TW201236678A - New non/low side-effect pharmaceutical composition of anti-tuberculosis drugs - Google Patents

Abstract

A new non/low side-effect pharmaceutical composition of anti-tuberculosis drugs, which contains a pharmaceutically effective dose of isoniazid (INH) in combination with a pharmaceutically effective dose of rifampin (RIF), or further in combination with a pharmaceutically effective dose of pyrazinamide (PZA), or further in combination with a pharmaceutically effective dose of Ethambutol (EMB), or further in combination with a pharmaceutically effective dose of other compound drugs, together with a pharmaceutically effective dose of a substance capable of reducing the side effect of anti-tuberculosis drugs. Furthermore, the new non/low side-effect pharmaceutical composition of anti-tuberculosis drugs according to this invention contains a pharmaceutically effective dose of pyrazinamide (PZA) in combination with a pharmaceutically effective dose of rifampin (RIF), or further in combination with a pharmaceutically effective dose of isoniazid (INH), or further in combination with a pharmaceutically effective dose of Ethambutol (EMB), together with a pharmaceutically effective dose of a substance capable of reducing the side effect of anti-tuberculosis drugs, so as to reduce the side effect, such as hepatotoxicity caused by pyrazinamide (PZA).

Description

201236678 六、發明說明： 【發明所屬之技術領域】 本發明係關於一種無/低副作用之一抗結核病藥物新複方，特別是指一 種將異终驗醯胺(INH)，或異菸鹼醯胺(麵）和立復黴素(rifampin)，或異终 驗酿胺(INH)和立復齡师)和丙基硫緋醯胺(pzA)，或丙基硫異終酿胺 (PZA)，合併使用細胞色素P45〇 2E1 (CYp2E1)抑制劑或醯胺水解酶㈣d㈣ 抑制劑’以降低由異终驗醯胺(聰）或丙基硫異於酿胺㈣㈤麵㈣所引 起之肝毒性等副作用之抗結核病藥物新複方。 【先前技術】 根據世界衛生組織(WH〇)估計，全敍約有三分之-的人㈣染肺結 核’每年約有八百萬新增賴；㈣騎登記的肺賴病患人數最近幾年 也不健升，每十萬人口有六衫人❹肺結核，但其巾只有大約四分之 三的人接受完整治療；根據衛生署的統計，台灣每天至少有4 2個人死於肺 結核；在這麼多接受肺結核藥物治療的病患中，臨床上最f見的藥物副作 用即為肝毒性和神經系統病變(如：聽神經和視神經病變），其中又以肝毒性 最為常見。再加上台灣又是B型及c断炎的盛行區，感染肺結核之肝炎 患者也不在錄，假鱗年有14，_錢增的騎核病患，粗略估計至少 有，〇〇〇名到3,〇〇〇名慢性肝病患者需接受抗結核藥物治療，因此在這些病 患身上所可能發生断毒性是吾人*可忽視的醫源性疾病。 多數的第-線抗結核藥物，例如：異於舰胺(isQniazid，俗稱敵療勉 星）、丙基硫異菸醯胺(pyrazinamide，俗稱敵癆新邁)及立復黴素(—pin)等 ^有導，肝毒性發生的潛在孩反應；其中異紐醯胺㈣一是目前最 效的早-抗結核藥物，也最容易引起服用者產生肝毒性；在6〇年代末期 ^續有異菸鹼醯胺(isoniazid)造成肝毒性的報告；祕驗酿胺(is〇n娜所造 成具有臨床症狀的肝毒性約〇.M%，而在购％的病患中，則可觀察到無 201236678 症狀的肝功能異常，這些肝功能異常通常於服藥後兩個月内發生。 如圖一所示’異菸鹼醯胺(isoniazid)在肝臟中主要經由氮-乙醯氨基轉移 酶(N-acetyltransferase，NAT)的幫助而乙醯化，產生的中間產物乙醯化異菸鹼 醯胺(acetylisoniazid)迅速被水解成乙醯化聯胺(acetylhydrazine);乙醯化聯胺 可以再經由氣-乙醯氣基轉移酶(N-acetyltransferase)被乙醢化成無毒性的雙 乙醯化聯胺(diacetylhydrazine)，或者經由細胞色素P450 2E1 (CYP450 2E1) 氧化成具有肝毒性的分子’其中包括乙酿化偶氣(aCetyldiaZene)、乙酿敍離 子(acetylonium ion)、乙醯自由基(acetyiradical)、乙烯酮(ketene)等，另外在 有氧及NADPH存在時’乙酿化聯胺會被細胞色素P45〇 2E1反應生成自由 基而造成氧化壓力，導致細胞死亡；此外，異菸鹼醯胺(isoniazid)亦可經由 醯胺水解酶(amidase)直接水解成有毒性的聯胺(hydrazine)，或者由上述乙醯 化聯胺(acetylhydrazine)經醯胺水解酶(amidase)水解成有毒性的聯胺 (hydrazine) 〇 近來有研究顯示，聯胺(而非異菸鹼醯胺或乙醯化聯胺)是在兔及鼠體内 造成異於驗醯胺引起之肝毒性(ΓΝΗ-induced hepatotoxicity)最可能的主因， 研究者認為異菸鹼醯胺引起之肝毒性的嚴重性與血漿中聯胺的濃度成正相 關；1999年Sarich等人的報導則認為對硝基苯盼填酸二酯 phosphate，BNPP ’為一種醢胺水解酶之抑制劑)可預防異菸驗醯胺引起之肝 毒性的傷害，其保護機制應是透過抑制異菸鹼醯胺產生聯胺。 細胞色素P450 2E1 (CYP2E1)在肝臟中會持續的表現，並負貴許多異物 質（如.肝毒素四氣化碳(CCU)以及乙醢氨盼(acetaminophen))的代謝生物 反應；然而’ CYP2E1在異菸驗醯胺引起之肝毒性中所扮演的角色並不明 確，異菸鹼醯胺本身即為CYP2E1的一種誘導物；有些研究認為肝臟内的 CYP2E1與異菸鹼醯胺引起之肝毒性的機制有關。在體外試驗中，雙硫舍 (disulfimm，DSF)及其代謝物二乙基二硫代氨基甲酸(diethyldithi〇carbamate) 均被確認為老鼠及人類肝臟微粒體CYP2E1的選擇性抑制劑(selective 201236678 mechanism-based inhibitors) ’ Brady等人的試驗則顯示老鼠服用單一 口服劑 量的雙硫w (DSF)後，會造成免疫反應肝容量(immun〇reactive hepatic c〇ntent) 以及CYP2E1催化活性快速且完全的下降。201236678 VI. Description of the Invention: [Technical Field of the Invention] The present invention relates to a new compound for anti-tuberculosis drugs with no/low side effects, in particular to a different final test amine (INH), or isonicotinium amide (face) and rifampin, or heterogeneous amine (INH) and eponymide) and propyl thioguanamine (pzA), or propyl thioisolanamide (PZA), Combination of cytochrome P45〇2E1 (CYp2E1) inhibitor or indoleamine hydrolase (iv) d(iv) inhibitor' to reduce side effects such as hepatotoxicity caused by heterologous prostamine (Cong) or propyl sulphide (Si) (5) face (4) A new compound for anti-tuberculosis drugs. [Prior Art] According to the World Health Organization (WH〇), there are about three-thirds of the total number of people (four) infected with tuberculosis 'about 8 million new liters per year; (four) the number of lung-dwelling patients registered in recent years also Not healthy, there are six people in every 100,000 people who have tuberculosis, but only about three-quarters of them have complete treatment. According to the statistics of the Department of Health, at least 42 people die from tuberculosis every day in Taiwan. Among the patients receiving tuberculosis drug treatment, the most clinical side effects of the drug are hepatotoxicity and neurological diseases (such as auditory nerve and optic neuropathy), of which hepatotoxicity is the most common. In addition, Taiwan is also a prevalent area of B-type and c-broken inflammation. The hepatitis patients infected with tuberculosis are not recorded. The fake scale year has 14, _ Qian Zeng's riding nuclear disease, roughly estimated at least, anonymous to 3, patients with chronic liver disease need to receive anti-tuberculosis drugs, so the possible toxicity in these patients is a iatrogenic disease that we can ignore. Most of the first-line anti-tuberculosis drugs, such as: isidamine (isQniazid, commonly known as miracle comet), propyl sulphonic acid (pyrazinamide, commonly known as chlorpyrifos) and levothinomycin (-pin) Etc., the potential child reaction of hepatotoxicity; among them, isotamide (4) is the most effective early-anti-tuberculosis drug, and is also the most likely to cause hepatotoxicity in the patients; in the late 6th century, there is a difference Report of hepatotoxicity caused by nicotinamide (isoniazid); secretive amine (the hepatotoxicity caused by clinical symptoms of is〇nna is about 〇M.%, while in the case of % of patients, no observation can be observed 201236678 Symptoms of abnormal liver function, which usually occur within two months after taking the drug. As shown in Figure 1, 'isoniazid' in the liver mainly through nitrogen-acetylaminotransferase (N- With the help of acetyltransferase, NAT), the intermediate product acetylisoniazid is rapidly hydrolyzed to acetylhydrazine; acetylated hydrazine can be passed through gas-B N-acetyltransferase is converted to non-toxic double B by acetamidine Diacetylhydrazine, or oxidized to a hepatotoxic molecule via cytochrome P450 2E1 (CYP450 2E1), including aCetyldiaZene, acetylonium ion, acetylene free radical (acetyiradical), ketene, etc., in addition, in the presence of aerobic and NADPH, the acetylated hydrazine will be reacted by cytochrome P45〇2E1 to generate free radicals, causing oxidative stress and causing cell death; in addition, isonicotine Isoniazid can also be directly hydrolyzed to a toxic hydrazine via amidase or hydrolyzed to a toxic by amidase by the above acetylhydrazine. Hydrazine 〇 Recently, studies have shown that hydrazine (rather than isoniazid guanamine or acetylated hydrazine) causes hepatotoxicity caused by prodamine in rabbits and mice (ΓΝΗ-induced) Hepatotoxicity) The most likely cause, the researchers believe that the severity of hepatotoxicity caused by isoniazid guanamine is positively correlated with the concentration of hydrazine in plasma; in 1999, Sarich et al. reported that nitrobenzene was expected to be filled with diester. Phosphate BNPP 'is an amine inhibitor of hydrolase minced) can prevent the damage of liver toxicity caused Amides isonicotinoyl test, the protection mechanism should be generated through hydrazine inhibition of growth niacinamide. Cytochrome P450 2E1 (CYP2E1) continues to behave in the liver and is responsible for the metabolic bioreactivity of many foreign substances such as hepatic toxin tetracarbonized carbon (CCU) and acetaminophen; however, 'CYP2E1 The role played by hepatotoxicity caused by isoniazid is not clear. Isoniazid amide itself is an inducer of CYP2E1; some studies suggest that hepatotoxicity caused by CYP2E1 and isonicotamine in liver Related to the mechanism. In vitro, disulfimm (DSF) and its metabolite diethyldithicarbamate were identified as selective inhibitors of mouse and human liver microsomes CYP2E1 (selective 201236678 mechanism -based inhibitors) 'The test by Brady et al. showed that mice taking a single oral dose of disulfide w (DSF) caused an immunoreactive hepatic c〇ntent and a rapid and complete decline in CYP2E1 catalytic activity. .

Sodhi等人則在1997年的報導指出，氧化壓力是造成幼鼠體内異菸鹼 醯胺及立復黴素引起之肝毒性的因素之一。有許多的研究想要找出適當的 生物標記(biomarker)以評估體内氧化傷害的速率，目前可能適用的生物標記 可分為三類，分別為對脂質、蛋白質、核酸氧化傷害的標記；8_異構*** 素F& (8-iso-prostaglandin Fi 8-iso-PGF2a)是一種自由基引起花生四烯酸 (arachidonic acid)發生脂質過氧化作用的產物，其化學性質穩定，8_is〇_pGF2a 含置可作為判斷活體内脂質過氧化的新指標，該脂質過氧化反映可能與體 内自由基的產生、乳化性的傷害(oxidative damage)及抗氧化劑的缺乏 (antioxidantdeficiency)有關；目前有許多方法可用來測量8_is〇_PGF2a含量， 包括酵素免疫分析法（enZyme immunoassay)、放射免疫分析法 (radioimmunoassay)、氣相層析質譜儀(gas_chromat〇graphy mass spectrometry) 以及液相層析質 s普儀(liquid chromatography mass spectrometry)等；此外，人 類尿液中的8-iso-PGF2a及其代謝物2,3-dinor-8-iso-PGF2a含量可利用C18固 相萃取(C18solid phase extraction，SPE)準備樣品後，再以液相層析串聯式質 譜儀(LC/MS/MS)分析。 利用侵入式及非侵入式方法測試大鼠(rat)肝功能，以監測肝損害的發展 以及篩選肝臟疾病’其中最常使用的方法包含測量血清中之天門冬氨酸轉 胺酶（aspartate aminotransferase, AST)、丙氨酸轉胺酶（alanine aminotransferase, ALT)以及驗性鱗酸酶(alkaline phosphatase)數值，以及測量 肝細胞產物如：膽紅素(biiimbin)、白蛋白(albumin)，以及利用量測前凝血 素時間(prothrombin time)來檢測凝血因子(coagulation factors)等；肝功能定 量測試是根據幾乎只經過肝臟代謝之受質在血清中的濃度而定，這些受質 的清除疋依肝門靜脈、肝動脈血流量以及由肝細胞對這些受質的作用而 201236678 定，肝臟金流量與提供給肝臟的受質量有關，反之，該受質的清除則決定· 於肝臟代謝的能力。 半乳糖(galactose)是一種具有高萃取率(extracti〇n她）、％%在肝臟中 代謝的醣類’在肝臟中’半乳糖是由半乳糖激酶(galact〇kinase)經過差向立 體異構化反應（epimerizaticm)，將之轉換成^磷酸葡萄糖 (Glucose-1-phosphate);半乳糖激酶的作用反應為肝細胞中半乳糖代謝途徑 的速率決定步驟(mte-Umiting step)。半乳糖的高萃取率使得依賴肝臟血流量 及肝臟功能的半乳糖代謝作用成為檢測肝功能最主要的方式，目前並無一 定的規則來評估大鼠之殘餘肝功能(1*以(11^11^^£1111比〇11)，量測一確切:合 物(如：半乳糖)之代謝能力，可推測肝臟中一代謝作用之速率決定步驟，: 可能取得殘餘肝功能之代表數質。 以半乳糖清除能力(galactose elimination capacity，GEC)作為人類肝功能 定量測試已行之有年’飾’半乳糖清除能力測試需取得多個血液樣本= 建立標準轉，姐床細上有其困歸，因此有料研究制半乳糖單 點法(Galactose Single Point，GSP)以評估人類肝功能；本案發明人以半乳糖 單點法測試慢斷炎、肝硬似及肝癌病患，結果顯科乳料點法可精 確測出這些肝臟疾病；半乳糖單點法已被成功的應用到測試肝病患者排除 如丙嗪(promazine)及抗生素頭孢酮(cefoperaz〇ne)等藥物之剩餘肝功能。此 外，半乳糖單點法已在美國食品藥物管理局(FDA)所出版的指南(咖_ for Industry)中成為建議採用測試肝功能的方法之一。 由此可見，上述習用抗結核藥物異於鹼醯胺(is〇niazid)仍有諸多缺失， 實非一良善之設計者，而亟待加以改良。 本案發明人鑑於上述習用抗結核藥物異於祕胺(is〇niazi撕導致肝毒 性等副作用的缺點，以及承襲本實驗室先前專利案（低副作用之麵新複 方，案號096101545)及(含異菸鹼醯胺(Isoniazid，_)之低副作用新複方 (2) ’案號097141522)之發明，乃亟思加以改良創新，並經多年苦心孤謅潛 201236678 心研究後，終於成功研發完成本件無/低副作用之抗結核病藥物新複方。另 外，在本案發明人先前專利申請案，PCT申請案號PCT/CS2〇〇8/〇〇1353 (低 副作用的异烟碱酰胺新复方）發明中，雖已揭示_單方併用部分本實驗 至揭露之CYP2E1抑制劑的藥學組合物，可降低所導致肝毒性等副作 用彳一後續發明人之研究，發現並不是任意的劑量組合都可以於體内達到 去除INH肝毒性的效果’例如，在動物體内實驗發現，小鼠每日合併使用 山奈酚(Kaempfer〇l) 3.78 mg/kg 與 INH/RIF 50 / 100 mg/kg 腹腔注射持續 3 週’可顯著抑制INH所導致之肝毒性，控制組(INH/RIF 5〇 / 1〇〇 mg/kg)之相Sodhi et al. reported in 1997 that oxidative stress is one of the factors that cause hepatotoxicity caused by isonicotinic acid guanamine and rifamycin in young rats. There are many studies that want to find appropriate biomarkers to assess the rate of oxidative damage in the body. Currently, biomarkers that may be applicable can be classified into three categories, which are markers for oxidative damage to lipids, proteins, and nucleic acids; _Isoprostaglandin F& (8-iso-prostaglandin Fi 8-iso-PGF2a) is a product of free radical-induced lipid peroxidation of arachidonic acid, which is chemically stable, 8_is〇_pGF2a Containing can be used as a new indicator to determine lipid peroxidation in vivo. The lipid peroxidation may be related to the production of free radicals, oxidative damage and antioxidant deficiency in the body. There are many methods. It can be used to measure the content of 8_is〇_PGF2a, including enZyme immunoassay, radioimmunoassay, gas-chromatography mass spectrometry, and liquid chromatography. Liquid chromatography mass spectrometry); in addition, 8-iso-PGF2a in human urine and its metabolite 2,3-dinor-8-iso-PGF2a The amount can be prepared by C18 solid phase extraction (SPE) and then analyzed by liquid chromatography tandem mass spectrometer (LC/MS/MS). Invasive and non-invasive methods are used to test rat liver function to monitor the development of liver damage and to screen for liver disease. The most commonly used method involves measuring aspartate aminotransferase in serum. AST), alanine aminotransferase (ALT), and alkaline phosphatase values, as well as measurements of hepatocyte products such as bilirubin (biiimbin), albumin (albumin), and utilization Prothrombin time is used to detect coagulation factors, etc.; liver function quantitative test is based on the concentration of serum in the liver that is almost exclusively metabolized by the liver, and the clearance of these receptors depends on the portal vein Hepatic arterial blood flow and the effect of hepatocytes on these receptors, 201236678, liver blood flux is related to the quality of the liver, and vice versa, the ability of the liver to metabolize. Galactose is a sugar with high extraction rate (extracti〇n her), %% metabolized in the liver 'in the liver' galactose is galactose kinase (galact〇kinase) through differential stereoisomerism The reaction (epimerizaticm) is converted to Glucose-1-phosphate; the action of galactose kinase is the mte-Umiting step of the galactose metabolic pathway in hepatocytes. The high extraction rate of galactose makes galactose metabolism, which depends on liver blood flow and liver function, the most important way to detect liver function. There is no rule to evaluate the residual liver function of rats (1*以(11^11) ^^£1111 〇11), the measurement is exact: the metabolic capacity of the compound (such as galactose), it can be speculated that the rate of a metabolic action in the liver determines the steps: The representative quality of residual liver function may be obtained. The galactose elimination capacity (GEC) has been used as a quantitative test for human liver function. It has been carried out for several years. The galactose clearance test requires multiple blood samples to be established. Therefore, it is expected to study the Galactose Single Point (GSP) method to evaluate human liver function; the inventor of this case tested the chronic bronchitis, liver cirrhosis and liver cancer patients by galactose single-point method, and the results showed that the dairy point was obvious. The method can accurately detect these liver diseases; the galactose single-point method has been successfully applied to test the residual liver function of patients with liver diseases such as promazine and cefoperaz〇ne. In addition, the galactose single-point method has become one of the recommended methods for testing liver function in the guidelines published by the US Food and Drug Administration (FDA). It can be seen that the above-mentioned anti-tuberculosis drugs are different. There are still many defects in is〇niazid, which is not a good designer, but needs to be improved. The inventors of the present invention have different side effects such as hepatotoxicity due to the use of anti-tuberculosis drugs. Disadvantages, as well as inheriting the previous patent case of the laboratory (new compound for low side effects, case number 096101545) and (new compound (2) 'case number 097141522) containing low side effects of isoniazid (Isoniazid, _) Invented, Nai Sisi made improvements and innovations, and after years of painstaking efforts, 201236678 heart research, finally successfully developed a new compound for anti-tuberculosis drugs with no/low side effects. In addition, the inventor's previous patent application, PCT application Case No. PCT/CS2〇〇8/〇〇1353 (New compound of isonicotinic acid amide with low side effects) In the invention, it has been revealed that the pharmacy group of CYP2E1 inhibitors which have been partially combined with this experiment has been disclosed. Compound, which can reduce the side effects caused by hepatotoxicity. A follow-up inventor's research found that not all dose combinations can achieve the effect of removing INH hepatotoxicity in vivo'. For example, in vivo experiments in mice Daily combination of kaempferol (Kaempfer〇l) 3.78 mg/kg and INH/RIF 50 / 100 mg/kg intraperitoneal injection for 3 weeks can significantly inhibit hepatotoxicity caused by INH, control group (INH/RIF 5〇/ 1〇〇mg/kg) phase

關肝功能指標 GOT、GPT 和 GSP 值為 571±295 U/L、364±192 U/L 和 866土 339 mg/L’ 而併用山奈盼 3.78 mg/kg 組之 GOT 值則為 89土 19 U/L、48±21U/L 和245±98mg/L，接近正常值，然而山奈酚使用劑量調整為189mg/kg後， 各項相關肝功能指標相較於控制組所下降的幅度均未達併用山奈酚3 78 mg/kg所獲得之成效。由以上動物試驗結果可知當合併使用cyp2Ei抑制劑 時’對INH所導断毒性確實具纽善絲，但必彡貞蚊其使關量因 此基於實驗結果，本發明著重於抑侧使關量之決定，可為新穎及進步 性發現。 【發明内容】 本發明之目的即在於提供—種無/制侧之抗結核病祕新複方藥物 組合’内含-藥學有效量之胁驗轉，或再個—藥學有 效量之立復齡(rifampin，RIF)，或再併用—藥學有效量之丙基硫異终酼胺 (pymzinamide，PZA) ’或再併用一藥學有效量之乙醯胺醇(Ethambut〇i， EMB) ’或再個-醉有效量之其他複方藥物，合併使用—藥學有效量之 、”田月l色素P450 2E1 (CYP2E1)抑制劑或醯胺水解酶(amidase)抑制劑，以降低 由異菸鹼醢胺(INH)所引起之肝毒性等副作用。 可達成上述發明目的之無/低副作用之抗結核病藥物新複方，内含一藥 201236678 學有效量之異菸鹼醯胺(isoniazid，INH)，或再併用一藥學有效量之其他複-方藥物，合併使用一藥學有效量之細胞色素P450 2E1(CYP2E1)抑制劑或醯 胺水解S^(amidase)抑制劑，其中該CYP 2E1抑制劑或amidase抑制劑係選 自於下列化合物所組成群組：正二羥癒瘡酸(Nordihydroguaiaretie add；)、 (-)-Epigallocetechin-3-gallate、茵陳色原酮（Capillarisin)、山奈盼 (Kaempferol)、根皮素(Phloretin)、橙皮素(Hesperetin)、6-薑辣醇(6-Gingerol)、 沒食子酸(gallic acid)、異甘草素(Isoliquritigenin)、柚皮素(Narigenin)、二氫 化槲皮素((+)-Taxifolin)、漢黃芩素(Wongonin)、原兒茶酸(protocatechuic acid)、兒茶素（(+)-Catechin)、yS -奈黃酮（β-naphthoflavone)、恩貝素 (Embelin)、反式肉桂酸(trans-Cinnamic acid)、表兒茶酚((-)_Epicatechin)、根 皮苷(Phloridzin)、Brij 58、Brij 76、Brij 35、Tween 20、Tween 80、Tween 40、 PEG 2000、PEG 400、Pluomic F68、PEG 4000、反式肉桂酸 (Trans-Cinnamaldehyde)、大豆甘元(Daidzein)、異牡荊素(lsovitexin)、香 葉烯(β-Myrcene)、槲皮素(Quercetin)、（+)-檸檬烯((+)_Limonene)、楊梅素 (Myricetin)、槲皮(Quercitrin)、木犀草素-7-葡萄糖苷（Luteolin-7-Glucoside)、 桑葉素（Morin)、新橙皮苷（Neohesperidin)、橙皮苷（Hesperidin)、 ((-)-Epigallocatechin)、木犀草素(Luteolin)、金絲桃苦(Hyperoside)、十四烧 酸乙酯(Ethyl Myristate)、禋柳素(Tamarixetin)、黃答素(Baicalein)、芸香素 (Rutin)、黃答（Baicalin)、芹菜素（Apigenin)、（+)-Epicatechin、 ㈠-Epicatechin-3-gallate、水飛薊賓(Silybin)、牡荊素(Vitexin)、金雀異黃酮 (Genistein)、異鼠李素(Isorhamnetin)、香葉木素(Diosmin)、葛根素(Puerarin)、 或傘形花内酯(Umbelliferone)、高良薑素(Galangin)、非瑟酮(fisetin)、 Cremophor EL ' Sodium Lauryl Sulfate ' Microcrystalline cellulose ' Dicalcium phosphate dihydrate、Mannitol、Cremophor RH40、Sucralose、Crospovidone、 Sodium starch glycolate、Crospovidone、Eudragit SI00、Croscarmellose sodium、Menthol、Saccharin、hydroxypropylcellulose、Pregelatinized starch、 8 201236678The GOT, GPT and GSP values of the liver function indicators were 571±295 U/L, 364±192 U/L and 866 soil 339 mg/L′, and the GOT value of the combination of Shannai 3.78 mg/kg was 89 soil 19 U. /L, 48±21U/L and 245±98mg/L, close to the normal value. However, after the dosage of kaempferol was adjusted to 189mg/kg, the relative liver function indexes were not compared with those of the control group. The effect of kaempferol 3 78 mg/kg. From the above animal test results, it can be seen that when the combination of cyp2Ei inhibitors is used, the inducing toxicity to INH does have a good-nose silk, but the insecticides are based on the experimental results, and the present invention focuses on the side-effects. Decisions can be novel and progressive discoveries. SUMMARY OF THE INVENTION The object of the present invention is to provide a non-tubular anti-tuberculosis secret new compound drug combination 'inclusion-pharmaceutical effective amount of stress test, or another - pharmaceutically effective amount of rifampin , RIF), or in combination - a pharmaceutically effective amount of pymzinamide (PZA) ' or a combination of a pharmaceutically effective amount of ethambutol (EMB) ' or another - drunk An effective amount of other combination drugs, combined with a pharmaceutically effective amount of "Tian Yue l pigment P450 2E1 (CYP2E1) inhibitor or indoleamine hydrolase (amidase) inhibitor to reduce by isoniazid amide (INH) Side effects of liver toxicity caused by the above-mentioned invention. A new compound of anti-tuberculosis drugs with no/low side effects, which contains a drug 201236678, an effective amount of isoniazidamine (INH), or a combination of pharmaceutically effective A further pharmaceutically effective amount of a cytochrome P450 2E1 (CYP2E1) inhibitor or a guanidine hydrolyzed S (amidase) inhibitor, wherein the CYP 2E1 inhibitor or amidase inhibitor is selected from the group consisting of Group of the following compounds: positive Hydrodihydroguaiaretie add;, (-)-Epigallocetechin-3-gallate, Capillarisin, Kaempferol, Phloetin, Hesperetin, 6 - 6-Gingerol, gallic acid, Isoliquritigenin, Nariginin, dihydroquercetin ((+)-Taxifolin), wogonin ( Wongonin), protocatechuic acid, catechin ((+)-Catechin), yS-naphthoflavone, embelin, trans-Cinnamic acid , epicatechin ((-)_Epicatechin), phloridzin (Phloridzin), Brij 58, Brij 76, Brij 35, Tween 20, Tween 80, Tween 40, PEG 2000, PEG 400, Pluomic F68, PEG 4000, anti Trans-Cinnamaldehyde, Daidzein, lsovitexin, β-Myrcene, Quercetin, (+)-Limonene ((+)_Limonene ), Myricetin, Quercitrin, Luteolin-7-Glucoside, Morin, Neohesperidin (Neoh Esperidin), Hesperidin, (---Epigallocatechin), Luteolin, Hyperoside, Ethyl Myristate, Tamarixetin , Baicalein, Rutin, Baicalin, Apigenin, (+)-Epicatechin, (I)-Epicatechin-3-gallate, Silybin, Vitexin ( Vitexin), Genistein, Isorhamnetin, Diosmin, Puerarin, Umbelliferone, Galangin, Non Fisetin, Cremophor EL 'Sodium Lauryl Sulfate 'Microcrystalline cellulose ' Dicalcium phosphate dihydrate, Mannitol, Cremophor RH40, Sucralose, Crospovidone, Sodium starch glycolate, Crospovidone, Eudragit SI00, Croscarmellose sodium, Menthol, Saccharin, hydroxypropylcellulose, Pregelatinized starch, 8 201236678

Dextrates NF hydrated、Citric acid、Aerosil 200、PEG 8000、Sorbic acid、Lemon oil' Hydroxy propylcellulose' Sodium benzoate' Acesulfame K' Hydroxypropyl methylcellulose、Hydroxy ethyl methylcellulose、Methyl cellulose、Sodium cyclamate、Lactose monohydrate、Maltodextrin、Glyceryl behenate、Oxide red、Dextrates NF hydrated, Citric acid, Aerosil 200, PEG 8000, Sorbic acid, Lemon oil' Hydroxy propylcellulose' Sodium benzoate' Acesulfame K' Hydroxypropyl methylcellulose, Hydroxy ethyl methylcellulose, Methyl cellulose, Sodium cyclamate, Lactose monohydrate, Maltodextrin, Glyceryl behenate, Oxide Red,

Glycerrin monostearate' Copovidone K28' Starch acetate' Magnesium stearate'Glycerrin monostearate' Copovidone K28' Starch acetate' Magnesium stearate'

Sodium lauryl sulfate、Povidone K-30、Benzyl alcohol、Methylparaben、 Propylparaben、Solutol HI5、Butylated hydroxyl anisol。 更進一步’本發明之無/低副作用之抗結核病藥物新複方包括一藥學有 效量之丙基硫異菸醯胺(PZA)，或再併用一藥學有效量之其他複方藥物，合 併使用一藥學有效量之醯胺水解酶(amidase)抑制劑，其中該amidase抑制劑 係選自於下列化合物所組成群組：槲皮素(Quercetin)、高良薑素(Galangin)、 桑葉素(Morin)、非瑟酮(fisetin)、異甘草素' 楊梅素(Myricetin)、木犀草素 (Luteolin)、山奈酴(Kaempferol)、茵陳色原酮(Capillarisin)、Cremophor EL、 Sodium Lauryl Sulfate、Tween 20、Brij58，以降低由丙基硫異菸醯胺(PZA) 所引起之肝毒性等副作用。 本發明所提供之無/低副作用之抗結核病藥物新複方，亦可加入藥學上 可接受之賦形劑至該複方，該賦形劑可為稀釋劑、填充劑、結合劑、崩解 劑、调滑劑專’例如：Tween 20、Tween 40、Tween 60、Tween 80、Brij 35、 Brij 58、Brij 76、Pluronic F68、Pluronic F127、（Poloxamer 407)、PEG 400、 PEG 2000、PEG 4000、Span 60、Span 80、Myri 52、PEG 8000、Acesulfame potassium、Aerosil 200、（Colloidal silicon dioxide)、Butylated hydroxyl aniso卜 Com starch、Crospovidone、Croscarmellose sodium、Dicalcium phosphate dihydrate ' EDTA 2 Na ' Lactose ' Lactose monohydrate ' Lactose S.G 'Sodium lauryl sulfate, Povidone K-30, Benzyl alcohol, Methylparaben, Propylparaben, Solutol HI5, Butylated hydroxyl anisol. Further, the new compound of the anti-tuberculosis drug of the present invention having no/low side effects includes a pharmaceutically effective amount of propyl sulphoidinamide (PZA), or a combination of a pharmaceutically effective amount of other compound drugs, and a combination of pharmaceutically effective drugs A dose of amidase inhibitor, wherein the amidase inhibitor is selected from the group consisting of quercetin (Quercetin), galangin (Galangin), mulberry (Morin), non- Fisetin, isoglycyrrhizin, Myricetin, Luteolin, Kaempferol, Capillarisin, Cremophor EL, Sodium Lauryl Sulfate, Tween 20, Brij58, To reduce side effects such as hepatotoxicity caused by propyl sulfisoinamide (PZA). The new compound of the anti-tuberculosis drug provided by the present invention with no/low side effects may also be added to the compound by adding a pharmaceutically acceptable excipient, which may be a diluent, a filler, a binder, a disintegrating agent, Slip agent special 'for example: Tween 20, Tween 40, Tween 60, Tween 80, Brij 35, Brij 58, Brij 76, Pluronic F68, Pluronic F127, (Poloxamer 407), PEG 400, PEG 2000, PEG 4000, Span 60 ,Span 80, Myri 52, PEG 8000, Acesulfame potassium, Aerosil 200, (Colloidal silicon dioxide), Butylated hydroxyl aniso, Com starch, Crospovidone, Croscarmellose sodium, Dicalcium phosphate dihydrate ' EDTA 2 Na ' Lactose ' Lactose monohydrate ' Lactose SG '

Low-substituted hydroxypropylcellulose、Maltodextrin、Mannitol、Menthol、 Propyl paraben、Methyl paraben、Microcrystalline cellulose'Guar gum、Xanthan gum、Pregelatinized starch、Povidone K-30、Sodium starch glycolate、Sodium 201236678 lauryl sulfate、Sucralose、Solutol H15、Cremophor EL、Cremophor RH40、 Sodium cyclamate、PVP K90F、Oxide red、Hydroxypropyl methylcellulose、 Cherry、Lemon oi卜 Sorbic acid、Benzyl alcohol、Glycerrin、Sodium benzolate、 Starch acetate、Citric acid、Sorbitol solution、Opady white、Dextrates，NF hydrate、Magnesium stearate、Alginic acid、Eudragit E90、Eeudragit E、Glyceryl behenate、Gelucire、kollidon VA64 (copovidone K28)、Hydrochoric acid、 Hydroxy ethyl methyl cellulose、Hydroxy propyl cellulose、Methyl cellulose、 Methacrylic acid copolymer type B (Eudragit 100)、Maltose、Methacrylic Eudragit SI00 acid copolymer、PEG 1450、Povidone K-90、phosphoric acid 85%' polyoxyl 40 hydrogenated castor oil (RH 40) ' Polyoxyl 35 castor oil (EL 35) ' sodium dihydrogen phosphate ' saccarin ' triethyl citrate ' Tri-Sodium Citrate 或其餘列於美國 FDA Generally Recognized as Safe (GRAS)中之常用 成分。 【實施方式】 本發明將就下列實施例作進一步說明，然該等實施例僅為例示說明之 用’而不應被解釋為實施本發明之限制。 實施例一、異菸鹼醯胺(INH)合併使用CYP2E1抑制劑雙硫侖(DSF)及/或硝 基苯酚磷酸二酯(BNPP)之動物試驗 一、材料與方法 1.試驗材料 所有的有機溶劑均為HPLC等級，購自Tedia有限公司(Fairfield，〇H,Low-substituted hydroxypropylcellulose, Maltodextrin, Mannitol, Menthol, Propyl paraben, Methyl paraben, Microcrystalline cellulose 'Guar gum, Xanthan gum, Pregelatinized starch, Povidone K-30, Sodium starch glycolate, Sodium 201236678 lauryl sulfate, Sucralose, Solutol H15, Cremophor EL , Cremophor RH40, Sodium cyclamate, PVP K90F, Oxide red, Hydroxypropyl methylcellulose, Cherry, Lemon oi, Sorbic acid, Benzyl alcohol, Glycerrin, Sodium benzolate, Starch acetate, Citric acid, Sorbitol solution, Opady white, Dextrates, NF hydrate, Magnesium Stearate, Alginic acid, Eudragit E90, Eeudragit E, Glyceryl behenate, Gelucire, kollidon VA64 (copovidone K28), Hydrochoric acid, Hydroxy ethyl methyl cellulose, Hydroxy propyl cellulose, Methyl cellulose, Methacrylic acid copolymer type B (Eudragit 100), Maltose, Methacrylic Eudragit SI00 acid copolymer, PEG 1450, Povidone K-90, phosphoric acid 85% 'polyoxyl 40 hydrogenated castor oil (RH 40) ' P Olyoxyl 35 castor oil (EL 35) 'sodium dihydrogen phosphate 'saccarin 'triethyl citrate ' Tri-Sodium Citrate or the other commonly used ingredients listed in the US FDA Generally Recognized as Safe (GRAS). The invention is further illustrated by the following examples, which are intended to be illustrative only and are not to be construed as limiting. Example 1. Animal test of isoniazid amide (INH) combined with CYP2E1 inhibitor disulfiram (DSF) and/or nitrophenol phosphate diester (BNPP) I. Materials and methods 1. Test materials All organic The solvents are all HPLC grades and are available from Tedia Ltd. (Fairfield, 〇H,

USA) ’ INH，BNPP, DSF以及玉米油則購自sigma化學公司(st. Louis，MO USA) ’ S-iso-PGFh以及放射線標定之8_iso_PGF2a_d4則得自㈣麵化學公司 (Ann Arbor，MI，USA)，半乳糖注射溶液由南光化學製藥股份有限公司製 201236678 備’係將4GG克半乳糖(Sigma)溶於α升含有適#緩衝溶液系統以及等張鹽 類之蒸顧水中，供作注射使用。 2. 試驗動物 體重為320-350公克之雄性SD(Sprague-Dawley)大鼠購自國家實驗動物 中心(台灣）’動物實驗係遵照國衛院動物實驗指南進行，所有的大鼠均置於 空氣/濕度調節環境下’光照與黑暗各12小時，水及飼料的供給不限，在試 驗期間大鼠體重均持續監測，所有的大鼠均以使用5〇毫克/公斤體重劑量之 戊巴比妥鈉（sodium pentobarbital)進行腹腔麻醉（intraperit〇neally anesthetized)，將聚乙烯導管置於大鼠右頸内靜脈(intema丨jugular vdn)内以 打半乳糖’導管係以切入穿刺(cut-d〇wn technique)***，該導管的末端係 置於大鼠頸後切口之皮膚下方，手術完成後，恢復期間使大鼠禁食一夜(約 16小時）’但水分照常供給。 3. 試驗處理 試驗動物隨機分成5組，每組包括3種處理，第一種處理為注射25 mg/kg BNPP或BNPP之基劑(vehicle, VEm，即食鹽水），BNPP係溶於加熱至6(rc 之食鹽水(0.9。/。NaCl) ’冷卻後以1 ml/kg的體積進行腹腔内注射至大鼠體 内；第二種處理為則注射l〇〇mg/kgDSF或DSF之基劑（VEH2，即玉求油）， DSF係溶於玉米油中’以1 mi/kg的體積進行腹腔内注射至大鼠體内；第三 種處理為注射150 mg/kg INH或INH之基劑(VEH3，即食鹽水），INH係溶於 食鹽水(0.9%NaCl)中’以1 ml/kg的體積進行腹腔内注射至大鼠體内；第一 組(BNPP或VEH1)較第三組(INH或VEH3)早30分鐘處理，第二組(DSF或 VEH2)比第三組(INH或VEH3)早I5分鐘處理。 上述5組試驗共包含： ⑴對照組(normal control group，NC，n=12):正常的大鼠每天注射1次 VEH1、VEH2以及VEH3(施行腹腔内注射)共21天； (2)INH組(INH，n=7):正常的大鼠每天注射1次INH、VEH1以及VEH2 201236678 (施行腹腔内注射)共21天； ⑶BNPP-INH組(BNPP-INH，n=7):正常的大鼠每天注射1次bnpp、INH 以及VEH2(施行腹腔内注射)共21天； (4) DSF-INH組(DSF-INH，n=7):正常的大鼠每天注射^DSF、jnh以 及VEH1(施行腹腔内注射)共21天；以及 (5) BNPP-DSF-INH組(BNPP-DSF-INH，n=7):正常的大鼠每天注射1次 BNPP、DSF以及INH(施行腹腔内注射)共21天； 半乳糖單點法於第21天處理後16小時進行測試。 4. 血液樣本 處理完畢後’大鼠以***麻醉犧牲’血液由大鼠背部主動脈抽取，置 於含有EDTA之試管中’血漿(plasma)以i3,〇〇〇g於4°C離心15分鐘，分離後 的血漿分裝到微量小管(Eppendorf tube)中並置於-80°C中儲存。 5. 生化分析 肝細胞損傷以量測血漿中天門冬氨酸轉胺酶(AST)與丙氨酸轉胺酶 (ALT)活性以進行定量，AST與alt活性是肝臟毒性常用的指標’係以 Synchron LXi 725 系統來量測(Beckman Instruments,美國）。 6. 光學顯微鏡與電子顯微鏡 大鼠犧牲後肝臟隨即進行組織學分析；肝臟樣本以10%磷酸緩衝液配製 之福馬林(phosphate-buffered formalin)固定，隨後脫水並包埋於石蠟(paraffin) 中’以5 μιη厚度切片，切片樣本以蘇木精(hemat〇xylin)與伊紅(eosin)染色， 並進行肝糖染色試驗(Periodic acid Schiff stain, PAS)，染色後以光學顯微鏡 進行組織學觀察；另外，肝臟切片以二曱胂緩衝液(cac〇dylate buffer，〇 1M PH 7.4)清洗，以20%四氧化娥水溶液(aqueous osmium tetroxide)後固定1小 時’以酒精連續脫水後包埋於Spun*樹脂(Spurr resin)中，並以鑽石刀切取超 薄切片，以醋酸鈾醯(uranylacetate)及檸檬酸鉛(leadcitrate)作雙重染色，並 以穿透式電子顯微鏡(Transmission Electron Microscope, Hitachi 600, Hitachi 12 201236678USA) 'INH, BNPP, DSF and corn oil were purchased from sigma chemical company (st. Louis, MO USA) 'S-iso-PGFh and radiation calibration 8_iso_PGF2a_d4 was obtained from (A) Facial Chemical Company (Ann Arbor, MI, USA) ), the galactose injection solution is made by Nanguang Chemical Pharmaceutical Co., Ltd. 201236678. The system is prepared by dissolving 4 GG g of galactose (Sigma) in α liter containing the appropriate buffer solution system and isotonic salt in steaming water for injection. . 2. Male SD (Sprague-Dawley) rats weighing 320-350 g were purchased from the National Laboratory Animal Center (Taiwan). The animal experiment was carried out in accordance with the guidelines of the National Animal Research Institute. All rats were placed in the air. / Humidity and darkness for 12 hours each, the supply of water and feed is not limited, the weight of the rats is continuously monitored during the test, and all rats are given pentobarbital at a dose of 5 mg/kg body weight. Sodium pentobarbital was intraperitally anesthetized (intraperit〇neally anesthetized), and a polyethylene catheter was placed in the right internal jugular vein (intema丨jugular vdn) of the rat to make a galactose 'catheter system for puncture (cut-d〇wn The insertion of the catheter was placed under the skin of the posterior cervical incision of the rat. After the surgery was completed, the rats were fasted overnight (about 16 hours) during the recovery period, but the water was supplied as usual. 3. Test treatment The test animals were randomly divided into 5 groups, each group consisting of 3 treatments. The first treatment was injection of 25 mg/kg BNPP or BNPP (vehicle, VEm, ready-to-feed saline), and BNPP was dissolved to 6 (Rc saline (0.9% NaCl) 'cooled and injected intraperitoneally into the rat in a volume of 1 ml/kg; the second treatment was a dose of l〇〇mg/kg DSF or DSF (VEH2, ie Yuqi oil), DSF is dissolved in corn oil 'intraperitoneal injection into the body in a volume of 1 mi / kg; the third treatment is the injection of 150 mg / kg INH or INH base (VEH3, ready-to-feed saline), INH is dissolved in saline (0.9% NaCl) and intraperitoneally injected into the rat in a volume of 1 ml/kg; the first group (BNPP or VEH1) is compared with the third group (BNPP or VEH1) INH or VEH3) was treated 30 minutes earlier, and the second group (DSF or VEH2) was treated 1 minute earlier than the third group (INH or VEH3). The above 5 groups of tests included: (1) Control group (normal control group, NC, n= 12): Normal rats were injected with VEH1, VEH2 and VEH3 once daily (administered intraperitoneally) for 21 days; (2) INH group (INH, n=7): normal rats were injected with INH and VEH1 once a day. And VE H2 201236678 (administration of intraperitoneal injection) for 21 days; (3) BNPP-INH group (BNPP-INH, n=7): normal rats were injected once daily for bnpp, INH and VEH2 (administered intraperitoneally) for 21 days; 4) DSF-INH group (DSF-INH, n=7): normal rats were injected daily with ^DSF, jnh and VEH1 (administered intraperitoneally) for 21 days; and (5) BNPP-DSF-INH group (BNPP) -DSF-INH, n=7): Normal rats were injected with BNPP, DSF and INH (administered intraperitoneally) once a day for 21 days; galactose single point method was tested 16 hours after treatment on day 21. 4 After the blood sample was processed, the rats were sacrificed by ether anesthesia. The blood was drawn from the rat aorta and placed in a test tube containing EDTA. The plasma was centrifuged at 4 ° C for 15 minutes at i°C. The separated plasma was dispensed into a small tube (Eppendorf tube) and stored at -80 ° C. 5. Biochemical analysis of hepatocyte damage to measure aspartate transaminase (AST) and alanine in plasma Aminease (ALT) activity is quantified, and AST and alt activity are commonly used indicators of liver toxicity' measured by the Synchron LXi 725 system (Beckman Instruments, USA). 6. Light microscopy and electron microscopy Rats were sacrificed for histological analysis immediately after sacrifice; liver samples were fixed with phosphate-buffered formalin in 10% phosphate buffer, then dehydrated and embedded in paraffin (paraffin) The samples were sliced at a thickness of 5 μηη, and the samples were stained with hemat〇xylin and eosin, and subjected to a liver glycosylation test (PAS). After staining, histological observation was performed by light microscopy; In addition, liver sections were washed with buffer buffer (cac〇dylate buffer, 〇1M PH 7.4), fixed with 20% aqueous osmium tetroxide for 1 hour, and continuously dehydrated with alcohol and embedded in Spun*. In the resin (Spurr resin), ultra-thin sections were cut with a diamond knife, double stained with uranylacetate and leadcitrate, and transmitted by electron microscope (Transmission Electron Microscope, Hitachi 600, Hitachi). 12 201236678

Co.，日本)觀察。 7· (S-ho-PG/^a的萃取與量測 所有PGF2a的同分異構物(isomers)均以適當體積之酒精溶解或稀釋以製 備原液，並分裝於小管中儲存於-70°C，取〇.5ml血漿至玻璃管中，加入i〇ng 内標準品(internal standard,即8-iso-PGF2a-d4)，混勻後之血聚以c 18固相萃取 管柱(Solid-Phase Extraction cartridge，J.T. Baker, MA，美國)純化，樣本流洗 液以氮氣蒸發乾燥後，以50μ1乙睛:水(acetonitrile: water, 15:85 v/v)溶液回溶 並震盪30秒，取10μ1回溶後的萃取物注射至LC/MS/MS系統進行分析。 8.液相層析串聯式質譜儀(LC/MS/MS)分析 HPLC 系統包括2個島津LC-lOADvP系(Shimadzu LC-10ADvP pumps)、1 個島津系統控制器（Shimadzu system control)以及1個島津自動樣本機 (Shimadzuautosampler)(島津科學儀器，曰本），以C18管柱(顆粒大小5-μιη， 内控50 X 2.1mm)進行HPLC分離，並使用含有2mM醋酸錢(ammonium acetate) 及乙睛(acetonitrile, ACN)之梯度流洗液(t = 0 min, 15% ACN; t = 6 min，70% ACN; t = 7 min，90% ACN; t = 8 min，90% ACN; t = 8.5 min, 15% ACN)流 洗，LC/MS/MS的流速均維持在200 μΐ/min，整個HPLC進行時間為13.5分 鐘；該HPLC系統與一三層四極質譜儀(triple stage quadrupole mass spectrometer，API3000, Applied Biosystem，Foster City, CA，美國）介接，配備 有一TurboIonSpray離子源(TurboIonSpray ionization source)，並使用負電電 喷霧(negative electrospray)作為電離(i〇nizati〇n)之方法；該質譜儀藉由擴散 2〇0 ng/ml 8-iso-PGF2ct或8-iso-PGF2ct-d4標準液以多重反應監測(multiple reaction monitoring, MRM)模式進行最佳化，m/z 353/193以及m/z 357/197離 子偶(ion pair)則個別用來監測8-iso-PGF2a以及8-iso-PGF2a-d4 ;測量後，計 算 6 個 8-iso-PGF2a 濃度(C)的線性標準曲線(linear calibration curve)對 8-iso-PGF2a比8-iso-PGF2a-d4比值之區域(γ)，得到相關係數(r，COITeiati〇n coefficient)值為0.999 ;血漿中8-iSO-PGF2a的線性範圍在0.1-2.5ng/ml之間， 13 201236678 其迴歸方程式(regression equation)為Y=-〇.〇517C +0.823 ng/ml ;所測得之結 果均對照重氫化8-iso-PGF2a (deuterated 8-iso-PGF2a)内標準品計算，標準曲 線之批間精密度以及準確度係以標準濃度樣品分別測試6次後，經由反向計 算法(Back-Calculation)來評估，其相對誤差㈣ative errors)範圍在_5 〇6%至 3.13%之間。 9. 肝功能之定量測試 所有的大鼠均進行半乳糖單點法(GSP)及半乳糖清除能力(GEC)測試， 大鼠接受在30秒内的快速靜脈注射，注射〇 4g/ml bW半乳糖溶液〇 5 g/kg ; 自注射後5、10、15、30、45以及60分鐘各採血一次，血液樣本取自尾部靜 脈’以半乳糖脫氫酶比色法(c〇l〇rimetric galact〇se dehydr〇genase)量測半乳糖 含量，測試濃度範圍為50至1，000 每個濃度的日内差異(within_day variation)係由標準偏差（standard deviati〇n)以及變異係數(c〇efficient variation，CV)百分比計算，最大容許的變異係數為1〇% cv ;日間差異 (day-to-day variation)則由比較校正曲線(ca驗i〇n cu叫之斜率及截距來 檢驗，半乳糖清除能力(GEC)係由下列公式計算，該公式係由Tygstmp,s方程 式修改而來：Co., Japan) observed. 7. (Extraction and measurement of S-ho-PG/^a All isoforms of PGF2a are dissolved or diluted in an appropriate volume of alcohol to prepare a stock solution, which is stored in a small tube and stored at -70 °C, take 55ml of plasma into the glass tube, add i〇ng internal standard (8-iso-PGF2a-d4), mix the blood after the accumulation of c 18 solid phase extraction column (Solid -Phase Extraction cartridge, JT Baker, MA, USA) Purification, sample stream washing was evaporated to dryness with nitrogen, and then reconstituted with 50 μl of acetonitrile: water (15:85 v/v) solution and shaken for 30 seconds. 10 μl of the re-dissolved extract was injected into the LC/MS/MS system for analysis. 8. Liquid Chromatography Tandem Mass Spectrometer (LC/MS/MS) Analytical HPLC System consisting of 2 Shimadzu LC-lOADvP lines (Shimadzu LC -10ADvP pumps), 1 Shimadzu system control and 1 Shimadzu autosampler (Shimadzuautosampler) (Shimadzu Scientific Instruments, 曰本), with C18 column (particle size 5-μιη, internal control 50 X 2.1 Mm) HPLC separation and use gradient flow wash containing 2 mM ammonium acetate and acetonitrile (ACN) = 0 min, 15% ACN; t = 6 min, 70% ACN; t = 7 min, 90% ACN; t = 8 min, 90% ACN; t = 8.5 min, 15% ACN) flow wash, LC/MS /MS flow rate was maintained at 200 μΐ / min, the entire HPLC time was 13.5 minutes; the HPLC system and a three-layer quadrupole mass spectrometer (API3000, Applied Biosystem, Foster City, CA, USA) Connected with a TurboIonSpray ionization source and a negative electrospray as a method of ionization (i〇nizati〇n); the mass spectrometer diffuses 2〇0 ng/ml 8-iso -PGF2ct or 8-iso-PGF2ct-d4 standard is optimized by multiple reaction monitoring (MRM) mode, m/z 353/193 and m/z 357/197 ion pair (ion pair) It was used to monitor 8-iso-PGF2a and 8-iso-PGF2a-d4; after measurement, the linear calibration curve of 6 8-iso-PGF2a concentrations (C) was calculated for 8-iso-PGF2a ratio 8- The area of the iso-PGF2a-d4 ratio (γ), the correlation coefficient (r, COITeiati〇n coefficient) is 0.999; the line of 8-iSO-PGF2a in plasma The range is between 0.1-2.5 ng/ml, 13 201236678, and the regression equation is Y=-〇.〇517C +0.823 ng/ml; the measured results are all compared with the heavy hydrogenated 8-iso-PGF2a (deuterated) 8-iso-PGF2a) internal standard calculation, the inter-assay precision and accuracy of the standard curve were tested by standard concentration samples after 6 times, and then evaluated by Back-Calculation, the relative error (4) ative errors The range is between _5 〇 6% and 3.13%. 9. Quantitative testing of liver function All rats were tested for galactose single point (GSP) and galactose clearance (GEC). Rats received rapid intravenous injection within 30 seconds, injected with 4 g/ml bW half. Lactose solution 〇 5 g / kg; blood was collected once every 5, 10, 15, 30, 45, and 60 minutes after injection, blood samples were taken from the tail vein's galactose dehydrogenase colorimetric method (c〇l〇rimetric galact 〇se dehydr〇genase) Measure the galactose content in the range of 50 to 1,000. The within_day variation is the standard deviation (standard deviati〇n) and the coefficient of variation (c〇efficient variation, CV) Percentage calculation, the maximum allowable coefficient of variation is 1〇% cv; the day-to-day variation is determined by comparing the calibration curve (ca) the slope and intercept of i〇n cu, galactose clearance The ability (GEC) is calculated by the following formula, which is modified by the Tygstmp, s equation:

D GEC= ~ (mg/kg * min)D GEC= ~ (mg/kg * min)

Tc=o + 7 其中D為半乳糖之注射f ; Τ(>〇為半乳糖濃度所需要的時間，係由注 射(通常為2.22 mmol/1)後2〇至60分鐘的血液濃度_時間曲線之線性迴歸推 得；7為讎躲正_不均自分布之校正值；半乳料點法(Gsp)則 為30秒注射停止後60分鐘時血液中半乳糖濃度。 10. 統計分析 所有的數據皆以平均土標準偏差(SD)表示，試驗結果以單因子變異數分 析(ANOVA)職法來計算是料有财上賴著Μ，使肋麻Μ 201236678Tc=o + 7 where D is the injection of galactose f; Τ(> 〇 is the concentration required for galactose concentration, blood concentration from 2〇 to 60 minutes after injection (usually 2.22 mmol/1) _ time The linear regression of the curve is derived; 7 is the correction value of the 雠 正 _ 不 ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; 半 半 半 半 半 半 半 半 半 半 半 半 半 半 半The data are expressed in terms of mean soil standard deviation (SD), and the results of the test are calculated by the single factor analysis of variance (ANOVA), which is expected to have financial resources, so that rib paralysis 201236678

Package of the Social Science program (Version 13, SPSS Inc.)套裝軟體來計 算；隨後使用事後比較(post hoc test)最小差異顯著性(least signiflcant difference)方法做多重比較’以確認族群間的顯著差異；族群平均之顯著差 異為P<0.05。 二、結果 1·生化分析結果 試驗結束時，測量試驗動物的體重及相對肝重量，與對照組動物相較 之下並無顯著差異；生化分析結果如圖二所示，只有INH組血聚中的天門冬 氨酸轉胺酶(AST)與丙氨酸轉胺酶(ALT)活性明顯高於對照組(對照組血衆 中的AST活性為116±11 IU/L ; INH組血漿中的AST活性為129±1〇 IU/L，p < 0.05 ;對照組血漿中的ALT活性為44±6IU/L ; INH組血漿中的ALT活性為52 ± 3 IU/L,/? < 0·05)，顯示INH組產生生化上的肝損傷；對照組、BNpp_INH、 DSF-INH以及BNPP-DSF-INH組血清中轉胺酶濃度則為正常。 2.組織病理學 經過為期三週施行腹腔注射150mg/kg/dayINH之大鼠，其體内成功的 產生肝毒性；相對的，在對照組大鼠體内的肝結構則較正常，如圖三A所 示，對照組大鼠肝實質(liver parenchyma)内的肝細胞係排列於自肝小葉中央 靜脈輻射排列的網狀平板内’肝血竇（hepatic sinusoids)則在兩肝板 (anastomosing plates)之間被發現；INH組大鼠的組織切片則如圖三b所示， INH組大鼠中央靜脈周圍的肝細胞則呈現碎裂及空泡化，然而並無看到肝 細胞壞死(necrosis)的徵兆；以電子顯微鏡觀察之結果顯示，相較於對照組(如 圖三C所示），INH組大鼠肝細胞内的粗内質網(rER)明顯增加(如圖三D所 示）。根據文獻報導，INH是一個強效的細胞色素P450 2E1 (CYP2E1)的誘 導物’而CYP2E1會導致超氧基(superoxide)以及氫氧自由基(hydr〇xyl radicals)的產生，並且會引發内質網的增加，因此本試驗之結果與先前研究 相符。而其他試驗組：BNPP-INH組、DSF-INH組、BNPP-DSF-INH組大 15 201236678 鼠的肝損害程度與對照組相較，並無明顯區別(未顯示結果）。 3. 血液樣本中的量測 在負電電喷霧模式下，S-iso-PGF^最大量之分子離子為質荷比(m/z)353 之離子’ 8-iso-PGF2a-d4最大量之分子離子為質荷比(ηι/ζ)357之離子，這些負 電荷分子離子係經過大量碰撞誘導而產生游離’這兩個目標化合物的分子 結構以及產生的離子光譜如圖四所示；除了 8_iso_PGF2〇rd4的子離子(daughter ions)恆較S-iso-PGF^的子離子高四個單位之外，8_is〇_pGF2a以及 8-iso-PGF2a-d4兩者的碎裂模式(fragmentati〇npatterns)很相似，這顯示大多數 穩定的子離子係由A鏈產生而來’該A鏈上標示有4個氘原子(deuterium atoms); 8-iso-PGF2a最密集之子離子為質荷比(„^2)193之離子，8-iso-PGF2a-d4 最密集之子離子為質荷比(m/z)197之離子。圖五所示為在多重反應監測模式 (MRM)偵測下，含有loopgS-iso-PGFae^aSOpg/mlS-iso-PGFh-dt的標準溶 液’以及一血液樣本的典型LC/MS/MS色譜，在注入lng8-iso-PGF2a-d4作為 内標準品後，該標準溶液與該血液樣本均經過相同的固相萃取(SpE)純化， 並以前述LC/MS/MS規程分析。 4. 血漿中8-iso-PGF2a的濃度Package of the Social Science program (Version 13, SPSS Inc.) is packaged with software; then post hoc test is used to make multiple comparisons by the least signiflcant difference method to confirm significant differences between groups; The significant difference in ethnic group average was P < 0.05. 2. Results 1. Biochemical analysis results At the end of the experiment, the body weight and relative liver weight of the test animals were measured, and there was no significant difference compared with the control animals. The results of biochemical analysis are shown in Figure 2, only in the blood pool of the INH group. The activity of aspartate transaminase (AST) and alanine transaminase (ALT) was significantly higher than that of the control group (the AST activity in the control group was 116±11 IU/L; the AST in the plasma of the INH group) The activity was 129±1〇IU/L, p <0.05; the ALT activity in the plasma of the control group was 44±6 IU/L; the ALT activity in the plasma of the INH group was 52 ± 3 IU/L, /? 05), showing biochemical liver damage in the INH group; the serum transaminase concentration in the control group, BNpp_INH, DSF-INH and BNPP-DSF-INH groups was normal. 2. Histopathology After three weeks of intraperitoneal injection of 150 mg/kg/day INH rats, liver toxicity was successfully produced in vivo; in contrast, the liver structure in the control group was normal, as shown in Figure 3. As shown in A, the hepatic cell lines in the liver parenchyma of the control group are arranged in a reticular plate arranged from the central venous lobes of the hepatic lobe. 'Hepatic sinusoids' are in anastomosing plates. The tissue sections of the rats in the INH group were as shown in Fig. 3b. The hepatocytes around the central vein of the INH group showed fragmentation and vacuolization, but no necrosis was observed. The signs of electron microscopy showed that the crude endoplasmic reticulum (rER) in the hepatocytes of the INH group was significantly increased compared with the control group (as shown in Figure 3C) (as shown in Figure 3D). . According to the literature, INH is a potent inducer of cytochrome P450 2E1 (CYP2E1), and CYP2E1 leads to the production of superoxide and hydr〇xyl radicals, and causes endoplasmic The net has increased, so the results of this trial are consistent with previous studies. In other test groups, the degree of liver damage in the BNPP-INH group, the DSF-INH group, and the BNPP-DSF-INH group was not significantly different from that of the control group (no results were shown). 3. Measurement in blood samples In the negative electrospray mode, the maximum molecular ion of S-iso-PGF^ is the mass of the mass-to-charge ratio (m/z) 353 ' 8-iso-PGF2a-d4 The molecular ions are ions of mass-to-charge ratio (ηι/ζ) 357. These negatively charged molecular ions are induced by a large number of collisions to generate free 'the molecular structure of the two target compounds and the resulting ion spectrum is shown in Figure 4; except 8_iso_PGF2 The daughter ions of 〇rd4 are always four units higher than the daughter ions of S-iso-PGF^, and the fragmentation patterns of both 8_is〇_pGF2a and 8-iso-PGF2a-d4 (fragmentati〇npatterns) Very similar, this shows that most stable daughter ions are produced by the A chain. 'The A chain is labeled with four deuterium atoms; the most dense daughter ion of 8-iso-PGF2a is the mass-to-charge ratio („^ 2) 193 ions, 8-iso-PGF2a-d4 The most dense daughter ions are mass-to-charge ratio (m/z) 197 ions. Figure 5 shows the detection of multiple reaction monitoring mode (MRM) with loopgS- A standard solution of iso-PGFae^aSOpg/mlS-iso-PGFh-dt and a typical LC/MS/MS chromatogram of a blood sample injected into lng8-iso-PGF2a-d4 After the standard, the standard solution with the same blood samples were obtained on solid phase extraction (SpE) purified, and in the LC / MS / MS analysis procedure. 4. The plasma concentrations of 8-iso-PGF2a of

血衆中的S-iso-PGF2^—種氧化壓力(〇xidative stress)的指標，如圖六所 示，相較於對照組，INH組大鼠血漿中8-iso-PGF2c^濃度明顯增加(INH組大 鼠血漿中8-iso-PGF2c^濃度為151±26pg/ml ;對照組大鼠血漿中8-iso-PGF2a 的濃度為110±15?咖1，；7<0.001);與鹏組相較，8]^?_臓組、1)兕_1仰 組、BNPP-DSF-INH組三組則明顯降低由INH引起肝臟的8-iso-PGF2a產生 (BNPP-INH組大鼠血漿中8-iS0_PGF2(^濃度為I28±29pg/ml ; DSF-INH組大 鼠血漿中8-iso-PGF2A濃度為126±20 pg/ml ; BNPP-DSF-INH組大鼠血漿中 8-iso-PGF2<^濃度為 123±17 pg/ml ; INH組大鼠血漿中 8-iso-PGF2o^濃度為 151±26pg/ml，/?<0.005);值得注意的是，對照組、BNPP-INH組、DSF-INH 組、BNPP-DSF-INH組四組之間，大鼠血漿中8_iS0_pGF2(^濃度無顯著差 201236678 異，與BNPP-INH組及DSF-INH組相較，INH合併施用BNPP與DSF並不會進 一步減少血漿中8-iso-PGF2a的濃度。 5.剩餘肝功能之量測 如圖七所示，對照組與INH組大鼠之半乳糖單點法(GSP)值具有高度的 顯著差異(對照組大鼠之GSP值為384±69 pg/ml ; INH組大鼠之GSP值為 565±87 pg/ml，p < 0.001)，此外，BNPP-INH 組、DSF-INH 組、BNPP-DSF-INH 組大鼠之 GSP 值各為 401±70 pg/m卜 449±45 pg/mb 388±53 pg/mL· 與 INH 組相較，ΒΝΡΡ-ΙΝΗ組、DSF-INH組、BNPP-DSF-INH組大鼠之GSP值各 與ΙΝΗ組大鼠具有高度的顯著差異(其ρ值各為夕< 0.001,ρ< 0.005, and/? < 0.001);單獨施用INH的大鼠之GSP值明顯增加；然而，在INH合併施用 BNPP或DSF或BNPP與DSF之大鼠則可抵抗這種改變；另一方面，與 DSF-INH組相較’ INH合併施用BNPP與DSF顯示可以降低INH引起的肝 毒性’雖然兩者之間的差異未達到統計上的差異(p=〇. 1)，而對照組、 BNPP-INH組、DSF-ΙΝΉ組、BNPP-DSF-INH組四組之間大鼠的GSP值無 顯著差異存在。 相似的結果在使用半乳糖清除能力(GEC)方法上也可觀察的到，如圖八 所示，與對照組相較，INH組大鼠之GEC值明顯減少(INH組大鼠之GEC 值為 3_4±0.6 mg/min.kg ;對照組大鼠之 GEC 值為 4.9±0.8 mg/min.kg，< 0.001)，此外，BNPP-INH 組、DSF-INH 組、BNPP-DSF-INH 組大鼠之 GEC 值各為 4·5±0.6 mg/min.kg、4·3±0.4 mg/min.kg、4.7±0·5 mg/min_kg ;與 INH 組相較’ BNPP-INH組、DSF-INH組、BNPP-DSF-INH組大鼠之GEC值各 與INH組大鼠具有高度的顯著差異(其尸值各為^ < 〇 〇〇5，卩< 〇 〇5, and^ < 0.005);單獨施用INH的大鼠之GEC值明顯減少；然而，在_合併施用 BNPP或DSF或BNPP與DSF之大鼠則可恢復這種改變；與組相 較，INH合併施用BNPP與DSF者有增加GEc值的傾向(dsf-jnh組與 BNPP-DSF-INH 組大鼠之GEC 值各為 4.3±〇.4 mg/min.kg、4.7士0.5 mg/min.kg， 17 201236678 P - 0.29)，此外’對照組、BNPP-INH 組、DSF-ΙΝΗ 組、BNPP-DSF-INH 組 四組之間大鼠的GEC值無顯著差異存在。 為了確定AST、ALT、血漿中8-iso-PGF2a的濃度，以及定量肝功能測 "式(如· GSP以及GEC)疋否相關，以數種相關分析計算後，發現Gsp值與 血漿中S-iso-PGFh的濃度具有高度相關(如九所示），相關係數為〇836 ; GSP值與GEC值具有高度相關(如圖十所示）& < 〇〇〇1)，相關係數為_ 0.822 ; GEC值也與血漿中8_iso_PGF2a的滚度具有高度相關(相關係數為_ 〇·743，ρ < 〇·〇〇卜如表一所示）；而GSP值、GEC值以及血榮中8 is〇 pGF2a 的濃度則與AST及ALT均無明顯相關(如表一所示）。 表一、GSP 、GEC以及8-iso-PGF2a與生化測試之相關性The S-iso-PGF2^- 氧化xidative stress index in the blood group, as shown in Figure 6, compared with the control group, the concentration of 8-iso-PGF2c^ in the plasma of the INH group was significantly increased ( The concentration of 8-iso-PGF2c in the plasma of rats in the INH group was 151±26pg/ml; the concentration of 8-iso-PGF2a in the plasma of the control group was 110±15? coffee1,;7<0.001); In comparison, 8]^?_臓 group, 1) 兕_1 仰 group, and BNPP-DSF-INH group significantly reduced 8-iso-PGF2a production in the liver caused by INH (BNPP-INH group in rat plasma) 8-iS0_PGF2 (concentration was I28±29pg/ml; 8-iso-PGF2A concentration in plasma of DSF-INH group was 126±20 pg/ml; 8-iso-PGF2<1> in plasma of BNPP-DSF-INH group The concentration of 8-iso-PGF2o^ in the plasma of INH group was 151±26pg/ml,/?<0.005); it is worth noting that the control group and BNPP-INH group In the DSF-INH group and the BNPP-DSF-INH group, the plasma concentration of 8_iS0_pGF2 in rats was not significantly different from 201236678. Compared with BNPP-INH group and DSF-INH group, INH combined with BNPP and DSF. It does not further reduce the concentration of 8-iso-PGF2a in plasma. 5. The measurement of residual liver function is shown in the figure. As shown, there was a significant difference in the galactose single-point (GSP) value between the control group and the INH group (GSP value of 384±69 pg/ml in the control group; GSP value in the INH group was 565). ±87 pg/ml, p < 0.001). In addition, the GSP values of the BNPP-INH group, the DSF-INH group, and the BNPP-DSF-INH group were 401±70 pg/m 449±45 pg/mb. 388±53 pg/mL· Compared with the INH group, the GSP values of the ΒΝΡΡ-ΙΝΗ group, the DSF-INH group, and the BNPP-DSF-INH group were significantly different from those of the sputum group (the ρ values were For the evening < 0.001, ρ < 0.005, and /? 0.001); the GSP value of the rats administered with INH alone was significantly increased; however, rats that were administered with BNPP or DSF or BNPP and DSF in INH were resistant to this. On the other hand, compared with the DSF-INH group, 'INH combined with BNPP and DSF showed reduced INH-induced hepatotoxicity' although the difference between the two did not reach a statistical difference (p=〇. 1) There were no significant differences in GSP values between the control group, BNPP-INH group, DSF-ΙΝΉ group, and BNPP-DSF-INH group. Similar results were also observed using the galactose clearance (GEC) method. As shown in Figure 8, the GEC values of the rats in the INH group were significantly reduced compared with the control group (the GEC values of the rats in the INH group). 3_4±0.6 mg/min.kg; the GEC value of the control rats was 4.9±0.8 mg/min.kg, < 0.001), in addition, the BNPP-INH group, the DSF-INH group, and the BNPP-DSF-INH group were large. The GEC values of the rats were 4. 5 ± 0.6 mg / min. kg, 4 · 3 ± 0.4 mg / min. kg, 4.7 ± 0 · 5 mg / min_kg; compared with the INH group 'BNPP-INH group, DSF- The GEC values of the rats in the INH group and the BNPP-DSF-INH group were significantly different from those in the INH group (the corpse values were each ^ < 〇〇〇 5, 卩 < 〇〇 5, and ^ <0.005); the GEC value of rats administered with INH alone was significantly reduced; however, this change was restored in rats with conjugated BNPP or DSF or BNPP and DSF; compared with the group, the combination of INH and BNPP and DSF There is a tendency to increase the value of GEc (the GEC values of the rats in the dsf-jnh group and the BNPP-DSF-INH group are 4.3±〇.4 mg/min.kg, 4.7±0.5 mg/min.kg, 17 201236678 P - 0.29 ), in addition, 'control group, BNPP-INH group, DSF-ΙΝΗ group, BNPP-DSF-INH group There was no significant difference in the GEC values of the rats between the four groups. In order to determine the concentration of 8-iso-PGF2a in AST, ALT, plasma, and quantitative liver function test (such as · GSP and GEC), the correlation between Gsp and plasma S was found after several correlation analyses. The concentration of -iso-PGFh is highly correlated (as shown in IX), and the correlation coefficient is 〇836; the GSP value is highly correlated with the GEC value (as shown in Figure 10) &<< 〇〇〇 1), the correlation coefficient is _ 0.822 ; GEC value is also highly correlated with the rolling degree of 8_iso_PGF2a in plasma (correlation coefficient is _ 〇 · 743, ρ < 〇·〇〇卜 as shown in Table 1); and GSP value, GEC value and blood Rongzhong The concentration of 8 is〇pGF2a was not significantly correlated with AST and ALT (as shown in Table 1). Table 1. Correlation between GSP, GEC and 8-iso-PGF2a and biochemical tests

實施例二、細胞色素P450 2E1 (CYP2E1)抑制劑之篩選_ cDNA合成微粒體細胞色素p45〇 2E1 一、材料與方法 1.試驗材料 本實施例係使用細胞色素P450 2E1 (CYP2E1)抑制劑之篩選套組 (CYP2E1 High Throughput Inhibitor Screening Kit，BD Bioscience，美國)針對 22種中藥藥引及i〇種賦形劑進行細胞色素p45〇2E1 (CYp2E1)抑制劑之篩 選；該CYP2E1抑制劑之篩選套組的作用原理為：在含有細胞色素ρ45〇2Ει (CYP2E1)以及其螢光性受質 mfc (7_Meth〇xy_4_triflu〇r〇methy丨 c〇umarin)的 201236678 環境下加入測試樣品作用後，再偵測CYP2E1代謝物標準品HFC (7-Hydroxy-4-trifluoromethyl coumarin)的生成量，並以對照組(control)的 HFC生成量為基準，計算測試樣品之CYP2E1抑制率。 各測試樣品均溶於乙腈(acentoitrile)，測試不同濃度之中藥藥引(66μΜ， 33μΜ，16·5μΜ)及賦形劑(0.167%，0.08%，0.042%，w/v)對 CYP2E1 之抑制 率’所測試之中藥藥引及結果如表三所列，所測試之賦形劑及結果如表四 所列。 另外，本實施例使用之細胞色素P450 2E1 (CYP2E1)抑制劑之篩選套組 (CYP2E1 High Throughput Inhibitor Screening Kit，BD Bioscience,美國)戶斤需 之藥劑如下： (1) CYP2E1 + P450 Reductase + Cytochrome b5 ： 100 mM potassium phosphate (pH 7.4)含有 1.3 nmol P450 以及 p-Nitropheno卜X解酶 〇 (2) Control Protein : 15 mg/mL Control Protein 溶於 100 mM PotassiumExample 2 Screening of cytochrome P450 2E1 (CYP2E1) inhibitors _ cDNA synthesis microsomal cytochrome p45 〇 2E1 I. Materials and methods 1. Test materials This example was screened using cytochrome P450 2E1 (CYP2E1) inhibitors. CYP2E1 High Throughput Inhibitor Screening Kit (BD Bioscience, USA) Screening of cytochrome p45〇2E1 (CYp2E1) inhibitors against 22 Chinese herbal medicines and excipients; screening kit for CYP2E1 inhibitors The principle of action is: after adding the test sample to the cytochrome ρ45〇2Ει (CYP2E1) and its fluorescent acceptor mfc (7_Meth〇xy_4_triflu〇r〇methy丨c〇umarin), the CYP2E1 is detected. The amount of production of the metabolite standard HFC (7-Hydroxy-4-trifluoromethyl coumarin) was calculated based on the amount of HFC produced by the control (control), and the CYP2E1 inhibition rate of the test sample was calculated. Each test sample was dissolved in acetonitrile (acentoitrile), and the inhibition rate of CYP2E1 was measured at different concentrations of the drug (66 μΜ, 33 μΜ, 16·5 μΜ) and excipients (0.167%, 0.08%, 0.042%, w/v). 'The results of the tested Chinese medicines are listed in Table 3. The tested excipients and results are listed in Table 4. In addition, the CYP2E1 High Throughput Inhibitor Screening Kit (BD Bioscience, USA) used in the present embodiment is as follows: (1) CYP2E1 + P450 Reductase + Cytochrome b5 : 100 mM potassium phosphate (pH 7.4) contains 1.3 nmol P450 and p-Nitropheno X X-olytic enzyme (2) Control Protein : 15 mg/mL Control Protein Dissolved in 100 mM Potassium

Phosphate (pH 7.4)中。 (3) Buffer Solution : 0.5 M Potassium Phosphate (pH 7.4)。 (4) Stop Solution : 0.5 M Tris Base。 (5) Cofactors ··含有 1.3 mM NADP+、66 mM MgCl2 以及 66 mM Glucose 6-Phosphate ° (6) Glucose 6-Phosphate Dehydrogenase : 40 units/ml 溶於 5 mM SodiumPhosphate (pH 7.4). (3) Buffer Solution : 0.5 M Potassium Phosphate (pH 7.4). (4) Stop Solution : 0.5 M Tris Base. (5) Cofactors ················

Citrate Buffer (pH 7.5)。 (7) MFC (7-Methoxy-4-trifluoromethyl coumarin)：螢光性受質 (fluorescence substrate) ’ 50 mM MFC 溶於乙腈(acetonitrile)。 (8) DDTC (Diethyldithiocarbamic add) : CYP2E1 選擇性抑制劑(陽性對 照組），20 mMDDTC 溶於乙腈(acentoitrile)。 (9) HFC (7-Hydroxy-4-trifluoromethyl coumarin) : CYP2E1 代謝物標準品 (metabolite standard)，0.25 mM HFC 溶於 0.1M Tris (pH 9.0)。 201236678 (10) NADPH-Cofactor Mix :於 14.56 ml 無菌水中加入 187.5 μΐ Cofactors、150 μΐ G6PDH (Glucose 6-Phosphate Dehydrogenase Solution)以及 100 μΐ Control Protein 〇 (11) Cofactor/ acetonitrile mix:於 9.93 ml NADPH-Cofactor Mix 中加入 66 μΐ Acetonitrile ° (12) Enzyme/Substrate Mix :於 4ml Buffer Soultion 中加入 5·94 ml 無菌 水、50μ1 HTS-706(CYP2E1，2 μΜ P450 content)以及 28 μΐ 50 mM MFC (7-Methoxy-4-trifluoromethyl coumarin，螢光性受質）。 2.細胞色素P450 2E1 (CYP2E1)抑制劑之筛選 使用細胞色素P450 2E1 (CYP2E1)抑制劑之篩選套組(cyp2ei High Throughput Inhibitor Screening Kit，BD Bioscience，美國）進行中藥藥引及賦 形劑之篩選，實驗步驟如下所述： (1) 製備對照組： a. 於96孔盤上第1孔井(weu)内加入以及 Ιμΐ 20mM DDTC並混合均勻； b. 於該96孔盤上第2至12孔井内各加人卿μ1邮咖/脱加麻 mix ’第1至8孔井為陽性對照組(p〇sitive c〇ntr〇1);第9與第⑴孔 井為對照組(eontml);第U與第12孔井為空白對照鄉驗）； c. 於該第1至8孔井内做連續稀釋動作：自第i孔井内取㈣液體加 入第2孔井内混勻，再自第2孔井内取5〇 μ1液體加入第3孔井内混 句’以此類推’至第8孔井時絲多餘的5G μ1液體，崎到連_ 釋濃度 66.6、22.2、7.4、2.47、0.82、0.27、0.091、0 〇3 Μ。 (2) 製備試驗組： ‘ μ ° a·於96孔盤上第1行的第 札开内各加入149 N卿H-C〇factorMix，以及_福中藥藥引測試樣品2 (w/v)賦形劑測試樣品，並混合均勻； 201236678 b.再自該第1行的第1及第2孔井内各取50 μΐ液體加入第3孔井内混 句（即每一測試樣品均為三重複）； (3)反應起始與終止： a•將上述對照組與試驗組置於37t靜置10分鐘； 匕除了 δ亥空白對照組之外，其他孔井内均加入lOOplEnzyme/Substrate Mix混勻； c. 將所有對照組與試驗組置於3r»c靜置4〇分鐘； d. 所有的孔井内均加入75 μ1 St〇p Soluti〇n混勻； e. 緊接著於該空白對照組内加入100 μ丨EnZyme/Substrat；e Mix混勻； f·將所有對照組與試驗組以螢冷光儀(Flu〇r〇skan Ascent fl，Thermo Electron Corporation，芬蘭)讀取結果，所使用之激發光(exdtati〇n) 波長為405 nm，發散光(emissi〇n)波長為538 nm。 (4)結果分析：測得之螢光訊號數值換算成為CYp2E1代謝物標準品 生成量(pmol)後’以對照組(contr〇i)為基準，即對照組之CYP2E1抑制 率為〇%,以下列公式計算各陽性對照組及試驗組之CYP2E1抑制率： CYP2E1 抑制率(％) = 樣品之HFC生成量 對照組(control)之HFC生成量 二、結果 I陽性對照組 陽性對照組(DDTC)所測出之CYP2E1抑制率如表二所示，由表二可知 當DDTC的濃度為66.6 μΜ (即為0.167 %，w/v)時，CYp 2m抑制率可達 97.55%，係以66_6 μΜ作為中藥藥引最高測試濃度，以〇 167 % (w/力作為 賦形劑最高測試濃度。 表二陽性對照組之CYP 2E1抑制率 21 201236678 g_DTC 濃度（μΜ) HFC 生成量（pm〇l) CYP2E1 抑^ 〇(對照組） 222.00 ----- 0 0.03 256.00 - 0.091 202.00 8.71 0.27 151.71 31.52 0.82 126.14 43.06 2.47 55.18 75.09 7.4 21.08 90.49 22.2 15.10 93.19 66.6 5.42 97.55 2.試驗組CYP 2E1抑制率 中藥藥引所測出之CYP 2E1抑制率如表三所示 ，由結果可知各中藥藥 引於不同濃度(66μΜ，33μΜ，16.5μΜ)的條件下，對細胞色素Ρ450 2Ε1具有 不同程度的抑制效果，其中以66 μΜ正二羥癒瘡酸(Nordihydroguaiare^ add) 抑制效果最佳(97.99±0.66 %)。 表三中藥藥引之CYP2E1抑制率 中藥藥引 CYP2E1 (〇/〇) 測試濃度 66 μΜ 33 μΜ 16.5 μΜ 最小 有效 劑量* (mg)Citrate Buffer (pH 7.5). (7) MFC (7-Methoxy-4-trifluoromethyl coumarin): Fluorescent substrate '50 mM MFC was dissolved in acetonitrile. (8) DDTC (Diethyldithiocarbamic add): CYP2E1 selective inhibitor (positive control group), 20 mM DDTC dissolved in acetonitrile (acentoitrile). (9) HFC (7-Hydroxy-4-trifluoromethyl coumarin): CYP2E1 metabolite standard, 0.25 mM HFC dissolved in 0.1 M Tris (pH 9.0). 201236678 (10) NADPH-Cofactor Mix: Add 187.5 μΐ Cofactors, 150 μΐ G6PDH (Glucose 6-Phosphate Dehydrogenase Solution) and 100 μΐ Control Protein® (11) Cofactor/ acetonitrile mix in 14.56 ml of sterile water: 9.93 ml NADPH-Cofactor Add 66 μΐ Acetonitrile ° (12) Enzyme/Substrate Mix to Mix: Add 5·94 ml sterile water, 50μ1 HTS-706 (CYP2E1, 2 μΜ P450 content) and 28 μΐ 50 mM MFC (7-Methoxy) to 4ml Buffer Soultion -4-trifluoromethyl coumarin, fluorescent receptor). 2. Screening of cytochrome P450 2E1 (CYP2E1) inhibitors The cytochrome P450 2E1 (CYP2E1) inhibitor screening kit (cyp2ei High Throughput Inhibitor Screening Kit, BD Bioscience, USA) was used for traditional Chinese medicine introduction and excipients. Screening, the experimental steps are as follows: (1) Prepare the control group: a. Add in the first well (weu) on the 96-well plate and Ιμΐ 20mM DDTC and mix well; b. On the 96-well plate, the second to In the 12-well well, each person's μ1 mailing coffee/de-mixed Ma mixed 'the first to eighth wells was the positive control group (p〇sitive c〇ntr〇1); the 9th and the (1) wells were the control group (eontml) The U and 12th wells are blank control townships); c. Continuous dilution in the 1st to 8th wells: take the liquid from the i-hole well (4) and add the liquid to the second well to mix well, then from the second 5 〇μ1 liquid in the well was added to the well of the third well, and so on to the 8th well, the excess 5G μ1 liquid, the concentration of 66.6, 22.2, 7.4, 2.47, 0.82, 0.27, 0.091, 0 〇 3 Μ. (2) Preparation test group: ' μ ° a · Add 149 N qing HC〇factorMix in the first row of the 96-well plate, and _ Fu Chinese Medicine Pharmacy Test Sample 2 (w/v) Test the sample and mix it evenly; 201236678 b. Add 50 μΐ of liquid from the first and second wells of the first row to the well of the third well (that is, each test sample is three replicates); 3) Start and stop of the reaction: a• The above control group and the test group were placed at 37t for 10 minutes; in addition to the δHai blank control group, other wells were added with lOOplEnzyme/Substrate Mix; c. All control groups and test groups were placed in 3r»c for 4 minutes; d. All wells were mixed with 75 μl St〇p Soluti〇n; e. Immediately after the addition of 100 μ丨 to the blank control group EnZyme/Substrat; e Mix; f· Read all the control and test groups with a fluorescent luminometer (Flu〇r〇skan Ascent fl, Thermo Electron Corporation, Finland), using the excitation light (exdtati〇n The wavelength is 405 nm and the divergence (emissi〇n) wavelength is 538 nm. (4) Analysis of results: After the measured value of the fluorescent signal was converted into the amount of CYp2E1 metabolite standard (pmol), the control group (contr〇i) was used as the reference, that is, the CYP2E1 inhibition rate of the control group was 〇%, below The column formula calculates the CYP2E1 inhibition rate of each positive control group and the test group: CYP2E1 inhibition rate (%) = HFC production amount of the sample HFC production amount of the control group II, result I positive control group positive control group (DDTC) The measured inhibition rate of CYP2E1 is shown in Table 2. It can be seen from Table 2 that when the concentration of DDTC is 66.6 μΜ (that is, 0.167 %, w/v), the inhibition rate of CYp 2m can reach 97.55%, which is 66_6 μΜ as a traditional Chinese medicine. The highest concentration of the drug was measured at 〇167% (w/force as the highest tested concentration of the excipient. Table 2 CYP 2E1 inhibition rate of the positive control group 21 201236678 g_DTC concentration (μΜ) HFC production amount (pm〇l) CYP2E1 suppression ^ 〇 (control group) 222.00 ----- 0 0.03 256.00 - 0.091 202.00 8.71 0.27 151.71 31.52 0.82 126.14 43.06 2.47 55.18 75.09 7.4 21.08 90.49 22.2 15.10 93.19 66.6 5.42 97.55 2. Test group CYP 2E1 inhibition rate measured by Chinese medicine CYP 2E1 inhibition rate as As shown in the third, it can be seen from the results that the various traditional Chinese medicines have different inhibitory effects on cytochrome Ρ450 2Ε1 under different conditions (66μΜ, 33μΜ, 16.5μΜ), among which 66 μΜPolydihydroxyguaiare^ Add) The best inhibitory effect (97.99±0.66 %). Table 3: CYP2E1 inhibition rate of traditional Chinese medicine cited CYP2E1 (〇/〇) Test concentration 66 μΜ 33 μΜ 16.5 μΜ Minimum effective dose* (mg)

陽性對照組(DDTC) --I - - - 97.55±1.862 正二經癒瘡酸 (Nordihydroguaiaretic acid) (-)-Epigallocetechin-3 -gallate 97.99±0.66 97.56土 0.18 92.36±2.20 96.47±0.64 76.52±3.86 17Positive control group (DDTC) --I - - - 97.55±1.862 Nordihydroguaiaretic acid (-)-Epigallocetechin-3 -gallate 97.99±0.66 97.56 soil 0.18 92.36±2.20 96.47±0.64 76.52±3.86 17

S 22 201236678 中藥藥引 CYP2E1抑制率（％) 最小 有效 劑量* (mg) 測試濃度 66 μΜ 33 μΜ 16.5 μΜ 茵陳色原酮 (Capillarisin) 76·12±1·89 60.54±5.91 49.05土 5.18 17 山奈盼 (Kaempferol) 70·63±2·53 70.04±3.75 71.87±1.14 16 根皮素 (Phloretin) 66.84土 4.79 54.69±2.84 42.04±3.63 15 雙硫侖 (disulfiram) 66.54±2.55 60.55±5.70 57.89±3.91 17 撥皮素 (Hesperetin) 54.75士 1.37 43.29土 0.82 32.10±5.80 33 6-薑辣醇 (6-Gingerol) 51.89±3.33 39.83±2.32 30.13±2.67 16 沒食子酸 (gallic acid) 48.24土 4.20 42_74±7.36 35.59±10.03 9 異甘草素 (Isoliquritigenin) 47.83±5.36 46.27±3.28 39.08士 2.75 18 柚皮素 (Narigenin) 41.84±3.51 36.82土 3.97 25.11 土7.60 9 二氫化槲皮素 ((+)-Taxifolin) 34.54±3.47 23.80土 5.84 22.58±11.69 17 漢黃芩素 (Wongonin) 23.48±2.59 21.87 士 1.90 15.64±7.82 16 原兒茶酸 (Protocatechuic acid) 22.75±4.07 19.95±8.95 25·66 土 12.74 8 兒茶素 ((+)-Catechin) 16.45 土 9_67 33.83±8.76 41.53±7.62 16 23 201236678 中藥藥引 CYP2E1 抑制率 最小 有效 劑量* (mg) 測試濃度 66 μΜ 33 μΜ 16.5 μΜ β-奈黄酮 (β-naphthoflavone) 15.40±12.94 16.83±〇.96 6.52±6.64 15 恩貝素 (Embelin) 13.54±11.64 ~----- 12.30±ΐ〇.24 5.95±7.48 16 反式肉桂酸 (trans-Cinnamic acid) 7.10±6.95 4.66±6.50 5·71±10·53 8 表兒茶酚 ((-)-Epicatechin) 2.57±11.60 卜 --- 15.02±5.50 18.27±9.34 16 根皮苷 (Phloridzin) 1.42±9.28 3·76±3.58 1.25±7.90 24 葛根素 (Puerarin) -12.86±2.75 ---- -4.64±3.47 0.43±2.31 23 傘形花内酯 (Umbelliferone)_ 1 A 厶：AU 且· _!=L It* * -1081·56±168.00 -571.97±117.56 -280.41±19.48 9 :最小有效劑量：最低篩選濃度（mg/L) X人肝腸體積(3L) 朗劑所測出之CYP2E1抑制率如表四所示，由結果可知各賦形劑於 不同濃度(0.167%，0.08%，0.042%，w/v)的條件下，對細胞色素以5〇 2E1具有 不同程度的抑制效果，其中u 〇.167% Brij 58的抑制效' (97.75±〇.66%) 〇 — _____藥引 CYP 2E1抑制率 .測成濃度（w/v) 0.167% _0.08% - 對照組 0 __ 0.042% 最小有致 劑量* (mg) 24 201236678 中藥藥引 '~~------ CYP 2E1抑制率 最小有效 劑量* (mg) 測試濃度（W/V) 陽性對照組 (DDTC) 0-167% 97.55±1·862 0.08% 0.042% Brij 58 97.75±0.66 96.58±0.40 96.02±0.17 1260 Brij 76 97.56±l.〇2 96·87±1·〇〇 94.76±0.47 1260 Brij 35 93.33±0.82 (測試濃度 0.025%) 89.45±0.68 (測試濃度 0.013%) 76.21±7.37 (測試濃度 0.006%) 180 Tween 20 87.20±1.29 82.80±1·71 71.77±4.48 1260 Tween 80 73.92±4.71 65.45±2.50 64.02土 12.54 1260 Tween 40 58.97±3.29 47.05±6.48 44.79±2.49 1260 PEG 2000 44.33±2.75 40.13±3.06 35.81 土 3,26 1260 PEG 400 42.33±5.25 39.10±0.73 31.98±5.97 1260 Pluomic F68 41.72±5.34 42.98±3.24 37.11±l〇.35 1260 PEG 4000 37.21±1.91 41.22±0.97 37.18±10.52 1260 *最小有效劑量：最低篩選濃度（mg/L) X人肝腸體積(3l) 實施例三、細胞色素P450 2E1 (CYP2E1)抑制劑的篩選-人肝微粒體細胞色 素 P450 2E1 一、材料與方法 1.試驗材料 本實施例是使用人類肝臟所製備微粒體，針對細胞色素P450 2E1 (CYP2E1)與39種中藥藥引及1〇種賦形劑進行細胞色素P450 2E1 (CYP2E1) 抑制劑的篩選，以篩選出對於人類肝臟之細胞色素P45〇 2E1 (CYP2E1)有效 抑制劑；該CYP2E1抑制劑的篩選作用原理為：係利用人類肝臟所製備微 25 201236678 粒體中細胞色素P450 2E1 (CYP2E1)與其受質Chlorzoxazone反應，加入測-試樣品作用後，再偵測CYP2E1代謝物標準品6-OH-CZX (6-Hydroxy-Chlorzoxazone)的生成量，並以對照組(control)的 6-OH-CZX 生 成量為基準，計算測試樣品的CYP2E1抑制率。 各測試樣品均溶於10%甲醇(methanol)或是二次水中，測試不同濃度的 中藥藥引(66μΜ，33μΜ，16_5μΜ)及賦形劑(0.167%，0.08%，0.042%, w/v)對 CYP2E1的抑制率’所測試的中藥藥引及結果如表六所列，所測試的賦形劑 及結果如表七所列。 本實施例所利用人類肝臟細胞色素P450 2E1 (CYP2E1)抑制劑筛選所 需的藥劑如下： (1) CYP2E1 : 100 mM potassium phosphate (pH 7.4)含有 1〇 mg/ml P450 protein concentration ° (2) Control Protein : 10 mg/mL P450 Protein 溶於 100 mM PotassiumS 22 201236678 Chinese medicine CYP2E1 inhibition rate (%) Minimum effective dose* (mg) Test concentration 66 μΜ 33 μΜ 16.5 μΜ Capillarisin 76·12±1·89 60.54±5.91 49.05 Soil 5.18 17 Shannai Kaempferol 70·63±2·53 70.04±3.75 71.87±1.14 16Phloretin 66.84 soil 4.79 54.69±2.84 42.04±3.63 15 disulfiram 66.54±2.55 60.55±5.70 57.89±3.91 17 Hesperetin 54.75士 1.37 43.29土 0.82 32.10±5.80 33 6-gingerol (6-Gingerol) 51.89±3.33 39.83±2.32 30.13±2.67 16 gallic acid 48.24 soil 4.20 42_74±7.36 35.59±10.03 9 Isoliquritigenin 47.83±5.36 46.27±3.28 39.08士2.75 18 Naricin 41.84±3.51 36.82 Soil 3.97 25.11 Soil 7.60 9 Dihydroquercetin ((+)-Taxifolin) 34.54± 3.47 23.80 soil 5.84 22.58±11.69 17 Wongonin 23.48±2.59 21.87 ± 1.90 15.64±7.82 16 Protocatechuic acid 22.75±4.07 19.95±8.95 25·66 Earth 12.74 8 Catechin ((+ )-Catechin) 16.45 Soil 9_67 33.83±8.7 6 41.53±7.62 16 23 201236678 Chinese medicine CYP2E1 inhibition rate minimum effective dose* (mg) test concentration 66 μΜ 33 μΜ 16.5 μΜ β-naphthoflavone (β-naphthoflavone) 15.40±12.94 16.83±〇.96 6.52±6.64 15 Embelin 13.54±11.64 ~----- 12.30±ΐ〇.24 5.95±7.48 16 trans-Cinnamic acid 7.10±6.95 4.66±6.50 5·71±10·53 8 Tea phenol ((-)-Epicatechin) 2.57±11.60 卜--- 15.02±5.50 18.27±9.34 16 Phloridzin 1.42±9.28 3·76±3.58 1.25±7.90 24 Puerrarin -12.86±2.75 ---- -4.64±3.47 0.43±2.31 23 Umbelliferone_ 1 A 厶:AU and · _!=L It* * -1081·56±168.00 -571.97±117.56 -280.41±19.48 9 : Minimum effective dose: Minimum screening concentration (mg/L) X human liver and intestine volume (3L) The CYP2E1 inhibition rate measured by the Langfang agent is shown in Table 4. From the results, the respective excipients were found at different concentrations (0.167%, 0.08). Under the condition of %, 0.042%, w/v), the cytochrome has different inhibitory effects on 5〇2E1, and the inhibition effect of u 〇.167% Brij 58' (97.75±〇.66) %) 〇— _____ drug induced CYP 2E1 inhibition rate. Measured concentration (w/v) 0.167% _0.08% - control group 0 __ 0.042% minimum injectable dose * (mg) 24 201236678 Chinese medicine medicine cited '~~- ----- CYP 2E1 inhibition rate minimum effective dose* (mg) test concentration (W/V) positive control group (DDTC) 0-167% 97.55±1·862 0.08% 0.042% Brij 58 97.75±0.66 96.58±0.40 96.02±0.17 1260 Brij 76 97.56±l.〇2 96·87±1·〇〇94.76±0.47 1260 Brij 35 93.33±0.82 (test concentration 0.025%) 89.45±0.68 (test concentration 0.013%) 76.21±7.37 (test concentration 0.006%) 180 Tween 20 87.20±1.29 82.80±1·71 71.77±4.48 1260 Tween 80 73.92±4.71 65.45±2.50 64.02 soil 12.54 1260 Tween 40 58.97±3.29 47.05±6.48 44.79±2.49 1260 PEG 2000 44.33±2.75 40.13±3.06 35.81 Soil 3,26 1260 PEG 400 42.33±5.25 39.10±0.73 31.98±5.97 1260 Pluomic F68 41.72±5.34 42.98±3.24 37.11±l〇.35 1260 PEG 4000 37.21±1.91 41.22±0.97 37.18±10.52 1260 *Minimum effective dose: Minimum screening concentration (mg/L) X human liver and intestinal volume (3l) Example III, cytochrome P450 2E1 (CYP2E1) inhibition Screening-Human Liver Microsomal Cytochrome P450 2E1 I. Materials and Methods 1. Test Materials This example is a microsome prepared by using human liver, and is directed to cytochrome P450 2E1 (CYP2E1) and 39 kinds of traditional Chinese medicines. Excipients were screened for cytochrome P450 2E1 (CYP2E1) inhibitors to screen for potent inhibitors of cytochrome P45〇2E1 (CYP2E1) in human liver; the principle of screening for this CYP2E1 inhibitor was: using human liver Preparation of micro 25 201236678 The cytochrome P450 2E1 (CYP2E1) in mitochondria reacted with its receptor Chlorzoxazone, and the detection of CYP2E1 metabolite standard 6-OH-CZX (6-Hydroxy-Chlorzoxazone) was detected after the test sample was added. The amount of CYP2E1 inhibition of the test sample was calculated based on the amount of 6-OH-CZX produced by the control. Each test sample was dissolved in 10% methanol or secondary water to test different concentrations of traditional Chinese medicine (66μΜ, 33μΜ, 16_5μΜ) and excipients (0.167%, 0.08%, 0.042%, w/v) The inhibition rate of CYP2E1's test results are shown in Table 6. The excipients and results tested are listed in Table 7. The reagents required for screening human liver cytochrome P450 2E1 (CYP2E1) inhibitors in this example are as follows: (1) CYP2E1: 100 mM potassium phosphate (pH 7.4) contains 1 mg/ml P450 protein concentration ° (2) Control Protein : 10 mg/mL P450 Protein Dissolved in 100 mM Potassium

Phosphate (pH 7.4)中。 (3) Buffer Solution : 0.5 M Potassium Phosphate (pH 7.4) 〇 Stop Solution : ice-acetonitrile。 (4) Cofactors :含有 100 mMNADP+以及 10 mM Glucose 6-Phosphate。 (5) Glucose 6-Phosphate Dehydrogenase : 2000 units/ml 溶于無菌水。 (6) Chlorzoxazone :受質(substrate)，16 mM Chlorzoxazone 溶於 1〇0/0 甲 醇(Methanol) 〇 ⑺ DDTC (Diethyldithiocarbamic add) ·· CYP2E1 選擇性抑制劑(陽性對照 組），20 mM DDTC 容於 10% 甲醇(Methanol)。 ⑻ NADPH-regenerating System:於 3.42 ml 中加入 530 μΐ Cofactors、40 μΐ G6PDH (Glucose 6-Phc«phate Dehydrogenase Solution)以及 100 μΐPhosphate (pH 7.4). (3) Buffer Solution : 0.5 M Potassium Phosphate (pH 7.4) 〇 Stop Solution : ice-acetonitrile. (4) Cofactors: Contains 100 mM NADP+ and 10 mM Glucose 6-Phosphate. (5) Glucose 6-Phosphate Dehydrogenase: 2000 units/ml Dissolved in sterile water. (6) Chlorzoxazone: Substrate, 16 mM Chlorzoxazone dissolved in 1〇0/0 Methanol 7(7) DDTC (Diethyldithiocarbamic add) ·· CYP2E1 selective inhibitor (positive control), 20 mM DDTC 10% methanol (Methanol). (8) NADPH-regenerating System: Add 530 μΐ Cofactors, 40 μΐ G6PDH (Glucose 6-Phc«phate Dehydrogenase Solution) and 100 μΐ to 3.42 ml

Control Protein ° s 26 201236678 2.細胞色素P450 2E1 (CYP2E1)抑制劑的篩選 使用人類肝臟微粒體細胞色素P450 2E1 (CYP2E1)進行細胞色素 P450 2E1 (CYP2E1)抑制劑篩選的實驗步驟如下所述： (1) 在4°C冰浴環境下，o.im磷酸緩衝液（pH = 7.4)包含0.5 mg/ml 人肝微粒體、5 mMMgCl2靜置15分鐘； (2) 此時實驗組加入細胞色素P450 2E1反應基質藥物16 mM Chlorzoxazone以及濃縮中藥藥引萃取液；對照組以曱醇：無菌水=1 : 1 取代中藥藥引；陽性對照組則以DDTC取代； (3) 最後加入輔酶1 mM NADP+、10 mM G6P與2 IU G6PD。將反應 液轉移至37°C水浴預溫（pre_incubati〇n) 1分鐘，活性測試實驗的反應時 間為30分鐘； (4) 反應完後以500 L acetonitrile終止反應，樣品靜置1分鐘後加入 内部標準品（5 g/mL 4-hydroxy-tobutamide)，離心後取上層液2〇 L以 甲醇·無菌水作稀釋十倍動作，取5 L之回溶液注入LC/MS/MS系統進 行分析。 (5)結果分析：將lc/MS/MS測得的訊號數值換算成為CYP2E1代謝 物標準品6-Hydr〇xy-Chl〇rzoxazone生成量(pm〇i)後，以對照組(c〇mr〇丨)為 基準’即對照組的CYP2E1抑制率為〇%，以下列公式計算各陽性對照組 及试驗組的CYP 2E1抑制率： CYP2E1抑制率(％)=卜——g組的^g^CZX生成量_ 對照組(control)的6-OH-CZX生成量 二、結果 1_陽性對照組 木陽性對照組(DDTC)所測出的CYP2E1抑制率如表五所示，由表二可知 冨D丁C的/農度為1〇〇 μΜ時，CYP2E1抑制率可達87.56¾。 27 201236678 __表五陽性對照組的c ΥΡ 2E1抑制率_ 卫gyc 濃度（_ 6_OH_czx 生成量（pm〇1) cyp 2m 〇 (對照組） ^ ^^ 50 1644.5 48.66 __87.56 2.試驗組CYP2E1抑制率 中藥藥引所測出的CYP2E1抑制率如表六所示，由結果可知各中藥藥 引於不同濃度(66μΜ，33μΜ, 16.5μΜ)的條件下，對細胞色素P45〇 2E1具有 不同程度的抑制縣，其㈣ 66 μΜ jLH^_dihydiOguaiaretic add) 抑制效果（96.98±0.19 %)及 66 μΜ 反式肉桂醛(Trans-Cinnamaldehyde)抑制 效果(92.81±0,53 %)最佳。 表六中藥藥引的CYP2E1抑制率 中藥藥引 CYP2E1抑制率ΓΜΛ 最小 有效 劑量 (mg) 測試濃度 66 μΜ 33 μΜ 16.5 μΜ 對照組 0 0 0 正二羥愈瘡酸 (Nordihydroguaiaretic acid) 96.98±〇·19 67.68. 士 2.24 49.81±2.42 17 反式肉桂醛 (Trans-Cinnamaldehyde) 92.81±〇.53 89.56±1.52 60.79±3.00 7 大豆甘元 (Daidzein) 86.77±1_〇4 76.33士 2.28 73.55±1.74 14 異牡荊素 (Isovitexin) 81.82±1.34 67.60土 3.24 59.82±1.41 24 山奈紛 (Kaempferol) 79.25±0.27 74.74±0.60 66.53±1.71 16 28 201236678 中藥藥引 CYP2E1抑制率（％) 最小 有效 劑量 (mg) 測試濃度 66 μΜ 33 μΜ 16.5 μΜ 雙硫余 (Disulfiram) 78.23±0.25 75.75±1.38 74.09±1.10 17 β-香葉烯 (β-Myrcene) 76.49±2.18 75.50土 2.14 53.40±4.93 8 槲皮素 (Quercetin) 73.32±1.57 53.02±2.17 46.40±4.68 16 (-)-Epigallocetechin-3-gallate 72.16±1.02 60.53士 2.06 50.19±1.89 25 (+)-檸檬烯 ((+)-Limonene) 63.64±2.74 38.05 士 1.95 13.77±1.96 7 楊梅素 (Myricetin) 61.60±0.88 59.21±1.27 42.21±2.55 17 槲皮 (Quercitrin) 61.04±5.88 53_77 土 3.51 33.51±4.29 24 木犀草素-7-葡萄糖苷 (Luteolin-7-Glucoside) 60_26±1.11 55.87±0.67 42.96士 5.10 24 桑葉素 (Morin) 60.26士 1.56 52.08±1.70 36.88±1.56 16 新橙皮苷 (Neohesperidin) 58.70±1.06 48.96±2.37 42.81±1.75 33 橙皮苷 (Hesperidin) 58.57士 3.78 50.91 士 2.81 45.32±1.57 33 茵陳色原嗣 (Capillarisin) 57.31±1.31 46.22±2.65 32.89±2.46 17 201236678 中藥藥引 CYP2E1抑制率（％) 最小 有效 劑量 (mg) 測試濃度 66 μΜ 33 μΜ 16.5 μΜ (-)-Epigallocatechin 57.08±1.85 36.40±2.18 38.95土 1.92 17 金絲桃苷 (Hyperoside) 53.51 士 1.20 35.58±3.68 -24.16士1.19 25 木犀草素 (Luteolin) 53.23土 1.78 43.40±4.74 39.15±3.42 16 十四烧酸乙S旨 (Ethyl Myristate) 51.95±2.38 41.04 士 4.76 22.08±0.78 14 禋柳素 (Tamarixetin) 50.91±3.12 47.79±2.81 37.40±1.96 17 根皮素 (Phloretin) 50.90士 2.09 39.78±3.28 29.60±3.21 15 黃芩素 (Baicalein) 50.13±5.11 47.79±3.40 35.32士 1.51 15 黃芩 (Baicalin) 49.30±2.26 35.61±3.09 22.51±2.24 24 芹菜素 (Apigenin) 47.51±3.66 36.80±1.98 28.89±1.54 15 柚皮素 (Naringenin) 45.16 土 4.43 28.45±2.21 19.50 士 2.02 9 橙皮素 (Hesperetin) 44.56士2.35 34.28±2.03 25.74±2.45 17 (+)-Epicatechin 44.32±1.25 52.32士 1.59 66.71±1.79 16Control Protein ° s 26 201236678 2. Screening of cytochrome P450 2E1 (CYP2E1) inhibitors The experimental procedure for screening for cytochrome P450 2E1 (CYP2E1) inhibitors using human liver microsomal cytochrome P450 2E1 (CYP2E1) is as follows: 1) o.im phosphate buffer (pH = 7.4) containing 0.5 mg/ml human liver microsomes and 5 mMMgCl2 for 15 minutes in an ice bath at 4 °C; (2) At this time, the experimental group was added with cytochrome P450. 2E1 reaction matrix drug 16 mM Chlorzoxazone and concentrated Chinese medicine extract; the control group was treated with sterol: sterile water = 1: 1 instead of Chinese medicine; the positive control group was replaced by DDTC; (3) finally added coenzyme 1 mM NADP+, 10 mM G6P and 2 IU G6PD. The reaction solution was transferred to a 37 ° C water bath pre-war (pre_incubati〇n) for 1 minute, and the reaction time of the activity test was 30 minutes; (4) After the reaction, the reaction was terminated with 500 L of acetonitrile, and the sample was allowed to stand for 1 minute and then added to the inside. Standard product (5 g / mL 4-hydroxy-tobutamide), after centrifugation, take 2 〇L of the upper layer and dilute with methanol·sterile water for 10 times. Take 5 L of the solution back into the LC/MS/MS system for analysis. (5) Analysis of results: The signal value measured by lc/MS/MS was converted into the CYP2E1 metabolite standard 6-Hydr〇xy-Chl〇rzoxazone production amount (pm〇i), and then the control group (c〇mr〇)丨) As the benchmark, the control group's CYP2E1 inhibition rate was 〇%, and the CYP 2E1 inhibition rate of each positive control group and the test group was calculated by the following formula: CYP2E1 inhibition rate (%) = Bu - g group ^g^ The amount of CZX produced _ 6-OH-CZX production amount of the control group 2, the result 1_ positive control group wood positive control group (DDTC) measured CYP2E1 inhibition rate as shown in Table 5, as shown in Table 2 When D/C is 1〇〇μΜ, the inhibition rate of CYP2E1 can reach 87.563⁄4. 27 201236678 __ Table 5 positive control group c ΥΡ 2E1 inhibition rate _ wei gyc concentration (_ 6_OH_czx production amount (pm 〇 1) cyp 2m 〇 (control group) ^ ^^ 50 1644.5 48.66 __87.56 2. Test group CYP2E1 The inhibition rate of CYP2E1 measured by the Chinese herbal medicine is as shown in Table 6. From the results, it can be seen that each Chinese medicine has different degrees of cytochrome P45〇2E1 under different conditions (66μΜ, 33μΜ, 16.5μΜ). Inhibition of the county, its (four) 66 μΜ jLH^_dihydiOguaiaretic add) inhibition effect (96.98 ± 0.19 %) and 66 μΜ trans-Cinnamaldehyde (Trans-Cinnamaldehyde) inhibition effect (92.81 ± 0, 53%) is best. Table 6 CYP2E1 inhibition rate of traditional Chinese medicine cited CYP2E1 inhibition rate 最小 Minimum effective dose (mg) Test concentration 66 μΜ 33 μΜ 16.5 μΜ Control group 0 0 0 Nordic hydroguaiaretic acid 96.98±〇·19 67.68士 2.24 49.81±2.42 17 Trans-Cinnamaldehyde 92.81±〇.53 89.56±1.52 60.79±3.00 7 Dairyzein 86.77±1_〇4 76.33士2.28 73.55±1.74 14 Isovitexin 81.82±1.34 67.60 soil 3.24 59.82±1.41 24 Kaempferol 79.25±0.27 74.74±0.60 66.53±1.71 16 28 201236678 Chinese medicine CYP2E1 inhibition rate (%) Minimum effective dose (mg) Test concentration 66 μΜ 33 μΜ 16.5 μΜ Disulfiram 78.23±0.25 75.75±1.38 74.09±1.10 17 β-Myrcene 76.49±2.18 75.50 Soil 2.14 53.40±4.93 8 Quercetin 73.32±1.57 53.02 ±2.17 46.40±4.68 16 (-)-Epigallocetechin-3-gallate 72.16±1.02 60.53 ± 2.06 50.19±1.89 25 (+)-limonene ((+)-Limonene) 63.64±2.74 38.05 ± 1.95 13.77±1.96 7 Yangmeisu ( Myriceti n) 61.60±0.88 59.21±1.27 42.21±2.55 17 Quercitrin 61.04±5.88 53_77 Soil 3.51 33.51±4.29 24 Luteolin-7-Glucoside 60_26±1.11 55.87±0.67 42.96士5.10 24 Morin 60.26±1.56 52.08±1.70 36.88±1.56 16 Neohesperidin 58.70±1.06 48.96±2.37 42.81±1.75 33 Hesperidin 58.57士3.78 50.91士2.81 45.32±1.57 33 Capillarisin 57.31±1.31 46.22±2.65 32.89±2.46 17 201236678 Chinese medicine CYP2E1 inhibition rate (%) Minimum effective dose (mg) Test concentration 66 μΜ 33 μΜ 16.5 μΜ (-)-Epigallocatechin 57.08± 1.85 36.40±2.18 38.95 soil 1.92 17 Hypericine 53.51 ± 1.20 35.58 ± 3.68 - 24.16 ± 1.19 25 Luteolin 53.23 soil 1.78 43.40 ± 4.74 39.15 ± 3.42 16 Ethyl Myristate) 51.95±2.38 41.04 ± 4.76 22.08±0.78 14 Tamarixetin 50.91±3.12 47.79±2.81 37.40±1.96 17Phloretin 50.90±2.09 39.78±3.28 29.60±3.21 15 Baicalein (B Aicalein) 50.13±5.11 47.79±3.40 35.32±1.51 15 Baicalin 49.30±2.26 35.61±3.09 22.51±2.24 24 Apigenin 47.51±3.66 36.80±1.98 28.89±1.54 15 Naringenin 45.16 Soil 4.43 28.45±2.21 19.50 ± 2.02 9 Hesperetin 44.56 ± 2.35 34.28 ± 2.03 25.74 ± 2.45 17 (+)-Epicatechin 44.32 ± 1.25 52.32 ± 1.59 66.71 ± 1.79 16

S 30 201236678 中藥藥引 測試濃度 芸香素 (Rutin) 66 μΜ 43.51i3.09 33 μΜ 30.13±1.62 16.5 μΜ 最小 有致 劑量i2|) 33 30.00±0.81 34.84±1.72 31.48±1.24S 30 201236678 TCM Drug Concentration Test Concentration Rutin 66 μΜ 43.51i3.09 33 μΜ 30.13±1.62 16.5 μΜ Minimum Intravenous dose i2|) 33 30.00±0.81 34.84±1.72 31.48±1.24

42.92±0.65 41.12土 0.92 38.96土 1.19 37.14±1.15 59.48±2.3442.92±0.65 41.12 soil 0.92 38.96 soil 1.19 37.14±1.15 59.48±2.34

38.7〇±1.62 30.65±〇.78 23.12±1.19 24 (-)-Epicatechin-3 -gal late 異甘草素 (Isoliquritigenin) 水飛薊賓 (Silybin) 牡荊素 (Vitexin) 金雀異黃酮 (Genistein) 異鼠李素 (Isorhamnetin) 沒食子酸 (gallic acid) 香葉木素 (Diosmin) 6-薑辣醇 (6-Gingerol) 36.88±1.56 36.31±1.59 27.96土 1.56 21.56±1.19 19.08±1.36 30.91±1.62 18.68±1.22 18.79±2.〇3 43.12±3.57 11·51±ι·〇2 43.90±2.06 12.06±1.〇6 10·50±1·12 60.00±1.96 7.84±0.92 15 14 9 :最小有效姆：最低f帛選濃度（mg/L) x人肝腸體積(3l) 賦形劑所測出的CYP2E1抑制率如表七所示，由結果可知各賦形劑於 不同濃度(0.167%，〇.〇8%，_2%，w/v)的條件下，對細胞色素p45〇加具有 31 201236678 不同程度的抑制效果，其中以0.167% Brij 58的抑制效果最佳(91·24±1·33 %)。 表七賦形劑的CYP2E1抑制率 賦形劑 CYP2E1抑制率（％) 最小 有效 劑量 (mg)* 測試濃度（w/v) 0.167% 0.08% 0.042% 對照組 0 - Brij 58 91.24 士 1.33 80.50±1.14 62.57±2.10 1260 Brij 76 86·15±1·02 75.71±1.61 68.99士 3.77 1260 Saccharin 78.5±2.1 (測試濃度66 uM) 51.2 ±0.9 (測試濃度33 uM) 29.4 士 2.7 (測試濃度16.5 uM) 10 Brij 35 77.28±1.02 (測試濃度 0.025%) 64.17±1.71 (測試濃度 0.013%) 42.37±1·78 (測試濃度 0.006%) 18 Tween 20 75.38±3.64 70.44±0.93 55.38±1.95 1260 PEG 400 64.17±1.53 54.78±3.53 26.42±1.81 1260 Microcrystalline cellulose 60.2±4.1 (測試濃度 0.025%) 54.4±3.8 (測試濃度 0.013%) 48.8±0.2 (測試濃度 0.006%) 180 Dicalcium phosphate dihydrate 60.U0.3 (測試濃度66 uM) 56.8±2.2 (測試濃度33 uM) 31.2 土 2.9 (測試濃度16.5 uM) 9 Sucralose 55.8 ±2.0 (測試濃度66 uM) 45.8 ±4.0 (測試濃度33 uM) 37.1 士 2.8 (測試濃度16.5 uM) 22 S. 32 201236678 賦形劑 CYP2E1抑制率（％) 最小 有效 劑量 (mg)* 測試濃度（w/v) 0.167% 0.08% 0.042% Mannitol 54.5 士 4.2 (測試濃度66 uM) 51.2±2.1 (測試濃度33 uM) 44.8±1.8 (測試濃度16.5 uM) 10 Cremophor RH40 50.4 土 1.1 43.2 士 3.1 30.2±2.8 1260 Sodium starch glycolate 50.3 土 1.9 51.3 ±2.2 34.7 ± 1.3 (測試濃度 0.00525%) 158 PEG 4000 47.11 士0.92 23.94±0.92 8.70 士 0.77 1260 PEG 2000 47.06士 1.53 41.43 士 1.60 22_25 士 1.93 1260 Crospovidone 44.1±0.9 40.3±2.1 34.8 ± 1.1 (測試濃度 0.00525%) 158 Tween 40 46.34±3.06 33.43±2.10 16.88 土 1.17 1260 Tween 80 39.14±2.40 40.56士 3.85 23.1 ± 3.0 (測試濃度 0.00525%) 158 Eudragit SI00 38.1 ±0.1 35.6 ±2.4 10.2 ± 0.3 (測試濃度 0.00525%) 158 Croscarmellose sodium 35.4 ±0.8 30.3 ±2.4 4.3 ± 0.3 (測試濃度 0.00525%) 158 Pluomic F68 31.46±1.60 17.39 士 1.07 7.93±0.27 1260 201236678 賦形劑 CYP2E1抑制率（％) 最小 有效 劑量 (mg)* 測試濃度（w/v) 0.167% 0.08% 0.042% Menthol 30.8 土 0.3 20.8 ±2.1 10.5 ±0.4 8 Hydroxypropylcellulose 22.1 ±0.4 (測試濃度 0.025%) 20.3 ± 1.1 (測試濃度 0.013%) 17.5 ±0.9 (測試濃度 0.006%) 158 Pregelatinized starch 18.3 ± 1·1 12.8 ±0.2 10.2 ±2.3 (測試濃度 0.00525%) 158 Dextrates NF hydrated 19.2 ± 1.1 14.4 ±3.2 10.6 ± 1.5 (測試濃度 0.00525%) 158 Citric acid 20.5 ±1.8 (測試濃度66 uM) 15.5 ±0.0 (測試濃度33 uM) 9.9 ±3.1 (測試濃度16.5 uM) 10 Cremophor EL 19.2 ±〇·5 15.2 土 2.2 2.4 士 0.3 (測試濃度 0.00525%) 158 Aerosil 200 15·4 士 U 17.8 士 2.1 4.3 ±0.1 (測試濃度 0.00525%) 158 Myrj 52 18.1 士 2.6 15.7 ±2.7 14.6 ±1.8 1260 PEG 8000 21.1 ±3.4 14.2 ±3.3 9.4 ± 0.2 1260 Sorbic acid 14.8 土 〇·1 (測試濃度66 uM) 10.9 士 2.1 (測試濃度33 uM) 8.4 ± 1.6 (測試濃度16.5 uM) 638.7〇±1.62 30.65±〇.78 23.12±1.19 24 (-)-Epicatechin-3 -gal late Isoliquritigenin Silybin Vitexin Genistein Isorhamnetin gallic acid geranin (Diosmin) 6-gingerol (6-Gingerol) 36.88±1.56 36.31±1.59 27.96 soil 1.56 21.56±1.19 19.08±1.36 30.91±1.62 18.68± 1.22 18.79±2.〇3 43.12±3.57 11·51±ι·〇2 43.90±2.06 12.06±1.〇6 10·50±1·12 60.00±1.96 7.84±0.92 15 14 9 : least effective m: minimum f Selection concentration (mg/L) x human liver and intestine volume (3l) The inhibition rate of CYP2E1 measured by the vehicle is shown in Table 7. From the results, it can be seen that each excipient is at different concentrations (0.167%, 〇.〇8%) Under the condition of _2%, w/v), the cytochrome p45 addition had different inhibitory effects on 31 201236678, among which 0.167% Brij 58 had the best inhibitory effect (91·24±1.33%). Table 7 Excipients CYP2E1 inhibition rate Excipient CYP2E1 inhibition rate (%) Minimum effective dose (mg)* Test concentration (w/v) 0.167% 0.08% 0.042% Control group 0 - Brij 58 91.24 ± 1.33 80.50 ± 1.14 62.57±2.10 1260 Brij 76 86·15±1·02 75.71±1.61 68.99士3.77 1260 Saccharin 78.5±2.1 (test concentration 66 uM) 51.2 ±0.9 (test concentration 33 uM) 29.4 士2.7 (test concentration 16.5 uM) 10 Brij 35 77.28±1.02 (test concentration 0.025%) 64.17±1.71 (test concentration 0.013%) 42.37±1·78 (test concentration 0.006%) 18 Tween 20 75.38±3.64 70.44±0.93 55.38±1.95 1260 PEG 400 64.17±1.53 54.78± 3.53 26.42±1.81 1260 Microcrystalline cellulose 60.2±4.1 (test concentration 0.025%) 54.4±3.8 (test concentration 0.013%) 48.8±0.2 (test concentration 0.006%) 180 Dicalcium phosphate dihydrate 60.U0.3 (test concentration 66 uM) 56.8 ±2.2 (test concentration 33 uM) 31.2 soil 2.9 (test concentration 16.5 uM) 9 Sucralose 55.8 ±2.0 (test concentration 66 uM) 45.8 ±4.0 (test concentration 33 uM) 37.1 ± 2.8 (test concentration 16.5 uM) 22 S. 32 201236678 Excipient CYP2E1 inhibition rate (%) Small effective dose (mg)* Test concentration (w/v) 0.167% 0.08% 0.042% Mannitol 54.5 ± 4.2 (test concentration 66 uM) 51.2 ± 2.1 (test concentration 33 uM) 44.8 ± 1.8 (test concentration 16.5 uM) 10 Cremophor RH40 50.4 Soil 1.1 43.2 ± 3.1 30.2 ± 2.8 1260 Sodium starch glycolate 50.3 Soil 1.9 51.3 ± 2.2 34.7 ± 1.3 (test concentration 0.00525%) 158 PEG 4000 47.11 ± 0.92 23.94 ± 0.92 8.70 ± 0.77 1260 PEG 2000 47.06 ± 1.53 41.43 ± 1.60 22_25士1.93 1260 Crospovidone 44.1±0.9 40.3±2.1 34.8 ± 1.1 (test concentration 0.00525%) 158 Tween 40 46.34±3.06 33.43±2.10 16.88 Soil 1.17 1260 Tween 80 39.14±2.40 40.56 ± 3.85 23.1 ± 3.0 (test concentration 0.00525%) 158 Eudragit SI00 38.1 ±0.1 35.6 ±2.4 10.2 ± 0.3 (test concentration 0.00525%) 158 Croscarmellose sodium 35.4 ±0.8 30.3 ±2.4 4.3 ± 0.3 (test concentration 0.00525%) 158 Pluomic F68 31.46±1.60 17.39 ±1.07 7.93±0.27 1260 201236678 Excipient CYP2E1 inhibition rate (%) Minimum effective dose (mg)* Test concentration (w/v) 0.167% 0.08% 0.042% Menthol 30.8 Soil 0.3 20.8 ± 2.1 10.5 ±0.4 8 Hydroxypropylcellulose 22.1 ±0.4 (test concentration 0.025%) 20.3 ± 1.1 (test concentration 0.013%) 17.5 ±0.9 (test concentration 0.006%) 158 Pregelatinized starch 18.3 ± 1·1 12.8 ±0.2 10.2 ±2.3 (test concentration 0.00525 %) 158 Dextrates NF hydrated 19.2 ± 1.1 14.4 ±3.2 10.6 ± 1.5 (test concentration 0.00525%) 158 Citric acid 20.5 ±1.8 (test concentration 66 uM) 15.5 ±0.0 (test concentration 33 uM) 9.9 ±3.1 (test concentration 16.5 uM) 10 Cremophor EL 19.2 ±〇·5 15.2 Soil 2.2 2.4 ± 0.3 (test concentration 0.00525%) 158 Aerosil 200 15·4 ± U 17.8 ± 2.1 4.3 ± 0.1 (test concentration 0.00525%) 158 Myrj 52 18.1 ± 2.6 15.7 ±2.7 14.6 ±1.8 1260 PEG 8000 21.1 ±3.4 14.2 ±3.3 9.4 ± 0.2 1260 Sorbic acid 14.8 Soil 〇·1 (test concentration 66 uM) 10.9 ± 2.1 (test concentration 33 uM) 8.4 ± 1.6 (test concentration 16.5 uM) 6

S 34 201236678 賦形劑 CYP2E1 抑制率(〇/〇) 測試濃度（w/v) 0.167% 0.08%S 34 201236678 Excipients CYP2E1 Inhibition rate (〇/〇) Test concentration (w/v) 0.167% 0.08%

Lemon oil 7.8 ± 0.3 9.8 ± 0.4 0.042% 22 ± 0.4 (測試濃度 0.00525%) 最小 有效 劑量(mgY 158Lemon oil 7.8 ± 0.3 9.8 ± 0.4 0.042% 22 ± 0.4 (test concentration 0.00525%) minimum effective dose (mgY 158

Span 60Span 60

SorbitolSorbitol

Sodium benzoateSodium benzoate

Acesulfame KAcesulfame K

Hydroxypropyl methylcelluloseHydroxypropyl methylcellulose

Hydroxy ethyl methylcellulose Methyl celluloseHydroxy ethyl methylcellulose Methyl cellulose

Span 80 17_4 士 0.9 13.9 ±0.7 12.4 ±2.3 1260 16.1 ±0.7 15.8 土 0.9 14.5 士 1.9 5.6 ±0.5 4.4 ± 1.7 (測試濃度 ^00525%) 7.1 ±2.0 3.9 ±2.76T±〇J (測試濃度 0.00525%) 158 7.8 ±4.1 13.9 ±2.2Span 80 17_4 ± 0.9 13.9 ±0.7 12.4 ±2.3 1260 16.1 ±0.7 15.8 Soil 0.9 14.5 ± 1.9 ± 5.6 ± 4.4 ± 1.7 (test concentration ^ 00525%) 7.1 ± 2.0 3.9 ± 2.76T ± 〇 J (test concentration 0.00525%) 158 7.8 ±4.1 13.9 ±2.2

11.6±0·9 10·2± 1.7 10.1 土 2.1 9.1 ± 2.6 (測試濃度66 13.2 ±0.6 5.5 ±0.5 6.2 ± 0.4 5.7±4.7 (測試濃度33 1.7 ±0.2 (測試濃度 0.00525%)~4Τ±Τ9^ (測試濃度 0.00525%) 5.9 ±0.311.6±0·9 10·2± 1.7 10.1 Soil 2.1 9.1 ± 2.6 (test concentration 66 13.2 ±0.6 5.5 ±0.5 6.2 ± 0.4 5.7±4.7 (test concentration 33 1.7 ±0.2 (test concentration 0.00525%)~4Τ±Τ9^ (Test concentration 0.00525%) 5.9 ±0.3

Sodium cyclamate 9.4 ±2.7 (測試濃度16.5Sodium cyclamate 9.4 ± 2.7 (test concentration 16.5)

Lactose monohydrate (測試濃度66 uM) (測試濃度33 uM) 7.8 ±2.2 (測試濃度16.5 _uM) 158 158 1260 10 18 35 201236678 賦形劑 CYP2E1 抑制率 最小 有效 劑量 (mg)* 測試濃度（w/v) 0.167% ---— 0.08% 0.042% Maltodextrin 8.5 ±2.8 5.9 士 2.1 7.2 士 1.2 (測試濃度 0.00525%) 158 Glyceryl behenate 8.2 ± 2.0 (測試濃度66 uM) 3.1 ±2.5 (測試濃度33 uM) 3.1 ±0.3 (測試濃度16.5 uM) 52 Oxide red 8.0 ±5.8 (測試濃度66 uM) 10.3 ±5.3 (測試濃度33 uM) 10.7 ±4.5 (測試濃度16.5 uM) 34 Glycerrin monostearate 6·9±3·8 7.4 ±2.9 5.8 ± 1.7 (測試濃度 0.00525%) 158 Copovidone K28 6.1 ±0.7 4.5 ± 0.5 6.4 士 0.5 (測試濃度 0.00525%) 158 Starch acetate 5.3 ± 0.7 4.9 ± 1.2 4.9 ± 1.4 (測試濃度 0.00525%) 158 Magnesium stearate 5.0 ± 1.6 (測試濃度66 uM) 3.0 ±0.7 (測試濃度33 uM) 2.0 ± 1.0 (測試濃度16.5 uM) 29 Sodium lauryl sulfate 4.9 ± 1.6 (測試濃度66 uM) 6.4 土 0.9 (測試濃度33 uM) 4.6± 1.1 (測試濃度16.5 uM) 14 Povidone K-30 3.2±0_2 (測試濃度66 uM) 2.2 ±0.1 (測試濃度33 uM) 4.7 ± 1.0 (測試濃度16.5 uM) 6 201236678 賦形劑 CYP2E1抑制率m、 最小 有效 劑量 (mg)* 測試濃度（w/v) 0.167% 0.08% 0.042% Benzyl alcohol -10.3 ±6.3 6.7 ± 1.0 7.7 ±2.6 (測試濃度 0.00525%) 158 Methylparaben Propylparaben -21.5 ±2.0 (測試濃度66 uM) -14.0 (測試濃度33 uM) 4.6 ±3.2 (測試濃度16.5 uM) 8 -27.3 土 3.7 (測試濃度66 uM) -17.2 ±2.4 (測試濃度33 uM) -4.1 ± 1.2 (測試濃度16.5 uM) 9 Solutol HI5 -21.0 ±4.8 (測試濃度 0.084%) -9.3 ±0.8 (測試濃度 0.042%) 2.7 ±0.3 (測試濃度 0.00525%) 158 Butylated hydroxyl anisol -85.5 土 3.9 (測試濃度66 uM) -47.1 ±5.1 (測試濃度33 uM) -16.8 土 0.5 (測試濃度16.5 uM) 9 最小有效劑量.最低篩選濃度（mg/L) χ人肝腸體積(3l) 實施例四、醯胺水解酶(amidase)抑制劑的篩選_鼠肝微粒體醯胺水解酶 (amidase) 一、材料與方法 1·試驗材料Lactose monohydrate (test concentration 66 uM) (test concentration 33 uM) 7.8 ± 2.2 (test concentration 16.5 _uM) 158 158 1260 10 18 35 201236678 Excipient CYP2E1 inhibition rate minimum effective dose (mg)* test concentration (w/v) 0.167% --- 0.08% 0.042% Maltodextrin 8.5 ±2.8 5.9 ± 2.1 7.2 ± 1.2 (test concentration 0.00525%) 158 Glyceryl behenate 8.2 ± 2.0 (test concentration 66 uM) 3.1 ± 2.5 (test concentration 33 uM) 3.1 ±0.3 (Test concentration 16.5 uM) 52 Oxide red 8.0 ± 5.8 (test concentration 66 uM) 10.3 ± 5.3 (test concentration 33 uM) 10.7 ± 4.5 (test concentration 16.5 uM) 34 Glycerrin monostearate 6·9±3·8 7.4 ±2.9 5.8 ± 1.7 (test concentration 0.00525%) 158 Copovidone K28 6.1 ±0.7 4.5 ± 0.5 6.4 ± 0.5 (test concentration 0.00525%) 158 Starch acetate 5.3 ± 0.7 4.9 ± 1.2 4.9 ± 1.4 (test concentration 0.00525%) 158 Magnesium stearate 5.0 ± 1.6 (test concentration 66 uM) 3.0 ± 0.7 (test concentration 33 uM) 2.0 ± 1.0 (test concentration 16.5 uM) 29 Sodium lauryl sulfate 4.9 ± 1.6 (test concentration 66 uM) 6.4 soil 0.9 (test concentration 33 uM) 4.6 ± 1.1 ( Test concentration 16.5 uM) 1 4 Povidone K-30 3.2±0_2 (test concentration 66 uM) 2.2 ±0.1 (test concentration 33 uM) 4.7 ± 1.0 (test concentration 16.5 uM) 6 201236678 Excipient CYP2E1 inhibition rate m, minimum effective dose (mg)* test Concentration (w/v) 0.167% 0.08% 0.042% Benzyl alcohol -10.3 ±6.3 6.7 ± 1.0 7.7 ±2.6 (test concentration 0.00525%) 158 Methylparaben Propylparaben -21.5 ±2.0 (test concentration 66 uM) -14.0 (test concentration 33 uM 4.6 ±3.2 (test concentration 16.5 uM) 8 -27.3 soil 3.7 (test concentration 66 uM) -17.2 ±2.4 (test concentration 33 uM) -4.1 ± 1.2 (test concentration 16.5 uM) 9 Solutol HI5 -21.0 ±4.8 (test Concentration 0.084%) -9.3 ±0.8 (test concentration 0.042%) 2.7 ±0.3 (test concentration 0.00525%) 158 Butylated hydroxyl anisol -85.5 soil 3.9 (test concentration 66 uM) -47.1 ±5.1 (test concentration 33 uM) -16.8 soil 0.5 (test concentration 16.5 uM) 9 minimum effective dose. minimum screening concentration (mg/L) human liver and intestine volume (3l) Example 4, screening of indoleamine hydrolase (amidase) inhibitor _ rat liver microsomal guanamine hydrolysis Enzyme (amidase) I. Materials and methods 1. Test materials

Isonicotinic acid以高效能液相層析儀(HPLC-UY)分析定量。所有的有機 溶劑均為 HPLC 等級，購自 Tedia 有限公司(Fairfield, OH, USA)，isoniazid、 isonicotinic acid 與 nicotinic acid (内在標準品，intemai standard)購自 Sigma 化學公司(St. Louis, MO USA)。 37 201236678 2·試樣處理 本實驗㈣統餘體歸料amidase酵素來源，isQniazid作為 aimdase代謝模式藥物’定量—經—代謝生成之代謝物 isonicotinic acid (INA)，作為amidase活性測量之標的，建立―脱體外活 性抑制劑篩選平台，HPLC系統包括i個島津似⑽泵(shimadzu LC-10ADpump)、津系統控制器(Shimadzusystemc〇miOi)以及 ι 個島 津自動樣本機(ShimadzuautosamplerX島津科學儀器，日本)，以⑽管柱(顆 粒大小5 μηι，内徑50 X 4.6 mm，25 cm)並使用含有70%甲醇和3〇%甲酸 銨(50mM，pH=2.5)之移動相進行hpLC分離，實驗步驟簡述如下·· (1) 鼠肝酵素微粒體溶液製備及蛋白濃度測定。 (2) 取鼠肝微粒體溶液15〇成加入1〇〇瓜isoniazid溶液(3應溶於）％此 67 mM磷酸鉀緩衝溶液(kH2P〇3,阳=7)及15吣amidase抑制劑(控制組 加入去離子水)混合均勻。 (3) 置於37°C水浴槽反應30分鐘。 ⑷加入300 pL乙晴(acetonitrile，ACN)混勻後靜置6分鐘。 (5) 加入30 pL過氯酸混勻後靜置6分鐘。 (6) 以13000g之轉速離心6分鐘。 (7) 取100 pL離心後上清液注射入hplc。 ⑻以甲醇：甲酸録(50mM，PH=2·5) = 70:30(V/V)為移動相，控制流速為 1 ml/min ’以波長270 nm紫外光檢測。 ⑼結果分析：將HPLC-UV測得的訊號數值換算成為amidase代謝物標準 品isonicotinicadd生成量(ng/mL)後，以對照組(control)為基準，即對照 組的amidase抑制率為〇% ’以下列公式計算各陽性對照組及試驗組的 amidase抑制率： 38 201236678 amidase 抑制率(％) 一實驗組的isonicotinic acid生成量 對照組(control)的 isonicotinic acid 生成量 結果 中樂純成份及賦形劑所測出之Ami—抑制率如表表八及表九所干 =果可知各中藥純成分及賦形_同心^^ 具有不同__效果，其中以刚_咖_抑制效果最佳 (75.5±2·2 %)。 表八 體外篩選中藥純成份所測出之Amidase抑制率 篩選成分\抑制濃度Isonicotinic acid was quantified by high performance liquid chromatography (HPLC-UY). All organic solvents were HPLC grades available from Tedia Limited (Fairfield, OH, USA), isoniazid, isonicotinic acid and nicotinic acid (integean standard) purchased from Sigma Chemical Company (St. Louis, MO USA) . 37 201236678 2·Sample treatment This experiment (4) is the source of amidase enzyme, isQniazid as aimdase metabolic model drug 'quantitative-metabolized metabolite ionogenicic acid (INA), as the target of amidase activity measurement, establish ― The in vitro activity inhibitor screening platform, the HPLC system includes i Shimadzu (10) pumps (shimadzu LC-10ADpump), Tsu system controller (Shimadzusystemc〇miOi), and ι Shimadzu automatic sample machine (ShimadzuautosamplerX Shimadzu Scientific Instruments, Japan) (10) Column (particle size 5 μηι, inner diameter 50 X 4.6 mm, 25 cm) and hpLC separation using mobile phase containing 70% methanol and 3% ammonium formate (50 mM, pH=2.5). The experimental steps are as follows: ·· (1) Preparation of rat liver enzyme microsome solution and determination of protein concentration. (2) Take the rat liver microsome solution 15 〇 into a 1 〇〇 mesaiazid solution (3 should be dissolved)% of this 67 mM potassium phosphate buffer solution (kH2P 〇 3, yang = 7) and 15 吣 amidase inhibitor (control Add deionized water to the group and mix well. (3) Place in a 37 ° C water bath for 30 minutes. (4) After adding 300 pL of acetonitrile (ACN), it was allowed to stand for 6 minutes. (5) Add 30 pL perchloric acid and mix for 6 minutes. (6) Centrifuge at 13,000 g for 6 minutes. (7) After centrifugation at 100 pL, the supernatant was injected into hplc. (8) Using methanol: formic acid recording (50 mM, pH = 2·5) = 70:30 (V/V) as the mobile phase, and controlling the flow rate to be 1 ml/min 'detected by ultraviolet light at a wavelength of 270 nm. (9) Analysis of results: After the signal value measured by HPLC-UV was converted into the amount of toidocitinaddadd (ng/mL) of the amidase metabolite standard, the control group was based on the control, that is, the amidase inhibition rate of the control group was 〇% ' The amidase inhibition rate of each positive control group and the test group was calculated by the following formula: 38 201236678 amidase inhibition rate (%) The amount of idiocotinic acid produced in the experimental group was controlled by the amount of idiocotinic acid in the control group. The Ami-inhibition rate measured by the agent is as shown in Tables 8 and 9 below. It can be seen that the pure components of each traditional Chinese medicine and the shape of the traditional Chinese medicine have different __ effects, among which the best effect is the best. ±2·2 %). Table 8 In vitro screening of Amidase inhibition rate measured by pure components of traditional Chinese medicine Screening component\inhibitory concentration

Amidase 活性抑制率(％)Amidase activity inhibition rate (%)

100 uM100 uM

10 uM10 uM

Positive Control (BNPP)Positive Control (BNPP)

1 uM 92_1 ± 8.7 最小有效 劑量 獬皮素 (Quercetin) 高良薑素 (Galangin) 桑葉素 (Morin) 異甘草素 (Isoliquirtigenin) 楊梅素 (Myricetin) 非瑟酮 (Fisetin) 雙硫命 (Disulfiram) 山奈酴 (Kaempferol) 75.5 ± 2.2 61.5 ± 2.7 59.8 ± 5.1 57.2 ± 8.5 56.4 ± 1.5 56.4 ± 2.9 50.2 ± 9.1 49.1 ± 8.6 62.3 ± 4.4 32.8 ±《4 9·! i 1.7 17.7 士 8.3 37·8 ± 8.4 38.3 ± 2 〇 42.1 ±《I 253 ± 7,8 48.1 ± 15.0 9.6 ± 9.9 16.6 土 4.8 -18.7 ± 26.0 8.0 土 4.9 -9.2 土 2.5 44.1 土 1.0 7.3 土 8.2 0.8 0.5 0.9 0.8 0.9 0.9 0.9 1.0 39 201236678 篩選成分\抑制濃度 Amidase活性抑制率(％) 最小有效 劑量 (mg) 100 uM 10 uM 1 uM 木犀草素 (Luteolin) 47.5 ± 6_2 23.0 ± 6.0 6.1 ± 9.1 0.6 茵陳色原酮 (Capillarisin) 4.2 45.7 ± ±3.7 18.2 ±8.1 -4.1 0.4 α-萘黃酮 (a-Naphthoflavone) 36.9 ± 2.7 19.3 ± 3.7 7.3 ± 7.9 1.8 雙氫槲皮素 ((+)-Taxifolin) 36.4 ± 3.4 32.2 ± 5.8 36.8 士 11.5 1.3 黃芩 (Baicalin) 34.8 ± 10.2 15.2 ± 6.3 2.1 ± 7.5 1.4 傘形酮 (Umbelliferone) 33.5 ± 8.2 -1.0 ± 13.2 24.1 ± 11.6 0.9 聖草次苷 (Eriocitrin) 32.8 ± 1.9 19.0 ± 3.0 -16.1 ± 12.8 0.8 異鼠李素 (Isorhamnetin) 31.3 ± 1.3 20.0 ± 4.6 13.8 ± 1.0 1.3 根皮素 (Phloretin) ±8.1 29.9 ±5.4 9.8 ±14.3 16.5 0.8 恩貝酸 (Embelin) 29.1 ± 1.2 6.6 土 8.6 5.3 ± 5.2 1.4 樘柳素 (Tamarixetin) 28.9 ± 4.2 27.9 ± 6.3 3.5 ± 10.5 1.8 齊墩果酸 (Oleanolic Acid) 4.0 24.5 ± ± 3_4 14.8 ±5.2 20.3 0.8 甘草酸 (Glycyrrhizin) 24.4 ± 12.7 4.3 ± 11.3 -1.3 ± 3.1 1.3 柚皮素 24.2 ± 7.6 13.5 ± 9.4 11.2 ± 6.5 1.01 uM 92_1 ± 8.7 Minimum effective dose Quercetin Galangin Morin Isoliquirtigenin Myricetin Fisetin Disulfiram Emp(Kaempferol) 75.5 ± 2.2 61.5 ± 2.7 59.8 ± 5.1 57.2 ± 8.5 56.4 ± 1.5 56.4 ± 2.9 50.2 ± 9.1 49.1 ± 8.6 62.3 ± 4.4 32.8 ± "4 9·! i 1.7 17.7 ± 8.3 37 · 8 ± 8.4 38.3 ± 2 〇42.1 ± "I 253 ± 7,8 48.1 ± 15.0 9.6 ± 9.9 16.6 Soil 4.8 -18.7 ± 26.0 8.0 Soil 4.9 -9.2 Soil 2.5 44.1 Soil 1.0 7.3 Soil 8.2 0.8 0.5 0.9 0.8 0.9 0.9 0.9 1.0 39 201236678 Filter components\ Inhibitory concentration Amidase activity inhibition rate (%) Minimum effective dose (mg) 100 uM 10 uM 1 uM Luteolin 47.5 ± 6_2 23.0 ± 6.0 6.1 ± 9.1 0.6 Capillarisin 4.2 45.7 ± ±3.7 18.2 ±8.1 -4.1 0.4 α-naphthoflavone 36.9 ± 2.7 19.3 ± 3.7 7.3 ± 7.9 1.8 Dihydroquercetin ((+)-Taxifolin) 36.4 ± 3.4 32.2 ± 5.8 36.8 ± 11.5 1.3 Baicalin ) 34.8 ± 10.2 15.2 ± 6.3 2.1 ± 7.5 1.4 Umbelliferone 33.5 ± 8.2 -1.0 ± 13.2 24.1 ± 11.6 0.9 Eriocitrin 32.8 ± 1.9 19.0 ± 3.0 -16.1 ± 12.8 0.8 Isorhamnetin 31.3 ± 1.3 20.0 ± 4.6 13.8 ± 1.0 1.3 Phloetin ± 8.1 29.9 ± 5.4 9.8 ± 14.3 16.5 0.8 Embelin 29.1 ± 1.2 6.6 Soil 8.6 5.3 ± 5.2 1.4 Tamarixetin 28.9 ± 4.2 27.9 ± 6.3 3.5 ± 10.5 1.8 Oleanolic Acid 4.0 24.5 ± ± 3_4 14.8 ± 5.2 20.3 0.8 Glycyrrhizin 24.4 ± 12.7 4.3 ± 11.3 -1.3 ± 3.1 1.3 Naringen 24.2 ± 7.6 13.5 ± 9.4 11.2 ± 6.5 1.0

S 40 201236678 篩選成分\抑制濃度 Amidase活性抑制率(％) 最小有效 劑量 (mg) 100 uM 10 uM 1 uM (Nariagenin) 金聖草黃素 (Chrysoeriol) 23.2 ± 1.2 24.9 土 11.2 7.7 ± 7.1 0.9 桉油醇 (Cineole) 22.9 ± 10.4 -4.7 ± 4.9 -16.5 ± 17.5 1.8 6-薑辣醇 6-Gingerol 22.6 ± 3.4 0.5 ± 0.7 -8.2 土 9.0 1.8 聖草酚 (Eriodictyol) 22_3 ± 4.7 6.5 ± 11.3 9.0 ± 4.7 1.4 異野櫻素 (Isosakuranetin) 22.2 ± 7.3 -8.9 ±11.2 -8.0 土 10.5 1.2 白楊素 (Chrysin) 21.5 ± 2.1 24.7 士 2.2 3_5 ± 6.1 1.1 金松雙黃酮 (Sciadopitysin) 20.6 ± 19.3 20.7 ± 6.0 10.3 士 3.8 1.3 異槲皮苷 (Isoquercitrin) 19.6 ± 5.9 5.1 ± 3.6 -3·7 士 1.4 0.4 橙皮素 (Hesperetin) 19.0 土 3_6 5_4 ± 2.5 -40.5 ± 24.8 1.7 異荭草素 (Homoorientin) 18.0 ± 2.6 3.5 ± 0_9 7.4 ± 3.8 0.5 葛根素 (Puerarin) 17.9 ± 7.2 33_0 ± 3_3 40_9 土 3_6 0.8 枸橘苦 (Poncirin) 17.3 ± 5.8 8.1 ± 11.7 10.8 土 3.8 0.9 大豆甘元 16.2 士 12.6 1.0 ± 7_4 -12·1 ± 6_3 1.4 201236678 篩選成分\抑制濃度 Amidase活性抑制率(％) 最小有效 劑量 (mg) 100 uM 10 uM 1 uM (Daidzein) 原兒茶酸 (Protocatechuic acid) 15.7 ± 8.1 10.9 ± 2.0 11.5 ± 4.0 0.9 黃芩素 (Baicalein) 15.6 ± 1.0 5.7 ± 2.6 2.8 ± 15.1 0.9 木犀草素-7-葡萄糖甙 (Luteolin-7-Glucoside) 12.9 ± 3.1 8.7 ± 1.9 3.2 ± 0.8 0.8 肉桂酸 (Trans-Cinnamic Acid) ±1.7 12.6 ±0.6 -1.4 ±2.3 -11.9 0.9 甘草甙 (Liquiritin) 12.5 ± 8.3 -7.5 ± 13.4 2.5 ± 2.9 0.8 佩蘭素 (Eupatorin) 12.0 ± 7.4 7.5 ± 0.7 0.9 ± 2.8 0.8 牡荊素 (Vitexin) 11.5 ± 1.0 6.3 ± 5.0 -0.6 ± 16.7 1.3 芫花素 (Genkwanin) 11.3 ± 1.3 2.1 士 10.0 1.6 ± 3.4 0.8 刺芒柄花素 (Formononetin) 9.8 ± 5.2 5.0 ± 0.4 -2.1 ± 5.0 0.5 甜橙黃酮 (Sinensetin) 9.7 ± 0.9 6.6 ± 0.5 -0.8 ± 3.0 1.1 薑黃素 (Curcumin) 9.7 ± 6.0 16.1 ± 0.4 16.9 ± 11.0 1.1 金絲桃甙 (Hyperoside) 8.4 ± 3.7 3.8 ± 4.4 4.6 ± 2.7 1.3 s. 42 201236678 篩選成分\抑制濃度 Amidase活性抑制率(％) 最小有效 劑量 (mg) 100 uM 10 uM 1 uM 大豆甙 (Daidzin) 7.4 ± 6.3 1.1 ± 5.4 3.6 ± 3.0 0.9 根皮甙 (Phloridzin) 7.1 ± 13.9 -3.1 ± 3.4 -3.2 ± 11.0 1.3 ㈩-棒檬烯 ((+)-Limonene) 6.2 ± 3.8 3.2 ± 8.6 5.1 ± 0.4 0.5 金雀異黃酮 (Genistein) 5.8 土 6.8 1.8 ± 7.6 -2.1 ± 46.2 0.5 月桂烯 (β-Myrcene) 5.8 ± 2.7 6.6 ± 4.1 1.7 ± 1.3 0.9 芸香素 (Rutin) 5.7 ± 8.2 -4.7 ± 4.7 -5.2 ± 1.2 0.9 松油醇 (Terpineol) 4.4 ± 5.2 7.0 ± 4.6 -0.7 士 5_0 1.7 月桂醇 (Lauryl Alcohol) 4.1 土 4.3 -0.7 ± 1.5 2.2 ± 3.1 1.8 (-)-Epicatechin 3.6 土 4.3 -4.8 ± 10.5 -25.8 ± 6.2 1.4 (-)-Epigallocatechin 2.2 ± 4.1 -7.3 ± 7.6 -1.2 ± 2.1 0.9 香葉木素 (Diosmin) 1.6 士 5.1 -0.7 ± 7.7 0.3 ± 8.1 1.2 槲皮 (Quercitrin) 1.6 ± 4.4 -14.4 士 5.3 -14.1 ± 12.1 1.3 兒茶素 ((+)-Catechin) 1.3 士 6.3 -11.8 ± 20.2 -2.1 ± 1.1 0.8 異牡荊素 (Isovitexin) 1.1 土 7.1 5.8 ± 2.7 13_9 ± 2_3 2.5 43 201236678 ---- 篩選成分\抑制濃度 Amidase活性抑制率⑽ 最小有效 — 劑量 100 uM 1〇 uM 1 uM 麥角固醇 (Ergosterol) --- (mg) 0.4 ± 3.6 -0.4 ± 1〇 7 4.3 ± 10·7 1.9 沒食子酸 (Gallic Acid) ---^-___ •20.2 ± 26.5 20.0 ± 5 j 12.0 ± 5.5 1.3 芹黃素 (Apigenin) 13.2 ± 3 4 -10.2 ± 20.3 1.2 *最小有效劑量：最低筛選濃度（mg/L) X人肝腸體積(3L) 表九體外篩選賦形劑所測出之Ajnidase抑制率 篩選成分\抑制濃度 ------- Ami dase活性抑制率(0/〇) 最小有 效劑量 (mg) 0.05% 0-005% 0 0005% Positive Control (BNPP) 92.1 ± 8.7 Sodium Lauryl Sulfate 66.1 ± 2.1 19.3 ± 2.7 9 6 土 5 0 17 Tween 20 64.4 ± 1.2 14-9 i 3.6 -47 4 士 14 1 17 Cremophor EL 56.4 ± 2_5 7.6 ± 9.6 X 3 士 S 1 17 Brij58 55.8 土 9.7 16-9 ± 5.5 14.3 ± 0.3 17 Acesulfame Potassium 24.3 ± 4.9 -167.2 ± 167.3 -12.4 土 27.4 17 Brij76 21.0 土 6·2 1·2 ± 6·6 -10 8 ± 5 7 17 Tween 80 16.7 ± 6.7 -3.3 ± 9.9 11 9 ± 2.1 17 Tween 40 Γ 15.4 ± 8.1 7.2 ± 7.4 3 1 ± 42 17 Mryi52__ 4·0 士 6.5 L5 ± 3.9 -3 4 ± 1 3 17 Mannitol 1.9 ± 6.0 52.8 ± 7.6 58.3 ± 4.3 17 Pluronic F68 1.2 ± 9.1 口 ± 6.8 -1 0 ± 4 7 17 PEG400 0.3 ± 5.9 -2.7 ± 7.9 1 0 ± 4 2 17 PEG2000 -7.0 ± 7.1 9-2 i 2.8 2.5 ± 12.8 17 Tween 60 -10.2 ± 17.4 -19.0 ± 23.3 Γ 4 1 士 8.1 17 Pluronic FI27 -13.7 ± 3.1 -8·0 i 5.1 -4·5 ± 2.2 17S 40 201236678 Screening Ingredients\Inhibitory Concentration Amidase Activity Inhibition Rate (%) Minimum Effective Dose (mg) 100 uM 10 uM 1 uM (Nariagenin) Chrysoeriol 23.2 ± 1.2 24.9 Soil 11.2 7.7 ± 7.1 0.9 Oyster sauce Cineole 22.9 ± 10.4 -4.7 ± 4.9 -16.5 ± 17.5 1.8 6-Gingerol 6-Gingerol 22.6 ± 3.4 0.5 ± 0.7 -8.2 Soil 9.0 1.8 Eriodictyol 22_3 ± 4.7 6.5 ± 11.3 9.0 ± 4.7 1.4 Isosakuranetin 22.2 ± 7.3 -8.9 ± 11.2 -8.0 Soil 10.5 1.2 Chrysin 21.5 ± 2.1 24.7 ± 2.2 3_5 ± 6.1 1.1 Sciadopitysin 20.6 ± 19.3 20.7 ± 6.0 10.3 3.8 1.3 Isoquercitrin 19.6 ± 5.9 5.1 ± 3.6 -3·7 ± 1.4 0.4 Hesperetin 19.0 Soil 3_6 5_4 ± 2.5 -40.5 ± 24.8 1.7 Homoorientin 18.0 ± 2.6 3.5 ± 0_9 7.4 ± 3.8 0.5 Puerarin 17.9 ± 7.2 33_0 ± 3_3 40_9 Soil 3_6 0.8 Poncirin 17.3 ± 5.8 8.1 ± 11.7 10.8 Soil 3.8 0.9 Soybean Gann 16.2 ± 12.6 1.0 ± 7_4 -12·1 ± 6_3 1.4 201236678 Screening component\Inhibitory concentration Amidase activity inhibition rate (%) Minimum effective dose (mg) 100 uM 10 uM 1 uM (Daidzein) Protocatechuic acid 15.7 ± 8.1 10.9 ± 2.0 11.5 ± 4.0 0.9 Baicalein 15.6 ± 1.0 5.7 ± 2.6 2.8 ± 15.1 0.9 Luteolin-7-Glucoside 12.9 ± 3.1 8.7 ± 1.9 3.2 ± 0.8 0.8 Trans-Cinnamic Acid ±1.7 12.6 ±0.6 -1.4 ±2.3 -11.9 0.9 Liquiritin 12.5 ± 8.3 -7.5 ± 13.4 2.5 ± 2.9 0.8 Eupatorin 12.0 ± 7.4 7.5 ± 0.7 0.9 ± 2.8 0.8 Vitexin 11.5 ± 1.0 6.3 ± 5.0 -0.6 ± 16.7 1.3 Genkwanin 11.3 ± 1.3 2.1 ± 10.0 1.6 ± 3.4 0.8 Formononetin 9.8 ± 5.2 5.0 ± 0.4 -2.1 ± 5.0 0.5 Sinensetin 9.7 ± 0.9 6.6 ± 0.5 -0.8 ± 3.0 1.1 Curcumin 9.7 ± 6.0 16.1 ± 0.4 16.9 ± 11.0 1.1 Hyperpodide 8.4 ± 3.7 3.8 ± 4.4 4.6 ± 2.7 1.3 s. 42 201236678 Screening Ingredients\Inhibitory Concentration Amidase Activity Inhibition Rate (%) most Effective dose (mg) 100 uM 10 uM 1 uM Daidzin 7.4 ± 6.3 1.1 ± 5.4 3.6 ± 3.0 0.9 Phloridzin 7.1 ± 13.9 -3.1 ± 3.4 -3.2 ± 11.0 1.3 (10)-Limene ( (+)-Limonene) 6.2 ± 3.8 3.2 ± 8.6 5.1 ± 0.4 0.5 Genistein 5.8 Soil 6.8 1.8 ± 7.6 -2.1 ± 46.2 0.5 Myrcene (β-Myrcene) 5.8 ± 2.7 6.6 ± 4.1 1.7 ± 1.3 0.9 Rutin 5.7 ± 8.2 -4.7 ± 4.7 -5.2 ± 1.2 0.9 Terpineol 4.4 ± 5.2 7.0 ± 4.6 -0.7 ± 5_0 1.7 Lauryl Alcohol 4.1 Soil 4.3 -0.7 ± 1.5 2.2 ± 3.1 1.8 (-)-Epicatechin 3.6 Soil 4.3 -4.8 ± 10.5 -25.8 ± 6.2 1.4 (-)-Epigallocatechin 2.2 ± 4.1 -7.3 ± 7.6 -1.2 ± 2.1 0.9 Diosmin 1.6 ± 5.1 ± 7.7 0.3 ± 8.1 1.2 Quercitrin 1.6 ± 4.4 -14.4 ± 5.3 -14.1 ± 12.1 1.3 catechin ((+)-Catechin) 1.3 ± 6.3 -11.8 ± 20.2 -2.1 ± 1.1 0.8 Isovitexin 1.1 Soil 7.1 5.8 ± 2.7 13_9 ± 2_3 2.5 43 201236678 ---- Screening component \ inhibitory concentration Amidase activity inhibition rate (10) Minimum effective - dose 100 uM 1〇uM 1 uM Ergosterol --- (mg) 0.4 ± 3.6 -0.4 ± 1〇7 4.3 ± 10·7 1.9 Gallic Acid --- ^-___ •20.2 ± 26.5 20.0 ± 5 j 12.0 ± 5.5 1.3 Apigenin 13.2 ± 3 4 -10.2 ± 20.3 1.2 *Minimum effective dose: Minimum screening concentration (mg/L) X Human liver and intestine volume (3L Table 9 In vitro screening of excipients measured by Ajnidase inhibition rate Screening component \ Inhibitory concentration ------- Ami dase activity inhibition rate (0 / 〇) Minimum effective dose (mg) 0.05% 0-005% 0 0005% Positive Control (BNPP) 92.1 ± 8.7 Sodium Lauryl Sulfate 66.1 ± 2.1 19.3 ± 2.7 9 6 Soil 5 0 17 Tween 20 64.4 ± 1.2 14-9 i 3.6 -47 4 ± 14 1 17 Cremophor EL 56.4 ± 2_5 7.6 ± 9.6 X 3 S 1 17 Brij58 55.8 Soil 9.7 16-9 ± 5.5 14.3 ± 0.3 17 Acesulfame Potassium 24.3 ± 4.9 -167.2 ± 167.3 -12.4 Soil 27.4 17 Brij76 21.0 Soil 6·2 1·2 ± 6·6 -10 8 ± 5 7 17 Tween 80 16.7 ± 6.7 -3.3 ± 9.9 11 9 ± 2.1 17 Tween 40 Γ 15.4 ± 8.1 7.2 ± 7.4 3 1 ± 42 17 Mryi52__ 4·0 6.5 L5 ± 3.9 -3 4 ± 1 3 17 Mannitol 1.9 ± 6.0 52.8 ± 7.6 58.3 ± 4.3 17 Pluronic F68 1.2 ± 9.1 Port ± 6.8 -1 0 ± 4 7 17 PEG400 0.3 ± 5.9 -2.7 ± 7.9 1 0 ± 4 2 17 PEG2000 -7.0 ± 7.1 9-2 i 2.8 2.5 ± 12.8 17 Tween 60 -10.2 ± 17.4 -19.0 ± 23.3 Γ 4 1 士 8.1 17 Pluronic FI27 -13.7 ± 3.1 -8·0 i 5.1 -4·5 ± 2.2 17

S 44 201236678 s fegQ300 -ZZTl -19.8 ：T 3.2「-24.7 ± 6.1 | 2.7 ± 9.7 | 17~ *最小有效劑量：最低篩選濃度（mg/L) X人肝腸體積(3L) 實施例五、丙基硫異菸醯胺(pZA)合併使用醯胺水解酶(amidase)抑制劑硝基 苯酚磷酸二酯(BNPP)之動物試驗 一、材料與方法 1. 試驗材料 所有的有機溶劑均為HPLC等級，購自Tedia有限公司(Fairfield，OH， USA) ’ PZA，BNPP則購自Sigma化學公司(St. Louis，MO USA)，确基苯酚磷 3文一®旨(BNPP)為一文獻普遍使用之酿胺水解酶(啦丨也记)抑制劑，半乳糖注 射溶液由南光化學製藥股份有限公司製備，係將4〇〇克半乳糖(Sigma)溶於j 公升含有適當緩衝溶液系統以及等張鹽類之蒸餾水中，供作注射使用。 2. 試驗動物 體重為320-350公克之雄性SD(Sprague-Dawley)大鼠購自國家實驗動物 中心(台灣）’動物實驗係遵照國衛院動物實驗指南進行，所有的大鼠均置於 空氣/濕度調節環境下，光照與黑暗各12小時，水及飼料的供給不限，在試 驗期間大鼠體重均持續監測。 3. 試驗處理 3式驗動物隨機分成3組，每組包括2種處理，第一種處理為注射5〇 mg/j^g BNPP或BNPP之基劑(vehicle, VKil，即食鹽水），BNPP係溶於加熱至6(rc 之食鹽水(0.9% NaCl)，冷卻後以1 ml/kg的體積進行腹腔内注射至大鼠體 内；第二種處理為注射500mg/kgPZA或PZA之基劑(VEH2,即食鹽水），PZA 係溶於食鹽水(0.9% NaCl)中，以1 ml/kg的體積進行腹腔内注射至大鼠體 内；第一種處理(BNPP或VEH1)較第二種處理(PZA或VEH2)早15分鐘處理。 上述3組試驗共包含： 45 201236678 (1) 對照組(normal control group，NC，n=l〇):正常的大鼠每天注 射1次VEH1及VEH2(施行腹腔内注射)共49天；S 44 201236678 s fegQ300 -ZZTl -19.8 :T 3.2"-24.7 ± 6.1 | 2.7 ± 9.7 | 17~ *Minimum effective dose: minimum screening concentration (mg/L) X human liver and intestine volume (3L) Example V, propyl Animal test of sulphonic isoniazid (pZA) combined with amidase inhibitor nitrophenol phosphate diester (BNPP) I. Materials and methods 1. Test materials All organic solvents are HPLC grade, purchased Since Tedia Co., Ltd. (Fairfield, OH, USA) 'PZA, BNPP was purchased from Sigma Chemical Company (St. Louis, MO USA), and phenolphthalein 3 (BNPP) is a commonly used amine in the literature. Hydrolase (Laoji) inhibitor, galactose injection solution prepared by Nanguang Chemical Pharmaceutical Co., Ltd., which dissolves 4 g of galactose (Sigma) in j liters containing appropriate buffer solution system and isotonic salts. Distilled water for injection. 2. Male SD (Sprague-Dawley) rats weighing 320-350 g were purchased from the National Experimental Animal Center (Taiwan)' Animal Experiments in accordance with the guidelines of the National Animal Institute. All rats were placed in an air/humidity environment Light and dark for 12 hours each, the supply of water and feed is not limited, and the weight of the rats is continuously monitored during the test. 3. Test treatment 3 The animals are randomly divided into 3 groups, each group consisting of 2 treatments, the first treatment For the injection of 5 〇mg/j^g BNPP or BNPP base (vehicle, VKil, ready-to-feed saline), BNPP is dissolved in 6 (rc of saline solution (0.9% NaCl), cooled to 1 ml / kg The volume was intraperitoneally injected into the rat; the second treatment was injection of 500 mg/kg PZA or PZA base (VEH2, ready saline), and PZA was dissolved in saline (0.9% NaCl) to 1 ml/kg. The volume was intraperitoneally injected into the rat; the first treatment (BNPP or VEH1) was treated 15 minutes earlier than the second treatment (PZA or VEH2). The above three groups of trials included: 45 201236678 (1) Control group (normal control group, NC, n=l〇): normal rats were injected with VEH1 and VEH2 once a day (administered intraperitoneally) for a total of 49 days;

(2) PZA組(ΙΝΉ，n=10).正常的大鼠每天注射1次pzA及vehI (施行腹腔内注射)共49天； (3) BNPP-PZA組(BNPP-PZA，n=l〇):正常的大鼠每天注射卜欠 BNPP及PZA (施行腹腔内注射)共49天； 半乳糖單點法於第49天處理後16小時進行測試。 4. 血液樣本 處理完畢後，大鼠以***麻醉犧牲，血液由大鼠背部主動脈抽取，置 於含有EDTA之試管中，血漿(plasma)以i3,〇〇〇g於4°C離心15分鐘，分離後 的血漿分裝到微量小管(Eppendorf tube)中並置於-8(TC中儲存。 5. 生化分析 肝細胞損傷以量測血漿中天門冬氨酸轉胺酶(AS乃與丙氨酸轉胺酶 (ALT)活性以進行定量，AST與alt活性是肝臟毒性常用的指標，係以(2) PZA group (ΙΝΉ, n=10). Normal rats were injected with pzA and vehI (administered intraperitoneally) once a day for 49 days; (3) BNPP-PZA group (BNPP-PZA, n=l〇) ): Normal rats were injected with BNPP and PZA (administered intraperitoneally) for a total of 49 days; galactose single-point method was tested 16 hours after treatment on day 49. 4. After the blood sample was processed, the rats were sacrificed by ether anesthesia. The blood was drawn from the rat aorta and placed in a test tube containing EDTA. The plasma was centrifuged at 4 ° C for 15 minutes at i3, 〇〇〇g. The separated plasma was dispensed into a small tube (Eppendorf tube) and stored in -8 (TC). 5. Biochemical analysis of hepatocyte damage to measure aspartate transaminase in plasma (AS is alanine) Transaminase (ALT) activity for quantification, AST and alt activity are commonly used indicators of liver toxicity,

SynchronLXi 725系統來量測(BeckmanInstruments，美國）。 6. 光學顯微鏡與電子顯微鏡 大鼠犧牲後肝臟隨即進行組織學分析；肝臟樣本以10%構酸緩衝液配製 之福馬林(phosphate-buffered formalin)固定’隨後脫水並包埋於石躐(paraffm) 中’以5 μηι厚度切片’切片樣本以蘇木精(hemat〇Xyiin)與伊紅(eosin)染色， 並進行肝糖染色試驗(Periodic acid Schiff stain，PAS)，染色後以光學顯微鏡 進行組織學觀察；另外，肝臟切片以二甲胂緩衝液(cac〇dylate buffer，0.1M pH 7.4)清洗’以20%四氧化餓水溶液(aqUe0US osmium tetroxide)後固定1小 時’以涵精連續脫水後包埋於Spurr樹脂(Spurrresin)中，並以鑽石刀切取超 薄切片，以醋酸鈾醯(uranyl acetate)及檸檬酸鉛(lead citrate)作雙重染色，並 以穿透式電子顯微鏡(Transmission Electron Microscope, Hitachi 600, Hitachi Co.,曰本)觀察8 s 46 201236678 7. 肝功能之定量測試 所有的大鼠均進行半乳糖單點法(GSP)測試，大鼠接受在3〇秒内的快速靜脈 注射’注射0.4g/ml BW半乳糖溶液〇.5 g/kg ;自注射後60分鐘採血一次，血 液樣本取自尾部靜脈；以半乳糖脫氫酶比色法(colorimetric galactose dehydrogenase)量測半乳糖含量，測試濃度範圍為50至woo μβ/Γη卜每個濃 度的日内差異(within-day variation)係由標準偏差(standard deviation)以及變 異係數(coefficient of variation，CV)百分比計算，最大容許的變異係數為1〇% CV ;日間差異(day-to-day variation)則由比較校正曲線(calibration curves)之 斜率及截距來檢驗；半乳糖單點法(GSP)為30秒注射停止後60分鐘時血液中 半乳糖濃度。 8. 統計分析 所有的數據皆以平均土標準偏差(SD)表示，試驗結果以單因子變異數分 析(ANOVA)測試法來計算是否具有統計上的顯著差異，使用Statistical Package of the Social Science program (Version 13, SPSS Inc.)套裝軟體來計 算；隨後使用事後比較(post hoc test)最小差異顯著性(least signiflcant difference)方法做多重比較，以確認族群間的顯著差異；族群平均之顯著差 異為P<0.05。 二、結果 1.生化分析結果 試驗結束時’測量試驗動物的體重及相對肝重量，與對照組動物相較 之下並無顯著差異；生化分析結果如圖—所示，PZA組血聚中的天門冬 氨酸轉胺酶(AST)與丙氨酸轉胺酶(ALT)活性明顯高於對照組(對照組血聚 中的AST活性為1〇9±27 IU/L ; PZA組血漿中的AST活性為179±1〇 IU/L，p < 0.005，對照組血漿中的ALT活性為43±9 IU/L ; PZA組血毁中的ALT活性為 91±11 IU/L，p<0.005) ’顯示PZA組產生生化上的肝損傷；BNPP_PZA血清 中轉胺酶濃度則顯著低於PZA組。 47 201236678 2. 組織病理學 經過為期7週施行腹腔注射500 mg/kg/day PZA之大鼠，其體内成功的 產生肝毒性；相對的，在對照組大鼠體内的肝結構則較正常，如圖十二A 所示’對照組大鼠肝實質(liver parenchyma)内的肝細胞係排列於自肝小葉中 央靜脈輻射排列的網狀平板内’肝血竇(hepatic sinus〇ids)則在兩肝板 (anastomosing plates)之間被發現；PZA組大鼠的組織切片則如圖十二b所 示’ PZA組大鼠中央靜脈周圍的肝細胞則呈現碎裂及空泡化，然而並無看 到肝細胞壞死(necrosis)的徵兆。而BNPP-PZA組大鼠的肝損害程度與對照 組相較，並無明顯區別(未顯示結果）。 3. 剩餘肝功能之量測 如圖十三所示，對照組與PZA組大鼠之半乳糖單點法(GSP)值具有高度 的顯著差異(對照組大鼠之GSP值為260土50 pg/ml ; PZA組大鼠之GSP值 為 776±65 Mg/ml，;? < 0.005)，此外，BNPP-PZA 組大鼠之 GSP 值為 293±61 pg/ml，與PZA組相較，具有高度的顯著差異②<〇.〇〇5);單獨施用PZA的 大鼠之GSP值明顯增加；然而，在PZA合併施用BNPP之大鼠則可抵抗這 種改變；而對照組與BNPP-PZA組之間大鼠的GSP值無顯著差異存在。 實施例六、異菸鹼酿胺(INH)及/或立復黴素(RIF)及/或丙基硫異菸醯胺(PZA) 合併使用CYP2E1抑制劑山奈盼(Kaempferol)或醯胺水解酶 (amidase)抑制劑槲皮素(Quercetin)之動物試驗 一、材料與方法 1.試驗材料 所有的有機溶劑均為HPLC等級，購自Tedia有限公司(Fairfield，OH， USA)，INH、RIF、PZA、Kaempfero卜 Quercetin購自 Sigma化學公司(st. Louis， MO USA)，半乳糖注射溶液由南光化學製藥股份有限公司製備，係將400 克半乳糖(Sigma)溶於1公升含有適當緩衝溶液系統以及等張鹽類之蒸餾水 48 § 201236678 中，供作注射使用。 2. 試驗動物 體重為18 25么克之129/SVaj、鼠是購自美國國家衛生研究院教⑧r,The SynchronLXi 725 system was measured (Beckman Instruments, USA). 6. Light microscopy and electron microscopy Rats were sacrificed for histological analysis immediately after sacrifice; liver samples were fixed with phosphate-buffered formalin in 10% acid buffer and subsequently dehydrated and embedded in paraffin Medium 'slices of 5 μηι thickness' slice were stained with hemat〇Xyiin and eosin, and subjected to periodic acid Schiff stain (PAS). After staining, histology was performed by light microscopy. In addition, the liver sections were washed with caffeine buffer (0.1 M pH 7.4) and fixed with 20% aqueous solution of aquaculture (aqUe0US osmium tetroxide) for 1 hour. In Spurr resin (Spurrresin), ultra-thin sections were cut with a diamond knife, double stained with uranyl acetate and lead citrate, and transmitted by electron microscope (Transmission Electron Microscope, Hitachi) 600, Hitachi Co., 曰本) Observation 8 s 46 201236678 7. Quantitative testing of liver function All rats were tested for galactose single point (GSP) and rats were received within 3 sec. Rapid intravenous injection 'injection of 0.4g/ml BW galactose solution 〇.5 g/kg; blood was taken once 60 minutes after injection, blood samples were taken from the tail vein; colorimetric galactose dehydrogenase was used. The galactose content is measured, and the test concentration range is 50 to woo μβ/Γη. The intra-day variation of each concentration is calculated from the standard deviation and the coefficient of variation (CV) percentage. The allowable coefficient of variation is 1〇% CV; the day-to-day variation is checked by comparing the slope and intercept of the calibration curves; the galactose single point method (GSP) is 30 seconds for the injection to stop. The concentration of galactose in the blood at 60 minutes later. 8. Statistical Analysis All data are expressed as mean soil standard deviation (SD). The test results are statistically significant using the Single Factor Variance Analysis (ANOVA) test method. The Statistical Package of the Social Science program is used. Version 13, SPSS Inc.) is a set of software to calculate; then a post hoc test is used to make multiple comparisons to confirm significant differences between ethnic groups; the significant difference in ethnic averages is P<;0.05. 2. Results 1. Biochemical analysis results At the end of the experiment, 'the body weight and relative liver weight of the test animals were measured, and there was no significant difference compared with the control animals. The results of biochemical analysis are shown in the figure. The activity of aspartate transaminase (AST) and alanine transaminase (ALT) was significantly higher than that of the control group (the AST activity in the blood group of the control group was 1〇9±27 IU/L; in the plasma of PZA group) The AST activity was 179±1〇IU/L, p < 0.005, the ALT activity in the control group was 43±9 IU/L; the ALT activity in the blood loss of the PZA group was 91±11 IU/L, p<0.005 'Showing that the PZA group produced biochemical liver damage; the concentration of transaminase in BNPP_PZA serum was significantly lower than that in the PZA group. 47 201236678 2. Histopathology After 5 weeks of intraperitoneal injection of 500 mg/kg/day PZA rats, hepatotoxicity was successfully produced in vivo; in contrast, the liver structure in the control group was normal. As shown in Figure 12A, the hepatic cell line in the liver parenchyma of the control group is arranged in the reticular plate arranged from the central vein of the hepatic lobules, and the hepatic sinus (id) is in the hepatic sinus The anastomosing plates were found between the two groups; the tissue sections of the PZA group were as shown in Figure 12b. 'The hepatocytes around the central vein of the PZA group showed fragmentation and vacuolization, but there was no See signs of necrosis of liver cells. The degree of liver damage in the BNPP-PZA group was not significantly different from that in the control group (no results were shown). 3. The measurement of residual liver function is shown in Figure 13. The galactose single point (GSP) value of the control group and the PZA group is highly significant (GSP value of 260 soil 50 pg in the control group) The GSP value of the rats in the PZA group was 776±65 Mg/ml, ;? < 0.005). In addition, the GSP value of the rats in the BNPP-PZA group was 293±61 pg/ml, compared with the PZA group. There was a significant difference in height 2 < 〇. 〇〇 5); the GSP value of rats administered PZA alone was significantly increased; however, rats in PZA combined with BNPP were resistant to this change; whereas the control group and BNPP-PZA There was no significant difference in GSP values between the groups. Example 6. Isonicotinic Anhydride (INH) and/or Ribomycin (RIF) and/or Propylsulfonamide (PZA) combined with CYP2E1 inhibitor Kaempferol or indoleamine hydrolase Animal test of (amidase) inhibitor quercetin (Quercetin) I. Materials and methods 1. Test materials All organic solvents were HPLC grade, purchased from Tedia Co., Ltd. (Fairfield, OH, USA), INH, RIF, PZA Kaempfero Bu Quercetin was purchased from Sigma Chemical Company (st. Louis, MO USA), and the galactose injection solution was prepared by Nanguang Chemical Pharmaceutical Co., Ltd., which dissolved 400 g of galactose (Sigma) in 1 liter of a system containing a suitable buffer solution. Isotonic salt distilled water 48 § 201236678 for injection. 2. Test animals 129/SVaj weighing 18 25 grams, the mouse was purchased from the National Institutes of Health, 8r,

Gonzalez(美國）’引進公鼠3隻，母鼠4隻後’自行配對繁殖，動物實驗係遵 照國細h動物實驗指南進行，所有的小氣均置於空氣/濕度調節環境下，光 ‘，、、與黑暗各I2小時，水及铺的供給不限，在試驗綱小鼠體重均持續監 測所有的小鼠均以使用***進行麻醉，並以眼寫方式進行半乳糖注射給 藥，60分鐘後由尾靜脈採血測Gsp值。 3. 試驗處理 試驗動物隨機分成7組，每組包括5種處理，第一種處理為注射Gonzalez (USA) introduced 3 male rats, 4 females, and then self-matched breeding. The animal experiment was carried out in accordance with the national animal experiment guide. All the small gas was placed in the air/humidity adjustment environment, For 12 hours with the darkness, the water and the supply of the shop were not limited. All the mice in the test group were continuously monitored for anesthesia with diethyl ether, and galactose injection was administered by eye writing, 60 minutes later. The Gsp value was measured by blood sampling from the tail vein. 3. Test treatment The test animals were randomly divided into 7 groups, each group consisting of 5 treatments, the first treatment was injection.

Kaempferol3.78mg/kg或其基劑(vehicle，VEH1，即食鹽水），以1 ml/kg的體 積進行腹腔内/主射至小鼠體内；第二種處理為則注射加似如 或其基劑（VEH2，即食鹽水），Quercetin係溶於食鹽水(〇 9% Na⑶中以i ml/kg的體積進行腹腔内注射至小鼠體内；第三種處理為注射 或INH之基劑(VEH3 ’即食鹽水），ink係溶於食鹽水(〇9%聽丨)中，以{ ml/kg的體積進行腹腔内注射至小鼠體内；第四種處理為注射励叫知聊 或其基劑(VEH4，即食鹽水），WF係溶於食鹽水(〇9%他⑶中，以1 m丨/kg 的體積進行腹腔内注射至小鼠體内；第五種處理為注射25〇mg/kgPZA或其 基劑(VEH5，即食鹽水），PZA係溶於食鹽水(〇 9%NaC丨)中，以丨ml/kg的體 積進行腹腔内注射至小鼠體内。 上述5組試驗共包含： (1) 對照組(normal control group，NC，n= 10):正常的小鼠每天注 射1次VEH1、VEH2、VEH3、VEH4以及VEH5(施行腹腔内 注射)共21天； (2) INH-RIF組(INH-RIF, n=10):正常的小鼠每天注射1次INH、 RIF、VEm、VEH2以及VEH5 (施行腹腔内注射)共21天； 201236678 (3) Kaempferol-INH-RIF組(Kaempferol-INH-RIF，n=10):正常的 小鼠每天注射 1 次 1(^11^€61*〇1、1>111、反117、\^112以及'\^^15(施 行腹腔内注射)共21天； (4) Quercetin-INH-RIF 組(Quercetin-INH-RIF, n=10):正常的小鼠 每天注射1次Quercetin、INH、RIF、VEH1以及VEH5(施行腹 腔内注射)共21天； (5) INH-RIF-PZA組(INH-RIF-PZA，n=10):正常的小鼠每天注射 1次INH、RIF、PZA、VEH1以及VEH2 (施行腹腔内注射)共 21天； (6) Kaempferol-INH-RIF-PZA 組（Kaempferol-INH-RIF-PZA， n=10):正常的小鼠每天注射1次Kaempferol、INH、RIF、PZA 以及VEH2(施行腹腔内注射)共21天； (7) Quercetin-INH-RIF-PZA組(Quercetin-INH-RIF-PZA，n=l〇): 正常的小鼠每天注射1次Quercetin、INH、RIF、PZA以及 VEH1(施行腹腔内注射)共21天； 4.血液樣本 處理完畢後，’小鼠以***麻醉，血液由小鼠心臟採血，置於含有Heparin 之試管中’血漿(plasma)以13，000g於4°C離心10分鐘，分離後的血漿分裝到 微量小管(Eppendorf tube)中並置於-80°C中儲存* 5·生化分析 肝細胞損傷以量測血漿中天門冬氨酸轉胺酶(AST)與丙氨酸轉胺酶 (ALT)活性以進行定量’ AST與ALT活性是肝臟毒性常用的指標，係以Kaempferol 3.78mg/kg or its base (vehicle, VEH1, ready-to-feed saline), intraperitoneal/mainly injected into the mouse in a volume of 1 ml/kg; the second treatment is injection plus or like Agent (VEH2, ready-to-feed saline), Quercetin is dissolved in saline (〇9% Na(3) in a volume of i ml/kg for intraperitoneal injection into mice; the third treatment is injection or INH base (VEH3) 'Present saline,' is dissolved in saline (〇9% 丨), injected intraperitoneally into mice in a volume of { ml/kg; the fourth treatment is injection stimulation or its basis (VEH4, ready-to-feed saline), WF is dissolved in saline (〇9% (3), intraperitoneal injection into mice in a volume of 1 m丨 / kg; the fifth treatment is injection 25 〇 mg / kgPZA or its base (VEH5, saline), PZA is dissolved in saline (〇9% NaC丨) and intraperitoneally injected into mice in a volume of 丨ml/kg. : (1) Control group (normal control group, NC, n= 10): normal mice were injected with VEH1, VEH2, VEH3, VEH4, and VEH5 (administered intraperitoneally) once a day for 21 days. (2) INH-RIF group (INH-RIF, n=10): Normal mice were injected with INH, RIF, VEm, VEH2, and VEH5 (administered intraperitoneally) once a day for 21 days; 201236678 (3) Kaempferol- INH-RIF group (Kaempferol-INH-RIF, n=10): Normal mice were injected once a day 1 (^11^€61*〇1, 1>111, anti117, \^112, and '\^^ 15 (administration of intraperitoneal injection) for 21 days; (4) Quercetin-INH-RIF group (Quercetin-INH-RIF, n=10): Normal mice were injected with Quecelin, INH, RIF, VEH1 and VEH5 once a day ( Intraperitoneal injection for a total of 21 days; (5) INH-RIF-PZA group (INH-RIF-PZA, n=10): normal mice were injected once daily with INH, RIF, PZA, VEH1, and VEH2 (peritoneal cavity) Intra-injection) for 21 days; (6) Kaempferol-INH-RIF-PZA group (Kaempferol-INH-RIF-PZA, n=10): Normal mice were injected with Kaempferol, INH, RIF, PZA and VEH2 once a day ( Intra-abdominal injection for a total of 21 days; (7) Quercetin-INH-RIF-PZA group (Quercetin-INH-RIF-PZA, n=l〇): Normal mice were injected once a day for Quercetin, INH, RIF, PZA And VEH1 (administered intraperitoneal injection) for 21 days; 4. After the blood sample is processed, 'mouse The mixture was anesthetized with ether, blood was collected from the heart of the mouse, and the plasma was placed in a Heparin-containing tube. The plasma was centrifuged at 13,000 g for 10 minutes at 4 ° C. The separated plasma was dispensed into a small tube (Eppendorf tube) and placed. Storage at -80 °C * 5 · Biochemical analysis of hepatocyte injury to measure plasma aspartate transaminase (AST) and alanine transaminase (ALT) activity for quantification 'AST and ALT activity is liver Commonly used indicators of toxicity

Synchron LXi 725 系統來量測(Beckman Instruments,美國）。 6.光學顯微鏡與電子顯微鏡 小鼠犧牲後肝臟隨即進行組織學分析；肝臟樣本以1〇%磷酸緩衝液配製 之福馬林(phosphate-bufferedformalin)固定，隨後脫水並包埋於石蠟 50 201236678 中以5叫厚度切片’切片樣本以蘇木精(hemat〇xylin)與伊紅(eosin)染色， 並進行肝糖染色試驗(Periodic acid Schiff stain，PAS)，染色後以光學顯微鏡 進行組織學觀察；另外，肝臟切片以二甲胂緩衝液(cac〇dylate buffer, 〇 1M pH 7.4)’青洗’以2〇%四氧化餓水溶液(叫此〇仍。啦⑴爪tetr〇xide)後固定1小 時’以酒精連續脫水後包埋於SpUrr樹脂(SpUrrresin)中，並以鑽石刀切取超 薄切片，以醋酸鈾醢(uranylacetate)及檸檬酸鉛(leadcitrate)作雙重染色，並 以穿透式電子顯微鏡(Transmission Electron Microscope, Hitachi 600, HitachiThe Synchron LXi 725 system was measured (Beckman Instruments, USA). 6. Light microscopy and electron microscopy The liver of the mice was sacrificed immediately after histological analysis; the liver samples were fixed with phosphate-buffered formalin in 1% phosphate buffer, then dehydrated and embedded in paraffin 50 201236678 to 5 Thick slice 'slice samples were stained with hemat〇xylin and eosin, and subjected to periodic acid Schiff stain (PAS), stained and histologically observed by light microscopy; Liver sections were fixed with caffeine buffer (cac〇dylate buffer, 〇1M pH 7.4), 'green wash' with 2%% tetrahydrogenated aqueous solution (called 〇 still. (1) claw tetr〇xide) and fixed for 1 hour. Alcohol was continuously dehydrated and embedded in SpUrr resin (SpUrrresin), and ultrathin sections were cut with a diamond knife. Double staining with uranylacetate and leadcitrate, and transmission electron microscopy (Transmission) Electron Microscope, Hitachi 600, Hitachi

Co.,曰本)觀察。 7. 肝功能之定量測試 所有的小鼠均進行半乳糖單點法(GSP)測試，小鼠接受在3〇秒内的快速 眼窩注射，注射〇.4g/ml BW半乳糖溶液〇_5 g/kg;自注射後60分鐘採血一次， 血液樣本取自尾部靜脈；以半乳糖脫氩酶比色法(c〇l〇rjmetric gaiact〇se dehydrogenase)量測半乳糖含量，測試濃度範圍為50至ιοοο μβ/ηι卜每個濃 度的日内差異(within-day variation)係由標準偏差(standard deviation)以及變 異係數(coefficientofvariation，CV)百分比計算，最大容許的變異係數為1〇% CV ;曰間差異(day-to-day variation)則由比較校正曲線(calibration curves)之 斜率及戴距來檢驗；半乳糖單點法(GSP)為30秒注射停止後60分鐘時血液中 半乳糖濃度。 8. 統計分析 所有的數據皆以平均士標準偏差(SD)表示，試驗結果以單因子變異數分 析(ANOVA)測試法來計算是否具有統計上的顯著差異，使用別此出㈤Co., 曰本) observation. 7. Quantitative test of liver function All mice were tested for galactose single point (GSP). The mice received rapid eye socket injection within 3 sec seconds, and injected with 〇4 g/ml BW galactose solution 〇5 g /kg; blood was taken once 60 minutes after injection, blood samples were taken from the tail vein; galactose content was measured by galactose de-arsenase colorimetric method (c〇l〇rjmetric gaiact〇se dehydrogenase), the test concentration range was 50 to Ιοοο μβ/ηι The intra-day variation of each concentration is calculated from the standard deviation and the coefficient of variation (CV). The maximum allowable coefficient of variation is 1〇% CV; The (day-to-day variation) was tested by comparing the slope of the calibration curves and the wearing distance; the galactose single point method (GSP) was the concentration of galactose in the blood at 60 minutes after the 30 second injection was stopped. 8. Statistical Analysis All data are expressed in terms of mean ± standard deviation (SD). The test results are calculated by the single factor analysis (ANOVA) test to determine whether there is a statistically significant difference.

Package of the Social Science program (Version 13, SPSS Inc.)套裝軟體來古十 算；隨後使用事後比較(post hoc test)最小差異顯著性〇east significant difference)方法做多重比較，以確認族群間的顯著差異；族群平均之顯著差 異為P<0.05。 201236678 一、結果 ι·生化分析結果 試驗結束時，糧試驗小鼠的體重及相對肝重量，與賴組動物相較 下並…、員著差異，生化分析結果如(圖十四及表十)所示，當小鼠以5〇/1〇〇 mg/kg/day連續給予3週厕後，麵瓣控制組血漿中的天門冬氨酸 轉胺酶(AST)與丙氨酸轉胺酶(ALT)活性明顯高於空白對照組(空白對照組 血漿中的AST活性為9〇±15 IU/L;INH/RIF控制組血漿中的AST活性為571 295 IU/L,p < 〇‘〇〇ι，空白對照組血漿中的Α|^τ活性為4〇 土 $ ιυ/L ; ^'^^控制組血漿中的八^活性為祕土脱阳几^⑽叫顯示 INH/RIF的確對小鼠造成生化程度上的肝損傷；而以5〇/1〇〇/25〇mg/kg/day 連續給予3週INH/RIF/PZA的小鼠，INH/RIF/PZA控制組血漿中的AST和 ALT活性分別為702 ± 172 IU/L及464 ± 72IU/L，顯著高於空白對照組及 INH/RIF控制組，顯示inh/RIF/PZA的確對小鼠造成生化程度上的肝損傷， 且損傷程度高於INH/RIF造成的傷害；同時給予CYP2E1抑制劑Kaempferol 或醯胺水解酶抑制劑Quercetin的Quercetin-INH-RIF、Package of the Social Science program (Version 13, SPSS Inc.) package software to the ancient ten calculation; then use the post hoc test significanteast significant difference) method to make multiple comparisons to confirm the significant between the groups Difference; significant difference in ethnic mean was P < 0.05. 201236678 I. Results ι·Biochemical analysis results At the end of the experiment, the body weight and relative liver weight of the grain test mice were compared with those of the Lai group, and the differences were observed. The biochemical analysis results were as shown in Fig. 14 and Table 10. As shown, aspartic acid transaminase (AST) and alanine transaminase in the plasma of the face flap control group were given to the mice at 5 〇/1 〇〇mg/kg/day for 3 weeks. ALT) activity was significantly higher than that of the blank control group (AST activity in the blank control group was 9〇±15 IU/L; AST activity in the plasma of the INH/RIF control group was 571 295 IU/L, p < 〇'〇 〇ι, the 对照组|^τ activity in the plasma of the blank control group was 4 〇 $ L ; ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ Mice caused biochemical liver damage; while mice given 3 weeks of INH/RIF/PZA at 5〇/1〇〇/25〇mg/kg/day, AST in plasma of INH/RIF/PZA control group The ALT activity was 702 ± 172 IU/L and 464 ± 72 IU/L, respectively, which was significantly higher than that of the blank control group and the INH/RIF control group, indicating that inh/RIF/PZA did cause biochemical liver damage in mice, and degree of damage Higher than the damage caused by INH/RIF; simultaneously given the CYP2E1 inhibitor Kaempferol or the indole hydrolase inhibitor Quercetin's Quercetin-INH-RIF,

Kaempferol-INH-RIF ' Quercetin-INH-RIF-PZA ' Kaempferol-INH-RJF-PZA 實驗組血清中轉胺酶濃度則接近正常值。 S. 52 201236678 表十、為對照組、ΙΝΗ-RIF 組、KH-INH-RIF 組、KM-INH-RIF 組、KL-INH-RIF 組、 MH-INH-RIF 組、MM-INH-RIF 組及 ML-INH-RIF 組 Total HAI score、天門冬 氨酸轉胺酶(AST)與丙氨酸轉胺酶(ALT)活性分析，數值之計算為mean ±SD。 Liver function parameters AST(IU/L) ALT(IU/L) Total HAI score Normal control (n=9) 80 土 13 46 土 10 0.0 + 0.0 ΙΝΗ-RIF (n=8) 420 + 66 358 + 67 5.3 ±2.2 KH-INH-RIF (n=8) 93 土 12*** 60 ±12*** 1.8+ 0.7* KM-INH-RIF (n=6) % ± 15*** 77士30*** 1.7 土 0.8* KL-INH-RIF (n=6) 111土27*** 128土36*** 2.8 ± 1.3* MH-INH-RIF(n=8) 93 土 12 *** 54 ±18*** 0.8 ±0.5*** MM-INH-RIF(n=6) 85 士 16*** 52 土12*** 0.7 土 0.8*** ML-INH-RIF(n=6) 154 + 62* 119 + 55** 2.0 土 0.6* Data are shown as mean 土 SD. */;<0.05, **/><0.01, ***p<0.005: Study compare to control group. 2.組織病理學 經過為期三週施行腹腔注射50/100 mg/kg/day INH/RIF及50/100/250 mg/kg/dayINH/RIF/PZA之小鼠，其體内確實成功的產生肝毒性；相對的， 在空白對照組小鼠體内的肝結構則較正常；相較於組，The concentration of transaminase in the serum of Kaempferol-INH-RIF 'Quercetin-INH-RIF-PZA ' Kaempferol-INH-RJF-PZA experimental group was close to normal. S. 52 201236678 Table 10, control group, sputum-RIF group, KH-INH-RIF group, KM-INH-RIF group, KL-INH-RIF group, MH-INH-RIF group, MM-INH-RIF group And ML-INH-RIF group Total HAI score, aspartate transaminase (AST) and alanine transaminase (ALT) activity analysis, the value is calculated as mean ± SD. Liver function parameters AST(IU/L) ALT(IU/L) Total HAI score Normal control (n=9) 80 Soil 13 46 Soil 10 0.0 + 0.0 ΙΝΗ-RIF (n=8) 420 + 66 358 + 67 5.3 ± 2.2 KH-INH-RIF (n=8) 93 Soil 12*** 60 ±12*** 1.8+ 0.7* KM-INH-RIF (n=6) % ± 15*** 77士30*** 1.7 Soil 0.8* KL-INH-RIF (n=6) 111 soil 27*** 128 soil 36*** 2.8 ± 1.3* MH-INH-RIF(n=8) 93 soil 12 *** 54 ±18** * 0.8 ±0.5*** MM-INH-RIF(n=6) 85 士16*** 52 土12*** 0.7 土0.8*** ML-INH-RIF(n=6) 154 + 62* 119 + 55** 2.0 soil 0.6* Data are shown as mean soil SD. */;<0.05, **/><0.01, ***p<0.005: Study compare to control group. 2. Histopathology After three weeks of intraperitoneal injection of 50/100 mg/kg/day INH/RIF and 50/100/250 mg/kg/day INH/RIF/PZA, the mice did successfully produce hepatotoxicity; in contrast, The liver structure in the blank control mice was normal; compared with the group,

Kaempferol-INH-RIF 組、Quercetin-INH-RIF、Kaempferol-INH-RIF-PZA 組 和Quercetih-INH-RIF-PZA組小鼠中央靜脈周圍肝細胞較為完整，空泡明顯 較少，發炎細胞亦較少。 在評估肝臟病理組織切片嚴重程度的HAZ—score方面，小鼠在連續給予 INH/RIF 或 INH/RIF/PZA 3 週後，確實在 intralobular Degeneration and Focal Necrosis與Inflammati〇n這兩個項目得到積分，且在及 INH-RIF-PZA 控制組中發現有 piecemeai necr〇sis，而 組、Quercetin-INH-RIF 、 Kaempferol-INH-RIF-PZA 組和 Quercetin-INH-RIF-PZA組與INH/RIF控制組相比則有明顯的改善（圖十 53 201236678 六、圖十七）。 1·剩餘肝功能之量測 INH-RIF及INH-RIF-ΡΖΑ控制組的半乳糖單點法(GSP)值有隨著 INH/RIF給藥時間愈長而愈高的趨勢；空白對照組與及 INH-RIF-PZA控制組小鼠之GSP值具有高度的顯著差異(空白對照組小鼠 之 GSP 值為 177 ± 22 mg/L ;給藥 3 週後，INH-RIF 及 INH-RIF-PZA 小鼠之 GSP 值各為 866 ± 339 mg/L，p < 0.001 與 858 ± 172 mg/L，;? < 0.00卜此外， 在 Kaempferol-INH-RIF 組、Quercetin-INH-RIF、Kaempferol-INH-RIF-PZA 組和 Quercetin-INH-RIF-PZA 組小鼠之 GSP 值為 401 ± 178 mg/L、203 士 76 mg/L、273 ± 61 mg/L、216 ± 67 mg/L，與 INH-RIF 及 INH-RIF-PZA 控制組 小鼠具有高度的顯著差異(p < 0.001);顯示施用INH-RIF及INH-RIF-PZA 控制組小鼠之GSP值明顯增加；然而，併用Kaempferol或Quercetin之小 鼠則可抵抗這種改變，空白對照組與Kaempferol或Quercetin實驗組相比 時，小鼠之間的GSP值無顯著差異存在(圖十五）。 實施例七、異菸鹼醯胺(INH)及立復黴素(RIF)合併使用CYP2E1抑制劑山奈 盼（Kaempferol)、Mannitol、Saccharin、Sucralose、Dicalcium phosphate、Crospovidone 之動物試驗 一、材料與方法 1.試驗材料 所有的有機溶劑均為HPLC專級’購自Tedia有限公司(Fairfield，OH， USA) > INH ' RIF ' Kaempferol' Mannitol' Saccharin ' Sucralose > Dicalcium phosphate、Crospovidone購自 Sigma化學公司(St. Louis，MO USA)，半乳糖注 射溶液由南光化學製藥股份有限公司製備’係將400克半乳糖(Sigma)溶於1 公升含有適當緩衝溶液系統以及等張鹽類之蒸館水中，供作注射使用。 S. 54 201236678 2. 試驗動物 體重為18-25公克之i29/sv小鼠是購自美國國家衛生研究院教授!^ Gonzalez (美國）’引進公鼠3隻，母鼠4隻後，自行配對繁殖’動物實驗係遵 照國衛院動物實驗指南進行，所有的錢均置於线/濕度靖環境下，光 照與黑暗各12小時，水及鋪的供給稀，在試驗姻械體重均持續監 測，所有的小鼠均以使用***進行麻醉，並以眼窩方式進行半乳糖注射給 藥’ 60分鐘後由尾靜脈採血測GSP值。 3. 試驗處理 試驗動物隨機分成13組，每組包括3種處理，第一種處理為口服 Kaempferol 1.67mg/kg ’ 以0.1 mi/kg的體積給藥；或為口服Kaempfer〇1427 mg/kg 以0_1 ml/kg的體積給藥，或為口服Kaempfer〇i 8.33 mg/kg，以0.1 ml/kg 的體積給藥；或為口服Mannitol 0.17 mg/kg，以0.1 ml/kg的體積給藥；或為 口服Mannitol 0.83 mg/kg，以0.1 ml/kg的體積給藥；或為口服Mannit〇i 167 mg/kg ’ 以0.1 ml/kg的體積給藥；或為 口 Hg_saccharin 0.83 mg/kg，以0.1 ml/kg 的體積給藥；或為口服Sucralose l_67mg/kg，以0.1 ml/kg的體積給藥；或為 口服 Saccharin 0.83 mg/kg+Mannitol 0.83 mg/kg ,或為口服Dicalcium phosphate 0.83 mg/kg ’ 或處理為口服Crospovidone 2_83 mg/kg，第二種處理 為注射50 mg/kg INH或INH之基劑(VEH1，即食鹽水），INH係溶於食鹽水 (0.9% NaCl)中，以1 ml/kg的體積進行腹腔内注射至小鼠體内；第三種處理 為注射100 mg/kg RIF或其基劑(VEH2，即食鹽水），RIF係溶於食鹽水(〇 9〇/0 NaCl)中’以1 ml/kg的體積進行腹腔内注射至小鼠體内。 上述5組試驗共包含： (!) 對照組(normal control group, NC，n=l0):正常的小鼠每天注 射1次VEm、VEH2(施行腹腔内注射)共21天； (2) INH-RIF組(INH-RIF，n=10):正常的小鼠每天注射1次inh、 RIF (施行腹腔内注射)共21天； 55 201236678 (3) KL-INH-RIF組(n=8).正常的小鼠母天口服一次Kaempferol 1.67mg/kg，注射1次INH、RIF (施行腹腔内注射)共21天； (4) KM-INH-RIF 組(n=6):正常的小鼠每天口 服一次Kaempferol 4.17 mg/kg，注射1次INH、RIF (施行腹腔内注射)共21天； (5) KH-INH-RIF 組(n=6):正常的小鼠每天口 服一次Kaempferol 8.33 mg/kg，注射1次INH、RIF (施行腹腔内注射)共21天； (6) ML-INH-RIF組(n=8):正常的小鼠每天口服一次Mannitol 0.17 mg/kg ’注射1次INH、RIF (施行腹腔内注射)共21天； (7) MM-INH-RIF組(n=6):正常的小鼠每天口服一次Mannitol 0.83 mg/kg，注射1次INH、RIF (施行腹腔内注射)共21天； (8) MH-INH-RIF組(n=6):正常的小鼠每天口服一次Mannitol 1.67 mg/kg，注射1次INH、RIF (施行腹腔内注射)共21天； (9) SA-INH-RIF 組(n=4) ··正常的小鼠每天口服一次 Saccharin 0.83 mg/kg，注射1次INH、RIF (施行腹腔内注射)共21天； (10) SU-INH-RIF 組(n=4) ♦正常的小鼠每天口服一次 Sucralose 1.67 mg/kg，注射1次INH、RIF (施行腹腔内注射)共21天； (11) SAM-INH-RIF組(11==4):正常的小鼠每天口服一次Saccharin 0.83 mg/kg+Mannitol 0.83 mg/kg，注射 1 次INH、RIF (施行腹 腔内注射)共21天； (12) D-INH-RIF組(n=4):正常的小鼠每天口服一次Dicalcium phosphate 0.83 mg/kg，注射1次INH、RIF (施行腹腔内注射) 共21天； (13) C-INH-RIF組(n=4):正常的小鼠每天口服一次Crospovidone 2.83 mg/kg，注射1次INH、RIF (施行腹腔内注射)共21天； 4.血液樣本 處理完畢後’，小鼠以乙謎麻醉’血·液由小鼠心臟採血’置於含有HeparinThe Kaempferol-INH-RIF group, the Quercetin-INH-RIF, the Kaempferol-INH-RIF-PZA group, and the Querceteh-INH-RIF-PZA group had relatively complete hepatocytes around the central vein, with less vacuoles and more inflammatory cells. less. In the HAZ-score aspect of assessing the severity of liver histological sections, the mice did score points in the intralobular Degeneration and Focal Necrosis and Inflammati〇n after 3 weeks of continuous administration of INH/RIF or INH/RIF/PZA. And piecemeai necr〇sis was found in the INH-RIF-PZA control group, while the group, Quercetin-INH-RIF, Kaempferol-INH-RIF-PZA group and Quercetin-INH-RIF-PZA group and INH/RIF control group Compared with the obvious improvement (Figure 10 53 201236678 VI, Figure 17). 1. Measurement of residual liver function The galactose single point method (GSP) values in the INH-RIF and INH-RIF-ΡΖΑ control groups have a higher tendency as the INH/RIF administration time is longer; the blank control group and The GSP values of the mice in the INH-RIF-PZA control group were highly significant (GSP values of 177 ± 22 mg/L in the blank control group; INH-RIF and INH-RIF-PZA after 3 weeks of administration) The GSP values of the mice were 866 ± 339 mg/L, p < 0.001 and 858 ± 172 mg/L, respectively; < 0.00 b, in the Kaempferol-INH-RIF group, Quercetin-INH-RIF, Kaempferol- The GSP values of the INH-RIF-PZA and Quercetin-INH-RIF-PZA mice were 401 ± 178 mg/L, 203 ± 76 mg/L, 273 ± 61 mg/L, 216 ± 67 mg/L, and The INH-RIF and INH-RIF-PZA control groups showed a highly significant difference (p <0.001); the GSP values of the mice administered the INH-RIF and INH-RIF-PZA control groups were significantly increased; however, Kaempferol was used in combination. Or Quercetin mice were resistant to this change. There was no significant difference in GSP values between the blank control group and the Kaempferol or Quercectin experimental group (Fig. 15). Example 7 Indoleamine (INH) and rifamycin (RIF) combined with CYP2E1 inhibitor Kaempferol, Mannitol, Saccharin, Sucralose, Dicalcium phosphate, Crospovidone animal test I. Materials and methods 1. Test materials All organic solvents All were HPLC grade 'purchased from Tedia Co., Ltd. (Fairfield, OH, USA) > INH ' RIF ' Kaempferol ' Mannitol ' Saccharin ' Sucralose > Dicalcium phosphate, Crospovidone was purchased from Sigma Chemical Company (St. Louis, MO USA) The galactose injection solution was prepared by Nanguang Chemical Pharmaceutical Co., Ltd., and 400 g of galactose (Sigma) was dissolved in 1 liter of steaming water containing a suitable buffer solution system and isotonic salts for injection. S. 54 201236678 2. The experimental animals weighed 18-25 grams of i29/sv mice were purchased from the National Institutes of Health professors! ^ Gonzalez (USA) 'Introduction of 3 male rats, 4 mothers, self-matching breeding 'animals The experiment was conducted in accordance with the guidelines of the National Animal Health Laboratory. All the money was placed in the line/humidity environment, and the light and darkness were each 12 hours. The water and the supply of the shop were thin. All the animals were continuously monitored for weight. All mice were anesthetized with ether and administered with galactose in the orbital manner. After 60 minutes, the GSP was measured by blood sampling from the tail vein. 3. Test treatment The test animals were randomly divided into 13 groups, each group consisting of 3 treatments, the first treatment was oral Kaempferol 1.67 mg/kg 'administered in a volume of 0.1 mi / kg; or the oral Kaempfer 〇 1427 mg / kg Dosage in a volume of 0_1 ml/kg, or oral administration of Kaempfer〇i 8.33 mg/kg, in a volume of 0.1 ml/kg; or oral Mannitol 0.17 mg/kg, in a volume of 0.1 ml/kg; or Oral Mannitol 0.83 mg / kg, administered in a volume of 0.1 ml / kg; or oral Mannit〇i 167 mg / kg ' in a volume of 0.1 ml / kg; or mouth Hg_saccharin 0.83 mg / kg, to 0.1 Dosage in ml/kg; or oral Sucralose l_67mg/kg in 0.1 ml/kg; or oral Saccharin 0.83 mg/kg+Mannitol 0.83 mg/kg, or oral Dicalcium phosphate 0.83 mg/kg ' Or treatment with oral Crospovidone 2_83 mg/kg, the second treatment is 50 mg/kg INH or INH base (VEH1, ready-to-feed saline), and INH is dissolved in saline (0.9% NaCl) to 1 ml The volume of /kg is intraperitoneally injected into mice; the third treatment is injection of 100 mg/kg RIF or its base (VEH2, ready to eat) In saline, RIF was dissolved in saline (〇 9〇/0 NaCl) and intraperitoneally injected into the mice in a volume of 1 ml/kg. The above five groups of tests included: (!) Control group (normal control group, NC, n=l0): Normal mice were injected with VEm and VEH2 once a day for intrauterine injection for 21 days; (2) INH- RIF group (INH-RIF, n=10): Normal mice were injected with inh, RIF (administered intraperitoneally) once a day for 21 days; 55 201236678 (3) KL-INH-RIF group (n=8). Normal mice were orally administered with Kaempferol 1.67 mg/kg once daily, 1 time with INH and RIF (administered intraperitoneally) for 21 days; (4) KM-INH-RIF group (n=6): normal mice daily Oral Kaempferol 4.17 mg/kg, injection of INH, RIF (administered intraperitoneally) for 21 days; (5) KH-INH-RIF group (n=6): normal mice were orally administered Kaempferol 8.33 mg/day. Kg, injection of INH, RIF (administered intraperitoneally) for 21 days; (6) ML-INH-RIF group (n=8): normal mice once daily oral Mannitol 0.17 mg / kg 'injection 1 INH RIF (administration of intraperitoneal injection) for 21 days; (7) MM-INH-RIF group (n=6): normal mice were orally administered Mannitol 0.83 mg/kg once a day, once with INH, RIF (peritoneal intraperitoneal) Injection) for 21 days; (8) MH-INH-RIF group (n=6): normal mice were taken orally every day Mannitol 1.67 mg/kg, 1 injection of INH, RIF (administered intraperitoneally) for 21 days; (9) SA-INH-RIF group (n=4) · Normal mice once daily oral Saccharin 0.83 mg / Kg, injection of INH, RIF (administration of intraperitoneal injection) for 21 days; (10) SU-INH-RIF group (n=4) ♦ normal mice once daily oral Sucralose 1.67 mg / kg, injection of INH once RIF (administration of intraperitoneal injection) for 21 days; (11) SAM-INH-RIF group (11==4): normal mice were given Saccharin 0.83 mg/kg+Mannitol 0.83 mg/kg once a day, once a day. INH, RIF (administration of intraperitoneal injection) for 21 days; (12) D-INH-RIF group (n=4): normal mice were given Oral Dicalcium phosphate 0.83 mg/kg once a day, once injection of INH, RIF (administration) Intra-abdominal injection) for 21 days; (13) C-INH-RIF group (n=4): normal mice were given Oral Crospovidone 2.83 mg/kg once a day, once with INH, RIF (administered intraperitoneally) 21 Day; 4. After the blood sample is processed, 'the mouse is anesthetized with a mystery 'Blood Liquid' from the heart of the mouse' placed in Heparin

S 56 201236678 之試管中，血漿(plasma)以13,000g於4°C離心10分鐘，分離後的血漿分裝到 微量小管(Eppendorftube)中並置於-80°C中儲存。 5.生化分析 肝細胞損傷以量測企漿中天門冬氨酸轉胺酶(AST)與丙氨酸轉胺酶 (ALT)活性以進行定量，AST與ALT活性是肝臟毒性常用的指標，係以 Synchron LXi 725 系統來量測(Beckman Instruments,美國）。 6. 光學顯微鏡與電子顯微鏡 小鼠犧牲後肝臟隨即進行組織學分析；肝臟樣本以10%磷酸緩衝液配製 之福馬林(phosphate-buffered formalin)固定，隨後脫水並包埋於石蠟(paraffin) 中’以5 μηι厚度切片’切片樣本以蘇木精(hematoxylin)與伊紅(eosin)染色， 並進行肝糖染色試驗(Periodic acid Schiff stain，PAS)，染色後以光學顯微鏡 進行組織學觀察，另外’肝臟切片以二甲肿緩衝液(cac〇dyiate buffer，0.1M pH 7·4)清洗，以20%四氧化餓水溶液(aqueous osmium tetroxide)後固定1小 時’以酒精連續脫水後包埋於Spurr樹脂(Spurrresin)中，並以鑽石刀切取超 薄切片，以醋酸鈾醯(uranylacetate)及檸檬酸鉛(leadcitrate)作雙重染色，並 以穿透式電子顯微鏡(Transmission Electron Microscope, Hitachi 600, Hitachi Co”日本)觀察。 7. 肝功能之定量測試 所有的小鼠均進行半乳糖單點法(GSP)測試，小鼠接受在3〇秒内的快速 眼窩注射，注射0.4g/mlBW半乳糖溶液〇.5g/kg;自注射後60分鐘採血一次， 血液樣本取自尾部靜脈；以半乳糖脫氫酶比色法(c〇1〇rimetric gahct〇se dehydrogenase)量測半乳糖含量，測試濃度範圍為別至^⑻叫加卜每個濃 度的曰内差異(within-day variation)係由標準偏差(standard deviati〇n)以及變 異係數(coefficient 〇fvariati〇n，Cv)百分比計算，最大容許的變異係數為1〇% CV ;日間差異(day-to-day variation)則由比較校正曲線㈣ibrati〇n curves)之 斜率及飾錄驗；|乳料點法(GSP)⑽秒錄停止後齡鐘時血液中 57 201236678 半乳糖濃度。 8.統計分析 所有的數據皆以平均土標準偏差(SD)表示，試驗結果以單因子變異數分 析(ANOVA)測試法來計算是否具有統計上的顯著差異，使用沿扯丨此㈤In a test tube of S 56 201236678, plasma was centrifuged at 13,000 g for 10 minutes at 4 ° C, and the separated plasma was dispensed into a small tube (Eppendorf tube) and stored at -80 ° C. 5. Biochemical analysis of hepatocyte injury to measure aspartate aminotransferase (AST) and alanine transaminase (ALT) activity in the laboratory for quantification, AST and ALT activity are commonly used indicators of liver toxicity, Measured with the Synchron LXi 725 system (Beckman Instruments, USA). 6. Light microscopy and electron microscopy Mice were sacrificed immediately after histological analysis of the liver; liver samples were fixed in phosphate-buffered formalin in 10% phosphate buffer, then dehydrated and embedded in paraffin (paraffin) The sliced samples were sliced with 5 μηι thickness and stained with hematoxylin and eosin, and subjected to periodic acid Schiff stain (PAS). After staining, histological observation was performed by light microscopy. The liver sections were washed with cac〇dyiate buffer (0.1 M pH 7.·4) and fixed with 20% aqueous osmium tetroxide for 1 hour. The alcohol was continuously dehydrated and embedded in Spurr resin. (Spurrresin), ultra-thin sections were cut with a diamond knife, double stained with uranylacetate and leadcitrate, and transmitted by electron microscopy (Transmission Electron Microscope, Hitachi 600, Hitachi Co) Japan) Observations 7. Quantitative testing of liver function All mice were tested for galactose single point (GSP) and mice received fast eye sockets within 3 sec. Shoot, inject 0.4g/ml BW galactose solution 〇.5g/kg; take blood once 60 minutes after injection, blood sample from tail vein; galactose dehydrogenase colorimetry (c〇1〇rimetric gahct〇se dehydrogenase Measure the galactose content, the concentration range of the test is different from ^(8). The in-day variation of each concentration is the standard deviation (standard deviati〇n) and the coefficient of variation (coefficient 〇fvariati〇n) , Cv) Percentage calculation, the maximum allowable coefficient of variation is 1〇% CV; the day-to-day variation is determined by the slope of the comparison correction curve (4) ibrati〇n curves); GSP) (10) seconds recorded in the blood at the age of 58. 201236678 galactose concentration 8. Statistical analysis All data are expressed in terms of mean soil standard deviation (SD), the test results are measured by the single factor analysis of variance (ANOVA) test Calculate whether there is a statistically significant difference, use the edge along this (five)

Package of the Social Science program (Version 13, SPSS Inc.)套裝軟體來叶 算；隨後使用事後比較(post hoc test)最小差異顯著性伽诚significam difference)方法做多重比較，以確認族群間的顯著差異；族群平均之顯著差 異為P<0.05。 二、結果 1 ·生化分析結果 試驗結束時，測量試驗小鼠的體重及相對肝重量，與對照組動物相較 之下並無顯著差異；生化分析結果如表十一所示，當小鼠以⑻ mg/kg/day連續給予3週!nh/RIF後，；[NH/rjf控制組血漿中的天門冬氨酸 轉胺酶(AST)與丙氨酸轉胺酶(ALT)活性明顯高於空白對照組(空白對照組 血漿中的AST活性為80±13 IU/L;INH/RIF控制組血漿中的AST活性為42〇 ± 66 IU/L，p < 0.001 ;空白對照組血漿中的alt活性為牝± ι〇刃几； INH/RIF控制組血衆中的ALT活性為358 ± 67 IU/L，p < 〇._)，顯示 INH/RIF的確對小鼠造成生化程度上的肝損傷；同時給^ cyp2Ei抑制劑Package of the Social Science program (Version 13, SPSS Inc.) package software to calculate the leaf; then use the post hoc test to make multiple comparisons to confirm significant differences between ethnic groups. The significant difference in the population average was P < 0.05. II. Results 1 · Biochemical analysis results At the end of the experiment, the body weight and relative liver weight of the test mice were measured, and there was no significant difference compared with the control animals. The biochemical analysis results are shown in Table 11. (8) mg/kg/day for 3 weeks in a row! After nh/RIF, [the activity of aspartate aminotransferase (AST) and alanine transaminase (ALT) in plasma of NH/rjf control group was significantly higher than that. The blank control group (the AST activity in the plasma of the blank control group was 80±13 IU/L; the AST activity in the plasma of the INH/RIF control group was 42〇±66 IU/L, p <0.001; in the plasma of the blank control group The alt activity is 牝± ι〇 几; the ALT activity in the blood of the INH/RIF control group is 358 ± 67 IU/L, p < 〇._), indicating that INH/RIF does biochemically affect mice. Liver injury; simultaneously give ^ cyp2Ei inhibitor

Kaempferd、Mannitol各劑量實驗組、血清中轉胺酶濃度皆顯著低於 INH/RIF控制組。 2.組織病理學 經過為期三週施行腹腔注射50/100 mg/kg/day 之小鼠，其體内 確實成功的產生肝雜；姆的，在空⑽小鼠體_輯構則較正 常；相較於INH-RIF組，Mannitol各劑量實驗組小鼠中央靜脈厢肝細胞 較為完整，空泡明顯較少，發炎細胞亦較少(圖十八）。 在評估肝臟病理組織切片嚴重程度的腕·re方面，小鼠在連續給予The concentrations of transaminase in the Kaempferd and Mannitol dose groups were significantly lower than those in the INH/RIF control group. 2. Histopathology After 50 weeks of intraperitoneal injection of 50/100 mg/kg/day mice, the mice were indeed successful in producing hepatic miscellaneous; in the empty (10) mice, the body composition was normal; Compared with the INH-RIF group, the central venous hepatocytes of the Mannitol dose group were more complete, with less vacuoles and less inflammatory cells (Fig. 18). In the wrist re-evaluation of the severity of liver pathological tissue sections, the mice were given continuously

58 S 201236678 INH/RIF 3週後’ Kaempfero卜Mannitol各劑量實驗組與ΙΝΗ/RIF控制組相 比則有明顯的改善。 3.剩餘肝功能之量測 INH-RIF控制組的半乳糖單點法(GSp)值有隨著INH/RIF給藥時間愈長 而愈高的趨勢；空白對照組與INH-RIF控制組小鼠之GSP值具有高度的顯 著差異(空白對照組小鼠3週之GSP值為192 ± 18 mg/L :給藥3週後， INH-RIF 小鼠之 GSP 值為 666 ± 126 mg/L,尸 < 0.001，然而，Kaempferol、 Mannito卜 Saccharin、Sucralose、Dicalcium phosphate 各劑量實驗組之小鼠則 可抵抗這種改變’其中空白對照組與KH-INH-RIF組、KM-INH-RIF組、 MH-INH-RIF 組、MM-INH-RIF 組、SU-INH-RIF 組相比時，小鼠之間的 GSP 值無顯著差異存在(如表十一）。 GSP (mg/L) 0 weeks 2 weeks 3 weeks Anova and LSD 0-2 0-3 2-3 Normal control (n=4) 197 ±16 186 ±19 192 ± 18 ND ND ND INH-RIF (n=8) 201 ±23 472 ±128 666 ±126 <0.005 < 0.005 <0.01 KH-INH-RIF (n=8) 199 ± 19 195 ±41 254 ± 34 ND ND ND KM-INH-RIF (n=6) 195 ±26 221 ±17 262 ±33 ND ND ND KL-INH-RIF (n=6) 212 ±34 290 ± 43 327 土 50 <0.005 < 0.005 ND MH-INH-RIF (n=8) MM-INH-RIF (n=6) ML-INH-RIF (n=6) 196 ±22 208 ± 26 252 ± 24 ND ND ND 201 ±17 240 ± 29 237 ± 30 ND ND ND 188 ±26 287 ±28 300 ± 40 <0.01 <0.01 ND SA-INH-RIF(n=4) 199 + 21 269 ±40 2邻 ±28 ND ND ND SU-INH-RIF(n=4) 203 ± 19 300 ±31 399 ±22 <0.005 <0.005 <0.05 SAM-INH-RIF(n=4) 196 ±22 240 ±38 223 ± 29 ND ND ND D-INH-RIF(n=4) 208 ±25 249 ±35 366 ± 77 ND < 0.005 <0.01 C-INH-RIF(n=4) 193 ±7 330 ± 56 459 ± 76 < 0.005 < 0.005 ND Data are shown as mean ± SD* *ρ<〇·〇5, **/^0.01, /><0.005: Study compare to control group* 表十一、為對照組、INH-RIF 組、KH-INH-RIF 組、KM-INH-RIF 組、KL-INH-RIF 組、 MH-INH-RIF 組、MM-INH-RIF 組、ML-INH-RIF 組、SA-INH-RIF 組、 SU-INH-RIF 組、SAM-INH-RIF 組、D-INH-RIF 組及 C-rNH-RIF 組小鼠半乳糖 _卓點法(GSP)值，數值之計算為mean 土 SD。 59 201236678 實施例八、異菸鹼醯胺(INH)及立復黴素(RIF)及丙基硫異於醯胺(pzA)合併 使用CYP2E1抑制劑Mannitol之動物試驗 一、材料與方法 1.試驗材料 所有的有機溶劑均為HPLC等級，購自Tedia有限公司(Fairfieid, 〇H USA) ’ INH、RIF、PZA、Mannitol購自 Sigma化學公司(St L〇uis，M〇 USA)， 半乳糖注射溶液由南光化學製藥股份有限公司製備，係將4〇〇克半乳糖 (Sigma)溶於1公升含有適當緩衝溶液系統以及等張鹽類之蒸餾水中，供作注 射使用。 2. 試驗動物 體重為18-25公克之129/sv小鼠是購自美國國家衛生研究院教授1> G_lez(美國）’引進公鼠3隻，母鼠4隻後，自行配職殖，動物實驗係遵 照國衛院動物貫驗指南進行’所有的小鼠均置於空氣/濕度調節環境下，光 照與黑暗各12小時，水及補的供給不限，在試驗顧傾體重均持續監 測’所有的小鼠均以使用乙魏行麻醉，並以眼窩方式進行半乳糖注射給 藥’ 60分鐘後由尾靜脈採血測GSP值。 3. 試驗處理 試驗動物隨機分成3組，每組包括4種處理，第一種處理為口服為口服 Mannitd L67mg/kg ’以(M ml/kg的體積給藥；第二種處理為注射5〇mg/kg INH或INK之基劑(VEH1，即食鹽水），卿系溶於食鹽水(〇 9%NaCi)中以 1邊g的體積進行腹誠注射至錢體内；第三種處理為注射⑽ mg/kg 祕戈其基劑(VEffi，即食鹽水)，聊係溶於食鹽水(〇 9%齡)中以i _ 的體積進械朗轉到、鼠_ ;第四_理躲脚或其 基劑(VEH3，即食鹽水）’ PZA係溶於食鹽水㈣趟⑶中以丨爪丨域體 積進行腹腔内注射至小鼠體内。 201236678 上述3組試驗共包含： (!) 對照組(normal control group, NC，n=10):正常的小鼠每天注 射1次VEm、VEH2、VEH3 (施行腹腔内注射)共21天； (2) INH-RIF-PZA組(INH-RIF，n=6):正常的小鼠每天注射i次 INH、RIF、PZA (施行腹腔内注射)共21天； (3) M-INH-RIF-PZA組(n=6):正常的小鼠每天口 服一次Mannitol 1.67 mg/kg，注射1次INH、RIF、PZA (施行腹腔内注射)共21 天。 4. 血液樣本 處理完畢後，，小鼠以***麻醉’血液由小鼠心臟採血，置於含有Heparin 之試管中，血漿(plasma)以13,000g於4。(：離心10分鐘，分離後的血漿分裝到 微量小管(Eppendorf tube)中並置於-80。匚中儲存。 5. 肝功能之定量測試 所有的小鼠均進行半乳糖單點法(GSP)測試’大鼠接受在3〇秒内的快速 眼窩注射’注射0.4g/ml BW半乳糖溶液0.5 g/kg ;自注射後60分鐘採血一次， 血液樣本取自尾部靜脈，以半乳糖脫氫酶比色法(c〇l〇rimetric gaiact〇se dehydrogenase)量測半乳糖含量’測試濃度範圍為⑽至丨/㈨μ§/ηι卜每個濃 度的日内差異(within-day variation)係由標準偏差(standard deviation)以及變 異係數(coefficientofvariation，CV)百分比計算，最大容許的變異係數為10〇/〇 CV ;日間差異(day-to-day variation)則由比較校正曲線(calibration curves)之 斜率及截距來檢驗；半乳糖單點法(GSP)為30秒注射停止後6〇分鐘時血液中 半乳糖濃度。 6. 統計分析 所有的數據皆以平均土標準偏差(SD)表示，試驗結果以單因子變異數分 析(ANOVA)測試法來計算是否具有統計上的顯著差異，使用Statistical58 S 201236678 INH/RIF After 3 weeks, the Kaempfero Mannitol dose test group showed a significant improvement compared with the ΙΝΗ/RIF control group. 3. Measurement of residual liver function The galactose single point method (GSp) value of the INH-RIF control group has a higher tendency as the INH/RIF administration time is longer; the blank control group and the INH-RIF control group are small. The GSP values of the rats were highly significant (the GSP value of the blank control mice at 3 weeks was 192 ± 18 mg/L: after 3 weeks of administration, the GSP value of INH-RIF mice was 666 ± 126 mg/L, The corpse was 0.001, however, the mice in the Kaempferol, Mannito, Saccharin, Sucralose, and Dicalcium phosphate groups were resistant to this change. The blank control group and the KH-INH-RIF group, the KM-INH-RIF group, There was no significant difference in GSP values between the MH-INH-RIF group, the MM-INH-RIF group, and the SU-INH-RIF group (see Table XI). GSP (mg/L) 0 weeks 2 weeks 3 weeks Anova and LSD 0-2 0-3 2-3 Normal control (n=4) 197 ±16 186 ±19 192 ± 18 ND ND ND INH-RIF (n=8) 201 ±23 472 ±128 666 ±126 <0.005 < 0.005 <0.01 KH-INH-RIF (n=8) 199 ± 19 195 ±41 254 ± 34 ND ND ND KM-INH-RIF (n=6) 195 ±26 221 ±17 262 ±33 ND ND ND KL-INH-RIF (n=6) 212 ±34 290 ± 43 32 7 soil 50 <0.005 < 0.005 ND MH-INH-RIF (n=8) MM-INH-RIF (n=6) ML-INH-RIF (n=6) 196 ±22 208 ± 26 252 ± 24 ND ND ND 201 ±17 240 ± 29 237 ± 30 ND ND ND 188 ±26 287 ±28 300 ± 40 <0.01 <0.01 ND SA-INH-RIF(n=4) 199 + 21 269 ±40 2 adjacent ±28 ND ND ND SU-INH-RIF(n=4) 203 ± 19 300 ±31 399 ±22 <0.005 <0.005 <0.05 SAM-INH-RIF(n=4) 196 ±22 240 ±38 223 ± 29 ND ND ND D-INH-RIF(n=4) 208 ±25 249 ±35 366 ± 77 ND < 0.005 <0.01 C-INH-RIF(n=4) 193 ±7 330 ± 56 459 ± 76 < 0.005 < 0.005 ND Data are shown as mean ± SD* *ρ<〇·〇5, **/^0.01, /><0.005: Study compare to control group* Table XI, for the control group, INH- RIF group, KH-INH-RIF group, KM-INH-RIF group, KL-INH-RIF group, MH-INH-RIF group, MM-INH-RIF group, ML-INH-RIF group, SA-INH-RIF Groups, SU-INH-RIF group, SAM-INH-RIF group, D-INH-RIF group and C-rNH-RIF group were tested for galactose-based point method (GSP) value, and the value was calculated as mean soil SD. 59 201236678 Example VIII, Animal test of isoniazid amide (INH) and rifamycin (RIF) and propyl sulphide (pzA) combined with CYP2E1 inhibitor Mannitol I. Materials and methods 1. Test Materials All organic solvents were HPLC grade, purchased from Tedia Co., Ltd. (Fairfieid, 〇H USA) 'INH, RIF, PZA, Mannitol purchased from Sigma Chemical Company (St L〇uis, M〇USA), galactose injection solution Prepared by Nanguang Chemical Pharmaceutical Co., Ltd., 4 g of galactose (Sigma) was dissolved in 1 liter of distilled water containing a suitable buffer solution system and isotonic salts for injection. 2. Test animals weighing 1-25sg of 129/sv mice were purchased from the National Institutes of Health Professor 1> G_lez (USA)'s introduction of 3 male rats, 4 female rats, self-supporting animals, animals The experimental department followed the guidelines of the Animal Health Examination of the National Guardian Hospital. All mice were placed in an air/humidity environment, and the light and darkness were each 12 hours. The supply of water and supplements was not limited. All the mice were anesthetized with Weiwei and administered by galactose injection in the orbital manner. After 60 minutes, the GSP value was measured by blood sampling from the tail vein. 3. Test treatment The test animals were randomly divided into 3 groups, each group consisting of 4 treatments. The first treatment was oral administration of Mannitd L67 mg/kg '(M ml/kg volume; the second treatment was 5 injections) The base of mg/kg INH or INK (VEH1, ready-to-use saline) is dissolved in saline (〇9% NaCi) and injected into the body in a volume of 1 side g; the third treatment is injection. (10) mg/kg gef base (VEFfi, ready-to-feed salt water), which is dissolved in salt water (〇 9% age), and is transferred to the volume of i _, and the mouse is _; The base (VEH3, salt water) 'PZA is dissolved in saline (4) 趟(3) and intraperitoneally injected into the mice in the volume of the scorpion scorpion. 201236678 The above three groups of tests include: (!) Normal control group, NC, n=10): Normal mice were injected with VEm, VEH2, and VEH3 once daily (administered intraperitoneally) for 21 days; (2) INH-RIF-PZA group (INH-RIF, n= 6): Normal mice were injected with INH, RIF, PZA (administered intraperitoneally) for 21 days; (3) M-INH-RIF-PZA group (n=6): normal mice were orally administered once a day. Mannitol 1.67 mg/kg, 1 injection INH, RIF, PZA (administration of intraperitoneal injection) for 21 days. 4. After the blood sample was processed, the mice were anesthetized with ether. Blood was collected from the heart of the mouse and placed in a test tube containing Heparin. The plasma was 13,000 g at 4. (: Centrifugation for 10 minutes, the separated plasma was dispensed into a small tube (Eppendorf tube) and placed in -80. Stored in sputum. 5. Quantitative test of liver function All mice were subjected to galactose Point method (GSP) test 'rat received rapid eye socket injection within 3 sec seconds' injection 0.4 g / ml BW galactose solution 0.5 g / kg; blood was taken 60 minutes after injection, blood samples taken from the tail vein, The galactose content was measured by the galactose dehydrogenase colorimetric method (c〇l〇rimetric gaiact〇se dehydrogenase). The test concentration range was (10) to 丨/(9) μ§/ηι. Within-day variation of each concentration (within-day variation) It is calculated from the standard deviation and the coefficient of variation (CV). The maximum allowable coefficient of variation is 10〇/〇CV; the day-to-day variation is determined by the calibration curves. Slope and cut The distance test was performed; the galactose single point method (GSP) was the concentration of galactose in the blood at 6 minutes after the 30 second injection was stopped. 6. Statistical analysis All data are expressed as mean soil standard deviation (SD). The test results are statistically significant using the single factor analysis (ANOVA) test. Use Statistical.

Package of the Social Science program (Version 13, SPSS Inc.)套裝軟體來計 201236678 算；隨後使用事後比較(post hoc test)最小差異顯著性(least signiflcant ' difference)方法做多重比較，以確認族群間的顯著差異；族群平均之顯著差 異為Ρ<0·05。 二、結果 1.剩餘肝功能之量測 INH-RIF-PZA控制組的半乳糖單點法(GSP)值有隨著ΓΝΗ/RIF給藥時間 愈長而愈高的趨勢；空白對照組與INH-RIF-PZA控制組小鼠之GSP值具有 南度的顯著差異(空白對照組小鼠3週之GSP值為570 ± 293 mg/L ;給藥3 週後 ’ INH-RIF-PZA 小鼠之 GSP 值為 948 ± 236 mg/L，/? < 0.001，然而， Mannitol實驗組之小鼠則可抵抗這種改變(如表十二）。 表十二、為對照組、INH-RIF-PZA組、Μ-INH-RIF-PZA組小鼠半乳糖單點法(GSP)值， 數值之計算為mean ± SD。 GSP(mg/L) NC (n=6) INH-RIF-PZA (n=8) M-INH-RIF-PZA (n=6) 0 weeks 344 ± 1% 372 ±172 356 ±144 2 weeks 381±157 431+103 283 ±178 3 weeks 570 ± 293 948 ± 236 296 ±102***Package of the Social Science program (Version 13, SPSS Inc.) package software to calculate 201236678; then use post hoc test least difference significant (least signiflcant ' difference) method to make multiple comparisons to confirm inter-ethnic Significant difference; the significant difference in ethnic averages is Ρ<0·05. 2. Results 1. Measurement of residual liver function The galactose single point method (GSP) value of the INH-RIF-PZA control group has a higher tendency as the sputum/RIF administration time is longer; blank control group and INH - The GSP value of the mice in the RIF-PZA control group was significantly different from the south (GSP values of 570 ± 293 mg/L in the blank control group for 3 weeks; after 3 weeks of administration - INH-RIF-PZA mice) The GSP value was 948 ± 236 mg/L, /? < 0.001, however, the mice in the Mannitol experimental group were resistant to this change (see Table 12). Table 12, for the control group, INH-RIF-PZA Group, Μ-INH-RIF-PZA group galactose single point method (GSP) value, the value is calculated as mean ± SD. GSP (mg / L) NC (n = 6) INH-RIF-PZA (n = 8) M-INH-RIF-PZA (n=6) 0 weeks 344 ± 1% 372 ±172 356 ±144 2 weeks 381±157 431+103 283 ±178 3 weeks 570 ± 293 948 ± 236 296 ±102** *

Data are shown as mean 土 SD. */7<0.05, **/><0.01, ***p<0.005: Study compare to control group. 實施例九、低副作用INH/RIF劑型於健康受試者體内對INH相關代謝酵素 之影響研究 一、材料與方法 1.試驗處理 利用 CYP2E1 phenoytping 藥物 Chlorzoxazone 500 mg 與 Rifamate (Isoniazid 150 mg/Rifampin 300 mg)併服CYP2E1 抑制劑Mannitol 100 mg，於 § 62 201236678 健康受試者進行藥動學比較研究。試驗過程中，監測受試者血跋中 Chlorzoxazone (CZX)及其代職的變化情形，並掌握似、ast及Gsp值等 生化值變化，進崎估健康受試者在有無併服CYP2E1抑侧下，研究 CYP2E1在健康受試者體内的活性變化情形。 2. 試驗分組 本試驗全程於三軍總醫院臨床研究中心執行，試驗共有2次階段性給 藥’每次試驗間隔一週。第i次給藥，口服給予國外原細色_(1咖咖 150 mg/Rifampin 300 mg)與Chlorzoxazone (500 mg)，第 1次給藥後一週，同 批又式者進行第2次給藥，給予國外原薇Rjfamate (is〇niazjd 15〇 mg/Rifampin 300 mg) + Mannitol (100 mg)與Chlorzoxazone (500 mg)。 3. 評估及統計方法 受試者的試驗數據及統計分析結果將會作一個整合性概述，藥物動 力學數據以平均值及標準差描述，試驗中所得到的藥動學參數及數據上 的顯著差異，將會以ONE WAY ANOVA或其他更適切的統計分析方法 進行分析。 二、結果 1.血液分析結果 已完成18人次臨床試驗，控制組(Chlorzoxazone 500 mg + Isoniazid 300 mg) 9 人次與實驗組(Chlorzoxazone 500 mg + Isoniazid 300 mg + HUCHE033 180 mg)9人次。結果顯示，併用HUCHE033組，Chlorzoxazone原型藥之 藥物動力學參數未有顯著影響，但其經CYP2E1代謝之代謝物6-OH Chlorzoxazone 之 Cmax 顯著較低，其 6-OH-Chlorzoxazone/ Chlorzoxazone 代 謝比亦顯著低於控制組（圖十九、圖二十及表十三）。 63 201236678Data are shown as mean soil SD. */7<0.05, **/><0.01, ***p<0.005: Study compare to control group. Example IX. Low side effects INH/RIF dosage form for healthy subjects Effects of INH-related metabolic enzymes in the body I. Materials and methods 1. Test treatment using CYP2E1 phenoytping drug Chlorzoxazone 500 mg and Rifamate (Isoniazid 150 mg/Rifampin 300 mg) and CYP2E1 inhibitor Mannitol 100 mg, § 62 201236678 Healthy subjects were compared to pharmacokinetic studies. During the test, the changes of Chlorzoxazone (CZX) and its substitutes in the blood stasis of the subjects were monitored, and the changes in biochemical values such as ast, ast and Gsp values were obtained, and the healthy subjects in the Qishen assessment were included in the presence or absence of CYP2E1. Next, study the changes in the activity of CYP2E1 in healthy subjects. 2. Trial group The whole trial was conducted at the Clinical Research Center of the Three Military General Hospital. The trial consisted of two phased medications, one week apart. For the i-th administration, the original fine color _ (1 coffee 150 mg/Rifampin 300 mg) and Chlorzoxazone (500 mg) were orally administered, one week after the first administration, and the same batch was given for the second time. The drug was given to Rjfamate (is〇niazjd 15〇mg/Rifampin 300 mg) + Mannitol (100 mg) and Chlorzoxazone (500 mg). 3. Assessment and statistical methods The experimental data and statistical analysis results of the subjects will be an integrated overview. The pharmacokinetic data are described by mean and standard deviation, and the pharmacokinetic parameters and data obtained in the test are significant. Differences will be analyzed using ONE WAY ANOVA or other more appropriate statistical analysis methods. II. Results 1. Results of blood analysis 18 clinical trials have been completed. The control group (Chlorzoxazone 500 mg + Isoniazid 300 mg) 9 times and the experimental group (Chlorzoxazone 500 mg + Isoniazid 300 mg + HUCHE033 180 mg) 9 times. The results showed that the pharmacokinetic parameters of the Chlorzoxazone prototype drug had no significant effect in the HUCHE033 group, but the Cmax of the 6-OH Chlorzoxazone metabolite CYP2E1 metabolized was significantly lower, and the 6-OH-Chlorzoxazone/Chlorzoxazone metabolic ratio was also significant. Below the control group (Figure 19, Figure 20 and Table 13). 63 201236678

表十三、為Chlorzoxazone+ Rifamate併服或不併服Mannitol，Chlorzoxazone及其代謝物 6·ΟΗ Chlorzoxazone於健康受試者之藥物動力學參數，數值之計算為mean士 SD FK pai^uneters Contnol (»=4) Mannitol (n-4) Folds CZX tl/2 (hr) L42 士 0.28 1.2S 土 0.26 0.8S Tm„ 〇ir) 2.00 ± 0.00 2.00 ± 0.00 1.00 Cug/mL) 16Λ5 ± 1.81 22J8 士 2.35 L.3? *»* AUC< Ou^us/inL) 61,72 ± 3.31 86.14 ± 6.17 1.40 *** AUCj (hr*u〇fmL) 62-21 3.20 87.65 dk 4.79 3L41 … 60H-CZX Tm« (hr) 5.50 ± 1.00 +50 ± 1.00 0M (u&n»L) 1.21 0Λ5 0.75 士 0.06 0.62 * AUC< (hi^ugimL) S.7$ ± 0.50 3,9$ ± 0.S3 0,59 AUC 丨 7Λ5 ± 4.15 士 0.55 0,58 Metabolic Ratio* 0.U db 0.01 0.05 dt 0.01 0*42 … Data lepr&.^ent meaj>±S.D.. 4p<0.05, “产0.01, “ k*pc〇.〇〇5 IVEct^botic AUCTt ^ AUCt 本發明所提供之含異於驗醯胺(Isoniazid，INH)之無/低副作用新複方，與 單獨使用異於驗醯胺(INH)及/或立復黴素(rifampin, RIF)及/或丙基硫異於醯 胺(pyrazinamide，PZA)之試驗結果相互比較時，在生化分析(ALT、AST值）、 病理學分析、剩餘肝功能之量測(GSP值、GEC值)以及氧化壓力的指標(血 漿中8-iso-PGF2a的濃度)等各方面之分析結果’都有明顯減少使用異終驗醢 胺(INH)所造成的肝毒性副作用的功效。 本發明所提供之含異菸鹼醯胺(Isoniazid，INH)之無/低副作用新複方，其 中亦提供可作為細胞色素P450 2E1 (CYP2E1)抑制劑或醯胺水解酶(amidase) 抑制劑之中藥藥引，相較習知細胞色素P450 2E1 (CYP2E1)抑制劑或酿胺水 解酶(amidase)抑制劑’本發明所提供者係從天然中藥藥引萃取者，較無生 理、化學毒性，且對於人類肝臟之細胞色素Ρ41〇2Ε1活性有明顯之抑制活 性。 上列詳細說明係針對本發明之一可行實施例之具體說明，惟該實施例 並非用以限制本發明之專利範圍，凡未脫離本發明技藝精神所為之等效實 施或變更，例如：異菸鹼醯胺(INH)、細胞色素P450 2E1抑制劑、酿胺水解 酶(amidase)抑制劑施用之濃度及比例，以及細胞色素p41〇2E1抑制劑或醯 64 1 201236678 胺水解酶(amidase)抑制劑選用之種類等變化之等效性實施例，均應包含於 本案之專利範圍中。 上列詳細說明係針對本發明之一可行實施例之具體說明’惟該實施例 並非用以限制本發明之專利範圍，凡未脫離本發明技藝精神所為之等效實 施或變更，均應包含於本案之專利範圍中。 > ’’’τ'上所述’本案不但在活性物質的合成上媒屬創新，並能較習用物品 :進上述夕項功效，應已充分符合_性及進步性之法定發明專利要件， w貴局核准本件發明專射請案，以勵發明，至感 65 201236678 【圖式簡單說明】 圖一為異菸鹼醯胺(INH)在肝臟中之代謝途徑圖。 圖二為對照組、INH 組、BNPP-INH 組、DSF-INH 組以及 BNPP-DSF-INH 組大鼠，天門冬氨酸轉胺酶(AST)與丙氨酸轉胺酶(ALT)活性分析，數值之 計算為mean土SD，*表示各試驗組與對照組比較後尸<〇〇5者。 圖三為對照組(圖三A及C)與INH組(圖三B及D)大鼠肝臟切片：圖三 A，對照組相對正常肝組織之型態(he染色，4〇〇χ);圖三b，INH組在周 圍中央靜脈(V)的肝細胞呈現碎裂及空泡化(HE染色，4〇〇χ);圖三c，以電 子顯微鏡檢視對照組大鼠肝切片，Nu :細胞核(9,000X);圖三D，以電子顯 微鏡檢視INH組大鼠肝切片，相較於圖三c對照組之肝細胞切片，_組 大鼠肝細胞之粗内質網(rER)明顯增加，Nu :細胞核(9,000X)。 圖四為8-iso-PGF2a-d4 (A)與8-iso-PGF2a (B)之分子結構以及子離子光 譜。 圖五為含有250 pg 8-iso-PGF2a-d4 (A)的内標準品溶液、含有1〇〇 pg 8-iso-PGF2a (B)的標準品溶液與空白樣本(c)，在多重反應監測模式(MRM) 4貞測下之液相層析串聯式質譜儀(LC/MS/MS)色譜，質荷比(m/z) 357/197以 及質荷比(m/z) 353/193之離子偶(i〇n pairs)分別被用來監測8_is〇_pGF2a_d4 (A)(作為内標準品)以及8-iso-PGF2a (B)(作為標準品）；波峰1 :空白血漿； 波峰2 :注入標準品之空白血漿。 圖六為對照組、INH組、BNPP-INH組、DSF-INH組以及BNPP-DSF-INH 組大鼠血漿中8-iso-PGF2a的濃度，數值之計算為mean±SD，*表示試驗組 與對照組比較後戶< 0.001者；#表示各試驗組與JNH組比較後户< 〇 〇5者。 圖七為對照組、INH組、BNPP-INH組'DSF-INH組以及BNPP-DSF-INH 組大鼠半乳糖單點法(GSP)值，數值之計算為mean± SD，*表示試驗組與對 照組比較後尸< 0.001者；#表示各試驗組與INH組比較後P < 0.001者；※ 表示各試驗組與INH組比較後尸< 0.005者。 66 201236678 圖八為對照組、INH組、BNPP-INH組、DSF-ΙΝΗ組以及BNPP-DSF-INH 組大鼠半乳糖清除能力(GEC)值，數值之計算為mean 士 SD，*表示試驗組 與對照組比較後/> < 0.001者;#表示各試驗組與組比較後p < 〇 〇〇5者； ※表示各試驗組與INH組比較後p < 0.05者。Table 13 is the pharmacokinetic parameters of Chlorzoxazone+ Rifamate with or without Mannitol, Chlorzoxazone and its metabolite 6·ΟΗ Chlorzoxazone in healthy subjects. The value is calculated as mean SD FK pai^uneters Contnol (»= 4) Mannitol (n-4) Folds CZX tl/2 (hr) L42 ± 0.28 1.2S soil 0.26 0.8S Tm„ 〇ir) 2.00 ± 0.00 2.00 ± 0.00 1.00 Cug/mL) 16Λ5 ± 1.81 22J8 ± 2.35 L.3 ? *»* AUC< Ou^us/inL) 61,72 ± 3.31 86.14 ± 6.17 1.40 *** AUCj (hr*u〇fmL) 62-21 3.20 87.65 dk 4.79 3L41 ... 60H-CZX Tm« (hr) 5.50 ± 1.00 +50 ± 1.00 0M (u&n»L) 1.21 0Λ5 0.75 ± 0.06 0.62 * AUC< (hi^ugimL) S.7$ ± 0.50 3,9$ ± 0.S3 0,59 AUC 丨7Λ5 ± 4.15 0.55 0,58 Metabolic Ratio* 0.U db 0.01 0.05 dt 0.01 0*42 ... Data lepr&.^ent meaj>±SD. 4p<0.05, “Production 0.01, “k*pc〇.〇〇5 IVEct^ Botic AUCTt ^ AUCt The present invention provides a new compound with no/low side effects different from Isoniazid (INH), and is used separately from indoleamine (INH) and/or rifampin (RIF). )and/ Or propyl sulfonamide (PZA) test results are compared with each other, in biochemical analysis (ALT, AST value), pathological analysis, measurement of residual liver function (GSP value, GEC value) and oxidative stress The results of the analysis of various indicators (concentration of 8-iso-PGF2a in plasma) have significantly reduced the efficacy of hepatotoxic side effects caused by the use of heterologous prostamine (INH). A new compound with no/low side effects of isoniazid (INH), which is also available as a cytochrome P450 2E1 (CYP2E1) inhibitor or amidase hydrolyzing enzyme (amidase) inhibitor, compared to conventional cells. Pigment P450 2E1 (CYP2E1) inhibitor or amylin hydrolase (amidase) inhibitors. The present invention provides those who are extracted from natural Chinese medicines, have no physiological and chemical toxicity, and have cytochrome 人类 41〇2Ε1 for human liver. The activity has significant inhibitory activity. The detailed description above is a detailed description of one of the possible embodiments of the present invention, and is not intended to limit the scope of the invention. Concentrations and ratios of base guanamine (INH), cytochrome P450 2E1 inhibitor, amidase inhibitor (amidase) inhibitor, and cytochrome p41〇2E1 inhibitor or 醯64 1 201236678 amine hydrolase (amidase) inhibitor Equivalent embodiments of changes in the type of selection, etc., should be included in the scope of the patent in this case. The detailed description of the preferred embodiments of the present invention is intended to be limited to the scope of the invention, and is not intended to limit the scope of the invention. The patent scope of this case. > '''τ' on the above] This case is not only innovative in the synthesis of active substances, but also in comparison with the customary items: into the above-mentioned effects, it should have fully complied with the statutory invention patent requirements of _ sex and progress. w You approved this invention special shot request, in order to invent, to the sense 65 201236678 [Simplified schematic] Figure 1 is the metabolic pathway of isonicotine guanamine (INH) in the liver. Figure 2 shows the activity of aspartate aminotransferase (AST) and alanine transaminase (ALT) in control, INH, BNPP-INH, DSF-INH and BNPP-DSF-INH rats. The numerical value is calculated as mean soil SD, and * indicates that each test group is compared with the control group and the corpse < Figure 3 shows the liver sections of the control group (Fig. 3A and C) and the INH group (Fig. 3B and D): Fig. 3A, the type of the control group relative to normal liver tissue (he staining, 4〇〇χ); In Figure 3b, hepatocytes in the peripheral central vein (V) of the INH group showed fragmentation and vacuolation (HE staining, 4〇〇χ); Figure 3c, the liver slices of the control group were examined by electron microscopy, Nu: The nucleus (9,000X); Fig. 3D, the liver sections of the rats in the INH group were examined by electron microscopy. Compared with the hepatocyte sections of the control group of the third group, the crude endoplasmic reticulum (rER) of the hepatocytes of the _ group was significantly increased. , Nu: Nuclei (9,000X). Figure 4 shows the molecular structure and ionization spectrum of 8-iso-PGF2a-d4 (A) and 8-iso-PGF2a (B). Figure 5 shows an internal standard solution containing 250 pg of 8-iso-PGF2a-d4 (A), a standard solution containing 1 〇〇pg 8-iso-PGF2a (B) and a blank sample (c) in multiplex reaction monitoring. Mode (MRM) 4 液相 liquid chromatography tandem mass spectrometry (LC/MS/MS) chromatography, mass-to-charge ratio (m/z) 357/197 and mass-to-charge ratio (m/z) 353/193 Io n pairs were used to monitor 8_is〇_pGF2a_d4 (A) (as internal standard) and 8-iso-PGF2a (B) (as standard); crest 1: blank plasma; crest 2: Inject blank plasma of the standard. Figure 6 shows the concentration of 8-iso-PGF2a in the plasma of the control group, INH group, BNPP-INH group, DSF-INH group and BNPP-DSF-INH group. The value is calculated as mean±SD, * indicates the test group and The control group was compared with the households <0.001;# indicates that each test group was compared with the JNH group and the household was < Figure 7 shows the galactose single point method (GSP) values of the control group, INH group, BNPP-INH group 'DSF-INH group and BNPP-DSF-INH group. The value is calculated as mean± SD, * indicates the test group and The control group was compared with the corpse <0.001;# indicates that each test group was compared with the INH group, P <0.001; ※ indicates that each test group was compared with the INH group after corpse < 0.005. 66 201236678 Figure VIII shows the galactose clearance capacity (GEC) values of the control group, INH group, BNPP-INH group, DSF-ΙΝΗ group and BNPP-DSF-INH group. The value is calculated as mean SD, * indicates the test group After comparison with the control group, ><0.001;# indicates that each test group was compared with the group p < 〇〇〇 5; * indicates that each test group was compared with the INH group p < 0.05.

圖九為對照組、INH組、BNPP-INH組、DSF-ΙΝΗ組以及BNPP-DSF-INH 組各組半乳糖單點法(GSP)值與血漿中8-iso-PGF2a的濃度具有高度相關之 統計圖。Figure 9 shows that the galactose single-point (GSP) values of the control group, the INH group, the BNPP-INH group, the DSF-ΙΝΗ group, and the BNPP-DSF-INH group are highly correlated with the concentration of 8-iso-PGF2a in plasma. summary graph.

圖十為對照組、INH組、BNPP-INH組、DSF-ΙΝΗ組以及BNPP-DSF-INH 組各組半乳糖單點法(GSP)值與半乳糖清除能力(GEC)值具有高度相關之統 計圖。 圖十一為對照組、PZA組、BNPP-PZA組及BNPP組大鼠，天門冬氨 酸轉胺酶(AST)與丙氨酸轉胺酶(ALT)活性分析，數值之計算為mean± SD， *表示各試驗組與對照組比較後p<〇 〇5者。 圖十二為對照組(圖十二A)與PZA組(圖十二B)大鼠肝臟切片：圖十二 A ’對照組相對正常肝組織之型態(he染色，4〇〇χ);圖十二b，pza組在 周圍中央靜脈(V)的肝細胞呈現碎裂及空泡化(he染色，400X)。 圖十三為、PZA組、BNPP-PZA組大鼠半乳糖單點法(GSP)值，數值之 計算為mean ± SD。 圖十四為對照組、INH-RIF 組、INH-RIF-PZA 組、Kaempferol-INH-RIF 組、Quercetin-INH-RIF 組、Kaempferol-INH-RIF-PZA 組及Figure 10 shows the high correlation between galactose single point (GSP) and galactose clearance (GEC) values in the control, INH, BNPP-INH, DSF-ΙΝΗ, and BNPP-DSF-INH groups. Figure. Figure 11 shows the activity of aspartate transaminase (AST) and alanine transaminase (ALT) in the control group, PZA group, BNPP-PZA group and BNPP group. The calculation of the value is mean± SD. , * indicates that each test group is compared with the control group, p<〇〇5. Figure 12 shows the liver sections of the control group (Fig. 12A) and the PZA group (Fig. 12B): Fig. 12A 'type of the control group relative to normal liver tissue (he staining, 4〇〇χ); Figure 12b, hepatocytes in the peripheral central vein (V) of the pza group showed fragmentation and vacuolation (he staining, 400X). Figure 13 shows the galactose single point method (GSP) values of the rats in the PZA group and the BNPP-PZA group. The numerical value is calculated as mean ± SD. Figure 14 shows the control group, INH-RIF group, INH-RIF-PZA group, Kaempferol-INH-RIF group, Quercetin-INH-RIF group, Kaempferol-INH-RIF-PZA group and

Quercetin-INH-RIF-PZA組小鼠，天門冬氨酸轉胺酶(AST)與丙氨酸轉胺酶 (ALT)活性分析，數值之計算為mean± SD。 圖十五為對照組、INH-RIF 組、INH-RIF-PZA 組、Kaempferol-INH-RIF 組、Quercetin-INH-RIF 組、Kaempferol-INH-RIF-PZA 組及In the Quercetin-INH-RIF-PZA group, aspartate transaminase (AST) and alanine transaminase (ALT) activity were analyzed and the values were calculated as mean±SD. Figure 15 shows the control group, the INH-RIF group, the INH-RIF-PZA group, the Kaempferol-INH-RIF group, the Quercetin-INH-RIF group, the Kaempferol-INH-RIF-PZA group, and

Quercetin-INH-RIF-PZA組小鼠半乳糖單點法(GSP)值，數值之計算為mean 士 SD。 67 201236678 圖十六為空白對照組（A)、INH-RIF-PZA控制組（B)、-Quercetin-INH-RJF-PZA 組(C)、Kaempferol-INH-RIF-PZA 組(D)，於實驗 3 週小鼠肝臟切片。 圖十七為空白對照組(A)、INH-RIF控制組(B)、Quercetin-INH-RIF組 (C)、Kaempferol-INH-RIF組(D)，於實驗3週小鼠肝臟切片。 圖十八為對照組（A)、INH-RIF 組（B)、MH-INH-RIF 組（C)、 MM-INH-RIF(D)組及ML-INH-RIF組(E)，於實驗3週小鼠肝臟切片。 圖十九為 Chlorzoxazone + Rifamate 併服或不併服 Mannitol， Chlorzoxazone於健康受試者血中濃度圖。實心方塊為Rifamate控制組，給予 Chlorzoxazone (5OOmg) + Rifamate (INH/RIF 150/300 mg);空心圓形為 HUCHE033 實驗組，給予 Chlorzoxazone (500mg) + Rifamate (INH/RIF 150/300 mg) + Mannitol 100 mg。 圖二十為 Chlorzoxazone + Rifamate 併服或不併服 Mannitol， Chlorzoxazone於健康受試者血中濃度圖。實心方塊為Rifamate控制組，給予 Chlorzoxazone (500mg) + Rifamate (INH/RIF 150/300 mg);空心圓形為 HUCHE033 實驗組，給予 Chlorzoxazone (500mg) + Rifamate (INH/RIF 150/300 mg) + Mannitol 100 mg 〇 【主要元件符號說明】 無The galactose single point method (GSP) value of the Quercetin-INH-RIF-PZA group was calculated as mean SD. 67 201236678 Figure 16 shows blank control group (A), INH-RIF-PZA control group (B), -Quercetin-INH-RJF-PZA group (C), Kaempferol-INH-RIF-PZA group (D), The liver slices of the mice were tested for 3 weeks. Figure 17 shows the blank control group (A), the INH-RIF control group (B), the Quercetin-INH-RIF group (C), and the Kaempferol-INH-RIF group (D). Figure 18 shows the control group (A), the INH-RIF group (B), the MH-INH-RIF group (C), the MM-INH-RIF (D) group, and the ML-INH-RIF group (E). 3 weeks mouse liver section. Figure 19 shows the concentration of Chlorzoxazone + Rifamate in the blood concentration of Mannitol, Chlorzoxazone in healthy subjects. The solid box is the Rifamate control group, given Chlorzoxazone (5OOmg) + Rifamate (INH/RIF 150/300 mg); the hollow round is the HUCHE033 experimental group, given Chlorzoxazone (500mg) + Rifamate (INH/RIF 150/300 mg) + Mannitol 100 mg. Figure 20 shows the concentration of Chlorzoxazone + Rifamate in the blood concentration of Mannitol, Chlorzoxazone in healthy subjects. The solid square is the Rifamate control group, given Chlorzoxazone (500mg) + Rifamate (INH/RIF 150/300 mg); the hollow round is HUCHE033 experimental group, given Chlorzoxazone (500mg) + Rifamate (INH/RIF 150/300 mg) + Mannitol 100 mg 〇【Main component symbol description】 None

S 68S 68

Claims (1)

201236678 七、申請專利範圍： 1. 一種無/低副作用之抗結核病藥物醫藥配方，包含： (a) —抗結核病藥物之組合，其係異於驗醢胺(isoniazid)、或立復徽素 (rifampin)、或丙基硫異於驗醯胺(pyrazinamide)、或乙醯胺醇(EMB)或前 述任兩種以上藥物之組成；以及 (b) 至少一種醫藥上可接受之降低抗結核病藥物副作用之物質。 2. 如申請專利範圍第1項之抗結核病藥物醫藥配方，其中該降低抗結核病 藥物副作用之物質係選自於下列化合物所組成的群組：正二羥癒瘡酸 (Nordihydroguaiaretic acid)(其含量為 17 毫克至 10 克）、 (-)-Epigallocetechin-3-gallate(其含量為 25 毫克至 1〇 克）、茵陳色原_ (Capillarisin)(其含量為17毫克至10克)、山奈酚(Kaempferol)(其含量為 16毫克至10克)、根皮素(Phloretin)(其含量為15毫克至1〇克)、橙皮素 (Hesperetin)(其含量為17毫克至10克）、6-薑辣醇(6-Gingerol)(其含量 為16毫克至10克）、沒食子酸(gallic acid)(其含量為9毫克至1〇克）、 異甘草素(Isoliquritigenin)(其含量為18毫克至1〇克)、柚皮素 (其含量為9毫克至10克)、二氫化槲皮素((+)-Taxifolin)(其含量為17毫 克至10克）、漢黃芩素(Wongonin)(其含量為16毫克至1〇克）、原兒茶 酸(Protocatechuic acid)(其含量為 8 毫克至 10 克）、兒茶素((+)_Catechin) (其含量為16毫克至10克）、/3-奈黃酮(β-naphthoflavone)(其含量為15 毫克至10克）、恩貝素(Embelin)(其含量為16毫克至1〇克）、反式肉桂 酸(trans-Cinnamic acid)(其含量為8毫克至1〇克）、表兒茶盼 ((-)-Epicatechin)(其含量為16毫克至10克）、根皮苦(Phk)ridzin)(其含量 為24毫克至10克）、反式肉桂酸(Trans-Cinnamaldehyde)(其含量為7毫 克至10克）、大豆甘元(Daidzein)(其含量為14毫克至10克）、異牡莉素 (Isovitexin)(其含量為24毫克至10克）、石-香葉烯(p_Myrcene)(其含量 為8毫克至10克）、槲皮素(Quercetin)(其含量為〇_9毫克至1〇克）、（+)_ 201236678 檸檬烯(⑴-Limonene)(其含量為7毫克至10克）、揚梅素(Myricetin)(其 含量為17毫克至10克）、槲皮(Quercitrin)(其含量為24毫克至10克）、 木犀草素-7-葡萄糖苦（Luteolin-7-Glucoside)(其含量為24毫克至10 克)、桑葉素(Morin)(其含量為16毫克至10克)、新撥皮苦·Weohesperidin) (其含量為33毫克至1〇克）、橙皮苷(Hesperidin)(其含量為33毫克至10 克)、((-)-Epigallocatechin)(其含量為 17 毫克至 1〇 克)、木犀草素(Luteolin) (其含量為16毫克至10克）、金絲桃苷(Hyperoside)(其含量為25毫克至 10克)、樘柳素(Tamarixetin)(其含量為17毫克至1〇克)、黃芩素(Baicalein) (其含量為15毫克至1〇克)、芸香素(Rutin)(其含量為15毫克至1〇克）、 黃芩(Baicalin)(其含量為％毫克至10克）、序菜素(Apigenin)(其含量為 15毫克至10克）、（+)-EpiCatechin (其含量為16毫克至1〇克）、 (-)-Epicatechin-3-gallate (其含量為24毫克至1〇克）、水飛薊賓⑸吵㈣ (其含量為26毫克至10克)、牡荊素(vitexin)(其含量為24毫克至1〇克)、 金雀異黃酮(Genistein)(其含量為15毫克至10克）、異鼠李素 (Isorhamnetin)(其含量為μ毫克至1〇克）、香葉木素(Di〇smin)(其含量 為33毫克至1〇克)、葛根素(Puerarin)(其含量為23毫克至1〇克）' 或傘 形花内酯(Umbelliferone)(其含量為9毫克至10克）、高良薑素 (Galangin)(其含量為〇_8毫克至1〇克）、非瑟酮(fisetin)(其含量為〇 8毫 克至10克）、Brij 58(其含量為1.4克至1〇克）、Brij 76(其含量為丨4克 至10克）、Brij 35(其含量為18毫克至1〇克）、Tween 20(其含量為1 4 克至10克）、Tween 80(其含量為17〇毫克至1〇克）、Tween 4〇(其含量為 1.4克至1〇克）、PEG 2000(其含量為ι·4克至1〇克）、PEG 4〇〇(其含量為 1.4克至1〇克）、piuomic F68(其含量為丨4克至1〇克）、pEG 4⑽〇(其含 量為1.4克至1〇克）、十四烷酸乙酯(Ethyl Myristate)(其含量為毫克 至10克）、Cremophor EL(其含量為丨7毫克至1〇克）、s〇dium Uuryl Sulfate(其含量為 17 毫克至 ι〇 克）、Micr〇cr^stauine ceuui〇se(其含量為 201236678 190 毫克至 10 克）、Dicalcium phosphate dihydrate(其含量為 9 毫克至 10 克）、Crem〇Ph〇rRH4〇(其含量為 I.% 克至 10 克）、Cr〇sp〇vid〇ne(其含量 為158毫克至1〇克）、Sodium starch glycolate(其含量為158毫克至10 克)、Eudmgit S100(其含量為 158 毫克至 1〇 克)、Croscarmdl〇se —(其 含量為158毫克至10克）、Menthol(其含量為8毫克至1〇克）、 hydroxypmpylcellulose(其含量為158毫克至1〇克卜加㈣—丨以 starch(其含量為158毫克至10克）'Mannitol(其含量為〇.1毫克至10 克）、〇加_8观1^恤(1(其含量為158毫克至10克）、(：丨价以(1(其含 量為10毫克至10克）、Aerosil 200(其含量為158毫克至10克）、PEG 8000(其含量為1.26克至10克）、Sorbic acid(其含量為6毫克至1〇克）、 Lemon oil(其含量為158毫克至10克）、Sodium benzoate(其含量為9毫 克至 10 克）、Acesulfame K(其含量為 1〇 毫克至 10 克）、Hydr〇xypr〇pyl methykellulose(其含量為 158 毫克至 1〇 克）、Hydr〇xy ethyl methylcellulose(其含量為158毫克至1〇克）、Saccharin(其含量為〇]毫 克至10克）、Methyl cellulose(其含量為158毫克至1〇克）、sodium cyclamate(其含量為 1〇 毫克至 1〇 克）、Lactose monohydrate(其含量為 18 毫克至10克）、Maltodextrin(其含量為158毫克至1〇克）、Glyceryi behenate(其含量為52毫克至1〇克）、Oxide red(其含量為34毫克至1〇 克）、Glycerrin monostearate(其含量為 M8 毫克至 1〇 克）、copovidone K28(其含量為158毫克至10克）、Starch acetate(其含量為158毫克至1〇 克）、Magnesium stearate(其含量為 29 毫克至 1〇 克）、Sodium huryl sulfate(其含量為14毫克至10克）、povidone K-30(其含量為6毫克至ι〇 克）、Sucralose(其含量為0.22毫克至10克）、Benzyl alcohol(其含量為 158毫克至10克）、Methylparaben(其含量為8毫克至1〇克）、 Propylparaben(其含量為9毫克至1〇克)、Solutol HI5(其含量為158毫克 至 10 克）、Butylatedhydroxyl anisol(其含量為 9 毫克至 1〇 克）。 201236678 3. 如申請專利範圍第1項之抗結核病藥物醫藥配方， 接受之賦形劑。 其中可加入藥學上可 4. 如申請專利範圍第3項之抗結核病藥物醫藥配方，其中該職 釋劑、填充劑、結合劑'崩解劑或潤滑劑。 … 5. 如申請專利範圍第1項之抗結核病藥物醫藥配方，其中該醫藥配方、 型為口服錠劑、膠囊劑、散劑、溶液劑、懸浮劑、乳劑、芳香水齊丨/ 漿劑、醑劑、酏劑、酊劑、流浸膏劑、軟膏、乳霜劑、糊劑'注射劑或 栓劑。 6. —種無/低副作用之抗結核病藥物醫藥配方，包含： 1. (a)—抗結核病化合物，其係選自於由異菸鹼醯胺(is〇niazid)、立復黴 素(rifampin)、丙基硫異菸鹼醯胺(pyrazinamide)以及乙醯胺醇(ΕΜβ)所組 成的群組；以及 2. (b)至少一種醫藥上可接受之降低抗結核病藥物副作用之物質。 7. 如申請專利範圍第6項之抗結核病藥物醫藥配方，其中該降低抗結核病 藥物副作用之物質係選自於下列化合物所組成的群組：正二羥癒瘡酸 (Nordihydroguaiaretic acid)(其含量為 17 毫克至 1〇 克）、 ㈠-Epigallocetechin-3-gallate(其含量為25毫克至1〇克）、茵陳色原酮 (Capillarisin)(其含量為17毫克至10克）、山奈盼(Kaempferol)(其含量為 16毫克至10克）、根皮素(Phloretin)(其含量為15毫克至1〇克）、燈皮素 (Hesperetin)(其含量為17毫克至10克)、6-薑辣醇(6-Gingerol)(其含量為 16毫克至10克）、沒食子酸(gallic acid)(其含量為9毫克至1〇克）、異甘 草素(Isoliquritigenin)(其含量為18毫克至10克）、柚皮素(Narigenin)(其 含量為9毫克至10克）、二氩化槲皮素((+)-Taxif〇lin)(其含量為π毫克 至1〇克）、漢黃芩素(Wongonin)(其含量為16毫克至10克）、原兒茶酸 (Protocatechuic acid)(其含量為 8 毫克至 10 克）、兒茶素((+)-Catechin)(其 含量為16毫克至10克）、β-奈黃酮(β-naphthoflavone)(其含量為15毫克 72 201236678 至10克）、恩貝素(Embelin)(其含量為16毫克至l〇克）、反式肉桂酸 (tmns-Cinnamic acid)(其含量為8毫克至10克)、表兒茶酚((今Epicatechin) (其含莖為16毫克至10克）、根皮普(phi〇ridzin)(其含量為24毫克至10 克）、反式肉桂醛(Trans-Cinnamaldehyde)(其含量為7毫克至1〇克）、大 豆甘元(Daidzein)(其含量為丨4毫克至1〇克)、異牡莉素(is〇vitexjn)(其含 量為24毫克至10克)、β-香葉烯(|3-Myrcene)(其含量為8毫克至1〇克）、 槲皮素(Quercetin)(其含量為0.9毫克至10克)、(+)·檸檬烯((+)_Lim〇nene) (其含望:為7毫克至10克）、楊梅素(Myricetin)(其含量為17毫克至10 克）、槲皮(Quercitrin)(其含量為24毫克至10克）、木犀草素_7_葡萄糖苷 (Luteolin-'Glucoside)(其含量為24毫克至10克）、桑葉素(Morin)(其含 量為16毫克至10克）、新橙皮苦(]sje〇heSperidin)(其含量為33毫克至1〇 克)、橙皮苦(Hesperidin)(其含量為 33 毫克至 10 *)、((_)_Epigall〇catechin) (其含量為17毫克至10克）、木犀草素(Lute〇lin)(其含量為毫克至 克）、金絲桃苷(Hyperoside)(其含量為25毫克至1〇克）、禋柳素 (Tamarixetin)(其含量為Π毫克至1〇克）、黃芩素(Baicalein)(其含量為 15毫克至10克)、芸香素(Rutin)(其含量為15毫克至1〇克)、黃芩(Baicalin) (其含ϊ：為24毫克至10克）、芹菜素(Apigenin)(其含量為15毫克至1〇 克）、（+)-Ερ—η (其含量為 16 毫克至 1〇 克）、（_)_Epicatechin_3_gallate (其含量為24毫克至10克）、水飛薊賓(silybin)(其含量為26毫克至1〇 克）、牡莉素(Vitexin)(其含量為24毫克至1〇克）、金雀異黃嗣(Genian) (其含量為15毫克至10克）、異鼠李素(isorhamnetin)(其含量為μ毫克 至10克)、香葉木素(Di_in)(其含量為33毫克至1〇克)、葛根素(p_rin) (其含量為23毫克至10克）、或傘形花内酯(UmbeUifer〇ne)(其含量為9 毫克至10克）、高良薑素(Galangin)(其含量為〇·8毫克至1〇克）、非瑟酮 (fisetin)(其含量為〇.8毫克至1〇克）、Brij 58(其含量為丨4克至1〇克）、 BHj 76(其含量為M克至10克)、Brij 35(其含量為18毫克至忉克)、丁 73 201236678 2〇(其含量為1.4克至10克)、Tween 80(其含量為170毫克至10克)、Tween 4〇(其含量為1.4克至10克）、PEG 2000(其含量為1.4克至1〇克）、peg 400(其含量為1.4克至10克)、Pluomic F68(其含量為1.4克至10克)、PEG 4000(其含量為1.4克至10克)、十四烧酸乙醋(EthylMyristate)(其含量為 14毫克至10克)、CremophorEL(其含量為17毫克至1〇克)、S〇diumLauryl Sulfate(其含量為 17 毫克至 10 克)、Microcrystalline cellulose(其含量為 190 毫克至 10 克）、Dicalcium phosphate dihydrate(其含量為 9 毫克至 1〇 克）、 Cremophor RH40(其含量為 1.26 克至 10 克）、Crospovidone(其含量為 158 毫克至10克）、Sodium starch glycolate(其含量為158毫克至1〇克）、 Eudragit S100(其含望:為 158 毫克至 1〇 克）、Croscarmellose sodium(其含 2：為158毫克至10克）、Menthol(其含量為8毫克至1〇克）、 hydroxypropylcellulose(其含量為158毫克至1〇 starch(其含罝為158毫克至1〇克）'Mannitol(其含量為〇·ι毫克至 克）、Dextrates NF hydrated(其含量為 w 毫克至 1〇 克）、Citric add(其含 量為10毫克至10克）、Aerosil 2Q0(其含量為1S8毫克至1〇克）、pEG 8000(其含量為1.26克至10克）、Sorbic acid(其含量為6毫克至1〇克）、 Lemon oil(其含量為158毫克至1〇克)、s〇dium benzoate(其含量為9毫克 至10克）、A_lfame K(其含量為10毫克至1〇克^办—卿㈣ methykeUubse(其含量為158毫克至1〇克）、 methylcellulose(其含量為⑸毫克至1〇克）、Saccharin(其含量為〇」毫 克至10克）、Methyl Cellul〇se(其含量為158毫克至1〇克卜歸⑽ cydamate(其含轉1G技至1G克）、L_e _却_(其含量為18 毫克至10克）、Malt〇dextrin(其含量為158毫克至1〇克）、__ behenate(其含量為π毫克至1〇克）、〇xide red(其含量為％毫克至川 ^ Glycerrin monostearate(^^t^ 158 1〇 , c〇povidone K28(其含量為158毫克至1G克）、Starch aeetate(其含量為158毫克至ι〇 201236678 克）、Magnesium stearate(其含量為 29 毫克至 10 克）、Sodium lauryl sulfate(其含量為14毫克至10克）、povid〇ne K-30(其含量為6毫克至ι〇 克）、Sucralose(其含量為0.22毫克至1〇克）、Benzyl aicohol(其含量為 I58毫克至10克）、Methylparaben(其含量為8毫克至1〇克）、 Propylparaben(其含量為9毫克至1〇克）、Solut〇1H15(其含量為158毫克 至 10 克）、Butylated hydroxyl anisol(其含量為 9 毫克至 1〇 克）。 8.如申請專利範圍第6項之抗結核病藥物醫藥配方，其中可加入—藥 可接受之賦形劑。 樂學上 9. 如申請專利第8項之抗結核鋪物醫藥财，其巾該賦形 釋劑、填充劑、結合劑、崩解劑或潤滑劑。 為稀 10, 如申請專利範圍第6項之抗結核病藥物醫藥配方，其中該醫藥配 型為口服鍵劑、膠囊劑、散劑、溶液劑、懸浮劑、乳劑、芳香^方之劑 聚劑、關、酿劑、西丁劑、流浸膏劑、軟膏、乳 =、掩 栓劑。 胃]_削或 75201236678 VII. Scope of application for patents: 1. A pharmaceutical formula for anti-tuberculosis drugs with no/low side effects, comprising: (a) a combination of anti-tuberculosis drugs, which is different from isoniazid or regulacin Rifampin), or propyl sulphide is different from pyrazinamide, or acetaminophen (EMB) or a composition of any two or more of the foregoing; and (b) at least one pharmaceutically acceptable side effect of reducing anti-tuberculosis drugs Substance. 2. For the anti-tuberculosis drug and pharmaceutical formula of claim 1, wherein the substance for reducing the side effects of the anti-tuberculosis drug is selected from the group consisting of: Nordic hydroguaiaretic acid (the content thereof is 17 mg to 10 g), (-)-Epigallocetechin-3-gallate (25 mg to 1 g), Capillarisin (17 mg to 10 g), kaempferol ( Kaempferol) (from 16 mg to 10 g), Phloetin (15 mg to 1 g), Hesperetin (17 mg to 10 g), 6- 6-Gingerol (content of 16 mg to 10 g), gallic acid (content of 9 mg to 1 g), isoliquritigenin (content of 18) MG to 1 gram), naringenin (9 mg to 10 g), dihydroquercetin ((+)-Taxifolin) (content 17 mg to 10 g), Wongonin (from 16 mg to 1 g), protocatechuic acid (from 8 mg to 10 g), catechin ((+)_Catec Hin) (content 16 mg to 10 g), /3-naphthoflavone (15 mg to 10 g), and embelin (16 mg to 1 g) ), trans-Cinnamic acid (content of 8 mg to 1 g), epiphysis ((-)-Epicatechin) (content of 16 mg to 10 g), root skin bitter ( Phk)ridzin) (from 24 mg to 10 g), trans-Cinnamaldehyde (from 7 mg to 10 g), and Daidzein (14 mg to 10 g) ), Isovitexin (content of 24 mg to 10 g), stone geranyl (p_Myrcene) (content of 8 mg to 10 g), Quercetin (content of 〇) _9 mg to 1 gram), (+) _ 201236678 Limonene ((1)-Limonene) (content of 7 mg to 10 g), Myricetin (content of 17 mg to 10 g), suede (Quercitrin) (content of 24 mg to 10 g), Luteolin-7-Glucoside (content of 24 mg to 10 g), Morin (content of 16 mg to 10 g), new skin pain · W Eohesperidin) (from 33 mg to 1 g), Hesperidin (content 33 mg to 10 g), ((-)-Epigallocatechin) (content 17 mg to 1 g), Luteolin (content 16 mg to 10 g), Hyperposide (25 mg to 10 g), Tamarixetin (17 mg to 1 〇) Gram), Baicalein (15 mg to 1 g), Rutin (15 mg to 1 g), Baicalin (% mg to 10 g) ), Apigenin (content 15 mg to 10 g), (+)-EpiCatechin (content 16 mg to 1 g), (-)-Epicatechin-3-gallate (content 24) MG to 1 gram), silibinin (5) noisy (four) (the content is 26 mg to 10 g), vitexin (the content is 24 mg to 1 gram), genistein (Genistein) (from 15 mg to 10 g), Isorhamnetin (from 1 mg to 1 g), Di〇smin (from 33 mg to 1 g), Puerarin (the content of Puerarin) 23 mg to 1 gram)' or Umbelliferone (content 9 mg to 10 g), Galangin (content 〇8 mg to 1 g), Fasher Ketone (content of 〇8 mg to 10 g), Brij 58 (content of 1.4 g to 1 g), Brij 76 (content of 丨4 g to 10 g), Brij 35 (content of 18 mg to 1 g), Tween 20 (14 g to 10 g), Tween 80 (17 mg to 1 g), Tween 4 (1.4 g to 1 g) ), PEG 2000 (its content is ι·4g to 1〇g), PEG 4〇〇 (its content is 1.4g to 1〇g), piuomic F68 (its content is 丨4g to 1〇g), pEG 4(10)〇 (content of 1.4 g to 1 g), Ethyl Myristate (mg of 10 to 10 g), Cremophor EL (content of 7 mg to 1 g), s 〇dium Uuryl Sulfate (content 17 mg to ι〇g), Micr〇cr^stauine ceuui〇se (content of 201236678 190 mg to 10 g), Dicalcium phosphate dihydrate (content 9 mg to 10 g), Crem 〇Ph〇rRH4〇 (content of I.% to 10 grams), Cr〇sp〇vid〇ne (content of 158 mg to 1 gram), Sodium starch glycolate (content of 158 mg to 10 grams) , Eudmgit S100 (content of 158 mg to 1 g), Croscarmld〇se (content of 158 mg to 10 g), Menthol (content of 8 mg to 1 g), hydroxypmpylcellulose (content of 158 mg) To 1 〇克卜加(四)—丨 star star star 其 其 其 其 158 158 158 Man Man Man Man Man Man Man Man Man Man Man Man Man Man Man Man Man Man Man Man Man Man Man Man Man Man Man Man Man Man Man Man Man Man Man Man Man Man Man Man Man 158 mg to 10 g), (: valence to (1 (content of 10 mg to 10 g), Aerosil 200 (content of 158 mg to 10 g), PEG 8000 (content of 1.26 g to 10 g) , Sorbic acid (content of 6 mg to 1 g), Lemon oil (content of 158 mg to 10 g), Sodium benzoate (content of 9 mg to 10 g), Acesulfame K (content of 1 mg) Up to 10 g), Hydr〇xypr〇pyl methykellulose (from 158 mg to 1 g), Hydr〇xy ethyl methylcellulose (content 158 mg) 1 gram), Saccharin (content is 〇) mg to 10 grams), Methyl cellulose (content 158 mg to 1 gram), sodium cyclamate (content of 1 gram to 1 gram), Lactose monohydrate ( It is from 18 mg to 10 g), Maltodextrin (from 158 mg to 1 g), Glyceryi behenate (from 52 mg to 1 g), and Oxide red (from 34 mg to 1 g) , Glycerrin monostearate (content of M8 mg to 1 g), copovidone K28 (content of 158 mg to 10 g), Starch acetate (content of 158 mg to 1 g), Magnesium stearate (content of 29 mg) Up to 1 gram), Sodium huryl sulfate (14 mg to 10 g), povidone K-30 (6 mg to ι), Sucralose (0.22 to 10 g), Benzyl alcohol (from 158 mg to 10 g), Methylparaben (from 8 mg to 1 g), Propylparaben (from 9 mg to 1 g), Solutol HI5 (from 158 mg to 10 g), Butylatedhydroxyl anisol (with a content of 9 mils) Gram to 1 gram). 201236678 3. If you are applying for the anti-tuberculosis drug and medicine formula in item 1 of the patent scope, accept the excipient. It can be added pharmaceutically acceptable. 4. For example, the anti-tuberculosis drug pharmaceutical formulation of claim 3, wherein the release agent, filler, binder "disintegrant or lubricant". 5. For example, the anti-tuberculosis drug and pharmaceutical formula of claim 1 of the patent scope, wherein the pharmaceutical formula, the form is an oral lozenge, a capsule, a powder, a solution, a suspension, an emulsion, an aroma water/slurry, a sputum Agents, tinctures, tinctures, fluid extracts, ointments, creams, pastes, injections or suppositories. 6. A pharmaceutical formula for anti-tuberculosis drugs with no/low side effects, comprising: 1. (a) an anti-tuberculosis compound selected from the group consisting of isoniazid and isoflavin a group consisting of pyrazinamide and acetaminophen (ΕΜβ); and 2. (b) at least one pharmaceutically acceptable substance that reduces the side effects of anti-tuberculosis drugs. 7. For the anti-tuberculosis drug and pharmaceutical formula of claim 6, wherein the substance for reducing the side effects of the anti-tuberculosis drug is selected from the group consisting of: Nordic hydroguaiaretic acid (the content thereof is 17 mg to 1 g), (i)-Epigallocetechin-3-gallate (25 mg to 1 g), Capillarisin (17 mg to 10 g), Kaempferol ) (from 16 mg to 10 g), Phloetin (15 mg to 1 g), Hesperetin (17 mg to 10 g), 6-ginger 6-Gingerol (content of 16 mg to 10 g), gallic acid (content of 9 mg to 1 g), Isoliquritigenin (content of 18 mg) Up to 10 g), Naricin (content 9 mg to 10 g), argon quercetin ((+)-Taxif〇lin) (content is π mg to 1 g), Han Wongonin (content of 16 mg to 10 g), protocatechuic acid (content of 8 mg to 10 g), Tea ((+)-Catechin) (content of 16 mg to 10 g), β-naphthoflavone (content 15 mg 72 201236678 to 10 g), Embelin (its content) The content is 16 mg to 1 g), trans-cinnamic acid (tmns-Cinnamic acid) (the content is 8 mg to 10 g), epicatechin ((Epicatechin) (the stem contains 16 mg to 10 g) ), phi〇ridzin (content of 24 mg to 10 g), trans-Cinnamaldehyde (content of 7 mg to 1 g), and soybean glycerin (Daidzein) The content is 丨4 mg to 1 gram), isoglucan (is〇vitexjn) (the content is 24 mg to 10 g), β-geranene (|3-Myrcene) (the content is 8 mg to 1) Quercetin, Quercetin (content 0.9 mg to 10 g), (+)·limonene ((+)_Lim〇nene) (its hope: 7 mg to 10 g), myricetin Myricetin) (content 17 mg to 10 g), Quercitrin (24 mg to 10 g), Luteolin-'Glucoside (24 mg to 10 g), Morin (content of 16 mg to 10) ), new orange peel (]sje〇heSperidin) (content of 33 mg to 1 g), Hesperidin (content of 33 mg to 10 *), ((_)_Epigall〇catechin) It is 17 mg to 10 g), Lute〇lin (mg to gram), Hyperposide (25 mg to 1 g), and tauroplatin ( Tamarixetin) (from Π mg to 1 gram), Baicalein (15 mg to 10 g), Rutin (15 mg to 1 gram), Baicalin ) (containing ϊ: 24 mg to 10 g), apigenin (15 mg to 1 g), (+)-Ερ-η (content 16 mg to 1 g), (_)_Epicatechin_3_gallate (with a content of 24 mg to 10 g), silybin (with a content of 26 mg to 1 g), and vitexin (with a content of 24 mg to 1 g) ), Genian (content of 15 mg to 10 g), isorhamnetin (content of μ mg to 10 g), and lignin (Di_in) (content of 33) MG to 1 gram), puerarin ( P_rin) (content of 23 mg to 10 g), or Umbe Uifer〇ne (content of 9 mg to 10 g), Galangin (content of 〇·8 mg to 1 gram), fisetin (the content is 〇.8 mg to 1 gram), Brij 58 (the content is 丨 4 gram to 1 gram), BHj 76 (the content is from M to 10)克), Brij 35 (content of 18 mg to gram), Ding 73 201236678 2〇 (content of 1.4 g to 10 g), Tween 80 (content of 170 mg to 10 g), Tween 4〇 (its The content is from 1.4 g to 10 g), PEG 2000 (content of 1.4 g to 1 g), peg 400 (content of 1.4 g to 10 g), Pluomic F68 (content of 1.4 g to 10 g), PEG 4000 (content of 1.4 g to 10 g), Ethyl Myristate (content of 14 mg to 10 g), Cremophor EL (content of 17 mg to 1 g), S〇diumLauryl Sulfate ( It is 17 mg to 10 g), Microcrystalline cellulose (190 mg to 10 g), Dicalcium phosphate dihydrate (9 mg to 1 g), Cremophor RH40 (1.26 g) Up to 10 g), Crospovidone (content 158 mg to 10 g), Sodium starch glycolate (content 158 mg to 1 g), Eudragit S100 (predicted: 158 mg to 1 g), Croscarmellose sodium (It contains 2: 158 mg to 10 g), Menthol (8 mg to 1 g), hydroxypropylcellulose (content 158 mg to 1 〇starch (containing 158 mg to 1 g) Mannitol (content of 〇·ι mg to gram), Dextrates NF hydrated (content of w mg to 1 gram), Citric add (content of 10 mg to 10 g), Aerosil 2Q0 (content of 1 S8 mg to 1 gram), pEG 8000 (content of 1.26 grams to 10 grams), Sorbic acid (content of 6 mg to 1 gram), Lemon oil (content of 158 mg to 1 gram), s〇dium benzoate (the content is 9 mg to 10 g), A_lfame K (the content is 10 mg to 1 gram ^ do - Qing (four) methykeUubse (the content is 158 mg to 1 gram), methylcellulose (the content is (5) mg to 1 〇克), Saccharin (its content is 〇" mg to 10 grams), Methyl Cellul〇se ( Its content is 158 mg to 1 gram of gram (10) cydamate (which contains 1G to 1G), L_e _ but _ (content of 18 mg to 10 g), Malt〇dextrin (content of 158 mg to 1) 〇克), __ behenate (its content is π mg to 1 gram), 〇xide red (its content is % mg to Sichuan ^ Glycerrin monostearate (^^t^ 158 1〇, c〇povidone K28 (its content is 158 Mg to 1G), Starch aeetate (from 158 mg to ι〇201236678 g), Magnesium stearate (29 mg to 10 g), Sodium lauryl sulfate (14 mg to 10 g), povid〇 Ne K-30 (content 6 mg to ι·g), Sucralose (0.22 mg to 1 g), Benzyl aicohol (I58 mg to 10 g), Methylparaben (8 mg to 1 gram), Propylparaben (content 9 mg to 1 gram), Solut 〇 1H15 (content 158 mg to 10 g), Butylated hydroxyl anisol (content 9 mg to 1 gram). 8. For the anti-tuberculosis drug and pharmaceutical formula of claim 6 of the patent application, an acceptable excipient can be added. Lexic 9. If you apply for anti-tuberculosis products and medicines in Article 8 of the patent, the towel, the filler, the binder, the disintegrant or the lubricant. Dilute 10, such as the anti-tuberculosis drug pharmaceutical formula of claim 6 of the patent scope, wherein the pharmaceutical formulation is an oral key agent, a capsule, a powder, a solution, a suspension, an emulsion, an aromatic agent, and a mixture. , brewing agent, siting agent, flow extract, ointment, milk =, esophage. Stomach]_cut or 75