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.
[email protected] • 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
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Nonrodent Models: Minipig Specialty Capabilities
[email protected] • www.criver.com © 2020, Charles River Laboratories International, Inc.
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