- 學位論文
光敏劑結合佐劑藥物於倉鼠口腔癌前病變動物模式之光動力診斷及治療研究
Combination therapy of photodynamic therapy and adjuvant studies for hamster buccal pouch premaligant lesions
摘要
光動力療法 (photodynamic therapy、PDT) 為非侵入式治療，將光敏劑或光敏劑前驅物以塗抹、口服或注射方式給藥，光敏劑在體內會選擇性地滯留於癌症或癌前病變組織內，以特定波長光線照射，感光劑會吸收特定波長的光能而被激發，啟動光化學反應產生自由基及單態氧造成細胞傷害，達到選擇性消滅癌細胞的作用，而其螢光特性也可用於腫瘤的診斷及定位。由於光的物理性質直接決定了治療的效果及治療的深度，因此選擇適當的感光劑、可吸收的光線波長及適合的治療時間點，是光動力療法成功的關鍵。 本研究使用已用於臨床治療之5-氨基酮戊酸 (5-aminolaevulinic acid、ALA) 光敏感劑前驅物質為治療基礎，並於倉鼠口腔癌前病變模式動物進行光動力治療之光源、功率、劑量比較，篩選出合適於倉鼠口腔癌前病變模式動物光動力治療之組合。並以局部塗抹方式比較甲氨蝶呤 (Methotrexate、MTX)、卡泊三醇 (1,24(OH)2-22-ene-24-cyclopropyl D3、calcipotriol) 兩種佐劑藥物對於ALA-PDT之增效，以及有效治療次數。最後利用人類舌鱗狀細胞癌SCC4細胞 (human tongue squamous cell carcinoma、SCC4) 探討增效機轉。 本實驗結果顯示，在倉鼠口腔癌前病變動物模式之ALA-PDT中，光功率在700mW/cm2能量為200焦耳 (joules、J)，治療週期為72hr之持續照射治療條件時，可於病變處產生治療效果，但會對患部表面造成破壞性傷口，並有少量熱效應。若以300mW/cm2能量100J，治療週期為1週之間歇性治療條件時，可減少熱效應，但對患部的傷害仍過大。而以200mW/cm2能量100J，治療週期為1週之光照條件，則無熱效應產生，成功針對病變產生有效治療反應。 本研究發現分別以佐劑MTX及calcipotriol預處理之倉鼠口腔癌前病變模式動物，於ALA光動力診斷 (photodynamic diagnosis、PDD) 研究發現，會因不同佐劑的預處理而有不同的最佳治療窗口。控制組之ALA-PDD治療時間點為20%ALA (w/w) 水膠，於患部局部塗抹後2.5hr； MTX水膠預處理之治療時間點為6.5hr；以calcipotriol預處理之治療時間點為3.5hr。根據以上PDD條件進行ALA-PDT的結果為控制組平均需經4.8次療程，以肉眼觀察可達完全治癒 (complete response、CR) 之療效；以MTX佐劑預處理後，進行ALA-PDT達CR，平均則需經2.3次療程；以calcipotriol佐劑預處理後，進行ALA-PDT達CR平均經3.8次療程。經佐劑增效的機轉研究，初步發現SCC4細胞經不同濃度之MTX以及calcipotriol預處理後，可發現合成具光敏功能之原紫環IX (protoporphyrin IX、PpIX)上游酵素糞卟啉原氧化酶 (coproporphyrinogen oxidase、CPOX) 有上升的表現；而同為上游酵素合成PpIX的原卟啉原氧化酶 (protoporphyrinogen oxidase、PPOX) 則無明顯變化；而下游代謝PpIX之亞鐵螯合酶 (ferrochelatase、FECH) 也無明顯變化，可說明無論以MTX或calcipotriol佐劑預處理，皆可以提高細胞CPOX酵素含量，進而導致ALA-PDT的療效增加。
並列摘要
Photodynamic therapy (PDT) is a non-invasive cancer therapy modality. Photosensitizer or its precursor is given by topical, oral or vein-injection administration. Then photosensitizer accumulates selectively in precancerous or cancerous lesions and is then activated after irradiatiing light. The activated photosensitizer reacts with oxygen to form reactive oxygen species (ROS), resulting in tissue destruction of precancerous and cancerous tissue. Photodynamic diagnosis (PDD) of topical 5-aminolaevulinic acid (ALA) was obtained for the optimal treatment schedule to deliver optimumn PDT therapeatic results. We established a hamster buccal pouch precancerous animal model to research on different adjuvant drugs including methotrexate (MTX) and calcipotriol for combination therapy with topical ALA-PDT in vivo. On the other hand, we used human tongue squamous cell carcinoma (SCC4) cell line, to investigate the ALA-PDT therapeutic effect with preconditioning adjuvant drugs. For ALA-mediated PDD, the optimal treatment schedule post topical ALA administration dependeds on different kind of preconditioning adjuvant. PDD time was determined for 2.5 hr without adjuvant, 6.5 hr after 72hr MTX preconditioning, and 3.5 hr after 72hr calcipotriol preconditioning. Our study found that there are 4.8 PDT cycles for no preconditioning adjuvant, 2.3 PDT cycles post 72hr MTX preconditioning, and 3.8 PDT cycles post 72hr calcipotriol preconditioning. In vitro, SCC4 cells were preconditioned with MTX at 0.001, 0.01, 0.1 and 1(mg/L), with calcipotriol at 10-8, 10-7, 10-6 and 10-5 M. Up-regulation of coproporphyrinogen III oxidase (CPOX), an upstream enzyme of PpIX synthesis, but there was no significant change in the regulation of protoporphyrinogen oxidase (PPOX), an enzyme co-regulating on PpIX synthesis, and ferrochelatase (FECH), the terminal enzyme in the biosynthesis of heme. Based on the above studies, it demonstrated that such combination therapy of adjuvants and ALA-PDT served a successful therapeutic modality on oral precancer therapy.