Biodistribution and Pharmacokinetics of RGD Conjugated and 188Re and Doxorubicin Encapsulated Liposome in U-87 MG Tumor Bearing Mice

RGD胜肽耦合與188Re及Doxorubicin包埋之微脂體在植有U-87 MG腫瘤小鼠之生物分佈與藥動研究

林翊翔(I-Hsiang Lin)

指導教授 ： 羅建苗 ； 李德偉

國立清華大學/原子科學院/生醫工程與環境科學系/碩士(2008年)

摘要

聚乙二醇微脂體小紅莓(Polyethylene glycolated (PEGylated) liposomal doxorubicin (LIPO-DOX®))為一臨床腫瘤之化療藥物。LIPO-DOX®係藉由滲透及滯留增強效應(enhanced permeability and retention (EPR) effect)傳輸doxorubicin (DOX)至腫瘤組織。將此包埋DOX之微脂體耦合具腫瘤特異性標靶之配位子(specific tumor targeting ligand)為一值得嘗試增加藥物累積至腫瘤組織之有用策略。此外，同時包埋188Re於此微脂體可增加放射治療及診斷造影之用途。
方法：首先將對整合素(Integrin) αvβ3具標靶特性之配位子RGD胜肽(c(RGDyK)及E[c(RGDyK)]2)耦合至PEGylated liposomes，再將188Re(以188Re-BMEDA形態)及DOX包埋至微脂體。分別得到兩種RGD胜肽耦合與188Re及DOX包埋之微脂體，簡稱c(RGDyK)-PL-DOX-188Re及E[c(RGDyK)]2-PL-DOX-188Re。同時亦將188Re包埋於LIPO-DOX®，簡稱LIPO-DOX-188Re。將c(RGDyK)-PL-DOX-188Re 和E[c(RGDyK)]2-PL-DOX-188Re連同未具RGD胜肽耦合之LIPO-DOX-188Re以U-87 MG進行細胞親和力試驗，進而以植有U-87 MG腫瘤小鼠，進行生物分佈、藥物動力學及造影等試驗及比較。
結果：在U-87MG親和力試驗，依此腫瘤細胞攝入DOX之量，顯示c(RGDyK)-PL-DOX和E[c(RGDyK)]2-PL-DOX分別比LIPO-DOX高出7倍和6倍之多。生物分佈試驗顯示c(RGDyK)-PL-DOX-188Re及E[c(RGDyK)]2-PL-DOX-188Re 與LIPO-DOX-188Re在植有U-87 MG腫瘤小鼠中，有相似腫瘤放射活性累積。但LIPO-DOX-188Re之腫瘤/血液比(tumor-to-blood ratio)卻小於c(RGDyK)-PL-DOX-188Re 或E[c(RGDyK)]2-PL-DOX-188Re之腫瘤/血液比。在生物分佈試驗中各個時間點，LIPO-DOX-188Re之脾臟放射活性累積皆甚高於c(RGDyK)-PL-DOX-188Re或E[c(RGDyK)]2-PL-DOX-188Re之脾臟放射活性累積。在血液藥物動力學試驗之排除半衰期(elimination half-life (T1/2))和平均滯留時間(mean residence time (MRT))顯示c(RGDyK)-PL-DOX-188Re(T1/2 = 25.94 h, MRT = 12.1 h) 和E[c(RGDyK)]2-PL-DOX-188Re(T1/2 = 26.79 h, MRT = 11.57 h)皆大於LIPO-DOX-188Re (T1/2 = 13.29 h, MRT = 11 h)。 c(RGDyK)-PL-DOX-188Re 和E[c(RGDyK)]2-PL-DOX-188Re在植有U-87 MG腫瘤小鼠，經靜脈注射之16及24小時後microSPECT/CT影像顯示有些微可見的腫瘤放射活性累積，但在同實驗時間點，LIPO-DOX-188Re之microSPECT/CT影像卻幾乎無腫瘤放射活性累積。
結論：RGD胜肽耦合之微脂體c(RGDyK)-PL-DOX-188Re及E[c(RGDyK)]2-PL-DOX-188Re，比較無RGD胜肽耦合之微脂體LIPO-DOX-188Re，由細胞試驗結果，有頗為顯著U-87 MG親和力，由生物分佈結果，在脾臟累積之量顯著降低(幾近一半)，由腫瘤/血液比顯示腫瘤累積量略有增加。本研究必須進一步在腫瘤抑制效力及在植有腫瘤老鼠之存活率進行試驗，以證實LIPO-DOX-188Re、c(RGDyK)-PL-DOX-188Re與E[c(RGDyK)]2-PL-DOX-188Re之治療能力。

關鍵字

聚乙二醇微脂體 ； c(RGDyK) ； E[c(RGDyK)]2 ； 小紅莓 ； 188Re ； 細胞攝入 ； 生物分佈 ； 藥物動力學 ； 微型單光子電腦斷層融合影像儀

並列摘要

The use of polyethylene glycolated (PEGylated) liposomal doxorubicin (LIPO-DOX®) for clinical chemotherapy of cancers is well known. While LIPO-DOX® can accumulate DOX in tumor tissues due to the enhanced permeability and retention (EPR) effect, further conjugation of specific tumor targeting ligand to the doxorubicin (DOX) encapsulated liposome may be a very useful strategy to improve the tumor uptake and antitumor efficacy. On the other hand, 188Re encapsulated liposomes may have potential applications in radiotherapy and diagnostic imaging.
Methods: In this study, Arg-Gly-Asp (RGD) peptides with a high affinity for αvβ3 integrin were conjugated to the PEGylated liposomes, where two types of RGD, c(RGDyK) and E[c(RGDyK)]2 were used. 188Re-BMEDA and DOX were subsequently encapsulated into the liposomes. The resulted liposomes are referred to c(RGDyK)-PL-DOX-188Re and E[c(RGDyK)]2-PL-DOX-188Re. 188Re-BMEDA was also encapsulated into LIPO-DOX to produce the liposome referred to LIPO-DOX-188Re. U-87 MG cells were used for in vitro bioactivity test for the three liposomes. For animal trial, the main works included investigation of biodistribution, pharmacokinetics and imaging of c(RGDyK)-PL-DOX-188Re and E[c(RGDyK)]2-PL-DOX-188Re in comparison with non RGD conjugated liposome, LIPO-DOX-188Re using female athymic nude mice bearing subcutaneous U-87 MG glioblastoma tumors.
Results: The cellular uptake of DOX for c(RGDyK)-PL-DOX and E[c(RGDyK)]2-PL-DOX in the U-87 MG cells were about seven-fold and six-fold higher than that for LIPO-DOX, respectively. The biodistribution studies indicated that the three liposomal formulations showed very close activity accumulation in the tumor, but tumor-to-blood ratio for LIPO-DOX-188Re being lower than that for c(RGDyK)-PL-DOX-188Re or E[c(RGDyK)]2-PL-DOX-188Re. The spleen uptake of LIPO-DOX-188Re was significantly higher than that of c(RGDyK)-PL-DOX-188Re and E[c(RGDyK)]2-PL-DOX-188Re at all time points examined. Pharmacokinetic studies revealed that the elimination half-life (T1/2) and mean residence time (MRT) of c(RGDyK)-PL-DOX-188Re (T1/2 = 25.94 h, MRT = 12.1 h) and E[c(RGDyK)]2-PL-DOX-188Re (T1/2 = 26.79 h, MRT = 11.57 h) in blood were longer than that of LIPO-DOX-188Re (T1/2 = 13.29 h, MRT = 11 h), respectively. The coronal microSPECT/CT images of c(RGDyK)-PL-DOX-188Re and E[c(RGDyK)]2-PL- DOX-188Re showed their significant accumulations in the tumor at 16 and 24 h postinjection. In contrast, the coronal microSPECT/CT image of LIPO-DOX-188Re became almost invisible in the tumor at 16 and 24 h postinjection.
Conclusion: The results suggest that the use of RGD conjugation to the 188Re and DOX encapsulated liposome is beneficial to pronouncedly reduce the accumulation in the spleen and simultaneously enhance slightly higher specific tumor accumulation. Further studies such as tumor inhibitory activities and mice survival prolongation in tumor-bearing mice are necessary to confirm therapeutic efficacy for LIPO-DOX-188Re, c(RGDyK)-PL-DOX-188Re and E[c(RGDyK)]2-PL-DOX-188Re.

並列關鍵字

PEGylated liposomes ； c(RGDyK) ； E[c(RGDyK)]2 ； doxorubicin ； 188Re ； cellular uptake ； biodistribution ； pharmacokinetics ； microSPECT/CT

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