Use of the anti-idiopathic pulmonary fibrosis drug nintedanib to treat 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] The present invention relates to a new use of drugs, in particular, the use of nintedanib for the manufacture of a drug for the treatment of tuberculosis. [Background technology]

[0002] Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (MTB) infection and remains one of the single most deadly infectious diseases. MTB infection generally induces pathological changes characterized by granulomatous inflammation, destruction of the lung parenchyma, and interstitial fibrosis. Existing chemotherapy has problems such as long treatment periods, many side effects, and poor efficacy. Standard anti-TB therapy is highly effective against drug-susceptible TB and can achieve microbiological cure, but after treatment, more than two-thirds of patients experience extensive lung structural changes and more than half experience permanent lung function damage. Secondary fibrosis from pulmonary tuberculosis is a common pulmonary impairment after TB (PIAT), and extensive pulmonary fibrosis not only severely affects patients' lung function, but also makes it difficult for drugs to completely kill the bacteria because the TB bacilli are encapsulated in fibers, making it prone to recurrence.

[0003] Mycobacterium tuberculosis has co-evolved with the human immune system and continues to live within infected cells through various pathways, causing severe histopathological damage to the host, which reduces the therapeutic efficacy of existing antibiotics and promotes the development of drug resistance. These factors have led to a strong demand for new therapeutic methods and drug combinations to combat Mycobacterium tuberculosis infection. Host-directed therapy (HDT) is one of the emerging therapeutic methods in the anti-infectious field and is a new and effective adjuvant therapy for tuberculosis. HDT therapy can (1) enhance the action of antibiotics, (2) shorten the duration of TB treatment, (3) prevent relapse, and (4) ameliorate TB-associated immunopathology such as matrix destruction and fibrosis that hinders the penetration and therapeutic efficacy of anti-TB 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) can mediate the progression of fibrosis and cause respiratory failure in patients with late-stage chronic tuberculosis. This highlights the need for HDT therapy against collagen deposition and fibrosis.

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

[0005] Nintedanib ethanesulfonate, currently available on the market in a soft capsule formulation, is clinically used 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. Nintedanib may inhibit various signaling receptors involved in the pathogenesis of fibrosis, including platelet-derived growth factor receptor (PDGFR), fibroblast growth factor receptor (FGFR), and vascular endothelial growth factor receptor (VEGFR). By inhibiting PDGFR, FGFR, and VEGFR, nintedanib limits the function of profibrotic mediators released from damaged ECM. Nintedanib functions as a VEGFR inhibitor and may therefore have dual beneficial effects of reducing fibrosis and angiogenesis in granulomas.

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

[0007] The present invention has conducted related research and found that nintedanib can be used to treat tuberculosis. Summary of the Invention

[0008] Therefore, the present invention provides the use of nintedanib or a pharmaceutical salt thereof alone or in combination with other anti-tuberculosis drugs for the manufacture of a therapeutic drug for tuberculosis.

[0009] In the use according to the present invention, the pharmaceutical salt of nintedanib is nintedanib ethanesulfonate.

[0010] The tuberculosis according to the present invention is an infectious disease caused by infection with Mycobacterium tuberculosis, where tuberculosis occurring within the lungs is pulmonary tuberculosis, and tuberculosis occurring outside the lungs is extrapulmonary tuberculosis.

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

[0012] In the use according to the invention, said other antituberculous drug is selected from rifampicin, isoniazid, pyrazinamide, ethambutol, fluoroquinolones, streptomycin, fluoroquinolones, kanamycin, amikacin, capreomycin, sodium 4-aminosalicylate, ethionamide, cycloserine, clofazimine, linezolid.

[0013] In the use according to the present invention, said combination includes the combination of three or more drugs, or the preparation of three or more drugs as a combined pharmaceutical preparation.

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

[0015] In the use according to the present invention, when used to treat 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, the pharmaceutical composition is prepared by mixing nintedanib ethanesulfonate with four drugs, namely, isoniazid, rifampicin, and pyrazinamide.

[0017] Through analysis and evaluation in vitro and in mouse tuberculosis models, the present invention unexpectedly found that nintedanib has a therapeutic effect against tuberculosis. In further experiments, the present invention 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 including simultaneous or sequential administration of both, the dosage being each effective dose, for example, 150 mg of nintedanib ethanesulfonate and 450-600 mg of rifampicin, taken once a day. In the case of a combination of nintedanib ethanesulfonate and isoniazid (H), rifampicin (R), and pyrazinamide (Z), each may be administered by a standard administration method.

[0019] The present invention provides a combined pharmaceutical preparation prepared by using nintedanib ethanesulfonate and rifampicin as active pharmaceutical ingredients for easy administration, and the combined pharmaceutical preparation may be in any form of oral preparation such as tablet, capsule, granule, or soft capsule form. In the unit dose preparation, the content of both is the effective dose, for example, 50-300 mg of nintedanib ethanesulfonate and 50-300 mg of rifampicin are contained per tablet. Preferably, 150 mg of nintedanib ethanesulfonate and 450-600 mg of rifampicin are contained per tablet.

[0020] The present invention further provides experimental results related to nintedanib. Effect of the Invention

[0021] The present invention has the following advantages over existing tuberculosis drug therapies: This finding is novel because no related reports have been found in China or abroad. This is the first time that nintedanib has been found to have antituberculous activity. The treatment period will be shortened. For drug-susceptible tuberculosis, it is expected to be shortened to four to five months. Good therapeutic effect. It has the same therapeutic effect as existing therapies. Low recurrence rate: The recurrence rate is even lower than with existing therapies. [Brief description of the drawings]

[0022] [Figure 1] FIG. 1 shows the results of CFU counts in lung tissue from each treatment group at different time points. [Diagram 2] FIG. 2 shows the hydroxyproline (HYP) content in each treatment group at different time points. [Diagram 3] FIG. 3 shows the alveolar inflammation scores after 4 weeks of treatment and after 8 weeks of treatment. [Figure 4] FIG. 4 shows the results of Masson staining in the BIBF1120+HRZ treatment group and the HRZ treatment group. [Diagram 5] FIG. 5 shows the positive area rate of Masson's staining. [Figure 6] Figure 6 shows the percentage of positive special stains in each treatment group. [Figure 7] FIG. 7 shows the results of immunohistochemical analysis showing that the expression level of CD31 was reduced in the BIBF1120+HRZ treatment group compared to the HRZ treatment group. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The present invention will now be described in detail with reference to the drawings and examples, which are intended to interpret the present invention but are not intended to limit it.

[0024] Experimental Example 1: In vitro activity of nintedanib against Mycobacterium tuberculosis The H37Rv strain was cultured until it reached logarithmic growth phase, and the bacterial suspension was diluted to 1 × 10 in 7H9 liquid medium for use. 6 CFU / mL. As described below, rows A-F are positive / negative control wells, INH, RFP, PFD, SC1011, and BIBF1120, respectively. Each drug well was serially diluted 2-fold in a 96-well plate. After 7 days of incubation at 37°C, 20μL of Alamar Blue and 12.5μL of 20% polysorbate 80 were added to each well, followed by 24 hours of incubation at 37°C. The color of each well was recorded, with blue representing no growth of the strain, red representing growth of the strain, and MIC representing the minimum drug concentration at which the color changes from blue to red. The experiment was repeated three times. The results showed that nintedanib had antibacterial activity, and the MIC value against the H37Rv standard strain was 24.567μg / mL.

[0025] Experimental Example 2: Effect of nintedanib on the antituberculous drug rifampicin The minimum drug concentration (MIC) at which a drug inhibits the growth of 90% of tuberculosis bacteria was determined using broth microdilution. 90 ) was measured, and then, from the MIC value when each drug was used alone, BIBF1120 and RFP were diluted to six concentrations of 2 × MIC to 1 / 16 × MIC by the 2-fold dilution method. 50 μL of drug A (BIBF1120) at 2 × MIC to 1 / 16 × MIC was added to the second column of the 96-well plate, 50 μL of drug B (RFP) at 2 × MIC to 1 / 16 × MIC was added to the first to seventh wells of the second to seventh rows, 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, and a positive control well (100 μL of diluted bacterial solution + 100 μL of 7H9) and a negative control well (200 μL of 7H9) were set up. The MICs of single drugs in drug combinations were determined by observing the color change in the 96-well plate (a change in color from blue to purple or pink indicates the presence of growth of the strain, whereas blue indicates the inhibition of the drug and the absence of growth of the strain). The effect of the combination was determined by the 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, respectively. 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 different concentrations of nintedanib within macrophages The anti-tuberculosis activities of BIBF1120 alone and the combination of BIBF1120 + RFP were evaluated for their 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 into a 48-well transparent microplate at 1 mL per well. After seeding was completed, it was placed in a 37 °C, 5% CO2 incubator and cultured for 24 hours. At MOI = 5:1, 2 × 10 6CFU / mL of H37Rv was co-cultured with macrophages. After 4 hours, the plates were washed twice with sterile PBS to remove extracellular tuberculosis bacteria, and new RPMI cell culture medium was added. The plates were placed in a 37℃, 5% CO2 incubator and cultured for 72 hours. After 3 days, the RPMI was discarded from the 48-well plate, 200μL of 0.1% SDS was added, the plates were placed in an incubator and left for 5-10 minutes, and 800μL of cell culture medium was added to each well and mixed evenly. After the cells were dissolved, 100μL was taken from each well and diluted 10x, 100x, 1000x, and 100000x, and 100μL was inoculated into 7H10 solid medium and spread evenly using a spreader. CFU were counted after 3 weeks. Table 1: Results of intracellular bactericidal activity of BIBF1120 alone [Table 1] Table 2: Bactericidal activity of the combination of BIBF1120+RFP in macrophages [Table 2]

[0027] In conclusion, we found that the combination of BIBF1120 and the antituberculous drug RFP showed synergistic bactericidal activity in macrophages at safe doses, whereas BIBF1120 alone had no significant antibacterial effect at a dose of 25 μg / mL (corresponding to the results of in vitro MIC experiments).

[0028] Experimental example 4: Effect of nintedanib in a mouse tuberculosis model H37Rv strain in logarithmic growth phase was diluted in 20 mL of 1x PBS to a bacterial concentration of 1x10 7The antibody was diluted to CFU / mL and infected 6-8 week-old female C57BL / 6 mice via aerosol. A mouse chronic tuberculosis model by aerosol infection was established, and the antibacterial activity of each treatment group was evaluated in the model. Three mice were randomly selected and killed on the 10th day after infection (D-32), and six mice were randomly selected and killed on the day of treatment (D0), dissected, and their spleen and lung tissues were collected and homogenized, and the colonies (CFU) in the spleen and lungs were counted on a 7H10 plate, thereby determining the baseline number of tuberculosis bacteria in the lungs and spleens of mice at the beginning of infection and at the start of treatment. Treatment was started 6 weeks after infection, and 4 and 8 weeks after treatment, 7-8 mice were randomly selected from each treatment group, killed, dissected, and their spleen and lung tissues were collected and homogenized, and the colonies (CFU) in the spleen and lungs were counted on a 7H10 plate. After 8 weeks of treatment and 12 weeks of rest, the recurrence of tuberculosis bacteria in the lungs and spleens of seven mice from each treatment group was observed. C57BL / 6 mice were infected for 6 weeks and then 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 stands for isoniazid, R stands for rifampicin, and Z stands for pyrazinamide. D-32 was the 10th day of infection, W4 was 4 weeks after treatment, W8 was 8 weeks after treatment, and W8+12W was relapse observation after 8 weeks of treatment and 12 weeks of drug rest. D was days and W was weeks. Isoniazid was 10 mg / kg / day, rifampicin was 10 mg / kg / day, pyrazinamide was 150 mg / kg / day, pirfenidone (PFD) was 100 mg / kg / day, and BIBF1120 was 50 mg / kg / day. Here, RFP was administered with at least 1 hour interval from other drugs.

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

[0030] 2. Compared with the HRZ treatment group, the BIBF1120+HRZ treatment group can reduce the recurrence rate of mice.

[0031] (Experimental Example 5) A HYP measurement kit was used. The content of hydroxyproline in the right lung tissue was measured. Fresh lung tissue was separated by dissection, weighed, and placed in a test tube, and exactly 1 mL of hydrolysate was added. The tissue was bathed in a water bath for 20 minutes. The pH was adjusted to approximately 6.0-6.8. Distilled water was then added to make 10 mL, and 4 mL of the diluted hydrolysate was taken and an appropriate amount of activated charcoal was added. After centrifugation at 3500 rpm for 10 minutes, 1 mL of the supernatant was taken and measured. The blank tube and standard tube represent distilled water and standard, respectively. Each reagent was added sequentially according to the instructions. Finally, the microplate reader was used to zero-adjust the absorbance A value of each tube at 550 nm using distilled water. The 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 as follows: A × 5 μg / mL (content in standard tube) × [10 (total amount of hydrolysate) / wet weight of lung tissue]. 測 is the absorbance of the measurement tube, and A ブランク is the absorbance of the blank tube, and A 標 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 result analysis: Pathological examination of lung tissue was performed as follows. Left lung tissue was taken from mice, trimmed, fixed with 4% paraformaldehyde, and confirmed to be in good condition. After washing with saline and fixing with 4% paraformaldehyde solution, normal paraffin embedding was performed. 3 μm paraffin sections were prepared in this way, and pathological changes in lung tissue were observed by HE staining. Pathological changes in lung tissue were observed under an optical microscope, and the degree of inflammatory infiltration was described to determine the pathological lung injury score. The degree of lung injury was evaluated by four indicators: 1. alveolar congestion, 2. alveolar hemorrhage, 3. infiltration or aggregation of neutrophils into the alveolar space or vascular wall, and 4. alveolar septal thickening. The score 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 of each item was the pathological lung injury score. Table 6: Alveolar inflammation scores after 4 weeks of treatment [Table 6] Table 7: Alveolar inflammation scores after 8 weeks of treatment [Table 7]

[0033] Masson staining was performed according to the method described in the kit's instructions. The sections were deparaffinized in water, immersed overnight in Masson staining solution A, immersed for 1 minute in an equal mixture of Masson staining solution B and Masson staining solution C, immersed for 6 minutes in Masson staining solution D, immersed for 1 minute in Masson staining solution E, and immersed for 2 to 30 seconds in Masson staining solution F. They were then rinsed with 1% glacial acetic acid to induce differentiation, dehydrated in absolute ethanol, cleared in xylene, mounted in neutral balsam, and observed under a normal optical microscope. As shown in Figures 5 and 6, the results showed that collagen fibers were displayed in blue, and muscle fibers, cellulose, and red blood cells were displayed in red. Images of the tissue-stained sections were acquired using an imaging system, and the tissue measurement area was automatically identified using analysis software, and the positive area and tissue area in the measurement area were calculated to obtain the positive area rate.

[0034] After the frozen sections were warmed, they were washed three times with PBS buffer and sodium citrate was added. After the sections were allowed to cool naturally and washed, 5% blocking goat serum was added to block the sections at room temperature for 2 hours. After blocking was completed, anti-CD31 antibody was added and incubated at 4°C overnight, with an antibody concentration of 1:200. The next 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, developed with DAB, heavily stained with hematoxylin, and the slides were sealed and photographed and recorded under a biological microscope. As shown in Figure 7, the immunohistochemical test results show that the expression of CD31 in the BIBF1120+HRZ treatment group was reduced compared with the HRZ treatment group. Therefore, it can be speculated that the adjunctive effect of BIBF1120 in treating tuberculosis is due to its suppression of abnormal angiogenesis, thereby promoting the delivery of small molecule compounds (anti-tuberculosis drugs) and enabling them to exert better anti-tuberculosis activity.

Claims

1. Use of nintedanib or a pharmaceutically acceptable salt thereof in the manufacture of a medicament against Mycobacterium tuberculosis (MTB).

2. The use according to claim 1, wherein the pharmaceutically acceptable salt of nintedanib is nintedanib ethanesulfonate.

3. The use according to claim 1, wherein the nintedanib or a pharmaceutically acceptable salt thereof can be used in combination with other anti-tuberculosis drugs.

4. The use according to claim 3, wherein the other anti-tuberculosis drugs are selected from rifampicin, isoniazid, pyrazinamide, ethambutol, fluoroquinolones, streptomycin, fluoroquinolones, kanamycin, amikacin, capreomycin, sodium 4-aminosalicylate, ethionamide, cycloserine, clofazimine, linezolid.

5. The use according to claim 3, wherein the combination includes using in combination with three or more other anti-tuberculosis drugs, or manufacturing a combined pharmaceutical preparation together with three or more other anti-tuberculosis drugs.

6. The use according to claim 5, wherein the combined pharmaceutical preparation is a combined pharmaceutical preparation manufactured by mixing four drugs, namely nintedanib ethanesulfonate, isoniazid, rifampicin, and pyrazinamide.

7. The use according to claim 2, wherein the dosage and administration method of the nintedanib ethanesulfonate are 50 - 300 mg / day, administered 1 - 3 times a day, and the treatment period is at least 4 - 6 months.

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