Hepatotoxicity Screening Sot Poster 2008 2009

1,630 views
1,443 views

Published on

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

0 Comments
1 Like
Statistics
Notes
  • Be the first to comment

No Downloads
Views
Total views
1,630
On SlideShare
0
From Embeds
0
Number of Embeds
10
Actions
Shares
0
Downloads
40
Comments
0
Likes
1
Embeds 0
No embeds

No notes for slide

Hepatotoxicity Screening Sot Poster 2008 2009

  1. 1. Screening for Mechanisms of Hepatotoxicity: Phospholipidosis, Steatosis, Apoptosis and Inflammatory Markers K.F. Marcoe, R. Keyser, P. TB. Nguyen, Yulia Ovechkina, and C. O’Day 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. O’Day MDS Pharma Services – Bothell, WA, USA
  2. 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. 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. 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. 5. Multiplexed High Content ScreeningHepatotoxicity 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. 6. Multiplexed In vitro Hepatotoxicity AssayIn 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., O’Brien 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. 7. Multiplexed In vitro Hepatotoxicity AssayMultiplexed 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 ½ 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. 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], MHepG2, 72 hr assay
  9. 9. Primary human hepatotoxicity assay: MTS and HCS comparison Amiodarone Valproic acid Amitriplyline 100 160 120 140 100 80 120MTS 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 microMHCS 24 hour compound treatment of human primary hepatocytes; High Content Screening approach (HCS)
  10. 10. Primary human hepatotoxicity assay:MTS and HCS comparison HCS % of attached MTS Viability IC50 Compound live cells IC50 (microM) (microM) Tamoxifen 36.7 ± 5.6 19.8 ± 0.7 Chlorpramazine 28.0 ± 6.9 26.4 ± 1.8 Amitriplyline 49.39 ± 4.06 62.3 ± 4.2 Amiodarone 125 ± 10 146 ± 22 Valproic acid >500 >500 Astemizole 6.39 ± 2.38 12.2 ± 2.1 Rosiglitazone 354 ± 133 413 ± 80 Troglitzqone 157 ± 6 122 ± 4224 hour compound treatment of human primary hepatocytes; High Content Screening approach (HCS)
  11. 11. Multiplexed In vitro Hepato-LipidAccumulation 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. 12. Multiplexed In vitro Hepato-LipidAccumulation AssayMultiplexed 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 ½ 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. 13. Multiplexed In vitro Hepato-Lipid Accumulation Assay (HepG2)HepG2, 48 hr assay
  14. 14. Multiplexed In vitro Hepato-Lipid Accumulation Assay (HepG2) Hepato-Neutral Lipid Accumulation Assay Labels: Nuclei - green; Neutral lipids - redHepG2, 48 hr assay
  15. 15. Multiplexed In vitro Hepato-Lipid Accumulation Assay (HepG2)HepG2, 48 hr assay
  16. 16. In vitro Hepato-Lipid Accumulation Assayusing primary human hepatocytes in 384 WP
  17. 17. In vitro Hepato-Lipid Accumulation Assayusing primary human hepatocytes in 384 WP Amiodarone Amitriplyline
  18. 18. Multiplexed In vitro Hepato-CytokineSecretion AssayMultiplexed Hepato-Cytokine Secretion Assay IN Cell xMAP™ Automated technology fluorescent using microscopy Luminex  imaging  Markers of cell count inflammation normalization
  19. 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 it’s 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. 20. Multiplexed In vitro Hepato-Cytokine SecretionAssay (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, TNFα, IL-1β and acetaminophen serially diluted ½ 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. 21. Multiplexed In vitro Hepato-Cytokine SecretionAssay HepG2 cells treated with LPS, TNFα, IL-1β and acetaminophen HepG2 cells Screened for the secretory presence of 30 human inflammatory markers: LPS, TNFα, IL-1β and acetaminophen IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p40, IL- 12-70, IL-13, INFγ, INFα2a, Fibrinogen, Apo AI, Apo AII, Apo B, IP-10, GM-CSF, G-CSF, CRP, Haptoglobin, Apo CII, Apo CIII and MCP-1, MIP-1α, MIP-1β, SAA Apo E TNFα, IL-1 receptor antagonist
  22. 22. Multiplexed In vitro Hepato-CytokineSecretion Assay (HepG2)
  23. 23. Early Safety Screening for Mechanismsof HepatotoxicityConclusion: 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

×