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Hepatoxicity Sot 2008 Poster
 

Hepatoxicity Sot 2008 Poster

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    Hepatoxicity Sot 2008 Poster Hepatoxicity Sot 2008 Poster Presentation Transcript

    • SCREENING FOR MECHANISMS OF HEPATOTOXICITY: PHOSPHOLIPIDOSIS, STEATOSIS, APOPTOSIS AND INFLAMMATORY MARKERS K.F. Marcoe, R. Keyser, P. TB. Nguyen, Y. Ovechkina, and C. O’Day MDS Pharma Services – Bothell, WA, USA INTRODUCTION MULTIPLEXED HEPATOTOXICITY ASSAY MULTIPLEXED HEPATO-LIPID ACCUMULATION ASSAY Drug-induced hepatotoxicity is difficult to predict and remains a major cause for failures during drug development. Predictive toxicology Hepato-Phospholipid Accumulation Assay A summary of Hepato-Lipid Accumulation Assay parameters for each compound tested, (n = 3). screening assays for identifying biomakers and providing mechanistic assessments allow earlier identification of potential liabilities and can result in recommendations to minimize these risks during lead optimization as well as understand their relevance in provoking clinical Relative cell count Relative cell count Phospholipid induction Neutral lipid induction hepatotoxicity. Development of an effective in vitro cell-based screening model to assess human hepatotoxicity potential of drugs requires Compound IC50, (microM) EC50, (microM) (microM) (microM) the use of multiplexed technologies that utilize human hepatocytes and measure parameters at the single cell level, morphological and biochemical, investigative of pre-lethal cytotoxic effects, representative of different mechanisms of toxicity and suitable for rapid throughput. Propranolol > 100 > 100 11.42 ± 1.13 95.43 ± 5.79 In this study we used multiplexed high content screening (HCS) with automated fluorescence microscopy and image analysis based technology (GE Healthcare INCell Analyzer 1000) to develop mechanistic cellular assays for assessment of hepatotoxicity in HepG2 cells and multiplexed Cyclosporin A 19.70 ± 5.66 8.73 ± 0.52 15.19 ± 1.10 9.40 ± 0.86 sandwich immunoassays based on flowmetric Luminex xMap™ technology to measure inflammatory markers in these same cells. These in vitro cell-based assays provide a robust and rapid screening system for testing compound effects on cell proliferation, apoptosis, cell Vehicle Vinblastine cycle, steatosis, phospholipidosis and cytokine secretion and other inflammatory markers. This cost-effective extensive multiplexed platform Vehicle Propranolol Erythromycin > 100 > 100 35.10 ± 9.98 — for predictive assessments delivers a more sensitive approach to detection of end-point-specific drug hepatotoxicities. Use of primary Hepatotoxicity Assay, representative images are shown. Labels: Nuclei - green; Apoptotic cells - blue; hepatocytes as the cell source for these assays is in development. Mitotic cells - red The relative cell count IC50 (half maximal inhibitory constant) and EC50 (half maximal effective constant) values measure cell proliferation. Compound concentrations are indicative of 5-fold phospholipid and neutral lipid induction over vehicle background. All values are given as the mean ± s.e.m. Ce l l Pr ol i f e r at i on Apopt osi s I n d u c t i o n Ce l l C y c l e B l o c k METHODS MULTIPLEXED HEPATO-CYTOKINE SECRETION ASSAY 160 100 6 Cell Culture Human hepatocellular carcinoma cell line (HepG2) was grown in MEM, 10% FBS, 1% NEAA, 1% Alanyl-L-Glutamine and 1% Percent of Control over Background over Background 140 Fold Induction Fold Induction 80 120 sodium pyruvate in tissue culture flasks, 75 mm2 and 225 mm2, in a humidified atmosphere of 5% CO2 at 37oC. Working stocks of log-phase 100 80 60 4 HepG2 cells were cryo-preserved using a standard slow cooling in 10% DMSO protocol. Cells were thawed from working stocks and either 60 40 2 A summary of detected cytokine secretion measured in HepG2 cells, (n =3). HepG2 cells were treated with LPS, TNFα, IL-1β and acetaminophen 40 passage once prior to seeding into plates or directly seeded into plates from cryo-preserved stocks. 20 20 and screened for the secretory presence of 30 human inflammatory markers including IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p40, IL- 0 -10 -9 -8 -7 -6 -5 -4 -3 0 -10 -9 -8 -7 -6 -5 -4 -3 0 -10 -9 -8 -7 -6 -5 -4 -3 Vehicle Erythromycin 12p70, IL-13, INFγ, INFα2a, IP-10, GM-CSF, G-CSF, MCP-1, MIP-1α, MIP-1β, TNFα, IL-1 receptor antagonist, Fibrinogen, CRP, Haptoglobin, SAA, Multiplexed Hepatotoxicity Assay Caspase-3 activation, a marker of apoptosis, phospho-histone-3, a marker of mitosis, and [Cyclosporin A], M [Cyclosporin A], M [Cyclosporin A], M Apo AI, Apo AII, Apo B, Apo CII, Apo CIII and Apo E. nuclear count, an index of cell proliferation, were measured. HepG2 cells were seeded at 2x103 cells per well with complete 160 100 6 Hepato-Phospholipid Accumulation Assay, representative images are shown. Labels: Nuclei - green; growth media into 384-well Collagen I coated optical plates (BD Sciences) and incubated in a humidified atmosphere of 5% Percent of Control over Background over Background Phospholipids - red 140 Fold Induction Fold Induction 5 80 120 CO2 at 37oC . Test compounds were serially diluted 3-fold over 10 concentrations and added 24 hours post cell seeding with 100 60 4 α α a final assay concentration of 0.5% DMSO. Following an additional 72 hour incubation in the humidified atmosphere of 5% 80 3 60 40 2 Cell Proliferation Phospholipid Induction Neutral Lipid Induction [LPS] µg/ml: where CO2 at 37oC, cells were fixed and immunolabeled with anti-active caspase-3 for detection of apoptosis and anti-phospho- 40 20 20 1 Cell Proliferation Phospholipid Induction Neutral Lipid Induction cytokine secretion _ _ _ _ _ _ histone-3 for detection of cell cycle and stained with a nuclei dye for cell proliferation quantification. Automated fluorescence 3-fold over background 120 260 60 0 0 0 240 (30 - 0.01 µg/ml) Percent of Control over Background over Background -10 -9 -8 -7 -6 -5 -4 -3 -10 -9 -8 -7 -6 -5 -4 -3 -10 -9 -8 -7 -6 -5 -4 -3 120 100 260 220 60 50 Maximum [secreted cytokine], Fold Induction Fold Induction 240 microscopy was carried out using a GE Healthcare INCell Analyzer 1000, and images were collected with a 4X objective. 200 _ _ _ _ _ _ Percent of Control [Propranolol], M [Propranolol], M [Propranolol], M over Background over Background 180 00 220 180 50 Fold Induction Fold Induction 40 80 60 200 160 1 80 140 40 30 pg/ml [TNFα] ng/ml: where 160 120 60 140 100 40 30 20 Multiplexed Hepato-Lipid Accumulation Assay Intracellular phospholipids, a marker of phospholipidosis, intracellular neutral 180 20 cytokine secretion 0.45 ± 0.08 0.29 ± 0.04 0.40 ± 0.05 0.110 ± 0.01 _ _ α 160 10 0 6 160 00 40 20 20 10 80 40 Percent of Control over Background over Background 140 lipid, a marker of steatosis, and nuclear count, an index of cell proliferation, were measured. HepG2 cells were seeded at 60 5 20 3-fold over background Fold Induction Fold Induction 80 20 0 10 (90 - 0.04 ng/ml) 120 40 0 0 4 -8 -7 -6 -5 -4 -3 20 -8 -7 -6 -5 -4 -3 -8 -7 -6 -5 -4 -3 100 0 0 0 Maximum [secreted cytokine], 4x103 cells per well with complete growth media into 384-well Collagen I coated optical plates (BD Sciences) and incubated in 60 80 3 -8 -7 [Pro6 ranolol], M -p -5 -4 -3 -8 -7 [Pro6 ranolol], M -p -5 -4 -3 -8 -7 [Pro6 ranolol], M -p -5 -4 -3 10.64 ± 1.75 6454 ± 458 26.19 ± 2.53 87.83 ± 8.31 _ 5955 ± 29 60 40 [Propranolol], M [Propranolol], M [Propranolol], M pg/ml a humidified atmosphere of 5% CO2 at 37oC for 24 hours. For detection of phospholipid accumulation a fluorescently-labeled 2 [IL-1β] ng/ml: where 40 20 1 20 phospholipid (Invitrogen, H34350) was added to the cells with test compounds serially diluted 3-fold over 10 concentrations cytokine secretion 0.016 ± 0.003 < 0.04 _ 0.002 ± 0.000 0.81 ± 0.45 0.15 ± 0.05 β 0 0 0 -12 -11 -10 -9 -8 -7 -6 -5 -12 -11 -10 -9 -8 -7 -6 -5 -12 -11 -10 -9 -8 -7 -6 -5 120 260 60 240 3-fold over background Percent of Control in a final assay concentration of 0.5% DMSO. Following an additional 48 hour incubation in a humidified atmosphere of 5% over Background over Background [Staurosporine], M [Staurosporine], M [Staurosporine], M 120 100 260 220 60 50 Fold Induction Fold Induction 240 200 (90 - 0.04 ng/ml) Percent of Control over Background over Background 00 180 220 180 50 Maximum [secreted cytokine], Fold Induction Fold Induction 40 CO2 at 37oC, cells were fixed and stained with a neutral lipid dye (Invitrogen, H34476) for neutral lipid detection and a nuclei 16.78 ± 0.78 5039 ± 39 _ 259 ± 12 6.64 ± 1.11 8.85 ± 1.29 200 160 80 180 140 pg/ml 60 40 30 160 120 dye for cell proliferation quantification. Automated fluorescence microscopy was carried out using a GE Healthcare INCell 60 140 100 40 30 160 100 6 40 20 120 80 100 60 20 20 [Acetam.] mM: where Analyzer 1000, and images were collected with a 4X objective. 80 40 10 cytokine secretion _ _ _ _ _ _ Percent of Control over Background over Background 140 60 Fold Induction Fold Induction 80 20 120 0 20 40 0 10 0 100 60 4 0 -8 -7 -6 -5 -4 -3 20 -8 0 -7 -6 -5 -4 -3 0 -8 -7 -6 -5 -4 -3 (1 mM - 450 nM) 3-fold over background [Eryt6 romyc5n], M -4 -h -i [Eryt6 romyc5n], M -4 -h -i [Eryt6 romyc5n], M -4 -h -i 80 -8 -7 -3 -8 -7 -3 -8 -7 -3 Maximum [secreted cytokine], Multiplexed Hepato-Cytokine Secretion Assay Cytokine secretion, as markers of inflammation, and nuclear count, as an index of cell 60 40 40 2 [Erythromycin], M [Erythromycin], M [Erythromycin], M pg/ml _ _ _ _ _ _ proliferation, were measured in HepG2 cells. HepG2 cells were seeded at 8x103 cells per well with complete growth media into 96-well 20 20 0 0 0 Collagen I coated optical plates (BD Sciences) and incubated in a humidified atmosphere of 5% CO2 at 37oC for 24 hours. Cells were treated -13 -12 -11 -10 [Vinblastine], M -9 -8 -7 -6 -13 -12 -11 -10 [Vinblastine], M -9 -8 -7 -6 -13 -12 -11 -10 [Vinblastine], M -9 -8 -7 -6 Hepato-Phospholipid Accumulation Assay, representative curves for cell proliferation and phospholipid and Concentrations for cytokine induction and secretion are given as the mean ± s.e.m. Lipopolysaccharide (LPS), Tumor necrosis factor (TNF), Interleukin (IL), Interferon with LPS (30 µg/ml), TNFα (90 ng/ml), IL-1β (90 ng/ml) and acetaminophen (1 mM) serially diluted 3-fold over 8 concentrations. Final assay neutral lipid accumulation are shown. (INF), Interferon-gamma-inducible protein (IP), Granulocyte-macrophage-colony-stimulating factor (GM-CSF), Granulocyte colony-stimulating factor (G-CSF), Monocyte chemoattractant protein (MCP ), Macrophage inflammatory protein (MIP), C-Reactive Protein (CRP), Serum Amyloid A (SAA), Apolipoprotein (Apo) concentration of DMSO for acetaminophen was 0.05%. Following an additional 48 hour incubation in a humidified atmosphere of 5% CO2 at 37oC, supernatants were collected and cytokine detection was carried out using multiplexed sandwich immunoassays based on Luminex 160 100 6 Percent of Control 140 Hepato-Neutral Lipid Accumulation Assay over Background over Background xMAP™ technology. To quantify cell proliferation, the monolayer of HepG2 cells remaining in each plate was immediately stained with Fold Induction Fold Induction 80 120 4 CONCLUSION 100 Hoechst nuclear dye for normalization, and incubated 15 min at 37°C. Images were collected using a GE Healthcare INCell Analyzer 1000 60 80 60 40 with a 10X objective. 40 20 2 20 0 0 0 Identification of drug-induced hepatotoxic potential early in the drug development cascade can create opportunities for ranking and prioritizing, Data Analysis For HCS 12 bit tiff images were acquired using the INCell Analyzer 1000 3.2 and analyzed with Developer Toolbox 1.6 software. -10 -9 -8 -7 -6 -5 -4 -3 -10 -9 -8 -7 -6 -5 -4 -3 -10 -9 -8 -7 -6 -5 -4 -3 [Erythromycin], M [Erythromycin], M [Erythromycin], M or developing alternatives with lower toxicity. We have developed a robust and rapid throughput screening system using HepG2 cells that EC50 and IC50 values were calculated using nonlinear regression to fit data to a sigmoidal 4 point, 4 parameter One-Site dose response allows early assessment of acute and chronic mechanisms of hepatotoxicity. Compounds with known hepatotoxicities were tested to model, where: y (fit) = A + [(B – A)/(1 + (C/x) ^ D))]. Cytokine and inducer concentrations were interpolated from standard curves obtained validate the capabilities of this multiparametric HCS system in identifying and quantifying toxicities relevant to cell proliferation, apoptosis, by 5 point parameter sigmoidal nonlinear regression analysis. Curve-fitting and EC50 and IC50 calculations were performed using XLFit™ Hepatotoxicity Assay, representative curves for cell proliferation, apoptosis induction, and cell cycle block are shown. cell cycle, steatosis, phospholipidosis. High concordance was found with the reported hepatotoxic profile for each compound tested. software (IDBS). Curves were generated with GraphPad Prism™ 3 software using a sigmoidal dose-response, variable slope model. Further, we evaluated cytokine secretion in HepG2 cells to identify measurable biomarkers of inflammation. Significant secretion levels for 6 of the cytokines tested were confirmed thus validating this multiplexed approach for quantifying indications of hepatic inflammation. Measured Parameters Cell proliferation was measured by the signal intensity of the incorporated nuclear dye. The cell proliferation output A summary of Hepatotoxicity Assay parameters for each compound tested, (n = 3). Vehicle Cyclosporin A These hepatotoxicity screening assays are sensitive and reproducible and provide results that previously only have been attainable in was referred to as the relative cell count. To determine the cell proliferation end point, the cell proliferation data output was transformed more complex in vivo models. Our cost-effective in vitro multiplexed HCS platform offers comprehensive predictive information allowing to percent of control (POC) using the following formula: Inhibition of Relative cell count Relative cell count Apoptosis Mitosis pre-selection of drug scaffold designs with long-term hepatotoxicity considerations and may even have more relevance when performed POC = relative cell count (compound wells) x100 mitosis (G1/S cell Hepato-Neutral Lipid Accumulation Assay, representative images are shown. Labels: Nuclei - green; in normal primary hepatocytes. Compound IC50 EC50 induction cell cycle block relative cell count (vehicle wells) cycle block) Neutral lipids - red. (microM) (microM) (microM) (microM) (microM) Relative cell count IC50 is the test compound concentration that produces 50% of the cell proliferation inhibitory response or 50% cytotoxicity Propranolol 62.81 ± 5.23 61.38 ± 5.38 55.28 ± 5.43 — — Cell Proliferation Phospholipid Induction Neutral Lipid Induction level. A relative cell count EC50 is the test compound concentration that produces 50% of the maximum effective response that occurs at the curve inflection point. The output of each biomarker is fold increase over vehicle background normalized to the relative cell count in each Staurosporine 0.036 ± 0.005 0.027 ± 0.003 0.214 ± 0.019 — 0.144 ± 0.018 Cell Proliferation Phospholipid Induction Neutral Lipid Induction REFERENCES well. Concentrations of test compound that cause a 5 or more fold induction in the caspase-3 signal were determined to induce significant Cyclosporin A 7.71 ± 0.76 5.43 ± 0.30 9.99 ± 0.29 — — 120 260 60 1. Dambach DM, Andrews BA, Moulin F. New technologies and screening srategies for hepatotoxicity: use on in vitro models. Toxicologic apoptotic induction. When the fold induction of the phospho-histone-3 signal over background is ~1, there was “no effect” on the cell 240 Percent of Control over Background over Background Vinblastine 0.002 ± 0.000 0.002 ± 0.000 0.003 ± 0.001 0.002 ± 0.000 — 120 260 220 60 Pathology, 2005;33:17-26. Fold Induction Fold Induction 100 240 Percent of Control 200 over Background over Background cycle. Two or more fold increase in phospho-histone-3 signal over vehicle background indicated significant test compound induction of 220 Fold Induction Fold Induction 180 00 180 40 2. Anderson N, Borlak J. Drug-induced phospholipidosis. FEBS Lett. 2006;580(23):5533-40. 260 1 00 mitotic block. Two or more fold decrease in the phospho-histone-3 signal may indicate G1/S block only when cytotoxicity levels are below Erythromycin > 100 > 100 — — — 80 60 180 140 160 120 140 100 40 3. Nioi P, Perry BK, Wang E, et al. In vitro detection of drug-induced phospholipidosis using gene expression and fluorescent phospholipid- 60 40 180 20 20 the measured relative cell count IC80. When 2 or more fold decrease in the phospho-histone-3 signal are observed at concentrations higher 40 20 160 00 80 40 20 than the relative cell count nuclear IC80, the decrease in mitotic cell counts are most likely due to a more general cytotoxicity effect rather 0 20 60 20 40 0 0 based methodologies. Toxicology Sciences, 2007;99(1):162-173. The relative cell count IC50 (half maximal inhibitory constant) and EC50 (half maximal effective constant) values measure cell -8 -7 -6 -5 -4 -3 20 -8 -7 -6 -5 -4 -3 -8 -7 -6 -5 -4 -3 4. Stonans I, Stonane E, RuBwurm S, et al. HepG2 human hepatoma cells express multiple cytokine genes. Cytokines, 1999;11(2):151-6. 0 0 0 than a true G1/S phase block. Wells with concentrations higher than the relative cell count nuclear IC80 are eliminated from the phospho- proliferation. Compound concentrations are indicative of a 5-fold apoptosis induction in activated caspase-3 signal and a -8 -7 [Cyc-l6 spori-5 A], M -4 o n [Cyclosporin A], M -3 -8 -7 [Cyc-l6 spori-5 A], M -4 o n [Cyclosporin A], M -3 -8 -7 [Cyc-l6 spori-5 A], M -4 o n [Cyclosporin A], M -3 histone-3 analysis. Concentrations of test compound that cause a 5 or more fold induction of the labeled-phospholipid signal represent 2-fold change in phospho-histone 3 signal (mitosis marker) over vehicle background. All values are given as the mean ± s.e.m. significant phospholipidosis induction. Concentrations of test compound which cause a 5 or more fold induction of the neutral lipid signal Hepato-Neutral Lipid Accumulation Assay, representative curves for cell proliferation, phospholipid and represent significant steatosis induction. Three or more fold increase in cytokine detection represents significant cytokine secretion. neutral lipid accumulation are shown.