Czerwin Ski M.,XenoTech LLC |
Kazmi F.,XenoTech LLC |
Parkinson A.,XPD Consulting |
Buckley D.B.,XenoTech LLC
Drug Metabolism and Disposition | Year: 2015
Like most infections and certain inflammatory diseases, some therapeutic proteins cause a cytokine-mediated suppression of hepatic drug-metabolizing enzymes, which may lead to pharmacokinetic interactions with small-molecule drugs. We propose a new in vitro method to evaluate the whole blood-mediated effects of therapeutic proteins on drug-metabolizing enzymes in human hepatocytes cocultured with Kupffer cells. The traditional method involves treating hepatocyte cocultures with the therapeutic protein, which detects hepatocyte- and macrophage-mediated suppression of cytochrome P450 (P450). The new method involves treating whole human blood with a therapeutic protein to stimulate the release of cytokines from peripheral blood mononuclear cells (PBMCs), after which plasma is prepared and added to the hepatocyte coculture to evaluate P450 enzyme expression. In this study, human blood was treated for 24 hours at 37°C with bacterial lipopolysaccharide (LPS) or ANC28.1, an antibody against human T-cell receptor CD28. Cytokines were measured in plasma by sandwich immunoassay with electrochemiluminescense detection. Treatment of human hepatocyte cocultures with LPS or with plasma from LPS-treated blood markedly reduced the expression of CYP1A2, CYP2B6, and CYP3A4. However, treatment of hepatocyte cocultures with ANC28.1 did not suppress P450 expression, but treatment with plasma from ANC28.1-treated blood suppressed CYP1A2, CYP2B6, and CYP3A4 activity and mRNA levels. The results demonstrated that applying plasma from human blood treated with a therapeutic protein to hepatocytes cocultured with Kupffer cells is a suitable method to identify those therapeutic proteins that suppress P450 expression by an indirect mechanism-namely, the release of cytokines from PBMCs. ©2014 by The American Society for Pharmacology and Experimental Therapeutics.
Shrestha S.P.,University of Colorado at Denver |
Shrestha S.P.,XenoTech LLC |
Thompson J.A.,University of Colorado at Denver |
Wempe M.F.,University of Colorado at Denver |
And 3 more authors.
Pharmaceutical Research | Year: 2012
Purpose: The 3,3″-di-O-galloyl ester of procyanidin B2 (B2G2) is a component of grape seed extract that inhibits growth of human prostate carcinoma cell lines. In preparation for studies in mice, its hepatic metabolism was examined in vitro and compared to B2 and the corresponding monomers, epicatechin (EC) and 3-O-galloyl-epicatechin (ECG). Methods: Compounds were incubated with liver microsomes or cytosol containing cofactors for glucuronidation, sulfation or methylation, and products analyzed by liquid chromatographymass spectrometry (LC-MS). B2G2 was administered orally to mice and plasma analyzed by LC-MS for unmodified procyanidin and metabolites. Results: Glucuronides and methyl ethers of B2 and B2G2 were formed in small amounts. In contrast, EC and ECG were largely or completely converted to glucuronides, sulfates and methyl ethers under the same incubation conditions. B2G2 given orally to mice was partially absorbed intact; no significant metabolites were detected in plasma. Conclusions: Glucuronidation and methylation of procyanidins B2 and B2G2 occurred but were minor processes in vitro. B2G2 was partially absorbed intact in mice after oral dosing and did not undergo significant metabolism. Unlike the flavanol monomers EC and ECG, therefore, B2G2 bioavailability should not be limited by metabolism. These results paved the way for ongoing pharmacokinetic and efficacy studies. © Springer Science+Business Media, LLC 2011.
System-dependent outcomes during the evaluation of drug candidates as inhibitors of cytochrome P450 (CYP) and uridine diphosphate glucuronosyltransferase (UGT) enzymes: Human hepatocytes versus liver microsomes versus recombinant enzymes
Parkinson A.,XenoTech LLC |
Kazmi F.,XenoTech LLC |
Buckley D.B.,XenoTech LLC |
Yerino P.,XenoTech LLC |
And 2 more authors.
Drug Metabolism and Pharmacokinetics | Year: 2010
The ability of a drug to cause clinically significant drug-drug interactions due to direct or metabolism-dependent inhibition of cytochrome P450 (CYP) can generally be predicted from in vitro studies with human liver microsomes (HLM) or recombinant CYP enzymes, as recommended by the FDA and other regulatory agencies. This review highlights some examples of system-dependent inhibition of CYP and uridine diphosphate glucuronosyltransferase (UGT) enzymes. In the case of CYP enzymes, examples are presented where in vitro studies with HLM under-predict or over-predict the degree of inhibition observed in the clinic and where the correct prediction comes from studies with human hepatocytes. Studies with HLM under-predict the ability of gemfibrozil and bupropion to cause clinically significant inhibition of CYP2C8 and CYP2D6, respectively, and over-predict the ability of ezetimibe to cause clinically significant inhibition of CYP3A4. Gemfibrozil and bupropion represent examples of glucuronidation-dependent and reduction-dependent activation to metabolites that inhibit CYP2C8 and CYP2D6, respectively, whereas ezetimibe represents an example of glucuronidation-dependent protection against metabolism-dependent inhibition of CYP3A4. This article illustrates why, when drug candidates are extensively metabolized by non-CYP enzymes, it would be prudent to use human hepatocytes in addition to HLM or recombinant enzymes to evaluate their ability to inhibit CYP enzymes.
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.