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

1. Early Safety Screening

2. 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.

3. “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

4. 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

5. 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

6. 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

7. 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.

8. 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

9. 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

10. 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

11. 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

12. HepG2 phospholipid accumulation assay
Labels: Nuclei - green; Phospholipids - red

13. HepG2 phospholipid accumulation assay

14. 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

15. 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

16. 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