WO2022135462A1 - Magl抑制剂的医药用途 - Google Patents

Magl抑制剂的医药用途 Download PDF

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WO2022135462A1
WO2022135462A1 PCT/CN2021/140424 CN2021140424W WO2022135462A1 WO 2022135462 A1 WO2022135462 A1 WO 2022135462A1 CN 2021140424 W CN2021140424 W CN 2021140424W WO 2022135462 A1 WO2022135462 A1 WO 2022135462A1
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maglz
hepatitis
group
liver
liver fibrosis
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PCT/CN2021/140424
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English (en)
French (fr)
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张贵民
孙成宏
潘丽红
李翔子
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鲁南制药集团股份有限公司
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics

Definitions

  • the invention belongs to the field of medicine, and relates to the use of a MAGL inhibitor in preparing a medicine for preventing or treating hepatitis or liver fibrosis.
  • Hepatitis is a general term for inflammation of the liver. Usually refers to the damage to liver cells caused by a variety of pathogenic factors - such as viruses, bacteria, parasites, chemical poisons, drugs, alcohol, autoimmune factors, etc. Abnormal liver function indicators.
  • Hepatitis can be divided into viral hepatitis, drug-induced hepatitis, chemical hepatitis, and autoimmune hepatitis according to the etiology. According to the duration of the disease, it can be divided into acute hepatitis, chronic hepatitis and so on. According to the severity of the disease, chronic hepatitis can be divided into mild, moderate, severe and so on.
  • Liver fibrosis refers to the excessive deposition of diffuse extracellular matrix (especially collagen) in the liver. It is a damage repair response of the body to chronic liver injury caused by various etiologies. Cell degeneration and necrosis, liver tissue structure and function are destroyed, resulting in abnormal liver function, affecting the prognosis of liver disease. Liver fibrosis may progress to decompensated cirrhosis and various end-stage liver disease complications if not treated in time. Liver cirrhosis and its complications have become the main causes of global morbidity and mortality. Liver fibrosis is a major cause of liver disease. The basis for the development of complex diseases in the end-stage stage, such as hepatic portal hypertension, hepatic ascites, synthetic dysfunction, impaired metabolic capacity, etc.
  • Western medicine bifendate has a rapid and significant enzyme-lowering effect, but it "rebounds" (that is, the abnormal liver function index doubles) after drug withdrawal, and it is difficult to say that the drug has an exact effect on the recovery of liver pathological tissue.
  • the more recognized effective drugs are interferon and its inducer vidarabine, etc., but they are expensive and have serious side effects, and they are harmful to the human nervous system, digestive system, cardiovascular system, urinary system and hematopoietic system. Can cause varying degrees of damage, as well as allergic reactions.
  • Clinical drugs include interferon, single Chinese medicine and Chinese medicine compound. Some of these drugs are expensive, some have toxic and side effects after long-term use, some have insignificant effects, poor reproducibility, and some have complex components, which are not conducive to in-depth research on the mechanism of action of the drugs. Therefore, the development of safe and effective drugs for the treatment of liver fibrosis has become one of the hot areas of current drug research.
  • the purpose of the present invention is to provide the application of the compound MAGLZ-II-11 of the present invention in the preparation of medicines for preventing or treating hepatitis or liver fibrosis, which has good therapeutic effect and high safety.
  • the purpose of the present invention is to provide the use of MAGLZ-II-11 in the preparation of medicines for preventing or treating hepatitis or liver fibrosis.
  • the hepatitis is immune hepatitis, drug-induced hepatitis, chemical hepatitis or viral hepatitis.
  • the drug-induced hepatitis is acetaminophen-induced drug-induced hepatitis.
  • the chemical hepatitis is chronic chemical hepatitis or acute chemical hepatitis.
  • the chemical hepatitis is organic solvent-induced hepatitis.
  • the liver fibrosis is chemical liver fibrosis or immune liver fibrosis.
  • the present invention studies the therapeutic effect of MAGLZ-II-11 on various hepatitis models.
  • MAGLZ-II-11 has effects on ConA-induced immune hepatitis, acetaminophen-induced drug-induced hepatitis, and carbon tetrachloride-induced acute chemical hepatitis. Hepatitis and viral hepatitis have significant therapeutic effects.
  • Immune hepatitis is a disease of liver inflammation and necrosis caused by immune cells attacking its own liver tissue after autoimmune dysfunction. rise. Liver pathology can be manifested as interface hepatitis, lymphocyte infiltration, rosette formation, etc. In the blood, autoimmune hepatitis antibodies can be found positive, such as antinuclear antibody positive, anti-liver kidney microsome antibody positive, anti-smooth muscle antibody positive Antibody positive etc.
  • the present invention provides the use of the MAGLZ-II-11 in the preparation of a medicament for preventing or treating hepatitis, wherein the hepatitis is drug-induced hepatitis.
  • Drug-induced hepatitis can manifest as any type of acute or chronic liver disease currently known. If acute drug-induced hepatitis is hepatocellular, it can be manifested as hepatitis or fatty liver. The clinical features are fatty liver, azotemia, and pancreatitis. It can also be manifested as intrahepatic cholestasis drug-induced hepatitis.
  • Chronic drug-induced hepatitis can have chronic active hepatitis or steatohepatitis, cholestatic hepatitis and other manifestations.
  • Drug-induced hepatitis is a series of liver inflammation caused by the use of prescription and over-the-counter chemical preparations, biological preparations, traditional Chinese medicines, health products and their metabolites.
  • the present invention provides the use of the MAGLZ-II-11 in the preparation of a medicament for preventing or treating hepatitis, wherein the hepatitis is chemical hepatitis, that is, hepatitis caused by chemical hepatotoxic substances.
  • the efficacy examples of the present invention show that MAGLZ-II-11 has a good therapeutic effect on hepatitis caused by chemical substances, and can significantly reduce alanine aminotransferase (ALT) and aspartate aminotransferase in hepatitis model (AST).
  • the chemical hepatitis is hepatitis caused by organic solvents or toxic hepatitis caused by organic solvents, and toxic hepatitis is caused by chemical poisons (such as phosphorus, arsenic, carbon tetrachloride, etc.), drugs or Hepatitis or liver disease caused by biological toxins.
  • Toxic hepatitis can be divided into two categories: acute and chronic toxic hepatitis according to the time and amount of exposure to poisons, and chronic hepatitis is more common. The onset of acute toxic hepatitis is rapid, often without prodromal symptoms, and symptoms usually appear within 24 to 48 hours of poisoning.
  • the efficacy examples of the present invention show that MAGLZ-II-11 has a good therapeutic effect on hepatitis caused by carbon tetrachloride.
  • the effect of MAGLZ-II-11 on CCl4-induced liver fibrosis in rats was studied, and it was found that MAGLZ-II-11 improved the survival rate of the liver fibrosis model, decreased the serum hyaluronic acid (HA), III Type procollagen (PCIII), improve the pathological changes of liver fibrosis in the model.
  • HA serum hyaluronic acid
  • PCIII III Type procollagen
  • the effect of MAGLZ-II-11 on immune hepatic fibrosis in rats was studied, and it was found that MAGLZ-II-11 significantly reduced the determination of serum total protein (TP), albumin (ALB), ALT, AST in rat serum , reducing the content of hydroxyproline (Hyp) in the liver of rats, and has a significant anti-hepatic fibrosis effect.
  • TP serum total protein
  • ALB albumin
  • ALT ALT
  • AST AST in rat serum
  • Hyp hydroxyproline
  • Oxidative stress response is an important cause of liver damage. Excessive production of intracellular ROS and weakened antioxidant prevention function can cause tissue and cell damage. Oxidative stress response plays an important role in the occurrence of liver fibrosis.
  • the present invention provides the use of MAGLZ-II-11 in the manufacture of a product for reducing alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in patients with hepatitis or liver fibrosis.
  • ALT alanine aminotransferase
  • AST aspartate aminotransferase
  • the invention provides the application of MAGLZ-II-11 in the preparation of a product for treating and/or preventing the increase of aspartate aminotransferase and/or alanine aminotransferase in the serum of liver fibrosis patients.
  • the invention provides the application of MAGLZ-II-11 in the preparation of a product for treating and/or preventing the increase of Hyp in the liver of a liver fibrosis diseased body.
  • the product is a nutraceutical or a drug.
  • the invention provides the application of MAGLZ-II-11 in the preparation of a product for improving the pathological changes of liver fibrosis in patients with liver fibrosis.
  • the dosage form of MAGLZ-II-11 of the present invention is not particularly limited, for example, it may be an oral dosage form or an injection dosage form.
  • the oral dosage form can be a liquid dosage form or a solid dosage form.
  • the oral dosage form is selected from hard capsules, soft capsules, slow-release capsules, compressed tablets, sugar-coated tablets, powders, granules, dropping pills, honeydew pills, syrup or oral liquid;
  • the injection dosage form is selected from solution type, suspension Liquid, emulsion or lyophilized powder.
  • the administration mode of the MAGLZ-II-11 for preventing and/or treating alcoholic fatty liver disease can be oral, drip or injection.
  • the tablet When preparing a solid preparation for oral use, after adding an excipient and optionally a binder, a disintegrating agent, a lubricant, a coloring agent, a flavoring agent, etc. to the main drug, the tablet can be prepared according to a conventional method. , coated tablets, granules, fine granules, powders, capsules, etc.
  • the excipient can be selected from one or more of lactose, corn starch, white sugar, glucose, sorbitol, crystalline cellulose, and silicon dioxide.
  • binder it can be selected from one or more of polyvinyl alcohol, ethyl cellulose, methyl cellulose, gum arabic, hydroxypropyl cellulose, and hydroxypropyl methyl cellulose.
  • magnesium stearate As the lubricant, one or more of magnesium stearate, talc and silicon dioxide can be selected.
  • cocoa powder As a flavoring agent, one or more of cocoa powder, menthol, aromatic acid, peppermint oil, borneol and cinnamon powder can be used.
  • the above-mentioned tablets and granules can also be coated with sugar coating, gelatin coating, and other necessary outer coatings.
  • pH adjusters, buffers, suspending aids, solubilizers, stabilizers, isotonic agents, preservatives, etc. can be added to the main drug as required, and then intravenous, subcutaneous, and intramuscular injections can be prepared according to conventional methods. . At this time, if necessary, a freeze-dried product can be prepared by a conventional method.
  • suspending aid it can be selected from one or more of methyl cellulose, Tween 80, hydroxyethyl cellulose, gum arabic, sodium carboxymethyl cellulose, polyoxyethylene sorbitan monolaurate .
  • solubilizer it can be selected from one or more of polyoxyethylene hydrogenated castor oil, Tween 80, nicotinamide, polyoxyethylene sorbitan monolaurate, polyethylene glycol, and castor oil fatty acid ethyl ester.
  • a stabilizer it can be selected from one or more of sodium sulfite and sodium metabisulfite; as a preservative, it can be selected from methylparaben, ethylparaben, sorbic acid, phenol, cresol, One or more of chlorocresol.
  • the MAGLZ-II-11 of the present invention is administered to a subject, which may be a mammal, such as a human, a rat, a rabbit, a sheep, a pig, a cow, a cat, a dog, a monkey, etc., preferably a human.
  • a subject which may be a mammal, such as a human, a rat, a rabbit, a sheep, a pig, a cow, a cat, a dog, a monkey, etc., preferably a human.
  • the preparation methods of the above-mentioned pharmaceutical preparations can be prepared by those skilled in the art by conventional preparation methods for preparing such dosage forms.
  • the content of the compound of the present invention contained in each preparation unit is 0.001 mg to 50 mg.
  • FIG. 1 is the pathological section diagram of hepatitis in each group of Example 2.
  • FIG. 1 is the pathological section diagram of hepatitis in each group of Example 2.
  • MAGLZ-II-2, MAGLZ-II-10, MAGLZ-II-11, and MAGLZ-II-17 were prepared according to the method in the invention patent with the application number of CN201910836454.5.
  • Concanavalin (ConA)-induced hepatitis is considered to be a good animal model to mimic human autoimmune hepatitis.
  • ConA is a mitogen that is widely used to activate T cells. It relies on helper T lymphocytes (Th) and macrophages to exert its effects.
  • ConA activates T lymphocytes and stimulates Th cells and macrophages to produce TNF- ⁇ . and cytokines such as IFN- ⁇ . TNF- ⁇ can directly damage hepatocytes, leading to apoptosis of hepatocytes.
  • ConA-induced mouse immune hepatitis experimental animal model its pathological characteristics are mainly through the activation of T lymphocytes to induce immune hepatitis. After intravenous injection of ConA into mice, most of it accumulated in the liver, indicating that the liver is the target organ for ConA to induce toxicity in vivo.
  • ICR mice weighing 33-39 g, all male.
  • the patients were administered once a day for 7 consecutive days. After 30 minutes of the last administration, except the control group, the other groups were injected with ConA-12 mg/kg via tail vein.
  • mice After modeling, the death of the mice was recorded. 24 hours after modeling, 70 mg/kg sodium pentobarbital was administered to anesthetize the mice. Blood was collected from the inferior vena cava, placed in a biochemical coagulation tube, centrifuged at 4000 rpm for 10 min, and the serum was separated. Serum liver function (ALT, AST) levels of mice were detected.
  • Each dose group of MAGLZ-II-11 can significantly reduce serum ALT and AST in mice, and its therapeutic effect on immune hepatitis is better than other single drug groups.
  • MAGLZ-II-2 decreased the serum ALT and AST levels in mice, but the effect was lower than that of MAGLZ-II-11.
  • MAGLZ-II-2 reduced the degree of liver necrosis, but the effect was lower than that of MAGLZ-II-11.
  • Acute hepatitis induced by acetaminophen is an important model for clinical study of drug-induced hepatitis.
  • APAP-induced acute hepatitis is related to the toxic substances produced by its metabolism in the liver. After being metabolized by CYP450 enzymes in hepatocytes, APAP can generate a highly active electrophile N-first-line p-benzoquinoneimine (NAPQI). NAPQI can cause oxidative stress and hepatocyte damage in hepatocytes, causing hepatocyte inflammation and even death.
  • Reference method 64 healthy male ICR mice were selected and randomly divided into 8 groups according to body weight, control group, model group, MAGLZ-II-17 group, MAGLZ-II-10 group, MAGLZ-II-2 group, MAGLZ- II-11 low-dose group, MAGLZ-II-11 medium-dose group, and MAGLZ-II-11 high-dose group.
  • MAGLZ-II-17 group 16mg/kg MAGLZ-II-17, gavage;
  • MAGLZ-II-10 group 16mg/kg MAGLZ-II-10, gavage;
  • MAGLZ-II-2 group 16mg/kg MAGLZ-II-2, gavage;
  • MAGLZ-II-11 low-dose group 4 mg/kg MAGLZ-II-11 by gavage;
  • MAGLZ-II-11 medium dose group 8 mg/kg MAGLZ-II-11, gavage;
  • MAGLZ-II-11 high-dose group 16 mg/kg MAGLZ-II-11 by gavage.
  • the rats were administered by intragastric administration once a day for 7 consecutive days. After the last administration for 60 minutes, except the control group, the other groups were administered APAP-300mg/kg by intragastric administration, and APAP was dissolved in 0.5% sodium carboxymethylcellulose.
  • mice 24h after modeling, the mice were anesthetized by intragastric administration of 70 mg/kg of sodium pentobarbital, blood was collected from the inferior vena cava, placed in a biochemical coagulation tube, centrifuged at 4000 rpm for 10 min, the serum was separated, and the serum liver of the mice was detected by a biochemical automatic analyzer.
  • Function (ALT, AST) levels the left outer lobe of liver was taken and fixed in paraformaldehyde, HE staining was performed, and the inflammatory infiltration, necrosis and pathological changes of liver tissue were observed.
  • Each dose group of MAGLZ-II-11 can significantly reduce serum ALT and AST in mice, and its therapeutic effect on drug-induced hepatitis is better than other single-drug groups.
  • MAGLZ-II-2 decreased the serum ALT and AST levels in mice, but the effect was lower than that of MAGLZ-II-11.
  • Carbon tetrachloride (CCl 4 ) has a strong affinity for the liver in vivo, and is metabolized by cytochrome P450-dependent mixed-function oxidase in the endoplasmic reticulum of hepatocytes to generate chloroform radicals, dichloromethane radicals and Peroxide trichloromethane free radicals can covalently bind to macromolecules in cell membranes, causing loss of enzyme function, lipid peroxidation of cell membranes, damage to the structure and function of liver cell membranes, and obstacles to protein synthesis; The activity of the Ca 2+ pump causes a large influx of Ca 2+ , which leads to the damage and inflammation of the liver cells, so that the intracytoplasmic transaminase penetrates into the blood.
  • ICR mice weighing 29-34 g, all male.
  • Reference method 64 healthy male ICR mice were selected and randomly divided into 8 groups according to body weight, control group, model group, MAGLZ-II-17 group, MAGLZ-II-10 group, MAGLZ-II-2 group, MAGLZ- II-11 low-dose group, MAGLZ-II-11 medium-dose group, and MAGLZ-II-11 high-dose group.
  • MAGLZ-II-17 group 16mg/kg MAGLZ-II-17, gavage;
  • MAGLZ-II-10 group 16mg/kg MAGLZ-II-10, gavage;
  • MAGLZ-II-2 group 16mg/kg MAGLZ-II-2, gavage;
  • MAGLZ-II-11 low-dose group 4 mg/kg MAGLZ-II-11 by gavage;
  • MAGLZ-II-11 medium dose group 8 mg/kg MAGLZ-II-11, gavage;
  • MAGLZ-II-11 high-dose group 16 mg/kg MAGLZ-II-11 by gavage.
  • the rats were administered by intragastric administration once a day for 7 consecutive days. After the last administration for 2 hours, except the control group, the other groups were intragastrically administered 0.1%-CCl 4 olive oil solution, 10 mL/kg.
  • mice 18 hours after modeling, the mice were anesthetized by gavage of 70 mg/kg sodium pentobarbital, blood was collected from the inferior vena cava, put into a biochemical coagulation tube, centrifuged at 4000 rpm for 10 min, the serum was separated, and the serum liver of the mice was detected by a biochemical automatic analyzer. Functional (ALT, AST) levels.
  • Each dose group of MAGLZ-II-11 can significantly reduce serum ALT and AST in mice, and its therapeutic effect on chemical hepatitis is better than other single drug groups.
  • MAGLZ-II-2 decreased the serum ALT and AST levels in mice, but the effect was lower than that of MAGLZ-II-11.
  • the invention adopts the experimental model recognized at home and abroad to carry out experiments in ducks infected with hepatitis B virus.
  • DHBV infection 32 ducklings were injected with DHBV-DNA positive duck serum via tibial vein, each 0.2ml. Blood was drawn 7 days after infection and serum was isolated. Store at 70°C for testing.
  • Drug treatment test 1-day-old Peking ducks infected with DHBV were randomly divided into groups, and each group was given intragastric administration.
  • the blank control group was replaced by 0.5% sodium carboxymethyl cellulose
  • Positive drug acyclovir group 50mg/kg
  • MAGLZ-II-11 low-dose group 4 mg/kg MAGLZ-II-11;
  • MAGLZ-II-11 high-dose group 8 mg/kg MAGLZ-II-11.
  • Detection method Take the above-mentioned duck serum to be tested, and spot the membrane at the same time for each batch to determine the dynamic level of DHBV-DNA in duck serum. According to the method of the nick translation kit, label DHBV-DNA probe with 32p, make duck serum spot hybridization, autoradiography patch spot, measure the OD value (490nm), calculate the serum DHBV-DNA density, and use the OD value of the hybridization spot as the Specimen DHBV-DNA level value.
  • DHBV-DNA inhibition rate (OD value before administration-OD value after administration)/OD value before administration ⁇ 100%.
  • CCl 4 is used as the inducer for the rat liver fibrosis model, and the long-term injection of CCl 4 in small doses (3 months) can cause the liver fibrosis model.
  • the other animals were fed with simple cornmeal (20% lard in the first two weeks), 0.5% cholesterol, 1% salt, and tap water.
  • CCl 4 stock solution 5ml/kg body weight was subcutaneously injected on the back, and then 40% CCl 4 -olive oil 3ml/kg body weight was subcutaneously injected every 3 days. After 6 weeks, the dosage of 40% CCl 4 -olive oil was reduced to 1.5 mg/kg body weight, while the animals were given a normal nutrient feed.
  • the modeling cycle was shortened to 6 weeks.
  • hepatic fibrosis rats were randomly divided into CCl4 model group, MAGLZ-II-11 low-dose group, MAGLZ-II-11 high-dose group, and colchicine group, 16 in each group. Only, half male and half male. Each group was given intragastric administration.
  • the doses are as follows:
  • MAGLZ-II-11 low-dose group 5 mg/kg MAGLZ-II-11.
  • MAGLZ-II-11 high-dose group 10 mg/kg MAGLZ-II-11.
  • Colchicine group 0.1 mg/kg colchicine.
  • the normal control group and the model group were given an equal volume of 0.5% sodium carboxymethylcellulose.
  • Therapeutic dosing was initiated once daily for one month. Continue to give CCl 4 stimulation, after 6 weeks, the injection dose of 40% CCl 4 -olive oil was reduced to 1.5ml/kg body weight, and the normal nutritional feed was restored at the same time. At the end of the experiment (8 weeks), the rats were anesthetized by intraperitoneal injection of 1% sodium pentobarbital, the whole blood was collected by amputation of the neck, the serum was collected by centrifugation, and the contents of HA and PCIII were determined.
  • Serum HA and PCIII levels were detected by Elisa kit.
  • the left lobe of the liver was excised and fixed in 10% neutral formaldehyde.
  • the materials were routinely collected, dehydrated, stained with HE, and the degree of liver fibrosis was observed under a microscope. According to the degree of fibrosis of the fibrous connective tissue, it was divided into grades 0-4.
  • Grade 0 No fibrosis
  • Grade 1 The fibrous connective tissue is limited to the portal area or the portal area is enlarged, and tends to develop to the lobule
  • Grade 2 The fibrous connective tissue hyperplasia is obvious, exceeding 2/3 of the lobule and accompanied by 1
  • Grade 3 The fibrous connective tissue enters around the central vein of the hepatic lobule
  • Grade 4 The fibrous connective tissue shows multiple diffuse hyperplasia in the whole lobule, with the formation of pseudolobules, and there are grade 3 changes.
  • the survival rate of animals in MAGLZ-II-11 groups was significantly higher than that in the model group after 1 month of treatment, indicating that MAGLZ-II-11 has a protective effect on liver fibrosis caused by CCl 4 .
  • the changes of serum HA and pcIII in the model control group were significantly different.
  • the MAGLZ-II-11 groups had significant differences in HA content and pcIII content.
  • MAGLZ-II-11 high-dose group had significant difference in HA content and pcIII content.
  • HE staining under light microscope showed that the hepatic lobule structure of the normal control group was complete, and the shape of hepatocytes was normal.
  • the center of the liver lobule was obviously necrotic, and fatty degeneration of hepatocytes could be seen around the necrotic area, with obvious punctate and focal necrosis.
  • the degree of liver lesions in the MAGLZ-II-11 high and low dose groups was significantly lighter than that in the model group.
  • the degree of liver fibrosis in the MAGLZ-II-11 administration group was reduced, and the degree of reduction was positively correlated with the dose.
  • the immune liver fibrosis model induced by xenogeneic serum is an important screening tool for the study of therapeutic drugs for liver fibrosis.
  • this model is closer to the pathological mechanism of human liver fibrosis, and the formation of liver fibrosis is stable, the damage to liver cells is less, and the overall condition of the animals is good, which is conducive to long-term research.
  • Wisata rats weighing 120-160 g, all male.
  • model rats were divided into model group, JZL184 group, MAGLZ-II-2 group, MAGLZ-II-10 group, MAGLZ-II-17 group, MAGLZ-II-11 low-dose group, MAGLZ-II-11 low-dose group, MAGLZ-II-11 group according to body weight. 11 Low-dose group, MAGLZ-II-11 medium-dose group, and MAGLZ-II-11 high-dose group.
  • the animals were fasted for 12-16 hours, anesthetized by intraperitoneal injection of 70 mg ⁇ kg -1 sodium pentobarbital solution, blood was collected from the abdominal main vein, centrifuged at 3500 r ⁇ min -1 for 10 minutes, and the upper serum was separated by automatic biochemical analysis. Serum AST, ALT, TP, and ALB contents were detected by instrument; the rats were killed by bloodletting, and the liver was quickly excised. After weighing, 0.5 g of liver tissue was clipped, and the content of Hyp was detected according to the kit instructions.
  • each MAGLZ-II-11 group could significantly reduce the levels of serum ALT and AST, and increase the levels of serum TP and ALB, which was statistically significant compared with the model group.
  • Each group of MAGLZ-II-11 can significantly reduce the serum TP, ALB, ALT, AST of rats, and the results show that its effect on liver fibrosis is better than other groups.
  • MAGLZ-II-11 middle and high dose groups decreased TP, ALB, ALT, AST, with significant difference.
  • each MAGLZ-II-11 group could significantly reduce the hepatic Hyp content, and the degree of reduction was positively correlated with the dose.
  • Each group of MAGLZ-II-11 can significantly reduce the content of Hyp in the liver of rats, and the results show that the effect of MAGLZ-II-11 in treating liver fibrosis is better than other groups.

Abstract

本发明请求保护MAGLZ-Ⅱ-11用于制备预防或治疗肝炎或肝纤维化药物的用途，属于医药领域。本发明的目的在于提供本发明化合物在制备预防或治疗肝炎药物中的应用，其治疗效果好、安全性高。本发明所述的MAGLZ-Ⅱ-11能够降低肝炎患者的丙氨酸氨基转移酶和天门冬氨酸氨基转移酶。本发明MAGLZ-Ⅱ-11用于免疫性肝炎、药物性肝炎、化学性肝炎或病毒性肝炎的治疗，治疗效果显著；对于肝纤维化有显著的治疗效果。

Description

MAGL抑制剂的医药用途 技术领域
本发明属于医药领域，涉及MAGL抑制剂在制备预防或治疗肝炎或肝纤维化药物中的用途。
背景技术
肝炎是肝脏炎症的统称。通常是指由多种致病因素--如病毒、细菌、寄生虫、化学毒物、药物、酒精、自身免疫因素等使肝脏细胞受到破坏，肝脏的功能受到损害，引起身体一系列不适症状，以及肝功能指标的异常。
肝炎根据病因，可以分为病毒性肝炎、药物性肝炎、化学性肝炎、自身免疫性肝炎等。根据病程长短，可以分为急性肝炎、慢性肝炎等。根据病情轻重程度，慢性肝炎可以分为轻度、中度、重度等。
肝纤维化是指肝脏内弥漫性细胞外基质(特别是胶原物质)过度沉积，是机体对各种病因引起的慢性肝损伤后的一种损伤修复反应，表现为肝内胶原纤维异常增生，肝细胞变性坏死，肝组织结构和功能遭到破坏，造成肝功能异常，影响肝病预后。肝纤维化如不及时治疗则可能进展成为失代偿期肝硬化并出现各种终末期肝病并发症，肝硬化及其并发症已经成了全球发病与死亡的主要因素，肝纤维化是很多肝病终末期阶段复杂病症发生的基础，如肝门静脉高压、肝腹水、合成功能紊乱、代谢能力受损等等。
西药联苯双酯有快速、显著的降酶作用，但停药后“反跳”(即肝功能异常指标加倍升高)，而且药物对肝脏病理组织的恢复也难说有确切效果。对病毒抑制方面，比较公认有效的药物是干扰素及其诱导剂阿糖腺苷等，但价格昂贵、副作用大，且对人体的神经系统、消化系统、心血管系统、泌尿系统及造血系统等均可造成不同程度的损害，还有过敏反应。
现代医学尚无安全、高效、廉价的抗肝纤维化药物问世，临床用药有干扰素、单味中药及中药复方等。这些药物有的价格昂贵，有的长期服用有毒副作用，有的效果不显著，重现性差，有的成分复杂，不利于深入研究药物的作用机制。因 此，研发安全有效的治疗肝纤维化药物已成为当前药物研究的热点领域之一。
发明内容
本发明的目的在于提供本发明化合物MAGLZ-Ⅱ-11在制备预防或治疗肝炎或肝纤维化药物中的应用，其治疗效果好、安全性高。
MAGLZ-Ⅱ-11结构式如下：
Figure PCTCN2021140424-appb-000001
本发明的目的是提供MAGLZ-Ⅱ-11在制备预防或治疗肝炎或肝纤维化药物中的用途。
在一个实施方式中，所述肝炎为免疫性肝炎、药物性肝炎、化学性肝炎或病毒性肝炎。
在一个实施方式中，所述药物性肝炎为对乙酰氨基酚导致的药物性肝炎。
在一个实施方式中，所述化学性肝炎为慢性化学性肝炎或急性化学性肝炎。
在一个实施方式中，所述化学性肝炎为有机溶剂所致的肝炎。
在一个实施方式中，所述肝纤维化是化学性肝纤维化或免疫性肝纤维化。
本发明研究了MAGLZ-Ⅱ-11对多种肝炎模型的治疗效果，MAGLZ-Ⅱ-11对ConA诱导的免疫性肝炎、对乙酰氨基酚导致的药物性肝炎、四氯化碳导致的急性化学性肝炎、病毒性肝炎均具有显著的治疗效果。
免疫性肝炎是由于自身免疫功能紊乱之后，免疫细胞攻击自身肝脏组织而导致肝脏炎症、坏死的一种疾病，它的主要特点就是表现为谷丙转氨酶、谷草转氨酶的升高，同时伴有球蛋白的升高。在肝脏病理学上可以表现为界面性肝炎、淋巴细胞浸润、玫瑰花环形成等，在血液中可以查到自身免疫性肝炎抗体阳性，如抗核抗体阳性、抗肝肾微粒体抗体阳性、抗平滑肌抗体阳性等。
本发明提供所述的MAGLZ-Ⅱ-11在制备预防或治疗肝炎药物中的用途，其 中所述肝炎为药物性肝炎。药物性肝炎可以表现为目前所知任何类型急性或慢性肝脏疾病。急性药物型肝炎若为肝细胞型，可表现为肝炎型，亦可表现为脂肪肝型，临床特点为脂肪肝、氮质血症和胰腺炎，还可表现为肝内胆淤型药物性肝炎，包括单纯淤胆型，淤胆伴炎症型肝炎，混合型药物性肝炎既有肝炎型的表现亦有胆汁淤积的表现。慢性药物性肝炎可以有慢性活动性肝炎或脂肪性肝炎、胆汁淤积性肝炎等表现。药物性肝炎是由于使用了处方和非处方类的化学制剂、生物制剂以及传统的中药、保健品及其代谢产物所引起的一系列肝脏炎症。
本发明提供所述的MAGLZ-Ⅱ-11在制备预防或治疗肝炎药物中的用途，其中所述肝炎为化学性肝炎，即由化学性肝毒性物质所造成的肝炎。本发明药效实施例显示，MAGLZ-Ⅱ-11对于化学物质所导致的肝炎具有很好的治疗效果，能够显著降低肝炎模型的丙氨酸氨基转移酶(ALT)和天门冬氨酸氨基转移酶(AST)。
在一个实施方式中，所述化学性肝炎为有机溶剂所致的肝炎或有机溶剂所致的中毒性肝炎，中毒性肝炎是由化学毒物(如磷、砷、四氯化碳等)、药物或生物毒素所引起的肝炎或所致的肝脏病变。中毒性肝炎按接触毒物的时间和量可分为急性和慢性中毒性肝炎两类，以慢性者多见。急性中毒性肝炎发病急骤，常无前驱症状，一般在中毒24～48小时出现症状。本发明药效实施例显示，MAGLZ-Ⅱ-11对四氯化碳导致的肝炎具有很好的治疗效果。
本发明实施例研究了MAGLZ-Ⅱ-11对CCl4所致大鼠肝纤维化的影响，发现MAGLZ-Ⅱ-11提高肝纤维化模型的存活率，降低大鼠血清透明质酸(HA)、III型前胶原(PCIII)，改善模型的肝纤维化病理变化。
本发明实施例研究了MAGLZ-Ⅱ-11对大鼠免疫性肝纤维化的影响，发现MAGLZ-Ⅱ-11显著降低大鼠血清血清总蛋白测定(TP)、白蛋白(ALB)、ALT、AST，降低大鼠肝脏羟脯氨酸(Hyp)含量，具有显著的抗肝纤维化效果。
化学性肝纤维化是肝细胞发生损伤后引起的，保护肝细胞，在一定程度上可以起到预防和治疗肝纤维化的作用。氧化应激反应是导致肝损伤的重要原因，细胞内ROS过度产生和抗氧化预防功能减弱，会造成组织、细胞的损伤，氧化应激反应在肝纤维化的发生过程中起到重要的作用。
在又一个方面，本发明提供MAGLZ-Ⅱ-11在制备降低肝炎或肝纤维化患者的丙氨酸氨基转移酶(ALT)和天门冬氨酸氨基转移酶(AST)的产品中的用途。
本发明提供MAGLZ-Ⅱ-11在制备治疗和/或预防肝纤维化患病机体血清中天冬氨酸氨基转移酶和/或丙氨酸氨基转移酶增加的产品中的应用。
本发明提供MAGLZ-Ⅱ-11在制备治疗和/或预防肝纤维化患病机体肝脏Hyp增加的产品中的应用。
在一个实施方式中，所述产品为保健品或药物。
本发明提供MAGLZ-Ⅱ-11在制备改善肝纤维化患病机体肝纤维化病理变化的产品中的应用。
对本发明的MAGLZ-Ⅱ-11的剂型没有特殊限制，例如可以是口服剂型或注射剂型。所述口服剂型可以是液体剂型，也可以是固体剂型。所述口服剂型选自硬胶囊、软胶囊、缓控释放胶囊、压片、糖衣片、粉剂、颗粒剂、滴丸、水蜜丸、糖浆或口服液；所述注射剂型选自溶液型、混悬液型、乳浊液型或冻干粉。
所述用于预防和/或治疗酒精性脂肪肝的MAGLZ-Ⅱ-11的给药方式可以为口服、滴注或注射。
制备口服用固体制剂时，可以在向主药中加入赋形剂以及视需要而定的粘合剂、崩解剂、滑润剂、着色剂、矫味剂等后，按照常规方法制成片剂、包衣片剂、颗粒剂、细粒剂、散剂、胶囊剂等。
作为赋形剂，可选自乳糖、玉米淀粉、白糖、葡萄糖、山梨糖醇、结晶纤维素、二氧化硅中的一种或几种。
作为粘合剂，可选自聚乙烯醇、乙基纤维素、甲基纤维素、阿拉伯胶、羟基丙基纤维素、羟基丙基甲基纤维素中的一种或几种。
作为滑润剂，可选自硬脂酸镁、滑石、二氧化硅中的一种或几种。
作为矫味剂，可使用可可粉、薄荷脑、芳香酸、薄荷油、龙脑、桂皮粉中的一种或几种。
当然，也可以在上述片剂、颗粒剂上包覆糖衣、明胶衣、以及其它的必要外衣。
制备注射剂时，可以根据需要向主药中添加pH调节剂、缓冲剂、悬助剂、增溶剂、稳定剂、等渗剂、防腐剂等，再按照常规方法制成静脉、皮下、肌肉内注射剂。此时，也可以根据需要，利用常规方法制成冷冻干燥物。
作为悬助剂，可选自甲基纤维素、吐温80、羟基乙基纤维素、阿拉伯胶、 羧甲基纤维素钠、聚氧乙烯山梨糖醇单月桂酸盐中的一种或几种。
作为增溶剂，可选自聚氧化乙烯氢化蓖麻油、吐温80、烟酰胺、聚氧乙烯山梨糖醇单月桂酸盐、聚乙二醇、蓖麻油脂肪酸乙酯中的一种或几种。
另外，作为稳定剂，可选自亚硫酸钠、偏亚硫酸钠中的一种或几种；作为防腐剂，可选自对羟基苯甲酸甲酯、对羟基苯甲酸乙酯、山梨酸、苯酚、甲酚、氯甲酚中的一种或几种。
将本发明的MAGLZ-Ⅱ-11施用于对象，所述对象可以是哺乳动物，例如可以是人、大鼠、兔、羊、猪、牛、猫、狗、猴等，优选为人。
上述药物制剂的制备方法均可采用本领域技术人员制备该种剂型常规使用制备方法制得。上述药物制剂中，每一制剂单位中含有本发明化合物的含量为0.001mg～50mg。
附图说明
图1为实施例2各组的肝炎病理切片图。
具体实施方式
为了使本领域技术人员充分了解本发明，以下通过具体的实施例进一步说明本发明，但本领域技术人员应该知晓，本发明实施例并不以任何方式限制本发明。
以下实施例中，MAGLZ-Ⅱ-2、MAGLZ-Ⅱ-10、MAGLZ-Ⅱ-11、MAGLZ-Ⅱ-17按照申请号为CN201910836454.5的发明专利中的方法制备得到。
实施例1化合物MAGLZ-Ⅱ-11对ConA诱导的小鼠免疫性肝炎的保护作用
刀豆蛋白(ConA)诱导的肝炎被认为是一种很好的模拟人类自身免疫性肝炎的动物实验模型。ConA是一种被广泛应用于可活化T细胞的有丝分裂原，依赖于辅助性T淋巴细胞(Th)及巨噬细胞发挥效应，ConA活化T淋巴细胞，刺激Th细胞及巨噬细胞产生TNF-α和IFN-γ等细胞因子。TNF-α可直接损伤肝细胞，导致肝细胞凋亡。ConA诱导的小鼠免疫性肝炎实验动物模型，其病理特点主要是通过活化T淋巴细胞而致免疫性肝炎。ConA静脉注射小鼠体内后，大部分在肝脏内聚集，表明肝脏是ConA体内诱导毒性的靶器官。
1材料
1.1实验动物
ICR小鼠，体重33～39g，全部雄性。
2方法
2.1动物分组及给药
参考文献方法，选取健康雄性ICR小鼠72只，按体重随机分为9组，对照组、模型组、JZL184组、MAGLZ-Ⅱ-17组、MAGLZ-Ⅱ-10组、MAGLZ-Ⅱ-2组、MAGLZ-Ⅱ-11低剂量组、MAGLZ-Ⅱ-11中剂量组、MAGLZ-Ⅱ-11高剂量组。
动物给药如下：
Figure PCTCN2021140424-appb-000002
Figure PCTCN2021140424-appb-000003
按上述给药方式和给药剂量，每天给药一次，连续7d，末次给药30min后，除对照组外，其余各组尾静脉注射ConA-12mg/kg。
2.2指标检测
造模后记录小鼠的死亡情况，造模24h后灌胃70mg/kg的戊巴比妥钠麻醉小鼠，下腔静脉取血，放入生化促凝管中，4000rpm离心10min，分离血清，检测小鼠血清肝功能(ALT、AST)水平。
2.3数据统计
数据用
Figure PCTCN2021140424-appb-000004
表示，采用SPSS 19.5软件进行分析，组间比较采用ANOVA统计学检验，P<0.05视为差异有统计学意义。
3结果
3.1化合物MAGLZ-Ⅱ-11对血清转氨酶ALT、AST的影响
(1)表1结果显示，与对照组相比，模型组血清ALT、AST含量明显升高，与模型组相比，给药后，除MAGLZ-Ⅱ-17组、MAGLZ-Ⅱ-10组外，其余各组均可显著降低血清ALT，AST的含量，与模型组比较有统计学意义。
(2)MAGLZ-Ⅱ-11各剂量组能够显著降低小鼠血清ALT、AST，其对免疫性肝炎的治疗效果优于其它各单药组。
(3)MAGLZ-Ⅱ-2降低小鼠血清ALT、AST含量，但效果低于MAGLZ-Ⅱ-11。
(4)与JZL184相比，MAGLZ-Ⅱ-11中、高剂量组降低小鼠血清ALT、AST，具有显著性差异。
表1 MAGLZ-Ⅱ-11对免疫性肝炎小鼠血清ALT和AST的影响
Figure PCTCN2021140424-appb-000005
Figure PCTCN2021140424-appb-000006
Figure PCTCN2021140424-appb-000007
与对照组比较， ###P<0.001；
与模型组比较，*P<0.05，**P<0.01，***P<0.001；
与JZL184组比较， ΩP<0.05， ΩΩP<0.01。
3.2化合物MAGLZ-Ⅱ-11对肝组织坏死程度的影响
(1)表2结果显示，与对照组相比，模型组肝脏坏死明显增多，评分显著升高。与模型组相比，给药后，除MAGLZ-Ⅱ-17组、MAGLZ-Ⅱ-10组外，其余各组肝脏坏死明显减少，评分显著下降，与模型组比较有统计学意义。
(2)MAGLZ-Ⅱ-11各剂量组肝脏坏死明显减少，评分显著下降，其对免疫性肝炎的治疗效果优于其它各单药组。
(3)MAGLZ-Ⅱ-2减少肝组织坏死程度，但效果低于MAGLZ-Ⅱ-11。
(4)与JZL184相比，MAGLZ-Ⅱ-11中、高剂量组肝脏坏死明显减少，评分显著下降，具有显著性差异。
表2 MAGLZ-Ⅱ-11对免疫性肝炎小鼠肝组织坏死程度的影响
Figure PCTCN2021140424-appb-000008
Figure PCTCN2021140424-appb-000009
与对照组比较， ###P<0.001；
与模型组比较，*P<0.05，**P<0.01，***P<0.001；
与JZL184组比较， ΩP<0.05， ΩΩP<0.01。
实施例2化合物MAGLZ-Ⅱ-11对对乙酰氨基酚致小鼠药物性肝炎的保护作用
对乙酰氨基酚(acetaminophen，APAP)所诱导的急性肝炎是临床上研究药物性肝炎的重要模型。APAP诱导的急性肝炎与其在肝脏内代谢产生的毒性物质有关，肝细胞内经CYP450酶代谢转化后，APAP可生成高活性的亲电子物N-一线对苯醌亚胺(NAPQI)。NAPQI可引起肝细胞内的氧化应激和肝细胞损伤，引起肝细胞炎症，甚至死亡。
1材料
1.1实验动物
昆明小鼠，体重22～26g，全部雄性。
2方法
2.1动物分组及给药
参考文献方法，选取健康雄性ICR小鼠64只，按体重随机分为8组，对照组、模型组、MAGLZ-Ⅱ-17组、MAGLZ-Ⅱ-10组、MAGLZ-Ⅱ-2组、MAGLZ-Ⅱ-11低剂量组、MAGLZ-Ⅱ-11中剂量组、MAGLZ-Ⅱ-11高剂量组。
对照组，等体积的0.5％羧甲基纤维素钠，灌胃；
模型组，等体积的0.5％羧甲基纤维素钠，灌胃；
MAGLZ-Ⅱ-17组：16mg/kg MAGLZ-Ⅱ-17，灌胃；
MAGLZ-Ⅱ-10组：16mg/kg MAGLZ-Ⅱ-10，灌胃；
MAGLZ-Ⅱ-2组：16mg/kg MAGLZ-Ⅱ-2，灌胃；
MAGLZ-Ⅱ-11低剂量组：4mg/kg MAGLZ-Ⅱ-11，灌胃；
MAGLZ-Ⅱ-11中剂量组：8mg/kg MAGLZ-Ⅱ-11，灌胃；
MAGLZ-Ⅱ-11高剂量组：16mg/kg MAGLZ-Ⅱ-11，灌胃。
按上述剂量，每天灌胃给药一次，连续7d，末次给药60min后，除对照组外，其余各组灌胃APAP-300mg/kg，APAP溶于温0.5％羧甲基纤维素钠中。
2.2指标检测
造模24h后灌胃70mg/kg的戊巴比妥钠麻醉小鼠，下腔静脉取血，放入生化促凝管中，4000rpm离心10min，分离血清，使用生化自动分析仪检测小鼠 血清肝功能(ALT、AST)水平；取肝左外叶固定于多聚甲醛中，进行HE染色，观察肝组织炎性浸润、坏死及病理学变化。
2.3数据统计
数据用
Figure PCTCN2021140424-appb-000010
表示，采用SPSS 19.5软件进行分析，组间比较采用ANOVA统计学检验，P<0.05视为差异有统计学意义。
3结果
3.1化合物MAGLZ-Ⅱ-11对血清转氨酶ALT、AST的影响
(1)表3结果显示，与对照组相比，模型组血清ALT、AST含量明显升高，与模型组相比，给药后，除MAGLZ-Ⅱ-17组、MAGLZ-Ⅱ-10组外，其余各组均可显著降低血清ALT，AST的含量，与模型组比较有统计学意义。
(2)MAGLZ-Ⅱ-11各剂量组能够显著降低小鼠血清ALT、AST，其对药物性肝炎的治疗效果优于其它各单药组。
(3)MAGLZ-Ⅱ-2降低小鼠血清ALT、AST含量，但效果低于MAGLZ-Ⅱ-11。
表3 MAGLZ-Ⅱ-11对对乙酰氨基酚致药物性肝炎小鼠血清ALT和AST的影响
Figure PCTCN2021140424-appb-000011
与对照组比较， ###P<0.001；
与模型组比较，*P<0.05，**P<0.01，***P<0.001。
3.2化合物MAGLZ-Ⅱ-11对肝脏病理变化的影响
图1结果表明，肝病理切片显示，模型组组肝中含有大量的脂肪粒、炎症坏死严重，而MAGLZ-Ⅱ-11各组脂肪粒较少、炎症较轻。
实施例3化合物MAGLZ-Ⅱ-11对四氯化碳致急性化学性肝炎的保护作用
四氯化碳(CCl 4)在体内对肝脏的亲和力很强，在肝细胞内质网中经细胞色素P450依赖性混合功能氧化酶的代谢，生成三氯甲烷自由基、二氯甲烷自由基和过氧化三氯甲烷自由基，可与细胞膜大分子共价结合，使酶的功能丧失，细胞膜发生脂质过氧化，破坏肝细胞膜的结构和功能完整，使蛋白质合成障碍；并通过抑制细胞膜上的Ca 2+泵的活性，使Ca 2+大量内流，导致肝细胞的损伤和炎症，从而使胞浆内转氨酶渗入血液。
1材料
1.1实验动物
ICR小鼠，体重29～34g，全部雄性。
2方法
2.1动物分组及给药
参考文献方法，选取健康雄性ICR小鼠64只，按体重随机分为8组，对照组、模型组、MAGLZ-Ⅱ-17组、MAGLZ-Ⅱ-10组、MAGLZ-Ⅱ-2组、MAGLZ-Ⅱ-11低剂量组、MAGLZ-Ⅱ-11中剂量组、MAGLZ-Ⅱ-11高剂量组。
对照组，等体积的0.5％羧甲基纤维素钠，灌胃；
模型组，等体积的0.5％羧甲基纤维素钠，灌胃；
MAGLZ-Ⅱ-17组：16mg/kg MAGLZ-Ⅱ-17，灌胃；
MAGLZ-Ⅱ-10组：16mg/kg MAGLZ-Ⅱ-10，灌胃；
MAGLZ-Ⅱ-2组：16mg/kg MAGLZ-Ⅱ-2，灌胃；
MAGLZ-Ⅱ-11低剂量组：4mg/kg MAGLZ-Ⅱ-11，灌胃；
MAGLZ-Ⅱ-11中剂量组：8mg/kg MAGLZ-Ⅱ-11，灌胃；
MAGLZ-Ⅱ-11高剂量组：16mg/kg MAGLZ-Ⅱ-11，灌胃。
按上述剂量，每天灌胃给药一次，连续7d，末次给药2h后，除对照组外，其余各组灌胃0.1％-CCl 4橄榄油溶液，10mL/kg。
2.2指标检测
造模18h后灌胃70mg/kg的戊巴比妥钠麻醉小鼠，下腔静脉取血，放入生化促凝管中，4000rpm离心10min，分离血清，使用生化自动分析仪检测小鼠血清肝功能(ALT、AST)水平。
2.3数据统计
数据用
Figure PCTCN2021140424-appb-000012
表示，采用SPSS 19.5软件进行分析，组间比较采用ANOVA统计学检验，P<0.05视为差异有统计学意义。
3结果
3.1化合物MAGLZ-Ⅱ-11对血清转氨酶ALT、AST的影响
(1)表4结果显示，与对照组相比，模型组血清ALT、AST含量明显升高，与模型组相比，给药后，除MAGLZ-Ⅱ-17组、MAGLZ-Ⅱ-10组外，其余各组均可显著降低血清ALT，AST的含量，与模型组比较有统计学意义。
(2)MAGLZ-Ⅱ-11各剂量组能够显著降低小鼠血清ALT、AST，其对化学性肝炎的治疗效果优于其它各单药组。
(3)MAGLZ-Ⅱ-2降低小鼠血清ALT、AST含量，但效果低于MAGLZ-Ⅱ-11。
表4 MAGLZ-Ⅱ-11对四氯化碳致急性化学性肝炎小鼠血清ALT和AST的影响
Figure PCTCN2021140424-appb-000013
Figure PCTCN2021140424-appb-000014
与对照组比较， ###P<0.001；
与模型组比较，*P<0.05,**P<0.01,***P<0.001。
实施例4化合物MAGLZ-Ⅱ-11对鸭体内乙型病毒性肝炎病毒感染的治疗效果
本发明采用国内外公认的实验模型，在乙型病毒性肝炎病毒感染的鸭体内进行实验。
1.DHBV感染:32只雏鸭经胫静脉注射DHBV-DNA阳性鸭血清，每只0.2ml。在感染后7天取血，分离血清。70℃保存待检。
2.药物治疗试验：DHBV感染1日龄北京鸭随机分组，各组灌胃给药。
空白对照组，以0.5％羧甲基纤维素钠代替药物；
阳性药物阿昔洛韦组：50mg/kg；
MAGLZ-Ⅱ-11低剂量组：4mg/kg MAGLZ-Ⅱ-11；
MAGLZ-Ⅱ-11高剂量组：8mg/kg MAGLZ-Ⅱ-11。
每天2次，连续给药10d。
给药前，给药第5d、10d和停药后3d，分别自鸭腿胫静脉取血，分离血清，于-70℃保存待检。
3.检测方法：取上述待检鸭血清，每批同时点膜，测定鸭血清中DHBV-DNA的动态水平。按缺口翻译试剂盒说明书方法，用32p标记DHBV-DNA探针，作鸭血清斑点杂交，放射自显影膜片斑点，测定OD值(490nm)，计算血清DHBV-DNA密度，以杂交斑点OD值作为标本DHBV-DNA水平值。
4.药效计算:计算每组不同时间点血清DHBV-DNA水平，各组用药前后比较采用配对t检验，计算DHBV-DNA抑制率，比较各组鸭血清DHBV-DNA抑制率的动态变化。给药组与病毒对照组比较，采用成组t检验。DHBV-DNA抑制率＝(给药前OD值-给药后OD值)/给前OD值×100％。
结果见表5。
表5 MAGLZ-Ⅱ-11对DHBV感染鸭血清DHBV-DNA水平的影响
Figure PCTCN2021140424-appb-000015
Figure PCTCN2021140424-appb-000016
与空白对照组比较， #P<0.05， ##P<0.01；
与阿昔洛韦组比较，*P<0.05，**P<0.01。
本实验通过鸭乙型病毒性肝炎模型证明了MAGLZ-Ⅱ-11具有更好的抗鸭乙型病毒性肝炎病毒活性，乙型病毒性肝炎病毒感染鸭灌胃给予MAGLZ-Ⅱ-11治疗，一天给药两次，MAGLZ-Ⅱ-11各治疗组对感染鸭血清DHBV-DNA水平均有显著抑制作用。
实施例5化合物MAGLZ-Ⅱ-11对CCl 4所致大鼠肝纤维化作用研究
1.动物及实验分组
选用180-220g健康Wistar大鼠，76只，雌雄各半。
随机分成正常对照组、CCl 4模型组、MAGLZ-Ⅱ-11低剂量组、MAGLZ-Ⅱ-11高剂量组及秋水仙碱组。
2.模型的建立
除正常对照组外，各组大鼠进行造模。方法如下：大鼠肝纤维化模型选用CCl 4为诱导剂，CCl 4小剂量长期注射(3个月)可造成肝纤维化模型。除正常对照组外，其余动物均饲以单纯玉米面(前两周加20％猪油)加胆固醇0.5％、食盐1％、饮自来水。实验第1天背部皮下注射CCl 4原液5ml/kg体重，以后每隔3天皮下注射40％CCl 4-橄榄油3ml/kg体重，6周后将40％CCl 4-橄榄油的用量降为1.5mg/kg体重，同时给动物恢复正常的营养饲料。
造模周期缩短为6周。
分别于实验的3周末、6周末和8周处死动物。取血离心，收集血清，定位取肝组织进行肝纤维化指标的测定及病理组织学检查。
3.对大鼠肝纤维化的治疗作用
用加速法成功造模31天后，将64只肝纤维化大鼠随机分成CCl 4模型组，MAGLZ-Ⅱ-11低剂量组，MAGLZ-Ⅱ-11高剂量组，秋水仙碱组，每组16只，雌雄各半。各组灌胃给药。剂量如下：
MAGLZ-Ⅱ-11低剂量组：5mg/kg MAGLZ-Ⅱ-11。
MAGLZ-Ⅱ-11高剂量组：10mg/kg MAGLZ-Ⅱ-11。
秋水仙碱组：0.1mg/kg秋水仙碱。
正常对照组、模型组给予等体积0.5％羧甲基纤维素钠。
开始进行治疗给药，每日一次，连续一个月。继续给予CCl 4刺激，6周后将 40％CCl 4-橄榄油的注射剂量下调为1.5ml/kg体重，同时恢复正常营养饲料。实验结束时(8周)大鼠腹腔注射1％戊巴比妥钠麻醉，断颈取全血，离心收集血清，测定HA，PCⅢ含量。
4.检测项目及指标
用Elisa试剂盒检测血清HA、PCⅢ水平。
摘取肝脏左叶放入10％中性甲醛中固定，常规取材、脱水，HE染色，显微镜下观察肝脏纤维化程度，根据纤维结缔组织的纤维化程度将其分为0～4级。
0级:无纤维化；1级:纤维结缔组织仅局限于汇管区或汇管区有扩大，有向小叶发展的倾向；2级:纤维结缔组织增生明显，超过小叶的2/3并伴有1级改变；3级:纤维结缔组织进入肝小叶中央静脉周围；4级:纤维结缔组织在全小叶呈多处弥漫性增生，有假小叶形成，并有3级改变。
5.实验结果
(1)一般观察及对肝纤维化大鼠存活率的影响
造模大鼠注射CCl 4后明显消瘦，毛色干枯。
各实验组均有动物状态不佳，甚至出现死亡。濒死动物剖腹可见有肝腹水。
实验期间各组动物的存活情况见表6。
表6 MAGLZ-Ⅱ-11对CCl 4所致肝纤维化大鼠存活率的影响
Figure PCTCN2021140424-appb-000017
治疗给药1月后MAGLZ-Ⅱ-11各组的动物存活率明显高于模型组组，该结果提示MAGLZ-Ⅱ-11对CCl 4造成的肝纤维化有保护作用。
(2)对肝纤维化大鼠血清HA、pcIII含量的影响
MAGLZ-Ⅱ-11治疗给药后对纤维化大鼠血清HA、pcIII的测定结果见表7。
表7 MAGLZ-Ⅱ-11对CCl 4所致肝纤维化大鼠血清指标的影响
Figure PCTCN2021140424-appb-000018
Figure PCTCN2021140424-appb-000019
Figure PCTCN2021140424-appb-000020
与正常对照组比较， #P<0.05， ##P<0.01， ###P<0.001；
与模型对照组比较，*P<0.05，**P<0.01，***P<0.001；
与秋水仙碱组比较， ΩP<0.05， ΩΩP<0.01。
与正常对照组相比，模型对照组血清HA、pcIII变化有显著性差异。与模型对照组相比，MAGLZ-Ⅱ-11各组HA含量、pcIII含量有显著性差异。与秋水仙碱组相比，MAGLZ-Ⅱ-11高剂量组HA含量、pcIII含量有显著性差异。
(3)对大鼠肝脏病理变化的影响
结果如表8所示。
表8 MAGLZ-Ⅱ-11对CCl 4所致肝纤维化大鼠肝纤维化的影响
Figure PCTCN2021140424-appb-000021
光镜下HE染色显示正常对照组大鼠的肝小叶结构完整，肝细胞形态正常。模型对照组肝脏小叶中心部明显坏死，其坏死区周围可见脂肪变性的肝细胞，有明显的点状和灶状坏死。而MAGLZ-Ⅱ-11高低剂量组大鼠肝脏病变程度较模型组均明显减轻。与模型对照组相比，MAGLZ-Ⅱ-11给药组肝纤维化程度有所减轻，减轻程度与剂量正相关。
实施例6化合物MAGLZ-Ⅱ-11对大鼠免疫性肝纤维化的影响
异种血清诱导的免疫性肝纤维化模型是研究肝纤维化治疗药物的重要筛选工具，其主要发病机制类似于Ⅲ过敏反应，与临床病毒性肝炎引起的肝纤维化类似，与化学性肝纤维化模型相比较，该模型与人类肝纤维化的病理机制更为接近，而且肝纤维化形成稳定，肝细胞受损较轻，动物总体状态良好，有利于进行长期的研究。
Wisata大鼠，体重120～160g，全部雄性。
大鼠免疫性肝纤维化模型的制备
参考文献方法，选取健康雄性Wisatr大鼠80只，留取8只作为正常对照，其余大鼠均多点皮下注射9mg·mL -1牛血清白蛋白(BSA)弗氏不完全佐剂乳剂，每次0.5mL，连续注射5次致敏，前两次注射间隔2周，后3次间隔1周。末次致敏后第10天，大鼠眼眶静脉丛采血检测抗体，取BSA抗体阳性大鼠尾静脉攻击注射BSA生理盐水溶液，每次0.4mL，每周2次，剂量由每次2.0mg递增至3.0mg，以后每次增加0.2mg，直至4mg，共攻击注射16次。正常对照组用生理盐水代替BSA。
72只造模大鼠按照体重分为模型组、JZL184组、MAGLZ-Ⅱ-2组、MAGLZ-Ⅱ-10组、MAGLZ-Ⅱ-17组、MAGLZ-Ⅱ-11低剂量组、MAGLZ-Ⅱ-11低剂量组、MAGLZ-Ⅱ-11中剂量组、MAGLZ-Ⅱ-11高剂量组。
动物给药如下：
Figure PCTCN2021140424-appb-000022
Figure PCTCN2021140424-appb-000023
指标测定
给药结束后动物禁食不禁水12～16h，70mg·kg -1戊巴比妥钠溶液腹腔注射麻醉，腹主静脉取血，3500r·min -1离心10min，分离上层血清用全自动生化分析仪检测血清AST、ALT、TP、ALB含量；放血处死大鼠，迅速摘取肝脏，称重后剪取0.5g肝组织，按照试剂盒说明书检测Hyp含量
统计处理
数据用
Figure PCTCN2021140424-appb-000024
表示，采用SPSS 17.5软件进行分析，组间比较采用ANOVA统计学检验，P<0.05视为差异有统计学意义。
结果
1.MAGLZ-Ⅱ-11对免疫性肝纤维大鼠血清蛋白和转氨酶的影响
(1)表9结果显示，与对照组相比，模型组血清TP、ALB含量明显降低，ALT、AST含量明显升高；
与模型组相比，MAGLZ-Ⅱ-11各组均可显著降低血清ALT、AST的含量，升高血清TP、ALB的含量，与模型组比较有统计学意义。
(2)MAGLZ-Ⅱ-11各组能够显著降低大鼠血清TP、ALB、ALT、AST，结果表明其治疗肝纤维化的效果优于其它给药组。
与JZL184相比，MAGLZ-Ⅱ-11中、高剂量组降低TP、ALB、ALT、AST，具有显著性差异。
表9 MAGLZ-Ⅱ-11对免疫性肝纤维化大鼠血清TP、ALB、ALT和AST的影响
Figure PCTCN2021140424-appb-000025
Figure PCTCN2021140424-appb-000026
与对照组比较， ##P<0.01， ###P<0.001；
与模型组比较，*P<0.05，**P<0.01，***P<0.001；
与JZL184组比较， ΩP<0.05， ΩΩP<0.01。
2.MAGLZ-Ⅱ-11对免疫性肝纤维化大鼠肝脏Hyp含量的影响
(1)表10结果显示，与对照组相比，模型组肝脏Hyp含量显著升高；
与模型组相比较，MAGLZ-Ⅱ-11各组能显著降低肝脏Hyp含量，降低程度与剂量呈正相关。
(2)MAGLZ-Ⅱ-11各组能够显著降低大鼠肝脏Hyp含量，结果表明其治疗肝纤维化的效果优于其它给药组。
与JZL184相比，MAGLZ-Ⅱ-11中、高剂量组降低Hyp含量，具有显著性差异。
表10 MAGLZ-Ⅱ-11对免疫性肝纤维化肝脏大鼠Hyp含量的影响
Figure PCTCN2021140424-appb-000027
Figure PCTCN2021140424-appb-000028
Figure PCTCN2021140424-appb-000029
与对照组比较， ##P<0.01， ###P<0.001；
与模型组比较，*P<0.05，**P<0.01，***P<0.001；
与JZL184组比较， ΩP<0.05。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (10)

  1. MAGLZ-Ⅱ-11在制备预防或治疗肝炎或肝纤维化药物中的用途。
  2. 如权利要求1所述的用途，其特征在于，所述肝炎为免疫性肝炎、药物性肝炎、化学性肝炎或病毒性肝炎。
  3. 如权利要求2所述的用途，其特征在于，所述药物性肝炎是由对乙酰氨基酚导致的药物性肝炎。
  4. 如权利要求1所述的用途，其特征在于，所述肝纤维化是免疫性肝纤维化或化学性肝纤维化。
  5. 如权利要求1所述的用途，其特征在于，所述MAGLZ-Ⅱ-11在制备降低肝炎或肝纤维化患者的丙氨酸氨基转移酶(ALT)和天门冬氨酸氨基转移酶(AST)的药物中的用途。
  6. 如权利要求1所述的用途，其特征在于，所述MAGLZ-Ⅱ-11在制备治疗和/或预防肝纤维化患病机体肝脏羟脯氨酸(Hyp)增加的药物中的应用。
  7. 如权利要求1所述的用途，其特征在于，所述MAGLZ-Ⅱ-11在制备改善肝纤维化患病机体肝纤维化病理变化的药物中的应用。
  8. 如权利要求1所述的用途，其特征在于，所述MAGLZ-Ⅱ-11的人用给药量为0.001mg/kg·d～50mg/kg·d；进一步地，人用给药量为0.01mg/kg·d～10mg/kg·d。
  9. 如权利要求1所述的用途，其特征在于，所述MAGLZ-Ⅱ-11为口服制剂或注射制剂；所述口服制剂为其片剂、胶囊剂或颗粒剂；所述注射制剂为溶液型、混悬液型、乳浊液型或冻干粉。
  10. 如权利要求8或9所述的用途，其特征在于，每一制剂单位中含有MAGLZ-Ⅱ-11的含量为0.001mg～50mg。
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