1. Non-invasive telemetry What else but ECG ? Pierre Lainee Claire Grant – Emily Bailey

2. Non invasive telemetry BP Why would we use it in Tox studies ? Because they give access to repeated dosing Because exposures are higher, potentially improving safety margins Because Phase 1 trials also include repeated administration Because small changes over long periods need attention If ECG is already collected with this method, limiting the need for additional jacket training Because incidence of BP effects in preclinical studies is significant 2 EMKA Presentation– 21 September 2011

3. Impact of repeated dosing Comparison of ECG findings beginning vs. end of studies 3 EMKA Presentation– 21 September 2011 Dog DRF studies (n=150)Dog 1-month studies (n=160) Isn’t it likely to be similar for BP effects ?

4. Current Methods 4 EMKA Presentation– 21 September 2011 Blood Pressure Invasive telemetry  Not suitable for tox studies Minimally invasive telemetry  Growing interest and rational  But still demanding and expensive Invasive ear catheter  Restraint/Stress  30s/twice a week Respiration Rodent WBP  This is an indirect measure of respiratory function  Limited in duration due to animal welfare constraints

5. Non-invasive Blood Pressure (NIBP) 5 EMKA Presentation– 21 September 2011  Utilises tail cuff  Detects changes in oscillations  Extended recording periods  Freely moving animals  Programmed to inflate/deflate every 4 min  Cuff inflated to 240 mmHg  Deflation takes ~ 30 s  Reported as an average of 5 inflations (time point ±10 min)  On average replace tail cuff ~ 1 per session  From our experience, we feel that the difficulty of keeping the cuff on is strongly related to the tail shape, e.g. some have very thick bases and the tail gets progressively thinner (cone- shaped)

6. Non-invasive Blood Pressure (NIBP) Tail is shaved (~ 10 cm) Animals are habituated to tail cuff (at the same time as jacket training). First training session just jacket, second session with tail cuff, third session with cuff inflating/deflating. Each session gets progressively longer. Wrap the tail cuff round the tail, approximately 3 cm from the base of the tail. Vetwrap is wrapped round the tail below the cuff to prevent the cuff from slipping (a ‘stopper’). Another layer of vetwrap is placed round the cuff and the ‘stopper’. Do not wrap too tightly as this will impair blood flow. The tubes coming out of the cuff are cut to correct length to and are attached to the transmitter. 6 EMKA Presentation– 21 September 2011 Animal preparation

7. Respiratory Inductive Plethysmography (RIP)  RIP utilizes specialized belts containing inductive wires placed around the thorax and abdomen.  Measures changes in inductance, which are proportional to cross sectional area and then indirectly to lung volume.  Enables calculation of breathing frequency (f), tidal volume (TV) and minute volume (MV).  To get volume values, a face mask calibration is required (approx. 30 sec measurement).  Good calibration performed pre-study and that calibration coefficient used for subsequent recordings for that dog.  Animal posture does not impact on calibration (let the dog be in a relaxed position). 7 EMKA Presentation– 21 September 2011

8. Analysis 8 EMKA Presentation– 21 September 2011 Blood Pressure  Pulse amplitude vs. Cuff pressure plotted  SBP and DBP derived from curve Respiration  Respiratory trace recording  Continuous acquisition and on-line analysis of ach breath (up to ~30,000 during a 24 hour recording)  Noisy data can easily be excluded Analysis  Signals are acquired and analysed by the EMKA analysis system.

9. Methods 9 EMKA Presentation– 21 September 2011  4 reference compounds tested  Theophylline:0, 5, 10 and 20 mg/kg  Clonidine:0, 0.03, 0.1 and 0.3 mg/kg  Verapamil:5, 10 and 15 mg/kg  L-NAME: 0 and 10 mg/kg  Single dose of vehicle followed by single dose of compound in ascending concentrations at intervals of 2-5 days  Dogs are part of the invasive telemetry colony, naïve to jacket training  SBP, DBP and ECG recorded by invasive telemetry (INV)  SBP, DBP, ECG, f, TV, MV recorded by NIBP and RIP  Blood Samples taken at pre-dose, 1, 3 and 24 hours  All animals observed via CCTV for abnormal clinical signs and vomiting

10. NIBP vs. INV 10 EMKA Presentation– 21 September 2011  Values for both SBP (-14%) and DBP(-10%) are lower with the NIBP system than the INV.  NIBP system measures pressure in the medial coccygeal artery  INV measures pressure directly in the descending aorta  Reference (i.e. zero) value not undefined for INV

11. Sensitivity of NIBP system Theophylline 11 EMKA Presentation– 21 September 2011 Graphs only display high dose, mean ± SEM System detected increase in BP Duration and magnitude similar to invasive telemetry. NIBP INV

12. Sensitivity of NIBP system L-NAME 12 EMKA Presentation– 21 September 2011 NIBP INV System detected increase in BP Duration and magnitude similar to invasive telemetry. Graphs only display high dose, mean ± SEM

13. Sensitivity of NIBP system Clonidine 13 EMKA Presentation– 21 September 2011 NIBP is less sensitive than INV for this decrease in BP Graphs only display high dose, mean ± SEM NIBP INV

14. Verapamil 14 EMKA Presentation – 21 September 2011 Graphs only display high dose, mean ± SEM System detected decrease in BP Duration and magnitude similar to invasive telemetry Effects on DBP are less marked NIBP INV Sensitivity of NIBP system

15. NIBP variability  Using 4 animals the statistical power of NIBP and INV to detect significant changes in blood pressure are comparable.  Using the NIBP system with 4 dogs a change of 24 (SBP) and 18 (DBP) mmHg is significant (vs. 29 and 18 for INV)  Most of the difference in sensitivity comes from the ability to select the best periods with invasive telemetry (i.e. continuous vs. repeated timepoints) 15 EMKA Presentation – 21 September 2011 Baseline values (mmHg) Significant Delta% (80% power, n=4)

16. Duration of recording Recording of NIBP has been limited to 6 h post-dose for welfare reasons and better protection of the equipment. A review of AstraZeneca INV telemetry studies from 2000 (~300 studies) was performed. 36 studies showed clear blood pressure related effects. -T max ≤ 6h 35 studies -Time of onset all below 4 h -Return to baseline ≤ 6h 16 studies If recording for 6 h post-dose an effect would have been detected in all studies. 16 EMKA Presentation – 21 September 2011

17. Sensitivity of RIP system 17 5 mg/kg10 mg/kg20 mg/kg BF+22%+18%+45% TV+18%+21%+25% MV+25%+41%+78% 0.03 mg/kg0.01 mg/kg0.3 mg/kg BF-21%-16%-28% Theophylline Clonidine EMKA Presentation – 21 September 2011 Graphs only display high dose

18. Sensitivity of RIP system 18 EMKA Presentation – 21 September 2011 10 mg/kg BF+32% TV-26% MV-28% L-NAME Graphs only display high dose. % change is max effect compared to time matched vehicle.

19. NIBP Practical considerations Pros and Cons  Although HDO offers a better outcome, it seems that SBP effects are still better detected than DBP ones  Long term recording but not continuous, which limits the selection of optimal period, and secondarily decreases sensitivity  Limitations in early studies due to small groups  Additional time for data analysis (but very valuable data)  Additional investment (but value for money)  Access to effects after repeated dosing with no need for additional study and no surgery  Easy to use with staff trained to external telemetry  Training is not a limitation as it takes place along jacket training  In a context where external telemetry is used routinely, NIBP and RIP definitely add value  In the hands of a SP group, reference compounds do show the expected effects  Sensitivity can be compensated by larger size groups in 1-month studies 19 EMKA Presentation – 21 September 2011 This validation work was convincing and NIBP will find a place in our CVS strategy

20. Testing Strategy  Include NIBP in repeat dose phase of dose range finding dog toxicology study when blood pressure liability is identified from  Project knowledge  Earlier CVS studies (anesthetised GP or rat telemetry) 3Rs incentive of more data without increasing animal numbers  Include RIP in GLP invasive telemetry study (need more data first).  Replace rodent whole body plethysmography study.  3Rs incentive of reduction of ~40 rodents per compound.  Assess multiple organ systems simultaneously in large animals to explore the pharmacological interaction in response to a drug. 20 EMKA Presentation – 21 September 2011

21. Conclusions 21 EMKA Presentation – 21 September 2011  10 minutes to jacket animal and perform 30 second calibration  BP – 6 hours, Respiration and ECG 24 hours  Detected changes in response to reference compounds  Ease of analysis – eliminates noisy data  No effect on quality of ECG measurements Future Work and Perspectives  Now being used in repeat-dose tox studies  Incorporation into ToE ?  Use of activity and video for clinical monitoring  Improves vomiting detection  Possible replacement of WBP with RIP  Bronchoconstriction – Carbachol  use of the shift between abdominal and thoracic signals)

22. Thanks To all staff involved in testing over the last year - Emily for her dedication along a placement year - Claire for supervising the work and organising the studies - Jackie for helping with the invasive telemetry acquisition and analysis - Clare and Kate for bringing the expertise and patience acquired from ECG recordings into this new method To the EMKA team which provided - the appropriate conditions and support for the required adjustments to be made - the training to facilitate data analysis optimisation 22 EMKA Presentation – 21 September 2011