CN113209070A - Application of N-acetylcysteine in preparation of product for treating biliary atresia and medicine for treating biliary atresia - Google Patents
Application of N-acetylcysteine in preparation of product for treating biliary atresia and medicine for treating biliary atresia Download PDFInfo
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- CN113209070A CN113209070A CN202110664249.2A CN202110664249A CN113209070A CN 113209070 A CN113209070 A CN 113209070A CN 202110664249 A CN202110664249 A CN 202110664249A CN 113209070 A CN113209070 A CN 113209070A
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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
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
- A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
- A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
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- 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
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Abstract
The invention provides application of N-acetylcysteine in preparing a product for treating biliary atresia and a medicine for treating biliary atresia, and relates to the technical field of disease treatment. The invention provides application of N-acetylcysteine in preparing a product for treating biliary atresia. The inventor researches to find that after N-acetylcysteine is administered to treat the patient suffering from biliary atresia, CD177 of the patient+The number and the cell activity (ROS level, mitochondrial level and extracellular trap level) of the neutrophils are obviously reduced, the direct bilirubin and the total bilirubin are obviously reduced, the N-acetylcysteine has an improvement effect on the treatment of biliary atresia, and the N-acetylcysteine is safe and reliable and can be used for preparing products for treating biliary atresia. The medicine for treating biliary atresia provided by the invention comprises N-acetylcysteine, and has a treatment effect on biliary atresia.
Description
Technical Field
The invention relates to the technical field of disease treatment, in particular to application of N-acetylcysteine in preparing a product for treating biliary atresia and a medicine for treating biliary atresia.
Background
Biliary Atresia (BA) is a common disease causing neonatal obstructive jaundice, and has poor prognosis, high fatality rate, and unknown etiology and pathogenesis. The basic pathological changes are progressive inflammation of the intrahepatic and extrahepatic bile ducts and hepatic fibrosis. China is a high-incidence area of biliary atresia diseases, and the incidence rate is 1/5000-1/10000. Most children suffer from death from liver failure within 1 and half of their lives, mostly due to failure to perform liver transplantation, at 1-3 months after birth. At present, effective drug treatment is lacked, so that finding a drug capable of effectively improving the clinical symptoms of biliary atresia has great clinical significance.
In view of the above, the present invention is particularly proposed.
Disclosure of Invention
The first purpose of the invention is to provide the application of N-acetylcysteine in preparing products for treating biliary tract occlusion.
The second purpose of the invention is to provide a medicine for treating biliary atresia, which comprises N-acetylcysteine, can improve the clinical symptoms of patients with biliary atresia and has a therapeutic effect on biliary atresia.
In a first aspect, the invention provides the use of N-acetylcysteine in the manufacture of a product for the treatment of biliary atresia.
As a further aspect, the product comprises a medicament.
In a second aspect, the present invention provides a medicament for the treatment of biliary atresia, said medicament comprising N-acetylcysteine.
As a further technical scheme, the medicine also comprises pharmaceutically acceptable auxiliary materials.
As a further technical solution, the pharmaceutically acceptable auxiliary material includes one or more of diluents, fillers, excipients, binders, wetting agents, disintegrating agents, absorption enhancers, surfactants, adsorption carriers, lubricants and flavors.
As a further technical scheme, the dosage form of the medicine comprises an oral preparation or an injection preparation.
As a further technical scheme, the oral preparation comprises tablets, capsules, granules, pills, syrups, oral solutions, oral suspensions or oral emulsions.
As a further technical scheme, the injection preparation comprises injection or powder injection.
According to a further technical scheme, the dosage of the medicine is 120-180 mg/kg-qd calculated by the mass of N-acetylcysteine.
As a further technical scheme, the dosage of the medicine is 150 mg/kg-qd calculated by the mass of N-acetylcysteine.
Compared with the prior art, the invention has the following beneficial effects:
the invention provides application of N-acetylcysteine in preparing a product for treating biliary atresia. The inventor researches to find that after N-acetylcysteine is administered to treat the patient suffering from biliary atresia, CD177 of the patient+The number and activity of neutrophils (ROS level, mitochondria level and extracellular trapping net level) are obviously reduced, and the direct bilirubin isAnd total bilirubin is obviously reduced, the N-acetylcysteine has an improvement effect on the treatment of biliary atresia, and the safety is reliable, so that the N-acetylcysteine can be used for preparing products for treating biliary atresia.
The invention provides a medicine for treating biliary atresia, which comprises N-acetylcysteine and has a treatment effect on biliary atresia.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.
FIG. 1 is a graph showing CD177 in each peripheral blood sample provided in example 1 of the present invention+The number of neutrophils in proportion;
FIG. 2 shows the results of intracellular ROS staining in each peripheral blood sample provided in example 1 of the present invention;
FIG. 3 shows CD177 of each peripheral blood sample provided in example 1 of the present invention+(ii) neutrophil mitochondrial immunofluorescence results;
FIG. 4 is a graph showing CD177 in each peripheral blood sample provided in example 1 of the present invention+Performing immunofluorescence of the neutrophil extracellular trapping net;
FIG. 5 shows direct bilirubin and total bile acid levels in various peripheral blood samples provided in example 1 of the present invention.
Detailed Description
Embodiments of the present invention will be described in detail below with reference to embodiments and examples, but those skilled in the art will understand that the following embodiments and examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention. Those who do not specify the conditions are performed according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
In a first aspect, the invention provides the use of N-acetylcysteine in the manufacture of a product for the treatment of biliary atresia.
In previous studies, the inventor unexpectedly found that N-acetylcysteine (NAC) has a certain therapeutic effect on BA. NAC has the effects of protecting liver and promoting bile excretion clinically, and the intravenous dosage form of the NAC is used for treating patients with early stage hepatic failure, and the use safety of the NAC is reported in diseases in pregnancy and newborn period. The inventor researches and discovers that CD177+Cells can cause Biliary Atresia (BA) by releasing extracellular trapping NETs (NETs), and the results of cell and animal studies show that N-acetylcysteine is an inhibitor of NETs, and the inhibition of NETs can block CD177 in vitro+The damage of the cells to the biliary epithelial cells has the function of inhibiting the formation process of biliary atresia of mice. Thus, the inventors tried to use NETs-inhibiting drugs in patients with biliary atresia.
Thus, the inventors tried to use NAC in BA patients by comparing peripheral blood CD177 before and after treatment with the drug group and between the drug-free groups+The safety and effectiveness of NAC in the clinical treatment of biliary atresia is explored by the number of cells, intracellular ROS levels, the number of mitochondria, the formation of extracellular traps, and the liver and kidney functions of patients, and the basic clinical signs and symptoms of patients. The research result shows that after the N-acetylcysteine is administrated to treat the children with biliary tract atresia, the CD177 of the children is+The number and the cell activity (ROS level, mitochondrial level and extracellular trap level) of the neutrophils are obviously reduced, the direct bilirubin and the total bilirubin are obviously reduced, the N-acetylcysteine has an improvement effect on the treatment of biliary atresia, and the N-acetylcysteine is safe and reliable and can be used for preparing products for treating biliary atresia.
As a further aspect, the product includes, but is not limited to, a pharmaceutical, or other therapeutically effective product known to those skilled in the art.
In a second aspect, the present invention provides a medicament for the treatment of biliary atresia, said medicament comprising N-acetylcysteine.
The invention provides a medicine for treating biliary atresia, which comprises N-acetylcysteine, so that the medicine has an improvement effect on the treatment of biliary atresia and can be used for treating patients with biliary atresia.
As a further technical scheme, the medicine can also comprise pharmaceutically acceptable auxiliary materials besides the N-acetylcysteine.
As a further technical solution, the pharmaceutically acceptable auxiliary material includes one or more of diluents, fillers, excipients, binders, wetting agents, disintegrating agents, absorption enhancers, surfactants, adsorption carriers, lubricants and flavors.
As a further technical scheme, the dosage form of the medicine comprises an oral preparation or an injection preparation.
As a further technical scheme, when the medicine is orally taken, the medicine can be prepared into any orally acceptable preparation form, such as, but not limited to, tablets, capsules, granules, pills, syrups, oral solutions, oral suspensions or oral emulsions.
As a further technical scheme, when the medicine is administered in the form of injection, the medicine can be prepared into any preparation form acceptable for injection, such as but not limited to injection solution or powder injection.
In a further embodiment, the dosage of the drug is 120 to 180 mg/kg.qd, for example, but not limited to, 120 mg/kg.qd, 130 mg/kg.qd, 140 mg/kg.qd, 150 mg/kg.qd, 160 mg/kg. qd, 170 mg/kg. qd, or 180 mg/kg. qd based on the mass of N-acetylcysteine.
The term "120-180 mg/kg-qd" refers to 120-180 mg of a drug taken by each kilogram of a person once a day.
As a further technical scheme, the dosage of the medicine is 150 mg/kg-qd calculated by the mass of N-acetylcysteine.
In the invention, N-acetylcysteine with the dose of 120-180 mg/kg-qd is adopted for administration, so that the medicine has a good improvement effect on the treatment of biliary atresia.
The invention is further illustrated by the following specific examples and comparative examples, but it should be understood that these examples are for purposes of illustration only and are not to be construed as limiting the invention in any way.
Example 1
1. Material
Experimental reagent: DCFDA/H2DCFDA-Cellular ROS Assay kit (ab113851), Assay Hoechst 33342 stabilizing Dye Solution (ab228551), 7-AAD (ab228563), PE fluorescently labeled Anti-Human CD177 antibody (ab69777, Abcam), Human Fc Block (564765), APC-Cy7 fluorescently labeled Anti-Human CD45 antibody (557833, BD Biosciences), Anti-PE microbeads (130-048-801, Miltenyi Biotec), MitockeTM Red CMXRos (M7512, thermo scientific), MitockerTM Green FM(M7514，thermo scientific)。
Experimental equipment: magnetic cell sorter (Miltenyi Biotec, Germany), laser confocal microscope (Leica DMI8, German), BD FACS Aria II flow cytometer (BD Biosciences, USA).
2. Experimental procedure
1) Grouping
12 patients with biliary atresia were randomly divided into two groups, namely NAC-treated group and non-NAC-treated group, 6 patients in each group, half of men and women, and informed consent was issued before treatment, and a normal control group (NC group, non-biliary atresia patients) was set. The children patients in the NAC treatment group are injected with N-acetylcysteine (the dosage is 150 mg/kg/day for N-acetylcysteine, namely 5ml of NAC is dissolved in 30ml of 10% glucose every day) for 1 week on the basis of the conventional clinical application of biliary atresia; the non-NAC treatment group was treated with conventional biliary atresia clinical medication. Peripheral blood samples of an NAC treatment group and a non-NAC treatment group before and after treatment and peripheral blood samples of a normal control group are respectively taken for detection, wherein the samples of the NAC treatment group before treatment are recorded as: BA; samples after treatment in the NAC treatment group were scored as: BA + NAC; the normal control group was noted as: and (5) NC. One week later, the above samples were sorted for peripheral blood CD177+Cells, detecting changes in their numbers, intracellular ROS levels, mitochondria and extracellular traps.
In order to further discuss the safety and the effectiveness of the medicine on the treatment of patients, 4 patients are treated for one week after the gexi operation, and the patients have no adverse reaction.
2) CD177 in peripheral blood+Cell sorting
Erythrocyte lysate is used for cracking erythrocytes in peripheral blood of children patients in NC group and NAC treatment group before and after treatment to prepare single cell suspension for standby. FcR blocking was added to the cell suspension separately and incubated at room temperature for 10min, anti-human PE conjugated CD177 antibody was added and incubated at 4 ℃ for 30min, flow staining buffer was added after washing twice, anti-PE Microbeads were added and incubated at 4 ℃ for 15min, flow staining buffer was added after washing twice, CD177 was performed on a magnetic cell sorter (Miltenyi Biotec, Germany) using an LD magnetic separation column+Sorting of cells, sorted CD177+Cells cell purity was examined on BD FACS Asia II (BD Biosciences, USA), and used>95% of the cells were subjected to the next experiment.
3) BA infant CD177+Cell analysis
Lysis of NAC-treated group blood samples into single cell suspensions using erythrocyte lysate according to manufacturer's instructions, taking 2) single cell suspensions, adding Human Fc Block to incubate for 10min at room temperature, adding anti-Human PE conjugated CD177, anti-Human APC-Cy7 conjugated CD45, incubating for 30min at 4 ℃, after centrifugation and washing, adding 7-AAD dye to stain dead cells, adding FACS buffer to reselect cells, performing detection analysis on BD FACS Aria II (BD Biosciences, USA) flow cytometer, and analyzing using Flowjo10.0, the analysis results are shown in FIG. 1.
As can be seen from FIG. 1, the patient with biliary atresia had CD177 in their peripheral blood before treatment+The cell ratio is 29.3%, and after one week of treatment with N-acetylcysteine, CD177 in peripheral blood of children patients+The cell proportion was 12.2%, which was close to the normal group (CD 177 in peripheral blood)+The cell ratio is 10.1 percent), which shows that the N-acetylcysteine can obviously reduce the content of the drug after being used for treating the patient with biliary tract atresiaCD177 in peripheral blood of children with low patient's risk+(CD45+CD177+) The number of cells.
4) Staining of intracellular ROS Using DCFDA reagents
a. Take 1.5X 1052) CD177 sorted from peripheral blood or liver tissue in step+A cell;
b. washing the cells once by centrifugation with 1Xbuffer 500g for 5min, removing the supernatant and leaving the cell precipitate;
c. DCFDA fluorescent staining solution was diluted to 20. mu.M using 1 Xbuffer;
d. adding 20 mu M DCFDA fluorescent staining solution into the cell sediment, uniformly mixing, and incubating for 30 minutes at 37 ℃ in the dark;
e. washing the cells once by centrifugation with 1Xbuffer 500g for 5min, removing the supernatant and leaving the cell precipitate;
f. 200ul 1Xbuffer was added to resuspend the cells, analyzed by flow cytometry, and the mean fluorescence intensity of DCFDA fluorochrome, which represents the intracellular ROS level, was measured using 488 laser. The results of the test are shown in FIG. 2 (in the figure, Negative Control).
As can be seen from FIG. 2, the mitochondrial ROS level in the peripheral blood of the patient with biliary atresia before treatment is higher, and after one week of treatment with N-acetylcysteine, the mitochondrial ROS level in the peripheral blood of the patient is remarkably reduced and is close to that of the normal group, which shows that after the patient with biliary atresia is treated with N-acetylcysteine, the mitochondrial ROS level in the peripheral blood of the patient can be remarkably reduced.
5)CD177+Intracellular mitochondrial fluorescent staining
Take 1.5X 1052) separation of CD177 from peripheral blood in step (a)+Cells were individually added 500nM MitoTrackerTMRed CMXRos dye and 150nM MitoTrackerTMGreen FM dye, in 37 ℃ cell culture box shading incubation for 45min, cell smear on polylysine coated slide glass, adding Dapi dye to stain cell nucleus, covering with cover glass and adding sealing piece, and laser confocal microscope observation and photograph. The immunofluorescence results are shown in FIG. 3 (Green fluorescence using M)itoTrackerTMGreen CMXRos, a mitochondrial specific dye; MitoTracker for Red fluorescenceTMRed CMXRos, mitochondrial specific dye).
As can be seen in FIG. 3, CD177 sorted in peripheral blood of choledocholithiasis patients after NAC treatment was compared to control and before administration to choledocks+The expression of the neutrophil mitochondria is significantly reduced.
6) BA infant CD177+Extracellular trapping net staining of neutrophils formed by cells
To evaluate each group of CD177+The NET level of cell production was 1.5X 1052) separation of CD177 from peripheral blood in step (a)+Cells were smeared onto polylysine-coated slides, and CD177 was applied to the slides using Hoechest3342 dye+The neutrophil extracellular trap formed by the cells is stained.
As can be seen in FIG. 4, CD177 sorted in peripheral blood of BA patients after treatment with NAC compared to control and pre-drug patients+Neutrophil Extracellular Traps (NETs) levels were significantly reduced.
7) Biochemical indexes of liver and gall are detected by Hitachi Pre-Analytical Process Automation System with 7600Clinical Analyzer in Clinical laboratory department of the hospital, and comprise the following steps: gamma-glutamyl transpeptidase (GGT), Direct Bilirubin (DBIL), Indirect Bilirubin (IBIL), Total Bilirubin (TBIL), Total Bile Acid (TBA), alkaline phosphatase (ALP), serum alanine Aminotransferase (ALT), aspartate Aminotransferase (AST), Albumin (ALB), Globulin (GLO), and total protein (TB). Statistics and comparison are carried out on blood routine and liver function indexes before and after treatment by the medicine group and the medicine group without the medicine group. The hepatic function results are shown in FIG. 5 (prior treatment; posttreatment; control group: non-NAC treated group; treatment group: NAC treated group).
As can be seen from fig. 5, after the conventional treatment, the direct bilirubin and total bile acid levels of the infant with biliary atresia are slightly reduced, while after the N-acetylcysteine treatment, the direct bilirubin and total bile acid levels of the infant with biliary atresia are obviously reduced, and the final direct bilirubin and total bile acid levels are lower than those of the conventional treatment, which indicates that the N-acetylcysteine treatment effect of the infant with biliary atresia is better.
8) Patients were closely observed for adverse reactions during dosing: blood routine, liver function, kidney function index, gastrointestinal tract reaction (nausea, vomiting, diarrhea), nervous system reaction (delirium, convulsion), skin and connective tissue (rash, edema), general condition of the infant (body temperature, mental state, heart rate, respiration) and anaphylaxis. The results show that all indexes of the patient are in a normal range during the medication period, and no adverse reaction and anaphylactic reaction exist, which indicates that NAC is safe and has no obvious side effect.
Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.
Claims (10)
- Use of N-acetylcysteine in the preparation of a product for the treatment of biliary atresia.
- 2. The use of claim 1, wherein the product comprises a medicament.
- 3. A medicament for the treatment of biliary atresia, wherein said medicament comprises N-acetylcysteine.
- 4. The medicament of claim 3, further comprising a pharmaceutically acceptable excipient.
- 5. The medicament of claim 4, wherein the pharmaceutically acceptable excipients comprise one or more of diluents, fillers, excipients, binders, humectants, disintegrants, absorption enhancers, surfactants, adsorptive carriers, lubricants, and flavoring agents.
- 6. The medicament of claim 3, wherein the dosage form of the medicament comprises an oral preparation or an injectable preparation.
- 7. The medicament of claim 6, wherein the oral formulation comprises a tablet, a capsule, a granule, a pill, a syrup, an oral solution, an oral suspension, or an oral emulsion.
- 8. The medicament of claim 6, wherein the injection preparation comprises injection solution or powder injection.
- 9. The medicament according to any one of claims 3 to 8, wherein the medicament is administered in an amount of 120 to 180 mg/kg-qd, based on the mass of N-acetylcysteine.
- 10. The medicament according to claim 9, wherein the medicament is administered in an amount of 150 mg/kg-qd, based on the mass of N-acetylcysteine.
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| PCT/CN2022/085877 WO2022262388A1 (en) | 2021-06-15 | 2022-04-08 | Application of n-acetylcysteine in preparation of product for treating biliary atresia, and drug for treating biliary atresia |
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| WO2022262388A1 (en) * | 2021-06-15 | 2022-12-22 | 广州市妇女儿童医疗中心 | Application of n-acetylcysteine in preparation of product for treating biliary atresia, and drug for treating biliary atresia |
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| CN105777856A (en) * | 2016-04-23 | 2016-07-20 | 陈昊 | Medicine composition with acetylcysteine and medical application of medicine composition |
| CN109419793A (en) * | 2017-08-24 | 2019-03-05 | 中国人民解放军第二军医大学东方肝胆外科医院 | N-acetylcystein is preparing the application in cancer treatment drug |
| WO2021167949A1 (en) * | 2020-02-18 | 2021-08-26 | Children's Hospital Medical Center | Compositions and methods for treating liver disease |
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| CN104758269A (en) * | 2015-02-12 | 2015-07-08 | 北京众盈汇丰科技发展有限责任公司 | Acetylcysteine effervescent tablet |
| CN113209070A (en) * | 2021-06-15 | 2021-08-06 | 广州市妇女儿童医疗中心 | Application of N-acetylcysteine in preparation of product for treating biliary atresia and medicine for treating biliary atresia |
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| CN105777856A (en) * | 2016-04-23 | 2016-07-20 | 陈昊 | Medicine composition with acetylcysteine and medical application of medicine composition |
| CN109419793A (en) * | 2017-08-24 | 2019-03-05 | 中国人民解放军第二军医大学东方肝胆外科医院 | N-acetylcystein is preparing the application in cancer treatment drug |
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| WO2022262388A1 (en) * | 2021-06-15 | 2022-12-22 | 广州市妇女儿童医疗中心 | Application of n-acetylcysteine in preparation of product for treating biliary atresia, and drug for treating biliary atresia |
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