therefore, all ionizable lipids with structures similar to 244-cis can also be included within the scope of the present invention as equivalents.
이에 제한되는 것은 아니나, 본 발명의 이온화 가능 지질은 하기 화학식 2로 표시되는 화합물일 수 있다.Although not limited thereto, the ionizable lipid of the present invention may be a compound represented by the following formula (2).
[화학식 2][Formula 2]
여기서, A는 
R1은 -C2-3알킬-NR2R3 또는 -C4-8알킬-(C=O)-Y-(CH2)xCH=CH-C5-6알킬이고,R 1 is -C 2-3 alkyl-NR 2 R 3 or -C 4-8 alkyl-(C=O)-Y-(CH 2 ) x CH=CH-C 5-6 alkyl,
R2 및 R3는 각각 독립적으로 -H, -C1-6알킬, 또는 -C4-8알킬-(C=O)-Y-(CH2)xCH=CH-C5-6알킬로부터 선택된 어느 하나이고,R 2 and R 3 are each independently selected from -H, -C 1-6 alkyl, or -C 4-8 alkyl-(C=O)-Y-(CH 2 ) x CH=CH-C 5-6 alkyl Which one is chosen,
Y는 -O- 또는 -NH-이고,Y is -O- or -NH-,
m은 1 내지 2의 정수이고,m is an integer from 1 to 2,
n은 3 내지 7의 정수이고,n is an integer from 3 to 7,
x는 1 내지 3의 정수이고,x is an integer from 1 to 3,
l은 3 내지 4의 정수이고, 및l is an integer from 3 to 4, and
p는 0 내지 2의 정수이다.p is an integer from 0 to 2.
또한, 본 발명의 구체예에 따르면, 상기 화학식 2로 표시되는 화합물은 하기 표 1에 기재된 화합물로 이루어진 군으로부터 선택된 것일 수 있다.Additionally, according to an embodiment of the present invention, the compound represented by Formula 2 may be selected from the group consisting of compounds shown in Table 1 below.
1
One
2
2
3
3
4
4
5
5
6
6
7
7
8
8
본 발명에 있어서, 용어 "알킬"은 다른 기재가 없는 한, 직쇄 또는 분지쇄의 비고리형 포화 탄화수소를 의미한다. 예를 들어, "C1-6알킬"은 탄소원자를 1 내지 6개 포함하는 알킬을 의미할 수 있다. 본 발명의 알킬 구조에서 단순한 치환기의 부가 등을 한 경우라도, 본 발명의 이온화 가능 지질과 동등한 효과를 가지는 한 모두 균등 범위로 본 발명의 범주에 포함된다.In the present invention, the term “alkyl” refers to a straight-chain or branched-chain acyclic saturated hydrocarbon, unless otherwise specified. For example, “C 1-6 alkyl” may mean alkyl containing 1 to 6 carbon atoms. Even in the case where a simple substituent is added to the alkyl structure of the present invention, as long as it has the same effect as the ionizable lipid of the present invention, it is included in the scope of the present invention to the extent of equivalents.
본 발명의 지질나노입자는 양이온성 지질 (cationic lipid)을 포함하는 것을 다른 하나의 특징으로 한다. 상기 양이온성 지질은 지질나노입자에서 헬퍼 지질 (helper lipid)에 해당하며 본 발명의 이온화 가능 지질과 함께 음이온성 약물이 폐 특이적으로 전달되도록 하는 역할을 수행한다.Another feature of the lipid nanoparticles of the present invention is that they contain cationic lipids. The cationic lipid corresponds to a helper lipid in lipid nanoparticles and, together with the ionizable lipid of the present invention, plays a role in delivering anionic drugs specifically to the lungs.
본 발명에 있어서, 용어 "헬퍼 지질 (helper lipid)"은 지질나노입자 내에서 이온화 가능 지질 및 약물이 상호 작용하여 형성된 코어를 감싸서 보호하는 역할을 수행하며, 타겟 세포의 인지질 이중층과 결합하여 약물의 세포 내 전달 시 세포막 통과 및 엔도좀 탈출 (endosomal escape)을 용이하게 한다. In the present invention, the term "helper lipid" serves to surround and protect the core formed by the interaction of ionizable lipids and drugs within lipid nanoparticles, and binds to the phospholipid bilayer of the target cell to protect the drug. When delivered intracellularly, it facilitates cell membrane passage and endosomal escape.
본 발명에 있어서, "양이온성 지질"은 DOTAP (1,2-dioleoyl-3-trimethylammonium propane) 또는 포스파티딜콜린 (phosphatidylcholine)일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the “cationic lipid” may be DOTAP (1,2-dioleoyl-3-trimethylammonium propane) or phosphatidylcholine, but is not limited thereto.
본 발명에 있어서, 용어 "포스파티딜콜린 (phosphatidylcholine)"은 인지질의 일종으로 헤드 그룹으로 콜린 (choline)이 포함된 화합물을 말한다. 동물, 식물, 효모, 및 곰팡이류에 널리 존재하며, 레시틴이라고도 한다. 포유동물의 막구성 인지질로서 주로 뇌수, 신경, 혈구, 난황 등에 들어 있다. 식물에서는 대두, 해바라기씨, 밀배아 등에 함유되어 있다. 일반적으로 글리세롤의 1번 위치에는 포화지방산, 2번 위치에는 불포화지방산이 결합되어 있는 것이 많다. 본 발명에서 포스파티딜콜린은 여러 구조의 포스파티딜콜린의 혼합물일 수 있고, 천연물 (예컨대, 난황, 대두 등)에서 분리된 것, 또는 화학적으로 합성된 것일 수 있다.In the present invention, the term "phosphatidylcholine" refers to a type of phospholipid and a compound containing choline as a head group. It is widely present in animals, plants, yeast, and molds, and is also called lecithin. It is a membrane phospholipid of mammals and is mainly contained in brain water, nerves, blood cells, and egg yolk. In plants, it is contained in soybeans, sunflower seeds, and wheat germ. In general, glycerol often has a saturated fatty acid bound to the 1st position and an unsaturated fatty acid bound to the 2nd position. In the present invention, phosphatidylcholine may be a mixture of phosphatidylcholine of various structures, may be isolated from natural products (e.g., egg yolk, soybeans, etc.), or may be chemically synthesized.
구체적인 하나의 양태로서, 본 발명의 지질나노입자는 DOTAP을 지질나노입자 전체를 기준으로 10 mol% 내지 70 mol%로 포함하는 것일 수 있고, 예를 들어 15 mol% 내지 70 mol%, 20 mol% 내지 70 mol%, 15 mol% 내지 65 mol%, 20 mol% 내지 65 mol%, 20 mol% 내지 60 mol%, 또는 20 mol% 내지 50 mol%일 수 있으나, 이에 제한되는 것은 아니다.In one specific embodiment, the lipid nanoparticles of the present invention may contain DOTAP in an amount of 10 mol% to 70 mol% based on the entire lipid nanoparticle, for example, 15 mol% to 70 mol%, 20 mol%. It may be from 70 mol%, 15 mol% to 65 mol%, 20 mol% to 65 mol%, 20 mol% to 60 mol%, or 20 mol% to 50 mol%, but is not limited thereto.
다른 구체적인 하나의 양태로서, 본 발명의 지질나노입자는 포스파티딜콜린을 지질나노입자 전체를 기준으로 10 mol% 내지 50 mol%, 15 mol% 내지 45 mol% 또는 20 mol% 내지 60 mol%로 포함하는 것일 수 있으나, 이에 제한되는 것은 아니다.In another specific embodiment, the lipid nanoparticles of the present invention contain 10 mol% to 50 mol%, 15 mol% to 45 mol%, or 20 mol% to 60 mol% based on the total lipid nanoparticles. However, it is not limited to this.
본 발명의 구체적인 실시예에서는 244-cis 이온화 가능 지질을 이용하여 지질나노입자를 제조하였을 때, 일반적으로는 거의 대부분 간으로 전달되지만 (도 4), DOTAP이나 포스파티딜콜린과 같은 양이온성 지질을 포함하였을 때에는 폐 특이적으로 전달되어 mRNA가 발현되는 것을 확인하였다 (도 5 내지 도 11). 따라서 본 발명의 화학식 1 (또는 화학식 2)로 표시되는 이온화 가능 지질 및 양이온성 지질을 포함하는 지질나노입자는 폐 조직에 특이적으로 전달되며, 이에 따라 높은 효율로 음이온성 약물을 폐에 특이적으로 전달할 수 있다.In a specific example of the present invention, when lipid nanoparticles are prepared using 244-cis ionizable lipids, they are generally mostly delivered to the liver (Figure 4), but when they contain cationic lipids such as DOTAP or phosphatidylcholine, It was confirmed that mRNA was expressed through lung-specific delivery (Figures 5 to 11). Therefore, lipid nanoparticles containing ionizable lipids and cationic lipids represented by Formula 1 (or Formula 2) of the present invention are specifically delivered to lung tissue, and thus deliver anionic drugs specifically to the lungs with high efficiency. It can be passed on.
본 발명의 지질나노입자는 구조적 지질 또는 PEG-지질을 더 포함할 수 있다.The lipid nanoparticles of the present invention may further include structural lipids or PEG-lipids.
상기 구조적 지질은 지질나노입자 내에서 입자 모양을 유지하고 나노입자의 코어 및 표면에 분산되어 나노입자의 안정성을 향상시키는 역할을 한다. 상기 구조적 지질은 예컨대, 콜레스테롤, 콜레스테놀, 스피나스테롤, 페코스테롤, 시토스테롤, 에르고스테롤, 에르고스테놀, 캄페스테롤, 스티그마스테롤, 브라시카스테롤, 토마티딘, 우르솔산, 알파-토코페롤 또는 이들의 혼합물일 수 있으나, 이에 제한되는 것은 아니다.The structural lipid maintains the particle shape within the lipid nanoparticle and is dispersed on the core and surface of the nanoparticle to improve the stability of the nanoparticle. The structural lipid is, for example, cholesterol, cholesterol, spinasterol, fecosterol, sitosterol, ergosterol, ergostenol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol or these. It may be a mixture of, but is not limited to this.
상기 PEG-지질은 지질과 PEG가 컨쥬게이트된 형태를 지칭하는 것으로, 친수성 중합체인 폴리에틸렌글리콜 중합체가 결합된 지질을 의미한다. 상기 PEG-지질은 지질나노입자 내에서 나노입자의 혈청 내 입자 안정성에 기여하며, 나노입자 간 응집을 막는 역할을 수행한다. 또한, PEG-지질은 분해효소로부터 핵산을 보호하여 핵산의 체내 안정성을 강화시키며, 나노입자 내 봉입된 약물의 반감기를 증가시킬 수 있다. 상기 PEG-지질은 예컨대, PEG-세라마이드, PEG-DMG, PEG-c-DOMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE 또는 이들의 혼합물일 수 있고, 구체적으로 C16-PEG2000 세라마이드이나, 이에 제한되는 것은 아니다.The PEG-lipid refers to a conjugated form of lipid and PEG, and refers to a lipid to which polyethylene glycol polymer, a hydrophilic polymer, is bound. The PEG-lipid within lipid nanoparticles contributes to particle stability in serum and plays a role in preventing aggregation between nanoparticles. In addition, PEG-lipids protect nucleic acids from degrading enzymes, enhance the stability of nucleic acids in the body, and can increase the half-life of drugs encapsulated in nanoparticles. The PEG-lipid may be, for example, PEG-ceramide, PEG-DMG, PEG-c-DOMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE or a mixture thereof, specifically C16-PEG2000 ceramide. However, it is not limited to this.
구체적인 하나의 양태로서, 본 발명의 이온화 가능 지질과 DOTAP, 구조적 지질 및 PEG-지질을 혼합하여 지질나노입자를 제조할 경우, 이온화 가능 지질 : DOTAP : 구조적 지질 : PEG-지질의 몰비는 10 내지 40 : 5 내지 80 : 5 내지 80 : 1 내지 5일 수 있다. 더 구체적으로, 상기 몰비는 20 내지 30 : 10 내지 70 : 10 내지 70 : 1 내지 5일 수 있으며, 더 구체적으로는 20 내지 30 : 15 내지 70 : 15 내지 70 : 1 내지 3일 수 있으나, 이에 제한되지 않는다.In one specific embodiment, when lipid nanoparticles are prepared by mixing the ionizable lipid of the present invention with DOTAP, structural lipid, and PEG-lipid, the molar ratio of ionizable lipid: DOTAP: structural lipid: PEG-lipid is 10 to 40. : 5 to 80 : 5 to 80 : 1 to 5. More specifically, the molar ratio may be 20 to 30:10 to 70:10 to 70:1 to 5, and more specifically, 20 to 30:15 to 70:15 to 70:1 to 3. Not limited.
다른 구체적인 하나의 양태로서, 본 발명의 이온화 가능 지질과 포스파티딜콜린, 구조적 지질 및 PEG-지질을 혼합하여 지질나노입자를 제조할 경우, 이온화 가능 지질 : 포스파티딜콜린 : 구조적 지질 : PEG-지질의 몰비는 10 내지 40 : 5 내지 80 : 5 내지 80 : 1 내지 5일 수 있다. 더 구체적으로, 상기 몰비는 20 내지 30 : 10 내지 50 : 30 내지 60 : 1 내지 5일 수 있으며, 더 구체적으로는 20 내지 30 : 20 내지 40 : 30 내지 60 : 1 내지 3일 수 있으나, 이에 제한되지 않는다.In another specific embodiment, when lipid nanoparticles are prepared by mixing the ionizable lipid of the present invention with phosphatidylcholine, structural lipid, and PEG-lipid, the molar ratio of ionizable lipid: phosphatidylcholine: structural lipid: PEG-lipid is 10 to 10. It may be 40:5 to 80:5 to 80:1 to 5. More specifically, the molar ratio may be 20 to 30:10 to 50:30 to 60:1 to 5, and more specifically, 20 to 30:20 to 40:30 to 60:1 to 3. Not limited.
본 발명의 지질나노입자는 산성 pH 조건에서 양전하를 나타내므로 음전하를 나타내는 핵산, 약물 등의 치료제와 정전기적 상호작용을 통해 용이하게 약물과의 복합체를 형성하여 높은 효율로 음이온성 약물을 봉입할 수 있으며, 약물의 세포 내 또는 생체 내 약물 전달 조성물로서 사용될 수 있다. 따라서 본 발명의 지질나노입자는 핵산뿐만 아니라 음이온을 띄는 모든 형태의 약물의 전달에 유용하게 사용될 수 있다. 즉, 본 발명의 지질나노입자는 최종적으로 음이온성 약물을 추가로 포함하는 형태 (봉입된 형태)로 제조될 수 있고, 봉입된 음이온성 약물을 폐에 특이적으로 전달할 수 있다.Since the lipid nanoparticles of the present invention exhibit a positive charge under acidic pH conditions, they can easily form a complex with the drug through electrostatic interaction with therapeutic agents such as nucleic acids and drugs that exhibit a negative charge, enabling the encapsulation of anionic drugs with high efficiency. It can be used as an intracellular or in vivo drug delivery composition. Therefore, the lipid nanoparticles of the present invention can be useful for the delivery of not only nucleic acids but also all types of drugs with anionic properties. That is, the lipid nanoparticles of the present invention can ultimately be manufactured in a form (encapsulated form) that additionally contains an anionic drug, and the encapsulated anionic drug can be specifically delivered to the lungs.
본 발명에서, 용어 "봉입 (encapsulation)"은 전달물질을 둘러싸서 효율적으로 생체 내로 함입시키기 위해 캡슐화하는 것을 말하고, 약물 봉입률 (캡슐화 효율, Encapsulation efficiency)은 제조에 사용된 전체 약물 함량에 대하여 지질나노입자 내에 봉입된 약물의 함량을 의미한다.In the present invention, the term "encapsulation" refers to encapsulating the delivery material to surround it and efficiently incorporate it into the body, and the drug encapsulation efficiency (encapsulation efficiency) refers to the lipid content relative to the total drug content used in manufacturing. It refers to the content of drug encapsulated in nanoparticles.
상기 음이온성 약물은 핵산, 저분자 화합물, 펩타이드, 단백질, 단백질-핵산 구조체 또는 음이온성 생체고분자-약물 접합체 등일 수 있으나, 본 발명의 이온화 가능 지질과 함께 지질나노입자를 형성하여 안정적이고 효율적으로 전달될 수 있는 한 이에 제한되는 것은 아니다.The anionic drug may be a nucleic acid, low molecular weight compound, peptide, protein, protein-nucleic acid structure, or anionic biopolymer-drug conjugate, but may be stably and efficiently delivered by forming lipid nanoparticles together with the ionizable lipid of the present invention. It is not limited to this as long as possible.
본 발명에서 상기 핵산은 소간섭 리보핵산 (siRNA), 리보좀 리보핵산 (rRNA), 디옥시리보핵산 (DNA), 상보성 디옥시리보핵산 (cDNA), 앱타머 (aptamer), 전령 리보핵산 (mRNA), 운반 리보핵산 (tRNA), sgRNA, 안티센스 올리고뉴클레오티드, shRNA, miRNA, 리보자임 (ribozyme), PNA, 및 DNAzyme, 또는 이들의 혼합물일 수 있으나, 이에 제한되는 것은 아니다.In the present invention, the nucleic acids include small interfering ribonucleic acid (siRNA), ribosomal ribonucleic acid (rRNA), deoxyribonucleic acid (DNA), complementary deoxyribonucleic acid (cDNA), aptamer, messenger ribonucleic acid (mRNA), and transport ribonucleic acid. It may be (tRNA), sgRNA, antisense oligonucleotide, shRNA, miRNA, ribozyme, PNA, and DNAzyme, or a mixture thereof, but is not limited thereto.
상기 지질나노입자에서 총 지질/핵산의 중량비는 1 내지 30일 수 있으며, 구체적으로 5 내지 27, 보다 구체적으로 10 내지 23일 수 있으나, 이에 제한되는 것은 아니다.The weight ratio of total lipid/nucleic acid in the lipid nanoparticles may be 1 to 30, specifically 5 to 27, and more specifically 10 to 23, but is not limited thereto.
본 발명의 다른 하나의 양태는, 상기 화학식 1의 이온화 가능 지질 및 양이온성 지질을 포함하는 지질나노입자, 및 음이온성 약물을 유효성분으로 포함하는, 폐 질환의 예방 또는 치료용 약학적 조성물이다.Another aspect of the present invention is a pharmaceutical composition for preventing or treating lung disease, comprising lipid nanoparticles containing the ionizable lipid and cationic lipid of Formula 1 above, and an anionic drug as an active ingredient.
본 발명의 또 다른 하나의 양태는, 상기 조성물을 이를 필요로 하는 개체에 투여하는 단계를 포함하는, 폐 질환의 치료 방법이다.Another aspect of the present invention is a method of treating lung disease, comprising administering the composition to an individual in need thereof.
본 발명의 또 다른 하나의 양태는, 폐 질환의 예방 또는 치료에 사용하기 위한 약학적 조성물, 또는 폐 질환의 예방 또는 치료를 위한 상기 조성물의 용도이다.Another aspect of the present invention is a pharmaceutical composition for use in the prevention or treatment of lung disease, or use of the composition for the prevention or treatment of lung disease.
지질나노입자 및 음이온성 약물에 대해서는 상기 설명한 바와 같다.Lipid nanoparticles and anionic drugs are as described above.
본 발명의 지질나노입자는 핵산 등의 음이온성 약물과 안정된 복합체를 형성하고 낮은 세포독성 및 효과적인 세포 흡수성을 나타내므로, 음이온성 약물을 전달하는데 효과적이다. 또한, 상기 지질나노입자는 투여 시 폐에 특이적으로 전달될 수 있으므로, 폐 질환의 예방 또는 치료에 유용하게 활용될 수 있다.The lipid nanoparticles of the present invention form a stable complex with anionic drugs such as nucleic acids and exhibit low cytotoxicity and effective cell absorption, so they are effective in delivering anionic drugs. In addition, since the lipid nanoparticles can be specifically delivered to the lungs when administered, they can be useful in preventing or treating lung diseases.
본 발명의 지질나노입자는 폐를 표적으로 하여 약물을 특이적으로 전달할 수 있으며, 본 발명의 목적 상 사용되는 음이온성 약물의 종류, 핵산의 종류, 핵산 서열에 따라 다양한 폐 질환에 대한 예방 또는 치료 효과를 기대할 수 있다. 즉, 본 발명에서 폐 질환의 종류는 제한되지 않는다. 따라서, 상기 폐 질환은 예를 들어, 폐기종, 천식, 폐렴, 결핵, 폐 고혈압, 폐암, 신생아 기관지폐 형성 장애증, 만성 폐쇄성 폐질환, 급성 기관지염, 만성 기관지염, 세기관지염, 기관지 확장증, 과민증, 폐실질 염, 급성 연기 흡입, 열에 의한 폐 손상, 낭포성 섬유증, 허파꽈리 단백증, 알파-1-프로테아제 결핍, 폐 염증성 장애, 급성 호흡 곤란 증후군, 급성 폐 손상, 특발성 호흡 곤란 증후군 및 폐 섬유증으로 이루어진 군에서 선택된 1종 이상일 수 있으며, 구체적으로는 폐 섬유증일 수 있으나, 이에 제한되지 않는다.The lipid nanoparticles of the present invention can specifically deliver drugs by targeting the lungs, and for the purpose of the present invention, prevent or treat various lung diseases depending on the type of anionic drug, type of nucleic acid, and nucleic acid sequence used. Effects can be expected. That is, in the present invention, the type of lung disease is not limited. Therefore, the lung diseases include, for example, emphysema, asthma, pneumonia, tuberculosis, pulmonary hypertension, lung cancer, neonatal bronchopulmonary dysplasia, chronic obstructive pulmonary disease, acute bronchitis, chronic bronchitis, bronchiolitis, bronchiectasis, hypersensitivity, lung parenchyma. A group consisting of salts, acute smoke inhalation, heat-induced lung injury, cystic fibrosis, alveolar proteinosis, alpha-1-protease deficiency, pulmonary inflammatory disorders, acute respiratory distress syndrome, acute lung injury, idiopathic respiratory distress syndrome, and pulmonary fibrosis. It may be one or more types selected from, and specifically may be pulmonary fibrosis, but is not limited thereto.
본 발명에서 용어, "치료"는 질병을 갖는 개개인 또는 세포의 천연 과정을 변경시키기 위해 개입하는 것을 지칭하고, 이는 병리 상태가 진행되는 동안 또는 이를 예방하기 위해 수행될 수 있다. 목적하는 치료 효과에는 질병의 발생 또는 재발을 예방하고, 증상을 완화시키며, 질병에 따른 모든 직접 또는 간접적인 병리학적 결과를 저하시키며, 전이를 예방하고, 질병 진행 속도를 감소시키며, 질병 상태를 경감 또는 일시적 완화시키며, 차도시키거나 예후를 개선시키는 것이 포함된다. 특히, 본 발명에서는 이온화 가능 지질 및 양이온성 지질을 포함하는 지질나노입자 및 음이온성 약물을 유효성분으로 포함하는 조성물의 투여로 폐 관련 질환의 경과를 호전시키는 모든 행위를 포함한다. 또한, 용어 "예방"은 상기 지질나노입자의 투여로 질병의 발병을 억제 또는 지연시키는 모든 행위를 말한다. 본 발명의 지질나노입자가 치료 또는 예방 목적으로 사용될 경우, 개체에 치료학적으로 유효한 양으로 투여된다.In the present invention, the term “treatment” refers to intervention to alter the natural processes of an individual or cell with a disease, which may be performed during the progression of the pathology or to prevent it. The desired therapeutic effects include preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing all direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, and alleviating the disease state. Or it includes temporary relief, remission, or improvement of prognosis. In particular, the present invention includes all actions to improve the course of lung-related diseases by administering a composition containing lipid nanoparticles containing ionizable lipids and cationic lipids and anionic drugs as active ingredients. Additionally, the term “prevention” refers to all actions that suppress or delay the onset of a disease by administering the lipid nanoparticles. When the lipid nanoparticles of the present invention are used for treatment or prevention purposes, they are administered to an individual in a therapeutically effective amount.
본 발명에서 사용되는 "치료학적으로 유효한 양"이라는 용어는 음이온성 약물 함유 지질나노입자의 유효한 양을 나타낸다. 구체적으로, "치료학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 개체 종류 및 중증도, 연령, 성별, 질병의 종류, 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 약학적 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 시판되는 치료제와는 순차적으로 또는 동시에 투여될 수 있다. 그리고 단일 또는 다중 투여될 수 있다. 상기 요소를 모두 고려하여 부작용없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 당업자에 의해 용이하게 결정될 수 있다. 본 발명의 약학적 조성물의 투여 용량은 환자의 상태, 연령, 성별 및 합병증 등의 다양한 요인에 따라 전문가에 의해 결정될 수 있다. 본 발명의 조성물의 유효성분은 안전성이 우수하므로, 결정된 투여 용량 이상으로도 사용될 수 있다.The term “therapeutically effective amount” used in the present invention refers to an effective amount of anionic drug-containing lipid nanoparticles. Specifically, "therapeutically effective amount" means an amount sufficient to treat the disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type and severity of the individual, age, gender, type of disease, It can be determined based on factors including the activity of the drug, sensitivity to the drug, time of administration, route of administration and excretion rate, duration of treatment, drugs used simultaneously, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with commercially available therapeutic agents. And it can be administered single or multiple times. Considering all of the above factors, it is important to administer an amount that can achieve maximum effect with the minimum amount without side effects, and can be easily determined by a person skilled in the art. The administered dose of the pharmaceutical composition of the present invention may be determined by an expert depending on various factors such as the patient's condition, age, gender, and complications. Since the active ingredient of the composition of the present invention has excellent safety, it can be used beyond the determined administration dose.
상기 지질나노입자를 포함하는 조성물은 경구, 근육, 정맥, 동맥, 피하, 복강, 폐, 및 비강 주사 등으로 투여될 수 있으며, 구체적으로 정맥 투여될 수 있으나, 이에 제한되지 않는다.The composition containing the lipid nanoparticles may be administered orally, intramuscularly, intravenously, arterially, subcutaneously, intraperitoneally, pulmonaryly, and intranasally, and may specifically be administered intravenously, but is not limited thereto.
본 발명의 조성물은 투여를 위해서 추가로 약학적으로 허용가능한 담체를 1 종 이상 더 포함할 수 있다. 약학적으로 허용 가능한 담체는 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로즈 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 하나 이상을 혼합하여 사용할 수 있으며, 필요에 따라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다.The composition of the present invention may further include one or more pharmaceutically acceptable carriers for administration. Pharmaceutically acceptable carriers can be saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol, and a mixture of one or more of these ingredients, and if necessary, antioxidants, buffer solutions, Other common additives such as bacteriostatic agents can be added.
본 발명의 이온화 가능 지질 및 양이온성 지질을 포함하는 지질나노입자는 폐 조직에 특이적으로 전달되고 생체 친화도가 우수하며, 고효율로 음이온성 약물을 전달할 수 있어 지질나노입자 매개 유전자 치료 등 관련 기술분야에 유용하게 활용될 수 있다.Lipid nanoparticles containing ionizable lipids and cationic lipids of the present invention are specifically delivered to lung tissue, have excellent biocompatibility, and can deliver anionic drugs with high efficiency, thereby enabling related technologies such as lipid nanoparticle-mediated gene therapy. It can be useful in this field.
도 1은 hEPO mRNA 봉입 244-cis 지질나노입자의 생체 내 전달을 확인하기 위하여 마우스에 정맥주사한 후 채혈하여 hEPO 수준을 분석한 결과이다.
도 2는 hEPO mRNA 봉입 244-cis 지질나노입자의 생체 내 전달효능을 확인하기 위하여 마우스에 정맥주사한 후 혈액내 EPO 단백질 수준 및 MCP-1 수치를 확인한 결과이다.
도 3은 hEPO mRNA 봉입 244-cis 지질나노입자의 DOTAP 함유 여부에 따른 MCP-1 분비 정도를 확인한 결과이다.
도 4는 fLuc mRNA가 봉입되고 DOPE를 포함하는 244-cis 지질나노입자의 생체발광을 확인한 결과이다.
도 5는 fLuc mRNA가 봉입되고 10 mol%의 DOTAP을 포함하는 244-cis 지질나노입자의 생체 내 전달효능을 확인하기 위하여 마우스에 정맥주사한 후 생체발광을 확인한 결과이다.
도 6은 fLuc mRNA가 봉입되고 20 mol%의 DOTAP을 포함하는 244-cis 지질나노입자의 생체 내 전달효능을 확인하기 위하여 마우스에 정맥주사한 후 생체발광을 확인한 결과이다.
도 7은 fLuc mRNA가 봉입되고 40 mol%의 DOTAP을 포함하는 244-cis 지질나노입자의 생체 내 전달효능을 확인하기 위하여 마우스에 정맥주사한 후 생체발광을 확인한 결과이다.
도 8은 fLuc mRNA가 봉입되고 60 mol%의 DOTAP을 포함하는 244-cis 지질나노입자의 생체 내 전달효능을 확인하기 위하여 마우스에 정맥주사한 후 생체발광을 확인한 결과이다.
도 9는 fLuc mRNA가 봉입된 지질나노입자의 DOTAP을 포함하는 비율에 따른 생체 내 전달효능을 확인하기 위하여 마우스에 정맥주사한 후 각 장기 별 발광 분포를 확인하여 비교한 결과이다.
도 10은 fLuc mRNA가 봉입되고 20 mol%의 포스파티딜콜린을 포함하는 244-cis 지질나노입자의 생체 내 전달효능을 확인하기 위하여 마우스에 정맥주사한 후 생체발광을 확인한 결과이다.
도 11은 fLuc mRNA가 봉입되고 40 mol%의 포스파티딜콜린을 포함하는 244-cis 지질나노입자의 생체 내 전달효능을 확인하기 위하여 마우스에 정맥주사한 후 생체발광을 확인한 결과이다.
도 12는 Cre mRNA 봉입 244-cis 지질나노입자를 LSL-tdTomato 마우스에 정맥주사 후 폐 세포별 형광발현을 비교한 결과이다.
도 13은 Cre mRNA 봉입 244-cis 지질나노입자를 폐 섬유화가 일어난 LSL-tdTomato 마우스에 정맥주사 후 폐 세포별 형광발현을 비교한 결과이다.
도 14는 Cre mRNA 봉입 244-cis 지질나노입자를 폐 섬유화가 일어난 LSL-tdTomato 마우스에 정맥주사 후 섬유아세포의 Sca-1+ 세포에서의 형광발현을 확인한 결과이다.Figure 1 shows the results of hEPO levels analyzed by intravenous injection into mice and blood collection to confirm in vivo delivery of hEPO mRNA-encapsulated 244-cis lipid nanoparticles.
Figure 2 shows the results of confirming the EPO protein level and MCP-1 level in the blood after intravenous injection into mice to confirm the in vivo delivery efficacy of hEPO mRNA-encapsulated 244-cis lipid nanoparticles.
Figure 3 shows the results of confirming the level of MCP-1 secretion depending on whether hEPO mRNA-encapsulated 244-cis lipid nanoparticles contain DOTAP.
Figure 4 shows the results of confirming bioluminescence of 244-cis lipid nanoparticles encapsulated with fLuc mRNA and containing DOPE.
Figure 5 shows the results of bioluminescence after intravenous injection into mice to confirm the in vivo delivery efficacy of 244-cis lipid nanoparticles encapsulated with fLuc mRNA and containing 10 mol% of DOTAP.
Figure 6 shows the results of bioluminescence after intravenous injection into mice to confirm the in vivo delivery efficacy of 244-cis lipid nanoparticles encapsulated with fLuc mRNA and containing 20 mol% DOTAP.
Figure 7 shows the results of bioluminescence after intravenous injection into mice to confirm the in vivo delivery efficacy of 244-cis lipid nanoparticles encapsulated with fLuc mRNA and containing 40 mol% of DOTAP.
Figure 8 shows the results of bioluminescence after intravenous injection into mice to confirm the in vivo delivery efficacy of 244-cis lipid nanoparticles encapsulated with fLuc mRNA and containing 60 mol% of DOTAP.
Figure 9 shows the results of comparing the distribution of luminescence in each organ after intravenous injection into mice to confirm the in vivo delivery efficacy according to the DOTAP content of lipid nanoparticles encapsulated with fLuc mRNA.
Figure 10 shows the results of bioluminescence after intravenous injection into mice to confirm the in vivo delivery efficacy of 244-cis lipid nanoparticles encapsulated with fLuc mRNA and containing 20 mol% phosphatidylcholine.
Figure 11 shows the results of bioluminescence after intravenous injection into mice to confirm the in vivo delivery efficacy of 244-cis lipid nanoparticles encapsulated with fLuc mRNA and containing 40 mol% phosphatidylcholine.
Figure 12 shows the results of comparing fluorescence expression in each lung cell after intravenous injection of Cre mRNA-encapsulated 244-cis lipid nanoparticles into LSL-tdTomato mice.
Figure 13 shows the results of comparing the fluorescence expression of each lung cell after intravenous injection of Cre mRNA-encapsulated 244-cis lipid nanoparticles into LSL-tdTomato mice with lung fibrosis.
Figure 14 shows the results of confirming fluorescence expression in Sca-1 + cells of fibroblasts after intravenous injection of Cre mRNA-encapsulated 244-cis lipid nanoparticles into LSL-tdTomato mice with lung fibrosis.
이하, 실시예를 통하여 본 발명의 구성 및 효과를 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것일 뿐 본 발명의 범위가 이들 실시예에 의해 제한되는 것은 아니다.Hereinafter, the configuration and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention and the scope of the present invention is not limited by these examples.
실시예 1: 경폐 전달용 지질나노입자의 제조Example 1: Preparation of lipid nanoparticles for transpulmonary delivery
이온화 가능 지질로서 244-cis (WO2023/136689 참조)를 준비하였으며, 하기의 반응식 1에 따라 합성하였다. 본 발명에서 244-cis를 포함하는 지질나노입자는 '244-cis 지질나노입자'로 명명하였다. 대조군으로 SM-102 또는 MC3를 사용하였다.244-cis (see WO2023/136689) was prepared as an ionizable lipid and synthesized according to Scheme 1 below. In the present invention, lipid nanoparticles containing 244-cis were named '244-cis lipid nanoparticles'. SM-102 or MC3 was used as a control.
[반응식 1][Scheme 1]
이온화 가능 지질, 양이온성 지질 (DOTAP, 1,2-dioleoyl-3-trimethylammonium propane; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; 또는 EPC, egg phosphatidylcholine (Avanti, 890704P), 콜레스테롤 (Cholesterol powder, BioReagent, 세포 배양용, ≥99%, sigma, 한국), 및 C16-PEG2000 세라마이드 (Avanti, 미국)를 에탄올에 26.5 : 10 내지 60 : 12 내지 62 : 1.5의 몰비 (표 2)로 용해시켰다.Ionizable lipids, cationic lipids (DOTAP, 1,2-dioleoyl-3-trimethylammonium propane; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; or EPC, egg phosphatidylcholine (Avanti, 890704P), cholesterol ( Cholesterol powder, BioReagent, for cell culture, ≥99%, Sigma, Korea), and C16-PEG2000 ceramide (Avanti, USA) were dissolved in ethanol at a molar ratio of 26.5:10 to 60:12 to 62:1.5 (Table 2). I ordered it.
이온화 가능 지질, 콜레스테롤, 양이온성 지질, 및 PEG-세라마이드 접합체가 용해된 에탄올 및 핵산 (mRNA)이 용해된 수성상 (아세테이트산 나트륨 또는 시트르산 나트륨)을 1 : 3의 부피비로 12 ml/min의 유속으로 미세유체 혼합 장치 (Benchtop Nanoassemblr; PNI, Canada)를 통해 혼합하여 지질나노입자를 제조하였다. mRNA : 이온화 가능 지질의 중량비는 1 : 20으로 하였다.Ionizable lipids, cholesterol, cationic lipids, and PEG-ceramide conjugates were dissolved in ethanol and nucleic acids (mRNA) were dissolved in aqueous phase (sodium acetate or sodium citrate) at a volume ratio of 1:3 at a flow rate of 12 ml/min. Lipid nanoparticles were prepared by mixing using a microfluidic mixing device (Benchtop Nanoassemblr; PNI, Canada). The weight ratio of mRNA:ionizable lipid was 1:20.
제조된 지질나노입자에서 에탄올 제거 및 체내의 pH와 지질나노입자의 pH를 맞추기 위해서, 3500 MWCO 투석 카세트를 사용하여 16 시간 동안 PBS에 대해 투석하였다.In order to remove ethanol from the prepared lipid nanoparticles and adjust the pH of the lipid nanoparticles to that of the body, they were dialyzed against PBS for 16 hours using a 3500 MWCO dialysis cassette.
실험예 1: 244-cis를 포함하는 지질나노입자의 전달 효능 확인Experimental Example 1: Confirmation of delivery efficacy of lipid nanoparticles containing 244-cis
실험예 1-1. mRNA 발현 확인Experimental Example 1-1. Confirmation of mRNA expression
hEPO mRNA (서열번호 1)가 봉입되고, 244-cis, DOPE (20 mol%), 콜레스테롤 및 PEG-지질을 포함하는 지질나노입자를 mRNA 기준 0.1 mg/kg 용량으로 7 주령 Balb/c 마우스에 정맥 주사하였다. 3, 6, 9, 24 및 48 시간 뒤에 혈액을 채취하여, ELISA (R&D systems, USA)를 사용하여 hEPO 단백질 발현을 확인하였다 (도 1).hEPO mRNA (SEQ ID NO: 1) was encapsulated, and lipid nanoparticles containing 244-cis, DOPE (20 mol%), cholesterol, and PEG-lipid were administered intravenously to 7-week-old Balb/c mice at a dose of 0.1 mg/kg based on mRNA. Injected. Blood was collected after 3, 6, 9, 24, and 48 hours, and hEPO protein expression was confirmed using ELISA (R&D systems, USA) (Figure 1).
그 결과, 244-cis를 포함하는 지질나노입자는 핵산을 생체 내로 효과적으로 전달하고 발현시켜 혈액 내에서 hEPO가 상당한 수준으로 검출되었다. 244-cis를 포함하는 지질나노입자는 대조군인 MC3 LNP 대비 현저히 높은 수준을 나타내며, SM-102 LNP와 동등한 수준을 나타내었다.As a result, lipid nanoparticles containing 244-cis effectively delivered and expressed nucleic acids in vivo, and hEPO was detected at a significant level in the blood. Lipid nanoparticles containing 244-cis showed a significantly higher level than the control MC3 LNP, and showed a level equivalent to that of SM-102 LNP.
실험예 1-2. MCP-1 분비 확인Experimental Example 1-2. Confirmation of MCP-1 secretion
상기 실험예 1-1.의 지질나노입자를 mRNA 기준 0.5 mg/kg 용량으로 7주령 Balb/c 마우스에 정맥 주사하고, 3 시간 뒤에 혈액을 채취하였다. ELISA (Invitrogen, USA)를 사용하여 MCP-1 단백질 발현을 확인하였다 (도 2).The lipid nanoparticles of Experimental Example 1-1 were injected intravenously into 7-week-old Balb/c mice at a dose of 0.5 mg/kg based on mRNA, and blood was collected 3 hours later. MCP-1 protein expression was confirmed using ELISA (Invitrogen, USA) (Figure 2).
그 결과, 도 2에 나타난 바와 같이, 244-cis를 포함하는 지질나노입자는 SM-102 LNP 및 MC3 LNP와 비교하여 현저히 낮은 MCP-1 발현을 나타내는 것을 확인하였다.As a result, as shown in Figure 2, it was confirmed that lipid nanoparticles containing 244-cis showed significantly lower MCP-1 expression compared to SM-102 LNP and MC3 LNP.
실험예 1-3. DOTAP에 의한 MCP-1 분비 확인Experimental Example 1-3. Confirmation of MCP-1 secretion by DOTAP
상기 실험예 1-1.의 244-cis 지질나노입자에서 DOPE를 DOTAP으로 교체한 지질나노입자를 mRNA 기준 0.5 mg/kg 용량으로 7 주령 Balb/c 마우스에 정맥 주사하고, 6 시간 뒤에 혈액을 채취하였다. ELISA (Invitrogen, USA)를 사용하여 MCP-1 단백질 발현을 확인하였다 (도 3).Lipid nanoparticles in which DOPE was replaced with DOTAP in the 244-cis lipid nanoparticles of Experimental Example 1-1 above were injected intravenously into 7-week-old Balb/c mice at a dose of 0.5 mg/kg based on mRNA, and blood was collected 6 hours later. did. MCP-1 protein expression was confirmed using ELISA (Invitrogen, USA) (Figure 3).
그 결과, 도 3에 나타난 바와 같이, DOPE를 DOTAP으로 대체하더라도 기존 244-cis 제형의 MCP-1 분비 정도와 통계적으로 유의미한 차이는 보이지 않았다.As a result, as shown in Figure 3, even if DOPE was replaced with DOTAP, there was no statistically significant difference from the level of MCP-1 secretion in the existing 244-cis formulation.
실험예 2: fLuc mRNA를 이용한 244-cis 지질나노입자의 폐 특이적 전달 확인Experimental Example 2: Confirmation of lung-specific delivery of 244-cis lipid nanoparticles using fLuc mRNA
실험예 2-1. DOTAP의 폐 전달 효능 확인Experimental Example 2-1. Confirmation of pulmonary delivery efficacy of DOTAP
fLuciferase mRNA (서열번호 2)가 봉입되고, 244-cis, DOTAP, 콜레스테롤 및 PEG-지질을 포함하는 지질나노입자를 표 2의 함량 별로 제조하고, mRNA 기준 0.2 mg/kg 용량으로 7 주령 C57BL/6 마우스에 정맥 주사하였다. 6 시간 뒤에 루시페린 (luciferin) 0.25 mg/kg을 복강 투여하여 IVIS (PerkinElmer, USA) 장비를 통해 탈체 기관 이미지 (ex vivo organ image)로 생체발광을 확인하였다 (도 4 내지 도 8). 대조군으로 DOPE 함유 지질나노입자를 사용하였다.fLuciferase mRNA (SEQ ID NO: 2) was encapsulated and lipid nanoparticles containing 244-cis, DOTAP, cholesterol, and PEG-lipid were prepared according to the contents in Table 2, and administered to 7-week-old C57BL/6 at a dose of 0.2 mg/kg based on mRNA. It was injected intravenously into mice. Six hours later, 0.25 mg/kg of luciferin was administered intraperitoneally, and bioluminescence was confirmed through ex vivo organ images using IVIS (PerkinElmer, USA) equipment (FIGS. 4 to 8). As a control, DOPE-containing lipid nanoparticles were used.
그 결과, 20 mol% DOPE를 포함하는 경우 간에서 높은 발광 강도를 나타내었으나 (도 4), DOPE를 DOTAP으로 대체한 경우 mRNA가 폐로 전달되어 발현되는 것을 확인할 수 있었다 (도 5 내지 도 9). 특히, DOTAP을 20 내지 60 mol%로 포함할 경우 폐 특이적으로 높은 발광 강도를 나타내었다 (도 6 내지 도 9). 이를 통해, 이온화 가능 지질 244-cis는 양이온성 지질로서 DOTAP을 포함하여 지질나노입자를 제조하였을 때 폐 특이적으로 전달된다는 것을 알 수 있었다.As a result, when 20 mol% DOPE was included, high luminescence intensity was shown in the liver (Figure 4), but when DOPE was replaced with DOTAP, it was confirmed that the mRNA was delivered to the lungs and expressed (Figures 5 to 9). In particular, when DOTAP was included at 20 to 60 mol%, high lung-specific luminescence intensity was observed ( FIGS. 6 to 9 ). Through this, it was found that the ionizable lipid 244-cis was delivered specifically to the lungs when lipid nanoparticles were prepared including DOTAP as a cationic lipid.
실험예 2-2. 포스파티딜콜린의 폐 전달 효능 확인Experimental Example 2-2. Confirmation of pulmonary delivery efficacy of phosphatidylcholine
244-cis 및 포스파티딜콜린 (EPC)을 함유하는 지질나노입자를 표 2의 함량 별로 제조하고, mRNA 0.1 mg/kg을 7 주령 C57BL/6 마우스에 정맥 주사하였다. 6 시간 뒤에 루시페린 (luciferin) 0.25 mg/kg을 복강 투여하여 IVIS (PerkinElmer, USA) 장비를 통해 탈체 기관 이미지 (ex vivo organ image)로 생체발광을 확인하였다 (도 10 및 도 11).Lipid nanoparticles containing 244-cis and phosphatidylcholine (EPC) were prepared according to the contents in Table 2, and 0.1 mg/kg of mRNA was injected intravenously into 7-week-old C57BL/6 mice. Six hours later, 0.25 mg/kg of luciferin was administered intraperitoneally, and bioluminescence was confirmed by ex vivo organ image using IVIS (PerkinElmer, USA) equipment (FIGS. 10 and 11).
그 결과, DOTAP과 마찬가지로 포스파티딜콜린을 포함하는 244-cis 지질나노입자는 폐 특이적으로 높은 발광 강도를 나타내었다.As a result, like DOTAP, 244-cis lipid nanoparticles containing phosphatidylcholine showed high lung-specific luminescence intensity.
실험예 3: Cre mRNA를 이용한 244-cis 지질나노입자의 폐 세포별 전달 효율 확인Experimental Example 3: Confirmation of delivery efficiency of 244-cis lipid nanoparticles to lung cells using Cre mRNA
본 발명의 지질나노입자의 폐 세포별 전달 효과를 확인하기 위해, Cre 단백질을 이용하여 Tomato 단백질을 발현하는 LSL-tdTomato 마우스 (The Jackson Laboratory #:007914)를 사용하였다. Cre mRNA (서열번호 3)가 봉입되고, 244-cis, DOTAP (20 mol%), 콜레스테롤 및 PEG-지질을 포함하는 지질나노입자를 mRNA 기준 0.3 mg/kg 용량으로 7 주령 LSL-tdTomato 마우스에 4 일 간격으로 2 번 정맥 주사하였다. 2 차 주사 2 일 후에 폐 조직 세포를 분리하여 유세포 분석 (LSRFortessa, BD)을 이용하여 폐 세포별 (내피 세포, 섬유아세포, 상피 세포, 면역 세포) tdTomato 형광 발현을 확인하였다 (도 12). 내피 세포 염색을 위해 APC 항-마우스 CD31 항체 (BioLegend; 102409), 면역 세포 염색을 위해 FITC 항-마우스 CD45.2 항체 (BioLegend; 109805), 상피 세포 염색을 위해 PE/Cyanine7 항-마우스 CD326 항체 (BioLegend; 118216), 섬유아세포 염색을 위해 PE/Cyanine7 항-마우스 CD140a 항체 (BioLegend; 135911)를 사용하였다.To confirm the delivery effect of the lipid nanoparticles of the present invention to lung cells, LSL-tdTomato mice (The Jackson Laboratory #: 007914), which express Tomato protein using Cre protein, were used. Cre mRNA (SEQ ID NO: 3) was encapsulated, and lipid nanoparticles containing 244-cis, DOTAP (20 mol%), cholesterol, and PEG-lipid were administered to 7-week-old LSL-tdTomato mice at a dose of 0.3 mg/kg based on mRNA. It was administered intravenously twice at daily intervals. Two days after the second injection, lung tissue cells were isolated and tdTomato fluorescence expression in each lung cell (endothelial cells, fibroblasts, epithelial cells, immune cells) was confirmed using flow cytometry (LSRFortessa, BD) (Figure 12). APC anti-mouse CD31 antibody (BioLegend; 102409) for staining endothelial cells, FITC anti-mouse CD45.2 antibody (BioLegend; 109805) for staining immune cells, and PE/Cyanine7 anti-mouse CD326 antibody (BioLegend; 109805) for staining epithelial cells. BioLegend; 118216), and PE/Cyanine7 anti-mouse CD140a antibody (BioLegend; 135911) was used to stain fibroblasts.
그 결과, 모든 유형의 폐 세포에서 대조군인 MC3 LNP에 비해 압도적으로 높은 발현을 나타내어, 본 발명의 지질나노입자의 우수한 폐 전달 효과를 확인하였다. 세포 유형 별로 보면, 내피 세포에서 80 % 이상의 tdTomato 발현을 확인하였으며, 상피 세포, 면역 세포, 섬유아세포에서 약 20 %의 tdTomato 발현을 확인하였다.As a result, all types of lung cells showed overwhelmingly higher expression than the control MC3 LNP, confirming the excellent lung delivery effect of the lipid nanoparticles of the present invention. By cell type, more than 80% of tdTomato expression was confirmed in endothelial cells, and about 20% of tdTomato expression was confirmed in epithelial cells, immune cells, and fibroblasts.
실험예 4: 폐 섬유화 모델에서 244-cis 지질나노입자의 폐 특이적 전달 확인Experimental Example 4: Confirmation of lung-specific delivery of 244-cis lipid nanoparticles in lung fibrosis model
질환 모델에서 본 발명의 지질나노입자의 효능을 확인하기 위하여, 블레오마이신 1.8 mg/kg을 처리하여 14 일 후 폐 섬유화가 일어난 7 주령 LSL-tdTomato 마우스를 준비하였다. 상기 실험예 3의 244-cis 지질나노입자를 폐 섬유화가 일어난 7 주령 LSL-tdTomato 마우스에 mRNA 기준 0.3 mg/kg 용량으로, 4 일 간격으로 2 번 정맥 주사하였다. 2 차 주사 2 일 후에 폐 조직 세포를 분리하고 유세포 분석 (LSRFortessa, BD)을 이용하여 폐 세포별 (내피 세포, 섬유아세포, 상피 세포, 면역 세포) tdTomato 형광 발현을 확인하였다 (도 13 및 도 14).In order to confirm the efficacy of the lipid nanoparticles of the present invention in a disease model, 7-week-old LSL-tdTomato mice, which developed lung fibrosis 14 days after treatment with 1.8 mg/kg of bleomycin, were prepared. The 244-cis lipid nanoparticles of Experimental Example 3 were injected intravenously twice at 4-day intervals at a dose of 0.3 mg/kg based on mRNA into 7-week-old LSL-tdTomato mice with lung fibrosis. Two days after the second injection, lung tissue cells were isolated and tdTomato fluorescence expression was confirmed in each lung cell (endothelial cells, fibroblasts, epithelial cells, immune cells) using flow cytometry (LSRFortessa, BD) (Figures 13 and 14 ).
세포 유형별 염색을 위해 상기 실험예 3과 같은 조건에 더하여, 섬유아세포 subtype 염색을 위해 Brilliant Violet 421™ 항-마우스 CD90.1 항체 (BioLegend; 202529), 및 FITC 항-마우스 Sca-1 항체 (BioLegend; 122505)를 사용하였다.In addition to the same conditions as in Experimental Example 3 for staining by cell type, Brilliant Violet 421™ anti-mouse CD90.1 antibody (BioLegend; 202529) and FITC anti-mouse Sca-1 antibody (BioLegend; 202529) were used for fibroblast subtype staining. 122505) was used.
그 결과, 섬유화 질환을 유발하지 않은 마우스에서와 마찬가지로 본 발명의 지질나노입자는 모든 유형의 폐 세포에서 대조군인 MC3 LNP에 비해 압도적으로 높은 발현을 나타내었다. 또한, 내피 세포에서 80 % 이상의 tdTomato 발현을 나타내었으며, 상피 세포, 면역 세포 및 섬유아세포에서도 약 30 % 수준의 tdTomato 발현을 나타내었다 (도 13). 섬유아세포에서 tdTomato의 발현 수준을 확인하였을 때 MC3 LNP의 경우 거의 발현이 나타나지 않았으나, 244-cis LNP의 경우 특히 Sca-1+ 섬유아세포에서 40 % 이상의 tdTomato 발현을 나타내었다 (도 14).As a result, as in mice that did not develop fibrotic disease, the lipid nanoparticles of the present invention showed overwhelmingly higher expression than the control MC3 LNP in all types of lung cells. In addition, endothelial cells showed over 80% tdTomato expression, and epithelial cells, immune cells, and fibroblasts also showed tdTomato expression at a level of about 30% (Figure 13). When the expression level of tdTomato was confirmed in fibroblasts, almost no expression was observed in MC3 LNPs, but in the case of 244-cis LNPs, tdTomato expression was shown by more than 40%, especially in Sca-1 + fibroblasts (Figure 14).
이는 본 발명의 244-cis LNP가 MC3 LNP와 비교하여 mRNA를 폐 특이적으로 전달하여 발현시킬 수 있고, 특히 폐 섬유화증 모델에서 섬유아세포에 mRNA를 표적 전달할 수 있다는 것을 시사한다.This suggests that the 244-cis LNP of the present invention can deliver and express mRNA specifically to the lung compared to the MC3 LNP, and can specifically deliver mRNA to fibroblasts in a lung fibrosis model.
이상의 설명으로부터, 본 발명의 속하는 기술분야의 당업자는 본 발명이 그 기술적 사상이나 필수적 특징을 변경하지 않고서 다른 구체적인 형태로 실시될 수 있다는 것을 이해할 수 있을 것이다. 이와 관련하여, 이상에서 기술한 실시예들은 모든 면에서 예시적인 것이며 한정적인 것이 아닌 것으로 이해해야만 한다. 본 발명의 범위는 상기 상세한 설명보다는 후술하는 특허 청구범위의 의미 및 범위 그리고 그 등가 개념으로부터 도출되는 모든 변경 또는 변형된 형태가 본 발명의 범위에 포함되는 것으로 해석되어야 한다.From the above description, those skilled in the art will understand that the present invention can be implemented in other specific forms without changing its technical idea or essential features. In this regard, the embodiments described above should be understood in all respects as illustrative and not restrictive. The scope of the present invention should be construed as including the meaning and scope of the patent claims described below rather than the detailed description above, and all changes or modified forms derived from the equivalent concept thereof are included in the scope of the present invention.
서열목록 전자파일 첨부Sequence list electronic file attached
Claims (12)
[화학식 1]
.
A composition for transpulmonary delivery of a drug, comprising lipid nanoparticles containing an ionizable lipid and a cationic lipid of the following formula (1):
[Formula 1]
.
The composition for transpulmonary delivery of a drug according to claim 1, wherein the cationic lipid is DOTAP (1,2-dioleoyl-3-trimethylammonium propane) or phosphatidylcholine.
The composition for transpulmonary delivery of a drug according to claim 1, wherein the lipid nanoparticles further include structural lipids or PEG-lipids.
4. The method of claim 3, wherein the structural lipids are cholesterol, cholesterol, spinasterol, fecosterol, sitosterol, ergosterol, ergostenol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid and alpha - A composition for transpulmonary delivery of a drug, which is at least one selected from the group consisting of tocopherol.
The method of claim 3, wherein the PEG-lipid is at least one selected from the group consisting of PEG-ceramide, PEG-DMG, PEG-c-DOMG, PEG-DLPE, PEG-DMPE, PEG-DPPC and PEG-DSPE. Composition for transpulmonary delivery of drugs.
The composition for transpulmonary delivery of a drug according to claim 3, wherein the DOTAP is contained in an amount of 10 mol% to 70 mol% based on the total lipid nanoparticles.
The composition for transpulmonary delivery of a drug according to claim 3, wherein the phosphatidylcholine is contained in an amount of 10 mol% to 50 mol% based on the total lipid nanoparticles.
[화학식 1]
.
i) Lipid nanoparticles containing ionizable lipids and cationic lipids of the following formula (1), and ii) Pharmaceutical compositions for the prevention or treatment of lung diseases, containing an anionic drug as an active ingredient:
[Formula 1]
.
The pharmaceutical composition for preventing or treating lung disease according to claim 8, wherein the cationic lipid is DOTAP (1,2-dioleoyl-3-trimethylammonium propane) or phosphatidylcholine.
The pharmaceutical composition for preventing or treating lung disease according to claim 8, wherein the anionic drug is a nucleic acid drug.
11. The method of claim 10, wherein the nucleic acid drug is small interfering ribonucleic acid (siRNA), ribosomal ribonucleic acid (rRNA), deoxyribonucleic acid (DNA), complementary deoxyribonucleic acid (cDNA), aptamer, messenger ribonucleic acid (mRNA). , a pharmaceutical composition for preventing or treating lung disease, which is at least one selected from the group consisting of transport ribonucleic acid (tRNA), sgRNA, antisense oligonucleotide, shRNA, miRNA, ribozyme, PNA, and DNAzyme.
The method of claim 8, wherein the lung disease includes emphysema, asthma, pneumonia, tuberculosis, pulmonary hypertension, lung cancer, neonatal bronchopulmonary dysplasia, chronic obstructive pulmonary disease, acute bronchitis, chronic bronchitis, bronchiolitis, bronchiectasis, hypersensitivity, lung parenchyma. A group consisting of salts, acute smoke inhalation, heat-induced lung injury, cystic fibrosis, alveolar proteinosis, alpha-1-protease deficiency, pulmonary inflammatory disorders, acute respiratory distress syndrome, acute lung injury, idiopathic respiratory distress syndrome, and pulmonary fibrosis. A pharmaceutical composition for preventing or treating lung disease, which is one or more types selected from.
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