Mycophenolate mofetil in combination with recombinant interferon alfa-2a in interferon-nonresponder patients with chronic hepatitis C

Journal of Hepatology 37 (2002) 843–847 www.elsevier.com/locate/jhep

Mycophenolate mofetil in combination with recombinant interferon alfa-2a in interferon-nonresponder patients with chronic hepatitis C Markus Cornberg 1, Holger Hinrichsen 2, Gerlinde Teuber 3, Thomas Berg 4, Uta Naumann 4, Christian Falkenberg 2, Stefan Zeuzem 3, Michael P. Manns 1,* 1

Department of Gastroenterology, Hepatology, and Endocrinology, Medizinische Hochschule Hannover, Carl Neuberg Strasse 1, D-30625 Hannover, Germany 2 I. Medizinische Klinik, Christian-Albrechts-Universitaet zu Kiel, Kiel, Germany 3 Medizinische Klinik II, Klinikum der Johann Wolfgang Goethe Universitaet Frankfurt, Frankfurt, Germany 4 Medizinische Klinik m. S. Hepatologie und Gastroenterologie, Charite´, Campus Virchow-Klinikum, Humboldt-Universitaet zu Berlin, Berlin, Germany

Background/Aims: Since ribavirin was able to improve the antiviral efficacy of interferon alfa in patients with chronic hepatitis C, several other adjuncts have been studied. It has been shown that mycophenolate mofetil (MMF) is a more potent inhibitor of the inosine 5 0 -monophosphate-dehydrogenase (IMPDH) than ribavirin. The present study is a pilot study evaluating the efficacy and safety of combination therapy with interferon alfa-2a and MMF in interferon alfa nonresponder patients. Methods: Thirty-eight adult patients with chronic hepatitis C who did not respond to a previous interferon alfa monotherapy were enrolled to receive 6 million units of interferon alfa-2a tiw in combination with MMF (1 week 500 mg/day, 1 week 1000 mg/day, 22 weeks 2000 mg/day). Results: An interim analysis of 29 patients after 12 weeks of therapy showed that only one patient had negative hepatitis C virus-RNA at this time point. There was no significant reduction of the viral load during therapy. Due to inefficacy the study was discontinued. Conclusions: Combination therapy of interferon alfa-2a and MMF is ineffective in improving virological response rates in nonresponder patients with chronic hepatitis C. These data suggest that inhibition of the IMPDH seems not to be the major mechanism of ribavirin in enhancing the antiviral effect of interferon alfa in chronic hepatitis C. q 2002 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. Keywords: Hepatitis C virus; Chronic hepatitis C; Interferon alfa-2a; Ribavirin; Mycophenolate mofetil; Therapy

1. Introduction Recent clinical trials have focussed on drugs that may enhance the efficacy of interferon alfa therapy such as ribavirin [1,2]. A large number of possible adjuncts have been evaluated, including ursodeoxycholic acid, nonsteroidal anti-inflammatory agents, thymosin alfa-1, histamine dihydrochloride, and amantadine. Most clinical trials were disappointing. Although some of these drugs may have an additional therapeutic effect, e.g. lead to transient reduction Received 3 April 2002; received in revised form 16 August 2002; accepted 19 August 2002 * Corresponding author. Tel.: 149-511-532-3305; fax: 149-511-5324896. E-mail address: [email protected] (M.P. Manns).

of biochemical markers, at present, none have been demonstrated to be superior to combination therapy of interferon alfa and ribavirin in respect to virological response [3–5]. Mycophenolate mofetil (MMF), a morpholinoethyl ester of mycophenolic acid (MPA), is currently used as an immunosuppressive agent. After oral administration, MMF is rapidly hydrolyzed to MPA, the active compound, which is a potent inhibitor of inosine 5 0 -monophosphate dehydrogenase (IMPDH) [6]. Interestingly, ribavirin also leads to an inhibition of the IMPDH, which had been shown in ascites tumor cells in vitro [7]. Moreover, it has been demonstrated that MMF is more effective than ribavirin or 5-ethynyl-1-bd-ribofuranosylimidazole-4-carboxamide (EICAR) in inhibiting the IMPDH [8]. IMPDH catalyses a key step in purine nucleotide biosynthesis. Therefore, inhibition of IMPDH

0168-8278/02/$20.00 q 2002 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved. PII: S 0168-827 8(02)00300-8

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results in depletion of the intracellular GTP and dGTP pools. Lack of guanosine nucleotides may lead to a reduction of de novo synthesis of viral RNA and DNA [9–13]. MPA has been shown to inhibit the replication of a number of viruses, including arena viruses (Junin and Tacaribe), reovirus-1, parainfluenza-3 virus, Coxsackie B4 virus, Epstein–Barr virus, human immunodeficiency virus, and the yellow fever virus [14–16]. Since the yellow fever virus belongs to the family of flaviviridae such as the hepatitis C virus (HCV), MMF might also display antiviral activity against HCV. In addition an inhibitory effect of MPA on HBV replication in primary human hepatocyte cultures was observed [8]. In a recent study in patients after liver transplantation, eight of 11 HCV positive patients who were switched from cyclosporine or FK506 to MMF showed a decline in HCV-RNA levels and a normalization of ALT levels [17]. Therefore, the aim of our study was to analyze the effect of combination therapy with interferon alfa-2a and MMF in patients with chronic hepatitis on HCV replication and safety. 2. Methods 2.1. Selection of patients Adult patients (18–65 years) with chronic hepatitis C infection were eligible for the study if they were virological nonresponders to a previous therapy with interferon alfa (HCV-RNA positive after at least 3 million units (MU) interferon tiw for 12 weeks). Nonresponse was further classified as flat nonresponse (,1 log reduction of HCV-RNA), flat partial response (.1 log ,2 log reduction of HCV-RNA), and partial response with a significant reduction of viral load (.2 log reduction of HCV-RNA). However, in a number of patients quantitative HCV-RNA levels of the previous therapy were not available, since this was not standard in former therapies. All patients were seropositive for HCV-RNA by testing with polymerase chain reaction by Roche Amplicor w and had elevated serum alanine aminotransferase activity (ALT). Patients were excluded if they had decompensated liver disease, liver diseases unrelated to HCV infection, anemia (hemoglobin concentration less than 12 g/dl for women and less than 13 g/dl for men), leukocytopenia (less than 3000/ml), thrombocytopenia (less than 100,000/ml), decompensated renal disease (serum creatinine above 130 mmol/l), decompensated thyroid disease, HIV or hepatitis B infection, psychiatric conditions, a history of seizures, poorly controlled autoimmune diseases, or previous organ transplantation. Ongoing intravenous drug (IVDA) or alcohol abuse were exclusion criteria. Patients were excluded if they were not abstinent for at least 12 months.

2.2. Study design Patients received 6 MU interferon alfa-2a (Roferon w, Hoffmann-La Roche AG) subcutaneously thrice weekly for 24 weeks. In addition the patients were treated with MMF (CellCept w, Hoffmann-La Roche AG) orally. The first week patients received 500 mg/day MMF, followed by 1 g/day for another week and 2 g/day for the last 22 weeks. We decided to use the high dose of 6 MU interferon alfa three times a week (tiw), because in 1999 the European Consensus was to use a higher dose of interferon alfa in interferon monotherapy compared to recommendations in the USA. The dose of MMF was chosen according to current standard immunosuppression protocols. The patients were evaluated as outpatients at weeks 0, 1, 2, 4, 12, 18, 24 during and 24 weeks after the end of therapy. Biochemical and hematological testing was performed by the laboratory of each center.

Serum HCV-RNA levels (copies/ml) were determined at each center with the Cobas Amplicor Hepatitis C Monitor Test (v2.0, Roche Diagnostics). Viral genotypes were determined at each site with the INNO-LiPA HCV II Kit (Innogenetics, Heiden). For central analysis serum aliquots were stored at 280 8C. This protocol was approved by ethics committees at the Medical School of Hannover, the University of Kiel, the University of Frankfurt, and the University of Berlin, and was conducted according to principles of the Helsinki Declaration. Written informed consent was obtained from all participants.

2.3. Assessment of efficacy The primary end point was sustained virologic response, defined as undetectable HCV-RNA in serum 24 weeks after the end of treatment. Secondary end points were absence of serum HCV-RNA at week 12, and at the end of therapy, and normalization of serum ALT. Therapy was stopped if the HCV-RNA was detectable at week 12 of treatment. In order to get an early estimation of the risk vs. benefit ratio in this pilot study, we performed an interim analysis. This analysis was done after the first 29 patients had been treated for 12 weeks. Since we observed no significant reduction in HCV-RNA levels in the first 29 patients who completed 12 weeks of treatment we decided to discontinue the study for ethical reasons. Therapy was stopped in all patients who were HCV-RNA positive. Patients who were negative for HCV-RNA were considered for treatment with the combination therapy of 6 MU interferon alfa-2a tiw and ribavirin (1000/1200 mg qd) for an additional 36 weeks.

2.4. Statistical analysis The biostatistical analysis was performed by WiSP GmbH, Langenfeld, Germany. According to the explorative nature of this pilot study, mainly descriptive statistical methods were used, giving rates, medians, quartiles and ranges. Within-group comparisons of viral load and blood counts Table 1 Baseline characteristics of the patients a N ¼ 38 (%) N ¼ 29 (%) Male patients Age (years ^ SD) HCV-genotype distribution HCV-genotype 1 HCV-genotype 2/3 HCV-genotype 4 HCV-genotype not available Mode of transmission Blood products/transfusion I.v. drug abuse Others Liver histology Fibrosis Transition to liver cirrhosis Liver cirrhosis Previous therapy Flat nonresponse (,1 log reduction of HCV-RNA) Flat partial response (.1 log ,2 log reduction of HCV-RNA) Significant HCV-RNA reduction (.2 log reduction of HCV-RNA) Only qualitative HCV-RNA available

30 (79) 26 (90) 44.5 ^ 11.8 42.0 ^ 11.1 30 (79) 2 (5) 2 (5) 4 (11)

34 (83) 0 (0) 2 (7) 3 (10)

14 (37) 4 (11) 20 (53)

11 (38) 3 (10) 15 (52)

20 (53) 3 (8) 6 (16)

12 (41) 3 (10) 6 (21)

20 (53)

19 (66)

8 (21)

6 (21)

2 (5)

0 (0)

8 (21)

4 (14)

a Thirty-eight patients were enrolled to receive interferon alfa and MMF. An interim analysis of 29 patients after 12 weeks of therapy showed inefficacy. Thereafter therapy was stopped in all patients.

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Fig. 1. HCV-RNA levels at treatment week (TW) 0, TW4, TW8, and TW12 of each of the 29 patient who had been treated for 12 weeks. Only patients 8 and 10 demonstrated a .2 log reduction of HCV-RNA and became HCV-RNA negative during 12 weeks of therapy, while only patient 10 remained HCV-RNA negative until TW12. Quantitative HCV-RNA levels at TW4 and TW8 are not available from four and two patients, respectively. before and after study treatment were performed using an exact version of the Wilcoxon–Pratt test for paired data. All P values reported were twosided.

3. Results 3.1. Patients Thirty-eight patients fulfilled inclusion criteria and were treated at four different sites (Hannover, Kiel, Frankfurt, and Berlin) from August 1999 until March 2000. All patients were Caucasian. The baseline characteristics are shown in Table 1. After 12 weeks of therapy an interim analysis of 29 patients was performed.

these patients had a .2 log viral decline and became HCVRNA negative (Fig. 1). However, only one patient remained HCV-RNA negative until week 12. Overall, 97% (28/29) of the patients had no .2 log reduction of HCV-RNA and were still HCV-RNA positive at treatment week 12 (Fig. 1, see Table 2). Due to inefficacy the trial was discontinued. The patient who was HCV-RNA negative at week 12 was changed to combination therapy with ribavirin (1200 mg qd). However, after 20 weeks of therapy a breakthrough occurred. When the trial was discontinued, nine additional patients had entered the study but were treated for only 2–8 weeks. None of these nine patients were HCV-RNA negative at this time point. In all nine patients therapy was stopped, and none were further treated with, for example, combination therapy of interferon alfa and ribavirin.

3.2. Efficacy 3.3. Safety Thirty-eight patients were enrolled to receive therapy with interferon alfa and MMF. After 12 weeks of therapy an interim analysis of 29 patients showed inefficacy of therapy. During the initial 12 week treatment phase three patients showed a .1 log reduction of HCV-RNA, while two of

Therapy was well tolerated and the spectrum of side effects was comparable to previous trials with interferon alfa monotherapy. There were no serious adverse events during therapy. We noted no increase of symptoms such

Table 2 HCV-RNA viral load during therapy ( £ 1000 copies/ml) a

Median Quartile Range a

TW0

TW1

TW2

TW3

TW4

TW8

TW12

1000 756.8–1742.5 123–10000

1000 527.5–1907.5 176–8470

787 472–2560 39–7550

723 378–1550 17–7930

677.5 311.2–1090 4–6050

806 458.5–2795 0–13000

1000 586.5–2630 0–8930

Interim analysis from 29 patients. P ¼ 0:78 (treatment week (TW) 0 vs. TW12, Wilcoxon–Pratt test).

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M. Cornberg et al. / Journal of Hepatology 37 (2002) 843–847

Table 3 White blood cells during therapy (/nl) a

Median Quartile Range a

TW0

TW1

TW2

TW3

TW4

TW8

TW12

5.7 4.8–7 3.1–12.8

4.6 3.5–6.2 2–12.1

4.6 3.6–5.8 2–7.9

4.6 3.2–5.7 2.3–9.2

4.2 3.2–5.6 1.9–7.6

4.6 3.3–5.8 2.1–11.9

4.6 3.5–5.4 2.5–11.4

Interim analysis from 29 patients. P , 0:0001 (TW0 vs. TW12, Wilcoxon–Pratt test).

as diarrhea, nausea, myalgia, and headache during the MMF treatment period. No signs of diminished liver function (as measured clinically and by coagulation function and serum albumin) were noted during therapy. During the course of therapy significant leukopenia (Table 3) and thrombocytopenia (Table 4) were observed. The leukopenia was not more severe compared to interferon alfa monotherapy.

4. Discussion The mechanism of action of ribavirin in combination with interferon alfa in patients with chronic hepatitis C remains unknown. Since MMF and ribavirin both display an effect on a key enzyme in nucleotide synthesis, it has been speculated that both substances may have similar antiviral effects [7,8]. Moreover, MMF displays in vitro antiviral activity to several viruses [14–16] and seems to reduce HCV viral load in patients after liver transplantation [17]. Therefore, we evaluated the potential efficacy of MMF in combination with interferon alfa in HCV positive interferon-nonresponder patients prospectively. An interim analysis revealed dramatically low response. Only one patient demonstrated a .2 log reduction in HCV-RNA after 12 weeks of treatment. Taking into account the low response data and the side effects of this therapy, we considered that it was clinically inappropriate to continue the study. Later it was shown that patients who did not become negative for HCV-RNA in serum or who did not show a 2 log reduction in HCV-RNA after 12 weeks of therapy with pegylated interferon alfa-2a and ribavirin had only a minimal chance to achieve a sustained response and were likely to be nonresponders [18]. Lack of HCV-RNA reduction appears to be an accurate predictor for nonresponse to interferon alfa-based therapies at an early stage. Thus, our study demonstrated that a combination of the potent IMPDH inhibitor MMF with interferon alfa-2a has no effect in patients with chronic hepatitis C who did not respond to previous interferon alfa

monotherapy. In contrast to observations in transplant patients with HCV infection who received MMF (without interferon alfa) for immunosuppression [17], no significant changes in HCV viremia was observed in this study, even with the combination of interferon alfa-2a. Even patients with a previous flat partial response demonstrated no significant HCV-RNA reduction. Although we treated only previous interferon-nonresponder patients, we would have expected a HCV-RNA decline if MMF displays antiviral activity against HCV. Thus, our data do not support evidence for direct antiviral activity of MMF against HCV. This is in agreement with a recent study of 14 HCV positive renal transplant patients who received cyclosporine as baseline therapy and MMF either in place of azathioprine or in addition to baseline therapy [19]. MMF therapy was even associated with a significant rise in HCV viremia after at least 1 year of therapy. We believe that the observed reduction of HCV-RNA in transplant patients after switching immunosuppression to MMF may be due to changes in the antiviral host responses directed against HCV. Cellular immune responses against HCV are detectable after liver transplantation [20], and the activity of hepatitis after liver transplantation is dependent on HCV-specific cellular immune responses [21]. MMF does not inhibit T-cell receptor-induced activation of virus-specific T-cells (such as cyclosporine or FK506), but modulates proliferation of Tcells. Thus, switching immunosuppression might lead to transient activation of antiviral T-cells and results in a decline of HCV-RNA. In contrast, when MMF is just added to cyclosporine (and not replaced), the viral load might not change or might even increase [19]. In summary, MMF in combination with interferon alfa is ineffective in nonresponder patients with chronic hepatitis C. The inhibition of IMPDH seems not to be the major mechanism of ribavirin in enhancing the antiviral effect of interferon alfa in chronic hepatitis C. However, sustained response data of ongoing studies with the combination of pegylated interferon alfa and MMF [22] and clinical data on

Table 4 Platelets during therapy (/nl) a

Median Quartile Range a

TW0

TW1

TW2

TW3

TW4

TW8

TW12

171 155–231 97–447

163 117.5–206.5 97–341

173.5 123.5–192.5 86–326

158 124–185 86–326

154 118.5–180 78–280

160 113–168 79–318

138 118–179 63–350

Interim analysis from 29 patients. P , 0:0001 (TW0 vs. TW12, Wilcoxon–Pratt test).

M. Cornberg et al. / Journal of Hepatology 37 (2002) 843–847

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