Composition comprising hypoxia-inducible factor prolyl hydroxylase inhibitor and antituberculosis drug, and use thereof

Abstract

A composition comprising a hypoxia-inducible factor prolyl hydroxylase inhibitor and an antituberculosis drug, and a use thereof in the preparation of medicines or health care products for preventing and treating chronic inflammatory anemia and infections. The obtained medicines or health care products can alleviate chronic inflammatory anemia caused by interference from mycobacterium tuberculosis, and can be used for treating tuberculosis.

Description

Combinations of hypoxia-inducible factor prolyl hydroxylase inhibitors and anti-tuberculosis drugs and their applications

[0001] This application claims priority to Chinese Patent Application No. 202411896437.8, filed on December 20, 2024, entitled “Composition of Hypoxia-Inducible Factor Prolyl Hydroxylase Inhibitor and Antituberculosis Drug and Its Application,” the entire contents of which are incorporated herein by reference. Technical Field

[0002] This application relates to the field of bioengineering technology, specifically to compositions and applications of hypoxia-inducible factor prolyl hydroxylase inhibitors and anti-tuberculosis drugs. Background Technology

[0003] Chronic inflammatory anemia, also known as inflammatory blood anemia, is one of the most common types of anemia in clinical practice. It is usually associated with chronic systemic inflammatory diseases, including those caused by Mycobacterium tuberculosis infection, human immunodeficiency virus infection, and chronic fungal infections. Chronic inflammatory anemia is characterized by hypoferemia, mainly due to increased hepcidin induced by inflammation, leading to iron accumulation in mononuclear macrophages and reduced iron absorption in the intestines, resulting in a decrease in recyclable iron.

[0004] The principle of treating chronic inflammatory anemia is to treat the underlying disease. However, infection and anemia interact, significantly exacerbating disease progression and patient mortality. This indicates that improving anemia is crucial for enhancing the efficacy of anti-infective therapy in clinical practice. In clinical cohorts, simply using nutritional iron supplementation not only fails to effectively reverse anemia and treat infection, but also further worsens the patient's condition.

[0005] Therefore, there is an urgent need to develop a new type of drug that can simultaneously improve anemia and enhance anti-infective efficacy. Technical issues

[0006] The purpose of this application is to provide a composition of hypoxia-inducible factor prolyl hydroxylase inhibitor and anti-tuberculosis drug and its application, aiming to solve the problem that there is no novel drug in the prior art that can simultaneously improve anemia and enhance anti-infection efficacy. Technical solutions

[0007] The technical solution adopted in the embodiments of this application is:

[0008] In a first aspect, this 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, dapoxetine, ennadustat, and valduxstat; and / or, the anti-tuberculosis drug includes at least one of isoniazid, rifampin, pyrazinamide, ethambutol, streptomycin, rifapentine, rifabutin, kanamycin, amikacin, ofloxacin, levofloxacin, moxifloxacin, cycloserine, para-aminosalicylic acid, linezolid, clofazimine, and bedaquinine.

[0009] Secondly, this application provides the use of a composition of hypoxia-inducible factor prolyl hydroxylase inhibitor and anti-tuberculosis drug in the preparation of a drug or health product for the prevention and treatment of chronic inflammatory anemia and infection.

[0010] In some embodiments, the medicine or health product comprises a combination of a hypoxia-inducible factor prolyl hydroxylase inhibitor and an anti-tuberculosis drug.

[0011] 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.

[0012] In some embodiments, the dosage form of the health supplement includes any one of tablets, capsules, pills, powders, granules, syrups, solutions, emulsions, injections, sprays, aerosols, gels, and creams.

[0013] In some embodiments, the content of the composition of hypoxia-inducible factor prolyl hydroxylase inhibitor and anti-tuberculosis drug in the drug or health product is 1 wt% to 99.5 wt%.

[0014] In some embodiments, the drug or health product may also include a carrier.

[0015] In some embodiments, the carrier includes at least one of a self-diluent, a wetting agent, an adhesive, a lubricant, a colorant, and a coating agent.

[0016] In some embodiments, chronic inflammatory anemia includes those caused by bacterial, human immunodeficiency virus, or fungal infections.

[0017] Thirdly, this application provides a drug for preventing and treating chronic inflammatory anemia and infection, the drug comprising an active pharmaceutical ingredient and a carrier, wherein the active pharmaceutical ingredient comprises a composition of a hypoxia-inducible factor prolyl hydroxylase inhibitor and an anti-tuberculosis drug. Beneficial effects

[0018] The first aspect of this application provides a composition of a hypoxia-inducible factor prolyl hydroxylase inhibitor and an anti-tuberculosis drug. The hypoxia-inducible factor prolyl hydroxylase inhibitor includes at least one of roxadustat, dapoxetine, ennadustat, and valdustat; the anti-tuberculosis drug includes at least one of isoniazid, rifampin, 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 mimicking one of the substrates of prolyl hydroxylase, ketoglutarate, thereby affecting its function in promoting the degradation of hypoxia-inducible factor and improving chronic inflammatory anemia caused by Mycobacterium tuberculosis interference. Furthermore, when used in combination with anti-tuberculosis drugs, it plays an effective synergistic role in the treatment of anemia and anti-infection. This composition has high safety and good development and application value.

[0019] The second aspect of this application provides the use of a composition of hypoxia-inducible factor prolyl hydroxylase inhibitor and anti-tuberculosis drug in the preparation of drugs or health products for the prevention and treatment of chronic inflammatory anemia and infection. Since the provided composition can play an effective synergistic role in the treatment of anemia and anti-infection, its application in the preparation of drugs or health products for the prevention and treatment of chronic inflammatory anemia and infection results in drugs or health products that can improve chronic inflammatory anemia caused by Mycobacterium tuberculosis interference and can be used to treat tuberculosis, demonstrating broad application effects.

[0020] The third aspect of this application provides a drug for preventing and treating chronic inflammatory anemia and infection. The drug includes an active pharmaceutical ingredient and a carrier. The active pharmaceutical ingredient includes a composition of a hypoxia-inducible factor prolyl hydroxylase inhibitor and an anti-tuberculosis drug. Because the provided active pharmaceutical 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 mycobacterium tuberculosis interference. Furthermore, the drug has high safety and good efficacy. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or exemplary technologies will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 is a schematic diagram of the results of detecting the effect of hypoxia-inducible factor prolyl hydroxylase inhibitor on stabilizing the expression of hypoxia-inducible factor HIF-1α in THP-1 macrophages induced by tuberculosis infection with PMA in this embodiment of the application.

[0023] Figure 2 is a schematic diagram of the results of detecting the cytotoxicity of hypoxia-inducible factor prolyl hydroxylase inhibitor and its ability to promote the clearance of Mycobacterium tuberculosis by THP-1 macrophages induced by Mycobacterium tuberculosis infection in the embodiments of this application.

[0024] Figure 3 is a schematic diagram showing the effects of tuberculosis infection on mice for four weeks and two weeks of intervention by gavage with hypoxia-inducible factor prolyl hydroxylase inhibitor on heme concentration, erythropoiesis, etc. in mice in the embodiments of this application.

[0025] Figure 4 is a schematic diagram of the results of hematoxylin-eosin staining of mouse lung sections after four weeks of tuberculosis infection in mice and two weeks of intervention by gavage with hypoxia-inducible factor prolyl hydroxylase inhibitor.

[0026] Figure 5 is a statistical graph showing the tuberculosis load in the lung and spleen tissues of mice after four weeks of tuberculosis infection and two weeks of intervention by gavage with hypoxia-inducible factor prolyl hydroxylase inhibitor in the embodiments of this application. Embodiments of the present invention

[0027] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the scope of this application.

[0028] It should be noted that when a component is referred to as "fixed to" or "set on" another component, it can be directly or indirectly attached to that other component. When a component is referred to as "connected to" another component, it can be directly or indirectly connected to that other component. The terms "upper," "lower," "left," "right," etc., indicate orientation or positional relationships based on the orientation or positional relationships shown in the accompanying drawings, and are for ease of description only, not to indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this application. Those skilled in the art can understand the specific meaning of the above terms according to the specific circumstances. The terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of technical features. "A plurality" means two or more, unless otherwise explicitly defined.

[0029] The first aspect of this 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, dapoxetine, ennadustat, and valdustat; and / or, the anti-tuberculosis drug includes at least one of isoniazid, rifampin, pyrazinamide, ethambutol, streptomycin, rifapentine, rifabutin, kanamycin, amikacin, ofloxacin, levofloxacin, moxifloxacin, cycloserine, para-aminosalicylic acid, linezolid, clofazimine, and bedaquinine.

[0030] The first aspect of this application provides a composition of a hypoxia-inducible factor prolyl hydroxylase inhibitor and an anti-tuberculosis drug. The hypoxia-inducible factor prolyl hydroxylase inhibitor includes at least one of roxadustat, dapoxetine, ennadustat, and valdustat; the anti-tuberculosis drug includes at least one of isoniazid, rifampin, 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 mimicking one of the substrates of prolyl hydroxylase, ketoglutarate, thereby affecting its function in promoting the degradation of hypoxia-inducible factor and improving chronic inflammatory anemia caused by Mycobacterium tuberculosis interference. Furthermore, when used in combination with anti-tuberculosis drugs, it plays an effective synergistic role in the treatment of anemia and anti-infection. This composition has high safety and good development and application value.

[0031] Hypoxia-inducible factor prolyl hydroxylase inhibitors are highly effective treatments for chronic renal anemia in adults, including roxadustat, daprodustat, enarodustat, and vadadustat. These drugs inhibit prolyl hydroxylase by mimicking ketoglutarate, a substrate of prolyl hydroxylase, thereby affecting its function in promoting the degradation of hypoxia-inducible factor and correcting anemia. Notably, hypoxia-inducible factor also plays a crucial role in host immune responses against infection. For example, knockout of hypoxia-inducible factor significantly increases susceptibility to tuberculosis in mice. Furthermore, cell model assessments have shown that staminodes stabilizing hypoxia-inducible factor, such as roxadustat, daprodustat, enarodustat, and vadadustat, significantly promote macrophage clearance of Mycobacterium tuberculosis. Therefore, in individuals with chronic infections, hypoxia-inducible factor prolyl hydroxylase inhibitors can improve inflammatory anemia and enhance anti-infective effects.

[0032] Anti-tuberculosis drugs include at least one of isoniazid, rifampin, pyrazinamide, ethambutol, streptomycin, rifapentine, rifabutin, kanamycin, amikacin, ofloxacin, levofloxacin, moxifloxacin, cycloserine, para-aminosalicylic acid, linezolid, clofazimine, and bedaquinine. Hypoxia-inducible factor prolyl hydroxylase inhibitors, in synergy with anti-tuberculosis drugs, can improve chronic inflammatory anemia caused by Mycobacterium tuberculosis interference and can be used to treat tuberculosis. This synergistic effect is beneficial.

[0033] The second aspect of this application provides the use of a composition of hypoxia-inducible factor prolyl hydroxylase inhibitor and anti-tuberculosis drug in the preparation of a drug or health product for the prevention and treatment of chronic inflammatory anemia and infection.

[0034] The second aspect of this application describes the use of a composition of hypoxia-inducible factor prolyl hydroxylase inhibitor and anti-tuberculosis drug in the preparation of drugs or health products for the prevention and treatment of chronic inflammatory anemia and infection. Since the provided composition can play an effective synergistic role in the treatment of anemia and anti-infection, its application in the preparation of drugs or health products for the prevention and treatment of chronic inflammatory anemia and infection results in drugs or health products that can improve chronic inflammatory anemia caused by Mycobacterium tuberculosis interference and can be used to treat tuberculosis, demonstrating broad application effects.

[0035] Optionally, hypoxia-inducible factor prolyl hydroxylase inhibitors and anti-tuberculosis drugs can be used together to improve chronic inflammatory anemia and infection. In practice, the dosage of each inhibitor and drug should be adjusted according to the clinical situation; any dosage is acceptable.

[0036] Optionally, a drug or health product composed of hypoxia-inducible factor prolyl hydroxylase inhibitor and anti-tuberculosis drug can be prepared for use to improve chronic inflammatory anemia and infection.

[0037] In some embodiments, the medicine or health product comprises a combination of a hypoxia-inducible factor prolyl hydroxylase inhibitor and an anti-tuberculosis drug.

[0038] 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.

[0039] In some embodiments, the dosage form of the health supplement includes any one of tablets, capsules, pills, powders, granules, syrups, solutions, emulsions, injections, sprays, aerosols, gels, and creams.

[0040] In some embodiments, the content of the composition of hypoxia-inducible factor prolyl hydroxylase inhibitor and anti-tuberculosis drug in the drug or health product is 1 wt% to 99.5 wt%.

[0041] In some embodiments, the drug or health product may also include a carrier.

[0042] In some embodiments, the carrier includes at least one of a self-diluent, a wetting agent, an adhesive, a lubricant, a colorant, and a coating agent.

[0043] In some embodiments, chronic inflammatory anemia includes those caused by bacterial, human immunodeficiency virus, or fungal infections.

[0044] A third aspect of this application provides a drug for preventing and treating chronic inflammatory anemia and infection. The drug includes an active pharmaceutical ingredient and a carrier, wherein the active pharmaceutical ingredient includes a composition of a hypoxia-inducible factor prolyl hydroxylase inhibitor and an anti-tuberculosis drug.

[0045] The third aspect of this application provides a drug for preventing and treating chronic inflammatory anemia and infection. The drug includes an active pharmaceutical ingredient and a carrier. The active pharmaceutical ingredient includes a composition of a hypoxia-inducible factor prolyl hydroxylase inhibitor and an anti-tuberculosis drug. Since the provided active pharmaceutical 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 mycobacterium tuberculosis interference. Furthermore, the drug has high safety and good efficacy.

[0046] The following description is based on specific embodiments.

[0047] Example 1

[0048] An experiment to stabilize hypoxia-inducible factor HIF-1α expression in THP-1 cells using a hypoxia-inducible factor prolyl hydroxylase inhibitor.

[0049] Hypoxia-inducible factor prolyl hydroxylase inhibitors are the latest small-molecule oral drugs used to treat chronic renal anemia in adults. They stabilize the expression level of hypoxia-inducible factor protein in vivo by inhibiting prolyl hydroxylase, thereby promoting the production and receptor expression of endogenous erythropoietin (EPO). To investigate the effect of hypoxia-inducible factor prolyl hydroxylase inhibitors on hypoxia-inducible factor HIF-1α expression in THP-1 cells, this example will conduct an in-depth study using cell biology and molecular biology techniques.

[0050] 1. Test materials

[0051] THP-1 cells (human monocytic leukemia cells) were purchased from ATCC (TIB-202) and cultured statically at 37°C in a 3% CO2 incubator using RPMI 1640 (10% FBS). Mycobacterium tuberculosis H37Rv strain was cultured in the Biosafety Level 3 Laboratory of the Eighth Affiliated Hospital of Guangzhou Medical University (Guangzhou Biosafety Level 3 Laboratory for Clinical Detection of Highly Pathogenic Infectious Diseases). In the initial stage of the experiment, H37Rv was inoculated into 7H9 liquid medium containing 10% OADC and shaken at 37°C and 110 rpm / min until the logarithmic growth phase. Other instruments and consumables, including 5×SDS lysis buffer, 8% SDS-PAGE pre-cast gel, 10× electrophoresis buffer, 10× transfer buffer, and the electrophoresis apparatus, were all domestically produced.

[0052] 2. Test reagents

[0053] Roxadustat (HY-13426), dapoxetine (HY-17608), ennadustat (HY-109057), valdulstat (HY-101277), and PMA (Phorbol 12-myristate 13-acetate, HY-18739) were all purchased from MCE. Hypoxia-inducible factor HIF-1α Rabbit mAb (catalog number: A22041) and β-ACTIN Rabbit mAb (AC038) were both purchased from ABclonal. OADC and 7H9 broth were purchased from BD.

[0054] Preparation of 7H9 medium (1L liquid medium):

[0055] 4.7g of 7H9 culture medium

[0056] Tween 80 2.5mL,

[0057] Dissolve in 900 mL ddH₂O and autoclave at 121 °C for 20 min. Cool and store at 4 °C for later use. Add OADC at a ratio of 10% when using.

[0058] 3. Experimental Methods

[0059] (1) Count the THP-1 cells and add 40 ng / mL PMA at a concentration of 5 × 10⁻⁶. 5 Seeds were placed into 12-well plates per well and induced to differentiate for 48 hours.

[0060] (2) Remove the culture medium and add 1 mL of 10% FBS RPMI 1640 medium to each well and continue culturing overnight.

[0061] (3) Take 1 mL of tuberculosis bacteria in the logarithmic growth phase into a 15 mL centrifuge tube, add 10 mL of PBS, centrifuge at 2700 rpm / min for 3 min, and resuspend in 1 mL of PBS. After measuring the OD value, dilute to RPMI 1640 (10% FBS) at an MOI of 5. At the same time, dilute various drugs to the corresponding concentrations, including roxadustat (100 uM), dapoxetine (50 uM), ennadustat (100 uM), and varduxatine (100 uM). Add 1 mL of culture medium to each well to infect THP-1 cells.

[0062] (4) After 24 hours of continuous infection, remove the culture medium, add 200 μL of 1×SDS loading buffer to each well to lyse the cells, and place them in a metal bath heater to boil at 100°C for 10 min, then centrifuge at 12000 rpm for 2 min.

[0063] (5) Take 8% precast gel and add 10 μL of sample or 1.5 μL of protein marker to each well. Add diluted 1× electrophoresis buffer and electrophoresis at 100V until the gel runs out of the loading buffer.

[0064] (6) Prepare a 1× transfer solution (methanol: 10× transfer solution: ddH2O = 2:1:7), and pre-activate the PVDF membrane by soaking it in methanol. Transfer at 100V for 100 min.

[0065] (7) After the transfer is completed, block with blocking solution prepared with 5% skim milk powder for 1 hour, add primary antibody dilution solution (prepared with 5% skim milk powder) diluted primary antibody, and incubate overnight on a shaker at 4°C.

[0066] (8) Wash with PBST three times the next day, 10 min each time.

[0067] (9) Immerse the PVDF membrane in a dilution buffer containing HRP-labeled secondary antibody (prepared with 5% skim milk powder) and incubate at room temperature on a shaker for 1 hour. Wash three times with PBST for 10 minutes each time.

[0068] (10) Chemiluminescence detection: Blot dry the washing solution with absorbent paper, lay the PVDF membrane flat on plastic wrap, and add the chemiluminescence substrate working solution. Place the membrane into the BIO-RAD ChemiDoc container. TM XRS+ chemiluminescence imager is used for continuous exposure development and photography.

[0069] 4. Experimental Results

[0070] Based on the above experimental steps, the experimental results are shown in Figure 1. We found that different hypoxia-inducible factor prolyl hydroxylase inhibitors could increase the expression of hypoxia-inducible factor HIF-1α in THP-1 cells in both uninfected and infected Mycobacterium tuberculosis conditions.

[0071] 5. Conclusion

[0072] Hypoxia-inducible factor prolyl hydroxylase inhibitors can stabilize the expression level of hypoxia-inducible factor HIF-1α protein in THP-1 cells.

[0073] Example 2

[0074] An experiment on the cytotoxicity of hypoxia-inducible factor prolyl hydroxylase inhibitors and their effect on promoting the clearance of tuberculosis bacteria by THP-1 macrophages.

[0075] In this embodiment, a lactate dehydrogenase (LDH) release assay was used to evaluate the cytotoxicity of hypoxia-inducible factor prolyl hydroxylase inhibitors; and colony-forming units (CFU) counting was used to evaluate the effect of hypoxia-inducible factor prolyl hydroxylase inhibitors on the ability of THP-1 cells to clear Mycobacterium tuberculosis.

[0076] 1. Experimental Materials

[0077] The culture of THP-1 cells and the culture of the virulent strain H37Rv of Mycobacterium tuberculosis are the same as in Example 1.

[0078] 2. Experimental reagents

[0079] LDH toxicity test kit (catalog number: C0017) was purchased from Beyotime International Co., Ltd. 7H10 agar medium was purchased from BD Biosciences.

[0080] Preparation of 7H10 agar plates (1L solid culture medium):

[0081] 21g of 7H10 culture medium

[0082] 5 mL of glycerol

[0083] L-Asparagine 1g,

[0084] Dissolve the culture medium completely in 900 mL ddH2O and autoclave at 121 °C for 8 min. Remove the culture medium and place it in a 56 °C incubator for at least 30 min. Then, incubate the OADC, which has been equilibrated to room temperature, in a 56 °C incubator for 5 min. After that, add 100 mL of OADC to the culture medium, mix well, and pour into a petri dish with 4.5 mL in each of the four quarters. Place the dish on a ventilated aseptic work surface for 4 h, wrap it in aluminum foil, and incubate it overnight in a 37 °C incubator. After confirming that there is no contamination, it can be stored at room temperature for no more than 1 month for later use.

[0085] 3. Experimental Methods

[0086] THP-1 cytotoxicity and CFU count

[0087] ① THP-1 cells were plated and induced to differentiate in the same manner as in Example 1. Mycobacterium tuberculosis was centrifuged, resuspended, and infected with THP-1 cells at MOI=1.

[0088] ② Six hours after infection, discard the culture supernatant, wash away bacteria that have not been phagocytosed by macrophages with 1 ml PBS, and add 1 ml of 1640 complete culture medium containing 100 uM roxadustat, 100 uM varduxat and 50 uM dapoxetine respectively, and incubate statically in a 37°C, 5% CO2 incubator.

[0089] ③ After culturing for 72 hours, collect the supernatant of the replicate medium, centrifuge at 400g / min, and measure the LDH release amount according to the instructions.

[0090] ④ Wash the cells twice with preheated PBS, add 0.1% SDS (prepared with PBS) to lyse them, aspirate them, and serially dilute them with PBS at a 10-fold ratio. Take 50 μL of the bacterial culture for each concentration, spread it on 7H10 plates, and incubate them in a constant temperature incubator at 37℃. Perform CFU counting after 2-3 weeks.

[0091] 4. Experimental Results

[0092] Based on the above experimental steps, the experimental results are shown in Figure 2. We found that roxadustat, dapoxetine, and varduxetine can enhance the ability of THP-1 cells to clear tuberculosis bacteria, and there is no cytotoxicity at the current experimental concentration.

[0093] 5. Conclusion

[0094] Hypoxia-inducible factor prolyl hydroxylase inhibitors effectively enhance the function of macrophages in clearing Mycobacterium tuberculosis.

[0095] Example 3

[0096] Effects of hypoxia-inducible factor prolyl hydroxylase inhibitor combined with isoniazid on heme and erythropoiesis in Mycobacterium tuberculosis-infected mice

[0097] In this embodiment, a wild-type mouse model of Mycobacterium tuberculosis infection was constructed, and hypoxia-inducible factor prolyl hydroxylase inhibitor and isoniazid were continuously administered by gavage for two weeks. The effects of hypoxia-inducible factor prolyl hydroxylase inhibitor on reversing the number of red blood cells and heme concentration in mice were observed.

[0098] 1. Experimental Materials

[0099] Female C57BL / 6 mice aged 8-10 weeks were housed in the Biosafety Level 3 laboratory of the Eighth People's Hospital of Guangzhou Medical University (Guangzhou Biosafety Level 3 Laboratory for Clinical Detection of Highly Pathogenic Infectious Diseases). The culture of the virulent strain H37Rv of Mycobacterium tuberculosis was the same as in Example 1.

[0100] 2. Experimental Methods

[0101] (1) Infection and administration to mice

[0102] C57BL / 6 mice were randomly divided into four groups: control group, isoniazid group, roxadustat group, and roxadustat combined with isoniazid group, with 8-9 mice in each group. These four groups of mice were infected with Mycobacterium tuberculosis (H37Rv) at an infectious dose of 200 CFU / mouse. Four weeks after infection, the treatment groups received isoniazid and roxadustat dissolved in PBS via gavage daily. The dose of roxadustat was 30 mg / kg, and the dose of isoniazid was 10 mg / kg. The control group received an equal volume of DMSO.

[0103] (2) Orbital blood sampling and routine blood test

[0104] Fourteen days after drug administration, 200 μL of orbital blood was collected from mice and mixed in an EDTA-anticoagulated vessel by inversion. A complete blood count was performed using a Mindray BC-7500CS hematology analyzer. With the instrument in the ready state (indicated by a solid green indicator light), the sample was placed in the probe solution tube, and the sample aspiration button was pressed to start the analysis process. The analyzer automatically performed the sample analysis. The measurement results were recorded as follows: Red blood cell count (RBC, 102). 12 (g / L), hemoglobin (Hb, g / L), hematocrit (HCT, %), and mean corpuscular volume (MCV, fl).

[0105] 3. Experimental Results

[0106] Based on the above experimental procedures, the experimental results are shown in Figure 3. Administration of hypoxia-inducible factor prolyl hydroxylase inhibitors significantly increased the red blood cell count (RBC, 102) in tuberculosis-infected mice. 12 The levels of hemoglobin (Hb, g / L), hematocrit (HCT, %), and mean corpuscular volume (MCV, fl).

[0107] 4. Conclusion

[0108] Hypoxia-inducible factor prolyl hydroxylase inhibitors can effectively reverse heme concentration and erythrocyte production in mice infected with Mycobacterium tuberculosis.

[0109] Example 4

[0110] Effects of hypoxia-inducible factor prolyl hydroxylase inhibitor combined with isoniazid on lung pathological damage in Mycobacterium tuberculosis infected mice

[0111] In this embodiment, the same as in Example 3, a mouse model of Mycobacterium tuberculosis infection was constructed and administered the drug to investigate the effect of hypoxia-inducible factor prolyl hydroxylase inhibitor on lung pathology in mice infected with Mycobacterium tuberculosis.

[0112] 1. Test materials

[0113] Same as Example 3

[0114] 2. Experimental Methods

[0115] (1) Mouse infection and administration

[0116] Same as Example 3.

[0117] (2) Take tissue

[0118] After euthanizing the mice, lung tissue was removed for pathological sectioning. The relevant steps are as follows:

[0119] One lobe of a mouse lung was taken, fixed in 10% formaldehyde for 24 hours, and routinely embedded in paraffin, sectioned, and stained with hematoxylin and eosin (HE) to examine the pathological damage of the lung.

[0120] 3. Experimental Results

[0121] Based on the above experimental steps, the results are shown in Figure 4. The lung pathological damage in mice treated with hypoxia-inducible factor prolyl hydroxylase inhibitors in combination with isoniazid was significantly reduced.

[0122] 4. Conclusion

[0123] Hypoxia-inducible factor prolyl hydroxylase inhibitors, when combined with isoniazid, can effectively reduce lung pathological damage in mice infected with Mycobacterium tuberculosis.

[0124] Example 5

[0125] Effects of hypoxia-inducible factor prolyl hydroxylase inhibitor combined with isoniazid on bacterial load in the lungs of Mycobacterium tuberculosis-infected mice

[0126] In this embodiment, a mouse model of Mycobacterium tuberculosis infection was constructed and administered the drug as in Example 3, to study the effect of hypoxia-inducible factor prolyl hydroxylase inhibitor combined with isoniazid on the bacterial load in the lungs of mice infected with Mycobacterium tuberculosis.

[0127] 1. Test materials

[0128] Same as Example 3.

[0129] 2. Experimental Methods

[0130] (1) Mouse infection and administration

[0131] Same as Example 3.

[0132] (2) Mouse lung tuberculosis CFU test

[0133] One-third of mouse lung tissue was taken and homogenized with 5 mL PBS. Serial dilutions were performed, with each concentration diluted 10-fold with PBS. 100 μL of the bacterial culture was plated onto 7H10 plates and incubated at 37°C. CFU counts were performed after 2-3 weeks.

[0134] 3. Experimental Results

[0135] Based on the above experimental steps, the experimental results are shown in Figure 5. The combination of hypoxia-inducible factor prolyl hydroxylase inhibitor and isoniazid significantly reduced the tuberculosis load in the lungs of mice.

[0136] 4. Conclusion

[0137] As can be seen from the above examples, the combination of hypoxia-inducible factor prolyl hydroxylase inhibitor and isoniazid can effectively reduce the degree of pathological damage to lung tissue in mice infected with Mycobacterium tuberculosis and reduce the viral load of Mycobacterium tuberculosis in lung tissue, thereby providing effective drug support for the treatment of tuberculosis.

[0138] In summary, the composition of hypoxia-inducible factor prolyl hydroxylase inhibitor and anti-tuberculosis drug provided in this application embodiment has an effective synergistic effect in the treatment of anemia and anti-infection. Therefore, when this composition is applied to the preparation of drugs or health products for the prevention and treatment of chronic inflammatory anemia and infection, the resulting drugs or health products can improve chronic inflammatory anemia caused by Mycobacterium tuberculosis interference and can be used to treat tuberculosis, with a wide range of application effects.

[0139] The above are merely optional embodiments of this application and are not intended to limit this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

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

2. The use of the composition of the hypoxia-inducible factor prolyl hydroxylase inhibitor and the anti-tuberculosis drug as described in claim 1 in the preparation of a drug or health product for the prevention and treatment of chronic inflammatory anemia and infection.

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

4. 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. Use according to claim 2, characterized in that, The dosage forms of the health products include any one of the following: tablets, capsules, pills, powders, granules, syrups, solutions, emulsions, injections, sprays, aerosols, gels, and creams.

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

7. Use according to claim 3, characterized in that, The drug or health product also includes a carrier.

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

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

10. A medicament for preventing and treating chronic inflammatory anemia and infection, characterized by comprising the compound according to claim 1. The drug comprises an active pharmaceutical ingredient and a carrier, wherein the active pharmaceutical ingredient comprises a composition of the hypoxia-inducible factor prolyl hydroxylase inhibitor and an anti-tuberculosis drug as described in claim 1.

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