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Antiviral therapy of hepatitis C — present and future
S130
Journal of Hepatology, 1993; 17 (Suppl. 3): S130-SI36 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. 0168-8278/93/$06.00
HEPAT 01313
Antiviral therapy of hepatitis C -
present and future
Jay H. Hoofnagle, Adrian M. Di Bisceglie and Michiko Shindo Liver Diseases Section, Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, United States of America
The current recommendations for therapy of chronic hepatitis C are a 6-month course of alpha-interferon in doses of 3 million units 3 times weekly. Patients should have compensated chronic liver disease with elevations in serum aminotransferases, serologic evidence of hepatitis C virus (HCV) infection and chronic hepatitis by liver biopsy. At present, a long-term beneficial response to alpha-interferon occurs in only 10-25% of patients. The modest long-term response rate and the restricted recommendations for use of interferon leave several unresolved issues regarding therapy of this disease. Do patients with atypical, severe or advanced disease warrant therapy? What is the optimal dose and duration of treatment? How can one increase the response rate to interferon? How can one predict which patients are likely to benefit from therapy? Which patients are likely to relapse if therapy is stopped? Ultimately, what is needed to answer these issues are better techniques to assess HCV infection and monitor therapy as well as more effective and better-tolerated agents that can be used alone or in combination with alpha-interferon.
Key words: Chronic hepatitis; Cirrhosis; Hepatitis C virus; Controlled trials; Antiviral therapy; Interferon; Ribavirin; Polymerase chain reaction
Pilot studies (1) followed by multiple randomized controlled trials (2-10) have documented that alphainterferon is effective in suppressing disease activity and inducing remissions in disease in a high proportion of patients with chronic hepatitis C. Treatment with doses of 3-5 million units (MU) of alpha-interferon subcutaneously thrice weekly has been associated with a serum biochemical response in 40-70% of patients and a longterm, sustained improvement in aminotransferases in 10-25% of patients. In several trials, this improvement in serum biochemical tests has been shown to correlate well with improvements in liver histology and function (3-6). More recently, retrospective analyses have shown that improvements in clinical features of disease are associated with a decrease or loss of serum hepatitis C virus (HCV) RNA from the serum and liver (1 I). These studies have provided the basis for the licensing and approval of alpha-interferon as therapy for chronic hepatitis C in most countries of the world. These initial controlled trials provided the basis for current recommendations for the use of alpha-interferon as therapy of chronic hepatitis C. Treatment is recommended for patients with compensated chronic hepati-
tis C if serum aminotransferases are at least 1.3 times the upper limit of normal and liver biopsy demonstrates chronic hepatitis disease activity. Interferon should be given at a dose of 3 MU subcutaneously thrice weekly for 6 months. If improvement in aminotransferases has not occurred after 2-3 months of treatment, therapy can be discontinued early. The dose of interferon can be increased to 5 MU thrice weekly if a partial response occurs. However, side effects of interferon are common, and are often dose-limiting. Indeed, a sizeable proportion of patients receiving 3 MU of interferon will require a decrease in dose because of intractable fatigue, anxiety, depression, leukopenia or other side effects. This approach to treatment should lead to improvements in approximately 50% of patients and sustained responses in 10-25%.
Unresolved issues Although alpha-interferon is now considered standard therapy for chronic hepatitis C, there are several unresolved issues concerning its use. Some major issues are
Correspondence to. Jay H. Hoofnagle, M.D., National Institutes of Health, Building 31, Room 9A23, Bethesda, MD 20892, U.S.A.
ANTIVIRAL THERAPY OF HEPATITIS C TABLE I Therapy in chronic hepatitis C: unresolved issues How to treat patients with atypical or severe disease? What is the optimal dose and duration of therapy? What are the predictors of a response to therapy? What are the predictors of a relapse after therapy? How can one increase the response rate?
TABLE 2 Alpha-interferon therapy in chronic hepatitis C: atypical patients Decompensated cirrhosis Children Atypical serology (anti-HCV-negative) Immunocompromised patients Organ transplant patients
(Table I): (1) How and whether to treat patients with atypical or complicated disease? (2) What is the optimal dose and duration of alpha-interferon therapy? (3) How can one predict which patients are likely to have a beneficial response to therapy? (4) How can one identify whether a relapse is likely to occur when therapy is stopped? (5) How can one increase t'he response rate?
Atypical patients Alpha-interferon is recommended largely for patients with well-compensated chronic hepatitis C who have serologic evidence of disease (anti-HCV or HCV-RNA), elevations in serum aminotransferase activities, chronic hepatitis by liver biopsy, and no other serious complicating illness. Not included in these recommendations are patients with clinically apparent cirrhosis, children, patients with atypical serologic patterns and patients who are immunocompromised either because of an immune deficiency or immunosuppressive therapy (Table 2). Patients with decompensated cirrhosis due to hepatitis C represent an important group that warrants some form of therapy short of liver transplantation. There have been no prospective randomized studies of alphainterferon in patients with advanced cirrhosis due to chronic hepatitis C, but anecdotal reports indicate that a proportion of such patients responds to treatment with improvements in aminotransferases and liver histology (12). However, the response rate to alpha-interferon is somewhat lower in patients with cirrhosis (5) and the improvements induced by therapy are difficult to sustain. A prospective, controlled trial of alpha-interferon in patients with early or mildly decompensated liver disease (Child's Class A or B) is needed. Chronic hepatitis C is rare in childhood. As a result
S131 there have been no controlled trials of alpha-interferon treatment in children. There is no reason to believe that children will respond differently to interferon from adults. Indeed, children tolerate interferon very well. Consequently, interferon therapy is indicated in children with chronic hepatitis C. Therapy of patients with suspected chronic hepatitis C and atypical serologic markers can be problematic. At least 80% of patients with chronic hepatitis C will have anti-HCV in serum using a first-generation assay and the remainder will have HCV-RNA in serum (I 1,13,14). A higher percentage will be reactive using a secondgeneration anti-HCV test (15). Unfortunately, secondgeneration anti-HCV tests and assays for HCV-RNA are not commercially available in all areas of the world. At present, most patients are diagnosed as having chronic hepatitis C based upon the finding of anti-HCV in serum by a first-generation ELISA along with elevations in serum aminotransferases and liver histology compatible with chronic hepatitis. However, this approach is not without shortcomings. Some patients with chronic hepatitis C will be non-reactive for antiHCV (false negatives) (11,13). In addition,.some patients with other forms of liver disease (autoimmune hepatitis or alcoholic liver disease) will test positive for anti-HCV by ELISA due to a non-specific reaction (false positives) (16,17). The history of an exposure to blood or blood products (transfusion, drug addiction, medical care exposure) is helpful in verifying the diagnosis of hepatitis C, but these features are not always present in the clinical history and are not always reliable. These difficulties become important because there is evidence that autoimmune chronic active hepatitis will worsen with alphainterferon therapy (18). For this reason, additional efforts should be made to exclude patients with autoimmune features of disease before using alpha-interferon, particularly if HCV-RNA testing is not available and if there is no history of exposure, or if clinical features of the disease are atypical. Autoimmune hepatitis should be considered in young patients, particularly women, if the disease is severe or there is no exposure history. In any case, alpha-interferon should be withdrawn rapidly if there is worsening of serum aminotransferases with alpha-interferon therapy (18,19). Severe worsening of disease and even fatalities due to exacerbation of the underlying hepatitis have been reported during alphainterferon therapy of hepatitis C. Another group of problematic patients are those who are immunocompromised, such as patients who have human immunodeficiency virus (HIV) infection, who have renal failure, who have received an organ transplant, or who are receiving immunosuppressive
S132
therapy. There are no reports available concerning treatment of such patients. Anecdotal results suggest that, in contrast to immunocompromised patients with chronic hepatitis B, those with chronic hepatitis C may benefit from alpha-interferon therapy (12). The role of interferon therapy in this group deserves further evaluation, particularly in view of new information suggesting that chronic hepatitis C can be severe following organ transplantation (20,21). A final group of patients in whom studies of therapy are needed are those with acute hepatitis C. Preliminary, small trials of alpha- and beta-interferon in acute hepatitis C have indicated that early treatment may prevent chronicity (22-24). In most of these studies, interferon was given for 6-12 weeks and patients were followed for the development of chronic hepatitis C for up to 1 year after treatment. In all 3 studies reported to date, the interferon-treated patients have demonstrated a more rapid decrease of aminotransferase levels into the normal range and a lower rate of chronicity at 6 months and 1 year. Overall, however, the decrease in chronicity with interferon treatment has been limited. Obviously, larger controlled trials with longer periods of follow-up evaluation are needed. At present alpha-interferon should not be used in acute hepatitis C outside of a controlled trial.
J.H. H O O F N A G L E et al.
100%
80%
60% 48%
o cL n.-
50%
40% 28":, 20%
6 months (21) Alpha
12 months (18) Interferon
(20) Placebo Di Bisceglie et a11990
Fig. I. Overall response rate a m o n g 41 patients with chronic hepatitis C who were entered into a randomized, controlled trial of alphainterferon and who were gwen interferon (2 MU) 01=21) or placebo (n = 20) thrice weekly for 6 months. Eighteen of the placebo recipients were then crossed over to receive interferon (2-5 MU) thnce weekly for 12 months. The upper bars show the overall response rate, the lower bars the sustained response rate associated with each treatment.
6-month course of treatment. The dose should be increased only if the response is partial.
Dose and duration of therapy The currently recommended regimen of alphainterferon for chronic hepatitis C is 3 MU subcutaneously thrice weekly for 6 months. However, this dose may not be high enough for some patients, and a 6-month course may be suboptimal in reliably inducing a sustained response. Several ongoing trials are comparing regimens of 3, 5 and 10 MU and courses of 6 and 12 months. In a randomized, placebo-controlled trial conducted at the National Institutes of Health, patients received either alpha-interferon (2 MU) or placebo thrice weekly for 6 months (4). Afterwards patients who received placebo were crossed over to receive interferon for up to 12 months in doses of 2-5 M U thrice weekly depending upon response in aminotransferase levels and tolerance. While the overall response rate was similar with both regimens, the sustained response rate was higher with the 12-month course of treatment (Fig. 1). Thus, at present, the optimal dose and duration of therapy are unclear. There also may be variation between patients in response to different doses. Until prospective, randomized controlled trials demonstrate a benefit for higher doses of alpha-interferon or for longer courses of therapy, one must recommend a dose of 3 MU and a
Predictors of response Only 50% of patients with chronic hepatitis C respond to alpha-interferon therapy. It would be very helpful if there were clinical or serologic features that would predict which patients were likely to respond to therapy and which were not. Unfortunately, retrospective analyses from the controlled trials of interferon have failed to identify any clinical, serum biochemical, serological or histological feature of disease that reliably predicted a response to treatment (3-6,25). Importantly, the presence or titers of anti-HCV or HCV-RNA in serum did not identify patients who were likely to have a response to interferon (11). These findings were in contrast to hepatitis B, where the height of serum aminotransferases and level of HBV-DNA are helpful in identifying patients who are likely to benefit from treatment (26). In some studies, patients who lacked anti-HCV and who had cirrhosis histologically were less likely to benefit from therapy (5). However, these correlations have been weak and cannot be used to decide on whether to use interferon or not.
ANTIVIRAL THERAPY OF HEPATITIS C Predictors of relapse
The optimal duration of therapy is not currently known. Different patients may require different lengths of treatment. What is needed is a means of monitoring treatment that would correctly identify when H C V has been cleared and a sustained clinical response could be expected. In this way, the duration of therapy would be based upon attainment of a complete response. This is the case in chronic hepatitis B, in which serological markers can identify when a sustained response is attained; the loss of H B V - D N A and HBeAg from serum indicates clearance of HBV replication (26). Several clinical, serum biochemical, serological and histological features have been analyzed as possible means of monitoring therapy in chronic hepatitis C (Table 3). Obviously, serum aminotransferase levels and liver histology, while correlating with a response to therapy, do not predict a sustained response. More recently, specific serologic markers have been analyzed for a possible role in monitoring treatment and identifying when a complete and lasting response in chronic hepatitis C has occurred. Most patients with chronic hepatitis C have anti-HCV in serum in titers ranging from 10° to 105. Retrospective analyses have shown that titers of anti-HCV as assessed by first-generation ELISA did not change in a consistent manner during alpha-interferon therapy (I 1,27). A preliminary report has suggested that IgM anti-HCV, which is frequently detectable in patients with chronic hepatitis C, decreases and often disappears during treatment in patients who have a sustained response but not i n those without a response or with a transient response (27). If these findings are confirmed, IgM anti-HCV testing might play an important role in monitoring patients and deciding how long to continue treatment. The technique of polymerase chain reaction (PCR) is a very sensitive means of detecting specific nucleic acid sequences and has been used to detect and quantify H C V - R N A in serum (!1,13,14). Most patients with chronic hepatitis C have H C V - R N A in serum in titers ranging from I0 ° to 105. Testing of stored serum from
S 133 patients treated with alpha-interferon has shown that levels of this viral marker decrease on treatment and H C V - R N A becomes undetectable in most patients with a beneficial response to treatment (Fig. 2). Levels decrease only slightly or not at all in patients with no or only a partial response. However, H C V - R N A reappears in patients with a relapse after therapy is stopped and loss of HCV-RNA, while correlating well with a
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Fig. 2. Serum levels of HCV-RNA in 39 patients who entered a randomized placebo controlled trial of alpha-interferon in chronic hepatitis C. HCV-RNA fell to undetectable levels in most patients who responded to treatment but in only rare patients without a response and in no patients who received placebo. HC
-1 0
2
4
6
8
10 12 Months
14
16
18
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Fig. 3. Course of a patient with chronic hepatitis C treated with alpha-
interferon. Serum ALT levels fell into the normal range and serum HCV-RNA became undetectable and remained undetectable even when therapy was stopped. HC
Tk,BLE 3 Alpha-interferon therapy in chronic hepatitis C: potential means for monitoring therapy Serum aminotransferase levels Liver histology Antibody to HCV IgM antibody to HCV HCV-RNA in serum HCV-RNA in liver HCV antigens in liver
-1 0
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6
8
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Fig. 4. Course of a patient with chronic hepatitis C treated with alphainterferon. Serum ALT levels fell into the normal range and serum HCV-RNA became undetectable. However, a relapse of disease and reappearance of serum HCV-RNA occurred when treatment was stopped.
S134 response to alpha-interferon treatment, does not predict a sustained response (Figs. 3 and 4). Nevertheless, these findings provide a virologic basis for assessing the efficacy of alpha-interferon in chronic hepatitis C. The major effect of interferon appears to be inhibition of viral replication, and thus long-term responses occur when there has been sustained clearance of virus. Obviously, better markers are needed to assess and monitor therapy and to provide guidance in when to initiate and when to stop therapy. Further assessments of serum antibody responses and evaluation of HCVRNA and HCV antigens in liver tissue (28) may yet provide the serologic tools to place therapy of hepatitis C on a more rational basis.
How to increase the response rate
While alpha-interferon has provided the first effective treatment for chronic hepatitis C, this treatment is effective in only 50% of patients and a long-term response occurs in only 10-25% of patients. Furthermore, interferon is difficult to administer, is quite expensive, and has multiple side effects which can be dose-limiting. Better tolerated and more effective therapies are needed. Use of repeated courses of alpha-interferon in patients with partial or temporary responses has been only partially beneficial. Characterization of the genome of HCV suggests that it is a flavi- or pesti-like virus. A tissue culture or simple animal model of hepatitis C would be extremely helpful in further characterizing this virus and to screen for antiviral agents of potential use. Until there is such an in vitro or in vivo method for monitoring HCV replication, however, antiviral studies will have to be done in human patients with this disease. For this reason, the majority of agents that have been used in chronic hepatitis C are those that have been previously evaluated for other conditions in man (Table 4). The agent that currently demonstrates the most promise is ribavirin. Ribavirin (1-fl-D-ribofuranosyl 1,2,4-triazole-3-carboxamide) is a guanosine analogue that has been evaluated extensively as therapy of respiratory syncytial virus and HIV infection in man (29). Ribavirin is especially attractive as it is absorbed orally and is well tolerated, its only significant side effect being a dose-related hemolysis that is generally mild, subclinical, and rapidly reversible when therapy is stopped. Ribavirin was initially tried in chronic hepatitis C based upon its safe clinical profile and its known activity against many RNA viruses including some flaviviruses. Weiland and co-workers from Sweden treated 10
J.H. HOOFNAGLE et al. TABLE 4 Therapy of chronic hepatitis C: potential agents Antivirals
Ribavirin, acyclovir, adenine arabinoside, foscarnet, dideoxynucleosides, azathymidine, gangiclovir, suramin Biologic response modifiers
Alpha-, beta- and gamma-interferon, interleukins 2, 4 and 6, colony-stimulating factors, tumor necrosis factor hnmunomodulat ors
Prednisone, thymosin, levamisole
patients with chronic hepatitis C with ribavirin for 12 weeks in doses of 1000-1200 mg per day (30). Serum aminotransferases decreased by 60% during therapy but rose to pre-treatment values soon after ribavirin was stopped. Changes in liver histology and levels of HCVRNA or anti-HCV were not reported. At the National Institutes of Health, we have completed a pilot study of oral ribavirin therapy in 13 patients with chronic hepatitis C (31). Ribavirin was administered in gradually escalating doses for 6 months. Serum aminotransferase levels fell in all 13 patients and became normal in 4 (28%). The average decrease in aminotransferases was 67%. However, even after 6 months of treatment, liver biopsies demonstrated no consistent improvement in degree of inflammation or hepatocellular necrosis. When ribavirin was stopped, serum aminotransferases rose to pre-treatment levels in most, but not all patients. On the other hand, ribavirin was well-tolerated even at the highest doses (1200 mg/d) and the only side effect noted was a mild, clinically silent, hemolytic anemia. Interesting differences were noted between the clinical and serologic responses in patients treated with ribavirin versus those treated with alpha-interferon. The serum aminotransferases fell much more slowly with ribavirin than with interferon therapy. However, the ultimate level of improvement in aminotransferases was the same with both drugs: with both there was a 60-70% decrease from pre-treatment levels (3,31). Another difference was that aminotransferase levels decreased in all patients treated with ribavirin but in only 50-70% of those treated with interferon. This fact underscores the peculiar variability in response to alpha-interferon therapy. Finally, serum levels of HCV-RNA decreased markedly in all patients who responded to alpha-interferon therapy (i 1); in contrast, HCV-RNA levels decreased only slightly during ribavirin therapy (3 I). The encouraging preliminary observations on the effects of ribavirin therapy in chronic hepatitis C have
ANTIVIRAL THERAPY OF HEPATITIS C
led to a prospective, randomized, double-blind controlled trial of ribavirin (1200 mg/d) versus placebo for 12 months that is being carried out at the National Institutes of Health (Fig. 5). This trial began in November 1991 and will include 50-60 patients entered over a 6-month period. Meanwhile, more studies are needed on the potential combination of ribavirin with alphainterferon as well as dose-finding studies based upon response of aminotransferases to ribavirin therapy.
S135
Liver Biopsy
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hepatitis C.
The future of therapy of hepatitis C Future research into therapy for hepatitis C will focus on more agents than alpha-interferon and ribavirin (Table 4) and on a broader spectrum of this disease. It is possible that multiple antiviral agents with activity against HCV will be identified. Indeed, initial results suggest that this virus is quite sensiti.ve to inhibition and that lowering the level of virus leads to amelioration of disease. In addition to a search for more agents to treat this disease, further studies of alpha-interferon and riba-
virin therapy are needed. Particularly important are studies in patients with clinically apparent cirrhosis, in children, in patients with unusual forms of the disease and immunocompromised patients. Thus, the future of antiviral therapy in chronic hepatitis C will help to fill in the gaps in knowledge about the correct use of alpha-interferon but will also focus on newer antiviral agents that alone or in combination with interferon promise to provide a relatively safe but highly effective therapy for all patients with livor disease due to hepatitis C.
References I Hoofnagle JH, Mullen KD, Jones DB, et al. Treatment of chronic non-A, non-B hepatitis with recombinant human alpha interferon: a preliminary report. N Engl J Mad 1987; 315: 1575-8. 2 Jacyna MR, Brooks MG, Loke RHT, Main J, Murray-Lyon IM, Thomas HC. Randomised controlled trial of interferon alfa (lymphoblastoid interferon) in chronic non-A, non-B hepatitis. Br Mad J 1989; 298: 80-2. 3 Davis GL, Balart LA, Schiff ER, et al. Treatment of chronic hepatitis C with recombinant interferon alfa: a multicenter randomized, controlled trial. N Engl J Mad 1989; 321: 1501-6. 4 Di Bisceglie AM, Martin P, Kassianides C, et al. Recombinant interferon alfa therapy for chronic hepatitis C: a randomized, double-blind, placebo-controlled trial. N Engl J Med 1989; 321: 1506-10. 5 Marcellin P, Boyer N, Giostra E, et al. Recombinant human alphainterferon in patients with chronic non-A, non-B hepatitis: a multicenter randomized controlled trial from France. Hepatology 1991; 13: 393-7. 6 Saracco G, Rosina F, Torrani Cerenzia MR, et al. A randomized controlled trial of interferon alfa-2b as therapy for chronic non-A, non-B hepatitis. J Hepatol 1990; I h $43-9. 7 Ferenci P, Vogel W, Pristautz H, et al. One-year treatment of chronic non-A, non-B hepatitis with interferon alfa-2b. J Hepatol 1990; 11: $50-6. 8 Weiland O, Schvarcz R, Wejstal R, Norkrans G, Fryden A. Therapy of chronic post-transfusion non-A, non-B hepatitis with interferon alfa-2b: Swedish experience. J Hepatol 1990; 1I: $57-62. 9 Gomez-Rubio M, Porres JC, Castillo I, Quiroga JA, Moreno A, Carrefio V. Prolonged treatment (I 8 months) of chronic hepatitis C with recombinant alpha-interferon in comparison with a control group. J Hepatol 1990; lh $63-7. 10 Realdi G, Diodata G, Bonetti P, Scaccabarozzi S, Alberti A,
11
12
13 14
15
16
17
18
19
20
Ruol A. Recombinant human interferon alfa-2a in communityacquired non-A, non-B chronic active hepatitis: preliminary results of a randomized, controlled trial. J Hepatol 1990; lh $68-71. Shindo M, Di Bisceglie AM, Cheung L, Shih W-K, Cristiano K, Feinstone SM, Hoofnagle JH. Decrease in serum hepatitis C viral RNA during alpha-interferon therapy for chronic hepatitis C. Ann Intern Med 1991; 115: 700-4. Martin P, Di Bisceglie AM, Kassianides C, Lisker-Melman M, Hoofnagle JH. Rapidly progressive non-A, non-B hepatitis in patients with human immunodeficiency virus infection. Gastroenterology 1989; 97: 1559-61. Weiner AJ, Kuo G, Bradley DW, et al. Detection of hepatitis C viral sequences in non-A, non-B hepatitis. Lancet 1990; 335: I-3. Farci P, Alter H J, Wong D, Miller RH, Shih JW, Gett B, Purcell RH. A long-term study of hepatitis C virus replication in non-A, non-B hepatitis. N Engl J Mad 1991: 325: 98-104. Van der Poel CL, Cuypers HTM, Reesink HW, et al. Confirmation of hepatitis C virus infection by new four-antigen recombinant immunoblot assay. Lalacet 1991; 337: 317-9. McFarlane IG, Smith HM, Johnson PJ, Bray GP, Vergani D, Williams R. Hepatitis C virus antibodies in chronic active hepatitis: pathogenic factor or false-positive result? Lancet 1990; 335: 754-7. Mendenhall CL, Seeff L, Diehl AM, et al. Antibodies to hepatitis B virus and hepatitis C virus in alcoholic hepatitis and cirrhosis: their prevalence and clinical relevance. Hepatology 1991; 14: 581-9. Papo T, Marcellin P, Bernuau J, Durand F, Poynard T, Benhamou J-P. Autoimmune chronic hepatitis exacerbated by alphainterferon. Ann Intern Mad 1992; 116: 51-3. Shindo M, Di Bisceglie AM, Hoofnagle JH. Acute exacerbation of liver disease during alpha interferon therapy for chronic hepatitis C. Gastroenterology 1992; 102: in press. Martin P, Munoz SJ, Di Bisceglie AM, et al. Recurrence of hepatitis C infection following orthotopic liver transplantation. Hepatology 1991; 13: 719-21.
S136 21 Pereira BJG, Milford EL, Kirkman RL, Levey AS. Transmission of hepatitis C virus by organ transplantation. N Engl J Med 1991; 325: 454-60. 22 Omata M, Yokosuka O, Takano S, et al. Resolution of acute hepatitis C after therapy with natural beta interferon. Lancet 1991; 338: 914-5. 23 Alberti A, Chemello L, Benvegnu L, Belussi F, Tagger A, Ruol A. Pilot study of interferon alpha-2a therapy in preventing chronic evoluation of acute hepatitis C. In: Hollinger FB, Lemon SM, Margolis H, Eds. Viral Hepatitis and Liver Disease. Baltimore: Williams and Wilkins, 1991; 656-8. 24 Viladomiu L, Gonzalez A, Lopez-Talavera JC, et al. A randomized controlled trial of alpha-interferon in acute posttransfusion C hepatitis (Abstract). J Hepatol 1990; I1: $64. 25 Lindsay KL, Davis GL, Bodenheimer HC, et al. Predictors of relapse and response to re-treatment in patients with an initial response to recombinant alpha interferon (rlFN) therapy for chronic hepatitis C (Abstract). Hepatology 1990; 12: 847.
J.H. HOOFNAGLE et al. 26 Perrillo RP, Schiff ER, Davis GL, et al. A randomized controlled trial of interferon alfa-2b alone and after prednisone withdrawal for the treatment of chronic hepatitis B. N Engl J Med 1990; 323: 295-301. 27 Brillianti S, Masci C, Ricci P, Paganelli GM, Miglioli M, Barbara L. Serum IgM antibody to HCV: a reliable marker of active hepatitis C (Abstract). Gastroenterology 1991; 100: A723. 28 Krawczynski K, Beach M J, Bradley DW, et al. Hepatitis C virus antigen in hepatocytes: immunomorphologic detection and identification. Gastroenterology 1992; 102: in press. 29 Gilbert BE, Knight V. Biochemistry and clinical applications of ribavirin. Antimicrob Agents Chemother 1986; 30: 200-5. 30 Reichard O, Andersson J, Schvarcz R, Weiland O. Ribavirin treatment for chronic hepatitis C. Lancet 1991; 337: 1058-61. 31 Di Bisceglie AM, Fong T-L, Fried MW, Swain MG, Bergasa NV, Shindo M, Hoofnagle JH. Ribavirin therapy for 6 months in patients with chronic hepatitis C (Abstract). Gastroenterology 1991; 100: A734.
Journal of Hepatology, 1993; 17 (Suppl. 3): S130-SI36 © 1993 Elsevier Scientific Publishers Ireland Ltd. All rights reserved. 0168-8278/93/$06.00
HEPAT 01313
Antiviral therapy of hepatitis C -
present and future
Jay H. Hoofnagle, Adrian M. Di Bisceglie and Michiko Shindo Liver Diseases Section, Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, United States of America
The current recommendations for therapy of chronic hepatitis C are a 6-month course of alpha-interferon in doses of 3 million units 3 times weekly. Patients should have compensated chronic liver disease with elevations in serum aminotransferases, serologic evidence of hepatitis C virus (HCV) infection and chronic hepatitis by liver biopsy. At present, a long-term beneficial response to alpha-interferon occurs in only 10-25% of patients. The modest long-term response rate and the restricted recommendations for use of interferon leave several unresolved issues regarding therapy of this disease. Do patients with atypical, severe or advanced disease warrant therapy? What is the optimal dose and duration of treatment? How can one increase the response rate to interferon? How can one predict which patients are likely to benefit from therapy? Which patients are likely to relapse if therapy is stopped? Ultimately, what is needed to answer these issues are better techniques to assess HCV infection and monitor therapy as well as more effective and better-tolerated agents that can be used alone or in combination with alpha-interferon.
Key words: Chronic hepatitis; Cirrhosis; Hepatitis C virus; Controlled trials; Antiviral therapy; Interferon; Ribavirin; Polymerase chain reaction
Pilot studies (1) followed by multiple randomized controlled trials (2-10) have documented that alphainterferon is effective in suppressing disease activity and inducing remissions in disease in a high proportion of patients with chronic hepatitis C. Treatment with doses of 3-5 million units (MU) of alpha-interferon subcutaneously thrice weekly has been associated with a serum biochemical response in 40-70% of patients and a longterm, sustained improvement in aminotransferases in 10-25% of patients. In several trials, this improvement in serum biochemical tests has been shown to correlate well with improvements in liver histology and function (3-6). More recently, retrospective analyses have shown that improvements in clinical features of disease are associated with a decrease or loss of serum hepatitis C virus (HCV) RNA from the serum and liver (1 I). These studies have provided the basis for the licensing and approval of alpha-interferon as therapy for chronic hepatitis C in most countries of the world. These initial controlled trials provided the basis for current recommendations for the use of alpha-interferon as therapy of chronic hepatitis C. Treatment is recommended for patients with compensated chronic hepati-
tis C if serum aminotransferases are at least 1.3 times the upper limit of normal and liver biopsy demonstrates chronic hepatitis disease activity. Interferon should be given at a dose of 3 MU subcutaneously thrice weekly for 6 months. If improvement in aminotransferases has not occurred after 2-3 months of treatment, therapy can be discontinued early. The dose of interferon can be increased to 5 MU thrice weekly if a partial response occurs. However, side effects of interferon are common, and are often dose-limiting. Indeed, a sizeable proportion of patients receiving 3 MU of interferon will require a decrease in dose because of intractable fatigue, anxiety, depression, leukopenia or other side effects. This approach to treatment should lead to improvements in approximately 50% of patients and sustained responses in 10-25%.
Unresolved issues Although alpha-interferon is now considered standard therapy for chronic hepatitis C, there are several unresolved issues concerning its use. Some major issues are
Correspondence to. Jay H. Hoofnagle, M.D., National Institutes of Health, Building 31, Room 9A23, Bethesda, MD 20892, U.S.A.
ANTIVIRAL THERAPY OF HEPATITIS C TABLE I Therapy in chronic hepatitis C: unresolved issues How to treat patients with atypical or severe disease? What is the optimal dose and duration of therapy? What are the predictors of a response to therapy? What are the predictors of a relapse after therapy? How can one increase the response rate?
TABLE 2 Alpha-interferon therapy in chronic hepatitis C: atypical patients Decompensated cirrhosis Children Atypical serology (anti-HCV-negative) Immunocompromised patients Organ transplant patients
(Table I): (1) How and whether to treat patients with atypical or complicated disease? (2) What is the optimal dose and duration of alpha-interferon therapy? (3) How can one predict which patients are likely to have a beneficial response to therapy? (4) How can one identify whether a relapse is likely to occur when therapy is stopped? (5) How can one increase t'he response rate?
Atypical patients Alpha-interferon is recommended largely for patients with well-compensated chronic hepatitis C who have serologic evidence of disease (anti-HCV or HCV-RNA), elevations in serum aminotransferase activities, chronic hepatitis by liver biopsy, and no other serious complicating illness. Not included in these recommendations are patients with clinically apparent cirrhosis, children, patients with atypical serologic patterns and patients who are immunocompromised either because of an immune deficiency or immunosuppressive therapy (Table 2). Patients with decompensated cirrhosis due to hepatitis C represent an important group that warrants some form of therapy short of liver transplantation. There have been no prospective randomized studies of alphainterferon in patients with advanced cirrhosis due to chronic hepatitis C, but anecdotal reports indicate that a proportion of such patients responds to treatment with improvements in aminotransferases and liver histology (12). However, the response rate to alpha-interferon is somewhat lower in patients with cirrhosis (5) and the improvements induced by therapy are difficult to sustain. A prospective, controlled trial of alpha-interferon in patients with early or mildly decompensated liver disease (Child's Class A or B) is needed. Chronic hepatitis C is rare in childhood. As a result
S131 there have been no controlled trials of alpha-interferon treatment in children. There is no reason to believe that children will respond differently to interferon from adults. Indeed, children tolerate interferon very well. Consequently, interferon therapy is indicated in children with chronic hepatitis C. Therapy of patients with suspected chronic hepatitis C and atypical serologic markers can be problematic. At least 80% of patients with chronic hepatitis C will have anti-HCV in serum using a first-generation assay and the remainder will have HCV-RNA in serum (I 1,13,14). A higher percentage will be reactive using a secondgeneration anti-HCV test (15). Unfortunately, secondgeneration anti-HCV tests and assays for HCV-RNA are not commercially available in all areas of the world. At present, most patients are diagnosed as having chronic hepatitis C based upon the finding of anti-HCV in serum by a first-generation ELISA along with elevations in serum aminotransferases and liver histology compatible with chronic hepatitis. However, this approach is not without shortcomings. Some patients with chronic hepatitis C will be non-reactive for antiHCV (false negatives) (11,13). In addition,.some patients with other forms of liver disease (autoimmune hepatitis or alcoholic liver disease) will test positive for anti-HCV by ELISA due to a non-specific reaction (false positives) (16,17). The history of an exposure to blood or blood products (transfusion, drug addiction, medical care exposure) is helpful in verifying the diagnosis of hepatitis C, but these features are not always present in the clinical history and are not always reliable. These difficulties become important because there is evidence that autoimmune chronic active hepatitis will worsen with alphainterferon therapy (18). For this reason, additional efforts should be made to exclude patients with autoimmune features of disease before using alpha-interferon, particularly if HCV-RNA testing is not available and if there is no history of exposure, or if clinical features of the disease are atypical. Autoimmune hepatitis should be considered in young patients, particularly women, if the disease is severe or there is no exposure history. In any case, alpha-interferon should be withdrawn rapidly if there is worsening of serum aminotransferases with alpha-interferon therapy (18,19). Severe worsening of disease and even fatalities due to exacerbation of the underlying hepatitis have been reported during alphainterferon therapy of hepatitis C. Another group of problematic patients are those who are immunocompromised, such as patients who have human immunodeficiency virus (HIV) infection, who have renal failure, who have received an organ transplant, or who are receiving immunosuppressive
S132
therapy. There are no reports available concerning treatment of such patients. Anecdotal results suggest that, in contrast to immunocompromised patients with chronic hepatitis B, those with chronic hepatitis C may benefit from alpha-interferon therapy (12). The role of interferon therapy in this group deserves further evaluation, particularly in view of new information suggesting that chronic hepatitis C can be severe following organ transplantation (20,21). A final group of patients in whom studies of therapy are needed are those with acute hepatitis C. Preliminary, small trials of alpha- and beta-interferon in acute hepatitis C have indicated that early treatment may prevent chronicity (22-24). In most of these studies, interferon was given for 6-12 weeks and patients were followed for the development of chronic hepatitis C for up to 1 year after treatment. In all 3 studies reported to date, the interferon-treated patients have demonstrated a more rapid decrease of aminotransferase levels into the normal range and a lower rate of chronicity at 6 months and 1 year. Overall, however, the decrease in chronicity with interferon treatment has been limited. Obviously, larger controlled trials with longer periods of follow-up evaluation are needed. At present alpha-interferon should not be used in acute hepatitis C outside of a controlled trial.
J.H. H O O F N A G L E et al.
100%
80%
60% 48%
o cL n.-
50%
40% 28":, 20%
6 months (21) Alpha
12 months (18) Interferon
(20) Placebo Di Bisceglie et a11990
Fig. I. Overall response rate a m o n g 41 patients with chronic hepatitis C who were entered into a randomized, controlled trial of alphainterferon and who were gwen interferon (2 MU) 01=21) or placebo (n = 20) thrice weekly for 6 months. Eighteen of the placebo recipients were then crossed over to receive interferon (2-5 MU) thnce weekly for 12 months. The upper bars show the overall response rate, the lower bars the sustained response rate associated with each treatment.
6-month course of treatment. The dose should be increased only if the response is partial.
Dose and duration of therapy The currently recommended regimen of alphainterferon for chronic hepatitis C is 3 MU subcutaneously thrice weekly for 6 months. However, this dose may not be high enough for some patients, and a 6-month course may be suboptimal in reliably inducing a sustained response. Several ongoing trials are comparing regimens of 3, 5 and 10 MU and courses of 6 and 12 months. In a randomized, placebo-controlled trial conducted at the National Institutes of Health, patients received either alpha-interferon (2 MU) or placebo thrice weekly for 6 months (4). Afterwards patients who received placebo were crossed over to receive interferon for up to 12 months in doses of 2-5 M U thrice weekly depending upon response in aminotransferase levels and tolerance. While the overall response rate was similar with both regimens, the sustained response rate was higher with the 12-month course of treatment (Fig. 1). Thus, at present, the optimal dose and duration of therapy are unclear. There also may be variation between patients in response to different doses. Until prospective, randomized controlled trials demonstrate a benefit for higher doses of alpha-interferon or for longer courses of therapy, one must recommend a dose of 3 MU and a
Predictors of response Only 50% of patients with chronic hepatitis C respond to alpha-interferon therapy. It would be very helpful if there were clinical or serologic features that would predict which patients were likely to respond to therapy and which were not. Unfortunately, retrospective analyses from the controlled trials of interferon have failed to identify any clinical, serum biochemical, serological or histological feature of disease that reliably predicted a response to treatment (3-6,25). Importantly, the presence or titers of anti-HCV or HCV-RNA in serum did not identify patients who were likely to have a response to interferon (11). These findings were in contrast to hepatitis B, where the height of serum aminotransferases and level of HBV-DNA are helpful in identifying patients who are likely to benefit from treatment (26). In some studies, patients who lacked anti-HCV and who had cirrhosis histologically were less likely to benefit from therapy (5). However, these correlations have been weak and cannot be used to decide on whether to use interferon or not.
ANTIVIRAL THERAPY OF HEPATITIS C Predictors of relapse
The optimal duration of therapy is not currently known. Different patients may require different lengths of treatment. What is needed is a means of monitoring treatment that would correctly identify when H C V has been cleared and a sustained clinical response could be expected. In this way, the duration of therapy would be based upon attainment of a complete response. This is the case in chronic hepatitis B, in which serological markers can identify when a sustained response is attained; the loss of H B V - D N A and HBeAg from serum indicates clearance of HBV replication (26). Several clinical, serum biochemical, serological and histological features have been analyzed as possible means of monitoring therapy in chronic hepatitis C (Table 3). Obviously, serum aminotransferase levels and liver histology, while correlating with a response to therapy, do not predict a sustained response. More recently, specific serologic markers have been analyzed for a possible role in monitoring treatment and identifying when a complete and lasting response in chronic hepatitis C has occurred. Most patients with chronic hepatitis C have anti-HCV in serum in titers ranging from 10° to 105. Retrospective analyses have shown that titers of anti-HCV as assessed by first-generation ELISA did not change in a consistent manner during alpha-interferon therapy (I 1,27). A preliminary report has suggested that IgM anti-HCV, which is frequently detectable in patients with chronic hepatitis C, decreases and often disappears during treatment in patients who have a sustained response but not i n those without a response or with a transient response (27). If these findings are confirmed, IgM anti-HCV testing might play an important role in monitoring patients and deciding how long to continue treatment. The technique of polymerase chain reaction (PCR) is a very sensitive means of detecting specific nucleic acid sequences and has been used to detect and quantify H C V - R N A in serum (!1,13,14). Most patients with chronic hepatitis C have H C V - R N A in serum in titers ranging from I0 ° to 105. Testing of stored serum from
S 133 patients treated with alpha-interferon has shown that levels of this viral marker decrease on treatment and H C V - R N A becomes undetectable in most patients with a beneficial response to treatment (Fig. 2). Levels decrease only slightly or not at all in patients with no or only a partial response. However, H C V - R N A reappears in patients with a relapse after therapy is stopped and loss of HCV-RNA, while correlating well with a
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Fig. 2. Serum levels of HCV-RNA in 39 patients who entered a randomized placebo controlled trial of alpha-interferon in chronic hepatitis C. HCV-RNA fell to undetectable levels in most patients who responded to treatment but in only rare patients without a response and in no patients who received placebo. HC
-1 0
2
4
6
8
10 12 Months
14
16
18
20
26
Fig. 3. Course of a patient with chronic hepatitis C treated with alpha-
interferon. Serum ALT levels fell into the normal range and serum HCV-RNA became undetectable and remained undetectable even when therapy was stopped. HC
Tk,BLE 3 Alpha-interferon therapy in chronic hepatitis C: potential means for monitoring therapy Serum aminotransferase levels Liver histology Antibody to HCV IgM antibody to HCV HCV-RNA in serum HCV-RNA in liver HCV antigens in liver
-1 0
2
4
6
8
10
12
Months
Fig. 4. Course of a patient with chronic hepatitis C treated with alphainterferon. Serum ALT levels fell into the normal range and serum HCV-RNA became undetectable. However, a relapse of disease and reappearance of serum HCV-RNA occurred when treatment was stopped.
S134 response to alpha-interferon treatment, does not predict a sustained response (Figs. 3 and 4). Nevertheless, these findings provide a virologic basis for assessing the efficacy of alpha-interferon in chronic hepatitis C. The major effect of interferon appears to be inhibition of viral replication, and thus long-term responses occur when there has been sustained clearance of virus. Obviously, better markers are needed to assess and monitor therapy and to provide guidance in when to initiate and when to stop therapy. Further assessments of serum antibody responses and evaluation of HCVRNA and HCV antigens in liver tissue (28) may yet provide the serologic tools to place therapy of hepatitis C on a more rational basis.
How to increase the response rate
While alpha-interferon has provided the first effective treatment for chronic hepatitis C, this treatment is effective in only 50% of patients and a long-term response occurs in only 10-25% of patients. Furthermore, interferon is difficult to administer, is quite expensive, and has multiple side effects which can be dose-limiting. Better tolerated and more effective therapies are needed. Use of repeated courses of alpha-interferon in patients with partial or temporary responses has been only partially beneficial. Characterization of the genome of HCV suggests that it is a flavi- or pesti-like virus. A tissue culture or simple animal model of hepatitis C would be extremely helpful in further characterizing this virus and to screen for antiviral agents of potential use. Until there is such an in vitro or in vivo method for monitoring HCV replication, however, antiviral studies will have to be done in human patients with this disease. For this reason, the majority of agents that have been used in chronic hepatitis C are those that have been previously evaluated for other conditions in man (Table 4). The agent that currently demonstrates the most promise is ribavirin. Ribavirin (1-fl-D-ribofuranosyl 1,2,4-triazole-3-carboxamide) is a guanosine analogue that has been evaluated extensively as therapy of respiratory syncytial virus and HIV infection in man (29). Ribavirin is especially attractive as it is absorbed orally and is well tolerated, its only significant side effect being a dose-related hemolysis that is generally mild, subclinical, and rapidly reversible when therapy is stopped. Ribavirin was initially tried in chronic hepatitis C based upon its safe clinical profile and its known activity against many RNA viruses including some flaviviruses. Weiland and co-workers from Sweden treated 10
J.H. HOOFNAGLE et al. TABLE 4 Therapy of chronic hepatitis C: potential agents Antivirals
Ribavirin, acyclovir, adenine arabinoside, foscarnet, dideoxynucleosides, azathymidine, gangiclovir, suramin Biologic response modifiers
Alpha-, beta- and gamma-interferon, interleukins 2, 4 and 6, colony-stimulating factors, tumor necrosis factor hnmunomodulat ors
Prednisone, thymosin, levamisole
patients with chronic hepatitis C with ribavirin for 12 weeks in doses of 1000-1200 mg per day (30). Serum aminotransferases decreased by 60% during therapy but rose to pre-treatment values soon after ribavirin was stopped. Changes in liver histology and levels of HCVRNA or anti-HCV were not reported. At the National Institutes of Health, we have completed a pilot study of oral ribavirin therapy in 13 patients with chronic hepatitis C (31). Ribavirin was administered in gradually escalating doses for 6 months. Serum aminotransferase levels fell in all 13 patients and became normal in 4 (28%). The average decrease in aminotransferases was 67%. However, even after 6 months of treatment, liver biopsies demonstrated no consistent improvement in degree of inflammation or hepatocellular necrosis. When ribavirin was stopped, serum aminotransferases rose to pre-treatment levels in most, but not all patients. On the other hand, ribavirin was well-tolerated even at the highest doses (1200 mg/d) and the only side effect noted was a mild, clinically silent, hemolytic anemia. Interesting differences were noted between the clinical and serologic responses in patients treated with ribavirin versus those treated with alpha-interferon. The serum aminotransferases fell much more slowly with ribavirin than with interferon therapy. However, the ultimate level of improvement in aminotransferases was the same with both drugs: with both there was a 60-70% decrease from pre-treatment levels (3,31). Another difference was that aminotransferase levels decreased in all patients treated with ribavirin but in only 50-70% of those treated with interferon. This fact underscores the peculiar variability in response to alpha-interferon therapy. Finally, serum levels of HCV-RNA decreased markedly in all patients who responded to alpha-interferon therapy (i 1); in contrast, HCV-RNA levels decreased only slightly during ribavirin therapy (3 I). The encouraging preliminary observations on the effects of ribavirin therapy in chronic hepatitis C have
ANTIVIRAL THERAPY OF HEPATITIS C
led to a prospective, randomized, double-blind controlled trial of ribavirin (1200 mg/d) versus placebo for 12 months that is being carried out at the National Institutes of Health (Fig. 5). This trial began in November 1991 and will include 50-60 patients entered over a 6-month period. Meanwhile, more studies are needed on the potential combination of ribavirin with alphainterferon as well as dose-finding studies based upon response of aminotransferases to ribavirin therapy.
S135
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-
I 0
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hepatitis C.
The future of therapy of hepatitis C Future research into therapy for hepatitis C will focus on more agents than alpha-interferon and ribavirin (Table 4) and on a broader spectrum of this disease. It is possible that multiple antiviral agents with activity against HCV will be identified. Indeed, initial results suggest that this virus is quite sensiti.ve to inhibition and that lowering the level of virus leads to amelioration of disease. In addition to a search for more agents to treat this disease, further studies of alpha-interferon and riba-
virin therapy are needed. Particularly important are studies in patients with clinically apparent cirrhosis, in children, in patients with unusual forms of the disease and immunocompromised patients. Thus, the future of antiviral therapy in chronic hepatitis C will help to fill in the gaps in knowledge about the correct use of alpha-interferon but will also focus on newer antiviral agents that alone or in combination with interferon promise to provide a relatively safe but highly effective therapy for all patients with livor disease due to hepatitis C.
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20
Ruol A. Recombinant human interferon alfa-2a in communityacquired non-A, non-B chronic active hepatitis: preliminary results of a randomized, controlled trial. J Hepatol 1990; lh $68-71. Shindo M, Di Bisceglie AM, Cheung L, Shih W-K, Cristiano K, Feinstone SM, Hoofnagle JH. Decrease in serum hepatitis C viral RNA during alpha-interferon therapy for chronic hepatitis C. Ann Intern Med 1991; 115: 700-4. Martin P, Di Bisceglie AM, Kassianides C, Lisker-Melman M, Hoofnagle JH. Rapidly progressive non-A, non-B hepatitis in patients with human immunodeficiency virus infection. Gastroenterology 1989; 97: 1559-61. Weiner AJ, Kuo G, Bradley DW, et al. Detection of hepatitis C viral sequences in non-A, non-B hepatitis. Lancet 1990; 335: I-3. Farci P, Alter H J, Wong D, Miller RH, Shih JW, Gett B, Purcell RH. A long-term study of hepatitis C virus replication in non-A, non-B hepatitis. N Engl J Mad 1991: 325: 98-104. Van der Poel CL, Cuypers HTM, Reesink HW, et al. Confirmation of hepatitis C virus infection by new four-antigen recombinant immunoblot assay. Lalacet 1991; 337: 317-9. McFarlane IG, Smith HM, Johnson PJ, Bray GP, Vergani D, Williams R. Hepatitis C virus antibodies in chronic active hepatitis: pathogenic factor or false-positive result? Lancet 1990; 335: 754-7. Mendenhall CL, Seeff L, Diehl AM, et al. Antibodies to hepatitis B virus and hepatitis C virus in alcoholic hepatitis and cirrhosis: their prevalence and clinical relevance. Hepatology 1991; 14: 581-9. Papo T, Marcellin P, Bernuau J, Durand F, Poynard T, Benhamou J-P. Autoimmune chronic hepatitis exacerbated by alphainterferon. Ann Intern Mad 1992; 116: 51-3. Shindo M, Di Bisceglie AM, Hoofnagle JH. Acute exacerbation of liver disease during alpha interferon therapy for chronic hepatitis C. Gastroenterology 1992; 102: in press. Martin P, Munoz SJ, Di Bisceglie AM, et al. Recurrence of hepatitis C infection following orthotopic liver transplantation. Hepatology 1991; 13: 719-21.
S136 21 Pereira BJG, Milford EL, Kirkman RL, Levey AS. Transmission of hepatitis C virus by organ transplantation. N Engl J Med 1991; 325: 454-60. 22 Omata M, Yokosuka O, Takano S, et al. Resolution of acute hepatitis C after therapy with natural beta interferon. Lancet 1991; 338: 914-5. 23 Alberti A, Chemello L, Benvegnu L, Belussi F, Tagger A, Ruol A. Pilot study of interferon alpha-2a therapy in preventing chronic evoluation of acute hepatitis C. In: Hollinger FB, Lemon SM, Margolis H, Eds. Viral Hepatitis and Liver Disease. Baltimore: Williams and Wilkins, 1991; 656-8. 24 Viladomiu L, Gonzalez A, Lopez-Talavera JC, et al. A randomized controlled trial of alpha-interferon in acute posttransfusion C hepatitis (Abstract). J Hepatol 1990; I1: $64. 25 Lindsay KL, Davis GL, Bodenheimer HC, et al. Predictors of relapse and response to re-treatment in patients with an initial response to recombinant alpha interferon (rlFN) therapy for chronic hepatitis C (Abstract). Hepatology 1990; 12: 847.
J.H. HOOFNAGLE et al. 26 Perrillo RP, Schiff ER, Davis GL, et al. A randomized controlled trial of interferon alfa-2b alone and after prednisone withdrawal for the treatment of chronic hepatitis B. N Engl J Med 1990; 323: 295-301. 27 Brillianti S, Masci C, Ricci P, Paganelli GM, Miglioli M, Barbara L. Serum IgM antibody to HCV: a reliable marker of active hepatitis C (Abstract). Gastroenterology 1991; 100: A723. 28 Krawczynski K, Beach M J, Bradley DW, et al. Hepatitis C virus antigen in hepatocytes: immunomorphologic detection and identification. Gastroenterology 1992; 102: in press. 29 Gilbert BE, Knight V. Biochemistry and clinical applications of ribavirin. Antimicrob Agents Chemother 1986; 30: 200-5. 30 Reichard O, Andersson J, Schvarcz R, Weiland O. Ribavirin treatment for chronic hepatitis C. Lancet 1991; 337: 1058-61. 31 Di Bisceglie AM, Fong T-L, Fried MW, Swain MG, Bergasa NV, Shindo M, Hoofnagle JH. Ribavirin therapy for 6 months in patients with chronic hepatitis C (Abstract). Gastroenterology 1991; 100: A734.