Use of the anti-idiopathic pulmonary fibrosis drug nintedanib for the treatment of tuberculosis.

Abstract

Use of nintedanib or a medicinal salt thereof for the manufacture of a drug for treating tuberculosis, wherein the nintedanib or a medicinal salt thereof is used in combination with another antituberculous drug to treat tuberculosis or as an adjunct to tuberculosis treatment, the other antituberculous drug being selected from rifampicin, isoniazid, pyrazinamide, ethambutol, fluoroquinolones, streptomycin, kanamycin, amikacin, capreomycin, sodium 4-aminosalicylate, ethionamide, cycloserine, clofazimine, and linezolid.

Description

[Technical Field]

[0001] This invention relates to novel uses of drugs, and more particularly to the use of nintedanib for the manufacture of a tuberculosis treatment drug. [Background technology]

[0002] Tuberculosis (TB) is a chronic infectious disease caused by infection with Mycobacterium tuberculosis (MTB) and remains one of the leading causes of death from a single infectious disease. Infection with Mycobacterium tuberculosis typically leads to pathological changes characterized by granulomatous inflammation, destruction of lung parenchyma, and interstitial fibrosis. Existing chemotherapy regimens suffer from problems such as long treatment durations, numerous side effects, and poor efficacy. Standard anti-tuberculosis therapy is highly effective against drug-susceptible tuberculosis, and microbiological cure is possible, but more than two-thirds of patients experience extensive structural changes in the lungs after treatment, and more than half experience permanent lung dysfunction. Secondary fibrosis of pulmonary tuberculosis (PIAT) is a common post-tuberculosis pulmonary impairment. Extensive pulmonary fibrosis not only severely impacts patients' lung function, but the fibrous structure makes it difficult for drugs to completely kill the bacteria, leading to a high risk of relapse.

[0003] Mycobacterium tuberculosis has evolved alongside the human immune system, surviving within infected cells through various pathways and causing severe histopathological damage to the host, thereby reducing the effectiveness of existing antibiotics and promoting the development of drug resistance. For these reasons, there is a strong need for new treatment methods and drug combinations to address Mycobacterium tuberculosis infection. Host-directed therapy (HDT) is one of the newly emerging treatment methods in the field of anti-infective medicine and is a new and effective adjunctive therapy for tuberculosis. HDT therapy can (1) enhance the effects of antibiotics, (2) shorten the duration of TB treatment, (3) prevent recurrence, and (4) improve immunopathology such as TB-related substrate destruction and fibrosis, which interferes with the penetration and therapeutic effect of anti-tuberculosis drugs. Several studies have revealed that cytokines such as tumor necrosis factor α (TNF-α), transforming growth factor β (TGFβ), and interleukin-1β (IL-1β) mediate the progression of fibrosis and may cause respiratory failure in patients with late-stage chronic tuberculosis. This highlights the need for HDT therapy for collagen deposition and fibrosis.

[0004] Nintedanib (BIBF1120) is a potent intracellular tyrosine kinase inhibitor, and its ethanesulfonate salt is used clinically. Its structural formula is as follows: [ka]

[0005] Nintedanib ethanesulfonate, currently available on the market, is a soft capsule formulation used clinically to treat idiopathic pulmonary fibrosis (IPF). Previous studies have shown that transforming growth factor (TGF)-β, platelet-derived growth factor (PDGF), epidermal growth factor, fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF) all play important roles in the progression of fibrosis. It is thought that nintedanib inhibits various signaling receptors involved in the pathogenesis of fibrosis, such as platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), and vascular endothelial growth factor receptor (VEGFR). Nintedanib restricts the function of fibrosis-promoting mediators released from damaged ECM by inhibiting PDGFR, FGFR, and VEGFR. Because nintedanib functions as a VEGFR inhibitor, it may have a dual beneficial effect of reducing fibrosis and angiogenesis in granulomas.

[0006] To date, there have been no reports, either in China or abroad, of the use of nintedanib for the treatment of tuberculosis.

[0007] This invention was developed based on related research that found nintedanib could be used to treat tuberculosis. [Overview of the project]

[0008] Therefore, the present invention provides a use for manufacturing tuberculosis treatment drugs using nintedanib or its medicinal salt alone or in combination with other anti-tuberculosis drugs.

[0009] In the use described in this invention, the medicinal salt of nintedanib is nintedanib ethanesulfonate.

[0010] The tuberculosis described in this invention is an infectious disease caused by infection with Mycobacterium tuberculosis, where tuberculosis occurring in the lungs is pulmonary tuberculosis, and tuberculosis occurring outside the lungs is extrapulmonary tuberculosis.

[0011] In the use described in the present invention, the drug is used alone or in combination with other anti-tuberculosis drugs to treat tuberculosis, or as an adjunct to the treatment of tuberculosis.

[0012] In the use described in the present invention, the other anti-tuberculosis drugs are selected from rifampicin, isoniazid, pyrazinamide, ethambutol, fluoroquinolones, streptomycin, fluoroquinolones, kanamycin, amikacin, capreomycin, sodium 4-aminosalicylate, ethionamide, cycloserine, clofazimine, and linezolid.

[0013] In the use described in the present invention, the combination includes the combination of three or more drugs, or the manufacture of three or more drugs as a compound pharmaceutical preparation.

[0014] In the use described in this invention, the dosage and administration of nintedanib ethanesulfonate is 50 to 300 mg / day, administered orally 1 to 3 times a day.

[0015] In the use described in this invention, when used for the treatment of tuberculosis, the treatment period is at least 4 to 6 months.

[0016] Another object of the present invention is to provide a pharmaceutical composition comprising nintedanib and other anti-tuberculosis drugs, specifically, one prepared by mixing nintedanib ethanesulfonate with four drugs: isoniazid, rifampicin, and pyrazinamide.

[0017] The present invention has unexpectedly found that nintedanib has a therapeutic effect on tuberculosis by analyzing and evaluating in vitro and in a mouse tuberculosis model. In further experiments, the present invention has found that the combination of nintedanib ethanesulfonate and rifampicin, or the combination of nintedanib ethanesulfonate and isoniazid (H), rifampicin (R), and pyrazinamide has a synergistic effect.

[0018] Therefore, preferably, the present invention provides a combination of nintedanib ethanesulfonate and rifampicin for treating tuberculosis, or a combination of nintedanib ethanesulfonate and isoniazid (H), rifampicin (R), and pyrazinamide. The combination includes simultaneous administration and sequential administration of both, and the dosage is the effective dosage of each. For example, 150 mg of nintedanib ethanesulfonate and 450 - 600 mg of rifampicin are taken once a day. In the case of the combination of nintedanib ethanesulfonate and isoniazid (H), rifampicin (R), and pyrazinamide (Z), they may be administered by standard administration methods respectively.

[0019] For easy administration, the present invention further provides a compound pharmaceutical preparation prepared with nintedanib ethanesulfonate and rifampicin as pharmaceutical active ingredients. The compound pharmaceutical preparation may be in the form of any oral preparation such as tablets, capsules, granules, or may be in the form of soft capsules. In unit dosage preparations, the content of both is the effective dosage. For example, each tablet contains 50 - 300 mg of nintedanib ethanesulfonate and 50 - 300 mg of rifampicin. Preferably, each tablet contains 150 mg of nintedanib ethanesulfonate and 450 - 600 mg of rifampicin.

[0020] The present invention further provides experimental results related to nintedanib.

Effects of the Invention

[0021] The present invention has the following advantages over existing tuberculosis drug therapies. Since no related reports have been found in China or overseas, it is novel. It was first found that nintedanib has anti-tuberculosis activity. The treatment period is shortened. In the case of drug-susceptible tuberculosis, it is expected to be shortened to 4 to 5 months. The treatment effect is good. It has the same treatment effect as existing therapies. The recurrence rate is low. Compared with existing therapies, the recurrence rate is even lower.

Brief Description of the Drawings

[0022] [Figure 1] Figure 1 shows the results of CFU counts in lung tissues of each treatment group at different time points. [Figure 2] Figure 2 shows the hydroxyproline (HYP) content of each treatment group at different time points. [Figure 3] Figure 3 shows the alveolar inflammation scores after 4 weeks of treatment and after 8 weeks of treatment. [Figure 4] Figure 4 shows the Masson staining results of the BIBF1120 + HRZ treatment group and the HRZ treatment group. [Figure 5] Figure 5 shows the positive area ratio of Masson staining. [Figure 6] Figure 6 shows the ratio of positive special staining of each treatment group. [Figure 7] Figure 7 shows the immunohistochemical examination results indicating that the expression level of CD31 in the BIBF1120 + HRZ treatment group is reduced compared to the HRZ treatment group.

Modes for Carrying Out the Invention

[0023] Hereinafter, the present invention will be described in detail with reference to the drawings and examples, which are for interpreting the present invention and not for limiting it.

[0024] Experimental Example 1: In Vitro Activity of Nintedanib Against Mycobacterium tuberculosis The H37Rv strain was cultured until it reached the logarithmic growth stage, and the bacterial suspension was prepared in 7H9 liquid medium in 1 × 10⁶ units for use. 6 The drugs were diluted to CFU / mL. Rows A-F were, in order, positive / negative control wells, INH, RFP, PFD, SC1011, and BIBF1120, as described below. Each drug well was sequentially diluted 2-fold in a 96-well plate. After incubation at 37°C for 7 days, 20 μL of Alamar Blue and 12.5 μL of 20% polysorbate 80 were added to each well, and the wells were incubated at 37°C for 24 hours. The color of each well was recorded, with blue indicating no bacterial growth and red indicating bacterial growth. The MIC represents the minimum drug concentration at which the color changed from blue to red. The experiment was repeated three times. The results showed that nintedanib has antibacterial activity and that its MIC against the H37Rv standard strain was 24.567 μg / mL.

[0025] Experimental Example 2: Effect of nintedanib on the anti-tuberculosis drug rifampicin Using the microdilution method, the minimum drug concentration (MIC) at which a drug inhibits 90% of the growth of Mycobacterium tuberculosis is determined. 90 The MIC values ​​were measured, and then, based on the MIC values ​​when each drug was used alone, BIBF1120 and RFP were diluted to six concentrations ranging from 2×MIC to 1 / 16×MIC using the 2-fold dilution method. 50 μL of drug A (BIBF1120) at concentrations ranging from 2×MIC to 1 / 16×MIC was added to the second column of a 96-well plate, and 50 μL of drug B (RFP) at concentrations ranging from 2×MIC to 1 / 16×MIC was added to the first seven wells of rows 2 to 7. 50 μL of 7H9 was added to each single-drug MIC measurement well, and finally, 100 μL of the diluted bacterial suspension was added to each well of the 96-well plate, creating a positive control well (100 μL of diluted bacterial suspension + 100 μL of 7H9) and a negative control well (200 μL of 7H9). The MIC of a single drug in a combination drug study was determined by observing the color change of a 96-well plate (a change from blue to purple or pink indicates bacterial growth, while a blue color indicates no bacterial growth due to drug inhibition). The effect of the combination was determined by FICI, where FICI = (MIC). A併用 / MIC A単独 )+(MIC B併用 / MICB単独 ) and MIC A単独 , MIC B単独 represent the MICs when drug A and drug B act on Mycobacterium tuberculosis alone, respectively. MIC A併用 , MIC B併用 represent the MICs of drug A and drug B corresponding to obtaining the same drug efficacy as the single use of the drug when drug A and drug B are used in combination. When FICI ≤ 0.5, the two drugs show a synergistic effect. When 0.5 < FICI < 1, the two drugs show a partial synergistic effect. When FICI = 1, the two drugs show an additive effect. When 1 < FICI < 4, the two drugs do not affect each other. When FICI > 4, the two drugs show an antagonistic effect. The results showed that the combination of the two drugs exhibited antibacterial activity.

[0026] Experimental Example 3: Bactericidal activity of nintedanib at different concentrations within macrophages The anti-tuberculosis activities of BIBF1120 alone and the combination of BIBF1120 + RFP were evaluated for bactericidal activity within macrophage cells. The specific procedure was as follows. J774A.1 macrophages were placed in a 50 mL centrifuge tube, and 100 μL of macrophages and 900 μL of cell culture medium were used, added to a 1.5 mL sterile centrifuge tube and diluted 10-fold. Counted under a microscope. The macrophages were diluted to 4 × 10 5 cells / mL and gently seeded at 1 mL per well in a 48-well transparent microplate. After the seeding was completed, it was placed in a 37 °C, 5% CO2 incubator and cultured for 24 hours. MOI = 5:1 with 2 × 10A 6H37Rv cells at CFU / mL were co-cultured with macrophages. After 4 hours, the cells were washed twice with sterile PBS to remove extracellular Mycobacterium tuberculosis, and fresh RPMI cell culture medium was added. The cells were incubated in a 37°C, 5% CO2 incubator for 72 hours. After 3 days, the RPMI was discarded from the 48-well plate, 200 μL of 0.1% SDS was added, and the plate was incubated for 5-10 minutes. 800 μL of cell culture medium was added to each well and mixed uniformly. After the cells had lysed, 100 μL was taken from each well and diluted 10-fold, 100-fold, 1000-fold, and 100,000-fold. 100 μL of each dilution was inoculated into 7H10 solid medium, spread uniformly using a spreader, and the CFU was counted after 3 weeks. Table 1: Results of intracellular bactericidal activity of BIBF1120 alone [Table 1] Table 2: Results of bactericidal activity of the BIBF1120 + RFP combination in macrophages [Table 2]

[0027] In conclusion, the following was found: In experiments within macrophages, the combination of BIBF1120 and the anti-tuberculosis drug RFP showed a synergistic bactericidal effect at safe dose concentrations. BIBF1120 did not show a clear antibacterial effect when administered alone at a dose of 25 μg / mL (corresponding to the results of in vitro MIC experiments).

[0028] Experimental Example 4: Effects of nintedanib in a mouse tuberculosis model H37Rv strain in the logarithmic growth phase was raised in 20 mL of 1 × PBS, resulting in a bacterial concentration of 1 × 10⁶. 7The solution was diluted to CFU / mL and used to infect 6-8 week old female C57BL / 6 mice via aerosol. An aerosol-induced mouse model of chronic tuberculosis was established, and the antibacterial activity of each treatment group was evaluated in this model. Three mice were randomly selected and killed on day 10 after infection (D-32), and six mice were randomly selected and killed on the day of administration (D0). The mice were dissected, spleen and lung tissue was collected and homogenized, and the colonies (CFUs) in the spleen and lungs were counted on 7H10 plates to determine the baseline number of Mycobacterium tuberculosis in the lungs and spleen of mice in the early stages of infection and at the start of treatment. Administration was started 6 weeks after infection, and 7-8 mice were randomly selected and killed from each treatment group 4 and 8 weeks after administration. The mice were dissected, spleen and lung tissue was collected and homogenized, and the colonies (CFUs) in the spleen and lungs were counted on 7H10 plates. After 8 weeks of treatment and 12 weeks of drug-free periods, the recurrence of Mycobacterium tuberculosis in the lungs and spleens of 7 mice in each treatment group was observed. After infecting C57BL / 6 mice for 6 weeks, they were randomly divided into three groups (see Table 3). Table 3: Results of CFU counts in lung tissue at different time points for each treatment group [Table 3] Table 4: Results of spleen CFU counts at different time points for each treatment group [Table 4] Here, H represents isoniazid, R represents rifampicin, and Z represents pyrazinamide. D-32 is day 10 of infection, W4 is 4 weeks after treatment, W8 is 8 weeks after treatment, and W8+12W is relapse observation after 8 weeks of treatment and 12 weeks of drug-free rest. D is day, and W is week. The doses were 10 mg / kg / day for isoniazid, 10 mg / kg / day for rifampicin, 150 mg / kg / day for pyrazinamide, 100 mg / kg / day for pirfenidone (PFD), and 50 mg / kg / day for BIBF1120. Here, RFP was administered at least 1 hour apart from the other drugs.

[0029] In conclusion, the following was found: 1. Both the BIBF1120 + HRZ treatment group and the HRZ treatment group showed significant bactericidal effects at 4 and 8 weeks after administration. Of these, the spleen and lungs became sterile after 8 weeks of BIBF1120 administration, and the addition of BIBF1120 can shorten the treatment time for pulmonary tuberculosis and achieve sterility earlier.

[0030] 2. Compared to the HRZ treatment group, the BIBF1120 + HRZ treatment group can reduce the relapse rate in mice.

[0031] (Experimental Example 5) A HYP measurement kit was used. The hydroxyproline content in right lung tissue was measured. Fresh lung tissue was isolated by dissection, weighed, placed in a test tube, and exactly 1 mL of hydrolysate was added. The test tube was bathed in a water bath for 20 minutes. The pH was adjusted to approximately 6.0-6.8. Next, distilled water was added to make 10 mL, 4 mL of diluted hydrolysate was taken out, and an appropriate amount of activated carbon was added. After centrifugation at 3500 rpm for 10 minutes, 1 mL of the supernatant was taken out and measured. The blank tube and standard tube represent distilled water and the standard, respectively. Each reagent was added sequentially according to the instructions. Finally, the zero was adjusted using distilled water at 550 nm using a microplate reader, and the absorbance A value of each tube was measured. Formula (μg / mg 湿重量 )=(A 測 -A ブランク ) / (A 標 -A ブランク The HYP content was calculated using the formula: ) × 5 μg / mL (content in standard tube) × [10 (total amount of hydrolysates) / wet weight of lung tissue]. Here, A 測 is the absorbance of the measuring tube, and A ブランク is the absorbance of the blank tube, and A 標 This was the absorbance of the standard tube. Table 5: HYP content in lung tissue at each treatment time point. [Table 5]

[0032] HE staining and results analysis: Pathological examination of lung tissue was performed as follows: Left lung tissue was collected from mice, trimmed, fixed with 4% paraformaldehyde, and after confirming its good condition, washed with physiological saline, fixed with 4% paraformaldehyde solution, and embedded in paraffin as usual. In this way, 3 μm paraffin sections were prepared, and pathological changes in the lung tissue were observed by HE staining. Pathological changes in the lung tissue were also observed under a light microscope, and the degree of inflammatory infiltration was described to determine the pathological lung injury score. The degree of lung injury was evaluated using four indicators: 1. alveolar congestion, 2. alveolar hemorrhage, 3. neutrophil infiltration or aggregation into the alveolar space or vascular wall, and 4. alveolar septal thickening. The scoring criteria were as follows: 0 points for no injury, 1 point for mild injury, 2 points for moderate injury, and 3 points for severe injury. The sum of the scores for each item was the pathological lung injury score. Table 6: Alveolar inflammation score after 4 weeks of treatment [Table 6] Table 7: Alveolar inflammation score after 8 weeks of treatment [Table 7]

[0033] Masson staining was performed according to the instructions in the kit. Sections were deparaffinized with water as usual, immersed overnight in Masson stain A, immersed for 1 minute in a mixture of equal parts Masson stain B and Masson stain C, immersed for 6 minutes in Masson stain D, immersed for 1 minute in Masson stain E, immersed for 2-30 seconds in Masson stain F, rinsed with 1% glacial acetic acid to induce differentiation, dehydrated with anhydrous ethanol, cleared with xylene, mounted in neutral balsam, and observed under a standard light microscope. As shown in Figures 5 and 6, collagen fibers appeared in blue, while muscle fibers, cellulose, and red blood cells appeared in red. Tissue stained section images were acquired using an imaging system, the tissue measurement area was automatically identified using analysis software, the positive area and tissue area within the measurement area were calculated, and the positive area percentage was obtained.

[0034] After rewarming the frozen sections, they were washed three times with PBS buffer and sodium citrate was added. The sections were allowed to cool naturally, washed, and then 5% goat serum for blocking was added, and the sections were blocked at room temperature for 2 hours. After blocking was complete, anti-CD31 antibody was added and incubated overnight at 4°C, with an antibody concentration of 1:200. The following day, biotin-labeled secondary antibody was added, incubated at room temperature for 1 hour, washed three times, horseradish peroxidase-labeled streptavidin working solution was added, incubated at 37°C for 15 minutes, stained with DAB, restained with hematoxylin, the slides were sealed, and photographs were taken under a biological microscope for recording. As shown in Figure 7, the immunohistochemical results show that the BIBF1120+HRZ treatment group has reduced CD31 expression compared to the HRZ treatment group. Therefore, it can be inferred that the adjunctive effect of BIBF1120 on tuberculosis treatment is due to its ability to suppress abnormal angiogenesis, thereby promoting the delivery of small molecule compounds (anti-tuberculosis drugs) and thus exhibiting better anti-tuberculosis activity.

Claims

1. Use of a compound pharmaceutical preparation in the manufacture of a drug containing Mycobacterium tuberculosis (MTB), The aforementioned compound pharmaceutical preparation is a compound pharmaceutical preparation manufactured by mixing four drugs: nintedanib ethanesulfonate, isoniazid, rifampicin, and pyrazinamide.

2. The use of the compound pharmaceutical preparation according to Claim 1, wherein the dosage and administration are 50 to 300 mg / day, administered once to three times a day, and the treatment period is at least four to six months.

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