8 September 2013 14.00 – 17.30 STRESA, ITALY

41ST ANNUAL MEETING OF

THE EUROPEAN

TERATOLOGY SOCIETY

COURSE SPONSORED BY: ETS Education Course: “Testing Strategies for Biopharmaceuticals”

ETS Education Course: “Testing Strategies for Biopharmaceuticals” 8 S E P T E M B E R 2 0 1 3

PROGRAMME

14.00 - 15.00: INTRODUCTION INTO BIOPHARMACEUTICALS

Manon Beekhuijzen, WIL Research Europe, the Netherlands

15.00 – 16.00: THE NON-CLINICAL TESTING OF VACCINES

Paul Barrow, Roche, Switzerland

16.00 - 16.30: COFFEE/TEA BREAK

16.30 - 17.30: USE OF NONHUMAN PRIMATES FOR BIOLOGICS DART

Gary Chellman, Charles River Laboratories, USA

SESSION CHAIRS:

Alan M. Hoberman

Manon Beekhuijzen

Note: Each topic will include ~15 minutes for questions and discussion

ETS Education Course: “Testing Strategies for Biopharmaceuticals”

Page 1

SPEAKER INFORMATION AND ABSTRACTS

INTRODUCTION INTO BIOPHARMACEUTICALS Speaker: Manon Beekhuijzen Affiliation: WIL Research Europe, the Netherlands After graduating as doctorandus in Medical Biology at the University of Utrecht and performing her traineeships and master thesis in the field of reproduction and developmental toxicology (DART), Manon started at the Toxicology department of WIL Research (then named NOTOX B.V.) in 1999. In 2001 she was appointed study director at WIL Research for all types of DART studies. She obtained her Masters degree in Applied Toxicology at the University of Surrey (England) in 2004. In 2007 she started a working group for Belgium and Dutch companies to discuss the technical aspects of non-clinical DART testing. From 2011, onwards she is a council member of the NVT (Nederlandse Vereniging voor Toxicologie) reprotox section and the ETS (European Teratology Society). ABSTRACT The principles of DART (developmental and reproductive toxicity) testing are the same as those for all preclinical toxicology studies: to identify potential hazards to patients. However, DART studies are somewhat unique in that controlled human clinical trials, which assess reproduction and development, are rarely performed. Therefore, preclinical DART testing forms a fundamental basis for risk assessment. A segmented approach to DART testing is most frequently conducted in which fertility, embryo/fetal development (EFD), and pre/postnatal development are assessed in three separate studies in the rat; an EFD study will also be performed in the rabbit. This most common option is described in ICH S5 (R2). However, the guidance document also gives suggested options for combining the three segments into one or two studies.

These principles, as described in the ICH S5 guideline, are also applicable for biopharmaceuticals. However, because of species- specificity alternate approaches may be needed. And in that case you can follow the ICH S6 guideline. In addition to these ICH guidelines, there is an FDA guidance for developmental toxicity testing of preventive and therapeutic vaccines.

In May 2012, the ICH S6 guidance for industry was updated with an addendum to clarify several subjects discussed in the guidance of 1997, including DART. The need for DART testing depends on the type of product, clinical indication and the intended patient population. The specific study design and dosing schedule may be modified based on issues related to species specificity, immunogenicity, nature of the product, mechanism of action, pharmacokinetic behaviour, and embryo-fetal exposure. The ICH S6 guidance mentions when the clinical candidate is pharmacologically active in rodents and rabbits, both species should be used for EFD studies. Developmental toxicity studies should only be conducted in NHP when they are the only relevant species. When no relevant animal species exists for testing the clinical candidate, the use of transgenic mice expressing the human target or homologous protein can be considered. A stepwise approach to selecting the optimal preclinical DART package for a biopharmaceutical will be discussed. First, the most relevant test species must be selected. The relevant test species needs to fulfill four criteria: acceptable species cross-reactivity, appropriate pharmacology/efficacy, acceptable immunogenicity and acceptable exposure. These criteria apply to the overall preclinical package, so it is not only applicable for

ETS Education Course: “Testing Strategies for Biopharmaceuticals”

Page 2

DART. However, there are certain specifics for DART that need to be taken into consideration when going through these criteria.

Based on the most relevant animal species, there are five possible approaches for DART testing of a biopharmaceutical: use of traditional animal species, non-traditional animal species, genetically modified animals or surrogate molecules, or to do no DART testing.

There is one additional approach which was developed especially for preventive and therapeutic vaccines. The EMEA guidance of 1997 mentions that embryo-fetal and/or perinatal studies may be necessary for vaccines that will be given to WOCBP or during pregnancy, but it gives no guidance on study design. Guidance was issued by the FDA in 2006, and is currently used as standard study design for vaccines. The reasons for the need of this adjusted study type are that vaccines are intended to give an immune response, vaccines are usually administered in limited, episodic dosages with months or even years between doses, and these may be adjuvanted.

In conclusion, a scientific-based case-by-case approach is considered the most appropriate strategy for preclinical safety assessment of biopharmaceuticals. There is not one standard approach applicable per biopharmaceutical subclass. While the stepwise approach for non-clinical safety assessment for general toxicity and DART is similar, there are DART specifics that need to be considered for safety testing.

THE NON-CLINICAL TESTING OF VACCINES Speaker: Paul Barrow Affiliation: Roche, Switzerland Paul Barrow has just started work in his fourth European country, having been recruited recently by Roche in Switzerland to take over as Global Head of Reproductive Toxicology when the current occupant retires in a few months. He previously worked as a regulatory toxicologist at Beecham Pharmaceuticals (UK), Roma Toxicology Centre (Italy), MDS (France) and CiToxLAB (France). Currently Vice President of the ETS, he will assume presidential office in 2014. Paul’s latest book on Teratogenicity Testing was published this year; he will probably try to sell you a copy (beware).

ABSTRACT Preventative and therapeutic vaccines are increasingly used during pregnancy and present special considerations for developmental toxicity testing. The various components of the vaccine formulation (i.e. protein or polysaccharide antigen, adjuvants and excipients) need to be assessed for direct effects on the developing conceptus. In addition, possible adverse influences of the induced antibodies on fetal and/or post-natal development need to be evaluated. A guidance document on the preclinical testing of preventative and therapeutic vaccines for developmental toxicity was issued by the FDA in 2006. Preclinical studies are designed to assess possible influences of vaccines on pre- and post-natal development. The choice of model animal for these experiments is influenced by species differences in 1) immunogenicity and 2) the timing and extent of transfer of the induced maternal antibodies to the fetus. The cross-placental transport of maternal immunoglobulins generally only occurs in late gestation and tends to be greater in humans and monkeys than in non-primate species. For many vaccines, the rabbit shows a greater rate of prenatal transfer of the induced antibodies than rodents. For biotechnology-derived vaccines that are not immunogenic in lower species, non-human primates may be the only appropriate models. It may be advisable to test new adjuvants using the ICH

ETS Education Course: “Testing Strategies for Biopharmaceuticals”

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study designs for conventional pharmaceuticals in addition to the developmental toxicity study with the final vaccine formulation. Possible effects of vaccines on fertility may also need to be considered. At present, there are no formal requirements for preclinical juvenile studies, even though the majority of vaccines are intended for children.

Reference: Barrow (ed.) 2013, Teratogenicity Testing: Methods and Protocols, DOI 10.1007/978-1-62703-131-8_7.

USE OF NONHUMAN PRIMATES FOR BIOLOGICS DART Speaker: Gary Chellman Program Director, Developmental and Reproductive Toxicology Affiliation: Charles River Nonclinical Services, Nevada USA

Gary earned his Ph.D. in toxicology from the University of Rochester in 1984, followed by postdoctoral training at the Chemical Industry Institute of Toxicology. Over the next 20+ years Gary has worked in pharmaceutical toxicology, with specialty in reproductive toxicology. From 1986-1996, he was employed by Syntex Pharmaceuticals where he advanced to Department Head of Reproductive Toxicology. For the next 3 years, he was Director of Toxicology at Sanofi Pharmaceuticals. Gary joined Sierra Biomedical (now Charles River Preclinical Services) in 1999, as Vice President of Toxicology. Since 2003, he has been the Program Director for Developmental and Reproductive Toxicology, specializing in the design, conduct and interpretation of nonhuman primate reproductive toxicology studies. Gary is a diplomat of the American Board of Toxicology and a member of the Society of Toxicology, the American College of Toxicology, and the Teratology Society. He has authored/co-authored numerous publications in reproductive toxicology, most recently related to optimizing these studies in nonhuman primates to support biopharmaceutical drug development.

ABSTRACT Conduct of reproductive toxicology studies in nonhuman primates has increased in recent years, especially for biopharmaceuticals based on considerations of pharmacologic responsiveness, immunogenicity, and the ability to test the clinical drug candidate. Initially these studies reflected standard reproductive toxicology designs and were conducted as stand-alone segmented studies. Recent changes in regulatory guidelines (ICH S6 (R1)) and growing emphasis on the 3Rs have resulted in new study designs that are improved scientifically while substantially reducing the number of animals required. This presentation will review these various study designs and discuss current trends in the conduct of NHP developmental and reproductive toxicology (DART) studies. Challenges in interpretation of NHP DART data will be discussed, including fetal loss rates and endpoints for which interpretation is complicated by background changes during pregnancy in cynomolgus monkeys, the most commonly used species. Some key safety findings from NHP repro testing will be presented. Specialty areas including developmental immunotoxicology (DIT) and juvenile toxicology will be covered, including the advantages and disadvantages of working with NHPs for these types of studies. The goal of this presentation will be to enhance the ability of course participants to approach NHP DART study design and implementation in an effective, case-by-case manner.

Introduction into

biopharmaceuticals and DART

Manon Beekhuijzen

ETS EDUCATION COURSE 2013

Aim of presentation

Overview of stepwise approach to follow on

preclinical DART testing for biopharmaceuticals

Regulations and considerations

Introduction DART

(ICH S5)

Introduction

Developmental

And

Reproductive

Toxicity

testing

→ Basis for risk assessment

Introduction DART

One complete life cycle

Premating to

Conception

Conception

to

Implantation

Implantation

to Closure

of hard

palate

Hard palate

closure to

End of

pregnancy

Birth to

Weaning

Weaning to

Sexual

maturity

DOSING

DOSING

DOSING

Traditional ‘segmented’ approach (ICH S5 (R2))

I. Fertility and early embryonic development (FEED)

II. Embryo-fetal development (EFD)

III. Pre- and postnatal development (PPND)

Introduction

biopharmaceuticals

Definition biopharmaceuticals

Biotechnology-derived pharmaceuticals

Biotechnology is:

the use of living systems and organisms to develop useful products,

or

any technological application that uses biological systems, living

organisms or derivatives thereof, to make or modify products or

processes for specific use

Range of biopharmaceutical classes

Biopharmaceutical class Example

Hormones Insulin

Blood products Albumin

Cytokines and growth factors Interferons

Antagonists/inhibitors Soluble receptors

Monoclonal antibodies Mouse, chimeric or humanized

Modified human proteins PEGylation

Vaccines Recombinant proteins

Gene-transfer products Viral delivery systems

Cell-based therapies Autologous, allogeneic and xenogenic

Tissue-engineered products Long-term implants

Advantages of biopharmaceuticals

High degree of specificity:

Highly effective and potent action

Fewer side effects

Cure disease rather than treat

symptoms

Small molecules versus Biopharmaceuticals

Species independent

Non-immunogenic

Metabolized

Short acting

Chronic daily dosing

Toxicity

Linear dose-response curve

Direct effects

Complex formulations

Oral route

Species specific

Immunogenic

Degraded

Long acting

Intermittent dosing

Exaggerated pharmacology

Bell-shaped dose-response curve

Complex temporal relationships

Simple formulations

Parenteral routes

Small molecules versus Biopharmaceuticals

ICH S6 update:

special DART considerations

Principles of DART testing

Follow ICH S5(R2) for biopharmaceuticals.

However, because of species specificity, alternate

approaches may be needed → follow ICH S6(R2).

FDA guidance (2006) for preventive and therapeutic

vaccines for infectious disease indications

ICH S6 addendum (2012)

Need for DART dependent upon:

Product

Clinical indication

Intended patient population

ICH S6 addendum (2012)

Specific study design and dosing schedule:

Species specificity

Immunogenicity

Nature of the product

Mechanism of action

Pharmacokinetic behavior

Embryo-fetal exposure

ICH S6 addendum (2012)

When clinical candidate

pharmacologically active in

rodents and rabbits

When only NHP relevant

species

When no relevant species

Use both species in EFD

Use NHP

Use genetically modified animals

or homologous protein

Selection of relevant species

Selection of relevant species

Four criteria:

A. Species cross-reactivity

B. Pharmacology/efficacy

C. Immunogenicity

D. Exposure

A. Species cross-reactivity

Acceptable level of reactivity

Similar tissue distribution

B. Pharmacology/efficacy

Theoretical hazards with established pharmacology

C. Immunogenicity

Anti-drug antibody (ADA) formation:

Clearing antibodies

Neutralizing antibodies

Cross-reactive neutralizing antibodies

Collection of ADA data:

Titer

Number of responding animals

Neutralizing activity

C. Immunogenicity

Successful strategy: ‘staggered window’ approach

May change dose response

or pattern of developmental

effects

Premating to

Conception

Conception

to

Implantation

Implantation

to Closure

of hard

palate

Hard palate

closure to

End of

pregnancy

Birth to

Weaning

Weaning to

Sexual

maturity

DOSING

DOSING

DOSING

D. Exposure

ICH S6 addendum → differences in placental transfer

High molecular weight proteins do not cross the

placenta by simple diffusion

Monoclonal antibodies use specific transport

mechanism (FcRn) → varies across species

D. Exposure – placental transfer IgG

DeSesso, 2012

D. Exposure – placental transfer IgG

Non-human Primates:

Comparable to humans

It might be questioned if EFD studies are of added value

Important to evaluate indirect result of maternal or

placental effects

D. Exposure – placental transfer IgG

D. Exposure – placental transfer IgG

Five possible approaches

Five possible approaches

When relevant species:

1. Traditional animal species

2. Non-traditional animal species

When no relevant species:

3. Genetically modified animals

4. Use of surrogate molecule

5. No DART testing

1. Traditional animal species

Ensure adequate exposure during developmentally-

sensitive periods

1. Traditional animal species

Mouse may not be appropriate for human mAbs

Evaluation of a mixture of nonspecific source of human

IgGs by Janssen R&D → loss of conceptuses during pre-

or very early post-implantation period

Implants Live young Implants Live young

Range finding study

(N = 4-7)

Main study

(N = 21-25)

Control 13.4 12.0 13.0 11.6

50 mg/kg 13.2 12.8 12.4 11.3

200 mg/kg 11.7 11.4 11.6 10.4

2. Non-traditional animal species

ICH S6 addendum → Fertility studies in NHP:

Mating studies are not practical

Evaluation reproductive tract in general toxicity study

If specific concern, add specialized assessments

If potential effect on conception/implantation,

then homologous product or transgenic model

2. Non-traditional animal species

ICH S6 addendum → EFD and PPND studies in NHP:

Combine these into an enhanced PPND (ePPND)

Stewart, 2009

3. Genetically modified animals

Knock-out (KO): lack an endogenous gene and

therefore fail to express the related protein(s)

Transgenic (Tg): overexpress a target protein

“Humanized” knock-in (KI): a human gene is inserted

(independently or with endogenous KO)

3. Genetically modified animals

Scientific justification:

Lack of available ‘normal’ relevant animal species

Ability to mimic disease and/or to assess worst-case

scenario

Can only identify potential hazards (not useful for

quantitative risk assessment)

3. Genetically modified animals

Evaluate available data

Worst-case

Safety margins

Compensatory mechanisms

Humanized KI mice

Consider mouse-human

protein interactions

4. Use of surrogate molecule

Scientific justification:

Lack of available ‘normal’ relevant animal species

Availability of comparable surrogate molecule

4. Use of surrogate molecule

High demand on resource requirements:

Thoroughly evaluation for similarity

→ target affinity, pharmacodynamics,

undesirable cross-reactivity

Development of new assays

→ biochemical characterization,

bioanalysis, immunogenicity

Separate manufacturing processes

→ characterization of purity and stability

4. Use of surrogate molecule

Can also be used to replace NHP testing:

Minimize use of NHP

Allow for evaluation of DART endpoints not easily

addressed in NHP

Well-established protocols and HCD

Improved statistical power

More experienced labs

More favorable fertility rates

Low spontaneous abortion rates

5. No DART testing

ICH S6 addendum (2012):

Products directed at foreign target

Suggestion adverse effect on fertility or pregnancy

outcome

More reasons:

Based on intended use

Life-saving therapy

Combination with other molecule with DART liability

Sufficient information available about product class

The ‘6th approach’ for

preventive and therapeutic vaccines

Vaccines – guidelines for DART

EMEA (1997).

Note for guidance on

preclinical pharmacological

and toxicological testing of

vaccines:

Embryo-fetal and/or perinatal

studies may be necessary for

vaccines that will be given to

WOCBP.

Vaccines - Developmental toxicity study

Distinguishable factors relevant to vaccines:

Intended vaccine-induced immune response

Administered in limited, episodic dosages

May be adjuvanted

Vaccines - Developmental toxicity study

Premating to

Conception

Conception

to

Implantation

Implantation

to Closure

of hard

palate

Hard palate

closure to

End of

pregnancy

Birth to

Weaning

Weaning to

Sexual

maturity

Summary and Conclusion

Summary (1)

DART ICH S5(R2): FEED, EFD, PPND

Species specificity biopharmaceuticals → ICH S6(R2)

Selection of relevant test species

Species cross-reactivity

Pharmacology/efficacy

Immunogenicity

Exposure

Summary (2)

Five approaches

Traditional animal species

Non-traditional animal species

Genetically modified animals

Use of surrogate molecule

No DART testing

Sixth approach for preventive and therapeutic

vaccines (FDA, 2006)

Conclusion

A scientific-based case-by-case approach is

considered the most appropriate for nonclinical safety

assessment of biopharmaceuticals.

There is not one standard approach applicable per

biopharmaceutical subclass

The stepwise approach is similar for general toxicity as for

DART, however DART specifics need to be considered

manon.beekhuijzen@wilresearch.com

References

Bailey G.P. et al. The mouse may not be an appropriate species for evaluation of reproductive and developmental toxicity for human monoclonal antibodies. Birth Defects Research (Part A) 97:339 P30 (2013).

Barrow P. Developmental and reproductive toxicity testing of vaccines. Journal of Pharmacological and Toxicological Methods 65: 58-63 (2012).

Baumann A. Foundation review: nonclinical development of biopharmaceuticals. Drug Discovery Today 14: 1112-1122 (2009).

Bowman C.J. et al. Embryo-fetal developmental toxicity of Figitumumab, an anti-insulin-like growth factor-1 receptor (IGF-1R) monoclonal antibody, in cynomolgus monkeys. Birth Defects Research (Part B) 89: 326-338 (2010).

Bowman C.J. et al. Embryo-fetal distribution of a biopharmaceutical IgG2 during rat organogenesis. Reproductive Toxicology 34: 66-72 (2012).

Bugelski P.J. and Martin P.L. Concordance of preclinical and clinical pharmacology and toxicology of monoclonal antibodies and fusion proteins: cell surface targets. British Journal of Pharmacology. 166: 823-846 (2012).

Bussiere J.L. et al. Alternative strategies for toxicity testing of species-specific biopharmaceuticals. International Journal of Toxicology 28 (3): 230-253 (2009).

Cavagnaro J.A. Preclinical safety evaluation of biotechnology-derived pharmaceuticals. Nature reviews drug discovery. Volume 1, June, 469-475 (2002).

Chaible L.M. et al. Genetically modified animals for use in research and biotechnology. Genetics and Molecular Research 9(3): 1469-1482 (2010).

References

Chapman K. et al. Preclinical development of monoclonal antibodies: considerations for the use of non-human primates. mAbs 1:5, 505-516 (2009).

DeSesso J.M. et al. The placenta, transfer of immunoglobulins, and safety assessment of bipharmaceuticals in pregnancy. Critical Reviews in Toxicology 42 (3): 185-210 (2012).

EMEA. Note for guidance on preclinical pharmacological and toxicological testing of vaccines (1997).

FDA, Guidance for Industry. Considerations for developmental toxicity studies for preventive and therapeutic vaccines for infectious disease indications (2006).

Herzyk D.J. et al. Practical aspects of including functional endpoints in developmental toxicity studies. Case study: immune function in HuCD4 transgenic mice exposed to anti-CD4 MAb in utero. Human & Experimental Toxicology 21: 507-512 (2002).

ICH S5 (R2), Guideline. Detection of toxicity to reproduction for medicinal products & toxicity to male fertility (2005).

ICH S6, Guidance for Industry. Preclinical safety evaluation of biotechnology-derived pharmaceuticals (1997).

ICH S6 (R2), Guidance for Industry. Addendum to preclinical safety evaluation of biotechnology-derived pharmaceuticals (2012).

Kooijman M. et al. 30 Years of preclinical safety evaluation of biopharmaceuticals: did scientific progress lead to appropriate regulatory guidance? Expert Opin Drug Saf 1-5 (2012).

References

Lebrec H. et al. Overview of the nonclinical quality and toxicology testing for recombinant biopharmaceuticals produced in mammalian cells. Journal of Applied Toxicology 30: 387-396 (2010).

Martin P.L. et al. Considerations in assessing the developmental and reproductive toxicity potential of biopharmaceuticals. Birth Defects Research (Part B) 86: 176-203 (2009).

Martin P.L. and Weinbauer G.F. Developmental toxicity testing of biopharmaceuticals in nonhuman primates: previous experience and future directions. International Journal of Toxicology 29(6): 552-568 (2010).

Pentšuk N. and van der Laan J.W. An interspecies comparison of placental antibody transfer: new insights into developmental toxicity testing of monoclonal antibodies. Birth Defects Research (Part B) 86: 328-344 (2009).

Ponce R. et al. Immunogenicity of biologically-derived therapeutics: assessment and interpretation of nonclinical safety studies. Regulatory Toxicology and Pharmacology 54: 164-182 (2009).

Stewart J. Developmental toxicity testing of monoclonal antibodies: an enhanced pre- and postnatal study design option. Reproductive Toxicology 28: 220-225 (2009).

Verdier F. et al. Reproductive toxicity testing of vaccines. Toxicology 185: 213-219 (2003).

The non-clinical testing of vaccinesETS Education Course, Stresa8 September 2013

Paul Barrowpaul.barrow.pb1@roche.com

Overview

• Why test vaccines for developmental toxicity?– possible specific risks to reproduction

• Testing strategy– methodology

– species selection

– FDA guidelines

– proposed study designs

– adjuvant testing

– biotech vaccines (primate studies)

– paediatric / juvenile studies

– fertility assessments

Disclaimer: Some views expressed herein are my own; others I’ve shamelessly stolen from far cleverer people. My employer really has no idea what I’m up to.

Advertising

ISBN 9781627031301

Current vaccine products

• Inactivated bacterial and viral vaccines • Cholera, influenza…

• Live attenuated vaccine strains • Measles

• Purified, recombinant or engineered proteins• Engerix hepatitis B vaccine

• Polysaccharide and conjugated vaccines • Bacterial meningitis

• DNA vaccines• EquineWest Nile Virus

• Therapeutic vaccines• Cancer, Alzheimer

• Veterinary vaccines

Adjuvants

• Inorganic salts• alum

• Oil emulsions

• MF59, AS03

• Lipid A fractions of LPS

• MPL

• Saponin-based mixtures

• QS-21

• Oligonucleotides

• CpG sequences.

Why test vaccines for developmental toxicity?

• Arguments against:– Infrequent administration

– Vaccines rarely used in pregnant women in the past

– No documented evidence to date of reprotoxic effects in humans of approved vaccines

– Too complex!

Why NOT test vaccines for developmental toxicity?

www.cdc.gov

• Arguments for:– Extremely long duration of action

• High likelihood of exposure to Abs or sensitised T-cells during pregnancy (even following paediatric use)

– Possible specific risks to development

– Very low acceptable risk threshold for preventative vaccines

– Vaccines now more frequently used in pregnancy• e.g. H1N1

POSSIBLE risks to development

• Changes in maternal immune system during pregnancy– shift towards Th2 (humoral) responses

• effects of immune stimulation on pregnancy not always predictable

• Potential immune targets during development– e.g. cell adhesion molecules

wikispaces.psu.edu

Immune influences on pregnancy

• Cytokines of macrophage origin may cause pregnancy loss– IL-1β, TNFα, IFNγ...

• BUT, non-specific immune stimulation may also protect against chemical-induced teratogenesis– influence on specific gene

expression

Effects of maternal immune stimulation

S Holladay et al.

Mouse

Valproic acid

500 mg/kg on GD8

Neural Tube Defects

In 18% of fetuses

Immune stimulation of mother before mating

GM CSF or IFNγ

Valproic acid

500 mg/kg on GD8

NTD

In 3% of fetuses

Cell adhesion glycoproteins

• Polysialiated forms of NCAMS play a crucial role throughout formation of the CNS (and other tissues, e.g. testis)– not normally prevalent in adult

• except in some pathological states and as a consequence of developmental toxicity

– similar isoforms in polysaccharide MenBvaccines• risk of auto-immunity?

www.med.unc.edu/embryo_images/

Group B Neisseria meningitides

• Polysaccharide structure of capsule well preserved between all strains of GBM– Potential antigenic target

– Immunogenicity improved by chemical modification and conjugation with tetanus toxoid.

– GBMP contains homopolimer of sialic acid

• Murine antibodies to GBMP cross react (weakly) with human polysialiated NCAMS– Coquillat et al., 2001

Vaccine safety data

- Immunogenicity

- Local tolerance and reactogenicity

- General (“repeat dose”) toxicity

- Safety pharmacology investigations ?

- Reproductive toxicity

- Biodistribution studies ?

- Reversion to virulence- Part of product quality control

- Mutagenicity studies not required

- Pharmacokinetics studies not required- Pharmacologic action mediated at injection sites or draining LN.

General toxicology: example study design

20♂, 20♀/group

10♂, 10♀/group

Early necropsy

10♂, 10♀/group

Late necropsy

Control + 1 human dose + adjuvant control (optional)

N vaccinations = human schedule + 1

Performed before first human exposure

Species selection for general toxicology

• Rodents and rabbits often preferred for vaccine safety studies:• Rabbit is sufficiently large to allow administration of human dose.

• The mouse can be useful when Cytotoxic T-Lymphocyte (CTL) responses are to be characterised, because of availability of reagents.

• The monkey is sometimes the only species that meets criteria for selection

• Genetically modified animals or alternative species are sometimes appropriate• Ferret, cotton rat.

ICH DART protocols not applicable

• Daily vaccination not feasible– overdose– desensitisation

• Several potential reproductive toxicants– protein/polysaccharide component (+ adjuvant, excipients, etc)

• vaccinate at least once during organogenesis

• Time to mount antibody response– vaccinate before mating

• Not limited to effects during organogenesis

GD0 GD6 GD17 GD20

4 groups of 24 mated females

NecropsyFetal examinations

Species selection

• Selected species should be responsive to the intended action of the vaccine:

– develop an immune response similar to that expected in humans • humoral and/or cell mediated

– demonstrate a similar effect of any adjuvant

– be susceptible to the pathogen or disease• reflecting infection in man, permitting evaluation of viremia / bacteremia

• Practical considerations

– previous experience with the pathogen

– feasibility of route of administration

– availability of naïve animals

– practicability of reproductive investigations

– availability of reagents for immune analysis

Comparative development

• Rodents immature at birth with respect to humans– may need to expose rat pups post-natally to cover

gesational events in human• kidney development, Ig transfer...

• May need to evaluate entire development

Placentation

www.vivo.colostate.edu

Horse, pig Ruminants

Primates, rodents Dog, cat

Fetal endothelial cells

Fetal connective tissue

Chorionic epithelial cells

Endometrial epithelial cells

Maternal connective tissue

Maternal endothelial cells

Types of placenta & antibody transfer

Placenta Relation to maternal tissue

IgG transfer

Primates Chorioallantoic Hemochorial +++

Lagomorphs Yolk sac, then Chorioallantoic

Endotheliochorial ++

Rodents Yolk sac, then Chorioallantoic

Hemochorial +

Carnivors Chorioallantoic Epitheliochorial ~0

Timing of maternal Ig transfer

From placenta

From colostrum (FcRn in gut)

Primates 100% (IgG) 0

Rodents 10% (IgG) 90% (IgG, IgA, IgM)

Lagomorphs 100% (IgG, IgM) 0

Dog, cat 5% (IgG) 95%

Pig, sheep 0 100% (IgG, IgA, IgM)

More work need to investigate FcRn ontogeny in the placenta/yolk sac across species (possible ILSI/HESI project)

Fetal:maternal IgG ratio

0

100

0.0 0.2 0.4 0.6 0.8 1.0

Proportion of gestation

% m

ater

nal l

og t

itre

Human

Monkey

Rabbit

Rat

Redrawn from Pentsuk & van der Laan, 2009

Guidance for Industry

– Considerations for Developmental Toxicity Studies for Preventive and Therapeutic Vaccines for Infectious Disease Indications. CBER February, 2006

• Single species acceptable–expected to show Ab response

• Episodic dosing preferable

• Dosing before mating

• Single dose level acceptable–1 human dose per animal if possible

• 20 females for caesarean, 20 for post-natal

• Sampling for Ab analysis–maternal Ab transfer / persistence in pups

• Assess post-natal development

Possible strategy

• Preliminary studies of Ab transfer– rat, mouse, rabbit

• Main developmental tox. study in most pertinent species

Preliminary study

• Groups of 12 females

• Treat before and during gestation– eg. 2 weeks before mating, G6, end of gestation

• Submit 6 females to caesarean– maternal & fetal blood samples for Ab analysis

• Terminate 6 females 1 week after birth– maternal & pup blood samples for Ab analysis

Example - HIV vaccine

IgG anti-gp 160 - % maternal titre

0

100

rat mouse rabbit

%(log titre)

fetus

LD11

Species selected

Vaccine Species Reason

HIV Mouse Maternal Ab transfer Tetanus/diptheria/pertussis Mouse Maternal Ab transfer Rabies Rabbit Maternal Ab transfer Meningitis Rabbit Immunogenicity

Main study

• Groups of 50 rodents or rabbits– sub-group for caesarean

• routine embryotox

– sub-group for post-natal examinations• routine developmental assessments up to weaning

– growth, physical development, behaviour...

• routine necropsy

Developmental toxicity study for vaccines

50 ♀ / group

25 ♀ / group post-natal

M-14d M-1dGD6

GD28

GD29

PND35

25 ♀ / group caesarean

Rabbit – post-natal investigations

Rabbit background data

• Historical control data from 15 studies: Barrow and Allais 2012, In: Barrow, P.C. (Ed). Teratogenicity Testing. 602p. Springer, Berlin. ISBN 9781627031301.

Number of females Mean Mean % of kits % of kits

Mated Pregnant With With kits gestation length number of liveborn surviving to

liveborn at weaning (days) kits born weaning

326 301 289 241 31.8 8,6 97 91

92% 89% 74%

Data: WIL Europe, Lyon

Adjuvants

• Separate study:– Following ICH S5(R2) embryotox guidelines

• No need for dosing before mating• 2 species preferable

• Or, add additional group to vaccine study– Also covers post-natal development

• Biomarkers of systemic inflammatory response?– e.g. serum IL-6, CRP, fibrinogen…

• Possible exaggerated pharmacology– e.g. TLR-9 agonists in rodents

Primates - considerations

• Poor availability of animals

• Poor mating performance– Vaccination prior to mating is impractical

– Ab exposure will ensue during pregnancy thanks to long gestation period

• Very limited number of offspring examined– Only one fetus per pregnancy

– Frequent fetal loss

• Study duration of 1.5 to 2 years– ~4 months necessary to mate all females

– 160 days of gestation, several months of post-natal development

• Long maturation period of offspring (4 to 5 years)

Proposed design - primates

Based on “Enhanced pre- and post-natal study design” proposed for Mabs.

Stewart, 2009, Reproductive Toxicology 28 220-5.

12-14 ♀ / group

PND160~GD160

Birth

GD20

Ultrasound scan

ePPND study - limitations

• Disadvantages– Few animals

• Even less offspring (~10/group)• Little or no statistical power

– No exposure before GD20 (pregnancy confirmed by ultrasound scan)• Not a major problem for mAbs

– No caesarean exams • Fetal examination by X-ray• Examination of babies at birth.

Assessment of immune system

• Clinical pathology

– e.g. lymphocyte subsets

• Organ weights & histopathology

– liver, spleen, lymph nodes...

• Functional tests of cellular & humoral immunity

– e.g. TDAR

Paediatric evaluations

• A paediatric evaluation (or waiver) is now mandatory for all New Drug Applications in North America or Marketing Authorisation Applications in Europe– FDA, EMA and MHLW (Japan) have issued guidelines on juvenile toxicity

studies

• Developmental toxicity studies are usually not necessary for vaccines indicated for immunization during childhood (yet!).– Even though most vaccines are given to children

Suggestion

• Perform repeat dose studies in juvenile animals– Examples of current RDS:

• Rats starting at 6 weeks old, rabbits starting at 12-14 weeks• 3 vaccinations 2 weeks apart• Half of animals retained for 2 weeks recovery

– Juvenile / RDS:• Start dosing 1 week after weaning (4 weeks for rat, 8 for rabbit)• Perform all examinations currently included in RDS• Both species will be adult at time of necropsy

–10.5 weeks for rat, 14.5 weeks for rabbit (recovery animals)

• Covers all developmental periods for vaccines given to infants or older– No loss of sensitivity for effects in adult

POSSIBLE effects on fertility

• Effects on reproductive organs should be detected in repeat dose studies– Provided that animals are adult at time of necropsy

– No test of mating ability

• Mating performance of the female can be assessed during developmental toxicity study– Not the main objective of the study

• E.g.: what if copulation is delayed by a few days in the vaccinated group, but females later prove to be fertile?

Effects on early embryo?

• Preimplantation embryo has no protection from immunoglobulins present in intrauterine or fallopian fluid.

• Toxic insult at this stage may result in embryonic death, but not normally in dysmorphogenesis.

Vaccines and Medicationsin Pregnancy Surveillance System (VAMPSS)

“Women are also asked about all vaccines they may have received, including those given in non-traditional settings such as health fairs or at the supermarket.”

Summary

• Most vaccines present a theoretical risk of developmental toxicity– no proven causal links demonstrated with marketed vaccines to date

• Classical testing strategies (ICH) are not applicable– species, dosing regime, post-natal evaluations– primate studies will be necessary for some biotech vaccines

• Potential hazards are not limited to use during pregnancy– need to think about paediatric studies– fertility?

• Pharmacosurveillance in humans is important

Use of Nonhuman Primates for Biologics DART

Gary J. Chellman, PhD, DABT Program Director, DART Charles River Preclinical Services, Reno, Nevada USA European Teratology Society Meeting Stresa, Italy 08 Sep 2013

Introduction of Topics

• Advantages and Disadvantages of NHP model

• Study Designs

• Current Trends

• Data Interpretation Challenges

• Developmental Immunotoxicology (DIT)

• Juvenile Toxicology

Advantages of NHP Model

• Biologics: pharmacology w/ minimal immunogenicity

• Allows testing of the clinical candidate (drug)

• Embryology/reproductive physiology similar to man

• Similarity in response to known human teratogens

• Pregnancy can be monitored by ultrasound

• Comprehensive endpoints can be evaluated

Disadvantages of NHP Model

• Small sample size (n = 12-20/group)

• Low conception rate (30-40%)

• Long duration (gestation = 5.5 months)

• High abortion rate (15-20% full term)

• Single offspring

• Limited availability of CROs with expertise

• Cost of studies

Study Designs

“Typical” NHP DART Study Designs

• Mating Studies

– Embryo-fetal development (EFD)

– Pre and postnatal (PPND)

• Non-mating Studies

– Male and/or female reproductive toxicity

– Juvenile

Think outside the box for study design!

Chellman et al. (2009) BDR 86:446-462

Reproductive Life Cycle – ICH S5 (Stages A to F)

Senescence

Embryo

Juvenile

Mating/fertility

Adolescence/puberty

Zygote

Fetus

Gametes (M/F)

A. Premating to Conception

F. Weaning to Sexual Maturity

B. Conception to Implantation

C. Implantation to Closure of Hard Palate

D. Hard Palate Closure to End of Pregnancy

E. Birth to Weaning

Source: Christian, M.S. (2001). In: Principles and Methods of Toxicology (4th Edition, A. Wallace

Hayes, editor), pp. 1301-1381.

Male Reproduction Stand-Alone Study (non-mating, sexually mature)

Treatment

Usually include: • Sperm count, motility and morphology • Testes volume • Testosterone Duration spermatogenesis ~45d

Recovery

Necropsy Organ weights Histopathology (option for spermatogenesis eval.)

Week -3 Day 1 Month 3 Month 4

Female Reproduction Stand-Alone Study (non-mating, sexually mature)

Observation Cycles

1

Treatment Cycles

2 3 6 7 4 5

Recovery (1+ cycle)

Synchronize dosing to menstrual cycle?

Average NHP cycle length 28-32 days

Necropsy Organ weights Histopathology

Usually include: • Menstrual cycles (vaginal swabs) • Hormones:

Blood samples every 2-3 days, 3 cycles (Pre, EOD, EOR) Measure E/P (LH/FSH optional)

Embryo-Fetal Developmental Study (EFD)

Cycle check

2 months

GD 0 GD 18-20 GD 50 GD 100

Treatment Pregnancy Monitoring

Confirm pregnancy (ultrasound)

Mating C-section • Placenta • Maternal blood • Cord blood • Amniotic fluid

Fetal exams • External • Visceral • Skeletal

End of major

organogenesis

Biologics: Immunologic Testing • Flow cytometry (mother) • Immunoglobulins (mother) • Immunohistochemistry (fetus)

Note: extend dosing?

(e.g., MAb’s)

Placental Transfer NHP (FcRn)

Although fetal IgG exposure is represented as being at or

below baseline prior to the second trimester, recent

information suggests some IgG may be present during the 1st

trimester at levels that could be pharmacologically or

toxicologically active (Dybdal,2010; Wang et al., 2011).

Pre-Postnatal Development Study (PPND)

Cycle checks

2-3 months

GD0 GD18-20 GD160 PP 3-12M

Treatment/ Pregnancy Monitoring

Confirm pregnancy (ultrasound)

Mating Infant necropsy Delivery • Mothers: blood/milk • Infants: blood, physical exams, neurobehavior

Nursing/Behavior

• Standard tox: clinical signs, BW, TK, clin path, necropsy/histo • Immunologic: flow cytometry, Ig’s, TDAR (KLH), NK cell, IHC • Specialized: fetal measures, infant behavior/learning

Juvenile NHP Toxicology Study (13-week duration common)

Dosing

• Clinical signs • Body weight • ECGs • Ophthalmology • Skeletal growth • Clinical pathology • Immunology • Behavioral • TK and ADA

Recovery

• Necropsy • Organ weights • Gross and histopathology • Specialized

• Immunohistochemistry • Bone densitometry

Week -2 Day 1 Week 13 Week 26

Typical age of juvenile NHP is 10/12M to 24M

Current Trends in NHP DART Streamlining (Consolidation) of Study Designs

– Instead of stand alone studies for M/F repro, combine

the endpoints into a chronic toxicology study

• ≥ 13 week dose duration

• Use sexually mature animals

– Instead of conducting separate EFD and PPND

studies, combine into one design known as the

enhanced PPND study (ePPND)

Each of these reduces the number of animals

required by one-half (3Rs)

Male Reproduction in Chronic Toxicity Study Per Current ICH S6 (R1) (3, 6, 9 Months)

Dosing Recovery

Week -3 Day 1 Month 3 Month 4

As needed (for cause): • Sperm count, motility and morphology (at necropsy?) • Testes volume • Testosterone Duration spermatogenesis ~45d

Necropsy (required) Organ weights Histopathology

Female Reproduction in Chronic Toxicity Study (3, 6, 9 Months)

Observation Cycles

1

Treatment Cycles

As needed (ICH S6): • Menstrual cycles (vaginal swabs) • Hormones:

Blood samples 3x/week for 6 weeks (Pre, EOD, EOR) Measure E/P (LH/FSH optional)

2 3 6 7 4 5

Recovery (1+ cycle)

Dosing not synchronized to menstrual cycle

Average NHP cycle length 28-32 days

Necropsy Organ weights Histopathology

Selection Criteria for Sexual Maturity: How successful is your strategy?

• Using males ≥5 years old and ≥5 kg has provided 97% mature [maturity confirmed by histopathology]

Regul Toxicol Pharmacol (2012) 63:391-400

Functional Assessment of Maturity Male Cynos

NHP Enhanced PPND Study Design (ePPND)

Cycle checks

2-3 months

GD0 GD18-20 GD160 PP 3-12M

Treatment/ Pregnancy Monitoring

Confirm pregnancy (ultrasound)

Mating Infant necropsy Delivery • Mothers: blood/milk • Infants: blood, physical exams, neurobehavior

Nursing/Behavior

External Assessment/ Morphometric Eval

(Birth, 1, 3, 6 Months)

Necropsy: External/ Visceral

Eval

Skeletal Eval (Radiograph) (1 Week or

1 Month)

2008-2012: Shift Towards ePPND Studies

ePPND studies have increased (with EFD’s decreased).

Chronic tox/M-F repro has also dramatically increased.

Additional Current Trends

Additional Trends in NHP DART Design ePPND

• Later dose start (GD50) if previous EFD

• Include placental transfer cohort C-sections

• Decrease group size (pregnancies + infants)

• Streamlining of endpoints to focus on infant

growth/development

Chronic Tox with M/F Repro

• Focus on ICH - organ weights/histo (fewer extras)

• Screen males and females functionally to verify

sexual maturity

Social Housing for ePPND Studies

• Can social housing reduce NHP abortion rate?

• How about infant losses?

• Is infant growth and development improved?

Use of Group (Social) Housing for NHP

• Trend is away from single

housing

• New caging allows 2 or

more NHPs to be together

• Stainless mesh and solid

pen panels to

accommodate infants on

DART studies

• Multiple perches, climbing

pole, exploratory balcony

and foraging devices

Data Interpretation Challenges with

NHP ePPND Studies

Historical Control Data (12 ePPNDs): Fetal and Infant Losses

Category Average (%) Range (%) Gestation length (days) 159 ± 7 130 -175

Abortions 1st trimester 8 0 - 15

2nd trimester 1 0 - 10

3rd trimester 14 0 - 29

Infant loss (BD1-28) 11 0 - 20

Data for 12 final ePPND studies (217 pregnancies, 161 births, 2008-2012)

Fetal Loss Interpretation

20

14.3

% loss (Article)

3

0

2

4

1

2

No. lost

27.3

0

16.7

30.8

10

18.2

% loss (Group)

15.0

24.0

14.3

% loss (Dose)

18.2

% loss (Study)

11 High B

9 High A

12 Low B

13 Low A

10 Control B

11 Control A

No. preg.

Group

14.3

20.0

18.2

Background Changes in Pregnancy: B-lymphocytes

Reprod Toxicol 2009 28:443-455

Background Changes in Pregnancy: Cholesterol

Test Article Effects in NHP ePPND’s

• Long lasting TK and PD (≥ 3M postpartum) • GD20-50 EFD monoclonal antibody →

malformations (placental transfer)

• Placental target (CTLA-4) → decreased gestation length + fetal/infant loss

• Metabolic target (RANK-L) → fetal/infant loss + skeletal alterations

• Strong maternal ADA response had major impact

on maternal (and infant) exposure

Developmental Immunotoxicology

DIT in Nonhuman Primate DART Studies Charles River – Nevada (2008-2012)

Inflammatory disease (8), immunomodulatory (5), metabolic (3), other (1)

NHP Infant Immunophenotyping

NHP Immunologic Evaluations (B-cell modulator)

BD7 BD28 BD91 BD3650

500

1000

1500

2000

2500

3000

3500

150 mg/kg

0 (Control)

5 mg/kg

Infant Age (Days)

CD

20+/

CD

21+

(Mat

ure

B-ly

mph

ocyt

es)

Infant Spleen Control

Lymphoid Follicle Lymphoid Follicle

Infant Spleen High dose

7 28 91 182 273 365 0 1 2 3 4 5 6 7 8 9

10 11 12

Days After Birth

*

7 28 91 182 273 365 0

250

500

750

1000 Control

Low (5 mg/kg)

High (150 mg/kg)

Days After Birth

* * * *

Monitoring Immunoglobulins in NHP Infants IgM IgG

Total IgG: transient decrease BD7 to BD91 transition of maternal to infant

source

Total IgM: dose-independent decrease BD7 and BD91 vs. controls, with

recovery

Imm

une

Res

pons

e

Adaptive

Memory

KLH Immunization #1

KLH Immunization #2

Juvenile Toxicology

Juvenile NHP Toxicology Study (13-week duration common)

Dosing

• Clinical signs • Body weight • ECGs • Ophthalmology • Skeletal growth • Clinical pathology • Immunology • Behavioral • TK and ADA

Recovery

• Necropsy • Organ weights • Gross and histopathology • Specialized

• Immunohistochemistry • Bone densitometry

Week -2 Day 1 Week 13 Week 26

Typical age of juvenile NHP is 10/12M to 24M

Juvenile Toxicity Study Purpose

• Evaluate possible effects on postnatal development when

exposure occurs in some/all of the neonatal to pre-adult period

Why not just use adult clinical data for pediatric safety?

• Children are not simply miniature adults

• Differences in physiology and metabolism can alter sensitivity

to drug effects

• These factors are constantly changing during maturation, along

with unique timelines for development of each organ system

• Therefore juvenile toxicity studies need careful design

– Factor in animal age + stage of organ system development

38

Physiology/Pharmacokinetics in Neonate/Infant Compared to Adult

39

Neonate/Infant

GI Motility/pH Lower GI motility ( GI absorption), higher pH ( absorption

of basic molecule, absorption of acidic molecule)

Protein Binding Typically lower, more free compound

Total Water Content Higher, affects volume of distribution

Metabolism Enzymes not fully developed, affecting pharmacokinetics

Glomerular Filtration Lower, may half-life

Biliary Excretion Lower, may half-life

Organ Systems of Potential Concern (and Maturation Periods in Humans) Behavior/cognition development up to adulthood

Immune system development up to twelve years

Reproduction development up to adulthood

Skeleton (growth) development up to adulthood

Renal development up to one year

Pulmonary development up to two years

Cardiovascular development up to five to seven years

Gastrointestinal development up to one to two years

Test what is pertinent (not everything)!

41

Non-Clinical Species/Ages to Support Pediatric Use

Designed on a case-by-

case basis, and the

dosing is designed so

that the organs of

concern would be at the

same maturational stage

in both the animals (likely

rats) and intended patient

population.

Challenges of Acquiring Juvenile NHPs • Can purchase 9M (weaning) to 12M old from vendors

• If need <9M: purpose breed ($$) or obtain from PPND

• Testing juveniles or infants?

• Is goal to close gap below standard age for tox?

– PPN (6M-1Y old) to tox study (2-3Y old)

– Use of sexually immature NHP’s in chronic toxicity

studies may eliminate the need for an additional study

in juvenile NHP’s

– Use of sexually mature NHPs for tox widens gap

• Not practical to use NHP for testing to sexual maturity (4-6

years old)

Conclusions • DART studies in NHP have become more

common due to the increasing number of

biopharmaceuticals in drug development.

• NHPs may be used when traditional species or

alternate models cannot be used (rodents,

rabbits, surrogate molecule).

• The ePPND design has become one of the most

frequently conducted NHP DART designs.

• Work with regulatory agency(s) & CRO to

determine what DART studies are needed and

timeline – plan ahead!

1. Chellman GJ, et al. (2009) Developmental and reproductive toxicology studies in nonhuman primates. Birth Defects Research (Part B) 86:446-462.

2. Rocca MS and Wehner NG. (2009) The guinea pig as an animal model for developmental and reproductive toxicology studies. Birth Defects Research (Part B) 86:92-97.

3. Morford LL, et al. (2011) Preclinical safety evaluations supporting pediatric drug development with biopharmaceuticals: strategy, challenges, current practices. Birth Defects Research (Part B) 92:359-380.

4. Collinge M, et al. (2012) Developmental immunotoxicity (DIT) testing of pharmaceuticals: current practices, state of the science, knowledge gaps, and recommendations. J Immunotoxicol 9(2):210-230.

5. Chapman K, et al. (2012) Pharmaceutical toxicology: Designing studies to reduce animal use, while maximizing human translation. Regulatory Toxicology and Pharmacology 66(1):88-103.

6. Dybdal, N. (2010) Impact of monoclonal antibody administration to pregnant cynomolgus monkeys on early fetal health and development. In: 16th Annual Charles River Biotech Symposium - Biotechnology Derived Therapeutics, Perspectives on Non-clinical Development, San Diego, CA

7. Wang H, et al. (2011) Assessment of placental transfer and the effect on embryo-fetal development of a humanized monoclonal antibody targeting lymphotoxin-α in non-human primates. The Toxicologist. Abstract No. 2772.

Publications Related to Biologics DART

ICH References Related to Biologics DART

• ICH M3(R2) Nonclinical Safety Studies for the Conduct of Human

Clinical Trials and Marketing Authorization for Pharmaceuticals (June

2009)

• ICH S3A Note for Guidance on Toxicokinetics: The Assessment of

Systemic Exposure in Toxicity Studies (Oct 1994)

• ICH S5(R2): Detection of Toxicity to Reproduction for Medicinal

Products & Toxicity to Male Fertility (Nov 2005)

• ICH S6(R1): Addendum to ICH S6: Preclinical Safety Evaluation of

Biotechnology-derived Pharmaceuticals (June 2011)

• ICH S9:Nonclinical Evaluation for Anticancer Pharmaceuticals (Oct

2009)

Thank You!

Questions?