Purpose: To develop an agent-based, discrete-event, synthetic model that integrates the existing knowledge about intestinal absorption and disposition of baicalein (Ba) and dynamically represents the pharmacokinetic properties of Ba. To validate the model by matching simulated observations to bi-directional Caco-2 transport profiles of Ba. / Methods: A 3D multi-agent system extending the previous 2D in silico analogue of Caco-2 cell monolayer was used to study the pharmacokinetic properties of Ba. The model specification was based on previous study findings. Our model consisted of three 3D spaces and two 2D membranes: apical space (S1), intracellular space (S2), basolateral space (S3), apical membrane (M1), and basolateral membrane (M2). Validated enzyme components (UGTs and SULTs) and binder components (BINDERs) were placed in S2. Validated efflux transporter components (BCRPs, MRP2s, MRP1s and MRP3s) were placed at M1 and M2 respectively. Initially, Solutes (BA) were loaded to either S1 or S3. From there, they penetrated into S2 according to a validated, passive transport algorithm. Within S2, BA could be transformed into Baicalein-Glucuronide (BG) by UGTs or to Baicalein-O-Sulfate (BS) by SULTs. These metabolites were pumped out to either S1 or S3 by the active transport of BCRPs, MRP2s, MRP1s and MRP3s. Simulated results were then compared to corresponding wet-lab data to assess similarity based on the pre-specified similarity criteria. An iterative refinement protocol combined with Monte-Carlo simulation was employed to parameterize the model. Finally, the in silico parameters had also been mapped to the classical pharmacokinetic parameters. / Results: The simulated results captured the preset qualitative and quantitative features of the wet-lab observations. The feasible parameter set showed that substrate inhibition happened in both conjugation pathway of Ba. The simulation results suggested that sulfation pathway was dominated at low concentrations and that SULT was more inclined to substrate inhibition than UGT. All these findings were consistent with the previous outcomes from a catenary model. In addition, a micro-hypothesis that BS’s apical efflux transporter could be inhibited by Ba at high loading concentration was implemented to address the changing preference of the distribution of BS. / Conclusion: The mechanisms represented by our model are plausible. Our novel modeling approach could dynamically represent the pharmacokinetics of bi-directional transport of Ba in Caco-2 system. Furthermore, through the model development, we suspect there is inhibition of BS’s apical efflux transporter at high loading concentration of Ba, which is worthy of further investigation. / 研究目的：建立一個基於離散事件，並由智慧單元體組成的組合模型。該模型將整合與黃芩素小腸吸收和處置相關的已有知識，從而動態呈現黃芩素的藥動學性質。黃芩素在Caco-2系統中進行的雙向轉運實驗資料將被用來對該模型進行校驗。 / 研究方法：一個新的三維多智慧體系拓展自已有的二維Caco-2單層細胞體系模型，將被用來研究黃芩素的藥動學特徵。模型的配置將基於已有的研究發現。該模型由三個三維的空間對象和兩個二維的膜對象組成，他們是：頂端空間（S1）,細胞內空間（S2）,基底空間（S3）,頂端膜（M1）,和基底膜（M2）。已校驗的酶物件（UGTs和SULTs）和結合蛋白物件（BINDERs）將被設置在空間物件S2內。已校驗的外排轉運體對象（BCRPs, MRP2s, MRP1s和MRP3s）將被分別設置在膜物件M1和M2上。起始階段，黃芩素物件(BA)將被載入到空間物件S1或S3中。從載入空間出發，BA將基於已校驗的被動轉運演算法進入細胞內空間S2。在S2內，BA將通過UDP-葡萄糖醛酸轉移酶對象（UGTs）被轉化為黃芩苷對象（BG），或是通過硫酸轉移酶對象（SULTs）轉化為硫酸黃芩素物件（BS）。這些代謝產物物件將通過乳腺癌耐藥蛋白物件（BCRPs）和多種多藥耐藥相關蛋白物件（MRP2s, MRP1s和MRP3s）主動泵出到空間S1或S3中。模擬結果將按照預先設定的相似性判斷標準與對應的實驗資料進行比對。我們將通過結合蒙特卡洛模擬的迴圈反覆運算優化方案進行模型的參數化。最終，該模型參數將與傳統的藥動學模型參數建立起相關映射關係。 / 研究結果：模擬結果能夠很好刻畫實驗觀察到的定性和定量特徵。可行的參數集顯示底物抑制效應在黃芩素的兩條代謝通路都有發生。模擬結果顯示在低濃度下硫酸化通路是主要代謝途徑，且硫酸轉移酶相較於UDP-葡萄糖醛酸轉移酶更易被底物抑制。所有這些發現都與之前鏈式模型得到的結論相吻合。此外，一個關於硫酸黃芩素的頂端外排轉運體能夠被高濃度黃芩素抑制的微假設被引入到模型中來解釋硫酸黃芩素分佈偏好的變化。 / 結論：模型所呈現的機制是可行的。我們新穎的模型化方法能夠動態地呈現黃芩素在Caco-2系統中雙向轉運的藥動學特徵。此外，通過模型，我們推測硫酸黃芩素的頂端外排轉運體可能存在被高濃度黃芩素抑制的現象，這值得進一步的深入研究。 / Zhu, Xiao. / Thesis M.Phil. Chinese University of Hong Kong 2015. / Includes bibliographical references (leaves 115-123). / Abstracts also in Chinese. / Title from PDF title page (viewed on 12, October, 2016). / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only.

Aim. Baicalein is a bioactive flavonoid component isolated from the root of Scutellaria baicalensis, which has been used as a traditional Chinese medicinal herb for the treatment of inflammation for centuries. Although various pharmacological effects of baicalein have been demonstrated, only limited studies in rats reported pharmacokinetic of baicalein, which exhibited a low oral bioavailability due to extensive first-pass metabolism. In addition, no investigation on human oral absorption or metabolic kinetic profile was reported previously. The current project conducted a series of mechanistic studies aiming to elucidate the intestinal absorption, metabolism and disposition of baicalein. Since glucuronidation plays an important role in the first-pass metabolism of flavonoids including baicalein, additional studies on the relationship between human intestinal glucuronidation activities and chemical structures of flavonoids have also been performed. / Conclusion. Baicalein is well absorbed at intestine but subjected to extensive intestinal glucuronidation resulting in low oral bioavailability. The glucuronidation of baicalein is catalyzed by multiple UGT isozymes. The disposition of baicalein 7-O-glucuronide, the major metabolite of baicalein in vivo, is mediated by the MRP and OATP transporters. The nucleophilicity and stereo-conformation of -OH substituents are crucial for the intestinal glucuronidation of flavonoids. / Methods. For investigation on intestinal absorption, metabolism and disposition of baicalein, human Caco-2 cell monolayer model, rat in situ intestinal perfusion model, and in vitro metabolism model were employed in the present study. For the further investigation on the position preference on glucuronidation of flavonoids at human intestine, the in vitro rates of glucuronidation among seven commercially available mono-hydroxyflavones, namely 3-, 5-, 6-, 7-, 2'-, 3'- and 4'-mono-hydroxyflavones were determined and compared. / Results. The satisfactory permeabilities of baicalein obtained from both Caco-2 cell model and rat intestinal perfusion model indicated its potential good absorption at gastrointestinal tract. Therefore, absorption should not be the rate-limiting factor causing the low oral bioavailability of baicalein. However, extensive glucuronidation occurred in the rat intestine perfusion model with over 90% of baicalein being metabolized after intestinal absorption. Consistent findings were also observed in the in vitro enzyme kinetic studies of baicalein. The biotransformation of baicalein to baicalein 7-O-glucuronide was extensive in human liver microsome, human jejunum microsome, rat liver microsome, and rat jejunum microsome with intrinsic clearances (Vmax/Km) of 618, 446, 436, 298 mul/min/mg, respectively, which are orders of magnitude greater than those of most of western drugs that share the same metabolic pathway. Further enzyme kinetic studies using human recombinant glucuronosyltransferases (UGT) isozymes showed that UGT 1A1, 1A3, 1A8, 1A9, 1A7 and 2B15 were involved in the glucuronidation of baicalein with different kinetic profiles. Mechanistic studies on the disposition of baicalein 7-O-glucuronide formed from a rapid glucuronidation of baicalein in intestine demonstrated that this intracellularly formed glucuronide of baicalein could be actively extruded to both the apical and basolateral sides (the so called efflux) in Caco-2 cell model as well as rat intestinal perfusion model. It was also found that the efflux of the baicalein 7-O-glucuronide followed saturable enzyme kinetics and was effectively inhibited by multi-drug resistance associated proteins (MRP) and organic anion transporters (OATP) inhibitors. Further study on the relationship between flavonoid structures and glucuronidation activities using seven monohydroxyflavones demonstrated that the conjugation rates of 6- and 3'-monohydroxyflavones (HF) were much greater than those of 3-, 4'-, 7-, 2'-HF, while 5HF was the lowest. / Zhang Li. / "August 2006." / Advisers: Zhong Joan Zuo; Ge Lin. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1587. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 186-223). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Methods. The intestinal absorption and metabolism of W and OA as well as the potential interactions among B, Wand OA were investigated at in vitro, in situ and in vivo levels. Various models were employed including Caco-2 cell monolayer model, in vitro enzymatic kinetics study, rat in situ single-pass intestinal perfusion model and in vivo pharmacokinetic study in rats. / Purpose. Scutellariae baicalensis Georgi is a medicinal plant widely distributed in Asia. Its dried root, Radix Scutellariae (RS), has been extensively used in Chinese and Japanese medicine. Six flavones including baicalein (B), wogonin (W), oroxylin A (OA) and their corresponding glucuronic acid conjugates (BG, WG, OAG) are the major bioactive components in RS. Our previous studies on B revealed an extensive first-pass metabolism during its absorption. Hence, it is expected that W and OA which have the similar structures as B, may share similar absorption and metabolic pathways as B. The present project aims to (1) establish an assay method for better quality control of RS; (2) provide further biopharmaceutic characterizations ofW and OA in RS; (3) investigate the potential pharmacokinetic interactions among B, Wand OA. / Results. Similar to B, Wand OA showed favorable permeability in both the Caco-2 cell and the rat in situ single-pass perfusion models. However, they experienced extensive first-pass metabolism, mainly in the form of glucuronidation. Intracellularly formed WG and OAG could be effluxed to both the apical side (lumen side) and basolateral side (mesenteric blood side) mainly by MRPs, which was confirmed by inhibition transport studies in Caco-2 cells and transfected MDCK cells. The glucuronidation rate of OA was higher than that of W, which was observed by enzymatic kinetics studies by sub-cellular fractions with intrinsic clearances (Vmax/K m, mul/min/mg) of 456 to 4170 for W and 509∼5038 for OA. UGT 1A9 was the most potent metabolic enzyme for hepatic glucuronidation, while UGTs 1A8 and 1AlO were responsible for the intestinal glucuronidation of W and OA. The in vivo rat pharmacokinetics studies showed that W and OA may be readily absorbed and extensively metabolized with no parent compound detectable in blood after oral administration of W and OA. A new metabolite of W was identified to be the glucuronic acid conjugate at 5-0H of W. After co-administration of B, W and OA, decreased formation of BG, WG and OAG was observed in in vitro enzymatic kinetics study. Further studies in absorption models of Caco-2 cell monolayer and rat in situ single-pass intestinal perfusion demonstrated the enhancement in absorption of B, W and OA and decrease of BG, WG and OAG after the co-administration of B, W and OA. The ultimate pharmacokinetics interaction study revealed that glucuronides were the predominant form in systemic circulation and the AUC of OAG significantly increased after co-administration of B, Wand OA. Conclusion: Similar to B, Wand OA may be well absorbed followed by extensive first-pass metabolism, which was mediated by various UGT isozymes. During absorption, the intracellularly formed WG and OAG were mainly effluxed by MRPs to both the lumen and mesenteric blood side of the intestine. Both in vitro and in situ models indicated that interactions among B, W and OA would lead to decreased glucuronidation and increased absorption of parent flavones. Due to extensive metabolism in vivo, only glucuronides appeared in systemic circulation after co-administration of B, W and OA in rats. The resulted increased systemic exposure of OAG indicated that the co-administration might lead to the enhancement of bioavailability for the studied flavones in the form of glucuronides. / Li, Chenrui. / Adviser: Zuo Zhong. / Source: Dissertation Abstracts International, Volume: 73-03, Section: B, page: . / Thesis (Ph.D.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 201-236). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [201-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.