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Ribavirin treatment for patients with chronic hepatitis C: results of a placebo-controlled study
Journalof Hepatology1996;25: 591-598 Printed in Denmark All rightsreserved
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Copyright0 EuropeanAssociation for the Studv of the Liver 1996 Journalof Hepatology ISSN0168.8278
Ribavirin treatment for patients with chronic hepatitis C: results of a placebo-controlled study Geoffrey Dusheiko’, Janice Main2, Howard Thomas2, Olle Reichard3, Christine Lee”, Amar Dhillon’, Suhra Rassam’, Aril Fryden4, Henk Reesink’, Margaret Bassendine6, Gunnar Norkrans7, Theo Cuypers*, Nice Lelie’, Paul Telfer”, Jon Watson’, Christine Weegink’, Peter Sillikens”and Ola Weiland’ Departments of Medicine and Pathology ‘Royal Free Hospital, and ‘St. Mary’s HospitalLondon, 6Freeman Hospital, Newcastle; “Haemophilia Center Royal Free Hospital, United Kingdom; ‘Division of Infectious Diseases, Danderyd Hospital, ‘Huddinge Hospital, and Karolinska Institute, “University Hospital, Linkoping, ‘Ostra Hospital, Goteborg, Sweden; ‘Vereenigen Voor Ziekenverpleging, ‘CLB, Amsterdam, “Organon Teknika Laboratories, The Netherlands
Background/Aims:
Small, uncontrolled studies of ribavirin for patients with chronic hepatitis C have reported efficacy in chronic hepatitis C. We have evaluated the efficacy and safety of a 24week course of oral ribavirin in patients with chronic hepatitis C, compared to placebo. Methods: A total of 114 patients were randomised to ribavirin or placebo. Ribavirin was administered in doses of 1000 or 1200 mg/day for 24 weeks. Efficacy was determined in the intentionto-treat population: 76 received ribavirin and 38 placebo. Results: Ribavirin was significantly more effective than placebo in reducing and normalising serum ALT levels: 42/76 (55%) of ribavirin-treated patients vs 2/38 (5 %) placebo recipients had either normalisation of the ALT levels or a reduction from baseline of at least 50% (~~0.001). ALT levels were normal in 22/76 (29%) of ribavirintreated patients vs O/38 placebo recipients (pcO.001). Twenty-four weeks after stopping ribavirin, the majority of patients had abnormal ALT levels. There was no difference between the treatment groups in reduction or disappearance of HCV-RNA levels. HCV RNA disappeared during
treatment in 3% of ribavirin-treated patients and 3% of placebo recipients. More ribavirin than placebo patients showed improvement in total Knodell score (45% vs 31%), but these differences were not statistically significant. Analysis of each component of a histology activity index revealed no statistically significant differences between treatment groups. Ribavirin patients had fewer lymphoid aggregates than did placebo recipients at the post-treatment assessment (p=O.O5).Ribavirin was associated with reversible haemolytic anaemia: a fall in haemoglobin occurred in 3% of placebo- and 32% (25/78) of ribavirin-treated patients, respectively (pcO.001). Conclusions: These data indicate that ribavirin was no more effective than placebo in reducing or eliminating HCV-RNA levels, and was not significantly more effective than placebo in improving hepatic histology after 6 months of treatment. The role of a 6-month treatment of chronic hepatitis C with ribavirin alone, without a significant effect on HCV RNA, is therefore limited.
P
sidious. Interferon-a treatment results in sustained biochemical responses (normal serum aminotransferases) in 15%-20% of those treated for 6 months (4,5). As side effects are not uncommon in treated patients, other therapies are therefore being sought to improve the treatment of chronic hepatitis C. Ribavirin (1 -Beta-D-ribofuranosyl1 H- 1,2,4-triazole-3-carboxamide) is a water-soluble nucleoside
with hepatitis C infection frequently develop chronic hepatitis (l-3). This may progress to cirrhosis, although this progression is usually inATIENTS
Received 25 August 1995; revised and accepted 9 January 1996
Correspondence: Prof. G. Dusheiko, Viral Hepatitis Study Group, Royal Free Hospital, Pond Street, London NW3 2QG, UK. Tel:+44 171 830 2993. Fax. +44 1714314581.
Key words: Antivirals; Chronic hepatitis; Hepatitis C antiviral therapy; Ribavirin.
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et al.
analogue of guanosine with antiviral activity against DNA and RNA viruses (6). The mechanism of action is unknown, but the drug may decrease intracellular pools of guanosine triphosphate. and result in synthesis of RNA with abnormal 5’ cap structures. Alternatively the drug may have a direct suppressive effect on viral RNA polymerase activity. Previous uncontrolled studies of the effect of ribavirin in patients with chronic hepatitis C have noted that the drug reduced serum aminotransferases concentrations and, in one study, a significant decrease in geometric mean titre of HCV RNA in serum was reported (7-9). This potentially useful drug therefore necessitated further study. We have undertaken a phase III multicentre, multinational, randomised double-blinded placebo-controlled study of oral ribavirin therapy in subjects with chronic hepatitis C infection. The study involved nine centres. The aim of the study was to evaluate the efficacy of ribavirin in reducing serum ALT concentrations, HCV RNA levels, and improving histological status when given for 24 weeks, comparing the effect of ribavirin with placebo.
Patients and Methods One hundred and eighteen male and female patients ranging in age from 18-70, with confirmed chronic hepatitis C, were randomised to receive either ribavirin (80) or placebo (38). All patients had proven chronic hepatitis C and were anti-HCV positive by second-generation ELISA and by RIBA, and/or had evidence of HCV RNA in plasma by polymerase chain reaction (PCR). All previously treated patients had stopped interferon at least 24 weeks before. Eligible patients had to have: evidence of elevated serum ALT (at least 1.5 times upper limit of normal) on at least three occasions in the 6-month period prior to the study, or evidence of elevated ALT on at least two occasions in the previous 6 months, plus PCR evidence of HCV RNA in plasma, and a previous liver biopsy showing evidence of chronic hepatitis or (compensated) cirrhosis (except patients with bleeding disorders). The protocol stipulated that all patients should have a baseline liver biopsy, unless a biopsy had been performed in the 24 weeks prior to study entry, at least 24 weeks after cessation of any previous treatment with interferon. A biopsy performed within 12 months of study entry was accepted in the case of otherwise eligible patients who refused a baseline liver biopsy, provided that the serum ALT had been monitored on a regular basis in the interval between the biopsy and the study entry, and there was not more than a four-fold 592
difference between the mean of at least two ALT levels measured within 3 months of the biopsy and the mean of at least two levels during the 3 months prior to the study entry. Two investigators included patients with bleeding disorders, who were not biopsied. All patients gave written informed consent to the procedure, except at the Swedish centres, where verbal consent was the norm. The study was conducted in accordance with the Declaration of Helsinki. Patients who consumed more than 80 g/day (males) and 60 g/day (females) of alcohol were excluded, as were HBsAg or anti-HIV-positive patients, known active intravenous drug abusers, or patients with pre-existing anaemia (haemoglobin less than 10 g/dl) or a known history of haemolytic anaemia. Patients with a history of clinical gout or currently elevated serum uric acid, who had received interferon or immuno-suppressive therapy within the previous 6 months, or who had significant systemic illness other than liver disease, or with decompensated liver disease, or known evidence of other forms of liver disease, or who had autoantibody titres greater than 1: 100 were also excluded. Two patients did not receive treatment, and therefore the intention-to-treat population comprised 116 patients. The efficacy population (i.e. excluding those who did not have a baseline and follow-up liver biopsy) comprised 65 patients: 21 in the placebo group and 44 in the ribavirin group. There was no evidence of differences between the intention to treat and efficacy group, and there was also no centre effect or bias. Therefore the results of the intention to treat group have been reported. The patient characteristics are summarised in Table 1. Twenty-two patients in the intention-to-treat group had previously received interferon. An ALT, virological, or histological response was noted in 15, 7 and 8 of these, respectively. Study design Patients who fulfilled the entry criteria were randomly assigned to ribavirin or placebo in a ratio of 2: 1, respectively. Unequal randomisation was chosen in order to minimise exposure to placebo, for ethical reasons. The total sample size of 120 patients was based on the ability to detect a 50% ALT response rate in the active treatment group as compared to 25% in the placebo recipients (80% power, 5% significance level). The study medication, supplied as 200-mg capsules or identical placebo capsules, was given orally in two divided doses of 1000 mg/day for patients
Ribavirin treatment for chronic hepatitis C
weighing less than 75 kg, and 1200 mg/day for patients weighing more than 75 kg for 24 weeks. Patients were monitored regularly for 6 weeks prior to initiation of treatment. All patients were monitored for a further 24 weeks after cessation of therapy. Efficacy was assessed by monitoring of serum ALT levels and plasma HCV RNA levels, and by repeat liver biopsy at the end of treatment. Safety was assessed by collection of adverse event data and by performing periodic haematology and biochemistry tests. HCV RNA measurement Serum for HCV RNA concentrations was measured immediately before the start of treatment, at the end of the active treatment, and at the end of the posttreatment phase. Ten millilitres of blood was drawn into a vacuum blood tube containing EDTA, centrifuged within 2 h and the plasma transferred into sterile cryotubes and stored at -70°C. PCR measurement was specified initially, and after completion of the study, it was decided to run a signal amplification method (bDNA) on retained samples. 1) Semi-quantitative measurement. The samples were transported in dry ice to the Netherlands Red Cross Blood Transfusion Service for analysis at the end of the study. HCV RNA was detected using PCR and measured semi-quantitatively by endpoint dilution. For each RNA extract the endpoint dilution for HCV RNA detection was determined, and the mean of the duplicate endpoint titres was calculated. Serial four-fold dilutions of each nucleic acid extract in water containing 100 pg/ml poly A were prepared. 7.5 ~1 was tested in a cDNA-PCR assay, in which amplification products were detected by oligomer hybridisation in liquid phase with the oligo Alx89. To guarantee the validity of the measured endpoint titres and to exclude inter-assay variation in sensitivity, a lo4 dilution of a donor panel proficiency was included in every analysis series. Assay results were only included when this control sample scored positive with the same intensity of the hybridisation signal as in preceding series. 2) Signal amplijication: Samples collected and stored as above were assayed by a chemoluminescent assay measuring signal amplification, using the quantitative bDNA signal amplification assay (Quantiplex HCV RNA assay, Chiron Corporation, Emeryville CA USA) according to the manufacturer’s instructions. (10) This test is based upon specific hybridisation of virus RNA in the sample by synthetic oligonucleotides to the highly conserved 5’-untranslated region and core gene of HCV RNA immobilised on
the surface of a microwell plate. Synthetic bDNA amplifier molecules and multiple copies of an alkaline phosphatase-linked probe are hybridised to the immobilised complex. The complex is incubated with a chemoluminescent substrate and the light emission is measured, the signal being proportional to the level of target nucleic acid. The quantity of HCV RNA in the sample is determined from a standard curve. The detection limit of the test is 350000 HCV RNA equivalents per ml. HCV genotyping HCV genotyping was performed by NASBA amplification of the 5’-untranslated region with universal primers and analysis of the amplification products with type specific oligonucleotides in a different hybridisation assay using electro-chemoluminescence to read the results (1 l-l 3). Liver biopsies Liver biopsies were assessed before and at end of treatment. The biopsies were examined at the end of the study by a nominated single pathologist who scored all the samples using the Knodell histological activity index numerical screening system. (14). The pathologist was blinded to treatment allocation and to whether each biopsy was from before or after treatment. Periportal necrosis, bridging necrosis, intralobular degeneration and focal necrosis, portal inflammation and fibrosis/cirrhosis were scored. Any other features relevant to HCV, such as lymphoid aggregates, were noted. Statistical analysis The results were analysed for the intention to treat population (defined as all patients who received study medication) and for an ‘efficacy’ subset (which excluded patients with major protocol violations). The difference between the proportion of patients with an ALT response in the two groups was examined using the Chi-squared test. The magnitude of change in ALT levels from the pre-treatment mean was assessed by analysis of variance. Logistic regression analysis was employed to determine responder status by age, sex, race duration of history of HCV infection, dose and mean pre-treatment ALT values for ribavirin-treated patients. The magnitude of change of HCV-RNA PCR titres during the treatment and follow-up periods was compared using the nonparametric Kruskal Wallis test. The change in the degree of inflammatory activity observed by liver biopsy at end of therapy compared to pre-treatment was determined using the total Knodell score (sum of 593
G. Dusheiko et al.
four components), and analysed by the Kruskall Wallis test. Adverse events were compared by Fisher’s exact test or Chi-squared, as appropriate.
300
Treatment phase
I I -u- Ribavirin
I
Results One hundred and eighteen patients were recruited. Two patients did not receive study medication and therefore the intent population comprised 116 patients. The efficacy population comprised 65 patients. The baseline characteristics of the patients are shown in Table 1. Efficacy variables The two primary efficacy variables were response based on normalisation of serum ALT levels during and after therapy and response based on reduction or disappearance of HCV RNA titres in plasma. The secondary endpoint was improvement in the degree of inflammatory activity observed by liver biopsy at end of therapy compared to pre-treatment. Data were also compared for other morphological features of chronic hepatitis C. ALT concentrations Ribavirin was significantly more effective than placebo in reducing or normalising serum ALT levels at the end of treatment (Fig. I), but this effect was not sustained. The time to normalisation of ALT results from the start of treatment, for those whose ALT did become normal, were analysed by time to event survival methods. We observed a relatively slow decline for patients who did achieve normal serum ALT. The median time to normalisation was 56 days. After approximately 2 months of treatment, 25% of ribavirin-treated patients had normal ALT. When factors related to outcome were analysed, based on ALT values at the last active treatment visit, only dose and duration of HCV infection were related to outcome. The proportion of patients with reduction TABLE 1 Patient characteristics
Number of females Number of males Mean age (years) Age range Previous interferon-o Median duration HCV Genotype 1 Genotype 2 Genotype 3 Genotype 4
Placebo (38)
Ribavirin (78)
9 (24) 29 (76) 44 23-69 7 (18.4%) 8.4 months 19 (54.2) 4 (11.4) 10 (28.5) 2 (5.7)
21 (27) 57 (73) 44.4 24-69 15 (19.2%) 10.5 months 36 (53.7) 14 (20.8) 22 (32) l(1.4)
The percentage of patients is shown in parenthesis,
594
01
,
,
,
I
I
0
4
8
12
IS
20
(I
,
,
I
I
,
I
24
28
32
36
40
44
48
Week
Fig. 1. Serum ALT concentrations in placebo and ribavirin treated patient (mean&standard deviation).
or normalisation of ALT was 68% for patients whose dose of ribavirin was greater than 15 mg/kg per day as compared to 43% for patients whose dose was less than 15 mg/kg per day (p
Ribuvirin treatmentfor
chronic hepatitis C
TABLE 2a Change in HCV RNA concentrations in ribavirin and placebo patients (semi-quantitative measurement) Ribavirin 67
Placebo 36
Number During
I
1
1 (3) 12 (33)
Disappearance Responder* Post-treatment
p>0.2
2 (3) 17 (25)
I
I
1 (3)
Disappearance Responder*
p-value
7 (22)
1 (2) 20 (36)
p>O.l
TABLE 2b Mean HCV RNA (bDNAxlOS) levels at each treatment phase Time
Placebo
Ribavirin
p-value
Initial Mid-treatment End of treatment End of follow up
34.69&56.2** 47.82k70.15 67.19i94.4 62.06S6.1
38.53k71.3 19.87zb29.8 28.2M37.9 51.17k75.75
0.784 0.005 0.002 0.552
Percentages are shown in brackets. * Definition: Responder status for HCV RNA was based on disappearance of HCV RNA or reduction by one or more PCR titre. ** Mea&standard deviation.
genotype 1 of HCV more efficiently, which could explain the observed difference. The mean percent decline in concentrations of HCV RNA was higher in type 1 ribavirin-treated patients, but this was not statistically significant (p=O.O9).
also had fewer lymphoid aggregates than did placebo patients (~~0.05, Wilcoxon test). However, there were no other statistically significant histological differences between the ribavirin versus the placebo recipients in histology from pre- to post-treatment.
Histology
Safety results
Ninety-four patients had a pre-treatment biopsy, and 77 had a post-treatment biopsy. There was a numerical trend in favour of ribavirin in the total score and each of the four component scores (Table 3). Ribavirin patients
There were no deaths during the study. Three patients in the ribavirin group (4%) and one patient in the placebo group (3%) withdrew from the study due to adverse events. Adverse events were reported by 37% (14/38) of placebo group patients and 59% (47/ 68) of ribavirin group patients (~~0.005). Anaemia was detected in 3% (l/38) and 32% (25/78) of placebo and ribavirin patients, respectively. The mean fall in haemoglobin in the ribavirin group was 1.73 g/ dl. The fall in haemoglobin was accompanied by a
400 1
.
3.5
Detection limit .a.._..Y.U. DDaoIIn
Placebo
TABLE 3 Histological change in ribavirin-and placebo-treated patients Category
Change
Placebo Ribavirin
p-value
Periportal bridging Necrosis
Improved Worse
0.16
Intralobular degeneration and focal necrosis
Improved Worse
4 (15.6) 17 (33.3) 3 (11.5) 5 (9.8) 4 (15.4) 13 (25.5) 2 (7.7) 5. (9.8)
Portal inflammation
Improved Worse
4 (15.4) 11 (21.6) 5 (19.2) 3 (5.9)
0.18
Fibrosis
Improved Worse
2 (7.7) 4 (15.6)
7 (13.7) 6 (11.8)
0.42
Total score
Improved
8 (30.8) 23 (45.1) 8 (30.8) 12 (23.5)
0.18
Ribavirin
Fig. 2. HCV RNA concentrations at the end of treatment in placebo- and ribavirin-treated patients. The horizontal bar indicates the mean value.
Worse
0.59
Percentages are shown in parentheses.
595
G. Dusheiko et al. TABLE 4 Laboratory values in ribavirin vs placebo recipients
Bilirubin (ymol/l)/l GGT (U/l) Uric acid @moWI) Haemoglobin (g/dl) Red cell countsx10’2 Lymphocyte countsxl0’ Monocyte countsxl0’ Platelet countsxl0’
Placebo
Ribavirin
p-value
-0.06 1 -1.6 U/L -9.07 -0.07 -0.03 -0.11 -0.03 -2.15
+ 4.42 -28.94 +20.39 - 1.73 - 0.55 - 0.52 - 0.04 +27.4
p
parallel fall in red cell count and a symmetrical rise in total bilirubin level, as is typical for haemolytic anaemia. These changes promptly reversed following withdrawal of ribavirin. Development of anaemia necessitated a reduction of the dose of ribavirin in nine patients and temporary discontinuation in two patients; all of these patients were able to complete the course of treatment. Skin disorders were reported by 14% of ribavirin patients and no placebo recipients. These disorders included pruritis, rash, alopecia, dry skin, eczema, herpes simplex, herpes zoster and hirsutism (p=O.O16, Fisher’s exact test). Nervous system disorders (depression, insomnia, nervousness, paraesthesia, somnolence, vertigo) were reported by 14% of ribavirin patients and no placebo recipients (~~0.05). The analysis of laboratory safety data is summarised in Table 4 in terms of mean changes from baseline to end of therapy by treatment group. Bilirubin concentrations decreased from a mean of 0.06 pmol/l for placebo and increased by a mean of 4.42 l_tmol/l for ribavirin recipients. The changes related to haemolysis are discussed above. The small rise in serum uric acid may also be related to metabolism of ribavirin to urates. No patient developed clinical gout or an elevated uric acid during the study. The rise in platelet counts has also been observed in rodents in toxicology studies. No patient developed an above normal count during the study.
Discussion Our data show that 6 months treatment with ribavirin is associated with an improvement in serum ALT concentrations during treatment, but no significant change in HCV RNA concentrations compared with placebo. There was a slight difference in levels of viraemia between ribavirin and placebo recipients at the end of treatment when measured by the bDNA assay. This difference is just statistically significant, and may reflect a weak antiviral effect of ribavirin However, the lower limit of detection of the bDNA 596
assay is 350000 equivalents per ml, and it is more appropriate to judge the antiviral effect using the more sensitive nested PCR technique. A similar effect has been noted in earlier studies, in which patients were treated for a shorter period (7,8). There was a trend toward improved histology after this relatively short course of treatment, but the differences were not significantly different from those treated with placebo. Thus our data suggest that 24 weeks of therapy of chronic hepatitis C confers little benefit for patients. Although the treatment was generally well tolerated, haemolysis and anaemia occurred more frequently than in placebo recipients. The effect on serum ALT differs somewhat from the effect seen in interferona-responsive patients, in whom the effect is usually more rapid (15-18). These data confirm that ALT measurements in the absence of measurements of viraemia may not be indicative of an antiviral response, but the improvement in serum ALT concentrations without elimination of viraemia needs further explanation. It is possible that minor reductions in HCV replication explain this phenomenon, or that this effect is a consequence of an effect of ribavirin on hepatic inflammation and necrosis. This phenomenon has not been well studied, however, and although ribavirin enhances macrophage function, our data do not further define an immunological or anti-inflammatory effect. The small fall in lymphocyte counts remains unexplained, although lymphoid atrophy and thymic lymphocyte depletion have been observed in animal studies. Earlier studies have shown that relapse was universal after 12 weeks of ribavirin treatment (8), a trend which was confirmed in this controlled trial. Longer-term safety and efficacy studies will be required to assess the effect of ribavirin in patients with chronic hepatitis C to assess the effect on the disease. The mechanism of action of interferon-a in hepatitis C is clearly complex, and a number of recent studies have suggested that viral load (level of viraemia), genotype, quasispecies diversity, and absence of cirrhosis are all important factors. (17,19-22) The mechanism of action of ribavirin appears equally complex, but the relatively high ALT response rates in patients with type 2 and 3 hepatitis C are interesting. A noteworthy number of patients with type 1 hepatitis C also had biochemical response, and this may explain in part an apparent synergistic improvement in response in patients treated with both ribavirin and interferon alpha.
Ribavirin treatment for chronic hepatitis C
The decline in haemoglobin was generally mild and a reduction in dose was usually followed by an immediate improvement in haemoglobin concentrations. The drug is concentrated in red blood cells, which may partly explain the mechanism of haemolysis. Bone marrow suppression and a delay in late phase erythroblast maturation may contribute to this. Haemoglobin levels normalised in most patients soon after stopping therapy. The role of ribavirin as a single agent is doubtful. The drug per se does not eliminate HCV RNA. It may perhaps be useful in patients with active cirrhosis, and thrombocytopaenia or leucopaenia, in whom interferon-a is often contraindicated, or in patients with an autoimmune diathesis or thyroid disease in whom the disease is aggravated by interferon-a. In these latter patients, ribavirin may be an alternative to starting therapy with corticosteroids. However, it is uncertain whether the natural history of the disease is changed by administration of this agent. Approximately 30% of patients had normal serum ALT at the end of therapy. Responsive patients may benefit from long-term therapy with ribavirin, but further histological study will be required to indicate any possible positive influence on the natural history of the disease. Evidence of the safety of this approach will also be required. The drug has the advantage of oral administration and it was well tolerated in the majority of patients. There are a number of small trials which suggest that combination therapy with interferon-a and ribavirin my be beneficial. If further controlled trials confirm the observations that combined therapy improves response rates in interferon non-responders, or compared to interferon-a treatment, ribavirin may yet be a useful adjunct for the management of selected patients with chronic hepatitis C (23).
3.
4.
5.
6.
7.
8.
9.
10.
11.
12. 13. 14.
Acknowledgements This study was supported by a grant from ICN Pharmaceuticals. We acknowledge the assistance of Dr John Gait and Dr Gordana Kosutic, ICN pharmaceuticals, Costa Mesa, California and Linda Capsey.
15.
16.
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G. Dusheiko et al. interferon therapy on hepatitis C viraemia in communityacquired chronic non-A, non-B hepatitis: a quantitative polymerase chain reaction study. J Med Virol 1991; 34: 13-1. 18. Shindo M, Di-Bisceglie AM, Cheung L, Shih JW, 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. 19. Kanai K, Kako M, Okamoto H. HCV genotypes in chronic hepatitis C and response to interferon. Lancet 1992; 339: 1543. 20. Okada S, Akahane Y, Suzuki H, Okamoto H, Mishiro S. The degree of variability in the amino terminal region of the E2/ NS 1 protein of hepatitis C virus correlates with responsiveness to interferon therapy in viremic patients. Hepatology 1992; 16: 619-4.
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21 McOmish F, Chan S-W, Dow BC, Gillon J, Frame WD, Crawford RJ, Yap P-L, Follett EAC, Simmonds I? Detection of three types of hepatitis C virus in blood donors: investigation of type-specific differences in serologic reactivity and rate of alanine aminotransferase abnormalities. Transfusion 1993; 33: 7-13. 22. Simmonds P, Holmes EC, Cha T-A, Chan S-W, McOmish F. Irvine B, Beall E, Yap PL, Kolberg J, Urdea MS. Classification of hepatitis C virus into six major genotypes and a series of subtypes by phylogenetic analysis of the NS-5 region. J Gen Virol 1993; 74: 2391-9. 23. Brillanti S, Garson J, Foli M, Whitby K, Deaville R, Masci C, Miglioli M, Barbara L. A pilot study of combination therapy with ribavirin plus interferon alfa for interferon alfa-resistant chronic hepatitis C. Gastroenterology 1994; 107: 812-7.
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Copyright0 EuropeanAssociation for the Studv of the Liver 1996 Journalof Hepatology ISSN0168.8278
Ribavirin treatment for patients with chronic hepatitis C: results of a placebo-controlled study Geoffrey Dusheiko’, Janice Main2, Howard Thomas2, Olle Reichard3, Christine Lee”, Amar Dhillon’, Suhra Rassam’, Aril Fryden4, Henk Reesink’, Margaret Bassendine6, Gunnar Norkrans7, Theo Cuypers*, Nice Lelie’, Paul Telfer”, Jon Watson’, Christine Weegink’, Peter Sillikens”and Ola Weiland’ Departments of Medicine and Pathology ‘Royal Free Hospital, and ‘St. Mary’s HospitalLondon, 6Freeman Hospital, Newcastle; “Haemophilia Center Royal Free Hospital, United Kingdom; ‘Division of Infectious Diseases, Danderyd Hospital, ‘Huddinge Hospital, and Karolinska Institute, “University Hospital, Linkoping, ‘Ostra Hospital, Goteborg, Sweden; ‘Vereenigen Voor Ziekenverpleging, ‘CLB, Amsterdam, “Organon Teknika Laboratories, The Netherlands
Background/Aims:
Small, uncontrolled studies of ribavirin for patients with chronic hepatitis C have reported efficacy in chronic hepatitis C. We have evaluated the efficacy and safety of a 24week course of oral ribavirin in patients with chronic hepatitis C, compared to placebo. Methods: A total of 114 patients were randomised to ribavirin or placebo. Ribavirin was administered in doses of 1000 or 1200 mg/day for 24 weeks. Efficacy was determined in the intentionto-treat population: 76 received ribavirin and 38 placebo. Results: Ribavirin was significantly more effective than placebo in reducing and normalising serum ALT levels: 42/76 (55%) of ribavirin-treated patients vs 2/38 (5 %) placebo recipients had either normalisation of the ALT levels or a reduction from baseline of at least 50% (~~0.001). ALT levels were normal in 22/76 (29%) of ribavirintreated patients vs O/38 placebo recipients (pcO.001). Twenty-four weeks after stopping ribavirin, the majority of patients had abnormal ALT levels. There was no difference between the treatment groups in reduction or disappearance of HCV-RNA levels. HCV RNA disappeared during
treatment in 3% of ribavirin-treated patients and 3% of placebo recipients. More ribavirin than placebo patients showed improvement in total Knodell score (45% vs 31%), but these differences were not statistically significant. Analysis of each component of a histology activity index revealed no statistically significant differences between treatment groups. Ribavirin patients had fewer lymphoid aggregates than did placebo recipients at the post-treatment assessment (p=O.O5).Ribavirin was associated with reversible haemolytic anaemia: a fall in haemoglobin occurred in 3% of placebo- and 32% (25/78) of ribavirin-treated patients, respectively (pcO.001). Conclusions: These data indicate that ribavirin was no more effective than placebo in reducing or eliminating HCV-RNA levels, and was not significantly more effective than placebo in improving hepatic histology after 6 months of treatment. The role of a 6-month treatment of chronic hepatitis C with ribavirin alone, without a significant effect on HCV RNA, is therefore limited.
P
sidious. Interferon-a treatment results in sustained biochemical responses (normal serum aminotransferases) in 15%-20% of those treated for 6 months (4,5). As side effects are not uncommon in treated patients, other therapies are therefore being sought to improve the treatment of chronic hepatitis C. Ribavirin (1 -Beta-D-ribofuranosyl1 H- 1,2,4-triazole-3-carboxamide) is a water-soluble nucleoside
with hepatitis C infection frequently develop chronic hepatitis (l-3). This may progress to cirrhosis, although this progression is usually inATIENTS
Received 25 August 1995; revised and accepted 9 January 1996
Correspondence: Prof. G. Dusheiko, Viral Hepatitis Study Group, Royal Free Hospital, Pond Street, London NW3 2QG, UK. Tel:+44 171 830 2993. Fax. +44 1714314581.
Key words: Antivirals; Chronic hepatitis; Hepatitis C antiviral therapy; Ribavirin.
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et al.
analogue of guanosine with antiviral activity against DNA and RNA viruses (6). The mechanism of action is unknown, but the drug may decrease intracellular pools of guanosine triphosphate. and result in synthesis of RNA with abnormal 5’ cap structures. Alternatively the drug may have a direct suppressive effect on viral RNA polymerase activity. Previous uncontrolled studies of the effect of ribavirin in patients with chronic hepatitis C have noted that the drug reduced serum aminotransferases concentrations and, in one study, a significant decrease in geometric mean titre of HCV RNA in serum was reported (7-9). This potentially useful drug therefore necessitated further study. We have undertaken a phase III multicentre, multinational, randomised double-blinded placebo-controlled study of oral ribavirin therapy in subjects with chronic hepatitis C infection. The study involved nine centres. The aim of the study was to evaluate the efficacy of ribavirin in reducing serum ALT concentrations, HCV RNA levels, and improving histological status when given for 24 weeks, comparing the effect of ribavirin with placebo.
Patients and Methods One hundred and eighteen male and female patients ranging in age from 18-70, with confirmed chronic hepatitis C, were randomised to receive either ribavirin (80) or placebo (38). All patients had proven chronic hepatitis C and were anti-HCV positive by second-generation ELISA and by RIBA, and/or had evidence of HCV RNA in plasma by polymerase chain reaction (PCR). All previously treated patients had stopped interferon at least 24 weeks before. Eligible patients had to have: evidence of elevated serum ALT (at least 1.5 times upper limit of normal) on at least three occasions in the 6-month period prior to the study, or evidence of elevated ALT on at least two occasions in the previous 6 months, plus PCR evidence of HCV RNA in plasma, and a previous liver biopsy showing evidence of chronic hepatitis or (compensated) cirrhosis (except patients with bleeding disorders). The protocol stipulated that all patients should have a baseline liver biopsy, unless a biopsy had been performed in the 24 weeks prior to study entry, at least 24 weeks after cessation of any previous treatment with interferon. A biopsy performed within 12 months of study entry was accepted in the case of otherwise eligible patients who refused a baseline liver biopsy, provided that the serum ALT had been monitored on a regular basis in the interval between the biopsy and the study entry, and there was not more than a four-fold 592
difference between the mean of at least two ALT levels measured within 3 months of the biopsy and the mean of at least two levels during the 3 months prior to the study entry. Two investigators included patients with bleeding disorders, who were not biopsied. All patients gave written informed consent to the procedure, except at the Swedish centres, where verbal consent was the norm. The study was conducted in accordance with the Declaration of Helsinki. Patients who consumed more than 80 g/day (males) and 60 g/day (females) of alcohol were excluded, as were HBsAg or anti-HIV-positive patients, known active intravenous drug abusers, or patients with pre-existing anaemia (haemoglobin less than 10 g/dl) or a known history of haemolytic anaemia. Patients with a history of clinical gout or currently elevated serum uric acid, who had received interferon or immuno-suppressive therapy within the previous 6 months, or who had significant systemic illness other than liver disease, or with decompensated liver disease, or known evidence of other forms of liver disease, or who had autoantibody titres greater than 1: 100 were also excluded. Two patients did not receive treatment, and therefore the intention-to-treat population comprised 116 patients. The efficacy population (i.e. excluding those who did not have a baseline and follow-up liver biopsy) comprised 65 patients: 21 in the placebo group and 44 in the ribavirin group. There was no evidence of differences between the intention to treat and efficacy group, and there was also no centre effect or bias. Therefore the results of the intention to treat group have been reported. The patient characteristics are summarised in Table 1. Twenty-two patients in the intention-to-treat group had previously received interferon. An ALT, virological, or histological response was noted in 15, 7 and 8 of these, respectively. Study design Patients who fulfilled the entry criteria were randomly assigned to ribavirin or placebo in a ratio of 2: 1, respectively. Unequal randomisation was chosen in order to minimise exposure to placebo, for ethical reasons. The total sample size of 120 patients was based on the ability to detect a 50% ALT response rate in the active treatment group as compared to 25% in the placebo recipients (80% power, 5% significance level). The study medication, supplied as 200-mg capsules or identical placebo capsules, was given orally in two divided doses of 1000 mg/day for patients
Ribavirin treatment for chronic hepatitis C
weighing less than 75 kg, and 1200 mg/day for patients weighing more than 75 kg for 24 weeks. Patients were monitored regularly for 6 weeks prior to initiation of treatment. All patients were monitored for a further 24 weeks after cessation of therapy. Efficacy was assessed by monitoring of serum ALT levels and plasma HCV RNA levels, and by repeat liver biopsy at the end of treatment. Safety was assessed by collection of adverse event data and by performing periodic haematology and biochemistry tests. HCV RNA measurement Serum for HCV RNA concentrations was measured immediately before the start of treatment, at the end of the active treatment, and at the end of the posttreatment phase. Ten millilitres of blood was drawn into a vacuum blood tube containing EDTA, centrifuged within 2 h and the plasma transferred into sterile cryotubes and stored at -70°C. PCR measurement was specified initially, and after completion of the study, it was decided to run a signal amplification method (bDNA) on retained samples. 1) Semi-quantitative measurement. The samples were transported in dry ice to the Netherlands Red Cross Blood Transfusion Service for analysis at the end of the study. HCV RNA was detected using PCR and measured semi-quantitatively by endpoint dilution. For each RNA extract the endpoint dilution for HCV RNA detection was determined, and the mean of the duplicate endpoint titres was calculated. Serial four-fold dilutions of each nucleic acid extract in water containing 100 pg/ml poly A were prepared. 7.5 ~1 was tested in a cDNA-PCR assay, in which amplification products were detected by oligomer hybridisation in liquid phase with the oligo Alx89. To guarantee the validity of the measured endpoint titres and to exclude inter-assay variation in sensitivity, a lo4 dilution of a donor panel proficiency was included in every analysis series. Assay results were only included when this control sample scored positive with the same intensity of the hybridisation signal as in preceding series. 2) Signal amplijication: Samples collected and stored as above were assayed by a chemoluminescent assay measuring signal amplification, using the quantitative bDNA signal amplification assay (Quantiplex HCV RNA assay, Chiron Corporation, Emeryville CA USA) according to the manufacturer’s instructions. (10) This test is based upon specific hybridisation of virus RNA in the sample by synthetic oligonucleotides to the highly conserved 5’-untranslated region and core gene of HCV RNA immobilised on
the surface of a microwell plate. Synthetic bDNA amplifier molecules and multiple copies of an alkaline phosphatase-linked probe are hybridised to the immobilised complex. The complex is incubated with a chemoluminescent substrate and the light emission is measured, the signal being proportional to the level of target nucleic acid. The quantity of HCV RNA in the sample is determined from a standard curve. The detection limit of the test is 350000 HCV RNA equivalents per ml. HCV genotyping HCV genotyping was performed by NASBA amplification of the 5’-untranslated region with universal primers and analysis of the amplification products with type specific oligonucleotides in a different hybridisation assay using electro-chemoluminescence to read the results (1 l-l 3). Liver biopsies Liver biopsies were assessed before and at end of treatment. The biopsies were examined at the end of the study by a nominated single pathologist who scored all the samples using the Knodell histological activity index numerical screening system. (14). The pathologist was blinded to treatment allocation and to whether each biopsy was from before or after treatment. Periportal necrosis, bridging necrosis, intralobular degeneration and focal necrosis, portal inflammation and fibrosis/cirrhosis were scored. Any other features relevant to HCV, such as lymphoid aggregates, were noted. Statistical analysis The results were analysed for the intention to treat population (defined as all patients who received study medication) and for an ‘efficacy’ subset (which excluded patients with major protocol violations). The difference between the proportion of patients with an ALT response in the two groups was examined using the Chi-squared test. The magnitude of change in ALT levels from the pre-treatment mean was assessed by analysis of variance. Logistic regression analysis was employed to determine responder status by age, sex, race duration of history of HCV infection, dose and mean pre-treatment ALT values for ribavirin-treated patients. The magnitude of change of HCV-RNA PCR titres during the treatment and follow-up periods was compared using the nonparametric Kruskal Wallis test. The change in the degree of inflammatory activity observed by liver biopsy at end of therapy compared to pre-treatment was determined using the total Knodell score (sum of 593
G. Dusheiko et al.
four components), and analysed by the Kruskall Wallis test. Adverse events were compared by Fisher’s exact test or Chi-squared, as appropriate.
300
Treatment phase
I I -u- Ribavirin
I
Results One hundred and eighteen patients were recruited. Two patients did not receive study medication and therefore the intent population comprised 116 patients. The efficacy population comprised 65 patients. The baseline characteristics of the patients are shown in Table 1. Efficacy variables The two primary efficacy variables were response based on normalisation of serum ALT levels during and after therapy and response based on reduction or disappearance of HCV RNA titres in plasma. The secondary endpoint was improvement in the degree of inflammatory activity observed by liver biopsy at end of therapy compared to pre-treatment. Data were also compared for other morphological features of chronic hepatitis C. ALT concentrations Ribavirin was significantly more effective than placebo in reducing or normalising serum ALT levels at the end of treatment (Fig. I), but this effect was not sustained. The time to normalisation of ALT results from the start of treatment, for those whose ALT did become normal, were analysed by time to event survival methods. We observed a relatively slow decline for patients who did achieve normal serum ALT. The median time to normalisation was 56 days. After approximately 2 months of treatment, 25% of ribavirin-treated patients had normal ALT. When factors related to outcome were analysed, based on ALT values at the last active treatment visit, only dose and duration of HCV infection were related to outcome. The proportion of patients with reduction TABLE 1 Patient characteristics
Number of females Number of males Mean age (years) Age range Previous interferon-o Median duration HCV Genotype 1 Genotype 2 Genotype 3 Genotype 4
Placebo (38)
Ribavirin (78)
9 (24) 29 (76) 44 23-69 7 (18.4%) 8.4 months 19 (54.2) 4 (11.4) 10 (28.5) 2 (5.7)
21 (27) 57 (73) 44.4 24-69 15 (19.2%) 10.5 months 36 (53.7) 14 (20.8) 22 (32) l(1.4)
The percentage of patients is shown in parenthesis,
594
01
,
,
,
I
I
0
4
8
12
IS
20
(I
,
,
I
I
,
I
24
28
32
36
40
44
48
Week
Fig. 1. Serum ALT concentrations in placebo and ribavirin treated patient (mean&standard deviation).
or normalisation of ALT was 68% for patients whose dose of ribavirin was greater than 15 mg/kg per day as compared to 43% for patients whose dose was less than 15 mg/kg per day (p
Ribuvirin treatmentfor
chronic hepatitis C
TABLE 2a Change in HCV RNA concentrations in ribavirin and placebo patients (semi-quantitative measurement) Ribavirin 67
Placebo 36
Number During
I
1
1 (3) 12 (33)
Disappearance Responder* Post-treatment
p>0.2
2 (3) 17 (25)
I
I
1 (3)
Disappearance Responder*
p-value
7 (22)
1 (2) 20 (36)
p>O.l
TABLE 2b Mean HCV RNA (bDNAxlOS) levels at each treatment phase Time
Placebo
Ribavirin
p-value
Initial Mid-treatment End of treatment End of follow up
34.69&56.2** 47.82k70.15 67.19i94.4 62.06S6.1
38.53k71.3 19.87zb29.8 28.2M37.9 51.17k75.75
0.784 0.005 0.002 0.552
Percentages are shown in brackets. * Definition: Responder status for HCV RNA was based on disappearance of HCV RNA or reduction by one or more PCR titre. ** Mea&standard deviation.
genotype 1 of HCV more efficiently, which could explain the observed difference. The mean percent decline in concentrations of HCV RNA was higher in type 1 ribavirin-treated patients, but this was not statistically significant (p=O.O9).
also had fewer lymphoid aggregates than did placebo patients (~~0.05, Wilcoxon test). However, there were no other statistically significant histological differences between the ribavirin versus the placebo recipients in histology from pre- to post-treatment.
Histology
Safety results
Ninety-four patients had a pre-treatment biopsy, and 77 had a post-treatment biopsy. There was a numerical trend in favour of ribavirin in the total score and each of the four component scores (Table 3). Ribavirin patients
There were no deaths during the study. Three patients in the ribavirin group (4%) and one patient in the placebo group (3%) withdrew from the study due to adverse events. Adverse events were reported by 37% (14/38) of placebo group patients and 59% (47/ 68) of ribavirin group patients (~~0.005). Anaemia was detected in 3% (l/38) and 32% (25/78) of placebo and ribavirin patients, respectively. The mean fall in haemoglobin in the ribavirin group was 1.73 g/ dl. The fall in haemoglobin was accompanied by a
400 1
.
3.5
Detection limit .a.._..Y.U. DDaoIIn
Placebo
TABLE 3 Histological change in ribavirin-and placebo-treated patients Category
Change
Placebo Ribavirin
p-value
Periportal bridging Necrosis
Improved Worse
0.16
Intralobular degeneration and focal necrosis
Improved Worse
4 (15.6) 17 (33.3) 3 (11.5) 5 (9.8) 4 (15.4) 13 (25.5) 2 (7.7) 5. (9.8)
Portal inflammation
Improved Worse
4 (15.4) 11 (21.6) 5 (19.2) 3 (5.9)
0.18
Fibrosis
Improved Worse
2 (7.7) 4 (15.6)
7 (13.7) 6 (11.8)
0.42
Total score
Improved
8 (30.8) 23 (45.1) 8 (30.8) 12 (23.5)
0.18
Ribavirin
Fig. 2. HCV RNA concentrations at the end of treatment in placebo- and ribavirin-treated patients. The horizontal bar indicates the mean value.
Worse
0.59
Percentages are shown in parentheses.
595
G. Dusheiko et al. TABLE 4 Laboratory values in ribavirin vs placebo recipients
Bilirubin (ymol/l)/l GGT (U/l) Uric acid @moWI) Haemoglobin (g/dl) Red cell countsx10’2 Lymphocyte countsxl0’ Monocyte countsxl0’ Platelet countsxl0’
Placebo
Ribavirin
p-value
-0.06 1 -1.6 U/L -9.07 -0.07 -0.03 -0.11 -0.03 -2.15
+ 4.42 -28.94 +20.39 - 1.73 - 0.55 - 0.52 - 0.04 +27.4
p
parallel fall in red cell count and a symmetrical rise in total bilirubin level, as is typical for haemolytic anaemia. These changes promptly reversed following withdrawal of ribavirin. Development of anaemia necessitated a reduction of the dose of ribavirin in nine patients and temporary discontinuation in two patients; all of these patients were able to complete the course of treatment. Skin disorders were reported by 14% of ribavirin patients and no placebo recipients. These disorders included pruritis, rash, alopecia, dry skin, eczema, herpes simplex, herpes zoster and hirsutism (p=O.O16, Fisher’s exact test). Nervous system disorders (depression, insomnia, nervousness, paraesthesia, somnolence, vertigo) were reported by 14% of ribavirin patients and no placebo recipients (~~0.05). The analysis of laboratory safety data is summarised in Table 4 in terms of mean changes from baseline to end of therapy by treatment group. Bilirubin concentrations decreased from a mean of 0.06 pmol/l for placebo and increased by a mean of 4.42 l_tmol/l for ribavirin recipients. The changes related to haemolysis are discussed above. The small rise in serum uric acid may also be related to metabolism of ribavirin to urates. No patient developed clinical gout or an elevated uric acid during the study. The rise in platelet counts has also been observed in rodents in toxicology studies. No patient developed an above normal count during the study.
Discussion Our data show that 6 months treatment with ribavirin is associated with an improvement in serum ALT concentrations during treatment, but no significant change in HCV RNA concentrations compared with placebo. There was a slight difference in levels of viraemia between ribavirin and placebo recipients at the end of treatment when measured by the bDNA assay. This difference is just statistically significant, and may reflect a weak antiviral effect of ribavirin However, the lower limit of detection of the bDNA 596
assay is 350000 equivalents per ml, and it is more appropriate to judge the antiviral effect using the more sensitive nested PCR technique. A similar effect has been noted in earlier studies, in which patients were treated for a shorter period (7,8). There was a trend toward improved histology after this relatively short course of treatment, but the differences were not significantly different from those treated with placebo. Thus our data suggest that 24 weeks of therapy of chronic hepatitis C confers little benefit for patients. Although the treatment was generally well tolerated, haemolysis and anaemia occurred more frequently than in placebo recipients. The effect on serum ALT differs somewhat from the effect seen in interferona-responsive patients, in whom the effect is usually more rapid (15-18). These data confirm that ALT measurements in the absence of measurements of viraemia may not be indicative of an antiviral response, but the improvement in serum ALT concentrations without elimination of viraemia needs further explanation. It is possible that minor reductions in HCV replication explain this phenomenon, or that this effect is a consequence of an effect of ribavirin on hepatic inflammation and necrosis. This phenomenon has not been well studied, however, and although ribavirin enhances macrophage function, our data do not further define an immunological or anti-inflammatory effect. The small fall in lymphocyte counts remains unexplained, although lymphoid atrophy and thymic lymphocyte depletion have been observed in animal studies. Earlier studies have shown that relapse was universal after 12 weeks of ribavirin treatment (8), a trend which was confirmed in this controlled trial. Longer-term safety and efficacy studies will be required to assess the effect of ribavirin in patients with chronic hepatitis C to assess the effect on the disease. The mechanism of action of interferon-a in hepatitis C is clearly complex, and a number of recent studies have suggested that viral load (level of viraemia), genotype, quasispecies diversity, and absence of cirrhosis are all important factors. (17,19-22) The mechanism of action of ribavirin appears equally complex, but the relatively high ALT response rates in patients with type 2 and 3 hepatitis C are interesting. A noteworthy number of patients with type 1 hepatitis C also had biochemical response, and this may explain in part an apparent synergistic improvement in response in patients treated with both ribavirin and interferon alpha.
Ribavirin treatment for chronic hepatitis C
The decline in haemoglobin was generally mild and a reduction in dose was usually followed by an immediate improvement in haemoglobin concentrations. The drug is concentrated in red blood cells, which may partly explain the mechanism of haemolysis. Bone marrow suppression and a delay in late phase erythroblast maturation may contribute to this. Haemoglobin levels normalised in most patients soon after stopping therapy. The role of ribavirin as a single agent is doubtful. The drug per se does not eliminate HCV RNA. It may perhaps be useful in patients with active cirrhosis, and thrombocytopaenia or leucopaenia, in whom interferon-a is often contraindicated, or in patients with an autoimmune diathesis or thyroid disease in whom the disease is aggravated by interferon-a. In these latter patients, ribavirin may be an alternative to starting therapy with corticosteroids. However, it is uncertain whether the natural history of the disease is changed by administration of this agent. Approximately 30% of patients had normal serum ALT at the end of therapy. Responsive patients may benefit from long-term therapy with ribavirin, but further histological study will be required to indicate any possible positive influence on the natural history of the disease. Evidence of the safety of this approach will also be required. The drug has the advantage of oral administration and it was well tolerated in the majority of patients. There are a number of small trials which suggest that combination therapy with interferon-a and ribavirin my be beneficial. If further controlled trials confirm the observations that combined therapy improves response rates in interferon non-responders, or compared to interferon-a treatment, ribavirin may yet be a useful adjunct for the management of selected patients with chronic hepatitis C (23).
3.
4.
5.
6.
7.
8.
9.
10.
11.
12. 13. 14.
Acknowledgements This study was supported by a grant from ICN Pharmaceuticals. We acknowledge the assistance of Dr John Gait and Dr Gordana Kosutic, ICN pharmaceuticals, Costa Mesa, California and Linda Capsey.
15.
16.
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