- 學位論文
建構非固醇類抗發炎藥穿皮吸收之結構與滲透定量關係式與使用高效液相層析串聯質譜儀分析神經醯胺與甘油磷脂及鞘脂質
Construction of a Quantitative Structure Permeability Relationship (QSPR) for the Transdermal Delivery of NSAIDs and Profiling of Ceramides and Glycerophospholipids and Sphingolipid by LC-MS
摘要
本研究將建立一非類固醇抗發炎藥物(non-steroidal anti-inflammatory drugs, NSAIDs)經皮傳遞之結構與滲透量化關係(quantitative structure permeability relationship, QSPR)的實驗模式來預測滲透係數(permeability coefficients, kp)。選取十三種不同非類固醇抗發炎藥物來進行裸鼠全皮穿皮體外試驗。而其相關的裸鼠皮生理參數經皮水分散失(transepidermal water loss, TEWL)、皮膚水分含量(hydration content, HD)、皮膚油脂含量(lipid content, SB)、皮膚纖維彈性及方向性 (elasticity and direction of the fibers, RVM)及皮膚彈性係數(elasticity, EL)也被測量。將全部藥物根據不同軟體系統所得到的藥物極性度分成三個資料庫且將這三個資料庫中的藥物再依極性度(clogP 或 logKo/w)分為大於2及小於2兩組。而後根據模式藥物有關的物理特性分子量及極性度或溶解因子(solubility parameter)在有或無考慮皮膚的生理參數下進行迴歸。結果顯示，不管有沒有考慮裸鼠皮的生理參數，clogP 或 logKo/w值小於2的藥物組，當加入溶解因子進行迴歸後其所得到的相關係數(adjusted R2)大於0.90以上，相較於只有使用clogP 或 logKo/w所得到的相關係數有顯著提升。這個結果說明藥物的溶解因子可能是一個較適合用來評估預測NSAIDs藥物穿皮吸收的影響因子。將觀察所得的滲透係數對經由QSPR預測的滲透係數作圖，其結果證明此關係式具有預測經皮傳遞滲透係數的能力。結論得知，將clogP 或 logKo/w值小於2的模式藥物加上溶解因子及裸鼠皮的生理參數下所得到的預測QSPR關係式可以得到最理想的預測結果。另一方面，一個快速、敏感度高且準確性佳的高效液相層析串聯質譜儀分析方法(LC-MS)將用來分析脂質包括裸鼠皮皮膚中重要且複雜的神經醯胺物質，及大腦內主要脂質甘油磷脂族群及鞘脂質成份。使用正相的二氧化矽層析管柱來分析裸鼠皮中的神經醯胺物質，流動相由100%庚烷至100%丙酮/丁醇(90:10 v/v)，以每分鐘2%進行梯度沖堤，所有溶劑皆含有體積百分比0.1%的三乙醇胺及0.1%的甲酸，流速為每分鐘0.8毫升。正相的LC-APCI-MS可以很理想的分離裸鼠皮角質層中神經醯胺的九個族群。而藉由分析神經醯胺Ceramide [NS]標準品，並確效建立一檢量線來量化所有類型的神經醯胺。而甘油磷脂族群及鞘脂質成份則使用逆相的層析管柱Inertsil 6 ODS-3 (4.6 × 150mm) 來分析，流速設定為1.0 mL/min，移動相組成以acetonitrile/methanol/triethylamine =550/1000/25 (w/w/w) ，所有溶劑皆含有體積百分比0.1%的三乙醇胺。逆相的LC-ESI-MS可以很理想的分析甘油磷脂族群及鞘脂質成份。未來可將分析神經醯胺及甘油磷脂及鞘脂質的方法應用於了解人類身體的變化，以期在預防醫學，治療醫學或新藥開發領域上有新的進展。
關鍵字
穿皮滲透 ； 結構與滲透定量關係式 ； 非固醇類抗發炎藥 ； 溶解因子 ； 生理參數 ； 正相液相層析串聯大氣壓化學電離質譜儀 ； 神經醯胺 ； 裸鼠皮膚 ； 逆相液相層析串聯電灑游離質譜儀 ； 甘油磷脂 ； 鞘脂質並列摘要
In this study, an empirical model of the quantitative structure permeability relationship (QSPR) of the transdermal delivery of non-steroidal anti-inflammatory drugs (NSAIDs) was constructed in an attempt to predict the permeability coefficients (kP). Thirteen model NSAIDs were selected, and their in vitro permeation through the full skin of nude mice was examined. The biological parameters of transepidermal water loss (TEWL), hydration content (HD), lipid content (SB), elasticity and direction of the fibers (RVM), and elasticity (EL) were measured. The permeability coefficients were obtained by using three kinds of datasets of clogP or logKo/w and all model drugs were grouped into two group that were clogP or logKo/w values > 2 and < 2; these datasets were regressed with respect to the physical characters of molecular weight (MW) and polarity factor (clogP or logKo/w) or the solubility parameter (δ) of the model drugs rationally chosen to replace the polarity factor with or without taking into consideration the biological parameters of the skin. Results demonstrated that δ could be greatly improved compared to clogP and logKo/w in the regression with an adjusted R2 of > 0.90 using the dataset of those drugs with clogP or logKo/w values of < 2, regardless of whether or not biological parameters were taken into consideration. This indicates that δ might rationally be a more-appropriate drug parameter for predicting the skin permeability of NSAIDs for transdermal delivery. A plot of observed kP versus predicted kP values by this simple empirical model of QSPR was validated to demonstrate the predictive capability of kP for transdermal delivery. In conclusion, an empirical model of QSPR to predict kp based on the hydrophilicity of the model drugs was statistically improved with δ and by taking the biological parameters of the skin into consideration. A sensitive and accurate liquid chromatography and mass spectrometry (LC- MS) for determining the standard ceramide [NS], glycerophospholipid and sphingolipid were developed. A normal-phase silica column was utilized with the gradient elution from 100% heptane to 100% acetone/butanol 90:10 v/v in 2%/min at 0.8 mL/min (all solvents contained 0.1% triethylamine and 0.1%formic acid v/v). Normal-phase LC-APCI-MS was optimized to separate the nine classes of ceramide presented in the stratum corneum (SC) of nude mouse skin. Then analysis of ceramide [NS] was validated and employed as the standard for constructing a calibration curve for quantitation of all classes of ceramides. Application of this method to profile the class and content of ceramides in the SC of nude mouse skin was conducted and proved to be workable. Reverse-phase LC-ESI-MS was optimized to analyze glycerophospholipid and sphingolipid, and Inertsil 6 ODS-3 (4.6 × 150mm) column was utilized with mobile phase : acetonitrile / methanol / triethylamine = 550 / 1000 / 25 (w/w/w) contained 0.1% triethylamine v/v。Flow rate was 1.0 mL/min. It was concluded that this improved method could be used to detect ceramide and glycerophospholipid and sphingolipid directly. Furthermore, the analysis method of ceramide and glycerophospholipid and sphingolipid could be applied to detect the physiology change of human in order to discover the preventive medicine, treatment medicine, or new drugs development field.
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