WO2023217239A1

WO2023217239A1 - Pharmaceutical composition and use thereof

Info

Publication number: WO2023217239A1
Application number: PCT/CN2023/093587
Authority: WO
Inventors: 陆宇; 祈雪婷; 付雷; 郑璐瑶; 张炜焱; 李永国; 李磊; 陈小宁
Original assignee: 广州嘉越医药科技有限公司; 首都医科大学附属北京胸科医院
Priority date: 2022-05-12
Filing date: 2023-05-11
Publication date: 2023-11-16
Also published as: CN117045657A

Abstract

Disclosed are a pharmaceutical composition and use thereof. The present invention provides a pharmaceutical combination or a pharmaceutical composition. Active components of the pharmaceutical combination or the pharmaceutical composition include substance A, substance B, substance C and substance D; substance A is the compound (I) or a pharmaceutically acceptable salt thereof; substance B is isoniazid (H) or a pharmaceutically acceptable salt thereof; substance C is rifampicin (R) or a pharmaceutically acceptable salt thereof; substance D is pyrazinamide (Z) or a pharmaceutically acceptable salt thereof. The pharmaceutical combination or the pharmaceutical composition of the present invention can reduce pulmonary alveolus inflammation, pulmonary tuberculosis inflammatory lung injury and pulmonary tuberculosis secondary fibrosis, and increase the permeation of anti-tuberculosis drugs at lesion sites to accelerate therapeutic responses and reduce the risk of recurrence.

Description

A drug combination and its application

This application claims the priority of Chinese patent application 2022105332915 with a filing date of 2022/5/12. This application cites the full text of the above-mentioned Chinese patent application.

Technical field

The present invention relates to a pharmaceutical combination and its use.

Background technique

Tuberculosis is one of the major infectious diseases caused by Mycobacterium tuberculosis infection that seriously threatens human health. The 2021 World Health Organization (WHO) tuberculosis report shows that in 2020, there were 9.87 million new tuberculosis patients worldwide, with an incidence rate of 127/100,000. Chemotherapy is the main means of controlling tuberculosis. Although the standard 6-month treatment regimen is highly effective against drug-sensitive tuberculosis, complete absorption of lung lesions is less likely, and more than half of people still have permanent lung function damage. Often a large number of fibrous lesions remain. Secondary fibrosis of pulmonary tuberculosis is a common complication of pulmonary tuberculosis. Extensive pulmonary fibrosis can seriously affect the patient's lung function. Secondary fibrosis of pulmonary tuberculosis is a common pulmonary impairment after TB (PIAT). At the same time, tuberculosis bacteria are The fiber coating makes it difficult for the drug to completely kill the bacteria and it is easy for them to relapse. Both "calcified nodules and fibrotic lesions" increase the risk of tuberculosis recurrence, and those with fibrotic lesions have a significantly higher risk of tuberculosis recurrence.

PFD (Pirfenidone) is a drug approved by the FDA in 2014 for the treatment of idiopathic fibrosis. Its effects are mainly reflected in antioxidant, anti-inflammatory and anti-fibrosis, and it has broad spectrum. It can inhibit the expression of transforming growth factor (TGF-β1), inhibit the release of a variety of inflammatory factors, and reduce inflammatory reactions.

To date, no drug combination containing pyridone derivatives containing heteroatom cyclobutane substituents has been used to reduce alveolar inflammation, tuberculosis inflammatory lung injury, and tuberculosis secondary fibrosis while increasing the penetration of anti-tuberculosis drugs at the lesion site. , to accelerate treatment response and reduce the risk of recurrence.

Contents of the invention

The technical problem to be solved by the present invention is that the existing drugs for treating tuberculosis are difficult to completely kill bacteria and control the recurrence of tuberculosis. Instead, the invention provides a pharmaceutical combination or pharmaceutical composition and its application. The pharmaceutical composition of the present invention can reduce alveolar inflammation, tuberculosis inflammatory lung damage and tuberculosis secondary fibrosis, and at the same time increase the penetration of anti-tuberculosis drugs in the lesion site to accelerate the treatment response and reduce the risk of recurrence.

The present invention solves the above technical problems through the following technical solutions.

The invention provides a pharmaceutical combination, the active components of which include substance A, substance B, substance C and substance D;

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

Substance B is isoniazid (H) or its pharmaceutically acceptable salt;

Substance C is rifampicin (R) or its pharmaceutically acceptable salt;

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

The structure of compound I is as follows:

In some embodiments, the pharmaceutical combination is a pharmaceutical composition.

In some embodiments, in the pharmaceutical combination, the weight ratio of the substance B and the substance A is (3:1)-(1:30), preferably 1:6.

In some embodiments, in the pharmaceutical combination, the weight ratio of the substance C and the substance A is (3:1)-(1:60), preferably 1:6.

In some embodiments, in the pharmaceutical combination, the substance D and the substance A are The weight ratio is (30:1)-(1:3), preferably 5:2.

In some embodiments, in the pharmaceutical combination, the active component consists of substance A, substance B, substance C and substance D.

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

In some embodiments, Substance A, Substance B, Substance C and Substance D may be administered simultaneously or separately.

Said "simultaneous administration", for example, substance A, said substance B, said substance C and said substance D includes simultaneous administration in separate pharmaceutical compositions; alternatively, "a separate pharmaceutical composition comprising substance A", "comprising substance The "separate pharmaceutical composition of B", the "separate pharmaceutical composition containing substance C" and the "separate pharmaceutical composition containing substance D" are administered simultaneously.

The "separate administration" is for example "a separate pharmaceutical composition containing substance A", "a separate pharmaceutical composition containing substance B", "a separate pharmaceutical composition containing substance C" and "a separate pharmaceutical composition containing substance D" Separately administered at different times, for example: "a separate pharmaceutical composition containing substance A", "a separate pharmaceutical composition containing substance B", "a separate pharmaceutical composition containing substance C" and "a separate pharmaceutical composition containing substance D" "One of them is administered first, the others later. The separate administrations may be close in time or far apart in time.

Regardless of whether they are administered simultaneously or separately, the administration regimen (including administration route, administration dose, administration interval, etc.) of the substance A, the substance B, the substance C and the substance D can be the same or different, which can be determined by those skilled in the art. Technicians make adjustments as needed to provide optimal treatment results.

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;

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

Substance B is isoniazid (H) or its pharmaceutically acceptable salt;

Substance C is rifampicin (R) or its pharmaceutically acceptable salt;

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

The structure of compound I is as follows:

The pharmaceutical composition X is a single pharmaceutical composition.

In some embodiments, the pharmaceutical composition X consists of the substance A, the substance B, the substance C, the substance D and pharmaceutical excipients.

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

In some embodiments, the pharmaceutical composition X is presented in an injectable dosage form.

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;

The first pharmaceutical composition includes substance A and pharmaceutical excipients; substance A is compound I or a pharmaceutically acceptable salt thereof;

The second pharmaceutical composition includes substance B and pharmaceutical excipients; substance B is isoniazid (H) or a pharmaceutically acceptable salt thereof;

The third pharmaceutical composition includes substance C and pharmaceutical excipients; substance C is rifampicin (R) or a pharmaceutically acceptable salt thereof;

The fourth pharmaceutical composition includes substance D and pharmaceutical excipients; substance D is pyrazinamide (Z) or a pharmaceutically acceptable salt thereof;

The structure of compound I is as follows:

The first pharmaceutical composition is a separate pharmaceutical composition; the second pharmaceutical composition is a separate pharmaceutical composition; the third pharmaceutical composition is a separate pharmaceutical composition; the fourth pharmaceutical composition is a separate pharmaceutical combination things.

In some embodiments, the pharmaceutical composition Y consists of a first pharmaceutical composition, a second pharmaceutical composition, a third pharmaceutical composition and a fourth pharmaceutical composition.

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

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

In some embodiments, the third pharmaceutical composition is presented in an oral dosage form or an injectable dosage form.

In some embodiments, the fourth pharmaceutical composition is presented in an oral dosage form or an injectable dosage form.

The present invention provides an application of the above pharmaceutical combination, pharmaceutical composition X or pharmaceutical composition Y in the preparation of drugs for the treatment and/or prevention of tuberculosis.

The present invention provides an application of the above pharmaceutical combination, pharmaceutical composition X or pharmaceutical composition Y in the preparation of drugs for treating and/or preventing alveolar inflammation; preferably, the alveolar inflammation is alveolar inflammation caused by tuberculosis.

The present invention provides an application of the above-mentioned pharmaceutical combination, pharmaceutical composition X or pharmaceutical composition Y in the preparation of medicines for treating and/or preventing inflammatory lung damage caused by tuberculosis.

The present invention provides the above pharmaceutical combination, pharmaceutical composition Prepared for application in medicines for treating and/or preventing pulmonary fibrosis; preferably, the pulmonary fibrosis is fibrosis secondary to tuberculosis.

The present invention provides an application of the above-mentioned pharmaceutical combination, pharmaceutical composition bacterial recurrence rate.

In some embodiments, the drug is used to reduce the recurrence rate of Mycobacterium tuberculosis in spleen tissue, such that the recurrence rate in spleen tissue is reduced by 80%.

In some embodiments, the drug is used to reduce the recurrence rate of tuberculosis bacteria in lung tissue, such that the recurrence rate in lung tissue is reduced by 40%-50%.

In another aspect, the present invention provides a method of treating and/or preventing tuberculosis, comprising administering to a patient in need thereof a therapeutically effective amount of a pharmaceutical combination, pharmaceutical composition X or pharmaceutical composition Y as described herein.

The present invention also provides a method for treating and/or preventing alveolar inflammation, which comprises administering a therapeutically effective amount of a pharmaceutical combination, pharmaceutical composition X or pharmaceutical composition Y as described herein to a patient in need thereof.

The present invention also provides a method for treating and/or preventing pulmonary tuberculosis inflammatory lung injury, which comprises administering a therapeutically effective amount of a pharmaceutical combination, pharmaceutical composition X or pharmaceutical composition Y as described herein to a patient in need thereof.

The present invention also provides a method for treating and/or preventing pulmonary fibrosis, which includes administering a therapeutically effective amount of a pharmaceutical combination, pharmaceutical composition X or pharmaceutical composition Y as described herein to a patient in need thereof; preferably Specifically, the pulmonary fibrosis is fibrosis secondary to tuberculosis.

The present invention provides a method for reducing the recurrence rate of tuberculosis, which includes administering a therapeutically effective amount of a pharmaceutical combination, pharmaceutical composition X or pharmaceutical composition Y as described herein to a patient in need thereof; preferably, the method uses To reduce the recurrence rate of tuberculosis bacteria in lung tissue and/or spleen tissue.

In some embodiments, the method is used to reduce the recurrence rate of Mycobacterium tuberculosis in splenic tissue, such that the recurrence rate in splenic tissue is reduced by 80%.

In some embodiments, the method is used to reduce the recurrence rate of tuberculosis bacteria in lung tissue, such that the recurrence rate in lung tissue is reduced by 40%-50%.

The substance A, the substance B, the substance C and the substance D may be administered simultaneously or separately.

The substance A, the substance B, the substance C and the substance D can be administered by any suitable route in the art, including oral administration, injection (eg intravenous, intramuscular, subcutaneous), etc.

The dosage of isoniazid can be administered according to the patient's weight, and non-limiting examples can range from 10 mg/kg to 30 mg/kg, such as 10 mg/kg or 25 mg/kg.

The dosage of rifampicin can be administered according to the patient's weight, and a non-limiting example range can be 5 mg/kg-30 mg/kg, such as 10 mg/kg.

The dosage of pyrazinamide can be administered according to the patient's weight, and non-limiting examples can range from 100 mg/kg to 300 mg/kg, such as 150 mg/kg.

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

As used herein, the term "treatment" refers to therapeutic therapy. When referring to a specific condition, treatment means: (1) alleviating the disease or one or more biological manifestations of the condition, (2) interfering with (a) one or more points in the biological cascade that causes or causes the condition or (b) ) one or more biological manifestations of a condition, (3) amelioration of 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) slow the progression of a condition or one or more biological manifestations of a condition.

The term "therapeutically effective amount" as used herein refers to an amount of a compound sufficient to effectively treat the disease or condition described herein when administered to a patient. The amount of a compound that constitutes a "therapeutically effective amount" will vary depending on the compound, the condition and its severity, and the age of the patient to be treated, but can be adjusted as necessary by one skilled in the art.

As used herein, the term "pharmaceutical composition" refers to a pharmaceutical composition containing the specified active ingredient and prepared in the same form. One dosage form composition.

The term "patient" as used herein refers to any animal, preferably a mammal, and most preferably a human, to which a compound or composition is or has been administered in accordance with embodiments of the present 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, humans, etc., with humans being the most preferred.

The term "pharmaceutical excipients" as used herein refers to the excipients and additives used in the production of pharmaceuticals and preparation of prescriptions, which are all substances included in pharmaceutical preparations except active ingredients. See the Pharmacopoeia of the People's Republic of China (2020 Edition), Part Four, or Handbook of Pharmaceutical Excipients (Raymond C Rowe, 2009 Sixth Edition).

The term "pharmaceutically acceptable" as used herein means that acids or bases (used to prepare salts), solvents, excipients, etc. are generally non-toxic, safe, and suitable for use by patients. The "patient" is preferably a mammal, more preferably a human.

As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a compound prepared with a relatively non-toxic, pharmaceutically acceptable acid or base. When compounds contain relatively acidic functional groups, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of a pharmaceutically acceptable base in pure solution or in a suitable inert solvent. Pharmaceutically acceptable base addition salts include, but are not limited to: lithium salts, sodium salts, potassium salts, calcium salts, aluminum salts, magnesium salts, zinc salts, bismuth salts, ammonium salts, and diethanolamine salts. When the compounds contain relatively basic functional groups, acid addition salts may be obtained by contacting the neutral form of such compounds with a sufficient amount of a pharmaceutically acceptable acid in neat solution or in a suitable inert solvent. The pharmaceutically acceptable acid includes inorganic acid, and the inorganic acid includes but is not limited to: hydrochloric acid, hydrobromic acid, hydroiodic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate Roots, phosphorous acid, sulfuric acid, hydrogen sulfate, etc. The pharmaceutically acceptable acids include organic acids, and the organic acids include but are not limited to: acetic acid, propionic acid, oxalic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, suberic acid , fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, salicylic acid, tartaric acid, methanesulfonic acid, isonicotinic acid, acid citric acid, oleic acid , tannic acid, pantothenic acid, hydrogen tartrate, ascorbic acid, gentisic acid, Fumaric acid, gluconic acid, sugar acid, formic acid, ethanesulfonic acid, pamoic acid (i.e. 4,4'-methylene-bis(3-hydroxy-2-naphthoic acid)), amino acids (such as glutamine acid, arginine), etc. When a compound contains relatively acidic and relatively basic functional groups, 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. Heinrich Stahl and Camille G. Wermuth, ed., Wiley-VCH, 2002).

On the basis of common sense in the field, the above preferred conditions can be combined arbitrarily to obtain preferred examples of the present invention.

The reagents and raw materials used in the present invention are all commercially available.

The positive and progressive effect of the present invention is that the pharmaceutical combination or pharmaceutical composition of the present invention can reduce alveolar inflammation, tuberculosis inflammatory lung damage and tuberculosis secondary fibrosis, and at the same time increase the penetration of anti-tuberculosis drugs in the lesion site to accelerate the treatment response. , reduce the risk of recurrence.

Description of the drawings

Figure 1 shows the results of HE staining and Masson staining of mouse lung tissue.

Figure 2 shows the alveolar inflammation score of HE stained lung tissue of mice after 4 weeks of drug treatment.

Figure 3 shows the alveolar inflammation score of HE stained lung tissue of mice after 8 weeks of drug treatment.

Figure 4 shows the Masson staining results of the lung tissue of mice after 4 weeks and 8 weeks of drug treatment.

Figure 5 shows the proportion of special staining positive areas in mice after 4 weeks of drug treatment.

Figure 6 shows the proportion of special staining positive areas in mice after 8 weeks of drug treatment.

Figure 7 shows the hydroxyproline content in the right lung tissue of each treatment group after 4 weeks of treatment.

Figure 8 shows the hydroxyproline content in the right lung tissue of each treatment group after 8 weeks of treatment.

Figure 9 shows the hydroxyproline content in the right lung tissue of each treatment group at different time points.

Figure 10 shows the CFU counts in the lung tissue of each group after 4 weeks of treatment.

Figure 11 shows the CFU count in the spleen of each treatment group after 8 weeks of treatment.

Figure 12 is a summary of CFU counts in lung tissue of each group at different treatment time points.

Figure 13 is a summary of CFU counts in the spleens of each group at different treatment time points.

Detailed ways

The present invention is further described below by means of examples, but the present invention is not limited to the scope of the described examples. Experimental methods that do not indicate specific conditions in the following examples should be selected according to conventional methods and conditions, or according to product specifications.

Example 1 Study on the protective effect of compound I in tuberculosis lung injury

1. Experimental materials:

1. Experimental strains

Laboratory standard MTB strain: H37Rv (ATCC 27294) is preserved and provided by Beijing Chest Hospital Affiliated to Capital Medical University/Beijing Key Laboratory of Drug-Resistant Tuberculosis.

2. Experimental animals

SPF grade 6-8 week old C57BL/6 female mice with a body weight of 16-19 g were purchased from Beijing Vitong Lever Experimental Animal Technology Co., Ltd.

3. Experimental drugs

Rifampicin (batch number: WXBB7288V, purchased from Sigma Company in the United States);

Isoniazid (batch number: MKBQ8553V, purchased from Sigma Company in the United States);

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

Pirfenidone (batch number: 53179-13-8 purchased from Hanxiang Biotechnology Company);

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

4.Experimental reagents

Middlebrook OADC nutrient solution was purchased from Becton Dickinson Company in the United States (batch number: 9043888);

Alamar Blue indicator was purchased from Bio-Rad Company of the United States (batch number: 154244);

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

Tween-80 was purchased from Beijing Solebao Technology Co., Ltd. (batch number: 1114D0123H);

Hydroxyproline determination kit was purchased from Nanjing Jiancheng Bioengineering Institute (batch number: A030-2-1);

Masson dye set (purchased from Servicebio G1006 company).

5. Experimental methods

(1) Microplate alamar blue assay (MABA) method to measure the minimum inhibitory concentration (Minimum inhibitory concentration (MIC) of PFD and compound I)

Cultivate the H37Rv strain to the logarithmic growth phase, and dilute the bacterial suspension in 7H9 liquid medium to 1×10 ⁶ CFU/ml for later use. Take a 96-well plate and add isoniazid (H), rifampicin (R), compound I, pirfenidone (PFD) + isoniazid (H), pirfenidone (PFD) + rifampicin in sequence. (R), Compound Ⅰ + Isoniazid (H), Compound Ⅰ + Rifampicin (R) dilutions. After culturing at 37°C for 7 days, add 20 μl of Alma Blue and 12.5 μl of 20% Tween-80 to each well, continue culturing at 37°C for 24 hours, and record the color of each well. Blue indicates no bacterial growth, red indicates bacterial growth, and MIC Indicates the lowest drug concentration that changes from blue to red. Repeat the test twice.

(2) Dilute the H37Rv strain in the logarithmic growth phase with 20 ml 1×PBS to a bacterial concentration of 1×10 ⁷ CFU/ml, and infect 75 6-8 week old female C57BL/6 mice by aerosol. A chronic mouse tuberculosis model of aerosol infection was established, and this model was used to evaluate the antibacterial activity of PFD+HRZ, compound I+HRZ, and HRZ treatment groups. On the 10th day after infection (D-32), 3 mice were randomly selected and sacrificed, and on the day of administration and treatment (D0), 6 mice were randomly selected and sacrificed. The spleen and lung tissues were dissected and homogenized, and the spleen and lung tissues were homogenized on 7H10 plates. Lung colony-forming unit (CFU) counts were performed to determine baseline numbers of Mycobacterium tuberculosis in the lungs and spleens of mice at the onset of infection and at the start of treatment. Administration was started 6 weeks after infection. The mice were weighed every week. The total mass and volume of the drug required each week was calculated based on the average weight of mice in each group, and the corresponding dosage concentration was configured. Isoniazid (H) is dissolved in sterile water, and the other drugs are dissolved in DMSO solution. The drug was administered 5 days a week and administered by intragastric administration at 8 o'clock in the morning every day. Mix isoniazid (H) and pyrazinamide (Z) and administer 0.2 ml to each mouse; the compound I+HRZ treatment group and the PFD+HRZ treatment group were given compound I 0.1 ml and PFD 0.1 to each mouse respectively. ml; Rifampicin (R) should be administered at least 1 hour after the end of all drug administration. Preheat and mix all drugs at 37°C before administration. 4 weeks and 8 weeks after administration Afterwards, 7-8 mice from each treatment group were randomly selected and sacrificed for dissection. The spleen and lung tissues were homogenized and then the spleen and lung colonies (CFU) were counted on 7H10 plates. After 8 weeks of treatment and 12 weeks of drug withdrawal, the recurrence of Mycobacterium tuberculosis in the lungs and spleens of 7 mice in each treatment group was observed. C57BL/6 mice were randomly divided into 3 groups 6 weeks after infection (see Table 1)

Table 1 C57BL/6 mouse grouping and dosage regimen

Remarks: Pirfenidone (PFD); Isoniazid (H); Rifampicin (R); Pyrazinamide (Z); D-32: 10 days of infection; W4: 4 weeks of treatment; W8: 8 weeks of treatment ; W8+12W: 8 weeks of treatment followed by 12 weeks of discontinuation to observe recurrence. D: day; W: week. Isoniazid: 10mg/kg/d; Rifampicin: 10mg/kg/d; Pyrazinamide: 150mg/kg/d; Pirfenidone PFD: 100mg/kg/d; Compound I: 60mg/kg/d .

Histopathological HE staining and alveolar inflammation score

Lung histopathological observation: The left lung tissue of the mouse was trimmed and fixed in 4% paraformaldehyde. After the fixation was in good condition, it was washed with physiological saline and fixed in 4% paraformaldehyde solution, embedded in conventional paraffin, and made into 3 μm Paraffin sections were sectioned and HE stained to observe the pathological changes of the lung tissue. The pathological changes of the lung tissue were observed under a light microscope. The degree of inflammatory infiltration was described and the pathological lung injury score was performed. Four indicators were used to evaluate the degree of lung injury: ① alveolar congestion; ② alveolar hemorrhage; ③ neutrophil infiltration or aggregation in the alveolar space or blood vessel wall; ④ alveolar septal thickening. Scoring standard: 0 points for no injury, 1 point for mild injury, 2 points for moderate injury, and 3 points for severe injury. The sum of each score is the lung injury pathological score.

Masson dyeing and special dyeing proportion

Masson staining was performed according to the instructions in the kit instructions. The sections were routinely dewaxed to water, soaked in Masson A solution overnight, mixed with Masson B solution and Masson C solution in equal proportions and soaked for 1 min, soaked in Masson D solution for 6 min, Masson E solution for 1 min, Masson F solution for 2-30 s, rinsed and differentiated with 1% glacial acetic acid , dehydrated with anhydrous ethanol, transparent with xylene, mounted with neutral gum, and observed with an ordinary optical microscope. Knot The results show that collagen fibers are blue; muscle fibers, cellulose, and red blood cells are red. An imaging system is used to collect images on tissue stained sections, and analysis software is used to automatically read the tissue measurement area, calculate the positive area and tissue area in the measurement area, and calculate the proportion of positive area.

Detection of hydroxyproline content in lung tissue by alkaline hydrolysis method

Assay using HYP detection kit. Determination of hydroxyproline content in right lung tissue. Dissect and separate fresh lung tissue, weigh it, place it in a test tube, and add exactly 1 ml of hydrolyzate. Water bath in water bath for 20 minutes. Adjust the pH to about 6.0-6.8. Then add distilled water to 10ml, take 4ml of the diluted hydrolyzate and add an appropriate amount of activated carbon. Centrifuge at 3500r/min for 10min, and take 1ml of the supernatant for testing. Blank tubes and standard tubes represent distilled water and standard products respectively. Add each reagent in sequence according to the instructions. Finally, use a microplate reader to zero in distilled water at 550 nm, and measure the absorbance A value of each tube. Use the following formula to calculate the HYP content (ug/mg wet weight) = (A test-A empty)/(A standard-A empty)*5ug/ml (standard tube content)*[10 (total volume of hydrolyzate)/lung tissue Ww]. A test: the absorbance of the measurement tube; A blank: the absorbance of the blank tube; A standard: the absorbance of the standard tube.

6. Statistical analysis

Data drawing uses GraphPad prism 8.0 software (GraphPad Company of the United States), and data statistics uses SPSS 22 software. The measurement data conformed to the normal distribution and were described by "x±s". The comparison of differences between multiple groups was performed by one-way analysis of variance. Further pairwise comparisons within groups were performed by t-test. P<0.05 was used to indicate statistical differences.

7.Results

1) MABA method to determine the MIC of PFD, compound I alone and in combination with H/R against the H37Rv standard strain

The microbroth dilution method was used to determine the MIC values of PFD and compound I alone and in combination with H/R against the H37Rv standard strain of Mycobacterium tuberculosis. The results showed that the MIC values of compound I and PFD were both >100ug/ml; 100ug/ml was measured simultaneously. MIC value of compound I/PFD and H/R combination drug in ml. The MIC value of isoniazid single drug is: 0.037ug/ml. The MIC value of rifampicin alone is: 0.038ug/ml. The MIC values of PFD+H and compound I+H are both 0.037ug/ml; the MIC values of PFD+R and compound I+R are both 0.038ug/ml. The results showed that the MIC value of the combination drug added with 100ug/ml PFD or compound I did not change significantly compared with the MIC value of the anti-tuberculosis drug alone. That is, PFD and compound I do not have antibacterial activity and do not affect the antibacterial activity of anti-tuberculosis drugs.

Table 2 MIC results

2) Pathological analysis of HE staining and Masson staining of mouse lung tissue

Observe the pathological sections of each group under a light microscope and score: On the day of treatment, 4 weeks, and 8 weeks after treatment, the left lung tissues of three mice in each treatment group were taken for HE staining and alveolar inflammation scoring. After 4 weeks of treatment, the PFD+HRZ treatment group and the compound I+HRZ treatment group could significantly reduce alveolar inflammation compared with the HRZ treatment group (P<0.05). The results of lung histopathology sections after 4 weeks of treatment showed that extensive mild thickening of the alveolar walls was seen in the lung tissue of the HRZ treatment group, accompanied by diffuse infiltration of lymphocytes and neutrophils, and inflammatory cell infiltration in rings around local blood vessels. Vascular cuffs were formed; a small amount of lymphocytes and macrophages infiltrated in the tissues of the PFD+HRZ treatment group; a small amount of lymphocytes and macrophages infiltrated in the tissues of the compound I+HRZ treatment group, and mild thickening of the alveolar walls was seen locally. The alveolar inflammation score after 8 weeks of treatment showed that the PFD+HRZ treatment group and the compound I+HRZ treatment group could reduce alveolar inflammation compared with the HRZ treatment group (P<0.05). The results are shown in Table 3.

The left lung tissues of three mice in each treatment group were taken for Masson staining at 4 and 8 weeks of treatment. The Masson staining results were collected through the imaging system on the tissue stained sections, and the analysis software was used to automatically read the tissue measurement area. Calculate the positive area and tissue area in the measurement area, and calculate the proportion of positive area. The results are shown in Table 4. The results show that: after 4 weeks of treatment, the PFD+HRZ treatment group and the compound I+HRZ treatment group are better than the HRZ treatment group. It can significantly reduce pulmonary tissue fibrosis (P<0.001); after 8 weeks of treatment, the PFD+HRZ treatment group and the Compound I+HRZ treatment group were better than HRZ The treatment group can still reduce lung tissue fibrosis (P<0.05); the results are shown in Figure 1-6, mouse lung tissue pathological observation, and the protective effect of the three treatment options on tuberculosis lung damage in mice was evaluated. (A in Figure 1 shows: the results of HE staining in the lungs of mice after 4 and 8 weeks of drug treatment. Figure 1 B shows: the results of Masson staining in the lungs of mice after 4 and 8 weeks of drug treatment. Figure 2 represents: HE staining alveolar inflammation score of lung tissue of mice after 4 weeks of drug treatment. Figure 3 represents: HE staining alveolar inflammation score of lung tissue of mice after 8 weeks of drug treatment. AB in Figure 4 represents: mice after 8 weeks of drug treatment Masson staining of lung tissue after 4 weeks and 8 weeks of drug treatment. Figure 5 shows the statistical chart of the proportion of special staining positive areas in mice after 4 weeks of drug treatment.ns. Figure 6 shows the mice after 8 weeks of drug treatment. , special staining positive area ratio statistical chart.ns, no statistical difference, *P＜0.05, **P＜0.01, ***P＜0.001, ****P＜0.0001).

Table 3 Alveolar inflammation scores of mice in each group

Table 4 Special staining positive area ratio (%) of each treatment group at different treatment time points

3) Hydroxyproline kit detects the degree of pulmonary fibrosis in mice in each treatment group

At the 4th and 8th week of treatment, 3 mice from each treatment group were sacrificed and dissected, and the right lung tissue of the mice was taken to detect the hydroxyproline content of the right lung tissue of each treatment group according to the instructions of the hydroxyproline assay kit. Experimental results show that despite HRZ treatment, as the infection cycle of Mycobacterium tuberculosis prolongs, the degree of fibrosis in the lung tissue continues to deepen. In addition, compared with the HRZ treatment group, the pirfenidone + HRZ treatment group and the compound I + HRZ treatment group could reduce the degree of fibrosis in the lung tissue of mice at 4 and 8 weeks of treatment, with fibrosis reduced at 8 weeks. The degree is more significant (P<0.05), and the results are shown in Table 5 and Figures 7-9 (alkaline hydrolysis method was used to measure the hydroxyproline content in the lung tissue of each treatment group, and the anti-fibrosis effect of the three treatment options was evaluated. Figure 7 shows: the content of hydroxyproline in the right lung tissue of each treatment group after 4 weeks of treatment; Figure 8 shows: the content of hydroxyproline in the right lung tissue of each treatment group after 8 weeks of treatment; Figure 9 shows: the content of hydroxyproline in the right lung tissue of each treatment group at different time points Hydroxyproline content in right lung tissue. ns, no statistical difference, *P＜0.05, **P＜0.01, ***P＜0.001).

Table 5 Hyp content in lung tissue at each treatment time point

4) Mouse chronic tuberculosis model to evaluate the anti-tuberculosis activity of each treatment group

On the 10th day of infection, on the day of treatment, 4 weeks of treatment, 8 weeks of treatment, and after 8 weeks of treatment and 12 weeks of drug withdrawal, a certain number of mice from each group were dissected, and the spleen and lung tissue homogenates were taken and homogenized at 7H10 Lung CFU counts were performed on the plate. The CFU count results of lung tissue at each time point are shown in Table 6 below. The CFU count results of spleen tissue at each time point are shown in Table 7 below. In the PFD+HRZ treatment group, two experimental mice died unexpectedly due to improper gastric administration. The mice in the remaining experimental groups tolerated the drug well during the infection and administration processes, and no deaths occurred. The CFU count result in the mouse lung tissue on the day of treatment (D0) was (5.27±0.07)lg (CFU+1), which was used as the baseline value of viable bacterial count in the mouse lung tissue at the beginning of treatment. After 4 weeks of treatment, the three treatment groups of PFD+HRZ, Compound Ⅰ+HRZ and HRZ could significantly reduce the number of viable bacteria in the lung tissue of mice by 2.75lg (CFU+1) and 2.54lg (CFU+1) respectively. , 2.70lg (CFU+1). After 8 weeks of treatment, the number of viable bacteria in the spleens of the PFD+HRZ, Compound Ⅰ+HRZ, and HRZ groups all reached sterility; after 8 weeks of treatment, the number of viable bacteria in the lungs of mice were 2/3, 3/5, and 3/5 respectively. Achieve sterility. The CFU counting results showed that in C57BL/6 mice, the addition of 100 mg/kg/d PFD or compound I had no significant impact on the antibacterial effect of existing anti-tuberculosis drugs, nor did it lead to further spread of Mycobacterium tuberculosis. Spread, the results are shown in Figure 10-13 (Evaluation of the antibacterial effect of 100 mg/kg/d PFD, compound I combined with HRZ in the tuberculosis mouse model (Figure 10 shows: CFU count in the lung tissue of each group after 4 weeks of treatment; Figure 11 Represents: CFU count in spleen of each treatment group after 8 weeks of treatment; Figure 12 shows: Summary of CFU count in lung tissue of each group at different treatment time points; Figure 13 shows: Summary of CFU count in spleen of each group at different treatment time points; Each point represents One mouse, CFU data represent the mean of organs from n ≥ 3 mice and are expressed as Mean ± SD; all lung CFU counts were tested using one-way ANOVA to determine statistical differences between groups).

Table 6 CFU counts in lung tissue of each group

Table 7 CFU counts in spleens of each group

5) In order to further evaluate whether the anti-inflammatory and anti-fibrotic effects of Compound I and PFD have an 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 in Table 8 shown.

Table 8 Relapse in spleen and lung tissue of mice in different treatment groups after 12 weeks of drug withdrawal

The results showed that the recurrence rates of tuberculosis bacteria in the lungs of mice in the PFD+HRZ, compound I+HRZ, and HRZ treatment groups were: 85.7%, 42.9%, and 71.4% respectively; the recurrence rates of the spleen were: 71.4%, 14.3%, and 71.4%, respectively. Among them, the spleen recurrence rate in the compound I+HRZ treatment group was significantly lower than that in the HRZ treatment group (by about 80%). Fisher's exact test showed statistical differences between the two groups (χ2=4.67v=1p<0.05). The recurrence rate of lung tissue in the compound I+HRZ treatment group was significantly lower than that in the HRZ treatment group (by about 40%); the recurrence rate of spleen tissue in the compound I+HRZ treatment group was significantly lower than that in the PFD+HRZ treatment group (by about 80%); The recurrence rate of lung tissue in the compound I+HRZ treatment group was significantly lower than that in the PFD+HRZ treatment group (reduced by about 50%). Through experimental data, it was not observed that the PFD+HRZ treatment group had the effect of reducing the recurrence rate in tuberculosis mice.

In the C57BL/6 mouse chronic tuberculosis model, all three treatment regimens had antibacterial effects, and there was no obvious intra-group difference in antibacterial effects. Compound I may have more outstanding advantages than PFD. Although it has no significant effect in reducing the number of viable bacteria in the lungs of mice, it can reduce the recurrence rate of tuberculosis mice in the long-term efficacy, especially for It has a significant effect on reducing the recurrence rate of mouse spleen.

Although specific embodiments of the present invention have been described above, those skilled in the art should understand that these are only examples, and various changes or changes can be made to these embodiments without departing from the principles and essence of the present invention. Revise. Accordingly, the scope of the present invention is defined by the appended claims.

Claims

A pharmaceutical combination, the active components of which include 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 rifampicin or its pharmaceutically acceptable salt;

Substance D is pyrazinamide or a pharmaceutically acceptable salt thereof;

The structure of compound I is as follows:
The pharmaceutical combination according to claim 1, characterized in that it meets one or more of the following conditions:

(1) In the pharmaceutical combination, the weight ratio of the substance B and the substance A is (3:1)-(1:30);

(2) In the pharmaceutical combination, the weight ratio of the substance C and the substance A is (3:1)-(1:60);

(3) In the pharmaceutical combination, the weight ratio of substance D and substance A is (30:1)-(1:3);

(4) The substance A, the substance B, the substance C and the substance D are administered orally or by injection;

(5) The substance A, the substance B, the substance C and the substance D are administered simultaneously or separately;

(6) The pharmaceutical combination is a pharmaceutical composition.
The pharmaceutical combination according to claim 2, characterized in that it meets one or more of the following conditions:

(1) In the pharmaceutical combination, the weight ratio of the substance B and the substance A is 1:6;

(2) In the pharmaceutical combination, the weight ratio of the substance C and the substance A is 1:6;

(3) In the pharmaceutical combination, the weight ratio of the substance D and the substance A is 5:2.
The pharmaceutical combination according to claim 1, characterized in that,

In the pharmaceutical combination, the active component consists of substance A, substance B, substance C and substance D.
A pharmaceutical composition X, which includes 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 (H) or its pharmaceutically acceptable salt;

Substance C is rifampicin (R) or its pharmaceutically acceptable salt;

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

The structure of compound I is as follows:
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 consists of the substance A, the substance B, the substance C, the substance D and pharmaceutical excipients;

(2) The pharmaceutical composition X is presented in an oral dosage form or an injection dosage form.
A pharmaceutical composition Y, which includes 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 pharmaceutical excipients; substance A is compound I or a pharmaceutically acceptable salt thereof;

The second pharmaceutical composition includes substance B and pharmaceutical excipients; substance B is isoniazid (H) or a pharmaceutically acceptable salt thereof;

The third pharmaceutical composition includes substance C and pharmaceutical excipients; substance C is rifampicin (R) or a pharmaceutically acceptable salt thereof;

The fourth pharmaceutical composition includes substance D and pharmaceutical excipients; substance D is pyrazinamide (Z) or a pharmaceutically acceptable salt thereof;

The structure of compound I is as follows:
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 consists of 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 dosage form or an injection dosage form;

(3) The second pharmaceutical composition is presented in an oral dosage form or an injection dosage form;

(4) The third pharmaceutical composition is presented in an oral dosage form or an injection dosage form;

(5) The fourth pharmaceutical composition is presented in an oral dosage form or an injection dosage form.
A pharmaceutical combination as claimed in any one of claims 1-4, as claimed in claim 5 or 6 The use of the pharmaceutical composition
A pharmaceutical combination according to any one of claims 1 to 4, a pharmaceutical composition and/or application in medicines for preventing alveolar inflammation; preferably, the alveolar inflammation is alveolar inflammation caused by tuberculosis.
A pharmaceutical combination according to any one of claims 1 to 4, a pharmaceutical composition and/or application in drugs to prevent inflammatory lung damage caused by tuberculosis.
A pharmaceutical combination as described in any one of claims 1-4, a pharmaceutical composition Application of drugs in tuberculosis recurrence rate.
A pharmaceutical combination according to any one of claims 1 to 4, a pharmaceutical composition and/or application in medicines for preventing pulmonary fibrosis; preferably, the pulmonary fibrosis is fibrosis secondary to tuberculosis.
A method for treating and/or preventing tuberculosis, which includes administering to a patient in need a therapeutically effective amount of a pharmaceutical combination according to any one of claims 1-4, or a pharmaceutical according to claims 5 or 6 Composition X or pharmaceutical composition Y according to claim 7 or 8.
A method for treating and/or preventing alveolar inflammation, which includes administering to a patient in need a therapeutically effective amount of the pharmaceutical combination as described in any one of claims 1-4, or as claimed in claim 5 or 6 Pharmaceutical composition X or pharmaceutical composition Y as claimed in claim 7 or 8.
A method for treating and/or preventing pulmonary tuberculosis inflammatory lung damage, which includes administering to a patient in need a therapeutically effective amount of a pharmaceutical combination as claimed in any one of claims 1-4, as claimed in claim 5 or 6 The pharmaceutical composition X or the pharmaceutical composition Y according to claim 7 or 8.
A method of treating and/or preventing pulmonary fibrosis, comprising administering to a patient in need thereof A pre-therapeutic effective amount of the pharmaceutical combination according to any one of claims 1 to 4, the pharmaceutical composition X according to claims 5 or 6, or the pharmaceutical composition Y according to claims 7 or 8.
A method for reducing the recurrence rate of tuberculosis, which includes administering to a patient in need a therapeutically effective amount of the pharmaceutical combination according to any one of claims 1-4, or the pharmaceutical composition according to claims 5 or 6 X or the pharmaceutical composition Y according to claim 7 or 8; preferably, the method is used to reduce the recurrence rate of tuberculosis bacteria in lung tissue and/or spleen tissue.
The method of claim 18, characterized in that it satisfies one or more of the following conditions:

(1) The method is used to reduce the recurrence rate of Mycobacterium tuberculosis in spleen tissue, reducing the recurrence rate of spleen tissue by 80%;

(2) The method is used to reduce the recurrence rate of tuberculosis bacteria in lung tissue, so that the recurrence rate in lung tissue is reduced by 40%-50%.
The application according to any one of claims 9-13 or the method according to any one of claims 14-19, characterized in that it satisfies one or more of the following conditions:

(1) The substance A, the substance B, the substance C and the substance D are administered simultaneously or separately;

(2) The substance A, the substance B, the substance C and the substance D are administered orally or by injection;

(3) The dosage of isoniazid is 10mg/kg-30mg/kg;

(4) The dosage of rifampicin is 5mg/kg-30mg/kg;

(5) The dosage of pyrazinamide is 100mg/kg-300mg/kg;

(6) The dosage of compound I is 10 mg/kg-300 mg/kg;

Preferably, it satisfies one or more of the following conditions:

(1) The dosage of isoniazid is 10 mg/kg or 25 mg/kg;

(2) The dosage of rifampicin is 10 mg/kg;

(3) The dosage of 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.