WO2018190680A1 - NUTLIN-3α FOR CONTROLLING INTRACELLULAR MYCOBACTERIUM TUBERCULOSIS, AND COMPOSITION AND METHOD FOR REGULATING EXPRESSION OF P53 - Google Patents
NUTLIN-3α FOR CONTROLLING INTRACELLULAR MYCOBACTERIUM TUBERCULOSIS, AND COMPOSITION AND METHOD FOR REGULATING EXPRESSION OF P53 Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/13—Amines
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
- A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
- A61K31/4409—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 4, e.g. isoniazid, iproniazid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
- A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
Definitions
- the present invention relates generally to the field of immunology and, in part, to methods of inhibiting survival and proliferation of Mycobacterium tuberculosis and to methods for providing Nutlin-3 ⁇ (neutrin-3 ⁇ ) and p53 expression control compositions.
- Tuberculosis is a chronic infectious disease caused by infection of Mycobacterium tuberculosis and other Mycobacterium species. Tuberculosis is a major disease in developing countries that kills about 8 million new infections each year, killing 3 million lives, and is also a problem in developed countries. While the infection may be asymptomatic for a significant period of time, tuberculosis is the most common symptom, indicating acute inflammation of the lung, resulting in fever and nonproductive cough. If left untreated, it usually results in serious complications and death.
- Tuberculosis can be treated using prolonged antibiotic therapy, but this treatment is not sufficient to prevent the spread of tuberculosis. Infected individuals do not exhibit symptoms but may be contagious for a period of time. In addition, it is difficult to control the patient's behavior even if the treatment regimen is strictly followed. Some patients do not complete the course of treatment, which can lead to ineffective treatment and development of drug resistance. Even when the entire course of treatment is completed, the TB germ infection is not eradicated from the infected individual and remains a latent infection that can still be reactivated.
- BCG Bacillus Calmette-Guerin
- PPD tuberculin proteinpurified derivative
- Antigen-specific T cell responses result in measurable degree induration at the site of infusion up to 48-72 hours post infusion, indicating exposure to mycobacterium antigens.
- the assay is problematic in sensitivity and specificity, and individuals vaccinated with BCG cannot be distinguished from those infected.
- p53 molecular weight 53 kDa
- PUMA molecular weight 53 kDa
- the P53 protein is labeled ubiquitin by MDM2 protein and degraded by proteasome.
- Nutlin-3 ⁇ (molecular weight 581.49), known as an anticancer agent, binds to this MDM2 and inhibits p53-MDM2 interaction to increase p53 pathway activity. to be. Therefore, as a method for inhibiting Mycobacterium tuberculosis with p53 as a target, Nutlin-3 ⁇ alone or in combination with existing anti-tuberculosis therapeutics may be used.
- the present invention contributes to the activation of macrophages using Nutlin-3 ⁇ , which induces expression of p53 or induces p53 activity, to provide a method for preventing and effectively treating tuberculosis, which has a different mechanism of action and relatively fewer side effects.
- the present invention is to provide a pharmaceutical composition for preventing and treating tuberculosis comprising a Nutlin-3 ⁇ compound as an active ingredient.
- the Nutlin-3 ⁇ compound thus provided is characterized by overexpressing p53, and the overexpressed p53 inhibits the growth of Mycobacterium tuberculosis by inducing apoptosis of macrophages infected with Mycobacterium tuberculosis.
- the anti-tuberculosis agent used herein may be at least one selected from the group consisting of Rifampicin, Isoniazid, Ethambutol, and Pyrazinamide.
- the present invention utilizes the function of Mycobacterium tuberculosis of Nutlin-3 ⁇ , which is not known at all, it can be expected to have a prophylactic and therapeutic effect against Mycobacterium tuberculosis having multidrug resistance with respect to existing drugs.
- the growth of latent Mycobacterium tuberculosis can be suppressed, so that latent Mycobacterium tuberculosis can be removed when used alone or in combination with a conventional medicine.
- Figure 2 is a protein expression electrophoresis picture by western blotting showing p53 expression and Caspase-3 activity according to Nutlin-3 ⁇ concentrations treated in Mycobacterium tuberculosis-infected macrophages.
- 3 is a graph showing that the number of Mycobacterium tuberculosis in macrophages is significantly reduced by Nutlin-3 ⁇ treatment.
- 4 and 5 are graphs showing that the number of Mycobacterium tuberculosis in macrophages is significantly reduced by the combination of antituberculosis therapeutic agents (isoniazid, rifampicin, pyrazinamide, ethambutol) and Nutlin-3 ⁇ .
- antituberculosis therapeutic agents isoniazid, rifampicin, pyrazinamide, ethambutol
- Nutlin-3 ⁇ are graphs showing that the number of Mycobacterium tuberculosis in macrophages is significantly reduced by the combination of antituberculosis therapeutic agents (isoniazid, rifampicin, pyrazinamide, ethambutol) and Nutlin-3 ⁇ .
- FIG. 6 is a graph showing a significant increase in Mycobacterium tuberculosis survival in mice lungs with p53-deficient macrophage.
- FIG. 7 is a graph showing that the number of Mycobacterium tuberculosis in lung tissue is significantly decreased when pneumoniae-infected mice are intraperitoneally injected with Nutlin-3a (10 mg / kg / day).
- FIG. 8 is a graph showing that the expression of p53 in the macrophage of tuberculosis patients is significantly lower than that of healthy macrophage.
- FIG. 9 is a graph showing that the number of Mycobacterium tuberculosis in cells is significantly reduced when p53 activity is increased through Nutlin-3a.
- the present invention was first identified that the Nutlin-3 ⁇ compound has an effect of preventing or treating Mycobacterium tuberculosis through p53 overexpression and acting on apoptosis of macrophages.
- the present invention comprises a Nutlin-3 ⁇ compound as an active ingredient. It is characterized by providing a composition for preventing or treating Mycobacterium tuberculosis.
- 'Nutlin-3 ⁇ ' is a small molecule compound that binds to MDM2 and inhibits binding between MDM2 and p53 protein, and is known for its anticancer effect, and its chemical structure is shown in FIG. 1. More specifically, Nutlin-3 ⁇ is a small molecule compound that binds to MDM2 and inhibits binding between MDM2 and p53 protein. It is an anticancer drug currently being developed for the treatment of solid cancer. In detail, the MDM2 protein binds to the transactivation site of p53, a gene that inhibits cancer, and blocks the activation ability. The Nutlin-3 ⁇ compound has a molecular structure similar to that of p53.
- Nutlin-3 ⁇ compound is only known as an anticancer drug that is currently being developed in the clinic, and there is no report on the effect that it has on the treatment of tuberculosis.
- the present inventors have completed the present invention by confirming that Nutlin-3 ⁇ compound can improve tuberculosis through a mechanism of activating p53.
- Example 1 of the present invention when the Nutlin-3 ⁇ compound was injected into Bone marrow-derived macrophages (BMDMs) cells, which are mouse-derived macrophages, the number of Mycobacterium tuberculosis bacteria was significantly reduced, resulting in an improvement in tuberculosis. It could be confirmed.
- BMDMs Bone marrow-derived macrophages
- Example 2 of the present invention when used in combination with the existing anti-tuberculosis agent was confirmed to show an effect of significantly higher tuberculosis bacteria compared to the use of the existing anti-tuberculosis agent alone.
- Nutlin-3 ⁇ compound is effective in improving tuberculosis
- the composition of the present invention comprising it as an active ingredient can be usefully used in the prevention or treatment for tuberculosis.
- Tuberculosis in the present invention includes eye tuberculosis, skin tuberculosis, adrenal tuberculosis, kidney tuberculosis, epididymal tuberculosis, lymphatic tuberculosis, laryngeal tuberculosis, middle ear tuberculosis, intestinal tuberculosis, multidrug-resistant tuberculosis, pulmonary tuberculosis, tuberculosis, bone tuberculosis, throat tuberculosis, lymphatic tuberculosis We can include ruins, breast tuberculosis or spinal tuberculosis.
- existing anti-tuberculosis agents may include Rifampicin, Isoniazid, Ethambutol or Pyrazinamide used in Example 2 of the present invention.
- the term 'treatment' unless stated otherwise, reverses, alleviates, inhibits, or prevents the disease or condition to which the term applies, or one or more symptoms of the disease or condition, As used herein, the term 'treatment' refers to the act of treating when 'treating' is defined as above.
- BMDMs Bone marrow-derived macrophages
- Mycobacterium tuberculosis H37Ra non-pathogenic Mycobacterium tuberculosis
- Figure 2 shows the expression level of p53 protein expressed during Mycobacterium tuberculosis infection and the apoptosis marker Caspase-3 activity as a result of Western blot method in accordance with the increased treatment concentration of Nutlin-3 ⁇ (Fig. 1), a p53 inducing agent in macrophages infected with Mycobacterium tuberculosis. Increasing p53 and Caspas-3 activity was observed. ( ⁇ -actin is an internal control in Figure 2)
- FIG. 3 was treated with Nutlin-3 ⁇ (30 uM) in large macrophages infected with Mycobacterium tuberculosis and after 24 hours, the number of viable Mycobacterium tuberculosis cells was cultured in 7H10 agar medium for 14-21 days. As a result, the number of survival of the intracellular pathogenic Mycobacterium tuberculosis and nonpathogenic Mycobacterium tuberculosis was significantly reduced in the macrophages treated with Nutlin-3 ⁇ (30 uM) compared with the control group.
- Figures 4 and 5 show anti-tuberculosis treatment of isoniazid (10 ng / ml), rifampicin (10 ng / ml), pyrazinamide (100 ng / ml) or etambutol (100 ng / ml) during the infection of Mycobacterium tuberculosis bacteria. And Nutlin-3 ⁇ (30 uM) in combination to inhibit the growth of mycobacterium tuberculosis.
- Macrophage cells infected with pathogenic Mycobacterium tuberculosis or non-pathogenic Mycobacterium tuberculosis were treated with antituberculosis treatment alone or in combination with Nutlin-3 ⁇ to determine the number of tuberculosis viable in cells after 24 hours.
- the experimental results showed that the number of intracellular tuberculosis bacteria decreased significantly when anti-TB treatment and Nutlin-3 ⁇ were used in combination with anti-TB treatment alone.
- FIG. 6 shows the number of viable Mycobacterium tuberculosis bacteria in lung tissue after intranasal infection of non-pathogenic Mycobacterium tuberculosis in a control (wild type) or macrophage-deficient p53 (p53 conditional knockout) mouse.
- wild type wild type
- macrophage-deficient p53 p53 conditional knockout
- FIG. 7 shows that the number of Mycobacterium tuberculosis in lung tissue was significantly reduced when p53 activity was increased by intraperitoneal injection of Nutlin-3a (10 mg / kg / day) into mice infected with non-pathogenic Mycobacterium tuberculosis via the nasal cavity.
- the expression of p53 in the macrophage of tuberculosis patients was significantly lower than that of healthy macrophage.
- Figure 9 was infected with non-pathogenic Mycobacterium tuberculosis in macrophage derived from the blood of healthy or tuberculosis patients, treated with Nutlin-3a (10 uM) and the number of intracellular Mycobacterium viability was measured. As a result, when the p53 activity was increased through Nutlin-3a, it was confirmed that the number of Mycobacterium tuberculosis in the cells was significantly reduced.
- the mycobacterium tuberculosis can be suppressed by increasing the expression of p53 in the cells. Therefore, when the composition according to the present invention (Nutlin-3 ⁇ ) is treated with Mycobacterium tuberculosis-infected cells as an active ingredient to control p53, that is, when the composition of the present invention is treated to Mycobacterium tuberculosis carriers, tuberculosis patients or patients with existing anti-tuberculosis drug resistance It can be expected to kill my tuberculosis bacteria.
- the statistical method used was Bonferroni's multiple comparison test, marked with * when p-value ⁇ 0.05, ** when p-value ⁇ 0.01, and *** when p-value ⁇ 0.001.
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Abstract
The present invention relates to a pharmaceutical composition for preventing or treating tuberculosis including a Nutlin-3α compound as an active ingredient. A Nutlin-3α compound, which is an active ingredient according to the present invention, has an effect of inhibiting the proliferation of Mycobacterium tuberculosis by inducing the apoptosis of macrophages that have been infected with Mycobacterium tuberculosis through the overexpression of p53. Thus, a method according to the present invention including the Nutlin-3α compound as an active ingredient may be usefully used for preventing or treating tuberculosis.
Description
본 발명은 전반적으로 면역학 분야에 관한 것이며, 부분적으로 결핵균의 생존 및 증식을 억제하는 방법에 관한 것이며 이를 위하여 Nutlin-3α (뉴트린-3α) 및 p53 발현 조절 조성물을 제공하는 방법에 관한 것이다. FIELD OF THE INVENTION The present invention relates generally to the field of immunology and, in part, to methods of inhibiting survival and proliferation of Mycobacterium tuberculosis and to methods for providing Nutlin-3α (neutrin-3α) and p53 expression control compositions.
결핵은 결핵균 및 다른 마이코박테리움(Mycobacterium)종의 감염에 의해 유발되는 만성 감염성 질병이다. 결핵은 매년 약 8백만명의 신규 감염자가 발생되며 3백만명의 목숨을 앗아가는 개발도상국의 주요 질병이며, 또한 선진국에서도 문제점으로 부각되고 있다. 상당한 기간 동안 감염은 무증상일 수 있으나, 결핵은 가장 공통적인 증상으로서 폐의 급성 염증을 나타내며, 그 결과 발열 및 마른 기침(nonproductive cough)을 유발시킨다. 치료하지 않으면, 일반적으로 심각한 합병증 및 사망을 초래한다. Tuberculosis is a chronic infectious disease caused by infection of Mycobacterium tuberculosis and other Mycobacterium species. Tuberculosis is a major disease in developing countries that kills about 8 million new infections each year, killing 3 million lives, and is also a problem in developed countries. While the infection may be asymptomatic for a significant period of time, tuberculosis is the most common symptom, indicating acute inflammation of the lung, resulting in fever and nonproductive cough. If left untreated, it usually results in serious complications and death.
결핵은 장기간에 걸친 항생제 요법을 이용하여 치료될 수 있으나, 이러한 치료법이 결핵의 확산을 막기에는 역부족이다. 감염된 개체들은 증상을 나타내지는 아니하나, 일정 기간 보균상태(contagious)일 수 있다. 게다가 치료 섭생을 엄격하게 따르더라도 환자의 행동을 통제하는 것은 어렵다. 일부 환자들은 치료과정을 끝마치지 못하는데, 이는 효험없는 치료와 약제 내성의 발달을 초래할 수 있다. 치료의 전 과정을 끝마친다 하더라도, 결핵균 감염은 감염된 개체로부터 근절되지 아니하며 여전히 재활성화될 수 있는 잠복성 감염으로 잔존하게 된다. Tuberculosis can be treated using prolonged antibiotic therapy, but this treatment is not sufficient to prevent the spread of tuberculosis. Infected individuals do not exhibit symptoms but may be contagious for a period of time. In addition, it is difficult to control the patient's behavior even if the treatment regimen is strictly followed. Some patients do not complete the course of treatment, which can lead to ineffective treatment and development of drug resistance. Even when the entire course of treatment is completed, the TB germ infection is not eradicated from the infected individual and remains a latent infection that can still be reactivated.
결핵의 확산을 통제하기 위해서는, 상기 질병에 대한 효과적인 예방접종(vaccination) 및 정확한 초기 진단이 가장 중요하다. 현재까지는 생균(live bacteria)을 이용한 예방접종이 보호 면역을 유도하기 위한 가장 효율적인 방법이다. 상기 목적을 위해 이용되는 가장 일반적인 마이코박테리움은 바실러스 칼메트-게링(BacillusCalmette-Guerin, BCG)인데, M. bovis의 비병원성 종이다. 그러나, BCG의 안정성 및 효능은 논쟁의 대상이 되고 있으며, 미국과 같은 일부 국가에서는 상기 제제를 사용한 일반 대중의 예방접종을 실시하지 않고 있다. 결핵의 진단은 일반적으로 피부 검정법을 이용하여 수행되는데, 상기 검정법은 투베르쿨린 PPD(proteinpurified derivative)에 대한 피내 노출을 포함한다. 항원-특이 T 세포 반응은 주입후 48-72시간 까지 주입 부위에서의 측정가능한 정도 경화(induration)를 초래하는데, 이는 마이코박테리움 항원에 대한 노출을 의미한다. 그러나 상기 검정법은 민감도 및 특이성에서 문제가 있으며, BCG로 예방접종된 개체들은 감염된 개체들과 구별될 수 없다.In order to control the spread of tuberculosis, effective vaccination and accurate initial diagnosis of the disease are of utmost importance. To date, vaccination with live bacteria is the most efficient way to induce protective immunity. The most common mycobacterium used for this purpose is Bacillus Calmette-Guerin (BCG), a non-pathogenic species of M. bovis . However, the stability and efficacy of BCG are controversial, and some countries, such as the United States, do not provide the public with the vaccine. Diagnosis of tuberculosis is generally carried out using a skin assay, which includes intradermal exposure to tuberculin proteinpurified derivative (PPD). Antigen-specific T cell responses result in measurable degree induration at the site of infusion up to 48-72 hours post infusion, indicating exposure to mycobacterium antigens. However, the assay is problematic in sensitivity and specificity, and individuals vaccinated with BCG cannot be distinguished from those infected.
p53 (분자량 53 kDa) 은 세포의 이상증식 및 돌연변이 억제 기능을 가진 대표적인 항암유전자이다. 또한, 전사인자로써 세포자사멸 (apoptosis) 에 관여하는 PUMA, Bax 등의 발현을 증가시켜, 암세포 및 세균 감염세포의 사멸 유도에 효과적인 단백질이다. 따라서, 대식세포의 결핵균 사멸과정에서 p53 역할이 중요할 것으로 여겨진다. p53 (molecular weight 53 kDa) is a representative anti-cancer gene with functions of cell proliferation and mutation suppression. In addition, by increasing the expression of PUMA, Bax and the like involved in apoptosis as a transcription factor, it is an effective protein for inducing death of cancer cells and bacterial infected cells. Therefore, the role of p53 in the killing of Mycobacterium tuberculosis is considered to be important.
P53 단백질은 MDM2 단백질에 의하여 유비퀴틴으로 표지되고 proteasome 에 의해 분해되는데, 항암제로 알려져 있는 Nutlin-3α (분자량 581.49)는 이러한 MDM2 와 결합하여 p53-MDM2 상호작용을 억제함으로써 p53 경로 활성을 증가시켜주는 화합물이다. 따라서, p53을 타겟으로 하여 결핵균 억제 방법으로써, Nutlin-3α 단독 또는 기존 항결핵 치료제와 병행하여 사용할 수 있을 것으로 사료된다. The P53 protein is labeled ubiquitin by MDM2 protein and degraded by proteasome. Nutlin-3α (molecular weight 581.49), known as an anticancer agent, binds to this MDM2 and inhibits p53-MDM2 interaction to increase p53 pathway activity. to be. Therefore, as a method for inhibiting Mycobacterium tuberculosis with p53 as a target, Nutlin-3α alone or in combination with existing anti-tuberculosis therapeutics may be used.
본 발명은 p53의 발현조절 또는 p53 활성을 유도하는 Nutlin-3α 를 이용하여 대식세포의 활성화에 기여하여, 기존 약제와 작용기전이 다르고 부작용이 상대적으로 적은 결핵 예방 및 효율적인 치료 방법을 제공하고자 한다. The present invention contributes to the activation of macrophages using Nutlin-3α, which induces expression of p53 or induces p53 activity, to provide a method for preventing and effectively treating tuberculosis, which has a different mechanism of action and relatively fewer side effects.
전술한 목적을 달성하기 위하여 본 발명은 Nutlin-3α 화합물을 유효성분으로 포함하는 결핵 예방 및 치료용 약학적 조성물을 제공하고자 한다. In order to achieve the above object, the present invention is to provide a pharmaceutical composition for preventing and treating tuberculosis comprising a Nutlin-3α compound as an active ingredient.
이렇게 제공된 Nutlin-3α 화합물은 p53 과발현 시키는 것을 특징으로 하며 이렇게 과발현된 p53은 결핵균에 감염된 대식세포의 세포자멸사(apoptosis)를 유도함으로써 결핵균의 증식을 억제하는 것을 특징으로 한다. The Nutlin-3α compound thus provided is characterized by overexpressing p53, and the overexpressed p53 inhibits the growth of Mycobacterium tuberculosis by inducing apoptosis of macrophages infected with Mycobacterium tuberculosis.
또한 Nutlin-3α에 기존의 항결핵 약제를 병용하여 사용하는 것을 포함할 수 있다. It may also include the use of a combination of existing anti-tuberculosis drugs in Nutlin-3α.
여기에서 사용되는 항결핵 약제는 리팜피신(Rifampicin), 아이소니아지드(Isoniazid), 에탐부톨(Ethambutol), 피라진아미드(Pyrazinamide)으로 이루어진 군으로부터 선택된 적어도 하나일 수 있다. The anti-tuberculosis agent used herein may be at least one selected from the group consisting of Rifampicin, Isoniazid, Ethambutol, and Pyrazinamide.
또한 상기 결핵은 안결핵, 피부 결핵, 부신 결핵, 신장결핵, 부고환 결핵, 림프선 결핵, 후두 결핵, 중이 결핵, 장결핵, 다제내성 결핵, 폐결핵, 담결핵, 골결핵, 인후결핵, 임파선 결핵, 폐허증, 유방 결핵 및 척추 결핵으로 이루어진 군으로부터 선택된 적어도 하나일 수 있다. In addition, the tuberculosis, tuberculosis, skin tuberculosis, adrenal tuberculosis, kidney tuberculosis, epididymal tuberculosis, lymphatic tuberculosis, laryngeal tuberculosis, middle ear tuberculosis, intestinal tuberculosis, multidrug-resistant tuberculosis, pulmonary tuberculosis, biliary tuberculosis, bone tuberculosis, throat tuberculosis, lymph node tuberculosis, lung , Breast tuberculosis and spinal tuberculosis.
상기 사용된 Nutlin-3α의 분자식은 C30H30Cl2N4O4 이다.The molecular formula of Nutlin-3α used above is C 30 H 30 Cl 2 N 4 O 4 .
이상과 같이 본 발명에 의하면 종래 전혀 알려져 있지 않던 Nutlin-3α의 결핵균에 대한 기능을 활용한 것이므로, 기존 약제에 대해 다약제내성이 있는 결핵균에 대해서도 예방 및 치료효과를 기대할 수 있다. 또한 본 발명에 의하면 세포내에 잠복하고 있는 결핵균의 생장을 억제할 수 있기 때문에 단독으로 사용하거나, 종래 약제와 병용하는 경우 잠복성 결핵균을 제거할 수 있게 된다.As described above, since the present invention utilizes the function of Mycobacterium tuberculosis of Nutlin-3α, which is not known at all, it can be expected to have a prophylactic and therapeutic effect against Mycobacterium tuberculosis having multidrug resistance with respect to existing drugs. In addition, according to the present invention, the growth of latent Mycobacterium tuberculosis can be suppressed, so that latent Mycobacterium tuberculosis can be removed when used alone or in combination with a conventional medicine.
도 1은 Nutlin-3α 구조도를 나타낸 사진이다. 1 is a photograph showing a Nutlin-3α structure diagram.
도 2는 결핵균이 감염된 대식세포에 처리된 Nutlin-3α 농도에 따른 p53 발현과 Caspase-3 활성을 보여주는 western blotting에 의한 단백질 발현 전기영동사진이다. Figure 2 is a protein expression electrophoresis picture by western blotting showing p53 expression and Caspase-3 activity according to Nutlin-3α concentrations treated in Mycobacterium tuberculosis-infected macrophages.
도 3은 Nutlin-3α 처리에 의해 대식세포 내 결핵균의 수가 유의하게 감소하는 것을 보여주는 그래프이다. 3 is a graph showing that the number of Mycobacterium tuberculosis in macrophages is significantly reduced by Nutlin-3α treatment.
도 4 및 도 5는 항결핵 치료제 (아이소니아지드, 리팜피신, 피라지나마이드, 에탐부톨) 와 Nutlin-3α 병용 처리에 의해 대식세포 내 결핵균의 수가 유의하게 감소하는 것을 보여주는 그래프이다. 4 and 5 are graphs showing that the number of Mycobacterium tuberculosis in macrophages is significantly reduced by the combination of antituberculosis therapeutic agents (isoniazid, rifampicin, pyrazinamide, ethambutol) and Nutlin-3α.
도 6은 p53이 결핍된 macrophage를 가진 ㅤ마우스 폐에서 결핵균 생존수가 현저히 증가하는 것을 보여주는 그래프이다. FIG. 6 is a graph showing a significant increase in Mycobacterium tuberculosis survival in mice lungs with p53-deficient macrophage.
도 7은 결핵균을 감염시킨 마우스에 Nutlin-3a (10mg/kg/day)를 복강 주입하여 p53 활성을 증가시켜주었을 때, 폐 조직 내의 결핵균 수가 현저히 감소하는 것을 보여주는 그래프이다. FIG. 7 is a graph showing that the number of Mycobacterium tuberculosis in lung tissue is significantly decreased when pneumoniae-infected mice are intraperitoneally injected with Nutlin-3a (10 mg / kg / day).
도 8은 건강인 macrophage에 비해 결핵환자의 macrophage에서 p53의 발현이 현저히 떨어지는 것을 보여주는 그래프이다. 8 is a graph showing that the expression of p53 in the macrophage of tuberculosis patients is significantly lower than that of healthy macrophage.
도 9는 Nutlin-3a를 통해 p53 활성을 증가시켜주었을 때, 세포 내의 결핵균 수가 현저히 감소하는 것을 보여주는 그래프이다. 9 is a graph showing that the number of Mycobacterium tuberculosis in cells is significantly reduced when p53 activity is increased through Nutlin-3a.
이하 첨부된 도면을 참조하여 본 발명을 보다 상세히 설명한다. 그러나 첨부된 도면은 본 발명의 기술적 사상의 내용과 범위를 쉽게 설명하기 위한 예시일 뿐, 이에 의해 본 발명의 기술적 범위가 한정되거나 변경되는 것은 아니다. 또한 이러한 예시에 기초하여 본 발명의 기술적 사상의 범위 안에서 다양한 변형과 변경이 가능함은 당업자에게는 당연할 것이다. Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings. However, the accompanying drawings are only examples for easily describing the content and scope of the technical idea of the present invention, and thus the technical scope of the present invention is not limited or changed. In addition, it will be apparent to those skilled in the art that various modifications and changes can be made within the scope of the present invention based on these examples.
본 발명은 Nutlin-3α 화합물이 p53 과발현시켜 대식세포의 세포자멸사를 작용시키는 것을 통하여 결핵균의 예방 또는 치료할 수 있는 효과가 있음을 최초로 규명하였으며, 따라서 본 발명은 Nutlin-3α 화합물을 유효성분으로 함유하는 결핵균의 예방 또는 치료용 조성물을 제공함에 그 특징이 있다. The present invention was first identified that the Nutlin-3α compound has an effect of preventing or treating Mycobacterium tuberculosis through p53 overexpression and acting on apoptosis of macrophages. Thus, the present invention comprises a Nutlin-3α compound as an active ingredient. It is characterized by providing a composition for preventing or treating Mycobacterium tuberculosis.
일반적으로 ‘Nutlin-3α’는 MDM2에 결합해 MDM2와 p53 단백질 간 결합을 저해하는 소형분자 화합물로서, 항암효과가 알려져 있으며, 화학구조는 하기 도 1과 같다. 좀 더 구체적으로 설명하자면 Nutlin-3α은 MDM2에 결합해 MDM2와 p53 단백질 간 결합을 저해하는 소형분자 화합물로서 고형암 등의 치료를 위해 현재 임상에서 개발 중인 항암 약물이다. 자세하게는, MDM2 단백질은 암을 억제하는 유전자로 알려진 p53의 전이활성(transactivation) 부위에 결합하여 활성화 능력을 차단시키는 역할을 하는데, Nutlin-3α 화합물의 경우 p53 과 유사한 분자 구조를 가지기 때문에 MDM2 단백질과 결합을 형성하며, 이로 인해 p53 단백질이 MDM2 단백질과 결합하지 못함에 따라 활성화 능력이 유지되어 p53이 과발현되게 되는 것이다. 그러나 상기 Nutlin-3α 화합물은 현재 임상에서 개발 중인 항암 약물로만 알려져 있으며, 결핵 치료와 관련하여 효과를 가진다는 내용에 대해서는 전혀 보고된 바가 없다. 이에 본 발명자들은 Nutlin-3α 화합물이 p53을 활성화시키는 기작을 통하여, 결핵을 개선시킬 수 있음을 확인함으로써 본 발명을 완성하게 되었다.In general, 'Nutlin-3α' is a small molecule compound that binds to MDM2 and inhibits binding between MDM2 and p53 protein, and is known for its anticancer effect, and its chemical structure is shown in FIG. 1. More specifically, Nutlin-3α is a small molecule compound that binds to MDM2 and inhibits binding between MDM2 and p53 protein. It is an anticancer drug currently being developed for the treatment of solid cancer. In detail, the MDM2 protein binds to the transactivation site of p53, a gene that inhibits cancer, and blocks the activation ability. The Nutlin-3α compound has a molecular structure similar to that of p53. It forms a bond, which causes the p53 protein to not bind to the MDM2 protein and thus maintains its activating ability, resulting in overexpression of p53. However, the Nutlin-3α compound is only known as an anticancer drug that is currently being developed in the clinic, and there is no report on the effect that it has on the treatment of tuberculosis. The present inventors have completed the present invention by confirming that Nutlin-3α compound can improve tuberculosis through a mechanism of activating p53.
또한 본 발명의 하기 실시예 1에 따르면, 마우스 유래 대식세포인 Bone marrow-derived macrophages (BMDMs) 세포에 Nutlin-3α 화합물을 주입한 경우 결핵균의 수가 유의적으로 감소되는 것으로 나타나 결핵이 개선되는 효과가 있음을 확인할 수 있었다.In addition, according to Example 1 of the present invention, when the Nutlin-3α compound was injected into Bone marrow-derived macrophages (BMDMs) cells, which are mouse-derived macrophages, the number of Mycobacterium tuberculosis bacteria was significantly reduced, resulting in an improvement in tuberculosis. It could be confirmed.
또한 본 발명의 하기 실시예 2에 따르면, 기존 항결핵제와 병용하여 사용시 기존 항결핵제 단독으로 사용한 것에 비하여 월등히 높은 결핵균 감소의 효과를 보임을 확인할 수 있었다. In addition, according to Example 2 of the present invention, when used in combination with the existing anti-tuberculosis agent was confirmed to show an effect of significantly higher tuberculosis bacteria compared to the use of the existing anti-tuberculosis agent alone.
따라서 Nutlin-3α 화합물은 결핵을 개선시키는 효과 있으므로, 이를 유효성분으로 포함하는 본 발명의 조성물은 결핵에 대한 예방 또는 치료에서 유용하게 사용될 수 있다. Therefore, Nutlin-3α compound is effective in improving tuberculosis, the composition of the present invention comprising it as an active ingredient can be usefully used in the prevention or treatment for tuberculosis.
본 발명에서 말하는 결핵에는 안결핵, 피부 결핵, 부신 결핵, 신장결핵, 부고환 결핵, 림프선 결핵, 후두 결핵, 중이 결핵, 장결핵, 다약제내성 결핵, 폐결핵, 담결핵, 골결핵, 인후결핵, 임파선 결핵, 폐허증, 유방 결핵 또는 척추 결핵등을 포함할 수 있다. Tuberculosis in the present invention includes eye tuberculosis, skin tuberculosis, adrenal tuberculosis, kidney tuberculosis, epididymal tuberculosis, lymphatic tuberculosis, laryngeal tuberculosis, middle ear tuberculosis, intestinal tuberculosis, multidrug-resistant tuberculosis, pulmonary tuberculosis, tuberculosis, bone tuberculosis, throat tuberculosis, lymphatic tuberculosis We can include ruins, breast tuberculosis or spinal tuberculosis.
또한 기존 항결핵제로서는 본 발명의 실시예 2에 사용된 리팜피신(Rifampicin), 아이소니아지드(Isoniazid), 에탐부톨(Ethambutol) 또는 피라진아미드(Pyrazinamide)등이 있을 수 있다. In addition, existing anti-tuberculosis agents may include Rifampicin, Isoniazid, Ethambutol or Pyrazinamide used in Example 2 of the present invention.
본 발명에서 상기 '치료'란, 달리 언급되지 않는 한, 상기 용어가 적용되는 질환 또는 질병, 또는 상기 질환 또는 질병의 하나 이상의 증상을 역전시키거나, 완화시키거나, 그 진행을 억제하거나, 또는 예방하는 것을 의미하며, 본원에서 사용된 상기 '치료'란 용어는 '치료하는'이 상기와 같이 정의될 때 치료하는 행위를 말한다. In the present invention, the term 'treatment', unless stated otherwise, reverses, alleviates, inhibits, or prevents the disease or condition to which the term applies, or one or more symptoms of the disease or condition, As used herein, the term 'treatment' refers to the act of treating when 'treating' is defined as above.
이하 좀 더 구체적인 실시예를 통해서 본 발명을 살펴보고자 한다. Hereinafter, the present invention will be described through more specific examples.
실시예 1 : p53 발현 조절을 통한 세포 내 결핵균의 생장 억제 효과 1Example 1 Growth Inhibition Effect of Mycobacterium Tuberculosis by Controlling p53 Expression 1
세포 내 결핵균의 감염 시 Nutlin-3α 처리에 의한 p53 발현 여부와, 이것이 어떠한 역할을 가지는지를 분석하기 위하여 결핵균을 감염시킨 대식세포로부터 p53 의 발현과 세포 내 결핵균 사멸 효과를 확인하였다. 마우스 유래 대식세포인 Bone marrow-derived macrophages (BMDMs) 세포를 사용하여, 병원성 결핵균(Mycobacterium tuberculosis H37Rv)과 비병원성 결핵균 (Mycobacterium tuberculosis H37Ra)을 세포당 균 수 1의 비율로 감염시키고 Nutlin-3α 를 처리한 후, 세포 내 결핵균의 생존 수를 측정하였다. In order to analyze the expression of p53 by Nutlin-3α treatment and its role in intracellular Mycobacterium tuberculosis infection, p53 expression and intracellular Mycobacterium tuberculosis killing effect were confirmed. Bone marrow-derived macrophages (BMDMs) cells, which are mouse-derived macrophages, were used to infect Mycobacterium tuberculosis H37Rv and non-pathogenic Mycobacterium tuberculosis (Mycobacterium tuberculosis H37Ra) at a rate of 1 per cell and treated with Nutlin-3α. Afterwards, the viable number of Mycobacterium tuberculosis was measured.
도 2는 결핵균 감염 시 발현되는 p53 단백질 발현 정도와 세포자멸사 마커인 Caspase-3 활성을 western blot 방법으로 확인한 결과로서 결핵균이 감염된 대식세포에서 p53 유도제인 Nutlin-3α (도 1) 처리 농도 증가에 따라 점점 p53 과 Caspas-3 의 활성이 증가하는 것을 보여준다. (도 2에서 β-actin은 내부콘트롤) Figure 2 shows the expression level of p53 protein expressed during Mycobacterium tuberculosis infection and the apoptosis marker Caspase-3 activity as a result of Western blot method in accordance with the increased treatment concentration of Nutlin-3α (Fig. 1), a p53 inducing agent in macrophages infected with Mycobacterium tuberculosis. Increasing p53 and Caspas-3 activity was observed. (Β-actin is an internal control in Figure 2)
도 3은 결핵균이 감염된 큰대식세포에 Nutlin-3α (30 uM) 를 처리하여 24 시간 경과 후, 세포 내 생존하고 있는 결핵균 수를 7H10 agar 배지에서 14~21일 동안 배양하여 산출하였다. 그 결과, 대조군에 비해 Nutlin-3α (30 uM) 를 처리한 대식세포에서 세포 내 병원성 결핵균, 비병원성 결핵균의 생존수가 현저히 감소된 것을 보여준다. FIG. 3 was treated with Nutlin-3α (30 uM) in large macrophages infected with Mycobacterium tuberculosis and after 24 hours, the number of viable Mycobacterium tuberculosis cells was cultured in 7H10 agar medium for 14-21 days. As a result, the number of survival of the intracellular pathogenic Mycobacterium tuberculosis and nonpathogenic Mycobacterium tuberculosis was significantly reduced in the macrophages treated with Nutlin-3α (30 uM) compared with the control group.
실시예 2 : p53 발현 조절을 통한 세포 내 결핵균의 생장 억제 효과 2Example 2 Growth Inhibition Effect of Mycobacterium Tuberculosis by Controlling p53 Expression 2
세포 내 결핵균의 감염 시 Nutlin-3α 처리에 의한 p53 발현 조절이 어떠한 역할을 가지는지를 분석하기 위하여 기존의 항결핵 치료제와 병용하여 사용한 후, 세포 내 결핵균의 수를 측정하였다.In order to analyze the role of p53 expression regulation by Nutlin-3α treatment in the infection of intracellular Mycobacterium tuberculosis, it was used in combination with an existing anti-tuberculosis therapeutic agent, and the number of Mycobacterium tuberculosis was measured.
도 4 및 도5는 대식세포내 결핵균 감염 시 항결핵 치료제인 아이소니아지드(10 ng/ml), 리팜피신(10 ng/ml), 피라지나마이드(100 ng/ml) 또는 에탐부톨(100 ng/ml) 과 Nutlin-3α (30 uM) 를 병용하여 처리하였을 때 세포 내 결핵균의 증식 억제효과를 확인한 그림이다. 병원성 결핵균 또는 비병원성 결핵균이 감염된 대식세포에 항결핵 치료제 단독 또는 Nutlin-3α 와 병용 처리하여 24 시간 경과 후, 세포 내에 생존하고 있는 결핵균의 수를 측정하였다. 실험 결과, 항결핵 치료제 단독사용에 비해 항결핵 치료제와 Nutlin-3α 를 병용하여 처리하였을 때 세포 내 결핵균 수가 유의하게 감소하는 것을 보여준다. Figures 4 and 5 show anti-tuberculosis treatment of isoniazid (10 ng / ml), rifampicin (10 ng / ml), pyrazinamide (100 ng / ml) or etambutol (100 ng / ml) during the infection of Mycobacterium tuberculosis bacteria. And Nutlin-3α (30 uM) in combination to inhibit the growth of mycobacterium tuberculosis. Macrophage cells infected with pathogenic Mycobacterium tuberculosis or non-pathogenic Mycobacterium tuberculosis were treated with antituberculosis treatment alone or in combination with Nutlin-3α to determine the number of tuberculosis viable in cells after 24 hours. The experimental results showed that the number of intracellular tuberculosis bacteria decreased significantly when anti-TB treatment and Nutlin-3α were used in combination with anti-TB treatment alone.
도 6은 대조군 (wild type) 또는 macrophage의 p53이 결핍된 (p53 conditional knockout) 마우스에 비병원성 결핵균을 비강 내 감염시킨 후, 폐 조직 내 생존하고 있는 결핵균 수를 측정하였다. 그 결과, 대조군에 비해 p53이 결핍된 macrophage를 가진 ㅤ마우스 폐에서 결핵균 생존수가 현저히 증가하는 것을 보여준다. ㅤFIG. 6 shows the number of viable Mycobacterium tuberculosis bacteria in lung tissue after intranasal infection of non-pathogenic Mycobacterium tuberculosis in a control (wild type) or macrophage-deficient p53 (p53 conditional knockout) mouse. As a result, the survival rate of Mycobacterium tuberculosis in mice with p53-deficient macrophage was significantly increased. ㅤ
도 7은 비강을 통해 비병원성 결핵균을 감염시킨 마우스에 Nutlin-3a (10mg/kg/day)를 복강 주입하여 p53 활성을 증가시켜주었을 때, 폐 조직 내의 결핵균 수가 현저히 감소하는 것을 나타낸다.FIG. 7 shows that the number of Mycobacterium tuberculosis in lung tissue was significantly reduced when p53 activity was increased by intraperitoneal injection of Nutlin-3a (10 mg / kg / day) into mice infected with non-pathogenic Mycobacterium tuberculosis via the nasal cavity.
도 8은 건강인(n=9) 또는 결핵환자(n=13)의 혈액에서 분리한 macrophage 에 비병원성 결핵균을 감염시킨 후, Real-time PCR 방법을 통해 p53 발현 양상을 확인하였다. 그 결과, 건강인 macrophage에 비해 결핵환자의 macrophage에서 p53의 발현이 현저히 떨어지는 것을 알 수 있다. FIG. 8 shows the expression of p53 by real-time PCR after infecting non-pathogenic Mycobacterium tuberculosis to macrophage isolated from blood of healthy subjects (n = 9) or tuberculosis patients (n = 13). As a result, the expression of p53 in the macrophage of tuberculosis patients was significantly lower than that of healthy macrophage.
도 9는 건강인 또는 결핵환자 혈액에서 유래한 macrophage에 비병원성 결핵균을 감염시킨 후, Nutlin-3a (10 uM)를 처리하고 세포 내 결핵균 생존 수를 측정하였다. 그 결과로, Nutlin-3a를 통해 p53 활성을 증가시켜주었을 때, 세포 내의 결핵균 수가 현저히 감소하는 것을 확인하였다.Figure 9 was infected with non-pathogenic Mycobacterium tuberculosis in macrophage derived from the blood of healthy or tuberculosis patients, treated with Nutlin-3a (10 uM) and the number of intracellular Mycobacterium viability was measured. As a result, when the p53 activity was increased through Nutlin-3a, it was confirmed that the number of Mycobacterium tuberculosis in the cells was significantly reduced.
상기 실시예에서는 세포 내 p53 의 발현을 증가시킴으로써 세포 내 결핵균을 생존을 억제시킬 수 있음을 보았다. 따라서 p53 을 조절하는 유효성분으로 본 발명에 의한 조성물 (Nutlin-3α) 을 결핵균 감염세포에 처리하는 경우 즉, 본 발명에 의한 조성물을 결핵균 보균자, 결핵환자 또는 기존 항결핵제 내성 환자에게 처치하는 경우에도 세포 내 결핵균을 사멸시킬 수 있음을 기대할 수 있다.In the above example, it was found that the mycobacterium tuberculosis can be suppressed by increasing the expression of p53 in the cells. Therefore, when the composition according to the present invention (Nutlin-3α) is treated with Mycobacterium tuberculosis-infected cells as an active ingredient to control p53, that is, when the composition of the present invention is treated to Mycobacterium tuberculosis carriers, tuberculosis patients or patients with existing anti-tuberculosis drug resistance It can be expected to kill my tuberculosis bacteria.
통계처리에 사용한 방법은 Bonferroni’s multiple comparison test로, p-값 < 0.05 일 때 *, p-값 < 0.01 일 때 **, p-값 < 0.001 일 때 ***로 표시하였다. The statistical method used was Bonferroni's multiple comparison test, marked with * when p-value <0.05, ** when p-value <0.01, and *** when p-value <0.001.
Claims (6)
- Nutlin-3α 화합물을 유효성분으로 포함하는 결핵 예방 및 치료용 약학적 조성물. Tuberculosis prevention and treatment pharmaceutical composition comprising a nutlin-3α compound as an active ingredient.
- 제1항에 있어서,The method of claim 1,상기 Nutlin-3α 화합물은 p53 과발현 시키는 것을 특징으로 하는 결핵 예방 및 치료용 약학적 조성물. The nutlin-3α compound is a p53 overexpression, characterized in that the pharmaceutical composition for preventing and treating tuberculosis.
- 제2항에 있어서, The method of claim 2,상기 p53 과발현은 결핵균에 감염된 대식세포의 세포자멸사(apoptosis)를 유도함으로써 결핵균의 증식을 억제하는 것을 특징으로 하는 결핵 예방 및 치료용 약학적 조성물. The p53 overexpression is a pharmaceutical composition for preventing and treating tuberculosis, characterized in that to inhibit the growth of Mycobacterium tuberculosis by inducing apoptosis (apoptosis) of macrophage infected with Mycobacterium tuberculosis.
- 제1항에 있어서,The method of claim 1,상기 Nutlin-3α 화합물에 항결핵 약제를 병용하여 사용하는 결핵 예방 및 치료용 약학적 조성물. Tuberculosis prevention and treatment pharmaceutical composition using an anti-tuberculosis agent in combination with the Nutlin-3α compound.
- 제4항에 있어서, The method of claim 4, wherein상기 항결핵 약제는 리팜피신(Rifampicin), 아이소니아지드(Isoniazid), 에탐부톨(Ethambutol) 또는 피라진아미드(Pyrazinamide)인 결핵 예방 및 치료용 약학적 조성물. The anti-tuberculosis drug is rifampicin (Rifampicin), isoniazid (Isoniazid), Ethambutol (Ethambutol) or pyrazinamide (Pyrazinamide) pharmaceutical composition for preventing and treating tuberculosis.
- 제1항 내지 제5항에 있어서, The method according to claim 1, wherein상기 결핵은 안결핵, 피부 결핵, 부신 결핵, 신장결핵, 부고환 결핵, 림프선 결핵, 후두 결핵, 중이 결핵, 장결핵, 다약제내성 결핵, 폐결핵, 담결핵, 골결핵, 인후결핵, 임파선 결핵, 폐허증, 유방 결핵 또는 척추 결핵인 결핵 예방 및 치료용 약학적 조성물. The tuberculosis may include eye tuberculosis, skin tuberculosis, adrenal tuberculosis, kidney tuberculosis, epididymal tuberculosis, lymphatic tuberculosis, laryngeal tuberculosis, middle ear tuberculosis, intestinal tuberculosis, multidrug-resistant tuberculosis, pulmonary tuberculosis, biliary tuberculosis, bone tuberculosis, throat tuberculosis, lymph node tuberculosis Pharmaceutical composition for the prevention and treatment of tuberculosis, tuberculosis or tuberculosis.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112575078A (en) * | 2020-12-30 | 2021-03-30 | 山东大学第二医院 | Application of lncRNAs as specific markers of active pulmonary tuberculosis |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20110063518A (en) * | 2008-09-03 | 2011-06-10 | 화이자 인코포레이티드 | Combination therapy for tuberculosis |
| KR20140032312A (en) * | 2012-09-06 | 2014-03-14 | 충남대학교산학협력단 | Pharmaceutical composition for treatment of tuberculosis |
| KR20150128731A (en) * | 2013-03-15 | 2015-11-18 | 보드 오브 리전츠, 더 유니버시티 오브 텍사스 시스템 | Inhibition of Pulmonary Fibrosis with Nutlin-3a and Peptides |
-
2018
- 2018-04-13 WO PCT/KR2018/004350 patent/WO2018190680A1/en active Application Filing
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20110063518A (en) * | 2008-09-03 | 2011-06-10 | 화이자 인코포레이티드 | Combination therapy for tuberculosis |
| KR20140032312A (en) * | 2012-09-06 | 2014-03-14 | 충남대학교산학협력단 | Pharmaceutical composition for treatment of tuberculosis |
| KR20150128731A (en) * | 2013-03-15 | 2015-11-18 | 보드 오브 리전츠, 더 유니버시티 오브 텍사스 시스템 | Inhibition of Pulmonary Fibrosis with Nutlin-3a and Peptides |
Non-Patent Citations (2)
| Title |
|---|
| CRANE, E. K.: "Nutlin-3a: A potential therapeutic opportunity for TP53 wild-type ovarian carcinomas", PLOS ONE, 2015, pages 1 - 13 * |
| MADENSPACHER, J. H.: "p53 integrates host defense and cell fate during bacterial pneumonia", THE JOURNAL OF EXPERIMENTAL MEDICINE, 2013, pages 891 - 904 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112575078A (en) * | 2020-12-30 | 2021-03-30 | 山东大学第二医院 | Application of lncRNAs as specific markers of active pulmonary tuberculosis |
| CN112575078B (en) * | 2020-12-30 | 2022-10-04 | 山东大学第二医院 | Application of lncRNAs as specific markers of active tuberculosis |
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