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研究生:蔡宜珊
研究生(外文):Yi-Shan Tsai
論文名稱:口服聚合微膠體包覆pMBP-LacZ之藥物動力學
論文名稱(外文):Pharmacokinetics of oral administered pMBP-Lac Z with polymeric micelles
指導教授:廖嘉鴻
學位類別:碩士
校院名稱:臺北醫學大學
系所名稱:藥學研究所
學門:醫藥衛生學門
學類:藥學學類
論文種類:學術論文
論文出版年:2007
畢業學年度:95
語文別:中文
論文頁數:43
中文關鍵詞:藥物動力學質體DNA
外文關鍵詞:pharmacokineticsplasmid DNA
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先前本實驗室曾使用非離子型聚合微膠體當作口服基因的載體,於48小時內投與六個劑量後,發現質體DNA不僅於腸胃道有表現,還能由血液分佈至較遠的組織及器官中,包括腦及脊髓。為了進一步了解質體DNA於體內分佈的情形;合適的藥物劑量與多劑量給藥之間距等因此本實驗分別由靜脈及口服投與單一劑量(40 μg/150μL) pMBP-Lac Z與同劑量經微膠體包覆的pMBP-Lac Z 至裸鼠體內,評估pMBP-Lac Z之分布及其藥物動力學。於不同時間點下採集裸鼠血液及組織(包括十二指腸,肝臟,脊髓,與腦) ,經過萃取得到total DNAs,使用同步定量PCR (Q-PCR)分析檢品中Lac Z的含量。本實驗採用絕對定量方式分析檢品中Lac Z DNA的含量,因此以濃度為10^2 copies/μL到10^8 copies/μL的純的pMBP-Lac Z DNA作為標準曲線。在此濃度範圍內,標準曲線的對數值呈線性關係,並以同日內及異日間的數值來評估本實驗分析方法的精確性與準確性,其變異係數均小於15%。實驗結果顯示,利用靜脈注射方式投與後,於血液檢品中發現,未包覆之pMBP-Lac Z的area under the curve (AUC)為1.37x10^8 (min x pg/mL),而微膠體包覆之pMBP-Lac Z於血AUC為3.88x10^7 (min x pg/mL),兩者間無統計上的差異。但經過微膠體包覆後的pMBP-Lac Z的排除半衰期由54.5分鐘延長為210.5分鐘。另一方面,在口服投與後於血液檢品中未包覆之pMBP-Lac Z其 AUC值為9.29 (min x pg/mL),使用聚合微膠體包覆者為6.09x10^3 (min x pg/mL)。此外可觀察到pMBP-Lac Z在體內的排除半衰期從46.6分鐘延長到257分鐘。由結果計算其不包覆與包覆之相對口服生體可用率時,約增加600倍左右。此外,聚合微膠體包覆pMBP-Lac Z經由靜脈注射後,其AUC於十二指腸、肝臟、脊髓及腦分別為170.6,108.3,1086.6,182.6 (min x pg/g gDNA)。未包覆者於上述四種器官中的AUC分別為127.6,199.0,463.8,187.4 (min x pg/g gDNA)。聚合微膠體包覆之pMBP-Lac Z經由口服投與後,於十二指腸、肝臟、脊髓及腦中的AUC分別為331.5,0.26,22.2,9.93 (min x pg/g gDNA),除了小腸之AUC外,其於組織的AUC均小於靜脈注射之值。口服投與未包覆pMBP-Lac Z於十二指腸,脊髓,與腦中的AUC,分別為57.5,1.39,1.8 (min x pg/g gDNA) ,其AUC值均小於相同投與途徑包覆之pMBP-Lac Z。綜合上述結果,聚合微膠體包覆pMBP-Lac Z能增加pMBP-Lac Z之口服生體可用率。此外,利用口服投與聚合微膠體包覆質體DNA,比未包覆者相比有較高的AUC值。然而,經靜脈投與後,於血液中pMBP-Lac Z 有最高的AUC值。
We have previously used the nonionic polymeric micelles (PM) as a carrier for oral gene delivery given at six doses within 48 hours, and the results showed that the delivered gene expression was detected in GI, plasma, and other tissues and organs, including brain and spinal cord. In order to optimize the ideal dose and dose regimen, we evaluate the distribution and pharmacokinetic profile of pMBP-Lac Z DNA (40 μg/150 μL) delivered with or without polymeric micelles to nude mice orally and intravenously. The plasma and tissue samples (duodenum, liver, spinal cord, and brain) were collected at various time points, and total DNAs were extracted for Lac Z DNA quantitation using real-time quantitative polymerase chain reaction (QPCR). To absolutely quantitate Lac Z DNA in the samples, a standard curve was generated using purified pMBP-Lac Z DNA in a range of 10^2-10^8 copies/μL. Within the log linear range of standard DNA at 10^2 to 10^8 copies/μL, we obtained the CV% of within- and between-day assays were all less than 15%, indicating the precision and accuracy of the method used in this study. After IV administration, the area under the curve (AUC) of pMBP-Lac Z and formulated pMBP-Lac Z in plasma was 1.37x10^8 (min x pg/mL) and 3.88x10^7 (min x pg/mL), respectively, which were no significant different. But the formulated pMBP-Lac Z had prolonged elimination half-lives (from 54.5 to 210.5 min). On the other hand, the area under the curve (AUC) of pMBP-Lac Z and formulated pMBP-Lac Z in plasma was 9.29 (min x pg/mL) and 6.09x10^3 (min x pg/mL), respectively, after orally administration. In addition, the formulated pMBP-Lac Z had prolonged elimination half-lives (from 46.6min to 257min). The results indicated that the relative oral bioavailability of formulated pMBP-Lac Z was 600 folds of that of the naked pMBP-Lac Z. Furthermore, after IV administration, the AUC values of formulated pMBP-Lac Z in duodenum, liver, spinal cord and brain were 170.6, 108.3, 1086.6, 182.6 (min x pg/g gDNA), respectively. For naked pMBP-Lac Z, the AUC values in the above organs were 127.6, 199.0, 463.8, 187.4 (min x pg/g gDNA), respectively. The AUC values of formulated pMBP-Lac Z via oral administration in duodenum, liver, spinal cord, and brain were 331.5, 0.26, 22.2, 9.93 (min x pg/g gDNA). Most of the AUC values were smaller than that detected after IV administration, except that detected in duodenum. For the naked pMBP-Lac Z in duodenum, spinal cord and brain, the AUC values were 57.5, 1.39, 1.8 (min x pg/g gDNA) and were lower than that detected with formulated DNA. Overall, these results indicated that PM formulation enhanced the bioavailability of pMBP-Lac Z. In addition, PM-formulated pMBP-Lac Z shows higher AUC than naked pMBP-Lac Z in all tissues after oral administration. However, the highest AUC level of pMBP-Lac Z was observed in the plasma after IV administration.
中文摘要 i
Abstract iii
附表目錄 v
附圖目錄 vii
壹、緒論 1
一、大分子藥物 1
1.1蛋白質藥物 1
1.2核酸藥物 2
二、非病毒型基因載體 3
2.1微脂粒 (Liposome) 4
2.2聚合物 5
2.2.1陽離子性聚合物 (cationic polymer) 5
2.2.2聚合微膠體(Polymeric micelles) 5
三、聚合微膠體在大分子藥物傳遞之運用 7
3.1蛋白質藥物 7
3.2核酸藥物 7
3.3抗癌藥物 7
四、基因藥物投與途徑的選擇 8
五、核酸藥物之藥物動力學 8
貳、研究目的 10
參、研究材料與方法 10
一、質體DNA的製備 10
1.1 pMBP-Lac Z質體之製備與純化 11
二、聚合微膠體之臨界微膠體濃度測定 11
三、質體/聚合微膠體 (plasmid/ polymeric micelles) 製備 (P/PM) 12
四、質體/聚合微膠體溶液之物化性質 12
4.1質體/聚合微膠體粒徑大小測定 12
4.2質體/聚合微膠體表面電位測定 12
五、pMBP-Lac Z之同步定量PCR (Real-time quantitative PCR)分析方法 12
5.1同步定量PCR之實驗流程 13
5.2標準檢量曲線之製作 14
5.3分析方法精確性及準確性試驗 14
5.4PCR 反應產物之確認 14
六、動物實驗 15
6.1裸鼠(BALB/c-nu)15
6.2口服及靜脈注射投與質體DNA/聚合微膠體溶液 (P/PM) 及質體DNA 15
七、血漿檢品中質體DNA之分離 16
八、組織中基因體DNA (genomic DNA, gDNA) 之分離 16
九、藥物動力學參數與統計分析 17
肆、研究結果與討論 18
一、聚合微膠體臨界膠體濃度之測定 18
二、聚合微膠體溶液之物化性質 18
2.1聚合微膠體溶液之粒徑 (Particle size) 18
2.2聚合微膠體溶液之表面電位 18
三、pMBP-Lac Z之QPCR分析方法之確效 18
3.1QPCR之標準檢量線 18
3.2QPCR分析方法之產物專一性確認 19
3.3分析方法精確性及準確性試驗 20
四、動物實驗之結果 20
4.1pMBP-Lac Z於裸鼠血液中之藥物動力學 20
4.2pMBP-Lac Z於裸鼠十二指腸中之藥物動力學 21
4.3pMBP-Lac Z於裸鼠肝臟中之藥物動力學 21
4.4pMBP-Lac Z於裸鼠脊髓中之藥物動力學 22
4.5pMBP-Lac Z於裸鼠腦中之藥物動力學 22
伍、結論 24
參考文獻 40
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