阅读说明：本技术 肌苷在制备预防和治疗结核病药物中的应用 (Application of inosine in preparing medicine for preventing and treating tuberculosis ) 是由 戈宝学 杨华 王菲 郭欣娅 于 2021-08-27 设计创作，主要内容包括：本发明提供了肌苷在制备预防和治疗结核病药物中的应用。本发明证实了肌苷显著降低结核分枝杆菌(Mtb)感染小鼠肺脏的荷菌量,减轻病理变化,抑制结核性肉芽肿恶化,并通过显著促进IL-1β和IL-6基因的表达,进而促进巨噬细胞对Mtb的清除,抑制Mtb的体内存活。因此,肌苷有望作为新的有效代谢分子用于制备预防和治疗结核病的药物,为治疗结核病提供新的策略。(The invention provides an application of inosine in preparing a medicament for preventing and treating tuberculosis. The invention proves that inosine obviously reduces the bacterial load of mycobacterium tuberculosis (Mtb) infected mouse lung, lightens pathological changes, inhibits the deterioration of tuberculous granuloma, and promotes the elimination of Mtb by macrophage and inhibits the survival of Mtb in vivo by obviously promoting the expression of IL-1 beta and IL-6 genes. Therefore, inosine is expected to be used as a new effective metabolic molecule for preparing the drugs for preventing and treating tuberculosis, and provides a new strategy for treating tuberculosis.)

1. The application of inosine in the preparation of the drugs for preventing and treating tuberculosis is characterized in that the drugs comprise inosine or pharmaceutically acceptable salts thereof as the main active ingredient.

2. Use of inosine for the preparation of a medicament for inhibiting the progression of tuberculous granuloma, wherein the medicament comprises inosine or a pharmaceutically acceptable salt thereof as a main active ingredient.

3. Use of inosine for the preparation of a medicament for promoting the clearance of mycobacterium tuberculosis by macrophages, wherein the medicament comprises inosine or a pharmaceutically acceptable salt thereof as a main active ingredient.

4. Use according to claim 3, wherein inosine promotes the clearance of M.tuberculosis by macrophages by promoting the expression of IL-1 β and IL-6 genes.

5. A medicament for the prevention and treatment of tuberculosis, characterized by comprising inosine or a pharmaceutically acceptable salt thereof as a main active ingredient.

6. The medicament of claim 5, further comprising a pharmaceutically acceptable carrier or excipient.

7. The medicine of claim 5, wherein the dosage form of the medicine is tablets, capsules, oral liquid, buccal agents, granules, pills, powder, paste, pellets, suspension, powder or injection.

8. The medicament of claim 5, wherein the administration route of the medicament is oral, transdermal, intramuscular, subcutaneous or intravenous injection.

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of inosine in preparing a medicine for preventing and treating tuberculosis.

Background

Tuberculosis (TB) is a chronic infectious disease caused by infection with mycobacterium Tuberculosis, and is still the second largest killer disease caused by a single infectious etiology. According to the report of the world health organization, about 1,000 million people newly develop active tuberculosis in 2019, and 145 million people die due to TB. A more serious challenge is that about one quarter of the world population is Latent tuberculosis infected (LTBI), of which about 5-10% may develop active tuberculosis, becoming a new source of infection, infecting more people. How to effectively control LTBI and prevent more people from infecting Mtb (Mycobacterium tuberculosis, Mtb) has important significance for turning the whole tuberculosis epidemic situation, and is very slow. On the other hand, multi-drug resistant tuberculosis, especially widely drug resistant tuberculosis, has become a new incurable disease, the epidemic situation of the drug resistant tuberculosis in China is more severe, the cure rate of the existing chemotherapy drugs is low, the death rate is high, and new treatment strategies and innovative drugs need to be developed urgently to meet the urgent requirements of the country for reducing the morbidity and the mortality of the tuberculosis.

Host-directed therapy (HDT) does not directly kill Mtb, but rather controls Mtb infection by modulating the Host's immune function. A plurality of clinical researches show that HDT drug adjuvant therapy can effectively shorten the medication time of patients, and especially has obvious curative effect on patients with multi-drug resistant tuberculosis. HDT is currently an emerging therapeutic modality in the tuberculosis field, and the drugs that have been reported and developed are still very limited. Therefore, finding more HDT medicines targeting host anti-tuberculosis immune pathways, especially targeting the development of granuloma is imminent, and has important significance for developing new treatment means in clinic. In recent years, more and more researches show that most diseases are caused and developed along with local or overall metabolic abnormalities, and the outcome of many diseases, especially cancer, can be directly influenced by intervening related metabolic pathways or artificially adding metabolites with protective effects, so that the method is possibly applied to clinic as a new treatment method. However, the influence of metabolites on the progress of tuberculosis diseases, particularly the development of granulomas, and the application thereof in anti-tuberculosis treatment are still lacking in relevant research and reports.

Inosine (Inosine), also known as Inosine, etc., formula C₁₀H₁₂N₄O₅It is a nucleoside compound formed by combining hypoxanthine and ribose, and is suitable for leukopenia, thrombocytopenia, various heart diseases, acute and chronic hepatitis, liver cirrhosis, etc. caused by various reasons, and can also be used for treating central retinitis, optic atrophy, etc. In the early stage, the lung and peripheral blood comparative metabonomics analysis of Mtb infected granuloma mice is carried out,the content of inosine in lung granuloma and peripheral blood of mice is found to be remarkably reduced, which suggests that inosine may participate in the process of the development of tuberculous granuloma, but the action and mechanism of the inosine in the development of the tuberculous granuloma are not clear, and particularly, the application of the inosine in the preparation of drugs for preventing and treating tuberculosis is not reported.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention provides the application of inosine in preparing the medicines for preventing and treating tuberculosis.

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

the first aspect of the present invention provides use of inosine in the preparation of a medicament for the prevention or treatment of tuberculosis, the medicament comprising inosine or a pharmaceutically acceptable salt thereof as a main active ingredient.

The second aspect of the present invention provides use of inosine for preparing a medicament for inhibiting the progression of tuberculous granuloma, which comprises inosine or a pharmaceutically acceptable salt thereof as a main active ingredient.

The third aspect of the present invention provides use of inosine for the preparation of a medicament for promoting the clearance of Mtb by macrophages, which comprises inosine or a pharmaceutically acceptable salt thereof as a main active ingredient.

Further, inosine promotes the clearance of Mtb by macrophages by promoting the expression of IL-1. beta. and IL-6 genes.

The fourth aspect of the present invention is to provide a medicament for preventing and treating tuberculosis, which comprises inosine or a pharmaceutically acceptable salt thereof as a main active ingredient.

Further, the medicine also comprises a pharmaceutically acceptable carrier or excipient.

Furthermore, the dosage form of the medicine is tablets, capsules, oral liquid, buccal agents, granules, medicinal granules, pills, powder, paste, pellets, suspensions, powder or injections.

Further, the administration route of the above drugs is oral, transdermal, intramuscular, subcutaneous or intravenous injection.

By adopting the technical scheme, compared with the prior art, the invention has the following technical effects:

the invention proves that inosine obviously reduces the bacterial load of the lung, lightens pathological changes, inhibits the deterioration of tuberculous granuloma, and further promotes the elimination of Mtb by macrophage and inhibits the survival of Mtb in vivo by obviously promoting the expression of IL-1 beta and IL-6 genes. Therefore, inosine is expected to be used as a new effective metabolic molecule for preparing the drugs for preventing and treating tuberculosis, and provides a new strategy for treating tuberculosis.

Drawings

FIG. 1 shows the result of the measurement of the amount of H37 Rv-infected mice in the lung after control or inosine treatment;

FIG. 2 shows the results of HE and acid fast staining of lungs after H37Rv infected mice were treated with control or inosine in one example of the present invention;

FIG. 3 shows the result of the measurement of the amount of Mycobacterium marinum infected adult fish after control or inosine treatment in accordance with an embodiment of the present invention;

FIG. 4 shows HE and acid-fast staining results of Mycobacterium marinum infected adult fish treated with control or inosine according to one embodiment of the present invention;

FIG. 5 is the intracellular CFU results of macrophage infection with H37Rv after control or inosine treatment in accordance with one embodiment of the present invention;

FIG. 6 shows the results of IL-1. beta. and IL-6 gene expression of macrophage infected with H37Rv after control or inosine treatment in accordance with one embodiment of the present invention;

FIG. 7 shows OD of HEK293, SH-SY5Y and HePG2 cells treated with different concentrations of inosine in one embodiment of the present invention₅₇₀And (4) light absorption value.

Detailed Description

The invention provides an application of inosine in preparing a medicament for preventing and treating tuberculosis, which mainly promotes the elimination of Mtb by macrophages, inhibits the in vivo survival of Mtb and inhibits the exacerbation of granuloma by remarkably promoting the expression of IL-1 beta and IL-6 genes.

The present invention also provides a medicament for the prevention and treatment of tuberculosis, which comprises inosine or a pharmaceutically acceptable salt thereof as a main active ingredient.

In a preferred embodiment of the present invention, the above-mentioned medicament further comprises a pharmaceutically acceptable carrier or excipient. The term "pharmaceutically acceptable" refers to substances that are suitable for use in humans without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio. The "carrier or excipient" includes one or more of binder, filler, diluent, tabletting agent, lubricant, disintegrating agent, coloring agent, flavoring agent, and humectant.

In a preferred embodiment of the present invention, the above-mentioned pharmaceutical composition is in the form of tablet, capsule, oral liquid, buccal agent, granule, pill, powder, paste, pellet, suspension, powder or injection.

In a preferred embodiment of the present invention, the above-mentioned drug is administered orally, transdermally, intramuscularly, subcutaneously or intravenously.

The present invention will be described in detail and specifically with reference to the following examples and drawings so as to provide a better understanding of the invention, but the following examples do not limit the scope of the invention.

In the examples, the conventional methods were used unless otherwise specified, and reagents used were those conventionally commercially available or formulated according to the conventional methods without specifically specified.

Example 1

In this example, C57BL/6 mice were infected with H37Rv model to analyze the effect of inosine on tuberculosis, and the specific experimental procedures and results are as follows:

female C57BL/6 mice, 6 per group, 6 at 6-8 weeks were infected with H37Rv strain, approximately 200 CFU/mouse, by nasal drip. After 3 weeks of infection, control group was added with 1% DMSO in the drinking water, inosine group was added with 1% inosine in the drinking water, and mice were sacrificed 4 weeks after administration. The effect of inosine on tuberculosis infection in mice was analyzed by testing the following criteria:

the lung load bacterial load: lungs were collected under aseptic conditions, homogenized with PBS, diluted 10-fold, inoculated on 7H10 agar medium, cultured at 37 ℃ for 4 weeks, observed for growth of H37Rv, and the lung burden was calculated, and as a result, it was found that the lung burden was significantly reduced after mice infected with H37Rv were administered inosine (fig. 1).

② lung histopathology detection: lungs were collected under sterile conditions, and a portion of lung tissue was fixed with 4% paraformaldehyde, dehydrated, wax-soaked, embedded, sliced, stained with H & E and acid-fast, and examined by microscope for pathological changes in lung tissue, and it was found that pathological changes in lung, and immune cell infiltration and lung tissue damage were significantly reduced in mice infected with inosine by drinking as compared to the control group (fig. 2).

The above results indicate that inosine can significantly improve the host's ability to resist Mtb.

Example 2

In this example, a zebrafish infection model was used to analyze the effect of inosine on mycobacteriosis, particularly granuloma, and the specific experimental procedures and results were as follows:

wild zebra fish (AB strain) was purchased from the China zebra fish resource center, raised in a circulating breeding system, transferred to a toxicology system for a Mycobacterium marinum infection test, while ensuring standard conditions for zebra fish raising and infection (water temperature about 28 ℃, pH about 7.4, conductivity about 1,500. mu.S). Adult zebrafish were anesthetized with 0.1% tricaine, infected with mycobacterium marinum by intraperitoneal injection, with an infectious dose of about 200CFU, 1 week later with DMSO administered orally to the control group and 0.01mg inosine administered orally to the inosine group. Sacrificed 1 week after administration, and the effect of inosine on the development of granuloma of adult fish of mycobacterium marinum-infected zebrafish was analyzed by the following criteria:

(1) the bacterial load: after infection administration, zebrafish were subjected to terminal anesthesia in 0.5% tricaine, homogenized in PBS, diluted 10 times, inoculated on 7H10 agar medium, cultured at 37 ℃ for 1-2 weeks, observed for growth of mycobacterium marinum of different groups, and counted for viable bacteria CFU number, and as a result, adult fish of the inosine orally-perfused group were found to have a lower bacterial load and to have dose dependence (FIG. 3).

(2) And (3) histopathological detection: after infection and administration, zebra fish is subjected to terminal anesthesia in 0.5% tricaine, whole fish is fixed in 4% neutral buffered paraformaldehyde solution for 72 hours, decalcification, wax dipping, embedding, slicing, H & E and acid-fast staining are carried out, the pathological changes of the whole fish, mainly the granulomatosis of kidney and liver tissues, are observed by a microscope, and the result shows that the granulomatosis of the adult fish in the oral inosine filling group is obviously reduced and the pathological changes are also obviously relieved (figure 4).

The results show that inosine can significantly improve the host's resistance to mycobacteria and reduce the formation and progression of granulomas.

Example 3

This example utilizes an in vitro macrophage infection model to analyze the effect of inosine on the survival of Mtb in macrophage cells, and the specific experimental procedures and results are as follows:

taking primary macrophages of wild mouse abdominal cavities, culturing the primary macrophages with complete 1640 culture medium (containing 10% FBS + 1% penicillin-streptomycin) at 37 ℃ for 4 hours, replacing the complete 1640 culture medium after cells are attached to the walls, adding inosine to enable the final concentration to be 50 mu M, and infecting H37Rv after 12 hours, wherein MOI is 5. After 3 hours of infection, the supernatant was removed, the cells were washed three times with PBS to remove extracellular bacteria, and CFU counts the number of entering cells. After another portion of the cells were washed, and cultured for 24 hours in 1640 medium containing DMSO or 50. mu.M inosine, the survival of intracellular bacteria was examined by CFU. As a result, inosine was found to significantly promote intracellular clearance of Mtb by macrophages, see fig. 5.

Example 4

In this example, a macrophage infection model was used to analyze the effect of inosine on macrophage immunoprotection, and the specific experimental procedures and results are as follows:

wild type mouse peritoneal macrophages were taken, treated with DMSO control or final concentration of 50 μ M inosine for 12 hours, and then given H37Rv infection with MOI 5. After 0, 3 and 6 hours of infection, respectively, removing supernatant, using 1ml Trizol to crack cells, extracting RNA, detecting the change of the expression level of protective inflammatory factors IL-1 beta and IL-6 genes by qRT-PCR, and determining the influence of inosine on the macrophage inflammatory factor. As a result, inosine was found to promote the expression of IL-1. beta. and IL-6 genes, as shown in FIG. 6.

Example 5

This example uses HEK293, SH-SY5Y and HePG2 cell models to analyze the effect of inosine on cytotoxicity, and the specific experimental procedures and results are as follows:

in vitro culture of HEK293, SH-SY5Y and HePG2 cell line, when the cell growth reaches 80%, adding 0, 0.001, 0.01, 0.1, 1,5, 10, 30, 100, 1000 and 10000 μ M inosine to treat for 24h, and using MTT kit to treat cytotoxicity OD₅₇₀The absorbance values were measured and found to be non-toxic to HEK293, SH-SY5Y and HePG2 cells, as shown in FIG. 7.

The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. It will be appreciated by those skilled in the art that any equivalent modifications and substitutions are within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.