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Beerse, Belgium

Hall L.,Drug Safety science | Laskin J.D.,Rutgers University | Laskin D.L.,Rutgers University
American Journal of Physiology - Lung Cellular and Molecular Physiology | Year: 2015

Nitrogen mustard (NM) is a vesicant that causes lung injury and fibrosis, accompanied by a persistent macrophage inflammatory response. In these studies we analyzed the spleen as a source of these cells. Splenectomized (SPX) and sham control rats were treated intratracheally with NM (0.125 mg/kg) or PBS control. Macrophage responses were analyzed 1–7 days later. Splenectomy resulted in an increase in lung macrophages expressing CCR2, but a decrease in ATR-1a+ cells, receptors important in bone marrow and spleen monocyte trafficking, respectively. Splenectomy was also associated with an increase in proinflammatory M1 (iNOS+, CD11b+CD43+) macrophages in lungs of NM-treated rats, as well as greater upregulation of iNOS and COX-2 mRNA expression. Conversely, a decrease in CD11b+CD43+ M2 macrophages was observed in SPX rats, with no changes in CD68+, CD163+, CD206+, or YM-1+ M2 macrophages, suggesting distinct origins of M2 subpopulations responding to NM. Macrophage expression of M2 genes including IL-10, ApoE, PTX-2, PTX-3, 5-HT2a, and 5-HT7 was also reduced in NM-treated SPX rats compared with shams, indicating impaired M2 activity. Changes in lung macrophages responding to NM as a consequence of splenectomy were correlated with exacerbated tissue injury and more rapid fibrogenesis. These data demonstrate that the spleen is a source of a subset of M2 macrophages with anti-inflammatory activity; moreover, in their absence, proinflammatory/ cytotoxic M1 macrophages predominate in the lung, resulting in heightened pathology. Understanding the origin of macrophages and characterizing their phenotype after vesicant exposure may lead to more targeted therapeutics aimed at reducing toxicity and disease pathogenesis. © 2015 the American Physiological Society.

Meermann B.,Ghent University | Bockx M.,Drug Safety science | Laenen A.,Drug Safety science | Van Looveren C.,Drug Safety science | And 2 more authors.
Analytical and Bioanalytical Chemistry | Year: 2012

The aim of this work was speciation analysis of metabolites in feces samples collected within a clinical study during which a bromine-containing anti-tuberculosis drug (TMC207) was administered to patients with multi-drug resistant tuberculosis infection. Owing to slow elimination of the drug, no 14C label was used within this study. Quantification of the bromine species was accomplished using high performance liquid chromatography coupled to inductively coupled plasma-mass spectrometry (HPLC/ICP-MS) in combination with on-line isotope dilution (on-line ID), while structural elucidation of the species was performed using HPLC coupled to electrospray ionization-mass spectrometry. The ICP-MS-based method developed shows a good intra- and inter-day reproducibility (relative standard deviation∈=∈3.5%, N∈=∈9); the limit of detection (1.5 mg TMC207 L -1) is of the same order of magnitude as that for HPLC/radiodetection; the dynamic range of the method covers more than two orders of magnitude. Furthermore, the column recovery was demonstrated to be quantitative (recoveries between 90.6% and 99.5%). Based on the excellent figures of merit, the "cold" HPLC/ICP-MS approach could be deployed for the actual human in vivo metabolism study, such that exposure of the human volunteers to the 14C radiolabel was avoided. [Figure not available: see fulltext.] © 2011 Springer-Verlag.

Cuyckens F.,Drug Safety science | Pauwels N.,Drug Safety science | Koppen V.,Drug Safety science | Leclercq L.,Drug Safety science
Bioanalysis | Year: 2012

Background: There is considerable interest in the determination of relative abundances of human metabolites in plasma (and potentially excreta) with reasonable accuracy early on in the drug development process in order to make scientifically sound decisions with regard to the presence of potentially active or toxic disproportionate metabolites. At this point, authentic metabolite standards are generally not available. Results: A new methodology is proposed for the estimation of metabolite concentrations in the absence of authentic standards. A reference sample containing radiolabeled metabolites of interest is produced by incubating the 14C-labeled drug in vitro, and mixed with a sample to be quantitated containing the unlabeled metabolites. The 12C/ 14C isotope ratio is measured with high-resolution ESI-MS for each metabolite, and used as a basis for quantitation of the cold metabolite based on the concentration of radioactive metabolite, determined from independent analysis of the radioactive sample with LC-radiochemical detection. The 14C-labeled metabolite serves as an isotopically labeled internal standard, which corrects for any variations in injection volume, sample preparation, MS intensity drift, matrix effects and/or saturation of electrospray ionization. The approach was validated by the analysis of solutions containing variable amounts of the analyte with a fixed amount of radioactive standard on a QToF Synapt ® G2 MS system. The same methodology was also successfully applied to first-in-human plasma samples analyzed on a LTQ-Orbitrap ®. Conclusion: The metabolite abundances obtained by 12C/ 14C isotope ratio measurements showed suitable accuracy and precision and were very close to those obtained with matrix mixing. The parent drug concentrations also corresponded well with the bioanalytical results obtained with a validated LC-MS/MS method. © 2012 Future Science Ltd.

Meermann B.,Ghent University | Hulstaert A.,Ghent University | Laenen A.,Drug Safety science | Looveren C.V.,Drug Safety science | And 3 more authors.
Analytical Chemistry | Year: 2012

During the development of a new drug compound, its metabolism needs to be unraveled. For quantification of the metabolites formed, the drug under investigation is traditionally synthesized with a radiolabel ( 14C or 3H) and the metabolites present in different matrixes (blood, urine, feces) upon drug administration are determined by means of high-performance liquid chromatography (HPLC) coupled to radiodetection. This approach allows for quantification of the metabolites formed and enables a straightforward distinction between exogenous (i.e., drug-related) and endogenous species (as only the radiolabeled species are detected). However, in some cases, the use of a radiolabeled compound in human in vivo studies is not advisible, e.g., for drug compounds or their metabolites showing a long plasma or tissue half-life. In cases where the candidate drug molecule contains an element detectable by means of inductively coupled plasma mass spectrometry (ICP-MS), HPLC/ICP-MS is a promising alternative approach. However, the method lacks specificity when a distinction between drug-related species and endogenous compounds containing the same target element needs to be accomplished. As a result, we have developed an HPLC/ICP-MS-based method combined with "reverse" online isotope dilution ("reverse" online ID) for metabolite quantification. The methodology was evaluated by the analysis of feces samples from rats dosed with a 81Br-labeled drug compound. The method allows for both (i) valid quantification of the drug metabolites and (ii) distinction among endogenous, exogenous, and "mixed" species, based on their isotopic "fingerprint". A good repeatability (relative standard deviation of 4.2%) and limit of detection (0.35 mg of drug compound L -1 of feces extract), of the same order of magnitude as those observed for "normal" online ID HPLC/ICP-MS and HPLC/radiodetection, were achieved. © 2012 American Chemical Society.

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