Oka T, Walkup R D, Tamada Y, Nakajima E, Tochigi A, Shearer T R, Azuma M
Kobe Creative Center, Senju Pharmaceutical Co., Ltd., 1-5-4 Murotani, Nishiku, Kobe, Hyogo 651-2241, Japan.
Neuroscience. 2006 Sep 15;141(4):2139-45. doi: 10.1016/j.neuroscience.2006.05.060. Epub 2006 Jul 14.
Our recent study suggested involvement of calpain-induced proteolysis in retinal degeneration and dysfunction in acute ocular hypertensive rats. The purpose of the present study was to determine if an orally available form of calpain inhibitor, ((1S)-1-((((1S)-1-benzyl-3-cyclopropylamino-2,3-di-oxopropyl)amino)carbonyl)-3-methylbutyl)carbamic acid 5-methoxy-3-oxapentyl ester (SNJ-1945), ameliorated retinal degeneration induced by acute hypertension in rats. To help extrapolate the effect of SNJ-1945 from the rat model to the human glaucomatous patient, in vitro inhibition of calpain-induced proteolysis by SNJ-1945 in monkey and human retinal proteins was compared with proteolysis in rat proteins.
Intraocular pressure (IOP) in rats was elevated to 110 mm Hg for 50 min. SNJ-1945 was administrated i.p. or orally before ocular hypertension. Retinal degeneration was evaluated by hematoxylin and eosin (H&E) staining and cell counting. Transcripts for calpains and calpastatin in rat, monkey, and human retinas were measured by quantitative RT-PCR. Calpain activities were determined by casein zymography. Soluble retinal proteins from rat, monkey, and humans were incubated with calcium to activate calpains, with or without SNJ-1945. Proteolysis of calpain substrate alpha-spectrin was analyzed by immunoblotting.
Elevated IOP caused retinal degeneration and proteolysis of alpha-spectrin. Both i.p. and oral administration of SNJ-1945 inhibited proteolysis of alpha-spectrin and ameliorated retinal degeneration. Transcript levels for calpain 1 and calpastatin were similar in rat, monkey, and human retinas. Calpain 2 transcript levels were higher in rats compared with monkey and human. Appreciable caseinolytic activities due to calpains were observed in monkey and human retinas. Incubation of retinal soluble proteins with calcium led to proteolysis of alpha-spectrin due to calpains in rat, monkey, and human samples. SNJ-1945 similarly inhibited proteolysis in all species.
Our results suggested that orally available calpain inhibitor SNJ-1945 might be a possible candidate drug for testing in preventing progression of glaucomatous retinal degeneration.
我们最近的研究表明，钙蛋白酶诱导的蛋白水解参与了急性高眼压大鼠的视网膜变性和功能障碍。本研究的目的是确定一种口服可用的钙蛋白酶抑制剂，即（（1S）-1-（（（1S）-1-苄基-3-环丙基氨基-2,3-二氧代丙基）氨基）羰基）-3-甲基丁基）氨基甲酸5-甲氧基-3-氧杂戊酯（SNJ-1945），是否能改善大鼠急性高血压诱导的视网膜变性。为了帮助将SNJ-1945在大鼠模型中的作用外推至人类青光眼患者，比较了SNJ-1945在猴和人类视网膜蛋白中对钙蛋白酶诱导的蛋白水解的体外抑制作用与在大鼠蛋白中的蛋白水解抑制作用。
将大鼠眼压升高至110 mmHg并持续50分钟。在眼压升高前腹腔注射或口服SNJ-1945。通过苏木精和伊红（H&E）染色及细胞计数评估视网膜变性。通过定量逆转录聚合酶链反应（RT-PCR）测量大鼠、猴和人类视网膜中钙蛋白酶和钙蛋白酶抑制蛋白 的转录本。通过酪蛋白酶谱法测定钙蛋白酶活性。将大鼠、猴和人类的可溶性视网膜蛋白与钙一起孵育以激活钙蛋白酶，有或没有SNJ-1945。通过免疫印迹分析钙蛋白酶底物α-血影蛋白的蛋白水解情况。
眼压升高导致视网膜变性和α-血影蛋白的蛋白水解。腹腔注射和口服SNJ-1945均抑制了α-血影蛋白的蛋白水解并改善了视网膜变性。大鼠、猴和人类视网膜中钙蛋白酶1和钙蛋白酶抑制蛋白的转录水平相似。与猴和人类相比，大鼠中钙蛋白酶2的转录水平更高。在猴和人类视网膜中观察到了由钙蛋白酶引起的明显酪蛋白水解活性。将视网膜可溶性蛋白与钙一起孵育导致大鼠、猴和人类样品中由于钙蛋白酶引起的α-血影蛋白的蛋白水解。SNJ-1945在所有物种中均类似地抑制了蛋白水解。
我们的结果表明，口服可用的钙蛋白酶抑制剂SNJ-1945可能是一种用于测试预防青光眼性视网膜变性进展的候选药物。
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