Oleanoic acid (OA), a pentacyclic triterpenoid compound, is commonly present in Chinese herbs, such as Fructus Ligustri Lucidi, Fructus Forsythiae and Radix Ginseng, etc. It has been shown to exhibit anti-inflammatory, antitumor, hypoglycemic and protecting liver activities. Oleanoic acid has been used as an OTC drug for oral administration to treat human liver disorders in China. The commonly used formulations are tablets, capsules and pills. Oleanoic acid belongs to a BCS IV active compound according to the biopharmaceutics classification system in FDA guidance, which shows poor aqueous solubility and low permeability across the intestinal membrane. It was reported that the solubility of OA was only \(4.61 \times 10^{-3} \, \mu g/ml\) at room temperature with slow dissolution rate. Furthermore, the permeability study across Caco-2 cell monolayer showed the \(P_{app}\) of OA was \(1.1 - 1.3 \times 10^{-6} \, cm/s\), which was similar to that of a low permeable marker atenolol (\(Papp = 0.25 \times 10^{-6} \, cm/s\)). The poor solubility and permeability lead to the low oral bioavailability (i.e., 0.7% at oral doses of 25 and 50 mg/kg in rats). The aim of the present study is to increase the oral bioavailability of OA by improving its dissolution rate as well as the membrane permeability by preparation of solid dispersions using Spray Freeze Drying (SFD) technology and salt formation methods. To enhance the dissolution of OA, spray freeze drying technology and salt formation methods were utilized to prepare the solid dispersion of OA. SFD is a relatively new technology used to prepare solid dispersion powder under low temperature conditions, which is especially suitable for heat-sensitive materials with poor aqueous solubility. Moreover, the salt formation is an effective method for improving the dissolution of poor water soluble drugs. To improve the permeability across the intestinal membrane of OA, absorption enhancer was used and evaluated by transport study on a Caco-2 cell monolayer model and in-vivo pharmacokinetic study. According to the dissolution results, PVP40 was selected as the excipient. To prepare SFD OA-PVP40 solid dispersion powder, the spray freeze drying apparatus was firstly installed. Power X-ray diffraction (PXRD) showed that all SFD powders lacked the characteristic crystalline peaks, indicating a completely amorphous OA had been prepared. Scanning electron microscopy (SEM) showed that SFD OA-PVP40 particles were porous particles composed of many small subunits with a geometric diameter of about 1-5 μm. The dissolution rate of OA raw material was slow. Only ~ 70% OA could be dissolved within 2 hours. On the contrary, ~ 90% OA could be dissolved within 10 mins for all of the SFD processed OA-PVP formulations. Accelerated stability test demonstrated both SFD OA-PVP40 at 1:1 and 1:9 ratios (w/w) were stable for 6 months under 40°C with 75% relative humidity. And no significant differences were detected by comparison the dissolution profiles at 1, 2, 3 and 6 months to the initial samples. Meanwhile, a LC-MS and a liquid-liquid extraction method for OA were established. The bioavailabilities of commercial OA tablet and SFD OA-PVP 1:1 were evaluated in SD rats. SFD powders displayed higher Cmax and AUC (153% and 268.56%, respectively) compared to the commercial OA tablets. However, there were no significantly differences between these two formulaes, which may suggest that the very low permeability of OA is the rate-limiting step for OA absorption. To prepare more bioavailable OA formulation, sodium caprate was used as a potential absorption enhancer. And the sodium salt of OA was prepared to enhance solubility of OA. The new formulations were SFD OA:PVP:SC 1:1:2 and SFD OANa:PVP:SC 1:1:2. PXRD and SEM revealed that both powders were completely amorphous and had a porous morphology with small columnar connections. The dissolution rate is higher than those of other formulations. More than 58% and 75% OA was dissolved in 10 min, 60% and 84% in 2 h, respectively. It has been demonstrated that sodium caprate can greatly increase the transported amount of OA from 3.68% to 10.16% in 2 hous. Compared to OA tablet, both two formulations had shorter Tmax (12.5 and 16 min, respectively) and higher Cmax (399.36 and 348.91 ng/ml, respectively), which are significantly different from those of the OA tablet. Furthermore, the relative bioavailability of SFD OANa:PVP:SC was 218%, compared to the OA tablet. In conclusion, the combined use of SFD technology, salt formation and absorption enhancer can successfully improve the dissolution rate, intestinal membrane permeability and oral bioavailability of OA. Keywords: Oleanolic acid; Solid dispersion; Spray freeze drying; Absorption enhancer; Caco-2 cell model; Bioavailability; Dissolution; Stability study

摘要 齊墩果酸是一種五環三萜類化合物，廣泛的分布于女貞子、連翹和人參等中藥中。齊墩果酸具有保肝護肝、降血糖、消炎和抗腫瘤等藥理作用，在臨床上被用作治療慢性肝硬化和傳染性急性黃疸型肝炎。目前主要劑型爲片劑、膠囊和丸劑等。 在 FDA 制定的生物藥劑學分類系統（Biopharmaceutics Classification System，BCS）中，齊墩果酸屬于 BCSIV類，即難溶于水，體內透過很差的一類藥物。研究表明，齊墩果酸在水中的溶解度極差，20℃時僅4.61×10^-3 μg/ml，且溶出緩慢；Caco-2細胞實驗表明，齊墩果酸原藥的 Papp 僅有1.1-1.3×10^-6 cm/s，與低透過藥物阿替洛爾（Papp=0.25×10^-6 cm/s）類似，證明 OA 腸細胞透過極差。以上原因直接導致齊墩果酸在體內的絕對生物利用度很低（小于0.7%）。因此，本實驗旨在用製劑的方法改造齊墩果酸藥物，提高其溶出和透過，以達到提高其在體內口服生物利用度的目的。 在本研究中，爲了提高齊墩果酸的溶出，使用了成鹽技術和噴霧冷凍乾燥技術（Spray Freeze Drying, SFD）製備固體分散體。噴霧冷凍乾燥方法是近幾年出現的一種新型固體分散體製備方法，是在溫度極低的環境中進行的，特別適合熱敏性和水溶性差的藥物；而成鹽技術也是一種有效的增加水難溶性藥物的溶解和溶出的手段。在透過方面，使用了吸收促進劑來提高齊墩果酸在小腸內的透過，并利用 Caco-2 細胞模型和大鼠體內實驗對劑型進行評價。 本研究首先成功設計和組裝了噴霧冷凍乾燥裝置并製備出齊墩果酸固體分散體。經過溶出實驗篩選，選用 PVP40 作爲輔料製備 SFD OA-PVP40 固體分散體，針對其進行一系列的表徵分析。粉末 X 射線衍射和掃描電鏡結果顯示，SFD OA-PVP40 1:1（w/w）固體分散體爲完全的非晶體，是由幾個微米的不規則微粒組成的多孔結構顆粒。在 1%的 SDS 溶出介質中，SFD OA-PVP40 固體分散體在 10 分鐘內完全溶出，達到了 90%以上的釋放；加速穩定性實驗表明，齊墩果酸的含量和溶出度在 6 個月內均沒有顯著的下落。這說明噴霧冷凍乾燥技術能够製備出具有多孔結構的非晶體顆粒，這些顆粒是由幾個微米的不規則微粒組成，有良好的分散狀態，溶出相對于齊墩果酸原藥均有顯著性提高。 同時，本實驗建立了齊墩果酸血液樣品的液液萃取方法及 LC-MS 分析方法，進一步對 SFD OA-PVP40 1:1 固體分散體進行了大鼠體內藥代動力學的研究，并與市售劑型進行了比較。結果表明，齊墩果酸固體分散體的生物利用度相對于市售齊墩果酸片有很大的提高（268.56%），Cmax的平均值也有 153%的提高，但是均無顯著性差异。這可能是因爲齊墩果酸在腸細胞膜的透過成爲其口服吸收的限速步驟，因此只解決溶出問題并不能提高其口服吸收。 經過進一步的探索，本實驗在配方中加入經典的吸收促進劑癸酸鈉來解決齊墩果酸的透過問題，幷將齊墩果酸改造成爲齊墩果酸鈉鹽，製備出的兩個處方爲 SFD OA:PVP:SC 1:1:2 和 SFD OANA:PVP:SC 1:1:2。對這兩種製劑處方進行表徵和體外評價，其粉末顆粒均爲完全的非晶體，而比表面積比之前的處方有所增加，掃描電鏡顯示顆粒爲不規則的形狀幷有柱狀的連接和突起。在水中的溶出比原藥和之前的處方有明顯的增加，在 10 分鐘內分別有 58%和 75%的釋放，幷最終達到 60%和 84%的釋放度。加入癸酸鈉的齊墩果酸在兩小時內 Caco-2細胞透過率由原來的 3.68%增加到 10.16%，說明癸酸鈉可以顯著的提高齊墩果酸在腸細胞的透過。大鼠體內藥動學實驗表明，相對于 OA 片劑，兩個處方的達峰時間更短，平均只有 12.5 和 16 min 血藥濃度更高。Cmax 分別爲 399.36 和 348.91 ng/ml（均有顯著性差異）。雖然處方 SFD OA:PVP:SC 1:1:2 的平均相對生物利用度與齊墩果酸片劑沒有顯著性差異，但是處方 SFD OANA:PVP:SC 1:1:2 平均相對生物利用度比齊墩果酸片劑提高了 218%，幷具有顯著性差異。表明使用噴霧冷凍乾燥技術，在配方中加入吸收促進劑癸酸鈉，幷將齊墩果酸改造成爲鈉鹽，可以成功的提高藥物的溶出度、透過和體內的血藥濃度，幷最終顯著地提高其在體內的生物利用度。 關鍵詞：齊墩果酸；固體分散體；噴霧冷凍乾燥；吸收促進劑；生物利用度；Caco-2 細胞模型；溶出度；穩定性