Authors’ Disclosure SRM & APB are employees of GlaxoSmithKline

1. Disclosures “Effects of Aggressive Versus Conventional Lipid-lowering Therapy by Atorvastatin on Inflammatory Activity in Human Carotid Atherosclerotic Plaques: A Prospective Randomised Double Blind Trial with USPIO-Enhanced High Resolution Magnetic Resonance Imaging” The following relationships exist related to this presentation:Sinerem™ is not licensed by the FDA Authors’ Disclosure SRM & APB are employees of GlaxoSmithKline JHG is a consultant to GlaxoSmithKline

2. AtorvastatinTherapy:EffectsonReductionOfMacrophageActivity.Evaluation using USPIO enhanced high resolution MR in carotid disease ATHEROMA TY Tang, SP Howarth, SR Miller, AJ Patterson, MJ Graves, JM U-King-Im, ZY Li, AP Brown, JR Boyle, ME Gaunt & JH Gillard University Department of Radiology, Cambridge, UK

3. Vascular beds • Coronary disease • Carotid disease • Aortic disease • Peripheral vascular disease • Intracranial disease

4. Assessing outcome • Coronary disease • Carotid disease • Aortic disease • Peripheral vascular disease • Intracranial disease • MI, death • Stroke, TIA, death

5. Carotid vs. coronary disease – are they the same?

6. The Vulnerable Plaque • Why do plaques rupture? • Morphological Characteristics • thin, fibrous cap • large, necrotic lipid core • Physiology • cytokine and angiogenic factors • inflammatory burden (activated macrophages) • Biomechanics • plaque geometry and composition. Atherosclerosis: The New View Peter Libby, Scientific American May 2002

7. MR plaque imaging 390 mm x 390 mm x 3 mm 100 x 100 x 3 mm, 256 x 256 ETL 24, 44 heart beats

8. 100 mm scale right left

9. Rudd et al., Circulation 2002;105 :2708- Imaging inflammatory activity with FDG-PET

10. Sinerem (Guerbet, Paris) ultrasmall superparamagnetic iron oxide USPIO - Imaging the Inflammation • Signal drop on T2* imaging • Taken up by macrophages into phagolysosomes • When clumped in phagolysosomes, T2 shortening effect predominates.

11. USPIO transit

12. USPIO transit

13. 10mm Pre-USPIO infusion 48hr post USPIO infusion MAC387/Perls MAC387

14. USPIO Longitudinal Study • Pre and post imaging at 0(A), 6(B) and 12(C) months • Plaque can be seen to take up more USPIO at 12 months • Pre-USPIO imaging remains very similar. • No USPIO uptake is seen at pre-imaging at 6 months

15. USPIO enhanced MR imaging

16. Low concentrations - signal enhancement. USPIO-enhanced MR imaging T1 effect at low concentrations – signal enhancement T2* effects at higher concentrations – signal loss

17. ATHEROMA • Patients screened for USPIO “positivity” • Randomised to 10mg or 80 mg atorvastatin • Imaged at 6 and 12 weeks as well as biomarkers and transcranial Doppler ultrasound • Endpoints: • Changes in USPIO determined inflammation • Changes in plaque stress and compliance • Changes in emboli counts

18. Study procedures Screening visit Clinical assessment including informed consent Blood tests (renal function, haematology, liver function) ECG ? suitable EXCLUDE no yes no V1 V2 (36 hours) ? 10% signal drop in 2 or more quadrants on T2* weighted sequences between V1 and V2 imaging • MR imaging • TCD • Biomarkers • USPIO infusion • MR imaging yes RANDOMISE

19. Recruitment • Screened: 64 • Enrolled: 47 • Withdrawal: 7 • Adverse event 2 (statin related) • Withdrawn consent 1 (co-incidental lymphoma) • Other 4 • Non randomised 17

20. Image analysis • Signal pre and post normalised to adjacent muscle signal and expressed as: Quadrant Signalpost Pre-USPIO Post-USPIO Quadrant Signalpre Quadrant Signalpost Muscle Signalpre Muscle Signalpost Muscle Signalpost Quadrant Signalpre Muscle Signalpre

21. Image Analysis (2)

22. Patient Demographics

23. Baseline Characteristics

24. Change in Lipid Profiles

25. Change of LDL-C from baseline

26. 12 week data – High Dose Patient Visit 1 (pre) Visit 2 (post) Visit 5 (post) Visit 6 (post)

27. 12 week data – High Dose Patient Lipid profiles Atorvastatin 10mg previously V2 (post 0 weeks) Total chol Chol/ HDL ratio 4.0 4.0 2.1 2.1 V6 (post 12 weeks) 3.1 1.6

28. 12 week data – High Dose Patient -0.12 -0.02 V2 (post 0 weeks) -0.09 -0.05 • Signal loss decreased • Majority of quadrants now enhancing V6 (post 12 weeks) 0.18 0.13 -0.06 0.05

29. 12 week data – Low Dose Patient Visit 1 (pre) Visit 2 (post) Visit 5 (post) Visit 6 (post)

30. 12 week data – Low Dose Patient Lipid profiles Atorvastatin 10mg previously V2 (post 0 weeks) Total chol Chol/ HDL ratio 4.8 4.8 4.8 4.8 V6 (post 12 weeks) 4.9 4.8

31. 12 week data – Low Dose Patient 0.14 0.18 V2 (post 0 weeks) 0.29 0.32 signal loss has increased V6 (post 12 weeks) -0.2 0.1 0.04 -0.13

32. No change in low dose case at 6 & 12 weeks 76 yr old female. Asymptomatic bilateral 60% ICA Chol 5.1 Chol 6.0 Previously Simvastatin 40mg (equivalent Atorvastatin 10mg)

33. Enhancement in low dose patient previously only on 2.5mg equivalent of atorvastatin 70 yr old male. Asymptomatic 55% R ICA Chol 3.9 Chol 3.4 Previously Simvastatin 10mg (equivalent Atorvastatin 2.5mg)

34. Change in Imaging Endpoints

35. Signal Change from Baseline

36. Clinical correlates?

37. Conclusions from ATHEROMA • Aggressive lipid-lowering therapy over 12 weeks is associated with significant reduction in USPIO-defined inflammation • Change in clinical correlate – reduction in Micro-emboli on TCD • USPIO-enhanced MRI methodology may be a useful imaging biomarker for the assessment of therapeutic response to “anti-inflammatory” interventions in patients with carotid atherosclerotic lesions • Enrichment of trial populations - so only individuals with active inflammation can be selected

38. Nagui Antoun Jean-Claude Baron Tim Baynes Richard Coulden Justin Cross Hayley Eales Tim Fryer Stewart Walsh Mike Gaunt Martin J Graves Martin Goddard Nick Higgins Simon Howarth Ilse Joubert Peter J Kirkpatrick Zhi-Yong Li Andrew Patterson Karin Muller Eoin O’Brien James Rudd Jonathan Boyle Jeremy Skepper Tjun Tang Rikin Trivedi Jean U-King-Im Kevin Varty Elizabeth Warburton Peter Weissberg NHS R&D ECR Research Foundation GE Medical Systems Acknowledgements Andrew Brown Paul Matthews George Quartey Paul Thompson Liqun Wang Andrew Zalewski Andrew Zambanini Russell Kinch Sam Miller HannsJoachim Weinmann Bruno Bonnemain Claire Corot Sophie Gaillars Eric Lancelot Michelle Schaefer

39. AtorvastatinTherapy:EffectsonReductionOfMacrophageActivity.Evaluation using USPIO enhanced high resolution MR in carotid disease ATHEROMA TY Tang, SP Howarth, SR Miller, AJ Patterson, MJ Graves, JM U-King-Im, ZY Li, AP Brown, JR Boyle, ME Gaunt & JH Gillard University Department of Radiology, Cambridge, UK

40. ATHEROMA Discussion Slides

41. Reproducibility ICC 0.91 2 experienced readers (SPSH & TT) Blinded to the subject and clinical information 10 patients 24 paired patient visits 118 slices 906 matched quadrants included

42. Repeatability (V1 – V3) ICC 0.85 1 experienced reader (TT) 10 patients 58 slices 232 matched serial quadrants included

43. Repeatability (V3 – V5) ICC 0.81 1 experienced reader (TT) 10 patients 58 slices 232 matched serial quadrants included

44. ATHEROMA – Power Calculation • No data available from which to estimate the within-subject standard deviation for repeated assessments of USPIO-enhanced MRI signal change over time. • Data available from a study comparing a group of symptomatic to a group of asymptomatic patients (Howarth et al Eur J Rad 2008). • The estimated difference between these groups was 0.097, with a between-subject standard deviation of 0.093. • A sample size of 40 patients randomized 1:1 to high (80mg) or low (10mg) doses of atorvastatin should provide 90% power overall to detect a difference (with 5% type-I error) between the groups USPIO-enhanced MRI signal change at 12 weeks • Gives approximately 0.078 times the within-subject standard deviation.

45. 0 hrs 36 hrs 48 hrs 56 hrs 72 hrs 96 hrs Imaging kinetics of USPIO: T1 and T2* T2* weighted QIR spiral acquisition maximal T2* effect

46. Imaging kinetics of USPIO: T1 and T2* 0 hrs 36 hrs 48 hrs 56 hrs 72 hrs 96 hrs T1 weighted QIR acquisition maximal T1 effect

47. Imaging kinetics of USPIO: T1 and T2* 27.1 20.8 21.3 13.3 14.1 28.5 20.5 19.6 13.6 20.4 19.9 21.9 18.6 23.3 16.4 15.4 23.3 16.5 19.3 15.2 15.6 19.2 14.0 14.7 T2* quantification 0 hrs 36 hrs 48 hrs 56 hrs 72 hrs 96 hrs

48. Spiral @ 3.0T A B 1.5T C D 3.0T

50. Plaque risk • Luminal stenosis • Fibrous cap thickness • Lipid core • Macrophage infiltration • Plaque stress