Application of dried blood spots combined with HPLC-MS/MS for the quantification of acetaminophen in toxicokinetic studies

A reversed phase HPLC-MS/MS method has been developed and validated for the quantitative bioanalysis of acetaminophen in dried blood spots (DBS) prepared from small volumes (15 microL) of dog blood. Samples were extracted for analysis with methanol. Detection was by positive ion TurboIonSpray ionisation combined with selected reaction monitoring MS. The analytical concentration range was 0.1-50 microg/mL. The intra-day precision and bias values were both less than 15%. Acetaminophen was stable in DBS stored at room temperature for at least 10 days. The methodology was applied in a toxicokinetic (TK) study where the data obtained from DBS samples was physiologically comparable with results from duplicate blood samples (diluted 1:1 (v/v) with water) analysed using identical HPLC-MS/MS conditions. This work demonstrates that quantitative analysis of a drug extracted from DBS can provide high quality TK data while minimising the volume of blood withdrawn from experimental animals, to an order of magnitude lower than is current practice in the pharmaceutical industry. This is the first reported application of DBS analysis to a TK study in support of a safety assessment study. The success of this and similar, related studies has led to the intent to apply DBS technology as the recommended analytical approach for the assessment of pharmacokinetics (PK)/TK for all new oral small molecule drug candidates, which have previously demonstrated a successful bioanalytical validation.     

Effects of multiple-bond ruptures on kinetic parameters extracted from force spectroscopy measurements: revisiting biotin-streptavidin interactions

Force spectroscopy measurements of the rupture of the molecular bond between biotin and streptavidin often results in a wide distribution of rupture forces. We attribute the long tail of high rupture forces to the nearly simultaneous rupture of more than one molecular bond. To decrease the number of possible bonds, we employed hydrophilic polymeric tethers to attach biotin molecules to the atomic force microscope probe. It is shown that the measured distributions of rupture forces still contain high forces that cannot be described by the forced dissociation from a deep potential well. We employed a recently developed analytical model of simultaneous rupture of two bonds connected by polymer tethers with uneven length to fit the measured distributions. The resulting kinetic parameters agree with the energy landscape predicted by molecular dynamics simulations. It is demonstrated that when more than one molecular bond might rupture during the pulling measurements there is a noise-limited range of probe velocities where the kinetic parameters measured by force spectroscopy correspond to the true energy landscape. Outside this range of velocities, the kinetic parameters extracted by using the standard most probable force approach might be interpreted as artificial energy barriers that are not present in the actual energy landscape. Factors that affect the range of useful velocities are discussed.     

Crystal structures of the G139A, G139A–NO and G143H mutants of human heme oxygenase-1. A finely tuned hydrogen-bonding network controls oxygenase versus peroxidase activity

Conserved glycines, Gly139 and Gly143, in the distal helix of human heme oxygenase-1 (HO-1) provide the flexibility required for the opening and closing of the heme active site for substrate binding and product dissociation during HO-1 catalysis. Earlier mutagenesis work on human HO-1 showed that replacement of either Gly139 or Gly143 suppresses heme oxygenase activity and, in the case of the Gly139 mutants, increases peroxidase activity (Liu et al. in J. Biol. Chem. 275:34501, 2000). To further investigate the role of the conserved distal helix glycines, we have determined the crystal structures of the human HO-1 G139A mutant, the G139A mutant in a complex with NO, and the G143H mutant at 1.88, 2.18 and 2.08 Å, respectively. The results confirm that fine tuning of the previously noted active-site hydrogen-bonding network is critical in determining whether heme oxygenase or peroxidase activity is observed.     

Mechanism of inhibition of human KSP by ispinesib

KSP, also known as HsEg5, is a kinesin that plays an essential role in the formation of a bipolar mitotic spindle and is required for cell cycle progression through mitosis. Ispinesib is the first potent, highly specific small-molecule inhibitor of KSP tested for the treatment of human disease. This novel anticancer agent causes mitotic arrest and growth inhibition in several human tumor cell lines and is currently being tested in multiple phase II clinical trials. In this study we have used steady-state and pre-steady-state kinetic assays to define the mechanism of KSP inhibition by ispinesib. Our data show that ispinesib alters the ability of KSP to bind to microtubules and inhibits its movement by preventing the release of ADP without preventing the release of the KSP-ADP complex from the microtubule. This type of inhibition is consistent with the physiological effect of ispinesib on cells, which is to prevent KSP-driven mitotic spindle pole separation. A comparison of ispinesib to monastrol, another small-molecule inhibitor of KSP, reveals that both inhibitors share a common mode of inhibition.     

Ueber den Aufbau der Gattung Trifolium

Ohne Zusammenfassung     

Individual Differences in Scotopic Visual Acuity and Contrast Sensitivity: Genetic and Non-Genetic Influences

Despite the large amount of variation found in the night (scotopic) vision capabilities of healthy volunteers, little effort has been made to characterize this variation and factors, genetic and non-genetic, that influence it. In the largest population of healthy observers measured for scotopic visual acuity (VA) and contrast sensitivity (CS) to date, we quantified the effect of a range of variables on visual performance. We found that young volunteers with excellent photopic vision exhibit great variation in their scotopic VA and CS, and this variation is reliable from one testing session to the next. We additionally identified that factors such as Circadian preference, iris color, astigmatism, depression, sex and education have no significant impact on scotopic visual function. We confirmed previous work showing that the amount of time spent on the vision test influences performance and that laser eye surgery results in worse scotopic vision. We also showed a significant effect of intelligence and photopic visual performance on scotopic VA and CS, but all of these variables collectively explain <30% of the variation in scotopic vision. The wide variation seen in young healthy volunteers with excellent photopic vision, the high test-retest agreement, and the vast majority of the variation in scotopic vision remaining unexplained by obvious non-genetic factors suggests a strong genetic component. Our preliminary genome-wide association study (GWAS) of 106 participants ruled out any common genetic variants of very large effect and paves the way for future, larger genetic studies of scotopic vision.     

Bemerkung überAjuga Hampeana A. Br. et Vatke

Ohne Zusammenfassung     

Phytographische Beiträge

Ohne Zusammenfassung