A pharmaceutical combination and uses thereof

Abstract

The application discloses a kind of drug combination and its application.The application provides a kind of drug combination or pharmaceutical composition, the active component of the drug combination or pharmaceutical composition includes material A, material B, material C and material D;Material A is compound I or its pharmaceutically acceptable salt;Material B is isoniazid (H) or its pharmaceutically acceptable salt;Material C is rifampicin (R) or its pharmaceutically acceptable salt;Material D is pyrazinamide (Z) or its pharmaceutically acceptable salt.The drug combination or pharmaceutical composition of the application can reduce alveolar inflammation, pulmonary tuberculosis inflammatory lung injury and pulmonary tuberculosis secondary fibrosis, while increasing the penetration of anti-tuberculosis drugs in the lesion site, to accelerate the treatment response, reduce the risk of recurrence.

Description

Technical Field

[0001] This invention relates to a drug combination and its application. Background Technology

[0002] Tuberculosis (TB) is one of the major infectious diseases that seriously threaten human health, caused by Mycobacterium tuberculosis. The 2021 World Health Organization (WHO) TB report showed that in 2020, there were 9.87 million new TB cases globally, with an incidence rate of 127 per 100,000. Chemotherapy is the main means of controlling TB. Although the standard 6-month treatment regimen is highly effective for drug-sensitive TB, the possibility of complete absorption of lung lesions is small, and more than half of patients still suffer from permanent lung function impairment, often leaving behind a large number of fibrotic lesions. Secondary fibrosis of pulmonary tuberculosis is a common complication of pulmonary tuberculosis. Extensive pulmonary fibrosis can severely affect the patient's lung function. Secondary fibrosis of pulmonary tuberculosis is a common post-TB lung disease (PIAT). Furthermore, the tuberculosis bacteria are encapsulated in fibrous tissue, making it difficult for drugs to completely eradicate the bacteria, leading to a high relapse rate. Both calcified nodules and fibrotic lesions increase the risk of TB relapse, with those having fibrotic lesions showing a significantly higher risk of relapse.

[0003] Pirfenidone (PDF) is a drug approved by the FDA in 2014 for the treatment of idiopathic fibrosis. Its main effects are anti-oxidation, anti-inflammation, and anti-fibrosis, exhibiting broad-spectrum activity. It can inhibit the expression of transforming growth factor-β1 (TGF-β1) and simultaneously inhibit the release of various inflammatory factors, thereby reducing the inflammatory response.

[0004] To date, there are no reports on the use of drug combinations containing pyridone derivatives with heteroatom cyclobutane substituents to reduce alveolar inflammation, inflammatory lung injury caused by tuberculosis, and secondary fibrosis caused by tuberculosis, while increasing the penetration of anti-tuberculosis drugs into the lesion site to accelerate treatment response and reduce the risk of recurrence. Summary of the Invention

[0005] The technical problem this invention aims to solve is the deficiency of existing tuberculosis treatment drugs in completely eradicating bacteria and controlling tuberculosis recurrence. This invention provides a drug combination or pharmaceutical composition and its application. The pharmaceutical composition of this invention can reduce alveolar inflammation, inflammatory lung injury caused by pulmonary tuberculosis, and secondary fibrosis caused by pulmonary tuberculosis, while increasing the penetration of anti-tuberculosis drugs into the lesion site to accelerate the treatment response and reduce the risk of recurrence.

[0006] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0007] The present invention provides a drug combination, wherein the active components of the drug combination include substance A, substance B, substance C and substance D;

[0008] Substance A is compound I or a pharmaceutically acceptable salt thereof;

[0009] Substance B is isoniazid (H) or a pharmaceutically acceptable salt thereof;

[0010] Substance C is rifampin (R) or a pharmaceutically acceptable salt thereof;

[0011] Substance D is pyrazinamide (Z) or a pharmaceutically acceptable salt thereof;

[0012] The structure of compound I is shown below:

[0013]

[0014] In some embodiments, the drug combination is a pharmaceutical composition.

[0015] In some embodiments, the weight ratio of substance B to substance A in the drug combination is (3:1) to (1:30), preferably 1:6.

[0016] In some embodiments, the weight ratio of substance C to substance A in the drug combination is (3:1) to (1:60), preferably 1:6.

[0017] In some embodiments, the weight ratio of substance D to substance A in the drug combination is (30:1) to (1:3), preferably 5:2.

[0018] In some embodiments, the active component in the drug combination comprises substance A, substance B, substance C, and substance D.

[0019] In some embodiments, substances A, B, C, and D can be administered by any suitable route in the art, including oral administration, injection (e.g., intravenous, intramuscular, subcutaneous), etc.

[0020] In some embodiments, substances A, B, C, and D may be applied simultaneously or separately.

[0021] The term "simultaneous administration" means, for example, that substance A, substance B, substance C, and substance D are included in a single pharmaceutical composition and administered simultaneously; or, that a "single pharmaceutical composition containing substance A," a "single pharmaceutical composition containing substance B," a "single pharmaceutical composition containing substance C," and a "single pharmaceutical composition containing substance D" are administered simultaneously.

[0022] The term "separate administration" refers to, for example, the administration of "a single pharmaceutical composition containing substance A," "a single pharmaceutical composition containing substance B," "a single pharmaceutical composition containing substance C," and "a single pharmaceutical composition containing substance D" at different times. For instance, one of these compositions may be administered first, followed by the others. The separate administration can be administered close together or far apart in time.

[0023] Whether administered simultaneously or separately, the administration regimens (including administration route, dosage, and administration interval) of substances A, B, C, and D can be the same or different, and can be adjusted by those skilled in the art as needed to provide optimal therapeutic effects.

[0024] On the other hand, the present invention provides a pharmaceutical composition X, which includes substance A, substance B, substance C, substance D and pharmaceutical excipients;

[0025] Substance A is compound I or a pharmaceutically acceptable salt thereof;

[0026] Substance B is isoniazid (H) or a pharmaceutically acceptable salt thereof;

[0027] Substance C is rifampin (R) or a pharmaceutically acceptable salt thereof;

[0028] Substance D is pyrazinamide (Z) or a pharmaceutically acceptable salt thereof;

[0029] The structure of compound I is shown below:

[0030]

[0031] The pharmaceutical composition X is a single pharmaceutical composition.

[0032] In some embodiments, the pharmaceutical composition X comprises substance A, substance B, substance C, substance D, and pharmaceutical excipients.

[0033] In some embodiments, the pharmaceutical composition X is presented in an oral dosage form.

[0034] In some embodiments, the pharmaceutical composition X is presented in an injectable form.

[0035] On the other hand, the present invention also provides a pharmaceutical composition Y, which includes a first pharmaceutical composition, a second pharmaceutical composition, a third pharmaceutical composition and a fourth pharmaceutical composition;

[0036] The first pharmaceutical composition includes substance A and a pharmaceutical excipient; substance A is compound I or a pharmaceutically acceptable salt thereof;

[0037] The second pharmaceutical composition comprises substance B and a pharmaceutical excipient; substance B is isoniazid (H) or a pharmaceutically acceptable salt thereof;

[0038] The third pharmaceutical composition comprises substance C and a pharmaceutical excipient; substance C is rifampin (R) or a pharmaceutically acceptable salt thereof;

[0039] The fourth pharmaceutical composition comprises substance D and a pharmaceutical excipient; substance D is pyrazinamide (Z) or a pharmaceutically acceptable salt thereof;

[0040] The structure of compound I is shown below:

[0041]

[0042] The first pharmaceutical composition is a single pharmaceutical composition; the second pharmaceutical composition is a single pharmaceutical composition; the third pharmaceutical composition is a single pharmaceutical composition; and the fourth pharmaceutical composition is a single pharmaceutical composition.

[0043] In some embodiments, the pharmaceutical composition Y comprises a first pharmaceutical composition, a second pharmaceutical composition, a third pharmaceutical composition, and a fourth pharmaceutical composition.

[0044] In some embodiments, the first pharmaceutical composition is presented in an oral or injectable form.

[0045] In some embodiments, the second pharmaceutical composition is presented in an oral or injectable form.

[0046] In some embodiments, the third pharmaceutical composition is presented in oral or injectable form.

[0047] In some embodiments, the fourth pharmaceutical composition is presented in oral or injectable form.

[0048] The present invention provides the use of the above-mentioned drug combination, drug composition X or drug composition Y in the preparation of a drug for treating and / or preventing tuberculosis.

[0049] The present invention provides the use of the above-mentioned drug combination, drug composition X or drug composition Y in the preparation of a drug for treating and / or preventing alveolar inflammation; preferably, the alveolar inflammation is alveolar inflammation caused by pulmonary tuberculosis.

[0050] This invention provides the use of the above-mentioned drug combination, drug composition X or drug composition Y in the preparation of a medicament for treating and / or preventing inflammatory lung injury in pulmonary tuberculosis.

[0051] The present invention provides the use of the above-mentioned drug combination, drug composition X or drug composition Y in the preparation of a drug for treating and / or preventing pulmonary fibrosis; preferably, the pulmonary fibrosis is secondary fibrosis of pulmonary tuberculosis.

[0052] The present invention provides the use of the above-mentioned drug combination, drug composition X or drug composition Y in the preparation of a drug for reducing the recurrence rate of tuberculosis; preferably, the drug is used to reduce the recurrence rate of tuberculosis in lung tissue and / or spleen tissue.

[0053] In some embodiments, the drug is used to reduce the recurrence rate of Mycobacterium tuberculosis in spleen tissue, thereby reducing the recurrence rate in spleen tissue by 80%.

[0054] In some embodiments, the drug is used to reduce the recurrence rate of tuberculosis in lung tissue, thereby reducing the recurrence rate by 40%-50%.

[0055] On the other hand, the present invention provides a method for treating and / or preventing tuberculosis, comprising administering to a patient in need a therapeutically effective amount of a combination of drugs, drug composition X or drug composition Y as described herein.

[0056] Substance A, substance B, substance C, and substance D may be applied simultaneously or separately.

[0057] The substances A, B, C, and D can be administered by any suitable route in the art, including oral administration, injection (e.g., intravenous, intramuscular, subcutaneous), etc.

[0058] The dosage of isoniazid can be administered based on the patient's weight, and the non-limiting example range is 10 mg / kg to 30 mg / kg, such as 10 mg / kg or 25 mg / kg.

[0059] The dosage of rifampin can be administered based on the patient's weight, and the non-limiting example range is 5 mg / kg to 30 mg / kg, for example, 10 mg / kg.

[0060] The dosage of the pyrazinamide can be administered according to the patient's weight, and a non-limiting example range is 100 mg / kg to 300 mg / kg, for example, 150 mg / kg.

[0061] The dosage of compound I can be administered according to the patient's weight, and the non-limiting example range is 10 mg / kg to 300 mg / kg, such as 25 mg / kg, 50 mg / kg, 60 mg / kg, 100 mg / kg, 150 mg / kg or 200 mg / kg.

[0062] As used in this article, “treatment” means therapeutic therapy. When a specific condition is involved, treatment means: (1) alleviating one or more biological manifestations of the disease or condition; (2) interfering with (a) one or more points in a biological cascade that causes or precipitates the condition or (b) one or more biological manifestations of the condition; (3) improving one or more symptoms, effects or side effects associated with the condition, or one or more symptoms, effects or side effects associated with the condition or its treatment; or (4) slowing the development of the condition or one or more biological manifestations of the condition.

[0063] As used herein, the term "therapeutic effective amount" refers to the amount of a compound that, when administered to a patient, is sufficient to effectively treat the disease or condition described herein. The amount of compound constituting a "therapeutic effective amount" will vary depending on the compound, the condition and its severity, and the age of the patient to be treated, but may be adjusted as needed by those skilled in the art.

[0064] As used in this article, "pharmaceutical composition" refers to a composition containing a specified active ingredient that can be prepared into the same dosage form.

[0065] As used herein, the term "patient" refers to any animal, preferably a mammal, that is about to be or has already been administered the compound or composition according to embodiments of the invention. As used herein, the term "mammal" includes any mammal. Examples of mammals include, but are not limited to, cattle, horses, sheep, pigs, cats, dogs, mice, rats, rabbits, guinea pigs, monkeys, and humans, with humans being the most preferred.

[0066] The term "pharmaceutical excipients" as used in this article refers to excipients and additives used in the production of pharmaceuticals and the dispensing of prescriptions. It includes all substances contained in pharmaceutical preparations, excluding the active ingredient. See the Pharmacopoeia of the People's Republic of China (2020 Edition), Volume IV, or the Handbook of Pharmaceutical Excipients (Raymond C. Rowe, 2009 Sixth Edition).

[0067] As used herein, the term "pharmaceutically acceptable" means that the acids or bases, solvents, excipients, etc. (used in the preparation of the salt) are generally non-toxic, safe, and suitable for patient use. The term "patient" preferably refers to mammals, and more preferably humans.

[0068] As used herein, the term "pharmaceutically acceptable salt" refers to a salt prepared by reacting a compound with a relatively non-toxic, pharmaceutically acceptable acid or base. When a compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of a pharmaceutically acceptable base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include, but are not limited to, lithium, sodium, potassium, calcium, aluminum, magnesium, zinc, bismuth, ammonium, and diethanolamine salts. When a compound contains a relatively basic functional group, an acid addition salt can be obtained by contacting the neutral form of such a compound with a sufficient amount of a pharmaceutically acceptable acid in a pure solution or a suitable inert solvent. The pharmaceutically acceptable acid includes inorganic acids, including but not limited to, hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, phosphorous acid, sulfuric acid, and hydrogen sulfate. The pharmaceutically acceptable acids include organic acids, including but not limited to: acetic acid, propionic acid, oxalic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, octanoic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, salicylic acid, tartaric acid, methanesulfonic acid, isonicotinic acid, acidic citric acid, oleic acid, tannic acid, pantothenic acid, hydrogen tartrate, ascorbic acid, gentic acid, fumaric acid, gluconic acid, succinic acid, formic acid, ethanesulfonic acid, dihydroxynaphthyl acid (i.e., 4,4'-methylene-bis(3-hydroxy-2-naphthylcarboxylic acid)), amino acids (e.g., glutamic acid, arginine), etc. When a compound contains functional groups with relatively acidic and relatively basic properties, it can be converted into a base addition salt or an acid addition salt. For details, see Berge et al., "Pharmaceutical Salts", Journal of Pharmaceutical Science 66: 1-19 (1977), or Handbook of Pharmaceutical Salts: Properties, Selection, and Use (P. HeinrichStahl and Camille G. Wermuth, ed., Wiley-VCH, 2002).

[0069] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0070] The reagents and raw materials used in this invention are all commercially available.

[0071] The positive and progressive effects of this invention are as follows: the drug combination or drug composition of this invention can reduce alveolar inflammation, inflammatory lung injury caused by pulmonary tuberculosis and secondary fibrosis caused by pulmonary tuberculosis, while increasing the penetration of anti-tuberculosis drugs into the lesion site, thereby accelerating the treatment response and reducing the risk of recurrence. Attached Figure Description

[0072] Figure 1 Pathological analysis of mouse lung tissue by HE staining and Masson staining.

[0073] Figure 2 The content of hydroxyproline in lung tissue of each treatment group was determined by alkaline hydrolysis.

[0074] Figure 3 To evaluate the antibacterial effects of each treatment group in a mouse model of tuberculosis. Detailed Implementation

[0075] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0076] Example 1: Study on the protective effect of compound I in tuberculous lung injury

[0077] I. Experimental Materials:

[0078] 1. Experimental strain

[0079] The laboratory standard MTB strain, H37Rv (ATCC 27294), was preserved and provided by Beijing Chest Hospital, Capital Medical University / Beijing Key Laboratory of Drug-Resistant Tuberculosis.

[0080] 2. Laboratory animals

[0081] SPF-grade 6-8 week old female C57BL / 6 mice, weighing 16-19g, were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0082] 3. Experimental drugs

[0083] Rifampin (lot number: WXBB7288V, purchased from Sigma, USA);

[0084] Isoniazid (lot number: MKBQ8553V, purchased from Sigma, USA);

[0085] Pyrazinamide (batch number: H1929213, purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.);

[0086] Pirfenidone (batch number: 53179-13-8, purchased from Hanxiang Biotechnology Co., Ltd.);

[0087] Compound I was provided by Guangzhou Jiayue Pharmaceutical Technology Co., Ltd.

[0088] 4. Experimental reagents

[0089] Middlebrook OADC nutrient solution was purchased from Becton Dickinson, USA (lot number: 9043888);

[0090] Alamar Blue indicator was purchased from Bio-Rad Laboratories, USA (lot number: 154244);

[0091] Dimethyl sulfoxide (DMSO) was purchased from Chengdu Chemical Reagent Factory (batch number: Y190601);

[0092] Twain-80 was purchased from Beijing Solarbio Technology Co., Ltd. (batch number: 1114D0123H);

[0093] The hydroxyproline assay kit was purchased from Nanjing Jiancheng Biotechnology Institute (batch number: A030-2-1);

[0094] Masson dye kit (purchased from Servicebio G1006).

[0095] 5. Experimental Methods

[0096] (1) Determination of the minimum inhibitory concentration (MIC) of PFD and compound I by microplate alamar blue assay (MABA)

[0097] The H37Rv strain was cultured to the logarithmic growth phase, and the bacterial suspension was diluted to 1×10⁻⁶ in 7H9 liquid medium. 6 CFU / ml was prepared for use. Isoniazid (H), rifampin (R), Compound I, and pirfenidone (PFD) + isoniazid (H), pirfenidone (PFD) + rifampin (R), Compound I + isoniazid (H), and Compound I + rifampin (R) were serially diluted in a 96-well plate. After incubation at 37°C for 7 days, 20 μl of Alma Blue and 12.5 μl of 20% Tween-80 were added to each well, and the plate was incubated at 37°C for another 24 hours. The color of each well was recorded; blue indicated no growth, and red indicated growth. MIC represents the minimum drug concentration at which blue turned red. The experiment was repeated twice.

[0098] (2) Dilute the H37Rv strain in the logarithmic growth phase with 20 ml of 1×PBS to a bacterial concentration of 1×10⁻⁶. 7Seventy-five 6-8 week old female C57BL / 6 mice were aerosol-infected to establish a chronic mouse model of tuberculosis caused by aerosol infection. The antibacterial activity of the PFD+HRZ, compound I+HRZ, and HRZ treatment groups was evaluated using this model. On day 10 post-infection (D-32), three mice were randomly sacrificed. On the day of treatment (D0), six mice were randomly sacrificed. Spleen and lung tissues were dissected, homogenized, and counted on 7H10 plates to determine the baseline number of Mycobacterium tuberculosis in the lungs and spleen at the initial infection and treatment start. Drug administration began 6 weeks after infection. Mouse body weight was measured weekly, and the total weekly drug mass and volume required were calculated based on the average body weight of each group to prepare the appropriate drug concentration. Isoniazid (H) was dissolved in sterile water, and all other drugs were dissolved in DMSO solution. Administration was performed 5 days a week, administered by gavage at 8:00 AM each day. Isoniazid (H) and pyrazinamide (Z) were mixed and administered to each mouse at a dose of 0.2 ml. The compound I + HRZ treatment group and the PFD + HRZ treatment group were administered compound I (0.1 ml) and PFD (0.1 ml) per mouse, respectively. Rifampin (R) was administered at least 1 hour after the completion of all drug administrations. All drugs were preheated to 37°C and mixed before administration. At 4 and 8 weeks post-treatment, 7-8 mice from each treatment group were randomly sacrificed and dissected. Spleen and lung tissues were homogenized and counted on 7H10 plates for colony-forming units (CFU). Relapse of Mycobacterium tuberculosis in the lungs and spleen of 7 mice in each treatment group was observed 12 weeks after drug withdrawal. C57BL / 6 mice were randomly divided into 3 groups 6 weeks after infection (see Table 1).

[0099] Table 1. Grouping and Dosing Regimens of C57BL / 6 Mice

[0100]

[0101] Notes: Pirfenidone (PFD); Isoniazid (H); Rifampin (R); Pyrazinamide (Z); D-32: 10 days after infection; W4: 4 weeks of treatment; W8: 8 weeks of treatment; W8+12W: 8 weeks of treatment followed by 12 weeks of observation for relapse after discontinuation. D: day; W: week. Isoniazid: 10 mg / kg / day; Rifampin: 10 mg / kg / day; Pyrazinamide: 150 mg / kg / day; Pirfenidone (PFD): 100 mg / kg / day; Compound I: 60 mg / kg / day.

[0102] Histopathological histology, HE staining, and alveolar inflammation score

[0103] Lung tissue pathological observation: Left lung tissue from mice was trimmed, fixed in 4% paraformaldehyde, and after good fixation, washed with physiological saline and fixed in 4% paraformaldehyde solution. It was then routinely embedded in paraffin, and 3μm paraffin sections were prepared. HE staining was performed to observe pathological changes in the lung tissue. The degree of inflammatory infiltration was described, and a lung injury pathological score was calculated. Four indicators were used to assess the degree of lung injury: ① alveolar congestion; ② alveolar hemorrhage; ③ neutrophil infiltration or aggregation in the alveolar spaces or blood vessel walls; ④ alveolar septal thickening. Scoring criteria: 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 indicator was the lung injury pathological score.

[0104] Masson staining and special staining percentages

[0105] Masson staining was performed according to the kit instructions. Sections were routinely dewaxed to water, soaked overnight in Masson A solution, then soaked for 1 min in a mixture of equal parts Masson B and Masson C solutions, followed by 6 min in Masson D solution, 1 min in Masson E solution, and 2-30 s in Masson F solution. After differentiation, sections were rinsed with 1% glacial acetic acid, dehydrated with anhydrous ethanol, cleared with xylene, mounted with neutral resin, and observed under a regular optical microscope. Results showed that collagen fibers appeared blue; muscle fibers, cellulose, and erythrocytes appeared red. Images of the stained tissue sections were acquired using an imaging system, and analysis software was used to automatically read the tissue measurement areas, calculate the positive area and tissue area within the measurement areas, and determine the percentage of positive area.

[0106] Alkaline hydrolysis method for detecting hydroxyproline content in lung tissue

[0107] The HYP assay kit was used to determine the hydroxyproline content in the right lung tissue. Fresh lung tissue was dissected, weighed, and placed in a test tube. 1 ml of hydrolysate was accurately added. The tube was incubated in a water bath for 20 min. The pH was adjusted to approximately 6.0-6.8. Then, distilled water was added to 10 ml, and 4 ml of the diluted hydrolysate was added with an appropriate amount of activated charcoal. The tube was centrifuged at 3500 rpm for 10 min, and 1 ml of the supernatant was used for detection. Blank and standard tubes represent distilled water and standards, respectively. Reagents were added sequentially according to the instructions. Finally, the absorbance (A value) of each tube was measured using a microplate reader at 550 nm with distilled water as the zero point. The HYP content (ug / mg wet weight) was calculated using the following formula: (Atest - Ablank) / (Astandard - Ablank) * 5 ug / ml (standard tube content) * [10 (total volume of hydrolysate) / wet weight of lung tissue]. Atest: absorbance of the test tube; Ablank: absorbance of the blank tube; Astandard: absorbance of the standard tube.

[0108] 6. Statistical Analysis

[0109] Data plotting was performed using GraphPad Prism 8.0 software (GraphPad Inc., USA), and statistical analysis was performed using SPSS 22 software. Quantitative data conformed to a normal distribution and were described using "x±s". One-way ANOVA was used to compare differences among multiple groups, and t-tests were used for further pairwise comparisons within groups. A p-value < 0.05 was considered statistically significant.

[0110] 7. Results

[0111] 1) MABA method for determining the MIC of PFD, Compound I alone and in combination with H / R against H37Rv standard strain.

[0112] The inhibitory concentrations (MICs) of PFD, Compound I alone, and in combination with H / R against Mycobacterium tuberculosis H37Rv standard strain were determined using the microbroth dilution method. The results showed that the MICs of Compound I and PFD were both >100 μg / ml. The MICs of the combination drug of Compound I / PFD and H / R at 100 μg / ml were also determined. The MIC of isoniazid alone was 0.037 μg / ml. The MIC of rifampin alone was 0.038 μg / ml. The MICs of PFD+H and Compound I+H were both 0.037 μg / ml; the MICs of PFD+R and Compound I+R were both 0.038 μg / ml. These results indicate that the MICs of adding 100 μg / ml PFD or Compound I in combination did not significantly change compared to the MICs of single anti-tuberculosis drugs. This suggests that PFD and Compound I do not possess antibacterial activity and do not affect the antibacterial activity of the anti-tuberculosis drugs.

[0113] Table 2 MIC Results

[0114] Serial Number drug MIC (ug / ml) 1 Isoniazid (H) 0.037 2 Rifampin(R) 0.038 3 Compound I >100 4 PFD >100 5 Compound I + rifampin(R) 0.038 6 PFD + Rifampin(R) 0.038 7 Compound I + isoniazid (H) 0.037 8 PFD + Isoniazid (H) 0.037

[0115] 2) Pathological analysis of mouse lung tissue by HE staining and Masson staining

[0116] Pathological sections of each group were observed and scored under an optical microscope: Left lung tissue from three mice in each treatment group was collected on the day of treatment, at 4 weeks, and at 8 weeks of treatment for HE staining and alveolar inflammation scoring. At 4 weeks of treatment, the PFD+HRZ treatment group and the Compound I+HRZ treatment group showed significantly reduced alveolar inflammation compared to the HRZ treatment group (P<0.05). Pathological results of lung tissue sections at 4 weeks of treatment showed: In the HRZ treatment group, extensive mild thickening of the alveolar walls was observed, accompanied by diffuse infiltration of lymphocytes and neutrophils; inflammatory cells formed rings around local blood vessels, creating vascular cuffs; a small number of lymphocytes and macrophages were observed in the tissue of the PFD+HRZ treatment group; a small number of lymphocytes and macrophages were observed in the tissue of the Compound I+HRZ treatment group, with mild thickening of the alveolar walls in some areas. After 8 weeks of treatment, alveolar inflammation scores showed that the PFD+HRZ treatment group and the compound I+HRZ treatment group had reduced alveolar inflammation compared to the HRZ treatment group (P<0.05), as shown in Table 3.

[0117] At weeks 4 and 8 of treatment, left lung tissue from three mice in each treatment group was collected for Masson staining. Images of the stained tissue sections were acquired using an imaging system, and analysis software automatically read the tissue measurement areas, calculated the positive area and tissue area within each area, and determined the percentage of positive area. The results are shown in Table 4. The results indicate that at week 4, the PFD+HRZ treatment group and the compound I+HRZ treatment group significantly reduced lung fibrosis compared to the HRZ treatment group (P<0.001); at week 8, the PFD+HRZ treatment group and the compound I+HRZ treatment group still reduced lung fibrosis compared to the HRZ treatment group (P<0.05). Figure 1 As shown, histopathological observation of mouse lung tissue evaluated the protective effect of three treatment regimens against lung injury caused by pulmonary tuberculosis in mice. Figure 1 A represents the HE staining results in the lungs of mice after 4 and 8 weeks of treatment. Figure 1 B indicates the results of Masson staining in the lungs of mice after 4 and 8 weeks of treatment. Figure 1 CD represents the alveolar inflammation score of lung tissue stained with HE after 4 weeks and 8 weeks of drug treatment in mice. Figure 1 EH represents the statistical percentage of positive Masson staining and special staining areas in lung tissue of mice after 4 and 8 weeks of drug treatment. ns, no statistical difference was found, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001).

[0118] Table 3 Alveolar inflammation scores of mice in each group

[0119]

[0120]

[0121] Table 4. Percentage of positive areas for special staining at different treatment time points in each treatment group (%)

[0122]

[0123] 3) Hydroxyproline reagent kit was used to detect the degree of pulmonary fibrosis in mice in each treatment group.

[0124] At 4 and 8 weeks of treatment, three mice from each treatment group were sacrificed, dissected, and their right lung tissue was collected to determine the hydroxyproline content in the right lung tissue according to the instructions of the hydroxyproline assay kit. The results showed that despite HRZ treatment, the degree of fibrosis in the lung tissue continued to increase with the duration of Mycobacterium tuberculosis infection. Furthermore, the pirfenidone + HRZ treatment group and the compound I + HRZ treatment group showed a reduction in the degree of fibrosis in the mouse lung tissue at both 4 and 8 weeks of treatment compared to the HRZ treatment group, with a more significant reduction at 8 weeks (P<0.05). The results are shown in Table 5. Figure 2 As shown (the alkaline hydrolysis method was used to determine the hydroxyproline content in the lung tissue of each treatment group to evaluate the anti-fibrotic effect of the three treatment regimens), Figure 2 A represents the hydroxyproline content in the right lung tissue of each treatment group after 4 weeks of treatment; Figure 2 B indicates the hydroxyproline content in the right lung tissue of each treatment group after 8 weeks of treatment; Figure 2 C represents the hydroxyproline content in the right lung tissue of each treatment group at different time points. (Note: There was no statistically significant difference (*P < 0.05, **P < 0.01, ***P < 0.001)).

[0125] Table 5. Hyp content in lung tissue at different treatment time points

[0126]

[0127] 4) Evaluation of anti-tuberculosis activity in each treatment group using a mouse model of chronic tuberculosis.

[0128] Mice in each group were dissected 10 days after infection, on the day of treatment, at 4 weeks of treatment, at 8 weeks of treatment, and 12 weeks after treatment (after drug withdrawal). Spleen and lung tissues were homogenized, and lung CFU counts were performed on 7H10 plates. The lung CFU count results at each time point are shown in Table 6. The spleen CFU count results at each time point are shown in Table 7. In the PFD+HRZ treatment group, two mice died unexpectedly due to improper gavage. Mice in the remaining experimental groups tolerated the infection and drug administration well, with no deaths. The CFU count in the lung tissue of mice on the day of treatment (D0) was (5.27±0.07)lg(CFU+1) as the baseline value for viable bacteria count in the lung tissue at the beginning of treatment. After 4 weeks of treatment, the PFD+HRZ, Compound I+HRZ, and HRZ treatment groups all significantly reduced the number of viable bacteria in mouse lung tissue, decreasing by 2.75lg(CFU+1), 2.54lg(CFU+1), and 2.70lg(CFU+1), respectively. After 8 weeks of treatment, the spleen in all three groups (PFD+HRZ, Compound I+HRZ, and HRZ) achieved sterilization; and 2 / 3, 3 / 5, and 3 / 5 of the mice, respectively, achieved sterilization of viable bacteria in their lungs after 8 weeks. CFU count results indicated that in C57BL / 6 mice, the addition of 100 mg / kg / d of PFD or Compound I had no significant effect on the antibacterial activity of existing anti-tuberculosis drugs, nor did it lead to further dissemination of Mycobacterium tuberculosis. (See attached results). Figure 3 Evaluation of the antibacterial effect of 100 mg / kg / d PFD and compound I combined with HRZ in a mouse model of tuberculosis. Figure 3 A indicates the CFU count in the lung tissue of each group after 4 weeks of treatment; Figure 3 The value of B indicates the CFU count in the spleen of each treatment group after 8 weeks of treatment. Figure 3 The "C" in the middle indicates the sum of CFU counts in lung tissue of each group at different treatment time points; Figure 3 D represents the sum of CFU counts in the spleen of each group at different treatment time points; each point represents one mouse, and the CFU data represents the mean of organs from n≥3 mice, expressed as Mean±SD; all lung CFU counts were determined using a one-way ANOVA test to determine statistical differences between groups.

[0129] Table 6. CFU counts in lung tissue of each group

[0130] Comparison HRZ+PFD HRZ+ compound I HRZ D-32 4.38±0.09 - - - D0 5.271±0.066 - - - 4W - 2.52±0.23 2.73±0.08 2.57±0.18 8W - 0.30(2 / 3) 0.48±0(3 / 5) 0.39±0.09(3 / 5)

[0131] Table 7. CFU count in spleen of each group

[0132] Comparison HRZ+PFD HRZ+ compound I HRZ D-32 2.15±0.11 - - - D0 3.496±0.257 - - - D4W - 1.82±0.08 1.91±0.05 1.79±0.17 D8W - 0 0 0

[0133] 5) To further evaluate whether the anti-inflammatory and anti-fibrotic effects of compounds I and PFD have any impact on the long-term prognosis of Mycobacterium tuberculosis infection, we observed each treatment group after 8 weeks of treatment and 12 weeks of drug withdrawal. The experimental results are shown in Table 8.

[0134] Table 8. Relapse rate of spleen and lung tissue in mice from different treatment groups 12 weeks after drug withdrawal.

[0135]

[0136] The results showed that the recurrence rates of Mycobacterium tuberculosis in the lungs of mice treated with PFD+HRZ, Compound I+HRZ, and HRZ were 85.7%, 42.9%, and 71.4%, respectively; the recurrence rates in the spleen were 71.4%, 14.3%, and 71.4%, respectively. The recurrence rate in the spleen was significantly lower in the Compound I+HRZ treatment group than in the HRZ treatment group (approximately 80% lower). Fisher's exact test showed a statistically significant difference between the two groups (χ² = 4.67, v = 1, p < 0.05). The recurrence rate in lung tissue was significantly lower in the Compound I+HRZ treatment group than in the HRZ treatment group (approximately 40% lower); the recurrence rate in spleen tissue was significantly lower in the Compound I+HRZ treatment group than in the PFD+HRZ treatment group (approximately 80% lower); and the recurrence rate in lung tissue was significantly lower in the Compound I+HRZ treatment group than in the PFD+HRZ treatment group (approximately 50% lower). The experimental data did not show that the PFD+HRZ treatment group had a recurrence-reducing effect in mice with tuberculosis.

[0137] In the C57BL / 6 mouse model of chronic tuberculosis, all three treatment regimens exhibited antibacterial activity, with no significant intra-group differences in efficacy. Compound I may have a more prominent advantage than PFD, although it did not significantly reduce the number of viable bacteria in the mouse lungs. However, in the long-term efficacy in tuberculosis mice, it could reduce the relapse rate, especially in reducing the relapse rate in the spleen.

[0138] While specific embodiments of the present invention have been described above, those skilled in the art should understand that these are merely illustrative examples, and various changes or modifications can be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A pharmaceutical composition, wherein the active components of the pharmaceutical composition comprise substance A, substance B, substance C and substance D; Substance A is compound I or a pharmaceutically acceptable salt thereof; Substance B is isoniazid or a pharmaceutically acceptable salt thereof; Substance C is rifampin or a pharmaceutically acceptable salt thereof; Substance D is pyrazinamide or a pharmaceutically acceptable salt thereof; The structure of compound I is shown below: 。 2. The pharmaceutical composition according to claim 1, characterized in that, It meets one or more of the following conditions: (1) In the pharmaceutical composition, the weight ratio of substance B to substance A is (3:1)-(1:30). (2) In the pharmaceutical composition, the weight ratio of substance C to substance A is (3:1)-(1:60). (3) In the pharmaceutical composition, the weight ratio of substance D to substance A is (30:1)-(1:3); (4) Substance A, substance B, substance C and substance D are administered orally or by injection; (5) The substances A, B, C and D are applied simultaneously or separately.

3. The pharmaceutical composition according to claim 2, characterized in that, It meets one or more of the following conditions: (1) In the pharmaceutical composition, the weight ratio of substance B to substance A is 1:6; (2) In the pharmaceutical composition, the weight ratio of substance C to substance A is 1:6; (3) In the pharmaceutical composition, the weight ratio of substance D to substance A is 5:

2.

4. The pharmaceutical composition according to claim 1, characterized in that, In the pharmaceutical composition, the active component is composed of substance A, substance B, substance C, and substance D.

5. A pharmaceutical composition X, comprising substance A, substance B, substance C, substance D and pharmaceutical excipients; Substance A is compound I or a pharmaceutically acceptable salt thereof; Substance B is isoniazid or a pharmaceutically acceptable salt thereof; Substance C is rifampin or a pharmaceutically acceptable salt thereof; Substance D is pyrazinamide or a pharmaceutically acceptable salt thereof; The structure of compound I is shown below: 。 6. The pharmaceutical composition X according to claim 5, characterized in that, It meets one or more of the following conditions: (1) The pharmaceutical composition X is composed of substance A, substance B, substance C, substance D and pharmaceutical excipients; (2) The pharmaceutical composition X is presented in oral or injectable form.

7. A pharmaceutical composition Y, comprising a first pharmaceutical composition, a second pharmaceutical composition, a third pharmaceutical composition, and a fourth pharmaceutical composition; The first pharmaceutical composition includes substance A and a pharmaceutical excipient; substance A is compound I or a pharmaceutically acceptable salt thereof; The second pharmaceutical composition comprises substance B and a pharmaceutical excipient; substance B is isoniazid or a pharmaceutically acceptable salt thereof; The third pharmaceutical composition comprises substance C and pharmaceutical excipients; substance C is rifampin or a pharmaceutically acceptable salt thereof; The fourth pharmaceutical composition comprises substance D and a pharmaceutical excipient; substance D is pyrazinamide or a pharmaceutically acceptable salt thereof; The structure of compound I is shown below: 。 8. The pharmaceutical composition Y according to claim 7, characterized in that, It meets one or more of the following conditions: (1) The pharmaceutical composition Y comprises a first pharmaceutical composition, a second pharmaceutical composition, a third pharmaceutical composition and a fourth pharmaceutical composition; (2) The first pharmaceutical composition is presented in an oral or injectable dosage form; (3) The second pharmaceutical composition is presented in oral or injectable form; (4) The third pharmaceutical composition is presented in oral or injectable form; (5) The fourth pharmaceutical composition is presented in oral or injectable form.

9. Use of a pharmaceutical composition as described in any one of claims 1-4, pharmaceutical composition X as described in claim 5 or 6, or pharmaceutical composition Y as described in claim 7 or 8 in the preparation of a medicament for treating and / or preventing tuberculosis.

10. Use of a pharmaceutical composition as described in any one of claims 1-4, pharmaceutical composition X as described in claim 5 or 6, or pharmaceutical composition Y as described in claim 7 or 8 in the preparation of a medicament for treating and / or preventing alveolar inflammation caused by pulmonary tuberculosis.

11. Use of a pharmaceutical composition as described in any one of claims 1-4, pharmaceutical composition X as described in claim 5 or 6, or pharmaceutical composition Y as described in claim 7 or 8 in the preparation of a medicament for treating and / or preventing inflammatory lung injury caused by pulmonary tuberculosis.

12. Use of a pharmaceutical composition as described in any one of claims 1-4, pharmaceutical composition X as described in claim 5 or 6, or pharmaceutical composition Y as described in claim 7 or 8 in the preparation of a medicament for treating and / or preventing pulmonary fibrosis.

13. The application as described in claim 12, characterized in that, The pulmonary fibrosis mentioned is secondary fibrosis caused by pulmonary tuberculosis.

14. Use of a pharmaceutical composition as described in any one of claims 1-4, pharmaceutical composition X as described in claim 5 or 6, or pharmaceutical composition Y as described in claim 7 or 8 in the preparation of a medicament for reducing the recurrence rate of tuberculosis.

15. The application as described in claim 14, characterized in that, The drug is used to reduce the recurrence rate of tuberculosis in lung and / or spleen tissues.

16. The application as described in any one of claims 9-15, characterized in that, It meets one or more of the following conditions: (1) Substance A, substance B, substance C and substance D are applied simultaneously or separately; (2) Substance A, substance B, substance C and substance D are administered orally or by injection; (3) The dosage of the isoniazid is 10 mg / kg-30 mg / kg; (4) The dosage of rifampicin is 5 mg / kg-30 mg / kg; (5) The dosage of the pyrazinamide is 100 mg / kg to 300 mg / kg; (6) The dosage of compound I is 10 mg / kg-300 mg / kg.

17. The application as described in claim 16, characterized in that, It meets one or more of the following conditions: (1) The dosage of the isoniazid is 10 mg / kg or 25 mg / kg; (2) The dosage of rifampicin is 10 mg / kg; (3) The dosage of the pyrazinamide is 150 mg / kg; (4) The dosage of compound I is 25 mg / kg, 50 mg / kg, 60 mg / kg, 100 mg / kg, 150 mg / kg or 200 mg / kg.

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