臺灣博碩士論文加值系統

第一部分
目的：為了使化學治療有更好的選擇性和功效以提高腫瘤的治療效果並降低正常組織毒性，發展出基因導引酵素前驅藥物治療。葡萄酸苷酶已被廣泛用於酵素標靶前驅藥物治療，因此發展監測轉植葡萄酸苷酶基因在腫瘤中的表現程度及持續性的方法十分重要，可於臨床上用於評估使用前驅藥物治療腫瘤的可行性。本研究使用放射性碘標幟酚酞葡萄糖酸苷做為評估表現於細胞膜上葡萄酸苷酶的放射性示蹤劑。
材料與方法：以124I及131I標記酚酞葡萄糖酸苷(phenolphthalein glucuronide, PhG)得到124/131I-phenolphthalein glucuronide (124/131I-PhG)，產率分別為70 %及80 %，放射化學純度皆大於95 %。細胞實驗中，給予124/131I-phenolphthalein glucuronide後15, 30, 60, 120, 240分鐘，分析在原生型大腸癌細胞(CT-26 cell)及經轉植基因、可表現葡萄糖酸苷酶於膜上的大腸癌細胞(CT-26/mβbG cell)的細胞積聚量及其代謝物。動物實驗中，於BALB/c小鼠右前脅下種入5×106 CT-26/mβG細胞，左前脅下種入5×106 CT-26細胞，並於腫瘤植入後第7天注射藥物進行微正子電腦斷層掃描、自動放射攝影、生物分布實驗、藥物動力學評估及體內代謝物分析。
結果與討論：細胞實驗結果顯示，131I-PhG對CT-26/mβG細胞具特異性，僅能被細胞膜上表現葡萄糖酸苷酶(mβG)的CT-26/mβG細胞代謝為高脂溶性的131I-phenolphthalein (131I-Ph)。由於細胞膜的脂雙層特性，高脂溶性的131I-Ph快速吸附於CT-26/mβG細胞膜上，並在約120分鐘時達成於水相(培養液)及脂相(細胞膜)的分布平衡。動物實驗中，微正子電腦斷層掃描、自動放射攝影及生物分布實驗結果均指出，尾靜脈注射124/131I-PhG後CT-26/mβG腫瘤有較高放射活性積聚，約為CT-26腫瘤的1.5倍，但比值隨著時間逐漸下降，顯示放射活性可能並非積聚在CT-26/mβG細胞內而僅是附著於細胞膜上，因此注射後20小時的微正子電腦斷層掃描影像中，腫瘤放射活性幾乎與背景相當；在腹腔中，注射後20小時仍有高度活性積聚，主要來自肝膽系統(肝臟、膽汁及腸道)。活體代謝產物分析結果顯示，注射131I-phenolphthalein 或131I-phenolphthalein glucuronide後，膽汁及血液中皆可發現131I-phenolphthalein glucuronide和碘離子的存在，且在尿液中皆存在碘離子，顯示131I-phenolphthalein glucuronide 及其經由表現葡萄糖酸苷酶(mβG)的腫瘤代謝生成的 131I-phenolphthalein會經由肝臟的葡萄糖醛酸反應及去碘作用由膽汁(胃腸道)及尿液排出體外。而藥物動力學評估中得到131I-phenolphthalein glucuronide分布相之半衰期(t1/2α)為9.10分鐘，排除相半衰期(t1/2β)為614分鐘，顯示藥物在血液中清除快速。
結論：124/131I-PhG對於能表現mβG於細胞膜上的腫瘤有特異性，故124/131I-PhG可做為在基因轉植傳遞系統中mβG基因在體內的表現示蹤劑，做為進行基因治療前的評估進而增進治療效果。
第二部分
背景：5-Iodo-2’-deoxyuridine (IUdR) 一向被視為極具潛力的腫瘤化療藥物和放射治療增敏藥物，然而IUdR在生物體內並不穩定，因此腫瘤組織不易積聚能達到有效治療濃度的IUdR。本研究製備親脂性較高的[*I]IUdR-3',5'-diacetyl ester ([*I]IUdR-(OAc)2)及[*I]IUdR-3',5'-dibenzoyl ester ([*I]IUdR-(OBz)2)做為[*I]IUdR的前趨藥物，評估二種前趨藥物的腫瘤治療潛力。
方法：IUdR與acetic anhydride和benzoic anhydride反應得到IUdR-(OAc)2和IUdR-(OBz)2，再分別與六丁基二錫作用可獲得Bu3SnUdR-(OAc)2及Bu3SnUdR-(OBz)2，經碘-131標定、純化後得到最終產物[131I]IUdR-(OAc)2、[131I]IUdR-(OBz)2。將[131I]IUdR、[131I]IUdR-(OAc)2及[131I]IUdR-(OBz)2與做為載體的lipiodol混合，經無菌過濾即成為[131I]IUdR/lipiodol、[131I]IUdR-(OAc)2/lipiodol及[131I]IUdR-(OBz)2/lipiodol注射液。與生理食鹽水輕微搖盪，測量藥物釋出於生理食鹽水的比例。[131I]IUdR、[131I]IUdR-(OAc)2及[131I]IUdR-(OBz)2加入培養液中觀察N1S1肝癌細胞在不同時間（0、2、4、8、24及48小時）中，細胞積聚藥物、DNA併入率的程度及代謝物分析。
結果：Bu3SnUdR-(OAc)2的化學合成產率大於40%，而Bu3SnUdR-(OBz)2的化學合成產率大於30%。標記效率方面，[131I]IUdR-(OAc)2及[131I]IUdR-(OBz)2分別大於98%及95%，化學純度也都大於98%。在藥物脂溶性測定中，[131I]IUdR、[131I]IUdR-(OAc)2及[131I]IUdR-(OBz)2的分布係數分別為0.86、2.73與21.88，且在溶出試驗中，二種前趨藥物自lipiodol釋放的程度小於[131I]IUdR/lipiodol，可知[131I]IUdR-(OAc)2、[131I]IUdR-(OBz)2脂溶性明顯大於[131I]IUdR。在細胞實驗中，24小時內[131I]IUdR-(OAc)2及[131I]IUdR-(OBz)2皆有穩定進入細胞及DNA的表現，但[131I]IUdR併入細胞及DNA的比例高於[131I]IUdR-(OAc)2及[131I]IUdR-(OBz)2。
結論：由於此二前趨藥物比[131I]IUdR具較高之親脂性，其由lipiodol釋出之速率比[131I]IUdR緩慢，預期二者在肝腫瘤應有更長之滯留時間，而[131I]IUdR-(OAc)2和[131I]IUdR-(OBz)2代謝後成為[131I]IUdR可進入DNA，因此肝腫瘤細胞對[131I]IUdR的攝取率以及腫瘤的輻射吸收劑量可望獲得提高。

Part One:
Objectives: β-glucuronidase has been widely used as a prodrug-activating enzyme for cancer therapy. The ability to monitor β-glucuronidase activity in living animals would further allowed both imaging and therapy of cancer. This study demonstrated that radioiodinated phenolphthalein glucuronide (*I-PhG) was a promising probe to monitor the membrane-anchored β-glucuronidase (mβG) gene expression in vitro and in vivo.
Methods: The radioiodinated glucuronide conjugate 124/131I-PhG was prepared with high radiochemical purity (>95%). The accumulation and metabolism of 131I-PhG in mβG gene expressing CT26/mβG colon carcinoma cells and the wild-type CT26 cells were studied. About 5x106 CT26/mβG and CT26 cells were inoculated in the right and left flanks of BLAB/c mice. The biodistribution study and scintigraphic imaging of 124/131I-PhG were performed with tumors-bearing mice at 7-10 days post tumor implantation.
Result: The hydrophilic 131I-PhG was metabolized by the β-glucuronidase to produce mainly 131I-Ph and a trace amount of 131I-iodide in the CT26/mβG cell culture medium. The radioactivity retained in CT26/mbG cells owing to the retention of lipophilic metabolite, 131I-Ph, in the cell membrane increased rapidly with time and reached plateau (46.2%) after 240 min incubation. The 131I-PhG was stable in the CT-26 cell culture, no 131I-Ph was found and only 2.0% radioactivity was accumulated in the cells after 240 min incubation. The biodistribution studies showed higher radioactivity accumulations in CT26/mβG tumors than those in CT-26 tumors in the whole experimental periods. The results of microPET imaging were consistent with those of biodistribution. The production of lipophilic metabolite *I-Ph from deglucuronidation of hydrophilic 131I-PhG might account for the high retention of radioactivity in abdomen region.
Conclusions: These results demonstrate that radioiodinated PhG can systemically monitor the membrane-anchored β-glucuronidase in vitro and in vivo. This targeting strategy may provide a valuable tool to estimate the efficacy and specificity of different gene delivery systems and optimize gene therapy protocols in the clinic.

Part Two:
Objectives: IUdR is long regarded as a highly potential tumor therapeutic drug and radiosensitizer. But IUdR is not very stable in vivo and high tumor uptake cannot be achieved easily. We prepare more lipophilic [*I]IUdR-3',5'-diacetyl ester ([*I]IUdR-(OAc)2) and [*I]IUdR -3',5'-dibenzoyl ester ([*I]IUdR-(OBz)2) as prodrugs of [*I]IUdR and evaluate their potency in hepatoma radiotherapy.
Methods: Starting from IUdR and acetic anhydride or benzoic anhydride, IUdR-(OAc)2 (or IUdR-(OBz)2) was obtained. After reacting with hexabutylditin followed by radioiodination, [131I]IUdR-(OAc)2 and [131I]IUdR-(OBz)2 can be prepared. [131I]IUdR, [131I]IUdR-(OAc)2 and [131I]IUdR-(OBz)2 mixed with lipiodol (act as vehicle) followed by sterile filtration with 0.22 micrometer membrane filter to afford [131I]IUdR/lipiodol, [131I]IUdR-(OAc)2/lipiodol and [131I]IUdR-(OBz)2/lipiodol injections. The release of IUdR or prodrugs from lipiodol was examined. The cellular uptake and DNA incorporation ratio of [131I]IUdR, [131I]IUdR-(OAc)2 and [131I]IUdR-(OBz)2 in N1S1 hepatoma cells has been determined after incubation for different time periods.
Results: The overall chemical yields in preparing Bu3SnUdR-(OAc)2 and Bu3SnUdR-(OBz)2 were 40 and 30%, individually. The radiochemical yield and radiochemical purity of [131I]IUdR-(OAc)2 and [131I]IUdR -(OBz)2 were both higher than 95%. The release rates of the two prodrugs from lipiodol were slower than that of IUdR, indicating the higher lipophilicity of the prodrugs. When incubated with [131I]IUdR, [131I]IUdR-(OAc)2 and [131I]IUdR-(OBz)2, the cellular uptakes and the incorporation of radioactivity into DNA all increased steadily within 24 h, but the DNA incorporation ratio of [131I]IUdR was higher than the two lipophilic prodrugs.
Conclusions: Due to the higher lipophilicity of the two prodrugs, their release from lipiodol was slower than that of IUdR. Since lipiodol as carrier will stay longer in hepatocyte carcinoma than in normal liver, both [131I]IUdR-(OAc)2 and [131I]IUdR-(OBz)2 will retain longer in hepatoma, higher tumor uptake, higher DNA incorporation ratio of [131I]IUdR and higher radiation dosimetry to hepatoma cells can thus be achieved.

第一部分目錄:
中文摘要 ………………………………………………………………1
英文摘要 ………………………………………………………………4
壹、前言 ………………………………………………………………6
貳、材料與方法 ………………………………………………………12
一.材料 ………………………………………………………………12
二.方法 ………………………………………………………………14
1.124/131I-phenolphthalein glucuronide之放射化學合成
2.131I-phenolphthalein之放射化學合成
3.CT-26及CT-26-mbG細胞培養
4.131I-phenolphthalein glucuronide細胞積聚實驗
5.131I-phenolphthalein glucuronide細胞代謝產物分析
6.荷腫瘤小鼠動物模式之建立
7.124I-phenolphthalein glucuronide於荷腫瘤小鼠之微正子電腦斷層造影
8.131I-phenolphthalein glucuronide於荷腫瘤小鼠之全身自動放射顯影
9.131I-phenolphthalein glucuronide於荷腫瘤小鼠之生物分布研究
10.131I-phenolphthalein glucuronide與131I-phenolphthalein於荷腫瘤小鼠之代謝產物分析
11.131I-phenolphthalein glucuronide於荷腫瘤小鼠之藥物動力學評估
參、結果………………………………………………………………23
1.124/131I-phenolphthalein glucuronide之放射化學合成
2.131I-phenolphthalein之放射化學合成
3.131I-phenolphthalein glucuronide細胞積聚實驗
4.131I-phenolphthalein glucuronide細胞代謝產物分析
5.124I-phenolphthalein glucuronide於荷腫瘤小鼠之微正子電腦斷層造影
6.131I-phenolphthalein glucuronide於荷腫瘤小鼠之全身放射自動顯影
7.131I-phenolphthalein glucuronide於荷腫瘤小鼠之生物分布研究
8.131I-phenolphthalein glucuronide與131I-phenolphthalein於荷腫瘤小鼠之代謝產物分析
9.131I-phenolphthalein glucuronide於荷腫瘤小鼠之藥物動力學評估
肆、討論………………………………………………………………37
伍、結論………………………………………………………………40
陸、參考文獻…………………………………………………………41
第二部分目錄:
目錄
中文摘要 ……………………………………………………………1
英文摘要 ……………………………………………………………3
壹、前言 ……………………………………………………………5
貳、材料與方法 ……………………………………………………10
一.材料 ………………………………………………………………10
二.方法 ………………………………………………………………12
1.[131I]IUdR前趨藥物的放射性標記前驅物Bu3SnUdR-(OAc)2及Bu3SnUdR-(OBz)2之合成
2.[131I]IUdR、[131I]IUdR-(OAc)2與[131I]IUdR-(OBz)2的放射合成
3.[131I]IUdR、[131I]IUdR-(OAc)2與[131I]IUdR-(OBz)2的分布係數測定
4.[131I]IUdR/lipiodol、[131I]IUdR-(OAc)2/lipiodol及[131I]IUdR-(OBz)2/lipiodol之製備及其對於normal saline之溶出比例分析 (水溶液溶出速率分析)
5.N1S1細胞株之細胞培養
6.N1S1肝腫瘤細胞對[131I] IUdR、[131I]IUdR-(OAc)2和[131I] IUdR-(OBz)2的攝取及DNA併入率測定
參、結果………………………………………………………………19
1.[131I]IUdR前趨藥物的放射性標記前驅物Bu3SnUdR-(OAc)2及Bu3SnUdR-(OBz)2之合成
2.[131I]IUdR、[131I]IUdR-(OAc)2與[131I]IUdR-(OBz)2的放射合成與放射化學純度分析
3.[131I]IUdR、[131I]IUdR-(OAc)2與[131I]IUdR-(OBz)2的分布係數測定
4.[131I]IUdR/lipiodol、[131I]IUdR-(OAc)2/lipiodol及[131I]IUdR-(OBz)2/lipiodol之製備及其對於normal saline之溶出比例分析
5.N1S1肝腫瘤細胞對[131I] IUdR、[131I]IUdR-(OAc)2和[131I]IUdR-(OBz)2的攝取及DNA併入率測定
肆、討論………………………………………………………………27
伍、結論………………………………………………………………28
陸、參考文獻…………………………………………………………29

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