A 2-part webinar for scientists interested in novel drug delivery methods for basic research, drug discovery and development. Learn about novel infusion technologies and how challenges in physiological monitoring and drug delivery are being overcome by implantable and programmable devices. Session 1: The harmony of gold standard physiological monitoring with novel infusion technology: Better quality of science in preclinical models ranging from mice to marmosets. Presenter: Christian Schnell, Novartis AG, Basel, Switzerland Mr. Schnell presents data from multiple species that show how differences in blood pressure, heart rate and acute stress impact drug activity and how dosing a pre-trained animal can influence hemodynamic parameters.

1. 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 Research
Novartis AG
Copyright 2015 C. Schnell, PrimeTech & InsideScientific. All Rights Reserved.

2. InsideScientific is an online educational environment
designed for life science researchers. Our goal is to aid in
the sharing and distribution of scientific information
regarding innovative technologies, protocols, research
tools and laboratory services.

3. Thank you to our event sponsor
Innovative drug infusion technology for laboratory animals.

4. What is good science?
Poole (1997)…
 Important problem that needs an answer
 Unambiguous results
 Variables not under investigation strictly controlled
 Valid, reliable, repeatable
4

5. • 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. 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. 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

8. • 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. 9
"He looks a little
stressed…
Do you think leads,
tethers, cuffs and
restraints will
compromise the
physiological
pressure data?"

10. New Method of Measurements: Radio-Telemetry Systems
10

11. 11
Telemetry
technology is
known since the
late 50th and was
used for large
animals only...

13. 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

14. Ciba-Geigy 1992

15. It is important to
note that radio-
telemetry recording
is not only reducing
stress in animals
...but also for our
technical staff!

16. 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…

17. 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

18. Measurements Of HR In Restrained And Freely Moving
Adult Marmosets In Different Laboratories
G
laxoN
ovartis
P
harm
a-LS
RR
oche
C
am
bridge
0
100
200
300
400
500Heartrate(bpm)
(168)
(55)
(33)
(7)
Restrained
(12)
18

19. G
laxoN
ovartis
P
harm
a-LS
RR
oche
C
am
bridgeN
ovartis
-C
H
G
öttingen
-D
Zürich
-C
H
R
oche
-Japan
P
fizer
-F
D
stl-U
K
C
E
R
B
-F
B
oehringer-U
S
0
100
200
300
400
500Heartrate(bpm)
(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. 20

21. 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

22. Can acute stress
influence drug activity?

23. Log Dose (mg/kg p.o.)
0.01 0.1 1 10 100
AUC(mmHgx6h)
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

24. Time (hours)
0 1 2 3 4 5 6
Time (hours)
0 1 2 3 4 5 6
DeltaBP(mmHg)
-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
Control
Renin 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

25. Time (hours)
0 1 2 3 4 5 6
Time (hours)
0 1 2 3 4 5 6
DeltaHR(bpm)
-150
-100
-50
0
50
100
150
Control
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

26. How can dosing a pre-trained
animal influence haemodynamic
parameters ?

27. 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
DiastolicBP(mmHg)
60
65
70
75
80
1 week
Feedingp.o.
(12)
(n)
27

28. Time of day (hours)
8:00 9:00 10:00 11:00 12:00 13:00 14:00 15:00 16:00 17:00
DiastolicBP(mmHg)
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.

29. Time of day (hours)
8:00 9:00 10:00 11:00 12:00 13:00 14:00 15:00 16:00 17:00
HeartRate(bpm)
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.

30. Time of day (hours)
8:00 9:00 10:00 11:00 12:00 13:00 14:00 15:00 16:00 17:00
HeartRate(bpm)
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.

31. Time of day (hours)
8:00 9:00 10:00 11:00 12:00 13:00 14:00 15:00 16:00 17:00
DeltaDiastolicBP(mmHg)
-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

32. 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
DeltaHeartRate(bpm)
-20
0
20
40
60
Per os.
Milk shake
Feeding
p.o.
Shake
* **
* *
** *
**
*
(12)
(7)
(n)
32

33. Baseline cardiovascular parameters (mean ± sem) in SHR rats recorded
by a telemetry device, indwelling arterial catheters, or indirect tail cuff
Tail cuff Catheters Telemetry
Systolicbloodpressure
(mmHg)
100
150
200
250
(n=33)
(n=10)
(n=18)
Tail cuff Catheters Telemetry
Heartrate
(bpm)
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)

34. Restraint Telemetry
MeanBloodPressure
(mmHg)
80
90
100
110
120
(n=30)
(n=14)
Restraint Telemetry
HeartRate
(bpm)
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)

35. 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. 36
The body of the
transmitter is
placed
subcutaneously
in the neck
region of the
rabbit
Ciba-Geigy 1996

37. IOP(mmHg)
10.5
11.0
11.5
12.0
12.5
Time (secondes)ECG(mvolts)
-0.2
-0.1
0.0
0.1
0.2
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

38. Restrain Box Cage
Intraocularpressure
(mmHg)
8
12
16
20
24
(n=175)
(n=9)
(n=9)
(PTG) (Telemetry)
38

39. Time (hours)
0 1 2 3 4 5 6
Time (hours)
0 1 2 3 4 5 6
DeltaIOP(mmHg)
-6
-4
-2
0
2
4
Control
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

40. Time (hours)
0 1 2 3 4 5 6
Time (hours)
0 1 2 3 4 5 6
DeltaIOP(mmHg)
-6
-4
-2
0
2
4
Control
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

41. 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

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
42

43. 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

44. 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%
IV
Age < 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

45. 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

46. U87MG-luc glioblastoma orthotopic tumor model:
5 x 104 cells are injected in the thalamus + iPRECIO® pump implantation
46 Courtesy of Primetech

47. 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

48. Time post U87MG cell injection (Days)
0 1 2 3 4 5 6 7 8 9 10 11 12
Changeinbodyweight
(%,mean±SEM)
-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

49. 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)

50. 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)

51. 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

52. Control
U87MG glioblastoma orthotopic tumor model:
Tumor description (brain pictures from an implanted rat 2-3 week post-surgery)
52

53. vehicle
U87MG glioblastoma orthotopic tumor model:
Tumor description (brain pictures from an implanted rat 2-3 week post-surgery)
53

54. U87MG glioblastoma orthotopic tumor model:
Tumor description (brain pictures from an implanted rat 2-3 week post-surgery)
54
treated

55. Bioluminescence measurements in-vivo
Detection of Glioma brain tumors implanted orthotopicaly
55

56. Time post implantation (Days)
20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36
FractionalTumorGrowth(mean±SEM)
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
FractionalLuminescencechange
(mean±SEM)
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.

57. Time post cell injection (Days)
12 13 14 15 16 17 18 19 20 21 22 23
FractionalLuminescencechange
(mean±SEM)
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
FractionalTumorGrowth(mean±SEM)
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)
*

58. Efficacy in U87MG-luc implanted orthotopically in nude rats:
Compound A 50 µg/kg brain/day intra-cranial infusion
Control
Treated
58

59. 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

60. 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

61. 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

62. 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 technology
provides unique opportunities for such assessments.
62

63. 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
christian.schnell@novartis.com