KR20150083466A - Inhibition of Elastase from Pseudomonas aeruginosa - Google Patents
Inhibition of Elastase from Pseudomonas aeruginosa Download PDFInfo
- Publication number
- KR20150083466A KR20150083466A KR1020140002754A KR20140002754A KR20150083466A KR 20150083466 A KR20150083466 A KR 20150083466A KR 1020140002754 A KR1020140002754 A KR 1020140002754A KR 20140002754 A KR20140002754 A KR 20140002754A KR 20150083466 A KR20150083466 A KR 20150083466A
- Authority
- KR
- South Korea
- Prior art keywords
- pseudomonas aeruginosa
- elastase
- dtpa
- expression
- present
- Prior art date
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Abstract
Description
본 발명은 슈도모나스 애루지노사(Pseudomonas aeruginosa)의 엘라스테이즈(elastase) 억제용 조성물 및 이를 이용한 슈도모나스 애루지노사 감염의 예방 또는 치료용 약제학적 조성물에 관한 것이다.
The present invention relates to a composition for inhibiting elastase of Pseudomonas aeruginosa and a pharmaceutical composition for preventing or treating Pseudomonas aeruginosa infection using the same.
슈도모나스 애루지노사(Pseudomonas aeruginosa)는 여러 생명 또는 비생명의 환경에서 편재하며, 인간의 면역 체계가 약해졌을 때만 발생하는 병원균이다. 이는 낭포성섬유증, 중이염, 각막염 및 화상 상처 감염에 걸린 환자들로부터 종종 분리되며, 또한 면역이 저하된 환자들에서 패혈증의 병인이 되기도 한다. 이 박테리아는 3세 이하의 어린이들에게 감염되어 폐의 기능에 심각한 영향을 줄 수 있다. 게다가, 슈도모나스 애루지노사은 맹장 파열 후 이차적으로 발생하는 패혈증, 복막염 증상의 건강한 어린이들에게서도 보통 발견된다. 슈도모나스 애루지노사에 의한 복막염은 복막 투석(continous ambulatory peritoneal dialysis, CAPD)을 받고 있는 환자들에게 심각한 위협(약 10%의 치사율)이 될 수 있다. 이러한 경우, 박테리아에 의한 감염은 종종 높은 사망률, CAPD의 실패 및 합병증을 유발하게 된다. 복막염의 병리생리학적 복잡성을 이해하기 위해 슈도모나스 애루지노사에 의한 복막염에 걸린 설치류 모델이 개발되었다. 병리학적 결과는 일반적으로 균혈증을 동반하며, 감염된 간에서는 감염 6시간 내에 인터류킨-6의 혈청 레벨이 증가하였고, 슈도모나스 애루지노사 종 및 감염량에 따라 다르지만, 48시간 내에 최종적으로 사망하였다.Pseudomonas aeruginosa is ubiquitous in many life or non-life environments, and only occurs when the human immune system is weakened. It is often isolated from patients with cystic fibrosis, otitis media, keratitis and burn wounds, and is also the etiologic agent of sepsis in immunocompromised patients. This bacterium is infected by children under three years of age and can seriously affect lung function. In addition, Pseudomonas aeruginosa is commonly found in healthy children with septicemia and peritonitis secondary to secondary appendicitis. Peritonitis by Pseudomonas aeruginosa can be a serious threat (about 10% mortality) to patients undergoing peritoneal dialysis (CAPD). In such cases, bacterial infections often lead to high mortality, failure of CAPD, and complications. To understand the pathophysiological complexity of peritonitis, a rodent model with peritonitis caused by Pseudomonas aeruginosa was developed. Pathologic results were generally bacteremic. Serum levels of interleukin-6 increased within 6 hours of infection in infected liver and eventually died within 48 hours, depending on the species and amount of Pseudomonas aeruginosa.
비록, 박테리아의 감염을 조절하는데 사용되는 항생물질이 다양하게 존재하지만, 항생물질에 대한 저항성이 부득이하게 발생할 수 있기 때문에 종종 그 항생 물질은 효과적이지 못할 수 있다. 박테리아들 간의 고유한 또는 획득한 항생물질에 대한 저항성 기작의 선택 및 보급은 지금까지 밝혀진 다양한 항생 물질을 포함한 화학적 치료 방법을 방해하고, 다수의 항생 물질 저항성을 갖는 박테리아종의 발생을 증진시킨다. 이러한 이유로 최근 박테리아에 의한 감염 환자의 사망률이 증가하고 있다. 따라서 박테리아에 의한 감염을 조절할 수 있는 새로운 치료 및 예방 전략의 개발이 강력하게 요구된다.Although there are a variety of antibiotics used to control bacterial infection, the antibiotics may often be ineffective because of the inevitable resistance to antibiotics. The selection and dissemination of inherent or acquired antimicrobial resistance mechanisms among bacteria interferes with the various chemical methods of treatment including antibiotics that have so far been discovered and promotes the development of many bacterial species with antibiotic resistance. For these reasons, the mortality rate of recent bacterial infections is increasing. Therefore, the development of new therapeutic and prophylactic strategies to control bacterial infection is strongly required.
항생 물질-저항성 미생물에 의해 야기되는 감염과 싸우기 위한 대체적인 및/또는 추가적인 항-감염 방법으로써, 특이적으로 숙주 박테리아의 감염을 목표로 할 수 있는 박테리오파아지가 다시 주목받고 있으며, 이를 파아지 치료 방법이라 불리운다. 파아지 치료 방법은 파아지를 생체물질로서 사용하는 방법으로 최초의 항생 물질인 페니실린이 발견되기 전인 1916년 Felix d'Herelle에 의해 처음으로 소개되었다. 파아지는 구소련 및 동유럽에서 박테리아 감염의 치료에 항생 물질 대신으로 사용되어왔다. 최근 더 많은 박테리아들이 항생 물질의 저항에 대해 빠른 속도로 진화하고 있기 때문에 파아지 치료 방법에 많은 관심을 두고 있다. 따라서 파아지 치료 방법은 항생 물질의 가치 있는 대안이 될 수 있으며, 특정한 경우에서는 항생 물질보다 의학적으로 우월하다는 것이 증명되었다.As a general and / or additional anti-infective method for combating infections caused by antibiotic-resistant microorganisms, bacteriophages capable of specifically targeting host bacterial infections are attracting attention, . The phage treatment method was first introduced by Felix d'Herelle in 1916, before the first antibiotic, penicillin, was discovered using the phage as a biomaterial. Phages have been used as antibiotics in the treatment of bacterial infections in the Soviet Union and Eastern Europe. Recently, more bacteria are interested in phage treatment methods because they are rapidly evolving against the resistance of antibiotics. Thus, the phage treatment method can be a valuable alternative to antibiotics, and in certain cases it has proven to be medically superior to antibiotics.
슈도모나스 애루지노사는 전통적인 항생 물질 치료 방법의 존재에도 불구하고 생태학적인 적성을 증진시키는 환경에 대한 적응력이 매우 강한 박테리아이다. 빠른 속도로 새로운 슈도모나스 애루지노사 종들이 발생하고, 항생 물질-저항성의 임상 분리 박테리아주들이 지속적으로 증가함에 따라 슈도모나스 애루지노사-매개성 감염을 치료하기 위한 파아지 치료 방법과 같은 대안이 강하게 대두되고 있다. 최근, 유전적으로 변형된 필라멘트형 파아지 Pf3R, 용해성 파아지 분리 집단 또는 파아지 칵테일을 이용한 파아지 치료 방법의 효능이 화상 감염 및 장-유래 패혈증을 포함하여 슈도모나스 애루지노사에 의해 감염된 다양한 실험용 마우스에 대해 연구가 진행 중에 있다. Pseudomonas aeruginosa is a highly adaptive environment-friendly bacteria that promotes ecological aptitude despite the presence of traditional antibiotic treatment methods. As new Pseudomonas aeruginosa species develop rapidly and clinically isolated bacterial strains of antibiotic-resistance continue to increase, alternatives such as phage treatment methods for treating Pseudomonas aeruginosa-mediated infections are strongly emerged have. In recent years, studies have been conducted on various experimental mice infected with Pseudomonas aeruginosa, including the effect of phage treatment using genetically modified filamentous phage Pf3R, soluble phage isolate group or phage cocktail, including burn infection and enteric- It is in progress.
한편, 슈도모나스 애루지노사의 분비 단백질인 엘라스테이즈는 인간 숙주에의 감염시 조직 손상 확대의 원인이 되는 것으로 알려진 주요한 독성 인자로서 그 역할에 대하여는 'Bengt Wretlind et al., Pseudomonas aeruginosa Elastase and Its Role in Pseudomonas Infections, REVIEWS OF INFECTIOUS DISEASES, 5(5):S998-1004(1983).'에 기재되어 있다.
On the other hand, Elastase, a secreted protein of Pseudomonas aeruginosa, is a major toxic factor known to cause tissue damage during infectious human host disease. Bengt Wretlind et al., Pseudomonas aeruginosa Elastase and Its Role in Pseudomonas Infections, REVIEWS OF INFECTIOUS DISEASES , 5 (5): S998-1004 (1983).
본 명세서 전체에 걸쳐 다수의 논문 및 특허문헌이 참조되고 그 인용이 표시되어 있다. 인용된 논문 및 특허문헌의 개시 내용은 그 전체로서 본 명세서에 참조로 삽입되어 본 발명이 속하는 기술 분야의 수준 및 본 발명의 내용이 보다 명확하게 설명된다.
Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.
본 발명자들은 슈도모나스 애루지노사(Pseudomonas aeruginosa)의 엘라스테이즈(elastase) 억제용 조성물을 개발하고자 예의 연구 노력하였다. 그 결과 펜테트산(Pentetic acid)을 유효성분으로 포함하는 조성물이 슈도모나스 애루지노사(Pseudomonas aeruginosa)의 엘라스테이즈(elastase)를 억제하는 것을 규명함으로써, 본 발명을 완성하게 되었다. The present inventors have made extensive efforts to develop a composition for inhibiting elastase of Pseudomonas aeruginosa . As a result, the present inventors have completed the present invention by confirming that a composition containing pentetic acid as an active ingredient inhibits elastase of Pseudomonas aeruginosa .
따라서, 본 발명의 목적은 슈도모나스 애루지노사(Pseudomonas aeruginosa)의 엘라스테이즈(elastase) 억제용 조성물을 제공하는데 있다. Accordingly, an object of the present invention is to provide a composition for inhibiting elastase of Pseudomonas aeruginosa .
본 발명의 다른 목적은 슈도모나스 애루지노사(Pseudomonas aeruginosa) 감염의 예방 또는 치료용 약제학적 조성물을 제공하는데 있다. It is another object of the present invention to provide a pharmaceutical composition for the prevention or treatment of Pseudomonas aeruginosa infection.
본 발명의 또 다른 목적은 슈도모나스 애루지노사(Pseudomonas aeruginosa)에 대한 정균용(bateriostatic) 약제학적 조성물을 제공하는데 있다. It is another object of the present invention to provide a bacteriostatic pharmaceutical composition for Pseudomonas aeruginosa .
본 발명의 또 다른 목적은 슈도모나스 애루지노사(Pseudomonas aeruginosa) 감염의 예방 또는 치료용 약물을 스크리닝 하는 방법을 제공하는데 있다.
It is another object of the present invention to provide a method for screening a drug for the prevention or treatment of Pseudomonas aeruginosa infection.
본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다.
Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.
본 발명의 일 양태에 따르면, 본 발명은 펜테트산(Pentetic acid)을 유효성분으로 포함하는 슈도모나스 애루지노사(Pseudomonas aeruginosa)의 엘라스테이즈(elastase) 억제용 조성물을 제공한다.
According to one aspect of the present invention, there is provided a composition for inhibiting elastase of Pseudomonas aeruginosa comprising, as an active ingredient, pentetic acid.
본 발명자들은 슈도모나스 애루지노사(Pseudomonas aeruginosa)의 엘라스테이즈(elastase) 억제용 조성물을 개발하고자 예의 연구 노력하였다. 그 결과 펜테트산(Pentetic acid)을 유효성분으로 포함하는 조성물이 슈도모나스 애루지노사(Pseudomonas aeruginosa)의 엘라스테이즈(elastase)를 억제하는 것을 규명하였다.The present inventors have made extensive efforts to develop a composition for inhibiting elastase of Pseudomonas aeruginosa . As a result, it was found that a composition containing pentetic acid as an active ingredient inhibits elastase of Pseudomonas aeruginosa .
슈도모나스 애루지노사는 여러 생명 또는 비생명의 환경에서 편재하며, 인간의 면역 체계가 약해졌을 때만 발생하는 병원균이고, 슈도모나스 애루지노사의 엘라스테이즈는 슈도모나스 애루지노사가 세포 외부로 분비하는 단백질로서, 균의 독성과 관계되어 있다. 본 발명의 조성물은 상기 슈도모나스 애루지노사 엘라스테이즈를 억제함으로써, 슈도모나스 애루지노사 감염을 예방 또는 치료할 수 있다. Pseudomonas aeruginosa is a ubiquitous pathogen in a variety of life or non-life environments, and only occurs when the human immune system is weakened. Pseudomonas aeruginosa Elastase is a protein secreted by Pseudomonas aeruginosa into the cell , And the toxicity of the bacteria. The composition of the present invention can prevent or treat Pseudomonas aeruginosa infection by inhibiting the Pseudomonas aeruginosaella stage.
본 발명은 유효성분으로서 펜테트산(pentetic acid)을 포함한다. 본 발명의 펜테트산은 디에틸렌트리아민 펜타아세트산(DTPA)을 지칭하는 것으로 5개의 카복시메틸기를 포함하는 디에틸렌트리아민 골격으로 구성되는 아미노폴리카복실산이며 하기 화학식 1로 표시된다. 펜테트산의 짝염기는 금속 양이온에 대한 높은 친화성을 갖는다. The present invention includes pentetic acid as an active ingredient. The pentatic acid of the present invention refers to diethylenetriaminepentaacetic acid (DTPA), which is an aminopolycarboxylic acid composed of a diethylene triamine skeleton containing five carboxymethyl groups and is represented by the following formula (1). The conjugated bases of pentetic acid have a high affinity for metal cations.
화학식 1
본 발명은 슈도모나스 애루지노사의 엘라스테이즈를 억제하는 펜테트산의 새로운 용도를 제공한다. The present invention provides a new use of pentactic acid to inhibit the erasites of Pseudomonas aeruginosa.
본 발명의 일 구현예에 있어서, 본 발명의 엘라스테이즈 억제는 슈도모나스 애루지노사의 엘라스테이즈의 생성을 억제함으로써 이루어진다. 슈도모나스 애루지노사 엘라스테이즈는 인간 숙주에의 감염시 조직 손상 확대의 원인이 되는 것으로 알려진 주요한 독성 인자이다. 본 발명의 펜테트산은 슈도모나스 애루지노사 엘라스테이즈의 생성을 억제함으로써 슈도모나스 애루지노사 감염을 억제할 수 있다.
In one embodiment of the present invention, the Elastase inhibition of the present invention is achieved by inhibiting the production of Elastinase of Pseudomonas aeruginosa. Pseudomonas aeruginosa Ella stasis is a major toxic factor known to cause tissue damage in infected human hosts. The pentatic acid of the present invention can inhibit Pseudomonas aeruginosa infection by inhibiting the production of Pseudomonas aeruginosaella stasis.
본 발명의 일 양태에 따르면, 본 발명은 (a) 펜테트산(Pentetic acid)의 약제학적 유효량; 및 (b) 약제학적으로 허용되는 담체를 포함하는 슈도모나스 애루지노사(Pseudomonas aeruginosa) 감염의 예방 또는 치료용 약제학적 조성물을 제공한다. According to one aspect of the present invention, there is provided a pharmaceutical composition comprising (a) a pharmaceutically effective amount of Pentetic acid; And (b) a pharmaceutically acceptable carrier. The present invention also provides a pharmaceutical composition for preventing or treating Pseudomonas aeruginosa infection.
본 명세서에서 용어 "슈도모나스 애루지노사 감염"은 녹농균에 의한 감염 그 자체, 녹농균의 감염에 의해 유발되거나 악화되는 질환 및 상기 질환에 의한 증상을 모두 포함한다. 상기 질환은, 예를 들면, 낭포성 섬유증, 중이염, 각막염, 내안구염(endophthalmitis), 균혈증, 화상 상처 감염, 폐렴, 뇌막염, 복막염 또는 패혈증이다. 본 명세서에서 용어 "예방"은 질환 또는 질병을 보유하고 있다고 진단된 적은 없으나, 이러한 질환 또는 질병에 걸리기 쉬운 경향이 있는 동물에서 질환 또는 질병의 발생을 억제하는 것을 의미한다. 본 명에서에서 용어 "치료"는 (ⅰ) 질환 또는 질병의 발전의 억제; (ⅱ) 질환 또는 질병의 경감; 및 (ⅲ) 질환 또는 질병의 제거를 의미한다. As used herein, the term " Pseudomonas aeruginosa infection "encompasses both the infection by P. aeruginosa itself, the disease caused or exacerbated by infection of P. aeruginosa, and the symptoms caused by the disease. Such diseases are, for example, cystic fibrosis, otitis media, keratitis, endophthalmitis, bacteremia, burn injury, pneumonia, meningitis, peritonitis or sepsis. As used herein, the term "prevention" refers to inhibiting the development of a disease or disease in an animal that has never been diagnosed as having a disease or condition, but tends to be susceptible to such disease or disease. As used herein, the term "treatment" refers to (i) inhibition of the development of a disease or disease; (Ii) relief of disease or disease; And (iii) elimination of the disease or disease.
본 발명의 약제학적 조성물에 포함되는 약제학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 본 발명의 약제학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. 적합한 약제학적으로 허용되는 담체 및 제제는 Remington's Pharmaceutical Sciences (19th ed., 1995)에 상세히 기재되어 있다.The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).
본 발명의 약제학적 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 따라 다양한 방법으로 처방될 수 있으며, 바람직하게는 1일 당 0.001-1000 mg/kg(체중)이다.The appropriate dosage of the pharmaceutical composition of the present invention may be determined by various methods depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient And preferably 0.001-1000 mg / kg (body weight) per day.
본 발명의 약제학적 조성물은 경구 또는 비경구로 투여할 수 있고, 비경구로 투여되는 경우, 정맥내 주입, 피하 주입, 근육 주입, 복강 주입, 경피 투여 등으로 투여할 수 있다.The pharmaceutical composition of the present invention can be administered orally or parenterally, and when administered parenterally, it can be administered by intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, transdermal administration, or the like.
본 발명의 조성물에 포함되는 유효성분인 펜테트산의 농도는 치료 목적, 환자의 상태, 필요 기간, 질환의 위중도 등을 고려하여 결정하며 특정 범위의 농도로 한정되지 않는다.The concentration of pentatic acid, which is an active ingredient contained in the composition of the present invention, is determined in consideration of the purpose of treatment, the condition of the patient, the period of time required, the severity of disease, and the like.
본 발명의 약제학적 조성물은 당해 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화 함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기내에 내입시켜 제조될 수 있다. 이때 제형은 오일 또는 수성 매질중의 용액, 현탁액 또는 유화액 형태이거나 엑스제, 분말제, 과립제, 정제 또는 캅셀제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다.
The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.
본 발명의 일 양태에 따르면, 본 발명은 (a) 펜테트산(Pentetic acid)의 약제학적 유효량; 및 (b) 약제학적으로 허용되는 담체를 포함하는 슈도모나스 애루지노사(Pseudomonas aeruginosa)에 대한 정균용(bateriostatic) 약제학적 조성물을 제공한다.According to one aspect of the present invention, there is provided a pharmaceutical composition comprising (a) a pharmaceutically effective amount of Pentetic acid; And (b) a pharmaceutically acceptable carrier. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 is a schematic diagram of a bacteriostatic pharmaceutical composition for Pseudomonas aeruginosa .
본 발명의 약제학적 조성물은 슈도모나스 애루지노사에 대한 정균제(bateriostatic)로 표현하였지만, 이는 슈도모나스 애루지노사에 대한 해독제(antidote)로 표현될 수도 있다.Although the pharmaceutical composition of the present invention is expressed as a bacteriostatic agent for Pseudomonas aeruginosa, it may be expressed as an antidote to Pseudomonas aeruginosa.
항생제는 크게 살균제와 정균제로 나뉜다. 정균제의 경우 세균의 증식을 중지시키거나 속도를 늦추는 작용을 하며, 살균제와 함께 투여하는 것이 효과적이다. 본 발명의 정균용 약제학적 조성물은 슈도모나스 애루지노사의 엘라스테이즈의 합성을 억제하여 감염 조직의 손상이 확장되는 것을 예방하거나 감염부위를 치료할 수 있고, 살균제와 병용 사용함으로써, 슈도모나스 애루지노사에 치료 효과를 향상시킬 수 있다. 본 발명인 정균제에 대하여 약제학적으로 허용되는 담체는 상술한 약제학적 조성물에 대한 담체와 공통되며, 본 발명의 정균제의 적합한 투여량은 상술한 약제학적 조성물과 마찬가지로 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 따라 다양한 방법으로 처방될 수 있으며, 바람직하게는 1일 당 0.001-1000 mg/kg(체중)이다.Antibiotics are largely divided into bactericides and bacteriostats. In the case of a bacteriostatic agent, it acts to stop or slow the growth of bacteria, and it is effective to administer it together with a bactericide. The pharmaceutical composition of the present invention can inhibit the synthesis of Pseudomonas aeruginosa Elastase to prevent the damage of the infected tissue from spreading or to treat the infected area and can be used in combination with a fungicide to treat Pseudomonas aeruginosa The effect can be improved. A suitable pharmaceutically acceptable carrier for the bacteriostatic agent of the present invention is the same as the carrier for the aforementioned pharmaceutical composition, and a suitable dose of the bacteriostatic agent of the present invention may be appropriately selected depending on the formulation method, administration method, May be prescribed in various ways depending on such factors as body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness, preferably 0.001-1000 mg / kg (body weight) per day .
본 발명의 약제학적 조성물의 적합한 투여방식은 상술한 약제학적 조성물과 공통되며, 명세서 기재의 과도한 복잡성을 피하기 위하여 다시 기재하지 않는다.Suitable modes of administration of the pharmaceutical compositions of the present invention are common with the pharmaceutical compositions described above and are not rewritten to avoid undue complexity of the description.
본 발명의 정균제에 포함되는 유효성분인 펜테트산의 농도는 치료 목적, 환자의 상태, 필요 기간, 질환의 위중도 등을 고려하여 결정하며 특정 범위의 농도로 한정되지 않는다.The concentration of pentatic acid, which is an effective ingredient contained in the bacteriostatic agent of the present invention, is determined in consideration of the purpose of treatment, the condition of the patient, the period of time required, the severity of disease, and the like.
본 발명의 일 양태에 따르면, 본 발명은 다음의 단계를 포함하는, 엘라스테이즈(elastase) 억제에 의한 슈도모나스 애루지노사(Pseudomonas aeruginosa) 감염의 예방 또는 치료용 약물을 스크리닝하는 방법을 제공한다:According to one aspect of the present invention, the present invention provides a method of screening for a drug for the prevention or treatment of Pseudomonas aeruginosa infection by elastase inhibition comprising the steps of:
(a) 시험물질을 슈도모나스 애루지노사에 처리하는 단계; 및 (a) treating the test substance to Pseudomonas aeruginosa; And
(b) 상기 시험물질이 슈도모나스 애루지노사의 LasB 유전자의 발현을 억제하는지 여부를 분석하는 단계로서; 상기 시험물질이 슈도모나스 애루지노사의 LasB 유전자의 발현을 억제하는 경우, 상기 시험물질은 엘라스테이즈 억제에 의한 슈도모나스 애루지노사 감염의 예방 또는 치료용 약물로 판정한다.(b) analyzing whether the test substance inhibits the expression of the LasB gene of Pseudomonas aeruginosa; When the test substance inhibits the expression of the LasB gene of Pseudomonas aeruginosa, the test substance is judged to be a drug for the prevention or treatment of Pseudomonas aeruginosa infection caused by Elastase inhibition.
본 발명의 LasB 유전자는 슈도모나스 애루지노사 엘라스테이즈를 코딩하는 유전자이다. LasB의 발현 양상을 분석하는 것에 의하여 후보 물질이 슈도모나스 애루지노사 엘라스테이즈의 발현을 억제하는지 여부를 측정할 수 있다. The LasB gene of the present invention is a gene encoding Pseudomonas aeruginosaella stasis. By analyzing the expression pattern of LasB, it can be determined whether the candidate substance inhibits the expression of Pseudomonas aeruginosa Elastase.
본 발명의 약물을 스크리닝하는 방법에 따르면, 우선, 단계(a)에서 시험물질을 슈도모나스 애루지노사에 처리하는 방법은 슈도모나스 애루지노사 균주 배양액에 후보 약물을 첨가하여 적합한 온도에서 항온 배양하여 실시할 수 있다. 본 발명의 약물을 스크리닝하는 방법에 있어서, 단계(b)의 LasB 유전자 발현 억제여부 분석 방법에는 특별한 제한이 없고, 당업계에 공지되어 있는 방법을 이용할 수 있다. 구체적으로 예를 들면, 리얼-타임 PCR을 이용하여 실시간으로 신속하게 유전자 발현을 측정할 수 있다. According to the method for screening a drug of the present invention, first, in the step (a), the method for treating Pseudomonas aeruginosa with a test substance is carried out by adding a candidate drug to the Pseudomonas aeruginosa culture medium and incubating at a suitable temperature . In the method for screening a drug of the present invention, there is no particular limitation on the method for analyzing the inhibition of LasB gene expression in step (b), and a method known in the art can be used. Specifically, for example, gene expression can be measured quickly in real time using real-time PCR.
본 발명의 일 구현예에 의하면, 본 발명의 스크리닝 하는 방법의 (b) 단계는 LasB 유전자의 발현을 억제하는지 여부를 분석함과 함께 LasI, LasR, rhlI 및 rshR 유전자의 발현을 억제하는지 여부를 추가적으로 더 분석한다: 상기 시험물질이 슈도모나스 애루지노사의 LasB 유전자의 발현을 억제하고, LasI, LasR, rhlI 및 rshR 유전자의 발현은 억제하지 않는 경우, 상기 시험물질은 엘라스테이즈 억제에 의한 슈도모나스 애루지노사 감염의 예방 또는 치료용 약물로 판정한다. LasI, LasR, rhlI 및 rshR 유전자는 슈도모나스 애루지노사의 쿼럼센싱 유전자들로서, 후보 약물이 상기의 유전자들에 대한 발현에는 영향을 주지 않고 LasB의 발현만을 억제하는 경우, 상술한 바와 같이 슈도모나스 애루지노사 엘라스테이즈에 대한 선택적인 억제제임을 알 수 있다. According to one embodiment of the invention, (b) step of the method for screening of the present invention additionally whether inhibiting the expression of genes LasI, LasR, rhlI and rshR with Analyze whether inhibiting the expression of the LasB gene Further analysis: When the test substance suppresses the expression of the LasB gene of Pseudomonas aeruginosa and does not inhibit the expression of the LasI, LasR, rhlI and rshR genes, the test substance is a Pseudomonas aeruginosa strain It is judged to be a drug for prevention or treatment of labor-management infection. The LasI, LasR, rhlI, and rshR genes are quorum sensing genes of Pseudomonas aeruginosa. When the candidate drug inhibits the expression of LasB without affecting the expression of the above genes, Pseudomonas aeruginosa Lt; RTI ID = 0.0 > of Elastase. ≪ / RTI >
본 발명의 일 구현예에 의하면, 본 발명의 약물은 슈도모나스 애루지노사에 대한 정균제(bateriostatics)이다. 본 발명인 슈도모나스 애루지노사에 대한 정균제에 대한 스크리닝 방법은, 상술한 슈도모나스 애루지노사에 대한 정균제를 스크리닝 하는 방법에 관한 것이므로, 본 명세서 기재의 과도한 복잡성을 피하기 위해 공통되는 내용은 중복하여 기재하지 않는다.
According to one embodiment of the present invention, the drug of the present invention is bacteriostatics for Pseudomonas aeruginosa. Since the screening method for the bacteriostatic agent for Pseudomonas aeruginosa of the present invention relates to the method for screening the above-mentioned bacterium against Pseudomonas aeruginosa, common contents are not described in duplicate in order to avoid the excessive complexity described in the present specification .
본 발명의 특징 및 이점을 요약하면 다음과 같다:The features and advantages of the present invention are summarized as follows:
(a) 본 발명은 슈도모나스 애루지노사(Pseudomonas aeruginosa)의 엘라스테이즈(elastase) 억제용 조성물을 제공한다.(a) The present invention provides a composition for inhibiting elastase of Pseudomonas aeruginosa .
(b) 본 발명은 슈도모나스 애루지노사(Pseudomonas aeruginosa) 감염의 예방 또는 치료용 약제학적 조성물을 제공한다.(b) The present invention provides a pharmaceutical composition for preventing or treating Pseudomonas aeruginosa infection.
(c) 본 발명은 슈도모나스 애루지노사(Pseudomonas aeruginosa)에 대한 정균용(bateriostatic) 약제학적 조성물을 제공한다.(c) The present invention provides a bacteriostatic pharmaceutical composition for Pseudomonas aeruginosa .
(d) 본 발명은 슈도모나스 애루지노사(Pseudomonas aeruginosa) 감염의 예방 또는 치료용 약물을 스크리닝 하는 방법을 제공한다.
(d) The present invention provides a method for screening a drug for the prevention or treatment of Pseudomonas aeruginosa infection.
도 1은 EDTA, DTPA 두 가지 약물에 대한 복용량-반응 관계(dose-response relationship)로서, 약물 농도에 따른 엘라스테이즈 활성(도 1의 A) 및 박테리아 개체수(도 1의 B)를 나타낸다.
도 2는 엘라스테이즈 발현을 면역 블랏으로 확인한 결과를 나타낸다.
도 3은 LasB 유전자 및 다른 쿼럼 센싱 유전자 LasI, LasR, rhlI 및 rshR의 발현에 있어서의 DTPA의 효과를 확인한 결과를 나타낸다.
도 4는 ZnCl2, CaCl2 및 FeCl3를 이용하여, 펜테트산에 의해 억제된 엘라스테이즈의 생산을 회복시키는 실험을 한 결과를 나타낸다.
도 5는 펜테트산이 PAO-1 유도의 세포사멸에 대한 보호능을 갖는지를 확인한 결과를 나타낸다. Figure 1 shows the dose-response relationship for two drugs with EDTA and DTPA, showing Elastase activity (Figure 1 A) and bacterial population (Figure 1 B) with drug concentration.
Fig. 2 shows the results of ELASTASE expression confirmed by immunoblot.
FIG. 3 shows the results of confirming the effect of DTPA on the expression of LasB gene and other quorum sensing genes LasI, LasR, rhlI and rshR.
FIG. 4 shows the results of experiments in which the production of elastase inhibited by pentetic acid was restored using ZnCl 2 , CaCl 2 and FeCl 3 .
Fig. 5 shows the results of confirming that pentetic acid has protective ability against PAO-1 induced cell death.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.
Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .
실시예Example
실험 재료 및 방법Materials and Methods
1. 슈도모나스 엘라스테이즈 억제제에 대한 약물 스크리닝 1. Drug Screening for Pseudomonas elastase Inhibitors
슈도모나스 애루지노사(PAO-1)(ATCC 15692)의 엘라스테이즈의 생산을 억제하는 화합물에 대하여 FDA 승인 약물의 라이브러리를 스크리닝하였다. 사용된 화합물 라이브러리는 마이크로소스 디스커버리사(MicroSource Discovery, Inc.)로부터 구입한"The spectrum collection"이었다(17). 10 mM DMSO 용액 내의 2320 화합물로 구성된 상기 라이브러리는 주로 FDA 승인된 인간 치료학적 약물, 약물-유사 화합물 및 천연물을 포함한다. 각각의 화합물을 DMSO 내에서 2.5 mM의 농도에 이를 때까지 희석하였다. 이것은 96 플레이트의 각각의 웰에 도입될 100× 화합물 용액으로서 사용하였다. 많은 양의 엘라스테이즈를 생산하고 분비하는 것으로 판별된 PAO1 균주를 실험에 사용하였다. 박테리아 균주는 1 ml의 LB 배지(broth)에서 25 μM의 각 화합물과 함께 배양하였다. 배양물은 5분간 원심분리하였고, 상청액은 이어지는 엘라스틴 콩고 레드(Elastin Congo red) 분석을 위해 회수하였다.
A library of FDA-approved drugs was screened for compounds that inhibit the production of Elastase in Pseudomonas aeruginosa (PAO-1) (ATCC 15692). The compound library used was "The spectrum collection" purchased from MicroSource Discovery, Inc. (17). The library consisting of 2320 compounds in 10 mM DMSO solution contains mainly FDA approved human therapeutic drugs, drug-like compounds and natural products. Each compound was diluted in DMSO to a concentration of 2.5 mM. This was used as a 100x compound solution to be introduced into each well of a 96-well plate. The PAO1 strain, which was determined to produce and secrete a large amount of ella - stase, was used in the experiment. Bacterial strains were incubated with 25 μM each compound in 1 ml of LB broth. The culture was centrifuged for 5 minutes and the supernatant was collected for subsequent Elastin Congo red analysis.
2. 엘라스테이즈 활성에 대한 엘라스틴 콩고 레드 분석 2. Elastin Congo Red assay for Elastase activity
이미 공지된 것과 같이(18) 엘라스틴 콩고 레드 분석을 수행하였다. 요컨대 박테리아 배양물 상청액은, 20 mg의 엘라스틴 콩고 레드(Sigma-Aldrich)를 포함하는 10 mM Na2HPO4(pH 7.0) 1ml를 이용하여, 37℃에서 4시간 동안 배양하였다. 반응은 5분간 원심분리하였고, 가수분해된 콩고 레드는 광학 밀도 495 nm(Versa Max Microplate Reader, Molecular Devices)에 의해 정량하였다.
Elastin Congolese analysis (18) was performed as previously known. Briefly, the bacterial culture supernatant was incubated at 37 ° C for 4 hours with 1 ml of 10 mM Na 2 HPO 4 (pH 7.0) containing 20 mg of elastin Congo red (Sigma-Aldrich). The reaction was centrifuged for 5 minutes and the hydrolyzed congo red was quantified by optical density 495 nm (Versa Max Microplate Reader, Molecular Devices).
3. 엘라스테이즈 웨스턴 블랏3. Ella Stays Western Blot
최종의 세포 밀도(3.0 이하의 OD600)와 유사하게 배양한 PAO1의 배양물의 분액(Aliquots) 1 mL는 13000 rpm에서 5분간 원심분리하였고, 상청액은 0.2 μm 아크로디스크(Acrodisc) 실린더 필터(Pall Life Science inc.)를 통과시키고 분석을 위해 저장하였다. 세포 펠릿은 100 μL의 B-PER 단백질 추출 시료(Thermo Fisher Scientific inc.)에서 재현탁하였고, 실온에서 10 분간 배양하였으며, 13000 rpm에서 10분간 원심분리하였고, 상청액을 획득하였다(세포 추출 분획). 배양물 상청액 20 μL, 세포 추출 분획 20 μg을 12% SDS-PAGE 겔 상에 로딩하였다. 이어서 웨스턴 블랏 분석은 다른 곳에서 설명된 바와 같이(8) 수행하였다. 항-엘라스테이즈 항체는 이스라엘 텔 아비브 대학(Tel Aviv University)의 Efrat Kessler로부터 제공받았다.
1 mL of aliquots of the culture of PAO1 cultured similar to the final cell density (OD 600 below 3.0) was centrifuged at 13000 rpm for 5 min and the supernatant was passed through a 0.2 μm Acrodisc cylinder filter Science inc.) And stored for analysis. The cell pellet was resuspended in 100 μL of B-PER protein extraction sample (Thermo Fisher Scientific inc.), Incubated at room temperature for 10 min, centrifuged at 13000 rpm for 10 min, and supernatant was obtained (cell extract fraction). 20 μL of the culture supernatant and 20 μg of the cell extract fraction were loaded onto a 12% SDS-PAGE gel. Western blot analysis was then performed as described elsewhere (8). Anti-Elastin antibodies were obtained from Efrat Kessler of Tel Aviv University, Israel.
4. qRT-PCR 분석, PQS 분석, CFU 측정4. qRT-PCR analysis, PQS analysis, CFU measurement
qRT-PCR 분석을 기존에 공지된 방법(8, 19)으로 수행하였다. rpoD 유전자의 전사 수준은 플레인(plain) LB, 또는 DTPA 또는 EDTA가 보충된 LB 내에서 배양한 세포에서와 유사하였고, 그러므로 정규화에 사용하였다. PQS 분석은 다른 곳에 기재된 방법(20)에 따라 수행하였다. 다양한 배양 조건에서 배양한 PAO1의 CFU(colony forming unit)는 순차적으로 희석한 박테리아 배양물의 콜로니 수를 측정하는 것에 의해 결정하였다.
qRT-PCR analysis was performed by a known method (8, 19). The transcription level of the rpoD gene was similar to that in cultured cells in plain LB, or LB supplemented with DTPA or EDTA, and was therefore used for normalization. The PQS analysis was performed according to the method described elsewhere (20). The colony forming unit (CFU) of PAO1 cultured under various culture conditions was determined by measuring the number of colonies of bacterial cultures that were sequentially diluted.
5. A59 세포 생존력 분석5. A59 cell viability analysis
A549 인간 기도 상피 세포의 유지 및 성장을 이미 공지된 바(8)와 같이 수행하였다. 처리를 위하여 A549 세포는 96 웰 플레이트(1×104 세포/웰) 내에 주사(inoculate)하였고, 플레이트는 일반적인 배양 조건(37 ℃ 및 5% CO2)에서 하루 동안 배양하였다. 처리에 앞서, A549 세포는 세럼-프리 배지에서 1시간 배양하였고, 동일한 세럼-프리 배지에서 재구성된(reconstituted) 박테리아 배양물 상청액은 A549 세포에 대하여 10배로 희석하였다. 필요한 경우, 지정된 농도의 DTPA를 포함하는 세럼-프리 배지를 사용하였다. 살아있는 박테리아 세포의 처리를 위하여, 100:1의 MOI의 세럼-프리 배지에서 재현탁된 PAO1 또는 그것의 △lasB 돌연변이로 A549 세포를 감염시켰다. 처리 6시간 후, A549 세포 생존력은 MTT 분석에 의해 측정하였고, 각각의 처리 후의 상대적인 생존은 무처리 A549 세포의 생존력(8)에 기초하여 계산하였다.
Maintenance and growth of A549 human airway epithelial cells was performed as previously described (8). For treatment, A549 cells were inoculated into 96-well plates (1 x 10 4 cells / well) and the plates were incubated for one day under normal culture conditions (37 ° C and 5% CO 2). Prior to treatment, A549 cells were incubated in serum-free medium for 1 hour, and reconstituted bacterial culture supernatants in the same serum-free medium were diluted 10-fold with respect to A549 cells. If necessary, serum-free medium containing DTPA at the indicated concentration was used. For the treatment of live bacterial cells, A549 cells were infected with PAOl or its ΔlasB mutant resuspended in a serum-free medium with an MOI of 100: 1. After 6 hours of treatment, A549 cell viability was measured by MTT assay and the relative survival after each treatment was calculated based on the viability (8) of untreated A549 cells.
6. 6. In vivoIn vivo 마우스 기도 감염 Mouse airway infection
42 마리의 B6 마우스(수컷, 7주령)는 3 그룹(그룹당 14 마우스)으로 나누었다. 첫 번째 그룹은 Δ lasB 돌연변이(30 μl, 5×106 CFU/마우스)로 감염시켰고, 5일 동안 무처리 하였다. 다른 두 그룹의 마우스는 동일 양의 PAO1(30 μl, 5×106 CFU/마우스)로 감염시켰고, 각각의 그룹은 살린(saline)(운반체 대조구) 또는 Ca-DTPA(100 mg/kg/일(day))로 각각 처리하였다. 박테리아 감염 및 약물 운반은 비강 경로를 통해 이루어졌다. 감염 및 약물 처리 전에, 마우스는 30 mg/kg의 졸레틸(Virbac) 및 10 mg/kg의 럼폰(Bayer)으로 복강 마취를 하였다.
Forty-two B6 mice (male, 7 weeks old) were divided into 3 groups (14 mice per group). The first group is Δ lasB Mutations (30 μl, 5 × 10 6 CFU / mouse) were infected and left untreated for 5 days. The other two groups of mice were infected with the same amount of PAO1 (30 μl, 5 × 10 6 CFU / mouse) and each group received either saline (vehicle control) or Ca-DTPA (100 mg / kg / day). Bacterial infections and drug delivery were through the nasal passages. Prior to infection and drug treatment, the mice were anesthetized with abdominal anesthesia with 30 mg / kg of Zoletil (Virbac) and 10 mg / kg of Lumon (Bayer).
7. 통계 분석7. Statistical Analysis
데이터를 분석하기 위해 짝 비교 t-검정(paired t-test)(statview; SAS)을 수행하였고, <0.05의 p 값은 통계적으로 유의한 것으로 간주하였다. 도 7에 나타낸 마우스 생존 분석의 통계적 유의성을 평가하기 위해 로그 랭크 분석을 수행하였다. 모든 실험은 재현성을 위해 반복하였다.
A paired t-test (SAS) was performed to analyze the data and a p value of <0.05 was considered statistically significant. A log rank analysis was performed to evaluate the statistical significance of the mouse survival analysis shown in FIG. All experiments were repeated for reproducibility.
실험 결과Experiment result
1. 슈도모나스 애루지노사 내의 엘라스테이즈 분비를 억제하는 화합물의 선별1. Selection of compounds that inhibit the release of Elastase in Pseudomonas aeruginosa
엘라스테이즈는 슈도모나스 애루지노사 내에서 주요한 독성 결정요인으로 간주되었다(8). 슈도모나스 애루지노사의 활성을 감소 조절할 수 있는 화합물을 식별하기 위해 2320개의 FDA 승인 약물들로 구성된 화합물 라이브러리를 스크리닝하였다. 콩고 레드 분석을 포함하는 초기 스크리닝에서 16 화합물이 PAO1 엘라스테이즈 활성을 억제하는 것을 발견하였다. 엘라스테이즈 활성은 무처리의 PAO1 세포와 비교하여 75% 이상 감소하였다. 그러나 그들 중 15 화합물은 항균 또는 항곰팡이성 활성을 나타내는 것으로 판별되었고, 엘라스테이즈활성의 억제는 박테리아 세포 사멸의 결과였다. 표 1에 나타낸바와 같이, 상기 15 화합물 각각을 처리했을 때, CFU 수는 현저히 감소하였다. Ella Stages was considered a major toxicity determinant in Pseudomonas aeruginosa (8). To identify compounds capable of reducing the activity of Pseudomonas aeruginosa, a library of compounds consisting of 2320 FDA-approved drugs was screened. In early screening involving Congo red analysis 16 compounds were found to inhibit PAO1 elastase activity. Elastase activity was reduced by 75% or more as compared to untreated PAO1 cells. However, 15 compounds among them were identified as exhibiting antibacterial or antifungal activity, and inhibition of the activity of elastase was the result of bacterial cell death. As shown in Table 1, when each of the above 15 compounds was treated, the number of CFU was remarkably decreased.
MK-366synthesis;
MK-366
Abbott-56619synthesis;
Abbott-56619
DNA 자이레이즈 억제제Antimicrobial activity,
DNA xylyase inhibitor
NSC-26980Streptomyces butyrylatus;
NSC-26980
RNA 합성 억제Antimicrobial activity,
RNA synthesis inhibition
SB-265805S,
LB-20304asynthesis;
SB-265805S,
LB-20304a
DTPAsynthesis;
DTPA
진단목적Chelating agents,
Diagnostic purpose
구충제Antimicrobial activity,
helminthic
(국소)Anti-infective
(Local)
중요하게도, 엘라스테이즈 분비의 상당한 수준의 억제를 유발하는 디에틸렌트리아민펜테트산(DTPA 또는 펜테트산)(참조: 도 1의 A)은 박테리아 생존에 대해 세포 독성 효과를 보이지 않는다. 25 μM DTPA의 존재하에서 생장한 PAO1의 엘라스테이즈 활성은 무처리 PAO1 세포에 비하여 오직 17.3%이고, DTPA 처리 후 생존 가능한 세포의 수는 무처리 PAO1보다 약간 더 높다(참조: 표 1). 가돌리늄(Gd)과의 접합 내의 DTPA는 MRI(magnetic resonance imaging)에서의 음영 촉진제로서 널리 사용되어 왔다(21, 22). 이것은 DTPA가 인체내에서 어떠한 세포독성 효과도 나타내지 않는다는 것은 분명히 말해준다. 게다가 DTPA는 다양한 다가 양이온에 대한 킬레이팅 능력을 갖는 것으로 보여졌고, 다량 사용시 박테리아 성장을 분명 억제할 수 있다(23). 그러나 본 발명의 스크리닝에서, 25 μM DPTA는 박테리아 성장에 영향을 주지 않고, 엘라스테이즈 분비를 특이적이고 극적으로 억제하였다.
Importantly, diethylenetriamine pentetate (DTPA or pentetate) (see Fig. 1 A), which causes a significant level of inhibition of the elastase secretion, does not show cytotoxic effects on bacterial viability. Elastase activity of PAO1 grown in the presence of 25 [mu] M DTPA is only 17.3% compared to untreated PAO1 cells and the number of viable cells after DTPA treatment is slightly higher than untreated PAO1 (see Table 1). DTPA in gadolinium (Gd) conjugation has been widely used as a shading promoter in magnetic resonance imaging (MRI) (21, 22). It is clear that DTPA does not show any cytotoxic effects in the human body. In addition, DTPA has been shown to have chelating abilities for a variety of multivalent cations and can inhibit bacterial growth in large doses (23). However, in the screening of the present invention, 25 [mu] M DPTA did not affect bacterial growth, and specifically and dramatically suppressed Elastase secretion.
2. DTPA는 EDTA보다 더욱 효과적으로 엘라스테이즈 활성을 억제한다.2. DTPA inhibits elastase activity more effectively than EDTA.
슈도모나스 애루지노사 엘라스테이즈는 메탈로프로티네이즈이고 아연은 그 촉매 메카니즘과 관련되어 있다(24, 25). 엘라스테이즈 분비의 억제자로서의 DTPA의 역할이 그것의 금속 킬레이팅 활성에 의해 매개되는 것으로 예상되므로, 그 엘라스테이즈 억제 활성을, 또 다른 우세한 금속 이온 킬레이터인 EDTA의 억제 활성과 비교하였다. 증가되는 양의 DTPA 또는 EDTA 처리로 배양된 PAO1의 엘라스테이즈 활성이 점차 감소하였지만, DTPA가 엘라스테이즈 활성을 억제하는데 더욱 효과적이었다. 20 μM 농도의 DTPA 처리에서, 엘라스테이즈 활성은 오직 무처리 세포에서 관찰된 것의 25% 이하였다. 대조적으로, 엘라스테이즈 활성은 20 μM EDTA 처리에 의해 감소하지 않았다(참조: 도 1의 B). 50 μM의 DTPA를 처리한 경우, 엘라스테이즈 활성은 콩고 레드 분석에서 완전히 사라졌다. 그러나 PAO1을 동일한 농도의 EDTA(50 μM)로 처리하는 경우, 원래의 엘라스테이즈 활성의 절반 가량은 여전히 검출되었다(참조: 도 1의 B). 이러한 결과들은 DTPA에 의한 엘라스테이즈 억제가 복용량 의존적인 방법으로 일어나고, 엘라스테이즈 활성을 억제하기 위한 DTPA의 능력이 EDTA보다 상당히 더 효과적이라는 것을 보여준다. Pseudomonas aeruginosa Ella stasis is a metalloproteinase and zinc is associated with its catalytic mechanism (24, 25). Since the role of DTPA as an inhibitor of elastase secretion is expected to be mediated by its metal chelating activity, its elastase inhibitory activity is compared to the inhibitory activity of EDTA, another predominant metal ion chelator. The elasstase activity of PAO1 cultured with increasing amounts of DTPA or EDTA treatment was gradually decreased, but DTPA was more effective in inhibiting the activity of elastase. In DTPA treatment at a concentration of 20 [mu] M, Elastase activity was only 25% or less of that observed in untreated cells. In contrast, Elastase activity did not decrease by treatment with 20 [mu] M EDTA (see Fig. 1B). When 50 μM of DTPA was treated, Elastase activity completely disappeared in the Congo red assay. However, when PAO1 was treated with the same concentration of EDTA (50 [mu] M), half of the original Elastase activity was still detected (see Fig. 1B). These results demonstrate that inhibition of ElaSTase by DTPA occurs in a dose-dependent manner and that the ability of DTPA to inhibit ElaSTase activity is significantly more effective than EDTA.
엘라스테이즈 생산은, 슈도모나스 애루지노사의 세포 밀도 의존 유전자 조절 메카니즘인 쿼럼 센싱(6)에 의해 제어된다. 엘라스테이즈 활성의 감소가 DTPA에 의한 성장 억제의 결과인 가능성을 제거하기 위해, DTPA 존재하에서의 PAO1의 박테리아 성장을 조사하였다. DTPA 또는 EDTA를 가지고 하루 동안 배양한 후 생존 세포의 수는 차이가 없었다(참조: 도 1의 C). 100 μM 이하 수준의 DTPA는 박테리아 성장 상에 어떠한 부정적인 효과도 가하지 않는다. 마찬가지로 최대 100 μM의 EDTA로 배양한 PAO1도 유사한 수의 생존 세포를 얻었다. 100 μM DTPA 또는 EDTA를 이용한 성장 곡선 실험은 최종 OD600 값이 대조구 성장에서의 값보다 다소 낮다는 것을 보여주었다. 그러나 박테리아 성장은 화합물의 존재에 의하여 지연되지 않았고, 성장 단계 발달은 서로 간에 유사하였다(참조: 도 1의 D).
Ella stasis production is controlled by quorum sensing (6), a cell density-dependent gene regulation mechanism of Pseudomonas aeruginosa. Bacterial growth of PAO1 in the presence of DTPA was investigated to eliminate the possibility that the decrease in Elastase activity was the result of growth inhibition by DTPA. There was no difference in the number of viable cells after one day incubation with DTPA or EDTA (cf. Fig. 1). DTPA levels below 100 μM do not have any negative effects on bacterial growth. Similarly, a similar number of viable cells were obtained in PAO1 cultured with EDTA at a maximum of 100 μM. Growth curve experiments with 100 μM DTPA or EDTA showed that final OD 600 values were somewhat lower than those in control growth. However, bacterial growth was not retarded by the presence of compounds, and growth stage development was similar to each other (see Fig. 1 D).
3. 엘라스테이즈 활성은 몇몇의 금속 이온들의 보충에 의해 회복된다. 3. Elastase activity is restored by supplementation of some metal ions.
Zn2+ 및 Ca2+와 같은 금속 이온들은 슈도모나스 애루지노사에 의한 엘라스테이즈의 효과적인 생산 및 처리에 필수적이라는 것이 알려졌다(26). DTPA의 억제의 방식을 더 잘 이해하기 위해, 엘라스테이즈 활성에 대한 DTPA의 억제 효과가 외부적으로 첨가된 금속 이온에 의해 완화될 수 있는지 여부를 조사하였다. ZnCl2(100 μM), CaCl2(2 mM), FeCl3(100 μM)의 효과를 시험하였다. DTPA 처리 없이, 각 금속 이온들의 존재 또는 부존재 하에서 성장한 PAO1 내에서 유사한 수준의 엘라스테이즈 활성이 관찰되었다(참조: 도 2의 좌측 도). 50 μM DTPA 및 100 μM ZnCl2를 처리하여 배양시, PAO1은 비슷한 수준의 엘라스테이즈를 생산하였고, Zn2+ 이온의 첨가가 엘라스테이즈 활성에 대한 DTPA의 억제 효과에 대해 거의 완전하게 길항작용을 한다는 것을 보여준다. 이와 유사하게 Ca2+ 이온 처리 배양 또한 DTPA 처리된 PAO1에서의 엘라스테이즈 활성을 회복시켰다(참조: 도 2의 우측 도). Fe3+ 이온의 첨가는 이러한 효과가 없었다.
It has been found that metal ions such as Zn 2+ and Ca 2+ are essential for the effective production and processing of elastase by Pseudomonas aeruginosa (26). To better understand the manner of inhibition of DTPA, we investigated whether the inhibitory effect of DTPA on Elastase activity could be mitigated by exogenously added metal ions. The effects of ZnCl 2 (100 μM), CaCl 2 (2 mM) and FeCl 3 (100 μM) were tested. A similar level of Elastase activity was observed in PAO1 grown under the presence or absence of each metal ion, without DTPA treatment (see left side of FIG. 2). When incubated with 50 μM DTPA and 100 μM ZnCl 2 , PAO1 produced a similar level of elastase, and the addition of Zn 2+ ion was almost completely antagonistic to the inhibitory effect of DTPA on elastase activity . Similarly, Ca 2+ ion-treated cultures also restored Elastase activity in DTPA-treated PAO1 (see right side of FIG. 2). Addition of Fe 3+ ions did not have this effect.
4. DTPA 처리 PAO1에서의 전사 수준에서 엘라스테이즈 활성의 감소가 조절된다. 4. Decrease in Elastase activity is regulated at the transcription level in DTPA-treated PAO1.
상기 결과들은 DTPA 처리에 의해 감소된 엘라스테이즈 활성이 금속 이온들의 제거로 인한 것이며, 엘라스테이즈 합성의 수준은 PAO1에서의 DTPA 처리에 의해 영향을 받지 않을 것이라는 것을 보여준다. 상기 이론을 뒷받침하기 위해, 엘라스테이즈 단백질 수준을 정량하였다. 도 3의 A는 DTPA 또는 EDTA의 다양한 농도를 처리한 PAO1의 배양물 상청액(CSs) 및 세포질 추출물의 웨스턴 블랏 분석 결과를 나타낸다. 배양 배지로의 엘라스테이즈의 분비는 10 μM DTPA 또는 EDTA를 처리하였을 때, 잘 이루어졌다(참조: 도 3의 A). 50 μM DTPA를 처리하였을때, 배양물 상청액에서 엘라스테이즈가 검출되지 않았다. 대조적으로 동일 농도의 EDTA를 처리한 PAO1의 배양물 상청액에서 엘라스테이즈는 감소된 수준이기는 하지만 검출되었다. 이러한 결과는, DTPA가 EDTA 보다 더욱 효과적으로 엘라스테이즈 활성을 억제할 수 있다는 본 발명의 도 1의 A에 나타낸 결과를 더욱 뒷받침한다. 100 μM DTPA 또는 EDTA로 처리한 PAO1은 엘라스테이즈를 생산하는데 실패하였다. 중요하게도 박테리아 세포 내부의 엘라스테이즈의 축적은 엘라스테이즈를 분비하지 않는 PAO1 세포에서 관찰되지 않았다. 이러한 결과는 DTPA 유도의 엘라스테이즈 활성의 감소가, 엘라스테이즈의 결함있는 분비가 아닌 엘라스테이즈 합성의 감소에서 기인한다는 것을 보여준다. 다음으로 엘라스테이즈를 코딩하는 lasB 유전자의 전사 수준을 측정하였다. DTPA의 증가되는 농도의 처리에 있어서, 점진적인 lasB 유전자 전사의 감소가 명백하게 관찰되었다. 그러나 EDTA 처리는 lasB 유전자 전사에 있어서 상당한 수준의 감소를 보이지는 않는다(참조: 도 3의 B). 이러한 결과들은 DTPA 유도의 엘라스테이즈 활성 억제는 전사 수준에서 조절된다는 것을 보여준다.
The results show that the reduced elastase activity by DTPA treatment is due to the removal of metal ions and that the level of Elastase synthesis will not be affected by DTPA treatment in PAO1. To support the theory, Elastase protein levels were quantified. Figure 3 A shows the results of Western blot analysis of culture supernatants (CSs) and cytoplasmic extract of PAO1 treated with various concentrations of DTPA or EDTA. Elastase secretion into the culture medium was well done when 10 [mu] M DTPA or EDTA was treated (Fig. 3A). When 50 [mu] M DTPA was treated, Elastase was not detected in the culture supernatant. In contrast, in the supernatant of the culture of PAO1 treated with the same concentration of EDTA, ELASTASE was detected at a reduced level. These results further support the results shown in Fig. 1A of the present invention, in which DTPA can inhibit elasstase activity more effectively than EDTA. PAO1 treated with 100 μM DTPA or EDTA failed to produce elastase. Importantly, the accumulation of elastase in bacterial cells was not observed in PAO1 cells that did not secrete elastase. These results demonstrate that the decrease in Elastase activity of DTPA induction is due to a decrease in Elastase synthesis rather than defective secretion of Elastase. Next, the transcription level of the lasB gene coding for Elastase was measured. In the treatment of increasing concentrations of DTPA, a gradual decrease in lasB gene transcription was clearly observed. However, EDTA treatment does not show a significant reduction in lasB gene transcription (see Fig. 3B). These results show that the inhibition of Elastase activity of DTPA induction is regulated at the transcription level.
5. PQS 쿼럼 센싱 시스템은 DTPA 처리된 PAO에서 억제된다. 5. The PQS quorum sensing system is suppressed in DTPA-treated PAOs.
슈도모나스 애루지노사에서, LasI/R, RhlI/R 및 PQS 쿼럼 센싱 시스템 모두는 lasB 유전자 발현을 활성화하기 위한 복합 신호 캐스케이드에 참여한다(8). DTPA 유도의 엘라스테이즈 합성 억제에 대한 각 쿼럼 센싱 시스템의 상대적인 기여의 아이디어를 얻기 위해, DTPA 처리에 대한 반응에서의 lasI, lasR, rhlI, rhlR 및 pqsA 유전자의 전사 수준을 측정하였다. 이러한 유전자들의 전사 수준은, 처리 과정 내내 유사한 정도의 발현을 보이는 rpoD 유전자의 전사 수준을 이용하여 정규화하였다. 도 4의 A에 나타낸 것처럼, lasI, lasR, rhlI, rhlR 유전자의 전사는 DTPA 처리에 대한 반응에서 중요하게 변화되지 않았다. 대조적으로 pqsA 유전자의 nRNA 수준은 DTPA 처리된 PAO1에서 눈에 띄게 감소하였다. 이 결과는 DTPA 처리가 PQS 쿼럼 센싱 시스템의 특이적 억제의 결과를 낳는다는 것을 보여준다. 이러한 개념과 부합하여, PQS 신호 화합물의 생산은, 본 발명의 TLC 분석에서의 DTPA의 증가하는 양으로의 처리에 반응하여, 점진적으로 감소하였다(참조: 도 4의 B).
In Pseudomonas aeruginosa, both the LasI / R , RhlI / R and PQS quorum sensing systems participate in a complex signal cascade to activate lasB gene expression (8). To obtain the idea of the relative contributions of each quorum sensing system to the inhibition of Elastase synthesis of DTPA induction, the transcription levels of las I , lasR, rhlI, rhlR and pqsA genes in response to DTPA treatment were measured. Transcription levels of these genes were normalized using the transcription level of the rpoD gene, which exhibits a similar degree of expression throughout the process. As shown in Fig . 4A, the transcription of lasI, lasR, rhlI, rhlR gene was not significantly changed in response to DTPA treatment. In contrast, the level of nRNA in the pqsA gene was significantly decreased in DTPA treated PAO1. This result shows that DTPA treatment results in a specific inhibition of the PQS quorum sensing system. Consistent with this concept, the production of the PQS signal compound was progressively reduced in response to treatment with increasing amounts of DTPA in the TLC assay of the present invention (Fig. 4B).
6. 배양된 상피성 세포에 대한 슈도모나스 애루지노사의 세포독성은 DTPA의 존재하에서 약화된다.6. Cytotoxicity of Pseudomonas aeruginosa to cultured epithelial cells is attenuated in the presence of DTPA.
A549 인간 기도 상피성 세포에 대한 슈도모나스 애루지노사의 엘라스테이즈 매개의 세포독성은 잘 알려져 있다(8). PAO1 배양물 상청액으로 처리하는 때에, A549 세포는 그들의 생존력을 상실한다. 도 5의 A에 나타낸것처럼, PAO1 배양물 상청액으로 6시간 동안 처리한 후 A549 세포내에서 정상적인 세포 형태는 완전히 파괴되었다. 대조적으로 ΔlasB 돌연변이의 배양물 상청액으로 배양한 A549 세포는 생존한채로 남아있었고, 그들의 상대적인 생존력은 무처리 A549 세포의 생존력에 비해 80% 이하였다(참조: 도 5의 B). 이러한 결과들은 엘라스테이즈가 슈도모나스 애루지노사 세포 독성에 대한 우세한 독성 결정인자라는 것을 더욱 뒷받침한다. 중요하게도 50 μM 또는 100 μM의 DTPA로 배양한 후 회수된 PAO1의 배양물 상청액은 A549 세포에 대하여 세포독성이 없다. A549 세포는 그들의 일반적인 세포 형태로 남아있고(도 5의 A), 상대적인 생존력은 무처리 A549 세포의 생존력과 비슷한 수준이다(도 5의 B). 다음으로 숙주 세포와 박테리아의 상호작용 동안에 DTPA 매개의 독성 약화가 발생하는지 여부를 시험하기 위하여, DTPA로 처리 또는 처리하지 않은 살아있는 박테리아로 A549 세포를 처리하였다. DTPA가 없는 배양 배지 내의 PAO1 세포와 배양시, A549 세포 생존력은 감소하였다(참조: 도 5의 D). DTPA가 50 μM 또는 100 μM 농도로 배지에 첨가되었을 때, PAO1 유도의 세포 독성 효과는 관찰되지 않았다. 중간수준의 세포독성은 10 μM의 DTPA가 사용되었을 때 관찰되었다(참조: 도 5의 D). ΔlasB 돌연변이 세포는 A549 세포 상에 어떠한 세포 독성 효과도 유도하지 않았다(참조: 도 5의 C 및 D). 종합적으로, 이러한 결과들은 DTPA 처리가 슈도모나스 애루지노사 병원성 잠재성을 숙주 기도 상피성 조직과 상호작용하는 동안에 감소 조절할 수 있다는 것을 보여준다.
The cytotoxicity of Pseudomonas aeruginosa elastase mediated to A549 human airway epithelial cells is well known (8). When treated with PAO1 culture supernatants, A549 cells lose their viability. As shown in Fig. 5A, normal cell morphology in A549 cells was completely destroyed after 6 hours of treatment with PAO1 culture supernatant. In contrast, A549 cells cultured with the supernatant of the ΔlasB mutant remained viable and their relative viability was less than 80% of the viability of untreated A549 cells (see FIG. 5B). These results further support that Elastase is the predominant toxic determinant for Pseudomonas aeruginosa cytotoxicity. Importantly, the culture supernatant of the recovered PAO1 after 50 μM or 100 μM of DTPA was not cytotoxic to A549 cells. A549 cells remain in their normal cell morphology (Fig. 5A) and the relative viability is comparable to the viability of untreated A549 cells (Fig. 5B). Next, A549 cells were treated with live bacteria that had not been treated or treated with DTPA in order to test whether DTPA mediated toxicity reduction occurred during host cell-bacterial interaction. When cultured with PAO1 cells in DTPA-free culture medium, A549 cell viability decreased (see Fig. 5D). When DTPA was added to the medium at a concentration of 50 μM or 100 μM, the cytotoxic effect of PAO1 induction was not observed. Intermediate cytotoxicity was observed when 10 μM of DTPA was used (see FIG. 5 D). The ΔlasB mutant cells did not induce any cytotoxic effect on A549 cells (see C and D in FIG. 5). Overall, these results show that DTPA treatment can modulate the Pseudomonas aeruginosa pathogenicity potential during interaction with host airway epithelial tissue.
7. 7. in vivoin vivo 독성에 대한 DTPA 처리의 효과 Effect of DTPA treatment on toxicity
마지막으로 DTPA 처리가 슈도모나스 애루지노사 감염 마우스의 병원성 증상을 약화시킬 수 있는지 여부를 시험하였다. ΔlasB 돌연변이로 감염된 마우스가 5일 동안 생존한채로 남아있었다는 것은 중요한 사실이다(참조: 도 6). 다시 말해서, 이 결과는 엘라스테이즈는 슈도모나스 애루지노사의 우세한 독성 결정인자라는 것을 강하게 뒷받침한다. PAO1 감염 마우스를 대조구 용액으로 처리하였을 때, 14 마리의 모든 마우스는 5일 내에 모두 사망하였다. 대조적으로 DTPA 처리 마우스는 PAO1 감염시 크게 향상된 생존력을 보였고, 14 마리 중 6마리의 마우스가 생존하였다(참조: 도 6).
Finally, we examined whether DTPA treatment could attenuate the pathogenic symptoms of Pseudomonas aeruginosa infected mice. It is important to note that mice infected with the ΔlasB mutation remained viable for 5 days (see FIG. 6). In other words, this result strongly supports that Elastane is the predominant toxicity determinant of Pseudomonas aeruginosa. When PAO1 infected mice were treated with the control solution, all 14 mice died within 5 days. In contrast, DTPA-treated mice showed significantly improved viability when infected with PAO1, and 6 out of 14 mice survived (see Figure 6).
이상으로 본 발명의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현 예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.
While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.
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Claims (7)
A composition for inhibiting elastase of Pseudomonas aeruginosa comprising pentetic acid as an active ingredient.
The composition according to claim 1, wherein said Elastase inhibition is accomplished by inhibiting the production of Elastinase of Pseudomonas aeruginosa.
(a) a pharmaceutically effective amount of pentetic acid; And (b) a pharmaceutically acceptable carrier. A pharmaceutical composition for preventing or treating Pseudomonas aeruginosa infection.
(a) a pharmaceutically effective amount of pentetic acid; And (b) Pseudomonas Ke containing a pharmaceutically acceptable carrier Rouge labor (Pseudomonas aeruginosa) jeonggyunyong (bateriostatic) a pharmaceutical composition for.
(a) 시험물질을 슈도모나스 애루지노사에 처리하는 단계; 및
(b) 상기 시험물질이 슈도모나스 애루지노사의 LasB 유전자의 발현을 억제하는지 여부를 분석하는 단계로서; 상기 시험물질이 슈도모나스 애루지노사의 LasB 유전자의 발현을 억제하는 경우, 상기 시험물질은 엘라스테이즈 억제에 의한 슈도모나스 애루지노사 감염의 예방 또는 치료용 약물로 판정한다.
A method of screening for a drug for the prevention or treatment of Pseudomonas aeruginosa infection by elastase inhibition comprising the steps of:
(a) treating the test substance to Pseudomonas aeruginosa; And
(b) analyzing whether the test substance inhibits the expression of the LasB gene of Pseudomonas aeruginosa; When the test substance suppresses the expression of the LasB gene of Pseudomonas aeruginosa, the test substance is judged to be a drug for the prevention or treatment of Pseudomonas aeruginosa infection caused by Elastase inhibition.
6. The method according to claim 5, wherein the step (b) further comprises analyzing whether or not the expression of the LasB gene is suppressed , and additionally further examining whether or not the expression of the LasI, LasR, rhlI and rshR genes is suppressed. When the test substance suppresses the expression of the LasB gene of Pseudomonas aeruginosa and does not inhibit the expression of the LasI, LasR, rhlI, and rshR genes, the test substance inhibits the Pseudomonas aeruginosa infection caused by the Elastase inhibition Or a therapeutic drug.
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| CN117257955A (en) * | 2023-11-17 | 2023-12-22 | 深圳大学 | Application of ferredoxin reductase Fpr as target point of pseudomonas aeruginosa treatment drug |
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| CN113430158B (en) * | 2021-06-21 | 2022-05-10 | 广东省科学院微生物研究所(广东省微生物分析检测中心) | Preparation of geraniol in promotion of pseudomonas aeruginosa 3OC12Application of HSL signal molecule synthesis preparation |
| CN117257955A (en) * | 2023-11-17 | 2023-12-22 | 深圳大学 | Application of ferredoxin reductase Fpr as target point of pseudomonas aeruginosa treatment drug |
| CN117257955B (en) * | 2023-11-17 | 2024-03-15 | 深圳大学 | Application of ferredoxin reductase Fpr as target point of pseudomonas aeruginosa treatment drug |
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