- 學位論文
玻尿酸口服給予之特性描述與體內分佈探討
Characterization and Biodistribution of Oral Administration of Hyaluronic Acid
摘要
玻尿酸為一多功能之生物相容性良好之大分子聚合物，其物化特性及生理功能會受本身之分子量大小及化學修飾的影響。目前在醫藥方面欲發展玻尿酸口服藥物傳遞系統，但關於探討口服玻尿酸其特性描述及生物體內分佈的文獻並不多，因此希望對不同分子量及修飾程度的玻尿酸口服後在體內的變化作一瞭解，以期在口服藥物傳遞系統上有新的進展。 實驗首先建立一套分析玻尿酸分子量之分析方法，根據實驗結果，以玻尿酸標準品及分子篩滲透層析儀為一簡便且準確及再現性高的玻尿酸分子量分析方法。 本研究利用酵素消化得到分子量各異的玻尿酸。實驗結果發現，酵素濃度是造成酵素反應所得消化產物差異的主要影響因子，在固定的時間及酸鹼值下，只需調整玻尿酸酶之濃度即可獲得一系列不同分子量的玻尿酸。 接著利用adipic acid dihydrazide (ADH) 得到一系列不同化學修飾程度的玻尿酸。玻尿酸的羧基官能基在1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) 的催化下被ADH取代，玻尿酸的修飾程度會隨反應時間及玻尿酸與ADH、EDC的莫耳數比而不同。 以具螢光特性之量子點 (QDots) 與玻尿酸接合形成量子點玻尿酸接合物 (QDot-HA conjugates)，藉由尾靜脈注射和口服方式投予動物，以即時顯像試驗討論分子量及化學修飾比例對於玻尿酸體內分佈的影響。結果顯示：(1) 分子量較小的玻尿酸及輕度修飾比例的玻尿酸注射後會較快由體內清除，且小分子量玻尿酸在老鼠的胸部及腹部分布的量比大分子多。(2) 修飾比例低的玻尿酸注射後有較專一性的分佈，明顯集中在肝臟；修飾比例高的玻尿酸，因其特性受修飾的影響，在體內組織有較廣泛的分佈。 口服方面：(1) 小分子量玻尿酸口服後較快被腸胃道吸收；大分子玻尿酸吸收比較慢也較慢被代謝及排除。(2) 高修飾比例的玻尿酸其代謝及排除比低修飾比例的慢，在體內的停留時間較久。在頭頸部及胸部，低修飾比例的玻尿酸口服後的分佈量比高修飾比例的玻尿酸多，高修飾比例的玻尿酸則於骨盆的地方比低修飾比例的多。 因此，分子量是影響玻尿酸經由系統性給藥後體內分佈的主要因子；而影響玻尿酸口服後體內分佈差異的主要因素則是化學修飾程度。根據所要傳遞藥物的特性，我們可選擇具有適合之分子量大小及化學修飾比例的玻尿酸應用於口服藥物傳遞系統中。
並列摘要
Hyaluronic acid (HA) is a multifunctional biocompatible polymer, its physical and chemical properties and physiological functions will be affected by its molecular weight (MW) and chemical modification degrees. In the medical fields, the current demand is the development of HA oral drug delivery system, but the literatures about the characteristics and bio-distribution of HA in the body after oral administration were only a few. So it is important to understand the changes of HA with different MW and modification degrees in the body after oral administration, with a view to have new progress in developing HA oral drug delivery systems. First, we established an experimental method to analyze the MW of HA. The results showed that, with the HA standards, the size exclusion chromatography was a simple, accurate and reproducible method for molecular weight analysis. Digested by the enzyme, HA molecules could degrade into HA fragments with different MW. It was found that the enzyme concentration was the main influencing factor that caused products obtained from enzyme reaction diverse. At a fixed time and pH value, we could simply adjust the concentration of hyaluronidase and get a series of HA fragments with different MW. Then we used adipic acid dihydrazide (ADH) to obtain a series of HA-ADH conjugates with different chemical modification degrees. Carboxyl functional groups on HA were replaced by ADH under the catalysis of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). The modification degrees of HA changed with the reaction time and molar degrees of HA, ADH, and EDC. Further, quantum dot conjugates HA-ADH-(QDot) with different characteristics (HA with different MW and modification degrees) were synthesized by conjugating HA-ADH with QDots. By ways of intravenous injection (IV) and oral administration, we studied the effects of MW and chemical modification degrees on the in vivo bio-distribution of HA using real-time imaging. The results showed that (i) HA with lower MW is metabolized rapidly after IV injection, while HA with higher MW could last longer time. The distributed amount of HA with lower MW in the chest and abdomen was more than HA with higher MW. [Lovvorn Iii, et al.] HA with lesser modification degrees showing the more specific distribution, accumulated in liver, and more rapid clearance after tail-vein injection; whereas HA with higher modification degrees, and its properties will be greatly affected, showing a more even tissue distribution in the body after IV injection. On the other hand, the results of oral application showed: (i) HA with lower MW absorbed more rapidly after oral administration, while HA with higher MW demonstrated slower absorption and clearance. [Lovvorn Iii, et al.] The clearance of HA with higher modification degrees was slower than HA with lesser modification degrees. In the head, neck, and chest, HA with lesser modification degrees showed more distribution amount than higher one after oral ingestion. In addition, the amount of HA with higher modification degrees distributed to pelvis was more than lesser one. It was concluded that the MW was the main factor that affected in vivo distribution of HA after IV injection, while the degree of chemical modification was the main factor that affected the bio-distribution of HA after oral administration. HA with suitable sizes and chemical modification degrees could be chosen for delivery of oral drugs with diverse characteristics.