Cellular Systems Biology Profiling in Drug Discovery and Development

Cellular Systems Biology Profiling in Drug Discovery and Development Kenneth A. Giuliano, Ph.D. Principal Scientist Cellumen, Inc. “Optimizing Drug Discovery, Development and Clinical Trials Through Cellular Systems Biology” FDA/NIST Sponsored Workshop In Vitro Analyses of Cell/Scaffold Products December 2007 © 2007 Cellumen, Inc. | CONFIDENTIAL 11 Agenda Introduction to Cellumen and Cellular Systems Biology Cytotoxicity profiling as an example application of cellular systems biology © 2007 Cellumen, Inc. | CONFIDENTIAL 21 Exploring Cells and Tissues as Systems “From Molecules to Man” © 2007 Cellumen, Inc. | CONFIDENTIAL 31 The Challenge: Cell is An Integrated, Interacting Network Gene Expression Cell Surface Attachments Cytoskeletal Assembly-Disassembly Membrane Receptors Membrane Pumps Cell Movements Signaling Pathways Post-translational Modifications Molecular Synthesis/ Degradation Energy Metabolism © 2007 Cellumen, Inc. | CONFIDENTIAL 41 Evolution of Cell-based Discovery Emergence of Cellular Systems Biology Cell population responses HTS methods Whole plate readers “Simple” HCS Automated cellular imaging 1st generation HCS readers/1-2 features measured Multiplexed HCS Automated cellular imaging 3-4 features with data management Cellular Systems Biology Optimally multiplexed cell and tissue imaging advanced reagents, >10 features & classifier informatics © 2007 Cellumen, Inc. | CONFIDENTIAL 51 Cellular Systems Biology (CSB)™ Approach The cell is an integrated and interacting network of genes, proteins & metabolic processes that gives rise to function Functional Biomarkers Cell Arrays “omics” Information Tissue MicroArrays © 2007 Cellumen, Inc. | CONFIDENTIAL 61 The Solution: Cellular Systems Biology Profiling Proprietary Cellular Biomarker Panels 75 95 46 18 88 31 83 68 86 71 © 2007 Cellumen, Inc. | CONFIDENTIAL 71 Proprietary Profile Database The Solution: Cellular Systems Biology Profiling Proprietary Cellular Biomarker Panels 75 95 46 18 88 31 83 68 86 71 Proprietary Profile Database 90 98 75 2 80 42 95 3 6 98 17 67 46 3 47 11 18 8 6 79 89 88 5 76 21 95 47 91 31 3 94 97 83 5 45 87 97 13 68 13 91 88 86 4 99 79 71 55 6 © 2007 Cellumen, Inc. | Classifiers CONFIDENTIAL 81 Tools of Cellular Systems Biology Existing Imagers • Cell “Models” • Patient Cells/ Tissues Reagents/Profiles Informatics/Classifiers © 2007 Cellumen, Inc. | Systems Knowledge & Decisions CONFIDENTIAL 91 Implementing Cellular Systems Biology (CSB)™ Improve efficacy and decrease toxicity of leads, clinical candidates & drugs Drug Discovery Drug Development Clinical Trials Cellular Models of Disease Cytotoxicity Profiling Patient Sample Profiling © 2007 Cellumen, Inc. | CONFIDENTIAL 101 Cellumen Solution Drug Discovery Drug Development Clinical Trials CellCiphr™ Cellular Models of Disease • Disease-relevant cellular models, biosensors, assays & profiles to improve quality and quantity of lead compounds • Profiles & tools for important, previously intractable targets • Flags off-target effects at the earliest stage © 2007 Cellumen, Inc. | CONFIDENTIAL 111 Cellumen Solution Drug Discovery Drug Development Clinical Trials CellCiphr™ Cytotoxicity Profiles •Identifies potential toxicity before entering expensive pre-clinical testing •Enables prioritization of lead compounds Partners CHA US EPA | CONFIDENTIAL 121 © 2007 Cellumen, Inc. Cellumen Solution Drug Discovery Drug Development Clinical Trials CellCiphr™ Patient Sample Profile R&D • Improves trial enrollment and therefore new drug candidate efficacy by stratifying patients with profiles of the patient’s own samples • Proprietary panels of cellular biomarkers can also serve as diagnostic tools to improve treatment selection Collaborative Partner © 2007 Cellumen, Inc. | CONFIDENTIAL 131 Cytotoxicity Profiling CellCiphr™ Cytotoxicity Profile CellCiphr™ Advantages: Systems Biology approach monitors multiple functions, time points and doses Fully leverages the sensitivity and throughput of HCS Validated to HTS standards with 384-well capacity & extensive QC Classifier software for simplification and predictivity Delivers insights on mechanism of action © 2007 Cellumen, Inc. | CONFIDENTIAL 141 Cytotoxicity Profiling CellCiphr™ Cytotoxicity Panel #1 Stress Pathway Activation Organelle Functions Panel 1 design* (Collaboration with Millipore) • Multiple mechanisms of toxicity • Acute, early & chronic exposure • 384-well capacity • HepG2 Oxidative Stress DNA Damage Cell Cycle CSK Integrity *Cellumen patents pending © 2007 Cellumen, Inc. | CONFIDENTIAL 151 Cytotoxicity Profiling CellCiphr™ Cytotox Panel #1 Features HepG2 Cells Measurement Cell Number DNA Content DNA Structure Nuclear Area Histone H2A.X Phosphorylation c-Jun Phosphorylati on Phosphorylatio p53 activation Mitochondrial memb. po tential memb. potential Mitochondrial mass Histone H3 Phosphorylation Microtubule stability Cell Features Cell Loss Cell Cycle Arrest DNA Degradation Nucl ear Size Nuclear Oxidative S tress St Stress Kina se Activation Kinase DNA Dama ge Response Damage Mitochondrial Function I Mitochondrial Function II Mitosis marker Microtubule CSK Reagents Hoech st 33342 Hoechst anti-phospho-histone H2A.X Antibo dy anti- phosphoAntibody anti-phospho-c-jun Antibo dy anti- phosphoAntibody anti-p53 Antibody antiMitoTracker Red MitoTracker Red anti-phospho-histone H3 Antibo dy anti- phosphoAntibod anti-α-tubulin Antibody anti- © 2007 Cellumen, Inc. | CONFIDENTIAL 161 Cytotoxicity Profiling CellCiphr Cytotoxicity Panel #2 Apoptosis Peroxisome Proliferation ™ Panel 2 design*: • Primary Rat Hepatocytes • Multiple mechanisms of toxicity • 11 parameters • Acute, Early, 48h exposure • Fixed endpoint, 384-well capacity Phospholipidosis Mitochondrial Functions Stress Pathway Activation CSK Integrity * Cellumen patents pending © 2007 Cellumen, Inc. | CONFIDENTIAL 171 Cytotoxicity Profiling CellCiphr™ Analysis Acute Profile Early Profile Chronic Profile Analysis of identifies Clusters of Additional measurements after 24 & 72 with a set of CellCiphr™ Cytotoxicity Profiling starts hours of Image analysis for a panel of Clustering a seriesPot. cellular functions compounds profiles that enhancing compounds characterize early & response capturecorrelate with cellular biomarkers selected to profile… the cellular produces a systems chronic responses,populates a based on cellular CellCiphr Profile CellCiphr sensitivityof cellular activity. CellCiphr Profile. the systemic response to toxic challenge.functions… a ‘fingerprint’ & specificity of the KinaseC-124 Oxidative Stress Profiles allowing classification. Database. Stress Induction 0.4 TERF CMPT PTXL 0.2 Comp.2 ETPS MNDN CHLQ T IN PROP SUCL VALP QUACR DI LN IMIP BU ICHPZSP NDO PARX ASTM BUPV CCCP FURA MEVA 0.0 MTRX -0.2 KTZL VBLN NOCD ANIS -0.4 AMIO MEVN -0.4 -0.2 0.0 0.2 0.4 Comp.1 Apoptosis Induction -10 -5 0 5 10 Mito 5 -10 -5 0 10 STSP Library of CellCiphr Profiles Principle Components Compound Classification Analysis to build classifier © 2007 Cellumen, Inc. | CONFIDENTIAL 181 Cytotoxicity Profiling CSB Imaging and Data Analysis W11 400 300 200 100 1x10 -8 A. Ox. Stress 1x10 M -5 11 features measured in EACH cell; 3 time points; 10 pt dose-response, curve fit the data Tabulate AC50 values B. Cmpd Plate Cell Loss Acute CC Arrest Acute DNA Degrad Acute Nuclear Size Acute Ox. Stress Acute Stress Kinase Acute DNA Damage Acute Mito Potential Acute Mito Mass Acute Mitotic Arrest Acute Microtube Stability Cell Loss CC Arrest Acute Early Early DNA Degrad Early Nuclear Size Early Ox. Stress Early Stress Kinase Early DNA Damage Early Mito Potential Early Mito Mass Early Mitotic Arrest Early Microtube Stability Cell Loss CC Arrest Early Chronic Chronic DNA Degrad Chronic Nuclear Size Chronic Ox. Stress Chronic Stress Kinase Chronic DNA Damage Chronic Mito Potential Chronic Mito Mass Chronic Mitotic Arrest Chronic Microtube Stability Chronic T42 R50 P10 S23 W11 O13 5.18 3.40 4.92 4.80 5.66 4.18 4.75 5.45 3.80 4.52 3.89 6.15 5.04 4.41 4.57 4.57 4.28 5.77 4.38 4.85 4.00 4.80 3.44 5.45 5.00 3.61 5.63 4.40 3.67 5.69 5.26 5.01 4.32 5.94 4.78 5.97 4.56 5.09 4.45 6.41 4.86 4.29 5.90 3.87 5.25 4.96 5.71 4.89 4.07 5.72 4.80 5.08 5.72 3.63 5.71 6.00 3.82 3.34 4.43 4.46 3.21 6.01 3.99 6.45 4.25 5.59 5.04 C. 10 9 Activity (-log(M)) 8 7 6 5 4 3 2 1 2 3 4 5 6 7 8 9 10 CellCiphr Profile Plot 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 Max w11 Quantitative and Visual Tools for Compound Profiling © 2007 Cellumen, Inc. | CONFIDENTIAL 191 Cytotoxicity Profiling CellCiphr™ Profiling Case Study Drug Safety Data for 137 compounds* Each compound scored on a scale of 0, 1, 2, 3, 4 for in vivo toxicity Compounds tested in CellCiphr HepG2 Panel 1 and CellCiphr Rat Hepatocyte Panel 2 CellCiphr 1 automated analysis provided similarity to known controls, the group toxicity rank order and a safety index * Includes 101 unknown (blinded) and 36 control compounds © 2007 Cellumen, Inc. | CONFIDENTIAL 201 Cytotoxicity Profiling Combining CellCiphr™ Features from HepG2 and Rat Hepatocytes Optimizes Toxin Detection P70 W81 D11 Total Activity X49 X39 nM μM mM Min Toxicity Significant Toxicity No Safety Data In Vivo Tox © 2007 Cellumen, Inc. | CONFIDENTIAL 211 Cytotoxicity Profiling CellCiphr™ Similarity Profile nM μM mM Compound H25 (blue) H25 (blue) Differences from Etoposide (red) - CC Arrest Acute - Stress Kinase Chronic © 2007 Cellumen, Inc. | CONFIDENTIAL 221 Cytotoxicity Profiling Classifier Design 137 compounds produced over 4500 doseresponse curves Difficult to apply manual scoring methodology to handle analysis Assay data from compounds was used with in vivo scorings to construct a classifier to rank compound toxicity First generation classifier had improved performance over simple cytotoxicity assay © 2007 Cellumen, Inc. | CONFIDENTIAL 231 Cytotoxicity Profiling Accuracy of CellCiphr Classifier 1 Toxicity Significant Moderate Minimal Overall accuracy In vivo n 47 66 24 137 24 hr cell loss 84% 65% 25% 61% CellCiphr Classifier 100% 80% 46% 82% © 2007 Cellumen, Inc. | CONFIDENTIAL 241 Cytotoxicity Profiling CellCiphr™ Report Compound: W11 Source: CHA Project Maximum Tolerate Dose Earliest Toxic Indicator Most Sensitive Toxic Indicator General Indicator of Toxicity Measurable Effects Mitochondrial Potential Nuclear Size Cell Loss Nuclear Size Oxidative Stress Stress Kinase DNA Damage Mitosis Arrest Microtube Stability Cell Cycle Arrest Mitochondrial Potential Mitochondrial Mass AC50 (M) n/a, 1.2E-5 7.07E-07 3.90E-07 1.5E-6, 1.3E-6 8.4E-6, 3.4E-7 1.9E-6, 2.2E-6, 36.8E-6 1.9E-6, 3.6E-6, 5.7E-6 993.0E-9, 982.4E-9 2.3E-6, 9.1E-6 2.0E-6, 16.0E-6, 1.9E-6 8.4E-6, 8.0E-6 7.07E-7 27.2E-6, 2.5E-6 Time (hrs) 1, 72 24 72 1, 72 1, 72 1, 24, 72 1, 24, 72 1, 72 1, 72 1, 24, 72 1, 72 24 24, 72 Mechanistic Indicators Correlation with Other Compounds Similarity with Compounds in set Compound C9 Vinblastine Nocodazole Colcemid 0.70 0.69 0.60 Correlation Coefficient 0.59 Similarity with Toxin Cassette Compounds CellCiphr™ Profile W11 (red), Vinblastine (blue) Rank Order 1/ Safety H I d © 2007 Cellumen, Inc. | CONFIDENTIAL 251 Cytotoxicity Profiling CellCiphr™ Panel 3: Rat Hepatobiliary • Highly differentiated rat hepatocytes (sandwich culture) • Chronic exposure (3 day +) • 384 well capacity • Functional assays for: • Cholestasis • Steatosis • Mitochondrial potential Monolayer culture overlay culture © 2007 Cellumen, Inc. | CONFIDENTIAL 261 Cytotoxicity Profiling CellCiphr™ Panels in Development Human and Rat 1° and Cell Lines • Tissue selectivity panels • Neuronal cells • Cardiomyocytes • Stem-cell derived cultures • Co-culture/tissue engineered array models • Kinetic, Live Cell Panels © 2007 Cellumen, Inc. | CONFIDENTIAL 271 Summary The CSB approach to cell based discovery is being implemented to improve efficacy and decrease toxicity of leads, clinical candidates & drugs Cellular models of disease Cytotoxicity profiling Patient sample profiling CSB based cytotoxicity profiling assays and classifiers are evolving to improve early decision making © 2007 Cellumen, Inc. | CONFIDENTIAL 281