J Gastroenterol 2007; 42:362367 DOI 10.

1007/s00535-007-2006-3

A randomized trial of induction doses of interferon alone or in combination with ribavirin or ribavirin plus amantadine for treatment of nonresponder patients with chronic hepatitis C
Annagiulia Gramenzi1, Pietro Andreone1, Carmela Cursaro1, Gabriella Verucchi2, Sergio Boccia3, Pier Luigi Giacomoni4, Silvia Galli5, Giuliano Furlini5, Maurizio Biselli1, Stefania Lorenzini1, Luciano Attard2, Fiorenza Bonvicini1, and Mauro Bernardi1
1 Dipartimento di Medicina Interna, Cardioangiologia ed Epatologia, Policlinico S. Orsola, University of Bologna, Via Massarenti, 9-40138 Bologna, Italy 2 Dipartimento di Medicina Clinica Specialistica e Sperimentale, University of Bologna, Bologna, Italy 3 Gastroenterologia, Azienda Ospedaliera Universitaria di Ferrara Arcispedale S. Anna, Ferrara, Italy 4 Lungodegenza, Ospedale di Lugo, Lugo (RA), Italy 5 Servizio di Microbiologia e Virologia, Policlinico S. Orsola-Malpighi, Bologna, Italy

Background. Efcacy and safety of interferon induction therapy alone or in combination with ribavirin or ribavirin plus amantadine were evaluated in chronic hepatitis C patients who were nonresponders to primary antiviral treatment. Methods. The study was designed to have 225 HCV nonresponder patients, but at an interim analysis the response rate difference between groups was lower than expected and the enrollment was stopped when 75 patients had been randomized to receive interferon-2a (group A, n = 26), interferon-2a plus 15 mg/kg per day of ribavirin (group B, n = 24), or interferon-2a plus ribavirin plus 200 mg/day of amantadine hydrochloride (group C, n = 25). Treatment duration was 48 weeks. The dose of interferon was 6 MU/day for 4 weeks followed by 3 MU/day for the remaining 44 weeks. Results. On intention-to-treat, the sustained virological response at 24 weeks of follow-up was 11.5% in group A, 12.5% in group B, and 12% in group C. Therapy was discontinued because of adverse effects in three patients in group A (11.5%), three in group B (12.5%), and two in group C (8%). Conclusions. Nonresponders with chronic hepatitis C may achieve a sustained virological response rate of approximately 12% if retreated with interferon induction treatment followed by administration of a daily dose. The addition of ribavirin or amantadine did not seem to improve the response rates.

Key words: chronic hepatitis C, high-dose interferon, nonresponders, amantadine, ribavirin

Introduction Although pegylated interferon- (PEG-IFN) plus ribavirin-based treatments for chronic hepatitis C have considerably increased the number of patients achieving a sustained virological response,1,2 30%50% of patients remain nonresponders to primary antiviral treatment. At present, there are no clear indications for therapy of nonresponder patients. Several strategies have been evaluated, including retreatment with IFN alone or in combination with ribavirin or amantadine, with disappointing results.311 In an effort to improve the sustained virological response rate in nonresponders to IFN alone or in combination with ribavirin or amantadine, more potent IFN regimens (such as induction and/or daily doses) as well as a triple combination of antiviral drugs have been employed. Preliminary studies also reported promising results with the use of a combination of IFN, ribavirin, and amantadine: patients not responding to previous antiviral treatment achieved sustained virologic response rates of up to 48%.1214 These results, however, have not been conrmed by subsequent studies.15,16 The aim of this randomized multicenter study was to evaluate the therapeutic efcacy and safety prole of induction therapy with IFN followed by daily doses of IFN alone or in combination with ribavirin and/or

Received: July 27, 2006 / Accepted: January 4, 2007 Reprint requests to: P. Andreone

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amantadine in patients with chronic hepatitis C not responding to previous antiviral treatment.

Subjects and methods Patients From January 2001 to January 2002, all consecutive patients who were histologically proven chronic hepatitis C nonresponders to a course of recombinant IFN with or without ribavirin were considered for admission to the present study. Failure to respond to previous antiviral treatment was dened as failure to clear hepatitis C virus RNA (HCV-RNA) from serum and to normalize serum alanine aminotransferase (ALT) during the treatment period. Only patients who had received a dose of 3 or 6 MU three times a week for at least 16 weeks and thereafter showed persistent positivity of HCV-RNA for at least 12 months were considered. All patients had undergone a treatment washout period of at least 12 months, had ALT levels persistently greater than 1.5 times the normal value during the follow-up period, and were positive for serum HCV-RNA. Criteria for exclusion were age <18 or 65 years; decompensated liver disease; serum human immunodeciency virus or hepatitis B surface antigen positivity; evidence of any cause of liver disease other than chronic hepatitis C; serum hemoglobin concentration less than 12 g/dl for women or 13 g/dl for men; white cell count of less than 3000/ mm3; neutrophil count of less than 1500/mm3; platelet count of less than 70 000/mm3; presence of hemoglobinopathy or hemolytic anemia; alcohol abuse; drug abuse; pregnancy; renal, cardiovascular, or pulmonary diseases; diabetes mellitus; epilepsy; or psychiatric disorders. Study design The study was a multicenter prospective open randomized trial and was conducted at four centers in Italy in accordance with the tenets of the Declaration of Helsinki. The study was approved by the ethics committee of each participating center, and all patients provided informed consent. Patients were randomly assigned to one of three different treatment groups: group A received IFN-2a (Roferon, Roche, Milano, Italy) alone, group B received IFN-2a plus oral ribavirin (kindly supplied by Roche) at the dose of 15 mg/kg per day, and group C received IFN-2a plus 15 mg/kg per day of oral ribavirin plus oral amantadine hydrochloride (kindly supplied by Roche) 200 mg/day administered in two doses of 100 mg. In all groups, the treatment duration was 48 weeks, and IFN was subcutaneously administered with an induction course of 4 weeks at the dose

of 6 MU daily followed by a dose of 3 MU/day for the remaining 44 weeks. After treatment cessation, patients were followed up for at least 24 weeks. During the rst 12 weeks of treatment, clinical and laboratory examinations were performed every 2 weeks and each month thereafter, or more frequently when indicated by clinical needs. Serum HCV-RNA was determined at baseline, after 12, 24, and 48 weeks of treatment, and after 24 weeks of follow-up. According to the study protocol, the interferon or ribavirin dose could be modied if an important adverse event occurred or in the case of important abnormalities in laboratory values, including hematologic toxicity as manifested by hemoglobin levels of less than 10 g/dl, a white cell count of less than 2500/mm3, granulocyte count of less than 1000/mm3, or a platelet count of less than 70 000/mm3. The end point of the study was sustained virological response (SVR), dened as undetectable serum HCV-RNA by qualitative polymerase chain reaction (PCR) 6 months after the end of treatment. According to the protocol, at week 12 only the patients with undetectable serum HCVRNA or a >2 log10 drop from baseline (on-treatment responders) completed the treatment period. The remaining patients were considered nonresponders and were followed up for 6 months. After treatment week 24, antiviral treatment was continued only in patients with undetectable serum HCV-RNA. All patients underwent liver biopsy within 18 months before entry; each patient was offered a second liver biopsy at the end of follow-up. Each liver biopsy was blindly reviewed by one pathologist according to the histological activity index proposed by Knodell et al.17 All hematologic, biochemical, and virologic parameters were determined in the local laboratories of each study center. Serologic assessment of HCV infection was made by third generation enzyme-linked immunosorbent assay (Ortho Diagnostic System, Raritan, NJ, USA). HCV genotype was determined by line probe assay (InnoLiPA; Innogenetics, Zwijndrecht, Belgium). Qualitative serum HCV-RNA was measured by PCR (second generation Amplicor HCV; Roche Diagnostic Systems, Basel, Switzerland; sensitivity limit, 50 copies/ ml), and quantitation was performed with a bDNA assay (Quantiplex 2.0; Chiron, Emeryville, CA, USA). Statistical analysis The primary objective of this study was the SVR. A sample size of 225 patients (75 in each treatment group) was estimated on the basis of an (type I) error of 0.05 and a (type II) error of 0.20, on the assumption of a SVR of 10% in group A and 35% in groups B and C. In order to get an early estimation of the risk/benet ratio, an interim analysis was planned after one-third of

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the enrolled patients had completed the treatment period. Since we observed a constant and lower than expected difference in the SVR rate among the three treatment groups, we decide to discontinue the study for ethical reasons. Data were evaluated according to the intention-totreat principle; that is, patients in whom a response could not be ascertained were considered to be nonresponders. The -squared and Fishers exact tests were used to analyze categorical variables. Continuous variables were analyzed by the Mann-Whitney test. A P value of less than 5% was considered to be signicant. All statistical analyses were performed using SPSS for Windows version 11.0.1 software packages (SPSS, North Chicago, IL, USA).

Results Response to treatment A total of 75 consecutive patients not responding to previous antiviral treatment were enrolled in the study. Twenty-six patients were randomized to group A, 24 to group B, and 25 to group C. The baseline characteristics of the patients are summarized in Table 1. There were no signicant differences in age, sex, genotype, baseline HCV-RNA, or ALT serum levels among the three groups. As expected for an IFN-resistant population, the majority of patients were infected with HCV genotype 1. Fifty-one patients of the 75 (68%) had previously received one course of antiviral treatment, and the remaining 24 (32%) had had two or more courses of antiviral treatment. More than half of the patients in each group had been previously treated with IFN in combination with ribavirin (Table 1). Duration of the antiviral course and the median total

cumulative dose of IFN were similar among the three groups. After 12 weeks of treatment, 12 patients in group A (46%), 11 in group B (46%), and 12 in group C (48%) were classied as on-treatment responders and continued treatment. Among them, seven in group A (27%), ve in group B (21%) and ve in group C (20%) had undetectable serum HCV-RNA, while the remaining showed a >2 log10 drop of HCV-RNA from baseline. As shown in Table 2, seven patients in group A (27%), six in group B (25%), and six in group C (24%) achieved an end-of-treatment response. However, an overall SVR was observed in 9 of 75 patients (12%) without any difference among the three treatment groups. A sustained biochemical response 6 months after cessation of treatment was achieved in four patients (15%) in group A, four (17%) in group B, and four (16%) in group C. There were no signicant differences concerning the SVR among the three groups with respect to baseline characteristics, such as genotype, viral load, stage of brosis, or type and schedule of previous antiviral treatment. In particular, a SVR was achieved in 4 of 58 patients (7%) with genotype 1b (two in group A, one in group B, and one in group C), in three of nine (33%) with genotype 2 (one in each group), and in two of three (67%) with genotype 3 (one in group B and one in group C). No statistically signicant difference in the basal mean viral load was observed between SVR patients and nonresponders (2.89 3.5 vs. 2.22 2.9 MEq/ ml, respectively; not signicant). Low basal viremia (2.0 MEq/ml) was present in six of the nine (67%) SVR patients and in 43 of the 66 (65%) nonresponders. Paired pre- and posttreatment liver biopsy specimens were available only from SVR patients. Given the limited number of paired liver biopsy specimens, we refrained from performing any comparison.

Table 1. Characteristics of patients at enrollment Group A No. of patients Sex (M/F) Age (years)a ALT (U/l)a Viral genotype, no. (%) 1 2 3 4 HCV-RNA (MEq/ml)a HCV-RNA >2.0 MEq/ml, no. (%) Cirrhosis, no. (%) Previous treatment with ribavirin, no. (%) 26 14/12 54.5 10.6 128.8 94.8 21 (81) 3 (11) 1 (4) 1 (4) 2.1 2.9 8 (31) 6 (23) 14 (54) Group B 24 16/8 49.7 11.3 120.8 77.9 18 (75) 3 (13) 2 (8) 1 (4) 2.3 2.9 8 (33) 5 (20) 17 (71) Group C 25 15/10 50.1 10.0 114.4 69.8 19 (76) 3 (12) 3 (12) 2.3 2.0 10 (40) 7 (29) 16 (64) P NS NS NS NS

NS NS NS NS

ALT, alanine aminotransferase; HCV, hepatitis C virus; NS, not signicant a Data are means SD

A. Gramenzi et al.: IFN, RBV, and AMD in HCV nonresponders Table 2. Virological and biochemical response Group A (n = 26) No. (%) Virological responsea Treatment week 12 Treatment week 24 Treatment week 48 End of follow-up Biochemical response Treatment week 12 Treatment week 24 Treatment week 48 End of follow-up
a

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Group B (n = 24) No. (%) 5 (21) 5 (21) 6 (25) 3 (12.5) 11 (46) 6 (25) 4 (17) 4 (17)

Group C (n = 25) No. (%) 5 (20) 6 (24) 6 (24) 3 (12) 12 (48) 8 (32) 4 (16) 4 (16)

P NS NS NS NS NS NS NS

7 (27) 9 (35) 7 (27) 3 (11.5) 8 (31) 10 (38) 8 (31) 4 (15)

Dened as undetectable hepatitis C virus RNA by qualitative polymerase chain reaction

Safety The most frequent adverse effects were u-like symptoms (32 patients, 43%), mild depression (28 patients, 37%), alopecia (16 patients, 21%), and weight loss (15 patients, 20%). Adverse effects were mostly mild and were similar in the three treatment groups. Only the rate of anemia (12 patients, 16%) was higher in the two groups receiving ribavirin. Amantadine was well tolerated and did not increase the frequency or the severity of side effects. Therapy was discontinued before the end of treatment in eight patients (11%), three in group A (11.5%), three in group B (12.5%), and two in group C (8%). The most frequent cause was severe fatigue (three patients), followed by severe anemia (two patients), depression (two patients), and sleep disturbances (one patient). None of the patients discontinued treatment during the 4-week induction regimen. A reduction in the dose of ribavirin because of hemolytic anemia was necessary in seven patients, three in group B (12.5%) and four in group C (16%). As a consequence of side effects, the IFN dose had to be reduced in ve patients (three for fatigue, one for neutropenia, and one for anxiety and irritability).

Discussion In this randomized multicenter clinical trial, we evaluated the efcacy and safety of IFN induction therapy alone or in combination with ribavirin or ribavirin plus amantadine in HCV patients resistant to previous antiviral treatments. The data of this study showed an overall SVR of 12%, without any difference among the three treatment groups. Surprisingly, in our study, a short induction period with a daily high dose (6 MU) of IFN followed by daily administration of 3 MU had the same efcacy whether administered alone or in combination

with ribavirin or ribavirin and amantadine. Our SVR rate obtained with retreatment with IFN alone is consistent with rates reported for retreatment with IFN and ribavirin, ranging from 12% to 18%.14,18 There is solid evidence that retreatment of nonresponders with IFN monotherapy is of no clinical value.19 Therefore, in our study, the efcacy of retreatment with IFN alone was likely increased by using induction doses followed by daily administration for 12 months. It has recently been shown that patients who were nonresponders to one or two courses of standard recombinant or lymphoblastoid IFN may achieve a response rate ranging from 23% to 33% if retreated with 5 MU of IFN plus ribavirin three times a week for 48 weeks with or without a short induction period.20 Unfortunately, in this trial, there was not a group of patients treated with a reinforced regimen of IFN alone. Concerning the use of triple therapy, our results were poor when compared with those of other studies reporting SVR rates higher than 20% in nonresponder patients with chronic hepatitis C retreated with the combination IFN plus ribavirin and amantadine.1214 It should be pointed out that the schedules of treatment of nonresponder patients utilized in published studies are extremely heterogeneous and hardly comparable. However, our disappointing response rates might be attributable, at least in part, to different study populations. First, our criteria of nonresponse were very strict. In fact, we enrolled only those patients who achieved neither a virological nor a biochemical response during previous antiviral treatment. This strategy ensured the exclusion of so-called partial responders, relapsers, or breakthroughs. Younossi et al.11 reported that retreatment of a strictly dened IFN nonresponder population with a 24-week course of IFN at standard doses plus ribavirin or amantadine was associated with very low, if any, sustained viral eradication.

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Furthermore, in our study, 32% of patients had been treated with more than one previous course of antiviral treatment, and 63% had received at least one course of IFN plus ribavirin. It has been shown that the number of previous antiviral treatments is an important response predictor for retreatment.14 This might explain the difference in SVR in a recently published clinical trial, which reported a SVR rate of 48% in patients who were nonresponders to only one course of interferon monotherapy retreated with IFN, ribavirin, and amantadine.12 Unfortunately, owing to the low number of both enrolled and sustained-responder patients, we were not able to nd any relationship between pretreatment host or viral factors such as number, schedule and type of previous antiviral treatments, viral load, genotype or brosis score, or response to treatment. An early virological response to IFN treatment has been shown to be a strong predictor of SVR.21 In our study, an early virological response (after 12 weeks of therapy) was seen in 7 of 26 patients in group A (27%), 5 of 24 in group B (21%), and 5 of 25 in group C (20%), but only three patients in each group attained a SVR. The incidence of side effects was similar in the three treatment groups, except for anemia, which was obviously more common in the two groups receiving ribavirin. The overall rate of treatment discontinuation for adverse events was 11%, without differences among the three arms of the study. The rate of discontinuation and the rate of dose reduction were comparable to those reported for either standard IFN plus ribavirin therapy in nave patients22,23 or high IFN doses plus ribavirin.20 In summary, our study demonstrated that patients with chronic hepatitis C who were nonresponders to previous antiviral treatments may achieve a sustained virologic response rate of approximately 12% if retreated with IFN induction treatment followed by daily dose administration. The addition of ribavirin or ribavirin plus amantadine did not result in an improvement in the sustained virologic response. At present, PEG-IFNs have replaced non-PEG IFNs for the treatment of chronic hepatitis C. However, the results for the use of the combination PEG-IFN plus ribavirin in nonresponder patients are still not conclusive, and were disappointing in patients with genotype 1.24 Unfortunately, patients with genotype 1 are predominant among nonresponders to previous therapies. Thus, to date there are no therapeutic guidelines for such patients. Considering the lower cost of non-PEG IFN, our results may help in planning future trials in nonresponder patients.
Acknowledgments. This study was in part supported by the Associazione per la Ricerca sulle Malattie Epatiche (ARME). The authors gratefully thank Dr. Marzia Margotti and Dr. Elisabetta Loggi for their helpful assistance.

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