CN111214495B

CN111214495B - Application of mycobacterium vaccae for injection in preparation of medicine for preventing and treating respiratory system RSV infection

Info

Publication number: CN111214495B
Application number: CN202010136371.8A
Authority: CN
Inventors: 李超乾; 姜晓红
Original assignee: First Affiliated Hospital of Guangxi Medical University
Current assignee: First Affiliated Hospital of Guangxi Medical University
Priority date: 2020-03-02
Filing date: 2020-03-02
Publication date: 2021-12-31
Anticipated expiration: 2040-03-02
Also published as: CN111214495A

Abstract

The invention belongs to the field of medicines, and particularly relates to application of mycobacterium vaccae for injection in preparation of a medicine for preventing and treating respiratory system RSV infection. The mycobacterium vaccae for injection can be used as a single preparation, can also be compatible with other medicines, is added with a proper amount of pharmaceutically acceptable auxiliary materials, and is prepared into a pharmaceutically acceptable preparation by adopting the prior art. The medicine prepared by the invention can adopt an atomization inhalation treatment mode, and the medicine directly enters an airway, takes effect quickly and has less side effect on the whole body through short-term atomization inhalation; short atomization time and good patient tolerance. Experiments prove that the Mycobacterium vaccae (microcard) for aerosol inhalation injection can obviously reduce RSV in lungs of mice; the micro-card aerosol inhalation can prevent respiratory system RSV infection. The new application of the mycobacterium vaccae for injection in preparing the medicine for preventing and treating respiratory system RSV infection provides a new way and scheme for safely and effectively preventing and treating respiratory system RSV infection in clinic.

Description

Application of mycobacterium vaccae for injection in preparation of medicine for preventing and treating respiratory system RSV infection

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of mycobacterium vaccae for injection in preparation of a medicine for preventing and treating respiratory system RSV infection.

Background

RSV (respiratory syncytial virus) is a common pathogen causing respiratory infections. Respiratory viral infection has always been the most important infectious disease in humans, and its prevention and treatment remains a major academic challenge. RSV is an RNA virus belonging to the family of paramyxoviridae and is transmitted by air entrainment and intimate contact. RSV is a main pathogen of lower respiratory tract infection of infants and adults in the whole world, and the early severe RSV infection of the infants is closely related to the development of bronchial asthma after adults; since 2000, the method and the effect of short-term aerosol inhalation of mycobacterium to adjust respiratory tract mucosal immunity and prevent and treat bronchial asthma are researched on animals and human bodies in sequence, the respiratory tract mucosal immunity is found to have local and aging uniqueness, and the aerosol inhalation of inactivated mycobacterium phlei can remarkably increase the respiratory tract Interferon (IFN) level for a plurality of days to correct Th1/Th2 imbalance of asthma, so that the airway responsiveness of patients with moderate asthma is remarkably reduced, the symptoms are remarkably relieved, and the aerosol inhalation of mycobacterium phlei has a prevention and treatment effect on asthma.

Interferon (IFN) is the most important and possibly the only natural non-specific antiviral natural component in the body. IFN is the most important non-specific antiviral component found and recognized in vivo at present, and is classified into type I (IFN-. beta.and IFN-. alpha.), type II (IFN-. gamma.) and type III (IFN-. lambda.1, IFN-. lambda.2, IFN-. lambda.3) IFN. IFN regulates the transcription of hundreds of related effect antiviral genes (ISG) by activating an intracellular JAK-STAT pathway of an infected part after being generated, thereby inhibiting virus proliferation, promoting the apoptosis of infected cells, enabling uninfected cells to enter an antiviral state, promoting the development and maturation of various natural immune cells, adaptive immune cells and effector cells and promoting the adaptive immune response to play an antiviral role. Type I and type III IFNs have antiviral effects, especially type III IFNs are critical in respiratory antiviral defense. After respiratory virus infection, IFN acts on an IFN receptor of an infected cell, and then through a JAK-STAT pathway, the expression of antiviral effect genes such as Mx1 and ISG15 is activated, the transcription and protein synthesis of the respiratory virus are inhibited, and the apoptosis of an airway epithelial cell infected by the virus is promoted to play an antiviral effect. Furthermore, Petersen E et al have shown that intranasal pretreatment of mice with poly (I.C) (TLR 3 agonist) and to a lesser extent CpG, R848 or lipopolysaccharide (TLR 9, TLR7/8 or TLR4 agonist) up-regulates the expression of interferon-beta (IFN-. beta.), IFN-. gamma., IL-1. beta. and Tumor Necrosis Factor (TNF) genes in the lung, exerting a protective effect against SARS coronavirus infection.

The mycobacterial agent (such as Bacillus Calmette-Guerin, inactivated Mycobacterium phlei, and injectable Mycobacterium vaccae) can increase interferon level. The mycobacterial preparation is a multifunctional immunomodulator, can be used for preventing and treating infection, mainly regulates the immune function of an organism by influencing immune response reaction, enhances the activity of Th cells, stimulates B cells to enter the proliferation and differentiation stages, and finally promotes the formation of specific antibodies. Studies such as Yangjing and the like show that the inactivated mycobacterium phlei can obviously improve the function of Th (including Th1 and Th2) cells in Peripheral Blood Mononuclear Cells (PBMC) of asthmatic patients, promote the PBMC to generate IFN-gamma and IL-4, particularly improve the Th1 type cell factor IFN-gamma, promote the maturation of Th1 and Th2 cells, simultaneously up-regulate the function of Th1 of asthmatic children, partially correct the Th2 dominant state of the asthmatic children, further improve the Th1/Th2 unbalanced state of the asthmatic children, and play an important role in asthma prevention and treatment.

Disclosure of Invention

The invention aims to provide application of mycobacterium vaccae for injection in preparation of a medicament for preventing and treating respiratory system RSV infection, and provides a new scheme for preventing and treating respiratory system RSV infection.

In order to achieve the purpose, the invention provides the following technical scheme:

the invention discloses application of mycobacterium vaccae for injection in preparation of a medicament for preventing and treating respiratory system RSV infection.

The application of the mycobacterium vaccae for injection in preparing the medicine for preventing and treating respiratory RSV infection comprises the active ingredient of the mycobacterium vaccae for injection.

The mycobacterium vaccae for injection can be used as a single preparation and can also be matched with other medicines, and a proper amount of pharmaceutically acceptable auxiliary materials are added to prepare a medicinal preparation by adopting the prior art.

The mycobacterium vaccae for injection is applied to preparation of medicines for preventing and treating respiratory system RSV infection, and the dosage forms of the medicines for preventing and treating respiratory system RSV infection comprise aerosol, aqua, powder and the like.

Further, the application of the mycobacterium bovis for injection in preparing the medicine for preventing and treating respiratory system RSV infection is that when the mycobacterium bovis for injection is used, the mycobacterium bovis for injection is prepared into a physiological saline mixed solution with the concentration of 2.25-6.75 mu g/ml for atomization and inhalation.

The properties and clinical effects of the drugs are described below:

mycobacterium vaccae for injection (microca), english name: mycobacterium vaccae for Injection; [ types of drugs ] biological products, [ major components ] Mycobacterium vaccae mycoprotein, [ properties ] white loose powder, [ specifications ] 22.5 μ g/bottle, [ pharmacological effects ] Mycobacterium vaccae for injection has bidirectional immunoregulation function, can improve the level of hydrogen peroxide and nitric oxide generated by normal mouse abdominal cavity macrophages and promote the proliferation reaction of T lymphocytes, can obviously inhibit the reaction of tubercle bacillus infected guinea pigs, and has obvious enhancement effect on lymphocyte transformation and macrophage phagocytic function of mice with low immune function; [ ADAPTIVE PROBLEMS ] the product is a bidirectional immunoregulation preparation, can be used as a combined medicine for adjuvant therapy of tuberculosis chemotherapy, and [ safety before clinic ] the product is injected into mice by muscle route in 3 different doses, wherein the doses are 5000 times, 2500 times and 1250 times of the human dose respectively, and the abnormal symptoms and death of the mice are not caused. The administration of the drug is 78 times and 1560 times of the dosage of a rat and 4 times and 100 times of the dosage of a clinical human of a test dog, and the experimental result shows that under the dosage, the test dog and the rat have no toxic reaction in the aspects of indexes such as general behaviors, hematology, biochemistry, pathology and the like; adverse reactions very individual patients may develop local rashes, induration or fever. [ contraindication ] patients with allergic or allergic constitution are disclosed. (2) Patients with severe heart disease, myocardial damage, significant vascular sclerosis, endocarditis. (3) Women in gestation period. (4) Extreme asthenia and severe anemia.

When the mycobacterium bovis for injection is used, the mycobacterium bovis for injection is prepared into a physiological saline mixed solution with the concentration of 2.25-6.75 mu g/ml for atomization and inhalation. The specific steps of using the medicament to prevent and treat respiratory system RSV infection are as follows: mycobacterium vaccae (microcard) for injection 22.5 microgram/bottle × 1+ normal saline 10ml, making into normal saline mixed solution, and atomizing and inhaling in a closed container through an atomizer, Qd, 5 days/treatment course.

The injection mycobacterium vaccae is applied to the preparation of the medicine for preventing and treating respiratory system RSV infection, the prepared medicine dosage forms comprise aerosol, aqueous solution, powder and the like, an aerosol inhalation treatment mode can be adopted, and the medicine directly enters an airway in the aerosol inhalation treatment mode and has quick response; the particles are uniform and easy to deposit in an airway, and the atomized and inhaled medicine directly acts on the diseased bronchus, bronchiole and lung tissues, so that the treatment effect of the medicine can be improved, and the side effect of the whole body is less; the atomization time is short, and the tolerance of a patient is good; simple operation and easy matching (suitable for both the old and the young). The medicine with the special application provided for the patient can improve the compliance of the patient through short-term atomization inhalation, so that the patient can take the medicine conveniently, the medical cost is reduced, and the market prospect is good.

Experiments prove that the Mycobacterium vaccae (microcard) for aerosol inhalation injection can obviously reduce RSV in lungs of mice; the micro-card aerosol inhalation can prevent respiratory system RSV infection, is beneficial to preventing and controlling respiratory system RSV infection, and is worthy of further clinical popularization and application.

The new application of the mycobacterium vaccae for injection in preparing the medicine for preventing and treating respiratory system RSV infection provides a new way and scheme for safely and effectively preventing and treating respiratory system RSV infection in clinic.

Drawings

FIG. 1 is a graph of airway responsiveness test results;

FIG. 2 is an immunofluorescence image of group A in animal experimental lung immunofluorescence assays;

FIG. 3 is an immunofluorescence image of group B in animal experimental lung immunofluorescence assays;

FIG. 4 is an immunofluorescence image of group C in animal experimental lung immunofluorescence assays;

FIG. 5 is an immunofluorescence image of group D in animal experimental lung immunofluorescence assays;

FIG. 6 is an immunofluorescence image of group E in animal experimental lung immunofluorescence assays;

FIG. 7 is an immunofluorescence image of group F in animal experimental lung immunofluorescence assays;

FIG. 8 is an immunofluorescence image of group G in animal experimental lung immunofluorescence assays;

as can be seen from the immunofluorescence images of the 7 different groups in FIGS. 2-8, the normal control group A is negative, the fluorescence of the groups B and E is strongly positive, the fluorescence of the group C, D, F, G is weakly positive, and there is no obvious difference between the different PFU infection groups and the different minicard groups;

FIG. 9 is an electron micrograph of group B among the groups of mice in animal experiment 7;

FIG. 10 is an electron micrograph of group C among the groups of mice in animal experiment 7;

FIG. 11 is an electron micrograph of group D among the groups of 7 mice in the animal experiment;

FIG. 12 is an electron micrograph of group E among the groups of mice in animal experiment 7;

FIG. 13 is an electron micrograph of group F among the groups of mice in animal experiment 7;

FIG. 14 is an electron micrograph of group G among the groups of 7 mice in animal experiment;

as can be seen from FIGS. 9-14, a large amount of RSV was observed under the electron microscope for group B (106 PFU-infected group), RSV was occasionally observed under the electron microscope for group E (107 PFU-infected group), no virus was observed under the microscope for group micro-truck intervention (group C, group D, group F, group G), and lamellar body destruction was observed under all the group microscopes.

Detailed Description

Examples

The microcard 22.5 microgram/bottle X1 + normal saline 10ml is atomized in a closed container by an atomizer to allow the experimental animal to inhale until all the liquid medicine is inhaled (about 20 minutes), Qd, 5 days/course of treatment.

The following are animal tests of the inventive medicament:

1. materials and methods

1.1 materials and equipment: experimental animals: healthy female Balb/c mice aged 4-6 weeks, having a weight of 20-25g and an SPF (specific pathogen free) grade, are fed in an air-conditioned room at a temperature of 23 +/-3 ℃ and a humidity of 55.5 +/-10% by using pellet feed and are fed with free drinking water. Mycobacterium vaccae for injection (trade name: microcard, component is Mycobacterium vaccae mycoprotein, Chinese medicine standard S200110003) is available from Biopharmaceutical Co., Ltd, Naja Najordan Temminck, Japan; RSV (Strain Long) from the American Type Culture Collection (ATCC), anti-RSV immunofluorescence kit from Abcam; the total RNA extraction kit, the transcription-simulating kit, the RT-PCR kit and the IFN-gamma mRNA primer are all from TaKaRa company; methacholine was from Sigma, usa; chloral hydrate is from Qingdao Yulong seaweed Co., Ltd, the Chinese medicine standard H37022673; the TBL4500 noninvasive small animal lung function instrument is from BUXCO company of America; the ultrasonic atomizer WH-2000 is from Guangdong Yuehua medical instruments factory; self-made atomizing suction box.

1.2 Experimental methods

1.2.1 animal grouping and intervention: 56 mice were randomly divided into 7 groups of 8 mice each, normal control group (group A), RSV10⁶Titer infection group (group B), mini-1 arm nebulization for prevention of RSV10⁶Titer infection group (group C), 3-arm microcard nebulization for prevention of RSV10⁶Titer infection group (group D), RSV10⁷Titer infection group (group E), mini-1 arm nebulization for prevention of RSV10⁷Titer infection group (group F), 3-arm microcard nebulization for prevention of RSV10⁷Titre infected groups (group G). Adding microcard into 10ml PBS solution 1 week before group virus infection, and performing ultrasonic atomization inhalation 1 time per day for 5 days, and performing virus infection on 7 days. Mice in the virus infection group are injected with hydrated aldehyde (0.35 mg/g body weight) into the abdominal cavity, then are respectively injected with 50 mul drops of RSV with different titers into the left nostril, 1 time per day for 3 consecutive days, and the mice are sacrificed after 4 days of virus infection by measuring the reactivity of the airway.

1.2.2 airway reactivity assay: the method comprises the steps of measuring the gas channel reactivity of a TBL4500 small animal noninvasive lung function instrument on the 4 th day after virus infection, calibrating the instrument, loading the instrument into an animal, opening bias ventilation, starting an experiment, respectively administering PBS (phosphate buffer solution) liquid and 20 mu l of Mch with the concentration of 6.25mg/ml, 12.5mg/ml and 25mg/ml after the mouse adapts for 5 minutes, carrying out atomization excitation on each Mch for 30 seconds, recording data for 3 minutes, recovering for 4 minutes, then carrying out atomization excitation on the next concentration, automatically outputting the data and a statistical result after the experiment is finished, and expressing the gas channel reactivity by using sRaw (Specific air resistance).

1.2.3 Electron microscopy: the mice were sacrificed immediately and fixed with glutaraldehyde from the upper left lung tissue for 2 hours and examined by electron microscopy.

1.2.4 lung immunofluorescence: after the section is fixed, a 1:80RSV fluorescent antibody is added, the section is incubated for 45 minutes at room temperature, and the section is washed and observed under a fluorescence inverted microscope after being sealed.

1.2.5 statistical methods: data to

Presentation, comparison between groups by one-way analysis of variance, P<A difference of 0.05 was significant. The plots were made using GraphPad Prism 5.0 software.

1.3 results

1.3.1 general case: no mouse died in the experiment, the mice in the control group infected by the virus are poor in spirit and easy to control when the 2 nd virus infection is carried out, and the mice in the micro-card atomization intervention group are good in spirit.

1.3.2 airway reactivity test results: after viral infection, airway reactivity is obviously improved, and the P value of each group is less than 0.05 compared with a normal control group; but the virus infection control group and the microcard atomization intervention group have no difference, and the P values are all more than 0.05; the comparison between different PFUs and different microcard intervention groups was not different, and the P values were all >0.05 (see FIG. 1).

1.3.3 immunofluorescence see FIGS. 2-8, which shows that the normal control group A is negative, the groups B and E are strongly positively expressed in fluorescence, the group C, D, F, G is weakly positively expressed, and there is no obvious difference between the different PFU infection groups and the different microcarboards.

1.3.4 Electron microscopy: a large amount of RSV can be seen under an electron microscope in the group B (106 PFU infection group), RSV can be occasionally seen under an electron microscope in the group E (107 PFU infection group), viruses can not be seen under the micro-card intervention group (group C, group D, group F and group G) and lamellar body destruction can be seen under all the groups of lenses (see figures 9-14).

1.4 discussion

The mycobacterial agent (such as Bacillus Calmette-Guerin, inactivated Mycobacterium phlei, and injectable Mycobacterium vaccae) can increase interferon level. The mycobacterial preparation is a multifunctional immunomodulator, can be used for preventing and treating infection, mainly regulates the immune function of an organism by influencing immune response reaction, enhances the activity of Th cells, stimulates B cells to enter the proliferation and differentiation stages, and finally promotes the formation of specific antibodies. The previous series of researches also prove that the inactivated mycobacteria are inhaled in an atomizing mode for the first time to adjust Th1/Th2 balance, so that the airway responsiveness of patients with mild and moderate bronchial asthma is obviously reduced, and the asthma symptoms are relieved. Meanwhile, the immunoreactivity of respiratory tract inhalation immunity and intradermal and subcutaneous injection immunity is found to have obvious difference, particularly the characteristics of quick and strong immunoreaction in the aspects of timeliness and local immunoreaction intensity, and obvious adverse reaction is not found in more than 500 cases of clinical application. (the series of research results successively obtain the second-class prize and the first-class prize of Guangxi scientific and technological progress).

The previous series of researches prove that the asthma can be well prevented and treated by inactivating mycobacteria through atomization inhalation, and the main mechanism is to quickly up-regulate the level of respiratory mucosa-lung tissue interferon and correct the imbalance of Th1/Th 2; in combination with the similarities between viral infection and asthma immunity (both Th2 dominant responses) and the clinical manifestations of asthma induced by respiratory viral infection, we speculate that: (1) the aerosol inhalation inactivated mycobacterium can be used for non-specifically preventing and treating respiratory virus infection. (2) The aerosol inhalation inactivated mycobacterium can increase I, III type IFN expression by regulating DC innate immunity, and can non-specifically prevent respiratory virus infection. (3) The Mycobacterium vaccae for aerosol inhalation injection can prevent respiratory system RSV infection. Based on the hypothesis, based on the previous research, an animal experiment for preventing mouse RSV infection by Mycobacterium vaccae (trade name: microca) for aerosol inhalation injection is carried out to evaluate the effectiveness and safety of the animal experiment, and a new way and a new scheme are provided for clinically preventing RSV infection.

Research results show that the reactivity of the mouse airway is increased after virus infection, the microcard atomization intervention group is reduced, but the statistical difference is not achieved compared with a virus infection control group, the phenomenon is basically consistent with the occurrence and development conditions of clinical asthma, and the significance of the statistical difference is not achieved, and the estimation is related to the change time lag of pathology; the virus infection control group shows strong positive expression in immunofluorescence examination, and the microcard atomization intervention group shows negative expression; a large amount of RSV can be seen under an electron microscope of 106PFU infection group, RSV is occasionally seen under a 107PFU infection group, RSV is not seen under a microcard intervention group electron microscope, and lamellar body destruction can be seen under all group electron microscopes. The immunofluorescence result is basically consistent with the result of electron microscope examination, the morphologic reality shows that RSV of the microcard atomization intervention group is obviously reduced or even can not be seen, the effect is obvious, and the effect is probably related to rapid high expression of I, III type IFN by respiratory immune cells induced by microcard.

And (4) conclusion: RSV infection can cause airway hyperreactivity, and the inhibition effect of short-term microcard aerosol inhalation on airway hyperreactivity caused by RSV infection is required to be further observed; the RSV in the lung can be obviously reduced by the microcard aerosol inhalation; the micro-card aerosol inhalation can prevent respiratory system RSV infection.

Claims

1. Application of mycobacterium vaccae for injection in preparing aerosol for preventing and treating respiratory system RSV infection is provided.