CN115177602A - Medicine for treating tuberculosis - Google Patents

Abstract

The invention discloses a medicine for treating tuberculosis, which belongs to the field of medicine. The present application proves that cannabidiol can significantly inhibit the symptoms of tuberculosis caused by Mycobacterium tuberculosis infection through infection experiments at the level of cells and animals, and has a significant inhibitory effect on tuberculosis caused by drug-resistant tuberculosis bacteria. Compared with clinically used anti-tuberculosis drugs isoniazid INH and bedaquiline BDQ, cannabidiol can not only inhibit tuberculosis infection, but also effectively maintain the body's inflammatory immune balance and inhibit the damage of excessive inflammatory response to the body. It has good clinical application prospects for anti-tuberculosis or other anti-infective diseases.

Description

a drug to treat tuberculosis

technical field

The invention relates to a medicine for treating tuberculosis, belonging to the field of medicine.

Background technique

Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (Mtb). According to the 2020 Global Tuberculosis Report released by the World Health Organization: in 2019, there were about 10 million new tuberculosis patients worldwide, and the number of deaths was as high as 1.41 million; at the same time, there were 460,000 new cases of drug-resistant tuberculosis in the world in 2019, and the number of deaths of drug-resistant tuberculosis patients was 21 10,000, the mortality rate is close to 50%. The current TB prevention and control capacity is far from the World Health Organization's goal of ending TB by 2030, so there is an urgent need to develop new anti-TB drugs or develop new anti-TB strategies to deal with the current severe TB (especially drug-resistant TB) situation.

Cannabidiol (CBD) is a pure natural ingredient extracted from the cannabis plant and is currently being used in the clinical treatment of many diseases. Cannabidiol has anti-inflammatory and analgesic effects, so it is often used clinically to relieve pain and inflammation; cannabidiol can inhibit nerve excitation, so it can be used for the treatment of epilepsy; cannabidiol can also improve mood by maintaining the level of cannabinoids in the human body , so it can be used for the treatment of depression. Additionally, cannabidiol has been found to relieve symptoms in Parkinson's patients.

SUMMARY OF THE INVENTION

The present invention provides a new use of cannabidiol in relieving and treating pulmonary tuberculosis caused by tuberculosis branch infection.

The first object of the present invention is to provide the application of cannabidiol in the preparation of a medicine for relieving and/or treating pulmonary tuberculosis caused by tuberculosis branch infection.

In one embodiment, the alleviation and/or treatment of tuberculosis caused by tuberculosis branch infection comprises at least one of the following functions:

(1) Recovery of lung tissue abnormalities caused by Mycobacterium tuberculosis;

(2) Reduce pulmonary inflammatory infiltration;

(3) Increase the level of pro-inflammatory cytokines TNF-α and/or IL-10;

(4) Inhibit the proliferation of Mycobacterium tuberculosis.

可单独作为药物的有效成分。In one embodiment, the cannabidiol structural formula is

It can be used alone as an active ingredient of a drug.

In one embodiment, the medicament further contains a pharmaceutically acceptable carrier.

In one embodiment, the medicament further contains an anti-tuberculosis medicinal ingredient.

In one embodiment, the anti-tuberculosis medicinal ingredients include but are not limited to isoniazid, rifampicin, ethambutol, pyrazinamide, kanamycin, amikacin, quinolones, parabens One or more of the first- and second-line anti-tuberculosis drugs such as aminosalicylic acid.

In one embodiment, the anti-tuberculosis medicinal ingredients include but are not limited to drugs such as bedaquiline against drug-resistant tuberculosis bacteria.

In one embodiment, the tuberculosis includes tuberculosis caused by common Mycobacterium tuberculosis infection and tuberculosis caused by drug-resistant Mycobacterium tuberculosis infection.

The second object of the present invention is to provide the application of cannabidiol and medicines containing cannabidiol in anti-tuberculosis and other infectious diseases.

Further, the infectious diseases include diseases caused by infections such as viruses, intracellular bacteria, and facultative intracellular bacteria.

In one embodiment, the application includes, but is not limited to, oral anti-tuberculosis drugs;

In one embodiment, the other infectious diseases include, but are not limited to, viral, intracellular, facultative intracellular, and extracellular infectious diseases.

The present invention also claims to contain cannabidiol and at least one selected from the group consisting of: isoniazid, rifampicin, ethambutol, pyrazinamide, kanamycin, amikacin, quinolones, p-aminosalicylic Drugs in acid, bedaquiline.

Beneficial effects: The present invention provides a new use of cannabidiol alone or in combination with other drugs in relieving and treating tuberculosis infection. The present application proves that cannabidiol can significantly inhibit the symptoms of tuberculosis caused by Mycobacterium tuberculosis infection through infection experiments at the level of cells and animals, and has a significant inhibitory effect on tuberculosis caused by drug-resistant tuberculosis bacteria. Compared with clinically used anti-tuberculosis drugs isoniazid INH and bedaquiline BDQ, cannabidiol can not only inhibit tuberculosis infection, but also effectively maintain the body's inflammatory immune balance and inhibit the damage of excessive inflammatory response to the body. It has good clinical application prospects for anti-tuberculosis or other anti-infective diseases.

Description of drawings

Figure 1 shows the changes in the level of autophagy marker protein LC3 in tuberculosis-infected macrophages detected by western blotting; ddH ₂ O, double distilled water; INH, isoniazid; CBD, cannabidiol; Non-infection, uninfected group; H37Rv, standard strain of Mycobacterium tuberculosis.

Figure 2(A) shows the appearance of lung tissue of mice fed with standard TB strain (H37Rv) and multidrug-resistant TB strain (MDR) infected with different anti-TB drugs; (B) is fed with standard TB strain (H37Rv) with different anti-TB drugs ) or multidrug-resistant tuberculosis (MDR)-infected mice; where, ddH ₂ O, double distilled water; INH, isoniazid; CBD, cannabidiol. INH+CBD, combination of isoniazid and cannabidiol; BDQ, bedaquiline; BDQ+CBD, combination of bedaquiline and cannabidiol; H37Rv, standard strain of Mycobacterium tuberculosis; MDR, multidrug-resistant Mycobacterium tuberculosis.

Figure 3(A) shows the results of hematoxylin-eosin staining of lung tissue of mice fed with standard TB strains with different anti-TB drugs; (B) shows the lungs of mice fed with MDR-TB strains infected with different anti-TB drugs Tissue hematoxylin-eosin staining results; wherein, ddH ₂ O, double distilled water; INH, isoniazid; CBD, cannabidiol. INH+CBD, combination of isoniazid and cannabidiol; BDQ, bedaquiline; BDQ+CBD, combination of bedaquiline and cannabidiol; H37Rv, standard strain of Mycobacterium tuberculosis; MDR, multidrug-resistant Mycobacterium tuberculosis.

Figure 4(A) is the transcriptional level of spleen cytokine Tnf gene in mice fed with different anti-tuberculosis drugs and infected with standard TB strains; (B) is the spleen cytokine Tnf in mice fed with different anti-tuberculosis drugs and infected with MDR-TB strain Gene transcription level; (C) is the spleen cytokine Il10 gene transcription level of mice fed with different anti-tuberculosis drugs and infected with standard tuberculosis strains; (D) is the spleen cells of mice fed with different anti-tuberculosis drugs and infected with MDR-TB strains Factor Il10 gene transcription level; wherein, ddH ₂ O, double distilled water; INH, isoniazid; CBD, cannabidiol; BDQ, bedaquiline; H37Rv, standard strain of Mycobacterium tuberculosis; MDR, multidrug-resistant tuberculosis Mycobacterium.

Figure 5 shows the TB load in lung tissue of standard or multidrug-resistant TB-infected mice fed with different anti-TB drugs; ddH ₂ O, double distilled water; INH, isoniazid; CBD, cannabidiol; among them, INH +CBD, combination of isoniazid and cannabidiol; BDQ, bedaquiline; BDQ+CBD, combination of bedaquiline and cannabidiol; H37Rv, standard strain of Mycobacterium tuberculosis; MDR, multidrug-resistant tuberculosis Mycobacterium.

Detailed ways

Example 1 Detection of changes in the level of autophagy marker protein LC3 in tuberculosis-infected macrophages by western blotting

1) Isolation and culture of mouse bone marrow-derived macrophages: C57BL/6 mice were sacrificed and the hind leg bones were removed; a syringe was inserted into the bone marrow cavity and the bone marrow was flushed out with DMEM medium containing 1% double antibody; bone marrow cells were collected by centrifugation; cells were added Resuspend in DMEM medium containing 20% fetal bovine serum, 1% double antibody, and 10 ng/mL M-CSF, and culture at 37 degrees in 5% CO ₂ for 7 days;

2) Evenly transfer the isolated and cultured DMEM cells to a 12-well cell culture plate. After the cells adhered to the wall, the infected group was added with Mycobacterium tuberculosis-infected cells equal to the number of cells. The uninfected cell group was used as a control; after 1 hour of tuberculosis infection of the cells, the cell culture medium of all well plates was removed, the cells were washed with PBS three times to remove residual tuberculosis bacteria, and fresh DMEM containing 20% fetal bovine serum was added for culture. basal culture, and different types of anti-tuberculosis drugs were added to the culture medium at the same time, and the ddH ₂ O treatment group was used as a control;

3) Collect the cell samples cultured in step 2) at 0, 8, 24, and 48 hours after infection, and add RIPA lysis solution (Biyuntian P0013B) to lyse the cells;

4) After the sample was placed in a 100-degree water bath for 10 minutes, the sample was loaded onto an SDS denatured protein gel, and the levels of different proteins in the sample were analyzed by Western blotting.

The results showed that compared with the uninfected group, the level of LC3 protein in CBD-treated TB-infected macrophages increased with the infection time, suggesting that CBD could enhance the autophagy level of TB-infected macrophages (Figure 1). . Since autophagy is an important way for macrophages to clear pathogenic infection and activate secondary immune responses, it can be speculated that CBD may potentially have the effect of clearing intracellular pathogenic bacteria (such as Mycobacterium tuberculosis) infection.

Example 2 Differences in the appearance of lung and spleen tissue of mice fed with different anti-tuberculosis drugs or infected with drug-resistant tuberculosis strains

1) Select 6-8 week old C57BL/6 mice, and infect standard tuberculosis strain H37Rv and multidrug-resistant tuberculosis strain MDR Mtb respectively by aerosolization, at a dose of 60-100 bacteria per mouse; after the aerosolization, put the mice back into the mice. Keep feeding in cages;

2) After 2 weeks of aerosol-infected tuberculosis bacteria, the mice were fed with drugs. The mice infected with standard tuberculosis strains were divided into four groups that were fed with ddH ₂ O, INH, CBD, and INH+CBD; those infected with drug-resistant tuberculosis strains Mice were divided into four groups fed with ddH ₂ O, BDQ, CBD, BDQ+CBD. The dosage of CBD is 10 mg/kg, once a day; the dosage of INH is 50 mg/kg body weight, once a day; the dosage of BDQ is 25 mg/kg body weight, once a day; each drug is used in combination The amount is the same as when administered alone. The administration time lasted for four weeks; when using, the drug was appropriately diluted, and the volume of each mouse was not more than 100ul; the administration method was gavage.

3) Mice in each group were sacrificed at 1, 2, 3 and 4 weeks of administration, respectively, and the changes in the appearance of lung and spleen of mice in different drug-treated groups at different times were compared.

The morphological differences of lung and spleen were compared between H37Rv-infected mice fed with ddH ₂ O, INH, CBD and CBD+INH, and MDR Mtb-infected mice fed with ddH ₂ O, BDQ, CBD and CBD+BDQ, respectively. The results showed that the H37Rv-infected mice fed with ddH ₂ O (negative control group) had obvious pathological reactions in the lungs and splenomegaly after 6 weeks of infection. However, the appearance of the lungs in the INH-fed group was normal, while the lungs in the CBD-fed group showed milder pathological phenomena, and the appearance of the lungs of the mice in the combined treatment group of INH and CBD was normal. The results suggest that CBD has an inhibitory effect on the intracellular survival of Mtb. For the MDR-infected mice fed with ddH ₂ O, CBD, BDQ and CBD+BDQ, the lungs of the mice fed with ddH ₂ O for 6 weeks had obvious pathological phenomena, and the spleen was enlarged, and its size was about 1.5% of that in the drug group times. The lung pathological phenomena of mice fed BDQ and CBD for 6 weeks were significantly weaker than those fed ddH ₂ O group, and the combined use of INH and CBD had better effects.

Example 3 Comparison of hematoxylin and eosin staining results of lung tissue of mice fed with different anti-tuberculosis drugs or with drug-resistant tuberculosis infection

1) The lung tissue of each group of mice after 4 weeks of administration obtained in Example 2 was fixed in formalin for 24 hours;

2) The fixed lung tissue was dehydrated, embedded in paraffin, sectioned, deparaffinized, stained, mounted, and other steps to obtain hematoxylin-eosin-stained lung tissue

The results showed that the H37Rv-infected mice (negative control group) fed with ddH ₂ O showed obvious inflammatory infiltration in the lungs 6 weeks after infection. However, the lungs of the INH-fed group were basically normal, the CBD-fed group showed a slight inflammatory infiltration phenomenon, and the lungs of the mice in the INH and CBD combined treatment group were normal. The results suggest that CBD has an inhibitory effect on the intracellular survival of Mtb. For MDR-infected mice fed _H2O , CBD, BDQ and CBD+BDQ, the lungs of mice fed with ddH2O for 6 weeks had obvious inflammatory infiltration, while the lungs of mice fed with BDQ and CBD for 6 weeks The inflammatory infiltration of the lungs was significantly weakened, and the lungs were basically normal when INH and CBD were used in combination, indicating that CBD had an inhibitory effect on drug-resistant tuberculosis infection, and when combined with BDQ, it could improve the ability to resist drug-resistant tuberculosis infection.

Example 4 Comparison of cytokine expression levels in spleen tissue of mice fed with different anti-tuberculosis drugs or drug-resistant tuberculosis-infected mice

1) Collect the mouse spleen tissue obtained in Example 2, fully grind and centrifuge to collect cells;

2) Use RNA extraction kit (R1061, Dongsheng Biotechnology) to extract total cell RNA;

3) using a reverse transcription kit to obtain cDNA by reverse transcription using the above total RNA as a template;

4) Fluorescence real-time quantitative PCR was used to detect the transcription level of cytokines, and Gapdh gene was used as an internal reference.

QRT-PCR primer sequences

The results showed that the levels of pro-inflammatory cytokine TNF-alpha in mice fed INH and BDQ were about 1-fold higher than those fed ddH ₂ O or CBD. In contrast, the levels of the anti-inflammatory cytokine IL-10 in the CBD-fed mice were higher than those in the mice fed with ddH ₂ O, INH, or BDQ. Among the mice infected with H37Rv bacteria, the spleen IL-10 level in the CBD group was about 1.5 and 2 times higher than that in the INH and ddH ₂ O groups, respectively, after 1 week of administration. The -10 levels were about 5 and 10 times higher than those in the INH and ddH ₂ O groups, respectively. MDR-infected mice were treated with MDR bacteria for 1 week, and the spleen IL-10 levels in the CBD group were about 4 and 1 times higher than those in the BDQ and ddH ₂ O groups, respectively; 10 levels were about 1.4 and 1.8 times higher than those in the groups fed BDQ and ddH ₂ O, respectively. The results suggest that both INH and BDQ clinically used anti-tuberculosis drugs have strong pro-inflammatory effects, which can easily lead to damage caused by excessive inflammation in the body, while CBD has a better inhibitory effect on inflammation and can prevent the body's inflammatory immunity Storm, better maintain the body's immune balance.

Example 5 Comparison of TB load in lung tissue of mice fed with different anti-tuberculosis drugs of standard or drug-resistant TB

1) Collect the mouse lung tissue obtained in Example 2, and grind it fully;

2) the above-mentioned grinding liquid is successively diluted 10 times, 100 times and 1000 times successively;

3) Spread the above dilution evenly on the 7H10 tuberculosis medium plate, and cultivate at 37 degrees for 2 weeks;

4) Calculate the Mycobacterium tuberculosis load of different lung tissues according to the number of colonies grown on the plate.

The results showed that after 4 weeks of administration, both INH and CBD could effectively inhibit the survival of standard tuberculosis strains in mice, which reduced the bacterial counts in mice to about 1.4% and 10% of those in the unmedicated group, respectively. The anti-tuberculosis effect is better after the combination of these drugs, which basically achieves the purpose of killing all of Mycobacterium tuberculosis; in addition, when BDQ is used alone in the treatment of drug-resistant tuberculosis infection, the amount of bacteria in mice can be reduced to 4 weeks after treatment. About 35% of the bacteria in the unmedicated group, CBD alone was slightly more effective in inhibiting drug-resistant bacteria than BDQ, which could reduce the bacteria in mice to about 14% of the bacteria in the unmedicated group. The drug has better inhibitory effect on Mycobacterium tuberculosis, and basically achieves the purpose of killing all Mycobacterium tuberculosis.

On the basis of Example 2, the inventor also tried to reduce the acting dose of the drug for the BDQ+CBD group of combined medication. The results showed that the combined medication of BDQ and CBD has a synergistic effect, and its effect is better than using one of the drugs alone. The sum of the effects achieved.

Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Anyone who is familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, The protection scope of the present invention should be defined by the claims.

SEQUENCE LISTING

<110> Institute of Microbiology, Chinese Academy of Sciences

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Claims (10)

1. Use of cannabidiol in the manufacture of a medicament for the alleviation and/or treatment of tuberculosis caused by a Mycobacterium tuberculosis infection.

2. The use according to claim 1, wherein said alleviating and/or treating tuberculosis caused by a mycobacterium tuberculosis infection comprises at least one of the following functions:

(1) Restoring lung tissue abnormality caused by mycobacterium tuberculosis;

(2) Reducing pulmonary inflammatory infiltration;

(3) Increasing the levels of proinflammatory cytokines TNF-alpha and IL-10;

(4) Inhibiting proliferation of Mycobacterium tuberculosis.

3. The use as claimed in claim 1 wherein cannabidiol is used alone as an active ingredient in a medicament.

4. Use according to claim 1, wherein the medicament further comprises a pharmaceutical agent against tuberculosis.

5. The use according to claim 4, wherein the anti-tuberculosis pharmaceutical agent comprises, but is not limited to, one or more of isoniazid, rifampin, ethambutol, pyrazinamide, kanamycin, amikana, quinolones, p-aminosalicylic acid, bedaquiline.

6. The use according to any one of claims 1 to 5, wherein the medicament further comprises a pharmaceutically acceptable carrier.

7. The use according to any one of claims 1 to 6, wherein the tuberculosis comprises tuberculosis caused by infection with Mycobacterium tuberculosis in general or tuberculosis caused by infection with drug-resistant Mycobacterium tuberculosis.

8. Application of cannabidiol or medicine containing cannabidiol component in resisting infectious diseases is provided.

9. The use of claim 7, wherein the infectious disease comprises a disease caused by infection with one or more of a virus, an intracellular bacterium, a facultative intracellular bacterium, and an extracellular bacterium.

10. A pharmaceutical composition characterized by comprising cannabidiol and at least one pharmaceutical ingredient selected from the group consisting of:

isoniazid, rifampin, ethambutol, pyrazinamide, kanamycin, amikana, quinolones, p-aminosalicylic acid, bedaquiline.

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