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Hepatotoxicity Screening Sot Poster 2008 2009

O
ovechkina

Screening for Mechanisms of Hepatotoxicity: Phospholipidosis, Steatosis, Apoptosis and Inflammatory Markers

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Screening for Mechanisms of Hepatotoxicity: Phospholipidosis, Steatosis, Apoptosis and Inflammatory Markers K.F. Marcoe, R. Keyser, P. TB. Nguyen, Yulia Ovechkina, and C. ODay MDS Pharma Services Bothell, WA, USA Multiparametric Hepatotoxicity Screening in HepG2 Cells with Comparison in Primary Hepatocytes K.F. Marcoe, Yulia Ovechkina, R. Keyser, P. TB. Nguyen, C. ODay MDS Pharma Services Bothell, WA, USA
Drug-Induce Hepatotoxicity Liver major site of metabolism for most drugs Based on safety, hepatotoxicity recognized as a leading cause for drug withdrawal Toxicity of new drug candidates routinely evaluated just prior to compounds moving into clinical trial Late stage In vivo toxicity studies have problems Costly (multiple animal species requirements) Large amounts of compounds Significant investment of resources tied to late findings In vitro early stage toxicity studies afford Identification of hepatotoxic potential earlier (cost and time savings) Opportunities for ranking and prioritizing or development of alternatives with lower toxicity Multiparameter high content cell-based screening methods in drug discovery contribute to better predictivity of human hepatotoxicity potential Early safety screening current priority in drug development
Early Safety Hepatotoxicity Screening Assays Development of effective in vitro cell-based screening models to assess human hepatotoxicity potential of drugs ideally requires: Use of high content multiplexed technologies Utilization of primary human cell and HepG2 cell line hepatocyte models Measurement of parameters At the single cell level Morphological and biochemical Investigative of pre-lethal cytotoxic effects Representative of different mechanisms of toxicity Suitable for rapid throughput 384 well plate format Minimal amount of compound for testing (1 - 2 mg)
Multiplexed High Content Screening Tools IN Cell 1000 Analyzer automated fluorescent microscopy imaging of live or fixed cells allows Subcellular localization AND quantitation of the cellular targets Multiplexing capabilities: multiple data points from a single assay well High sensitivity (nuclear staining allows for normalization of cellular signals against cell number) Measurement of individual cell responses in the heterogeneous cell populations Customized protocols for cell image quantitation (IN Cell Developer Software) xMAP technology using Luminex Flow based multiplexed microsphere assay system Multi-analyte protein analysis in the same well Nuclei staining with IN Cell imaging allows normalization of cellular signals against cell number
Multiplexed High Content Screening Hepatotoxicity Early Safety Platform HCS Hepatotoxicity Early Safety Platform Hepato-toxicity (cell proliferation, apoptosis, mitosis) Hepato-Lipid Accumulation (cell proliferation, phospholipidosis, neutral lipids) Hepato-Cytokine Secretion (cell proliferation, inflammatory markers)
Multiplexed In vitro Hepatotoxicity Assay In vitro hepatotoxicity assessment Cultured HepG2 cells (human hepatocellular carcinoma cell line) useful screening reagent Evaluation of toxicity window / safety margin and mechanism of death helps determine dosing and cost/benefit analysis of therapeutic agent based on prediction of in vivo toxicity potential In vitro cell-based safety margin = cytotoxic concentration on-target potency concentration (cell-based efficacy) Higher values predict higher in vivo safety margins In vitro cell-base safety margins use to rank compounds based on hepatotoxicity potential in humans 80% correlation between actual in vivo and in vitro cell-based toxicity results have been demonstrated (Shrivastava R, et al., OBrien PJ, et al., Vivek C, et al.) Other factors contributing to toxicity profiles: drug properties, concentrations, protein binding and transport, pharmacokinetic characteristics Provides information on the relative toxicities of candidate drugs within particular compound families to aid selection of lead candidates. Offers insight into drug toxicity mechanism Provides end-point-specific drug hepatotoxicities
Multiplexed In vitro Hepatotoxicity Assay Multiplexed Hepatotoxicity Assay HepG2 cells seeded in 384-well Collagen I coated optical plates, incubated 24 hrs Cells incubated 72 hrs with test compounds serially diluted 遜 log over 10 concentrations Post 72 hrs incubation cells fixed and immunolabeled with: Anti-active Caspase-3 for detection of apoptosis Anti-phospho-Histone-3 for detection of cell cycle Stained with a nuclear dye for cell proliferation quantification Automated fluorescence microscopy carried out using a GE Healthcare IN Cell Analyzer 1000 Images collected with a 4X objective
Multiplexed In vitro Hepatotoxicity Assay Vehicle Vinblastine Labels: Nuclei - green; Apoptotic cells - blue; Mitotic cells - red Cell Proliferation Apoptosis Induction Cell Cycle Block Percent of Control over Background over Background Fold Induction 160 100 6 Fold Induction 140 80 120 100 60 4 80 60 40 2 40 20 20 0 0 -13 -12 -11 -10 -9 -8 -7 -6 -13 -12 -11 -10 -9 -8 -7 -6 0 -13 -12 -11 -10 -9 -8 -7 -6 [Vinblastine], M [Vinblastine], M [Vinblastine], M HepG2, 72 hr assay
Primary human hepatotoxicity assay: MTS and HCS comparison Amiodarone Valproic acid Amitriplyline 100 160 120 140 100 80 120 MTS 80 100 POC POC POC 60 80 60 40 60 40 40 20 20 20 0 0 0 0.01 1 100 0.01 1 100 0.0 1 100 Concentration microM Concentration microM 1 Concentration microM HCS 24 hour compound treatment of human primary hepatocytes; High Content Screening approach (HCS)
Primary human hepatotoxicity assay: MTS and HCS comparison HCS % of attached MTS Viability IC50 Compound live cells IC50 (microM) (microM) Tamoxifen 36.7 賊 5.6 19.8 賊 0.7 Chlorpramazine 28.0 賊 6.9 26.4 賊 1.8 Amitriplyline 49.39 賊 4.06 62.3 賊 4.2 Amiodarone 125 賊 10 146 賊 22 Valproic acid >500 >500 Astemizole 6.39 賊 2.38 12.2 賊 2.1 Rosiglitazone 354 賊 133 413 賊 80 Troglitzqone 157 賊 6 122 賊 42 24 hour compound treatment of human primary hepatocytes; High Content Screening approach (HCS)
Multiplexed In vitro Hepato-Lipid Accumulation Assay In vitro hepato-lipid accumulation assessment Cultured HepG2 cells (human hepatocellular carcinoma cell line) Phospholipidosis accumulation of excess phospholipids in cells Cationic amphiphilic drugs often induce phospholipidosis in vivo Toxic effect due to drug or metabolite accumulation in affected tissue, leads to acute and chronic disease Liver and lung common targets Neutral lipid accumulation Steatosis accumulation of fatty acids Other mechanisms of lipid accumulation Can cause enlargement of the liver and irreversible cell damage Flags drug candidate hepatotoxicity potential in the lead optimization stage of drug discovery End-point-specific drug-induced mechanism of hepatotoxicity
Multiplexed In vitro Hepato-Lipid Accumulation Assay Multiplexed Hepato-Lipid Accumulation Assay HepG2 cells seeded in 384-well Collagen I coated optical plates, incubated 24 hrs Cells incubated for 48 hrs with Fluorescently-labeled phospholipid (Invitrogen, H34350) for phospholipid accumulation detection Test compounds serially diluted 遜 log over 10 concentrations Post 48 hrs incubation cells fixed and stained with Neutral lipid dye (Invitrogen, H34476) for neutral lipid detection Nuclear dye for cell proliferation quantification Automated fluorescence microscopy carried out using a GE Healthcare INCell Analyzer 1000 Images were collected with a 4X objective.
Multiplexed In vitro Hepato-Lipid Accumulation Assay (HepG2) HepG2, 48 hr assay
Multiplexed In vitro Hepato-Lipid Accumulation Assay (HepG2) Hepato-Neutral Lipid Accumulation Assay Labels: Nuclei - green; Neutral lipids - red HepG2, 48 hr assay
Multiplexed In vitro Hepato-Lipid Accumulation Assay (HepG2) HepG2, 48 hr assay
In vitro Hepato-Lipid Accumulation Assay using primary human hepatocytes in 384 WP
In vitro Hepato-Lipid Accumulation Assay using primary human hepatocytes in 384 WP Amiodarone Amitriplyline
Multiplexed In vitro Hepato-Cytokine Secretion Assay Multiplexed Hepato-Cytokine Secretion Assay IN Cell xMAP Automated technology fluorescent using microscopy Luminex imaging Markers of cell count inflammation normalization
xMAP technology-Multiple Analytes /Well Multiplexing: Up to 100 analytes/well Analytes cytokines or other inflammatory markers Flow based assay system. Uses beads loaded with different concentrations of 2 dyes. Each bead has its own unique spectral signature (100 possible), antibodies are derivitized to unique bead Beads are incubated with test sample Sandwich assay performed with a biotinylated second antibody (mouse) Streptavidin labeled with phycoerythrin (PE) used for detection Beads are run individually (Flow) through a laser which detects the exact bead and then determines whether PE is associated
Multiplexed In vitro Hepato-Cytokine Secretion Assay (HepG2) Multiplexed Hepato-Cytokine Secretion Assay Biomarker secretion, as markers of inflammation Nuclear count, analyte normalization to cell number HepG2 cells seeded into 96-well Collagen I coated optical plates incubated 24 hrs Cells treated with LPS, TNF留, IL-1硫 and acetaminophen serially diluted 遜 log over 8 concentrations incubated 48 hrs Post 48 hrs incubation supernatants collected, cytokine detection was carried out using Luminex xMAP technology To quantify cell proliferation the monolayer of HepG2 cells remaining in each plate was immediately stained with nuclear dye for normalization Images were collected using a GE Healthcare INCell Analyzer 1000
Multiplexed In vitro Hepato-Cytokine Secretion Assay HepG2 cells treated with LPS, TNF留, IL-1硫 and acetaminophen HepG2 cells Screened for the secretory presence of 30 human inflammatory markers: LPS, TNF留, IL-1硫 and acetaminophen IL-1留, IL-1硫, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p40, IL- 12-70, IL-13, INF粒, INF留2a, Fibrinogen, Apo AI, Apo AII, Apo B, IP-10, GM-CSF, G-CSF, CRP, Haptoglobin, Apo CII, Apo CIII and MCP-1, MIP-1留, MIP-1硫, SAA Apo E TNF留, IL-1 receptor antagonist
Multiplexed In vitro Hepato-Cytokine Secretion Assay (HepG2)
Early Safety Screening for Mechanisms of Hepatotoxicity Conclusion: We have developed a robust and rapid throughput screening system using HepG2 cells that allows early assessment of acute and chronic mechanisms of hepatotoxicity Compounds with known hepatotoxicities tested in validating the capabilities of this multiparametric HCS system in identifying and quantifying toxicities relevant to cell proliferation, apoptosis, cell cycle, steatosis/cholestasis and phospholipidosis demonstrated high concordance with reported hepatotoxic profile for each compound tested Evaluation of cytokine secretion in HepG2 cells to identify measurable biomarkers of inflammation demonstrated significant secretion levels for 6 of the cytokines tested thus validating this multiplexed approach for quantifying indications of hepatic inflammation These hepatotoxicity screening assays are sensitive and reproducible and provide results that previously only have been attainable in more complex in vivo models Our cost-effective in vitro multiplexed HCS platform offers comprehensive predictive information allowing pre-selection of drug scaffold designs with long-term hepatotoxicity considerations and may even have more relevance when performed in normal primary hepatocytes

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Hepatotoxicity Screening Sot Poster 2008 2009

  • 1. Screening for Mechanisms of Hepatotoxicity: Phospholipidosis, Steatosis, Apoptosis and Inflammatory Markers K.F. Marcoe, R. Keyser, P. TB. Nguyen, Yulia Ovechkina, and C. ODay MDS Pharma Services Bothell, WA, USA Multiparametric Hepatotoxicity Screening in HepG2 Cells with Comparison in Primary Hepatocytes K.F. Marcoe, Yulia Ovechkina, R. Keyser, P. TB. Nguyen, C. ODay MDS Pharma Services Bothell, WA, USA
  • 2. Drug-Induce Hepatotoxicity Liver major site of metabolism for most drugs Based on safety, hepatotoxicity recognized as a leading cause for drug withdrawal Toxicity of new drug candidates routinely evaluated just prior to compounds moving into clinical trial Late stage In vivo toxicity studies have problems Costly (multiple animal species requirements) Large amounts of compounds Significant investment of resources tied to late findings In vitro early stage toxicity studies afford Identification of hepatotoxic potential earlier (cost and time savings) Opportunities for ranking and prioritizing or development of alternatives with lower toxicity Multiparameter high content cell-based screening methods in drug discovery contribute to better predictivity of human hepatotoxicity potential Early safety screening current priority in drug development
  • 3. Early Safety Hepatotoxicity Screening Assays Development of effective in vitro cell-based screening models to assess human hepatotoxicity potential of drugs ideally requires: Use of high content multiplexed technologies Utilization of primary human cell and HepG2 cell line hepatocyte models Measurement of parameters At the single cell level Morphological and biochemical Investigative of pre-lethal cytotoxic effects Representative of different mechanisms of toxicity Suitable for rapid throughput 384 well plate format Minimal amount of compound for testing (1 - 2 mg)
  • 4. Multiplexed High Content Screening Tools IN Cell 1000 Analyzer automated fluorescent microscopy imaging of live or fixed cells allows Subcellular localization AND quantitation of the cellular targets Multiplexing capabilities: multiple data points from a single assay well High sensitivity (nuclear staining allows for normalization of cellular signals against cell number) Measurement of individual cell responses in the heterogeneous cell populations Customized protocols for cell image quantitation (IN Cell Developer Software) xMAP technology using Luminex Flow based multiplexed microsphere assay system Multi-analyte protein analysis in the same well Nuclei staining with IN Cell imaging allows normalization of cellular signals against cell number
  • 5. Multiplexed High Content Screening Hepatotoxicity Early Safety Platform HCS Hepatotoxicity Early Safety Platform Hepato-toxicity (cell proliferation, apoptosis, mitosis) Hepato-Lipid Accumulation (cell proliferation, phospholipidosis, neutral lipids) Hepato-Cytokine Secretion (cell proliferation, inflammatory markers)
  • 6. Multiplexed In vitro Hepatotoxicity Assay In vitro hepatotoxicity assessment Cultured HepG2 cells (human hepatocellular carcinoma cell line) useful screening reagent Evaluation of toxicity window / safety margin and mechanism of death helps determine dosing and cost/benefit analysis of therapeutic agent based on prediction of in vivo toxicity potential In vitro cell-based safety margin = cytotoxic concentration on-target potency concentration (cell-based efficacy) Higher values predict higher in vivo safety margins In vitro cell-base safety margins use to rank compounds based on hepatotoxicity potential in humans 80% correlation between actual in vivo and in vitro cell-based toxicity results have been demonstrated (Shrivastava R, et al., OBrien PJ, et al., Vivek C, et al.) Other factors contributing to toxicity profiles: drug properties, concentrations, protein binding and transport, pharmacokinetic characteristics Provides information on the relative toxicities of candidate drugs within particular compound families to aid selection of lead candidates. Offers insight into drug toxicity mechanism Provides end-point-specific drug hepatotoxicities
  • 7. Multiplexed In vitro Hepatotoxicity Assay Multiplexed Hepatotoxicity Assay HepG2 cells seeded in 384-well Collagen I coated optical plates, incubated 24 hrs Cells incubated 72 hrs with test compounds serially diluted 遜 log over 10 concentrations Post 72 hrs incubation cells fixed and immunolabeled with: Anti-active Caspase-3 for detection of apoptosis Anti-phospho-Histone-3 for detection of cell cycle Stained with a nuclear dye for cell proliferation quantification Automated fluorescence microscopy carried out using a GE Healthcare IN Cell Analyzer 1000 Images collected with a 4X objective
  • 8. Multiplexed In vitro Hepatotoxicity Assay Vehicle Vinblastine Labels: Nuclei - green; Apoptotic cells - blue; Mitotic cells - red Cell Proliferation Apoptosis Induction Cell Cycle Block Percent of Control over Background over Background Fold Induction 160 100 6 Fold Induction 140 80 120 100 60 4 80 60 40 2 40 20 20 0 0 -13 -12 -11 -10 -9 -8 -7 -6 -13 -12 -11 -10 -9 -8 -7 -6 0 -13 -12 -11 -10 -9 -8 -7 -6 [Vinblastine], M [Vinblastine], M [Vinblastine], M HepG2, 72 hr assay
  • 9. Primary human hepatotoxicity assay: MTS and HCS comparison Amiodarone Valproic acid Amitriplyline 100 160 120 140 100 80 120 MTS 80 100 POC POC POC 60 80 60 40 60 40 40 20 20 20 0 0 0 0.01 1 100 0.01 1 100 0.0 1 100 Concentration microM Concentration microM 1 Concentration microM HCS 24 hour compound treatment of human primary hepatocytes; High Content Screening approach (HCS)
  • 10. Primary human hepatotoxicity assay: MTS and HCS comparison HCS % of attached MTS Viability IC50 Compound live cells IC50 (microM) (microM) Tamoxifen 36.7 賊 5.6 19.8 賊 0.7 Chlorpramazine 28.0 賊 6.9 26.4 賊 1.8 Amitriplyline 49.39 賊 4.06 62.3 賊 4.2 Amiodarone 125 賊 10 146 賊 22 Valproic acid >500 >500 Astemizole 6.39 賊 2.38 12.2 賊 2.1 Rosiglitazone 354 賊 133 413 賊 80 Troglitzqone 157 賊 6 122 賊 42 24 hour compound treatment of human primary hepatocytes; High Content Screening approach (HCS)
  • 11. Multiplexed In vitro Hepato-Lipid Accumulation Assay In vitro hepato-lipid accumulation assessment Cultured HepG2 cells (human hepatocellular carcinoma cell line) Phospholipidosis accumulation of excess phospholipids in cells Cationic amphiphilic drugs often induce phospholipidosis in vivo Toxic effect due to drug or metabolite accumulation in affected tissue, leads to acute and chronic disease Liver and lung common targets Neutral lipid accumulation Steatosis accumulation of fatty acids Other mechanisms of lipid accumulation Can cause enlargement of the liver and irreversible cell damage Flags drug candidate hepatotoxicity potential in the lead optimization stage of drug discovery End-point-specific drug-induced mechanism of hepatotoxicity
  • 12. Multiplexed In vitro Hepato-Lipid Accumulation Assay Multiplexed Hepato-Lipid Accumulation Assay HepG2 cells seeded in 384-well Collagen I coated optical plates, incubated 24 hrs Cells incubated for 48 hrs with Fluorescently-labeled phospholipid (Invitrogen, H34350) for phospholipid accumulation detection Test compounds serially diluted 遜 log over 10 concentrations Post 48 hrs incubation cells fixed and stained with Neutral lipid dye (Invitrogen, H34476) for neutral lipid detection Nuclear dye for cell proliferation quantification Automated fluorescence microscopy carried out using a GE Healthcare INCell Analyzer 1000 Images were collected with a 4X objective.
  • 13. Multiplexed In vitro Hepato-Lipid Accumulation Assay (HepG2) HepG2, 48 hr assay
  • 14. Multiplexed In vitro Hepato-Lipid Accumulation Assay (HepG2) Hepato-Neutral Lipid Accumulation Assay Labels: Nuclei - green; Neutral lipids - red HepG2, 48 hr assay
  • 15. Multiplexed In vitro Hepato-Lipid Accumulation Assay (HepG2) HepG2, 48 hr assay
  • 16. In vitro Hepato-Lipid Accumulation Assay using primary human hepatocytes in 384 WP
  • 17. In vitro Hepato-Lipid Accumulation Assay using primary human hepatocytes in 384 WP Amiodarone Amitriplyline
  • 18. Multiplexed In vitro Hepato-Cytokine Secretion Assay Multiplexed Hepato-Cytokine Secretion Assay IN Cell xMAP Automated technology fluorescent using microscopy Luminex imaging Markers of cell count inflammation normalization
  • 19. xMAP technology-Multiple Analytes /Well Multiplexing: Up to 100 analytes/well Analytes cytokines or other inflammatory markers Flow based assay system. Uses beads loaded with different concentrations of 2 dyes. Each bead has its own unique spectral signature (100 possible), antibodies are derivitized to unique bead Beads are incubated with test sample Sandwich assay performed with a biotinylated second antibody (mouse) Streptavidin labeled with phycoerythrin (PE) used for detection Beads are run individually (Flow) through a laser which detects the exact bead and then determines whether PE is associated
  • 20. Multiplexed In vitro Hepato-Cytokine Secretion Assay (HepG2) Multiplexed Hepato-Cytokine Secretion Assay Biomarker secretion, as markers of inflammation Nuclear count, analyte normalization to cell number HepG2 cells seeded into 96-well Collagen I coated optical plates incubated 24 hrs Cells treated with LPS, TNF留, IL-1硫 and acetaminophen serially diluted 遜 log over 8 concentrations incubated 48 hrs Post 48 hrs incubation supernatants collected, cytokine detection was carried out using Luminex xMAP technology To quantify cell proliferation the monolayer of HepG2 cells remaining in each plate was immediately stained with nuclear dye for normalization Images were collected using a GE Healthcare INCell Analyzer 1000
  • 21. Multiplexed In vitro Hepato-Cytokine Secretion Assay HepG2 cells treated with LPS, TNF留, IL-1硫 and acetaminophen HepG2 cells Screened for the secretory presence of 30 human inflammatory markers: LPS, TNF留, IL-1硫 and acetaminophen IL-1留, IL-1硫, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p40, IL- 12-70, IL-13, INF粒, INF留2a, Fibrinogen, Apo AI, Apo AII, Apo B, IP-10, GM-CSF, G-CSF, CRP, Haptoglobin, Apo CII, Apo CIII and MCP-1, MIP-1留, MIP-1硫, SAA Apo E TNF留, IL-1 receptor antagonist
  • 22. Multiplexed In vitro Hepato-Cytokine Secretion Assay (HepG2)
  • 23. Early Safety Screening for Mechanisms of Hepatotoxicity Conclusion: We have developed a robust and rapid throughput screening system using HepG2 cells that allows early assessment of acute and chronic mechanisms of hepatotoxicity Compounds with known hepatotoxicities tested in validating the capabilities of this multiparametric HCS system in identifying and quantifying toxicities relevant to cell proliferation, apoptosis, cell cycle, steatosis/cholestasis and phospholipidosis demonstrated high concordance with reported hepatotoxic profile for each compound tested Evaluation of cytokine secretion in HepG2 cells to identify measurable biomarkers of inflammation demonstrated significant secretion levels for 6 of the cytokines tested thus validating this multiplexed approach for quantifying indications of hepatic inflammation These hepatotoxicity screening assays are sensitive and reproducible and provide results that previously only have been attainable in more complex in vivo models Our cost-effective in vitro multiplexed HCS platform offers comprehensive predictive information allowing pre-selection of drug scaffold designs with long-term hepatotoxicity considerations and may even have more relevance when performed in normal primary hepatocytes