www.criver.com

EVERY STEP OF THE WAY

SAFETY ASSESSMENT

Nonrodent Models: Minipig Specialty CapabilitiesThe Göttingen minipig has gained favor in pharmaceutical

development programs due to its physiological similarity

to humans (skin structure, digestive, cardiovascular, and

urinary systems), ready availability, known disease status,

and feasibility of genetic and phenotypic manipulation1.

Minipigs also experience fewer species-specific adverse

reactions to compound classes than other large animal

models, making them a viable alternative in those cases1.

As such, regulatory authorities accept the minipig as a

suitable nonrodent species for use in safety evaluation

assessment and efficacy studies of pharmaceutical

products when scientifically appropriate.

Studies using minipigs as a nonrodent species for toxicity

testing have been conducted and submitted to regulatory

authorities worldwide by Charles River Laboratories for

more than 20 years2,3. These include pharmacokinetic,

safety pharmacology, and toxicity studies, including DART

and juvenile, using various dose routes including oral,

dermal, subcutaneous, intravenous, target tissue, intranasal,

ocular, wound healing, and intrathecal.

Intravenous AdministrationIntravenous administration can be performed as bolus

injections via ear vein or implanted catheter, or as

intravenous infusions using cannulated methods for

continuous or intermittent infusion regimens for up to

six months4.

Cannulated methods typically utilize cannulation of the

vena cava, performed with entry via the femoral vein

(a procedure comparable to that used in rodent and

other nonrodent species). While the cannula is in place,

continuous saline infusion is used to maintain patency

(no anticoagulant is used) and historical background has

shown infection rates maintained at < 1%.

Intrathecal AdministrationIntrathecal administration is an invasive procedure;

however, we have has maintained a very low rate of

complications using two approaches in the minipig. With

the first approach, we can surgically implant a catheter

via a microlaminectomy followed by a durotomy at the

lumbar level. Once the catheter is secured on the dura,

SummaryCharles River supports drug

discovery and development

programs with extensive

capabilities and experience in

studies that utilize minipigs as a

nonrodent model.

Nonrodent Models: Minipig Specialty Capabilities

it is connected to an access port which is placed in the

subcutaneous space on the dorsal aspect of the animal.

This approach allows dosing on a conscious animal, and is

particularly well suited for repeat dose studies. The second

method is performed under anesthesia by direct injection in

the intrathecal space. A spinal needle is slowly inserted in

between two vertebrae at the lumbar level; once the dura is

performed, a flow of cerebrospinal fluid (CSF) in the hub of

the needle confirms correct placement. The syringe with the

formulation is then connected to the needle and the dose

is administered slowly. This method is less invasive than

the catheter-based approach, and is ideal for single dose

studies. We can also perform collection of CSF using direct

puncture under anesthesia or via the lumbar catheter.

Subcutaneous AdministrationSubcutaneous administration can be performed as

injections or infusions using bolus injections or a temporary

indwelling cannula combined with rotation of sites. This

model may be of marked interest for evaluation of the

irritancy potential of formulations intended for subcutaneous

administration. In particular, it allows for evaluation of

reactions from the epidermis to the subcutis following

single administrations at various levels/sites, or repeat

administrations at given site(s) within the same animal

to mimic or surpass the clinical plan. A poster detailing a

subcutaneous infusion model which utilizes a rotation of

sites was presented at the annual meeting of the American

College of Toxicology in 20114.

Wound HealingWound healing assessment in the minipig closely resembles

that in humans, based on the similar characteristics between

minipig and human skin and wound healing process as

discussed above5. Specific types of wounds can vary based

on specific need, and wounds ranging from partial thickness

(thermal, incisional, or excisional) to full thickness (incisional

or excisional) can be created on the dorsal surface. Dosing

of the material of interest follows the intended route of clinical

exposure (topical, subcutaneous, intravenous). Macroscopic

evaluation throughout the study interval and microscopic

evaluation at termination allow a full assessment throughout

the healing process. Specialized staining and tensile strength

(incisional wounds only) can be incorporated.

Intranasal AdministrationA clinical device or commercially available nasal spray

pump device is used. Alternatively, when the dose needs

to be administered based on the animal’s body weight,

a calibrated micropipette or a 1 cc syringe could also be

used to deliver the dose in each nostril. The dose volumes

that can be administered are similar to those used in other

nonrodent models (0.1 to 1.0 mL/nostril) and dosing can be

performed several times per day.

Fundus photographs of the Göttingen minipig illustrating normal optic nerve head and major retinal vessels.

Intraocular AdministrationOf the species used for ocular safety evaluations, the

minipig’s eye most closely approximates the size of the

human eye. The minipig also has similar innervation and

vascular structure to humans, making it one of the most

suitable eyes for intraocular administration in preclinical

ocular toxicology studies. The larger eye size permits the

implantation of medical devices and conduct of clinically

relevant surgical procedures and facilitates translation of

preclinical results to the development and support of

clinical protocols6-8.

Safety PharmacologyAs the use of the minipig in preclinical safety testing is

increasing, it follows that this species should also be used

in safety pharmacology testing. Minipig models are now

available for use in cardiovascular safety pharmacology

testing, and data demonstrate that the minipig constitutes a

suitable model for telemetry9-13.

askcharlesriver@criver.com • www.criver.com

We use jacketed external telemetry (JET™, Data Sciences

International, St. Paul, MN) for the collection of continuous

electrocardiographic and hemodynamic data in large animal

stand-alone and repeat-dose settings. JET employs surface

electrodes for ECG collection, and implanted probes for

blood pressure measurement. The JET transmitter has a

direct connection to the ECG sensors, and radio frequency

communication to the optional blood pressure probe. The

jacketed device then sends both types of data wirelessly via

Bluetooth® to a licensed acquisition system.

This system provides a unique signal for each animal and

allows for continuous collection of electrocardiograms and

real-time visualization of trends. Measurements are on alert,

unrestrained animals, improving sensitivity to detect changes

in the absence of the sympathetic stimulation of manually

restrained approaches. Recordings can be limited to study

days with minimal room entry to provide hours of undisturbed

data. The technology offers a viable alternative to both

snapshot techniques and fully implantable telemetry and is

amenable to use in long-term repeat-dose (toxicity) studies.

DART and Juvenile ToxicologyWhen it comes to regulatory teratogenicity and juvenile

toxicology, the minipig once again has several advantages

over the more traditional species14-16. They have a relatively

large litter size, which reduces the number of pregnant

females required in the overall study. Furthermore, they

reach sexual maturity earlier, and have a shorter cycle

length when compared with other large animal models.

The species also exhibits the same teratogenic sensitivities

as humans, demonstrating susceptibility to similar fetal

malformations induced by thalidomide, hydroxyurea,

pyrimethamine, and ethanol, amongst others.

MetabolismFor certain enzyme systems, the use of the minipig is

favored over more traditional species. Minipigs are the first

non-primate choice when the drug candidate is metabolized

by aldehyde oxidase, which is practically non-existent

in dogs but present in pigs17. This would also apply to

substrates of N-acetyltransferases (NAT1 and NAT2), which

are practically absent in the dog liver18. Therefore, minipigs

should be included in any in vitro or in vivo assessment of

metabolism of a compound.

Safety guidelines include studies to investigate tissue

distribution. Quantitative whole-body autoradiography

(QWBA) is a powerful tool in determining whole-body

tissue distribution as well as distribution to and localization

within specific tissues. QWBA has, however, been

traditionally restricted to the investigation of distribution in

smaller species. In what appears to be the first application

for such a type of investigation, Charles River used

QWBA techniques to investigate the tissue distribution of

radioactivity in the minipig. The work was presented at the

national meeting of the International Society for the Study of

Xenobiotics in 201219.

DiabetesAlthough a variety of rodent models for type I diabetes

exist, such models have genuine limitations and poor

predictability due to the stark differences between rodents

and humans. In many respects, diabetic minipig models

more closely resemble the human condition; for example,

human insulin and porcine insulin differ by only a single

amino acid on the B-chain.

The type 1 diabetic minipig can be induced with

streptozotocin. Monitoring and control of diabetes is

facilitated with daily monitoring of blood glucose and

appropriate insulin injection to reduce blood sugar

fluctuations. This has allowed animals to be maintained for

greater than 365 days in the laboratory setting20.

Standard preclinical testing can be conducted in the diabetic

minipig, along with specialized protocols for specific

endpoints (oral glucose tolerance test, etc.).

An Alternative ApproachThe use of the minipig as a nonrodent species has been

well documented, and in 2010 was the subject of the

RETHINK European FP6 project. The RETHINK project

evaluated the potential impact of toxicity testing in the

minipig and established the use of this species as a valid

alternative approach in regulatory toxicity testing that can

contribute to the replacement, refinement and reduction of

animal testing (3Rs)21.

The species demonstrates numerous practical advantages

over other large animal models in the preclinical

toxicology space:

1. Minipigs tolerate NSAIDs, which can cause

gastrointestinal lesions in other species.

2. Minipigs tolerate sympathicomimetic and

antihypertensive drugs, which cause cardiotoxicity even

at low doses in the traditional large animal model.

3. Minipigs do not develop arteriopathy with endothelin

receptor antagonists.

4. Minipigs are not prone to vomiting, nor are they

susceptible to anaphylactic reactions as a result

of a histamine release, which can occur following

administration of some vehicles/excipients.

5. Minipigs are not sensitive to sex hormones with

estrogenic activity, which can result in aplastic anemia in

other large animal models.

ConclusionThe minipig is recognized as a suitable alternate nonrodent

species associated with efficacy and safety assessment

of certain types of pharmaceutical products, and its

prevalence and relevance in preclinical testing are growing.

There is a good supply of high-quality animals of known

disease status with sufficient background data now

available to allow unequivocal data interpretation. Minipigs

offer certain advantages over the traditional species in

relation to specific responses to particular drug classes,

and therefore can also offer advantages over the traditional

species in relation to the ethical considerations and cost of

animals in biomedical research.

References

1. Ganderup NC, Harvey W, Mortensen JT, Harrouk W.

The minipig as nonrodent species in toxicology –

Where are we now? Intl J of Toxicol. 2012 Nov/Dec;

31:507528

2. Weaver ML, Grossi AB, Schützsack J, et al. Vehicle

Systems and Excipients Used in Minipig Drug

Development Studies. Toxicol Pathol. 2016;44(3):367-

372. https://pubmed.ncbi.nlm.nih.gov/26674803/

3. Bernier L, Mansell P, Copeman C. Background data in

the instrumented (Cannulated) and non-Instrumented

Göttingen Minipig (2011).

4. Prefontaine A, Trudel Y, Caron C, Copeman C.

A novel approach for continuous or intermittent

subcutaneous infusion using single or multiple sites

in minipigs. (2011).

5. Testing dermal wound care products in the Göttingen

Minipig Ellegaard Newsletter 50. Winter 2017 https://

minipigs.dk/fileadmin/filer/Newsletters/Newsletter_50.

pdf p8-9 Accessed June 2020

6. Vézina M, Washer G. Electroretinogram (ERG) and

intravitreal injection in Göttingen minipigs: post-

acquisition filtration method comparison. Presented at:

Annual Meeting of the Society of Toxicology, 2001.

7. Patel A, Vézina M. Electroretinogram (ERG) recording

in rodent and nonrodent species. Paper presented

at: 6th Annual Meeting of the Safety Pharmacology

Society, September 28, 2006, San Diego, CA.

Nonrodent Models: Minipig Specialty Capabilities

askcharlesriver@criver.com • www.criver.com © 2020, Charles River Laboratories International, Inc.

8. Patel A, Vézina M. Electroretinogram (ERG) Recording

in rodent and nonrodent species. Paper Presented at

the Annual Meeting of the Society of Toxicology, March

26-29, 2007, Charlotte, NC.

9. Authier S, Gervais G, Fournier S, Gauvin D, Maghezzi

S, Troncy EJ. Cardiovascular and respiratory

safety pharmacology in Göttingen minipigs:

pharmacological characterization. Pharmacol Toxicol

Methods. 2011 Jul/Aug; 64(1):53-9. doi: 10.1016/j.

vascn.2011.04.007 (Epub May 3, 2011).

10. Markert M, Stubhan M, Mayer K, Trautmann T, Klumpp

A, Schuler-Metz A, Schumacher K, Guth B. Validation

of the normal, freely moving Göttingen minipig for

pharmacological safety testing. J Pharmacol Toxicol

Methods. 2009 Jul/Aug 60(1):79-87. doi: 10.1016/j.

vascn.2008. 12.004 (Epub May 8, 2009).

11. Vézina M, Patel A, Wise S. Short-term EEG recording

in conscious Göttingen Minipigs for seizure liability

testing as an alternative to traditional large animal

models. Presented at: 8th Annual Meeting of the

Safety Pharmacology Society, September 22-25 2008,

Madison, WI.

12. Kirouac A, Norton K. Jacketed external telemetry (JET)

assessment in the Göttingen minipig (2013).

13. Diehl L, Nungester W, Regalia D. Effects of

isoproterenol and moxifloxacin hydrochloride on

cardiovascular, ECG and cardiovascular parameters

assessed via jacketed external telemetry (JET) in the

freely moving minipig (2014).

14. McAnulty PA, Barrow P, Marsden E. Reproductive

system including studies in juvenile minipigs. The

Minipig in Biomedical Research (2011), Chapter 17 p

263–276 CRC Press/Taylor & Francis Group

15. Ellemann-Laursen S, Marsden E, Peter B, Downes

N, Coulby D, Grossi AB. The incidence of congenital

malformations and variations in Göttingen minipigs.

Reproductive Toxicology, Developmental Basis of

Health and Disease, volume 64, September 2016,

pages 162-168.

16. Feyen B, Penard L, Van Heerden M, Fant P, Marsden

E, De Jonghe S, Desmidt M, Maki Mousa S, Bailey GP.

“All pigs are equal” Does the background data from

juvenile Göttingen minipigs support this? Reproductive

Toxicology, Developmental Basis of Health and Disease,

volume 64, September 2016, pages 105-115.

17. Pryde DC, Dalvie D, Hu Q, Jones P, Obach RS,

Tran T-D (2009). Aldehyde oxidase: An enzyme of

emerging importance in drug discovery. J. Med.

Chem. 2009 Feb; (53):8441–8460.

18. Dalgaard L. Comparison of minipig, dog, monkey and

human drug metabolism and disposition. J Pharmacol

Toxicol Methods. 2015;74:80-92. https://pubmed.

ncbi.nlm.nih.gov/25545337/

19. Madden S, Patterson A, Stevenson K. The utilisation

of quantitative whole body autoradiography (QWBA)

methods to investigate whole body tissue distribution

in the minipig (2012).

20. Smedley J, Cornicelli J, Lucke L, Merriman T.

Induction and long-term management of type I

diabetes in Gottingen minipigs. Presented at: Annual

Meeting of the Society of Toxicology, March 22-26,

2015, San Diego, CA.

21. Forster R, Bode G, Ellegaard L, van der Laan JW. The

RETHINK Project on minipigs in the toxicity testing

of new medicines and chemicals: conclusions and

recommendations. J. Pharmacol Toxicol Methods.

2010 Nov/Dec; 62(3):236–42.