建立軟骨保護口服藥物以及關節注射藥物對骨性關節炎實驗動物模式治療效果的評估

Therapeutic effects evaluation of oral chondroprotective and intra-articular modified hyaluronic acid injection on experimental rabbit osteoarthritis model

呂憲宗(Hsien-Tsung Lu)

指導教授 ： 李建和 ； 陳志華 ； 李桓昇

臺北醫學大學/醫學院/臨床醫學研究所/博士(2015年)

https://doi.org/10.6831/TMU.2015.00074

摘要

骨性關節炎（OA）是最常見的慢性關節疾病。同時由於現今的醫療科技發達，平均年齡增加，社會的人口結構高齡化的現象非常的快速，由於這些族群常常和合併一些內科相關特別是心血管疾病，造成治療關節炎的手術可能會併發高危險的併發症，所以關節炎的治療以及研究一直持續不斷進行，而且仍然有必要開發特殊的藥物具有高效力和低副作用以及低侵犯性，用於治療退化性關節炎。目前研究治療關節炎藥物的其中一個方向在於進一步修飾已知傳統藥物之官能基，使其成為具有改善連結組織結構(CTSMAs)以及減緩關節炎症狀之藥物(DMODs) 。在我們一系列的動物實驗當中，我們評估了I:口服的海藻萃取物CTX II:結合去氧羥四環黴素(Doxycycline)以及玻尿酸(HA)的HA-DOX水膠體的關節注射液在紐西蘭兔動物關節炎實驗模型的治療效果。
在第一項的研究實驗評估了海藻萃取物CTX在人軟骨肉瘤SW1353細胞模型和紐西蘭兔動物關節炎實驗模型保護軟骨的效果。CTX是一種利用藻酸鹽裂解酶水解海藻酸鈉之後然後與果膠混合的聚合物。經過高效液相色譜法分析CTX的成分之後，經過白細胞介素1β處理的人軟骨肉瘤SW1353細胞用來研究命名為CTX海藻萃取物對關節軟骨細胞的炎症和合成代謝的影響。就算CTX濃度高達1000微克/毫升的環境當中也只會產生相當輕微的細胞毒性。在此濃度下CTX抑制促炎細胞基質金屬蛋白酶MMP-1, MMP-3, MMP-13的表達和增加構成關節軟骨的主要蛋白聚醣(Aggregan)合成。
動物實驗以十三週齡之紐西蘭白兔進行了前十字韌帶切斷手術，並分組口服生理鹽水，葡萄糖胺CTX共7週。實驗動物紐西蘭兔膝關節股骨髁和脛骨髁軟骨經過巨觀和組織學變化進行檢查。結果口服CTX為30毫克/公斤/天的劑量的實驗組比其他對照組的膝關節樣本承受較輕度關節僵直和軟骨組織損傷。
結論:由實驗細胞基因的表達和實驗動物的關節軟骨進行的檢查表現出海藻萃取物CTX可以成為一種關節炎治療的藥劑。

在第二項的研究中我們連結去氧羥四環黴素Doxycycline (DOX)及玻尿酸(hyaluronic acid, HA)，製造成含有Doxycycline的玻尿酸HA水凝膠( HA-DOX gel)並且以手術引發關節炎並且將HA-DOX水凝膠進行關節腔直接注射來驗證HA-DOX水凝膠是否具備協同之治療效果。
實驗利用十三週齡紐西蘭白兔進行部分半月板和腓骨韌帶切斷手術並且分組成生理鹽水（NT），HA，DOX或HA-DOX水凝膠在第0，3，6，9和12天進行注射不同的藥劑;術後的檢查包括每三天的疼痛評估。手術後第14天進行取樣用於進一步的組織病理學評估。
結果:在疼痛的程度評估方面HA和HA-DOX水凝膠組別在第7天治療後，疼痛顯著的減輕。在取下來的標本關節軟骨方面的外觀，HA-DOX水凝膠組比較單獨HA或DOX治療呈現比較光滑的關節軟骨表面，關節沒有或無潰瘍而且只產生少量的磨損裂縫及骨刺。在HA-DOX水凝膠組雖然某些關節面略有OA的變化，但是當地的軟骨細胞的發展出較為正常的分佈，表現出軟骨的再生。此外HA-DOX水凝膠也有通過保護關節軟骨層的損傷以及提供較佳之黏彈性質減緩關節炎的惡化。
結論:不論是在巨觀或者是顯微結構下HA-DOX水凝膠表現出對兔關節軟骨的關節炎治療具有長期的療效。

關鍵字

骨關節炎 ； 前十字韌帶切除 ； ，白介素1β ； 基質金屬蛋白酶蛋白多醣 ； 海藻萃取物

並列摘要

Osteoarthritis (OA) is the most common chronic joint disorder. Advanced medical technology promote the older segment of the population to be the fastest growing. Although related arthritis surgery are popular, however, situation presents the orthopaedic surgeon with many medical comorbidities especially cardiovascular disease which make surgical arthritis treatment more difficult and risky. Research has been carried out continuously, and there is still an unmet need to develop specific drugs with high efficacy with mild invasive and low side effects for the treatment of degenerative arthritis. Some of conducted arthritis researches have modified known arthritis medicine to be the connective tissue structure-modifying agents (CTSMAs) and disease-modifying osteoarthritis drugs (DMODs). In our series of animal experiments, we evaluated I: Alterative effects of an oral alginate extract on experimental rabbit osteoarthritis and II: Injectable hyaluronic-acid-doxycycline hydrogel therapy in experimental rabbit osteoarthritis

I: Alterative effects of an oral alginate extract on experimental rabbit osteoarthritis

In this study, we evaluated the effects of the alginate extract named CTX in cell and rabbit OA models.
Methods and Results. CTX was formulated by hydrolyzing sodium alginate polymers with alginate lyase and then mixing with pectin. HPLC was used to analyze the CTX content. Human chondrosarcoma SW1353 cells treated with interleukin-1β were used as OA model cells to investigate the effects of CTX on chondrocyte inflammation and anabolism. CTX at concentrations up to 1000 μg/ml exerted low cytotoxicity. It inhibited the gene expression of proinflammatory matrix metalloproteinases (MMPs) including MMP1, MMP3 and MMP13 in a dose-dependent manner and increased the mRNA level of aggrecan, the major proteoglycan in articular cartilage, at 1000 μg/ml. Thirteenweek-old New Zealand White rabbits underwent a surgical anterior cruciate ligament transection and were orally treated with normal saline, glucosamine or CTX for up to 7 weeks. Examinations of the rabbit femur and tibia samples demonstrated that the rabbits taking oral CTX at a dosage of 30 mg/kg/day suffered lesser degrees of articular stiffness and histological cartilage damage than the control rabbits.
Conclusions. The gene expression profiles in the cell and the examinations done on the rabbit cartilage suggest that the alginate extract CTX is a pharmaco-therapeutic agent applicable for OA therapy.

II: Injectable hyaluronic-acid-doxycycline hydrogel therapy in experimental rabbit osteoarthritis

We evaluated the therapeutic effects of intra-articular injections of hydrogels containing hyaluronic acid (HA) and doxycycline (DOX) in a rabbit OA model.
Methods and Results: Thirteen week old New Zealand White rabbits undergone a partial meniscectomy and unilateral fibular ligament transection were administered with either normal saline (NT), HA, DOX or HA-DOX hydrogels on day 0, 3, 6, 9 and 12; animals were also examined the pain assessment in every three days. The joint samples were taken at ay 14 post-surgery for further histopathological evaluation. The degree of pain was significantly attenuated after ay 7 post-treatment with both HA and HA-DOX hydrogels. In macroscopic appearance, HA-DOX hydrogel group showed a smoother cartilage surface, no or minimal signs of ulceration, smaller osteophytes, and less fissure formation in compare to HA or DOX treatment alone. In the areas with slight OA changes, HA-DOX hydrogel group exhibited normal distribution of chondrocytes, indicating the existence of cartilage regeneration. In addition, HA-DOX hydrogels also ameliorated the progression of OA by protecting the injury of articular cartilage layer and restoring the elastoviscosity.
Conclusion: both macroscopic and microscopic data of this study indicate the injectable HA-DOX hydrogels presented as a long-lasting pharmacotherapeutic agent to apply for OA therapy.

並列關鍵字

Osteoarthritis, anterior cruciate ligament transection, Interleukin-1β, matrix metalloproteinases, aggrecan, CTX

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