Ricerca Early Safety Assays
Upcoming SlideShare
Loading in...5
×
 

Ricerca Early Safety Assays

on

  • 1,167 views

Cardiac toxicity (hERG and hNav1.5 inhibition) ...

Cardiac toxicity (hERG and hNav1.5 inhibition)
HepG2 liver cell line cytotoxicity (cell proliferation, apoptosis and necrosis induction)
HepG2 liver cell line lipidosis assay (phospholipidosis and neutral lipid induction)
Genetic toxicity (in vitro micronucleus assay, Ames test)
High throughput aqueous solubility screening by nephelometry

Statistics

Views

Total Views
1,167
Views on SlideShare
1,164
Embed Views
3

Actions

Likes
1
Downloads
12
Comments
0

2 Embeds 3

http://www.linkedin.com 2
http://www.slideshare.net 1

Accessibility

Categories

Upload Details

Uploaded via as Adobe PDF

Usage Rights

© All Rights Reserved

Report content

Flagged as inappropriate Flag as inappropriate
Flag as inappropriate

Select your reason for flagging this presentation as inappropriate.

Cancel
  • Full Name Full Name Comment goes here.
    Are you sure you want to
    Your message goes here
    Processing…
Post Comment
Edit your comment

Ricerca Early Safety Assays Ricerca Early Safety Assays Presentation Transcript

  • Early Safety Screening
  • Why Early Safety Screening? • Drug development costs have risen to an estimated average of $800,000,000 per approved drug (e.g., $1 billion for Taxol and $250 million for human growth hormone) • Drug development timeline has stretched to 10–15 years • Expensive late stage failure would be avoided if toxic compounds were identified earlier. • Early testing needs to be done quickly, in a cost-effective and high throughput manner.
  • “Fail early, fail cheaply" using our early safety screening • In vitro Cellular Toxicity » Cardiac toxicity (hERG and hNav1.5 inhibition) » HepG2 liver cell line cytotoxicity (cell proliferation, apoptosis and necrosis induction) » HepG2 liver cell line lipidosis assay (phospholipidosis and neutral lipid induction) » Genetic toxicity (in vitro micronucleus assay, Ames test) • In Vitro ADME » Solubility/Stability by LC/MS » High throughput aqueous solubility screening by nephelometry » Lipophilicity Profile (Log D pH 7.4) » Metabolic CYP Pathway Identification » Metabolic Stability and Metabolite Profiling » Cytochrome P-450 Inhibition » Plasma Protein Binding and Cell Permeability
  • Weed out bad compounds sooner to save time and money • In vitro Cellular Toxicity » Multiplexed cytotoxicity assay (cell proliferation, apoptosis and necrosis) » Multiplexed lipidosis assay (phospholipidosis and neutral lipid induction) » Genetic Toxicity » In vitro micronucleus assay measures a chromosomal damage potential in a high throughput and cost-effective manner. » Ames test provides a sensitive evaluation of mutagenicity
  • Multiplexed HepG2 cytotoxicity assay • Cell proliferation (relative cell count) - blue • Apoptosis (activated-caspase-3 marker) - green • Mitosis (phospho-histone-3 marker) - red • Necrosis (cell membrane permeability marker) 3000 2500 200 100 2000 150 POC POC POC 1500 50 100 1000 50 500 Cell proliferation Apoptosis Mitosis 0 0 0 -12 -11 -10 -9 -8 -7 -12 -11 -10 -9 -8 -7 -12 -11 -10 -9 -8 -7 log [Vinblastine], M log [Vinblastine], M log [Vinblastine], M
  • Multiplexed cytotoxicity assay features • Quantitation of cell proliferation, apoptosis and mitosis in one assay well over 10 concentrations, n=3 • Accelerated throughput screening (800 compounds per week) • Experience with 300 unique human cell lines and primary cells
  • In vitro micronucleus assay using mammalian cells, CHO- K1, with and without metabolic stimulation (S9) • Nuclei green; Micronuclei white; Mitotic cells red; apoptotic cells blue. • Mitotic and apoptotic cell micronuclei are identified in circles and excluded. • Scored micronuclei are indicated by arrows. The micronucleus detection in mitotic and apoptotic cells would result in a false positive signal unless excluded.
  • Multiplexing micronucleus assay with cell proliferation assay minimizes counting of micronuclei in dying or dead cells High Cytotoxicity 30 2 % of cells with MNs Growth Index, GI 20 1 10 50% Cytotoxicity 0 0 -9 -8 -7 -6 -5 log [Etoposide], M Growth Index, GI % cells with MNs
  • In vitro micronucleus assay features • Micronuclei induction, apoptosis and cell proliferation mutiplexed outputs from one assay well over 10 concentrations, n=3 • Evaluation of test compounds in the absence and presence of in vitro metabolic activation system (S9) in pre-validated mammalian cell line • Multiplexing the micronucleus assay with the apoptosis assay reduces false-positives by excluding apoptotic and mitotic cells from micronuclei scoring • Accelerated throughput screening (200 compounds per week) • Minimum compound consumption for 384-well plate format and acoustic based compound addition system, Labcyte® Echo™ 550
  • High throughput aqueous solubility screening by nephelometry • Aqueous solubility is determined by measuring fold induction of scattered light intensity of a sample concentration over that of the solvent. • Insolubility is defined as the concentration at which the fold induction is significantly greater than that of the solvent. Fold induction in intensity of scattered light by laser nephelometry 90 80 70 60 50 40 30 20 10 0 1 10 100 Log [Ketoconazole], microM
  • Multiplexed lipidosis assay (phospholipidosis and neutral lipid induction) HepG2 HepG2 Published HepG2 Neutral Published Relative cell Phospholipido Neutral lipid induction Phospholipido count IC50 sis Induction lipid Compound (microM) sis Induction (microM) (microM) Induction 48hr (microM) 48hr 48hr (Positive) Acetaminophen > 500 N/A N/A > 8005 Staurosporine 0.009 ± 0.001 N/A N/A Methotrexate 0.04 ± 0.01 N/A N/A Isoproterenol 207 ± 50 N/A N/A Valproic acid > 500 N/A 433 ± 67 > 8005 positive8,9 Cerivastatin Na 0.8 ± 0.2 N/A 0.16 ± 0.06 Cyclosporin A 11.9 ± 1.5 N/A 7.97 ± 2.03 positive8 Troglitazone 26.5 ± 6.8 N/A 52.3 ± 44.5 positive9 Propranolol 71.2 ± 14.3 12.1 ± 0.2 N/A 12.927 Rosiglitazone 222 ± 36 131 ± 27 N/A amitriplyline HCL 30.3 ± 2.0 6.3 ± 0.4 14.9 ± 5.1 12.55, 12.27 Astemizole 3.9 ± 0.4 0.8 ± 0.0 1.0 ± 0.05 Chlorpromazine 13.0 ± 3.8 2.6 ± 0.1 4.74 ± 1.59 45, 8.36, 4.17 Aminodarone 14.4 ± 2.8 2.4 ± 0.1 2.62 ± 0.24 165, 8.36, 4.87 positive9 Tamoxifen 13.9 ± 1.7 2.4 ± 0.7 13.75 ± 10.61 165, 8.36, 23.37 Terfenadine 2.9 ± 0.5 0.6 ± 0.0 1.34 ± 0.25
  • HepG2 phospholipid accumulation assay Labels: Nuclei - green; Phospholipids - red
  • HepG2 phospholipid accumulation assay
  • Cardiac toxicity • Radioligand binding assays » hERG binding » Sodium channel, Site 2 » Calcium Channel L-Type • The patch clamp ion channel inhibition cellular assays » hERG (Kv11.1) » hNav1.5 • In vivo assay » Cardiovascular, QTc Interval
  • Cardiac toxicity using the patch clamp PatchXpress® 7000A Inhibition of hERG or hNav1.5 causes undesirable changes to the QT interval Astemizole-mediated hERG inhibition +20 mV -50 mV -80 mV Control 300 nM Astemizole
  • hERG PatchXpress: Consistent peak currents, accelerated throughput and high quality recordings High agreement of PatchXpress with conventional patch clamp data Spearman r = 0.99, p <0.001 8 Pimozide hERG Conventional Patch Astemizole E4031 pIC50 (Conventional Patch) Haloperidol Terfenadine 7 Cisapride Risperidone 6 Quinidine Verapamil Ketoconazole 5 Moxifloxacin 4 4 5 6 7 8 pIC50 (PatchXpress) hERG PatchXpress