Non-Clinical Safety Assessment of New Drugs – Reproductive Toxicology

1. Non-Clinical Safety Assessment of New Drugs – Reproductive Toxicology

2. OUTLINE OF PRESENTATION • Regulatory Guidelines and Principles • Animal Studies • Support of Clinical Trials – What and When • Risk assessment • The label

3. ICHS5 Guideline

5. ICH S5 (R2)

8. THE ‘ICH’ APPROACH • Emphasis on detection of effects (hazards) • Flexible study designs acceptable in all territories • Investigation/characterisation of effects • Refinement and reduction of animal usage

9. How are effects on reproduction and development detected when the target tissues are constantly changing in form and function throughout the reproductive process?

10. Pre-Mating to conception Conception to implantation Implantation and organ formation Organ formation to end of pregnancy Birth to Weaning Weaning to sexual maturity (ICH S5A Guideline) Breaking the process into an integrated sequence………for convenience

11. Reproductive Cycle Premating Sexual Maturity Conception Weaning Implantation Birth

12. APPROACH TO DETECTION Break process into targets Define endpoints Identify appropriate test systems Practical study designs

13. TARGETS • Adult male and female reproductive functions • Development and maturation of gametes • Mating behaviour • Fertilisation • Pre-implantation development • Implantation • Embryonic development • Major organ formation • Fetal development and growth • Organ development and growth • Neonate adaptation to extrauterine life • Preweaning development and growth • Adaptation to independent life • Attainment of full sexual function

14. pre-coital interval proportion mated proportion siring pregnancy pathology of reproductive organs semen analysis oestrous cycles pre-coital interval proportion mated proportion pregnant pre-implantation loss post-implantation loss (early/late) fetal bodyweight sex ratio fetal pathology gestation length proportion of litters born litter size/weight developmental markers behavioural assessments onset of puberty mating to provide F2 generation • pre-coital interval • proportion mated • proportion siring pregnancy • pathology of reproductive organs • semen analysis • oestrous cycles • pre-coital interval • proportion mated • proportion pregnant • pre-implantation loss • post-implantation loss (early/late) • fetal bodyweight • sex ratio • fetal pathology • gestation length • proportion of litters born • litter size/weight • developmental markers • behavioural assessments • onset of puberty • mating to provide F2 generation ENDPOINTS for TARGETS Males Females

15. “TEST SYSTEMS” Less commonly…… Mice, Domestic and/or mini pigs, Non-human primates, Hamsters • In vitro (for special investigations) • eg: rat embryo culture and embryonic stem cells

16. Fertility and early embryonic development Embryo-fetal development Pre- and postnatal development ICH PREFERRED OPTIONS • Although the ICHS5 guideline allows flexibility, • for most medicinal products a 3-study design is used

17. Fertility and early embryonic development Pre- and postnatal development TARGETED STUDY DESIGNS Adult male and female reproductive functions Development and maturation of gametes Mating behaviour Fertilisation Pre-implantation development Implantation Embryonic development Major organ formation Fetal development and growth Organ development and growth Embryo-fetal development Neonate adaptation to extrauterine life Preweaning development and growth Adaptation to independent life Attainment of full sexual function

18. Fertility and Early Embryonic Development Study Segment I Study Fertility Study (male and female) Male or Female Fertility Studies Usually conducted in rats

19. Female Fertility And Early Embryonic Development Study (Rat) Group size = 25 Females AutopsyCaesarean & External Fetal Exam Start Dosing Mating with untreated males Last Dose 14 Days 14 Days Day 6 pc (implantation) Day 0 pc Day 20 pc Dosing period

20. Rat Oestrous Cycle Pro-oestrus oestrus dioestrus

21. Embryofetal Development Study Teratology Study Segment II Study Usually conducted in rats and rabbits

22. Critical Periods In Embryogenesis [Rat] Limbs Palate Face Eye Heart Nervous System 10 11 13 18 19 21 7 8 9 12 14 15 16 17 20 Day of Pregnancy Term = Day 22

23. Embryofetal Development Study(Rat) Group size = 22 Female Rat & Rabbit, 24 Mouse Mating with untreated males Start Dosing Last Dose AutopsyCaesarian & Fetal Exam Day 0 post coitum Day 21 pc Day 17 pc Day 6 pc Dosing period MOUSE dosing period Days 6 - 15 pc RABBIT dosing period Days 7 - 19 pc

24. METHODOLOGY: EMBRYO-FETAL DEVELOPMENT • Embryonic development, and fetal growth and development • pre- and post-implantation embryo-fetal loss • number of live/dead fetuses • crown-rump length • fetal bodyweight, placental weight • sex ratio • Major organ formation, organ development and growth FETAL PATHOLOGY • external examination • skeletal examination • visceral examination

25. Post mortem Examinations in Females

28. RRat Fetus _Day 21 gestation Bouin’s fixation to allow examination of viscera by serial sectioning

29. RRat Fetus _Day 21 gestation. ‘Double Staining’ Processed and stained. Alizarin Red (ossified) & Alcian Blue (cartilaginous) R

30. Rat fetus – Alizarin Red Staining Examples of skeletal abnormalities – Sternum

31. MALE FERTILITY ASSESSMENT • Males dosed for 2 weeks (or more) prior to mating • Semen analysis not routinely required [Semen analysis = sperm number, motility and morphology]

32. Male Fertility (rat) Group size = 25 males Start Dosing Mating with untreated females Last Dose & Autopsy 2, 4 or 10 weeks Longer duration if repro organ changes in toxicology AutopsyCaesarean & External Fetal Exam Females (untreated) Day 0 pc Day 20 pc Dosing period

33. METHODOLOGY: FERTILITY(Treated Male) • Mating behaviour - pre-coital interval. copulation plugs (rat) • Fertility- proportion mated vs proportion pregnant/siring pregnancy • Reproductive function, and development and maturation of gametes • Pathology of reproductive organs • (Semen analysis – case by case) • Fertilisation, pre-implantational development, implantation • proportion of ova implanted (pre-implantation loss)

34. Rat – Testis showing seminiferous tubules

35. General ToxicologySPERMATOGENESIS STAGING • In a transverse section of a seminiferous tubule, up to 5 different cell types may be present, but the cellular associations remain constant • These cellular associations can be divided into 14 stages • Disruption of this pattern indicates testicular toxicity

36. Pre and Post Natal Development Study Peri/post natal study Segment III study Usually rat

37. Pre and Post Natal Development Study(Rat ) Group size = 24 females Matingwith untreated males Start Dosing Parturition Weaning Fo Last Dose & Autopsy 4 weeks Exposure via milk Day 21 pc Day 21 pp Day 18 pp Day 0 pc Day 6 pc F1 Autopsy F1 Parturition F2 Autopsy Mate F1 Offspring F2 Day 7 pp F1 Day 73+ pp F1 Day 95+ pp

38. METHODOLOGY: POST-NATAL DEVELOPMENT • Adult female reproductive function - parturition (gestation length), proportion of litters born • Neonate adaptation to extrauterine life, preweaning development and growth - clinical signs, litter size/weight • Adaptation to independent life - developmental markers, behavioural assessment • Attainment of full sexual function - onset of puberty, mating to provide F2 generation

40. Evaluation of motor co-ordinationusing a rotating rod

41. Evaluation of learning and memoryusing mazes

42. Evaluation of activityusing automated, in cage, beam detectors

43. Biotechnology derived Products ICH S6 (R1) addendum

44. Post-partum Treatment Delivery~160 days after mating Mating Pregnancy confirmed20 days after mating End of studyInfants 6 months old “Enhanced pre- and post-natal study” • Mother and Infant • Plasma TK X4 • Milk TK X3 • Antibodies X2 • Immunoglobulins • Clin pathology X3 • TK profile X2 • TK pre-dose X5 • Antibodies X3 • Immunoglobulins X3 • Clinical pathology X5 • Monitoring of pregnancy • [Fetal growth] Numerous pre-experimental assessments • Infant • Neurobehavioural X3 • Morphological X5 • Skeletal exam X2 • Immune function X2 • ECG, opthals X1

45. CLINICAL TRIALS

46. REPRODUCTION STUDIES RELATIVE TO CLINICAL TRIALS Men • Men can be included in Phase I (volunteers) and Phase II (patients) trials before the conduct of the male fertility study – reproductive organs are assessed in general toxicology studies. • A male fertility study should be completed before the initiation of large scale or long duration clinical trials – Phase III Women • Women not of childbearing potential can be included without reproduction studies if reproductive organs are assessed in general toxicology studies. • For women of childbearing potential there are two options: • Conduct appropriate reproduction studies and take appropriate precautions • Do no definitive reproduction studies but take precautions to prevent pregnancy by pregnancy testing, use of highly effective methods of birth control and entry to trials only after a confirmed menstrual period.

47. REPRODUCTION STUDIES RELATIVE TO CLINICAL TRIALS Women of childbearing potential • In USA assessment of embryo-fetal development (EFD) and female fertility can be deferred to Phase III with use of adequate precautions to prevent pregnancy. • In EU and Japan appropriate preliminary EFD studies are required in 2 species. Definitive studies can be deferred until studies of >3 months or >150 patients are required (normally Phase III). • Based on low rate of pregnancy in clinical trials • Adequacy of preliminary studies to detect hazard • Female fertility studies can be deferred to Phase III • In all regions the pre-and postnatal study is required for marketing approval Clinicians should always be aware of any class history of reproductive hazard of the test material

48. Biotechnology Derived Products

49. How to communicate the outcome? Statutory and regulatory documents (IB, CTA, IND etc) Marketing Applications Product Labelling and SMPC Data that can provide reassurance that the medicine is safe and guide the clinical community and patients on its use.

50. THE LABEL Different approaches in USA and Europe. Reviews of processes ongoing