眼角膜與脈絡膜之抗新生血管治療研究

Studies in anti-angiogenesis therapy of cornea and choroid

吳佩昌(Pei-Chang Wu)

指導教授 ： 辛錫璋

共同指導教授 ： 戴明泓(Ming-Hong Tai)

高雄醫學大學/醫學院/醫學研究所/博士(2008年)

https://doi.org/10.6832/KMU.2008.00041

摘要

新生血管是一個重要的生理過程，如胎兒的生長、女性的生殖週期及傷口的癒合。未控制的新生血管會導致多種病變，如腫瘤的生長及轉移、發炎、退化及眼科的疾病。新生血管的實驗方法包括測試刺激及抑制劑，而且需要體外及活體實驗來測試有效性。本研究針對兩種不同機轉之抗新生血管藥物，一為未上市之抗新生血管重組蛋白vasostatin，及另一為上市藥物-癌思停(AvastinTM)在於抑制眼科新生血管之效果及其相關之藥物動力學。
首先，我們研究新的重組蛋白vasostatin在體外及活體抑制新生血管的效果，在體外實驗，重組蛋白vasostatin在不論血管生長因子存在與否都能顯著抑制內皮細胞的生長，顯示其主要抑制新生血管的機轉是抑制血管內皮細胞的生長，在活體實驗方面，成功的建立大白鼠角膜新生血管的動物模式，發現不論是直接植入vasostatin或點vasostatin眼藥水，都可以有效抑制大白鼠角膜新生血管，其眼藥水的劑量效應在0.1ug/ml可以相當有效的抑制角膜新生血管。接續研究Vasostatin眼藥水的眼內藥物動力學，來測試是否能夠到達眼睛內部，而是否可能治療眼睛後極部的新生血管，以質譜儀液態層析、基質輔助雷射脫附游離飛行時間質譜儀分析前房液，及西方點漬法分析眼角膜及視網膜組織，結果發現vasostatin眼藥水無法在兔眼的前房液及玻璃體液偵測到，但在西方點漬法卻可以在視網膜偵測到vasostatin 的訊號，顯示vasostatin眼藥水可能經由結膜鞏膜眼外血管而到視網膜，有潛力去抑制黃斑新生血管。
高度近視黃斑新生血管出血是導致青壯年人口失明的主因，高度近視定義為高於600度以上，在高度近視的病人之中，約有百分之五到十會有脈絡膜新生血管。雖然發現vasostatin抗新生血管效果相當好，但臨床上仍為上市，無法使用，於是使用已上市的重組蛋白VEGF抗體-癌思停(AvastinTM) 對於高度近視之黃斑脈絡膜新生血管的患者眼內注射，評估六個月的療效，使用癌思停眼內注射六個月的追蹤可以發現視力顯著地從0.26進步到0.95 (P=0.02)，而且網膜厚度也從264 um恢復到223 um。接續研究癌思停在4℃冰箱六個月的安全性、穩定性及有效性，以便臨床上的應用，癌思停藥水冰在4℃冰箱六個月，細菌培養檢測其安全性，並使用質譜儀分析穩定性，再使用VEGF與之結合來鑑定其有效性，結果發現癌思停藥水冰在4℃冰箱六個月並沒有微生物污染，而且六個月內的有效性仍達到90%以上。
本研究的結果顯示Vasostatin能有效率的抑制眼角膜新生血管，但是不能經由前房液或玻璃體液到達視網膜，可能經由其他路徑到達視網膜，如結膜或鞏膜血管，未來在治療眼部的新生血管相當有潛力。在高度近視併發症中黃斑脈絡膜新生血管的患者，以癌思停眼內注射效果相當好，是目前最佳的治療方法，在臨床應用上，癌思停藥水可以安全的存放在4℃冰箱六個月其效果仍然存在。

關鍵字

眼角膜 ； 脈絡膜 ； 新生血管 ； 抗新生血管 ； 癌思停 ； 高度近視

並列摘要

Angiogenesis, the formation of new blood vessels, is an important part for normal processes such as fetal development, female reproductive cycle, and tissue repair. In contrast, uncontrolled angiogenesis promotes numerous pathologies ranging from tumor growth and metastasis to inflammation and ocular diseases. Angiogenesis assays are used to test efficacy of both pro and antiangiogenic agents. Most studies of angiogenesis inducers and inhibitors rely on various models both in vitro and in vivo as indicators of efficacy.
We use these models to investigate a new recombinant vasostatin protein in anti-angiogenesis. Recombinant protein (vasostain) was studied on endothelial cells proliferation, the inhibitory effect of rat corneal neovascularization. Recombinant protein vasostatin potently inhibited the growth of endothelial cells in the absence or presence of angiogenic factors such as bFGF or VEGF. In the rat corneal micropocket assay, concurrent incorporation of vasostatin abolished the bFGF induced neovascularization. Topical application of VS eye drops potently inhibited bFGF induced neovascularization in rat corneas and there was a dose-dependent manner. We further investigate the pharmacokinetics of topical vasostatin eye drop within the eye. In the studies of pharmacokinetics, liquid chromatography mass spectrometry and matrix-assisted laser desorption time-of-flight mass spectrometry were used for analysis of aqueous humor and vitreous. Western blot analysis of retinal tissue and cornea. The result was that vasostatin eyedrop could not be detected in rabbit aquous and vitreous. The detectable level of vasostatin in retina by Western blot analysis might be through outer eyeball routine such as conjunctival or scleral vessels.
High myopia with choroidal neovascularization is the major cause of blindness in young people. Although vasostatin has good effect on experimental corneal angiogenesis and good potential to inhibit angiogenesis, it is still an experimental protein. We investigate effect of choroical neovascularization (CNV) treated with recombinant anti-VEGF antibody- bevacizumab (AvastinTM) in high myopic patients. Patients with high myopia macular neovascularization were treated with intravitreous injection of avastin. Final vision and lesion were assessed. In myopic CNV treated with avastin, the mean initial VA was 0.26, which significantly improved to 0.95 at the end of follow-up (p＝0.02). The mean foveal thickness was 264 μm before treatment, which significantly decreased to 223 μm at the end of follow-up.For the clinical application, we further investigate the safety, stability and efficacy of bevacizumab (Avastin) stored in the original, repeatedly pierced vials under 4℃ within 6 months. Avastin was left in the vials refrigerated at 4°C for 1 month through 6months. All samples were prepared for analysis of microbial growth and MALDI-TOF MS (Matrix-assisted laser desorption ionization/time-of-flight mass spectrometry analysis). At each time period, the efficacy of Avastin was represented by measuring concentrations of active VEGF-165 in the mixture of Avastin and VEGF-165 in a determined condition. The results showed that no growth was obtained from any of the avastin samples stored at 4℃ during 6 months. All samples from 1 month to 6 months remained stable in the MALDI-TOF MS analysis. There were no molecular changes among all samples. And it showed less than 10% degradation within 6 months.
Local application of VS could inhibit corneal angiogenesis and possible therapy in macular neovascularization. High myopia with CNV treated with intravitreal bevacizumab is promising. For clinical application, avastin could be stable stored at 4°C remain sterile and effective for at least 6 months.

並列關鍵字

cornea ； choroid ； neovascularization ； anti-angiogenesis ； avastin ； high myopia ； vasostatin

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