1. Overview
Aspects of Postnatal Growth:
Ontogeny of Organ Systems
Joseph F. Holson
WIL Research Laboratories

2. Acknowledgements

WIL Research Laboratories
SmithKline Beecham
• John M. DeSesso
• Catherine F. Jacobson
• Amy L. Lavin
• Bennett J. Varsho
• James L. Schardein
• Patrick J. Wier

3. Organization
• Prenatal Models and Ontogeny
• Concept of Physiologic Time
• Review of Adolph’s Seminal Work
• Difficulties in a priori Selection of Models
for Nonclinical Pediatric Toxicity
• Strengths and Weaknesses of Current
Safety Designs

4. Why are we interested
in organ system maturation?
It is essential for comparing
postnatal toxicity among species.

5. Prenatal Models and Ontogeny

6. Attributes of Successful Models
for Safety Assessment
•
•
•
•
Validity
Sensitivity
Reproducibility
Practicability

7. Differentiation Increases with Age
of Developing Organisms

8. Animal: Human Concordance Studies
for Prenatal Toxicity
Authors
Attributes
Holson et al., 1981
(Tox Forum)
Kimmel et al., 1984
(NCTR Report)
Interdisciplinary team
Criteria for acceptance of data/conclusions
Concept of multiple developmental
toxicology endpoints
No measures of internal dose
Nisbet & Karch, 1983
Many chemicals
Relied on authors’ conclusions
Emphasis on fertility
No measures of internal dose

9. Animal: Human Concordance Studies
for Prenatal Toxicity
Authors
Attributes
Hemminki &
Vineis, 1985
Interspecies inhalatory doses adjusted
Relied on authors’ conclusions
23 occupational chemicals and mixtures
No measures of internal dose
Newman et al.,
1993
Provided detailed information
Only 4 drugs
Emphasis on morphology
Focus on NOAELs
No measures of internal dose
Schardein, 1995
Many chemicals
Relied on authors’ conclusions
No measures of internal dose

10. Ontogeny Recapitulates Phylogeny
(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

11. Ontogeny Recapitulates Phylogeny - 1

12. Ontogeny Recapitulates Phylogeny - 2

13. Ontogeny Recapitulates Phylogeny - 3

14. Ontogeny Recapitulates Phylogeny - 4

15. 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.

16. Concept of Physiologic Time
Comparisons among Species
Comparisons among Developmental Stages

17. Maturational Data for Various
Species
Human
Gestation
(days)
Minimal
Breeding
Age
(weeks)
Human to
Animal
Life Span
Syrian
Hamster
Mouse
Rat
Rabbit
Guinea
Pig
Rhesus
Monkey
267
16
20
22
32
63
167
7
10.5
44
33
728
1.0
5
6.5
66
28
32
12
4
10
17
218
4.4

18. Background
• Adolph (1949) showed that metabolic rates scale
across species according to (body weight)0.73.
• Boxenbaum (1982) demonstrated that the
disposition kinetics of xenobiotics in species is
scaled by the same relationship.
• These concepts led to the mathematical
relationships that are used to standardize
experimental dose regimens and to scale across
species in PBPK models.

19. Physiologic Time
A method for scaling the lifespan of different
species so that comparable stages of maturation
are congruent, regardless of chronological age
An example of the concept of physiologic time that
is intrinsic to PBPK models:
0.25
T1/2 =
Body Weight rat
Body Weight human

20. Time to Develop Adult
Characteristics
100
Rat
Human
%
Adult
Status
0
0
5
10
Age (years)
15
20

21. Relationship Between Extent of Maturation
and Birth in Rats and Humans
100%
Adult
Status
= Birth
Maturation
Human
Rat
Conception
Physiologic Time

22. Review of Adolph’s Seminal Work

23. Ontogeny of Physiologic Regulation
in Selected Mammals
Hamster Rat Rabbit Cat Pig Human
Stagemarks
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
4
After Adolph 1970
8 10
20
40
80 100
Days After Conception
200
400

24. Perinatal Changes in Fetal Water
and Fat Content
Fat-Free Water Fraction
Gestation
Duration,
Days
Hamster
Rat
Rabbit
Cat
Guinea
Pig
Human
Age at
90% H2O
Days for
Transit to
80% H2O
15*
17*
23*
23
19
20
38*
56*
96*
27
72
170
16
21
32
65
67
114
266
* Prenatal
Body Fat Fraction
Age at
2% Fat
Days for
Transit to
6% Fat
20
22
32
65
39*
116
210*
17
6
8
10
21
3
27
After Adolph and Heggeness, 1971

25. Comparative Perinatal Water
Content
95
Pig
Rabbit
90
% Water
In a
Fat-Free
Body
85
*
GP
Rat
= Birth
Human
*
*
Water fraction
decreases with
age in all
species
*
Hamster
*
*
80
*
75
10
30
100
Days After Conception
300
After Adolph and Heggeness, 1971

26. Comparative Water Content at Birth
95
90
% Water
In a
Fat-Free
Body
Hamster
Rabbit
Rat
Do
g
85
Mouse
Pig
Human
Cat
80
Longer gestation
develops drier
(“denser”)
animals
Guinea
Pig
75
10
30
100
Days After Conception
300
After Adolph and Heggeness, 1971

27. Comparative Ontogeny of Fat Content
30
Hamster
Rat
Rabbit
25
Guinea Pig
Cat
% Fat
In
Body
20
Fetal Guinea Pig
and human deposit
fat prior to birth
Pig
Human
*
15
*
= Birth
*
10
5
0
10
* * *
30
*
*
100
Days After Conception
300
After Adolph and Heggeness, 1971

28. Comparative Perinatal Fat Content
15
10
Hamster
% Fat
In
Body
Guinea Pig
Birth
Birth
5
0
10
20
30
40
50
60
Days After Conception
After Adolph and Heggeness, 1971
70
80

29. Difficulties in a priori Selection
of Models for Preclinical Pediatric Toxicity

30. Relationship Between Development
and Phenotypic Diversity
Extent of Differentiation
Embryonic
Period
Fetal
Period
Postnatal
Period
Degree of
Phenotypic
Variability
Birth
Time in Development (Age)

31. Presence of Enzymes During Embryonic (E),
Fetal (F), and Neonatal (N) Periods
CYP1A1
CYP1A2
CYP1B1
CYP2E1
CYP3A4
CYP3A5
CYP3A7
CYP2C8
CYP2C9
CYP2D6
Flavin-containing monooxygenase
Prostaglandin synthetase
Lipoxygenase
Perosidase
Epoxide hydrase
GSH-S-transferase
UDP-glucuronyltranferase
Sulfotransferases
E
+
–
+
–
–
–
Rat
F N
+ +
– +
+
+
–
–
–
Mouse
E F N
+ + +
–
+
+ +
+
– –
– –
– –
+
+
Hamster
E F N
Rabbit
E F N
G. Pig
E F N
+
+
+
–
+
+
–
+
+
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
+ + –
– – +
+ +
+ + +
– – +
+ + +
+ + –
+ +
– – +
+ +
+
+
+
+
+
+
+

32. Selected Milestones of Reproductive
Development in Rats and Humans
Event
Rat
Human
gd 13
gd 35-37
gd 13-14
gd 40-42
gd 17
gd 60-70
gd 15 - pnd 16
fetal - to puberty?
Oocytes initiate meiosis
gd 17
gd 84
Arrest of meiosis in females
pnd 5
by pnd 56
Testes descend into scrotum
pnd 21
gd 220-225
Pubertal period: females
pnd 30-38
12-13 years
Pubertal period: males
pnd 35-60
13-15 years
Germ cells in genital ridges
Gonads begin sexual differentiation
Leydig cells differentiate
Sertoli cells proliferate

33. Comparison of Times
in Male Sexual Development
Birth
Conception
Genital Tubercle
Genital Development Static
Formation
Rat
Human
Adult Status
Secondary Sexual
Characteristics
3 Days
19 Days
50 Days
14 Days
8 Months
14 Years

34. Challenges of “Mining” the Literature
• Limited attention given to the issue of postnatal
models for safety assessment
• There is a paucity of reviews / data compilations
• Isolated key information is embedded in papers
addressing other concerns
• Analysis requires interdisciplinary expertise and
commitment of resources
• Many and substantial data gaps (species and
organ systems) exist

35. Strengths and Weaknesses
of Current Safety Designs
Suggested Modifications
Additional Strategies

36. Effects on Prenatal and Postnatal
Development Including Maternal Function
ICH 4.1.2 (Segment
III)
GD 6
Female
(Rat)
PND 20
Gestation
Lactation
(Macroscopic Pathology)
F1
Denotes Treatment Period
Denotes Possible Transfer Via Milk
Weaning Growth
PN day 21 9 wks
PN day 17
Mating
2 wks
Gestation
3 wks
PN day 80
Behavioral/Anatomic Measures
Motor Activity
Auditory Startle
Water Maze
Developmental Landmark
Vaginal Patency
Preputial Separation
F2

37. Comparison of Prenatal
and Postnatal Toxicity Profiles
Maternal
Toxicity
Developmental
Log of Dose
Prenatal – valid and insightful
– Embryonic exposure
– Mode of action
Postnatal – valid only
– when xenobiotic level is
measured in both mother and
offspring

38. Comparison of Prenatal and Postnatal
Modes of Exposure
Prenatal
Embryo/Fetus
Placenta
Treatment
Mother
Prenatal
Postnatal
Mammae
Neonate
Postnatal
Drug Transfer to
Offspring
Nearly all transferred
Apparent selectivity (“barrier”)
Drug Levels in Offspring
Cmax and AUC measured
Not routinely measured
Maternal Blood vs.
Offspring Levels
Maternal often a surrogate
Maternal levels probably NOT
a good predictor
Exposure Route to
Offspring
Modulated IV exposure,
via placenta
Oral, via immature GI tract
Commentary
Timing of exposure is
critical
Extent of transfer to milk and
neonatal bioavailability is key to
differentiating indirect (maternal)
effects
from neonatal sensitivity

39. Critical Periods for Structural
and Functional Effects
Structural
Development
Sensitivity
Functional
Development
Organogenesis
Time

40. ACE Inhibition-Induced Fetopathy
(Human)
ACEinh
Fetal
Hypotension
Renal
Compromise
(Anuria)
Calvarial
Hypoplasia
Oligohydramnios
Neonatal Anuria
IUGR
Death
• Organogenesis (classically defined) is unaffected
• Effects are severe
• Risk is low
• Caused by ACEinh that cross placenta

41. ACE Inhibition in Developing Rats
• RAS (renin-angiotensin system) matures around
GD17
• No ‘apparent’ effect in initial reproductive
studies
• Subsequent postnatal studies with direct
administration to pups
– Growth retardation
– Renal alterations (anatomic and functional)
– Death

42. Examples of Perinatal/Juvenile
Toxicants
• The following examples are not the result of an exhaustive
literature search.
• In most instances, the cause of postnatal morbidity/
mortality has not been investigated or is not known.
• The absence of standard blood biochemistry/hematology
assays and target organ pathology hinders the identification
of sites and modes of action.

43. Examples of Perinatal/Juvenile (?)
Developmental Toxicants
Toxicant
Exposure
Period
Species
Endpoint
Estrogen
PND1-5
mouse
cervical/vaginal
cancer
adult
Dunn & Green, 1963;
Takasagi & Bern, 1964
DES
prenatal
human
vaginal cancer/
reprod. tract effects
pubescence
Herbst & Skully, 1970
DES
Sex hormone
PND1-5
PND1-5
mouse
mouse
vaginal adenosis
vaginal adenosis/
cancer
adult
adult
Forsberg, 1976
GD15, 16, 17
mouse
vaginal adenosis,
transverse ridges
adult
Walker, 1980
(DES)
DES
Time of
Manifestation
(14 mo.)
Reference
Bern et al., 1976

44. Selective Juvenile Toxicity of Quinilones
Drug
Ofloxacin
(and other
quinilones)
Species &
Treatment
Effects
Remarks
Multiple Species,
postnatal exposure.
20mg/kg (dog, 3 mo.)
600mg/kg (rat, 5 wk)
Chondrotoxic
effects. Cartilage
erosion in weightbearing joints.
Human relevance
unknown; drugs
contraindicated in juvenile
patients.
Gait alterations in
juvenile dogs only.
Mechanism: Probable
deficiency of bioavailable
Mg2+ in cartilage
(quinilones chelate
divalent cations).
No effect in routine
segment III studies.
Modified from Stahlmann et al., 1997.

45. Reasons for Increased Attention to
Juvenile Toxicity
• New Trends in Drug Discovery
– Chiral molecules
– Rational, structure-based molecular design
– Targeted pharmacology
• Attention to Sensitive Subpopulations in Human Risk
Assessment
– Food Quality Protection Act
– FDA Modernization Act

46. Challenges
• Identifying and managing risks
– Modulation of growth
– Alteration of functional maturation
• Examples:
– EGF, TGF, Leptin, KGF, CRF

47. Pediatric Classifications
• Neonates
Birth to 1 month
• Infants
1 month to 2 years
• Children
2 to 12 years
• Adolescents
12 to <16 Years
• Comparable categories for animal species dependent on
individual organ or system

48. Non-Human Developmental Classifications
Category
Neonate
Dog
Rat (days)
(wks)
Primate
(mos)
Mini-Pig
(wks)
<10
<3
<0.5
<2
Infant
10-21
3-6
0.5-6
2-4
Child
21-35
6-20
6-36
4-14
Adolescent
35-60
20-28
36-48
14-26

49. Primary Reasons that Experimental
Models
Appear to be Invalid
• Findings at, or extrapolated to, exaggerated doses
• Exposure to and internal dose of noxious agent not
measured
• Timing of exposure does not coincide with the
appearance of the developmental target
• Duration of exposure not scaled to physiologic time
• Incorrect / unvalidated endpoints assessed
• Too little knowledge / data concerning mode of action

50. Conclusions
• Parallelism exists among species regardless of
lifespan.
• Additional measurements and changes to current
guidelines could increase our ability to predict
postnatal toxicity.
• Molecular biology and genomics have influenced
pharmaceutical development toward agents with
increasing specificity.
• For novel, selective pharmaceutical agents,
nonclinical testing must be preceded by literature
mining and analysis.