Application of APX-115 free base in the fight against Mycobacterium tuberculosis infection

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of APX-115 free base in anti-mycobacterium tuberculosis infection. The application finds that APX-115 free base has a biological effect in anti-mycobacterium tuberculosis infection, the MIC of APX-115 free base to a standard strain of mycobacterium tuberculosis can reach 2.5 muM, and the MIC distribution of APX-115 free base to a drug-resistant mycobacterium tuberculosis clinical isolate is 5 muM-10 muM. In conclusion, in anti-mycobacterium tuberculosis infection, APX-115 free base can become a new treatment drug. The application has guiding significance for clinic, and APX-115 free base is simple to obtain, low in cost, fast in effect when made into a drug, and has a practical production application prospect. APX-115 free base is high in safety, small in side effect, and good in patient compliance.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of APX-115 free base in anti-Mycobacterium tuberculosis infection. BACKGROUND

[0002] Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (M.tb) infection. According to the report released by the World Health Organization (WHO), there were 108 million new TB patients worldwide in 2023, 8.2 million confirmed patients, and 1.25 million deaths. The number of multidrug-resistant TB patients is growing rapidly, with 400,000 new patients with multidrug-resistant / rifampicin-resistant TB (MDR / RR-TB) in 2023. The incidence of TB is not optimistic (WHO. The Global Tuberculosis Report [J]. 2024.).

[0003] The classic tuberculosis treatment strategy is anti-tuberculosis chemotherapy targeting pathogens, but antibiotic resistance has become a major obstacle to responding to the global public health crisis, which has promoted the development of new therapies. In 2019, WHO recommended bedaquiline (BDQ) and clofazimine (CFZ) as group A and group B drugs for the treatment of MDR / RR-TB. Studies have shown that BDQ treatment of M.tb and extensively drug-resistant tuberculosis (XDR-TB) helps improve the negative conversion rate of sputum smears and sputum cultures, has a high success rate, good safety, and promotes the absorption of imaging lesions in patients (KOIRALA S, BORISOV S, DANILA E, et al. Outcome of treatment of MDR-TB or drug-resistant patients treated with bedaquiline and delamanid: Results from a large global cohort [J]. Pulmonology, 2021, 27 (5): 403-412.). Treatment regimens containing CFZ can also significantly shorten the treatment time required to cure drug-sensitive tuberculosis, significantly improve the sputum negative conversion rate and lesion absorption rate of XDR-TB patients (DUAN H, CHEN X, LI Z, et al. Clofazimine improves clinical outcomes in multidrug-resistant tuberculosis: a randomized controlled trial[J]. ClinMicrobiol Infect, 2019, 25(2): 190-195.), and reduce patients' adverse reactions, with good safety and tolerability (Wan Qiu, Yang Fuping, Tang Lixin. Efficacy and safety analysis of clofazimine-containing combination regimens for the treatment of multidrug-resistant tuberculosis[J / CD]. Chinese Journal of Lung Diseases (Electronic Edition), 2023, 16(2): 275-277.). Despite the development of new drugs such as BDQ, CFZ, and pretomanid (PA-824), the treatment of tuberculosis, particularly multidrug-resistant tuberculosis (MDR-TB), still requires months of treatment and is associated with adverse drug reactions, undoubtedly increasing the burden on patients. Tuberculosis drug resistance poses a significant threat to human health worldwide, and the identification of new drug targets is urgently needed to assist in the clinical treatment of drug-resistant TB.

[0004] APX-115 free base (Ewha-18278 free base) is an effective, orally active, non-selective Nox inhibitor developed by Aptabio, mainly used for the treatment of Diabetic Kidney Disease (DKD). APX-115 free base can effectively prevent kidney damage in diabetic mice. At present, there is no relevant report on the anti-tuberculosis infection of APX-115 free base drug molecules. SUMMARY

[0005] The present application finds that APX-115 free base has the effect of inhibiting the activity of Mycobacterium tuberculosis, and based on this, the present application is completed.

[0006] In a first aspect, the present application provides a cleaning agent, which comprises APX-115 free base.

[0007] Further, the cleaning agent inhibits the activity of Mycobacterium tuberculosis.

[0008] Still further, the Mycobacterium tuberculosis comprises Mycobacterium tuberculosis standard strains and / or Mycobacterium tuberculosis clinical isolates.

[0009] In a second aspect, the present application provides an anti-Mycobacterium tuberculosis infection drug, which comprises APX-115 free base.

[0010] Further, the Mycobacterium tuberculosis comprises Mycobacterium tuberculosis standard strains and / or Mycobacterium tuberculosis clinical isolates.

[0011] Further, one or more pharmaceutically acceptable carriers or excipients can be added to the drug.

[0012] Still further, the carrier material comprises one or more of water-soluble carrier materials, poorly soluble carrier materials, and / or enteric carrier materials, but is not limited to them.

[0013] Further, the water-soluble carrier material comprises one or more of polyethylene glycol, polyvinylpyrrolidone, and / or organic acid, but is not limited to them.

[0014] Further, the poorly soluble carrier material comprises one or more of ethyl cellulose and / or cholesteryl stearate, but is not limited to them.

[0015] Further, the enteric carrier material comprises one or more of cellulose acetate phthalate and / or carboxymethyl cellulose, but is not limited to them.

[0016] Further, the medicament can be prepared in various dosage forms, including but not limited to one or more of tablets, capsules, dripping pills, aerosols, pills, powders, solutions, suspensions, granules, liposomes, transdermal preparations, buccal tablets, suppositories, and / or lyophilized powder injections.

[0017] Further, the preparation can be one or more of ordinary preparations, sustained-release preparations, controlled-release preparations, and / or various microparticle drug delivery systems.

[0018] Further, the tablets can widely use various carriers known in the art, including one or more of diluents and absorbents, wetting agents and binders, disintegrants, disintegration inhibitors, absorption promoters, and / or lubricants.

[0019] Further, the diluents and absorbents include but are not limited to one or more of starch, dextrin, calcium sulfate, lactose, mannitol, sucrose, sodium chloride, glucose, urea, calcium carbonate, kaolin, microcrystalline cellulose, and / or aluminum silicate.

[0020] Further, the wetting agents and binders include but are not limited to one or more of water, glycerol, polyethylene glycol, ethanol, propanol, starch paste, dextrin, sugar syrup, honey, glucose solution, acacia paste, gelatin paste, sodium carboxymethyl cellulose, shellac, methyl cellulose, potassium phosphate, and / or polyvinylpyrrolidone.

[0021] Further, the disintegrants include but are not limited to one or more of dry starch, alginate, agar powder, alginic acid, sodium bicarbonate and citric acid, calcium carbonate, polyoxyethylene, sorbitol fatty acid ester, sodium lauryl sulfate, methyl cellulose, and / or ethyl cellulose.

[0022] Further, the disintegration inhibitors include but are not limited to one or more of sucrose, glycerol triestearate, cocoa butter, and / or hydrogenated oil, etc.

[0023] Further, the absorption promoters include but are not limited to one or more of quaternary ammonium salts and / or sodium lauryl sulfate.

[0024] Further, the lubricants include but are not limited to one or more of talc, silicon dioxide, corn starch, stearate, boric acid, liquid paraffin, and / or polyethylene glycol.

[0025] Further, the tablets can also be further prepared into coated tablets, including sugar-coated tablets, film-coated tablets, enteric-coated tablets, double-layer tablets, and multi-layer tablets.

[0026] Further, the pills can widely use various carriers known in the art, including diluents and absorbents, binders, and / or disintegrants.

[0027] Still further, the diluents and absorbents include, but are not limited to, one or more of dextrose, lactose, starch, cocoa butter, hydrogenated vegetable oil, polyvinylpyrrolidone, Gelucire, kaolin, and / or talc.

[0028] Still further, the binders include, but are not limited to, one or more of acacia, tragacanth, gelatin, alcohol, honey, sirup, rice paste, and / or gruel.

[0029] Still further, the disintegrants include, but are not limited to, one or more of agar powder, dried starch, alginic acid, sodium lauryl sulfate, methyl cellulose, and / or ethyl cellulose.

[0030] Still further, the suppositories can employ as a carrier any of the various carriers known in the art, including, but not limited to, one or more of polyethylene glycol, lecithin, cocoa butter, higher alcohols, esters of higher alcohols, gelatin, and / or semi-synthetic glycerides.

[0031] Still further, the injectable formulations include, but are not limited to, one or more of solutions, lyophilized powders, and / or suspensions.

[0032] Still further, the injectable formulations can employ as diluents all of the diluents commonly used in the art, including, but not limited to, one or more of water, ethanol, polyethylene glycol, 1,3-propanediol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, and / or polyoxyethylene sorbitol fatty acid esters.

[0033] Still further, the injectable formulations can be made isotonic for injection by adding to the injectable formulations an appropriate amount of one or more of sodium chloride, dextrose, glycerin, conventional solubilizing agents, buffers, and / or pH adjusting agents.

[0034] Still further, the various formulations can also include, if desired, coloring agents, preservatives, flavoring agents, flavor correctants, sweetening agents, or other materials.

[0035] In a third aspect, the present application provides the use of APX-115 free base in the manufacture of a medicament for the treatment of Mycobacterium tuberculosis infection, said medicament comprising at least one of the following uses:

[0036] the treatment of Mycobacterium tuberculosis infection;

[0037] the prevention and / or treatment of tuberculosis.

[0038] Still further, the Mycobacterium tuberculosis includes Mycobacterium tuberculosis standard strains and / or Mycobacterium tuberculosis clinical isolates.

[0039] Still further, the medicament for the treatment of Mycobacterium tuberculosis infection can also include one or more pharmaceutically acceptable carriers or excipients.

[0040] Further, the carrier is selected from a water-soluble carrier, a poorly water-soluble carrier or an enteric carrier.

[0041] Further, the dosage form of the drug against Mycobacterium tuberculosis infection is selected from a tablet, a capsule, an aerosol, a pill, a powder, a solution, a suspension, a granule, a liposome, a transdermal agent, a suppository or a lyophilized powder injection.

[0042] Further, the drug against Mycobacterium tuberculosis infection is selected from one or more of a general preparation, a sustained-release preparation, a controlled-release preparation and / or various microparticle drug delivery systems.

[0043] Further, the various preparations can also add a coloring agent, a preservative, a flavoring agent, a sweetener or other materials to the drug preparation, if necessary.

[0044] Further, the drug against Mycobacterium tuberculosis infection can be introduced into the body such as muscle, intradermal, subcutaneous, intravenous or mucosal tissue by injection, penetration, absorption, physical or chemical mediated methods; or is mixed or wrapped with other substances and introduced into the body.

[0045] Beneficial effects

[0046] The present application finds that APX-115 free base has a biological effect against Mycobacterium tuberculosis infection, and the MIC of APX-115 free base against Mycobacterium tuberculosis standard strains can reach 2.5 μM, and the MIC distribution of APX-115 free base against drug-resistant Mycobacterium tuberculosis clinical isolates is 5 μM-10 μM. In summary, APX-115 free base can become a new therapeutic drug against Mycobacterium tuberculosis infection. It has guiding significance for clinical use; and APX-115 free base is easy to obtain, low in cost, fast in effect after being made into a drug, and has practical production and application prospects. APX-115 free base is high in safety, small in side effects, and good in patient compliance. BRIEF DESCRIPTION OF DRAWINGS

[0047] Figure 1 Figure 2 is the result of the antibacterial activity detection of APX-115 free base against multi-drug resistant Mycobacterium tuberculosis clinical isolates.

[0048] Figure 2 Figure 4 is the result of the cytotoxicity experiment of APX-115 free base on THP-1 cells. DETAILED DESCRIPTION

[0049] The specific embodiments of the present application will be further described below. It should be noted that the description of these embodiments is intended for the purpose of aiding in the understanding of the present application and is not intended to be limiting of the present application. Moreover, the technical features involved in the embodiments described below can be combined with each other as long as there is no conflict.

[0050] The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all commercially available unless otherwise specified.

[0051] Terminology

[0052] The "Mycobacterium tuberculosis standard strain" mentioned in the present application is H37Rv (ATCC 27294) purchased from the American Type Culture Collection (ATCC).

[0053] The "human blood-derived monocyte THP1 cell line" mentioned in the present application is purchased from the Chinese Academy of Sciences Cell Bank.

[0054] The "APX-115 free base" mentioned in the present application has the molecular formula of C 17 H 17 N3O; a molecular weight of 279.34 g / mol, and a CAS number of 1270084-92-8, and has the following structural formula:

[0055]

[0056] Example 1: Detection of the antibacterial activity of the Mycobacterium tuberculosis standard strain

[0057] A. Preparation of Mycobacterium tuberculosis standard strain suspension

[0058] Mycobacterium tuberculosis (ATCC 27294) was cultured to the logarithmic phase, and the logarithmic phase strain was scraped and ground uniformly in a grinding bottle. The bacterial suspension was diluted with 7H9 culture medium and turbidimetrically to 1 McFarland, and then added to 7H9 liquid culture medium at a ratio of 1:20 and mixed uniformly to obtain the Mycobacterium tuberculosis suspension.

[0059] B. Minimum inhibitory concentration (MIC)

[0060] (1) 7H9 liquid culture medium was added, and then Mycobacterium tuberculosis suspension was added, so that the final volume in each well was 200 μl, and the final concentration of the bacterial solution was 4 x 10 5 CFU / mL. The final drug concentration in each well is shown in Table 1.

[0061] (2) The culture well plate was incubated for 7 days.

[0062] (3) Add color developing solution and observe the color change of the well plate;

[0063] (4) Read the Minimal inhibitory concentration (MIC).

[0064] Table 1. Drug concentration of APX-115 free base

[0065] 1 2 3 4 5 6 7 8 9 10 11 12 A Negative Positive 40 μM 20 μM 10 μM 5 μM 2.5 μM 1.25 μM 0.625 μM 0.3125 μM 0.1563 μM 0.078 μM B Negative Positive 40 μM 20 μM 10 μM 5 μM 2.5 μM 1.25 μM 0.625 μM 0.3125 μM 0.1563 μM 0.078 μM C Negative Positive 40 μM 20 μM 10 μM 5 μM 2.5 μM 1.25 μM 0.625 μM 0.3125 μM 0.1563 μM 0.078 μM D Negative Positive 40 μM 20 μM 10 μM 5 μM 2.5 μM 1.25 μM 0.625 μM 0.3125 μM 0.1563 μM 0.078 μM E Negative Positive 40 μM 20 μM 10 μM 5 μM 2.5 μM 1.25 μM 0.625 μM 0.3125 μM 0.1563 μM 0.078 μM F Negative Positive 40 μM 20 μM 10 μM 5 μM 2.5 μM 1.25 μM 0.625 μM 0.3125 μM 0.1563 μM 0.078 μM G Negative Positive 40 μM 20 μM 10 μM 5 μM 2.5 μM 1.25 μM 0.625 μM 0.3125 μM 0.1563 μM 0.078 μM H Negative Positive 40 μM 20 μM 10 μM 5 μM 2.5 μM 1.25 μM 0.625 μM 0.3125 μM 0.1563 μM 0.078 μM

[0066] MIC reading method: MIC is the drug concentration that can inhibit 90% of the colony growth. Blue wells are sterile growth, pink wells are bacterial growth, the lowest drug concentration that prevents the color change from blue to pink is the MIC that can inhibit bacterial growth.

[0067] The negative control is the medium without drug and bacteria, i.e. column 1. The positive control is the medium with bacteria without drug, i.e. column 2; when the positive control is pink and the negative control is blue, the MIC data of this batch is valid.

[0068] C. Test results

[0069] The MIC of APX-115 free base against the standard strain of Mycobacterium tuberculosis is 2.5 μM.

[0070] Example 2. In vitro bacteriostatic activity test of clinical isolates of multi-drug resistant Mycobacterium tuberculosis strains

[0071] A. Test method

[0072] According to the method in Example 1, the in vitro bacteriostatic activity of APX-115 free base against 12 clinical isolates of Mycobacterium tuberculosis was tested. The final drug concentration in each well is shown in Table 2.

[0073] The activity of APX-115 free base against clinical isolates of multi-drug resistant strains, i.e. MDR-1, MDR-2, MDR-3, MDR-4, MDR-5, MDR-6, MDR-7, MDR-8, MDR-9, MDR-10, MDR-11 and MDR-12, was determined by the well plate micro-dilution method.

[0074] Table 2. Distribution of APX-115 free base concentrations in 96 well plates

[0075]

[0076] B. Test results

[0077] As shown in Table 3 and Figure 1As shown, the MIC results of APX-115 free base against 12 clinical multi-drug resistant strains were 5 uM-10 uM. APX-115 free base had a good inhibitory effect on Mycobacterium tuberculosis in vitro.

[0078] Table 3. MIC results of APX-115 free base against 12 clinical multi-drug resistant strains

[0079]

[0080]

[0081] Example 3. APX-115 free base inhibits Mycobacterium tuberculosis cytotoxicity test

[0082] A. Test method

[0083] (1) THP1 cells were cultured in RPMI 1640 complete medium, and passaged every 2 days;

[0084] (2) Take well-grown cells, add PMA, inoculate 1 x 10 5 cells / well and culture for 24 hours to induce adherent macrophages;

[0085] (3) Discard the old culture medium, add culture medium to the culture plate, and dilute the final concentration of APX-115 free base to 40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM; the negative control wells do not contain drugs, and the cell plates are incubated;

[0086] (4) Then discard the culture medium, follow the CCK-8 cell proliferation / cytotoxicity kit instructions to detect the cytotoxicity of the drug;

[0087] (5) Calculate the survival rate (%) of THP1 cells after stimulation by APX-115 free base = (OD450nm [test group]-OD450nm [negative control group] / OD450nm [positive control group]-OD450nm [negative control group]) x 100%.

[0088] B. Test results

[0089] The experimental results showed that APX-115 free base had low cytotoxicity to THP-1 cells. When the drug concentration was 20 uM, the survival rate of THP-1 cells was 96±1.63% and 105±2.83% after 24 hours and 48 hours of treatment, respectively (see Figure 2 ).

[0090] Based on the experimental results, it is shown that the APX-115 free base has good antibacterial activity against Mycobacterium tuberculosis and can significantly inhibit Mycobacterium tuberculosis. In addition, the APX-115 free base has high THP-1 cell survival rate. Therefore, the APX-115 free base can be used as a drug for preparing an anti-Mycobacterium tuberculosis drug.

[0091] It is found through a series of tests that not all non-selective Nox inhibitors, diabetes drugs (such as insulin, metformin, etc.) and similar compounds have an effect on Mycobacterium tuberculosis.

Claims

1. Use of APX-115 free base in the preparation of a drug for treating Mycobacterium tuberculosis infection, wherein the drug comprises at least one of the following uses: Against Mycobacterium tuberculosis infection; Prevent and / or treat tuberculosis.

2. The use according to claim 1, characterized in that The Mycobacterium tuberculosis includes Mycobacterium tuberculosis standard strains and / or Mycobacterium tuberculosis clinical isolates.

3. The use according to claim 1, characterized in that The drug for resisting Mycobacterium tuberculosis infection is introduced into the body through physical or chemical methods, such as into muscle, intradermal, subcutaneous, intravenous or mucosal tissues; or is mixed or wrapped with other substances and then introduced into the body.

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