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.

• 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

“Fail early, fail cheaply" using our early safety screening

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)

-12 -11 -10 -9 -8 -70

50

100

log [Vinblastine], M

POC

Cell proliferation

-12 -11 -10 -9 -8 -70

500

1000

1500

2000

2500

3000

log [Vinblastine], M

POC

Apoptosis

-12 -11 -10 -9 -8 -70

50

100

150

200

log [Vinblastine], M

POC

Mitosis

• 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

Multiplexed cytotoxicity assay features

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

-9 -8 -7 -6 -50

10

20

30

0

1

2

log [Etoposide], M

% of cells with MNs G

rowth Index, GI

% cells with MNsGrowth Index, GI

High Cytotoxicity

50% Cytotoxicity

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.

Log [Ketoconazole], microM

Fold induction in intensity of scattered

light by laser nephelometry

1 10 100

0102030405060708090

Multiplexed lipidosis assay (phospholipidosis and neutral

lipid induction)

1.34 ± 0.250.6 ± 0.02.9 ± 0.5Terfenadine

165, 8.36, 23.3713.75 ± 10.612.4 ± 0.713.9 ± 1.7Tamoxifen

positive9165, 8.36, 4.872.62 ± 0.242.4 ± 0.114.4 ± 2.8Aminodarone

45, 8.36, 4.174.74 ± 1.592.6 ± 0.1 13.0 ± 3.8Chlorpromazine

1.0 ± 0.050.8 ± 0.03.9 ± 0.4Astemizole

12.55, 12.2714.9 ± 5.16.3 ± 0.430.3 ± 2.0amitriplyline HCL

N/A131 ± 27222 ± 36Rosiglitazone

12.927N/A12.1 ± 0.271.2 ± 14.3Propranolol

positive952.3 ± 44.5N/A26.5 ± 6.8Troglitazone

positive87.97 ± 2.03N/A11.9 ± 1.5Cyclosporin A

0.16 ± 0.06 N/A0.8 ± 0.2Cerivastatin Na

positive8,9> 8005 433 ± 67N/A> 500 Valproic acid

N/AN/A207 ± 50Isoproterenol

N/AN/A0.04 ± 0.01Methotrexate

N/AN/A0.009 ± 0.001Staurosporine

> 8005N/AN/A> 500Acetaminophen

Published

Neutral

lipid

Induction

(Positive)

Published

Phospholipido

sis Induction

(microM)

HepG2 Neutral

lipid induction

(microM)

48hr

HepG2

Phospholipido

sis Induction

(microM)

48hr

HepG2

Relative cell

count IC50 (microM)

48hr

Compound

1.34 ± 0.250.6 ± 0.02.9 ± 0.5Terfenadine

165, 8.36, 23.3713.75 ± 10.612.4 ± 0.713.9 ± 1.7Tamoxifen

positive9165, 8.36, 4.872.62 ± 0.242.4 ± 0.114.4 ± 2.8Aminodarone

45, 8.36, 4.174.74 ± 1.592.6 ± 0.1 13.0 ± 3.8Chlorpromazine

1.0 ± 0.050.8 ± 0.03.9 ± 0.4Astemizole

12.55, 12.2714.9 ± 5.16.3 ± 0.430.3 ± 2.0amitriplyline HCL

N/A131 ± 27222 ± 36Rosiglitazone

12.927N/A12.1 ± 0.271.2 ± 14.3Propranolol

positive952.3 ± 44.5N/A26.5 ± 6.8Troglitazone

positive87.97 ± 2.03N/A11.9 ± 1.5Cyclosporin A

0.16 ± 0.06 N/A0.8 ± 0.2Cerivastatin Na

positive8,9> 8005 433 ± 67N/A> 500 Valproic acid

N/AN/A207 ± 50Isoproterenol

N/AN/A0.04 ± 0.01Methotrexate

N/AN/A0.009 ± 0.001Staurosporine

> 8005N/AN/A> 500Acetaminophen

Published

Neutral

lipid

Induction

(Positive)

Published

Phospholipido

sis Induction

(microM)

HepG2 Neutral

lipid induction

(microM)

48hr

HepG2

Phospholipido

sis Induction

(microM)

48hr

HepG2

Relative cell

count IC50 (microM)

48hr

Compound

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

-80 mV-50 mV

+20 mV

Control

300 nM Astemizole

Astemizole-mediated hERG inhibition

Inhibition of hERG or hNav1.5 causes undesirable changes to the QT

interval

hERG PatchXpress: Consistent peak currents, accelerated

throughput and high quality recordings

PimozideAstemizoleE4031

Terfenadine

Cisapride

Haloperidol

Risperidone

VerapamilQuinidine

Ketoconazole

Moxifloxacin

4

5

6

7

8

4 5 6 7 8

pIC50 (PatchXpress)

pIC50 (Conventional Patch)

Spearman r = 0.99, p <0.001

High agreement of PatchXpress with conventional patch clamp data

hERG Conventional Patch

hERG PatchXpress