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Anti-fibrotic Effects of New Transforming Growth Factor β1 Type I Receptor Kinase (ALK5) Inhibitors on Hepatic Fibrosis and Pulmonary Fibrosis

Title
Anti-fibrotic Effects of New Transforming Growth Factor β1 Type I Receptor Kinase (ALK5) Inhibitors on Hepatic Fibrosis and Pulmonary Fibrosis
Authors
이미연
Issue Date
2004
Department/Major
대학원 약학과
Publisher
이화여자대학교 대학원
Degree
Master
Advisors
김화정
Abstract
Transforming growth factor β1 (TGF-β1) 는 다양한 생물학적 활동을 조절하는 cytokine으로 세포 외 기질의 축적으로 발생되는 조직 경화증의 주요 조절인자로 알려져 있다. 경화증은 간, 폐, 신장 등에서 수십 년에 걸쳐 진행, 유발된다. 따라서 병적인 상태에서 나타나는 TGF-β1의 과잉 신호전달 체계를 억제할 수 있는 약물의 개발이 시급하나 실제로 아직까지 실험 단계에 놓여져 있다. 본 연구에서는 TGF-β1의 type I 수용체인 ALK5를 억제하는 기전으로 경화증을 치료하는 새롭게 합성된 IN-1139과 IN-1130·HCl의 효과를 간과 폐 경화 동물 모델에서 테스트 하였다. 또한 최근에 같은 기전으로 경화증을 치료한다고 보고된 SB-431542와 SB-505124·HCl를 비교약물로 사용하였다. 우선, 간 경화증을 동물 모델에서 유발시키기 위해 담관결찰술을 수컷 rats에 시행하고, 폐 경화증을 동물 모델에서 유발시키기 위해 블레오마이신을 수컷 mice의 기관지에 삽입하는 시술을 시행하였다. Sham-operated 동물은 수술 후 4주 후에 모두 생존하였으나, 경화증을 유발시킨 rats과 mice의 생존율은 각각 64%와 72%로 감소하였다. 경화증의 정도 (degree) 는 hydroxyproline와 β-smooth muscle actin (β-SMA)를 지표로 하여 측정하였다. 담관결찰술이 시행된 뒤 8주 후에 얻어진 rats의 간에서는 외관상 micronodular cirrhosis의 특징이 관찰되었고, hydroxyproline와 β-smooth muscle actin (β-SMA) 발현도 유의적으로 증가하였다. 블레오마이신을 기관지에 삽입하는 시술을 시행한 뒤 8주 후에 얻어진 mouse의 폐에서는 외관상 interstitial fibrosis의 특징이 관찰되었고, 폐 fibroblast activation의 지표인 β-SMA 발현도 유의적으로 증가하였다. 다음으로, 새로 개발된 IN-1139와 IN-1130·HCl의 효과를 간과 폐 경화증 동물 모델에서 테스트 하였다. 담관결찰술을시행하고 블레오마이신을 삽입하여 만들어진 동물 모델에서 4주간 경화증을 유발시킨 후, 다음 4주간 일주일에 3번씩 약물을 투여하였다. 간 경화증 동물 모델에 IN-1139를 투여하였더니 생존율이 약물 투여 후 3주째 까지는 개선되었으나, 4주째에는 약물을 투여하지 않은 간 경화증 동물 모델과 비슷해졌다. IN-1139와 SB-431542를 4주간 투여한 간 경화증 모델 rats의 hydroxyproline와 β-SMA 발현은 비슷하게 감소되었고, 이는 이 두 약물이 콜라겐의 생성과 hepatic stellate cell (HSC) 의 활성화 억제에 효과가 있음을 나타낸다. 폐 경화증 동물 모델에서는 IN-1139 (20 mg/kg)를 투여하였더니 생존율은 약물을 투여하지 않은 폐 경화증 동물 모델과 비교해 보았을 때 (55%), 67%로 증가하였다. Connective tissue growth factor (CTGF) mRNA와 β-SMA 단백질 발현 정도는 투여된 약물의 농도 의존적으로 개선되었다. IN-1130·HCl과 SB-505124·HCl는 IN-1139와 SB-431542와는 달리 수용성이 높은 약물이다. 간 경화증 모델에서 IN-1130·HCl (30 mg/kg)를 4주간 투여한 rats의 생존율은 약물을 투여하지 않은 간 경화증 모델의 생존율인 48%와 비교하여 60%로 증가하였고, 같은 몰 농도인 SB-505124·HCl 를 12, 24 mg/kg로 4주간 투여한 그룹의 생존율은 각각 37%와 19%에 그쳤다. 담관결찰술에 의해 증가된 hydroxyproline 발현은 IN-1130·HCl 투여에 의해 유의적으로 감소되었다. 또한, 담관결찰술에 의해 증가된 CTGF mRNA 와 β-SMA 발현도 역시 IN-1130·HCl (30 mg/kg) 투여에 의해 감소되었다. Hematoxyline and eosin (H&E) 염색과 β-SMA를 이용한 면역 염색 실험법으로 조직학적인 변화를 관찰한 결과, IN-1130·HCl 투여는 콜라겐의 축적과 β-SMA positive area를 약물을 투여하지 않은 간 경화증 동물 모델에 비하여 감소시켰다. 담관결찰술에 의해 증가된 proliferation cell nuclear antigen (PCNA)의 발현도 IN-1130·HCl과 SB-505124·HCl 투여에 의해 억제되었다. 폐 경화증 동물 모델에서 IN-1130·HCl (15, 30 mg/kg)를 4주간 투여한 mice의 생존율은 83%로 약물을 투여하지 않은 폐 경화증 동물 모델의 생존율인 66%와 비교하여 증가하였다. 반면 SB-505124·HCl를 4주간 투여한 폐 경화증 동물 모델의 생존율은 50%였고, sham operated mice의 생존율도 50%였다. 폐 경화증 유발로 인해 증가된 CTGF와 fibronectin mRNA 발현은 IN-1130·HCl 투여에 의해 유의적으로 감소되었다. 그러나 H&E 염색과 β-SMA를 이용한 면역 염색의 실험법에 의해서는 IN-1130·HCl을 투여한 폐 경화증 동물 모델과 약물을 투여하지 않은 폐 경화증 동물 모델에서의 조직학적인 변화가 뚜렷하게 관찰되지 않았다. 본 연구 결과로부터, ALK5 저해제로 새롭게 합성된 IN-1130·HCl과 대조 약물로 사용된 SB-505124·HCl는 본 연구에서 이용된 약물농도에서 상대적으로 약한 효과를 나타내는 것으로 보인다. 또한 IN-1130·HCl은 최근에 알려진 치료 후보 약물인 SB-505124·HCl보다 더 안전한 것으로 보이므로 조직 경화증 치료에 임상적인 약물로 발전할 가능성이 있는 것으로 사료된다.;Transforming growth factor β1 (TGF-β1) regulates diverse biological activities and is believed to be a key mediator of tissue fibrosis as a consequence of extracelluar matrix accumulation. This cytokine was related to induction of a variety of human fibrotic diseases including liver cirrhosis, lung fibrosis, and diabetic nephropathy. Thus, there is a great interest in development of specific small molecule inhibitors of TGF-β1 signaling to control the pathological states, however, therapeutic agents for fibrosis are still at an experimental stage. The purpose of this study is to investigate anti-fibrotic effects of newly developed small molecule inhibitors (IN-1139 and IN-1130βHCl) of TGF-β1 type I receptor serine/threonine kinase known as an activin-like kinase (ALK) 5 in in vivoanimal models of hepatic and pulmonary fibrosis. SB-431542 and SB-505124 which have recently been reported as selective ALK5 inhibitors were also tested to compare their anti-fibrotic effects to those of IN-1139 and IN-1130·HCl. We induced liver fibrosis by bile duct ligation (BDL) of rats, and pulmonary fibrosis by intratracheal injection of bleomycin to ICR mice. The survival rate of BDL rats and bleomycin injected mice were decreased to 64% and 72%, respectively, whereas all of sham operated animals survived at 4 week after surgery. The degrees of fibrosis were examined by examining organ morphology and detecting hydroxyproline or β-smooth muscle actin (β-SMA) levels. In surviving BDL rats after 8 weeks of BDL surgery, liver fibrosis was represented by the entire granular texture of the liver, a characteristic of micronodular cirrhosis, and significantly increased levels of hydroxylproline and β-SMA expression, indicatives of collagen accumulation and hepatic stellate cell (HSC) activation. Also, in surviving bleomycin injected mice after 4 weeks of surgery, varying degrees of interstitial fibrotic morphology in lung tissue were examined, and the expression of β-SMA, a representative index of lung fibroblast activation, was significantly increased in lung homogenates. Next, the effects of new anti-fibrotic agents were investigated in these in vivo hepatic and lung fibrosis animal models. Drugs were administrated to animals three times per week for next 4 weeks after 4 weeks of BDL surgery or bleomycin treatment. Treatment of IN-1139 showed an improved survival rate of BDL rats during 3 weeks after drug treatment, but produceda similar survival rate at 4 weeks after drug treatment compared with that in vehicle treated BDL rats. Treatment of either IN-1139 or SB-431542 for 4 weeks in surviving BDL rats similarly decreased the levels of liver hydroxyproline and β-SMA expression, representing reduced collagen deposition and HSC activation. In a pulmonary fibrosis model, the survival rate was increased up to 67% in IN-1139 (20 mg/kg) treated bleomycin injected mice compared with that of vehicle treated mice (55%). The levels of connective tissue growth factor (CTGF) mRNA and β-SMA protein enhanced by bleomycin injection were dose-dependently decreased by treatment with IN-1139. Unlike IN-1139 and SB-431542, both IN-1130 and SB-505124 could form salts with an acid, and IN-1130·HCl and SB-505124·HCl are highly soluble in an agents solution (>100 mg/kg). Treatment of IN-1130·HCl (30 mg/kg) improved the survival rate of these rats to 60% compared with 48% in vehicle-treated BDL rats. On the other hand, same molar concentration of SB-505124·HCl (12 and 24 mg/kg) markedly reduced the survival rate to 37% and 19%, respectively. The hydroxylproline level increased by BDL surgery as significantly decreased by treatment of IN-1130·HCl. The increased levels of CTGF mRNA and β-SMA protein expression induced by BDL were also significantly decreased by treatment of IN-1130βHCl. Treatment of IN-1130βHCl can effectively inhibit the liver fibrosis induced by BDL, as further evidenced by improved histological outstanding findings by hematoxyline and eosin staining and β-SMA immunostaining. A proliferation cell nuclear antigen (PCNA) expression was increased by BDL surgery, and it was inhibited by treatment of either SB-505124βHCl or IN-1130βHCl. Treatment of IN-1130·HCl (15 and 30 mg/kg) also improved the survival rate of bleomycin induced lung fibrosis mice model to 83% compared with the vehicle treated control bleomycin injected mice (66%). On the other hand, SB-505124·HCl markedly reduced the survival rate to 50%, which was similar in sham operated control mice group. The levels CTGF and fibronectin mRNA expression were significantly decreased by treatment of IN-1130·HCl. However, no obvious histological changes between drug treated and vehicle treated bleomycin injected mice were observed in hematoxyline and eosin staining and β-SMA immunostaining. These results indicate that both IN-1130·HCl and SB-505124·HCl produce effective anti-fibrotic effects in BDL induced liver fibrosis and less effective effects in lung fibrosis. A novel ALK5 inhibitor IN-1130·HCl appears to be a much safer and effective therapeutic agent than SB-505124·HCl, and has a high potential to be developed as a new anti-fibrotic agent.
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