Evaluating Toxicology in Special Populations

The Pregnant, Unborn and Children as Sensitive

Subpopulations Per Se

Joseph F. Holson, Ph.D., DABFEWIL Research Laboratories, LLC

8:40 A.M. – 9:20 P.M.Monday, November 7, 2005Williamsburg Marriott Hotel

Williamsburg, VA

Primary Causes of Healthcare Costs at WIL, LLC – 2005 to Date

Case Predominant Diagnosis Description % of Total Cost1 Other Congenital Musculoskeletal Anaomalies 22.3%2 Vascular Insufficiency of Intestine 12.0%3 Other and Ill-Defined Conditions Originating in the Prenatal Period 10.8%4 Acute Myocardial Infarction 5.4%5 Complications Peculiar to Certain Specified Procedures 5.4%6 Antepartum Hemorrhage, Abruptio Placentae, and Placenta Previa 3.1%7 Uterine Leiomyoma 2.9%8 Strabismus and other Disorders of Binocular Eye Movements 2.8%9 Malposition and Malpresentation of Fetus 22.0%10 Acute Appendicitis 2.1%11 Abnormality of Organs and Soft Tissues of Pelvis 2.1%12 Other Cellulitis and Abcess 2.0%13 Diabetes Mellitus 1.9%14 Conduction Disorders 1.8%15 Neoplasm of Uncertain Behavior 1.8%16 Chronic Bronchitis 1.8%

Total 100.0%

Sensitive Population Subtotal (7 of 16 cases) 74.9%

Comparison of Overall Spontaneous Malformation Rates in Different Species

Species Mean % Range (%) N

Rat 0.33 0-1.6 9643

Mouse 1.2 0-3 5207

Rabbit 3.2 0-10 4708

Dog 5.5 5.3-5.7 167

Human 4.0 3-9 Multiple Surveys

WIL Research Laboratories, LLC Historical Control Database

Recent Public Awareness

• 1996 – Food Quality Protection Act (FQPA)• Tolerance levels are appropriately protective of children • Apply an additional safety factor of 10 to account for the

potential for pre- and post-natal toxicity, unless EPA determines that another factor is adequately protective

• 2000 – Children’s Health Act (CHA)• Authorized National Children’s Study to study environmental

influences

• 2002 – Best Pharmaceuticals for Children Act (BPCA)• Reauthorized FDAMA 6 mos. patent extension

• Authorized FDA request for study (generic and patent)

Sensitive Subpopulation Timeline

1997

FDAMA

1996

FQPA

1966

Wilson’s Principles

1979

NCTR Collaborative

Behavioral Study

1775

Pott Describes Scrotal Cancer

in Chimney Sweeps

1934

Phenylketonuria Discovered

1973

EPA Begins Lead Phase-out from

Gasoline

1990

CAA Amendment Banned Lead & Lead Additives in Gasoline

1978

CPSC Bans Use of Lead Paints in

Housing

Chloramphenicol- Gray Baby Syndrome

1971

Hexachlorophene Effects on Rat

Brains Reported

DES(Herbst & Scully)

1957

1972

Hexachlorophene Human Effects

1951

Retinopathy of Prematurity Linked to

Oxygen Therapy

1956

Sulfa Drugs Associated with

Kernicterus

1982

Benzyl Alcohol Induces “Gasping Syndrome”

Isotretinoin Approved

1980

Reye’s Syndrome Linked to Aspirin

1991

First FIFRA DNT

Requirement

1904

Paint Dust Identified as

Major Source of Lead in Children

2004

CERHR Publishes Fluoxetine

Report

1998

FDA Pediatric

RuleIssued

2002

BPCA

2000

Children’s Health Act

2003

Pediatric Research Equity Act

1962

Thalidomide Epidemic 1963

Conference on Prenatal Drug

Effects

Ongoing MeHg and DES Exposures

1981

First ACE-Fetopathy Case Report

NCTR Concordance Study

NCTR Collaborative

Study Reported

1985

1993

“ACE-Fetopathy” Coined

Rubella Epidemic(Gregg)

1941

~ Fractional (Percentage) Temporal Risks – Lifestages of US Population

Preterm Infant Child Adolescent

9 Mos. 24 Mos. 10 Years 4 Years

% of 85-Year

Lifespan1% 2% 12% 5%

Adult69 Years

80%

~ 300 Million

~ 4 MillionBirths/ Year (2003 Data)

~ 1 in 5 lost before pregnancy recognized;

additional 10-15% spontaneously aborted after

recognition

1%

A B C D E F

Premating to Conception

Conception to Implantation

Implantation to Closure of Hard Palate

Hard-Palate Closure to End of Pregnancy

Birth to Weaning Weaning to Sexual Maturity

Parturition Litter Size Landmarks of Sexual DevelopmentGestation Length Pup Viability Neurobehavioral Assessment F1 Mating and Fertility Pup Weight Acoustic Startle Response

Organ Weights Motor Activity Learning & Memory

ParturitionGestation Length Pup Viability Litter SizeLandmarks of Sexual Development Pup WeightNeurobehavioral Assessment Organ Weights Acoustic Startle Response F1 Mating and Fertility Motor Activity Hormonal Analyses Learning & Memory Ovarian QuantificationHistopathology Premature Senescence

Postimplantation LossViable FetusesMalformations & VariationsFetal Weight

Postimplantation LossViable FetusesMalformationsVariationsFetal Weight

Estrous Cyclicity Mating Corpora Lutea Fertility Implantation SitesPre-Implantation Loss Spermatogenesis

Estrous CyclicityMatingFertilityCorpora LuteaImplantation SitesPre-Implantation LossSpermatogenesis

Denotes Dosing Period

Standard DART Study Designs

Single- and Multigenerational

Satellite Phase

OECD 415, OECD 416, OPPTS 870.3800, FDA Redbook I, NTP RACB

F1

F2 ????????????????

????????????????

Pre- and Postnatal Development

F1

ICH 4.1.2F0

????????????????

Prenatal DevelopmentICH 4.1.3 OECD 414

OPPTS 870.3600 870.3700

Fertility StudyICH 4.1.12W4W

ƒ CMAX

AUC

ƒ CMAX

AUC

10W

Holson, et al. (2005)

Maternal Physiologic Changes during Pregnancy

Parameter Early Middle Term

Heart Rate ↑ 10 BPM

Cardiac Output ↑ 25-50% of NP ↑ 35-60% of NP ↑ 35-60% of NP

Plasma Volume ? ↑ 30-70% of NP ↑ 40-90% of NP

Plasma Protein ↓ 60% in Plasma Albumin Level; ↑ Free Drug

Renal Blood Flow ↑ 50% of NP ↑ 50% of NP Declines in last mo.

GFR ↑ 45% of NP* ↑ 60% of NP ↔

GI Motility ↓ ↓ ↓

GI Emptying Time ↑ ↑ ↑

Mean Blood Pressure ↓

Tidal Volume ↑ 40% of NP

Mean O2 Consumption ↑ 20% of NP

Metabolism ↓ Hepatic Drug Metabolism (?)

Page et al. (1972)* = Earliest Physiological Change

Pregnancy:Tissue/Fluid Compartments

• Uterus• Placenta• Embryo/fetus• Mammary glands• Liver (40-50%)• Fat Stores (variable)

• Maternal Plasma Volume (PV)

• ECF Volume

Tissues which Increase in Volume with Pregnancy

Page et al. (1972)

Pregnancy-Related Mortality

• The risk of death from complications of pregnancy has decreased approximately 99% during the twentieth century, from approximately 850 maternal deaths per 100,000 live births in 1900 to 7.5 in 1982

• However, since 1982, no further decrease has occurred in maternal mortality in the United States

MMWR (2003)

Maternal Death and Pregnancy Outcome

MMWR (2003)

Causes of Maternal Mortality

MMWR (2003)

Case Study: Dystocia, Extended Parturition and/or Pregnancy

• 2-generation with second mating phase of F1, vapor inhalation, used industrially, OTC pharmaceutically

PPM 0 70 300 500 700

F0 0 0 0 2/24 3/26

F1-1st 0 0 0 0 1/17

F1-2nd 0 0 1/21 1/18 0/12

• HC then: 2/333 = 0.60%• HC now: 4/1100 = 0.36%

Holson, et al.Holson, et al. (2005)

What is Meant by Maternal Toxicity?

• No universally accepted definition• Varies among laboratories and regulatory agencies

• Broad range of severity in endpoints• Maternal deaths (~10%)

• Minimal changes in weight gain (as low as 5%)

• Endpoints used to define should vary depending on species• Rat vs. rabbit

OECD Workshop:Maternal Toxicity in Experimental Animals

• Criteria and endpoints that define maternal toxicity remain rather crude

• Maternal toxicity is expected in standard in guideline animal studies

• Maternal toxicity often occurs concomitantly with developmental toxicity

• Maternal toxicity may secondarily affect the normal development of the embryo/fetus

Welsch (2004)

Hypothetical Comparison of Maternaland Developmental Toxicity Profiles

Toxicity

Log of Dose

Maternal

Developmental

The position of these curves is determined by the relative sensitivities of the maternal and developing

organism to xenobiotic Holson, et al. (2005)

Maternal Influence on Development

Mother

Lactation (Neonate/Infant)

Toddler

Child

Adolescent

Gestation(Embryo/Fetus)

More Maternal Influence

Less Maternal Influence

Holson, et al. (2005)

Examples of Maternally Mediated Developmental Toxicity

• Embryonic Hypoxia• Decreased uterine blood flow (Vasopressin)• Anemia (Diflunisal)• Altered oxygen-carrying capacity of blood (CO)

• Zinc Deficiency• Metallothionein induction

Daston (2004)

OECD Workshop Summary

• Measuring endpoints of acute phase dose-response could define how physiological changes in the mother might be related to developmental toxicity

• Acute maternal responses may remain undetected using current, limited endpoints

• Some past investigations may indicate potential directions to reduce uncertainties about maternal toxicity and possible links to developmental toxicity

Welsch (2004)

Offspring Sensitivity to Xenobiotics

• Qualitative Differences• Effects more persistent than adults• Effects more severe than adults

• Quantitative Differences• Effects occurring at lower exposures than in adults

Ladics et al. (2003)

Relationship Between Developmentand Phenotypic Diversity

Phenotypic Expression

and Diversity

Time in Development (Age)

EmbryonicPeriod

FetalPeriod

PostnatalPeriod

Extent of Differentiation

Birth

Holson, et al. (2005)

Examples of Lifestage-Specific Xenobiotic Concerns

Embryo Fetus Neonate Infant Child Adolescent

Pregnant Mother Lactating Mother

Diabetes/Epilepsy

Lead/Organochlorine Stores

Adult

DES

Coal Tar

HCP

Lead

CAP

Litigation continues - Involves forseeability

Medical community believed DES promoted

progesterone synthesis by FPU

First randomized control study with placebo: safe

but no efficacy

Present

(Walker - Mouse) Numerous experimental

studies to develop a good animal model

Litigation

Karnaby Clinical Studies Apparent safety at high doses

Green, et al., Intersexuality in

mice

SYNTHESISfirst orally active estrogen mimic

DES-Vaginal CCA link in 8 young women (15-22 years)

Debate about DES use in cattle and residues in meat

1971Herbst & Scully

Not considered a teratogenKalter & Warkany

Prescribed to prevent spontaneous abortion

Based on Smith

Gabriel-RobezCleft Palates and

heart defects in mice

Juvenile Toxicity Studies

• Generally measure same endpoints as adult studies, just starting at earlier age – developmental measures• Clinical Findings, Weight, Food, Clinical and Anatomic

Pathology• However, organ systems not completely developed

at time of treatment can also be evaluated• Sexual Maturation• Reproductive Function• CNS (Neurobehavioral Paradigms, Brain Morphometry)• Immune Function• Renal Function• Pulmonary Function

Comparison of Prenatal and Postnatal Modes of Exposure

Drug Transfer to Offspring

Drug Levels in Offspring

Maternal Blood vs.Offspring Levels

Exposure Route toOffspring

Commentary

Prenatal

Nearly all transferred

Cmax and AUC measured

Maternal often a surrogate

Modulated IV exposure, via placenta

Timing of exposure is critical

Postnatal

Apparent selectivity (“barrier”)

Not routinely measured

Maternal levels probably NOT a good predictor

Oral, via immature GI tract

Extent of transfer to milk and neonatal bioavailability is key to differentiating indirect (maternal) effects from neonatal sensitivity

Prenatal Treatment Postnatal

Embryo/Fetus Placenta Mother Mammae Neonate

Holson, et al. (2005)

Case Study: Functional Alteration Example ACE Inhibition-Induced Fetopathy (Human)

• Organogenesis (classically defined) is unaffected

• Effects are severe

• Risk is low

• Caused by ACEinh that cross placenta

ACEinhFetal

Hypotension

RenalCompromise

(Anuria)Oligohydramnios

Calvarial Hypoplasia

Neonatal Anuria

IUGR

Death

• RAS (renin-angiotensin system) matures around GD 17

• No ‘apparent’ effect in initial reproductive studies• Nonstatistically significant increase in postnatal mortality

(~8%)

• Subsequent postnatal studies with direct administration to pups• Growth retardation

• Renal alterations (anatomic and functional)

• Mortality increased to more than 30%

Case Study: Functional Alteration Example ACE Inhibition in Developing Rats

Holson, Teratology Society Meeting, 1999

References

• Holson, et al. (2005) Significance, Reliability, and Interpretation of Developmental and Reproductive Toxicity Study Findings in: Developmental and Reproductive Toxicology – A Practical Approach, R.D. Hood, Editor. Taylor & Francis, Boca Raton, FL.

• Ladics, GS, Chapin, RE, Hastings, KL, Holsapple, MP, Makris, SL, Sheets, LP, Woolhiser, MR and LA Burns-Naas (2005) Developmental Toxicology Evaluations – Issues with Including Neurotoxicology and Immunotoxicology Assessments in Reproductive Toxicology Studies. Toxicological Sciences 88:24-29.

• Page, EW, Villee, CA, and DB Villee (1972) Human Reproduction. WB Saunders Company. Philadelphia, PA.