Over the recent years, bile acids are no longer only recognized as lipid solubilizing molecules in the intestine but also identified as complex signaling hormones contributing to the regulation of many metabolic pathways. In the liver, the sodium taurocholate co-transporting polypeptide (NTCP) functions as the main uptake transporter of conjugated bile acids and inhibition or deficiency of this transporter leads to (temporary) elevated bile acid levels. Additionally, NTCP is the entry receptor for the hepatitis B and delta virus. In the first part of this thesis, a screening platform was developed to identify novel NTCP inhibitors. Using this method, 5 hits were identified that inhibited NTCP-mediated bile acid transport as well as hepatitis B and delta infection in cell lines. Next, we studied the molecular interaction between NTCP and the NTCP inhibitor Myrcludex B. We show that NTCP-bound Myrcludex B dissociates and relocates to newly formed NTCP molecules and thereby prolonging its presence, and thus its inhibitory potential, on the plasma membrane. In the second part of this thesis, the metabolic consequences of temporary elevated bile acid levels were studied using NTCP knockout (KO) mice and the NTCP inhibitor Myrcludex B. On a high fat diet, NTCP KO mice or mice treated with Myrcludex B had a lower body weight and decreased fat content in the liver compared to control animals. Mechanistically, decreased intestinal fat absorption and increased energy expenditure by brown adipose tissue played an important role. Therefore, NTCP is an attractive target in the treatment of metabolic diseases.
To predict the absorption, distribution, metabolism and excretion (ADME) profile of candidate drugs a variety of preclinical models can be applied. The ADME and toxicological behavior of newly developed drugs are often investigated prior to assessment in humans, which is associated with long time-lines and high costs. Therefore, good predictions of ADME profiles earlier in the drug development process are very valuable. Good prediction of intestinal absorption and renal and biliary excretion remain especially difficult, as there is an interplay of active transport and metabolism involved. To study these processes, including enterohepatic circulation, ex vivo tissue models are highly relevant and can be regarded as the bridge between in vitro and in vivo models. In this review the current in vitro, in vivo and in more detail ex vivo models for studying pharmacokinetics in health and disease are discussed. Additionally, we propose novel models, i.e., perfused whole-organs, which we envision will generate valuable pharmacokinetic information in the future due to improved translation to the in vivo situation. These machine-perfused organ models will be particularly interesting in combination with biomarkers for assessing the functionality of transporter and CYP450 proteins.
This study investigated two aspects of the perception of bronchoconstriction ("sensitivity" and "absolute perceptual magnitude") in asthmatic patients and identified which clinical characteristics are related to these two aspects of perception of bronchoconstriction. The perception of histamine induced bronchoconstriction was measured in 128 asthmatic patients. Subjects quantified their breathlessness on a Visual Analogue Scale (VAS) before forced expiratory volume in one second (FEV1 was measured after each inhalation of histamine. The perceptive "sensitivity" for changes in FEV1 was analysed by the "VAS percentage fall in FEV1" slope. The "absolute perceptual magnitude" was determined by the VAS value at a 20% fall in FEV1. Spearman correlations were used for analysis between the two aspects of perception and asthma symptoms, peak flow variability, bronchial responsiveness and FEV1 % predicted. Patients with a low "sensitivity" for changes in FEV1 were more likely to show a frequent peak flow variability (Rs=-0.21; p<0.05), a high bronchial responsiveness (Rs= 0.37; p<0.001) and a low baseline FEV1 % pred (Rs=0.22; p<0.05). Patient's "absolute perceptual magnitude" correlated positively with symptoms during daily life (significant correlations varied 0.21-0.32) but not with the lung function parameters. The severity of asthma reflected by a low lung function and a high bronchial responsiveness, is associated with a low "sensitivity" for changes in forced expiratory volume in one second. A patient's "absolute perceptual magnitude" is positively related with asthma symptoms during daily life.
The majority of screening and predictive models do not reflect the physiology of the human intestinal tract since they show major limitations to include the processes that determine the oral bioavailability1,2. A major drawback of current intestinal models is the use of single cell lines and the static environment, which is in contrast with the dynamic processes in vivo2. Here we overcome these shortcomings by combining ex vivo models and organ on a chip technology.
Background: The gut and its microbiome have a major impact on many aspects of health and are therefore also an attractive target for drug- or food-based therapies. Here, we report on the added value of combining a microbiome screening model, the i-screen, with fresh intestinal tissue explants in a microfluidic gut-on-a-chip model, the Intestinal Explant Barrier Chip (IEBC). Methods: Adult human gut microbiome (fecal pool of 6 healthy donors) was cultured anaerobically in the i-screen platform for 24 h, without and with exposure to 4 mg/mL inulin. The i-screen cell-free culture supernatant was subsequently applied to the luminal side of adult human colon tissue explants (n = 3 donors), fixed in the IEBC, for 24 h and effects were evaluated. Results: The supplementation of the media with inulin promoted the growth of Anaerostipes , Bifidobacterium , Blautia , and Collinsella in the in vitro i-screen, and triggered an elevated production of butyrate by the microbiota. Human colon tissue exposed to inulin-treated i-screen cell-free culture supernatant or control i-screen cell-free culture supernatant with added short-chain fatty acids (SCFAs) showed improved tissue barrier integrity measured by a 28.2%-34.2% reduction in FITC-dextran 4000 (FD4) leakage and 1.3 times lower transport of antipyrine. Furthermore, the release of pro-inflammatory cytokines IL-1β, IL-6, IL-8, and TNF-α was reduced under these circumstances. Gene expression profiles confirmed these findings, but showed more profound effects for inulin-treated supernatant compared to SCFA-supplemented supernatant. Conclusion: The combination of i-screen and IEBC facilitates the study of complex intestinal processes such as host-microbial metabolite interaction and gut health.
Over the past decade, microfluidic intestine-on-a-chip models have been emerging as a novel platform to study intestinal function in health and disease. These microphysiological systems surpass conventional in vitro intestinal model systems, as they add microenvironmental context in the form of mechanical cues or by the incorporation of multiple cell types and/or gut microbiome, thereby better reflecting intestinal architecture and physiology. This review summarizes the current intestine-on-a-chip models with a distinction between cell- or organoid-based models and models that apply ex vivo tissue biopsies, as well as describing the progress and hurdles to overcome when applying intestine-on-a-chip models to study host-microbe interactions and intestinal diseases.
