- 學位論文
比麗明之藥效研究：以體外及其白內障疾病動物模式為藥物活性測試系統
Effects of pirenoxine: Using an in vitro and in vivo cataract animal model as assay systems
摘要
Pirenoxine（Catalin®； Px）於1958年在日本上市，臨床上被用於治療白內障，但仍缺乏足夠證據確定其具療效與否，台灣臨床上至今仍廣泛使用。為了釐清Px藥理與藥效，本研究分別以體外或白內障疾病動物模式進行Px藥效分析。體外實驗部分，利用過量鈣（50 mM）或亞硒酸鈉（10 mM、30 mM、50 mM）所誘發水晶體蛋白質混濁，同時以波長405nm偵測五日混濁度(turbidity)前後變化情形；結果顯示不同濃度之Px（1.6 μM，16 μM，32 μM，80μM）對第五日水晶體蛋白質混濁度皆具劑量依隨效應（dose-dependent）之抑制作用。其中，含不同濃度之Px（0，32、80μM）對鈣誘導之水晶體蛋白混濁度改變情形分別為2.9±0.3、1.9±0.2、0.9±0.1倍；亞硒酸鈉模式中，含有不同濃度Px（0，16、32、80μM ）對亞硒酸鈉（10 mM）所誘導之水晶體蛋白混濁度改變情形分別為8.8 ± 1.4及4.5 ± 1.1、2.2 ± 0.7、0.1 ± 0.3倍；對亞硒酸鈉（30mM）則為4.5±0.6及2.7±0.2、1.9±0.2、1.2±0.1倍；至於亞硒酸鈉（50 mM）則分別為5.7±0.5與3.6±0.4、2.7±0.3、1.5±0.1倍，且具統計上顯著差異（p< 0.05）。前述測試後之水晶體蛋白質經SDS-電泳（SDS-PAGE）分析顯示，各個過量鈣之測試組蛋白質分佈情形無明顯差異，然而亞硒酸鈉組中，含Px實驗組則隨濃度愈高與正常組的結果愈相近。 動物活體試驗則先進行預防給藥模式之測試。首先於出生後第9日之Sprague-Dawley幼鼠每日皮下給予Px（5 mg/ kg）連續11天，在出生後第12日以亞硒酸鈉（19µmole/kg）皮下注射誘導白內障生成後，每日觀察水晶體變化，並依混濁嚴重程度每日記錄其白內障積分（opacity score），於第19日實驗結束時犧牲一部分實驗鼠取其水晶體以SDS-電泳來判別水晶體蛋白質變化情形；結果顯示Px（5 mg/ kg）對白內障生成之進程有延緩效果，但無預防作用且其蛋白質分解情形較控制組輕微。此外，續以藥物治療模式進行測試，進行方式則於兩眼皆形成白內障之實驗鼠連續三日於玻璃體注射（intravitreal）10μl之Px（1mg/ml），觀察注射後每日水晶體混濁程度之變化，結果顯示本研究使用的Px劑量在此白內障疾病動物模式無治療效果。 綜論本研究結果，Px於體外實驗具延緩水晶體蛋白混濁效果，但本研究所使用之疾病動物體劑量僅能在初期延緩病程並無治療成效；臨床使用Px於白內障預防或治療實證醫學，仍需更多研究來證實。
並列摘要
Pirenoxine (Catalin®; Px) was first introduced in 1958 in Japan as an anti-cataractogenic agent. In spite of insufficient evidence, it is still widely used for cataract treatment in Taiwan now. To elucidate the pharmacology and effects of Px, in vitro and in vivo cataract animal model were used to analyze the effects of Px in current study. In in vitro screening system, excess calcium (50mM) or sodium selenite (10, 30, 50mM)-induced lens protein turbidity were established. The possible protective effects of Px in inhibiting or delaying the turbidity formation were determined by daily measurements of the optical density (OD) at 405 nm for 5 consecutive days. All endpoint results showed that various concentrations of Px (1.6μM, 16μM, 32 μM, 80 μM) possesses dose-dependent effect in protecting lens proteins. In calcium-induced lens turbidity system, the changes of OD for of Px (0, 32, 80 μM) treatments, compared to the day 0, were 2.9 ± 0.3, 1.9 ± 0.2, 0.9 ± 0.1 in fold, respectively. As to selenite (10mM)-induced groups, the changes of OD (fold) for Px (0, 16, 32, 80 μM) treatments were 8.8 ± 1.4、4.5 ± 1.1、2.2 ± 0.7、0.1 ± 0.3, respectively. In selenite (30mM)-induced groups, the changes of OD (fold) for test groups of Px (0, 16, 32, 80 μM) were 4.5 ± 0.6, 2.7 ± 0.2, 1.9 ± 0.2, 1.2 ± 0.1, respectively. In selenite (50mM)-induced groups, the changes of OD (fold) for test groups of Px (0, 16, 32, 80 μM) were 5.7 ± 0.5 versus 3.6 ± 0.4, 2.7 ± 0.3, 1.5 ± 0.1 respectively. All above treatment groups showed significant statistically differences compared to the controls without Px incubations (p<0.05). The incubated lens proteins of each group were then analyzed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. There was no difference between control and the Px-treated groups in calcium-induced turbidity system; however, patterns of lens proteins in groups with higher Px resulted in more similar to the normal ones in selenite-induced turbidity system and suggested Px tends to protect lens proteins against conformation changes induced by selenite. The in vivo study was designed to test the preventive use of Px from cataractogenesis. Postnatal day 9 rat pups (Sprague-Dawley) were pretreated with Px (5 mg/kg, subcutaneously) for three days and continuously for the next seven days. After giving overdoses of sodium selenite (19 µmole/kg, subcutaneously) to induce cataract formation at postnatal day 12, the degrees of cataract formation were scored daily until the eye lenses were collected at postnatal day 19 for protein analyzing by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that there was no significant difference in opacity score among the groups at endpoint; however, Px （5 mg/ kg） delayed the progression of cataract formation during the development. SDS-PAGE showed fewer degraded proteins in the rats treated with higher dose of Px. Intravitreal injections of Px (10μl, 1 mg/ml) for 3 consecutive days were then followed to test therapeutic effects of Px against cataract but no curative effect was observed. In conclusion, our in vitro experimental results suggest that Px delays turbidity. Even Px can delay cataract progression at first; nevertheless, the dose used for animal model has no protecting effects. Clinical uses of Px for cataract prevention or treatment still require further investigations.