Use of atractylodes lactone ii in multiple drug-resistant bacteria infectious diseases

Abstract

The present application relates to the application of atractylodes lactone II in the preparation of a drug for treating multiple drug-resistant bacteria infectious diseases, the multiple drug-resistant bacteria being non-tuberculous mycobacteria, Mycobacterium tuberculosis and methicillin-resistant Staphylococcus aureus. The present application also provides the application of atractylodes lactone II in the preparation of a drug for treating abscess mycobacterium pulmonary infection disease and the application of atractylodes lactone II in the preparation of a drug for treating methicillin-resistant Staphylococcus aureus chronic infection disease. Atractylodes lactone II has considerable clinical application prospect in treating non-tuberculous mycobacteria, Mycobacterium tuberculosis and methicillin-resistant Staphylococcus aureus chronic infection diseases. It provides scientific basis and support for multiple drug-resistant bacteria infectious diseases.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and more specifically, to the application of atractylodes lactone II in multidrug-resistant bacterial infections. Background Technology

[0002] Multidrug-resistant organisms (MDROs) are bacteria that exhibit resistance to three or more classes of antibiotics used clinically. Common MDROs include methicillin-resistant Staphylococcus aureus (MRSA), multidrug-resistant / pan-drug-resistant Pseudomonas aeruginosa, multidrug-resistant Mycobacterium tuberculosis (MTB), and nontuberculous mycobacteria (NTMs). NTM pulmonary infections are often caused by Mycobacterium abscessus (MAB) and Mycobacterium avium (MAC). Infections caused by MDROs are characterized by complexity and difficulty in treatment. Coupled with factors such as pathogen immune escape, insufficient intracellular clearance efficacy of antibiotics, long treatment cycles, severe side effects, and a high recurrence rate, chronic MDRO infections pose a significant clinical challenge.

[0003] After pathogens infect the host, macrophages, as the main immune sentinel cells, first recognize and engulf the invading pathogens. The invading pathogens can develop a variety of strategies to evade macrophage killing and adapt to the host cell environment, replicating and surviving in the cell[1]. Staphylococcus aureus / MRSA, as a representative of Gram-positive bacteria, is traditionally considered to be a typical extracellular bacteria. Its pathogenicity is mainly caused by exotoxins and other factors that cause inflammatory reactions of varying degrees. After effective antibacterial drugs kill the pathogens, the infection can be quickly controlled. In November 2015, Nature published a new discovery by Lehar SM's team—the traditional extracellular bacteria MRSA can survive in tissue cells / phagocytes and escape / colonize. Vancomycin's MIC (>100 μg / ml) for clearing intracellular MRSA bacteria is more than 100 times that for clearing extracellular bacteria (1 μg / ml)[2]. Mycobacterium tuberculosis (MTB) and nontuberculous mycobacteria (NTM) are facultative intracellular parasites that survive long-term within host cells by preventing phagosome acidification and maturation or escaping from phagosomes into the nutrient-rich cytoplasm [3]. Intracellular replication and survival promote recurrent MDRO infections or exacerbations.

[0004] Atractylodes macrocephala is a traditional Chinese medicine belonging to the category of tonifying qi and strengthening the body. It is the dried rhizome of Atractylodes macrocephala, a plant of the Asteraceae family. It has a fragrant aroma, is warm in nature, and tastes bitter and sweet. It enters the spleen and stomach meridians and has the effects of strengthening the spleen and strengthening qi, drying dampness and promoting diuresis, stopping sweating and calming the fetus [4]. Atractylenolides (AT) are sesquiterpenoid compounds, including AT-I, AT-II and AT-III. As the main bioactive components of Atractylodes macrocephala, they have a variety of biological effects. In vitro studies have shown that AT-I and AT-III have an inhibitory effect on Staphylococcus aureus, but no studies have reported the antibacterial activity of AT-II [5]. The antitumor effect of AT-II has been extensively studied. AT-II administration can improve the efficacy of chemotherapy for colorectal cancer and melanoma [6,7], and may be a potential strategy for the clinical treatment of endometrial cancer [8]. AT-II inhibits M2 tumor-associated macrophages and reduces tumor metastasis [9]. In addition, studies have shown that AT-II reduces hepatic sn-1,2-DAG levels, inhibits PKCε activity, and improves obesity-induced hyperlipidemia, hepatic steatosis, and insulin resistance

[10] . AT-II inhibits radiation-induced lung injury by regulating the MAPKP38 / NRF2 signaling pathway

[11] . AT-II may inhibit oxidative stress in myocardial fibrosis and spontaneous hypertension

[12] . AT-II inhibits platelet activation and may be a potential candidate drug for preventing thrombosis

[13] . Although the pharmacological effects of AT-II have been extensively studied, its effect in inhibiting chronic MDRO infection remains unexplained, and exploring its new uses is of great significance.

[0005] Chinese patent document CN110585435A discloses a composition with anticancer effects and its application. The active ingredients of the composition include atractylodes lactone II and a Chk1 inhibitor. The composition with anticancer effects of the present invention combines atractylodes lactone II and a Chk1 inhibitor, and through their synergistic effect, they exert a significant synergistic effect, which is much better than the anticancer effect of atractylodes lactone or Chk1 inhibitor alone.

[0006] Chinese patent document CN110548025A discloses the use of atractylodes lactone II in the preparation of a drug to improve insulin resistance and glucose-lipid metabolism disorders caused by obesity. After high-fat feeding, mice showed increased body weight, significantly reduced respiratory entropy, and decreased energy expenditure, indicating that obese mice shifted from predominantly glucose metabolism to predominantly lipid metabolism, exhibiting adipose tissue lesions and increased lipid droplets. Simultaneously, plasma and liver indicators confirmed liver damage and insulin resistance in the mice. This invention found that after treatment with atractylodes lactone II, the body weight and glucose-lipid metabolism of obese mice were improved, liver lipid droplets decreased, and insulin resistance was restored. Therefore, atractylodes lactone II can effectively improve insulin resistance and glucose-lipid metabolism disorders caused by obesity.

[0007] There are currently no reports on the application of atractylodes lactone II in multidrug-resistant bacterial infections. Summary of the Invention

[0008] The purpose of this invention is to address the shortcomings of the prior art by providing the application of atractylodes lactone II in multidrug-resistant bacterial infections.

[0009] In a first aspect, the present invention provides the use of atractylodes lactone II in the preparation of a drug for treating infectious diseases caused by multidrug-resistant bacteria, wherein the multidrug-resistant bacteria are Mycobacterium abscessis, Mycobacterium tuberculosis, and methicillin-resistant Staphylococcus aureus.

[0010] As a preferred example, the atractylodes lactone II is prepared into a pharmaceutical formulation by adding pharmaceutically acceptable excipients and a carrier, wherein the excipients include at least one of fillers, disintegrants, binders, excipients, diluents, lubricants, sweeteners, and colorants.

[0011] Secondly, the present invention provides the use of atractylodes lactone II in the preparation of drugs for treating infectious diseases caused by multidrug-resistant bacteria, wherein the drug-resistant bacteria are selected from one, two, or three of Mycobacterium abscessus, Mycobacterium tuberculosis, or methicillin-resistant Staphylococcus aureus.

[0012] As a preferred example, the atractylodes lactone II is prepared into a pharmaceutical formulation by adding pharmaceutically acceptable excipients and a carrier, wherein the excipients include at least one of fillers, disintegrants, binders, excipients, diluents, lubricants, sweeteners, and colorants.

[0013] Thirdly, the present invention provides the use of atractylodes lactone II in the preparation of a drug for treating lung infections caused by Mycobacterium abscessis.

[0014] As a preferred example, the atractylodes lactone II is prepared into a pharmaceutical formulation by adding pharmaceutically acceptable excipients and a carrier, wherein the excipients include at least one of fillers, disintegrants, binders, excipients, diluents, lubricants, sweeteners, and colorants.

[0015] Fourthly, the present invention provides the use of atractylodes lactone II in the preparation of drugs for treating chronic infections caused by methicillin-resistant Staphylococcus aureus.

[0016] As a preferred example, the chronic infection refers to one or more of the lungs, liver, kidneys, or abdominal cavity.

[0017] As a preferred example, the atractylodes lactone II is prepared into a pharmaceutical formulation by adding pharmaceutically acceptable excipients and a carrier, wherein the excipients include at least one of fillers, disintegrants, binders, excipients, diluents, lubricants, sweeteners, and colorants.

[0018] The advantages of this invention are as follows: Experiments have shown that atractylodes lactone II significantly reduces the intracellular survival of MAB, H37Rv, and MRSA in macrophages. Atractylodes lactone II significantly reduces the bacterial load in MAB-infected lungs in vivo. Atractylodes lactone II significantly reduces the bacterial load in the lungs, liver, and kidneys of mice infected with MRSA; simultaneously, 60 mg / kg and 120 mg / kg of atractylodes lactone II significantly reduce the total, extracellular, and intracellular bacterial load in mouse peritoneal lavage fluid; atractylodes lactone II significantly reduces the bacterial load in chronic MRSA infection in vivo. In summary, atractylodes lactone II has considerable clinical application potential in treating chronic infections caused by nontuberculous mycobacteria, Mycobacterium tuberculosis, and methicillin-resistant Staphylococcus aureus. It provides scientific evidence and support for the treatment of multidrug-resistant bacterial infections. Attached Figure Description

[0019] Figure 1 Intracellular survival of macrophages infected with multidrug-resistant organisms (MDROs). (A) Mycobacterium abscessus MAB infection (MOI=3); (B) Mycobacterium tuberculosis H37Rv infection (MOI=5); (C) Methicillin-resistant Staphylococcus aureus (MRSA) infection (MOI=20).

[0020] Figure 2 Bacterial load in the lungs of C57BL / 6 mice infected with Mycobacterium abscessus.

[0021] Figure 3 MRSA-infected C57BL / 6 mice were tested for bacterial load. (AC) Bacterial load in the lungs, liver, and kidneys. (DF) Total, extracellular, and intracellular bacterial load in peritoneal lavage fluid. Detailed Implementation

[0022] The present invention will be further described below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, it should be understood that after reading the description of this invention, those skilled in the art can make various alterations or modifications to the invention, and these equivalent forms also fall within the scope defined by the appended claims.

[0023] Example 1

[0024] 1. AT II method for inhibiting intracellular survival of multidrug-resistant organisms (MDRO) infection: (1) Primary peritoneal macrophages were extracted from the peritoneal cavity of C57BL / 6J mice. Three days before extraction, 2 ml of 3% sterile Brewer modified thioglycolate broth was injected into the peritoneum of each mouse. The mice were placed in a supine position in a laminar flow hood for extraction. The abdominal skin tissue of the mice was cut along the midline of the abdomen to expose the peritoneum. 10 ml of sterile 1640 culture medium was injected into the peritoneum of the mice with a syringe. The needle was carefully removed, and the peritoneum was slowly lifted and massaged. ～ 2 min. Using a 10 ml syringe inserted below the peritoneum, slowly aspirate the cell suspension from the peritoneal cavity and collect it into a sterile 50 ml centrifuge tube. Centrifuge at 1000 rpm for 5 min, discard the supernatant, and retain the white cell pellet. Resuspend the cells in 20 ml of RPMI 1640 complete medium containing 10% fetal bovine serum and 1% penicillin-dextrose antibody, count the cells, and plate them.

[0025] (2) Mycobacterium abscessus (MAB) infection: 1.5 × 10⁻⁶ cells were injected into the abscess at an MOI (bacteria:cells) ratio of 3. 6 Add the bacterial culture to the well plate, mix well in a cross shape, and incubate in a cell culture incubator for 2 hours to kill extracellular bacteria. Wash 2-3 times with sterile PBS, then add 200 μg / ml amikacin culture medium and incubate in a cell culture incubator for 1 hour to kill extracellular bacteria. Add 20 μM AT-II or 2.5 μg / ml clarithromycin (10×MIC), co-incubate with cells for 24 hours, discard the supernatant, and wash 2 times with sterile PBS. ～ Three times, add 1 ml of 0.1% Triton X-100 to lyse macrophages and release intracellular bacteria phagocytosed by the macrophages. Dilute serially and plate on 7H10 agar plates. Mycobacterium tuberculosis (H37Rv) infection: Infect peritoneal macrophages with H37Rv (MOI=5). After 4 hours of infection, discard the bacterial culture medium and wash twice with sterile PBS to remove extracellular bacteria. Add 20 μM AT-II or 0.5 μg / ml isoniazid (INH) and co-incubate with cells for 24 hours. Discard the supernatant, wash 2-3 times with sterile PBS, add 1 ml of 0.1% Triton X-100 to lyse macrophages and release intracellular bacteria phagocytosed by the macrophages. Dilute serially and plate on 7H10 agar plates.

[0026] Methicillin-resistant Staphylococcus aureus (MRSA infection): RAW264.7 macrophages were plated at 2 × 10⁶ cells per well. 5Cells were cultured at MOI = 20, with a phagocytosis time of 1 hour. Extracellular MRSA residues were thoroughly removed using lysostaphin. Cells were cultured in an incubator for 24 hours to simulate the intracellular colonization environment of MRSA. Subsequently, AT-II 20 μM or IFN-γ (40 ng / mL) prepared using DMEM infection medium was added to the corresponding wells, and the cells were cultured for another 24 hours. The supernatant was discarded, and the cells were washed twice with sterile PBS. ～ Three times, add 1 ml of 0.1% Triton X-100 to lyse macrophages and release intracellular bacteria phagocytosed within the macrophages. Perform serial dilutions and plate onto LB agar plates.

[0027] Result: As Figure 1 As shown, AT-II significantly reduced the intracellular survival of MAB, H37Rv, and MRSA in macrophages.

[0028] 2. AT II Inhibition of Mycobacterium abscessus Lung Infection Method: C57BL / 6J mice were administered dexamethasone (5 mg / kg) via gavage starting 7 days before infection and continuing until day 3 post-infection. Mice were infected via tracheal inhalation on day 0, with an infection dose of 5 × 10⁻⁶ mg / kg per mouse. 7 CFU. On day 3 post-infection, mice were administered different doses of AT-II (60 mg / kg, 120 mg / kg) or clarithromycin (150 mg / kg) via gavage, with AT-II administered twice daily and clarithromycin once daily. After 14 days of continuous intervention, mice were sacrificed, and lung homogenates were collected, diluted, and plate-spread for counting.

[0029] Result: The results show ( Figure 2 Compared with the model group, AT-II at 60 mg / kg and 120 mg / kg significantly reduced the bacterial load in the lungs of infected mice, and the in vivo antibacterial effect of AT-II at 120 mg / kg was not significantly different from that of the positive control antibiotic, indicating that AT-II can significantly reduce the bacterial load of Mycobacterium abscessis in the lungs in vivo.

[0030] 3. Method for AT-II to inhibit chronic infection by methicillin-resistant Staphylococcus aureus: Mice were infected with methicillin-resistant Staphylococcus aureus (MRSA) via intraperitoneal injection, with an infection dose of 5 × 10⁻⁶ per mouse. 8CFU. Mice were administered linezolid (10 mg / kg) by gavage 2 hours before infection, once daily for 7 days post-infection. Linezolid administration was discontinued on day 8 post-infection. On day 9 post-infection, mice were administered different doses of AT-II (60 mg / kg or 120 mg / kg) or linezolid (10 mg / kg) by gavage, with AT-II administered once daily and linezolid once daily for 7 days. Mice were sacrificed on day 15 post-infection, and peritoneal lavage fluid, lungs, liver, and kidneys were collected. The peritoneal lavage fluid was divided into three aliquots. Total bacterial load was determined by direct dilution and plate counting. Extracellular bacterial load was determined by dilution and plate counting of the supernatant after low-speed centrifugation (300g, 3min). Intracellular bacterial load was determined by collecting the lower cell pellet after low-speed centrifugation (300g, 3min), thoroughly killing any remaining extracellular bacteria with lysin, lysing the cells with Triton-X 100, followed by dilution and plate counting. Lung, liver, and kidney samples were homogenized, diluted, and plated for counting.

[0031] Result: The results show ( Figure 3 Compared with the model group, AT-II at 60 mg / kg and 120 mg / kg significantly reduced the bacterial load in the lungs, liver, and kidneys of mice. Figure 3 AC); meanwhile, AT-II at 60 mg / kg and 120 mg / kg significantly reduced the total, extracellular, and intracellular bacterial load in mouse peritoneal lavage fluid (AC); Figure 3 (DF). In summary, this suggests that AT-II can significantly reduce the bacterial load in chronic MRSA infection in vivo.

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[0051] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. The application of atractylodes lactone II in the preparation of drugs for treating infectious diseases caused by multidrug-resistant bacteria, characterized in that, The multidrug-resistant bacteria mentioned are Mycobacterium abscessus, Mycobacterium tuberculosis, and methicillin-resistant Staphylococcus aureus.

2. The application of atractylodes lactone II in the preparation of drugs for treating multidrug-resistant bacterial infections, characterized in that, The drug-resistant bacteria are selected from one or more of Mycobacterium abscessus, Mycobacterium tuberculosis, or methicillin-resistant Staphylococcus aureus.

3. The application of atractylodes lactone II in the preparation of drugs for treating infectious diseases caused by multidrug-resistant bacteria, characterized in that, The multidrug-resistant bacteria are nontuberculous mycobacteria, tuberculous mycobacteria, and methicillin-resistant Staphylococcus aureus.

4. Application of atractylodes lactone II in the preparation of drugs for treating lung infections caused by Mycobacterium abscessus.

5. Application of atractylodes lactone II in the preparation of drugs for treating chronic infections caused by methicillin-resistant Staphylococcus aureus.

6. The application according to claim 5, characterized in that, The chronic infection refers to one or more of the lungs, liver, kidneys, or abdominal cavity.

7. The application according to any one of claims 1-4, characterized in that, The atractylodes lactone II is prepared into a pharmaceutical formulation by adding pharmaceutically acceptable excipients and carriers, wherein the excipients include at least one of fillers, disintegrants, binders, excipients, diluents, lubricants, sweeteners, and colorants.

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