Application of bleomycin in resisting multi-drug-resistant tubercle bacillus (MDR-TB) and extensive drug-resistant tubercle bacillus (XDR-TB)

Abstract

The bleomycin disclosed by the invention can inhibit the normal growth of multi-drug-resistant tubercle bacillus (MDR-TB) and extensive drug-resistant tubercle bacillus (XDR-TB), and can be regarded as a lead compound or a potential drug for resisting the MDR-TB and the extensive drug-resistant tubercle bacillus (XDR-TB), so that the treatment effect for resisting the multi-drug-resistant tubercle bacillus infection diseases is better.

Description

Technical Field

[0001] The present invention involves a breakthrough study in the field of pharmacognosy, namely the potential application of bleomycin sulfate in combating multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) infections. Background Art

[0002] Tuberculosis is a major public health problem worldwide. According to the World Health Organization (WHO), about a quarter of the world's population is infected with Mycobacterium tuberculosis, and there are about 10 million new cases each year. In China, the tuberculosis epidemic situation remains severe. In recent years, although the epidemic has shown a downward trend through strengthening tuberculosis prevention and control work, it is still one of the infectious diseases that my country focuses on controlling.

[0003] According to the World Health Organization, the estimated number of tuberculosis cases in the world in 2022 is 10.6 million, of which multidrug-resistant tuberculosis (MDR-TB) accounts for 3.3%. China is a country with a high burden of drug-resistant tuberculosis. It is estimated that the number of new patients in 2022 will be 748,000, and the number of patients with multidrug-resistant / rifampicin-resistant tuberculosis will be 30,000. The treatment cycle of multidrug-resistant tuberculosis is long, usually taking 18-24 months or even longer, while drug-sensitive tuberculosis generally only takes 6-9 months. Its treatment requires the use of a variety of second-line anti-tuberculosis drugs, and the side effects of the drugs are large. The range of options for some drugs is limited, which often makes it difficult for patients to tolerate and affects treatment compliance.

[0004] Extensively drug-resistant tuberculosis (XDR-TB) is a more serious type of tuberculosis than multidrug-resistant tuberculosis (MDR-TB). In addition to resistance to the two main first-line anti-tuberculosis drugs, isoniazid and rifampicin, it is also resistant to any fluoroquinolone and at least one second-line injectable drug (such as kanamycin, amikacin, and capreomycin). The World Health Organization (WHO) estimates that there are about 30,000 new cases worldwide each year. China is also facing the threat of extensively drug-resistant tuberculosis. The treatment of extensively drug-resistant tuberculosis is very difficult. Due to resistance to multiple first-line and second-line drugs, the choice of effective drugs is very limited. Treatment plans usually require the use of some drugs that have not been widely verified or have significant side effects. The treatment period is longer than that of ordinary tuberculosis and multidrug-resistant tuberculosis, generally requiring 20-24 months or even longer.

[0005] The main mechanism of drug resistance in tuberculosis is gene mutation. The main target of isoniazid is the enoyl reductase (InhA) of Mycobacterium tuberculosis, which is involved in the synthesis of mycolic acid in the bacterial cell wall. When the gene encoding InhA mutates, it will cause changes in the structure and function of the InhA protein. For example, mutations in the promoter region of the inhA gene can cause overexpression of the InhA protein, thereby reducing the inhibitory effect of isoniazid on Mycobacterium tuberculosis. In addition, the catalase-peroxidase encoded by the katG gene is also related to the mechanism of action of isoniazid. This enzyme can activate isoniazid to a form with bactericidal activity. When the katG gene mutates, such as the common katG315 site mutation, it will cause the enzyme activity to be lost or reduced, so that isoniazid cannot be activated normally, thereby producing drug resistance. Rifampicin mainly acts on the RNA polymerase β subunit of Mycobacterium tuberculosis (encoded by the rpoB gene). Mutations in the rpoB gene are the main cause of rifampicin resistance, the most common of which is mutations in specific regions of the rpoB gene (such as the 81bp rifampicin resistance determining region). These mutations change the structure of RNA polymerase, making it unable to bind to rifampicin normally, resulting in rifampicin being unable to inhibit bacterial RNA synthesis. There are also other mechanisms such as enhanced drug efflux pump mechanisms, reduced cell wall barriers and drug uptake, and changes in drug metabolizing enzymes.

[0006] There are significant differences in the drug treatments for common tuberculosis, multidrug-resistant tuberculosis, and extensively drug-resistant tuberculosis, as shown in the following table: Drug selection Treatment Course Drug combination Treatment monitoring Common tuberculosis First-line anti-TB drugs, such as isoniazid, rifampin, pyrazinamide, and ethambutol 6-9 months Quadruple drug (isoniazid, rifampin, pyrazinamide, ethambutol) Symptoms (such as whether cough, sputum, fever, night sweats, etc. have been alleviated) and whether sputum bacteria have turned negative Multidrug-resistant tuberculosis Second-line drugs include injectable drugs (such as amikacin, capreomycin, etc.), fluoroquinolones (such as moxifloxacin, levofloxacin, etc.) and oral second-line anti-tuberculosis drugs (such as ethionamide, prothionamide, etc.) 18-24 months 4-6 second-line anti-TB drugs In addition to routine symptoms and sputum tests, it is also necessary to pay close attention to the adverse reactions of second-line drugs. Extensively drug-resistant tuberculosis New anti-TB drugs (such as bedaquiline, delamanid), clofazimine, etc., combined with drugs still under study or new use of old drugs 20-24 months In addition to conventional second-line drugs and new drugs, adjuvant drugs are also added Comprehensively monitor the patient's physical condition, including symptoms, signs, various laboratory tests (such as blood routine, liver and kidney function, electrolytes, etc.) and imaging tests (such as chest CT, etc.)

[0007] Bleomycin is an alkaline glycopeptide antitumor antibiotic extracted from Streptomyces verticillus. It is a cell cycle non-specific drug, mainly used to treat a variety of malignant tumors. Bleomycin can form a complex with copper or iron ions, and then this complex can convert oxygen molecules into oxygen free radicals. These oxygen free radicals can directly act on DNA, causing single-strand and double-strand breaks in DNA, thereby interfering with DNA replication and transcription. Summary of the invention

[0008] The present invention provides a research strategy of bleomycin in fighting multi-drug resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB).

[0009] The bleomycin sulfate used in the present invention has a CAS number of 9041-93-4 and is purchased from Shanghai Yuanye. Bacterial level experiments have shown that bleomycin can inhibit the normal growth of the standard strain of Mycobacterium tuberculosis H37Rv and clinical multi-drug resistant strains, especially for clinical multi-drug resistant strains.

[0010] The present invention provides a lead compound for preventing or treating multidrug-resistant mycobacterium tuberculosis or extensively drug-resistant mycobacterium tuberculosis, wherein the active ingredient is bleomycin, which can be used by other carriers for the research of medicine and preparation. The carrier is a conventional carrier for pharmacognosy research--diluent, excipient, filler, adhesive, wetting agent, disintegrant, absorption promoter, surfactant, adsorption carrier, lubricant and synergist, etc. Bleomycin can be made into a dosage form of one of injection, tablet, pill, capsule, suspension or emulsion. The administration route of bleomycin is mostly intramuscular or intravenous injection, so it is mostly an injection.

[0011] The present invention has the advantage that it can effectively inhibit the growth of multi-drug resistant tuberculosis (MDR-TB) and extensively drug resistant tuberculosis (XDR-TB) at the bacterial level. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 This is a table showing the results of the minimum inhibitory concentration (MIC) of bleomycin against the standard strain H37Rv of Mycobacterium tuberculosis and clinical multidrug-resistant strains.

[0013] Figure 2 It is a schematic table of drug sensitivity of the Mycobacterium tuberculosis strains used in the present invention.

[0014] How the experiment works In order to better illustrate the research content of the present invention, the specific experimental operation of the present invention will be described in detail below. The present invention uses the Almar Blue microplate method to determine the minimum inhibitory concentration MIC of bleomycin. 1. Bacterial liquid culture: Different tuberculosis strains (standard strain H37Rv and clinical multidrug-resistant strains) are inoculated into liquid 7H9 culture medium and cultured in a 37°C incubator to the logarithmic growth phase. 2. Bleomycin concentration gradient: When used, follow the 4-fold dilution method to adjust the final bleomycin concentration gradient as follows: 112μg / mL, 28μg / mL, 7μg / mL, 1.76μg / mL, 0.44μg / mL, 0.11μg / mL. 3. Plating experiment: Add 150μL of 7H9 culture medium containing bleomycin to a 96-well plate, and perform gradient dilution simultaneously, then add 50μL of bacterial liquid to make the final bacterial concentration 5*10 5 / mL. Place the plate in a 37℃ incubator for one week. 4. AlmarBlue experiment: Take out the 96-well plate, add 70μL of the prepared AlmarBlue solution in the biosafety cabinet, and then continue to incubate in a 37℃ incubator for 24 hours. 5. Observation: Observe the color development. Pink is a positive result, and blue is a negative result. The size of the MIC can be inferred from this.

[0015] The present invention relates to the technical field of pharmacy, and specifically refers to the research on bleomycin in the fight against multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB), and is expected to become a potential therapeutic drug or lead compound.

[0016] The methods used above are commonly used methods in the art unless otherwise specified.

[0017] The above are only specific implementation modes of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be within the protection scope of the present invention.

[0018] References [1]WHO.Global tuberculosis report 2022[R].WHO, 2022. [2] Zhao Yanlin, Pang Yu. (2015). Laboratory testing procedures for tuberculosis. Beijing: People’s Medical Publishing House.

Claims

1. Research on the application of bleomycin in the fight against multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) infections.

2. The structural formula of bleomycin is:

3. The drug (bleomycin) for multi-drug resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB), the compound of which can be used in salt form, including sulfate (bleomycin sulfate) and hydrochloride (bleomycin hydrochloride).

4. Drugs against multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) (bleomycin) must be able to use one or more common carriers in pharmacognosy; the types of these carriers are as follows: excipients, fillers, binders, disintegrants, diluents, surfactants and lubricants.

5. Drugs (bleomycin) for multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB), in the form of injection, tablet, pill, capsule, suspension or emulsion.

Images