Use of an alkaloid compound in combination therapy for treating mycobacterial infections

Abstract

This invention belongs to the field of pharmaceutical technology, specifically relating to the application of an alkaloid compound in combination therapy for treating mycobacterial infections. Currently, commonly used antimycobacterial drugs generally suffer from significant toxic side effects and are prone to drug resistance. Single-drug therapy is insufficient to meet the increasingly serious clinical treatment needs for drug-resistant tuberculosis and non-tuberculous mycobacterial infections. This invention combines an alkaloid compound with an antimycobacterial drug, resulting in a significant synergistic antibacterial effect. This combination can effectively reduce the dosage of antimycobacterial drugs while maintaining equal or better efficacy, thereby mitigating adverse reactions such as liver toxicity, bone marrow suppression, and nervous system damage caused by these drugs, while simultaneously delaying the development of mycobacterial resistance. The combination therapy provided by this invention offers a highly effective, low-toxicity, and drug-resistant new strategy for the treatment of mycobacterial infections.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of an alkaloid compound in combination therapy for treating mycobacterial infections. More specifically, this invention relates to a class of alkaloid compounds with anti-mycobacterial activity, and synergistic pharmaceutical compositions of such compounds with mycobacterial infection treatment drugs. Background Technology

[0002] In 2024, approximately 1.23 million tuberculosis (TB) deaths occurred globally, with nearly one-third related to treatment failure. The prevalence of multidrug-resistant tuberculosis (MDR-TB), caused by Mycobacterium tuberculosis, further exacerbates the disease burden. However, the clinical relevance of nontuberculous mycobacteria (NTMs) within the mycobacterial genus is also increasingly prominent. In recent years, the incidence of NTM infection has been rising globally, and its morbidity and mortality rates have remained at high levels for a long time, making it a significant public health issue. Notably, in some developed countries, the incidence of NTM infection has exceeded that of new TB cases (Baker, AW, et al., 2025; Dahl, VN, et al., 2022, 125, 120-131).

[0003] Clinical drug resistance is the core bottleneck restricting the treatment efficacy of mycobacterial infections. For Mycobacterium tuberculosis, MDR-TB treatment typically requires a combination of three to five drugs, which often have significant toxicity and serious adverse reactions, leading to decreased patient adherence (Aguilar-Pérez, C., et al. 2025, 16, 9344). Although newer drugs such as bedaquiline, delamani, and putomani have shown some efficacy against MDR-TB and extensively drug-resistant tuberculosis (XDR-TB), the development of drug resistance and adverse reactions remain prominent issues. In comparison, the drug resistance situation for NTM infections is even more severe: NTM has inherent resistance to most commonly used anti-tuberculosis drugs, and traditional long-course chemotherapy regimens easily induce multidrug-resistant strains; in addition, the resistance patterns of different bacterial species vary significantly, and the incidence of adverse reactions and relapse rates are high, resulting in a generally high clinical treatment failure rate.

[0004] Faced with these challenges, combination therapy has become an essential choice for treating mycobacterial infections. Single-drug therapy not only struggles to completely eradicate pathogens but also readily identifies drug-resistant mutants. Combination therapy offers multiple advantages: firstly, by rationally combining drugs with different mechanisms of action, it can simultaneously target multiple key processes such as cell wall synthesis, protein synthesis, and nucleic acid replication, achieving multi-target synergistic inhibition. This makes it difficult for bacteria to escape drug efficacy through single-site mutations, thus significantly enhancing antibacterial activity and delaying the development of drug resistance (Clinical Guidelines, 2024); secondly, synergistic effects allow individual drugs to achieve equivalent inhibitory levels at lower doses, helping to reduce dose-related adverse reactions and improve long-term tolerability and adherence. For mycobacterial infections requiring long-term treatment, shortening the treatment course and reducing adverse reactions directly impact patient treatment completion rates and clinical prognosis.

[0005] Against this backdrop, developing anti-mycobacterial compounds with synergistic sensitizing effects and combining them with existing clinical drugs to form combination therapy regimens has significant scientific and practical value. Based on this, this invention explores the synergistic anti-mycobacterial effect of combining alkaloid compounds with drugs for treating mycobacterial infections, aiming to provide a new drug combination strategy for the treatment of mycobacterial infections. Summary of the Invention

[0006] The purpose of this invention is to provide a new application of alkaloid compounds in the preparation of combination therapy drugs for mycobacteria.

[0007] To achieve the above objectives, in a first aspect, the present invention provides the application of an alkaloid compound in the preparation of a combined therapeutic agent for mycobacterial infections, wherein the mycobacteria are selected from one or more of Mycobacterium tuberculosis, Mycobacterium marineum, Mycobacterium smegmatis, Mycobacterium ulcerativeale, Mycobacterium abscessum, Mycobacterium avium complex, Mycobacterium guilloché, and Mycobacterium kansasus; the composition comprises an active ingredient (a) and a therapeutic agent for mycobacterial infections (b), wherein (a) is one or more of an alkaloid compound, and (b) is three or more therapeutic agents for mycobacterial infections. The weight ratio of (a) to (b) in the composition is from 1:10 to 10:1.

[0008] Thirdly, the composition of the present invention can produce a synergistic effect compared with the individual administration of each drug, thereby improving the antibacterial effect, and can reduce the dosage of the combined drugs in combination therapy, achieving the therapeutic benefit of reduced toxicity and increased efficacy.

[0009] Specifically, this invention relates to the use of an alkaloid compound in combination therapy for treating mycobacterial infections, wherein the alkaloid compounds 1-20 have the following structures:

[0010] .

[0011] In some embodiments of the present invention, the mycobacteria are selected from one or more of Mycobacterium tuberculosis, Mycobacterium marineum, Mycobacterium smegmatis, Mycobacterium ulcerans, Mycobacterium abscessus, Mycobacterium avium complex, Mycobacterium guilloché, and Mycobacterium kansasii.

[0012] In some embodiments of the present invention, the composition is used in combination with three or more clinical treatment agents for mycobacterial infections to prepare a drug, wherein the clinical treatment agents are selected from three or more of isoniazid, rifapentine, pyrazinamide, moxifloxacin, rifampin, ethambutol, bedaquiline, putomani, linezolid, delamani, levofloxacin, clofazimine, and streptomycin.

[0013] Furthermore, the drug combination schemes include the following five: (1) Isoniazid + Rifapentine + Pyrazinamide + Moxifloxacin (1~3:1~3:1~3:1~3); (2) Isoniazid + Rifapentine + Moxifloxacin (1~3:1~3:1~3); (3) Isoniazid + Rifampin + Pyrazinamide + Ethambutol (1~3:1~3:1~3:1~3); (4) Bedaquiline + Putomomani + Linezolid + Moxifloxacin (1~3:1~3:1~3:1~3); (5) Bedaquiline + Delamani + Linezolid + Levofloxacin + Clofazimine (1~3:1~3:1~3:1~3:1~3), where the ratio refers to the relative weight parts of each component.

[0014] Furthermore, the alkaloid compounds and clinical therapeutic drugs are administered separately without any specific order, or the alkaloid compounds and clinical therapeutic drugs are administered simultaneously.

[0015] Furthermore, the pharmaceutical composition may also contain one or more pharmaceutically acceptable excipients, carriers, adjuvants, or solvents.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] Currently used anti-mycobacterial drugs in clinical practice generally suffer from drawbacks such as significant toxic side effects and easy development of drug resistance. To address these technical problems, this invention provides an anti-mycobacterial combination drug composition containing alkaloid compounds. The composition comprises an alkaloid compound (a) and a mycobacterial infection treatment drug (b), wherein the weight ratio of (a) to (b) is 1:10 to 10:1. Through the synergistic combination of (a) and (b), the composition significantly enhances the antibacterial activity against mycobacteria (FICI < 0.5), while simultaneously reducing the total dosage of the mycobacterial infection treatment drug (b) by 30% to 75% while maintaining equivalent efficacy. This significantly reduces adverse reactions such as hepatotoxicity, bone marrow suppression, and neuropathy caused by drugs like linezolid and pyrazinamide, and effectively delays the development of mycobacterial drug resistance. The technical solution provided by this invention combines the advantages of high efficiency, low toxicity, and resistance to drug resistance, and has significant clinical translational value. Detailed Implementation

[0018] To better illustrate the purpose, technical solution, and advantages of this invention, the following detailed description, in conjunction with specific embodiments, aims to provide a thorough understanding of the invention, rather than limiting it. Unless otherwise specified, the experimental reagents, raw materials, and instruments involved in the embodiments of this invention are all commonly used reagents, raw materials, and instruments. For simplicity, this invention names the clinical drug combination for treating mycobacterial infections Combination-X, and its specific formulation is as follows:

[0019] Combination-1: Isoniazid + Rifampin + Pyrazinamide + Ethambutol (1:1:1:1, by weight).

[0020] Combination-2: Isoniazid + Rifapentine + Pyrazinamide + Moxifloxacin (1:1:1:1, by weight).

[0021] Combination-3: Bedaquiline + Putomomani + Linezolid + Moxifloxacin (3:3:1:3, by weight).

[0022] Example 1: Drug susceptibility test of the compound of the present invention with Combination-1~2

[0023] I. Materials and Methods

[0024] 1. Strain Information

[0025] Specific experiments were conducted using Mycobacterium tuberculosis, Mycobacterium marineum, Mycobacterium smegmatis, Mycobacterium ulcerans, Mycobacterium abscessus, Mycobacterium avium complex, Mycobacterium guilloché, and Mycobacterium kansasus.

[0026] 2. Preparation of drug stock solution

[0027] Weigh 1 mg of the compound of the present invention and clinical treatment drugs (isoniazid, rifampin, pyrazinamide, ethambutol, rifapentine, moxifloxacin), add DMSO to prepare 100 mM stock solution, which will be further diluted for use in subsequent experiments.

[0028] 3. Drug MIC 90 Detection methods

[0029] 198 μL of mycobacterial bacterial suspension diluted 1000 times (OD) was inoculated into the test plate. 550 The value was approximately 0.225–0.275, then 2 μL of the prepared compound working solution was added. The detection plate was incubated at 37°C for 10–12 days. Then, 12.5 μL of 7H9 + 20% Tween 80 medium and 20 μL of Alamar blue solution were added, and the plate was incubated for another 24 hours. DMSO was used as a solvent and growth control. 200 μL of medium was placed in each well of the detection plate as a medium control. Rifampin and isoniazid were used as positive controls, and the assay was performed in triplicate.

[0030] 4. Procedure for combined drug susceptibility testing

[0031] The combined drug susceptibility test was performed using the checkerboard method, with the MICs of the compound of this invention and various mycobacterial infection treatment drugs as the basis. 90 Based on the data, the checkerboard dilution method was used to dilute the two drugs at 4×MIC concentrations in a 96-well plate containing bacterial culture. 90 2×MIC 90 MIC 90 1 / 2×MIC 90 1 / 4×MIC 90 1 / 8×MIC 90 and 1 / 16×MIC 90 Use a 7×7 combination, and repeat the measurement three times for each well.

[0032] 5. Results Analysis

[0033] The inhibition rate and MIC90 value can be determined by visually observing whether Alamar blue changes color or by using fluorescence detection (Ex / Em, 530nm / 590nm).

[0034] The results of combined drug susceptibility testing were evaluated using the fractional inhibitory concentration index (FICI). The lowest inhibitory concentration (MIC) when drug A is used in combination with other drugs is denoted as... A The minimum inhibitory concentration (MIC) when drugs B are used in combination is denoted as MIC. BLet A and B be the MICs of the two drugs when used alone. The formula for calculating FICI is as follows: FICI = MIC A / A+MIC B / B.

[0035] The criteria for determining synergistic effects are as follows: ① FICI ≤ 0.5, synergistic effect; ② 0.5 < FICI ≤ 1, additive effect; ③ 1 < FICI ≤ 2, unrelated; ④ FICI > 2, antagonistic effect.

[0036] II. Results

[0037] Table 1. In vitro activity of compound 1 in combination with Combination-1 against mycobacteria at different ratios.

[0038]

[0039] Note 1: In the table, "A+" in Mycobacterium tuberculosis activity indicates MIC. 90 Values ​​less than or equal to 0.1 μM, “A” indicates MIC. 90 Values ​​range from 0.1 to 1 μM, where “B” indicates MIC. 90 Values ​​range from 1 to 15 μM; for Mycobacterium marineum, Mycobacterium abscessus, and Mycobacterium avium complex, "A+" indicates MIC. 90 Values ​​less than or equal to 10 μM, “A” indicates MIC 90 Values ​​range from 10 to 40 μM, where “B” indicates MIC. 90 Values ​​range from 40 to 80 μM; among Mycobacterium smegmatis, Mycobacterium ulcerans, Mycobacterium guilloché, and Mycobacterium kansasii, "A+" indicates MIC. 90 Values ​​less than or equal to 1 μM, “A” indicates MIC 90 Values ​​range from 1 to 15 μM, where “B” indicates MIC. 90 The values ​​ranged from 15 to 30 μM. Furthermore, the activity of compounds 2 to 20 against more than one mycobacterium was tested in parallel in this invention.

[0040] Note 2: "**" means FICI ≤ 0.5, "*" means 0.5 < FICI ≤ 1, "#" means 1 < FICI ≤ 2, and "##" means FICI > 2.

[0041] Table 2 shows the in vitro activity of compound 1 in combination with Combination-2 against mycobacteria at different ratios.

[0042]

[0043] Note 1: In the table, "A+" in Mycobacterium tuberculosis activity indicates MIC.90 Values ​​less than or equal to 0.1 μM, “A” indicates MIC. 90 Values ​​range from 0.1 to 1 μM, where “B” indicates MIC. 90 Values ​​range from 1 to 15 μM; for Mycobacterium marineum, Mycobacterium abscessus, and Mycobacterium avium complex, "A+" indicates MIC. 90 Values ​​less than or equal to 10 μM, “A” indicates MIC 90 Values ​​range from 10 to 40 μM, where “B” indicates MIC. 90 Values ​​range from 40 to 80 μM; among Mycobacterium smegmatis, Mycobacterium ulcerans, Mycobacterium guilloché, and Mycobacterium kansasii, "A+" indicates MIC. 90 Values ​​less than or equal to 1 μM, “A” indicates MIC 90 Values ​​range from 1 to 15 μM, where “B” indicates MIC. 90 The values ​​ranged from 15 to 30 μM. Furthermore, the activity of compounds 2 to 20 against more than one mycobacterium was tested in parallel in this invention.

[0044] Note 2: "**" means FICI ≤ 0.5, "*" means 0.5 < FICI ≤ 1, "#" means 1 < FICI ≤ 2, and "##" means FICI > 2.

[0045] This invention tested similar compounds a1-a8 as controls in parallel, and their structures are as follows.

[0046]

[0047] in conclusion:

[0048] As shown in Table 1, when compound 1 is used in combination with Combination-1~2 in a ratio of 1:5, FICI ≤ 0.5, indicating a synergistic effect.

[0049] The above results indicate that compound 1, when used in combination with drugs for treating mycobacterial infections in a specific ratio, can produce a significant synergistic anti-mycobacterial effect, suggesting that this combination has the potential to be developed into an anti-mycobacterial combination therapy.

[0050] Similar to the aforementioned checkerboard microdilution method, 20 alkaloid compounds (compounds 1-20) were combined with Combinations 1-5, and the FICI values ​​of each combination were determined to screen for synergistic effects. A total of 100 combinations were tested. The results showed that, using FICI ≤ 0.5 as the synergistic criterion, 42 combinations exhibited synergistic effects, accounting for 42.0% of the total combinations. Among them, 21 combinations showed strong synergistic effects with FICI ≤ 0.3.

[0051] This invention also screened compounds a1-a8 as controls, but none of these compounds showed significant synergistic effects when used in combination with drugs for the treatment of mycobacterial infections.

[0052] The combined screening results showed that the alkaloid compounds described in this invention can produce synergistic effects when used in combination with existing mycobacterial infection treatment drugs. Among them, the combination of compounds 2, 7, 12, and 16 with mycobacterial infection treatment drugs has the most significant synergistic effect (FICI ≤ 0.35, clinical drug dosage reduction ≥75%).

[0053] Based on the above screening results, this study further selected compounds 2, 7, 12, and 16, which had the best synergistic effect, and combined them with low-dose regimens of pyrazinamide and linezolid, respectively (see Experimental Example 2).

[0054] Example 2: Screening for anti-mycobacterial activity of compound 2 combined with Combination-3

[0055] I. Materials and Methods

[0056] Same as Example 1.

[0057] Tested strains: Mycobacterium tuberculosis H37Ra, Mycobacterium marinum, Mycobacterium smegmatis, Mycobacterium ulcerans, Mycobacterium abscessus, Mycobacterium avium complex, Mycobacterium guilloché, Mycobacterium kansasii.

[0058] Test drugs: Compounds 2, 7, 12, and 16, which were screened based on the results of Example 1 and showed the best synergistic effect, as well as bedaquiline, putomomani, linezolid, and moxifloxacin, which are used to treat mycobacterial infections.

[0059] II. Experimental Methods

[0060] Based on the combined experiment in Example 1, the antibacterial activity of low-dose linezolid combination was further investigated. The specific protocol was the same as in Example 1.

[0061] III. Experimental Results

[0062] The FICI values ​​and evaluation results of the combination of compound 2 and combination-3 in different ratio ranges are shown in Table 3.

[0063] Table 3. Antimycobacterial activity of compound 2 and combination-3

[0064]

[0065] Note 1: In the table, "A+" in Mycobacterium tuberculosis activity indicates MIC. 90 Values ​​less than or equal to 1 μM, “A” indicates MIC 90Values ​​range from 1 to 15 μM; Mycobacterium marineum, Mycobacterium abscessus, Mycobacterium avium complex, activity, "A+" indicates MIC. 90 Values ​​range from 10 to 40 μM, where “A” indicates MIC. 90 Values ​​range from 40 to 80 μM, where "B" indicates MIC. 90 Values ​​range from 80 to 100 μM; among Mycobacterium smegmatis, Mycobacterium ulcerans, Mycobacterium guilloché, and Mycobacterium kansasii, "A+" indicates MIC. 90 Values ​​range from 1 to 15 μM, where “A” indicates MIC. 90 Values ​​range from 15 to 30 μM, where “B” indicates MIC. 90 The value is between 30-50 μM.

[0066] Note 2: "**" means FICI ≤ 0.5, "*" means 0.5 < FICI ≤ 1, "#" means 1 < FICI ≤ 2, and "##" means FICI > 2.

[0067] in conclusion:

[0068] Table 3 shows that when compound 2 and combination-3 are used in combination, they exhibit a certain synergistic or additive effect. When the two are used in a 1:5 ratio, FICI ≤ 0.5, demonstrating a synergistic effect, the overall dosage of the drug for treating mycobacterial infections is reduced by 75%. Based on this scheme, this invention also tested a drug combination scheme with reduced pyrazinamide dosage, and the results showed that this combination scheme can still synergistically kill bacteria.

[0069] In addition, this invention also tested combination therapy regimens of compounds 2, 7, 12, 16 and clinical therapeutic agents. The effects were essentially equivalent, and due to space limitations, they will not be shown further.

[0070] In summary, this invention, through a systematic screening of 20 alkaloid compounds combined with five clinical treatment drugs for mycobacterial infections, has demonstrated the significant potential of these alkaloid compounds as adjuvants in combination therapy. This composition effectively enhances the efficacy against mycobacteria while significantly reducing the dosage of highly toxic drugs such as linezolid and pyrazinamide, thereby mitigating their toxic side effects. This invention provides a novel, highly effective, and low-toxicity combination therapy strategy for the treatment of mycobacterial infections, and has promising prospects for clinical translational applications.

Claims

1. A pharmaceutical composition for treating mycobacterial infections, characterized in that, Contains active components (a) and (b): (a) Selected from any one or more of alkaloid compounds 1 to 20 or their pharmaceutically acceptable salts; (b) Three or more medications for treating mycobacterial infections; The weight ratio of (a) to (b) is 1:10 to 10:

1.

2. The pharmaceutical composition according to claim 1, characterized in that, The alkaloid compounds 1-20 have the following structures: 。 3. The pharmaceutical composition according to claim 1, characterized in that, The drugs for treating mycobacterial infections are selected from three or more of the following: isoniazid, rifapentine, pyrazinamide, moxifloxacin, rifampin, ethambutol, bedaquiline, putomani, linezolid, delamani, levofloxacin, clofazimine, and streptomycin.

4. The pharmaceutical composition according to claim 3, characterized in that, The following five formulations of anti-mycobacterial positive drugs are included: (1) Isoniazid + Rifapentine + Pyrazinamide + Moxifloxacin (1~3:1~3:1~3:1~3); (2) Isoniazid + Rifapentine + Moxifloxacin (1~3:1~3:1~3); (3) Isoniazid + Rifampin + Pyrazinamide + Ethambutol (1~3:1~3:1~3:1~3); (4) Bedaquiline + Putomomani + Linezolid + Moxifloxacin (1~3:1~3:1~3:1~3); (5) Bedaquiline + Delamani + Linezolid + Levofloxacin + Clofazimine (1~3:1~3:1~3:1~3:1~3), where the ratio refers to the relative weight fractions of each component.

5. The pharmaceutical composition according to any one of claims 1 to 4, characterized in that, It also includes one or more pharmaceutically acceptable excipients, carriers, adjuvants, and solvents.

6. Use of the pharmaceutical composition according to any one of claims 1 to 5 in the preparation of a medicament for treating mycobacterial infections.

7. The use according to claim 6, characterized in that, The mycobacterial infections mentioned include those caused by Mycobacterium tuberculosis, Mycobacterium marineum, Mycobacterium smegmatis, Mycobacterium ulcerans, Mycobacterium abscessus, Mycobacterium avium complex, Mycobacterium guildrums, and Mycobacterium kansasii.

8. The use according to claim 6 or 7, characterized in that, The combined administration produces a synergistic effect compared to monotherapy, achieving one or more effects such as improving antibacterial efficacy and reducing the dosage of combined therapeutic drugs.

9. The use according to claim 6 or 7, characterized in that, Alkaloids and clinical treatment drugs are administered separately without any order of administration, or alkaloids and clinical treatment drugs are administered simultaneously.

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