KR101254752B1 - Synthesis of 5-aminolevulinic acid trimer using citric acid for photodynamic therapy - Google Patents

Synthesis of 5-aminolevulinic acid trimer using citric acid for photodynamic therapy Download PDF

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KR101254752B1
KR101254752B1 KR1020110018008A KR20110018008A KR101254752B1 KR 101254752 B1 KR101254752 B1 KR 101254752B1 KR 1020110018008 A KR1020110018008 A KR 1020110018008A KR 20110018008 A KR20110018008 A KR 20110018008A KR 101254752 B1 KR101254752 B1 KR 101254752B1
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acid
trimer
aminolevlynic
photodynamic therapy
citric acid
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KR20120098210A (en
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강대환
정영일
정정욱
김시현
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부산대학교 산학협력단
부산대학교병원
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0057—Photodynamic therapy with a photosensitizer, i.e. agent able to produce reactive oxygen species upon exposure to light or radiation, e.g. UV or visible light; photocleavage of nucleic acids with an agent
    • A61K41/0061—5-aminolevulinic acid-based PDT: 5-ALA-PDT involving porphyrins or precursors of protoporphyrins generated in vivo from 5-ALA
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid, pantothenic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2121/00—Preparations for use in therapy

Abstract

본 발명은 광증감제를 사용하는 광역동치료(photodynamic therapy PDT)에 관한 것으로, 시트릭산(citric acid)의 카르복실그룹에 아미노레블리닉산이 공유결합되어 이루어지는 구연산을 이용한 아미노레블리닉산 트라이머를 제공하는 것으로 순도 90%이상으로 이루어지도록 하고, 50 ~ 300nm 크기의 나노입자 형태로 이루어지도록 한다. 본 발명에 따르면 종양조직 및 암세포에 변형된 아미노레블리닉산 광증감제의 흡수효율이 높아짐에 따라 보다 적은 양의 약제사용으로 인체 부작용의 최소화 및 빠른 환자치료가 가능해지는 효과를 가질 수 있다.BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to photodynamic therapy (PDT) using a photosensitizer, wherein an aminolevininic acid trimer using citric acid covalently bonded to aminocarboxylic acid to a carboxyl group of citric acid. To provide a purity of 90% or more, and 50 to 300nm size of the nanoparticles to be made. According to the present invention, as the absorption efficiency of the modified aminolevlynic acid photosensitizer increases in tumor tissues and cancer cells, the use of a smaller amount of the drug may have the effect of minimizing human side effects and enabling rapid patient treatment.

Description

광역동치료의 광증감제로 사용하기 위한 구연산을 이용한 아미노레불리닉산 트라이머{Synthesis of 5-aminolevulinic acid trimer using citric acid for photodynamic therapy}Synthesis of 5-aminolevulinic acid trimer using citric acid for photodynamic therapy

본 발명은 광증감제를 사용하는 광역동치료(photodynamic therapy PDT)에 관한 것으로, 보다 상세하게는 필요로 하는 세포 혹은 특정조직에 보다 약물의 침투가 잘 이루어지도록 하는 구연산을 이용한 아미노레불리닉산 트라이머에 관한 것이다. The present invention relates to photodynamic therapy (PDT) using a photosensitizer, and more specifically, aminolevulinic acid using citric acid to better infiltrate drugs into cells or specific tissues that are needed. It's about mer.

광증감제를 사용하는 광역동치료 (photodynamic therapy, PDT)는 기존의 암 치료법인 수술, 방사선요법, 약물요법의 부작용 및 암치료 이후의 후유증 문제를 해결할 수 있는 대체치료법으로 주목받고 있다. PDT에 사용되는 광증감제 (photosensitizer)는 빛에 노출되지 않으면 높은 농도에서도 세포 독성을 거의 나타내지 않다가 특정 파장의 빛에 의해 여기 되어 반응성 산소종 (일항산소, 산소라디칼, 수퍼옥사이드, 폐록사이드)을 생성하여 세포의 사멸을 유도한다. 일항 산소는 세포내 구성성분들 (예를 들어, 불포화 지방산, 콜레스테롤, 단백질, guanine 등)과 화학적으로 반응하여 손상을 입힘에 따라 암세포는 아포토시스(apoptosis)나 네오크로시스(necrosis)를 보이는데, 손상의 정도가 커질수록 아포토시스(apoptosis)보다는 네오크로시스(necrosis)를 통하여 죽는 세포의 비율이 증가하게 된다. 즉 광증감제를 정맥 주사 후 일정 시간이 지나면 암조직에 광증감제가 선택적으로 축적되고, 이 후 특정 파장의 빛을 조사하면 암세포만 선택적으로 사멸되며 빛을 쪼여주지 않은 다른 조직은 영향을 받지 않는다.Photodynamic therapy (PDT) using a photosensitizer is attracting attention as an alternative treatment that can solve the side effects of surgery, radiation therapy, drug therapy, and the sequelae after cancer treatment. Photosensitizers used in PDT show little cytotoxicity even at high concentrations when not exposed to light, but are excited by light of a specific wavelength and react with reactive oxygen species (monooxygen, oxygen radicals, superoxides, and lungoxides). Induces cell death. Singlet oxygen chemically reacts with intracellular components (e.g. unsaturated fatty acids, cholesterol, proteins, guanine, etc.) and damages, resulting in cancer cells showing apoptosis or necrosis. As the degree increases, the percentage of cells dying through neocrosis rather than apoptosis increases. That is, after a certain period of time after intravenous injection of photosensitizers, photosensitizers are selectively accumulated in cancer tissues, and after irradiation with light of a specific wavelength, only cancer cells are selectively killed, and other tissues that do not emit light are not affected. .

항암제의 경우 암세포뿐만 아니라 정상세포에서 심각한 강한 독성효과를 보이는 반면, 광역동치료(PDT)에 사용되는 광증감제는 암에 선택적으로 축적되는 암세포 특이성을 지니며 빛을 쪼여준 부위에서만 독성을 나타내므로 광역동치료(PDT) 후 항암제 치료시 보이는 머리카락 빠짐, 면역기능 저하, 구토 등의 부작용이 나타나지 않는다. 또한 광역동치료(PDT) 시술시 고통이 거의 수반되지 않으며 부작용이 거의 없기 때문에 광역동치료(PDT)는 반복 시술이 가능하여 말기 전이암 환자에게도 반복치료를 통해 최소한의 고통으로 생명을 연장시킬 수 있다. 따라서 광역동치료(PDT)는 환자의 생명연장과 삶의 질을 향상시킬 수 있는 장점이 있다. 광역동치료(PDT)는 또한 수술이 불가능한 환자의 치료에도 효과적이다. 폐암, 담관암 등에서 수술을 할 수 없는 암환자나 체력이 허약하여 수술이나 항암제 치료가 불가한 노약자, 또는 수술을 거부하는 환자의 경우에도 수술을 대체하여 광역동치료(PDT)를 수행할 수 있다.Anti-cancer drugs show severe toxic effects not only on cancer cells but also on normal cells, while the photosensitizers used for photodynamic therapy (PDT) have cancer cell specificities that selectively accumulate in cancer and are toxic only in the lighted areas. Therefore, after photodynamic therapy (PDT), there are no side effects such as hair loss, immune function, and vomiting when treated with anticancer drugs. In addition, photodynamic therapy (PDT) is rarely accompanied by pain, and since there are few side effects, photodynamic therapy (PDT) can be repeated to prolong life with minimal pain through repeated treatment even for terminal metastatic cancer patients. have. Therefore, photodynamic therapy (PDT) has the advantage of improving the life extension and quality of life of patients. Photodynamic therapy (PDT) is also effective in treating non-surgical patients. Cancer patients who cannot perform surgery on lung cancer, bile duct cancer, or the elderly who cannot perform surgery or chemotherapy due to weak physical strength, or patients who refuse the operation can perform photodynamic therapy (PDT) in place of surgery.

한편 광역동치료(PDT)를 위해 사용되는 일반적인 광증감제는 크게 포피린을 기반(porphyrin-based)으로 한 것과 그 외의 것으로 나눌 수 있는데, 임상 사용이 허가된 광증감제의 거의 모두가 포피린을 기반으로 한 것이다. 프로토포피린IX(Protoporphyrin IX (PpIX))는 포피린 기반 광증감제로 아미노레불리닉산(5-Aminolevulinic acid (ALA))로부터 생합성된다. 아미노레불리닉산은 미토콘드리아내에서 헴(heme) 생합성 과정의 중간 물질인 프로토포피린IX(PpIX)로 전환되며, 이물질은 임상에서 좋은 결과를 보여 왔기 때문에 지난 20년 동안 커다란 관심의 대상이었다.On the other hand, the general photosensitizers used for photodynamic therapy (PDT) can be broadly divided into porphyrin-based and others. Almost all of the photosensitizers licensed for clinical use are porphyrin-based. I did it. Protoporphyrin IX (PpIX) is biosynthesized from 5-Aminolevulinic acid (ALA) as a porphyrin-based photosensitizer. Aminolevulinic acid is converted into protopophyrin IX (PpIX), an intermediate in the heme biosynthesis process in the mitochondria, which has been of great interest for the past 20 years because it has shown good results in clinical practice.

암세포의 경우, 프로토포피린IX(PpIX) 생합성에 필요한 레이트리미팅 효소(rate-limiting enzyme)인 포르포빌리노겐 디아미나제(porphobilinogen deaminase)가 증가되어 있을 뿐 아니라 프로토포피린IX(PpIX)를 헴(heme)으로 전화시켜주는 효소인 페로킬라타제(ferrochelatase)가 정상세포에 비해서 감소되어 있는 것으로 알려져 있다. 따라서 외부에서 고농도의 아미노레불리닉산을 투여하게 되면 정상세포보다는 암세포에서 효율적으로 프로토포피린(PpIX)의 생성이 촉진되고, 이후에 특정 파장의 빛을 쪼여주면 암세포를 선택적으로 죽일 수 있는 것으로 추정된다. 아미노레불리닉산의 경우 포피린 계열의 다른 광증감제와는 달리, 아미노레불리닉산으로 부터 생성된 프로토포피린IX(PpIX)가 24-48시간 이후에는 전부 제거되기 때문에 장기간 피부 광 민감성의 위험이 적다는 큰 장점이 있다. In the case of cancer cells, not only is there an increase in porphobilinogen deaminase, a rate-limiting enzyme required for protopophyrin IX (PpIX) biosynthesis, but also heme (Proppophyrin IX (PpIX)) It is known that ferrochelatase, an enzyme that converts to), is reduced compared to normal cells. Therefore, the administration of high concentrations of aminolevulinic acid from the outside promotes the production of protopopyrine (PpIX) in cancer cells more efficiently than normal cells, and can subsequently kill cancer cells by irradiating with specific wavelengths of light. . Unlike other porphyrin-based photosensitizers, aminolevulinic acid is less prone to long-term skin light sensitivity because protoporphyrin IX (PpIX) produced from aminolevulinic acid is completely removed after 24-48 hours. Has a big advantage.

그러나, 이러한 아미노레불리닉산은 광역동 치료 효과는 뛰어나지만 아미노레불리닉산의 친수성 성질 때문에 세포 혹은 조직내로의 흡수가 방해되는 단점이 있기 때문에 아미노레불리닉산의 세포, 조직내 흡수를 증가시키기 위한 연구가 활발히 진행되고 있다. 예를 들자면, 지방친화성을 도입한 에스테르(ester)-아미노레불리닉산, 약물의 흡수를 도와주는 증강제(enhancer), 리포좀과 같은 약물전달체 등을 사용하거나 파고사이토시스(pagocytosis) 혹은 엔도사이토시스(endosytosis)를 통해 아미노레불리닉산을 흡수시키고자 아미노레불리닉산-덴드리머(dendrimer)를 대상으로 활발히 연구가 진행되고 있다.However, such aminolevulinic acid has excellent photodynamic therapeutic effect, but due to the hydrophilic nature of aminolevulinic acid, the absorption of the aminolevulinic acid into the cell or tissue is impeded. Research is actively underway. For example, ester-aminolevulinic acid incorporating fat affinity, an enhancer that aids in drug absorption, drug delivery agents such as liposomes, or pagocytosis or endocytosis. In order to absorb aminolevulinic acid through endosytosis, active studies are being conducted on aminolevulinic acid-dendrimers.

그러나, 현재 까지 연구되고 있는 다양한 방법들은 많은 노력이 수반되는 반면에 아미노레불리닉산의 세포 흡수 효율이 높지 않은 문제점을 안고 있다.However, the various methods that have been studied up to now require a lot of effort, but have a problem in that the efficiency of cell absorption of aminolevulinic acid is not high.

본 발명자들 역시 이와 같이 생체조직 내로 아미노레블리닉산의 흡수 효율을 높이는 다양한 연구를 수행하던 중 흡수효율을 높이는 방안의 하나로 아미노레블리닉산의 구조를 변경시킴으로써 보다 기존 보다 효율을 높일 수 있음을 착안하여 본 발명을 완성하게 되었다.The present inventors also focused on improving the efficiency of the aminolevlinic acid by changing the structure of the aminolevlinic acid as one of the methods of increasing the absorption efficiency while conducting various studies to increase the absorption efficiency of the aminolevlynic acid into the biological tissue. The present invention was completed.

따라서 본 발명은 상기 요구에 부응하기 위하여 고안된 것으로서, 본 발명의 목적은 암세포 및 종양조직에 광역동치료(PDT)를 위한 광증감제로 사용되는 아미노레블리닉산을 악성종양조직 및 암세포에 보다 효율적으로 흡수되도록 생체내 물질인 구연산(citric acid)을 이용하여 이를 아미노레블리닉산과 결합시켜 구조를 변경함으로서 이루어지는 구연산을 이용한 아미노레블리닉산 트라이머를 제공하는 것을 목적으로 한다.Therefore, the present invention is designed to meet the above demands, and an object of the present invention is to efficiently use aminolevlynic acid used as a photosensitizer for photodynamic therapy (PDT) in cancer cells and tumor tissues in malignant tumor tissues and cancer cells. It is an object of the present invention to provide an aminolevlynic acid trimer using citric acid, which is formed by combining citric acid, which is an in vivo substance, to be absorbed with aminolevlynic acid to change its structure.

이와 같은 목적을 달성하기 위한 본 발명의 일 양태에 따르면, 광역동치료(photodynamic therapy PDT)에 사용되는 아미노레블리닉산 광증감제에 있어서, 아미노레블리닉산 광증감제는, 시트릭산(citric acid)의 카르복실그룹에 아미노레블리닉산이 공유결합되어 이루어지는 구연산을 이용한 아미노레블리닉산 트라이머를 제공하는데 특징이 있다. According to one aspect of the present invention for achieving the above object, in the aminolevlynic acid photosensitizer used in photodynamic therapy PDT, the aminolevlynic acid photosensitizer is citric acid The present invention is characterized in providing an aminolevlynic acid trimer using citric acid formed by covalent bonding of aminolevlynic acid to a carboxyl group.

바람직하기로는, 상기 아미노레블리닉산 트라이머는, 구연산 (Citric acid) 1M을 5 ml의 증류수에 녹인 용액과; 디메틸아미노프로필-에틸카르보디이미드-하이드로클로라이드(N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC)) 3M과, 하이드록시숙시미드(N-hydroxysuccimide) 3M과 아미노레블리닉산(5-aminolevulinic acid) 3M을 테트라히드로푸란(tetrahydrofuran (THF)/H20 (8/2)) 95 ml에 녹여 서로 혼합한 용액;의 비율로 혼합하여 암실에서 약 24시간 동안 교반한 뒤 용매를 제거하고 동결건조하여 메탄올에 침전 시킨 후 재결정하여 실온에서 건조하고 플래쉬크로마토그래피(flash chromatography) (3/1=ethylacetate/hexane)로 정제한 뒤, 디클로로메탄/헥산(dichloromethane (DCM) /hexane)으로 재결정 형성되는 것으로 순도 90%이상으로 이루어지도록 하고, 50 ~ 300nm 크기의 나노입자 형태로 이루어지도록 한다.Preferably, the aminolevlynic acid trimer comprises: a solution of 1M of citric acid dissolved in 5 ml of distilled water; Dimethylaminopropyl-ethylcarbodiimide-hydrochloride (N- (3-Dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (EDC)) 3M, hydroxysuccimide 3M and aminolevlynic acid (5- Aminolevulinic acid) 3M was dissolved in 95 ml of tetrahydrofuran (THF) / H 2 0 (8/2) and mixed with each other; the mixture was mixed at a ratio of about 24 hours in a dark room, and then the solvent was removed. Lyophilized, precipitated in methanol, recrystallized, dried at room temperature, purified by flash chromatography (3/1 = ethylacetate / hexane), and then recrystallized with dichloromethane (DCM) / hexane. It is to be made of more than 90% purity, and to be made in the form of nanoparticles of 50 ~ 300nm size.

본 발명에 있어서는 종양조직 및 암세포에 변형된 아미노레블리닉산 광증감제의 흡수효율이 높아짐에 따라 보다 적은 양의 약제 사용으로 인체 부작용의 최소화 및 빠른 환자치료가 가능해지는 효과를 가질 수 있다.In the present invention, as the absorption efficiency of the modified aminolevlynic acid photosensitizer in tumor tissues and cancer cells is increased, the use of a smaller amount of the drug may have the effect of minimizing human side effects and enabling rapid patient treatment.

또한, 본 발명은 적은 양으로 보다 높은 효율을 달성할 수 있으므로 환자치료의 비용부담을 절감할 수 있는 다른 효과도 있다.In addition, the present invention can achieve higher efficiency in a small amount, so there are other effects that can reduce the cost of patient care.

도 1은 본 발명에 따른 시트릭산을 이용한 아미노레블리닉산 트라이머의 합성과정을 나타낸 도이다.
도 2는 본 발명에 따른 구연산(a) 아미노레블리닉산(b) 아미노레블리닉산 트라이머(c)의 수소 핵자기공명 스펜트럼의 모습을 보인도이다.
도 3은 본 발명에 따른 아미노레블리닉산 트라이머와 대조군인 아미노레블리닉산의 세포독성 평가 그래프를 나타낸 도이다.
도 4는 본 발명에 따른 아미노레블리닉산 트라이머와 대조군인 아미노레블리닉산을 HuCC-T1 세포에 처리한 뒤 생합성된 프로토포피린IX(PpIX)의 양의 변화를 나타내는 그래프를 보인 도이다.1 is a view showing the synthesis process of aminolevlynic acid trimer using citric acid according to the present invention.
Figure 2 is a view showing the state of the hydrogen nuclear magnetic resonance spectrum of the citric acid (a) aminolevynic acid (b) aminolevlynic acid trimer (c) according to the present invention.
Figure 3 is a diagram showing a cytotoxicity evaluation graph of the aminolevlynic acid trimer and the control aminolevlynic acid according to the present invention.
Figure 4 is a graph showing a change in the amount of biosynthesized protoporphyrin IX (PpIX) after treatment with HuCC-T1 cells and aminolevlynic acid trimer and a control aminolevlynic acid according to the present invention.

실시예Example

1. 아미노레블리닉산 트라이머의 합성1. Synthesis of Aminolevynic Acid Trimmers

먼저, 본 발명에 따라, 광증감제인 아미노레블리닉산의 세포 및 조직내로 침투효율을 높이기 위하여 구조체를 변경시킨다. 생체조직인 구연산을 이용하여 아미노레블리닉산이 구연산과 결합되어 트라이머 구조로 이루어지도록 한다.First, according to the present invention, the structure is changed in order to increase the penetration efficiency into the cells and tissues of the amino senblinic acid as a photosensitizer. Using amino acid citric acid, aminolevlynic acid is combined with citric acid to form a trimer structure.

도 1은 아미노레불리닉산 트라이머의 합성방법을 나타낸 것으로써, 도시된 바와 같이 구연산의 카르복실그룹에 아미노레불리닉산을 공유결합시켜 아미노레불리닉산 트라이머를 합성하였으며 합성물을 고성능액체크로마토그래피(HPLC)기기로 분석한 결과 모노머(monomer)는 3 % (w/w), 다이머(dimer)는 12 % (w/w), 트라이머(trimer)는 83 % (w/w), 그 외 구연산 등이 2 %를 차지하였다. 이 경우 원하는 트라이머(trimer)가 많은 비율로 합성되었으나, 이를 컬럼을 이용하여 다시 정제한 결과 모노머(monomer)는 1 % (w/w), 다이머(dimer)는 3 % (w/w), 트라이머(trimer)는 96 % (w/w)의 표준품을 얻을 수 있었다.1 shows a method for synthesizing an aminolevulinic acid trimer. As shown, an aminolevulinic acid trimer was synthesized by covalently binding an aminolevulinic acid to a carboxyl group of citric acid. As a result of analysis by (HPLC) device, monomer was 3% (w / w), dimer was 12% (w / w), trimer was 83% (w / w), and others. Citric acid and so on accounted for 2%. In this case, the desired trimer was synthesized in a large proportion, but the result of re-purification using the column was 1% (w / w) for monomers, 3% (w / w) for dimers, Trimers were able to obtain 96% (w / w) standard.

상기 과정을 좀 더 자세히 설명하면, 구연산 (Citric acid) 1M을 5 ml의 증류수에 녹인 후, 디메틸아미노프로필-에틸카르보디이미드-하이드로클로라이드(N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC)) 3M과, 하이드록시숙시미드(N-hydroxysuccimide) 3M과 아미노레블리닉산(5-aminolevulinic acid) 3M을 테트라히드로푸란(tetrahydrofuran (THF)/H20 (8/2)) 95 ml에 녹여 서로 혼합한다. 암실에서 약 24시간 동안 교반한 뒤 회전증발기(rotary evaporator)로 용매를 제거하고 동결건조하여 메탄올에 침전 시킨 후 재결정하여 실온에서 3일간 건조하고 플래쉬크로마토그래피(flash chromatography) (3/1=ethylacetate/hexane)로 정제한 뒤, 디클로로메탄/헥산(dichloromethane (DCM /hexane) 으로 재결정하여 흰색의 고체분말을 얻었다. In more detail, 1M citric acid is dissolved in 5 ml of distilled water, and then dimethylaminopropyl-ethylcarbodiimide-hydrochloride (N- (3-Dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride ( EDC)) 3M, hydroxysuccimide 3M and 5-aminolevulinic acid 3M in 95 ml of tetrahydrofuran (THF) / H 2 0 (8/2)) Melt and mix with each other. After stirring in the dark for about 24 hours, the solvent was removed by rotary evaporator, lyophilized, precipitated in methanol, recrystallized, dried for 3 days at room temperature, and flash chromatography (3/1 = ethylacetate / After purification with hexane, and recrystallized with dichloromethane (DCM / hexane) to give a white solid powder.

2. 아미노레블리닉산 트라이머의 분석2. Analysis of Aminolevulinic Acid Trimmers

1에서 합성한 아미노레불리닉산 트라이머가 원하는 결과대로 합성되었는지 여부와 모노머(monomer), 다이머(dimer), 트라이머(trimer)의 비율을 고성능액체크로마토그래피(High performance lliquid chromatography)로 분석하였고, 화학구조는 수소핵자기공명기( 1H nuclear magnetic resonance)로 분석하였다. 도 2는 위에서 합성한 아미노레불리닉산 트라이머의 수소 핵자기 공명 (1H NMR) 스펙트럼이다. 그림에서 보는 바와 같이 약 2.6 ppm에서 구연산의 특징 피크가 나타나며 (도2(a)) 아미노레불리닉산은 2.7 ppm, 3.8 ppm에서 특정 피크를 나타낸다 (도 2(b)). 아미노레불리닉산 트라이머는 2.6, 2.7, 3.8 ppm에서 모두 특정 피크가 나타남으로써 아미노레불리닉산 트라이머가 잘 합성되었음을 알 수 있었다. The aminolevulinic acid trimer synthesized in step 1 was synthesized as desired and the ratios of monomer, dimer and trimer were analyzed by high performance lliquid chromatography. The structure was analyzed by 1 H nuclear magnetic resonance. 2 is a hydrogen nuclear magnetic resonance ( 1 H NMR) spectrum of the aminolevulinic acid trimer synthesized above. As shown in the figure, a characteristic peak of citric acid appears at about 2.6 ppm (Fig. 2 (a)) and aminolevulinic acid shows a specific peak at 2.7 ppm and 3.8 ppm (Fig. 2 (b)). The aminolevulinic acid trimers showed specific peaks at 2.6, 2.7, and 3.8 ppm, indicating that aminolevulinic acid trimers were well synthesized.

3. 나노입자의 제조 및 분석3. Preparation and Analysis of Nanoparticles

상기 2에서 얻은 아미노레블리닉산 트라이머를 pH 4.5의 버퍼 용액 또는 증류수에 녹인 후 (또는 증류수 +DMSO) pH 8.5의 버퍼용액에 떨어뜨려 나노입자를 제조하였고, 필요에 따라 증류수에 대하여 투석을 하여 염을 제거하였다. 이러한 나노입자의 제조는 당업계에서 일반적인 제조과정이므로 이하 자세한 설명은 생략하기로 한다. 나노입자가 제대로 제조되었는지 확인하기 위하여 아미노레블리닉산 트라이머를 고성은 액체 크로마토그래피(High performance lliquid chromatography)와 수소 핵자기공명기(1H nuclear magnetic resonance)로 분석하였다 (500 MHz Superconducting FT-NMR Spectrometer, Unity-Inova 500). 나노파티클의 모양 (형태)은 주사 전자 현미경을 사용하여 분석하였다 (TEM, JEOL JEM-2000 FX Ⅱ, Japan). The aminolevlynic acid trimer obtained in 2 was dissolved in a buffer solution of pH 4.5 or distilled water (or distilled water + DMSO) and then dropped in a buffer solution of pH 8.5 to prepare nanoparticles, and dialysis was performed on distilled water as needed. The salt was removed. Since the preparation of such nanoparticles is a general manufacturing process in the art, the following detailed description will be omitted. In order to confirm that the nanoparticles were prepared properly, aminolevininic acid trimers were analyzed by high performance lliquid chromatography and 1 H nuclear magnetic resonance (500 MHz Superconducting FT-NMR Spectrometer). , Unity-Inova 500). The shape (morphology) of the nanoparticles was analyzed using a scanning electron microscope (TEM, JEOL JEM-2000 FX II, Japan).

나노입자 형태 제작과정을 간단히 설명하면, 나노파티클 용매 한 방울을 copper grid위가 코팅된 탄소 필름에 올려놓는다. Phosphotungstic acid (0.05 % (w/w))를 음성 염색에 사용하였다. 나노파티클의 관찰은 80 kV에서 측정하였다. 나노파티클의 크기는 dynamic laser scattering (DLS-7000, Otsuka Electonics Co. Japan)로 측정되었고, 입자 크기(concentration: 1 mg/ml)를 결정하기 위해서 샘플용액은 투석방법을 통해 준비하였다. 이와 같은 방법으로 나노파티클의 형성여부를 확인한 바 약 50 nm~300nm 전후의 나노입자가 형성된 것을 알 수 있었으며, 이는 대조군인 아미노레블리닉산의 기존에 사용되는 크기와 대동소이한 것으로써 원하는 적정한 형태의 크기의 나노입자가 형성됨을 알 수 있었다. Briefly describing the nanoparticle morphology, a drop of nanoparticle solvent is placed on a carbon film coated on a copper grid. Phosphotungstic acid (0.05% (w / w)) was used for negative staining. Observation of nanoparticles was measured at 80 kV. The size of the nanoparticles was measured by dynamic laser scattering (DLS-7000, Otsuka Electonics Co. Japan), and the sample solution was prepared by dialysis to determine the particle size (concentration: 1 mg / ml). As a result of confirming the formation of nanoparticles in this manner, it was found that the nanoparticles were formed around 50 nm to 300 nm, which is similar to the size of the conventional aminolevlinic acid as a control group, which is desired. It was found that nanoparticles of size were formed.

4. 세포배양 및 세포독성 실험4. Cell culture and cytotoxicity experiment

HSRRB (일본)에서 구입한 담도암 세포주인 HuCC-T1세포를 사용하여 세포 실험을 진행하였다. 세포는 RPMI1640배지를 이용하여 CO2 배양기에서 37oC에서 배양하였다. HuCC-T1 세포를 6웰에 각 웰당 10,000 개의 세포를 깔고 하루밤 동안 배양한 뒤, serum이 없는 배지로 교환한 뒤 대조군인 아미노레불리닉산과 본 발명에 의한 아미노레불리닉산 트라이머를 각 농도에 따라 처리하였다. 24시간 동안 암조건에서 배양한 후 10 % FBS가 들어 있는 RPMI배지로 교환해주고 24시간동안 배양한 후 MTT분석방법으로 세포의 생존율을 구하였다.Cell experiments were performed using HuCC-T1 cells, a biliary tract cancer cell line purchased from HSRRB (Japan). Cells were cultured at 37 ° C. in a CO 2 incubator using RPMI 1640 medium. HuCC-T1 cells were placed in 6 wells for 10,000 cells per well and incubated overnight, and then exchanged with serum-free medium. The aminolevulinic acid control group and the aminolevulinic acid trimer according to the present invention were added to each concentration. Treated accordingly. After culturing for 24 hours in dark conditions, the cells were exchanged with RPMI medium containing 10% FBS, and cultured for 24 hours, and cell viability was determined by MTT assay.

도 3은 대조군인 아미노레불리닉산과 본 발명에 의한 아미노레불리닉산 트라이머의 세포독성을 평가한 그래프이다. 도시된 바와 같이, 2mM 의 농도 까지 약물을 HuCC-T1 세포에 처리한 결과 대조군인 아미노레불리닉산과 본 발명에 의한 아미노레불리닉산 트라이머를 처리한 군에서 별다른 세포독성의 차이를 볼 수 없었다. 즉, 본 발명에 의한 아미노레블리닉산 트라이머 역시 인체에 어떠한 부작용을 야기 시키지 않으므로 광증감제로써 사용이 가능함을 확인할 수 있었다. Figure 3 is a graph evaluating the cytotoxicity of the aminolevulinic acid as a control and the aminolevulinic acid trimer according to the present invention. As shown, when the drug was treated to HuCC-T1 cells up to a concentration of 2 mM, there was no difference in cytotoxicity between the control group aminolevulinic acid and the aminolevulinic acid trimer according to the present invention. . That is, the aminolevlynic acid trimer according to the present invention also did not cause any adverse effects on the human body, so it could be confirmed that it can be used as a photosensitizer.

5. 프로토포피린IX(PpIX) 측정5. ProtopophyrinIX (PpIX) Measurement

대조군인 아미노레불리닉산과 본 발명에 의한 아미노레불리닉산 트라이머의 처리에 따른 HuCC-T1세포 내에 프로토포피린IX(PpIX)의 축적은 다음과 같이 실험하였다. 아미노레불리닉산과 아미노레불리닉산 트라이머를 각 농도에 따라 처리하고 24시간 암조건에서 배양한 뒤, 세포용해버퍼(cell lysis buffer)로 세포를 용해시킨 후 세포내에 축적된 프로토포피린IX(PpIX)양을 마이크로플레이트 리더(microplate reader (Infinite M200 pro, Tecan, Austria))로 측정하였다. 프로토포피린IX(PpIX)에 대한 여기파장길이(excitation wavelength)는 485nm였고, 방출파장길이(emission wavelength)는 635nm 였다. 측정된 프로토포피린IX(PpIX)양은 단백질양에 대하여 보정하였다. 도 4는 아미노레불리닉산과 아미노레불리닉산 트라이머를 HuCC-T1 세포에 동일 농도로 처리한 후 세포내에서 전환된 PpIX의 양을 정량한 것이다. 도시된 바와 같이, 1mM의 농도에서 아미노레불리닉산 트라이머를 처리한 세포군에서 약 20 % 증가한 PpIX 생성량을 보였다. 이 결과로 같은 농도의 아미노레불리닉산을 세포에 처리했을 때 자유로운 형태의 대조군인 아미노레불리닉산에 비해 아미노레불리닉산 트라이머의 경우 세포내 흡수가 증가된 것을 간접적으로 알 수 있었다. 따라서 본 본 발명에 의한 아미노레르블리닉산 트라이머가 기존 에 사용되는 아미노레블리닉산 보다 세포 및 조직내로 흡수가 잘되어 프로토포피린IX(PpIX)의 생성을 보다 원활하게 유도하는 반면에 부작용을 야기하는 독성은 나타나지 않으므로 보다 개량된 물질임을 알 수 있다. Accumulation of Protopophyrin IX (PpIX) in HuCC-T1 cells following treatment of aminolevulinic acid as a control and aminolevulinic acid trimer according to the present invention was tested as follows. Aminolevulinic acid and aminolevulinic acid trimers were treated at different concentrations and incubated in dark conditions for 24 hours. After lysing the cells with a cell lysis buffer, Protopophyrin IX (PpIX) accumulated in the cells was accumulated. The amount was measured with a microplate reader (Infinite M200 pro, Tecan, Austria). The excitation wavelength for Protoporphyrin IX (PpIX) was 485 nm and the emission wavelength was 635 nm. The measured amount of Protopophyrin IX (PpIX) was corrected for the amount of protein. Figure 4 quantifies the amount of PpIX converted intracellularly after treatment with aminolevulinic acid and aminolevulinic acid trimer at the same concentration in HuCC-T1 cells. As shown, PpIX production increased by about 20% in the cell group treated with aminolevulinic acid trimer at a concentration of 1 mM. As a result, when the same concentration of aminolevulinic acid was treated to cells, it was indirectly observed that the aminolevulinic acid trimer increased intracellular uptake compared to aminolevulinic acid, which is a free form control. Therefore, the aminollevulinic acid trimer according to the present invention is more readily absorbed into cells and tissues than the aminolevlynic acid used in the present invention, thereby inducing the production of protopopyrine IX (PpIX) more smoothly and causing side effects. Since it does not appear, it can be seen that it is a more improved material.

6. 광역동치료 (Photodynamic therapy, PDT) 효과 확인6. Confirmation of Photodynamic Therapy (PDT) Effect

광역동치료를 위해 LED램프 (SH system, Korea)를 사용하였으며 파장은 635 nm였고 빛의 강도는 0.25 J/cm2,였다 (photo-radiometer로 측정 (DeltaOhm, Italy)). HuCC-T1세포에 대조군인 아미노레불리닉산과 본 발명에 의한 아미노레불리닉산 트라이머를 농도에 따라 처리한 후 24시간동안 암조건에서 배양하고 635 nm, 0.25J/cm2 로 처리하였다. 배지를 10 % FBS 배지로 교환하고 24시간동안 배양한 뒤 MTT 분석방법으로 세포의 생존능을 분석하였다. 도 5는 아미노레불리닉산과 아미노레불리닉산 트라이머를 HuCC-T1 세포에 농도별로 처리한 뒤 빛을 조사하여 세포사멸을 평가한 것이다. 도 4에서 확인한 바와 같이 생성된 프로토포피린IX(PpIX)의 양이 증가할수록 세포 사멸 또한 증가하는 양상을 아미노레불리닉산 트라이머를 처리한 실험군에서 확인할 수 있었다. 따라서 본 발명에 의한 구연산을 이용한 아미노레블리닉산 트라이머는 암세포 및 종양조직내로의 선택적 흡수가 보다 원활히 이루어지고, 이에 따라 보다 많은 양의 프로토포피린IX(PpIX)의 생성을 유도하며 생성된 프로토포피린IX(PpIX)은 광조사에 의해 활성화되어 흡수된 암세포 및 암조직의 특정부위에서 세포사멸을 유도함으로써 보다 획기적인 광증감제로써의 역할을 수행할 수 있게 됨을 알 수 있다.LED lamp (SH system, Korea) was used for photodynamic therapy. The wavelength was 635 nm and the intensity of light was 0.25 J / cm 2 (measured by photo-radiometer (DeltaOhm, Italy)). After treatment with aminolevulinic acid as a control group and aminolevulinic acid trimer according to the present invention, HuCC-T1 cells were cultured in dark conditions for 24 hours and treated with 635 nm and 0.25 J / cm 2 . After replacing the medium with 10% FBS medium and incubated for 24 hours, the viability of the cells was analyzed by MTT assay. 5 is treated with aminolevulinic acid and aminolevulinic acid trimer concentrations in HuCC-T1 cells and then irradiated with light to evaluate apoptosis. As shown in FIG. 4, the cell death was also increased as the amount of Protopophyrin IX (PpIX) produced was increased in the experimental group treated with the aminolevulinic acid trimer. Therefore, the aminolevlynic acid trimer using citric acid according to the present invention facilitates selective absorption into cancer cells and tumor tissues, thereby inducing the production of higher amounts of protopophyrin IX (PpIX) and resulting protopophyrin IX. (PpIX) can be seen to be able to play a role as a more innovative photosensitizer by inducing apoptosis in specific areas of cancer cells and cancer tissues that are activated and absorbed by light irradiation.

그리고, " 본 연구는 보건복지부 보건의료연구개발사업의 지원에 의하여 이루어진 것임을 밝힙니다. (과제 고유번호 : A091047) "And, "This research was made with the support of the Ministry of Health and Welfare's medical research and development project. (Task ID: A091047)"

Claims (3)

광역동치료(photodynamic therapy PDT)에 사용되는 아미노레블리닉산 광증감제에 있어서,
아미노레블리닉산 광증감제는, 시트릭산(citric acid)의 카르복실그룹에 아미노레블리닉산이 공유결합되어 이루어지는 아미노레블리닉산 트라이머를 특징으로 하는 것을 광역동치료의 광증감제로 사용하기 위한 구연산을 이용한 아미노레블리닉산 트라이머.In aminolevulinic acid photosensitizers used for photodynamic therapy PDT,
Aminolevulinic acid photosensitizers are characterized by aminolevlynic acid trimers in which aminolevlynic acid is covalently bonded to a carboxyl group of citric acid for use as a photosensitizer for photodynamic therapy. Aminolevynic acid trimer using citric acid. 제1항에 있어서,
상기 아미노레블리닉산 트라이머는, 구연산 (Citric acid) 1M을 5 ml의 증류수에 녹인 용액과; 디메틸아미노프로필-에틸카르보디이미드-하이드로클로라이드(N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC)) 3M과, 하이드록시숙시미드(N-hydroxysuccimide) 3M과 아미노레블리닉산(5-aminolevulinic acid) 3M을 테트라히드로푸란(tetrahydrofuran (THF)/H20 (8/2)) 95 ml에 녹여 서로 혼합한 용액;의 비율로 혼합하여 암실에서 약 24시간 동안 교반한 뒤 용매를 제거하고 동결건조하여 메탄올에 침전 시킨 후 재결정하여 실온에서 건조하고 플래쉬크로마토그래피(flash chromatography) (3/1=ethylacetate/hexane)로 정제한 뒤, 디클로로메탄/헥산(dichloromethane (DCM) /hexane)으로 재결정 형성되는 것으로 순도 90%이상으로 이루어지는 것을 특징으로 하는 광역동치료의 광증감제로 사용하기 위한 구연산을 이용한 아미노레블리닉산 트라이머.The method of claim 1,
The aminolevlynic acid trimer may include a solution of 1 M of citric acid dissolved in 5 ml of distilled water; Dimethylaminopropyl-ethylcarbodiimide-hydrochloride (N- (3-Dimethylaminopropyl) -N'-ethylcarbodiimide hydrochloride (EDC)) 3M, hydroxysuccimide 3M and aminolevlynic acid (5- Aminolevulinic acid) 3M was dissolved in 95 ml of tetrahydrofuran (THF) / H 2 0 (8/2) and mixed with each other; the mixture was mixed at a ratio of about 24 hours in a dark room, and then the solvent was removed. Lyophilized, precipitated in methanol, recrystallized, dried at room temperature, purified by flash chromatography (3/1 = ethylacetate / hexane), and then recrystallized with dichloromethane (DCM) / hexane. An aminolevininic acid trimer using citric acid for use as a photosensitizer for photodynamic therapy, characterized in that the purity is made up to 90% or more. 제 1항 또는 제 2항에 있어서,
상기 광증감제는, 50 ~ 300nm 크기의 나노입자 형태로 이루어짐을 특징으로 하는 광역동치료의 광증감제로 사용하기 위한 구연산을 이용한 아미노레블리닉산 트라이머.3. The method according to claim 1 or 2,
The photosensitizer, aminolevlynic acid trimer using citric acid for use as a photosensitizer for photodynamic therapy, characterized in that the nanoparticles of the size of 50 ~ 300nm.
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