玻尿酸修飾之尿酸酶對於尿酸結晶誘導痛風之影響

本研究製備含有玻尿酸修飾之尿酸酶關節腔注射劑，探討玻尿酸於關節腔內降解後形成小片段的玻尿酸-尿酸酶複合物能進入軟骨組織促進尿酸結晶溶解，以達到治療痛風的目的。首先，利用酸降解方法製備不同分子量玻尿酸，探討玻尿酸分子量效應於發炎反應與生物體分佈情形。在引起發炎反應方面，利用小鼠巨噬細胞(RAW264.7)進行體外測試(in vitro)。結果顯示，低分子量玻尿酸(8.6x10^3Da)會引起細胞嚴重發炎反應產生，而高分子量玻尿酸(4.8x10^4、2.3x10^5、1.1x10^6Da)則無此現象。在生物體分佈方面，將不同分子量玻尿酸個別修飾有機螢光染料(5-AMF)探討玻尿酸於關節腔的停留時間及模擬玻尿酸-尿酸酶複合物進入關節軟骨組織內部之可行性。在注入螢光玻尿酸(F-HA)四天時間後，螢光玻尿酸在關節腔內可通過軟骨組織表層進入到軟骨組織內部。此外，玻尿酸修飾之尿酸酶可保有64.83%的活性。此玻尿酸-尿酸酶(HA-Uricase) 複合物能有效溶解尿酸單鈉鹽結晶(MSU crystal)，並能減緩尿酸結晶引起之發炎反應。本研究合理推測玻尿酸-尿酸酶複合物於關節腔內能有效溶解尿酸結晶，且小片段的玻尿酸-尿酸酶亦能成功進入軟骨組織內部溶解尿酸結晶。此玻尿酸-尿酸酶複合物具有相當之潛力可繼續開發成為關節腔注射劑，以應用於溶解沉積在關節軟骨組織中尿酸結晶之痛風治療。

關鍵字

痛風；玻尿酸；尿酸酶關節軟骨

並列摘要

In the present study, we developed hyaluronan modified uricases (HA-uricase) for gout therapy. Smaller sized HA-uricases, formation after their decomposition in the joints, were able to get into articular cartilage to dissolve the deposited urate crystals. Hyluronans were firstly fragmented to varied sizes by acid digestions. The effects of these hyaluronans with different molecular weights were then examined on elicited inflammation and their biodistribution. Results indicated that hyaluronans with lower molecular weights (8.6x10^3Da) induced significant inflammatory responses, whereas hyaluronans with higher molecular weights (1.1x10^6Da) did not. The fluorescent dye (5-AMF)-labeled hyaluronans remained in the joints for at least 4 days, and were able to reach to articular cartilage. The activity of hyaluronan modified uricase was around 64.83%, which could efficiently dissolve monosodium urate (MSU) crystals and reduce the inflammation induced by them. Taken together, the HA-uricases can dissolve urate crystals in the joints, and likely in articular cartilage. This ability makes HA-uricases a potential strategy for gout therapy, especially for the urate crystals deposited in articular cartilage.