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Tools such as genome resequencing and genome-wide association studies have recently been used to uncover a number of variants that affect drug toxicity and efficacy, as well as potential drug targets. But how much closer are we to incorporating pharmacogenomics into routine clinical practice? Five experts discuss how far we have come, and highlight the technological, informatics, educational and practical obstacles that stand in the way of realizing genome-driven medicine. [ABSTRACT FROM AUTHOR]

ATP-binding cassette(ABC) transporters comprise one of the largest membrane bound protein families. They are involved in transport of numerous compounds. These proteins transport substrates against a concentration gradient with ATP hydrolysis as a driving force across the membrane. Mammalian ABC proteins have important physiological, pharmacological and toxicological functions including the transport of lipids, bile salts, drugs, toxic and environmental agents. The efflux pumps serve both as natural defense mechanisms and influence the bioavailability and disposition of drugs. In general terms, the transporters remove xenobiotics from the cellular environment. For example, in cancer cells, over expression of these molecules may confer to multidrug resistance against cytostatic drugs. In addition, based on diverse structural characteristics and a broad substrate specifity, ABC transport proteins alter the intracellular concentration of a variety of therapeutically used compounds and toxicologically relevant agents. We review the function of the human multidrug resistance protein MDR1,(P-glycoprotein, ABCB1) and the multidrug resistance protein MRP2(ABCC2). We focus on four topics namely 1) structure and physiological functions of these transporters, 2) substrates e.g., drugs, xenotoxins, and environmental toxicants including their conjugates, 3) drug–drug interactions, and the role of chemosensitizers which may be able to reverse drug resistance, and 4) pharmacologically and toxicologically relevant genetic polymorphisms in transport proteins and their clinical implications. [ABSTRACT FROM AUTHOR]

Background: Genetic factors as predictor of the individual outcome of drug therapy is one aim of personalized medicine approaches. Case presentation: We report a drug metabolism based analysis of genetic polymorphisms in a Caucasian patient receiving fluvastatin and telmisartan experiencing myotoxicity (myalgia and moderate creatine kinase elevation). Conclusions: The obtained findings suggest that heterocygocity of cytochrome P450 CYP2C9*3 variant in combination with multidrug resistance-associated protein MRP2 -24C > T functions as risk factor predisposing to experience drug-drug interaction combing those drugs. [ABSTRACT FROM AUTHOR]

Platelets store signaling molecules (eg, serotonin and ADP) within their granules. Transporters mediate accumulation of these molecules in platelet granules and, on platelet activation, their translocation across the plasma membrane. The balance between transporter-mediated uptake and elimination of signaling molecules and drugs in platelets determines their intracellular concentrations and effects. Several members of the 2 major transporter families, ATP-binding cassette (ABC) transporters and solute carriers (SLCs), have been identified in platelets. An example of an ABC transporter is MRP4 (ABCC4), which facilitates ADP accumulation in dense granules. MRP4 is a versatile transporter, and various additional functions have been proposed, notably lipid mediator release and a role in aspirin resistance. Several other ABC proteins have been detected in platelets with functions in glutathione and lipid homeostasis. The serotonin transporter (SERT, SLC6A4) in the platelet plasma membrane represents a well-characterized example of the SLC family. Moreover, recent experiments indicate expression of OATP2B1 (SLC02B1), a high affinity transporter for certain statins, in platelets. Changes in transporter localization and expression can affect platelet function and drug sensitivity. This review summarizes available data on the physiologic and pharmacologic role of transporters in platelets. [ABSTRACT FROM AUTHOR]

renal elimination of a number of cationic compounds is thought to be mediated by the organic cation transporter 2 (OCT2, SLC22A2), a drug uptake transporter expressed at the basolateral domain of renal tubular cells. Recently, the key efflux transporter for the secretion organic cations was identified as an electroneutral H/organic cation exchanger termed the multidrug and toxin extrusion (MATE)-type transporter 1 (MATE1, SLC47AJ). The key goals of this study were to assess the interplay between the renal cationic transporters OCT2 and MATE 1 and the functional assessment of genetic variation in human MA TEl. First, the ability of various agents to interact with OCT2- or MATE1-mediated transport was determined using a recombinant vaccinia expression system. We were able to identify several drugs in clinical use with a divergent inhibitory capacity for these transporters. Subsequently, we further assessed the effect of those com- pounds on the cellular accumulation of shared substrates using OCT2 and MATE1 double-transfected cells. Consistent with data obtained using single transporter transfected cells, compounds that exhibited preferential inhibition of MATE1 such as rapamycin and mitoxantrone induced significant cellular accumulation of cationic substrates. We next assessed the functional relevance of MATE1 genetic polymorphisms. Significant loss of transport activity for metformin and tetraethylarnmonium was noted for two nonsynonymous single nucleotide polymorphisms (SNPs), c.404T>C (p.159T>M) and c.1012G>A (p.338V>A). The c.404T>C was only seen in Asian subjects with an allele frequency of 1%, and the c. 101 2G>A SNP was much more common, especially among those of African descent. In conclusion, we show that coordinate function of MATE1 with OCT2 likely contributes to the vectorial renal elimination of organic cationic drugs and that altered activity of MATE1, whether by drugs or polymorphisms, should be considered as an important determinant of renal cationic drug elimination. [ABSTRACT FROM AUTHOR]

Heart failure represents the composite endpoint of various cardiovascular disorders. Advanced pharmacotherapy resulted in significant improvement of overall survival, however with highly variable outcome, possibly due to genetic modification of drug disposition and action. This review highlights the role of genetic polymorphisms in systems responsible for disposition of drugs, used in heart failure patients (e.g. the polymorphic drug metabolizing enzymes such as cytochrome P450 enzymes, as well as polymorphic ATP-membrane transporters like P-glycoprotein (P-gp)). In addition, genetic variants in physiological systems, being target of drug action, particularly beta-adrenergic receptors, the renin–angiotensin–aldosterone system (RAAS)- and endothelin system, and the endothelial nitrogene monoxide (NO) synthase are reviewed. The current situation in pharmacogenomics of heart failure with respect to drug disposition and action is characterized by multiple studies investigating single components of a complex system. Therefore, overall conclusions regarding treatment and/or outcome of heart failure patients based on individual genetic traits require large prospective trials allowing for simultaneous assessment of multiple genetic variants in different systems. Using advanced screening technologies, such trials can be carried out in the near future. [Copyright &y& Elsevier]

The present investigation addressed the role of verapamil for oral pharmacokinetics of morphine-6-β-glucuronide (M6G). Male Sprague–Dawley rats received 62.5 mg kg−1 M6G-dihydrate orally w/wo pre-treatment with 70 mg kg−1 verapamil. Intravenous M6G (3.9 mg kg−1 ) and oral morphine (52.7 mg kg−1 morphine–hydrochloride) were also employed. Oral bioavailability of M6G and the fraction of M6G deglucuronidated to morphine were estimated from areas under the plasma-concentration vs. time curves (AUC) of morphine and its glucuronides. As initial results pointed towards inhibition of glucuronidases by verapamil, its capability to specifically inhibit E. coli and/or rat intestinal β-glucuronidase was assessed using altered cleavage of the model substrate 4-methylumbelliferyl-β-d-glucuronide (MUG). Oral bioavailability of M6G was 2.1%; 13% of oral M6G was deglucuronidated to morphine. Co-administration of verapamil did not increase the AUC of M6G. AUCs of morphine and morphine-3-glucuronide were smaller in the verapamil group than in controls. Verapamil co-administration decreased the fraction of M6G deglucuronidated to morphine to 4.6%. In vitro experiments provided evidence that verapamil inhibits β-glucuronidase from E. coli with an ic50 of 30 μM, whereas no inhibition of the rat β-glucuronidase from small intestine was seen. In conclusion, verapamil decreased intestinal deglucuronidation of M6G by inhibiting E. coli β-glucuronidase. This indicates that verapamil is not suited as P-gp inhibitor in experiments involving glucuronides. An increase in the intestinal absorption of M6G due to P-gp-inhibition was not observed at the verapamil dose studied. [Copyright &y& Elsevier]

The solute carrier (SLC) group of membrane transport proteins includes about 400 members organized into more than 50 families. The SLC family that comprises nucleoside-sugar transporters is referred to as SLC35. One of the members of this family is SLC35F1. The function of SLC35F1 is still unknown; however, recent studies demonstrated that SLC35F1 mRNA is highly expressed in the brain and in the kidney. Therefore, we examine the distribution of Slc35f1 protein in the murine forebrain using immunohistochemistry. We could demonstrate that Slc35f1 is highly expressed in the adult mouse brain in a variety of different brain structures, including the cortex, hippocampus, amygdala, thalamus, basal ganglia, and hypothalamus. To examine the possible roles of Slc35f1 and its subcellular localization, we used an in vitro glioblastoma cell line expressing Slc35f1. Co-labeling experiments were performed to reveal the subcellular localization of Slc35f1. Our results indicate that Slc35f1 neither co-localizes with markers for the Golgi apparatus nor with markers for the endoplasmic reticulum. Time-lapse microscopy of living cells revealed that Slc35f1-positive structures are highly dynamic and resemble vesicles. Using super-resolution microscopy, these Slc35f1-positive spots clearly co-localize with the recycling endosome marker Rab11. [ABSTRACT FROM AUTHOR]

The anthracycline-mediated cardiotoxicity is still not completely understood. To examine the impact of cholesterol metabolism and transport in this context, cholesterol and oxysterol levels as well as the expression of the cholesterol transporters ABCA1 and ABCG1 were analyzed in doxorubicin-treated HL-1 murine cardiomyocytes as well as in mouse model for acute doxorubicin-induced cardiotoxicity. Doxorubicin-treated HL-1 cells exhibited enhanced cholesterol (153 ± 20% of control), oxysterol (24 S-hydroxycholesterol: 206 ± 29% of control) and cholesterol precursor levels (lathosterol: 122 ± 12% of control; desmosterol: 188 ± 10% of control) indicating enhanced cholesterol synthesis. Moreover, abca1 and abcg1 were upregulated on mRNA, protein and functional level caused by a doxorubicin-mediated activation of the nuclear receptor LXR. In addition, the oxysterols not only induced the abca1 and abcg1 in HL-1 cells but also enhanced the expression of endothelin-1 and transforming growth factor-β, which have already been identified as important factors in doxorubicin-induced cardiotoxicity. These in vitro findings were verified in a murine model for acute doxorubicin-induced cardiotoxicity, demonstrating elevated cardiac (2.1 ± 0.2 vs. 3.6 ± 1.0 ng/mg) and systemic cholesterol levels (105.0 ± 8.4 vs. 130.0 ± 4.3 mg/dl), respectively, as well as enhanced oxysterol levels such as cardiac 24 S-hydroxycholesterol (2.1 ± 0.2 vs. 3.6 ± 1.0 ng/mg). In line with these findings cardiac mRNA expression of abca1 (303% of control) and abcg1 (161% of control) was induced. Taken together, our data demonstrate enhanced cholesterol and oxysterol levels by doxorubicin, resulting in a LXR-dependent upregulation of abca1 and abcg1. In this context, the cytotoxic effects of oxysterols and their impact on cardiac gene expression should be considered as an important factor in doxorubicin-induced cardiotoxicity. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented in response to an article about first-in-human clincial trial in the December 9, 2016 issue.

Renal tubular handling of urate is realized by a network of uptake and efflux transporters, including members of drug transporter families such as solute carrier proteins and ATP-binding cassette transporters. Solute carrier family 2, member 9 (SLC2A9), is one key factor of this so called "urate transportosome." The aim of the present study was to understand the transcriptional regulation of SLC2A9 and to test whether identified factors might contribute to a coordinated transcriptional regulation of the transporters involved in urate handling. In silico analysis and cell-based reporter gene assays identified a hepatocyte nuclear factor (HNF)4α-binding site in the promoter of SLC2A9 isoform 1, whose activity was enhanced by transient HNF4α overexpression, whereas mutation of the binding site diminished activation. HNF4α over-expression induced endogenous SLC2A9 expression in vitro. The in vivo role of HNF4α in the modulation of renal SLC2A9 gene expression was supported by findings of quantitative real-time RT-PCR analyses and chromatin immunoprecipitation assays. Indeed, mRNA expression of SLC2A9 and HNF4α in human kidney samples was significantly correlated. We also showed that in renal clear cell carcinoma, downregulation of HNF4α mRNA and protein expression was associated with a significant decline in expression of the transporter. Taken together, our data suggest that nuclear receptor family member HNF4α contributes to the transcriptional regulation of SLC2A9 isoform 1. Since HNF4α has previously been assumed to be a modulator of several urate transporters, our findings support the notion that there could be a transcriptional network providing synchronized regulation of the functional network of the urate transportosome. [ABSTRACT FROM AUTHOR]

Multidrug resistance protein 4 (MRP4/ABCC4) has been established as an independent regulator of cyclic AMP (cAMP) levels particularly in vascular smooth muscle cells and in hematopoietic cells. Here, we assessed whether cAMP in turn regulates MRP4. A significant upregulation of MRP4 mRNA and protein by long-term treatment with cAMP-enhancing agents was observed in HeLa cells, smooth muscle cells, and megakaryoblastic leukemia M07e cells. This upregulation was not affected by inhibition of protein kinase A, but could be reverted by inhibitors and siRNA of an alternative cAMP-signaling route involving exchange proteins activated by cyclic AMP (EPAC) and mitogen-activated protein kinases. A selective EPAC activator could equally induce MRP4. The transcriptional regulation was confirmed in a luciferase reporter gene assay using a vector containing a 1494-bp fragment of the promoter region of the MRP4/ABCC4 gene. Our results suggest that enhanced cAMP levels upregulate MRP4 expression, which can result in increased cAMP efflux. [ABSTRACT FROM AUTHOR]

Background: L-Arginine and its dimethylated derivatives asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) have been associated with cardiovascular (CV) and all-cause mortality in populations at risk. The present study aimed to investigate the prognostic value of L-arginine and its derivatives in the general population. Methods and Results: We evaluated 3,952 individuals (1,936 men and 2,016 women) aged 20–81 (median (IQR) 51 (37; 64) years) from the population-based Study of Health in Pomerania (SHIP). Associations of continuous [per standard deviation (SD) increase] and categorized (age- and sex-specific tertiles) serum L-arginine, ADMA, and SDMA concentrations with all-cause and cause-specific mortality were analysed. During a median (IQR) follow-up period of 10.1 (9.3; 10.8) years (38,476 person-years), 426 deaths (10.8%) were observed, including 139 CV deaths (3.5%), and 150 cancer deaths (3.8%). After multivariable adjustment, we revealed a positive association of SDMA with all-cause [hazard ratio (HR) per SD increase: 1.16, 95% confidence interval (CI): 1.07–1.25] and CV mortality [HR: 1.19, 95% CI: 1.05–1.35]. In contrast, we did not observe any association of SDMA with cancer mortality. Neither L-arginine nor ADMA were associated with all-cause or CV mortality. Conclusion: SDMA, but not ADMA, is an independent predictor of all-cause and CV mortality in a large population-based cohort of European ancestry. [ABSTRACT FROM AUTHOR]

Organic anion transporting polypeptide OATP1B3 is a membrane-bound drug transporter that facilitates cellular entry of a variety of substrates. Most of the previous studies focused on its hepatic expression and function in hepatic drug elimination. In this study, we report expression of OATP1B3 in human pancreatic tissue, with the abundance of the transporter localized in the islets of Langerhans. Transport studies using OATP1B3-overexpressing MDCKII cells revealed significant inhibition of the cellular uptake of the known substrate cholecystokinin-8 in the presence of the insulinotropic antidiabetes compounds tolbutamide, glibenclamide, glimepiride, and nateglinide and identified glibenclamide as a novel substrate of OATP1B3. Sulfonylurea derivatives exert their insulinotropic effect by binding to the SUR1 subunit of the KATP channels inducing insulin secretion in [beta]-cells. Here, we show that transient overexpression of human OATP1B3 in a murine [beta]-cell line (MIN6)-which exhibits glucose and glibenclamide-sensitive insulin secretion-significantly enhances the insulinotropic effect of glibenclamide without affecting glucose-stimulated insulin secretion. Taken together, our data provide evidence that the drug transporter OATP1B3 functions as a determinant of the insulinotropic effect of glibenclamide on the tissue level. Changes in transport activity based on drug-drug interactions or genetic variability may therefore influence glibenclamide efficacy. [ABSTRACT FROM AUTHOR]

Organic anion transporting polypeptide OATP1B3 is a membrane-bound drug transporter that facilitates cellular entry of a variety of substrates. Most of the previous studies focused on its hepatic expression and function in hepatic drug elimination. In this study, we report expression of OATP1B3 in human pancreatic tissue, with the abundance of the transporter localized in the islets of Langerhans. Transport studies using OATP1B3-overexpressing MDCKII cells revealed significant inhibition of the cellular uptake of the known substrate cholecystokinin-8 in the presence of the insulinotropic antidiabetes compounds tolbutamide, glibenclamide, glimepiride, and nateglinide and identified glibenclamide as a novel substrate of OATP1B3. Sulfonylurea derivatives exert their insulinotropic effect by binding to the SUR1 subunit of the KATP channels inducing insulin secretion in β-cells. Here, we show that transient overexpression of human OATP1B3 in a murine b-cell line (MIN6)--which exhibits glucose and glibenclamide-sensitive insulin secretion--significantly enhances the insulinotropic effect of glibenclamide without affecting glucose-stimulated insulin secretion. Taken together, our data provide evidence that the drug transporter OATP1B3 functions as a determinant of the insulinotropic effect of glibenclamide on the tissue level. Changes in transport activity based on drug-drug interactions or genetic variability may therefore influence glibenclamide efficacy. [ABSTRACT FROM AUTHOR]

Objectives: This study sought to evaluate the role of protease-activated receptor-2 (PAR2) in coxsackievirus B3 (CVB3)-induced myocarditis.Background: An infection with CVB3 leads to myocarditis. PAR2 modulates the innate immune response. Toll-like receptor-3 (TLR3) is crucial for the innate immune response by inducing the expression of the antiviral cytokine interferon-beta (IFNβ).Methods: To induce myocarditis, wild-type (wt) and PAR2 knockout (ko) mice were infected with 10(5) plaque-forming units CVB3. Mice underwent hemodynamic measurements with a 1.2-F microconductance catheter. Wt and PAR2ko hearts and cardiac cells were analyzed for viral replication and immune response with plaque assay, quantitative polymerase chain reaction, Western blot, and immunohistochemistry.Results: Compared with wt mice, PAR2ko mice and cardiomyocytes exhibited a reduced viral load and developed no myocarditis after infection with CVB3. Hearts and cardiac fibroblasts from PAR2ko mice expressed higher basal levels of IFNβ than wt mice did. Treatment with CVB3 and polyinosinic:polycytidylic acid led to higher IFNβ expression in PAR2ko than in wt fibroblasts and reduced virus replication in PAR2ko fibroblasts was abrogated by neutralizing IFNβ antibody. Overexpression of PAR2 reduced the basal IFNβ expression. Moreover, a direct interaction between PAR2 and Toll-like receptor 3 was observed. PAR2 expression in endomyocardial biopsies of patients with nonischemic cardiomyopathy was positively correlated with myocardial inflammation and negatively with IFNβ expression and left ventricular ejection fraction.Conclusions: PAR2 negatively regulates the innate immune response to CVB3 infection and contributes to myocardial dysfunction. The antagonism of PAR2 is of therapeutic interest to strengthen the antiviral response after an infection with a cardiotropic virus. [ABSTRACT FROM AUTHOR]

Objectives: This study sought to evaluate the role of protease-activated receptor-2 (PAR2) in coxsackievirus B3 (CVB3)–induced myocarditis. Background: An infection with CVB3 leads to myocarditis. PAR2 modulates the innate immune response. Toll-like receptor-3 (TLR3) is crucial for the innate immune response by inducing the expression of the antiviral cytokine interferon-beta (IFNβ). Methods: To induce myocarditis, wild-type (wt) and PAR2 knockout (ko) mice were infected with 105 plaque-forming units CVB3. Mice underwent hemodynamic measurements with a 1.2-F microconductance catheter. Wt and PAR2ko hearts and cardiac cells were analyzed for viral replication and immune response with plaque assay, quantitative polymerase chain reaction, Western blot, and immunohistochemistry. Results: Compared with wt mice, PAR2ko mice and cardiomyocytes exhibited a reduced viral load and developed no myocarditis after infection with CVB3. Hearts and cardiac fibroblasts from PAR2ko mice expressed higher basal levels of IFNβ than wt mice did. Treatment with CVB3 and polyinosinic:polycytidylic acid led to higher IFNβ expression in PAR2ko than in wt fibroblasts and reduced virus replication in PAR2ko fibroblasts was abrogated by neutralizing IFNβ antibody. Overexpression of PAR2 reduced the basal IFNβ expression. Moreover, a direct interaction between PAR2 and Toll-like receptor 3 was observed. PAR2 expression in endomyocardial biopsies of patients with nonischemic cardiomyopathy was positively correlated with myocardial inflammation and negatively with IFNβ expression and left ventricular ejection fraction. Conclusions: PAR2 negatively regulates the innate immune response to CVB3 infection and contributes to myocardial dysfunction. The antagonism of PAR2 is of therapeutic interest to strengthen the antiviral response after an infection with a cardiotropic virus. [ABSTRACT FROM AUTHOR]

Aim To identify loci associated with chronic periodontitis through a genome-wide association study ( GWAS). Materials and Methods A GWAS was performed in 4032 individuals of two independent cross-sectional studies of West Pomerania ( SHIP n = 3365 and SHIP- TREND n = 667) with different periodontal case definitions. Samples were genotyped with the Affymetrix Genome-Wide Human SNP Array 6.0 or the Illumina Human Omni 2.5 array. Imputation of the HapMap as well as the 1000 Genome-based autosomal and X-chromosomal genotypes and short insertions and deletions ( INDELs) was performed in both cohorts. Finally, more than 17 million SNPs and short INDELs were analysed. Results No genome-wide significant associations were found for any periodontitis case definition, regardless of whether individuals aged >60 years where excluded or not. Despite no single SNP association reached genome-wide significance, the proportion of variance explained by additive effects of all common SNPs was around 23% for mean proximal attachment loss. Excluding subjects aged >60 years increased the explained variance to 34%. Conclusions No single SNPs were found to be genome-wide significantly associated with chronic periodontitis in this study. [ABSTRACT FROM AUTHOR]

The primary goals of personalized medicine are to optimize diagnostic and treatment strategies by tailoring them to the specific characteristics of an individual patient. In this Review, we summarize basic concepts and methods of personalizing cardiovascular medicine. In-depth characterization of study participants and patients in general practice using standardized methods is a pivotal component of study design in personalized medicine. Standardization and quality assurance of clinical data are similarly important, but in daily practice imprecise definitions of clinical variables can reduce power and introduce bias, which limits the validity of the data obtained as well as their potential clinical applicability. Changes in statistical methods with personalized medicine include a shift from dichotomous outcomes towards continuously measured variables, predictive modelling, and individualized medical decisions, subgroup analyses, and data-mining strategies. A variety of approaches to personalized medicine exist in cardiovascular research and clinical practice that might have the potential to individualize diagnostic and therapeutic procedures. For some of the emerging methods, such as data mining, the most-efficient way to use these tools is not yet fully understood. In addition, the predictive models--although promising--are far from mature, and are likely to be greatly improved by using available large-scale data sets. [ABSTRACT FROM AUTHOR]

Background Resistance of the highly aggressive glioblastoma multiforme (GBM) to drug therapy is a major clinical problem resulting in a poor patient's prognosis. Beside promoter methylation of the O6-methylguanine-DNA-methyltransferase (MGMT) gene the efflux transporters ABCB1 and ABCG2 have been suggested as pivotal factors contributing to drug resistance, but the methylation of ABCB1 and ABCG2 has not been assessed before in GBM. Methods Therefore, we evaluated the proportion and prognostic significance of promoter methylation of MGMT, ABCB1 and ABCG2 in 64 GBM patient samples using pyrosequencing technology. Further, the single nucleotide polymorphisms MGMT C-56 T (rs16906252), ABCB1 C3435T (rs1045642) and ABCG2 C421A (rs2231142) were determined using the restriction fragment length polymorphism method (RFLP). To study a correlation between promoter methylation and gene expression, we analyzed MGMT, ABCB1 and ABCG2 expression in 20 glioblastoma and 7 non-neoplastic brain samples. Results Despite a significantly increased MGMT and ABCB1 promoter methylation in GBM tissue, multivariate regression analysis revealed no significant association between overall survival of glioblastoma patients and MGMT or ABCB1 promoter methylation. However, a significant negative correlation between promoter methylation and expression could be identified for MGMT but not for ABCB1 and ABCG2. Furthermore, MGMT promoter methylation was significantly associated with the genotypes of the MGMT C-56 T polymorphism showing a higher methylation level in the T allele bearing GBM. Conclusions In summary, the data of this study confirm the previous published relation of MGMT promoter methylation and gene expression, but argue for no pivotal role of MGMT, ABCB1 and ABCG2 promoter methylation in GBM patients' survival. [ABSTRACT FROM AUTHOR]

Abstract: Previously, the steroid hormone progesterone has been demonstrated to stimulate OATP2B1-mediated transport of estrone-3-sulphate (E1S), dehydroepiandrosterone sulphate (DHEAS) and pregnenolone sulphate (PS), which may influence the uptake of precursor molecules for steroid hormone synthesis. However, it is unclear whether OATP2B1 drug substrates like atorvastatin or glibenclamide are also affected by this phenomenon. In addition, it has not been studied so far if this stimulatory effect is specific for OATP2B1. To address these questions, we examined the influence of progesterone on OATP2B1-mediated atorvastatin and glibenclamide uptake and studied the impact of steroid hormones on the transport activity of OATP1A2, OATP1B1 and OATP1B3. Comparison of the substrate spectrum of the investigated OATPs revealed that DHEAS and atorvastatin are substrates of all transporters, while E1S was only significantly transported by OATP1A2, OATP2B1 and OATP1B1. Glibenclamide uptake was limited to OATP1A2, OATP1B1 and OATP2’B1. Subsequent interaction studies indicated that progesterone only increases OATP2B1-mediated E1S and DHEAS transport, whereas uptake of BSP, atorvastatin and glibenclamide was either inhibited or not affected. Moreover, the steroid hormone effect was specific for OATP2B1; neither OATP1B1, OATP1B3 nor OATP1A2 function was stimulated in the presence of progesterone. Similar to progesterone, the glucocorticoide dexamethasone stimulated OATP2B1-mediated transport of E1S and DHEAS (EC50 for E1S: 10.2±5.6μM and 17.9±15.4μM for DHEAS). In conclusion, our data demonstrate that among the tested compounds the stimulatory effect of progesterone is specific for OATP2B1 and restricted to sulphated steroids like E1S and DHEAS while the OATP-mediated drug transport is not enhanced. [Copyright &y& Elsevier]

The clinical course of patients with dilated cardiomyopathy (DCM) varies from cardiac recovery to end stage heart failure. The etiology of this variability is largely unknown. In this study, we investigated the impact of coding polymorphisms of the innate immune protein Toll-like receptor 4 (TLR4) on left ventricular performance in patients with DCM. Two variants of TLR4 (rs4986790, TLR4 c.1187A→G, p.299D→G and rs4986791,TLR4 c.1487C→T, p.T399I) were investigated in 158 patients with DCM. Other reasons for heart failure were excluded by coronary angiography, myocardial biopsy, and echocardiography. Risk factors, age, gender, or treatment did not differ among the groups. At the follow-up evaluation (median 4.0 -5.4 months), patients carrying the TLR4 wild type gene displayed cardiac recovery under intense medical heart failure therapy indexed by reduced left ventricular dilation, improved left ventricular ejection fraction, and reduced NT-probrain natriuretic peptide blood level when compared with the initial evaluation. In contrast, patients carrying both the rs4986790 and the rs4986791 variant showed significantly reduced improvement of left ventricular ejection fraction (p = 0.006) and left ventricular dilation (p = 0.015) at the follow-up evaluation when compared with carriers of the wild type gene under the same treatment conditions. In addition, NT-probrain natriuretic peptide level in carriers of both TLR4 variants did not change significantly at the follow up when compared with the first evaluation. Among patients with DCM, the presence of the TLR4 variants rs4986790 and rs4986791 predicts impaired cardiac recovery independently of medical treatment or cardiac risk factors. [ABSTRACT FROM AUTHOR]

Background: Impaired vascular compliance is associated with cardiovascular mortality. The effects of heart rate on vascular compliance are unclear. Therefore, we characterized effects of heart rate reduction (HRR) by I(f) current inhibition on aortic compliance and underlying molecular mechanisms in apolipoprotein E-deficient (ApoE-/-) mice. Methods: ApoE-/- mice fed a high-cholesterol diet and wild-type (WT) mice were treated with ivabradine (20 mg/kg/d) or vehicle for 6 weeks. Compliance of the ascending aorta was evaluated by MRI. Results: Ivabradine reduced heart rate by 113 ± 31 bpm (∼19%) in WT mice and by 133 ± 6 bpm (∼23%) in ApoE-/- mice. Compared to WT controls, ApoE-/- mice exhibited reduced distensibility and circumferential strain. HRR by ivabradine increased distensibility and circumferential strain in ApoE-/- mice but did not affect both parameters in WT mice. Ivabradine reduced aortic protein and mRNA expression of the angiotensin II type 1 (AT1) receptor and reduced rac1-GTPase activity in ApoE-/- mice. Moreover, membrane translocation of p47phox was inhibited. In ApoE-/- mice, HRR induced anti-inflammatory effects by reduction of aortic mRNA expression of IL-6, TNF-alpha and TGF-beta. Conclusion: HRR by ivabradine improves vascular compliance in ApoE-/- mice. Contributing mechanisms include downregulation of the AT1 receptor, attenuation of oxidative stress and modulation of inflammatory cytokine expression. Copyright © 2012 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: Objective: Lower levels of anabolic hormones in older age are well documented. Several studies suggested that low insulin-like growth factor I (IGF-I) or testosterone levels were related to increased mortality. The aim of the present study was to investigate the combined influence of low IGF-I and low testosterone on all-cause mortality in men. Methods and results: From two German prospective cohort studies, the DETECT study and SHIP, 3942 men were available for analyses. During 21,838 person-years of follow-up, 8.4% (n =330) of men died. Cox model analyses with age as timescale and adjusted for potential confounders revealed that men with levels below the 10th percentile of at least one hormone [hazard ratio (HR) 1.38 (95% confidence-interval (CI) 1.06–1.78), p =0.02] and two hormones [HR 2.88 (95% CI 1.32–6.29), p <0.01] showed a higher risk of all-cause mortality compared to men with non-low hormones. The associations became non-significant by using the 20th percentile as cut-off showing that the specificity increased with lower cut-offs for decreased hormone levels. The inclusion of both IGF-I and total testosterone in a mortality prediction model with common risk factors resulted in a significant integrated discrimination improvement of 0.5% (95% CI 0.3–0.7%, p =0.03). Conclusions: Our results prove that multiple anabolic deficiencies have a higher impact on mortality than a single anabolic deficiency and suggest that assessment of more than one anabolic hormone as a biomarker improve the prediction of all-cause mortality. [Copyright &y& Elsevier]

The administration of drugs using biodegradable polymer nanoparticles as carriers has generated immense interest due to their excellent biocompatibility and the prolonged drug release. The scope of this work was to determine the applicability of sirolimus-loaded biodegradable poly(D,L-lactide) (PDLLA) nanoparticles as drug carriers to prevent restenotic processes after stent implantation. The average 250 nm sized 20%(w/w) sirolimus-loaded nanoparticles were extensively characterized with regard to in vitro degradation, biocompatibility and in vitro drug release. The particles show biphasic release kinetics consisting of a short burst release of 50%(w/w) sirolimus payload, followed by a longer, slower release phase, which are desirable for the application as a drug delivery carrier. All presented results exhibit the potential of sirolimus-loaded PDLLA nanoparticles as promising local and sustained drug delivery systems administered intraluminally to reduce in-stent restenosis after stent implantation. [ABSTRACT FROM AUTHOR]

Abstract: The recently introduced vessel-simulating flow-through cell offers new possibilities to examine the release from drug-eluting stents in vitro. In comparison with standard dissolution methods, the additional compartment allows for the examination of distribution processes and creates dissolution conditions which simulate the physiological situation at the site of implantation. It was shown previously that these conditions have a distinct influence on the release rate from the stent coating. In this work, different preparation techniques were developed to examine the spatial distribution within the compartment simulating the vessel wall. These methods allowed for the examination of diffusion depth and the distribution resulting in the innermost layer of the compartment simulating the vessel wall. Furthermore, the in vitro release and distribution examined experimentally were modelled mathematically using finite element (FE) methods to gain further insight into the release and distribution behaviour. The FE modelling employing the experimentally determined diffusion coefficients yielded a good general description of the experimental data. The results of the modelling also provided important indications that inhomogeneous coating layer thicknesses around the strut may result from the coating process which influence release and distribution behaviour. Taken together, the vessel-simulating flow-through cell in combination with FE modelling represents a unique method to analyse drug release and distribution from drug-eluting stents in vitro with particular opportunities regarding the examination of spatial distributions within the vessel-simulating compartment. [Copyright &y& Elsevier]

Abstract: The aim of the study is to characterize the signal transduction leading to interstitial fibrosis in the pathogenesis of atrial fibrillation (AF) and atrial remodeling. Samples of the left atrial appendage (LA) from patients with AF showed higher collagen content (73±5 vs. 38±2μg/mg protein) and 2.5-fold increased collagen crosslinking compared to patients with sinus rhythm (SR). Affymetrix-assays, RT-PCR and western Blot analysis revealed that LA of AF patients are characterized by increased lysyl oxidase (LOX) mRNA (218±42%) and protein (253±11%) expression. This was associated with increased expression of connective tissue growth factor (CTGF), fibronectin and Rac1 activity compared to SR. In neonatal cardiac fibroblasts, the Rac1 specific small molecule inhibitor NSC23766 prevented angiotensin II (AngII) induced upregulation of LOX (214±16%) expression. Inhibition of CTGF by siRNA transfections completely inhibited AngII induced LOX expression. The LOX specific small molecule inhibitor BAPN prevented AngII and CTGF induced fibronectin expression. Left atria of transgenic mice with cardiac overexpression of Rac1 (RacET) that develop AF at high age exhibited upregulation of CTGF as well as LOX (187±7%) and fibronectin (627±146%) expression. Atria of RacET showed increased collagen content (28±2μg/mg protein) and crosslinking (10±0.7) compared to wildtypes (20±0.4μg/mg protein; 5±0.9). Left atrial myocardium of patients with atrial fibrillation is characterized by increased lysyl oxidase and fibronectin expression as well as collagen cross-linking. In cardiac fibroblasts, Rac1 GTPase mediates upregulation of fibronectin via LOX and CTGF. Inhibition of this signaling pathway may therefore represent a target for the prevention of fibrotic atrial remodeling. [Copyright &y& Elsevier]

Background.- An association between the 677C>T polymorphism (rs1801133) in the methylenetetrahydrofolate reductase gene (MTHFR) and cluster headache is plausible, but has not been investigated. Objective.- To investigate this association among Caucasians. Methods.- Case-control study among 147 cluster headache patients and 599 population-based age- and gender-matched controls. Cluster headache was diagnosed according to the criteria of the International Headache Society. Genotypes of the MTHFR 677C>T polymorphism were detected by restriction fragment length polymorphism analysis. We used logistic regression analysis to investigate the association between cluster headache and genotypes with additive, dominant, and recessive models. We considered a Bonferroni-corrected P value <.004 as significant. Results.- Mean age at study entry among patients was 44.9 years (SD 11.4), of whom 76.2% were men. The genotype distribution among controls and patients was in Hardy-Weinberg equilibrium. The genotype and allele distribution did not differ between patients with any cluster headache and controls. We also did not find an association when assuming additive, dominant or recessive genetic models. When we looked at subgroups, the effect estimates suggested an increased risk for chronic cluster headache (dominant model: odds ratio = 2.82; 99.6% confidence interval = 0.72-11.07; P = .03). Conclusions.- Data from our case-control study do not indicate an association between genotypes of the MTHFR 677C>T polymorphism and cluster headache overall. Subgroup analyses suggested that carriers of the MTHFR 677T allele may have an increased risk for chronic cluster headache. This may be regarded as hypothesis-generating and should be further investigated in independent studies. [ABSTRACT FROM AUTHOR]

( Headache 2011;51:201-207) An association between the 677C>T polymorphism (rs1801133) in the methylenetetrahydrofolate reductase gene ( MTHFR) and cluster headache is plausible, but has not been investigated. To investigate this association among Caucasians. Case-control study among 147 cluster headache patients and 599 population-based age- and gender-matched controls. Cluster headache was diagnosed according to the criteria of the International Headache Society. Genotypes of the MTHFR 677C>T polymorphism were detected by restriction fragment length polymorphism analysis. We used logistic regression analysis to investigate the association between cluster headache and genotypes with additive, dominant, and recessive models. We considered a Bonferroni-corrected P value <.004 as significant. Mean age at study entry among patients was 44.9 years (SD 11.4), of whom 76.2% were men. The genotype distribution among controls and patients was in Hardy-Weinberg equilibrium. The genotype and allele distribution did not differ between patients with any cluster headache and controls. We also did not find an association when assuming additive, dominant or recessive genetic models. When we looked at subgroups, the effect estimates suggested an increased risk for chronic cluster headache (dominant model: odds ratio = 2.82; 99.6% confidence interval = 0.72-11.07; P = .03). Data from our case-control study do not indicate an association between genotypes of the MTHFR 677C>T polymorphism and cluster headache overall. Subgroup analyses suggested that carriers of the MTHFR 677T allele may have an increased risk for chronic cluster headache. This may be regarded as hypothesis-generating and should be further investigated in independent studies. [ABSTRACT FROM AUTHOR]

Neurovascular dysfunction is an important component of Alzheimer's disease, leading to reduced clearance across the blood-brain barrier and accumulation of neurotoxic β-amyloid (Aβ) peptides in the brain. It has been shown that the ABC transport protein P-glycoprotein (P-gp, ABCB1) is involved in the export of Aβ from the brain into the blood. To determine whether Aβ influences the expression of key Aβ transporters, we studied the effects of 1-day subcutaneous Aβ1-40 and Aβ1-42 administration via Alzet miniosmotic pumps on P-gp, BCRP, LRP1, and RAGE expression in the brain of 90-day-old male FVB mice. Our results demonstrate significantly reduced P-gp, LRP1, and RAGE mRNA expression in mice treated with Aβ1-42 compared to controls, while BCRP expression was not affected. The expression of the four proteins was unchanged in mice treated with Aβ1-40 or reverse-sequence peptides. These findings indicate that, in addition to the age-related decrease of P-gp expression, Aβ1-42 itself downregulates the expression of P-gp and other Aβ-transporters, which could exacerbate the intracerebral accumulation of Aβ and thereby accelerate neurodegeneration in Alzheimer's disease and cerebral β-amyloid angiopathy. [ABSTRACT FROM AUTHOR]

We have identified the ATP-binding cassette (ABC) transporter ABCC4 as an active constituent of mediator-storing granules in human platelets. In addition to multidrug resistance protein 4, other ABC-type transport proteins may contribute to platelet secretory function as well as determine intended or adverse effects of drugs. Here, we provide a comprehensive expression profiling of ABC transporters in human platelets based on a novel screening approach by combining the TaqMan low-density array RNA screening platform with a recently developed liquid chromatography/mass spectrometry (MS)/MS method for the simultaneous detection of membrane proteins. Transcripts of 25 ABC transporters were detected and showed differential expression compared with megakaryocytic progenitor cells. On the protein level ABCA7, ABCB4, ABCC1, ABCC3 and ABCC4 were identified by liquid chromatography/MS/MS and localized by immunofluorescence microscopy. Their functions may be related to glutathione and lipid homeostasis, secretion of lipid mediators, cell protection as well as drug transport. [ABSTRACT FROM AUTHOR]

Members of the organic anion transporting polypeptide (OATP) family are involved in various pharmacological, pathophysiological, and physiological processes, such as hepatic drug uptake, progress of cancer, or transport of hormones. Although variability in expression and function of OATPs has been investigated in detail, data concerning regulation are rather limited. Here, we report a novel mechanism for rapid regulation of OATP2B1 mediated by protein kinase C (PKC) resulting in significant changes of transport activity. PKC activation by the phorbol ester (phorbol 12-myristate 13-acetate, PMA) resulted in increased phosphorylation of OATP2B1 as well as reduced OATP2B1 transport activity with a decrease in Vmax of E1S uptake (288 ± 21 (control) versus 165 ± 16 pmol/min/mg of protein (PMA)). This effect was sensitive to the PKC inhibitor bisindolylmaleimide I (BIM-1). Confocal microscopy, fluorescence-based internalization assay, and live-cell imaging using green fluorescent protein-tagged OATP2B1 revealed that transport inhibition was due to internalization of the transporter. Furthermore, colocalization with LAMP-2 and chloroquine-sensitive degradation of OATP2B1 suggest that the internalized protein is targeted to a lysosomal degradation pathway. With regard to the underlying mechanism inhibition of caveolin/lipid raft-mediated endocytosis failed to prevent OATP2B1 internalization, whereas inhibition of clathrin-mediated processes blocked OATP2B1 sequestration. However, small interfering RNA-mediated clathrin knock-down affected general trafficking of OATP2B1 and resulted in intracellular accumulation in the absence of PMA. In conclusion, our data demonstrate that OATP2B1 function is regulated by PKCmediated, clathrin-dependent internalization and followed by lysosomal degradation. Furthermore, internalization could be shown in an ex vivo placenta perfusion. Our findings represent a new, rapid mechanism in regulation of human OATPs. [ABSTRACT FROM AUTHOR]

Objectives: We studied the signal transduction of atrial structural remodeling that contributes to the pathogenesis of atrial fibrillation (AF). Background: Fibrosis is a hallmark of arrhythmogenic structural remodeling, but the underlying molecular mechanisms are incompletely understood. Methods: We performed transcriptional profiling of left atrial myocardium from patients with AF and sinus rhythm and applied cultured primary cardiac cells and transgenic mice with overexpression of constitutively active V12Rac1 (RacET) in which AF develops at old age to characterize mediators of the signal transduction of atrial remodeling. Results: Left atrial myocardium from patients with AF showed a marked up-regulation of connective tissue growth factor (CTGF) expression compared with sinus rhythm patients. This was associated with increased fibrosis, nicotinamide adenine dinucleotide phosphate oxidase, Rac1 and RhoA activity, up-regulation of N-cadherin and connexin 43 (Cx43) expression, and increased angiotensin II tissue concentration. In neonatal rat cardiomyocytes and fibroblasts, a specific small molecule inhibitor of Rac1 or simvastatin completely prevented the angiotensin II–induced up-regulation of CTGF, Cx43, and N-cadherin expression. Transfection with small-inhibiting CTGF ribonucleic acid blocked Cx43 and N-cadherin expression. RacET mice showed up-regulation of CTGF, Cx43, and N-cadherin protein expression. Inhibition of Rac1 by oral statin treatment prevented these effects, identifying Rac1 as a key regulator of CTGF in vivo. Conclusions: The data identify CTGF as an important mediator of atrial structural remodeling during AF. Angiotensin II activates CTGF via activation of Rac1 and nicotinamide adenine dinucleotide phosphate oxidase, leading to up-regulation of Cx43, N-cadherin, and interstitial fibrosis and therefore contributing to the signal transduction of atrial structural remodeling. [Copyright &y& Elsevier]

We aimed to evaluate the association of non-response to antiepileptic pharmacotherapy with the frequency of variant alleles in the drug transporter genes ABCB1 and ABCC2 or in the CYP2C locus in young patients with epilepsy and an independent cohort of adults with drug-refractory epilepsy.A total of 221 pediatric or adolescent Caucasian patients with epilepsy (105 females; age: 14.5±6.54 years) were genotyped for nine putatively functionally relevant ABCB1, ABCC2, CYP2C8, CYP2C9, and CYP2C19 polymorphisms. In addition, 70 adult patients (35 females, age: 41.9±11.5 years) with drug-refractory epilepsy who had earlier undergone neurosurgical therapy were genotyped and partly (n = 22) investigated for hippocampal ABCB1 and ABCC2 mRNA expression. Finally, 242 healthy volunteers (167 females, age: 27.0±6.77 years) from the same region were included as controls.The young cohort consisted of 103 (46.6%) responders and 118 (53.4%) non-responders to the first-line anticonvulsant. Carriers of the putatively low-expression ABCC2 -24T variant were significantly overrepresented among non-responders [odds ratio (OR) 2.15 (1.16-3.99); P = 0.016)]. This overrepresentation was confirmed by comparing young responders with adult drug-refractory patients [OR 3.36 (1.71-6.59); P<0.001]. Conversely, ABCB1 genotype distribution did not significantly differ between young responders and non-responders or adult drug-refractory patients. Excluding patients with febrile convulsions, heterozygous CYP2C8∗4 [OR 0.35 (0.13-0.95); P = 0.038] and CYP2C9∗3 [OR 0.34 (0.14-0.81); P = 0.015] variant allele carriers were underrepresented among non-responders. ABCC2 -24C>T genotype did not affect hippocampal ABCC2 expression, but was associated with increased ABCB1 expression (P = 0.034).These data suggest a higher risk of antiepileptic drug failure in ABCC2 -24T allele carriers possibly because of compensatory upregulation of ABCB1. [ABSTRACT FROM AUTHOR]

We tested the effect of kidney-specific multidrug resistance-related protein (MRP2, ABCC2) deficiency on renal organic solute disposition as well as on renal protein and gene expression. Furthermore, we investigated whether a particular kidney donor ABCC2 genotype is associated with delayed graft function in patients.A new MRP2-deficient rat strain was established. Renal cross-transplantations were performed between congenic MRP2-deficient and wild-type rats. Renal disposition of MRP2 substrates was investigated in native and transplanted rats. Proteomic analyses and transcriptional profiling were performed in rat kidney graft cortices. Ninety-eight human kidney donor-recipient pairs were genotyped for five ABCC2 polymorphisms. The relationship between delayed graft function and ABCC2 genetic variants in donors and recipients was analyzed by backward stepwise logistic regression.In rats, the absence of renal MRP2 reduced renal bilirubin glucuronide excretion at pathologic plasma concentrations, modified renal p-aminohippurate excretion and did not affect renal morphine-6-glucuronide excretion. Renal MRP2 deficiency led to renal cortical protein or mRNA upregulation of glutathione transferase isoenzymes, glutaredoxin 2, and heme oxygenase-1. In patients, a particular donor ABCC2 genotype was associated with an increased incidence of delayed graft function.Kidney graft-specific MRP2 deficiency has mild effects on the renal excretion of some organic solutes under experimental conditions and induces a protein and gene expression pattern indicative of activated antioxidant defense mechanisms. This suggests that MRP2 is a determinant of the redox status in tubular epithelial cells and thus of the susceptibility to renal damage under conditions of treatment with multiple drugs and increased oxygen radical formation. [ABSTRACT FROM AUTHOR]

Abstract: The interaction of thrombocyte vesicles with the surface of metal electrodes, i.e., mercury, gold and gold electrodes modified with self assembled monolayers (SAM), was studied with the help of chronoamperometry, atomic force microscopy, and quartz crystal microbalance measurements. The experimental results show that the interaction of the thrombocyte vesicles with the surface of the electrodes depends on the hydrophobicity of the latter: whereas on very hydrophobic surfaces (mercury and gold functionalized with SAM) the thrombocyte vesicles disintegrate and form a monolayer of lipids, on the less hydrophobic gold surface a bilayer is formed. The chronoamperometric measurements indicate the possibility of future applications to probe membrane properties of thrombocytes. [Copyright &y& Elsevier]

Abstract: A novel in vitro dissolution test for drug-eluting stents (DES) based on the compendial flow-through cell was developed. The model contains an additional compartment simulating the vessel wall enabling the examination of drug release and distribution. The compartment consists of alginate hydrogel containing a central aperture forming the lumen for stent placement and media flow. The method was tested utilizing stents coated with hydrophilic (fluorescein sodium) and hydrophobic (triamterene) fluorescent model substances as well as the cytostatic drug doxorubicin hydrochloride and compared to standard dissolution methods. The results show the suitability of the developed method to observe drug release and distribution. The determination of model substance content in the compartments media, hydrogel and stent yielded differing distribution patterns for the model compounds and enabled the observation of redistribution processes. The dissolution profiles differed from the results of compendial dissolution testing. Besides lower endpoints and slower rises of media concentrations due to distribution into the hydrogel, the release rates from the stent coatings were altered. These findings emphasize the necessity to adapt dissolution testing for DES to the unique conditions influencing delivery to the vessel wall to learn more about local distribution and to anticipate the in vivo performance of DES. [Copyright &y& Elsevier]

Genetic variability in hepatic uptake was recently shown to influence the disposition and cholesterol-lowering effects of statins. Ezetimibe, an inhibitor of the intestinal cholesterol uptake protein Niemann-Pick C 1 like 1, is another drug for which genetic polymorphisms of hepatic organic anion transporting polypeptides (OATPs) are expected to be of clinical relevance because ezetimibe undergoes intensive enterohepatic circulation for which hepatic uptake transporters may be rate-limiting determinants.Using OATP1B3-, OATP2B1-, and OATP1B1-transfected HEK cells, including the OATP1B1 variants OATP1B1∗1b and OATP1B1∗5, we measured the uptake of ezetimibe and its glucuronide and we analyzed the competition with the common OATP-substrate bromosulfophthalein. Disposition and sterol-lowering effects of 20-mg ezetimibe were measured in 35 healthy participants genotyped for OATP1B1, ABCB1, ABCC2, and UGT1A1.Ezetimibe glucuronide inhibited bromosulfophthalein uptake in all OATP-transfected cells (50% inhibitory concentration (IC50): 0.14-0.26 μmol/l) whereas ezetimibe was 30-100 times less potent. Only the glucuronide was accumulated significantly in cells expressing OATP1B1 and OATP2B1. Its uptake in cells expressing OATP1B1∗1b and ∗5 was reduced. In-vivo studies showed there was a gene-dose-dependent decrease in the area under the curve of ezetimibe in participants with the OATP1B1∗1b protein (∗1a/∗1a, N=12, 112±66 ng×h/ml vs. ∗1a/∗1b, N=8, 88±39 ng×h/ml vs. ∗1b/∗1b, N=5, 55±18 ng×h/ml; Jonkheere-Terpstra, P=0.041) and a tendency for increased glucuronide exposure (704±296 vs. 878±369 vs. 1059±363 ng×h/ml; P=0.092). Fecal ezetimibe excretion was significantly decreased whereas renal glucuronide excretion was increased in carriers of ∗1b/∗1b. Fecal excretion was also diminished in carriers of OATP1B1∗5 and ∗15. The sterol-lowering effect of ezetimibe was not influenced by OATP1B1 polymorphisms.Pharmacokinetics of ezetimibe is influenced by OATP1B1 polymorphisms in healthy participants after single dose administration. [ABSTRACT FROM AUTHOR]

Abstract: Background: Cardiac inflammation and generation of oxidative stress are known to contribute to doxorubicin (Dox)-induced cardiomyopathy. Toll-like receptors (TLRs) are a part of the innate immune system and are involved in cardiac stress reactions. Since TLR4 might play a relevant role in cardiac inflammatory signalling, we investigated whether or not TLR4 is involved in Dox-induced cardiotoxicity. Methods and results: Five days after a single injection of Dox (20 mg/kg; i.p.), left ventricular pressure–volume loops were measured in wild-type and TLR4-deficient mice (TLR4−/−) Dox-treated and control mice. Analyses of possible pathophysiological mechanisms were performed in left ventricular tissue and isolated myocytes, respectively. Dox injection resulted in an impairment of left ventricular function and neurohumoral activation, indexed by increased ET-1 expression. This was further associated with an increase in cardiac oxidative stress, inflammation and apoptosis, as indicated by an up-regulation of cardiac lipid peroxidation, TNF-α expression and enhanced content of TUNEL-positive cells. In contrast, TLR4−/−Dox mice showed improved left ventricular function with reduced oxidative and inflammatory stress response including reduced cardiac apoptosis. These results were found to be associated with an increase of GATA-4 expression. Conclusions: TLR4 deficiency improves left ventricular function and attenuates pathophysiological key mechanisms in Dox-induced cardiomyopathy. [Copyright &y& Elsevier]

Abstract: Functional analysis of intracellular structures requires isolation and purification of these cellular compartments. With regard to platelet function both delta and alpha granules are relevant target structures. However, the availability of sufficient purification protocols for these structures is rather limited and restricted to density gradient centrifugation. Because this method is time-consuming and the resulting products are often of limited purity, we designed a new purification method based on immunolabeling followed by magnetic sorting. We directly compared this new method with the conventional method of ultracentrifugation. We were able to get highly purified subcellular fractions of human platelets using several antibodies against specific markers for dense granules (LAMP2), alpha granules (P-selectin) and the plasma membrane (GPIIb/IIIa) in combination with antibody-coated magnetic beads. In the respective fractions the marker proteins used for isolation as well as further independent, structure specific markers (for example MRP4 for dense granules, von Willebrand factor (vWF) for alpha granules and protein disulfide isomerase, PDI and GPIbβ, for plasma membrane) could be detected by Western blotting. The method describes purification of membranal structures of human platelets such as the plasma membrane and both types of granules. Therefore, studies requiring highly purified material (e.g. identification of specific transporters and receptors) will benefit from these results. [Copyright &y& Elsevier]

Cardiac autoantibodies play a pathogenic role in dilated cardiomyopathy (DCM). Removal of antibodies by immunoadsorption (IA) induces hemodynamic improvement in DCM patients. The present study investigated the effects of IA on myocardial gene expression of the intermediate cytoskeletal filament desmin, which is upregulated in heart failure. RNA was isolated from five explanted non-failing hearts and five explanted failing hearts of DCM patients, and myocardial gene expression of desmin was estimated by real-time polymerase chain reaction (PCR). In a case-control study in six DCM patients (LVEF < 40%, NYHA II-III), IA and subsequent IgG substitution were performed at monthly intervals until month 3. Endomyocardial biopsies (EMBs) were obtained before and after IA (after 3–6 months). From six DCM patients without IA therapy (controls), EMBs were also obtained over a comparable time interval. Expression of the desmin gene was analyzed in these EMBs by real-time PCR. In failing explanted hearts, expression of desmin was significantly increased (0.88 ± 0.12 vs 0.45 ± 0.15 in non-failing hearts, P < 0.05). After IA, myocardial gene expression of desmin was significantly decreased (from 0.26 ± 0.05 [baseline] to 0.14 ± 0.04 [ P < 0.05] vs baseline and controls). Removal of antibodies by IA modulates myocardial gene expression of desmin in DCM patients. [ABSTRACT FROM AUTHOR]

Amyloid-β (Aβ) is the major component of the insoluble amyloid plaques that accumulate intracerebrally in patients with Alzheimer’s disease (AD). It has been suggested that MDR1-P-glycoprotein (ABCB1, P-gp) plays a substantial role in the elimination of Aβ from the brain. In the present study, MDR1-transfected LLC cells growing in a polarized cell layer were used to characterize the interaction of Aβ1-40/1-42 with P-gp. In this system, P-gp-mediated transport can be followed by the efflux of the fluorescent dye rhodamine-123, or of Aβ itself from the cells into the apical extracellular space. Aβ significantly decreased the apical efflux of rhodamine-123, and the transcellular transport of Aβ1-40 and Aβ1-42 into the apical chamber could be demonstrated using both ELISA and fluorescence (FITC)-labeled peptides. This transport was inhibited by a P-gp modulator. Furthermore, ATP-dependent, P-gp-mediated transport of the fluorescence-labeled peptides could be demonstrated in isolated, inside-out membrane vesicles. Our data support the concept that P-gp is important for the clearance of Aβ from brain, and thus may represent a target protein for the prevention and/or treatment of neurodegenerative disorders such as AD. [ABSTRACT FROM AUTHOR]

Adenosine triphosphate-binding cassette (ABC)-type transport proteins were initially described for their ability to reduce intracellular concentrations of anticancer compounds, thereby conferring drug resistance. In recent years, expression of this type of proteins has also been reported in numerous cell types under physiological conditions; here, these transporters are often reported to alter systemic and local drug disposition (e.g. in the brain or the gastrointestinal tract). In this context, peripheral blood cells have also been found to express several ABC-type transporters. While erythrocytes mainly express multidrug resistance protein (MRP) 1, MRP4 and MRP5, which are discussed with regard to their involvement in glutathione homeostasis (MRP1) and in the efflux of cyclic nucleotides (MRP4 and MRP5), leukocytes also express P-glycoprotein and breast cancer resistance protein. In the latter cell types, the main function of efflux transporters may be protection against toxins, as these cells demonstrate a very high turnover rate. In platelets, only two ABC transporters have been described so far. Besides MRP1, platelets express relatively high amounts of MRP4 not only in the plasma membrane but also in the membrane of dense granules, suggesting relevance for mediator storage. In addition to its physiological function, ABC transporter expression in these structures can be of pharmacological relevance since all systemic drugs reach their targets via circulation, thereby enabling interaction of the therapeutic agent with peripheral blood cells. Moreover, both intended effects and unwanted side effects occur in peripheral blood cells, and intracellular micropharmacokinetics can be affected by these transport proteins. The present review summarises the data available on expression of ABC transport proteins in peripheral blood cells. [ABSTRACT FROM AUTHOR]

Identification and quantification of proteins from human tissue by electrophoresis and subsequent mass spectrometry (MS) has been mainly restricted to high abundance, non-membrane-bound molecules. Pharmacologically relevant structures such as cytochrome P450 enzyme, drug transporters and receptors were not accessible by this technology. We developed a method to identify an integral membrane-bound protein (ABCB1) from human placenta using Fourier transform ion cyclotron (FTICR)-MS. Apical and basal membrane fractions were enriched from term human placenta using differential centrifugation. Following SDS-page these fractions were cleaved with trypsin, separated by nano-HPLC and subjected to FTICR-MS. We identified a total of 70 and 89 proteins in the apical and basal membranes, respectively. Among these proteins, and restricted to the apical membrane, was the transport protein ABCB1, with 10 peptides identified by MS, covering a total of 10% of the entire protein. The study describes a method suitable for direct monitoring of membrane-bound proteins from human tissue using FTICR-MS. [ABSTRACT FROM AUTHOR]

The abnormal conformation and assembly of proteins in the central nervous system is increasingly thought to be a critical pathogenic mechanism in neurodegenerative disorders such as Creutzfeldt–Jakob disease (CJD) and Alzheimer’s disease (AD). CJD is marked primarily by the buildup of misfolded prion protein (PrPSc) in brain, whereas the accrual of β-amyloid protein (Aβ) and tau protein are characteristic for AD. Prior studies have shown that the ATP-binding cassette transporter P-glycoprotein (P-gp) is a cellular efflux pump for Aβ, and that age-associated deficits in P-gp may be involved in the pathogenesis of Alzheimer’s disease. In the present study, we investigated the relationship between P-gp and idiopathic CJD, and found that CJD, like AD, is associated with a decrease in the expression of cerebrovascular P-gp. In some instances, Aβ and PrP deposits coexist in cases of CJD, suggesting the possibility of pathogenic interactions. Since there is, to date, no evidence that PrP itself is a substrate for P-gp, we hypothesize that the age-related deficits in P-gp could promote the accumulation of PrPSc either by promoting the buildup of Aβ (which could act as a seed for the aggregation of PrPSc), or by overloading the ubiquitin-proteasomal catabolic system, and thereby facilitating the accumulation of PrP. Alternatively, the loss of P-gp could be a non-specific response to neurodegenerative changes in the central nervous system. In either case, dysfunction of this critical toxin-elimination pathway in CJD and AD suggests that selectively increasing cerebrovascular P-gp function could open new therapeutic pathways for the prevention and/or treatment of a number of proteopathic disorders of the central nervous system. [ABSTRACT FROM AUTHOR]

The deleterious consequences of total β-glucuronidase deficiency, leading to symptomatic mucopolysaccharidosis type VII (MPS VII), have been firmly established. However, the question of whether sequence variations in β-glucuronidase of non-MPS VII patients affect expression of the enzyme, thereby explaining the wide inter-individual expression, has not been addressed in a systematic manner. In the present study, a population of 965 subjects were screened for enzyme activity and relevant fractions of the β-glucuronidase gene were sequenced in those individuals belonging to the highest or lowest decile of activity. The study showed a substantial inter-individual variability of β-glucuronidase in plasma (range 0.5-150.2 μmol/min) and confirmed the association of β-glucuronidase activity with gender (P<0.001), age (r=0.218; P<0.001) and body mass index (r=0.311; P<0.001). We were able to identify six β-glucuronidase single base substitutions (-1026A>G, -72G>T, -12G>A, +7728C>T, +14 209C>T and +14 604A>G) in 193 non-MPS VII patients at a rate of one single nucleotide polymorphism (SNP) per 520 bp sequenced. The GG genotype of +14 604A>G and the CT genotype of +14 209C>T were associated with higher β-glucuronidase activity (P<0.05). Subsequently, reporter gene assays were carried out to elucidate the effects of the SNPs -1026A>G, -72G>T and -12G>A, and the combined genotype -1026A>G and -12G>A observed in one of the subjects. Variant -12G>A reduced the promoter activity (75%; 95% confidence interval 70-84%, P<0.05). The present study demonstrates that three of the described SNPs influence the activity and/or expression of β-glucuronidase. Taken together, the data indicate a rather limited influence of genetic factors on inter-individual variability in β-glucuronidase activity. [ABSTRACT FROM AUTHOR]

Aim Gingival overgrowth is a common undesired side-effect in patients taking calcium channel blockers. Different reports have suggested that the drug-induced gingival hyperplasia may aggravate inflammatory periodontal disease. However, representative epidemiological data are lacking. We investigated the association between the intake of calcium antagonists and periodontitis in a population-based analysis including the most important risk factors of periodontitis. Methods In a cross-sectional epidemiological investigation involving 4290 subjects aged 20–80 years, we recorded periodontal risk factors and identified participants using calcium antagonists. Periodontal parameters, attachment loss, probing depth and number of teeth were assessed. In a subgroup analysis with matched pairs, 456 subjects using calcium antagonists and 456 without were compared for periodontal status. Results Subjects treated with calcium antagonistic drugs had significantly deeper gingival pockets than their drug-free counterparts. This was observed in the total population of 4290 and confirmed by logistic regression analyses ( P < 0.001) controlled for the known risk factors of periodontitis (age, sex, smoking, education). In the matched-pair analysis only the probing depth was increased: extent probing depth ≥ 4 mm median 23.5 vs. 17.0% ( P < 0.001); mean probing depth 3.0 ± 0.8 vs. 2.7 ± 0.9 mm ( P < 0.001). No differences were found in extent and severity of clinical attachment loss and in the number of teeth. The risk of gingival overgrowth was aggravated in smokers. Conclusion In the general population, treatment with calcium antagonists leads to gingival overgrowth without an aggravation of periodontal disease. Interaction with smoking indicates the multifactorial background of the undesired effect of calcium antagonists. [ABSTRACT FROM AUTHOR]

Like other members of the multidrug resistance protein (MRP)/ABCC subfamily of ATP-binding cassette transporters. MRP4 (AHCC4) and MRP5 (ABCC5) are organic anion transporters. They have, however, the outstanding ability to transport nucleotides and nucleotide analogs. In vitro experiments using drug-selected or -transfected cells indicated that these transport proteins, when overexpressed, can lower the intracellular concentration of nucleoside/nucleotide analogs, such as the antiviral compounds PMEA (9-(2-phosphonylmethoxyethyl/adenine) or ganciclovir, and of anticancer nucleobase analogs, such as 6-mercaptopurine, after their conversion into the respective nucleotides. This may lead lo an impaired ability of these compounds to inhibit virus replication or cell proliferation. It remains to be tested whether antiviral or anticancer chemotherapy based on nucleobase. nucleoside, or nucleotide precursors can be modulated by inhibition of MRP4 and MRP5. MRP4 also seems to be able to mediate the transport of conjugated steroids, prostaglandins, and glutathione. Furthermore, cyclic nucleotides (cyclic adenosine monophosphate and cyclic guanine monophosphate) are exported from cells by MRP4 and MRP5. This may modulate the intracellular concentration of these important mediators, besides the action of phosphodiesterases, as well as provide extracellular nucleotides for a possible paracrine action. In this line, tissue distribution and subcellular localization of MRP4 and MRP5 specifically in smooth muscle cells (MRP5), platelet-dense granules (MRP4), and nervous cells (MRP4 and MRP5), besides the capillary endothelium, point not only to a possible function of these transporters as exporters in cellular defense, but also to a physiological function in signaling processes. [ABSTRACT FROM AUTHOR]