KR100811744B1 - Pharmaceutical composition containing arazyme for the prevention and treatment of cancer - Google Patents
Pharmaceutical composition containing arazyme for the prevention and treatment of cancer Download PDFInfo
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- KR100811744B1 KR100811744B1 KR1020060136707A KR20060136707A KR100811744B1 KR 100811744 B1 KR100811744 B1 KR 100811744B1 KR 1020060136707 A KR1020060136707 A KR 1020060136707A KR 20060136707 A KR20060136707 A KR 20060136707A KR 100811744 B1 KR100811744 B1 KR 100811744B1
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- Prior art keywords
- arazyme
- cancer
- pharmaceutical composition
- aranicola
- preventing
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Abstract
Description
도 1은 인간 폐암 세포주(A549)가 이식된 누드(Nude) 마우스에 아라자임을 투여한 후, 체중의 변화를 나타낸 그래프이다:1 is a graph showing changes in body weight after administration of arazyme to nude mice implanted with human lung cancer cell line (A549):
대조군: 아라자임 0 ㎎/㎏, 경구투여; T1: 아라자임 150 ㎎/㎏, 경구투여; T2: 아라자임 300 ㎎/㎏, 경구투여; 및 T3: 아라자임 600 ㎎/㎏, 경구투여. Control:
도 2는 인간 폐암 세포주(A549)가 이식된 누드 마우스에 아라자임을 투여한 후, 종양크기를 나타낸 그래프이다:2 is a graph showing the tumor size after administration of arazyme to a nude mouse transplanted with a human lung cancer cell line (A549):
대조군: 아라자임 0 ㎎/㎏, 경구투여; T1: 아라자임 150 ㎎/㎏, 경구투여; T2: 아라자임 300 ㎎/㎏, 경구투여; T3: 아라자임 600 ㎎/㎏, 경구투여; *: p<0.05; 및 **: p<0.01.Control:
도 3은 인간 폐암 세포주(A549)가 이식된 누드 마우스에 아라자임을 투여한 후의 조직학적 사진이다(13배율 및 33배율): Figure 3 is a histological picture after administration of arazyme to nude mice implanted with human lung cancer cell line (A549) (13 and 33):
대조군: 아라자임 0 ㎎/㎏, 경구투여; T1: 아라자임 150 ㎎/㎏, 경구투 여; T2: 아라자임 300 ㎎/㎏, 경구투여; 및 T3: 아라자임 600 ㎎/㎏, 경구투여. Control:
도 4는 인간 폐암 세포주(A549)가 이식된 누드 마우스에 아라자임을 투여한 후, MMP-9의 면역조직학적 사진이다(33배율 및 66배율):Figure 4 is an immunohistograph of MMP-9 after administration of arazyme to nude mice implanted with human lung cancer cell line (A549) (33 and 66):
대조군: 아라자임 0 ㎎/㎏, 경구투여; T1: 아라자임 150 ㎎/㎏, 경구투여; T2: 아라자임 300 ㎎/㎏, 경구투여; 및 T3: 아라자임 600 ㎎/㎏, 경구투여. Control:
도 5는 인간 폐암 세포주(A549)가 이식된 누드 마우스에 아라자임을 투여한 후, MMP-9의 면역 조직화학적 발현 정도를 나타낸 그래프이다:5 is a graph showing the degree of immunohistochemical expression of MMP-9 after administration of arazyme to nude mice transplanted with human lung cancer cell line (A549):
대조군: 아라자임 0 ㎎/㎏, 경구투여; T1: 아라자임 150 ㎎/㎏, 경구투여; T2: 아라자임 300 ㎎/㎏, 경구투여; 및 T3: 아라자임 600 ㎎/㎏, 경구투여. Control:
도 6은 인간 폐암 세포주(A549)가 이식된 누드 마우스에 아라자임을 투여한 후, NF-κB의 면역조직학적 사진이다(33배율 및 66배율):FIG. 6 is an immunohistograph of NF-κB after administration of arazyme to nude mice implanted with human lung cancer cell line (A549) (33-fold and 66-fold):
대조군: 아라자임 0 ㎎/㎏, 경구투여; T1: 아라자임 150 ㎎/㎏, 경구투여; T2: 아라자임 300 ㎎/㎏, 경구투여; 및 T3: 아라자임 600 ㎎/㎏, 경구투여. Control:
도 7은 인간 폐암 세포주(A549)가 이식된 누드 마우스에 아라자임을 투여한 후, NF-κB의 면역 조직화학적 발현 정도를 나타낸 그래프이다:7 is a graph showing the immunohistochemical expression level of NF-κB after administration of arazyme to nude mice transplanted with human lung cancer cell line (A549):
대조군: 아라자임 0 ㎎/㎏, 경구투여; T1: 아라자임 150 ㎎/㎏, 경구투여; T2: 아라자임 300 ㎎/㎏, 경구투여; 및 T3: 아라자임 600 ㎎/㎏, 경구투여. Control:
도 8은 인간 폐암 세포주(A549)가 이식된 누드 마우스에 아라자임을 투여한 후, PCNA의 면역조직학적 사진이다(66배율 및 132배율):8 is an immunohistograph of PCNA after administration of arazyme to nude mice implanted with human lung cancer cell line (A549) (66 and 132 times):
대조군: 아라자임 0 ㎎/㎏, 경구투여; T1: 아라자임 150 ㎎/㎏, 경구투 여; T2: 아라자임 300 ㎎/㎏, 경구투여; 및 T3: 아라자임 600 ㎎/㎏, 경구투여. Control:
도 9는 인간 폐암 세포주(A549)가 이식된 누드 마우스에 아라자임을 투여한 후, PCNA의 면역 조직화학적 발현 정도를 나타낸 그래프이다:9 is a graph showing the degree of immunohistochemical expression of PCNA after administration of arazyme to nude mice implanted with human lung cancer cell line (A549):
대조군: 아라자임 0 ㎎/㎏, 경구투여; T1: 아라자임 150 ㎎/㎏, 경구투여; T2: 아라자임 300 ㎎/㎏, 경구투여; 및 T3: 아라자임 600 ㎎/㎏, 경구투여. Control:
본 발명은 아라자임을 유효성분으로 하는 암 예방 및 치료용 약학적 조성물에 관한 것으로, 더욱 상세하게는 아라니콜라 프로테오리티쿠스(Aranicola proteolyticus)가 생산하는 아라자임(arazyme)을 함유하는 폐암 예방 및 치료용 약학적 조성물에 관한 것이다. The present invention relates to a pharmaceutical composition for preventing and treating cancer comprising arazyme as an active ingredient, and more particularly to preventing lung cancer containing arazyme (arazyme) produced by Aranicola proteolyticus . It relates to a therapeutic pharmaceutical composition.
폐암은 예후가 매우 불량하며 최근에 전 세계적으로 발생빈도가 급격히 증가하는 추세에 있으며, 국내에서도 이미 암 사망률 제 2위를 차지하고 있다(Bae JM, et al ., Cancer Res Treat, 34:319-325, 2002). 그중에서 폐암의 75% 이상을 차지하는 비소세포 폐암은 진단될 때 65% 이상에서 이미 절제 수술이 불가능한 상태로 발견된다(Scientific Committee of Korean Academy of Tuberculosis and Respiratory disease, Tubere Respir Dis, 46:455-465. 1999). 최근 외과적 수술 및 방사선요법의 발전, 새로운 항암요법의 개발과 여러 치료법의 복합 적용으로 인하여 더불어 암 환자의 일부 생존율을 향상시키고 있다. 그러나 방사선요법의 경우 피부에 직접적인 영향을 주어 방사선 피부염을 일으킬 수 있으며, 이미 개발된 대부분의 항암제의 경우 강한 항암 치료효과는 가지지만 독성으로 인한 부작용이 크며 재발의 위험이 높은 것으로 알려져 있다. 그러므로 부작용이 없거나 적으며 암의 치료효과가 큰 물질을 찾고자 하는 연구가 활발하게 진행되고 있으며, 이러한 목적으로 천연물의 생리활성에 대하여 국내외적으로 많은 연구들이 진행되고 있다. Lung cancer has a very poor prognosis, and the incidence rate is increasing rapidly around the world, and it is already the second largest cancer death rate in Korea (Bae JM, et. al ., Cancer Res Treat , 34: 319-325, 2002). Among them, non-small cell lung cancer, which accounts for more than 75% of lung cancers, is already found to be inoperable in over 65% when diagnosed (Scientific Committee of Korean Academy of Tuberculosis and Respiratory disease, Tubere). Respir Dis , 46: 455-465. 1999). Recent advances in surgical and radiotherapy, the development of new anti-cancer therapies, and the combined application of several therapies have also improved the survival rate of some cancer patients. However, radiation therapy has a direct effect on the skin and can cause radiation dermatitis. Most of the previously developed anticancer drugs have a strong anti-cancer effect, but the side effects due to toxicity are high and the risk of recurrence is known. Therefore, researches are actively conducted to find a substance having little or no side effects and a large therapeutic effect of cancer, and for this purpose, many studies at home and abroad have been conducted on the biological activity of natural products.
암의 발병에는 유전적 소인도 깊이 관여하지만 환경적 요인 또한 큰 영향을 미치며 선진국일수록 암의 발병이 증가하는 경향을 보이고 있다. 이에 관해 가능한 원인으로는 농약, 살충제 등의 사용량 및 식품 내 잔유량 증가, 식품 보존제, 방부제, 착색제 등이 첨가된 가공식품의 소비 증가, 수질, 토양, 대기오염의 증가, 현대인의 스트레스 증가, 활동량의 감소 그리고 풍요로운 식생활을 통한 비만 등이 보고되어 있다(kim HJ, et al ., Liv Sci of Tj Univ. 3:99-130, 1997; Jacobs MM, Nutr Tod, 28(3):19-23, 1993). Genetic predisposition is also deeply involved in the development of cancer, but environmental factors also have a significant impact. In advanced countries, the incidence of cancer is increasing. Possible causes include: increased use of pesticides and pesticides, increased residues in foods, increased consumption of processed foods with added food preservatives, preservatives, and colorants, increased water quality, soil, air pollution, increased stress in modern people, and increased activity. Reduction and obesity through abundant diets have been reported (kim HJ, et. al ., Liv Sci of Tj Univ . 3: 99-130, 1997; Jacobs MM, Nutr Tod , 28 (3): 19-23, 1993) .
종양이 성장과 전이를 하기 위해서는 신생혈관 형성이 필수 과정이다(Folkman J, J Natl Cancer Inst, 82:4-6, 1990). 신생혈관 형성은 증식하는 종양에 영양분과 산소를 공급해 줄 뿐만 아니라 원발병소로부터 혈관벽을 통한 종양세포의 관통과 원격장기에 전이를 가능하게 한다(Kim KJ, et al., Nature, 362:841-844, 1993). 폐암은 조직학적으로 정상 기관지 상피세포가 증 식(hyperplasia), 화생(metaplasia), 이형성(dysplasia), 상피내 암종(carcinoma in situ)의 변화를 거쳐서 암으로 발생하면서(Mulshine JL, et al., Oncology, 5:25-32, 1991) 점진적으로 많은 대립유전자들의 소실이 증가하는 것으로 잘 알려져 있다(Wistuba II, et al., Oncogene, 18:643-650, 1991).Neovascularization is essential for tumor growth and metastasis (Folkman J, J Natl). Cancer Inst , 82: 4-6, 1990). Angiogenesis not only supplies nutrients and oxygen to the proliferating tumors, but also allows the penetration of tumor cells through the vascular wall from the primary path and metastases to distant organs (Kim KJ, et al ., Nature , 362: 841-844). , 1993). Lung cancer occurs histologically as cancer develops through changes in hyperplasia, metaplasia, dysplasia, and carcinoma in situ (Mulshine JL, et al. al ., Oncology , 5: 25-32, 1991) It is well known that progressively more alleles are lost (Wistuba II, et. al ., Oncogene , 18: 643-650, 1991).
한편, 본 발명자들은 새로운 단백질 분해효소를 개발하고자 노력한 결과, 한국산 무당거미(Nephila clavata)로부터 신규 미생물인 아라니콜라 프로테오리티쿠스(Aranicola proteolyticus) HY-3 균주(수탁번호 : KCTC 0268BP; WO 01/57222호)를 분리하였으며, 본 균주로부터 신규한 단백질 분해효소인 아라자임(arayzme)을 분리하였다. 아라자임(arazyme)은 저온과 높은 염 농도에서 안정된 단백질 분해 활성을 나타낼 뿐 만 아니라 사람의 체온인 37℃에서 가장 높은 활성을 보였으며 넓은 pH 범위에서도 안정된 활성을 나타내는 것을 발견하고 본 단백질 분해효소의 유전자를 확인하였다(WO 01/57222호). On the other hand, the present inventors have tried to develop a new proteolytic enzyme, a new microorganism Aranicolaa Proteoricicus ( Aranicola ) from the Nephila clavata of Korea proteolyticus ) HY-3 strain (Accession No .: KCTC 0268BP; WO 01/57222) was isolated, and arazyme (arayzme), a novel protease, was isolated from the strain. Arazyme not only showed stable proteolytic activity at low temperatures and high salt concentrations, but also showed the highest activity at human body temperature of 37 ° C and stable activity over a wide pH range. Genes were identified (WO 01/57222).
본 발명자들은 아라니콜라 프로테오리티쿠스(Aranicola proteolyticus) 균주에서 유래한 아라자임(arazyme)의 효과를 조사한 결과, 아라자임이 인간 폐암 세포주(A549)를 이식한 누드(Nude) 마우스에서 체중의 증가와 종양세포 억제작용을 일으키며 MMP-9, NF-κB 및 PCNA의 발현양상을 감소시키는 것을 확인하고 암 예방 및 치료용 약학적 조성물로 유용하게 이용될 수 있음을 밝힘으로써 본 발명을 완성하였다.The inventors have found that Aranicola Investigation of the effects of arazyme derived from proteolyticus strains revealed that arazyme causes weight gain and tumor cell suppression in nude mice transplanted with human lung cancer cell line (A549). The present invention has been completed by confirming that the expression patterns of -κB and PCNA are reduced and revealing that they can be usefully used as pharmaceutical compositions for preventing and treating cancer.
본 발명의 목적은 폐암 예방 및 치료용 약학적 조성물의 유효성분으로서 아라니콜라 프로테오리티쿠스(Aranicola proteolycius)가 생산한 아라자임(arazyme)의 새로운 용도를 제공하는 것이다.An object of the present invention is to provide a new use of arazyme (arazyme) produced by Aranicola proteolycius as an active ingredient in the pharmaceutical composition for preventing and treating lung cancer.
상기 목적을 달성하기 위하여, 본 발명은 아라자임(arazyme)을 유효성분으로 함유하는 암 예방 및 치료용 약학적 조성물을 제공한다.In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing and treating cancer containing arazyme (arazyme) as an active ingredient.
또한, 아라니콜라 프로테오리티쿠스의 배양액 또는 이로부터 분리된 아라자임을 유효성분으로 함유하는 암 예방용 건강식품을 제공한다.In addition, the present invention provides a health food for cancer prevention, containing the culture solution of aranicola proteoricicus or arazyme isolated therefrom as an active ingredient.
또한, 아라자임을 유효성분으로 함유하는 암전이 예방 및 억제용 조성물을 제공한다.The present invention also provides a composition for preventing and inhibiting cancer metastasis containing arazyme as an active ingredient.
또한, 아라니콜라 프로테오리티쿠스의 배양액 또는 이로부터 분리된 아라자임을 유효성분으로 함유하는 암전이 예방용 건강식품을 제공한다.In addition, the present invention provides a cancer metastasis prevention health food containing the culture solution of aranicola proteoricicus or an arazyme isolated from it.
또한, 아라자임을 유효성분으로 함유하는 MMP-9 발현 억제제를 제공한다.In addition, it provides an MMP-9 expression inhibitor containing arazyme as an active ingredient.
또한, 아라자임을 유효성분으로 함유하는 NF-κB 발현억제제를 제공한다.In addition, it provides an NF-κB expression inhibitor containing arazyme as an active ingredient.
아울러, 아라자임을 유효성분으로 함유하는 PCNA 발현억제제를 제공한다.In addition, it provides a PCNA expression inhibitor containing arazyme as an active ingredient.
이하, 본 발명을 상세히 설명한다.Hereinafter, the present invention will be described in detail.
본 발명은 아라자임(arazyme)을 유효성분으로 함유하는 암 예방 및 치료용 약학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing and treating cancer containing arazyme as an active ingredient.
상기 암은 폐암인 것을 특징으로 한다.The cancer is characterized in that lung cancer.
본 발명의 아라자임은 아라니콜라 프로테오리티쿠스 균주가 생산하는 효소로서, 1) 아라니콜라 프로테오리티쿠스 균주를 배양하여 배양액을 얻는 단계; 2) 상기 배양액을 여과하여 상등액을 얻는 단계; 및 3) 상등액내 포함된 상기 아라자임을 수지를 이용하여 정제하는 단계로 제조할 수 있다(WO 01/57222호). 아라자임을 생산하는 미생물로서 아라니콜라 프로테오리티쿠스 균주를 이용하는 것이 바람직하며, 한국생명공학연구원 유전자은행에 1996년 7월 29일자로 기탁된 수탁번호 KCTC 0268BP호의 아라니콜라 프로테오리티쿠스 HY-3를 이용하는 것이 더욱 바람직하나, 이에 한정되는 것은 아니다. 아라니콜라 프로테오리티쿠스 HY-3 균주는 거미 장내에서 분리한 호기성의 그람 음성 세균으로서, 0.5 ~ 0.8 ㎜ 크기의 구형이고 운동성을 가지고 있으며 카탈라아제(catalase)에는 양성 반응을, 옥시다아제(oxidase)에는 음성반응을 나타낸다(대한민국 특허 제 0220091호). 본 발명에서는 상기와 같은 방법으로 수득된 아라자임을 이용하였다.The arazyme of the present invention is an enzyme produced by the aranicola proteoricicus strain, comprising the steps of: 1) culturing the aranicola proteoricicus strain to obtain a culture solution; 2) filtering the culture solution to obtain a supernatant; And 3) purifying the arazyme contained in the supernatant using a resin (WO 01/57222). It is preferable to use aranicoli proteoricicus strain as a microorganism producing arazyme, and Aranicola proteoricicus HY-3 of Accession No. KCTC 0268BP, deposited on July 29, 1996 with the Korea Biotechnology Research Institute Gene Bank. More preferably, but is not limited thereto. Aranicola Proteoricicus HY-3 strain is an aerobic Gram-negative bacterium isolated from the intestine of the spider's intestine, and has a spherical and motility of 0.5 to 0.8 mm in size, positive for catalase, and negative for oxidase. Reaction (Korean Patent No. 0220091). In the present invention, arazyme obtained by the above method was used.
본 발명의 아라자임은, (a) 서열번호 1로 기재되는 아미노산 서열을 포함하는 폴리펩티드; (b) 서열번호 1과 70% 이상 상동성을 가지며, (a)의 폴리펩티드와 생물학적으로 동등한 기능을 갖는 폴리펩티드; 및 (c) 서열번호 1로 기재되는 아미노산 서열에서 하나 또는 복수의 아미노산이 치환, 결실, 부가 및/또는 삽입된 아미노산 서열로 이루어진 폴리펩티드 서열을 가지나, (a)의 폴리펩티드와 생물학적 으로 동등한 폴리펩티드를 포함하는 단백질이어도 무방하다. Arazyme of the present invention, (a) a polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 1; (b) a polypeptide having at least 70% homology with SEQ ID NO: 1 and having a biologically equivalent function as the polypeptide of (a); And (c) has a polypeptide sequence consisting of an amino acid sequence in which one or a plurality of amino acids in the amino acid sequence set forth in SEQ ID NO: 1 is substituted, deleted, added and / or inserted, but which is biologically equivalent to the polypeptide of (a) The protein may be used.
상기 아라자임(arazyme)의 폐암에 대한 항암 효능을 검정하기 위하여, 인간 폐암 세포주(A549)를 이식한 누드(Nude) 마우스를 대조군(아라자임 0 ㎎/㎏ 투여), T1(아라자임 150 ㎎/㎏ 투여), T2(아라자임 300 ㎎/㎏ 투여) 및 T3(아라자임 600 ㎎/㎏ 투여)의 4개의 그룹으로 나누어 아라자임을 반복 경구 투여하였다(표 1 참조). To assay the anticancer efficacy of the arazyme against lung cancer, nude mice transplanted with human lung cancer cell line (A549) were treated with a control (administered 0 mg / kg of arazyme), T1 (arazyme 150 mg / Kg administration), T2 (administered 300 mg / kg of arazyme) and T3 (administration of arazyme 600 mg / kg) were divided into four groups, and oral administration of arazyme was repeated (see Table 1).
아라자임은 멸균 주사용수에 용해시켜 주 6회 경구 투여용 존대(zonde)를 이용하여 강제 투여를 실시하였다. 아라자임 투여 후 체중 측정 및 일반 증상을 관찰하였으며 종양의 발생정도를 주 1회씩 관찰하고, 그 크기를 측정하였고 아라자임 투여 12주 후 부검을 실시하였다. 육안 병리 소견에서는 전 장기에서 특별한 이상 소견이 관찰되지 않았고, 현미경 관찰에 있어서도 아라자임 투여에 의한 실질장기의 이상소견은 모든 개체에서 관찰되지 않았다. 체중은 아라자임을 300 ㎎/㎏(T2) 및 600 ㎎/㎏(T3) 투여한 그룹에서 증가하였다(표 2 및 도 1참조). 인간 폐암 세포주(A549)를 이식한 누드 마우스에서 채취한 종양조직의 크기측정 및 헤마톡살린-에오신 염색(Hematoxylin-eosin staining, H&E 염색)으로 면역 조직화학적 관찰을 실시한 결과 300 ㎎/㎏(T2) 및 600 ㎎/㎏(T3)의 아라자임을 투여한 그룹에서 아라자임의 농도 의존성으로 종양발생 억제 효과 및 종양세포 침윤현상의 감소가 관찰되었다(도 2 및 도 3 참조).Arazyme was dissolved in sterile water for injection and subjected to forced administration using a zonde for oral administration six times a week. Body weight and general symptoms were observed after arazyme administration. Tumor incidence was observed once a week, its size was measured, and autopsy was performed 12 weeks after arazyme administration. In gross pathological findings, no abnormal abnormalities were observed in all organs, and even in microscopic observation, no abnormal findings of parenchyma by arazyme administration were observed in all individuals. Body weight was increased in the groups administered arazyme 300 mg / kg (T2) and 600 mg / kg (T3) (see Table 2 and FIG. 1). 300 mg / kg (T2) as a result of immunohistochemical observation by tumor size measurement and hematoxylin-eosin staining (H & E staining) obtained from nude mice transplanted with human lung cancer cell line (A549) In the group administered 600 mg / kg (T3) of arazyme, the arazyme concentration-dependent inhibition of tumorigenicity and tumor cell infiltration were observed (see FIGS. 2 and 3).
매트릭스 매탈로프로테인아제(matrix metalloproteinases; MMPs)는 세포외 기질(extracellular matrix; ECM)을 분해하는 단백분해 효소로서(Matrisan LM, Trends Genet, 6:121-125, 1990) MMPs의 생성이 증가하면 염증과 더불어 여러 종류의 암에서 침윤도와 전이가 증가하는 것으로 알려져 있다(Stearns MA, et al., Cancer Res, 53:878-883, 1993). 종양의 전이과정 중 혈관 기저막의 가장 중요한 구조물인 콜라겐 타입(collagen type) Ⅳ의 분해가 이루어져야 비로소 체내 혈류로의 전이가 가능하며 이러한 콜라겐 타입 Ⅳ의 분해는 MMPs 중에서 주로 MMP-9 및 MMP-2에 의해서 이루어진다(Liotta LA, et al., Cell, 64:327-336, 1991). MMP는 또한 신생혈관형성에서 혈관 내피세포의 이동과 발아에 필요한 물질로 인식되고 있다(Fisher C, et al., Devel Biol, 162:499-510, 1994). MMP의 활성은 1) MMP 유전자의 전사 조절; 2) 전구체의 활성; 3) 지질특이성의 차이; 및 4) 알파-매크로 글로블린(a-macroglobulins)과 조직 매트릭스 매탈로프로테인아제 저해제(tissue matrix metalloproteinases inhibitor; TIMPs)와 같은 MMP 억제물질 등에 의해 조절된다(Birkedal-Hansen. H, J periodontol, 64:474-484, 1993). MMP-9의 발현을 조절하는 상위 신호전달 기전에는 미토겐-활성 단백질 키나아제(mitogen-activated protein kinases; MAPKs)를 포함하는 성장인자, 사이토카인, 종양촉진인자 등이 관여한다. 이 중 p38 키나아제(kinase) 및 세포 외부의 신호에 의해 조절된 단백질 키나아제(extracellular signal-regulated protein kinase; ERK)는 염증 사이토카인이나 아포토시스 관련신호 및 성장인자 등에 의하여 활성화된다. 활성화된 MAPKs는 핵으로 이동하여 MMP 시발체 주변의 전사인자 결합부와 반응하여 다양한 전사인자를 활성화시킴으로써 하위 표적 단백질의 발현을 조절한다. 대부분 암은 암세포의 기질 침습, 원발병소에서 혈관계로의 이동, 혈관에서 이차 병소로의 이동, 이차 종양의 형성이라는 과정을 통하여 원발병소에서 이차병소로 전이된다. 암세포의 기질 침습에는 단백 분해효소에 의한 세포 외기질의 분해가 요구되는데 MMPs는 세포 외기질을 분해하는 가장 핵심적인 효소이다. 다양한 MMPs중에서 MMP-2 및 MMP-9는 각각 72 및 92 kDa의 콜라겐아제(collagenase)로서 세포의 기저막을 형성하는 콜라겐을 분해하여 암의 침습과 전이를 촉진한다(Himelstein BP, et al ., Invasion Metastasis, 14:246-258, 1994).Matrix metalloproteinases (MMPs) are proteolytic enzymes that degrade the extracellular matrix (ECM) (Matrisan LM, Trends Genet , 6: 121-125, 1990) Increased production of MMPs is known to increase inflammation and invasion and metastasis in many cancer types (Stearns MA, et. al ., Cancer Res , 53: 878-883, 1993). During the tumor metastasis, collagen type IV, the most important structure of the vascular basement membrane, must be degraded before it can be transferred into the bloodstream. The degradation of collagen type IV is mainly caused by MMP-9 and MMP-2. (Liotta LA, et al ., Cell , 64: 327-336, 1991). MMP is also recognized as a necessary material for the migration and germination of vascular endothelial cells in angiogenesis (Fisher C, et. al ., Devel Biol , 162: 499-510, 1994). The activity of MMPs is 1) to regulate transcription of the MMP gene; 2) activity of precursors; 3) differences in lipid specificity; And 4) MMP inhibitors such as a-macroglobulins and tissue matrix metalloproteinases inhibitors (TIMPs) and the like (Birkedal-Hansen. H, J periodontol , 64: 474). -484, 1993). Higher signaling mechanisms that regulate the expression of MMP-9 involve growth factors including mitogen-activated protein kinases (MAPKs), cytokines, and tumor promoters. Among them, p38 kinase and extracellular signal-regulated protein kinase (ERK) regulated by extracellular signals are activated by inflammatory cytokines or apoptosis related signals and growth factors. Activated MAPKs move into the nucleus and react with transcription factor binding sites around the MMP primers to regulate the expression of downstream target proteins by activating various transcription factors. Most cancers are metastasized from primary to secondary by processes such as matrix invasion of cancer cells, migration from primary to vascular system, migration from vascular to secondary lesions, and formation of secondary tumors. Invasion of cancer cells requires the breakdown of extracellular matrix by protease. MMPs are the most important enzymes that break down extracellular matrix. Among the various MMPs, MMP-2 and MMP-9 are 72 and 92 kDa collagenases, which break down the collagen that forms the cell's basement membrane to promote cancer invasion and metastasis (Himelstein BP, et. al ., Invasion Metastasis , 14: 246-258, 1994).
본 발명에서는 암의 침습과 전이를 촉진하는 MMP-9의 발현이 아라자임에 의해 감소됨을 확인할 수 있었고(도 4 및 도 5 참조) 이는 아라자임의 항암 효능을 나타낸다. In the present invention, it was confirmed that the expression of MMP-9, which promotes cancer invasion and metastasis, was reduced by arazyme (see FIGS. 4 and 5), indicating the anticancer efficacy of arazyme.
핵 전사 인자-카파B(nuclear transcription factor-κB; NF-κB)는 다양한 환경적 자극에 대한 반응으로 활성화되는 유도전사 인자이다( Baeuerle, PA and Baichwal, VR, Adv Immunol 65:111-137, 1997; Auphan, N, et al ., Science, 270:286-290, 1995). 잠정적 형태의 NF-κB는 세포질(cytoplasm)에 있는 억제단백질, I-κB에 결합되어 있다. 다양한 NF-κB 활성인자, 즉 종양괴사인자(tumor necrosis factor), IL-1, 리포다당체(lipopolysaccharides) 및 UV(ultraviolet) 등의 자극은 억제단백질인 I-κB의 인산화반응(phosphorylation) 및 유비퀴틴이 표지되는 과정(ubiquitination)을 통하여 분해(degradation)를 유도하게 된다. 이러한 과정은 I-κB와 분리된 NF-κB를 핵으로 이동시켜 표적 유전자의 프로모터 부위에 있는 NF-κB-결합 자리(NF-κB-binding site)와의 결합을 가능하게 하여, 표적 유전자의 발현을 촉진시킨다(Baldwin, AS, Annu Rev Immunol, 14:649-683, 1996; Huxford T, et al ., Cell, 95:759-770, 1998). NF-κB는 약물 불응성의 또 다른 영향인자이다. 이는 사이토카인 합성의 중요한 조절인자이며, 면역반응이나 염증반응에 관여하는 다양한 유전자들의 발현을 증가시키는 전사인자로 활성화되면 핵 내로 이동하여 염증 유전자의 발현을 증가시킨다. Nuclear transcription factor-kappaB (NF-κB) is an induced transcription factor that is activated in response to various environmental stimuli (Baeuerle, PA and Baichwal, VR, Adv). Immunol 65: 111-137, 1997; Auphan, N, et al ., Science , 270: 286-290, 1995). The tentative form of NF-κB is bound to the inhibitory protein, I-κB, in the cytoplasm. Stimulation of various NF-κB activators, such as tumor necrosis factor, IL-1, lipopolysaccharides, and UV (ultraviolet), caused phosphorylation and ubiquitin of the inhibitory protein I-κB. Degradation is induced through ubiquitination. This process transfers the NF-κB separated from I-κB into the nucleus to allow binding to the NF-κB-binding site at the promoter site of the target gene, thereby reducing the expression of the target gene. (Baldwin, AS, Annu Rev Immunol , 14: 649-683, 1996; Huxford T, et al ., Cell , 95: 759-770, 1998). NF-κB is another influencer of drug refractory. It is an important regulator of cytokine synthesis, and when activated as a transcription factor that increases the expression of various genes involved in immune or inflammatory reactions, it moves into the nucleus and increases the expression of inflammatory genes.
본 발명에서는 이러한 NF-κB의 발현이 아라자임의 투여에 의해 감소되었으며(도 6 및 도 7 참조) 이는 아라자임에 의한 종양세포의 감소를 의미한다.In the present invention, the expression of such NF-κB was reduced by administration of arazyme (see FIGS. 6 and 7), which means the reduction of tumor cells by arazyme.
PCNA는 세포주기 중 G0 단계(G0 phase)에는 존재하지 않으며, G1 단계(G1 phase) 동안에 증가하기 시작해서 S 단계(S phase)에 가장 많이 합성되고 G2 단계(G2 phase)에 감소되는, 36 kDa의 세포내 DNA 폴리머라제 델타(DNA polymerase delta)의 보조적인 단백질로, 세포의 DNA 합성과 세포의 증식에 필수적인 것으로 알려져 있다(Miyagawa S, et al ., J Invest Dematol, 93:678-681, 1989). 일반적으로 PCNA의 조직면역형광법은 종양조직에서 성장에 관여하는 부분이 얼마 만큼인지 알려주는 척도로 작용한다. PCNA 발현은 각질세포의 증식상태, 신경교증, 착색된 피부병변, 폐의 비소세포성암의 조직 병리학적 등급과 관련 있다고 알려져 왔다(Underhill C, J Cell Sci, 103:293-298, 1992). PCNA does not exist in the G0 phase of the cell cycle, but begins to increase during the G1 phase, most synthesized in the S phase and decreased in the G2 phase, 36 kDa As a supplementary protein of DNA polymerase delta, it is known to be essential for cell DNA synthesis and cell proliferation (Miyagawa S, et. al ., J Invest Dematol , 93: 678-681, 1989). In general, tissue immunofluorescence of PCNA acts as a measure of how much of the tumor tissue is involved in growth. PCNA expression has been associated with histopathological grades of keratinocyte proliferation, gliosis, pigmented skin lesions, and non-small cell carcinoma of the lung (Underhill C, J Cell). Sci , 103: 293-298, 1992).
아라자임의 투여에 의해 PCNA의 발현이 감소하는 것을 확인할 수 있었으며(도 8 및 도 9 참조) 종양조직의 성장이 감소하였음을 의미한다. It was confirmed that the expression of PCNA decreased by the administration of arazyme (see FIGS. 8 and 9), which means that the growth of tumor tissues was reduced.
인간 폐암 세포주(A549)를 이식한 누드 마우스에 본 발명의 아라자임(arazyme)을 투여한 결과 체중의 증가와 종양부피가 감소되는 양상을 보임으로써 항암 효과를 나타냈으며 조직병리학적으로는 종양세포의 침윤현상이 약한 것을 관찰 할 수 있었고 면역 조직화학적으로는 MMP-9, NF-κB 및 PCNA의 발현양상이 감소된 것을 관찰할 수 있었다. When arazyme of the present invention was administered to nude mice transplanted with a human lung cancer cell line (A549), the weight gain and tumor volume were reduced, and the anticancer effect was shown. Weak infiltration was observed and immunohistochemically, the expression of MMP-9, NF-κB and PCNA was decreased.
이상의 결과는 아라자임이 항암 및 암 예방 효능의 잠재력을 가지고 있음을 의미한다. 또한 아라자임을 암컷 위스터(Wistar) 랫트에 경구 투여하여 독성 실험을 수행한 결과, 0, 1250, 2500 및 5000 ㎎/㎏ 농도에서 일반증상과 육안 병리 소견에서 아무런 이상 소견이 나타나지 않았다. 이상과 같이 경구 투여 독성시험에 의한 50% 치사량(LD50)은 적어도 5,000 ㎎/㎏ 이상인 안전한 물질로 판단된다. 그러므로 본 발명의 아라자임은 암 예방 및 치료용 약학적 조성물로서 제공될 수 있다.The above results indicate that arazyme has potential for anticancer and cancer prevention efficacy. Toxicity experiments were performed by oral administration of arazyme to female Wistar rats. There were no abnormalities in general symptoms and gross pathological findings at concentrations of 0, 1250, 2500 and 5000 mg / kg. As described above, the 50% lethal dose (LD 50 ) by the oral administration toxicity test is considered to be a safe substance of at least 5,000 mg / kg or more. Therefore, the arazyme of the present invention may be provided as a pharmaceutical composition for preventing and treating cancer.
본 발명의 아라자임에 추가로 동일 또는 유사한 기능을 나타내는 유효성분을 1종 이상 함유할 수 있다. 투여를 위해서는 추가로 약제학적으로 허용 가능한 담체를 1종 이상 포함하여 제조할 수 있다. 약제학적으로 허용 가능한 담체는 식염수, 멸균수, 링거액, 완충 식염수, 덱스트로스 용액, 말토 덱스트린 용액, 글리세롤, 에탄올 및 이들 성분 중 1 성분 이상을 혼합하여 이용할 수 있으며, 필요에 따 라 항산화제, 완충액, 정균제 등 다른 통상의 첨가제를 첨가할 수 있다. 또한, 희석제, 분산제, 계면활성제, 결합제 및 윤활제를 부가적으로 첨가하여 수용액, 현탁액, 유탁액 등과 같은 주이용 제형, 산제, 정제, 캡슐제, 환, 과립 또는 주사액제로 제제화 할 수 있다. 더 나아가 당 분야의 적정한 방법으로 또는 Remington's Pharmaceutical Science(Mack Publishing Company, Easton PA, 18th, 1990)에 개시되어 있는 방법을 이용하여 각 질환에 따라 또는 성분에 따라 바람직하게 제제화할 수 있다.In addition to the arazyme of the present invention, it may contain one or more active ingredients exhibiting the same or similar functions. For administration, it may be prepared further comprising one or more pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers may be used in combination with saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components, if necessary And other conventional additives such as bacteriostatic agents can be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate main formulations, powders, tablets, capsules, pills, granules or injections, such as aqueous solutions, suspensions, emulsions and the like. Furthermore, it may be preferably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).
본 발명의 암 예방 및 치료용 약학적 조성물은 목적하는 방법에 따라 비경구 투여(예를 들어 정맥 내, 피하, 복강 내 또는 국소에 적용)하거나 경구 투여할 수 있으며, 투여량은 환자의 체중, 연령, 성별, 건강상태, 식이, 투여시간, 투여방법, 배설율 및 질환의 중증도 등에 따라 그 범위가 다양하다. 본 발명에 따른 아라자임의 일일 투여량은 0.01 ~ 5000 ㎎/㎏ 이며, 바람직하게는 0.01 ~ 10 ㎎/㎏ 이며, 하루 일회 내지 수회에 나누어 투여하는 것이 더욱 바람직하다. The pharmaceutical composition for preventing and treating cancer of the present invention may be administered parenterally (for example, intravenously, subcutaneously, intraperitoneally, or topically) or orally according to a desired method. The range varies depending on age, sex, health condition, diet, administration time, administration method, excretion rate and severity of disease. The daily dose of arazyme according to the present invention is 0.01 to 5000 mg / kg, preferably 0.01 to 10 mg / kg, and more preferably administered once to several times a day.
또한, 아라니콜라 프로테오리티쿠스의 배양액 또는 이로부터 분리된 아라자임을 유효성분으로 함유하는 암 예방용 건강식품을 제공한다.In addition, the present invention provides a health food for cancer prevention, containing the culture solution of aranicola proteoricicus or arazyme isolated therefrom as an active ingredient.
본 발명의 아라니콜라 프로테오리티쿠스의 배양액 또는 이로부터 분리된 아라자임을 식품 첨가물로 이용할 경우, 아라니콜라 프로테오리티쿠스의 배양액 또는 상기의 아라자임을 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 이용될 수 있고, 통상적인 방법에 따라 적절하게 이용될 수 있다. 아라자임은 상기와 같은 동일한 방법으로 수득하여 이용한다. 유효 성분의 혼합양은 이용 목적(예방, 건강 또는 치료적 처치)에 따라 적합하게 결정될 수 있다. 일반적으로, 식품 또는 음료의 제조 시에는 본 발명의 아라니콜라 프로테오리티쿠스의 배양액 또는 이로부터 분리된 아라자임은 원료에 대하여 0.01 ~ 10 중량부, 바람직하게는 0.05 ~ 1 중량부의 양으로 첨가된다. 그러나 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간의 섭취의 경우에는 상기 양은 상기 범위 이하일 수 있으며, 안전성 면에서 아무런 문제가 없기 때문에 유효성분은 상기 범위 이상의 양으로도 이용될 수 있다.When using the culture solution of aranicola proteoricicus of the present invention or the arazyme separated from it as a food additive, the culture medium of aranicola proteoricicus or the arazyme as described above is used as it is or used with other food or food ingredients. It may be used and may be appropriately used according to a conventional method. Arazyme is obtained and used in the same manner as described above. The mixed amount of the active ingredient may be appropriately determined depending on the purpose of use (prevention, health or therapeutic treatment). In general, in the preparation of food or beverage, the culture solution of aranicola proteoricicus of the present invention or arazyme separated therefrom is added in an amount of 0.01 to 10 parts by weight, preferably 0.05 to 1 part by weight based on the raw materials. . However, in the case of long-term intake for health and hygiene or health control, the amount may be below the above range, and the active ingredient may be used in an amount above the above range because there is no problem in terms of safety. .
상기 식품의 종류에는 특별한 제한은 없다. 상기 물질을 첨가할 수 있는 식품의 예로는 육류, 소세지, 빵, 쵸코렛, 캔디류, 스넥류, 과자류, 피자, 라면, 기타 면류, 껌류, 아이스크림류를 포함한 낙농제품, 각종 스프, 음료수, 차, 드링크제, 알코올음료 및 비타민 복합제 등이 있으며, 통상적인 의미에서의 건강식품을 모두 포함한다.There is no particular limitation on the kind of food. Examples of the food to which the substance can be added include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, drinks, Alcoholic beverages and vitamin complexes, etc., includes all of the health food in the conventional sense.
본 발명의 건강음료 조성물은 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물은 포도당, 과당과 같은 모노사카라이드, 말토스, 수크로오스와 같은 디사카라이드, 및 덱스트린, 사이클로덱스트린과 같은 폴리사카라이드, 자일리톨, 소르비톨, 에리트리톨 등의 당알코올이다. 감미제로서는 타우마틴, 스테비아 추출물과 같은 천연 감미제나, 사카린, 아스파르탐과 같은 합성 감미제 등을 이용할 수 있다. 상기 천연 탄수화물의 비율은 본 발명의 음료 조성물 100 ㎖당 일반적으로 약 0.01 ~ 0.04 g, 바람직하게는 약 0.02 ~ 0.03 g 이다.The health beverage composition of the present invention may contain various flavors or natural carbohydrates, etc. as additional components, as in the general beverage. The natural carbohydrates described above are glucose, monosaccharides such as fructose, disaccharides such as maltose and sucrose, and polysaccharides such as dextrin, cyclodextrin, sugar alcohols such as xylitol, sorbitol, and erythritol. As the sweetener, natural sweeteners such as tautin and stevia extract, synthetic sweeteners such as saccharin, aspartame, and the like can be used. The proportion of the natural carbohydrate is generally about 0.01 to 0.04 g, preferably about 0.02 to 0.03 g per 100 ml of the beverage composition of the present invention.
상기 외에 아라니콜라 프로테오리티쿠스의 배양액 또는 이로부터 분리된 아라자임은 여러 가지 영양제, 비타민, 전해질, 풍미제, 착색제, 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산 음료에 이용되는 탄산화제 등에 첨가할 수 있다. 그밖에 천연 과일쥬스, 과일쥬스 음료 및 야채 음료의 제조를 위한 과육에도 아라니콜라 프로테오리티쿠스의 배양액 또는 이로부터 분리된 아라자임을 첨가할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 이용할 수 있다. 이러한 첨가제의 비율은 크게 중요하진 않지만 본 발명의 아라니콜라 프로테오리티쿠스의 배양액 또는 이로부터 분리된 아라자임 100 중량부 당 0.01 ~ 0.1 중량부의 범위에서 선택되는 것이 일반적이다.In addition to the above, Aranicola proteoricicus culture medium or arazyme isolated therefrom is a variety of nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners, pH It can be added to regulators, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks and the like. In addition to the pulp for the production of natural fruit juices, fruit juice beverages and vegetable beverages can be added to the culture solution of aranicola proteoricicus or arazyme separated therefrom. These components can be used independently or in combination. The ratio of such additives is not critical, but is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the culture solution of aranicola proteoricicus of the present invention or arazyme separated therefrom.
또한, 아라자임을 유효성분으로 함유하는 암전이 예방 및 억제용 약학적 조성물을 제공한다.The present invention also provides a pharmaceutical composition for preventing and inhibiting cancer metastasis containing arazyme as an active ingredient.
또한, 아라니콜라 프로테오리티쿠스의 배양액 또는 이로부터 분리된 아라자임을 유효성분으로 함유하는 암전이 예방용 건강식품을 제공한다.In addition, the present invention provides a cancer metastasis prevention health food containing the culture solution of aranicola proteoricicus or an arazyme isolated from it.
또한, 아라자임을 유효성분으로 함유하는 MMP-9 발현 억제제를 제공한다.In addition, it provides an MMP-9 expression inhibitor containing arazyme as an active ingredient.
또한, 아라자임을 유효성분으로 함유하는 NF-κB 발현억제제를 제공한다.In addition, it provides an NF-κB expression inhibitor containing arazyme as an active ingredient.
아울러, 아라자임을 유효성분으로 함유하는 PCNA 발현억제제를 제공한다.In addition, it provides a PCNA expression inhibitor containing arazyme as an active ingredient.
이하 본 발명을 실시예, 실험예 및 제제예에 의해 상세히 설명한다.Hereinafter, the present invention will be described in detail by Examples, Experimental Examples and Formulation Examples.
단, 하기 실시예, 실험예 및 제제예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예, 실험예 및 제제예에 한정되는 것은 아니다.However, the following Examples, Experimental Examples, and Formulation Examples are merely illustrative of the present invention, and the content of the present invention is not limited to the following Examples, Experimental Examples, and Formulation Examples.
<< 실시예Example 1> 1> 아라자임Arazyme (( arazymearazyme )의 제조Manufacturing
본 발명의 유효성분인 아라자임(arazyme)을 제조하기 위하여, 아라니콜라 프로테오리티쿠스 HY-3 균주(KCTC 0268BP)를 배양배지(박토 트립톤 0.5 %, 효모 추출물 0.5 %, 염화 나트륨 0.1 %, 염화 칼륨 0.05 %, 염화 칼슘 0.02 %, 황산 마그네슘 0.02 %)에서 22℃로 18 시간 동안 배양하였다. 배양액을 2 ㎛ 막여과를 이용해서 균체와 상등액을 분리한 다음 분리된 상등액을 10 kDa의 막 여과를 이용하여 농축하였다. 본 발명의 아라자임은 기본적으로 음이온의 특성을 가지므로 농축액을 50 mM 트리스-염산 완충액(pH 7.6)으로 전처리한 DEAE-셀룰로오즈(DEAE-cellulose)를 이용한 이온 교환 수지(ion exchange resin)와 20 mM 트리스-염산 완충액(pH 7.6)으로 전처리한 세파덱스 G-75(Sephadex G-75)를 이용한 젤 여과 교환수지(gel filtration exchange resin)를 이용해 정제를 했다. 정제된 효소액은 10% SDS-PAGE(Sodium dodecyl sulfate-polyacrylamide gel)로 전기영동(electrophoresis)을 하여 밴드 경향을 확인하였는데 그 결과 본 발명의 아라자임은 소단위를 갖지 않는 단일체(monomer)로서 약 51.5 kDa의 분자량을 갖는 밴드를 나타냄을 확인하였다. 단, 아라자임 생산을 위한 아라니콜라 프로테오리티쿠수 HY-3 균주(KCTC 0268BP)는 다양한 산업용 배지에서 생산을 할 수 있으며 생산된 배양액 또한 다양한 방법으로 분리 및 정제하여 이용할 수 있다. 본 발명의 아라자임은 서열번호 1로 기재되는 폴리펩티드 서열을 가지며, 서열번호 2로 기재되는 폴리뉴클레오티드 서열을 가진다.In order to prepare arazyme (arazyme), the active ingredient of the present invention, Aranicola proteoricicus HY-3 strain (KCTC 0268BP) was cultured in a culture medium (bacto tryptone 0.5%, yeast extract 0.5%, sodium chloride 0.1%, 0.05% potassium chloride, 0.02% calcium chloride, 0.02% magnesium sulfate) was incubated at 22 ° C. for 18 hours. The culture solution was separated from the cells and the supernatant using 2 μm membrane filtration, and the separated supernatant was concentrated using membrane filtration of 10 kDa. Since the arazyme of the present invention basically has anion properties, the ion exchange resin and the ion exchange resin using DEAE-cellulose, which have been pretreated with 50 mM tris-hydrochloric acid buffer (pH 7.6), and 20 mM Purification was carried out using a gel filtration exchange resin using Sephadex G-75 pretreated with Tris-HCl buffer (pH 7.6). The purified enzyme solution was subjected to electrophoresis with 10% SDS-PAGE (Sodium dodecyl sulfate-polyacrylamide gel), and the band tendency was confirmed. As a result, the arazyme of the present invention was a monomer having no subunit and was approximately 51.5 kDa. It was confirmed that a band having a molecular weight of? However, Aranicola proteoritikkusu HY-3 strain (KCTC 0268BP) for the production of arazyme can be produced in a variety of industrial media, and the produced culture solution can also be isolated and purified by various methods. The arazyme of the present invention has a polypeptide sequence as set out in SEQ ID NO: 1 and a polynucleotide sequence as set out in SEQ ID NO: 2.
<< 실험예Experimental Example 1> 1> 시험군의Test group 구성 및 투여 Composition and Administration
본 발명에서는 CAnN.Cg-Foxn1nu/CrljBgi 계통의 5주령 특정병원체부재(SPF) 누드(Nude) 마우스(오리엔트, 한국) 평균체중 15 g인 것을 이용하였다. In the present invention, an average body weight of 15 g of five-week-old specific pathogen-free (SPF) nude mice (Orient, Korea) of CAnN.Cg-Foxn1nu / CrljBgi strain was used.
마우스는 입수 후 동물실에서 약 7일간 검역 및 순화시켰으며, 순화기간 중 일반증상을 관찰하여 건강한 동물만을 선발하여 시험에 이용하였다. Mice were quarantined and purified for about 7 days in the animal room after acquisition, and only healthy animals were selected and used for testing by observing general symptoms during the acclimation period.
본 발명에서 이용된 마우스의 경우, 온도 22± 3℃, 상대습도 50± 10%, 조명시간 12 시간(08:00 점등 ~ 20:00 소등)으로 설정된 자동 온습도 조절 장치가 설치된 동물실에서 순화 및 사육되었다. 환경조건은 정기적(3개월 1회)으로 측정되었다. 환경 측정의 결과, 시험에 영향을 미칠 것으로 사료되는 변동은 인정되지 않았다. 전 시험기간 동안 폴리카보네이트제 사육상자[240 W × 390 L × 175 H (mm)]에 5마리 이하씩 수용하였다. 개별 식별은 유성 매직펜을 이용한 피부표식법과 사육상자별 개체식별 카드표시법을 이용하였다. 사료는 실험동물용 고형사료(PMI Nutrition International, 505 North 4th Street Richmond, in 47374, USA)를 방사선 조사(13.2 kGy) 멸균하여 자유 섭취시켰다. 물은 카본 필터를 통해 정화된 상수도수를 물병을 이용하여 자유 섭취 시켰다. In the case of the mouse used in the present invention, the temperature of 22 ± 3 ℃, relative humidity 50 ± 10%,
일주일의 순화 기간이 경과한 뒤, 인간 페암 세포(Human Lung Adenocarcinoma cell)인 A549 세포주를 1ⅹ107 세포(Cell)로 마우스의 왼쪽 겨드랑이 피하에 이식시켜 암을 유발시킨 동시에 4개의 그룹(대조군, T1, T2, T3 및 표 1)으로 분리한 후, 12주간 아라자임(arazyme)을 0 ㎎/㎏(대조군), 150 ㎎/㎏(T1), 300 ㎎/㎏(T2) 및 600 ㎎/㎏(T3)의 농도로 주 6회 존대(zonde)를 이용하여 경구 투여하였다. 전 실험기간 중 체중측정 및 일반 증상 관찰을 실시하고 12주 후 부검을 실시하였다. After a week of acclimatization, human Lung Adenocarcinoma cells, A549 cell line, were transplanted into 1x10 7 cells subcutaneously in the left armpit of the mouse to induce cancer and at the same time in four groups (control, T1, After separation into T2, T3 and Table 1), arazyme was administered for 12 weeks at 0 mg / kg (control), 150 mg / kg (T1), 300 mg / kg (T2) and 600 mg / kg (T3). Oral administration with a zonde six times a week. Body weights and general symptoms were observed during the entire experimental period, and necropsy was performed 12 weeks later.
실험군의 구성은 아래의 표 1과 같다.The composition of the experimental group is shown in Table 1 below.
<< 실험예Experimental Example 2> 2> 아라자임Arazyme 투여가 체중변화에 미치는 영향 조사 Investigate the effects of dosing on weight change
실험예 1에서와 같이 인간 폐암 세포주(A549)를 누드 마우스에 이식한 후, 1주일에 한 번씩 체중을 측정함으로써 체중변화를 관찰하고 동물의 일반 증상을 관찰하였다. As in
전 실험기간(12주)을 거쳐 아라자임 투여군은 체중이 증가되는 양상을 보였는데 이중 특히 300 ㎎/㎏ 투여군(T2) 및 600 ㎎/㎏ 투여군(T3)에서 체중의 증가가 두드러졌다(표 2 및 도 1).The body weight gain of the arazyme group was increased over the entire experimental period (12 weeks), especially in the 300 mg / kg administration group (T2) and 600 mg / kg administration group (T3). And FIG. 1).
대조군: 아라자임 0 ㎎/㎏ 경구투여, Control: arazyme 0 mg / kg orally,
T1: 아라자임 150 ㎎/㎏ 경구투여, T1: arazyme 150 mg / kg oral administration,
T2: 아라자임 300 ㎎/㎏ 경구투여, T2: oral administration of arazyme 300 mg / kg,
T3: 아라자임 600 ㎎/㎏ 경구투여. T3: oral administration of arazyme 600 mg / kg.
<< 실험예Experimental Example 3> 3> 아라자임Arazyme 투여가 종양 성장에 미치는 영향 Effect of dosing on tumor growth
실험예 1에서와 같이 인간 폐암 세포주(A549)를 누드 마우스에 이식한 후, 5주째부터 육안적으로 종양크기를 확인할 수 있었으며 이후부터 8주 동안 일주일에 한번씩 캘리퍼(caliper)를 이용하여 각각의 마우스에서의 종양의 크기를 측정하였다. 종양의 부피는 종양의 장경과 단경을 측정하여 다음 식 1과 같이 계산하였다. As in
<식 1><
종양의 부피(㎣) = (장경 × 단경2)/2 Tumor volume = (long diameter × short diameter 2 ) / 2
측정한 결과 전 실험기간동안 아라자임을 300 ㎎/㎏(T2) 및 600 ㎎/㎏(T3) 투여한 군에서 대조군에 비해 종양크기가 감소하였으며 통계학적 유의성이 관찰되었다(도 2).As a result, the tumor size was reduced and a statistical significance was observed in the arazyme administered 300 mg / kg (T2) and 600 mg / kg (T3) groups compared to the control group for the entire experimental period (FIG. 2).
<실험예 4> 폐암조직의 조직병리학적 관찰Experimental Example 4 Histopathological Observation of Lung Cancer Tissues
누드 마우스에 인간 폐암 세포주를 이식한 12주 후에 에틸에테르에 마취시켜 희생시키고 종양 및 실질장기를 채취하였다. 채취한 종양조직의 무게를 측정하였으며 심장, 간장, 폐장, 췌장, 신장, 비장 등 장기를 채취하였다. 채취한 종양과 장기는 10% 중성 포르말린에 24 시간 고정시킨 후 자동 조직 처리기구(automatic tissue processor)로 조직 처리를 수행하였다. 조직 처리과정을 거친 종양과 장기는 파라핀 포매 후 4 ㎛의 두께로 박절하여 헤마톡살린-에오신 염색(Hematoxylin-eosin staining, H&E 염색)을 실시하여 광학현미경으로 관찰하였다. Twelve weeks after transplanting human lung cancer cell lines to nude mice, anesthesia was sacrificed by ethyl ether, and tumors and parenchyma were collected. The tumor tissues were weighed and organs such as heart, liver, lung, pancreas, kidney and spleen were collected. The collected tumors and organs were fixed in 10% neutral formalin for 24 hours and then treated with an automatic tissue processor. After treatment with tissues, tumors and organs were cut to 4 μm thickness after embedding paraffin and subjected to hematoxylin-eosin staining (H & E staining).
그 결과, 아라자임 투여군은 대조군에 비해 종양세포침윤이 약하게 진행되고 있었다(도 3). As a result, the arazyme-administered group had a weaker tumor cell infiltration than the control group (FIG. 3).
<실험예 5> MMP-9의 면역 조직화학적 관찰Experimental Example 5 Immunohistochemical Observation of MMP-9
MMP-9의 면역 조직화학적 관찰을 위하여, 포르말린에 고정한 후 제작된 파라핀 포매조직을 4 ㎛의 두께로 박절하여 탈파라핀시킨 후 내인성 과산화효소의 활성을 억제시키기 위해 3% H2O2에 30분간 처리하고 0.01 M 인산완충식염수(phosphate-buffer saline, PBS)로 세척한 다음, 조직의 항원 발현을 증가시키기 위하여 0.01 M 구연산염(citrate) 완충액으로 처리하고, 마이크로파(microwave)로 고열처리 하였다. 비특이적 반응을 억제하기 위하여 20 g/㎖ 프로테이나제(proteinase) K로 37℃에서 10분간 처리한 후 인산완충식염수로 세척 후 1 시간 동안 차단(blocking)시켰다. 그 후, 항-래빗(rabbit) 1차 항체인 MMP-9(1:100, Santa Cruz Bio, USA)을 4℃에서 하룻밤 동안 처리한 다음 PBS 완충액으로 5분간 3회 세척하고 비오틴(biotin)과 결합시켰다. 비오틴과 결합된 1차 항체를 완충액으로 세척한 후 스트렙트아비딘(Streptavidin)으로 접합된 HRP(Horse-Rradish Peroxidase)를 부착 시키고, Histostantin-plus bulk kit(Zymed Laboratories Inc, USA)로 37℃에서 30분간 반응시켜 충분히 세척한 후 3,3-디아미노벤지딘 테트라하이드로클로라이드(3,3-diaminobenzidine tetrahydrochloried; DAB, Zymed Laboratories Inc, USA) 용액으로 발색시키고 증류수로 세척한 다음 Meyer's hematoxylin(Research genetics, USA)으로 대조 염색을 실시하여 광학현미경으로 관찰하였다.For immunohistochemical observation of MMP-9, paraffin embedded tissues fixed in formalin were cut to 4 μm thickness and deparaffinized, followed by 30 minutes in 3% H 2 O 2 to inhibit endogenous peroxidase activity. After treatment and washing with 0.01 M phosphate-buffer saline (PBS), and then treated with 0.01 M citrate buffer to increase the antigen expression of the tissues, and was subjected to high heat treatment with microwave (microwave). In order to inhibit the nonspecific reaction, 20 g / ml proteinase K was treated for 10 minutes at 37 ° C., and then washed with phosphate buffered saline and then blocked for 1 hour. Subsequently, the anti-rabbit primary antibody MMP-9 (1: 100, Santa Cruz Bio, USA) was treated overnight at 4 ° C., then washed three times with PBS buffer for 5 minutes, followed by biotin and Combined. After washing the primary antibody bound with biotin in buffer, attach HRP (Horse-Rradish Peroxidase) conjugated with streptavidin, and at 37 ° C with Histostantin-plus bulk kit (Zymed Laboratories Inc, USA). After sufficient reaction and washing for 3 minutes, the solution was developed with 3,3-diaminobenzidine tetrahydrochloried (DAB, Zymed Laboratories Inc, USA) solution, washed with distilled water, and then washed with Meyer's hematoxylin (Research genetics, USA). Control staining was carried out and observed with an optical microscope.
그 결과, 아라자임 투여군은 대조군에 비해 MMP-9의 발현양상이 감소하였다(도 4 및 도 5). As a result, the expression pattern of MMP-9 was reduced in the arazyme administered group compared to the control group (Figs. 4 and 5).
MMPs(matrix metalloproteases)의 생성의 증가시 염증과 더불어 여러 종류의 암에서 침윤도와 전이가 증가하는 것으로 알려져 있다. 또한 MMPs는 주로 MMP-9 및 MMP-2에 의해서 이루어진다. 특히 MMP-9의 발현을 조절하는 상위 신호전달 기전에는 MAPKs를 포함하는 성장인자, 사이토카인, 종양촉진인자 등이 관여한다. MMP-9의 발현양상의 감소는 암의 침윤과 전이가 억제되고 있는 것을 의미한다.Increased production of matrix metalloproteases (MMPs) is known to increase inflammation and invasion and metastasis in many cancers. MMPs are also mainly made up of MMP-9 and MMP-2. In particular, higher signaling mechanisms that regulate the expression of MMP-9 include growth factors, cytokines, and tumor promoters including MAPKs. Decreased expression of MMP-9 means that cancer invasion and metastasis are suppressed.
<< 실험예Experimental Example 6> 6> NFNF -κB의 면역 조직화학적 관찰Immunohistochemical Observation of -κB
NF-κB의 면역 조직화학적 관찰을 위하여, 상기 실험예 5와 같은 과정으로 수행하였으며 1차 항체는 MMP-9 대신에 항-래빗(rabbit) 1차 항체인 NF-κB(1:200, Cell Signaling Technology Inc, USA)를 이용하였다.For immunohistochemical observation of NF-κB, In the same manner as in Experimental Example 5, the primary antibody was NF-κB (1: 200, Cell Signaling Technology Inc, USA), which is an anti-rabbit primary antibody instead of MMP-9.
그 결과, 아라자임 투여군은 대조군에 비해 NF-κB의 발현양상이 감소하였다(도 6 및 도 7). As a result, the expression of NF-κB was reduced in the arazyme administered group compared to the control group (FIGS. 6 and 7).
NF-κB는 다양한 환경적 인자에 대한 반응으로 활성화되는 유도전사 인자이다. 또한 약물 불응성의 또 다른 영향인자이기도 하다. 이는 사이토카인 합성의 중요한 인자이며, 면역반응이나 염증반응에 관여하는 다양한 유전자들의 발현을 증가시키는 전사인자로 활성화되면 핵 내로 이동하여 염증 유전자의 발현을 증가시킨다. NF-κB의 발현 감소는 암이 억제되고 있는 것을 의미한다. NF-κB is an induced transcription factor that is activated in response to various environmental factors. It is also another factor of drug refusal. It is an important factor in cytokine synthesis, and when activated as a transcription factor that increases the expression of various genes involved in immune or inflammatory reactions, it moves into the nucleus and increases the expression of inflammatory genes. Decreased expression of NF-κB means that cancer is inhibited.
<실험예 7> PCNA의 면역 조직화학적 관찰Experimental Example 7 Immunohistochemical Observation of PCNA
PCNA의 면역 조직화학적 관찰을 위하여, 상기 실험예 5와 같은 과정으로 수행하였으며 1차 항체는 MMP-9 대신에 항-마우스 1차 항체인 PCNA(1:800, Santa Cruz Bio, USA)를 이용하였다. For immunohistochemical observation of PCNA, The same procedure as in
그 결과, 아라자임 투여군은 대조군에 비해 PCNA의 발현양상이 감소하였다(도 8 및 도 9). As a result, the expression pattern of PCNA was reduced in the arazyme administered group compared to the control group (Figs. 8 and 9).
PCNA는 DNA 폴리머라제 델타(polymerase delta)의 보조적인 단백질로, PCNA의 발현은 각질세포의 증식상태, 신경교증, 착색된 피부병변, 폐의 비소세포성암의 조직 병리학적 등급과 관련이 있다고 알려져 왔기에, PCNA의 조직면역형광법은 종양조직에서의 성장에 관여하는 부분이 얼마 만큼인지 알려주는 척도로 작용한다. PCNA의 발현의 감소는 종양조직의 성장이 억제되고 있음을 의미한다.PCNA is a complementary protein of the DNA polymerase delta. PCNA expression has been known to be associated with histopathological grades of keratinocyte proliferation, neurogliosis, pigmented skin lesions and lung non-small cell cancer. Tissue immunofluorescence of PCNA acts as a measure of how much of the tumor tissue is involved in growth. Reduction of the expression of PCNA means that the growth of tumor tissue is inhibited.
<실험예 8> 아라자임의 급성 독성실험Experimental Example 8 Acute Toxicity Test of Arazyme
본 발명의 아라자임에 대한 급성 독성을 알아보기 위하여, 하기와 같은 실험을 수행하였다.In order to determine the acute toxicity for arazyme of the present invention, the following experiment was performed.
위스터(Wistar) 계통의 9주령 특정병원체부재(SPF) 암컷 랫트((주)오리엔트, 대한민국, 서울)에 대해 각각 4마리씩 4개군으로 나누어, 온도 22± 3℃, 습도 55± 10%, 조명 12L/12D 조건의 동물실 내에서 사육하였다. 랫트 입수 후 동물실에서 1 주일간 검역 및 순화과정을 거쳤다. 전 시험기간 동안 폴리카보네이트제 사육상자[240 W X 390 L X 175 H (mm)]에서 5마리 이하씩 수용하였다. 개체식별은 유성 매직펜을 이용한 피부표식법과 사육상자별 개체식별 카드표시법을 이용하였다. 실험 동물용 고형사료(PMI Nutrition International, 미국)는 멸균하였으며, 물은 상수도수를 물병을 이용하여 자유 섭취 시켰다.Four rats of 9-week-old SPF female rats (Orient Co., Ltd., Seoul, Korea) of Wistar system were divided into four groups, with
상기 실시예의 아라자임을 0, 1250, 2500 및 5000 ㎎/㎏ 농도로 주사용 멸균주사용수에 용해하여 10 ㎖/㎏의 용량으로 조재하여 랫트에 경구투여용 존대(zonde)를 이용하여 단회 경구 투여하였다. 일주일간의 순화 기간을 거쳐서 4개의 그룹으로 분리 한 후 10 주령의 랫트에 아라자임 농도별로 투여한 후 일반 증상 관찰을 실시한 후 아라자임의 실질 장기에 대한 독성을 평가하기 위해서 24 시간 후 부검을 실시하였다. Arazyme of the above example was dissolved in sterile injectable water for injection at concentrations of 0, 1250, 2500 and 5000 mg / kg, prepared at a dose of 10 ml / kg, and orally administered to rats using a zonde for oral administration. It was. After a week-long purifying period, the rats were divided into four groups, administered to arazyme concentrations in 10-week-old rats, followed by general symptoms observation, and autopsies were performed 24 hours later to evaluate the toxicity of arazyme to the organs. .
암컷 랫트에 아라자임을 경구 투여한 후 시험물질 투여에 따른 일반증상을 정확하게 관찰하고 독성을 평가하기 위해서 동물의 행동, 외관, 기능에 대한 관찰을 실시하였다. Oral administration of arazyme to female rats was performed to observe the behavior, appearance and function of animals in order to accurately observe the general symptoms and to evaluate toxicity.
실험결과, 아라자임 투여 후 동물의 활동성, 보행, 기질, 경련 등에 있어서는 아무런 이상 증상이 관찰되지 않았으며, 피모 상태, 안구주변, 귀, 생식기, 사지 상태, 꼬리 상태 등의 동물의 외관 관찰 및 호흡 상태, 타액분비, 분변, 구토 등의 기능 관찰에서도 아무런 이상 증상이 관찰되지 않았다. 아라자임 투여 24 시간 후에 부검을 실시하였다. 육안부검 소견에서도 전장기에서 아라자임 투여에 의한 아무런 이상도 관찰되지 않았다. 아라자임 치사랑(LD50)은 5000 ㎎/㎏이상의 고농도인 것으로 판단된다. 현미경학적 관찰에 있어서 간, 심장, 폐 및 췌장 등의 실질 장기에서는 아무런 병리학적 이상 소견이 관찰되지 않았다.As a result, no abnormal symptoms were observed in the activity, gait, temperament and spasms of the animals after arazyme administration, and the appearance and breathing of the animals such as the skin condition, periphery, ears, genitals, limbs, tail, etc. No abnormal symptoms were observed in the observation of status, saliva secretion, feces, and vomiting. An autopsy was performed 24 hours after arazyme administration. In the gross autopsy findings, no abnormalities were observed in the long-term. Arazyme chisarang (LD 50 ) is believed to be a high concentration of 5000 mg / kg or more. Microscopic observations showed no pathological abnormalities in parenchymal organs such as liver, heart, lung and pancreas.
하기에 본 발명의 조성물을 위한 제제예를 예시한다.Examples of preparations for the compositions of the present invention are illustrated below.
<< 제제예Formulation example 1> 약학적 제제의 제조 1> Preparation of Pharmaceutical Formulations
<1-1> <1-1> 산제의Powder 제조 Produce
아라자임 2 g2 g of arazyme
유당 1 g1 g lactose
상기의 성분을 혼합한 후, 기밀포에 충진하여 산제를 제조하였다.After mixing the above components, the airtight cloth was filled to prepare a powder.
<1-2> 정제의 제조<1-2> Preparation of Tablet
아라자임 100 ㎎Arazyme 100 mg
옥수수전분 100 ㎎Corn starch 100 mg
유 당 100 ㎎Lactose 100 mg
스테아린산 마그네슘 2 ㎎2 mg magnesium stearate
상기의 성분을 혼합한 후, 통상의 정제의 제조방법에 따라서 타정하여 정제를 제조하였다.After mixing the above components, tablets were prepared by tableting according to a conventional method for producing tablets.
<1-3> 캡슐제의 제조 <1-3> Preparation of Capsule
아라자임 100 ㎎Arazyme 100 mg
옥수수전분 100 ㎎Corn starch 100 mg
유 당 100 ㎎Lactose 100 mg
스테아린산 마그네슘 2 ㎎2 mg magnesium stearate
상기의 성분을 혼합한 후, 통상의 캡슐제의 제조방법에 따라서 젤라틴 캡슐에 충전하여 캡슐제를 제조하였다.After mixing the above components, the capsule was prepared by filling in gelatin capsules according to the conventional method for producing a capsule.
<1-4> 환의 제조<1-4> Preparation of the ring
아라자임 1 g1 g of arazyme
유당 1.5 gLactose 1.5 g
글리세린 1 g1 g of glycerin
자일리톨 0.5 gXylitol 0.5 g
상기의 성분을 혼합한 후, 통상의 방법에 따라 1환 당 4 g이 되도록 제조하였다.After mixing the above components, it was prepared to be 4 g per ring in a conventional manner.
<1-5> 과립의 제조<1-5> Preparation of Granules
아라자임 150 ㎎
대두추출물 50 ㎎Soy extract 50 mg
포도당 200 ㎎
전분 600 ㎎
상기의 성분을 혼합한 후, 30% 에탄올 100 ㎎을 첨가하여 섭씨 60℃에서 건조하여 과립을 형성한 후 포에 충진하였다.After mixing the above components, 100 mg of 30% ethanol was added and dried at 60 ° C. to form granules, which were then filled into fabrics.
<1-6> 주사액제의 제조<1-6> Preparation of Injection Solution
아라자임 10 ㎍/㎖Arazyme 10 μg / ml
묽은 염산 BP pH 7.6로 될 때까지Dilute hydrochloric acid BP until pH 7.6
주이용 염화나트륨 BP 최대 1 ㎖Main sodium chloride BP up to 1 ml
적당한 용적의 주이용 염화나트륨 BP 중에 아라자임을 용해시키고, 생성된 용액의 pH를 묽은 염산 BP를 이용하여 pH 7.6로 조절하고, 주이용 염화나트륨 BP를 이용하여 용적을 조절하고 충분히 혼합하였다. 용액을 투명 유리로 된 5 ㎖ 타입 I 앰플 중에 충전시키고, 유리를 용해시킴으로써 공기의 상부 격자하에 봉입시키고, 120℃에서 15분 이상 오토클래이브시켜 살균하여 주사액제를 제조하였다.Arazyme was dissolved in a suitable volume of main sodium chloride BP, the pH of the resulting solution was adjusted to pH 7.6 with dilute hydrochloric acid BP, and the volume was adjusted with a main volume of sodium chloride BP and thoroughly mixed. The solution was filled into a 5 ml Type I ampoule made of clear glass, encapsulated under an upper grid of air by dissolving the glass, and sterilized by autoclaving at 120 ° C. for at least 15 minutes to prepare an injection solution.
<< 제제예Formulation example 2> 식품의 제조 2> Manufacture of food
상기 제제예 1의 산제, 정제, 캡슐제, 환 및 과립을 식품에 응용하여도 무방하며, 상기의 아라니콜라 프로테오리티쿠스의 배양액 또는 이로부터 분리된 아라자임을 포함하는 식품들을 다음과 같이 제조하였다.The powders, tablets, capsules, pills, and granules of Formulation Example 1 may be applied to foods, and foods containing the culture solution of aranicola proteoritus or arazyme separated therefrom are prepared as follows. It was.
<2-1> 밀가루 식품의 제조<2-1> Preparation of Flour Food
아라니콜라 프로테오리티쿠스의 배양액 또는 이로부터 분리된 아라자임 0.1 ~ 10.0 중량부를 밀가루에 첨가하고, 이 혼합물을 이용하여 통상의 방법으로 빵, 케이크, 쿠키, 크래커 및 면류를 제조하여 건강 증진용 식품을 제조하였다.Foods for health promotion by adding 0.1 to 10.0 parts by weight of the culture solution of Aranicola proteoriticus or the arazyme separated therefrom to flour, and preparing breads, cakes, cookies, crackers and noodles using the mixture in a conventional manner. Was prepared.
<2-2> <2-2> 스프soup 및 육즙( And juicy ( graviesgravies )의 제조Manufacturing
아라니콜라 프로테오리티쿠스의 배양액 또는 이로부터 분리된 아라자임 0.1 ~ 1.0 중량부를 스프 및 육즙에 첨가하여 통상의 방법으로 건강 증진용 육가공 제품, 면류의 수프 및 육즙을 제조하였다.0.1-1.0 parts by weight of the culture solution of Aranicola proteoriticus or arazyme isolated therefrom was added to soups and broth to prepare meat products for health promotion, soups of noodles and broth in a conventional manner.
<2-3> 그라운드 <2-3> ground 비프(ground beef)의Ground beef 제조 Produce
아라니콜라 프로테오리티쿠스의 배양액 또는 이로부터 분리된 아라자임 10 중량부를 그라운드 비프에 첨가하여 통상의 방법으로 건강 증진용 그라운드 비프를 제조하였다.10 parts by weight of the culture solution of Aranicola proteoricicus or arazyme isolated therefrom was added to the ground beef to prepare ground beef for health promotion in a conventional manner.
<2-4> 유제품(<2-4> dairy products ( dairydairy productsproducts )의 제조Manufacturing
아라니콜라 프로테오리티쿠스의 배양액 또는 이로부터 분리된 아라자임 0.1 ~ 1.0 중량부를 우유에 첨가하고, 상기 우유를 이용하여 통상의 방법으로 버터 및 아이스크림과 같은 다양한 유제품을 제조하였다.0.1-1.0 parts by weight of the culture solution of Aranicola proteoricicus or arazyme isolated therefrom was added to milk, and various dairy products such as butter and ice cream were prepared in a conventional manner using the milk.
<2-5> <2-5> 선식의Linear 제조 Produce
현미, 보리, 찹쌀, 율무를 공지의 방법으로 알파화시켜 건조시킨 것을 배전한 후 분쇄기로 입도 60 메쉬의 분말로 제조하였다.Brown rice, barley, glutinous rice, and yulmu were alphad by a known method, and then dried and roasted to prepare a powder having a particle size of 60 mesh.
검정콩, 검정깨, 들깨도 공지의 방법으로 쪄서 건조시킨 것을 배전한 후 분쇄기로 입도 60 메쉬의 분말로 제조하였다.Black beans, black sesame seeds, and perilla were also steamed and dried by a known method, and then ground to a powder having a particle size of 60 mesh.
아라니콜라 프로테오리티쿠스의 배양액 또는 이로부터 분리된 아라자임을 진공 농축기에서 감압, 농축하고, 분무, 열풍건조기로 건조하여 얻은 건조물을 분쇄기로 입도 60 메쉬로 분쇄하여 건조분말을 얻었다.Araniacola proteoricicus medium or arazyme separated therefrom was decompressed and concentrated in a vacuum concentrator, dried by spraying and drying with a hot air dryer, and then pulverized with a particle size of 60 mesh to obtain a dry powder.
상기에서 제조한 곡물류, 종실류 및 아라니콜라 프로테오리티쿠스의 배양액 또는 이로부터 분리된 아라자임의 건조분말을 다음의 비율로 배합하여 통상의 방법으로 제조하였다.The grains, seeds and the culture solution of Aranicola proteoriticus prepared above or the dry powder of arazyme separated therefrom were combined in the following ratio to prepare a conventional method.
곡물류(현미 30 중량부, 율무 15 중량부, 보리 20 중량부),Cereals (30 parts by weight brown rice, 15 parts by weight brittle, 20 parts by weight of barley),
종실류(들깨 7 중량부, 검정콩 8 중량부, 검정깨 7 중량부),Seeds (7 parts by weight perilla, 8 parts by weight black beans, 7 parts by weight black sesame seeds),
아라니콜라 프로테오리티쿠스의 배양액 또는 이로부터 분리된 아라자임의 건조분말(1 중량부),Aranicola proteoricicus culture or dry powder of arazyme isolated therefrom (1 part by weight),
영지(0.5 중량부),Ganoderma lucidum (0.5 parts by weight),
지황(0.5 중량부)Foxglove (0.5 part by weight)
<< 제제예Formulation example 3> 음료의 제조 3> Manufacture of beverage
아라니콜라 프로테오리티쿠스의 배양액 또는 이로부터 분리된 아라자임을 포함하는 음료를 다음과 같이 제조하였다.A beverage comprising a culture solution of Aranicola proteoricicus or arazyme isolated therefrom was prepared as follows.
<3-1> <3-1> 건강음료의Health drink 제조 Produce
액상과당(0.5 중량부), 올리고당(2 중량부), 설탕(2 중량부), 식염(0.5 중량부), 물(75 중량부)과 같은 부재료와 아라니콜라 프로테오리티쿠스의 배양액 또는 이로부터 분리된 아라자임(0.5 중량부)을 균질하게 배합하여 순간 살균을 한 후, 이를 유리병, 패트병 등 소포장 용기에 포장하여 건강음료를 제조하였다.Substances such as liquid fructose (0.5 parts by weight), oligosaccharides (2 parts by weight), sugar (2 parts by weight), salt (0.5 parts by weight), water (75 parts by weight), and a culture medium of Aranicola proteoricicus or After the separate arazyme (0.5 parts by weight) homogeneously blended for instant sterilization, it was packaged in small packaging containers such as glass bottles and plastic bottles to prepare a healthy beverage.
<3-2> <3-2> 야채쥬스의Vegetable juice 제조 Produce
아라니콜라 프로테오리티쿠스의 배양액 또는 이로부터 분리된 아라자임 0.5 g을 토마토 또는 당근 등의 야채의 쥬스 1,000 ㎖에 가하여 통상의 방법으로 건강 증진용 야채쥬스를 제조하였다.A culture solution of aranicola proteoricicus or 0.5 g of arazyme isolated therefrom was added to 1,000 ml of a vegetable juice such as tomato or carrot to prepare a vegetable juice for health promotion in a conventional manner.
<3-3> <3-3> 과일쥬스의Fruit juice 제조 Produce
아라니콜라 프로테오리티쿠스의 배양액 또는 이로부터 분리된 아라자임 0.1 g을 사과 또는 포도 등의 과일의 쥬스 1,000 ㎖에 가하여 통상의 방법으로 건강 증진용 과일쥬스를 제조하였다.A culture solution of Aranicola proteoricicus or 0.1 g of arazyme isolated therefrom was added to 1,000 ml of fruit juices such as apples or grapes to prepare fruit juice for health promotion in a conventional manner.
상기에서 살펴본 바와 같이, 본 발명의 아라니콜라 프로테오리티쿠스 (Aranicola proteolyticus)가 생산하는 아라자임(arazyme)은 인간 폐암 세포주가 이식된 누드 마우스의 체중을 증가시키고 종양의 크기 및 침윤현상은 감소시키며, MMP-9, NF-κB 및 PCNA의 발현양상도 감소시키므로 폐암 예방 및 치료용 약학적 조성물로 유용하게 이용될 수 있다.As described above, arazyme produced by Aranicola proteolyticus of the present invention increases the weight of nude mice transplanted with human lung cancer cell lines and reduces tumor size and invasion. Since it reduces the expression of MMP-9, NF-κB and PCNA, it can be usefully used as a pharmaceutical composition for preventing and treating lung cancer.
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AT07851819T ATE536745T1 (en) | 2006-12-28 | 2007-12-28 | COMPOSITION WITH ARAZYM FOR THE PREVENTION AND TREATMENT OF CANCER |
US12/521,676 US20090317374A1 (en) | 2006-12-28 | 2007-12-28 | Composition containing arazyme for the prevention and treatment of cancer |
PCT/KR2007/006938 WO2008082182A1 (en) | 2006-12-28 | 2007-12-28 | A composition containing arazyme for the prevention and treatment of cancer |
CN2007800514751A CN101663045B (en) | 2006-12-28 | 2007-12-28 | A composition containing arazyme for the prevention and treatment of cancer |
JP2009543953A JP4980429B2 (en) | 2006-12-28 | 2007-12-28 | Cancer prevention and treatment composition comprising arazyme as active ingredient |
EP07851819A EP2114431B1 (en) | 2006-12-28 | 2007-12-28 | A composition containing arazyme for the prevention and treatment of cancer |
US13/225,317 US8399223B2 (en) | 2006-12-28 | 2011-09-02 | Composition containing arazyme for the prevention and treatment of cancer |
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KR100879962B1 (en) | 2000-02-03 | 2009-01-23 | 한국생명공학연구원 | Digestive enzyme composition, thrombolytic agent, food solubilizing agent, food softening agent, meat modifier, aniaml feed additive, food additive and oil/fat seperator containing protease |
KR100887334B1 (en) | 2007-07-30 | 2009-03-06 | 한국생명공학연구원 | Feed additive for fish or crustacean breeding containing arazyme as effective ingredient |
KR100909998B1 (en) | 2007-03-29 | 2009-07-29 | 주식회사 인섹트 바이오텍 | Pharmaceutical composition for breast cancer prevention and treatment with arazyme as an active ingredient |
KR100945960B1 (en) | 2008-01-11 | 2010-03-05 | 주식회사 인섹트 바이오텍 | A composition containing Arazyme for the prevention and treatment of arthritis |
KR101191599B1 (en) | 2011-01-03 | 2012-10-15 | 제주대학교 산학협력단 | Composition comprising Polyopes lancifolia Harvey kawaguchi et wang extract for protecting cell or tissue against radiation |
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KR101540319B1 (en) * | 2013-10-01 | 2015-07-30 | 한국생명공학연구원 | Pharmacological composition containing a cyb5R3 protein or a polynucleotide encoding cyb5R3 for the prevention and treatment of cancer |
KR102320280B1 (en) * | 2018-05-31 | 2021-11-29 | 주식회사 센트릭스바이오 | Pharmaceutical composition comprising inhibitors of CD300c's expression or activation for preventing or treating of cancer |
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KR19980069206A (en) * | 1997-02-27 | 1998-10-26 | 박원훈 | Novel Microorganisms Isolated from the Intestines of Korean Sugarless Spiders and Proteolytic Enzymes Produced therefrom |
KR20010077591A (en) * | 2000-02-03 | 2001-08-20 | 복성해 | A novel metalloprotease and a gene thereof derived from Aranicola proteolyticus |
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Cited By (5)
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KR100879962B1 (en) | 2000-02-03 | 2009-01-23 | 한국생명공학연구원 | Digestive enzyme composition, thrombolytic agent, food solubilizing agent, food softening agent, meat modifier, aniaml feed additive, food additive and oil/fat seperator containing protease |
KR100909998B1 (en) | 2007-03-29 | 2009-07-29 | 주식회사 인섹트 바이오텍 | Pharmaceutical composition for breast cancer prevention and treatment with arazyme as an active ingredient |
KR100887334B1 (en) | 2007-07-30 | 2009-03-06 | 한국생명공학연구원 | Feed additive for fish or crustacean breeding containing arazyme as effective ingredient |
KR100945960B1 (en) | 2008-01-11 | 2010-03-05 | 주식회사 인섹트 바이오텍 | A composition containing Arazyme for the prevention and treatment of arthritis |
KR101191599B1 (en) | 2011-01-03 | 2012-10-15 | 제주대학교 산학협력단 | Composition comprising Polyopes lancifolia Harvey kawaguchi et wang extract for protecting cell or tissue against radiation |
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