Use of delavirdine

Abstract

The application discloses a use of delavirdine, and the new use is that the delavirdine is applied to resisting mycobacterium tuberculosis, and relates to the technical field of medicines, in particular to a use of delavirdine, which provides a new medicine for resisting mycobacterium tuberculosis. The delavirdine is found to have the human macrophage level anti-tuberculosis effect for the first time, and has good clinical application value.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to the use of deraviridine. Background Technology

[0002] Tuberculosis (TB) is the leading cause of death worldwide from a single infectious source. In 2021, there were approximately 10.6 million new TB cases and about 1.6 million deaths globally. AIDS patients are immunocompromised and often have multiple opportunistic infections, with Mycobacterium tuberculosis (MTB) being a common pathogen. The disease is often progressive; HIV-infected individuals with latent Mycobacterium tuberculosis infection (LTBI) are 30 times more likely to progress to active TB than HIV-negative individuals. Therefore, HIV and MTB interact and influence each other, making diagnosis and treatment complex and challenging. my country is one of the 30 countries with a high burden of HIV / MTB co-infection worldwide. Therefore, controlling the spread of TB, preventing new HIV infections, and standardizing the diagnosis and treatment of co-infected individuals are important tasks and challenges in my country's disease prevention and control efforts. In 2017, the AIDS Group of the Infectious Diseases Branch of the Chinese Medical Association and the AIDS Group of the Tropical Diseases and Parasitology Branch of the Chinese Medical Association jointly formulated and released the first edition of the "Expert Consensus on the Diagnosis and Treatment of HIV Co-infection with Mycobacterium tuberculosis". In 2021, the HIV Co-infection and Tuberculosis Professional Committee of the China Association for STD and AIDS Prevention organized relevant experts to revise and update the first edition of the consensus. The new guidelines emphasize that early identification and detection of tuberculosis (including LTBI and active tuberculosis) and the development of effective anti-tuberculosis and antiviral therapy (ART) regimens are the most effective key measures to reduce the risk of HIV / MTB co-infection. Summary of the Invention

[0003] The main objective of this invention is to propose a use for deraviridine, aiming to provide a new drug for the treatment of Mycobacterium tuberculosis.

[0004] To achieve the above objectives, the present invention proposes a use of deraviridine, the use of which is in the treatment of Mycobacterium tuberculosis.

[0005] Optionally, the Mycobacterium tuberculosis is Mycobacterium tuberculosis at the macrophage level.

[0006] Optionally, the Mycobacterium tuberculosis includes H37Rv and H37Ra.

[0007] Optionally, the MIC concentration of deraviridine in anti-tuberculosis mycobacterium H37Rv is greater than or equal to 250 μM.

[0008] Optionally, the deraviridine described herein has an inhibition rate of 62-65% against Mycobacterium tuberculosis H37Rv in the human macrophage cell line THP-1.

[0009] Optionally, the deraviridine has an inhibition rate of 64-66% against Mycobacterium tuberculosis H37Ra in the human macrophage cell line THP-1.

[0010] Optionally, the anti-tuberculosis effect of deraviridine is achieved by targeting the host and regulating the host's immune response, thereby inhibiting the intracellular survival of Mycobacterium tuberculosis.

[0011] This invention proposes a use of depraviridine, namely, its application in the treatment of Mycobacterium tuberculosis, providing a new drug for the treatment of Mycobacterium tuberculosis. Attached Figure Description

[0012] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the structures shown in these drawings without creative effort.

[0013] Figure 1 The inhibitory effect of 50 μM deraviridine on Mycobacterium tuberculosis H37Rv at the THP-1 macrophage level, as provided in an embodiment of the present invention;

[0014] Figure 2 The inhibitory effect of 50 μM deraviridine on Mycobacterium tuberculosis H37Ra at the THP-1 macrophage level, as provided in an embodiment of the present invention;

[0015] Figure 3 The dose-response curve of deraviridine on THP-1 cells for the use of deraviridine according to an embodiment of the present invention.

[0016] The realization of the objective, functional features and advantages of the present invention will be further explained in conjunction with the embodiments and with reference to the accompanying drawings. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Where the manufacturers of reagents or instruments are not specified, they are all conventional products that can be purchased commercially. Furthermore, the meaning of "and / or" throughout the text includes three parallel solutions; for example, "A and / or B" includes solution A, or solution B, or a solution where both A and B are satisfied simultaneously. In addition, the technical solutions of the various embodiments can be combined with each other, but this must be based on the ability of those skilled in the art to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be considered that such a combination of technical solutions does not exist and is not within the scope of protection claimed by the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0018] Tuberculosis (TB) is the leading cause of death worldwide from a single infectious source. In 2021, there were approximately 10.6 million new TB cases and about 1.6 million deaths globally. AIDS patients are immunocompromised and often have multiple opportunistic infections, with Mycobacterium tuberculosis (MTB) being a common pathogen. The disease is often progressive; HIV-infected individuals with latent Mycobacterium tuberculosis infection (LTBI) are 30 times more likely to progress to active TB than HIV-negative individuals. Therefore, HIV and MTB interact and influence each other, making diagnosis and treatment complex and challenging. my country is one of the 30 countries with a high burden of HIV / MTB co-infection worldwide. Therefore, controlling the spread of TB, preventing new HIV infections, and standardizing the diagnosis and treatment of co-infected individuals are important tasks and challenges in my country's disease prevention and control efforts. In 2017, the AIDS Group of the Infectious Diseases Branch of the Chinese Medical Association and the AIDS Group of the Tropical Diseases and Parasitology Branch of the Chinese Medical Association jointly formulated and released the first edition of the "Expert Consensus on the Diagnosis and Treatment of HIV Co-infection with Mycobacterium tuberculosis". In 2021, the HIV Co-infection and Tuberculosis Professional Committee of the China Association for STD and AIDS Prevention and Control organized relevant experts to revise and update the first edition of the consensus. The new guidelines emphasize that early identification and detection of tuberculosis (including latent and active tuberculosis) and the development of effective anti-tuberculosis and antiviral therapy (ART) regimens are the most effective key measures to reduce the risk of HIV / MTB co-infection.

[0019] Delavirdine (Del) is a potent, orally active non-nucleoside reverse transcriptase inhibitor. Delavirdine selectively inhibits HIV-1 reverse transcriptase (HIV-1RT) (IC50 = 0.26 μM), with greater selectivity than DNA polymerase α (IC50 = 440 μM) and DNA polymerase δ (IC50 > 550 μM). There are currently no reported uses of delavirdine for treating Mycobacterium tuberculosis.

[0020] This invention proposes a use of deraviridine in the treatment of Mycobacterium tuberculosis, providing a new drug for this purpose. This invention verifies the use of deraviridine in the treatment of Mycobacterium tuberculosis through the following methods:

[0021] Mycobacterium tuberculosis infection: THP-1 cells were used in 24-well plates at a density of 2.5 × 10⁶ cells per well. 5 The number of cells was induced into macrophages using the method described above. One hour before infection, cells were pretreated with 50 μM deraviridine. Mycobacterium tuberculosis H37Rv was used to infect the cells at a multiplicity of infection (MOI) of 10. Four hours later, the cells were washed three times with PBS (pH 7.4) and then cultured in RPMI-1640 medium containing 10% FBS, 0.05 mM β-mercaptoethanol, 1 mM sodium pyruvate, and 10 mM HEPES. The cells were incubated at 37°C in a 5% CO2 incubator, with continuous addition of deraviridine at a concentration of 50 μM throughout the process.

[0022] CFU count by plating: 4 and 72 hours after infection, cells were lysed with 0.025% SDS (sodium dodecyl sulfate) and counted at 10... 2 10 3 Dilute the sample and plate it. Incubate at 37°C for approximately three weeks to count CFU.

[0023] The inhibitory effect of deraviridine on Mycobacterium tuberculosis H37Ra in the human macrophage cell line THP-1 was detected using the same experimental method described above.

[0024] Figure 1 The study investigated the inhibitory effect of 50 μM delavidin on Mycobacterium tuberculosis H37Rv at the THP-1 macrophage level. The results showed that delavidin could inhibit the growth of Mycobacterium tuberculosis H37Rv in macrophages. Compared with the DMSO (dimethyl sulfoxide) control group, the survival rate of Mycobacterium tuberculosis H37Rv was significantly reduced after the addition of delavidin. The inhibition rate of delavidin on Mycobacterium tuberculosis in the human macrophage line THP-1 was approximately 62-65%.

[0025] Figure 2The study investigated the inhibitory effect of 50 μM delavidin on Mycobacterium tuberculosis H37Ra at the THP-1 macrophage level. The results showed that delavidin could inhibit the growth of Mycobacterium tuberculosis H37Ra in macrophages. Compared with the DMSO (dimethyl sulfoxide) control group, the survival rate of Mycobacterium tuberculosis H37Ra was significantly reduced after the addition of delavidin. The inhibition rate of delavidin on Mycobacterium tuberculosis in the human macrophage line THP-1 was approximately 64-66%.

[0026] Furthermore, the Mycobacterium tuberculosis is Mycobacterium tuberculosis at the macrophage level. The role of deraviridine in the anti-tuberculosis process is currently unknown. This invention has found that deraviridine inhibits the intracellular survival of Mycobacterium tuberculosis at the macrophage level, indicating that deraviridine has the potential as an anti-tuberculosis drug and has important application value in the clinical treatment of HIV combined with MTB infection.

[0027] Furthermore, the MIC concentration of deraviridine against Mycobacterium tuberculosis is 250 μM. The MIC concentration of deraviridine against Mycobacterium tuberculosis can be determined as follows:

[0028] Minimum inhibitory concentration (MIC) assay: Take a 96-well plate. Add 100 μL of ddH2O (ultrapure water) to each well on the outermost edge. Add 98 μL of 7H9-OADC complete culture medium to each well of B2-G2, and 50 μL of 7H9-OADC complete culture medium to each of the remaining wells. Add 2 μL of deraviridine to each well of B2-D2, and 2 μL of isoniazid (INH) to each well of E2-G2 as positive controls. Dilute B2-G2 to B10-G10 by two-fold. B11-G11 are negative control wells without drug treatment. Finally, add 2 × 10⁻⁶ ppm of ddH2O to each well (except the ddH2O wells). 5 CFU / 50 μL of Mycobacterium tuberculosis H37Rv bacterial suspension was sealed with sealing film and incubated in a 37°C bacterial incubator for 10-14 days. After incubation, the bacterial inhibition at different drug concentrations was observed, and the MIC values ​​were recorded. It should be noted that B2-D2, B10-G10, and B11-G11 are the well numbers of the 96-well plate; the method of well numbering is not limited in this invention.

[0029] Table 1 shows the minimum inhibitory concentrations (MICs) of deraviridine and INH against Mycobacterium tuberculosis H37Rv. The results indicate that the minimum inhibitory concentration of deraviridine against Mycobacterium tuberculosis in vitro is 250 μM, significantly higher than the 50 μM concentration used at the cellular level. This suggests that the anti-tuberculosis effect of deraviridine at the macrophage level does not act directly on bacteria as an antibiotic, but rather inhibits intracellular survival of Mycobacterium tuberculosis by targeting the host and regulating the host's immune response. INH, as a positive control, showed a minimum inhibitory concentration of 0.02 μg / mL against Mycobacterium tuberculosis, consistent with the concentration range reported in the literature, thus demonstrating the reliability of the experimental data.

[0030] Table 1. MICs of deraviridine and INH against Mycobacterium tuberculosis H37Rv

[0031] dilavin 250μM INH 0.02 μg / mL

[0032] Furthermore, the IC50 concentration of deraviridine against Mycobacterium tuberculosis is 89.96 μM. The IC50 concentration of deraviridine against Mycobacterium tuberculosis can be determined as follows:

[0033] Differentiation of THP-1 macrophages: THP-1 cells were purchased from the Cell Bank of the Chinese Academy of Sciences and cultured in 1640 medium containing 10% fetal bovine serum at 37°C in a cell culture incubator with 5% CO2. Cells were cultured in 96-well plates at a density of 2.5 × 10⁶ cells per well. 4 The cells were seeded into plates and stimulated overnight with PMA (phorbol 12-tetradecanoate 13-acetate) at a final concentration of 50 ng / mL to differentiate into macrophages. After 24 hours, the medium was replaced with complete medium.

[0034] Cell viability determination: After culturing the induced macrophages for another 24 h, deraviridine was added at concentrations of 5 μM, 10 μM, 25 μM, 50 μM, 75 μM, 100 μM, 150 μM, and 200 μM, with three replicates for each concentration. DMSO (dimethyl sulfoxide) was used as the control well, and cell-free medium was used as the blank well. The cells were cultured at 37°C and 5% CO2 for another 48 h. Cell viability was detected using the CCK-8 (Cell Counting Kit-8) kit. 10 μL of CCK-8 was added to each well, and the cells were incubated for 2 h. The absorbance at OD450 nm was then measured using a microplate reader.

[0035] Cell viability is calculated using the following formula:

[0036] Cell viability = [(As-Ab) / (Ac-Ab)] × 100%;

[0037] As: Experimental wells (containing cell culture medium, CCK-8, and different concentrations of deraviridine);

[0038] Ac: Control (culture medium containing cells, CCK-8, DMSO);

[0039] Ab: Blank wells (cell-free culture medium, CCK-8);

[0040] After calculating the cell viability at different concentrations of deraviridine, dose-response curves were plotted and IC50 was calculated using GraphPad software.

[0041] Figure 3The dose-response curve for deraviridine shows that the IC50 concentration of deraviridine in THP-1 cells is 89.96 μM.

[0042] Furthermore, the anti-tuberculosis effect of deraviridine is achieved by targeting the host and regulating the host's immune response, thereby inhibiting the intracellular survival of Mycobacterium tuberculosis.

[0043] This invention is the first study to discover that deraviridine has anti-tuberculosis effects at the human macrophage level, and has good clinical application value.

[0044] The above are merely preferred embodiments of the present invention and do not limit the patent scope of the present invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the patent protection scope of the present invention.

Claims

1. The use of deraviridine in the preparation of drugs against Mycobacterium tuberculosis, characterized in that, Delavoridine is the sole active ingredient, and the Mycobacterium tuberculosis is Mycobacterium tuberculosis at the macrophage level; the anti-tuberculosis effect of delavoridine is to inhibit the intracellular survival of Mycobacterium tuberculosis by targeting the host and regulating the host's immune response.

2. The application as described in claim 1, characterized in that, The tuberculosis mycobacteria include H37Rv and H37Ra.

3. The application as described in claim 1, characterized in that, The MIC concentration of the deraviridine in the anti-tuberculosis mycobacterium H37Rv is greater than or equal to 250 μM.

4. The application as described in claim 1, characterized in that, The inhibitory rate of deraviridine against Mycobacterium tuberculosis H37Rv in the human macrophage cell line THP-1 was 62-65%.

5. The application as described in claim 1, characterized in that, The inhibitory rate of deraviridine against Mycobacterium tuberculosis H37Ra in the human macrophage cell line THP-1 was 64-66%.

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