1 Reproduction, Development, and Pathology: Finding Common GroundPoints to Consider for the Inclusion of Reproductive and Pathology Endpoints for Assessment of Reproductive and Developmental Toxicity in Pharmaceutical Drug Development Wendy Halpern, DVM, PhD, DACVP Principal Scientist/Pathologist Genentech, A Member of the Roche Group Christopher J. Bowman, PhD, DABT Associate Research Fellow Pfizer Worldwide R & D

2 Integration of results from General Toxicity and DART studies, including Pathology, is necessary to identify Hazards to Development and Reproduction Clinical and Anatomic Pathology General Toxicity Studies Acute Toxicity Subacute Toxicity Chronic Toxicity Special Toxicity Studies Immunotoxicity Local Tolerance Phototoxicity Carcinogenicity Juvenile Toxicity Developmental and Reproductive Toxicity Reproductive Studies Fertility studies Embryofetal Development Pre-and Postnatal development Developmental Studies Juvenile toxicity

3 Guidance for Pathology and DART Assessments‘Generally…toxicity studies should be designed to evaluate hematology, clinical chemistry, necropsy and histopathology data’ (ICH M3) STP Best Practices for Pathology Assessments in General Toxicity Studies have been established (Bregman et al. 2003, Sellers et al. 2007) Reproductive tract and function are discussed in guidelines for: Development of drugs (ICH M3) Drugs for use in advanced cancer (ICH S9) Biopharmaceuticals (ICH S6) Specific DART Guidance (ICH S5) is currently under revision Development of a new guideline for nonclinical testing in support of pediatrics (ICH S11) is underway

4 Considerable flexibility in existing guidance…Ultimate goal is to evaluate any potential risk to human patients based on the available information, and communicate this risk to physicians & patients When should DART endpoints be added to general toxicity studies? When should pathology endpoints be added to DART studies? What are the current practices? What are the limitations and concerns? STP commissioned an Expert Working Group (EWG) composed of Anatomic Pathologists, Clinical Pathologists, and Reproductive Toxicologists EWG conducted a survey through the STP, but distributed broadly to both toxicologic pathology and reproductive toxicology communities

5 2014 Survey of Pathology and DART EndpointsSurvey Respondents included Academic, Industry and Contract Research Organizations Global Distribution (North America, Europe, Africa, Asia) Individuals and Groups (One response submitted per organization) Pathology, Clinical Pathology and Reproductive Toxicity Groups No enumeration of total individuals or of the total number of studies Several opportunities for respondents to add comments Survey Results enabled identification of: Areas of common practice Areas of variable/inconsistent practice Areas of limited experience Integrated Assessment Existing Guidance Survey Responses Review of Literature EWG Member Experience

6 2016 Reference

7 DART Studies Overlap with General Toxicity TestingPre- and Post-Natal Development Embryo-Fetal Development Male Fertility Female Fertility Juvenile Toxicity

8 Routine Pathology Endpoints in General Toxicity Studies that Contribute to DART AssessmentOrgan Weights Collected Routinely Clinical Pathology Hematology, Clinical Chemistry, Coagulation Hormones only ‘for cause’ Anatomic Pathology Pituitary Gland Testes Epididymides Prostate Gland Seminal Vesicles Ovaries Uterus Vagina % of Respondents Stage-aware histologic evaluation of reproductive tissues: Spermatogenic progression and transit in mature males Cycle stage in mature females

9 Assessment of MaturityCan be important for non-rodents as part of test-system characterization Often, initial nonclinical studies supporting FIH are conducted with pre- or peripubertal dogs or monkeys Normal for the individual animal, so should not be recorded as a microscopic pathology finding BUT nice to know if study could have identified effects on the mature reproductive tissues in that species Typically, at least 1 study of at least 3 months duration should be conducted in mature males to detect potential testicular toxicity

10 Recording of Sexual Maturity in General Toxicity StudiesConfidence of Pathologist Group (Majority) in Histologic Evaluation of Sexual Maturity Females

11 Inclusion of Dedicated DART Endpoints in General Toxicity StudiesRat NHP Dog Rat NHP Dog Rat NHP Dog Rat NHP Dog Sperm Cyclicity Hormones Mating

12 Opportunities and ‘Watch Outs’ for Reproductive Endpoints in General Toxicity StudiesMost repeat dose toxicity studies include a gross and histologic assessment of the male and female reproductive tract Mammary gland assessment can also be useful, especially in cycling females Specialized reproductive assessments, are infrequently included, but may be added for cause May include sperm assessments, monitoring cyclicity, hormone evaluations, and/or mating trials ‘Stage aware’ assessment of male and female reproductive tissues in mature animals Documentation of sexual maturity should be considered Use caution in interpretation of findings in the reproductive tract Easily confounded by stress or body weight loss Endpoints should not be interpreted in isolation

13 Additional ConsiderationsDiscussion of NHP Test Systems Challenges in determining maturity Challenges of environmental stress and social hierarchy Challenges of high variability and low ‘n’ Evaluation of the Mammary Gland in General Toxicity Studies (all species) Not a standard/required tissue, but typically evaluated Characteristic changes during cycle in mature females Sensitive to hormonal disruption First tissue to mature during puberty in NHP Tiered approach to assessment of Ovaries Initial assessment of single section, both ovaries Step sectioning, with follicle counts, as a second tier for cause May require additional investigative study

14 Integrated Assessment-General Toxicity StudiesSubset Species Fertility Clinical Pathology, Organ Weights, Gross and Histopathology Males Rodent Sperm at necropsy and/or hormones for cause Complete (Standard Endpoints) Record Maturity Dog Semen and/or hormones for cause NHP Females Cycling (vaginal smears) and/or hormones for cause Record estrous/menstrual cycle stage if needed to clarify or interpret results Rarely Assessed Cycling for cause; hormones rare

15 Pathology endpoints in DART studiesFlexibility in available guidelines ICH S5(R2) All DART studies: Tissues with macroscopic findings for possible histology Fertility study: preserve testes, epididymides, ovaries, & uteri for possible histology (then discard) Organ weights not specifically required ICH S6(R1) NHP-only programs may rely on reproductive tissue weights & pathology from repeat dose study in mature animals in lieu of fertility study, with additional endpoints added for cause Other FDA Guidance (2015) Testicular Toxicity Recommends histopathology in fertility study if adverse findings in repeat- dose toxicity study FDA Guidance (2011) Integrating study results to assess concerns

16 Survey: Path Endpoints in Fertility StudiesRodent NHP Male Female Clin Path Organ Weights Histopath

17 Pathology Endpoints in Fertility StudiesLimited experience with NHP fertility studies Clinical pathology not typically added without scientific justification, but majority of respondents would include if useful based on previous findings or pharmacological relevance Organ weights routinely collected by a majority of respondents Histopathology done either routinely or conducted for cause by a majority of respondents Experience indicates that tissues are typically collected for possible future examination but NOT routinely evaluated if no effects or pathology data already available from repeat-dose studies in that species and dose range Overall, general practice based on the survey results appear consistent with current regulatory guidance

18 Survey: Path Endpoints in EFD StudiesRodent NHP Dam Fetus Clin Path Organ Weights Histopath

19 Pathology Endpoints in EFD StudiesLimited experience with NHP EFD studies Few respondents routinely add path endpoints (clin path, organ weight, histopath) to EFD studies Addition of path endpoints to define maternal toxicity (slightly different question) 30% respondents yes 48% respondents if no other signs were anticipated Biologics (S6R1) may use path endpoints to justify high dose Most respondents indicated potential challenges of including path endpoints during gestation or development did not impede interpretation Lack of historical data/familiarity Changes with pregnancy/development Effects and/or NOAEL may be different than tox studies

20 Pathology Endpoints in EFD StudiesClinical pathology not routinely evaluated (dams or fetuses), but majority of respondents would include for dams if useful based on previous findings or pharmacological relevance Organ weights from dams not routinely collected (40%) or triggered on case-by-case basis (40%), although acknowledged that gravid uterine weights collected per guideline Fetal organ weights not routinely collected in rodents (63%) but collection is variable in NHP (36% No, 36% Maybe, 28% Yes) Only 6-19% respondents routinely include maternal or fetal histopathology but 47-60% respondents would trigger histopathology ….however, it is unclear how often this would actually happen across studies or programs

21 Survey: Pathology Endpoints in Pre- and Post-Natal Development (PPND) StudiesRodent NHP Dam Pup Clin Path Organ Weights Histopath

22 Pathology Endpoints in PPND StudiesSimilar to EFD studies, few respondents routinely add path endpoints (clin path, organ weight, histopath) to PPND studies but they can be incorporated into the maternal or pup aspects when deemed appropriate based on previous findings or pharmacological relevance 47% respondents add path endpoints case-by-case (ie, pharmacodynamic effects on development) 37% respondents do not add these path endpoints in this way In general, it appeared slightly more respondents would consider path endpoints on NHP studies compared to rodent Different dose rationale for NHP studies (often biologics)? Maximize use of NHP tissues? Most respondents indicated potential challenges of including path endpoints during gestation or development did not impede interpretation Lack of historical data/familiarity Changes with pregnancy/development Effects and/or NOAEL may be different than tox studies

23 Pathology Endpoints in PPND StudiesLike EFD studies, clinical pathology not routinely evaluated (dams or pups), but majority of respondents would include for dams if useful based on previous findings or pharmacological relevance Organ weights from dams not routinely collected (42-46%) but % indicated would trigger based on cause Fetal organ weights not routinely collected in rodents (42%) but may be triggered (37%) or routinely collected (37%) in NHP 47-54% respondents would trigger maternal or fetal histopathology ….however, it is unclear how often this would actually happen across studies or programs Relative to rodent, slightly more respondents would consider endpoints in NHP Maternal & offspring clin path Offspring organ weight & histopath

24 Integrated Assessment-FertilitySpecies Subset Clinical Pathology Organ Weights Histopathology Rodent Male Targeted1 Yes, typically limited to reproductive organs Yes, if reproductive organs are not already characterized. Otherwise preserve for possible future evaluation Female NHP Yes, typically limited to reproductive organs2 Routinely evaluated2 but not driven by guidance 1 limited to endpoints to evaluate efficacy markers, pharmacologic endpoints or specific toxicity concerns 2 when part of general toxicity study a standard panel of tissues including reproductive tissues

25 Integrated Assessment-EFDSpecies Subset Clinical Pathology Organ Weights Histopathology Rodent Dam Targeted1 Targeted1 (uterine weights routine) Fetus Not typical (not typical) No NHP Yes 1 limited to endpoints to evaluate efficacy markers, pharmacologic endpoints or specific toxicity concerns that may contribute to defining maternal toxicity when no other signs of maternal tox are anticipated

26 Integrated Assessment-PPNDSpecies Subset Clinical Pathology Organ Weights Histopathology Rodent Dam Targeted1 (Not typical) Pup NHP Offspring Yes/targeted1 (may depend on age/blood volume) Routinely evaluated but not driven by guidance 1 limited to endpoints to evaluate efficacy markers, pharmacologic endpoints or specific toxicity concerns

27 Nonclinical Studies Supporting PediatricsCurrently regional juvenile toxicity testing guidance is in effect in the US, EU and Japan, and there is an active ICH working group to develop harmonized guidance for nonclinical safety testing in support of development of pediatric medicines (ICH S11) Goal is to assess potential effects on development Focus on organ systems that undergo postnatal development Studies tend to be complex and logistically challenging Each species grows and develops at a different rate …and each organ system can mature at a different rate May be difficult to discriminate between a cumulative toxicity vs exposure during a developmental window of susceptibility

28 Experience with Juvenile Toxicity Studies…is still somewhat limited Relatively low survey response rate (only responding organizations out of the ~100 participating organizations) Comments indicated that some responding organizations lacked direct experience General willingness of survey respondents to include anatomic and clinical pathology endpoints Generally positive experience with inclusion of pharmacodynamic endpoints on a ‘case-by-case’ basis Some concern with availability of historical control data Importance of concurrent controls Challenges of unscheduled necropsies

29 Current Practices for Pathology Endpoints in Juvenile Toxicity Studies

30 What pediatric-relevant information is already available?..from General Toxicity Studies? General effects on growth Body Weight Gain Histology of bone, including growth plate (if open) Effects on tissues of developmental concern Nonrodents are often sexually immature or pubertal …from DART studies? Fertility and EFD studies are not directly supportive May identify target tissues of developmental concern PPND studies can be useful Late gestation and lactation period exposure of offspring May provide ‘worst-case’ scenario for youngest patients Must understand exposure of offspring Consider inclusion of pathology endpoints in offspring

31 Integrated Assessment-Juvenile ToxicitySpecies Subset Fertility Clin Path, Organ Wt, Gross and Histopath Record Maturity Rodent Pre-Weaning Not Applicable Complete/Standard (typically after post-natal day 70) Yes, both sexes Post-weaning to Mature Sperm at necropsy Hormones for cause once mature Mating trials possible NHP Immature Complete (typically after 6-12 months) Dog (typically after 3-6 months)

32 Key aspects of integrating pathology and reproductive endpoints in safety assessmentGeneral toxicity studies/endpoints provide baseline safety assessment May inform fertility assessment (repro tissues) May inform potential risks in pediatrics Do not adequately inform other life stages DART studies/endpoints Functional effects on fertility, pregnancy, lactation Developmental toxicity (pre/postnatal) Juvenile toxicity studies As needed to complement available data

33 Summary Across general toxicity and DART studies should integrate findings from all available information (including general toxicity and DART studies) to identify any hazards to development and reproduction Ultimate goal is to evaluate any potential risk to human patients based on the available information, and communicate this risk to physicians & patients

34 Acknowledgements Working Group Member Affiliations Survey ParticipantsGovernment (NIEHS/National Toxicology Program, Food Safety Commission of Japan) Biopharmaceutical Industry (Bristol-Myers Squibb, Genentech, GlaxoSmithKline, Incyte, Pfizer) Contract Research and Consulting (Covance, Charles River, Regan Path/Tox) Includes diplomates or members of the STP, ABT, ACT, ACVP, ASVCP, SOT, Teratology Society, and the ILSI/HESI DART Technical Committee Survey Participants Broadly distributed to DART and Toxicologic Pathology Community Approximately 100 responding organizations Reviewers Executive Committee of the STP Scientific and Regulatory Policy Committee of the SRPC Reproductive Pathology Interest Group of the STP Regulatory Affairs Committee of the American College of Veterinary Clinical Pathologists Drs. D. Dixon, J. Vidal, M. Cline, and R. Chapin ACT, STP and AIM Support for this Webinar

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36 Presenter Bio Wendy G Halpern, DVM, PhD, DACVPPrincipal Scientist/Pathologist, Safety Assessment Genentech, South san Francisco, CA Dr. Halpern is a veterinary pathologist with more than 17 years of drug development experience in the biopharmaceutical industry. She earned her DVM from the Ohio State University, and also completed an MSc at Ohio State in Veterinary Pathobiology. This was followed by a PhD in Biomedical Sciences from the University of New Mexico focusing on molecular characterization of myeloid leukemias, after which she joined Human Genome Sciences in 2000, and then Genentech in Wendy is a Diplomate of the American College of Veterinary Pathologists, and is also a member of the Society of Toxicologic Pathology, the Safety Pharmacology Society, the Teratology Society, and the ILSI/HESI DART Technical Committee. She has contributed to 24 publications, 4 book chapters, and has presented at more than 30 national and international scientific meetings. She is the current Chair of the Society of Toxicologic Pathology Scientific and Regulatory Policy Committee, is a member of the BioSafe Leadership committee, and represents BIO on the ICH S11 Working Group. She also supports nonclinical strategy and safety assessment for pediatric oncology drug development at Genentech, and leads internal Working Groups for reproductive and developmental toxicology.

37 Presenter Bio (EXAMPLE—CB and WH will be added)Christopher J Bowman, PhD, DABT Associate Research Fellow, Pfizer, Groton, CT Chris completed his PhD in 2001 from the University of Florida.  For the next 2 years as a postdoc at CIIT, Centers for Health Research (Research Triangle Park, NC) he worked on anti-androgens.  After his post-doc, Chris worked at WIL Research Laboratories (Ashland, OH) for ~5 years serving as study director of developmental & reproductive toxicity (DART) studies.  Since 2008 Chris has been part of the senior staff in the Pfizer DART group (Groton, CT) being actively involved in regulatory & investigative strategies evaluating DART, nonclinical support of pediatric development, & serving as a drug safety representative.  Chris has also co-mentored a post-doc in DART.  Over the past 15 years Chris has chaired sessions, presented, & participated at meetings of the Society of Toxicology (SOT), Teratology Society, European Teratology Society, BioSafe (part of BIO), ILSI-HESI (DART), & several regional meetings including lecturing at University of Rhode Island.  He is a member of the BioSafe Leadership Committee and was recently President of the Reproductive & Developmental Toxicology Specialty Section of SOT.  Chris has been a Diplomate of the American Board of Toxicology since 2005.  Chris has over 35 publications in peer-reviewed journals & numerous book chapters.  He has been & continues to be a leader & member of several working groups tackling various challenges associated with DART study strategy, design, interpretation and risk assessment.