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CARDIOVASCULAR AND NEUROBEHAVIORAL PRECLINICAL DRUG SAFETY ENDPOINTS IN A GÖTTINGEN MINIPIG MODEL
Metea, M.R.; Burke, A.S.; Setser, J.J.; Gleason, T.R.; Landis, K.L.;
Shellhammer, L.J.; Turchyn, L.; Allis, A.; Enama, T.T.; Atterson, P.R.
WIL Research Japan K.K. • Tokyo 105-0004Phone 81-(0)3-5776-5234 • Fax 81-(0)3-5776-2624
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CARDIOVASCULAR AND NEUROBEHAVIORAL PRECLINICAL DRUG SAFETY ENDPOINTS IN A GÖTTINGEN MINIPIG MODEL
Metea, M.R.; Burke, A.S.; Setser, J.J.; Gleason, T.R.; Landis, K.L.; Shellhammer, L.J.; Turchyn, L.; Allis, A.; Enama, T.T.; Atterson, P.R.WIL Research Laboratories, LLC, 1407 George Road, Ashland, OH 44805
SUMMARY
The Göttingen minipig presents advantages as an alternative non-rodent model for toxicology
and safety pharmacology studies due to its numerous similarities to humans in the anatomy
of organs (such as the skin, heart or brain), as well as the function of all major physiological
systems (Bode, 2010). The heart of the minipig is similar to the human heart anatomically (i.e.
coronary artery distribution, Purkinje fi bers) and functionally (i.e. susceptibility to infarction,
response to standard drugs affecting cardiovascular parameters, etc). A large database of
knowledge exists on the swine brain, which is extensively used as a model in neuroscience and
is increasingly accepted for regulatory toxicology requiring neurobehavioral studies. In general,
the swine is a pharmacological model more similar in function to humans for both cardiovascular
and neurological testing and is recommended in cases where either species-specifi c toxicity is
encountered in non rodent models, emesis is prohibitive (especially in dogs), or the metabolical
differences are signifi cant. In the present study we evaluated the feasibility of this model for
neurological and cardiovascular evaluation in support of preclinical studies. In particular, we
developed a Functional Observational Battery (FOB) consisting of neurological evaluations and
behavioral assessments of normal physiological functions. Further, we evaluated the parameters
included in the FOB; validated the procedures; and demonstrated the inter-observer reliability
following the administration of neuro-modulating chemicals (d-amphetamine and clonidine).
For the cardiovascular component of the study, we tested implanted and external telemetry
devices and validated their use and sensitivity for measurements of ECG or hemodynamic
variables, based on changes elicited with moxifl oxacin or propanolol.
METHODSCardiovascular evaluation
Six male Göttingen minipigs implanted with telemetry transmitters and also equipped with
Jacketed External (JET) Devices were dosed according to a crossover design with vehicle
(0.5% methylcellulose and 0.1% polysorbate 80), moxifl oxacin at 100 mg/kg, and propanolol
at 20 mg/kg, and data were continuously collected for 22 hours postdose (ECG and
hemodynamic parameters).
The implanted telemetry system emulated Lead II confi guration and consisted of large animal
transmitters (TL11M2-D70-PCT with arterial pressure, electrocardiographic waveform and body
temperature capabilities) confi gured to monitor each animal at a sampling rate of 500 Hz for
ECG and pressure signals, and 50 Hz for body temperature signals. The implanted telemetry
data were recorded by DSI PONEMAH Physiology Platform (P3 Plus) version 4.80 SP 2 with
Dataquest™ OpenART™ Platinum, version 3.11, and analyzed by DSI PONEMAH Physiology
Platform (P3 Plus), version 4.80 SP 2 with ECG Template Analysis Option. The external telemetry
system consisted of the Jacketed External (JET) Devices (3ETA-EXP), a JET Bluetooth receiver
and jacket. The system was confi gured to monitor each animal at a sampling rate of 500 Hz.
ECG waveforms were recorded by DSI PONEMAH Physiology Platform (P3 Plus) version 4.7
and analyzed by DSI PONEMAH Physiology Platform (P3 Plus), version 4.80 SP 2 with ECG
Template Analysis Option. Templates were selected separately for each system. Qualitative
assessments of ECG waveforms were performed by trained personnel for disturbances in
rhythm and waveform morphology, for both paradigms. Heart rate-corrected QT (QTc) values
were calculated with the following formulas: QTcV = QT - 0.087*(RR-1) (Van de Water et al.,
1989; Spence et al., 1998), QTcB = QT interval divided by the square root of the RR interval
(Bazett, 1920), and QTcF = QT interval divided by the cube root of the RR interval (Fridericia,
1920). ECG parameters were analyzed with a repeated measure analysis of covariance for each
acquisition method, and the results compared.
Neurobehavioral evaluation
FOB parameters deemed appropriate for the minipigs were initially established (Table 2).
Subsequent FOB evaluations with these parameters were conducted in 3 consecutive phases.
Phase I: FOBs of untreated animals. 5 males and 5 females, 7-10 months old and 10-14 kg were
selected for the training of personnel.
Phase II: FOBs of positive control-treated animals. Two neuroactive substances, d-amphetamine
sulfate (0.7 mg/kg) or clonidine hydrochloride (0.03 mg/kg) were administered as single doses by
intramuscular injection (0.2 mL/kg) to 1 male and 1 female. FOB assessments were performed
prior to dosing and at approximately 0.5 and 1 hour postdose.
Phase III: Assessment of the inter-observer reliability. The following criteria were used to evaluate
the reliability of individual observers in comparison to the most experienced observer (Table 4):
equal to or greater than 95% concordance with all parameters of the FOB as a whole; a mean
difference of approximately 20% or less for quantitative parameters; a difference of no more
than one grade for graded parameters; agreement on qualitative parameters that were graded
either present or absent; equal to or greater than 70% concordance for qualitative parameters.
During this phase, 9 animals/sex were administered a single intramuscular injection of
d-amphetamine or clonidine hydrochloride or a concurrent control, 0.9% sodium chloride. Doses
were administered a total of 3 days with each dosing day consisting of 6 previously untreated
animals (1 animal/sex/group) receiving one of the 3 treatments. FOB assessments were again
performed prior to dosing and at approximately 0.5 and 1 hour after dose administration. Testing
was performed by the technicians without knowledge of the animals’ group assignment.
RESULTS
Fig. 1: ECG waveforms. a) Example of waveforms collected with each system. Notable is the
short amplitude R wave and the large S wave, typical to Lead II in this species. b) Comparison
between dog and pig ECG waveforms with Lead II confi guration.
Fig. 2: Changes in cardiovascular parameters over the data collection period.
Table 1: Statistical Analysis of Data. ECG data was analyzed with a repeated measure
analysis of covariance, based on light/dark cycle-related phases.
Merged cells imply analysis was conducted across the pooled time intervals
NS: Not statistically signifi cant
↑ (↓) Dose-related increase (decrease) for moxifl oxacin
Table 2: FOB parameters selected for use in the minipig.
Table 3: Summary of positive functional observations at 30 and 60 minutes postdose.
Table 4: Inter-observer reliability testing. The comparison met the criteria used to evaluate
the reliability of individual observers in comparison to the most experienced observer
(observer B).
CONCLUSIONS
The data support the use of the Göttingen minipig model for assessment of cardiovascular and
neurobehavioral function in safety pharmacology and toxicology studies. For cardiovascular
studies, the model was appropriate for use with both invasive and non-invasive telemetry
methods. Quality ECG waveforms were obtained with both the implanted and external systems,
and a procedure was developed to mark the minipig-specifi c waveforms for quantitative
analysis. QT prolongation with moxifl oxacin was detected adequately and was comparable
between the two telemetry methods for all standard correction factors tested. Statistically
signifi cant changes in hemodynamic variables were elicited with propanolol, validating the
sensitivity of the telemetry system to detect these changes.
For neurobehavioral assessment, the FOB parameters selected were deemed to be practical
for measurement, reasonable for this species, and provided an adequate assessment of the
motor, sensory, and autonomic nervous function based on the sensitivity of the test in capturing
the positive control effects at appropriate postdose timepoints. The inter-observer reliability
was high, supporting the use of this assay in standard preclinical settings, where standard
processes are needed.
Overall, this model is recommended for use as an alternative large animal species for toxicology
and pharmacology studies.
REFERENCES
Bazett, HC. An analysis of the time-relations of the electrocardiograms. Heart, 7, pp.353-370.
1920.
Bode, G., Clausing, P., Gervais, F., Loegsted, J., Luft, J., Nogues, V., Sims, J. The utility of the
minipig as an animal model in regulatory toxicology. J. Pharmacol. Methods, 62, pp.196-220. 2010.
Fridericia, L.S. The duration of systole in an electrocardiogram in normal humans and in patients
with heart disease. Acta Medica Scandinavica, 53, pp.469-486. 1920.
SAS Institute, Inc. SAS® Proprietary Software Release, Version 9.1, SAS Institute, Inc., Cary, NC.
2002-2003.
Spence, S., Soper, K., Hoe, C-M. and Coleman, J. The heart rate-corrected QT interval of conscious
Beagle dogs: A formula based on analysis of covariance. Toxicological Sciences, 45, pp.247-258.
1998.
Van de Water, A., Verheyen, J., Xhonneux, R. and Reneman, R.S. An improved method to correct
the QT interval of the electrocardiogram for changes in heart rate. J. Phamacol. Methods, 22(3),
pp.207-217. 1989.
ACKNOWLEDGMENTS
The authors acknowledge the excellent technical support provided by the staff of WIL
Research Laboratories, LLC.
NOVEMBER 2010
TABLE 1 Statistical Analysis QTc IntervalsPhase Time (hour) QTcB1 QTcB2 QTcF1 QTcF2 QTcV1 QTcV2
1 1 ↑ NS ↑ NS ↑ NS2 ↑ ↑ ↑ ↑ ↑ ↑3 ↑ ↑ ↑ ↑ ↑ ↑4 ↑ ↑ ↑ ↑ ↑ ↑5 NS NS NS NS NS NS6 ↑ ↑ ↑ ↑ ↑ ↑
Overall ↑ ↑ ↑ ↑ ↑ ↑2 7-8
↑ ↑ ↑ ↑ ↑ ↑
9-1011-1213-1415-1617-18
Overall1 – External telemetry data 2 – Implanted telemetry data
TABLE 2 Functional Observational BatteryHome Cage Observations Open Field Table Top Observations
General Appearance Time to First Step Respiration Rate/Pattern Capillary Refi ll Time1
Behavior Gait Perineal Refl ex Cliff Avoidance
Posture and Stance Behavior Body Temperature Righting Refl ex
Head Posture Heart Rate Proprioceptive Positioning
Body Symmetry Auditory Response Wheel Barrowing3
Convulsions/Tremors Pinna Sensitivity Hemistanding/Hemiwalking
Salivation Menace Response Toe Sensitivity2
Lacrimation Palpebral Fissure Triceps/Patellar Refl ex
Excreta Pupillary Size/Light Refl ex
Emesis Pathologic Nystagmus1 When evaluating capillary refi ll time for the minipigs a blunt instrument is needed to apply pressure to the snout as opposed to applying pressure to the gums of the teeth as in other species such as the dog.
2 The sensitivity response was also performed by prodding the interdigital skin between the hooves with a blunt needle as opposed to prodding in between the toes of an animal with paws as in other species such as the dog.
3 The wheelbarrowing of the animal while holding the front legs can only be performed by walking the animal backwards as opposed to walking the animal both forward and backward as in other species such as the dog so this parameter was modifi ed to only include the observation of the minipig to walk backwards. Parameters performed in other species such as posterior extension thrust (holding the animal from the rear under the front legs (axillary area) and slowly lowering it toward the fl oor to observe whether hind legs extend as fl oor nears) and gag refl ex were not considered for evaluation in the minipig FOB due to the lack of feasibility.
TABLE 3 Summary of Positive Functional Observations30 Minutes Post-Dose 60 Minutes Post-Dose
Amphetamine Clonidine Amphetamine Clonidine
General Appearance Abnormal Abnormal
Behavior Hyperactive Hypoactive Hyperactive Hypoactive
Posture and Stance Abnormal Abnormal
Head Posture Drooped Drooped
Salivation Abnormal Dryness Abnormal Dryness
Respiration Rate Increased Decreased Increased Decreased
Respiration Pattern Rapid Slow Rapid Slow
Heart Rate Decreased Increased Decreased
Auditory Response Absent
Capillary Refi ll Time Prolonged
Toe Sensitivity Absent Absent
TABLE 4 Inter-Observer Reliability of the Functional Observational Battery in the MinipigOBSERVER CONCORDANCE (%)
NUMBER OF MINIPIGS 18 12 12 6 6 6
aOBSERVER B B B B B B
OBSERVER A D G E F C
General Appearance 100 100 100 100 100 100
Behavior 100 100 100 100 100 100
Posture and Stance 100 100 100 100 100 100
Head Posture 100 100 100 100 100 100
Body Symmetry 100 100 100 100 100 100
Convulsions/Tremors 100 100 100 100 100 100
Salivation 83 100 83 100 100 100
Lacrimation 89 92 100 100 100 100
Excreta 100 100 100 100 100 100
Emesis 100 100 100 100 100 100
Gait 100 100 100 100 100 100
Behavior 100 100 100 100 100 100
Respiration Rate 94 88 84 95 79 100
Respiration Pattern 89 83 83 83 83 100
Perineal Refl ex 100 92 100 100 100 100
Heart Rate 93 96 95 97 93 93
Auditory Response 89 92 100 100 100 100
Pinna Sensitivity 100 100 100 100 100 100
Menace Response 100 100 100 100 100 100
Palpebral Fissure 100 100 100 100 100 100
Pupillary Size 100 100 100 100 100 100
Pupillary Light Refl ex 100 100 100 100 100 67
Pathologic Nystagmus 100 100 100 100 100 100
Capillary Refi ll Time 100 92 83 100 67 67
Cliff Avoidance 100 100 100 100 100 100
Righting Refl ex 100 100 100 100 100 100
Proprioceptive Positioning 100 100 100 100 100 100
Wheelbarrowing - Forelimbs 100 100 100 100 100 100
Wheelbarrowing - Hindlimbs 100 100 100 100 100 100
Hemistanding/Hemiwalking - Left Side 100 100 100 100 100 100
Hemistanding/Hemiwalking - Right Side 100 100 100 100 100 100
Toe Sensitivity 100 100 100 100 100 100
Triceps Refl ex - Right Forelimb 100 100 100 100 100 100
Triceps Refl ex - Left Forelimb 100 100 100 100 100 100
Patellar Refl ex - Right Hindlimb 100 100 100 100 100 100
Patellar Refl ex - Left Hindlimb 100 100 100 100 100 100
aAll observers were compared to observer B.
ECG Waveforms from an Implanted Telemetry Transmitter
a) External vs Implanted Telemetry (Pig)
b) Comparison between Dog and Pig ECG Waveforms
Fig. 1 ECG Waveforms
ECG Waveforms from Jacketed External Telemetry Leads
ECG Waveforms from an Implanted Telemetry Transmitter (Pig)
ECG Waveforms from an Implanted Telemetry Transmitter (Dog)
MEAN ARTERIAL PRESSURE-IMPLANTEDMean ± SEM
BA
SE
LIN
E 1 2 3 4 5 6
7-8
9-10
11-1
2
13-1
4
15-1
6
17-1
8
19-2
0
21-2
2
60
80
100
120
140
160
180
0 mg/kg
100 mg/kg MOXI
20 mg/kg PROP Time (Hours)
Mea
n A
rter
ial P
ress
ure
(mm
Hg)
DIASTOLIC BLOOD PRESSURE-IMPLANTEDMean ± SEM
BA
SE
LIN
E 1 2 3 4 5 6
7-8
9-10
11-1
2
13-1
4
15-1
6
17-1
8
19-2
0
21-2
2
50
70
90
110
130
150
0 mg/kg
100 mg/kg MOXI
20 mg/kg PROP Time (Hours)
Dia
stol
ic B
lood
Pre
ssur
e (m
mH
g)
SYSTOLIC BLOOD PRESSURE-IMPLNATEDMean ± SEM
BA
SE
LIN
E 1 2 3 4 5 6
7-8
9-10
11-1
2
13-1
4
15-1
6
17-1
8
19-2
0
21-2
2
100
150
200
0 mg/kg
100 mg/kg MOXI
20 mg/kg PROPTime (Hours)
Sys
tolic
Blo
od P
ress
ure
(mm
Hg)
PULSE PRESSURE-IMPLANTEDMean ± SEM
BA
SE
LIN
E 1 2 3 4 5 6
7-8
9-10
11-1
2
13-1
4
15-1
6
17-1
8
19-2
0
21-2
2
20
30
40
50
60
70
0 mg/kg
100 mg/kg MOXI
20 mg/kg PROP Time (Hours)
Pul
se P
ress
ure
(mm
Hg)
BODY TEMPERATURE-IMPLANTEDMean ± SEM
BA
SE
LIN
E 1 2 3 4 5 6
7-8
9-10
11-1
2
13-1
4
15-1
6
17-1
8
19-2
0
21-2
2
34
36
38
40
42
0 mg/kg
100 mg/kg MOXI
20 mg/kg PROP Time (Hours)
Bod
y T
empe
ratu
re (°C
)
HEART RATE-IMPLANTEDMean ± SEM
BA
SE
LIN
E 1 2 3 4 5 6
7-8
9-10
11-1
2
13-1
4
15-1
6
17-1
8
19-2
0
21-2
2
0
50
100
150
200
100 mg/kg MOXI
0 mg/kg
20 mg/kg PROP Time (Hours)
Hea
rt R
ate
(bp
m)
PR INTERVAL-IMPLANTEDMean ± SEM
BA
SE
LIN
E 1 2 3 4 5 6
7-8
9-10
11-1
2
13-1
4
15-1
6
17-1
8
19-2
0
21-2
2
50
100
150
100 mg/kg MOXI
0 mg/kg
20 mg/kg PROP Time (Hours)
PR
Inte
rval
(m
sec)
QRS COMPLEX-IMPLANTEDMean ± SEM
BA
SE
LIN
E 1 2 3 4 5 6
7-8
9-10
11-1
2
13-1
4
15-1
6
17-1
8
19-2
0
21-2
2
25
30
35
40
45
50
100 mg/kg MOXI
0 mg/kg
20 mg/kg PROP Time (Hours)
QR
S C
ompl
ex (
mse
c)
QT INTERVAL-IMPLANTEDMean ± SEM
BA
SE
LIN
E 1 2 3 4 5 6
7-8
9-10
11-1
2
13-1
4
15-1
6
17-1
8
19-2
0
21-2
2
200
250
300
350
400
450
100 mg/kg MOXI
0 mg/kg
20 mg/kg PROP Time (Hours)
QT
Inte
rval
(m
sec)
QTcB INTERVAL-IMPLANTEDMean ± SEM
BA
SE
LIN
E 1 2 3 4 5 6
7-8
9-10
11-1
2
13-1
4
15-1
6
17-1
8
19-2
0
21-2
2
200
250
300
350
400
450
100 mg/kg MOXI
0 mg/kg
20 mg/kg PROP Time (Hours)
QT
cB In
terv
al (
mse
c)
QTcF INTERVAL-IMPLANTEDMean ± SEM
BA
SE
LIN
E 1 2 3 4 5 6
7-8
9-10
11-1
2
13-1
4
15-1
6
17-1
8
19-2
0
21-2
2
200
250
300
350
400
450
100 mg/kg MOXI
0 mg/kg
20 mg/kg PROP Time (Hours)
QT
cF In
terv
al (
mse
c)
QTcV INTERVAL-IMPLANTEDMean ± SEM
BA
SE
LIN
E 1 2 3 4 5 6
7-8
9-10
11-1
2
13-1
4
15-1
6
17-1
8
19-2
0
21-2
2
200
250
300
350
400
450
100 mg/kg MOXI
0 mg/kg
20 mg/kg PROP Time (Hours)
QT
cV In
terv
al (
mse
c)
RR INTERVAL-IMPLANTEDMean ± SEM
BA
SE
LIN
E 1 2 3 4 5 6
7-8
9-10
11-1
2
13-1
4
15-1
6
17-1
8
19-2
0
21-2
2
0
500
1000
1500
100 mg/kg MOXI
0 mg/kg
20 mg/kg PROP Time (Hours)
RR
Inte
rval
(m
sec)
HEART RATE-EXTERNALMean ± SEM
BA
SE
LIN
E 1 2 3 4 5 6
7-8
9-10
11-1
2
13-1
4
15-1
6
17-1
8
19-2
0
21-2
2
0
50
100
150
200
100 mg/kg MOXI
0 mg/kg
20 mg/kg PROP Time (Hours)
Hea
rt R
ate
(bp
m)
PR INTERVAL-EXTERNALMean ± SEM
BA
SE
LIN
E 1 2 3 4 5 6
7-8
9-10
11-1
2
13-1
4
15-1
6
17-1
8
19-2
0
21-2
2
50
100
150
100 mg/kg MOXI
0 mg/kg
20 mg/kg PROP Time (Hours)
PR
Inte
rval
(m
sec)
QRS COMPLEX-EXTERNALMean ± SEM
BA
SE
LIN
E 1 2 3 4 5 6
7-8
9-10
11-1
2
13-1
4
15-1
6
17-1
8
19-2
0
21-2
2
25
30
35
40
45
50
100 mg/kg MOXI
0 mg/kg
20 mg/kg PROP Time (Hours)
QR
S C
ompl
ex (
mse
c)
QT INTERVAL-EXTERNALMean ± SEM
BA
SE
LIN
E 1 2 3 4 5 6
7-8
9-10
11-1
2
13-1
4
15-1
6
17-1
8
19-2
0
21-2
2
200
250
300
350
400
450
100 mg/kg MOXI
0 mg/kg
20 mg/kg PROP Time (Hours)
QT
Inte
rval
(m
sec)
QTcB INTERVAL-EXTERNALMean ± SEM
BA
SE
LIN
E 1 2 3 4 5 6
7-8
9-10
11-1
2
13-1
4
15-1
6
17-1
8
19-2
0
21-2
2
200
250
300
350
400
450
100 mg/kg MOXI
0 mg/kg
20 mg/kg PROP Time (Hours)
QT
cB In
terv
al (
mse
c)
QTcF INTERVAL-EXTERNALMean ± SEM
BA
SE
LIN
E 1 2 3 4 5 6
7-8
9-10
11-1
2
13-1
4
15-1
6
17-1
8
19-2
0
21-2
2
200
250
300
350
400
450
100 mg/kg MOXI
0 mg/kg
20 mg/kg PROP Time (Hours)
QT
cF In
terv
al (
mse
c)
QTcV INTERVAL-EXTERNAL
Mean ± SEM
BA
SE
LIN
E 1 2 3 4 5 6
7-8
9-10
11-1
2
13-1
4
15-1
6
17-1
8
19-2
0
21-2
2
200
250
300
350
400
450
100 mg/kg MOXI
0 mg/kg
20 mg/kg PROPTime (Hours)
QT
cV In
terv
al (
mse
c)
RR INTERVAL-EXTERNALMean ± SEM
BA
SE
LIN
E 1 2 3 4 5 6
7-8
9-10
11-1
2
13-1
4
15-1
6
17-1
8
19-2
0
21-2
2
0
200
400
600
800
1000
100 mg/kg MOXI
0 mg/kg
20 mg/kg PROP Time (Hours)
RR
Inte
rval
(m
sec)
Fig. 2 Changes in Cardiovascular ParametersFig. 1 ECG Waveforms