OverviewOverviewAspects of Postnatal Growth: Aspects of Postnatal Growth: Ontogeny of Organ SystemsOntogeny of Organ Systems

Joseph F. HolsonJoseph F. HolsonWIL Research LaboratoriesWIL Research Laboratories

AcknowledgementsAcknowledgements

• John M. DeSessoJohn M. DeSesso• Catherine F. JacobsonCatherine F. Jacobson• Amy L. LavinAmy L. Lavin

• Bennett J. VarshoBennett J. Varsho

• James L. SchardeinJames L. SchardeinWIL Research LaboratoriesWIL Research Laboratories

SmithKline BeechamSmithKline Beecham • Patrick J. WierPatrick J. Wier

OrganizationOrganization

• Prenatal Models and OntogenyPrenatal Models and Ontogeny

• Concept of Physiologic TimeConcept of Physiologic Time

• Review of Adolph’s Seminal WorkReview of Adolph’s Seminal Work

• Difficulties in Difficulties in a prioria priori Selection of Models Selection of Models for Nonclinical Pediatric Toxicityfor Nonclinical Pediatric Toxicity

• Strengths and Weaknesses of Current Strengths and Weaknesses of Current Safety DesignsSafety Designs

It is essential for comparing postnatal toxicity among species.

Why are we interestedWhy are we interestedin organ system maturation?in organ system maturation?

Prenatal Models and OntogenyPrenatal Models and Ontogeny

Attributes of Successful ModelsAttributes of Successful Modelsfor Safety Assessmentfor Safety Assessment

• ValidityValidity

• SensitivitySensitivity

• ReproducibilityReproducibility

• PracticabilityPracticability

Differentiation Increases with AgeDifferentiation Increases with Ageof Developing Organismsof Developing Organisms

Animal: Human Concordance StudiesAnimal: Human Concordance Studiesfor Prenatal Toxicityfor Prenatal Toxicity

Authors

Holson et al., 1981 (Tox Forum)Kimmel et al., 1984 (NCTR Report)

Attributes

Interdisciplinary team Criteria for acceptance of data/conclusionsConcept of multiple developmental

toxicology endpoints No measures of internal dose

Many chemicalsRelied on authors’ conclusionsEmphasis on fertilityNo measures of internal dose

Nisbet & Karch, 1983

Animal: Human Concordance StudiesAnimal: Human Concordance Studiesfor Prenatal Toxicityfor Prenatal Toxicity

Authors

Hemminki & Vineis, 1985

Newman et al., 1993

Schardein, 1995

Attributes

Interspecies inhalatory doses adjustedRelied on authors’ conclusions23 occupational chemicals and mixtures No measures of internal dose

Provided detailed informationOnly 4 drugsEmphasis on morphologyFocus on NOAELsNo measures of internal dose

Many chemicalsRelied on authors’ conclusionsNo measures of internal dose

Ontogeny Recapitulates PhylogenyOntogeny Recapitulates Phylogeny(von Baer, 1828)(von Baer, 1828)

• General features appear earlier in embryos than do specialized features

• Embryos of higher animals pass through stages that are similar to those of embryos of lower species

Ontogeny Recapitulates Phylogeny - 1Ontogeny Recapitulates Phylogeny - 1

Ontogeny Recapitulates Phylogeny - 2Ontogeny Recapitulates Phylogeny - 2

Ontogeny Recapitulates Phylogeny - 3Ontogeny Recapitulates Phylogeny - 3

Ontogeny Recapitulates Phylogeny - 4Ontogeny Recapitulates Phylogeny - 4

Ontogeny, Inc., based in Cambridge, MA, applies recent discoveries in developmental biology to the treatment of human diseases. Ontogeny has proprietary rights to a number of molecules known to induce cell differentiation, including several members of the hedgehog gene family that play a role in disorders involving the central nervous system, bone and cartilage, fertility and cancer. Ontogeny has signed a collaborative agreement with Biogen and with Genetics Institute to develop hedgehog proteins for neurological disorders, and Boehringer Mannheim in bone development and repair.

Concept of Physiologic TimeConcept of Physiologic Time

Comparisons among Species

Comparisons among Developmental Stages

Maturational Data for Various Maturational Data for Various SpeciesSpecies

Human toAnimal

Life Span

267 20 22 32 63 16716

Human Mouse Rat RabbitGuinea

PigRhesusMonkey

SyrianHamster

MinimalBreeding

Age(weeks)

Gestation(days)

728 7 10.52832

410 218

56.5

1.0 44 33 12 17 4.466

BackgroundBackground

• Adolph (1949) showed that metabolic rates scale Adolph (1949) showed that metabolic rates scale across species according to (body weight)across species according to (body weight)0.730.73..

• Boxenbaum (1982) demonstrated that the Boxenbaum (1982) demonstrated that the disposition kinetics of xenobiotics in species is disposition kinetics of xenobiotics in species is scaled by the same relationship.scaled by the same relationship.

• These concepts led to the mathematical These concepts led to the mathematical relationships that are used to standardize relationships that are used to standardize experimental dose regimens and to scale across experimental dose regimens and to scale across species in PBPK models.species in PBPK models.

Physiologic TimePhysiologic Time

A method for scaling the lifespan of different A method for scaling the lifespan of different species so that comparable stages of maturation species so that comparable stages of maturation are congruent, regardless of chronological ageare congruent, regardless of chronological age

An example of the concept of physiologic time that An example of the concept of physiologic time that is intrinsic to PBPK models:is intrinsic to PBPK models:

TT1/21/2 == Body Weight Body Weight ratrat

Body Weight Body Weight humanhuman

0.250.25

Time to Develop Adult Time to Develop Adult CharacteristicsCharacteristics

%Adult Status

Age (years)

0

Rat

Human

100

0 2015105

Relationship Between Extent of MaturationRelationship Between Extent of Maturationand Birth in Rats and Humansand Birth in Rats and Humans

Maturation

Physiologic Time

Conception

= Birth

Rat

Human

100%Adult Status

Review of Adolph’s Seminal WorkReview of Adolph’s Seminal Work

Implantation

First Heart Beat

Exterioception

Hemoglobin 8% in Blood

Body Weight 1gm

Thyroid Iodine

Lung Surfactant

Liver Glycogen 0.05%

Birth

Water 85% of Fat-free

Na/K one gm/gm

Anoxia Tolerance 10 min.

Body Fat 5%

Arterial Pr. 50 mm/Hg

Lethal Temp Shift

Resistance to Cooling

Ontogeny of Physiologic RegulationOntogeny of Physiologic Regulationin Selected Mammalsin Selected Mammals

Stagemarks

4

Days After Conception

Hamster Rat Rabbit Cat Pig Human

8 10 20 40 80 100 200 400

After Adolph 1970

Perinatal Changes in Fetal WaterPerinatal Changes in Fetal Waterand Fat Contentand Fat Content

After Adolph and Heggeness, 1971

16 23 20 1715*

GestationDuration,

Days

Days forTransit to80% H2O

Age at2% Fat

Days forTransit to

6% FatAge at

90% H2O

HamsterRat

Rabbit

Guinea Pig

Cat

PigHuman

21 19 22 617*32 20 32 823*

67 27 39* 2138*

114 72 116 356*266 170 210* 2796*

65 65 10

Fat-Free Water Fraction Body Fat Fraction

* Prenatal

Comparative Perinatal Water Comparative Perinatal Water ContentContent

After Adolph and Heggeness, 1971

Hamster

Rat

Rabbit

GP

PigHuman

= Birth

% WaterIn a

Fat-FreeBody

95

90

85

75

Days After Conception

80

10 30 100 300

Water fraction decreases with age in all species

*

*

*

**

*

*

Comparative Water Content at BirthComparative Water Content at Birth

Longer gestation develops drier (“denser”) animals

% WaterIn a

Fat-FreeBody

95

90

85

75

Days After Conception

80

Hamster

RatRabbit

Guinea Pig

Cat

Pig Human

10 30 100 300

Mouse

Dog

After Adolph and Heggeness, 1971

Comparative Ontogeny of Fat ContentComparative Ontogeny of Fat Content

Fetal Guinea Pig and human deposit fat prior to birth

% FatIn

Body

30

25

20

15

10

5

010 30 100 300

Days After Conception

Hamster

Rat

Rabbit

Guinea Pig

Cat

Pig

Human

= Birth

After Adolph and Heggeness, 1971

*

*

*****

*

Comparative Perinatal Fat ContentComparative Perinatal Fat Content

After Adolph and Heggeness, 1971

% FatIn

Body

15

10

5

0

10 30 40 50

Days After Conception

60 70 8020

BirthBirth

Hamster Guinea Pig

Difficulties in Difficulties in a prioria priori Selection Selection

of Models for Preclinical Pediatric Toxicityof Models for Preclinical Pediatric Toxicity

Relationship Between DevelopmentRelationship Between Developmentand Phenotypic Diversityand Phenotypic Diversity

Degree of Phenotypic Variability

Time in Development (Age)

EmbryonicPeriod

FetalPeriod

PostnatalPeriod

Extent of Differentiation

BirthBirth

Presence of Enzymes During Embryonic (E),Presence of Enzymes During Embryonic (E),Fetal (F), and Neonatal (N) PeriodsFetal (F), and Neonatal (N) Periods

Data extracted from Juchau et al., Kulkarni, 1997; Miller et al., 1996; Oesterheld, 1998; Raucy and Carpenter, 1993. CYP=cytochrome P450

Human

E F N

G. Pig

E F N

Rabbit

E F N

Hamster

E F N

Mouse

E F N

Rat

E F NCYP1A1

CYP1A2

CYP1B1

CYP2E1

CYP3A4

CYP3A5

CYP3A7

CYP2C8

CYP2C9

CYP2D6

Flavin-containing monooxygenase

Prostaglandin synthetase

Lipoxygenase

Perosidase

Epoxide hydrase

GSH-S-transferase

UDP-glucuronyltranferase Sulfotransferases

+–+

–––

++

+–++–––

–+

++

+–+

–––

+

+

++–––

+

–

++

+

+

++

++

+–++–++

–

+–++–+++–++

++++++

–+

+++–+++

Event

Germ cells in genital ridges

Gonads begin sexual differentiation

Leydig cells differentiate

Sertoli cells proliferate

Oocytes initiate meiosis

Arrest of meiosis in females

Testes descend into scrotum

Pubertal period: females

Pubertal period: males

Rat

gd 13

gd 13-14

gd 17

gd 15 - pnd 16

gd 17

pnd 5

pnd 21

pnd 30-38

pnd 35-60

Human

gd 35-37

gd 40-42

gd 60-70

fetal - to puberty?

gd 84

by pnd 56

gd 220-225

12-13 years

13-15 years

Selected Milestones of Reproductive Selected Milestones of Reproductive Development in Rats and HumansDevelopment in Rats and Humans

Comparison of TimesComparison of Timesin Male Sexual Developmentin Male Sexual Development

3 Days 50 Days19 Days

Human

Rat

14 Days 14 Years8 Months

Genital TubercleFormation

Conception

Genital Development StaticSecondary Sexual

Characteristics

BirthAdult Status

Challenges of “Mining” the LiteratureChallenges of “Mining” the Literature

• Limited attention given to the issue of postnatal Limited attention given to the issue of postnatal models for safety assessmentmodels for safety assessment

• There is a paucity of reviews / data compilationsThere is a paucity of reviews / data compilations

• Isolated key information is embedded in papers Isolated key information is embedded in papers addressing other concernsaddressing other concerns

• Analysis requires interdisciplinary expertise and Analysis requires interdisciplinary expertise and commitment of resourcescommitment of resources

• Many and substantial data gaps (species and Many and substantial data gaps (species and organ systems) existorgan systems) exist

Strengths and WeaknessesStrengths and Weaknessesof Current Safety Designsof Current Safety Designs

Suggested ModificationsSuggested ModificationsAdditional StrategiesAdditional Strategies

Effects on Prenatal and Postnatal Effects on Prenatal and Postnatal Development Including Maternal FunctionDevelopment Including Maternal Function

ICH 4.1.2 (Segment ICH 4.1.2 (Segment III)III)

Denotes Treatment PeriodDenotes Treatment Period

GD 6GD 6 PND 20PND 20

GestationGestation LactationLactation

WeaningWeaning GrowthGrowth MatingMating GestationGestationPN day 21PN day 21 9 wks9 wks 2 wks2 wks 3 wks3 wks

FF 11

FF 22

Female Female (Rat)(Rat) (Macroscopic Pathology)(Macroscopic Pathology)

PN day 17PN day 17 PN day 80PN day 80

Behavioral/Anatomic MeasuresBehavioral/Anatomic Measures

Motor ActivityMotor ActivityAuditory StartleAuditory StartleWater MazeWater MazeDevelopmental LandmarkDevelopmental Landmark

Vaginal PatencyVaginal PatencyPreputial SeparationPreputial Separation

Denotes Possible Transfer Via MilkDenotes Possible Transfer Via Milk

Comparison of PrenatalComparison of Prenataland Postnatal Toxicity Profilesand Postnatal Toxicity Profiles

Toxicity

Log of Dose

Maternal

Developmental

Prenatal – valid and insightful – Embryonic exposure – Mode of action

Postnatal – valid only – when xenobiotic level is

measured in both mother and offspring

Comparison of Prenatal and PostnatalComparison of Prenatal and PostnatalModes of ExposureModes 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) effectsfrom neonatal sensitivity

Prenatal Treatment Postnatal

Embryo/Fetus Placenta Mother Mammae Neonate

Critical Periods for StructuralCritical Periods for Structuraland Functional Effectsand Functional Effects

Sensitivity

Time

Organogenesis

Structural Development

Functional Development

ACE Inhibition-Induced Fetopathy ACE Inhibition-Induced Fetopathy (Human)(Human)

• Organogenesis (classically defined) is unaffectedOrganogenesis (classically defined) is unaffected

• Effects are severeEffects are severe

• Risk is lowRisk is low

• Caused by ACECaused by ACE inhinh that cross placenta that cross placenta

ACEACEinhinhFetal

Hypotension

RenalCompromise

(Anuria)Oligohydramnios

Calvarial Hypoplasia

Neonatal Anuria

IUGR

Death

ACE Inhibition in Developing RatsACE Inhibition in Developing Rats

• RAS (renin-angiotensin system) matures around RAS (renin-angiotensin system) matures around GD17GD17

• No ‘apparent’ effect in initial reproductive No ‘apparent’ effect in initial reproductive studiesstudies

• Subsequent postnatal studies with direct Subsequent postnatal studies with direct administration to pupsadministration to pups

– Growth retardationGrowth retardation

– Renal alterations (anatomic and functional)Renal alterations (anatomic and functional)

– DeathDeath

Examples of Perinatal/Juvenile Examples of Perinatal/Juvenile ToxicantsToxicants

• The following examples are not the result of an exhaustive The following examples are not the result of an exhaustive literature search.literature search.

• In most instances, the cause of postnatal morbidity/ In most instances, the cause of postnatal morbidity/ mortality has not been investigated or is not known.mortality has not been investigated or is not known.

• The absence of standard blood biochemistry/hematology The absence of standard blood biochemistry/hematology assays and target organ pathology hinders the identification assays and target organ pathology hinders the identification of sites and modes of action.of sites and modes of action.

Examples of Perinatal/Juvenile (?) Examples of Perinatal/Juvenile (?) Developmental Toxicants Developmental Toxicants

Exposure Time ofToxicant Period Species Endpoint Manifestation Reference

Estrogen PND1-5 mouse cervical/vaginal adult Dunn & Green, 1963;cancer Takasagi & Bern, 1964

DES prenatal human vaginal cancer/ pubescence Herbst & Skully, 1970

reprod. tract effects

DES PND1-5 mouse vaginal adenosis adult Forsberg, 1976

Sex hormone PND1-5 mouse vaginal adenosis/ adult Bern et al., 1976

(DES) cancer

DES GD15, 16, 17 mouse vaginal adenosis, adult Walker, 1980

transverse ridges (14 mo.)

Selective Juvenile Toxicity of QuinilonesSelective Juvenile Toxicity of Quinilones

Drug

Ofloxacin (and other quinilones)

Modified from Stahlmann et al., 1997.

Species &Treatment

Multiple Species,postnatal exposure.20mg/kg (dog, 3 mo.)600mg/kg (rat, 5 wk)

Effects

Chondrotoxic effects. Cartilage erosion in weight-bearing joints.

Gait alterations in juvenile dogs only.

Remarks

Human relevance unknown; drugs contraindicated in juvenile patients.

Mechanism: Probable deficiency of bioavailable Mg2+ in cartilage (quinilones chelate divalent cations).

No effect in routine segment III studies.

Reasons for Increased Attention toReasons for Increased Attention toJuvenile ToxicityJuvenile Toxicity

• New Trends in Drug DiscoveryNew Trends in Drug Discovery

– Chiral moleculesChiral molecules

– Rational, structure-based molecular designRational, structure-based molecular design

– Targeted pharmacologyTargeted pharmacology

• Attention to Sensitive Subpopulations in Human Risk Attention to Sensitive Subpopulations in Human Risk AssessmentAssessment

– Food Quality Protection ActFood Quality Protection Act

– FDA Modernization ActFDA Modernization Act

ChallengesChallenges

• Identifying and managing risksIdentifying and managing risks– Modulation of growthModulation of growth– Alteration of functional maturationAlteration of functional maturation

• Examples:Examples:– EGF, TGF, Leptin, KGF, CRFEGF, TGF, Leptin, KGF, CRF

Pediatric ClassificationsPediatric Classifications

12 to <16 Years12 to <16 Years• AdolescentsAdolescents

2 to 12 years2 to 12 years• ChildrenChildren

1 month to 2 years1 month to 2 years• InfantsInfants

Birth to 1 monthBirth to 1 month• NeonatesNeonates

• Comparable categories for animal species dependent on individual organ or system

Non-Human Developmental ClassificationsNon-Human Developmental Classifications

14-2614-2636-4836-4820-2820-2835-6035-60AdolescentAdolescent

4-144-146-366-366-206-2021-3521-35ChildChild

2-42-40.5-60.5-63-63-610-2110-21InfantInfant

<2<2<0.5<0.5<3<3<10<10NeonateNeonate

Mini-Pig Mini-Pig (wks)(wks)

Primate Primate (mos)(mos)

Dog Dog (wks)(wks)Rat (days)Rat (days)CategoryCategory

Primary Reasons that Experimental Primary Reasons that Experimental ModelsModels

Appear to be InvalidAppear to be Invalid

• Findings at, or extrapolated to, exaggerated dosesFindings at, or extrapolated to, exaggerated doses

• Exposure to and internal dose of noxious agent not Exposure to and internal dose of noxious agent not measuredmeasured

• Timing of exposure does not coincide with the Timing of exposure does not coincide with the appearance of the developmental targetappearance of the developmental target

• Duration of exposure not scaled to physiologic time Duration of exposure not scaled to physiologic time

• Incorrect / unvalidated endpoints assessedIncorrect / unvalidated endpoints assessed

• Too little knowledge / data concerning mode of actionToo little knowledge / data concerning mode of action

ConclusionsConclusions

• Parallelism exists among species regardless of Parallelism exists among species regardless of lifespan.lifespan.

• Additional measurements and changes to current Additional measurements and changes to current guidelines could increase our ability to predict guidelines could increase our ability to predict postnatal toxicity.postnatal toxicity.

• Molecular biology and genomics have influenced Molecular biology and genomics have influenced pharmaceutical development toward agents with pharmaceutical development toward agents with increasing specificity.increasing specificity.

• For novel, selective pharmaceutical agents, For novel, selective pharmaceutical agents, nonclinical testing nonclinical testing mustmust be preceded by literature be preceded by literature mining and analysis.mining and analysis.