Development and Application of In Vitro Models

for Screening Drugs and Environmental
Chemicals that Predict Toxicity in Animals and
Humans

By: Jim McKim, Ph.D., DABT
Founder and Chief Science Officer

Presentation Topics

 Introduction to CeeTox Laboratories

 Overview of services

 Description of our Systemic Toxicity
Model
 Predicting an in vitro LD50

CeeTox ,Inc.: Is a Specialty Contract Research
Laboratory Located in Kalamazoo Michigan at the
Southwest Michigan Innovation Center

Founded 2003

Focus on in vitro toxicity

 Currently 32 employees

 Owned by NAMSA, Inc 2008

CeeTox Customer Base

Small and midsized Pharma
Personal care companies
Household product companies
Medical device
Petroleum companies
Government agencies
Proprietary to CeeTox, Inc.

CeeTox has a World Class Cell Culture and In
Vitro Toxicology Laboratory

Proprietary to CeeTox, Inc.

 Inc. .Art Laboratory Equipment to Process Samples Proprietary to CeeTox.CeeTox Utilizes Robotics and State-of-the.

CeeTox Has a Wide Range of Assay Capabilities all of Which can be Done in a High Throughput Mode  Roche 480 thermocycler 96.and 384-well head  BioTek plate reader  Perkin Elmer Fusion  Roche xCelligence system for cardiac toxicity  Beckman Coulter Biomek Robotics Station .

CeeTox Intellectual Property CeeTox Patents New In Vitro Screening Method Issued CardioTox Issued Anti-Tumor Predictive Tox Pending Skin Sensitization Pending Multi-Organ Prediction Pending .

CeeTox Overview of Services … a wide range of in vitro toxicology and safety assessment services  Systemic Toxicity Services  Organ Specific Toxicity  CTOX Panel® Models  Acute Toxicity Screen  Heart (CardioTox®)  In Vitro LD50  Kidney  Dermal Toxicity  Liver  Percutaneous Absorption  ADME Serviced  Corrosion  CYP Induction/Inhibition  Irritation  Caco2 absorption  Sensitization  Pgp transport  Photo Toxicity  Metabolism  Metabolic stability  Ocular Toxicity  BSEP inhibition  Endocrine Disruption  Drug / Drug Interaction  Estrogen receptor (ER) binding  Estrogen transcriptional activation  Target / Pathway Assays  Androgen receptor (AR) binding  Acute Inflammatory Response   Steroidogenesis  Apoptosis   Aromatase  Cell Proliferation   Cell Viability   Glutathione Homeostasis   Hepatobiliary Toxicity   Lipidosis   Membrane Integrity   Metabolism   Mitochondrial Function  .

. DABT Chief Science Officer .D. Ph.Building In Vitro Models for Predicting  In Vivo Toxicity The Road From Pharmaceuticals to Environmental Chemicals By: James McKim.

 LD50. organ effects • Subacute systemic toxicity Repeated‐dose study. and short exposure time Intrinsic toxicity of a chemical. typically 28 and 90 days Organ specific effects Establish NOAEL and LOAEL Regulatory implications FDA and EPA • Chronic systemic toxicity . typically 14 day Information on toxicity following repeated exposure Helps establish doses for subchronic studies  • Subchronic systemic toxicity Repeated‐dose.What is Systemic Toxicity and What do We Want From Alternative Methods?  Toxicity that occurs after a chemical is absorbed into general circulation • Acute systemic toxicity Single dose.

The Goal is to Predict Human Systemic Toxicity Predicts Toxicity Realistically What data are required for risk assessment NOAEL. Benchmark dose  Ideally Predicts NOAEL . LOAEL RfD.

. Building an In Vitro Model to Predict Repeat Dose Systemic Toxicity is Too Complex to Achieve Within the Next 10 Years! Most models  Alternative approach is focus on  to identify a plasma concentration predicting   that results in general systemic toxicity organ specific  toxicity BARRIER to SUCCESS Focus on the type of data needed to make decisions Proprietary to CeeTox. Inc.

In Vitro Models Should Provide Data That Can be Used to Make Decisions What data are required from an in vitro method in order to make decisions on systemic toxicity? Minimum dose that results in toxicity Maximally tolerated dose Plasma concentration that results in toxicity LOAEL NOAEL Margin of Safety .

. then a relationship between the animal response data and the in vitro response data can be explained mathematically. Defining a Working Hypothesis If the in vivo (animal) plasma concentration versus time and toxicity curves can be related to In vitro concentration versus toxicity response curves for a large number of compounds.

Relating Plasma Concentration to Toxicity  Start with pharmaceuticals • Intended for internal exposure at higher doses for shorter time • Environmental chemicals typically not intended to be ingested. exposure to lower doses. but for longer times  Develop a means to estimate the plasma concentration where general toxicity would occur • Develop a relationship between concentration response curves in vitro and Cmax in vivo at the dose where toxicity was observed in rat 14-day repeated oral dose studies  Clinical chemistry  Histopathology .

The Relationship Between In Vivo Plasma Concentration (Bioavailability) and Toxicity Provides the Basic Model In vivo and In vitro data JL Stevens (2006) Chem Res Toxicol 19. . Inc. 1393‐1401 Toxicity increases with potency = target  Cell models that lack target show toxicity = chemistry Proprietary to CeeTox.

Building the Predictive Model Based on Empirical Data Sets  Acquired in vivo data for pharmacokinetic parameters  Developed concentration response data in vitro  Based on multiple endpoint analysis developed a  relationship between in vivo and in vitro data sets  Test the model with small number of drugs  Validate model in large blinded study  Evaluate relevance of method for determining effects in humans .

Focus on Cell Biology and Physical Chemical Properties Improves the Model Biochemical Function Membrane Integrity Mitochondrial Function Cell Proliferation Redox‐State Oxidative Stress Apoptosis Physical-Chemical Properties Solubility Partition coefficients Pka Protein binding Metabolic stability Metabolic activation Pharmacology Transporter interaction CNS receptors Pharmacokinetic Parameters Cardiovascular receptors and ion  Clearance channels Bioavailability Volume of distribution .

Pharmacokinetic Data are Essential for Building In Vitro Models That Have In Vivo Relevance  Metabolic stability  Clearance  Absorption  Protein binding (Kd = on vs off kinetics)  Metabolic activation  Bioavailability  Volume of distribution .

Selecting a Cell Model That Consistently Provides Accurate Data  Rat hepatoma (H4IIe) Cell line • Retains oxidative metabolism • Low constitutive metabolism • Accepts a wide range of serum concentrations • Human hepatocyte model (HepaRG) .

Determining the Relationship Between In Vitro Concentration-Response and In Vivo Plasma Levels  Multiple endpoints improve interpretation • Membrane integrity • Cell number or mass • Mitochondrial function  Analysis and comparison to in vivo plasma Cmax/AUC  Development of weighting factors for each endpoint  Result was an estimate of the plasma concentration where toxicity would be expected to occur (Ctox) .

Multiple Endpoints Are Essential For Correct Interpretation of In Vitro Data CMPD H4IIE 24HR 20050817 Cell# MemTox 140 MTT ATP 120 GSH 100 % Control 80 60 40 20 0 1 10 100 1000 FIG. A: Exposure Concentration (µM) .

Important Take Home Points  Multiple endpoints based on cellular function are important  Understanding the cell model  Knowing what each assay is actually measuring .

Determining Weighting Factors  Membrane integrity • Highest weight = cell death • Concentration response and potency • Time adjustment   Mitochondrial function • Moderate weighting • Concentration response and potency • Time adjustment .

Evaluating Assumptions Against Rat In Vivo Data 100 R2 = 0.85 80 60 Keto (rat) 40 20 0 0 20 40 60 80 100 120 Predicted Concentration (uM) .

1 10 1000 FIG.001 0. KETOCONAZOLE H4IIE 24HR 80 Human actual Ctox = 1‐3 M 140 60 Cell# Ctox = 55 M 120 MemTox 40 MTT 20 100 ATP % Control 0 0. In Vitro Toxicity Data Correlates With Adverse Effects in Animals and Humans CHLOROQUINE H4IIE 24HR 20041006 140 Cell# Ctox = 5M MemTox 120 MTT ATP 100 Rat actual Ctox = 8 M.1 1 10 100 1000 80 Exposure Concentration (µM) 60 Cell# METHOTREXATE H4IIE 24HR MemTox 40 20060118. A: Exposure Concentration .  ATP 140 Ctox = 1 20 Human actual Ctox = 30 M 120 Rat actual Ctox = 0. 20060329 MTT Rat Actual Ctox = 60 M. Human actual Ctox = 0.88 M 0 100 0.1 1 10 100 1000 80 Exposure Concentration (µM) 60 40 20 0 0.7 M.

A: Exposure Concentration (µM) FIG. A: Exposure Concentration (µM) Proprietary to CeeTox. Velcade is an Approved Drug With High In Vitro Toxicity? Low Concentration High Concentration 24 hr Exposure 24 hr Exposure 140 140 Cell# Cell# 120 MemTox 120 MemTox MTT MTT 100 ATP 100 ATP BrDU BrDU % Control % Control 80 80 60 60 40 40 20 20 0 0 0. .001 0. Inc.1 10 1000 1 10 100 1000 FIG.

A: Exposure Concentration (µM) Proprietary to CeeTox. In Rat Primary (non-dividing) Hepatocytes Velcade Has Low Toxicity VELCADE (LOW) RAT HEPATOCYTE 24HR 20061205 Cell# 140 MemTox 120 MTT ATP 100 WST-1 % Control 80 60 40 20 0 0.001 0.1 10 1000 FIG. . Inc.

. Protein Binding Affinity Can Impact Toxicity CMPD1 H4IIE 48HR 10% SERUM HIGH DOSE CMPD1 H4IIE 48HR 20% SERUM Cell# 140 HIGH DOSE MemTox 120 MTT Cell# ATP 140 100 MemTox % Control 80 120 MTT 60 ATP 100 40 % Control 20 80 0 100 1000 10000 60 Exposure Concentration (µM) 40 CMPD1 H4IIE 48HR 5% SERUM 20 HIGH DOSE Cell# 140 0 MemTox 120 MTT 100 1000 10000 ATP 100 Exposure Concentration (µM) % Control 80 60 40 20 0 100 1000 10000 Exposure Concentration (µM) Proprietary to CeeTox. Inc.

.1 1 10 100 1000 Exposure Concentration (µM) Exposure Concentration (µM) Considered safe in IND enabling safety studies Considered safe in clinical trials Why is it toxic in vitro? Proprietary to CeeTox. Inc. Metabolic Stability is Key for Correct Interpretation of In Vitro Data TERFENADINE H4IIE 6HR 20040929 TERFENADINE H4IIE 24HR 20040728 140 Cell# 140 Cell# MemTox MemTox 120 MTT 120 MTT ATP ATP 100 100 % Control % Control 80 80 60 60 40 40 20 20 0 0 0.1 1 10 100 1000 0.

 Inc. Importance of Metabolic Stability for Identifying False Positives  Terfenadine is a prodrug  Terfenadine undergoes first‐pass metabolism  Primary metabolite is efficacious not toxic  In Vivo blood levels of terfenadine are low Proprietary to CeeTox. .

0 20.0 60.0 % GSH relative 100.5 hr incubation with induced microsomes 140.0 No Drug Flutamide NEM Acetaminophen Tacrine Test Article Full Reaction -NADPH reaction .0 40.0 0.Metabolic Activation Identifies False Negative Results  Tacrine = liver  Flutamide = liver  Acetaminophen = liver GSH Depletion relative to no drug control after 3.0 80.0 120.

Proprietary to CeeTox. Inc. "Building a tiered approach to in vitro predictive toxicity screening: a focus on assays with in vivo relevance. Determining Relevance to Human Systemic Toxicity Ctox (M) 100 10 Azathioprine 1 1e-8 1e-7 1e-6 1e-5 1e-4 1e-3 max therapeutic [plasma] (log M) Ctox = 5 M MTPC = 8 M    In vitro margin of safety <= 1 McKim.. Jr. J. . M."  Comb Chem High Throughput Screen 13(2): 188‐206.

B: Exposure Concentration (µM) FIG. A: Exposure Concentration (µM) AZATHIOPRINE H4IIE 24HR 20051012 500 Caspase 3 400 Fluorescence Units 300 200 Extremely Toxic 100 Ctox = < 5 µM 0 1 10 100 1000 FIG. . Azathioprine: Inhibits Purine Synthesis and Produces Toxicity Under Clinical Conditions AZATHIOPRINE H4IIE 24HR 20051012 AZATHIOPRINE H4IIE 24HR 20051012 140 140 50 GSH Cell# ISO 120 MemTox 120 MTT 40 8-Isoprostane (pg/ml) 100 ATP 100 GSH % Control % Control 80 30 80 60 60 20 40 40 10 20 20 0 0 0 1 10 100 1000 1 10 100 1000 FIG. C: Exposure Concentration (µM) Proprietary to CeeTox. Inc.

There is Good Concordance Between Predicted Toxicity Validated to Rat Data and Human Clinical Data Ctox (M) 100 Thioridazine 10 Azathioprine 1 1e-8 1e-7 1e-6 1e-5 1e-4 1e-3 max therapeutic [plasma] (log M) .

83 .1 1 10 100 1000 Exposure Concentration (µM) Ctox = 11 M MTPC = 6 M    In vitro margin of safety = 1. THIORIDAZINE H4IIE 24HR 20041006 140 Cell# MemTox 120 MTT ATP 100 80 60 40 20 0 0.

Drug Potency Can Influence Plasma Concentration and Toxicity Valproic acid Ctox (M) 100 Thioridazine 10 Paroxetine Chloroquine Azathioprine 1 1e-8 1e-7 1e-6 1e-5 1e-4 1e-3 max therapeutic [plasma] (log M) .

3 M      In Vitro Margin of Safety = 50 .Antidepressant Paxil (SSRI) Considered Safe Under Appropriate Use: Rare Incidences of Hepatotoxicity Cell# Paroxetine H4IIE 24HR 20051108 MemTox MTT 140 ATP 120 100 80 60 40 20 0 1 10 100 1000 FIG. A: Exposure Concentration Ctox = 15 M MTPC = 0.

. 1120. Predicting Bioavailability of a New Drug and Maximum Therapeutic Plasma Concentration • In Vitro CLint (human microsomes) • Scaled in vitro CLint • Calculate bioavailability (%f) – f=1–E – E = fu x CLint / QH Kuhnz and Gieschen (1998) Drug Metab Disp 26.

 organ effects  Subacute systemic toxicity  Repeated‐dose study.What is Systemic Toxicity and What do We Want From Alternative Methods?  Toxicity that occurs after a chemical is absorbed into general circulation  Acute systemic toxicity  Single dose. typically 28 and 90 days  Organ specific effects  Establish NOAEL and LOAEL  Regulatory implications FDA and EPA  Chronic systemic toxicity . typically 14 day  Information on toxicity following repeated exposure  Helps establish doses for subchronic studies   Subchronic systemic toxicity  Repeated‐dose. LD50. and short exposure time  Intrinsic toxicity of a chemical.

 SOT 2010  Results show that an integrated in vitro approach can provide good estimates of an LD50 . Predicting a Rat Acute Oral LD50 Dose  Collaborative research effort with LOREAL Paris  Integrative or systems biology + Computational Toxicology • Multiple endpoints related to cell health • Receptor binding data (pharmacology) • Physical Chemical parameters • Approach has been evaluated with more than 200  chemicals • Posters at the World Congress in Rome 2009.

 1‐2‐3 Cat. 5 Toxicity Oral < 300 >300 > 2000 (mg/Kg) < 500 . 5 Toxicity Oral <  5 >5  > 50 > 300 Safe (mg/Kg) < 50 < 300 < 2000 >2000 Revised Categories Acute  Cat.Global Harmonization System for Acute Toxicity Standard Categories Acute  Cat. 1 Cat. 4 Cat.    2 Cat. 3 Cat. 4 Cat.

 2010 Presented at the World Congress on Alternatives to Animal Testing  Montréal. R et al. August 2011 . Addition of Pharmacology and Physical Chemistry Properties Improves Model Performance Performance of the standard model at the Performance of the integrated model at the LD50 threshold of 500 mg/kg LD50 threshold of 500 mg/kg REDUCTION OF THE FALSE NEGATIVE RATE (yellow area) Note.

An In Vitro Acute LD50 Toxicity Model for Cosmetic Industry Note. 2010 Presented at the World Congress on Alternatives to Animal  Testing Montréal.  . August 2011 . R et al.

Application of the Optimized Version of the In Vitro Acute LD50 Assay  Early screening with specific LD50 thresholds: case 1  Classification and labeling purposes (GHS categorization): case 2&3  Change current classification systems for acute oral toxicity as suggested in the final document of the FP6 AcuteTox program: case 3 .

Conclusions  It is currently possible to predict in vivo toxicity (animals) with  a cell‐based model  Approach  includes functional endpoints for cell health.  physical and chemical and pharmacokinetic properties  Models are based initially on plasma concentrations that result  in toxicity  High level of concordance with in vivo toxicity  An acute LD50 can be determined .

Future Improvements to the Model  Develop a drug/chemical database with animal 28 or 90 day data. in vitro toxicity data. chemical properties  Determine a calculation for NOAEL in humans  Broaden the chemical space .

CeeTox Overview of Services … a wide range of in vitro toxicology and safety assessment services  Systemic Toxicity Services  Organ Specific Toxicity – CTOX Panel® Models ‒ Acute Toxicity Screen – Heart (CardioTox®)  Kidney  In vitro LD50 (acute tox)  Liver  CYP Induction/Inhibition  Dermal Toxicity   Percutaneous Absorption Corrosion  Drug / Drug Interaction   Irritation Sensitization  Target / Pathway Assays  Acute Inflammatory Response   Photo Toxicity  Apoptosis   Cell Proliferation   Ocular Toxicity  Cell Viability   Glutathione Homeostasis   Hepatobiliary Toxicity   Kidney Collecting Duct Toxicity   Endocrine Disruption  Kidney Distal Tubular Toxicity   Estrogen receptor (ER) binding  Kidney Proximal Tubule Toxicity   Estrogen transcriptional activation  Lipidosis   Androgen receptor (AR) binding  Membrane Integrity   Steroidogenesis  Metabolism   Aromatase  Mitochondrial Function  .

Acknowledgments CeeTox staff EPA and ToxCast Program .