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Combining Cardiovascular, Respiratory and Neurobehavioral Endpoints for Efficient Study Design
Brian M. RochePhD, DSP, DABT
Director of Safety Pharmacology,Charles River Laboratories
Jason D. Payseur
Senior Scientist – IVIVT, MSD Safety Pharmacology USGlaxoSmithKline
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Combining plethysmography, telemetry and Allay™ restraint technology for the use in safety pharmacology and inhalation toxicology environments
Brian M. Roche PhD, DSP, DABT
Director of Safety Pharmacology,Charles River Laboratories,Ashland, OH
Copyright 2016 B.M. Roche, Data Sciences International and InsideScientific. All Rights Reserved.
Agenda
1. Allay Restraint Technology
• Overview of device
• Acclimation process
2. Allay restrainers vs. nose only cone restraint tubes
• Acclimation data
• Hemodynamic telemetry data
3. Plethysmography and Telemetry: Allay vs Head Out
• Hemodynamic assessment
• Respiratory function
• Resistance and compliance
4. Dosimetry approaches with Allay plethysmography
5. What we’ve learned from conducting this pilot study
Allay Restraint AcclimationThe Process
Allay Restraint Technology
1 Allay restraint tube
2 Neck clip
3 Plethysmography bell
4 Nose seal1
2 3
4
Important to allow for adequate time prior to study to acclimate the rats to the restraint device
Introducing the allay restraint tube and the rat either through placing the restraint tube or “sled” portion of the device into the home cage and/or manually placing and holding the rat inside the device.
Allay Restraint and Acclimation
After the initial familiarity with the device, the next step was to introduce the restraint portion of the tube.
This was conducted by manually placing and holding the rat in the device without the neck clip.
Allowing the rat to extend its head and neck out through the front of the device while it explored its surroundings.
Allay Restraint and Acclimation
The next step is to determine the appropriately sized “clip” for the size of the rat’s neck. This process starts as an extension of the manual restraint of the rat within the device.
Each time the rat extends it’s neck through the “clip” area, slowly place the neck clip over the rat’s neck. This step requires patience as restraining the rat too early may set you back a step in acclimation.
Allay Restraint and Acclimation
Once the appropriate sized neck clip is in place the rat will begin acclimating to this step of the procedure.
Allay Restraint and Acclimation
Once the rat has been introduced and the appropriate neck clip has been determined, acclimation to the allay restraint technology for respiratory function beings. Depending upon your study design, the next step in acclimation is to add the plethysmography bell.
This step is required if collecting respiratory function. At this point the restraint is similar to a head out chamber without a neck seal. This step is critical in the acclimation process for tail placement and temperature regulation.
Allay Restraint and Acclimation
Nose and mouth seal.
The nose seal can also be placed on the restraint tube without the plethysmography bell for inhalation delivery of test articles/chemicals.
The rat will react once the face whiskers are stimulated but will calm down. Additionally, the nose will be positioned directly in the center of the nose seal.
Allay Restraint and Acclimation
Putting it all together.
The device is functioning as a nose only plethysmography chamber for inhalation delivery. This piece is also critical for placement on the inhalation tower.
The acclimation will be conducted in steps, with increasing duration. Once the entire Allay restraint device is assembled around the rat, start building up acclimation time to cover the time that the rat will be restrained on study.
Allay Restraint and Acclimation
Acclimation: Allay restrainers vs. nose only restraint coneHD-S21 Telemetry – Hemodynamic data
Allay Restraint vs. Nose Only Restraint Cone
Heart rate reduced initially in Allay restrainers
No change in blood pressure between the two methods of restraint
EVERY STEP OF THE WAY16
Slight increase in body temperature, expected with no bias flow and tail enclosed in Allay restraint
Plethysmography and Telemetry: Allay vs. Head Out RestraintHemodynamic Assessment, Respiratory Function, Resistance and Compliance
Allay Restraint vs. Head Out Plethysmograph
Reduction in heart rate through first 60 minutes for Allay restraint
Allay Head Out
No change in systolic blood pressure
Allay Head Out
Slight reduction in diastolic blood pressure for initial 60 minutes in Allay restraint
Allay Head Out
Slight reduction in mean arterial blood pressure for Allay restraint
Allay Head Out
Slight reduction in body temperature for the Allay restrain
Allay Head Out
Decrease in respiratory rate for Allay restraint
Allay Head Out
Increase in tidal volume with Allay restraint
Allay Head Out
No change in minute volume
Allay Head Out
No change in lung compliance
Allay Head Out
No change in lung resistance
Allay Head Out
Dosimetry Approaches With Allay PlethysmographyInhalation drug delivery
Standard fit for the Allay restraint system with a conventional nose only inhalation system.
Inhalation tower designed with a support ring to handle the size and weight of the Allay restraint.
Allay Technology and Inhalation Tower
Telemetry hemodynamic and intrathoracic pressure data were collected in combination with respiratory function by placing a DSI RPC-1 small animal receiver in close proximity to the animal instrumented with HD-S21 transmitter.
Adequate separation of the animals throughout the inhalation tower, reduces the risk of cross-talk.
Allay Restraint and Telemetry
What we’ve learned from conducting this pilot study…
Pros… Ability to combine inhalation and dosimetry
Ability to dose via inhalation (nose only cone)
Ability to collect dosimetry (head out chamber)
Potentially less stressful (nose cone and head out)
Plethysmography - no thoracic compaction of rat vs head out
Tight seal of device when mounted on inhalation tower
Spacing collar for proper placement of head chamber and nose seal
No physical obstruction against the throat (head out chamber)
Dual chamber (reference) reduces variability of ambient pressure changes in the collection environment (head out chamber)
Cons… Nose seal
o Destruction by rat
o Ability to replace nose seal (loss of data)
Acclimation process is time consuming
Design
o Heavy (ring design of inhalation tower)
o Rolls (acclimation)
o Position of pneumotach and transducer ports (Obstructed by tail/urine/feces)
Acute Respiratory Disorders
• Respiratory Depression
• Respiratory Syncytial Virus (RSV)
• Acute Respiratory Distress Syndrome (ARDS)
• Mucociliary Clearance and Dysfunction
• Pneumonia
• Cough
• Tuberculosis (TB)
• Bronchiolitis/ Bronchitis
COPD
• Emphysema
• Chronic Bronchitis
• Model Development
• Treatment Assessment
Asthma
• Model Development
• Treatment Assessment
Lung Fibrosis
• Pulmonary Fibrosis
• Cystic Fibrosis
Safety Assessment
• Safety Pharmacology
• Toxicology
Select any of the links below to learn more about Respiratory Solutions offered by DSI.
www.datasci.com
Combined Cardiovascular, Respiratory, and Neurobehavioral Telemetry Model in the Conscious Rat:
Jason D. PayseurSenior Scientist – IVIVT, MSD Safety Pharmacology USGlaxoSmithKline
Copyright 2016 J.D. Payseur, Data Sciences International and InsideScientific. All Rights Reserved.
A Novel Approach to Study the Acute Physiological Effects of Caffeine and Chlorpromazine Following Oral Administration
Background
• Rodent studies historically focused on a single physiological system
• Integration of cardiovascular, respiratory, and neurobehavioral assessments would allow for more insight into how changes in one physiological system impact the others
• Advances in recent years have allowed combined models in large animals (i.e. JET/EMKA jacketed models)
• The release of a dual pressure catheter in a small animal model implant (model HD-S21) by Data Sciences International (DSI) opens up an opportunity to test a combined rodent model
Surgical Procedure
Surgical Procedure
• Prior to study, rats were implanted with a telemetry device (Model No. HD-S21, Data Sciences International (DSI), St. Paul MN).
• 1 pressure catheter was inserted into the abdominal aorta and advanced to a position caudal to the renal bifurcation. Another pressure catheter was inserted under the serosal surface of the esophagus in the thoracic cavity.
• 4x4 Latin square cross-over design [7 days between each treatment]
• Male rats (CRL:WI(HAN)) instrumented with telemetry devices for the measurement of arterial pressure, heart rate, ECG, respiratory rate, respiratory pressure, activity, and body temperature
• Each rat received oral doses of vehicle (distilled water) and caffeine at 3, 12, and 24 mg/kg
• Each rat received oral doses of vehicle (distilled water) and chlorpromazine at 2, 8, and 16 mg/kg
• Arterial pressures, ECG waveforms, respiratory rate, respiratory pressure, and activity were monitored continuously for 2 hours prior to dosing and up to 24 hours post-dose
Study Design
Dose dependant increase in heart rate at all dose levels up to 2 hours post dose.
Ab
solu
te D
ata
Ch
ange
Fro
m P
re-d
ose
Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
Hea
rt R
ate
(be
ats
/min
ute
)
250
300
350
400
450
500
Vehicle
Caffeine (3 mg/kg)
Caffeine (12 mg/kg)
Caffeine (24 mg/kg)
Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
H
ea
rt R
ate
(be
ats
/min
ute
)-50
0
50
100
150
Vehicle
Caffeine (3 mg/kg)
Caffeine (12 mg/kg)
Caffeine (24 mg/kg)
24mpk: 67.8 bpm; 19.3%
12mpk: 18.7%
3mpk: 10.2%
Effect of Caffeine on Heart Rate
Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
Resp
ira
tory
Ra
te(b
reath
s/m
inu
te)
30
60
90
120
150
180
210
240Vehicle
Caffeine (3 mg/kg)
Caffeine (12 mg/kg)
Caffeine (24 mg/kg)
Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
R
esp
ira
tory
Ra
te(b
reath
s/m
inu
te)
-50
-25
0
25
50
75
100
125
Vehicle
Caffeine (3 mg/kg)
Caffeine (12 mg/kg)
Caffeine (24 mg/kg)
24mpk: 61.9 bpm; 54.9%
12mpk: 30.0%
3mpk: 22.3%
Dose dependant increase in respiratory rate at all dose levels up to 2 hours post dose.
Ab
solu
te D
ata
Ch
ange
Fro
m P
re-d
ose
Effect of Caffeine on Respiratory Rate
Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
Re
sp
ira
tory
Pre
ssu
re(m
mH
g)
-10
-5
0
5
10
15
20
25
Vehicle
Caffeine (3 mg/kg)
Caffeine (12 mg/kg)
Caffeine (24 mg/kg)
Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
R
esp
ira
tory
Pre
ssu
re(m
mH
g)
-6
-4
-2
0
2
4
6 Vehicle
Caffeine (3 mg/kg)
Caffeine (12 mg/kg)
Caffeine (24 mg/kg)
No statistically significant changes in pressure at any dose level.
Effect of Caffeine on Respiratory Pressure
Ab
solu
te D
ata
Ch
ange
Fro
m P
re-d
ose
Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
Activity
(AU
C)
0
2000
4000
6000
8000
10000 Vehicle
Caffeine (3 mg/kg)
Caffeine (12 mg/kg)
Caffeine (24 mg/kg)
Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
Activity
(AU
C)
-4000
-2000
0
2000
4000
6000
8000
Vehicle
Caffeine (3 mg/kg)
Caffeine (12 mg/kg)
Caffeine (24 mg/kg)
Effect of Caffeine on Activity
24mpk: 400%
12mpk: 489%
3mpk: 257%
Significant increases in activity in a dose dependant manner up to 3 hours post dose.
Effect of Caffeine on Activity
Ab
solu
te D
ata
Ch
ange
Fro
m P
re-d
ose
Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
Activity
(AU
C)
0
2000
4000
6000
8000
10000 Vehicle
Caffeine (3 mg/kg)
Caffeine (12 mg/kg)
Caffeine (24 mg/kg)
Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
Activity
(AU
C)
-4000
-2000
0
2000
4000
6000
8000
Vehicle
Caffeine (3 mg/kg)
Caffeine (12 mg/kg)
Caffeine (24 mg/kg)
Effect of Caffeine on Activity
24mpk: 400%
12mpk: 489%
3mpk: 257%
Significant increases in activity in a dose dependant manner up to 3 hours post dose.
Effect of Caffeine on Activity
Ab
solu
te D
ata
Ch
ange
Fro
m P
re-d
ose
Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
Bo
dy T
em
pe
ratu
re(D
eg
C)
32
34
36
38
40
42
Vehicle
Caffeine (3 mg/kg)
Caffeine (12 mg/kg)
Caffeine (24 mg/kg)
Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
B
od
y T
em
pe
ratu
re(D
eg
C)
-5
-3
-1
1
3
5 Vehicle
Caffeine (3 mg/kg)
Caffeine (12 mg/kg)
Caffeine (24 mg/kg)
Small increases in body temperature in the mid and high doses up to 2 hours post dose, most likely due to the large increase in activity at those times.
Effect of Caffeine on Body Temperature
Ab
solu
te D
ata
Ch
ange
Fro
m P
re-d
ose
Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
He
art
Ra
te(b
ea
ts/m
inu
te)
250
300
350
400
450
500
Vehicle
Chlorpromazine (2 mg/kg)
Chlorpromazine (8 mg/kg)
Chlorpromazine (16 mg/kg)
Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
H
ea
rt R
ate
(be
ats
/min
ute
)-50
0
50
100
150
Vehicle
Chlorpromazine (2 mg/kg)
Chlorpromazine (8 mg/kg)
Chlorpromazine (16 mg/kg)
16mpk: 66.1 bpm; 54.9%
8mpk: 66.7%
An unexpected increase in heart rate in both the mid and high doses for up to 8 hours post dose (the duration of the light cycle after dosing). Heart rate was comparable to vehicle once lights went out and rats became more active.
Effect of Chlorpromazine on Heart Rate
Ab
solu
te D
ata
Ch
ange
Fro
m P
re-d
ose
Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
Re
sp
ira
tory
Ra
te(b
rea
ths/m
inu
te)
30
60
90
120
150
180
210
240Vehicle
Chlorpromazine (2 mg/kg)
Chlorpromazine (8 mg/kg)
Chlorpromazine (12 mg/kg)
Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
R
esp
ira
tory
Ra
te(b
rea
ths/m
inu
te)
-50
-25
0
25
50
75
100
125
Vehicle
Chlorpromazine (2 mg/kg)
Chlorpromazine (8 mg/kg)
Chlorpromazine (12 mg/kg)
No changes in respiratory rate at any dose level. Very unexpected!
Effect of Chlorpromazine on Respiratory Rate
Ab
solu
te D
ata
Ch
ange
Fro
m P
re-d
ose
Pressure
Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
Re
sp
ira
tory
Pre
ssu
re(m
mH
g)
-10
-5
0
5
10
15
20
25
Vehicle
Chlorpromazine (2 mg/kg)
Chlorpromazine (8 mg/kg)
Chlorpromazine (12 mg/kg)
Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
R
esp
ira
tory
Pre
ssu
re(m
mH
g)
-6
-4
-2
0
2
4
6 Vehicle
Chlorpromazine (2 mg/kg)
Chlorpromazine (8 mg/kg)
Chlorpromazine (12 mg/kg)
No significant changes in respiratory pressure immediately post dose, though we do start to see some depressed pressures during the night cycle (hours 9-21 post dose), especially in the high dose.
Effect of Chlorpromazine on Respiratory Pressure
Ab
solu
te D
ata
Ch
ange
Fro
m P
re-d
ose
Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
Activity
(AU
C)
0
2000
4000
6000
8000
10000 Vehicle
Chlorpromazine (2 mg/kg)
Chlorpromazine (8 mg/kg)
Chlorpromazine (12 mg/kg)
Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
Activity
(AU
C)
-4000
-2000
0
2000
4000
6000
8000
Vehicle
Chlorpromazine (2 mg/kg)
Chlorpromazine (8 mg/kg)
Chlorpromazine (12 mg/kg)
16mpk: -231%
Once again we see no changes in activity until the night cycle, were we see some depression in the high dose.
Effect of Chlorpromazine on Activity
Ab
solu
te D
ata
Ch
ange
Fro
m P
re-d
ose
Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
Body T
em
pera
ture
(Deg
C)
32
34
36
38
40
42
Vehicle
Chlorpromazine (2 mg/kg)
Chlorpromazine (8 mg/kg)
Chlorpromazine (16 mg/kg)
Time After Dosing (Hours)
0 2 4 6 8 10 12 14 16 18 20 22 24
B
ody T
em
pera
ture
(Deg
C)
-5
-3
-1
1
3
5 Vehicle
Chlorpromazine (2 mg/kg)
Chlorpromazine (8 mg/kg)
Chlorpromazine (16 mg/kg)
A dose dependant decrease in core body temperature from hours 2-14 post dose.
Effect of Chlorpromazine on Temperature
Ab
solu
te D
ata
Ch
ange
Fro
m P
re-d
ose
Results (Caffeine)
• Rats dosed with 24 mg/kg of caffeine showed the expected transient increase in heart rate (67.8 bpm; 19.3%) as well as increases in respiratory rate (61.9 bpm; 54.9%), and activity (400%). There was no apparent change in respiratory pressure.
• Rats given 3 and 12 mg/kg of caffeine also showed increases in heart rate (10.2%; 18.7%), respiratory rate (22.3%; 30.0%), and activity (257%; 489%) in a dose responsive manner
• Rats given chlorpromazine showed an increase in heart rate (66.1 bpm; 19.9%) and the expected decrease in activity (-231%) at a dose of 16 mg/kg.
• There was no apparent change in respiratory parameters following administration of chlorpromazine at any dose.
Results (Chlorpromazine)
Conclusions…
• Surgically feasible model
• Continuous 24 hour respiratory monitoring (time consuming)
• Reliable cardiovascular and neurobehavioral results for both caffeine and chlorpromazine
• Expected changes in respiratory parameters seen in caffeine dosed rats
• Unexpectedly, a decrease in respiratory parameters was not seen in chlorpromazine treated rats, thus further studies must be conducted to determine if the this combined model is able to detect decreases in respiratory rate with confidence.
Acknowledgements & References
• Dennis J. Murphy
• Safety Pharmacology staff at GlaxoSmithKline
• All studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed by the Institutional Animal Care and User Committee either at GSK or by the ethical review process at the institution where the work was performed.
1. Sgoifo, A., et al., “Electrode positioning for reliable telemetry ECG recordings during social stress in unrestrained rats.” Physiology and Behavior. 1996 Mar; 60(6):1397-1401. LINK
2. Lynch III, J. et al., “Comparison of methods for the assessment of locomotor activity in rodent safety pharmacology studies”, Journal of Pharmacological and Toxicological Methods, 64 (2011) 74-80. LINK
Thank You:
Brian M. RochePhD, DSP, DABT
Director of Safety Pharmacology,Charles River Laboratories
Jason D. Payseur
Senior Scientist – IVIVT, MSD Safety Pharmacology USGlaxoSmithKline
For additional information on the solutions presented in this webinar please visit www.datasci.com