TW201016216A - New Isoniazid-containing compound with low side effects - Google Patents

Abstract

A new Isoniazid-containing compound with low side effects (2) includes a pharmaceutically effective amount of isoniazid (INH), and at least a pharmaceutically effective amount of cytochrom P450 2E1 (CYP2E1) inhibitor, in which the CYP 2E1 inhibitor is selected from a group consisting of: Trans-Cinnamaldehyde,Daidzein, Isovitexin, β -Myrcene, Quercetin, (+)-Limonene, Myricetin, Quercitrin, Luteolin-7-Glucoside, Morin, Neohesperidin, Hesperidin, (-)-Epigallocatechin, Luteolin, Hyperoside, Ethyl Myristate, Tamarixetin, Baicalein, Rutin, Baicalin, Apigenin, (+)-Epicatechin, (-)-Epicatechin-3-gallate, Silybin, Vitexin, Genistein, Isorhamnetin, Diosmin, Puerarin,and Umbelliferone, in order to reduce side effects, such as liver toxicity, caused by INH. Furthermore, the present invention also provides a novel cytochrom P450 2E1 inhibitor.

Description

201016216 • Nine, invention description: [Technical field to which the invention pertains] The present invention relates to a novel compound of low side effects containing isoniazine (INH) (2) 'especially refers to an isonianic acid amide ( INH) A new combination of isoniazid and guanamine containing a novel cytochrome p45〇2E1 (CYP2E1) inhibitor to reduce hepatotoxicity caused by isoniazid amide (INH).肇[First Technology] According to 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 also been in recent years. Keep climbing. More than 60 people per 100,000 people 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 every day in Taiwan from the lungs. Among the many patients receiving tuberculosis medication, the most common clinical side effects are hepatotoxicity and neurological diseases (such as auditory nerve and optic neuropathy), among which hepatotoxicity is the most common. In addition, Taiwan is also a prevalent area for hepatitis B and c, and hepatitis for tuberculosis. There are also 14 new tuberculosis patients with a number of 'false ^^ years. It is estimated that at least 3,000 patients with chronic liver disease need to receive anti-tuberculosis drugs, so the liver may occur in these patients. Toxicity is a iatrogenic disease that we can't. The first-line anti-tuberculosis drugs in the township, for example: different from the testisamine (isQniazid, commonly known as the enemy (4)), the pyrazmamide (the common round of the enemy) and the Lixin (the other) There are potential adverse reactions leading to liver preparation; among them, isoniazid is currently the most effective single anti-tuberculosis drug in 201016216, and it is also the most likely to cause hepatotoxicity in the patients; it is different in the late 6th century. Nicotinamide (i-) is a report of hepatotoxicity; it is different from the clinical symptoms of hepatotoxicity caused by toniazid (about OW%) '2, and in 1 2% of patients, Asymptomatic liver dysfunction can be observed 3, these liver function abnormalities usually occur within two months after taking the drug. - As shown in Figure 1, 'isoiazid' in the liver mainly through nitrogen-acetamidine With the help of N-acety丨transferase (NAT), it is acetylated, and the intermediate product, acetylisoniazid, is rapidly hydrolyzed to acetyihydrazine; The hydrazine can be further brewed via N-acetyltransferase Non-toxic diacetylhydrazine, or emulsified into hepatotoxic molecules via cytochrome P45 〇2E1 (CYP 450 2E1), including aCetyldiaZene, acetylonium Ion), acety!radicai, ketene, etc. In addition, in the presence of aerobic and NADPH, acetylated hydrazine is reacted by cytochrome P45〇2E1 to form free radicals and is Oxidative pressure, leading to cell death; in addition, is〇niazid can also be directly hydrolyzed to toxic hydrazine via 醯fe hydrolysis S# (amidase) or by the above-mentioned acetamidine Amine (hydretylhydrazine) is hydrolyzed to a toxic hydrazine by ajnidase. Recently, studies have shown that 'biamine (rather than isoniaceine guanamine or acetylated hydrazine) is in rabbits and rats. The most likely cause of hepatotoxicity (INH_induced hepatotoxicity) caused by anthocyanin in the body'' The investigator believes that the severity of hepatotoxicity caused by the detection of guanamine is positively correlated with the concentration of hydrazine in the gold paste; 1999 The report of Sarich6 et al. Bis-/Miitrophenyl phosphate (ΊΒΝΡΡ, is an inhibitor of indoleamine hydrolase) can prevent the different 201016216 * in the detection of guanamine caused by liver poisoning _ injury #, its protective mechanism should be through the weaving The amine produces a hydrazine. Cytochrome P450 2E! (CYP2E1) will continue to behave in the liver and is responsible for the metabolic bioreactivity of many heterologous hepatotonics, four gasifications, and acetamin〇phen 7'8; however, CYP2E1 The role played by hepatotoxicity caused by the detection of guanamine is not clear. It is different from the testin itself as an inducer of CYP2E1. 9 Some studies suggest that CYP2E1 in the liver and hepatotoxicity caused by silkworm (4) The heart of the machine. . In vitro test, disulfide #dis (DSF) and its metabolite diethyldithiocarbamidine (diethyidi-amate) were identified as selective inhibitors of mouse and human liver microsomes cYp2Ei (selective mechanism_based (d) 丨 (4) „·13, Β _13 et al ^ show that the mice take a single-oral dose of disulfide remaining _), resulting in immunoreactive liver capacity (immunoreactive hepatic content) and CYP2E1 catalytic activity decreased rapidly and completely.

Sodhi丨4 et al. reported in the year-old report that oxidative stress is one of the factors that cause hepatotoxicity caused by S! amine and rifamycin in young rats. There are many studies to find appropriate biomarkers to assess the rate of oxidative damage in the body. Biomarkers that may be applicable at present can be divided into three categories, which are markers for oxidative damage to lipids, proteins, and nucleic acids; 8_Iso-prostaglandin F2a (8-is〇-prostaglandin F2a, 8_iso_PGF2a) is a product of free radical-induced lipid peroxidation of arachidonic acid, which is chemically stable, and the content of 8_is〇pGF2a can be used as Judging the activity of lipid peroxidation, the lipid peroxidation may be related to the production of free radicals, oxidative d_ge and antioxidant deficiency in the body. There are many methods available. Measurement of 8 is〇pGF2a containing 201016216, including enzyme immunoassay 17, radioimmunoassay 18, gas-chromatography mass spectrometry 19 and liquid chromatography mass spectrometer (liquid chromatography mass spectrometry) M, etc. In addition, 8-iso-PGF2a and its metabolite 2,3-dinor-8 in human urine The -iso-PGF2a content can be prepared by C18 solid phase extraction (SPE) and then analyzed by liquid chromatography tandem mass spectrometry (Lc/ms/MS). Invasive and non-invasive methods are used to test rat liver function to monitor the development of liver damage and to screen for liver disease. The most commonly used method involves measuring aspartate aminotransferase in serum. , AST) 'Alanine aminotransferase (ALT) and aikaline phosphatase values, as well as measuring hepatocyte products such as bilirubin, albumin, and utilization The prothrombin time is measured to detect coagUiati〇n fact〇rs, etc. The liver and function quantitative tests are based on the concentration of the serum in the serum that is almost exclusively metabolized by the liver. The removal is based on the hepatic arterial blood flow of the hepatic portal and the effect of hepatocytes on these receptors. The blood flow of the liver is related to the quality of the liver. In contrast, the clearance of the receptor is determined by the ability of the liver to metabolize. twenty three. Galactose An auditory that has two extraction rates (extracti〇n rati〇) and is metabolized in the liver. In the liver, galactose is differentially stereoisomerized by galact〇kinase. Reaction (epimerization), which is converted to Glucose-1-phosphate. The action of galactose kinase is the rate-determining step of the galactose metabolic pathway in hepatocytes (rate-limiting galactose The high extraction rate makes the hepatic blood flow in 201016216 and the galactose metabolism side of the liver Wei become the most important way of detecting function. At present, there is no fine sputum to estimate the residual liver function age (eidual liver fu (10) ion), measurement - The metabolic ability of the exact compound (eg, galactose) can be used to determine the rate of the liver towel-metabolic side, and it is also possible to obtain the representative quality of residual liver function M,25. The galactose eliminatiQn eapaeity (GEC) has been used as a quantitative test for human liver function for 26 years. However, the galactose clearance test requires multiple blood samples to establish a standard curve. Its heterogeneity, so many studies use Galactose Single P〇int (GSP) to assess human liver function; the inventors tested chronic hepatitis, cirrhosis and liver cancer patients by galactose single-point method, The results show that the galactose single-point method can accurately detect these liver diseases. 27 The galactose single-point method has been successfully applied to test the residual liver of patients with liver diseases such as promazine and cef0perazone. Function 28·30. In addition, the galactose single-point method has become one of the recommended methods for testing liver function in the guidelines published by the US Food and Drug Administration (FDA). It can be seen that the above-mentioned conventional anti-tuberculosis drugs are different from the test. There are still many shortcomings in the amine (isoniazid), which is not a good designer, but needs to be improved. Therefore, the inventor of this case applied for a new compound of Isoniazid (INH) with low side effects in the patent No. 200831087 of China, which was published on January 16, 2007, which revealed a low side effect. A new compound of isoniazid (INH) comprising a pharmaceutically effective amount of isoniazid (INH) in combination with a pharmaceutically effective amount of disulfiram (DFF), or recombination A pharmaceutically effective amount of bis-p-nitrophenyl phosphate (BNPP); in addition to the cited case also provides a low side effect of isonicotinic acid amide (INH) Xinfu 201016216 prescription including a pharmaceutically effective The amount of isonicotinicinamide (INH) is combined with a pharmaceutically effective amount of a cytochrome P450 2E1 (CYP2E1) inhibitor to reduce side effects such as hepatotoxicity caused by isoniazidamine (INH). However, in this case, only the cDNA synthesis of microsomal cytochrome p45〇 2E1 for screening for possible cytochrome P450 2E1 inhibitors was less able to screen for inhibitors that inhibit cytochrome P450 2E1 activity in humans. Therefore, the inventors of the present invention prepared a cytochrome P45 2E1 inhibitor by preparing microsomes rich in cytochrome P450 2E1 in human liver to screen for effective inhibition of cytochrome p45〇2E1 in human liver. Active Day Suppressor. In addition, the inventors of the present invention also found novel cytochrome P450 2E1 inhibitors from traditional Chinese medicines. Therefore, the present invention provides a low-side side effect of Isoniazid (INH). The new compound (2) is known to be a healthy and beneficial compound for the world to provide a natural and novel compound. In view of the above-mentioned shortcomings of side effects such as hepatotoxicity caused by testisamine (isoni^id), the inventors of the present invention have improved and innovated after the research and development of the anti-tuberculosis drug, and after years of painstaking research, finally succeeded in research and development. It is a low-side side effect compound (2). It is an object of the present invention to provide a new compound (I) which is different from the base amide (Is〇niazid). 'The inhibitor of cytochrome p450 2E1 (CYP2E1) is used in combination with an inhibitor of cytochrome p450 2E1 (CYP2E1) to reduce side effects such as hepatotoxicity caused by isoniazidamine (INH). The second object of the present invention is Providing a novel cytochrome P450 2E1 (CYP2E1) inhibitor containing isoniazid amide (Is〇niazkJ, a low side effect compound (2)' which also provides a cytochrome P450 2E1 (CYP2E1) inhibitor (The side caused by sputum riding is toxic and other side effects. In order to achieve the above object of the invention, it is different from the test amine (Is〇niazid, the low side effect of the new complex 201016216 ' square (7), the present invention first with isoniazid amide (INH) Induction of rat (4) hepatotoxicity as a pattern To study the cytochrome P450 2E1 (CYP2E1) inhibitor double dredging (DSF), and the indoleamine hydrolase (amidase) inhibitor nitrophenol phosphate diester (BNpp) on the liver induced by isonicotinicin in rats The effects of toxicity; in addition to the use of general hepatotoxicity markers, galactose single point method (Gsp) and galactose clearance (GEC) for quantitative measurement of residual liver function in rats, the inventors of the present invention use improved liquid layer The tandem mass spectrometer (LC/MS/MS) was used to measure the concentration of S-iso-PGFh in rat plasma to further judge whether "~-yang sputum is related to the toxicity of liver sputum induced by INH in rats. Further screening of possible cytochrome inhibitors (CYP2E1) inhibitors with human liver microsomes. A new compound (2) with low side effects containing the indoleamine inH) which achieves the above object, includes one A pharmaceutically effective amount of isoniazidamine (INH) in combination with at least one pharmaceutically effective amount of a cytochrome P450 2E1 (CYP2E1) inhibitor, wherein the cytochrome P450 2E1 (CYP2E1) inhibitor is selected from Group consisting of the following compounds: trans-cinnamaldehyde (T rans-Cinnamaldehyde), Daidzein, Isovitexm, p_Myrcene, Quercetin, (+)-limonene ((+)-Limonene), myricetin (Myricetin), Quercitrin, Luteolin-'Glucoside, Morin, Neohesperidin, Hesperidin, (- )-Epigallocatechin), Luteolin, Hyperoside, Ethyl Myristate, Tamarixetin, Baicalein, Rutin , Baicalin, Apigenin, (+)-Epicatechin, (_)_Epicatechin-3-gallate, Silybin, Vitexin 201016216 (Vitexin) Genistein (Genistein) , isorhamnetin, dioxin (Puerarin), or Umbrate lactone (Umbemfer〇ne). The low-field effect of this month is different from the new compound of is〇niazid (inh), and can also be added - pharmaceutically acceptable only to paste the compound, the system can be a lion agent, a filler , binders, disintegrants, lubricants, etc. [Embodiment] The present invention is to be construed as a exemplification of the present invention, and is not to be construed as limiting the scope of the present invention. Example 1 Isoniaciline guanamine (INH) combined with animal test using CYP2E1 inhibitor disulfiram (DSF>a/ or nitrophenol phosphate diester (BNPP) I. Materials and methods 1. Test materials All organic solvents All HPLC grades, purchased from Tedia Ltd. (Fairfieid, 〇H,

USA) 'INH, BNPP, DSF and corn oil were purchased from sigma chemical company (St. Louis, MO USA) ' 8-is〇-PGF2a and radiation calibration 8_iso_PGF2a_d4 was obtained from (A) Facial Chemical Company (Ann Arbor, MI, USA), a 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. Male SD (Sprague-Dawley) rats weighing 320-350 g were purchased from the National Laboratory Animal Center (Taiwan). The animal experiments were carried out in accordance with the guidelines of the National Animal Research Institute. All rats were placed in 12 201016216 Under the air/humidity adjustment environment, the light and the darkness were each 12 hours, and the supply of water and feed was not limited. The weight of the rats was continuously monitored during the test. All the rats were dosed with 50 mg/kg body weight of pentobarbital. Sodium pentobarbital was intraperitally anesthetized (intraperit〇neally anesthetized), and the polyethylene catheter was placed in the right internal jugular vein (intemal detonated vene) of the rat to add a galactose 'catheter to cut into the puncture (cut_d〇 wntechniqUe) was inserted, and the end of the catheter was placed under the skin of the posterior cervical incision of the rat. After the surgery was completed, the rats were fasted for one night (about 16 hours) during the recovery, but the water was supplied as usual. I 3. Test treatment The test animals were randomly divided into 5 groups, each group consisting of 3 treatments. The first treatment was injection of 25 mg/kg BNPP or BNPP (vehicle, VEm 'ready saline), and BNPP was dissolved in heating. Saline solution (0.9% NaCl) to 60 ° C, after cooling, intraperitoneal injection into the body in a volume of 1 ml / kg; the second treatment is to inject l〇〇mg / kg DSF or 'DSF base Agent (VEH2, corn oil), DSF is dissolved in corn oil and injected intraperitoneally into the body in a volume of 1 ml/kg; the third treatment is the injection of 150 mg/kg INH or INH. (VEH3, ready-to-feed saline), INH is dissolved in barium (0.9°/. NaCl) and injected intraperitoneally into the body in a volume of 1 ml/kg; the first group (BNPP or VEH1) is the first Three groups (INH or VEH3) were treated 30 minutes earlier and the second group (DSF or VEH2) was treated 15 minutes earlier than the third group (INH or VEH3). The above five groups of experiments included: (1) Control group (normal control group, NC, n=l2): 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 once daily with INH, VEH1 and VEH2 13 201016216 • (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; the galactose single-point method was tested 16 hours after the 21st day of treatment. 4. Blood sample After the treatment, the rats were sacrificed by ether anesthesia. The gold solution was taken from the rat aorta and placed in a test tube containing EDTA. The blood piasma was i3 and 〇〇〇g was 4. The crucible was centrifuged for 15 minutes, and the separated crucible was dispensed into a small tube (Eppendorf tube) and placed at -80. (3 in storage. 5. Biochemical analysis · Hepatocyte injury to measure plasma aspartate transaminase (AST) and alanine transaminase spring (ALT) activity for quantification, AS1 ALT activity is liver Commonly used indicators of toxicity

The Synchron LXi 725 system was measured (Beckman Instruments, USA). 6. Light microscopy and electron microscopy Rats were sacrificed for histological analysis immediately after sacrifice; liver samples were fixed with phosphate-buffered formalin in 1% linonic acid buffer, then dehydrated and embedded in paraffin (paraffin) The sliced samples were sliced with 5 μπι thickness and stained with hematium (XyKn) and eosin (e〇sin), and subjected to a liver glycan staining test (Peri〇dic acid Schiff stain, pAS), followed by staining with an optical microscope 14 201016216 • Histological observation; in addition, liver sections were washed with caC〇 dylate buffer (0.1 M pH 7.4) and fixed with 20% aqueous osmium tetroxide for 1 hour. Alcohol was continuously dehydrated and embedded in Spurr resin (Spurrresin), and ultra-thin sections were cut with a stone knife. Double staining with uranylacetate and leadcitrate, and transmission electron microscopy (Transmission) Electron Microscope, Hitachi 600, Hitachi Co., Japan). Extraction and measurement φ All PGFk isomers are 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, taking 5 5 ml of plasma to glass In the tube, i〇ng internal standard (8-iso-PGF2a-d4) was added, and the mixed plasma was subjected to C18 solid phase extraction cartridge (JT Baker, MA, USA). Purification, the sample flow washing solution was evaporated and evaporated in a random atmosphere, and then dissolved in 50 μl of acetonitrile: water (15:85 v/v) solution and shaken for 30 seconds. The extract after 10 μl of the solution was injected into the LC. /MS/MS system for analysis. 8. Liquid Chromatography Tandem Mass Spectrometer (LC/MS/MS) The HPLC system consists of two Shimadzu LC-10OvvP pumps (Shimadzu LC-10ADvP pumps) and one Shimadzu system control. 1 Shimadzu autosampler (Shimadzu Scientific sampler, Japan), HPLC separation with C18 column (particle size 5·μηι, inner diameter 50 X 2_lmm), and containing 2 mM ammonium acetate and B Gradient flow wash of acetonitrile (ACN) (t = 〇min, 15% ACN; t = 6 min, 70% ACN; t = 7 min, 90% ACN; t = 8 min, %% ACN; t = 8.5 min, 15% ACN) flow wash, LC/MS/MS flow rate is maintained at 2〇ρμ1/πήη, the entire HPLC time is 13 5 minutes; 15 201016216 • The HPLC system and a three-layer quadrupole mass spectrometer (triple Stage quadrupole mass spectrometer, API3000, Applied Biosystem, Foster City, CA, USA) interfaced with a TurboIonSpray ionization source and negative electrospray as ionization method; Mass spectrometer by diffusion of 200 ng/ml 8-iso-PG The F2a or 8-iso-PGF2a-d4 standard was optimized in a multiple reaction monitoring (MRM) mode, and m/z 353/193 and m/z 357/197 ion couples (ionpair) were used individually. Monitoring 8-iso-PGF2α and 8-iso_PGF2α-d4;

Calculate the correlation coefficient (r, correlation coefficient) of the region of the 8-iso-PGF2a concentration (C) by the linear calibration curve for the region of the 8-iso-PGF2a-d4 ratio (7)'. The value is 0.999; the linear range of 8-iso-PGF2a in plasma is between 〇.l-2.5 ng/ml, and the regression equation is Y=-0.0517C +0_823 ng/ml; the measured result All were calculated according to the standard of re-hydrogenated 8-iso-PGF2〇t (deuterated 8-iso-PGF2a). The inter-assay precision and accuracy of the standard curve were tested 6 times after the standard concentration samples, respectively. Back-Calculation was used to evaluate that the relative errors ranged from -5.06% to ❷3.13%. * 9. Quantitative testing of liver function All rats were tested for galactose single point (GSP) and galactose clearance (GEC). Rats received rapid intravenous injection within 30 seconds, injection of 0 4g/ml bW Galactose solution 〇·5 g/kg; blood was collected once every 5, 10, 15, 30, 45, and 60 minutes after the main shot, and the blood sample was taken from the tail vein's colorimetric galactose colorimetric method (colorimetric galactose) Dehydrogenase) The galactose content was measured at concentrations ranging from 5 〇 to i, 〇〇〇pg/mi, and intraday differences in each concentration (within_day 16 201016216 ^ .variation) were determined by standard deviation (standard deviati〇n) and variation The coefficient (c〇efficient 〇f variation, CV) is calculated as the percentage of the maximum allowable coefficient of variation is 1〇y❶cv; the day-to-day variation is calculated by comparing the slope and intercept of the caibration curves. Test; galactose clearance capacity (GEC) is calculated by the following formula, which is modified by Tygstmp, s equation 32: ratio L = '~ - (mg / kg · min) A c=o + 7 ❹ where D Is the amount of galactose injected; tc=<) is the concentration of galactose reaching 〇 The time required is derived from the linear regression of the blood concentration-time curve from 20 to 60 minutes after injection (usually 2.22 mmol/1), and 7 is the correction value for correcting the uneven distribution in the body according to the rule of thumb; galactose The point method (Gsp) is the concentration of galactose in the blood at 60 minutes after the 30 second injection is stopped. 10. Statistical analysis All data are expressed in terms of mean soil standard deviation (SD). The test results are calculated by the single factor analysis of variance (ANOVA) test method to determine whether there is a statistically significant difference, using s as in (5) Guangdong Package of the The Social Science program (Version π, SPSS Inc) sets the software to calculate; then uses the post hoc test to make multiple comparisons to confirm the significant differences between the groups; The significant difference was the average of PO.05 〇 2. Results 1 · Biochemical analysis results At the end of the experiment, the weight of the sloping secrets and (4) the liver weight, the squamous animals were not significantly different from the 17 201016216; As shown in the second, only the aspartate aminotransferase (AST) and alanine transaminase (ALT) activities in the plasma of the INH group were significantly higher than those of the control group (the AST activity in the plasma of the control group was U6 ± ll IU / The AST activity in the plasma of the INH group was 129±1〇IU/L, p <〇.〇5; the ALT activity in the plasma of the control group was 44 ± 6 IU / L; the ALT activity in the plasma of the INH group was 52 ± 3 IU/L, p < 〇·〇5) 'Show INH group Students on biochemical liver injury; control group, BNPP-INH, DSF-INH and BNPP-DSF-INH group transfer of serum aminotransferase concentration was normal. 2. Histopathology φ Rats were intraperitoneally injected with 150 mg/kg/day INH for three weeks, and the hepatotoxicity was successfully produced in vivo. In contrast, the liver structure in the control group was normal. The liver cell line in the liver parenchyma of the control group indicated by the third A is arranged in the reticular plate arranged from the central vein of the hepatic lobules. The hepatic sinusoids are in the two liver plates (anastomosing). The plates were found between the plates; the tissue sections of the rats in the INH group are shown in Figure 3b.

The hepatocytes around the central vein of the INH group showed fragmentation and vacuolization. However, no necrosis was observed. The results of electron microscopy showed that compared with the control group (such as Guangdong). Figure 3C shows that the crude endoplasmic reticulum (rER) in the liver cells of the INH group is significantly increased (as shown in Figure 3d). According to the literature, INH is a potent inducer of cytochrome P450 2E1 (CYP2E1) 33 ' and CYP2E1 leads to the production of superoxide and hydr〇Xyi radicals 34 and can cause The endoplasmic reticulum increased by 35, so the results of this trial are consistent with previous studies. In other test groups, the degree of liver damage in the BNPP-INH group, the DSF-INH group, and the BNPP-DSF-INH group was not significantly different from that of the control group (no results were shown). 3. Measurement of blood sample center 18 201016216 In the negative electric power Confucian mode, (4) + the team's maximum amount of molecules is rarely corrected. (4) Yang ion 8 iso-PGFh-d4 The maximum amount of molecular ions is mass-to-charge ratio (m /z) 357 ions, these negative 4 knives ion system (four) induced by a large number of collisions to produce free, the molecules of the two target compounds, '. The structure and the generated ion spectrum are shown in Fig. 4; except for 8 isG_pGF2a_(10) daughter ions (daughter urns) are always four units higher than S-iso-PGF^, and 8_is〇pGF2a and 8 iso PGFh-d4 The fragmentation mode ((4) is similar to that of the grief gamma (4), which shows that most stable daughter ions are produced by the A bond, which is labeled with 4 atmosphere atoms (demerium ❹atoms); S-iso-PGFh The most dense daughter ion is the mass-to-charge ratio (m/z) 193 ion, and the last ion of the 8_is〇pGF2a mountain is the ion of the mass charge tb(m/z)197. Figure 5 shows the multi-reaction monitoring mode (M A typical LC/MS/MS chromatogram containing a standard solution of l〇〇pg8_is〇_pGF2a and 25〇pg/ml8 is〇pGF2a_d4 and a blood sample was injected into ing8-iso-PGF2a-d4 as measured by abdominal hernia. After the internal standard, the standard solution and the blood sample were purified by the same solid phase extraction (spE) and analyzed by the aforementioned LC/MS/MS protocol. 4. The concentration of 8-iso-PGF2a in plasma ❹ in plasma 8-iso~PGF2〇^ — an indicator of oxidative stress, as shown in Figure 6.

Compared with the control group, the concentration of 8-iso-PGF2W in the plasma of INH group was significantly increased (the concentration of 8-iso-PGF2 in the plasma of INH group was 151±26pg/ml; The concentration of -iso-PGF2a was 11〇±15 pg/ml,;? <0.001); compared with the INH group, the bnPP-INH group, the DSF-INH group, and the BNPP-DSF-INH group were significantly reduced. INH caused 8-iso-PGF2a production in the liver (the concentration of S-iso-PGFzW in the plasma of rats in the BNPP-INH group was 128±29 pg/ml; the concentration of 8-iso-PGF2〇 in the plasma of the DSF-INH group was 126). ±20 pg/ml ; BNPP-DSF-INH group blood group 19 201016216 • 8-iso-PGF2A concentration was 123 soil 17 pg/ml; INH group rats plasma 8-iso-PGF2〇^ concentration was 151 ±26 pg/ml,;? <0.005); notable is the control group, BNPP-INH group, DSF-INH group 'BNPP-DSF-INH group between the four groups, rat blood 'slurry in the 8- The iso-PGF2 (no significant difference in concentration) was compared with the BNPP-INH group and DSF-INH, and the combination of BNPP and DSF in INH did not further reduce the concentration of 8-iso-PGF2a in plasma. The measurement of function is shown in Figure 7. 'The galactose single point method (GSP) value of the control group and the INH group has a high value. Significant difference in degree (GSP value of 384±69 pg/ml in control rats; GSP value in 565±87 pg/ml in INH group; ≥ 0.001), in addition to 'BNPP-INH group, The GSP values of the DSF-INH group and the BNPP-DSF-INH group were 401±70 pg/m, 449±45 pg/m, 388±53 pg/m, compared with the INH group, and the BNPP-INH group. The GSP values of the rats in the DSF-INH group and the BNPP-DSF-INH group were significantly different from those in the INH group (the corpse values were p < 〇.〇〇i, p < 0.005, and_p <0.001); GSP values were significantly increased in rats administered INH alone; however, rats that received BNPP or DSF or BNPP and DSF in INH were resistant to this change; on the other hand, to _ DSF-INH group In contrast, INH combined with BNPP and DSF showed a reduction in hepatotoxicity induced by INH, although the difference between the two did not reach a statistical difference (p = 〇.l), while the control group, BNPP-INH group, DSF-INH There was no significant difference in GSP values between the four groups in the BNPP-DSF-INH group. Similar results were also observed using the galactose clearance (G'EC) method. As shown in Figure 8, the GEC values of the INH group were significantly reduced compared to the control group (GEC of the INH group). The value was 3·4±0·6 mg/min.kg; the GEC value of the control rats was 4.9 ± 0.8 mg/min.kg, p < 20 201016216 -0.001), in addition, the BNPP-INH group, DSF- The GEC values of the rats in the ΙΝΗ group and the BNPP-DSF-INH group were 4.5 soils, 0.6], 11%, and 111111.] <^, 4.3±0.411^/11 enterprises 1.1^, 4.7±0.5 11^/111^1.1^ Compared with the 1>^[ group, the GEC 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 < 0.005, p < 0.05, and jp <0.005); the GEC value of rats administered INH alone was significantly reduced; however, rats in the combination of INH with BNPP or DSF or BNPP and DSF recovered this change; Compared with the DSF-INH group, the combination of INH and BNPP舆DSF had a tendency to increase the GEC value (the GEC values of the DSF-INH group and the ❹BNPP-DSF-INH group were 4.3±0.4 mg/min.kg, 4.7, respectively. ±0.5 mg/min.kg, = 0.29); in addition, the control group, BNPP-INH group, DSF-INH group, BN PP-DSF-INH group There was no significant difference in GEC values between the four groups. To determine whether AST, ALT, plasma 8-iso-PGF2a concentrations, and quantitative liver function tests (eg, GSP and GEC) were correlated, GSP values were found in 8-iso-PGF2a in plasma after several correlation analyses. The concentration is highly correlated (as shown in Figure 9), the correlation coefficient is 0.836; the GSP value is highly correlated with the GEC value (as shown in Figure 10)? < 0.001), the correlation coefficient is -❹0.822; GEC value It is also highly correlated with the concentration of 8-iso-PGF2a in plasma (correlation coefficient _ 〇.743, ρ < 〇 · 〇〇ι, as shown in Table 1); and GSP value, GEC value and plasma 8-iso The concentration of -PGF2a was not significantly correlated with A ST and ALT (as shown in Table 1). Table 1 Correlation between GSP, GEC and 8-iso-PGF2a and biochemical tests ___GSP . GEC 8-iso-PGF2a AST r = 0.114 r = -0.111 r = 0.217 ALT r = 0.016 r = 0.039 r = 0.035 21 201016216 8 -iso-PGF2a_r = 0.836* r =-0.743* r=i* Calculated by Pearson's correlation coefficient, * household <〇·〇〇ι Example 2 Cytochrome Ρ45〇2Ε1 (CYP2E1 Screening of inhibitors _ cDNA synthesis microsomal cytochrome P450 2E1 I. Materials and methods * 1. Test materials This example uses a cytochrome P450 2E1 (CYP2E1) inhibitor screening kit (CYP2E1 High Throughput Inhibitor Screening) Kit, BD Bioscience, USA) Screening of cytochrome p45〇2E1 (CYP2E1) inhibitors against 22 Chinese herbal medicines and l-type excipients; the principle of the CYP2E1 inhibitor screening kit is: CYP2E1 metabolite standard HFC (7-Hydroxy-4-) was added to the test sample after the pigment p45〇2m (CYP2E1) and its fluorescent substance MFC (7-Methoxy-4-trifluoromethyl coumarin) were added to the test sample. The amount of trifluoromethyl coumarin), And in the control group ((7) the amount of HFC produced by blood, the CYP2E1 inhibition rate of the test sample was calculated. Each test sample was dissolved in acetonitrile (acentoitrile) 'tested with different concentrations of Chinese medicine (66 μΜ, 33 μΜ, 16.5 μΜ) and The inhibition rate of CYP2E1 by excipients (〇·167%, 0·08°/., 0.042%, w/v), the results of the tested drugs and the results listed in Table 3, the tested excipients and The results are shown in Table 4. In addition, the reagents required for the CYP2E1 High Throughput Inhibitor Screening Kit (BD Bioscience, USA) used in this example are as follows: - (1) CYP2E1 + P450 Reductase + Cytochrome b5 : 100 mM potassium 22 201016216 phosphate (pH 7_4) contains i_3 nmol P450 and p-Nitrophenol hydrolase. (2) Control Protein: 15 mg/ujL Control Protein is dissolved in 100 mM Potassium Phosphate (pH 7.4). (3) Buffer Solution : 0.5 M Potassium Phosphate (pH 7.4). (4) Stop Solution : 0.5 M Tris Base. (5) Cofactors: contains U mM NADP+ ' 66 mM MgCl2 and 66 mM Glucose 6-Phosphate ° ❻ (6) Glucose 6-Phosphate Dehydrogenase : 40 units/ml Dissolved in 5 mM Sodium

Citrate Buffer (pH 7.5) 〇 (7) MFC (7-Methoxy-4-trifluoromethyl coumarin): Fluorescent substrate, 50 mM MFC dissolved in acetonitrile. (8) DDTC (Diethyldithiocarbamic acid): CYP2E1 selective inhibitor (positive control group), 20 mM DDTC dissolved in acetonitrile (acentoitrile). (9) HFC (7-Hydroxy-4-trifluoromethyl coumarin): CYP2E1 metabolite standard, 0.25 mM HFC dissolved in 0.1 M Tris (pH 9.0) 〇 (10) NADPH-Cofactor Mix: at 14.56 ml 187.5 μΐ Cofactors ' 150 μΐ G6PDH (Glucose 6-Phosphate Dehydrogenase Solution) and 100 μΐ Control Protein were added to the sterile water. (11) Cofactor/ acetonitrile mix: 66 μΐ in a 9_93 ml NADPH-Cofactor Mix Acetonitrile 0 (12) Enzyme/Substrate Mix: Add 5.94 ml of sterile 23 201016216 water, 50μ1 HTS-706 (CYP2E1) to 4ml Buffer Soultion , 2 μΜ P450 content) and 28 μΐ 50 mM MFC (7-Methoxy-4-trifluoromethyl coumarin, fluorescent substrate). 2. Screening of cytochrome P45〇2El (CYP: 2E1) inhibitors using the cytochrome P450 2E1 (CYP2E1) inhibitor screening kit (CYP2E1 High Throughput Inhibitor Screening Kit, BD Bi〇Science, USA) And the screening of the excipients, the experimental steps are as follows: (1) Preparation of the control group: ❹ a• Add 149pl NADPH-CofactorMix and lpl20mMDDTC in the first well on the 96-well plate and mix well; b. In the wells of the 2nd to 12th wells of the 96-well plate, 1〇〇μ1 c〇fact〇r/Cai (10) she mixed, the wells 1 to 8 were positive control group (p〇sitive c〇mr〇1); 9 and the first 〇 well are the control group (co coffee 1); the U and 12 wells are the blank control group (blartk); c. in the first to eighth wells to do the continuous dilution action: from the i The % ^ liquid in the well is added to the well of the second well to be mixed, and then the liquid is added from the well of the second well. (4) The liquid is added to the well of the third well, and the excess 5 〇 μ1 liquid is removed from the well of the eighth well. Continuous dilution concentrations of 66.6, 22.2, 7, 4, 2.47, 〇·82, 〇·27, 〇〇91, 〇〇3 _ were obtained. (2) Preparation test group: 149 149 μl NADPH Yang coffee Mix' and 1μ12〇_ Chinese medicine drug test sample or (w/v) shape were added to the first and second wells in the first row of the 96-well plate. Test the sample and mix it evenly; b. Then take 5〇μ1 liquid from the third and second wells of the i-th row and add it to the third well. 24 201016216 Evenly (ie, each test sample is three replicates) (3) Reaction initiation and termination: a· The above control group and the test group were placed in 3r>c for 丨〇 minutes; b. In addition to the blank control group, 1 giEnzyme/ was added to other wells. Substrate Mix; c, place all control and test groups at 37. (: Allow to stand for 40 minutes; d. Add 75 μl St〇ps〇luti〇n to all wells; e_ immediately add 1 〇〇μΐ Enzyme/Substrate Mix to the blank control group; f. All the control and test groups were read with a luminescence spectrometer (Fluoroskan Ascent FL, Thermo Electron Corporation, Finland) using an excitation light (excitati〇n) wavelength of 405 nm 'emission wavelength' 538 nm. (4) Analysis of results: The measured value of the fluorescent signal was converted to the amount of HFC produced by the CYP2E1 metabolite standard (pmol), which was based on the control group (contr〇i), ie, the cyp2e1 inhibition rate of the control group. 〇°/., the CYp2E1 inhibition rate of each positive control group and the test group was calculated by the following formula: CYP2m inhibition rate (%) = HFC production* of the control group - HFC production amount of the control group 2, result 1. Positive The inhibition rate of CYP2E1 measured in the control group (DDTC) is shown in Table 2. It can be seen from Table 2 that when the concentration of DDTC is 66.6 μΜ (ie, 〇167%, w/v), the inhibition rate of cyp 2ei can be Up to 97.55%, with 66.6 μΜ as the highest test concentration of traditional Chinese medicine, to 〇167 coffee v) 25201016216 to 'the highest concentration tested excipients. Table, positive control group cΥΡ 2Ε1 inhibition rate

The amount of production (pm〇n 222.00 gYP2El inhibition rate is 0 DDTC concentration (μΜ) 0 (control group) 0.03 0.091 0.27 0.82 2.47 7.4 22.2 66.6 256.00 202.00 151.71 126.14 55.18 21.08 15.10 5.42 8.71 31.52 43.06 75.09 90.49 93.19 97.55

2. Test group CYP2E1 inhibition rate The CYP2E1 inhibition rate measured by Chinese medicine drug cited is shown in Table 3. From the results, it can be seen that each Chinese medicine drug is condensed to different concentrations (66μΜ, 33μΜ, 16'5μΜ) under conditions of cytochrome p45 〇2m has a different degree of inhibitory effect, of which 66-positive acne acid ____ _ inhibits the best results (97.99zh0.66 %). Table 3: CYP2E1 inhibition rate of traditional Chinese medicine cited drug--------- CYP2E1 inhibition rate (know---' test tangtang_66 μΜ 33 μΜ 16.5 μΜ - control group 0 0 0 ^ sex control group (DDTC) 97.55+1.862 ------ 26 201016216 CYP2E1 inhibition rate of Chinese medicine (%) Test concentration 66 μΜ 33 μΜ 16.5 μΜ Nordic hydroguaiaretic acid 97.99±0.66 92.36±2.20 76.52±3.86 ( -)-Epigallocetechin-3-gallate 97.56±0.18 96.47±0.64 92.56+0.46 Capillarisin 76.12+1.89 60.54±5.91 49.05±5.18 Kaempferol 70.6312.53 70.04±3.75 71.87±1.14 Phloetin 66.84±4.79 54.69±2.84 42.0413.63 disulfiram 66.54+2.55 60.55±5.70 57.89±3.91 Hesperetin 54.75±1.37 43.29±0.82 32.10±5.80 6-Gingerol ( 6-Gingerol) 51.89±3.33 39.83±2.32 30.13±2.67 gallic acid ★ 48.24±4.20 42.74±7.36 35.59±10.03 Isoliquritigenin 47.83±5.36 46.27±3.28 39.08±2.75 Naringenin ( Narigenin) 41.84+3.51 36.82±3.97 25.11±7.60 Dihydrogen Quercetin ((+)-Taxifolin) 34.54+3.47 23.80±5.84 22.58±11.69 Wongonin 23.48±2.59 21.87±1.90 15.64±7.82 Protocatechuic acid 22.75+4.07 19.95+8.95 25.66+ 12.74 catechin ((+)-Catechin) 16.45+9.67 33.83±8.76 41.53+7.62 27 201016216 Chinese medicine induces CYP 2E1 inhibition rate (〇/〇) Test concentration 66 μΜ 33 μΜ 16.5 μΜ β-naphthoflavone (β-naphthoflavone 15.40±12.94 16.83±0.96 6.52±6.64 Embelin 13.54±11.64 12·30±10·24 5.95±7.48 trans-Cinnamic acid Epicatechin ((-)-Epicatechin) Phloridzin 7.10+6.95 4.66±6.50 5.71±10_53 2.57±11.60 1.42±9.28 15.02+5.50 3.76±3.58 18.27±9.34 1.25±7.90 Puerarin -12.8612.75 -4.64+3.47 0.43±2.31 Umbrella Umbelliferone -1081.56 soil 168.00 -571.97±117.56 -280.41±19.48 The inhibition rate of CYP2E1 measured by the excipient is shown in Table 4. From the results, it can be seen that the excipients are at different concentrations (0.167%). , 0.08%, 0. (M2%, w/v) under the medium, on the cytochrome? 45〇 2m has different degrees of inhibitory effect in Guangdong, among which 167.167% Brij 58 has the best inhibitory effect (97 75±〇 66%). Table 4, CYP2E1 inhibition rate of excipients CYP2E1 rate (〇/〇) 0.08%

-SSi^^DDTC) 97.55±1.862 drug index _ test concentration (w/v) control group ———__97.75±0.66__96.58+0.40 96.02±0.17

28 201016216 Chinese medicine medicine cited CYP2E1 inhibition rate f%) Test concentration (w / v) 0.167% 0.08% 0.042% Brij 35 93.33 +0.82 89.45 ± 0.68 76.21 ± 7.37 (test concentration 0.025%) (test concentration 0.013%) (test concentration 0.006%) Tween 20 87.20+1.29 82.80il.71 71.7714.48 Tween 80 73.92+4.71 65.4512.50 64.02±12.54 Tween 40 58.97+3.29 47.05+6.48 44.79±2.49 PEG 2000 44.33+2.75 40.13±3.06 35.81±3.26 PEG 400 42.33+5.25 39.10+0.73 31.98+5.97 Pluomic F68 41.72+5.34 42.98±3.24 37.11+10.35 PEG 4000 37.21+1.91 41.22±0.97 37.18±10.52 Example 3 Screening of cytochrome P45〇2E1 (CYP2E1) inhibitors - human liver Microsomal cytochrome P450 2E1 I. Materials and methods 1. Test materials

In this example, microsomes prepared by human liver were used to screen for cytochrome p45〇2E1 (CYp2E1) ❸ inhibitors against cytochrome p45〇2E1 (CYP2E1) and 39 kinds of traditional Chinese medicines and one type of excipient. The effective inhibitor of cytochrome P45〇2ei (CYP2E1) for human liver was screened; the principle of screening of CYP2E1 inhibitor was: cytochrome P450 2E1 (CYP2E1) and its receptor, Chlorzoxazone reaction in microsomes prepared by human liver After the test sample was added, the amount of 6-OH-CZX (6-Hydroxy-Chlorzoxazone) produced by the CYP2E1 metabolite standard was detected, and the amount of 6-OH-CZX produced by the control group (c〇ntr〇1) was measured. Calculate the CYP2E1 inhibition rate of the test sample for the benchmark '. Each test sample was dissolved in 10% methanol or secondary water, and tested at different concentrations of Chinese medicine (66 μΜ, 33 μΜ, 16.5 μΜ) and excipients (〇1670/〇, 0.08%, 0.042%, w/v) The inhibition rate of CYP2E1, the results of the tested traditional Chinese medicines are listed in Table 3, the tested excipients 29 201016216 and the results are listed in Table 4. The agents required for screening human liver cytochrome Ρ45〇 2Ε1 (CYP2E1) inhibitors in this example are as follows: (1) CYP2E1: 1〇〇 niM potassium phosphate (pH 7.4) contains 1〇 ρ450 protein concentration. (2) Control Protein : 10 mg/mL P450 Protein Dissolved in 100 mM Potassium

Phosphate (pH 7.4) (3) Buffer Solution : 0.5 M Potassium Phosphate (pH 7.4) 〇 Stop Solution : ice-acetonitrile o φ (4) Cofactors: Contains 100 mM NADP+ and 10 mM Glucose 6-Phosphate. (5) Glucose 6-Phosphate Dehydrogenase: 2000 units/ml Dissolved in sterile water. (6) Chlorzoxazone: Substrate, 16 mM Chlorzoxazone is dissolved in 10% methanol (Methanol). (7) DDTC (Diethyldithiocarbamic acid): CYP2E1 selective inhibitor (positive control group), 20 mM DDTC is contained in 1〇. /0 Methanol (8) NADPH-regenerating System: 530 μΐ Cofactors, 40 μΐ G6PDH (Glucose 6-Phosphate Dehydrogenase Solution) and 100 μΐ Control Protein ° Lu 2·Cytochrome P45〇 in 3·42 ml Screening of 2El (CYP2El) Inhibitors The experimental procedures for screening for cytochrome Ρ450 2Ε1 (CYP2E1) inhibitors using human liver microsomal cytochrome Ρ50(E) (CYP2E1) are as follows: (1) In an ice bath at 4 °C, 0.1Μ phosphate buffer (pH = 7.4) containing 0.5 mg/ml human liver microsomes and 5 mMMgCl2 for 15 minutes; (2) At this time, the experimental group was added with cytochrome P450 2E1 reaction matrix drug 16 mM Chlorzoxazone and concentrated Chinese medicine Extract; control group with methanol: sterile water = 1 : 1 201016216 » * replaced with traditional Chinese medicine; positive control group was replaced by ddtc; (3) finally added coenzyme 1 mM NADP +, 10 mM G6P and 2 IU G6PD. Transfer the reaction to 37. (: pre-incubatkm for 1 minute, reaction time for activity test is 30 minutes; (4) Stop reaction with 500pL acetonitrile after the reaction, and leave the sample for 1 minute and then add internal standard (5 pg/mL4) -hydroxy-tobutamide), after centrifugation, take 20pL of the supernatant solution and dilute it with methanol: sterile water for 10 times. Take 5pL of the solution back into the LC/MS/MS system.

analysis. (5) Analysis of results: The signal value measured by LC/MS/MS was converted into the amount of 6-Hydroxy-Chlorzoxazone (pmol) of the CYP2E1 metabolite standard, which was based on the control group (contr〇i), ie, the control group. The CYP2E1 inhibition rate was 〇%, and the 6-OH-CZX production amount of each positive control group and the CΥΡ 2E1 inhibition rate of the test group CYP 2E1 inhibition rate (%) = 1 - control group were calculated by the following formula 6-OH-CZX production amount 1. Result φ 1· Positive control group The positive control group (DDTC) measured the inhibition rate of CYP2E1 as shown in Table 2. It can be seen from Table 2 that the agricultural degree of § DDTC is 1〇〇μΜ When the 'CYP2E1 inhibition rate can reach 87.56%. gpTC 'agricultural degree (μΜ)__fOH-CZX production amount (pmol) cγρ 2Ε1 inhibitory seat (10) 〇 (control group) 3207.5 〇5〇1644'5 48.66 ---__ 87.56_ L test group CYP2E1 inhibition rate 31 201016216 • towel medicine The inhibition rate of CYP2E1 from the drug cited is shown in Table 3. From the knots to the different concentrations (66μΗ 33μΜ, 16.5μΜ), the cytochrome ρ45〇°2Ει = different degrees of inhibition, of which 66 The optimal inhibitory effect of N〇rdmydr〇guaiaretic (96·98±0.19 %) and 66 μΜ Trans-Cinnamaldehyde (92·81±0.53%) was the best. Inhibition rate of CYP2E1 in Chinese medicine (〇/0) Test concentration 66 μΜ 33 μΜ 16.5 μΜ Control group 0 0 0 Nordic hydroguaiaretic acid 96.98±0.19 67·68.±2·24 49.81 ± 2.42 Trans Cinnamaldehyde (Trans-Cinnamaldehyde) 92.81±0.53 89.56±1.52 60.79±3_00 Soybean Gan (Daidzein) 86.77±1.04 76.33±2.28 73.55±1.74 Isovitexin 81.82±1_34 67.60±3.24 59.82 Soil 1.41 Shan Nai Pan ( Kaempferol) 79.25±0.27 74.74±0.60 66.53±1.71 double Disulfiram 78_23 Soil 0.25 75.75±1.38 74.09±1.10 β-Zerene (β-Myrcene) 76.49 Soil 2.18 75.50±2.14 53.40 Soil 4.93 Quercetin 73.32±1.57 53.02±2.17 46.40 Soil 4.68 (-) -Epigallocetechin-3 -gallate 72.16±1.02 60.53±2.06 50.19±1.89 32 201016216 CYP2E1 inhibition rate of Chinese medicine (%) Test concentration 66 μΜ 33 μΜ 16.5 μΜ (+)-limonene ((+)-Limonene) 63.64±2.74 38 ·05±1·95 13.77 ± 1.96 mericetin 61.60±0.88 59.21±1.27 42.21±2.55 Quercitrin 61.04±5.88 53.77±3.51 33.51±4.29 Luteolin-7-glucoside (Luteolin-7 -Glucoside) 60.26士1_11 55_87士0.67 42.96±5.10 Morin 60.26±1.56 52.08±1.70 36.88±1.56 Neohesperidin 58.70士1.06 48.96±2.37 42.81士1.75 Hesperidin 58.57 Soil 3.78 50.91±2.81 45.32 soil 1.57 Capillarisin 57.31 ± 1.31 46.22 ± 2.65 32.89 ± 2.46 (-)-Epigallocatechin 57.08 soil 1.85 36.40 ± 2.18 38.95 soil 1.92 Hypericine (Hyperoside) 53.51 ± 1.20 35.58 ±3. 68 -24.16±1.19 Luteolin 53.23±1.78 丄43.40±4.74 39_15±3·42 Ethyl Myristate 51.95±2.38 41.04±4.76 22.08±0.78 Tamarixetin 50.91 士士3.12 47.79士2.81 37.40±1.96 33 201016216 CYP2E1 inhibition rate of Chinese medicine (%) Test concentration 66 μΜ 33 μΜ 16.5 μΜ Phloretin 50.90±2.09 ♦ 39.78±3.28 29.60±3.21 Baicalein 50.13±5.11 47.79士 3.40 35.32±1.51 Baicalin 49.30±2.26 35.61 ±3.09 22.51±2.24 Apigenin 47.51±3.66 36.80±1.98 28.89 soil 1.54 Naringenin 45.16±4.43 28.45±2.21 19.50 ± 2.02 Orange peel Hesperetin 44_56±2.35 34.28士2.03 25.74±2.45 (+)-Epicatechin 44.32±1.25 52.32±1.59 66.71±1.79 Rutin 43.51 Soil 3_09 30.13士1.62 30.00士0.81 (-)-Epicatechin-3-gallate 42.92±0.65 34.84±1.72 30.31±1.27 Isoliquritigenin 41.12±0.92 31.48±1.24 21.18±1.96 Silybin 38.96±1.19 37.14±1.15 59.48±2.34 Vitex Vitexin 38.70 soil 1.62 30.65±0.78 23.12 soil 1.19 genistein 36.88 soil 1_56 30.91±1.62 43.90±2.06 34 201016216 test concentration isorhamnetin CYP2E1 33 μΜ 18.68±1·22 Chinese medicine Galic acid 66 μΜ 36.31±1.59 27.96±1.56 18.79±2.03 16.5 μΜ 12.06±1.〇6 ------ 1〇.50±ΐ.ΐ2 香 cedar lignin Diosmin 6-ginger Spicy alcohol (6-Gingerol) 21.56±1.19 43.12±3.57 60.〇〇±ι.96 19.08±1.36 11·51±1.〇2 7.84±〇.92 The CYP2m inhibition rate measured by the excipient is shown in Table IV. As shown by the results, it can be seen that under the conditions of fine different concentrations (0.167%, 〇.08%, 0.042%, w/v), the cytochrome p45 具有 has different degrees of inhibitory effect, of which 167167% Brij 58 has the best inhibition effect). ·... TCM 2E1 inhibition rate of CYP2E1 inhibition rate (%) Reference test concentration (w/v) Control group 0.167% 0.08% 0.042%

Brij 58 91.24±1.33 80.50±1.14 62.57士2.10

Brij 76 86.15 土 1.02 75.71±1.61 68.99士 3 ·77

Brij 35 77.28士 1.02 64.17±1.71 2 test concentration 0.025%) (test concentration 0.013%) (42.37 ± 1.78: test concentration 0.006%)

Tween 20 75·38±3.64 70.44±0.93 55.38±1.95 PEG 400 64.17±1.53 54.78±3.53 26_42 Soil 1.81 PEG 4000 47.11 ± 0.92 23.94±0.92 8.70 Soil 0.77 35 201016216 Chinese medicine CYP2E1 inhibition rate (〇/〇) Test concentration (w/v) PEG 2000 0.167% 47.06±1.53 0.08% 41.43±1.60 0.042% 22.25±1.93

The present invention provides a new compound (7) with low side effects different from the amine (IS0niazid), and when compared with the test results of isoniazid (INH) alone, in biochemical analysis (ALT, ast ❹ value) ), pathological analysis, measurement of residual liver function (Gsp value, GEC value), and analysis of oxidative stress (concentration of S-iso-PGFh in plasma), etc., have significantly reduced the use of different The efficacy of hepatotoxic side effects caused by nicotinamide (INH). The invention provides a novel side effect of isoniazid amide (Isoniazid (INH)) (7), which also provides cytochrome P450 2E1 (CYP2E1) sedative medicine, she knows cytochrome P450 2E1 (CYP2E1) Inhibitors The present invention provides those who are extracted from natural Chinese medicines, have no physiological and chemical toxicity, and have significant inhibitory activity against cytochrome p45〇2E1 activity in human liver. 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. The concentration and ratio of alkali guanamine (mH), cytochrome p45〇2m inhibitor, disulfiram (DSF), nitrophenol phosphate diester (BNpp), and the type of cytochrome p45〇2E1 inhibitor Equivalent embodiments are to be included in the scope of the patent. 36 201016216 0 In summary, this case is not only innovative in cytochrome P450 2E1 inhibitors, but also can effectively reduce the side effects of hepatotoxicity caused by the use of isonicotinic acid amide (INH), which should fully meet the novelty and progress. The statutory invention patent requirements for sex, 提出 apply in accordance with the law, and ask your bureau to approve the application for this invention patent, in order to invent invention, to the sense of virtue. [Simplified illustration] Figure 1 shows the metabolic pathway of isonicotinic acid amide (INH) in the liver; Figure 2 shows the control group, INH group, BNPP-INH group, DSF-INH group and BNPP-DSF-INH ® The rats in the group were analyzed for the activity of aspartate aminotransferase (AST) and alanine transaminase (ALT). The values were calculated as mean soil SD, and * indicates that each test group was compared with the control group. Figure 3 shows the liver sections of the control group (Fig. 3A and C) and the INH group (Fig. 3B and D): Fig. 3A 'type of the control group relative to normal liver tissue (he staining, 400X); In the three B, INH group, the hepatocytes in the peripheral central vein (V) showed fragmentation and vacuolation (he staining, 400X); Fig. 3C, the liver slices of the control group were observed by electron microscopy, Nu: nuclei (9,三); Figure 3D, the liver sections of the rats in the INH group were examined by electron microscopy. Compared with the hepatocyte sections of the control group of the third group, the crude endoplasmic reticulum (rER) of the hepatocytes of the group 9 was significantly increased. Nu: Nuclei (9,000Χ); Figure 4 shows the molecular structure and daughter ion spectrum of 8-iso-PGF2a-d4 (Α) and 8-iso-PGF2a (Β); Figure 5 contains 250 pg 8-iso-PGF2a -d4 (A) internal standard Liquid, liquid chromatography-tandem mass spectrometer (LC/MS/MS) chromatography with standard solution of 100 pg 8-iso-PGF2a (B) and blank sample (c), detected by multiplex reaction monitoring mode, Mass-to-charge ratio (111/2) 357/197 and mass-to-charge ratio (m/z) 35V193 ion couples (i〇n pairs) were used to monitor 8_is〇_pGF2a_d4 37 201016216 9 (A) (as internal standard) ) and 8-iso-PGF2a (B) (as a standard); peak i: blank plasma; peak 2: blank plasma injected into the standard; Figure 6 is the control group, INH group, BNPP-INH group, DSF-INH group And the concentration of 8-iso-PGF2a in the plasma of the BNPP-DSF-INH group, the value was calculated as mean soil SD, * indicates that the test group was compared with the control group P <0.001;# indicates each test group and INH group After comparison, /^ <0.05; Figure 7 is the control group, INH group, BNPP-INH group, DSF-INH group and BNPP-DSF-INH ❹ group galactose single point method (GSP) value, the calculation of the value For mean ± sd, * indicates that the test group is compared with the control group, ρ < 〇 · 〇〇 1; # indicates that each test group is compared with the _ group after p < 0 001; ※ indicates that each test group is compared with the INH group &lt ; 0.005; 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 values are calculated as mean soil SD, and * indicates the test group and control. After the group comparison /><0·001;# indicates that each test group is compared with the INH group p < 〇 005; 》 indicates that each test group is compared with the INH group Ρ < 〇 · 〇 5;

❹ Figure IX shows that the galactose single point method (GSP) values in the control group, sputum group, BNPP-INH group, DSF-INH group and BNPP-DSF-INH group are highly correlated with the concentration of 8-iso-PGF2a in plasma. a chart; and

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. 38 201016216 '[Main component symbol description] None [References] 1. Kopanoff DE et al., Isoniazid-related hepatitis: a US Public Health Service cooperative surveillance study., 1978. Am. Rev RespirDis 117:991-1001. Nolan CM et al., Hepatotoxicity associated with isoniazid preventive therapy: a 7-year survey from a public health tuberculosis clinic. 1999. JAMA 281: 1014. 3. Steele MA et al., Toxic hepatitis with isoniazid and rifampin: A meta -analysis. 1991. Chest. φ 99:465. 4. Sarich TC, Youssefi M, Zhou T, Adams SP, Wall RA, Wright JM. Role of hydrazine in the mechanism of isoniazid hepatotoxicity in rabbits. 1996. Arch Toxicol 70: 835-840. 5. Yue J, Peng RX, Yang J, Kong R, Liu J. CYP2E1 mediated isoniazid-induced hepatotoxicity in rats. 2004. Acta Pharmacol Sin. 25: 699-704. 6. Sarich TC, Adams SP, Petricca G, Wright JM. Inhibition of isoniazid-induced hepatotoxicity in rabbits by pretreatment with an amidase inhibitor. 1999. J Pharmacol Exp Ther. 289: 695-702.

7. Lee SS, Buters JT, Pineau T, Fernandez-Salguero P, Gonzalez FJ. Role of CYP2E1 in the hepatotoxicity of acetaminophen. 1996. J Biol Chem 271: 12063-12067. 8. Wong FW, Chan WY, Lee SS. Resistance to carbon tetrachloride-induced hepatotoxicity in mice which lack CYP2E1 expression. 1998. Toxicol Appl Pharmacol. 153: 109-118. 9. Ramaiah SK, Apte U, Mehendale HM. Cytochrome P4502E1 induction increases thioacetamide liver injury in diet-restricted rats. 2001. Drug Metab Dispos. 29: 1088-1095. 10. Huang YS, Chem HD, Su WJ, Wu JC, Chang SC, Chiang CH, Chang FY, et al. Cytochrome P450 2E1 genotype and the susceptibility to antituberculosis drug-induced 2003. Hepatology 37: 924-930. 11. Guengerich FP, Kim DH, Iwasaki M. Role of human cytochrome P-450 IIE1 in the oxidation of many low molecular weight cancer suspects. 1991. Chem Res Toxicol. 4: 39 201016216 Ψ 168-179. 12. Hunter AL, Neal RA. Inhibition of hepatic mixed-function oxidase activity in vitro and in vivo by various thio No-sulfur-containing compounds. 1975. Biochem Pharmacol. 24: 2199-2205. 13· Brady JF, Xiao F, Wang MH, Li Y, Ning SM, Gapac JM, Yang CS. Effects of disuLfiram on hepatic P450IIE1, other microsomal Enzymes, and hepatotoxicity in rats. 1991. Toxicol Appl Pharmacol. 108: 366-373. 14. Sodhi CP9 Rana SV, Mehta SKs Vaiphei K, Attari S5 Mehta S. Study of oxidative-stress in isoniazid-rifampicin induced hepatic injury in young Rats. 1997. Drug Chem Toxicol 20: 255-269. Reference 15. Morrow JD, Hill KE, Burk RF, Nammour TM, Badr KF, Roberts LJ, 2nd. A series of prostaglandin F2-like compounds are produced in vivo in humans 1990. Proc. Natl. Acad. Sci. USA 87: 9383-9387. 16. Morrow JD. The isoprostanes: their quantification as an index of oxidant stress status in vivo. 2000. Drug Metab Rev. 32: 377-385. 17. Devaraj S, Hirany SV, Burk RF, Jialal I. Divergence between LDL oxidative susceptibility and urinary F(2)-isoprostanes as mea 2001. Clin. Chem. 47: 1974-1979. 18. Helmersson J, Basu S. F2-isoprostane excretion rate and diurnal variation in human urine. 1999. Prostaglandins Leukot. Essent. Fatty Acids 61: 203-205. 19. Morrow JD, Roberts LJ? 2nd. Mass spectrometric quantification of F2-isoprostanes in biological fluids and tissues as measure of oxidant stress. 1999. Methods Enzymol. 300: 3-12. 20. Li H, Lawson JA, Reilly M, Adiyaman M, Hwang SW, Rokach J, FitzGerald GA. Quantitative high performance liquid chromatography/tandem mass spectrometric analysis of the four classes of F(2)-isoprostanes in human urine. 1999. Proc. Natl. Acad Sci. USA 96: 13381-13386. 21. Liang Y5 Wei P3 Duke RW, Reaven PD, Harman SM? Cutler RGS Heward CB. 40 201016216 ,

22. Quantification of 8-iso-prostaglandin-F2a and 2,3-dinor-8-iso-prostaglandin-F2a in human urine using liquid chromatography-tandem mass spectrometry. 2003. Free Radic. Biol. Med 34:409-418. Carlisle R, Galambos JT, Warren WD. The relationship between conventional liver tests, quantitative function tests, and histopathology in cirrhosis. 1979. Dig. Dis. Sci. 24: 358-362. 23. Hero Id C, Heinz R, Niedobitek G , Schneider T, Hahn EQ Schuppan D. Quantitative testing of liver function in relation to fibrosis in patients with chronic hepatitis B and C. 2001. Liver 21: 260-265. 24. Keiding S, Johansen S, Tonnesen K. Kinetics of ethanol Inhibition of galactose elimination in perfused pig liver. 1977. Scand J. Clin. Lab Invest. 37: 487-494. 25. Keiding S, Johansen S3 Winkler K. Hepatic galactose elimination kinetics in the intact pig. 1982. Scand J. Clin Lab Invest 42: 253-259. 26. Lindskov J. The quantitative liver function as^ measured by the galactose elimination capacity. I. Diagnostic Value and relations to clinical, biochemical, and histological findings in patients with steatosis and patients with cirrhosis. 1982. Acta Med. Scand. 212: 295-302. 27. Tang HS, Hu OY. Assessment of liver function using a novel galactose single 1992. Digestion 52: 222-231. 28. Hu OY, Tang HS, Chang CL. The influence of chronic lobular hepatitis on pharmacokinetics of cefoperazone—a novel galactose single-point method as a measure of residual liver function. 1994 Phy 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 。 Liver dysfunction. 1995. J. Pharm. Sci. 84:231-235. 30. Hu OY, Tang HS? Sheeng TY3 Chen TC, Curry SH. Pharmacokinetics of promazine in patients with hepatic cirrhosis—correlation with a novel galactose single point method 1995. J. Pharm. Sci. 84: 111-114. 41 201016216 « 31. FDA Center for Drug Evaluation and Research (CDER) Pharmacokinetics in patients with impaired hepatic function: Study design, data analysis, and impact on dosing and labeling. Guidance for Industry, US Department of Health and Human Service. 2003 pp5 32. Tygstrup N. The Galactose Elimination Capacity in Control Subjects and in Patients with Cirrhosis of the Liver. 1964. Acta Med. Scand 175: 281-289. 33. Ryan DE, Ramanathan L, Iida S, Thomas PE, Haniu M , Shively JE, Lieber CS, et al. Characterization of a major form of rat hepatic microsomal cytochrome P-450 induced by isoniazid. 1985. J. Biol. Chem. 260: 6385-6393. 34. Ekstrom G, Ingelman-Sundberg M Rat liver microsomal NADPH-supported oxidase activity and lipid peroxidation dependent on ethanol-inducible cytochrome P-450 〇(P-450IIE1). 1989. Biochem. Pharmacol. 38: 1313-1319. 35. Sodhi CP, Rana SV5 Mehta SK5 Vaiphei K, Attri S3 Thakur S5 Mehta S, Study of oxidative stress in isoniazid-induced hepatic Injury in young rats with and without protein-energy malnutrition. 1996. J Biochem Toxicol. 11: 139-146. 42

Claims (1)

201016216 'X. Patent application scope: 1. A new compound (2) with low side effects of isoniazid (INH), including a pharmaceutically effective amount of isoniazid (INH), combined use At least one pharmaceutically effective amount of a cytochrome P450 2E1 (CYP2E1) inhibitor. 2. A new compound (2) having a low side effect different from the isoniazid (INH) according to the scope of claim 1, wherein the cytochrome P450 2E1 (CYP2E1) inhibitor is selected from the following compounds; Groups: Trans-Cinnamaldehyde, Daidzein, Isovitexin, β-Myrcene, Quercetin, (+ )-Limonene ((+)-Limonene), Myricetin, Quercitrin, Luteolin-7-Ghicoside, Morin, New Orange Peel Neohesperidin, Hesperidin, (---Epigallocatechin), Luteolin, Hyperoside, Ethyl Myristate 'Salvain' Tamarixetin), Baicalein, Rutm, Baicalin, Apigenin, (1)-Epicatechin ' Φ (_)_Epicatechin_3_gaUate, Shuifei, Silybin, Vitexin , Gemstem, Isorhamnetin, Di〇smin, Puerarin, or Umbelliferous Lactone (Umbemfer〇ne). 3. A new side effect of a low side effect of isoniazidamine (Is〇niazid, leg) as described in item 1 of the scope of the patent application. (2) A pharmaceutically acceptable excipient may be added to the compound. . 4. If the application of the patent scope 帛3 is different, the compensation is low, the side-effect compound (), and the medium-based excipient can be a diluent, a filler, a binder, disintegration. Agent, lubrication 43 201016216 *agents, etc. 5. A cytochrome P450 2E1 (CYP2E1) inhibitor, wherein the cytochrome Ρ450 2Ει (CYP2E1) inhibitor is selected from the group consisting of trans-Cinnamaldehyde, ugly gamma (Daidzein), isovitexin, β-Myrcene, Quercetin, (+)-lean ((+)_Limonene), mericetin, suede (Quercitrin), luteolin-7_Glucoside, Morin, Neohesperidin, Hesperidin, (I)-Epigallocatechin, Luteolin ( Luteolin), Hypercoside, Ethyl Myristate, Tamarixetin, Baicalein, Rutin, Baicalin, Apigenin ) ' (+)-Epicatechin, (-)-Epicatechin-3-gallate, Silybin, Vitexin, Genistein, Isorhamnetin Diosmin, Puerarin, or Umbelliferone. 44