TWI360418B - - Google Patents

Description

U6〇4i8 IX. Description of the invention: .. [Technical field to which the invention pertains]. The present invention relates to a new compound of isoniazid indamine (INH) with low side effects, in particular, a heterologous prostamine (INH) combined with cytochrome P450 2E1 (CYP2E1) inhibitor, or combined with nitrophenol phosphate diester (BNPP) to reduce side effects such as hepatotoxicity caused by isoniazid amide (INH) A new compound of alkali amide. [Prior Art] According to estimates by the World Health Organization (WHQ), about three-thirds of the world's population is infected with tuberculosis, with about 8 million new cases each year; and the number of newly registered tuberculosis patients in Taiwan has not been in recent years. Nasaki 'more than 60 people per 100,000 population _ tuberculosis, but only about a quarter of them - received 70 full 'Lin' According to the statistics of the Department of Health, at least 4.2 people die of tuberculosis every day in Taiwan; Among the many patients receiving tuberculosis medication, the most common clinical side effects are hepatotoxicity and neurological diseases (such as auditory nerve and optic neuropathy), among which hepatotoxicity is the most common. In addition, Taiwan is also a prevalent area for hepatitis B and hepatitis C. The number of hepatitis patients infected with tuberculosis is not in the minority' assumption that there are 14 new tuberculosis patients in the coffee field every year, roughly estimated at least #2,_name to 3, _ Qianshou patients with miscellaneous red knot _ secret treatment, the liver toxicity that may occur in these diseases and patients is a natural disease that we can not ignore. • The first line of anti-tuberculosis drugs in the township, for example: different from the test of guanamine (Μ, commonly known as the enemy raisin star), propyl sulphide is different from guanamine (four) - Na, commonly known as the enemy treatment Xinmai) and rifamycin Time (4) and other potential adverse reactions leading to hepatotoxicity; which is different from the test of amine (4) is also the most effective single-anti-tuberculosis drugs, and most of the patients produce hepatotoxicity; in the late 6's 1360418 is different from the report of hepatotoxicity caused by isoniazid; it is about 0J_1%|,2, and is less than 1J2% of the clinical symptoms caused by is〇niazid. In patients, asymptomatic liver dysfunction 3 can be observed, and these abnormal liver functions usually occur within two months after taking the drug. As shown in Figure 1, isoniazid in the liver is mainly acetylated by the help of N-acetyltransferase (NAT), and the intermediate product is acetylated isonianic acid. The acetonide (acetylisoniazid) is rapidly hydrolyzed to the acetyihydrazine; the hydrazine is brewed and then converted to a non-toxic double-branched hydrazine via the nitrogen-ethyl hydrazine-transferase. Diacetylhydrazine, or oxidized to cytotoxic molecules via cytochrome p45〇2E1 (CYP 450 2E1), including acetylonium ion, acetyiradicai, ethylene Ketone (ketene), etc., in the presence of aerobic and NADPH, acetaminophen will be reacted by cytochrome P45〇2E1 to generate free radicals, causing oxidative stress to cause cell death; in addition, 'different to the amine (isoni^) Id) can also be directly hydrolyzed to a toxic hydrazine via amidolytic hydrolyzed amidase, or hydrolyzed to a toxic hydrazine by amidase hydrolyzate (amidase) from the above-mentioned acetylhydrazine (hydrazine). Recently, studies have shown that hydrazine (rather than isoniazid guanamine or acetylated hydrazine) is the most likely cause of causing hepatotoxicity (INH-induced hepatotoxicity) in rabbits and mice. 4 '5, the researchers believe that the severity of hepatotoxicity caused by the determination of amines is positively correlated with the concentration of hydrazine in blood polycondensation; in 1999, Sarich6 et al reported that p-nitrophenol phosphate (bis-p- Nitrophenyl phosphate, BNPP 'is an inhibitor of indoleamine hydrolase, prevents hepatotoxicity caused by isoniazid amide, and its protective mechanism should be through inhibition of isoniazid guanamine production ί S ) 6 1360418 hydrazine. Cytochrome P450 2E1 (CYP2E1) is sustained in the liver and is responsible for the metabolic biological reactions of many foreign substances such as hepatic toxin carbon tetrachloride (CCU) and acetaminophen 7,8; However, the role of CYP2E1 in the hepatotoxicity caused by isonicotine amide is not clear. Isoniazid guanamine itself is an inducer of CYP2E1 9; some studies suggest that CYP2E1 and isonicotamine in the liver The mechanism of liver toxicity caused by 5'1〇. In vitro, disulfiram (DSF) and its metabolite diethyldithiocarbamate have been identified as selective inhibitors of mouse and human liver microsomes CYP2E1 (selective mechanism-based inhibitors) ) n, 13, Brady 丨 3 and other tests showed that mice taking a single oral dose of disulfiram (DSF), resulting in immune response liver capacity (immun 〇 reactive hepatic content) and CYP2E1 catalytic activity rapid and complete decline .

Sodhi14 et al. reported in 1997 that oxidative stress is one of the factors that cause hepatotoxicity caused by isonicotinicin and rifamycin in young rats. There are many studies that want to find appropriate biomarkers to assess the rate of oxidative damage in the body. Currently, biomarkers that may be applicable can be classified into three categories, which are markers for oxidative damage to lipids, proteins, and nucleic acids; _Iso-prostaglandin FZa (S-iso-PGFa) is a product of free radical-induced lipid peroxidation of arachidonic acid, which is chemically stable, 8_is〇_pGF2a I can be used as a new indicator to determine lipid peroxidation in vivo. The lipid peroxidation may be related to the production of free radicals, oxidative damage and antioxidant deficiency in the body 15'16 There are currently many methods for measuring 8_is〇_pGF2c^1 including enzyme immunoassay 17, radioimmunoassay 7 1360418 (radioimmunoassay), gas-chromatography mass spectrometry I9 and Liquid chromatography mass spectrometer curry _ 〇 卿 卿 spect spect spect spect spect spect spect spect 20 20 20 · · · · · · 人类 人类 人类 人类 人类 人类 人类 人类 人类 人类 人类 人类 人类 人类 人类 人类 人类 人类 人类 人类-8-is〇-PGF2a content can be determined by C18 solid phase extraction (α8 s〇lid detachment extracti〇n, * . SPE) after preparation of the sample 're-liquid chromatography tandem mass spectrometer (LC/MS/MS) analysis. Using invasive and Lai-style methods Lin rats (four) liver function, to monitor the development of damage • and liver refractory disease, its towel most often (four) method side of the serum towel asparagine acid transaminase (aspartate aminotransferase , AST), alanine aminotransferase (ALT), and auxin luciferase (exciting & nejne ph〇Sphatase) values, as well as measuring * liver cell products such as bilirubin llububin, albumin (albumin), and the use of prothrombin time to detect coagulation factors (c〇agulati〇n such as (d), etc. a; liver function quantitative test 疋根谭 almost only through the liver metabolism of the receptor in serum In terms of the concentration, the removal of these receptors is based on the hepatic portal vein, hepatic artery blood flow, and the effect of hepatocytes on these receptors. The liver blood flow is provided and the quality of the liver is impaired. The removal of the receptor is determined by the ability of the liver to metabolize. 23. Galactose, a sugar with high extraction rate (extraeti〇n rati〇), metabolized in the liver 'in the liver' is galactose Kinactin Ase) undergoes epimerization to convert it to Glucose-1-phosphate; the action of galactose kinase is the rate-determining step of the galactose metabolic pathway in hepatocytes (rate- Limiting step)24 The high extraction rate of galactose makes galactose metabolism, which is dependent on hepatic blood flow and liver function, the most important way to detect liver function. There is currently no rule of 1360418 来 to assess the residual liver function of rats ( Residuai iiver, like ^), measurement - the exact compound (such as: half-new) generation of Wei Li, can be fine-skin towel - the metabolic side of the fresh decision step, can also obtain the representative liver quality of the representative quality 24,2 The ability to remove galactose π (galactose eliminati〇n, GEC) as a test for human liver function tests 26 has been carried out for a year, half-clearing ability, and multiple liquid samples to establish a standard curve 'in clinical There are many difficulties in application, so many studies use Galactose Single Point (GSP) to evaluate human liver function; the inventor of this case used galactose single-point method to slow down, "like and occupational patients" Silk display (4) single point method can accurately detect these liver diseases 27; galactose Lai has been used in the field of liver disease patients to exclude such drugs as promazine and antibiotic cefotaxone (cef〇peraz〇ne) The remaining liver function is 28-30 〇l L. The galactose single-point method has become one of the recommended methods for testing liver function in the guidelines published by the US Food and Drug Administration (FDA) (Guidance for Industry). Seeing that the above-mentioned anti-tuberculosis drugs are different from is〇niazid, there are still many defects, which is not a good designer, but needs to be improved. The inventor of this case secreted the above-mentioned system of anti-resistance threats _sQniazid. The shortcomings of the action of detoxification and other fields were improved and innovated, and after years of painstaking efforts and research, ~ successfully developed the low side of this piece. Trace inspection _ (- d, willing) new compound. SUMMARY OF THE INVENTION The object of the present invention is to provide a low side effect of the different final amine (4) ^ d, face) 'combination of the different final amine (dirty) combined with the use of cytochrome P45 〇 2ei (cy? 2ei) Inhibitors to reduce side effects such as hepatotoxicity caused by isoniazidamine (INH). 1360418 A second object of the present invention is to provide a novel compound of isoniazid amide (INH) with low side effects, which is selective for inhibition of guanamine (INH) combined with cytochrome p45〇2El (CYP2E1). Disulfiram (DSF), and inhibitor of guanamine hydrolase, nitrophenol phosphate diester (BNPP), to reduce side effects such as hepatotoxicity caused by different final amine (INH). In order to achieve the low side effect of the above-mentioned invention, a new compound of isoniazid amide (is〇niazid, _), the present invention firstly induced hepatotoxicity in rats by isoniazidamine (INH) as a model, research cells Pigment P450 2E1 (CYP2E1) inhibitor Disulfiram (DSF), and indoleamine hydrolase (amidase) inhibitor nitrophenol phosphodiester (BNPP) on liver induced by isonicotinicinamide (iNH) in rats The effects of toxicity; in addition to the use of general hepatotoxicity markers, galactose single point method (GSP) and galactose clearance capacity (GEC) for quantitative measurement of residual liver function in rats, the inventors of the present invention use improved liquid layer The concentration of 8-iso-PGF2a in rat plasma was measured by tandem mass spectrometry (LC/MS/MS) to further determine whether 8-iso-PGF2〇^ was associated with hepatic toxicity induced by INH in rats. A new compound of isoniazid (INH) which achieves the low side effects of the above object of the invention includes a pharmaceutically effective amount of isoniazid (INH) in combination with a pharmaceutically effective amount of cytochrome P450 2E1 (CYP2E1) inhibitor. Wherein the cytochrome P450 2E1 (CYP2E1) inhibitor is selected from the group consisting of disulfiram (DSF), naringin (Narigenin), hesperetin, quercetin ), Nordic hydroguaiaretic acid, Capillarisin, protocatechuic acid, gainc acid, ursolic acid, (-)- Epigallocetechin, (-)-Epicetechin-3-gallate, (a)-Epigallocetechin-3-gallate, Tween 20, Tween 40, Tween 60, Tween 80, 10 1360418

Bnj 35, Brij 58 ' Brij 76, PEG 2000, PEG 4000 ·. The low side effect of achieving the above-mentioned object is different from the new compound of the testisamine (INH), which includes a learning effective amount different from the wake-up Amines (such as "anal fistula small jjsjh"), combined with a pharmaceutically effective amount of disulfiram (DSF), and a pharmaceutically effective amount of nitrophenol phosphate diester (bis-/?-nitrophenyl phosphate) , BNPP) The low side effect of the present invention is different from the new compound of the test amine (is〇niazjld, j]^H), and a pharmaceutically acceptable excipient may also be added to the compound. The agent may be a diluent, a filler, a compound #1, a disintegrant, a lubricant, etc. [Embodiment] - The present invention will be further illustrated by the following examples, but the examples are for illustrative purposes only. It should not be construed as limiting the practice of the invention. Example 1 isoniaceine guanamine (INH) combined with €:¥1>21:1 inhibitor disulfiram (DSF) and/or nitrophenol phosphate II Animal Test of Ester (BNPP) I. Materials and Methods • 1. All organic solvents of the test materials are HPLC grade. Tedia Co., Ltd. (Fairfldd, 〇H, USA) 'INH, BNPP, DSF and corn oil are gambling from the local coffee chemical company (9). This, m〇USA) '8_ls〇-PGF2« and radiation-calibrated 8-iso- PGF2a-d4 was obtained from Cayman Chemical Company (Ann Arbor, MI'USA), and the galactose injection solution was prepared by Nanguang Chemical Pharmaceutical Co., Ltd. '400 g of galactose (Sigm mixed with α liter containing appropriate buffer system and etc. Distilled water in salt, for injection.

11 1360418 2. Male SD (Sprague-Dawley) rats weighing 320-350 g were purchased from the National Laboratory Animal Center (Taiwan). The animal experiments were carried out in accordance with the guidelines of the National Animal Research Institute. All rats were placed. Under the air/humidity adjustment environment, the light and the darkness were each 12 hours, and the supply of water and feed was not limited. The weight of the rats was continuously monitored during the test. All the rats were dosed with 50 mg/kg body weight of pentobarbital. Sodium pentobarbital was intraperitoneally anesthetized, and a polyethylene catheter was placed in the rat internal jugular vein to galactose. The catheter was inserted by a cut-down technique. The end of the catheter was placed under the skin of the posterior cervical incision of the rat. After the surgery was completed, the rats were fasted overnight (about 16 hours) during the recovery period, but the water was supplied as usual. 3. Test treatment The test animals were randomly divided into 5 groups. Each group included 3 treatments. The first treatment was injection of 25 mg/kg BNPP or BNPP (vehicle, VEH1 'ready saline), and BNPP was dissolved to 6 Hey. Salined saline (0.9% NaCl), after cooling, was intraperitoneally injected into the rat in a volume of 1 ml/kg; the second treatment was to inject 100 mg/kg of DSF or DSF (VEH2, corn oil) DSF is dissolved in corn oil and injected intraperitoneally into the body in a volume of 1 ml/kg; the third treatment is a 150 mg/kg INH or INH base (VEH3 'ready saline). INH was dissolved in saline (0.9% NaCl) by intraperitoneal injection into the rat in a volume of 1 ml/kg; the first group (BNPP or VEH1) was 30 minutes earlier than the third group (INH or VEH3). Treatment, the second group (DSF or VEH2) was processed 15 minutes earlier than the third group (INH or VEH3). The above five groups of experiments included: 12 1360418 (1) Control group (normal control group, NC, n=12): normal rats were injected once a day for VEH1, VEH2 and VEH3 (administered intraperitoneally) for 2i days; 2) INH group (INH, n=7): normal rats were injected once a day, y£H1 and (administered intraperitoneally) for 21 days; (3) BNPP-INH group (BNPP-INH, n=7) ): Normal rats were injected with 丨 & BNPP and VEH2 (administered intraperitoneally) daily for 21 days; (4) DSF-INH group (DSF-INH, n=7): normal rats were injected daily with ^DSF , legs and VEH1 (administered intraperitoneally) for a total of 21 days; and (5) BNPP-DSF-INH group (BNPP-DSF-INH, n=7): normal rats were injected once a day with BNPP, DSF and INH ( A total of 21 days were administered intraperitoneally; the galactose single point method was tested 16 hours after the 21st day of treatment. 4. After the blood sample was processed, the rats were sacrificed by ether anesthesia. The blood was taken from the rat aorta and placed in a test tube containing EDTA. The plasma was centrifuged at 13,000 g for 15 minutes at 4 ° C. The blood was collected after separation. Dispense into a small tube (Eppendorf tube) and store at -80 °C. 5. Biochemical analysis of hepatocyte injury to measure aspartate aminotransferase (AST) and alanine transaminase (ALT) activity in plasma for quantification 'AST and ALT activity are commonly used indicators of liver toxicity, The Synchron LXi 725 system was measured (Beckman Instruments, USA). 6. Light microscopy and electron microscopy Rats were sacrificed for histological analysis immediately after sacrifice; liver samples were fixed with 13 (S) phosphate-buffered formalin in 10% phosphate buffer, then dehydrated and embedded in paraffin ( Paraffm) 'Skin sliced at 5 μηι thickness' slice was stained with hematoxylin and eosin, and subjected to periodic acid Schiff stain (PAS), stained and histologically studied by light microscopy Observe, in addition, the liver sections were washed with buffer buffer (cac〇dyiate buffer, 0.1 Μ pH 7.4) and fixed with 20% aqueous osmium tetroxide for 1 hour. In Spurr resin (Spurrresin), ultra-thin sections were cut with diamond knives, double stained with uranylacetate and leadcitrate, and transmitted by electron microscopy (Transmission Electron Microscope, Hitachi 600, Hitachi). Co., Japan) observed. Extraction and measurement of 7.8-iso-PGF2a All isoforms of PGFh were dissolved or diluted in an appropriate volume of alcohol to prepare stock solution 'packed in small tubes and stored at -70 ° C, 0.5 ml Plasma to glass tube, add i〇ng internal standard (8-iso-PGF2a-d4) 'mixed blood _ slurry with C18 solid phase extraction cartridge (Solid-Phase Extraction cartridge, JT Baker , MA, USA) Purified, the sample stream washing solution was evaporated to dryness with nitrogen, and then dissolved in 50 μl of acetonitrile: water (15:85 v/v) solution and shaken for 30 seconds. After 1 〇μ1 was dissolved. The extract was injected into an LC/MS/MS system for analysis. 8. Liquid Chromatography Tandem Mass Spectrometer (LC/MS/MS) Analytical HPLC system consisting of 2 Shimadzu LC-10ADvP pumps (Shimadzu LC-10ADvP pumps), 1 Shimadzu system control, and 1 Shimadzu autosampler (Shimadzu autosampler) (Shimadzu Scientific Instruments, 曰本), HPLC separation with C18 column (particle size 5-μιη, inner diameter 50 X 2.1mm), and using 2mM acetate acetate 1360418 and Gradient flow wash of acetonitrile (ACN) (t = 0 min, 15% ACN; t = 6 min, 70% ACN; t = 7 min, 90% ACN; t = 8 min, 90% ACN; t = 8.5 min, 15% ACN) The flow rate of '1^/]\48/1'48 was maintained at 2〇0b1/111丨11, and the entire 11?1^ time was 13.5 minutes; The system is interfaced with a triple stage quadrupole mass spectrometer (API3000, Applied Biosystem, Foster City, CA, USA).

TurboIonSpray ionization source, and use negative electrospray as ionization method; the mass spectrometer diffuses 200 ng/ml 8-iso-PGF2ct or 8-iso-PGF2a- The d4 standard was optimized by multiple reaction monitoring (MRM) mode, m/z 353/193 and m/z 357/197 ion couples (丨〇11卩3丨〇 were used to monitor 8-7 individually) 〇-?0?2〇1 and 8-丨8〇~?0?2〇1-(14; after measurement, calculate the linear calibration curve of six 8-iso-PGF2a concentrations (C) 8-iso-PGF2a is in the region of 8-iso-PGF2a-d4 ratio (Y), and the correlation coefficient (r, correlation coefficient) is 0.999. The linear range of plasma t8-iso-PGF2a is 0.1-2.5 ng/ml. The regression equation is Y=-0.0517C +0.823 ng/ml; the measured results are calculated according to the standard of the hydrogenated 8-iso-PGF2a (deuterated 8-iso-PGF2a) standard curve. The inter-batch precision and accuracy were evaluated by standard-concentration samples after 6 times, and then evaluated by Back-Calculation. The relative error rs) ranged from -5J6% to 3.13%. 9. Quantitative testing of liver function All rats were tested for galactose single point (GSP) and galactose clearance (GEC) tests. Rats received a rapid intravenous injection within 30 seconds of injection of 4.4g/ml BW galactose solution 0.5 g/kg; 15 (S) 1360418 Blood was collected from 5, 10, 15, 30, 45 and 60 minutes after injection. The blood sample was taken from the tail vein. The galactose content was measured by the colorimetric galactose dehydrogenase. The test concentration ranged from 50 to 1,000 pg/ml, and the intra-orbital difference of each concentration (within-day) Variation) is calculated from the standard deviation and the coefficient of variation (coe cient 〇f variation, CV). The maximum allowable coefficient of variation is i〇% cv; the day-to-day variation is determined by The slope and intercept of the calibration curves are compared to test; the galactose clearance capacity (GEC) is calculated by the following formula, which is modified by Tygstrup, s Equation 32:

D GEC ---(mg / kg · min) Τ〇=〇+ 7 where D is the amount of galactose injected; tc=0 is the time required for the galactose concentration to reach 〇 by injection (usually 2.22 mmol/ 1) The linear regression of the blood concentration-time curve after 20 to 60 minutes is derived; 7 is the correction value for correcting the uneven distribution in the body according to the rule of thumb; the galactose single point method (GSp) is 30 seconds after the injection is stopped. The concentration of galactose in the blood at the minute. 10. Statistical Analysis All data are expressed as mean soil standard deviation (SD). The test results are calculated by the one-way variance analysis (ANOVA) test to determine whether there is a statistically significant difference.

The package of the Social Science program (Version 13, SPSS Inc.) is packaged with software; then multiple comparisons are made using the post hoc test of the least significant difference method to confirm significant differences between groups; The significant difference in ethnic group average was P < 0.05. 16 1360418 II. Results 1. Biochemical analysis results At the end of the experiment, the body weight and relative liver weight of the test animals were measured, and there was no significant difference compared with the control animals. The biochemical analysis results are shown in Figure 2, only the plasma of the INH group. The activity of aspartate transaminase (AST) and alanine transaminase (ALT) was significantly higher than that of the control group (the AST activity in the blood group of the control group was ii6±il iu/L; in the plasma of the INH group) The AST activity was 129±10 IU/L, pc 〇·〇5; the ALT activity in the plasma of the control group was 44 ± 6 IU/L; the ALT activity in the plasma of the INH group was 52 ± 3 IU/L, p < 〇5), showing biochemical liver damage in the INH group; the serum transaminase concentration in the control group, DSF-INH and BNPP-DSF-INH groups was normal. 2. Histopathology After three weeks of intraperitoneal injection of 150 mg/kg/day INH rats, liver toxicity was successfully produced in vivo; in contrast, the liver structure in the control group was normal, as shown in Figure 3. The liver cell line in the liver parenchyma of the control group indicated by A is arranged in the reticular plate arranged from the central vein of the hepatic lobules. The hepatic sinusoids are in the two liver plates (anastomosing plates). The tissue sections of the rats in the INH group were as shown in Fig. 3b. The hepatocytes around the central vein of the INH group showed fragmentation and vacuolization, but no hepatocyte necrosis was observed. The signs of electron microscopy showed that the crude endoplasmic reticulum (rER) in the hepatocytes of the INH group was significantly increased compared with the control group (as shown in Fig. 3C) (as shown in Fig. 3D). ). According to the literature, INH is a potent inducer of cytochrome P450 2E1 (CYP2E1), and CYP2E1 causes superoxide and hydroxyl radicals to produce 34' and induce endoplasmic The net increase of 35, so the results of this test is consistent with the previous 17 1360418 study. In other test groups, the degree of liver damage in the BNPP-INH group, the DSF-ΙΝΗ group, and the BNPP-DSF-INH group was not significantly different from that of the control group (no results were shown). 3. Measurement in the blood sample In the negative electrospray mode, the maximum molecular ion of 8-iso-PGF2a is the maximum molecular weight of ion 8-iS0-PGF2a-d4 of mass-to-charge ratio (m/z) 353. The ions are ions of mass-to-charge ratio (rn/z) 357. These negatively charged molecular ions are freed by a large number of collisions. The molecular structure of the two target compounds and the resulting ion spectra are shown in Figure 4; except for 8_is〇 The _pGF2a_d4& daughter ion is better than 8-iso-PGF2. The fragmentation patterns of 8-iso-PGF2a and 8_iso_PGF2a_d4 are similar except that the daughter ion is four units higher, which shows that most stable daughter ions are produced from the A chain, which is derived from the A chain. There are 4 deuterium atoms; the most dense daughter ions of S-iso-PGFh are ions with mass-to-charge ratio (m/z) 193, and the most dense daughter ions of 8_is〇_PGF2a_d4 are mass-to-charge ratio (m/z). 197 ions. Figure 5 shows a standard solution containing l〇〇Pg8-iso-PGF2c^250pg/ml8-iso-PGF2a-d4 and a typical LC/MS/ of a blood sample under multiplex reaction monitoring mode (MRM) detection. MS chromatography, after injecting lng8-iso-PGF2a-d4 as an internal standard, the standard solution and the blood sample were purified by the same solid phase extraction (spE) and analyzed by the aforementioned LC/MS/MS protocol. 4. Concentration of 8-iso-PGF2a in plasma

8-iso-PGF2a in blood pooling is an indicator of oxidative stress, as shown in Figure 6. Compared with the control group, the concentration of 8-iS0-PGF2 in the plasma of INH group (the concentration of ^(I) is significantly increased ( The concentration of 8-iso-PGF2a in the plasma of INH group was 151 ±26 pg/ml; the concentration of 8-iso-PGF2a in the plasma of control group was 110±15 pg/ml,;?&lt;0.001); Compared with the INH group, the BNPP-INH group, the DSF-INH 1360418 group, and the BNPP-DSF-INH group significantly reduced 8-iS0-PGF2a production in the liver caused by xnh (8-iso in the plasma of BNPP-INH group). -PGF2(^ concentration was 128±29pg/ml; the concentration of 8-iso-PGF2〇 in plasma of DSF-INH group was 126±20 pg/ml; 8-iso- in plasma of BNPP-DSF-INH group) The concentration of PGF2W was 123±17 pg/ml; the concentration of 8-iso-PGF2 in the plasma of INH group was 151±26 pg/ml, p &lt;0.005; it is worth noting that the control group and BNPP-INH group There was no significant difference in plasma 8-iS0-PGF2c^ concentration between the four groups of DSF-INH group and BNPP-DSF-INH group. Compared with BNPP-INH group and DSF-INH group, INH combined with BNPP and DSF does not further reduce the concentration of 8-丨3〇屮0?2 in plasma (1. 5. Measurement of residual liver function) As shown in Figure 7, the galactose single point method (GSP) values of the control group and the INH group were highly significant (GSP value of 384±69 pg/ml in the control group; GSP in the INH group) The value was 565 ± 87 pg/ml, p &lt; 0.001). In addition, the GSP values of the BNPP-INH group, the DSF-INH group, and the BNPP-DSF-INH group were 401 soils, 70 pg/m, 449±45. Pg/ml' 388±53 pg/m b Compared with the INH group, the GSP values of the BNPP-INH group, the DSF-INH group, and the BNPP-DSF-INH group were significantly different from those of the INH group ( The p-values were ^<O.OOljsO.Oi^and/x 0.001); the GSP values of rats administered with INH alone were significantly increased; however, rats in combination with BNPP or DSF or BNPP and DSF in INH were resistant. On the other hand, compared with the DSF-INH group, INH combined with BNPP and DSF showed reduced hepatotoxicity induced by inH' although the difference between the two did not reach a statistical difference (ρ=〇· 1), but there was no significant difference in GSP values between the control group, the sputum-sputum group, the DSF-INH group, and the BNPP-DSF-INH group.

(S 19 136 〇 418 similar results can be observed using the galactose clearance capacity (GEC) method as shown in Figure 8. Compared with the control group, the GEC value of the INH group was significantly reduced (INH group) The GEC value of the rat was 3.4±0.6 mg/min.kg; the GEC value of the control rats was 4.9±0.8 mg/min.kg, &lt; 0.001), in addition, the BNPP-INH group and the DSF-INH group The GEC values of the BNPP-DSF-ΙΝΉ group were 4.5 soil 0.6 mg/min.kg, 4.3 ± 0.4 mg/min.kg, and 4.7±0.5 mg/min.kg; compared with the INH group, BNPP-INH The GEC values of the rats in the DSF-INH group and the BNPP-DSF-INH group were significantly different from those in the INH group (the 7 values were ρ &lt; 〇·〇〇5, ρ &lt; 0.05, And/? &lt;0.005); GEC values were significantly reduced in rats administered INH alone; however, rats in either INH with either BNPP or DSF or BNPP and DSF recovered this change; compared with the DSF-INH group ' INH combined with BNPP and DSF had a tendency to increase GEC values (GEC values of DSF-INH group and BNPP-DSF-INH group were 4.3±0.4 mg/min.kg, 4.7±0.5 mg/min.kg, respectively) , /? = 0.29); In addition, the control group, BNPP-INH group, DSF-INH group, BNPP-DSF-INH There were no significant differences in GEC values between the four groups. To determine whether AST, ALT, plasma 8-iso-PGF2a concentrations, and quantitative liver function tests (eg, GSP and GEC) were related to several After correlation analysis, it was found that the gsp value was highly correlated with the concentration of 8-iso-PGF2a in blood stasis (as shown in Figure IX), and the correlation coefficient was 〇836; the GSP value was highly correlated with the GEC value (as shown in Figure 10). 2&lt; 〇〇〇 1), the correlation coefficient is _ 〇 _822; the GEC value is also highly correlated with the concentration of S-iso-PGFh (the relevant silk is _ 〇.743, ρ &lt; 0.0 (Π, 如Table 1 shows); Gsp values, GEC values, and concentrations of 8 is〇pGF2a in plasma were not significantly correlated with AST and ALT (as shown in Table 1). 20 1360418 Table 1 GSP, GEC, and 8-iso- Correlation between PGF2a and biochemical tests GSP GEC 8-iso-PGF2a AST r = 0.114 r = -0.111 r = 0.217 ALT r = 0.016 r = 0.039 r = 0.035 8-iso-PGF2a r = 0.836* r =-0.743* r = 1* with Pearson's correlation coefficient as a statistical calculation, *p&lt;0.001 Example 2 Cytochrome P450 2E1 (CYP2E1) inhibitor A screening φ, Materials and Methods 1. Test materials

In this example, a cytochrome P450 2E1 (CYP2E1) inhibitor screening kit (CYP2E1 High Throughput Inhibitor Screening Kit, BD Bioscience, USA) was used to perform cytochrome P45〇2E1 on 22 kinds of Chinese medicines and 10 kinds of excipients. Screening of CYP2E1) inhibitors; the screening set of the CYP2E1 inhibitor works by containing cytochrome P45〇2E1 (CYP2E1) and its fluorescent acceptor MFC (7-Methoxy-4-trifluoromethyl coumarin) After adding the test sample to the environment, the amount of CYP2E1 metabolite standard HFC (7-Hydroxy-4-trifluoromethyl coumarin) was detected, and the test sample was calculated based on the HFC production of the control group. Inhibition rate. Each test sample was dissolved in acetonitrile (acent〇itrile), and tested at different concentrations of the drug (66 μΜ, 33 μΜ, 16.5 μΜ) and excipients (0.167%, 〇.〇8%, 0.042%, w/v) The inhibition rate of CYP2E1, the measured results of the s-type drug and the results listed in Table 3, the tested excipients and results are listed in Table 4. In addition, the screening kit 21 1360418 (CYP2E1 High Throughput Inhibitor Screening Kit, BD Bioscience, USA) used in the cytochrome P45〇2El (CYP2E1) inhibitor of the present embodiment is as follows: (1) CYP2E1 + P450 Reductase + Cytochrome b5 ^ 100 mM potassium phosphate (pH 7.4) contains 1.3 nmol P450 and p-Nitrophenol hydrolase. (2) Control Protein: 15 mg/mL Control Protein is dissolved in 100 mM Potassium Phosphate (pH 7.4). (3) Buffer Solution : 0.5 M Potassium Phosphate (pH 7.4).

(4) Stop Solution : 0·5 M Tris Base. (5) Cofactors: containing 1.3 mM NADP+, 66 mM MgCl2 and 66 mM Glucose 6-Phosphate o (6) Glucose 6-Phosphate Dehydrogenase: 40 units/ml Dissolved in 5 mM Sodium

Citrate Buffer (pH 7.5) ° (7) MFC (7-Methoxy-4-trifluoromethyl coumarin) · Fluorescent substrate ' 50 mM MFC dissolved in acetonitrile. (8) DDTC (Diethyldithiocarbamic acid): CYP2E1 selective inhibitor (positive control group), 20 mM DDTC dissolved in acetonitrile (acentoitrile). (9) HFC (7-Hydroxy-4-trifluoromethyl coumarin): CYP2E1 metabolite standard, 0.25 mM HFC dissolved in 0.1 M Tris (pH 9.0). (10) NADPH-Cofactor Mix: Add 18.7.5 μΐ Cofactors ' 150 μΐ G6PDH (Glucose 6-Phosphate Dehydrogenase Solution) and 100 μΐ Control Protein to 14.56 ml of sterile water.

(S 22 1360418 (11) Cofactor/ acetonitrile mix: Add 66 μΐ Acetonitrile to 9.93 ml NADPH-Cofactor Mix. (12) Enzyme/Substrate Mix: Add 5.94 ml sterile water, 50μ1 HTS-706 (CYP2E1) to 4ml Buffer Soultion 2 μΜ P450 content) and 28 μΐ 50 mM MFC (7-Methoxy-4-trifluoromethyl coumarin, camping substrate) 2. Screening for cytochrome P45〇2El (CYP2El) inhibition 剤 using cytochrome P450 2E1 (CYP2E1 The CYP2E1 High Throughput Inhibitor Screening Kit (BD Bioscience, USA) was used to screen Chinese herbal medicines and excipients. The experimental procedures were as follows: (!) Preparation of control group: . on 96-well plate Add 149μ1ΝΑ〇ρΗ (:〇ί^〇ΓΜ^ and 1 μΐ 20mM DDTC to the first well and mix well; • Add 1〇〇μ1 c to the wells of the 2nd to 12th wells on the 96-well plate. 〇facW acet〇n.trik mix was the positive control group (4); the 9th and 7th wells were the control group (_!); the u and u wells were blank control group (_; The 1st to 8th axis continuous dilution operation: 5M liquid is taken into the second well from the (10) Evenly, add 5〇μι of liquid from the 2nd well of the second brother to the well of the 3rd hole, and so on. After the 8th hole is spit + open, remove the excess 5〇μΐ liquid to obtain the serial dilution concentration of 66.6. 22.2, 7.4, 2 47 λ ·47, 〇·82, 0.27, 0.091, 0.03 μΜ. 23 1360418 (2) Preparation test group: a. Add in the first and second wells of the first row on the 96-well plate 149 μΐ NADPH-Cofactor Mix' and Ιμΐ 20 mM Chinese medicine test sample or ΐμΐ 25% (w/v) excipient test sample, and mix well; b. From the first and second wells of the first row Each 50 μΐ liquid was added to the well of the third well to mix (that is, each test sample was three replicates); (3) Start and stop of the reaction: a. The above control group and the test group were placed in 3r&gt; 〇 min; b. In addition to the blank control group, add 1 〇〇 Mix to mix in other wells; c. Place all control and test groups at 37*&gt;c for 4 〇 minutes; 丄 all Add 75 μ丨stop S〇iution to the wells; e. Immediately add 1〇〇wEnzyme/SubstrateMix to the blank control group; f. The control group and the test group read the results using a fluorescent luminometer (Flu〇r〇skan Ascent FL, Thermo Eleetron Corporation, Finland). The excitation light used was 405 11111 'Emission wavelength. Analysis of 538 nm 〇(4) results: followed by the control group (c〇ntr〇〇), which is the cyp2E1 inhibition rate of the control group, after the red 峨 鲜 鲜 fresh silk cyp2E1 generation standard 赃 production amount (pmol) %, the cyp2E1 inhibition rate of each positive control group and the test group was calculated by the following formula: C YP 2E1 inhibition rate (%) = HFC production amount of the sample HFC production amount of the control group 24 1360418 II. Results ι·positive control The inhibition rate of CYP2E1 measured by the group positive control group (DDTC) is shown in Table 2. From Table 2, the inhibition rate of cyp 2Ei when the DDTC is 66.6 μΜ (that is, 0.167 %, w/v). Up to 97.55%, with 66.6 μΜ as the highest test concentration of traditional Chinese medicine, to 〇i67 〇/. (Parties) as the highest tested concentration of the excipient. __ Table 2 % control group CYP2E1 inhibition rate gpTC concentration (μΜ)-gFC production amount (pmol) CYP2E1 inhibition rate 222.00 0 256.00 202.00 8.71 151.71 31.52 126.14 43.06 55.18 75.09 21.08 90.49 15.10 93.19 5.42 97,55 0 (control group 0.03 0.091 ' 0.27 0.82 2.47 7.4 φ 22.2 66.6 2. The inhibition rate of CYP2E1 in the test group The CYP2E1 inhibition rate measured by the Chinese medicine medicine is shown in Table 3. From the results, it can be seen that the various Chinese medicines are introduced at different concentrations (66 μΜ, 33 μΜ, 16.5). Under the conditions of μΜ), the cytochrome P45〇2E1 had different inhibitory effects, and the inhibitory effect of 66-dihydrohydroguaiaretic acid was the best (97.99±0.66%).

(S 25 1360418

Table 3: CYP2E1 inhibition rate of traditional Chinese medicine cited CYP2E1 inhibition rate (%) Test concentration 66 μΜ 33 μΜ 16.5 μΜ Control group 0 0 0 Positive control group (DDTC) 97.55+1.862 Nordic hydroguaiaretic acid 97.99 ±0.66 92.36±2.20 76.52+3.86 (-)-Epigallocetechin-3 -gallate 97.56+0.18 96.47±0.64 92.56±0.46 Capillarisin 76.12+1.89 60.54±5.91 49.05±5.18 Kaempferol 70.63+ 2.53 70.04±3.75 71.87±1.14Phloretin 66.8414.79 54.69+2.84 42.04±3.63 disulfiram 66.54+2.55 60.55+5.70 57.89+3.91 Hesperetin 54.75+1.37 43.29±0.82 32.10 ±5.80 6-Gingerol (5-Gingerol) 51.89+3.33 39.83+2.32 30.13+2.67 gallic acid 48.24+4.20 42.74±7.36 35.59±10.03 Isoliquritigenin 47.83±5.36 46.27+3.28 39.08+2.75 Naricin 41.84+3.51 36.82±3.97 25.11+7.60 Dihydroquercetin ((+)-Taxifolin) 34.54+3.47 23.80+5.84 22.58±11.69 Wongonin 23.48±2.59 21.87+ 1.90 15.64+7.82 26 &lt;S ) 1360418 Chinese medicine drug induced CYP2E1 inhibition rate (〇 / n, test concentration 66 μΜ 33 μΜ 16.5 μΜ Protocatechuic acid 22.75±4.07 19.95+8.95 25.66±12.74 catechin ((+)-Catechin 16.45+9.67 33.83±8.76 41.53±7.62 β-naphthoflavone 15.40+12.94 16.8310.96 6.52+6.64 Embelin 13.54+11.64 ------ 12.30+10.24 5.95+7.48 Anti Trans-Cinnamic acid 7.10±6.95 4.66±6.50 5.71+10.53 Epicatechin ((-)-Epicatechin) 2.57±11.60 15.02±5.50 18.27±9.34 Phloridzin 1.42±9.28 3.76±3.58 1.25±7.90 Puerarin -12.86±2.75 -4.64±3.47 0·43±2.31 Umbelliferone -1081.56 soil 168.00 -571.97+117.56 -280.41+19.48 CYP2E1 inhibition measured by excipient The rate is shown in Table 4. From the results, it can be seen that the various excipients are cytochrome at different concentrations (0.167%, 0.08%, 0.042%, \\^). 45〇2 ugly 1 has different degrees of inhibitory effect, among which 0.167% Brij 58 has the best inhibitory effect (97.75±0.66%)» &lt; S ) 27 1360418 Table 4 excipient CYP2E1 inhibition rate Chinese medicine CYP2E1 inhibition Rate test concentration (w/v) 0.167% 0.08% 0.042% Control group 0 Positive control group (DDTC) 97.55±1.862 Brij 58 97.75±0.66 96.58±0.40 96.02±0.17 Brij 76 97.56±1.02 96.87±l.〇〇94.76± 0.47 Brij 35 93.3310.82 89.45±0.68 76.21+7.37 (test concentration 0.025%) (test concentration 0.013%) (test concentration 0.006%) Tween 20 87.20±1.29 82.80±1.71 71.77±4.48 Tween 80 73.92±4.71 65.45±2.50 64.02 +12.54 Tween 40 58.97±3.29 47.05+6.48 44.79+2.49 PEG 2000 44.33±2.75 40.13±3.06 35.81±3.26 PEG 400 42.33+5.25 39.10±0.73 31.98±5.97 Pluomic F68 41.72i5.34 42.98±3.24 37.11+10.35 PEG 4000 37.21 + 1.91 41.22±0.97 37.18±10.52 The low side-effect of the present invention provides a new compound, the biochemical analysis of the new compound, when compared with the test results of isoniazidamine (INH) alone. (MT, AST value), pathology Analyze, measure the residual liver function (Gsp value, GEC value), and the index of oxidative stress (concentration of 8-iso-PGF2a in blood), etc. (INH) The efficacy of hepatotoxic side effects. The detailed description above is a detailed description of one of the possible embodiments of the present invention, but the embodiment is not intended to limit the scope of the present invention, and equivalent implementations or modifications of the present invention (4), such as: Concentrations and ratios of alkali guanamine, cytochrome p45〇2E1 inhibitor, disulfiram 28 丄·418 (DSF), nitrophenol phosphatidyl phosphate (ΒΝΡΡ), and the type of cytochrome Ρ450 2Ε1 inhibitor Examples of variations of equivalence should be included in the scope of the patent in this case. * « In summary, this case is not only innovative in the application of disulfiram (DSF), but also can effectively reduce the side effects of hepatotoxicity caused by the use of heterologous amines (face), which should be fully consistent with novelty. And progress 'steep legal patent requirements for the invention, love to apply in accordance with the law, I ask you to approve the invention patent ^ application, in order to invent invention, to the sense of convenience" [simplified description of the diagram] Figure 1 is isoniazid guanamine ( INH) map of metabolic pathways in the liver;

Figure 1 shows the control group, the INH group, the group, the group, and the BNpp DsF I1^H 'group. The activity of transaminase (N) and alanine transaminase (ALT) was analyzed in Tianmen, and the value was calculated as mean soil. SD, * indicates that each test group is compared with the control group and the household is &lt; 〇〇 5; Figure 3 is the control group (Fig. 3A and C) and the INH group (Fig. 3B and D) rat liver section: Fig. 3 A 'control group relative to normal liver tissue type (HE staining, 4〇〇χ); Figure 3B, surface [group of hepatocytes in the surrounding central vein (v) showed fragmentation and vacuolization (ΗΕ staining, 4 〇〇χ); Figure 3 c, the liver slices of the control group were observed by electron microscopy, Nu: nucleus (9, _ χ); Figure 3 d, with electronic display 'microscopic examination of the leg group big eye mail' she in Figure 3 Hepatic cell sections of the C Lai group, crude endoplasmic reticulum ER) of the hepatocytes of the sputum group _ increased, Nu: nucleus (9, _ χ); Figure 4 is 8-is〇-PGF2 «_d4 (Α) and 8 7 The molecular structure of the unknown (Β) and the spectrum of the product ions; Figure 5 shows the internal standard solution containing 250 pg 8-is〇-PGF2a_d4 (Α) containing 1〇〇4·(S) 29 1360418 8-iso-PGF2tt (B) Standard solution and blank sample (〇, in multiple Liquid Chromatography Tandem Mass Spectrometry (LC/MS/MS) with MMP detection, mass-to-charge ratio (m/z) 357/197 and mass-to-charge ratio (〇1^) 353/193 The ion couples (丨〇111) are also used to monitor 8 〇 _1 &gt; (3172 (1_(14 (A) (as internal standard) and 8-iso-PGF2a (8) (as standard); Bee 1: blank blood; peak 2: blank plasma injected into the standard; Figure 6 is the 8-iso- in the plasma of the control group, INH group, BNPP-INH group, DSF-INH group and BNPP-DSF-INH group. The concentration of PGF2a was calculated as mean soil SD, * indicates that the test group was compared with the control group /&gt;&lt;〇_〇〇1;# indicates that each test group was compared with the XNH group, p &lt; 0 05; Figure 7 shows the galactose single point method (GSP) values of the control group, INH group, BNPP-INH group, DSF-INH group and BNPP-DSF-INH group. The value is calculated as mean soil SD, * indicates the test group and The control group was compared with the households &lt;〇·〇〇1;# indicates that each test group was compared with the INH group/&gt;&lt;〇.〇〇1;& indicates that each test group was compared with the INH group&lt;〇.〇〇5; Figure VIII is the control group, INH group, BNPP-INH group, DSF-INH And the galactose clearance ability (GEC) value of the rats in the BNPP-DSF-INH group, the value is calculated as mean ± SD, * indicates that the test group is compared with the control group and the household is &lt;0.001;# indicates that each test group is compared with the INH group After P &lt;0.005;&quot;&quot; indicates that each test group was compared with the INH group (0.05); Figure 9 is the control group, INH group, BNPP-INH group, DSF-INH group and BNPP-DSF-INH The galactose single point method (GSP) values of each group were highly correlated with the concentration of 8-iso-PGF2a in plasma, and

Figure 10 is a high correlation between the galactose single point method (GSP) values and the galactose clearance capacity (GEC) values in the control group, INH group, BNPP-INH group, DSF-INH group, and BNPP-DSF-INH 30 1360418 group. The chart. [Explanation of main component symbols] None [References] 1. Kopanoff DE et al., Isoniazid-related hepatitis: a U.S. Public Health Service cooperative surveillance study., 1978. Am. RevRespirDis 117:991-1001.

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Claims (1)

1360418 X. The scope of application for patents: 1. A new compound of isoniazid (INH) with low side effects, including a pharmaceutically effective amount of isoniazid (INH), combined with a pharmaceutically effective amount of cells a pigment P450 2E1 (CYP2E1) inhibitor; wherein the cytochrome P450 2E1 (CYP2E1) inhibitor is selected from the group consisting of: Nordic hydroguaiaretic acid, (-)-Epigallocetechin-3-gallate , Capillarisin, Kaempferol, Phloetin, Hesperetin, 6-Gingero1, gallic acid, different Isoliquritigenin, Nariginin, dihydroquercetin ((+)_Taxif〇Un), scutellarin), protocatechuic acid, catechin (( +)_catechin), β-naphthoflavone, Embelin, trans-Cinnamic acid, epiphylaxis ((-)-Epicatechin), phloridzin (Phl 〇ridzin), Brij 58, Brij 76, Brij 35, Tween 20 'Tween 80, Tween 40, PEG 2000, PEG 400 , Pluomic F68, PEG 4000. 2. If the claim has been patented, the low side effects described in item i are different from the test, although a new compound, in which a pharmaceutically acceptable excipient can be added to the compound. 3. If _«^ specializes in the second phase of the Wei-gj, the secret of the remedy (four) Xincheng 匪), the excipient can be a diluent, a filler, a binder, a disintegrating agent, Lubricants, etc.