硼化RGD胜肽之合成與硼中子捕獲治療之生物評估

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研究生：	劉育村 Yu-Tsuen Liu
論文名稱：	硼化RGD胜肽之合成與硼中子捕獲治療之生物評估 Synthesis of boronated RGD peptides and biological evaluation for BNCT
指導教授：	羅建苗 Jem-Mau Lo
口試委員:
學位類別：	碩士 Master
系所名稱：	原子科學院 - 生醫工程與環境科學系 Department of Biomedical Engineering and Environmental Sciences
論文出版年：	2007
畢業學年度：	95
語文別：	中文
論文頁數：	69
中文關鍵詞：	硼中子捕獲治療、胜肽、腦瘤、癌症
外文關鍵詞：	RGD, integrin, BNCT, U87-MG
相關次數：	點閱：4 下載：0
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硼中子捕獲治療(Boron Neutron Capture Therapy, 簡稱BNCT)是一種特殊而有效之腦瘤或其他腫瘤治療術。此治療術係以一適當之含硼藥物注入人體為腫瘤所吸收，再以熱中子或超熱中子照射，藉由10B與熱中子之核反應產生高線性能量轉移(LET)之α及7Li帶電粒子，殺死腫瘤細胞達到治療的效果。含硼藥物為正常組織細胞吸收之量應儘可能抑低(腫瘤/正常細胞吸收比至少大於4)，則正常組織的傷害可降至最低。
Integrin αvβ3為一與腫瘤血管新生及癌細胞轉移密切相關之細胞黏著受器(cell adhesion receptor)，活絡表現於腫瘤新生血管或其他新生血管之內皮細胞。基於具Arg-Gly-Asp (RGD)胺基酸序列之胜肽與此受器具有高度特異性親和力，本研究選擇一種單分子胜肽cRGDyK 及另一種雙分子胜肽E[cRGDyK]2，利用有機合成技術製備硼化RGD胜肽化合物，進而探討發展為硼中子捕獲治療腫瘤藥物之可行性。
本實驗包含幾個過程，4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan- 2-yl)benzoic aicd c(RGDyK) (簡稱TDB-c(RGDyK))及4-(4,4,5,5- tetramethyl-1,3,2-dioxaborolan-2-yl)benzoic aicd E[c(RGDyK)]2 (簡稱TDB-E[c(RGDyK)]2)二硼化RGD胜肽化合物之合成，TDB-c(RGDyK)及TDB-E[c(RGDyK)]2之產率分別為65%及48%；131I標誌硼化RGD化合物，131I-[TDB-c(RGDyK)]及131I-{TDB-E[c(RGDyK)]2}之標誌產率分別為96%及97%；穩定度測試，131I-[TDB-c(RGDyK)]及131I-{TDB-E[c(RGDyK)]2}貯存於PBS中48小時之放化純度分別維持在91%與89%，貯存於血清中48小時之放化純度則均維持在92%；脂溶性測試，131I-[TDB-c(RGDyK)]及131I-{TDB-E[c(RGDyK)]2}之logP值分別為-1.44±0.006及-1.67±0.016；對U87-MG細胞之化學毒性測試，加入TDB-c(RGDyK)及TDB-E[c(RGDyK)]2培養48小時之LD50分別為2.75 nM及2.57 nM；對U87-MG細胞αvβ3 integrin特異性結合試驗，加入131I-[TDB-c(RGDyK)]及131I-{TDB-E[c(RGDyK)]2}培養3.5小時之結合百分比分別為12.96±2.63% 及14.49±0.24%，經加入大量TDB-c(RGDyK)及TDB-E[c(RGDyK)]2抑制(blocking assay)後，結合百分比分別降至2.47±0.06%及6.16±0.87%；利用131I-E[(RGDK)]2為基準之競爭實驗，TDB-c(RGDyK)及TDB-E[c(RGDyK)]2之IC50分別為101.88 nM及61.62 nM。植有硼化RGD化合物之U87-MG細胞之中子照射效應試驗，加有500 nM (攝取為28%)TDB-c(RGDyK)之U87-MG細胞經熱中子 (熱中子通量為2.686×1012 n/cm2)照射後細胞存活率為27.97±5.75%；加有500 nM (攝取為28%) TDB- E[c(RGDyK)]2之U87-MG細胞經熱中子 (熱中子通量為2.686×1012 n/cm2)照射後存活率為24.10±2.84%。

Boron neutron capture therapy (BNCT) is a unique binary cancer therapy modality that a drug of boronated compound is injected in a patient and accumulated trap in a tumor followed by bombardment with thermal neutrons. Being RGD peptide able to specifically bind to αvβ3 integrin receptor that can be highly expressed in brain tumors such as glioblastoma, we have attempted to develop boronated RGD compounds as to be BNCT drugs for treatment of brain tumors. To incorporate RGD peptide with boron, 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2- yl)benzoic acid (STDBA) was synthesized and then conjugated to monomeric RGD peptide, c(RGDyK) and dimeric RGD peptide, E[c(RGDyK)]2 to produce the two boronated RGD peptides, TDB-c(RGDyK) and TDB-E[c(RGDyK)]2. The yields of TDB-c(RGDyK) and TDB-E[c(RGDyK)]2 were 65% and 48%, respectively. By 131I labeling, the labeled yields of the two boronated RGD compounds were both over 95%. The stabilities of 131I-[TDB-c(RGDyK)] and 131I-{TDB- E[c(RGDyK)]2} were high, being maintained at about 90% when stored either in both PBS and serum at ambient temperature for 48 h. 131I-[TDB- c(RGDyK)] and 131I-{TDB-E[c(RGDyK)]2} were found to be hydrophilic with log P = -1.44 ± 0.01 and -1.67 ± 0.02, respectively. In toxicity study, LD50 values of TDB-c(RGDyK) and TDB-E[c(RGDyK)]2 to U87-MG cells were found to be 2.57 nM and 2.75 nM for incubation of 48 h. The binding percentages for U87-MG cells of 131I-[TDB-c(RGDyK)] and 131I-{TDB-E[c(RGDyK)]2} were 12.96 ± 2.63% and 14.49 ± 0.24%, respectively for incubation of 3.5 h. By blocking with the large amounts of TDB-c(RGDyK) and TDB-E[c(RGDyK)]2, the binding percentages decreased to 2.47 ± 0.06% and 6.16 ± 0.87%, respectively. In competitive binding with 131I-E[c(RGDyK)]2, the obtained IC50 values for TDB- c(RGDyK) and TDB-E[c(RGDyK)]2 were 101.88 nM and 61.62 nM, respectively. Upon thermal neutron irradiation with a fluence of 2.69×1012 n•cm-2, the survival fractions of U87-MG cells incubated with 500 nM TDB-c(RGDyK) and 500 nM TDB-E[c(RGDyK)]2 were 27.97 ± 5.75% and 24.10 ± 2.84%, respectively, in contrast with 58.93 ± 5.50% in the case of irradiation of the cells alone.

目            錄

第一章  緒論                                              1
1.1  核子醫學簡介    1
1.2  硼中子捕獲治療術(BNCT)    1
1.3  腫瘤細胞之血管新生作用(angiogenesis)    5
1.4  RGD胜肽藥物之簡介                              9
1.5  研究方向與目的    11
第二章  材料與方法                                        14
2.1  試藥          14
2.2  儀器與材料    15
2.3  有機合成                                        16
2.3.1  Succinimidyl-4-(4,4,5,5-tetramethyl-1,3,2-
    dioxaborolan-2-yl)benzoic acid(STDBA)之合成    16
      2.3.2  STDBA與c(RGDyK)及E[c(RGDyK)]2之耦合反應17
           ● 反應步驟                                   18
           ● 高效能液相層析                             19
     2.4  放射性標誌                                      19
       2.4.1  131I標誌TDB-c(RGDyK)及TDB-E[c(RGDyK)]2    19
       2.4.2  131I-[TDB-c(RGDyK)]及131I-{TDB-E[c(RGDyK)]2}
之穩定度測試                               20
● 在PBS中之穩定度測試                      20
● 在血清中之穩定度                          21
2.4.3  131I-[TDB-c(RGDyK)]及131I-{TDB-E[c(RGDyK)]2}
之脂溶性測試                               21
    2.5  神經膠質瘤細胞之生長曲線                         22
       2.5.1  MEM培養基之配製                           22
       2.5.2  腫瘤細胞之培養                              22
       2.5.3  腫瘤細胞生長曲線測定                        23
    2.6  活體外細胞實驗 ( In vitro tests)                      23
     2.6.1  硼化RGD化合物對癌細胞之化學毒性測試(Cytotoxicity)                                23
       2.6.2  硼化RGD化合物對U87-MG細胞特異性結合
試驗                                       25
       2.6.3  硼化RGD化合物對U87-MG細胞結合競爭實驗   26
    2.7  熱中子束之照射實驗                               26
       2.7.1  含不同硼化RGD化合物之細胞經中子照射之存
活率測試                                      26
2.7.2  接受不同熱中子劑量之含硼化RGD化合物細胞
之存活率測試                               28
第三章  結果與討論    30
    3.1  有機合成                                         30
3.1.1  Succinimidyl-4-(4,4,5,5-tetramethyl-1,3,2-
      dioxaborolan-2-yl)benzoic acid(STDBA)之合成      30
    3.1.2  STDBA與c(RGDyK)及E[c(RGDyK)]2之耦合
反應                                         33
  3.2  放射性標誌                                       35
3.2.1  131I標誌TDB-c(RGDyK)及TDB-E[c(RGDyK)]2     35
      3.2.2  131I-[TDB-c(RGDyK)]及131I-{TDB-E[c(RGDyK)]2}
之穩定度測試                                  36
3.2.3  131I-[TDB-c(RGDyK)]及131I-{TDB-E[c(RGDyK)]2}
之脂溶性測試                                  36
    3.3  神經膠質瘤細胞之生長曲線                          37
    3.4 活體外細胞實驗 ( In vitro tests)                        37
      3.4.1  硼化RGD化合物對癌細胞之化學毒性測試
(Cytotoxicity)                                   37
      3.4.2  硼化RGD化合物對U87-MG細胞特異性結合
試驗                                          39
      3.4.3  硼化RGD化合物對U87-MG細胞結合競爭實驗    39
    3.5  熱中子束之照射效應                               40
      3.5.1  含不同硼化RGD化合物之細胞經中子照射之存
活率測試                                        40
3.5.2    接受不同熱中子劑量之含硼化RGD化合物細胞
之存活率測試                                  42
第四章 結論                                                65
第五章 參考文獻                                           66


























表目錄

表3-1. 131I-[TDB-c(RGDyK)]之穩定度                         46
表3-2. 131I-{TDB-E[c(RGDyK)]2}之穩定度                     46
表3-3. U87-MG細胞株資料表                                46
表3-4. 植有含硼化合物之腫瘤細胞經中子照射後存活率之比較   47
表3-5. 接受不同熱中子通量下腫瘤細胞之存活率 (%)           47













圖目錄

圖1-1. 硼中子捕獲治療基本原理                              2圖1-2. 腫瘤細胞藉血管新生作用遷移及其造成之免疫系統破壞    8
圖1-3. RGD藥物與αvβ3 integrin之鍵結力                9
圖1-4. cRGD藥物之基本結構圖                              11
圖1-5.    RGD胜肽藥物較生物體內之細胞外基質蛋白有更強之
αvβ3 integrin親和力                                 13
圖1-6.    RGD胜肽藥物當作腫瘤追蹤造影劑或治療劑之示意圖    13
圖3-1. STDBA之1H核磁共振光譜圖                         48
圖3-2. STDBA之13C核磁共振光譜圖                         49
圖3-3. STDBA之DEPT核磁共振光譜圖                      50
圖3-4. STDBA之ESI+質譜圖                                51
圖3-5. STDBA之HPLC圖 (以UV254nm偵測)                     52
圖3-6. STDBA之HPLC圖 (以UV280nm偵測)                   52
圖3-7. c(RGDyK)之HPLC圖 (以UV254nm偵測)                 53
圖3-8. c(RGDyK)之HPLC圖 (以UV280nm偵測)                 53
圖3-9. E[c(RGDyK)]2之HPLC圖 (以UV254nm偵測)             54
圖3-10. E[c(RGDyK)]2之HPLC圖 (以UV280nm偵測)            54
圖3-11. TDB-c(RGDyK)之HPLC圖 (以UV254nm偵測)           55
圖3-12. TDB-c(RGDyK)之HPLC圖 (以UV280nm偵測)           55
圖3-13. TDB-E[c(RGDyK)]2之HPLC圖 (以UV254nm偵測)        56
圖3-14. TDB-E[c(RGDyK)]2之HPLC圖 (以UV280nm偵測)        56
圖3-15. TDB-c(RGDyK)之MALDI-TOF-MS圖譜               57
圖3-16. TDB-E[c(RGDyK)]2MALDI-TOF-MS圖譜               58
圖3-17. 131I-[TDB-c(RGDyK)]之TLC分析圖                    59
圖3-18. 131I-{TDB-E[c(RGDyK)]2}之TLC分析圖                59
圖3-19. [131I]NaI之TLC分析圖                              60
圖3-20. U87-MG細胞之生長曲線                             60
圖3-21. c(RGyD)、E[c(RGDyK)]2、TDB-c(RGyD)及TDB- E[c(RGDyK)]2 對U87-MG細胞之簡略毒性測試        61
圖3-22. TDB-c(RGD)及TDB-E[c(RGDyK)]2 對U87-MG細胞之
毒性測試曲線                                     61
圖3-23. 131I-[TDB-c(RGDyK)]對具αvβ3 integrin表現之U87-MG細胞特異性結合試驗                                   62
圖3-24. 131I-{TDB-E[c(RGDyK)]2}對具αvβ3 integrin表現之U87-MG細胞特異性結合試驗                               62
圖3-25. TDB-c(RGDyK)及TDB-[c(RGDyK)]2與131I-E[c(RGDyK)]2競爭U87-MG細胞結合曲線                           63
圖3-26. 含不同濃度硼化RGD化合物細胞經中子照射之存活率   63
圖3-27. 接受不同中子劑量之含硼化RGD化合物細胞存活率     64

                                

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