1. Iron Chelator Basics: Emerging Data Beyond Transfusion-Dependent Iron Overload Antonello Pietrangelo, MD, PhD Professor of Internal Medicine Center for Hemochromatosis University Hospital of Modena Modena, Italy Alberto Piperno, MD Professor of Internal Medicine Department of Clinical Medicine and Prevention University of Milano-Bicocca Monza, Italy

2. Beyond Transfusion-Dependent Iron Overload Hereditary haemochromatosis Chronic liver diseases –Chronic hepatitis C –Chronic hepatitis B –Nonalcoholic fatty liver disease and metabolic syndrome –Other

3. Beyond Transfusion-Dependent Iron Overload Hereditary Haemochromatosis Antonello Pietrangelo, MD, PhD

4. Iron Overload in Hereditary Haemochromatosis 1,2 Autosomal recessive disorder characterized by excessive iron uptake through intestinal absorption If untreated, tissue iron overload may lead to toxicity and disease in an appreciable proportion of patients Liver cirrhosis, hepatocellular carcinoma, diabetes, cardiomyopathy, arthropathy, and hypogonadism are common clinical features of fully expressed disease The paradigmatic and most common form is associated with homozygosity for the C282Y polymorphism of HFE Phlebotomy is the standard of care in hereditary haemochromotosis –Reduces iron burden and prevents clinical complications 1. Pietrangelo A. N Engl J Med. 2004;350:2383-2397. 2. Beutler E, et al. Annu Rev Med. 2006.57:331-347.

5. Phlebotomy in HH—Limitations Some patients may: –Not be able to tolerate the induced mild anaemic state (eg, elderly patients) –Be poor candidates due to underlying medical conditions (eg, severe heart disease or anaemia) and/or poor venous access –Experience hypovolaemia –Experience discomfort of needle puncture Low acceptance of the procedure may lead to a decline in compliance over time and treatment failure

6. Iron Chelation in HH—An Option The use of iron chelators in HH has only been reported in case studies 1–4 Despite approval of iron chelators for posttransfusional iron overload, the extent and rate of iron accumulation in HH is substantially different, which may affect safety and efficacy Deferoxamine is approved in the European Union for use in HH but rarely used due to very low patient acceptance 1. Franchini M, et al. Blood. 2000;95:2776-2779. 2. Barton JC, et al. Am J Hematol. 2005;78:83. 3. Polo- Romero FJ. Am J Hematol. 2006;81:225-226. 4. Nielsen P, et al. Br J Haematol. 2003;123:952-953.

7. 1. Pietrangelo A, et al. Blood. 2007; 110:2680. 2 Exjade. Summary of Product Characteristics. West Sussex, UK: Novartis Europharm Ltd; 2006. 3. Galanello R, et al. Haematologica. 2006;91:1343-1351. 4. Piga A, et al. Haematologica. 2006;91:873-880. 5. Cappellini MD, et al. Blood. 2006;107:3455-3462. 6. Porter J, et al. Eur J Haematol. 2008;80:168-176. 7. Vichinsky E, et al. Br J Haematol. 2007;136:501-508. Iron Chelation Therapy with Deferasirox in HH A phase I/II dose-escalation trial with deferasirox is the first trial of iron chelation in a nontransfusional iron overloaded population 1 Deferasirox is a once-daily oral iron chelator under investigation as a possible additional treatment option Deferasirox is approved for the treatment of transfusional hemosiderosis in patients >2 years of age 2 –Daily therapy results in dose-dependent reduction in iron load as indicated by liver iron concentration and serum ferritin 2 –Efficacy and safety in a variety of transfusion-dependent anaemias 3–7

8. Deferasirox in HFE-Related HH A Phase I/II, Open-Label, Dose-Escalation Trial Primary objective: To explore the safety of deferasirox in iron-overloaded adult patients with HFE C282Y homozygosity Secondary objective: To explore the effect of deferasirox on serum ferritin 2-part study –Core study: 24-weeks duration –Extension study: patients completing the core study were eligible to continue receiving deferasirox for a further 24 weeks with appropriate dose adjustments Pietrangelo A, et al. Blood. 2007; 110:2680.

9. Key Inclusion/Exclusion Criteria Inclusion criteria –Male and female patients aged ≥18 years with HFE C282Y homozygous HH documented by molecular diagnostic testing –Serum ferritin levels of 300 ng/mL to 2000 ng/mL with transferrin saturation ≥45% Exclusion criteria –Haemoglobin <13 g/dL for males and <12 g/dL for females –History of blood transfusion during the 6 months prior to study entry –Serum creatinine > the upper limit of normal (ULN) –Alanine aminotransferase (ALT) levels of ≥2 x ULN Pietrangelo A, et al. Blood. 2007; 110:2680.

10. Study Design 1,2 49 patients enrolled Doses explored –5 mg/kg/day –10 mg/kg/day –15 mg/kg/day 1. Pietrangelo A, et al. Blood. 2007; 110:2680. 2. Pietrangelo A, et al. J Hepatol. 2009;50:S4.

11. Slide # 11 Most Common Adverse Events (AE) (>10%) AE, n (%)5 mg/kg/day (n = 11) 10 mg/kg/day (n = 15) 15 mg/kg/day (n = 23) All Patients (n = 49) Diarrhea2 (18.2)6 (40.0)10 (43.5)18 (36.7) Headache1 (9.1)2 (13.3)7 (30.4)10 (20.4) Nausea–2 (13.3)6 (26.1)8 (16.3) Abdominal pain–3 (20.0)4 (17.4)7 (14.3) Back pain1 (9.1)4 (26.7)2 (8.7)7 (14.3) Increased blood creatinine –3 (20.0)4 (17.4)7 (14.3) Increased ALT–3 (20.0)3 (13.0)6 (12.2) Rash1 (9.1)1 (6.7)4 (17.4)6 (12.2) Flatulence2 (18.2)1 (6.7)2 (8.7)5 (10.2) Nasopharyngitis1 (9.1)1 (6.7)3 (13.0)5 (10.2) No deaths or serious AEs occurred during the course of this study Pietrangelo A, et al. J Hepatol. 2009;50:S4. Graphic courtesy of Dr. Antonello Pietrangelo.

12. Serum Ferritin by Visit and Dose Cohort BL, baseline 859 322 634 205 512 356 Abbreviation: SF, seum ferritin. Pietrangelo A, et al. J Hepatol. 2009;50:S4. Graphic courtesy of Dr. Antonello Pietrangelo.

13. Conclusions of the Core Trial—Safety The results from the core trial suggest that deferasirox has an acceptable safety profile in patients with HH Adverse events are consistent with the known safety profile of deferasirox and previous data acquired in transfusion-dependent iron overload Pietrangelo A, et al. J Hepatol. 2009;50:S4.

14. Deferasirox doses of 5, 10, and 15 mg/kg/day effectively reduced serum ferritin in HH (nontransfusional iron overload population) 1 Efficacy was demonstrated at lower doses of deferasirox in this population compared with doses in the current label for the transfusional overload population 1 The 6-month extension phase to the trial is now complete and data will be presented at the 2009 ASH Annual Meeting 2 Conclusions of the Core Trial—Efficacy 1. Pietrangelo A, et al. J Hepatol. 2009;50:S4. 2. Phatak PD, et al. Presented at: 2009 ASH Annual Meeting. New Orleans, LA; December 5-8, 2009. Abstract 1514.

15. Beyond Transfusion-Dependent Iron Overload Iron Overload and Chronic Liver Disease Iron Overload and Chronic HCV Iron Overload and Insulin Resistance Alberto Piperno, MD

16. Local Iron Overload—The Liver Liver Iron Overload Viral hepatitis Alcoholic liver disease End-stage liver disease Porphyria cutanea tarda NAFLD DIOS The liver is the central regulator of iron homeostasis and the main site of iron storage –Probably more exposed to iron accumulation because of the first-pass effect of the portal circulation Beyond systemic forms of iron overload, there are many other disorders associated with mild-moderate hepatic iron overload Abbreviation: DIOS, dismetabolic iron overload syndrome; NAFLD, nonalcoholic fatty liver disease. Graphic courtesy of Dr. Alberto Piperno.

17. Iron Overload in Chronic Liver Diseases 1-4 Many patients with chronic liver diseases (CLD) have increased serum iron indices –Mild-moderate amount of haemosiderin iron is present in a significant proportion of liver biopsies of patients with CLD –The amount of iron associated with these conditions would be generally considered to be at nontoxic levels, certainly much lower than in hereditary haemochromatosis or in thalassaemia What are the mechanisms of iron accumulation in CLD? May the combination of slightly-moderately increased iron with an added insult act synergistically to increase the risk of progression of liver damage? Consequently, may iron removal be useful in the management of CLD and in improving the natural history of these disorders? 1. Chapman RW, et al. Dig Dis Sci. 1982;27:909-916. 2. Martinelli AL, et al. J Gastroenterol Hepatol. 2004;19:1036-1041. 3. Bugianesi E, et al. Hepatology. 2004;39:179-187. 4 Piperno A, et al. Hepatology. 1998;28:1105-1109

18. Genetic background Mutations of HH genes Increased iron absorptionHepatocyte damage and inflammation Phagocytosis of damaged hepatocytes Cytokine-mediated iron retention in macrophages Increased iron and ferritin release to extracellular fluid and plasma Increased NTBI Fe Macrophage Fe Hepatocyte Chronic Liver DiseaseHepatic Iron Overload Mechanisms of Iron Accumulation in CLD Abbreviations: HH; hereditary haemochromatosis; NTBI, nontransferrin-bound iron. Graphics courtesy of Dr. Alberto Piperno.

19. Oxidative Stress at the Interface of Iron- and Other Hepatoxin-Induced Liver Damage Oxidative stress in the liver can be induced by alcohol, hepatitis C virus (HCV) or insulin resistance associated with obesity 1-3 –Excess iron in the liver may exacerbate this oxidative stress and promote liver injury and fibrosis and, in some cases, carcinogenesis Abbreviation: C/EBPa, CCAAT/enhancer binding protein-alpha; PCT, porphyria cutanea tarda; UROD, uroporphyrinogen decarboxylase. 1. Wallace DF, et al. Biochim Biophys Acta. 2009;1790:663-670. 2. Harrison-Findik DD. World J Gastroenterol. 2007;13:4925-4930. 3. Miura K, et al. Hepatology. 2008;48:1420-1429. 4. Girelli D, et al. J Hepatol. 2009;51:845-852. 5. Nishina S, et al. Gastroenterology. 2008;134:226-238. Graphic courtesy of Dr. Alberto Piperno. Oxidative stress can depress hepcidin expression in hepatocytes via reduced activity of C/EBP- , as shown in animal models of HCV infection and alcoholic liver disease 1,4,5 –This may increase iron absorption and accumulation in the liver, contributing further to oxidative stress. AlcoholHCV Insulin resistance Obesity Oxidative stress Cell death Liver fibrosis Carcinogenesis ‘Labile’ or ‘free’ iron

20. Chronic Liver Diseases, Iron, and Progression to Liver Damage + Chronic Liver Disease Liver Damage (fibrosis, cirrhosis, HCC) Marked Iron Overload 1. Adams PC, et al. Hepatology. 1996;23:724-727. 2. Powell EE, et al. Gastroenterology. 2005;129:1937-1943. 3. Fletcher LM, et al. Gastroenterology. 2002;122:281-289. 4. Piperno A, et al. J Hepatol. 1992;16:364-368. 5. Diwakaran HH, et al. J Hepatol. 2002;36:687-691. 6. Angelucci E, et al. Blood. 2002;100:17-21. 7. Fargion S, et al. Hepatology. 1992;15:655-659. Graphic courtesy of Dr. Alberto Piperno. Synergistic Effect Alcohol intake promotes liver damage in patients with haemochromatosis 1-3 Chronic HCV and HBV infection promotes liver damage in patients with haemochromatosis and thalassaemia 4-6 NAFLD promotes liver damage in patients with haemochromatosis 7

21. 1. Theurl I, et al. J Infect Dis. 2004;190:819-825. 2. Fillebeen C, et al. J Biol Chem. 2005;280:9049-9057. 3. Mueller S, et al. Gastroenterology. 2006;130:2229-2234. 4. Alexander J, et al. Liver Int. 2007;27:268-273. 5. Kato J, et al. Cancer Res. 2001;61:8697-8702. 6. Rulyak SJ, et al. Am J Gastroenterol. 2005;100:332- 337. 7. Sievert W, et al. Am J Gastroenterol. 2002;97:982-987. Iron, Chronic HCV Infection, and Disease Progression—A Controversial Issue The effect of iron on HCV replication Iron has been shown to either increase expression of HCV genes, suppress HCV replication, or to have no effect on HCV transgene expression in vitro 1-3 Clinical data indicate that iron status does not significantly influence HCV replication in vivo 3 The effect of iron on disease progression Phlebotomies significantly decrease serum ALT 4,5 –In small pilot studies, phlebotomies improve markers of hepatic fibrosis and liver histology in untreated patients with chronic HCV 5 Phlebotomies may improve the response rate to interferon alone 4 –Uncertain whether it may improve response rate to interferon-ribavirin combination therapy 6,7

22. Chronic Hepatitis B Virus (HBV) Infection and Hepatic Iron Overload There are very few data available on iron metabolism in patients with chronic HBV infection 1-5 A relationship between serum iron indices and outcome of HBV infection and clearance of virus was reported in the past but has not been confirmed 1,2 Mild abnormalities in serum iron indices and increased liver iron deposits have been described in about 30% of HBV-infected patients 3 In contrast with chronic HCV, hepatic oxidative damage in chronic HBV is not related to body iron 4 Reduction of ferritin by iron depletion therapy improved the likelihood of response to interferon therapy in a single small study 5 Iron depletion inhibits HBV secretion in HBV stably-transfected cells 6 –This may not reflect a decrease of viral replicative form, but could be associated with active viral DNA synthesis 1. Felton C, et al. Proc Natl Acad Sci U S A. 1979;76:2438-2441. 2. Blumberg BS, et al. Proc Natl Acad Sci U S A. 1981;78:3222-3224. 3. Martinelli AL, et al. J Gastroenterol Hepatol. 2004;19:1036-1041. 4. Fujita N, et al. J Viral Hepat. 2008;15:498-507. 5. Bayraktar Y, et al. Hepatogastroenterology. 1998;45:2322-2327. 6. Chouteau P, et al. J Hepatol. 2001;34:108-113.

23. Severe Fibrosis/Cirrhosis Is More Frequent in Chronic HCV with Hepatic Iron Overload 1-5 Some studies report an association between HFE mutations and iron overload, and between HFE mutations and fibrosis –Others have found no such association Some studies however, found an association between hepatic iron or increased serum iron indices and more severe disease independent of HFE mutations 1. Hézode C, et al. J Hepatol. 1999;31:979-984. 2. Corengia C, et al. Am J Clin Pathol. 2005;124:846-853. 3. Guyader D, et al. J Hepatol. 2007;46:587-95. 4. Tung BY, et al. Gastroenterology. 2003;124:318-326. 5. Negro F, et al. J Med Virol. 2000;60:21-27. Graphic courtesy of Dr. Alberto Piperno; data from unpublished meta-analysis of references 1, 2, and 3. 10 20 30 40 50 60 No Iron DepositsWith Iron Deposits 23.1 2 17 1 16 3 9.6 2 3.3 1 29 3 85/659 (12.9%)68/287 (23.7%) P <.001 Patients with Severe Fibrosis/Cirrhosis (%)

24. Interactions Between Hepatic Iron Overload and Cirrhosis 1-4 1. Deugnier Y, et al. Am J Surg Pathol. 1997;21:669-675. 2. Ludwig J, et al. Gastroenterology. 1997;112:882-888. 3. Guyader D, et al. J Hepatol. 2007;46:587-595. 4. Tsukamoto H, et al. Am J Physiol. 1999;277(6 Pt 1):G1240-G1250. Graphic courtesy of Dr. Alberto Piperno. Hepatic CirrhosisIron Overload Mechanisms of Iron Load Decreased transferrin synthesis Spontaneous porto-systemic shunts Ineffective erythropoiesis Chronic hemolysis Reduced hepcidin synthesis Hepatocellular necrosis (sideronecrosis) Amplification and propagation of fibrogenesis in concert with other hepatotoxins Induction of fibrogenesis through activation of hepatic stellate cells Mechanisms of Fibrogenesis HSC Activated HSC Injured Hepatocyte Activated Kupffer cell IRON Hepatotoxins Liver Fibrosis

25. NASH—An Emerging Cause of Cirrhosis O'Leary JG, et al. Gastroenterology. 2008;134:1764-1776. Nonalcoholic steato-hepatitis (NASH) is the predominant cause of cryptogenic cirrosis –Represents the severe end of the NAFLD spectrum –Associated with metabolic syndrome and insulin resistance Proportion of Liver Transplants for Specific Etiologies; US, 1992 to 2007

26. NAFLD/NASH and Iron Overload—An Intriguing and Controversial Association 1-6 Increased serum ferritin is frequent in patients with NAFLD, not always associated with hepatic iron overload In fact, hepatic iron is generally present in not more than half of patients with hyperferritinaemia Some studies have shown that increased ferritin levels, but not iron overload, are markers of severe histologic damage and that iron burden does not contribute to hepatic fibrosis in patients with NAFLD Others studies have shown that hyperferritinemia or hepatic iron concentration are associated with increased risk of fibrosis in NASH, or increased risk of NASH Some studies demonstrated an association between HFE mutations, fibrosis and iron overload in NASH, but others failed to find this association 1. Trombini P, et al. J Hepatol. 2007;46:549-552. 2. Bugianesi E, et al. Hepatology. 2004;39:179-187. 3. Fargion S, et al. Am J Gastroenterol. 2001;96:2448-2455. 4. George DK, et al. Gastroenterology. 1998;114:311-318. 5. Nelson JE, et al. Hepatology. 2007;46:723-729. 6. Chitturi S, et al. Hepatology. 2002;36:142-149.

27. NAFLD/NASH and Iron Overload—The Dismetabolic Iron Overload Syndrome A new syndrome termed insulin resistance hepatic iron overload (IR-HIO) or dismetabolic iron overload (DIOS) 1-4 –Mild to moderate hepatic iron overload is associated with features of insulin resistance or metabolic syndrome –Most common form of iron overload in Western countries (at least 10 times more common than haemochromatosis) Increased prevalence of HFE mutations and of NAFLD or NASH have been observed in DIOS 5 Physiopathologic mechanism of iron overload in DIOS is unclear 6 A link between iron and the progression of NAFLD have been supported by the observation that iron removal by phlebotomy can improve insulin resistance, metabolic indices, and liver function in patients with NAFLD or DIOS 7,8 1. Mendler MH, et al. Gastroenterology. 1999;117:1155-1163. 2. Riva A, et al. World J Gastroenterol. 2008;14:4745-4752. 3. Facchini FS, et al. Gastroenterology. 2002;122:931-939. 4. Le Guenno G, et al. Diabetes Res Clin Pract. 2007;77:363-370. 5. Barisani D, et al. J Hepatol. 2008;49:123-133. 6. Ruivard M, et al. J Hepatol. 2009;50:1219-1225. 7. Valenti L, et al. Am J Gastroenterol. 2007;102:1251-1258. 8. Piperno A, et al. Liver Int. 2004;24:471-476.

28. Iron and Hepatocellular Carcinoma Patients with haemochromatosis have significantly increased risk for Hepatocellular Carcinima (HCC) 1-3 HCC has been reported in the absence of cirrhosis and after reversal of cirrhosis, suggesting iron might be direct carcinogen 4,5 In transgenic mice expressing HCV poliprotein, iron overload increases risk of HCC by potentiating mitochrondrial injury via oxidative stress 6 Recent data suggest that, in patients with alcoholic cirrhosis, but not HCV-related cirrhosis, liver iron overload (whatever its cause) is major risk factor for HCC 7 1. Niederau C, et al. N Engl J Med. 1985;313:1256-1262. 2. Bradbear RA, et al. J Natl Cancer Inst. 1985;75:81-84. 3. Hsing AW, et al. Int J Cancer. 1995;60:160-162. 4. Kew MD. Hepatology. 1990;11:1086- 1087. 5. Blumberg RS, et al. Gastroenterology. 1988;95:1399-1402. 6. Nishina S, et al. Gastroenterology. 2008;134:226-238. 7. Nahon P, et al. Gastroenterology. 2008;134:102-110.

29. Iron Removal Therapy in CLD—Reality, Myth, and Perspectives In patients with CLD for whom there is a suspicion of hepatic iron overload, the decision to remove iron should rely firstly on demonstration of hepatic iron overload –Noninvasive (quantitative MR, SQUID) or invasive (liver biopsy) methods There is some evidence that the association of CLD and high levels of hepatic iron contribute to more rapid progression to hepatic fibrosis and cirrhosis –iron removal is recommended as part of the therapeutic options If hepatic iron is mildly/moderately increased, treatment to reduce excessive iron burden can be considered before, together, or after specific therapies (antiviral, alcohol abstinence, lifestyle modification) However, at this time there is insufficient evidence to elevate iron removal therapies to the status of a proven therapy in these scenarios The availability of new iron chelators (ie, able to remove iron from specific sites) may open new perspectives in this exiting area of research This issue needs to be addressed in large cohorts of patients in carefully designed studies