Genetic Variation in the Proximal Promoter of ABC and SLC Superfamilies: Liver and Kidney Specific Expression and Promoter Activity Predict Variation

Membrane transporters play crucial roles in the cellular uptake and efflux of an array of small molecules including nutrients, environmental toxins, and many clinically used drugs. We hypothesized that common genetic variation in the proximal promoter regions of transporter genes contribute to observed variation in drug response. A total of 579 polymorphisms were identified in the proximal promoters (−250 to +50 bp) and flanking 5′ sequence of 107 transporters in the ATP Binding Cassette (ABC) and Solute Carrier (SLC) superfamilies in 272 DNA samples from ethnically diverse populations. Many transporter promoters contained multiple common polymorphisms. Using a sliding window analysis, we observed that, on average, nucleotide diversity (π) was lowest at approximately 300 bp upstream of the transcription start site, suggesting that this region may harbor important functional elements. The proximal promoters of transporters that were highly expressed in the liver had greater nucleotide diversity than those that were highly expressed in the kidney consistent with greater negative selective pressure on the promoters of kidney transporters. Twenty-one promoters were evaluated for activity using reporter assays. Greater nucleotide diversity was observed in promoters with strong activity compared to promoters with weak activity, suggesting that weak promoters are under more negative selective pressure than promoters with high activity. Collectively, these results suggest that the proximal promoter region of membrane transporters is rich in variation and that variants in these regions may play a role in interindividual variation in drug disposition and response

ATP-Binding Cassette Efflux Transporters and Passive Membrane Permeability in Drug Absorption and Disposition

Transport into and across the cells of the human body is a prerequisite for the pharmacological action of drugs. Passive membrane permeability and active transport mechanisms are major determinants of the intestinal absorption of drugs, as well as of the distribution to target tissues and the subsequent metabolism and excretion from the body. In this thesis, the role of ATP-binding cassette (ABC) transporters and passive permeability on drug absorption and disposition was investigated. Particular emphasis was placed on defining the molecular properties important for these transport mechanisms. The influence of different transport pathways on predictions of intestinal drug absorption was investigated using experimental models of different complexity. Experimental models that include the paracellular pathway gave improved predictions of intestinal drug absorption, especially for incompletely absorbed drugs. Further, the inhibition of the ABC transporters breast cancer resistance protein (BCRP/ABCG2) and multidrug-resistance associated protein 2 (MRP2/ABCC2) was experimentally investigated using structurally diverse datasets that were representative of orally administered drugs. A large number of previously unknown inhibitors were identified among registered drugs, but their clinical relevance for drug-drug interactions and drug-induced toxicity remains to be determined. The majority of the inhibitors affected all three major ABC transporters BCRP, MRP2 and P-glycoprotein (P gp/ABCB1), and these multi-specific inhibitors were found to be enriched in highly lipophilic weak bases. To summarize, the present work has led to an increased knowledge of the molecular features of importance for ABC transporter inhibition and passive membrane permeability. Previously unknown ABC transporter inhibitors were identified and predictive computational models were developed for the different drug transport mechanisms. These could be valuable tools to assist in the prioritization of experimental efforts in early drug discovery

Due Diligence - En resurskrävande process : En checklista som mindre privata aktiebolag kan följa innan de ska utföra en Due Diligence

Abstract                “Due diligence - A resource demanding process” Date:                     June 8th 2012 Level:                    Master thesis in business economics, 15 ECTS Institution:           School of Sustainable development of society and technology, Mälardalen University Authors:                Erdinc Kirik                                  Pär Matsson                                11th January 1987                      31th May 1985 Title:                      Due diligence - A resource demanding process Tutor:                     Staffan Boström Keywords:             Due diligence, the acquisition process, acquisitions, business valuation Research questions:              What are the main factors that Bank X, Nordea and Almi is focused on an examination of a due diligence for an acquisition, focusing on private limited companies? Does the approach between the various banks and Almi differ? Purpose:                The purpose of this paper is to create a checklist for private limited companies to use in the context of application for funding Method:                 The study was based on a qualitative study, based on primary data in terms of interviews in the subject of due diligence. Secondary data used in the study consisted of relevant literature and scientific articles. Conclusion:            The study showed that the respondents in general focuses on the same areas within Due diligence. The most important point which the respondents agreed on was profitability and repayment capacity. In overall, all elements in a Due diligence process that has an impact on profitability is important to examine. Including management, especially in smaller private companies, has an important role. They must perform in the business plan and convince the respondents that there is a repayment capacity.Sammanfattning    ”Due diligence – En resurskrävande process” Datum:                   24 maj, 2012 Nivå:                       Magisteruppsats i företagsekonomi, 15 ECTS Institution:            Akademin för hållbar samhälls- och teknikutveckling, HST, Mälardalens Högskola Författare:             Erdinc Kirik                                  Pär Matsson                                 11 januari 1987                          31 maj 1985 Titel:                       Due diligence – En resurskrävande process Handledare:            Staffan Boström Nyckelord:              Due diligence, förvärsprocessen, företagsförvärv, företagsvärdering Frågeställning:       Vilka är de viktigaste faktorerna som Bank X, Nordea och Almi fokuserar på vid granskning av en Due diligence inför ett företagsförvärv, med fokus på privata aktiebolag? Skiljer sig tillvägagångssättet mellan de olika bankerna och Almi? Syfte:                     Syftet med uppsatsen är att skapa en checklista som privata aktiebolag kan använda sig utav i samband med ansökan om finansiering. Metod:                    Uppsatsen gjordes utifrån en kvalitativ undersökning och baserades på primärdata i form av intervjuer inom ämnet Due diligence. Sekundärdata som använts i uppsatsen bestod av relevant litteratur samt vetenskapliga artiklar. Slutsats:                 Uppsatsen visade att respondenterna i stort fokuserar på samma områden inom Due diligence. Den allra viktigaste punkten som respondenter var eniga om var lönsamhet och återbetalningsförmåga. I det stora hela är, alla faktorer i en Due diligence som har en inverkan på lönsamheten viktiga att granska. Även företagsledningen, speciellt i mindre privata aktiebolag, har en viktig roll. De måste prestera i affärsplanen och kunna övertyga respondenterna om att återbetalningsförmågan finns

Computational modeling to predict the functions and impact of drug transporters

Transport proteins are important mediators of cellular drug influx and efflux and play crucial roles in drug distribution, disposition and clearance. Drug-drug interactions have increasingly been found to occur at the transporter level and, hence, computational tools for studying drug-transporter interactions have gained in interest. In this short review, we present the most important transport proteins for drug influx and efflux. Computational tools for predicting and understanding the substrate and inhibitor interactions with these membrane-bound proteins are discussed. We have primarily focused on ligand-based and structure-based modeling, for which the state-of-the-art and future challenges are also discussed

A Cell-Free Approach Based on Phospholipid Characterization for Determination of the Cell Specific Unbound Drug Fraction (f(u,cell))

Purpose The intracellular fraction of unbound compound (f(u,cell)) is an important parameter for accurate prediction of drug binding to intracellular targets. f(u,cell) is the result of a passive distribution process of drug molecules partitioning into cellular structures. Initial observations in our laboratory showed an up to 10-fold difference in the f(u,cell) of a given drug for different cell types. We hypothesized that these differences could be explained by the phospholipid (PL) composition of the cells, since the PL cell membrane is the major sink of unspecific drug binding. Therefore, we determined the f(u,cell) of 19 drugs in cell types of different origin. Method The cells were characterized for their total PL content and we used mass spectrometric PL profiling to delineate the impact of each of the four major cellular PL subspecies: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI). The cell-based experiments were compared to cell-free experiments that used beads covered by PL bilayers consisting of the most abundant PL subspecies. Results PC was found to give the largest contribution to the drug binding. Improved correlations between the cell-based and cell-free assays were obtained when affinities to all four major PL subspecies were considered. Together, our data indicate that f(u,cell) is influenced by PL composition of cells. Conclusion We conclude that cellular PL composition varies between cell types and that cell-specific mixtures of PLs can replace cellular assays for determination of f(u,cell) as a rapid, small-scale assay covering a broad dynamic range

Intracellular drug bioavailability : a new predictor of system dependent drug disposition

Intracellular drug exposure is influenced by cell-and tissue-dependent expression of drug-transporting proteins and metabolizing enzymes. Here, we introduce the concept of intracellular bioavailability (F-ic) as the fraction of extracellular drug available to bind intracellular targets, and we assess how Fic is affected by cellular drug disposition processes. We first investigated the impact of two essential drug transporters separately, one influx transporter (OATP1B1; SLCO1B1) and one efflux transporter (P-gp; ABCB1), in cells overexpressing these proteins. We showed that OATP1B1 increased Fic of its substrates, while P-gp decreased Fic. We then investigated the impact of the concerted action of multiple transporters and metabolizing enzymes in freshly-isolated human hepatocytes in culture configurations with different levels of expression and activity of these proteins. We observed that Fic was up to 35-fold lower in the configuration with high expression of drug-eliminating transporters and enzymes. We conclude that Fic provides a measurement of the net impact of all cellular drug disposition processes on intracellular bioavailable drug levels. Importantly, no prior knowledge of the involved drug distribution pathways is required, allowing for high-throughput determination of drug access to intracellular targets in highly defined cell systems (e.g., single-transporter transfectants) or in complex ones (including primary human cells)

Cardiovascular complications following cesarean section and vaginal delivery : a national population-based study

Introduction: Rates of cesarean section are rising in both developed and developing countries and while pregnancy and cesarean section are established as risk factors for thromboembolism and stroke, large population-based investigations focusing on all types of cardiovascular complication after delivery is missing. The aim was to analyze the risk of severe cardiovascular complications in the post-partum period following delivery by cesarean section. We also had a control group of vaginal deliveries and a reference group with nulliparas. Materials and Methods: This Swedish population-based study used three national registers between 2005 and 2017 and comprised a total of 1 165 684 individuals. Unselected register data was cross-linked and cardiovascular adverse events were identified by ICD diagnosis codes. 140 128 women (209 391 deliveries) were included in the cesarean group and 614 355 women (973 429 deliveries) in the vaginal control group. The reference group comprised 411 201 age-matched nulliparous women. The primary analysis was the risk of severe cardiovascular complications within 42 days of cesarean section or vaginal delivery. The secondary analysis evaluated risk factors for cardiovascular complications. Results: In the cesarean section group, 410 (0.20%) had a serious cardiovascular event within 42 days after delivery, and in the vaginal control group the number was 857 (0.09%). The risk of having an adverse cardiovascular event was significantly greater in the cesarean group (OR 2.23, CI 1.98 to 2.51) for all types of cardiovascular events. Risk factors were high BMI, preeclampsia, greater maternal age, tobacco use and acute cesarean delivery. Conclusions: The absolute numbers on severe maternal morbidity after delivery are low. However, since almost half of the world’s population are affected and the frequency of elective cesarean section continues to rise, a doubling of the risk for a severe cardiovascular event within 42 days of delivery is important to consider globally

Intracellular Drug Bioavailability : Effect of Neutral Lipids and Phospholipids

Intracellular unbound drug concentrations are the pharmacologically relevant concentrations for targets inside cells. Intracellular drug concentrations are determined by multiple processes, including the extent of drug binding to intracellular structures. The aim of this study was to evaluate the effect of neutral lipid (NL) and phospholipid (PL) levels on intracellular drug disposition. The NL and/or PL content of 3T3-L1 cells were enhanced, resulting in phenotypes (in terms of morphology and proteome) reminiscent of adipocytes (high NL and PL) or mild phospholipidosis (only high PL). Intracellular bioavailability (F-ic) was then determined for 23 drugs in these cellular models and in untreated wild-type cells. A higher PL content led to higher intracellular drug binding and a lower F-ic. The induction of NL did not further increase drug binding but led to altered F-ic due to increased lysosomal pH. Further, there was a good correlation between binding to beads coated with pure PL and intracellular drug binding. In conclusion, our results suggest that PL content is a major determinant of drug binding in cells and that PL beads may constitute a simple alternative to estimating this parameter. Further, the presence of massive amounts of intracellular NLs did not influence drug binding significantly

Quantifying the impact of transporters on cellular drug permeability.

The conventional model of drug permeability has recently been challenged. An alternative model proposes that transporter-mediated flux is the sole mechanism of cellular drug permeation, instead of existing in parallel with passive transmembrane diffusion. We examined a central assumption of this alternative hypothesis; namely, that transporters can give rise to experimental observations that would typically be explained with passive transmembrane diffusion. Using systems-biology simulations based on available transporter kinetics and proteomic expression data, we found that such observations are possible in the absence of transmembrane diffusion, but only under very specific conditions that rarely or never occur for known human drug transporters