評估插管小鼠經靜脈及口服多次劑量投與質體 DNA (pMBP-Lac Z) 聚合微膠體溶液之藥物動力學

1. 評估插管小鼠經靜脈及口服多次劑量投與質體DNA (pMBP-Lac Z) 聚合微膠體溶液之藥物動力學 • 發展一個新的基因載體，評估其質體DNA 及質體DNA 與載體之複合物，在體內之組織分佈情形是必要的。而本次實驗所利用的質體DNA，為帶有寡樹突神經細胞上的特殊蛋白質基因 - 髓鞘基質蛋白質 (myelin basic protein, MBP)基因的啟動子之pMBP-Lac Z，評估經由靜脈及口服投與三個劑量pMBP-Lac Z (40?g DNA/150 ?L) 與pMBP-Lac Z 聚合微膠體溶液到插管小鼠，其體內 pMBP-Lac Z 之藥物動力學參數及體內之分佈情形。血液及組織檢品利用即時聚 合酶連鎖反應法 (Q-PCR) 分析檢品中pMBP-Lac Z 含量。實驗初步結果顯示， 在頸靜脈投與(IVJ)三次總劑量後之血液檢品中發現，pMBP-Lac Z (P) 之曲線下 面積(area under the curve, AUC) 為1.56 x 1012 (min x copies/?L)，投與pMBP-Lac Z 聚合微膠體溶液 (P/PM) 之AUC 為5.29 x 1011 (min x copies/?L)。而排除半衰 期的部份，投與P 為436 分鐘，投與P/PM 為344 分鐘。經由尾靜脈投與 (IVT) P/PM 後的血中濃度曲線圖發現， AUC 為5.12 x 1011 (min x copies/?L)，其半衰 期為97 分鐘。口服投與三個劑量的P/PM，其AUC 為1.73 x 107 (min x copies/?L) ，半衰期為58 分鐘。利用IVJ 和IVT 投與單一劑量P 和P/PM 的5, 30 和60 分鐘後，觀察到DNA 主要分佈於心臟、肺臟、腦、脊髓和腎臟等高血流 的組織，若經由口服投與P 和P/PM 後，以十二指腸及胃部組織為主要分佈組織。 此外，體外DNase 安定性試驗發現，PM 存在下確實能對DNA 有保護效果，而 pMBP-Lac Z 在體外的血漿安定性試驗發現，不論是P 或P/PM 之安定性，在20 分鐘都已觀察不到完整之pMBP-Lac Z 存在，若是胃沖洗液的安定性試驗，則是 P 或P/PM 皆在60 分鐘才開始出現DNA 片段。綜合以上結果，體內實驗發現， 不同投與途徑下確實會影響其體內的組織分佈情形。

2. Evaluation of pharmacokinetics of pMBP-Lac Z with polymeric micelles via multiple-dose intravenous and oral administrations in cannulated mice • A clear understanding of the tissue distribution of plasmid DNA and its complexes with nonviral vectors is a prerequisite for a strategy for developing novel in vivo gene transfer methods. Therefore the aim of this study was to evaluate the pMBP-Lac Z plasmid DNA, which is driven by specific, myelin basic protein（MBP） gene promoter into oligodendrocytes, with or without polymeric micelles (PM) in the systemic distribution of pharmacokinetics (PK) profiles via 3 dose (40 ug DNA/150 uL) of jugular vein injection (IVJ), tail vein injection (IVT) or oral administration in cannulated mice. The distribution of pharmacokinetics profiles were quantitated using real-time quantitation polymerase chain reaction (Q-PCR). After 3 dose of multiple jugular vein injection (IVJ) , the area under the curve (AUC) of pMBP-Lac Z (P) and with polymeric micelles (P/PM) in plasma were 1.56 x 10^12 (min x copies/μL) and 5.29 x 10^11 (min x copies/uL), respectively. And the elimination half-lives of pMBP-Lac Z (P) and with PM (P/PM) in plasma were 436 min and 344 min, respectively. For 3 dose of multiple tail vein injection (IVT), the AUC of P/PM in plasma was 5.12 x 10^11 (min x copies/uL), and the elimination half-lives was 97 min. After 3 dose of multiple oral administrations, the AUC was 1.73 x 10^7 (min x copies/uL), and the elimination half-lives was 58 min. At 5, 30 and 60 min after single IVJ or IVT of P or P/PM, the higher tissue distribution was found in heart, lung, brain, spinal cord and kidney tissues which were high blood flow tissues. Then, after single oral administration, the duodenum and stomach were the major distributed tissues. The in vitro DNAse stability test of pMBP-Lac Z was confirmed that the PM can protect the DNA, and the in vitro serum stability of pMBP-Lac Z were indicated that both of with or without formulated were not detectable within 20 min. And the in vitro gastric washing fluid stability of pMBP-Lac Z were indicated that both of with or without PM had degradation at 60 min. The different administration routes suggest influence the biodistribution of plasmid.