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Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Veronika Řepová Supervisor: Prof. PharmDr. Petr Pávek, Ph.D., Prof. Ramiro Jover Atienza, Ph.D. Title of diploma thesis: Alterations in gene expression of hepatobiliary transporters as potential mechanisms for drug-induced cholestasis by amoxicillin and clavulanic acid The combination of amoxicillin and clavulanic acid (AMO/CLA) represents one of the most frequent causes of the idiosyncratic type of drug-induced liver injury (DILI) nowadays. Despite difficulties in diagnosis and causality assessment, the clinical features have already been reported and in most of the cases categorized as cholestatic damages. Number of descriptions of the molecular mechanisms of drug-induced cholestasis has been rising recently and the role of hepatobiliary transporters has turned out to be crucial in the pathogenesis. However, the mechanisms of AMO/CLA-induced DILI at the molecular level still remain indistinct. In order to investigate the hepatotoxic effects of AMO/CLA and AMO alone in vitro, HepG2 and human Upcyte hepatocytes were used as hepatocellular models. The mRNA levels of key bile acid (BA) transporters, enzymes and nuclear receptors (NRs) were measured by quantitative real-time polymerase chain...

Le carcinome hépatocellulaire (CHC) et le cholangiocarcinome (CCA) sont 2 types de cancer primitif du foie. Le CHC est le plus fréquent, cependant l’incidence du CCA augmente partout dans le monde avec un diagnostic difficile, un mauvais pronostic et des thérapies très limitées. Ce travail avait pour objectif d'identifier des cibles pour le diagnostic et la thérapeutique du CCA. Il est basé sur l'étude de la septine 9 et des phosphoinositides (PIs). La septine 9 appartient à une famille de GTPases qui participent à l’organisation des microtubules et du cytosquelette d’actine. Les septines sont impliquées dans la cytokinèse, le trafic vésiculaire et la polarité cellulaire, elles sont aussi des partenaires importantes des PIs. Pour déterminer le rôle de la septine 9 dans le CCA nous nous sommes intéressés à son interaction avec les PIs et avec l’inhibiteur de l’inducteur et activateur de la transcription 1 (PIAS1) qui a été décrite comme une protéine pouvant agir comme une SUMO ligase pour les septines. Nous avons étudié l’expression de la septine 9 et de PIAS1 dans le CCA et le CHC. Nous avons mis en évidence un mécanisme original par lequel, la production du PtdIns5P (Phosphatidylinositol -5-phosphate) permet un recrutement de la septine 9, la stabilisation des microtubules et le transport de PIAS1 du cytoplasme vers le noyau. Il démontre un rôle important des septines en association avec les PIs dans le trafic. De plus, nous avons montré que la septine 9 est un régulateur de la signalisation de l’interféron γ qui agit au niveau de la phosphorylation de STAT1 et l’entrée de PIAS1 dans le noyau. Ce travail peut constituer une nouvelle piste pour la recherche des thérapies ciblées en immunothérapie dans le traitement de ce cancer. / Hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA) are two types of primary liver cancer. HCC is the most frequent, however the incidence of CCA increases throughout the world with a difficult diagnosis, poor prognosis and very limited therapies. The objective of this work was to identify targets for the diagnosis and treatment of CCA. It is based on the study of septin 9 and phosphoinositides (PIs). Septin 9 belongs to a family of GTPases that participate in the organization of microtubules and the actin cytoskeleton. Septins are involved in cytokinesis, vesicular trafficking and cellular polarity and are also important partners of PIs. To determine the role of septin 9 in the CCA, we investigated its interaction with PIs and with Protein inhibitory of activated STAT1 (PIAS1), which has been described as a SUMO ligase for septins. We studied the expression of septin 9 and PIAS1 in CCA and CHC. We have demonstrated an original mechanism by which la production of PtdIns5P allows the recruitment of septin 9, the stabilization of microtubules and the transport of PIAS1 from the cytoplasm to the nucleus. It demonstrates an important role of the septins in association with the PIs in trafficking. Besides, we have shown that septin 9 is a regulator of interferon γ signaling which acts at the level of the phosphorylation of STAT1 and the entry of PIAS1 into the nucleus. This work can constitute a new avenue for the research of targeted immunotherapy for this cancer.

Hepatobiliary disease can sometimes be difficult to diagnosis due to non-specific clinical signs, and diagnostic imaging is a vital tool in diagnosing these diseases. Multi-slice computed tomographic cholangiography (MSCTC) is a non-invasive way to obtain high quality images of the hepatobiliary system. Our objectives were to determine the best technique for performing MSCTC in normal dogs with regards to contrast agent, dose, and optimal time to imaging. Our test subjects included eight normal adult hounds. Four dogs were administered Cholografin and the other four Biliscopin. Two dose groups were established with four dogs receiving 0.5mL/kg and four receiving 1 mL/kg. Our results demonstrated that MSCTC is feasible in normal dogs and produces high quality images of the hepatobiliary system. The contrast agent Biliscopin at the higher dose subjectively produced the best quality images. The optimal time to image patients following contrast administration varied between contrast agents (15-60 minutes).

() is a commensal organism of the gastrointestinal tract. It rarely causes hepatobiliary infections; however, in the presence of underlying inflammation, immunosuppression, or malignancy, it can cause hepatobiliary infection via an ascending route. Herein, we report a case of pyogenic liver abscess secondary to associated with cholangiocarcinoma, which was treated with ceftriaxone and metronidazole.

The Relationship Between Central Venous Catheter and Post-operative Complications in Patients Undergoing Hepatic Resection David C. O’Connor, Ph.D., DNAP, CRNA A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at Virginia Commonwealth University Virginia Commonwealth University, 2018 Dissertation Chair: Clarence J. Biddle, Ph.D., CRNA Hepatic resection is indicated for primary and secondary malignancies. Use of a low central venous pressure technique is associated with decreased blood loss in these cases. This technique has evolved; central venous catheters and high dose morphine are no longer used, and patients are extubated earlier. The purpose of this study is to assess a relationship between these changes and outcomes. Central venous pressure has fallen out of favor as an accurate fluid measurement. Central venous catheters are associated with many complications. Outcomes in patients undergoing hepatic resection have improved over 20 years at one high volume institution. Guided by Donabedian’s theory of measuring outcomes, a non-randomized, non-experimental, retrospective, cohort design was conducted. The independent variables were intraoperative insertion of a central venous catheter, use of morphine, and time of extubation. The dependent variables were superficial and deep wound infections, number and severity of complications. The population sample is patients who submitted to partial hepatectomy at Memorial Sloan Kettering Cancer Center from 2007-2016. Data was obtained from hepatobiliary and anesthesia databases at Memorial Sloan Kettering Cancer Center. Data of 2518 from a possible 3903 patients were analyzed with chi square, univariate, Poisson and multivariate regressions. Univariate analysis for presence of CVC was significant for 90-day mortality (p 0.013). Use of morphine was significant for superficial wound infection (p 0.035), and a decrease in complications (p <.001). Amount of morphine was associated with fewer severe complications (p <.001). Incidental findings included a relationship between gender, total amount of fluids and number of segments resected. The significance of CVC with 90-day mortality was eliminated with stepwise multivariate regression. The findings support the change in anesthetic practice with clinical significance. Incidental findings regarding fluids and segments are supported in the literature. Future research should include goal directed fluid therapy and investigation of the relationship between gender and outcomes.

Conventional ‘well-stirred’ extrapolation methodology using intrinsic metabolic clearance data from rat liver microsomes poorly predicts in vivo clearance for approximately half of drug discovery compounds. The aim of this present study was to gain a more detailed understanding of the hepatobiliary disposition pathways which influence drug clearance. A set of 77 new chemical entities (NCEs), demonstrating a range of physicochemical properties and in vitro-in vivo clearance correlations (IVIVC), were employed to explore relationships between hepatobiliary disposition pathways in rat and physicochemical, structural and molecular properties of the NCEs. Primary rat hepatocytes with >80% cell viability were successfully isolated from male Han Wistar rats and used to establish in vitro models of drug uptake and biliary efflux. Preliminary studies with cultured primary rat hepatocytes indicated that uptake of d8-taurocholic acid and pitavastatin was time, concentration and temperature dependent. Initial studies with sandwich cultured primary rat hepatocytes demonstrated that cellular accumulation and biliary efflux of [3H]-Taurocholic acid was time and concentration dependent. These in vitro rat hepatocyte models were then used to investigate drug uptake and biliary efflux for all NCEs. In general, NCEs with high (passive) permeability showed better IVIVC and a lower incidence of active uptake and biliary efflux compared to NCEs with lower permeability, suggesting permeability is a key property influencing hepatobiliary drug disposition in rat. Preliminary in silico models analysing structural and molecular descriptors of substrates of active transport in rat hepatocytes were developed and indicated modest potential to highlight clearance pathways beyond hepatic metabolism but further follow up work with larger, more diverse compound sets is warranted to gain confidence in these models. Extended clearance models were investigated to estimate the effect of hepatic transporters on clearance and to predict the overall hepatic clearance of the NCEs. None of these models resulted in a 1 to 1 correlation but in general, improvements in clearance predictions were made when drug transport processes were accounted for. In vivo excretion studies using bile duct cannulated rats demonstrated that NCEs with high permeability and good IVIVC were not directly eliminated in bile or urine as unchanged drug, whereas NCEs with lower permeability and poor IVIVC (> 3-fold under predicted) were all directly eliminated unchanged indicating key drivers of clearance beyond metabolism. In conclusion these investigations confirmed a role for hepatic transporters in clearance but the complex nature of active transport mechanisms and a lack of robust in vitro tools create challenges in the quantitative prediction of hepatobiliary clearance. However, one of the key findings from this research, which is highly applicable in early drug discovery, was to identify the existence of disposition permeability relationships. These can be anticipated by observing physicochemical parameters of NCEs in conjunction with conventional IVIVC, since NCEs that are not highly permeable, possess some hydrophobic characteristics, and which are poor substrates of cytochrome P450 enzymes are more likely to be good substrates of transporters and be directly eliminated in bile and/or urine. The present study focused on exploring hepatobiliary disposition pathways using rat as the investigative species. Whilst there is no guarantee that pathways relevant to rat will be similar to other preclinical species or even humans, an early diagnosis of dominant clearance pathways can guide a more efficient use of the ADME-PK toolbox.

Primary Sclerosing Cholangitis (PSC) is a progressive cholestatic liver disease with outcomes that include hepatobiliary malignancy and liver transplantation. The pathogenesis of PSC is incompletely understood and, as a result, few markers of disease progression have been identified. Vitamin D is associated with the development and treatment of multiple cancers as well as the progression of inflammatory bowel disease, making it a possible candidate as a biomarker associated with PSC outcomes. In this study, we retrospectively and prospectively collected complete laboratory results and outcome datapoints on 179 patients with PSC to determine the association between total 25(OH)-vitamin D levels, vitamin D supplementation, and both hepatobiliary malignancy and liver transplantation. Through survival analysis, we found that history of vitamin D supplementation was significantly associated with increased hepatobiliary malignancy-free and liver transplantation-free survival (p=0.025 and p=0.042, respectively). These results indicate that vitamin D is a promising factor associated with the progression of PSC to transplantation and malignancy. Future studies on this registry cohort as it increases in size and age may provide more conclusive data on the relationship between vitamin D and PSC.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20083号 / 医博第4176号 / 新制||医||1018(附属図書館) / 33199 / 京都大学大学院医学研究科医学専攻 / (主査)教授 増永 慎一郎, 教授 妹尾 浩, 教授 松田 道行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The studies presented here evaluate the biliary, parenchymal and vascular enhancement effects of two T1-shortening liver-specific contrast agents, Gd-BOPTA and Gd-EOB-DTPA, in Magnetic Resonance Imaging (MRI) of healthy subjects and of patients. Ten healthy volunteers were examined with both contrast agents in a 1.5 T MRI system using three-dimensional gradient echo sequences for dynamic imaging until five hours after injection. The enhancement of the common hepatic duct in contrast to the liver parenchyma was analyzed in the first study. This was followed by a study of the image contrasts of the hepatic artery, portal vein and middle hepatic vein versus the liver parenchyma. While Gd-EOB-DTPA gave an earlier and more prolonged enhancement and image contrast of the bile duct, Gd-BOPTA achieved higher maximal enhancement and higher image contrast for all vessels studied during the arterial and portal venous phases. There was no significant difference in the maximal enhancement obtained in the liver parenchyma. In a third study, another 10 healthy volunteers were examined with the same protocol in another 1.5 T MRI system. Using signal normalization and a more quantitative, pharmacokinetic analysis, the hepatocyte-specific uptake of Gd-EOB-DTPA and Gd-BOPTA was calculated. A significant between-subjects correlation of the uptake estimates was found and the ratio of these uptake rates was of the same magnitude as has been reported in pre-clinical studies. The procedure also enabled quantitative analysis of vascular enhancement properties of these agents. Gd-BOPTA was found to give higher vessel-to-liver contrast than Gd-EOB-DTPA when recommended doses were given. In the final study, retrospectively gathered datasets from patients with hepatobiliary disease were analyzed using the quantitative estimation of hepatic uptake of Gd-EOB-DTPA described in the third study. The uptake rate estimate provided significant predictive ability in separating normal from disturbed hepatobiliary function, which is promising for future evaluations of regional and global liver disease. In conclusion, the differing dynamic enhancement profiles of the liver-specific contrast agents presented here can be beneficial in one context and challenging in another. Diseases of the liver and biliary system may affect the vasculature, parenchyma or biliary excretion, or a combination of these. The clinical context in terms of the relative importance of vascular, hepatic parenchymal and biliary processes should therefore determine the contrast agent for each patient and examination. A quantitative approach to analysis of contrast-enhanced liver MRI examinations is feasible and may prove valuable for their interpretation.