Application of acivicin in the preparation of drugs against mycobacterium tuberculosis

Abstract

The present invention discloses the use of acivicin in preparing anti-mycobacterium tuberculosis drugs, belonging to the technical field of biomedicine. The present invention discloses the use of acivicin in preparing anti-mycobacterium tuberculosis and anti-tuberculosis drugs, and the present invention confirms through in vitro experiments that acivicin has a good effect of inhibiting Mtb H37Rv and Mtb H37Ra. Therefore, acivicin can be used to prepare anti-mycobacterium tuberculosis or anti-tuberculosis drugs alone, and can also be used as an auxiliary drug for combined use with other anti-tuberculosis drugs. That is, the present invention broadens the types of anti-tuberculosis drugs and is expected to shorten the treatment course of tuberculosis. At the same time, the present invention belongs to "new uses of old drugs", thus saving drug research and development costs and treatment costs, and has obvious economic benefits and clinical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to an application of acivicin in the preparation of a drug for resisting mycobacterium tuberculosis. Background Art

[0002] Tuberculosis (TB) is a chronic infectious disease caused by Mycobacterium tuberculosis (Mtb). The emergence of drug-resistant Mtb, particularly multidrug-resistant and extensively drug-resistant strains, has made TB treatment more difficult. According to the World Health Organization's "2022 Global Tuberculosis Report," the current global treatment success rate for drug-resistant TB is 60%, which remains low. Therefore, in the face of increasingly severe challenges, the continued development of new anti-TB drugs, especially those targeting drug-resistant TB, is urgent.

[0003] Acivicin is a natural product produced by Streptococcus suis. It is a γ-glutamyl transpeptidase inhibitor. Its chemical name is (αS,5S)-α-amino-3-chloro-2-isoxazoline-5-acetic acid, its molecular formula is C5H7ClN2O3, and its molecular weight is 178.574. Its structural formula is as follows. Acivicin can cross the blood-brain barrier and has anti-cancer and anti-parasitic properties.

[0004] Summary of the Invention

[0005] The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide a method for preparing a drug that effectively inhibits Mycobacterium tuberculosis and thus achieves effective anti-tuberculosis effect.

[0006] To achieve the above objectives, in a first aspect of the present invention, the present invention provides use of Acivicin in the preparation of anti-Mycobacterium tuberculosis drugs.

[0007] The researchers of the present invention have found that Acivicin has excellent anti-Mycobacterium tuberculosis effect and can be used in the preparation of anti-Mycobacterium tuberculosis drugs.

[0008] As a preferred embodiment of the application of the present invention, the anti-Mycobacterium tuberculosis includes Mtb H37Rv and MtbH37Ra.

[0009] The inventors have found that in in vitro experiments, Acivicin can effectively inhibit the proliferation of Mtb H37Rv and MtbH37Ra. Specifically, in in vitro experiments, it was found that the minimum inhibitory concentration (MIC) of Acivicin against Mtb H37Ra was 0.25 μg / mL, and the MIC against Mtb H37Rv was 0.25-0.5 μg / mL.

[0010] In the second aspect of the present invention, the present invention provides the use of Acivicin in the preparation of anti-tuberculosis drugs.

[0011] As a preferred embodiment of the use of the present invention, the tuberculosis is tuberculosis caused by Mtb H37Rv and / or Mtb H37Ra.

[0012] The inventors have found that Acivicin has a good inhibitory effect on Mtb H37Rv and Mtb H37Ra in vitro, thereby being able to inhibit tuberculosis caused by Mtb H37Rv and / or Mtb H37Ra.

[0013] In a third aspect of the present invention, the present invention provides an anti-tuberculosis pharmaceutical preparation, comprising Acivicin or a pharmaceutically acceptable salt, isomer, prodrug, polymorph or solvate thereof.

[0014] As a preferred embodiment of the anti-tuberculosis pharmaceutical preparation of the present invention, the pharmaceutical preparation further comprises a pharmaceutically acceptable carrier.

[0015] As a preferred embodiment of the anti-tuberculosis pharmaceutical preparation of the present invention, the pharmaceutical preparation is any one of tablets, pills, capsules and injections.

[0016] In a fourth aspect, the present invention further provides an anti-tuberculosis pharmaceutical composition, comprising Acivicin or a pharmaceutically acceptable salt, isomer, prodrug, polymorph or solvate thereof, and a common anti-tuberculosis drug.

[0017] As a preferred embodiment of the anti-tuberculosis pharmaceutical composition of the present invention, the common anti-tuberculosis drugs include any one of rifampicin, isoniazid, pyrazinamide, ethambutol, streptomycin, capreomycin, and quinolones.

[0018] As a preferred embodiment of the anti-tuberculosis pharmaceutical composition of the present invention, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] The present invention discloses the application of Acivicin in the preparation of anti-tuberculosis drugs and anti-tuberculosis drugs, and the present invention confirms through in vitro experiments that Acivicin has a good inhibitory effect on Mtb H37Rv and Mtb H37Ra; specifically, in the in vitro experiments, it was found that the MIC of Acivicin against Mtb H37Ra was Lux The MIC of Acivicin against MtbH37Rv is 0.25 μg / mL, and the MIC of Acivicin against MtbH37Rv is 0.25-0.5 μg / mL. Therefore, Acivicin can be used alone to prepare an anti-Mycobacterium tuberculosis or anti-tuberculosis drug, and can also be used as an adjuvant drug for combined use with other anti-tuberculosis drugs. That is, the present invention broadens the types of anti-tuberculosis drugs and is expected to shorten the course of tuberculosis treatment. At the same time, the present invention belongs to "new uses of old drugs", thus saving drug research and development costs and treatment costs, and has obvious economic benefits and clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a graph showing the in vitro time-kill curve of different concentrations of Acivicin against Mtb H37Ra in Example 1 of the present invention;

[0022] Figure 2 This is a graph showing the effects of different concentrations of Acivicin on the in vitro activity of Mtb H37Rv detected using the microporous Alamar Blue assay (MABA method) in Example 2 of the present invention. DETAILED DESCRIPTION

[0023] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0024] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0025] The present invention adopts a relative luminescence unit (RLU) method to determine the activity of Acivicin against Mtb H37Ra; and adopts a microporous Alamar Blue assay (MABA) method to determine the activity of Acivicin against Mtb H37Rv.

[0026] Example 1

[0027] The present invention explores the effect of Acivicin on the activity of Mtb H37Ra, specifically comprising the following steps:

[0028] 1. Preparation of drugs at different concentrations

[0029] Under sterile conditions, Acivicin and Rifampicin (RIF) were prepared into a 10 mg / mL solution with DMSO. After fully dissolved, the Acivicin solution was diluted with DMSO into 8 concentration gradients, making the drug concentrations as follows: 200 μg / mL, 100 μg / mL, 50 μg / mL, 25 μg / mL, 12.5 μg / mL, 6.25 μg / mL, and 3.125 μg / mL; the RIF solution was diluted The concentration of the drug was 100 μg / mL. 4 μL of the drug in the above concentration gradient was added to the Eppendorf tube sterilized at high temperature and high pressure, so that the final concentrations of the drug were: 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, 0.12 μg / mL, 0.06 μg / mL, and 0.03 μg / mL. 4 μL of RIF and DMSO were added to the Eppendorf tubes respectively as the positive control group and the negative control group. Three replicates were set for each concentration gradient.

[0030] 2. Preparation of Autoluminescent Mtb H37Ra (AlRa) Bacterial Solution

[0031] Take out AlRa from -80℃ freezer, inoculate into 5mL 7H9 liquid culture medium (containing 0.05% Tween 80), add glass beads to break up the bacterial solution, and when the OD 600 When the concentration reaches 0.8, dilute the bacterial solution by 10 -6 , take 0.5mL and spread it on 7H11 solid culture medium, and culture it at 37℃ for 28 days; pick a single colony from the plate and use a microplate reader to detect RLU, inoculate the confirmed luminescent single colony into 50mL 7H9 liquid culture medium (containing 0.05% Tween 80) for culture, and add glass beads to break it up; when the RLU of 1mL bacterial solution reaches 2 million, dilute the bacterial solution with 7H9 liquid culture medium (without Tween 80) so that the RLU of 0.2mL of the diluted bacterial solution is in the range of 2000-5000; add 196μL of the diluted bacterial solution to the Ep tube to which the drug solution has been added, then cover the Ep tube, mark it and immediately detect the initial RLUs of each Ep tube; place it in a constant temperature incubator at 37℃ for culture, detect the RLU of each Ep tube every 12h, and detect it continuously for 72h; use time (Time (hours)) as the horizontal axis and the logarithm of the detected RLU (Log 10 RLU / mL was used as the vertical axis for plotting analysis, and the minimum inhibitory concentration (MIC) of the drug was Lux ) is defined as the minimum concentration at which the RLU logarithm value decreases by 1 Log value compared with the negative control group;

[0032] 3. Results Analysis

[0033] The RLU of each Ep tube at different time points is shown in Table 1. The Time (hours)-Log is plotted according to Table 1. 10 RLU / mL line graph Figure 1 As shown, the MIC Lux , the results are expressed as mean value and standard deviation;

[0034] Table 1

[0035]

[0036]

[0037] From Table 1 and Figure 1 It can be seen that Acivicin has a strong inhibitory effect on AlRa. The MIC of Acivicin on AlRa is Lux 0.25μg / mL.

[0038] Example 2

[0039] The present invention explores the effect of Acivicin on the activity of Mtb H37Rv, specifically comprising the following steps:

[0040] 1. Preparation of drugs at different concentrations

[0041] Under sterile conditions, acivicin was prepared into a 10 mg / mL solution using DMSO. After sufficient dissolution, the acivicin solution was diluted to 32 μg / mL using 7H9 liquid culture medium (without Tween 80). The 32 μg / mL acivicin solution was added to the first column of a 96-well white plate, with 200 μL per well. 100 μL of 7H9 liquid culture medium (without Tween 80) was added to columns 2-12, and then 100 μL of the solution from the first column was added to the second column for dilution. After mixing, 100 μL of the solution was added to the third column for dilution. This dilution procedure was continued until the eleventh column, and then 100 μL was discarded. The twelfth column served as a negative control.

[0042] 2. Preparation of Mtb H37Rv bacterial solution

[0043] Take out the Mtb H37Rv from the -80℃ freezer, inoculate into 5mL 7H9 liquid medium (containing 0.05% Tween80), and add glass beads to disperse the bacteria. 600 When the concentration reaches 0.8, dilute the bacterial solution by 10 -6, 0.5 mL was spread on 7H11 solid medium and cultured at 37 ° C for 28 days; a single colony was picked from the plate and inoculated into 50 mL 7H9 liquid medium (containing 0.05% Tween 80), glass beads were added to break it up, and it was placed in a constant temperature shaking incubator at 37 ° C to the logarithmic growth phase (OD 600 =0.6-0.8), and then dilute the bacterial solution in the logarithmic growth phase to OD 600 =0.01, 100 μL of the diluted bacterial solution was added to each well of the above 96-well plate to make the final concentrations of the drug test 16 μg / mL, 8 μg / mL, 4 μg / mL, 2 μg / mL, 1 μg / mL, 0.5 μg / mL, 0.25 μg / mL, 0.125 μg / mL, 0.063 μg / mL, 0.031 μg / mL, 0.016 μg / mL, and 0 μg / mL, respectively, and each drug concentration was repeated 3 times; after the addition of drugs, the 96-well white plate was placed in a 37°C incubator and cultured for 7 days; after 7 days, 32.5 μL of a freshly prepared 8:5 mixture of alamar blue and 20% Tween 80 was added to each well, and the color change of each well was observed after another incubation at 37°C for 24 h. The MIC was defined as the lowest drug concentration at which no color change (blue) was observed;

[0044] 3. Results Analysis

[0045] The color conditions at different concentrations are as follows Figure 2 As shown, from Figure 2 As can be seen from the data, the MIC of Acivicin against MtbH37Rv is 0.25-0.5 μg / mL.

[0046] Finally, it should be noted that the above embodiments are intended to illustrate the technical solutions of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. Use of acivicin in the preparation of a drug for treating Mycobacterium tuberculosis; the anti-Mycobacterium tuberculosis includes MtbH37Rv and Mtb H37Ra.

2. Use of acivicin in the preparation of anti-tuberculosis drugs; the tuberculosis is tuberculosis caused by Mtb H37Rv and / or Mtb H37Ra.

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