As a critical parameter in pharmacokinetics, prediction of clearance is an integral aspect of drug discovery programmes. Since the liver is the major site of xenobiotic biotransformation, accurate prediction of hepatic clearance (CLh) is vital. The use of cellular and subcellular in vitro systems for this purpose is common practice; however, prediction accuracy tends to be poor. The aim of this thesis was to explore potential contributing factors to the underprediction of in vivo clearance, specifically with relation to the in vitro methodology of hepatocyte clearance assays, which is largely unstandardised. Literature data analyses highlighted an overall clearance-dependent trend of underprediction in both human and rat hepatocytes, indicating a fundamental in vitro system bias which is independent of species. During initial investigation of incubation conditions, the format of hepatocyte assays (suspension in microcentrifuge tubes, 96-well plates, 24-well plates and short-term monolayer culture) was demonstrated to influence determined intrinsic clearance (CLint). Differences in midazolam CLint were observed not only between suspended and short-term cultured hepatocytes, but also between suspended hepatocytes in different vessels/plate formats. The applicability of 1 µM as a generic substrate incubation concentration for determination of CLint by substrate depletion was evaluated in rat hepatocytes using nine well-characterised drugs. For seven of the nine drugs, a statistically significantly (p < 0.05) higher CLint was observed in determinations of 0.1 µM substrate as opposed to 1 µM, highlighting the potential for false determinations using current practices. Cofactor depletion in isolated hepatocytes was investigated based on previous speculation as the cause of clearance-dependent underprediction of in vivo clearance. Although moderate increases in CLint were observed with the addition of NADPH to hepatocyte incubations, this was subsequently attributed to the replenishment of NADPH in membrane-damaged hepatocytes. Retained functionality of metabolic enzymes in cells which would generally be considered non-viable by trypan blue exclusion was indicated in comparisons of unpurified and Percoll-purified cryopreserved hepatocytes. This phenomenon was conclusively demonstrated in incubations of permeabilised hepatocytes supplemented with NADPH, revealing a need for re-evaluation of the use of plasma membrane integrity (trypan blue exclusion) as a measure of viability in metabolic studies. ATP content was considered as a potential alternative measure; however no significant correlations were found between ATP content, trypan blue exclusion and the CLint of nine drugs in associated preparations. The effect of shaking on CLint in rat hepatocytes was also examined. For 10 out of 12 drugs, CLint determined at 900 rpm was significantly (p < 0.05) higher than in static incubations. Three potential mechanisms were hypothesised: plasma membrane damage, increased substrate distribution throughout the bulk medium and reduction in the depth of the unstirred water layer (UWL) surrounding cells. Shaking of saponin-permeabilised hepatocytes (supplemented with NADPH to maintain metabolism) also increased the determined CLint of saquinavir, indicating a rate-limitation other than membrane permeation. However, shaking of ultra-sonicated hepatocytes in which the plasma membrane was entirely destroyed (also supplemented with NADPH) did not change the determined CLint of saquinavir, revealing the rate-limitation of UWL permeation in both intact and permeabilised cells. The depth of such an UWL in vitro is likely to be artificially greater than in vivo; therefore reduction of UWL depth through incubation shaking is proposed as a physiologically sound approach by which to increase in vitro CLint. In addition, a framework of experiments and related equations is presented by which intact and permeabilised hepatocytes in static and shaken conditions may be utilised to identify the rate-determining process and contribution of individual processes to the in vitro CLint of a drug. The effects of substrate concentration and shaking were also evaluated in human hepatocytes. Significant increases in determined CLint of drugs with use of 0.1 µM substrate (as opposed to 1 µM) and shaking at 900 rpm were demonstrated, confirming equivalent potential in vitro sources of underprediction between rat and human. These highly significant findings reveal the existing limitations of in vitro assays and potential flaws in current practice in in vitro determinations of CLint. Appropriate consideration of the properties of in vitro systems, including the presence of the UWL, should lead to improved predictions of in vivo clearance.

Dihydromyricetin (DHM), also ampelopsin, is a flavonoid compound which exhibits a broad spectrum of positive effects on the human body. Herbal extracts containing this compound have been widely used in traditional Chinese medicine mainly for their hepatoprotective properties. DHM also helps with alcohol intoxication and reduces the signs of hangover or abstinence. Given the fact that the mechanism of DHM effects on the ethanol metabolism has not been clarified yet, the effect of dihydromyricetin on the expression and activity of alcohol dehydrogenase (ADH), one of the most important enzymes involved in ethanol metabolism, was therefore studied in this thesis. The cultivation conditions of primary hepatocytes which were isolated from unpretreated and ethanol-pretreated rats and subsequently exposed to EtOH and DHM were optimized. While determining the degree of cell damage caused by EtOH in the presence of DHM, no significant trend in the protective effect of DHM was found. On the other hand, the protective effect of ethanol in hepatocytes cultivated in EtOH and DHM was detected by technique of ELISA (the determination of alanine transaminase). The Western blot technique followed by immunodetection did not detect the induction of ADH expression in hepatocytes. Furthemore, the modulation effect of...

Diss. (sammanfattning) Stockholm : Karol. inst., 2003. / Härtill 6 uppsatser.

The production of very low density lipoprotein and high density lipoprotein particles by the liver is a tightly regulated process, which begins with synthesis and assembly of core protein components in the rough endoplasmic reticulum. Factors influencing the production and metabolism of these particles are of immediate medical relevance, as their malfunction or hyperactivity can lead to an assortment of disease states. Hepatic lipase is a secreted liver enzyme, with many previously described roles in the metabolism and clearance of both high and low density lipoproteins. Increased production and assembly of this enzyme is an indicator of metabolic dysfunction, while its absence or insufficiency leads to pre-mature atherosclerosis and death. The present study shows that this enzyme’s role in lipoprotein metabolism is not confined to the degradation and clearance of these particles after they have been secreted. Experiments using co-immunoprecipitation targeted at hepatic lipase demonstrate that this protein interacts with ApoA1 and ApoB100, the core protein components of HDL and VLDL respectively, at the ER level in hepatocytes, as part of an enormous multi-subunit protein complex. This interaction with ApoA1 leads to decreased competence of hepatocytes to secrete HDL, which confers a pro-atherogenic phenotype. Analysis of ER to Golgi VLDL transport vesicles, produced with a cell-free in vitro budding assay, has revealed that hepatic lipase is co-secreted between these compartments with immature VLDL particles. Further analysis of cytosol isolated from hepatocytes demonstrates an interaction between hepatic lipase and the LDL-receptor related protein in a post-Golgi vesicle; the significance of which will be investigated in future studies.

No description available.

Thesis (Ph. D.)--University of Washington, 2001. / Vita. Includes bibliographical references (leaves 81-96).

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2006. / Härtill 4 uppsatser.

La phosphoinositide 3-kinase p110δ (PI3Kδ) est principalement exprimée dans les cellules hématopoïétiques et son inhibiteur, l'idélalisib est approuvé pour le traitement de la leucémie et du lymphome. Cependant, la fonction de cette isoforme dans les cellules non hématopoïétiques reste insaisissable. Dans cette étude, nous rapportons que la formation des canalicules biliaires est dépendante de l’activité de la PI3Kδ lors de la culture en 3D de cellules de carcinome hépatocellulaire. Une étude in-vitro reposant sur la différenciation de cellules souches embryonnaires humaines en hépatocytes révèle que la PI3Kδ est enrichie dans les cellules souches et que son expression diminue au fur et à mesure que les cellules se différencient. Lorsqu’elle est surexprimée, la PI3Kδ reprogramme ces cellules en cellules ressemblant à des cellules souches formant des rosettes polarisées et perdant des marqueurs d’hépatocytes pour acquérir des traits de cholangiocytes. Dans le foie murin, la réexpression de la PI3Kδ entraine des modifications de la morphologie de cellules jouxtant la veine porte, ainsi que l’augmentation de l’expression du gène codant pour EpCAM. Cette reprogrammation dépendante de la PI3Kδ est associée à l'activation de la voie de Notch et requiert l'activation de la protéine Src. Enfin, la PI3Kδ est exprimée dans les lignées cellulaires dérivées d’hépatoblastome humain et le traitement des souris par l’idélalisib diminue la taille des tumeurs formées par les PDX d’hépatoblastome. La PI3Kδ représente donc une cible thérapeutique prometteuse dans ce cancer pédiatrique avec des caractéristiques de cellules souches. / The phosphoinositide 3-kinase p110δ (PI3Kδ) is primarily expressed in the hematopoietic cells and its inhibitor, Idelalisib, is approved for leukemia and lymphoma treatments. Nevertheless, the function of PI3Kδ in the non-hematopoietic cells is still elusive. Here we report that the formation of bile canaliculi is dependent on the PI3Kδ activity using hepatocellular carcinoma (HCC) cells grown in a 3D culture. In-vitro study based on hepatocytes differentiation from human Embryonic Stem Cell (hESC) highlights that PI3Kδ is enriched in hESC and increasingly reduces over time while cells are differentiating. When PI3Kδ is overexpressed, it reprograms those cells into stem-like cells forming polarized rosette structures and losing hepatocyte markers to gain cholangiocyte characteristics. These changes were observed in mice liver overexpressing PI3Kδ. The aforementioned reprogramming, dependent on PI3Kδ, is associated with the Notch pathway activation and requires the Src protein activation. Finally,PI3Kδ is expressed in hepatoblastoma cell lines and idelalisib efficiently reduces tumor size formed by patient derived xenograft (PDX). Therefore, PI3Kδ represents a promising therapeutic target for this pediatric cancer with stem cell features.

Primary human hepatocytes are a scarce resource with variable function, which diminishes with time in culture. As a consequence their use in tissue modelling and therapy is restricted. Human embryonic stem cells (hESCs) could provide a stable source of human tissue due to their properties of self-renewal and their ability to give rise to all three germ layers. hESCs have the potential to provide an unlimited supply of hepatic endoderm (HE) which could offer efficient tools for drug discovery, disease modelling and therapeutic applications. In order to create a suitable environment to enhance HE formation, hESC culture needed to be standardised. As such, a media trail was carried out to define serum free media capable of maintaining hESC in a pluripotent undifferentiated state. We also ensured hESC cultured in the various media could be directly differentiated to HE in a reproducible and efficient manner. The project then focused on the effect of post-translational modifications (PTMs), specifically SUMOylation, in hepatocyte differentiation and its subsequent manipulation to enhance HE viability. SUMOylation is a PTM known to modify a large number of proteins that play a role in various cellular processes including: cell cycle regulation, gene transcription, differentiation and cellular localisation. We hypothesised that SUMO modification may not only regulate hESC self renewal, but also maybe required for efficient hESC differentiation. We therefore interrogated the role of SUMOylation in hESC differentiation to hepatic endoderm (HE). hESC were differentiated and the cellular lysates were analysed by Western blotting for key proteins which modulate the conjugation and de conjugation of SUMO. We demonstrate that peak levels of SUMOylation were detectable in hESC populations and during cellular differentiation to definitive endoderm (DE), day 5. Following commitment to DE we observed a decrease in the level of SUMO modified proteins during cellular specialisation to a hepatic fate, corresponding with an increase in SENP 1, a SUMO deconjugation enzyme. We also detected reduced levels of hepatocyte nuclear factor 4 α (HNF4α), a critical regulator of hepatic status and metabolic function, as SUMOylation decreased. As a result, we investigated if HNF4α was SUMOylated and if this process was involved in modulating HNF4α’s critical role in HE. HNF4α is an important transcription factor involved in liver organogenesis during development and is a key regulator for efficient adult liver metabolic functions. We observed a decreasing pattern of HNF4α expression at day 17 of our differentiation protocol in conjunction with a decrease in SUMO modified proteins. In order to further investigate and validate a role of SUMOylation on HNF4α stability Immunoprecipitation (IP) was employed. HNF4α protein was pulled down and probed for SUMO 2. Results show an increase in the levels of SUMO2 modification as the levels of HNF4α decrease. Through deletion and mutation analysis we demonstrated that SUMO modification of HNF4α was restricted to the C-terminus on lysine 365. Protein degradation via the proteasome was responsible for the decrease in HNF4α, demonstrated by the use of a proteasome 26S inhibitor MG132. Additionally, a group at the University of Dundee has shown that polySUMOylation of promyelocytic leukaemia protein (PML) leads to its subsequent ubiquitination via RNF4, an ubiquitin E3 ligase, driving its degradation. Using an in vitro ubiquitination assay, we show that polySUMOylated HNF4α is preferentially ubiquitinated in the presence of RNF4. Overall polySUMOylation of HNF4α may reduce its stability by driving its degradation, hence regulating protein activity. In conclusion, polySUMOylation of HNF4α is associated with its stability. HNF4α is subsequently important for HE differentiation both driving the formation of the hepatocytes and in maintaining a mature phenotype, in agreement with a number of different laboratories. Creating the ideal environment for sustaining mature functional hepatocytes, primary and those derived from hESCs and iPSCs, is essential for further use in applications such as drug screening, disease modelling and extracorporeal devices.

The liver is a multi-functional and highly regenerative organ. While resilient, the liver is susceptible to organ damage and failure. In both the acute and chronic settings liver disease has dire consequences for health. A common cause of liver damage is adverse reactions to drugs which can lead to drug induced liver injury (DILI). This creates major problems for patients, clinicians, the pharmaceutical industry and regulatory authorities. In the context of drug overdose or serious adverse reactions, liver failure can be acute and life threatening, and in some cases require orthotopic liver transplantation. While transplantation is highly successful, such an approach has limitations and justifies basic science attempts to develop better human models to study liver injury and to develop scalable intervention strategies. With this in mind, we have studied the importance of microRNAs (miRs) in regulating human drug metabolism in pluripotent stem cell – derived hepatocytes and their potential to reduce liver toxicity in response to toxic levels of paracetamol. miRs are small non-coding RNAs that are approximately 20 - 24 nucleotides long and their major function is to fine tune gene expression of their target genes. Recently, it has been demonstrated that microRNAs play a role in regulating the first phase of drug metabolism however the second phase of drug metabolism, drug conjugation, has not been studied in detail. Drug conjugation is a crucial stage in human drug metabolism, and any alterations in this process can lead to changes in compound pharmacology, including therapeutic dose and clearance from the body. To test the importance of miRs in regulating phase II drug metabolism we opted to study the metabolism of a common used analgesic, paracetamol. When taken in the appropriate amounts paracetamol is modified by sulfotransferases (SULTs) and UDP - glucuronosyltransferases (UGTs) and removed from the body without organ damage. However, when paracetamol is taken above the recommended dose it is metabolised by phase I enzymes to generate a toxic intermediate N-acetyl-p-benzoquinone imine (NAPQI), which if untreated can lead to massive hepatocyte cell death and liver failure, placing the patient in a life threatening situation. In order to promote non-toxic drug metabolism, in the context of drug overdose, we employed candidate miRs to regulate different parts of the paracetamol metabolism pathway. In summary, we have focused on studying human drug metabolism in the major metabolic cell type of the liver, the hepatocyte. We have identified a novel microRNA (called miR-324-5p) which regulates phase II drug metabolism and reduces cell cytotoxicity. Additionally, a supportive role of anti-microRNA- 324 in response to fulminant plasma collected from paracetamol overdose patients is also observed. The findings of this project are novel, provide proof of concept and exemplify the power of stem cell based models to identify new approaches to treating human liver damage.