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Ovcharenko E.,University of Sfax | Hansen M.K.,Wyeth Research | Goltsman I.,University of Sfax | Abu-Saleh N.,University of Sfax | And 5 more authors.
Nephron - Physiology | Year: 2010

Background/Aims: Patients treated with peroxisome proliferator-activated receptor analogs (PPAR) α or α/γ may develop a transient and reversible increase in serum creatinine, the mechanism of which remains obscure. This study evaluates whether treatment with either PPAR-α or -α/γ analogs, fenofibrate or tesaglitazar, may cause deterioration in renal hemodynamics or exert direct tubular or glomerular nephrotoxic effects in rats. Methods: Male Sprague-Dawley rats (300-320 g) were treated per os with fenofibrate (300 mg/kg/day), tesaglitazar (1.2 mg/kg/day) or vehicle, for 14 days. Glomerular filtration rate (GFR) and renal blood flow (RBF) were measured by inulin clearance and ultrasonic flowmetry, and cumulative excretion of sodium and creatinine were assessed. Biomarkers of glomerular and tubular injury were measured, including urinary albumin excretion and renal mRNA levels of kidney injury molecule 1 (Kim-1), lipocalin 2 (Lcn2), and osteopontin (Spp1). Results: Fenofibrate and tesaglitazar improved the lipid profile, but caused no detectable decrease in GFR or RBF compared with vehicle-treated rats. Furthermore, the cumulative excretions of sodium and creatinine were not altered by the drugs. Finally, there was no significant difference between drug- and vehicle-treated groups in urinary albumin excretion or in the expression of renal injury biomarkers. Conclusions: In the rat, no direct nephrotoxic effect or deterioration in renal hemodynamics and function were observed following treatment with fenofibrate or tesaglitazar. Copyright © 2010 S. Karger AG, Basel.

Hurko O.,Translational Medicine Research Collaboration
Molecular Genetics and Metabolism | Year: 2010

Drug discovery and development has evolved significantly over the last century. Extrapolation from current practice invites speculation about the future in four areas of particular interest. These include the range of therapeutic modalities; the methods for selection of drug targets; the interjection of translational medicine in between the traditional discovery and development phases; and the relationships between institutions. A major focus for the latter three developments is the shortcomings of current target validation. This personal view will be given from perspectives gained in large integrated pharmaceutical companies that are primarily focused on common disorders. Where appropriate, comparisons will be made to the development of treatments for rare Mendelian disorders. © 2010 Elsevier Inc. All rights reserved.

Atrih A.,Translational Medicine Research Collaboration | Turnock D.,University of Dundee | Sellar G.,Pfizer | Thompson A.,University of Dundee | And 4 more authors.
Journal of Proteome Research | Year: 2010

The Ptdlns-3-kinase (PI3-K) signaling pathway plays a vital role in cell survival, proliferation, apoptosis and differentiation in normal cells, as well as in diseases such as cancer and diabetes. Quantification of phospho-Akt is a standard way of assessing the activity of the PI3-K signaling pathway in both cells and tumors. This measurement is traditionally performed semiquantitatively using immunoassays such as Western blot. Here we report an LC-MS method to accurately measure the stoichiometry of Akt phosphorylation in biological samples. The procedure includes immunoprecipitation, gel electrophoresis, in-gel digestion, addition of isotopicaly labeled internal standards and LC-MS/MS. Two proteolytic enzymes, chymotrypsin and trypsin, were used to generate suitable peptide fragments for measuring Thr308 and Ser473 phosphorylation, respectively. The interday imprecision was estimated to be 3.8% and 2.3% for Thr308 and Ser473, respectively. This method has been tested on human T-cells grown in presence and absence of pervanadate and with or without a PI3-K inhibitor and on human glioblastoma cells (U-87 MG) grown in presence and absence of wortmannin (PI3-K inhibitor).The results of T cells suggest that the levels of Akt phosphorylation in untreated cells were below 1% for both phosphorylation sites. Pervanadate treatment provoked an 18-fold increase in phosphorylation of Thr308 and the PI3-K inhibitor partially reversed the increase. A comparison between LC-MS/MS and Western blotting suggests that the LC-MS based method is of comparable sensitivity and provides a more accurate phosphorylation stoichiometry, a wider dynamic range and more in-depth information. The application of the new method and its utility to providing predictive markers of response to targeted therapies is discussed. © 2010 American Chemical Society.

Albarbarawi O.,Translational Medicine Research Collaboration | Albarbarawi O.,Taibah University | Barton A.,Translational Medicine Research Collaboration | Miller D.,Pfizer | And 8 more authors.
Bioanalysis | Year: 2013

Background: Desmosine/isodesmosine (DES/IDS) is a promising biomarker for estimating activity of elastin degradation. Results/methodology: A stable isotope dilution LC-MS/MS method for measuring serum/plasma DES/IDS was developed and validated. The reportable range of this assay was 0.1-160 ng/ml. Serum/plasma DES/IDS level was stable at room temperature or 4°C for 20 h, and for three freeze-thaw cycles. Interferences from endogenous compounds and ion suppression/enhancing effect were also evaluated. Our results suggest the absolute necessity of using an IS in the measurement. We found that serum/plasma DES/IDS levels from patients with chronic obstructive pulmonary disease and cystic fibrosis were significantly higher compared with healthy smokers. Conclusion: These results demonstrate that the LC-MS/MS method provides sensitive, reproducible and accurate quantification of total serum/plasma DES/IDS. © 2013 Future Science Ltd.

Burch L.R.,University of Dundee | Donnelly L.A.,University of Dundee | Doney A.S.F.,University of Dundee | Brady J.,Translational Medicine Research Collaboration | And 6 more authors.
Journal of Clinical Endocrinology and Metabolism | Year: 2010

Context: Previous studies have identified a single-nucleotide polymorphism in the gene encoding peroxisome proliferator-activated receptor-δ (PPARD), rs2016520, that is associated with changes in metabolic disease in some but not all studies, which suggests that PPARD agonists may have therapeutic benefits for the treatment of metabolic disorders, including dyslipidemia, type 2 diabetes, and obesity. Objective: The objective of the study was to determine whether rs2016520 or other single-nucleotide polymorphism in the PPARD locus influenced the risk of developing various characteristics of metabolic disease. Design: Haplotype tagging analysis across PPARD was performed in 11,074 individuals from the Welcome Trust U.K. Type 2 Diabetes Case Control Collection. Results: In subjects with and without type 2 diabetes, rs2016520 was associated with body mass index, high-density lipoprotein cholesterol, leptin, and TNFα and was dependent on gender. Conclusion: The current results suggest differential effects of PPARδ in males and females. Copyright © 2010 by The Endocrine Society.

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