- 學位論文
新型肝癌治療劑之研究-C/GP智慧型凝膠合併Doxorubicin和錸-188膠體之探討
A novel pharmaceutical in hepatic tumor research – the study of used C/GP intelligent gel combined Doxorubicin and 188Re-colloid
摘要
目的：利用直接腫瘤注射(I.T.)方式，將188Re-Dox-C/GP凝膠藥物固定於肝腫瘤，進行治療肝癌動物模型療效評估，與探討相關C/GP凝膠特性研究及放射凝膠藥物於體內滯留評估之研究。 材料與方法：一、C/GP凝膠時間研究：針對C/GP凝膠與Dox-C/GP凝膠藥物(25 µg & 0.4 mg/mL)，在Tg凝膠溫度(37℃)，經由UV分光度計量測，探討形成凝膠之時間點；二、體外藥物釋放研究：利用透析膜管(Spectra/pro®)，封裝Dox sol.、Dox-C/GP及188Re-Dox-C/GP凝膠藥物，置於裝有PBS溶液燒杯中，於不同時間點採集樣品，經由螢光光度計分析Dox藥物含量；三、細胞毒性研究：將完成製備之C/GP凝膠與N1-S1肝癌細胞進行培養，評估C/GP凝膠對抑制N1-S1生長能力。四、平面閃爍&生物分佈研究：動物麻醉後，以I.T.給藥方式，將188Re-Dox-C/GP凝膠藥注射於病灶處，分別在給藥後0、24和48小時進行平面閃爍造影及定量分析其錸-188膠體於體內之滯留率；相同時間點，犧牲老鼠取其主要器官，透過γ-counter分析放射藥物活度；五、治療療效評估研究：荷肝腫瘤之大鼠，接受不同配方藥物注射後，監測體重變化在治療後第0、2及4週。同時間點，將同批老鼠，利用超音波掃描量測腫瘤之大小。療效存活評估，則是老鼠接受治療後60天，記錄實驗老鼠存活天數，經由生物統計分析是否具有顯著意義。完成存活時間測試後，針對188Re-Dox-C/GP凝膠藥物治療之肝癌老鼠，藉由CT掃描作比對是否與超音波診斷結果相符。隨即，將老鼠犧牲，取出主要來源器官，送往病理部門分析，評估藥物凝膠於肝腫瘤之治療效果。 結果：凝膠時間研究顯示C/GP凝膠與Dox-C/GP凝膠藥物(25 µg & 0.4 mg/mL)，在Tg溫度下，所測得凝膠時間相近約4.5分鐘。但是，對於形成之凝膠特性則有所差異。在體外藥物釋放研究，以188Re-Dox-C/GP熱凝膠延緩Dox藥物釋放效果最佳，在藥物釋放後48小時，僅觀察到51±0.88 %Dox藥物被釋出。平面閃爍及生物分佈研究，表示I.T.注射188Re-Dox-C/GP凝膠溶液，能有效將錸-188膠體固定於病灶處，維持48無明顯擴散，其藥物滯留率為39%±0.132。體內生物分佈研究結果，證實以I.T.注射188Re-Dox-C/GP藥物，其錸-188放射藥物累積於腫瘤最高且對正常肝臟影響較小，唯有少量藥物活度累積於肺臟於注射後24小時。細胞毒性與治療肝腫瘤之療效研究，發現C/GP凝膠本身具抑制腫瘤能力。在CT造影與病理切片查驗，我們發現在C/GP凝膠合併放療及化療藥物的組織切片，其治療腫瘤之功效比188Re-C/GP凝膠藥物好，並伴隨有鈣化現象產生。 討論&結論：C/GP系統合併188Re-Tin colloid與Doxorubicin，經由I.T.注射給藥途徑，局部治療腫瘤，除了具備延緩藥物釋放能力及合併不同治療模式，提高治療肝腫瘤之功效外；凝膠本身亦具備抑制腫療生長能力，更大幅加強其治療效果且適用於未來臨床研究，針對無法切除腫瘤之肝癌病患，作為另一項選擇。
並列摘要
Objective: Intratumoral (I.T.) injected 188Re-Dox-C/GP gel into hepatic tumor, to execute treatment efficacy evaluation of liver cancer animal model and explore related characteristic of C/GP (chitosan/β-glycerophosphate) gel and biodistribution study. Materials and methods: gelation time study: C/GP gel and Dox-C/GP gel (25 µg & 0.4 mg/mL) were measured at 37℃ by UV-spectrophotometer from flowable liquid to semi-solid gel. (2) In vitro of drug released study: used the dialysis tube to encapsulate of doxorubicin (Dox) sol., Dox-C/GP, and 188Re (colloid)-Dox-C/GP gel, then them were immersed in phosphate buffer solution (PBS, 0.01M, pH =7.4) at different time, sample of PBS were withdrawn by fluorescence spectrometer analysis. (3) Cytotoxic study: the C/GP gel was prepared and co-incubation with N1-S1 cells for evaluation the survival cells in different time (4) Planar scintigraphy & biodistribtuion study: animals were anesthetized to perform and calculation of 188Re-Tin colloid retention rate on whole rat using planar scintigraphy at 1, 24, and 48 h drug delivery. At the same time point, the tumor-bearing rats were sacrificed to obtain source organs with radioactive of the obtained tissues were quantitatively analyzed by auto-γ-counter. (5)Therapeutic efficacy studies: the tumor-bearing rats were treated through 0.1 mL of normal saline, C/GP gel, 188Re-C/GP (37 MBq) and 188Re-Dox-C/GP (0.5 mg Dox and 37 MBq of 188Re-Tin colloid) gel-drug by I.T. injection for comparative treatment efficacy of hepatic tumor on body weight, tumor size, survival time and histological examinations. Results: the gelation time of different ration of Dox-C/GP gel and C/GP gel at 37℃ was 4.5 min. In vitro released of Dox study from 188Re-Dox-C/GP gel has the best of control drug delivery ability, only 51%±0.88 of accumulate drug were observed at 48 h. Observation 188Re of radioactivity on planar scintigrams at selected time points showed no obvious diffusion in injection site even 48h after injections with retention rate was 39%±0.132. In biodistibution study demonstrated that 188Re of radioactivity accumulated in tumor are highest than other organ, but still a part of 188Re-colloid distributed in lung were observed at 24h. I.T. injection of normal saline and different formulations gel-drug in treatment efficacy evaluations and in vitro of cytotoxic test, it found C/GP gel had inhibited tumor growth effect. Although, it can’t comparative both of 188Re-C/GP gel and 188Re-Dox-C/GP gel in tumor growth curve and survival time test by calculation tumor size and biostatistics analysis. However, for CT image and histology H&E stain analysis on 188Re-Dox-C/GP gel-group showed more calcification and apoptosis than 188Re-C/GP gel-group. Discussion/Conclusions: The C/GP system combined 188Re-Tin colloid and Doxorubicin by I.T. injections to treatment hepatic. Not only has prolonged drug delivery time but also radio-and chemotherapy model merge, for raised the treatment effects; the C/GP itself also has ability of inhibited tumor growth with more drastic increase in treatment efficacy. Perhaps this study will offer development of the potentiality for drug and a novel treatment model.
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