KR20240002134A - Composition for preventing, improving or treating nonalcoholic steatohepatitis comprising tenofovir alafenamide - Google Patents
Composition for preventing, improving or treating nonalcoholic steatohepatitis comprising tenofovir alafenamide Download PDFInfo
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- KR20240002134A KR20240002134A KR1020220174657A KR20220174657A KR20240002134A KR 20240002134 A KR20240002134 A KR 20240002134A KR 1020220174657 A KR1020220174657 A KR 1020220174657A KR 20220174657 A KR20220174657 A KR 20220174657A KR 20240002134 A KR20240002134 A KR 20240002134A
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Classifications
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- A—HUMAN NECESSITIES
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- A—HUMAN NECESSITIES
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- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2250/00—Food ingredients
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Abstract
본 발명은 테노포비르 알라페나미드(tenofovir alafenamide, TAF) 또는 이의 약학적으로 허용 가능한 염을 포함하는 비알콜성 지방간 질환, 특히 비알콜성 지방간염 예방, 개선 또는 치료용 조성물에 대한 것이다.
본 발명의 상기 조성물은, 비알콜성 지방간염 특이적으로 간 손상 지표인 AST 및 ALT 의 수치와 염증성 대식세포의 수치를 유의적으로 감소시킬 수 있으므로, 비알콜성 지방간 질환, 특히 비알콜성 지방간염 예방, 개선 또는 치료용 조성물로서 효과적으로 사용될 수 있다.The present invention relates to a composition for preventing, improving or treating non-alcoholic fatty liver disease, particularly non-alcoholic steatohepatitis, containing tenofovir alafenamide (TAF) or a pharmaceutically acceptable salt thereof.
The composition of the present invention can significantly reduce the levels of AST and ALT, which are indicators of liver damage, and the levels of inflammatory macrophages, specifically in non-alcoholic steatohepatitis, and thus, non-alcoholic fatty liver disease, especially non-alcoholic fatty liver disease. It can be effectively used as a composition for preventing, improving, or treating hepatitis.
Description
본 발명은 테노포비르 알라페나미드(tenofovir alafenamide, TAF) 또는 이의 약학적으로 허용 가능한 염을 포함하는 비알콜성 지방간 질환, 특히 비알콜성 지방간염 예방, 개선 또는 치료용 조성물에 대한 것이다.The present invention relates to a composition for preventing, improving or treating non-alcoholic fatty liver disease, particularly non-alcoholic steatohepatitis, containing tenofovir alafenamide (TAF) or a pharmaceutically acceptable salt thereof.
비알코올성 지방간염(NASH, Nonalcoholic fatty liver disease)은 간 지방증, 간세포 손상 및 간 염증을 특징으로 하며, 간경화 또는 간세포 암종을 포함하여 더 심각한 단계로 진행된다. NASH는 매우 널리 퍼져 있지만 현재 이 질병에 대한 효과적인 치료법은 없다. NASH 치료제는 현재 다양한 타깃을 대상으로 개발되고 있으며 여러 임상시험이 진행 중이다. 소수의 약물만이 3상 시험에 있는 반면 다른 약물은 1상 또는 2상 시험에 남아 있다. 미국 식품의약국은 아직 NASH 치료를 위한 특정 약물을 승인하지 않았다. 따라서 새롭고 개선된 치료제 개발이 필수적이다.Nonalcoholic fatty liver disease (NASH) is characterized by hepatic steatosis, hepatocyte damage, and liver inflammation, progressing to more severe stages including cirrhosis or hepatocellular carcinoma. NASH is very prevalent, but there is currently no effective treatment for this disease. NASH treatments are currently being developed for various targets, and several clinical trials are in progress. Only a few drugs are in phase 3 trials, while others remain in phase 1 or 2 trials. The U.S. Food and Drug Administration has not yet approved any specific medications for the treatment of NASH. Therefore, the development of new and improved treatments is essential.
간세포, 간 성상 세포(HSC, hepatic stellate cells) 및 다양한 면역 세포 사이의 복잡한 혼선은 NASH 진행 중에 활성화된다. 이 과정에서 염증 신호는 간에서 단핵 식세포(MP, mononuclear phagocyte) 풀 크기를 증가시킨다. NASH 마우스 간의 단일 세포 RNA 시퀀싱은 정상 마우스 간과 비교하여 증가된 MP 클러스터 크기를 보여주었다. 최근 연구는 NASH 마우스 모델에서 MP의 다양성에 초점을 맞췄다. 간내 MP는 전통적으로 Kupffer 세포(KC)와 단핵구 유래 대식세포(MoMF)의 두 집단으로 분류된다. KC는 간 상주 식세포이다. MoMF는 순환에서 간 손상 부위로 모집된다. 단핵구는 간에 침투하고 모집된 단핵구는 모집된 간내 MP로 분화하여 만성적으로 염증이 있는 간에서 HSC 활성화를 촉진한다. 최근의 단일 세포 분석은 또한 모집된 MP와 간내 MP가 NAFLD 진행 동안 뚜렷한 염증 표현형을 나타낸다는 것을 보여주었다.A complex crosstalk between hepatocytes, hepatic stellate cells (HSCs), and various immune cells is activated during NASH progression. In this process, inflammatory signals increase the size of the mononuclear phagocyte (MP) pool in the liver. Single-cell RNA sequencing of NASH mouse liver showed increased MP cluster size compared to normal mouse liver. Recent studies have focused on the diversity of MPs in mouse models of NASH. Intrahepatic MPs are traditionally classified into two populations: Kupffer cells (KC) and monocyte-derived macrophages (MoMF). KCs are liver-resident phagocytes. MoMF is recruited from the circulation to sites of liver injury. Monocytes infiltrate the liver, and recruited monocytes differentiate into recruited intrahepatic MPs, promoting HSC activation in the chronically inflamed liver. A recent single cell analysis also showed that recruited MPs and intrahepatic MPs display distinct inflammatory phenotypes during NAFLD progression.
인간 면역결핍 및 B형 간염 바이러스 감염을 치료하기 위해 2개의 테노포비르 전구약물이 현재 이용가능하다. TDF(Tenofovir disoproxil fumarate)와 TAF(tenofovir alafenamide)는 간 섬유화 퇴행을 유발하는 강력한 항바이러스 활성을 가진 일반적으로 사용되는 항바이러스제이다. 그러나 현재 TAF 치료가 NASH 퇴행에 미치는 영향을 보여주는 보고는 없었다. Two tenofovir prodrugs are currently available to treat human immunodeficiency and hepatitis B virus infection. Tenofovir disoproxil fumarate (TDF) and tenofovir alafenamide (TAF) are commonly used antiviral drugs with potent antiviral activity that induce liver fibrotic regression. However, there are currently no reports showing the effect of TAF treatment on NASH regression.
본 발명은 효과적인 비알콜성 지방간 질환 치료용 조성물을 제공하고자 예의 노력한 결과, 테노포비르 알라페나미드를 이용하는 경우 적은 양으로도 간 손상 내지 간 염증 관련 수치를 유의적으로 감소시킬 수 있음을 확인하고 본 발명을 완성하였다. The present invention has made diligent efforts to provide an effective composition for treating non-alcoholic fatty liver disease, and as a result, it has been confirmed that when using tenofovir alafenamide, liver damage and liver inflammation-related values can be significantly reduced even in small amounts. The present invention has been completed.
따라서, 본 발명의 목적은 테노포비르 알라페나미드(tenofovir alafenamide, TAF) 또는 이의 약학적으로 허용 가능한 염을 포함하는 비알콜성 지방간 질환, 특히 비알콜성 지방간염 예방, 개선 또는 치료용 조성물을 제공하는 것이다.Therefore, the object of the present invention is to provide a composition for preventing, improving or treating non-alcoholic fatty liver disease, especially non-alcoholic steatohepatitis, containing tenofovir alafenamide (TAF) or a pharmaceutically acceptable salt thereof. It is provided.
본 발명은 테노포비르 알라페나미드(tenofovir alafenamide, TAF) 또는 이의 약학적으로 허용 가능한 염을 포함하는 비알콜성 지방간 질환 예방 또는 치료용 약학적 조성물을 제공한다. The present invention provides a pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease containing tenofovir alafenamide (TAF) or a pharmaceutically acceptable salt thereof.
본 발명의 바람직한 일실시예에 따르면, 상기 비알콜성 지방간 질환은 비알콜성 지방간염인 것이다. According to a preferred embodiment of the present invention, the non-alcoholic fatty liver disease is non-alcoholic steatohepatitis.
본 발명의 바람직한 일실시예에 따르면, 상기 조성물은 비알콜성 지방간 질환의 염증을 억제하는 것이다. According to a preferred embodiment of the present invention, the composition suppresses inflammation in non-alcoholic fatty liver disease.
본 발명의 바람직한 일실시예에 따르면, 상기 염증은 정상대조군에 비하여 증가한 염증성 대식세포에 의한 염증인 것이다. According to a preferred embodiment of the present invention, the inflammation is caused by increased inflammatory macrophages compared to the normal control group.
본 발명의 바람직한 일실시예에 따르면, 상기 조성물은 정상대조군에 비하여 증가한 혈액 내 간 효소 마커 AST(aspartate aminotransferase), ALT(alanine aminotransferase) 및 트리글리세라이드(triglyceride) 로 이루어진 군에서 선택되는 어느 하나 이상의 수치를 감소시키는 것이다. According to a preferred embodiment of the present invention, the composition has an increased level of liver enzyme markers AST (aspartate aminotransferase), ALT (alanine aminotransferase), and triglyceride in the blood compared to the normal control group. is to reduce.
또한, 본 발명은 테노포비르 알라페나미드 푸마르산(tenofovir alafenamide, TAF) 또는 이의 약학적으로 허용 가능한 염을 포함하는 비알콜성 지방간 질환 예방 또는 개선용 건강기능식품 조성물을 제공한다.In addition, the present invention provides a health functional food composition for preventing or improving non-alcoholic fatty liver disease containing tenofovir alafenamide fumaric acid (TAF) or a pharmaceutically acceptable salt thereof.
본 발명의 테노포비르 알라페나미드 푸마르산을 포함하는 조성물은, 비알콜성 지방간염 특이적으로 간 손상 지표인 AST 및 ALT 의 수치와 염증성 대식세포의 수치를 유의적으로 감소시킬 수 있으므로, 비알콜성 지방간 질환, 특히 비알콜성 지방간염 예방, 개선 또는 치료용 조성물로서 효과적으로 사용될 수 있다.The composition containing tenofovir alafenamide fumaric acid of the present invention can significantly reduce the levels of AST and ALT, which are indicators of liver damage, and the levels of inflammatory macrophages, specifically for non-alcoholic steatohepatitis, and thus, non-alcoholic It can be effectively used as a composition for preventing, improving or treating fatty liver disease, especially non-alcoholic steatohepatitis.
도 1a 는 테노포비르 알라페나미드(TAF) 처리는 비알코올성 지방간염(NASH) 마우스 모델에서 간 손상을 개선하는 것으로서, 스트렙토조토신(STZ) 주사 및 고지방, 고콜레스테롤(HFHC) 식이를 사용한 NASH 마우스 모델에 대한 실험 일정을 나타낸다. C57BL/6J 마우스에게 STZ 0.2 mg을 주사하고 HFHC 식이 또는 탄수화물(CHO) 식이를 공급하고 모의 또는 TAF로 처리했다. NASH는 STZ 피하 주사 후 4주(n = 6-10) 동안 HFHC 식이 요법으로 인해 발생했다.
도 1b 는 테노포비르 알라페나미드(TAF) 처리는 비알코올성 지방간염(NASH) 마우스 모델에서 간 손상을 개선하는 것으로서, 모의 또는 TAF로 처리된 STZ, HFHC-유도 NASH 실험 동물의 혈청 알라닌 아미노전이효소(ALT), 아스파르테이트 아미노전이효소(AST) 및 트리글리세라이드(TRIG) 수준을 나타낸다.
도 1c 는 테노포비르 알라페나미드(TAF) 처리는 비알코올성 지방간염(NASH) 마우스 모델에서 간 손상을 개선하는 것으로서, STZ 주입된 모의 처리된 HFHC-식이 및 STZ 주입된 TAF-처리된 HFHC 식이 마우스의 간을 나타낸다. 원본 배율: 50×.
도 1d 는 테노포비르 알라페나미드(TAF) 처리는 비알코올성 지방간염(NASH) 마우스 모델에서 간 손상을 개선하는 것으로서, 모의 또는 TAF로 처리된 STZ, HFHC-유도된 NASH 실험 동물 사이의 LPS 또는 TLR4 양성 세포의 빈도의 비교를 나타낸다. 시리우스 레드로 염색된 간 절편의 형태계측 분석은 그룹당 5개의 간 절편의 3개 필드에서 수행되었다. 각 처리군 간 마우스 간 표본의 NASH 활동 점수(NAS)를 비교하였다.
도 1e 는 테노포비르 알라페나미드(TAF) 처리는 비알코올성 지방간염(NASH) 마우스 모델에서 간 손상을 개선하는 것으로서, CDAHF 식이 처리 모의 또는 TAF를 사용한 NASH 마우스 모델에 대한 실험 일정을 나타낸다. C57BL/6J 마우스에게 CDAHF 식이 또는 CHO 식이를 6주 동안 공급했다(n = 7-10).
도 1f 는 테노포비르 알라페나미드(TAF) 처리는 비알코올성 지방간염(NASH) 마우스 모델에서 간 손상을 개선하는 것을 것으로서, 모의 또는 TAF로 처리된 CDAHF-유도된 NASH 실험 동물의 혈청 ALT, AST 및 TRIG 수준을 나타낸다. *p<0.05, **p<0.001, ***p<0.001.
도 2a 는 테노포비르 알라페나미드(TAF) 처리는 비알코올성 지방간염(NASH) 마우스 모델에서 간 손상을 개선하는 것을 나타낸다. 체중과 임의의 포도당 수치를 2주 간격으로 측정한 결과, STZ 주입된 모의 처리된 HFHC-식이로 인해 NASH가 유도되었음을 확인하였다. 또한 TAF 처리된 HFHC 식이를 먹인 쥐의 총 간 무게는 모의 처리된 쥐보다 낮았다.
도 2b 는 테노포비르 알라페나미드(TAF) 처리는 비알코올성 지방간염(NASH) 마우스 모델에서 간 손상을 개선하는 것을 나타낸다. 체중과 임의의 포도당 수치를 2주 간격으로 측정한 결과, CDAHF-식이로 인해 NASH가 유도되었음을 확인하였다. 또한 TAF 처리된 CDAHF 식이를 먹인 쥐의 총 간 무게는 모의 처리된 쥐보다 낮았다.
도 2c 는 테노포비르 알라페나미드(TAF) 처리는 비알코올성 지방간염(NASH) 마우스 모델에서 간 손상을 개선하는 것을 나타낸다. 포름알데하이드로 고정된 간 절편의 H&E 염색은 TAF 처리된 CDAHF-식이 요법 그룹의 간에서 모의 처리된 간과 비교하여 간세포 괴사와 동모양혈관의 울혈(sinusoidal congestion)이 유의하게 감소되었다.
도 3a 는 유동 세포 계측법을 사용하여 간내 활성화된 MP를 정량화하기 위한 게이팅 전략을 나타낸다.
도 3b 는 간 무게 1g당 활성화된 CD11b+ F4/80+ MP의 수는 모의 처리 그룹보다 TAF 처리 HFHC 식이 그룹에서 유의하게 감소했다.
도 3c 는 간 무게 1g당 활성화된 CD11b+ F4/80+ MP의 수는 모의 처리 그룹보다 TAF 처리 CDAHF 식이 그룹에서 유의하게 감소했다.
도 4a 는 TAF는 STZ, HFHC 식이 유발 NASH 마우스 간에서 활성화된 단핵 식세포(MP)의 수를 감소시키는 것으로서, 모집/상주 MP의 비율을 설명하는 대표적인 도트 플롯을 나타낸다. 데이터는 5개의 독립적인 실험을 대표한다.
도 4b 는 TAF는 STZ, HFHC 식이 유발 NASH 마우스 간에서 활성화된 단핵 식세포(MP)의 수를 감소시키는 것으로서, 모집 MP(CD11bhigh F4/80low) 및 상주 MP(CD11blow F4/80high) 간 중량 1g당 MP 세포 수 및 모의 또는 TAF 처리된 STZ, HFHC 식이 유발 NASH 마우스 모델에서 세포 백분율을 유세포 분석기를 사용하여 계산했다.
도 4c 는 TAF는 STZ, HFHC 식이 유발 NASH 마우스 간에서 활성화된 단핵 식세포(MP)의 수를 감소시키는 것으로서, CD11b+ F4/80+ 세포에서 프로그래밍된 사멸 리간드 1(PD-L1), IAIE 및 MER 원발암 유전자 및 티로신 키나제(MerTK)의 평균 형광 강도(MFI)를 나타낸다.
도 4d 는 TAF는 STZ, HFHC 식이 유발 NASH 마우스 간에서 활성화된 단핵 식세포(MP)의 수를 감소시키는 것으로서, 모의 또는 TAF를 처리한 STZ, HFHC 식이 유도 NASH 마우스 모델에서 PD-L1+/IAIE+/MerTK+ 간내 MP 세포의 빈도를 나타낸다.
도 4e 는 TAF는 STZ, HFHC 식이 유발 NASH 마우스 간에서 활성화된 단핵 식세포(MP)의 수를 감소시키는 것으로서, 모의 또는 TAF 처리된 NASH 마우스 간에서 염증 유전자의 상대적 mRNA 발현을 나타낸다. *p<0.05, **p<0.001, ***p<0.001.
도 5a 는 TAF는 CDAHF 식이 유발 NASH 마우스 간에서 활성화된 MP의 수를 감소시키는 것으로서, 모집/상주 MP의 비율을 설명하는 대표적인 도트 플롯을 나타낸다. 데이터는 5개의 독립적인 실험을 대표한다.
도 5b 는 TAF는 CDAHF 식이 유발 NASH 마우스 간에서 활성화된 MP의 수를 감소시키는 것으로서, 모집 MP(CD11bhigh F4/80low) 및 상주 MP(CD11blow F4/80high) 간 중량 1g당 MP 세포 수 및 모의 또는 TAF 처리된 CDAHF 식이 유발 NASH 마우스 모델에서 세포 백분율을 유세포 분석기를 사용하여 계산했다.
도 5c 는 TAF는 CDAHF 식이 유발 NASH 마우스 간에서 활성화된 MP의 수를 감소시키는 것으로서, CD11b+ F4/80+ 세포에서 PD-L1, IAIE 및 MerTK의 MFI를 나타낸다.
도 5d 는 TAF는 CDAHF 식이 유발 NASH 마우스 간에서 활성화된 MP의 수를 감소시키는 것으로서, 모의 또는 TAF를 처리한 CDAHF 식이 유발 NASH 마우스 모델에서 PD-L1+/IAIE+/MerTK+ 간내 MP 세포의 빈도를 나타낸다. *p<0.05, **p<0.001, ***p<0.001.
도 6a 는 티오아세트아미드(TAA) 유도 간 손상 마우스 모델의 간에서 분리된 MP에 대한 TAF의 ex vivo 효과로서, TAA 주입을 사용한 간 손상 마우스 모델 실험 일정(n = 5)을 나타낸다. 마우스에 100 mg/kg 및 150 mg/kg TAA를 일주일에 세 번 복강내 주사했다.
도 6b 는 티오아세트아미드(TAA) 유도 간 손상 마우스 모델의 간에서 분리된 MP에 대한 TAF의 ex vivo 효과로서, TAA 처리 마우스 간에서 H&E, 시리우스 레드로 염색된 간 절편의 대표적인 조직학적 분석 결과를 나타낸다. 원본 배율: 50×. 시리우스 레드로 염색된 간 절편의 형태계측 분석은 그룹당 5개의 간 절편의 3개 필드에서 수행되었다.
도 6c 는 티오아세트아미드(TAA) 유도 간 손상 마우스 모델의 간에서 분리된 MP에 대한 TAF의 ex vivo 효과로서, 모의 또는 TAF 처리된 간 손상 마우스 모델로부터 분리된 MP의 ex vivo 실험 계획을 나타낸다. 마우스 간을 관류시키고, 구배 원심분리를 사용하여 MP를 분리하였다.
도 6d 는 티오아세트아미드(TAA) 유도 간 손상 마우스 모델의 간에서 분리된 MP에 대한 TAF의 ex vivo 효과로서, 모의 또는 TAF 처리 후 분리된 CD11b+F4/80+ 세포 중 PD-L1+ 세포의 빈도 및 MFI를 나타낸다. *p<0.05, **p<0.01, ***p<0.001.
도 7a 는 TAF는 단핵구/MP에서 AKT의 인산화를 차단하는 것으로서, 모의 또는 TAF 처리된 STZ, HFHC-유도된 NASH 마우스 간 조직에서의 AKT 및 mTOR의 발현을 웨스턴 블롯팅을 사용하여 분석하였다. 인산화된 단백질의 상대적인 발현은 전체 단백질 발현으로 정규화되었다. 데이터는 3개의 독립적인 실험을 대표한다. 오른쪽 그래프는 정량적 밀도 측정 면역블롯팅 분석 결과를 보여준다.
도 7b 는 TAF는 단핵구/MP에서 AKT의 인산화를 차단하는 것으로서, 분리된 말초 CD14+ 단핵구의 실험 계획을 나타낸다. 세포를 6시간 동안 TAF(1, 5μM)로 전처리하고 24시간 동안 1μg/mL LPS로 자극했다. 모의 또는 TAF로 처리된 자극된 단핵구에서 표면 인간 백혈구 항원-DR 이소타입(HLA-DR) 및 PD-L1 수준의 도트 플롯. 데이터는 3개의 독립적인 실험을 대표한다.
도 7c 는 TAF는 단핵구/MP에서 AKT의 인산화를 차단하는 것으로서, 모의 또는 TAF로 처리된 활성화된 CD14+ 단핵구 세포에서 PD-L1+/HLA-DR+ 세포의 빈도를 나타낸다.
도 7d 는 TAF는 단핵구/MP에서 AKT의 인산화를 차단하는 것으로서, 모의 또는 TAF 처리 후 활성화된 CD14+ 단핵구 세포에서 PD-L1 및 HLA-DR의 MFI를 나타낸다.
도 7e 는 TAF는 단핵구/MP에서 AKT의 인산화를 차단하는 것으로서, 모의 또는 TAF 처리된 활성화된 말초 CD14+ 단핵구에서 AKT 및 pAKT 발현의 대표적인 도트 플롯은 유동 세포측정법을 사용하여 분석되었다. 모의 또는 TAF 처리된 활성화된 말초 CD14+ 단핵구에서 pAKT의 MFI는 유동 세포측정법을 사용하여 분석되었다.
도 7f 는 TAF는 단핵구/MP에서 AKT의 인산화를 차단하는 것으로서, NASH 간에서 TAF 역할의 도식적 표현을 나타낸다. *p<0.05, **p<0.001, ***p<0.001.
도 8은 In vivo STZ, HFHC 유도 NASH 마우스 모델 처리된 모의 또는 TAF에서 TAF에 의해 차단된 신호 전달 경로를 결정하기 위해 수행한 면역블롯팅의 전체 이미지를 나타낸다. Figure 1A shows that tenofovir alafenamide (TAF) treatment improves liver damage in a non-alcoholic steatohepatitis (NASH) mouse model using streptozotocin (STZ) injection and a high-fat, high-cholesterol (HFHC) diet. The experimental schedule for the mouse model is shown. C57BL/6J mice were injected with 0.2 mg of STZ, fed HFHC diet or carbohydrate (CHO) diet, and treated with mock or TAF. NASH occurred due to HFHC diet for 4 weeks (n = 6-10) after subcutaneous injection of STZ.
Figure 1b shows that tenofovir alafenamide (TAF) treatment improves liver damage in a non-alcoholic steatohepatitis (NASH) mouse model, and serum alanine transamination of STZ and HFHC-induced NASH experimental animals treated with sham or TAF. Enzyme (ALT), aspartate aminotransferase (AST) and triglyceride (TRIG) levels are shown.
Figure 1C shows that tenofovir alafenamide (TAF) treatment ameliorate liver damage in a non-alcoholic steatohepatitis (NASH) mouse model in STZ-injected mock-treated HFHC-diet and STZ-injected TAF-treated HFHC diet. Represents the liver of a mouse. Original magnification: 50×.
Figure 1D shows that tenofovir alafenamide (TAF) treatment ameliorate liver damage in a non-alcoholic steatohepatitis (NASH) mouse model, compared with STZ, HFHC-induced NASH experimental animals treated with sham or TAF or LPS. A comparison of the frequencies of TLR4 positive cells is shown. Morphometric analysis of liver sections stained with Sirius red was performed in three fields of five liver sections per group. NASH activity scores (NAS) of mouse liver samples were compared between each treatment group.
Figure 1E shows that tenofovir alafenamide (TAF) treatment improves liver damage in a non-alcoholic steatohepatitis (NASH) mouse model, showing the experimental schedule for the NASH mouse model using sham CDAHF diet treatment or TAF. C57BL/6J mice were fed CDAHF diet or CHO diet for 6 weeks (n = 7-10).
Figure 1f shows that tenofovir alafenamide (TAF) treatment improves liver damage in a non-alcoholic steatohepatitis (NASH) mouse model, showing serum ALT and AST in CDAHF-induced NASH experimental animals treated with sham or TAF. and TRIG level. *p<0.05, **p<0.001, ***p<0.001.
Figure 2A shows that tenofovir alafenamide (TAF) treatment improves liver damage in a non-alcoholic steatohepatitis (NASH) mouse model. Body weight and random glucose levels were measured at two-week intervals, and it was confirmed that NASH was induced by the STZ-injected mock-treated HFHC-diet. Additionally, the total liver weight of rats fed the TAF-treated HFHC diet was lower than that of mock-treated rats.
Figure 2B shows that tenofovir alafenamide (TAF) treatment improves liver damage in a non-alcoholic steatohepatitis (NASH) mouse model. As a result of measuring body weight and arbitrary glucose levels at two-week intervals, it was confirmed that NASH was induced by the CDAHF-diet. Additionally, the total liver weight of mice fed the TAF-treated CDAHF diet was lower than that of mock-treated mice.
Figure 2C shows that tenofovir alafenamide (TAF) treatment improves liver damage in a non-alcoholic steatohepatitis (NASH) mouse model. H&E staining of formaldehyde-fixed liver sections showed that hepatocyte necrosis and sinusoidal congestion were significantly reduced in the livers of the TAF-treated CDAHF-diet group compared to the mock-treated livers.
Figure 3A shows the gating strategy for quantifying intrahepatic activated MPs using flow cytometry.
Figure 3b shows that the number of activated CD11b+ F4/80+ MPs per gram of liver weight was significantly reduced in the TAF-treated HFHC diet group than in the mock-treated group.
Figure 3C shows that the number of activated CD11b+ F4/80+ MPs per gram of liver weight was significantly reduced in the TAF-treated CDAHF diet group than in the mock-treated group.
Figure 4A shows that TAF reduces the number of activated mononuclear phagocytes (MPs) in STZ, HFHC diet-induced NASH mouse liver, showing a representative dot plot illustrating the ratio of recruited/resident MPs. Data are representative of five independent experiments.
Figure 4B shows that TAF reduces the number of activated mononuclear phagocytes (MPs) in STZ, HFHC diet-induced NASH mouse livers, showing the recruitment of recruited MPs (CD11b high F4/80 low ) and resident MPs (CD11b low F4/80 high ). MP cell counts per gram of weight and cell percentages in mock- or TAF-treated STZ, HFHC diet-induced NASH mouse model were calculated using flow cytometry.
Figure 4C shows that TAF reduces the number of activated mononuclear phagocytes (MP) in STZ, HFHC diet-induced NASH mouse livers, expressing programmed death ligand 1 (PD-L1), IAIE and MER on CD11b + F4/80 + cells. Mean fluorescence intensity (MFI) of proto-oncogene and tyrosine kinase (MerTK) is shown.
Figure 4d shows that TAF reduces the number of activated mononuclear phagocytes (MP) in the STZ, HFHC diet-induced NASH mouse liver, showing PD-L1+/IAIE+/MerTK+ in the STZ, HFHC diet-induced NASH mouse model treated with mock or TAF. Indicates the frequency of intrahepatic MP cells.
Figure 4e shows that TAF reduces the number of activated mononuclear phagocytes (MP) in the liver of STZ, HFHC diet-induced NASH mice, showing the relative mRNA expression of inflammatory genes in the liver of mock- or TAF-treated NASH mice. *p<0.05, **p<0.001, ***p<0.001.
Figure 5A shows a representative dot plot illustrating the ratio of recruited/resident MPs as TAF reduces the number of activated MPs in CDAHF diet-induced NASH mouse livers. Data are representative of five independent experiments.
Figure 5B shows that TAF reduces the number of activated MPs in CDAHF diet-induced NASH mouse livers, showing the number of recruited MPs (CD11b high F4/80 low ) and resident MPs (CD11b low F4/80 high ) MP cells per gram of liver weight. and cell percentages in the mock- or TAF-treated CDAHF diet-induced NASH mouse model were calculated using flow cytometry.
Figure 5C shows MFI of PD-L1, IAIE and MerTK in CD11b + F4/80 + cells, showing that TAF reduces the number of activated MPs in CDAHF diet-induced NASH mouse liver.
Figure 5D shows that TAF reduces the number of activated MPs in CDAHF diet-induced NASH mouse liver, showing the frequency of PD-L1+/IAIE+/MerTK+ intrahepatic MP cells in the CDAHF diet-induced NASH mouse model treated with mock or TAF. *p<0.05, **p<0.001, ***p<0.001.
Figure 6a shows the ex vivo effect of TAF on MP isolated from the liver of a thioacetamide (TAA)-induced liver injury mouse model, showing the experimental schedule (n = 5) of the liver injury mouse model using TAA injection. Mice were injected intraperitoneally with 100 mg/kg and 150 mg/kg TAA three times a week.
Figure 6b shows the ex vivo effect of TAF on MP isolated from the liver of a thioacetamide (TAA)-induced liver injury mouse model, showing representative histological analysis results of liver sections stained with H&E and Sirius Red from TAA-treated mouse liver. indicates. Original magnification: 50×. Morphometric analysis of liver sections stained with Sirius red was performed in three fields of five liver sections per group.
Figure 6c shows the ex vivo effect of TAF on MP isolated from the liver of a thioacetamide (TAA)-induced liver injury mouse model, showing the ex vivo experimental plan of MP isolated from a mock- or TAF-treated liver injury mouse model. Mouse livers were perfused and MPs were isolated using gradient centrifugation.
Figure 6d shows the ex vivo effect of TAF on MPs isolated from the liver of a mouse model of thioacetamide (TAA)-induced liver injury, showing the frequency of PD-L1+ cells among CD11b+F4/80+ cells isolated after mock or TAF treatment. and MFI. *p<0.05, **p<0.01, ***p<0.001.
Figure 7a shows that TAF blocks the phosphorylation of AKT in monocytes/MPs, and the expression of AKT and mTOR in mock- or TAF-treated STZ, HFHC-induced NASH mouse liver tissue was analyzed using Western blotting. Relative expression of phosphorylated proteins was normalized to total protein expression. Data are representative of three independent experiments. The graph on the right shows the results of quantitative densitometric immunoblotting analysis.
Figure 7b shows the experimental plan of isolated peripheral CD14+ monocytes as TAF blocks the phosphorylation of AKT in monocytes/MP. Cells were pretreated with TAF (1, 5 μM) for 6 h and stimulated with 1 μg/mL LPS for 24 h. Dot plot of surface human leukocyte antigen-DR isotype (HLA-DR) and PD-L1 levels in stimulated monocytes treated with mock or TAF. Data are representative of three independent experiments.
Figure 7c shows the frequency of PD-L1+/HLA-DR+ cells in activated CD14+ monocytes treated with mock or TAF, as TAF blocks phosphorylation of AKT in monocytes/MP.
Figure 7d shows the MFI of PD-L1 and HLA-DR in activated CD14+ monocyte cells after mock or TAF treatment, as TAF blocks the phosphorylation of AKT in monocytes/MP.
Figure 7E: TAF blocks phosphorylation of AKT in monocytes/MP. Representative dot plots of AKT and pAKT expression in mock- or TAF-treated activated peripheral CD14+ monocytes were analyzed using flow cytometry. The MFI of pAKT in mock- or TAF-treated activated peripheral CD14+ monocytes was analyzed using flow cytometry.
Figure 7f shows a schematic representation of the role of TAF in NASH liver, as TAF blocks phosphorylation of AKT in monocytes/MP. *p<0.05, **p<0.001, ***p<0.001.
Figure 8 shows full images of immunoblotting performed to determine signaling pathways blocked by TAF in mock or TAF treated in vivo STZ, HFHC-induced NASH mouse model.
이하, 본 발명을 보다 상세히 설명한다. Hereinafter, the present invention will be described in more detail.
본 발명의 '예방' 이란, 본 발명의 TAF 또는 이의 약학적으로 허용 가능한 염으로 인해 비알콜성 지방간 질환이 억제되거나 그 발병이 지연되도록 하는 모든 행위를 의미한다. 'Prevention' of the present invention refers to any action that suppresses or delays the onset of non-alcoholic fatty liver disease due to TAF or a pharmaceutically acceptable salt thereof of the present invention.
본 발명의 '개선' 또는 '치료' 란, 본 발명의 TAF 또는 이의 약학적으로 허용 가능한 염으로 인해 비알콜성 지방간 질환과 관련된 파라미터, 예를 들면 증상의 정도가 호전되거나 이롭게 되도록 하는 모든 행위를 의미한다.'Improvement' or 'treatment' of the present invention refers to any action that improves or benefits parameters related to non-alcoholic fatty liver disease, such as the degree of symptoms, due to TAF or a pharmaceutically acceptable salt thereof of the present invention. it means.
본 발명의 '정상대조군' 은 비알콜성 지방간 질환을 보유하지 않은 대조군을 의미할 수 있다. The 'normal control group' of the present invention may refer to a control group that does not have non-alcoholic fatty liver disease.
본 발명에서는 2개의 다른 in vivo NASH 마우스 모델이 구축하였다: 스트렙토조토신(STZ, streptozotocin; 0.2 mg)을 피하 주사하고 마우스에게 HFHC(high-fat, high-cholesterol) 식이 또는 CDAHF(choline-deficient, L-amino acid-defined, high-fat diet) 식이를 공급. 그 결과, TAF 처리된 NASH 마우스의 혈청 알라닌 아미노트랜스퍼라제 및 트리글리세라이드 수치는 모의 처리된 것보다 현저히 낮았다. TAF 처리된 NASH 마우스의 간은 모의 처리된 것의 간과 비교하여 단핵 식세포(MP, mononuclear phagocyte) 침윤이 약화된 것으로 나타났다. TAF 처리된 NASH 마우스는 활성화된 MP(IAIE+/PD-L1+/MerTK+)의 간 침윤 감소를 나타냈다. LPS(lipopolysaccharide) 및/또는 TAF로 처리된 분류된 인간 CD14+ 단핵구를 사용한 ex vivo 실험에서 본 발명자들은 모의 처리된 것과 비교하여 TAF 처리된 LPS로 자극된 단핵구에서 인산화된 AKT의 감소된 수준을 확인했다. 마우스 간 면역블롯팅은 AKT의 인산화 수준이 모의 처리된 그룹보다 TAF 처리된 NASH 그룹에서 유의하게 더 낮았다는 것을 보여주었다.In the present invention, two different in vivo NASH mouse models were constructed: streptozotocin (STZ, 0.2 mg) was injected subcutaneously and mice were fed a high-fat, high-cholesterol (HFHC) diet or a choline-deficient (CDAHF) diet. L-amino acid-defined, high-fat diet) diet. As a result, serum alanine aminotransferase and triglyceride levels in TAF-treated NASH mice were significantly lower than those in mock-treated mice. Livers from TAF-treated NASH mice showed attenuated mononuclear phagocyte (MP) infiltration compared to livers from mock-treated ones. TAF-treated NASH mice showed reduced liver infiltration of activated MPs (IAIE+/PD-L1+/MerTK+). In ex vivo experiments using sorted human CD14+ monocytes treated with lipopolysaccharide (LPS) and/or TAF, we identified reduced levels of phosphorylated AKT in TAF-treated LPS-stimulated monocytes compared to mock-treated ones. . Mouse liver immunoblotting showed that the phosphorylation level of AKT was significantly lower in the TAF-treated NASH group than in the mock-treated group.
즉, 본 발명자들은 TAF는 간내 활성화 MP에서 AKT 인산화를 약화시켜 NASH 간에서 항염증 효과를 발휘하므로, TAF는 NASH에 대한 새로운 치료 옵션이 될 수 있음을 확인하고 본 발명을 완성하였다. That is, the present inventors completed the present invention by confirming that TAF can be a new treatment option for NASH because TAF exerts an anti-inflammatory effect in NASH liver by attenuating AKT phosphorylation in activated MP in the liver.
따라서, 본 발명은 테노포비르 알라페나미드(tenofovir alafenamide, TAF) 또는 이의 약학적으로 허용 가능한 염을 포함하는 비알콜성 지방간 질환 예방 또는 치료용 약학적 조성물을 제공할 수 있다. Accordingly, the present invention can provide a pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease containing tenofovir alafenamide (TAF) or a pharmaceutically acceptable salt thereof.
본 발명의 바람직한 일실시예에 따르면, 상기 비알콜성 지방간 질환은 비알콜성 지방간염일 수 있다. According to a preferred embodiment of the present invention, the non-alcoholic fatty liver disease may be non-alcoholic steatohepatitis.
본 발명의 바람직한 일실시예에 따르면, 상기 조성물은 비알콜성 지방간 질환의 염증을 억제하는 것일 수 있다. According to a preferred embodiment of the present invention, the composition may suppress inflammation in non-alcoholic fatty liver disease.
본 발명의 바람직한 일실시예에 따르면, 상기 염증은 정상대조군에 비하여 증가한 염증성 대식세포에 의한 염증인 것일 수 있다. According to a preferred embodiment of the present invention, the inflammation may be inflammation caused by increased inflammatory macrophages compared to the normal control group.
본 발명의 바람직한 일실시예에 따르면, 상기 조성물은 정상대조군에 비하여 증가한 혈액 내 간 효소 마커 AST(aspartate aminotransferase), ALT(alanine aminotransferase) 및 트리글리세라이드(triglyceride) 로 이루어진 군에서 선택되는 어느 하나 이상의 수치를 감소시키는 것일 수 있다. According to a preferred embodiment of the present invention, the composition has an increased level of liver enzyme markers AST (aspartate aminotransferase), ALT (alanine aminotransferase), and triglyceride in the blood compared to the normal control group. It may be to reduce .
본 발명의 약학적 조성물은 경구 또는 비경구의 여러 가지 제형일 수 있다. 상기 조성물을 제형화할 경우에는 하나 이상의 완충제(예를 들어, 식염수 또는 PBS), 항산화제, 정균제, 킬레이트화제(예를 들어, EDTA 또는 글루타치온), 충진제, 증량제, 결합제, 아쥬반트(예를 들어, 알루미늄 하이드록사이드), 현탁제, 농후제 습윤제, 붕해제 또는 계면활성제, 희석제 또는 부형제를 사용하여 조제될 수 있다. The pharmaceutical composition of the present invention may be in various oral or parenteral dosage forms. When formulating the composition, one or more buffering agents (e.g., saline or PBS), antioxidants, bacteriostatic agents, chelating agents (e.g., EDTA or glutathione), fillers, extenders, binders, adjuvants (e.g., Aluminum hydroxide), suspending agents, thickening agents, wetting agents, disintegrants or surfactants, diluents or excipients.
경구투여를 위한 고형제제에는 정제, 환제, 산제, 과립제, 캡슐제 등이 포함되며, 이러한 고형제제는 하나 이상의 화합물에 적어도 하나 이상의 부형제 예를 들면, 전분(옥수수 전분, 밀 전분, 쌀 전분, 감자 전분 등 포함), 칼슘카보네이트(calcium carbonate), 수크로스(sucrose), 락토오스(lactose), 덱스트로오스, 솔비톨, 만니톨, 자일리톨, 에리스리톨 말티톨, 셀룰로즈, 메틸 셀룰로즈, 나트륨 카르복시메틸셀룰로오즈 및 하이드록시프로필메틸-셀룰로즈 또는 젤라틴 등을 섞어 조제된다. 예컨대, 활성성분을 고체 부형제와 배합한 다음 이를 분쇄하고 적합한 보조제를 첨가한 후 과립 혼합물로 가공함으로써 정제 또는 당의정제를 수득할 수 있다. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc. These solid preparations contain one or more compounds and at least one excipient, such as starch (corn starch, wheat starch, rice starch, potato). starch, etc.), calcium carbonate, sucrose, lactose, dextrose, sorbitol, mannitol, xylitol, erythritol maltitol, cellulose, methyl cellulose, sodium carboxymethylcellulose and hydroxypropylmethyl. -It is prepared by mixing cellulose or gelatin. For example, tablets or dragees can be obtained by combining the active ingredient with solid excipients, grinding them, adding suitable auxiliaries, and processing them into a granule mixture.
또한, 단순한 부형제 이외에 스테아린산 마그네슘, 탈크 등과 같은 윤활제들도 사용된다. 경구투여를 위한 액상 제제로는 현탁제, 내용액제, 유제 또는 시럽제 등이 해당되는데, 흔히 사용되는 단순 희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제 또는 보존제 등이 포함될 수 있다. 또한, 경우에 따라 가교결합 폴리비닐피롤리돈, 한천, 알긴산 또는 나트륨 알기네이트 등을 붕해제로 첨가할 수 있으며, 항응집제, 향료, 유화제, 가용화제, 분산제, 향미제, 항산화제, 포장제, 안료 및 방부제 등을 추가로 포함할 수 있다.Additionally, in addition to simple excipients, lubricants such as magnesium stearate, talc, etc. are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, or syrups. In addition to the commonly used simple diluents such as water and liquid paraffin, they may contain various excipients such as wetting agents, sweeteners, fragrances, or preservatives. You can. In addition, in some cases, cross-linked polyvinylpyrrolidone, agar, alginic acid, or sodium alginate can be added as a disintegrant, and anti-coagulants, flavoring agents, emulsifiers, solubilizers, dispersants, flavoring agents, antioxidants, and packaging agents. , pigments and preservatives may be additionally included.
비경구 투여를 위한 제제에는 멸균된 수용액, 비수성용제, 현탁용제, 유제, 동결건조제제 또는 좌제 등이 포함된다. 비수성용제 및 현탁용제로는 프로필렌글리콜(propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등이 사용될 수 있다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세롤, 젤라틴 등이 사용될 수 있다.Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, or suppositories. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate. As a base for suppositories, witepsol, macrogol, tween 61, cacao, laurel, glycerol, gelatin, etc. can be used.
본 발명의 조성물은 경구 또는 비경구로 투여될 수 있으며, 비경구 투여시 복강내, 직장, 정맥, 근육 또는 피하의 형태로 당업계에 공지된 방법에 따라 제형화할 수 있다.The composition of the present invention can be administered orally or parenterally, and when administered parenterally, it can be formulated in the form of intraperitoneal, rectal, intravenous, intramuscular or subcutaneous administration according to methods known in the art.
상기 주사제의 경우에는 반드시 멸균되어야 하며 박테리아 및 진균과 같은 미생물의 오염으로부터 보호되어야 한다. 주사제의 경우 적합한 담체의 예로는 이에 한정되지는 않으나, 물, 에탄올, 폴리올(예를 들어, 글리세롤, 프로필렌 글리콜 및 액체 폴리에틸렌 글리콜 등), 이들의 혼합물 및/또는 식물유를 포함하는 용매 또는 분산매질일 수 있다. 보다 바람직하게는, 적합한 담체로는 행크스 용액, 링거 용액, 트리에탄올 아민이 함유된 PBS (phosphate buffered saline) 또는 주사용 멸균수, 10% 에탄올, 40% 프로필렌 글리콜 및 5% 덱스트로즈와 같은 등장 용액 등을 사용할 수 있다. 상기 주사제를 미생물 오염으로부터 보호하기 위해서는 파라벤, 클로로부탄올, 페놀, 소르빈산, 티메로살 등과 같은 다양한 항균제 및 항진균제를 추가로 포함할 수 있다. 또한, 상기 주사제는 대부분의 경우 당 또는 나트륨 클로라이드와 같은 등장화제를 추가로 포함할 수 있다.The above injections must be sterilized and protected from contamination by microorganisms such as bacteria and fungi. For injections, examples of suitable carriers include, but are not limited to, solvents or dispersion media including water, ethanol, polyols (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, etc.), mixtures thereof, and/or vegetable oils. You can. More preferably, suitable carriers include Hanks' solution, Ringer's solution, phosphate buffered saline (PBS) containing triethanolamine, or isotonic solutions such as sterile water for injection, 10% ethanol, 40% propylene glycol, and 5% dextrose. etc. can be used. In order to protect the injection from microbial contamination, it may additionally contain various antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, sorbic acid, thimerosal, etc. Additionally, in most cases, the injection may additionally contain an isotonic agent such as sugar or sodium chloride.
본 발명의 조성물은 약제학적으로 유효한 양으로 투여한다. 약제학적으로 유효한 양은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효용량 수준은 환자의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명의 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 순차적 또는 동시에 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 즉, 본 발명의 조성물의 총 유효량은 단일 투여량(single dose)으로 환자에게 투여될 수 있으며, 다중 투여량(multiple dose)으로 장기간 투여되는 분할 치료 방법(fractionated treatment protocol)에 의해 투여될 수 있다. 상기한 요소들을 모두 고려하여 부작용없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The composition of the present invention is administered in a pharmaceutically effective amount. A pharmaceutically effective amount refers to an amount sufficient to treat a disease with a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level refers to the type of patient's disease, severity, activity of the drug, sensitivity to the drug, and administration time. , route of administration and excretion rate, duration of treatment, factors including concurrently used drugs, and other factors well known in the field of medicine. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiple times. That is, the total effective amount of the composition of the present invention can be administered to the patient in a single dose, or by a fractionated treatment protocol in which multiple doses are administered over a long period of time. . Considering all of the above factors, it is important to administer an amount that can achieve the maximum effect with the minimum amount without side effects, and this can be easily determined by a person skilled in the art.
본 발명의 약학적 조성물의 투여량은 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설율 및 질환의 중증도에 따라 그 범위가 다양할 수 있다.The dosage of the pharmaceutical composition of the present invention may vary depending on the patient's weight, age, gender, health condition, diet, administration time, administration method, excretion rate, and severity of disease.
본 발명의 조성물은 단독으로, 또는 수술, 방사선 치료, 호르몬 치료, 화학 치료 및 생물학적 반응 조절제를 사용하는 방법들과 병용하여 사용할 수 있다.The composition of the present invention can be used alone or in combination with surgery, radiation therapy, hormone therapy, chemotherapy, and methods using biological response modifiers.
또한, 본 발명은 테노포비르 알라페나미드(tenofovir alafenamide, TAF) 또는 이의 약학적으로 허용 가능한 염을 포함하는 비알콜성 지방간 질환 예방 또는 개선용 건강기능식품 조성물을 제공할 수 있다. In addition, the present invention can provide a health functional food composition for preventing or improving non-alcoholic fatty liver disease containing tenofovir alafenamide (TAF) or a pharmaceutically acceptable salt thereof.
상기 비알콜성 지방간 질환은 상기 약학적 조성물에서 대상으로 하는 질환과 동일하므로 설명은 상기 기재로 대신한다. Since the non-alcoholic fatty liver disease is the same as the disease targeted in the pharmaceutical composition, the description is replaced with the above description.
본 발명에 따른 건강기능식품 조성물은 당업계에 공지된 통상적인 방법에 따라 다양한 형태로 제조할 수 있다. 일반 식품으로는 이에 한정되지 않지만 음료(알콜성 음료 포함), 과실 및 그의 가공식품(예: 과일통조림, 병조림, 잼, 마아말레이드 등), 어류, 육류 및 그 가공식품(예: 햄, 소시지 콘비이프 등), 빵류 및 면류(예: 우동, 메밀국수, 라면, 스파게이트, 마카로니 등), 과즙, 각종 드링크, 쿠키, 엿, 유제품(예: 버터, 치이즈 등), 식용식물 유지, 마아가린, 식물성 단백질, 레토르트 식품, 냉동식품, 각종 조미료(예: 된장, 간장, 소스 등) 등에 본 발명의 TAF 또는 이의 약학적으로 허용 가능한 염을 첨가하여 제조할 수 있다. 또한, 영양보조제로는 이에 한정되지 않지만 캡슐, 타블렛, 환 등에 본 발명의 TAF 또는 이의 약학적으로 허용 가능한 염을 첨가하여 제조할 수 있다. 또한, 건강기능식품으로는 이에 한정되지 않지만 예를 들면, 본 발명의 TAF 또는 이의 약학적으로 허용 가능한 염 자체를 차, 쥬스 및 드링크의 형태로 제조하여 음용(건강음료)할 수 있도록 액상화, 과립화, 캡슐화 및 분말화하여 섭취할 수 있다. 또한, 본 발명의 TAF 또는 이의 약학적으로 허용 가능한 염을 식품 첨가제의 형태로 사용하기 위해서는 분말 또는 농축액 형태로 제조하여 사용할 수 있다. 또한, 본 발명의 TAF 또는 이의 약학적으로 허용 가능한 염과 비알콜성 지방간 질환 예방 또는 개선 효과가 있다고 알려진 공지의 활성 성분과 함께 혼합하여 조성물의 형태로 제조할 수 있다.The health functional food composition according to the present invention can be manufactured in various forms according to conventional methods known in the art. General foods include, but are not limited to, beverages (including alcoholic beverages), fruits and their processed foods (e.g. canned fruit, bottled foods, jam, mamalade, etc.), fish, meat and their processed foods (e.g. ham, sausages, etc.) corned beef, etc.), bread and noodles (e.g. udon, buckwheat noodles, ramen, spagate, macaroni, etc.), fruit juice, various drinks, cookies, taffy, dairy products (e.g. butter, cheese, etc.), edible vegetable oil, margarine , vegetable proteins, retort foods, frozen foods, various seasonings (e.g., soybean paste, soy sauce, sauce, etc.), etc. can be produced by adding the TAF of the present invention or a pharmaceutically acceptable salt thereof. In addition, nutritional supplements are not limited to this, but can be prepared by adding TAF of the present invention or a pharmaceutically acceptable salt thereof to capsules, tablets, pills, etc. In addition, health functional foods are not limited to this, but for example, TAF of the present invention or its pharmaceutically acceptable salt itself can be manufactured in the form of tea, juice, and drinks and liquefied and granulated for drinking (health drinks). It can be consumed in form, encapsulation, and powder. Additionally, in order to use TAF or a pharmaceutically acceptable salt thereof of the present invention in the form of a food additive, it can be prepared and used in the form of a powder or concentrate. In addition, it can be prepared in the form of a composition by mixing TAF of the present invention or a pharmaceutically acceptable salt thereof with a known active ingredient known to be effective in preventing or improving non-alcoholic fatty liver disease.
본 발명의 TAF 또는 이의 약학적으로 허용 가능한 염을 건강음료로 이용하는 경우, 상기 건강음료 조성물은 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로 함유할 수 있다. 상술한 천연 탄수화물은 포도당, 과당과 같은 모노사카라이드; 말토스, 슈크로스와 같은 디사카라이드; 덱스트린, 사이클로덱스트린과 같은 폴리사카라이드; 자일리톨, 소르비톨, 에리트리톨 등의 당알콜일 수 있다. 감미제는 타우마틴, 스테비아 추출물과 같은 천연 감미제; 사카린, 아스파르탐과 같은 합성 감미제 등을 사용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 조성물 100 mL 당 일반적으로 약 0.01 ~ 0.04 g, 바람직하게는 약 0.02 ~ 0.03 g 이다.When using TAF or a pharmaceutically acceptable salt thereof of the present invention as a health drink, the health drink composition may contain various flavoring agents or natural carbohydrates as additional ingredients like a conventional drink. The above-mentioned natural carbohydrates include monosaccharides such as glucose and fructose; Disaccharides such as maltose and sucrose; polysaccharides such as dextrins and cyclodextrins; It may be a sugar alcohol such as xylitol, sorbitol, or erythritol. Sweeteners include natural sweeteners such as thaumatin and stevia extract; Synthetic sweeteners such as saccharin and aspartame can be used. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g, per 100 mL of the composition of the present invention.
또한, 본 발명의 TAF 또는 이의 약학적으로 허용 가능한 염은 비알콜성 지방간 질환 예방 또는 개선용 건강기능식품 조성물의 유효성분으로 함유될 수 있는데, 그 양은 비알콜성 지방간 질환 예방 또는 개선 작용을 달성하기에 유효한 양으로 특별히 한정되는 것은 아니나, 전체 조성물 총 중량에 대하여 0.01 내지 100 중량%인 것이 바람직하다. 본 발명의 건강기능식품 조성물은 TAF 또는 이의 약학적으로 허용 가능한 염과 함께 비알콜성 지방간 질환에 효과가 있는 것으로 알려진 다른 활성 성분과 함께 혼합하여 제조될 수 있다.In addition, TAF or a pharmaceutically acceptable salt thereof of the present invention may be contained as an active ingredient in a health functional food composition for preventing or improving non-alcoholic fatty liver disease, and the amount is sufficient to achieve the effect of preventing or improving non-alcoholic fatty liver disease. The effective amount is not particularly limited, but is preferably 0.01 to 100% by weight based on the total weight of the entire composition. The health functional food composition of the present invention can be prepared by mixing TAF or a pharmaceutically acceptable salt thereof with other active ingredients known to be effective in non-alcoholic fatty liver disease.
상기 외에 본 발명의 건강기능식품은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산, 펙트산의 염, 알긴산, 알긴산의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올 또는 탄산화제 등을 함유할 수 있다. 그 밖에 본 발명의 건강기능식품은 천연 과일주스, 과일주스 음료, 또는 야채 음료의 제조를 위한 과육을 함유할 수 있다. 이러한 성분은 독립적으로 또는 혼합하여 사용할 수 있다. 이러한 첨가제의 비율은 크게 중요하진 않지만 본 발명의 조성물 100 중량부당 0.01 ~ 0.1 중량부의 범위에서 선택되는 것이 일반적이다.In addition to the above, the health functional food of the present invention includes various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, and preservatives. , may contain glycerin, alcohol, or carbonating agent. In addition, the health functional food of the present invention may contain pulp for the production of natural fruit juice, fruit juice beverage, or vegetable beverage. These ingredients can be used independently or in combination. The ratio of these additives is not very important, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.
이하, 실시예를 통하여 본 발명을 보다 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의하여 제한되는 것으로 해석하지 않는 것은 해당 기술분야에서 통상의 지식을 가진 자에 있어서 자명한 것이다. Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it is obvious to those skilled in the art that the scope of the present invention should not be construed as limited by these examples.
말초 인간 CD14+ 단핵구의 분리 및 in vitro 실험Isolation and in vitro experiments of peripheral human CD14+ monocytes.
Ficoll-Hypaque 밀도 구배 원심분리를 사용하여 건강한 성인 기증자로부터 말초 혈액 단핵 세포를 분리했다. CD14+ 단핵구(anti-CD14 microbeads, MACS, Miltenyi Biotec, Bergisch Gladbach, Germany)는 OctoMACS 분리기 및 시작 키트(MACS, Miltenyi Biotec)를 사용하여 말초 혈액 단핵 세포에서 분리되었다. 분리된 인간 CD14+ 단핵구를 무혈청 Roswell Park Memorial Institute 1640 배지에 1 × 106 cells/mL의 밀도로 90 × 20 mm 세포 배양 접시(SPL Life Sciences, Pochon, Korea)에 도말하였다. 배지를 TAF로 6시간 동안 전처리하였다. 대조군은 동량의 DMSO(dimethyl sulfoxide)로 처리하였다. 배지를 1 μg/mL LPS 또는 모의로 24시간 동안 새로 고쳤다. 그런 다음 세포를 37 ℃에서 5% CO2로 배양했다. 그런 다음 형광 염색(AKT, CD45, 인간 백혈구 항원-DR 이소타입[HLA-DR], 포스포-AKT, 프로그래밍된 사멸-리간드 1[PD-L1] 및 LIVE/DEAD 염료)을 수행했다. 연구 프로토콜은 헬싱키 선언을 준수했다. 서울성모병원 기관심사위원회(KC20TISI0817)에서 이 연구를 승인했다. 모든 참가자로부터 사전 동의를 얻었다.Peripheral blood mononuclear cells were isolated from healthy adult donors using Ficoll-Hypaque density gradient centrifugation. CD14+ monocytes (anti-CD14 microbeads, MACS, Miltenyi Biotec, Bergisch Gladbach, Germany) were isolated from peripheral blood mononuclear cells using the OctoMACS separator and starter kit (MACS, Miltenyi Biotec). Isolated human CD14+ monocytes were plated on 90 × 20 mm cell culture dishes (SPL Life Sciences, Pochon, Korea) at a density of 1 × 106 cells/mL in serum-free Roswell Park Memorial Institute 1640 medium. The medium was pretreated with TAF for 6 hours. The control group was treated with the same amount of dimethyl sulfoxide (DMSO). Media were refreshed with 1 μg/mL LPS or mock for 24 h. The cells were then cultured at 37°C with 5% CO 2 . Fluorescent staining (AKT, CD45, human leukocyte antigen-DR isotype [HLA-DR], phospho-AKT, programmed death-ligand 1 [PD-L1], and LIVE/DEAD dye) was then performed. The study protocol complied with the Declaration of Helsinki. The institutional review board of Seoul St. Mary's Hospital (KC20TISI0817) approved this study. Informed consent was obtained from all participants.
In vivo 마우스 모델 구축 Construction of an in vivo mouse model
<2-1> HFHC 유도 NASH 마우스 모델 in vivo 구축<2-1> Construction of HFHC-induced NASH mouse model in vivo
임신 16일 된 암컷 C57BL/6J 마우스(Jackson Laboratory, Japan)는 The Jackson Laboratory(Japan)에서 구입했다. STZ(0.2 mg; Sigma-Aldrich, St. Louis, MO, USA)를 3일 된 수컷 C57BL/6J 마우스에 피하 주사하였다. STZ는 투여 전에 0.1M 시트르산나트륨 완충액(pH 4.5)에 용해시켰다. 한 달 후, 수컷 마우스를 무작위로 4개 그룹으로 나누었다: 정상, STZ 주입 정상 탄수화물(Teklad Global 18% Protein Rodent Diet; TD 2018C, Envigo, Indianapolis, IN, USA) 식이, STZ 주입 HFHC(high-fat, high-cholesterol; Western Diet) ; D12079B, 41kcal% 지방, 43kcal% 탄수화물, Research Diets, New Brunswick, NJ, USA) 식이, STZ 주입 TAF 처리 HFHC (TAF, 5 mg/kg, Gilead Sciences, Foster City, CA, USA) 식이 그룹. TAF는 4주 동안 매일 투여되었다. 2주마다 안와정맥총에서 혈액을 채취하여 임의의 포도당 수치를 분석하였다. 제조사의 프로토콜(Vettest 8008 Chemistry Analyzer; IDEXX Laboratories, Westbrook, ME, USA)에 따라 화학 분석기를 사용하여 혈청 알라닌 아미노전이효소(ALT), 아스파르테이트 아미노전이효소(AST) 및 트리글리세라이드(TRIG) 수치를 측정했다. 마우스를 Rompun(10 mg/kg) 및 Zoletil(40 mg/kg)로 복강내 마취하고 간을 적출하였다. 마우스 간은 해리 키트(MACS, Miltenyi Biotec) 및 gentleMACS Dissociator(MACS, Miltenyi Biotec)를 사용하여 해리되었다. 그런 다음 형광 염색을 수행했다(CD11b, F4/80, CD45, Ly6G, PD-L1, IAIE, MerTK 및 LIVE/DEAD 염료).Female C57BL/6J mice (Jackson Laboratory, Japan), 16 days pregnant, were purchased from The Jackson Laboratory (Japan). STZ (0.2 mg; Sigma-Aldrich, St. Louis, MO, USA) was injected subcutaneously into 3-day-old male C57BL/6J mice. STZ was dissolved in 0.1 M sodium citrate buffer (pH 4.5) before administration. One month later, male mice were randomly divided into four groups: normal, STZ-injected normal carbohydrate (Teklad Global 18% Protein Rodent Diet; TD 2018C, Envigo, Indianapolis, IN, USA) diet, STZ-infused high-fat HFHC (Teklad Global 18% Protein Rodent Diet; TD 2018C, Envigo, Indianapolis, IN, USA) diet. , high-cholesterol; Western Diet) ; D12079B, 41 kcal% fat, 43 kcal% carbohydrate, Research Diets, New Brunswick, NJ, USA) diet, STZ-injected TAF-treated HFHC (TAF, 5 mg/kg, Gilead Sciences, Foster City, CA, USA) diet group. TAF was administered daily for 4 weeks. Blood was collected from the orbital venous plexus every 2 weeks and random glucose levels were analyzed. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and triglyceride (TRIG) levels using a chemistry analyzer according to the manufacturer's protocol (Vettest 8008 Chemistry Analyzer; IDEXX Laboratories, Westbrook, ME, USA). was measured. Mice were anesthetized intraperitoneally with Rompun (10 mg/kg) and Zoletil (40 mg/kg), and livers were removed. Mouse liver was dissociated using a dissociation kit (MACS, Miltenyi Biotec) and gentleMACS Dissociator (MACS, Miltenyi Biotec). Fluorescent staining was then performed (CD11b, F4/80, CD45, Ly6G, PD-L1, IAIE, MerTK, and LIVE/DEAD dyes).
식품의약품안전청 한국우수동물실험실은 2007년 가톨릭대학교 송의캠퍼스 동물실험실 운영위원회(IACUC)와 동물실험실(DOLA)로부터 인가를 받았으며, 2018년 실험실 동물 관리 국제 인증에 의한 평가 및 인증을 획득하였다. 모든 동물 관리 및 실험 프로토콜은 기관 동물 관리 및 사용 위원회에 따라 수행되었으며 가톨릭 대학교 의과학 센터의 승인을 받았다(2021-0329-02).The Korea Excellent Animal Laboratory of the Korea Food and Drug Safety Administration received approval from the Catholic University Songui Campus Animal Laboratory Management Committee (IACUC) and the Laboratory Animal Laboratory (DOLA) in 2007, and obtained evaluation and certification by the International Accreditation for Laboratory Animal Management in 2018. All animal care and experimental protocols were performed in accordance with the Institutional Animal Care and Use Committee and were approved by the Catholic University Medical Science Center (2021-0329-02).
<2-2> CDAHF 식이 유도 NASH 마우스 모델 in vivo 구축<2-2> Construction of CDAHF diet-induced NASH mouse model in vivo
6주령 수컷 C57BL/6 마우스(Orient Bio, Inc., Seoul, Korea)를 무작위로 세 그룹으로 나누었다: 정상 탄수화물 식이 그룹, CDAHF(choline-deficient, L-amino acid-defined, high-fat diet; A06071302, Research Diets, New Brunswick, NJ, USA) 식이, TAF 처리, CDAHF 식이 공급 그룹. CDAHF 식이는 2주 먼저, TAF는 4주 동안 매일 투여하였다. 2주마다 안와정맥총에서 혈액을 채취하여 임의의 포도당 수치를 분석하였다. 제조사의 프로토콜(Vettest 8008 Chemistry Analyzer; IDEXX)에 따라 화학 분석기를 사용하여 혈청 ALT, AST 및 TRIG 수치를 측정했다. 그런 다음 형광 염색을 수행했다(CD11b, F4/80, CD45, Ly6G, PD-L1, IAIE, MerTK 및 LIVE/DEAD 염료). 모든 동물 관리 및 실험 프로토콜은 기관 동물 관리 및 사용 위원회에 따라 수행되었으며 가톨릭 대학교 의과학 센터의 승인을 받았다(2021-0329-02).Six-week-old male C57BL/6 mice (Orient Bio, Inc., Seoul, Korea) were randomly divided into three groups: normal carbohydrate diet group, CDAHF (choline-deficient, L-amino acid-defined, high-fat diet; A06071302) , Research Diets, New Brunswick, NJ, USA) diet, TAF-treated, and CDAHF diet-fed groups. The CDAHF diet was administered 2 weeks first, and TAF was administered daily for 4 weeks. Blood was collected from the orbital venous plexus every 2 weeks and random glucose levels were analyzed. Serum ALT, AST, and TRIG levels were measured using a chemistry analyzer according to the manufacturer's protocol (Vettest 8008 Chemistry Analyzer; IDEXX). Fluorescent staining was then performed (CD11b, F4/80, CD45, Ly6G, PD-L1, IAIE, MerTK, and LIVE/DEAD dyes). All animal care and experimental protocols were performed in accordance with the Institutional Animal Care and Use Committee and were approved by the Catholic University Medical Science Center (2021-0329-02).
<2-3> TAA 유발 간손상 마우스 모델 in vivo 구축<2-3> Construction of a TAA-induced liver damage mouse model in vivo
6주령의 수컷 BALB/c 마우스(Orient Bio, Inc., Seoul, Korea)에 간 손상을 유발하기 위해 TAA(Thioacetamide; Sigma-Aldrich, St. Louis, MO, USA)를 투여하였다. TAA 주사는 4주 동안 3일마다 100 mg/kg, 4주 동안 2일마다 150 mg/kg으로 투여되었다. 간은 0.5mM EGTA, 2.5mM CaCl2 및 0.1% IV형 콜라게나아제를 포함하는 Hank의 평형 염 용액으로 간문맥에 삽입된 카테터(직경 2mm)를 사용하여 관류되었다. 그런 다음 형광 염색을 수행했다(CD11b, F4/80, CD45, Ly6G, PD-L1 및 LIVE/DEAD 염료).TAA (Thioacetamide; Sigma-Aldrich, St. Louis, MO, USA) was administered to 6-week-old male BALB/c mice (Orient Bio, Inc., Seoul, Korea) to induce liver damage. TAA injections were administered at 100 mg/kg every 3 days for 4 weeks and 150 mg/kg every 2 days for 4 weeks. The liver was perfused with Hank's balanced salt solution containing 0.5mM EGTA, 2.5mM CaCl 2 and 0.1% type IV collagenase using a catheter (2 mm diameter) inserted into the portal vein. Fluorescent staining was then performed (CD11b, F4/80, CD45, Ly6G, PD-L1, and LIVE/DEAD dyes).
모든 동물 관리 및 실험 프로토콜은 기관 동물 관리 및 사용 위원회에 따라 수행되었으며 가톨릭 대학교 의과학 센터의 승인을 받았다(2020-0300-05).All animal care and experimental protocols were performed in accordance with the Institutional Animal Care and Use Committee and were approved by the Catholic University Medical Science Center (2020-0300-05).
TAF 처리에 따른 간 손상 개선 효과Liver damage improvement effect due to TAF treatment
상기 <실시예 2> 에서 STZ 및 HFHC 식이를 사용하여 구축한 NASH 마우스 모델을 이용하여 NASH에서 TAF의 역할을 평가하고자 하였다(도 1A). NASH 모델과 TAF 효과를 확인하기 위해 혈청 ALT, AST, 트리글리세라이드(TRIG, triglyceride) 수치를 측정하여 간기능 검사를 시행하였다. We attempted to evaluate the role of TAF in NASH using the NASH mouse model established using the STZ and HFHC diet in <Example 2> (Figure 1A). To confirm the effectiveness of the NASH model and TAF, liver function tests were performed by measuring serum ALT, AST, and triglyceride (TRIG) levels.
파라핀 내장 블록을 절단하여 실란 처리된 유리 슬라이드로 옮겼다. 절편을 Sirius Red(Pico Sirius Red 염색 키트, Abcam, Cambridge, UK) 및 헤마톡실린 및 에오신으로 염색하였다. 항-LPS 및 항-toll-like 수용체 4(TLR-4)(Abcam, Cambridge, UK)로 항체를 사용하였다.Paraffin-embedded blocks were cut and transferred to silanized glass slides. Sections were stained with Sirius Red (Pico Sirius Red staining kit, Abcam, Cambridge, UK) and hematoxylin and eosin. Antibodies were used as anti-LPS and anti-toll-like receptor 4 (TLR-4) (Abcam, Cambridge, UK).
ALT, AST 및 TRIG 수준은 모의 처리된 그룹보다 TAF 처리된 HFHC 식이 그룹에서 더 낮았다(도 1B). 체중과 임의의 포도당 수치를 2주 동안 측정했다. TAF 처리된 HFHC 식이를 먹인 쥐의 총 간 무게는 모의 처리된 쥐보다 낮았다 (도 2A). 파라핀 포매된 간 절편의 헤마톡실린 및 에오신(H&E) 염색은 간세포 괴사와 사인 울혈이 모의 처리된 것보다 TAF 처리된 HFHC-식이 그룹의 간에서 유의하게 감소되었음을 보여주었다. 시리우스 레드(Sirius red) 염색은 모의 처리군에 비해 TAF 처리된 HFHC 식이그룹에서 간에서 콜라겐 침착 면적이 감소되었음을 보여주었다. 또한, IHC 결과는 LPS 및 TLR4(toll-like receptor 4)-양성 세포가 TAF 처리군보다 모의 처리된 HFHC 식이군에서 더 자주 검출됨을 보여주었다(도 1C). TLR4- 또는 LPS-양성 세포 빈도는 모의 처리 그룹보다 TAF 처리 HFHC 식이 그룹에서 더 낮았다. 염색된 콜라겐의 면적 비율은 모의 처리군보다 TAF 처리된 HFHC 식이군에서 더 낮았다. NASH 활동 점수(NAS)는 모의 처리 그룹에 비해 TAF 처리 HFHC 식이 그룹의 간에서 감소했다(도 1D).ALT, AST, and TRIG levels were lower in the TAF-treated HFHC diet group than in the mock-treated group (Figure 1B). Body weight and random glucose levels were measured for 2 weeks. The total liver weight of TAF-treated rats fed the HFHC diet was lower than that of mock-treated rats (Figure 2A). Hematoxylin and eosin (H&E) staining of paraffin-embedded liver sections showed that hepatocellular necrosis and sinus congestion were significantly reduced in the livers of the TAF-treated HFHC-diet group than in the mock-treated ones. Sirius red staining showed that the area of collagen deposition in the liver was reduced in the TAF-treated HFHC diet group compared to the mock-treated group. Additionally, IHC results showed that LPS and toll-like receptor 4 (TLR4)-positive cells were detected more frequently in the mock-treated HFHC diet group than in the TAF-treated group (Figure 1C). The frequency of TLR4- or LPS-positive cells was lower in the TAF-treated HFHC diet group than in the mock-treated group. The area percentage of stained collagen was lower in the TAF-treated HFHC diet group than in the mock-treated group. NASH activity score (NAS) was reduced in the liver of the TAF-treated HFHC diet group compared to the sham-treated group (Figure 1D).
또한, 본 발명자들은 CDAHF(choline-deficient, L-amino acid-defined, high-fat) 식단을 사용하여 NASH 마우스 모델을 확립했다(도 1E). ALT, AST 및 TRIG 수준은 TAF 처리 CDAHF-식이 요법 그룹에서 모의 처리 그룹보다 낮았다(도 1F). 체중과 임의의 포도당 수치를 2주 동안 측정했다. TAF 처리 CDAHF 식이 그룹의 총 간 무게는 모의 처리 그룹보다 낮았다(도 2B). 파라핀 포매된 간 절편의 H&E 염색은 TAF 처리된 CDAHF-식이 요법 그룹의 간에서 모의 처리된 간과 비교하여 간세포 괴사와 동모양혈관의 울혈(sinusoidal congestion)이 유의하게 감소되었음을 보여주었다(도 2C).Additionally, we established a NASH mouse model using a choline-deficient, L-amino acid-defined, high-fat (CDAHF) diet (Figure 1E). ALT, AST, and TRIG levels were lower in the TAF-treated CDAHF-diet group than in the mock-treated group (Figure 1F). Body weight and random glucose levels were measured for 2 weeks. The total liver weight of the TAF-treated CDAHF diet group was lower than that of the mock-treated group (Figure 2B). H&E staining of paraffin-embedded liver sections showed that hepatocyte necrosis and sinusoidal congestion were significantly reduced in the livers of the TAF-treated CDAHF-diet group compared to the mock-treated livers (Figure 2C).
HFHC 식이 유도 NASH 마우스 모델에서 간내 MP의 역할Role of intrahepatic MPs in the HFHC diet-induced NASH mouse model
모의 또는 TAF가 처리된 STZ 및 HFHC 식이 마우스의 간을 사용하여 NASH 마우스 모델에서 간내 MP의 역할을 확인하기 위해 마우스 간 소화 후 유세포 분석을 수행했다. 유동 세포 계측법을 사용하여 간내 MP를 정량화하기 위한 게이팅 전략이 [도 3A]에 나와 있다. 간내 모집된 MP(CD11bhigh F4/80low) 및 상주 MP(CD11blow F4/80high) 에 대한 대표적인 게이팅 전략이 [도 4A]에 나와 있다. To determine the role of intrahepatic MPs in a NASH mouse model using livers from mock- or TAF-treated STZ and HFHC diet mice, flow cytometry analysis was performed after mouse liver digestion. The gating strategy for quantifying intrahepatic MPs using flow cytometry is shown in [Figure 3A]. A representative gating strategy for intrahepatic recruited MPs (CD11b high F4/80 low ) and resident MPs (CD11b low F4/80 high ) is shown in Figure 4A.
mRNA 발현을 검출하기 위해 cDNA 합성 및 TaqMan RT-qPCR 분석을 수행했다. TaqMan 유전자 발현 분석(Applied Biosystems, Foster City, CA, USA)으로 표적 유전자 mRNA 수준을 결정하였다. 각 유전자의 분석 ID는 액틴(Mm02619580_g1), 케모카인(C-C 모티프) 리간드 2(Mm00441242_m1), 종양 괴사 인자(Mm00443258_m1), 인터루킨-1 베타(Mn00434228-m1)이었다. qRT-PCR은 Light Cycle 480 Probes Master Reaction Mix(Roche)와 함께 Light Cycler 480 II(Roche Diagnostics)를 사용하여 총 반응 부피 20μL에서 수행되었다.cDNA synthesis and TaqMan RT-qPCR analysis were performed to detect mRNA expression. Target gene mRNA levels were determined by TaqMan gene expression assay (Applied Biosystems, Foster City, CA, USA). The analysis IDs of each gene were actin (Mm02619580_g1), chemokine (C-C motif) ligand 2 (Mm00441242_m1), tumor necrosis factor (Mm00443258_m1), and interleukin-1 beta (Mn00434228-m1). qRT-PCR was performed in a total reaction volume of 20 μL using a Light Cycler 480 II (Roche Diagnostics) with Light Cycle 480 Probes Master Reaction Mix (Roche).
TAF 처리된 HFHC-식이 요법 그룹은 모의 처리된 것보다 유의하게 감소된 간내 모집 MP(CD11bhigh F4/80low)를 보였다. 그러나 상주(CD11blow F4/80high) MP(도 4B)는 모의 처리된 MP와 비교하여 유의한 차이가 없었다. 프로그래밍된 사멸 리간드 1(PD-L1), IAIE, MER 원암 유전자 및 티로신 키나제(MerTK)를 CD11b+F4/80+ MP 활성화 표면 마커로 사용했다. 간내 IAIE+ 및 MerTK+ MP 평균 형광 강도(MFI) 값은 모의 처리 그룹보다 TAF 처리 HFHC 식이 그룹에서 유의하게 낮았다(도 4C). 간내 PD-L1+/IAIE+/MerTK+-양성 MP 수치는 TAF 처리 HFHC 식이군에서 모의 처리군보다 유의하게 감소하였다(도 4D). 또한 간 무게 1g당 CD11b+ F4/80+ MP의 수는 모의 처리 그룹보다 TAF 처리 HFHC 식이 그룹에서 유의하게 감소했다(도 3B). 도 4E는 종양 괴사 인자-α, 케모카인(C-C 모티프) 리간드 2 및 인터루킨-1 베타 mRNA 발현 수준이 모의 처리 그룹보다 TAF 처리 HFHC 식이 그룹에서 유의하게 낮았으며 염증이 감소했음을 나타낸다.The TAF-treated HFHC-diet group showed significantly reduced intrahepatic recruitment of MPs (CD11b high F4/80 low ) compared to the mock-treated group. However, resident (CD11b low F4/80 high ) MPs (Figure 4B) were not significantly different compared to mock-treated MPs. Programmed death ligand 1 (PD-L1), IAIE, MER proto-oncogene, and tyrosine kinase (MerTK) were used as surface markers for CD11b + F4/80 + MP activation. Intrahepatic IAIE+ and MerTK+ MP mean fluorescence intensity (MFI) values were significantly lower in the TAF-treated HFHC diet group than in the mock-treated group (Figure 4C). Intrahepatic PD-L1 + /IAIE + /MerTK + -positive MP levels were significantly reduced in the TAF-treated HFHC diet group compared to the mock-treated group ( Fig. 4D ). Additionally, the number of CD11b+ F4/80+ MPs per gram of liver weight was significantly reduced in the TAF-treated HFHC diet group than in the mock-treated group (Figure 3B). Figure 4E shows that tumor necrosis factor-α, chemokine (CC motif) ligand 2, and interleukin-1 beta mRNA expression levels were significantly lower in the TAF-treated HFHC diet group than in the mock-treated group, indicating reduced inflammation.
CDAHFD 식이 유도 NASH 마우스 모델에서 간내 MP의 역할Role of intrahepatic MPs in the CDAHFD diet-induced NASH mouse model
모의 또는 TAF가 처리된 CDAHF 식이 마우스의 간을 사용하여 NASH 마우스 모델에서 간 내 MP의 역할을 확인하기 위해 마우스 간 분쇄 후 유세포 분석을 수행했다. 간내 모집 MP(CD11bhigh F4/80low) 및 상주 MP(CD11blow F4/80high) 에 대한 대표적인 게이팅 전략이 [도 5A]에 나와 있다. To determine the role of intrahepatic MPs in a NASH mouse model using livers from mock- or TAF-treated CDAHF diet mice, flow cytometry analysis was performed after pulverizing mouse livers. A representative gating strategy for intrahepatic recruited MPs (CD11b high F4/80 low ) and resident MPs (CD11b low F4/80 high ) is shown in Figure 5A.
TAF 처리된 CDAHF 식이요법 그룹은 모의 처리된 그룹보다 유의하게 감소된 간내 모집 MP(CD11bhigh F4/80low)를 보였다. 그러나 상주 MP(CD11blow F4/80high)는 모의 처리된 MP와 비교하여 유의한 차이가 없었다(도 4B). 간 내 IAIE+ 및 MerTK+ MP MFI 값은 모의 처리 그룹보다 TAF 처리 CDAHF 식이 그룹에서 유의하게 낮았다(도 5C). 간 내 PD-L1+/IAIE+/MerTK+ 양성 MP 수치는 TAF 처리된 HFHC 식이 그룹에서 유의하게 감소했다(도 5D). 또한 간 무게 1g당 CD11b+ F4/80+ MP의 수는 모의 처리 그룹보다 TAF 처리 CDAHF 식이 그룹에서 유의하게 감소했다(도 3C).The TAF-treated CDAHF diet group showed significantly reduced intrahepatic recruitment of MPs (CD11b high F4/80 low ) compared to the mock-treated group. However, resident MPs (CD11b low F4/80 high ) were not significantly different compared to mock-treated MPs (Figure 4B). The intrahepatic IAIE + and MerTK + MP MFI values were significantly lower in the TAF-treated CDAHF diet group than in the mock-treated group (Figure 5C). The levels of PD-L1 + /IAIE + /MerTK + positive MPs in the liver were significantly reduced in the TAF-treated HFHC diet group ( Figure 5D ). Additionally, the number of CD11b + F4/80 + MPs per gram of liver weight was significantly reduced in the TAF-treated CDAHF diet group than in the mock-treated group (Figure 3C).
활성화된 간내 MP에 대한 ex vivo TAF 처리의 효과Effect of ex vivo TAF treatment on activated intrahepatic MPs
본 발명자들은 염증이 있는 간내 MP에 대한 ex vivo TAF 효과를 확인하기 위해 동물 TAA 유발 간 손상 모델을 구축했다. TAA를 8주 동안 복강 내 주사하였다(도 6A). We established an animal TAA-induced liver injury model to determine the effect of TAF ex vivo on inflamed intrahepatic MP. TAA was injected intraperitoneally for 8 weeks (Figure 6A).
파라핀 포매된 간 절편의 H&E 염색은 간세포 괴사 및 동모양혈관의 울혈 이 TAA 처리되지 않은 간보다 TAA 처리된 간에서 유의하게 더 높았음을 보여주었다. 시리우스 레드 염색은 TAA 처리군이 TAA 처리하지 않은 군에 비해 간에서 콜라겐 침착 면적이 증가함을 보여주었다(도 6B). TAA로 유도된 손상된 간은 콜라게나아제로 관류되었고 MP는 구배 원심분리를 사용하여 분리되었다. 분리된 MP는 다른 TAF 농도로 처리되었다(도 6C). TAF 또는 모의 처리된 MP는 유동 세포 계측법을 사용하여 분석되었다. 분석된 전체 MP 중 PD-L1+ 세포의 수는 TAF 농도 의존적으로 감소하였고, TAF 처리는 CD11b+F4/80+ 간 내 MP에서 PD-L1의 MFI를 감소시켰다(도 6D).H&E staining of paraffin-embedded liver sections showed that hepatocyte necrosis and congestion of sinusoids were significantly higher in TAA-treated livers than in non-TAA-treated livers. Sirius red staining showed that the area of collagen deposition in the liver of the TAA-treated group was increased compared to the non-TAA-treated group (Figure 6B). TAA-induced injured livers were perfused with collagenase and MPs were isolated using gradient centrifugation. Isolated MPs were treated with different TAF concentrations (Figure 6C). TAF or mock-treated MPs were analyzed using flow cytometry. The number of PD-L1 + cells among the total analyzed MPs decreased in a TAF concentration-dependent manner, and TAF treatment reduced the MFI of PD-L1 in CD11b + F4/80 + intrahepatic MPs ( Fig. 6D ).
MP에서 TAF의 AKT 인산화 차단 효과Effect of TAF on blocking AKT phosphorylation in MP
In vivo STZ, HFHC 유도 NASH 마우스 모델 처리된 모의 또는 TAF에서 TAF에 의해 차단된 신호 전달 경로를 결정하기 위해 면역블롯팅을 수행하였다. Immunoblotting was performed to determine the signaling pathways blocked by TAF in mock or TAF treated in vivo STZ, HFHC-induced NASH mouse model.
면역 블롯팅을 위하여 NASH 마우스 모델 및 정상 마우스 간 조직을 프로테아제 억제제 칵테일(Roche, Basel, Switzerland) 및 포스파타제 억제제 칵테일(100x)(Sigma-Aldrich)을 함유하는 RIPA 완충액(pH 7.5의 20mM Tris-HCl, 150mM NaCl, 1% Triton X-100, 1% 소듐 데옥시콜레이트 및 0.1% 소듐 도데실 설페이트)에서 용해시켰다. 각 용해물의 단백질은 소듐 도데실 설페이트-폴리아크릴아미드 겔 전기영동을 사용하여 분리하고, 단백질을 니트로셀룰로오스 막(Schleicher & Schuell, Dassel, Germany)으로 옮겼다. Cell Signaling Technology(Danvers, MA, USA)에서 구입한 항체들을 면역블롯팅에 사용하였다: 토끼 단클론 항-GAPDH, 토끼 다클론 항-AKT 및 토끼 단클론 항-포스포-AKT(Ser473), 토끼 다클론 항-mTOR, 토끼 다클론 항-포스포-mTOR(Ser2448).For immunoblotting, NASH mouse model and normal mouse liver tissues were incubated in RIPA buffer (20mM Tris-HCl at pH 7.5; 150mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, and 0.1% sodium dodecyl sulfate). Proteins from each lysate were separated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and proteins were transferred to nitrocellulose membranes (Schleicher & Schuell, Dassel, Germany). Antibodies purchased from Cell Signaling Technology (Danvers, MA, USA) were used for immunoblotting: rabbit monoclonal anti-GAPDH, rabbit polyclonal anti-AKT and rabbit monoclonal anti-phospho-AKT (Ser473), rabbit polyclonal. anti-mTOR, rabbit polyclonal anti-phospho-mTOR (Ser2448).
NASH 마우스 간에서 p-AKT, AKT, p-mTOR, mTOR 및 GAPDH 단백질 수준이 검출되었다. p-AKT 및 p-mTOR 수준은 STZ 주입 모의 처리 HFHC-식이 마우스와 비교하여 STZ 주입 TAF-처리 HFHC-식이 마우스의 간에서 감소하였다(도 7A, 도 8).Protein levels of p-AKT, AKT, p-mTOR, mTOR and GAPDH were detected in NASH mouse liver. p-AKT and p-mTOR levels were reduced in the liver of STZ-injected TAF-treated HFHC-diet mice compared with STZ-injected sham-treated HFHC-diet mice (Figure 7A, Figure 8).
본 발명자들은 단핵구가 손상된 간으로 모집되고 모집된 MP로 분화되기 때문에 인간 단핵구에 대한 TAF의 영향을 조사했다. 본 발명자들은 말초 혈액 단핵 세포에서 CD14+ 단핵구를 분류했다. 분류된 인간 CD14+ 단핵구를 TAF로 6시간 동안 전처리한 다음 LPS를 사용하거나 사용하지 않고 24시간 동안 자극했다. CD14+ 단핵구 집단은 유동 세포측정법을 사용하여 결정되었다. We investigated the effect of TAF on human monocytes because monocytes are recruited to the injured liver and differentiate into recruited MPs. We sorted CD14 + monocytes from peripheral blood mononuclear cells. Sorted human CD14 + monocytes were pretreated with TAF for 6 h and then stimulated with or without LPS for 24 h. CD14 + monocyte populations were determined using flow cytometry.
세포내 단백질을 염색하고, 자극된 CD14+ 세포를 세포내 단백질 염색을 위해 Fix 완충액 I 및 Perm/Wash 완충액 I(BD Biosciences, Franklin Lakes, NJ, USA)로 고정 및 투과화시켰다.Intracellular proteins were stained, and stimulated CD14 + cells were fixed and permeabilized with Fix buffer I and Perm/Wash buffer I (BD Biosciences, Franklin Lakes, NJ, USA) for intracellular protein staining.
PD-L1+ 및 HLA-DR(human leukocyte antigen-DR isotype)+ 개체군은 모의 그룹에 비해 TAF 농도가 증가함에 따라 감소했다(도 7B). HLA-DR+/PD-L1+ 세포수는 LPS 처리 후 증가하였으나 TAF 자극군에서는 농도 의존적으로 감소하였다(도 7C). 더욱이, TAF 처리는 모의 그룹에 비해 활성화된 CD14+ 단핵구에서 HLA-DR 및 PD-L1 MFI를 감소시켰다(도 7D). 모의 또는 TAF 처리된 활성화된 인간 CD14+ 단핵구에서 대표적인 AKT 및 p-AKT 수준 플롯을 유동 세포측정법을 사용하여 분석했다. 인산화된 AKT 개체군은 p-AKT MFI 값을 기반으로 활성화된 인간 CD14+ 단핵구에 TAF를 추가한 후 크게 감소했다. AKT 인산화는 TAF 처리에 의해 차단되었다(도 7E). 도 7F 는 NASH 간에서 TAF의 역할에 대한 도식적 표현을 나타낸다. PD-L1 + and human leukocyte antigen-DR isotype (HLA-DR) + populations decreased with increasing TAF concentration compared to the sham group ( Figure 7B ). The number of HLA-DR + /PD-L1 + cells increased after LPS treatment, but decreased in a concentration-dependent manner in the TAF-stimulated group (Figure 7C). Moreover, TAF treatment reduced HLA-DR and PD-L1 MFI in activated CD14 + monocytes compared to the sham group ( Figure 7D ). Representative AKT and p-AKT level plots in mock- or TAF-treated activated human CD14 + monocytes were analyzed using flow cytometry. The phosphorylated AKT population was significantly reduced after addition of TAF to activated human CD14 + monocytes based on p-AKT MFI values. AKT phosphorylation was blocked by TAF treatment (Figure 7E). Figure 7F shows a schematic representation of the role of TAF in NASH liver.
[통계 분석][Statistical analysis]
모든 통계 분석은 GraphPad Prism 버전 7(GraphPad, Inc., San Diego, CA, USA)을 사용하여 수행되었다. 연속 변수는 독립적인 t-테스트를 사용하여 평가되었다. 두 매개 변수 간의 관계는 Pearson의 상관 테스트를 사용하여 조사되었다. 통계적 유의성은 p <0.05로 설정되었다.All statistical analyzes were performed using GraphPad Prism version 7 (GraphPad, Inc., San Diego, CA, USA). Continuous variables were evaluated using independent t-tests. The relationship between two parameters was investigated using Pearson's correlation test. Statistical significance was set at p < 0.05.
Claims (6)
A pharmaceutical composition for preventing or treating non-alcoholic fatty liver disease comprising tenofovir alafenamide (TAF) or a pharmaceutically acceptable salt thereof.
The pharmaceutical composition according to claim 1, wherein the non-alcoholic fatty liver disease is non-alcoholic steatohepatitis.
The pharmaceutical composition according to claim 1, wherein the composition inhibits inflammation in non-alcoholic fatty liver disease.
The pharmaceutical composition according to claim 3, wherein the inflammation is caused by increased inflammatory macrophages compared to the normal control group.
The method of claim 1, wherein the composition reduces the level of one or more liver enzyme markers selected from the group consisting of AST (aspartate aminotransferase), ALT (alanine aminotransferase), and triglyceride, which are increased in the blood compared to the normal control group. Characterized pharmaceutical composition.
A health functional food composition for preventing or improving non-alcoholic fatty liver disease containing tenofovir alafenamide (TAF) or a pharmaceutically acceptable salt thereof.
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