8 September 2013
14.00 – 17.30 STRESA, ITALY
41ST
ANNUAL
MEETING OF
THE
EUROPEAN
TERATOLOGY
SOCIETY
COURSE SPONSORED BY:
E...
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: INT...
ETS Education Course: “Testing Strategies for Biopharmaceuticals”
Page 1
SPEAKER INFORMATION AND ABSTRACTS
INTRODUCTION IN...
ETS Education Course: “Testing Strategies for Biopharmaceuticals”
Page 2
DART. However, there are certain specifics for DA...
ETS Education Course: “Testing Strategies for Biopharmaceuticals”
Page 3
study designs for conventional pharmaceuticals in...
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
 Regulati...
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
pr...
Traditional ‘segmented’ approach (ICH S5 (R2))
I. Fertility and early embryonic development (FEED)
II. Embryo-fetal develo...
Introduction
biopharmaceuticals
Definition biopharmaceuticals
 Biotechnology-derived pharmaceuticals
 Biotechnology is:
 the use of living systems and ...
Range of biopharmaceutical classes
Biopharmaceutical class Example
Hormones Insulin
Blood products Albumin
Cytokines and g...
Advantages of biopharmaceuticals
High degree of specificity:
 Highly effective and potent action
 Fewer side effects
 C...
Small molecules versus Biopharmaceuticals
 Species independent
 Non-immunogenic
 Metabolized
 Short acting
 Chronic d...
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
...
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...
ICH S6 addendum (2012)
 When clinical candidate
pharmacologically active in
rodents and rabbits
 When only NHP relevant
...
Selection of relevant species
Selection of relevant species
Four criteria:
A. Species cross-reactivity
B. Pharmacology/efficacy
C. Immunogenicity
D. Exp...
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 ne...
C. Immunogenicity
Successful strategy: ‘staggered window’ approach
 May change dose response
or pattern of developmental...
D. Exposure
ICH S6 addendum → differences in placental transfer
High molecular weight proteins do not cross the
placenta...
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 a...
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 n...
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 ...
2. Non-traditional animal species
ICH S6 addendum → Fertility studies in NHP:
 Mating studies are not practical
 Evalua...
2. Non-traditional animal species
ICH S6 addendum → EFD and PPND studies in NHP:
 Combine these into an enhanced PPND (e...
3. Genetically modified animals
Knock-out (KO): lack an endogenous gene and
therefore fail to express the related protein...
3. Genetically modified animals
Scientific justification:
 Lack of available ‘normal’ relevant animal species
 Ability ...
3. Genetically modified animals
Evaluate available data
 Worst-case
 Safety margins
 Compensatory mechanisms
Humanize...
4. Use of surrogate molecule
Scientific justification:
 Lack of available ‘normal’ relevant animal species
 Availabilit...
4. Use of surrogate molecule
High demand on resource requirements:
 Thoroughly evaluation for similarity
→ target affini...
4. Use of surrogate molecule
Can also be used to replace NHP testing:
 Minimize use of NHP
 Allow for evaluation of DAR...
5. No DART testing
ICH S6 addendum (2012):
 Products directed at foreign target
 Suggestion adverse effect on fertility...
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 - Developmental toxicity study
Distinguishable factors relevant to vaccines:
 Intended vaccine-induced immune r...
Vaccines - Developmental toxicity study
Premating to
Conception
Conception
to
Implantation
Implantation
to Closure
of hard...
Summary and Conclusion
Summary (1)
DART ICH S5(R2): FEED, EFD, PPND
Species specificity biopharmaceuticals → ICH S6(R2)
Selection of relevant ...
Summary (2)
Five approaches
 Traditional animal species
 Non-traditional animal species
 Genetically modified animals
...
Conclusion
A scientific-based case-by-case approach is
considered the most appropriate for nonclinical safety
assessment ...
manon.beekhuijzen@wilresearch.com
References
 Bailey G.P. et al. The mouse may not be an appropriate species for evaluation of
reproductive and development...
References
 Chapman K. et al. Preclinical development of monoclonal antibodies: considerations
for the use of non-human p...
References
 Lebrec H. et al. Overview of the nonclinical quality and toxicology testing for
recombinant biopharmaceutical...
The non-clinical testing of vaccines
ETS Education Course, Stresa
8 September 2013
Paul Barrow
paul.barrow.pb1@roche.com
O...
Advertising
ISBN 9781627031301
Current vaccine products
• Inactivated bacterial and viral vaccines
• Cholera, influenza…
• Live attenuated vaccine strain...
Why test vaccines for developmental toxicity?
• Arguments against:
– Infrequent administration
– Vaccines rarely used in p...
POSSIBLE risks to development
• Changes in maternal immune system during
pregnancy
– shift towards Th2 (humoral) responses...
Effects of maternal immune stimulation
S Holladay et al.
Mouse
Valproic acid
500 mg/kg on GD8
Neural Tube Defects
In 18% o...
Group B Neisseria meningitides
• Polysaccharide structure of capsule well preserved
between all strains of GBM
– Potential...
General toxicology: example study design
20♂, 20♀/group
10♂, 10♀/group
Early necropsy
10♂, 10♀/group
Late necropsy
Control...
ICH DART protocols not applicable
• Daily vaccination not feasible
– overdose
– desensitisation
• Several potential reprod...
Comparative development
• Rodents immature at birth with respect to humans
– may need to expose rat pups post-natally to c...
Types of placenta & antibody transfer
Placenta Relation to
maternal tissue
IgG
transfer
Primates Chorioallantoic Hemochori...
Fetal:maternal IgG ratio
0
100
0.0 0.2 0.4 0.6 0.8 1.0
Proportion of gestation
%maternallogtitre
Human
Monkey
Rabbit
Rat
R...
Possible strategy
• Preliminary studies of Ab transfer
– rat, mouse, rabbit
• Main developmental tox. study in most
pertin...
Example - HIV vaccine
IgG anti-gp 160 - % maternal titre
0
100
rat mouse rabbit
%
(log titre)
fetus
LD11
Species selected
...
Main study
• Groups of 50 rodents or rabbits
– sub-group for caesarean
• routine embryotox
– sub-group for post-natal exam...
Rabbit – post-natal investigations
Rabbit background data
• Historical control data from 15 studies: Barrow and Allais 201...
Adjuvants
• Separate study:
– Following ICH S5(R2) embryotox guidelines
• No need for dosing before mating
• 2 species pre...
Proposed design - primates
Based on “Enhanced pre- and post-natal study
design” proposed for Mabs.
Stewart, 2009, Reproduc...
Assessment of immune system
• Clinical pathology
– e.g. lymphocyte subsets
• Organ weights & histopathology
– liver, splee...
Suggestion
• Perform repeat dose studies in juvenile animals
– Examples of current RDS:
• Rats starting at 6 weeks old, ra...
Effects on early embryo?
• Preimplantation embryo has no protection from
immunoglobulins present in intrauterine or fallop...
Vaccines and Medications
in Pregnancy Surveillance
System (VAMPSS)
“Women are also asked about all
vaccines they may have ...
Use of Nonhuman Primates for
Biologics DART
Gary J. Chellman, PhD, DABT
Program Director, DART
Charles River Preclinical S...
Introduction of Topics
• Advantages and Disadvantages of NHP model
• Study Designs
• Current Trends
• Data Interpretation ...
Advantages of NHP Model
• Biologics: pharmacology w/ minimal immunogenicity
• Allows testing of the clinical candidate (dr...
Disadvantages of NHP Model
• Small sample size (n = 12-20/group)
• Low conception rate (30-40%)
• Long duration (gestation...
Study Designs
“Typical” NHP DART Study Designs
• Mating Studies
– Embryo-fetal development (EFD)
– Pre and postnatal (PPND)
• Non-mating...
Reproductive Life Cycle – ICH S5 (Stages A to F)
Senescence
Embryo
Juvenile
Mating/fertility
Adolescence/puberty
Zygote
Fe...
Male Reproduction Stand-Alone Study
(non-mating, sexually mature)
Treatment
Usually include:
• Sperm count, motility
and m...
Female Reproduction Stand-Alone Study
(non-mating, sexually mature)
Observation
Cycles
1
Treatment
Cycles
2 3 6 74 5
Recov...
Embryo-Fetal Developmental Study (EFD)
Cycle
check
2 months
GD 0 GD 18-20 GD 50 GD 100
Treatment
Pregnancy Monitoring
Conf...
Placental Transfer NHP (FcRn)
Although fetal IgG exposure is represented as being at or
below baseline prior to the second...
Pre-Postnatal Development Study (PPND)
Cycle
checks
2-3 months
GD0 GD18-20 GD160 PP 3-12M
Treatment/
Pregnancy Monitoring
...
Juvenile NHP Toxicology Study
(13-week duration common)
Dosing
• Clinical signs
• Body weight
• ECGs
• Ophthalmology
• Ske...
Current Trends in NHP DART
Streamlining (Consolidation) of Study Designs
– Instead of stand alone studies for M/F repro, c...
Male Reproduction in Chronic Toxicity Study
Per Current ICH S6 (R1) (3, 6, 9 Months)
Dosing Recovery
Week -3 Day 1 Month 3...
Female Reproduction in Chronic Toxicity Study
(3, 6, 9 Months)
Observation
Cycles
1
Treatment
Cycles
As needed (ICH S6):
•...
Selection Criteria for Sexual Maturity:
How successful is your strategy?
• Using males ≥5 years old and ≥5 kg has provided...
NHP Enhanced PPND Study Design
(ePPND)
Cycle
checks
2-3 months
GD0 GD18-20 GD160 PP 3-12M
Treatment/
Pregnancy Monitoring
...
2008-2012: Shift Towards ePPND Studies
ePPND studies have increased (with EFD’s decreased).
Chronic tox/M-F repro has also...
Additional Current Trends
Additional Trends in NHP DART Design
ePPND
• Later dose start (GD50) if previous EFD
• Include placental transfer cohort C...
Social Housing for ePPND Studies
• Can social housing reduce NHP abortion rate?
• How about infant losses?
• Is infant gro...
Use of Group (Social) Housing for NHP
• Trend is away from single
housing
• New caging allows 2 or
more NHPs to be togethe...
Data Interpretation Challenges with
NHP ePPND Studies
Historical Control Data (12 ePPNDs):
Fetal and Infant Losses
Category Average (%) Range (%)
Gestation length (days) 159 ± ...
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
...
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 →
malform...
Developmental Immunotoxicology
DIT in Nonhuman Primate DART Studies
Charles River – Nevada (2008-2012)
Inflammatory disease (8), immunomodulatory (5), me...
NHP Infant Immunophenotyping
NHP Immunologic Evaluations (B-cell modulator)
BD7 BD28 BD91 BD365
0
500
1000
1500
2000
2500
3000
3500
150 mg/kg
0 (Contro...
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 ...
ImmuneResponse
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
• Ske...
Juvenile Toxicity Study
Purpose
• Evaluate possible effects on postnatal development when
exposure occurs in some/all of t...
Physiology/Pharmacokinetics in
Neonate/Infant Compared to Adult
39
Neonate/Infant
GI Motility/pH
Lower GI motility ( GI a...
Organ Systems of Potential Concern
(and Maturation Periods in Humans)
Behavior/cognition development up to adulthood
Immun...
41
Non-Clinical Species/Ages to Support Pediatric Use
Designed on a case-by-
case basis, and the
dosing is designed so
tha...
Challenges of Acquiring Juvenile NHPs
• Can purchase 9M (weaning) to 12M old from vendors
• If need <9M: purpose breed ($$...
Conclusions
• DART studies in NHP have become more
common due to the increasing number of
biopharmaceuticals in drug devel...
1. Chellman GJ, et al. (2009) Developmental and reproductive toxicology studies
in nonhuman primates. Birth Defects Resear...
ICH References Related to Biologics DART
• ICH M3(R2) Nonclinical Safety Studies for the Conduct of Human
Clinical Trials ...
Thank You!
Questions?
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2013 ets ce course materials (stresa italy)

  1. 1. 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”
  2. 2. 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
  3. 3. 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
  4. 4. 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
  5. 5. ETS Education Course: “Testing Strategies for Biopharmaceuticals” Page 3 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.
  6. 6. Introduction into biopharmaceuticals and DART Manon Beekhuijzen ETS EDUCATION COURSE 2013
  7. 7. Aim of presentation Overview of stepwise approach to follow on preclinical DART testing for biopharmaceuticals  Regulations and considerations
  8. 8. Introduction DART (ICH S5)
  9. 9. Introduction Developmental And Reproductive Toxicity testing → Basis for risk assessment
  10. 10. Introduction DART One complete life cycle
  11. 11. 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
  12. 12. Traditional ‘segmented’ approach (ICH S5 (R2)) I. Fertility and early embryonic development (FEED) II. Embryo-fetal development (EFD) III. Pre- and postnatal development (PPND)
  13. 13. Introduction biopharmaceuticals
  14. 14. 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
  15. 15. 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
  16. 16. Advantages of biopharmaceuticals High degree of specificity:  Highly effective and potent action  Fewer side effects  Cure disease rather than treat symptoms
  17. 17. 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
  18. 18. Small molecules versus Biopharmaceuticals
  19. 19. ICH S6 update: special DART considerations
  20. 20. 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
  21. 21. ICH S6 addendum (2012) Need for DART dependent upon:  Product  Clinical indication  Intended patient population
  22. 22. 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
  23. 23. 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
  24. 24. Selection of relevant species
  25. 25. Selection of relevant species Four criteria: A. Species cross-reactivity B. Pharmacology/efficacy C. Immunogenicity D. Exposure
  26. 26. A. Species cross-reactivity Acceptable level of reactivity Similar tissue distribution
  27. 27. B. Pharmacology/efficacy Theoretical hazards with established pharmacology
  28. 28. 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
  29. 29. 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 W DOSING DOSING
  30. 30. 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
  31. 31. D. Exposure – placental transfer IgG DeSesso, 2012
  32. 32. 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
  33. 33. D. Exposure – placental transfer IgG
  34. 34. D. Exposure – placental transfer IgG
  35. 35. Five possible approaches
  36. 36. 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
  37. 37. 1. Traditional animal species Ensure adequate exposure during developmentally- sensitive periods
  38. 38. 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
  39. 39. 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
  40. 40. 2. Non-traditional animal species ICH S6 addendum → EFD and PPND studies in NHP:  Combine these into an enhanced PPND (ePPND) Stewart, 2009
  41. 41. 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)
  42. 42. 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)
  43. 43. 3. Genetically modified animals Evaluate available data  Worst-case  Safety margins  Compensatory mechanisms Humanized KI mice  Consider mouse-human protein interactions
  44. 44. 4. Use of surrogate molecule Scientific justification:  Lack of available ‘normal’ relevant animal species  Availability of comparable surrogate molecule
  45. 45. 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
  46. 46. 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
  47. 47. 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
  48. 48. The ‘6th approach’ for preventive and therapeutic vaccines
  49. 49. 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.
  50. 50. Vaccines - Developmental toxicity study Distinguishable factors relevant to vaccines:  Intended vaccine-induced immune response  Administered in limited, episodic dosages  May be adjuvanted
  51. 51. 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
  52. 52. Summary and Conclusion
  53. 53. 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
  54. 54. 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)
  55. 55. 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
  56. 56. manon.beekhuijzen@wilresearch.com
  57. 57. 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).
  58. 58. 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).
  59. 59. 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).
  60. 60. The non-clinical testing of vaccines ETS Education Course, Stresa 8 September 2013 Paul Barrow paul.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.
  61. 61. Advertising ISBN 9781627031301
  62. 62. 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.
  63. 63. 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
  64. 64. 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
  65. 65. 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 MenB vaccines • risk of auto-immunity? www.med.unc.edu/embryo_images/
  66. 66. 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.
  67. 67. 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.
  68. 68. 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 Necropsy Fetal 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
  69. 69. 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
  70. 70. 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)
  71. 71. Fetal:maternal IgG ratio 0 100 0.0 0.2 0.4 0.6 0.8 1.0 Proportion of gestation %maternallogtitre 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
  72. 72. 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
  73. 73. 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
  74. 74. 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-1d GD6 GD28 GD29 PND35 25 ♀ / group caesarean
  75. 75. 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
  76. 76. 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)
  77. 77. 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.
  78. 78. 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
  79. 79. 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?
  80. 80. 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.
  81. 81. Vaccines and Medications in 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
  82. 82. 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
  83. 83. Introduction of Topics • Advantages and Disadvantages of NHP model • Study Designs • Current Trends • Data Interpretation Challenges • Developmental Immunotoxicology (DIT) • Juvenile Toxicology
  84. 84. 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
  85. 85. 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
  86. 86. Study Designs
  87. 87. “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
  88. 88. 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.
  89. 89. 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
  90. 90. Female Reproduction Stand-Alone Study (non-mating, sexually mature) Observation Cycles 1 Treatment Cycles 2 3 6 74 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)
  91. 91. 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)
  92. 92. 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).
  93. 93. 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 necropsyDelivery • 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
  94. 94. 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
  95. 95. 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)
  96. 96. 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
  97. 97. 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 74 5 Recovery (1+ cycle) Dosing not synchronized to menstrual cycle Average NHP cycle length 28-32 days Necropsy Organ weights Histopathology
  98. 98. 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
  99. 99. 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 necropsyDelivery • 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)
  100. 100. 2008-2012: Shift Towards ePPND Studies ePPND studies have increased (with EFD’s decreased). Chronic tox/M-F repro has also dramatically increased.
  101. 101. Additional Current Trends
  102. 102. 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
  103. 103. Social Housing for ePPND Studies • Can social housing reduce NHP abortion rate? • How about infant losses? • Is infant growth and development improved?
  104. 104. 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
  105. 105. Data Interpretation Challenges with NHP ePPND Studies
  106. 106. 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)
  107. 107. 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) 11High B 9High A 12Low B 13Low A 10Control B 11Control A No. preg. Group 14.3 20.0 18.2
  108. 108. Background Changes in Pregnancy: B-lymphocytes Reprod Toxicol 2009 28:443-455
  109. 109. Background Changes in Pregnancy: Cholesterol
  110. 110. 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
  111. 111. Developmental Immunotoxicology
  112. 112. DIT in Nonhuman Primate DART Studies Charles River – Nevada (2008-2012) Inflammatory disease (8), immunomodulatory (5), metabolic (3), other (1)
  113. 113. NHP Infant Immunophenotyping
  114. 114. NHP Immunologic Evaluations (B-cell modulator) BD7 BD28 BD91 BD365 0 500 1000 1500 2000 2500 3000 3500 150 mg/kg 0 (Control) 5 mg/kg Infant Age (Days) CD20+/CD21+ (MatureB-lymphocytes) Infant Spleen Control Lymphoid Follicle Lymphoid Follicle Infant Spleen High dose
  115. 115. 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 IgMIgG 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
  116. 116. ImmuneResponse Adaptive Memory KLH Immunization #1 KLH Immunization #2
  117. 117. Juvenile Toxicology
  118. 118. 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
  119. 119. 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
  120. 120. 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
  121. 121. 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)!
  122. 122. 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.
  123. 123. 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)
  124. 124. 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!
  125. 125. 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
  126. 126. 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)
  127. 127. Thank You! Questions?

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