WO2016208880A1 - 어류의 정액 또는 정소로부터 분리된 dna 단편 혼합물을 포함하는 허혈성 장염의 예방 또는 치료용 조성물 - Google Patents

어류의 정액 또는 정소로부터 분리된 dna 단편 혼합물을 포함하는 허혈성 장염의 예방 또는 치료용 조성물 Download PDF

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WO2016208880A1
WO2016208880A1 PCT/KR2016/005528 KR2016005528W WO2016208880A1 WO 2016208880 A1 WO2016208880 A1 WO 2016208880A1 KR 2016005528 W KR2016005528 W KR 2016005528W WO 2016208880 A1 WO2016208880 A1 WO 2016208880A1
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Prior art keywords
dna fragment
composition
ischemic
growth
treatment
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PCT/KR2016/005528
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English (en)
French (fr)
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김익수
백승걸
김창주
전정원
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주식회사 파마리서치프로덕트
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Priority claimed from KR1020160054666A external-priority patent/KR20170001567A/ko
Application filed by 주식회사 파마리서치프로덕트 filed Critical 주식회사 파마리서치프로덕트
Priority to US15/739,646 priority Critical patent/US11065281B2/en
Priority to EP16814593.6A priority patent/EP3315135B1/en
Priority to JP2017565766A priority patent/JP6530828B2/ja
Priority to CN201680036573.7A priority patent/CN107810007B/zh
Publication of WO2016208880A1 publication Critical patent/WO2016208880A1/ko
Priority to US17/348,279 priority patent/US20210308192A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/52—Sperm; Prostate; Seminal fluid; Leydig cells of testes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/60—Fish, e.g. seahorses; Fish eggs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/20—Pills, tablets, discs, rods
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate

Definitions

  • composition for the prevention or treatment of ischemic growthitis comprising DNA fragment mixture isolated from the semen or testis of fish
  • the present invention relates to a composition for the prevention or treatment of ischemic growth salts comprising DNA fragment mixtures isolated from the semen or testes of fish.
  • Ischemic colitis is the most common form of vascular disease involving the large intestine and accounts for about 50 to 60 ⁇ of all ischemic gastrointestinal diseases (Suh DC et al., 2007). Severe cases: Nonspecific eye symptoms, such as abdominal pain and diarrhea, often improve symptoms before visiting the hospital. When visiting a hospital, hematologic diarrhea and abdominal pain usually improve with main symptoms or conservative treatment.
  • Ischemia In older adults, the mesenteric artery is blocked by thrombosis or embolism, or due to myocardial infarction or heart failure due to contraction of the intestinal artery. , 1981; Hunter GC et al, 1988). Ischemia is divided into three pathologies. The first ischemia only in mucosa and submucosa.
  • Korean Patent Registration No. 0818752 discloses a composition for treating ischemic disease, which contains siRNA that inhibits human FAF1 protein, but no DNA fragment mixture is mentioned.
  • the object of the present invention is to provide a composition for the prevention or treatment of ischemic growth infections comprising DNA fragment mixtures isolated from the semen or testes of fish.
  • the present invention is for the prevention or treatment of ischemic growth infections, including DNA fragment complexes.
  • the DNA fragment mixture may be a DNA fragment mixture isolated from the semen or testis of fish.
  • the fish may be a salmon family.
  • the DNA fragment complex may have a molecular weight of 30 kDa to 2,500 kDa.
  • the DNA fragment complex refers to a DNA corresponding to a living polymer composed of phosphoric acid, four kinds of bases, and 'deoxyribose', and refers to the presence of a fragment having a reduced molecular weight.
  • the fish may be a fish with salmon, preferably salmon or trout, and most preferably salmon.
  • the DNA fragment complex may have a molecular weight of 30 kDa to 2,500 kDa, preferably 40 kDa to 2,000 kDa, most preferably 50 kDa to l, 500 kDa.
  • the present invention also provides a pharmaceutical composition for the prevention or treatment of ischemic enteritis, comprising the DNA fragment complex and the pharmaceutical excipient.
  • the DNA fragment complex is preferably 0.001 to 50 based on the total weight of the total composition. Weight% , More
  • the pharmaceutical composition is oral formulation, external preparation, suppository, such as powder, granule, tablet, capsule, suspension, emulsion, syrup, aerosol, etc.
  • Carriers, excipients, and diluents that may be included in the pharmaceutical composition may be used as lactose, textrose, sucrose, sorbitan, manny, xili, erythritol, malty, Starch, acacia rubber, alginate, gelatin, calce phosphate, calce silicate, celrose, methyl salose, microcrystalline Cellulose, polyvinylpyridone, water, methylhydroxybenzoate,
  • Propyl hydroxybenzoate, talc, magnesium stearate and mineral oil are usually used.
  • diluents or excipients such as layering agents, extenders, binders, wetting agents, disintegrating agents and surfactants are usually used. .
  • compositions for the prevention or treatment of ischemic growthitis comprising DNA fragment complexes according to the present invention are preferably administered orally and by injection.
  • Solid preparations for oral administration include sperm, pills, powders, granules, capsules
  • Such solid preparations are prepared by mixing at least one excipient, for example, starch, carbonate, sucrose or lactose, gelatin, etc., with an extract or compound of the present invention.
  • Lubricants such as talc are also used.
  • Liquid oral preparations for oral use include suspensions, solvents, emulsions, syrups, and other simple diluents, liquid paraffin, and other excipients, for example wetting agents, sweeteners, fragrances, and preservatives. And the like. :
  • Injectables can be formulated in the form of sterile strains in accordance with the usual methods. Preferably, they can be used as sterile strains, dissolved in water for injection (KP).
  • KP water for injection
  • the dosage of the therapeutic composition of the present invention will vary depending on the age, sex, weight, pathology of the disease, the severity of the pathology, the route of administration and the judgment of the prescriber.
  • the DNA fragment complex may be administered at a dose of 2 mg / kg to 25 mg / kg.
  • the DNA fragment complex may be administered at a concentration of 2 mg / kg to 16 mg / kg.
  • the DNA fragment complex may be administered once a day or divided into several times.
  • the therapeutic composition of the present invention can be administered in a variety of routes to mammals such as rats, livestock and humans for the prevention or treatment of ischemic growth infections. All methods of administration can be expected, for example, oral, It may be administered by rectal or intravenous, muscle, subcutaneous, endometrial dura, or cerebrovascular injection.
  • the present invention relates to a composition for the prevention or treatment of ischemic growth salts comprising DNA fragment complexes isolated from the semen or testes of fish.
  • the composition of the present invention has an excellent effect on the prevention or treatment of ischemic growth infections.
  • the composition for preventing or treating ischemic growth inflammation of the present invention was safe even when administered for a long time and almost no side effects.
  • FIG. 2 shows the intestine following administration of DNA fragment complexes in mice with ischemic growth infection
  • FIG. 3 shows the histological changes of the intestine following H & E staining following administration of DNA fragment complexes in rats with ischemic growth infection.
  • (A) is a normal group without ischemic growth infection
  • (B) is a group with ischemic enteritis
  • (C) is a group with 2rag / kg of DNA fragment complex
  • D) represents the group administered 4 mg / kg of the DNA fragment mixture
  • E) represents the group administered 8 mg / kg of the DNA fragment mixture
  • F represents the group administered 16 mg / kg of the DNA fragment mixture.
  • the experimental animals were S / D (Sprague Dawley) male rats aged 7 weeks of age weighing 180g 5g.
  • mice freely consumed solid feed and water, and the humidity ranged from 22 to 24 ° C.
  • Examples 1 to a rat raised on were anesthetized by intraperitoneal injection of Zoletil 50 ®.
  • Incision mouse operation is completed After sealing, the solution was sterilized with 1% iodine solution and then recovered in a thermo-hygrostat.
  • DNA fragment complex molecular weight 50kDa ⁇ 1500kDa
  • DNA fragment mixtures were administered in a dose of 2, 4, 8, 16 mg / kg intraperitoneally at a dose of 500 ⁇ once a day for a total of 21 days, in which the control group and DNA fragments that did not cause ischemic growth infection were used as controls.
  • a group of ischemic growth salts without administration of the compound was used.
  • FIG. 1 it is shown that the ischemic growth inflammation is increased in all the experimental groups except the normal group (A) after induction, and the secondary test results in a decrease in the temperature of the complex depending on the concentration of the DNA fragment complex. In particular.
  • necrosis was increased in the ischemic colon area, and vascular hemorrhage in the intestine was also observed.
  • Necrosis caused by ischemia improved in (C, D, E, and F), and the colorectal shape was similar to that of normal group (A).
  • Example 5 tissues were extracted using mice photographed of intestinal tissues. Some of the tissues were dissolved in 4% paraformaldehyde in 100 mM phosphate buffer. Tissue fixation was carried out by fixation for 24 hours. Afterwards, 70%, 80%, 90% and 100% ethane was added in order to dehydrate and precipitated with xylene, followed by hard paraffin (Leica, USA) solution was used to infiltrate the paraffin into the tissue through a three-step process. After the invasion, the paraffin tissue was prepared using a paraffin slicer (Shandon Finesse 325, Thermo Electron Co.,
  • Example 6-2 Hematoxylin and eosin dyeing
  • Example 6-1 The tissue sections prepared in Example 6-1 were reacted with Mayer's hematoxyline (Sigma, USA) solution for 30 seconds and washed in flowing water for 10 minutes. Afterwards, the tissue sections were reacted for 3 seconds in eosin (Sigma, USA) solution. Dyeing Completed 3 ⁇ 4 tissue was dehydrated and encapsulated in Permount ® (Fischer scientific, USA). H & E stained tissue sections were observed using a microscope to confirm tissue changes.
  • the ischemia-inflammation-induced group (B) was observed to damage tissues including contractions and divisions in the colonic mucosa compared to the normal group (A).
  • D, E, and F showed decreased contraction and division of the colonic mucosa due to ischemia, indicating that the concentration of DNA fragment complexes administered was dependent.
  • Example 6-1 the paraffin-embedded tissue was deposited on xylene to remove paraffin, hydrated, and treated in Bouin's solution (Sigma, USA) for 1 hour. Thereafter, Weigner's iron hematoxylin The solution (Sigma, USA) was treated for 10 minutes and then washed, and Biebrich scarlet-acid fuchsin solution (Sigma, USA) was treated for 2 minutes and washed. The washed tissue was treated with phosphomolybdic-phosphotungstic acid solution (Sigma, USA) for 10 to 15 minutes, and then treated with aniline blue solution (Sigma, USA) for 5 minutes and light green solution for 1 minute, and washed with acetic solution. After treatment with 95% ethanol and xylene, it was sealed with Peraiount ® (Fischer scientific, USA). The operation stained with Mason Trichrome was observed using a microscope and the results are shown in FIG.
  • Example 5 The aim of this study was to determine the expression of apoptosis-related factors in relation to cell necrosis following ischemic growth.
  • the protein was extracted from the colon tissue obtained from the intestine tissue, and then centrifuged to obtain a supernatant, followed by concentration of the protein using Bio-Rad protein quantitative reagent. Quantification yielded 50 proteins.
  • Proteins were purified using nitrocellulose membranes (nitrocellulose membrane).
  • the protein-transferred membranes were blocked with 5% skim milk and the primary antibodies against Bax or Bcl-2 were treated at 4 ° C. for 16-18 hours. At this time, GAPDH antibody was used as a quantitative control group.
  • the primary antibody-treated membrane was washed three times for 10 minutes with TBST solution (Tris-buffered saline with 0.05% tween 20). The washed membrane was placed in a secondary antibody against each primary antibody and the phase was kept for 1 hour at After reaction, the resultant was washed three times with TBST solution for 15 minutes each time.
  • the washed membrane was treated with ECL solution (enhanced chemiluminescence solution) to confirm the protein band. The results are shown in FIG.

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Abstract

본 발명은 어류의 정액 또는 정소로부터 분리된 DNA 단편 혼합물을 포함하는 허혈성 장염의 예방 또는 치료용 조성물에 관한 것으로, 본 발명의 조성물이 허혈성 장염의 예방 또는 치료에 우수한 효과가 있음을 확인하였다.또한, 본 발명의 허혈성 장염 예방 또는 치료용 조성물은 장기간 투여 시에도 안전하고 부작용이 거의 없음을 확인하였다. 이에따라, 본 발명의 허혈성 장염 예방 또는 치료용 조성물을 이용하여 안전하고 부작용이 없으면서 치료 효과가 뛰어난 허혈성 장염 치료제를 개발함으로써 허혈성 장염 치료에 큰 도움을 줄 수 있을 것으로 기대된다.

Description

명세서
발명의명칭:어류의정액또는정소로부터분리된 DNA단편 흔합물을포함하는허혈성장염의 예방또는치료용조성물 기술분야
[1] 본발명은어류의정액또는정소로부터분리된 DNA단편흔합물을포함하는 허혈성장염의예방또는치료용조성물에관한것이다.
배경기술
[2] 산멉사회의발전으로인해각종스트레스와환경적요인에의하여소화기계통 질환의환자가증가하고있는추세이다.소화기계통에서많이 일어나는 질병으로는허혈 (ischemic)이 있으며，이는혈관의수축또는폐색에의해 유발되는신체기관，조직또는부위로의혈액공급의감소상태를말한다.허혈 후에는혈액의재관류 (reperfusion)가일어나면서신경세포가손상되는등 다양한후유증이야기된다.허혈은궁극적으로비가역적인손상，즉세포및 조직의괴사로이루어지면서，허혈성장염과같은질환들이나타날수있다.
[3] 허혈성장염 (ischemic colitis, IC)은대장을침범하는혈관성질환증가장흔한 형태이며전체허혈성위장관질환의 약 50~60< 를차지한다 (Suh D.C. et al., 2007).허혈성장염은:심하지않은경우：복통과설사와같은비특이적안 증상으로대부분병원을찾기전에증상이호전되는경우가많고，병원을찾는 경우에는대개혈성설사와복통이주증상이나보존적치료로호전되는경우가 많다.
[4] 허혈성장염의원인은대부분확인하기어려운경우가많으나，주로
장년층에서장간막동맥이혈전증이나색전증으로막히거나또는심근 경색이나심부전으로내장동맥이수축되어발생한다.장간막정맥혈전증， 혈액의과웅고나저혈압을유발하는약물，혈관염이원인이되기도한다 (Reinus J.F. et al., 1981 ; Hunter G.C. et al, 1988).허혈성장염은병리학적으로크게세 가지로나뉜다.첫번째는점막과점막하조직에만허혈을알으켜
보존치료만으로도회:복이가능한일과성의가역형，두번째는
내측원형근육까지허혈을일으킨만성형，세번째는전벽층에급격한허혈을 일으키는전격형이있다 (Toursarkissian B et al., 1997; Bower T.C., 1993; Deana D.G. et al., 1995).허혈성장염의절반정도는자연치유될수있는일과성의 가역형이므로보존적치료만으로도충분하나나머지절반은장전층괴사나 장천공에이를수있고，이경우사망률이높아수술을염두에둔적극적인 치료가필요하다 (Green B.T. et al. 2005; Jung S.H. et al., 2008).
[5] 이에,본발명자는어류의정액또는정소에서추출한 DNA단편흔합물을
이용하여연구를진행하던중허혈성장염마우스모델에서: DNA단편흔합물이 세포의괴사를억제한다는것을확인함으로써본발명을완성할수있었다. [6] 종래선행기술로서한국등록특허제 0818752호에서는인간 FAF1단백질을 억제하는 siRNA를유효성분으로하는허혈성질환치료용조성물을개시하고 있으나 DNA단편흔합물이언급된바가없다.또한한국공개특허
제 2003-0001370호에서는 EGF활성을갖는펩타이드및성장호르몬분비촉진 핵사펩타이드를이용하여내장허혈을치료할수있음을개시하고있으나본 발명의 DNA단편흔합물의구성과는차이가있다.
발명의상세한설명
기술적과제
[7] 본발명의목적은어류의정액또는정소로부터분리된 DNA단편흔합물을 포함하는허혈성장염의 예방또는치료용조성물을제공하는데있다.
과제해결수단
[8] 본발명은 DNA단편흔합물을포함하는허혈성장염의 예방또는치료용
조성물에관한것이다.
[9] 상기 DNA단편혼합물은어류의정액또는정소로부터분리한 DNA단편 흔합물일수있다.
[10] 상기어류는연어과일수있다.
[11] 상기 DNA단편흔합물은분자량이 30kDa내지 2,500kDa일수있다.
[12] 이하본발명을상세하게설명한다.
[13] 상기 DNA단편흔합물이란,인산, 4종류의염기, ' 데옥시리보오스 (deoxyribose)로이루어진생체고분자에해당하는 DNA로, 분자량이 저감된단편형태로흔합되어존재하는것을지칭한다.
[14] 상기어류는연어과의어류일수있고，바람직하게는연어또는송어일수있고 가장바람직하게는연어일수있다ᅳ
[15] 상기 DNA단편흔합물은분자량이 30kDa내지 2,500kDa일수있고, 40kDa 내지 2,000kDa인것이바람직하며, 50kDa내지 l,500kDa인것이가장
바람직하다.
[16] 또한，본발명은상기 DNA단편흔합물및약제학적부형제를포함하는허혈성 장염의 예방또는치료용약학조성물을제공한다.상기 DNA단편흔합물은 전체조성물총중량에대하여바람직하게는 0.001내지 50중량 %，더
바람직하게는 0.001내지 40중량 %,가장바람직하게는 0.001내지 30중량 %로 하여첨가될수있다. ,
[17] 상기약학조성물은,각각통상의방법에따라산제，과립제，정제，캡슐제， 현탁액，에멀견，시럽，에어로졸등의경구형제형，외용제，좌제및
멸균주사용액의형태로제형화하여사용될수있다.상기 약학조성물에포함될 수있는담체，부형제및희석제로는락토즈，텍스트로즈，수크로스，솔비를， 만니를，자일리를,에리스리틀，말티를，전분，아카시아고무,알지네이트， 젤라틴,칼슴포스페이트,칼슴실리케이트,셀를로즈，메틸샐를로즈，미정질 셀를로스,폴리비닐피를리돈,물,메틸히드록시벤조에이트，
프로필히드록시벤조에이트，탈크，마그네슴스테아레이트및광물유를들수 있다.제제화할경우에는보통사용하는층진제，증량제，결합제，습윤제， 붕해제，계면활성제등의희석제또는부형제를사용하여조제된다.
[18] 본발명에따른 DNA단편흔합물을포함하는허혈성장염의예방또는치료용 조성물은경구및주사제로투여하는것이바람직하다.
[19] 경구투여를위한고형제제에는정게，환제，:산제，과립제，캡슐제둥이
포함되며，이러한고형제제는본발명의추출물또는화합물에적어도하나 이상의부형제,예를들면，전분，탄산칼슴，수크로스또는락토즈，젤라틴등을 섞어조제된다.또한단순한부형제이외에마그네슴스테아레이트，탈크같은 윤활제들도사용된다.경구를위한액상제제로는현탁제，내용액제，유제, 시럽제등이해당되는데흔히사용되는단순희석제인물,리퀴드파라핀이외에 여러가지부형제，예를들면습윤제，감미제，방향제，보존제등이포함될수 있다. :
[20] 주사제는통상의방법에따라멸균주사용액의형태로제형화하여사용할수 있다.바람직하게는주사용수 (대한약전규격)에녹여멸균주사용액으로이용될 수있다.
[21] 본발명의치료용조성물의투여량은치료받을대상의연령，성별，체중， 질환의병리상태，병리상태의심각도,투여경로및처방자의판단에따라 달라질것이다.
[22] 상기 DNA단편흔합물은 2mg/kg내지 25mg/kg을투여할수있다ᅳ바람작하게는 2mg/kg내지 16mg/kg의농도로투여할수있다.투여농도가 2mg/kg미만일 경우에는치료효과를얻기어렵고， 25mg/kg를초과할경우에는투여량대비 치료효과의상승정도가크지않다.
[23] 상기 DNA단편흔합물은 1일 1회또는수회로나누어투여될수있다.
[24] 본발명의치료용조성물은허혈성장염의 예방또는치료를위하여쥐 ,가축및 인간과같은포유동물에다양한경로로투여될수있다.투여의모든방식은 예상될수있는데，예를들면，경구,직장또는정맥,근육，피하，자궁내경막 또는뇌혈관내주사에의해투여될수있다.
발명의효과
[25] 본발명은어류의정액또는정소로부터분리된 DNA단편흔합물을포함하는 허혈성장염의예방또는치료용조성물에관한것으로，본발명의조성물이 허혈성장염의예방또는치료에우수한효과가있음을확인하였다.또한,본 발명의허혈성장염예방또는치료용조성물은장기간투여시에도안전하고 부작용이거의없음을확인하였다.
[26] 이에따라，본발명의허혈성장염예방또는치료용조성물을아용하여
안전하고부작용이없으면서치료효과가뛰어난허혈성장염치료제를 개발함으로써허혈성장염치료에큰도움을줄수있을것으로기대된다. 도면의간단한설명
[27] 도 1은허혈성장염의발병에따른환부의온도변화를보여주고있다.
[28] 도 2는허혈성장염이유발된쥐에서 DNA단편흔합물의투여에따른장
조직의변화를육안으로관찰한결과를보여주고있다.
[29] 도 3은허혈성장염이유발된쥐에서 DNA단편흔합물의투여에따른장의 조직학적변화를 H&E염색을통해보여주고있다.
[30] 도 4는하혈성장염이유발된쥐에서 DNA단편흔합물의투여에따른대장 점막의분열을메이슨트리크름염색을통해보여주고있다.
[31] 도 5는허혈성장염이유발된쥐에서 DNA단편흔합물의투여에따른
세포사멸관련인자의단백질발현을보여주고있다.
[32] 각각의도면에서 (A)는허혈성장염이유발되지않은정상군을, (B)는허혈성 장염이유발된군을， (C)는 DNA단편흔합물을 2rag/kg투여한군을， (D)는 DNA 단편흔합물을 4mg/kg투여한군을, (E)는 DNA단편혼합물을 8mg/kg투여한 군을， (F)는 DNA단편흔합물을 16mg/kg투여한군을나타낸다.
발명의실시를위한최선의형태
[33] 이하본발명의바람직한실시예를상세히설명하기로한다.그러나，본발명은 여기서설명되는실시예에한정되지않고다른형태로구체화될수도있다. 오히려，여기서소개되는내용이철저하고완전해지고，당업자에게본발명의 사상을충분히전달하기위해제공하는것이다.
[34] <실시예 1.실험동물의준비 >
[35] 실험동물은 180土 5g무게의 7주령이된 S/D(Sprague Dawley)계열수컷흰쥐를 이용하였다.
[36] 모든쥐는고형사료와물을자유롭게섭취하였으며， 22~24°C에서습도가
60%가되도록유지하며，밤낮주기가 12시간간격으로조절되는실험실 환경에서사육하였다.사육한쥐는하기표 1의실험군으로나누어，실험군당 10마리씩사용하였다.
[37]
Figure imgf000006_0001
[38] <실시예 2.허혈성장염동물모델의제조 >
[39] 실시예 1에서사육한쥐를 Zoletil 50®을복강주사하여마취시켰다.마취한 쥐의복부중앙을절개하여개복한후하행결장 4cm범위의주변부혈관을 결찰시켰다.수술이끝난쥐는절개부분을봉합한뒤 1%요오드액으로소독한 후항온항습장치내에서회복시켰다.
[40] <실시예 3.약물투여>
[41] 실시예 2의허혈성장염동물모델에본발명의 DNA단편흔합물을투여하여 허혈성장염의치료효과를확인하였다. '
[42] DNA단편흔합물 (분자량 50kDa~1500kDa)의투여는허혈성장염유발수술이 끝난후 48시간이후부터실시하였다. DNA단편흔합물은 2, 4， 8, 16mg/kg의 농도로 500 ^씩복강에 1일 1회로총 21일동안주사하여투여하였다.이때 대조군으로는허혈성장염을유발하지않은정상군과 DNA단편흔합물을 투여하지않은허혈성장염모델군을이용하였다ᅳ
[43] <실시예 4.환부의은도측정 >
[44] 허혈성장염유발의확인은염증이 일어난환부에서열이증가되는것에
기인하여레이저온도계 (MT6, Raytek Co., CA. US)를이용하여허혈성장염유발 전과유발후 1주단위로 3번총 4회실시하였고,그결과를도 1에나타내었다.
[45] 도 1을살펴보면，허혈성장염을유발후의환부온도가정상군 (A)을제외한 모든실험군에서중가하는:것으로나타났으며， 2차검사에사는 DNA단편 흔합물농도의존적으로환부온도가낮아지는것으로나타났다.특히나
16mg/kg의 DNA단편흔합물을투여한군 (F)에서가장높은감소효과를보였다. DNA단편흔합물투여 3주후와측정검사에서도 DNA단편흔합물을투여한 집단의환부은도가통계적으로의미 있게감소하는것올확인하였다ᅳ
[46] <실시예 5.장관찰 >
[47] DNA단편흔합물투여 21일후에쥐를 lOmg/kg의 Zoletil 50®을복강내
주사하여마취시킨후복강을열고허혈성장염을유발시킨장조직을노출시킨 후동일한각도와거리에서장조직을촬영하였고그결과를도 2에나타내었다.
[48] 도 2에서보여주듯이,허혈성장염을유발시킨군 (B)의경우에는허혈시킨 대장부위에괴사가증가되는것으로나타났으며，장외부의혈관출혈도 관찰되었다.반면 DNA단편흔합물을투여한경우 (C, D, E, F)에는허혈로인한 괴사가호전되어정상군 (A)의상태와비슷한대장형태를유지하고있음을알수 있었다.
[49] 이를통해， DNA단편흔합물의투여가허혈성장염으로유발된대장부위의 괴사를억제또는치료할수있음을알수있었다.
[50] <실사예 6.조직염색>
[51] 실시예 6-1.조직처리
[52] 실시예 5에서장조직을촬영한쥐를이용하여조직을적출하였다.이중일부 조직을 lOOmM인산완충액에녹인 4%파라포름알데히드 (paraformaldehyde) 고정액에 24시간동안넣어조직고정을실시하였디-.이후 70%， 80%, 90%및 100%에탄을에순서대로넣어탈수분시켰으며자일렌 (xylene)으로침착시킨후 경질파라핀 (Leica, USA)용액을이용하여 3단계의과정을거쳐조직에파라핀을 침습시켰다.침습이끝난조직은절편을제작하기위해블록을만들고고정된 파라핀조직을파라핀절편기 (Shandon Finesse 325, Thermo Electron Co.,
England)를이용하여 5； 두께로절편을조직하였다.조직절편을코팅
슬라이드에붙인후 370C슬라이드오븐에서 16-18시간방치하였다.
[53] 실시예 6-2. H&E(Hematoxylin and eosin)염색
[54] 실시예 6-1에서제작한조직절편을 Mayer's hematoxyline(Sigma, USA)용액에 30초간반응시킨후，흐르는물에서 10분간세척하였다.이후 eosin(Sigma, USA) 용액에 3초간반웅시켰다.염색아완료 ¾조직은탈수과정을거쳐 Permount® (Fischer scientific, USA)로봉입하였다. H&E염색한조직절편을현미경을 이용하여관찰하여，조직의변화를확인하였고，이를도 3에나타내었다.
[55] 도 3에서보여주듯이，허혈성장염유발군 (B)은정상군 (A)에 비해대장내점막 부위에서웅축과분열을비롯한조직의손상이관찰되었다.그러나 DNA단편 흔합물을투여한군 (C, D, E, F)에서는허혈로인해야기된대장내점막의웅축 및분열이감소되는것으로나타났으며，이는투여한 DNA단편흔합물농도 의존적인것을알수있었다.
[56] 실시예 6-3.메이슨트리크롬 (Masson's trichrome)염색
[57] 허혈성장염유발후 DNA단편흔합물투여에따른대장점막의분열정도를 확인하기위한방법으로총콜라겐섬유 (total collagen fiber)의발현을확인하고자 하였다.이에따라，메이슨트리크름염색을실시하였다.
[58] 실시예 6-1에서파라핀에포매된조직을자일렌 (xylene)에침착시켜파라핀을 제거하고，수화시킨후 Bouin's용액 (Sigma, USA)에서 1시간동안처리하였다.그 후 Weigner's iron hematoxylin용액 (Sigma, USA)에 10분간처리한후세척하고， Biebrich scarlet-acid fuchsin용액 (Sigma, USA)에 2분간처리하여세척하였다. 세척한조직을 phosphomolybdic-phosphotungstic acid용액 (Sigma, USA)어】 10~15분간처리한후 aniline blue용액 (Sigma, USA)에 5분간, light green용액에 1분간처리하고세척한후 acetic용액에 5분간처리후 95%에탄올과자일렌으로 처리한후 Peraiount®(Fischer scientific, USA)로봉입하였다.메이슨트리크롬으로 염색한조작을현미경을이용하여관찰하였고，그결과를도 4에나타내었다.
[59] 도 4에서보여주듯이，정상군 (A)에비해허혈상장염유발군 (B)은허혈로인해 대장점막의분열이증가된반면에， DNA단편흔합물을투여한군 (C, D, E,
F)에서는허혈로인해야기된대장점막의분열이 DNA단편흔합물농도 의존적으로감소하는것을관찰할수있었다ᅳ
[60] <실시예 7.세포사멸관련인자의단백질발현확인 >
[61] 허혈성장염에따른세포괴사와관련하여세포사멸관련인자의발현정도를 확인하고자하였다ᅳ [62] 실시예 5에서장조직을촬영한쥐로부터적출한대장조직을용해버퍼 (lysis buffer)로단백질을추출한후원심분리하여상층액을얻은뒤 Bio-Rad의단백질 정량시약을사용해단백질농도를정량하여 50 의단백질을얻었다.
[63] 상기에서확보한단백질 50 zg을 10% SDS-PAGE로전기영동시킨후
니트로샐를로스멤브레인 (nitrocellulose membrane)으로단백질을
트랜스퍼 (tranfer)시켰다.단백질이트랜스퍼된멤브레인을 5%탈지분유 (skim milk)로블로킹 (blocking)시킨다음 Bax또는 Bcl-2에대항하는일차항체를 4°C에서 16~18시간동안처리하였다.이때정량대조군으로 GAPDH항체를 이용하였다. 1차항체를처리한멤브레인을 TBST용액 (Tris-buffered saline with 0.05% tween 20)으로 10분씩 3회세척하였다.세척한멤브레인에각각의일차 항체에대항하는 2차항체를넣고상은에서 1시간동안반웅시킨후다시 TBST 용액으로 15분씩 3회세척하였다.세척한멤브레인에 ECL용액 (enhanced chemiluminescence solution)을처리하여단백질밴드를확인하였다.그결과를도 5에나타내었다.
[64] 도 5에서보여주듯이，세포사멸인자 (apoptotic factor)인 Bax의경우정상군 (A)에 비해허혈성장염이유발된군 (B)에서그발현이증가하는것을관찰하였다. 2mg/kg및 4mg/kg의 DNA단편흔합물을투여한경우에는 Bax의발현이 증가하였으나， 8mg/kg및 16mg/kg의 DNA단편흔합물을투여한경우에는농도 의존적으로 Bax의발현이감소하는것을알수있었다.반면，
항세포사멸인자 (anti-apoptotic factor)인 Bcl-2의경우에는허혈성장염
유발군 (B)와비교하였을때, DNA단편흔합물을투여한군 (C, D, E, F)에서 발현이증가하는것을알수있었다.
[65] 이를통해, DNA단편혼합물이허혈성장염이발생한부위의세포괴사를 억제함으로써허혈성장염을치료할수있음을예측할수있었다.

Claims

청구범위
[청구항 1] DNA단편흔합물을유효성분으로포함하는허혈성장염의 예방또는 치료용조성물.
[청구항 2] 제 1항에 있어서，
상기 DNA단편흔합물은어류의정액또는정소로부터분리한것을 특징으로하는허혈성장염의 예방또는치료용조성물.
[청구항 3] 제 2항에 있어서,
상기어류는연어과인것을특징으로하는허혈성장염의 예방또는 치료용조성물.
[청구항 4] 제 i항내지제 2항의어느한항에있어서，
상기 DNA단편흔합물은분자량이 30kDa내지 2,500kDa인것을 특징으로하는허혈성장염의 예방또는치료용조성물.
[청구항 5] 제 4항에있어서,
상기 DNA단편흔합물은분자량이 40kDa내지 2,000kDa인것을 특징으로하는허혈성장염의예방또는치료용조성물.
[청구항 6] 제 5항에있어서;
상기 DNA단편흔합물은분자량이 50kDa내지 l,500kDa인것을 특징으로하는허혈성장염의 예방또는치료용조성물.
PCT/KR2016/005528 2015-06-26 2016-05-25 어류의 정액 또는 정소로부터 분리된 dna 단편 혼합물을 포함하는 허혈성 장염의 예방 또는 치료용 조성물 WO2016208880A1 (ko)

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US15/739,646 US11065281B2 (en) 2015-06-26 2016-05-25 Composition for preventing or treating ischemic enteritis containing DNA fragment mixture isolated from sperm or testis of fish
EP16814593.6A EP3315135B1 (en) 2015-06-26 2016-05-25 Composition for preventing or treating ischemic enteritis containing dna fragment mixture isolated from sperm or testis of fish
JP2017565766A JP6530828B2 (ja) 2015-06-26 2016-05-25 魚類の精液または精巣から分離されたｄｎａ断片混合物を含む虚血性腸炎の予防または治療用組成物
CN201680036573.7A CN107810007B (zh) 2015-06-26 2016-05-25 包含从鱼类的精液或精巢分离出来的dna片段混合物的缺血性肠炎的预防或治疗用组合物
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