TW201707694A - Compound for removing hepatotoxicity of acetaminophen (APAP) drug to liver especially using cytochrome P4502E1(CYP2E1) enzyme inhibitor to reduce side effect of toxicity of acetaminophen (APAP) to liver - Google Patents

Abstract

The invention provides a compound for the removal of hepatotoxicity of acetaminophen (APAP) drug, which is selected from the following groups: Tween 20, microcrystalline cellulose, dicalcium phosphate dihydrate, polyoxyethylene 23 lauryl ether, saccharin, mannitol, polyoxyethylene alkyl ether, sucralose, pyrrolidone, sodium starch glycolate, acrylic resin S100, carboxymethyl cellulose, polyoxyethylene polyoxypropylene, menthol, low-substituted hydroxypropyl cellulose, pregelatinized starch, Dextrates NF hydrated, citric acid, polyoxyethylene castor oil, colloidal silica, polyethylene glycol monostearate aliphatic ester, sorbic acid, lemon oil, hydroxypropyl cellulose, sorbitol, acesulfame potassium, hydroxypropyl methylcellulose, lactose monohydrate, maltodextrin, Brij 58, Brij 76, Tween 80, Tween 40, PEG 400, PEG 4000, PEG 2000, and the like, so as to reduce the side effect of the toxicity caused by acetaminophen to the liver.

Description

Use of a compound for removing hepatic toxicity of acetaminophen (APAP) drug

The present invention relates to a use of a compound for removing hepatic toxicity of Acetaminophen (APAP) drug, in particular, using a cytochrome P450 2E1 (CYP2E1) enzyme inhibitor to reduce hepatotoxicity to acetaminophen And other side effects.

Acetaminophen, also known as paraceamol or N-acetyl-para-aminophenol, referred to as APAP, is the most commonly used antipyretic analgesic in the market. There are often many improper use of APAP every year. Poisoning or cases of suicide occur. Liver damage caused by APAP is the most important cause of severe illness and death. There are many clinical experiences that can prove that hepatotoxicity of APAP can be prevented. Early diagnosis and immediate administration of antidote N-acetylcysteine referred to as NAC can effectively prevent hepatotoxicity.

Early detection of an overdose of acetaminophen is necessary because the best prognostic effect can be achieved by administering an antidote within 8 hours after the poisoning. Early signs of drug poisoning include physical discomfort, nausea, and vomiting, but some patients have a blood concentration of acetaminophen that is toxic and has abnormal liver function, and is initially asymptomatic (first phase), hepatotoxic. Signs such as abdominal pain, persistent vomiting, jaundice, pain in the right upper quadrant, etc., become more obvious 24 to 48 hours after a large intake (second phase). Serum transaminase usually begins to rise 16 hours after taking it, with clinical symptoms beginning to appear. The third phase begins about 3-4 days after taking it, and the extent and prognosis of liver damage can be estimated. Symptoms of hepatotoxicity can range from mild symptoms with increased liver function (AST > 1,000 IU / L) to severe violent hepatitis with metabolic acidosis, jaundice, hypoglycemia, AST > 10,000 IU / L, abnormal blood clotting And liver and brain lesions. The fourth phase can cause oliguric renal failure, which can lead to death.

Some patients with acetaminophen poisoning have very mild liver damage, but have severe nephrotoxicity, mainly due to the metabolism of cytochrome P450s (CYPs) directly on the renal tubules caused by APAP. toxicity. However, acute renal failure may also be caused by acute liver failure. In this case, the fractional excretion of Na (FeNa) can be used to distinguish primary kidney injury (FeNa>1) or liver and kidney. Syndrome (FeNa <1). Formula for calculating FeNa: (Sodiumurinary ÷ Creatinineurinary) ÷ (Sodiumplasma ÷ Creatinineplasma) × 100.

The acetaminophen can reach peak blood concentration 1 to 2 hours after oral administration, most of which is metabolized from the liver, and more than 90% is combined with glucuronide and sulfate to form non-toxic metabolites. Only less than 5% are metabolized via different CYPs, including: CYP2E1, CYP1A2, and CYP3A4, of which CYP2E1 and CYP1A2 are major metabolic enzymes. The metabolite N-acetyl-p-benzoquinoneimine produced by the metabolism of these enzymes, abbreviated as NAPQI (Fig. 1), is a highly active electrophile. Under normal conditions, NAPQI reacts immediately with intracellular glutathione to form a non-toxic mercaptide. When the excess of acetaminophen is used, the rate of glutathione consumption in the cell is greater than the rate of synthesis. When the intracellular glutathione content is lower than 30% of normal, NAPQI binds to the macromolecule or nucleotide containing cysteine in the cell. , causing liver cell damage. Staining from cell tissue can demonstrate that NAPQI forms a covalent bond with the thiol group of cysteine in the central region of the hepatic lobules prior to hepatocyte necrosis.

For patients with liver disease, alcohol abuse, or taking drugs that induce liver cytochrome P450 enzyme activity, such as: Carbamazepine, Ethanol, Isoniazid, Phenobarbital (possibly other barbiturates), Phenytoin, Sulfinpyrazone, Sulfonylureas, Rifampin, Primidone, etc. A high risk group that causes severe liver toxicity of APAP. At this time, if the patient has complications such as adult respiratory distress syndrome, cerebral edema, uncontrolled bleeding, infection, or multiple tube failure, it is easy to cause death. Taking alcohol as an example, alcohol is mainly metabolized by CYP2E1 in the liver. The mechanism of induced APAP poisoning can be divided into three stages. The first stage is the receptor for alcohol and APAP competing for liver CYP2E1. At this time, the concentration of NAPQI will decrease first. The second stage is that alcohol will increase the half-life of CYP2E1 from 7 hours to 37 hours, which will increase the content of CYP2E1 in the liver, and the concentration of NAPQI will slowly rise. The third stage is when the alcohol is absent, the liver has more CYP2E1 metabolizes acetaminophen, resulting in a significant increase in toxic metabolites of acetaminophen, causing liver cell damage. In recent years, studies have confirmed that the use of diallyl sulfide can effectively prevent the hepatotoxic damage caused by acetaminophen in mice, and further discover that diallyl sulfide inhibits the activity of CYP2E1 enzyme, and infers that diallyl sulfide prevents acetaminophen. The protective mechanism of hepatotoxicity caused by inhibition of the production of the active intermediate NAPQI by acetaminophen.

Invasive and non-invasive methods are used to test liver function in rats to monitor the development of liver damage and to screen for liver disease. The most commonly used method involves measuring aspartate aminotransferase (AST) in serum, Alanine aminotransferase (ALT) and alkaline phosphatase values, as well as measurements of hepatocyte products such as bilirubin, albumin, and protonectin Prothrombin time to detect coagulation factors, etc.; liver function quantitative test is based on the concentration of serum in the liver that is almost exclusively metabolized by the liver. The clearance of these receptors is based on hepatic portal vein and hepatic artery blood flow. And by the effect of hepatocytes on these receptors, the blood flow of the liver is related to the quality of the liver, and vice versa, the clearance of the receptor is determined by the ability of the liver to metabolize.

Galactose is a sugar with a high extraction ratio and 90% metabolism in the liver. In the liver, galactose is subjected to differential stereoisomerization by galactokinase (epimerization). ), which is converted to Glucose-1-phosphate; the action of galactose kinase is a rate-limiting step of the galactose metabolic pathway in hepatocytes. The high extraction rate of galactose makes galactose metabolism, which depends on liver blood flow and liver function, the most important way to detect liver function. There is no rule to evaluate the residual liver function of rats. The metabolic ability of an exact compound (eg, galactose) can be presumed to be a rate-determining step in the liver, and it is also possible to obtain a representative quality of residual liver function.

The inventors tested chronic hepatitis, cirrhosis and liver cancer patients by galactose single-point method. The results showed that the galactose single-point method can accurately detect these liver diseases; the galactose single-point method has been successfully applied to test patients with liver disease. Residual liver function of drugs such as promazine and antibiotic cefoperazone. In addition, the galactose single-point method has become one of the recommended methods for testing liver function in the guidelines published by the US Food and Drug Administration (FDA).

It can be seen that the above-mentioned conventional use of acetaminophen still has many defects, which is not a good designer, but needs to be improved.

In view of the above-mentioned shortcomings of the above-mentioned conventional side effects such as hepatotoxicity caused by acetaminophen, the inventors of the present invention have improved and innovated, and after years of research, finally successfully developed a compound for removing acetaminophen ( Acetaminophen, APAP) The use of drugs for hepatotoxicity.

It is an object of the present invention to provide a compound for use in removing hepatotoxicity against a drug of Acetaminophen (APAP).

To achieve the foregoing object, the compound is selected from at least one of the following groups or any combination thereof: polyoxyethylene (20) sorbitan monolaurate (Tween 20), microcrystalline cellulose (Microcrystalline cellulose), Dicalcium phosphate dihydrate, Polyoxyethylene 23 lauryl ether (Brij 35), Saccharin, Mannitol, Cremophor RH40, III Sucralose, Crospovidone, Sodium starch glycolate, Acrylic S100 (Eudragit S100), Croscarmellose sodium, Polyoxyethylene polyoxypropylene (Pluronic F68) ), Menthol, Low-substituted hydroxypropyl cellulos, Pregelatinized starch, Dextrates NF hydrated, Citric acid, Castor oil polyoxyethylene ether (Cremophor) EL), colloidal cerium oxide (Aerosil 200), polyethylene glycol monostearate (Myrj 52), sorbic acid (Sorbic acid), lemon oil (Lemon oil), hydroxypropyl fiber Hydroxypropyl cellulose, sorbitol, Acesulfame potassium, Hydroxypropyl methylcellulose, Lactose monohydrate, Maltodextrin ), Brij 58, Brij 76, Tween 80, Tween 40, PEG 400, PEG 4000, PEG 8000, Span 60, Sodium benzoate, Hydroxyethylmethylcellulose, Cellulose Methylcellulose, Span 80, Sodium cyclamate, Glyceryl behenate, Oxide red, Glycerin monostearate, Copovidone K28, Starch acetate Starch acetate, Magnesium stearate, Sodium lauryl sulfate, Provideone K30, and PEG 2000; all of the above compounds are enzyme P450 inhibitors.

To achieve the foregoing object, the compound composition is selected from at least one of the following groups or any combination thereof, and defines an effective dosage of the composition: polyoxyethylene (20) sorbitan monolaurate (Polyethylene glycol) Sorbitan monolaurate; Tween 20) content of 0.001 ~ 55 grams, microcrystalline cellulose (Microcrystalline cellulose) content of 0.001 ~ 55 grams, Dicalcium phosphate dihydrate (Dicalcium phosphate dihydrate) content of 0.001 ~ 55 grams, polyoxyethylene 23 lauryl ether (Brij 35) content of 0.001 ~ 55 grams, saccharin (Saccharin) content of 0.001 ~ 55 grams, Mannitol (Mannitol) content of 0.001 ~ 55 grams, polyoxyethylene alkyl ether (Cremophor RH40 The content is 0.001~55g, the content of Sucralose is 0.001~55g, the content of Crospovidone is 0.001~55g, and the content of sodium starch glycolate is 0.001~55克, acrylic resin S100 (Eudragit S100) content of 0.001 ~ 55 grams, sodium carboxymethyl cellulose (Croscarmellose sodium) content of 0.001 ~ 55 grams, polyoxyethylene polyoxypropylene (Pl Uronic F68) 0.001-55 g, menthol (Menthol) 0.001-55 g, low-substituted hydroxypropyl cellulos, 0.001-55 g, pregelatinized starch Pregelatinized starch) is 0.001~55g, Dextrates NF hydrated is 0.001~55g, Citric acid is 0.001~55g, and castor oil Cremophor EL is 0.001. ~55 grams, colloidal cerium oxide (Aerosil 200) content of 0.001 ~ 55 grams, polyethylene glycol monostearate (Myrj 52) of which 0.001 ~ 55 grams, sorbic acid (Sorbic acid) It is 0.001~55g, lemon oil is 0.001~55g, hydroxypropylcellulose is 0.001~55g, and sorbitol is 0.001~55g. The content of Acesulfame potassium is 0.001~55g, the content of Hydroxypropylmethylcellulose is 0.001~55g, and the content of Lactose monohydrate is 0.001~ 55 grams, maltodextrin (Ma Ltodextrin) is 0.001~55g, Brij 58 is 0.001~55g, Brij 76 is 0.001~55g, Tween 80 is 0.001~55g, Tween 40 is 0.001~55g, PEG 400 is 0.001~55g, PEG 4000 is 0.001~55g, PEG 8000 is 0.001~55g, Span 60 is 0.001~55g, Sodium benzoate is 0.001. ~55 mg, Hydroxyethylmethylcellulose, 0.001-55 g, Methylcellulose, 0.001-55 g, Span 80, 0.001-55 g Sodium cyclamate is 0.001~55g, Glyceryl behenate is 0.001~55g, Oxide red is 0.001~55g, glycerol is hard. The content of Glycerin monostearate is 0.001~55g, the content of Copovidone K28 is 0.001~55g, the content of Starch acetate is 0.001~55g, and the content of Magnesium stearate. For 0.001~55 grams, laurel Sulfate (Sodium lauryl sulfate) wherein 0.001 to 55 g, Providone K30 wherein 0.001 to 55 g, wherein the content of PEG 2000 and 0.001 to 55 g.

To achieve the aforementioned object, the compound is selected from Mannitol and is preferably combined with Sucalose.

To achieve the aforementioned object of the invention, the compound is used separately, simultaneously or sequentially with the p-acetamidophenol.

In order to achieve the aforementioned object, the compound is a gel, a spray, a pasty, a lozenge, a dispersible tablet, a capsule, a soft capsule, a pill, a suspension, a microsphere, an oral implant, an emulsified injection or Implants and pharmaceutically acceptable forms are administered.

To achieve the aforementioned object of the invention, the compound is included in a pharmaceutical pack, kit or patient pack.

In view of the above-mentioned shortcomings of the above-mentioned conventional side effects such as hepatotoxicity caused by acetaminophen, the inventors of the present invention have improved and innovated, and after years of painstaking research, finally succeeded in research and development of a compound for removing acetaminophen. The use of phenol (Acetaminophen, APAP) drug for hepatotoxicity.

In order to achieve the above-mentioned object of the invention, the new compound of acetaminophen has no hepatotoxic side effects, and the present invention firstly studies the hepatotoxicity induced by the new compound of acetaminophen in rats, and studies the combination of CYP2E1 using one or any combination thereof. The effect of inhibitors on hepatotoxicity induced by acetaminophen in rats; except for the use of general hepatotoxicity markers and pathological tissue sections, the quantitative amount of residual liver function in rats was measured by galactose single point method (GSP). Test, further evaluation.

All of the technical and scientific terms described in this specification, unless otherwise defined, are intended to be common to those of ordinary skill in the art.

As used herein, the term "combination", when applied to an amount of two or more compounds and/or agents (also referred to herein as ingredients), is intended to define A compound or a combination of compounds/agents. The terms "combined" and "combining" are used herein to describe the terms.

Pharmaceutical kits, pharmaceutical packs, or patient packs in which two or more compounds/agents are co-packaged or co-presented (eg, as a unit batch) Part).

The present invention is exemplified by the following examples, but the present invention is not limited by the following examples. The drugs and biological materials used in the present invention are commercially available and are readily available. The following are only examples of available pipelines.

The present invention is exemplified by the following examples, but the present invention is not limited by the following examples.

Embodiment 1

A combination of acetaminophen in a safe or pharmaceutically acceptable excipient of one or any combination thereof for improving drug hepatotoxicity in an animal test.

Materials and Methods

Test material

All organic solvents were HPLC grade, purchased from Tedia Co., Ltd. (Fairfield, OH, USA), APAP was purchased from Sigma Chemical Company (St. Louis, MO USA), and galactose injection solution was prepared by Nanguang Chemical Pharmaceutical Co., Ltd. 400 g of galactose (Sigma) was dissolved in 1 liter of distilled water containing a suitable buffer solution system and isotonic salts for injection.

2. Test animals

Male SD (Sprague-Dawley) rats weighing 175-280 g were purchased from the National Laboratory Animal Center (Taiwan). Animal experiments were carried out in accordance with the guidelines of the National Animal Research Institute. All rats were placed in an air/humidity environment. Under the light and dark for 12 hours, the supply of water and feed is not limited. During the test period, the body weight of the rats is continuously monitored, and the water is supplied as usual.

3. Test treatment

The CYP2E1 effective inhibitor was selected for in vitro screening, and the APAP was not combined or combined with an inhibitor to improve the animal toxicity induced by APAP. Hepatotoxicity test animals were administered a single dose of 1000 mg of APAP per kilogram of body weight of the rat to produce hepatotoxicity. Liver protection experimental animals were administered 1.67 mg of dicalcium phosphate per kilogram of body weight of the rat; or 1.67 mg of mannitol per kg of body weight tube; or 1.67 mg of menthol per kg of body weight tube; or per kg of body weight tube Feed 1.67 mg of HUEXC041; or 1.67 mg of mannitol and 1.67 mg of sucralose per kg of body weight tube; or 0.83 mg of mannitol and 0.83 mg of sucralose per kg of body weight tube; or nectar per kg of body weight tube 0.42 mg of alcohol and 0.42 mg of sucralose; or 0.17 mg of mannitol and 0.17 mg of sucralose per kg of body weight tube, and a single dose of 1000 mg of APAP per kg of body weight tube. To measure hepatic inflammation by measuring aspartate aminotransferase (AST) and alanine transaminase (ALT) in plasma; measuring galactose single point method (GSP) before administration and 16 hours after administration Analyze the residual liver function of rats; analyze the pathological changes of liver tissue in each group of rats and evaluate the mechanism of liver damage or liver protection.

4. Blood sample

After the treatment, the rats were sacrificed by ether anesthesia, the blood was taken from the rat tail artery, placed in a test tube containing EDTA, and the plasma was centrifuged at 13,000 g for 15 minutes at 4 ° C. The separated plasma was dispensed into trace amounts. Store in a small tube (Eppendorf tube) at -80 ̊C.

5. Biochemical analysis

Hepatocyte injury to measure aspartate aminotransferase (AST) and alanine transaminase (ALT) activity in plasma for quantification, AST and ALT activity are commonly used indicators of liver toxicity, with Synchron LXi 725 system To measure (Beckman Instruments, USA).

6. Optical microscope

The rat liver was sacrificed immediately after histological analysis; the liver samples were fixed with phosphate-buffered formalin in 10% phosphate buffer, then dehydrated and embedded in paraffin, sliced at 5 μm thickness, sliced The samples were stained with hematoxylin and eosin, and subjected to a glycine staining test (PAS). After staining, histological observation was performed by light microscopy.

7. Quantitative testing of liver function

At the end of the experiment, all rats were tested for galactose single point (GSP). Rats received a rapid intravenous injection within 30 seconds, and injected 0.4 g/ml BW galactose solution 0.5 g/kg; Blood was collected at 10, 15, 30, 45, and 60 minutes, and blood samples were taken from the tail vein; galactose content was measured by colorimetric galactose dehydrogenase at a concentration range of 50 to 1,000 μg/ Ml, the within-day variation of each concentration is calculated from the standard deviation and the coefficient of variation (CV) percentage. The maximum allowable coefficient of variation is 10% CV; daytime difference (day- To-day variation is tested by comparing the slope and intercept of the calibration curves; the galactose single point method (GSP) is the concentration of galactose in the blood at 60 minutes after the 30 second injection is stopped.

8. Statistical analysis

All data are expressed as mean ± standard deviation (SD), and the test results are calculated by the single factor analysis of variance (ANOVA) test to determine whether there is a statistically significant difference. Using the Statistical Package of the Social Science program (Version 13, SPSS) Inc.) The software was used to calculate; then multiple comparisons were made using the post hoc test for the least significant difference method to confirm significant differences between ethnic groups; the significant difference in population mean was p < 0.05.

result

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 normal control animals. The results of blood biochemical analysis are shown in Table 1, except for the aspartic acid in the plasma of the APAP hepatotoxic group. The activities of transaminase (AST) and alanine transaminase (ALT) were significantly higher than those of the normal control group (the AST activity in the plasma of the normal control group was 202±34 IU/L; the AST activity in the plasma of the APAP hepatotoxic group was 499±112 IU/L, p <0.005; ALT activity in plasma of normal control group was 56±14 IU/L; plasma ALT activity in APAP hepatotoxic group was 368±71 IU/L, p < 0.005, indicating The APAP hepatotoxic group produced biochemical liver damage; except for the dicalcium phosphate test group, which was not expected, this liver injury phenomenon can be improved by using common safety excipients such as mannitol, menthol, and sucralose. The measured indicators of liver inflammation, AST, ALT, GSP and Total HAI-score, which represent the severity of liver lesions on pathological sections, were significantly decreased. The experimental data are shown in Table 1, in which mannitol was combined with sucralose. Best, no different from the normal control group.

Table 1, normal control group, APAP hepatotoxicity group and liver protection test group mice such as dicalcium phosphate, mannitol, menthol and sucralose, galactose single point method (GSP) after tube feeding single dose test Analysis, aspartate transaminase (AST), alanine transaminase (ALT) activity and pathological tissue section Total HAI-score, the value is calculated as mean ± SD

2. Histopathology

The improved results were also reflected in the corresponding liver tissue. In rats fed a single oral dose of 1000 mg/kg APAP, hepatotoxicity was successfully produced in vivo, and liver tissue sections of rats in the APAP hepatotoxic group were surrounded by central veins. Hepatocytes showed fragmentation and vacuolization, and there were few nuclei and even necrosis symptoms in some hepatocytes. Compared with the normal control group, hepatocyte damage was severe (as shown in Figure 2B); In the normal control group, the liver structure of the rats was normal, and the liver cells were intact and neatly arranged without cavitation (as shown in Figure 2A). The liver protection test group such as mannitol, menthol and sucralose The liver tissue sections of the rats showed that the liver cells were intact, the nuclei were obvious and the vacuoles were less (as shown in Fig. 2, C, D, F, G, and H), showing liver protection such as mannitol, menthol and sucralose. The liver tissue of the experimental group was closer to the liver tissue of normal rats. The combination of mannitol and sucralose had the best protective effect and was positively correlated with the dose. The higher the dose, the better the protective effect.

3. Measurement of residual liver function

As shown in Table 1, the galactose single-point (GSP) value of the normal control group and the APAP hepatotoxicity group was highly significant (GSP value of 289±38 mg/L in the normal control group; APAP liver) The GSP value of the rats in the toxic group was 848±123 mg/L, p < 0.005. In addition, the GSP values of the rats in the liver protection test group such as dicalcium phosphate, mannitol, menthol and sucralose were 444±60. Mg/L, 253±29 mg/L, 289±20 mg/L, 218±31 mg/L, compared with APAP hepatotoxicity group, liver protection test group rats such as mannitol, menthol and sucralose GSP values were highly different from those in the APAP hepatotoxic group ( p <0.005); GSP values in rats with hepatotoxicity were significantly increased by administration of APAP alone; however, mannitol, menthol and sucralose were administered in combination with APAP. Rats in the liver protection test group of the same excipients were resistant to this change.

Embodiment 2

Screening of cytochrome P450 2E1 (CYP2E1) inhibitors - murine liver microsomes and human liver microsomes.

Materials and Methods

Test material

In this example, rat and human liver preparation microsomes were used for in vitro screening of CYP2E1 inhibitors, and 55 common and safe edible excipients were screened for cytochrome P450 2E1 (CYP2E1) inhibitors. A potent inhibitor of CYP2E1 in mouse or human liver; the screening principle of the CYP2E1 inhibitor is: CYP2E1 in the microsomes prepared from the liver of the species is reacted with its specific receptor Chlorzoxazone (CZX), and the test sample is added to detect CYP2E1 metabolism. The amount of the standard 6-OH-CZX (6-Hydroxy-Chlorzoxazone) was calculated, and the CYP 2E1 inhibition rate of the test sample was calculated based on the amount of 6-OH-CZX produced by the control.

Each test sample was dissolved in 10% methanol or secondary water and tested for inhibition of CYP2E1 by various concentrations of excipients (66 uM, 33 uM, 16.5 uM; 0.167%, 0.08%, 0.042%, w/v). Rate, the results of the excipients tested are listed in Table 2.

The reagents required for screening cytochrome CYP2E1 inhibitors using rat liver or human liver microsomes in this example are as follows: (1) CYP2E1: 100 mM potassium phosphate (pH 7.4) contains 10 mg/mL P450 protein concentration. (2) Control Protein: 10 mg/mL P450 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: ice-acetonitrile. (5) Cofactors: Contains 100 mM NADP+ and 10 mM Glucose 6-Phosphate. (6) Glucose 6-Phosphate Dehydrogenase: 2000 units/ml dissolved in sterile water. (7) Chlorzoxazone: Substrate, 16 mM Chlorzoxazone dissolved in 10% methanol. (8) DDTC (Diethyldithiocarbamic acid): CYP2E1 selective inhibitor (positive control), 20 mM DDTC dissolved in 10% methanol (methanol). (9) NADPH-regenerating System: 530 uL Cofactors, 40 uL G6PDH (Glucose 6-Phosphate Dehydrogenase Solution) and 100 uL Control Protein were added to 3.42 mL.

Screening of cytochrome P450 2E1 (CYP2E1) inhibitors

The experimental procedure for screening for cytochrome CYP2E1 inhibitors using rat liver or human liver microsomes is as follows: (1) 0.1 M phosphate buffer (pH 7.4) containing 0.5 mg/mL mouse in a 4 ^o C ice bath environment Liver or human liver microsomes, 5 mM MgCl _{2 were} allowed to stand for 15 minutes (2) At this time, the experimental group was added with cytochrome P450 2E1 reaction matrix drug 16 mM Chlorzoxazone and the compound to be screened; the control group was replaced by methanol: sterile water = 1:1. The positive control group was replaced by DDTC; (3) Coenzyme 1 mM NADP+, 10 mM G6P and 2 IU G6PD were added. The reaction solution was transferred to a 37 ° C water bath for pre-incubation for 1 minute, and the reaction time of the activity test was 30 minutes. (4) After the reaction, the reaction was terminated with 500 μL of acetonitrile, and the sample was allowed to stand for 1 minute and then added to the internal standard. (5 ug/mL 4-hydroxy-tobutamide), after centrifugation, take 20 uL of the supernatant, dilute with methanol: sterile water for ten times, and take 5 uL of the solution back into the LC/MS/MS system for analysis. (5) Analysis of results: After converting the signal value measured by LC/MS/MS into the amount of 6-Hydroxy-Chlorzoxazone (pmol) of the CYP2E1 metabolite standard, the control group was based on the CYP 2E1 inhibition rate of 0%. The CYP 2E1 inhibition rate of each positive control group and test group was calculated by the following formula:

result

Positive control group

The inhibition rate of CYP 2E1 measured by the positive control group (DDTC) is shown in Table 2. It can be seen from the table that when the concentration of DDTC is 100 μM, the inhibition rate of CYP 2E1 can reach 89.2%.

2. Test group CYP 2E1 inhibition rate

The inhibitory rate of CYP 2E1 measured by the excipient in rat liver microsomes is shown in Table 2. From the results, it can be seen that each excipient is at different concentrations (66 μM, 33 μM, 16.5 μM; 0.167%, 0.08%, 0.042%, w Under the conditions of /v), the cytochrome P450 2E1 had different degrees of inhibitory effects, among which 0.167% Brij 58 had the best inhibitory effect (100.0±0.00%).

Table 2. Inhibition rate of CYP 2E1 inhibitors in vitro by vehicle excipients in rat liver microsomes

The inhibition rate of CYP 2E1 measured by human excretion agent in human liver microsomes is shown in Table 3. From the results, it can be seen that each excipient is at different concentrations (66 μM, 33 μM, 16.5 μM; 0.167%, 0.08%, 0.042%, w Under the conditions of /v), the cytochrome P450 2E1 had different degrees of inhibitory effects, among which 0.167% Brij 58 had the best inhibitory effect (91.2±1.3%).

Table 3. Inhibition rate of CYP 2E1 inhibitors in vitro by human excipients in human liver microsomes

A new combination of noxious side effects to improve the hepatotoxicity of Acetaminophen (APAP) drug, the effective dosage range of each excipient at different concentrations (66 μM, 33 μM, 16.5 μM) It is: Polyethylene glycol sorbitan monolaurate (Tween 20), the content of which is 0.17~5.5g, Microcrystalline cellulose, the content of which is 100~1000mg, hydrogen phosphate II Dicalcium phosphate dihydrate is 10~250mg, polyoxyethylene 23 lauryl ether (Brij 35) is 100~1000mg, Saccharin is 10~40mg, Mannitol The content is 10~250mg, the content of Cremophor RH40 is 0.17~5.5g, the content of Sucralose is 10~250mg, and the content of Crospovidone is 0.17~5.5. Sodium starch glycolate (Sodium starch glycolate) content of 0.17 ~ 5.5 grams, acrylic resin S100 (Eudragit S100) content of 0.17 ~ 5.5 grams, sodium carboxymethyl cellulose (Croscarmellose sodium) Medium content is 0.17~5.5g, polyoxyethylene polyoxypropylene (Pluronic F68) content is 1.4~5.5g, menthol (Menthol) content is 8~34mg, low-substituted hydroxypropyl Cellulos) is 0.19~0.82g, pregelatinized starch is 1.7~5.5g, Dextrates NF hydrated is 0.17~5.5g, Citric acid is 10~42mg. Castor oil Cremophor EL content of 1.7 ~ 5.5 grams, colloidal cerium oxide (Aerosil 200) content of 0.17 ~ 5.5 grams, polyethylene glycol monostearate (Myrj 52) The content is 1.4~5.5g, the content of sorbic acid (Sorbic acid) is 6~24mg, the content of lemon oil is 0.17~5.5g, and the content of Hydroxypropyl cellulose is 0.17~5.5. The content of gram and sorbitol (Sorbitol) is 0.17~5.5g, the content of Acesulfame potassium is 1.4~5.5g, and the content of Hydroxypropylmethylcellulose is 0.17~ 5.5 g, lactose monohydrate (Lactose monohydra Te) the content is 6~24 mg, maltodextrin (Maltodextrin) is 0.17~5.5 g, Brij 58 is 0.17~5.5 g, Brij 76 is 0.17~5.5 g, Tween 80 is 0.17 ~5.5g, Tween 40, the content is 1.4~5.5g, PEG 400 is 1.4~5.5g, PEG 4000 is 1.4~5.5g, PEG 8000 is 1.4~5.5g, Span 60 is 1.4. ~5.5g, sodium benzoate, 2.9~11.9mg, Hydroxyethylmethylcellulose, 0.17~5.5g, Methylcellulose 0.17~5.5g, Span 80 is 1.4~5.5g, Sodium cyclamate is 3.3~13.2mg, Glyceryl behenate is 17.4~69.9mg, iron oxide red ( Oxide red) is 11.3~45.2mg, Glycerin monostearate is 1.4~5.5g, Copovidone K28 is 0.17~5.5g, Starch acetate is 0.17. ~5.5 grams, magnesium stearate (Ma Gnesium stearate) is 9.7~39.0mg, sodium sulphate (Sodium lauryl sulfate) is 4.7~19.0mg, Providone K30 is 0.18~0.73mg, and PEG 2000 is 1.4~5.5g.

The biochemical analysis (ALT, AST value), pathological analysis and residual liver are compared with the results of the test for the non-hepatic side effects of the new compound of acetaminophen and the acetaminophen alone. The results of various aspects such as measurement of function (GSP value) have significantly reduced the efficacy of hepatotoxic side effects caused by acetaminophen.

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, which is equivalent to the embodiment of the invention. The guanamine phenol, cytochrome P450 2E1 inhibitor, cytochrome P450 2E1 inhibitor selection type, application concentration and ratio, and equivalent variations of the equivalent embodiments are all included in the patent scope of the present application.

In summary, this case is not only innovative in the application of acetaminophen, but also can be used to improve the hepatotoxic side effects caused by the use of acetaminophen with a common and safe common excipient. It should have fully complied with the statutory invention patent requirements of novelty and progressiveness, and file an application according to law. You are requested to approve the application for this invention patent to encourage invention.

no

Figure 1 shows the metabolic pathway of acetaminophen (APAP) in the liver; Figure 2 shows (A) normal control group, (B) APAP hepatotoxicity group, (C) dicalcium phosphate, (D) nectar Alcohol, (E) menthol, (F) sucralose, (G) mannitol + sucralose (1.67 + 1.67 mg / kg) and (H) mannitol + sucralose (0.83 + 0.83 mg / kg) In the liver protection test group, the liver tissue sections of the rats after feeding a single dose were: (A) the liver tissue type of the normal control group; (B) the hepatocytes of the peripheral central vein (V) showed fragmentation and inflammatory cells were produced. Necrosis and vacuolization occurred; compared with the APAP hepatotoxicity group, in the liver protection test group, except for the dicalcium phosphate test group, the rats in the other experimental groups had complete hepatocytes around the central vein, and the nuclei were obvious, and Less vacuoles (D, E, F, G, H), and (F), (G) closest to normal rat liver tissue (H&E staining, 200X).

no

Claims (19)

A use of a compound for the manufacture of a medicament for ameliorating or removing hepatotoxicity of a drug, wherein the compound is selected from the group consisting of polyoxyethylene (20) sorbitan monolaurate (Tween 20). ), microcrystalline cellulose, Dicalcium phosphate dihydrate, polyoxyethylene 23 lauryl ether (Brij 35), saccharin (Saccharin), mannitol (Mannitol), polyoxyethylene alkyl ether (Cremophor RH40), Sucralose, Crospovidone, Sodium starch glycolate, Acrylic S100 (Eudragit S100), Croscarmellose sodium, Polyoxyethylene Polyoxene (Pluronic F68), Menthol, Low-substituted hydroxypropyl cellulose, Pregelatinized starch, Dextrates NF hydrated, Citric acid, Castor oil Cremophor EL, colloidal cerium oxide (Aerosil 200), polyethylene glycol monostearate (Myrj 52), sorbic acid (Sorbic acid), Lemon oil, Hydroxypropyl cellulose, Sorbitol, Acesulfame potassium, Hydroxypropyl methylcellulose, Monohydrate lactose (Lactose monohydrate), maltodextrin (Maltodextrin), Brij 58, Brij 76, Tween 80, Tween 40, PEG 400, PEG 4000, PEG 8000, Span 60, sodium benzoate, hydroxymethylcellulose citrate Hydroxy ethylmethylcellulose, Methylcellulose, Span 80, Sodium cyclamate, Glyceryl behenate, Oxide red, glyceryl monostearate (Glycerin monostearate), Copovidone K28, Starch acetate, Magnesium stearate, Sodium lauryl sulfate, Provideone K30, PEG 2000, and any combination thereof. The use of the compound according to claim 1, wherein the effective amount of the single dose of the compound is as follows: polyoxyethylene (20) sorbitol monolaurate (Tween 20) is 0.001 ~55 grams, microcrystalline cellulose (Microcrystalline cellulose) content of 0.001 ~ 55 grams, Dicalcium phosphate dihydrate content of 0.001 ~ 55 grams, polyoxyethylene 23 lauryl ether (Brij 35) content It is 0.001~55g, the content of saccharin (Saccharin) is 0.001~55g, the content of Mannitol is 0.001~55g, the content of polyoxyethylene alkyl ether (Cremophor RH40) is 0.001~55g, three The content of Sucralose is 0.001-55 g, the content of Crospovidone is 0.001-55 g, the content of sodium starch glycolate is 0.001-55 g, and the resin S100 (Eudragit S100) The content is 0.001 to 55 g, the content of croscylellose sodium is 0.001 to 55 g, the content of polyoxyethylene polyoxypropylene (Pluronic F68) is 0.001 to 55 g, and menthol (Menthol) The content is 0.001~55g, low The content of Low-substituted hydroxypropyl cellulose is 0.001-55 grams, the content of pregelatinized starch is 0.001-55 grams, the content of Dextrates NF hydrated is 0.001-55 grams, and citric acid (Citric) The content of acid) is 0.001~55g, the content of cremophor EL is 0.001~55g, the content of colloidal cerium oxide (Aerosil 200) is 0.001~55g, polyethylene glycol single The content of stearate (Myrj 52) is 0.001-55 g, the content of sorbic acid is 0.001-55 g, and the content of lemon oil is 0.001-55 g, hydroxypropyl cellulose ( The content of Hydroxypropyl cellulose is 0.001-55 g, the content of sorbitol (Sorbitol) is 0.001-55 g, the content of Acesulfame potassium is 0.001-55 g, hypromellose phthalate (Hydroxypropyl methylcellulose) is 0.001~55g, Lactose monohydrate is 0.001~55g, maltodextrin is 0.001~55g, Brij 58 is 0.001~55g The content of Brij 76 is 0.001~55g, and the content of Tween 80 is 0.001~55g. The content of Tween 40 is 0.001-55 g, the content of PEG 400 is 0.001-55 g, the content of PEG 4000 is 0.001-55 g, the content of PEG 8000 is 0.001-55 g, and the content of Span 60 is 0.001-55 g. The content of sodium benzoate is 0.001-55 g, the content of Hydroxyethylmethylcellulose is 0.001-55 g, and the content of Methylcellulose is 0.001-55. The content of gram and Span 80 is 0.001~55g, the content of Sodium cyclamate is 0.001~55g, the content of Glyceryl behenate is 0.001~55g, Oxide red The content is 0.001~55g, the content of Glycerin monostearate is 0.001~55g, the content of Copovidone K28 is 0.001~55g, and the content of Starch acetate is 0.001~55g. Magnesium stearate is 0.001-55 g, sodium lauryl sulfate is 0.001-55 g, Provideone K30 is 0.001-55 g, and PEG 2000 is 0.001. ~55 grams. The use of claim 1, wherein the compound is selected from the group consisting of Brij 58, Brij 76, saccharin, Brij 35, Tween 20, PEG 400, microcrystalline cellulose, dicalcium phosphate, Sucralose, mannitol, polyoxyethylene alkyl ether, sodium starch glycolate, PEG2000, PEG4000, Tween 40, pyrrolidone, Tween 80, acrylic resin S100, sodium carboxymethyl cellulose, polyoxyethylene polyoxypropylene, Menthol and any combination thereof. The use of claim 1, wherein the compound is selected from the group consisting of dicalcium phosphate, menthol, mannitol, sucralose, and any combination thereof. The use of claim 1, wherein the compound is selected from the group consisting of mannitol, sucralose, and combinations thereof. The use of any one of claims 1 to 5, wherein the compound is used separately, simultaneously or sequentially with the drug. The use according to any one of claims 1 to 5, wherein the compound is selected from any two or more of the group, and the compounds are each separable, simultaneously or sequentially. The use according to any one of claims 1 to 5, wherein the compound is a gel, a spray, a pasty, a tablet, a dispersible tablet, a capsule, a soft capsule, a pill, a suspension , microspheres, oral implants, emulsified injections, implants or other pharmaceutically acceptable forms are administered. The use of any one of claims 1 to 5, wherein the compound and the drug are in the form of a pharmaceutical pack, kit or patient pack. The use of any one of claims 1 to 5, wherein the drug is Acetaminophen (APAP). The use of any one of claims 1 to 5, wherein the drug is alcohol. A compound combination for improving or eliminating the hepatotoxicity of a drug, comprising the drug and the compound according to any one of claims 1 to 5, wherein the amount of the compound present in the combination is effective to improve or Remove the hepatotoxicity produced by the drug. The combination according to claim 12, wherein the compound and the drug are in the form of a medical kit, a kit or a patient pack. The combination according to claim 12, wherein the amount of the drug present in the combination is higher than the dose when the drug is used alone. The combination according to claim 12, wherein the amount of the drug present in the combination is lower than the dose when the drug is used alone. The combination of any one of claims 12 to 15, wherein the pharmaceutical product comprises Acetaminophen (APAP). The combination of any one of claims 12 to 15, wherein the pharmaceutical product comprises alcohol. For example, in the combination of claim 16 of the patent scope, a single dose of acetaminophen can be used up to 10 grams. For example, in the combination of claim 16, the single-use dosage of acetaminophen is more than 500 mg to 10 g.