The proposed human intestinal absorption prediction model is applied to over 900 pharmaceuticals and has about 82.5% true prediction power. This study will provide a screening tool that can differentiate well absorbed and poorly absorbed drugs in the early stage of drug discovery and development. This model is based on fundamental physicochemical properties and can be applied to virtual compounds. The maximum well-absorbed dose (i.e., the maximum dose that will be more than 50 percent absorbed) calculated using this model can be utilized as a guideline for drug design, synthesis, and pre-clinical studies.

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第13346号 / 論農博第2889号 / 新制||農||1080(附属図書館) / 学位論文||R2||N5253(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 菅原 達也, 教授 佐藤 健司, 教授 澤山 茂樹 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

No description available.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第23940号 / 農博第2489号 / 新制||農||1090(附属図書館) / 学位論文||R4||N5375(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 菅原 達也, 教授 佐藤 健司, 教授 松井 徹 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM

Bi-directional transport studies in SimBioDASRTM and Caco-2 cells were employed to examine the transport profiles of Buph, Ligs and SenA. Apical to basolateral (A-B) transport studies of the tested compounds revealed high intestinal permeability and predicted human absorption of over 98%. Permeability ratio of B-A/A-B of Buph (0.7-1.3) and Ligs (0.8-1.2) indicated that they were transported by passive transcellular and paracellular pathways while the low B-A/A-B ratio of SenA may imply possible involvement of other transport mechanisms. One metabolite (M-1) generated from hydration of Buph was observed in Caco-2 cells and the fraction of metabolism was 12.5% (A-B). / In conclusion, Buph, Ligs and SenA were predicted to have good intestinal absorptions in human and rat. However, extensive hepatic and intestinal first-pass metabolism of Buph in rat and human were found to cause its low oral bioavailability. On the other hand, certain degree of hepatic first-pass metabolism of Ligs and SenA may account for the partial loss of drugs via oral administration to rat. Therefore, other routes of delivery, such as sublingual administration, are worth to be considered to improve the therapeutic effects of chuanxiong. / In the rat SPIP, permeability calculated from the appearance of Buph in mesenteric blood (Pblood) was 6.0+/-1.7 x 10-4 cm/s while the fraction of formation of M-1 was about 7.1%. Together with the in vitro results, it is proposed that first-pass metabolism of Buph was present in human and rat small intestine. Moreover, Ligs and SenA had high Pblood values of 4.2+/-1.2 x 10-3 cm/s and 3.8+/-2.8 x 10-3 cm/s, respectively, indicated that they were highly permeable across rat intestinal mucosa. No metabolism of Ligs was observed. But several metabolites of SenA were detected despite they were not quantified in the present study. / In vitro metabolic studies of Buph demonstrated that major metabolite M-1, which was also found in Caco-2 cells and SPIP, formed mainly in intestine and liver cytosol in rat and human. The intrinsic clearance (Vmax/Km) of Buph was extensive and similar in both organs, and its extent in human was comparable to that in rat. The sum of the estimated in vivo extraction ratio of Buph by liver (48.3%) and intestine (55.0%) was higher the loss via oral administration to rat (77%). On the other hand, several metabolites of Ligs and SenA were found in rat and human liver microsome but not in intestinal preparations. The estimated in vivo extraction ratio by liver of rat was 47.3% (Ligs) and 22.9% (SenA), respectively, which were less than the corresponding loss via oral administration to rat (Ligs: 92.2% and SenA: 97.7%), suggesting that first-pass effect other than metabolism of these two compounds in intestine also contributed to their low oral bioavailability. / Rhizoma chuanxiong is commonly prescribed orally for improving blood circulation and treating cardiovascular disorders in China. Like other traditional Chinese medicines, chuanxiong has been used for thousands of years in China but its chemical basis, pharmacological effects and pharmacokinetic fates of the active ingredients, especially absorption, are poorly understood. Recently, seventeen compounds such as 3-butylidenephthalide (Buph), Z-ligustilide (Ligs), senkyunolide A (SenA), vanillin (Vani), ferulic acid (Fera), senkyunolide I (SenI), senkyunolide H (SenH), coniferyl ferulate (ConFer), sedanolide (Sdan), riligustilide (Rili) and levistolide A (LevA) have been isolated and recognized as the main constituents of chuanxiong by our research team (Li et al., 2003). Moreover, it has been demonstrated that Buph, Ligs and SenA are bioactive components of chuanxiong for vasodilatation and anti-thromboembolism (Chan, 2005) though their oral bioavailability in rat are very low (2.3, 7.8 and 23% respectively) (Yan, 2005). Therefore, the present study aims at investigating the intestinal permeability of the major ingredients of chuanxiong and characterizing the intestinal absorption and first-pass metabolism of Buph, Ligs and SenA by in vitro Caco-2 cell monolayers, SimBioDASRTM , in situ single-pass intestinal perfusion (SPIP) in rat and in vitro metabolism using rat and human intestine and liver subcellular fractions respectively. / Using the in vitro cell monolayers of SimBioDAS RTM, the intestinal permeability of major components of chuanxiong ranged from 12.2+/-1.6 x 10-6 cm/s to 70.6+/-9.6 x 10-6 cm/s with a rank order of Fera < Buph < Ligs < Sdan < SenH < SenI < SenA < Vani. They were predicted to have over 70% absorption in human. However, ConFer, Rili and LevA were estimated to have poor human oral absorption. / Ko Nga Ling. / "September 2005." / Adviser: Ge Lin. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1577. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 215-239). / 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.

Two specific goals were addressed for this dissertation. First to investigate and identify the mechanistic profile of ketoconazole (KT)-induced hepatotoxicity by utilizing in vivo and in vitro approaches determining the mechanism of action for the hepatotoxicity incurred. To date, there has not been a mechanistic determination of the hepatotoxicity associated with KT in vivo. This dissertation evaluates the possible metabolic bioactivation of KT by cytochrome-P450 (CYP) or flavin-containing monooxygenases (FMO) resulting in covalent binding with hepatic macromolecules. The hypothesis of this study was to reveal whether covalent binding by the parent compound, KT, and/or reactive metabolites produces hepatic damage associated with increased serum alanine aminotransaminase (ALT) release and decreased hepatic glutathione (GSH). The first objective was determination of in vivo covalent binding in a dose-time response comparison in Sprague-Dawley (SD) rat ALT and GSH levels. Increased ALT and reduced hepatic GSH levels occurred. The second objective was an in vitro comparison of covalent binding with GSH levels utilizing SD microsomal protein with incubations of KT. Covalent binding decreased with added GSH to microsomal incubations. Thirdly, correlate in vivo with in vitro findings. Covalent binding of KT in vivo and in vitro occurred with increased doses and time. The final objective was to determine the bioactivation pathway utilizing heat inactivation and no NADPH in vitro. Covalent binding of KT decreased in the absence of NADPH and deactivation of FMO. The second goal was to determine and quantitate in vitro the presence of FMO isozymes in microsomes of the human intestinal duodenum, jejunum, ileum, and colon as well as the Caco-2 (HTB-37), epithelial intestinal (CCL-241) and colon (CRL1790) cell lines. The presence of FMO could result in a first-pass effect decreasing the bioavailability of soft nucleophiles or a toxicity effect due to inhibition or modulation of the enzyme from co-administration. To date, this is the first evaluation of FMO isoforms in human intestine and cell lines. Western blot techniques were utilized for detection of human FMO1, FMO3, and FMO5 using human FMO-expressed recombinant cDNA from a baculovirus system. / Graduation date: 2003

[Formulae and special characters can only be approximated here. Please see the pdf version of the abstract for an accurate reproduction.] Iron is vital for almost all living organisms by participating in a wide variety of metabolic processes, including oxygen transport, DNA synthesis, and electron transport. However, iron concentrations in body tissues must be tightly regulated because excessive iron leads to tissue damage, as a result of formation of free radicals. In mammals since no controlled means of eliminating unwanted iron has evolved, body iron balance is maintained by alterations in dietary iron intake. This occurs in the duodenum where most dietary iron is absorbed. Absorption involves at least two steps, uptake of iron from the intestinal lumen and then its transport into the body, processes that occur at the apical and basal membranes of enterocytes, respectively. In chapter one of this thesis the background information relevant to iron absorption is described. Despite numerous studies, the role of these proteins in iron absorption remains unclear, partly because many studies have reported them in non-enterocyte cell lines where the expression of the proteins involved in iron absorption is unlikely and therefore the physiological significance of the findings uncertain. Therefore, the study of iron absorption would value from additional cell lines of intestinal origin being used, preferably derived from a species used to comprehensively study this process in vivo, namely the rat. Validation of such a model would enable comparisons to be made from a molecular level to its relevance in the whole organism. In chapter 3 of this thesis, the rat intestinal cell line 6 (IEC-6) was examined as a model of intestinal iron transport. IEC-6 cells expressed many of the proteins involved in iron absorption, but not the ferrireductase Dcytb, sucrase or αvβ3 integrin. In addition, in IEC-6 cells the expression of the apical transporter divalent metal transporter 1 (DMT1), the iron storage protein ferritin, the uptake of Fe(II) and Fe(III) were regulated by cellular iron stores as is seen in vivo. This suggests that IEC-6 cells are of a lower villus enterocyte phenotype. Presented in chapter 4 is the study of the uptake of iron from Fe(II):ascorbate and Fe(III):citrate by IEC-6 cells in the presence of a blocking antibody to the putative basolateral transporter ferroportin1 and of colchicine and vinblastine, different pHs, and over-expression of DMT1. It was shown that optimal Fe(II) uptake required a low extracellular pH and was dependent on DMT1. Uptake of Fe(III) functioned optimally at a neutral pH, did not require surface ferrireduction, and was increased during over-expression of DMT1. These observations suggest that intravesicular ferrireduction takes place before transport of Fe(II) to the cytoplasm by DMT1. This pathway was not blocked by a functional antibody against αvβ3 integrin but was inhibited by competition with unlabeled iron citrate or citrate alone. Surprisingly, a functional antibody against ferroportin1 had no effect on efflux but significantly reduced (p<0.05) uptake of Fe(II) by 40-50% and Fe(III) by 90%, indicating two separate pathways for the uptake of iron from Fe(II)-ascorbate and from Fe(III)-citrate in IEC-6 cells. Presented in chapter 5 is the development and validation of a technique for the removal of freshly isolated enterocytes from the rat duodenum and their use to study iron transport processes that enabled comparisons to be made between these cells, IEC-6 cells and the human enterocyte cell line Caco-2 cells. In chapter 6 a blocking antibody to ferroportin1 was shown to inhibit uptake of Fe(II) but not release of iron in freshly isolated duodenal enterocytes from rats and Caco-2 cells supporting the findings obtained with IEC-6 cells described in chapter 4. Fe(II) uptake was reduced only when the antibody was in contact with the apical membrane indicating its expression at the microvillus membrane. Confirming this, ferroportin1 was shown along the microvillus membrane of Caco-2 cells, in enriched microvillus membrane preparations and in enterocytes of duodenum tissue of rats where it co-localised with lactase. The significant findings to emerge from this thesis are that the IEC-6 cell is a valid model to study iron absorption producing results consistent with those found in freshly isolated enterocytes and in human enterocyte-like cells. In particular, ferroportin1 functions in the uptake of iron at the apical membrane possibly by modulating surface binding of Fe(II) to DMT1 or the activity of DMT1. In addition to this in Fe(II) uptake from Fe(III) ferroportin1 may also affect the number of Fe(III): citrate binding sites. Preliminary studies further characterizing the function of ferroportin1 at the apical membrane and at intracellular sites of IEC-6 cells along with integration of these data are discussed in chapter 7.

Orientador: José Antonio Rocha Gontijo / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-21T06:22:30Z (GMT). No. of bitstreams: 1 Possignolo_LuizFernando_M.pdf: 4767580 bytes, checksum: b843458e4c7049fc29741e4aca9f918d (MD5) Previous issue date: 2012 / Resumo: Devido ao maior consumo de alimentos ricos em gordura e um estilo de vida mais sedentário, houve um aumento na incidência de desordens metabólicas relacionadas ao metabolismo lipídico como a resistência à insulina, dislipidemias, e sua associação com doenças cardiovasculares. A alimentação materna desequilibrada, durante a gestação e lactação, pode predispor a prole à doenças durante a vida adulta. Alguns transportadores como o Scavenger Receptor class B type I (SR-BI) e ATP-binding cassette transporter A1 (ABCA1) são descritos como responsáveis pela captação de colesterol e transporte deste a partir de lipoproteínas, principalmente no transporte reverso de colesterol, sendo denominados receptores antiaterogênicos podendo modificar seu padrão de expressão frente a uma dieta hiperlipídica. Ratas Wistar receberam dieta hiperlipídica (DHL) ou dieta padrão (CTL) desde o desmame, até a lactação. Após o desmame a prole de machos recebeu a dieta padrão até a 16ª semana de vida. Nas mães quanto e na prole foram analisados os seguintes parâmetros: ingestão alimentar, ganho ponderal, perfil lipídico e glicídico. Na prole foi estudada a expressão e localização de ABCA1 e SR-BI na placenta, no rim, fígado, e intestino delgado em animais com 17 dias pré-natal (E17), 12 dias pós-natal (PN12d) com 8 e 16 semanas pós-natal (PN8s e PN16s). As mães DHL apresentaram: 1) maior ingestão calórica com menor ganho ponderal; 2) aumento glicêmico associado à menor produção de insulina nos três períodos estudados e, 3) aumento nos níveis séricos de triglicérides em M8s. A prole de mães DHL apresentaram menor massa corporal desde E17 até PN8s, sem que tenha havido diferenças ponderais e na ingestão de ração. PN8s e PN16S apresentaram menor captação tissular de glicose associada à hiperinsulinemia, e aumento nos níveis séricos de triglicérides com PN16S. Não houve alterações nos níveis de colesterol e HDLcolesterol. Não foi observada alteração na expressão de SR-BI e ABCA1 no intestino delgado, placenta enquanto no fígado houve uma queda tempo-dependente para ambos transportadores. No rim da prole DHL aos PN12d e PN16s observou-se maior expressão de ABCA1. Este estudo mostrou que o consumo materno crônico de uma dieta hiperlipídica causa alterações metabólicas nas mães e predispõe a prole, a modificações no metabolismo lipídico e glicídico, além de elevação da expressão de ABCA1 no rim / Abstract: Due to the abundance and accessibility to foods high in fat and a more sedentary lifestyle, there is an increased incidence of metabolic disorders related to lipid metabolism such as insulin resistance, dyslipidemia, and a high correlation with cardiovascular disease. The unbalanced maternal diet during pregnancy and lactation predisposes the offspring to diseases during adult life. Some carriers such as scavenger receptor class B type I (SR-BI) and ATP-binding cassette transporter A1 (ABCA1) are described as responsible for raising cholesterol and transporting it to and from lipoproteins, due the participation in the reverse cholesterol transport, these receptors are called antiatherogenic and are subject to change its pattern of expression when exposed to a high fat diet. Female Wistar rats were fed a diet (HFD) or a standard chow (CTL) from weaning, during pregnancy and lactation. After weaning the male offspring was exposed to a standard chow until the 16th week of life. Both the dams and the offsprings food intake were monitored, weight gain, lipid profile and glucose level. It was analyzed in the offspring the expression and localization of ABCA1 and SR-BI in the placenta, kidney, liver, and small intestine in animals at 17º prenatal day (E17), 12 days post-natal (PN12d), 8 and 16 postnatal weeks (PN8s PN16s respectively). DHL dams had a higher intake of calories in the diet, but the weight was smaller, they had higher blood glucose due to decreased production of insulin in the pre-pregnancy (m8s), during pregnancy (M17g) and lactation (M15l) and a higher triglyceride level in m8s. The offspring of dam fed a high fat diet had lower weight since E17 until PN8s, with no differences in weight gain and food intake. PN8s and PN16s had lower glucose uptake and hyperinsulinemia, and high triglycerides with PN16s, no changes were observed in cholesterol and HDL-cholesterol levels. There were no changes in the expression of SR-BI and ABCA1 in the small intestine, placenta and liver, however there was a decrease over the age for both receptors, and kidney of the offspring and DHL PN12d PN16s showed higher expression of ABCA1. The present study showed that chronic consumption of high fat diet causes metabolic changes in dams and predisposes offspring to changes in lipid and glucose metabolism of the offspring, increasing the expression of ABCA1 in the kidney / Mestrado / Biologia Estrutural, Celular, Molecular e do Desenvolvimento / Mestre em Fisiopatologia Médica

This study was designed to determine whether feeding an Aspergillus product would influence growth or feed utilization and intestinal mucosal cell function as indicated by uptake of methionine and glucose by isolated intestinal brush border membrane vesicles (BBMV). In Experiment 1, 24 weanling pigs were paired by sex, BW, and litter and were allotted, within pairs, to either an 18% CP corn-soy diet (control) or the same diet supplemented (.15%) with an Aspergillus product. There were no differences (P > .05) in ADG, daily feed intake, or feed/gain between the two groups. In Experiment 2, 24 weanling wether lambs were paired by BW and were randomly assigned within pair to a 14% CP diet containing 61.1 % cracked corn, 17.3% soybean meal, and 15% ground orchard grass hay (control) or the same diet fortified (.07%) with an Aspergillus product. There were no differences (P > .05) in ADG, daily feed intake, or feed/gain between the two groups. Enrichment of alkaline phosphatase in BBMV used in transport studies were 12.7-fold higher in pigs and 5.6-fold higher in lambs over the original homogenate. / Ph. D.

Master of Science / Department of Animal Sciences and Industry / Duane L. Davis / Intestinal absorption of colostral leukocytes (CL), peripheral blood mononuclear cells (PBMC), and porcine umbilical cord matrix stem cells (PUC) was analyzed in neonatal pigs. Maternal CL have previously been demonstrated in pigs, and maternal PBMC have been observed in calves to enter neonatal circulation after ingestion. PUC are primitive stem cells that are easily isolated from Wharton's jelly of the porcine umbilical cord. These cells do not have an immunogenic effect on the host upon initial transplantation. The general characteristics of PUC may allow them to serve as a delivery system to the neonate. Cellular migration through the duodenum, jejunum, and ileum was assessed using confocal microscopy. In vitro experiments utilized an organ explant culture system to determine the trafficking of labeled cells. Small-intestine tissue was collected from stillborn and sacrificed neonates. All three cell types (CL, PBMC, and PUC) were detected below the luminal surface, after 72 h of culture with media, and regardless of whether explants were from stillborns or live-born pigs. In vivo trafficking was assessed using neonatal pigs that were fed PBMC isolated from their mother or PUC from an unrelated pig. The effect of prior exposure to 25% acellular colostrum (AC) in medium was evaluated for both cell types. Piglets were euthanized 8 h or 24 h post feeding and sections of the small intestine collected. Both PBMC and PUC were found in all intestinal samples. Exposure to AC had no detected effect on the ability of either cell type to attach and migrate into the tissue. Labeled PUC were detected on the surface of the epithelium and in the lamina propria 8 h post treatment. PBMC were observed on the surface of the epithelium, in the lamina propria, and superficial submucosa 8 h following ingestion. In neonates sacrificed 24 h post treatment, both PUC and PBMC were observed on the surface of the epithelium, in the lamina propria, superficial submucosa, and deep submucosa of the small intestine. PUC and PBMC were noted at the apex, intermediate between the apex and the base, or at the base of the villus.