TWI360418B - - Google Patents

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TWI360418B
TWI360418B TW96101545A TW96101545A TWI360418B TW I360418 B TWI360418 B TW I360418B TW 96101545 A TW96101545 A TW 96101545A TW 96101545 A TW96101545 A TW 96101545A TW I360418 B TWI360418 B TW I360418B
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bnpp
cyp2e1
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U6〇4i8 九、發明說明: .. 【發明所屬之技術領域】 .. 本發明係關於一種低副作用之異菸鹼醯胺(isoniazid, INH)新複方,特別 疋指一種將異終驗醢胺(INH)合併使用細胞色素P450 2E1 (CYP2E1)抑制 劑,或再合併使用硝基苯酚磷酸二酯(BNPP),以降低由異菸鹼醯胺(INH) 所引起之肝毒性等副作用之異菸鹼醯胺新複方。 【先前技術】 鲁 根據世界衛生組織(WHQ)估計,全球大約有三分之_的人口感染肺結 核,每年約有八百萬新增病例;而台灣新登記的肺結核病患人數最近幾年 -也不停崎’每十萬人口有六十多人_肺結核,但其中只有大約四分之 ' —的人接文70整’麟’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,臨床上最常見的藥物副作 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 鲁最為常見。再加上台灣又是B型及c型肝炎的盛行區,感染肺結核之肝炎 患者也不在少數’假設每年有14,咖名新增的肺結核病患,粗略估計至少 # 2,_名到3,_錢性職患者雜紅結_祕療,目此在這些病 •患身上所可能發生的肝毒性是吾人不可忽視的歸性疾病。 • 乡數的第一線抗結核藥物,例如:異於驗醯胺(Μ,俗稱敵賴 •星)、丙基硫異於醯胺㈣—娜,俗稱敵療新邁)及立復黴素时啦㈣等 都有導致肝毒性發生的潜在不良反應;其中異於驗酿胺㈣也邮是目前最 有效的單-抗結核藥物,也最_起服用者產生肝毒性;在6〇年代末期 1360418 陸續有異於驗酿胺(isoniazid)造成肝毒性的報告;異於驗酿胺(is〇niazid)所造 成具有臨床症狀的肝毒性約0J_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(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)迅速被水解成乙酿化聯胺(acetyihydrazine);乙酿化聯胺 了以再經由氮-乙醯氣基轉移酶^ltransferase)被乙酿化成無毒性的雙 乙酿化聯胺(diacetylhydrazine),或者經由細胞色素p45〇 2E1 (CYP 450 2E1) 氧化成具有肝毒性的分子,其中包括乙醯化偶乙醯銨離 子(acetylonium ion)、乙醢自由基(acetyiradicai)、乙稀酮(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’異於驗酿胺(isoni^id)亦可經由 酿胺水解扭(amidase)直接水解成有毒性的聯胺(hydrazine),或者由上述乙酿 化聯胺(acetylhydrazine)經醯胺水解酶(amidase)水解成有毒性的聯胺 (hydrazine)。 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 造成異於驗醢胺引起之肝毒性(INH-induced hepatotoxicity)最可能的主因 4’5,研究者認為異於驗酿胺引起之肝毒性的嚴重性與血聚中聯胺的濃度成正 相關;1999年Sarich6等人的報導則認為對硝基苯酚磷酸二酿 (bis-p-nitrophenyl phosphate,BNPP ’為一種醯胺水解酶之抑制劑)可預防異 菸鹼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 ί S ) 6 1360418 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(CCU)以及乙醯氨紛(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5’1〇。在體外試驗中,雙硫 侖(disulfiram,DSF)及其代謝物二乙基二硫代氨基甲酸 (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)n·13,Brady丨3 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 (immun〇reactive hepatic content)以及CYP2E1催化活性快速且完全的下降。U6〇4i8 IX. Description of the invention: .. [Technical field to which the invention pertains]. The present invention relates to a new compound of isoniazid indamine (INH) with low side effects, in particular, a heterologous prostamine (INH) combined with cytochrome P450 2E1 (CYP2E1) inhibitor, or combined with nitrophenol phosphate diester (BNPP) to reduce side effects such as hepatotoxicity caused by isoniazid amide (INH) A new compound of alkali amide. [Prior Art] According to estimates by the World Health Organization (WHQ), about three-thirds of the world's population is infected with tuberculosis, with about 8 million new cases each year; and the number of newly registered tuberculosis patients in Taiwan has not been in recent years. Nasaki 'more than 60 people per 100,000 population _ tuberculosis, but only about a quarter of them - received 70 full 'Lin' According to the statistics of the Department of Health, at least 4.2 people die of tuberculosis every day in Taiwan; Among the many patients receiving tuberculosis medication, the most common clinical side effects are hepatotoxicity and neurological diseases (such as auditory nerve and optic neuropathy), among which hepatotoxicity is the most common. In addition, Taiwan is also a prevalent area for hepatitis B and hepatitis C. The number of hepatitis patients infected with tuberculosis is not in the minority' assumption that there are 14 new tuberculosis patients in the coffee field every year, roughly estimated at least #2,_name to 3, _ Qianshou patients with miscellaneous red knot _ secret treatment, the liver toxicity that may occur in these diseases and patients is a natural disease that we can not ignore. • The first line of anti-tuberculosis drugs in the township, for example: different from the test of guanamine (Μ, commonly known as the enemy raisin star), propyl sulphide is different from guanamine (four) - Na, commonly known as the enemy treatment Xinmai) and rifamycin Time (4) and other potential adverse reactions leading to hepatotoxicity; which is different from the test of amine (4) is also the most effective single-anti-tuberculosis drugs, and most of the patients produce hepatotoxicity; in the late 6's 1360418 is different from the report of hepatotoxicity caused by isoniazid; it is about 0J_1%|,2, and is less than 1J2% of the clinical symptoms caused by is〇niazid. In patients, asymptomatic liver dysfunction 3 can be observed, and these abnormal liver functions usually occur within two months after taking the drug. As shown in Figure 1, isoniazid in the liver is mainly acetylated by the help of N-acetyltransferase (NAT), and the intermediate product is acetylated isonianic acid. The acetonide (acetylisoniazid) is rapidly hydrolyzed to the acetyihydrazine; the hydrazine is brewed and then converted to a non-toxic double-branched hydrazine via the nitrogen-ethyl hydrazine-transferase. Diacetylhydrazine, or oxidized to cytotoxic molecules via cytochrome p45〇2E1 (CYP 450 2E1), including acetylonium ion, acetyiradicai, ethylene Ketone (ketene), etc., in the presence of aerobic and NADPH, acetaminophen will be reacted by cytochrome P45〇2E1 to generate free radicals, causing oxidative stress to cause cell death; in addition, 'different to the amine (isoni^) Id) can also be directly hydrolyzed to a toxic hydrazine via amidolytic hydrolyzed amidase, or hydrolyzed to a toxic hydrazine by amidase hydrolyzate (amidase) from the above-mentioned acetylhydrazine (hydrazine). Recently, studies have shown that hydrazine (rather than isoniazid guanamine or acetylated hydrazine) is the most likely cause of causing hepatotoxicity (INH-induced hepatotoxicity) in rabbits and mice. 4 '5, the researchers believe that the severity of hepatotoxicity caused by the determination of amines is positively correlated with the concentration of hydrazine in blood polycondensation; in 1999, Sarich6 et al reported that p-nitrophenol phosphate (bis-p- Nitrophenyl phosphate, BNPP 'is an inhibitor of indoleamine hydrolase, prevents hepatotoxicity caused by isoniazid amide, and its protective mechanism should be through inhibition of isoniazid guanamine production ί S ) 6 1360418 hydrazine. Cytochrome P450 2E1 (CYP2E1) is sustained in the liver and is responsible for the metabolic biological reactions of many foreign substances such as hepatic toxin carbon tetrachloride (CCU) and acetaminophen 7,8; However, the role of CYP2E1 in the hepatotoxicity caused by isonicotine amide is not clear. Isoniazid guanamine itself is an inducer of CYP2E1 9; some studies suggest that CYP2E1 and isonicotamine in the liver The mechanism of liver toxicity caused by 5'1〇. 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) ) n, 13, Brady 丨 3 and other tests showed that mice taking a single oral dose of disulfiram (DSF), resulting in immune response liver capacity (immun 〇 reactive hepatic content) and CYP2E1 catalytic activity rapid and complete decline .

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_異構前列腺 素^ (8-iSO-prostaglandin FZa,S-iso-PGFa )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_pGF2a 含I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(〇xidatjve damage)及抗氧化劑的缺乏 (antioxidant deficiency)有關15’16;目前有許多方法可用來測量8_is〇_pGF2c^ 1包括酵素免疫分析法(enzyme immunoassay) 17、放射免疫分析法 7 1360418 (radioimmunoassay)、氣相層析質譜儀(gas-chromatography mass spectrometry) I9以及液相層析質譜儀咖〇_〇啡卿脱沾 spectrometry) 20等’·此外,人類尿液中的8 is〇 pGF2a及其代謝物 • 2,3-din〇r-8-is〇-PGF2a含量可利用 C18 固相萃取(α8 s〇lid 沖脱 extracti〇n, * . SPE)準備樣品後’再以液相層析串聯式質譜儀(LC/MS/MS)分析。 利用侵入式及賴人式方法麟大鼠㈣肝功能,以監断損害的發展 •以及轉肝難病’其巾最常使㈣方法包侧量血清巾之天門冬教酸轉 胺酶(aspartate aminotransferase,AST)、丙氨酸轉胺酶(alanine aminotransferase, ALT)以及驗性鱗酸酶(激&丨jne ph〇Sphatase)數值,以及測量 *肝細胞產物如.膽紅飾llirubin)、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(c〇agulati〇n如㈣等a;肝功能定 量測试疋根谭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 .定,肝臟血流量與提供、給肝臟的受質量有冑,反之,該受質的清除則決定 於肝臟代謝的能力23。 半乳糖(galactose)疋一種具有高萃取率(extraeti〇n rati〇)、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(epimerization),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(rate-limiting step)24 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 1360418 疋的規則來a平估大鼠之殘餘肝功能(residuai iiver如似^),量測—確切化 合物(如:半紐)之代魏力,可細聰巾―代謝侧之鲜決定步輝, 亦可能取得殘餘肝功能之代表數質24,2、 以半乳糖π除能力(galactose eliminati〇n哪扣办,GEC)作為人類肝功能 疋量測試26已行之有年H半雛清除能力職需取得多個i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 單點法(Galactose Single Point,GSP)以評估人類肝功能;本案發明人以半乳 糖單點法概慢崎《、職似及職病患,絲顯科㈣單點法可 精確測出這些肝臟疾病27;半乳糖賴法已彳域功的朗酬試肝病患者排 除如丙嗪(promazine)及抗生素頭孢酮(cef〇peraz〇ne)等藥物之剩餘肝功能 28-30 〇 l L 一 夕,半乳糖單點法已在美國食品藥物管理局(FDA)所出版的指南 (GuidanceforIndustry)中成為建議採用測試肝功能的方法之一 31。 了見上述各用抗結核藥物異於驗醯胺(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。 本案發明人祕上述制抗結補物胁祕_sQniazid)所導断毒 等田i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, 〜成功研發完成本件低副侧之跡驗_(—d,願)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终驗醯胺㈣^d,麵) '复方將異終驗酿胺(臟)合併使用細胞色素P45〇2ei (cy?2ei)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。 1360418 本發明之次一目的係在於提供一種低副作用之異菸鹼醯胺(isoniazid, INH)新複方,將異於驗醯胺(INH)合併使用細胞色素p45〇2El (CYP2E1)之 選擇性抑制劑雙硫侖(;DSF),以及醯胺水解酶之抑制劑硝基苯酚磷酸二酯 (BNPP) ’以降低由異終驗酿胺(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P450 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(iNH)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8-iso-PGF2a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菸鹼醯胺(isoniazid,INH)新複 方,係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(disulfiram, DSF)、柚皮素(Narigenin)、撥皮素(Hesperetin)、 槲皮素(quercetin)、正二羥癒瘡酸(Nordihydroguaiareticacid)、茵陳色原酮 (Capillarisin)、原兒茶酸(protocatechuic acid)、沒食子酸(gainc acid)、熊果 酸 (ursolic acid)、(-)-Epigallocetechin、(-)-Epicetechin-3-gallate、 ㈠-Epigallocetechin-3-gallate、Tween 20、Tween 40、Tween 60、Tween 80、 10 1360418Sodhi14 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 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; _Iso-prostaglandin FZa (S-iso-PGFa) is a product of free radical-induced lipid peroxidation of arachidonic acid, which is chemically stable, 8_is〇_pGF2a I 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 15'16 There are currently many methods for measuring 8_is〇_pGF2c^1 including enzyme immunoassay 17, radioimmunoassay 7 1360418 (radioimmunoassay), gas-chromatography mass spectrometry I9 and Liquid chromatography mass spectrometer curry _ 〇 卿 卿 spect spect spect spect spect spect spect spect 20 20 20 · · · · · · 人类 人类 人类 人类 人类 人类 人类 人类 人类 人类 人类 人类 人类 人类 人类 人类 人类 人类 人类 人类-8-is〇-PGF2a content can be determined by C18 solid phase extraction (α8 s〇lid detachment extracti〇n, * . SPE) after preparation of the sample 're-liquid chromatography tandem mass spectrometer (LC/MS/MS) analysis. Using invasive and Lai-style methods Lin rats (four) liver function, to monitor the development of damage • and liver refractory disease, its towel most often (four) method side of the serum towel asparagine acid transaminase (aspartate aminotransferase , AST), alanine aminotransferase (ALT), and auxin luciferase (exciting & nejne ph〇Sphatase) values, as well as measuring * liver cell products such as bilirubin llububin, albumin (albumin), and the use of prothrombin time to detect coagulation factors (c〇agulati〇n such as (d), etc. a; liver function quantitative test 疋根谭 almost only through the liver metabolism of the receptor in serum In terms of the concentration, the removal of these receptors is based on the hepatic portal vein, hepatic artery blood flow, and the effect of hepatocytes on these receptors. The liver blood flow is provided and the quality of the liver is impaired. The removal of the receptor is determined by the ability of the liver to metabolize. 23. Galactose, a sugar with high extraction rate (extraeti〇n rati〇), metabolized in the liver 'in the liver' is galactose Kinactin Ase) undergoes epimerization to convert it to Glucose-1-phosphate; the action of galactose kinase is the rate-determining step of the galactose metabolic pathway in hepatocytes (rate- Limiting 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 1360418 来 to assess the residual liver function of rats ( Residuai iiver, like ^), measurement - the exact compound (such as: half-new) generation of Wei Li, can be fine-skin towel - the metabolic side of the fresh decision step, can also obtain the representative liver quality of the representative quality 24,2 The ability to remove galactose π (galactose eliminati〇n, GEC) as a test for human liver function tests 26 has been carried out for a year, half-clearing ability, and multiple liquid samples to establish a standard curve 'in clinical There are many difficulties in application, so many studies use Galactose Single Point (GSP) to evaluate human liver function; the inventor of this case used galactose single-point method to slow down, "like and occupational patients" Silk display (4) single point method can accurately detect these liver diseases 27; galactose Lai has been used in the field of liver disease patients to exclude such drugs as promazine and antibiotic cefotaxone (cef〇peraz〇ne) The remaining liver function is 28-30 〇l L. 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) (Guidance for Industry). Seeing that the above-mentioned anti-tuberculosis drugs are different from is〇niazid, there are still many defects, which is not a good designer, but needs to be improved. The inventor of this case secreted the above-mentioned system of anti-resistance threats _sQniazid. The shortcomings of the action of detoxification and other fields were improved and innovated, and after years of painstaking efforts and research, ~ successfully developed the low side of this piece. Trace inspection _ (- d, willing) new compound. SUMMARY OF THE INVENTION The object of the present invention is to provide a low side effect of the different final amine (4) ^ d, face) 'combination of the different final amine (dirty) combined with the use of cytochrome P45 〇 2ei (cy? 2ei) Inhibitors to reduce side effects such as hepatotoxicity caused by isoniazidamine (INH). 1360418 A second object of the present invention is to provide a novel compound of isoniazid amide (INH) with low side effects, which is selective for inhibition of guanamine (INH) combined with cytochrome p45〇2El (CYP2E1). Disulfiram (DSF), and inhibitor of guanamine hydrolase, nitrophenol phosphate diester (BNPP), to reduce side effects such as hepatotoxicity caused by different final amine (INH). In order to achieve the low side effect of the above-mentioned invention, a new compound of isoniazid amide (is〇niazid, _), the present invention firstly induced hepatotoxicity in rats by isoniazidamine (INH) as a model, research cells Pigment P450 2E1 (CYP2E1) inhibitor Disulfiram (DSF), and indoleamine hydrolase (amidase) inhibitor nitrophenol phosphodiester (BNPP) on liver induced by isonicotinicinamide (iNH) in rats The effects of toxicity; in addition to the use of general hepatotoxicity markers, galactose single point method (GSP) and galactose clearance capacity (GEC) for quantitative measurement of residual liver function in rats, the inventors of the present invention use improved liquid layer The concentration of 8-iso-PGF2a in rat plasma was measured by tandem mass spectrometry (LC/MS/MS) to further determine whether 8-iso-PGF2〇^ was associated with hepatic toxicity induced by INH in rats. A new compound of isoniazid (INH) which achieves the low side effects of the above object of the invention includes a pharmaceutically effective amount of isoniazid (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 disulfiram (DSF), naringin (Narigenin), hesperetin, quercetin ), Nordic hydroguaiaretic acid, Capillarisin, protocatechuic acid, gainc acid, ursolic acid, (-)- Epigallocetechin, (-)-Epicetechin-3-gallate, (a)-Epigallocetechin-3-gallate, Tween 20, Tween 40, Tween 60, Tween 80, 10 1360418

Bnj 35、Brij 58 ' Brij 76、PEG 2000、PEG 4000 ·. 了達成上述發明目的之低副作用之異於驗酿胺(isoniazid,INH)新複 方,係包括一樂學有效量之異於驗醒胺(丨如“肛丨小jjsjh),合併使用一藥學有 * 效量之雙硫侖(disuIflram,DSF),以及一藥學有效量之硝基苯酚磷酸二酯 • (bis-/?-nitrophenyl phosphate, BNPP) 〇 本發明所k供之低副作用之異於驗酿胺(is〇niazjld,j]^H)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#1、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。 實施例一異菸鹼醢胺(INH)合併使用€:¥1>21:1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(Fairfldd,〇H, USA) ’ INH,BNPP,DSF以及玉米油則賭自地咖化學公司⑼几。此,m〇 USA) ’ 8_ls〇-PGF2«以及放射線標定之8-iso-PGF2a-d4則得自Cayman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 備’係將400克半乳糖(Sigm雜於α升含有適當緩衝雜系統以及等張鹽 類之蒸餾水中,供作注射使用。Bnj 35, Brij 58 ' Brij 76, PEG 2000, PEG 4000 ·. The low side effect of achieving the above-mentioned object is different from the new compound of the testisamine (INH), which includes a learning effective amount different from the wake-up Amines (such as "anal fistula small jjsjh"), combined with a pharmaceutically effective amount of disulfiram (DSF), and a pharmaceutically effective amount of nitrophenol phosphate diester (bis-/?-nitrophenyl phosphate) , BNPP) The low side effect of the present invention is different from the new compound of the test amine (is〇niazjld, j]^H), and a pharmaceutically acceptable excipient may also be added to the compound. The agent may be a diluent, a filler, a compound #1, a disintegrant, a lubricant, etc. [Embodiment] - The present invention will be further illustrated by the following examples, but the examples are for illustrative purposes only. It should not be construed as limiting the practice of the invention. Example 1 isoniaceine guanamine (INH) combined with €:¥1>21:1 inhibitor disulfiram (DSF) and/or nitrophenol phosphate II Animal Test of Ester (BNPP) I. Materials and Methods • 1. All organic solvents of the test materials are HPLC grade. Tedia Co., Ltd. (Fairfldd, 〇H, USA) 'INH, BNPP, DSF and corn oil are gambling from the local coffee chemical company (9). This, m〇USA) '8_ls〇-PGF2« and radiation-calibrated 8-iso- PGF2a-d4 was obtained from Cayman Chemical Company (Ann Arbor, MI'USA), and the galactose injection solution was prepared by Nanguang Chemical Pharmaceutical Co., Ltd. '400 g of galactose (Sigm mixed with α liter containing appropriate buffer system and etc. Distilled water in salt, for injection.

11 1360418 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 戊巴比妥鈉(sodium pentobarbital)進行腹腔麻醉(intraperitoneally anesthetized),將聚乙烯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)插入,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,VEH1 ’即食鹽水),BNPP係溶於加熱至6〇。〇 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100mg/kgDSF或DSF之基劑(VEH2,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15分鐘處理。 上述5組試驗共包含: 12 1360418 (1) 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2i天; (2) INH組(INH,n=7):正常的大鼠每天注射1次、y£H1以及 (施行腹腔内注射)共21天; (3) BNPP-INH組(BNPP-INH,n=7):正常的大鼠每天注射丨&BNPP、 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、腿以 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,大鼠以乙醚麻醉犧牲,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13,000g於4eC離心15分鐘,分離後 的血聚分裝到微量小管(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 13 (S ) 之福馬林(phosphate-buffered formalin)固定,隨後脫水並包埋於石蠟(paraffm) 中’以5 μηι厚度切片’切片樣本以棘木精(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,肝臟切片以二曱胂緩衝液(cac〇dyiate buffer, 0.1 Μ pH 7.4)清洗,以20%四氧化蛾水溶液(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’以醋酸轴酿(uranylacetate)及檸檬酸錯(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。 7.8-iso-PGF2a的萃取與量測 所有PGFh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0.5ml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_漿以C18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LC-10ADvP泵(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, 内徑50 X 2.1mm)進行HPLC分離,並使用含有2mM醋酸錄(ammonium acetate) 1360418 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’1^/]\48/1'48的流速均維持在2〇0卜1/111丨11,整個11?1^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,配備有一11 1360418 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 the air/humidity adjustment environment, the light and the darkness were each 12 hours, and the supply of water and feed was not limited. The weight of the rats was continuously monitored during the test. All the rats were dosed with 50 mg/kg body weight of pentobarbital. Sodium pentobarbital was intraperitoneally anesthetized, and a polyethylene catheter was placed in the rat internal jugular vein to galactose. The catheter was inserted by a cut-down technique. 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 The test animals were randomly divided into 5 groups. Each group included 3 treatments. The first treatment was injection of 25 mg/kg BNPP or BNPP (vehicle, VEH1 'ready saline), and BNPP was dissolved to 6 Hey. Salined saline (0.9% NaCl), after cooling, was intraperitoneally injected into the rat in a volume of 1 ml/kg; the second treatment was to inject 100 mg/kg of DSF or DSF (VEH2, 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 a 150 mg/kg INH or INH base (VEH3 'ready saline). INH was dissolved in saline (0.9% NaCl) by intraperitoneal injection into the rat in a volume of 1 ml/kg; the first group (BNPP or VEH1) was 30 minutes earlier than the third group (INH or VEH3). Treatment, the second group (DSF or VEH2) was processed 15 minutes earlier than the third group (INH or VEH3). The above five groups of experiments included: 12 1360418 (1) Control group (normal control group, NC, n=12): normal rats were injected once a day for VEH1, VEH2 and VEH3 (administered intraperitoneally) for 2i days; 2) INH group (INH, n=7): normal rats were injected once a day, y£H1 and (administered intraperitoneally) for 21 days; (3) BNPP-INH group (BNPP-INH, n=7) ): Normal rats were injected with 丨 & BNPP and VEH2 (administered intraperitoneally) daily for 21 days; (4) DSF-INH group (DSF-INH, n=7): normal rats were injected daily with ^DSF , legs and VEH1 (administered intraperitoneally) for a total of 21 days; and (5) BNPP-DSF-INH group (BNPP-DSF-INH, n=7): normal rats were injected once a day with BNPP, DSF and INH ( A total of 21 days were administered intraperitoneally; the galactose single point method was tested 16 hours after the 21st day of treatment. 4. After the blood sample was processed, the rats were sacrificed by ether anesthesia. The blood was taken from the rat aorta and placed in a test tube containing EDTA. The plasma was centrifuged at 13,000 g for 15 minutes at 4 ° C. The blood was collected after separation. Dispense into a small tube (Eppendorf tube) and store at -80 °C. 5. Biochemical analysis of hepatocyte injury to measure aspartate aminotransferase (AST) and alanine transaminase (ALT) activity in plasma for quantification 'AST and ALT activity are commonly used indicators of liver toxicity, The Synchron LXi 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 13 (S) phosphate-buffered formalin in 10% phosphate buffer, then dehydrated and embedded in paraffin ( Paraffm) 'Skin sliced at 5 μηι thickness' slice was stained with hematoxylin and eosin, and subjected to periodic acid Schiff stain (PAS), stained and histologically studied by light microscopy Observe, in addition, the liver sections were washed with buffer buffer (cac〇dyiate buffer, 0.1 Μ pH 7.4) and fixed with 20% aqueous osmium tetroxide for 1 hour. In Spurr resin (Spurrresin), ultra-thin sections were cut with diamond knives, double stained with uranylacetate and leadcitrate, and transmitted by electron microscopy (Transmission Electron Microscope, Hitachi 600, Hitachi). Co., Japan) observed. Extraction and measurement of 7.8-iso-PGF2a All isoforms of PGFh were dissolved or diluted in an appropriate volume of alcohol to prepare stock solution 'packed in small tubes and stored at -70 ° C, 0.5 ml Plasma to glass tube, add i〇ng internal standard (8-iso-PGF2a-d4) 'mixed blood _ slurry with C18 solid phase extraction cartridge (Solid-Phase Extraction cartridge, JT Baker , MA, USA) Purified, the sample stream washing solution was evaporated to dryness with nitrogen, 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 an LC/MS/MS system for analysis. 8. Liquid Chromatography Tandem Mass Spectrometer (LC/MS/MS) Analytical HPLC system consisting of 2 Shimadzu LC-10ADvP pumps (Shimadzu LC-10ADvP pumps), 1 Shimadzu system control, and 1 Shimadzu autosampler (Shimadzu autosampler) (Shimadzu Scientific Instruments, 曰本), HPLC separation with C18 column (particle size 5-μιη, inner diameter 50 X 2.1mm), and using 2mM acetate acetate 1360418 and Gradient flow wash of acetonitrile (ACN) (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) The flow rate of '1^/]\48/1'48 was maintained at 2〇0b1/111丨11, and the entire 11?1^ time was 13.5 minutes; The system is interfaced with a triple stage quadrupole mass spectrometer (API3000, Applied Biosystem, Foster City, CA, USA).

TurboIonSpray離子源(TurboIonSpray ionization source),並使用負電電喷霧 (negative electrospray)作為電離(ionization)之方法;該質譜儀藉由擴散200 鲁 ng/ml 8-iso-PGF2ct 或 8-iso-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(丨〇11卩3丨〇則個別用來監測8七〇-?0?2〇1以及8-丨8〇~?0?2〇1-(14;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(Y),得到相關係數(r, correlation coefficient)值為0.999 ;血漿 t8-iso-PGF2a的線性範圍在0.1-2.5ng/ml之間, 其迴歸方程式(regression 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(reiative em)rs)範圍在-5J6%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0.5 g/kg ; 15 (S ) 1360418 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(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(coe伍cient 〇f variation,CV)百分比計算’最大容許的變異係數為i〇% cv ;曰間差異 (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:TurboIonSpray ionization source, and use negative electrospray as ionization method; the mass spectrometer diffuses 200 ng/ml 8-iso-PGF2ct or 8-iso-PGF2a- The d4 standard was optimized by multiple reaction monitoring (MRM) mode, m/z 353/193 and m/z 357/197 ion couples (丨〇11卩3丨〇 were used to monitor 8-7 individually) 〇-?0?2〇1 and 8-丨8〇~?0?2〇1-(14; after measurement, calculate the linear calibration curve of six 8-iso-PGF2a concentrations (C) 8-iso-PGF2a is in the region of 8-iso-PGF2a-d4 ratio (Y), and the correlation coefficient (r, correlation coefficient) is 0.999. The linear range of plasma t8-iso-PGF2a is 0.1-2.5 ng/ml. The regression equation is Y=-0.0517C +0.823 ng/ml; the measured results are calculated according to the standard of the hydrogenated 8-iso-PGF2a (deuterated 8-iso-PGF2a) standard curve. The inter-batch precision and accuracy were evaluated by standard-concentration samples after 6 times, and then evaluated by Back-Calculation. The relative error rs) ranged from -5J6% to 3.13%. 9. Quantitative testing of liver function All rats were tested for galactose single point (GSP) and galactose clearance (GEC) tests. Rats received a rapid intravenous injection within 30 seconds of injection of 4.4g/ml BW galactose solution 0.5 g/kg; 15 (S) 1360418 Blood was collected from 5, 10, 15, 30, 45 and 60 minutes after injection. The blood sample was taken from the tail vein. The galactose content was measured by the colorimetric galactose dehydrogenase. The test concentration ranged from 50 to 1,000 pg/ml, and the intra-orbital difference of each concentration (within-day) Variation) is calculated from the standard deviation and the coefficient of variation (coe cient 〇f variation, CV). The maximum allowable coefficient of variation is i〇% cv; the day-to-day variation is determined by The slope and intercept of the calibration curves are compared to test; the galactose clearance capacity (GEC) is calculated by the following formula, which is modified by Tygstrup, s Equation 32:

D GEC ---(mg / kg · min) Τ〇=〇 + 7 其中D為半乳糖之注射量;tc=0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分鐘的血液濃度-時間曲線之線性迴歸推 得;7為依經驗法則修正體内不均勻分布之校正值;半乳糖單點法(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)測試法來計算是否具有統計上的顯著差異,使用D GEC ---(mg / kg · min) Τ〇=〇+ 7 where D is the amount of galactose injected; tc=0 is the time required for the galactose concentration to reach 〇 by injection (usually 2.22 mmol/ 1) The linear regression of the blood concentration-time curve after 20 to 60 minutes is derived; 7 is the correction value for correcting the uneven distribution in the body according to the rule of thumb; the galactose single point method (GSp) is 30 seconds after the injection is stopped. The concentration of galactose in the blood at the minute. 10. Statistical Analysis All data are expressed as mean soil standard deviation (SD). The test results are calculated by the one-way variance analysis (ANOVA) test to determine whether there is a statistically significant difference.

Package of the Social Science program (Version 13, SPSS Inc.)套裝軟體來計 算;隨後使用事後比較(post hoc test)最小差異顯著性(least 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。 16 1360418 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)活性明顯高於對照組(對照組血聚 中的AST活性為ii6±il iu/L ; INH組血漿中的AST活性為129±10 IU/L,p c 〇·〇5 ;對照組血漿中的ALT活性為44土6 IU/L ; INH組血漿中的ALT活性為52 ± 3 IU/L,p < 〇.〇5),顯示INH组產生生化上的肝損傷;對照組、、 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 靜脈輕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33,而CYP2E1會導致超氧基(superoxide)以及氫氧自由基(hydroxyl 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 1360418 研究相符。而其他試驗組:BNPP-INH組、DSF-ΙΝΗ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.血液樣本中尽的量測 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-iS0-PGF2a-d4最大量之分子離子為質荷比(rn/z)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_is〇_pGF2a_d4&子離子(daughter ions)悝較8-iso-PGF2。的子離子高四個單位之外,8-iso-PGF2a以及 8_iso_PGF2a_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); S-iso-PGFh最密集之子離子為質荷比(m/z)193之離子,8_is〇_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 l〇〇Pg8-iso-PGF2c^250pg/ml8-iso-PGF2a-d4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The package of the Social Science program (Version 13, SPSS Inc.) is packaged with software; then multiple comparisons are made using the post hoc test of the least significant difference method to confirm significant differences between groups; The significant difference in ethnic group average was P < 0.05. 16 1360418 II. 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 biochemical analysis results are shown in Figure 2, only the plasma 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 blood group of the control group was ii6±il iu/L; in the plasma of the INH group) The AST activity was 129±10 IU/L, pc 〇·〇5; 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, p < 〇5), showing biochemical liver damage in the INH group; the serum transaminase concentration in the control group, 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. 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 vein of the hepatic lobules. The hepatic sinusoids are in the two liver plates (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 hepatocyte 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 Fig. 3C) (as shown in Fig. 3D). ). According to the literature, INH is a potent inducer of cytochrome P450 2E1 (CYP2E1), and CYP2E1 causes superoxide and hydroxyl radicals to produce 34' and induce endoplasmic The net increase of 35, so the results of this test is consistent with the previous 17 1360418 study. In other test groups, the degree of liver damage in the BNPP-INH group, the DSF-ΙΝΗ group, and the BNPP-DSF-INH group was not significantly different from that of the control group (no results were shown). 3. Measurement in the blood sample In the negative electrospray mode, the maximum molecular ion of 8-iso-PGF2a is the maximum molecular weight of ion 8-iS0-PGF2a-d4 of mass-to-charge ratio (m/z) 353. The ions are ions of mass-to-charge ratio (rn/z) 357. These negatively charged molecular ions are freed by a large number of collisions. The molecular structure of the two target compounds and the resulting ion spectra are shown in Figure 4; except for 8_is〇 The _pGF2a_d4& daughter ion is better than 8-iso-PGF2. The fragmentation patterns of 8-iso-PGF2a and 8_iso_PGF2a_d4 are similar except that the daughter ion is four units higher, which shows that most stable daughter ions are produced from the A chain, which is derived from the A chain. There are 4 deuterium atoms; the most dense daughter ions of S-iso-PGFh are ions with mass-to-charge ratio (m/z) 193, and the most dense daughter ions of 8_is〇_PGF2a_d4 are mass-to-charge ratio (m/z). 197 ions. Figure 5 shows a standard solution containing l〇〇Pg8-iso-PGF2c^250pg/ml8-iso-PGF2a-d4 and a typical LC/MS/ of a blood sample under multiplex reaction monitoring mode (MRM) detection. MS chromatography, after injecting lng8-iso-PGF2a-d4 as an 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. Concentration of 8-iso-PGF2a in plasma

血聚中的8-iso-PGF2a是一種氧化屋力(oxidative stress)的指標,如圖六所 不,相較於對照組’ INH組大鼠血漿中8-iS0-PGF2(^濃度明顯增加(INH組大 鼠血漿中8-iso-PGF2a的濃度為151 ±26 pg/ml ;對照組大鼠血漿中8-iso-PGF2a 的濃度為 110±15 pg/ml,;?&lt; 0.001);與INH組相較,BNPP-INH組、DSF-INH 1360418 組、BNPP-DSF-INH組三組則明顯降低由xnh引起肝臟的8-iS0-PGF2a產生 (BNPP-INH組大鼠血漿中8-iso-PGF2(^濃度為 128±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 8-iso-PGF2W濃度為 123±17 pg/ml ; INH組大鼠血漿中8-iso-PGF2(^濃度為 151±26 pg/ml,p &lt; 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-PGF2c^濃度無顯著差 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-丨3〇屮0?2(1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士87 pg/ml,p &lt; 0.001),此外,BNPP-INH 組、DSF-INH 組、BNPP-DSF-INH 組大鼠之 GSP 值各為 401 土70 pg/m卜 449±45 pg/ml' 388±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p值各為^〈O.OOljsO.Oi^and/x 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计上的差異(ρ=〇· 1 ),而對照組、 ΒΝΡΡ-ΙΝΗ組、DSF-INH組、BNPP-DSF-INH組四組之間大鼠的GSP值無 顯著差異存在。8-iso-PGF2a in blood pooling is an indicator of oxidative stress, as shown in Figure 6. Compared with the control group, the concentration of 8-iS0-PGF2 in the plasma of INH group (the concentration of ^(I) is significantly increased ( The concentration of 8-iso-PGF2a in the plasma of INH group was 151 ±26 pg/ml; the concentration of 8-iso-PGF2a in the plasma of control group was 110±15 pg/ml,;?&lt;0.001); Compared with the INH group, the BNPP-INH group, the DSF-INH 1360418 group, and the BNPP-DSF-INH group significantly reduced 8-iS0-PGF2a production in the liver caused by xnh (8-iso in the plasma of BNPP-INH group). -PGF2(^ concentration was 128±29pg/ml; the concentration of 8-iso-PGF2〇 in plasma of DSF-INH group was 126±20 pg/ml; 8-iso- in plasma of BNPP-DSF-INH group) The concentration of PGF2W was 123±17 pg/ml; the concentration of 8-iso-PGF2 in the plasma of INH group was 151±26 pg/ml, p &lt;0.005; it is worth noting that the control group and BNPP-INH group There was no significant difference in plasma 8-iS0-PGF2c^ concentration between the four groups of DSF-INH group and BNPP-DSF-INH group. Compared with BNPP-INH group and DSF-INH group, INH combined with BNPP and DSF does not further reduce the concentration of 8-丨3〇屮0?2 in plasma (1. 5. Measurement of residual liver function) As shown in Figure 7, the galactose single point method (GSP) values of the control group and the INH group were highly significant (GSP value of 384±69 pg/ml in the control group; GSP in the INH group) The value was 565 ± 87 pg/ml, p &lt; 0.001). In addition, the GSP values of the BNPP-INH group, the DSF-INH group, and the BNPP-DSF-INH group were 401 soils, 70 pg/m, 449±45. Pg/ml' 388±53 pg/m b Compared with the INH group, the GSP values of the BNPP-INH group, the DSF-INH group, and the BNPP-DSF-INH group were significantly different from those of the INH group ( The p-values were ^<O.OOljsO.Oi^and/x 0.001); the GSP values of rats administered with INH alone were significantly increased; however, rats in combination with BNPP or DSF or BNPP and DSF in INH were resistant. On the other hand, compared with the DSF-INH group, INH combined with BNPP and DSF showed reduced hepatotoxicity induced by inH' although the difference between the two did not reach a statistical difference (ρ=〇· 1), but there was no significant difference in GSP values between the control group, the sputum-sputum group, the DSF-INH group, and the BNPP-DSF-INH group.

(S 19 136〇418 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,&lt; 0.001),此外,BNPP-INH 組、DSF-INH 組、BNPP-DSF-ΙΝΉ 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±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;7值各為ρ &lt; 〇·〇〇5,ρ &lt; 0.05, and/? &lt; 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DSF-INH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-DSF-INH組大鼠之GEC值各為 4.3±0.4 mg/min.kg、4.7±0.5 mg/min.kg, /? = 0.29);此外,對照組、BNPP-INH 組、DSF-INH 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 试(如:GSP以及GEC)是否相關’以數種相關分析計算後,發現gsp值與 血滎中8-iso-PGF2a的濃度具有高度相關(如圖九所示),相關係數為〇 836 ; GSP值與GEC值具有高度相關(如圖十所示)②&lt; 〇 〇〇1),相關係數為_ 〇_822;GEC值也與血聚巾S-iso-PGFh的濃度具有高度相關(相關絲為_ 〇.743,ρ&lt;0.0(Π,如表一所示);而Gsp值、GEC值以及血浆中8 is〇 pGF2a 的濃度則與AST及ALT均無明顯相關(如表一所示)。 20 1360418 表一 GSP、GEC以及8-iso-PGF2a與生化測試之相關性 GSP GEC 8-iso-PGF2a AST r = 0.114 r = -0.111 r = 0.217 ALT r = 0.016 r = 0.039 r = 0.035 8-iso-PGF2a r = 0.836* r =-0.743* r= 1* 以皮爾森氏相關係數(Pearson’s correlation coefficient)作為統計計算,*p&lt;0.001 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(S 19 136 〇 418 similar results can be observed using the galactose clearance capacity (GEC) method as shown in Figure 8. Compared with the control group, the GEC value of the INH group was significantly reduced (INH group) The GEC value of the rat was 3.4±0.6 mg/min.kg; the GEC value of the control rats was 4.9±0.8 mg/min.kg, &lt; 0.001), in addition, the BNPP-INH group and the DSF-INH group The GEC values of the BNPP-DSF-ΙΝΉ group were 4.5 soil 0.6 mg/min.kg, 4.3 ± 0.4 mg/min.kg, and 4.7±0.5 mg/min.kg; compared with the INH group, BNPP-INH The GEC values of the rats in the DSF-INH group and the BNPP-DSF-INH group were significantly different from those in the INH group (the 7 values were ρ &lt; 〇·〇〇5, ρ &lt; 0.05, And/? &lt;0.005); GEC values were significantly reduced in rats administered INH alone; however, rats in either INH with either BNPP or DSF or BNPP and DSF recovered this change; compared with the DSF-INH group ' INH combined with BNPP and DSF had a tendency to increase GEC values (GEC values of DSF-INH group and BNPP-DSF-INH group were 4.3±0.4 mg/min.kg, 4.7±0.5 mg/min.kg, respectively) , /? = 0.29); In addition, the control group, BNPP-INH group, DSF-INH group, BNPP-DSF-INH There were no significant differences in GEC values between the four groups. To determine whether AST, ALT, plasma 8-iso-PGF2a concentrations, and quantitative liver function tests (eg, GSP and GEC) were related to several After correlation analysis, it was found that the gsp value was highly correlated with the concentration of 8-iso-PGF2a in blood stasis (as shown in Figure IX), and the correlation coefficient was 〇836; the GSP value was highly correlated with the GEC value (as shown in Figure 10). 2&lt; 〇〇〇 1), the correlation coefficient is _ 〇 _822; the GEC value is also highly correlated with the concentration of S-iso-PGFh (the relevant silk is _ 〇.743, ρ &lt; 0.0 (Π, 如Table 1 shows); Gsp values, GEC values, and concentrations of 8 is〇pGF2a in plasma were not significantly correlated with AST and ALT (as shown in Table 1). 20 1360418 Table 1 GSP, GEC, and 8-iso- Correlation between PGF2a and biochemical tests GSP GEC 8-iso-PGF2a AST r = 0.114 r = -0.111 r = 0.217 ALT r = 0.016 r = 0.039 r = 0.035 8-iso-PGF2a r = 0.836* r =-0.743* r = 1* with Pearson's correlation coefficient as a statistical calculation, *p&lt;0.001 Example 2 Cytochrome P450 2E1 (CYP2E1) inhibitor A screening φ, Materials and Methods 1. Test materials

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10種賦型劑進行細胞色素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P45〇 2E1 (CYP2E1)以及其螢光性受質 MFC (7-Methoxy-4-trifluoromethyl coumarin)的 • 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(acent〇itrile),測試不同濃度之中藥藥引(66μΜ, 33μΜ,16.5μΜ)及賦形劑(0.167%,〇.〇8%,0.042%,w/v)對 CYP2E1 之抑制 率,所測s式之中藥藥引及結果如表三所列,所測試之賦型劑及結果如表四 所列。 另外,本實施例使用之細胞色素P45〇2El (CYP2E1)抑制劑之篩選套組 21 1360418 (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-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)。In this example, a cytochrome P450 2E1 (CYP2E1) inhibitor screening kit (CYP2E1 High Throughput Inhibitor Screening Kit, BD Bioscience, USA) was used to perform cytochrome P45〇2E1 on 22 kinds of Chinese medicines and 10 kinds of excipients. Screening of CYP2E1) inhibitors; the screening set of the CYP2E1 inhibitor works by containing cytochrome P45〇2E1 (CYP2E1) and its fluorescent acceptor MFC (7-Methoxy-4-trifluoromethyl coumarin) After adding the test sample to the environment, the amount of CYP2E1 metabolite standard HFC (7-Hydroxy-4-trifluoromethyl coumarin) was detected, and the test sample was calculated based on the HFC production of the control group. Inhibition rate. Each test sample was dissolved in acetonitrile (acent〇itrile), and tested at different concentrations of the drug (66 μΜ, 33 μΜ, 16.5 μΜ) and excipients (0.167%, 〇.〇8%, 0.042%, w/v) The inhibition rate of CYP2E1, the measured results of the s-type drug and the results listed in Table 3, the tested excipients and results are listed in Table 4. In addition, the screening kit 21 1360418 (CYP2E1 High Throughput Inhibitor Screening Kit, BD Bioscience, USA) used in the cytochrome P45〇2El (CYP2E1) inhibitor of 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-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。 (5) Cofactors :含有 1.3 mM NADP+、66 mM MgCl2 以及 66 mM Glucose 6-Phosphate o (6) Glucose 6-Phosphate Dehydrogenase : 40 units/ml 溶於 5 mM Sodium(4) Stop Solution : 0·5 M Tris Base. (5) Cofactors: containing 1.3 mM NADP+, 66 mM MgCl2 and 66 mM Glucose 6-Phosphate o (6) Glucose 6-Phosphate Dehydrogenase: 40 units/ml Dissolved in 5 mM Sodium

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 acid) : CYP2E1 選擇性抑制劑(陽性對 照組),20mMDDTC 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)。 (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。Citrate Buffer (pH 7.5) ° (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) HFC (7-Hydroxy-4-trifluoromethyl coumarin): CYP2E1 metabolite standard, 0.25 mM HFC dissolved in 0.1 M Tris (pH 9.0). (10) NADPH-Cofactor Mix: Add 18.7.5 μΐ Cofactors ' 150 μΐ G6PDH (Glucose 6-Phosphate Dehydrogenase Solution) and 100 μΐ Control Protein to 14.56 ml of sterile water.

(S 22 1360418 (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,營光性受質)。 2.細胞色素P45〇2El(CYP2El)抑制剤之篩選 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)進行中藥藥引及賦 型劑之篩選,實驗步驟如下所述: (!)製備對照組: .於96孔盤上第1孔井(well)内加入149μ1ΝΑ〇ρΗ (:〇ί^〇ΓΜ^以及 1 μΐ 20mM DDTC並混合均勻; •於該96孔盤上第2至12孔井内各加人1〇〇 μ1 c〇facW acet〇n.trik mix第至8孔井為陽性對照組㈣出;第9與第⑺孔 井為對照組(_!);第u與第u孔井為空白對照組(_ ; 於該第1至8孔軸連續稀釋動作:自第⑽内取5M液體加 入第2孔井内混勻,再自第 弟2孔井内取5〇μι液體加入第3孔井内混 勻,以此類推,至第8孔祙吐+ 开時去除多餘的5〇 μΐ液體,以得到連續稀 釋濃度 66.6、22.2、7.4、2 47 λ ·47、〇·82、0.27、0.091、0.03 μΜ。 23 1360418 (2)製備試驗組: a. 於96孔盤上第1行的第1及第2孔井内各加入149 μΐ NADPH-Cofactor Mix’以及Ιμΐ 20mM中藥藥引測試樣品或ΐμΐ 25% (w/v)賦形劑測試樣品,並混合均勻; 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 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3r&gt;c靜置1〇分鐘; b. 除了該空白對照組之外’其他孔井内均加入1〇〇 Mix混勻; c.將所有對照組與試驗組置於37*&gt;c靜置4〇分鐘; 丄所有的孔井内均加入75 μ丨stop S〇iution混勻; e. 緊接著於該空白對照組内加入1〇〇wEnzyme/SubstrateMix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 Eleetron Corporation,芬蘭)讀取結果’所使用之激發光(excitati〇n) 波長為405 11111 ’發散光(emission)波長為538 nm 〇 (4)結果分析:繼之紅峨紐鮮絲cyp2E1代纖標準品赃 生成量(pmol)後,以對照組(c〇ntr〇〇為基準,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: C YP 2E1 抑制率(%)= 樣品之HFC生成量 對照組(control)之HFC生成量 24 1360418 二、結果 ι·陽性對照组 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的漠度為66·6 μΜ (即為0.167 %,w/v)時,cyp 2Ei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 i67 〇/。(諸)作為 賦型劑最高測試濃度。 __表二%性對照組之CYP2E1抑制率 gpTC 濃度(μΜ)-gFC 生成量(pmol) CYP2E1 抑制率 222.00 0 256.00 202.00 8.71 151.71 31.52 126.14 43.06 55.18 75.09 21.08 90.49 15.10 93.19 5.42 97,55 0 (對照組) 0.03 0.091 ' 0.27 0.82 2.47 7.4 φ 22.2 66.6 2.試驗组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 不同程度的抑制效果,其中以66 正二羥癒瘡酸(Nordihydroguaiaretic acid) 抑制效果最佳(97.99±0.66 %)。(S 22 1360418 (11) Cofactor/ acetonitrile mix: Add 66 μΐ Acetonitrile to 9.93 ml NADPH-Cofactor Mix. (12) Enzyme/Substrate Mix: Add 5.94 ml sterile water, 50μ1 HTS-706 (CYP2E1) to 4ml Buffer Soultion 2 μΜ P450 content) and 28 μΐ 50 mM MFC (7-Methoxy-4-trifluoromethyl coumarin, camping substrate) 2. Screening for cytochrome P45〇2El (CYP2El) inhibition 剤 using cytochrome P450 2E1 (CYP2E1 The CYP2E1 High Throughput Inhibitor Screening Kit (BD Bioscience, USA) was used to screen Chinese herbal medicines and excipients. The experimental procedures were as follows: (!) Preparation of control group: . on 96-well plate Add 149μ1ΝΑ〇ρΗ (:〇ί^〇ΓΜ^ and 1 μΐ 20mM DDTC to the first well and mix well; • Add 1〇〇μ1 c to the wells of the 2nd to 12th wells on the 96-well plate. 〇facW acet〇n.trik mix was the positive control group (4); the 9th and 7th wells were the control group (_!); the u and u wells were blank control group (_; The 1st to 8th axis continuous dilution operation: 5M liquid is taken into the second well from the (10) Evenly, add 5〇μι of liquid from the 2nd well of the second brother to the well of the 3rd hole, and so on. After the 8th hole is spit + open, remove the excess 5〇μΐ liquid to obtain the serial dilution concentration of 66.6. 22.2, 7.4, 2 47 λ ·47, 〇·82, 0.27, 0.091, 0.03 μΜ. 23 1360418 (2) Preparation test group: a. Add in the first and second wells of the first row on the 96-well plate 149 μΐ NADPH-Cofactor Mix' and Ιμΐ 20 mM Chinese medicine test sample or ΐμΐ 25% (w/v) excipient test sample, and mix well; b. From the first and second wells of the first row Each 50 μΐ liquid was added to the well of the third well to mix (that is, each test sample was three replicates); (3) Start and stop of the reaction: a. The above control group and the test group were placed in 3r&gt; 〇 min; b. In addition to the blank control group, add 1 〇〇 Mix to mix in other wells; c. Place all control and test groups at 37*&gt;c for 4 〇 minutes; 丄 all Add 75 μ丨stop S〇iution to the wells; e. Immediately add 1〇〇wEnzyme/SubstrateMix to the blank control group; f. The control group and the test group read the results using a fluorescent luminometer (Flu〇r〇skan Ascent FL, Thermo Eleetron Corporation, Finland). The excitation light used was 405 11111 'Emission wavelength. Analysis of 538 nm 〇(4) results: followed by the control group (c〇ntr〇〇), which is the cyp2E1 inhibition rate of the control group, after the red 峨 鲜 鲜 fresh silk cyp2E1 generation standard 赃 production amount (pmol) %, the cyp2E1 inhibition rate of each positive control group and the test group was calculated by the following formula: C YP 2E1 inhibition rate (%) = HFC production amount of the sample HFC production amount of the control group 24 1360418 II. Results ι·positive control The inhibition rate of CYP2E1 measured by the group positive control group (DDTC) is shown in Table 2. From Table 2, the inhibition rate of cyp 2Ei when the DDTC is 66.6 μΜ (that is, 0.167 %, w/v). Up to 97.55%, with 66.6 μΜ as the highest test concentration of traditional Chinese medicine, to 〇i67 〇/. (Parties) as the highest tested concentration of the excipient. __ Table 2 % control group CYP2E1 inhibition rate gpTC concentration (μΜ)-gFC production amount (pmol) CYP2E1 inhibition rate 222.00 0 256.00 202.00 8.71 151.71 31.52 126.14 43.06 55.18 75.09 21.08 90.49 15.10 93.19 5.42 97,55 0 (control group 0.03 0.091 ' 0.27 0.82 2.47 7.4 φ 22.2 66.6 2. The inhibition rate of CYP2E1 in the test group The CYP2E1 inhibition rate measured by the Chinese medicine medicine is shown in Table 3. From the results, it can be seen that the various Chinese medicines are introduced at different concentrations (66 μΜ, 33 μΜ, 16.5). Under the conditions of μΜ), the cytochrome P45〇2E1 had different inhibitory effects, and the inhibitory effect of 66-dihydrohydroguaiaretic acid was the best (97.99±0.66%).

(S 25 1360418(S 25 1360418

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 33 μΜ 16.5 μΜ 對照組 0 0 0 陽性對照組(DDTC) 97.55+1.862 正二經癒瘡酸 (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 49.05±5.18 山奈酚 (Kaempferol) 70.63+2.53 70.04±3.75 71.87±1.14 根皮素 (Phloretin) 66.8414.79 54.69+2.84 42.04±3.63 雙硫余 (disulfiram) 66.54+2.55 60.55+5.70 57.89+3.91 橙皮素 (Hesperetin) 54.75+1.37 43.29±0.82 32.10±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) 23.48±2.59 21.87+1.90 15.64+7.82 26 &lt; S ) 1360418 中藥藥引 CYP2E1 抑制率(〇/n、 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±12.74 兒茶素 ((+)-Catechin) 16.45+9.67 33.83±8.76 41.53±7.62 β-奈黃酮 (β-naphthoflavone) 15.40+12.94 16.8310.96 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 5.71+10.53 表兒茶酚 ((-)-Epicatechin) 2.57±11.60 15.02±5.50 18.27±9.34 根皮苦 (Phloridzin) 1.42±9.28 3.76±3.58 1.25±7.90 葛根素 (Puerarin) -12.86±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CYP2E1抑制率如表四所示,由結果可知各賦型劑於 不同濃度(0.167%,0.08%,0.042%,\\^)的條件下,對細胞色素?45〇2丑1具有 不同程度的抑制效果,其中以0.167% Brij 58的抑制效果最佳(97.75±0.66%)» &lt; S ) 27 1360418 表四賦型劑之CYP2E1抑制率 中藥藥引 CYP2E1抑制率 測試濃度(w/v) 0.167% 0.08% 0.042% 對照組 0 陽性對照組(DDTC) 97.55±1.862 Brij 58 97.75±0.66 96.58±0.40 96.02±0.17 Brij 76 97.56±1.02 96.87±l.〇〇 94.76±0.47 Brij 35 93.3310.82 89.45±0.68 76.21+7.37 (測試濃度0.025%) (測試濃度0.013%) (測試濃度0.006%) Tween 20 87.20±1.29 82.80±1.71 71.77±4.48 Tween 80 73.92±4.71 65.45±2.50 64.02+12.54 Tween 40 58.97±3.29 47.05+6.48 44.79+2.49 PEG 2000 44.33±2.75 40.13±3.06 35.81±3.26 PEG 400 42.33+5.25 39.10±0.73 31.98±5.97 Pluomic F68 41.72i5.34 42.98±3.24 37.11+10.35 PEG 4000 37.21 + 1.91 41.22±0.97 37.18±10.52 本發明所提供之低副作用之異菸驗醯胺㈣血城腿)新複方,與單獨 使用異菸鹼醯胺(INH)之試驗結果相互比較時,在生化分析(MT、AST值)、 病理學分析、剩餘肝功能之量測(Gsp值、GEC值)以及氧化麼力的指標(血 裝中8-iso-PGF2a的濃度)等各方面之分析结果,都有嘯減少使用異於鹼醯 胺(INH)所造成的肝毒性副作用的功效。 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補範圍,凡未_本發明技藝㈣所為之等效實 施或變更,例如:異菸鹼醯胺、細胞色素p45〇 2E1抑制劑、雙硫侖 28 丄·418 (DSF)、硝基笨酚磷酸二酯(ΒΝΡΡ)施用之濃度及比例,以及細胞色素Ρ450 2Ε1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 使用異终驗醯胺(麵)所造成的肝毒性副作用,應已充分符合新穎性及進步 ’陡之法定發明專利要件,爱依法提出申請,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;Table 3: CYP2E1 inhibition rate of traditional Chinese medicine cited CYP2E1 inhibition rate (%) Test concentration 66 μΜ 33 μΜ 16.5 μΜ Control group 0 0 0 Positive control group (DDTC) 97.55+1.862 Nordic hydroguaiaretic 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 49.05±5.18 Kaempferol 70.63+ 2.53 70.04±3.75 71.87±1.14Phloretin 66.8414.79 54.69+2.84 42.04±3.63 disulfiram 66.54+2.55 60.55+5.70 57.89+3.91 Hesperetin 54.75+1.37 43.29±0.82 32.10 ±5.80 6-Gingerol (5-Gingerol) 51.89+3.33 39.83+2.32 30.13+2.67 gallic 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 Dihydroquercetin ((+)-Taxifolin) 34.54+3.47 23.80+5.84 22.58±11.69 Wongonin 23.48±2.59 21.87+ 1.90 15.64+7.82 26 &lt;S ) 1360418 Chinese medicine drug induced CYP2E1 inhibition rate (〇 / n, test concentration 66 μΜ 33 μΜ 16.5 μΜ Protocatechuic acid 22.75±4.07 19.95+8.95 25.66±12.74 catechin ((+)-Catechin 16.45+9.67 33.83±8.76 41.53±7.62 β-naphthoflavone 15.40+12.94 16.8310.96 6.52+6.64 Embelin 13.54+11.64 ------ 12.30+10.24 5.95+7.48 Anti Trans-Cinnamic acid 7.10±6.95 4.66±6.50 5.71+10.53 Epicatechin ((-)-Epicatechin) 2.57±11.60 15.02±5.50 18.27±9.34 Phloridzin 1.42±9.28 3.76±3.58 1.25±7.90 Puerarin -12.86±2.75 -4.64±3.47 0·43±2.31 Umbelliferone -1081.56 soil 168.00 -571.97+117.56 -280.41+19.48 CYP2E1 inhibition measured by excipient The rate is shown in Table 4. From the results, it can be seen that the various excipients are cytochrome at different concentrations (0.167%, 0.08%, 0.042%, \\^). 45〇2 ugly 1 has different degrees of inhibitory effect, among which 0.167% Brij 58 has the best inhibitory effect (97.75±0.66%)» &lt; S ) 27 1360418 Table 4 excipient CYP2E1 inhibition rate Chinese medicine CYP2E1 inhibition Rate test concentration (w/v) 0.167% 0.08% 0.042% Control group 0 Positive control group (DDTC) 97.55±1.862 Brij 58 97.75±0.66 96.58±0.40 96.02±0.17 Brij 76 97.56±1.02 96.87±l.〇〇94.76± 0.47 Brij 35 93.3310.82 89.45±0.68 76.21+7.37 (test concentration 0.025%) (test concentration 0.013%) (test concentration 0.006%) Tween 20 87.20±1.29 82.80±1.71 71.77±4.48 Tween 80 73.92±4.71 65.45±2.50 64.02 +12.54 Tween 40 58.97±3.29 47.05+6.48 44.79+2.49 PEG 2000 44.33±2.75 40.13±3.06 35.81±3.26 PEG 400 42.33+5.25 39.10±0.73 31.98±5.97 Pluomic F68 41.72i5.34 42.98±3.24 37.11+10.35 PEG 4000 37.21 + 1.91 41.22±0.97 37.18±10.52 The low side-effect of the present invention provides a new compound, the biochemical analysis of the new compound, when compared with the test results of isoniazidamine (INH) alone. (MT, AST value), pathology Analyze, measure the residual liver function (Gsp value, GEC value), and the index of oxidative stress (concentration of 8-iso-PGF2a in blood), etc. (INH) The efficacy of hepatotoxic side effects. The detailed description above is a detailed description of one of the possible embodiments of the present invention, but the embodiment is not intended to limit the scope of the present invention, and equivalent implementations or modifications of the present invention (4), such as: Concentrations and ratios of alkali guanamine, cytochrome p45〇2E1 inhibitor, disulfiram 28 丄·418 (DSF), nitrophenol phosphatidyl phosphate (ΒΝΡΡ), and the type of cytochrome Ρ450 2Ε1 inhibitor Examples of variations of equivalence should 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 the side effects of hepatotoxicity caused by the use of heterologous amines (face), which should be fully consistent with novelty. And progress 'steep legal patent requirements for the invention, love to apply in accordance with the law, I ask you to approve the invention patent ^ application, in order to invent invention, to the sense of convenience" [simplified description of the diagram] Figure 1 is isoniazid guanamine ( INH) map of metabolic pathways in the liver;

圖一為對照組、INH 組、組、組以及 BNpp DsF I1^H '組域,天門冬驗轉胺酶(紐)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户&lt;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,麵[組在周 圍中央靜脈(v)的肝細胞呈現碎裂及空泡化(ΗΕ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,_χ);圖三d,以電子顯 ' 微鏡檢視腿組大睛邮’她於圖三C賴組之肝細胞切片,ΙΝΗ組 大鼠肝細胞之粗内質晴ER)_增加,Nu :細胞核(9,_χ); 圖四為8-is〇-PGF2«_d4 (Α)與8七0挪知(Β)之分子結構以及子離子光 譜; 圖五為含有250 pg 8-is〇-PGF2a_d4 (Α)的内標準品溶液含有1〇〇四 ·( S ) 29 1360418 8-iso-PGF2tt (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(〇1^) 353/193之離子偶(丨〇111)也8)分別被用來監測8七〇_1&gt;(3172(1_(14 (A)(作為内標準品)以及8-iso-PGF2a⑻(作為標準品);波蜂1 :空白血衆; 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 土 SD,*表示試驗 組與對照組比較後/&gt; &lt; 〇_〇〇1者;#表示各試驗組與XNH組比較後p &lt; 0 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 土 SD,*表示試驗組與 對照組比較後户&lt;〇·〇〇1者;#表示各試驗組與INH組比較後/&gt;&lt;〇.〇〇1者; &amp;表示各試驗組與INH組比較後户&lt;〇.〇〇5者; 圖八為對照組、INH組、BNPP-INH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 ± SD,*表示試驗組 與對照組比較後户&lt; 0.001者;#表示各試驗組與INH組比較後P &lt; 0.005者; &quot;&quot;表示各試驗組與INH組比較後尸&lt; 0.05者; 圖九為對照組、INH組、BNPP-INH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s十圖,以及Figure 1 shows the control group, the INH group, the group, the group, and the BNpp DsF I1^H 'group. The activity of transaminase (N) and alanine transaminase (ALT) was analyzed in Tianmen, and the value was calculated as mean soil. SD, * indicates that each test group is compared with the control group and the household is &lt; 〇〇 5; Figure 3 is the control group (Fig. 3A and C) and the INH group (Fig. 3B and D) rat liver section: Fig. 3 A 'control group relative to normal liver tissue type (HE staining, 4〇〇χ); Figure 3B, surface [group of hepatocytes in the surrounding central vein (v) showed fragmentation and vacuolization (ΗΕ staining, 4 〇〇χ); Figure 3 c, the liver slices of the control group were observed by electron microscopy, Nu: nucleus (9, _ χ); Figure 3 d, with electronic display 'microscopic examination of the leg group big eye mail' she in Figure 3 Hepatic cell sections of the C Lai group, crude endoplasmic reticulum ER) of the hepatocytes of the sputum group _ increased, Nu: nucleus (9, _ χ); Figure 4 is 8-is〇-PGF2 «_d4 (Α) and 8 7 The molecular structure of the unknown (Β) and the spectrum of the product ions; Figure 5 shows the internal standard solution containing 250 pg 8-is〇-PGF2a_d4 (Α) containing 1〇〇4·(S) 29 1360418 8-iso-PGF2tt (B) Standard solution and blank sample (〇, in multiple Liquid Chromatography Tandem Mass Spectrometry (LC/MS/MS) with MMP detection, mass-to-charge ratio (m/z) 357/197 and mass-to-charge ratio (〇1^) 353/193 The ion couples (丨〇111) are also used to monitor 8 〇 _1 &gt; (3172 (1_(14 (A) (as internal standard) and 8-iso-PGF2a (8) (as standard); Bee 1: blank blood; peak 2: blank plasma injected into the standard; Figure 6 is the 8-iso- in the plasma of the control group, INH group, BNPP-INH group, DSF-INH group and BNPP-DSF-INH group. The concentration of PGF2a was calculated as mean soil SD, * indicates that the test group was compared with the control group /&gt;&lt;〇_〇〇1;# indicates that each test group was compared with the XNH group, p &lt; 0 05; 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 soil SD, * indicates the test group and The control group was compared with the households &lt;〇·〇〇1;# indicates that each test group was compared with the INH group/&gt;&lt;〇.〇〇1;& indicates that each test group was compared with the INH group&lt;〇.〇〇5; Figure VIII is the control group, INH group, BNPP-INH group, DSF-INH And the galactose clearance ability (GEC) value of the rats in the BNPP-DSF-INH group, the value is calculated as mean ± SD, * indicates that the test group is compared with the control group and the household is &lt;0.001;# indicates that each test group is compared with the INH group After P &lt;0.005;&quot;&quot; indicates that each test group was compared with the INH group (0.05); Figure 9 is the control group, INH group, BNPP-INH group, DSF-INH group and BNPP-DSF-INH The galactose single point method (GSP) values of each group were highly correlated with the concentration of 8-iso-PGF2a in plasma, and

圖十為對照組、INH組、BNPP-INH組、DSF-INH組以及BNPP-DSF-INH 30 1360418 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。 【主要元件符號說明】 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 is a high correlation between the galactose single point method (GSP) values and the galactose clearance capacity (GEC) values in the control group, INH group, BNPP-INH group, DSF-INH group, and BNPP-DSF-INH 30 1360418 group. The chart. [Explanation of main component symbols] 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 T, Adams SP, Wall RA, Wright JM. Role of hydrazine in the mechanism of isoniazid hepatotoxicity in rabbits. 1996. Arch Toxicol 70: 835-840. 5. Yue J, Peng RX, Yang J, Kong R, Liu J. CYP2E1 mediated isoniazid-induced hepatotoxicity in rats. 2004. Acta Pharmacol Sin. 25: 699-704. 6. Sarich TC, Adams SP, Petricca G, 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 T, 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 U, Mehendale HM. Cytochrome P4502E1 induction increases thioacetamide liver injury in diet-restricted rats. 2001. Drug Metab Dispos. 29: 1088-1095. 10. Huang YS, Chern 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 1360418 11. Guengerich FP, Kim DH, 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 thiono-sulfur-containing compounds. 1975. Biochem Pharmacol. 24: 2199-2205. 13. Brady JF, 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: 366-373.2. 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 T, Adams SP, Wall RA, Wright JM. Role of hydrazine in the mechanism of isoniazid hepatotoxicity in rabbits. 1996. Arch Toxicol 70: 835-840. 5. Yue J, Peng RX, Yang J, Kong R, Liu J. CYP2E1 mediated isoniazid-induced hepatotoxicity in rats. 2004. Acta Pharmacol Sin. 25: 699-704. 6. Sarich TC, Adams SP, Petricca G, 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 T, 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 hepat 1998. Toxicol Appl Pharmacol. 153: 109-118. 9. Ramaiah SK, Apte U, Mehendale HM. Cytochrome P4502E0 induction increases thioacetamide liver injury in diet-restricted rats. 2001. Drug Metab Dispos. 29: 1088-1095. 10. Huang YS, Chern 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 1360418 11. Guengerich FP, Kim DH, 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 thiono-sulfur-containing compounds. 1975. Biochem Pharmacol. 24: 2199-2205. 13. Brady JF, 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 JD, Hill KE, Burk RF, Nammour TM, 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 S, Hirany SV, Burk RF, Jialal I. Divergence between LDL oxidative susceptibility and urinary F(2)-isoprostanes as measures of oxidative stress in type 2 diabetes. 2001. Clin.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 JD, Hill KE, Burk RF, Nammour TM, 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 S, Hirany SV, Burk RF, Jialal I. 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.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 1360418 96: 13381-13386. 21. Liang Y, Wei P, Duke RW, Reaven PD, Hannan SM, Cutler RQ He ward 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 fixnction 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 1360418 96: 13381-13386. 21. Liang Y, Wei P, Duke RW, Reaven PD, Hannan SM, Cutler RQ He ward 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 fixnction tests, and histopathology in cirrhosis. 1979. Dig. Dis. Sci. 24: 358-362.

23. Hero Id C, Heinz R, Niedobitek G, Schneider T, Hahn EQ 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 function 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. Biopharm 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 dysfunction. 1995. J. Pharm. Sci. 84: 231-235. 30. Hu OY,Tang US, Sheeng TY,Chen TC,Curry SH. Pharmacokinetics of promazine in 33 1360418 patients with hepatic cirrhosis-correlation with a novel galactose single point method. 1995. J. Pharm. Sci. 84: 111-114. 31. FDA Center for Drug Evaluation 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. 33. Ryan DE, Ramanathan L, Iida S, Thomas PE, Haniu M, Shively JE, Lieber CS, et al.23. Hero Id C, Heinz R, Niedobitek G, Schneider T, Hahn EQ 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 function 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 pharmacokinet Ics of cefoperazone-a novel galactose single-point method as a measure of residual liver function. 1994. Biopharm 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 dysfunction. 1995. J. Pharm. Sci. 84: 231-235. 30. Hu OY, Tang US, Sheeng TY, Chen TC Curry SH. Pharmacokinetics of promazine in 33 1360418 patients with hepatic cirrhosis-correlation with a novel galactose single point method. 1995. J. Pharm. Sci. 84: 111-114. 31. FDA Center for Drug Evaluation 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 Cirrhosis of the Liver. 1964. Acta Med. Scand 175: 281-289. 33. Ryan DE, Ramanathan L, Iida S, Thomas PE, Haniu M, Shively JE, Lieber CS, et al.

Characterization of a major form of rat hepatic microsomal cytochrome P-450 induced byCharacterization of a major form of rat hepatic microsomal cytochrome P-450 induced by

isoniazid. 1985. J. Biol. Chem. 260: 6385-6393. 34. Ekstrom Q 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 SV, Mehta SK? Vaiphei K3 Attri S5 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.Isoniazid. 1985. J. Biol. Chem. 260: 6385-6393. 34. Ekstrom Q 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 SV, Mehta SK? Vaiphei K3 Attri S5 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)

13604181360418 十、申請專利範圍: 1. 一種低副作用之異菸鹼醯胺(isoniazid,INH)新複方,包括一藥學有效量 之異於驗酿胺(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:正二經癒瘡酸(Nordihydroguaiaretic acid)、 (-)-Epigallocetechin-3-gallate、茵陳色原 _ (Capillarisin)、山奈酌· (Kaempferol)、根皮素(Phloretin)、橙皮素(Hesperetin)、6-薑辣酵 (6_Gingero1)、沒食子酸(gallic acid)、異甘草素(Isoliquritigenin)、柚皮素 (Narigenin)、二氫化槲皮素((+)_Taxif〇Un)、漢黃芩素^的明此)、原兒茶 酸(Protocatechuic acid)、兒茶素((+)_catechin)、β-奈黄酮 (β-naphthoflavone)、恩貝素(Embelin)、反式肉桂酸(trans-Cinnamic acid)、 表兒命齡((-)-Epicatechin)、根皮苷(Phl〇ridzin)、Brij 58、Brij 76、Brij 35、 Tween 20 ' Tween 80、Tween 40、PEG 2000、PEG 400、Pluomic F68、 PEG 4000。 2·如申明專利$已圍第i項所述之低副作用之異於驗醯雖,願)新 複方,其中可加入-藥學上可接受之賦形劑至該複方。 3.如_«^專魏gj第2項所述之调相之異祕賴㈣心城匪)新 複方,、中該賦形劑可為稀釋劑、填充劑、結合劑、崩解劑、潤滑劑等。X. The scope of application for patents: 1. A new compound of isoniazid (INH) with low side effects, including a pharmaceutically effective amount of isoniazid (INH), combined with a pharmaceutically effective amount of cells a pigment P450 2E1 (CYP2E1) inhibitor; wherein the cytochrome P450 2E1 (CYP2E1) inhibitor is selected from the group consisting of: Nordic hydroguaiaretic acid, (-)-Epigallocetechin-3-gallate , Capillarisin, Kaempferol, Phloetin, Hesperetin, 6-Gingero1, gallic acid, different Isoliquritigenin, Nariginin, dihydroquercetin ((+)_Taxif〇Un), scutellarin), protocatechuic acid, catechin (( +)_catechin), β-naphthoflavone, Embelin, trans-Cinnamic acid, epiphylaxis ((-)-Epicatechin), phloridzin (Phl 〇ridzin), Brij 58, Brij 76, Brij 35, Tween 20 'Tween 80, Tween 40, PEG 2000, PEG 400 , Pluomic F68, PEG 4000. 2. If the claim has been patented, the low side effects described in item i are different from the test, although a new compound, in which a pharmaceutically acceptable excipient can be added to the compound. 3. If _«^ specializes in the second phase of the Wei-gj, the secret of the remedy (four) Xincheng 匪), the excipient can be a diluent, a filler, a binder, a disintegrating agent, Lubricants, etc.
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