USE OF EPOETIN BETA DURING COMBINATION THERAPY OF INFECTION WITH HEPATITIS C VIRUS WITH RIBAVIRIN IMPROVES A SUSTAINED VIRAL RESPONSE Katia Falasca, Claudio Ucciferri, Paola Mancino, Valeria Gorgoretti, Eligio Pizzigallo, Jacopo Vecchiet

To cite this version:

Katia Falasca, Claudio Ucciferri, Paola Mancino, Valeria Gorgoretti, Eligio Pizzigallo, et al.. USE OF EPOETIN BETA DURING COMBINATION THERAPY OF INFECTION WITH HEPATITIS C VIRUS WITH RIBAVIRIN IMPROVES A SUSTAINED VIRAL RESPONSE. Journal of Medical Virology, Wiley-Blackwell, 2009, 82 (1), pp.49. ￿10.1002/jmv.21657￿. ￿hal-00541149￿

HAL Id: hal-00541149 https://hal.archives-ouvertes.fr/hal-00541149 Submitted on 30 Nov 2010

HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. Journal of Medical Virology

USE OF EPOETIN BETA DURING COMBINATION THERAPY OF INFECTION WITH HEPATITIS C VIRUS WITH RIBAVIRIN IMPROVES A SUSTAINED VIRAL RESPONSE For Peer Review

Journal: Journal of Medical Virology

Manuscript ID: JMV-09-1316.R2

Wiley - Manuscript type: Research Article

Date Submitted by the 05-Aug-2009 Author:

Complete List of Authors: Falasca, Katia; Clinic of Infectious Diseases, Department of Medicine and Science of Ageing. “G. d’Annunzio” University Ucciferri, Claudio; Clinic of Infectious Diseases, Department of Medicine and Science of Ageing. “G. d’Annunzio” University Mancino, Paola; Clinic of Infectious Diseases, Department of Medicine and Science of Ageing. “G. d’Annunzio” University Gorgoretti, Valeria; Clinic of Infectious Diseases, Department of Medicine and Science of Ageing. “G. d’Annunzio” University Pizzigallo, Eligio; Clinic of Infectious Diseases, Department of Medicine and Science of Ageing. “G. d’Annunzio” University Vecchiet, Jacopo; Clinic of Infectious Disease, Department of Medicine and Science of Ageing. "G.d'Annunzio" University

Keywords: epoetin, HCV, sustained viral response, /ribavirin ,

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1 2 3 Figure 1: Comparison of end of treatment response (ETR) and sustained 4 viral response (SVR) between the two groups 5 6 ETR SVR 7 25 8 18 16 P=0.03 9 20 10 14 11 15 12 12 Negative 10 viral load 13 10 8 Positive 14 6 viral load 15 5 4 16 2 17 0 ß ß 0 18 Epoietin beta SOC group Epoietin beta group SOC group 19 group 20 For Peer Review 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 Figure 2: Spearman’s correlation between serum levels of ferritin at 4 5 T0 and MCV value at T-pre-change, T-1-month and SVR 6 7

8 300 300

9 r= -0,45 r= -0,43 10 p=0,03 p=0,04 11 12 200 200 13 14 15 16 100 100 17 18

19 0 FerritinT0 0 FerritinT0 20 70 80 90 100 110 80 90 100 110 120 ß For Peer Reviewß 21 MCV T pre change MCV T 1 month 22 23 300 24 25

26 r= 0,58

27 200 p=0,005 28 29 30

31 100 32 33 34 0 35 FerritinT0 86 88 90 92 94 96 98 100 36 37 MCV SVR 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 USE OF EPOETIN BETA DURING COMBINATION THERAPY OF 4 5 INFECTION WITH HEPATITIS C VIRUS WITH RIBAVIRIN IMPROVES A 6 SUSTAINED VIRAL RESPONSE 7 8 9 Katia Falasca, Claudio Ucciferri, Paola Mancino, Valeria Gorgoretti, Eligio Pizzigallo, 10 Jacopo Vecchiet. 11 12 13 14 Infectious Diseases Clinic – Department of Medicine and Science of Ageing. “G. 15 d’Annunzio” University Chieti-Pescara– Italy 16 17 18 19 20 For Peer Review 21 22 Running title: Benefits of epoetin beta in anti-HCV therapy 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 Correspondence to: 44 Prof. Jacopo Vecchiet 45 46 Infectious Diseases Clinic 47 Dept. of Medicine and Science of Ageing 48 “G. d’Annunzio” University, School of Medicine 49 Via dei Vestini, 66100 Chieti, Italy 50 E-mail: [email protected] 51 52 53 54 55 56 57 58 59 60

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1 2 3 ABSTRACT 4 5 6 The aim of the study was to evaluate the effects of epoetin-beta on anaemia and 7 8 sustained viral response in patients with chronic hepatitis C receiving treatment with 9 10 pegylated interferon and ribavirin. 11 12 13 Forty-two Caucasian patients with chronic hepatitis C infection, treated with pegylated 14 15 interferon α-2a or α-2b plus ribavirin, who experienced at least a 2 log decline in HCV- 16 17 18 RNA in the first month of therapy and a ≥ 2.5 g/dl haemoglobin drop from baseline, 19 20 were recruited. TheyFor were divided Peer into two Review groups: 22 patients received epoetin-beta 21 22 30.000U administered s.c. q.w. (group A) and 20 patients received a reduced ribavirin 23 24 25 dose of 600 mg daily (group B). 26 27 The end-of-treatment response was 95.4%(21/22) in group A and 80% (16/20) (p=0.2) 28 29 in group B. Sustained viral response in group A was 81.8% (18/22), statistically higher 30 31 32 than in group B (45%, 9/20) (p=0.03). Mean corpuscular volume of erythrocytes was 33 34 statistically lower in group A than in group B 4 weeks after starting epoetin-beta or 35 36 reduced ribavirin dose (p<0.001), end-of-treatment (p<0.001) and after 6 months 37 38 39 follow-up (p<0.001). A negative correlation between the levels of ferritin serum was 40 41 found in group A at the baseline and mean corpuscular volume value after 1 month of 42 43 44 combination antiviral therapy (r= –0.45; p=0.35), 4 weeks after starting epoetin-beta (r= 45 46 –0.43; p=0.04) and after 6 months follow-up (r= –0.45; p=0.03). 47 48 Administration of epoetin-beta increases sustained viral response rates among patients 49 50 51 developing anaemia, because the standard dose of ribavirin is maintained, thereby 52 53 reducing the side-effects of antiviral treatment. 54 55 56 57 58 Key-words: epoetin, HCV, anaemia, interferon/ribavirin, sustained viral response, 59 60 growth factor

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1 2 3 INTRODUCTION 4 5 6 7 8 Infection with hepatitis C virus (HCV) is an increasing epidemic with over 180 million 9 10 people infected worldwide [WHO, 2007]. HCV is the most important cause of chronic 11 12 13 liver disease in Italy and is found in nearly 70% of cases [Sagnelli et al, 2005]. Infection 14 15 with HCV persists in about 75% of cases and causes various degrees of liver 16 17 18 inflammation and fibrosis; in time it may lead to cirrhosis and hepatocellular carcinoma 19 20 [Poynard et al, 2003;For Kumada Peer et al, 2009]. Review The primary aim of anti-HCV therapy is 21 22 permanent eradication of the virus or a sustained viral response. A sustained viral 23 24 25 response is defined as undetectable plasma HCV-RNA 6 months after the end of 26 27 treatment, which lasts typically 6-12 months. This leads to a long-term clearance of the 28 29 virus in 98.3% of patients [McHutchison et al, 2001]. The most effective treatment for 30 31 32 chronic HCV infection at present is the combination of pegylated interferon (PEG-IFN) 33 34 α-2a or α-2b and ribavirin (RBV). Both drugs have a significant effect on virological 35 36 and histological responses and this combined therapy provides a sustained viral 37 38 39 response rate of 40% to 50% in patients with HCV genotype 1 and of 80% in patients 40 41 with HCV genotypes 2 or 3 in randomized controlled trials [Manns et al, 2001; Fried et 42 43 44 al, 2002 ]. However, the sustained viral response rate decreases dramatically when 45 46 adherence to treatment is not optimal . Data derived from clinical trials indicate that 47 48 compliance during treatment and the dose of RBV are important factors for achieving a 49 50 51 sustained viral response, particularly among “difficult-to-treat” patients, such as those 52 53 infected with HCV genotype 1 and those with high baseline levels of HCV-RNA 54 55 [McHutchinson et al, 2002; Hadziyannis et al, 2004; Akuta et al, 2006]. The challenging 56 57 58 side-effects of both PEG-IFN and RBV reduce adherence to treatment and also result in 59 60 high rates of dose reduction and discontinuance of treatment in clinical trials and in

practice.

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1 2 3 The single most common adverse event of antiviral treatment is anaemia, defined as 4 5 6 haemoglobin < 12 g/dL [Shiffman, 2004]. During the first 4 weeks of combination 7 8 treatment, mean haemoglobin levels decrease by an average of 2 to 3 g/dl, with an 9 10 impaired compensatory reticulocytosis [De Franceschi et al, 2000; Manns et al, 2001; 11 12 13 Fried et al, 2002]. Haemoglobin concentrations have been reported to decrease to below 14 15 12 g/dl (mean decrease 3.7 g/dl) in 52% of patients receiving combination treatment 16 17 18 with PEG-IFN α-2a plus RBV [Pegasys -package insert- 2004]. Significant anaemia (ie, 19 20 haemoglobin < 10For g/dL) has Peer been observed Review in up to 9-13% of patients receiving 21 22 combination therapy with IFN and RBV [Manns et al, 2001]. Moderate anaemia 23 24 25 (haemoglobin < 11 g/dL) may be seen in 30% [PEG-Intron -package insert- 2003]. 26 27 The etiology of the anaemia induced by HCV therapy is multifactorial. It is a “mixed 28 29 anaemia”, in which both haemolysis and bone marrow suppression occur 30 31 32 simultaneously [Kowdley, 2005]. Clinically, however, the contribution of RBV to 33 34 anaemia during combination treatment overshadows the effects of IFN on the bone 35 36 marrow. RBV induces a dose-dependent haemolytic anaemia which is reversible within 37 38 39 4-8 weeks of drug discontinuation. RBV-induced anaemia is the main reason for dose 40 41 reduction and discontinuance [Sulkowski et al, 2004]. 42 43 44 The standard-of-care management for patients who develop anaemia during HCV 45 46 therapy with IFN/RBV is RBV dose reduction to 600 mg/d for haemoglobin levels < 10 47 48 g/dl and drug discontinuation when haemoglobin levels drop to < 8,5 g/dl [Pegasys - 49 50 51 package insert- 2004]. However, maintaining the RBV dose is crucial to the outcome in 52 53 patients with chronic hepatitis C who are treatment naive or who did not respond to a 54 55 previous course of therapy [Shiffman, 2004]. Anaemia-related dose reduction or 56 57 58 discontinuance may adversely affect outcome since it reduces the probability of 59 60 achieving long-term sustained viral response [Dieterich and Spivak, 2003].

Increasing evidence now supports the use of recombinant human

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1 2 3 (rHuEPO), such as (EPO-α), epoetin beta (EPO-β) and , to 4 5 6 manage anaemia in these patients, with the objective of maintaining the RBV dose 7 8 [Sherman et al, 2006]. Unfortunately, no official guidelines currently exist for treating 9 10 antiviral therapy-associated anaemia, and many issues remain unresolved. 11 12 13 These agents appear to be effective in increasing haemoglobin level, allowing 14 15 maintenance of the RBV dose and improving the quality of life for the period of time 16 17 18 they are used. However, their effects on early and sustained viral response remain 19 20 uncertain, as do theFor duration Peer of treatment, Review optimal dose, and reference level of 21 22 haemoglobin. 23 24 25 It is widely assumed that the positive effects of utilizing a haematologic growth factor 26 27 will result in a sustained viral response advantage, although no relevant data are 28 29 available to date. Only one prospective, randomized, controlled trial has evaluated the 30 31 32 effect on virological response of EPO-α administered at the onset of therapy. It 33 34 concluded that the use of EPO-α in all subjects at the beginning of PEG-IFN/RBV 35 36 treatment will not enhance sustained viral response [Shiffman et al, 2007]. However, in 37 38 39 this study the mean RBV dose was not statistically different among the treated groups. 40 41 The pharmacokinetic and pharmacodynamic properties and efficacy of EPO-β are 42 43 44 generally better than those of EPO-α [Halstenson et al, 1991]. Data about the use of 45 46 EPO-β in this setting and its effects on sustained viral response are very limited. 47 48 The aim of this study was to evaluate the use of EPO-β to manage anaemia induced by 49 50 51 PEG-IFN/RBV therapy in patients with chronic hepatitis C and the effects on sustained 52 53 viral response. 54 55 56 57 58 59 60

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1 2 3 MATERIALS AND METHODS 4 5 6 7 8 Study Design 9 10 This was an observational study regarding outpatients attending the Clinic of Infectious 11 12 13 Diseases at the SS. Annunziata Hospital of the “G. d’Annunzio” University of Chieti- 14 15 Pescara, Italy from January 2006 to February 2007. 16 17 18 19 20 Patients For Peer Review 21 22 The subjects recruited were 42 Caucasian patients with chronic hepatitis C treated with 23 24 25 PEG-IFN α-2a or α-2b plus RBV who experienced at least a 2 log decline in HCV-RNA 26 27 during the first month of therapy and a ≥ 2.5 g/dl haemoglobin drop from baseline and a 28 29 haemoglobin level lower than 11 g/dl. The patients were divided into an EPO-β 30 31 32 treatment group (A) (n=22, 17 females and 5 males, mean age 47.5±10.6 years) and a 33 34 standard care (RBV dose reduction) treatment group (B) (n=20, 13 females and 7 males, 35 36 mean age 48.6±15.0 years). All patients were naïve to antiviral treatment. 37 38 39 PEG-IFN α-2a was administered in a once-weekly subcutaneous injection of 180 g and 40 41 PEG-IFN α-2b in a once-weekly subcutaneous injection of 1.5 g/Kg; RBV was given 42 43 44 orally in a dose of 1000-1200 mg for genotype 1 and of 800 mg for genotype 2-3 per 45 46 day administered in two split doses. There was no genotype 4. 47 48 49 Patients randomized to the EPO-β group (A) received EPO-β 30.000 U administered s.c. 50 51 q.w. EPO-β treatment was started at haemoglobin levels of 11 g/dl or less, to prevent a 52 53 54 worsening of anaemia (haemoglobin ≤ 10 g/dl), and the EPO-β dosage was adjusted 55 56 according to the haemoglobin response. Patients randomized to the standard care group 57 58 (B) received a reduced RBV dose of 600 mg daily. 59 60

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1 2 3 Four weeks after therapy changes, eight patients in group A who did not show an 4 5 6 improvement of haemoglobin levels were administered EPO-β increased to 30.000 U 7 8 s.c. twice a week. 9 10 The diagnosis of HCV infection was based on the usual biochemical data and detection 11 12 13 of anti-HCV antibodies (Abbott Axsym HCV-3 and Ortho HCV 3.0 Elisa) plus HCV 14 15 RNA in the serum by the polymerase chain reaction (PCR; Amplicor method - Roche 16 17 18 Mol. Diagn. Milan, Italy) with detection limit ≥ 600 HCV RNA copies/mL plasma. 19 20 The total study For population Peer was negative Review for other viral hepatitis and human 21 22 23 immunodeficiency virus infection. None of the patients showed clinical signs of 24 25 advanced liver disease (prothrombin time and albumin and total bilirubin levels were 26 27 28 within normal ranges). Additional exclusion criteria included: contraindication to EPO- 29 30 β therapy (e.g. known hypersensitivity to human albumin or mammalian cell-derived 31 32 products); serum ferritin levels less than 50 g/ml; uncontrolled hypertension (diastolic 33 34 35 pressure > 100 mm Hg); significant atherosclerotic heart disease (a 36 37 contraindication to RBV therapy); primary haematological disease; uncontrolled seizure 38 39 disorder; life expectancy less than 12 months. Current active substance abusers, 40 41 42 pregnant women, women who were breastfeeding and women of childbearing potential 43 44 who were not using adequate birth control measures were also excluded. 45 46 47 Biochemical and virological parameters were evaluated at baseline (T0), after 1 month 48 49 of combination antiviral therapy (T-pre-change), 4 weeks after therapy changes 50 51 52 (introduction of EPO-β or standard care) (T-1-month), at the end of treatment (end-of- 53 54 treatment response), and after 6 months follow-up (sustained viral response). Adverse 55 56 event reports were collected at each study visit. 57 58 59 Blood samples were taken to measure serum levels of aspartate aminotransferases 60 (AST), alanine aminotransferases (ALT), gamma-glutamyltranspeptidase ( γ-GT),

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1 2 3 fasting glucose, total cholesterol and triglycerides (automated enzymatic method - 4 5 6 Ortho-Clinical Diagnostics, Rochester, NY, USA), serum iron and ferritin (LIAISON 7 8 Ferritin, DiaSorin, Vercelli, Italy), haemoglobin, haematocrit (Hct), mean corpuscular 9 10 volume of erythrocytes (MCV) and platelet count (PLT). 11 12 13 All subjects gave written informed consent and the study was approved by the Medical 14 15 16 Ethics Committee of the “G. d’Annunzio” University Medical School. 17 18 19 20 Statistical AnalysisFor Peer Review 21 22 23 Data are presented as mean ± standard deviation (SD). Statistical significance was 24 25 assessed by Student’s t test for paired data and χ2 test for frequence variables. 26 27 28 Spearman’s correlation coefficient between serum levels of ferritin at baseline and 29 30 MCV value at baseline, T-pre-change, T-1-month and after 6 months follow-up was 31 32 computed. A p value < 0.05 was required. 33 34  TM 35 All the statistical analysis was performed with the SPSS Advanced Statistical 7.5 36 37 Software (1997, Chicago, IL, USA). 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 RESULTS 4 5 6 7 8 Forty-two patients with chronic hepatitis C (22 patients treated with EPO-β and 20 9 10 patients with standard care) were enrolled in the study. There were no drop outs during 11 12 13 the treatment and study compliance was complete. 14 15 No statistically significant differences were found at T0 between the EPO-β group (A) 16 17 18 and standard care group (B) for age, gender, weight, antiviral treatment, genotype, 19 20 HCV-RNA, serumFor iron, serum Peer ferritin, serumReview cholesterol, serum triglycerides and 21 22 fasting glucose (Table I), as well as for AST, ALT, γGT, haemoglobin, Hct, MCV and 23 24 25 PLT. 26 27 Group A achieved an end-of-treatment response of 95.4% (21/22), whereas that of 28 29 group B was 80% (16/20) (p=0.2). Evaluation after 6 months follow-up showed that 30 31 32 group A had a sustained viral response of 81.8% (18/22), statistically higher than group 33 34 B (45%, 9/20; p=0.03) (Figure 1). 35 36 MCV was statistically lower in group A than in group B at T-1-month (91.5±4.2 vs 37 38 39 97.9±6.4; p<0.001), at the end of treatment (90.0±6.8 vs 97.7±9.1; p<0.001) and after 6 40 41 months follow-up (83.6±32.9 vs 91.1±4.1; p<0.001). No significant differences were 42 43 44 found between the two groups regarding the platelet count. 45 46 In group A, ALT, AST, haemoglobin, Hct and PLT were statistically lower, while MCV 47 48 was statistically higher at T-pre-change in comparison with T0. In this group 49 50 51 haemoglobin, Hct and MCV proved to be increased with statistical significance at T-1- 52 53 month versus T-pre-change. But no significant differences were observed in group A for 54 55 all biochemical parameters between T-1-month and the end of treatment. Finally, in this 56 57 58 group after 6 months follow-up, haemoglobin, Hct and MCV were statistically different 59 60 from T-pre-change, T-1-month and the end of treatment. AST and ALT were higher

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1 2 3 after 6 months follow-up versus the end of treatment but only the ALT increase was 4 5 6 statistically significant (Table II). 7 8 The patients in this group did not show adverse effects of WHO 3-4 degrees, even those 9 10 patients treated with EPO-β doses of 30.000 IU twice weekly. Furthermore, no patient 11 12 13 in group A showed adverse events other than those due to antiviral therapy. 14 15 Group B showed a statistically significant decrease in all biochemical parameters at T- 16 17 18 pre-change versus T0. At T-1-month in these patients haemoglobin and Hct had 19 20 decreased with littleFor statistical Peer significance Review (p<0.05). There were no significant 21 22 differences for all biochemical parameters between T-1-month and the end of treatment. 23 24 25 After 6 months follow-up with respect to the end of treatment, group B showed a 26 27 significant increase in haemoglobin, Hct and PLT, whereas MCV decreased (Table III). 28 29 Finally, in group A a negative correlation was shown between ferritin serum levels at 30 31 32 T0 and MCV value at T-pre-change (r= –0.45 and p=0.35), at T-1-month (r= –0.43 and 33 34 p=0.04) and after 6 months follow-up (r= –0.45 and p=0.03) (Figure 2). 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 DISCUSSION 4 5 6 7 8 The use of EPO-β allowed an optimal PEG-IFN and standard RBV dose to be 9 10 maintained, thus reducing the side-effects of antiviral treatment, and permitted a higher 11 12 13 sustained viral response rate to be obtained. As was to be expected, the sustained viral 14 15 response in the standard care group was statistically lower. 16 17 18 EPO-β treatment was well tolerated in this patient population. The occurrence of 19 20 adverse events wasFor identical Peer between the Review two study groups and similar to those 21 22 expected with antiviral therapy. Adverse events related to rHuEPO are rare and consist 23 24 25 of hypertension, headache, reaction at injection site, increased numbers of platelets in 26 27 the blood and an increased risk of thrombosis [Rizzo et al, 2008]. The occurrence of 28 29 pure red cell aplasia, an uncommon disorder associated with the presence of anti-EPO 30 31 32 antibodies, was reported recently in a small number of patients with HCV infection and 33 34 subjects with chronic renal failure treated with EPO-α [Stravitz et al, 2005; McKoy et 35 36 al, 2008]. This phenomenon was not observed in the present study. Patients treated with 37 38 39 EPO-β did not show thrombotic complications. Generally, after a doubling of the initial 40 41 EPO-β dose, a further increment of the dose is not advisable because it increases the 42 43 44 risk of thrombosis. However, these data originate mainly from rHuEPO employment in 45 46 cancer patients, who show different characteristics of response and have a greater risk of 47 48 thrombosis than PEG-IFN/RBV treated patients [Bennett et al, 2008]. Patients with 49 50 51 chronic HCV infection do not have a pro-coagulative profile and need a different 52 53 rHuEPO treatment duration. In this study the haemoglobin value during EPO-β 54 55 administration was never above 12 g/dl, determining a lower risk of adverse events. 56 57 58 Finally, the presence of good iron tissue deposits seems to be significantly correlated to 59 60 a smaller increase in MCV during EPO-β treatment, which is likely to be due to a lower

immission of immature red blood cells in the circulation.

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1 2 3 RBV achieves an extensive accumulation in erythrocytes subsequent to active 4 5 6 unidirectional transmembraneous transport. Its activation to RBV triphosphate leads to a 7 8 marked depletion of adenosine triphosphate (ATP). Because erythrocytes lack enzymes 9 10 to hydrolyze RBV triphosphate, it accumulates in these cells. ATP deficiency impairs 11 12 13 the antioxidant defence, allowing oxidative membrane damage to occur. This leads to an 14 15 accelerated extravascular haemolysis by the reticulo-endothelial system [De Franceschi 16 17 18 et al, 2000; McHutchinson et al, 2006]. RBV-independent haematologic effects may 19 20 also occur in HCVFor patients receivingPeer combination Review treatment. To a lesser extent, IFN 21 22 contributes to anaemia by suppressing bone marrow function [Peck-Radosavljevic et al, 23 24 25 2002], limiting erythroid-progenitor-cell proliferation, increasing apoptosis of erythroid 26 27 cells, promoting autoimmune haemolytic reactions, reducing renal function and 28 29 impairing compensatory reticulocytosis to RBV-related haemolytic anaemia [Kowdley, 30 31 32 2005; McHutchinson et al, 2007]. Hence, the combined actions of these drugs result in a 33 34 “mixed” anaemia. 35 36 The anaemia of chronic diseases can be observed in patients with chronic HCV 37 38 39 infection. Its pathogenesis is complex, involving impaired iron reutilization, low-grade 40 41 haemolysis, shortened (RBC) lifespan, hyposecretion of erythropoietin 42 43 44 (EPO) and tissue hyporesponsiveness to EPO. These effects are thought to result from 45 46 the actions of inflammatory , which, among other effects, increase the 47 48 production of hepcidin [Vecchiet et al, 2005; Falasca et al, 2006; Falasca et al, 2007; 49 50 51 Falasca et al, 2008]. 52 53 A reduced production of EPO complicates the pathophysiology of anaemia. EPO is an 54 55 endogenous glycoprotein hormone which is the primary regulator of the rate of 56 57 58 erythropoiesis [Krantz, 1991]. It binds to specific receptors on the cell surface of RBC 59 60 precursors in the bone marrow, stimulating the formation of enucleated reticulocytes,

which rapidly mature into RBCs, causing an increase in the circulating red blood cell

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1 2 3 mass [Elliott et al, 2008]. Normally, a decrease in the haemoglobin level is accompanied 4 5 6 by an increase in the serum EPO level, which will ultimately normalize the 7 8 haemoglobin level [Spivak, 2000]. The relationship between haemoglobin and EPO is 9 10 less apparent in patients with chronic diseases, such as cancer and chronic viral 11 12 13 infections. HCV-infected patients who were treated with PEG-IFN/RBV appeared to 14 15 have inappropriately low levels of endogenous EPO for their degree of anaemia. 16 17 18 Although serum levels of endogenous EPO increased during the combination treatment, 19 20 the haemoglobin levelFor did notPeer return to Review normal, suggesting that the increase in 21 22 endogenous EPO was not sufficient to fully compensate for the degree of anaemia 23 24 25 [Trivedi and Trivedi, 2004]. In addition, even though EPO is synthesized primarily in 26 27 the kidney, other organs, such as the liver [Koury et al, 1991] and brain, also produce it. 28 29 An inadequate EPO production by HCV-infected hepatocytes in the liver also may 30 31 32 contribute to a subnormal rise in EPO. Thus the erythropoietic response in HCV- 33 34 infected patients appears to be decreased [Trivedi and Trivedi, 2004; Balan et al, 2005]. 35 36 Comparing the two groups in the current study, the higher MCV value in standard care 37 38 39 treated patients seems to be the expression of an impaired compensation of anaemia. 40 41 Patients treated with combination treatment for chronic hepatitis C may therefore 42 43 44 require earlier intervention compared with other populations with anaemia. 45 46 Many physicians have begun using rHuEPO “off-label” to manage anaemia in patients 47 48 with chronic hepatitis C. Administration of rHuEPO stimulates red blood cell 49 50 51 production and predictably increases haemoglobin concentrations; it has also proved to 52 53 be remarkably well tolerated and highly effective [Sowade et al, 1998]. However, 54 55 because of the time required for erythroid progenitors to mature and be released into the 56 57 58 circulation, a significant increase in haemoglobin concentration does not usually occur 59 60 for 2 weeks and may take up to 6 weeks.

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1 2 3 There are currently three rHuEPO agents on the market: EPO-α, EPO-β (in Europe 4 5 6 only) and darbepoetin alpha, a structurally different synthetic longer-acting analogue of 7 8 EPO-α. They are approved for the treatment of anaemia associated with chronic renal 9 10 failure, cancer, HIV infection and in the surgical setting, to reduce allogeneic blood 11 12 13 transfusion [Sowade et al, 1998; Elliott et al, 2008]. EPO-α and EPO-β, both produced 14 15 by Chinese hamster ovary cells, share the same sequence as endogenous 16 17 18 EPO and have the same physiological effects. However, differences in the 19 20 manufacturing processFor between Peer the two Review glycoproteins reflect differences in their 21 22 carbohydrate moieties [Storring et al, 1998], which determine differences in the 23 24 25 pharmacokinetic and pharmacodynamic properties between these agents. EPO-β has a 26 27 prolonged half-life following subcutaneous administration and seems to induce a greater 28 29 absolute reticulocyte response than EPO-α after subcutaneous administration 30 31 32 [Halstenson et al, 1991]. 33 34 A consensus is growing that EPO-α is useful for maintaining the RBV dose, particularly 35 36 when the patient is symptomatic during an acute fall in haemoglobin level. Results of 37 38 39 recent clinical trials have shown that the use of EPO-α can reduce the incidence and 40 41 severity of anaemia induced by PEG-IFN/RBV treatment [Dieterich et al, 2003; Afdhal 42 43 44 et al, 2004]. Administration of EPO-α at 40.000 IU once weekly increases the 45 46 haemoglobin concentration, maintains the RBV dose and improves the quality of life 47 48 [Brau, 2004]. 49 50 51 Whereas few studies have used EPO-β [ Lebray et al, 2005; Lindahl et al, 2005 ], most 52 53 practitioners use EPO-β because of its pharmacokinetic and pharmacodynamic 54 55 properties and efficacy. In a French retrospective study on the use of growth factors for 56 57 58 treatment of chronic hepatitis C, EPO-β was the main rHuEPO molecule prescribed in a 59 60 mean dose of 30.000 UI weekly (range 2.000-80.000 UI) [Thevenot et al, 2007].

Recently, an anecdotal report has shown that the use of a high dose of EPO-β in an

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1 2 3 HCV-infected patient with severe RBV-induced anaemia was well-tolerated and 4 5 6 allowed PEG-IFN/RBV treatment to be completed, limiting its adverse effects and an 7 8 optimal viral response to be achieved [Ucciferri et al, 2007] 9 10 On the basis of the results of the current study, EPO-β seems to be promising for the 11 12 13 treatment of anaemia in HCV-infected patients receiving PEG-IFN/RBV combination 14 15 therapy. One month after the introduction of EPO-β, haemoglobin was increased by 16 17 18 about 1 g/dl (p=0,001) in group A. The increase in haemoglobin and Hct, the small 19 20 change in MCV andFor the stabilization Peer of Review these parameters during the treatment in 21 22 patients receiving EPO-β show the positive haematologic effects of using this growth 23 24 25 factor. Moreover, rHuEPO has been shown to enhance platelet reactivity and platelet 26 27 counts in patients with alcoholic cirrhosis, an interesting feature during antiviral therapy 28 29 in thrombopenic HCV-positive patients with cirrhosis [ Homoncik et al, 2004 ]. 30 31 32 However, patients treated with EPO-β did not show significant changes in platelet count 33 34 in this study. 35 36 EPO-β therefore appears to be effective in maintaining haemoglobin and the standard 37 38 39 dose of RBV for the period of time they are used, but the duration of treatment, optimal 40 41 dose, and reference level of haemoglobin remain uncertain. It is evident that official 42 43 44 guidelines are needed. Disadvantages of this agent are that it adds another parenteral 45 46 drug to the treatment regimen, thereby increasing the costs, inconvenience and potential 47 48 side-effects. The rHuEPO agents are costly, but when compared with standard care, its 49 50 51 use has been shown recently to be cost-effective for managing HCV, by increasing 52 53 therapeutic compliance, improving the quality of life and avoiding the complications of 54 55 chronic liver disease [Spiegel et al, 2005]. 56 57 58 The results of the current study show for the first time that administration of EPO-β in 59 60 patients infected with HCV receiving combination PEG-IFN/RBV treatment increases

sustained viral response rates among patients developing anaemia by means of the

15 John Wiley & Sons Journal of Medical Virology Page 18 of 30

1 2 3 delivery of optimal dosages of RBV and decreased discontinuance rates for adverse 4 5 6 effects. 7 8 There are many factors that might have confounded the results, including the duration of 9 10 antiviral therapy and small sample size. If EPO levels had been measured in plasma 11 12 13 samples, a more comprehensive assessment of the haematological changes would have 14 15 been obtained. Future studies will attempt to assess these hypotheses. 16 17 18 In conclusion, current results indicate that the use of EPO-β in selected patients allows a 19 20 higher sustained viralFor response Peer rate to be obtained Review limiting the adverse effects of PEG- 21 22 IFN/RBV treatment. The improvement of sustained viral response is due to the 23 24 25 maintenance of the RBV standard dose, which could be obtained only in patients treated 26 27 with EPO-β. It is remarkable that during EPO-β therapy side-effects did not emerge 28 29 even with double doses, and this confirms that it is a highly manageable molecule. 30 31 32 Well-designed clinical trials with larger numbers of patients would be useful to confirm 33 34 the benefit of EPO-β therapy in conjunction with combination PEG-IFN/RBV 35 36 treatment, as well as the optimal dose and frequency of administration of EPO-β. 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 REFERENCES 4 5 6 7 8 Afdhal NH, Dieterich DT, Pockros PJ, Schiff ER, Shiffman ML, Sulkowski MS, Wright 9 10 T, Younossi Z, Goon BL, Tang KL, Bowers PJ; Proactive Study Group. 2004. Epoetin 11 12 13 alfa maintains ribavirin dose in HCV-infected patients: A prospective, double-blind, 14 15 randomized controlled study. Gastroenterology 126:1302–1311. 16 17 18 19 20 Akuta N, Suzuki F,For Sezaki H, PeerSuzuki Y, Hosaka Review T, Someya T, Kobayashi M, Saitoh S, 21 22 Watahiki S, Sato J, Kobayashi M, Arase Y, Ikeda K, Kumada H. 2006. Predictive 23 24 25 factors of virological non-response to interferon-ribavirin combination therapy for 26 27 patients infected with hepatitis C virus of genotype 1b and high viral load. J Med Virol 28 29 78:83–90. 30 31 32 33 34 Balan V, Schwartz D, Wu GY, Muir AJ, Ghalib R, Jackson J, Keeffe EB, Rossaro L, 35 36 Burnett A, Goon BL, Bowers PJ, Leizt GJ. 2005. Erythropoietic response to anemia in 37 38 39 chronic hepatitis C patients receiving combination pegylated interferon/ribavirin. Am J 40 41 Gastroenterol 100:299–307. 42 43 44 45 46 Bennett CL, Silver SM, Djulbegovic B, Samaras AT, Blau CA, Gleason KJ, Barnato 47 48 SE, Elverman KM, Courtney DM, McKoy JM, Edwards BJ, Tigue CC, Raisch DW, 49 50 51 Yarnold PR, Dorr DA, Kuzel TM, Tallman MS, Trifilio SM, West DP, Lai SY, Henke 52 53 M. 2008. Venous thromboembolism and mortality associated with recombinant 54 55 erythropoietin and darbepoetin administration for the treatment of cancer associated 56 57 58 anemia. JAMA 299(8): 914–924. 59 60

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1 2 3 Brau N. 2004. Epoetin alfa treatment for acute anaemia during interferon plus ribavirin 4 5 6 combination therapy for chronic hepatitis C. J Viral Hepat 11:191–197. 7 8 9 10 De Franceschi L, Fattovich G, Turrini F, Ayi K, Brugnara C, Manzato F, Noventa F, 11 12 13 Stanzial AM, Solero P, Corrocher R. 2000. Hemolytic anemia induced by ribavirin 14 15 therapy in patients with chronic hepatitis C infection: role of membrane oxidative 16 17 18 damage. Hepatology 31:997–1004. 19 20 For Peer Review 21 22 Dieterich DT, Spivak JL. 2003. Hematologic disorders associated with hepatitis C virus 23 24 25 infection and their management. Clin Infect Dis 37: 533–541. 26 27 28 29 Dieterich DT, Wasserman R, Brau N, Hassanein TI, Bini EJ, Bowers PJ, Sulkowski 30 31 32 MS. 2003. Once-weekly epoetin-alfa improves anemia and facilitates maintenance of 33 34 ribavirin dosing in hepatitis C virus-infected patients receiving ribavirin plus interferon 35 36 alfa. Am J Gastroenterol 98:2491–2499. 37 38 39 40 41 Elliott S, Pham E, Macdougall IC. 2008. : a common mechanism of 42 43 44 action. Exp Hematol 36(12):1573–1584. 45 46 47 48 Falasca K, Ucciferri C, Dalessandro M, Zingariello P, Mancino P, Petrarca C, Pizzigallo 49 50 51 E, Conti P, Vecchiet J. 2006. patterns correlate with liver damage in patients 52 53 with chronic hepatitis B and C. Ann Clin Lab Scie 36:144–150. 54 55 56 57 58 Falasca K, Mancino P, Ucciferri C, Dalessandro M, Zingariello P, Lattanzio FM, 59 60 Petrarca C, Martinotti S, Pizzigallo E, Conti P, Vecchiet J. 2007. Inflammatory

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1 2 3 cytokines and S-100b protein in patients with hepatitis C infection and 4 5 6 cryoglobulinemias. Clin Invest Med 30:E167–176. 7 8 9 10 Falasca K, Ucciferri C, Mancino P, Vitacolonna E, De Tullio D, Pizzigallo E, Conti P, 11 12 13 Vecchiet J. 2008. Treatment with silybin-vitamin E-phospholipid complex in patients 14 15 with hepatitis C infection. J Med Virol 81:446–451. 16 17 18 19 20 Fried MW, ShiffmanFor ML, Reddy Peer KR, Smith Review C, Marinos G, Goncales FL Jr, Haussinger 21 22 D, Diago M, Carosi G, Dhumeaux D, Craxi A, Lin A, Hoffman J, Yu J. 2002. 23 24 25 Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N Engl J 26 27 Med 347:975–982. 28 29 30 31 32 Hadziyannis SJ, Sette H, Morgan T, Balan V, Diago M, Marcellin P, Ramadori G, 33 34 Bodenheimer H Jr, Bernstein D, Rizzetto M, Zeuzem S, Pockros PJ, Lin A, Ackrill AM; 35 36 PEGASYS International Study Group. 2004. Peginterferon alfa-2a and ribavirin 37 38 39 combination therapy in chronic hepatitis C: a randomized study of treatment duration 40 41 and ribavirin dose. Ann Intern Med 140:346–355. 42 43 44 45 46 Halstenson CE, Macres M, Katz SA, Schnieders JR, Watanabe M, Sobota JT, Abraham 47 48 PA. 1991. Comparative pharmacokinetics and pharmacodynamics of epoetin alfa and 49 50 51 epoetin beta. Clin Pharmacol Ther 50:702–712. 52 53 54 55 Homoncik M, Jilma-Stohlawetz P, Schmid M, Ferlitsch A, Peck-Radosavljevic M. 56 57 58 2004. Erythropoietin increases platelet reactivity and platelet counts in patients with 59 60 alcoholic liver cirrhosis: a randomized, doubleblind, placebo-controlled study. Aliment

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1 2 3 4 5 6 Kowdley KV. 2005. Hematologic side effects of interferon and ribavirin therapy. J Clin 7 8 Gastroenterol 39(Suppl 1):S3–8. 9 10 11 12 13 Krantz SB. 1991. Erythropoietin. Blood 77:419–434. 14 15 16 17 18 Kumada T, Toyoda H, Kiriyama S, Sone Y, Tanikawa M, Hisanaga Y, Kanamori A, 19 20 Atsumi H, Takagi M,For Nakano PeerS, Arakawa T, Review Fujimori M. 2009. Long-term follow-up of 21 22 patients with hepatitis C with a normal alanine aminotransferase. J Med Virol 81:446– 23 24 25 451. 26 27 28 29 Lebray P, Nalpas B, Vallet-Pichard A, Broissand C, Sobesky R, Serpaggi J, Fontaine H, 30 31 32 Pol S. 2005. The impact of haematopoietic growth factors on the management and 33 34 efficacy of antiviral treatment in patients with hepatitis C virus. Antivir Ther 10:769 – 35 36 776. 37 38 39 40 41 Lindahl K, Stahle L, Bruchfeld A, Schvarcz R. 2005. High-dose ribavirin in 42 43 44 combination with standard dose peginterferon for treatment of patients with chronic 45 46 hepatitis C. Hepatology 41:275 –279. 47 48 49 50 51 Manns MP, McHutchison JG, Gordon SC, Rustgi VK, Shiffman M, Reindollar R, 52 53 Goodman ZD, Koury K, Ling M, Albrecht JK. 2001. Peginterferon alfa-2b plus 54 55 ribavirin compared with -2b plus ribavirin for initial treatment of chronic 56 57 58 hepatitis C: a randomised trial. Lancet 358:958–965. 59 60

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1 2 3 McHutchinson JG, Davis GL, Esteban-Mur R. 2001. Durability of sustained viral 4 5 6 response in patients with chronic hepatitis C after treatment with interferon alfa-2b 7 8 alone or in combination with ribavirin. Hepatology 34:244A. 9 10 11 12 13 McHutchinson JG, Manns M, Patel K, Lindsay KL, Poynard T, Trepo C, Dienstag J, 14 15 Lee WM, Mak C, Garaud JJ, Albrecht JK; International Hepatitis Interventional 16 17 18 Therapy Group. 2002. Adherence to combination therapy enhances sustained response 19 20 in genotype-1-infectedFor patients Peer with chronic Review hepatitis C. Gastroenterology 123:1061– 21 22 1069. 23 24 25 26 27 McHutchison JG, Manns MP, Longo DL. 2006. Definition and management of anemia 28 29 in patients infected with hepatitis C virus. Liver Int 26:389–398. 30 31 32 33 34 McHutchison JG, Manns MP, Brown RS, Reddy KR, Shiffman ML, Wong JB. 2007. 35 36 Strategies for managing anemia in hepatitis C patients undergoing antiviral therapy. Am 37 38 39 J Gastroenterol 102:880–889. 40 41 42 43 44 McKoy JM, Stonecash RE, Cournoyer D, Rossert J, Nissenson AR, Raisch DW, 45 46 Casadevall N, Bennet CL. 2008. Epoetin-associated pure red cell aplasia: past, present, 47 48 and future considerations. Transfusion 48:1754–1762. 49 50 51 52 53 Peck-Radosavljevic M, Wichlas M, Homoncik-Kraml M, Kreil A, Hofer H, Jessner W, 54 55 Gangl A, Ferenci P. 2002. Rapid suppression of hematopoiesis by standard or pegylated 56 57 58 interferon-alpha. Gastroenterology 123:141–151. 59 60

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1 2 3 4 5 6 PEG-Intron [package insert]. Kenilworth, NJ:Schering-Plough Corporation, 2003. 7 8 9 10 Poynard T, Yuen MF, Ratziu V, Lai CL. 2003. Viral hepatitis C. Lancet 362:2095– 11 12 13 2100. 14 15 16 17 18 Rizzo JD, Somerfield MR, Hagerty KL, Seidenfeld J, Bohlius J, Bennett CL, Cella DF, 19 20 Djulbegovic B, GoodeFor MJ, Jakubowski Peer AA, Review Rarick MU, Regan DH, Lichtin AE. 2008. 21 22 Use of epoetin and darbepoetin in patients with cancer: 2007 American Society of 23 24 25 Hematology/American Society of Clinical Oncology clinical practice guideline update. 26 27 Blood 111:25–41. 28 29 30 31 32 Sagnelli E, Stroffolini T, Mele A, Almasio P, Coppola N, Ferrigno L, Scolastico C, 33 34 Onofrio M, Imaprato M, Filippini P. 2005. The importance of HCV on the burden of 35 36 chronic liver disease in Italy: a multicenter prevalence study of 9,997 cases. J Med Virol 37 38 39 75:522–527. 40 41 42 43 44 Sherman M, Cohen L, Cooper MA, Elkashab M, Feinman V, Fletcher D, Girgrah N, 45 46 Heathcote J, Levstik M, McNaull WB, Wong D, Wong F, Yim C. 2006. Clinical 47 48 recommendations for the use of recombinant human erythropoietin in patients with 49 50 51 hepatitis C virus being treated with ribavirin. Can J Gastroenterol 20:479–485. 52 53 54 55 Shiffman ML. 2004. Side effects of medical therapy for chronic hepatitis C. Ann 56 57 58 Hepatol 3:5–10. 59 60

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1 2 3 Shiffman ML. 2004. Management of patients with chronic hepatitis C virus infection 4 5 6 and previous nonresponse. Rev Gastroenterol Disord 4: S22–30. 7 8 9 10 Shiffman ML, Salvatore J, Hubbard S, Price A, Sterling RK, Stravitz RT, Luketic VA, 11 12 13 Sanyal AJ. 2007. Treatment of chronic hepatitis C virus genotype 1 with peginterferon, 14 15 ribavirin, and epoetin alpha. Hepatology. 46:371–379. 16 17 18 19 20 Sowade B, SowadeFor O, Mocks Peer J, Franke W, Review Warnke H. 1998. The safety of treatment 21 22 with recombinant human erythropoietin in clinical use: A review of controlled studies. 23 24 25 Int J Mol Med 1:303–314. 26 27 28 29 Spiegel BM, Chen K, Chiou CF, Robbins S, Younossi ZM. 2005. Erythropoietic growth 30 31 32 factors for treatment-induced anemia in hepatitis C: a cost-effectiveness analysis. Clin 33 34 Gastroenterol Hepatol 3:1034 –1042. 35 36 37 38 39 Spivak JL. 2000. The blood in systemic disorders. Lancet 355:1707–1712. 40 41 42 43 44 Storring PL, Tiplady RJ, Gaines Das RE, Stenning BE, Lamikanra A, Rafferty B, Lee J. 45 46 1998. Epoetin alfa and beta differ in their erythropoietin isoform compositions and 47 48 biological properties. Br J Haematol, 100:79–89. 49 50 51 52 53 Stravitz RT, Chung H, Sterling RK, Luketic VA, Sanyal AJ, Price AS, Purrington A, 54 55 Shiffman ML. 2005. Antibody-mediated pure red cell aplasia due to epoetin alfa during 56 57 58 antiviral therapy of chronic hepatitis C. Am J Gastroenterol 100:1415–1419. 59 60

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1 2 3 Sulkowski MS, Wasserman R, Brooks L, Ball L, Gish R. 2004. Changes in 4 5 6 haemoglobin during interferon alpha-2b plus ribavirin combination therapy for chronic 7 8 hepatitis C virus infection. J Viral Hepat 11:243–250. 9 10 11 12 13 Thevenot T, Cadranet JF, Di Martino V, Pariente A, Causse X, renou C, Hagege H, 14 15 Denis J, Lunel-Fabiani F. 2007. A National French survey on the use of growth factors 16 17 18 as adjuvant treatment of chronic hepatitis C. Hepatology 45:377–383. 19 20 For Peer Review 21 22 Trivedi HS, Trivedi M. 2004. Subnormal rise of erythropoietin in patients receiving 23 24 25 interferon and ribavirin combination therapy for hepatitis C. J Clin Gastroenterol 26 27 38:595–598. 28 29 30 31 32 Ucciferri C, Falasca K, Mancino P, De Tullio D, Pizzigallo E, Vecchiet J. 2007. High 33 34 dose of erythropoietin in management of interferon/ribavirin induced anemia. 35 36 HepatoGastroenterology 54: 2181–2183. 37 38 39 40 41 Vecchiet J, Falasca K, Cacciatore P, Zingariello P, Dalessandro M, Marinopiccoli M, 42 43 44 D'Amico E, Palazzi C, Petrarca C, Conti P, Pizzigallo E, Guagnano MT. 2005. 45 46 Association between plasma interleukin-18 levels and liver injury in chronic hepatitis C 47 48 virus infection and non-alcoholic fatty liver disease. Ann Clin Lab Sci 35:415–422. 49 50 51 52 53 54 World Health Organization (WHO). Weekly epidemiologicaly record. World Health 55 56 57 Organization 2007. 58 59 60

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1 2 3 Table I: Clinical and biochemical characteristics of the two study populations 4 5 6 Parameter (and units) Epoetin Beta Standard care p 7 group group 8 (22 pz) (20 pz) 9

10 Age (yrs) 47.5 ± 10.6 48.6 ± 15.0 11 ns 12 13 Gender (%) 14 male 5 (22.7%) 7 (35%) 15 female 17 (77.3%) 13 (65%) ns 16 17 18 Genotype (%) 1 15 (68.2%) 15 (75%) 19 ns 20 non-1 For Peer7 (31.84%) Review 5 (25%) 21 22 23 24 Weight (Kg) 64.8 ± 9.5 65.4 ± 9.3 ns 25 26 Treatment (%) 27 Peginterferon α 2a + RBV 12 (54.5%) 13 (65%) ns 28 Peginterferon α 2b + RBV 10 (45.5%) 7 (35%) 29 30 4 31 HCV-RNA (x 10 )(cp/ml) 67.9±37.2 69.5±46.2 ns 32 33 Serum iron (µg/dl) 92.04±35.9 100.8±28.1 ns 34 35 Serum ferritin ( g/L) 152.0±62.7 166.3±147.1 ns 36 37 38 Serum cholesterol (mg/dl) 176.1±36.8 169.9±39.0 ns 39 40 Serum triglycerides (mg/dL) 130.5±58.0 129.2±81.0 ns 41 42 Fasting glucose (mg/dl) 86.5±23.7 89.6±34.8 ns 43 44 45 46 Ns: not significant 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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1 2 3 4 5 Table II: Biochemical characteristics of the Epoetin beta group (n=22) 6 7 Parameter (and units) End of 6 months T0 T-pre-change T-1-month 8 treatment follow-up 9 10 Serum AST activity (U/L) 108.0±158.4 40.3±18.8* 37.6±17.4 35.2±17.0† 57.6±69.4 11 # †† ● 12 Serum ALT activity (U/L) 116.5±131.2 47.6±21.3** 43.0±20.5 36.3±22.3 56.0±53.9 # † 13 Serum γ-GT activity (U/L) 56.5±56.1For Peer 43.3±32.0 33.0±19.3Review 34.1±23.4 31.9±11.8 14 Serum ferritin ( g/L) 152.0±62.7### 350.2±34.7*** 270.2±36.8§§ 295.6±184.8†† 91.5±48.7●●● 15 Haemoglobin (g/dl) 13.5±1.1 10.1±0.8*** 11.2±1.0§§§ 11.4±1.2††† 13.1±0.7●●● 16 Hct (%) 40.4±2.8# 32.0±3.8*** 35.2±3.6§§ 35.5±3.7††† 38.9±2.2●●● 17 ### §§ ††† ●● 18 MCV (fl) 87.0±4.4 91.5±6.9** 97.9±6.4 97.7±9.1 91.1±4.1 † ● 19 Platelets (x10 3/µl) 194.6±58.4 158.6±67.0* 157.1±57.2 155.5±55.1 189.0±52.7 20 21 22 23 T0 vs T-pre-change *p<0.05 **p<0.001 ***p<0.0001 24 T-pre-change vs T-1-month §p<0.05 §§p<0.001 §§§p<0.0001 25 T0 vs End of treatment †p<0.05 ††p<0.001 †††p<0.0001 26 27 T0 vs 6 months follow-up #p<0.05 ##p<0.001 ###p<0.0001 28 End of treatment vs 6 months follow-up ●p<0.05 ●● p<0.001 ●●● p<0.0001 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 26 45 John Wiley & Sons 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 29 of 30 Journal of Medical Virology

1 2 3 4 5 Table III: Biochemical characteristics of the Standard care group (n=20) 6 7 Parameter (and units) End of 6 months T0 T-pre-change T-1-month 8 treatment follow-up 9 10 Serum AST activity (U/L) 77.0±43.6## 49.1±21.9** 42.7±19.9 35.5±18.7††† 39.2±27.3 11 ## ††† 12 Serum ALT activity (U/L) 117.4±87.7 57.2±28.1** 48.5±21.2 42.1±28.5 51.7±73.6 ## † 13 Serum γ-GT activity (U/L) 78.9±64.5For Peer 49.8±33.4* 45.1±35.2Review 41.9±44.7 34.4±35.1 14 Serum ferritin ( g/L) 166.3±147.1 340.3±123.3*** 360.3±298.6 459.0±637.3† 100.9±61.1● 15 Haemoglobin (g/dl) 13.9±1.1# 10.5±0.8*** 10.9±0.9§ 11.3±1.3††† 13.2±1.4●●● 16 Hct (%) 40.8±2.8# 32.6±2.5*** 34.1±2.0§ 34.7±3.1††† 38.8±2.9●●● 17 # † ●●● 18 MCV (fl) 86.1±4.2 91.0±5.0*** 91.5±4.2 90.0±6.8 83.6±5.4 † ●●● 19 Platelets (x10 3/µl) 172.1±49.0 136.1±39.5** 135.7±41.6 140.4±30.6 176.2±32.9 20 21 22 23 T0 vs T-pre-change *p<0.05 **p<0.001 ***p<0.0001 24 T-pre-change vs T-1-month §p<0.05 §§p<0.001 §§§p<0.0001 25 T0 vs End of treatment †p<0.05 ††p<0.001 †††p<0.0001 26 27 T0 vs 6 months follow-up #p<0.05 ##p<0.001 ###p<0.0001 28 End of treatment vs 6 months follow-up ●p<0.05 ●● p<0.001 ●●● p<0.0001 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 27 45 John Wiley & Sons 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Journal of Medical Virology Page 30 of 30

1 2 3 4 5 6 7 8 9 10 11 12 13 For Peer Review 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 28 45 John Wiley & Sons 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60