XenoTech LLC | Date: 2017-04-04
The invention provides cryopreserved compositions of cells, wherein the compositions are advantageously in the form of self-sustaining bodies that can be individually handled and combined independently of a container, allowing for easy customization of the eventual pooled preparation. The invention also provides pre-pooled stacks of the self-sustaining cryopreserved compositions for eventual thawing to produce pooled preparations of cells, and methods of creating the same.
Kandel S.E.,XenoTech LLC |
Lampe J.N.,University of Kansas Medical Center
Chemical Research in Toxicology | Year: 2014
Through their unique oxidative chemistry, cytochrome P450 monooxygenases (CYPs) catalyze the elimination of most drugs and toxins from the human body. Protein-protein interactions play a critical role in this process. Historically, the study of CYP-protein interactions has focused on their electron transfer partners and allosteric mediators, cytochrome P450 reductase and cytochrome b5. However, CYPs can bind other proteins that also affect CYP function. Some examples include the progesterone receptor membrane component 1, damage resistance protein 1, human and bovine serum albumin, and intestinal fatty acid binding protein, in addition to other CYP isoforms. Furthermore, disruption of these interactions can lead to altered paths of metabolism and the production of toxic metabolites. In this review, we summarize the available evidence for CYP protein-protein interactions from the literature and offer a discussion of the potential impact of future studies aimed at characterizing noncanonical protein-protein interactions with CYP enzymes. © 2014 American Chemical Society.
Barbara J.E.,XenoTech LLC |
Kazmi F.,XenoTech LLC |
Parkinson A.,XPD Consulting LLC |
Buckley D.B.,XenoTech LLC
Drug Metabolism and Disposition | Year: 2013
Metabolism-dependent inhibition (MDI) of cytochrome P450 (P450) enzymes has the potential to cause clinically relevant drug-drug interactions. In the case of several alkylamine drugs, MDI of P450 involves formation of a metabolite that binds quasi-irreversibly to the ferrous heme iron to form a metabolic intermediate (MI) complex. The specific metabolites coordinately bound to ferrous iron and the pathways leading to MI complex formation are the subject of debate. We describe an approach combining heme iron oxidation with potassium ferricyanide and metabolite profiling to probe the mechanism of MI complex-based CYP3A4 inactivation by the secondary alkylamine drug lapatinib. Ten metabolites formed from lapatinib by CYP3A4-mediated heteroatom dealkylation, Chydroxylation, N-oxygenation with or without further oxidation, or a combination thereof, were detected by accurate mass spectrometry. The abundance of one metabolite, the N-dealkylated nitroso/ oxime lapatinib metabolite (M9), correlated directly with the prevalence or the disruption of the MI complex with CYP3A4. Nitroso/ oxime metabolite formation from secondary alkylamines has been proposed to occur through two possible pathways: (1) sequential N-dealkylation,N-hydroxylation, and dehydrogenation (primary hydroxylamine pathway) or (2) N-hydroxylation with dehydrogenation to yield a nitrone followed by N-dealkylation (secondary hydroxylamine pathway). All intermediates for the secondary hydroxylamine pathway were detected but the primary N-hydroxylamine intermediate of the primary hydroxylamine pathway was not. Our findings support the mechanism of lapatinib CYP3A4 inactivation as MI complex formation with the nitroso metabolite formed through the secondary hydroxylamine and nitrone pathway, rather than by N-dealkylation to the primary amine followed by N-hydroxylation and dehydrogenation as is usually assumed. © 2013 by The American Society for Pharmacology and Experimental Therapeutics.
Amunom I.,XenoTech LLC
Current protocols in toxicology / editorial board, Mahin D. Maines (editor-in-chief) ... [et al.] | Year: 2011
This protocol describes the procedure for measuring the relative rates of metabolism of the α,β-unsaturated aldehydes 9-anthracene aldehyde (9-AA) and 4-hydroxy-trans-2-nonenal (4-HNE). More specifically, these assays measure the aldehyde reduction reactions of cytochrome P450s (CYPs). They can be performed using liver microsomal or other tissue fractions, spherosome preparations of recombinant CYPs, or recombinant CYPs from other sources. The method for reduction of 9-AA (a model α,β-unsaturated aldehyde) by CYPs was adapted from an assay for 9-anthracene oxidation published by Marini et al. (2003). For reduction of the endogenous aldehyde 4-HNE, the substrate was incubated with CYP in the presence of oxygen and NADPH, and the metabolites were separated by HPLC, using an adaptation of the method by Srivastava et al. (2010). For both 9-AA and 4-HNE, the first step involves incubation of the substrate with the CYP in an appropriate medium. This is followed by quantification of metabolites through by spectrofluorometry (9-AA) or HPLC coupled with a radiometric assay (4-HNE). Metabolite identification can be achieved by HPLC GC/MS analysis. Inhibitors of cytochrome P450 can be utilized to show the role of the hemoprotein or other enzymes in these reduction reactions. The reduction of CYPs is not inhibited by either anaerobiosis or inclusion of CO in the gaseous phase of the reaction mixture. These characteristics are similar to those reported for some cytochrome P450-catalyzed azo reduction reactions. © 2011 by John Wiley & Sons, Inc.
Barbara J.E.,XenoTech LLC |
Castro-Perez J.M.,Waters Corporation
Rapid Communications in Mass Spectrometry | Year: 2011
Electrophilic reactive metabolite screening by liquid chromatography/mass spectrometry (LC/MS) is commonly performed during drug discovery and early-stage drug development. Accurate mass spectrometry has excellent utility in this application, but sophisticated data processing strategies are essential to extract useful information. Herein, a unified approach to glutathione (GSH) trapped reactive metabolite screening with high-resolution LC/TOF MS E analysis and drug-conjugate-specific in silico data processing was applied to rapid analysis of test compounds without the need for stable- or radio-isotope-labeled trapping agents. Accurate mass defect filtering (MDF) with a C-heteroatom dealkylation algorithm dynamic with mass range was compared to linear MDF and shown to minimize false positive results. MS E data-filtering, time-alignment and data mining post-acquisition enabled detection of 53 GSH conjugates overall formed from 5 drugs. Automated comparison of sample and control data in conjunction with the mass defect filter enabled detection of several conjugates that were not evident with mass defect filtering alone. High- and low-energy MS E data were time-aligned to generate in silico product ion spectra which were successfully applied to structural elucidation of detected GSH conjugates. Pseudo neutral loss and precursor ion chromatograms derived post-acquisition demonstrated 50.9% potential coverage, at best, of the detected conjugates by any individual precursor or neutral loss scan type. In contrast with commonly applied neutral loss and precursor-based techniques, the unified method has the advantage of applicability across different classes of GSH conjugates. The unified method was also successfully applied to cyanide trapping analysis and has potential for application to alternate trapping agents. © 2011 John Wiley & Sons, Ltd.
Barbara J.E.,XenoTech LLC |
Buckley D.B.,XenoTech LLC |
Horrigan M.J.,XenoTech LLC
Bioanalysis | Year: 2013
Background: The utility of high-resolution MS (HRMS) with post-acquisition data mining in DMPK goes much further than the now established approach to simultaneously acquire quantitative and qualitative information for lead compounds at the discovery stage. Indeed, HRMS has promise for addressing multiple complex drug-development applications in a single experiment. In the present study, one HRMS dataset acquired for in vitro incubations of the model compound dasatinib was mined post-acquisition to address four different issues: stability, metabolite profiling, glutathione conjugate analysis, and endogenous lipid profiling. Results & Conclusion: The derived results demonstrated that HRMS has potential for generating high information content datasets that can be stored and mined as needed to answer numerous complex development-stage questions without the need for additional sample generation or analysis. © 2013 Future Science Ltd.
Xenotech Llc | Date: 2010-08-30
The invention provides cryopreserved compositions of cells, wherein the compositions are advantageously in the form of self-sustaining bodies that can be individually handled and combined independently of a container, allowing for easy customization of the eventual pooled preparation. The invention also provides pre-pooled stacks of the self-sustaining cryopreserved compositions for eventual thawing to produce pooled preparations of cells. A mold and methods for forming the self-sustaining bodies are also provided. The invention is also concerned with methods of forming pooled preparations of cells using single-cryopreserved compositions of cells.
Xenotech Llc | Date: 2012-05-25
A method of evaluating the effect of a xenobiotic on biomarkers, such as drug transporters and drug-metabolizing enzymes in hepatocytes is provided. The method comprises the formation of a xenobiotic-stimulated biological sample, such as whole blood, which contains a plurality of cytokines. A portion of xenobiotic-stimulated biological sample is cultured with hepatocytes. The hepatocytes are then analyzed to evaluate the activity of drug transporters and/or drug-metabolizing enzymes, or other biomarkers to determine the effect of the xenobiotic on drug metabolism in the hepatocytes.
XenoTech LLC. | Date: 2014-08-19
XenoTech LLC. | Date: 2014-08-19
Cells for scientific, laboratory or medical research.