Roche Template - ivf

In vitro and in vivo models in reproductive toxicology
Nicole Clemann, PhD
F. Hoffmann-La Roche AG, Basel
Master Course in Toxicology
Section: Reproductive Toxicology
May 10, 2016
Swiss Center of Applied Human Toxicology
What do we do and when?
Drug discovery and development process
– In silico tools
– In vitro systems
– In vivo systems
• Embryo-fetal development
• Fertility studies
• Pre- and postnatatal studies
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Costs: $ 1000 Mio.
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In vitro
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Market
Registration
Phase III
Phase II
Phase I
Phase 0
Lead
Optimization
and
Clinical
Candidate
Selection
(CCS)
Lead
Identification
Target
Assessment
Proposal
Process
Drug Discovery and Development Process
What are we doing and when?
~ 14.2 years
In silico
In vivo
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Reproductive Cycle
Growth &
development
Post-natal
adaptation
Parturition
Fetal growth
Sexual
maturation
Gamete
production
Release of
gametes
Interaction
of gametes
Transport of
zygote
♀
Organogenesis
Implantation
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5
In silico tools
e.g. DEREK, CASE/MCASE (Multi Computer Automated Structure Evaluation), VITEC:
-Knowledge-based computer system (program) designed
for organizing
biological/toxicological data obtained
from evaluation of diverse
chemicals.
These programs:
- can automatically identify biological hazards based on
substructures
molecular
- based on learning set positive and negative compounds
- MCASE use the concept of biophores and modulators.
* Windows based Structure-Activity Relationship (SAR) automated expert system
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What do we do and when?
Drug discovery and development process
– In silico tools
– In vitro systems
– In vivo systems
• Embryo-fetal development
• Fertility studies
• Pre- and postnatatal studies
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In vitro test systems in reproductive toxicology
• Female fertility
– Ovary cell cultures
(e.g. granulosa, thecal, stromal cell cultures)
• Male fertility
– Testicular cell cultures (e.g. Sertoli cells, germ cells, Leydig cell)
• Embryo-fetal development
– Whole embryo cultures (rats, chick, frog……)
– Primary cell cultures or organ cultures (limb bud……)
– Cell lines (embryonic stem cells)
– Zebrafish (Danio rerio)
• Pre- and postnatal toxicity: ???
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Rat Whole Embryo Culture
Principle: 9.5 days embryo (serum), incubate for 48 h
Endpoints: Growth, differentiation, dysmorphogenesis, vascularization of yolksac, Haematopoesis, DNA-content…
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Embryonic Stem Cell Culture
Principle: Omnipotent (mouse) stem cells differentiate into cardiomyocytes
Endpoints: Cytotoxicity, inhibition of differentiation, gene expression
http://www.csa.com/discoveryguides/stemcell/images/pluri.jpg
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Zebrafish assay
1 cell
5 hpf
18 hpf
Day 1
Blastula
Before hatching
After hatching
body plan, brain, sense
organs, beating heart
Day 2-4
Most organs developed (e.g. gut,
liver, jaw)
Day 5
End of embryogenesis
Free swimming larva
Principle
• Development of a zebrafish embryo in 5 days (from egg to larvae)
Endpoints
• Growth, differentiation, dysmorphogenesis
Chapin, 2008
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Limitations of alternative tests
• Closed system
– No maternal metabolic activation
• Preincubation with S9 is possible
• No maternal influence
– Kinetics
– Physiology
• E.g. nifedine – hypotensive action
• Limited tissue types and/or duration of development
• Feasibility of tests often limited by solubility or cytotoxicity
– May give equivocal results
Useful as early screening tools and eg mechanistic tools
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Guidelines
ICH M3(R2): “Guidance on Nonclinical Safety Studies for the Conduct of Human Clinical Trials and
Marketing Authorization for Pharmaceuticals” (Feb 2012, Step 5)
Purpose: “...to recommend international standards for, and promote harmonisation of, the nonclinical safety
studies recommended to support human clinical trials of a given scope and duration and marketing
authorization…”
ICH S5(R2): “Detection of Toxicity to Reproduction for Medicinal Products &
(November 2005)
Toxicity to Male Fertility”
Purpose: “… to reveal any effect of one or more active substances on mammalian reproduction
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What do we do and when?
Drug discovery and development process
– In silico tools
– In vitro systems
– In vivo systems
• Embryo-fetal development
• Fertility studies
• Pre- and postnatatal studies
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Test for embryo-fetal development (teratology)
(rat & rabbit)
Mating
Gametogenesis
Birth
Gestation
Organogenesis
Implantation
Start with mated animals
treatment
Weaning
Lactation
A
u
t
o
p
s
y
Gametogenesis F1
Puberty
Parameter
•
•
•
•
•
•
•
•
General toxicity dam (body weight, food consumption…..)
Exposure dam (kinetic)
Exposure fetus
Number and quality of implantations
Intrauterine mortality (embryo or fetus)
Fetal weight, sex
Anomalies external, visceral, skeletal
Histopathology
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Evaluation of Rat Fetuses (day 21)
external
skeletal
visceral
(N=800; 4 g; 3.5cm)
N=400
N=400
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What do we do and when?
Drug discovery and development process
– In silico tools
– In vitro systems
– In vivo systems
• Embryo-fetal development
• Fertility studies
• Pre- and postnatatal studies
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Study for Fertility and Early Embryonic Development
Mating
Gametogenesis
Birth
Gestation
male
2-9 weeks
female
2 weeks
implantation
Gametogenesis F1
Weaning
Lactation
A
u
t
o
p
s
y
(rat/mouse)
Puberty
Parameter
• Parental toxicity (body weight, food consumption,
clinical signs)
• Estrus cycle
• time to mating
• mating success
• number and quality of implantations
• sperm parameters (CASA)
• histopathology (testes/ovaries)
Start with mated animals
treatment
Rats are hyperfertile !
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Sperm evaluation
Motility
Count number of sperm:
Ultrasonic resistant sperm heads from
homogenized testis tissue
Sperm from epididymis
dilution
slide
Microscope
CASA (Computer Assisted Semen Analyzer)
motility, velocity, direction…
• motile and /
immotile
• morphology
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What do we do and when?
Drug discovery and development process
– In silico tools
– In vitro systems
– In vivo systems
• Embryo-fetal development
• Fertility studies
• Pre- and postnatatal studies
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Pre- and postnatal development
Influence during intrauterine development and lactation may have impact on:
• Change from intrauterine to extrauterine life (birth)
• Metabolism, endocrine function…..
• Neurologic function (e.g. reflexes)
• Intellectual ability (learning ability, behavior)
• Ability to reproduction
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Study for pre- and postnatal development
(rats/mice)
Birth
Mating
Gametogenesis
Gestation
Weaning
Lactation
Puberty
Gametogenesis (mating
F1)
Parameter
Implantation
Treatment
Start with mated animals
Rearing behavior
Pup mortality
Body Weight
Reflexes
Developmental landmarks
Sex ratio
Behavior
Motor activity
Sexual development
A
u
t
o
p
s
y
Fertility of
F1 generation
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Summary / General Consideration
• Assess potential hazard and risk of a drug for humans on reproduction
• In-silico tools and in vitro tests allow a first hazard assessment (screening)
• Approach in animals studies is to assess:
– Fertility and early embryonic development until implantation
– Embryo-fetal development
– Pre- and Postnatal Development
• Species are: Rat < Rabbit < Mouse < Cynomolgus monkey
• Timing and Design of studies are regulated by internationally harmonized Guidelines (ICH)
• Timing and Design of studies also depend on indication and target
• Studies have to comply with Good Laboratory Practice (GLP)
• Conduct of studies requires a high degree of spezialisation and experience
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Acknowledgements
Acknowledgment
Heinrich Bürgin
Samia Khalil
Georg Schmitt
Paul Barrow
Alessandro Brigo
Claudia McGinnis
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Questions?
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References
• Chapin R. et al (2008). State of the art in developmental toxicity screening methods and a way forward: a
meeting report addressing embryonic stem cells, whole embryo culture, and zebrafish. Birth Def. Res. 83 (4),
446-456
• Flint O.P. (1984). An in vitro assay for teratogens with cultures of rat embryo midbrain and limb cells. Tox
Appl. Pharm. 76, 383-395
• New D.A.T. (1978). Whole-embryo culture and the study of mammalian embryos during organogenesis. Biol.
Rev. 53, 81-122
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