Novel In Vivo Approaches for Integrative Pharmacology and

Novel In Vivo Approaches for Integrative Pharmacology and Toxicology Studies: Combining Radio Telemetry with Automated Sampling/Delivery Systems Tuesday, July 13 th, 2010 We will begin at 10: 00 AM

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Novel In Vivo Approaches for Integrative Pharmacology and Toxicology Studies: Combining Radio Telemetry with Automated Sampling/Delivery Systems 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 4 Moderator Derek Leishman, Ph. D. Global Head of Safety Pharmacology derek_leishman@lilly. com 1 -317 -433 -2578 Safety Pharmacology Society Webinar, July 13, 2010

Presentation outline • Background • Integrative Pharmacology – definition and objective • Advantages to combining models • Statistical • 3 R’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 • • 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 • 10 Programmable sampling from conscious freely moving rodents 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: 3 R’s • Improved safety for animal and researcher • Automated data recordings and analysis • Adherence to guidelines and standards • e. g. , ICH S 7 A 12

DSI radio telemetry: Small animal system Physio. Tel® C 50 -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 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

Comparison of stress levels 21

Acclimation period optimization: CV and body temperature 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

Integrative pharmacology model: Renal parameters DSI Telemetry (C 50 -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 p. H Volume: ABST ± p. H: 24

Characterization of urinary parameters: Urinary electrolytes and biomarkers of injury ABST Mean ± SD; n = 4 • Electrolytes and renal biomakers of injury were similar (p=N. S. ) in ABST and metabolic cage systems 25

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 30

PK-PD distribution delay model: Baclofen-induced increases in MABP and HR Concentration Effect Relationship 31

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? [AZXXX 2 (μM)] Vehicle AZXXX 2 [AZXXX 2 (µM)]

Case study #1: Exploiting data in ABST Noisy data was informative! • Only possible with population PK AZXXX 2 MABP AZXXX 2 HR [AZXXX 2 (μM)] Outlier AZXXX 2 MABP xxx 2 34 Outlier AZXXX 2 HR Outlier [AZXXX 2 (μM)]

Case study #2: Value of multiparametric analyses This compound appears to effect CV function, but in ABST… Mean arterial blood pressure 35 Heart rate Vehicle AZDXXXX

Case study #2: Value of multiparametric analyses …EEG effects precede CV collapse 36

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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