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Sonee M.,Drug Safety science | Bogdan N.,Drug Safety science | Hall L.,Drug Safety science | Bryant S.,Drug Safety science | Vinken P.,Janssen Pharmaceutical
Birth Defects Research Part B - Developmental and Reproductive Toxicology | Year: 2013

BACKGROUND: This study was conducted as part of an ILSI-HESI International Life Sciences Institute-Health & Environmental Sciences Institute consortium effort to assess the utility of circulating Inhibin B as an early biomarker of Sertoli cell-specific testicular toxicity in rats. 1, 3-Dinitrobenzene (1,3-DNB) was selected as a testicular toxicant in this study as it is known to target Sertoli cells. METHODS: 1,3-DNB (2 and 6 mg/kg/day) or control (corn oil) was administered orally to male rats for two or five consecutive days. Blood was collected from rats treated for 2 days on days 1 and 2 and from rats treated for 5 days on days 1, 3, and 5. The resulting serum was evaluated for Inhibin B and follicle stimulating hormone. At the end of the treatment periods, the testes were removed, weighed, and examined histopathologically. RESULTS: Daily administration of 1,3-DNB resulted in decreased testis weight only on day 5 and only at the high dose (6 mg/kg/day). There was a time-dependent increase in incidence and severity of testicular findings characterized by degeneration of the germinal epithelium with loss of pachytene spermatocytes and vacuolization of the Sertoli cells in the seminiferous tubules at the high dose. Inhibin B levels in 1,3-DNB-treated animals were decreased with treatment only on day 5 at the high dose; there were no associated changes in follicle stimulating hormone. CONCLUSIONS: Changes in serum Inhibin B levels were detected only in association with moderate or severe testicular toxicity as evidenced by histopathology and is therefore considered to be of limited value as a biomarker for Sertoli cell toxicity. © 2013 Wiley Periodicals, Inc.


Sunil V.R.,Rutgers University | Vayas K.N.,Rutgers University | Cervelli J.A.,Rutgers University | Malaviya R.,Rutgers University | And 5 more authors.
Experimental and Molecular Pathology | Year: 2014

Nitrogen mustard (NM) is a toxic alkylating agent that causes damage to the respiratory tract. Evidence suggests that macrophages and inflammatory mediators including tumor necrosis factor (TNF)α contribute to pulmonary injury. Pentoxifylline is a TNFα inhibitor known to suppress inflammation. In these studies, we analyzed the ability of pentoxifylline to mitigate NM-induced lung injury and inflammation. Exposure of male Wistar rats (150-174g; 8-10weeks) to NM (0.125mg/kg, i.t.) resulted in severe histopathological changes in the lung within 3d of exposure, along with increases in bronchoalveolar lavage (BAL) cell number and protein, indicating inflammation and alveolar-epithelial barrier dysfunction. This was associated with increases in oxidative stress proteins including lipocalin (Lcn)2 and heme oxygenase (HO)-1 in the lung, along with pro-inflammatory/cytotoxic (COX-2+ and MMP-9+), and anti-inflammatory/wound repair (CD163+ and Gal-3+) macrophages. Treatment of rats with pentoxifylline (46.7mg/kg, i.p.) daily for 3d beginning 15min after NM significantly reduced NM-induced lung injury, inflammation, and oxidative stress, as measured histologically and by decreases in BAL cell and protein content, and levels of HO-1 and Lcn2. Macrophages expressing COX-2 and MMP-9 also decreased after pentoxifylline, while CD163+ and Gal-3+ macrophages increased. This was correlated with persistent upregulation of markers of wound repair including pro-surfactant protein-C and proliferating nuclear cell antigen by Type II cells. NM-induced lung injury and inflammation were associated with alterations in the elastic properties of the lung, however these were largely unaltered by pentoxifylline. These data suggest that pentoxifylline may be useful in treating acute lung injury, inflammation and oxidative stress induced by vesicants. © 2014 Elsevier Inc.


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.


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.


Groves A.M.,Rutgers University | Gow A.J.,Rutgers University | Massa C.B.,Rutgers University | Hall L.,Drug Safety science | And 2 more authors.
American Journal of Physiology - Lung Cellular and Molecular Physiology | Year: 2013

In these studies we determined whether progressive pulmonary inflammation associated with aging in surfactant protein D (Sftpd)-/- mice leads to an exacerbated response to ozone. In Sftpd^/^ mice, but not wild-type (WT) mice, age-related increases in numbers of enlarged vacuolated macrophages were observed in the lung, along with alveolar wall rupture, type 2 cell hyperplasia, and increased bronchoalveolar lavage protein and cell content. Numbers of heme oxygenase + macrophages also increased with age in Sftpd-/- mice, together with classically (iNOS +) and alternatively (mannosereceptor+, YM-1 +, orgalectin-3 +) activated macrophages. In both WT and Sftpd-/- mice, increasing age from 8 to 27 wk was associated with reduced lung stiffness, as reflected by decreases in resistance and elastance spectra; however, this response was reversed in 80-wk-old Sftpd-/- mice. Ozone exposure (0.8 ppm, 3 h) caused increases in lung pathology, alveolar epithelial barrier dysfunction, and numbers of iNOS+ macrophages in 8- and 27-wk-old Sftpd-/-, but not WT mice at 72 h postexposure. Conversely, increases in alternatively activated macrophages were observed in 8-wk-old WT mice following ozone exposure, but not in Sftpd-/- mice. Ozone also caused alterations in both airway and tissue mechanics in Sftpd-/- mice at 8 and 27 wk, but not at 80 wk. These data demonstrate that mild to moderate pulmonary inflammation results in increased sensitivity to ozone; however, in senescent mice, these responses are overwhelmed by the larger effects of age-related increases in baseline inflammation and lung injury. © 2013 the American Physiological Society.


Templeton I.,Drug Safety science | Eichenbaum G.,Drug Safety science | Sane R.,Boehringer Ingelheim Pharmaceuticals | Zhou J.,Boehringer Ingelheim Pharmaceuticals
Methods in Molecular Biology | Year: 2014

New molecular entities (NMEs) are evaluated using a rigorous set of in vitro and in vivo studies to assess their safety and suitability for testing in humans. Regulatory health authorities require that therapeutic and supratherapeutic doses be administered, by the intended route of administration, to two nonclinical species prior to human testing (ICH Expert Working Group. The international conference on harmonization of technical requirements for registration of pharmaceuticals for human use (ICH); Multidisciplinary guidelines; Nonclinical safety studies (M3). http://www.ich.org/fileadmin/ Public-Web-Site/ICH-Products/Guidelines/Multidisciplinary/M3-R2/Step4/ M3-R2-Guideline.pdf, 2009). The purpose of these studies is to identify potential target organ toxicity and to determine if the effects are reversible. Liver is a potential site for toxicity caused by orally administered NMEs due to high exposure during first pass after oral administration. A range of clinical chemistry analytes are routinely measured in both nonclinical and clinical studies to evaluate and monitor for hepatotoxicity. While bilirubin itself circulates within a wide range of concentrations in many animal species and humans, without causing adverse effects and possibly providing benefits (Sedlak and Snyder. Pediatrics 113(6):1776-1782, 2004), bilirubin is one of the few readily monitored circulating biomarkers that can provide insight into liver function. Therefore, any changes in plasma or urine bilirubin levels must be carefully evaluated. Changes in bilirubin may occur as a result of adaptive nontoxic changes or severe toxicity. Examples of adaptive nontoxic changes in liver function, which may elevate direct (conjugated) and/or indirect (unconjugated) bilirubin above baseline levels, include reversible inhibition of UGT1A1-mediated bilirubin metabolism and OATP1B1-, OATP1B3-, or MRP2-mediated transport (Keogh. Adv Pharmacol 63:1-42, 2012). Alternatively, hepatocellular necrosis, hypoalbuminuria, or cholestasis may also lead to elevation of bilirubin; in some cases, these effects may be irreversible (FDA/CDER. Guidance for industry drug-induced liver injury: premarketing clinical evaluation. http://www.fda.gov/downloads/Drugs/.../Guidances/UCM174090.pdf, 2012). This chapter aims to demonstrate application of enzyme kinetic principles in understanding the risk of bilirubin elevation through inhibition of multiple processes - involving both enzymes and transporters. In the sections that follow, we first provide a brief summary of bilirubin formation and disposition. Two case examples are then provided to illustrate the enzyme kinetic studies needed for risk assessment and for identifying the mechanisms of bilirubin elevation. Caveats of methods and data interpretation are discussed in these case studies. The data presented in this chapter is unpublished at the time of compilation of this book. It has been incorporated in this chapter to provide a sense of complexities in enzyme kinetics to the reader. © Springer Science+Business Media, LLC 2014.


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.


Xu Y.,Drug Safety science | Yang Y.,University of Illinois at Chicago | Smith L.,Drug Safety science | Edom R.,Drug Safety science | And 5 more authors.
Journal of Pharmaceutical and Biomedical Analysis | Year: 2013

A liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) assay was developed and qualified for analyzing 4β-hydroxycholesterol and cholesterol in 5. μl of human and mouse plasma. Stable isotope-labeled d7-analogs of both analytes were used as internal standards and 4.2% (w/v) human serum albumin in phosphate-buffered saline was used as the surrogate matrix for preparation of calibration curves and QCs. The assay is capable of quantification of 4β-hydroxycholesterol and cholesterol from 5 to 500. ng/ml and 50 to 2000. μg/ml, respectively, with acceptable accuracy and precision following evaluation of recovery of analytes, autosampler stability and potential contribution of chemical oxidation to the formation of 4β-hydroxycholesterol. The final reconstituted solution was diluted for quantification of cholesterol typically present at 1000 fold higher concentration than 4β-hydroxycholesterol in the same samples used for 4β-hydroxycholesterol quantification. The successful quantification using a low plasma volume was achieved by quantification of total forms (free and conjugated) of both analytes after alkaline hydrolysis, followed by derivatization to form electrospray ionization-sensitive picolinyl esters, which upon collision-induced dissociation gave high mass precursor-product ion pair for selective detection by multiple reaction monitoring. In addition, chromatographic separation using a 16-min reversed phase gradient elution on a 1.9. μm particle size, C18 column, overcame interference from other isobaric plasma oxysterols during detection by multiple-reaction monitoring. This assay was compared to an orthogonal enzymatic assay for cholesterol and all samples, but one, provided values that were within 10% of each other. In addition, this assay passed the incurred sample tests for both analytes in human and mouse plasma samples according to reported acceptance criteria for incurred sample reanalysis.The quantification of both analytes permitted the determination of 4β-hydroxycholesterol compared to its ratio to cholesterol as an endogenous biomarker for CYP3A4/5 activity. The LC-ESI-MS/MS assay was also successfully applied to quantification of 4β-hydroxycholesterol and cholesterol in plasma samples from untreated human and mice including FRG™ KO C57Bl/6 chimeric mice with humanized livers. The preliminary data indicated that the plasma 4β-hydroxycholesterol concentrations or their ratio to cholesterol from mice including chimeric mice were higher than those from human. © 2013 Elsevier B.V.


PubMed | Drug Safety science
Type: | Journal: Methods in molecular biology (Clifton, N.J.) | Year: 2014

New molecular entities (NMEs) are evaluated using a rigorous set of in vitro and in vivo studies to assess their safety and suitability for testing in humans. Regulatory health authorities require that therapeutic and supratherapeutic doses be administered, by the intended route of administration, to two nonclinical species prior to human testing (ICH Expert Working Group. The international conference on harmonization of technical requirements for registration of pharmaceuticals for human use (ICH); Multidisciplinary guidelines; Nonclinical safety studies (M3). http://www.ich.org/fileadmin/Public_Web_Site/ICH_Products/Guidelines/Multidisciplinary/M3_R2/Step4/M3_R2__Guideline.pdf , 2009). The purpose of these studies is to identify potential target organ toxicity and to determine if the effects are reversible. Liver is a potential site for toxicity caused by orally administered NMEs due to high exposure during first pass after oral administration. A range of clinical chemistry analytes are routinely measured in both nonclinical and clinical studies to evaluate and monitor for hepatotoxicity. While bilirubin itself circulates within a wide range of concentrations in many animal species and humans, without causing adverse effects and possibly providing benefits (Sedlak and Snyder. Pediatrics 113(6):1776-1782, 2004), bilirubin is one of the few readily monitored circulating biomarkers that can provide insight into liver function. Therefore, any changes in plasma or urine bilirubin levels must be carefully evaluated. Changes in bilirubin may occur as a result of adaptive nontoxic changes or severe toxicity. Examples of adaptive nontoxic changes in liver function, which may elevate direct (conjugated) and/or indirect (unconjugated) bilirubin above baseline levels, include reversible inhibition of UGT1A1-mediated bilirubin metabolism and OATP1B1-, OATP1B3-, or MRP2-mediated transport (Keogh. Adv Pharmacol 63:1-42, 2012). Alternatively, hepatocellular necrosis, hypoalbuminuria, or cholestasis may also lead to elevation of bilirubin; in some cases, these effects may be irreversible (FDA/CDER. Guidance for industry drug-induced liver injury: premarketing clinical evaluation. http://www.fda.gov/downloads/Drugs//Guidances/UCM174090.pdf , 2012).This chapter aims to demonstrate application of enzyme kinetic principles in understanding the risk of bilirubin elevation through inhibition of multiple processes-involving both enzymes and transporters. In the sections that follow, we first provide a brief summary of bilirubin formation and disposition. Two case examples are then provided to illustrate the enzyme kinetic studies needed for risk assessment and for identifying the mechanisms of bilirubin elevation. Caveats of methods and data interpretation are discussed in these case studies. The data presented in this chapter is unpublished at the time of compilation of this book. It has been incorporated in this chapter to provide a sense of complexities in enzyme kinetics to the reader.


Jian W.,Drug Safety science | Edom R.W.,Drug Safety science | Huang M.-Q.,Drug Safety science | Weng N.,Drug Safety science
Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences | Year: 2013

Leukotriene B4 (LTB4) is a potent mediator of inflammation and has been recognized as an important target for therapeutic intervention for treatment of diseases such as asthma. In the current work, a highly selective and sensitive UPLC-MS/MS assay was developed for quantitation of LTB4 in human sputum as a biomarker for LTB4 biosynthesis inhibition. A fit-for-purpose strategy for method development, assay qualification, and study support was adopted for this biomarker project. A surrogate matrix (protein buffer) was used for preparation of calibration samples and certain levels of quality control (QC) samples to avoid interference from endogenous analyte, while the low QC was prepared in authentic matrix, human sputum. The analytical methodology utilized a liquid-liquid extraction procedure in 96-well plate format. Chromatographic separation was achieved with a reversed-phase ultra high pressure liquid chromatography (UPLC) column using gradient elution, and the run time was 4.5min per sample. The lower limit of quantitation (LLOQ) was 0.2ng/mL, and the calibration curve range was 0.2-20ng/mL. Acceptable accuracy, precision, linearity, specificity, recovery, and matrix effect was obtained. Bench-top stability (6h), freeze-thaw stability (3 cycles at -20°C), and autosampler stability (97h at ambient temperature) all met acceptance criteria. Frozen long-term stability for 166 days at -20°C in sputum did not meet acceptance criteria by showing only ≥75% of nominal concentration and the information was taken into consideration for study support. Two important observations in the current work were: (1) LTB4 was unstable in sputum in the presence of liquification reagent dithiothreitol (DTT). Therefore, a non-DTT treatment method for sputum processing was developed and applied to the bioanalytical assay and clinical study support; and (2) chromatographic separation of LTB4 from its three non-enzymatically derived isomers, i.e. 6-trans-LTB4, 12-epi-LTB4, and 6-trans-12-epi-LTB4, was achieved. This assay was successfully applied to a Phase II clinical study for proof-of-concept of a LTA4 hydrolase inhibitor for treatment of asthma. © 2013 Elsevier B.V.

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