Division of Neurotoxicology/NCTR:Division of Neurotoxicology/NCTR:Neurotoxicological Research in Neurotoxicological Research in

Support of Science-Based DecisionsSupport of Science-Based Decisions

William Slikker, Jr., Ph.D.William Slikker, Jr., Ph.D.

Director, Division of NeurotoxicologyDirector, Division of Neurotoxicology

National Center for Toxicological ResearchNational Center for Toxicological Research

Food and Drug AdministrationFood and Drug Administration

Jefferson, Arkansas 72079Jefferson, Arkansas 72079

Mission StatementMission Statement

The mission of the Division of Neuro-The mission of the Division of Neuro-toxicology is to develop and validate toxicology is to develop and validate quantitative biomarkers of neurotoxicity quantitative biomarkers of neurotoxicity and utilize them to elucidate toxic and utilize them to elucidate toxic mechanisms and increase the certainty of mechanisms and increase the certainty of assumptions underlying risk assessment assumptions underlying risk assessment for neurotoxicants.for neurotoxicants.

NeurotoxicityNeurotoxicity

Neurotoxicity may be defined as any adverse effect on the Neurotoxicity may be defined as any adverse effect on the structure or function of the central and/or peripheral structure or function of the central and/or peripheral nervous system by a biological, chemical, or physical agent.nervous system by a biological, chemical, or physical agent.

Adverse effects can include both unwanted effects and any Adverse effects can include both unwanted effects and any alteration from baseline that diminishes the ability of an alteration from baseline that diminishes the ability of an organism to survive, reproduce or adapt to its environment.organism to survive, reproduce or adapt to its environment.

Neurotoxic effects may be permanent or reversible, Neurotoxic effects may be permanent or reversible, produced by neuropharmacological or neuro-degenerative produced by neuropharmacological or neuro-degenerative properties of a neurotoxicant, or the result of direct or properties of a neurotoxicant, or the result of direct or indirect actions on the nervous system.indirect actions on the nervous system.

Incidence of brain related Incidence of brain related disorders: Social Impactdisorders: Social Impact

One out of four Americans will sufferOne out of four Americans will suffer

from a brain-related disorder during from a brain-related disorder during their lifetime.their lifetime.

One out of ten school-age childrenOne out of ten school-age children

has a functional deficit.has a functional deficit.

Incidence of brain-related Incidence of brain-related disorders: Economic Impactdisorders: Economic Impact

In the U.S., brain-related disorders In the U.S., brain-related disorders account for more hospitalizations than account for more hospitalizations than any other major disease group.any other major disease group.

Estimated cost of treatment, rehabilitation, Estimated cost of treatment, rehabilitation, and related consequences is $400 billion and related consequences is $400 billion each year.each year.

Thus, brain-related toxicity results in long-Thus, brain-related toxicity results in long-lasting costs to human health and our lasting costs to human health and our national economy.national economy.

Multidisciplinary Research ApproachMultidisciplinary Research Approach

Many of the relevant effects can be Many of the relevant effects can be measured by neurochemical, measured by neurochemical, neurophysiological, neuropathological or neurophysiological, neuropathological or behavioral techniques.behavioral techniques.

A multidisciplinary approach is necessary A multidisciplinary approach is necessary to assess neurotoxicity because of the to assess neurotoxicity because of the complex and diverse functions of the complex and diverse functions of the nervous system.nervous system.

Molecular

Neurochemical

Morphological

Physiological

Behavioral

Psychological

Discipline - Continuum Approach

Genomics, Proteomics

Computational M

odeling

Research ApproachResearch Approach

Evaluation of Available EndpointsEvaluation of Available Endpoints

Neuropathology Neurobiology Neurophysiology BehaviorNeuropathology Neurobiology Neurophysiology Behavior

Light microscopyLight microscopyHistochemistryHistochemistryImmunohistochem.Immunohistochem.Electron Electron MicroscopyMicroscopy

NeurochemistryNeurochemistryMolecular Biol.Molecular Biol.MicrodialysisMicrodialysisProtein Biochem.Protein Biochem.Cell CultureCell Culture

Neurotoxicity ProfileNeurotoxicity Profile

EEGEEGEvoked PotentialsEvoked PotentialsSingle-unitSingle-unit RecordingsRecordings

SpontaneousSpontaneousSchedule-Schedule- controlledcontrolled““Challenge”Challenge”

Division of Neurotoxicology Senior StaffDivision of Neurotoxicology Senior Staff

Syed Ali, Ph.D.Syed Ali, Ph.D. Neurochemical ToxicologyNeurochemical Toxicology

Zbigniew Binienda, D.V.M., Ph.D.Zbigniew Binienda, D.V.M., Ph.D. Neurophysiological Neurophysiological

ToxicologyToxicology

John Bowyer, Ph.D.John Bowyer, Ph.D. Neurobiological ToxicologyNeurobiological Toxicology

Sherry Ferguson, Ph.D.Sherry Ferguson, Ph.D. Developmental Neuro-Developmental Neuro- behavioral Toxicology behavioral Toxicology

Merle Paule, Ph.D.Merle Paule, Ph.D. Behavioral NeurotoxicologyBehavioral Neurotoxicology

Andy Scallet, Ph.D.Andy Scallet, Ph.D. Experimental NeurohistologyExperimental Neurohistology

Larry Schmued, Ph.D.Larry Schmued, Ph.D. Neuroanatomical Histo-Neuroanatomical Histo- chemistry chemistry

NCTR/FDA Nonhuman NCTR/FDA Nonhuman Primate CenterPrimate Center

CapabilityCapability

– General toxicology and pharmacokinetics General toxicology and pharmacokinetics – Breeding colonyBreeding colony– Neurotoxicology Neurotoxicology – BehaviorBehavior

Extramural FundingExtramural Funding– Averaged over one-half million dollars per year during Averaged over one-half million dollars per year during

last 13 yearslast 13 years ProductivityProductivity

– Over ten publications per year, unique to FDAOver ten publications per year, unique to FDA

Learning Task: Accuracy

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 380

10

20

30

40

50

60

70

CONTROL 0.1 mg/kg MK-801 1.0 mg/kg MK-801

20 mg/kg REMACEMIDE 50 mg/kg REMACEMIDE

BLOCK OF TEST SESSIONS

OV

ER

ALL

AC

CU

RA

CY

Cases of comparable behavioral effects of drugs in humans and Cases of comparable behavioral effects of drugs in humans and animals (monkeys)animals (monkeys)

Acute Effects CitationsDrug Primary Effect Monkey Human

THC over-estimation oftime

Schulze, et al.,1998

Hicks, et al.,1984

MJ smoke short-term memoryimpairment

Schulze, et al.,1989

Darley, et al.,1974

Chlorpromazine decrease responseinitiation

Ferguson &Paule, 1992

Tecce, et al.,1975

Diazepam learning and memoryimpairments

Schulze et al.,1989

Ghoneim, et al.,1984

Morphine decrease responserates

Schulze & Paule,1991

Goldberg, et al.,1982

Atropine learning disruption Schulze, et al.,1992

Higgins, et al.,1989

Chronic Effects CitationsDrug Primary Effect Monkey Human

MJ smoke amotivationalsyndrome

Paule, et al.,1992

Lantner, 1982

Evaluation of Pediatric Anticonvulsants/ NMDA AntagonistsEvaluation of Pediatric Anticonvulsants/ NMDA Antagonists

NCTR, CFSAN and CDER are collaborating on a volumetric MRI imaging approach to detect drug effects on apoptosis in the developing brain.

Domoic Acid NeurotoxicityDomoic Acid Neurotoxicity

Acceptable doses of domoic acid based on silver Acceptable doses of domoic acid based on silver stain grain counts in hippocampus (CA2 region)stain grain counts in hippocampus (CA2 region)

Method Intravenousdose (mg/kg)

Oral dosea

(mg/kg)Concentration in200g of seafood

(PPM)b

LOAEL 0.00017 0.034 12.0

Benchmark 0.0009 0.018 6.4

Risk=10-4 0.0014 0.028 10.0

aBased on 5% absorption of oral dosebBased on 70 kg person

ConclusionsConclusions

Quantitative biomarkers of neurotoxicity can be Quantitative biomarkers of neurotoxicity can be determined.determined.

Continuous (nonquantal) data can be used in Continuous (nonquantal) data can be used in quantitative risk assessment.quantitative risk assessment.

Outcome from several risk assessment Outcome from several risk assessment approaches can be compared.approaches can be compared.

The locations of the brain regions sensitive to substituted The locations of the brain regions sensitive to substituted amphetamine-induced somatic neurodegeration can be amphetamine-induced somatic neurodegeration can be used as a map for the areas in which:used as a map for the areas in which:

More subtle types of long-term effects can be examined More subtle types of long-term effects can be examined

(and possibly used as biomarkers for neurotoxicity):(and possibly used as biomarkers for neurotoxicity):

– mRNA species changes as detected by cDNA arrays.mRNA species changes as detected by cDNA arrays.

– Changes in protein species detected by proteomics.Changes in protein species detected by proteomics. Lower doses of substituted amphetamines, that do not Lower doses of substituted amphetamines, that do not

produce overt signs of neurotoxicity, may produce long-produce overt signs of neurotoxicity, may produce long-term ultrastructural and neurochemical changes.term ultrastructural and neurochemical changes.

RNA from the Substantia Nigra 14 days Post Dosing

Control (Saline) Amphetamine

Publication and Publication and Review Review

ActivitiesActivities

Peer-reviewed PublicationsPeer-reviewed Publications Book ChaptersBook Chapters 40 per year40 per year

Reviews: FDA Guidance and Reviews: FDA Guidance and Harmonization DocumentsHarmonization Documents

Red Book II (CFSAN)Red Book II (CFSAN) Reproductive and Developmental Reproductive and Developmental

Toxicity (CDER)Toxicity (CDER) USEPA Food Quality Protection Act, USEPA Food Quality Protection Act,

10X Factor10X Factor OECD Guidance Document for OECD Guidance Document for

Neurotoxicity TestingNeurotoxicity Testing WHO, IARC Working Group on WHO, IARC Working Group on

Pharmaceutical AgentsPharmaceutical Agents

A Diversified Portfolio of Leveraging is ImportantA Diversified Portfolio of Leveraging is Important

CRADA/UALR - Effects of Prenatal Cocaine on Behavioral CRADA/UALR - Effects of Prenatal Cocaine on Behavioral Plasticity - Paule - $250,000 - 5 yearsPlasticity - Paule - $250,000 - 5 years

NTP/IAG- Endocrine Disruptors (Ongoing) - Ferguson and NTP/IAG- Endocrine Disruptors (Ongoing) - Ferguson and ScalletScallet

EPA/ IAG - Quantitative Procedures for Neurotoxicity Risk EPA/ IAG - Quantitative Procedures for Neurotoxicity Risk Assessment - Slikker - $200,000 - 2 yearsAssessment - Slikker - $200,000 - 2 years

CRADA/AstraZeneca - Development of a Nonhuman Primate CRADA/AstraZeneca - Development of a Nonhuman Primate Model for Studying the Consequences of Long-term Model for Studying the Consequences of Long-term Anticonvulsant Medication on Complex Brain Functions - Anticonvulsant Medication on Complex Brain Functions - Paule - $1.1 million - 5 yearsPaule - $1.1 million - 5 years

Ongoing Approved ProtocolOngoing Approved Protocol

Multiple cDNA Array Analysis of the Multiple cDNA Array Analysis of the Temporal Changes in mRNA Species after Temporal Changes in mRNA Species after Neurotoxic EventsNeurotoxic Events

John F. Bowyer, John Tor-Agbidye, A.W. John F. Bowyer, John Tor-Agbidye, A.W. Tank (Univ. Rochester), James P. Tank (Univ. Rochester), James P. O’Callaghan (NIOSH), Willard Freeman and O’Callaghan (NIOSH), Willard Freeman and Kent Vrana (Wake Forest Univ.), Syed Ali, Kent Vrana (Wake Forest Univ.), Syed Ali, Angela Harris, William Slikker, Jr.Angela Harris, William Slikker, Jr.

Ongoing Approved ProtocolOngoing Approved Protocol

CRADA

NCTR/ AstraZeneca

Development of a nonhuman primate model for studying the consequences of long-term anticonvulsant medication on complex brain functions: Rodent equivalent

Merle G. Paule (NCTR) and Edwin Pearson (AstraZeneca)

Protocol Under ReviewProtocol Under Review

Neurotoxicity Assessment of Ephedra-Neurotoxicity Assessment of Ephedra-Containing Dietary Supplements: Containing Dietary Supplements: Application of cDNA Array and Application of cDNA Array and Neurochemical ApproachesNeurochemical Approaches

Syed F. Ali, Bill Gurley (UAMS), Syed Z. Imam, Syed F. Ali, Bill Gurley (UAMS), Syed Z. Imam, Tucker Patterson, Helen Duhart, Saber Hussain Tucker Patterson, Helen Duhart, Saber Hussain (Mantech), Glenn Newport, Angela Harris, John (Mantech), Glenn Newport, Angela Harris, John Bowyer, Zbigniew Binienda, William Slikker, Jr.Bowyer, Zbigniew Binienda, William Slikker, Jr.

Concept Papers ApprovedConcept Papers Approved

Study on Alteration of the Programming of Study on Alteration of the Programming of the 5-HT System by Nicotine Exposure the 5-HT System by Nicotine Exposure During DevelopmentDuring Development

Zengjun Xu, Theodore Slotkin and Frederic J. Zengjun Xu, Theodore Slotkin and Frederic J. Seidler (Duke University), John F. Bowyer, Seidler (Duke University), John F. Bowyer, William Slikker, Jr.William Slikker, Jr.

Concept Papers ApprovedConcept Papers Approved

Enhancement of mitochondrial energy Enhancement of mitochondrial energy metabolism by carnitines and related metabolism by carnitines and related dietary supplements: Application of cDNA dietary supplements: Application of cDNA microarrays, neurophysiological, and microarrays, neurophysiological, and neurochemical approachesneurochemical approaches

Zbigniew Binienda (NCTR), Franco Gaetani and M. Ashraf Virmani (Sigma-Tau Health Science, S.P.A.), Chung S. Kim (CFSAN), Syed Ali and William Slikker, Jr. (NCTR)

Future DirectionsFuture Directions

Thimerosal (CBER, CDER)Thimerosal (CBER, CDER)

Nutritional Supplements, e.g. infant Nutritional Supplements, e.g. infant formula (CFSAN)formula (CFSAN)

Therapeutics, e.g. anticonvulsants, Therapeutics, e.g. anticonvulsants, neuroprotectants (CDER, CFSAN)neuroprotectants (CDER, CFSAN)

Future Directions-Resource DevelopmentFuture Directions-Resource Development

Imaging (CFSAN, CDER, CDRH)Imaging (CFSAN, CDER, CDRH)

Animal models, e.g. transgenic, nonhuman Animal models, e.g. transgenic, nonhuman primates (CFSAN, CBER, CDER)primates (CFSAN, CBER, CDER)

Genomics and proteomics (CDER, CBER, Genomics and proteomics (CDER, CBER, CFSAN)CFSAN)

FDA Intercenter Neurotoxicity Working GroupFDA Intercenter Neurotoxicity Working Group The FDA is a science-based regulatory agency with broad The FDA is a science-based regulatory agency with broad

responsibilities for promoting and protecting public health.responsibilities for promoting and protecting public health.

The credibility of and confidence in the FDA’s regulatory decisions are The credibility of and confidence in the FDA’s regulatory decisions are based on the agency’s ability to apply sound science in its review and based on the agency’s ability to apply sound science in its review and regulatory process.regulatory process.

Efforts are needed to ensure that regulators have the appropriate Efforts are needed to ensure that regulators have the appropriate expertise and necessary information in various scientific disciplines for expertise and necessary information in various scientific disciplines for making appropriate decisions based on high quality science.making appropriate decisions based on high quality science.

To assist the FDA in addressing regulatory concerns related to To assist the FDA in addressing regulatory concerns related to neurotoxicity, personnel from the Centers within the agency, who are neurotoxicity, personnel from the Centers within the agency, who are actively involved in neuroscience research or regulatory activities, have actively involved in neuroscience research or regulatory activities, have organized an Inter-center Neurotoxicity Working Group.organized an Inter-center Neurotoxicity Working Group.