Ellegaard_Minipigs_Newsletter_52

EllegaardGoettingenMinipigs

52 AUTUMN 2018

NEWSLETTER

••

••

••

••

••

••

Towards 3Rs: Multiplex protein profiling from minipig,

cyno, dog and rat samples, to get the most out of preclinical

safety studies page 3

Experience with mixed groups of intact female and

castrated male Göttingen Minipigs for pharmacokinetic

studies in drug discovery research – summary of

behavioral observations and collected back-ground data page 6

Anti-cancer drug development: Comparison of toxicity

in Göttingen Minipigs and mouse page 10

Body surface area measurement in Göttingen Minipigs

using a computed tomography scanner page 14

The Göttingen Minipig for the study of buccal and sublingual

products page 17

Update from our partners pages 24-26

••

New scientific publications on the Göttingen Minipig page 27

••

Join the CONFIRM Initiative page 28

See

where you

can meet us

in 2018

Clean pigs

for clear results

page 28


Dear

Reader

In Denmark, summertime this year has been

amazing with high temperatures and the sun

shining from early morning almost every day;

something quite unusual for the Scandinavian

part of the world. I hope that like our employees

you have been able to take some time off to enjoy

the summer.

The recent Minipig Research Forum 2018 in Barcelona was a

remarkable success, and I am pleased to report that a group of

attendees decided to prepare a scientific paper based on one

of the very popular topic themes discussed this year, namely

species selection. You can read a summary from the MRF on

page 20-22; The Steering Committee is already working hard to

organize yet another great MRF on 22-24 May 2019 in Vienna;

I will ask you already now to mark your calendar for this event.

At Ellegaard Göttingen Minipigs we entered the vacation period

with the positive announcement that Göttingen Minipigs are

now available for biomedical research also in India, as we are

now able to export Göttingen Minipigs from our Danish barrier

breeding facility to customers in India and thus to support the

rapidly growing Indian pharma and CRO industry with high-

quality and well-defined Göttingen Minipigs. This is good news,

as over the last decades we have experienced an extended use

of Göttingen Minipigs as non-rodent species in Europe, Asia and

US; not only in toxicology and safety assessment, but also in

pharmacological studies; all in line with the high translational

value and global regulatory acceptance.

To mark this important milestone, we will be present at two

major scientific conferences in India this Autumn to share our

deep knowledge about Göttingen Minipigs and related research

developments. Please consult the meeting calendar 2018 at the

last page for further information and please feel free to contact

us in advance if you want to set up a meeting with us during one

of the conferences. This of course goes for all conferences and

exhibitions that we attend throughout the year: We are always

very interested to meet with people who share our interest in

Göttingen Minipigs.

Last, but certainly not least; on the edge of the editorial deadline

for this Newsletter, we received the great news that the export

certificate for China will most likely be finalized and signed

during the visit by the Danish Minister for Environment and

Food to China in September 2018! We very much look forward

to announcing the availability of Göttingen Minipigs in China.

Enjoy your reading!

Lars Friis Mikkelsen

CEO

Ellegaard Göttingen Minipigs

Follow us on !

Join the Ellegaard Göttingen Minipigs company page to

stay updated on useful and interesting information about

Göttingen Minipigs and our company

Ellegaard Göttingen Minipigs

Research Foundation Grant 2018

We are very pleased to announce, that the Scientific Board of

Ellegaard Göttingen Minipigs Research Foundation has granted

€35,000 to the project “Speedgene targeted knock out of the

PCSK-9 gene in Göttingen Minipigs” led by Dr. Jaya Krishnan

and Dr. Jonathan Ward, Genome Biologics, together with Dr.

Roy Forster, Citoxlab. The exciting project is based on the knock

down of the PCSK-9 gene using an adeno associated viral vector

containing a short hairpin sequence to produce the knockdown

effect in Göttingen Minipigs. This technique has previously

been performed in other animal species, but never in Göttingen

Minipigs and will, if successful, be groundbreaking for the development

and characterization of transgenic Göttingen Minipigs.

The Ellegaard Göttingen Minipigs Research Foundation grants

annually €50,000 in supporting scientific research aimed at the

characterization of Göttingen Minipigs and the promotion of the

development of Göttingen Minipigs disease models, or projects

oriented toward improving animal welfare, optimizing handling

or research techniques and enhancing educational activities

related to the use of Göttingen Minipigs.

Pramila Singh,

Director of

Toxicology at

Citoxlab France,

received the

diploma from

Lars Friis

Mikkelsen on

behalf of the

winning project

team

CONTENTS

Towards 3Rs: Multiplex protein profiling from minipig,

cyno, dog and rat samples, to get the most out of preclinical

safety studies . . . . . . . . . . . . . . . . . . . . . . . . . . . 3

Experience with mixed groups of intact female and castrated

male Göttingen Minipigs for pharmacokinetic studies in drug

discovery research – summary of behavioral observations

and collected back-ground data . . . . . . . . . . . . . . . . . 6

Anti-cancer drug development: Comparison of toxicity

in Göttingen Minipigs and mouse . . . . . . . . . . . . . . . 10

Body surface area measurement in Göttingen Minipigs

using a computed tomography scanner . . . . . . . . . . . . 14

The Göttingen Minipig for the study of buccal and

sublingual products . . . . . . . . . . . . . . . . . . . . . . . 17

Minipig Research Forum 2018 . . . . . . . . . . . . . . . . . 20

MRF 2018 Best Poster . . . . . . . . . . . . . . . . . . . . . 23

Update from Oriental Yeast Co., Ltd., Japan . . . . . . . . . . 24

Update from WOOJUNGBIO, Korea . . . . . . . . . . . . . . 25

Update from Marshall BioResources, North America . . . . . 26

New scientific publications on Göttingen Minipigs . . . . . . 27

Join the CONFIRM Initiative . . . . . . . . . . . . . . . . . . 28

Meeting Calendar Autumn 2018 . . . . . . . . . . . . . . . . 28

2


Towards 3Rs:

Multiplex protein profiling from minipig,

cyno, dog and rat samples, to get the most

out of preclinical safety studies

By Gerrit Erdmann 1 , Julia Schneider 1 , Anja Briese 1 , Ewa Breitinger 2 , Yvonne Beiter 2 , Markus F. Templin 2 , Marian Raschke 3 , Christoph

Sachse 1

1

NMI TT Pharmaservices, Berlin, Germany

2

NMI Natural and Medical Sciences Institute at the University of Tübingen, Germany

3

Bayer AG, Berlin, Germany

Introduction

The increasing use of minipigs in preclinical safety studies has

produced countless refinements of pharmacological intervention

techniques and assays. While genomic approaches have for

many years primarily been focused on mouse models, minipigs

are nowadays also subject to several genomics-driven efforts

including genome-editing (1) , thus paving the way for toxicogenomics

and other -omics studies that will ultimately undoubtedly

benefit the richness of animal experiments in minipigs by

facilitating genetic and protein-based biomarker assays.

Our own activities in proteomics and protein profiling have led

to the establishment of a multiplex technology called DigiWest

(Treindl et al. 2016 (2) ), which allows for comprehensive studies

on up to 800 protein analytes per sample (see Figure 1) in human

and mouse materials. This antibody-based immuno-assay

technology has already proven its value in numerous studies on

cell signaling processes, drug modes of action, drug resistance

mechanisms and in biomarker discovery (2,3,4,5) . The following describes

how the DigiWest platform was utilized to characterize

Figure 1

hundreds of commercially available antibodies for their use

in sample materials from rat, dog, cynomolgus and minipig

origin.

Methodology

Total protein lysates from liver and kidney samples of untreated

rat, dog, cynomolgus and Göttingen Minipig were purchased

from Zyagen (San Diego, USA). Protein concentration of lysates

was adjusted by in-gel quantification, using a FluoOrange gel

staining. Afterwards, protein lysates were subjected to DigiWest

protein profiling, as described by Treindl et al. (2) .

In brief, gel electrophoresis and Western blotting onto PVDF

membranes was conducted. Blots were biotinylated on the

membrane; then, each sample lane was cut into 96 molecular

weight fractions and proteins were eluted in 96 well plates.

Proteins from each molecular weight fraction were loaded

onto one distinct color of neutravidin coated MagPlex beads

(Luminex) and afterwards pooled. Aliquots of these bead mixes

were then aliquoted into 96 well plates, and one primary antibody

was added per well. Next day, phycoerythrin-labelled

secondary antibodies were added prior to the assay readout of

plates on a Luminex FlexMAP 3D device. For quantification of

antibody-specific signals, a dedicated analysis tool was used to

identify peaks and to calculate peak areas.

A total of 722 primary antibodies from various commercial

sources (primarily from Cell Signaling Technology, but also

Cells, tissues,

sections,

xenografts

SDS-PAGE,

Western blot,

fractionation

Proteins from

96 size fractions

loaded to 96

Luminex bead IDs

Incubation with up

to 800 primary

antibodies

1

2

3

4

5

Assay: Luminex Flexmap 3D

Figure 1: Assay principle of DigiWest

multiplex protein profiling (modified

from Treindl et al. (2) ). Through a

combination of Western blotting and

Luminex bead-based multiplexing,

several hundreds of proteins per

sample are analyzed in parallel.


3


Figure 3


from Epitomics/Abcam, Biorbyt, RnD, Millipore, Acris, BD, and

others), which have been previously validated in human and

mouse samples (2) were tested. For comparative analyses, protein

expression values were normalized to the total protein.

Figure 2

Results and discussion

Expression of 722 protein analytes in DigiWest assays of liver

and kidney samples from untreated rat, dog, cynomolgus and

Göttingen Minipig was evaluated. As illustrated in Figure 2, immuno-assay

signals were individually checked one-by-one for

their expected molecular weight in the various tissues, and performance

of antibodies was scored individually, applying commonly

accepted standards (6) . The outcome of these qualification

experiments, stratified by species derivation, is summarized in

Figure 3. Out of the 722 antibodies tested, 440 were scored as

usable in rat, 401 in dog, 341 in cyno and 446 in minipig.

It appears quite likely that some of the negative antibody validation

data were due to the lack of expression in liver and kidney

rather than insufficient antibody quality, and that we would

Figure 3: Overall statistics of DigiWest antibody validation in samples from

six different species. A total of 446 antibodies were positively validated in

Göttingen Minipig liver and/or kidney tissues. Human and mouse data are from

a previous study (2) . Antibody scores were used as proposed (6) . Antibody lists

are available via (7) .

Arginase 1, minipig liver

AMPKa, minipig liver

Arginase 1, minipig kidney

AMPKa, minipig kidney

Figure 2: Sample data for DigiWest antibody

validation in Göttingen Minipig tissue samples.

Altogether, 722 antibodies were tested, targeting

total proteins and post-translational modifications

including phospho epitopes. As the molecular weight

resolution is retained, DigiWest results are very

similar to classical Western blot patterns. Peaks

of expected molecular weight are marked in dark

grey. Interpretation examples: Arginase 1 was found

to be expressed in minipig liver (with one specific

peak for the antibody) but not in kidney. HNF4

phospho-Ser313 was expressed in both minipig

liver and kidney, with a specific antibody signal plus

a few minor side peaks (due to the lower expression

level especially in kidney).

eEF2 p-Thr56, minipig liver

eEF2 p-Thr56, minipig kidney

HNF4a p-Ser313, minipig liver

HNF4a p-Ser313, minipig kidney

4


Figure 4

Figure 4: Hierarchical clustering of

DigiWest protein expression data

derived from a selection of 160

antibodies that were positively

validated across liver and kidney

samples from rat, dog, cynomolgus

and minipig.

have identified even more suitable antibodies if we had tested

other tissue types too (heart, muscle, brain, etc.). However, the

existing qualified antibody lists already represent an extensive

resource for various protein profiling approaches – using the

DigiWest platform (whose strength is in multiplex assays of >80

analytes), but also for individual Western blot experiments.

From the antibodies that were analyzed in the present study,

a subgroup of 160 antibodies were found to yield expression

values above threshold in liver and kidney samples across all

four species. Those analytes were subjected to a hierarchical

clustering analysis as depicted in Figure 4, which – as expected

– illustrates clustering of cross-species protein expression

signatures in liver vs kidney tissues. This dataset also highlights

that large-scale protein profiling experiments do allow for

valuable differential expression studies whenever two states of

treatment or disease are compared (treated vs untreated, toxic

vs non-toxic, sensitive vs resistant, diseased vs healthy, etc.),

and as such, the technology qualifies for in vivo pharmacology

and preclinical safety alike.

Bioinformatics analyses on all antibodies were performed to

categorize the different protein analytes into panels that cover

various cellular pathways and processes; an overview of those

panels is shown in Figure 5 for minipig. As the DigiWest platform

is to a major extend relying on classical Western blotting,

additional Western-compatible antibodies are constantly being

brought in for validation, to enlarge the antibody list and to

further broaden the range of possible applications.

Conclusion

Our parallelized protein expression effort has qualified hundreds

of antibodies for their use in future protein profiling studies

from rat, dog, cynomolgus and minipig samples. The results are

expected to contribute to the 3R aims by enabling comprehensive

protein-based analyses of cell signaling pathways and for biomarker

discovery, to eventually enhance the output, data depth

and conclusiveness of animal experiments on efficacy and safety.

Author contributions

MFT, YB, EW developed and optimized the DigiWest technology;

MR conceived the current study; CS, GE planned and supervised

the study; JS, AB, EB, YB, GE conducted the experiments; GE,

YB, MFT analyzed the data; AB performed all bioinformatics

analyses; CS wrote the manuscript.

Figure 5

Figure 5: Pathway panels, derived

via bioinformatics categorization

of the 446 antibodies identified

as suitable for minipig samples

(all panels are available via (7) ).

Note: Pathway assignments are

not mutually exclusive, i.e. one

analyte can be part of more

than one panel.

References

1 Forster R. et al. The minipig as a platform for new technologies

in toxicology. J Pharmacol Toxicol Methods 62; 3, 227-35.

DOI: 10.1016/j.vascn.2010.05.007 (2010).

2 Treindl F. et al. A bead-based western for high-throughput

cellular signal transduction analyses. Nature Communications

7, 12852, DOI:10.1038/ncomms12852 (2016).

3 Kissel M. et al. Antitumor effects of regorafenib and sorafenib

in preclinical models of hepatocellular carcinoma. Oncotarget

8; 63, 107096-107108 (2017).

4 Augustin I. et al. Autocrine Wnt regulates the survival and

genomic stability of embryonic stem cells. Science Signaling

10; 461, eaah6829, DOI: 10.1126/scisignal.aah6829 (2017).

5 Lomnytska M. et al. Platelet protein biomarker panel for ovarian

cancer diagnosis. Biomarker Research 6; 2, DOI 10.1186/

s40364-018-0118-y (2018).

6 Björling E., Uhlén M. Antibodypedia, a portal for sharing

antibody and antigen validation data. Mol Cell Proteomics 7;

10, 2028-37. DOI: 10.1074/mcp.M800264-MCP200 (2008).

7 www.digiwest.de

5


Experience with mixed groups of intact female and castrated

male Göttingen Minipigs for pharmacokinetic studies in drug

discovery research – summary of behavioral observations and

collected back-ground data

By Clemens Braun 1 and Miriam Wetzel 2

Boehringer Ingelheim Pharma GmbH & Co. KG, Drug Discovery Sciences 1 and Biological Laboratory Service ² , Germany

Introduction

For many years, Göttingen Minipigs have been used at Boehringer

Ingelheim (BI) drug discovery as non-rodent species for

pharmacokinetic (PK) investigations and cardiovascular telemetry

studies. In contrast to the drug development phase, where

studies in both sexes are often a regulatory requirement, this is

not required for the research phase.

For the last decade, only female Minipigs have been used for

research pharmacokinetic studies. The reasons are related to

animal health, handling, personal safety and social hierarchy of

group housed minipigs:

• Less fighting and mounting behavior lead to reduced number

of injuries, i.e. of the anogenital area and hence less drop outs

• Aversion to the experimental procedures in male pigs makes

procedures more difficult and unsafe

• The more prominent tusks of male pigs are considered as a

relevant source of injuries for the technicians, i.e. during oral

administration via gavage and during fixation of the head in

supine position for blood collection

• Less stressed and more compliant animals are observed in a

calmer housing environment

In the pharmaceutical industry female Minipigs are also preferred

in juvenile and in embryofetal developmental studies,

in studies involving the urinary tract, and in studies of longer

duration and settings where group housing is required. Thus,

slightly more female Minipigs than male Minipigs are sold by the

breeder in periods, and it would be optimal if slightly more male

animals could be used.

Positive experiences with castrated male Göttingen Minipigs

were made in the last years by Ellegaard Göttingen Minipigs A/S

(EGM) and another customer. The castrated male minipigs appeared

suitable for group housing with intact females at any age

as they fight less, have less male behavior, and appear calmer.

Castrated males and females do get tusks; however, tusk growth

is slower in castrated males and females and may not need to be

trimmed. Castrated males furthermore have the advantage that

they don’t show estrous, which often causes variation in food

intake and behavior in the female Minipigs.

Project proposal

In a collaboration project with EGM, we evaluated the use of

male castrated along with female intact Minipigs in our drug

discovery pharmacokinetic research setting. The focus was on

a.) feasibility of housing mixed groups, b.) behavioral observations

in housing and study environment, c.) collection of background

data (i.e. body weight, clinical chemistry, hematology,

tusk growth) and d.) impact on the pharmacokinetic read-outs in

drug testing. The preparation of a full publication with focus on

the PK data in a peer-reviewed journal is ongoing.

Animals

4 male Göttingen Minipigs were castrated before sexual maturity

at age of about 6 weeks at EGM. At this age the animals

were weaned and strong enough to be castrated and moved.

For instance, clinical signs like diarrhea are limited when the

animals reach 6 weeks of age, which is 2 weeks after weaning.

The surgery was done in balanced anesthesia and analgesia

(including meloxicam 3 days post-operatively). 4 age matched

castrated male and 4 intact female Minipigs from the same

barrier were provided by EGM to BI end of 2017 and housed

together in a group of 8 animals.

Housing

After delivery, the animals undergo an acclimatization and

training period of 6 to 10 weeks. After the training the 6 to 8

months old Minipigs are used in pharmacokinetic studies for 12

up to maximally 24 months. Animals are housed in groups in a

state of the art, modern facility with up to 36 m² indoor area

per group (up to 8 animals, usually 4) with open access to an

approximately 35 m² outdoor area during day time. They are

fed with a standard minipig diet from SDS once a day. Straw,

bedding, tubs, balls, brushes, chains and regular training are

part of the enrichment program.

Experimental procedures

During the PK studies the Minipigs are kept in metabolism cages

for up to 48 hours to enable urine sampling in parallel to plasma

sampling. Usually 9 to 12 blood samples are collected in supine

position from the venous plexus in the neck. Serum samples for

the monitoring of clinical chemistry parameters are prepared

at the last blood sampling time point. A wash-out and recovery

period of minimal 2 weeks is introduced after a study and a

veterinarian has to confirm good health status before animals

will be used in a further PK study.

Results and Discussion

Housing of mixed groups – Behavioral observations

Sleeping behavior, mounting behavior and fighting wounds

were documented daily by BI animal technicians within the

first 4 months (118 days) after arrival. The 4 intact female and

4 male castrated Göttingen Minipigs were housed in a mixed

group right from the start. Four days after arrival they started

to build up their social hierarchy allowing the animal technicians

to discriminate between more dominant and lower ranking

individuals but with little observed fighting. Every single day,

6


100

90

100%

A

behavior observed (%)

80

70

60

50

40

30

20

10

9% 4%*

0

slee ping

huddled

fighting

wounds

m ounting

B

C

Figure 1: Behavioral observations of a group of 4 intact female and 4 castrated

male Göttingen Minipigs. Favored sleeping behavior (green), undesired

mounting and fighting wounds (red) were observed over a period of 118 days.

*Mounting was observed in females only.

Figure 2: A Physical appearance of head and tusks of a 2 year old castrated

male Göttingen Minipig. B Tusks of a 2-year-old castrated male and C of a

1-year-old intact male Göttingen Minipig.

especially in the morning when the technicians entered the

room for the first time of the day, all 8 animals were detected to

cuddle up together as a group (Figure 1). Together with the low

rate of superficial fighting wounds of 9% this reflects a stable

social hierarchy. Only once in the whole period of mixed group

housing (20 month) veterinary treatment of a fighting wound

was necessary in one sow. Mounting behavior was observed

very rarely (4%, representing 5 days) and was evident only

in the 4 female Göttingen Minipigs, but not in the 4 castrated

male ones. As excessive mounting in intact boars can result in

a clear constraint of animal welfare, this is a fact that has to be

highlighted.

Secondary gender characteristics

In castrated males the tusk growth was reduced in comparison

to two uncastrated males. Thus, the tusk size of castrated male

minipigs in length and caliber at an age of 2 years was comparable

to tusk size of uncastrated males at an age of 1 (Figure

2). In the uncastrated males we noticed a fast development of

secondary gender characteristics starting at approximately the

age of 8-9 months such as tusk growth, broader shape of skull

and muscle growth especially in the area of the scapulae. The

castrated minipigs at the age of 2 years still showed very moderate

male secondary gender characteristics almost comparable

to female Minipigs. As mentioned before we consider secondary

gender characteristics, i.e. tusk size, in male minipigs as relevant

parameter for safety at work. In our experience it was safe to

work in pharmacokinetic studies with castrated males at the age

of 6 to 22 month.

Handling observations during PK studies

Behavioral differences between the intact female and castrated

male Minipigs were small and driven by the individual temperament

and not the sex of an animal. From the beginning and

throughout, no differences in handling during the PK studies

were observed from the caretakers and technicians for any

procedures. Main procedures were placement and restraining in

a hammock for intra-venous administrations in the ear veins,

restraining for oral administrations by gavage, placement in

supine position and restraining for blood collection from the

venous plexus of the neck. In contrast to intact male Minipigs

which developed aversion to the experimental procedures, the

castrated male Göttingen Minipigs got, as known from the female

animals, calmer when they got accustomed to the procedures

in the experimental setting. Also, no specific differences were

observed during their stay in the metabolism cages, i.e. activity,

interaction with staff, vocalization, food and water intake. All

animals enjoyed interaction with staff and playing with toys.

Back-ground data

Body weight

The 4 castrated male and 4 intact female Göttingen Minipigs

were delivered to BI at the age of 10 weeks. The animals

obtained a standard minipig diet from SDS once a day in the

morning and had free access to water. The same diet is fed

at EGM. The animals were weighed regularly and additionally

before the PK studies were performed. Bodyweight data were

pruned to adjust for different weighing datum. There was no

difference between castrated male and intact female Göttingen

Minipigs mean body weight development observed (Figure 3).

35

30

25

W eight (kg)

20

15

10

castrated m ale

5

intact fem ale

0

2 4 6 8 10 12 14 16 18 20 22 24

Age (month)

Figure 3: Growth curve of 4 intact female and 4 castrated male Göttingen

Minipigs delivered to BI at age of 10 weeks. Mean ± 95% CI


7


Parameter

Unit

Females (N=15)

Castrated males (N=4)

Mean SD N Mean SD N

Gamma glutamyltransferase (GGT) IU/L 52 4.2 238 55 5.2 62

Alanine aminotransferase (ALT) IU/L 53 6.7 241 64 9.0 57

Aspartate aminotransferase (AST) IU/L 26 7.2 246 29 8.4 63

Glutamatdehydrogenase (GLDH) U/L 0.48 0.23 239 0.67 0.32 50

Creatinin (Creat) µmol/L 90 9.6 247 97 10 61

CystatinC (CysC) mg/L 0.33 0.09 238 0.33 0.08 63

Creatinekinase (CK) U/L 923 537 246 1243 747 62

Lactate dehydrogenase (LDH) IU/L 447 61 246 574 105 62

Total protein (TP) g/L 63 4.8 248 62 4.3 63

Cholesterol (Chol) mg/dL 67 8.5 248 68 10 63

Triglycerides (Trig) mg/dL 38 11 248 28 6.8 63

Glucose (Gluc) mM 4.2 0.72 248 4.3 0.67 63

Phosphorous (Phos) mg/dL 6.6 0.80 248 7.0 0.90 63

Sodium (Na + ) mM 142 3.3 248 143 3.2 63

Potassium (K + ) mM 4.7 0.49 248 5.1 0.51 63

Chloride (Cl - ) mM 101 3.1 248 101 2.7 63

Table 1: Clinical chemistry data were obtained from 15 healthy intact female and 4 castrated male Göttingen Minipigs at an age of 6 to 20 months. Measurements

were performed at the end of the pharmacokinetic studies, i.e. the last blood collection time point. N, total number of measurements.

Test

Unit

Intact female

Castrated male

Median Range N Median Range N

WBC 10e3/µL 6.2 5.0 - 8.8 4 7.6 5.6 - 9.0 4

RBC 10e6/µL 7.2 6.4 - 9.3 4 7.0 6.4 - 8.1 4

HGB g/dL 14 12 - 17 4 14 14 - 15 4

HCT % 41 35 - 53 4 43 39 - 47 4

MCV fL 57 52 - 61 4 58 55 - 73 4

MCH pg 18 18 - 21 4 20 18 - 24 4

MCHC g/dL 33 31 - 35 4 34 32 - 35 4

CHCM g/dL 32 31 - 33 4 33 31 - 34 4

RDW % 16 15 - 20 4 15 14 - 16 4

HDW g/dL 1.8 1.7 - 1.9 4 1.7 1.4 - 1.8 4

PLT 10e3/µL 343 329 - 370 4 397 299 - 438 4

MPV fL 7.4 7.1 - 8 4 7.6 6.6 - 10 4

NEUT % 22 12 - 60 4 35 19 - 48 4

LYMPH % 64 35 - 74 4 59 44 - 70 4

MONO % 4.8 2.1 - 5.6 4 3.3 3.0 - 3.5 4

EOS % 6.4 1.60 - 7.7 4 3.9 1.6 - 5.1 4

BASO % 0.40 0.17 - 0.50 4 0.10 0.00 - 0.40 4

LUC % 1.3 0.50 - 2.1 4 1.1 0.40 - 2.5 4

NEUT 10e3/µL 1.3 0.67 - 5.3 4 2.5 1.2 - 4.3 4

LYMPH 10e3/µL 3.4 3.1 - 4.8 4 4.2 3.0 - 5.7 4

MONO 10e3/µL 0.26 0.19 - 0.36 4 0.25 0.19 - 0.27 4

EOS 10e3/µL 0.36 0.140 - 0.45 4 0.25 0.15 - 0.42 4

BASO 10e3/µL 0.025 0.01 - 0.040 4 0.010 0.00 - 0.020 4

LUC 10e3/µL 0.075 0.040 - 0.120 4 0.075 0.030 - 0.16 4

RETIC % 0.49 0.13 - 2.5 4 0.79 0.35 - 1.1 4

RETIC 10e9/µL 34 8.5 - 233 4 52 24 - 92 4

RBC-RETI 10e6/µL 7.2 6.6 - 9.0 4 6.6 5.9 - 7.8 4

Table 2: Haematology data were obtained from 4 healthy intact female and 4 castrated male Göttingen Minipigs at the age of 16 to 22 months.

8


The variability was higher in the females, but the number is too

low to take a general conclusion.

Clinical Chemistry and Haematology

In the BI research pharmacokinetics group clinical chemistry is

generally performed at the end of a study to test for compound

related effects in particular on liver and kidney parameters (Table

1). Clinical chemistry data shown (Konelab analyser, Thermo)

were obtained from 15 healthy intact female (11 historic and

4 actual) and 4 castrated male Göttingen Minipigs at age of 6

to 20 month. The measured values were within the range of

historic BI and published data. Data where drug related effects

were assumed were excluded from this analysis. No relevant

differences between intact female and castrated male Göttingen

were observed. Complete blood counts were done at the age

of 16 to 3522 month in context of a routine exam (ADVIA 2120i,

Siemens). Data for the 4 intact female and 4 castrated male

Göttingen 30 Minipigs are shown in Table 2. Relevant differences

on the haematological parameters were not observed.

25

W eight (kg)

Urine volume

The renally 20 excreted amount of an administered drug can be

Conclusion

Housing, study performance and training of the mixed group of

4 intact female and 4 castrated male Göttingen Minipigs turned

out to be very uncomplicated and showed no sex differences.

Moreover, the impression was that the presence of the 4 castrated

Göttingen Minipigs had a beneficial effect on the group

stability leading to an overall reduced stress level, compared to

female only groups. We consider this relevant for animal welfare.

Further, this could also have a positive impact on quality

of data and operating efficiency, such as time savings for study

procedures, regrouping and veterinary care of fighting wounds.

Handling and behavior during the PK studies was the same for

the intact females and castrated male animals. Typical behavior

of intact male Minipigs as increasing aversion towards the procedures

was not observed. Observed differences in behavior

were small and rather related to the individual temperament of

an animal. The collected back-ground data on growth, clinical

chemistry, hematology and urine volume revealed no relevant

differences between the intact females and castrated males.

The tusk growth in the castrated male pigs was delayed for

approximately one year. Since the castrated males are easier

to handle compared to age matched intact males the safety at

work is still improved compared to intact male Minipigs. However,

tusk growth must be considered depending on the intended

used to calculate the renal clearance of a drug. Therefore, the

15

urine is collected during the period the animals stay in the metabolism

cages – usually 24 to maximally 48 hours. At the end procedures. Overall the use of mixed groups of intact female and

10

of sampling the urine volume is noted, and the respective drug castrated male Göttingen Minipigs can be recommended in the

concentration is measured to determine the excreted amount castrated of mdescribed ale pharmaceutical industry setting.

5

drug. Although the drinking water is offered in a rack making intact it fem ale

difficult to spill, it cannot be excluded that the collected volume Acknowledgements

0

is solely urine, for instance in the case of Micky (Figure 4). In We would like to thank the colleagues in the animal house for

2 4 6 8 10 12 14 16 18 20 22 24

some cases, no or only low volumes of urine were collected in dedicated care and training, as well as performing the daily

the 24-hour period. In these cases, usually Age (month) the urine is retained behavioural observations. We would also like to thank the

in the bladder until the Minipigs are put back in the transport colleagues who conducted and supported the PK studies in an

carts for transport to the housing area. This behavior was excellent way. Further we would like to thank Henrik Duelund

mostly observed with 2 of the 4 female pigs, but our historic Pedersen and Ellegaard Göttingen Minipigs for the supply of the

observations show that this is not a general problem associated minipigs and for supporting this collaboration with BI.

with female animals.

24 h urine volume/BW

(m L/kg)

140

120

100

80

60

40

20

*

***

10% percentile

overall m ean

90% percentile

Figure 4: Urine volumes collected

from Göttingen Minipigs of 6 to

18 months of age during 24 hours

of housing in metabolism cages.

Urine volumes are corrected for

bodyweight. Red, intact female;

blue castrated male Göttingen

Minipigs.

0

M o tte

M a th ild e

W ilm a

W o lk e

M a x

M ic k y

W illi

W h is k y

9


Anti-cancer drug development:

Comparison of toxicity

in Göttingen Minipigs and mouse

By Sally-Anne Reynolds and Malcolm Blackwell, Sequani Limited, Ledbury, Herefordshire, UK.

Overview

In this article we present a brief overview of anti-cancer drug

development and discuss how the selection of the most appropriate

preclinical species can help in predicting safe but potentially

efficacious clinical starting doses. Using data from recent

toxicity studies in the mouse and minipig on a novel anti-cancer

therapeutic, we describe how results obtained from studies in

Göttingen Minipigs appeared to better predict potential adverse

effects in humans, in terms of the nature of the effects and the

dose levels at which they occurred.

Introduction

Nearly everyone has been or will be affected by cancer either

directly or through friends or relatives. This has led to huge

efforts and allocation of substantial resources to the development

of novel therapies. Anticancer drugs are now the biggest

therapy group among drugs currently in development with a

7.6 % increase in the last year; a growth rate 3 times that of the

overall drug pipeline such that over a third of drugs currently

in development have at least one oncology indication (Pharmaprojects®,

January 2018).

Statistics show that cancer survival has doubled over the last

40 years and there clearly has been success in the development

of effective new drugs. Nevertheless, the mainstays of cancer

treatment remain the cytotoxic therapies that have been in use

for decades. Huge improvements have been made in targeting

and combining these existing drugs which have resulted in much

of the increased survival but numbers of truly novel therapies

reaching market approval, with some notable exceptions, are

disappointingly small. Furthermore, a disproportionate number

of potential cancer therapies fail in clinical trials due to poor

efficacy or an unacceptable safety profile not adequately predicted

in preclinical studies.

The ready availability of clinical trial subjects drawn from the

population of terminally ill cancer patients has historically allowed

progression of drugs into early stage clinical trials based

on limited preclinical data. However, as a result of improving

treatment regimes, these patients can now expect a longer

survival and deserve a greater emphasis on ensuring that the

novel treatments they volunteer for are administered at doses

that are safe and have the greatest opportunity for efficacy.

This concept was recognised in the adoption of ICH Guideline

S9 on Nonclinical Evaluation for Anticancer Pharmaceuticals

(finalised in 2009). It allows for a reduced preclinical testing

strategy for pharmaceuticals intended for treatment of patients

with “serious and life-threatening malignancies” but moves

away from the historical reliance on preclinical studies conducted

only in rodent species. Studies are now expected, at least

for non-genotoxic drugs, to also be conducted in non-rodents

so that the rodent and non-rodent approach required for other

small molecule pharmaceuticals is followed. There is also an

increased regulatory emphasis on ensuring that animal models

used in preclinical studies are relevant and this is even more

important where only a single species is used.

The choice of non-rodent species for anticancer pharmaceuticals

is subject to similar considerations as those for other types of

drugs with the primary species being dogs, non-human primates

or minipigs. However, it is important to also consider the nature

of the expected effects and for many anticancer therapeutics

this includes substantial effects on rapidly dividing cells, especially

in the bone marrow. Indeed it is commonly a marked

neutropenic effect that results in limitation of clinical doses or

the early termination of clinical treatment. For this reason, we

should be seeking a non-clinical species that can predict these

effects and also the dose/exposure levels at which they are

expected.

NHP

• Likely similar bone marrow sensitivity to man

• Expensive

• Ethical concerns

• Disease status (immunosuppression)

Dog

• Possibly similar bone marrow sensitivity to man

• Prone to emesis

• Ethical concerns (charities)

Minipig

• Likely similar bone marrow sensitivity to man

• Less prone to emesis

• High throughput – cost effective

• Reduced ethical concern

As a result of regulatory pressure to include a non-rodent investigation

in the preclinical evaluation of a novel small molecule

anticancer drug, we had the opportunity to compare the toxicity

findings in mouse and minipig and to make an assessment regarding

predictivity of effects in man.

Studies Conducted

The following studies were conducted and study outcome and

data compared.

• Mouse – Preliminary and 14 Day Study

• Göttingen Minipig - MTD and Range-finding Study followed

by a 28 Day study with 28 Day Treatment-Free Period.

Standard endpoints were measured for both species and the

drug was administered by oral gavage in a twice daily (b.i.d.)

treatment regimen.

10


Mouse

Minipig

Clinical Observations post-dose and daily post-dose and daily

Body weights twice weekly, daily weekly

Food consumption

twice weekly

Ophthalmoscopy

acclimatisation and end of study

Electrocardiograms

acclimatisation and end of study

Haematology

end of study

acclimatisation and end of study

(additional 0.1 mL taken twice weekly)

Blood Chemistry end of study acclimatisation and end of study

Urinalysis

at necropsy, by cystocentesis

Proof of Absorption/TK end of study Day 1 and Day 28

Organ weights end of study end of study

Pathology end of study end of study

A particular immediate advantage of the minipig was the ability

to take additional blood samples for frequent monitoring during

the studies, enabling early detection of effects on blood count

and potential adjustment of dose levels or early termination to

preserve data collection.

Results

Tolerated Dose Levels

Mouse

Minipig

Dose Level

mg/kg BID

Low 75 3

High 125 6

Dose levels in the minipig were similar to those expected in

humans and more than 20-fold lower than those tolerated in

mice.

Minipig

Minipigs were continuously exposed for the full 10 hours

between the first and second sub-doses, after both single and

repeat administrations. Total systemic exposure was higher in

females than males with accumulation in both sexes. Individual

variations in exposure appeared to be in line with observed

toxicity, with females being terminated from study prematurely.

By the end of the study low dose females had similar exposure

to high dose males.

Plasma Profile Day 28

Concentration (ng/mL)

12000

10000

8000

6000

4000

2000

0

E

Mo

Exposure

Mouse

No difference in systemic exposure, 1 hour after the first subdose,

between the dose groups and sexes in the mouse; however,

there was an increase in the drug levels seen in the liver,

particularly for females (up to 22-fold higher).

Male Low

Female Lo

Concentration (ng/mL)

12000

10000

8000

6000

4000

2000

0

End of Study

Mouse - plasma

Mouse Liver

Plasma Profile Day 28

Male Low Dose

Female Low Dose

Male High dose

Female High Dose


11


Clinical Observations/Body weights

The clinical signs seen in the minipig were more representative

of those expected in humans: tremors, vomiting and subdued

behaviour. The mice showed piloerection, pale extremities,

decreased activity and hunched posture. Both species showed

body weight loss at the higher dose levels.

Bone Marrow Smears

The bone marrow of mice treated at the high dose was only slightly

less cellular than the Controls. The Minipig, however, showed a

marked response similar to that expected in humans; reversible in

surviving animals and fatal in some individuals. In minipigs, the

bone marrow was considered to be the main target organ.

Clinical Pathology

Although some haematology effects were seen in mice, the

effect was more prominent in the minipig and could be monitored

throughout the course of the study by taking additional

blood samples, hence treatment could be adjusted accordingly.

The effect in the minipig was typified by a marked and reversible

neutropenia similar to that expected in humans.

Mouse

Control

Neutrophil counts 10 3 µL -

Males

Neutrophil counts 10 3 High Dose

µL -

Females

8

7

6

5

4

3

2

1

0

Mouse - Mouse -

low dose high dose

Minipig

Control

Minipig - Minipig -

Low doseHigh dose

4

3,5

3

2,5

2

1,5

1

0,5

0

Minipig

Control

Mouse - Mouse -

Low dose High

dose

Minipig -

Control

Minipig -

Low dose

Minipig -

High

dose

Background/Pre-dose

End of Treatment

Background/Pre-dose

End of Treatment

End of Treatment-Free

End of Treatment-Free

-

Neutrophil counts 10 3 µL -

Females

High Dose

4

3,5

3

2,5

2

1,5

inipig -

igh dose

1

0,5

0

Mouse - Mouse -

Low dose High

dose

Minipig -

Control

Minipig -

Low dose

Minipig -

High

dose

Early Decedent

ent

Background/Pre-dose

End of Treatment-Free

End of Treatment

12


Mouse

Control Mouse

- duodenum

Histopathology

In the mouse the main pathology findings were in the GI tract

(villous/epithelial atrophy, increased apoptosis, epithelial dysplasia,

cryptal hyperplasia) and this was the main target organ.

In contrast the minipig showed effects primarily confined to the

large intestine and these were only seen in early decedents,

including: surface focal erosions, mucosal and villous atrophy

leading to surface epithelial ulceration in some cases.

Treated Mouse

- duodenum

Control Mouse

- ileum

Treated Mouse

- ileum

Conclusions

The mouse has historically been the most frequently used model

for preclinical toxicology of anticancer drugs; however, this can

lead to frequent disappointments when moving into clinical

trials due to poor prediction of safe and efficacious starting

doses. Clearly, this represents a huge cost both financially and

in human terms if a clinical trial fails.

The work described here demonstrated that the Göttingen Minipig

was a much better model of human effects than the mouse.

In the mouse the main target organ was the small intestine with

much higher dose levels achievable in this species than could

be tolerated in humans. The main pathology in humans has

been shown to be associated with bone marrow depletion and

consequent haematological effects characterised by pronounced

neutropenia. The findings in our minipig studies closely correlate

with the effects in humans and, furthermore, they were

seen at similar dose levels. It is also worthy of note that in the

minipig studies there was significant variation in individual

response including responders and non-responders; again, this

is a similar pattern to that seen in humans.

An overriding principle for species selection in preclinical studies

should be that of human relevance and this is enshrined in

many relevant regulatory guidelines, including the ICH S9 guideline

on Nonclinical Evaluation for Anticancer Pharmaceuticals.

It is acknowledged that the initial cost of conducting studies in

minipigs is much higher than for mice (or rats) but this would

be easily outweighed if the prediction of clinical safety and

efficacy could be improved.

Minipig

Minipig

- caecum

Minipig

- colon

13


Body surface area measurement

in Göttingen Minipigs using a

computed tomography scanner

Tadashi Itoh 1 , Mifumi Kawabe 2 , Takahiko Nagase 1 , Katsumi Endo 1 , Masafumi Miyoshi 3 , and Kazuro Miyahara 3

1

Nihon Bioresearch Inc., 6-104, Majima, Fukujucho, Hashima, Gifu 501-6251, Japan

2

Animal Medical Center, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan

3

Veterinary Medical Center, Obihiro University of Agriculture and Veterinary Medicine, Inadacho, Obihiro, Hokkaido 080-8555, Japan

This report is based on the results about Göttingen Minipigs in

“Body surface area measurement in laboratory miniature pigs using a computed tomography scanner”

in J. Toxicol. Sci., 41: 637-644 (2016).

1. Introduction

The body surface area (BSA) of an organism is one of the parameters

used for evaluating physiological functions. BSA has been

also used as a criterion for drug dosage determination since the

1950s (Pinkel, 1958). In drug development, the Food and Drug

Administration Center for Drug Evaluation and Research (CDER)

guidance recommends the use of BSA to estimate the starting

dose in the initial clinical trials for therapeutics in volunteer

subjects (CDER, 2005). Specifically, the no observed adverse effect

levels (NOAELs) in laboratory animal species are converted

to human equivalent doses (HEDs) using scaling factors. Normalization

by BSA (i.e., conversion of a dosage from mg/kg to mg/

m 2 ) is an appropriate method for extrapolating doses between

species. Thus, for most systemically administered therapeutics,

NOAELs in the laboratory animal species are divided by the appropriate

BSA-conversion factors to calculate HEDs. In addition,

the procedures for assessing dermal toxicity are described in the

guidelines issued by the Organization for Economic Cooperation

and Development (OECD) and the guidelines recommend that

the test substance should be applied on not less than 10% of the

total BSA (OECD, 1981a, 1981b, 1987). Thus, it is apparent that

an accurate measurement of the BSA of laboratory animals is

extremely important.

The BSA of animals is generally estimated by multiplying a

constant by 2/3 of the power of the body weight (BW) (Meeh,

1879). The values of K 2/3 (100 × BSA [m 2 ] = K 2/3 × BW [kg] 2/3 ) that

have been determined in multiple species (Spector, 1956) differ

among these subject species, and have been used to extrapolate

doses between species (CDER, 2005). Thus, obtaining an accurate

BSA is important for determining species-specific factors.

Using BSA determined by classical methods (such as skinning,

triangulation, surface integrator, and paper cover), the K 2/3 for

domestic pigs was determined to be 7.77-15.3 in four studies

(for references, please consult our original article). In addition to

these determinations, several other BSA formulas for domestic

pigs have been proposed to date. For laboratory miniature pigs,

Wachtel’s formula is as follows: BSA [m 2 ] = 0.121 × BW [kg] 0.575 .

This formula has been defined employing a geometric design, i.e.,

by a classical method (Wachtel et al, 1972). Myers et al (2016)

described the most applicable formula for miniature pigs in current

research as probably being Wachtel’s formula. Furthermore,

the K 3/4 (1000 × BSA [m 2 ] = K 3/4 × BW [kg] 3/4 ) was calculated to be

70 based on the theory that animals have a basal metabolism

of 70 kcal/metabolic BW: BW 3/4 and produce 1000 kcal of heat

per square meter (Bollen et al, 2010). Furthermore, the HED was

calculated with the standard k m (k m = 100/K 2/3 × BW [kg] 1/3 = BW

[kg]/BSA [m 2 ]) to convert the dose in mg/kg to the dose in mg/

m 2 for each species including laboratory miniature pigs (CDER,

2005). The k m for micro-pigs (working BW ranges: 10-33 kg) was

determined to be 27 and the k m for mini-pigs (working BW ranges:

25-64 kg) was determined to be 35 in the guidance issued

by the CDER. However BSA is difficult to measure because of the

complex architecture of the animals. Therefore, it appears that

the accuracy and reproducibility of the classical methods have

limitations. Although laboratory miniature pigs are increasingly

being used as non-rodent species for safety testing of chemicals

and drugs (Bollen et al, 2010), verification of the BSA of laboratory

miniature pigs is not as yet sufficient.

The computed tomography (CT) scanner can obtain detailed 3

dimensional (3D) images of an object, and analysis of these

CT images is expected to determine BSA more precisely than

classical measuring techniques. In this study, we measured the

BSA of laboratory miniature pigs using a CT scanner and then

analyzed the CT images. Because the CT images, unlike classical

measuring techniques, do not depend on the skills of the person

performing the measurement, the method used in this study

is expected to be a reproducible method. Additionally, we calculated

the values of K 2/3 , K 0.575 (BSA [m 2 ] = K 0.575 × BW [kg] 0.575 ),

K 3/4 , and k m for laboratory miniature pigs from the BSA and

the BW.

2. Materials and Methods

2.1. Animals

Göttingen Minipigs were obtained from Oriental Yeast Company

Ltd, Tokyo, Japan. The Göttingen Minipig is mainly used for

regulatory toxicity studies, and is available worldwide (Köhn,

2012).

The BSA of 20 Göttingen Minipigs (11 males and 9 females) that

had been used in other non-clinical studies and euthanized by

anesthesia with sodium pentobarbital according to the protocols

of the previous studies were measured. No critical abnormalities

in clinical signs or BW changes had been noted in these animals

14


during the survival period. All of the studies were conducted in

compliance with the guidelines for the management and welfare

of experimental animals of Nihon Bioresearch Inc.

2.2. CT scanning

The body of each miniature pig was set in the prone position

for whole-body CT scanning on the day of euthanasia. Images

were obtained using a Multislice CT scanner (Alexion TSX 033A,

Toshiba Medical Systems Co., Ltd., Tochigi, Japan) with a slice

thickness of 5 mm (tube voltage: 120 kV, tube current: 150 mA,

helical pitch: 5.5). The BSA was determined from the CT images

(Fig. 1) using high-speed 3D analysis software (TRI-3D/VOL,

Ratoc System Engineering Co., Ltd., Tokyo, Japan). Analysis of

the CT images was based on the computer graphics algorithm

known as Marching Cubes. After the BSA had been determined,

the K values (K 2/3 , K 0.575 , K 3/4 , and k m ) were calculated.

3. Results and Discussion

3.1. BSA and K values of the Göttingen Minipig

As shown in Table 1, the BWs of the 20 Göttingen Minipigs

ranged from 12.7 to 37.0 kg (mean: 19.9 kg), and their ages

ranged from 6 to 22 months (mean: 10.4 months). The BSA

values ranged from 0.4358 to 0.8356 m 2 .

The mean value ± S.D., coefficient of variation (C.V.), and the

correlation coefficient between the BW (r) of the K 2/3 value were

calculated to be 7.94 ± 0.21, 2.65%, and -0.163, respectively,

and those of the K 0.575 value were 0.104 ± 0.004, 3.74%, and

0.667, and those of the K 3/4 value were 62.2 ± 2.5, 3.99%, and

-0.771. Additionally, the k m values ranged from 29.1 to 44.3

(mean ± S.D.; 33.8 ± 4.0, C.V.; 11.92%, r; 0.976).

3.2. Estimating formula for BSA of laboratory miniature pigs

According to the present results, the values of the C.V. of the

Sex BW (kg) Age (month) BSA (m 2 ) K 2/3 K 0.575 K 3/4 km

Male 12.7 6 0.4358 8.01 0.101 64.8 29.1

Female 13.1 6 0.4417 7.95 0.101 64.1 29.7

Male 13.3 6 0.4497 8.01 0.102 64.6 29.6

Female 14.3 6 0.4691 7.96 0.102 63.8 30.5

Female 14.4 6 0.4583 7.74 0.099 62.0 31.4

Male 14.5 6 0.4714 7.93 0.101 63.4 30.8

Female 14.8 6 0.4639 7.70 0.099 61.5 31.9

Male 15.0 6 0.4826 7.93 0.102 63.3 31.1

Male 15.7 11 0.5202 8.30 0.107 66.0 30.2

Female 16.2 10 0.5008 7.82 0.101 62.0 32.3

Male 17.1 11 0.5516 8.31 0.108 65.6 31.0

Female 20.8 10 0.5761 7.62 0.101 59.1 36.1

Male 21.7 12 0.6249 8.03 0.106 62.2 34.7

Female 22.2 10 0.6142 7.78 0.103 60.1 36.1

Male 23.0 13 0.6389 7.90 0.105 60.8 36.0

Male 26.3 12 0.7080 8.01 0.108 61.0 37.1

Male 26.6 14 0.7092 7.96 0.108 60.5 37.5

Male 27.3 12 0.7536 8.31 0.113 63.1 36.2

Female 32.0 22 0.8051 7.99 0.110 59.8 39.7

Female 37.0 22 0.8356 7.53 0.105 55.7 44.3

Mean 19.9 10.4 0.5755 7.94 0.104 62.2 33.8

S.D. 7.0 4.9 0.1287 0.21 0.004 2.5 4.0

C.V. 2.65% 3.74% 3.99% 11.92%

r -0.163 0.667 -0.771 0.976

r: the correlation coefficient between the BW

100 × BSA (m 2 ) = K 2/3 × BW (kg) 2/3

BSA (m 2 ) = K 0.575 × BW (kg) 0.575

1000 × BSA (m 2 ) = K 3/4 × BW (kg )3/4

k m = 100/K 2/3 × BW (kg) 1/3 = BW (kg)/BSA (m 2 )

Table 1. BW, age, BSA, and K values in Göttingen Minipigs


15


A

B

Fig. 1. CT images of Göttingen Minipigs.

BSA was determined from CT images using high-speed 3D analysis software. (A) Göttingen Minipig in dorsal aspect. (B) In lateral side.

K 2/3 were smaller than those of the other K values (K 0.575 , K 3/4 ,

and k m ). The K 2/3 values were not the variable most affected by

the BW among the K values calculated in this study. In fact, the

absolute values of r of the K 2/3 were smaller than those of the

other parameters determined. The K 2/3 is also useful for extrapolating

doses between species. Furthermore, we measured

the BSAs of 20 (14 males and 6 females) Nippon Institute for

Bio logical Science miniature pigs (NIBS miniature pigs) employing

the same method, as in the case of the Göttingen Minipigs.

The NIBS miniature pig represents the results of mating three

distinct porcine breeds, Pitman-Moore, Chinese native (Shortear

pig of Taiwan) and Göttingen since 1993, and is a breed of

laboratory miniature pig available in Japan (Nunoya et al, 2007).

The mean value ± S.D. of the K 2/3 value of NIBS miniature pigs

was calculated to be 8.01 ± 0.36. The K 2/3 values of Göttingen

Minipigs and NIBS miniature pigs were approximately equal

(p value of Aspin-Welch’s t-test was 0.45). The mean value of

K 2/3 of 40 laboratory miniature pigs (20 Göttingen Minipigs and

20 NIBS miniature pigs) was 7.98. We propose the following

estimating formula for BSA of laboratory miniature pigs: 100 ×

BSA [m 2 ] = 7.98 × BW [kg] 2/3 .

References

Bollen P J A, Hansen A K, Olsen Alstrup A K (2010): Experimental

techniques. In the laboratory swine., pp.86-87, CRC press, New

York.

CDER (2005): Estimating the maximum safe starting dose in

initial clinical trials for therapeutics in adult healthy volunteers.

In guidance for industry.

Köhn F (2012): History and development of miniature, microand

minipigs. In the minipig in biomedical research, pp.3-15,

CRC press, New York.

Meeh K (1879): Oberflächenmessungen des menschlichen

Körpers. Z. Biol., 15, 425-458.

Myers Jr. D D, Lester P, Conte M L, Swindle M M (2016): Cardiovascular

catheterization, electrophysiology, and imaging

laboratory procedures. In swine in the laboratory third edition,

pp.337-382, CRC press, New York.

Nunoya T, Shibuya K, Saitoh T, Yazawa H, Nakamura K, Baba Y,

Hirai T (2007): Use of miniature pig for biomedical research, with

reference to toxicologic studies. J. Toxicol. Pathol., 20, 125-132.

OECD (1981a): Test No. 410: Repeated Dose Dermal Toxicity:

21/28-day Study. In OECD Guidelines for the Testing of Chemicals,

Section 4.

OECD (1981b): Test No. 411: Subchronic Dermal Toxicity:

90-day Study. In OECD Guidelines for the Testing of Chemicals,

Section 4.

OECD (1987): Test No. 402: Test No. 402: Acute Dermal

Toxicity. In OECD Guidelines for the Testing of Chemicals,

Section 4.

Pinkel D (1958): The use of body surface area as a criterion of

drug dosage in cancer chemotherapy. Cancer Res., 18, 853-856.

Spector W S (1956): Constants for estimating surface area:

mammals. In handbook of biological data, p.175, W. B. Saunders

Company, Philadelphia.

Wachtel T L, McCahan G R Jr., Watson W I, Gorman M (1972):

Determining the surface areas of miniature swine and domestic

swine by geometric design—A comparative study. USAARL

Report, 73-5, Fort Rucker, Alabama.

16


The Göttingen Minipig for the study

of buccal and sublingual products

Anette Blak Grossi 1 , Marianne Jochumsen 1 , Catherine Thirion-Delalande 2 , Andrew Makin 1

1

Citoxlab Scantox, Ejby, Denmark

2

Citoxlab France, Evreux Cedex, France

Introduction

The buccal and sublingual mucosa are attractive sites for drug

and vaccine delivery; among others, because the oral mucosa is

highly vascularised, which allows fast uptake of drugs and the

possibility of both local and systemic drug delivery.

Buccal absorption of drugs is very much dependent on the physiological

barrier functions, such as thickness of the epithelium,

keratinization, mucus and lipid content. These characteristics

can be very different from one species to another and need to

be taken into consideration when choosing the animal model.

In addition, it is important to consider drug delivery system

and study design as parameters such as flow rate of saliva and

blood may affect, for example, the buccal barrier properties and

dissolution of oral films and tablets.

-

Advantages of buccal and sublingual mucosal vaccine and

drug delivery

• Fast uptake of drugs

• Non-invasive systemic drug delivery

• The hepatic first pass metabolism is bypassed.

• Enzymatic and acidic degradation in the gastro-intestinal

tract are avoided.

• Oral mucosal drug delivery may be used in patients with

difficulties in swallowing.

• Both local immune responses at the site of pathogen entry

and systemic immunity can be obtained.

Histological characteristics of the minipig oral mucosa

In a previous study, the oral mucosa from different regions of

the oral cavity in various species (mice, rats, hamsters, guinea

pigs, rabbits, dogs, monkeys and minipigs) was characterized

histologically (Thirion-Delalande, C. et al.). The study showed that

minipigs and monkeys are the species with most similarities to

the oral mucosa of humans with respect to structure and composition.

In contrast to rodents, minipigs display a non-keratinized

epithelium with large rete ridges. The epithelia of minipigs is

also thicker (15-40 cell layers, depending of region) and closer

to that of humans, than that which is found in dogs and rodents.

(Table 1). Connective tissue density in the laminae propria and

number/size of blood vessels are comparable among species

(Fig 1-3). The study also showed that Göttingen Minipigs have

comparable immune cell subpopulations to that of other laboratory

animal species.

Technical considerations

Anatomy

The size and anatomy of the Göttingen Minipig oral cavity

allow for testing drug delivery systems intended for sublingual

and buccal application of similar size and shape as those used

in humans. I.e. in a 15 kg minipig, it is possible to apply an

oval buccal patch of 5.5 cm 2 . Because of limited elasticity and

space between the lips and the gingiva in the minipig, only small

patches can be applied in the gingival site. Buccal and sublingual

application of oral patches and inspection of dosing sites is

Species Area Keratin

No. of epithelial

layers

Rete ridges

Vascularisation

Rat

Dog

Minipig

Human

Sublingual* + 8-12 ++ +++

Cheek + 12-18 + ++

Sublingual* - 8-12 +/- +++

Cheek - 15-25 ++ +++

Sublingual* - 15-20 +++ ++

Cheek - 30-40 +++ +++

Sublingual* - 20-30 ++ +++

Cheek NA NA NA NA

*the ventral surface of the tongue.

NA= Not available

(-) absent, (-/+) minimal, (+) slight, (++) moderate, (+++) marked

Table 1: Histology of oral tissues.


17


A B

Fig 1 : Histology of the ventral surface of the tongue from minipig (a) and human (b) embedded in paraffin and stained with hematoxylin-eosin : the minipigs have

non-keratinized epithelium and rete ridges similar to the ones observed in humans, although less thick. (magnification x200)

A

B

Fig 2 : Collagen fiber density in the mucosa of the ventral surface of the tongue from minipig (a) and human (b) embedded in paraffin and stained with Masson

trichrome : the density of the connective tissue and number/size of blood vessels are similar in both species. (magnification x200)

A

B

Fig 3 : Histology of the ventral surface of the cheek (a) and mouth floor (b) from minipig embedded in paraffin and stained with hematoxylin-eosin : the epithelium

of the cheek is thicker than the epithelium of the mouth floor. Both are thicker than the epithelium of the ventral surface of the tongue (Fig 1, a) and display higher

rete ridges and larger squamous cell size. (magnification x200)

18


Fig 4: Flash release film 4 cm 2

applied on the buccal mucosa in a 20

kg Göttingen Minipig. The mobility

and elasticity of the jaws and lips

are limited in minipigs compared

with other species. This makes

application of patches and inspection

of dosing sites challenging and often

anaesthesia is needed for correct

application

nevertheless challenging in the minipig, because of the limited

mobility and elasticity of the jaws and lips, respectively as well

as the lack of collaboration from the animal when attempting to

open the mouth (Fig 4).

Examples of buccal and sublingual administration in Göttingen

Minipigs are given by Meng-Lund, E. et al.

Application of test item

Application of oral patches in conscious Göttingen Minipigs is

possible, but often a brief anaesthesia is necessary to ensure

correct placement and proper adhesion of the patches. Prolonged

anaesthesia may also be necessary in minipigs with sustained

release systems to allow sufficient time for drug delivery

and prevent that the pig would swallow, loose or destroy the

product. Such precautions would be expected in other animal

species also.

One issue observed in anesthetised minipigs, especially when

of longer duration is reduced salivation and dry mouth, which

impair adhesion of patches and reduce dissolution of e.g. films

and tablets. Thus, the choice of anaesthetics and possibly

addition of humidifiers to the oral cavity should be taken into

consideration.

Conclusion

The oral mucosa of humans and minipigs are similar. Application

of sublingual and buccal films of comparable surface area to

those used in humans is feasible in Göttingen Minipigs. Anaesthesia

is recommended for proper application and drug delivery

especially for slow release drug delivery systems.

References

Thirion-Delalande, C. et al. Comparative analysis of the oral mucosae

from rodents and non-rodents: Application to the nonclinical

evaluation of sublingual immunotherapy products. PLoS ONE

12(9): e0183398. https://doi.org/10.1371/journal.pone.0183398

Meng-Lund, E. et al.. Conscious and anaesthetised Göttingen

minipigs as an in-vivo model for buccal absorption of metoprolol

from bioadhesive tablets. Drug development and industrial

Pharmacy 40:5, 604-610

Meng-lund, E. et al. Buccal absorption of diazepam is improved

when administered in bioadhesive tablets—An in vivo study in

conscious Göttingen mini-pigs. International Journal of Pharmaceutics

515 (2016) 125–131

19


A report from the 12th

Minipig Research Forum 2018

From 16 to 18 May 2018, 101 participants from 16 countries met in Barcelona for the 12th Minipig Research Forum (MRF). Traditionally,

the MRF was kicked-off on the first evening with a cosy get-together dinner & drinks reception where old friends met and

greeted, and new acquaintances were made.

Before the actual MRF, the CONFIRM Initiative hosted a well-attended pre-congress meeting on Wednesday afternoon with three

presentations. The CONFIRM Initiative is a network aiming at generating evidence that minipigs are a relevant non-rodent species for

the immunological safety evaluation of human pharmaceuticals. Read more about the CONFIRM Initiative and how to join on page 28.

Thursday morning, the actual scientific MRF programme set off with a total of 16 inspiring speakers who presented recent scientific

results and shared their deep knowledge and experiences on topics within one of the four key sessions:

• Species selection

• Analytical methods & biomarkers

• Imaging & devices

• Disease models

In the afternoon, the attendees divided into three workshops and participated actively in the discussions on ‘tips & tricks’, ‘species

selection’ and ‘analytical methods’.

Twelve posters were presented during the MRF 2018 and their authors participated in the competition for the best poster. The happy

best poster award winner was Charlotte Bunnage, who is a student worker at Comparative and Translational Sciences at GSK, UK.

Charlotte presented the poster, co-authored by Fiona McClure. We look forward to welcoming Charlotte at the MRF 2019 with a free

registration fee! See the winning poster on page 23.

The speaker presentations including those from the CONFIRM pre-congress meeting as well as summaries of the workshops are

available for Minipig Research Forum members at the MRF website. If you are not a member yet, you are welcome to apply for free

membership at https://minipigresearchforum.org/.

We thank our generous sponsors for supporting this year’s MRF meeting and all participants for yet another successful MRF meeting.

We look forward to seeing you again next year!

The MRF get-together-evening is really about networking: Saying hello to old friends and making new ones; this year while enjoying Spanish tapas inside the venue

hotel and later drinks outside on the terrace in the pleasant Catalonian evening

20


The scientific MRF programme

in action; the first speaker on

stage was Susanne Mohr of F.

Hoffmann-La Roche, Switzerland

presenting Non-rodent species

selection to assure human

relevant safety assessment

Poster viewing is a very

popular part of the program:

Next year we will ensure

more space for this!

Feedback

from

participants:

“ Appreciated

the diversity

of topics and

numerous opportunities

to network



My first MRF:

Impressed

how friendly

and inclusive

all members were



21


Best poster award winner, Charlotte

Bunnage, GSK, UK, with her diploma

congratulated by Ofer Doron, Lahav CRO,

Israel, on behalf of the MRF Steering

Committee.

See the poster on page 23.

Seafood at the gala dinner night

on Thursday at the Olympic

Harbour of Barcelona: Delicious

food, however maybe “a little”

too much: Three more servings

went down! (Less food in 2019:

we promise…!)

Mark your calendar:

MRF 2019

The 13th MRF

from 22 to 24 May 2019

in Vienna, Austria

Follow MRF on

The MRF group on LinkedIn is an informative and useful platform where minipig users connect, interact and share results &

experiences. Apply for MRF LinkedIn group membership by sending us an email: contact@minipigresearchforum.org or click

here: https://www.linkedin.com/groups/4219925

22


MRF 2018 Best Poster

Investigating the the mechanical fragility of of minipigs

erythrocytes, in in comparison with other species

Charlotte Bunnage and Fiona McClure, Comparative and Translational Sciences (CTS), Translational Medicine and Comparative Pathophysiology (TMCP), UK

Charlotte Bunnage and Fiona McClure, Comparative and Translational Sciences (CTS), Translational Medicine and Comparative Pathophysiology (TMCP), UK

All animal studies were ethically reviewed and carried out in accordance with Animals (Scientific Procedures) Act 1986 and the GSK Policy on the Care, Welfare and Treatment of Animals

The All human animal biological studies were samples ethically were reviewed sourced and ethically carried and out their in accordance research use with was Animals in accord (Scientific with the Procedures) terms of the Act informed 1986 and consents the GSK under Policy an on IRB/EC the Care, approved Welfare protocol. and Treatment of Animals

The human biological samples were sourced ethically and their research use was in accord with the terms of the informed consents under an IRB/EC approved protocol.

May 2018

May 2018

Introduction

Introduction

Mechanical fragility of red blood cells (RBCs) refers to the degree

or Mechanical proportion of fragility haemolysis of red that blood occurs cells (RBCs) when RBC’s refers are to the degree

subjected or proportion to mechanical of haemolysis stress. that It is occurs thought when that RBC’s minipigs, are when

being subjected handled to for mechanical in vivo research, stress. It undergo is thought a degree that minipigs, of stress when

which being leads handled to a decreased for in vivo research, resistance undergo and therefore a degree higher of stress

fragility which of leads their to RBCs a decreased [1,2]. The resistance increased and use therefore of minipigs higher in

research fragility corresponds of their RBCs to [1,2]. an observed The increased increase use in of the minipigs number in of

haemolysed research corresponds blood samples to an (figure observed 1) received increase for in clinical the number of

pathology haemolysed analyses blood compared samples (figure with dogs 1) received and rats. for Haemolysis clinical is

known pathology to interfere analyses with compared measurement with dogs of chemistry and rats. biomarkers Haemolysis is

including known sorbitol interfere dehydrogenase with measurement (SDH) of and chemistry Aldolase biomarkers (ALD)

(table including 2). sorbitol dehydrogenase (SDH) and Aldolase (ALD)

Published (table 2). data shows a difference between pig, sheep, human,

cow Published and rabbit data erythrocyte shows a difference fragility [1,2] between and has pig, been sheep, focused human, on

osmotic cow and fragility. rabbit Some erythrocyte investigations fragility [1,2] date and back has to 1969 been focused [3] and on

may osmotic not be fragility. consistent Some with investigations current scientific date rigour. back to No 1969 recent [3] and

published may not data be consistent could be found with current showing scientific a comparison rigour. No of the recent

mechanical published fragility data could of multiple be found species, showing which a comparison could explain of the the

incidence mechanical of haemolysed fragility of multiple samples species, observed. which could explain the

incidence of haemolysed samples observed.

The aim of this experiment is to compare the mechanical fragility

of The multiple aim species, of this experiment to propose is an to answer compare to the the mechanical question, are fragility

minipig of multiple red blood species, cells to propose more fragile an answer than to other question, species? are This

will

minipig

be achieved

red blood

by inducing

cells more

haemolysis

fragile than

over

other

a 30 minute

species?

period

This

via

will

agitation

be achieved

with a

by

steel

inducing

ball and

haemolysis

measuring

over

the haemolysis

a 30 minute

both

period

visually

via agitation

and by

with

measuring

a steel

the

ball

concentration

and measuring

of

the

plasma

haemolysis both

haemoglobin

visually and

(PHb)

by measuring

on the Advia

the concentration

1800 chemistry

of plasma

analyser.

haemoglobin (PHb) on the Advia 1800 chemistry analyser.

Figure 1. Haemolysis index from Advia 1800 showing incidence

Figure 1. Haemolysis index from Advia 1800 showing incidence

and degree of haemolysis from minipig study samples.

and degree of haemolysis from minipig study samples.

Materials



5ml

5ml

lithium

lithium

heparin

heparin

samples

samples

obtained

obtained

from

from

each

each

of

of

3

3

minipigs,

minipigs,

3 rats,

3 rats,

3 human

3 human

and

and

3 dogs

3 dogs

from

from

GSK

GSK

Stevenage/Ware

Stevenage/Ware

UK.

UK.



Denley

Denley

TechSpiramix

TechSpiramix

5:

5:

Used

Used

to

to

rock

rock

500ul

500ul

aliquots

aliquots

of

of

blood

blood

with

with

or

or

without

without

a steel

a steel

ball.

ball.



Steel

Steel

ball

ball

bearing:

bearing:

6.5mm

6.5mm

diameter

diameter

steel

steel

ball

ball

used

used

to

to

mechanically

mechanically

haemolyse

haemolyse

samples.

samples.

• • Pasteur Pasteur pipette: pipette: used used in in preference preference to to pipettes, pipettes, due due to to their their

wider wider based based tips, tips, minimising minimising the the potential potential for for haemolysis haemolysis

caused caused when when transferring transferring samples samples through through restricted restricted space. space.

• • Heraeus Heraeus multifuge multifuge 3 L-R: 3 L-R: After After being being rocked rocked for for the the relevant relevant

time time period period at at a speed a speed of of 3000g 3000g for for 10 10 minutes, minutes, samples samples were were

spun spun

• • Siemens Siemens Advia Advia 1800 1800 Chemistry Chemistry system: system: Used Used to to measure measure

plasma plasma haemoglobin haemoglobin levels, levels, indicating indicating level level of of haemolysis haemolysis

• • Siemens Siemens Advia Advia 2120 2120 Haematology analyser: analyser: Used Used to to

measure measure haematocrit haematocrit (Hct) (Hct) and and Mean Mean Cell Cell Volume Volume (MCV) (MCV) of of

each each baseline baseline sample. sample.

Methods

Samples Samples were were split split in in to to 7 x 7500ul x 500ul aliquots.

Aliquots Aliquots were were rocked rocked with with one one 6.5mm 6.5mm steel steel ball ball for for pre-specified time time

points: points: 2, 2, 5, 5, 10, 10, 20, 20, 30 30 minutes. At At the the appropriate timepoint, an an

aliquot aliquot from from each each animal animal was was removed from from the the rocker rocker and and

centrifuged for for 10 10 minutes at at 3000g, following which which the the plasma was was

separated into into Starstedt tubes, tubes, assessed visually and and measured for for

plasma plasma haemoglobin.

A base A base line line sample (no (no rocking) and and a a control sample (rocked for for 30 30

minutes with with no no steel steel ball) ball) were were also also included.

Visual Inspect ion: ion:

Figure 2. 2. Haemolysis grading sheet, used used to to

visually inspect samples (0-7) (0-7)

Plasma haemoglobin: ADVIA 1800 1800 Chemistry Instrument, a a

quantitative measure. All All samples, standards and and Quality Controls

(QCs) (QCs) were were lysed lysed with with saponin. Hb Hb oxidised to to MetHbin in the the presence

of of Drabkin's alkaline potassium ferricyanide:

MetHb MetHb + KCN + KCN

CyanmetHb

The The absorbance of of the the sample at at 540 540 nm nm is is proportional to to Hb Hb

concentration.

Haematocrit and MCV values: measured on the Advia 2120. Hct

ranging Haematocrit from 37.8% and MCV to 48.5%. values: Samples measured were on not the normalised Advia 2120. for Hct

Haematocrit. ranging from Mean 37.8% MCV to 48.5%. values Samples are also were shown not (table normalised 1). for

Haematocrit. Mean MCV values are also shown (table 1).

Clinical Chemistry: Clinical chemistry analysis was performed on the

plasma Clinical samples Chemistry: using Clinical the Siemens chemistry Advianalysis 1800 Chemistry was performed system on the

(A1800), plasma with samples methodology using the available Siemens [5]. Advia 1800 Chemistry system

Parameters (A1800), with included: methodology aldolase, available alanine [5]. aminotransferase, aspartate

aminotransferase, Parameters included: glutamate aldolase, dehydrogenase, alanine aminotransferase, lactate dehydrogenase, aspartate

urea, aminotransferase, SDH, Na, K, Cl glutamate and plasma dehydrogenase, haemoglobin. lactate dehydrogenase,

QCs urea, included SDH, Na, BL1, K, BL3 Cl and (manufactured plasma haemoglobin. by Randox Laboratories).

SDH1 QCs and included SDH2 BL1, (manufactured BL3 (manufactured by Sekisui by Diagnostics) Randox Laboratories). were QCs for

SDH SDH1 measures and SDH2 and (manufactured pre-specified PHb by Sekisui concentrations Diagnostics) (0.2, were 1, QCs 5 g/ul) for

(manufactured SDH measures in-house) and pre-specified were QCs PHb for haemoglobin concentrations measurements.

(0.2, 1, 5 g/ul)

(manufactured in-house) were QCs for haemoglobin measurements.

Results

Results

Plasma Haemoglobin Mean Fold Change

Plasma Haemoglobin Mean Fold Change

16,00

16,00

14,00

14,00

12,00

12,00

10,00

10,00

8,00

8,00

6,00

6,00

4,00

4,00

2,00

2,00

0,00

0,00 Baseline 2 Min 5 Min 10 Min 20 Min 30 Min

Baseline 2 Min 5 Min 10 Min 20 Min 30 Min

Minipig Rat Dog Human

Minipig Rat Dog Human

Figure 3. Mean plasma haemoglobin fold change comparison between

minipig,

Figure

rat,

3. Mean

dog and

plasma

human

haemoglobin

rocked at varying

fold change

time

comparison

points

between

minipig, rat, dog and human rocked at varying time points

5

Plasma Haemoglobin (g/L)

Plasma Haemoglobin (g/L)

4

3

2

1

0

5

Plasma Haemoglobin (g/L)

Plasma Haemoglobin (g/L)

4

3

2

1

0

Plasma Haemoglobin Raw Change Occasion 1

Plasma Haemoglobin Raw Change Occasion 1

5

4

3

2

1

0

Baseline 2 min 5 min 10 min 20 min 30 min

Baseline 2 min 5 min 10 min 20 min 30 min

Dog 1 Dog 2 Dog 3

Dog 1 Dog 2 Dog 3

Plasma

Plasma

Haemoglobin

Haemoglobin

Raw

Raw

Change

Change

Occasion

Occasion

2

2

5

4

3

2

1

0

Baseline 2 Min 5 Min 10 Min 20 Min 30 Min

Baseline 2 Min 5 Min 10 Min 20 Min 30 Min

Dog 1 Dog 2 Dog 3

Dog 1 Dog 2 Dog 3

Figure Figure 4/5. 4/5. Mean Mean plasma plasma haemoglobin haemoglobin raw raw change change of of dog dog 1, 1, dog dog 22

and and dog dog 3 3 rocked rocked at at varying varying time time points points on on occasion occasion 11 and and 22

Animal Animal

MCV MCV

PHb PHbfold

fold

change change at at 30 30 mins mins

Human Human 89.1 89.1 14.2 14.2

Dog Dog 69.3 69.3 11.0 11.0

Minipig Minipig 51.4 51.4 8.8 8.8

Rat Rat 51.1 51.1 5.6 5.6

Table Table 1. 1. Correlation between mean species MCV and and PHbfold change

at at 30 30 minutes time time point point

Example of of positive and negative interference as as a a result of of

haemolysis in in minipig blood sample.

Animal

Parameter

0 0 min min

no. no.

(U/L)

22 min 55 min 10 10 min 20 20 min 30 30 min

1 1 ALD 14 14 15 15 17 17 21 21 33 33 33 33

2 2 ALD 13 13 14 14 18 18 26 26 70 70 55 55

3 3 ALD ALD 14 14 15 15 17 17 22 22 28 28 29 29

1 1 SDH 5.3 5.3 -2.5 -2.5 -10.3 -20.1 -32.8 -53.5

2 2 SDH SDH 2.4 2.4 -3.6 -3.6 -8.1 -8.1 -11.5 -17.4 -30.3

3 3 SDH SDH 4.2 4.2 -1.3 -1.3 -6.3 -6.3 -12.6 -27.7 -34.6

Table 2. 2. Aldolase (ALD) and and Sorbitol Dehydrogenase (SDH) activities

from from minipig blood samples at at varying time time points of of rocking

Discussion

Discussion

This investigation compared the haemolysis of erythrocytes from

minipig, This investigation rat, dog and compared humans the which haemolysis underwent of erythrocytes mechanical stress from

for minipig, up to 30 rat, minutes dog and using humans Plasma which Hbunderwent and visual mechanical inspection as stress

endpoints. for up to 30 Other minutes chemistry using Plasma parameters Hb and were visual measured inspection but not as all

presented endpoints. here. Other This chemistry investigation parameters showed were that measured minipig but not all

erythrocytes presented here. were This not investigation more susceptible showed to mechanical that minipig stress than

dogs erythrocytes although were they not appear more to susceptible be more susceptible to mechanical than stress rat than

erythrocytes. dogs although Human they appear samples to be appeared more susceptible to be most than susceptible rat to

mechanical erythrocytes. stress. Human Susceptibility samples appeared to mechanical to be most fragility susceptible appeared to

to mechanical correlate with stress. MCV Susceptibility values (table1), to mechanical i.e. small erythrocytes

fragility appeared

appear to correlate to be with less MCV fragile values than (table1), larger ones, i.e. small which erythrocytes

has been noted

in appear the literature to be less previously fragile than [4]. larger Plasma ones, Hb results which has from been the Advia noted

1800 in the and literature visual previously haemolysis [4]. scoring Plasma correlated, Hb results making from the results Advia

more 1800 reliable and visual and haemolysis confirm that scoring visual correlated, assessment making is adequate results for

determination more reliable and of degree confirm of that haemolysis. visual assessment Due to technical is adequate issues, for

human determination results presented of degree of were haemolysis. not performed Due to on technical the same issues, day as

the human other results species. presented However, were the not same performed dog samples on the were same day as

measured the other species. on both occasions: However, the Figures same 4 dog and samples 5 demonstrates were the

consistency measured on between both occasions: occasions Figures for the 4 dog. and 5 demonstrates the

consistency between occasions for the dog.

Table 2 shows the minipig ALD and SDH results when you induce

haemolysis Table 2 shows in samples. the minipig This ALD table and emphasises SDH results the when interference you induce

that haemolysis haemolysis in samples. can have This on table chemistry emphasises endpoints the reiterating interference the

importance

that haemolysis

of minimising

can have

disruption

on chemistry

in these

endpoints

samples.

reiterating

Other

the

chemistry

importance

parameters

of minimising

also

disruption

showed interference

in these samples.

effects

Other

but have

not

chemistry

been presented

parameters

here.

also showed interference effects but have

not been presented here.

From results displayed and previous processing investigations,

looking

From results

at standard

displayed

centrifugation

and previous

and

processing

storage conditions,[6]

investigations,

the

prevalence

looking at standard

of haemolysis

centrifugation

in minipig

and

samples

storage

received

conditions,[6]

for analysis

the

prevalence of haemolysis in minipig samples received for analysis

cannot be attributed to the fragility of the minipig erythrocyte or

cannot be attributed to the fragility of the minipig erythrocyte or

post sampling processes.

post sampling processes.

Conclusions

• Red blood cells of minipigs were found not to be more fragile

• Red blood cells of minipigs were found not to be more fragile

than dogs or humans but more fragile than rats, consistent with

than dogs or humans but more fragile than rats, consistent with

previous literature [1,2]

previous literature [1,2]

• Fragility of erythrocytes correlates with the MCV consistent

• Fragility of erythrocytes correlates with the MCV consistent

with previous literature [4].

with previous literature [4].

• There is still further investigation required to reduce the

• There is still further investigation required to reduce the

incidence

incidence

of

of

haemolysis

haemolysis

observed

observed

in

in

minipig

minipig

blood

blood

samples,

samples,

possible

possible

investigations

investigations

could

could

include:

include:



Influence

Influence

of

of

bleed

bleed

sites

sites



Correlation

Correlation

of

of

incidence

incidence

of

of

haemolysis

haemolysis

with

with

cortisol

cortisol

or

or

adrenaline.

adrenaline.



Influence

Influence

of

of

acclimatisation

acclimatisation

and

and

training

training

Acknowledgements

Aubrey Swain, Rebecca Grice, Mitul Gandhi - Clinical Pathology and

Diagnostics, TMCP, IBPS, GSK UK

Neil Young, Matt Gillespie, Matt L'Affineur – In Vivo Science Delivery

Team, IBPS, GSK UK

Blood Donation Unit, GSK Stevenage

References

[1] Oyewale, J. J. (1992). Changes in Osmotic Resistance of Erythrocytes

of of Cattle, Pigs, Rats and Rabbits During Variation in Temperature and

pH. Journal of Veterinary Medicine Series A, 39(1-10), pp.98-104.

[2] Brzezinska-slebodzinska,E., 2003. Species differences in the

susceptibility of erythrocytes exposed to free radicals in vitro. Veterinary

Research Communications, 27 (3), 211-217

[3] Coldman MF, Gent M, Good W (1969) The osmotic fragility of

mammalian erythrocytes in hypotonic solutions of sodium chloride.

Comp Biochem Physiol. 1969 Nov 15;31(4):605-9

GSK Internal Communication- Charlotte Bunnage and Fiona McClure

[4] Betticher DC1, Geiser J Resistance of mammalian red blood cells of

different size to hypertonic milieuComp Biochem Physiol A Comp

Physiol. 1989;93(2):429-32

[5] GSK SOP: Operation and Maintenance of the Siemens

Clinical Chemistry System (Advia 1800), UK

Clinical Pathology and Diagnostics SOP_49272

[6] GSK poster: Charlotte Bunnage/ Fiona McClure, "Effect of

processing conditions on erythrocyte haemolysis in minipigs"

Created by: Digital Media Services

23


Update

from Japan

Oriental Yeast Co., Ltd. (OYC) started to breed Göttingen Minipigs in Japan in 2013 in close cooperation with EGM.

Despite the distance between Denmark and Japan, we enjoy the fruitful relations with EGM which have helped OYC

to become a professional and successful breeder supplying Göttingen Minipigs to Japan and Taiwan. We are very

proud to report that our facility has been AAALAC fully re-accredited in March 2018, and we are indeed honored to

be highly reputed in the formal notification from AAALAC International regarding the efforts of our staff concerning

animal welfare:

Our facility especially attaches great emphasis on socialization of the animals and the amount of bedding given to

the animals. This is because socialization does not only facilitate animal handling and health observation, but also

has a good influence on their health and welfare. In addition, providing bedding to pigs also can help the animals to

promote their intrinsic behavior and keep the pens in a clean condition.

24


Update

from Korea

WOOJUNGBIO (which changed its name from WOOJUNGBSC in April 2018) has been the distributor of Göttingen

Minipigs in Korea since 2015 in partnership with EGM and is working closely with EGM on e.g. training and education

of the staff handling the animals. WOOJUNGBIO also organizes scientific seminars and practical workshops in

Korea on the use of Göttingen Minipigs for the benefit of scientists from all over the country.

There is a growing interest in and demand for Göttingen Minipigs in Korea from pharmaceutical companies as well

as CROs and universities as the pharmaceutical industry in Korea is rapidly developing; WOOJUNGBIO has a close

relation to KIT, The Korea Institute of Toxicology, which opened a new full barrier minipig SPF facility in October

2017 and started to support preclinical CRO and research services for a large number of the Korean pharmaceutical

companies. This facility will also be used for the quarantine of Göttingen Minipigs when imported to Korea with a

high focus on the animal welfare. Feel free to contact WOOJUNGBIO directly for information about the supply of

Göttingen Minipigs to the Korean market.

KIT Minipig Research Center

Capacity: 108 minipigs

25


Update

from North America

2018 is a strong year for joint exhibition participation between Marshall BioResources and Ellegaard Göttingen

Minipigs: Thus, in March we jointly hosted two well attended sessions at the SOT Annual Meeting held in San

Antonio, Texas, and during the Autumn, we will be present together at the SPS Annual Meeting at the beginning

of October to be held in Washington DC, as well as the ACT Annual Meeting in November in Palm Springs, Florida.

Please come and visit our booth!

Furthermore, our third Göttingen Minipig Symposium (GMS) took place on 15 and 16 August in Chicago, Illinois with

a total of 60 attendees. We had a very exciting program this year which featured topics such as minipig models

of cardiomyopathy and heart failure, minipig models of metabolic disease, gene and cell therapy delivery models,

and new technology for continuous glucose monitoring in large animals. We also had discussion about enrichment,

clicker training, and surgical techniques. CEO Lars Friis Mikkelsen represented Ellegaard Göttingen Minipigs at the

Symposium and held two talks on the recent scientific results and development within lifestyle diseases and transgenic

Göttingen Minipigs. We are also very thankful to Experimur who provided a history covering their experience

with Göttingen Minipigs for over almost 20 years, and who provided tours of their state-of-the-art facilities, along

with demonstrations for jacketed external telemetry and external catheterization in Göttingen Minipigs.

Our 2018 Reference Data Guides are now available for Göttingen Minipigs®, as well as our Marshall Beagles®,

and Mongrels & Hounds. You can access them on our website, https://www.marshallbio.com/, by logging into your

account and then selecting the corresponding animal page.

If you have not yet registered on our website for access to our background data and educational materials, you can

register for an account or update your profile at https://www.marshallbio.com/auth/profile. You can also contact us

at marketing@marshallbio.com to request a copy.

Marshall BioResources, from the left:

Michelle Salerno, Lars Friis Mikkelsen (EGM), Nicole Navratil, Karen Simpson

26


New scientific publications on Göttingen Minipigs

Ellegaard Göttingen Minipigs A/S gives high priority to collaborative projects that aim to better characterize and validate the

Göttingen Minipig as a translational animal model and which facilitate and refine the use of the minipig in research projects and

safety testing. Please contact us if you have an idea for such a collaborative project. Below is a list of a few recent articles on

Göttingen Minipigs.

• Christensen AB, Sørensen JCH, Ettrup KS, Orlowski D, Bjarkam CR Pirouetting pigs: A large non-primate animal model based on

unilateral 6-hydroxydopamine lesioning of the nigrostriatal pathway. Brain Res Bull. 2018 May;139:167-173. doi: 10.1016/j.

brainresbull.2018.02.010. Epub 2018 Feb 17.

• Lillethorup TP, Glud AN, Alstrup AKO, Mikkelsen TW, Nielsen EH, Zaer H, Doudet DJ, Brooks DJ, Sørensen JCH, Orlowski D,

Landau AM. Nigrostriatal proteasome inhibition impairs dopamine neurotransmission and motor function in minipigs.

Exp Neurol. 2018 May;303:142-152. doi: 10.1016/j.expneurol.2018.02.005. Epub 2018 Feb 8.

• Landau AM, Alstrup AK, Audrain H, Jakobsen S, Simonsen M, Møller A, Videbech P, Wegener G, Gjedde A, Doudet DJ. Elevated

dopamine D1 receptor availability in striatum of Göttingen minipigs after electroconvulsive therapy. J Cereb Blood Flow

Metab. 2018 May;38(5):881-887. doi: 10.1177/0271678X17705260. Epub 2017 May 16.

• Descotes J, Allais L, Ancian P, Pedersen HD, Friry-Santini C, Iglesias A, Rubic-Schneider T, Skaggs H, Vestbjerg P. Nonclinical

evaluation of immunological safety in Göttingen Minipigs: The CONFIRM initiative. Regul Toxicol Pharmacol. 2018 Apr;94:271-

275. doi: 10.1016/j.yrtph.2018.02.015. Epub 2018 Feb 23.

• Dincer Z, Piccicuto V, Walker UJ, Mahl A, McKeag S. Spontaneous and Drug-induced Arteritis/Polyarteritis in the Göttingen

Minipig. Review. Toxicol Pathol. 2018 Feb;46(2):121-130. doi: 10.1177/0192623318754791.

• Rasmussen BS, Sørensen CL, Vester-Glowinski PV, Herly M, Kurbegovic S, Ørholt M, Svalgaard JD, Kølle ST, Kristensen AT,

Talman MM, Drzewiecki KT, Fischer-Nielsen A. A Novel Porcine Model for Future Studies of Cell-enriched Fat Grafting. Plast

Reconstr Surg Glob Open. 2018 Apr 4;6(4):e1735. doi: 10.1097/GOX.0000000000001735. eCollection 2018 Apr.

• Coelho PG, Pippenger B, Tovar N, Koopmans SJ, Plana NM, Graves DT, Engebretson S, van Beusekom HMM, Oliveira PGFP,

Dard M. Effect of Obesity or Metabolic Syndrome and Diabetes on Osseointegration of Dental Implants in a Miniature Swine

Model: A Pilot Study. J Oral Maxillofac Surg. 2018 Mar 1. pii: S0278-2391(18)30185-X. doi: 10.1016/j.joms.2018.02.021.

[Epub ahead of print]

• O'Neill L, Harrison J, Chubb H, Whitaker J, Mukherjee RK, Bloch LØ, Andersen NP, Dam H, Jensen HK, Niederer S, Wright M,

O'Neill M, Williams SE. Voltage and pace-capture mapping of linear ablation lesions overestimates chronic ablation gap size.

Europace. 2018 Apr 26. doi: 0.1093/europace/euy062. [Epub ahead of print]

• Shrader SM, Greentree WF. Göttingen Minipigs in Ocular Research. Toxicol Pathol. 2018 Jun;46(4):403-407. doi:

10.1177/0192623318770379. Epub 2018 Apr 22.

• Henze LJ, Griffin BT, Christiansen M, Bundgaard C, Langguth P, Holm R. Exploring gastric emptying rate in minipigs: Effect of

food type and pre-dosing of metoclopramide. Eur J Pharm Sci. 2018 Jun 15;118:183-190. doi: 10.1016/j.ejps.2018.03.017.

Epub 2018 Mar 17.

• Renner S, Blutke A, Dobenecker B, Dhom G, Müller TD, Finan B, Clemmensen C, Bernau M, Novak I, Rathkolb B, Senf S, Zöls S,

Roth M, Götz A, Hofmann SM, Hrabĕ de Angelis M, Wanke R, Kienzle E, Scholz AM, DiMarchi R, Ritzmann M, Tschöp MH, Wolf

E. Metabolic syndrome and extensive adipose tissue inflammation in morbidly obese Göttingen minipigs. Mol Metab. 2018

Jun 28. pii: S2212-8778(18)30300-4. doi: 10.1016/j.molmet.2018.06.015. [Epub ahead of print]

• Pique C, Marsden E, Quesada P, Blondel A, Mikkelsen LF. A Shortened Study Design for Embryo-Fetal Development Studies

in the Minipig. Reprod Toxicol. 2018 Jun 22. pii: S0890-6238(18)30096-0. doi: 10.1016/j.reprotox.2018.06.009. [Epub ahead

of print]

27


Join the CONFIRM Initiative

The CONFIRM Initiative is aimed at generating evidence that minipigs are a relevant non-rodent

species for the immunological safety evaluation of human pharmaceuticals by setting up, promoting

and coordinating a COllaborative Network For Immunological safety Research in Minipigs, abbreviated

into ‘CONFIRM’.

The CONFIRM Initiative is intended to:

• serve as a trigger for immunological safety research in Göttingen Minipigs

• assist and synergize fundamental, translational and regulatory investigative efforts relevant to immunological safety

evaluation in Göttingen Minipigs

• spread current knowledge and new findings to the scientific and regulatory toxicology community

The first major meeting of the CONFIRM Initiative was held at the Minipig Research Forum in Barcelona in May and included

three presentations which can be read at https://minipigresearchforum.org/members-area/the-mrf-meeting-2018/. We call

on you to actively participate in the Initiative. Learn more at https://minipigs.dk/knowledge-base/the-confirm-initiative/ and

please fill in the application form online If you are interested in joining. You can also apply for membership of our LinkedIn

Group at https://www.linkedin.com/groups/13544253

52 AUTUMN 2018

NEWSLETTER

••

••

••

••

••

••

••

••

••

••

Towards 3Rs: Multiplex protein profiling

from minipig, cyno, dog and rat samples,

to get the most out of preclinical safety

studies page 3

Experience with mixed groups of intact

female and castrated male Göttingen Minipigs

See

where you

can meet us

in 2018

page 28

Minipig

Research

Forum 2018

page 20

for pharmacokinetic studies in drug discovery research

– summary of behavioral observations and collected

back-ground data page 6

Anti-cancer drug development: Comparison of toxicity

in Göttingen Minipigs and mouse page 10

Body surface area measurement in Göttingen Minipigs

using a computed tomography scanner page 14

The Göttingen Minipig for the study of buccal and sublingual

products page 17

Update from Oriental Yeast Co., Ltd., Japan page 24

Update from WOOJUNGBIO, Korea page 25

Update from Marshall BioResources North America page 26

New scientific publications on the Göttingen Minipig page 27

Join the CONFIRM Initiative page 28

Clean pigs

for clear results

The next Newsletter delivered

directly to your inbox

Our scientific newsletter is published three times a year. We welcome new and former

subscribe that we might have lost contact with during the recent GDPR process, and

ask you to sign up at https://minipigs.dk/contact-us/sign-up-for-our-newsletter/.

If you have suggestions for topics and/or author, for a comming issue of the Newsletter,

please email the editor: Søs Pihl-Poulsen at spp@minipigs.dk.

Meeting Calendar Autumn 2018

Name Date Location

EUROTOX 2 - 5 September Brussels, Belgium

ESTP 11 - 14 September Copenhagen, Denmark

GV-SOLAS + IGTP 12 - 14 September Munich, Germany

SPS 30 September - 3 October Washington, Columbia, USA

LAVA-ESLAV-ECLAM 15 - 16 October Barcelona, Spain

STP-I 26 - 28 October Hyderabad, India

ACT 4 - 7 November Palm Beach, Florida, USA

AFLAS 28 November - 1 December Bangalore, India

Europe and Asia

Ellegaard Göttingen Minipigs A/S

Sorø Landevej 302,

DK-4261 Dalmose,

Denmark

Tel.: +45 5818 5818

ellegaard@minipigs.dk

North America

Marshall BioResources

North Rose, NY 14516, USA

Tel.: +1 315 587 2295

Fax: +1 315 587 2109

infous@marshallbio.com

Japan & Taiwan

Oriental Yeast Co. Ltd.

3-6-10, Azusawa, Itabashi-ku

Tokyo, 174-8505, Japan

Tel.: +81 3 3968 1192

Fax: +81 3 3968 4863

fbi@oyc.co.jp

Korea

WOOJUNGBIO

B-3F, 145 Gwanggyo-ro,

Yeongtong-gu, Suwon, Korea

Tel.: +82 31 888 9369

Fax: +82 31 888 9368

wj@woojungbsc.co.kr

www.minipigs.dk

JANNERUP GRAFISK

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