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Novel In Vivo Approaches for Integrative Pharmacology and Toxicology Studies:Combining Radio Telemetry with Automated Sampling/Delivery Systems
Tuesday, July 13th, 2010We will begin at 10:00 AM
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Novel In Vivo Approaches for Integrative Pharmacology and Toxicology Studies:
Presenter Russell Bialecki, Ph.D.
Director, Safety Pharmacology North America
1-302-886-5356
Safety Pharmacology Society Webinar, July 13, 2010
Combining Radio Telemetry with Automated Sampling/Delivery Systems
ModeratorDerek Leishman, Ph.D.
Global Head of Safety Pharmacology
1-317-433-2578
Safety Pharmacology Society Webinar, July 13, 2010
5
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
6
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
• 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
7
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
8
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 ()
• 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
9
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
10
Available technology: BASi Culex® automated blood sampler
• 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
11
BASi Culex® components
Cage
Metabolic waste collection
Infusion pump
Sampling pump
Sample storage1
2
3
4
5
1
2
3
4
5
1
2
12
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
Available technology: Radio telemetry
13
DSI radio telemetry: Small animal system
PhysioTel® C50-PXT
Small Animal Transmitter
14
Our approach…
To maximize the utility of both systems, we needed an engineering solution combining BASi Culex® Automated Blood Sampling with DSI radio Telemetry.
15
Antenna bioengineering
• Obtained a mutual non-disclosure agreement with DSI and input on design modification
• Defined the radiation pattern of the transmitter
16
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)
17
The modified radio telemetry receiver…
18
Combined sytems: BASi Culex® and DSI radio telemetry
19
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
20
System optimization: Body weight gain
Effect of Different Acclimation Procedures on Body Weight Gain
Bod
y W
eigh
t (g)
200
250
300
350
400 ControlMetabolic cageCulex
Hydration Gelovernight
Hydration & Nutrition Gel
Fastovernight
*
-7.2%
*-5.1%
-5.5%-2.8%
+2.4%
Mean ± SD; n > 12
Bod
y W
eigh
t (g)
0
100
200
300
400
500
5 6 7 8 9 10 11 12 13 14 15 16
Age (weeks)
Comparison of Body Weight Gain
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
21
Comparison of stress levels
Stress hormones in ABSTvs tail bled and home cage rats
ACTH (p
g/m
l)
Cortic
oste
rone
(ng/
ml)
Insu
lin (u
IU/m
l)Pro
lact
in (n
g/m
l)
0
25
50
75
100
125
150
175
200
225Tail bleedABST Home cage
**
* * *
** p< .0001* p< .05
**
Lev
els
of
Ho
rmo
nes
22
Acclimation period optimization: CV and body temperature
Heart Rate
1 2 3 4 5 6300
350
400
450
500
550
9 10
Days
Bea
ts p
er m
inu
te (
min
)
Body Temperature
1 2 3 4 5 634
35
36
37
38
39
40
41
42
9 10
Days
Tem
p (
0C
)
Systolic Blood Pressure
1 2 3 4 5 6100
110
120
130
140
150
9 10
Days
Sys
toli
c B
P (
mm
Hg
)
Diastolic Blood Pressure
1 2 3 4 5 680
90
100
110
120
9 10
Days
Dia
sto
lic
BP
(m
m H
g)
Mean Arterial Blood Pressure
1 2 3 4 5 690
100
110
120
130
9 10
Days
MA
P (
mm
Hg
)
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
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)
24
Volume:
pH:
±
Characterization of urinary parameters: Urine volume and pH
pH
Time (day)
pH
0
2
4
6
8
10
0
2
4
6
8
10Metabolic cageCulex
1 3 7
Mean SD; n = 4
5.3
7.4
Ha
rlan
His
troric
al c
on
trol
Volume
Time (day)
Vo
lum
e (
mL
)
0
10
20
30
40
50
0
10
20
30
40
50Metabolic cageCulex
1 3 7
Mean SD; n = 4
3.2
28.0 Ha
rlan
His
troric
al c
on
trol (m
L)
ABST
25
U r in e C h e m is t r y
N a K C l P r o t e in C a P h o s U r e a
Cre
atin
ine
Ra
tio
0 .1
1
1 0
1 0 0
1 0 0 0
1 0 0 0 0 M e ta b o l ic c a g eC u le x
M e a n S D ; n = 4
D a y 1
D a y 3
D a y 7
Mean ± SD; n = 4
U r i n a r y B i o m a r k e r s
Cre
ati
nin
e R
ati
o
0 . 0 0 1
0 . 0 1
0 . 1
1
1 0
1 0 0
1 0 0 0
1 0 0 0 0 M e t a b o l i c c a g eC u l e x
M e a n S D ; n = 4
D a y 1
D a y 3
D a y 7
Mean ± SD; n = 4
Characterization of urinary parameters: Urinary electrolytes and biomarkers of injury
• Electrolytes and renal biomakers of injury were similar (p=N.S.) in ABST and metabolic cage systems
ABST ABST
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
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
Hours
27
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
28
Validation example: Baclofen effects on heart rate
Home Cage Animals ABST Animals
Time (min)
29
Validation example: Baclofen effects on mean arterial blood pressure
MA
BP
Home Cage Animals ABST Animals
30
PK-PD modeling: Baclofen-induced MABP changes
10
20
30
40
50
60
70
80
0 10 20 30 40 50 60 70 80
Ce Cp
Ce
Cp
31
Concentration- Effect
Relationship
PK-PD distribution delay model:Baclofen-induced increases in MABP and HR
32
PK-PD turnover model: Baclofen-induced decrease in temperature
Stimulation
Turnover Model
Oscillation in temp
Temp turnover
PK
Temp
Kin Kout
[AZ
XX
X2 (μ
M)]
[AZ
XX
X2 (μ
M)]
[AZ
XX
X2 (μ
M)]
Vehicle
AZXXX2
[AZXXX2 (µM)]
Case study #1: Exploiting data in ABST Noisy outcomes?
34
Case study #1: Exploiting data in ABST Noisy data was informative!
Outlier [AZXXX2 (μM)]
Outlier AZXXX2 MABP
Outlier AZXXX2 HR
xxx2
AZXXX2 MABP
AZXXX2 HR
[AZXXX2 (μM)]
• Only possible with population PK
35
Case study #2: Value of multiparametric analyses This compound appears to effect CV function, but in ABST…
VehicleAZDXXXX
Mean arterial blood pressure Heart rate
36
Case study #2: Value of multiparametric analyses …EEG effects precede CV collapse
-60 0 60 1200
50
100
150
200
250
300
350
400
450
500
550
600
MABP
HR
05101520253035404550
[AZDXXXX]
150 mg/kg po50 mg/kg po
51
76
101
126
151
176
150 165 180 195 210 225 240
interictal activity
spike & wavecontinuous seizure
Time post 1st oral dose (minutes)
bea
ts/m
in
mm
Hg
or [C
MP
D X
( M
)]
37
Case study #2: Value of multi-parametric analyses Spike & wave seizure seen before CV effects
38
EEG Spectrogram
39
Variables that can be measured in one ABST animal
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)
ABST
40
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
41
Acknowledgements
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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