Composition of hypoxia-inducible factor prolyl hydroxylase inhibitor and antituberculous drug and application thereof

Abstract

The invention relates to the technical field of bioengineering, in particular to a composition of a hypoxia inducible factor prolyl hydroxylase inhibitor and an antituberculosis drug and application of the composition. The composition of the hypoxia-inducible factor prolyl hydroxylase inhibitor and the antituberculous drug is applied to preparation of drugs or health-care products for preventing and treating chronic inflammatory anemia and infection, and the obtained drugs or health-care products can improve chronic inflammatory anemia caused by interference of mycobacterium tuberculosis. The medicine can be applied to treatment of tuberculosis and has a wide application effect.

Description

Technical Field

[0001] The present application belongs to the field of bioengineering technology, and in particular relates to a composition of a hypoxia-inducible factor prolyl hydroxylase inhibitor and an anti-tuberculosis drug and an application thereof. Background Art

[0002] Chronic inflammatory anemia, also known as inflammatory anemia, is one of the most common anemias in clinical practice and is usually associated with chronic systemic inflammatory diseases, including infection with Mycobacterium tuberculosis, infection with human immunodeficiency virus, and chronic fungal infection. Chronic inflammatory anemia is characterized by hypoiremia, mainly due to inflammation-induced increase in hepcidin, which causes iron accumulation in mononuclear macrophages and reduced intestinal absorption of iron, resulting in a reduction in recyclable iron.

[0003] The principle of treating anemia of chronic inflammation is to treat the underlying disease. However, infection and anemia affect each other, significantly aggravating disease progression and patient mortality. This shows that improving anemia in clinical practice is crucial to improving the anti-infection efficacy of patients. In the clinical cohort, the use of nutritional iron supplementation alone not only failed to effectively reverse anemia and treat infection, but also further worsened the patient's condition.

[0004] Therefore, there is an urgent need to develop a new drug that can simultaneously improve anemia and enhance anti-infection efficacy. Summary of the invention

[0005] The purpose of the present application is to provide a combination of a hypoxia-inducible factor prolyl hydroxylase inhibitor and an anti-tuberculosis drug and its application, aiming to solve the problem that there is no new drug in the prior art that can simultaneously improve anemia and enhance anti-infection efficacy.

[0006] In order to achieve the above application purpose, the technical solution adopted in this application is as follows:

[0007] In the first aspect, the present application provides a composition of a hypoxia-inducible factor prolyl hydroxylase inhibitor and an anti-tuberculosis drug, wherein the hypoxia-inducible factor prolyl hydroxylase inhibitor includes at least one of roxadustat, daptostat, enadostat, and valdostat; and / or, the anti-tuberculosis drug includes at least one of isoniazid, rifampicin, pyrazinamide, ethambutol, streptomycin, rifapentine, rifabutin, kanamycin, bucloxacillin, ofloxacin, levofloxacin, moxifloxacin, cycloserine, para-aminosalicylic acid, linezolid, clofazimine, bedaquinine and the like.

[0008] In a second aspect, the present application provides the use of a combination of a hypoxia-inducible factor prolyl hydroxylase inhibitor and an anti-tuberculosis drug in the preparation of a drug or health product for preventing and treating chronic inflammatory anemia and infection.

[0009] In some embodiments, the medicine or health product comprises a combination of a hypoxia-inducible factor prolyl hydroxylase inhibitor and an anti-tuberculosis drug.

[0010] In some embodiments, the dosage form of the drug includes any one of tablets, capsules, pills, powders, granules, syrups, solutions, emulsions, injections, sprays, aerosols, gels and creams.

[0011] In some embodiments, the dosage form of the health care product includes any one of tablets, capsules, pills, powders, granules, syrups, solutions, emulsions, injections, sprays, aerosols, gels and creams.

[0012] In some embodiments, in the medicine or health product, the content of the combination of hypoxia-inducible factor prolyl hydroxylase inhibitor and anti-tuberculosis drug is 1 wt % to 99.5 wt %.

[0013] In some embodiments, the medicine or health product further includes a carrier.

[0014] In some embodiments, the carrier comprises at least one of a self-diluent, a wetting agent, a binder, a lubricant, a colorant, and a coating agent.

[0015] In some embodiments, anemia of chronic inflammation is caused by bacterial, human immunodeficiency virus, or fungal infection.

[0016] In a third aspect, the present application provides a drug for preventing and treating chronic inflammatory anemia and infection, the drug comprising an active ingredient and a carrier, wherein the active ingredient comprises a combination of a hypoxia-inducible factor prolyl hydroxylase inhibitor and an anti-tuberculosis drug.

[0017] The first aspect of the present application provides a composition of a hypoxia-inducible factor prolyl hydroxylase inhibitor and an anti-tuberculosis drug, wherein the hypoxia-inducible factor prolyl hydroxylase inhibitor includes at least one of roxadustat, daptostat, enadolstat, and valdostat; the anti-tuberculosis drug includes at least one of isoniazid, rifampicin, pyrazinamide, ethambutol, streptomycin, rifapentine, rifabutin, kanamycin, bucloxacillin, ofloxacin, levofloxacin, moxifloxacin, cycloserine, para-aminosalicylic acid, linezolid, clofazimine, and bedaquinine. Among them, the hypoxia-inducible factor prolyl hydroxylase inhibitor inhibits prolyl hydroxylase by simulating ketoglutarate, one of the substrates of prolyl hydroxylase, affecting its function of promoting the degradation of hypoxia-inducible factor, improving chronic inflammatory anemia caused by interference of Mycobacterium tuberculosis, and, when used in combination with anti-tuberculosis drugs, plays an effective synergistic role in the treatment of anemia and anti-infection. The combination is highly safe and has good development and application value.

[0018] The second aspect of the present application provides a composition of a hypoxia-inducible factor prolyl hydroxylase inhibitor and an anti-tuberculosis drug for use in the preparation of a medicine or health product for preventing and treating chronic inflammatory anemia and infection; since the provided composition can play an effective synergistic role in the treatment of anemia and anti-infection, the composition is used in the preparation of a medicine or health product for preventing and treating chronic inflammatory anemia and infection. The obtained medicine or health product can improve chronic inflammatory anemia caused by interference of Mycobacterium tuberculosis, and can be used to treat tuberculosis, and has a wide range of application effects.

[0019] The third aspect of the present application provides a drug for preventing and treating chronic inflammatory anemia and infection, the drug comprising an active ingredient and a carrier, wherein the active ingredient comprises a combination of a hypoxia-inducible factor prolyl hydroxylase inhibitor and an anti-tuberculosis drug; since the provided active ingredient can play an effective synergistic role in the treatment of anemia and anti-infection, the obtained drug for preventing and treating chronic inflammatory anemia and infection can significantly improve chronic inflammatory anemia and infection caused by interference with anti-tuberculosis Mycobacterium tuberculosis, and the drug has high safety and good use effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present application. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 This is a schematic diagram of the results of detecting the effect of a hypoxia-inducible factor prolyl hydroxylase inhibitor on stabilizing the expression of hypoxia-inducible factor HIF-1α in THP-1 macrophages induced by phorbol ester (PMA) infection with tuberculosis in the examples of the present application;

[0022] Figure 2 This is a schematic diagram of the results of detecting the cytotoxicity of hypoxia-inducible factor prolyl hydroxylase inhibitors and the ability of macrophages to clear tuberculosis bacteria in THP-1 macrophages induced by PMA infected with tuberculosis in the examples of the present application;

[0023] Figure 3 This is a schematic diagram of the effect of tuberculosis-infected mice for four weeks and given hypoxia-inducible factor prolyl hydroxylase inhibitors by oral gavage for two weeks on the hemoglobin concentration, erythropoiesis, etc. of the mice in the embodiment of the present application;

[0024] Figure 4 This is a schematic diagram of the results of hematoxylin-eosin staining of lung sections of mice infected with tuberculosis for four weeks in the embodiment of the present application, after oral administration of a hypoxia-inducible factor prolyl hydroxylase inhibitor for two weeks;

[0025] Figure 5 This is a statistical graph of the tuberculosis load in the lung and spleen tissues of mice infected with tuberculosis for four weeks in the examples of the present application and given oral administration of a hypoxia-inducible factor prolyl hydroxylase inhibitor for two weeks. DETAILED DESCRIPTION

[0026] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present application more clearly understood, the present application is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and are not used to limit the present application.

[0027] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can mean: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural. The character " / " generally indicates that the associated objects are in an "or" relationship.

[0028] In this application, "at least one" means one or more, and "plurality" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single items or plural items. For example, "at least one of a, b, or c", or "at least one of a, b, and c" can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, c can be single or multiple, respectively.

[0029] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0030] The terms used in the embodiments of the present application are only for the purpose of describing specific embodiments, and are not intended to limit the present application. The singular forms "a", "an" and "the" used in the embodiments of the present application and the appended claims are also intended to include plural forms, unless the context clearly indicates other meanings.

[0031] The weight of the relevant components mentioned in the embodiment description of the present application can not only refer to the specific content of each component, but also represent the proportional relationship between the weights of the components. Therefore, as long as the content of the relevant components is proportionally enlarged or reduced according to the embodiment description of the present application, it is within the scope disclosed in the embodiment description of the present application. Specifically, the mass in the embodiment description of the present application can be μg, mg, g, kg and other mass units known in the chemical industry.

[0032] The terms "first" and "second" are used only for descriptive purposes to distinguish objects such as substances from each other, and should not be understood as indicating or implying relative importance or implicitly indicating the number of indicated technical features. For example, without departing from the scope of the embodiments of the present application, the first XX may also be referred to as the second XX, and similarly, the second XX may also be referred to as the first XX. Thus, features defined as "first" and "second" may explicitly or implicitly include one or more of the features.

[0033] In a first aspect, an embodiment of the present application provides a composition of a hypoxia-inducible factor prolyl hydroxylase inhibitor and an anti-tuberculosis drug, wherein the hypoxia-inducible factor prolyl hydroxylase inhibitor includes at least one of roxadustat, daptostat, enadostat, and valdostat; and / or the anti-tuberculosis drug includes at least one of isoniazid, rifampicin, pyrazinamide, ethambutol, streptomycin, rifapentine, rifabutin, kanamycin, bucloxacillin, ofloxacin, levofloxacin, moxifloxacin, cycloserine, para-aminosalicylic acid, linezolid, clofazimine, and bedaquinine.

[0034] The first aspect of the embodiment of the present application provides a composition of a hypoxia-inducible factor prolyl hydroxylase inhibitor and an anti-tuberculosis drug, wherein the hypoxia-inducible factor prolyl hydroxylase inhibitor includes at least one of roxadustat, daptostat, enadolstat, and valdostat; the anti-tuberculosis drug includes at least one of isoniazid, rifampicin, pyrazinamide, ethambutol, streptomycin, rifapentine, rifabutin, kanamycin, amikacin, ofloxacin, levofloxacin, moxifloxacin, cycloserine, para-aminosalicylic acid, linezolid, clofazimine, and bedaquinine. Among them, the hypoxia-inducible factor prolyl hydroxylase inhibitor inhibits prolyl hydroxylase by simulating ketoglutarate, one of the substrates of prolyl hydroxylase, affecting its function of promoting the degradation of hypoxia-inducible factor, improving chronic inflammatory anemia caused by interference of Mycobacterium tuberculosis, and, when used in combination with anti-tuberculosis drugs, plays an effective synergistic role in the treatment of anemia and anti-infection. The combination is highly safe and has good development and application value.

[0035] Hypoxia-inducible factor prolyl hydroxylase inhibitors are specific drugs for the treatment of chronic renal anemia in adults, including roxadustat, daprodustat, enarodustat and vadadustat. This type of drug inhibits prolyl hydroxylase by simulating ketoglutarate, one of the substrates of prolyl hydroxylase, affecting its function of promoting the degradation of hypoxia-inducible factor, thereby correcting anemia. It is worth noting that hypoxia-inducible factor also plays a very important role in host anti-infection immunity. For example, knocking out hypoxia-inducible factor significantly increases the susceptibility of mice to tuberculosis. In addition, through the evaluation of cell models, it was found that statin drugs such as roxadustat, daprodustat, enarodustat and vadadustat, which stabilize hypoxia-inducible factor, significantly promote the clearance of tuberculosis by macrophages. Therefore, in chronically infected people, hypoxia-inducible factor prolyl hydroxylase inhibitors can improve inflammatory anemia and resist infection.

[0036] Anti-tuberculosis drugs include at least one of isoniazid, rifampicin, pyrazinamide, ethambutol, streptomycin, rifapentine, rifabutin, kanamycin, amikacin, ofloxacin, levofloxacin, moxifloxacin, cycloserine, para-aminosalicylic acid, linezolid, clofazimine, and bedaquinine. Hypoxia-inducible factor prolyl hydroxylase inhibitors can work in conjunction with anti-tuberculosis drugs to improve chronic inflammatory anemia caused by interference from Mycobacterium tuberculosis and can be used to treat tuberculosis. This is conducive to achieving synergistic effects.

[0037] The second aspect of the embodiments of the present application provides the use of a combination of a hypoxia-inducible factor prolyl hydroxylase inhibitor and an anti-tuberculosis drug in the preparation of a drug or health product for preventing and treating chronic inflammatory anemia and infection.

[0038] The second aspect of the embodiment of the present application provides a composition of a hypoxia-inducible factor prolyl hydroxylase inhibitor and an anti-tuberculosis drug for use in the preparation of a medicine or health product for preventing and treating chronic inflammatory anemia and infection; since the provided composition can play an effective synergistic role in the treatment of anemia and anti-infection, the composition is used in the preparation of a medicine or health product for preventing and treating chronic inflammatory anemia and infection. The obtained medicine or health product can improve chronic inflammatory anemia caused by interference of Mycobacterium tuberculosis, and can be used to treat tuberculosis, and has a wide range of application effects.

[0039] Optionally, providing a hypoxia-inducible factor prolyl hydroxylase inhibitor and an anti-tuberculosis drug for use together can improve chronic inflammatory anemia and infection. In the specific use process, the dosage of the hypoxia-inducible factor prolyl hydroxylase inhibitor and the anti-tuberculosis drug is specifically adjusted according to the clinical situation, and any dosage is acceptable.

[0040] Optionally, a medicine or health product composed of a hypoxia-inducible factor prolyl hydroxylase inhibitor and an anti-tuberculosis drug is prepared and used to improve chronic inflammatory anemia and infection.

[0041] In some embodiments, the medicine or health product comprises a combination of a hypoxia-inducible factor prolyl hydroxylase inhibitor and an anti-tuberculosis drug.

[0042] In some embodiments, the dosage form of the drug includes any one of tablets, capsules, pills, powders, granules, syrups, solutions, emulsions, injections, sprays, aerosols, gels and creams.

[0043] In some embodiments, the dosage form of the health care product includes any one of tablets, capsules, pills, powders, granules, syrups, solutions, emulsions, injections, sprays, aerosols, gels and creams.

[0044] In some embodiments, in the medicine or health product, the content of the combination of hypoxia-inducible factor prolyl hydroxylase inhibitor and anti-tuberculosis drug is 1 wt % to 99.5 wt %.

[0045] In some embodiments, the medicine or health product further includes a carrier.

[0046] In some embodiments, the carrier comprises at least one of a self-diluent, a wetting agent, a binder, a lubricant, a colorant, and a coating agent.

[0047] In some embodiments, anemia of chronic inflammation is caused by bacterial, human immunodeficiency virus, or fungal infection.

[0048] A third aspect of an embodiment of the present application provides a drug for preventing and treating chronic inflammatory anemia and infection, the drug comprising an active ingredient and a carrier, wherein the active ingredient comprises a combination of a hypoxia-inducible factor prolyl hydroxylase inhibitor and an anti-tuberculosis drug.

[0049] A third aspect of an embodiment of the present application provides a drug for preventing and treating chronic inflammatory anemia and infection, the drug comprising an active ingredient and a carrier, wherein the active ingredient comprises a combination of a hypoxia-inducible factor prolyl hydroxylase inhibitor and an anti-tuberculosis drug; since the provided active ingredient can play an effective synergistic role in the treatment of anemia and anti-infection, the obtained drug for preventing and treating chronic inflammatory anemia and infection can significantly improve chronic inflammatory anemia and infection caused by interference with anti-tuberculosis Mycobacterium tuberculosis, and the drug has high safety and good use effect.

[0050] The following describes the invention in conjunction with specific embodiments.

[0051] Example 1

[0052] Experiment on the stabilization of HIF-1α expression by hypoxia-inducible factor prolyl hydroxylase inhibitor in THP-1 cells

[0053] Hypoxia-inducible factor prolyl hydroxylase inhibitors are the latest small molecule oral drugs for the treatment of chronic renal anemia in adults. They can stabilize the expression level of hypoxia-inducible factor protein in the body by inhibiting prolyl hydroxylases, thereby promoting the production of endogenous erythropoietin (EPO) and receptor expression. In order to study the effect of hypoxia-inducible factor prolyl hydroxylase inhibitors on the expression of hypoxia-inducible factor HIF-1α in THP-1 cells, this example will conduct in-depth research through cell biology and molecular biology methods.

[0054] 1. Test materials

[0055] THP-1 cells (human monocytic leukemia cells) were purchased from ATCC (TIB-202) and cultured in a 37°C, 5% CO2 incubator using RPMI1640 (10% FBS) culture medium. Mycobacterium tuberculosis H37Rv strain was cultured in the biosafety level 3 laboratory of the Eighth Affiliated Hospital of Guangzhou Medical University (Guangzhou Highly Pathogenic Infectious Disease Clinical Detection Biosafety Level 3 Laboratory). In the early stage of the experiment, H37Rv was inoculated into 7H9 liquid culture medium containing 10% OADC and placed in a shaker at 37°C and 110rpm / min to the logarithmic growth phase for use. Other instrument consumables: 5×SDS lysis buffer, 8% SDS-PAGE precast gel, 10× electrophoresis buffer, 10× transfer buffer, electrophoresis instrument, etc. are all domestically produced.

[0056] 2. Test Reagents

[0057] Roxadustat (HY-13426), daptostat (HY-17608), enadolstat (HY-109057), valdostat (HY-101277), PMA (Phorbol 12-myristate 13-acetate, HY-18739) were purchased from MCE. Hypoxia-inducible factor HIF-1α Rabbit mAb (Cat. No. A22041) and β-ACTIN Rabbit mAb (AC038) were purchased from ABclonal. OADC and 7H9 broth culture medium were purchased from BD.

[0058] Configuration of 7H9 medium (1L liquid medium):

[0059] 7H9 culture medium 4.7g,

[0060] Tween 80 2.5mL,

[0061] Dissolve in 900 mL ddH2O and heat at 121℃ for 20 min. Cool and store in a refrigerator at 4℃ for later use. Add OADC at a ratio of 10% when using.

[0062] 3. Experimental Methods

[0063] [1] Count THP-1 cells and add 40 ng / mL PMA at 5×10 5 The cells were plated into 12-well plates and induced to differentiate for 48 h.

[0064] [2] Aspirate the culture medium and add 1 mL of RPMI1640 culture medium containing 10% FBS to each well and continue culturing overnight.

[0065] [3] Take 1 mL of tuberculosis bacteria in the logarithmic growth phase and place it in a 15 mL centrifuge tube. Add 10 mL of PBS.

[0066] Centrifuge at 2700 rpm / min for 3 min, add 1 mL PBS to resuspend. After measuring the OD value, dilute to RPMI1640 (10% FBS) at a concentration of MOI=5, and dilute various drugs to the corresponding concentrations, including rosuvastatin (100uM), daptostat (50uM), enadostatin (100uM) and valdostat (100uM). Add 1 mL of culture medium to each well to infect THP-1 cells.

[0067] [4] After 24 hours of continuous infection, the culture medium was aspirated, 200 μl of 1× SDS loading buffer was added to each well to lyse the cells, and the cells were placed in a metal bath heater and boiled at 100°C for 10 min, 12,000 rpm / min, and centrifuged for 2 min.

[0068] [5] Take an 8% precast gel and add 10 μL of sample or 1.5 μL of protein marker to each well.

[0069] [6] Add diluted 1× electrophoresis buffer and run electrophoresis at 100 V until the loading buffer runs out of the gel.

[0070] [7] Prepare 1× transfer buffer (methanol: 10× transfer buffer: ddH20 = 2:1:7). Activate the PVDF membrane by soaking it in methanol in advance. 100V, transfer for 100 minutes.

[0071] [8] After transfer, the membrane was blocked with a blocking buffer prepared with 5% skim milk powder for 1 hour, and the primary antibody diluted with primary antibody diluent (prepared with 5% skim milk powder) was added and incubated overnight at 4°C in a shaker.

[0072] [9] The next day, wash the cells three times with PBST, each time for 10 min.

[0073]

[10] Soak the PVDF membrane in HRP-labeled secondary antibody dilution solution (prepared with 5% skimmed milk powder) and incubate at room temperature for 1 hour on a shaker.

[0074]

[11] Wash three times with PBST, 10 min each time.

[0075]

[12] Chemiluminescence detection: Use absorbent paper to absorb the washing solution, lay the PVDF membrane flat on plastic wrap, and add the chemiluminescent substrate working solution. Place the BIO-RAD ChemiDoc TM XRS+ chemiluminescence imager was used for continuous exposure, development and photography.

[0076] 4. Experimental Results

[0077] According to the above test steps, the test results are as follows Figure 1 As shown in the results, we found that treatment with different hypoxia-inducible factor prolyl hydroxylase inhibitors could increase the expression of hypoxia-inducible factor HIF-1α in THP-1 cells both without and with Mycobacterium tuberculosis infection.

[0078] 5. Conclusion

[0079] Hypoxia-inducible factor prolyl hydroxylase inhibitor can stabilize the expression level of hypoxia-inducible factor HIF-1α protein in THP-1 cells.

[0080] Example 2

[0081] Experiment on the cytotoxicity of hypoxia-inducible factor prolyl hydroxylase inhibitor and its promotion of THP-1 macrophages to clear tuberculosis bacteria

[0082] In this example, lactate dehydrogenase (LDH) release assay was used to evaluate the cytotoxicity of hypoxia-inducible factor prolyl hydroxylase inhibitors; and colony-forming units (CFU) counts were used to evaluate the effect of hypoxia-inducible factor prolyl hydroxylase inhibitors on the ability of THP-1 cells to eliminate tuberculosis bacteria.

[0083] 1. Experimental Materials

[0084] The culture of THP-1 cells and the culture of the virulent strain of Mycobacterium tuberculosis H37Rv were the same as in Example 1.

[0085] 2. Experimental Reagents

[0086] LDH toxicity detection kit (Cat. No. C0017) was purchased from Beyotime, Inc. 7H10 agar medium was purchased from BD Company.

[0087] Configuration of 7H10 agar plate (1L solid medium):

[0088] 7H10 medium 21g,

[0089] Glycerol 5mL,

[0090] L-Asparagine 1g,

[0091] Fully dissolve in 900mL ddH2O, and autoclave at 121℃ for 8min. Take out the culture medium and place it in a 56℃ incubator for at least 30min; then place the OADC equilibrated to room temperature in a 56℃ incubator for 5min, then take 100mL OADC and add it to the culture medium, mix well and pour it into a flat plate, 4.5mL per grid in the four-divided grid, place it on a ventilated sterile operating table for 4h, wrap it in tin foil and place it in a 37℃ incubator for overnight culture. After testing for contamination, it can be placed at room temperature for no more than 1 month for future use.

[0092] 3. Experimental Methods

[0093] THP-1 cytotoxicity and CFU count

[0094] ① THP-1 cell plating and differentiation induction were the same as in Example 1. Mycobacterium tuberculosis was centrifuged and resuspended, and THP-1 cells were infected at MOI=1.

[0095] ② After 6 hours of infection, discard the culture supernatant, wash away the bacteria not phagocytosed by macrophages with 1 ml PBS, and add 1 ml of 1640 complete culture medium containing 100 uM rosuvastatin, 100 uM valdustat and 50 uM daptostat, respectively, and place in a 37°C, 5% CO2 incubator for static culture.

[0096] ③ After 72 hours of culture, collect the supernatant of the duplicate culture medium, centrifuge at 400g / min, and take the supernatant to measure the LDH release according to the instructions.

[0097] ④ The cells were washed twice with preheated PBS, lysed by adding 0.1% SDS (PBS), aspirated, and diluted with PBS 10 times. 50ul of bacterial solution of each concentration was spread on 7H10 plates for culture, and cultured in a 37℃ constant temperature incubator. CFU was counted after 2-3 weeks.

[0098] 4. Experimental Results

[0099] According to the above test steps, the test results are as follows Figure 2 As shown, we found that roxadustat, daptostat, and valdustat could enhance the ability of THP-1 cells to clear tuberculosis bacteria and had no cytotoxicity at the current test concentrations.

[0100] 5. Conclusion

[0101] Hypoxia-inducible factor prolyl hydroxylase inhibitors effectively enhance the function of macrophages in clearing tuberculosis bacteria.

[0102] Example 3

[0103] Effects of hypoxia-inducible factor prolyl hydroxylase inhibitor combined with isoniazid on hemoglobin and erythropoiesis in mice infected with Mycobacterium tuberculosis

[0104] In this example, a mouse model was used to construct a wild-type mouse model of tuberculosis infection, and hypoxia-inducible factor prolyl hydroxylase inhibitor and isoniazid were continuously administered by oral gavage for two weeks to observe the effect of hypoxia-inducible factor prolyl hydroxylase inhibitor on reversing the number of red blood cells and hemoglobin concentration in mice.

[0105] 1. Experimental Materials

[0106] 8-10 week old female C57BL / 6 mice were raised in the biosafety level 3 laboratory of the Eighth People's Hospital affiliated to Guangzhou Medical University (Guangzhou Highly Pathogenic Infectious Disease Clinical Detection Biosafety Level 3 Laboratory). The culture of virulent Mycobacterium tuberculosis strain H37Rv was the same as in Example 1.

[0107] 2. Experimental Methods

[0108] [1] Infection and drug administration in mice

[0109] C57BL / 6 mice were divided into four groups, namely control group, isoniazid group, roxadustat group, and roxadustat combined with isoniazid group, with 8-9 mice in each group. The four groups of mice were infected with Mycobacterium tuberculosis (H37Rv), and the infection dose was 200 CFU / mouse. Four weeks after infection, the drug-treated group was respectively dissolved in PBS with isoniazid and roxadustat, and the drug was administered daily by gavage, with the dose of roxadustat being 30 mg / kg and the dose of isoniazid being 10 mg / kg, and the control group was given an equal volume of DMSO.

[0110] [2] Orbital blood sampling and routine blood test

[0111] 14 days after administration, 200ul of mouse orbital blood was collected and placed in an EDTA anticoagulation tube and mixed by inversion. A routine blood test was performed using a Mindray BC-7500CS blood cell analyzer. When the instrument is ready for counting (and the instrument indicator light is always green), place the sample in the probe liquid test tube position, press the sample suction key on the instrument to start the sample analysis process, and the analyzer automatically performs sample analysis. Record the measurement results: red blood cell count (RBC, 10 12 / L), hemoglobin (Hb, g / L), hematocrit (HCT, %), and mean corpuscular volume (MCV, fl).

[0112] 3. Experimental results

[0113] According to the above test steps, the test results are as follows Figure 3 As shown in Figure 2, administration of hypoxia-inducible factor prolyl hydroxylase inhibitor significantly increased the red blood cell count (RBC, 1012 / L), hemoglobin (Hb, g / L), hematocrit (HCT, %), and mean corpuscular volume (MCV, fl) levels.

[0114] 4. Conclusion

[0115] Hypoxia-inducible factor prolyl hydroxylase inhibitors can effectively reverse hemoglobin concentration and erythropoiesis in mice infected with Mycobacterium tuberculosis.

[0116] Example 4

[0117] Effects of hypoxia-inducible factor prolyl hydroxylase inhibitor combined with isoniazid on lung pathological damage in mice infected with Mycobacterium tuberculosis

[0118] In this example, the tuberculosis infection model of mice was constructed and drug administration was performed in the same manner as in Example 3 to study the effect of the hypoxia-inducible factor prolyl hydroxylase inhibitor on the lung pathology of tuberculosis infected mice.

[0119] 1. Test materials

[0120] Same as Example 3

[0121] 2. Experimental Methods

[0122] [1] Mouse infection and drug administration

[0123] Same as Example 3.

[0124] [2] Take the organization

[0125] After killing the mice, the lung tissue was removed for pathological sectioning. The relevant steps are as follows:

[0126] One lobe of the mouse lung was taken, fixed with 10% formaldehyde for 24 hours, and routinely embedded in paraffin, sectioned, and stained with HE to examine the pathological damage of the lung.

[0127] 3. Experimental results

[0128] According to the above test steps, the results are as follows Figure 4 As shown, the pathological damage in the lungs of mice treated with hypoxia-inducible factor prolyl hydroxylase inhibitor combined with isoniazid was significantly alleviated.

[0129] 4. Conclusion

[0130] Hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors can be combined with isoniazid to effectively reduce lung pathological damage in mice infected with Mycobacterium tuberculosis.

[0131] Example 5

[0132] Effects of hypoxia-inducible factor prolyl hydroxylase inhibitor combined with isoniazid on lung bacterial load in mice infected with Mycobacterium tuberculosis

[0133] In this example, a mouse tuberculosis infection model was constructed and administered in the same manner as in Example 3 to study the effect of a hypoxia-inducible factor prolyl hydroxylase inhibitor combined with isoniazid on the bacterial load in the lungs of mice infected with tuberculosis.

[0134] 1. Test materials

[0135] Same as Example 3.

[0136] 2. Experimental Methods

[0137] [1] Mouse infection and drug administration

[0138] Same as Example 3.

[0139] [2] Mycobacterium tuberculosis CFU test in mice lungs

[0140] Take one-third of the mouse lung tissue and grind it with 5mL PBS. Perform gradient dilutions with 10-fold dilutions with PBS. Take 100ul of bacterial solution at each concentration and spread it on a 7H10 plate for culture. Place it in a 37℃ constant temperature incubator and count the CFU after 2-3 weeks.

[0141] 3. Experimental results

[0142] According to the above test steps, the test results are as follows Figure 5 As shown, hypoxia-inducible factor prolyl hydroxylase inhibitor combined with isoniazid significantly reduced the tuberculosis load in the lungs of mice.

[0143] 4. Conclusion

[0144] It can be seen from the above examples that the combination of hypoxia-inducible factor prolyl hydroxylase inhibitor and isoniazid can effectively reduce the degree of pathological damage in the lung tissue of mice infected with tuberculosis and reduce the tuberculosis load in the lung tissue, thereby providing effective drug support for the treatment of tuberculosis.

[0145] In summary, the composition of the hypoxia-inducible factor prolyl hydroxylase inhibitor and the anti-tuberculosis drug provided in the embodiments of the present application plays an effective synergistic role in the treatment of anemia and anti-infection; therefore, the composition is used in the preparation of medicines or health products for preventing and treating chronic inflammatory anemia and infection, and the obtained medicines or health products can improve chronic inflammatory anemia caused by interference of Mycobacterium tuberculosis, and can be used to treat tuberculosis, and have a wide range of application effects.

[0146] The above description is only a preferred embodiment of the present application and is not intended to limit the present application. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present application should be included in the protection scope of the present application.

Claims

1. A composition of a hypoxia-inducible factor prolyl hydroxylase inhibitor and an anti-tuberculosis drug, characterized in that: The hypoxia-inducible factor prolyl hydroxylase inhibitor includes at least one of roxadustat, daptostat, enadostat, and valdostat; and / or the anti-tuberculosis drug includes at least one of isoniazid, rifampicin, pyrazinamide, ethambutol, streptomycin, rifapentine, rifabutin, kanamycin, amikacin, ofloxacin, levofloxacin, moxifloxacin, cycloserine, para-aminosalicylic acid, linezolid, clofazimine, and bedaquinine.

2. Use of the combination of the hypoxia-inducible factor prolyl hydroxylase inhibitor and an anti-tuberculosis drug as claimed in claim 1 in the preparation of a medicine or health product for preventing and treating chronic inflammatory anemia and infection.

3. The use according to claim 2, characterized in that: The medicine or the health product comprises a combination of a hypoxia-inducible factor prolyl hydroxylase inhibitor and an anti-tuberculosis drug.

4. The use according to claim 2, characterized in that: The dosage form of the drug includes any one of tablets, capsules, pills, powders, granules, syrups, solutions, emulsions, injections, sprays, aerosols, gels and creams.

5. The use according to claim 2, characterized in that: The dosage form of the health care product includes any one of tablets, capsules, pills, powders, granules, syrups, solutions, emulsions, injections, sprays, aerosols, gels and creams.

6. The use according to claim 3, characterized in that: In the medicine or the health product, the content of the combination of the hypoxia-inducible factor prolyl hydroxylase inhibitor and the anti-tuberculosis drug is 1wt% to 99.5wt%.

7. The use according to claim 3, characterized in that: The medicine or the health product also includes a carrier.

8. The use according to claim 7, characterized in that: The carrier includes at least one of a self-diluent, a wetting agent, a binder, a lubricant, a colorant, and a coating agent.

9. The use according to claim 2, characterized in that: The chronic inflammatory anemia includes those caused by bacterial, human immunodeficiency virus or fungal infection.

10. A drug for preventing and treating chronic inflammatory anemia and infection, characterized in that: The medicine comprises an effective pharmaceutical ingredient and a carrier, wherein the effective pharmaceutical ingredient comprises a combination of the hypoxia-inducible factor prolyl hydroxylase inhibitor according to claim 1 and an anti-tuberculosis drug.

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