Co-administrered formulation for alleviating Anti-tuberculosis drug-induced toxicity comprising lactobacillus sakei CVL-001 or lactobacillus reuteri nchbl-005, and pharmaceutical composition for preventing or treating tuberculosis comprising same

Abstract

The present invention relates to a co-administered formulation for alleviating anti-tuberculosis drug-induced toxicity comprising dead cells, and a pharmaceutical composition for preventing or treating tuberculosis comprising same. By co-administering Lactobacillus sakei CVL-001 or Lactobacillus reuteri NCHBL-005 as the dead cells with the anti-tuberculosis drug, the present invention exhibits excellent effects in alleviating drug toxicity (hepatotoxicity and nephrotoxicity) caused by the anti-tuberculosis drug, thereby increasing drug adherence of the anti-tuberculosis drug to obtain the effect of inhibiting the emergence of multidrug-resistant tuberculosis and recurrence of tuberculosis. In addition, the present invention can be used as an adjuvant for co-administration with drugs that have high efficacy despite significant side effects, such as bedaquiline or linezolid, with the expected effect of reducing the treatment period of tuberculosis, thereby being effectively used in the treatment process of tuberculosis.

Description

Combination formulation for alleviating antituberculosis drug toxicity induced by Lactobacillus sakei CVL-001 or Lactobacillus reuteri NCHBL-005, and pharmaceutical composition for preventing or treating tuberculosis comprising the same

[0001] This patent application claims priority to Republic of Korea Patent Application No. 10-2023-0145764, filed with the Korean Intellectual Property Office on October 27, 2023, the disclosure of which is incorporated herein by reference.

[0002] The present invention relates to a combination administration preparation for alleviating drug toxicity induced by an antituberculosis agent, including kimchi-derived Lactobacillus sakei CVL-001 or honeybee-derived Lactobacillus reuteri NCHBL-005, and a pharmaceutical composition for preventing or treating tuberculosis, including the same. More specifically, the present invention relates to a therapeutic technique for alleviating drug toxicity induced by an antituberculosis agent by using killed cells of Lactobacillus sakei CVL-001 or Lactobacillus reuteri NCHBL-005 in combination with the antituberculosis agent.

[0003] Tuberculosis (TB) is one of the three major infectious diseases threatening human health, designated by the World Health Organization (WHO). The primary causative agent of TB is Mycobacterium tuberculosis (Mtb), and approximately one-third of the world's population has been exposed to Mtb infection. According to the WHO's [Global TB Report 2022], there are an estimated 5.8 million new cases of TB worldwide.

[0004] The most common drugs used to treat tuberculosis are isoniazid, rifampin, ethambutol, and pyrazinamide. These drugs work by killing or stopping the growth of tuberculosis bacteria. The specific combination of drugs and the duration of treatment vary depending on the type of tuberculosis infection, whether it is drug-resistant, and the patient's overall health. However, a quadruple therapy is commonly used, consisting of isoniazid, rifampin, ethambutol, and pyrazinamide for two months after diagnosis. This is followed by four months of isoniazid and rifampin.

[0005] To completely cure the infection and prevent the development of drug-resistant tuberculosis and relapse, it is crucial to take anti-tuberculosis medications exactly as prescribed for the entire treatment period. However, some patients fail to adhere to anti-tuberculosis medications due to side effects such as nausea, vomiting, liver damage, kidney damage, and vision loss.

[0006] In particular, antibiotics used to treat drug-resistant tuberculosis, such as bedaquiline and linezolid, are effective but have fatal side effects, so they are prescribed only after close monitoring.

[0007] The present inventors conducted research to alleviate hepatotoxicity and nephrotoxicity, among the various side effects that inevitably accompany antituberculosis drugs, and discovered that when specific bacteria were administered together, indicators of hepatotoxicity and nephrotoxicity were restored.

[0008] Accordingly, the inventors of the present invention confirmed that, by using killed cells of Lactobacillus sakei CVL-001 or Lactobacillus reuteri NCHBL-005 in combination with an anti-tuberculosis drug, these killed cells have an excellent effect in alleviating drug toxicity caused by the anti-tuberculosis drug.

[0009] Accordingly, the purpose of the present invention is to provide a combination administration preparation for alleviating antituberculosis drug toxicity, which comprises at least one strain selected from the group consisting of Lactobacillus sakei and Lactobacillus reuteri.

[0010] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating tuberculosis, comprising at least one strain selected from the group consisting of Lactobacillus sakei and Lactobacillus reuteri; and an anti-tuberculosis agent.

[0011] Another object of the present invention relates to the use of at least one strain selected from the group consisting of Lactobacillus sakei and Lactobacillus reuteri in combination with an antituberculosis agent for the treatment of tuberculosis.

[0012] The present invention relates to a combination formulation for alleviating drug toxicity induced by an antituberculosis agent, including Lactobacillus sakei CVL-001 or Lactobacillus reuteri NCHBL-005, and a pharmaceutical composition for preventing or treating tuberculosis, including the same. The combination formulation and pharmaceutical composition according to the present invention alleviate drug toxicity induced by an antituberculosis agent and exhibit an effect capable of treating tuberculosis.

[0013] The present inventors confirmed that the combined use of killed cells of Lactobacillus sakei CVL-001 or Lactobacillus reuteri NCHBL-005 with an antituberculosis drug has an excellent effect in alleviating drug toxicity caused by the antituberculosis drug.

[0014] Hereinafter, the present invention will be described in more detail.

[0015] One aspect of the present invention is a combination administration preparation for alleviating antituberculosis-induced drug toxicity, comprising at least one strain selected from the group consisting of Lactobacillus sakei and Lactobacillus reuteri.

[0016] In the present invention, the Lactobacillus sakei may be the Lactobacillus sakei CVL-001 strain deposited under the accession number KCTC13816BP.

[0017] The daily dose of the above Lactobacillus sakei is 5 X 10 per 1 kg of subject body weight. 7 5 X 10 13 It may be CFU, preferably 5 X 10 7 5 X 10 12 CFU, 5 X 10 7 5 X 10 11 CFU, 5 X 10 7 5 X 10 10 CFU, 5 X 10 8 5 X 10 13 CFU, 5 X 10 8 5 X 10 12 CFU, 5 X 10 8 5 X 10 11 CFU, 5 X 10 8 5 X 10 10 CFU, 5 X 10 9 5 X 10 13 CFU, 5 X 10 9 5 X 10 12 CFU, 5 X 10 9 5 X 10 11 CFU, 5 X 10 9 5 X 10 10 CFU, 5 X 10 10 5 X 10 13 CFU, or 5 X 10 10 5 X 10 12It could be CFU, for example, 5 X 10 10 5 X 10 11 It may be, but is not limited to, CFU.

[0018] In the present invention, the Lactobacillus reuteri may be the Lactobacillus reuteri NCHBL-005 strain deposited under accession number KCTC15449BP, which has the effect of improving the migration and proliferation of fibroblasts.

[0019] The above Lactobacillus reuteri NCHBL-005 strain may include a 16S rRNA base sequence represented by sequence number 1.

[0020] The daily dose of the above Lactobacillus reuteri is 5 X 10 per 1 kg of subject body weight. 6 5 X 10 12 It may be CFU, preferably 5 X 10 6 5 X 10 11 CFU, 5 X 10 6 5 X 10 10 CFU, 5 X 10 6 5 X 10 9 CFU, 5 X 10 7 5 X 10 12 CFU, 5 X 10 7 5 X 10 11 CFU, 5 X 10 7 5 X 10 10 CFU, 5 X 10 7 5 X 10 9 CFU, 5 X 10 8 5 X 10 12 CFU, 5 X 10 8 5 X 10 11 CFU, 5 X 10 8 5 X 10 10 CFU, 5 X 10 8 5 X 10 9 CFU, 5 X 10 9 5 X 10 12CFU, or 5 X 10 9 5 X 10 11 It could be CFU, for example, 5 X 10 9 5 X 10 10 It may be, but is not limited to, CFU.

[0021] In the present invention, the strain may be a killed cell. The killed cell may be a killed cell produced by performing one or more methods selected from the group consisting of heat killing, ultraviolet treatment, radiation irradiation, ultrasonic pulverization, pressure control, freeze-drying, tyndallization, crushing, and chemical treatment on the strain, and for example, it may be a killed cell produced by heat treatment, but is not limited thereto.

[0022] The above heat treatment may be performed by applying a temperature of 70 to 120°C to the strain for 15 to 60 minutes, preferably by applying a temperature of 80 to 110°C for 20 to 45 minutes, and for example, by applying a temperature of 90 to 100°C for 25 to 30 minutes, but is not limited thereto.

[0023] The above antituberculosis drug toxicity may be selected from, but is not limited to, the group consisting of iatrogenic hepatotoxicity, nephrotoxicity and inflammatory reactions associated with antituberculosis drugs.

[0024] In the present invention, the antituberculosis agent may be at least one selected from the group consisting of isoniazid, rifampin, ethambutol, pyrazinamide, bedaquiline, and linezolid, preferably at least two selected from the group consisting of isoniazid and rifampin, or bedaquiline and linezolid, but is not limited thereto.

[0025] Another aspect of the present invention is a pharmaceutical composition for preventing or treating tuberculosis, comprising at least one strain selected from the group consisting of Lactobacillus sakei and Lactobacillus reuteri; and an anti-tuberculosis agent.

[0026] In the present invention, the strain and antituberculosis agent may be administered to the subject simultaneously or sequentially, but are not limited thereto.

[0027] In the present invention, the Lactobacillus sakei may be the Lactobacillus sakei CVL-001 strain deposited under the accession number KCTC13816BP.

[0028] The daily dose of the above Lactobacillus sakei is 5 X 10 per 1 kg of subject body weight. 7 5 X 10 13 It may be CFU, preferably 5 X 10 7 5 X 10 12 CFU, 5 X 10 7 5 X 10 11 CFU, 5 X 10 7 5 X 10 10 CFU, 5 X 10 8 5 X 10 13 CFU, 5 X 10 8 5 X 10 12 CFU, 5 X 10 8 5 X 10 11CFU, 5 X 10 8 5 X 10 10 CFU, 5 X 10 9 5 X 10 13 CFU, 5 X 10 9 5 X 10 12 CFU, 5 X 10 9 5 X 10 11 CFU, 5 X 10 9 5 X 10 10 CFU, 5 X 10 10 5 X 10 13 CFU, or 5 X 10 10 5 X 10 12 It could be CFU, for example, 5 X 10 10 5 X 10 11 It may be, but is not limited to, CFU.

[0029] In the present invention, the Lactobacillus reuteri may be the Lactobacillus reuteri NCHBL-005 strain deposited under accession number KCTC15449BP, which has the effect of improving the migration and proliferation of fibroblasts.

[0030] The above Lactobacillus reuteri NCHBL-005 strain may include a 16S rRNA base sequence represented by sequence number 1.

[0031] The daily dose of the above Lactobacillus reuteri is 5 X 10 per 1 kg of subject body weight. 6 5 X 10 12 It may be CFU, preferably 5 X 10 6 5 X 10 11 CFU, 5 X 10 6 5 X 10 10 CFU, 5 X 10 6 5 X 10 9 CFU, 5 X 10 7 5 X 10 12 CFU, 5 X 10 7 5 X 10 11CFU, 5 X 10 7 5 X 10 10 CFU, 5 X 10 7 5 X 10 9 CFU, 5 X 10 8 5 X 10 12 CFU, 5 X 10 8 5 X 10 11 CFU, 5 X 10 8 5 X 10 10 CFU, 5 X 10 8 5 X 10 9 CFU, 5 X 10 9 5 X 10 12 CFU, or 5 X 10 9 5 X 10 11 It could be CFU, for example, 5 X 10 9 5 X 10 10 It may be, but is not limited to, CFU.

[0032] In the present invention, the strain may be a dead cell. The dead cell may be a dead cell produced by performing one or more methods selected from the group consisting of heat treatment, ultraviolet treatment, radiation irradiation, ultrasonic grinding, pressure control, freeze-drying, tyndalization, crushing, and chemical treatment on the strain, and for example, it may be a dead cell produced by heat treatment, but is not limited thereto.

[0033] The above heat treatment may be performed by applying a temperature of 70 to 120°C to the strain for 15 to 60 minutes, preferably by applying a temperature of 80 to 110°C for 20 to 45 minutes, and for example, by applying a temperature of 90 to 100°C for 25 to 30 minutes, but is not limited thereto.

[0034] In the present invention, the antituberculosis agent may be at least one selected from the group consisting of isoniazid, rifampicin, ethambutol, pyrazinamide, bedaquiline, and linezolid, preferably at least two selected from the group consisting of isoniazid and rifampicin, or bedaquiline and linezolid, but is not limited thereto.

[0035] In the present invention, the daily dosage of the anti-tuberculosis agent may be 0.1 to 20 g per 1 kg of body weight of the subject.

[0036] Preferably, the antituberculosis agent may be isoniazid, and the daily dosage of isoniazid may be 0.1 to 15 g, 0.1 to 10 g, 0.1 to 5 g, 0.1 to 2 g, 0.5 to 20 g, 0.5 to 15 g, 0.5 to 10 g, 0.5 to 5 g, 0.5 to 2 g, 1 to 20 g, 1 to 15 g, 1 to 10 g, 1 to 5 g, 1 to 2 g, 2 to 20 g, 2 to 15 g, or 2 to 10 g, for example, but is not limited thereto.

[0037] Preferably, the antituberculosis agent may be rifampin, and the daily dosage of rifampin may be 0.1 to 15 g, 0.1 to 10 g, 0.1 to 5 g, 0.1 to 2 g, 0.1 to 1 g, 0.5 to 20 g, 0.5 to 15 g, 0.5 to 10 g, 0.5 to 5 g, 0.5 to 2 g, 0.5 to 1 g, 1 to 20 g, 1 to 15 g, 1 to 10 g, or 1 to 5 g per 1 kg of the subject's body weight, for example, but is not limited thereto.

[0038] In the present invention, the tuberculosis may be selected from the group consisting of middle ear tuberculosis, adrenal tuberculosis, ocular tuberculosis, laryngeal tuberculosis, epididymal tuberculosis, lymph node tuberculosis, renal tuberculosis, skin tuberculosis, intestinal tuberculosis, multidrug-resistant tuberculosis, pulmonary tuberculosis, biliary tuberculosis, lymph node tuberculosis, pharyngeal tuberculosis, bone tuberculosis, pulmonary tuberculosis, breast tuberculosis, and spinal tuberculosis, but is not limited thereto.

[0039] The present invention relates to a combination preparation for alleviating drug toxicity induced by an anti-tuberculosis drug, including killed cells, and a pharmaceutical composition for preventing or treating tuberculosis, including the same. By using Lactobacillus sakei CVL-001 or Lactobacillus reuteri NCHBL-005 as the killed cells in combination with an anti-tuberculosis drug, the combination preparation is excellent in alleviating drug toxicity (hepatotoxicity and nephrotoxicity) induced by the anti-tuberculosis drug, thereby increasing the adherence of the anti-tuberculosis drug, and thereby suppressing the emergence of multi-drug resistant tuberculosis and the recurrence of tuberculosis. In addition, by using the combination preparation as an adjuvant with drugs with significant side effects but good effects, such as bedaquiline or linezolid, the combination preparation is expected to have the effect of shortening the treatment period for tuberculosis, and thus can be effectively utilized in the treatment process of tuberculosis.

[0040] Figure 1 is a schematic diagram showing the process of confirming infection and treatment effects on a tuberculosis model mouse according to one embodiment of the present invention.

[0041] Figure 2a is a graph showing a decrease in the CFU value of tuberculosis bacteria in the lungs of a tuberculosis model mouse administered with a dead cell according to one embodiment of the present invention.

[0042] Figure 2b is a graph showing changes in alanine aminotransferase (ALT) measurement values ​​from a tuberculosis model mouse administered with a dead cell according to one embodiment of the present invention.

[0043] Figure 2c is a graph showing changes in the measured value of uric acid (UA), an indicator, from a tuberculosis model mouse administered with a dead cell according to one embodiment of the present invention.

[0044] Figure 3 is a graph showing changes in the secretion amount of interleukin-10 from immune cells in the lungs of a tuberculosis model mouse administered with a dead cell according to one embodiment of the present invention.

[0045] Figure 4 is a graph showing changes in the count of regulatory T cells in the lungs in a tuberculosis model mouse administered with a combination of dead cells according to one embodiment of the present invention.

[0046] Figure 5a is a graph showing that the proportion of alveolar macrophages further increases in the lungs of a tuberculosis model mouse administered with a dead cell according to one embodiment of the present invention.

[0047] Figure 5b is a graph showing that the number of alveolar macrophages further increases in the lungs of a tuberculosis model mouse administered with a combination of dead cells according to one embodiment of the present invention.

[0048] Figure 6a shows a multifunctional CD4 cell expressing interferon gamma (IFN-γ) in a single cell lung of a tuberculosis model mouse administered with a dead cell according to one embodiment of the present invention. + This is a graph showing a decrease in the number of T cells.

[0049] Figure 6b shows a multifunctional CD4 expressing tumor necrosis factor alpha (TNF-α) in a single cell lung of a tuberculosis model mouse administered with a dead cell according to one embodiment of the present invention. + This is a graph showing a decrease in the number of T cells.

[0050] Figure 6c shows a multifunctional CD4 expressing both TNF-α and IFN-γ in a single cell lung of a tuberculosis model mouse co-administered with a dead cell according to one embodiment of the present invention. + This is a graph showing a decrease in the number of T cells.

[0051] The present invention relates to a combination administration preparation for alleviating antituberculosis drug toxicity, comprising at least one strain selected from the group consisting of Lactobacillus sakei and Lactobacillus reuteri.

[0052] Hereinafter, the present invention will be described in more detail with reference to the following examples. However, these examples are only intended to illustrate the present invention, and the scope of the present invention is not limited by these examples.

[0053] Throughout this specification, "%" used to indicate the concentration of a particular substance is (wt / wt)% for solid / solid, (wt / vol)% for solid / liquid, and (vol / vol)% for liquid / liquid, unless otherwise stated.

[0054]

[0055] Preparation Example 1: Isolation and identification of Lactobacillus ruteri NCHBL-005 strain

[0056] To isolate lactic acid bacteria from the gut microbiota of honeybees, first, target insects were collected in the Gwangju area, placed in sterilized collection containers, and refrigerated for 10 minutes for each individual. The gastrointestinal tracts were aseptically removed using microsurgical instruments on a green bench. The honeybee gut microbiota was obtained by suspending the extracted gastrointestinal contents in 10 ml of sterile Tween 80-added 0.1% peptone physiological saline (0.9% w / v NaCl, 0.1% w / v Tween 80, 0.1% w / v peptone).

[0057] Lactic acid bacteria were serially diluted with 500 μl of a suspension of the pulverized intestines using a liquid medium (DifcoTM Lactobacilli MRS Broth, BD, USA), spread on a solid medium (BBLTM LBS Agar, BD, USA) using a platinum plate, and cultured at 37°C with BD GasPakTM EZdp until bacterial colonies were confirmed.

[0058] Bacterial colonies identified on LBS solid medium were identified as Lactobacillus based on the positive Gram stain result and the negative catalase reaction confirmed when mixed with 3% hydrogen peroxide. The isolated field isolate Lactobacillus was stored at -20℃ in MRS medium containing 15% glycerol and used in the following experiments.

[0059] Phylogenetic identification of Lactobacillus reuteri among the isolated strains was performed through base sequence analysis of the 16S rRNA gene. Specifically, based on the similarity of the primary and secondary structures of the 16S rRNA gene using the PHYDIT program, it was compared with the 16S rRNA genes of Lactobacillus strains and named Lactobacillus reuteri NCHBL-005, and its 16S rRNA base sequence is as shown in Table 1 below.

[0060] 서열번호명칭서열1LactobacillusreuteriNCHBL-00516S rRNACTGATTGATGGTGCTTGCACCTGATTGACGATGGATCACCAGTGAGTGGCGGACGGGTGAGTAACACGTAGGTAACCTGCCCCGGAGCGGGGGATAACATTTGGAAACAGATGCTAATACCGCATAACAACAAAAGCCACATGGCTTTTGTTTGAAAGATGGCTTTGGCTATCACTCTGGGATGGACCTGCGGTGCATTAGCTAGTTGGTAAGGTAACGGCTTACCAAGGCGATGATGCATAGCCGAGTTGAGAGACTGATCGGCCACAATGGAACTGAGACACGGTCCATACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCACAATGGGCGCAAGCCTGATGGAGCAACACCGCGTGAGTGAAGAAGGGTTTCGGCTCGTAAAGCTCTGTTGTTGGAGAAGAACGTGCGTGAGAGTAACTGTTCACGCAGTGACGGTATCCAACCAGAAAGTCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTATCCGGATTTATTGGGCGTAAAGCGAGCGCAGGCGGTTGCTTAGGTCTGATGTGAAAGCCTTCGGCTTAACCGAAGAAGTGCATCGGAAACCGGGCGACTTGAGTGCAGAAGAGGACAGTGGAACTCCATGTGTAGCGGTGGAATGCGTAGATATATGGAAGAACACCAGTGGCGAAGGCGGCTGTCTGGTCTGCAACTGACGCTGAGGCTCGAAAGCATGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGAGTGCTAGGTGTTGGAGGGTTTCCGCCCTTCAGTGCCGGAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGACCGCAAGGGTTGAAACTCAA

[0061]

[0062] 준비예 2: 사균체의 준비

[0063] Lactobacillus sakei, a lactic acid bacterium derived from kimchi, and Lactobacillus reuteri, a lactic acid bacterium derived from bees, are types of lactic acid bacteria commonly found in the human intestine as well as in dairy products and fermented foods.

[0064] Heat-killed cells were prepared from the Lactobacillus sakei CVL-001 strain deposited with the accession number KCTC13816BP and the Lactobacillus reuteri NCHBL-005 strain deposited with the accession number KCTC15449BP identified in Preparation Example 1 above.

[0065] Specifically, Lactobacillus sakei CVL-001 and Lactobacillus rhuteri NCHBL-005 stocks were plated on MRS agar, a selective medium for lactic acid bacteria, and cultured in an incubator at 30°C and 37°C, respectively, for 24 hours. After 24 hours, a single colony from the MRS agar was extracted and placed into a new MRS broth. The MRS broth was then pre-cultured in an incubator at 30°C and 37°C, respectively, with a rotation speed of 150 rpm for 16 hours.

[0066] The above pre-cultured bacteria were cultured in fresh MRS broth at 30°C and 37°C, respectively, and 150 rpm for 4 hours in a culture incubator. The cultured bacteria were centrifuged at 3,000 rpm for 15 minutes, and the centrifuged bacteria were washed with PBS and quantified by measuring absorbance. Afterwards, the quantified bacteria were suspended in a certain amount of PBS and heated in a double boiler at 100°C for 30 minutes using a heating block to kill the bacteria, and then pre-frozen at -80°C.

[0067] The pre-frozen fungi were dried for 72 hours in a freeze dryer under conditions of a chamber temperature of -80℃ and a vacuum pressure of 40 vacuum.

[0068] The above Lactobacillus reuteri NCHBL-005 strain was smeared on BBLTM LBS agar from the gastrointestinal tract of a honeybee, and the strain showing activity under anaerobic conditions was selected through Gram staining and catalase negative reaction, thereby isolating it as Lactobacillus, and identifying it as Lactobacillus reuteri based on the results of base sequence analysis of the 16s rRNA gene.

[0069]

[0070] Preparation Example 3: Production of a tuberculosis model mouse and administration of killed bacteria to a tuberculosis treatment model

[0071] Six-week-old C57BL6 / J female mice (weighing approximately 20 g) were infected with the clinical strain HN878 via airborne infection. Four weeks after infection, antituberculosis drugs, isoniazid 0.2 g / L and rifampin 0.1 g / L, were orally administered at 200 μl per day each, and the killed cells of Preparation Example 2 were administered simultaneously.

[0072] The above “HN878” is a highly pathogenic tuberculosis strain belonging to the Beijing family. In other words, it is more pathogenic than the laboratory standard strain, Mycobacterium tuberculosis H37Rv.

[0073] The killed cells were diluted to 9 mg / ml using sterile PBS as a solvent and administered orally to mice at a dose of 200 μl, administering 1.8 mg of the killed cells per mouse once a day. The amount of the killed cells administered at this time was 1 X 10 of the CVL-001 strain. 9 CFU / mouse, and the dead cells of NCHBL-005 strain are 1 X 10 8 It was applied as CFU / mouse.

[0074] The entire group was divided into a non-infected group (Naive) and an infected group (Infection) according to infection status, and an anti-tuberculosis drug administration group (Tx) and an anti-tuberculosis drug and killed bacteria combination administration group (Tx+CVL-001, Tx+NCHBL-005) according to the administered substance, and the experiment was conducted.

[0075] The first autopsy was performed at week 4 to determine whether tuberculosis infection had been properly established and to establish a baseline for future autopsies. A second autopsy was performed at week 7, effectively assessing the extent to which tuberculosis bacteria were eliminated from the host after antituberculosis treatment, to assess the effects of antituberculosis drugs and killed bacteria.

[0076]

[0077] Example 1: Confirmation of CFU from the lungs of tuberculosis model mice

[0078] The CFU of tuberculosis bacteria obtained from the lungs of the tuberculosis model mouse produced in the above Preparation Example 3 and autopsied for the second time in the 7th week was confirmed.

[0079] Specifically, the left lung, the largest of the five lobes of the infected mouse lung, was removed and transferred to an EP-tube, then ground in 1 mL of PBS solution to obtain a cell lysate. Fifty microliters of the cell lysate was inoculated onto 7H10 (OADC 10%) solid medium. The cultures were placed in a 37°C constant-temperature incubator, and the number of colonies emerging for 28 days after inoculation was counted to determine the colony-forming units (CFU).

[0080] InfectionInfection+CVL-001TxTx+CVL-001CFU / ml(log)6.56636.35275.56055.3636

[0081] As can be seen in Table 2 and Figure 2a above, the number of tuberculosis bacteria in the lungs was confirmed to be reduced in the group administered a combination of dead cells and an anti-tuberculosis drug compared to the group administered an anti-tuberculosis drug alone.

[0082]

[0083] Example 2: Confirmation of toxicity indicators from serum of tuberculosis model mice

[0084] Blood obtained from a tuberculosis model mouse produced in the above Preparation Example 3 and autopsied at week 7 was separated into plasma and serum using a centrifuge, and hepatotoxicity and nephrotoxicity indices were confirmed through the serum.

[0085] NaiveInfectionTxTx+CVL-001Tx+NCHBL-005ALT(U / L)13.525.528.8571923.125UA(mg / dl)1.23.12.6141.6251.788

[0086] As confirmed in Table 3 above and Figures 2b and 2c, serum analysis results confirmed that alanine aminotransferase (ALT), an indicator of hepatotoxicity, and uric acid (UA), an indicator of nephrotoxicity, increased in the group administered anti-tuberculosis drugs compared to the non-infected group. Here, alanine aminotransferase and uric acid decreased in both groups administered a combination of killed bacteria and anti-tuberculosis drugs.

[0087] Through the above results, it was found that the hepatotoxicity caused by antituberculosis drugs and the increased uric acid after tuberculosis infection were significantly reduced by the combined administration of killed bacteria and antituberculosis drugs.

[0088]

[0089] Example 3: Confirmation of immune activity from lung immune cells of tuberculosis model mice.

[0090] The lungs were removed from the tuberculosis model mouse produced in the above Preparation Example 3 and autopsied at week 7, and the tissue was ground and separated into single cells.

[0091] Specifically, cells were seeded at 1 X 10 in a 96-well plate. 6cells / ml concentration, and for a specific response by tuberculosis bacteria, ESAT-6, a virulence factor of tuberculosis, was added at a concentration of 1 ug / ml to stimulate. After culturing for 6 hours in a CO2 incubator, the supernatant was collected. The cytokine interleukin-10 was quantitatively analyzed from the supernatant obtained using multiplex (biolegend).

[0092] NaiveTxTx+CVL-001Tx+NCHBL-005IL-10(pg / ml)82.523516.019655.863791.291

[0093] As can be seen in Table 4 and Figure 3 above, the Multiplex analysis results confirmed that when the dead cells were administered together, the amount of interleukin-10 secreted from immune cells in the mouse lungs increased further.

[0094] The above results suggest that the inflammatory response in the lungs of tuberculosis model mice can be alleviated by the combined administration of killed bacteria and anti-tuberculosis drugs, as interleukin-10, an anti-inflammatory cytokine, was increased in mice co-administered with killed bacteria.

[0095]

[0096] Example 4: Determination of pulmonary regulatory T cell levels in tuberculosis model mice.

[0097] The increase or decrease in specific immune cells was confirmed using a flow cytometer on single cells from the lungs of the tuberculosis model mouse obtained in Example 3 above.

[0098] Specifically, CD25 regulatory T cells among total live cells + foxp3 + CD4 + T cells were quantified.

[0099] InfectionTxTx+CVL-001Tx+NCHBL-005Treg(%)0.370.4330.520.587

[0100] As can be seen in Table 5 and Figure 4 above, CD25 regulatory T cells + foxp3 + CD4 + It was confirmed that T cells significantly increased in all mice co-administered with killed strains of CVL-001 and NCHBL-005.

[0101] Through the above results, it was confirmed that the killed cells according to the present invention increase the number of regulatory T cells that suppress excessive immunity, and from this, it is possible to infer the relationship between cytotoxicity caused by tuberculosis infection and anti-tuberculosis drug administration and the anti-inflammatory response induced by the killed cells.

[0102]

[0103] Example 5: Confirmation of recovery of alveolar macrophages from tuberculosis model mice.

[0104] The recovery of alveolar macrophages was confirmed using a flow cytometer to analyze single cells from the lungs of the tuberculosis model mouse obtained in Example 3 above.

[0105] Specifically, in mice exposed to Mycobacterium tuberculosis via airborne infection, alveolar macrophages are initially infected. If the infection persists, these macrophages die, allowing the bacteria to spread to other immune cells. Treatment of tuberculosis leads to the recovery of alveolar macrophages. The impact of killed bacteria on this recovery was compared and analyzed.

[0106] InfectionTxTx+CVL-001Tx+NCHBL-005Alveolar macrophages / lung (%)0.2540.7221.0020.946Cell count(X 10 5 )0.921.482.721.86

[0107] As can be seen in Table 6 above and Figures 5a and 5b, the number of alveolar macrophages in the lungs (lung live cells) was confirmed to increase further in mice administered with the dead cells.

[0108]

[0109] Example 6: CD4 from tuberculosis model mice + Check T cell cytokine expression levels

[0110] CD4 was stimulated with tuberculosis antigen in single cells of the lung of the tuberculosis model mouse obtained in Example 3 above. + The cytokine levels of T cells specifically expressing tuberculosis bacteria among T cells were confirmed using a flow cytometer.

[0111] Specifically, single cells from the lungs of Mycobacterium tuberculosis-infected mice were stimulated with ESAT-6 protein, a representative secretory antigen of Mycobacterium tuberculosis, at a concentration of 1 ug / ml for 6 hours. After cytokine accumulation in T cells by adding a protein transport inhibitor, Brefeldin A, CD4 specific for tuberculosis antigen stimulation was stimulated. + Cytokine expression in T cells was measured using a flow cytometer.

[0112] NaiveInfectionInfection+CVL-001TxTx+CVL-001IFNγ / CD4+(%)0.157514.4613.929.1886.69TNFα / CD4+(%)0.40259.687.668.3925.82TNFα IFNγ / CD4+(%)0.1949.687.9468.065.65

[0113] As can be seen in Table 7 above and Figures 6a to 6c, the group administered with the combination of the killed cells and the anti-tuberculosis drug expressed more interferon gamma (IFN-γ), the inflammatory cytokine tumor necrosis factor alpha (TNF-α), and multifunctional CD4 cells that all expressed more than the group administered the anti-tuberculosis drug alone. + It was confirmed that all T cells were reduced.

[0114] In summary, the above results suggest that the killed cells of the present invention reduce the number of tuberculosis bacteria, mitigate hepatotoxicity caused by antituberculosis drugs, reduce pulmonary inflammation in infected mice, and promote the recovery of lung tissue cells. Therefore, administering the killed cells as an adjuvant alongside antituberculosis drugs is expected to provide more flexible tuberculosis treatment methods.

[0115] [Accession number]

[0116] Name of depositor: Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC)

[0117] Accession number: KCTC13816BP

[0118] Date of acceptance: February 19, 2019

[0119]

[0120] Name of depositor: Korea Research Institute of Bioscience and Biotechnology, Biological Resource Center (KCTC)

[0121] Accession number: KCTC15449BP

[0122] Date of acceptance: 20230519

[0123] The present invention relates to a combination administration preparation for alleviating drug toxicity induced by an antituberculosis agent, including kimchi-derived Lactobacillus sakei CVL-001 or honeybee-derived Lactobacillus reuteri NCHBL-005, and a pharmaceutical composition for preventing or treating tuberculosis, including the same. More specifically, the present invention relates to a therapeutic technique for alleviating drug toxicity induced by an antituberculosis agent by using killed cells of Lactobacillus sakei CVL-001 or Lactobacillus reuteri NCHBL-005 in combination with the antituberculosis agent.

[0124]

[0125]

Claims

1. A combination administration preparation for alleviating antituberculosis drug toxicity, comprising at least one strain selected from the group consisting of Lactobacillus sakei and Lactobacillus reuteri.

2. A combination administration preparation for alleviating anti-tuberculosis drug toxicity, wherein the Lactobacillus sakei in paragraph 1 is the Lactobacillus sakei CVL-001 strain deposited under the accession number KCTC13816BP.

3. A combination administration preparation for alleviating anti-tuberculosis drug toxicity, wherein the Lactobacillus reuteri in paragraph 1 is the Lactobacillus reuteri NCHBL-005 strain deposited under the accession number KCTC15449BP.

4. A combination administration preparation for alleviating anti-tuberculosis drug toxicity, wherein the strain is a dead cell in the first paragraph.

5. A pharmaceutical composition for preventing or treating tuberculosis, comprising at least one strain selected from the group consisting of Lactobacillus sakei and Lactobacillus reuteri; and an anti-tuberculosis agent.

6. A pharmaceutical composition for preventing or treating tuberculosis, wherein the strain and the anti-tuberculosis agent are administered to the subject simultaneously or sequentially in paragraph 5.

7. A pharmaceutical composition for preventing or treating tuberculosis, wherein the Lactobacillus sakei in paragraph 5 is the Lactobacillus sakei CVL-001 strain deposited under the accession number KCTC13816BP.

8. In the fifth paragraph, the daily dose of the Lactobacillus sakei is 5 X 10 per 1 kg of the subject's body weight. 7 5 X 10 13 A pharmaceutical composition for the prevention or treatment of tuberculosis, which comprises CFU.

9. A pharmaceutical composition for preventing or treating tuberculosis, wherein the Lactobacillus reuteri in paragraph 5 is the Lactobacillus reuteri NCHBL-005 strain deposited under the accession number KCTC15449BP.

10. In the fifth paragraph, the daily dose of Lactobacillus reuteri is 5 X 10 per 1 kg of body weight of the subject. 6 5 X 10 12 A pharmaceutical composition for the prevention or treatment of tuberculosis, which comprises CFU.

11. A pharmaceutical composition for preventing or treating tuberculosis, wherein the strain is a dead cell in the fifth paragraph.

12. A pharmaceutical composition for preventing or treating tuberculosis, wherein the anti-tuberculosis agent in paragraph 5 is at least one selected from the group consisting of isoniazid, rifampin, ethambutol, pyrazinamide, bedaquiline, and linezolid.

13. A pharmaceutical composition for preventing or treating tuberculosis, wherein the daily dosage of the anti-tuberculosis agent in paragraph 5 is 0.1 to 20 g per 1 kg of body weight of the subject.

14. A pharmaceutical composition for preventing or treating tuberculosis, wherein the tuberculosis in paragraph 5 is selected from the group consisting of middle ear tuberculosis, adrenal tuberculosis, ocular tuberculosis, laryngeal tuberculosis, epididymal tuberculosis, lymphatic tuberculosis, renal tuberculosis, skin tuberculosis, intestinal tuberculosis, multidrug-resistant tuberculosis, pulmonary tuberculosis, biliary tuberculosis, lymphatic tuberculosis, pharyngeal tuberculosis, bone tuberculosis, pulmonary tuberculosis, breast tuberculosis, and spinal tuberculosis.

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