clean pigs for clear results newsletter 36

Clean pigs for clear results

www. minipigs.dk

Refinement: Take blood samples

from minipigs in a

sling !

PEG Catheter

placement in the

Göttingen Minipig

Stem Cell Research in minipigs: a Potential Model for Mimicking

Human Disease

transgenic pig models for biomedical research

Posters: • Survey of minipig clinical biochemistry,

coagulation, and urinalysis parameters

• Use of pig, dog and NHP in biomedical research

GÖTTINGEN MINIPIGS A/S

Newsletter 36 Autumn 2011

Soroe Landevej 302 · DK-4261 Dalmose · Denmark · Tel. +45 5818 5818 · Fax +45 5818 5880 · [email protected] · www.minipigs.dkFor Göttingen Minipigs in North America please contact Marshall BioResources – [email protected] Göttingen Minipigs in Japan please contact Oriental Yeast Co., Ltd. - [email protected]

Göttingen minipig genome sequenced !Microarray for mRNA profiling in development

2 Newsletter 36 Autumn 2011

deAR ReAdeR

Content

➤➤

3 Socialising

3 Scand-LAS 2011 Minipig Handling Workshop

4 Poster: Percutaneous Endoscopic Gastrostomy

6 PEG catheter placement in the Göttingen Minipig

6 Auxiliary equipment

7 Refinement: Taking Blood Samples from Minipigs in a Sling

8 Pig models of early-stage cardiovascular disease

9 Minipig Genomics at Roche: State of the Art

I sincerely hope you have enjoyed your summer, despite the changeable summer weather we have seen in Northern Europe. Over the summer, we have experienced greater interest than nor-mal in this period from existing and potential customers, and we are pleased to note that this year’s demand for minipigs currently exceeds last year’s demand.

This growth in minipig sales shows that an increasing part of the pharmaceutical industry has acknowledged the minipig as an established and suitable animal model.

One of our more recent customers – a large pharmaceutical company – has shared with us that the process of implementing the Göttingen Minipig has been very successful. This particular customer has benefited from our advice on optimal design of a minipig facility and from participating in our Handling & Dosing Course and our Surgery Course. Generally, we have closely collaborated with them during the process of implementing the Göttingen Minipig as an animal model. We value our customers highly and give high priority to closely cooperating with them, espe-cially when they are preparing for and establishing the Göttingen Minipig as a new animal research model.

We hope that more of our customers will make use of our expertise and experience and, at the same time, we are also very interested in hearing about your work with minipigs.

Although we have many years of experience with minipigs, we realise that we can learn even more about minipigs by working closely with our customers and minipig users around the world.

Later this year the book “The Minipig in Biomedical Research” will be published. We look forward to the release of the updated

book about the minipig, and we are convinced that many will find it interesting and useful. New information about minipigs is needed and this new book provides minipig information that has been lacking but which now will enable readers to get an overview of the key minipig strengths and to compare minipigs to other non-rodent species.

In this newsletter you will find an interesting article from F. Hoffmann-La Roche AG that describes how the Göttingen Minipig genome has been investigated and sequenced. The availability of genomes for the Göttingen Minipig makes it possible to perform a thorough and justified species selection. Again, our well defined genetics shows its importance. This newsletter also provides you with informative articles about stem cell research in minipigs and transgenic pig models.

We are very pleased that researchers from many different phar-maceutical companies, CROs and universities are willing to share their expertise and knowledge with us and other minipig users. Hopefully, you will value the interesting articles in this newsletter as much as we do.

If you need information about Göttingen Minipigs, you are wel-come to visit our website. Please contact us if you cannot find the information you are looking for or if you have any other questions relating to minipigs.

Sincerely, Jens Ellegaard, CEO, Ellegaard Göttingen Minipigs A/S

If you prefer to receive this newsletter by e-mail, please send an e-mail to [email protected] or write to us at the address provided below. To correct mailing list problems, please send an e-mail to: [email protected] or write to us at this address:Ellegaard Göttingen Minipigs A/SSoroe Landevej 302, DK-4261 Dalmose

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10 Stem Cell Research in Minipigs: a Potential Model for Mimicking Human Disease

11 Meeting calendar 12 Poster: Use of pigs, dogs and NHPs in

biomedica

14 Poster: Survey of minipig clinical biochemistry

16 Reformulating Agricultural Organophosphorus Pesticides to Reduce Global Suicide Rates: a Göttingen Minipig Model

17 Recent Progress of Transgenic Pig Models for Biomedicine and Pharmaceutical Research

3Newsletter 36 Autumn 2011

Animal welfare is our main priority and we continuously adjust our procedures in order to provide the minipigs with the optimal conditions. In the early weaning section we socialise with the pigs every day to accustom them to human contact. At first, minipigs can be shy and do not want to be patted but once they feel safe and comfortable around humans, they love to be scratched behind the ear or on the back. They will also appreci-ate a gentle belly rub. Socialising will improve the welfare for the minipigs and the working conditions for the staff.

This picture shows Martin who is socialising with the minipigs in the early weaning section. He sits on the floor and makes no abrupt movements that might scare the minipigs. Most minipigs are curious and want to investigate new things or people in the pen.

Martin picks up one minipig at a time and his calm manner shows that they can feel safe with him. The female minipig on his lap obviously feels safe, is very relaxed and enjoys every moment.

Please contact us if you would like to know more about how to benefit from socialising with the minipigs.

Socialising

The annual Scand-LAS meeting was held at the University of Copenhagen on 25–28 May 2011

On Thursday 26 May, Ellegaard Göttingen Minipigs conducted a minipig handling workshop attended by participants from several pharmaceutical companies and universities.

To maximise the benefits for the participants, they were divided into four teams of 3–4 people.

Adrian Zeltner, our Laboratory Technician, has many years of experience in handling minipigs which he shared with the partici-pants during the workshop.

Anne, Lise and Mette are staff members working within our barriers and they assisted Adrian with their knowledge and practi-cal experience. Four minipigs were used to give the participants

the opportunity to get practical experience in handling and dosing minipigs.

During the workshop, participants showed a strong interest in the handling and dosing of minipigs, and feedback from the work-shop has been very positive.

Over the years, we have provided many of our customers with training courses in basic handling of minipigs, and you are welcome to contact us if you would like to attend a course or if you are interested in receiving further information.

We can run the course at your best convenience at our facility or your own facility. Please contact us for further information.

We also exhibited at the Scand-LAS symposium and had good conversations with many people who showed an interest in the Göttingen Minipig.

Scand-LAS 2011 Minipig Handling Workshop

Pe

rcutan

eous End

oscopic Gastrostomy (PEG

) cathe

ter placem

ent

for intragastric dosing stud

ies in the

Göttingen

minipig

Pe

ter Bo

llen', H

enrik Saxtorph

', Helle Loren

tsen

" & Adrian Zeltn

er"

') Biom

edical Laboratory, University

of Sou

thern Den

mark, Ode

nse, Den

mark

") Ellegaard Göttin

gen Minipigs, Dalmose, Den

mark

Oral dosing is widely used

to te

st com

poun

ds on Göttin

gen minipigs. The

procedu

re of o

ral dosing by

gavage is stressful fo

r the

animals and may req

uire up

to 3‐4 te

chnicians to perform

. Som

e of th

ese stud

ies are chronic do

sing

studies with

multip

le daily dosing. In

hum

ans, PEG

(Percutane

ous Endo

scop

ic

Gastrostomy) cathe

ters are com

mon

ly used to fe

ed patients who

are not able to ingest fo

od, and

whe

re th

e placem

ent o

f a nasal intragastric cathe

ter is

not p

ossible. W

e suggested that oral dosing stud

ies in m

inipigs wou

ld be easier to

perform

, and

wou

ld cause less distress for the

animals, whe

n using PEG

catheters, due

to th

e absence of th

e ne

ed fo

r restraint. For th

is reason, we investigated

if PEG

cathe

ters cou

ld be placed

and

maintaine

d in Göttin

gen minipigs,

and we exam

ined

the de

gree

of tissue reactio

n three weeks after placemen

t.

Metho

ds

Two male Göttin

gen minipigs with

a bod

y weight

of 6 kg were brou

ght into gene

ral ane

asthesia by

administration of 0.04 mg/kg m

edetom

idine, 0.2

mg/kg m

idazolam

and

0.05 mg/kg atrop

ine IM

, followed

by IV adm

inistration of 3‐5 m

g/kg

prop

ofol IV

. Anaesthesia was m

aintaine

d by

3.5%

sevoflu

rane

. Via a gastric end

oscope

, the

ventricle was insufflated with

CO

2, and the

mucosa of th

e ventricle was inspected for

optim

al placemen

t of th

e PEG cathe

ter. The

catheter (N

utricia Flow

care) w

as placed through

the mou

th, by retractin

g a transabd

ominal snare.

The security rings were attached

, and

the minipig

was re

covered from

anaesthesia. The

animals

were ho

used

post‐op

eratively for three weeks,

after w

hich th

ey were eu

thanized

for

pathological examination.

Figure 1: Placemen

t of the

end

oscope

through the mou

th and

oe

soph

agus into th

e ventricle.

Figure 2: The

site

of P

EG cathe

ter p

lacemen

t is clearly visible by

translum

ination.

Figure 3: A

n en

doscop

ic view of the

inserted

cannu

la (w

hite) and

pu

lling

snare (b

lue).

Results

PEG placemen

t was perform

ed with

out

complications, and

the anim

als recovered

uneven

tful from

anaesthesia. A

ppetite

was fine

, and a no

rmal weight g

ain was observed. In

one

anim

al, no visible signs of infla

mmation

(erythem

a, exudatio

n) were ob

served

. In on

e anim

al a slight fo

rmation of clear exudate was

observed

in com

binatio

n of light red

dening

of

the skin. Patho

logical inspe

ction revealed

a she

et

of con

nective tissue arou

nd th

e catheter, w

ith

slight infla

mmation in both anim

als. In

the

anim

al with

exterior exud

ation, a small (1x1 cm

) adhe

sion

to th

e spleen

was observed. In

both

anim

als the ventricle, intestines and

liver were

mob

ile.

Figure 4: Skin reactio

n to cathe

ter p

lacemen

t in tw

o Göttin

gen

minipigs. M

inipig A had

no erythe

ma and exud

ation. M

inipig B

had slight erythem

a and exud

ation.

Figure 5: The

cathe

ter encapsulated

in con

nective tissue in th

e abdo

minal cavity, w

ith vascularisatio

n and slight infla

mmation.

Figure 6: Schem

atic placemen

t of a

Flowcare PEG

cathe

ter.

Discussion

The stud

y de

mon

strated that PEG

cathe

ter

placem

ent in the Göttin

gen minipig is possible

with

out m

ajor com

plications. A

part from

mild

tissue reactio

ns to

the foreign bo

dy, no major

infla

mmation was observed. The

animals

tolerated the PEG cathe

ter w

ell, and had a

norm

al bod

y weight g

ain. Correct placemen

t of

the PEG cathe

ter is essential. Th

e adhe

sion

to

the spleen

was possibly du

e to dam

age of th

e splenic capsule. The

refore, extra atten

tion

shou

ld be paid to

the correct p

lacemen

t of the

catheter, and

the catheter sho

uld be

placed as

far a

s po

ssible from

palpable and visible organs,

like spleen

and

liver. This stud

y investigated

only

acute tissue reactio

n to PEG

cathe

ter placem

ent,

and a follow‐up stud

y over a longer period

shou

ld be pe

rformed

for investig

ating chronic

tissue reactio

ns, and

the effect of growth on PEG

catheter placemen

t.

Figure 7: The

loose en

d of th

e catheter is kep

t in place by

a net

stocking. This anim

al started

eating with

in 15 minutes after

recovery from

anaesthesia.

Ackno

wledgmen

ts

Associate professor Lars Ra

smussen MD, O

dense University

Hospital, is kindly acknow

ledged

for instructin

g the PEG

placem

ent p

rocedu

re. A

ssociate professor Jo

hn Che

mnitz M

D,

conservator An

nette Møller Dall PhD

and

con

servator assistant

Brian Ke

nneth Hansen, Dep

t. of A

natomy, University

of Sou

thern

Den

mark, are kindly acknow

ledged

for p

reparing

the plastin

ates.

AB


Investigations concerning whether PEG catheters can be placed and maintained in Göttingen Minipigs have been carried out in close cooperation with Peter Bollen and Henrik Saxtorph of the Biomedical Laboratory, University of Southern Denmark.

At Ellegaard, we find this procedure beneficial because placing PEG catheters can eliminate oral dosing, thus improving animal welfare.

Peter Bollen and Henrik Saxtorph are experienced in this field

PEG catheter placement in the Göttingen Minipig

and would like to share their knowledge with other minipig users. They possess the equipment required for placing PEG catheters and make this equipment available for hire. Furthermore you are welcome to contact Peter Bollen and Henrik Saxtorp to have them assist you in placing the catheter.

Peter Bollen can be contacted by email: [email protected] or by phone: +45 6550 3798.

We offer various types of

equipment that can make your

work with minipigs easier.

You are welcome to contact

us if you need any auxiliary

equipment.

Auxiliary equipment

Bite bar

Sling frame

Restraint chair

for gavaging

Restraint bench for

blood sampling

Minipig scale

If you would like a demonstration of the equipment, you can sign up

for one of our Handling and dosing courses


If you are considering this blood sampling method, there are few things to be aware of:• If you have a sling, you

need to cut an extra hole to expose the insertion site. This can be done easily with scissors.

• Before placing the mini-pig in the sling, you can mark the insertion site as the skin tends to curl up a little due to pressure from the sling.

• After taking the blood

sample, remember to apply pressure to the site of the vein puncture.

• Also consider the ergonomic aspects. The best solution is if the height of the sling can be adjusted to make the insertion site readily accessible.

Apart from training the minipigs to feel comfortable in the sling, atten-tion should also be paid to training personnel for the procedure.

You are always welcome to attend one of our handling and dosing courses at our facility in Dalmose, Denmark.

We can also arrange a visit to your facility where the method can be demonstrated. Please do not hesitate to contact us if you have any ques-tions or need further information regarding this blood sampling procedure.

We appreciate having good contact with you in any matter con-cerning a successful experience with the minipig.

If you already have taken blood samples from minipigs in a sling, we would like to hear your experiences and comments.

Helle Lorentsen, Head of Veterinary Services, Ellegaard Göttingen Minipigs A/[email protected]

At our handling and dosing courses, we teach participants how to handle the minipig while taking blood samples from its jugular vein.

Many minipig users are probably familiar with the method of using a blood bench and rolling the mini-pig on its back. This proce-dure requires 2 to 4 per-sons, depending on the size and temperament of the minipig.

Some minipigs struggle during fixation – most likely because being manipulated on the back is an unnatural posture for an animal.

Therefore, taking blood samples while the minipig rests in a sling is an alternative method worth considering.

One of the advantages of taking blood samples while a minipig is in a sling is that the procedure is less stressful for minipig (refinement) and person-nel alike. Also fewer peo-ple need to be involved in the procedure.

At Ellegaard we have tried this method on untrained minipigs at dif-ferent ages. Even when minipigs were placed in the sling for the first time, it was possible to perform stress-free blood sampling on each animal. Some of the minipigs were even so relaxed that one person could do the procedure alone.

Helle Lorentsen

Refinement: Taking Blood Samples from Minipigs in a Sling

Did you know that it is possible to take blood samples from unanaesthetised minipigs almost without restraint ?

Smaller animals are usually easy to handle in the blood bench

Taking blood samples from a minipig in a sling

For large blood volumes, you can use an extension which makes the sampling easier

You can cut an extra hole in the sling for taking blood samples


According to the World Health Organization, cardiovascular disease (CVD) is the leading cause of death worldwide. This PhD thesis focuses on atherosclerosis, which is an important contributor to the growing burden of CVD. In order to reduce the number of people suffering from atherosclerosis, risk factors that may initiate and progress atherosclerosis have been identified. Conventional risk factors such as smoking, hypertension, diabetes, obesity and hypercholesterolemia are not the only factors that can contribute to the development of atherosclerosis which can then cause CVD. Inflammation has been shown to play an important role in the development of atherosclerosis and this has increased the focus on infection as a possible risk factor. In early life, infection often causes inflammation and, hence, infection may have an impact on the initiation of atherosclerosis.

The aim of this PhD project was to develop a pig model with endothelial dysfunction and early atherosclerosis lesions based upon infection-induced inflammation.

The results are presented in three articles:– Infection-induced coronary dysfunction and systemic

inflammation in piglets are dampened in hypercholester-olemic milieu Malene M. Birck,1 Erkki Pesonen,2 Michal Odermarsky,2 Axel K. Hansen,1 Kenneth Persson,3 Henriette Frikke-Schmidt,1 Peter M. H. Heegaard,4 and Petru Liuba2 (2011) 1Department of Veterinary Disease Biology, Faculty of Life Sciences, University of Copenhagen, Frederiksberg, Denmark; 2Division of Paediatric Cardiology, Department of Paediatrics, Skåne University Hospital, Lund and 3Department of Microbiology, Skåne University Hospital, Malmö, Sweden; and 4Innate Immunology Group, National Veterinary Institute, Technical University of Denmark, Copenhagen, Denmark. This article has been published in American Journal of Physiology – Heart and Circulatory Physiology, Vol. 300 (5), H1595-H1601.

– Intimal changes in the coronary artery of infected hyper-cholesterolemic minipigs Malene M. Birck,1 Antti Saraste,2 Poul Hyttel,3 Michal Odermarsky,4 Petru Liuba,4 Pekka Saukko,5 Axel K. Hansen,1 and Erkki Pesonen4 1Department of Veterinary Disease Biology, Faculty of Life Sciences (LIFE), University of Copenhagen, Denmark; 2Department of Medicine, University of Turku, Finland; 3Department of Basic Animal and Veterinary Sciences, LIFE, University of Copenhagen, Denmark; 4Division of Paediatric Cardiology, Department of Paediatrics, Skåne University Hospital, Lund, Sweden; and 5Department of Forensic Medicine, University of Turku, FinlandThis article has been submitted.

– A novel method for trans-uterine identification of piglets Malene M. Birck*, T. Iburg†, M. Schmidt‡, P. T. Sangild§ and A. K. Hansen* (2008)

*Division of Laboratory Animal Science and Welfare, Department of Veterinary Pathobiology, Faculty of Life Sciences; †Section of Pathology, Department of Veterinary Pathobiology, Faculty of Life Sciences; ‡Veterinary Reproduction and Obstetrics, Department of Large Animals Sciences, Faculty of Life Sciences; §Department of Human Nutrition, Faculty of Life Sciences, University of Copenhagen, Denmark This article has been published in Laboratory Animals, Vol. 42, 331-337.

The thesis substantiates that the pig is a good animal model for atherosclerosis research.

The results show that repeated infection is associated with endothelial dysfunction and that the degree of impairment is related to the pathogen burden. Hypercholesterolemia seems to modulate the infection-induced inflammatory response, reducing inflamma-tion and lessening severe dysfunction. In the morphological study, the combination of hypercholesterolemia and infection seemed to speed up atherogenesis as increased numbers of foam cells were present in the subendothelial space of infected animals, compared to non-infected animals fed a cholesterol diet. Infection was also associated with increased endothelial cell death and degenera-tive changes. Further studies are needed to take a closer look at the interaction between infection, inflammation and altered lipid metabolism and its role in atherogenesis. In addition, the somewhat contradictory findings between the functional and the morphologi-cal studies deserve further investigation.

This thesis has shed light on some new aspects of athero-sclerosis and also stresses the importance of applying preventive measures early in life. The results reveal new and interesting knowledge about the dynamics of infection-induced inflamma-tion, hypercholesterolemia and endothelial dysfunction and should hopefully encourage additional studies about this topic.

Two additional articles based on this PhD project have been published:– Expression studies of the obesity candidate gene FTO in

pig. M. B. Madsen, M. M. Birck, M. Fredholm, S. Cirera (2010 Biotechnol. Vol. 21 (1), 51-63

– Expression profile of miR-122 and its target CAT-1 in high-cholesterol fed minipigs (Sus scrofa) S. Cirera, M. M. Birck, P. K. Busk, M. Fredholm (2010) Comparative Medicine Vol. 60 (2), 136-141.

Pig models of early-stage cardiovascular diseaseMalene Muusfeldt Birck, PhD thesis 2011


Today, minipigs are becoming increasingly appreciated as animal models in drug research and predictive drug safety and toxicology. Their physiology and metabolism as well as responses to toxicants and medicines resemble the human situation in many aspects. By contrast with more established animal models like the mouse, rat or the cynomolgus monkey, only limited sequence information is available for many gene families of interest, like the cytochromes p450, drug transporters or cytokines. In addition, the choice of minipig-specific diagnostic assays and kits for clinical chemistry or histopathology is limited. As a result of the Macaca fascicularis genome project, we realised that knowledge of all mRNA and pro-tein sequences is highly beneficial for the prediction of cross-reac-tivity of tests and reagents originally designed for use in humans. In addition, the deciphering of the cynomolgus genome enabled us to establish a variety of chip-based applications for genotyping, mRNA profiling, and copy-number variation analysis.

To decipher the genome of the Göttingen minipig, we based our work on the efforts of a large pig genome consortium consisting of 38 institutions which deciphered the farm-pig genome to advance research for animal production and breeding (http://piggenome.org/).

At the time of writing, an estimated 95–97% of the Göttingen minipig genome is known and assembled at Roche with about 18-fold sequence coverage. This genome version is already sufficient to deduce the minipig orthologues of virtu-ally all human proteins, provided they are present in the minipig genome. When comparing conservation among protein-coding genes between human beings and the cynomolgus monkey, the overall sequence identity is about 93%. Based on available data, we estimate an average sequence similarity in the range of 85% for human and minipigs. This highlights the need for careful validation of available human assays and development of minipig-specific protein detec-tion kits and reagents. The availability of the genome is an impor-tant step towards this goal because the cross-reactivity of human assays becomes predictable. TNF-a is considered a biomarker for systemic inflammation. If we wish to use a human ELISA kit to measure inflammatory responses in minipigs, for example, we can now align the TNF-a sequences of both species. In case the sequence identity is above 95%, the likelihood of cross-reactivity is high whilst identities below 85% are critical, especially when mono-clonal antibodies are used for detection. Similarly, it is possible to predict whether a given therapeutic antibody cross-reacts with the minipig based on target identity.

Pharmacological activity as well as adverse events depend not only on sequence similarity but also on gene expression in the target tissue. For this purpose we applied next generation RNA sequencing to profile mRNA expression in heart, liver, spleen, blood, kidney and lung. A highly sensitive analytical method of this nature is required, especially for blood profiling. For example,

genes that are only expressed by a small subpopulation of white blood cells have low-expression mRNA levels which are likely to escape detection by microarrays.

For more standard mRNA profiling applications like toxico-genomics, we are currently designing microarrays for mRNA pro-filing in collaboration with Roche-NimbleGen (Madison, USA). This technology is particularly suited for the design of prototype arrays since errors are easily corrected. Based on control hybridisations, we will assess performance and eliminate bad performing probes if necessary. An initially validated version of this array should be available in the autumn of this year, with probes for about 18,000 transcripts. Although the sensitivity of deep sequencing is con-siderably higher, microarrays are still the most robust tools for routine tissue expression analysis, backed up by well-established bioinformatics tools for data processing and analysis. Since pharmaceutical research mainly focuses on protein targets, micro-arrays will remain valuable tools for studying gene expression at

different stages of preclinical drug development. It is likely that the pig genome community will also appreciate the availability of these arrays. Good compatibility for both organisms is more than likely based on the genome project and the fact that pig and minipig belong to the same species.

In summary, minipig genomics support the decision-making drug-safety process on rational grounds. Sequence and gene expression information will largely inform the decision of whether the minipig is a responder species for the assessment of novel drugs. Consequently, “trial-and-error” experiments are avoided which will ultimately lead to a significant reduction in animal usage with a concomitant improvement of drug safety assessment. Genomics in general represents a very useful tool for refining and reducing animal experiments concordant with the 3R principles. With the availability of the genomes for mouse, rat, dog, minipig, and cynomolgus we can perform highly sophisticated tox-species selection which will not only reduce the number of animals required but also improve the prediction of drug safety in humans. Finally, novel minipig-specific tests will allow more reliable and specific measurement of safety parameters.

1Non-clinical Safety and 2Translation Research Sciences, pREDF. Hoffmann-La Roche AG, CH-4070 Basel, Switzerland

Minipig Genomics at Roche: State of the ArtProf. Dr Ulrich Certa,1 Dr Martin Ebeling,2 Dr Thomas Weiser1 and Dr Thomas Singer1

“With the availability of the genomes for mouse,

rat, dog, minipig, and cynomolgus we can per-

form highly sophisticated tox-species selection

which will not only reduce the number of animals

required but also improve the prediction of drug

safety in humans.”


Stem cell research has developed into a huge research topic that has grown and widened significantly in the past few years. This is evident by the expansion of stem cell research conferences and increasing numbers of delegates, as well as an increase in the number of scientific publications. This expansion is largely due to promising developments in the transplantation of adult stem cells for disease treatment, and a relatively new breakthrough: the pro-duction of a new type of stem cell, the induced pluripotent stem cell. These latter cells are stem cells that look and behave very similarly to embryonic stem cells. The advantage of induced pluri-potent stem cells is that they do not come from an early embryo. In fact, they can be made from almost any type of adult cell in the body. These cells are “reprogrammed” into embryonic stem cells by introducing a number of key genes into the cells, which changes their cell identity and turns them into an embryonic stem cell. These cells overcome the ethical barrier of embryonic stem cells whereby an embryo must be sacrificed in order to produce them. This remains a controversial issue in many countries around the world and has now been apparently and potentially resolved.

Why are embryonic and induced pluripotent stem cells so impor-tant for researchers? Because they have the ability to form any cell type of the body. This can be performed simply by adding a few specific cues when culturing these cells. This means that these cells are able to produce beating cardiomyocytes for treating car-diac disease, to produce insulin cells for treating diabetes, and to produce brain cells for potentially treating a number of brain-related diseases. Therefore, these cells have the capability to be used for cell transplantation to replace diseased or lost cells in the body. In fact, there are phase 1 clinical trials currently in progress using embryonic stem cells for treating spinal cord injury and for treating Stargardt’s macular dystrophy. These remarkable induced pluri-potent stem cells are the current object of focus of my research, which is primarily performed using the pig as a model.

Many people often question why I use the pig as a research model, since stem cell research is traditionally performed using either mice or human-derived cell lines. I think the best answer to this is that in Denmark we love pigs! There are considerably more pigs in Denmark than human beings. In fact, pigs outnumber

humans 5 to 1. As Denmark has an enormous export production of pork and pork products, Danes prize their pigs, which also play an important economic role for the country’s GDP. The pig is also considered a good, alternative model to the human. It is a large mammal and more similar to human beings (in many organs, both histologically and physiologically), than the well-characterised small mammal, the mouse.

The current focus of my research is to produce induced pluri-potent stem cells from a transgenic minipig. This minipig was pro-duced by cloning (also known as somatic cell nuclear transfer), and was designed to carry a human gene, which is inherited by a cohort of people who develop an early onset of Alzheimer’s disease. The hope is that this animal will develop Alzheimer’s in a manner similar to humans. This transgenic animal model was first produced in 2007 by a number of Danish researchers from the Foulum Research Centre (Faculty of Agricultural Sciences, University of Aarhus), together with researchers from the Department of Human Genetics (University of Aarhus) and the University of Copenhagen. The main research principle is to study pathological features of the disease in the stem cells and the cells derived from them (i.e. neu-rons) in order to determine whether these cells carry clues about the underlying mechanism of the disease. Can we see how these cells are unhealthy and die after we guide them from being stem cells into neurons? Therefore can we understand more about the disease, which may lead to breakthroughs for better treatment in the future? If these cells do display and recapitulate typical cha-racteristics of the disease, can these cells be used in screening for new and better drug compounds and medicines? These questions remain the hallmark of the current research. Moreover, we are working with non-transgenic pigs and minipigs, to produce induced pluripotent stem cell lines from animals considered “healthy” to determine whether these cells can form the specific neurons that are lost in Alzheimer’s disease. Another key research question being investigated is whether these neurons display typical cha-racteristics of the neurons found in the brain. If we can produce identical or similar neurons, these could be envisioned for future cell transplantation into the aging transgenic minipigs (or other models of Alzheimer’s disease).

Stem Cell Research in minipigs: a Potential Model for Mimicking Human Disease

Dr Vanessa Hall, University of Copenhagen, Faculty of Life Sciences, Department of Basic Animal and Veterinary Sciences


One of the problems entailed in producing good animal models of Alzheimer’s disease has been that, despite the ability to integrate the known human mutated genes which trigger the disease, these animals tend to lack the full pathological features of the disease. Traditionally, mouse models of Alzheimer’s disease have been produced. Zebra fish and rabbits have also been used, but less widely. Aging in mice is also significantly different from aging in humans. The lifespan of a mouse is roughly two years. Whether these animals are a suitable organism for studying the process of aging is hotly debated by researchers. Given the fact that it has been difficult to produce an animal model displaying all the hall-mark features of the disease, it has been quite difficult to identify

Stem Cell ReSeARCh in miniPiGS➤

Cell line And Cell bACkGRound PiG bReed SenioR AuthoR And loCAtion

iPSC from fetal fibroblasts Pig (breed unknown) Michael Roberts, University of Missouri (USA)

iPSC from embryonic fibroblasts Tibetan minipig Duanquing Pei, Guangzhou Institutes of Biomedicine and Health (China)

iPSC from adult ear fibroblasts Danish Landrace Lei Xiao, Shanghai Institutes for Biological Sciences (China)

iPSC from adult ear fibroblasts White landrace x Large white Juan Carlos Izpisua Belmonte, Salk Institute for Biological Studies (USA)

iPSC from mesenchymal stem cells Pig (breed unknown) Steven Stice, University of Georgia (USA)

the disease mechanisms, thereby limiting the extent of potential breakthroughs for new and better treatments.

Alzheimer’s disease is the most common form of dementia and no cures are currently available. It is therefore important to look to better, alternate disease models, which may mimic the disease more closely to that observed in humans. The pig (being a large mammal) is therefore a good alternative candidate to consider. In so doing, we may just be able to gain a little more insight into Alzheimer’s disease, which could lead to the development of better medicines and possibly breakthroughs, as well as pave the way for developing a cure.

Induced pluripotent stem cells (iPSC) have been produced successfully in the pig and minipig by a number of different research groups.

Meeting calendar

Name Date Place

ACt 6-9 September Phoenix, AZ

minipig Research forum - Annual meeting 28-29 november frankfurt, Germany

5th Annual Preclinical Safety and efficacy testing for new drugs

30 november – 1 december Zürich, Switzerland

bPS Winter meeting 13-15 december london, uk

Use

of p

ig, d

og a

nd N

HP

in b

iom

edic

al re

sear

ch in

Can

ada,

the

Eur

opea

n U

nion

(EU

), Ja

pan,

and

US

A N

C G

ande

rup

Nie

ls-C

hris

tian

Gan

deru

p [n

cg@

min

ipig

s.dk

], C

hief

Sci

entif

ic O

ffice

r, E

llega

ard

Göt

tinge

n M

inip

igs

A/S

, Dal

mos

e, D

enm

ark.

AB

STR

AC

T: D

ata

for p

igs,

dog

s, a

nd n

on-h

uman

prim

ates

(NH

P) i

s re

porte

d fo

r the

regi

ons

and,

whe

re p

ossi

ble,

are

a of

sci

entif

ic u

se is

pro

vide

d. T

he to

tal u

se (a

ll re

gion

s co

mbi

ned)

of p

igs

and

NH

Ps

has

incr

ease

d (1

5% a

nd 3

3% r

espe

ctiv

ely)

whi

le th

e us

e of

dog

s ha

s de

crea

sed

(2%

) fro

m 2

002

to 2

008.

The

re a

re s

triki

ng

diffe

renc

es b

etw

een

the

patte

rns

of u

se o

f the

thre

e sp

ecie

s am

ong

regi

ons.

Jap

an is

uni

que

in th

at d

ogs

are

used

to a

gre

ater

ext

ent t

han

pigs

and

NH

Ps.

Dog

use

ha

s de

crea

sed

dram

atic

ally

ove

r tw

o de

cade

s (3

8,91

5 in

199

1 vs

. 12,

376

in 2

007)

and

has

not

bee

n re

plac

ed b

y pi

gs o

r NH

Ps.

The

US

A ha

s a

cons

tant

use

of t

he

thre

e sp

ecie

s. T

hree

obs

erva

tion

can

be m

ade:

(1) t

he u

se o

f dog

s is

fairl

y co

nsta

nt, (

2) th

e us

e of

pig

s ha

s go

ne d

own,

(3) t

he u

se o

f NH

P ha

s in

crea

sed.

Can

ada

and

the

EU

hav

e si

mila

r pat

tern

s of

use

of t

he th

ree

spec

ies;

pig

s be

ing

used

mos

t fre

quen

tly, f

ollo

wed

by

the

dog

and

NH

P. In

the

EU

the

use

of p

igs

has

incr

ease

d an

d th

e us

e of

dog

s ha

s de

crea

sed,

whi

le th

e us

e of

NH

Ps

is c

onst

ant.

The

ratio

P =

[pig

/(dog

+NH

P)]

of p

ig to

dog

and

NH

P va

ries

with

regi

on a

nd y

ear:

Can

ada

[1.9

-4.0

], E

U [1

.4-3

.0],

Japa

n [0

.01-

0.16

], U

SA

[0.4

-0.6

]. E

.g. f

or e

ach

one

(1) c

ombi

ned

dog

and

NH

P 1.

9 pi

g w

as u

sed

(Can

ada

2007

). Th

us, C

anad

a an

d th

e E

U

has

grea

ter u

sage

(and

may

be a

ccep

tanc

e) o

f the

pig

as

a m

odel

in b

iom

edic

al re

sear

ch.

DAT

A C

OLL

ECTI

ON

& P

RO

VISO

S: D

ata

for t

he n

umbe

r of l

abor

ator

y an

imal

s us

ed in

thre

e re

gion

s (N

orth

Am

eric

a, E

urop

ean

Uni

on, J

apan

) of s

igni

fican

t pha

rmac

eutic

al in

dust

ry a

ctiv

ity is

pre

sent

ed.

Reg

istra

tion

of th

is d

ata

is fr

agm

ente

d: ty

pe o

f inf

orm

atio

n, le

vel o

f det

ail a

s w

ell a

s fre

quen

cy o

f col

lect

ion

is in

cons

iste

nt b

etw

een

regi

ons.

The

latte

r is

the

reas

on w

hy d

iffer

ent y

ears

are

repo

rted

for

the

diffe

rent

regi

ons,

and

in th

e ov

ervi

ew o

nly

sele

cted

yea

rs a

ppea

r. So

urce

s us

ed a

re C

anad

a: th

e C

anad

ian

Cou

ncil

on A

nim

al C

are’

s w

ebsi

te1 .

EU

: Rep

orts

from

the

Eur

opea

n C

omm

issi

on o

n th

e us

e of

exp

erim

enta

l ani

mal

s in

the

Eur

opea

n U

nion

2 . U

SA

: A

nim

al a

nd P

lant

Hea

lth I

nspe

ctor

ate

unde

r th

e U

SD

A3 .

Japa

n: J

apan

ese

Ass

ocia

tion

for

Labo

rato

ry A

nim

al S

cien

ce4 .

Prov

isos

: th

e un

derly

ing

reas

on w

hy th

is is

a v

alid

app

roac

h is

that

a s

peci

es is

onl

y us

ed if

it s

cien

tific

ally

rele

vant

and

like

ly to

gen

erat

e th

e in

form

atio

n so

ught

. Im

plic

itly

this

incl

udes

asp

ects

suc

h re

sem

blan

ce to

hu

man

ana

tom

y, p

hysi

olog

y, b

ioch

emis

try, a

s w

ell a

s su

itabi

lity

and

pred

ictiv

ity o

f the

spe

cies

sel

ecte

d. T

here

are

sev

eral

lim

itatio

ns: t

hese

dat

a do

not

spe

cify

pur

pose

aim

of t

he e

xper

imen

tal (

exce

pt

EU

); th

ey d

o no

t dis

tingu

ish

betw

een

pigs

and

min

ipig

s (e

xcep

t Can

ada)

; fre

quen

cy o

f dat

a co

llect

ion

varie

s gr

eatly

bet

wee

n th

e fo

ur re

gion

s, fu

rther

mor

e, th

e E

U h

as e

xpan

ded

in s

ize

(from

12

to 2

7 co

untri

es)

whi

ch in

and

of

itsel

f sk

ews

data

, Fr

ance

rep

orts

dat

a on

e ye

ar e

arlie

r th

an t

he r

est

of t

he E

U,

and

final

ly n

ot a

ll co

untri

es h

ave

repo

rted

sinc

e E

uros

tat

(EU

’s S

tatis

tics

Offi

ce)

mad

e it

man

dato

ry. C

ount

ries

like

Chi

na, I

ndia

, Pak

ista

n us

e a

size

able

num

ber

of d

ogs,

(m

ini)p

igs

and

NH

Ps

for

biom

edic

al p

urpo

ses,

but

suc

h da

ta is

, at b

est,

diffi

cult

to a

cces

s, a

nd a

ccur

acy

cann

ot b

e as

certa

ined

, hen

ce th

ey a

re n

ot in

clud

ed. D

espi

te o

f the

se s

hortc

omin

gs th

e co

mpa

rison

of u

sage

of t

he th

ree

mai

n no

n-ro

dent

spe

cies

stil

l pro

vide

s in

sigh

ts to

the

exte

nt o

f the

ir us

e in

thos

e re

gion

s.

REF

EREN

CES

(1

) CC

AC

. Can

adia

n C

ounc

il on

Ani

mal

Car

e [w

ww

.cca

c.ca

/en_

/pub

licat

ions

/aud

f]. 1

5-1-

2011

. (2

) Eur

opea

n C

omm

issi

on. R

epor

ts fr

om th

e C

omm

issi

on to

the

Cou

ncil

and

the

Eur

opea

n P

arlia

men

t on

the

Sta

tistic

s on

the

num

ber o

f ani

mal

s us

ed fo

r exp

erim

enta

l and

oth

er s

cien

tific

pu

rpos

es in

the

mem

ber s

tate

s of

the

Eur

opea

n U

nion

[h

ttp://

ec.e

urop

a.eu

/env

ironm

ent/c

hem

ical

s/la

b_an

imal

s/re

ports

_en.

htm

]. 2

011.

(3

) Uni

ted

Sta

tes

Dep

artm

ent o

f Agr

icul

ture

(US

DA

) AP

HIA

. Rep

orts

on

Ann

ual U

se o

f Lab

orat

ory

Ani

mal

s, A

nnua

l Rep

orts

of E

nfor

cem

ent [

ww

w.a

phis

.usd

a.go

v/an

imal

_wel

fare

/pub

s_re

ports

.sht

ml].

20

11.

(4) J

LAS

. Jap

anes

e A

ssoc

iatio

n fo

r Lab

orat

ory

Ani

mal

Sci

ence

[h

ttp://

ww

w.ja

las.

jp/e

nglis

h/en

_pol

icy.

htm

l]. 2

011.

(5

) Dec

lara

tion

of th

e E

urop

ean

Par

liam

ent o

n pr

imat

es in

sci

entif

ic e

xper

imen

ts, E

P re

fere

nce

num

ber:

DC

L-00

40/2

007

/ P6_

TA-P

RO

V(2

007)

0040

7, E

urop

ean

Par

liam

ent,

(200

7).

(6) E

urop

ean

Par

liam

ent.

Dire

ctiv

e 20

10/6

3/E

U o

f the

Eur

opea

n P

arlia

men

t and

of t

he c

ounc

il of

22

Sep

tem

ber 2

010

on th

e pr

otec

tion

of a

nim

als

used

for s

cien

tific

pur

pose

s. E

urop

ean

Dire

ctiv

e 20

10.

(7) F

ELA

SA

. FE

LAS

A st

atem

ent o

n th

e E

urop

ean

Par

liam

ent "

Writ

ten

decl

arat

ion

on p

rimat

es in

sc

ient

ific

expe

rimen

ts".

FELA

SA

offic

ial s

tate

men

t 200

7.

(8) W

ebst

er J

, Bol

len

P, G

rimm

H, J

enni

ngs

M. E

thic

al im

plic

atio

ns o

f usi

ng th

e m

inip

ig in

regu

lato

ry

toxi

colo

gy s

tudi

es. J

Pha

rmac

ol T

oxic

ol M

etho

ds 2

010;

62(3

):160

-166

. (9

) Car

lsso

n H

E, S

chap

iro S

J, F

arah

I, H

au J

. Use

of p

rimat

es in

rese

arch

: a g

loba

l ove

rvie

w. A

m J

P

rimat

ol 2

004;

63(4

):225

-237

. A

CK

NO

WLE

DG

EMEN

TS

The

auth

or w

ishe

s to

than

k D

r Nao

ki H

ayas

hi (O

YC

, Jap

an) f

or e

xtra

ctin

g d

ata

for e

xper

imen

tal

anim

al u

se in

Jap

an.

RES

ULT

S &

DIS

CU

SSIO

N: D

ata

for p

igs,

dog

s, a

nd n

on-h

uman

prim

ates

(NH

P) i

s re

porte

d by

regi

on (C

anad

a, E

U, J

apan

, U

SA

) see

bot

tom

, and

as

a to

tal f

or a

ll re

gion

s w

ith d

istri

butio

n am

ong

the

thre

e sp

ecie

s (s

ee ri

ght;

num

bers

insi

de c

olum

ns

are

per c

ent o

f tot

al fo

r a g

iven

yea

r). T

he to

tal u

se (a

ll re

gion

s co

mbi

ned)

of p

igs

and

NH

Ps

has

incr

ease

d by

15%

and

33%

, re

spec

tivel

y, w

hile

the

use

of d

ogs

has

decr

ease

d ne

glig

ibly

(2%

) fro

m 2

002

to 2

008.

No

conc

rete

reas

ons

for t

hose

cha

nges

ca

n be

pro

vide

d; o

ne m

ight

spe

cula

te t

hat

the

incr

ease

in

use

of N

HP

’s i

s dr

iven

by

the

incr

easi

ng r

esea

rch

and

deve

lopm

ent e

fforts

in b

ioph

arm

aceu

tical

s. P

igs

and

min

ipig

s ar

e us

ed in

gre

ater

num

bers

than

bot

h do

gs a

nd N

HP

s. T

here

ar

e st

rikin

g di

ffere

nces

bet

wee

n th

e pa

ttern

s of

use

of t

he th

ree

spec

ies

amon

g re

gion

s. C

anad

a an

d th

e E

U h

ave

sim

ilar

patte

rns

of u

se o

f the

thre

e sp

ecie

s, w

hile

Jap

an a

nd th

e U

SA

have

alto

geth

er d

istin

ct p

atte

rns.

In

Can

ada

the

use

of p

igs

fluct

uate

s w

hile

a s

tead

y in

crea

se in

dog

use

is o

bser

ved.

Whe

n lo

okin

g at

the

seco

nd h

alf o

f th

e 19

90’ie

s an

d 20

00’s

res

pect

ivel

y, i

t ap

pear

s th

at m

ore

NH

P’s

are

use

d in

the

sec

ond

half

the

2000

’s.

Aga

in,

biop

harm

aceu

tical

s m

ay b

e dr

ivin

g th

is in

crea

se. I

n th

e EU

the

use

of p

igs

has

incr

ease

d an

d th

e us

e of

dog

and

NH

P is

m

ore

or le

ss c

onst

ant.

The

EU

has

incr

ease

d pr

essu

re to

redu

ce th

e us

e of

NH

P’s

whi

ch le

ad to

a p

ropo

sal f

or b

anni

ng th

e us

e of

Gre

at A

pes

in 2

0075

and

to th

e ad

optio

n of

the

new

Dire

ctiv

e 20

10/6

3/E

U6

publ

ishe

d in

201

0 w

here

by g

ivin

g m

embe

r st

ates

unt

il 20

13 to

com

ply.

The

sci

entif

ic a

nd e

thic

al p

rinci

ples

mot

ivat

ing

the

dire

ctiv

e ha

s be

en c

omm

ente

d by

FE

LAS

A7

poin

ting

out l

aten

t iss

ues

and

shor

tcom

ings

in th

e sc

ient

ific

basi

s of

the

dire

ctiv

e. T

he d

irect

ive

proh

ibits

the

use

of G

reat

A

pes

for

stud

ies

not p

ertin

ent t

o th

e co

ntin

ues

exis

tenc

e of

the

spec

ies

and

thus

doe

s no

t dire

ctly

impa

ct th

e us

e of

oth

er

NH

Ps

in d

rug

deve

lopm

ent,

but

the

new

dire

ctiv

e in

crea

ses

requ

irem

ents

for

sci

entif

ic s

crut

iny

befo

re a

llow

ing

the

use

of

othe

r N

HP

s, a

tre

nd w

hich

may

wel

l gro

w s

trong

er m

akin

g th

e us

e of

NH

Ps

in t

he E

U le

ss a

ttrac

tive

to r

esea

rche

rs a

nd

phar

mac

eutic

al in

dust

ry a

like.

Pre

ssur

e to

mov

e aw

ay fr

om th

e do

g, a

com

pani

on a

nim

al, i

s ris

ing

in E

urop

e w

hich

is e

vide

nt

thro

ugh

the

incr

easi

ng re

quire

men

ts fo

r hou

sing

, wel

fare

in re

latio

n to

wor

king

with

dog

s. T

he m

inip

ig is

not

sub

ject

to s

uch

pres

sure

as

it is

vie

wed

as

a fo

od a

nim

al. T

his

is n

ot to

say

that

it is

of l

ess

ethi

cal c

once

rn to

use

min

ipig

s, it

is n

ot. T

heir

capa

city

to fo

r pai

n an

d su

fferin

g is

the

sam

e as

e.g

. the

dog

or t

he N

HP

as d

escr

ibed

in W

ebst

er e

t al8 .

Jap

an is

uni

que

in

that

dog

s ar

e us

ed to

a m

uch

grea

ter

exte

nt th

an p

igs

and

NH

Ps.

Dog

use

has

dec

reas

ed d

ram

atic

ally

ove

r tw

o de

cade

s (3

8,91

5 in

199

1 vs

. 12,

376

in 2

007)

and

has

not

bee

n re

plac

ed b

y pi

gs o

r NH

Ps.

The

re is

no

clea

r tre

nd in

the

use

of N

HP

s in

Jap

an. F

rom

198

5 th

e us

e of

NH

Ps

fell

to a

n al

l-tim

e lo

w in

199

8, o

nly

to in

crea

se a

gain

afte

r 199

8 pe

akin

g at

an

all-t

ime

high

in 2

007.

The

re is

no

obvi

ous

expl

anat

ion

for t

his

patte

rn a

lthou

gh b

ioph

arm

aceu

tical

s m

ay, a

s m

entio

ned

befo

re, p

lay

a ro

le. P

igs

are

only

use

d in

ver

y sm

all n

umbe

rs in

Jap

an, t

he re

ason

for t

his

is n

ot k

now

n. L

imite

d kn

owle

dge

abou

t the

mod

el

and

its m

erits

com

bine

d w

ith a

trad

ition

al a

ppro

ach

to s

peci

es s

elec

tion

in e

xper

imen

ts a

re p

ossi

bilit

ies.

For

the

USA

thre

e ob

serv

atio

ns c

an b

e m

ade:

(1) w

ith th

e ex

cept

ion

of 2

007

and

2008

the

use

of d

ogs

is fa

irly

cons

tant

, (2)

the

use

of p

igs

has

gone

dow

n 15

% f

rom

200

2 to

200

9, (

3) t

he u

se o

f N

HP

s ha

s in

crea

sed

by 3

5% o

ver

the

sam

e pe

riod;

it is

see

n th

at a

n in

vers

ion

of t

he r

elat

ive

prop

ortio

ns o

f pi

g an

d N

HP

use

has

take

n ha

ppen

ed f

rom

200

2 to

200

9. N

o ex

plan

atio

n fo

r th

e de

crea

sed

pig

use

can

be o

ffere

d; th

e in

crea

se in

NH

P us

e lik

ely

driv

en b

y bi

opha

rmac

eutic

als.

A m

ore

deta

iled

anal

ysis

of

NH

P us

e, s

imila

r to

Car

lsso

n et

al.9

, cou

ld a

ddre

ss th

is q

uest

ion,

but

that

is b

eyon

d th

e sc

ope

of th

is p

oste

r.

CO

NC

LUSI

ON

: pi

gs a

nd m

inip

igs

are

used

in g

reat

er n

umbe

rs t

han

dogs

and

NH

Ps.

In

Can

ada

and

EU

the

pat

tern

s of

re

lativ

e us

e ar

e co

mpa

rabl

e; f

urth

erm

ore

the

use

of N

HP

s in

the

EU

is

cons

tant

, as

opp

osed

to

Can

ada,

whe

re i

t ha

s in

crea

sed.

In th

e U

SA

pig

use

has

decr

ease

d w

hile

the

use

of N

HP

s ha

s in

crea

sed;

use

of d

ogs

is fa

irly

cons

tant

. Jap

an h

as

seen

a m

arke

d dr

op in

the

use

of d

ogs,

whi

le th

e us

e of

pig

s an

d N

HP

s re

mai

ns a

t low

leve

ls. T

he o

vera

ll pa

ttern

sug

gest

s th

at th

e in

crea

se in

use

of N

HP

s co

uld

be e

xpla

ined

by

biop

harm

aceu

tical

pro

duct

s be

com

ing

mor

e nu

mer

ous

in d

isco

very

an

d de

velo

pmen

t in

phar

mac

eutic

al in

dust

ry.

P

oste

r pre

sent

ed a

t The

Sw

ine

in B

iom

edic

al R

esea

rch

conf

eren

ce J

uly

2011

, Chi

cago

, IL,

US

A

DIS

CU

SSIO

N

RES

ULT

S

Sur

vey

of m

inip

ig c

linic

al b

ioch

emis

try, c

oagu

latio

n, a

nd u

rinal

ysis

par

amet

ers

in th

e E

urop

ean

phar

mac

eutic

al in

dust

ry

- cur

rent

pra

ctic

e an

d ch

alle

nges

for t

he fu

ture

dev

elop

men

t of b

iom

arke

rs o

f tox

icity

in th

e m

inip

ig

N.C

. Gan

deru

p1, L

. Ear

l2 , C

. Bou

chez

3 , S

. Mhe

dhbi

4 , A

. Gib

bs5 ,

P. G

leru

p6, G

. Itte

r7, P

. Cla

usin

g8

1: E

llega

ard

Göt

tinge

n M

inip

igs,

DK

[ncg

@m

inip

igs.

dk].

2: H

untin

gdon

Life

Sci

ence

, UK.

3: C

iTox

LAB

CiT

, Fra

nce.

4: G

alde

rma

R&

D, F

ranc

e. 5

: Cov

ance

Lab

orat

orie

s, U

K. 6

: Cito

xLA

B S

cant

ox, D

enm

ark.

7: S

anof

i-Ave

ntis

IPH

, Ger

man

y. 8

: Ber

lin, G

erm

any

A

BST

RA

CT:

The

use

of m

inip

igs

in re

gula

tory

saf

ety

test

ing

has

incr

ease

d si

gnifi

cant

ly th

e pa

st d

ecad

e. W

ith th

e in

crea

sed

use

of m

inip

igs

esta

blis

hing

bes

t pra

ctic

es fo

r bio

mar

kers

(clin

ical

-che

mis

try,

haem

atol

ogy,

coa

gula

tion

para

met

ers,

and

urin

alys

is) i

s es

sent

ial t

o en

sure

pre

dict

ive

safe

ty a

sses

smen

t stu

dies

, with

pat

ient

saf

ety

and

heal

th a

s th

e ul

timat

e go

al. M

uch

effo

rt is

inve

sted

in d

evel

opin

g an

d va

lidat

ing

biom

arke

rs in

min

ipig

s an

d be

tter u

nder

stan

ding

of c

urre

nt p

ract

ices

may

pro

ve m

ore

effic

ient

and

ben

efit

both

the

rese

arch

ers

and

rese

arch

inst

itutio

ns. A

sur

vey

of in

dust

ry w

ith th

e fo

llow

ing

obje

ctiv

e w

as c

ondu

cted

: (1)

col

late

info

rmat

ion

on c

urre

nt ro

utin

e bi

omar

kers

and

iden

tify

thei

r val

ue in

the

min

ipig

; (2)

iden

tify

biom

arke

rs u

nder

dev

elop

men

t/con

side

ratio

n to

add

ress

gen

eral

and

spe

cific

ne

eds

as m

arke

rs o

f tox

icity

in th

e m

inip

ig, a

nd (3

) lea

rn w

hat t

he m

inip

ig u

ser c

omm

unity

thin

ks th

e pr

ospe

cts

and

chal

leng

es a

re fo

r dev

elop

ing

and

usin

g ne

w b

iom

arke

rs in

the

min

ipig

.

Man

y st

anda

rd b

iom

arke

rs u

sed

in to

xico

logy

are

est

ablis

hed

in m

inip

igs.

The

ir de

velo

pmen

t is

prim

arily

driv

en b

y th

e ph

arm

aceu

tical

indu

stry

’s n

eeds

in re

gula

tory

saf

ety

asse

ssm

ent s

tudi

es a

nd w

hile

th

ere

is p

ublis

hed

liter

atur

e on

bio

mar

kers

ava

ilabl

e th

ey d

o no

t nec

essa

rily

mee

t the

nee

ds o

f tox

icol

ogis

t. Th

ere

appe

ars

to b

e an

inte

rest

in c

reat

ing

a pl

atfo

rm to

sha

re d

ata

and

info

rmat

ion

abou

t exi

stin

g bi

omar

kers

as

wel

l as

biom

arke

rs u

nder

dev

elop

men

t. S

uch

a da

taba

se w

ould

ben

efit

the

use

of m

inip

igs

in s

afet

y as

sess

men

t as

wel

l as

incr

ease

the

acce

ssib

ility

and

val

ue o

f min

ipig

bio

mar

kers

. Thi

s su

rvey

was

con

duct

ed b

y M

inip

ig R

esea

rch

Foru

m [M

RF]

Ste

erin

g G

roup

und

er th

e au

spic

es o

f the

MR

F.

INTR

OD

UC

TIO

N: T

he ro

le o

f bio

mar

kers

in p

harm

aceu

tical

dev

elop

men

t is

of im

porta

nce

to re

gula

tors

and

indu

stry

alik

e. T

his

is

evid

ence

d by

the

atte

ntio

n th

ey re

ceiv

e fro

m b

oth

“sid

es o

f the

tabl

e”. A

pplic

atio

ns a

nd c

halle

nges

in b

iom

arke

r qua

lific

atio

n is

dis

cuss

ed b

y th

e ph

arm

aceu

tical

indu

stry

(Gue

rrei

o et

al.

2003

; ; L

ee e

t al.

2005

; Lüh

e et

al.

2005

; Gau

ghan

200

6; M

arre

r and

Die

terle

200

7). R

egul

ator

y ag

enci

es

(mai

nly

the

US

FD

A) o

ffer t

heir

pers

pect

ive

on h

ow to

qua

lify

geno

mic

bio

mar

kers

in a

n in

dust

ry g

uida

nce

docu

men

t (U

S F

DA

) and

in s

cien

tific

lit

erat

ure

(Goo

dsai

d an

d Fr

ueh

2007

). Fu

rther

mor

e, U

S F

DA

also

pro

vide

s a

list o

f val

id g

enom

ic b

iom

arke

rs (U

S F

DA

2010

). Th

e ta

ke h

ome

mes

sage

is th

at q

ualif

ied

biom

arke

rs a

re o

f val

ue in

the

deve

lopm

ent o

f new

med

icin

es a

nd c

once

rted

effo

rts w

ill le

ad to

fast

er q

ualif

icat

ion.

The

use

of m

inip

igs

in re

gula

tory

saf

ety

test

ing

has

incr

ease

d si

gnifi

cant

ly th

e pa

st d

ecad

e. W

ith th

e in

crea

sed

use

of m

inip

igs

esta

blis

hing

be

st p

ract

ices

for b

iom

arke

rs s

uch

as c

linic

al b

ioch

emis

try, c

oagu

latio

n pa

ram

eter

s, a

nd u

rinal

ysis

is e

ssen

tial t

o en

sure

pre

dict

ive

safe

ty

asse

ssm

ent s

tudi

es, w

ith p

atie

nt s

afet

y an

d he

alth

as

the

defin

itive

obj

ectiv

es. M

uch

effo

rt is

inve

sted

in d

evel

opin

g an

d va

lidat

ing

biom

arke

rs, a

be

tter u

nder

stan

ding

of c

urre

nt p

ract

ices

may

pro

ve m

ore

effic

ient

and

ben

efit

both

the

rese

arch

ers

and

rese

arch

inst

itutio

ns.

M

ore

spec

ifica

lly, t

he p

urpo

se o

f thi

s su

rvey

can

be

sum

mar

ized

as

follo

ws:

(1) c

olla

te in

form

atio

n on

the

curr

ent r

outin

e cl

inic

al

bioc

hem

istry

, coa

gula

tion

and

urin

alys

is a

naly

tes/

para

met

ers

and

iden

tify

the

valu

e of

sai

d in

the

min

ipig

. (2)

Iden

tify

new

bio

mar

kers

abo

ut to

be

deve

lope

d to

add

ress

gen

eral

and

spe

cific

nee

ds a

s m

arke

rs o

f tox

icity

in th

e m

inip

ig. (

3) L

earn

wha

t the

min

ipig

use

r com

mun

ity th

inks

the

pros

pect

s an

d ch

alle

nges

are

for d

evel

opin

g an

d us

ing

new

bio

mar

kers

in th

e m

inip

ig. T

his

surv

ey w

as c

ondu

cted

by

the

Eur

opea

n M

inip

ig

Res

earc

h Fo

rum

Ste

erin

g G

roup

. Res

pond

ents

are

than

ked

for y

our v

alua

ble

inpu

t.

Th

e M

inip

ig R

esea

rch

Foru

m in

Eur

ope

is th

e us

er g

roup

whi

ch s

eeks

to p

rovi

de in

form

atio

n to

min

ipig

use

rs to

ena

ble

them

to u

se o

ther

pe

ople

’s e

xper

ienc

e to

refin

e an

d pr

ogre

ss th

eir r

esea

rch

in a

n ef

fect

ive

and

ethi

cal w

ay a

nd p

rovi

des

a fo

rum

to e

nabl

e ex

perie

nced

use

rs to

giv

e th

e re

st o

f us

the

bene

fit o

f the

ir ex

perie

nce.

Lea

rn m

ore

at w

ww

.min

ipig

rese

arch

foru

m.o

rg.

REF

EREN

CES

(B

acqu

evill

e, D

., B

oiss

on, M

., M

avon

, A.,

2007

. Lac

tate

Deh

ydro

gena

se is

a R

elev

ant E

ndpo

int B

iom

arke

r to

Stu

dy Ir

ritat

ion

and

Sol

ar R

adia

tion-

Indu

ced

Cel

lula

r Dam

ages

in P

ig S

kin

Org

an C

ultu

re. M

eetin

g P

oste

r Abs

tract

: 68

th A

nnua

l Mee

ting

of th

e S

ocie

ty fo

r Inv

estig

ativ

e D

erm

atol

ogy

(SID

200

7), H

yatt

Reg

ency

Cen

tury

Pla

za H

otel

, Los

Ang

eles

, Cal

iforn

ia (U

SA

), 9-

12 M

ay 2

007.

Fr

antz

, N.Z

., Fr

iese

n, K

.G.,

And

rew

s, G

.A.,

et a

l., 2

010.

Use

of s

erum

bio

mar

kers

to p

redi

ct th

e de

velo

pmen

t and

sev

erity

of o

steo

chon

dros

is le

sion

s in

the

dist

al p

ortio

n of

the

fem

ur in

pig

s. A

mer

ican

jour

nal o

f vet

erin

ary

rese

arch

, Aug

ust 0

1, 7

1(8)

:946

-952

. G

erha

rdy,

C.,

Rei

nich

e, A

., B

ouch

eix,

O.,

et a

l.,20

09. C

ardi

ac b

iom

arke

r eva

luat

ion

follo

win

g Is

opre

nalin

e ad

min

istra

tion

in th

e m

inip

ig. T

oxic

olog

y Le

tters

, Vol

. 189

, S15

7.

Ichi

nose

, T.,

Mill

er, M

.G.,

Shi

bam

oto,

T.,

1994

. Det

erm

inat

ion

of F

ree

Mal

onal

dehy

de F

orm

ed in

Liv

er M

icro

som

es u

pon

CC

l sub

(4) O

xida

tion.

Jou

rnal

of A

pplie

d To

xico

logy

[J. A

ppl.

Toxi

col.]

. Vol

. 14,

no.

6, 4

53 p

. Nov

199

4.

Link

o, A

.M.,

Ala

stai

r B.,

Kam

al-E

ldin

, A.,

et a

l., 2

006.

Kin

etic

s of

the

appe

aran

ce o

f cer

eal a

lkyl

reso

rcin

ols

in p

ig p

lasm

a. T

he B

ritis

h jo

urna

l of n

utrit

ion,

95(

2):2

82-2

87

Loft,

S.,

Fisc

her-

Nie

lsen

, A.,

Jedi

ng, I

.B.,

et a

l., 1

993.

8-H

ydro

xyde

oxyg

uano

sine

as

a ur

inar

y bi

omar

ker o

f oxi

dativ

e D

NA

dam

age.

Jou

rnal

of T

oxic

olog

y an

d E

nviro

nmen

tal H

ealth

, vol

. 40,

no.

2-3

. 199

3.

Sac

o Y,

Fra

ile L

, Gim

enez

M, e

t al.,

201

0. H

apto

glob

in s

erum

con

cent

ratio

n is

a s

uita

ble

biom

arke

r to

asse

ss th

e ef

ficac

y of

a fe

ed a

dditi

ve in

pig

s. A

nim

al v

ol. 4

is

sue:

9

page

s: 1

561-

1567

. S

tahl

, T.S

., Za

mzo

w, J

.B.,

Wan

g, D

., et

al.

2007

. Rel

atio

nshi

p of

Isop

rost

anes

, Bio

mar

ker o

f Oxi

dativ

e S

tress

, and

Pig

Pro

duct

ivity

. 200

7 Jo

int A

nnua

l Mee

ting

of th

e A

mer

ican

Dai

ry S

cien

ce A

ssoc

iatio

n, P

oultr

y S

cien

ce

Ass

ocia

tion,

Aso

ciac

ion

Mex

ican

a de

Pro

ducc

ion

Ani

mal

and

the

Am

eric

an S

ocie

ty o

f Ani

mal

Sci

ence

, San

Ant

onio

, Tex

as (U

SA

), 8-

12 J

ul 2

007

Te P

as, M

., K

euni

ng, E

., K

ruijt

, L.,

et a

l., 2

009.

Bio

mar

ker d

evel

opm

ent f

or re

cove

ry fr

om s

tress

in p

ig m

uscl

es. 6

0th

Ann

ual M

eetin

g of

the

Eur

opea

n A

ssoc

iatio

n fo

r Ani

mal

Pro

duct

ion,

Fira

de

Bar

celo

na, B

arce

lona

, 24-

27 A

ug

2009

. Va

n de

r Bur

ght,

A.S

.A.M

., Ty

sklin

d, M

., A

nder

sson

, P.L

., et

al.,

200

0. S

truct

ure

depe

nden

t ind

uctio

n of

CYP

1A b

y po

lych

lorin

ated

bip

heny

ls in

hep

atoc

ytes

of m

ale

cast

rate

d pi

gs. C

hem

osph

ere,

vol

. 41,

no.

10,

pp.

169

7-17

08.

Wol

f, A

., K

utz,

A.,

Plo

ttner

, S.,

et a

l., 2

005.

The

Effe

ct o

f Ben

zo(a

)pyr

ene

on P

orci

ne U

rinar

y B

ladd

er E

pith

elia

l Cel

ls a

naly

zed

for t

he E

xpre

ssio

n of

Sel

ecte

d G

enes

and

Cel

lula

r Tox

icol

ogic

al E

ndpo

ints

. Tox

icol

ogy,

vol

. 207

, no.

2,

255

p. 1

4 Fe

b 20

05.

Xia

, J.,

Sec

kin,

E.,

Xia

ng, Y

., et

al.,

200

8. P

ositr

on-e

mis

sion

tom

ogra

phy

imag

ing

of th

e an

giot

ensi

n II

subt

ype

1 re

cept

or in

sw

ine

rena

l arte

ry s

teno

sis.

Hyp

erte

nsio

n, 5

1(2)

:466

-473

. U

S F

DA

2010

. Val

id G

enom

ic B

iom

arke

rs in

the

Con

text

of A

ppro

ved

Dru

g La

bels

. ht

tp://

ww

w.fd

a.go

v/D

rugs

/Sci

ence

Res

earc

h/R

esea

rchA

reas

/Pha

rmac

ogen

etic

s/uc

m08

3378

.htm

t acc

esse

d N

ovem

ber 2

6th

2010

. U

S F

DA

. Gui

danc

e fo

r ind

ustry

. E16

Gen

omic

Bio

mar

kers

Rel

ated

to D

rug

Res

pons

e: C

onte

xt, S

truct

ure,

and

For

mat

of Q

ualif

icat

ion

Sub

mis

sion

s.

Gue

rreio

, N.,

Sta

edtle

r, F.

, Gre

net,

O.,

2003

. Tox

icog

enom

ics

in D

rug

Dev

elop

men

t. To

xico

logi

c P

atho

logy

, 31:

471–

479.

Lü

he, A

., S

uter

, L.,

Rue

pp, S

., et

al.,

200

5. T

oxic

ogen

omic

s in

the

phar

mac

eutic

al in

dust

ry: H

ollo

w p

rom

ises

or r

eal b

enef

it? M

utat

ion

Res

earc

h 57

5 (2

005)

102

–115

. M

arre

r, E

., an

d D

iete

rle, F

., 20

07. P

rom

ises

of B

iom

arke

rs in

Dru

g D

evel

opm

ent –

A R

ealit

y C

heck

. Che

mic

al B

iolo

gy &

Dru

g D

esig

n, 6

9: 3

81–3

94.

Lee,

J.W

., W

eine

r, R

.S.,

Sai

lsta

d, J

.M.,

et a

l. 20

05. M

etho

d Va

lidat

ion

and

Mea

sure

men

t of B

iom

arke

rs in

Non

clin

ical

and

Clin

ical

Sam

ples

in D

rug

Dev

elop

men

t: A

Con

fere

nce

Rep

ort.

Pha

rmac

eutic

al R

esea

rch,

Vol

. 22,

No.

4,

pp. 4

99-5

11.

Goo

dsai

d, F

., an

d Fr

ueh,

F.,

2007

. Bio

mar

ker Q

ualif

icat

ion

Pilo

t Pro

cess

at t

he U

S F

ood

and

Dru

g A

dmin

istra

tion.

The

AA

PS

Jou

rnal

200

7; 9

(1) A

rticl

e 10

(http

://w

ww.

aaps

j.org

). G

augh

an, A

., 20

06. B

ridgi

ng th

e di

vide

: the

nee

d fo

r tra

nsla

tiona

l inf

orm

atic

s. P

harm

acog

enom

ics,

7(1

), pp

.117

-22.

MAT

ERIA

LS A

ND

MET

HO

DS:

A s

erie

s of

que

stio

ns w

ere

form

ulat

ed a

nd c

onde

nsed

into

sur

vey.

The

sur

vey

cons

iste

d of

ten

ques

tions

with

repl

y op

tions

and

/or b

lank

fiel

ds. F

or c

oagu

latio

n, b

ioch

emis

try, a

nd u

rinal

ysis

(Q1-

3) re

spon

dent

s co

uld

repl

y w

heth

er th

ey w

ere

perfo

rmin

g th

e an

alys

is u

nder

Non

-GLP

con

ditio

ns (N

on-G

LP),

unde

r GLP

con

ditio

ns (G

LP),

or n

ot

perfo

rmed

at a

ll (N

o) fo

r wel

l-kno

wn

para

met

ers

in to

xici

ty s

tudi

es. F

or th

e re

mai

ning

que

stio

ns (4

-10)

bla

nk fi

elds

wer

e pr

ovid

ed

for f

ree

from

repl

ies.

Sur

vey

Mon

key

® w

as u

sed

as s

urve

y en

gine

to s

end

out,

colle

ct a

nd m

anag

e th

e re

sults

of t

he s

urve

y.

Sev

eral

are

as w

ere

inve

stig

ated

with

the

spec

ific

obje

ctiv

es to

iden

tify:

•

Whi

ch ro

utin

e an

d ne

w c

linic

al b

ioch

emis

try, c

oagu

latio

n an

d ur

inal

ysis

par

amet

ers

used

with

in th

e E

urop

ean

phar

mac

eutic

al

indu

stry

in d

rug

deve

lopm

ent?

•

Whi

ch a

naly

tes/

para

met

ers

are

bein

g/ha

ve b

een

valid

ated

as

biom

arke

rs in

toxi

city

stu

dies

in th

e m

inip

ig?

•W

hich

ana

lyte

s/pa

ram

eter

s ha

ve c

ause

d di

fficu

lties

in a

ssay

dev

elop

men

t and

wha

t has

bee

n th

e ou

tcom

e?

•C

omm

erci

ally

ava

ilabl

e as

say

kits

whi

ch h

ave

been

inte

rnal

ly v

alid

ated

for u

se in

toxi

city

stu

dies

? •

Wha

t cha

lleng

es a

nd p

rosp

ects

dire

ct th

e de

velo

pmen

t of b

iom

arke

rs o

f tox

icity

in th

e m

inip

ig?

Exp

erie

nced

min

ipig

s us

ers,

aca

dem

ic re

sear

cher

s an

d sp

ecia

lists

in th

e fie

ld w

ere

aske

d to

com

plet

e th

e qu

estio

nnai

re. 3

35

emai

ls w

ere

sent

out

and

18

resp

onde

d gi

ving

a re

spon

se ra

te o

f 5 %

. All

resp

onse

s ar

e pr

esen

ted

anon

ymou

s.

RES

ULT

S (B

ELO

W L

EFT)

: Res

pons

es to

eac

h of

the

ten

ques

tions

(1-1

0) p

osed

in th

e su

rvey

are

pro

vide

d be

low

. Fo

r rel

evan

t par

amet

ers

the

num

ber o

f res

pond

ents

(Res

pond

ents

) for

eac

h in

divi

dual

par

amet

er is

pro

vide

d. F

or Q

1-3

the

per

cent

dis

tribu

tion

of p

aram

eter

s co

nduc

ted

unde

r “N

on-G

LP”,

“GLP

”, or

not

at a

ll (N

o) c

ondi

tions

is p

rese

nted

. Fre

e fo

rm

resp

onse

s (Q

4-10

) are

col

late

d an

d ha

ve u

nder

gone

onl

y m

inor

edi

ting

and

rem

oval

of d

uplic

ate

resp

onse

s.

CO

NC

LUSI

ON

S A

ND

REC

OM

MEN

DAT

ION

S: M

any

stan

dard

bio

mar

kers

use

d in

toxi

colo

gy a

re

esta

blis

hed

in m

inip

igs.

Bio

mar

kers

in u

se in

the

min

ipig

use

r com

mun

ity is

prim

arily

driv

en b

y th

e ph

arm

aceu

tical

in

dust

ry’s

nee

ds in

regu

lato

ry s

afet

y as

sess

men

t stu

dies

and

whi

le th

ere

is p

ublis

hed

liter

atur

e on

bio

mar

kers

av

aila

ble

they

do

not n

eces

saril

y m

eet t

he n

eeds

of t

oxic

olog

ist.

Ther

e se

ems

to b

e an

inte

rest

in c

reat

ing

a pl

atfo

rm

for m

inip

igs

user

s to

sha

re d

ata

and

info

rmat

ion

abou

t exi

stin

g bi

omar

kers

as

wel

l as

biom

arke

rs u

nder

dev

elop

men

t. Id

entif

ying

and

ove

rcom

ing

exis

ting

or p

oten

tial h

urdl

es to

min

ipig

use

rs c

an b

e do

ne b

y in

itiat

ing

a su

rvey

with

the

sam

e de

sign

targ

etin

g do

gs a

nd n

on-h

uman

prim

ates

(NH

P).

If th

e ov

eral

l pic

ture

obs

erve

d is

sim

ilar f

or d

ogs

and

NH

P’s,

as

it is

for m

inip

igs,

this

wou

ld in

dica

te th

at h

urdl

es fa

ced

by th

e m

inip

ig is

the

sam

e as

dog

s an

d N

HPs

, or

vice

ver

sa, i

.e. i

t wou

ld h

elp

pinp

oint

spe

cific

focu

s ar

eas

wer

e w

ork

mus

t be

done

to b

ring

the

tool

box

on

par w

ith

wha

t is

avai

labl

e fo

r use

in d

ogs

and

NH

Ps.

Pos

ter p

rese

nted

at T

he S

win

e in

Bio

med

ical

Res

earc

h co

nfer

ence

Jul

y 20

11, C

hica

go, I

L, U

SA

Tabl

e 1:

Pub

lishe

d bi

omar

kers

em

ploy

ed in

min

ipig

s B

iom

arke

rs in

(min

i)pig

s ar

e de

scrib

ed in

the

publ

ishe

d lit

erat

ure,

in

clud

e bi

omar

kers

of d

isea

se s

tate

s an

d to

xici

ty; b

ut b

iom

arke

rs

used

in fo

od s

afet

y an

d nu

tritio

n ar

e kn

own.

B

iom

arke

r R

efer

ence

S

erum

bio

mar

kers

of c

artil

age

and

bone

m

etab

olis

m to

pre

dict

ost

eoch

ondr

osis

lesi

ons

Ger

hard

y et

al.

2009

Car

diac

trop

onin

I (c

TnI)

in n

orm

al p

igs.

Fr

antz

et a

l. 20

10

Ang

iote

nsin

II s

ubty

pe 1

rece

ptor

(AT(

1)R

) is

intro

duce

d as

a d

iagn

ostic

bio

mar

ker o

f re

nova

scul

ar d

isea

se.

Xia

et a

l. 20

09

8-hy

drox

ydeo

xygu

anos

ine

as a

non

-inva

sive

ur

inar

y bi

omar

ker o

f oxi

dativ

e D

NA

dam

age

in

hum

ans,

pig

s, a

nd o

ther

labo

rato

ry a

nim

al

spec

ies.

Loft

et a

l. 19

93

Lact

ate

dehy

drog

enas

e as

a re

leva

nt b

iom

arke

r to

ass

ess

skin

inju

ry in

ex

vivo

por

cine

ski

n or

gan

cultu

re.

Bac

quev

ille e

t al.

2007

Form

atio

n of

mal

onda

ldeh

yde

as a

pot

entia

l in

vitro

bio

mar

ker o

f car

bon

tetra

chlo

ride

indu

ced

hepa

toto

xici

ty

Ichi

nose

et a

l. 19

94

In v

itro

expo

sure

of b

enzo

(a)p

yren

e on

por

cine

ur

inar

y bl

adde

r epi

thel

ial c

ells

indi

cate

d C

YP1A

1 m

RN

A ex

pres

sion

as

a po

tent

ial b

iom

arke

r

Wol

f et a

l. 20

05

Pol

ychl

orin

ated

bip

heny

ls m

edia

ted

indu

ctio

n of

C

YP1A

act

ivity

as

biom

arke

r for

Ah-

rece

ptor

m

edia

ted

resp

onse

s.

Van

der B

urgh

t et

al. 2

000

Alk

ylre

sorc

inol

s m

ay b

e a

usef

ul b

iom

arke

r of

who

legr

ain

whe

at a

nd ry

e in

take

Li

nko

et a

l. 20

06

Bio

mar

kers

for v

ario

us p

aram

eter

s re

leva

nt fo

r co

mm

erci

al p

ig (m

eat)

prod

uctio

n.

Sta

hl e

t al.

2007

; Te

Pas

et a

l. 20

09

Hap

togl

obin

as

a bi

omar

ker t

o m

onito

r var

ious

pr

oduc

tion

para

met

ers

incl

udin

g us

e of

Auj

eszk

y m

odifi

ed li

ve v

acci

ne in

pro

duct

ion

pigs

.

Sac

o et

al.

2010

Q1:

WH

ICH

CO

AG

ULA

TIO

N P

AR

AM

ETE

RS D

O Y

OU

C

UR

RE

NTL

Y M

EA

SU

RE A

S R

OU

TIN

E H

AE

MAT

OLO

GY

AN

ALY

TES/P

AR

AM

ETE

RS O

N T

OX

ICIT

Y S

TUD

IES IN

M

INIP

IGS?

Par

amet

er

Non

-G

LP

GLP

N

o R

espo

nde

nts

Fibr

inog

en

1 6

8 15

A

ctiv

ated

par

tial

thro

mbo

plas

tin ti

me

(AP

TT)

1 8

6 15

Pro

thro

mbi

n tim

e 2

7 6

15

Tiss

ue p

lasm

inog

en

activ

ator

(tPA

) 1

0 11

12

D-D

imer

0

0 11

11

Th

rom

bin-

antit

hrom

bin

com

plex

es (T

AT)

0 0

11

11

% o

f tot

al p

aram

eter

s

6%

27%

67

%

Q2:

WH

ICH

BLO

OD

BIO

CH

EM

ISTR

Y PA

RA

ME

TER

S D

O

YOU

CU

RR

EN

TLY

ME

AS

UR

E A

S R

OU

TIN

E C

LIN

ICA

L B

IOC

HE

MIS

TRY

AN

ALY

TES/P

AR

AM

ETE

RS O

N T

OX

ICIT

Y S

TUD

IES IN

MIN

IPIG

S?

Par

amet

er

Non

-G

LP

GLP

N

o R

espo

nden

ts

Alk

alin

e ph

osph

atas

e 2

7 2

11

Glu

cose

2

7 2

11

Ure

a 2

7 2

11

Sod

ium

2

7 2

11

Pot

assi

um

2 7

2 11

A

lani

ne a

min

otra

nsfe

rase

1

7 2

10

Asp

arta

te a

min

otra

nsfe

rase

1

7 2

10

Gam

ma

glut

amyl

trans

fera

se

2 5

3 10

C

reat

inin

e 2

6 2

10

Tota

l pro

tein

1

7 2

10

Alb

umin

1

7 2

10

Chl

orid

e 1

7 2

10

Cal

cium

1

7 2

10

Tota

l bilir

ubin

1

6 2

9 D

irect

bilir

ubin

– T

otal

ch

oles

tero

l 1

6 2

9

Trig

lyce

rides

2

5 2

9 A

lbum

in/g

lobu

lin ra

tio

0 7

2 9

Cre

atin

e 1

4 3

8 G

amm

a gl

obul

ins

0 4

4 8

Inor

gani

c ph

osph

orus

0

6 2

8 La

ctat

e de

hydr

ogen

ase

2 2

3 7

HD

L-ch

oles

tero

l 2

1 4

7 LD

L-ch

oles

tero

l 2

1 4

7 La

ctat

e 2

1 4

7 A

lpha

-1 g

lobu

lins

0 3

4 7

Alp

ha-2

glo

bulin

s 0

3 4

7 M

agne

sium

0

4 3

7 C

reat

ine

kina

se

1 3

2 6

Glu

tam

ic d

ehyd

roge

nase

1

2 3

6 A

myl

ase

1 1

4 6

Pho

spho

lipid

s 1

1 4

6 U

ric a

cid

1 1

4 6

Bile

aci

ds

1 1

4 6

Lipa

se

0 1

4 5

Leuc

ine

amin

opep

tidas

e 0

0 5

5 5’

Nuc

leot

idas

e 0

0 5

5 In

sulin

1

0 4

5 Fr

ee fa

tty a

cids

0

1 4

5 Iro

n 0

1 4

5 To

tal i

ron

bind

ing

capa

city

0

1 4

5 Tr

ansf

errin

0

0 5

5 B

eta

glob

ulin

s 0

3 1

4 %

of t

otal

par

amet

ers

12%

48

%

39%

Q3:

WH

ICH

UR

INA

LYS

IS P

AR

AM

ETE

RS D

O Y

OU

CU

RR

EN

-TL

Y M

EA

SU

RE A

S R

OU

TIN

E C

LIN

ICA

L B

IOC

HE

MIS

TRY

AN

A-

LYTE

S/P

AR

AM

ETE

RS O

N T

OX

ICIT

Y S

TUD

IES IN

MIN

IPIG

S?

Par

amet

er N

on-

GLP

G

LP

No

Res

pon

dent

s pH

2

7 1

10

Spe

cific

gra

vity

2

7 1

10

Pro

tein

2

7 1

10

Glu

cose

2

7 1

10

Ket

one

2 6

2 10

B

ilirub

in (b

ile p

igm

ents

) 2

6 2

10

App

eara

nce

1 7

1 9

Volu

me

1 7

1 9

Blo

od p

igm

ents

2

5 2

9 U

robi

linog

en

0 6

2 8

Ure

a 1

3 2

6 S

odiu

m

0 4

1 5

Pot

assi

um

0 4

1 5

Cal

cium

0

3 2

5 M

agne

sium

0

3 2

5 C

hlor

ide

0 4

1 5

Inor

gani

c ph

osph

orus

0

3 2

5 N

-ace

tyl D

-glu

cosa

min

idas

e 0

3 2

5 G

amm

a gl

utam

yl tr

ansf

eras

e 0

2 3

5 La

ctat

e de

hydr

ogen

ase

0 2

3 5

Cre

atin

ine

0 3

2 5

Uric

aci

d 1

1 3

5 A

myl

ase

0 1

3 4

Per

cen

t of t

otal

par

amet

ers

11%

63

%

26%

D

EP

OS

IT A

FTE

R C

EN

TRIF

UG

ATIO

N E

XA

MIN

ED

FO

R:

Leuc

ocyt

es

0 6

2 8

Ery

thro

cyte

s 0

6 2

8 C

ryst

als

1 6

1 8

Epi

thel

ial c

ells

0

6 1

7 S

perm

atoz

oa a

nd p

recu

rsor

s 0

5 2

7 C

asts

0

6 1

7 O

ther

abn

orm

al c

ompo

nent

s 0

6 1

7 %

of t

otal

par

amet

ers

2%

80%

18

%

4: W

HIC

H P

AR

AM

ETER

S (O

THER

TH

AN

TH

E R

OU

TIN

E ID

ENTI

FIED

AB

OVE

) HAV

E B

EEN

INVE

STIG

ATED

AS

POTE

NTI

AL

BIO

MA

RK

ERS

OF

TOXI

CIT

Y A

ND

WH

Y W

AS

IT F

ELT

NEC

ESSA

RY

TO D

EVEL

OP

THE

ASS

AY?

Res

pond

ents

gav

e th

e fo

llow

ing

exam

ples

. Rep

lies

are

in n

o pa

rticu

lar o

rder

; any

repe

titio

ns

have

bee

n re

mov

ed.

•S

erum

am

yloi

d A

, Tr

opon

in I,

faec

al o

ccul

t blo

od (t

o ch

eck

bloo

d lo

ss in

the

gast

roin

test

inal

tra

ct),

insu

lin, S

-100

and

NS

E (i

ndic

ator

s of

bra

in d

amag

e), u

rinar

y el

ectro

lyte

s, o

steo

calc

in,

thyr

oid

horm

ones

(T3,

T4,

TS

H; t

o ch

eck

thyr

oid

toxi

city

), m

etha

emog

lobi

n, b

one-

spec

ific

alka

line

phos

phat

ase,

test

oste

rone

, his

tam

ine,

hap

togl

obin

, thr

ombi

n-an

tithr

ombi

n co

mpl

exes

(T

AT),

C-re

activ

e pr

otei

n (d

ue to

com

poun

d sp

ecifi

c to

xici

ties)

, Fac

tor X

a P

aram

eter

7 C

3.

5: W

HAT

CH

ALL

ENG

ES D

ID Y

OU

FA

CE

IN D

EVEL

OPI

NG

TH

E A

SSAY

FO

R T

HE

PAR

AM

ETER

S IN

TH

E Q

UES

TIO

N A

BO

VE?

Res

pond

ents

repl

ied

with

a n

umbe

r of c

halle

nges

. The

y ar

e lis

ted

in n

o pa

rticu

lar o

rder

; re

petit

ions

hav

e be

en re

mov

ed.

•La

ck o

f spe

cies

spe

cific

kits

. •

Diff

eren

ces

in p

lasm

a/se

rum

from

diff

eren

t min

ipig

sou

rces

. •

Mai

n ch

alle

nge

(ass

umin

g ad

equa

te s

ampl

e co

llect

ion

& a

ppro

pria

te ti

me

poin

ts) i

s th

e ad

optio

n of

app

ropr

iate

refe

renc

e m

ater

ials

for p

ig (i

.e. t

o pr

ove

we

are

mea

surin

g w

hat w

e th

ink

we

are

mea

surin

g). T

he a

ppro

ach

is to

inve

stig

ate

pig

mat

rix in

terfe

renc

e an

d w

ork

with

th

e gi

ven

qual

ity c

ontro

ls.

•R

are

use

of im

mun

olum

inom

etric

ass

ay

•Fi

ndin

g a

suita

ble

thyr

oid-

stim

ulat

ing

horm

one

ELI

SA

kit a

nd p

erfo

rmin

g co

mpl

ete

valid

atio

n.

Pro

duci

ng in

tern

al q

ualit

y co

ntro

l. •

Find

ing

suita

ble

Trop

onin

I E

LIS

A ki

t and

per

form

ing

com

plet

e va

lidat

ion.

Pro

duci

ng in

tern

al

qual

ity c

ontro

l. 6:

WH

AT W

AS

THE

OU

TCO

ME

OF

THE

ASS

AY D

EVEL

OPM

ENT

AN

D/O

R P

ERC

EIVE

D V

ALU

E O

F TH

E B

IOM

AR

KER

? R

espo

nden

ts g

ave

som

e ex

ampl

es o

f out

com

e an

d pr

edic

tive

valu

e. T

hey

are

liste

d in

no

parti

cula

r ord

er; a

ny re

petit

ions

hav

e be

en re

mov

ed.

•M

ost a

ssay

s ha

ve b

een

valid

ated

and

hav

e pe

rform

ed w

ell a

s bi

omar

kers

. Som

e as

says

ha

ven'

t wor

ked

so w

ell e

.g. p

arat

hyro

id h

orm

one.

•

For t

hrom

bin-

antit

hrom

bin

com

plex

es, s

ucce

ssfu

l val

idat

ion

of th

is a

ssay

and

use

of t

his

biom

arke

r ser

ves

as a

mea

sure

of i

n vi

vo c

oagu

latio

n ac

tivat

ion

and

refle

cts

thro

mbi

n fo

rmat

ion.

•

Gen

eral

ly g

ood,

but

larg

e st

anda

rd d

evia

tion

of m

easu

rem

ents

. •

Thyr

oid

horm

one

biom

arke

rs: c

halle

ngin

g to

set

-up

and

valid

ate.

Goo

d in

dica

tor o

f thy

roid

to

xici

ty.

•Tr

opon

in I:

cha

lleng

ing

to s

et-u

p an

d va

lidat

e. G

ood

indi

cato

r of c

ardi

ac to

xici

ty.

7: W

HIC

H C

OM

MER

CIA

LLY

AVA

ILA

BLE

ASS

AY K

ITS

DO

YO

U U

SE F

OR

MEA

SUR

ING

TH

E PA

RA

MET

ERS?

(O

THER

TH

AN

RO

UTI

NE

CLI

NIC

AL

BIO

CH

EMIS

TRY

AN

ALY

TES)

PLE

ASE

DES

CR

IBE

AN

Y PR

OB

LEM

S W

ITH

D

EVEL

OPI

NG

/VA

LID

ATIN

G A

MIN

IPIG

SPE

CIF

IC A

SSAY

. R

espo

nden

ts re

plie

d w

ith a

num

ber o

f com

mer

cial

kits

whi

ch h

ave

been

use

d. T

hey

are

liste

d in

no

par

ticul

ar o

rder

; any

repe

titio

ns h

ave

been

rem

oved

. •

SE

RU

M A

MY

LOID

AS

SAY

kit

(AbC

ys S

A).

•

Sie

men

s In

sulin

TK

IN1.

•

LIA

ISO

N S

angt

ec.

•

ELI

SA

kits

: the

pro

blem

is th

e la

ck o

f com

mer

cial

Qua

lity

Con

trol,

the

lack

of p

ositi

ve c

ontro

l, th

e lo

w s

ensi

tivity

, the

abs

ence

of s

hare

d hi

stor

ical

dat

a.

•B

TI m

id-ta

ct h

uman

ost

eoca

lcin

BT-

480.

•

LIA

ISO

N S

angt

ec.

•Q

uide

l Cor

p B

AP

8012

.

•N

eoge

n H

ista

min

e 40

9010

. •

Whe

re p

ossi

ble

spec

ies-

spec

ific

com

mer

cial

ly a

vaila

ble

kits

hav

e be

en u

sed;

if n

ot p

ossi

ble,

hu

man

or m

ulti-

spec

ies

kits

are

ofte

n us

ed, a

nd v

alid

atio

ns o

f sai

d w

ould

be

desc

ribed

as

fit

for p

urpo

se.

8: P

LEA

SE C

OM

MEN

T O

N T

HE

PRO

SPEC

TS F

OR

TH

E D

EVEL

OPM

ENT

OF

BIO

MA

RK

ERS

OF

TOXI

CIT

Y IN

TH

E M

INIP

IG.

Res

pons

es a

re li

sted

in n

o pa

rticu

lar o

rder

; any

repe

titio

ns h

ave

been

rem

oved

. •

Hop

eful

ly p

rosp

ects

are

goo

d as

mor

e an

d m

ore

com

pani

es s

eem

to b

e in

tere

sted

in u

sing

th

e m

inip

ig a

s a

non-

rode

nt u

sed

in s

afet

y as

sess

men

t. R

espo

nden

t has

see

n on

ly o

ne a

ssay

fa

il.

•Th

e de

velo

pmen

t of n

ew c

ardi

ac a

nd k

idne

y bi

omar

kers

cou

ld b

e of

gre

at h

elp

in th

e no

n-cl

inic

al to

xico

logi

cal s

tudy

. •

For t

he im

med

iate

futu

re w

e w

ill fo

llow

the

know

n m

ains

tream

toxi

citie

s in

dev

elop

ing

pred

ictiv

e bi

omar

kers

. Wha

t wou

ld b

e id

eal i

s a

chea

p to

ol, s

uch

as c

linic

al c

hem

istry

m

etab

olom

ics,

whe

re k

ey s

impl

e ch

ange

s ar

e fla

gged

to g

uide

dev

elop

men

t of f

urth

er

biom

arke

r ana

lysi

s.

•

Onc

e av

aila

ble

they

will

pro

vide

a c

onve

nien

t sol

utio

n, s

avin

g tim

e an

d ef

fort.

9: W

HAT

DO

YO

U F

EEL

AR

E TH

E C

HA

LLEN

GES

WE

FAC

E IN

DEV

ELO

PIN

G P

RED

ICTI

VE B

IOM

AR

KER

S O

F TO

XIC

ITY

IN T

HE

MIN

IPIG

? R

espo

nses

are

list

ed in

no

parti

cula

r ord

er; r

epet

ition

s ha

ve b

een

rem

oved

. •

Spe

cies

spe

cific

ity.

•To

dev

elop

bio

mar

ker w

ith g

ood

pred

ictiv

ity a

nd s

uita

ble

for t

rans

latio

nal t

oxic

olog

y.

•S

till a

n ev

olvi

ng s

cien

ce -

the

mor

e w

e ap

proa

ch in

vest

igat

ion

of b

iom

arke

rs in

min

ipig

s th

e m

ore

we

will

hav

e to

offe

r and

add

to th

e w

ider

sci

entif

ic a

nd c

ontra

ct re

sear

ch c

omm

unity

. •

If th

e fu

ture

use

dim

inis

hes

that

will

redu

ce in

cent

ive

to d

evel

op n

ew b

iom

arke

rs.

10: D

O Y

OU

HAV

E A

NY

SUG

GES

TIO

NS

FOR

OVE

RC

OM

ING

TH

E C

HA

LLEN

GES

? S

ugge

stio

ns a

re li

sted

in n

o pa

rticu

lar o

rder

; rep

etiti

ons

have

bee

n re

mov

ed.

•P

ersu

ade

kit m

anuf

actu

rers

to p

rovi

de s

peci

es s

peci

fic k

its.

•S

hare

info

rmat

ion

and

expe

rienc

e in

ord

er to

iden

tify

and

cros

s-qu

alify

new

saf

ety

biom

arke

rs.

•A

dver

tisin

g as

muc

h as

pos

sibl

e w

hen

the

deve

lopm

ent o

f a n

ew b

iom

arke

r is

achi

eved

.

Tabl

e 2:

Sum

mar

y of

ope

ratio

nal s

tand

ards

for c

oagu

latio

n,

bioc

hem

istr

y an

d ur

inal

ysis

. Th

e de

tails

of t

he q

ualit

ativ

e qu

estio

ns a

re re

view

ed. K

ey p

oint

s ar

e th

e la

ck o

f spe

cies

spe

cific

ass

ays/

kits

, new

car

diac

and

ki

dney

bio

mar

kers

of t

oxic

ity a

re s

ough

t afte

r, as

are

met

hods

to

help

ens

ure

qual

ity c

ontro

l inc

ludi

ng p

ositi

ve c

ontro

ls. S

harin

g of

in

form

atio

n (in

clud

ing

data

) on

new

bio

mar

kers

, as

wel

l as

adve

rtisi

ng th

eir e

xist

ence

is p

oint

ed o

ut a

s pi

vota

l to

mov

e fo

rwar

d th

is fi

eld

in a

n ef

ficie

nt m

anne

r. Th

is p

ath

may

wel

l als

o be

the

best

way

to p

ersu

ade

kit m

anuf

actu

rers

to d

evel

op m

inip

ig

spec

ific

kits

so

the

tech

nolo

gies

bec

ome

avai

labl

e in

a c

onve

nien

t an

d co

st e

ffect

ive

way

Pa

ram

eter

N

on-G

LP

GLP

N

o U

rinal

ysis

, dep

osits

afte

r ce

ntrif

ugat

ion

2%

80%

18

%

Urin

alys

is

11%

63

%

26%

B

lood

Bio

chem

istry

12

%

48%

39

%

Coa

gula

tion

6%

27%

67

%

Que

stio

ns 1

-3 a

re c

over

ed fi

rst,

follo

wed

by

key

poin

ts id

entif

ied

in th

e qu

alita

tive

ques

tions

(Q4-

10).

Exa

mpl

es o

f bio

mar

kers

from

the

publ

ishe

d lit

erat

ure

are

pres

ente

d (T

able

1) p

oint

ing

the

read

er to

war

ds

perti

nent

pub

licat

ions

dis

cuss

ing

biom

arke

r val

idat

ion

from

an

indu

stry

and

regu

lato

ry p

ersp

ectiv

e.

Am

ong

coag

ulat

ion,

bio

chem

istry

and

urin

alys

is

ther

e is

a c

lear

tren

d (T

able

2) t

hat t

hese

inve

stig

atio

n ar

e no

t con

duct

ed u

nder

Non

-GLP

con

ditio

ns, t

hat i

s to

say

, the

y ar

e ei

ther

per

form

ed u

nder

GLP

or n

ot a

t al

l. Th

is c

orre

late

d w

ith th

e de

mog

raph

ics

of s

urve

y re

spon

dent

s be

ing

rela

ted

pred

omin

antly

to

phar

mac

eutic

al d

evel

opm

ent w

here

GLP

is th

e no

rm.

Urin

alys

is o

f dep

osits

afte

r cen

trifu

gatio

n ha

s th

e hi

ghes

t pro

porti

on o

f GLP

ope

ratio

nal s

tand

ards

, fo

llow

ed b

y ur

inal

ysis

. If t

his

diffe

renc

e is

bec

ause

the

anal

ytic

al m

etho

ds a

pplie

d ar

e ve

ry s

tand

ardi

zed

and

easy

to u

se; w

heth

er th

is is

indi

cativ

e of

a ti

ck-b

ox-

appr

oach

, or,

if th

ere

is a

n un

derly

ing

scie

ntifi

c ra

tiona

le to

exp

lain

this

pat

tern

is u

ncle

ar.

Giv

en th

e ea

se w

ith w

hich

blo

od c

an b

e ob

tain

ed

(com

pare

d to

e.g

. urin

e) a

nd th

e nu

mbe

r of

para

met

ers

whi

ch c

an b

e m

onito

red

in th

is m

atrix

it is

no

tew

orth

y, th

at le

ss th

an h

alf (

48%

) of a

ll bl

ood

bioc

hem

istry

par

amet

ers

are

perfo

rmed

und

er G

LP.

Mat

rix e

ffect

s by

ser

um/p

lasm

a/w

hole

blo

od c

an m

ake

assa

y de

velo

pmen

t mor

e ch

alle

ngin

g m

ay p

artly

be

to

reas

on fo

r thi

s lo

w n

umbe

r as

esta

blis

hing

and

va

lidat

ing

new

met

hods

is c

ostly

it is

don

e on

ly w

hen

abso

lute

ly n

eces

sary

; dev

elop

ing

a ne

w b

iom

arke

r is

driv

en b

y ne

ed-to

-do,

rath

er th

an n

ice

to k

now

. A

noth

er e

xpla

natio

n m

ay a

lso

be th

at th

e de

velo

pmen

t was

aba

ndon

ed b

ecau

se o

f hur

dles

, e.g

. te

chni

cal,

whi

ch c

ould

not

be

over

com

e.

With

rega

rds

to c

oagu

latio

n pa

ram

eter

s th

ere

appe

ars

to b

e a

stan

dard

set

use

d in

min

ipig

stu

dies

, as

all

resp

onde

nts

anal

ysed

fibr

inog

en, a

ctiv

ated

pa

rtial

thro

mbo

plas

tin ti

me,

and

pro

thro

mbi

n tim

e un

der G

LP.

For t

he g

roup

s ab

ove

a de

taile

d fo

llow

-up

with

in

divi

dual

resp

onde

nts

may

she

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Organophosphorus (OP) insecticide self-poisoning is responsible for about one-quarter of global suicides. Treatment classically focuses on the fact that OP compounds inhibit the enzyme acetyl-cholinesterase, causing overstimulation of cholinergic receptors in central and autonomic nervous systems and in the neuromuscular junction. Poisoned patients die from respiratory failure. However, drugs that reactivate the acetyl-cholinesterase enzyme have been found to provide little benefit to OP-poisoned humans.

Part of the reason may be that humans ingest formulated ‘emulsifiable concentrate’ (EC) pesticides, containing solvents and surfactants as well as the OP active ingredient (AI), rather than pure OP AI. The authors of this paper therefore studied the role of solvent co-formulants in OP toxicity, developing a novel Göttingen minipig model of agricultural OP poisoning with the widely used insecticide dimethoate. This species was selected based on the similarity of its cardiorespiratory physiology and drug metabolism with humans and on its size, which allows collection of multiple blood samples and the use of human monitors.

Gottingen minipigs under terminal anaesthesia were orally poi-soned with a clinically relevant dose of the agricultural EC formula-tion of dimethoate, the dimethoate active ingredient (AI) alone, the solvents cyclohexanone and xylene, or a control. The severity of poisoning was recorded by monitoring the heart, lung and nerve function and by measuring the poisons’ effects in the blood.

Poisoning with agricultural dimethoate EC40, but not saline control, caused respiratory arrest within 30 minutes, very low blood pressure, and nerve dysfunction, that was highly similar to human poisoning. Mean arterial lactate concentration rose to 15.6 [1.1] in poisoned pigs compared to 1.4 [0.4] in control pigs. By contrast, only moderate toxicity resulted from poisoning with dimethoate AI alone or the major solvent cyclohexanone, compared to dimethoate EC40. Combining dimethoate AI with cyclohexanone reproduced

severe poisoning. These results indicated that the solvent co-formulant cyclohexanone was essential for full toxicity. This has not previously been considered.

The main manufacturer of dimethoate, Cheminova, then pro-vided an experimental formulation of dimethoate EC that did not contain cyclohexanone. This showed markedly less mammalian toxicity in this model than the usual agricultural formulation.

These results indicate that solvents play a crucial role in dimethoate toxicity. If companies were to reformulate agricultural dimethoate and other toxic OP insecticides, they would probably be much less toxic to humans. Safer formulations of dimethoate and other OP insecticides should rapidly reduce global suicide numbers. Further studies are required to determine how to change the formulations to increase human safety while maintaining agri-cultural efficacy.

1 Clinical Pharmacology Unit and 7 Endocrinology Unit, University/BHF Centre for Cardiovascular Science, 3 Department of Anaesthesia, and 4 Veterinary Pathology Unit, Royal (Dick) School of Veterinary Sciences, and 5 Roslin Institute, University of Edinburgh, UK 2 National Poisons Information Service - Edinburgh, Royal Infirmary, Edinburgh, UK 6 Centre for Statistics in Medicine, Wolfson College, University of Oxford, UK 8 Bundeswehr Institute of Pharmacology and Toxicology, Munich, Germany 9 Medical Toxicology Centre and Institute of Neuroscience, Newcastle University, Newcastle upon Tyne, UK

Reformulating Agricultural Organophosphorus Pesticides to Reduce Global Suicide Rates: a Göttingen minipig model

Michael Eddleston,1,2 Jonathan M Street,1 Ian Self,3 Adrian Thompson,1

Tim King,5 Nicola Williams,6 Gregorio Naredo,7 Kosala Dissanayake,1 Franz Worek,8 Harald John,8 Sionagh Smith,3,4 Horst Thiermann,8 John B Harris,9 and R Eddie Clutton.3


AbstractThe first transgenic pigs were produced by the microinjection of foreign DNA into zygotic pronuclei in 1985. Since then, the me-thodological repertoire for porcine transgenesis was expanded to somatic cell nuclear transfer, lentiviral transgenesis and, recently, cytoplasmic plasmid injection. The major impact of transgenic pigs and minipigs took place in the fields of humanised pig models and biomedical disease models, whereas agricultural applications did not find broad acceptance. The recent release of the porcine whole genome sequence and parallel developments of highly spe-cific enzymes and RNAs now make it possible to perform precise genetic modifications and fully exploit the advantages of this large animal model. We anticipate that genetically modified pigs and minipigs will increasingly complement the commonly used small-animal models in biomedical research, since several aspects of disease progression, physiology, metabolism and aging cannot properly be mirrored in small-animal models.

introductionThe production of transgenic pigs is labour-intensive and cost-intensive and depends on advanced techniques in molecular biolo-gy and the micromanipulation of gametes and zygotes. At present, progress in reproductive techniques and gene-transfer methods has allowed targeted modifications of the porcine genome (glos-sary box), albeit the overall success rates are still low (Clark and

Whitelaw, 2003; Niemann and Kues, 2007; Robl et al., 2007). A bottleneck for porcine transgenesis is the lack of authentic pluri-potent stem cells that are suitable for blastocyst complementation experiments (Brevini et al., 2008; Kues et al., 2010a). The semi-nal development of induced pluripotent stem cells (iPS) in mice and humans (Takahashi and Yamanaka, 2006) provides a new approach to this end. The results of the first attempts to generate porcine iPS cells were published recently (Esteban et al., 2009; Wu et al., 2009; Ezashi et al., 2009), yet the potential of current porcine iPS cells to contribute to chimera formation seems to be limited (West et al., 2010).

This paper briefly discusses the current progress of transgenic pig models for biomedical research. Comprehensive overviews about transgenic pigs and livestock are available elsewhere (Clark and Whitelaw, 2003; Robl et al., 2007; Kues and Niemann, 2011; Whyte and Prather, 2011).

basic and biomedical applications of transgenic pigsIn the last few years, an expanded methodological repertoire for porcine gene transfer has been developed (Table 1), resulting in an increasing number of transgenic approaches (Whyte and Prather, 2011). At least 90% of genetically modified pigs are generated for biomedical studies (Fig. 1A). Sequencing and annotation of the porcine genome are important milestones for accelerating the

Recent Progress of Transgenic Pig Models for Biomedicine and Pharmaceutical ResearchAuthors: Wiebke Garrels, Heiner Niemann. Corresponding author: Wilfried A. Kues

key words: Domestic animals, disease model, humanised, genome, large animal model

Fig.1. Increasing scientific interest in transgenic pig modelsA) Scientific interest in porcine transgenesis. Depicted are the numbers of total citations per year, as extracted from Thomson Reuters ISI Web of Knowledge for topic search terms “transgenic” and “pig model”. B) Transgenic boar exhibiting ubiquitous expression of the Venus fluorophor gene (Garrels et al., 2011). The boar is shown under specific excitation condi-tions of Venus, in front of the boar an autofluorescent toy is visible. Almost all somatic and germ cells are fluorescent.


generation of transgenic models, even if the porcine genome assembly still has gaps (annotated porcine genome data can be found at: www.ensembl.org and www.pubmed.org). Since pig and minipig physiology, anatomy, pathology, genome organisa-tion, body weight and life span are more similar to humans than are rodents, the domesticated pig represents a more appropriate biomedical model (Table 2).

For certain biomedical therapies, such as xenotransplanta-tion (transplantation of organs from one species to another (e.g. porcine-to-human)), transgenic pigs are the only reasonable spe-cies (Niemann and Kues, 2003). Xenotransplantation seems to be one option for closing the widening gap between demand and availability of appropriate human organs (Yang and Sykes, 2007). The prerequisites for potential porcine–human xenotransplanta-tion are: (i) overcoming immunological hurdles; (ii) preventing the transmission of porcine pathogens to human recipients; and (iii) the compatibility of porcine organs with human physiology.

The suppression of hyperacute rejection of porcine xenografts has been achieved by transgenic expression of human regula-tors of complement activity (RCA) (Tucker et al., 2002) and a gene knockout of the porcine alpha, 1,3-galactosyltransferase gene (Dai et al., 2002; Lai et al., 2002; Phelps et al., 2003). Maximal survival rates of up to 3–6 months have been achieved

with porcine alpha-galactosyltransferase knockout organs (kidney or heart) transplanted to baboons (Kuwaki et al., 2005; Yamada et al., 2005).

Extensive research has been conducted to reduce the risk of porcine endogenous retrovirus (PERV) transmission to human patients (Switzer et al., 2001; Irgang et al., 2003). RNA interfe-rence (RNAi) is a promising method for knocking down the PERV expression. RNAi is based on small RNAs, either small interfering RNA (siRNA) or short hairpin RNAs (shRNA). In the cytoplasm, small RNA molecules are incorporated into an RNA-induced silencing complex (RISC) and targets binding to a complementary transcript sequence, resulting in mRNA degradation (Plasterk, 2002; Dallas and Vlassow, 2006). The efficacy of RNAi for redu-cing PERV expression has been demonstrated in cloned piglets (Dieckhoff et al., 2008; Ramsoondar et al., 2009).

For several approaches, a conditional gene expression is desi-rable over a constitutive transgenic expression. Initial animal mod-els carrying the first generation of conditional promoter elements suffered from high basal-expression levels and pleiotropic effects (Miller et al., 1989). Recent expression systems responsive to exogenous tetracycline resulted in more tightly controlled expres-sion. In pigs, a tetracycline-controlled transgenic expression was achieved with a bicistronic expression cassette (Kues et al., 2006)

Table 1. Progress of technologies for transgenesis in pigs and minipigs

develoPment StRAteGy RefeRenCe

First transgenic pigs PNI Hammer et al., 1985

Somatic cloning of transgenic pigs SCNT using transgenic donor cells Park et al., 2001

Sperm-mediated gene transfer SMGT Lavitrano et al., 2002; Chang et al., 2002

Knock-out in pigs Homologous recombination in somatic cells and SCNT

Dai et al., 2002; Lai et al., 2002

Homozygous gene knockout Homozygous knockout Phelps et al., 2003

Lentiviral transgenesis Perivitelline injection of lentiviruses Hofmann et al., 2003; Whitelaw et al., 2004

SMGT / ICSI combination SMGT and ICSI Kurome et al, 2006

Conditional transgenesis PNI Kues et al., 2006

Episomal transgenesis SMGT and episomal plasmid Manzini et al., 2006; Giovannoni et al., 2010

Gene knock-down Knock-down of PERV genes with siRNA and SCNT

Dieckhoff et al., 2008; Ramsoondar et al., 2009

Transposon transgenesis Sleeping Beauty transposition in zygotic genome by CPI

Garrels et al., 2010; Kues et al., 2010b

Transposon transgenesis Sleeping Beauty transposition in somatic cells and SCNT

Jacobsen et al., 2011; Carlson et al. 2011

Targeted gene knockout Zinc finger nuclease-catalysed gene deletion in primary cells and SCNT

Whyte et al., 2011; Yang et al., 2011; Hauschild et al., 2011

Targeted integration Recombination-mediated cassette exchange in primary cells and SCNT

Garrels et al., 2011

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that was designed to give ubiquitous expression of human RCAs. Crossbreeding of lines with two cassettes was necessary to over-come epigenetic silencing and to achieve tetracycline-sensitive RCA expression.

Transgenic pigs have been shown to mimic human diseases such as atherosclerosis, non-insulin-dependent diabetes, cystic fibrosis, cancer, ophthalmological and neurodegenerative disorders (Kues and Niemann, 2004; Kragh et al., 2010; Rogers et al., 2008; Yang et al., 2010; Luo et al., 2011). An important exam-ple is the minipig cystic fibrosis model, which develops disease phenotypes that are highly similar to human patients (Rogers et al., 2008), whereas transgenic mouse models failed to exhibit lung, pancreatic and intestinal obstructions. Huntington’s disease is a neurodegenerative disorder characterised by the expression of mutated huntingtin with expanded polyglutamine tracts. The misfolded protein accumulates in neurons and is suspected of trig-gering apoptosis. Whereas genetic mouse models often failed to replicate overt neurodegeneration and apoptosis, a minipig model expressing the N-terminal huntingtin with a polyglutamine tract seems to do so (Yang et al., 2010).

Truncation mutations in the elongation of a very long-chain fatty-acids-4 (ELOVL4) gene cause macular dystrophy. Photoreceptor topography in the pig retina is more similar to that in humans as it includes cone-rich, macula-like area centralis, whereas mice lack a macular. Transgenic pigs expressing disease-causing ELOVL4 mutations were generated by PNI and SCNT (Sommer et al., 2011). A detailed analysis showed photoreceptor loss, disorga-nised inner and outer segments, and diminished electroretinogra-phy responses, suggesting that the transgenic pigs mirror macular degeneration and provide a unique model for therapeutic interven-

tion. Recently, the first immunodeficient pigs were cloned by SCNT (Mendicino et al., 2010; Ramsoondar et al., 2011), promising to serve as large-animal models for cell transplantation experiments.

Conventional gain-of-function transgenesis is based on random integration of the transgene at sites of spontaneous double-strand breaks of chromosomal DNA. The frequency of DNA double-strand breaks at a defined locus can be considerably increased by introducing specifically designed endonuclease enzymes (Urnov et al., 2005; Arnould et al., 2007). The artificial endonucleases are based on the DNA recognition sites of zinc finger transcription factors, meganuclei or transcription factor like elements (TALE), and they can be designed to bind highly specifically to a single, predetermined sequence in the genome. Double-strand break-repair pathways often create small deletions and, thus, designed endonucleases allow efficient gene knockouts. The proof-of-prin-ciple to generate knockout pigs by synthetic zinc finger nucleases has been demonstrated by the inactivation of enhanced green fluorescent protein (EGFP), peroxisome proliferator-activated receptor (PPAR gamma) and alpha-galactosyltransferase (Whyte et al., 2011; Yang et al., 2011; Hauschild et al., 2011) in primary somatic cells and the subsequent use of knockout cells for SCNT, respectively. Thus current lack of authentic porcine ES cells can be circumvented for the purpose of generating knockout pigs.

DNA-based transposons are mobile genetic elements that move in the genome via a “cut-and-paste” mechanism. Most DNA transposons are simply organised: they encode a transposase protein flanked by inverted terminal repeats (ITRs), which carry transposase binding sites, and it has been possible to separate the transposase coding sequence from ITR sequences. Any DNA flanked by ITRs will be recognised by the transposase and will

Table 2. Selected pig and minipig models for biomedicine and pharmaceutical research

model Comment RefeRenCe

Xenotransplantation knockout of alpha-galactosyltransferase Lai et al., 2002; Dai et al., 2002

Xenotransplantation expression of tumour necrosis factor ligand Klose et al., 2005

Xenotransplantation expression of human leukocyte antigen Weiss et al., 2009

Xenotransplantation PERV-knock down Dieckhoff et al., 2008

Xenotransplantation expression of human thrombomodulin Petersen et al., 2009

Xenotransplantation expression of human A20 (anti-apoptotic gene) Oropeza et al., 2009

Cystic fibrosis pig knockout of cystic fibrosis transmembrane conductance receptor Rogers et al., 2008

Diabetes model expression of mutated hepatocyte nuclear factor-1 Umeyama et al., 2009

Diabetes model expression of mutated insulin 2 Renner et al., 2010

Immunodeficient pig knockout of light chain Ramsoondar et al., 2010

Immunodeficient pig knockout of joining gene cluster Mendicino et al., 2010

Huntington model expression of mutated huntingtin with polyglutamine tract Yang et al., 2010

Alzheimer model expression of mutated human amyloid precursor protein Kragh et al., 2010

Breast cancer knockout of BRCA1 gene Luo et al., 2011

Macular degeneration introduced deletion in ELOVL4 gene Sommer et al., 2011


ReCent PRoGReSS of tRAnSGeniC PiG modelS foR biomediCine And PhARmACeutiCAl ReSeARCh

become enzymatically integrated into nuclear DNA. In a two-component system, the transposon is integrated solely by the trans-supplementation activity of transposase. The first transposon sufficiently active for use in vertebrates was the Sleeping Beauty (SB) transposon (Ivics et al., 1997; Clark et al., 2007). Many drawbacks of classical transgenic methods can be overcome by transposition-catalysed gene delivery, which increases the efficiency of chromosomal integration and facilitates single-copy (monomeric) insertion events. An additional advantage of trans-poson-catalysed transgenesis is that the integration of monomeric transgene units is directed to accessible euchromatic regions. Transposon transgenic pigs have been generated (Kues et al., 2010b; Garrels et al., 2011) by CPI (Iqbal et al., 2009), as well as by SCNT (Jakobsen et al., 2010; Carlson, 2011; Garrels, 2011). Ubiquitous expression of a fluorescent Venus protein, a derivative of the commonly used EGFP, was found in somatic and germ cells (differentiated spermatozoa) in own experiments (Fig. 1B, Garrels et al., 2011) for all integrations sites, strongly supporting the hypothesis that transposase preferentially integrates DNA into euchromatic regions. The robust transgenic expression of Venus is strictly copy-number dependent and facilitates cell-tracking experi-ments in cell-therapy approaches. The identification of integrations sites revealed that most transposon integration sites were found in intergenic regions of the porcine genome (Fig. 2). This approach made it possible to identify loci, which are suitable for transgenic

expression. Importantly, transposon-tagged loci can be read-dressed by recombination-mediated cassette exchange (RMCE) in cell culture. Via SCNT, the RMCE cells can be used to generate vital piglets carrying a targeted integration into a “safe harbour” locus (Garrels et al., 2011).

Since integrated transposons can be remobilised in the pre-sence of a transposase enzyme, these animals can provide the basis for performing whole genome mutagenesis screens in the pig. For the SB transposon, the phenomenon of local hopping after mobilisation has been described. The majority of secondary integrations take place at a distance of up to 5 megabases from the original integration. Figure 2 depicts one integration site on the gene-rich X chromosome. The neighbouring porcine genes are the von Hippel-Lindau binding gene (VBP1) and a novel gene, both about 10,000 base pairs away from the integration site. After mobilisation, the integration site can be screened for integration events in neighbouring genes, such as the VBP1. The VBP1 gene is of potential interest as an animal model, and the gene product is assumed to form a complex with the von Hippel-Lindau tumor sup-pressor (VHL). The von Hippel-Lindau syndrome is a dominantly inherited cancer syndrome predisposing carriers to several malig-nant and benign tumours. Thus, transposon transgenic pigs can be employed for performing unbiased and biased mutagenic events. It is anticipated that mutagenic screens with more advanced con-structs will be applied in the near future.

Fig.2. Applications of transposon transgenesis Depicted is one integration site of a Venus transposon on chromosome X (red arrow). By means of targeted cassette exchange (via the Cre/loxP system), the Venus reporter gene can be replaced by a gene of choice (I), thus introducing a transgene in a pretested locus (Garrels et al., 2011) suitable for expression, and avoiding integration into heterochromatic regions or inser-tional mutagenesis. Alternatively, by supplying the SB transposase in trans, a remobilisation (II) of the transposon can be induced. The annotated pig genome sequence was extracted from www.ensembl.org.

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ConclusionsMethodological improvements for gene transfer into the pig genome and a rapidly increasing list of biomedical pig models have been developed in recent years. Together with more accurate genome data and highly specific designed enzymes and RNAs, precise genetic modifications have become feasible. It is anticipa-ted that authentic pluripotent cells of the pig will be generated in the near future. Thus, porcine transgenesis will become a routine tool for generating relevant humanised porcine models. The most obvious application of transgenic pigs will be as disease models and biomedical therapies, which are not well-reflected in small rodent models. The progress expected in porcine transgenesis (increased success rates and decreasing costs), however, will make the pig an attractive complementary model for advanced approaches in biomedical research.

AcknowledgmentsThe expert technical support of Ms S. Holler, Ms Barg-Kues, Ms Herrmann and Ms Ziegler, and the financial support of the Deutsche Forschungsgemeinschaft (DFG) are gratefully acknow-ledged.

Conflicts of interestThe authors declare no conflicts of interest.

Wiebke Garrels, Heiner NiemannFriedrich-Loeffler-InstituteMariensee, DE-31535 Neustadt, Germany

Wilfried A. KuesFriedrich-Loeffler-InstituteInstitute of Farm Animal GeneticsMariensee, DE-31535 Neustadt, Germany0049 – (0)5034 871 1200049 – (0)5034 871 101

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Petersen B, Ramackers W, Tiede A, Lucas-Hahn A, Herrmann D, Barg-Kues B, Schuettler W, Friedrich L, Schwinzer R, Winkler M, Niemann H. 2009. Pigs transgenic for human thrombomodulin have elevated production of activated protein C. Xenotransplantation. 2009 Nov-Dec;16(6):486-95.

Phelps CJ, Koike C, Vaught TD, Boone J, Wells KD, Chen SH, Ball S, Specht SM, Polejaeva IA, Monahan JA, Jobst PM, Sharma SB, Lamborn AE, Garst AS, Moore M, Demetris AJ, Rudert WA, Bottino R, Bertera S, Trucco M, Starzl TE, Dai Y, Ayares DL (2003) Production of alpha 1,3-galactosyltransferasedeficient pigs. Science 299: 411–414

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Ramsoondar J, Vaught T, Ball S, Mendicino M, Monahan J, Jobst P, Vance A, Duncan J, Wells K, Ayares D. Production of transgenic pigs that express porcine endogenous retrovirus small interfering RNAs. Xenotransplantation. 2009 May-Jun;16(3):164-80.

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ellegaard Göttingen minipigs dvds available:

• The Göttingen Minipig – Handling and dosing This DVD contains an English, German and

French version. A unique tool for those who work with the Göttingen Minipig.

Price each ¤ 90 excl. shipping and handling.

• The Göttingen Minipig – Histology This DVD allows users to familiarize themselves with the normal histology of the Göttingen Minipig. Price each ¤ 65 excl. shipping and handling.

Please contact us for further information at [email protected] or phone +45 5818 5818.


The annual meeting of the Minipig Research Forum will take place on 28-29 November 2011. This year’s venue is Lindner Hotel & Residence Main Plaza in Frankfurt (Germany), situated just 6 km from Frankfurt Airport.

The main topics for the meeting will be Juvenile and Embryofoetal Development Studies and neurobehavioural assessment in minipigs.

By joining the meeting you will have a unique opportunity to meet minipig users from all over Europe. At the previous meetings we have experienced that besides listening to and

Register now

for the annual meeting of

the minipig Research forumlearning from the many presenters, the par-ticipants exploit this opportunity to exchange minipig knowledge, ideas and experiences.

You can now register for the meeting at the website www.minipigresearchforum.org

Besides access to an interesting two-day pro-gramme, the ¤300 registration fee includes lunch and meeting dinner on the first day and take-away lunch on day 2.

For further information please contact the Minipig Research Forum: [email protected]

book discusses the regulatory uses of minipigs in the evalua-tion of human and veterinary pharmaceuticals, medical devices, and other classes of xenobiotics. It describes features of normal minipig health, normal laboratory values, and common diseases. It also carefully elucidates ethical and legal considerations in the breeding, housing, and trans-port of minipigs. The result is an all-inclusive and up-to-date manual about the experimental uses of the minipig that describes ‘How to’ and ‘Why’ and ’What to expect under normal circumstances’, combining enthusiasm and experience with a critical assessment of the minipig’s values and potential problems.

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The

Minipigin

biomedical ResearchThe Minipig in Biomedical Research is a comprehensive resource for research scientists concerning the potential and use of the minipig in basic and applied biomedical research and in the devel-opment of drugs and chemicals. Written by acknowledged experts in the field and drawing on the authors’ global contacts and experi-ence with regulatory authorities and the pharmaceutical industry and other industries, this accessible manual covers a wide range of topics including the biological, scientific, and practical uses of the minipig in the laboratory. It also covers the minipig’s origins, anatomy, genetics, immunology, and physiology, as well as minipig welfare, health, and husbandry; practical dosing and examination procedures; surgical techniques; and all areas of toxicity testing and the uses of the minipig as a disease model. Regulatory aspects of minipig use are also considered.

The reader will find extensive theoretical and practical information in the pharmacology, ADME and toxicology chapters, which will help scientists and managers in deciding which species to use in basic research; drug discovery and pharmacology; and toxicology studies of chemicals, biotechnology products and devices. The

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