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New High Conten t Screening Methods Applied to in vitro Toxico logy Analys is
Bette r dec is ions . Earlie r in the game.
Anthony D Baxter, CEO
Business overviewA uniquely packaged ADMET solution
The leading specialist Contract Research Organisation (“CRO”) providing ADME-Toxscreening technology and information Evaluates and optimises Absorption, Distribution, Metabolism, Excretion, Toxicity (“ADMET”)
and pharmacokinetic properties of potential drug candidates
A portfolio of technologies to identify potential problems Assisting at the drug discovery stage and to help reduce the attrition of the drug development
process Genuine competitive advantages in different steps of the process
Cyprotex offers highly automated in-vitro and in-silico screening Designed to significantly accelerate and improve the cost-effectiveness of the process from
“hit-to-lead” in drug discovery Enormous global market opportunity – big pharma re-organisations
Commercial success Cloe® integrated product offering (Cloe® Screen, Cloe® Select and Cloe® Predict) Toxicology assays (Cyprotox) CellciphrTM (Cellumen, High content screening)
Company Overview
Founded in 1999. Publicly traded since 2002
Acquired Apredica in Aug 2010
Laboratories in UK (near Manchester) and US (near Boston)
100% focus on ADME-Tox/PK Services
No internal drug discovery programs
Over 500 customers worldwide (typically 50 at any one time)
Exceptional client retention rate
60+ staff
High PhD scientist ratio
Revenue and profitab ility
Group Revenues up 18.4% to £5.92 million (2009: £5.00 million)
UK revenues alone up 7.5% to £5.38 million
Additional Apredicarevenues of £0.54 million (5 months)
Profit/(loss)for the year
Acquis ition of Apredica
Purchase of Apredica meant that we acquired a respected rival ADMET CRO in Boston, MA, USA (August 2010)
Apredica gave us:• World renowned CSO in Dr Katya Tsaioun• Many new assays in customised ADME• Three year head start in in vitro toxicology assays• Access to high content toxicology assays acquired from Cellumen• A bigger share of the US ADME-Tox market• Access to the largest global market for ADME-Tox• Additional highly experienced scientists – further strengthening our team
Prec lin ica l ADME Tox s ervices
ServicesHigh-throughput ADME screening Customised ADME assaysBioanalysis and PK analysisIn silico QSAR and PBPK modelling In vitro Toxicology• Mechanisms of Toxicity• Genotoxicity• Metabolism Related Toxicity• High Content Toxicology
Our ADME As s ays – core bus ines s
AbsorptionCaco-2 PermeabilityMDR1-MDCKPAMPA (Standard and BBB)Transporter Studies (P-gp and BCRP)Dermal AbsorptionIntranasal and Lung Absorption
MetabolismMetabolic Stability (microsomes, hepatocytes, S9, plasma)CYP450 Inhibition (IC50 and Ki)CYP450 Time Dependent InhibitionCYP450 InductionCYP450 Reaction PhenotypingUGT1A1 InhibitionMAO InhibitionMetabolite Profiling
DistributionPlasma Protein BindingBrain Tissue BindingBlood to Plasma RatioMicrosomal Binding
PK and BioanalysisPharmacokinetic AnalysisBioanalytical Method Development and Validation
Physicochemical PropertiesTurbidimetric SolubilityThermodynamic SolubilitypKa DeterminationLipophilicity (CHI, LogP, LogD)Chemical Stability
Our ADME & PK Predic tion Services
PB-PK Modelling• Pharmacokinetic predictor
(Cloe® PK)
• Human intestinal absorption predictor (Cloe® HIA)
• Customized chemistry-specific models
• Animal-to-human in vivo extrapolator
• PK/PD modelling
QSARModel building and predictionNovel auto-QSAR techniques
Current Collaborative ProjectsOSIRIS - Risk assessment of industrial chemicalsTB Alliance – PK predictionIMI – European standards for data and model results access
Our Toxic ity As s ays
General CytotoxicityMTTNeutral RedLDH Release
Mechanisms of ToxicityMitochondrial ToxicityhERG inhibitionHaemolysisPhospholipidosisPhototoxicitySkin IrritationGene Regulation (qRT-PCR)
High Content ToxicologyCytotoxicity Screening Panel (Tier 1)CellCiphrTM Toxicity Profiling (Tier 2)
Metabolism-Related ToxicityReactive Metabolite AssessmentDrug-Drug InteractionsMultispecies Metabolite Profiling
GenotoxicityAMESGreenScreen HCTM
In vitro MNTIn vitro Comet
Why inves t in toxico logy?
Alle Ding' sind Gift, und nichts ohn' Gift; allein die Dosis macht, daß ein Ding kein Gift ist.
"All things are poison and nothing is without poison, only the dose permits something not to be poisonous."
Paracelsus (Phillippus Aureolus Theophrastus Bombastus von Hohenheim)
1493 – 1541 Switzerland
The Grandfather of toxicology
Why inves t in toxico logy?
In 1990s drug metabolism activities increasingly moved earlier, from development to discovery
Led to lower attrition in the clinic due to DMPK problems
Success in early ADME screening means that toxicology is now the ‘enemy of successful drug discovery’
Kola & Landis (2004)
Clinica
l Safe
ty
Efficac
y
Formulat
ion
ADME-PK
Commercial
Toxicology
Cost of G
oodsOther
0
10
20
30
40
5019912000
Criterion
Attr
ition
(%)
How compounds fail in the clinic
!
Toxic ity is a major reas on for drug a ttrition
Over 40% of all drugs fail preclinical or clinical testing (2010 data) because of apparent or suspected drug toxicities
Cost of toxicity-related drug failures is ~$2 billion per year
DM&D Market Analysis Report, 2003
Conventional animal toxicity studies fail to predict idiosyncratic human hepatotoxicity
http://www.fda.gov/cder/livertox/stateArt.htm
Toxicity is still a late-stage issue for many companies
Why High Content Toxicology?
Often drug toxicity is combination of multiple mechanismsA single experimental approach may not be predictive of the complex steps involved in toxicological effectsHCS captures multiple mechanistic parameters which cover a wide spectrum of cytopathological changesParameters can be measured in same cell populations in the same well, reducing inter-assay differences
Two tiers of toxicity profilingProfiling: Predict Preclinical Outcome
Organ-specific Panel Profiles
General Cytotoxicity Assay Sets
Mechanism-specific Assay Sets
Tier 1: Screening
Tier 2: Full profiling
Rationale of Selected Parameters: Tier 1
O’Brien et al, Arch. Toxicol (2006) 80:580-604: Pfizer
Assay Sensitivity Specificity
Cell ViabilityMembrane IntegrityApoptosis (Caspase 3)Protein SynthesisOxidative Stress (superoxide induction)Oxidative Stress (glutathione depletion)DNA Synthesis
Cell Viability or GSH or DNA synthesisRegulatory animal toxicity testing
10% 2%5%4%1%
19%10%
25%52%
92%99%95%97%97%85%92%
83%-
611 compounds (retrospective)
Cellomics “Quad Probe Assay (Tier 1)” 93% 98%
Sensitivity = % of hepatotoxic compounds detected i.e. 1:10 known hepatotoxicants in test set detected (10%)Specificity = % of correctly identified hepatotoxic compounds i.e. 9:10 positive compounds are hepatotoxicants (90%)
High Content Screening Technology for Cellu la r Toxic ity As s es s ment
State-of-the-art Thermo ArrayScan VTIAutomated fluorescence imaging and cellular analysisMulti-parametric indicators of cell toxicityDetection of cell death and mechanisms of cell death
Screening: Validated channels/colours
Customization is possible!Measuring other features is possible if reagents are commercially available
nm
Cell Health/Death Signalling
High Content Toxicology: Tier 1 Screening Panel
Cell lossNuclear areaDNA structure
1µM Paclitaxel
Nuclear morphology Cell permeability
1µM Cerivastatin
Mitochondrial potential
1µM Paclitaxel
0.2% DMSO
Cytochrome c
10µM Amiodarone
0.2% DMSO0.2% DMSO 0.2% DMSO
4x
4x
1µM Paclitaxel
Nuclear morphology Cell permeability
1µM Cerivastatin
Mitochondrial potential
1µM Paclitaxel
0.2% DMSO
Cytochrome c
10µM Amiodarone
0.2% DMSO0.2% DMSO 0.2% DMSO
4x
4x
24hr exposure of HepG2 cells
Increased cell permeability
Mitochondrial massMitochondrial potential loss
Cytochrome C release
Observations from single cell population
Control:
Treated:
Blue Green Red Far Red
High Content Toxicology CellCiphrTM Organ Specific Toxicity Panels (Tier 2)
Cytotoxicity ProfilingHepG2 cells (Human)
Human cell lineInsights into toxicity to cycling cells
Hepatotoxicity ProfilingRat Primary Hepatocytes
Primary cells with metabolic capacityInvestigate hepatocyte-specific toxicities
HepaRG (in development)Human cell lineMetabolic competencyCo-development opportunities
Cardiotoxicity ProfilingH9C2 cardiomyocytes (Rat)
Hypertrophy and cardiomyocyte-specific toxicities
Hepatocytes
Cardiom
yocytes
Animal & Human cell models
High Content Toxicology CellCiphrTM: Tier 2 Toxicity Panels
Cell Loss Nuclear Size DNA DamageMitochondrial Potential
Cell Cycle Arrest Cytoskeletal DisruptionOxidative Stress Mitochondrial MassMitosis Marker Stress Kinase Activation
Cytotoxicity Profiling -HepG2
CardiotoxicityProfiling – H9C2Cell Loss Nuclear Size DNA Damage Mitochondrial Potential
Apoptosis Steatosis
Hypertrophy Reactive Oxygen
Hepatotoxicity Profiling – 1o RatCell Loss Nuclear Size DNA Damage Mitochondrial Potential
ApoptosisSteatosis
DNA Fragmentation Phospholipidosis
CellCiphr® CSB™ Tier 2 HepG2 Panel
DMSO
HepG2 cells. Blue – nucleiGreen – microtubule stability markerRed – mitochondrial function markerMagenta – mitotic arrest marker.
0.2 μM paclitaxelDMSO
Cell Loss Nuclear Size DNA DamageMitochondrial PotentialCell Cycle Arrest Cytoskeletal DisruptionOxidative Stress Mitochondrial MassMitosis Marker Stress Kinase Activation
Cytotoxicity Profiling -HepG2
CellCiphr® CSB™ Tier 2 Primary Rat Hepatocyte Panel
Phospholipidosis
Cell loss & Nuclear Size
Apoptosis
DNA Damage
Mitochondrial Potential
Steatosis
Hepatotoxicity Profiling – 1o RatCell Loss Nuclear Size DNA Damage Mitochondrial PotentialApoptosisDNA Fragmentation PhospholipidosisSteatosis
Customer Cpds
Tier 2: High Content Toxicology CellCiphrTM
Database Source
Safety Toxicology
Physical Properties
580 Reference CpdsCommercial Libraries, FDA, EPA, and Cellumen
CellCiphr Analysis
PharmaPendiumEPA, FDA, others
Customer Proprietary Compounds
Safety Alerts
Rank Order
Similarity Profiles
Data Sources Database
Summary
Cyprotex is an ADME-Tox Expert
Cyprotex’s ServicesRoutine, custom & novel ADME-Tox assays
Process automation consultancy
Bioanalysis and PK analysis
In silico PBPK modelling
Cyprotex Advantage• Experienced technical staff
• Superior client communication
• Collaborative, flexible approach
Unrivaled process, speed, and quality
Cyprotex15 Beech LaneMacclesfieldCheshireSK10 2DRUK
Apredica , a Cyprotex company313 Pleasant StreetWatertown, MA 02472USA
Tel (UK): +44 (0) 1625 505115
Tel (US): +1-617-923-1466
Email: [email protected]
Website: www.cyprotex.com