17ß-estradiol 應用於腦部缺血治療之奈米控釋劑型研發

Novel rate-controlled nanoparticles of 17ß-estradiolfor cerebral ischemia therapy

鄭鈞任(Chun-Jen Cheng)

指導教授 ： 蔡東榮

高雄醫學大學/藥學院/天然藥物研究所碩士班/碩士(2019年)

摘要

腦部缺血藉由外力傷害或藉由興奮毒物、發炎、自由基過度產生及神經細胞凋亡等內部保護性機制引起腦部傷害。近年來，性荷爾蒙17ß-estradiol被證實具有神經保護及再生的效果。就藥品本身特性而言，其具有高度親脂性以至於在周邊給藥後能穿透血腦屏障並於腦中產生高藥品濃度。然而estradiol無法在腦內維持穩定的濃度。近年來有研究指出，該藥品保護機制與劑量呈現相關。除此之外，estradiol半衰期少於一小時且缺血性中風治療窗僅有四小時。從以上觀點來看，投藥時間與治療劑量是缺血性腦中風在治療上最大的阻礙。因此，本研究設計一具有藥品緩釋效果之奈米粒子以改善以上缺失。
在此使用胎牛血清白蛋白作為奈米粒子之載體，且粒子藉由水包油之乳化技術及運用戊醛對蛋白產生之化學性交叉連結特性來修飾粒子表面。爲獲得最佳處方，乃運用試驗設計法以評估處方變數產生之影響效應。在此選用效應曲面法之中心複合設計，以二變因五階次模式進行評估。其自變因子為水相之酸鹼值及戊醛的添加量；應變因子為藥品含量、粒子大小、粒子多分散係數、粒子表面電荷、藥品包埋率、初始突釋效應、治療窗時間內所釋出藥品百分比、最初十二小時藥品釋出量、百分之五十釋藥量所需時間以及釋出百分之七十五藥量之所需時間。
從以上應變數獲得之多項式套用各模式計算得最佳處方在水相pH值調整至9.98且戊醛添加量為1611.89 µl。該處方所製備之粒子顯示71.17±0.24%的藥量在治療窗內釋出，且在治療窗過後至二十四小時期間其藥品釋出呈現零階次模式。
結果顯示，最佳處方其預期與實際值所獲得結果差異極小亦即相關性良好而足以運用本設計模式詮釋其粒子特性。除此之外，腦部微透析研究亦證實17ß-estradiol之奈米粒子具有穿透血腦屏障的能力且本劑型設計可增加藥品在腦內的濃度並且維持一穩定濃度超過十二小時。

關鍵字

雌激素 ； 腦部缺血 ； 奈米粒子 ； 實驗設計法 ； 微透析

並列摘要

Cerebral ischemia leaded to brain damage caused by damaging and endogenous protective mechanism included excitotoxicity, inflammation, overproduction of free radicals and nerve cell apoptosis. Recently, 17ß-estradiol, a sex hormone, has been demonstrated the neuroprotective and neuroregenerative effect for cerebral ischemia. Base on its lipophilic character, it can penetrate the blood-brain barrier and achieve high central levels after peripheral administration. However, estradiol is poorly retained within the brain. According to recent study, the protective effects of estradiol were dose-dependent. Moreover, estradiol has short half-life (1hr) and the time of therapeutic window for ischemia therapy was 4hr. From these viewpoints, the time of remedy and drug dosage were the obstacles for cerebral ischemia therapy. The purpose of this study was to improve these disadvantages by the nanoparticles with sustained release character.
In our researsh, bovine serum albumin was chosen as the carrier of the nanoparticles and particles were prepared by the oil-in-water emulsion technique and chemical cross-linking with glutaraldehyde. To obtain the optimum formulation, statistical experimental design was applied to evaluate the influence of some formulation variables and a two-factor five-level central composite design of response surface methodology was chosen as the model. The independent variables were the pH value of aqueous phase and the amount of glutaraldehyde. The dependent variables were drug content, particle sizes, polydispersity index, particle zeta potential, drug encapsulation efficiency, initial burst percentage, cumulative release percentage within the time of therapeutic window, cumulative release percentage of the first 12hr, time of 50% drug release and time 75% drug release .
The optimum formulation (when pH value of aqueous phase was 9.98; 5% glutaraldehyde was added to 1611.89 µl. ) was developed from those response equation of each fitted model. The release profiles reveal that 71.17±0.24% drug was released within the time of therapeutic window and then follow therelationship of zero-order release model (from the 4hr to 24hr).
The results demonstrated that the little differences between predicated and actual result of optimum formulation revealed high correlation and the application of two-factor five-level response surface methodology design resulted a useful tool for the optimization of 17ß-estradiol nanoparticles. Besides, the brain microdialysis study demonstrated that 17ß-estradiol nanoparticles with the ability to penetrate blood-brain barrier and obviously increased and maintained drug concentration in the brain over 12hr.

並列關鍵字

17ß ； -estradiol ； cerebral ischemia ； nanoparticle ； statistical experimental design ； microdialysis

參考文獻

[1] R.R. Leker, E. Shohami, Cerebral ischemia and trauma-different etiologies yet similar mechanisms: neuroprotective opportunities, Brain Res. Rev. 39 (2002) 55-73.

[2] M.A. Petty, J.G. Wettstein, Elements of cerebral microvascular ischemia,Brain Res. Rev. 36 (2001) 23-34.

[3] F.X. Guix, I. Uribesalgo, M. Coma, F.J. Munoz, The physiology and pathophysiology of nitric oxide in the brain, Prog. Neurobiol. 76 (2005)126-152.

[4] R.R. Leker, A. Teichner, H. Ovadia, E. Keshet, E. Reinherz, T. Ben-Hur, Expression of endothelial nitric oxide synthase in the ischemic penumbra: relationship to expression of neuronal nitric oxide synthase and vascular

endothelial growth factor, Brain Res. 909 (2001) 1-7.