Use of sulfaclozine in the treatment of mycobacterium tuberculosis infection

Abstract

The application belongs to the technical field of biological medicine, and particularly relates to application of Sulfaclozine and salts thereof in anti-mycobacterium tuberculosis infection. The application finds that Sulfaclozine has good bacteriostatic activity on standard strains of mycobacterium tuberculosis and multi-drug resistant mycobacterium tuberculosis clinical isolates, and has good safety within a concentration of 40 uM, and is expected to become a new drug for anti-mycobacterium tuberculosis infection.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of Sulfaclozine and salts thereof in anti-Mycobacterium tuberculosis infection. BACKGROUND

[0002] Mycobacterium tuberculosis (Mtb) is the pathogen causing Tuberculosis (TB), belongs to the genus Mycobacterium, and is an acid-fast obligate aerobic bacillus. It mainly spreads through the respiratory tract and can invade multiple tissues and organs of the body, such as the lungs, lymph nodes, bones, and kidneys, and cause TB in the corresponding parts. Pulmonary tuberculosis is the main form of manifestation, and patients often have symptoms such as cough, hemoptysis, chest pain, and dyspnea after developing pulmonary tuberculosis. In addition to pulmonary tuberculosis, patients can also have extrapulmonary infections, which can or can not be accompanied by pulmonary symptoms, and the most common form is lymphadenitis.

[0003] Tuberculosis is an ancient disease, and human cases have been recorded as early as 1700 BC. It remains one of the major challenges to global public health. Epidemiological data shows that about one-quarter of the world's population is infected with active or latent tuberculosis, of which about 56 million people are newly infected and have a high risk of disease progression. Mycobacterium tuberculosis can remain dormant in the host's body for a long time without any signs of disease, and many people become asymptomatic carriers (non-active tuberculosis) in this case. However, when the host's immune function is impaired, such as combined human immunodeficiency virus (HIV) infection, the latent pathogen can be reactivated and cause active tuberculosis. In addition to maintaining a subclinical infection state for a long time, Mycobacterium tuberculosis also constantly adapts and changes during co-evolution with the host, with strong tolerance and host adaptation ability. At the same time, due to its slow growth characteristics and latent infection, the treatment period of tuberculosis is relatively long. These unique biological characteristics and complex adaptive mechanisms pose great challenges to the epidemiological prevention and control and clinical treatment of tuberculosis. Currently, there are mainly the following categories of drugs for treating tuberculosis, such as first-line oral antibiotics represented by isoniazid and rifampicin, second-line injectable drugs represented by kanamycin and amikacin, fluoroquinolone drugs represented by ofloxacin and moxifloxacin, and new drugs represented by linezolid and bedaquiline. However, due to the continuous emergence of drug resistance, some multi-drug resistant Mycobacterium tuberculosis strains have resistance to most of the above drugs, so it is urgent to develop new anti-tuberculosis drugs to cope with drug-resistant tuberculosis.

[0004] Sulfaclozine (SCZ), a sulfonamide derivative, is an anti-inflammatory and antibacterial drug for poultry, mainly used for the treatment of coccidiosis (caecal coccidiosis), chicken cholera and typhoid fever, etc. in chickens, rabbits and sheep. There are multiple potential metal ion coordination sites in the molecule of SCZ. The amide group in the molecule is connected with the chloropyrazine aromatic ring, and the chloropyrazine ring structure can significantly enhance the biological activity characteristics of the drug compared with the traditional sulfonamide group. Sulfachloropyrazine sodium is a salt of Sulfaclozine, which has antibacterial and anticoccidial effects. It is commonly used for the study of various poultry diseases (especially colibacillosis, avian cholera and coccidiosis). Currently, there is no report on the inhibition of Mycobacterium tuberculosis by Sulfaclozine. SUMMARY

[0005] The present application finds that Sulfaclozine has the effect of inhibiting Mycobacterium tuberculosis, and accordingly completes the present application.

[0006] In a first aspect, the present application provides the use of Sulfaclozine or a salt thereof in the preparation of a product for inhibiting Mycobacterium tuberculosis.

[0007] Further, the Sulfaclozine salt is Sulfachloropyrazine sodium.

[0008] Further, the Mycobacterium tuberculosis includes Mycobacterium tuberculosis standard strain, Mycobacterium tuberculosis clinical isolate or Mycobacterium tuberculosis carried by patients infected with Mycobacterium tuberculosis.

[0009] Further, the product for inhibiting Mycobacterium tuberculosis includes a medical product or a non-medical product.

[0010] Further, the medical product refers to a medical drug or a pharmaceutical excipient, and the non-medical product refers to an experimental reagent or a bacteriostatic agent.

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

[0012] Further, the carrier material includes one or more of water-soluble carrier material, poorly soluble carrier material and / or enteric carrier material.

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

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

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

[0016] In a second aspect, the present application provides a use of Sulfaclozine or a salt thereof in the preparation of a medicament for preventing and / or treating a disease caused by Mycobacterium tuberculosis infection, wherein the Sulfaclozine and the salt thereof exert their effects by inhibiting the activity of Mycobacterium tuberculosis.

[0017] Further, the Sulfaclozine salt is Sulfaclozine sodium.

[0018] Further, the Mycobacterium tuberculosis includes Mycobacterium tuberculosis standard strain, Mycobacterium tuberculosis clinical isolate, or Mycobacterium tuberculosis carried by a patient infected with Mycobacterium tuberculosis.

[0019] Further, one or more pharmaceutically acceptable carriers or excipients can be added to the medicament.

[0020] Further, the carrier material includes one or more of water-soluble carrier material, poorly soluble carrier material, and / or enteric carrier material.

[0021] Further, the medicament can be prepared into various dosage forms, including but not limited to one or more of tablets, capsules, aerosols, pills, powders, solutions, suspensions, granules, liposomes, transdermal preparations, and / or suppositories.

[0022] Further, the preparation can be one or more of ordinary preparation, sustained-release preparation, controlled-release preparation, and / or various microparticle drug delivery systems.

[0023] Further, the various preparations can also add coloring agents, preservatives, fragrances, flavoring agents, sweeteners, or other materials to the pharmaceutical preparation, if necessary.

[0024] Further, the medicament is introduced into the body, such as muscle, intradermal, subcutaneous, intravenous, or mucosal tissue, by injection, penetration, absorption, physical or chemical mediation; or is introduced into the body after being mixed or wrapped with other substances.

[0025] In a third aspect, the present application provides a pharmaceutical composition comprising Sulfaclozine or a salt thereof, and another drug against Mycobacterium tuberculosis infection, wherein the pharmaceutical composition has at least one of the following effects:

[0026] a) inhibiting the activity of Mycobacterium tuberculosis;

[0027] b) resisting Mycobacterium tuberculosis infection;

[0028] c) preventing and / or treating a disease caused by M. tuberculosis.

[0029] Further, the Sulfaclozine salt is Sulfaclozine sodium.

[0030] Further, the M. tuberculosis includes M. tuberculosis standard strain, M. tuberculosis clinical isolates or M. tuberculosis carried by patients infected with M. tuberculosis.

[0031] Further, the another anti-M. tuberculosis infection drug includes one or more of antibiotics and other drugs capable of helping to inhibit or kill M. tuberculosis or provide resistance to patients.

[0032] Further, the antibiotic includes one or more of rifampicin, streptomycin, ethambutol, moxifloxacin, clarithromycin and / or amikacin; and the other drug includes one or more of vitamins, amino acids, proteins and / or minerals.

[0033] Further, one or more pharmaceutically acceptable carriers can be added to the pharmaceutical composition.

[0034] Further, the pharmaceutical composition can be prepared into one or more of tablets, capsules, aerosols, pills, powders, solutions, suspensions, granules, liposomes, transdermal preparations and / or suppositories.

[0035] Further, the pharmaceutical composition 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 mixed or wrapped with other substances and introduced into the body.

[0036] Advantages

[0037] Sulfaclozine sodium has good bacteriostatic activity on M. tuberculosis standard strain and multi-drug resistant tuberculosis clinical isolates, and the MIC of Sulfaclozine sodium on M. tuberculosis standard strain can reach 10 μM, and the MIC distribution of Sulfaclozine sodium on multi-drug resistant tuberculosis clinical isolates is 1.25-20 μM.

[0038] Sulfaclozine sodium has no obvious toxicity, and is expected to become a new anti-M. tuberculosis infection drug. BRIEF DESCRIPTION OF DRAWINGS

[0039] Figure 1 Survival rate of THP-1 cells treated with Sulfaclozine sodium. DETAILED DESCRIPTION

[0040] The specific embodiments of the present application are further described below. It is to be understood that the description of these embodiments is intended to help understand the present application and is not intended to limit the present application. In addition, the technical features involved in the following described embodiments can be combined with each other as long as they do not conflict with each other.

[0041] The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all commercially available unless otherwise specified.

[0042] Material Description

[0043] 1. Test strains and drugs

[0044] Mycobacterium tuberculosis standard strain H37Rv: ATCC 27294;

[0045] Mycobacterium fortuitum standard strain: ATCC 6481.

[0046] Sulfaclozine sodium: purchased from MedchemExpress, CAS number 23307-72-4; molecular formula C 10 H8ClN4NaO2S, the structure is as follows:

[0047]

[0048] 2. Conversion of test drugs

[0049] The molecular weight of Sulfaclozine sodium is 306.70 g / mol. 6.13 mg of Sulfaclozine sodium was added to 1 mL of H2O to prepare a drug stock solution with a final concentration of 20 mM. The working solution with a concentration of 320 μM was diluted according to the ratio of 1:62.5.

[0050] Example 1 Detection of the antibacterial activity of Sulfaclozine sodium on Mycobacterium tuberculosis / Mycobacterium fortuitum standard strains

[0051] 1.1 Method

[0052] 1. Preparation of standard bacterial suspension

[0053] Mycobacterium tuberculosis standard strain (ATCC 27294) was inoculated in neutral Löwenstein-Jensen medium and cultured to the logarithmic growth phase. The colonies were collected, ground and adjusted to OD 600 0.3 (about 3 x 10 7 CFU / mL). Diluted with Middlebrooks 7H9 medium at a ratio of 1:20 for standby.

[0054] Inoculate Mycobacterium fortuitum standard strain (ATCC 6481) into Middlebrooks 7H9 medium and incubate to log phase. Collect the colonies, grind and adjust the bacterial solution OD to 0.3 (about 3 x 10 600 CFU / mL). Dilute 1:200 with Middlebrooks 7H9 medium and keep for use. 7

[0055] 2. Drug gradient dilution

[0056] Add 100 μL Middlebrooks 7H9 medium to each well of a 96-well plate, then add 100 μL Sulfaclozine sodium working solution with a concentration of 320 μM to the wells in column 3, mix and take 100 μL to the next well in turn to column 12, and mix and discard 100 μL from the last well. Add 100 μL diluted bacterial solution, and the final drug concentration gradient is shown in Table 1.

[0057] Table 1. Drug concentration of Sulfaclozine sodium

[0058] Column number 1 2 3 4 5 6 7 8 9 10 11 12 Drug concentration (μM) 0 0 80 40 20 10 5 2.5 1.25 0.625 0.3125 0.1563

[0059] 3. Inoculation and incubation

[0060] Control group: Column 1 is the negative control (containing 7H9 medium);

[0061] Column 2 is the positive control group (containing 7H9 medium and diluted bacterial solution);

[0062] Experimental group: Columns 3-12 contain drugs and bacterial solution with different concentrations as shown in Table 1.

[0063] 4. Color reaction

[0064] After incubation, first add resazurin color developing solution to each well of the positive control plate, and if the solution in the well changes from blue to pink, it indicates bacterial growth. At this time, add the same amount of color developing agent to the experimental plate and continue to incubate for 24 hours to observe the color change.

[0065] 5. Result determination

[0066] MIC definition: The lowest drug concentration that maintains blue color (sterile growth), i.e., the drug concentration that inhibits 90% of bacterial growth.

[0067] Quality control standard: The negative control (column 1) should be blue, and the positive control (column 2) should be pink, otherwise the experiment is invalid. (Note: The color change of resazurin color developing agent is used to visually determine bacterial activity, blue color indicates no growth, and pink color indicates growth.)

[0068] 1.2 Results ​

[0069] The MIC of Sulfaclozine sodium against M. tuberculosis standard strain was 10 μM, and the MIC of Sulfaclozine sodium against M. tuberculosis outbreak strain was >80 μM. It showed that even if they belonged to the same Mycobacterium, the inhibitory effect of Sulfaclozine sodium on different strains still had great difference.

[0070] Example 2 Inhibition activity of Sulfaclozine sodium against multidrug-resistant clinical isolates of M. tuberculosis

[0071] Clinical isolates: 21 strains of M. tuberculosis were isolated and cultured from sputum samples of patients infected with multidrug-resistant M. tuberculosis, and were identified as M. tuberculosis by 16S rRNA, hsp65, rpoB, and 16-23S rRNA intergenic region sequencing, and were M. tuberculosis strains that were resistant to at least two most effective first-line anti-tuberculosis drugs, isoniazid (INH) and rifampicin (RIF).

[0072] According to the method in Example 1, the in vitro inhibition activity of Sulfaclozine sodium against 21 strains of multidrug-resistant clinical isolates of M. tuberculosis was detected.

[0073] 2.2 Test results

[0074] The MIC of Sulfaclozine sodium against multidrug-resistant clinical isolates of M. tuberculosis (MDR-TB) is shown in Table 2, and the optimal MIC of Sulfaclozine sodium against multidrug-resistant clinical isolates of M. tuberculosis (MDR-TB) was only 1.25 μM.

[0075] Table 2 MIC of 21 strains of multidrug-resistant clinical isolates of M. tuberculosis

[0076]

[0077]

[0078] Example 3 Sulfaclozine sodium inhibits the cytotoxicity of M. tuberculosis

[0079] CCK-8 (Cell Counting Kit-8) experiment is a colorimetric method for detecting cell proliferation and activity based on WST-8 (tetrazolium salt). Its core principle is that the mitochondrial dehydrogenase of living cells catalyzes WST-8 to produce soluble orange formazan dye, and the metabolic activity of cells is reflected by measuring the absorbance (OD value) at 450 nm.

[0080] 3.1 Experimental method

[0081] 1. Cell plating and differentiation

[0082] Logarithmic growth phase THP-1 cells were collected, centrifuged, and resuspended in RPMI-1640 medium containing 10% FBS, and the cell density was adjusted to 5 x 10 5 / mL. PMA was added (final concentration 100 ng / mL) to induce cell differentiation into macrophages, and the cells were plated (100 μL / well) and cultured for 24 hours to adhere.

[0083] 2. Drug treatment

[0084] After the cells adhered, the culture medium was discarded, and the cells were treated as follows:

[0085] Blank control group (column 2): only RPMI-1640 medium (no cells, no drugs);

[0086] Positive control group (column 3): RPMI-1640 medium and cell suspension (no drugs);

[0087] Experimental group (columns 4-11): RPMI-1640 medium containing different concentrations of Sulfaclozine sodium (40 μM-0.3125 μM, 2-fold gradient dilution).

[0088] Each group was incubated for 24 h and 48 h, and the drug treatment was as follows:

[0089]

[0090]

[0091] 3. Cell viability detection

[0092] After incubation, the old culture medium was discarded, and fresh culture medium (10% FBS) containing CCK-8 (volume ratio 1:10) was added to each well. The cells were further cultured, and the absorbance (OD value) at 450 nm was measured using a microplate reader to calculate the cell survival rate.

[0093] 4. Cell survival rate calculation formula

[0094] The survival rate (%) of THP-1 cells after stimulation by Sulfaclozine sodium was calculated as follows: (OD 450nm [experimental group]-OD 450nm [negative control group] / OD 450nm [positive control group]-OD 450nm [negative control group]) x 100%.

[0095] 3.2 Experimental results

[0096] Sulfaclozine sodium was not toxic to THP-1 cells at a concentration of 0.3125-40 μM for 24 hours, and the cell viability was about 100%; after co-incubation for 48 hours, no cytotoxicity occurred (cell viability was about 100%), indicating that Sulfaclozine sodium had good safety within a concentration of 40 μM Figure 1 ).

Claims

1. Application of Sulfaclozine sodium in the preparation of drugs to inhibit multidrug-resistant Mycobacterium tuberculosis.

2. The application as described in claim 1, wherein the medicament comprises one or more pharmaceutically acceptable carriers.

3. The use of Sulfaclozine sodium in the preparation of medicaments for the prevention and / or treatment of diseases caused by multidrug-resistant Mycobacterium tuberculosis infection, wherein the Sulfaclozine sodium exerts its effect by inhibiting the activity of multidrug-resistant Mycobacterium tuberculosis.

4. The application as described in claim 3, wherein the medicament comprises one or more pharmaceutically acceptable carriers.

5. The application as described in claim 3, wherein the dosage form of the drug comprises one or more of tablets, capsules, aerosols, pills, powders, solutions, suspensions, granules, transdermal preparations, and / or suppositories.

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