開發含有雌激素類似物之硫醇乙酸修飾幾丁聚醣用於舒緩陰道萎縮

Abstract

停經後的女性會因為體內雌激素的濃度劇烈下降，而有像熱潮紅、盜汗、失眠的全身性的症狀，以及陰道萎縮、乾燥搔癢等局部不適的問題，除身體不舒服外還會嚴重降低生活素質，也可能因為情緒不定影響周遭親友。 目前臨床上最普遍使用於治療陰道乾燥萎縮的方式為塗抹陰道軟膏，但由於軟膏的黏膜貼附效果不佳，需要頻繁的塗抹給藥才能維持陰道的雌激素濃度，造成使用者的不便。再來是目前市面上所使用的有效成分皆為雌二醇及其衍生物，屬於處方藥物，須經醫師看診才能取得，無法在藥局臨櫃購買，會造成部分不願意上醫院看病的更年期女性無法受益。 本研究使用幾丁聚醣做為水膠主成分，以BDDE交聯、接上含硫醇乙酸增加黏膜貼覆性，動物黏膜表面的musin含有半胱胺酸未鍵結的 -SH基，可與實驗製備的含硫醇基水膠形成較穩固的雙硫鍵鍵結，並利用黏膜表面對硫的氧化還原特性達到將水膠緩慢代謝排出體外的目的。水膠內會加入雌激素類似物Di (2-ethylhexyl) phthalate (DEHP) 或Genistein以取代現有雌激素軟膏中的作用成分。 本實驗實驗材料合成後以Ellman's assay、FTIR及NMR檢測官能基及含硫量，能成功的將Chitosan接上硫基，加入DEHP、Genistein後利用HPLC測量兩者的釋放曲線。In vitro 則根據ISO 10993，以L929進行WST-1及Live Dead，確認材料沒有細胞毒性，以Caco2細胞進行微流道實驗，確認修飾後材料對黏膜的貼附能力提升。In vivo以去除雙側卵巢的SD大鼠模擬停經女性，於陰道給予含有DEHP或Genistein的水膠一個月後犧牲，透過量測體重、量測陰道子宮濕重量、製作陰道抹片、計算陰道成熟指數，及觀察組織切片HE染色後之表皮厚度，確認材料能部分緩解陰道萎縮的症狀。

Vagina atrophy is a common symptom in women after menopause due to lowering of estrogen level. Discomforts such as itch, dryness, irritation and soreness can bother for years. The most general treatment is estrogen cream, which is cheap and flexible in dosing. However, the cream contains the prescription drug, estrogen, which is not available at over-the-counter pharmacy. The other disadvantage is the weak adhesion to the vagina mucus, which means that frequent application is needed daily. In this study, we used BDDE to crosslink and conjugate chitosan and thiol glycolic acid to improve the mucoadhesive property of chitosan. The results of Ellman’s assay, FTIR and NMR confirm the functional groups and chemical structure of thiolated chitosan, and the release profile of DEHP/ genistein was checked by HPLC. WST-1 assay and Live Dead assay both showed that our material had low cytotoxicity. The mucoadhesive property was checked by microfluidic channel cultured with Caco2 cells and FITC-labeled material, and showed that thiolated chitosan is about two fold stronger against the constant flow compared with the unmodified one. The animal model was built up one month after bilateral ovariectomy of SD rats and divided into five groups, control group (C), ovariectomy group (OVX), estradiol group (E), thiolated chitosan with genistein (CT-G), and thiolated chitosan with DEHP (CT-D). By checking body weight, vagina smear, blood tests and histology, the results showed that our material can reverse about 50% of the epithelium thickness of atrophic vagina and had no toxicity to liver nor kidney.