TW200831087A - Novel isoniazid compound prescription with a low incidence of side effects - Google Patents

Novel isoniazid compound prescription with a low incidence of side effects Download PDF

Info

Publication number
TW200831087A
TW200831087A TW96101545A TW96101545A TW200831087A TW 200831087 A TW200831087 A TW 200831087A TW 96101545 A TW96101545 A TW 96101545A TW 96101545 A TW96101545 A TW 96101545A TW 200831087 A TW200831087 A TW 200831087A
Authority
TW
Taiwan
Prior art keywords
group
inh
isoniazid
dsf
effective amount
Prior art date
Application number
TW96101545A
Other languages
Chinese (zh)
Other versions
TWI360418B (en
Inventor
You-Pu Hu
Dong-Han Yang
Original Assignee
You-Pu Hu
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by You-Pu Hu filed Critical You-Pu Hu
Priority to TW96101545A priority Critical patent/TW200831087A/en
Publication of TW200831087A publication Critical patent/TW200831087A/en
Application granted granted Critical
Publication of TWI360418B publication Critical patent/TWI360418B/zh

Links

Abstract

A novel isoniazid compound prescription with a low incidence of side effects is provided, including an effective amount of isoniazid (INH) in medicine for merging with an effective amount of disulfiram (DSF) in medicine, or for further merging with an effective amount of bis-p-nitrophenyl phosphate (BNPP) in medicine. Moreover, this invention further comprises an effective amount of isoniazid (INH) in medicine for merging with an effective amount of P450 2E1 (CYP2E1) inhibitor in medicine, wherein CYP 2E1 inhibitor is selected from the group consisting of disulfiram, Nordihydroguaiaretic acid, (-)-Epigallocetechin-3-gallate, Capillarisin, Kaempferol, Phloretin, disulfiram, Hesperetin, 6- Gingerol, gallic acid, Isoliquritigenin, Narigenin, (+)-Taxifolin, Wongonin, Protocatechuic acid, (+)-Catechin, β-naphthoflavone, Embelin, trans-Cinnamic acid, (-)-Epicatechin, Phloridzin, Brij 58, Brij 76, Brij 35, Tween 20, Tween 80, Tween 40, PEG 2000, PEG 400, Pluornic F68, and PEG 4000, so as to inhibit the side effects of isoniazid-induced hepatotoxicity.

Description

200831087 九、發明說明： 【發明所屬之技術領域】 本舍明係關於-種低副作用之異於驗醯师麵新複方，特別 '疋才日種將異於驗醯胺(INH)合併使用細胞色素P45〇 2E1 (CTOEl)抑制 :4，或再合诚㈣基料觀二6旨(BNPP)，崎低由胁祕胺(_) 所引起之肝毒性等副作用之異於鹼醯胺新複方。 _ 【先前技術】 姆世界衛生_(WHQ)估計，全球大_三分之-的以感染肺結 核’每年約有人百萬新増齡】；㈣騎魏_結核減人數最近幾年 _也不_升，每十萬人σ有六十多人感染肺結核但其中只有大約四分之 :=人搂受完整、冶療；根據衛生署的統計，㈣每天至少有a個人死於肺 結核’·在這麼多接受肺結賴物治療的病料，臨床上最常見的藥物副作 、：^骨〖生和狀系統病變(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型肝炎的盛行區，感染肺結核之㈣ :患者也不在少數’假設每年有卿名新·肺結核病患，粗略料至少 f ’ 0名£丨3，_騎性肝病患者需接受抗結核藥物治療，因此在這些病 忠身上所可能發生的肝毒性是吾人不可忽視的醫源性疾病。 多數㈣4抗結核藥物，例如：胁鹼_(i咖㈣，俗稱敵_ 星）、丙基硫異於醯轉細麵ide，俗稱敵療新邁)及立復黴修 有效的單-抗結鋪物，也最容㈣起服財纽料性；在⑼年代末期 5 200831087 陸續有異於鹼醯胺(isoniazid)造成肝毒性的報告；異菸鹼醯胺(is〇niazid)所造 成具有臨床症狀的肝毒性約0.1-1%1，2，而在1〇_2〇%的病患中，則可觀察到 無症狀的肝功能異常3，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(is〇niazid)在肝臟中主要經由氮_乙醯氨基轉移 S#(N-acetyltransferase，NAT)的幫助而乙酿化，產生的中間產物乙醯化異於驗 醯胺(acetylisoniazid)迅速被水解成乙酸化聯胺(acetylhydrazine);乙醯化聯胺 可以再經由氮-乙酿氣基轉移酶(N-aCe|yltransjperase)被乙醯化成無毒性的雙 乙醯化聯胺(diacetylhydrazine)，或者經由細胞色素p45〇 2E1 (CYP 450 2E1) 氧化成具有肝毋性的分子’其中包括乙酸化偶氮(acetyMiazene)、乙醯銨離 子(acetylomum ion)、乙醯自由基(acetyiradical)、乙烯酮(ketene)等，另，外在 有氧及NADPH存在時，乙醯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，異於驗醯胺亦可經由 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(而非異於驗醯胺或乙醯化聯胺)是在兔及鼠體内 造成異彝驗醢胺引起之肝毒性(INH-induced hepatotoxicity)最可能的车因 ’3’研究者認為異於驗驢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Satich6等:人的報導則認為對硝基苯盼磷:酸二酯 (bisfnitrophenyl phosphate，BNPP，為一種醯胺水解酶之抑制劑)可預防異 菸鹼酸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 200831087 聯胺。 細胞色素P450 2E1 (CYP2E1)在肝臟中會持續的表現，並負責許多異物 質（如：肝毒素四氯化碳(CC14)以及乙醯氨酚(acetaminophen))的代謝生物 反應7,8;然而，CYP2E1在異菸鹼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9 ;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5，1G。在體外試驗中，雙疏 侖（disulfiram，DSF)及其代謝物二乙基二硫代氨基曱酸 (diethyldithiocarbamate)均被確認為老鼠及人類肝臟微粒體CYP2E1的選擇 性抑制劑(selective mechanism-based inhibitors)11·13，Brady13 等人的試驗則顯 示老鼠服用單一口服劑量的雙硫侖(DSF)後，會造成免疫反應肝容量 (immunoreactive hepatic content)以及CYP2E1催化活性快速且完全的下降。200831087 IX. INSTRUCTIONS: [Technical field to which the invention belongs] This description is based on the fact that the low side effects are different from the new prescriptions of the sputum tester, and in particular, the 疋 日 日 将 将 将 将 IN IN IN IN IN Pigment P45〇2E1 (CTOEl) inhibition: 4, or re-supplement (4) base material view 2 6 (BNPP), low side effects caused by hepatotoxicity caused by serotonin (_) is different from alkali amide new compound . _ [Previous technology] World Health _ (WHQ) estimates that the world's big _ three-points to infected tuberculosis 'about one million new age each year】; (four) riding Wei _ tuberculosis reduction in recent years _ not _ Li, every 100,000 people have more than 60 people infected with tuberculosis, but only about a quarter of them: = people are completely treated, treatment; according to the statistics of the Department of Health, (4) at least one person dies every day from tuberculosis' More to receive the treatment of lung lysate, the most common drug side effects in clinical, ^ bone 〗 〖Biology and systemic lesions (such as: auditory nerve and optic neuropathy), which is the most common liver toxicity #. In addition, Taiwan is also a prevalent area of hepatitis B and hepatitis C, infected with tuberculosis (4): patients are not in the minority 'assuming that there is a new name for new tuberculosis patients every year, roughly expected at least f '0 丨 丨 3, _ riding Patients with liver disease need to be treated with anti-tuberculosis drugs, so the hepatotoxicity that may occur in these patients is a iatrogenic disease that cannot be ignored. Most (four) 4 anti-tuberculosis drugs, such as: sulphate _ (i coffee (four), commonly known as enemy _ star), propyl sulphate is different from 醯 细 fine face ide, commonly known as venom treatment new ) and 复 霉 修 修 effective single-anti-knot The paving, also the most suitable (four) to serve the financial properties; in the late (9) period 5 200831087, there are reports of hepatotoxicity caused by isoniazid; isoniazid (is〇niazid) caused by clinical Symptomatic hepatotoxicity is about 0.1-1%1,2, and in patients with 1〇2〇%, asymptomatic liver function abnormalities can be observed. 3 These liver function abnormalities usually occur within two months after taking the drug. occur. As shown in Figure 1, isoniazid in the liver is mainly brewed by the help of N-acetyltransferase (NAT), and the intermediate product is produced by acetylation. Different from acesulfame (acetylisoniazid) is rapidly hydrolyzed to acetylhydrazine; acetylated hydrazine can be converted to non-toxic by acetylation of N-aCe|yltransjperase Diacetylhydrazine, or oxidized to a hepatic sputum molecule via cytochrome p45〇2E1 (CYP 450 2E1), including acety-mispene, acetylomum ion, Acetyl radical, ketene, etc. In addition, in the presence of external aerobic and NADPH, acetylated hydrazine is formed by the reaction of cytochrome P45〇2E1: free radicals cause oxidative stress, leading to cells Death; in addition, it can be directly hydrolyzed to a toxic hydrazine via amidase or a hydrazine hydrolase (amidase) Hydrolyzed to toxic hydrazine Recently, studies have shown that 'biamine (rather than diamine or acetamidine) is the most likely cause of inducing hepatotoxicity (INH-induced hepatotoxicity) in rabbits and mice. '3' researchers believe that the severity of hepatotoxicity caused by testin is positively correlated with the concentration of hydrazine in plasma. In 1999, Satich6 et al.: Human reports suggest that p-nitrophenyl phosphate: bisfnitrophenyl Phosphate, BNPP, an inhibitor of indoleamine hydrolase, prevents hepatotoxicity caused by isonicotinic acid amines, and its protective mechanism should be to produce 200831087 hydrazine by inhibiting isonicotinic acid amide. Cytochrome P450 2E1 (CYP2E1) continues to behave in the liver and is responsible for the metabolic bioreactivity of many foreign substances such as hepatic toxins carbon tetrachloride (CC14) and acetaminophen7,8; The role of CYP2E1 in the hepatotoxicity caused by isoniazid amide is not clear. Isoniazid amide itself is an inducer of CYP2E1 9; some studies suggest that CYP2E1 and isonicotamine in the liver cause The mechanism of hepatotoxicity is related to 5,1G. In vitro, disulfiram (DSF) and its metabolite diethyldithiocarbamate have been identified as selective inhibitors of mouse and human liver microsomes CYP2E1 (selective mechanism-based) Inhibitors 11·13, Brady13 et al. showed that mice taking a single oral dose of disulfiram (DSF) caused an immunoreactive hepatic content and a rapid and complete decline in CYP2E1 catalytic activity.

Sodhi14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Fm (8-iso-pn)staglandin Fm j-isoPGFk )是一種自由基引起花生四烯酸 (amchidonic acid)發生脂質過氧化作用的產物，其化學性質穩定，8_is〇_pGFh 含量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、氧化性的傷害damage)及抗氧化劑的缺之 (_ioxidantdeficiency)有關如6土目前有許多方法可用來測量8_is〇jpGI^含 里’包括酵素免疫么析法(e.確yme imi?llm〇assay) 1*7、放射免疫分析法 200831087 (radioimmunoassay) 18、氣相層析質譜儀（gas-chromatography mass spectrometry) 19 以及液相層析質譜儀（iiqUid chromatography mass spectrometry) 2G等；此外，人類尿液中的8_iso_PGF2a及其代謝物 2,3-dinor-8-iso-PGF2〇^3：可利用 C18 固相萃取(Cl8 solid 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η。 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（aianine aminotransferase，ALT)以及驗性填酸酶(浙迎如數值，以及測量 肝細胞產物如·膽紅素(bilirubin)、白蛋白(albumin)，以及利用量測前凝血 素時間(Prcthr〇mbin time)來檢測凝血因子(c〇aguM〇n fact〇rs)等22;肝功能定 量測试是根據幾乎只經過肝臟代謝之受質在血清中的濃度而定5這些受質 的清除是依肝門靜脈、肝動脈血流量以及由肝細胞對這些受質的作用而 φ 定，肝臟血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23 〇 半乳糖(galactose)是一種具有高萃取率(extracti〇n涵〇)、9佩在肝臟中代 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（dimerization)，將之轉換成μ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_4imiting step)24。半乳糖的高萃取率使得依賴肝臟血流 篁及肝臟功忐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 8 200831087 疋的規則來評估大鼠之殘餘肝功能(residual liver function)，量測一確切化 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， 亦可能取得殘餘肝功能之代表數質24,25。 : 以半乳糖清除能力(明lactose elimination capacity, GEC)作為人類肝功能 ' 定$測試％已行之有年，然而，半乳糖清除能力測試需取得多個血液樣本 以建立標準曲線，在臨床應用上有其困難度，因此有許多研究使用半乳糖 φ 單點法(Galactose Single Point，GSP)以評估人類肝功能；本案發明人以半乳 糖單點法測試慢性肝炎、肝硬化以及肝癌病患，結果顯示半乳糖單點法可 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(cefoperazone)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 (Gmdance for Industry)中成為建議採用測試肝功能的方法之一 μ。 由此可見，上述習用抗結核藥物異菸鹼醯胺(is〇niazi句仍有諸多缺失， 貫非良善之^又计者，而盈待加以改良。 j 本案务明人鐘於上述習用抗結核藥物異於驗醯胺(isoniazid)所導致肝毒 f生等田彳作用的缺點，乃亟思加以改良創新，並經多年苦心孤詣潛心研究後， 終於成功研發完成本件低副作用之異菸鹼醯胺新複方。 【發明内容】 本發明之目的即在於提供一種低副作用之異菸鹼醯胺(isoniazid，INH) 新複方’將異於鹼醯胺(INH)合併使用細胞色素P450 2E1 (CYP2E1)之抑制 劑’以降低由異菸鹼醯胺(INH)所引起之肝毒性等副作用。. 9 200831087 本發明之次-目的係在於提供一種低副作用之異終驗醯胺 )新複方將異終驗醯胺(j^)合併使用細胞色素(cyp2ei)之 選擇性抑制劑雙硫侖(DSF)，以及醯胺水解酶之抑麵硝基苯義酸二酯 (BNPP)，以降低由異菸鹼醯胺(INH)所引起之肝毒性等副作用。 為達成上述發明目的之低副作用之異菸鹼醯胺(is〇niazid，新複 方，本發明首先以異菸鹼醯胺(INH)誘導大鼠(rat)產生肝毒性為模式，研究 細胞色素P45〇 2E1 (CYP2E1)抑制劑雙硫侖(DSF)，以及醯胺水解酶(amidase) 抑制劑硝基苯酚磷酸二酯(BNPP)對大鼠體内異菸鹼醯胺(_)引發之肝毒 性的影響；除了使用一般肝毒性標記、半乳糖單點法(GSp)以及半乳糖清除 能力(GEC)進行大鼠的殘餘肝功能之定量量測外，本案發明人更利用改良式 液相層析串聯式質譜儀(LC/MS/MS)分析量測大鼠血漿中§七0_pgf2(x濃 度’以進一步判斷8-iso-PGF2〇^否與大鼠體内INH引發之肝毒性有關。 可達成上述發明目的之低副作用之異於驗酿胺(isoniazid，旧)新複 方’係包括一藥學有效量之異终驗醯胺(isoniazid，INH)，合併使用一藥學有 效量之細胞色素P450 2E1 (CYP2E1)抑制劑。 其中該細胞色素P450 2E1 (CYP2E1)抑制劑係選自於下列化合物所組 成群組:雙硫侖(disumram，DSF)、柚皮素（Narigenin)、橙皮素(Hesperetin)、 栴[皮素（quercetin)、正二經癒瘡酸（Nordihydroguaiaretic acid)、茵陳色原酮 (Capillarisin)、原兒茶酸（protocatechuic acid)、沒食子酸（gallic acid)、熊果 酸(ursolic acid)、（+Epigallocetechin、㈠-Epicetechin-3-gallate、 (-)-Epigallocetechin，3-gallate、Tween 20、Tween 40、Tween 60、Tween 80、 200831087Sodhi14 et al. reported in 1997 that oxidative stress is one of the factors that cause hepatotoxicity caused by isonicotinicin and rifamycin in young rats. There are a number of _ studies that want to find appropriate biomarkers to assess the rate of oxidative damage in the body. Biomarkers that may be applicable at present may be divided into three categories, which are markers of oxidative damage to lipids, proteins, and nucleic acids; - isoform prostaglandin Fm (8-iso-pn) staglandin Fm j-isoPGFk is a product of free radical-induced lipid peroxidation of arachidonic acid, its chemical stability, 8_is〇_pGFh content It can be used as a new indicator to judge lipid peroxidation in vivo. The lipid peroxidation may be related to the generation of free radicals in the body, oxidative damage (damage) and the lack of antioxidants (_ioxidantdeficiency). To measure 8_is〇jpGI^里里' including enzyme immunoassay (e. yme imi?llm〇assay) 1*7, radioimmunoassay 200831087 (radioimmunoassay) 18, gas chromatography mass spectrometer (gas-chromatography) Mass spectrometry) 19 and liquid chromatography mass spectrometry (iiqUid chromatography mass spectrometry) 2G; in addition, 8_iso_PGF2a in human urine and its metabolite 2,3-dinor-8-iso- PGF2〇^3: Samples can be prepared by C18 solid phase extraction (SPE), and then analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS). Rats are tested for liver function using invasive and non-invasive methods 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 auxinic acidase (Zheyue Ruyi, and measurement of hepatocyte products such as bilirubin, albumin, and utilization) Pre-clotting time (Prcthr〇mbin time) to detect coagulation factors (c〇aguM〇n fact〇rs), etc. 22; liver function quantitative test is based on the concentration of serum in almost only the liver metabolism The clearance of these receptors is determined by the hepatic portal vein, hepatic artery blood flow, and the effect of hepatocytes on these receptors. The blood flow of the liver is related to the quality of the liver, and vice versa. The ability to determine the metabolism of the liver 23 galactose is a sugar with a high extraction rate (extracti〇n 〇), 9 meta-metabolism in the liver, in the liver, galactose is caused by galactose kinase (gala Ct〇kinase) is converted to Glucose-1-phosphate by differential stereoisomerization; the action of galactose kinase is the rate determining step of the galactose metabolic pathway in hepatocytes _ 4imiting step)24. The high extraction rate of galactose makes galactose metabolism, which is dependent on hepatic blood flow and liver function, the most important way to detect liver function. There is currently no rule of 200831087 疋 to assess residual liver function in rats (residual liver function) ), measuring the metabolic capacity of an exact compound (such as galactose), can be presumed to be a rate-determining step in the liver, and may also represent the representative quality of residual liver function24,25. : The lactose elimination capacity (GEC) has been used as a human liver function. It has been a year of testing. However, the galactose clearance test requires multiple blood samples to establish a standard curve for clinical application. There are many difficulties in this, so many studies use Galactose Single Point (GSP) to evaluate human liver function; the inventors tested chronic hepatitis, cirrhosis and liver cancer patients by galactose single point method, The results show that the galactose single-point method can accurately detect these liver diseases. The galactose single-point method has been successfully applied to the test of liver disease patients - except for the residual liver function of drugs such as Promazine and cefoperazone. " . In addition, the galactose single-point method has become one of the recommended methods for testing liver function in the US Food and Drug Administration (FDA) guidelines (Gmdance for Industry). It can be seen that the above-mentioned anti-tuberculosis drug isoniazid amide (the is〇niazi sentence still has many defects, and the non-goodness is counted again, and the profit is improved. j This case is clear to the people in the above-mentioned anti-tuberculosis The drug is different from the shortcomings caused by isoniazid, which is caused by hepatotoxicity, such as hepatotoxicity, and is improved and innovated. After years of painstaking research, he finally succeeded in researching and developing this low-side side-effect isoniazid guanamine. [Explanation] The object of the present invention is to provide a low side effect of isoniazid amide (isoniazid, INH) new compound 'will be combined with guanamine (INH) using cytochrome P450 2E1 (CYP2E1) Inhibitors' to reduce side effects such as hepatotoxicity caused by isoniazidamine (INH). 9 200831087 The second objective of the present invention is to provide a low-side side effect of a different final amine. Indoleamine (j^) is combined with cytochrome (cyp2ei), a selective inhibitor of disulfiram (DSF), and a guanamine hydrolase inhibitor, nitrobenzate diester (BNPP) to reduce isonianic acid Side effects such as hepatotoxicity caused by indoleamine (INH)Isonianidamide (is〇niazid, a new compound for the purpose of achieving the above-mentioned object of the present invention, the present invention first studies the hepatotoxicity induced by isoniazidamine (INH) in rats, and studies cytochrome P45 〇2E1 (CYP2E1) inhibitor disulfiram (DSF), and guanidin hydrolase (amidase) inhibitor nitrophenol phosphate diester (BNPP) on hepatotoxicity induced by isonicotinic acid (_) in rats In addition to the use of general hepatotoxicity markers, galactose single point method (GSp) and galactose clearance (GEC) for quantitative measurement of residual liver function in rats, the inventors of the present invention further improved liquid chromatography Tandem mass spectrometry (LC/MS/MS) analysis of §7_pgf2 (x concentration' in rat plasma to further determine whether 8-iso-PGF2〇^ is associated with hepatotoxicity induced by INH in rats. The low side effect of the above object of the invention is different from the testis amine (isoniazid, old) new compound's system comprising a pharmaceutically effective amount of isoamizid (INH) in combination with a pharmaceutically effective amount of cytochrome P450 2E1 ( CYP2E1) inhibitor, wherein the cytochrome P450 2E1 (CYP2E1) inhibitor is selected From the group consisting of: disumram (DSF), naringin (Narigenin), hesperetin, quercetin, Nordic hydroguaiaretic acid, Capillarisin, protocatechuic acid, gallic acid, ursolic acid, (+Epigallocetechin, (a)-Epicetechin-3-gallate, (-) -Epigallocetechin, 3-gallate, Tween 20, Tween 40, Tween 60, Tween 80, 200831087

Brij 35、Brij 58、Brij 76、PEG 2000、PEG 4000 可達成上述發明目的之低副作用之異菸鹼醯胺(isoniazid，INH)新複 方，係包括一藥學有效量之異於鹼醯胺(isoniazid，INH)，合併使用一藥學有 ： 效量之雙硫侖(disulfiram，DSF)，以及一藥學有效量之硝基苯酚構酸二酯 . (bis-/>nitropheiiyl phosphate，BNPP)。 本發明所提供之低副作用之異於驗醯胺(isoniazid，INH)新複方，亦可加 ^ 入一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、潤滑劑等。 【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 φ 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，BNPP，DSF以及玉米油則購自sig麵化學公司（St L〇uis，M0 USA)，8-isc>PGF2a以及放射線標定之8-is〇-PGF2〇rd4則得自Cayman.化學公司 (Ann AAot，MI，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。 11 200831087 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50毫克/公斤體重劑量之 戊巴比妥納（sodium pentobarbital)進行腹腔麻醉（intraperitoneally anesthetized)，將聚乙稀導管置於大鼠右頸内靜脈(internal jugular vein)内以 施打半乳糖，導管係以切入穿刺(cut-downtechnique)***，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Brij 35, Brij 58, Brij 76, PEG 2000, PEG 4000 A new compound of isoniazid (INH) which achieves the low side effects of the above object of the invention, and comprises a pharmaceutically effective amount of isoguanide (isoniazid) , INH), a combination of pharmaceutics: a dose of disulfiram (DSF), and a pharmaceutically effective amount of nitrophenolic acid diester. (bis-/> nitropheiiyl phosphate, BNPP). The low side effect of the present invention is different from the new compound of isoniazid (INH), and a pharmaceutically acceptable excipient may be added to the compound, which may be a diluent or a filler. , binders, disintegrants, lubricants, etc. The invention is further described in the following examples, which are intended to be illustrative only and not to be construed as limiting the invention. Example 1 Isoniaciline guanamine (INH) combined with animal test using CYP2E1 inhibitor disulfiram (DSF) and/or nitrophenol phosphate diester (BNPP) φ I. Materials and methods 1. Experimental materials All organic The solvents were all HPLC grades and were purchased from Tedia Co., Ltd. (Fairfield, OH, USA) 'INH, BNPP, DSF and corn oil were purchased from sig 化学 s (M0 USA), 8-isc > PGF2a and Radioactivity calibration of 8-is〇-PGF2〇rd4 was obtained from Cayman. Chemical Company (Ann AAot, MI, USA) 'Lactose injection solution prepared by Nanguang Chemical Pharmaceutical Co., Ltd.'. 400 g of galactose liters with appropriate buffer The solution system and the distilled water of isotonic salts are used for injection. 11 200831087 2. Male SD (Sprague-Dawley) rats weighing 320-350 g were purchased from the National Laboratory Animal Center (Taiwan). The animal experiments were carried out in accordance with the guidelines of the National Animal Research Institute. All rats were placed. Under: In the air/humidity adjustment environment, the light and the darkness are each 12 hours, and the supply of water and feed is not limited. The weight of the rats is continuously monitored during the test. All rats are dosed with 50 mg/kg body weight. Sodium pentobarbital was intraperitoneally anesthetized, and the polyethylene catheter was placed in the internal jugular vein of the rat to galactose. The catheter was cut by puncture (cut- The end 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

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，VEH1，即食鹽水），BNPP係溶於加熱至6(TC • 之食鹽水(〇·9% 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1〇〇1^/1^08?或08[之基劑（\^112，即玉米油）， DSF係溶於玉米油中，以1 ml/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氣體内；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15分鐘處理。 上述5組試驗共包含： 12 200831087 ⑴對照組(normal control group，NC，n=12):正常的大鼠每天注射1次 VEm、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、VEH1以及 . (施行腹腔内注射)共21天；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, VEH1, ready-to-feed saline), and BNPP was dissolved to 6 (TC • Saline solution (〇·9% NaCl), after cooling, was intraperitoneally injected into the rat in a volume of 1 ml/kg; the second treatment was to inject 1〇〇1^/1^08? or 08[ Base (\^112, ie corn oil), DSF is dissolved in corn oil and injected intraperitoneally into the body in a volume of 1 ml/kg; the third treatment is injection of 150 mg/kg INH or INH Base (VEH3, saline), INH is dissolved in saline (0.9% NaCl), intraperitoneally injected into the atmosphere in a volume of 1 ml / kg; the first group (BNPP or VEH1) is the third The group (INH or VEH3) was treated 30 minutes earlier, and the second group (DSF or VEH2) was treated 15 minutes earlier than the third group (INH or VEH3). The above 5 groups of tests included: 12 200831087 (1) Control group (normal control group, NC, n=12): Normal rats were injected with VEm, VEH2, and VEH3 once daily (administered intraperitoneally) for 21 days; (2) INH group (INH, n=7): normal rats were injected daily, VEH1 and. Intraperitoneal injection) for 21 days;

(3) BNPP-INH組(ΒΝΡΡ-ΙΝΗ，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1次DSF、INH以 及VEH1(施行腹腔内注射)共21天；以及 (5) BNPP_DSF-INH組(BNPP-DSF-INH，ηθ):正常的大鼠每天注射以 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 I 於含有EDTA之試管中，血漿(plasma)以13,000g於4°C離心15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 13 200831087(3) BNPP-INH group (ΒΝΡΡ-ΙΝΗ, n=7): Normal rats were injected with BNPP, INH and VEH2 once daily (administered intraperitoneally) for 21 days; (4) DSF-INH group (DSF- INH, n=7): normal rats were injected with DSF, INH and VEH1 once daily (administered intraperitoneally) for 21 days; and (5) BNPP_DSF-INH group (BNPP-DSF-INH, ηθ): normal Rats were injected daily with BNPP, DSF, and INH (administered intraperitoneally) for 21 days; the 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 taken from the rat aorta, placed in a test tube containing EDTA, and the plasma was centrifuged at 13,000 g for 15 minutes at 4 ° C. After separation, the cells were separated. The plasma was dispensed into microtubules (Eppendorf tube) and stored at -80 °C. 5. Biochemical analysis - Hepatocyte injury to measure plasma aspartate aminotransferase (AST) and alanine transaminase (ALT) activity 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 The liver was sacrificed immediately after sacrifice in rats; liver samples were prepared in 10% phosphate buffer 13 200831087

之福馬林(phosphate-buffered formalin)固定，隨後脫水並包埋於石壤(paraffm) 中，以5 μ·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，肝臟切片以二甲胂緩衝液(cacodylate buffer，0.1M pH 7·4)清洗，以20%四氧化锇水溶液(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It酸轴醢(uranyl acetate)及檸檬酸錯(lead citrate)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· 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-7(TC，取〇.5ml血漿至玻璃管中，加入l〇ng 内標準品(internal standard，即8-iso-PGF2a-d4)，混勻後之血漿以C18固相萃取， _ 管柱(Solid-Phase Extraction cartridge，LT· Baker，MA，美國）純化，樣本流洗 液以氣氣蒸發乾燥後，以50μ1乙睛:水(acetonitrile: water，15:85 v/v)溶液回溶 並震盪30秒，取1〇μ1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 LOlOADvP 泵(Shimadzu LCMOADvP 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-μπι， 内徑50 X 2.1mm)進行HPLC分離,，並使用含有2mM醋酸鏔(ammonium acetate) 14 200831087 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2〇ΡμΙ/πώι，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 quadmpole mass ^>ectrometer， 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200 ® ng/ml 8-iso-PGF2(x 或 8-is〇-PGF2a-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(丨〇叩也)則個別用來監測8七〇氺〇?2〇1以及84〇孑0?2〇1-(14;測量後，計 算 6個 8-iscKPGF2cx濃度(〇 的線性標準曲線(linear calibration curve)對 8-iso-PGF2a比8-iS〇-PGF2a-d4比值之區域⑺，得到相關係數(r，correlation coefficient)值為0.999 ;血漿中8-is〇PGF2a的線性範圍在〇.l-2.5ng/ml之間， ^ 其迴歸方程式Egression equation)為Y=-0.0517C + 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(relative errors)範圍在一5.06%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 ; 15 200831087 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’以半乳糖脫鼠酶比色法(colorimetric galactose dehydrogenase)量測半乳糖 含量，測試濃度範圍為50至1，000 pg/ml，每個濃度的曰内差異(within-day variation)係由標準偏差(standard deviation)以及變異係數(coefflcient 0f 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(calibrati〇n curyes)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Tygstrup，s方程 式32修改而來： GEC 二The phosphate-buffered formalin was fixed, then dehydrated and embedded in the soil (paraffm), sliced at 5 μ·thick thickness, sliced with hematoxylin and eosin, and stained Hepatic glucose staining test (PAS), stained and observed by light microscopy: histological observation; in addition, liver sections were washed with cacodylate buffer (0.1 M pH 7.·4) to 20%. After the aqueous osmium tetroxide was fixed for 1 hour, it was continuously dehydrated with alcohol and embedded in Spurr resin (Spurrresin). Ultrathin sections were cut with a diamond knife to form uranyl acetate and citric acid. The lead citrate was double-stained and observed with a transmission electron microscope (Hitachi 600, Hitachi Co., Japan). Extraction and measurement of all PGF2a isomers are dissolved or diluted in an appropriate volume of alcohol to prepare a stock solution, and stored in a small tube stored in -7 (TC, 〇. 5ml plasma to In the glass tube, l〇ng internal standard (8-iso-PGF2a-d4) was added, and the mixed plasma was extracted with C18 solid phase, _ column (Solid-Phase Extraction cartridge, LT·Baker, MA, USA) Purified, the sample stream washing solution was evaporated by air, and then dissolved in 50 μl of acetonitrile: water (15:85 v/v) solution and shaken for 30 seconds. After 1 〇μ1 was dissolved. The extract was injected into the LC/MS/MS system for analysis. 8. Liquid Chromatography Tandem Mass Spectrometer (LC/MS/MS) Analytical HPLC System including 2 Shimadzu LODOADvP pumps (Shimadzu LCMOADvP pumps)' 1 Shimadzu system The controller (Shimadzu system control) and a Shimadzu autosampler (Shimadzuautosampler) (Shimadzu Scientific Instruments, 曰本) were separated by HPLC on a C18 column (particle size 5-μπι, inner diameter 50 X 2.1 mm), and Use a ladder containing 2 mM ammonium acetate 14 200831087 and acetonitrile (ACN) Flow wash (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) Flow washing, LC/MS/MS flow rate was maintained at 2〇ΡμΙ/πώι, and the entire HPLC time was 13.5 minutes; the HPLC system and a three-layer quadrupole mass spectrometer (triple stage quadmpole mass ^>ectrometer, API3000, Applied Biosystem, Foster City, CA, USA) interfaced with a TurboIonSpray ionization source and a negative electrospray as a method of ionization (i〇nizati〇n); Diffusion 200 ® ng/ml 8-iso-PGF2 (x or 8-is〇-PGF2a-d4 standard was optimized in multiple reaction monitoring (MRM) mode, m/z 353/193 and m/ z 357/197 ion couples (丨〇叩 also) were used to monitor 8 〇氺〇 〇氺〇 2 〇 1 and 84 〇孑 0 〇 2 〇 1- (14; after measurement, calculate the concentration of 6 8-iscKPGF2cx ( The linear calibration curve of 〇 is the ratio of 8-iso-PGF2a to 8-iS〇-PGF2a-d4 ratio (7), and the correlation coefficient is obtained (r, correlation coe The fficient) value is 0.999; the linear range of 8-is〇PGF2a in plasma is between 〇.l-2.5 ng/ml, and its regression equation Egression equation is Y=-0.0517C + 0.823 ng/ml; The results were calculated on the standard of re-hydrogenated 8-iso-PGF2a (deuterated 8-iso-PGF2a). The inter-assay precision and accuracy of the standard curve were tested 6 times after the standard concentration samples, respectively. The algorithm (Back-Calculation) evaluates that the relative errors range from 5.06% to 3.13%. 9. Quantitative test of liver function All rats were tested for galactose single point (GSP) and galactose clearance (GEC). Rats received rapid intravenous injection within 30 seconds, and injected with 〇4g/ml BW half. Lactose solution 〇·5 g/kg; 15 200831087 Blood was collected from 5, 10, 15, 30, 45 and 60 minutes after injection, and blood samples were taken from the tail vein. Colorimetric galactose dehydrogenase The galactose content is measured at a concentration ranging from 50 to 1,000 pg/ml, and the within-day variation of each concentration is determined by the standard deviation and the coefficient of variation (coefflcient 0f variation, CV). Percentage calculation, the maximum allowable coefficient of variation is 1〇% cv; the day-to-day variation is determined by the slope and intercept of the calibration curve (calibrati〇n curyes); galactose clearance ability ( GEC) is calculated by the following formula, which is modified by Tygstrup, s Equation 32: GEC II

(mg / kg · min) 其中D為半乳糖之注射量；丁為半乳糖濃度達到〇所需要的時間，係由注 射(通常為2.22 mmol/1)後20至60分鐘的錢濃度_時間曲線之線性迴歸推 得；7為依經驗法正體料均Μ布之校正值；半乳鱗點邮犯)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士標準偏差(SD)表示，試驗結果以單因子變異數分 析_0VA)賴法辑算是轉有輯上㈣_異，_她咖 Package of the Social Science program (Versi〇n 13, sm 算；隨後使用事後比較㈣hoc㈣最小差異顯著性細t鋼酬 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PO.05。 16 200831087 二、結果 1·生化分析結果 4驗^束4 ’測量試驗義的體重及相断重量，麟照組動物相較 : 下:員著差異，生化分析結果如圖二所示，只有INH組血漿中的天門冬 : 祕獅離ST)與賴轉麟(慰)雜麵級舰組(韻組血漿 中的AST’f⑨為U6±li iu/L ;簡組血漿㈣AST活性為129±1() IU/L，户< _ 〇.〇5，對照組血漿中的从极性為44土6肌；丽組血藥中的从丁活性為^ 士 3 ΐυ/Ι^<〇·〇5)，顯示臟組產纽化上的肝損傷；對照組、BNpp_iNH、 dsf-inh以及BNPP①SF4NH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(liverparenchyma)内的肝細胞係排列於自肝小葉中央 修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；_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)，_組大鼠肝細胞内的粗内質網(rER)明顯增加(如圖三0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33，而CYP2E1會導致超氧基(superoxide)以及氫氧自由基(hydr〇Xyi radicals)的產生34，並且會引發内質網的增加35，因此本試驗之結果與先前 17 200831087 研究相符。而其他試驗組：BNPP4NH組、DSF-INH組、BNPP-DSF-INH 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·血液樣本中5-/⑽的量測 : 在負電電喷霧模式下，8-iso-PGF2a最大量之分子離子為質荷比(m/z)353 • 之離子，8-ls〇-PGF2a-d4最大量之分子離子為質荷比(^)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 馨結構以及產生的離子光譜如圖四所示；除了84so-PGF2(rd4的子離子(daughter ions)恆較8-iso-PGF2a的子離子高四個單位之外，8-iso_PGF2a以及 8-isoPGF2a-d4兩者的碎裂模式(fragmentation patterns)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_is〇-PGF2a最密集之子離子為質荷比^2^93之離子，S-iso-PGFh-cU 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 (MRMK貞測下，含有 100Pg84so-PGF2c^250pg/ml8-iso_PGF2(rd4的標準溶 φ 液，以及一血液樣本的典型LC/MS/MS色譜，在注入ing S-isoPGFid4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(mg / kg · min) where D is the amount of galactose injected; the time required for the galactose concentration to reach 〇 is the concentration of _ time from 20 to 60 minutes after injection (usually 2.22 mmol/1) The linear regression is derived; 7 is the correction value of the normal body material according to the empirical method; the semi-milk scale point postal criminal) is the blood galactose concentration at 60 minutes after the 30 seconds of injection stop. 10. Statistical analysis All data are expressed in terms of mean ± standard deviation (SD), and the results of the test are analyzed by single-factor variability. _0VA) Lai's calculation is a combination of (four) _, _ she coffee Package of the Social Science program (Versi〇n 13, sm calculation; then use post hoc comparison (four) hoc (four) minimum difference significant thin steel steel differential method) to make multiple comparisons to confirm significant differences between ethnic groups; significant difference in ethnic group average is PO.05. 16 200831087 Second, the results 1 · Biochemical analysis results 4 test ^ bundle 4 'measure the weight of the test and the weight of the phase difference, compared with the Lin Zhao group of animals: The following: the difference between the members, biochemical analysis results shown in Figure 2, only INH Group of Asparagus in plasma: Secret lion from ST) and Lai Chenglin (comfort) miscellaneous class ship group (AST'f9 in plasma of rhyme group is U6±li iu/L; Jane group plasma (IV) AST activity is 129±1 () IU / L, household < _ 〇. 〇 5, the control group in the plasma from the polarity of 44 soil 6 muscle; Li group blood from the butyl activity is ^ 3 ΐυ / Ι ^ < 〇 · 〇5), showing liver damage on the visceral group; the concentration of transaminase in the serum of the control group, BNpp_iNH, dsf-inh and BNPP1SF4NH 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. The liver cell line in the liver parenchyma of the control group indicated by A is arranged in the reticular plate arranged from the central lobular vein of the hepatic lobules, and the hepatic sinusoids are in the anastomosing plates. The tissue sections of the rats in the _ group were as shown in Figure 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 _ group was significantly increased compared with the control group (as shown in Figure 3c) (as shown in Figure 30). . 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Xyi radicals34 and can trigger The endoplasmic reticulum increased by 35, so the results of this trial are consistent with the previous 17 200831087 study. In other test groups, the degree of liver damage in the BNPP4NH 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 of 5-/(10) in blood samples: In negative electrospray mode, the maximum molecular ion of 8-iso-PGF2a is mass-to-charge ratio (m/z) 353 • ions, 8-ls〇- The maximum molecular ion of PGF2a-d4 is the ion of mass-to-charge ratio (^) 357. These negatively charged molecular ions are freed by a large number of collisions. The molecular structure of the two target compounds and the ion spectrum produced are shown in Figure 4. Shown; Fragmentation mode of both 8-iso_PGF2a and 8-isoPGF2a-d4 except for 84so-PGF2 (daughter ions are always four units higher than the daughter ions of 8-iso-PGF2a) The patterns are very similar, which shows that most of the stable product ions are produced by the A chain, which is labeled with four deuterium atoms; the most dense daughter ion of 8_is〇-PGF2a is the mass-to-charge ratio ^2 The ion of ^93, the most dense daughter ion of S-iso-PGFh-cU is the ion of mass-to-charge ratio (m/z) 197. Figure 5 shows the multi-reaction monitoring mode (MRMK贞, containing 100Pg84so-PGF2c^ 250pg/ml8-iso_PGF2 (rd4 standard solution φ solution, and a typical LC/MS/MS chromatogram of a blood sample, injected into ing S-isoPGFid4 After the internal standard, the standard solution and the blood sample were purified by the same solid phase extraction (SPE), and analyzed by the aforementioned LC/MS/MS protocol. • 4 The concentration of 8_iso-PGF2a in plasma

血漿中的8-iso-PGF2〇^—種氧化壓力(oxidative stress)的指標，如圖六所 示’相較於對照組’ INH組大鼠血漿中8-is〇-PGF2o^濃度明顯增加(INH組大 鼠血漿中liso-PGF』濃度為151 土26 pg/ml ;對照組大鼠血漿中各iso-PGF2a 的濃度為 110士 15 pg/ml，;? < 0·001);與INH組相較，bNPP-INH組、DSF-INH 18 200831087The 8-iso-PGF2〇-oxidative stress index in plasma showed a significant increase in the plasma concentration of 8-is〇-PGF2o^ in the INH group compared with the control group. The plasma concentration of liso-PGF in the INH group was 151 ± 26 pg/ml; the concentration of iso-PGF2a in the plasma of the control group was 110 ± 15 pg/ml, ?? <0·001); and INH Compared with the group, bNPP-INH group, DSF-INH 18 200831087

組、BNPP-DSF-INH組三組則明顯降低由jnh引起肝臟的LpGF2a產生 (BNPP-INH組大鼠血漿中g-iso-PGF』濃度為 128士29pg/ml ; DSF-INH組大 鼠血漿中8-iso-PGF2〇^濃度為126±20 pg/ml ; BNPP-DSF-INH組大鼠血漿中 tiso-PGF』濃度為123士 17 pg/ml ; INH組大鼠血漿中8-iso-PGF2a的濃度為 151士26 pg/ml，;? < 0.005);值得注意的是，對照組、BNPP4NH組、DSF4NH 組、BNPP-DSF4NH組四組之間，大鼠血漿中8-is(>PGF2c^濃度無顯著差 異’與BNPP4NH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-PGF2c^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土69 pg/ml ; INH組大鼠之GSP值為 565士87 pg/ml，p < 〇·〇〇 1)，此外，BNPP4NH 組、DSF-INH 組、BNPP-DSF4NH 組大鼠之 GSP 值各為 401士70 pg/m卜 449土45 pg/mi、388士53 pg/m卜與 INH 組相較，BNPP-INH組、DSF-INH組、BNPP-DSF-INH組大鼠之GSP值各 與INH組大鼠具有高度的顯著差異(其夕值各為ρ < 0·001，ρ < 〇 〇〇5, and/? < 〇·〇〇1);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，ink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(ρ=〇·1)，而對照組、 ΒΝΡΡ4ΚΗ組、DSF-INH組，BNPP-DSF-INH組四組之間大鼠的GSP值無 顯著差異存在。 19 200831087 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土0·6 mg/min.kg、4·3士0·4 mg/min.kg、4·7±〇·5 mg/min.kg ;與 INH 組相較，BNPP-INH組、DSF4NH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p值各為夕< 〇 〇〇5，户< 〇 〇5, and^ < 0.005);單獨施用INH的大鼠之GEC值明顯減少；然而，在iNH合併施用 BNPP或DSF或BNPP與DSF之大鼠則可恢復這種改變；與DSF4NH組相 較，INH合併施用BNPP與DSF者有增加GEC值的傾向(DSF-INH組與 BNPP-DSF4NH 組大鼠之GEC 值各為屯3进4 mg/min.kg、4.7±0.5 mg/min.kg5 P 二 〇·29);此外，對照組、BNPP_INH 組、DSF4NH 組、BNpp DSF ][NH 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-iscKPGF2a的濃度，以及定量肝功能測 4(如’ GSP以及GEC)是否相，以數種相關分析計算後，發現GSp值與 血漿中8-!so-PGF2a的濃度具有高度相關(如圖九所示），相關係數為㈣6 ,· GSP值與GEC值具有高度相關(如圖十所示）^ < 〇綱，相關係數為” 〇助’ GEC值也與血針8‘呢匕的濃度具有高度_(侧係數為-〇.7心<〇._ .如表一所示）;而Gsp值、gec值以及金裝中㈣凝 的濃度則與AST及ALT均無_相關(如表_獅）。 20 200831087 表一 GSP、GEC以及8-iso-PGFh與生化測試之相關性 GSP GEC 8-iso-PGF2a AST r = 0.114 π〇·111 r — 0.217 ALT r = 0.016 r = 0.039 r 二 0.035 8-iso-PGF2a r 二 0.836* r--0.743* r 二 1* 以皮爾森氏相關係數(Pearson’s correlation coefficient)作為統計計算，* p<〇 〇〇 ^ 實施例二細胞色素P450 2E1 (CYP2E1)抑制劑之篩選 Φ 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)抑制劑之篩選套级 (CW2El High ^oughpm Inhibitor Screening Kit5 BD Bioscience，美國)針對 - ：； 22種中藥藥引及i〇種賦型劑進行細胞色素p45〇 2E1 (CYP2E1)抑制劑之筛 選；該CYP2E1抑制劑之篩選套組的作用原理為：在含有細胞色素p45〇 2泣 MFC (7-Methoxy^trifluoromethyI coumarin)^ B 環i兄下加入測試樣品作用後，再偵測CYP2ei代謝物標準品Ηρ。 (7御droxy-4^_methyl c〇_rin)的生成量，並以對照組(_加丨)的 HFC生成量為基準5計算測試樣品之cyp2E1抑制率。 各測試棣品均溶於乙腈(acent〇itrile)，測試不同濃度之中藥藥引 33μΜ，16·5μΜ)及賦形劑(0167%，〇〇8%，〇 〇42%，w/v樹 率，所_試之+藥藥引及結果如表三所列，所測試之賦型齊後結果如表四 所列。 另外，本實施例使用之細胞色素p45〇 2E1 (CYp2E1)抑制劑之篩選套組 21 200831087 (CW2E1 High Throughput Miibitor 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_Nitrophenol 水解酶。 (2) Control Protein : 15 mg/mL Control Protein 溶於 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 〇 (5) Cofactors ··含有 1_3 mM NADP+、66 mM MgCl2 &&66mMGlucose 6-Phosphate o (6) Glucose 6-Phosphate Dehydrogenase ·· 40 units/ml 溶於 5 mM Sodium Citrate Buffer (pH 7·5) oThe group and the BNPP-DSF-INH group significantly reduced the LpGF2a production in the liver caused by jnh (the concentration of g-iso-PGF in the plasma of the BNPP-INH group was 128 ± 29 pg/ml; the plasma of the DSF-INH group) The concentration of 8-iso-PGF2〇 was 126±20 pg/ml; the concentration of tiso-PGF in the plasma of BNPP-DSF-INH group was 123±17 pg/ml; the plasma of INH group was 8-iso- The concentration of PGF2a was 151 ± 26 pg/ml, ;? <0.005); it is worth noting that in the control group, BNPP4NH group, DSF4NH group, BNPP-DSF4NH group, 8-is (> There was no significant difference in the concentration of PGF2c^. Compared with the BNPP4NH group and the DSF-INH group, the combination of BNPP and DSF in INH did not further reduce the concentration of 8-iso-PGF2c in plasma. 5. The measurement of residual liver function As shown in the seven, there was a highly significant difference in the galactose single point method (GSP) between the control group and the INH group (GSP value of 384 soil 69 pg/ml in the control group; GSP value of the INH group) 565 士 87 pg/ml, p < 〇·〇〇1), in addition, the GSP values of the BNPP4NH group, the DSF-INH group, and the BNPP-DSF4NH group were 401 ± 70 pg / m 449 soil 45 pg / Mi, 388 g 53 pg/m b compared with the INH group, BNPP- The GSP values of the INH group, the DSF-INH group, and the BNPP-DSF-INH group were significantly different from those of the INH group (the eve values were ρ < 0·001, ρ < 〇〇〇 5 , and/? <〇·〇〇1); GSP values were significantly increased in rats administered INH alone; however, rats in combination with BNPP or DSF or BNPP and DSF in INH were resistant to this change; In contrast, compared with the DSF-INH group, ink combined with BNPp and DSF showed reduced hepatotoxicity induced by INH, although the difference between the two did not reach statistical difference (ρ = 〇 · 1), while the control group, There were no significant differences in GSP values between the four groups of the ΒΝΡΡ4ΚΗ group, the DSF-INH group, and the BNPP-DSF-INH group. 19 200831087 Similar results were also observed using the galactose clearance capacity (GEC) method. As shown in Figure 8, compared with the control group, the GEC value of the rats in the INH group was significantly reduced (the GEC value of the rats in the INH group was 3.4±0·6 mg/min.kg; the GEC value of the rats in the control group). 4.9±0.8 mg/min.kg, < 0.001), in addition, the GEC values of the BNPP-INH group, the DSF-INH group, and the BNPP-DSF-INH group were 4.5 soil 0·6 mg/min.kg. , 4·3士0·4 mg/mi N.kg, 4·7±〇·5 mg/min.kg; compared with the INH group, the GEC values of the BNPP-INH group, the DSF4NH group, and the BNPP-DSF-INH group were higher than those of the INH group. Significant differences (the p values are each eve < 〇〇〇 5, household < 〇〇 5, and ^ <0.005); the GEC values of the rats administered with INH alone were significantly reduced; however, the combined use of BNPP in iNH Rats with either DSF or BNPP and DSF could restore this change; compared with the DSF4NH group, INH combined with BNPP and DSF had a tendency to increase GEC values (GEC values for DSF-INH and BNPP-DSF4NH rats) Each was 进3 to 4 mg/min.kg, 4.7±0.5 mg/min.kg5 P 〇·29); in addition, the control group, BNPP_INH group, DSF4NH group, BNpp DSF][NH group between the four groups of rats There was no significant difference in the GEC values. In order to determine the concentration of 8-iscKPGF2a in AST, ALT, plasma, and quantitative liver function test 4 (such as 'GSP and GEC), after several correlation analysis, GSp value and plasma 8-!so-PGF2a were found. The concentration is highly correlated (as shown in Figure IX), and the correlation coefficient is (4)6. · The GSP value is highly correlated with the GEC value (as shown in Figure 10). ^ < 〇, the correlation coefficient is "〇助" GEC value also The concentration of 8' 与 with blood needle has a height _ (the side coefficient is -〇.7 heart <〇._. as shown in Table 1); and the Gsp value, gec value, and the concentration of (4) condensed in the gold package are There is no _ correlation between AST and ALT (eg Table _ lion). 20 200831087 Table 1 Correlation between GSP, GEC and 8-iso-PGFh and biochemical tests GSP GEC 8-iso-PGF2a AST r = 0.114 π〇·111 r — 0.217 ALT r = 0.016 r = 0.039 r two 0.035 8-iso-PGF2a r two 0.836* r--0.743* r two 1* with Pearson's correlation coefficient as a statistical calculation, * p<〇〇〇 ^ Example 2 Screening of Cytochrome P450 2E1 (CYP2E1) Inhibitors Φ I. Materials and Methods 1. Test Materials This example uses cell color C4502E1 (CYP2E1) inhibitor screening kit (CW2El High ^oughpm Inhibitor Screening Kit5 BD Bioscience, USA) for - :; 22 kinds of traditional Chinese medicines and i〇 kinds of excipients for cytochrome p45〇2E1 (CYP2E1) Screening of inhibitors; the principle of the screening set of the CYP2E1 inhibitor is: after adding the test sample to the cytochrome p45〇2 weeping MFC (7-Methoxy^trifluoromethyI coumarin) ^ B ring i brother, then re-detect The production amount of CYP2ei metabolite standard Ηρ. (7 Royal droxy-4^_methyl c〇_rin) was measured, and the cyp2E1 inhibition rate of the test sample was calculated based on the HFC production amount of the control group (_ plus 丨). The tested products were all dissolved in acetonitrile (acent〇itrile), tested at different concentrations of the drug, 33μΜ, 16·5μΜ) and excipients (0167%, 〇〇8%, 〇〇42%, w/v tree rate, The results of the test and the drug are listed in Table 3. The results of the tests are as shown in Table 4. In addition, the screening kit for the cytochrome p45〇2E1 (CYp2E1) inhibitor used in this example Group 21 200831087 (CW2E1 High Throughput Miibitor Screening Kit, BD Bioscience, USA) The agents need pounds as follows: (1) CYP2E1 + P450 Reductase + Cytochrome b5: 100 mM potassium phosphate (pH 7 · 4) containing 1.3 nmol P450, and p_Nitrophenol hydrolase. (2) Control Protein: 15 mg/mL Control Protein is dissolved in 100 mM Potassium, Phosphate (pH 7.4). (3) Buffer Solution : 0.5 M Potassium Phosphate (pH 7.4) ° (4) Stop Solution : 0.5 M Tris Base 〇 (5) Cofactors ·· Contains 1_3 mM NADP+, 66 mM MgCl2 &&66mMGlucose 6-Phosphate o ( 6) Glucose 6-Phosphate Dehydrogenase ·· 40 units/ml Dissolved in 5 mM Sodium Citrate Buffer (pH 7·5) o

(7) MFC (7-Methoxy-4-trifluoromethyl coumarin)：螢光性受質 (fluorescence substrate)，50 mM MFC 溶於乙腈(acetonitrile)。 ⑻ DDTC (Diethyldithiocarbamic acid) : CYP2E1 選擇性抑制劑(陽性對 照組），20 mM DDTC 溶於乙腈(acentoitrile)。 (9) OTC (7-Hydroxy-4-trifluoromethyl coumarin) ·· CYP2E1 代謝物標準品 (metabolite standard)，0·25 mM HFC 溶於 0.1M TYis (pH 9·0) 〇 (H^NADPH-Cofactor Mix :於 14.56 ml 無菌水中加入 187.5 μΐ Cofactors - 150 μΐ G6PDH (Glucose 6-Phosphate Dehydrogenase(7) MFC (7-Methoxy-4-trifluoromethyl coumarin): Fluorescent substrate, 50 mM MFC dissolved in acetonitrile. (8) DDTC (Diethyldithiocarbamic acid): CYP2E1 selective inhibitor (positive control group), 20 mM DDTC dissolved in acetonitrile (acentoitrile). (9) OTC (7-Hydroxy-4-trifluoromethyl coumarin) ·· CYP2E1 metabolite standard, 0·25 mM HFC dissolved in 0.1M TYis (pH 9·0) 〇 (H^NADPH-Cofactor Mix : Add 187.5 μΐ Cofactors - 150 μΐ G6PDH (Glucose 6-Phosphate Dehydrogenase) to 14.56 ml of sterile water.

Solution)以及 100 μΐ Control Protein。 22 200831087 (11) Cofactor/ acetonitrile mix:於 9·93 mi NADPH-Cofactor Mix 中加入 όό μΐ Acetonitrile ο (12) Enzyme/Substrate Mix :於 4ml Buffer Soultion 中加入 5.94 ml 無菌 水、50μ1 HTS-706(CYP2E15 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IMSO 2E1 (CYP2E1)抑制劑之筛選 使用細胞色素P450 2E1 (CY?2E1)抑制劑之篩選套組(CYP2E1 High Throughput Inhibitor Screening Kit，BD Bioscience，美國)進行中藥藥引及賦 型劑之篩選，實驗步驟如下所:述： (1)製備對照組： a·於96孔盤上第1孔井(well)内加入149 (^NADPH-CofactorMix以及 lW20mMDDTC並混合均勻； b·於《亥96孔盤上弟2至12孔井内各加入1〇〇 μΐ c〇fact〇r/ acetonitrile 聰，第1至8孔井為陽性對照組(pGsitive⑺ntn)1);第9與第1〇孔 井為對照組(control);第U與第12孔井為空白對照組(blank); c•於该第1至8孔井内做連續稀釋動作：自第丨孔井内取5〇叫液體加 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 勻，以此類推，至第8孔井時去除多餘的5〇 μ1液體，以得到連續稀 釋辰度 66.6、22.2、7·4、2.47、0·82、〇·27、0.091、0.03 μΜ。 23 200831087 (2) 製備試驗組： a·於％孔盤上第1行的第1及第2孔井内各加入149 μΐ NADPH-CofactorMix，以及1μ120ιπΜ中藥藥引測試樣品或1μ125% (w/v)賦形劑測試樣品7並混合均勻； b·再自該第1行的第1及第2孔井内各取50焯液體加入第3孔井内混 勻（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37°c靜置10分鐘； b.除了該空白對照組之外，其他孔井内均加入1〇〇 piEnzyme/Substrate Mix混勻； c·將所有對照組與試驗組置於37ι靜置4〇分鐘； d·所有的孔井内均加入75 μ1 St〇p s〇luti〇n混勻； e•緊接著於该空白對照組内加入卿μΐ EnZyme/Substrate 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 % Thermo、 Electron Corporation，芬蘭)讀取結果，所使用之激發光(excitati〇n) 波長為4〇5 nm ’發散光(emissi〇n)波長為538腿。 ⑷結果分析：測得之螢光訊號數值換算成為cyp2Ei代謝物標準品取匸 生成里(pmol)後，以對照組(e〇ntr〇]l)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cyp2Ei抑制率： CYP2E1抑制率(％)= t__樣品之HFC生成f 對照組(control)之HFC生成量 24 200831087 二、結果 1·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 當咖C的濃度為66.6 _ (即為〇 167 %，w/v)時’ cyp 抑制衬° 9娜’係以66.6 μΜ作為中刚最高測試濃度，以❹戰 賦型劑最高測試濃度。 ^Solution) and 100 μΐ Control Protein. 22 200831087 (11) Cofactor/ acetonitrile mix: 9 μΐ Acetonitrile ο (9) Enzyme/Substrate Mix in 9.93 mi NADPH-Cofactor Mix: 5.94 ml sterile water, 50μ1 HTS-706 (CYP2E15) in 4ml Buffer Soultion 2 μΜ P450 content) and 28 μΐ 50 mM MFC (7-Methoxy-4-trifluoromethyl coumarin, fluorescent receptor). 2. Screening of cytochrome IMDO 2E1 (CYP2E1) inhibitors The cytochrome P450 2E1 (CY 2E1) inhibitor screening kit (CYP2E1 High Throughput Inhibitor Screening Kit, BD Bioscience, USA) was used for Chinese medicine introduction and prototyping. Screening of the agent, the experimental steps are as follows: (1) Prepare the control group: a· Add 149 (^NADPH-CofactorMix and lW20mMDDTC in the first well on the 96-well plate and mix well; b· in 1 〇〇μΐ c〇fact〇r/ acetonitrile Cong, 2 to 12 wells in the 96-well plate, 1 to 8 wells as the positive control group (pGsitive(7)ntn) 1); 9th and 1st well For the control group; the U and 12 wells are blank control group (blank); c• The serial dilution operation is performed in the wells 1 to 8: 5 〇 from the first boring well is called liquid added to the second Mix well in the well, then add 5〇μ1 liquid from the second well to the well of the third well, and so on. After the 8th well, remove the excess 5〇μ1 liquid to obtain the serial dilution 66.6. 22.2, 7. 4, 2.47, 0·82, 〇·27, 0.091, 0.03 μΜ. 23 200831087 (2) Preparation test group: a· Add 149 μΐ NADPH-CofactorMix and 1μ120ιπΜ Chinese medicine test sample or 1μ125% (w/v) in the first and second wells of the first row on the % well plate. Excipient test sample 7 and mix well; b· take 50 焯 liquid from each of the first and second wells of the first row and add it to the third well to mix (ie, each test sample is three replicates); (3) Reaction initiation and termination: a· The above control group and the test group were allowed to stand at 37 ° C for 10 minutes; b. In addition to the blank control group, 1 〇〇 piEnzyme/Substrate Mix was added to other wells. Mix; c·Place all control and test groups at 37 静 for 4 〇 minutes; d· Add 75 μl St〇ps〇luti〇n to all wells; e• immediately follow the blank control group Add the μμ EnZyme/Substrate Mix to the mixture; f. Read all the control and test groups with a fluorescent luminometer (Flu〇r〇skan Ascent % Thermo, Electron Corporation, Finland), using the excitation light (excitati) 〇n) The wavelength is 4〇5 nm 'emissi〇n' wavelength is 538 legs. (4) Analysis of the results: The measured value of the fluorescent signal was converted into the cyp2Ei metabolite standard. After taking the pmol, the control group (e〇ntr〇]l) was used as the reference, that is, the cyp2E1 inhibition rate of the control group was 〇. %, the cyp2Ei inhibition rate of each positive control group and the test group was calculated by the following formula: CYP2E1 inhibition rate (%) = t__HFC production of the sample f HFC production amount of the control group 24 200831087 2. Results 1 · Positive control The inhibition rate of cyp2E1 measured by the group positive control group (DDTC) is shown in Table 2. When the concentration of C is C6 is 66.6 _ (that is, 〇167%, w/v), 'cyp inhibition lining ° 9 Na' The highest test concentration of 66.6 μΜ was used as the highest test concentration of the medium-sized excipient. ^

表二陽性對照組之CYP2E1抑制率Table 2 positive control group CYP2E1 inhibition rate

DDTC 濃度（μΜ) 〇(對照組） 0.03 0.091 0.27 0.82 2.47 7.4 22.2 66.6 jgc生成量（pm〇l) CYP2E1鈿申丨_ 222.00 256.00 202.00 151.71 126.14 55.18 21.08 8.71 31.52 43.06 75.09 90.49 (%) 15.10 5.42 93.19 97.55 2·試驗組CYP2E1抑制率 中藥藥引所測出之CYP2E1抑制率如表三所示:由結果可知各_藥藥 引於不同濃度(66μΜ，33μΜ，16·5μΜ)的條件下，對細胞色素P45〇 2m具有 不同程度的抑制效果，其中以66 μΜ正二羥癒瘡酸(Nordihydroguaiaretie add) 抑制效果最佳(97·99±0·66 %)。 - 25 200831087 表三中藥藥引之CYP2E1抑制率 中藥藥引 CYP2E1抑制率（％) 測試濃度 66 μΜ ,3 3 μΜ 16.5 μΜ 對照組 0 0 0 陽性對照組(DDTC) 97.55土 1.862 正二羥癒瘡酸 (Nordihydroguaiaretic acid) 97.99±0.66 92.36±2.20 76.52+3.86 (-)-Epigallocetechin-3-gallate 97.56±0.18 96.47±0.64 92.56±0.46 茵陳色原酮 (Capillarisin) 76.12+1.89 60.54±5.91 49.0515.18 山奈酚 (Kaempferol) 70.63±2.53 70.04土 3.75 71·87±1.14 根皮素 (Phloretin) 66.84±4.79 54.69±2.84 42·04±3·63 . 雙硫侖 (disulfiram) 66.54±2.55 60·55±5·70 57·89±3·91 橙皮素 (Hesperetin) 54/75土 1.37 43.29±0.82 32.10 土 5.80 6-薑辣醇 (6-Gingerol) 51.89±3.33 39.83+2.32 30·13±2·67 沒食子酸 (gallic acid) 48.24+4.20 42.74±7.36 35.59+10.03 異甘草素 (Isoliquritigenin) 47.83±5.36 46.27±3.28 39.08±2.75 柚皮素 (Narigenin) 41.84+3.51 36.82±3.97 25·11±7·60 二氫化槲皮素 ((+)-Taxifolin) 34.54±3.47 23.80±5.84 22.58±11.69 漢黃芩素 (Wongonin) 23.4812.59 21.87±1.90 15.64±7.82 26 200831087DDTC concentration (μΜ) 〇 (control group) 0.03 0.091 0.27 0.82 2.47 7.4 22.2 66.6 jgc production amount (pm〇l) CYP2E1钿 application丨 222.00 256.00 202.00 151.71 126.14 55.18 21.08 8.71 31.52 43.06 75.09 90.49 (%) 15.10 5.42 93.19 97.55 2. The inhibition rate of CYP2E1 in the test group The CYP2E1 inhibition rate measured by the Chinese medicine drug cited is shown in Table 3: From the results, it can be seen that each _ drug is condensed at different concentrations (66 μΜ, 33 μΜ, 16·5 μΜ) for cytochrome P45〇2m has different degrees of inhibitory effect, among which 66 μΜNordihydroguaiaretie add has the best inhibition effect (97·99±0.66%). - 25 200831087 Table 3 CYP2E1 inhibition rate of traditional Chinese medicine cited CYP2E1 inhibition rate (%) Test concentration 66 μΜ, 3 3 μΜ 16.5 μΜ Control group 0 0 0 Positive control group (DDTC) 97.55 soil 1.862 Ortho-hydroxyl-acid (Nordihydroguaiaretic acid) 97.99±0.66 92.36±2.20 76.52+3.86 (-)-Epigallocetechin-3-gallate 97.56±0.18 96.47±0.64 92.56±0.46 Capillarisin 76.12+1.89 60.54±5.91 49.0515.18 Kaempferol (Kaempferol) 70.63±2.53 70.04 soil 3.75 71·87±1.14 Phloretin 66.84±4.79 54.69±2.84 42·04±3·63 . Disulfiram 66.54±2.55 60·55±5·70 57·89±3·91 Hesperetin 54/75 soil 1.37 43.29±0.82 32.10 Soil 5.80 6-Gingerol (6-Gingerol) 51.89±3.33 39.83+2.32 30·13±2·67 Galic acid 48.24+4.20 42.74±7.36 35.59+10.03 Isoliquritigenin 47.83±5.36 46.27±3.28 39.08±2.75 Naricin 41.84+3.51 36.82±3.97 25·11±7·60 Dihydrogenation Quercetin ((+)-Taxifolin) 34.54±3.47 23.80±5.84 22.58±11.69 Hanaxanthin (Wongonin) 23.4812.59 21.87±1.90 15.64±7.82 26 200831087

中藥藥引 CYP2E1抑制率（％、 測試濃度 66 μΜ 33 μΜ 16.5 μΜ 原兒茶酸 . (Protocatechuic acid) 22.75±4.07 19·95±8·95 25·66 土 12.74 兒茶素 ((+)-Catechin) 16.45+9.67 33.83i8.76 41·53±7.62 β-奈黃酮 (β-naphthoflavone) 15.40±12.94 16.83i0.96 6·52±6·64 恩貝素' — (Embelin) 13.54±11.64 12.30+10.24 5.95±7.48 反式肉桂酸 (trans-Cinnamic acid) 7.10±6·95 4.66±6.50 5.71110.53 表兒茶酚 — ((-)-Epicatechin) 2.57±11.60 15.02+5.50 18.27±9.34 根皮苷 -_ (Phloridzin) 1.42±9.28 3·76±3·58 1.25±7·90 葛根素 ^_ (Puerarin) -12.86 土 2.75 4·64±3.47 0·43±2·31 傘形花内酯 _ (Umbelliferone)_ -1081.56土 168.00 ------- -571.97+117.56 -280.41±19.48 賦型劑所測出之CYP2m抑制率如表四所示，由結果可知各賦麵於 不同/辰度(0.167%，0.08%, 0.042%，w/v)的條件下，對細胞色素p45〇 2E1具有 同度的抑制效果，其中以G.l67% Brij 58的抑制效果最佳(97 75±Q 66%)。 27 200831087 表四賦型劑之CYP 2E1抑制率 中藥藥引 CYP2E1 (〇/❹) 測試濃度（w/v) 對照組 陽性對照組(DDTC)CYP2E1 inhibition rate of Chinese medicine (%, test concentration 66 μΜ 33 μΜ 16.5 μΜ Protocatechuic acid. 22.75±4.07 19·95±8·95 25·66 Soil 12.74 catechin ((+)-Catechin 16.45+9.67 33.83i8.76 41·53±7.62 β-naphthoflavone 15.40±12.94 16.83i0.96 6·52±6·64 Enbesin — — (Embelin) 13.54±11.64 12.30+10.24 5.95±7.48 trans-Cinnamic acid 7.10±6·95 4.66±6.50 5.71110.53 Epicatechin — ((-)-Epicatechin) 2.57±11.60 15.02+5.50 18.27±9.34 phloridin-_ (Phloridzin) 1.42±9.28 3·76±3·58 1.25±7·90 Puerarin^_ (Puerarin) -12.86 Soil 2.75 4·64±3.47 0·43±2·31 Umbelliferone _ (Umbelliferone) _ -1081.56 soil 168.00 ------- -571.97+117.56 -280.41±19.48 The CYP2m inhibition rate measured by the excipient is shown in Table 4. From the results, it can be seen that each surface is different in different lengths (0.167%). Under the conditions of 0.08%, 0.042%, w/v), the cytochrome p45〇2E1 had the same inhibitory effect, and the inhibitory effect of G.67% Brij 58 was the best (97 75±Q 66%). 27 200831087 Table 4 Fu Inhibition of CYP 2E1 agent of traditional Chinese medicine of CYP2E1 primer (square / ❹) Test concentration (w / v) positive control group (DDTC)

Brij 58Brij 58

Brij 76Brij 76

Brij 35Brij 35

Tween 20Tween 20

Tween 80Tween 80

Tween 40 0.167% 0.08% 0.042% 97.55+1.862 97.75+0.66 97.56±1.〇2 933310.82 87.20+1.29 73.92+4.71 96.58+040 96.87+1.00 89.45+0.68 (測試濃度0.013%) 82.80+1.71 65.45±2.50 96.02±0.17 94.76±0.47 76.21+7.37 (測試濃度0.006%) 71.77±4.48 64.02±12.54 58.97+3.29 PEG 2000 47.05i6.48 44.79±2.49 44.33+2 7S 4〇.13±3.06 35.81±3.26Tween 40 0.167% 0.08% 0.042% 97.55+1.862 97.75+0.66 97.56±1.〇2 933310.82 87.20+1.29 73.92+4.71 96.58+040 96.87+1.00 89.45+0.68 (test concentration 0.013%) 82.80+1.71 65.45±2.50 96.02± 0.17 94.76±0.47 76.21+7.37 (test concentration 0.006%) 71.77±4.48 64.02±12.54 58.97+3.29 PEG 2000 47.05i6.48 44.79±2.49 44.33+2 7S 4〇.13±3.06 35.81±3.26

本發明所提供之低副作用之異於驗醯胺 ，INH)新複方，與單獨 使用柊鹼醯胺(INH)之试驗結果相互比較時，在生化分析(ALT、AW值）、 病理學分析、麵肝功能之制(Gsp值、GEC值)以及氧化壓力的指標(血 衆中的濃度)等各方面之分析結果，都有明顯減少使用異於鹼醯 胺(INH)所祕断侧㈣效。 、上列雜說明係針對本發明之—可行實施例之具體說明，惟該實施例 ^、_本^明之專利範圍，凡未脫離本發明技藝精神所為之等效實 或隻更例如"異於驗醯胺(麵)、細胞色素剛Β抑制劑、雙硫余 28 200831087 (DSF)、硝基苯酚磷酸二酯(BNPP)施用之濃度及比例，以及細胞色素p45〇 2E1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。 / 綜上所述，本案不但在雙硫侖(DSF)的應用上確屬創新，並能確實減少 / 使用異菸鹼醯胺(_)所造成的肝毒性副作用，應已充分符合新穎性及進步 性之法定發明專利要件，爰依法提出申請，懇請貴局核准本件發明專利 ^ 申請案，以勵發明，至感德便。 .【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'ΪΝΗ組、BNPP-INH組、DSF4NH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 计异為mean 士 SD，*表示各試驗組與對照組比較後户<〇 〇5者； 圖三為對照組(圖三A及C)與INH組(圖三B及0)大鼠肝臟切片：圖三 φ A ’對照組相對正常肝組織之型態(HE染色，40OX);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，400X);圖三C，以電The low side effects provided by the present invention are different from the test compound, INH), and the biochemical analysis (ALT, AW value), pathological analysis when compared with the test results of indole amide (INH) alone. The results of various aspects such as the system of facial liver function (Gsp value, GEC value) and the index of oxidative stress (concentration in the blood group) have significantly reduced the use of the different side of the indoleamine (INH) (4) effect. The above description is for the specific description of the possible embodiments of the present invention, but the scope of the patents of the present invention is not limited to the equivalent of the technical spirit of the present invention or only more such as " Concentrations and ratios of guanamine (surface), cytochrome rhodium inhibitor, disulfide 28 200831087 (DSF), nitrophenol phosphate diester (BNPP), and types of cytochrome p45〇2E1 inhibitors Equivalent embodiments of the variations are to be included in the scope of the patent in this case. / In summary, this case is not only innovative in the application of disulfiram (DSF), but also can effectively reduce / use hepatotoxic side effects caused by isoniazid amide (_), should fully meet the novelty and The progressive statutory invention patent requirements, 提出 apply in accordance with the law, and ask your bureau to approve the invention patent ^ application, in order to invent invention, to the sense of virtue. [Simplified Schematic] Figure 1 shows the metabolic pathway of isonicotinicinamide (INH) in the liver; - Figure 2 shows the control group 'ΪΝΗ group, BNPP-INH group, DSF4NH group and BNPP_DSF-INH. Rats, aspartate transaminase (AST) and alanine transaminase (ALT) activity analysis, the value of the difference is mean SD, * indicates that each test group compared with the control group after the household 5; Figure 3 is the liver section of the control group (Fig. 3A and C) and the INH group (Fig. 3B and 0): Fig. 3 φ A 'type of control group relative to normal liver tissue (HE stain, 40OX) Figure 3B, hepatocytes in the peripheral central vein (V) of the INH group showed fragmentation and vacuolation (HE staining, 400X); Figure III C, electricity

I 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，INH組 大鼠肝細胞之粗内質網(rER)明顯增加，Nu :細胞核(9,〇〇〇X); 圖四為8-iso-PGF2a-d4 (A)與8-is〇-PGF2a (B)之分子結構以及子離子光 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 29 200831087 8-iso-PGEh (B)的標準品溶液與空白樣本(〇，在多重反應監測模式(mrm) 偵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(ion pairs)分別被用來監測8-iso-PGF2crd4 (A)(作為内標準品)以及8-is〇-PGF2a (B)(伊為標準品）；波峰1 :空白血漿； 波峰2 :沣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jk浆中8-iso-PGF2ct的濃度，數值之計算為mean 土 SD，*表示試驗 組與對照組比較後P < 0.001者；#表示各試驗組與XNH組比較後户< 〇.05 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/><〇·〇〇1者；#表示各試驗組與XNH組比較後7><0.001者、； %表示各試驗組與則Η組比較後Ρ < 0.005者； 圖八為對照組、ΙΝΗ組、ΒΝΡΡ-ΙΝΗ組、DSF-INH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 土 SD，*表示試驗組 與對照組比較後/> < 0.001者;#表示各試驗組與!ΝΗ組比較後P < 〇·〇〇5者； %表示各試驗組與ΙΝΗ組比較後/> < 〇.〇5者； 圖九為對照組、ΙΝΗ組、ΒΝΡΡ-ΙΝΗ組、DSF-INH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；以及I submicroscopic examination of the liver sections of the control group, Nu: nuclei (9,000X); Figure 3D, the liver sections of the rats in the INH group were examined by electron microscopy, compared with the hepatocytes of the control group of the third group, the INH group The crude endoplasmic reticulum (rER) of rat hepatocytes increased significantly, Nu: nuclei (9, 〇〇〇X); Figure 4 shows 8-iso-PGF2a-d4 (A) and 8-is〇-PGF2a (B) Molecular structure and product ionogram 5 is an internal standard solution containing 250 pg 8-iso-PGF2a-d4 (A), a standard solution containing 1〇〇pg 29 200831087 8-iso-PGEh (B) and a blank sample (〇, liquid chromatography tandem mass spectrometry (LC/MS/MS) chromatography with multiple reaction monitoring mode (mrm) detection, mass-to-charge ratio (m/z) 357/197 and mass-to-charge ratio (m/) z) The ion pairs of 353/193 were used to monitor 8-iso-PGF2crd4 (A) (as internal standard) and 8-is〇-PGF2a (B) (Ie standard); crest 1 : blank plasma; crest 2: blank plasma into the standard; Figure 6 is the 8-iso-PGF2ct in the jk slurry of the control group, INH group, BNPP-INH group, DSF-INH group and BNPP-DSF-INH group. The concentration, the value is calculated as mean soil SD, * indicates the test group and the pair After the group comparison, P <0.001;# indicates that each test group is compared with the XNH group after the household <〇.05; Figure 7 is the control group, INH group, BNPP-INH group, DSF-INH group and BNPP-DSF- The galactose single point method (GSP) value of the INH group was calculated as mean ± SD, * indicates that the test group was compared with the control group /><〇·〇〇1;# indicates each test group and XNH After the group comparison, 7 >< 0.001, % means that each test group is compared with the sputum group Ρ <0.005; Figure 8 is the control group, sputum group, sputum sputum group, DSF-INH group and BNPP-DSF The galactose clearance ability (GEC) value of the rats in the -INH group was calculated as mean soil SD, * indicates that the test group was compared with the control group /><0.001;# indicates that each test group was compared with the !ΝΗ group P <〇·〇〇5; % means that each test group is compared with the sputum group /><〇.〇5; Figure IX is the control group, sputum group, sputum-sputum group, DSF-INH group and The galactose single point method (GSP) values of the BNPP-DSF-INH group were highly correlated with the concentration of 8-iso-PGF2a in plasma;

圖十為對照組、INH組、BNPP-INH組、DSF-INH組以及BNPP-DSF-INH 200831087 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。 【主要元件符號説明】 無 。 【參考文獻】 1. Kopanoff DE et al., Isoniazid-related hepatitis: a U.S. Public Health Service cooperative surveillance study., 1978. Am. RevRespirDis 117:991-1001.Figure 10 shows that the galactose single point (GSP) value is highly correlated with the galactose clearance (GEC) value of the control group, the INH group, the BNPP-INH group, the DSF-INH group, and the BNPP-DSF-INH 200831087 group. summary graph. [Main component symbol description] None. [References] 1. Kopanoff DE et al., Isoniazid-related hepatitis: a U.S. Public Health Service cooperative surveillance study., 1978. Am. RevRespirDis 117:991-1001.

2. Nolan CM et al.? Hepatotoxicity associated with isoniazid preventive therapy: a 7-year survey from a public health tuberculosis clinic. 1999. JAMA 281: 1014. 3. Steele MA et al., Toxic hepatitis with isoniazid and rifampin: A meta-analysis. 1991. Chest. 99: 465. 4. Sarich TC, Youssefi M, Zhou T5 Adams SP^ Wall RA5 Wright JM. Role of hydrazine in the mechanism of isoniazid hepatotoxicity in rabbits. 1996. Arch Toxicol 70: 835-840. Λ 5. Yue J? Peng RX, Yang J5 Kong R5 Liu J. CYP2E1 mediated isoniazid-induced hepatotoxicity in rats. 2004. Acta Pharmacol Sin. 25: 699-704. 6. Sarich TC, Adams SP, Petricca (¾ Wright JM. Inhibition of isoniazid-induced hepatotoxicity in rabbits by pretreatment with an amidase inhibitor. 1999. J Pharmacol Exp Ther. 289: 695-702. 7. Lee SS, Buters JT? Pineau Ί, Femandez-Salguero P, Gonzalez FJ. Role of CYP2E1 in the hepatotoxicity of acetaminophen. 1996. J Biol Chem 271: 12063-12067. 8. Wong FW, Chan WY? Lee SS. Resistance to carbon tetrachloride-induced hepatotoxicity in mice which lack CYP2E1 expression. 1998. Toxicol Appl Pharmacol. 153: 109-118. 9. Ramaiah SK? Apte U5 Mehendale HM. Cytochrome P4502E1 induction increases thioacetamide liver injury in diet-restricted rats. 2001. Drug Metab Dispos. 29: 1088-1095. 10. Huang YS5 Chem HD，Su WJ，Wu JC，Chang SC，Chiang CH，Chang FY，et al· Cytochrome P450 2E1 genotype and the susceptibility to antituberculosis drug-induced hepatitis. 2003. Hepatology 37: 924-930. 31 200831087 11. Guengeiich FP, Kim DH5 Iwasaki M. Role of human cytochrome P-450 IIE1 in the oxidation of many low molecular weight cancer suspects. 1991. Chem Res Toxicol. 4: 168-179. 12. Hunter AL? Neal RA. Inhibition of hepatic mixed-function oxidase activity in vitro and in vivo by various tMono-sulfur-containing compounds. 1975. Biochem Pharmacol. 24: 2199-2205. 13. Brady JF5 Xiao F? Wang MH? Li Y, Ning SM? Gapac JM? Yang CS. Effects of disulfiram on hepatic P450IIE1, other microsomal enzymes, and hepatotoxicity in rats. 1991. Toxicol Appl Pharmacol. 108: 366-373. 14. Sodhi CP，Rana SV，Mehta SK，Vaiphei K，Attari S，Mehta S. Study of oxidative-stress in isoniazid-rifampicin induced hepatic injury in young rats. 1997. Drug Chem Toxicol 20: 255-269. 15. Morrow JD5 Hill KE, Burk RF, Nammour TM5 Badr KF, Roberts LJ? 2nd. A series of prostaglandin F2-like compounds are produced in vivo in humans by a non-cyclooxygenase, free radical-catalyzed mechanism. 1990. Proc. Natl. Acad. Sci. USA 87: 9383-9387. 16. Morrow JD. The isoprostanes: their quantification as an index of oxidant stress status in vivo. 2000. Drug Metab Rev. 32: 377-385.2. Nolan CM et al.? Hepatotoxicity associated with isoniazid preventive therapy: a 7-year survey from a public health tuberculosis clinic. 1999. JAMA 281: 1014. 3. Steele MA et al., Toxic hepatitis with isoniazid and rifampin: A Meta-analysis. 1991. Chest. 99: 465. 4. Sarich TC, Youssefi M, Zhou T5 Adams SP^ Wall RA5 Wright JM. Role of hydrazine in the mechanism of isoniazid hepatotoxicity in rabbits. 1996. Arch Toxicol 70: 835- 840. J 5. Yue J? Peng RX, Yang J5 Kong R5 Liu J. CYP2E1 mediated isoniazid-induced hepatotoxicity in rats. 2004. Acta Pharmacol Sin. 25: 699-704. 6. Sarich TC, Adams SP, Petricca (3⁄4 Wright JM. Inhibition of isoniazid-induced hepatotoxicity in rabbits by pretreatment with an amidase inhibitor. 1999. J Pharmacol Exp Ther. 289: 695-702. 7. Lee SS, Buters JT? Pineau Ί, Femandez-Salguero P, Gonzalez FJ. Role of CYP2E1 in the hepatotoxicity of acetaminophen. 1996. J Biol Chem 271: 12063-12067. 8. Wong FW, Chan WY? Lee SS. Resistance to carbon tetrachloride-induced 1998. Toxicol Appl Pharmacol. 153: 109-118. 9. Ramaiah SK? Apte U5 Mehendale HM. Cytochrome P4502E1 induction increases thioacetamide liver injury in diet-restricted rats. 2001. Drug Metab Dispos. 29 : 1088-1095. 10. Huang YS5 Chem HD, Su WJ, Wu JC, Chang SC, Chiang CH, Chang FY, et al· Cytochrome P450 2E1 genotype and the susceptibility to antituberculosis drug-induced hepatitis. 2003. Hepatology 37: 924 -930. 31 200831087 11. Guengeiich FP, Kim DH5 Iwasaki M. Role of human cytochrome P-450 IIE1 in the oxidation of many low molecular weight cancer suspects. 1991. Chem Res Toxicol. 4: 168-179. 12. Hunter AL Neal RA. Inhibition of hepatic mixed-function oxidase activity in vitro and in vivo by various tMono-sulfur-containing compounds. 1975. Biochem Pharmacol. 24: 2199-2205. 13. Brady JF5 Xiao F? Wang MH? Li Y, Ning SM? Gapac JM? Yang CS. Effects of disulfiram on hepatic P450IIE1, other microsomal enzymes, and hepatotoxicity in Rats. 1991. Toxicol Appl Pharmacol. 108: 366-373. 14. Sodhi CP, Rana SV, Mehta SK, Vaiphei K, Attari S, Mehta S. Study of oxidative-stress in isoniazid-rifampicin induced hepatic injury in young rats. 1997. Drug Chem Toxicol 20: 255-269. 15. Morrow JD5 Hill KE, Burk RF, Nammour TM5 Badr KF, Roberts LJ? 2nd. A series of prostaglandin F2-like compounds are produced in vivo in humans by a non-cyclooxygenase Free radical-catalyzed mechanism. 1990. Proc. Natl. Acad. Sci. USA 87: 9383-9387. 16. Morrow JD. The isoprostanes: their quantification as an index of oxidant stress status in vivo. 2000. Drug Metab Rev. 32: 377-385.

17. Devaraj S5 Hirany SV5 Burk RF, Jialal L Divergence between LDL oxidative susceptibility and urinary F(2)-isoprostanes as measures of oxidative stress in type 2 diabetes. 2001. Clin. Chem. 47: 1974-1979. 18. Helmersson J, Basu S. F2-isoprostane excretion rate and diurnal variation in human urine. 1999. Prostaglandins Leukot. Essent Fatty Acids 61: 203-205. 19. Morrow JD, Roberts LJ, 2nd. Mass spectrometric quantification of F2-isoprostanes in biological fluids and tissues as measure of oxidant stress. 1999. Methods EnzymoL 300: 3-12.17. Devaraj S5 Hirany SV5 Burk RF, Jialal L Divergence between LDL oxidative susceptibility and urinary F(2)-isoprostanes as measures of oxidative stress in type 2 diabetes. 2001. Clin. Chem. 47: 1974-1979. 18. Helmersson J , Basu S. F2-isoprostane excretion rate and diurnal variation in human urine. 1999. Prostaglandins Leukot. Essent Fatty Acids 61: 203-205. 19. Morrow JD, Roberts LJ, 2nd. Mass spectrometric quantification of F2-isoprostanes in biological fluids And tissues as measure of oxidant stress. 1999. Methods EnzymoL 300: 3-12.

20. Li H，Lawson JA，Reilly M，Adiyaman M，Hwang SW，Rokach J，FitzGerald GA· Quantitative high performance liquid chromatography/tandem mass spectrometric analysis of the four classes of F(2)-isoprostanes in human urine. 1999. Proc. Natl. Acad. Sci. USA 32 200831087 96: 13381-13386. 21. Liang Y，Wei P，Duke RW，Reaven PD，Harman SM，Cutler RQ Heward CB· Quantification of 8-iso-prostaglandin-F2a and 2?3-dinor-8-iso-prostaglandin-F2a in human urine using liquid chromatography-tandem mass spectrometry. 2003. Free Radic. Biol. Med 34: 409-418. 22. Carlisle R, Galambos JT, Warren WD. The relationship between conventional liver tests, quantitative function tests, and histopathology in cirrhosis. 1979. Dig. Dis. Sci. 24: 358-362.20. Li H, Lawson JA, Reilly M, Adiyaman M, Hwang SW, Rokach J, FitzGerald GA· Quantitative high performance liquid chromatography/tandem mass spectrometric analysis of the four classes of F(2)-isoprostanes in human urine. 1999. Proc. Natl. Acad. Sci. USA 32 200831087 96: 13381-13386. 21. Liang Y, Wei P, Duke RW, Reaven PD, Harman SM, Cutler RQ Heward CB· Quantification of 8-iso-prostaglandin-F2a and 2 ?3-dinor-8-iso-prostaglandin-F2a in human urine using liquid chromatography-tandem mass spectrometry. 2003. Free Radic. Biol. Med 34: 409-418. 22. Carlisle R, Galambos JT, Warren WD. The relationship Between conventional liver tests, quantitative function tests, and histopathology in cirrhosis. 1979. Dig. Dis. Sci. 24: 358-362.

23. Herold C? Heinz R5 Niedobitek (¾ Schneider T, Hahn EG, Schuppan D. Quantitative testing of liver function in relation to fibrosis in patients with chronic hepatitis B and C. 2001. Liver 21:260-265. 24. Keiding S，Johansen S，Tonnesen K. Kinetics of ethanol inhibition of galactose elimination in perfused pig liver.. 1977. Scand J. Clin. Lab Invest. 37: 487-494. 25. Keiding S, Johansen S, Winkler K. Hepatic galactose elimination kinetics in the intact pig. 1982. Scand J. Clin. Lab Invest. 42: 253-259. 26. Lindskov J. The quantitative liver fimction as measured by the galactose elimination capacity. I. Diagnostic value and relations to clinical, biochemical, and histological findings in patients with steatosis and patients with cirrhosis. 1982. Acta Med. Scand. 212: 295-302. 27. Tang HS, Hu OY. Assessment of liver function using a novel galactose single point method. 1992. Digestion 52: 222-231. 28. Hu OY，Tang HS，Chang CL. The influence of chronic lobular hepatitis on pharmacokinetics of cefoperazone—a novel galactose single-point method as a measure of residual liver function. 1994. Biopharai Drug Dispos 15: 563-576. 29. Hu OY? Hu TM, Tang HS. Determination of galactose in human blood by high-performance liquid chromatography: comparison with an enzymatic method and application to the pharmacokinetic study of galactose in patients with liver dysfimction. 1995. J. Pharm. Sci. 84: 231-235. 30. Hu OY, Tang HS, Sheeng TY, Chen TC, Curry SH. Pharmacokinetics of promazine in 33 200831087 patients with hepatic cirrhosis-correlation with a novel galactose single point method. 1995. J. Pharm. Sci. 84: 111-114. 31· FDA Center for Drug Evalu这tion and Research (CDER) Pharmacokinetics in patients with impaired hepatic function: Study design, data analysis, and impact on dosing and labeling. Guidance for Industry, U.S. Department of Health and Human Service. 2003 pp5. 32. Tygstrup N. The Galactose Elimination Capacity in Control Subjects and in Patients with Cirrhosis of the Liver. 1964. Acta Med. Scand 175: 281-289.23. Herold C? Heinz R5 Niedobitek (3⁄4 Schneider T, Hahn EG, Schuppan D. Quantitative testing of liver function in relation to fibrosis in patients with chronic hepatitis B and C. 2001. Liver 21:260-265. 24. Keiding S , Johansen S, Tonnesen K. Kinetics of ethanol inhibition of galactose elimination in perfused pig liver.. 1977. Scand J. Clin. Lab Invest. 37: 487-494. 25. Keiding S, Johansen S, Winkler K. Hepatic galactose elimination Kinetics in the intact pig. 1982. Scand J. Clin. Lab Invest. 42: 253-259. 26. Lindskov J. The quantitative liver fimction as measured by the galactose elimination capacity. I. Diagnostic value and relations to clinical, biochemical, And histological findings in patients with steatosis and patients with cirrhosis. 1982. Acta Med. Scand. 212: 295-302. 27. Tang HS, Hu OY. Assessment of liver function using a novel galactose single point method. 1992. Digestion 52: 222-231. 28. Hu OY,Tang HS,Chang CL. The influence of chronic lobular hepatitis on pharmacok Inetics of cefoperazone—a novel galactose single-point method as a measure of residual liver function. 1994. Biopharai Drug Dispos 15: 563-576. 29. Hu OY? Hu TM, Tang HS. Determination of galactose in human blood by high- Performance liquid chromatography: comparison with an enzymatic method and application to the pharmacokinetic study of galactose in patients with liver dysfimction. 1995. J. Pharm. Sci. 84: 231-235. 30. Hu OY, Tang HS, Sheeng TY, Chen TC , Curry SH. Pharmacokinetics of promazine in 33 200831087 patients with hepatic cirrhosis-correlation with a novel galactose single point method. 1995. J. Pharm. Sci. 84: 111-114. 31· FDA Center for Drug Evalu thistion and Research ( CDER) Pharmacokinetics in patients with impaired hepatic function: Study design, data analysis, and impact on dosing and labeling. Guidance for Industry, US Department of Health and Human Service. 2003 pp5. 32. Tygstrup N. The Galactose Elimination Capacity in Control Subjects And in Patients with Cirrhos Is of the Liver. 1964. Acta Med. Scand 175: 281-289.

33· Ryan DE，Ramanathan L，Iida S，Thomas PE，Haniu M，Shively JE，Lieber CS5 et al. Characterization of a major form of rat hepatic microsomal cytochrome P-450 induced by isoniazid. 1985. J. Biol. Chem. 260: 6385-6393. 34. Ekstrom G, Ingelman-Sundberg M. Rat liver microsomal NADPH-supported oxidase activity and lipid peroxidation dependent on ethanol-inducible cytochrome P-450 (P-450IIE1). 1989. Biochem. Pharmacol. 38: 1313-1319. 35. Sodhi CP5 Rana SV5 Mehta SK； Vaiphei K；.Attri S, Thakur S, Mehta S. Study of oxidative stress in isoniazid-induced hepatic injury in young rats with and without protein-energy malnutrition. 1996. J Biochem Toxicol. 11: 139-146.33. Ryan DE, Ramanathan L, Iida S, Thomas PE, Haniu M, Shively JE, Lieber CS5 et al. Characterization of a major form of rat hepatic microsomal cytochrome P-450 induced by isoniazid. 1985. J. Biol. Chem. 260: 6385-6393. 34. Ekstrom G, Ingelman-Sundberg M. Rat liver microsomal NADPH-supported oxidase activity and lipid peroxidation dependent on ethanol-inducible cytochrome P-450 (P-450IIE1). 1989. Biochem. Pharmacol. 38: 1313-1319. 35. Sodhi CP5 Rana SV5 Mehta SK; Vaiphei K;. Attri S, Thakur S, Mehta S. Study of oxidative stress in isoniazid-induced hepatic injury in young rats with and without protein-energy malnutrition. 1996. J Biochem Toxicol. 11: 139-146.

3434

Claims (1)

200831087 十、申請專利範圍：： 1· 一種低副作用之異辂驗醢胺(isoniazid，INH)新複方，包括一藥學有效量 之異菸鹼醯胺(isoniazid，JNH)，合併使用一藥學有效量之雙硫侖 (disulfimm，DSF)，以及一藥學有效量之硝基苯酚磷酸二酯 (bis-p-nitrophenyl phosphate，BNPP)。 2_如申請專利範圍第1項所述之低副作用之異菸鹼醯胺(18—，1]^)新 複方’其中可加入一藥學上可接受之賦形劑至該複方。 3·如申請專利範圍第1項所述之低副作用之異菸鹼醯胺(is〇niazid，丽)新 複方’其中該賦形劑可為稀釋劑、填充劑、結合劑、崩解劑、潤滑劑等。 4· 一種低副作用之異於驗醢胺(isoniazid，新複方，包括一藥學有效量 之異於驗醯胺(isoniazid，INH) ’合併使用一藥學有效量之細胞色素p45〇 2E1 (CYP2E1)抑制劑。 5·如申請專利範圍第4項所述之低副作用之異菸鹼醯胺(is〇niazid，新 複方其中5亥細胞色素P450 2E1 (CYP2E1)抑制劑為雙硫命(disuifjram， DSF) 〇 6·如申請專利範圍第4項所述之低副作用之異菸鹼醯胺(is〇niazid，jnh)新 複方’其中該細胞色素P450 2E1 (CYP2E1)抑制劑係選自於下列化合物 所組成群組·正^一經癒瘡酸（NordihydiOguaiaretic acid)、 (_)-Epigallocetechin-3-gallat?、茵陳色原酮（CapiUarisin)、山奈酚 (Kaempferol) ' 根皮素（Phloretin)、雙硫侖（disulfimm)、橙皮素 (Hesperetin)、6-薑辣醇(6-Gingerol)、沒食子酸(ganic acid)、異甘草素 35 200831087 (Isoliquritigenin)、柚皮素(Narigenin)、二氫化槲皮素((+)-Taxifolin)、漢黃 答素(他哗011111)、原兒茶‘(?1*〇1：€^16(：1111^^(^(!)、兒茶素((+)-€&16〇1^11)、 β·奈黄酮（P-ttaphthoflaVone)、恩貝素（Embelin)、反式肉桂酸 (trans-Cinnamic acid)、表兒茶紛((-)-Epicatechin)、根皮苦(Phloridzin)、Brij 58、Brij 76、Brij 35、Tween 20、Tween 80、Tween 40、PEG 2000、PEG 400、Pluomic F68、PEG 4000。 7. 如申清專利範圍第4項所述之低副作用之異菸鹼醯胺(is〇niazid，j^)新 複方’其中可加入一藥學上可接受之賦形劑至該複方。 8. 如申明專利範圍第了項所述之低副作用之異於驗醯離—城腿所 複方其中該賦形劑可為稀釋劑、填充劑、結合劑、崩解劑、潤滑劑等。200831087 X. Patent application scope: 1. A new compound of low-side side effects testin (isoniazid, INH), including a pharmaceutically effective amount of isoniazid amide (isoniazid, JNH), combined with a pharmaceutically effective amount Disulfimm (DSF), and a pharmaceutically effective amount of bis-p-nitrophenyl phosphate (BNPP). 2_ The isonicotinic acid amide (18-, 1]^) new compound as described in the first paragraph of the patent application, wherein a pharmaceutically acceptable excipient can be added to the compound. 3. The ascorbic acid amide (is〇niazid, Li) new compound as described in the first paragraph of the patent application scope, wherein the excipient may be a diluent, a filler, a binder, a disintegrating agent, Lubricants, etc. 4. A low side effect is different from testisamine (isoniazid, a new compound, including a pharmaceutically effective amount of isoniazid (INH)' combined with a pharmaceutically effective amount of cytochrome p45〇2E1 (CYP2E1) inhibition 5. As a nicotine guanamine (is〇niazid) as described in the fourth paragraph of the patent application scope, the new compound of which 5 cytochrome P450 2E1 (CYP2E1) inhibitor is disulfide (disuifjram, DSF) 〇6· The new side of the isoniazid amide (isnniazid, jnh) as described in the fourth paragraph of the patent application, wherein the cytochrome P450 2E1 (CYP2E1) inhibitor is selected from the following compounds Group·NordihydiOguaiaretic acid, (_)-Epigallocetechin-3-gallat?, CapiUarisin, Kaempferol 'Phloretin, Disulfiram (disulfimm), Hesperetin, 6-Gingerol, ganic acid, isoglycyrrhizin 35 200831087 (Isoliquritigenin), naringenin (Narigenin), indane Peel ((+)-Taxifolin), Hanhuang Ai (he 哗011111), original '(?1*〇1:€^16(:1111^^(^(!), catechin ((+)-€&16〇1^11), β-naphthoflavone (P-ttaphthoflaVone), Embelin, trans-Cinnamic acid, epiphyllo ((-)-Epicatechin), Phloridzin, Brij 58, Brij 76, Brij 35, Tween 20, Tween 80, Tween 40, PEG 2000, PEG 400, Pluomic F68, PEG 4000. 7. As shown in the fourth paragraph of the patent scope, the low side effects of isoniazidamine (is〇niazid, j^) new compound ' A pharmaceutically acceptable excipient may be added to the compound. 8. The low side effect as described in the scope of the patent claim is different from the test of the leg. The excipient may be a diluent, Fillers, binders, disintegrants, lubricants, and the like. 3636
TW96101545A 2007-01-16 2007-01-16 Novel isoniazid compound prescription with a low incidence of side effects TW200831087A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
TW96101545A TW200831087A (en) 2007-01-16 2007-01-16 Novel isoniazid compound prescription with a low incidence of side effects

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
TW96101545A TW200831087A (en) 2007-01-16 2007-01-16 Novel isoniazid compound prescription with a low incidence of side effects

Publications (2)

Publication Number Publication Date
TW200831087A true TW200831087A (en) 2008-08-01
TWI360418B TWI360418B (en) 2012-03-21

Family

ID=44818481

Family Applications (1)

Application Number Title Priority Date Filing Date
TW96101545A TW200831087A (en) 2007-01-16 2007-01-16 Novel isoniazid compound prescription with a low incidence of side effects

Country Status (1)

Country Link
TW (1) TW200831087A (en)

Also Published As

Publication number Publication date
TWI360418B (en) 2012-03-21

Similar Documents

Publication Publication Date Title
Groß et al. K+ efflux-independent NLRP3 inflammasome activation by small molecules targeting mitochondria
Zhou et al. Drug bioactivation covalent binding to target proteins and toxicity relevance
Ogburn et al. Efavirenz primary and secondary metabolism in vitro and in vivo: identification of novel metabolic pathways and cytochrome P450 2A6 as the principal catalyst of efavirenz 7-hydroxylation
Begum et al. NADPH oxidase family proteins: signaling dynamics to disease management
Hartai et al. Kynurenine metabolism in multiple sclerosis
US8552050B2 (en) Activators of pyruvate kinase M2 and methods of treating disease
US8969312B2 (en) Low side effect pharmaceutical composition containing antituberculosis drugs
US8304394B2 (en) Low side effect pharmaceutical composition containing isoniazid
Pulkoski-Gross et al. Novel sphingosine kinase-1 inhibitor, LCL351, reduces immune responses in murine DSS-induced colitis
Hua et al. P7C3‐A20 alleviates fatty liver by shaping gut microbiota and inducing FGF21/FGF1, via the AMP‐activated protein kinase/CREB regulated transcription coactivator 2 pathway
KR101881074B1 (en) New acetaminophen compound composition without side effect to liver
Vaajanen et al. Angiotensin receptors in the eyes of arterial hypertensive rats
Bruderer et al. Investigation of potential pharmacodynamic and pharmacokinetic interactions between selexipag and warfarin in healthy male subjects
KR20180034466A (en) ADO-resistant cysteamine analogs and uses thereof
JP2023519386A (en) Identification and use of compounds in the treatment or prevention of severe acute respiratory syndrome coronavirus 2
Gunay et al. Protective effects of Y-27632 on acute dichlorvos poisoning in rats
JP5901770B2 (en) A pharmaceutical composition for preventing or treating diabetes or fatty liver, comprising a CYP4A inhibitor as an active ingredient.
TW200831087A (en) Novel isoniazid compound prescription with a low incidence of side effects
Fornstedt-Wallin et al. 3-Hydroxyanthranilic acid accumulation following administration of the 3-hydroxyanthranilic acid 3, 4-dioxygenase inhibitor NCR-631
Sehitoglu et al. The effects of apomorphine on paracetamol-induced hepatotoxicity in rats
Xu et al. Inhibitory effect of propafenone on vortioxetine metabolism in vitro and in vivo
TWI552748B (en) The use of a compound for the removal of hepatotoxicity against Acetaminophen (APAP)
TW201016216A (en) New Isoniazid-containing compound with low side effects
TWI666013B (en) Use of compound for removing hepatotoxicity of Acetaminophen (APAP) medicine
JP6858729B2 (en) New acetaminophen complex composition with no side effects on the liver