Page 1 1 FDA Advisory Committee Meeting “Safety Considerations in the Development of Ultrasound Contrast Agents” June 24, 2008 Nonclinical Development Program: SonoVue® Patricia D. Williams, PhD Chief Operating Officer Summit Drug Development, LLC Rockville, MD

Page 2 2 Nonclinical Development Program: SonoVue  Pharmacology studies  Toxicology studies  Studies on concurrent SonoVue administration and ultrasound exposure at high acoustic pressure  Retrospectively (after anaphylactoid reactions were observed in humans) potential mechanisms of these reactions were studied in vitro and in vivo

Page 3 3 Pharmacology Studies: SonoVue  Imaging Studies to define the expected human dose of SonoVue (pig, dog)  Safety pharmacology studies at multiples of human imaging doses (MHDbsa) 3 (mouse), 5 (rat), 10 (rabbit), 18 (dog), 200 (monkey): - Cardiovascular (dog, monkey) - Respiratory (rat, rabbit) - CNS (mouse) - Gastrointestinal (rat) - Renal (rat)

Page 4 4 Key Findings Safety Pharmacology Studies: SonoVue  CONSCIOUS DOGS: - No cardiovascular effects at 0.3 mL/kg (10-times clinical dose) - At 1.0 mL/kg transient hypotension in 2/7 dogs  ANESTHETIZED DOGS (Pulmonary Hypertension model): - Transient and minimal (2.5 ± 1.3 mmHg) increase in PAP at 1 mL/kg  No other significant Cardiovascular, CNS, Respiratory, GI or Renal findings in mouse, rats, rabbits, monkeys

Page 5 5 Toxicology Studies: SonoVue Intravenous bolus administration Clinical formulation used Max doses times the human dose (MHDbsa)  Single dose studies (rat, monkey)  4-week repeated dose studies (rat, monkey)  Genetic toxicology  Reproductive toxicology (rat, rabbit)  Other studies (local tolerance; blood compatibility)

Page 6 6 Key Findings Toxicology Studies: SonoVue  No significant findings in single and repeat dose studies up to the dose of 5 mL/kg (rat, monkey)  Repeat dose NOEL in monkeys 5 mL/kg (50 MHDbsa)  Cecum lesions in rats: considered rodent specific and observed with other contrast agents; otherwise NOEL 5 mL/kg  No signs of immunological reactions in either species (thymus, spleen, lymph nodes)  No lung lesions or emboli in either species  No brain lesions after direct injection in carotid artery in rats (1 mL/kg)

Page 7 7 Studies With SonoVue and Concurrent Ultrasound Exposure  RATS: No histological lesions in organs with SonoVue (1 and 5 mL/kg) and exposed to ultrasound at high acoustic pressure (up to MI 1.9)  DOGS: No effect of ultrasound exposure (up to MI 1.2) on ECG (QTc) and on heart histopathology in conscious dogs administered with SonoVue (up to 1 mL/kg)

Page 8 8 Conclusion of the Nonclinical Animal Studies on SonoVue  SonoVue was well tolerated in standard toxicological and safety pharmacology studies when administered alone or with concurrent ultrasound exposure  Cardiovascular effects (transient hypotension) observed only at very high doses in dogs (1 mL/kg)  Nonclinical study results corroborated the overall safety profile of SonoVue in humans

Page 9 9 Mechanism of Anaphylactoid Reactions  Low incidence (~ 0.01%) of allergic-like (anaphylactoid) reactions in humans found in post-marketing surveillance  Additional in vitro and animals studies were designed to investigate potential mechanism(s) of these reactions

Page Mechanism of Anaphylactoid Reactions Hypothesis: allergic-like (anaphylactoid) reactions are related to the particulate nature of ultrasound contrast agents  Cardiopulmonary studies at imaging dose in pigs  Cardiovascular studies at very high doses in rats  In vitro complement activation (pig, human)  In vitro basophil activation (human)

Page The Use of the Pig: Pros & Cons  Common large animal used in echocardiography (eg. size of heart ~ human)  Known to have severe reactions to injection of particulates  Have high concentrations of pulmonary intravascular macrophages (PIMs) relative to other species including humans  Imaging studies of UCA in pigs are routinely done with pretreatment with indomethacin or aspirin  While useful for imaging, pig not considered as an animal model for safety pharmacology studies due to its over-reaction to all injected particles, however…  Based on rare human reactions, Bracco pursued studies in naïve pigs to possibly gain insight into the mechanism of these anaphylactoid reactions

Page Changes in PAP and SAP in Pigs Following SonoVue, and other UCAs at Imaging Doses

Page Marked Individual Variations of the CV Response to SonoVue in Pigs

Page Thromboxane Release Parallels PAP Increase with SonoVue and another UCA Kinetics of pulmonary arterial pressure and TXB2 changes following SonoVue® or another UCA (Product B) injections in the pig at the human imaging dose (mean of 5 injections)

Page Key Findings Mechanistic Studies: Pig Model  SonoVue and other marketed UCAs were tested on naïve, anesthetized pigs  Doses in the range of 1- 4x human imaging dose  ↓ SAP ↑ PAP ↑ HR  ↑ Airway resistance ↓ Lung compliance  Effects are dose and injection rate dependent  ↑ Plasma Thromboxane B2  No detectable increases in C3a/C5a in vivo  Effects blocked by aspirin pretreatment  Effects similar to other injected particulates (liposomes, micellar lipids, etc)

Page Key Findings Mechanistic Studies: Pig Model  Marked individual variation but consistent response in pigs at imaging dose of UCA  Pig shows sensitivity to SonoVue not seen in humans  Symptoms & cardiovascular effects resemble anaphylactoid reactions in humans  Release of vasoactive mediators considered key event in pigs  Relevance to humans unknown

Page Key Findings Mechanistic Studies: Rat Model  Transient hypotension observed  5 mL/kg (25 MHDbsa)  ↑ Plasma thromboxane B2 (similar to pigs), however…  Hypotension NOT blocked by aspirin pretreatment (contrary to pigs)  Hypotension blocked by PAF-antagonist (ABT-491) pretreatment (contrary to pigs)  Hypotension blocked by complement depletion with cobra venom factor (CVF)  Rats & pig mechanisms may differ in mediators or target cells involved

Page Hypotension in Rats only at High Dose Levels Systemic arterial blood pressure changes induced by a single administration of SonoVue in non-anesthetized rats (Dose levels correspond to 100 – 300 MHDbw or 17 – 50 MHDbsa)

Page Key Findings Mechanistic Studies: In Vitro Complement and Basophil Activation  SonoVue and another UCA tested at very high dose levels show similar findings after incubation in vitro  Dose-dependent increase in C3a/C5a in pig plasma  Dose-dependent increase in C3a/C5a/SC5b-9 in human serum  No marked differences between pigs & humans in vitro  No effects on human basophil activation (CD203c)

Page Summary of Mechanistic Studies: SonoVue  Symptoms observed in pigs are similar to human anaphylactoid reactions (cardiopulmonary changes)  Incidence of anaphylactoid reactions in pigs >>> humans  Sensitivity of pigs may be due to high density of PIMs relative to other species  Rats show hypotension at very high dose  Complement activation could be one of the mechanisms involved in the reactivity in rats, pigs and humans

Page SonoVue: Lessons Learned for Future  SonoVue was well tolerated in nonclinical studies and this was corroborated by the clinical trials  The lack of cardiovascular effects of SonoVue in safety pharmacology studies at doses relevant to humans correlates with the lack of anaphylactoid reactions in clinical trials  Anaphylactoid reactions similar to humans are seen in naive pigs with various classes of particulate agents including UCA  Reactions in pigs attributed to high density of PIMs  The relevance of the findings in pigs to humans is unknown

Page SonoVue: Lessons Learned for Future  In vitro complement activation may be an early triggering event in the reactions observed in humans and represents a potential screening tool  Bracco is incorporating in vitro and in vivo testing in selection of next generation products  Results in in vitro and in vivo models may be useful qualitatively but not quantitatively for risk assessment  The rare anaphylactoid reactions may be reduced through these screening efforts

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