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Species selection in toxicology studies
Generally, rats and dogs have been the rodent and non-rodent
species of choice in safety evaluation
For biologicals non-human primates are often used for safety
evaluation
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Criteria for the selection of the non-rodent toxicology model
Pharmacological activity comparable to human
Pharmacokinetic and metabolic parameters comparable to human
Comparable sensitivity and profile of reactions in test species
Comparative physiology to select for certain end point/target organ(s)
Practical issues
Case-by-case decision, justification needed
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4
The pig closely resembles man in many features of its anatomy,
physiology, biochemistry and life style. In particular, the cardio-
vascular system, skin and gastro-intestinal tract resemble the human
situation better than that in other species
Because of these similarities the toxic effects of chemicals and drugs
in pigs may resemble the effects in man more closely than do some
other commonly used laboratory animals
* See the report of the RETHINK project published in a special issue of the Journal of Pharmacol Toxicol Methods, vol 62, 2010.
Minipig as a model for safety testing*
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Easy to house
Less zoonotic diseases than nonhuman primates
Easy to breed (large litter sizes and quick development time)
Defined health status
Ample genetic and genomic background data available
Growing body of literature and background data
Will be more acceptable for the community as animal test species
than dog/primate
Minipig: a non rodent model for safety testing*
* See the report of the RETHINK project published in a special issue of the Journal of Pharmacol Toxicol Methods, vol 62, 2010.
Safety and toxicity studies
Safety pharmacology
Toxicokinetic and pharmacokinetic studies
General toxicity (acute, repeat dose, chronic toxicity)
Local tolerance
Immunotoxicity
Phototoxicity testing
Genotoxicity
Reproduction toxicity including juvenile toxicity
6 ICH guideline M3(R2) on non-clinical safety studies for the conduct of human clinical trials
and marketing authorization for pharmaceuticals, Step 5
Safety and toxicity studies
Safety pharmacology
Toxicokinetic and pharmacokinetic studies
General toxicity (acute, repeat dose, chronic toxicity)
Local tolerance
Immunotoxicity
Phototoxicity testing
Genotoxicity
Reproduction toxicity including juvenile toxicity
7 ICH guideline M3(R2) on non-clinical safety studies for the conduct of human clinical trials
and marketing authorization for pharmaceuticals, Step 5
Safety pharmacology in minipigs
Cardiovascular function by telemetry
Heart rate and blood pressure comparable to humans
QT interval slightly longer in minipig but does not preclude its use
Respiratory function
Tidal volume, respiratory rate, minute volume
Central nervous system function
FOB
Cognitive testing
8 See: Authier et. al. 2011 and Gieling et. al. 2013
Safety and toxicity studies
Safety pharmacology
Toxicokinetic and pharmacokinetic studies (N. Cnubben)
General toxicity (acute, repeat dose, chronic toxicity)
Local tolerance
Immunotoxicity
Phototoxicity testing
Genotoxicity
Reproduction toxicity including juvenile toxicity
9 ICH guideline M3(R2) on non-clinical safety studies for the conduct of human clinical trials
and marketing authorization for pharmaceuticals, Step 5
Safety and toxicity studies
Safety pharmacology
Toxicokinetic and pharmacokinetic studies
General toxicity (acute, repeat dose, chronic toxicity)
Local tolerance
Immunotoxicity
Phototoxicity testing
Genotoxicity
Reproduction toxicity including juvenile toxicity
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General toxicity studies, administration routes
Oral
Dermal
Intraveneous
Inhalation
Intranasal
Subcutaneous
Intramuscular
Intradermal
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General toxicity studies, observations
All standard observations possible
A.o. Clinical signs, body weight, physiological parameters,
ophthalmoscopy, hematology, clinical chemistry, histology
High blood sampling volume (no satellite groups necessary)
Local tolerance can be included
Immunological parameters can be included
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• Hematological changes (leukocytosis, lymphopenia)
• Alterations in immune system organ weights and/or histology
• Changes in serum globulins might be an indication for changes
in immunoglobulins
• Increased incidence of infections
• Increased occurrence of tumors (sign of immunosuppression?)
Quantitative immunotoxicity testing: Factors to consider in Standard Toxicity Studies*
ICH S8 “Immunotoxicity studies for Human pharmaceuticals” (ICH step 4, in operation from April 2006)
T cell dependent antibody response
Lymphocyte subset analysis
NK activity
Lymphocyte proliferation
Delayed type hypersensitivity
Cytokine production
Host resistance studies
ICH S8 “Immunotoxicity studies for Human pharmaceuticals” (ICH step 4, in operation from April 2006)
Qualitative (functional) immunotoxicity testing
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Vehicle
Cyclosporin A (20 mg/kg/day)
Dexamethasone (0.4 mg/kg/day)
4 ♂ and 4 ♀ per group
• No treatment related clinical observations
Quantitative and functional immunotoxicity in minipigs: a demonstrator study
Penninks and van Mierlo, In: The minipig in Biopharmaceutical Research. 2012, 397- 411.
van Mierlo et. al. Clinical and Experimental Pharmacology, 2013. doi: 10.417/2161-1459.S4-006.
Immunotoxicity study in minipigs
Quantitative endpoints
Hematology
Immune sytem organ weights and histology
Qualitative (functional) endpoints
Ex vivo lymphocyte proliferation
NK cell activity
Delayed type hypersensitivity reaction (DTH)
Lymphocyte subset analysis (still needs attention)
T cell dependent antibody response (TDAR)
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Were overall sucessfully implemented in minipigs.
The effects observed with CsA and DEX were generally in line with those
described in other species
and
Spleen weight
Males Females 0
5
10
15
20
25 Vehicle
CsA
DEX
Sp
leen
weig
ht
(g)
Thymus weight
Males Females 0
5
10
15
20
25
Th
ym
us w
eig
ht
(g) **
**
** P<0.01
Decreased thymus weight by Dexamethasone and Cyclosporin A
Vehicle treated Dexamethasone treated
Vehicle
Cyclosporin A
Dexamethasone
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18
**
** p<0.01
ConA
-1 13 27 0
50000
100000
150000
200000 Vehicle
Cyclosporin A
Dexamethasone
CP
M
**
Daynumber
Males and females combined
In vitro proliferation assay
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Group PBS 2 mg KLH
Vehicle 0 11.4
Cyclosporin A 0 2.2
Dex 0 9.2
Group PBS 2 mg KLH
Vehicle 0 9.1
CyclosporinA 1.1 2.1
Dex 0 9.0
Females
Males
DTH response 72 hours Mean skin reaction (diameter (mm)
Safety and toxicity studies
Safety pharmacology
Toxicokinetic and pharmacokinetic studies
General toxicity (acute, repeat dose, chronic toxicity)
Local tolerance
Immunotoxicity
Phototoxicity testing
Genotoxicity
Reproduction toxicity including juvenile toxicity
20
ICH guideline M3(R2) on non-clinical safety studiesfor the
conduct of human clinical trials and marketing authorisation
for pharmaceuticals, Step 5
Yoshikawa T et al. Toxicol Pathol 2012;41:109-113 Copyright © by Society of Toxicologic Pathology
Minipig in phototoxicity testing
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Safety and toxicity studies
Safety pharmacology
Toxicokinetic and pharmacokinetic studies (N. Cnubben)
General toxicity (acute, repeat dose, chronic toxicity)
Local tolerance
Immunotoxicity
Phototoxicity testing
Genotoxicity (in vitro)
Reproduction toxicity including juvenile toxicity
22
ICH guideline M3(R2) on non-clinical safety studiesfor the
conduct of human clinical trials and marketing authorisation
for pharmaceuticals, Step 5
Safety and toxicity studies
Safety pharmacology
Toxicokinetic and pharmacokinetic studies (N. Cnubben)
General toxicity (acute, repeat dose, chronic toxicity)
Local tolerance
Immunotoxicity
Phototoxicity testing
Genotoxicity
Reproduction toxicity including juvenile toxicity
23
ICH guideline M3(R2) on non-clinical safety studiesfor the
conduct of human clinical trials and marketing authorisation
for pharmaceuticals, Step 5
Minipigs in reproduction toxicity Polyoestrus with short cycle 19-21days (dog mono-estrous with long
interval), oestrus can be synchronised
Gestation period 114 days (dog 59-67 days, NHP ca. 150 days)
Fetal exposure of small molecules resembles that in humans
No placental transfer of antibodies, so less useful for biologicals
(mAbs)
Ca. 5 offspring/litter (dog ca. 6, NHP 1-2)
Cross-fostering and bottle-feeding possible
Size of piglets permits all dosing routes from an early age
Size of piglets permits several investigations (blood sampling, ECG,
opthalmoscopy etc.)
Rapid development to sexual maturity, 3-5 months (entire juvenile
period can be covered).
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25 25
Aim
To get more knowledge about the following parameters during development of
minipigs to 6 months of age:
physical and sensory development
(developmental landmarks)
hematology and clinical chemistry
immunological parameters
the weight and histopathology of 26 main organs
Necropsy
4 ♀ and 4 ♂ minipigs per necropsy day
aged 1-4 days, 7 days, 14 days, 4 wks,
2, 3 and 6 months .
Piglet body weight
0
500
1000
1500
2000
2500
3000
3500
0 5 10 15 20 25 30
days PN
mean
BW
(g
)
Males
Females
males+females
Juvenile development Göttingen Minipigs
Penninks et.al. In: Pedriatric Nonclinical Drug Testing: Priciples, Requirements,
and Practices, 2012, p231-254.
Kuper et. al. Toxicological pathology. 2012, 40:656-666.
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The minipig pig is born relatively mature regarding general
physical and sensory development. So, limited developmental
landmarks available
Comparable development of the lymphoid organs in minipigs
and other species (inverted morphology of lymph nodes)
The minipig is especially suitable for evaluation of toxicity on
development of the nervous-, reproductive- and immune system
Juvenile development of the minipig
Minipigs, what can we offer?
Standard toxicity studies (STS); oral, i.m., s.c., i.d., dermal by patch
testing or semi-occlusive treatment
Kinetics and Metabolism
We generated special experience in the following areas
Immunotoxicity testing
Immunogenicity
Juvenile (immune)development
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Our minipig publications
Book chapters:
Penninks A.H. and Van Mierlo G.J.D. Immunotoxicology: studies in the Minipig. In: The minipig in
Biopharmaceutical Research, Eds PA McAnulty, A Dayan, NC Ganderup and KH Hastings. Taylor & Francis
Group, CRC Press. 2012, 397- 411.
Penninks A.H., van Mierlo, G.J.D., Kuper, C.F., Snel C., Ganderup, N-C, Wolterbeek A.P.M. Juvenile
immunodevelopment in minipigs. In Pedriatric Nonclinical Drug Testing: Priciples, Requirements, and
Practices, Eds Alan M. Hoberman and Elise M Lewis, John Wiley & Sons, Hoboken, New Jersey, 2012,
p231-254.
Scientific journals:
Kuper C.F., Ernst H., Van Oostrum E.C.M., Rittinghausen S., Penninks A.H., Ganderup N-C., Wolterbeek
A.P.M. Nasal passages of Gottingen Minipigs from the neonatal Period to Young Adult. Toxicological
pathology. 2012, 40:656-666.
Van Mierlo G.J.D., Cnubben N.H.P., Wolthoorn J., Ganderup N-C., Penninks A.H. The Gottingen minipig as
alternative non-rodent species for immunogenicity testing: a demonstrater study using the IL-1 receptor
antagonist Anakinra. J Immunotoxicol. 2013;10(1):96-105.
Van Mierlo G.J.D., Cnubben N.H.P., Wouters D., Wolthoorn J., Wolbink G.J., Ganderup N-C, Aarden L.,
Penninks A.H. The minipig as alternative non-rodent model for immunogenicity testing using the TNF α
blockers Adalimumab and Infliximab. J Immunotoxicol. 2013, doi:10.3109/1547691X.2013.796023.
Van Mierlo G.J.D., Kuper C.F., De Zeeuw-Brouwer H.M.H., Schijf, M.A., Otto, M., Ganderup N-C., Penninks
A.H. A subacute immunotoxicity study in Gottingen minipigs with the classical immunosuppressive
compounds Cyclosporin A and Dexamethason. Clinical and Experimental Pharmacology, 2013. doi:
10.417/2161-1459.S4-006.
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