SCREENING FOR MECHANISMS OF HEPATOTOXICITY: PHOSPHOLIPIDOSIS, STEATOSIS,
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Hepatoxicity Sot 2008 Poster

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Hepatoxicity Sot 2008 Poster

  1. 1. 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

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