Novel In Vivo Approaches for Integrative Pharmacology and Toxicology Studies:Combining Radio Telemetry with Automated Sampling/Delivery Systems

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4

Novel In Vivo Approaches for Integrative Pharmacology and Toxicology Studies:

Presenter Russell Bialecki, Ph.D.

Director, Safety Pharmacology North America

russell.bialecki@astrazeneca.com

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

derek_leishman@lilly.com

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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