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研究生:溫季琦
研究生(外文):Chi-chi Wen
論文名稱:HPW98-1活性代謝物之研究
論文名稱(外文):Studies on the Active Metabolites of HPW98-1
指導教授:王惠珀王惠珀引用關係
學位類別:碩士
校院名稱:長庚大學
系所名稱:天然藥物研究所
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:2003
畢業學年度:91
語文別:中文
中文關鍵詞:活性代謝物
外文關鍵詞:active metabolites
相關次數:
  • 被引用被引用:2
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本實驗室先前合成的HPW98-1不僅能選擇性毒殺ras-基因變異引起的癌細胞包括直腸癌、胰臟癌及攝護腺癌等而不傷害正常細胞,且具有抗血管增生之作用。但鑒於HPW98-1之半衰期短卻具強力抗癌療效,推測其可能原因為於體內代謝成活性代謝物。因而本研究藉由分析鑑定投予大白鼠HPW98-1後尿液中之代謝物,及合成可能之代謝物,分別與投予HPW98-1之大白鼠血液比對,進而探討HPW98-1及其代謝物之藥物動力學行為。
腹腔注射給予Wistar雄性大白鼠HPW98-1 (40 mg/kg),分段收集尿液,經LC/MS/MS分離鑑定後,確認兩種代謝物之結構,並分別命名為HPW071X001 glucuronides,HPW071X004 glucuronides/sulfates。另外,合成三種可能代謝物HPW071X001, HPW071X002及HPW071X003,分別進行體外及體內之生物活性評估。利用MTT assay分別對於HL-60細胞株及SW480人類直腸癌細胞株做細胞毒性測試。結果顯示,只有HPW071X002對此兩株細胞之抑制效果具統計上之意義(P < 0.05)。另外,於抗血管增生之活體試驗,大白鼠分別口服三種合成代謝物皆明顯抑制VEGF,但只有HPW071X001於低劑量(10 mg/kg)即有明顯的作用。
  基於HPW071X001為HPW98-1之有效活性代謝物,進一步探討其於鼠體內之藥物動力學並與其原型藥HPW98-1比較之。給予大白鼠口服HPW98-1 (70 mg/kg),依時間點心臟採血。血漿檢品以高效液相層析法(HPLC)分析,採用Cosmosil RP-18 管柱,偵測波長為280 nm,HPW98-1之移動相組成為0.1M醋酸銨緩衝溶液:乙腈(4:6),而HPW071X001則為由0.1M醋酸銨緩衝溶液、乙腈、甲醇組成的梯度沖提溶液。兩者之同日內及異日間之確效結果顯示精密度及準確度良好,皆在15%以內,回收率在85%以上。定量極限(LOQ)分別為0.02及0.04 g/mL,偵測極限(LOD)分別為0.01及0.02 g/mL。所得血漿濃度,利用WINNONLIN軟體以非室性模式分析,求得藥動學參數。HPW98-1與HPW071X001之AUC0-t分別為796.5±649.7與248.1±80.5 min*nmol/mL,Cmax分別為3.6±1.1與0.7±0. 2 nmol/mL,T1/2分別為135.1±27.2與671.2±206.3 min。基於AUC0-t之比較,約31% 之HPW98-1代謝成HPW071X001。
本研究完成HPW98-1之部分代謝物的鑑定、合成、定量方法之建立以及HPW98-1代謝成HPW071X001之動力學研究,並發現此等代謝物亦具抗血管增生活性,深具開發為抗癌及防治心血管疾病藥物之潛力。另外,對於其他之可能代謝物如HPW071X004等,亦值得日後進一步探討,可提供更完整之HPW98-1活性代謝物應用之可能性。
HPW98-1, a bioactive compound synthesized by our laboratory, showed not only selectively killed mutant ras-gene induced cancer cells, including colon, pancreatic and prostate cancer without injury normal cells, but also demonstrated anti-angiogenesis effects both in in vitro HUVEC culture and in vivo mice model. However, in view of its short half-life, the potent anticancer efficacy might be resulted from its possible bioactive metabolites. Therefore, in this study, the metabolites in urine were identified after administration of HPW98-1 to Wistar rats and three possible metabolites were synthesized. They were compared with blood withdrawn from rat dosing with HPW98-1, furthermore, the pharmacokinetics of HPW98-1 and its metabolite were investigated.
After ip administration of HPW98-1 (40 mg/kg) to rats, urine were collected at pre-determined time points and then identified by LC/MS/MS. The metabolites were named as HPW071X001 glucuronides, and HPW071X004 glucuronides/sulfates. Three possible metabolites of HPW98-1, HPW071X001, HPW071X002, and HPW071X003 were synthesized and their biological activities were evaluated. The cytotoxicity test (MTT assay) were performed on HL-60 and SW480 cell lines. The results indicated that only HPW071X002 showed significant inhibition (P<0.05) for both cell lines. Using in vivo mice model, the anti-angiogenesis effect was shown for all three drugs whereas only HPW071X001 showed significant effect at low dose (10 mg/kg, oral).
As a potent bioactive metabolite of HPW98-1, HPW071X001 was further investigated for its pharmacokinetics in rats and comparision was made with its parent compound after oral administration of HPW98-1 (70 mg/kg). The blood were withdrawn via cardiopuncture at pre-determined time points and then assayed by HPLC method. The analysis of HPW98-1 and HPW071X001 were performed on Cosmosil RP-18 column with an UV detection at 280 nm. The mobile phase for HPW98-1 was consisted of 0.1M ammonia acetate: acetonitrile (4:6) whereas that of gradient elution for HPW071X001 was consisted of 0.1M ammonia acetate, acetonitrile and methanol. The precision and accuracy of both methods for intra-day and inter-day assays were less than 15%,the recovery was higher than 85%. The limit of quantitation (LOQ) was 0.02 and 0.04 g/mL, respectively. The limit of detection (LOD) was 0.01 and 0.02 g/mL, respectively. Pharmacokinetics parameters were calculated from plasma concentrations by WINNOLIN using non-compartment model. The AUC0-t of HPW98-1 and HPW071X001 were 796.5±649.7and 248.1±80.5 min*nmol/mL , the Cmax were 3.6±1.1 and 0.7±0. 2 nmol/mL, and the T1/2 were 135.1±27.2 and 671.2±206.3 min, respectively. About 31% of HPW98-1 was metabolized into HPW071X001 when calculated on their AUC0-t basis.
In this study, some metabolites of HPW98-1 were identified/synthesized. The determination methods for HPW98-1 and HPW071X001 were developed and their pharmacokinetics were investigated. Because their anti-angiogenesis effect was also demonstrated, the potential of being developed as an anticancer or cardiovascular disease preventive/therapeutic drugs was expected. Moreover, other possible metabolites are worthy of investigation, in order to provide more complete and possible application of bioactive HPW98-1 metabolites.
壹、研究背景及動機…………………………………………………….1
貳、研究方法………………………………………………………….....4
一、HPW98-1可能之代謝途徑……………………………………4
二、活性代謝物的合成…………………………………………….5
三、細胞生長抑制實驗…………………………………………….6
參、實驗儀器與材料…………………………………………………….7
一、儀器與材料…………………………………………………….7
二、溶液之配製…………………………………………………….9
三、實驗動物………………………………………………………10
肆、實驗設計與方法……………………………………………………11
一、代謝物之合成…………………………………………………11
二、代謝試驗………………………………………………..,……14
1、動物實驗…………………………………………………14
2、高效液相層析串聯質譜儀 (LC-MS-MS)分析條件
之建立…………………………………………………..15
三、代謝物之細胞毒性…………………………………………..16
四、HPW98-1之代謝動力學研究……………………………….20
1、動物口服給藥藥物動力學實驗………………………..20
2、HPW98-1, HPW071X001之分析條件與分析方法
之確效…………………………………………………..22
3、數據分析及統計方法…………………………………..24
伍、結果與討論…………………………………………………………27
一、代謝試驗……………………………………………..………..27
二、代謝物之細胞毒性結果………………………………………34
三、HPW98-1活性代謝物HPW071X001之藥物動力學……….. 38
1、HPW98-1分析方法之確效………………………………40
2、HPW071X001分析方法之確效…………………………44
3、HPW98-1及HPW071X001於鼠體內之藥物動力學……48
陸、結論…………………………………………………………………53
柒、參考文獻……………………………………………………………54
捌、圖譜…………………………………………………………………59
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