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Kafe 바로가기주관연구기관 | 전남대학교 산학협력단 Chonnam National University |
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보고서유형 | 2단계보고서 |
발행국가 | 대한민국 |
언어 | 한국어 |
발행년월 | 2014-03 |
과제시작연도 | 2013 |
주관부처 | 미래창조과학부 Ministry of Science, ICT and Future Planning |
과제관리전문기관 | 한국연구재단 National Research Foundation of Korea |
등록번호 | TRKO201500002491 |
과제고유번호 | 1711003654 |
사업명 | 바이오·의료기술개발 |
DB 구축일자 | 2015-05-16 |
키워드 | 바이러스.치료제.동물모델.효능.안전성.검증.산업화.Virus.Medicine.Animal model.Efficacy.Safety.Evaluation.Industrialization. |
DOI | https://doi.org/10.23000/TRKO201500002491 |
1차년도에서는 로타바이러스 치료후보물질의 효능 및 안전성 검증을 위해 동물모델을 확립하였으며, 치료후보물질인 EC를 다양한 농도별로 로타바이러스성 장염이 유발된 동물모델에 투여한 결과 항로타바이러스 효능은 있었지만 실험동물이 폐사하는 등 안전성에 문제가 있었음. 2차년도에서는 SW-100 및 JSC를 로타바이러스성 장염이 유발된 동물모델에 다양한 농도별로 각각 혹은 혼합물 형태로 투여한 결과 SW-100은 치료효과가 없었고, JSC는 로타바이러스성 장염의 개선효과가 있었으며, 이들 혼합물은 로타바이러스성 장염을 완벽히 치료하였음.
1차년도에서는 로타바이러스 치료후보물질의 효능 및 안전성 검증을 위해 동물모델을 확립하였으며, 치료후보물질인 EC를 다양한 농도별로 로타바이러스성 장염이 유발된 동물모델에 투여한 결과 항로타바이러스 효능은 있었지만 실험동물이 폐사하는 등 안전성에 문제가 있었음. 2차년도에서는 SW-100 및 JSC를 로타바이러스성 장염이 유발된 동물모델에 다양한 농도별로 각각 혹은 혼합물 형태로 투여한 결과 SW-100은 치료효과가 없었고, JSC는 로타바이러스성 장염의 개선효과가 있었으며, 이들 혼합물은 로타바이러스성 장염을 완벽히 치료하였음. 3차년도에서는 메탄올 추출물 형태의 KW-200 및 KW-200 분획물을 동물모델에 투여한 결과 완벽한 치료효과가 있었으며 랫트 및 자돈에서 안전성이 높았음. 하지만 KW-200의 주요 성분인 glycyrrhizin은 그 효능이 KW-200에 비해 낮았음. 제4차년도에서는 압착 생추출물 형태의 KW-200을 동물모델에 투여한 결과 완벽한 치료 효과와 랫트와 자돈에서 안전성이 높았음. ST-100 및 JSC 혼합제제에 대한 효능 및 안전성을 재검증한 결과, 제2차년도의 결과와 유사하게 매우 높았음. 또한 메탄올 추출물 형태인 KW-200에 대한 효능을 재검증한 결과 제3차년도와 유사하게 매우 높았음. 제5차년도에서는 KW-200의 송아지에서의 로타바이러스, 코로나바이러스, 로타바이러스와 코로나바이러스 혼합감염에 대한 효능을 검증한 결과 설사 완화효과가 높았음. 이상의 결과 2차년도-5차년도에서 검증된 로타바이러스 및 코로나바이러스 치료 후보물질은 돼지 로타바이러스성 장염 및 소 로타바이러스 및 코로나바이러스의 치료제로 가축에서 바로 활용할 수 있을 것이며, 인간의 로타바이러스 치료제 전임상 연구결과로 사용할 수 있을 것임.
Ⅳ. Results
▣ 1st year
● Development of rotavirus animal model: Before evaluating the efficacy and safety of antirotavirus drug candidates, we established rotavirus animal models with piglets and calves. Col-D piglets obtained from sows by hysterectomy were inoculated with a porcine rotavirus s
Ⅳ. Results
▣ 1st year
● Development of rotavirus animal model: Before evaluating the efficacy and safety of antirotavirus drug candidates, we established rotavirus animal models with piglets and calves. Col-D piglets obtained from sows by hysterectomy were inoculated with a porcine rotavirus strain. Piglets inoculated experimentally with porcine rotavirus showed severe diarrhea, fecal virus shedding, and histological changes in intestine. Col-D calves inoculated experimentally with bovine rotavirus also exhibited severe diarrhea, fecal virus shedding, and histological changes in intestine. These results concluded that the piglets and calves can be useful animal models for evaluating the efficacy and safety of antirotavirus drug candidates.
● Evaluation of efficacy and safety of antirotavirus drug candidate, EC: To evaluate the efficacy and safety of antirotavirus drug candidate, EC, the piglets after induction of rotavirus diarrhea were treated with various concentration of EC drug candidate. Even the piglets treated with drug candidate showed the alleviation of clinical signs, histological changes of intestine, fecal virus shedding, and antigen localization, during experimental period, some piglets died. These results implied that this candidate had toxic effect to piglets so that could not be used for drug due to safety problem.
● Evaluation of virus-host interaction: To evaluate the virus-host interaction, microarray system was used. Significant changes (either increase or decrease) of host mRNA expression levels were detected in many genes from virus inoculated or drug-treated cells. These data indicate that the increase or decrease of host genes can be used as the points acted by drugs.
● Analysis of full-length 11 genomic segments of rotaviruses: In order to analyze full-length 11 genomic segments of rotaviruses to be used for establishing rotavirus animal model, eleven genomic segments of various rotavirus strains were sequenced after RT-PCR and 5' and 3' RACE. Various genotypes and mutations were detected, indicating that different therapeutics might be applied for each different strain even in animals or humans.
▣ 2nd year
● Evaluation of efficacy and safety of SW-100 in animal model as an antirotavirus drug candidate: The piglets treated with SW-100 alone did not show any improvement of diarrhea score, fecal virus shedding, and small intestinal lesion in comparison with those of virus-inoculated and mock-treated piglets. However, this compound did not cause any toxic effects to piglets including the abnormal clinical signs, mortality, histological changes in any organ or tissue, etc.
● Evaluation of efficacy and safety of JSC in animal model as an antirotavirus drug candidate: Dose-dependently, JSC treated groups showed gradual decrease of diarrhea score, and improvement of fecal virus shedding and small intestinal lesion, but did not completely cure diarrhea. Moreover, this candidate did not cause any toxic effects to piglets including the abnormal clinical signs, mortality, histological changes in any organ or tissue, etc.
● Evaluation of efficacy and safety, and synergistic effect by combination therapy of various mixtures of JSC and SW-100 in animal model as an antirotavirus drug candidate: Combination therapy of JSC and SW-100 completely cured diarrhea at post-inoculation day 8. Furthermore, it markedly improved the small intestinal lesion and fecal virus shedding. Moreover, this combination therapy did not cause any toxic effects to piglets including the abnormal clinical signs, mortality, histological changes in any organ or tissue, etc. These data indicate that the combination therapy of JSC and SW-100 can be used to livestock and humans as an anti-rotavirus drug.
▣ 3rd year
● Evaluation of efficacy and safety of methanol-extracted KW-200 in animal model as an antirotavirus drug candidate: At 100 mg/ml and 200 mg/ml concentrations of KW-200, fecal consistency, fecal virus shedding and histological changes of intestinal lesions did not improved in piglets after rotaviral diarrhea induction. Interestingly, 400 mg/ml of KW-200 halted diarrhea at post-inoculation day 8 and markedly improved small intestinal lesion score and fecal virus shedding. Moreover, this candidate did not cause any toxic effects to piglets including the abnormal clinical signs, mortality, histological changes in any organ or tissue, etc.
● Evaluation of anti-proimflammatory efficacy of methanol-extracted KW-200: Decrease levels for mRNA expression for IL-8, IL-10, TNF-α, IFN-β, IFN-γ, NF-kB, p38, and JNK were accounted for those piglets that have received treatment in comparison to the virus-inoculated and mock-treated group.
● Overall, methanol-extracted KW-200 exerted anti-rotavirus effects as well as anti-proimflammatory efficacy. These data implied that KW-200 can be used to livestock and humans as an anti-rotavirus drug.
● Safety assessment of methanol-extracted KW-200 in mice: Compared to mock-treated group, orally or intraperitoneally administrated groups with various concentrations of methanol extracted KW-200 showed any abnormality in body weight, organ weight, blood cells as well as blood chemistries including HCT, MCH, MCHC, MCV, PLT, etc. These data indicate that methanol-extracted KW-200 were safe in mice.
● Safety assessment of methanol-extracted KW-200 in piglets: Compared to mock-treated group, orally or intraperitoneally administrated groups with various concentrations of methanol extracted KW-200 showed any abnormality in body weight, organ weight, blood cells as well as blood chemistries including HCT, MCH, MCHC, MCV, PLT, etc. These data indicate that methanol-extracted KW-200 were safe in piglets.
● Evaluation of efficacy and safety of KW-200 fraction in animal model as an anti-rotavirus drug candidate: At 10 mg/ml and 25 mg/ml concentrations of KW-200 fraction, fecal consistency, fecal virus shedding and histological changes of intestinal lesions did not improved in piglets after rotaviral diarrhea induction. Interestingly, 50 mg/ml of KW-200 fraction halted diarrhea at post-inoculation day 8 and improved small intestinal lesion score and fecal virus shedding.
● Evaluation of anti-proimflammatory efficacy of KW-200 fraction: Decrease levels for mRNA expression for NF-kB, IL8, IFN-β and TNF-α were accounted for those piglets that have received treatment in comparison to the virus-inoculated and mock-treated group. This candidate did not cause any toxic effects to piglets including the abnormal clinical signs, mortality, histological changes in any organ or tissue, etc.
● Evaluation of efficacy and safety of glycyrrhizin in animal model as an anti-rotavirus drug candidate: Compared to mock-treated and virus-inoculated group, glycyrrhizin-administrated group showed any improvement on diarrhea score, fecal virus shedding and histopathological intestinal lesion. These data indicate that glycyrrhizin might not be drug candidate for curring rotavirus enteritis.
● Development of a new animal model with G9 bearing porcine rotavirus strain: Development of a new animal model with G9 bearing porcine rotavirus strain: In order to develop rotavirus animal model with G9 bearing strain, G9P[7] and G9P[23] strains isolated in swine were inoculated into Col-D piglets produced in sows by hysterotomy, and intestinal and extra-intestinal pathogenicity were evaluated. The piglets inoculated with each strain showed typical diarrhea and histological changes in the intestinal and systemic organs, indicating these strains to be valuable to evaluate the efficacy and safety of antirotavirus drug candidates not only for animals but also humans.
▣ 4th year
● Evaluation of efficacy and safety of compression-extracted KW-200 in animal model as an antirotavirus drug candidate: At 100 mg/ml and 200 mg/ml concentrations of KW-200, fecal consistency, fecal virus shedding and histological changes of intestinal lesions did not improved in piglets after rotaviral diarrhea induction. Interestingly, 400 and 600 mg/ml of KW-200 cured diarrhea at post-inoculation day 8 and markedly improved small intestinal lesion score and fecal virus shedding. Moreover, 800 mg/kg shortened cure date to post-inoculation day 6 and improved small intestinal lesion score and fecal virus shedding more than those by lower concentration. In additon, this candidate did not cause any toxic effects to piglets including the abnormal clinical signs, mortality, histological changes in any organ or tissue, etc.
● Evaluation of anti-proimflammatory efficacy of compression-extracted KW-200: Decrease levels for mRNA expression for IL-8, IL-10, TNF-α, IFN-β, IFN-γ, NF-kB, p38, and JNK were accounted for those piglets that have received treatment in comparison to the virus-inoculated and mock-treated group.
● Overall, compression-extracted KW-200 exerted stronger anti-rotavirus effects as well as anti-proimflammatory efficacy than those by methanol-extracted KW-200. These data implied that compression-extracted KW-200 can be used to livestock and humans as an anti-rotavirus drug.
● Safety assessment of compression-extracted KW-200 in mice: Compared to mock-treated group, orally or intraperitoneally administrated groups with various concentrations of compression- extracted KW-200 showed any abnormality in body weight, organ weight, blood cells as well as blood chemistries including HCT, MCH, MCHC, MCV, PLT, etc. These data indicate that like methanol-extracted KW-200, compression-extracted KW-200 were also safe in mice.
● Safety assessment of compression-extracted KW-200 in piglets: Compared to mock-treated group, orally or intraperitoneally administrated groups with various concentrations of compression extracted KW-200 showed any abnormality in body weight, organ weight, blood cells as well as blood chemistries including HCT, MCH, MCHC, MCV, PLT, etc. These data indicate that like methanol-extracted KW-200, compression-extracted KW-200 were safe in piglets.
● Reevaluation of efficacy and safety, and synergistic effect by combination therapy of various mixtures of JSC and SW-100 in animal model as an antirotavirus drug candidate: In order to address the points raised by reviewers, the efficacy and safety of anti-rotavirus drug candidate were reevaluated using Col-D piglets. Combination therapy of JSC and SW-100 completely cured diarrhea at post-inoculation day 8. Furthermore, it markedly improved the small intestinal lesion and fecal virus shedding. Moreover, this combination therapy did not cause any toxic effects to piglets including the abnormal clinical signs, mortality, histological changes in any organ or tissue, etc. These data indicate that like the data shown in the part of 2nd year results, the combination therapy of JSC and SW-100 can be used to livestock and humans as an anti-rotavirus drug.
● Safety assessment of various mixtures of JSC and SW-100 in mice: Compared to mock-treated group, orally or intraperitoneally administrated groups with various concentrations of JSC and SW-100 mixtures showed any abnormality in body weight, organ weight, blood cells as well as blood chemistries including HCT, MCH, MCHC, MCV, PLT, etc. These data indicate that various combinations of JSC and SW-100 were safe in mice.
● Safety assessment of various mixtures of JSC and SW-100 in piglets: Compared to mock-treated group, orally or intraperitoneally administrated groups with various concentrations of JSC and SW-100 mixtures showed any abnormality in body weight, organ weight, blood cells as well as blood chemistries including HCT, MCH, MCHC, MCV, PLT, etc. These data indicate that various combinations of JSC and SW-100 were safe in piglets.
● Reevaluation of efficacy and safety of methanol-extracted KW-200 in animal model as an antirotavirus drug candidate: In order to address the points raised by reviewers, the efficacy and safety of anti-rotavirus drug candidate were reevaluated using Col-D piglets. At 100 mg/ml and 200 mg/ml concentrations of KW-200, fecal consistency, fecal virus shedding and histological changes of intestinal lesions did not improved in piglets after rotaviral diarrhea induction. Interestingly, 400 mg/ml of KW-200 halted diarrhea at post-inoculation day 8 and markedly improved small intestinal lesion score and fecal virus shedding. Moreover, this candidate did not cause any toxic effects to piglets including the abnormal clinical signs, mortality, histological changes in any organ or tissue, etc.
● Revaluation of anti-proimflammatory efficacy of methanol-extracted KW-200: In order to address the points raised by reviewers, the efficacy and safety of anti-rotavirus drug candidate were reevaluated using Col-D piglets. Decrease levels for mRNA expression for IL-8, IL-10, TNF-α, IFN-β, IFN-γ, NF-kB, p38, and JNK were accounted for those piglets that have received treatment in comparison to the virus-inoculated and mock-treated group.
● Overall, the anti-rotavirus efficacy and safety of methanol-extracted KW-200 were also demonstrated in the second trial. Moreover, anti-proimflammatory efficacy was also proved. These data implied that KW-200 can be used to livestock and humans as an anti-rotavirus drug.
▣ 5th year
● Evaluation of efficacy of methanol-extracted KW-200 in calf model as an anti-rotavirus drug candidate: Bovine rotavirus is one of the major enteric pathogens in calves worldwide. To evaluate whether methanol-extracted KW-200 exerted anti-rotavirus efficacy, Col-D calves were administered with methanol-extracted KW-200 after experimental induction of rotavirus diarrhea. Compared to mock-treated but virus-inoculated calves, KW-200 alleviated rotavirus diarrhea at post-inoculation day 7. Moreover, KW-200 obtunded fecal virus shedding and histopathological intestinal lesions. These data indicated that like cure effects in piglets, KW-200 can be used as drug to calves for curing the rotavirus diarrhea.
● Evaluation of efficacy of methanol-extracted KW-200 in calf model as an anti-coronavirus drug candidate: Among the enteric pathogens for calf diarrhea, bovine coronavirus is one of the major enteric pathogens in calves worldwide. To evaluate whether methanol-extracted KW-200 exerted anti-coronavirus efficacy, Col-D calves were administered with methanol-extracted KW-200 after experimental induction of coronavirus diarrhea. Compared to mock-treated but virus-inoculated calves, KW-200 alleviated coronavirus diarrhea at post-inoculation day 7. Moreover, KW-200 obtunded fecal virus shedding and histopathological intestinal lesions. These data indicated that like cure effects for rotavirus diarrhea in calves, KW-200 can be used as drug to calves for curing the coronavirus diarrhea.
● Evaluation of efficacy of methanol-extracted KW-200 in calf model as an anti-rotavirus and anti-coronavirus drug candidate: In the field calves, calf diarrhea can be caused by two more pathogens in particular with coinfection with rotavirus and coronavirus. Therefore, anti-viral medicine should exert its effects to more than 2 viral pathogens. To fulfill this condition, Col-D calves were co-infected with bovine rotavirus and coronavirus. At the point of diarrhea induced by both enteric viruses, methanol-extracted KW-200 were administrated. Accordingly, KW-200 alleviated diarrhea score as well as fecal virus shedding and intestinal lesion score. These data indicate that KW-200 can be used as drug for curing diarrhea due to coinfection of both rotavirus and coronavirus.
● Development of a new animal model with G5P[7] bearing porcine and bovine rotavirus strains: To choose the rotavirus strain which can induce diarrhea to piglets and calves, G5P[7] bearing porcine and bovine rotavirus strains were selected and then inoculated into Col-D piglets and Col-D calves. Porcine G5P[7] strain only induced diarrhea and intestinal and extra-intestinal pathology in piglets but not in calves. However, bovine G5P[7] strain induced diarrhea and intestinal and extra-intestinal pathology in piglets and calves. These data indicate that bovine G5P[7] rotavirus strain is useful for evaluating efficacy and safety of anti-rotavirus drug candidate in piglets and calves.
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