Toxicology for Industrial and Regulatory Scientists

Reproductive & DevelopmentalReproductive & Developmental Toxicology Studies

Kok Wah Hew, Ph.D., DABT, ,Takeda Pharmaceutical Company

Deerfield, IL 60015kok ah he @takeda comkok-wah.hew@takeda.com

April 29 2015April 29, 2015

Reproductive Toxicity Testing for Pharmaceuticals

Goals

Testing for Pharmaceuticals

Overview of reprotox study design & evaluation endpoints Allow better communication with reprotox study director Understand and interpret reprotox data Allow better communication of reprotox data to Project

Team Clinicians Regulators etcTeam, Clinicians, Regulators, etc.

Importance of nonclinical reprotox studies Difficult/unethical to perform these studies in humans

(lack of human data to assess risk to reproduction) Data will be used for risk assessment in product label Data will be used for risk assessment in product label

2

Reproductive Toxicity Testing for PharmaceuticalsTesting for Pharmaceuticals

OutlineOutline Biology/hormonal regulation of mammalian

male and female reproductive systemsmale and female reproductive systems

Mammalian reproductive cycle and development

Fertility and early embryonic development study

Embryo-fetal development studies

Pre- and postnatal development study

Juvenile animal study Juvenile animal study

3

Male Reproductive Systemp y

Rodent (Frontal View) Human (Frontal View)

4

Male Reproductive System: Testisp y

S ti f i if t b lSection of seminiferous tubule

Spermatozoa (n)

Spermatid (n)

Secondary spermatocyte (n)

Primary spermatocyte (2n)

Leydig cell

Sertoli cell

Spermatogonium (2n)Tight junction*

Sertoli cell* Forms the blood-testis barrier

5

Cycle of Seminiferous EpitheliumVI VII VIII IX X XI

Seminiferoustubule

y p

Rats ≈ 60 days

ba

b

yMen ≈ 74 days

Transit of sperm from testis to cauda epididymis

Rats ≈ 4 days

Cycle Figure from Russell et al., Histological and Histopathological Evaluation of the Testis

a

yMen ≈ 2 days

Histopathological Evaluation of the Testis, Cache River Press, Clearwater, FL, 1990, with permission.

6

Rat Testis: Cross-SectionRat Testis: Cross Section

7Roman numeral in lumen indicates stage of spermatogenesis

Hormonal Control of Testicular Functions

Hypothalamus GnRH

Inhibin Testosterone Testosterone

Pituitary

T t t

LHFSH

Leydig cellsSeminiferous

tubule

Testosterone

ABPDHT

Stimulation/secretion

Negative

Male sexual development,protein synthesis, Negative

feedback

ABP: androgen-binding protein

p y ,cell growth, etc.

DHT: dihydro-testosterone 8

Male Reproductive Systemp y Hypothalamus: receives input from the CNS and rest of the

body; secretes GnRH to stimulate the pituitarybody; secretes GnRH to stimulate the pituitary.

Pituitary: responds to GnRH by releasing LH and FSH.

Testis: responds to LH/FSH produces sperm secretes Testis: responds to LH/FSH, produces sperm, secretes Testosterone from Leydig cells

Androgen-binding protein and inhibin by Sertoli cellsd og b d g p o a d b by o

Epididymis*: a single tube that modifies sperm to confer motility and fertilizing ability.

Accessory Sex Organs (Prostate & Seminal Vesicles)*: contribute fluid that carry sperm, provide beneficial environment for sperm motility and transport.environment for sperm motility and transport.

9

* Functions regulated by testosterone and dihydro-testosterone

Female Reproductive Systemp y

Uterus Uterine tube

OvaryCervix

Vagina

Rodent (Frontal View) Human (Frontal View)

10

Ovulation, Fertilization, & Implantation, , p

Oocyte

Granulosa cells (stimulated by FSH,Granulosa cells (stimulated by FSH, produce & secrete estradiol)

Theca cells (stimulated by LH, participate in estradiol synthesis)

Ovarian follicle

11

participate in estradiol synthesis)

Female Reproductive System: Hormonal Control

Hypothalamus

Hormonal ControlGnRH

-/+

Uterus Uterine tube PituitaryLH

-/+

Granulosa cells

Theca cells

Ovary

yFSHLH

-/+

Vagina

OvaryCervix -/+

Oocyte

Corpus luteum Oocyte

Ovulation

Ovarian follicleStimulation/

secretion

Progesterone EstradiolOvulation

12*In rats, mice, hamster

Negative/positivefeedback-/+

Female Reproductive Systemp y

Hypothalamus: receives input from the CNS and rest of the body; secretes GnRH to stimulate the pituitary.

Pituitary: responds to GnRH by releasing LH and FSH.

Ovary: responds to LH/FSH, releasing oocytes, secretes

Estradiol from follicular cellsEstradiol from follicular cells

Progesterone and inhibin from corpus luteum

Oviduct Uterus Cervix Vagina*: Oocyte “handling” Oviduct, Uterus, Cervix, Vagina*: Oocyte handling , facilitate and enable egg and sperm transport, and maintain pregnancy.

13

* Functions of these organs regulated by estradiol and progesterone

Hormonal Changes During Human Menstrual Cycle and PregnancyMenstrual Cycle and Pregnancy

0 5 10 15 20 25 Days28/0 5 10 15 20 25

GonadotropinsLH

FSH

Ovary/FolliclesFollicular LutealPregnancy

yFollicular Luteal

ProgesteronehCG Ovarian Steroids

g

EstradiolhCG

14

P. Foster with permission hCG: human chorionic gonadotropin

Temporal Comparison of Menstrual vs. Estrous CyclesMenstrual vs. Estrous Cycles

Ovulation

Follicle

Menses

Corpus Luteum

man

Menses

Day 1 14 28

Hum

Day 1

Diestrus(48 hr)

Proestrus(18 hr)

Estrus(28 hr)

Metestrus(6 hr)t (48 hr) (18 hr) (28 hr) (6 hr)

Day 1 2 3 4

Rat

15

Reproductive Cycle & Reproductive Toxicity Studies

MatingGamete♂

p odu o y ud

Dosing period

Delivery

in uteroDevelopment

F1Maturation

F0

♂

Mating

Development

Growth/DevelopmentF1Gamete

Maturation♀ Delivery

Sexual Maturation F1

GestationSegmented approach to evaluate reproductive toxicity for pharmaceuticals

F2Seg. I

Seg. II

Fertility & early embryonic development study

Embryo-fetal development study

Seg. III Pre- & postnatal development study

16

Fertility & Early Embryonic y y yDevelopment Study

17

Fertility & Early Embryonic Development StudyDevelopment Study

Purposep To assess effects of compound on fertility and early embryonic

development when exposure occurred during gamete development, fertilization & before implantationp

Timing of study during drug development [ICH M3(R2)] Required before Phase III for subjects with reproductive capabilities Required before Phase III for subjects with reproductive capabilities Not needed for inclusion of subjects with reproductive potential in

Phase I/II if adequate microscopic exams were performed in reproductive organs in repeat-dose study of at least 2 weeks inreproductive organs in repeat-dose study of at least 2 weeks in duration

Not needed for drugs intended to treat patients with advanced cancer [ICH S9] Use general tox data to assess effects on reproductive[ICH S9]. Use general tox data to assess effects on reproductive organs

18

Fertility & Early Embryonic Development Study (Segment I)Development Study (Segment I)

MatingT t t P i dIn life

n=20/sex/group

Gamete

MaturationF1

Treatment Period

♂In-life

Termination Male: 2-10 weeks before cohabitation, during cohab. & 2-3 weeks post-mating

F l 2 k b f h bit ti

Gamete

in utero

DevelopmentF0

Female: 2 weeks before cohabitation, during cohab. & until GD 6/7 (monitor estrous cycle until successful mating)

Gamete

Maturation♀

Evaluate effects on• F0 gamete maturation• F0 mating behavior & fertilityF0 mating behavior & fertility• F1 implantation & pre-implantation embryos GD: Gestation Day

19

Reproductive Endpoints for MalesReproductive Endpoints for Males

Fertility and fecundity (ability to produce litters, b f i l t b /litt )number of implants or embryos/litter)

Mating behavior, time to mate, etc.

Organ weights (testis and accessory organs)

Sperm indicesd d d ( d ) Sperm count in testis and epididymis (sperm production)

Sperm motility (sperm function) Sperm morphology (sperm quality)p p gy ( p q y)

Histopathology (typically performed in repeat-dose study) Effects on spermatogenesis (most sensitive endpoint in male)

20

Reproductive Endpoints for Females

Successful fertilization, implantation, and gestation

Reproductive Endpoints for Females

, p , g

Estrous cyclicity (sensitive marker of hormonal changes)

Number of implants embryo/fetus numbers pregnant Number of implants, embryo/fetus, numbers pregnant per group

Organ weights and histologyg g gy

21

Fertility & Early Embryonic Development Study: Evaluation Endpoints

Estrous cyclicity

Study: Evaluation Endpoints

Precoital interval

Mating and fertility indicesg y

Cesarean section parameters

Organ weights Organ weights

Sperm parameters

Histopathology Histopathology

22

Fertility & Early Embryonic Development Study: Case StudyStudy: Case Study

Control Low Dose Mid-Dose High doseControl Low Dose Mid Dose High dose

Male Fertility 100 100 92 46**Index (%) 100 100 92 46**

% Motile% Motile Sperm 90 88 66** 16**

E idid lEpididymal Sperm Count (106/g cauda epididymis)

510 508 440** 220**

23** Statistically different from control value

epididymis)

Fertility & Early Embryonic Development Study: Case Study

Interpretation

Study: Case Study

p Male fertility affected at the high dose

Sperm parameters affected at the mid- and high doseSperm parameters affected at the mid and high dose Rodent has large sperm reserve and can tolerate some

effects on sperm without effect on fertility

Same is not true for humans

For potential risk on sperm parameters in men, needs to evaluate exposure margin between mid-dose andevaluate exposure margin between mid dose and therapeutic dose

24

Embryo-Fetal yDevelopment Study

25

Embryo-Fetal Development Studyy p y

PurposeT ff t f d b f t l d l t h To assess effects of compound on embryo-fetal development when exposure occurred in utero

Timing of study during drug development [ICH M3(R2)]Timing of study during drug development [ICH M3(R2)] Required before exposure to women of childbearing potential (WOCBP) Not needed for inclusion of WOCBP if

Short term trials (e g 2 weeks) with intensive control of pregnancy risk Short term trials (e.g., 2 weeks) with intensive control of pregnancy risk Trials must include WOCBP, but with sufficient precautions to prevent pregnancy

WOCBP (up to n=150, with adequate birth control) may be included in Phase I/II (up to 3 months) if data from preliminary embryo-fetalPhase I/II (up to 3 months) if data from preliminary embryo fetal development studies (2 species) are available

For anticancer drug [ICH S9] Not needed if genotoxic or has class effect on developmental tox If required, complete by marketing application (NDA/MAA)

26

Overview of Human DevelopmentFertilization I l t tiFertilization Implantation

Period of Organogenesis Modified from Moore 198827

Window of Sensitivity is Greatest during OrganogenesisOrganogenesis

vity Fertilization

I l t ti

High

Sens

itiv

Growth & differentiation of organs

Implantation

Organogenesisree

of S

BirthOrganogenesis

0 5 9 10 11 12 13 14 21-22

Deg

Low

Rat Gestation (Days)0 5 9 10 11 12 13 14 21 22

Period of Treatment Period for

Modified from Wilson (1973)

Period of Organogenesis

Treatment Period for Segment II Study=

28

Stages of in utero DevelopmentFertilization

ImplantationBirth

Embryonic Period Fetal Period

(Establishment of body (Growth/differentiation of

Segment II Treatment Period

(Establishment of body form, Organogenesis)

( /organ systems)* Days from fertilization

Implantation* Organogenesis Ends* Birth*

Mouse 5 15 19-20

Rat 5-6 16 21-22

Rabbit 6-7 19 30-32

Monkey 9** 44-45 166

Human 6-7 50-56 266** Dosing typically starts on GD 20 due to the need to confirm pregnancy by ultrasound on GD 18-20 29

Embryo-Fetal Development Study(Segment II or Teratology*)(Segment II or Teratology )

F0 MatingIn life

F1♂

Treatment Period

In-lifeTermination

in utero

Development* Study of Terata (Monster)

or birth defects

♀

Evaluate effects on• Viability of F1 embryos & fetuses• Growth of F1 embryos & fetusesGrowth of F1 embryos & fetuses• Structural development of F1 embryos & fetuses

30

Embryo-Fetal Developmental Study DesignEmbryo Fetal Developmental Study Design

Objective: Assess test article toxicity on maternal gestation an embryonic and fetaldevelopmentdevelopment

– Conventionally one rodent species and a non-rodent species (rabbit)– Exposure post-implantation to end of organogenesis– Endpoints: evaluate maternal toxicity, embryo/fetal death, fetal external, soft tissue and p y, y / , ,

skeletal alterations

FemaleCesareanSection +Fetal ExamsGD 0n=20/group

* *

Rat GD 6Rabbit GD 6/7

Rat GD 20-21Rabbit GD 29-30

Fetal ExamsGD 0n 20/group

Rat GD 16Rabbit GD 20

GD 0=Gestation Day 0, day when mating is confirmed

* Blood samples are typically obtained from satellite animals for toxicokinetics

Treatment Period

Range-finding study is usually performed to select doses for the definitive study:typically 4-5 dose groups + controls, n=5-6/group, limited fetal exam (external)

31

Preliminary Embryo-Fetal Developmental Study Designy y p y g

Objective: Assess drug toxicity on maternal gestation & embryonic andfetal development in order to allow inclusion of women offetal development, in order to allow inclusion of women ofchildbearing potential (up to 150, using adequate birth controlmethods) for trials of up to 3 months [ICH M3(R2)]

– One rodent species and a non-rodent species (rabbit)One rodent species and a non rodent species (rabbit)– Exposure from post-implantation to end of organogenesis– Endpoints: evaluate maternal toxicity, embryo/fetal death, fetal external

and soft tissue alterationsand soft tissue alterations

Femalen=6-8/group

CesareanSection +Fetal ExamsGD 0

Rat GD 6Rabbit GD 6/7

Rat GD 20-21Rabbit GD 29-30

Fetal ExamsGD 0

Rat GD 16Rabbit GD 20

GD 0=Gestation Day 0, day when mating is confirmed

32

Treatment Period

Endpoints in Embryo-Fetal Development StudiesEndpoints in Embryo Fetal Development Studies

Maternal endpoints Clinical signs, abortion (rabbit only) Food consumption, body weights, body weight gains

Fetal endpoints Fetal endpoints Number, sex ratio, viability, weight

External examination (all live fetuses)

Rats Half get examined for internal soft tissue structure, using conventional and

commonly-accepted dissection methodscommonly accepted dissection methods

Other half get examined for hard tissue structure (Alizarin Red staining for calcified tissue, sometimes Alcian Blue staining for cartilage)

Rabbits all animals get e amined fo soft tiss e st t es

33

Rabbits: all animals get examined for soft tissue structuresfollowed by hard tissue stains and examination

Endpoints of Embryo-Fetal DevelopmentEndpoints of Embryo Fetal Development

Altered Survival (live/dead embryos or fetuses) Pre-implantation loss Post-implantation loss*

Structural Changes (external, visceral, skeletal) Structural Changes (external, visceral, skeletal) Variation* Malformation*

D l l D l ll bl Developmental Delays – usually recoverable Growth (body weight, size)* Skeletal development (ossification)*p ( )

Functional Deficits – not evaluated in Segment II Biochemical

B h i l Behavioral

34* Evaluated and used to assess risk of exposure

Malformations and VariationsMalformations and Variations

Malformation is a permanent structural change that maystructural change that may adversely affect survival, development or function

Spina bifidaHydrocephaly Spina bifida

Variation is a divergence gbeyond the usual range of structural constitution that may not adversely

7th Cervical rib

that may not adversely affect survival or health

Incomplete centrum ossification

35

Data Interpretationp

Litter is the statistical unit

Maternal toxicity can affect fetal development (e.g., low fetal weight, supernumerary ribs)

S h bl ft bi th Some changes are recoverable after birth (e.g., wavy ribs, delayed ossification)

Read changes against concurrent controls and against the Read changes against concurrent controls and against the historical control database in the lab

To tell if something is noise or really treatment-induced Look for patterns Dose-relationship Greater number of dams affected

36

Greater number of dams affected (rather than many pups from 1 dam)

Litter Mean Calculation Litter No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 Total MeanNo.

No. affected fetuses

0 0 0 0 0 0 0 0 0 4 0 1 0 0 1 6

No fetuses/litter

7 10 8 10 7 8 8 7 9 14 4 8 4 9 8 121 5%

% affected fetuses/litter

0 0 0 0 0 0 0 0 0 28.6 0 12.5 0 0 12.5

Total no affected fetuses 6

53.6% 3.6%

Total no. affected fetuses=6Total no. fetuses evaluated=121% affected fetuses/litter=(6/121) x 100 5%*

Correct value53.6%15 3.6%

37

* Incorrect value because did not consider individual litter as an experimentalunit, i.e., each litter contains different no. of fetuses.

Integrated Approach in Data InterpretationIntegrated Approach in Data Interpretation

Spontaneous vs. treatment-related pmalformations/variations

Dose-response relationshipDose response relationship

Presence/absence of maternal toxicity

C i d Cross-species concordance

Similarities/differences in pharmacokinetics (test species vs. humans)

Exposure ratio (test species/humans)

38

Embryo-Fetal Development StudyCase Study

Control Low Dose Mid-Dose High dose

Case Study

Maternal Body Weight Normal Normal 4% less than 18% less than Body Weight Gain

Normal Normal control value control value**

Mean Fetal Weight (g) 3.5 3.8 3.4 2.9**

% Fetus With Delayed Ossification

0 0 1 5**Ossification

39** Statistically different from control value

Embryo-Fetal Development StudyCase Study

Interpretation

Case Study

Interpretation Maternal toxicity at the high dose

Fetal effects observed at the high dose Low fetal weight and delayed ossification are signs

f th t d tiof growth retardation

Fetal effects likely due to maternal toxicity (l i ht i ) t th hi h d(low weight gain) at the high dose

40

Pre- & Postnatal Development Study

41

Pre- and Postnatal Development Studyp y

Purposep To assess effects of compound on pre- and postnatal

development when exposure occurred in utero and during postnatal development (via mother’s milk)

Timing of study during drug development [ICH M3(R2)]Timing of study during drug development [ICH M3(R2)] Required for marketing approval submission (NDA/MAA)

Not needed for drugs intended to treat patients with g padvanced cancer [ICH S9]

42

Pre- & Postnatal Development Study (Segment III)(Segment III)

Treatment PeriodDeliveryF1

♂

in utero

DevelopmentM ti

F0

F1

In-life

GD 6 – LD 20 (n=20/group)

Growth/Development

Sexual Maturation

MatingF1

F1

♀Termination

Ⓐ

Evaluate effects on

Sexual Maturation F1

Gestation

Ⓐ

Ⓑ• Maternal gestation, parturition, nursing behavior & lactation • F1 viability and growth• F1 functional development & reproductive capability

F2

GD=Gestation Day, day of mating confirmation is GD 0LD=Lactation Day, day of delivery is LD 0

Ⓐ F1 GD 13Ⓑ F1 LD 4

43

Endpoints of Pre-/Postnatal Development StudyEndpoints of Pre /Postnatal Development Study

Maternal (F0) parameters( ) p

Clinical signs, food consumption, body weights, body weight gains

Gestation length

Parturition, nesting & nursing behavior

Litter size

Lactation (presence/absence of milk in the stomach of offspring)

44

Endpoints of Pre-/Postnatal Development Study

F1 Parameters

Endpoints of Pre /Postnatal Development Study

Viability (usually culled to 4/sex/litter on Lactation Day 4) Growth (body weight) Physical development Physical development

Pinna detachment Incisor eruption Eyelid opening Eyelid opening Vaginal opening Preputial separation

Reflexological & Sensory Development Reflexological & Sensory Development Righting reflex Negative geotaxis Pupillary reflex

45

Pupillary reflex Preyer reflex

Endpoints of Pre-/Postnatal Development Study

F1 Parameters (cont.)

Endpoints of Pre /Postnatal Development Study

Motor activity functions Open field test

Fi “8” Figure “8” maze

Learning and memory tests Passive avoidance Passive avoidance Water maze

Reproductive performance F1 Fertility F1 Gestation F2 viability

46

F2 viability

Data InterpretationData Interpretation

Litter is the statistical unit (where appropriate) Maternal toxicity can affect growth and development of offspring Endpoints ranked by sensitivity

Viable litter size Viable litter size Neonatal growth (body weight is the most sensitive indicator) & survival Gestation length

Landma ks of se al mat it Landmarks of sexual maturity Functional maturation, learning & memory capability

Evaluate all study data as a whole, not individual endpoints in i l tiisolation

To tell if something is noise or really treatment-related Dose-relationship

47

p Greater number of dams affected

Pre- and Postnatal Development StudyCase StudyCase Study

Landmarks of Sexual Development

Control Low Dose Mid-dose High Dose

Preputial 43 44 47** 47**pSeparation (PND) 43 44 47** 47**

Body weight (g) 236 235 218** 215**

Control Low Dose Mid-dose High Dose

Vaginal OpeningVaginal Opening (PND) 32 32 33 36**

Body weight (g) 111 108 115 95**

48

PND: Postnatal Day** Statistically different from control value

Pre- and Postnatal Development StudyCase Study

Interpretation

Case Study

Interpretation Sexual maturation was delayed in males and females

Body weights in the affected groups were Body weights in the affected groups were correspondingly lower than controls

Delay in sexual maturation was due to growth Delay in sexual maturation was due to growth retardation

49

Range-finding StudyRange finding Study

Fertility & early embryonic development study Range-finding study often not necessary

Typically use data from repeat-dose studies to select doses

Embryo-fetal development studies Rat: best to do a range-finding study

Rabbit: range-finding study a must

Pre- & postnatal development study Range-finding study often not necessary

Typically use data from embryo-fetal development study to select dosesselect doses

50

Dose Selection

High dose

Dose Selection

Should produce maternal or parental toxicity

In the absence of maternal toxicity, limit dose or i f ibl d b dmaximum feasible dose may be used

Sufficient exposure should be demonstrated

In the absence of parental toxicity or sufficient exposure In the absence of parental toxicity or sufficient exposure, alternative vehicle, route, dosing regimen, or species should be considered

Low dose

Should be the No-Observed-Adverse-Effect Level (NOAEL) Exposure at multiples of clinical efficacious dose, if possible

51

Toxicokinetics in Reproductive Toxicity Studies

Generally not determined in

Reproductive Toxicity Studies

y Fertility and early embryonic development study

Pre- and postnatal development study

Embryo-fetal development studies Not a requirement but generally performed in dam/doe to

determine maternal exposure

Useful for new drug, to provide exposure margin over human exposure (assessment of human risk in drug label)exposure (assessment of human risk in drug label)

Generally not determined in fetus due to

Technical difficulties in obtaining sufficient fetal blood samples

No comparative information in humans

52

Assessment of Risk in Reproduction & Pregnancy

US Label (until June 29, 2015): 8.1 (Pregnancy), 8.3 (Nursing Mothers), 13 1 (Carcinogenesis Mutagenesis Impairment of Fertility)13.1 (Carcinogenesis, Mutagenesis, Impairment of Fertility)

Pregnancy CategoriesA: Not teratogenic in humansA: Not teratogenic in humansB: Not teratogenic in animal & no controlled human dataC: Teratogenic in animals & no controlled human dataD: Teratogenic in humans but benefit to mother may outweigh risk

to fetusX: Teratogenic in humans risk of use during pregnancy outweighsX: Teratogenic in humans, risk of use during pregnancy outweighs

any possible benefit

EU: Guideline on Risk Assessment of Medicinal Products on Human

53

EU: Guideline on Risk Assessment of Medicinal Products on Human Reproduction and Lactation: From Data to Labelling (2009)

Pregnancy & Lactation Labeling Rule (PLLR)*

8.1 Pregnancy Pregnancy exposure registryg y p g y Risk summary (required information) Clinical considerations Data

8.2 Lactation Risk summary (required information)

Cli i l id ti Clinical considerations Data

8.3 Females and males of reproductive potential Pregnancy testing Contraception Infertility

54

y* Final Rule: Content and Format of Labeling for Human Prescription Drug and Biological Products; Requirements for Pregnancy and Lactation Labeling (effective June 30, 2015)

Reproductive/Developmental p / pToxicity Studies for Chemicals

55

Reproductive/Developmental Toxicity Studies for Chemicals*

Prenatal developmental toxicity studyE f t ti d (GD) 6/7 th h d f (GD 20 i

Toxicity Studies for Chemicals

Exposure from gestation day (GD) 6/7 through end of pregnancy (GD 20 in rats and GD 28/29 in rabbits)

C-section on the day before delivery, perform fetal exams similar to Segment II studies for drugsSegment II studies for drugs

Reproduction and fertility effect study (2-generation reproduction study) Exposure 10 weeks (males/females) before cohabitation, during

cohabitation pregnancy and lactationcohabitation, pregnancy and lactation Exposure continues for selected F1 offspring that are mated and allowed to

deliver and maintain their (F2) pups to weaningAssess effects on reproductive development for 2 generations Assess effects on reproductive development for 2 generations

Developmental neurotoxicity study Exposure from GD 6 to postnatal day 10 or 21 Evaluate developing nervous system

56* See guidelines listed in Slide No. 75.

Juvenile Animal Studyy

57

Juvenile Animal Studyy

Purposep To assess effects of compound on postnatal growth

and development when (direct) exposure occurred from neonatal to pre-adult period

Timing of study during drug development [ICH M3(R2)]Timing of study during drug development [ICH M3(R2)] Prior to long-term pediatric study Generally not needed to support short-term pharmaco-y pp p

kinetic studies in pediatric population (e.g., 1 to 3 doses)

58

Different Age Groups in Pediatric Population & Juvenile AnimalsPopulation & Juvenile Animals

Human Rat Dog Cynomolgus Monkeyg Monkey

Neonate Birth – 1 month Birth – PND 7 Birth – 3 weeks Birth – 4 months

Infant 1 – 24 months PND 7 – 21 3 – 6 weeks 4 – 6 months

Child 2 – 12 years PND 21 – 35 6 – 20 weeks 6 – 36 months

Adolescent 12 – 16 years PND 35 – 60 5 – 8 months 3 – 5 years

PND=Postnatal Day

Pediatric population has different subgroups each showing a

Adolescent 12 – 16 years PND 35 – 60 5 – 8 months 3 – 5 years

59

Pediatric population has different subgroups, each showing a different stage/rate of growth and organ/system maturation

Physiology/Pharmacokinetics in Neonate/Infant Compared to AdultNeonate/Infant Compared to Adult

Neonate/Infant

GI Motility/pH Lower GI motility ( GI absorption), higher pH ( absorption of basic molecule, absorption of acidic molecule)

P t i Bi di T i ll l f dProtein Binding Typically lower, more free compound

Total Water Content Higher, affects volume of distribution

Metabolism Enzymes not fully developed, affecting pharmacokinetics

Biliary Excretion Lower, may half-life

60

Glomerular Filtration Lower, may half-life

Organ/System Maturation Period in Human

Urinary System

g / yGI: Gastrointestinal

Dosing period in juvenile animal study ill be dete mined b ta get o gans

Pulmonary/GI Systems

ywill be determined by target organs that are undergoing development

during clinical exposure

Cardiovascular/Immune Systems

Central Nervous/Reproductive Systems

Biotransformation Enzymes

Skeletal System

Central Nervous/Reproductive Systems

61Birth 2 years 12 years 16 years

y

Non-Clinical Studies Performed to Support Pediatric Use

Animals should beAnimals should be treated throughout the stages of development that are comparable to pthe timing of exposure in the intended pediatric population

62

Adult vs. Juvenile Animal Study

Evaluate drug’s effect on developmental stage

y

and effect of developmental stage on drug

Repeat-Dose Studies J il A i l St diRepeat Dose Studies in Adult Animals Juvenile Animal Studies

A 1 t D Y d l Varies, depending onAge at 1st Dose Young adult Varies, depending on pediatric regimen

Typically standard e g Varies depending onDosing Period Typically standard, e.g.,4-, 13-week

Varies, depending on pediatric regimen

Study

63

Study Endpoints Typically standard Study-specific

Factors InfluencingJuvenile Animal Study Design

Pediatric indication

Juvenile Animal Study Design

Pediatric population Age range

D ti f Duration of exposure

Toxicity of drug in adult human/animal

Wh th t t id tifi d i d lt d Whether target organs identified in adults undergo significant postnatal development

Differences in pharmacology pharmacokinetics and Differences in pharmacology, pharmacokinetics, and toxicity profiles between adult and pediatric population

64

Juvenile Animal Study Design (1)

Step-wise Approach in Designing Juvenile Animal Study

y g ( )

1. Determine dosing regimen in pediatric population Dosing begins from age X Dosing extends from age X to YDosing extends from age X to Y

2. Identify appropriate Species Target organs (observed in adult humans and animals) Developing organs/systems in humans at age X to Y, and corresponding

developing intervals for these and target organs in test species

3. Determine age at 1st dose and dosing period based on #1 and #2

4. Study should include evaluation of effects onO / t i #2 Organs/systems in #2

Growth, functional & structural development, as appropriate65

Juvenile Animal Study Design (2)

Step-wise Approach in Designing Juvenile Animal Study

y g ( )

5. Dose route Intended route of clinical exposure, as appropriate Use labs whose technicians are competent in dosing young pupsUse labs whose technicians are competent in dosing young pups

6. Essential to perform pilot/range-finding study

7. Study should includey Toxicokinetic evaluation Recovery assessment

B th t d d i i ll li t d i lBecause the study design is usually complicated, uses many animals, takes a long time to complete and expensive, important to design the study appropriately

Where possible, get concurrence on study design from regulatory agencies

66

SummarySummary

Important to understand biology/timing of development

Embryo/fetus depends on dams for growth & development

Important to distinguish between direct fetal toxicity & fetal changes due to maternal toxicity

Litter is the unit for evaluation/comparison

U i t t d h t l t d ti t i it Use integrated approach to evaluate reproductive toxicity

Fetal/pup weight is the most sensitive indicator for growth & development& development

For juvenile animal studies Need to know the age range and duration of exposure, target organs, g g p , g g ,

timing of organ/system development in animals vs. humans

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ICI 204,636: Segment I Studies in Alpk:ApfSK (Wistar-derived) Rats

Male Fertility StudyTreatment Period

GD: Gestation Day11 weeksMating*

♂C-section on GD 21 to

perform uterine/fetal exam

F1 in uteroDevelopment

F0

♀ e e op e♀

* Control and high-dose males mated twice, (1) after 11 weeks of dosing [for up to 3 weeks], (2) 2 weeks after the end of dosing (at the end of 1st mating period)Lo nd mid do e m le ifi ed t Week 17 18 high do e m le ifi ed t

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Low and mid-dose males sacrificed at Week 17-18, high-dose males sacrificed at the end of 2nd pairing

ICI 204,636: Segment I Studies in Alpk:ApfSK (Wistar-derived) Rats

Treatment PeriodFemale Fertility StudyGD: Gestation DayLD: Lactation Day

Mating

y y

C-section on GD 21 to perform uterine/fetal exam

F1 i t

F0

♂ Dosing completed

LD 22-24 F1 males sacrificed at th d f tiF1 in utero

Development♀2 weeks

the end of mating

F1 growth/development

F1 females sacrificed

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on GD 21 or LD 22-24

ICI 204,636: Embryo-Fetal Development Studies

F0 Mating

Treatment Period

Alpk ApfSK rats GD 6 15F0 Mating

F1

♂In-life

Termination

Alpk:ApfSK rats: GD 6-15

Dutch Belted rabbits: GD 6-18

GD: Gestation Day

in utero

Development

F1 GD: Gestation Day

♀

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ICI 204,636: Peri- & Postnatal Development Study

T t t P i d

Alpk:ApfSK (Wistar-derived) Rats

Treatment Period

Delivery♂ GD: Gestation Day

LD: Lactation Day

GD 16

F1 in uteroDevelopment

F0Termination at LD 21

F1♀

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AbbreviationsAbbreviations

ABP Androgen-binding protein CNS Central nervous system DHT Dihydro-testosterone FSH Follicle-stimulating hormoneg GD Gestation day GnRH Gonadotropin-releasing hormone LD Lactation day LD Lactation day LH Luteinizing hormone MAA Marketing Authorisation Application

NDA New Drug Application NDA New Drug Application NHP Nonhuman primate PND Postnatal day WOCBP Women of childbearing potential

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References (1)

1. ICH M3(R2) Nonclinical Safety Studies for the Conduct of Human Clinical Trials and Marketing Authorization for Pharmaceuticals (June 2009).

2. ICH S5(R2): Detection of Toxicity to Reproduction for Medicinal Products & Toxicity to Male Fertility (Nov. 2005).

3. ICH S6(R1): Addendum to ICH S6: Preclinical Safety Evaluation of Biotechnology-derived Pharmaceuticals (Draft Guidance, Oct. 2009).

4. ICH S9:Nonclinical Evaluation for Anticancer Pharmaceuticals (March 2010).5. FDA Guidance for Industry: Nonclinical Safety Evaluation of Pediatric Drug Products (Feb. 2006).6. CHMP Guideline on Risk Assessment of Medicinal Products on Human Reproduction and Lactation:

From Data to Labelling (Jan. 2009).7. CHMP Guideline on the Need for Non-Clinical Testing in Juvenile Animals on Pharmaceuticals for

Paediatric Indications (Aug. 2008).8. PMDA Guideline on the Non-clinical Safety Study in Juvenile Animals for Pediatric Drugs (Oct.

2012).9. Lerman SA, Hew KW, Stewart J, Stump DG, Wise LD. 2009. The nonclinical fertility study design

for pharmaceuticals. Birth Defects Research Part B: Developmental and Reproductive Toxicology86(6):429-436.

10. Wise LD, Buschmann J, Feuston MH, Fisher JE, Hew KW, Hoberman AM, Lerman SA, Ooshima Y, St DG 2009 E b f t l d l t l t i it t d d i f h ti l Bi th

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Stump DG. 2009. Embryo-fetal developmental toxicity study design for pharmaceuticals. Birth Defects Research Part B: Developmental and Reproductive Toxicology 86(6):418-428.

References (2)

11. Bailey GP, Wise LD, Buschmann J, Hurtt M, Fisher JE. 2009. Pre- and postnatal developmental toxicity study design for pharmaceuticals. Birth Defects Research Part B: Developmental and Reproductive Toxicology 86(6):437-445.

12. Chellman GJ, Bussiere JL, Makori N, Martin PL, Ooshima Y, Weinbauer GF. 2009. Developmental and reproductive toxicology studies in nonhuman primates. Birth Defects Research Part B: Developmental and Reproductive Toxicology 86(6):446-462.

13 C GD B il GP B h J F t MH Fi h JE H KW H b AM O hi13. Cappon GD, Bailey GP, Buschmann J, Feuston MH, Fisher JE, Hew KW, Hoberman AM, Ooshima Y, Stump DG, Hurtt ME. 2009. Juvenile animal toxicity study designs to support pediatric drug development. Birth Defects Research Part B: Developmental and Reproductive Toxicology86(6):463-469.

14. Martin PL. 2008. Reproductive toxicity testing for biopharmaceuticals. In: J.A. Cavagnaro (Ed.), p y g p g ( ),Preclinical Safety Evaluation of Biopharmaceuticals. John Wiley & Sons, Inc., Hoboken, NJ, pp. 357-377.

15. Morford LL, Bowman CJ, Blanset DL, Bøgh IB, Chellman GJ, Halpern WG, Weinbauer GF, Coogan TP. 2011. Preclinical safety evaluations supporting pediatric drug development with biopharmaceuticals: strategy challenges current practices Birth Defects Research Part B:biopharmaceuticals: strategy, challenges, current practices. Birth Defects Research Part B: Developmental and Reproductive Toxicology 92:359-380.

16. Developmental and Reproductive Toxicology, 3nd Edition (R.D. Hood, Ed.) Informa Healthcare, London, 2012.

17. Teratogenicity Testing: Methods and Protocols (P.C. Barrow, Ed.) Humana Press, New York,

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17. Teratogenicity Testing: Methods and Protocols (P.C. Barrow, Ed.) Humana Press, New York, 2013.

18. Link to ICH Safety Guidelines: http://www.ich.org/products/guidelines/safety/article/safety-guidelines.html

Reproductive Toxicity Testing Guidelines for Chemicals

United States Environmental Protection Agency (US EPA) Health Effects Test Guidelines OPPTS 870.3700, Prenatal Developmental Toxicity Study, August 1998. [OECD Guideline

Guidelines for Chemicals

, p y y, g [414, January 2001]

US EPA Health Effects Test Guidelines OPPTS 870.3800, Reproduction and Fertility Effects, August 1998. [Two-Generation Reproduction Toxicity Study OECD Guideline 416, January 2001]2001]

US EPA Health Effects Test Guidelines OPPTS 870.6300, Developmental Neurotoxicity Study, August 1998. [OECD Guideline 426, October 2007]

Organisation for Economic Cooperation and Development (OECD) Guideline for Testing of Chemicals: 415, One-Generation Reproduction Toxicity Study, May 1983.

OECD Guideline for the Testing of Chemicals, 443, Extended One-Generation Reproductive Toxicity Study July 2012Toxicity Study, July 2012.

OECD Guideline for Testing of Chemicals: 421, Reproduction/Developmental Toxicity Screening Test, July 1995. [EPA OPPTS 870.3550, July 2000]

O C G id li f i f Ch i l 22 C bi d d i i S d OECD Guideline for Testing of Chemicals: 422, Combined Repeated Dose Toxicity Study with the Reproduction/Developmental Toxicity Screening Test, March 1996. [EPA OPPTS 870.3650, July 2000]

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Glossary of Reproductive Indices/Terms (1)Glossary of Reproductive Indices/Terms (1)

Precoital Intervals (Days) = Number of days to mate successfully

Male Mating Index (%) = Number of ♂ with evidence of mating

Total number of ♂ used for matingX100

Male Fertility Index (%) = Number of ♂ siring at least 1 litter

Total number of ♂ with evidence of matingX100

Female Mating Index (%) = Number of ♀ with evidence of mating

Total number of ♀ used for matingX100

Female Fertility Index (%) = Number of ♀ with confirmed pregnancy

Total number of ♀ with evidence of matingX100

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Gestation Index (%) = Number of ♀ with live pup

Number of ♀ with evidence of pregnancyX100

Glossary of Reproductive Indices/Terms (2)Glossary of Reproductive Indices/Terms (2)

Pre-implantation Loss (%) =Number of corpora lutea – Number of implants

X100Pre implantation Loss (%) = Total number of corpora lutea

X100

P i l i L (%)Number of implants – Number of viable embryos

X100

Number of pups born alive

Post-implantation Loss (%) = p y

Total number of implantsX100

Live Birth Index (%) = Number of pups born alive

Total number of pups bornX100

Number of live pups (at a given time point)Survival Index (%) =

Number of live pups (at a given time point)

Total number of pups bornX100

Number of pups with evidence of milk in stomach

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Lactation Index (%) = Number of pups with evidence of milk in stomach

Total number of pups bornX100

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