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The harmony of gold standard physiological monitoring with novel infusion technology: Better quality of science in pre-clinical models ranging from mice to marmosets
Christian Schnell
Lab Head in-vivo Pharmacology,
Oncology ResearchNovartis AG
Copyright 2015 C. Schnell, PrimeTech & InsideScientific. All Rights Reserved.
InsideScientific is an online educational environment designed for life science researchers. Our goal is to aid in
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Innovative drug infusion technology for laboratory animals.
What is good science?
Poole (1997)…
Important problem that needs an answer
Unambiguous results
Variables not under investigation strictly controlled
Valid, reliable, repeatable
4
• Marmoset species (Genus Callithrix) are New World monkeys
• The Common Marmoset (Callithrix jacchus) is the species, that is preferentially kept in laboratories
• Widely used for CVS research in the early 80’s
5
Marmosets
Ciba-Geigy 1992
6
Conventional Methods: Tail-cuff
Ciba-Geigy 1992
• Animal needs to be restrained
• Only offers periodic sampling
• Is a subjective measurement
• Non-invasive method
7
Conventional Methods: Tail-cuff
• Animal needs to be restrained
• Only offers periodic sampling
• Is a subjective measurement
• Non-invasive method
Argggghhhhhh!!!!Ciba-Geigy 1992
• Patency of vascular catheters difficult to maintain (constant inf.)
• Risk of infection
• Tethers have been documented to be a stressor in lab animals
• Measurements in conscious relatively freely moving animals
• Allows recordings around-the-clock
8
Conventional Methods:Tethering systems
Ciba-Geigy 1992
9
"He looks a little stressed…
Do you think leads, tethers, cuffs and restraints will compromise the physiological pressure data?"
New Method of Measurements: Radio-Telemetry Systems
10
11
Telemetry technology is known since the late 50th and was used for large animals only...
TA11PA-C40:Weight : 8.5 g
2% body weight
BP, HR, Act.; Cath. : 1 mm
TL11M2-C50-PXT:Weight : 10.5 g
3% body weight
BP, HR, ECG, Temp, Act.; Cath. : 1 mm
Technical Data
Adult animal: 350 g
Pregnancy: 20% body weight
13 Ciba-Geigy 1992
Ciba-Geigy 1992
It is important to note that radio-telemetry recording is not only reducing stress in animals ...but also for our technical staff!
Measurements of Blood Pressure (BP) & Heart Rate (HR) in Conscious Marmosets
Values are means ± SD
16
Marmosets chronically implanted with vascular catheters or pressure transducers…
Distribution of Mean Blood Pressure (MBP) & Heart Rate (HR) in Normotensive Restrained (==; n=55) or Freely Moving (—; n=67) Marmosets .
Lower baseline BP and HR > less intra- and inter-animal variation > enhanced statistical power
> REDUCTION IN ANIMAL USE17
Measurements Of HR In Restrained And Freely Moving Adult Marmosets In Different Laboratories
Gla
xo
Novart
is
Pharma-L
SR
Roche
Cam
bridge
0
100
200
300
400
500H
ea
rt r
ate
(b
pm
)
(168)(55)
(33)
(7)
Restrained
(12)
18
Gla
xo
Novart
is
Pharma-L
SR
Roche
Cam
bridge
Novart
is -
CH
Götti
ngen - D
Zürich -
CH
Roche -
Japan
Pfizer -
F
Dstl
- UK
CER
B - F
Boehrin
ger-US
0
100
200
300
400
500H
ea
rt r
ate
(b
pm
)
(168)
(9)
(6)
(6)
(4)
(5)
(67)
(7)
(55)
(33)
(5)
(7)
Restrained freely moving via telemetry
(12)
19
Measurements Of HR In Restrained And Freely Moving Adult Marmosets In Different Laboratories
20
21
• Circadian and circa-septal rhythms observed in MAP, HR and ACT are very reproducible over time each animal can be used as it’s own control
• Low salt diet is not inducing chronic hypertension in marmosets
Can acute stress influence drug activity?
Log Dose (mg/kg p.o.)
0.01 0.1 1 10 100
AU
C (
mm
Hg
x 6
h)
0
50
100
150
200
AII Antagonist in restraint animals
AII Antagonist in unrestraint animals
IC50
23
Influence of restrain on Angiotensin II antagonist hypotensive profile.
• IC50 in restraint marmosets: 0.3 mg/kg
• IC50 in unrestraint marmosets: 8 mg/kg
Factor ~ 30
Time (hours)
0 1 2 3 4 5 6
Time (hours)
0 1 2 3 4 5 6
De
lta
BP
(m
mH
g)
-40
-30
-20
-10
0
10
20
Restraint Unrestraint
(7)
(7)
(9)
(5)
(n)
p.o. p.o.
Initial BP: 93 ± 6 mmHg Initial BP: 75 ± 5 mmHg
ControlRenin inhibitor 10 mg/kg p.o.
24
Effects on Blood Pressure (Mean ± Sem) After Oral Administration of a Renin Inhibitor Recorded in Conscious Restrained or Unrestrained Radio-
telemetry Implanted Salt-depleted Marmosets
Time (hours)
0 1 2 3 4 5 6
Time (hours)
0 1 2 3 4 5 6
De
lta
HR
(b
pm
)
-150
-100
-50
0
50
100
150Control
Renin inhibitor 10 mg/kg p.o.
Restraint Unrestraint
(7)
(7)
(9)
(5)
(n)
p.o. p.o.
Initial HR: 390 ± 18 bpm Initial HR: 205 ± 13 bpm
25
Effects on Blood Pressure (Mean ± Sem) After Oral Administration of a Renin Inhibitor Recorded in Conscious Restrained or Unrestrained Radio-
telemetry Implanted Salt-depleted Marmosets
How can dosing a pre-trained animal influence haemodynamic parameters ?
Effects on diastolic blood pressure of different administration protocols in conscious unrestrained radio-telemetry implanted marmosets.
Time of day (hours)
8:00 9:00 10:00 11:00 12:00 13:00 14:00 15:00 16:00 17:00
Dia
sto
lic
BP
(m
mH
g)
60
65
70
75
80
1 week
Feedingp.o.
(12)
(n)
27
Time of day (hours)
8:00 9:00 10:00 11:00 12:00 13:00 14:00 15:00 16:00 17:00
Dia
sto
lic
BP
(m
mH
g)
60
65
70
75
80
1 week
3 week
Feedingp.o.
(12)
(n)
*
28
Effects on diastolic blood pressure of different administration protocols in conscious unrestrained radio-telemetry implanted marmosets.
Time of day (hours)
8:00 9:00 10:00 11:00 12:00 13:00 14:00 15:00 16:00 17:00
Hea
rt R
ate
(b
pm
)
170
190
210
230
250
1 week
Feedingp.o.
(12)
(n)
29
Effects on diastolic blood pressure of different administration protocols in conscious unrestrained radio-telemetry implanted marmosets.
Time of day (hours)
8:00 9:00 10:00 11:00 12:00 13:00 14:00 15:00 16:00 17:00
Hea
rt R
ate
(b
pm
)
170
190
210
230
250
1 week
3 week
Feedingp.o.
(12)
(n)
***
30
Effects on diastolic blood pressure of different administration protocols in conscious unrestrained radio-telemetry implanted marmosets.
Time of day (hours)
8:00 9:00 10:00 11:00 12:00 13:00 14:00 15:00 16:00 17:00
De
lta
Dia
sto
lic
BP
(m
mH
g)
-5
0
5
10
15
Per os.
Milk shake
Feeding
p.o.
Shake
*** *
** ****
(12)
(7)
(n)
31
Effects on Heart Rate of Different Administration Protocols In Conscious Unrestrained Radio-telemetry Implanted Marmosets
Effects on Heart Rate of Different Administration Protocols In Conscious Unrestrained Radio-telemetry Implanted Marmosets
Time of day (hours)
8:00 9:00 10:00 11:00 12:00 13:00 14:00 15:00 16:00 17:00
Delt
a H
eart
Rate
(b
pm
)
-20
0
20
40
60
Per os.
Milk shake
Feeding
p.o.
Shake
***
* *
** *
**
*
(12)
(7)
(n)
32
Baseline cardiovascular parameters (mean ± sem) in SHR rats recorded by a telemetry device, indwelling arterial catheters, or indirect tail cuff
Tail cuff Catheters Telemetry
Sy
sto
lic
blo
od
pre
ss
ure
(m
mH
g)
100
150
200
250
(n=33)
(n=10)
(n=18)
Tail cuff Catheters Telemetry
Heart
rate
(b
pm
)
100
200
300
400
500
(n=33)
(n=10)
(n=18)
33(Data obtained from Dr. R. Webb, Ciba-Geigy, East Hanover, U.S.A)
Restraint Telemetry
Me
an
Blo
od
Pre
ss
ure
(m
mH
g)
80
90
100
110
120
(n=30)
(n=14)
Restraint Telemetry
He
art
Ra
te
(b
pm
)
100
150
200
250
300
(n=30)
(n=14)
34
Comparative baseline cardiovascular parameters (mean ± sem) recorded in conscious restrained or unrestrained radio-telemetry implanted cynomolgus macaques.
(Data obtained from Dr. Y.Fradin, Aventis, France)
Intraocular pressure (IOP) measurements performed via radio-telemetry: Placement of the sensing catheter
Pressure radio-transmitter Model: TA11PA-C40
(Data Sciences, St. Paul, MN)
35
36
The body of the transmitter is placed subcutaneously in the neck region of the rabbit
Ciba-Geigy 1996
IOP
(m
mH
g)
10.0
10.2
10.4
10.6
10.8
Col 4
Col 5
Time (secondes)
EC
G (
mvo
lts)
-0.2
-0.1
0.0
0.1
0.2
Col 7 Col 4
0 1 2
IOP
(mm
Hg
)
10.5
11.0
11.5
12.0
12.5
0.06%
Col 4
Time (secondes)E
CG
(mvo
lts)
-0.2
-0.1
0.0
0.1
0.2
Col 7 Col 4
0 1 2
right eye lead II
Time (secondes)
0 1 2
37
Simultaneous intraocular pressure (IOP) and ECG measurements by telemetry performed in conscious unrestrained rabbits
Restrain Box Cage
Intr
ao
cu
lar
pre
ssu
re (
mm
Hg
)
8
12
16
20
24(n=175)
(n=9)
(n=9)
(PTG) (Telemetry)
38
Time (hours)
0 1 2 3 4 5 6
Time (hours)
0 1 2 3 4 5 6
De
lta
IO
P (
mm
Hg
)
-6
-4
-2
0
2
4Control
Timolol maleate 0.5%
Restraint Unrestraint
(5) (7)
(n)
Inst. Inst.
Initial IOP: 17.4 ± 0.7 mmHg Initial IOP: 12.8 ± 0.6 mmHg
Effect of timolol maleate on IOP in albino rabbits:
20 publications
47 % decrease of IOP
53 % no effect on IOP
?39
Time (hours)
0 1 2 3 4 5 6
Time (hours)
0 1 2 3 4 5 6
De
lta
IO
P (
mm
Hg
)
-6
-4
-2
0
2
4Control
Timolol maleate 0.5%
Restraint Unrestraint
(5) (7)
(n)
Inst. Inst.
Initial IOP: 17.4 ± 0.7 mmHg Initial IOP: 12.8 ± 0.6 mmHg
40
Implanted radio-telemetry - Pro -
• Eliminates artifact from restraint stress home-cage exp.
• Eliminate risk of infection from exit sites
• No special maintenance required
• Allows for automated data collection around-the-clock
• Implanted animals can be re-used in successive studies
• Reduces animal use in research
• Available for a wide range of species
• Improves data quality compared to those collected using conventional measurements techniques
41
Implanted radio-telemetry - Contra -
• Requires surgery (pre-invasive method)
• High initial costs
• Suitable for monitoring in a confined area only short transmitting range
• Possibility of long-term drift with some types of measurements (blood pressure)
• Battery life
42
Implanted radio-telemetry - Contra -
• Requires surgery (pre-invasive method)
• High initial costs
• Suitable for monitoring in a confined area only short transmitting range
• Possibility of long-term drift with some types of measurements (blood pressure)
• Battery life
• Still stress related to drug administration
43
RPA class Definition Median Survival 1-Year Survival 3-Year Survival 5-Year Survival
III Age < 50, KPS ≥ 90 17.1 months 70% 20% 14%
IVAge < 50, KPS < 90
11.2 months 46% 7% 4%Age > 50, KPS ≥ 70, surgical removal with good neurologic function
V + VI
Age ≥ 50, KPS ≥ 70, surgical removal with poor neurologic function
7.5 months 28% 1% 0%Age ≥ 50, KPS ≥ 70, no surgical removal
Age ≥ 50, KPS < 70
Glioblastoma Multiforme (GBM) represents the most common primary malignant brain tumor in both adults and children...
44
Glioblastoma Multiforme (GBM) represents the most common primary malignant brain tumor in both adults and children...
45
A major barrier to progress in treatment is the relative inaccessibility of brain tumors to chemotherapeutic agents
U87MG-luc glioblastoma orthotopic tumor model:
5 x 104 cells are injected in the thalamus + iPRECIO® pump implantation
46 Courtesy of Primetech
U87MG-luc glioblastoma orthotopic tumor model:
Implantation description (pictures from an implanted rat 1 week post-surgery)
• The iPRECIO® pump was implanted through a small incision made at the base of the neck in a subcutaneous pocket located on the flank of the animal
• Intracranial administration was achieved via connection of the pump to a specially designed, commercially available brain infusion kit (Alzet)
47
Time post U87MG cell injection (Days)
0 1 2 3 4 5 6 7 8 9 10 11 12
Ch
an
ge in
bo
dy w
eig
ht
(%
, m
ean
± S
EM
)
-25
-20
-15
-10
-5
0
5
10
15
20
25
U87MG intra-cranial inoculation only (n=6)
U87MG intra-cranial + iPRECIO pump (n=6)
Surgery
Subcutaneous implantation of the iPRECIO® pump did not affect body weight gain when compared to corresponding tumor bearing control rats (historical data)
U87MG glioblastoma orthotopic tumor model: Behavior and BW change following concomitant tumor cells and pump implantation
Effect on body weight:
48
Intracranial administration was achieved via connection of the pump to a specially designed, commercially available brain infusion kit (Alzet)
49
U87MG glioblastoma orthotopic tumor model: Implantation description (pictures from an implanted rat 1 week post-surgery)
At the end of the experiments in nearly all implanted rats, we observed a disconnection between the pump and the brain infusion kit
50
U87MG glioblastoma orthotopic tumor model: Implantation description (pictures from an implanted rat 1 week post-surgery)
U87MG glioblastoma orthotopic tumor model: Implantation description (pictures from an implanted rat 1 week post-surgery)
51
This problem could be solved by adding a loop in the catheter between the pump and the brain infusion kit
Control
U87MG glioblastoma orthotopic tumor model: Tumor description (brain pictures from an implanted rat 2-3 week post-surgery)
52
vehicle
U87MG glioblastoma orthotopic tumor model: Tumor description (brain pictures from an implanted rat 2-3 week post-surgery)
53
U87MG glioblastoma orthotopic tumor model: Tumor description (brain pictures from an implanted rat 2-3 week post-surgery)
54
treated
Bioluminescence measurements in-vivoDetection of Glioma brain tumors implanted orthotopicaly
55
Time post implantation (Days)
20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
Fra
cti
on
al T
um
or
Gro
wth
(m
ea
n ±
SE
M)
0
1
2
3
4
5
6
7
8
9
10
Vehicle (n=8)
Start treatment
Compound A 60 mg/kg p.o. qd (n=7)
*
Time post cell injection (Days)
15 16 17 18 19 20 21 22 23 24 25 26
Fra
cti
on
al L
um
ines
cen
ce
ch
an
ge
(m
ea
n ±
SE
M)
0
5
10
15
20
25
30
Vehicle (n=14)
Start treatment
Compound A 60 mg/kg/day p.o. (n=14)
56
Efficacy in U87MG-luc implanted s.c. or orthotopically in nude rats:Compound A 60 mg/kg/day per os.
Time post cell injection (Days)
12 13 14 15 16 17 18 19 20 21 22 23
Fra
cti
on
al L
um
ines
cen
ce
ch
an
ge
(m
ea
n ±
SE
M)
0
5
10
15
20
25
30
35
Vehicle (n=8)
Start treatment
Compound A 50 µg/kg/day intra-cranial (n=5)
*
57
Efficacy in U87MG-luc implanted s.c. or orthotopically in nude rats:Compound A 60 mg/kg/day per os.
Time post implantation (Days)
20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
Fra
cti
on
al T
um
or
Gro
wth
(m
ea
n ±
SE
M)
0
1
2
3
4
5
6
7
8
9
10
Vehicle (n=8)
Start treatment
Compound A 60 mg/kg p.o. qd (n=7)
*
Efficacy in U87MG-luc implanted orthotopically in nude rats:Compound A 50 µg/kg brain/day intra-cranial infusion
Control
Treated
58
U87MG glioblastoma orthotopic tumor model: Conclusion of the validation study
• The iPRECIO pump was tested in tumor bearing nude rats. No adverse effects where observed after the implantation procedure. Behavior and body weight gain was not impaired when compared to control tumor bearing animals without a subcutaneous pump
• The initial problem of catheter disconnection at the exit site of the pump was solved by increasing the catheter length and the addition of a loop
59
U87MG glioblastoma orthotopic tumor model: Conclusion of the validation study
We could identify several putative advantages when compared to existing infusion methods:
1. only one surgery is required for both, the tumor cell injection and the pump implantation causing less distress to the animals
2. the infusion protocol can include a recovery period phase where only saline is infused to keep the catheter patent. Then at an operator defined time (related to tumor size), infusion of the therapeutic can be started
3. there will be much less limitations in relation to the solubility of the substance, the prepared concentration of the substance, and the limited range of infusion rates with this pump
60
Our results demonstrate that the iPRECIO® micro infusion pump is a viable alternative to existing infusion methods which will allow
flexible methods to investigate new dosage regimens in orthotopic GBM tumor models in rats
61
The Harmony of gold standard physiological monitoring with novel infusion technology: Lessons learned and take home message...
Any approach (training, refinements of measurements techniques and drug delivery systems,…) that reduces research animal distress should answer both the need for valid data and the call for improved care and welfare of the animals.
Telemetry technologies combined with novel infusion technologyprovides unique opportunities for such assessments.
62
Thank You!For additional information on iPrecio infusion pumps and Innovative drug infusion technologies for laboratory animals please visit:
http://www.iprecio.com/Christian Schnell
