TWI517848B - New low side effect pharmaceutical composition containing antituberculosis drugs - Google Patents

Description

無/低副作用之抗結核病藥物新複方 New compound for anti-tuberculosis drugs without/low side effects

本發明係關於一種無/低副作用之一抗結核病藥物新複方，特別是指一種將異菸鹼醯胺(INH)，或異菸鹼醯胺(INH)和立復黴素(rifampin)，或異菸鹼醯胺(INH)和立復黴素(RIF)和吡嗪醯胺(PZA)，或吡嗪醯胺(PZA)，合併使用細胞色素P450 2E1(CYP2E1)抑制劑或醯胺水解酶(amidase)抑制劑，以降低由異菸鹼醯胺(INH)或吡嗪醯胺(pyrazinamide)所引起之肝毒性等副作用之抗結核病藥物新複方。 The present invention relates to a novel compound for anti-tuberculosis drugs which has no/low side effects, in particular to an isonianic acid amide (INH), or isonicotino amide (INH) and rifampin, or Isoniazid amide (INH) and rifamycin (RIF) and pyrazinamide (PZA), or pyrazinamide (PZA), combined with cytochrome P450 2E1 (CYP2E1) inhibitor or guanamine hydrolase (amidase) inhibitor, a new compound for anti-tuberculosis drugs that reduces side effects such as hepatotoxicity caused by isoniazidamine (INH) or pyrazinamide.

根據世界衛生組織(WHO)估計，全球大約有三分之一的人口感染肺結核，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爬升，每十萬人口有六十多人感染肺結核，但其中只有大約四分之三的人接受完整治療；根據衛生署的統計，台灣每天至少有4.2個人死於肺結核；在這麼多接受肺結核藥物治療的病患中，臨床上最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14,000名新增的肺結核病患，粗略估計至少有2,000名到3,000名慢性肝病患者需接受抗結核藥物治療，因此在這些病患身上所可能發生的肝毒性是吾人不可忽視的醫源性疾病。 According to estimates by the World Health Organization (WHO), about one-third of the world's population is infected with tuberculosis, and there are about 8 million new cases each year. The number of newly registered tuberculosis patients in Taiwan has continued to climb in recent years, every ten. More than 60 people in the 10,000 population are infected with tuberculosis, but only about three-quarters of them receive complete treatment. According to the statistics of the Department of Health, at least 4.2 people die from tuberculosis every day in Taiwan; in so many patients receiving tuberculosis medication Among them, the most common clinical side effects are hepatotoxicity and neurological diseases (such as auditory nerve and optic neuropathy), of which hepatotoxicity is the most common. In addition, Taiwan is also a prevalent area for hepatitis B and C. There are also a small number of hepatitis patients infected with tuberculosis. It is assumed that there are 14,000 new tuberculosis patients every year. It is estimated that at least 2,000 to 3,000 patients with chronic liver disease need to Treated with anti-tuberculosis drugs, the hepatotoxicity that may occur in these patients is an iatrogenic disease that cannot be ignored.

多數的第一線抗結核藥物，例如：異菸鹼醯胺(isoniazid，俗稱敵癆剋星)、吡嗪醯胺(pyrazinamide，俗稱敵癆新邁)及立復黴素(rifampin)等都有導致肝毒性發生的潛在不良反應；其中異菸鹼醯胺(isoniazid)是目前最有效的單一抗結核藥物，也最容易引起服用者產生肝毒性；在60年代末期陸續有異菸鹼醯胺(isoniazid)造成肝毒性的報告；異菸鹼醯胺(isoniazid)所造成具有臨床症狀的肝毒性約0.1-1%，而在10-20%的病患中，則可觀察到無症狀的 肝功能異常，這些肝功能異常通常於服藥後兩個月內發生。 Most first-line anti-tuberculosis drugs, such as isoniazid (commonly known as dipyridamole), pyrazinamide (commonly known as chlorpyrifos), and rifampin, have led to Potential adverse effects of hepatotoxicity; isoniazid is the most effective single anti-tuberculosis drug, and is most likely to cause hepatotoxicity in patients; in the late 1960s, isoniazid guanamine (isoniazid) Reports of hepatotoxicity; isoniazid is caused by clinical symptoms of hepatotoxicity of about 0.1-1%, while in 10-20% of patients, asymptomatic Abnormal liver function, which usually occurs within two months after taking the drug.

如圖一所示，異菸鹼醯胺(isoniazid)在肝臟中主要經由氮-乙醯氨基轉移酶(N-acetyltransferase,NAT)的幫助而乙醯化，產生的中間產物乙醯化異菸鹼醯胺(acetylisoniazid)迅速被水解成乙醯化聯胺(acetylhydrazine)；乙醯化聯胺可以再經由氮-乙醯氨基轉移酶(N-acetyltransferase)被乙醯化成無毒性的雙乙醯化聯胺(diacetylhydrazine)，或者經由細胞色素P450 2E1(CYP 450 2E1)氧化成具有肝毒性的分子，其中包括乙醯化偶氮(acetyldiazene)、乙醯銨離子(acetylonium ion)、乙醯自由基(acetylradical)、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P450 2E1反應生成自由基而造成氧化壓力，導致細胞死亡；此外，異菸鹼醯胺(isoniazid)亦可經由醯胺水解酶(amidase)直接水解成有毒性的聯胺(hydrazine)，或者由上述乙醯化聯胺(acetylhydrazine)經醯胺水解酶(amidase)水解成有毒性的聯胺(hydrazine)。 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 acetylhydrazine; the acetylated hydrazine can be converted to a non-toxic bis-hydrazide via acetonitrile via N-acetyltransferase. Diacetylhydrazine, or oxidized to cytotoxic molecules via cytochrome P450 2E1 (CYP 450 2E1), including acetyldiazene, acetylonium ion, acetylradical ), ketene, etc., in the presence of aerobic and NADPH, acetylated hydrazine is reacted by cytochrome P450 2E1 to generate free radicals, causing oxidative stress, leading to cell death; in addition, isonicotinic acid amide ( Isoniazid) can also be directly hydrolyzed to a toxic hydrazine via amidase or from the above acetylhydrazine to a toxic hydrazine by amidase (amidase) (hydrazine).

近來有研究顯示，聯胺(而非異菸鹼醯胺或乙醯化聯胺)是在兔及鼠體內造成異菸鹼醯胺引起之肝毒性(INH-induced hepatotoxicity)最可能的主因，研究者認為異菸鹼醯胺引起之肝毒性的嚴重性與血漿中聯胺的濃度成正相關；1999年Sarich等人的報導則認為對硝基苯酚磷酸二酯(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聯胺。 Recently, studies have shown that hydrazine (rather than isoniazid guanamine or acetylated hydrazine) is the most likely cause of hepatotoxicity caused by isoniazid in rabbits and mice. It is believed that the severity of hepatotoxicity caused by isonicotinamide is positively correlated with the concentration of hydrazine in plasma; in 1999, Sarich et al. reported that bis- p- nitrophenyl phosphate (BNPP, It is an inhibitor of indoleamine hydrolase to prevent hepatotoxicity caused by isoniazid amide, and its protective mechanism should be to produce hydrazine by inhibiting isonicotinic acid amide.

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(CCl₄)以及乙醯氨酚(acetaminophen))的代謝生物反應；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；有些研究認為肝臟內的CYP2E1與異菸鹼醯胺引起之肝毒性的機制有關。在體外試驗中，雙硫侖(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)，Brady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。 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 (CCl ₄ ) and acetaminophen; however, CYP2E1 The role played by heminic acid induced by isoniazid guanamine is not clear. Isoniazid amide itself is an inducer of CYP2E1; some studies suggest that hepatotoxicity caused by CYP2E1 and isonicotinic acid in liver Related to the mechanism. 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) ), Brady et al's trial 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.

Sodhi等人則在1997年的報導指出，氧化壓力是造成幼鼠體內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)以評估體內氧化傷害的速率，目前可能適用的生物標記可分為三類，分別為對脂質、蛋白質、核酸氧化傷害的標記；8-異構***素F_2α(8-iso-prostaglandin F_2α,8-iso-PGF_2α)是一種自由基引起花生四烯酸(arachidonic acid)發生脂質過氧化作用的產物，其化學性質穩定，8-iso-PGF_2α含量可作為判斷活體內脂質過氧化的新指標，該脂質過氧化反映可能與體內自由基的產生、氧化性的傷害(oxidative damage)及抗氧化劑的缺乏(antioxidant deficiency)有關；目前有許多方法可用來測量8-iso-PGF_2α含量，包括酵素免疫分析法(enzyme immunoassay)、放射免疫分析法(radioimmunoassay)、氣相層析質譜儀(gas-chromatography mass spectrometry)以及液相層析質譜儀(liquid chromatography mass spectrometry)等；此外，人類尿液中的8-iso-PGF_2α及其代謝物2,3-dinor-8-iso-PGF_2α含量可利用C18固相萃取(C18 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。 Sodhi 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; The isoform prostaglandin F _2α (8-iso-prostaglandin F _2α , 8-iso-PGF _2α ) is a product of free radical-induced lipid peroxidation of arachidonic acid, which is chemically stable, 8- The iso-PGF _2α content can be used as a new indicator for determining lipid peroxidation in vivo. The lipid peroxidation reaction may be related to the production of free radicals, oxidative damage and antioxidant deficiency in the body; There are a number of methods for measuring 8-iso-PGF _2α levels, including enzyme immunoassay, radioimmunoassay, gas-chromatography mass spectrometry, and liquid chromatography. In addition, the concentration of 8-iso-PGF _2α and its metabolite 2,3-dinor-8-iso-PGF _{2α in} human urine can be determined by liquid chromatography mass spectrometry The samples were prepared by C18 solid phase extraction (SPE) and analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS).

利用侵入式及非侵入式方法測試大鼠(rat)肝功能，以監測肝損害的發展以及篩選肝臟疾病，其中最常使用的方法包含測量血清中之天門冬氨酸轉胺酶(aspartate aminotransferase,AST)、丙氨酸轉胺酶(alanine aminotransferase,ALT)以及鹼性磷酸酶(alkaline phosphatase)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(prothrombin time)來檢測凝血因子(coagulation factors)等；肝功能定量測試是根據幾乎只經過肝臟代謝之受質在血清中的濃度而定，這些受質的清除是依肝門靜脈、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關，反之，該受質的清除則決定於肝臟代謝的能力。 Rat liver function is tested by 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 alkaline phosphatase values, as well as measurements of hepatocyte products such as bilirubin, albumin, and utilization measurements Prothrombin time to detect coagulation factors, etc.; liver function quantitative test is based on the concentration of serum in the liver that is almost exclusively metabolized by the liver. The clearance of these receptors is based on the portal vein. Hepatic artery blood flow and the effect of hepatocytes on these receptors It is determined that the blood flow of the liver is related to the quality of the liver, and vice versa, the clearance of the receptor is determined by the ability of the liver to metabolize.

半乳糖(galactose)是一種具有高萃取率(extraction ratio)、90%在肝臟中代謝的醣類，在肝臟中，半乳糖是由半乳糖激酶(galactokinase)經過差向立體異構化反應(epimerization)，將之轉換成1-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)。半乳糖的高萃取率使得依賴肝臟血流量及肝臟功能的半乳糖代謝作用成為檢測肝功能最主要的方式，目前並無一定的規則來評估大鼠之殘餘肝功能(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。 Galactose is a sugar with a high extraction ratio and 90% metabolism in the liver. In the liver, galactose is subjected to differential stereoisomerization by galactokinase (epimerization). ), which is converted to Glucose-1-phosphate; the action of galactose kinase is a rate-limiting step of the galactose metabolic pathway in hepatocytes. The high extraction rate of galactose makes galactose metabolism, which depends on liver blood flow and liver function, the most important way to detect liver function. There is no rule to evaluate the residual liver function of rats. The metabolic ability of an exact compound (eg, galactose) can be presumed to be a rate-determining step in the liver, and it is also possible to obtain a representative quality of residual liver function.

以半乳糖清除能力(galactose elimination capacity,GEC)作為人類肝功能定量測試已行之有年，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(Guidance for Industry)中成為建議採用測試肝功能的方法之一。 The galactose elimination capacity (GEC) has been used as a quantitative test for human liver function for many years. However, the galactose clearance test requires multiple blood samples to establish a standard curve, which is difficult in clinical application. Therefore, many studies have used Galactose Single Point (GSP) to evaluate human liver function; the inventors tested chronic hepatitis, cirrhosis, and liver cancer patients by galactose single-point method, and the results showed that galactose single point The liver disease can be accurately measured by the method; the galactose single-point method has been successfully applied to test the residual liver function of patients with liver diseases 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 guidelines published by the US Food and Drug Administration (FDA).

由此可見，上述習用抗結核藥物異菸鹼醯胺(isoniazid)仍有諸多缺失，實非一良善之設計者，而亟待加以改良。 It can be seen that the above-mentioned anti-tuberculosis drug isoniazid is still lacking, and it is not a good designer, but needs to be improved.

本案發明人鑑於上述習用抗結核藥物異菸鹼醯胺(isoniazid)所導致肝毒性等副作用的缺點，以及承襲本實驗室先前專利案(低副作用之INH新複方，案號096101545)及(含異菸鹼醯胺(Isoniazid,INH)之低副作用新複方(2)，案號097141522)之發明，乃亟思加以改良創新，並經多年苦心孤詣潛 心研究後，終於成功研發完成本件無/低副作用之抗結核病藥物新複方。另外，在本案發明人先前專利申請案，PCT申請案號PCT/CS2008/001353(低副作用的异烟碱酰胺新复方)發明中，雖已揭示INH單方併用部分本實驗室揭露之CYP2E1抑制劑的藥學組合物，可降低INH所導致肝毒性等副作用，但後續發明人之研究，發現並不是任意的劑量組合都可以於體內達到去除INH肝毒性的效果，例如，在動物體內實驗發現，小鼠每日合併使用山奈酚(Kaempferol)3.78mg/kg與INH/RIF 50/100mg/kg腹腔注射持續3週，可顯著抑制INH所導致之肝毒性，控制組(INH/RIF 50/100mg/kg)之相關肝功能指標GOT、GPT和GSP值為571±295U/L、364±192U/L和866±339mg/L，而併用山奈酚3.78mg/kg組之GOT值則為89±19U/L、48±21U/L和245±98mg/L，接近正常值，然而山奈酚使用劑量調整為1.89mg/kg後，各項相關肝功能指標相較於控制組所下降的幅度均未達併用山奈酚3.78mg/kg所獲得之成效。由以上動物試驗結果可知當合併使用CYP2E1抑制劑時，對INH所導致肝毒性確實具有改善效果，但必須限定其使用劑量，因此基於實驗結果，本發明著重於抑制劑使用劑量之決定，可為新穎及進步性發現。 The inventors of the present invention have the disadvantages of side effects such as hepatotoxicity caused by the above-mentioned anti-tuberculosis drug isoniazidamine (isoniazid), and inherited the previous patent case of the laboratory (INH new compound with low side effects, case number 096101545) and The invention of the low side effect of nicotinic acid amide (Isoniazid, INH) (2), case number 097141522), is the improvement and innovation of 亟思思, and has been painstaking for many years. After the heart research, I finally successfully developed a new compound for anti-tuberculosis drugs with no/low side effects. In addition, in the inventor's prior patent application, PCT Application No. PCT/CS2008/001353 (low side-effect isonicotinic acid amide new compound) invention, it has been revealed that INH unilaterally combines some of the CYP2E1 inhibitors disclosed in the laboratory. The pharmaceutical composition can reduce side effects such as hepatotoxicity caused by INH, but subsequent studies by the inventors have found that not all dose combinations can achieve the effect of removing INH hepatotoxicity in vivo, for example, in vivo in mice. Daily combination of kaempferol 3.78 mg/kg and INH/RIF 50/100 mg/kg for 3 weeks can significantly inhibit hepatotoxicity caused by INH, control group (INH/RIF 50/100 mg/kg) The relevant liver function indicators GOT, GPT and GSP values were 571±295U/L, 364±192U/L and 866±339mg/L, while the GOT value of the combination of kaempferol 3.78mg/kg was 89±19U/L. 48±21U/L and 245±98mg/L, close to the normal value. However, after the dosage of kaempferol was adjusted to 1.89mg/kg, the relative liver function indexes were less than the control group and the kaempferol was used. 3.78mg/kg of the results achieved. From the above animal test results, it can be seen that when the CYP2E1 inhibitor is used in combination, the hepatotoxicity caused by INH does have an improvement effect, but the dose to be used must be limited. Therefore, based on the experimental results, the present invention focuses on the determination of the dose of the inhibitor, which may be Novel and progressive discoveries.

本發明之目的即在於提供一種無/低副作用之抗結核病藥物新複方藥物組合，內含一藥學有效量之異菸鹼醯胺(isoniazid,INH)，或再併用一藥學有效量之立復黴素(rifampin,RIF)，或再併用一藥學有效量之吡嗪醯胺(pyrazinamide,PZA)，或再併用一藥學有效量之其他複方藥物，合併使用一藥學有效量之細胞色素P450 2E1(CYP2E1)抑制劑或醯胺水解酶(amidase)抑制劑，以降低由異菸鹼醯胺(INH)所引起之肝毒性等副作用。 The object of the present invention is to provide a new combination of anti-tuberculosis drugs with no/low side effects, comprising a pharmaceutically effective amount of isoniazid (INH), or a combination of a pharmaceutically effective amount of A pharmaceutically effective amount of cytochrome P450 2E1 (CYP2E1), or a combination of a pharmaceutically effective amount of pyrazinamide (PZA), or a combination of other pharmaceutically effective amounts of other combination drugs An inhibitor or amidase inhibitor to reduce side effects such as hepatotoxicity caused by isoniazidamine (INH).

可達成上述發明目的之無/低副作用之抗結核病藥物新複方，內含一藥學有效量之異菸鹼醯胺(isoniazid,INH)，或再併用一藥學有效量之其他複 方藥物，合併使用一藥學有效量之細胞色素P450 2E1(CYP2E1)抑制劑或醯胺水解酶(amidase)抑制劑，其中該CYP 2E1抑制劑或amidase抑制劑係選自於下列化合物所組成群組：正二羥癒瘡酸(Nordihydroguaiaretic acid)、(-)-Epigallocetechin-3-gallate、茵陳色原酮(Capillarisin)、山奈酚(Kaempferol)、根皮素(Phloretin)、橙皮素(Hesperetin)、6-薑辣醇(6-Gingerol)、沒食子酸(gallic acid)、異甘草素(Isoliquritigenin)、柚皮素(Narigenin)、二氫化槲皮素((+)-Taxifolin)、漢黃芩素(Wongonin)、原兒茶酸(Protocatechuic acid)、兒茶素((+)-Catechin)、β-奈黄酮(β-naphthoflavone)、恩貝素(Embelin)、反式肉桂酸(trans-Cinnamic acid)、表兒茶酚((-)-Epicatechin)、根皮苷(Phloridzin)、Brij 58、Brij 76、Brij 35、Tween 20、Tween 80、Tween 40、PEG 2000、PEG 400、Pluornic F68、PEG 4000、反式肉桂醛(Trans-Cinnamaldehyde)、大豆甘元(Daidzein)、異牡荊素(Isovitexin)、β-香葉烯(β-Myrcene)、檞皮素(Quercetin)、(+)-檸檬烯((+)-Limonene)、楊梅素(Myricetin)、檞皮(Quercitrin)、木犀草素-7-葡萄糖苷(Luteolin-7-Glucoside)、桑葉素(Morin)、新橙皮苷(Neohesperidin)、橙皮苷(Hesperidin)、((-)-Epigallocatechin)、木犀草素(Luteolin)、金絲桃苷(Hyperoside)、十四烷酸乙酯(Ethyl Myristate)、檉柳素(Tamarixetin)、黃芩素(Baicalein)、芸香素(Rutin)、黃芩(Baicalin)、芹菜素(Apigenin)、(+)-Epicatechin、(-)-Epicatechin-3-gallate、水飛薊賓(Silybin)、牡荊素(Vitexin)、金雀異黃酮(Genistein)、異鼠李素(Isorhamnetin)、香葉木素(Diosmin)、葛根素(Puerarin)、或傘形花內酯(Umbelliferone)、高良薑素(Galangin)、非瑟酮(fisetin)、Cremophor EL、Sodium Lauryl Sulfate、Microcrystalline cellulose、Dicalcium phosphate dihydrate、Mannitol、Cremophor RH40、Sucralose、Crospovidone、Sodium starch glycolate、Crospovidone、Eudragit S100、Croscarmellose sodium、Menthol、Saccharin、hydroxypropylcellulose、Pregelatinized starch、Dextrates NF hydrated、Citric acid、Aerosil 200、PEG 8000、Sorbic acid、Lemon oil、Hydroxy propylcellulose、Sodium benzoate、Acesulfame K、Hydroxypropyl methylcellulose、Hydroxy ethyl methylcellulose、Methyl cellulose、Sodium cyclamate、Lactose monohydrate、Maltodextrin、Glyceryl behenate、Oxide red、Glycerin monostearate、Copovidone K28、Starch acetate、Magnesium stearate、Sodium lauryl sulfate、Povidone K-30、Benzyl alcohol、Methylparaben、Propylparaben、Solutol H15、Butylated hydroxyl anisol。 A new compound of anti-tuberculosis drugs having no/low side effects which can achieve the above object, containing a pharmaceutically effective amount of isoniazid (INH), or a combination of a pharmaceutically effective amount of other compound drugs, and combining one A pharmaceutically effective amount of a cytochrome P450 2E1 (CYP2E1) inhibitor or an amidase inhibitor, wherein the CYP 2E1 inhibitor or amidase inhibitor is selected from the group consisting of: dihydrogen acne (Nordihydroguaiaretic acid), (-)-Epigallocetechin-3-gallate, Capillarisin, Kaempferol, Phloetin, Hesperetin, 6-Gingerol ( 6-Gingerol), gallic acid, Isoliquritigenin, Nariginin, dihydroquercetin ((+)-Taxifolin), Wongonin, Yuaner Protocatechuic acid, catechin ((+)-Catechin), β -naphthoflavone, Embelin, trans-Cinnamic acid, epicatechin ((-)-Epicatechin), Phloridizin, Brij 58, Brij 76, Brij 35, Tween 20 Tween 80, Tween 40, PEG 2000 , PEG 400, Pluornic F68, PEG 4000, trans-cinnamaldehyde (Trans-Cinnamaldehyde), soybean Gan element (Daidzein), iso Vitexin (Isovitexin), β - myrcene (beta] -Myrcene), Quercetin, (+)-Limonene ((+)-Limonene), Myricetin, Quercitrin, Luteolin-7-Glucoside ), Morin, Neohesperidin, Hesperidin, (---Epigallocatechin), Luteolin, Hyperposide, Tetradecane Ethyl Myristate, Tamarixetin, Baicalein, Rutin, Baicalin, Apigenin, (+)-Epicatechin, (-)-Epicatechin-3 -gallate, Silybin, Vitexin, Genistein, Isorhamnetin, Diosmin, Puerarin, or Umbrella Umbelliferone, Galangin, fisetin, Cremophor EL, Sodium Lauryl Sulfate, Microcrystalline cellulose, Dicalcium phosphate Dihydrate, Mannitol, Cremophor RH40, Sucralose, Crospovidone, Sodium starch glycolate, Crospovidone, Eudragit S100, Croscarmellose sodium, Menthol, Saccharin, hydroxypropylcellulose, Pregelatinized starch, Dextrates NF hydrated, Citric acid, Aerosil 200, PEG 8000, Sorbic acid, Lemon oil Hydroxy propylcellulose, Sodium benzoate, Acesulfame K, Hydroxypropyl methylcellulose, Hydroxy ethyl methylcellulose, Methyl cellulose, Sodium cyclamate, Lactose monohydrate, Maltodextrin, Glyceryl behenate, Oxide red, Glycerin monostearate, Copovidone K28, Starch acetate, Magnesium stearate, Sodium lauryl sulfate, Povidone K-30, Benzyl alcohol, Methylparaben, Propylparaben, Solutol H15, Butylated hydroxyl anisol.

更進一步，本發明之無/低副作用之抗結核病藥物新複方包括一藥學有效量之吡嗪醯胺(PZA)，或再併用一藥學有效量之其他複方藥物，合併使用一藥學有效量之醯胺水解酶(amidase)抑制劑，其中該amidase抑制劑係選自於下列化合物所組成群組：檞皮素(Quercetin)、高良薑素(Galangin)、桑葉素(Morin)、非瑟酮(fisetin)、異甘草素、楊梅素(Myricetin)、木犀草素(Luteolin)、山奈酚(Kaempferol)、茵陳色原酮(Capillarisin)、Cremophor EL、Sodium Lauryl Sulfate、Tween 20、Brij58，以降低由吡嗪醯胺(PZA)所引起之肝毒性等副作用。 Furthermore, the novel anti-tuberculosis drug combination of the present invention comprises a pharmaceutically effective amount of pyrazinamide (PZA), or a combination of a pharmaceutically effective amount of other compound drugs, in combination with a pharmaceutically effective amount of hydrazine. An amidase inhibitor, wherein the amidase inhibitor is selected from the group consisting of quercetin (Quercetin), galangin (Galangin), mulberry (Morin), and fisetin ( Fisetin), isoglycyrrhizin, myricetin, Luteolin, Kaempferol, Capillarisin, Cremophor EL, Sodium Lauryl Sulfate, Tween 20, Brij58 Side effects such as hepatotoxicity caused by pyrazinamide (PZA).

本發明所提供之無/低副作用之抗結核病藥物新複方，亦可加入藥學上可接受之賦形劑至該複方，該賦形劑可為稀釋劑、填充劑、結合劑、崩解劑、潤滑劑等，例如：Tween 20、Tween 40、Tween 60、Tween 80、Brij 35、Brij 58、Brij 76、Pluronic F68、Pluronic F127、Poloxamer 407、PEG 400、PEG 2000、PEG 4000、Span 60、Span 80、Myri 52、PEG 8000、Acesulfame potassium、Aerosil 200、Colloidal silicon dioxide、Butylated hydroxyl anisol、Corn starch、Crospovidone、Croscarmellose sodium、Dicalcium phosphate dihydrate、EDTA 2 Na、Lactose、Lactose monohydrate、Lactose S.G、Low-substituted hydroxypropylcellulose、Maltodextrin、Mannitol、Menthol、Propyl paraben、Methyl paraben、Microcrystalline cellulose、Guar gum、Xanthan gum、Pregelatinized starch、Povidone K-30、Sodium starch glycolate、Sodium lauryl sulfate、Sucralose、Solutol H15、Cremophor EL、Cremophor RH40、 Sodium cyclamate、PVP K90F、Oxide red、Hydroxypropyl methylcellulose、Cherry、Lemon oil、Sorbic acid、Benzyl alcohol、Glycerrin、Sodium benzolate、Starch acetate、Citric acid、Sorbitol solution、Opady white、Dextrates,NF hydrate、Magnesium stearate、Alginic acid、Eudragit E90、Eeudragit E、Glyceryl behenate、Gelucire、kollidon VA64(copovidone K28)、Hydrochoric acid、Hydroxy ethyl methyl cellulose、Hydroxy propyl cellulose、Methyl cellulose、Methacrylic acid copolymer type B(Eudragit 100)、Maltose、Methacrylic Eudragit S100 acid copolymer、PEG 1450、Povidone K-90、phosphoric acid 85%、polyoxyl 40 hydrogenated castor oil(RH 40)、Polyoxyl 35 castor oil(EL 35)、sodium dihydrogen phosphate、saccarin、triethyl citrate、Tri-Sodium Citrate或其餘列於美國FDA Generally Recognized as Safe(GRAS)中之常用成分。 The new compound of the anti-tuberculosis drug provided by the present invention with no/low side effects may also be added to the compound by adding a pharmaceutically acceptable excipient, which may be a diluent, a filler, a binder, a disintegrating agent, Lubricants and the like, for example: Tween 20, Tween 40, Tween 60, Tween 80, Brij 35, Brij 58, Brij 76, Pluronic F68, Pluronic F127, Poloxamer 407, PEG 400, PEG 2000, PEG 4000, Span 60, Span 80 , Myri 52, PEG 8000, Acesulfame potassium, Aerosil 200, Colloidal silicon dioxide, Butylated hydroxyl anisol, Corn starch, Crospovidone, Croscarmellose sodium, Dicalcium phosphate dihydrate, EDTA 2 Na, Lactose, Lactose monohydrate, Lactose SG, Low-substituted hydroxypropylcellulose, Maltodextrin, Mannitol, Menthol, Propyl paraben, Methyl paraben, Microcrystalline cellulose, Guar gum, Xanthan gum, Pregelatinized starch, Povidone K-30, Sodium starch glycolate, Sodium lauryl sulfate, Sucralose, Solutol H15, Cremophor EL, Cremophor RH40, Sodium cyclamate, PVP K90F, Oxide red, Hydroxypropyl methylcellulose, Cherry, Lemon oil, Sorbic acid, Benzyl alcohol, Glycerrin, Sodium benzolate, Starch acetate, Citric acid, Sorbitol solution, Opady white, Dextrates, NF hydrate, Magnesium stearate, Alginic acid , Eudragit E90, Eeudragit E, Glyceryl behenate, Gelucire, kollidon VA64 (copovidone K28), Hydrochoric acid, Hydroxy ethyl methyl cellulose, Hydroxy propyl cellulose, Methyl cellulose, Methacrylic acid copolymer type B (Eudragit 100), Maltose, Methacrylic Eudragit S100 acid Copolymer, PEG 1450, Povidone K-90, phosphoric acid 85%, polyoxyl 40 hydrogenated castor oil (RH 40), Polyoxyl 35 castor oil (EL 35), sodium dihydrogen phosphate, saccarin, triethyl citrate, Tri-Sodium Citrate or others Commonly used ingredients in the US FDA Generally Recognized as Safe (GRAS).

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。 The invention is further illustrated by the following examples, which are intended to be illustrative only and not to be construed as limiting.

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 Example 1. Animal test of isoniazid amide (INH) combined with CYP2E1 inhibitor disulfiram (DSF) and/or nitrophenol phosphate diester (BNPP) 一、材料與方法 First, materials and methods 1.試驗材料 Test material

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Sigma化學公司(St.Louis,MO USA)，8-iso-PGF_2α以及放射線標定之8-iso-PGF_2α-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(Sigma)溶於1公升含有適當緩衝溶液系統以及等張鹽 類之蒸餾水中，供作注射使用。 All organic solvents were HPLC grades, available from Tedia Co., Ltd. (Fairfield, OH, USA), INH, BNPP, DSF and corn oil from Sigma Chemical Company (St. Louis, MO USA), 8-iso-PGF _2α and radiolabeled 8-iso-PGF _2α -d ₄ were obtained from Cayman Chemical Company (Ann Arbor, MI, USA), and galactose injection solution was prepared by Nanguang Chemical Pharmaceutical Co., Ltd., which was 400 g of galactose (Sigma). ) Dissolved in 1 liter of distilled water containing a suitable buffer solution system and isotonic salts for injection.

2.試驗動物 2. Test animals

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內靜脈(internal jugular vein)內以施打半乳糖，導管係以切入穿刺(cut-down technique)***，該導管的末端係置於大鼠頸後切口之皮膚下方，手術完成後，恢復期間使大鼠禁食一夜(約16小時)，但水分照常供給。 Male SD (Sprague-Dawley) rats weighing 320-350 g were purchased from the National Laboratory Animal Center (Taiwan). Animal experiments were carried out in accordance with the guidelines of the National Animal Health Laboratory. All rats were placed in an air/humidity environment. Under the light and dark for 12 hours, the supply of water and feed was not limited. The weight of the rats was continuously monitored during the test. All rats were given sodium pentobarbital at a dose of 50 mg/kg. Intraperitoally anesthetized, a polyethylene catheter was placed in the rat's right jugular vein to galactose, and the catheter was inserted with a cut-down technique. Placed under the skin of the rat's posterior incision, the rats were fasted overnight (about 16 hours) during the recovery, but the water was supplied as usual.

3.試驗處理 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25mg/kg BNPP或BNPP之基劑(vehicle,VEH1，即食鹽水)，BNPP係溶於加熱至60℃之食鹽水(0.9% NaCl)，冷卻後以1ml/kg的體積進行腹腔內注射至大鼠體內；第二種處理為則注射100mg/kg DSF或DSF之基劑(VEH2，即玉米油)，DSF係溶於玉米油中，以1ml/kg的體積進行腹腔內注射至大鼠體內；第三種處理為注射150mg/kg INH或INH之基劑(VEH3，即食鹽水)，INH係溶於食鹽水(0.9% NaCl)中，以1ml/kg的體積進行腹腔內注射至大鼠體內；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。 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 in saline heated to 60 °C ( 0.9% NaCl), after cooling, intraperitoneal injection into the rat in a volume of 1ml / kg; the second treatment is to inject 100mg / kg DSF or DSF base (VEH2, corn oil), DSF is soluble In corn oil, intraperitoneal injection into the body was performed in a volume of 1 ml/kg; the third treatment was injection of 150 mg/kg of INH or INH (VEH3, ready-to-feed saline), and INH was dissolved in saline (0.9%). In NaCl, intraperitoneal injection into the rat was performed in a volume of 1 ml/kg; the first group (BNPP or VEH1) was treated 30 minutes earlier than the third group (INH or VEH3), and the second group (DSF or VEH2) was compared. The third group (INH or VEH3) was processed 15 minutes earlier.

上述5組試驗共包含：(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(施行腹腔內注射)共21天；(2)INH組(INH,n=7)：正常的大鼠每天注射1次INH、VEH1以及VEH2(施行腹腔內注射)共21天； (3)BNPP-INH組(BNPP-INH,n=7)：正常的大鼠每天注射1次BNPP、INH以及VEH2(施行腹腔內注射)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(施行腹腔內注射)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(施行腹腔內注射)共21天；半乳糖單點法於第21天處理後16小時進行測試。 The above five groups of tests included: (1) control group (normal control group, NC, n=12): normal rats were injected with VEH1, VEH2 and VEH3 (administered intraperitoneally) once a day for 21 days; (2) INH group (INH, n=7): normal rats were injected with INH, VEH1 and VEH2 once daily (administered intraperitoneally) for 21 days; (3) BNPP-INH group (BNPP-INH, 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, n=7 ): Normal rats were injected with BNPP, DSF, and INH (administered intraperitoneally) once a day for 21 days; galactose single-point method was tested 16 hours after treatment on day 21.

4.血液樣本 4. Blood sample

處理完畢後，大鼠以***麻醉犧牲，血液由大鼠背部主動脈抽取，置於含有EDTA之試管中，血漿(plasma)以13,000g於4℃離心15分鐘，分離後的血漿分裝到微量小管(Eppendorf tube)中並置於-80℃中儲存。 After the treatment, the rats were sacrificed by ether anesthesia, blood was taken from the rat aorta, placed in a test tube containing EDTA, and plasma was centrifuged at 13,000 g for 15 minutes at 4 ° C. The separated plasma was dispensed into trace amounts. Store in a small tube (Eppendorf tube) at -80 °C.

5.生化分析 5. Biochemical analysis

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)。 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, with Synchron LXi 725 system To measure (Beckman Instruments, USA).

6.光學顯微鏡與電子顯微鏡 6. Optical microscope and electron microscope

大鼠犧牲後肝臟隨即進行組織學分析；肝臟樣本以10%磷酸緩衝液配製之福馬林(phosphate-buffered formalin)固定，隨後脫水並包埋於石蠟(paraffin)中，以5μm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%四氧化鋨水溶液(aqueous osmium tetroxide)後固定1小時，以酒精連續脫水後包埋於Spurr樹脂(Spurr resin)中，並以鑽石刀切取超薄切片，以醋酸鈾醯(uranyl acetate)及檸檬酸鉛(lead citrate)作雙重染色，並以穿透式電子顯微鏡(Transmission Electron Microscope,Hitachi 600,Hitachi Co.，日本)觀察。 The rat liver was sacrificed immediately after histological analysis. The liver samples were fixed with phosphate-buffered formalin in 10% phosphate buffer, then dehydrated and embedded in paraffin, sliced at 5 μm thickness, and sliced. Hematoxylin and eosin staining, and performing a glycoside staining test (PAS), staining and histological observation by light microscopy; in addition, liver sections were treated with dimethylhydrazine buffer. (cacodylate buffer, 0.1M pH 7.4), fixed with 20% aqueous osmium tetroxide for 1 hour, continuously dehydrated with alcohol, embedded in Spurr resin, and cut with diamond knife Thin sections were double-stained with uranyl acetate and lead citrate, and observed by a transmission electron microscope (Hitachi 600, Hitachi Co., Japan).

7. 8-iso-PGF2α的萃取與量測 7. Extraction and measurement of 8-iso-PGF2α

所有PGF_2α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℃，取0.5ml血漿至玻璃管中，加入10ng內標準品(internal standard，即8-iso-PGF_2α-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50μl乙睛：水(acetonitrile：water,15：85 v/v)溶液回溶並震盪30秒，取10μl回溶後的萃取物注射至LC/MS/MS系統進行分析。 All isoforms of PGF _2α were dissolved or diluted in an appropriate volume of alcohol to prepare a stock solution, and stored in a small tube and stored at -70 ° C, 0.5 ml of plasma was taken into a glass tube, and 10 ng was added. The internal standard (8-iso-PGF _2α -d ₄ ), the mixed plasma was purified by C18 solid phase extraction cartridge (JT Baker, MA, USA), and the sample flow was washed with nitrogen. After evaporative drying, the solution was reconstituted with 50 μl of acetonitrile:water (15:85 v/v) solution and shaken for 30 seconds, and 10 μl of the re-dissolved extract was injected into an LC/MS/MS system for analysis.

8.液相層析串聯式質譜儀(LC/MS/MS)分析 8. Liquid chromatography tandem mass spectrometer (LC/MS/MS) analysis

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(Shimadzu autosampler)(島津科學儀器，日本)，以C18管柱(顆粒大小5-μm，內徑50×2.1mm)進行HPLC分離，並使用含有2mM醋酸銨(ammonium acetate)及乙睛(acetonitrile,ACN)之梯度流洗液(t=0min,15% ACN；t=6min,70% ACN；t=7min,90% ACN；t=8min,90% ACN；t=8.5min,15% ACN)流洗，LC/MS/MS的流速均維持在200μl/min，整個HPLC進行時間為13.5分鐘；該HPLC系統與一三層四極質譜儀(triple stage quadrupole mass spectrometer,API3000,Applied Biosystem,Foster City,CA，美國)介接，配備有一TurboIonSpray離子源(TurboIonSpray ionization source)，並使用負電電噴霧(negative electrospray)作為電離(ionization)之方法；該質譜儀藉由擴散200ng/ml 8-iso-PGF2α或8-iso-PGF2α-d₄標準液以多重反應監測(multiple reaction monitoring,MRM)模式進行最佳化，m/z 353/193以及m/z 357/197離子偶(ion pair)則個別用來監測8-iso-PGF2α以及8-iso-PGF2α-d₄；測量後，計算6個8-iso-PGF2α濃度(C)的線性標準曲線(linear calibration curve)對8-iso-PGF2α比8-iso-PGF2α-d₄比值之區域(Y)，得到相關係數(r,correlation coefficient)值為0.999；血漿中8-iso-PGF2α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+0.823ng/ml；所測得之結 果均對照重氫化8-iso-PGF2α(deuterated 8-iso-PGF2α)內標準品計算，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-5.06%至3.13%之間。 The HPLC system consists of two Shimadzu LC-10ADvP pumps (Shimadzu LC-10ADvP pumps), one Shimadzu system control, and one Shimadzu autosampler (Shimadzu Scientific Instruments, Japan) to C18. The column (particle size 5-μm, inner diameter 50×2.1 mm) was separated by HPLC, and a gradient flow wash containing 2 mM ammonium acetate and acetonitrile (ACN) was used (t=0 min, 15%). ACN; t=6min, 70% ACN; t=7min, 90% ACN; t=8min, 90% ACN; t=8.5min, 15% ACN) flow wash, LC/MS/MS flow rate is maintained at 200μl/ Min, the entire HPLC time was 13.5 minutes; the HPLC system was interfaced with a triple stage quadrupole mass spectrometer (API3000, Applied Biosystem, Foster City, CA, USA) equipped with a TurboIonSpray ionization (TurboIonSpray ionization) Source), and using negative electrospray as ionization method; the mass spectrometer is monitored by multiple reactions by diffusion of 200 ng/ml 8-iso-PGF2α or 8-iso-PGF2α-d ₄ standard solution ( Multiple reaction monitoring (MRM) mode for optimization, m/z 353/193 and m /z 357/197 ion pair (ion pair) was used to monitor 8-iso-PGF2α and 8-iso-PGF2α-d ₄ ; after measurement, calculate the linear standard curve of 6 8-iso-PGF2α concentration (C) (linear calibration curve) for the region of 8-iso-PGF2α ratio 8-iso-PGF2α-d ₄ ratio (Y), the correlation coefficient (r, correlation coefficient) is 0.999; the linear range of 8-iso-PGF2α in plasma Between 0.1-2.5 ng/ml, the regression equation is Y=-0.0517C+0.823 ng/ml; the measured results are all compared with the heavy hydrogenated 8-iso-PGF2α (deuterated 8-iso-PGF2α). In the internal standard calculation, the inter-assay precision and accuracy of the standard curve are evaluated by the standard concentration sample after 6 times, and then evaluated by Back-Calculation. The relative errors range from - Between 5.06% and 3.13%.

9.肝功能之定量測試 9. Quantitative testing of liver function

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秒內的快速靜脈注射，注射0.4g/ml BW半乳糖溶液0.5g/kg；自注射後5、10、15、30、45以及60分鐘各採血一次，血液樣本取自尾部靜脈；以半乳糖脫氫酶比色法(colorimetric galactose dehydrogenase)量測半乳糖含量，測試濃度範圍為50至1,000μg/ml，每個濃度的日內差異(within-day variation)係由標準偏差(standard deviation)以及變異係數(coefficient of variation,CV)百分比計算，最大容許的變異係數為10% CV；日間差異(day-to-day variation)則由比較校正曲線(calibration curves)之斜率及截距來檢驗；半乳糖清除能力(GEC)係由下列公式計算，該公式係由Tygstrup’s方程式修改而來： 其中D為半乳糖之注射量；T_C=0為半乳糖濃度達到0所需要的時間，係由注射(通常為2.22mmol/l)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內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。 All rats were tested for galactose single point (GSP) and galactose clearance (GEC). The rats received rapid intravenous injection within 30 seconds, and injected 0.4g/ml BW galactose solution 0.5g/kg; Blood was collected from 5, 10, 15, 30, 45, and 60 minutes after injection, and blood samples were taken from the tail vein; galactose content was measured by colorimetric galactose dehydrogenase, and the test concentration range was 50 to 1,000 μg/ml, the intra-day variation of each concentration is calculated from the standard deviation and the coefficient of variation (CV) percentage, and the maximum allowable coefficient of variation is 10% CV; The day-to-day variation is checked by comparing the slope and intercept of the calibration curves; the galactose clearance capacity (GEC) is calculated by the following formula, which is modified by Tygstrup's equation: Where D is the amount of galactose injected; T _{C = 0} is the time required for the galactose concentration to reach 0, which is a linear regression of the blood concentration-time curve from 20 to 60 minutes after injection (usually 2.22 mmol/l). 7; according to the rule of thumb to correct the correction value of the uneven distribution in the body; galactose single point method (GSP) is the concentration of galactose in the blood at 60 minutes after the 30 seconds of injection stop.

10.統計分析 10. Statistical analysis

所有的數據皆以平均±標準偏差(SD)表示，試驗結果以單因子變異數分析(ANOVA)測試法來計算是否具有統計上的顯著差異，使用Statistical Package of the Social Science program(Version 13,SPSS Inc.)套裝軟體來計 算；隨後使用事後比較(post hoc test)最小差異顯著性(least 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。 All data are expressed as mean ± standard deviation (SD), and the test results are calculated by the single factor analysis of variance (ANOVA) test to determine whether there is a statistically significant difference. Using the Statistical Package of the Social Science program (Version 13, SPSS) Inc.) package software to calculate Then, multiple comparisons were made using the post hoc test for the least significant difference method to confirm significant differences between the groups; the significant difference in the group average was P < 0.05.

二、結果 Second, the results 1.生化分析結果 1. Biochemical analysis results

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(對照組血漿中的AST活性為116±11IU/L；INH組血漿中的AST活性為129±10IU/L,p<0.05；對照組血漿中的ALT活性為44±6IU/L；INH組血漿中的ALT活性為52±3IU/L,p<0.05)，顯示INH組產生生化上的肝損傷；對照組、BNPP-INH、DSF-INH以及BNPP-DSF-INH組血清中轉胺酶濃度則為正常。 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 aspartate transaminase in the plasma of the INH group ( AST) and alanine transaminase (ALT) activity were significantly higher than the control group (AST activity in the control group was 116 ± 11 IU / L; AST activity in the plasma of the INH group was 129 ± 10 IU / L, p < 0.05 The ALT activity in the plasma of the control group was 44±6 IU/L; the ALT activity in the plasma of the INH group was 52±3 IU/L, p <0.05), indicating biochemical liver damage in the INH group; control group, BNPP-INH The concentrations of transaminase in the serum of the DSF-INH and BNPP-DSF-INH groups were normal.

2.組織病理學 2. Histopathology

經過為期三週施行腹腔注射150mg/kg/day INH之大鼠，其體內成功的產生肝毒性；相對的，在對照組大鼠體內的肝結構則較正常，如圖三A所示，對照組大鼠肝實質(liver parenchyma)內的肝細胞係排列於自肝小葉中央靜脈輻射排列的網狀平板內，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組大鼠肝細胞內的粗內質網(rER)明顯增加(如圖三D所示)。根據文獻報導，INH是一個強效的細胞色素P450 2E1(CYP2E1)的誘導物，而CYP2E1會導致超氧基(superoxide)以及氫氧自由基(hydroxyl radicals)的產生，並且會引發內質網的增加，因此本試驗之結果與先前研究相符。而其他試驗組：BNPP-INH組、DSF-INH組、BNPP-DSF-INH組大鼠的肝損害程度與對照組相較，並無明顯區別(未顯示結果)。 Rats who underwent intraperitoneal injection of 150 mg/kg/day INH for three weeks successfully produced hepatotoxicity in vivo; in contrast, the liver structure in the control group was normal, as shown in Figure 3A. The hepatic cell line in the liver parenchyma of the rat is arranged in a reticular plate arranged from the central vein of the hepatic lobules, and the hepatic sinusoids are found between the anastomosing plates; INH The tissue sections of the rats in the group were as shown in Figure 3B. The hepatocytes around the central vein of the rats in the INH group showed fragmentation and vacuolization, but no signs of necrosis were observed. The results of the observation showed that the crude endoplasmic reticulum (rER) in the hepatocytes of the INH group was significantly increased as compared with the control group (as shown in Fig. 3C) (as shown in Fig. 3D). According to the literature, INH is a potent cytochrome P450 2E1 (CYP2E1) inducer, and CYP2E1 leads to the production of superoxides and hydroxyl radicals, and causes endoplasmic reticulum Increased, so the results of this trial are consistent with previous studies. In other test groups, the degree of liver damage in the BNPP-INH group, the DSF-INH group, and the BNPP-DSF-INH group was not significantly different from that of the control group (no results were shown).

3.血液樣本中8-iso-PGF2α的量測 3. Measurement of 8-iso-PGF2α in blood samples

在負電電噴霧模式下，8-iso-PGF_2α最大量之分子離子為質荷比(m/z)353之離子，8-iso-PGF_2α-d₄最大量之分子離子為質荷比(m/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-iso-PGF_2α-d₄的子離子(daughter ions)恆較8-iso-PGF_2α的子離子高四個單位之外，8-iso-PGF_2α以及8-iso-PGF_2α-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-iso-PGF_2α最密集之子離子為質荷比(m/z)193之離子，8-iso-PGF_2α-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100pg 8-iso-PGF_2α與250pg/ml 8-iso-PGF_2α-d₄的標準溶液，以及一血液樣本的典型LC/MS/MS色譜，在注入1ng 8-iso-PGF_2α-d₄作為內標準品後，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。 In the negative electrospray mode, the maximum molecular ion of 8-iso-PGF _2α is the ion of mass-to-charge ratio (m/z) 353, and the maximum molecular ion of 8-iso-PGF _2α -d ₄ is the mass-to-charge ratio ( m/z) 357 ions, 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 spectrum are shown in Figure 4; except for 8-iso-PGF _2α -d ions (daughter ions) ₄ more than a constant 8-iso-PGF _{2 [alpha} ions four units high, 8-iso-PGF _2α and 8-iso-PGF _2α -d ₄ both fragmentation pattern (fragmentation Patterns) are very similar, which shows that most stable daughter ions are produced by the A chain, which is labeled with four deuterium atoms; the most dense daughter ion of 8-iso-PGF _2α is the mass-to-charge ratio. (m/z) ion of 193, the most dense daughter ion of 8-iso-PGF _2α -d ₄ is an ion of mass-to-charge ratio (m/z) 197. Figure 5 shows a standard solution containing 100 pg of 8-iso-PGF _2α and 250 pg/ml of 8-iso-PGF _2α -d ₄ and a typical LC/ of a blood sample under multiplex monitoring mode (MRM) detection. MS/MS chromatography, after injecting 1 ng of 8-iso-PGF _2α -d ₄ as an internal standard, the standard solution and the blood sample were purified by the same solid phase extraction (SPE), and the above LC/MS/MS Protocol analysis.

4.血漿中8-iso-PGF2α的濃度 4. The concentration of 8-iso-PGF2α in plasma

血漿中的8-iso-PGF_2α是一種氧化壓力(oxidative stress)的指標，如圖六所示，相較於對照組，INH組大鼠血漿中8-iso-PGF_2α的濃度明顯增加(INH組大鼠血漿中8-iso-PGF_2α的濃度為151±26pg/ml；對照組大鼠血漿中8-iso-PGF_2α的濃度為110±15pg/ml,p<0.001)；與INH組相較，BNPP-INH組、DSF-INH組、BNPP-DSF-INH組三組則明顯降低由INH引起肝臟的8-iso-PGF_2α產生(BNPP-INH組大鼠血漿中8-iso-PGF_2α的濃度為128±29pg/ml；DSF-INH組大鼠血漿中8-iso-PGF_2α的濃度為126±20pg/ml；BNPP-DSF-INH組大鼠血漿中8-iso-PGF_2α的濃度為123±17pg/ml；INH組大鼠血漿中8-iso-PGF_2α的濃度為151±26pg/ml,p<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-iso-PGF_2α的濃度無顯著差異，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-iso-PGF_2α的濃度。 8-iso-PGF _2α in plasma is an indicator of oxidative stress. As shown in Figure 6, the concentration of 8-iso-PGF _2α in the plasma of the INH group is significantly higher than that of the control group (INH). The concentration of 8-iso-PGF _2α in the plasma of the rats was 151±26pg/ml; the concentration of 8-iso-PGF _2α in the plasma of the control rats was 110±15pg/ml, p <0.001); more, BNPP-INH group, DSF-INH group, BNPP-DSF-INH three groups was significantly lower 8-iso-PGF _{2 [alpha} liver caused by the generation INH (BNPP-INH plasma of rats 8-iso-PGF _2α concentration of 128 ± 29pg / ml; concentration in plasma 8-iso-PGF DSF-INH group _{2 [alpha} rats was 126 ± 20pg / ml; concentration in plasma BNPP-DSF-INH group rats 8-iso-PGF _2α of The concentration of 8-iso-PGF _2α in the plasma of INH group was 151±26pg/ml, p <0.005); it is worth noting that the control group, BNPP-INH group and DSF-INH group There was no significant difference in the concentration of 8-iso-PGF _2α in the plasma between the four groups in the BNPP-DSF-INH group. Compared with the BNPP-INH group and the DSF-INH group, the combination of INH and BNPP did not. The concentration of 8-iso-PGF _2α in plasma is further reduced.

5.剩餘肝功能之量測 5. Measurement of residual liver function

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±69μg/ml；INH組大鼠之GSP值為565±87μg/ml,p<0.001)，此外，BNPP-INH組、DSF-INH組、BNPP-DSF-INH組大鼠之GSP值各為401±70μg/ml、449±45μg/ml、388±53μg/ml，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p<0.001,p<0.005,and p<0.001)；單獨施用INH的大鼠之GSP值明顯增加；然而，在INH合併施用BNPP或DSF或BNPP與DSF之大鼠則可抵抗這種改變；另一方面，與DSF-INH組相較，INH合併施用BNPP與DSF顯示可以降低INH引起的肝毒性，雖然兩者之間的差異未達到統計上的差異(p=0.1)，而對照組、BNPP-INH組、DSF-INH組、BNPP-DSF-INH組四組之間大鼠的GSP值無顯著差異存在。 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 μg/ml in the control group; GSP value of the rats in the INH group) 565±87μg/ml, p <0.001). In addition, the GSP values of BNPP-INH group, DSF-INH group and BNPP-DSF-INH group were 401±70μg/ml, 449±45μg/ml, 388. ±53μg/ml, 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 p <0.001, p <0.005, and p <0.001); GSP values were significantly increased in rats administered INH alone; however, rats administered either BNPP or DSF or BNPP and DSF in INH were resistant to this change; In contrast, INH combined with BNPP and DSF showed a reduction in hepatotoxicity induced by INH compared with the DSF-INH group, although the difference between the two did not reach a statistical difference ( p = 0.1), whereas the control group, BNPP- There were no significant differences in GSP values between the INH group, the DSF-INH group, and the BNPP-DSF-INH group.

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±0.6mg/min.kg；對照組大鼠之GEC值為4.9±0.8mg/min.kg,p<0.001)，此外，BNPP-INH組、DSF-INH組、BNPP-DSF-INH組大鼠之GEC值各為4.5±0.6mg/min.kg、4.3±0.4mg/min.kg、4.7±0.5mg/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p值各為p<0.005,p<0.05,and p<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±0.4mg/min.kg、4.7±0.5mg/min.kg,p=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。 Similar results were also observed using the galactose clearance (GEC) method. As shown in Figure 8, the GEC values of the rats in the INH group were significantly reduced compared with the control group (the GEC values of the rats in the INH group). 3.4±0.6mg/min.kg; the GEC value of the control group was 4.9±0.8mg/min.kg, p <0.001). In addition, the BNPP-INH group, the DSF-INH group and the BNPP-DSF-INH group were large. The GEC values of the rats were 4.5±0.6 mg/min.kg, 4.3±0.4 mg/min.kg, and 4.7±0.5 mg/min.kg; compared with the INH group, the BNPP-INH group, the DSF-INH group, and the BNPP group. The GEC values of the rats in the -DSF-INH group were significantly different from those in the INH group ( p values of p < 0.005, p < 0.05, and p < 0.005, respectively); GEC values of rats administered with INH alone Significantly reduced; 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 (DSF- The GEC values of the INH group and the BNPP-DSF-INH group were 4.3±0.4 mg/min.kg, 4.7±0.5 mg/min.kg, p =0.29); in addition, the control group, BNPP-INH group, DSF There were no significant differences in GEC values between the four groups in the -INH group and the BNPP-DSF-INH group.

為了確定AST、ALT、血漿中8-iso-PGF_2α的濃度，以及定量肝功能測試(如：GSP以及GEC)是否相關，以數種相關分析計算後，發現GSP值與血漿中8-iso-PGF_2α的濃度具有高度相關(如圖九所示)，相關係數為0.836；GSP值與GEC值具有高度相關(如圖十所示)(p<0.001)，相關係數為-0.822；GEC值也與血漿中8-iso-PGF_2α的濃度具有高度相關(相關係數為-0.743,p<0.001，如表一所示)；而GSP值、GEC值以及血漿中8-iso-PGF_2α的濃度則與AST及ALT均無明顯相關(如表一所示)。 To determine whether AST, ALT, plasma 8-iso-PGF _2α concentrations, and quantitative liver function tests (eg, GSP and GEC) were correlated, GSP values were found in 8-iso- in plasma after several correlation analyses. The concentration of PGF _2α is highly correlated (as shown in Figure 9) with a correlation coefficient of 0.836; the GSP value is highly correlated with the GEC value (as shown in Figure 10) ( p < 0.001), and the correlation coefficient is -0.822; the GEC value is also It is highly correlated with the concentration of 8-iso-PGF _2α in plasma (correlation coefficient -0.743, p <0.001, as shown in Table 1); and GSP value, GEC value, and plasma concentration of 8-iso-PGF _2α There was no significant correlation with AST and ALT (as shown in Table 1).

實施例二、細胞色素P450 2E1(CYP2E1)抑制劑之篩選-cDNA合成微粒體細胞色素P450 2E1 Example 2 Screening of Cytochrome P450 2E1 (CYP2E1) Inhibitors - cDNA Synthesis of Microsomal Cytochrome P450 2E1 一、材料與方法 First, materials and methods 1.試驗材料 Test material

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種賦形劑進行細胞色素P450 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P450 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CYP 2E1抑制率。 In this example, a cytochrome P450 2E1 (CYP2E1) inhibitor screening kit (CYP2E1 High Throughput Inhibitor Screening Kit, BD Bioscience, USA) was used to induce cytochrome P450 2E1 (CYP2E1) for 22 kinds of Chinese medicines and 10 excipients. Screening of inhibitors; the screening kit of the CYP2E1 inhibitor works by adding cytochrome P450 2E1 (CYP2E1) and its fluorescent acceptor MFC (7-Methoxy-4-trifluoromethyl coumarin) After testing the sample, detect the CYP2E1 metabolite standard HFC The amount of (7-Hydroxy-4-trifluoromethyl coumarin) was calculated, and the CYP 2E1 inhibition rate of the test sample was calculated based on the amount of HFC produced by the control.

各測試樣品均溶於乙腈(acentoitrile)，測試不同濃度之中藥藥引(66μM,33μM,16.5μM)及賦形劑(0.167%,0.08%,0.042%,w/v)對CYP2E1之抑制率，所測試之中藥藥引及結果如表三所列，所測試之賦形劑及結果如表四所列。 Each test sample was dissolved in acetonitrile (acentoitrile), and the inhibition rate of CYP2E1 was tested at different concentrations of the drug (66 μM, 33 μM, 16.5 μM) and the vehicle (0.167%, 0.08%, 0.042%, w/v). The results of the tested Chinese medicines are listed in Table 3. The tested excipients and results are listed in Table 4.

另外，本實施例使用之細胞色素P450 2E1(CYP2E1)抑制劑之篩選套組(CYP2E1 High Throughput Inhibitor Screening Kit,BD Bioscience，美國)所需之藥劑如下： In addition, the reagents required for the cytochrome P450 2E1 (CYP2E1) inhibitor screening kit (CYP2E1 High Throughput Inhibitor Screening Kit, BD Bioscience, USA) used in the present example are as follows:

(1)CYP2E1+P450 Reductase+Cytochrome b5：100mM potassium phosphate(pH 7.4)含有1.3nmol P450以及p-Nitrophenol水解酶。 (1) CYP2E1+P450 Reductase+Cytochrome b5: 100 mM potassium phosphate (pH 7.4) contains 1.3 nmol of P450 and p-Nitrophenol hydrolase.

(2)Control Protein：15mg/mL Control Protein溶於100mM Potassium Phosphate(pH 7.4)中。 (2) Control Protein: 15 mg/mL Control Protein was dissolved in 100 mM Potassium Phosphate (pH 7.4).

(3)Buffer Solution：0.5M Potassium Phosphate(pH 7.4)。 (3) Buffer Solution: 0.5M Potassium Phosphate (pH 7.4).

(4)Stop Solution：0.5M Tris Base。 (4) Stop Solution: 0.5M Tris Base.

(5)Cofactors：含有1.3mM NADP⁺、66mM MgCl₂以及66mM Glucose 6-Phosphate。 (5) Cofactors: containing 1.3 mM NADP ⁺ , 66 mM MgCl ₂ and 66 mM Glucose 6-Phosphate.

(6)Glucose 6-Phosphate Dehydrogenase：40units/ml溶於5mM Sodium Citrate Buffer(pH 7.5)。 (6) Glucose 6-Phosphate Dehydrogenase: 40 units/ml was dissolved in 5 mM Sodium Citrate Buffer (pH 7.5).

(7)MFC(7-Methoxy-4-trifluoromethyl coumarin)：螢光性受質(fluorescence substrate)，50mM MFC溶於乙腈(acetonitrile)。 (7) MFC (7-Methoxy-4-trifluoromethyl coumarin): a fluorescent substrate, 50 mM MFC dissolved in acetonitrile.

(8)DDTC(Diethyldithiocarbamic acid)：CYP2E1選擇性抑制劑(陽性對照組)，20mM DDTC溶於乙腈(acentoitrile)。 (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代謝物標準品(metabolite standard)，0.25mM HFC溶於0.1M Tris(pH 9.0)。 (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：於14.56ml無菌水中加入187.5μl Cofactors、150μl G6PDH(Glucose 6-Phosphate Dehydrogenase Solution)以及100μl Control Protein。 (10) NADPH-Cofactor Mix: Add 187.5 μl to 14.56 ml of sterile water. Cofactors, 150 μl G6PDH (Glucose 6-Phosphate Dehydrogenase Solution) and 100 μl Control Protein.

(11)Cofactor/acetonitrile mix：於9.93ml NADPH-Cofactor Mix中加入66μl Acetonitrile。 (11) Cofactor/acetonitrile mix: 66 μl of Acetonitrile was added to 9.93 ml of NADPH-Cofactor Mix.

(12)Enzyme/Substrate Mix：於4ml Buffer Soultion中加入5.94ml無菌水、50μl HTS-706(CYP2E1,2μM P450 content)以及28μl 50mM MFC(7-Methoxy-4-trifluoromethyl coumarin，螢光性受質)。 (12) Enzyme/Substrate Mix: 5.94 ml of sterile water, 50 μl of HTS-706 (CYP2E1, 2 μM P450 content) and 28 μl of 50 mM MFC (7-Methoxy-4-trifluoromethyl coumarin, fluorescent substrate) were added to 4 ml Buffer Soultion. .

2.細胞色素P450 2E1(CYP2E1)抑制劑之篩選 2. Screening of cytochrome P450 2E1 (CYP2E1) inhibitors

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)進行中藥藥引及賦形劑之篩選，實驗步驟如下所述： The cytochrome P450 2E1 (CYP2E1) inhibitor screening kit (CYP2E1 High Throughput Inhibitor Screening Kit, BD Bioscience, USA) was used to screen Chinese herbal medicines and excipients. The experimental procedures are as follows:

(1)製備對照組：a.於96孔盤上第1孔井(well)內加入149μl NADPH-Cofactor Mix以及1μl 20mM DDTC並混合均勻；b.於該96孔盤上第2至12孔井內各加入100μl Cofactor/acetonitrile mix，第1至8孔井為陽性對照組(positive control)；第9與第10孔井為對照組(control)；第11與第12孔井為空白對照組(blank)；c.於該第1至8孔井內做連續稀釋動作：自第1孔井內取50μl液體加入第2孔井內混勻，再自第2孔井內取50μl液體加入第3孔井內混勻，以此類推，至第8孔井時去除多餘的50μl液體，以得到連續稀釋濃度66.6、22.2、7.4、2.47、0.82、0.27、0.091、0.03μM。 (1) Prepare a control group: a. Add 149 μl of NADPH-Cofactor Mix and 1 μl of 20 mM DDTC to the first well on a 96-well plate and mix well; b. Wells 2 to 12 on the 96-well plate 100 μl of Cofactor/acetonitrile mix was added to each, and the first to eighth wells were positive control; the 9th and 10th wells were control; the 11th and 12th wells were blank control ( Blank); c. Perform serial dilution in the wells 1 to 8: 50 μl of liquid from the first well is added to the well of the second well, and 50 μl of liquid is added from the second well to the third Mix wells in wells, and so on. Remove excess 50 μl of liquid into Well 8 to obtain serial dilutions of 66.6, 22.2, 7.4, 2.47, 0.82, 0.27, 0.091, 0.03 μM.

(2)製備試驗組：a.於96孔盤上第1行的第1及第2孔井內各加入149μl NADPH-Cofactor Mix，以及1μl 20mM中藥藥引測試樣品或1μl 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孔井內各取50μl液體加入第3孔井內混 勻(即每一測試樣品均為三重複)； (2) Preparation test group: a. Add 149 μl of NADPH-Cofactor Mix in the first and second wells of the first row on the 96-well plate, and 1 μl of 20 mM Chinese medicine test sample or 1 μl 25% (w/v) Excipients test the sample and mix it evenly; b. Add 50 μl of liquid from the first and second wells of the first row to the third well. Evenly (ie, each test sample is three replicates);

(3)反應起始與終止：a.將上述對照組與試驗組置於37℃靜置10分鐘；b.除了該空白對照組之外，其他孔井內均加入100μl Enzyme/Substrate Mix混勻；c.將所有對照組與試驗組置於37℃靜置40分鐘；d.所有的孔井內均加入75μl Stop Solution混勻；e.緊接著於該空白對照組內加入100μl Enzyme/Substrate Mix混勻；f.將所有對照組與試驗組以螢冷光儀(Fluoroskan Ascent FL,Thermo Electron Corporation，芬蘭)讀取結果，所使用之激發光(excitation)波長為405nm，發散光(emission)波長為538nm。 (3) Start and stop of the reaction: 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, 100 μl of Enzyme/Substrate Mix was added to the wells. c. All control and test groups were allowed to stand at 37 ° C for 40 minutes; d. All wells were mixed with 75 μl Stop Solution; e. Immediately after the addition of 100 μl of Enzyme/Substrate Mix to the blank control group Mixing; f. All the control and test groups were read with a luminometer (Fluoroskan Ascent FL, Thermo Electron Corporation, Finland) using an excitation wavelength of 405 nm and an emission wavelength of 538nm.

(4)結果分析：測得之螢光訊號數值換算成為CYP2E1代謝物標準品HFC生成量(pmol)後，以對照組(control)為基準，即對照組之CYP 2E1抑制率為0%，以下列公式計算各陽性對照組及試驗組之CYP 2E1抑制率： (4) Analysis of results: After the measured value of the fluorescent signal was converted into the amount of HFC produced by the CYP2E1 metabolite standard (pmol), the control group was based on the control, that is, the CYP 2E1 inhibition rate of the control group was 0%, The column formula calculates the CYP 2E1 inhibition rate of each positive control group and test group:

二、結果 Second, the results 1.陽性對照組 Positive control group

陽性對照組(DDTC)所測出之CYP 2E1抑制率如表二所示，由表二可知當DDTC的濃度為66.6μM(即為0.167%,w/v)時，CYP 2E1抑制率可達97.55%，係以66.6μM作為中藥藥引最高測試濃度，以0.167%(w/v)作為賦形劑最高測試濃度。 The inhibition rate of CYP 2E1 measured by the positive control group (DDTC) is shown in Table 2. It can be seen from Table 2 that when the concentration of DDTC is 66.6 μM (that is, 0.167%, w/v), the inhibition rate of CYP 2E1 can reach 97.55. %, with 66.6 μM as the highest tested concentration of traditional Chinese medicine, with 0.167% (w/v) as the highest tested concentration of excipient.

2.試驗組CYP 2E1抑制率 2. Test group CYP 2E1 inhibition rate

中藥藥引所測出之CYP 2E1抑制率如表三所示，由結果可知各中藥藥引於不同濃度(66μM,33μM,16.5μM)的條件下，對細胞色素P450 2E1具有不同程度的抑制效果，其中以66μM正二羥癒瘡酸(Nordihydroguaiaretic acid)抑制效果最佳(97.99±0.66%)。 The inhibition rate of CYP 2E1 measured by traditional Chinese medicine is shown in Table 3. From the results, it can be seen that different Chinese medicines have different inhibitory effects on cytochrome P450 2E1 under different concentrations (66 μM, 33 μM, 16.5 μM). Among them, 66 μM of orthohydroguaiaretic acid had the best inhibitory effect (97.99±0.66%).

賦形劑所測出之CYP 2E1抑制率如表四所示，由結果可知各賦形劑於不同濃度(0.167%,0.08%,0.042%,w/v)的條件下，對細胞色素P450 2E1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%)。 The inhibition rate of CYP 2E1 measured by the vehicle was as shown in Table 4. From the results, it was found that each excipient was cytochrome P450 2E1 under different concentrations (0.167%, 0.08%, 0.042%, w/v). It has different degrees of inhibitory effect, among which 0.167% Brij 58 has the best inhibitory effect (97.75±0.66%).

實施例三、細胞色素P450 2E1(CYP2E1)抑制劑的篩選-人肝微粒體細胞色素P450 2E1 Example 3: Screening of cytochrome P450 2E1 (CYP2E1) inhibitors - human liver microsomal cytochrome P450 2E1 一、材料與方法 First, materials and methods 1.試驗材料 Test material

本實施例是使用人類肝臟所製備微粒體，針對細胞色素P450 2E1(CYP2E1)與39種中藥藥引及10種賦形劑進行細胞色素P450 2E1(CYP2E1)抑制劑的篩選，以篩選出對於人類肝臟之細胞色素P450 2E1(CYP2E1)有效抑制劑；該CYP2E1抑制劑的篩選作用原理為：係利用人類肝臟所製備微 粒體中細胞色素P450 2E1(CYP2E1)與其受質Chlorzoxazone反應，加入測試樣品作用後，再偵測CYP2E1代謝物標準品6-OH-CZX(6-Hydroxy-Chlorzoxazone)的生成量，並以對照組(control)的6-OH-CZX生成量為基準，計算測試樣品的CYP 2E1抑制率。 In this example, microsomes prepared by human liver were used to screen for cytochrome P450 2E1 (CYP2E1) inhibitors against cytochrome P450 2E1 (CYP2E1) and 39 kinds of Chinese medicines and 10 excipients to screen for humans. A potent inhibitor of cytochrome P450 2E1 (CYP2E1) in the liver; the principle of screening of the CYP2E1 inhibitor is: micro-preparation using human liver The cytochrome P450 2E1 (CYP2E1) in the mitochondria reacted with its receptor Chlorzoxazone, and after the test sample was added, the amount of 6-OH-CZX (6-Hydroxy-Chlorzoxazone) of the CYP2E1 metabolite standard was detected and used as a control group. The CYP 2E1 inhibition rate of the test sample was calculated based on the amount of 6-OH-CZX produced by (control).

各測試樣品均溶於10%甲醇(methanol)或是二次水中，測試不同濃度的中藥藥引(66μM,33μM,16.5μM)及賦形劑(0.167%,0.08%,0.042%,w/v)對CYP2E1的抑制率，所測試的中藥藥引及結果如表六所列，所測試的賦形劑及結果如表七所列。 Each test sample was dissolved in 10% methanol or secondary water. Different concentrations of traditional Chinese medicine (66μM, 33μM, 16.5μM) and excipients (0.167%, 0.08%, 0.042%, w/v) were tested. The inhibition rate of CYP2E1, the results of the Chinese medicines tested are listed in Table 6. The excipients and results tested are listed in Table 7.

本實施例所利用人類肝臟細胞色素P450 2E1(CYP2E1)抑制劑篩選所需的藥劑如下： The agents required for screening human liver cytochrome P450 2E1 (CYP2E1) inhibitors in this example are as follows:

(1)CYP2E1：100mM potassium phosphate(pH 7.4)含有10mg/ml P450 protein concentration。 (1) CYP2E1: 100 mM potassium phosphate (pH 7.4) contained 10 mg/ml P450 protein concentration.

(2)Control Protein：10mg/mL P450 Protein溶於100mM Potassium Phosphate(pH 7.4)中。 (2) Control Protein: 10 mg/mL P450 Protein was dissolved in 100 mM Potassium Phosphate (pH 7.4).

(3)Buffer Solution：0.5M Potassium Phosphate(pH 7.4)。Stop Solution：ice-acetonitrile。 (3) Buffer Solution: 0.5M Potassium Phosphate (pH 7.4). Stop Solution: ice-acetonitrile.

(4)Cofactors：含有100mM NADP⁺以及10mM Glucose 6-Phosphate。 (4) Cofactors: containing 100 mM NADP ⁺ and 10 mM Glucose 6-Phosphate.

(5)Glucose 6-Phosphate Dehydrogenase：2000units/ml溶于無菌水。 (5) Glucose 6-Phosphate Dehydrogenase: 2000 units/ml is dissolved in sterile water.

(6)Chlorzoxazone：受質(substrate)，16mM Chlorzoxazone溶於10%甲醇(Methanol)。 (6) Chlorzoxazone: Substrate, 16 mM Chlorzoxazone dissolved in 10% methanol (Methanol).

(7)DDTC(Diethyldithiocarbamic acid)：CYP2E1選擇性抑制劑(陽性對照組)，20mM DDTC容於10%甲醇(Methanol)。 (7) DDTC (Diethyldithiocarbamic acid): CYP2E1 selective inhibitor (positive control group), 20 mM DDTC was contained in 10% methanol (Methanol).

(8)NADPH-regenerating System：於3.42ml中加入530μl Cofactors、40μl G6PDH(Glucose 6-Phosphate Dehydrogenase Solution)以及100μl Control Protein。 (8) NADPH-regenerating System: 530 μl of Cofactors, 40 μl of G6PDH (Glucose 6-Phosphate Dehydrogenase Solution), and 100 μl of Control Protein were added to 3.42 ml.

2.細胞色素P450 2E1(CYP2E1)抑制劑的篩選 2. Screening of cytochrome P450 2E1 (CYP2E1) inhibitors

使用人類肝臟微粒體細胞色素P450 2E1(CYP2E1)進行細胞色素P450 2E1(CYP2E1)抑制劑篩選的實驗步驟如下所述： The experimental procedure for screening for cytochrome P450 2E1 (CYP2E1) inhibitors using human liver microsomal cytochrome P450 2E1 (CYP2E1) is as follows:

(1)在4℃冰浴環境下，0.1M磷酸緩衝液(pH=7.4)包含0.5mg/ml人肝微粒體、5mM MgCl₂靜置15分鐘； (1) 0.1 M phosphate buffer (pH=7.4) containing 0.5 mg/ml human liver microsomes and 5 mM MgCl _{2 was} allowed to stand for 15 minutes in an ice bath environment at 4 ° C;

(2)此時實驗組加入細胞色素P450 2E1反應基質藥物16mM Chlorzoxazone以及濃縮中藥藥引萃取液；對照組以甲醇：無菌水=1：1取代中藥藥引；陽性對照組則以DDTC取代； (2) At this time, the experimental group was added with cytochrome P450 2E1 reaction matrix drug 16 mM Chlorzoxazone and concentrated Chinese medicine extract; the control group was replaced by methanol: sterile water = 1:1; the positive control group was replaced by DDTC;

(3)最後加入輔酶1mM NADP⁺、10mM G6P與2IU G6PD。將反應液轉移至37℃水浴預溫(pre-incubation)1分鐘，活性測試實驗的反應時間為30分鐘； (3) Finally, coenzyme 1 mM NADP ⁺ , 10 mM G6P and 2 IU G6PD were added. The reaction solution was transferred to a 37 ° C water bath pre-incubation for 1 minute, and the reaction time of the activity test experiment was 30 minutes;

(4)反應完後以500L acetonitrile終止反應，樣品靜置1分鐘後加入內部標準品(5 g/mL 4-hydroxy-tobutamide)，離心後取上層液20L以甲醇：無菌水作稀釋十倍動作，取5L之回溶液注入LC/MS/MS系統進行分析。 (4) After the reaction, the reaction was terminated with 500 L of acetonitrile. After the sample was allowed to stand for 1 minute, the internal standard (5 g/mL 4-hydroxy-tobutamide) was added. After centrifugation, 20 L of the supernatant was taken and diluted with methanol: sterile water for ten times. Take 5L of the solution back into the LC/MS/MS system for analysis.

(5)結果分析：將LC/MS/MS測得的訊號數值換算成為CYP2E1代謝物標準品6-Hydroxy-Chlorzoxazone生成量(pmol)後，以對照組(control)為基準，即對照組的CYP 2E1抑制率為0%，以下列公式計算各陽性對照組及試驗組的CYP 2E1抑制率： (5) Analysis of results: After converting the signal value measured by LC/MS/MS into the amount of 6-Hydroxy-Chlorzoxazone (pmol) of the CYP2E1 metabolite standard, the control group was used as the reference, that is, the CYP of the control group. The 2E1 inhibition rate was 0%, and the CYP 2E1 inhibition rate of each positive control group and the test group was calculated by the following formula:

二、結果 Second, the results 1.陽性對照組 Positive control group

陽性對照組(DDTC)所測出的CYP 2E1抑制率如表五所示，由表二可知當DDTC的濃度為100μM時，CYP 2E1抑制率可達87.56%。 The inhibition rate of CYP 2E1 measured by the positive control group (DDTC) is shown in Table 5. It can be seen from Table 2 that when the concentration of DDTC is 100 μM, the inhibition rate of CYP 2E1 can reach 87.56%.

2.試驗組CYP 2E1抑制率 2. Test group CYP 2E1 inhibition rate

中藥藥引所測出的CYP 2E1抑制率如表六所示，由結果可知各中藥藥引於不同濃度(66μM,33μM,16.5μM)的條件下，對細胞色素P450 2E1具有不同程度的抑制效果，其中以66μM正二羥愈瘡酸(Nordihydroguaiaretic acid)抑制效果(96.98±0.19%)及66μM反式肉桂醛(Trans-Cinnamaldehyde)抑制效果(92.81±0.53%)最佳。 The inhibition rate of CYP 2E1 measured by Chinese herbal medicines is shown in Table 6. From the results, it can be seen that the various Chinese medicines have different degrees of inhibition on cytochrome P450 2E1 under different concentrations (66 μM, 33 μM, 16.5 μM). Among them, 66 μM of nordihydroguaiaretic acid inhibitory effect (96.98±0.19%) and 66 μM trans-Cinnamaldehyde (Trans-Cinnamaldehyde) inhibitory effect (92.81±0.53%) were the best.

賦形劑所測出的CYP 2E1抑制率如表七所示，由結果可知各賦形劑於不同濃度(0.167%,0.08%,0.042%,w/v)的條件下，對細胞色素P450 2E1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(91.24±1.33%)。 The inhibition rate of CYP 2E1 measured by the vehicle was as shown in Table 7. From the results, it was found that each excipient was cytochrome P450 2E1 under different concentrations (0.167%, 0.08%, 0.042%, w/v). have Different degrees of inhibition effect, among which 0.167% Brij 58 had the best inhibition effect (91.24±1.33%).

實施例四、醯胺水解酶(amidase)抑制劑的篩選-鼠肝微粒體醯胺水解酶(amidase) Example 4: Screening of indoleamine hydrolase (amidase) inhibitor - mouse liver microparticle indoleamine hydrolase (amidase) 一、材料與方法 First, materials and methods 1.試驗材料 Test material

Isonicotinic acid以高效能液相層析儀(HPLC-UV)分析定量。所有的有機溶劑均為HPLC等級，購自Tedia有限公司(Fairfield,OH,USA)，isoniazid、isonicotinic acid與nicotinic acid(內在標準品，internal standard)購自Sigma化學公司(St.Louis,MO USA)。 Isonicotinic acid was quantified by high performance liquid chromatography (HPLC-UV) analysis. All organic solvents were HPLC grades, purchased from Tedia Co., Ltd. (Fairfield, OH, USA), isoniazid, isicotinic acid and nicotinic acid (internal standard) from Sigma Chemical Company (St. Louis, MO USA) .

2.試樣處理 2. Sample processing

本實驗擬以鼠肝微粒體溶液作為amidase酵素來源，isoniazid作為amidase代謝模式藥物，定量isoniazid經amidase代謝生成之代謝物isonicotinic acid(INA)，作為amidase活性測量之標的，建立amidase體外活性抑制劑篩選平台，HPLC系統包括1個島津LC-10AD泵(Shimadzu LC-10AD pump)、1個島津系統控制器(Shimadzu system control)以及1個島津自動樣本機(Shimadzu autosampler)(島津科學儀器，日本)，以C18管柱(顆粒大小5μm，內徑50×4.6mm，25cm)並使用含有70%甲醇和30%甲酸銨(50mM,pH=2.5)之移動相進行HPLC分離，實驗步驟簡述如下： In this experiment, we used rat liver microsome solution as the source of amidase enzyme, and isoniazid as the amidase metabolic model drug, and quantified the isonicotinic acid (INA), which is produced by amidase metabolism by amiazid, as the target of amidase activity measurement, and established amidase in vitro activity inhibitor screening. The platform, the HPLC system includes a Shimadzu LC-10AD pump (Shimadzu LC-10AD pump), a Shimadzu system control, and a Shimadzu autosampler (Shimadzu Scientific Instruments, Japan). HPLC separation was carried out with a C18 column (particle size 5 μm, inner diameter 50 × 4.6 mm, 25 cm) and using a mobile phase containing 70% methanol and 30% ammonium formate (50 mM, pH = 2.5). The experimental steps are briefly described as follows:

(1)鼠肝酵素微粒體溶液製備及蛋白濃度測定。 (1) Preparation of rat liver enzyme microsome solution and determination of protein concentration.

(2)取鼠肝微粒體溶液150μL加入100μL isoniazid溶液(3mM溶於)35μL 67mM磷酸鉀緩衝溶液(KH₂PO₃,pH=7)及15μL amidase抑制劑(控制組加入去離子水)混合均勻。 (2) 150 μL of rat liver microsome solution was added to 100 μL isoniazid solution (3 mM dissolved) 35 μL 67 mM potassium phosphate buffer solution (KH ₂ PO ₃ , pH=7) and 15 μL amidase inhibitor (control group added deionized water) mixed evenly .

(3)置於37℃水浴槽反應30分鐘。 (3) Place in a 37 ° C water bath for 30 minutes.

(4)加入300μL乙晴(acetonitrile,ACN)混勻後靜置6分鐘。 (4) After adding 300 μL of acetonitrile (ACN), the mixture was allowed to stand for 6 minutes.

(5)加入30μL過氯酸混勻後靜置6分鐘。 (5) After adding 30 μL of perchloric acid, the mixture was allowed to stand for 6 minutes.

(6)以13000g之轉速離心6分鐘。 (6) Centrifuge at 13,000 g for 6 minutes.

(7)取100μL離心後上清液注射入HPLC。 (7) After centrifugation at 100 μL, the supernatant was injected into HPLC.

(8)以甲醇：甲酸銨(50mM,pH=2.5)=70：30(V/V)為移動相，控制流速為1ml/min，以波長270nm紫外光檢測。 (8) Using methanol: ammonium formate (50 mM, pH = 2.5) = 70:30 (V/V) as the mobile phase, controlling the flow rate to 1 ml/min, and detecting with ultraviolet light having a wavelength of 270 nm.

(9)結果分析：將HPLC-UV測得的訊號數值換算成為amidase代謝物標準品isonicotinic acid生成量(ng/mL)後，以對照組(control)為基準，即對照組的amidase抑制率為0%，以下列公式計算各陽性對照組及試驗組的amidase抑制率： (9) Analysis of results: After converting the signal value measured by HPLC-UV into the amount of toidocotine acid (ng/mL) of the amidase metabolite standard, the amidase inhibition rate of the control group was based on the control (control). 0%, the amidase inhibition rate of each positive control group and test group was calculated by the following formula:

二、結果 Second, the results

中藥純成份及賦形劑所測出之Amidase抑制率如表表八及表九所示，由結果可知各中藥純成分及賦形劑於不同濃度的條件下，對Amidase酵素具有不同程度的抑制效果，其中以100μM HUCHE033抑制效果最佳(75.5±2.2%)。 The Amidase inhibition rate measured by the pure ingredients and excipients of traditional Chinese medicines is shown in Tables 8 and IX. From the results, it can be seen that the pure components and excipients of various traditional Chinese medicines have different degrees of inhibition on Amidase enzyme under different concentrations. The effect was that the inhibition was best with 100 μM HUCHE033 (75.5±2.2%).

實施例五、吡嗪醯胺(PZA)合併使用醯胺水解酶(amidase)抑制劑硝基苯酚磷酸二酯(BNPP)之動物試驗 Example 5, animal test of pyrazinamide (PZA) combined with amidase inhibitor nitrophenol phosphate diester (BNPP) 一、材料與方法 First, materials and methods 1.試驗材料 Test material

所有的有機溶劑均為HPLC等級，購自Tedia有限公司(Fairfield,OH,USA)，PZA,BNPP則購自Sigma化學公司(St.Louis,MO USA)，硝基苯酚磷酸二酯(BNPP)為一文獻普遍使用之醯胺水解酶(amidase)抑制劑，半乳糖注射溶液由南光化學製藥股份有限公司製備，係將400克半乳糖(Sigma)溶於1公升含有適當緩衝溶液系統以及等張鹽類之蒸餾水中，供作注射使用。 All organic solvents were HPLC grade, purchased from Tedia Co., Ltd. (Fairfield, OH, USA), PZA, BNPP was purchased from Sigma Chemical Company (St. Louis, MO USA), and nitrophenol phosphate diester (BNPP) was A melamase inhibitor commonly used in the literature. The galactose injection solution is prepared by Nanguang Chemical Pharmaceutical Co., Ltd., which dissolves 400 g of galactose (Sigma) in 1 liter of a system containing an appropriate buffer solution and isotonic salt. Distilled water of the type used for injection.

2.試驗動物 2. Test animals

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。 Male SD (Sprague-Dawley) rats weighing 320-350 g were purchased from the National Laboratory Animal Center (Taiwan). Animal experiments were carried out in accordance with the guidelines of the National Animal Health Laboratory. All rats were placed in an air/humidity environment. Under the light and dark for 12 hours, the supply of water and feed was not limited, and the body weight of the rats was continuously monitored during the test.

3.試驗處理 3. Test treatment

試驗動物隨機分成3組，每組包括2種處理，第一種處理為注射50mg/kg BNPP或BNPP之基劑(vehicle,VEH1，即食鹽水)，BNPP係溶於加熱至60℃之食鹽水(0.9% NaCl)，冷卻後以1ml/kg的體積進行腹腔內注射至大鼠體內；第二種處理為注射500mg/kgPZA或PZA之基劑(VEH2，即食鹽水)，PZA係溶於食鹽水(0.9% NaCl)中，以1ml/kg的體積進行腹腔內注射至大鼠體內；第一種處理(BNPP或VEH1)較第二種處理(PZA或VEH2)早15分鐘處理。 The test animals were randomly divided into 3 groups, each group consisting of 2 treatments. The first treatment was injection of 50 mg/kg BNPP or BNPP (vehicle, VEH1, ready-to-feed saline), and BNPP was dissolved in saline solution heated to 60 °C. 0.9% NaCl), after cooling, intraperitoneal injection into the rat in a volume of 1ml / kg; the second treatment is the injection of 500mg / kg PZA or PZA base (VEH2, salt water), PZA is soluble in saline ( In 0.9% NaCl), intraperitoneal injection was performed into the rat in a volume of 1 ml/kg; the first treatment (BNPP or VEH1) was treated 15 minutes earlier than the second treatment (PZA or VEH2).

上述3組試驗共包含： (1)對照組(normal control group,NC,n=10)：正常的大鼠每天注射1次VEH1及VEH2(施行腹腔內注射)共49天；(2)PZA組(INH,n=10)：正常的大鼠每天注射1次PZA及VEH1(施行腹腔內注射)共49天；(3)BNPP-PZA組(BNPP-PZA,n=10)：正常的大鼠每天注射1次BNPP及PZA(施行腹腔內注射)共49天；半乳糖單點法於第49天處理後16小時進行測試。 The above three groups of tests include: (1) Control group (NC, n=10): Normal rats were injected with VEH1 and VEH2 once daily (administered intraperitoneally) for a total of 49 days; (2) PZA group (INH, n=10) : Normal rats were injected with PZA and VEH1 once daily (administered intraperitoneally) for a total of 49 days; (3) BNPP-PZA group (BNPP-PZA, n=10): normal rats were injected with BNPP and PZA once a day. (Intra-abdominal injection) for a total of 49 days; galactose single-point method was tested 16 hours after treatment on day 49.

4.血液樣本 4. Blood sample

處理完畢後，大鼠以***麻醉犧牲，血液由大鼠背部主動脈抽取，置於含有EDTA之試管中，血漿(plasma)以13,000g於4℃離心15分鐘，分離後的血漿分裝到微量小管(Eppendorf tube)中並置於-80℃中儲存。 After the treatment, the rats were sacrificed by ether anesthesia, blood was taken from the rat aorta, placed in a test tube containing EDTA, and plasma was centrifuged at 13,000 g for 15 minutes at 4 ° C. The separated plasma was dispensed into trace amounts. Store in a small tube (Eppendorf tube) at -80 °C.

5.生化分析 5. Biochemical analysis

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)。 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, with Synchron LXi 725 system To measure (Beckman Instruments, USA).

6.光學顯微鏡與電子顯微鏡 6. Optical microscope and electron microscope

大鼠犧牲後肝臟隨即進行組織學分析；肝臟樣本以10%磷酸緩衝液配製之福馬林(phosphate-buffered formalin)固定，隨後脫水並包埋於石蠟(paraffin)中，以5μm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%四氧化鋨水溶液(aqueous osmium tetroxide)後固定1小時，以酒精連續脫水後包埋於Spurr樹脂(Spurr resin)中，並以鑽石刀切取超薄切片，以醋酸鈾醯(uranyl acetate)及檸檬酸鉛(lead citrate)作雙重染色，並以穿透式電子顯微鏡(Transmission Electron Microscope,Hitachi 600,Hitachi Co.，日本)觀察。 The rat liver was sacrificed immediately after histological analysis. The liver samples were fixed with phosphate-buffered formalin in 10% phosphate buffer, then dehydrated and embedded in paraffin, sliced at 5 μm thickness, and sliced. Hematoxylin and eosin staining, and performing a glycoside staining test (PAS), staining and histological observation by light microscopy; in addition, liver sections were treated with dimethylhydrazine buffer. (cacodylate buffer, 0.1M pH 7.4), fixed with 20% aqueous osmium tetroxide for 1 hour, continuously dehydrated with alcohol, embedded in Spurr resin, and cut with diamond knife Thin sections were double-stained with uranyl acetate and lead citrate, and observed by a transmission electron microscope (Hitachi 600, Hitachi Co., Japan).

7.肝功能之定量測試 7. Quantitative testing of liver function

所有的大鼠均進行半乳糖單點法(GSP)測試，大鼠接受在30秒內的快速靜脈注射，注射0.4g/ml BW半乳糖溶液0.5g/kg；自注射後60分鐘採血一次，血液樣本取自尾部靜脈；以半乳糖脫氫酶比色法(colorimetric galactose dehydrogenase)量測半乳糖含量，測試濃度範圍為50至1,000μg/ml，每個濃度的日內差異(within-day variation)係由標準偏差(standard deviation)以及變異係數(coefficient of variation,CV)百分比計算，最大容許的變異係數為10% CV；日間差異(day-to-day variation)則由比較校正曲線(calibration curves)之斜率及截距來檢驗；半乳糖單點法(GSP)為30秒注射停止後60分鐘時血液中半乳糖濃度。 All rats were tested for galactose single point (GSP). Rats received rapid intravenous injection within 30 seconds, 0.5 g/ml BW galactose solution 0.5 g/kg; blood was collected once 60 minutes after injection. Blood samples were taken from the tail vein; galactose content was measured by colorimetric galactose dehydrogenase at concentrations ranging from 50 to 1,000 μg/ml, within-day variation for each concentration. Calculated from the standard deviation and the coefficient of variation (CV) percentage, the maximum allowable coefficient of variation is 10% CV; the day-to-day variation is determined by the calibration curves. The slope and intercept were tested; the galactose single point method (GSP) was the concentration of galactose in the blood at 60 minutes after the 30-second injection was stopped.

8.統計分析 8. Statistical analysis

所有的數據皆以平均±標準偏差(SD)表示，試驗結果以單因子變異數分析(ANOVA)測試法來計算是否具有統計上的顯著差異，使用Statistical Package of the Social Science program(Version 13,SPSS Inc.)套裝軟體來計算；隨後使用事後比較(post hoc test)最小差異顯著性(least 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。 All data are expressed as mean ± standard deviation (SD), and the test results are calculated by the single factor analysis of variance (ANOVA) test to determine whether there is a statistically significant difference. Using the Statistical Package of the Social Science program (Version 13, SPSS) Inc.) software was used to calculate; then multiple comparisons were made using the post hoc test for the least significant difference method to confirm significant differences between ethnic groups; the significant difference in ethnic mean was P < 0.05.

二、結果 Second, the results 1.生化分析結果 1. Biochemical analysis results

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；生化分析結果如圖十一所示，PZA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(對照組血漿中的AST活性為109±27IU/L；PZA組血漿中的AST活性為179±10IU/L,p<0.005；對照組血漿中的ALT活性為43±9IU/L；PZA組血漿中的ALT活性為91±11IU/L,p<0.005)，顯示PZA組產生生化上的肝損傷；BNPP-PZA血清中轉胺酶濃度則顯著低於PZA組。 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 11. The aspartate transaminase in the plasma of the PZA group ( AST) and alanine transaminase (ALT) activity were significantly higher than the control group (AST activity in the control group was 109 ± 27 IU / L; AST activity in the plasma of the PZA group was 179 ± 10 IU / L, p < 0.005 The ALT activity in the plasma of the control group was 43±9 IU/L; the ALT activity in the plasma of the PZA group was 91±11 IU/L, p <0.005), indicating biochemical liver damage in the PZA group; BNPP-PZA serum transfer The concentration of aminase was significantly lower than that of the PZA group.

2.組織病理學 2. Histopathology

經過為期7週施行腹腔注射500mg/kg/day PZA之大鼠，其體內成功的產生肝毒性；相對的，在對照組大鼠體內的肝結構則較正常，如圖十二A所示，對照組大鼠肝實質(liver parenchyma)內的肝細胞係排列於自肝小葉中央靜脈輻射排列的網狀平板內，肝血竇(hepatic sinusoids)則在兩肝板(anastomosing plates)之間被發現；PZA組大鼠的組織切片則如圖十二B所示，PZA組大鼠中央靜脈周圍的肝細胞則呈現碎裂及空泡化，然而並無看到肝細胞壞死(necrosis)的徵兆。而BNPP-PZA組大鼠的肝損害程度與對照組相較，並無明顯區別(未顯示結果)。 Rats who received an intraperitoneal injection of 500 mg/kg/day PZA for 7 weeks successfully produced hepatotoxicity in vivo; in contrast, the liver structure in the control group was normal, as shown in Figure 12A. The hepatic cell lines in the liver parenchyma of the group were arranged in a reticular plate arranged from the central vein of the hepatic lobules, and the hepatic sinusoids were found between the anastomosing plates; The tissue sections of the PZA group were as shown in Fig. 12B. The hepatocytes around the central vein of the PZA group showed fragmentation and vacuolization, but no signs of necrosis were observed. The degree of liver damage in the BNPP-PZA group was not significantly different from that in the control group (no results were shown).

3.剩餘肝功能之量測 3. Measurement of residual liver function

如圖十三所示，對照組與PZA組大鼠之半乳糖單點法(GSP)值具有高度的顯著差異(對照組大鼠之GSP值為260±50μg/ml；PZA組大鼠之GSP值為776±65μg/ml,p<0.005)，此外，BNPP-PZA組大鼠之GSP值為293±61μg/ml，與PZA組相較，具有高度的顯著差異(p<0.005)；單獨施用PZA的大鼠之GSP值明顯增加；然而，在PZA合併施用BNPP之大鼠則可抵抗這種改變；而對照組與BNPP-PZA組之間大鼠的GSP值無顯著差異存在。 As shown in Figure 13, the galactose single point method (GSP) values of the control group and the PZA group were highly significantly different (GSP value of 260 ± 50 μg/ml in the control group; GSP in the PZA group) The value was 776±65 μg/ml, p <0.005. In addition, the GSP value of the BNPP-PZA group was 293±61 μg/ml, which was highly significant ( p < 0.005) compared with the PZA group; The GSP value of PZA rats was significantly increased; however, rats with PZA combined with BNPP were resistant to this change; whereas there was no significant difference in GSP values between the control group and the BNPP-PZA group.

實施例六、異菸鹼醯胺(INH)及/或立復黴素(RIF)及/或吡嗪醯胺(PZA)合併使用CYP2E1抑制劑山奈酚(Kaempferol)或醯胺水解酶(amidase)抑制劑檞皮素(Quercetin)之動物試驗 Example 6. Isoniazid amide (INH) and/or rifamycin (RIF) and/or pyrazinamide (PZA) combined with CYP2E1 inhibitor Kaempferol or indole hydrolase (amidase) Animal test of inhibitor quercetin (Quercetin) 一、材料與方法 First, materials and methods 1.試驗材料 Test material

所有的有機溶劑均為HPLC等級，購自Tedia有限公司(Fairfield,OH,USA)，INH、RIF、PZA、Kaempferol、Quercetin購自Sigma化學公司(St.Louis,MO USA)，半乳糖注射溶液由南光化學製藥股份有限公司製備，係將400克半乳糖(Sigma)溶於1公升含有適當緩衝溶液系統以及等張鹽類之蒸餾水 中，供作注射使用。 All organic solvents were HPLC grade, purchased from Tedia Co., Ltd. (Fairfield, OH, USA), INH, RIF, PZA, Kaempferol, Quercettin from Sigma Chemical Company (St. Louis, MO USA), galactose injection solution from Nanguang Chemical Pharmaceutical Co., Ltd. prepared by dissolving 400 g of galactose (Sigma) in 1 liter of distilled water containing a suitable buffer solution system and isotonic salts. For use in injections.

2.試驗動物 2. Test animals

體重為18-25公克之129/sv小鼠是購自美國國家衛生研究院教授Dr.Gonzalez(美國)，引進公鼠3隻，母鼠4隻後，自行配對繁殖，動物實驗係遵照國衛院動物實驗指南進行，所有的小鼠均置於空氣/濕度調節環境下，光照與黑暗各12小時，水及飼料的供給不限，在試驗期間小鼠體重均持續監測，所有的小鼠均以使用***進行麻醉，並以眼窩方式進行半乳糖注射給藥，60分鐘後由尾靜脈採血測GSP值。 129/sv mice weighing 18-25 grams were purchased from Dr. Gonzalez (USA) of the National Institutes of Health, and 3 male rats were introduced. After 4 female rats, they were paired and breeding. The animal experiment was in accordance with the National Health In the animal experiment guide, all the mice were placed in an air/humidity environment, and the light and darkness were each 12 hours. The supply of water and feed was not limited. The mice were continuously monitored during the test, and all the mice were monitored. Anesthesia was performed using diethyl ether, and galactose injection was administered in an orbital manner, and 60 minutes later, blood was taken from the tail vein to measure the GSP value.

3.試驗處理 3. Test treatment

試驗動物隨機分成7組，每組包括5種處理，第一種處理為注射Kaempferol 3.78mg/kg或其基劑(vehicle,VEH1，即食鹽水)，以1ml/kg的體積進行腹腔內注射至小鼠體內；第二種處理為則注射Quercetin 3.02mg/kg或其基劑(VEH2，即食鹽水)，Quercetin係溶於食鹽水(0.9% NaCl)中，以1ml/kg的體積進行腹腔內注射至小鼠體內；第三種處理為注射50mg/kg INH或INH之基劑(VEH3，即食鹽水)，INH係溶於食鹽水(0.9% NaCl)中，以1ml/kg的體積進行腹腔內注射至小鼠體內；第四種處理為注射100mg/kg RIF或其基劑(VEH4，即食鹽水)，RIF係溶於食鹽水(0.9% NaCl)中，以1ml/kg的體積進行腹腔內注射至小鼠體內；第五種處理為注射250mg/kg PZA或其基劑(VEH5，即食鹽水)，PZA係溶於食鹽水(0.9% NaCl)中，以1ml/kg的體積進行腹腔內注射至小鼠體內。 The test animals were randomly divided into 7 groups, each group consisting of 5 treatments. The first treatment was injection of Kaempferol 3.78 mg/kg or its base (vehicle, VEH1, saline), and intraperitoneal injection was performed in a volume of 1 ml/kg. In the mouse; the second treatment is to inject Quercetin 3.02mg / kg or its base (VEH2, salt water), Quercetin is dissolved in saline (0.9% NaCl), intraperitoneal injection in a volume of 1ml / kg to In mice, the third treatment was injection of 50 mg/kg INH or INH (VEH3, ready-to-feed saline), and INH was dissolved in saline (0.9% NaCl) and injected intraperitoneally in a volume of 1 ml/kg. In mice, the fourth treatment is injection of 100 mg/kg RIF or its base (VEH4, saline), and RIF is dissolved in saline (0.9% NaCl) and injected intraperitoneally in a volume of 1 ml/kg. In the mouse; the fifth treatment is injection of 250 mg/kg PZA or its base (VEH5, ready saline), PZA is dissolved in saline (0.9% NaCl), and intraperitoneally injected into the mouse in a volume of 1 ml/kg. in vivo.

上述5組試驗共包含：(1)對照組(normal control group,NC,n=10)：正常的小鼠每天注射1次VEH1、VEH2、VEH3、VEH4以及VEH5(施行腹腔內注射)共21天；(2)INH-RIF組(INH-RIF,n=10)：正常的小鼠每天注射1次INH、RIF、VEH1、VEH2以及VEH5(施行腹腔內注射)共21天； (3)Kaempferol-INH-RIF組(Kaempferol-INH-RIF,n=10)：正常的小鼠每天注射1次Kaempferol、INH、RIF、VEH2以及VEH5(施行腹腔內注射)共21天；(4)Quercetin-INH-RIF組(Quercetin-INH-RIF,n=10)：正常的小鼠每天注射1次Quercetin、INH、RIF、VEH1以及VEH5(施行腹腔內注射)共21天；(5)INH-RIF-PZA組(INH-RIF-PZA,n=10)：正常的小鼠每天注射1次INH、RIF、PZA、VEH1以及VEH2(施行腹腔內注射)共21天；(6)Kaempferol-INH-RIF-PZA組(Kaempferol-INH-RIF-PZA,n=10)：正常的小鼠每天注射1次Kaempferol、INH、RIF、PZA以及VEH2(施行腹腔內注射)共21天；(7)Quercetin-INH-RIF-PZA組(Quercetin-INH-RIF-PZA,n=10)：正常的小鼠每天注射1次Quercetin、INH、RIF、PZA以及VEH1(施行腹腔內注射)共21天； The above five groups of experiments included: (1) control group (normal control group, NC, n=10): normal mice were injected once a day for VEH1, VEH2, VEH3, VEH4, and VEH5 (administered intraperitoneally) for 21 days. (2) INH-RIF group (INH-RIF, n=10): normal mice were injected with INH, RIF, VEH1, VEH2, and VEH5 (administered intraperitoneally) once a day for 21 days; (3) Kaempferol-INH-RIF group (Kaempferol-INH-RIF, n=10): normal mice were injected with Kaempferol, INH, RIF, VEH2 and VEH5 (administered intraperitoneally) once a day for 21 days; (4 Quercetin-INH-RIF group (Quercetin-INH-RIF, n=10): Normal mice were injected with Quecelin, INH, RIF, VEH1, and VEH5 (administered intraperitoneally) once a day for 21 days; (5) INH -RIF-PZA group (INH-RIF-PZA, n=10): Normal mice were injected with INH, RIF, PZA, VEH1, and VEH2 once daily for 21 days; (6) Kaempferol-INH -RIF-PZA group (Kaempferol-INH-RIF-PZA, n=10): Normal mice were injected with Kaempferol, INH, RIF, PZA and VEH2 (administered intraperitoneally) once a day for 21 days; (7) Quercectin -INH-RIF-PZA group (Quercetin-INH-RIF-PZA, n=10): Normal mice were injected with Quecelin, INH, RIF, PZA, and VEH1 (administered intraperitoneally) once a day for 21 days;

4.血液樣本 4. Blood sample

處理完畢後，，小鼠以***麻醉，血液由小鼠心臟採血，置於含有Heparin之試管中，血漿(plasma)以13,000g於4℃離心10分鐘，分離後的血漿分裝到微量小管(Eppendorf tube)中並置於-80℃中儲存。 After the treatment, the mice were anesthetized with ether, blood was collected from the heart of the mouse, placed in a test tube containing Heparin, plasma was centrifuged at 13,000 g for 10 minutes at 4 ° C, and the separated plasma was dispensed into microtubules ( Store in Eppendorf tube) at -80 °C.

5.生化分析 5. Biochemical analysis

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)。 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, with Synchron LXi 725 system To measure (Beckman Instruments, USA).

6.光學顯微鏡與電子顯微鏡 6. Optical microscope and electron microscope

小鼠犧牲後肝臟隨即進行組織學分析；肝臟樣本以10%磷酸緩衝液配製之福馬林(phosphate-buffered formalin)固定，隨後脫水並包埋於石蠟(paraffin) 中，以5μm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%四氧化鋨水溶液(aqueous osmium tetroxide)後固定1小時，以酒精連續脫水後包埋於Spurr樹脂(Spurr resin)中，並以鑽石刀切取超薄切片，以醋酸鈾醯(uranyl acetate)及檸檬酸鉛(lead citrate)作雙重染色，並以穿透式電子顯微鏡(Transmission Electron Microscope,Hitachi 600,Hitachi Co.，日本)觀察。 The liver was sacrificed immediately after histological analysis; the liver samples were fixed with phosphate-buffered formalin in 10% phosphate buffer, then dehydrated and embedded in paraffin The sections were sliced at a thickness of 5 μm, and the samples were stained with hematoxylin and eosin, and subjected to a liver glycosylation test (PAS). After staining, histological observation was performed by light microscopy; The liver sections were washed with cacodylate buffer (0.1 M pH 7.4), fixed with 20% aqueous osmium tetroxide for 1 hour, and continuously dehydrated with alcohol and embedded in Spurr resin. In the middle, the ultrathin section was cut with a diamond knife, double stained with uranyl acetate and lead citrate, and transmitted by electron microscope (Hitachi 600, Hitachi Co.). , Japan) observation.

7.肝功能之定量測試 7. Quantitative testing of liver function

所有的小鼠均進行半乳糖單點法(GSP)測試，小鼠接受在30秒內的快速眼窩注射，注射0.4g/ml BW半乳糖溶液0.5g/kg；自注射後60分鐘採血一次，血液樣本取自尾部靜脈；以半乳糖脫氫酶比色法(colorimetric galactose dehydrogenase)量測半乳糖含量，測試濃度範圍為50至1,000μg/ml，每個濃度的日內差異(within-day variation)係由標準偏差(standard deviation)以及變異係數(coefficient of variation,CV)百分比計算，最大容許的變異係數為10% CV；日間差異(day-to-day variation)則由比較校正曲線(calibration curves)之斜率及截距來檢驗；半乳糖單點法(GSP)為30秒注射停止後60分鐘時血液中半乳糖濃度。 All mice were tested for galactose single point (GSP). The mice received rapid eye socket injection within 30 seconds, 0.5 g/ml BW galactose solution 0.5 g/kg; blood was collected once 60 minutes after injection. Blood samples were taken from the tail vein; galactose content was measured by colorimetric galactose dehydrogenase at concentrations ranging from 50 to 1,000 μg/ml, within-day variation for each concentration. Calculated from the standard deviation and the coefficient of variation (CV) percentage, the maximum allowable coefficient of variation is 10% CV; the day-to-day variation is determined by the calibration curves. The slope and intercept were tested; the galactose single point method (GSP) was the concentration of galactose in the blood at 60 minutes after the 30-second injection was stopped.

8.統計分析 8. Statistical analysis

所有的數據皆以平均±標準偏差(SD)表示，試驗結果以單因子變異數分析(ANOVA)測試法來計算是否具有統計上的顯著差異，使用Statistical Package of the Social Science program(Version 13,SPSS Inc.)套裝軟體來計算；隨後使用事後比較(post hoc test)最小差異顯著性(least 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。 All data are expressed as mean ± standard deviation (SD), and the test results are calculated by the single factor analysis of variance (ANOVA) test to determine whether there is a statistically significant difference. Using the Statistical Package of the Social Science program (Version 13, SPSS) Inc.) software was used to calculate; then multiple comparisons were made using the post hoc test for the least significant difference method to confirm significant differences between ethnic groups; the significant difference in ethnic mean was P < 0.05.

二、結果 Second, the results 1.生化分析結果 1. Biochemical analysis results

試驗結束時，測量試驗小鼠的體重及相對肝重量，與對照組動物相較之下並無顯著差異；生化分析結果如(圖十四)所示，當小鼠以50/100mg/kg/day連續給予3週INH/RIF後，INH/RIF控制組血漿中的天門冬氨酸轉胺酶(AST)與丙氨酸轉胺酶(ALT)活性明顯高於空白對照組(空白對照組血漿中的AST活性為90±15IU/L；INH/RIF控制組血漿中的AST活性為571±295IU/L,p<0.001；空白對照組血漿中的ALT活性為40±5IU/L；INH/RIF控制組血漿中的ALT活性為364±192IU/L,p<0.001)，顯示INH/RIF的確對小鼠造成生化程度上的肝損傷；而以50/100/250mg/kg/day連續給予3週INH/RIF/PZA的小鼠，INH/RIF/PZA控制組血漿中的AST和ALT活性分別為702±172IU/L及464±72IU/L，顯著高於空白對照組及INH/RIF控制組，顯示INH/RIF/PZA的確對小鼠造成生化程度上的肝損傷，且損傷程度高於INH/RIF造成的傷害；同時給予CYP2E1抑制劑Kaempferol或醯胺水解酶抑制劑Quercetin的Quercetin-INH-RIF、Kaempferol-INH-RIF、Quercetin-INH-RIF-PZA、Kaempferol-INH-RIF-PZA實驗組血清中轉胺酶濃度則接近正常值。 At the end of the experiment, the body weight and relative liver weight of the test mice were measured, and there was no significant difference compared with the control animals. The biochemical analysis results were as shown in Fig. 14 and when the mice were 50/100 mg/kg/ After 3 weeks of continuous administration of INH/RIF, the activities of aspartate aminotransferase (AST) and alanine transaminase (ALT) in the plasma of INH/RIF control group were significantly higher than those of the blank control group (blank control plasma). The AST activity was 90±15 IU/L; the AST activity in the plasma of the INH/RIF control group was 571±295 IU/L, p <0.001; the ALT activity in the plasma of the blank control group was 40±5 IU/L; INH/RIF The ALT activity in the control group was 364 ± 192 IU/L, p < 0.001, indicating that INH/RIF did cause biochemical liver damage in mice; and continued for 3 weeks at 50/100/250 mg/kg/day. In the INH/RIF/PZA mice, the AST and ALT activities in the INH/RIF/PZA control group were 702±172 IU/L and 464±72 IU/L, respectively, which were significantly higher than the blank control group and the INH/RIF control group. It shows that INH/RIF/PZA does cause biochemical liver damage in mice, and the damage is higher than that caused by INH/RIF; at the same time, CYP2E1 inhibitor Kaempferol or indoleamine hydrolase is inhibited. The agent Quercetin Quercetin-INH-RIF, Kaempferol-INH-RIF, Quercetin-INH-RIF-PZA, Kaempferol-INH-RIF-PZA experimental group transfer of serum aminotransferase concentration is close to normal.

2.組織病理學 2. Histopathology

經過為期三週施行腹腔注射50/100mg/kg/day INH/RIF及50/100/250mg/kg/day INH/RIF/PZA之小鼠，其體內確實成功的產生肝毒性；相對的，在空白對照組小鼠體內的肝結構則較正常；相較於INH-RIF-PZA組，Kaempferol-INH-RIF組、Quercetin-INH-RIF、Kaempferol-INH-RIF-PZA組和Quercetin-INH-RIF-PZA組小鼠中央靜脈周圍肝細胞較為完整，空泡明顯較少，發炎細胞亦較少。 After three weeks of intraperitoneal injection of 50/100 mg/kg/day INH/RIF and 50/100/250 mg/kg/day INH/RIF/PZA, the mice did successfully produce hepatotoxicity; in contrast, in the blank The liver structure of the control mice was normal compared with the INH-RIF-PZA group, the Kaempferol-INH-RIF group, the Quercetin-INH-RIF group, the Kaempferol-INH-RIF-PZA group, and the Quercetin-INH-RIF- In the PZA group, the hepatocytes around the central vein were relatively intact, with less vacuoles and fewer inflammatory cells.

在評估肝臟病理組織切片嚴重程度的HAI-score方面，小鼠在連續給予INH/RIF或INH/RIF/PZA 3週後，確實在Intralobular Degeneration and Focal Necrosis與Inflammation這兩個項目得到積分，且在INH-RIF及INH-RIF-PZA控制組中發現有Piecemeal necrosis，而Kaempferol-INH-RIF組、Quercetin-INH-RIF、Kaempferol-INH-RIF-PZA組和Quercetin-INH-RIF-PZA組與INH/RIF控制組相比則有明顯的改善(圖十六、圖十七)。 In terms of HAI-score assessing the severity of liver histological sections, mice were scored in the Intralobular Degeneration and Focal Necrosis and Inflammation programs after 3 weeks of continuous administration of INH/RIF or INH/RIF/PZA. Piecemeal necrosis was found in the INH-RIF and INH-RIF-PZA control groups, while the Kaempferol-INH-RIF group, the Quercetin-INH-RIF group, the Kaempferol-INH-RIF-PZA group, and the Quercectin-INH-RIF-PZA group and INH were found. Compared with the /RIF control group, there was a significant improvement (Figure 16 and Figure 17).

1.剩餘肝功能之量測 1. Measurement of residual liver function

INH-RIF及INH-RIF-PZA控制組的半乳糖單點法(GSP)值有隨著INH/RIF給藥時間愈長而愈高的趨勢；空白對照組與INH-RIF及INH-RIF-PZA控制組小鼠之GSP值具有高度的顯著差異(空白對照組小鼠之GSP值為177±22mg/L；給藥3週後，INH-RIF及INH-RIF-PZA小鼠之GSP值各為866±339mg/L,p<0.001與858±172mg/L,p<0.001，此外，在Kaempferol-INH-RIF組、Quercetin-INH-RIF、Kaempferol-INH-RIF-PZA組和Quercetin-INH-RIF-PZA組小鼠之GSP值為401±178mg/L、203±76mg/L、273±61mg/L、216±67mg/L，與INH-RIF及INH-RIF-PZA控制組小鼠具有高度的顯著差異(p<0.001)；顯示施用INH-RIF及INH-RIF-PZA控制組小鼠之GSP值明顯增加；然而，併用Kaempferol或Quercetin之小鼠則可抵抗這種改變，空白對照組與Kaempferol或Quercetin實驗組相比 時，小鼠之間的GSP值無顯著差異存在(圖十五)。 The galactose single-point (GSP) values in the INH-RIF and INH-RIF-PZA control groups were higher with the longer INH/RIF administration time; the blank control group and INH-RIF and INH-RIF- The GSP values of the mice in the PZA control group were highly significant (GSP values in the blank control group were 177±22 mg/L; after 3 weeks of administration, the GSP values of the INH-RIF and INH-RIF-PZA mice were each 866 ± 339 mg / L, p < 0.001 and 858 ± 172 mg / L, p < 0.001, in addition, in the Kaempferol-INH-RIF group, Quercetin-INH-RIF, Kaempferol-INH-RIF-PZA group and Quercetin-INH- The GSP values of the RIF-PZA group were 401±178mg/L, 203±76mg/L, 273±61mg/L, 216±67mg/L, and the height of the mice in the INH-RIF and INH-RIF-PZA control groups. Significant differences ( p <0.001); showed a significant increase in GSP values in mice administered the INH-RIF and INH-RIF-PZA control groups; however, mice treated with Kaempferol or Quercet were resistant to this change, with blank controls and There was no significant difference in GSP values between the mice when compared to the Kaempferol or Quercectin experimental groups (Figure 15).

實施例七、異菸鹼醯胺(INH)及立復黴素(RIF)合併使用CYP2E1抑制劑山奈酚(Kaempferol)、Mannitol、Saccharin、Sucralose、Dicalcium phosphate、Crospovidone之動物試驗 Example VII, Isonicotinicinamide (INH) and Rifumycin (RIF) combined with animal test of CYP2E1 inhibitor Kaempferol, Mannitol, Saccharin, Sucralose, Dicalcium phosphate, Crospovidone 一、材料與方法 First, materials and methods 1.試驗材料 Test material

所有的有機溶劑均為HPLC等級，購自Tedia有限公司(Fairfield,OH,USA)，INH、RIF、Kaempferol、Mannitol、Saccharin、Sucralose、Dicalcium phosphate、Crospovidone購自Sigma化學公司(St.Louis,MO USA)，半乳糖注射溶液由南光化學製藥股份有限公司製備，係將400克半乳糖(Sigma)溶於1公升含有適當緩衝溶液系統以及等張鹽類之蒸餾水中，供作注射使用。 All organic solvents were HPLC grades available from Tedia Co., Ltd. (Fairfield, OH, USA), INH, RIF, Kaempferol, Mannitol, Saccharin, Sucralose, Dicalcium phosphate, Crospovidone from Sigma Chemical Company (St. Louis, MO USA). The galactose injection solution was prepared by Nanguang Chemical Pharmaceutical Co., Ltd., and 400 g of galactose (Sigma) was dissolved in 1 liter of distilled water containing a suitable buffer solution system and isotonic salts for injection.

2.試驗動物 2. Test animals

體重為18-25公克之129/sv小鼠是購自美國國家衛生研究院教授Dr.Gonzalez(美國)，引進公鼠3隻，母鼠4隻後，自行配對繁殖，動物實驗係遵照國衛院動物實驗指南進行，所有的小鼠均置於空氣/濕度調節環境下，光照與黑暗各12小時，水及飼料的供給不限，在試驗期間小鼠體重均持續監測，所有的小鼠均以使用***進行麻醉，並以眼窩方式進行半乳糖注射給藥，60分鐘後由尾靜脈採血測GSP值。 129/sv mice weighing 18-25 grams were purchased from Dr. Gonzalez (USA) of the National Institutes of Health, and 3 male rats were introduced. After 4 female rats, they were paired and breeding. The animal experiment was in accordance with the National Health In the animal experiment guide, all the mice were placed in an air/humidity environment, and the light and darkness were each 12 hours. The supply of water and feed was not limited. The mice were continuously monitored during the test, and all the mice were monitored. Anesthesia was performed using diethyl ether, and galactose injection was administered in an orbital manner, and 60 minutes later, blood was taken from the tail vein to measure the GSP value.

3.試驗處理 3. Test treatment

試驗動物隨機分成13組，每組包括3種處理，第一種處理為口服Kaempferol 1.67mg/kg，以0.1ml/kg的體積給藥；或為口服Kaempferol 4.27mg/kg，以0.1ml/kg的體積給藥；或為口服Kaempferol 8.33mg/kg，以0.1ml/kg的體積給藥；或為口服Mannitol 0.17mg/kg，以0.1ml/kg的體積給藥；或為口服Mannitol 0.83mg/kg，以0.1ml/kg的體積給藥；或為口服Mannitol 1.67 mg/kg，以0.1ml/kg的體積給藥；或為口服Saccharin 0.83mg/kg，以0.1ml/kg的體積給藥；或為口服Sucralose 1.67mg/kg，以0.1ml/kg的體積給藥；或為口服Saccharin 0.83mg/kg+Mannitol 0.83mg/kg，或為口服Dicalcium phosphate 0.83mg/kg，或處理為口服Crospovidone 2.83mg/kg，第二種處理為注射50mg/kg INH或INH之基劑(VEH1，即食鹽水)，INH係溶於食鹽水(0.9% NaCl)中，以1ml/kg的體積進行腹腔內注射至小鼠體內；第三種處理為注射100mg/kg RIF或其基劑(VEH2，即食鹽水)，RIF係溶於食鹽水(0.9% NaCl)中，以1ml/kg的體積進行腹腔內注射至小鼠體內。 The test animals were randomly divided into 13 groups, each group consisting of 3 treatments, the first treatment was oral Kaempferol 1.67 mg/kg, administered in a volume of 0.1 ml/kg; or the oral Kaempferol 4.27 mg/kg, 0.1 ml/kg. Dosing in volume; or oral administration of Kaempferol 8.33 mg/kg in a volume of 0.1 ml/kg; or oral Mannitol 0.17 mg/kg in a volume of 0.1 ml/kg; or oral Mannitol 0.83 mg/ Kg, administered in a volume of 0.1 ml/kg; or oral Mannitol 1.67 Mg/kg, administered in a volume of 0.1 ml/kg; or oral Saccharin 0.83 mg/kg, administered in a volume of 0.1 ml/kg; or oral Sucralose 1.67 mg/kg in a volume of 0.1 ml/kg Or oral Saccharin 0.83mg/kg+Mannitol 0.83mg/kg, or oral Dicalcium phosphate 0.83mg/kg, or oral Cospovidone 2.83mg/kg, the second treatment is injection 50mg/kg INH or INH Base (VEH1, ready saline), INH is dissolved in saline (0.9% NaCl), intraperitoneally injected into mice in a volume of 1ml/kg; the third treatment is injection of 100mg/kg RIF or its base The agent (VEH2, saline) was dissolved in saline (0.9% NaCl) and intraperitoneally injected into the mouse in a volume of 1 ml/kg.

上述5組試驗共包含：(1)對照組(normal control group,NC,n=10)：正常的小鼠每天注射1次VEH1、VEH2(施行腹腔內注射)共21天；(2)INH-RIF組(INH-RIF,n=10)：正常的小鼠每天注射1次INH、RIF(施行腹腔內注射)共21天；(3)KL-INH-RIF組(n=8)：正常的小鼠每天口服一次Kaempferol 1.67mg/kg，注射1次INH、RIF(施行腹腔內注射)共21天：(4)KM-INH-RIF組(n=6)：正常的小鼠每天口服一次Kaempferol 4.17mg/kg，注射1次INH、RIF(施行腹腔內注射)共21天；(5)KH-INH-RIF組(n=6)：正常的小鼠每天口服一次Kaempferol 8.33mg/kg，注射1次INH、RIF(施行腹腔內注射)共21天；(6)ML-INH-RIF組(n=8)：正常的小鼠每天口服一次Mannitol 0.17mg/kg，注射1次INH、RIF(施行腹腔內注射)共21天；(7)MM-INH-RIF組(n=6)：正常的小鼠每天口服一次Mannitol 0.83mg/kg，注射1次INH、RIF(施行腹腔內注射)共21天；(8)MH-INH-RIF組(n=6)：正常的小鼠每天口服一次Mannitol 1.67mg/kg，注射1次INH、RIF(施行腹腔內注射)共21天；(9)SA-INH-RIF組(n=4)：正常的小鼠每天口服一次Saccharin 0.83mg/kg，注射1次INH、RIF(施行腹腔內注射)共21天；(10)SU-INH-RIF組(n=4)：正常的小鼠每天口服一次Sucralose 1.67mg/kg，注射1次INH、RIF(施行腹腔內注射)共21天；(11)SAM-INH-RIF組(n=4)：正常的小鼠每天口服一次Saccharin 0.83mg/kg+Mannitol 0.83mg/kg，注射1次INH、RIF(施行腹腔內注射)共21天；(12)D-INH-RIF組(n=4)：正常的小鼠每天口服一次Dicalcium phosphate 0.83mg/kg，注射1次INH、RIF(施行腹腔內注射)共21天；(13)C-INH-RIF組(n=4)：正常的小鼠每天口服一次Crospovidone 2.83mg/kg，注射1次INH、RIF(施行腹腔內注射)共21天； The above five groups of experiments included: (1) control group (NC, n=10): normal mice were injected with VEH1 and VEH2 once daily (administered intraperitoneally) for 21 days; (2) INH- RIF group (INH-RIF, n=10): normal mice were injected with INH, RIF (administered intraperitoneally) once a day for 21 days; (3) KL-INH-RIF group (n=8): normal The mice were orally administered with Kaempferol 1.67 mg/kg once a day, once with INH and RIF (administered intraperitoneally) for 21 days: (4) KM-INH-RIF group (n=6): normal mice were orally administered Kaempferol once a day. 4.17mg/kg, 1 injection of INH, RIF (administered intraperitoneally) for 21 days; (5) KH-INH-RIF group (n=6): normal mice were given orally once a day Kaempferol 8.33mg/kg, injection 1 time INH, RIF (administration of intraperitoneal injection) for 21 days; (6) ML-INH-RIF group (n=8): normal mice were orally administered Mannitol 0.17mg/kg once a day, and injected once with INH and RIF ( Intra-abdominal injection for a total of 21 days; (7) MM-INH-RIF group (n=6): normal mice were orally administered Mannitol 0.83mg/kg once a day, once with INH, RIF (administered intraperitoneally) 21 days; (8) MH-INH-RIF group (n=6): normal mice were orally administered Mannitol 1.67 mg/kg once a day, injection 1 INH, RIF (intraperitoneal injection purposes) for 21 days; (9) SA-INH-RIF group (n = 4): Normal mice orally once daily Saccharin 0.83mg/kg, 1 injection of INH, RIF (administered intraperitoneally) for 21 days; (10) SU-INH-RIF group (n=4): normal mice were given oral Sucralose 1.67mg/kg once a day, injection 1 time INH, RIF (administration of intraperitoneal injection) for 21 days; (11) SAM-INH-RIF group (n=4): normal mice received Saccharin 0.83mg/kg+Mannitol 0.83mg/kg once a day, injection 1 time INH, RIF (administered intraperitoneal injection) for 21 days; (12) D-INH-RIF group (n=4): normal mice were given Oral Dicalcium phosphate 0.83mg/kg once a day, 1 time INH, RIF injection (Perioperative intraperitoneal injection) for 21 days; (13) C-INH-RIF group (n=4): Normal mice were given Crospovidone 2.83 mg/kg once a day, once with INH, RIF (administered intraperitoneally) 21 days in total;

4.血液樣本 4. Blood sample

處理完畢後，，小鼠以***麻醉，血液由小鼠心臟採血，置於含有Heparin之試管中，血漿(plasma)以13,000g於4℃離心10分鐘，分離後的血漿分裝到微量小管(Eppendorf tube)中並置於-80℃中儲存。 After the treatment, the mice were anesthetized with ether, blood was collected from the heart of the mouse, placed in a test tube containing Heparin, plasma was centrifuged at 13,000 g for 10 minutes at 4 ° C, and the separated plasma was dispensed into microtubules ( Store in Eppendorf tube) at -80 °C.

5.生化分析 5. Biochemical analysis

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)。 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, with Synchron LXi 725 system To measure (Beckman Instruments, USA).

6.光學顯微鏡與電子顯微鏡 6. Optical microscope and electron microscope

小鼠犧牲後肝臟隨即進行組織學分析；肝臟樣本以10%磷酸緩衝液配製之福馬林(phosphate-buffered formalin)固定，隨後脫水並包埋於石蠟(paraffin)中，以5μm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%四氧化鋨水溶液(aqueous osmium tetroxide)後固定1小 時，以酒精連續脫水後包埋於Spurr樹脂(Spurr resin)中，並以鑽石刀切取超薄切片，以醋酸鈾醯(uranyl acetate)及檸檬酸鉛(lead citrate)作雙重染色，並以穿透式電子顯微鏡(Transmission Electron Microscope,Hitachi 600,Hitachi Co.，日本)觀察。 The liver was sacrificed immediately after histological analysis. The liver samples were fixed with phosphate-buffered formalin in 10% phosphate buffer, then dehydrated and embedded in paraffin, sliced at 5 μm thickness, and sliced. Hematoxylin and eosin staining, and performing a glycoside staining test (PAS), staining and histological observation by light microscopy; in addition, liver sections were treated with dimethylhydrazine buffer. (cacodylate buffer, 0.1M pH 7.4), fixed with 20% aqueous osmium tetroxide In the case of continuous dehydration with alcohol, it is embedded in Spurr resin, and ultrathin sections are cut with a diamond knife, and double stained with uranyl acetate and lead citrate. Observed by a transmission electron microscope (Hitachi 600, Hitachi Co., Japan).

7.肝功能之定量測試 7. Quantitative testing of liver function

所有的小鼠均進行半乳糖單點法(GSP)測試，小鼠接受在30秒內的快速眼窩注射，注射0.4g/ml BW半乳糖溶液0.5g/kg；自注射後60分鐘採血一次，血液樣本取自尾部靜脈；以半乳糖脫氫酶比色法(colorimetric galactose dehydrogenase)量測半乳糖含量，測試濃度範圍為50至1,000μg/ml，每個濃度的日內差異(within-day variation)係由標準偏差(standard deviation)以及變異係數(coefficient of variation,CV)百分比計算，最大容許的變異係數為10% CV；日間差異(day-to-day variation)則由比較校正曲線(calibration curves)之斜率及截距來檢驗；半乳糖單點法(GSP)為30秒注射停止後60分鐘時血液中半乳糖濃度。 All mice were tested for galactose single point (GSP). The mice received rapid eye socket injection within 30 seconds, 0.5 g/ml BW galactose solution 0.5 g/kg; blood was collected once 60 minutes after injection. Blood samples were taken from the tail vein; galactose content was measured by colorimetric galactose dehydrogenase at concentrations ranging from 50 to 1,000 μg/ml, within-day variation for each concentration. Calculated from the standard deviation and the coefficient of variation (CV) percentage, the maximum allowable coefficient of variation is 10% CV; the day-to-day variation is determined by the calibration curves. The slope and intercept were tested; the galactose single point method (GSP) was the concentration of galactose in the blood at 60 minutes after the 30-second injection was stopped.

8.統計分析 8. Statistical analysis

所有的數據皆以平均±標準偏差(SD)表示，試驗結果以單因子變異數分析(ANOVA)測試法來計算是否具有統計上的顯著差異，使用Statistical Package of the Social Science program(Version 13,SPSS Inc.)套裝軟體來計算；隨後使用事後比較(post hoc test)最小差異顯著性(least 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。 All data are expressed as mean ± standard deviation (SD), and the test results are calculated by the single factor analysis of variance (ANOVA) test to determine whether there is a statistically significant difference. Using the Statistical Package of the Social Science program (Version 13, SPSS) Inc.) software was used to calculate; then multiple comparisons were made using the post hoc test for the least significant difference method to confirm significant differences between ethnic groups; the significant difference in ethnic mean was P < 0.05.

二、結果 Second, the results 1.生化分析結果 1. Biochemical analysis results

試驗結束時，測量試驗小鼠的體重及相對肝重量，與對照組動物相較之下並無顯著差異；生化分析結果如表十所示，當小鼠以50/100mg/kg/day連續給予3週INH/RIF後，INH/RIF控制組血漿中的天門冬氨酸轉胺酶(AST) 與丙氨酸轉胺酶(ALT)活性明顯高於空白對照組(空白對照組血漿中的AST活性為80±13IU/L；INH/RIF控制組血漿中的AST活性為420±66IU/L,p<0.001；空白對照組血漿中的ALT活性為46±10IU/L；INH/RIF控制組血漿中的ALT活性為358±67IU/L,p<0.001)，顯示INH/RIF的確對小鼠造成生化程度上的肝損傷；同時給予CYP2E1抑制劑Kaempferol、Mannitol各劑量實驗組、血清中轉胺酶濃度皆顯著低於INH/RIF控制組。 At the end of the experiment, the body weight and relative liver weight of the test mice were measured, and there was no significant difference compared with the control animals; the biochemical analysis results are shown in Table 10, and the mice were continuously administered at 50/100 mg/kg/day. After 3 weeks of INH/RIF, the aspartate aminotransferase (AST) and alanine transaminase (ALT) activities in the plasma of the INH/RIF control group were significantly higher than those of the blank control group (AST in the plasma of the control group) The activity was 80±13 IU/L; the AST activity in the plasma of the INH/RIF control group was 420±66 IU/L, p <0.001; the ALT activity in the plasma of the blank control group was 46±10 IU/L; the plasma of the INH/RIF control group The ALT activity was 358±67 IU/L, p <0.001), indicating that INH/RIF did cause biochemical liver damage in mice; CYP2E1 inhibitor Kaempferol, Mannitol dose test group, serum transaminase The concentrations were significantly lower than the INH/RIF control group.

2.組織病理學 2. Histopathology

經過為期三週施行腹腔注射50/100mg/kg/day INH/RIF之小鼠，其體內確實成功的產生肝毒性；相對的，在空白對照組小鼠體內的肝結構則較正常；相較於INH-RIF組，Mannitol各劑量實驗組小鼠中央靜脈周圍肝細胞較為完整，空泡明顯較少，發炎細胞亦較少(圖十八)。 After three weeks of intraperitoneal injection of 50/100 mg/kg/day INH/RIF, the mice did successfully produce hepatotoxicity in vivo; in contrast, the liver structure in the blank control group was normal; In the INH-RIF group, the mice in the Mannitol dose group had complete hepatocytes around the central vein, with less vacuoles and fewer inflammatory cells (Fig. 18).

在評估肝臟病理組織切片嚴重程度的HAI-score方面，小鼠在連續給予INH/RIF 3週後，Kaempferol、Mannitol各劑量實驗組與INH/RIF控制組相比則有明顯的改善。 In terms of HAI-score assessing the severity of liver histological sections, the mice were significantly improved in the Kaempferol and Mannitol dose groups compared with the INH/RIF control group after 3 weeks of continuous administration of INH/RIF.

3.剩餘肝功能之量測 3. Measurement of residual liver function

INH-RIF控制組的半乳糖單點法(GSP)值有隨著INH/RIF給藥時間愈長而愈高的趨勢；空白對照組與INH-RIF控制組小鼠之GSP值具有高度的顯著差異(空白對照組小鼠3週之GSP值為192±18mg/L；給藥3週後，INH-RIF小鼠之GSP值為666±126mg/L,p<0.001，然而，Kaempferol、Mannitol、Saccharin、Sucralose、Dicalcium phosphate各劑量實驗組之小鼠則可抵抗這種改變，其中空白對照組與KH-INH-RIF組、KM-INH-RIF組、MH-INH-RIF組、MM-INH-RIF組、SU-INH-RIF組相比時，小鼠之間的GSP值無顯著差異存在(如表十一)。 The galactose single-point (GSP) value of the INH-RIF control group has a higher tendency as the INH/RIF administration time is longer; the GSP values of the blank control group and the INH-RIF control group are highly significant. Difference (GSP value of blank control mice for 3 weeks was 192±18 mg/L; after 3 weeks of administration, the GSP value of INH-RIF mice was 666±126 mg/L, p < 0.001, however, Kaempferol, Mannitol, Mice in the Saccharin, Sucralose, and Dicalcium phosphate dose groups were resistant to this change, including the blank control group and the KH-INH-RIF group, the KM-INH-RIF group, the MH-INH-RIF group, and the MM-INH- There was no significant difference in GSP values between the mice in the RIF group and the SU-INH-RIF group (see Table 11).

實施例八、異菸鹼醯胺(INH)及立復黴素(RIF)及吡嗪醯胺(PZA)合併使用CYP2E1抑制劑Mannitol之動物試驗 Example VIII. Animal test of isoniazid amide (INH) and rifamycin (RIF) and pyrazinamide (PZA) combined with CYP2E1 inhibitor Mannitol 一、材料與方法 First, materials and methods 1.試驗材料 Test material

所有的有機溶劑均為HPLC等級，購自Tedia有限公司(Fairfield,OH,USA)，INH、RIF、PZA、Mannitol購自Sigma化學公司(St.Louis,MO USA)，半乳糖注射溶液由南光化學製藥股份有限公司製備，係將400克半乳糖(Sigma)溶於1公升含有適當緩衝溶液系統以及等張鹽類之蒸餾水中，供作注射使用。 All organic solvents were HPLC grade, purchased from Tedia Co., Ltd. (Fairfield, OH, USA), INH, RIF, PZA, Mannitol from Sigma Chemical Company (St. Louis, MO USA), galactose injection solution from Nanguang Chemical Prepared by Pharmaceutical Co., Ltd., 400 g of galactose (Sigma) was dissolved in 1 liter of distilled water containing a suitable buffer solution system and isotonic salts for injection.

2.試驗動物 2. Test animals

體重為18-25公克之129/sv小鼠是購自美國國家衛生研究院教授Dr.Gonzalez(美國)，引進公鼠3隻，母鼠4隻後，自行配對繁殖，動物實驗係遵照國衛院動物實驗指南進行，所有的小鼠均置於空氣/濕度調節環境下，光照與黑暗各12小時，水及飼料的供給不限，在試驗期間小鼠體重均持續監測，所有的小鼠均以使用***進行麻醉，並以眼窩方式進行半乳糖注射給藥，60分鐘後由尾靜脈採血測GSP值。 129/sv mice weighing 18-25 grams were purchased from Dr. Gonzalez (USA) of the National Institutes of Health, and 3 male rats were introduced. After 4 female rats, they were paired and breeding. The animal experiment was in accordance with the National Health In the animal experiment guide, all the mice were placed in an air/humidity environment, and the light and darkness were each 12 hours. The supply of water and feed was not limited. The mice were continuously monitored during the test, and all the mice were monitored. Anesthesia was performed using diethyl ether, and galactose injection was administered in an orbital manner, and 60 minutes later, blood was taken from the tail vein to measure the GSP value.

3.試驗處理 3. Test treatment

試驗動物隨機分成3組，每組包括4種處理，第一種處理為口服為口服Mannitol 1.67mg/kg，以0.1ml/kg的體積給藥；第二種處理為注射50mg/kg INH或INH之基劑(VEH1，即食鹽水)，INH係溶於食鹽水(0.9% NaCl)中，以1ml/kg的體積進行腹腔內注射至小鼠體內；第三種處理為注射100mg/kg RIF或其基劑(VEH2，即食鹽水)，RIF係溶於食鹽水(0.9% NaCl)中，以1ml/kg的體積進行腹腔內注射至小鼠體內；第四種處理為注射250mg/kg PZA或其基劑(VEH3，即食鹽水)，PZA係溶於食鹽水(0.9% NaCl)中，以1ml/kg的體積進行腹腔內注射至小鼠體內。 The test animals were randomly divided into 3 groups, each group consisting of 4 treatments. The first treatment was oral administration of Mannitol 1.67 mg/kg orally in a volume of 0.1 ml/kg; the second treatment was injection of 50 mg/kg INH or INH. The base (VEH1, ready to use saline), INH is dissolved in saline (0.9% NaCl), intraperitoneally injected into the mouse in a volume of 1ml / kg; the third treatment is injection of 100mg / kg RIF or Base (VEH2, saline), RIF is dissolved in saline (0.9% NaCl), intraperitoneally injected into mice in a volume of 1ml / kg; the fourth treatment is injection of 250mg / kg PZA or its base The agent (VEH3, ready-to-serve saline), PZA was dissolved in saline (0.9% NaCl), and intraperitoneally injected into the mouse in a volume of 1 ml/kg.

上述3組試驗共包含：(1)對照組(normal control group,NC,n=10)：正常的小鼠每天注射1次VEH1、VEH2、VEH3(施行腹腔內注射)共21天；(2)INH-RIF-PZA組(INH-RIF,n=6)：正常的小鼠每天注射1次INH、RIF、PZA(施行腹腔內注射)共21天；(3)M-INH-RIF-PZA組(n=6)：正常的小鼠每天口服一次Mannitol 1.67mg/kg，注射1次INH、RIF、PZA(施行腹腔內注射)共21天。 The above three groups of experiments included: (1) control group (normal control group, NC, n=10): normal mice were injected once a day for VEH1, VEH2, VEH3 (administered intraperitoneally) for 21 days; (2) INH-RIF-PZA group (INH-RIF, n=6): normal mice were injected with INH, RIF, PZA (administered intraperitoneally) once a day for 21 days; (3) M-INH-RIF-PZA group (n=6): Normal mice were orally administered with Mannitol 1.67 mg/kg once a day, and injected with INH, RIF, and PZA (intraperitoneal injection) for 21 days.

4.血液樣本 4. Blood sample

處理完畢後，，小鼠以***麻醉，血液由小鼠心臟採血，置於含有Heparin之試管中，血漿(plasma)以13,000g於4℃離心10分鐘，分離後的血漿分裝到微量小管(Eppendorf tube)中並置於-80℃中儲存。 After the treatment, the mice were anesthetized with ether, blood was collected from the heart of the mouse, placed in a test tube containing Heparin, plasma was centrifuged at 13,000 g for 10 minutes at 4 ° C, and the separated plasma was dispensed into microtubules ( Store in Eppendorf tube) at -80 °C.

5.肝功能之定量測試 5. Quantitative testing of liver function

所有的小鼠均進行半乳糖單點法(GSP)測試，大鼠接受在30秒內的快速眼窩注射，注射0.4g/ml BW半乳糖溶液0.5g/kg；自注射後60分鐘採血一次，血液樣本取自尾部靜脈；以半乳糖脫氫酶比色法(colorimetric galactose dehydrogenase)量測半乳糖含量，測試濃度範圍為50至1,000μg/ml，每個濃度的日內差異(within-day variation)係由標準偏差(standard deviation)以及變異係數(coefficient of variation,CV)百分比計算，最大容許的變異係數為10% CV；日間差異(day-to-day variation)則由比較校正曲線(calibration curves)之斜率及截距來檢驗；半乳糖單點法(GSP)為30秒注射停止後60分鐘時血液中半乳糖濃度。 All mice were tested for galactose single point (GSP). Rats received rapid eye socket injection within 30 seconds, 0.5 g/ml BW galactose solution 0.5 g/kg; blood was collected once 60 minutes after injection. Blood samples were taken from the tail vein; galactose content was measured by colorimetric galactose dehydrogenase at concentrations ranging from 50 to 1,000 μg/ml, within-day variation for each concentration. Calculated from the standard deviation and the coefficient of variation (CV) percentage, the maximum allowable coefficient of variation is 10% CV; the day-to-day variation is determined by the calibration curves. The slope and intercept were tested; the galactose single point method (GSP) was the concentration of galactose in the blood at 60 minutes after the 30-second injection was stopped.

6.統計分析 6. Statistical analysis

所有的數據皆以平均±標準偏差(SD)表示，試驗結果以單因子變異數分析(ANOVA)測試法來計算是否具有統計上的顯著差異，使用Statistical Package of the Social Science program(Version 13,SPSS Inc.)套裝軟體來計算；隨後使用事後比較(post hoc test)最小差異顯著性(least 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。 All data are expressed as mean ± standard deviation (SD), and the test results are calculated by the one-way variance analysis (ANOVA) test to determine whether there is a statistically significant difference, using Statistical 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 the groups; The significant difference in ethnic mean was P < 0.05.

二、結果 Second, the results

1.剩餘肝功能之量測 1. Measurement of residual liver function

INH-RIF-PZA控制組的半乳糖單點法(GSP)值有隨著INH/RIF給藥時間愈長而愈高的趨勢；空白對照組與INH-RIF-PZA控制組小鼠之GSP值具有高度的顯著差異(空白對照組小鼠3週之GSP值為570±293mg/L；給藥3週後，INH-RIF-PZA小鼠之GSP值為948±236mg/L,p<0.001，然而，Mannitol實驗組之小鼠則可抵抗這種改變(如表十二)。 The galactose single-point (GSP) value of the INH-RIF-PZA control group has a higher tendency as the INH/RIF administration time is longer; the GSP value of the blank control group and the INH-RIF-PZA control group mice There was a significant difference (the GSP value of the blank control group mice was 570±293 mg/L at 3 weeks; after 3 weeks of administration, the GSP value of INH-RIF-PZA mice was 948±236 mg/L, p < 0.001, However, mice in the Mannitol experimental group were resistant to this change (see Table 12).

實施例九、低副作用INH/RIF劑型於健康受試者體內對INH相關代謝酵素之影響研究 Example 9: Effect of low side effects INH/RIF dosage form on INH related metabolic enzymes in healthy subjects 一、材料與方法 First, materials and methods 1.試驗處理 Test treatment

利用CYP2E1 phenoytping藥物Chlorzoxazone 500mg與Rifamate (Isoniazid 150mg/Rifampin 300mg)併服CYP2E1抑制劑Mannitol 100mg，於健康受試者進行藥動學比較研究。試驗過程中，監測受試者血漿中Chlorzoxazone(CZX)及其代謝物的變化情形，並掌握ALT、AST及GSP值等生化值變化，進而評估健康受試者在有無併服CYP2E1抑制劑下，研究CYP2E1在健康受試者體內的活性變化情形。 Using CYP2E1 phenoytping drug Chlorzoxazone 500mg with Rifamate (Isoniazid 150 mg/Rifampin 300 mg) and a CYP2E1 inhibitor Mannitol 100 mg were administered for pharmacokinetic comparison studies in healthy subjects. During the test, the changes of plasma plasma Chlorzoxazone (CZX) and its metabolites were monitored, and changes in biochemical values such as ALT, AST and GSP values were obtained, and the healthy subjects were evaluated for the presence or absence of CYP2E1 inhibitors. The change in the activity of CYP2E1 in healthy subjects was studied.

2.試驗分組 2. Test grouping

本試驗全程於三軍總醫院臨床研究中心執行，試驗共有2次階段性給藥，每次試驗間隔一週。第1次給藥，口服給予國外原廠Rifamate(Isoniazid 150mg/Rifampin 300mg)與Chlorzoxazone(500mg)，第1次給藥後一週，同一批受試者進行第2次給藥，給予國外原廠Rifamate(Isoniazid 150mg/Rifampin 300mg)+Mannitol(100mg)與Chlorzoxazone(500mg)。 The whole trial was carried out at the Clinical Research Center of the General Hospital of the Three Armies. The trial consisted of two stages of administration, one week apart. For the first administration, the foreign original Rifamate (Isoniazid 150mg/Rifampin 300mg) and Chlorzoxazone (500mg) were orally administered. One week after the first administration, the same batch of subjects was given the second dose, and the foreign original was given. Plant Rifamate (Isoniazid 150 mg/Rifampin 300 mg) + Mannitol (100 mg) and Chlorzoxazone (500 mg).

3.評估及統計方法 3. Assessment and statistical methods

受試者的試驗數據及統計分析結果將會作一個整合性概述，藥物動力學數據以平均值及標準差描述，試驗中所得到的藥動學參數及數據上的顯著差異，將會以ONE WAY ANOVA或其他更適切的統計分析方法進行分析。 The experimental data and statistical analysis results of the subjects will be an integrated overview. The pharmacokinetic data will be described by mean and standard deviation. The significant differences in pharmacokinetic parameters and data obtained in the test will be ONE. WAY ANOVA or other more appropriate statistical analysis methods for analysis.

二、結果 Second, the results 1.血液分析結果 1. Blood analysis results

已完成18人次臨床試驗，控制組(Chlorzoxazone 500mg+Isoniazid 300mg)9人次與實驗組(Chlorzoxazone 500mg+Isoniazid 300mg+HUCHE033 180mg)9人次。結果顯示，併用HUCHE033組，Chlorzoxazone原型藥之藥物動力學參數未有顯著影響，但其經CYP2E1代謝之代謝物6-OH Chlorzoxazone之Cmax顯著較低，其6-OH-Chlorzoxazone/Chlorzoxazone代 謝比亦顯著低於控制組(圖十九、圖二十及表十三)。 Eighteen clinical trials have been completed, the control group (Chlorzoxazone 500mg + Isoniazid 300mg) 9 people and the experimental group (Chlorzoxazone 500mg + Isoniazid 300mg + HUCHE033 180mg) 9 times. The results showed that the pharmacokinetic parameters of the Chlorzoxazone prototype drug had no significant effect in the HUCHE033 group, but the Cmax of the 6-OH Chlorzoxazone metabolite CYP2E1 metabolized was significantly lower, and its 6-OH-Chlorzoxazone/Chlorzoxazone generation Shebi is also significantly lower than the control group (Figure 19, Figure 20 and Table 13).

本發明所提供之含異菸鹼醯胺(Isoniazid,INH)之無/低副作用新複方，與單獨使用異菸鹼醯胺(INH)及/或立復黴素(rifampin,RIF)及/或吡嗪醯胺(pyrazinamide,PZA)之試驗結果相互比較時，在生化分析(ALT、AST值)、病理學分析、剩餘肝功能之量測(GSP值、GEC值)以及氧化壓力的指標(血漿中8-iso-PGF_2α的濃度)等各方面之分析結果，都有明顯減少使用異菸鹼醯胺(INH)所造成的肝毒性副作用的功效。 The present invention provides a new compound with no/low side effects of isoniazid (INH), and isoniaminin (INH) and/or rifampin (RIF) and/or When the test results of pyrazinamide (PZA) are compared with each other, biochemical analysis (ALT, AST value), pathological analysis, measurement of residual liver function (GSP value, GEC value), and index of oxidative stress (plasma) The analysis results in various aspects such as the concentration of 8-iso-PGF _2α have significantly reduced the side effects of hepatotoxicity caused by the use of isonicotinic acid amide (INH).

本發明所提供之含異菸鹼醯胺(Isoniazid,INH)之無/低副作用新複方，其中亦提供可作為細胞色素P450 2E1(CYP2E1)抑制劑或醯胺水解酶(amidase)抑制劑之中藥藥引，相較習知細胞色素P450 2E1(CYP2E1)抑制劑或醯胺水解酶(amidase)抑制劑，本發明所提供者係從天然中藥藥引萃取者，較無生理、化學毒性，且對於人類肝臟之細胞色素P450 2E1活性有明顯之抑制活性。 The invention provides a new compound with no/low side effects of isoniazid (INH), which also provides a cytochrome P450 2E1 (CYP2E1) inhibitor or amidase hydrolyzing enzyme (amidase) inhibitor. Compared with the conventional cytochrome P450 2E1 (CYP2E1) inhibitor or indoleamine hydrolase (amidase) inhibitor, the present invention provides those who are extracted from natural Chinese medicines, have less physiological and chemical toxicity, and The cytochrome P450 2E1 activity of human liver has significant inhibitory activity.

上列詳細說明係針對本發明之一可行實施例之具體說明，惟該實施例並非用以限制本發明之專利範圍，凡未脫離本發明技藝精神所為之等效實施或變更，例如：異菸鹼醯胺(INH)、細胞色素P450 2E1抑制劑、醯胺水解 酶(amidase)抑制劑施用之濃度及比例，以及細胞色素P450 2E1抑制劑或醯胺水解酶(amidase)抑制劑選用之種類等變化之等效性實施例，均應包含於本案之專利範圍中。 The detailed description above is a detailed description of one of the possible embodiments of the present invention, and is not intended to limit the scope of the invention. Alkaline guanamine (INH), cytochrome P450 2E1 inhibitor, guanamine hydrolysis Equivalent examples of the concentration and ratio of the administration of the amidase inhibitor, and the type of the cytochrome P450 2E1 inhibitor or the amidase inhibitor, etc., should be included in the patent scope of the present application. .

上列詳細說明係針對本發明之一可行實施例之具體說明，惟該實施例並非用以限制本發明之專利範圍，凡未脫離本發明技藝精神所為之等效實施或變更，均應包含於本案之專利範圍中。 The detailed description of the preferred embodiments of the present invention is intended to be limited to the scope of the invention, and is not intended to limit the scope of the invention. The patent scope of this case.

綜上所述，本案不但在活性物質的合成上確屬創新，並能較習用物品增進上述多項功效，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。 In summary, this case is not only innovative in the synthesis of active substances, but also can enhance the above-mentioned multiple functions compared with conventional articles. It should fully comply with the statutory invention patent requirements of novelty and progressiveness, and apply for it according to law. Approved this invention patent application, in order to invent invention, to the sense of virtue.

圖一為異菸鹼醯胺(INH)在肝臟中之代謝途徑圖。 Figure 1 shows the metabolic pathway of isonicotinicinamide (INH) in the liver.

圖二為對照組、INH組、BNPP-INH組、DSF-INH組以及BNPP-DSF-INH組大鼠，天門冬氨酸轉胺酶(AST)與丙氨酸轉胺酶(ALT)活性分析，數值之計算為mean±SD，*表示各試驗組與對照組比較後P<0.05者。 Figure 2 shows the activity of aspartate transaminase (AST) and alanine transaminase (ALT) in control, INH, BNPP-INH, DSF-INH and BNPP-DSF-INH rats. The value was calculated as mean±SD, and * indicates that P < 0.05 after comparison between the test groups and the control group.

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，400X)；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組大鼠肝細胞之粗內質網(rER)明顯增加，Nu：細胞核(9,000X)。 Figure 3 shows the liver sections of the control group (Fig. 3A and C) and the INH group (Fig. 3B and D): Fig. 3A, the type of the control group relative to normal liver tissue (HE staining, 400X); Fig. 3B In the INH group, the hepatocytes in the peripheral central vein (V) showed fragmentation and vacuolation (HE staining, 400X); in Figure 3C, the liver sections of the control group were examined by electron microscopy, Nu: nuclei (9,000X); In Figure 3D, the liver sections of the INH group were examined by electron microscopy. The crude endoplasmic reticulum (rER) of the hepatocytes of the INH group was significantly increased compared with the hepatocyte sections of the control group of Fig. 3, Nu: nuclei (9,000 X).

圖四為8-iso-PGF_2α-d₄(A)與8-iso-PGF_2α(B)之分子結構以及子離子光譜。 Figure 4 shows the molecular structure and product ion spectra of 8-iso-PGF _2α -d ₄ (A) and 8-iso-PGF _2α (B).

圖五為含有250pg 8-iso-PGF_2α-d₄(A)的內標準品溶液、含有100pg 8-iso-PGF_2α(B)的標準品溶液與空白樣本(C)，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-PGF_2α-d₄(A)(作為內標準品)以及8-iso-PGF_2α(B)(作為標準品)；波峰1：空白血漿；波峰2：注入標準品之空白血漿。 Figure 5 shows an internal standard solution containing 250 pg of 8-iso-PGF _2α -d ₄ (A), a standard solution containing 100 pg of 8-iso-PGF _2α (B) and a blank sample (C) in a multiplex reaction monitoring mode. (MRM) detection of liquid chromatography tandem mass spectrometry (LC/MS/MS) chromatography, mass-to-charge ratio (m/z) 357/197 and mass-to-charge ratio (m/z) 353/193 ion couple (ion pairs) were used to monitor 8-iso-PGF _2α -d ₄ (A) (as internal standard) and 8-iso-PGF _2α (B) (as standard); peak 1: blank plasma; peak 2: Inject blank plasma of the standard.

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-PGF_2α的濃度，數值之計算為mean±SD，*表示試驗組與對照組比較後P<0.001者；^#表示各試驗組與INH組比較後P<0.05者。 Figure 6 shows the concentration of 8-iso-PGF _2α in the plasma of the control group, INH group, BNPP-INH group, DSF-INH group and BNPP-DSF-INH group. The value is calculated as mean±SD, and * indicates the test group. P < 0.001 after comparison with the control group; ^# indicates that each test group was compared with the INH group, P < 0.05.

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P<0.001者；^#表示各試驗組與INH組比較後P<0.001者；^※表示各試驗組與INH組比較後P<0.005者。 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 numerical value is calculated as mean±SD, * indicates the test group and after the control group P <0.001 by; ^# represents in each test group and the INH group P <0.001 by; ^※ after comparison indicates P <0.005 for each test group and by INH group.

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P<0.001者；^#表示各試驗組與INH組比較後P<0.005者；^※表示各試驗組與INH組比較後P<0.05者。 Figure 8 shows the galactose clearance capacity (GEC) values of the control group, INH group, BNPP-INH group, DSF-INH group and BNPP-DSF-INH group. The value is calculated as mean±SD, and * indicates the test group and control. After the group comparison, P <0.001;^# indicates that each test group was compared with the INH group, P <0.005; ^* indicates that each test group was compared with the INH group, P < 0.05.

圖九為對照組、INH組、BNPP-INH組、DSF-INH組以及BNPP-DSF-INH組各組半乳糖單點法(GSP)值與血漿中8-iso-PGF_2α的濃度具有高度相關之統計圖；y=0.2173x+33.727，r=0.836，n=40。 Figure 9 shows that the galactose single point method (GSP) values in the control group, INH group, BNPP-INH group, DSF-INH group and BNPP-DSF-INH group are highly correlated with the concentration of 8-iso-PGF _2α in plasma. Statistical chart; y = 0.2173x + 33.727, r = 0.836, n = 40.

圖十為對照組、INH組、BNPP-INH組、DSF-INH組以及BNPP-DSF-INH組各組半乳糖單點法(GSP)值與半乳糖清除能力(GEC)值具有高度相關之統計圖；y=-0.0069x+7.3828，r=-0.822，n=40。 Figure 10 shows the high correlation between galactose single point (GSP) and galactose clearance (GEC) values in the control, INH, BNPP-INH, DSF-INH, and BNPP-DSF-INH groups. Figure; y = -0.0069x + 7.3828, r = -0.822, n = 40.

圖十一為對照組、PZA組、BNPP-PZA組及BNPP組大鼠，天門冬氨酸轉胺酶(AST)與丙氨酸轉胺酶(ALT)活性分析，數值之計算為mean±SD，*表示各試驗組與對照組比較後P<0.05者。PZA控制組血漿中的天門冬氨酸轉胺酶(AST)與丙氨酸轉胺酶(ALT)活性皆明顯高於空白對照組，顯示大鼠因PZA之給藥而造成肝損傷；然而，PZA併用BNPP實驗組之大鼠則可抵抗這種肝傷害。單獨使用BNPP組其肝指數與空白對照組無異，顯示BNPP並不會造成肝臟損傷。 Figure 11 shows the activity of aspartate aminotransferase (AST) and alanine transaminase (ALT) in the control group, PZA group, BNPP-PZA group and BNPP group. The calculation of the value is mean±SD. , * indicates that each test group was compared with the control group, P < 0.05. The activities of aspartate transaminase (AST) and alanine transaminase (ALT) in the plasma of PZA control group were significantly higher than those of the blank control group, indicating that the rats were infected with liver caused by PZA; however, Rats with PZA and BNPP experimental groups were resistant to this liver injury. The liver index of the BNPP group alone was no different from that of the blank control group, indicating that BNPP did not cause liver damage.

圖十二為對照組(圖十二A)與PZA組(圖十二B)大鼠肝臟切片：圖十二A，對照組相對正常肝組織之型態(HE染色，400X)；圖十二B，PZA組在周圍中央靜脈(V)的肝細胞呈現碎裂及空泡化(HE染色，400X)。 Figure 12 shows the liver sections of the control group (Fig. 12A) and the PZA group (Fig. 12B): Fig. 12A, the type of the control group relative to normal liver tissue (HE staining, 400X); Fig. B, hepatocytes in the peripheral central vein (V) of the PZA group showed fragmentation and vacuolation (HE staining, 400X).

圖十三為、PZA組、BNPP-PZA組大鼠半乳糖單點法(GSP)值，數值之計算為mean±SD。PZA控制組的半乳糖單點法(GSP)值有隨著PZA給藥時間愈長而愈高的趨勢，顯示大鼠肝功能因PZA給藥而變差；然而，併用BNPP之實驗組大鼠則可抵抗這種肝功能受損的改變。 Figure 13 shows the galactose single point method (GSP) values of the rats in the PZA group and the BNPP-PZA group. The numerical value is calculated as mean±SD. The galactose single-point method (GSP) value of the PZA control group has a tendency to increase with the administration time of PZA, indicating that the liver function of rats is deteriorated by PZA administration; however, the experimental group rats of BNPP are used together. It can resist this change in liver function damage.

圖十四為對照組、INH-RIF組、INH-RIF-PZA組、Kaempferol-INH-RIF 組、Quercetin-INH-RIF組、Kaempferol-INH-RIF-PZA組及Quercetin-INH-RIF-PZA組小鼠，天門冬氨酸轉胺酶(AST)與丙氨酸轉胺酶(ALT)活性分析，數值之計算為mean±SD。INH/RIF控制組和INH/RIF/PZA控制組血漿中的天門冬氨酸轉胺酶(AST)與丙氨酸轉胺酶(ALT)活性皆明顯高於空白對照組，顯示小鼠因isoniazid和rifampin(INH/RIF)或isoniazid、rifampin、pyrazinamide(INH/RIF/PZA)之給藥而造成肝損傷；然而，併用Kaempferol和Quercetin各劑量實驗組之小鼠則可抵抗INH/RIF或INH/RIF/PZA造成之肝傷害。 Figure 14 shows the control group, INH-RIF group, INH-RIF-PZA group, Kaempferol-INH-RIF Group, Quercetin-INH-RIF, Kaempferol-INH-RIF-PZA and Quercetin-INH-RIF-PZA mice, aspartate transaminase (AST) and alanine transaminase (ALT) activity For analysis, the value is calculated as mean±SD. The aspartate aminotransferase (AST) and alanine transaminase (ALT) activities in the plasma of the INH/RIF control group and the INH/RIF/PZA control group were significantly higher than those of the blank control group, indicating that the mice were due to isoniazid Hepatic injury was caused by administration of rifampin (INH/RIF) or isoniazid, rifampin, pyrazinamide (INH/RIF/PZA); however, mice in the experimental group with Kaempferol and Quercetin were resistant to INH/RIF or INH/ Liver damage caused by RIF/PZA.

圖十五為對照組、INH-RIF組、INH-RIF-PZA組、Kaempferol-INH-RIF組、Quercetin-INH-RIF組、Kaempferol-INH-RIF-PZA組及Quercetin-INH-RIF-PZA組小鼠半乳糖單點法(GSP)值，數值之計算為mean±SD。 Figure 15 shows the control group, the INH-RIF group, the INH-RIF-PZA group, the Kaempferol-INH-RIF group, the Quercetin-INH-RIF group, the Kaempferol-INH-RIF-PZA group, and the Quercectin-INH-RIF-PZA group. The mouse galactose single point method (GSP) value, the value is calculated as mean ± SD.

圖十六為空白對照組(A)、INH-RIF-PZA控制組(B)、Quercetin-INH-RIF-PZA組(C)、Kaempferol-INH-RIF-PZA組(D)，於實驗3週小鼠肝臟切片。 Figure 16 shows blank control group (A), INH-RIF-PZA control group (B), Quercectin-INH-RIF-PZA group (C), Kaempferol-INH-RIF-PZA group (D), 3 weeks after the experiment. Mouse liver sections.

圖十七為空白對照組(A)、INH-RIF控制組(B)、Quercetin-INH-RIF組(C)、Kaempferol-INH-RIF組(D)，於實驗3週小鼠肝臟切片。 Figure 17 shows the blank control group (A), the INH-RIF control group (B), the Quercetin-INH-RIF group (C), and the Kaempferol-INH-RIF group (D).

圖十八為對照組(A)、INH-RIF組(B)、MH-INH-RIF組(C)、MM-INH-RIF(D)組及ML-INH-RIF組(E)，於實驗3週小鼠肝臟切片。 Figure 18 shows the control group (A), the INH-RIF group (B), the MH-INH-RIF group (C), the MM-INH-RIF (D) group, and the ML-INH-RIF group (E). 3 weeks mouse liver section.

圖十九為Chlorzoxazone+Rifamate併服或不併服Mannitol，Chlorzoxazone於健康受試者血中濃度圖。實心方塊為Rifamate控制組，給予Chlorzoxazone(500mg)+Rifamate(INH/RIF 150/300mg)；空心圓形為HUCHE033實驗組，給予Chlorzoxazone(500mg)+Rifamate(INH/RIF 150/300mg)+Mannitol 100mg。 Figure 19 shows the blood concentration of Chlorzoxazone+Rifamate with or without Mannitol and Chlorzoxazone in healthy subjects. The solid squares were the Rifamate control group, given Chlorzoxazone (500 mg) + Rifamate (INH/RIF 150/300 mg); the hollow circle was the HUCHE033 experimental group, and Chlorzoxazone (500 mg) + Rifamate (INH/RIF 150/300 mg) + Mannitol 100 mg was administered.

圖二十為Chlorzoxazone+Rifamate併服或不併服Mannitol， Chlorzoxazone於健康受試者血中濃度圖。實心三角形為Rifamate控制組，給予Chlorzoxazone(500mg)+Rifamate(INH/RIF 150/300mg)；空心菱形為HUCHE033實驗組，給予Chlorzoxazone(500mg)+Rifamate(INH/RIF 150/300mg)+Mannitol 100mg。 Figure 20 shows Chlorzoxazone+Rifamate with or without Mannitol. Chlorzoxazone is a blood concentration map of healthy subjects. The solid triangle was a Rifamate control group, given Chlorzoxazone (500 mg) + Rifamate (INH/RIF 150/300 mg); the open diamond was the HUCHE033 experimental group, and Chlorzoxazone (500 mg) + Rifamate (INH/RIF 150/300 mg) + Mannitol 100 mg was administered.

Claims (10)

一種無/低副作用之抗結核病藥物醫藥配方，包含：(a)一抗結核病藥物之化合物，其係異菸鹼醯胺(isoniazid)、或吡嗪醯胺(pyrazinamide)；以及(b)至少一種醫藥上可接受之降低抗結核病藥物副作用之物質，該物質係選自於下列化合物所組成的群組：高良薑素(Galangin)、非瑟酮(fisetin)、蓖麻油聚氧乙烯醚EL(Cremophor EL)、月桂硫酸納(Sodium Lauryl Sulfate)、微晶纖維素(Microcrystalline cellulose)、磷酸氫鈣二水物(Dicalcium phosphate dihydrate)、聚氧乙烯40氫化蓖麻油(Cremophor RH40)、交聯聚維酮(Crospovidone)、羧甲基澱粉鈉(Sodium starch glycolate)、丙烯酸樹脂S100(Eudragit S100)、交聯羧甲基纖維素鈉(Croscarmellose sodium)、薄荷腦(Menthol)、羥丙基纖維素(hydroxypropylcellulose)、預糊化澱粉(Pregelatinized starch)、甘露醇(Mannitol)、水和葡萄糖結合劑NF(Dextrates NF hydrated)、檸檬酸(Citric acid)、膠態二氧化矽(Aerosil 200)、聚乙二醇8000(PEG 8000)、山梨酸(Sorbic acid)、檸檬油(Lemon oil)、苯甲酸鈉(Sodium benzoate)、乙鮮舒泛鉀(Acesulfame K)、羥丙基甲基纖維素(Hydroxypropyl methylcellulose)、羥乙基甲基纖維素(Hydroxy ethyl methylcellulose)、糖精(Saccharin)、甲基纖維素(Methyl cellulose)、環己烷氨基磺酸鈉(Sodium cyclamate)、單水合乳糖(Lactose monohydrate)、麥芽糖糊精(Maltodextrin)、甘油山崳酸酯(Glyceryl behenate)、氧化鐵紅(Oxide red)、單硬脂酸甘油酯(Glycerin monostearate)、共聚維酮K28(Copovidone K28)、澱粉乙酸酯(Starch acetate)、硬脂酸鎂(Magnesium stearate)、聚維酮K-30(Povidone K-30)、三氯半乳蔗糖(Sucralose)、苯甲醇(Benzyl alcohol)。 An anti-tuberculosis drug pharmaceutical formulation having no/low side effects, comprising: (a) a compound of a primary anti-tuberculosis drug, which is isoniazid or pyrazinamide; and (b) at least one A pharmaceutically acceptable substance that reduces the side effects of an anti-tuberculosis drug, which is selected from the group consisting of Galangin, fisetin, and castor oil polyoxyethylene ether EL (Cremophor) EL), Sodium Lauryl Sulfate, Microcrystalline cellulose, Dicalcium phosphate dihydrate, Polyoxyethylene 40 hydrogenated castor oil (Cremophor RH40), crospovidone (Crospovidone), Sodium starch glycolate, Acrylic resin S100 (Eudragit S100), Croscarmellose sodium, Menthol, Hydroxypropylcellulose Pregelatinized starch, Mannitol, water and glucose binder NF (Dextrates NF hydrated), citric acid (Citric acid), colloidal cerium oxide (Aerosil 200) ), polyethylene glycol 8000 (PEG 8000), sorbic acid, lemon oil, sodium benzoate, Acesulfame K, hydroxypropyl methylcellulose (Hydroxypropyl methylcellulose), Hydroxyethyl methylcellulose, Saccharin, Methyl cellulose, Sodium cyclamate, Lactose monohydrate ), Maltodextrin, Glyceryl behenate, Oxide red, Glycerin monostearate, Copovidone K28, Starch acetate Starch acetate, Magnesium stearate, Povidone K-30, Sucralose, Benzyl alcohol. 如申請專利範圍第1項之抗結核病藥物醫藥配方，其中該降低抗結核病 藥物副作用之物質的最小有效劑量為：高良薑素(Galangin)(0.5毫克)、非瑟酮(fisetin)(0.9毫克)、蓖麻油聚氧乙烯醚EL(Cremophor EL)(158毫克)、月桂硫酸納(Sodium Lauryl Sulfate)(14毫克)、微晶纖維素(Microcrystalline cellulose)(180毫克)、磷酸氫鈣二水物(Dicalcium phosphate dihydrate)(9毫克)、聚氧乙烯40氫化蓖麻油(Cremophor RH40)(1.26克)、交聯聚維酮(Crospovidone)(158毫克)、羧甲基澱粉鈉(Sodium starch glycolate)(158毫克)、丙烯酸樹脂S100(Eudragit S100)(158毫克)、交聯羧甲基纖維素鈉(Croscarmellose sodium)(158毫克)、薄荷腦(Menthol)(8毫克)、羥丙基纖維素(hydroxypropylcellulose)(158毫克)、預糊化澱粉(Pregelatinized starch)(158毫克)、甘露醇(Mannitol)(10毫克)、水和葡萄糖結合劑NF(Dextrates NF hydrated)(158毫克)、檸檬酸(Citric acid)(10毫克)、膠態二氧化矽(Aerosil 200)(158毫克)、聚乙二醇8000(PEG 8000)(1.26克)、山梨酸(Sorbic acid)(6毫克)、檸檬油(Lemon oil)(158毫克)、苯甲酸鈉(Sodium benzoate)(9毫克)、乙鮮舒泛鉀(Acesulfame K)(10毫克)、羥丙基甲基纖維素(Hydroxypropyl methylcellulose)(158毫克)、羥乙基甲基纖維素(Hydroxy ethyl methylcellulose)(158毫克)、糖精(Saccharin)(10毫克)、甲基纖維素(Methyl cellulose)(158毫克)、環己烷氨基磺酸鈉(Sodium cyclamate)(10毫克)、單水合乳糖(Lactose monohydrate)(18毫克)、麥芽糖糊精(Maltodextrin)(158毫克)、甘油山崳酸酯(Glyceryl behenate)(52毫克)、氧化鐵紅(Oxide red)(34毫克)、單硬脂酸甘油酯(Glycerin monostearate)(158毫克)、共聚維酮K28(Copovidone K28)(158毫克)、澱粉乙酸酯(Starch acetate)(158毫克)、硬脂酸鎂(Magnesium stearate)(29毫克)、聚維酮K-30(Povidone K-30)(6毫克)、三氯半乳蔗糖(Sucralose)(22毫克)、苯甲醇(Benzyl alcohol)(158毫克)。 For example, the anti-tuberculosis drug and medicine formula of claim 1 of the patent scope, wherein the anti-tuberculosis is reduced The minimum effective dose of the drug side effects is: Galangin (0.5 mg), fisetin (0.9 mg), castor oil EL (Cremophor EL) (158 mg), lauric sulfate Sodium Lauryl Sulfate (14 mg), Microcrystalline cellulose (180 mg), Dicalcium phosphate dihydrate (9 mg), polyoxyethylene 40 hydrogenated castor oil (Cremophor RH40) (1.26 g), Crospovidone (158 mg), Sodium starch glycolate (158 mg), Acrylic resin S100 (Eudragit S100) (158 mg), cross-linked carboxymethyl Croscarmellose sodium (158 mg), menthol (8 mg), hydroxypropylcellulose (158 mg), pregelatinized starch (158 mg), nectar Mannitol (10 mg), water and glucose binder NF (Dextrates NF hydrated) (158 mg), Citric acid (10 mg), colloidal cerium oxide (Aerosil 200) (158 mg), Polyethylene glycol 8000 (PEG 8000) (1.26 g), sorbic acid (6 mg), Lemon oil (158 mg), sodium benzoate (9 mg), Acesulfame K (10 mg), Hydroxypropyl methylcellulose (158 mg) ), Hydroxyethyl methylcellulose (158 mg), Saccharin (10 mg), Methyl cellulose (158 mg), sodium cyclamate (Sodium) Cyclamate) (10 mg), Lactose monohydrate (18 mg), maltodextrin (158 mg), Glyceryl behenate (52 mg), iron oxide red (Oxide red) (34 mg), Glycerin monostearate (158 mg), Copovidone K28 (158 mg), Starch acetate (158 mg), stearic acid Magnesium stearate (29 mg), povidone K-30 (6 mg), sucralose (22 mg), Benzyl alcohol (158 mg) . 如申請專利範圍第1項之抗結核病藥物醫藥配方，其中可加入藥學上可 接受之賦形劑。 For example, the anti-tuberculosis drug and medicine formula of the first application of the patent scope can be added to the pharmacy. Accepted excipients. 如申請專利範圍第3項之抗結核病藥物醫藥配方，其中該賦形劑可為稀釋劑、填充劑、結合劑、崩解劑或潤滑劑。 An anti-tuberculosis drug pharmaceutical formulation according to claim 3, wherein the excipient can be a diluent, a filler, a binder, a disintegrant or a lubricant. 如申請專利範圍第1項之抗結核病藥物醫藥配方，其中該醫藥配方之劑型為口服錠劑、膠囊劑、散劑、溶液劑、懸浮劑、乳劑、芳香水劑、糖漿劑、醑劑、酏劑、酊劑、流浸膏劑、軟膏、乳霜劑、糊劑、注射劑或栓劑。 For example, the anti-tuberculosis drug pharmaceutical formulation of the scope of the patent application, wherein the pharmaceutical formulation is an oral tablet, a capsule, a powder, a solution, a suspension, an emulsion, an aromatic agent, a syrup, an expectorant, an expectorant. , tinctures, fluid extracts, ointments, creams, pastes, injections or suppositories. 一種無/低副作用之抗結核病藥物醫藥配方，包含：(a)一抗結核病化合物，其係選自於異菸鹼醯胺(isoniazid)、立復黴素(rifampin)及吡嗪醯胺(pyrazinamide)的至少兩者之組合；以及(b)至少一種醫藥上可接受之降低抗結核病藥物副作用之物質，該物質係選自於下列化合物所組成的群組：正二羥癒瘡酸(Nordihydroguaiaretic acid)、(-)表沒食子兒茶素-3-沒食子酸酯((-)-Epigallocetechin-3-gallate)、茵陳色原酮(Capillarisin)、山奈酚(Kaempferol)、根皮素(Phloretin)、橙皮素(Hesperetin)、6-薑辣醇(6-Gingerol)、沒食子酸(gallic acid)、異甘草素(Isoliquritigcnin)、柚皮素(Narigenin)、二氫化槲皮素((+)-Taxifolin)、漢黃芩素(Wongonin)、原兒茶酸(Protocatechuic acid)、兒茶素((+)-Catechin)、β-奈黄酮(β-naphthoflavone)、恩貝素(Embelin)、反式肉桂酸(trans-Cinnamic acid)、表兒茶酚((-)-Epicatechin)、反式肉桂醛(Trans-Cinnamaldehyde)、大豆甘元(Daidzein)、異牡荊素(Isovitexin)、β-香葉烯(β-Myrcene)、檞皮素(Quercetin)、(+)-檸檬烯((+)-Limonene)、楊梅素(Myricetin)、檞皮(Quercitrin)、木犀草素-7-葡萄糖苷(Luteolin-7-Glucoside)、桑葉素(Morin)、新橙皮苷(Neohesperidin)、橙皮苷(Hesperidin)、(-)-表沒食子兒茶素((-)-Epigallocatechin)、木犀草素(Luteolin)、金絲桃苷(Hyperoside)、檉柳素(Tamarixetin)、黃芩素(Baicalein)、芸香素(Rutin)、 黃芩(Baicalin)、芹菜素(Apigenin)、(+)-表兒茶素((+)-Epicatechin)、(-)表兒茶素-3-沒食子酸酯((-)-Epicatechin-3-gallate)、水飛薊賓(Silybin)、牡荊素(Vitexin)、金雀異黃酮(Genistein)、異鼠李素(Isorhamnetin)、香葉木素(Diosmin)、葛根素(Puerarin)、或傘形花內酯(Umbelliferone)、高良薑素(Galangin)、非瑟酮(fisetin)、聚氧乙烯醚(Brij 58)、聚氧乙烯10硬酯基醚(Brij 76)、(聚氧乙烯23月桂基醚)Brij 35、聚山梨酯20(Tween 20)、聚山梨酯80(Tween 80)、聚山梨酯40(Tween 40)、聚乙二醇2000(PEG 2000)、聚乙二醇400(PEG 400)、泊洛沙姆188(Pluronic F68)、聚乙二醇4000(PEG 4000)、十四烷酸乙酯(Ethyl Myristate)、蓖麻油聚氧乙烯醚EL(Cremophor EL)、月桂硫酸納(Sodium Lauryl Sulfate)、微晶纖維素(Microcrystalline cellulose)、磷酸氫鈣二水物(Dicalcium phosphate dihydrate)、聚氧乙烯40氫化蓖麻油(Cremophor RH40)、交聯聚維酮(Crospovidone)、羧甲基澱粉鈉(Sodium starch glycolate)、丙烯酸樹脂S100(Eudragit S100)、交聯羧甲基纖維素鈉(Croscarmellose sodium)、薄荷腦(Menthol)、羥丙基纖維素(hydroxypropylcellulose)、預糊化澱粉(Pregelatinized starch)、甘露醇(Mannitol)、水和葡萄糖結合劑NF(Dextrates NF hydrated)、檸檬酸(Citric acid)、膠態二氧化矽(Aerosil 200)、聚乙二醇8000(PEG 8000)、山梨酸(Sorbic acid)、檸檬油(Lemon oil)、苯甲酸鈉(Sodium benzoate)、乙鮮舒泛鉀(Acesulfame K)、羥丙基甲基纖維素(Hydroxypropyl methylcellulose)、羥乙基甲基纖維素(Hydroxy ethyl methylcellulose)、糖精(Saccharin)、甲基纖維素(Methyl cellulose)、環己烷氨基磺酸鈉(Sodium cyclamate)、單水合乳糖(Lactose monohydrate)、麥芽糖糊精(Maltodextrin)、甘油山崳酸酯(Glyceryl behenate)、氧化鐵紅(Oxide red)、單硬脂酸甘油酯(Glycerin monostearate)、共聚維酮K28(Copovidone K28)、澱粉乙酸酯(Starch acetate)、硬脂酸鎂(Magnesium stearate)、聚維酮K-30(Povidone K-30)、三氯半乳蔗糖(Sucralose)、苯甲醇(Benzyl alcohol)。 An anti-tuberculosis drug pharmaceutical formulation having no/low side effects, comprising: (a) a primary anti-tuberculosis compound selected from the group consisting of isoniazid, rifampin, and pyrazinamide And (b) at least one pharmaceutically acceptable substance that reduces the side effects of the anti-tuberculosis drug, the substance being selected from the group consisting of: Nordic hydroguaiaretic acid , (-) epigallocatechin-3-gallate ((-)-Epigallocetechin-3-gallate), capillarisin (Capillarisin), kaempferol (Kaempferol), phloretin ( Phloretin), Hesperetin, 6-Gingerol, gallic acid, Isoliquritigcnin, Nariginin, dihydroquercetin (+) - Taxifolin), wogonin (Wongonin), protocatechuic acid (protocatechuic acid), catechins ((+) - catechin), β - Chennai flavonoids (β-naphthoflavone), Enbei hormone (Embelin) , trans-Cinnamic acid, epicatechin ((-)-Epicatechin), trans-Cinnamaldehyde, soybean Element (Daidzein), iso Vitexin (Isovitexin), β - myrcene (β-Myrcene), quercetin (Quercetin), (+) - limonene ((+) - Limonene), myricetin (Myricetin), Quercitrin, Luteolin-7-Glucoside, Morin, Neohesperidin, Hesperidin, (-)- Essence of catechin ((-)-Epigallocatechin), Luteolin, Hyperposide, Tamarixetin, Baicalein, Rutin, Baicalin , Apigenin, (+)-epicatechin ((+)-Epicatechin), (-) epicatechin-3-gallate ((-)-Epicatechin-3-gallate), Silybin, Vitexin, Genistein, Isorhamnetin, Diosmin, Puerarin, or Umbelliferone (Umbelliferone), Galangin, fisetin, polyoxyethylene ether (Brij 58), polyoxyethylene 10 hard ester ether (Brij 76), (polyoxyethylene 23 lauryl ether) Brij 35, polysorbate 20 (Tween 20), polysorbate 80 (Tween 80), poly mountain Ester 40 (Tween 40), polyethylene glycol 2000 (PEG 2000), polyethylene glycol 400 (PEG 400), poloxamer 188 (Pluronic F68), polyethylene glycol 4000 (PEG 4000), tetradecane Ethyl Myristate, castor oil EL (Cremophor EL), sodium lauryl Sulfate, microcrystalline cellulose, Dicalcium phosphate dihydrate, Polyoxyethylene 40 hydrogenated castor oil (Cremophor RH40), Crospovidone, sodium starch glycolate, acrylic resin S100 (Eudragit S100), croscarmellose sodium (Croscarmellose) Sodium), menthol, hydroxypropylcellulose, pregelatinized starch, Mannitol, water and glucose binder NF (Dextrates NF hydrated), citric acid (Citric acid) ), colloidal cerium oxide (Aerosulf 200), polyethylene glycol 8000 (PEG 8000), sorbic acid (Sorbic acid), lemon oil (Lemon oil), sodium benzoate (Sodium benzoate), Ethyl sulphate (Acesulfame) K), Hydroxypropyl Methyl Cellulose (Hydroxypropyl meth Ylcellulose), Hydroxyethyl methylcellulose, Saccharin, Methyl cellulose, Sodium cyclamate, Lactose monohydrate, Maltodextrin, Glyceryl behenate, Oxide red, Glycerin monostearate, Copovidone K28, Starch acetate Starch acetate), Magnesium stearate, Povidone K-30, Sucralose, Benzyl alcohol. 如申請專利範圍第6項之抗結核病藥物醫藥配方，其中該降低抗結核病藥物副作用之物質的最小有效劑量為：正二羥癒瘡酸(Nordihydroguaiaretic acid)(17毫克)、(-)表沒食子兒茶素-3-沒食子酸酯((-)-Epigallocetechin-3-gallate)(25毫克)、茵陳色原酮(Capillarisin)(17毫克)、山奈酚(Kaempferol)(16毫克)、根皮素(Phloretin)(15毫克)、橙皮素(Hesperetin)(33毫克)、6-薑辣醇(6-Gingerol)(16毫克)、沒食子酸(gallic acid)(9毫克)、異甘草素(Isoliquritigenin)(18毫克)、柚皮素(Narigenin)(9毫克)、二氫化槲皮素((+)-Taxifolin)(17毫克)、漢黃芩素(Wongonin)(16毫克)、原兒茶酸(Protocatechuic acid)(8毫克)、兒茶素((+)-Catechin)(16毫克)、β-奈黄酮(β-naphthoflavone)(15毫克)、恩貝素(Embelin)(16毫克)、反式肉桂酸(trans-Cinnamic acid)(8毫克)、表兒茶酚((-)-Epicatechin)(16毫克)、反式肉桂醛(Trans-Cinnamaldehyde)(7毫克)、大豆甘元(Daidzein)(14毫克)、異牡荊素(Isovitexin)(24毫克)、β-香葉烯(β-Myrcene)(8毫克)、檞皮素(Quercetin)(16毫克)、(+)-檸檬烯((+)-Limonene)(7毫克)、楊梅素(Myricetin)(17毫克)、檞皮(Quercitrin)(24毫克)、木犀草素-7-葡萄糖苷(Luteolin-7-Glucoside)(24毫克)、桑葉素(Morin)(16毫克)、新橙皮苷(Neohesperidin)(33毫克)、橙皮苷(Hesperidin)(33毫克)、(-)-表沒食子兒茶素((-)-Epigallocatechin)(17毫克)、木犀草素(Luteolin)(16毫克)、金絲桃苷(Hyperoside)(25毫克)、檉柳素(Tamarixetin)(17毫克)、黃芩素(Baicalein)(15毫克)、芸香素(Rutin)(33毫克)、黃芩(Baicalin)(24毫克)、芹菜素(Apigenin)(15毫克)、(+)-表兒茶素((+)-Epicatechin)(16毫克)、(-)表兒茶素-3-沒食子酸酯((-)-Epicatechin-3-gallate)(24毫克)、水飛薊賓(Silybin)(26毫克)、牡荊素(Vitexin)(24毫克)、金雀異黃酮(Genistein)(15毫克)、異鼠李素(Isorhamnetin)(14毫克)、香葉木素(Diosmin)(33毫克)、葛根素(Puerarin)(0.8毫克)、或傘形花內酯(Umbelliferone)(0.9毫克)、 高良薑素(Galangin)(0.5毫克)、非瑟酮(fisetin)(0.9毫克)、聚氧乙烯醚(Brij 58)(1.26克)、聚氧乙烯10硬酯基醚(Brij 76)(1.26克)、聚氧乙烯23月桂基醚(Brij 35)(18毫克)、聚山梨酯20(Tween 20)(1.26克)、聚山梨酯80(Tween 80)(1.26克)、聚山梨酯40(Tween 40)(1.26克)、聚乙二醇2000(PEG 2000)(1.26克)、聚乙二醇400(PEG 400)(1.26克)、泊洛沙姆188(Pluronic F68)(1.26克)、聚乙二醇4000(PEG 4000)(1.26克)、十四烷酸乙酯(Ethyl Myristate)(14毫克)、蓖麻油聚氧乙烯醚EL(Cremophor EL)(158毫克)、月桂硫酸納(Sodium Lauryl Sulfate)(14毫克)、微晶纖維素(Microcrystalline cellulose)(180毫克)、磷酸氫鈣二水物(Dicalcium phosphate dihydrate)(9毫克)、聚氧乙烯40氫化蓖麻油(Cremophor RH40)(1.26克)、交聯聚維酮(Crospovidone)(158毫克)、羧甲基澱粉鈉(Sodium starch glycolate)(158毫克)、丙烯酸樹脂S100(Eudragit S100)(158毫克)、交聯羧甲基纖維素鈉(Croscarmellose sodium)(158毫克)、薄荷腦(Menthol)(8毫克)、羥丙基纖維素(hydroxypropylcellulose)(158毫克)、預糊化澱粉(Pregelatinized starch)(158毫克)、甘露醇(Mannitol)(10毫克)、水和葡萄糖結合劑NF(Dextrates NF hydrated)(158毫克)、檸檬酸(Citric acid)(10毫克)、膠態二氧化矽(Aerosil 200)(158毫克)、聚乙二醇8000(PEG 8000)(1.26克)、山梨酸(Sorbic acid)(6毫克)、檸檬油(Lemon oil)(158毫克)、苯甲酸鈉(Sodium benzoate)(9毫克)、乙鮮舒泛鉀(Acesulfame K)(10毫克)、羥丙基甲基纖維素(Hydroxypropyl methylcellulose)(158毫克)、羥乙基甲基纖維素(Hydroxy ethyl methylcellulose)(158毫克)、糖精(Saccharin)(10毫克)、甲基纖維素(Methyl cellulose)(158毫克)、環己烷氨基磺酸鈉(Sodium cyclamate)(10毫克)、單水合乳糖(Lactose monohydrate)(18毫克)、麥芽糖糊精(Maltodextrin)(158毫克)、甘油山崳酸酯(Glyceryl behenate)(52毫克)、氧化鐵紅(Oxide red)(34毫克)、單硬脂酸甘油酯(Glycerin monostearate) (158毫克)、共聚維酮K28(Copovidone K28)(158毫克)、澱粉乙酸酯(Starch acetate)(158毫克)、硬脂酸鎂(Magnesium stearate)(29毫克)、聚維酮K-30(Povidone K-30)(6毫克)、三氯半乳蔗糖(Sucralose)(22毫克)、苯甲醇(Benzyl alcohol)(158毫克)、。 For example, the anti-tuberculosis drug and pharmaceutical formula of claim 6 of the patent scope, wherein the minimum effective dose of the substance for reducing the side effects of the anti-tuberculosis drug is: Nordic hydroguaiaretic acid (17 mg), (-) epiphytic Catechin-3-gallate ((-)-Epigallocetechin-3-gallate) (25 mg), Capillarisin (17 mg), Kaempferol (16 mg), Phloetin (15 mg), Hesperetin (33 mg), 6-Gingerol (16 mg), gallic acid (9 mg), Isoliquritigenin (18 mg), Naricin (9 mg), dihydroquercetin ((+)-Taxifolin) (17 mg), Wongonin (16 mg), Protocatechuic acid (8 mg), catechin ((+)-Catechin) (16 mg), β-naphthoflavone (15 mg), Embelin (16) Mg), trans-Cinnamic acid (8 mg), epicatechin ((-)-Epicatechin) (16 mg), trans-Cinnamaldehyde (7 mg), soy Daidzein (14 mg), different vitex (Isovitexin) (24 mg), β-Myrcene (8 mg), Quercetin (16 mg), (+)-limonene ((+)-Limonene) (7 mg) , Myricetin (17 mg), Quercitrin (24 mg), Luteolin-7-Glucoside (24 mg), Morin (16 mg) ), Neohesperidin (33 mg), Hesperidin (33 mg), (-)-epigal catechin ((-)-Epigallocatechin) (17 mg), hibiscus Luteolin (16 mg), Hyperposide (25 mg), Tamarixetin (17 mg), Baicalein (15 mg), Rutin (33 mg) , Baicalin (24 mg), Apigenin (15 mg), (+)-epicatechin ((+)-Epicatechin) (16 mg), (-) epicatechin-3- Gallic acid ester ((-)-Epicatechin-3-gallate) (24 mg), silybin (26 mg), vitexin (24 mg), genistein (Genistein) (15 mg), Isorhamnetin (14 mg), Diosmin (33 mg), Puerarin (0.8 mg), or Umbelliferone (Umbelliferone) (0.9 mg), Galangin (0.5 mg), fisetin (0.9 mg), polyoxyethylene ether (Brij 58) (1.26 g), polyoxyethylene 10 stearate (Brij 76) (1.26 g) ), polyoxyethylene 23 lauryl ether (Brij 35) (18 mg), polysorbate 20 (Tween 20) (1.26 g), polysorbate 80 (Tween 80) (1.26 g), polysorbate 40 (Tween) 40) (1.26 g), polyethylene glycol 2000 (PEG 2000) (1.26 g), polyethylene glycol 400 (PEG 400) (1.26 g), poloxamer 188 (Pluronic F68) (1.26 g), poly Ethylene glycol 4000 (PEG 4000) (1.26 g), Ethyl Myristate (14 mg), castor oil Ethyl ether EL (Cremophor EL) (158 mg), sodium lauryl sulfate (Sodium Lauryl) Sulfate) (14 mg), Microcrystalline cellulose (180 mg), Dicalcium phosphate dihydrate (9 mg), polyoxyethylene 40 hydrogenated castor oil (Cremophor RH40) (1.26 g) ), Crospovidone (158 mg), Sodium starch glycolate (158 mg), Acrylic resin S100 (Eudragit S100) (158 mg), croscarmellose sodium (Croscarmellose sodium) (158 mg) , Menthol (8 mg), hydroxypropylcellulose (158 mg), Pregelatinized starch (158 mg), Mannitol (10 mg), water and glucose Binding agent NF (Dextrates NF hydrated) (158 mg), Citric acid (10 mg), colloidal cerium oxide (Aerosil 200) (158 mg), polyethylene glycol 8000 (PEG 8000) (1.26 g) ), Sorbic acid (6 mg), Lemon oil (158 mg), Sodium benzoate (9 mg), Acesulfame K (10 mg), Hydroxyl Hydroxypropyl methylcellulose (158 mg), Hydroxyethyl methylcellulose (158 mg), Saccharin (10 mg), Methyl cellulose (Methyl cellulose) 158 mg), sodium cyclamate (10 mg), Lactose monohydrate (18 mg), maltodextrin (158 mg), glyceryl behenate (Glyceryl) Behenate) (52 mg), Oxide red (34 mg), Glycerin monostearate (158 mg), Copovidone K28 (158 mg), Starch acetate (158 mg), Magnesium stearate (29 mg), Povidone K-30 (Povidone K-30) (6 mg), sucralose (22 mg), Benzyl alcohol (158 mg). 如申請專利範圍第6項之抗結核病藥物醫藥配方，其中可加入一藥學上可接受之賦形劑。 For example, the anti-tuberculosis drug pharmaceutical formulation of claim 6 can be added with a pharmaceutically acceptable excipient. 如申請專利範圍第8項之抗結核病藥物醫藥配方，其中該賦形劑可為稀釋劑、填充劑、結合劑、崩解劑或潤滑劑。 An anti-tuberculosis drug pharmaceutical formulation according to claim 8 wherein the excipient can be a diluent, a filler, a binder, a disintegrant or a lubricant. 如申請專利範圍第6項之抗結核病藥物醫藥配方，其中該醫藥配方之劑型為口服錠劑、膠囊劑、散劑、溶液劑、懸浮劑、乳劑、芳香水劑、糖漿劑、醑劑、酏劑、酊劑、流浸膏劑、軟膏、乳霜劑、糊劑、注射劑或栓劑。 For example, the anti-tuberculosis drug pharmaceutical formulation of claim 6 of the patent scope, wherein the pharmaceutical formulation is an oral tablet, a capsule, a powder, a solution, a suspension, an emulsion, an aromatic agent, a syrup, an expectorant, an expectorant. , tinctures, fluid extracts, ointments, creams, pastes, injections or suppositories.