Novel In Vivo Approaches for Integrative
Pharmacology and Toxicology Studies:
Combining Radio Telemetry with Automated Sampling/Delivery Systems
Tuesday, July 13th, 2010
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Novel In Vivo Approaches for Integrative
Pharmacology and Toxicology Studies:
Combining Radio Telemetry with Automated
Sampling/Delivery Systems
Presenter
Moderator
Russell Bialecki, Ph.D.
Director, Safety Pharmacology
North America
russell.bialecki@astrazeneca.com
1-302-886-5356
Derek Leishman, Ph.D.
Global Head of Safety
Pharmacology
derek_leishman@lilly.com
1-317-433-2578
Safety Pharmacology Society Webinar, July
13,
2010
4
Safety Pharmacology Society Webinar, July
13, 2010
Presentation outline
• Background
• Integrative Pharmacology – definition and objective
• Advantages to combining models
• Statistical
• 3R’s
• Cost, Quality and Speed
• Available technology
• BASi Culex®
• Data Sciences International (DSI) radio telemetry
• Our approach – integrating equipment
• The engineering solution
• Demonstrating signal capture
• System optimization and validation
• Case studies
• Summary and conclusions
5
Integrative pharmacology
•
Defined as the simultaneous evaluation of multiple parameters to
assess interrelationships of various physiological and pathological
processes in response to drugs
•
Integrative (i.e., in vivo) pharmacology remains an essential part of
drug discovery
•
Regulatory authorities require the pharmaceutical industry to
demonstrate in vivo pharmacokinetic (PK), pharmacodynamic (PD),
and safety properties of new drug candidates
•
The PK-PD relationship allows a determination of:
1. Therapeutic actions of drug exposure
2. Safety margins correlating therapeutic and toxic effects of a compound
6
•
The PK-PD relationship can be derived in many ways
•
Thus far, there has been no ideal way to determine PK-PD in a
single animal
Objective
• To describe a novel in vivo model for simultaneous
measurement of multiple organ system functions and PK
by combining automated blood sampling technology and
radio telemetry
7
Combined model:
Statistical advantages
‘Within’ experimental design
• Every animal serves as it’s own control
• Better control of multiple independent variables
• e.g., weight, age, food intake, environment, etc.
• Variability less than ‘between’ designs
• Fewer animals for the same test power (b)
• Comparison information across organ systems
• Establish correlative relationships
• Time-dependent vs. time-independent effects
• Exposure-dependent vs. dose-dependent
• Identification of outliers
• Data collected after dosing error still useful
• e.g., exposure comparisons post-dose
• Exposure differences used to your advantage
8
Combined model:
Animal welfare and other advantages
Measuring multiple organ functions simultaneously:
• Reduces animal use vs. singlet studies
• Refines data comparisons
• Better control of study variables
• Paired-wise studies vs. separate control groups
• Better evaluation of multivariate relationships
• Replaces use of higher mammals with sentient species
• Earlier stop decision on toxic compounds
• Decreases cost vs. singlet studies
• Increases speed of delivery vs. sequential singlet studies
• Facilitates better and earlier decision-making
9
Available technology:
BASi Culex® automated blood sampler
10
•
Programmable sampling from
conscious freely moving rodents
•
Up to 1.5 ml collected into chilled
vials; volume replaced with saline
•
4 rats sampled simultaneously
•
Little/no animal handling reduces
animal stress
•
Infusion system available for drug
delivery
•
Includes metabolic waste collection
system
BASi Culex® components
1
Cage
2
Metabolic waste
collection
3
Infusion pump
4
Sampling pump
5
Sample storage
1
4
3
5
1
2
11
2
Available technology:
Radio telemetry
Benefits
• Reduced Stress for animal
• Reduced Stress for the researcher!
• True, physiological, reproducible data
• Better animal welfare: 3R’s
• Improved safety for animal and researcher
• Automated data recordings and analysis
• Adherence to guidelines and standards
• e.g., ICH S7A
12
DSI radio telemetry:
Small animal system
PhysioTel® C50-PXT
Small Animal
Transmitter
13
Our approach…
To maximize the utility of both systems, we needed
an engineering solution combining BASi Culex®
Automated Blood Sampling with DSI radio
Telemetry.
14
Antenna bioengineering
• Obtained a mutual non-disclosure agreement with DSI and
input on design modification
• Defined the radiation pattern of the transmitter
15
Antenna bioengineering:
Demonstrating signal capture
• Constructed a series of antennas leading to an optimal design
D. Litwin et al. An Integrative Pharmacological Approach to Radiotelemetry and Blood Sampling in Pharmaceutical Drug
Discovery and Safety Assessment Engineering in Medicine and Biology (In review)
16
The modified radio telemetry receiver…
17
Combined sytems:
BASi Culex® and DSI radio telemetry
18
Presently measured parameters in ABST
• Cardiovascular
•
•
•
•
•
HR
MABP
Systolic BP
Diastolic BP
Pulse pressure
• CNS
• EEG
• Activity counts (telemetry and Culex®)
• Body temperature
• GFR/RPF, urinary electrolytes, biomarkers of injury
• Plasma drug exposures
• Blood constituents
19
System optimization:
Body weight gain
Comparison of Body Weight Gain
500
400
Body Weight (g)
350
Control
Metabolic cage
Culex
+2.4%
400
Body Weight (g)
Effect of Different Acclimation Procedures on
Body Weight Gain
300
200
100
Mean ± SD; n > 12
0
5
6
7
8
9
10
11
12
Age (weeks)
300
-5.5%
-7.2%
*
-2.8%
-5.1%
*
250
200
Fast
overnight
Hydration Gel
overnight
Hydration &
Nutrition Gel
H. Kamendi et al. Combining Radio Telemetry and Automated Blood Sampling: A Novel Approach for Integrative
Pharmacology
and Toxicology Studies. J. Pharmacol. Toxicol. Methods (2010) doi: 10.1016/j.vascn.2010.04.014
20
13
14
15
16
Comparison of stress levels
225
200
175
150
125
100
75
50
25
0
**
** p< .0001
* p< .05
*
(n
g/
m
l)
ac
tin
(u
I
in
In
su
l
Pr
ol
l)
(n
g/
m
m
os
te
ro
ne
(p
g/
Co
rti
c
U/
m
l)
* *
AC
TH
21
Tail bleed
ABST
Home cage
**
l)
Levels of Hormones
Stress hormones in ABST
vs tail bled and home cage rats
Acclimation period optimization:
CV and body temperature
ABST
Home cage Telemetry
Mean Arterial Blood Pressure
Systolic Blood Pressure
Diastolic
Blood Pressure
120
110
100
90
120
140
130
120
110
100
1
2
3
4
5
6 9
10
Diastolic BP (mm Hg)
120
1
2
3
Days
4
Days
5
Diastolic BP (mm Hg)
Systolic BP (mm Hg)
150
110
100
110
100
90
80
6 9
10
1
2
80
0 Body
1 Temperature
2
3
4
5
Days
41
500
4
Temp ( 0C)
40
450
400
39
38
37
36
350
35
34
300
1
2
3
4
Days
5
6 9
10
1
5
6 9
Days
42
550
3
90
Heart Rate
Beats per minute (min)
MAP (mm Hg)
130
Diastolic Blood Pressure
2
3
4
Days
H. Kamendi et al. Combining Radio Telemetry and Automated Blood Sampling: A Novel Approach for Integrative
Pharmacology
and Toxicology Studies. J. Pharmacol. Toxicol. Methods (2010) doi: 10.1016/j.vascn.2010.04.014
22
5
6 9
10
6
7
8
10
Integrative pharmacology model:
Renal parameters
DSI Telemetry
(C50-PXT)
• EEG
• Blood Pressure
• Heart rate
• Body temperature
• Activity
Renal Function
• Urinary Biomarkers
• GFR
• RPF/RBF
• Urinalysis
• Filtration fraction
(GFR/RPF)
• Renal vascular
resistance (MAP/RBF)
Characterization of urinary parameters:
Urine volume and pHVolume
Volume:
50
50
Metabolic cage
Culex
ABST
Mean ± SD; n = 4
40
30
28.0
30
20
20
10
10
pH
10
0
pH:
Metabolic cage
Culex
1
Mean
SD; n = 4
3
Time (day)
3.2
Harlan
Histrorical control (mL)
Volume (mL)
40
10
0
7
8
8
7.4
6
pH
5.3
4
4
2
2
0
24
0
1
3
Time (day)
7
Harlan
Histrorical control
6
Characterization of urinary parameters:
Urinary electrolytes and biomarkers of injury
Urine Chemistry
10000
Urinary Biomarkers
10000
Metabolic cage
Culex
ABST
Metabolic cage
ABST
Culex
1000
Mean± SD;
Mean
44
SD;n n= =
Mean± SD;
Mean
44
SD;n n= =
100
Day 7
100
Creatinine Ratio
Day 3
Day 1
10
Day 7
10
Day 3
Day 1
1
0.1
1
0.01
0.1
po
eo
st
O
•
25
Electrolytes and renal biomakers of injury were similar (p=N.S.)
in ABST and metabolic cage systems
1
im
K
nt
in
lin
ca
po
um
in
Li
lu
Al
b
st
er
in
PA
C
Urea
R
Phos
Yb
Ca
ST
Protein
G
Cl
ST
K
1
0.001
Na
aG
Creatinine Ratio
1000
Estimates of renal function in vivo
 Glomerular filtration rate (GFR) and renal plasma flow (RPF) were validated using
a continuous infusion method (i.v., FITC-Inulin and PAH) in conscious,
unrestrained Han Wistar rats
 No differences (p=N.S.) were noted in GFR or RPF estimates using plasma
clearance [Fick principle] vs. traditional urinary clearance methods
 Plasma clearance methods allow simple, rapid and reproducible evaluation of renal
function vs. traditional approaches
Hours
Y. Chen et al. Renal function evaluation in a cardio-renal pharmacology model using Culex Emphis® automated
infusion system and radiotelemetry. EB 2010 FASEB J. Abstr 5437
Validation example:
Baclofen effects on body temperature
Home Cage Animals
ABST Animals
• ABST rats (right) show similar responses to traditional telemetry (left) but
allow for blood sampling from the same subject
• Satellite animals used in traditional telemetry were required to avoid handling
artifact in temperature, CV and other parameters during manual blood draws
27
Validation example:
Baclofen effects on heart rate
Home Cage Animals
Time (min)
28
ABST Animals
Validation example:
Baclofen effects on mean arterial blood pressure
MABP
Home Cage Animals
29
ABST Animals
PK-PD modeling:
Baclofen-induced MABP changes
80
70
60
50
Ce
40
Cp
30
20
10
0
10
20
30
40
Ce Cp
30
50
60
70
80
PK-PD distribution delay model:
Baclofen-induced increases in MABP and HR
Ka
PK Compartment
Volume of distribution
Concentration of drug
in the Effect
Compartment (Ce)
ConcentrationEffect
Relationship
Effect/Time
Compartment
Cl*F
Mean Arterial Pressure
180
100.0
100.0
170
170
160
160
150
10.0
150
10.0
140
140
F1 Observ ed
130
F1 Predicted
F2 Observ ed
120
1.0
120
110
110
100
0.1
0.1
0
100
200
300
400
500
600
700
800
90
0
100
200
300
timec (min)
400
500
600
700
800
130
F2 Predicted
1.0
0
100
200
300
timec (min)
400
500
600
700
100
0.01
800
0.10
1.00
Time
10.00
100.00
Ce (uM)
Heart Rate
430
425
100.0
100.0
460
420
440
10.0
10.0
415
410
420
F1 Observ ed
400
405
F1 Predicted
F2 Observ ed
1.0
1.0
400
F2 Predicted
380
395
360
0.1
0.1
0
100
200
300
400
timec (min)
31
500
600
700
800
390
385
340
0
100
200
300
400
timec (min)
500
600
700
800
0
100
200
300
400
Time (min)
500
600
700
800
0
5
10
15
20
Ce (uM)
25
30
35
40
PK-PD turnover model:
Baclofen-induced decrease in temperature
Turnover Model
Kin
Oscillation in
temp
Temp
turnover
Kout
Stimulation
PK
Temp
32
Case study #1: Exploiting data in ABST
Noisy outcomes?
[AZXXX2 (μM)]
[AZXXX2
[AZXXX2 (μM)]
(μM)]
Vehicle
AZXXX2
[AZXXX2 (µM)]
Case study #1: Exploiting data in ABST
Noisy data was informative!
• Only possible with population PK
AZXXX2
MABP
AZXXX2
HR
[AZXXX2 (μM)]
Outlier AZXXX2 MABP
xxx2
34
Outlier AZXXX2 HR
Outlier [AZXXX2 (μM)]
Case study #2: Value of multiparametric analyses
This compound appears to effect CV function, but
in ABST…
Arterialblood
Blood Pressure
MeanMean
arterial
pressure
Heartrate
Rate
Heart
160
160
150
140
140
130
130
120
120
110
110
100
100
90
90
Vehicle
80
AZ13032174
% baseline
% baseline
150
80
70
60
60
50
40
40
30
30
20
20
10
10
0
30 60 90 120 150 180 210 240
Time post 1st oral dose (minutes)
50 mg/kg dose at t=0 minutes
150 mg/kg dose at t = 180 minutes
Vehicle
AZ13032174
70
50
0
-120 -90 -60 -30
35
Vehicle
AZDXXXX
0
-120 -90 -60 -30
0
30 60 90 120 150 180 210 240
Time post 1st oral dose (minutes)
50 mg/kg dose at t=0 minutes
150 mg/kg dose at t = 180 minutes
Case study #2: Value of multiparametric analyses
…EEG effects precede CV collapse
spike & wave
continuous seizure
600
interictal activity
550
176
500
151
101
beats/min
400
76
350
300
250
200
150
100
50
0
-60
0
60
51
50
45
40
35
30
25
20
15
10
5
0
120 150 165 180 195 210 225 240
Time post 1st oral dose (minutes)
50 mg/kg po
36
150 mg/kg po
mm Hg or [CMPD X (  M)]
126
450
MABP
HR
[AZDXXXX]
Case study #2: Value of multi-parametric analyses
Spike & wave seizure seen before CV effects
37
EEG Spectrogram
38
Variables that can be measured
in one ABST animal
ABST
D. Litwin et al. An Integrative Pharmacological Approach to Radiotelemetry and Blood Sampling in Pharmaceutical Drug
Discovery and Safety Assessment Engineering in Medicine and Biology (In review)
39
Summary and conclusions
• We have successfully combined automated blood sampling with
radio telemetry
• The ABST system enables measurement of multiple parameters
(e.g., CV, temperature, various biopotential signals, renal, etc)
while collecting blood and urine samples in real time
• Baclofen PK and PD variables were qualitatively and
quantitatively similar in traditional radio telemetry and ABST
systems
• The ABST system offers unique advantages over traditional
approaches including decreased cost, decreased live phase
study time, refined data sets and reduced animal use
40
Acknowledgements














41
Dennis Litwin
David Lengel
Harriet Kamendi
Yafei Chen
Carlos Fonck
Ray Rothstein
Michael Quirk
Khanh Bui
Mary Jo Bock
Mary Ann Gorko
Beth Bell
Chengwei Fang
Jean-Pierre Valentin
Silvana Lindgren











David Brott
Lindsey Kegelman
Patricia Bentley
Nicole Gilper
Susan Wartel
Perry Mills (DSI)
Candace Rohde (BASi)
Matthew Ruiter (SAI)
Andy Andler (SAI)
Harlan Laboratories
Charles River
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