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
Thank youPLEASE NOTE: Ellegaard Göttingen Minipigs A/S will not release or give away a sub-scriber’s e-mail address, name or any other information provided.
If you receive
Ellegaard Göttingen
Minipigs A/S
Newsletter by mail ...
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
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
6 Newsletter 36 Autumn 2011
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
7Newsletter 36 Autumn 2011
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
8 Newsletter 36 Autumn 2011
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
9Newsletter 36 Autumn 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.”
10 Newsletter 36 Autumn 2011
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
11Newsletter 36 Autumn 2011
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
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
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. 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
d lig
ht o
n th
e re
ason
s fo
r the
obs
erve
d di
strib
utio
ns a
nd a
ppar
ent
diffe
renc
es.
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
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Ichi
nose
, T.,
Mill
er, M
.G.,
Shi
bam
oto,
T.,
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. 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.]
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. 14,
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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
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16 Newsletter 36 Autumn 2011
The article is still under review
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
17Newsletter 36 Autumn 2011
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.
18 Newsletter 36 Autumn 2011
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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19Newsletter 36 Autumn 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
20 Newsletter 36 Autumn 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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21Newsletter 36 Autumn 2011
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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Kues WA, Schwinzer R, Wirth D, Verhoeyen E, Lemme E, Herrmann D, Barg-Kues B, Hauser H, Wonigeit H, Niemann H (2006) Epigenetic silencing and tissue independent expression of a novel tetracycline inducible system in double-transgenic pigs. FASEB Journal Express doi: 10.1096/fj.05-5415fje; printed short version: FASEB J 20: 1200-1202
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Lai L, Kolber-Simonds D, Park KW, Cheong HT, Greenstein JL, Im GS, Samuel M, Bonk A, Rieke A, Day BN, Murphy CN, Carter DB, Hawley RJ, Prather RS (2002) Production of a1, 3-galacto-syltransferase knockout pigs by nuclear transfer cloning. Science 295:1089-1092
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23Newsletter 36 Autumn 2011
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ellegaard Göttingen minipigs dvds available:
• The Göttingen Minipig – Handling and dosing This DVD contains an English, German and
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24 Newsletter 36 Autumn 2011
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