The minipig in toxicity testing Geertje van Mierlo

24 September 2014

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

2

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

17

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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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

Juvenile development Ellegaard® Minipig

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Day 3 Day 91

Development Mesenteric Lymph node

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Day 10 Day 91

Development spleen in minipig

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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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Geertje.vanmierlo@tno.triskelion.nl