Application of phenothiazine compounds in preparation of antituberculosis drugs

Abstract

The invention discloses application of phenothiazine compounds in preparation of antituberculosis drugs, and belongs to the technical field of biomedicine. The invention discloses application of phenothiazine compounds in preparation of antituberculous drugs, phenothiazine compounds X79 serve as a photosensitizer to mediate photodynamic therapy to generate active oxygen to achieve host-oriented treatment, a brand new treatment strategy is provided for infection of intracellular bacteria such as mycobacterium tuberculosis, biocompatibility is excellent, functional modification is easy to achieve, and the phenothiazine compounds X79 can be applied to preparation of antituberculous drugs. Good application prospects are realized in the field of tuberculosis treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and more specifically to the application of phenothiazine compounds in the preparation of anti-tuberculosis drugs. Background Technology

[0002] Tuberculosis (TB) is a highly contagious and fatal disease caused by Mycobacterium tuberculosis (Mtb). According to the World Health Organization's Global Tuberculosis Report 2024, 1.25 million people worldwide died from TB in 2023. Besides the lungs, TB can affect other parts of the body, with extrapulmonary tuberculosis accounting for 8-24% of these cases. Of these cases, cutaneous tuberculosis (CTB) accounts for 1.5-3% of extrapulmonary tuberculosis. Based on the route of infection and the host's immune status, CTB can be divided into true cutaneous tuberculosis and tuberculous rash. Mtb is not detected in tuberculous rash, but an immune response to tuberculosis antigens is present. In true cutaneous tuberculosis, Mtb is usually detectable, and common manifestations include tuberculous ulcers and verrucous tuberculosis of the skin. Furthermore, it can act as a source of dissemination, spreading through the blood or lymphatic system, leading to multi-system involvement of the lungs, kidneys, bones, joints, and other organs. In immunocompromised individuals, it can progress to acute miliary tuberculosis, which has an extremely high mortality rate. In terms of treatment, the treatment regimen for CTB is consistent with other forms of tuberculosis, employing a four-drug combination therapy including isoniazid, rifampin, pyrazinamide, and ethambutol. The initial phase requires continuous medication for two months, followed by continued use of isoniazid in combination with rifampin. The total treatment duration is typically 4 to 12 months. Notably, CTB presents with a variety of clinical manifestations similar to those of various common skin diseases, leading to frequent misdiagnosis in clinical practice. Furthermore, traditional treatment regimens face challenges due to their long duration, the potential for drug resistance with prolonged use, and the possibility of various toxic side effects from the drugs themselves. These factors collectively present significant challenges to clinical diagnosis and treatment. Therefore, there is an urgent need to develop new anti-tuberculosis drugs to cure and curb the spread of tuberculosis.

[0003] Macrophages play a crucial role as the main innate immune cells in the fight against tuberculosis, and macrophage autophagy helps clear Mycobacterium tuberculosis. However, in the process of long-term coexistence with the human host, Mycobacterium tuberculosis has evolved a variety of immune escape strategies, such as inhibiting macrophage maturation, lysosomal acidification, oxidative stress, apoptosis, and autophagy, thereby achieving long-term survival in the host. Therefore, developing drugs that promote macrophage autophagy to block the immune escape of Mycobacterium tuberculosis is of great significance in combating tuberculosis and CTB.

[0004] Photodynamic therapy (PDT) is a non-invasive treatment that relies on the accumulation of a photosensitizer (ps) at the lesion site. When the photosensitizer is activated by light, it generates reactive oxygen species (ROS), thereby inducing cytotoxicity. This treatment is simple, painless, and highly acceptable to patients. Furthermore, it directly disrupts key structures and components of Mycobacterium tuberculosis, exerting its anti-tuberculosis effect. Unlike traditional antibiotics that rely on specific targets for action, PDT prevents the development of drug resistance at the mechanistic level. PDT shows great potential in antibacterial applications.

[0005] Porphyrins, as the first generation of photosensitizers approved for clinical use, have been applied in the treatment of esophageal and bladder cancers. However, this tetrapyrrole-based molecular structure has limitations such as difficult preparation, strong skin toxicity, and poor water solubility. Furthermore, studies have shown that this structure has limited bactericidal efficacy against Gram-negative bacteria. This may stem from the dense negative charge on the outer membrane of Gram-negative bacteria, which is rich in lipopolysaccharides and anionic phospholipids. Traditional tetrapyrrole-based macrocyclic compounds are mainly neutral or weakly anionic, making it difficult to penetrate this outer membrane. This limitation restricts their ability to kill Mycobacterium tuberculosis, which also has a negatively charged outer membrane. Therefore, developing a novel photosensitizer that overcomes this limitation is a crucial problem that urgently needs to be solved.

[0006] Therefore, providing the application of phenothiazine compounds in the preparation of anti-tuberculosis drugs is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0007] In view of this, the present invention provides the use of phenothiazine compounds in the preparation of anti-tuberculosis drugs.

[0008] Phenothiazines, as positively charged photosensitizers, are considered promising candidates for the treatment of tuberculosis. Methylene blue, a commonly used phenothiazine compound, has demonstrated antibacterial activity against clinically isolated Mycobacterium tuberculosis (MTB) in in vitro studies, confirming the antibacterial potential of this class of compounds. This invention utilizes the novel phenothiazine compound X79 as a photosensitizer in photodynamic therapy to achieve anti-tuberculosis effects. This compound not only exhibits excellent biocompatibility and low effective concentrations (nanomolar levels), but also effectively inhibits the growth of Mycobacterium tuberculosis, thus providing a new direction for the development of anti-tuberculosis drugs.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: The application of phenothiazine compounds in the preparation of anti-tuberculosis drugs, wherein the phenothiazine compound is 9-(diethylamino)-3-methyl-5-(propylamino)pyrido[3,2-a]phenothiazine-7-onium chloride, and its structural formula is as follows: .

[0011] Furthermore, the application of phenothiazine compounds in the preparation of drugs for inhibiting Mycobacterium tuberculosis infection, wherein the phenothiazine compound is 9-(diethylamino)-3-methyl-5-(propylamino)pyrido[3,2-a]phenothiazine-7-onium chloride, and its structural formula is as follows: .

[0013] 9-(diethylamino)-3-methyl-5-(propylamino)pyrido[3,2-a]phenthiazine-7-onium chloride, see compound 14 of Example 9 in patent 201710447838.9. In this invention, it is abbreviated as "phenthiazine compound X79".

[0014] This invention investigates the molecular mechanism by which the phenothiazine compound X79 activates the autophagy pathway; and explores the antibacterial effects of the phenothiazine compound X79 on H37Ra both intracellularly and extracellularly.

[0015] As can be seen from the above technical solution, compared with the prior art, the present invention discloses the application of phenothiazine compounds in the preparation of anti-tuberculosis drugs, which has the following beneficial effects: (1) Phenothiazine compound X79 can effectively kill Mycobacterium tuberculosis at concentrations as low as nanomolar and is safe and non-toxic, which provides a new strategy and method for inhibiting MTB infection in vivo.

[0016] (2) Unlike traditional antibiotics that rely on specific targets to exert their effects, phenothiazine compound X79 utilizes PDT to exert its bactericidal effect. At the mechanism level, it naturally possesses the characteristic of preventing the development of drug resistance, which solves the current problems of poor efficacy, long treatment course, large side effects and easy drug resistance of anti-tuberculosis drugs. It will lay an important foundation for the development of new technologies for tuberculosis prevention and control. Attached Figure Description

[0017] 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 embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0018] Figure 1 This is the structural formula of a phenothiazine compound.

[0019] Figure 2The study investigated the cytotoxic effects of the phenothiazine compound X79. Specifically, (A) the cytotoxic effects of different concentrations of X79 on THP-1 macrophages; and (B) the photocytotoxic effects of X79 on THP-1 macrophages (630 nm, 1 min, 106 mW / cm²). 2 (C) Photocytotoxicity of X79 on H37Ra-infected THP-1 macrophages.

[0020] Figure 3 This study investigates the macrophage targeting of the phenothiazine compound X79. The images show: (A) Flow cytometry images of mouse spleen-derived T cells, B cells, and macrophages after 2 hours of X79 treatment; (B) Statistical analysis of cell uptake by mouse spleen-derived T cells, B cells, and macrophages after 2 hours of X79 treatment; (C) Flow cytometry images of THP-1 macrophages after 0.5, 1, and 2 hours of X79 treatment; and (D) Statistical analysis of cell uptake by THP-1 macrophages after 0.5, 1, and 2 hours of X79 treatment.

[0021] Figure 4 To detect ROS generation in THP-1 macrophages after X79 treatment by flow cytometry.

[0022] Figure 5 X79-mediated photodynamic therapy enhances autophagy by modulating signaling pathways in macrophages infected with Mycobacterium tuberculosis. (A) Immunoblotting images of LC3B, P62, ATG16L, and ATG4B in THP-1 macrophages infected with MTB after co-incubation of the phenothiazine compound X79 for 24 h. (BE) Statistical analysis of the immunoblotting data of LC3B, ATG16L, P62, and ATG4B in THP-1 macrophages infected with MTB after co-incubation of the phenothiazine compound X79 for 24 h.

[0023] Figure 6 Immunofluorescence detection of LC3 spots. (A) Typical fluorescence imaging of LC3B staining in H37Ra-infected THP-1 macrophages before and after treatment with the phenothiazine compound X79, scale bar: 10 μm. (B) Statistical results of LC3B spots in H37Ra-infected THP-1 macrophages before and after treatment with the phenothiazine compound X79.

[0024] Figure 7Transcriptomic sequencing results are shown. (A) Volcano plot of transcriptomic data between the MTB-infected THP-1 macrophage group and the group treated with phenothiazine compound X79 under light conditions (FC ≥ 2, p < 0.05). Red nodes indicate upregulated metabolites, and blue nodes indicate downregulated metabolites. (B) GO enrichment analysis of differentially expressed genes between the MTB-infected THP-1 macrophage group and the group treated with phenothiazine compound X79 under light conditions.

[0025] Figure 8 X79-mediated photodynamic therapy significantly inhibited both extracellular and intracellular Mycobacterium tuberculosis in infected macrophages. (A) The direct inhibitory effect of the phenothiazine compound X79 on H37Ra, as shown in the image on a 7H11 culture plate after a 10-fold dilution. (B) Statistical results of the direct inhibitory effect of the phenothiazine compound X79 on H37Ra (n=3). * P <0.05. (C) Antibacterial effect of phenothiazine compound X79 on H37Ra in THP-1 macrophages, image of the sample diluted 10-fold on a 7H11 culture plate. (D) Statistical results of the antibacterial effect of phenothiazine compound X79 on H37Ra in THP-1 macrophages (n=3). * P <0.05. (E) Antibacterial effect of phenothiazine compound X79 on H37Ra in mouse spleen-derived macrophages, typical image of the sample diluted 10-fold on a 7H11 culture plate. (F) Statistical results of the antibacterial effect of phenothiazine compound X79 on H37Ra in mouse spleen-derived macrophages (n=3). * P <0.05.

[0026] Figure 9 This study investigates the therapeutic effect of X79-mediated photodynamic therapy on a mouse model of cutaneous tuberculosis. (A) Typical images of different treatment groups after treatment for cutaneous tuberculosis in mice. (B) Size of the tuberculous tumor in mice on day 12 post-treatment (n=6) in different treatment groups. (C) Changes in mouse body weight during the experiment in different treatment groups. (D) Spleen weight in different treatment groups at the end of experiment 12.

[0027] Figure 10 This study examines the therapeutic effect of X79-mediated photodynamic therapy on a mouse cutaneous tuberculosis model. (A) Statistical analysis of mouse skin abrasive suspensions on 7H11 culture plates between different treatment groups (n=6). (B) Classic images of undiluted mouse skin abrasive solution, 10-fold dilution, and 100-fold dilution on 7H11 tri-plates between different treatment groups; the undiluted solution is at the top, the 10-fold dilution is at the bottom right, and the 100-fold dilution is at the bottom left. Detailed Implementation

[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. 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.

[0029] Example 1: In vitro cell uptake, intracellular reactive oxygen species generation, and in vitro cell uptake The structural formula of phenothiazine compound X79 is as follows: Figure 1 As shown.

[0030] (a) Cytotoxic effects of phenothiazine compound X79 Cytotoxicity: Drug group: THP-1 cells in logarithmic growth phase were injected at a rate of 5 × 10⁻⁶. 3 Cells / well were seeded in 96-well plates and stimulated with PMA (50 ng / mL) for 24 h; then different concentrations (10 nM, 20 nM, 50 nM, 100 nM) of phenothiazine compound X79 were added to the 96-well plates and incubated for 24 h.

[0031] Control group: RPMI 1640 medium; Control group: THP-1 cells in logarithmic growth phase were injected at a rate of 5 × 10⁻⁶ cells / year. 3 Cells / well were seeded into 96-well plates and stimulated with PMA (50 ng / mL) for 24 h; an equal volume of RPMI 1640 medium was added to replace the phenothiazine compound X79, and the plates were incubated for 24 h.

[0032] Afterwards, 10 µl of 5 mg / mL MTT solution was added to each group, and after 2 h, 100 µl of SDS-Tris-HCl was added and incubated for 22 h. The absorbance was measured at 570 nm using a microplate reader. The percentage of cell viability was calculated using the following formula: Cell viability = 100% - (OD drug - OD blank) / (OD control - OD blank) × 100%.

[0033] Photocytotoxicity: Photocytotoxic effect of X79 on THP-1 macrophages: THP-1 cells in logarithmic growth phase were injected with 5 × 10⁻⁶ cells. 3 Cells / well were seeded into 96-well plates and stimulated with PMA (50 ng / mL) for 24 hours. Different concentrations of phenothiazine compound X79 (10 nM, 20 nM, 50 nM, 100 nM) were added to the 96-well plates, and after 1 hour, the plates were irradiated with an LED light source (+Light) for 1 minute (630 nm, 106 mW / cm²). 2The non-light group (-Light) received no treatment and continued incubation for 23 hours. Cell viability was assessed using MTT assay on the second day.

[0034] X79 photocytotoxicity of H37Ra-infected THP-1 macrophages: Logarithmic growth phase THP-1 cells were injected with 5 × 10⁻⁶ cells. 3 Cells / well were seeded into 96-well plates and stimulated with PMA (50 ng / mL) for 24 hours. The next day, THP-1 macrophages were infected with H37Ra at an MOI of 1 (cells:bacteria = 1:1) and incubated overnight. Afterwards, extracellular H37Ra was washed away with PBS, and different concentrations of the phenothiazine compound X79 (10 nM, 20 nM, 50 nM, 100 nM) were added to the 96-well plates. One hour later, the plates were irradiated with an LED light source (+Light) for 1 minute (630 nm, 106 mW / cm²). 2 The non-light group (-Light) received no treatment and continued incubation for 23 hours. Cell viability was assessed using MTT assay on the second day.

[0035] To investigate the effect of X79 on THP-1 macrophages, the MTT assay was used to assess the effect of different concentrations of X79 on the survival rate of THP-1 macrophages. The results showed that it had dose-dependent cytotoxicity on THP-1 macrophages. Figure 2 A). Further investigation was conducted on the photocytotoxicity of X79. Under light irradiation, at a concentration of 20 nM, the viability of both normal THP-1 macrophages and H37Ra-infected THP-1 macrophages remained above 80%. Figure 2 BC). 20 nM was chosen as the concentration for subsequent experiments because it does not damage infected THP-1 macrophages while maintaining antibacterial activity.

[0036] (II) Macrophage targeting of phenothiazine compound X79 In mouse spleen lymphocytes: A complete spleen was removed from 6-week-old BABL / C mice. The spleen was cut into small segments and ground using a grinding mesh. The filtrate was then filtered through a 70µm sieve into a 50ml centrifuge tube. The supernatant was collected by centrifugation (4℃, 350g, 10min). 5ml of erythrocyte lysis buffer was added, and the mixture was incubated on ice for 15min to lyse the erythrocytes. After 15min, 30ml of PBS was added to stop the reaction. The supernatant was collected by centrifugation (4℃, 350g, 10min). 10ml of PBS was added and mixed well. The mixture was then filtered through a 30µm sieve into a 50ml centrifuge tube, and the filtrate was collected. The supernatant was collected by centrifugation (4℃, 350g, 10min). The remaining precipitate was identified as monocytes (mouse spleen lymphocytes).

[0037] Purified mouse spleen lymphocytes (1×10⁻⁶)7 Cells were seeded in 12-well plates (each well containing 20 nM of drug X79) and incubated for 2 h. Cells were then collected. After washing with PBS containing 2% FBS and 2 mM EDTA, the cells were stained with antibodies (PerCP anti-CD14, APC anti-CD3e, PE anti-CD19). 1 µl of antibody was added to each sample, and the staining was performed at 4 °C for 30 min. After washing with PBS containing 2% FBS and 2 mM EDTA, the uptake of T cells, B cells, and macrophages was analyzed by flow cytometry.

[0038] In THP-1 macrophages: the phenothiazine compound X79 exhibits red fluorescence at approximately 630 nm. THP-1 cells in logarithmic growth phase were cultured at 5 × 10⁻⁶ cells per cell line. 5 Cells were seeded per well in 12-well plates and stimulated with PMA (50 ng / mL) for 24 hours. The following day, 20 nM X79 was added to the wells at specified time points (0.5 h, 1 h, 2 h). Free drug was then washed away with PBS. Cells were digested with 600 µl of trypsin for 8 min, followed by the addition of 600 µl of RMPI 1640 medium containing 10% fetal bovine serum to stop digestion. The medium was collected and centrifuged (600 g, 10 min), the supernatant was discarded, and the cells were resuspended in PBS. This process was repeated three times. Fluorescence intensity was analyzed using flow cytometry to monitor X79 uptake.

[0039] To investigate the targeting effect of X79 on macrophages, monocytes were isolated from mouse spleens and co-cultured with X79 for 2 hours. Anti-T cell, anti-B cell, and anti-macrophage antibodies were then added. Flow cytometry was used to assess the uptake of X79 by different cell types. The results showed that macrophages uptake X79 significantly higher than B cells and T cells. Figure 3 (AB) confirmed that X79 can effectively target macrophages. To further verify the targeting efficacy, THP-1 macrophages were used as the research subject. The results showed that X79 could enter THP-1 macrophages (AB) within half an hour after administration. Figure 3 X79 (CD) levels peaked after 1 hour. These results indicate that X79 has potential application value in host-guided therapy for tuberculosis.

[0040] (III) Intracellular ROS generation level of phenothiazine compound X79 Intracellular reactive oxygen species (ROS) production was detected using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA). Logarithmic growth phase THP-1 cells were cultured at 5 × 10⁻⁶ cells / year. 5Cells were seeded per well in 12-well plates and stimulated with PMA (50 ng / mL) for 24 hours. The next day, cells were incubated with 20 nM X79 for 30 minutes, then the culture medium was discarded, and the cells were washed three times with PBS. The control group received no treatment. Each group was then incubated with DCFH-DA (10 µM) for 15 minutes. Subsequently, cells were incubated under an LED light source (630 nm, 106 mW / cm²). 2 Irradiate cells for 1 minute. Continue incubation for 15 minutes; the non-illuminated group received no treatment. After 15 minutes, wash away extracellular DCFH-DA with PBS. Digest cells with 600 µl trypsin for 8 minutes, then add 600 µl of RPMI 1640 medium containing 10% fetal bovine serum to stop digestion. Collect the medium, centrifuge (600 g, 10 min), discard the supernatant, resuspend in PBS, and repeat this process three times. Finally, detect fluorescence signals by flow cytometry.

[0041] The DCFH-DA probe was used to detect intracellular ROS production levels. DCFH-DA itself is non-fluorescent. After entering the cell, the probe is readily oxidized and dehydrogenated by reactive oxygen species, converting into the fluorescent compound DCF. Figure 4 As shown, no reactive oxygen species (ROS) were detected when X79 acted on macrophages alone. However, high levels of ROS generation were observed under light irradiation. This indicates that X79 absorbs photon energy under light irradiation, ultimately inducing ROS production.

[0042] Example 2: X79-mediated photodynamic therapy enhances autophagy by modulating signaling pathways in macrophages infected with Mycobacterium tuberculosis. Promoting autophagy can still inhibit the growth of Mycobacterium tuberculosis in both in vitro and in vivo environments. Therefore, drugs that activate the autophagy pathway have broad therapeutic prospects. To this end, the function of X79 in regulating macrophage autophagy was investigated.

[0043] (a) Phenothiazine compound X79 induces autophagy in THP-1 macrophages THP-1 cells in logarithmic growth phase were injected with 5 × 10⁻⁶ cells. 5 Cells were seeded per well in a 6-well plate and stimulated with PMA (50 ng / mL) for 24 hours. Then, a bacterial suspension of H37Ra was added to each well at MOI=1 and incubated for 24 hours. Extracellular H37Ra was washed away three times with PBS. The cells were then incubated with 20 nM X79 for 1 hour, followed by LED illumination for 1 minute. After 23 hours, protein was extracted and the protein concentration was determined using the BCA method. The samples were then mixed with 5× loading buffer and heated at 100°C for 10 minutes. The samples can then be stored at -20°C for later use.

[0044] Prepare SDS-PAGE gels and load total cellular proteins and marker molecular weight markers into the wells of the SDS-PAGE gel. Run the stacking gel at a constant voltage of 80V, increasing the voltage to 120V when reaching the separating gel, maintaining this constant voltage until all samples have been processed. After electrophoresis, remove the SDS-PAGE gel containing the separated protein samples from the glass plate and wash it in transfer buffer. Then, sandwich the gels in the following order: fiber pad, filter paper, PVDF membrane, gel, filter paper, fiber pad. Electrolyze at a constant current of 300 mA for 120 min, then remove the PVDF membrane and block it with TBST containing 5% skim milk on a shaker at room temperature for 1 h to reduce non-specific antibody binding. After 1 h of blocking, wash the PVDF membrane three times with TBST for 5 min each time. Then add the corresponding rabbit anti-LC3B, rabbit anti-ATG16L, rabbit anti-P62, and rabbit anti-ATG4B primary antibodies and incubate overnight at 4°C on a shaker. The next day, the PVDF membrane was washed three times with TBST for 5 min each time. Then, the membrane was sealed with horseradish peroxidase-labeled goat anti-rabbit IgG (H+L) antibody diluted with the corresponding amount of TBST and incubated at room temperature for 2 h. The membrane was washed three times with TBST for 5 min each time, and ECL was used for imaging. The membrane was then placed in a chemiluminescence imaging system for photography and image saving. The effects of X79 on autophagy-related proteins LC3, P62, ATG16L, and ATG4B in macrophages infected with Mycobacterium tuberculosis, as well as potential related signaling pathways and mechanisms, were investigated.

[0045] (II) Immunofluorescence detection of LC3 spots THP-1 cells in logarithmic growth phase were injected with 5 × 10⁻⁶ cells. 5 Cells / well were seeded in confocal dishes and stimulated with PMA (50 ng / mL) for 24 h. Then, H37Ra bacterial suspension was added to each well at MOI=1 and incubated for 24 h. Extracellular H37Ra was washed away three times with PBS. After incubation with 20 nM X79 for 1 h, the cells were then incubated under an LED light source (630 nm, 106 mW / cm²). 2 Cells were irradiated for 1 minute, while the control group received no treatment. Incubation continued. After 24 hours, the culture medium was removed, cells were washed three times with PBST, fixed with 4% paraformaldehyde at room temperature for 30 minutes, and washed three times with PBST. Cells were permeabilized with pre-cooled methanol at -20°C for 15 minutes, washed three times with PBST, and blocked overnight with 5% BSA. Cells were washed three times with PBST and incubated overnight at 4°C with anti-LC3B antibody. Cells were washed three times with PBST, and incubated with fluorescent secondary antibody (1:1000 dilution) in the dark for 2 hours. Cells were washed three times with PBST in the dark. Then, DAPI was added to stain the nuclei for 15 minutes, followed by three washes with PBST. The number of LC3 fluorescent spots was detected using a laser scanning confocal microscope.

[0046] (III) Transcriptomics sequencing THP-1 cells in logarithmic growth phase were fed at a rate of 2 × 10⁻⁶. 6 Cells were seeded per well in confocal dishes, stimulated with PMA (50 ng / mL) for 24 hours and cultured overnight. Then, H37Ra bacterial suspension was added to each well at MOI=1 and incubated for 24 hours. Extracellular H37Ra was washed away three times with PBS. After incubation with 20 nM X79 for 1 hour, followed by LED illumination for 1 minute, and then incubation for another 23 hours. Next, 1 mL of TRIzol reagent was added to each well, and the cells were repeatedly pipetted to ensure thorough contact and digestion. The solution was then transferred to cryovials, and the cells were repeatedly pipetted using a disposable syringe until no cell clumps were visible, resulting in a clear and non-viscous solution. The solution was then flash-frozen in liquid nitrogen and sent for analysis.

[0047] result The expression level of autophagy protein LC3 in macrophages was detected. The results showed that, compared with the control group, the X79+L group exhibited increased LC3-II levels and decreased LC3-I expression (…). Figure 5 A). As a marker protein of autophagy, LC3 exists in two different forms during this process: During autophagy, ATG4B cleaves the precursor LC3 (pro-LC3) into LC3-I. Cytoplasmic soluble LC3-I is converted into the lipid-bound membrane-associated form LC3-II, which promotes autophagosome membrane formation. The LC3-II to LC3-I ratio is often used as a criterion for determining whether autophagy has occurred. In this process, ATG16L forms a protein complex with Atg12 and Atg5, promoting LC3 lipidation. The results showed that X79 could increase the expression levels of LC3-II / LC3-I and ATG16L under light conditions. Figure 5 BC), while reducing ATG4B expression ( Figure 5 E), thereby promoting autophagy. Confocal LC3 fluorescence spots also confirmed its upregulation (E). Figure 6 AB). P62 / SQSTM1 acts as an autophagy substrate / cargo receptor, mediating the degradation of ubiquitinated proteins and further regulating autophagy. Typically, protein expression levels decrease during autophagy. The results showed that P62 expression was indeed reduced after X79 treatment ( ). Figure 5 (D) This confirms that autophagosomes successfully degraded their contents. Furthermore, transcriptome analysis compared the X79+L group and the infected group, identifying 283 significantly different features, including 130 upregulated and 153 downregulated features. Figure 7 A). Differential GO enrichment analysis showed a consistent enrichment trend for lysosomal / autophagy-related cellular components, suggesting that the lysosomal-autophagy axis may be preferentially activated at the transcriptional level. Figure 7 B).

[0048] Example 3: X79-mediated photodynamic therapy significantly inhibited extracellular and intracellular Mycobacterium tuberculosis infecting macrophages. (I) The bactericidal effect of phenothiazine compound X79 on macrophages infected with Mycobacterium tuberculosis. Direct bactericidal effect: 500µl containing 4×10 5 CFU / ml H37Ra in 7H9 liquid medium (a suspension containing H37Ra) and different treatment groups were co-cultured in 1.5ml EP tubes for 24 hours. The samples were then sequentially diluted 10-fold, 100-fold, and 1000-fold. 100μl of each dilution was inoculated onto 7H11 agar plates. Colony counting was performed after incubation at 37℃ for 3-4 weeks. The treatments for each group during co-culture were as follows: (1) Control group: 500 µl of 7H9 liquid culture medium was added to the suspension containing H37Ra; (2) Control+Light group: 500 µl of 7H9 liquid culture medium was added to the suspension containing H37Ra and incubated for 1 h, then the suspension was heated with an LED light source (630 nm, 106 mW / cm²). 2 (2) Irradiate for 1 minute; (3) Rif group: Add 500µl of Rif solution (20ng / ml) to the suspension containing H37Ra; (4) Rif+Light group: Add 500µl of Rif solution (20ng / ml) to the suspension containing H37Ra and incubate for 1 hour, then use an LED light source (630nm, 106mW / cm²) to irradiate for 1 minute; (5) Rif+Light group: Add 500µl of Rif solution (20ng / ml) to the suspension containing H37Ra and incubate for 1 hour, then use an LED light source (630nm, 106mW / cm²) to irradiate for 1 minute; (6) Rif+Light group: Add 500µl of Rif solution (20ng / ml) to the suspension containing H37Ra and incubate for 1 hour, then use an LED light source (630nm, 2 (5) X79 group: Add 500µl of X79 solution (40nM) to the suspension containing H37Ra; (6) X79+Light group: Add 500µl of X79 solution (40nM) to the suspension containing H37Ra; After co-incubation for 1h, use LED light source (630nm, 106mW / cm) 2 Irradiate for 1 minute.

[0049] Intracellular bactericidal effect: THP-1 cells were cultured to the logarithmic growth phase and then subjected to intracellular sterilization at a concentration of 5 × 10⁻⁶ cells / cells. 5THP-1 cells were seeded at a density of 2 mL / mL in sterile 6-well plates, with 2 mL of RPMI 1640 medium added to each well. The cells were then stimulated with 50 ng / mL PMA solution to induce differentiation and adhesion. After cell adhesion, a suspension of Mycobacterium tuberculosis (H37Ra) was added, with a multiplicity of infection (MOI) of 1. After culturing at 37°C and 5% CO2 for 24 hours, the medium in each well was discarded, and the cells were washed three times with sterile PBS. Following Mycobacterium tuberculosis infection, 20 nM of phenothiazine compound X79 and 10 ng / mL of Rif were added, respectively. The control group received an equal volume of RPMI 1640 medium. The light-treated group was treated with an LED light source (630 nm, 106 mW / cm²) 1 hour after drug treatment. 2 Irradiate for 1 minute, then continue culturing; the non-light-treated group receives no treatment. After co-culturing for 24 hours, lyse cells with 0.1% Triton X-100 and collect the supernatant. Dilute the samples sequentially 10-fold, 100-fold, and 1000-fold with PBS. Inoculate 100 μl of each dilution onto 7H11 agar plates. Incubate at 37°C for 3-4 weeks, then perform colony counting.

[0050] (II) The bactericidal effect of X79 on Mtb in mouse spleen mononuclear cells Purified mouse spleen lymphocytes were seeded into 12-well plates and cultured for 48 hours. After washing away suspended mononuclear cells, adherent cells were digested with trypsin and counted. Cells were infected with H37Ra at an MOI of 1 for 24 hours. Extracellular H37Ra was washed away with PBS, and 20 nM of phenothiazine compound X79 and 10 ng / ml of Rif were added. The control group received an equal volume of RPMI 1640 medium. The light-treated group was exposed to an LED light source (630 nm, 106 mW / cm²) 1 hour after drug treatment. 2 Irradiate for 1 minute, then continue culturing; the non-light-treated group receives no treatment. After co-incubation for 24 hours, lyse cells with 0.1% Triton X-100 and collect the supernatant. Dilute the samples sequentially 10-fold, 100-fold, and 1000-fold with PBS. Inoculate 100 μl of each dilution onto 7H11 agar plates. Incubate at 37°C for 3-4 weeks, then perform colony counting.

[0051] To further verify the anti-tuberculosis therapeutic potential of X79, its direct killing effect on H37Ra strain and its efficacy in clearing H37Ra within macrophages were evaluated. (See 7H11 plate colony images.) Figure 8 A), X79 alone showed a bactericidal trend against H37Ra, but this was not statistically significant. However, after light exposure, the bactericidal effect of X79 was significantly enhanced. Figure 8B). This phenomenon may be related to the generation of light-induced reactive oxygen species (ROS), as ROS can damage the cellular structure of Mycobacterium tuberculosis, ultimately leading to its death. Mycobacterium tuberculosis, as a typical intracellular parasite, primarily infects macrophages. Most antibiotics have poor permeability, making it difficult to effectively penetrate cells, which is a key reason for prolonged treatment cycles and limited vaccine efficacy. Therefore, the intracellular bactericidal activity of X79 against the H37Ra strain was further evaluated. The results showed that the bactericidal effect of rifampin was significantly reduced intracellularly, while X79 irradiated with LEDs significantly inhibited the proliferation of H37Ra in macrophages and mouse spleen-derived macrophages. Figure 8 CF).

[0052] Example 4: Therapeutic effect of X79-mediated photodynamic therapy on a mouse model of cutaneous tuberculosis A CTB mouse model was established to further examine its anti-tuberculosis effect. Eight-week-old BALB / c mice were acclimatized in the laboratory for two weeks and then divided into six groups. One group (uninfected) received a subcutaneous injection of 50 µl of physiological saline. The remaining five groups received subcutaneous injections of physiological saline containing H37Ra (50 µl, 4 × 10⁻⁶ ml). 8 After one week of infection observation (CFU / ml), the six groups of mice were treated as follows: (1) Uninfected group (100 μL normal saline); (2) Infected group (100 μL normal saline); (3) Rifampicin group (100 μL, 1.04 μg / kg); (4) Rifampicin group (100 μL, 1.04 μg / kg); 106 mW / cm 2 LED light irradiation for 10 minutes; (5) X79 group (100 μL, 2 mg / kg); (6) X79 group (100 μL, 2 mg / kg); 106 mw / cm 2 The drug was administered under LED light for 10 minutes. Laser irradiation was performed 1 hour after injection. Body weight and skin cysts were monitored after drug administration. Mice were sacrificed on day 12, and orbital blood and organs were collected. Except for the uninfected group, the skin cysts of other groups were homogenized, spread on 7H11 culture plates, and counted after 3-4 weeks.

[0053] To further investigate the anti-tuberculosis therapeutic effect of X79-mediated photodynamic therapy in vivo, a mouse skin tuberculosis model was constructed, and different treatment groups were observed for 7 days. Results showed that, compared with the control group, the size of the skin lesions in the X79+Light group was significantly reduced. Figure 9 AB); and throughout the entire experimental period, the body weight of mice in each group did not fluctuate significantly, demonstrating that the drug treatment did not cause significant acute toxicity or severe physiological stress in the mice ( ). Figure 9C). After the experiment, the spleen weight of mice in the drug-treated group increased, possibly because the drug activated the spleen's immune response to combat Mtb (C). Figure 9 D). Subsequently, the growth of H37Ra among different treatment groups was statistically analyzed ( Figure 10 A), the results showed that X79 exhibited excellent bactericidal effects under light conditions. On 7H11 culture plates, compared to the control group, the colony count in the X79+Light group was significantly lower (…). Figure 10 B), and its bactericidal effect is stronger than that of the rifampicin group and the X79 group.

[0054] This invention utilizes the novel phenothiazine compound X79 in photodynamic therapy to achieve anti-tuberculosis effects. This compound not only exhibits excellent biocompatibility and low effective concentrations at the nanomolar level, but also inhibits intracellular Mycobacterium tuberculosis by activating the autophagy pathway. By combining direct and host-directed therapy, it more effectively kills and eliminates Mycobacterium tuberculosis in macrophages, providing a new direction for anti-tuberculosis drug development.

[0055] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The application of phenothiazine compounds in the preparation of antituberculosis drugs, characterized in that, The phenothiazine compound is 9-(diethylamino)-3-methyl-5-(propylamino)pyrido[3,2-a]phenothiazine-7-onium chloride, with the following structural formula: 。 2. The application of phenothiazine compounds in the preparation of drugs for inhibiting Mycobacterium tuberculosis infection, characterized in that, The phenothiazine compound is 9-(diethylamino)-3-methyl-5-(propylamino)pyrido[3,2-a]phenothiazine-7-onium chloride, with the following structural formula: 。

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