Application of chaetocin or derivatives thereof in preparation of drugs

Abstract

The invention provides application of chaetocin or a derivative thereof in preparation of a medicine. The medicine is used for (1) inhibiting growth or proliferation of mycobacteria; (2) killing or killing mycobacteria; (3) inhibiting mycobacterium infection; and / or (4) an anti-tuberculosis drug combination, the chaetocin has significant inhibitory activity on sensitive and drug-resistant mycobacterium tuberculosis and nontuberculous mycobacterium, has the advantages of high activity and low production cost, and provides a new choice for treatment of tuberculosis and nontuberculous mycobacterium diseases.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to the application of chaetocin or its derivatives in the preparation of medicines. Background Art

[0002] Mycobacterium tuberculosis (M. tuberculosis, MTB) and nontuberculous mycobacteria (NTM) belong to the genus Mycobacterium. Tuberculosis, caused by M. tuberculosis, is an ancient and highly lethal chronic infectious disease that claims over one million lives annually. It is not only a major public health issue that severely endangers human health, but also a serious social and economic problem. Tuberculosis, especially drug-resistant tuberculosis, has a long treatment course, a low treatment success rate, and a high mortality rate, which is a major challenge in the prevention and control of tuberculosis. It is urgent to accelerate the development of new anti-tuberculosis drugs with new mechanisms and structures.

[0003] Non-tuberculous mycobacteria (NTM) refer to mycobacteria other than Mycobacterium tuberculosis complex and Mycobacterium leprae. There are a wide variety of species, approximately 200 in total, and most of them are found in the environment and are opportunistic pathogens. A few strains are pathogenic. Non-tuberculous mycobacteria are resistant to commonly used anti-tuberculosis drugs, such as isoniazid, rifampicin, and streptomycin, all of which have varying degrees of resistance. In recent years, infections caused by NTM have been on the rise, posing a serious threat to human health. Therefore, it is imperative to find drugs with better NTM treatment effects. Summary of the Invention

[0004] One object of the present invention is to provide the use of chaetocin or its derivatives in preparing medicines.

[0005] Another object of the present invention is to provide a pharmaceutical composition for treating mycobacterial infection.

[0006] In one aspect, the present invention provides the use of chaetocin or its derivatives in the preparation of a medicament, wherein the medicament is used for:

[0007] (1) Inhibit the growth or proliferation of mycobacteria;

[0008] (2) Kill or injure mycobacteria;

[0009] (3) inhibiting mycobacterial infection; and / or

[0010] (4) Combination use of anti-tuberculosis drugs.

[0011] Chaetocin, molecular formula is C30 H 28 N6O6S4 is a natural product from the genus Chaetomium. It is an epithiodiketopyrazine complex. Chaetomium is a specific inhibitor of the histone lysine 9 trimethylation (H3K9-Me3) transferase SU(VAR)3-9. It can regulate gene transcription and expression by modulating histone methylation. In some tumor cells, chaetomium can affect the expression of several genes closely related to tumor growth, thus having potential clinical value in tumor treatment. To date, there are no reports on the use of chaetomium in the treatment of mycobacterial infections.

[0012] In the above application, preferably, the combination of anti-tuberculosis drugs is the use of two or more drugs that have a synergistic effect on killing or injuring mycobacteria. More preferably, chaetocin is used in combination with clofazimine and / or putomanid to kill or injure mycobacteria.

[0013] In the above application, preferably, the structural formula of chaetocin is as shown in formula (I):

[0014]

[0015] In the above application, preferably, the derivative is a salt, stereoisomer or deuterated product of chaetocin.

[0016] More preferably, the salt of chaetocin is an acid addition salt, more preferably a hydrochloride, sulfate, acetate, maleate or fumarate.

[0017] In the above application, preferably, the mycobacterium is Mycobacterium tuberculosis or non-tuberculosis mycobacteria, and preferably, the non-tuberculosis mycobacteria is Mycobacterium abscessus, Mycobacterium avium, Mycobacterium intracellulare or Mycobacterium kansasii.

[0018] In the above application, preferably, the raw materials for preparing the drug for inhibiting mycobacterial infection further include pharmaceutically acceptable carriers and / or excipients.

[0019] More preferably, the carrier and / or excipient includes one or a combination of two or more of a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, an adsorption carrier, a lubricant and a synergist.

[0020] In the above application, preferably, the dosage form of the drug for inhibiting mycobacterial infection is an oral preparation or an injection. Preferably, the oral preparation includes tablets, capsules, powders, granules, oral liquid preparations, suspensions or emulsions. Preferably, the injection includes powder injection or injection solution.

[0021] In the above application, preferably, the dosage of chaetocin or its derivatives is 0.01 mg-1000 mg.

[0022] According to some specific embodiments of the present invention, in the above application, the chaetocin or its derivatives and pharmaceutically acceptable salts thereof inhibit mycobacterial infection by inhibiting the growth or proliferation of mycobacteria.

[0023] According to some specific embodiments of the present invention, in the above application, chaetocin is mainly used to inhibit mycobacteria in vitro.

[0024] On the other hand, the present invention also provides a pharmaceutical composition for treating mycobacterial infection, wherein the pharmaceutical composition contains a therapeutically effective amount of chaetocin or its derivatives and one or more pharmaceutically acceptable carriers.

[0025] According to some specific embodiments of the present invention, in the above pharmaceutical composition, the mycobacterium is Mycobacterium tuberculosis or non-tuberculosis mycobacteria.

[0026] According to some specific embodiments of the present invention, in the above pharmaceutical composition, the non-tuberculosis mycobacterium is Mycobacterium abscessus, Mycobacterium avium, Mycobacterium intracellulare or Mycobacterium kansasii.

[0027] According to some specific embodiments of the present invention, in the above-mentioned pharmaceutical composition, the carrier is selected from one or a combination of two or more of diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants and synergists.

[0028] According to some specific embodiments of the present invention, in the above-mentioned pharmaceutical composition, the dosage form of the pharmaceutical composition is an oral preparation or an injection.

[0029] According to some specific embodiments of the present invention, in the above-mentioned pharmaceutical composition, the oral preparation is selected from tablets, capsules, powders, granules, oral liquid preparations, suspensions or emulsions.

[0030] According to some specific embodiments of the present invention, in the above-mentioned pharmaceutical composition, the injection is selected from powder injection or injection solution.

[0031] The chaetocin or its derivatives of the present invention can be administered as the sole active ingredient or in combination with other anti-mycobacterial drugs.

[0032] According to some specific embodiments of the present invention, in the above pharmaceutical composition, the pharmaceutical composition further contains other anti-mycobacterial compounds.

[0033] According to some specific embodiments of the present invention, in the above-mentioned pharmaceutical composition, the other anti-mycobacterial compound is selected from one or a combination of two or more of isoniazid, rifampicin, ethambutol, amikacin, linezolid, clofazimine, bedaquiline, moxifloxacin and putamanid.

[0034] According to some specific embodiments of the present invention, in the above-mentioned pharmaceutical composition, the mycobacterial infection is tuberculosis caused by MTB or NTM disease caused by NTM.

[0035] According to some specific embodiments of the present invention, in the above pharmaceutical composition, the derivative of chaetocin is a salt of chaetocin.

[0036] According to some specific embodiments of the present invention, in the above pharmaceutical composition, the salt of chaetocin is an acid addition salt.

[0037] According to some specific embodiments of the present invention, in the above pharmaceutical composition, the salt of chaetocin is hydrochloride, sulfate, acetate, maleate or fumarate.

[0038] The mycobacterium used to illustrate the present invention may be a standard strain known in the art or a clinical strain isolated from a mycobacterium-infected patient.

[0039] According to some specific embodiments of the present invention, in the above-mentioned pharmaceutical composition, the dosage of chaetocin or its derivatives in the pharmaceutical composition is 0.01mg-1000mg (for example, 0.01mg-10mg, 0.01mg-50mg, 0.01mg-100mg, 0.01mg-500mg, 0.01mg-1000mg, 10mg-50mg, 10mg-100mg, 10mg-500mg, 10mg-1000mg, 50mg-100mg, 50mg-500mg, 50mg-1000mg, 100mg-500mg, 100mg-1000mg or 500mg-1000mg).

[0040] The dosage mentioned in the present invention is the dosage meaning commonly understood in the art, for example, it refers to the mass of chaetocin or its derivatives contained in each dose of the pharmaceutical composition; and the "per dose" mentioned here refers to each dosage unit, such as each tablet, each capsule, and each bottle of injection, and its meaning is understood by those skilled in the art.

[0041] The technical solution of the present invention has the following beneficial technical effects:

[0042] The present invention provides the use of chaetocin or its derivatives and pharmaceutically acceptable salts thereof in the treatment of mycobacterial infections. The results show that it has significant inhibitory activity against sensitive and resistant Mycobacterium tuberculosis and NTM, has the advantages of high activity and low production cost, and provides a new option for the treatment of tuberculosis and NTM diseases. DETAILED DESCRIPTION

[0043] In order to have a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention is now described in detail below, but it should not be understood as limiting the scope of implementation of the present invention.

[0044] Example 1

[0045] In vitro anti-Mycobacterium tuberculosis activity test:

[0046] The anti-Mycobacterium tuberculosis activity of the chaetocins of the present invention is demonstrated by measuring their minimum inhibitory concentration (MIC, μg / mL) against the standard strain H37Rv (ATCC 27294) and clinically drug-resistant strains of Mycobacterium tuberculosis. In this test, isoniazid and rifampicin were used as control drugs. The MIC was determined as follows: A sterile 96-well plate (specialized for rapid drug sensitivity testing of Mycobacterium tuberculosis) was plated with the drug diluted in a 2x concentration culture medium (modified Michaelis 7H9 liquid medium) to each well. Chaetocins were prepared to an appropriate initial concentration, diluted with the 2x concentration culture medium to twice the concentration of each compound used, and then added to the 96-well plate, 100 μL per well, to final concentrations of 3.2, 1.6, 0.8, 0.4, 0.2, 0.1, 0.05, 0.025, and 0.0125 μg / mL, respectively. 100 μL of the standard strain H37Rv (ATCC 27294) and clinical isolates were inoculated into each well, and the final concentration of the bacterial solution was 1×10 5CFU / ml. Each plate is equipped with two positive growth control wells without antibiotics and two negative growth control wells with distilled water instead of culture medium. The 96-well culture plate is placed in a 5% CO2 incubator at 37°C and cultured for 7 days. After 7 days, add Alamar Blue (Setotec). Operate in a biosafety cabinet away from light. Mix 20μl of Alamar Blue and 12.5μl of 20% Tween-80 per well, then use an eight-channel pipette to draw 32.5μl and add it to each well. At this point, the color of each well is blue, and continue to culture in a 5% CO2 incubator at 37°C for 24 hours. After 24 hours, observe the color changes of each well. The color of the well with bacterial proliferation turns pink, and the color of the well with bacterial inhibition growth remains blue. Alamar blue fluorescence values ​​in 96-well culture plates were then measured using a multifunctional microplate reader (Tecan Infinite m200) at wavelengths (530 nm and 590 nm). The inhibition rate for each drug-containing well was calculated using the formula (1 - value of drug-containing well / value of control well) × 100. The minimum inhibitory concentration (MIC) of the drug was determined to achieve a 90% inhibition rate. The results of the MIC (μg / ml) determination of chaetocin and the control drug against Mycobacterium tuberculosis are shown in Table 1.

[0047] Table 1

[0048]

[0049] Note: RIF, rifampicin; INH, isoniazid; EMB, ethambutol; BDQ, bedaquiline; LZD, linezolid; LVX, levofloxacin; MXF, moxifloxacin.

[0050] The results showed that the MIC of chaetocin against standard strains of Mycobacterium tuberculosis and clinically isolated drug-resistant strains was 0.05-0.1 μg / mL. Chaetocin's anti-TB activity was comparable to that of rifampicin, isoniazid, and bedaquiline. Furthermore, chaetocin did not induce cross-resistance with first-line anti-TB drugs, drugs for drug-resistant TB, or new anti-TB drugs.

[0051] Example 2

[0052] In vitro anti-nontuberculous mycobacteria (NTM) activity test:

[0053] Mycobacterium avium (ATCC 25291), Mycobacterium intracellulare (ATCC 13950), Mycobacterium abscessus (ATCC 19977), and Mycobacterium kansasii (ATCC 12478) were cultured to the logarithmic growth phase and the bacterial suspension was diluted to 5 × 10 5CFU / mL is set aside. Take a 96-well plate and dilute chaetocin and the control drug clarithromycin in multiple ratios and add them in sequence. After 7 days of culture at 5% CO2 and 37°C (3 days for Mycobacterium abscessus), operate in a biosafety cabinet away from light, add 20μL of Alamar blue and 12.5μL of 20% Tween-80 to each well, and continue to culture at 5% CO2 and 37°C for 24 hours. Record the color of each well. Blue indicates no growth of the strain, and red indicates bacterial growth. The drug concentration in the well that changes from blue to red is defined as the minimum drug concentration (MIC). Repeat the test 3 times. The results of the in vitro MIC (μg / mL) of chaetocin against NTM Mycobacterium are shown in Table 2.

[0054] Table 2

[0055]

[0056] The results showed that chaetocin has in vitro activity against Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium kansasii, and Mycobacterium abscessus, providing a new approach for inhibiting mycobacterial infections.

[0057] Example 3

[0058] Checkerboard method for determining drug combination effects:

[0059] Chaetocin and anti-tuberculosis drugs were prepared in 96-well plates with a final concentration of 2MIC to 1 / 32MIC solution (100 μL), and the standard strain H37Rv (ATCC 27294) was inoculated into each well. The bacterial solution was diluted to a final concentration of 1×10 5 CFU / ml. Each plate was equipped with two positive growth control wells without antibiotics and two negative growth control wells with distilled water replacing the culture medium. The 96-well culture plate was incubated in a 5% CO2 incubator at 37°C for 7 days. After 7 days, Alamar blue was added. Working in a biosafety cabinet, 20 μl of Alamar blue and 12.5 μl of 20% Tween-80 were mixed uniformly per well. Then, 32.5 μl was pipetted into each well. At this point, all wells were blue. The plates were incubated in a 5% CO2 incubator at 37°C for 24 hours. After 24 hours, the color of the wells was observed. Wells showing bacterial growth turned pink, while wells showing bacterial growth inhibition remained blue. The fractional inhibitory concentration index (FICI) was used as an evaluation metric. The FICI value was calculated using the formula FICI = (MIC for drug A combined with MIC / MIC for drug A alone) + (MIC for drug B combined with MIC / MIC for drug B alone). The FICI values ​​of chaetocin and other anti-tuberculosis drugs are shown in Table 3 .

[0060] Table 3

[0061] Drug combinations Combined FICI Combined action Chaetocin / INH 0.625 Partial collaboration Chaetocin / RIF 0.625 Partial collaboration Chaetocin / LZD 0.75 Partial collaboration Chaetocin / EMB 1 Additive effect Chaetocin / LVX 0.75 Partial collaboration Chaetocin / BDQ 0.75 Partial collaboration Chaetocin / CFZ 0.5 Synergy Chaetocin / PA-824 0.5 Synergy

[0062] Note: INH, isoniazid; RIF, rifampicin; LZD, linezolid; EMB, ethambutol; LVX, levofloxacin; BDQ, bedaquiline; CFZ, clofazimine; PA-824, putomanib.

[0063] The results showed that the combination of chaetocin with PA-824 and CFZ had a synergistic effect on the strain, and a partially synergistic or additive effect when combined with other drugs. This could provide a new approach for inhibiting mycobacterial infections.

Claims

1. Use of chaetocin or its derivatives in the preparation of medicines, wherein the medicines are used for: (1) Inhibit the growth or proliferation of mycobacteria; (2) Kill or injure mycobacteria; (3) inhibiting mycobacterial infection; and / or (4) Combination use of anti-tuberculosis drugs.

2. The use according to claim 1, wherein The anti-tuberculosis drug combination is the use of two or more drugs that have a synergistic effect on killing or injuring mycobacteria. Preferably, chaetocin, clofazimine and / or putomanib are used in combination to kill or injure mycobacteria.

3. The use according to claim 1, wherein: The structural formula of the chaetocin is shown in formula (I):

4. The use according to claim 1, wherein The derivative is a salt, stereoisomer or deuterated product of chaetocin.

5. The use according to claim 4, wherein the salt of chaetocin is an acid addition salt, more preferably hydrochloride, sulfate, acetate, maleate or fumarate.

6. The use according to claim 1, wherein: The mycobacterium is Mycobacterium tuberculosis or non-tuberculosis mycobacteria. Preferably, the non-tuberculosis mycobacteria is Mycobacterium abscessus, Mycobacterium avium, Mycobacterium intracellulare or Mycobacterium kansasii.

7. The use according to claim 1, wherein: The raw materials for preparing the drug for inhibiting mycobacterial infection also include pharmaceutically acceptable carriers and / or excipients.

8. The use according to claim 7, wherein: The carrier and / or auxiliary material includes one or a combination of two or more of a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, an adsorption carrier, a lubricant and a synergist.

9. The use according to claim 1, wherein: The dosage form of the drug for inhibiting mycobacterial infection is an oral preparation or an injection. Preferably, the oral preparation includes tablets, capsules, powders, granules, oral liquid preparations, suspensions or emulsions. Preferably, the injection includes powder injection or injection solution.

10. The use according to claim 1, wherein: The dosage of chaetocin or its derivatives is 0.01 mg-1000 mg.

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