臺灣博碩士論文加值系統

惡性黑色素瘤是具有早期轉移、高增生能力的惡性癌症之一，其治療方法多依賴外科手術切除，而轉移病灶則輔以放射治療及化學治療，若在癌症晚期則缺少有效治療的方法，因此開發出早期診斷之核醫造影藥劑是有其重要性的。
目標：
合成新的放射性鎝標誌的苯甲醯胺衍生物(5-Tc、9-Tc、12-Tc)，並進行藥物特性測試、體外細胞攝取實驗，以評估此類新型造影藥物之有效性。
方法：
經由多步驟合成的苯甲醯胺衍生物配位基，藉由放射性標誌鎝-99m金屬標誌形成單光子放出斷層造影劑。此些放射性標誌藥物進行穩定度試驗、油水分配係數試驗、體外黑色素結合試驗、並選用的細胞株為 B16F0 與 A375 惡性黑色素瘤細胞株體外細胞藥物攝取試驗及後續生物分佈實驗。
結果：
成功合成放射藥物5-Tc、9-Tc及12-Tc，而其放射性標誌的產率分別為56%、53%、89%。在藥物穩定度的測試中，藥物可在PBS及DMEM細胞培養液中於37℃之下持續六小時而無分解的現象。其油水分配係數的試驗，5-Tc為 -0.72 ± 0.03，9-Tc 為0.02 ± 0.03，12-Tc 為0.00 ± 0.02，顯示著其結構之差異造成的藥物極性之改變。在體外黑色素結合試驗，此些藥物與黑色素均有相當高結合能力(>80%)。
在體外細胞藥物攝取試驗，於培養四小時，5-Tc 被B16F0所攝取量為 0.10 ± 0.00% ID/106 cells；9-Tc 被B16F0所攝取量同為 0.08 ± 0.00%ID/106 cells；12-Tc 被B16F0所攝取量同為 0.03 ± 0.00%ID/106 cells。在活體生物分布實驗，藥物9-Tc以尾靜脈注射於種有B16F0的C57BL/6小鼠體內，藥物很快速的在體內被清除，主要為代謝器官之積聚。

結論：
此些新型的一價鎝金屬苯甲醯胺衍生物在黑色素結合試驗中與黑色素具有有高的結合能力，但於細胞藥物攝取實驗中呈現著低攝取的結果，並於活體生物試驗中亦呈現著腫瘤低攝取的狀況。此些生物實驗雖未有相當好的結果，但提供了改進的方向，期望在未來能改變藥物結構以增進藥物進入細胞的能力。

Malignant melanoma is one of the most lethal cancers due to its high cellular proliferation rate and the early occurrence of metastases. Melanoma is usually treated by surgery and support with radiotherapy and chemotherapy with metastasis, and late stage melanoma is lack of effective treatment, the development of new melanoma imaging probes is quite essential.
Purpose:
To synthesize 99mTc labeled benzamide derivatives, 5-Tc, 9-Tc and 12-Tc, are characterized to exhibit high binding to melanin and high hydrophilic for imaging melanoma tumors.
Methods:
The labeling precursors (5, 9, 12) were prepared via a multi-step synthesis. These tridentate ligands were applied to chelating [Tc(H2O)3(CO)3]+ core and form these 99mTc labeled compounds 5-Tc, 9-Tc,and 12-Tc were obtained. The stability, partition coefficient and binding affinity with synthetic melanin were evaluated with these imaging probes. Further, the biological characteristics of radiopharmaceuticals were evaluated by in vitro melanoma cell (B16F0, A375) uptake assay and in vivo biodistribution.
Results:
Three benzamide derivatives chelating 99mTc labeled tricarbonyl complesex were synthesized successfully and radio-chemical yields were higher than 50%. These radiopharmaceuticals were stable in PBS and DMEM medium in 37 ℃ during 6hr without decompose. The partition coefficient in PBS and 1-octanol for the three probes 5-Tc, 9-Tc and 12-Tc were -0.72 ± 0.03, 0.02 ± 0.03 and 0.00 ± 0.02, respectively. In melanin binding assay, these probes were showed high binding affinity (>80%) with synthetic melanin.
In vitro cellular uptake study, all of three probes were revealed the low uptake value (<0.10 ± 0.00%ID/106 cells) at 4hr incubation in B16F0 cell line. In vivo biodistribution study for probe 9-Tc in C57BL/6 mice bearing B16F0 tumor, the radio-compound was aggregated majorly in metabolized organs (liver and kidney) and the nonspecific binding was observed in tumor uptake.
Conclusions:
These novel monovalent tricarbonyl Tc labeling complexes of benzamide derivatives were showed high binding affinity with synthetic melanin. However, In vitro and In vivo study, the nonspecific tumor uptakes of these probes were observed from cellular uptake and tumor bearing mice. These probes were not suitable to using in melanoma imaging. According to these data, other structure modification should be improved to increase the melanoma cell uptake for future study.

目錄
中文摘要…………………………………………………………………………………i
英文摘要…………………………………………………………………..……………iii
目錄………………………………………………………………..…………………….v
圖目錄………………………………………………………………..………………...vii
表目錄………………………………………………………………..………………..viii
壹、 前言………………………………………………………………..………….1
貳、 結果………………………………………………………………..………...16
2.1 標誌前驅物5與非放射性標準品5-Re 合成………………………....16
2.2 標誌前驅物9與非放射性標準品9-Re 合成…………………………18
2.3 標誌前驅物12與非放射性標準品12-Re 合成……………………....20
2.4 放射性鎝-99m 同位素標誌………………………………………...…22
2.5 藥物水溶性測試…………………………………………………….….28
2.6 藥物與黑色素結合測試……………………………………………..…29
2.7 藥物穩定性測試………………………………………………..………30
2.8 體外細胞藥物攝取實驗………………………………………………..31
2.9 藥物9-Tc於添加2mM Tyrosine 促進黑色素生成之B16F0、A375黑色素瘤細胞株之細胞攝取實驗…………………………………………….33
2.10 荷皮下B16F0黑色素瘤之C57BL/6小鼠施打放射性藥物後之生物
分布研究………………………………………………………………….....35
參、 討論……………………………………………………………..………...…38
肆、 結論…………………………………………………………..………...……47
伍、 材料與方法………………………………………………………………….48
5.1 材料……………………………………………………………………..48
5.2 方法……………………………………………………………………..51
5.2.1藥物前驅物及藥物標準品合成………………………………...51
5.2.2放射性鎝-99m 標誌…………………..………………………..67
5.2.3評估藥物之脂溶性及分配係數(logP)測定……………………68
5.2.4 藥物穩定性試驗…………………………………………...…..69
5.2.5 黑色素結合試驗…………………………………………...…..69
5.2.6 B16F0與A375黑色素瘤細胞培養……………………………70
5.2.7 藥物於B16F0與A375黑色素瘤細胞株之細胞攝取實驗…...71
5.2.8 藥物於添加2mM Tyrosine 促進黑色素生成之B16F0、A375黑色素瘤細胞株之細胞攝取實驗…………………………………...72
5.2.9 荷皮下B16F0黑色素瘤之C57BL/6小鼠施打放射性藥物
後之生物分布研究…………………………………………………...73
陸、 附錄………………………………………………………..………...………74
柒、 參考文獻………………………………………………………………….101
圖目錄
圖 一、[125I]BZA 之結構 2
圖 二、Mohamme 等人合成之碘-131標誌苯醯胺類似物結構示意圖 3
圖 三、Titsch 等人合成之鎝-99m N2S2螯合苯醯胺類似物結構示意圖。 5
圖 四、Matthias Friebe 等人合成之鎝-99m 標誌’3+1螯合苯醯胺類似物結構示意圖。 7
圖 五、Matthias Friebe等人合成的N2S2螯合的合鎝標誌的苯醯胺類似物Tc-1[20][21]。 8
圖 六、Philippe Auzeloux 等人合成之鎝-99m N2S2螯合苯醯胺類似物結構示意圖。 9
圖 七、Harmel W. Peindy N'Dongo 等人合成之鎝-99m cyclopentadienyl螯合苯醯胺類似物結構示意圖。 10
圖 八、Carolina Moura等人合成之鎝-99m fac-螯合苯醯胺類似物結構示意圖，M = Re/99mTc。 12
圖 九、藥物 5-Tc 、9-Tc 、12-Tc 之結構示意圖。 15
圖 十：苯醯胺類似物藥物5-Tc之標準品全程合成示意圖。 16
圖 十一：苯醯胺類似物藥物9-Tc 之標準品全程合成示意圖。 18
圖 十二：苯醯胺類似物藥物12-Tc之標準品全程合成示意圖。 20
圖 十三：苯醯胺類似物藥物5-Tc、9-Tc、12-Tc 放射性藥物合成示意圖。 22
圖 十四：[Tc(CO)3(H2O)3]+ 與TcO4-之radio-HPLC 分析圖。 24
圖 十五： 藥物5-Tc與5-Re之HPLC 分析圖。 25
圖 十六：藥物9-Tc與9-Re之HPLC 分析圖。 26
圖 十七：藥物12-Tc與12-Re之HPLC 分析圖。 27
圖 十八：藥物5-Tc、9-Tc、12-Tc 與人工合成黑色素結合之試驗。 29
圖 十九：藥物5-Tc、9-Tc、12-Tc於B16F0與A375細胞株之藥物攝取試驗。 32
圖 二十：藥物9-Tc於外加Tyrosine之DMEM培養液之B16F0與A375細胞株之藥物攝取試驗。 34
圖 二十一、藥物9-Tc 於皮下種植B16F0之C57BL/6小鼠生物分布結果(%ID/g)。 37
圖 二十二、藥物5-Tc、9-Tc、12-Tc 與其他文獻報導之化合物(C1 [6-7]、C5 [6]、Tc-1 [20-21]、B1 [3]、B2 [3])之結構。 45


表目錄
表 一、Mohamme 等人合成之碘-131標誌苯醯胺類似物之結構差異及該藥物於生物分布試驗中與腫瘤、血液中的積聚(% ID/g)。 3
表 二、Matthias Friebe 等人合成之鎝-99m 標誌’3+1螯合苯醯胺類似物之結構差異及pKa值、分配係數。 7
表 三、Matthias Friebe 等人合成之鎝-99m 標誌’3+1螯合苯醯胺類似物於生物分布試驗中與腫瘤、血液、肝、腎中的積聚(% ID/g)。 7
表 四、Carolina Moura等人合成之鎝-99m fac-螯合苯醯胺類似物之分配係數、與人工黑色素結合比例。 13
表 五、Carolina Moura等人合成之鎝-99m fac-螯合苯醯胺類似物之生物分布。 13
表 六、藥物9-Tc 於皮下種植B16F0之C57BL/6小鼠生物分布結果(%ID/g)。 36
表 七：藥物5-Tc、9-Tc、12-Tc藥物化學特性。 40
表 八、藥物5-Tc、9-Tc、12-Tc 與其他文獻報導之化合物化學特性、細胞攝取實驗之比較。 44
表 九、藥物9-Tc 於皮下種植B16F0之C57BL/6小鼠生物生物分布實驗之腫瘤對器官之比值。 46

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