Application of rupatadine fumarate in the preparation of drugs for inhibiting mycobacteria

Abstract

The present invention belongs to the field of biomedicine and specifically relates to the use of rupatadine fumarate in the preparation of a drug for inhibiting mycobacteria. The present invention is the first to discover that the commercial second-generation H1-antihistamine rupatadine fumarate exhibits excellent inhibitory effects on both Mycobacterium tuberculosis and non-tuberculosis mycobacteria, with a minimum inhibitory concentration of 4 μg / mL. It also has a synergistic effect in inhibiting mycobacterial activity with existing antibiotics such as pratomanid (PA824), clofazimine, and TB47.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the application of rupatadine fumarate in the preparation of a drug for inhibiting mycobacteria. Background Art

[0002] Allergic rhinitis is a highly prevalent and common disease worldwide, affecting at least 10-25% of the general population worldwide, affecting the social life of patients, and the cost of treatment is huge. Histamine plays an important role in the pathogenesis of allergic rhinitis, mainly through the histamine H1 receptor. Platelet-activating factor (PAF) is another important inflammatory factor. Histamine and PAF promote the release of each other in different tissues and cells. Rupatadine Fumarate (RTF, chemical structure see Figure 1 ) is a commercially available second-generation H1 antihistamine. While most clinically available drugs inhibit a single inflammatory factor, RTF has dual affinity for both histamine H1 and PAF receptors. It is indicated for the treatment of seasonal allergic rhinitis (SAR), perennial allergic rhinitis (PAR), and chronic idiopathic urticaria (CIU) in patients aged 12 years and older. It has a rapid onset and a long duration of action. First-generation H1 antihistamines can cause side effects such as drowsiness, fatigue, headache, memory and learning impairment, and visual impairment, but RTF does not cause these side effects.

[0003] Mycobacterium tuberculosis (Mtb), the most virulent member of the Mycobacterium genus, is the pathogen that causes tuberculosis (TB). Mtb has been studied for a century, but eradication remains elusive. This is compounded by the emergence and spread of drug-resistant mutants due to the misuse and overuse of antibiotics, which has rendered traditionally effective treatments ineffective. Therefore, the most pressing task in treating TB is to identify more effective anti-Mtb drugs. Repurposing existing drugs for new uses may be the most feasible approach. Since these drugs are already clinically used for other diseases, this could significantly shorten the time it takes to commercialize TB treatments. In addition to Mtb, approximately 150 species of non-tuberculous mycobacteria (NTM) are also prevalent. These less virulent species are opportunistic pathogens that can cause lung and skin infections in adults and cervical lymphadenitis in children. NTM are characterized by a thin peptidoglycan layer surrounded by a thick, lipid-rich outer layer, which enables them to adhere to rough surfaces and is resistant to antibiotics and disinfectants, helping them survive hypoxia and other adverse conditions. Furthermore, many NTM, such as Mycobacterium abscessum (Mab), possess natural drug resistance and are therefore naturally resistant to clinical drugs, making their treatment challenging. The screening and development of effective drugs is currently a major challenge. Summary of the Invention

[0004] In response to the problems existing in the prior art of anti-mycobacterial drug screening, the present invention aims to provide the use of rupatadine fumarate in the preparation of drugs that inhibit mycobacteria. Rupatadine fumarate exhibits a good inhibitory effect on mycobacteria and has a synergistic inhibitory effect on mycobacteria with existing antibiotics and other drugs.

[0005] Based on the above purpose, the technical solution adopted by the present invention is as follows:

[0006] In a first aspect, the present invention provides use of rupatadine fumarate in the preparation of a drug for inhibiting mycobacteria.

[0007] The present invention discovered for the first time that the commercialized second-generation H1-antihistamine rupatadine fumarate exhibits excellent inhibitory effects on mycobacteria, especially Mycobacterium tuberculosis, indicating that RTF, as an active drug, has high application potential in the research and development of drugs against Mycobacterium tuberculosis and non-tuberculous mycobacteria.

[0008] Preferably, the mycobacteria include Mycobacterium tuberculosis and non-tuberculous mycobacteria; the non-tuberculous mycobacteria include Mycobacterium marinum, Mycobacterium abscessum and Mycobacterium smegmatis.

[0009] Preferably, Mycobacterium tuberculosis is Mtb H 37 Ra.

[0010] Preferably, the minimum inhibitory concentrations of rupatadine fumarate against Mycobacterium tuberculosis, Mycobacterium marinum, Mycobacterium abscessum, and Mycobacterium smegmatis are 4 μg / mL, 8 μg / mL, 32 μg / mL, and 16 μg / mL, respectively.

[0011] In a second aspect, the present invention provides a drug for inhibiting mycobacteria, comprising rupatadine fumarate.

[0012] Preferably, the mycobacteria include Mycobacterium tuberculosis and non-tuberculous mycobacteria; the non-tuberculous mycobacteria include Mycobacterium marinum, Mycobacterium abscessum and Mycobacterium smegmatis.

[0013] Preferably, the drug further comprises one of putomanib (PA824), clofazimine, and TB47.

[0014] Preferably, rupatadine fumarate has a partial synergistic effect with pratomanid, clofazimine or TB47 against mycobacteria.

[0015] In a third aspect, the present invention provides use of the above-mentioned drug in preparing a drug for treating tuberculosis.

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

[0017] The present invention discovered for the first time that the commercialized second-generation H1-antihistamine rupatadine fumarate exhibits excellent inhibitory effects on both Mycobacterium tuberculosis and non-tuberculosis mycobacteria, with a minimum inhibitory concentration of 4 μg / mL, and has a synergistic inhibitory effect on mycobacterial activity with existing antibiotics such as pratomanid, clofazimine, and TB47. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is the chemical structural formula of rupatadine fumarate;

[0019] Figure 2 Rupatadine fumarate anti-Mtb H 37 Activity curve of Ra;

[0020] Figure 3 Rupatadine fumarate anti-Mtb H 37 Activity curve of Rv;

[0021] Figure 4 This is a bar graph showing the changes in luminescence values of lung tissue grinding fluid. DETAILED DESCRIPTION

[0022] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. Those skilled in the art should understand that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0023] Unless otherwise specified, the experimental methods used in the examples are conventional methods; the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0024] Example 1

[0025] Experiment 1 Screening of anti-Mycobacterium tuberculosis H-type drugs from the anti-allergic drug library 37 The steps for drug experiments on Ra activity are as follows:

[0026] The compounds in the antiallergic drug library were dissolved in dimethyl sulfoxide (DMSO) to prepare a stock solution with a concentration of 10 mg / mL, which was then diluted to 5 mg / mL, 0.5 mg / mL, and 0.05 mg / mL using DMSO. 37 Ra(Autoluminescent MtbH 37Ra, referred to as AlRa), has been experimentally verified that the growth trend of self-luminous bacteria and their sensitivity to various clinical drugs are consistent with those of non-luminous wild-type mycobacteria, so the construction of AlRa can be used for screening of antibacterial active compounds. Single colonies were picked from the plate and the relative light units (RLU) were measured using a luminescence detector. The single colonies that were confirmed to be luminous were inoculated into 5mL 7H9 liquid culture medium (with Tween 80) for culture. When the RLU of 200μL bacterial solution reached 10 6 When the bacterial solution is diluted with 7H9 liquid culture medium (without Tween 80), the RLU of 200 μL diluted bacterial solution is in the range of 3000-5000. 4 μL of drug solution and 196 μL of diluted AlRa are added to a 96-well plate, so the final concentrations of drugs such as RTF are 100 μg / mL, 10 μg / mL and 1 μg / mL. At the same time, a positive control (rifampicin) and a negative control group (DMSO) are set up, and 3 parallels are made for each concentration. The 96-well plate is placed in a 37°C incubator, and the luminescence value is detected using a luminescence detector on the 5th day. MIC is defined as the minimum compound concentration that can reduce the luminescence value to less than 10% of the luminescence value of the DMSO control group.

[0027] Initial screening identified RTF with an MIC between 1 and 10 μg / mL. The RTF and fumarate stock solutions were then diluted to 0.8 mg / mL, 0.4 mg / mL, 0.2 mg / mL, 0.1 mg / mL, and 0.05 mg / mL, respectively. Four μL of the drug solution and 196 μL of the diluted AlRa were added to a sterilized 1.5 mL EP tube, with final concentrations of RTF and fumarate at 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, and 2 μg / mL, respectively. A positive control of rifampicin (RIF) and a negative control (DMSO) were also established, with triplicate replicates performed for each concentration. The EP tubes were placed in a 37°C incubator, and luminescence was measured using a luminometer on days 0, 1, 3, and 5.

[0028] The experimental results are as follows Figure 2 As shown in the results, it can be seen that RTF is anti-Mtb H 37 The MIC of Ra was 4 μg / mL, and fumarate was not found to have bactericidal or antibacterial activity. This indicates that rupatadine is the bactericidal or antibacterial agent in RTF, so rupatadine is effective against Mtb H. 37 The MIC of Ra was 3.13 μg / mL, showing obvious antibacterial activity and having certain application prospects.

[0029] Experiment 2: Detection of RTF anti-mycobacterial activity

[0030] The mycobacterial species used in the experiment are as follows:

[0031] Mycobacterium marinum (Mm), Mycobacterium abscessum (Mab), Mycobacterium smegmatis (Ms), Mycobacterium fortuitum (Mf).

[0032] The experimental steps are as follows:

[0033] Our laboratory has previously successfully constructed self-luminous bacteria Mab, Ms, and Mm without resistance markers. Experimental verification shows that the growth trend and sensitivity of self-luminous bacteria to various clinical drugs are consistent with those of non-luminous wild-type mycobacteria. Therefore, the constructed self-luminous bacteria can be used for screening of antibacterial active compounds. Single colonies were picked from the plate and RLU was detected using a luminescence detector. The single colonies that were confirmed to be luminous were inoculated into 5mL 7H9 liquid culture medium (with Tween 80) for culture. When the RLU value of 200μL bacterial solution was 10 7 At the same time, the bacterial suspension was diluted with 7H9 liquid medium (without Tween 80). A 10 mg / mL stock solution of RTF and fumarate was diluted with DMSO to 6.4 mg / mL, 3.2 mg / mL, 1.6 mg / mL, 0.8 mg / mL, 0.4 mg / mL, 0.2 mg / mL, 0.1 mg / mL, and 0.05 mg / mL. 4 μL of the drug solution and 196 μL of the diluted mycobacterial suspension were added to the same sterile 1.5 mL EP tube, resulting in final RTF and fumarate concentrations of 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, and 1 μg / mL, respectively. Positive controls (linezolid for Ms and clarithromycin for Mab and Mm) and negative controls (DMSO) were also established, with triplicates performed for each concentration. The EP tube was placed in a 37°C incubator and luminescence was measured using a luminescence detector at different time points. The MIC was defined as the minimum compound concentration that could reduce the luminescence value to less than 10% of the luminescence value of the DMSO control group.

[0034] At present, the laboratory has not constructed self-luminescent Mf, so the Mf single colony was picked from the plate and transferred to 5mL 7H9 liquid culture medium (with Tween 80) for culture; when the OD 600When the concentration reached approximately 0.8, the bacterial suspension was diluted 1000-fold with 7H9 liquid medium (without Tween 80). The RTF and fumarate stock solutions (50 mg / mL) were diluted to 0.256 mg / mL. 200 μL of the 0.256 mg / mL RTF solution was added to the second column of a transparent 96-well plate. 100 μL of culture medium was added to columns 3 through 11. The plates were then diluted two-fold from column 2 to column 10, and finally, 100 μL of the diluted bacterial suspension was added to each column. The RTF concentrations in columns 2 through 10 were 128 μg / mL, 64 μg / mL, 32 μg / mL, 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, and 0.5 μg / mL, respectively. Column 11 served as a control with only bacteria. 100 μL of culture medium was added every four weeks to prevent contamination. The 96-well plate was incubated at 37°C for 3 days and the results were observed. The lowest concentration corresponding to the wells where no bacterial growth was observed was defined as the MIC of RTF.

[0035] The experimental results are shown in Table 1. The MICs of RTF against Mm, Mab, Mf, and Ms were 8 μg / mL, 32 μg / mL, 128 μg / mL, and 16 μg / mL, respectively. No bactericidal or bacteriostatic activity was detected in the fumarate salt. Therefore, the bactericidal or bacteriostatic component of rupatadine fumarate is rupatadine. After conversion, the MICs of rupatadine against Mm, Mab, Mf, and Ms were 6.25 μg / mL, 25.02 μg / mL, 100.08 μg / mL, and 12.51 μg / mL, respectively. This indicates that RTF, as an active drug, has certain potential in the development of anti-nontuberculous mycobacteria drugs.

[0036] Table 1 Summary of RTF antimycobacterial activity

[0037] Chinese name scientific name MIC (μg / mL) Mycobacterium marinum Mycobacterium marinum 8 Mycobacterium abscessus Mycobacterium abscessum 32 Mycobacterium fortuitum Mycobacterium fortuitum 128 Mycobacterium smegmatis Mycobacterium smegmatis 16

[0038] Experiment 3: Detection of the combined activity of RTF and anti-Mycobacterium tuberculosis drugs

[0039] The experimental steps are as follows:

[0040] Pick up AlRa single colonies from the plate, use a luminescence detector to detect RLU, inoculate the confirmed luminescent single colonies into 5 mL 7H9 liquid culture medium (with Tween 80) for culture, add glass beads to break up; when the RLU of 200 μL bacterial solution reaches 10 6At the same time, the bacterial suspension was diluted with 7H9 liquid medium (without Tween 80) to obtain a RLU of 3000-5000 in 200 μL of the diluted bacterial suspension. RTF and nine selected anti-TB drugs were diluted two-fold with DMSO, with six different concentrations of each drug prepared. Six columns (B2-G2 to B7-G7) of an opaque 96-well plate were loaded with RTF at varying concentrations (from high to low concentration, with the same RTF solution added to each column). Six rows (B2-B7 to G2-G7) of an opaque 96-well plate were loaded with anti-TB drugs at varying concentrations (from high to low concentration, with the same drug concentration in each row). Finally, 4 μL of drug and 196 μL of the diluted test bacterial suspension were added to each well. A positive control (rifampicin) and a negative control (DMSO) were also set up. After 7 days of incubation at 37°C, the RLU values were measured using a fluorometer. The resulting data were analyzed to determine whether the combination of RTF and the nine anti-TB drugs exhibited activity.

[0041] The experimental results are shown in Table 2. The FICIs of RTF with putomanib, clofazimine, and TB47 were 0.5625, 0.75, and 0.75, respectively, indicating that RTF has partial synergistic effects with these three drugs. The FICIs with the remaining six drugs were all greater than or equal to 1, indicating that the two drugs have additive or unrelated effects. This suggests that RTF may become a new option for combination therapy against Mycobacterium tuberculosis.

[0042] Table 2 Summary of the combined activity of RTF and antimycobacterial drugs

[0043]

[0044] a, RTF; b, antimycobacterial compound; FICI, Fractional Inhibitory Concentration; FICI ≤ 0.5, synergistic effect; 0.5 < FICI < 1, partial synergistic effect; FICI = 1, additive effect; 1 < FICI < 2, indifferent effect; FICI > 2, antagonistic effect.

[0045] Experiment 4: Detection of RTF against Mycobacterium tuberculosis Mtb H 37 Rv activity

[0046] The experimental steps are as follows:

[0047] Our laboratory has previously successfully constructed an autonomous luminescent Mtb H 37 Rv(AutoluminescentMtb H 37Rv, referred to as AlRv), has been experimentally verified to have the same growth trend and sensitivity to various clinical drugs as wild-type mycobacteria that do not emit light. Therefore, the constructed AlRv can be used to screen antibacterial compounds. AlRv was transferred to 15mL 7H9 (+ Tween 80) medium and placed in a 37℃ constant temperature shaker to culture until the RLUs of 200μL bacterial solution reached 10 6 At 4 hr, dilute the test solution with 7H9 (Tween 80-free) medium (RLUs / 200 μL between 3000 and 5000). Use DMSO to dilute RTF and fumarate stock solutions (10 mg / mL) to 0.8 mg / mL, 0.4 mg / mL, 0.2 mg / mL, 0.1 mg / mL, and 0.05 mg / mL. Add 4 μL of the drug solution and 196 μL of the diluted AlRv to the same sterilized 1.5 mL EP tube, giving final RTF concentrations of 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, and 1 μg / mL. A positive control (rifampicin, abbreviated as RIF) and a negative control (DMSO) were also set up, with triplicate replicates for each concentration. The EP tubes were placed in a 37°C incubator, and luminescence was measured using a luminometer on days 0, 1, 3, and 5.

[0048] The experimental results are as follows Figure 3 As shown, RTF anti-Mtb H 37 The MIC of Rv was 4 μg / mL, and fumarate was not found to have bactericidal or antibacterial activity. Therefore, the bactericidal or antibacterial activity of rupatadine fumarate was rupatadine. After conversion, the activity of rupatadine against Mtb H 37 The MIC of Rv was 3.13 μg / mL, showing obvious antibacterial activity and having certain application prospects.

[0049] Experiment 5: Detection of RTF anti-Mtb H in mice 37 Rv activity

[0050] The experimental process is as follows:

[0051] Self-luminescent Mycobacterium tuberculosis H without resistance marker 37 Rv (UAlRv), add 7H9 liquid medium and culture until OD 600 The value reaches 0.8-1.0 and RLU / 200μL is 2×10 6Female BALB / c mice aged 4-6 weeks were selected and allowed to acclimate to the laboratory for 3 days. UAlRv bacterial suspension in late logarithmic growth phase was used to infect mice via aerosol inoculation, a method characterized by excellent infection uniformity and close proximity to natural infection. On day 16 post-infection, the mice were tested for luminescence and randomly divided into groups of 3 mice. RTF at a concentration of 3.84 mg / mL (corresponding to a dose of 38.4 mg / kg for rupatadine at a concentration of 3 mg / mL, corresponding to a dose of 30 mg / kg) and rifampicin at a concentration of 1 mg / mL (corresponding to a dose of 10 mg / kg) were prepared and administered orally for 5 days (200 μL / day per mouse). Mice were killed on days 0 and 5, and lungs were ground in 2 mL of phosphate buffer, and 200 μL of the sample was collected for luminescence analysis. Dosage and grouping information are shown in Table 3 below.

[0052] Table 3

[0053]

[0054]

[0055] Table 4

[0056]

[0057] The experimental results are shown in Table 4. After the data of the luminescence value of the lung tissue suspension were sorted, a bar graph was made as shown in Table 4. Figure 4 As shown in the figure, ns indicates no statistical difference; * indicates p < 0.05; the dosages (mg / kg) of rupatadine and rifampicin were 30 mg / kg and 10 mg / kg, respectively.

[0058] The results in Tables 3 and 4 show that the luminescence value of the lung tissue grinding fluid in the rupatadine treatment group was significantly lower than that in the solvent group after 5 days of administration, and there was no significant difference in luminescence value between the rupatadine treatment group and the rifampicin positive control group, indicating that rupatadine can effectively inhibit Mtb H 37 The growth of Rv. Rupatadine at a dose of 30 mg / kg and rifampicin at a dose of 10 mg / kg had an in vivo effect on the anti-Mtb H 37 The activity of Rv is comparable and has certain application prospects.

[0059] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. Use of rupatadine fumarate in the preparation of a drug for inhibiting mycobacteria, wherein the mycobacteria are non-tuberculous mycobacteria; the non-tuberculous mycobacteria are Mycobacterium marinum ( Mycobacterium marinum ), Mycobacterium abscessus ( Mycobacterium abscessum ) and Mycobacterium smegmatis ( Mycobacterium smegmatis ).

2. The use according to claim 1, characterized in that Rupatadine fumarate has an effect on Mycobacterium marinum ( Mycobacterium marinum ), Mycobacterium abscessus ( Mycobacterium abscessum ) and Mycobacterium smegmatis ( Mycobacterium smegmatis ) were 8 μg / mL, 32 μg / mL and 16 μg / mL, respectively.

3. A drug for inhibiting Mycobacterium tuberculosis, characterized in that: The drug contains rupatadine fumarate and further comprises one of pratomanid, clofazimine, and TB47.

4. The drug for inhibiting Mycobacterium tuberculosis according to claim 3, characterized in that The rupatadine fumarate has a partial synergistic effect with pratomanid, clofazimine or TB47 against Mycobacterium tuberculosis.

5. Use of the drug according to claim 3 or 4 in the preparation of drugs for inhibiting Mycobacterium tuberculosis.

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