Application of sevoflurane in the prevention of Mycobacterium tuberculosis infection

Abstract

This invention relates to the field of biomedical technology, specifically disclosing the application of an inhaled anesthetic in the treatment of Mycobacterium tuberculosis infection. Sevoflurane in this invention exhibits excellent antibacterial effects against Mycobacterium tuberculosis, with a MIC of 1.25% against the standard strain (ATCC27294). Treatment of Mycobacterium tuberculosis with 6% and 9% sevoflurane resulted in a decrease in bacterial load in sputum samples after 1 hour, and a significant decrease in the bacterial load of the standard strain after 8 hours. Sevoflurane exhibits low cytotoxicity and good safety, making it a potential candidate drug for the development of anti-Mycobacterium tuberculosis drugs. For surgical treatment of tuberculosis patients, sevoflurane and its combinations can serve as both general anesthetics and, while exerting anesthetic effects, inhibit the growth of Mycobacterium tuberculosis in the airway.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and in particular relates to application of an inhaled anesthetic in resisting Mycobacterium tuberculosis infection. Background Art

[0002] Tuberculosis is a chronic infectious disease caused by the invasion of Mycobacterium tuberculosis into the body, either primary or secondary, which seriously endangers human health (Mac Neil A, Glaziou P, Sismanidis C, et al. Global Epidemiology of Tuberculosis and Progress Toward Meeting Global Targets-Worldwide, 2018. MMWR Morb Mortal Wkly Rep, 2020, 69 (11): 281-285.). According to the latest report from the World Health Organization, the mortality rate caused by tuberculosis in 2022 is second only to coronavirus infection (Taghreed Adam ABMB. Global tuberculosis report 2023. Geneva: World Health Organization, 2023.). And studies have shown that since 2020, the prevalence and incidence of tuberculosis have been rising year by year (Williams PM, Pratt RH, Walker WL, et al. Tuberculosis-United States, 2023. MMWR Morb Mortal Wkly Rep, 2024, 73 (12): 265-270.), and there will be about 740,000 new tuberculosis patients in my country in 2022. Among them, pulmonary tuberculosis is the most common type of disease. Pulmonary tuberculosis can infect multiple tissues and organs of humans. Except for hair, nails and teeth, tuberculosis has been reported in all other tissues and organs. In addition, about 5-10% of patients infected with Mycobacterium tuberculosis develop extrapulmonary tuberculosis. my country is a country with a high burden of extrapulmonary tuberculosis. Patients with extrapulmonary tuberculosis account for 24.6% of all tuberculosis patients, which is significantly higher than the world average (16%) (Li T, Yan X, Du X, et al. Extrapulmonary tuberculosis in China: a national survey. Int J Infect Dis, 2023, 128: 69-77.). Extrapulmonary tuberculosis can exist independently or simultaneously with pulmonary tuberculosis.In most cases, extrapulmonary tuberculosis is caused by direct spread of pulmonary tuberculosis, lymphatic circulation or blood transmission (Pang Y, An J, Shu W, et al. Epidemiology of Extrapulmonary Tuberculosis among Inpatients, China, 2008—2017. Emerg Infect Dis, 2019, 25(3): 457-464.), mainly involving the intestines, meninges, lymph nodes, bones, joints, kidneys and skin (Acharya B, Acharya A, Gautam S, et al. Advances indiagnosis of Tuberculosis: an update into molecular diagnosis of Mycobacteriumtuberculosis. Mol Biol Rep, 2020, 47(5): 4065-4075.). Among them, lymph node tuberculosis and bone and joint tuberculosis are the most common. Pulmonary tuberculosis is mainly treated with combined chemotherapy, but some patients with pulmonary tuberculosis need to undergo surgery to remove tuberculosis lesions due to repeated failure of chemotherapy or the formation of large cavitary lesions in the lungs. Bone tuberculosis can affect bone development and easily cause disability. It is the main site of extrapulmonary tuberculosis accumulation (Lv Jing. Analysis of the application effect of high-quality nursing in postoperative rehabilitation of patients with bone tuberculosis [J]. Chinese Journal of Disability Medicine, 2021, 29(8): 62-63; He Yingfang. Effect of comprehensive rehabilitation nursing measures on the quality of life and prognosis of patients with bone tuberculosis [J]. Medical Theory and Practice, 2020, 33(13): 2224-2225.), and often occurs in the human hip joint, lumbar spine, knee joint and other parts. Lymph node tuberculosis patients often cause lymph node enlargement and suppuration, and can form pus bags containing a large amount of fluid. Therefore, surgical treatment is crucial for the treatment of extrapulmonary tuberculosis. In order to ensure the smooth progress of the operation, effectively reduce the patient's pain, and maintain the stability of the patient's vital signs, reliable anesthesia is very necessary. Anesthesia can not only eliminate or greatly reduce the pain felt by patients during surgery, thereby improving their comfort and cooperation, but also keep them still during surgery to avoid movement caused by pain, thus ensuring the accuracy and safety of the surgery. In addition, anesthesia can also reduce the tension and anxiety of patients, reduce stress reactions during surgery, and facilitate their postoperative recovery.

[0003] Sevoflurane is one of the new halogenated general anesthetics. Sevoflurane was first synthesized by Regan in 1968, and its physicochemical properties, pharmacology and toxicology were evaluated by Wallin et al. in 1975. It was first officially approved for clinical use in Japan in 1990, and was launched in my country in 2005 (Wang SQ, Fang F, Xue ZG, et al. Neonatal sevofluraneanesthesia induces long-term memory impairment and decreases hippocampal PSD-95 expression without neuronal loss [J]. Eur Rev Med Pharmacol Sci, 2013, 17 (7): 941-950.). Sevoflurane enters the human body by inhalation to achieve the effects of induction and maintenance of clinical anesthesia. It has the advantages of low blood gas distribution coefficient, rapid induction and awakening, little effect on liver function, and significant sedative and analgesic effects (Alifier M, Olsson B, Andreasson U, et al. Cardiac Surgery is Associated with Biomarker Evidence of Neuronal Damage [J]. J Alzheimers Dis, 2020, 74 (4): 1211-1220.), and the drug can be combined with a variety of drugs to achieve intravenous-inhalation composite induction, so it is widely used in surgical operations. Usually, sevoflurane is used to induce anesthesia at a concentration of 2% to 4% and maintained at 3%. For pediatric patients, the induction concentration can be as high as 8%. At present, there are no reports on the use of sevoflurane against tuberculosis infection. Summary of the invention

[0004] The present invention found that the anesthetic drug sevoflurane has good antibacterial activity against Mycobacterium tuberculosis in vitro and in sputum specimens, and has low cytotoxicity and good safety. Based on this, the present invention was completed.

[0005] In a first aspect, the present invention provides use of sevoflurane in preparing a product for inhibiting Mycobacterium tuberculosis, wherein the product contains sevoflurane.

[0006] Furthermore, the Mycobacterium tuberculosis includes clinical isolates of Mycobacterium tuberculosis, standard strains of Mycobacterium tuberculosis or Mycobacterium tuberculosis carried by patients infected with Mycobacterium tuberculosis.

[0007] Furthermore, the product for inhibiting Mycobacterium tuberculosis includes a medical product or a non-medical product.

[0008] Furthermore, the medical product refers to medical drugs, and the non-medical product refers to experimental reagents or antibacterial agents.

[0009] Furthermore, one or more pharmaceutically acceptable carriers or excipients may be added to the product.

[0010] Furthermore, the carrier material includes, but is not limited to, one or more of a water-soluble carrier material, a poorly soluble carrier material and / or an enteric carrier material.

[0011] Furthermore, the water-soluble carrier material includes, but is not limited to, one or more of polyethylene glycol, polyvinyl pyrrolidone and / or organic acid.

[0012] Furthermore, the poorly soluble carrier material includes but is not limited to one or more of ethyl cellulose and / or cholesterol stearate.

[0013] Furthermore, the enteric carrier material includes but is not limited to one or more of cellulose acetate phthalate and / or carboxymethyl ethyl cellulose.

[0014] Furthermore, the product can be prepared into a variety of dosage forms, including but not limited to one or more of tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories and / or lyophilized powder injections.

[0015] Furthermore, the preparation can be one or more of a conventional preparation, a sustained-release preparation, a controlled-release preparation and / or various microparticle delivery systems.

[0016] Furthermore, the tablets may widely use various carriers known in the art, including one or more of diluents and absorbents, wetting agents and binders, disintegrants, disintegration inhibitors, absorption promoters and / or lubricants.

[0017] Furthermore, the diluent and absorbent include, but are not limited to, one or more of starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose and / or aluminum silicate.

[0018] Furthermore, the wetting agent and adhesive include, but are not limited to, one or more of water, glycerol, polyethylene glycol, ethanol, propanol, starch slurry, dextrin, syrup, honey, glucose solution, acacia gum slurry, gelatin slurry, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate and / or polyvinyl pyrrolidone.

[0019] Furthermore, the disintegrant includes, but is not limited to, one or more of dry starch, alginate, agar powder, brown algae starch, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitan fatty acid ester, sodium dodecyl sulfate, methyl cellulose and / or ethyl cellulose.

[0020] Furthermore, the disintegration inhibitor includes but is not limited to sucrose, tristearin, cocoa butter and / or hydrogenated oil.

[0021] Furthermore, the absorption enhancer includes but is not limited to one or more of quaternary ammonium salts and / or sodium lauryl sulfate.

[0022] Furthermore, the lubricant includes, but is not limited to, one or more of talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin and / or polyethylene glycol.

[0023] Furthermore, the tablets can be further made into coated tablets, including sugar-coated tablets, film-coated tablets, enteric-coated tablets, double-layer tablets and multi-layer tablets.

[0024] Furthermore, the pills may be prepared using a wide variety of carriers known in the art, including diluents and absorbents, binders and / or disintegrants.

[0025] Furthermore, the diluent and absorbent include, but are not limited to, one or more of glucose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinyl pyrrolidone, Gelucire, kaolin and / or talc.

[0026] Furthermore, the adhesive includes, but is not limited to, one or more of gum arabic, gum tragacanth, gelatin, ethanol, honey, liquid sugar, rice paste and / or batter.

[0027] Furthermore, the disintegrant includes, but is not limited to, one or more of agar powder, dry starch, alginate, sodium dodecyl sulfate, methyl cellulose and / or ethyl cellulose.

[0028] Furthermore, the suppository may be made of a wide variety of carriers known in the art, including but not limited to one or more of polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin and / or semi-synthetic glycerides.

[0029] Furthermore, the injectable preparation includes, but is not limited to, one or more of a solution, an emulsion, a lyophilized powder injection and / or a suspension.

[0030] Furthermore, the injectable preparation may use all diluents commonly used in the art, including but not limited to one or more of water, ethanol, polyethylene glycol, 1,3-propylene glycol, ethoxylated isostearyl alcohol, polyoxygenated isostearyl alcohol and / or polyoxyethylene sorbitan fatty acid esters.

[0031] Furthermore, in order to prepare an isotonic injection solution, the injection preparation may be added with an appropriate amount of one or more of sodium chloride, glucose, glycerol, conventional cosolvents, buffers and / or pH regulators.

[0032] Furthermore, the various preparations may also add colorants, preservatives, spices, flavoring agents, sweeteners or other materials to the pharmaceutical preparations as needed.

[0033] In a second aspect, the present invention provides the use of sevoflurane in the preparation of a drug for preventing and / or treating a disease caused by Mycobacterium tuberculosis infection, wherein the drug contains sevoflurane, and the sevoflurane exerts its effect by inhibiting the activity of Mycobacterium tuberculosis.

[0034] Furthermore, the Mycobacterium tuberculosis includes clinical isolates of Mycobacterium tuberculosis, standard strains of Mycobacterium tuberculosis or Mycobacterium tuberculosis carried by patients infected with Mycobacterium tuberculosis.

[0035] Furthermore, one or more pharmaceutically acceptable carriers or excipients may be added to the drug for preventing and / or treating diseases caused by Mycobacterium tuberculosis infection.

[0036] Furthermore, the carrier material includes, but is not limited to, one or more of a water-soluble carrier material, a poorly soluble carrier material and / or an enteric carrier material.

[0037] Furthermore, the drug for preventing and / or treating diseases caused by Mycobacterium tuberculosis infection can be prepared into a variety of dosage forms, including but not limited to one or more of tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, lozenges, suppositories and / or lyophilized powder injections.

[0038] Furthermore, the preparation can be one or more of a conventional preparation, a sustained-release preparation, a controlled-release preparation and / or various microparticle delivery systems.

[0039] Furthermore, the various preparations may also add colorants, preservatives, spices, flavoring agents, sweeteners or other materials to the pharmaceutical preparations as needed.

[0040] Furthermore, the drug for preventing and / or treating diseases caused by Mycobacterium tuberculosis infection can be administered via injection, cavity administration, respiratory tract administration or mucosal administration.

[0041] Furthermore, the injection administration includes subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection, etc.; the cavity administration includes rectal or vaginal administration; and the respiratory tract administration includes nasal administration.

[0042] In a third aspect, the present invention provides a pharmaceutical composition, comprising sevoflurane and another active ingredient for resisting Mycobacterium tuberculosis infection, wherein the pharmaceutical composition has at least one of the following effects:

[0043] a) inhibiting the activity of Mycobacterium tuberculosis;

[0044] b) Anti-Mycobacterium tuberculosis infection;

[0045] c) Prevention and / or treatment of diseases caused by Mycobacterium tuberculosis.

[0046] Furthermore, the Mycobacterium tuberculosis includes clinical isolates of Mycobacterium tuberculosis, standard strains of Mycobacterium tuberculosis or Mycobacterium tuberculosis carried by patients infected with Mycobacterium tuberculosis.

[0047] Furthermore, the other active ingredient against Mycobacterium tuberculosis infection includes one or more of antibiotics and other active ingredients that can be beneficial to inhibiting or killing Mycobacterium tuberculosis or providing resistance to patients.

[0048] Furthermore, the antibiotics include one or more of rifampicin, ethambutol, isoniazid, streptomycin, pyrazinamide, thiosemicarbazide, para-aminosalicylic acid, prothionamide, amikacin, capreomycin, rifapentine, rifabutin, bedaquiline, delamanid, linezolid and / or clofazimine.

[0049] Furthermore, the other drugs include one or more of vitamins, amino acids, proteins and / or minerals.

[0050] Furthermore, one or more pharmaceutically acceptable carriers may be added to the pharmaceutical composition.

[0051] Furthermore, the carrier material includes, but is not limited to, one or more of a water-soluble carrier material, a poorly soluble carrier material and / or an enteric carrier material.

[0052] Preferably, the carrier material is a water-soluble carrier material.

[0053] Furthermore, the pharmaceutical composition can be prepared into a variety of dosage forms, including but not limited to one or more of tablets, capsules, pellets, aerosols, pills, powders, solutions, suspensions, emulsions, granules, liposomes, transdermal agents, buccal tablets, suppositories and / or lyophilized powder injections.

[0054] Furthermore, the preparation can be one or more of a conventional preparation, a sustained-release preparation, a controlled-release preparation and / or various microparticle delivery systems.

[0055] Furthermore, in order to prepare an isotonic injection solution, the injection preparation may be added with an appropriate amount of one or more of sodium chloride, glucose, glycerol, conventional cosolvents, buffers and / or pH regulators.

[0056] Furthermore, the various preparations may also add colorants, preservatives, spices, flavoring agents, sweeteners or other materials to the pharmaceutical preparations as needed.

[0057] Furthermore, the pharmaceutical composition can be administered by injection, cavity administration, respiratory tract administration or mucosal administration.

[0058] Furthermore, the injection administration includes subcutaneous injection, intravenous injection, intramuscular injection and intracavitary injection, etc.; the cavity administration includes rectal or vaginal administration; and the respiratory tract administration includes nasal administration.

[0059] Beneficial Effects

[0060] 1. The sevoflurane in the present invention has a good antibacterial effect on Mycobacterium tuberculosis. The MIC of sevoflurane to the standard strain of Mycobacterium tuberculosis (ATCC27294) is 1.25%. After 8 hours of treatment with 3%, 6% and 9% sevoflurane, the colony count of Mycobacterium tuberculosis can be significantly reduced. After 24 hours of treatment with 9% sevoflurane, the original load of Mycobacterium tuberculosis can be reduced by 1 Log.

[0061] 2. The bacterial load of Mycobacterium tuberculosis in sputum specimens decreased after 1 hour of treatment with 6% and 9% sevoflurane. After 4 hours of treatment, the bacterial load in the 9% sevoflurane group was significantly lower than that in the control group (P<0.01).

[0062] 3. Sevoflurane has low cytotoxicity and good safety, and can be used as a candidate drug in the development of drugs against Mycobacterium tuberculosis infection. When surgical treatment is performed on tuberculosis patients, sevoflurane and its compositions can be used as general anesthetics, and can also inhibit the growth of Mycobacterium tuberculosis in the airways while exerting an anesthetic effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0063] Figure 1 To test the antibacterial activity of sevoflurane against standard strains of Mycobacterium tuberculosis.

[0064] Figure 2 To evaluate the antibacterial activity of sevoflurane against standard strains of Mycobacterium tuberculosis at different time periods.

[0065] Figure 3 To evaluate the anti-Mycobacterium tuberculosis activity of sevoflurane in sputum suspension.

[0066] Figure 4 To verify the cytotoxicity of sevoflurane on THP1 cells. DETAILED DESCRIPTION

[0067] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0068] The experimental methods in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0069] Material Description:

[0070] Bacteria: The standard strain of Mycobacterium tuberculosis H37Rv (ATCC27294) was purchased from ATCC Culture Collection, USA.

[0071] Sevoflurane: molecular formula C4H3F7O: molecular weight is 200.05, CAS number is 28523-86-6, and its structural formula is as follows:

[0072]

[0073] Example 1. Detection of the antibacterial activity of sevoflurane against standard strains of Mycobacterium tuberculosis

[0074] 1.1 Preparation of sevoflurane solution

[0075] Sevoflurane (purchased from Shanghai Hengrui Medicine Co., Ltd., item number: 28523-86-6, national medicine standard number: H20070172), take 2 ml of the stock solution and add it to 10 mL of 7H9 culture medium to make a 10% solution. Add 200 μl of 20% sevoflurane solution to the second column of the 96-well plate, mix it by pipetting, then aspirate 100 μl from the second column to the third column, dilute it to the 12th column in sequence, aspirate 100 μl and discard it; add 100 μl of 7H9 culture medium to the first column as a positive control well. Set 4 replicate wells for each concentration, and repeat the experiment 3 times.

[0076] 1.2 Preparation of bacterial solution

[0077] Mycobacterium tuberculosis (ATCC27294) was cultured in a neutral Roche medium to the logarithmic phase, the strain in the logarithmic phase was scraped, the colony was placed in a grinding bottle and ground evenly, the bacterial suspension was diluted with 7H9 medium and the turbidity was measured to 1 McFarland, and then added to the 7H9 medium at a ratio of 1:20 and mixed to obtain a Mycobacterium tuberculosis suspension.

[0078] 1.3 Micropore method for measuring the minimum inhibitory concentration of sevoflurane against standard strains of Mycobacterium tuberculosis

[0079] 1. Add 100 μl of Mycobacterium tuberculosis suspension to each well in columns 1-11, so that the final volume in each well is 200 μl and the final concentration of the bacterial suspension is 4×10 5 CFU / mL, at this time, the final drug concentrations in the wells in columns 2-10 are 10%, 5%, 2.5%, 1.25%, 0.625%, 0.3125%, 0.16%, 0.08%, 0.04%, 0.02% and 0.01%, respectively, and a positive control well (column 1, bacterial culture medium without drug) is set up, 4 replicate wells are set for each concentration, and the experiment is repeated 3 times.

[0080] 2. Place the 96-well plate in a 37°C incubator for 7 days.

[0081] 3. Add 20 μL of Alamar Blue (purchased from Biorad, catalog number: BUF012B) and 50 μL of 5% Tween-80 (purchased from Sigma, catalog number: P4780-500ML) premixed color development solution to the microplate, continue to culture at 37°C for 24 hours, and observe the color change of the 96-well plate.

[0082] 1.4 Criteria

[0083] The blue wells represent sterile growth, the pink wells represent bacterial growth, and the lowest drug concentration that prevents the color from changing from blue to pink is recorded as the minimum inhibitory concentration (MIC) that can inhibit the growth of Mycobacterium tuberculosis (ATCC27294).

[0084] 1.5 Results

[0085] like Figure 1 As shown, the MIC of sevoflurane against Mycobacterium tuberculosis is 1.25%.

[0086] Example 2: Evaluation of the antibacterial activity of sevoflurane against standard strains of Mycobacterium tuberculosis

[0087] 2.1 Preparation of sevoflurane solution

[0088] 10% OADC enrichment agent was added to 7H9 medium as solvent. 0.6 mL, 1.2 mL and 1.8 mL of sevoflurane stock solution were taken, and 9.4 mL, 8.8 mL and 8.2 mL of 7H9 medium were added to prepare sevoflurane solutions with final concentrations of 6%, 12% and 18%.

[0089] 2.2 Evaluation of the antibacterial activity of different concentrations of sevoflurane against standard strains of Mycobacterium tuberculosis

[0090] The Mycobacterium tuberculosis bacterial solution in Example 1 and 1 mL of the above-mentioned sevoflurane solutions of different concentrations were mixed at a ratio of 1:1 to prepare sevoflurane solutions with final concentrations of 3%, 6%, and 9%, respectively (the final concentration of the bacterial solution was 4×10 5 CFU / mL), and cultured in 15mL shaking tubes. At the same time, a medium containing bacterial solution without drugs was used as a positive control. The shaking tubes were cultured in a 37°C incubator for 8h, 24h and 48h, respectively, with 3 replicates for each concentration and culture time point.

[0091] 2.3 Bacterial colony count (CFU)

[0092] At the corresponding culture time point, take 100 μL of Mycobacterium tuberculosis suspension containing or not containing different concentrations of sevoflurane, add 900 μL Middlebrook 7H9 (7H9) culture medium (containing 10% Middlebrook OADC enrichment solution) and mix well to obtain 10× diluted bacterial suspension, and then perform gradient dilution in sequence. Take 100 μL of the corresponding bacterial suspension and inoculate it in Middlebrook 7H10 (7H10) solid culture medium (containing 10% Middlebrook OADC enrichment solution), spread it evenly with a coating rod, and set 3 replicates for each tube of bacterial solution; place the 7H10 solid culture plate in a 37°C constant temperature incubator and culture for 3 to 4 weeks; take out the 7H10 solid culture plate, count the colonies, and calculate the colony forming unit (CFU) of the original bacterial solution.

[0093] 2.4 Results

[0094] like Figure 2 As shown in the figure, after 8 hours of treatment with 3%, 6% and 9% sevoflurane, the colony count of Mycobacterium tuberculosis can be significantly reduced. Treatment with 9% sevoflurane for 24 hours can lead to a 1 Log decrease in the original bacterial load.

[0095] Example 3. Evaluation of the anti-mycobacterium tuberculosis activity of sevoflurane in sputum suspension

[0096] 3.1 Preparation of sputum bacterial suspension

[0097] Fresh sputum specimens that were positive for Mycobacterium tuberculosis by GeneXpert MTB / RIF were collected from the National Tuberculosis Clinical Laboratory of Beijing Chest Hospital, Capital Medical University. The sputum was evenly spread on a six-well plate (200 μL / well) and placed in a clean bench for UV sterilization for 24 h to remove foreign bacteria. Subsequently, the sterilized sputum was diluted with culture medium to 10% (v / v) sputum-containing 7H9 medium (containing 10% OADC enrichment solution), and the sputum-containing 7H9 medium was diluted with sputum-containing 7H9 medium to form a 10% (v / v) sputum-containing medium in the logarithmic growth phase (OD 600The standard strain of Mycobacterium tuberculosis H37Rv was adjusted to OD 600 =0.3, and the sputum bacterial suspension is ready.

[0098] 3.2 Determination of the antibacterial effect of sevoflurane in sputum bacterial suspension

[0099] The antibacterial effect of sevoflurane on Mycobacterium tuberculosis in sputum suspension was compared by measuring the CFU of Mycobacterium tuberculosis under co-incubation conditions with different concentrations of sevoflurane within a certain period of time.

[0100] The sputum suspension was further diluted 20 times with sterile 7H9 medium, and sevoflurane was added at final concentrations of 3%, 6%, and 9%, respectively, and placed in a 37°C incubator for static culture. No drug was added as a negative control, and 3 replicates were set for each concentration. After 0, 1, 2, and 4 hours of culture, 200 μL of the culture fluid was taken and diluted 10 times with sterile PBS buffer (10 -1 -10 -5 Finally, 10 μL of each dilution was dripped onto Middlebrook 7H10 selective agar medium (containing 10% OADC enrichment solution, 50 μg / mL carbenicillin, 200 U / mL polymyxin B, 20 μg / mL trimethoprim and 10 μg / mL amphotericin B), and incubated at 37°C for 14 days before counting the CFU and comparing the results.

[0101] 3.3 Statistical analysis

[0102] GraphPad Prism 9 software was used for statistical analysis and graphing. Two-way ANOVA was used to compare the colony counts of Mycobacterium tuberculosis after co-culture with different concentrations of sevoflurane. P < 0.05 was considered statistically significant (*, P < 0.05; **, P < 0.01).

[0103] 3.4 Results

[0104] like Figure 3 As shown in the figure, the bacterial load of Mycobacterium tuberculosis in sputum samples decreased after 1 hour of treatment with 6% and 9% sevoflurane. After 4 hours of treatment, the bacterial load in the 9% sevoflurane group was significantly lower than that in the control group (P<0.01). This suggests that when patients with pulmonary tuberculosis or pulmonary tuberculosis combined with bone tuberculosis undergo surgery, sevoflurane can be used as a general anesthetic to inhibit the growth of Mycobacterium tuberculosis in the airway while exerting an anesthetic effect.

[0105] Case study 4: CCK-8 assay to detect the cytotoxicity of sevoflurane on THP1 cells

[0106] 4.1 Cell culture

[0107] Human blood-derived mononuclear cell THP1 cell line was purchased from the cell bank of the Chinese Academy of Sciences. THP1 cells were cultured in RPMI1640 (containing 10% FBS) complete medium, placed in a 37°C incubator, and passaged every 2 days. Cells with good growth were selected and PMA was added at a final concentration of 100 ng / mL. 5 Cells / well were seeded in 96-well plates and cultured for 48 hours to induce adherent macrophages.

[0108] 4.2 Verification of the cytotoxicity of sevoflurane on THP1 cells

[0109] THP1 cells were stimulated with DMSO and sevoflurane (final concentrations of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%) for 1, 2, and 4 hours (corresponding to the effective inhibition time of tuberculosis in Example 3), respectively. Five replicates were performed for each concentration.

[0110] The culture medium was discarded and the cells were washed with PBS buffer. 1640 complete culture medium containing 10% CCK-8 was added to the corresponding wells and the cells were incubated for 2 hours. The OD was measured by microplate reader. 450nm Absorbance.

[0111] Calculate the survival rate (%) of THP1 cells stimulated by sevoflurane = (OD 450nm [Experimental group]-OD 450nm [Negative control group] / OD 450nm [Positive control group]-OD 450nm [Negative control group])×100%.

[0112] 4.3 Results

[0113] like Figure 4 As shown, within 4 hours of incubation with THP1 cells, when the sevoflurane concentration was ≤3%, the cell survival rate was greater than 90%, and when the sevoflurane concentration was ≤9%, the cell survival rate was higher than 80%, indicating that it has low cytotoxicity and good safety.

Claims

1. Application of sevoflurane in the preparation of drugs for inhibiting Mycobacterium tuberculosis.

2. The use according to claim 1, wherein the Mycobacterium tuberculosis comprises a clinical isolate of Mycobacterium tuberculosis, a standard strain of Mycobacterium tuberculosis, or Mycobacterium tuberculosis carried by a patient infected with Mycobacterium tuberculosis.

3. The use according to claim 1, wherein the medicine further comprises one or more pharmaceutically acceptable carriers or excipients.

4. The use according to claim 1, wherein the dosage form of the drug is tablet, capsule, aerosol, pill, powder, solution, suspension, emulsion, granule, liposome, transdermal agent or suppository.

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