Search

3D spheroids of primary human hepatocytes (3D PHH) retain a differentiated phenotype with largely conserved metabolic function and proteomic fingerprint over weeks in culture. As a result, 3D PHH are gaining importance as a model for mechanistic liver homeostasis studies and in vitro to in vivo extrapolation (IVIVE) in drug discovery. However, the kinetics and regulation of drug transporters have not yet been assessed in 3D PHH. Here, we used organic cation transporter 1 (OCT1/SLC22A1) as a model to study both transport kinetics and the long-term regulation of transporter activity via relevant signalling pathways. The kinetics of the OCT1 transporter was studied using the fluorescent model substrate 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP+) and known OCT1 inhibitors in individual 3D PHH. For long-term studies, 3D PHH were treated with xenobiotics for seven days, after which protein expression and OCT1 function were assessed. Global proteomic analysis was used to track hepatic phenotypes as well as prototypical changes in other regulated proteins, such as P-glycoprotein and Cytochrome P450 3A4. ASP+ kinetics indicated a fully functional OCT1 transporter with a Km value of 14 ± 4.0µM as the mean from three donors. Co-incubation with known OCT1 inhibitors decreased the uptake of ASP+ in the 3D PHH spheroids by 35–52%. The long-term exposure studies showed that OCT1 is relatively stable upon activation of nuclear receptor signalling or exposure to compounds that could induce inflammation, steatosis or liver injury. Our results demonstrate that 3D PHH spheroids express physiologically relevant levels of fully active OCT1 and that its transporter kinetics can be accurately studied in the 3D PHH configuration. We also confirm that OCT1 remains stable and functional during the activation of key metabolic pathways that alter the expression and function of other drug transporters and drug-metabolizing enzymes. These results will expand the range of studies that ca, These authors contributed equally: Evgeniya Mickols, Alina Meyer and Niklas Handin.

Magnetic hyperthermia holds significant therapeutic potential, yet its clinical adoption faces challenges. One obstacle is the large-scale synthesis of high-quality superparamagnetic iron oxide nanoparticles (SPIONs) required for inducing hyperthermia. Robust and scalable manufacturing would ensure control over the key quality attributes of SPIONs, and facilitate clinical translation and regulatory approval. Therefore, we implemented a risk-based pharmaceutical quality by design (QbD) approach for SPION production using flame spray pyrolysis (FSP), a scalable technique with excellent batch-to-batch consistency. A design of experiments method enabled precise size control during manufacturing. Subsequent modeling linked the SPION size (6–30 nm) and composition to intrinsic loss power (ILP), a measure of hyperthermia performance. FSP successfully fine-tuned the SPION composition with dopants (Zn, Mn, Mg), at various concentrations. Hyperthermia performance showed a strong nonlinear relationship with SPION size and composition. Moreover, the ILP demonstrated a stronger correlation to coercivity and remanence than to the saturation magnetization of SPIONs. The optimal operating space identified the midsized (15–18 nm) Mn0.25Fe2.75O4 as the most promising nanoparticle for hyperthermia. The production of these nanoparticles on a pilot scale showed the feasibility of large-scale manufacturing, and cytotoxicity investigations in multiple cell lines confirmed their biocompatibility. In vitro hyperthermia studies with Caco-2 cells revealed that Mn0.25Fe2.75O4 nanoparticles induced 80% greater cell death than undoped SPIONs. The systematic QbD approach developed here incorporates process robustness, scalability, and predictability, thus, supporting the clinical translation of high-performance SPIONs for magnetic hyperthermia.

Detailed metabolic imaging of specific brain regions in early aging may expose pathophysiological mechanisms and indicate effective neuropharmacological targets in the onset of cognitive decline. Comprehensive imaging of brain aging and drug-target effects is restricted using conventional methodology. We simultaneously visualized multiple metabolic alterations induced by normal aging in specific regions of mouse brains by integrating Fourier-transform ion cyclotron resonance mass spectrometry imaging and combined supervised and unsupervised machine learning models. We examined the interplay between aging and the response to tacrine-induced acetylcholinesterase inhibition, a well-characterized therapeutic treatment against dementia. The dipeptide carnosine (β-alanyl-l-histidine) and the vitamin α-tocopherol were significantly elevated by aging in different brain regions. l-Carnitine and acetylcholine metabolism were found to be major pathways affected by aging and tacrine administration in a brain region-specific manner, indicating altered mitochondrial function and neurotransmission. The highly interconnected hippocampus and retrosplenial cortex displayed different age-induced alterations in lipids and acylcarnitines, reflecting diverse region-specific metabolic effects. The subregional differences observed in the hippocampal formation of several lipid metabolites demonstrate the unique potential of the technique compared to standard mass spectrometry approaches. An age-induced increase of endogenous antioxidants, such as α-tocopherol, in the hippocampus was detected, suggesting an augmentation of neuroprotective mechanisms in early aging. Our comprehensive imaging approach visualized heterogeneous age-induced metabolic perturbations in mitochondrial function, neurotransmission, and lipid signaling, not always attenuated by acetylcholinesterase inhibition.

Acamprosate is an anionic drug substance widely used in treating symptoms of alcohol withdrawal. It was recently shown that oral acamprosate absorption is likely due to paracellular transport. In contrast, little is known about the eliminating mechanism clearing acamprosate from the blood in the kidneys, despite the fact that studies have shown renal secretion of acamprosate. The hypothesis of the present study was therefore that renal organic anion transporters (OATs) facilitate the renal excretion of acamprosate in humans. The aim of the present study was to establish and apply OAT1 (gene product of SLC22A6) and OAT3 (gene product of SLC22A8) expressing cell lines to investigate whether acamprosate is a substrate or inhibitor of OAT1 and/or OAT3. The studies were performed in HEK293-Flp-In cells stably transfected with SLC22A6 or SLC22A8. Protein and functional data showed that the established cell lines are useful for studying OAT1- and OAT3-mediated transport in bi-laboratory studies. Acamprosate inhibited OAT1-mediated p-aminohippuric acid (PAH) uptake but did not inhibit substrate uptake via OAT3 expressing cells, neither when applied concomitantly nor after a 3 h preincubation with acamprosate. The uptake of PAH via OAT1 was inhibited in a competitive manner by acamprosate and cellular uptake studies showed that acamprosate is a substrate for OAT1 with a K-m-value of approximately 700 mu M. Probenecid inhibited OAT1-mediated acamprosate uptake with a K-i-value of approximately 13 mu M, which may translate into an estimated clinically significant DDI index. In conclusion, acamprosate was identified as a substrate of OAT1 but not OAT3.

In drug development, estimating fraction absorbed (Fa) in man for permeability-limited compounds is important but challenging. To model Fa of such compounds from apparent permeabilities (Papp) across filter-grown Caco-2 cell monolayers, it is central to elucidate the intestinal permeation mechanism(s) of the compound. The present study aims to refine a computational permeability model to investigate the relative contribution of paracellular and transcellular routes to the Papp across Caco-2 monolayers of the permeability-limited compound acamprosate having a bioavailability of ∼11%. The Papp values of acamprosate and of several paracellular marker molecules were measured. These Papp values were used to refine system-specific parameters of the Caco-2 monolayers, that is, paracellular pore radius, pore capacity, and potential drop. The refined parameters were subsequently used as an input in modeling the permeability (Pmodeled) of the tested compounds using mathematical models collected from two published permeability models. The experimental data show that acamprosate Papp across Caco-2 monolayers is low and similar in both transport directions. The obtained acamprosate Papp, 1.56 ± 0.28 × 10-7 cm·s-1, is similar to the Papp of molecular markers for paracellular permeability, namely, mannitol (2.72 ± 0.24 × 10-7 cm·s-1), lucifer yellow (1.80 ± 0.35 × 10-7 cm·s-1), and fluorescein (2.10 ± 0.28 × 10-7 cm·s-1), and lower than that of atenolol (7.32 ± 0.60 × 10-7 cm·s-1; mean ± SEM, n = 3-6), while the end-point amount of acamprosate internalized by the cell monolayer, Qmonolayer, was lower than that of mannitol. Acamprosate did not influence the barrier function of the monolayers since it altered neither the Papp of the three paracellular markers nor the transepithelial electrical resistance (TEER) of the cell monolayer. The Pmodeled for all the paracellular markers and acamprosate was dominated by the Ppara component and matched the experimentally obtained Papp. Furthe

Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9), i.e., CRISPR-Cas9, has been extensively used as a gene-editing technology during recent years. Unlike earlier technologies for gene editing or gene knockdown, such as zinc finger nucleases and RNA interference, CRISPR-Cas9 is comparably easy to use, affordable, and versatile. Recently, CRISPR-Cas9 has been applied in studies of drug absorption, distribution, metabolism, and excretion (ADME) and for ADME model generation. To date, about 50 papers have been published describing in vitro or in vivo CRISPR-Cas9 gene editing of ADME and ADME-related genes. Twenty of these papers describe gene editing of clinically relevant genes, such as ATP-binding cassette drug transporters and cytochrome P450 drug-metabolizing enzymes. With CRISPR-Cas9, the ADME tool box has been substantially expanded. This new technology allows us to develop better and more predictive in vitro and in vivo ADME models and map previously underexplored ADME genes and gene families. In this mini-review, we give an overview of the CRISPR-Cas9 technology and summarize recent applications of CRISPR-Cas9 within the ADME field. We also speculate about future applications of CRISPR-Cas9 in ADME research.

The purpose of this study, written in Swedish, is to study two of the most popular sportsevents in Sweden; Vasaloppet and Göteborgsvarvet (Gothenburg Half Marathon) and theaffect the events has on the host destinations and also how the organizers use the destinationsfor their development.The study also shows how municipalities and public authorities connected to destinationdevelopment handles these events for their own purpose in their destination marketing todevelop their destination and improve the destination image.To achieve the purpose of the study, the following research questions has been formulated:“How does the municipality work with these events to improve the development of thedestination?” and “How does Vasaloppet and Göteborgsvarvet affect the destinations and howdoes the race events use their destination for their development?”Big events related to tourism is often organized by independent actors without any supportfrom the government controlled authorities. Despite that, the municipality or governmentcontrolled organizations will always have an an impact on these big events since they providethe essential infrastructure, transport, etc.One of the tools used in this study is semi structured interviews with 19 different, for thecases, relevant people, including the leaders within the organizing committees of Vasaloppetand Göteborgsvarvet. Interviews has also been made with the municipalities and otherpartners to the organizers. As a complement to these interviews, we have also interviewed afew participants from the two races. The study is also complemented with media articles andother previous research in the subject.The study identifies a strong dependency between the organizers and destinations that goesboth ways. The conclusion clearly shows the needs of cooperation and support between all theinvolved organizations to be able to achieve the best possible result and development. Thestudy reveals how all the involved organizations cooperates and works, Syftet med den här studien är att kartlägga hur två av Sveriges största folkliga evenemang,Vasaloppet och Göteborgsvarvet, påverkar de destinationer de äger rum i och hur använder desig av respektive ort. Även hur aktuella kommuner och offentliga instanser kopplade tilldestinationsutveckling arbetar för att kapitalisera på dessa evenemang, dels ekonomiskt menframförallt i marknadsförande och imagelyftande syfte. För att uppnå studiens syfte harföljande forskningsfrågor utformats: “Hur arbetar kommunen, kopplat till destinationsutveckling,för att kapitalisera på dessa evenemang, dels ekonomiskt men framförallt imarknadsförande och imagelyftande syfte?” och “Hur påverkar Vasaloppet och Göteborgsvarvetsina respektive destinationer och hur använder evenemangen sig i sin tur av orten?”Stora evenemang relaterade till turistnäringen arrangeras ofta av självständiga aktörer utaninblandning från offentlig verksamhet. Dock kommer det offentliga alltid påverkaförutsättningarna för arrangemanget genom exempelvis att förse evenemanget med nödvändiginfrastruktur, transportmedel, markupplåtelse med mera, vilket är något som staten,kommunen, landstinget eller myndigheter ansvarar över (Svensk Turism 2010).Forskningsfrågorna har besvarats med hjälp utav semistrukturerade intervjuer med 19personer som vi ansett vara relevanra. Dessa är: Informant vid Mora kommun, Älvdalenskommun och Malung-Sälens kommun samt informant vid Sälens turistbyrå, Eva-Lena Frick(Vasaloppets VD) och Bo Edsberger (Göteborgsvarvets general).Vidare har även sponsorer avevenemangen och deltagare intervjuats. Intervjuerna gavs innebörd genom meningskoncentrering.Detta betyder att information som framkommit i intervjuerna delas in i olikateman. Intervjutexterna har sedan analyserats med hjälp av vår referensram som innehåller decentrala begreppen: Turistdestination, Evenemangsmarknadsföring, Event branding ochSportevenemang. Vidare kompletterades faktan från intervjuerna med information från mediasom behandlat de

Supplement: 1, Meeting Abstract: P143

Isolated human hepatocytes are commonly used to predict transporter-mediated clearance in vivo. Such predictions, however, do not provide estimations of transporter contributions and consequently do not allow predictions of the outcome resulting from a change in the activity of a certain transporter, e.g., by inhibition or a genetic variant with reduced function. Pitavastatin is a drug that is heavily dependent on hepatic transporters for its elimination and it is mainly excreted as unchanged drug in the bile. For this reason, pitavastatin was used as a model drug to demonstrate the applicability of a bottom-up approach to predict transporter-mediated disposition in sandwich-cultured human hepatocytes (SCHH), allowing for the estimation of transporter contributions. Transport experiments in transfected HEK293 cells and inverted membrane vesicles overexpressing each of the relevant transport proteins were used to generate parameter estimates for the mechanistic model. By adjusting for differences in transporter abundance between the in vitro systems and individual SCHH batches, the model successfully predicted time profiles of medium and intracellular accumulation. Our predictions of pitavastatin bile accumulation could, however, not be confirmed due to a very low biliary excretion of pitavastatin in relation to the hepatic uptake in our SCHH. This study is, to our knowledge, the first to successfully simulate transporter-mediated processes in a complex system such as SCHH at the level of individual transport proteins using a bottom-up approach.

In this chapter the transport proteins and enzymes of importance for drug clearance are discussed. The primary organ for drug metabolism is the liver and to reach the intracellular compartment of hepatocytes, orally administered drugs must cross both the intestinal wall and the cell membrane of the liver cells. Transport proteins present in the cellular membrane may facilitate or hinder the compounds crossing these cellular barriers and hence will influence to what extent compounds will reach the enzymes. Here, the enzymes and transport proteins of importance for drug clearance are discussed. The molecular features of importance for drug interactions with transport proteins and enzymes are analyzed and the possibility to predict molecular features vulnerable to enzymatic degradation is discussed. From detailed analysis of the current literature it is concluded that for interaction, both with transport proteins and enzymes, lipophilicity plays a major role. In addition to this property, molecular properties such as hydrogen bond acceptors and donors, charge, aromaticity and molecular size can be used to distinguish between routes of clearance.

Sustained autophagy contributes to the metabolic adaptation of cancer cells to hypoxic and acidic microenvironments. Since cells in such environments are resistant to conventional cytotoxic drugs, inhibition of autophagy represents a promising therapeutic strategy in clinical oncology. We previously reported that the efficacy of hydroxychloroquine (HCQ), an autophagy inhibitor under clinical investigation is strongly impaired in acidic tumor environments, due to poor uptake of the drug, a phenomenon widely associated with drug resistance towards many weak bases. In this study we identified salinomycin (SAL) as a potent inhibitor of autophagy and cytotoxic agent effective on several cancer cell lines under conditions of transient and chronic acidosis. Since SAL has been reported to specifically target cancer-stem cells (CSC), we used an established model of breast CSC and CSC derived from breast cancer patients to examine whether this specificity may be associated with autophagy inhibition. We indeed found that CSC-like cells are more sensitive to autophagy inhibition compared to cells not expressing CSC markers. We also report that the ability of SAL to inhibit mammosphere formation from CSC-like cells was dramatically enhanced in acidic conditions. We propose that the development and use of clinically suitable SAL derivatives may result in improved autophagy inhibition in cancer cells and CSC in the acidic tumor microenvironment and lead to clinical benefits.

Freshly isolated human hepatocytes are considered the gold standard for in vitro studies of liver functions, including drug transport, metabolism, and toxicity. For accurate predictions of the in vivo outcome, the isolated hepatocytes should reflect the phenotype of their in vivo counterpart, i.e., hepatocytes in human liver tissue. Here, we quantified and compared the membrane proteomes of freshly isolated hepatocytes and human liver tissue using a label-free shotgun proteomics approach. A total of 5144 unique proteins were identified, spanning over 6 orders of magnitude in abundance. There was a good global correlation in protein abundance. However, the expression of many plasma membrane proteins was lower in the isolated hepatocytes than in the liver tissue. This included transport proteins that determine hepatocyte exposure to many drugs and endogenous compounds. Pathway analysis of the differentially expressed proteins confirmed that hepatocytes are exposed to oxidative stress during isolation and suggested that plasma membrane proteins were degraded via the protein ubiquitination pathway. Finally, using pitavastatin as an example, we show how protein quantifications can improve in vitro predictions of in vivo liver clearance. We tentatively conclude that our data set will be a useful resource for improved hepatocyte predictions of the in vivo outcome.