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Effect of ribavirin on hepatitis C viral kinetics in patients treated with pegylated interferon
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Hepatology, June 2003, Volume 37, Number 6
Eva Herrmann1,2, Jung-Hun Lee3, George Marinos4, Marlene Modi5
Stefan Zeuzem1. Medizinische Klinik und Poliklinik, Innere Medizin II, UniversitŠtskliniken des Saarlandes, Homburg/Saar, Germany; 2Fachbereich Mathematik, Technische UniversitŠt Darmstadt, Darmstadt, Germany; 3Medizinische Klinik II, Klinikum der Johann Wolfgang Goethe-UniversitŠt Frankfurt a.M., Germany; 4Department of Gastroenterology, Prince of Wales Hospital, Randwick, Australia; and 5Hoffmann-La Roche, Nutley, NJ.
Some have felt that ribavirin had no effect on early treatment response but was effective after the end of treatment in preventing relapse and thus a sustained response. The 2 studies described below address this question and an Editorial follows by Jay Hoofnagle and Glen Lutchman (Liver Diseases Section, Digestive Diseases Branch, National Institute of Diabetes and
Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD).
Chronic infection with hepatitis C virus (HCV) is characterized by a dynamic equilibrium between virus production and clearance.1,2 A characteristic biphasic or multiphasic initial decline of serum HCV RNA is observed when this equilibrium is disturbed by interferon alfa and can be analyzed mathematically. Viral kinetic models estimated a very short half-life of free hepatitis C virions in vivo (<5 hours) and suggested that a rapid first phase (day 1) of viral decline relates to the decay of free viral particles, whereas the much slower second phase (days 2 to 14) of viral decline reflects the clearance of productively infected cells. Moreover, a typical biphasic decay could be explained if interferon has a single therapeutic effect of partially blocking viral production. Kinetic analyses were central to our current
understanding of antiviral therapy and have influenced approaches toward treatment of patients with chronic hepatitis C. New results indicate that a third phase of viral decay can be present and may be due to suppression of the immune response during chronic HCV infection and a restoration of the cellular immune response that occurs when the serum viral load declines below an individual threshold.
Wide fluctuations in the plasma concentration of interferon are seen because of its short elimination half-life (4 to 10 hours) and render mathematical modeling of HCV kinetics more difficult. Furthermore, suboptimal interferon plasma concentrations may lead to intermittent increases of serum HCV RNA between interferon alfa injections. Covalent attachment of polyethylene glycol to interferon alfa results in a more sustained absorption from the subcutaneous injection site, decreased
clearance, and increased serum half-life compared with interferon alfa itself. Once per week, application of pegylated (40 kd) interferon -2a leads to almost constant serum levels with very small fluctuations in the peak-to-trough concentrations and conceptually sustained antiviral activity, which not only facilitates the modeling of HCV dynamics but could also improve the chance of achieving a sustained virologic response. Viral kinetics of patients treated with pegylated interferon also showed a typical interferon-like profile of viral decline.
The synthetic purine nucleoside ribavirin has no antiviral activity against HCV in monotherapy; however, in combination with interferon alfa, the
end-of-treatment and sustained virologic response rates are substantially enhanced. The antiviral mechanisms of ribavirin in patients with chronic hepatitis C when used in combination with an alfa interferon are unknown, and both direct antiviral and immunologically mediated effects have been suggested. Mathematical modeling of viral kinetics can assist in elucidating the mechanism of action of ribavirin. Therefore, the effect of ribavirin on HCV dynamics was studied here in patients chronically infected with hepatitis C virus who were treated with long-acting peginterferon -2a alone or in combination with ribavirin. Only patients infected with genotype HCV-1 were studied because viral kinetics are genotype dependent and because the synergistic antiviral effect of ribavirin is most pronounced in HCV-1-infected patients.
ABSTRACT: A dynamic equilibrium between viral production and clearance characterizes untreated chronic hepatitis C viral infection. After initiating antiviral treatment, a typical multiphasic decay of viremia can be observed and analyzed using mathematical models. To elucidate the antiviral mechanism of ribavirin when used in combination with (pegylated) interferon alfa, we investigated kinetic parameters in patients with chronic hepatitis C treated with either peginterferon alfa-2a with or without ribavirin and standard interferon -2b plus ribavirin for 48 weeks. Serum HCV RNA was measured frequently before, during, and at the end-of-treatment and the follow-up period. By using an appropriate model for viral dynamics, kinetic parameters were derived from nonlinear, least square fitting of serum HCV RNA quantifications. The first phase of viral decay (day 1) and the second phase of viral decay (days 2 to 21) were similar for all treatment groups. After about 7 to 28 days, a third phase of viral decay was seen in several patients, and this phase of decay was significantly faster in patients treated with peginterferon alfa-2a plus ribavirin compared with those treated with peginterferon alfa-2a alone. The decay of this third phase was associated with the virologic end-of-treatment response and sustained virologic response. In conclusion, the third-phase decay of initial viral kinetics, which
may represent a treatment-enhanced degradation of infected cells, was more pronounced in patients treated with peginterferon alfa-2a plus ribavirin. This finding suggests that combination treatment leads to a better restoration of the patient's immune response.
Patients and methods
Male and female patients aged 18 or older with elevated serum alanine aminotransferase (ALT) levels, consistent detection of HCV RNA above 5,000 IU/mL, and compensated, histologically proven chronic HCV infection not previously treated with any form of interferon were eligible for enrollment. Patients were excluded if they were positive for hepatitis B surface antigen, IgM antibody to the hepatitis B virus core antigen, or antibody to the human immunodeficiency virus. The present kinetic study is an investigator lead part of 2, phase III, randomized, multicenter trials. At 2 centers (Frankfurt and Randwick), 34 patients infected with HCV genotype 1 met the criteria and were all enrolled both into the global trial and into the present kinetic study.
Patients were randomly assigned to receive subcutaneously either 180 µg peginterferon -2a (PEGASYS; Hoffmann-La Roche, Basel, Switzerland) once weekly for 48 weeks with (n1 = 10) or without ribavirin (n2 = 17) or 3 million units (MU) standard interferon -2b 3 times per week plus ribavirin (Rebetron; Schering-Plough, Kenilworth, NJ) for 48 weeks (n3 = 7). Ribavirin was given orally twice a day for a total dose of 1,000 mg (body weight <75 kg) or 1,200 mg (body weight >75 kg) per day. All patients were evaluated twice before initiation of treatment and at day 1 (6 and 12 hours after the first dose) and days 2 to 5, 8, 11, 15, 22, 29, 43, and 57 and subsequently every 4 weeks during treatment and the 24-week follow-up period. The primary efficacy end point for the study was defined as undetectable serum HCV RNA levels 24 weeks after treatment.
RESULTS
All study patients were genotype 1; age 43-49; mostly male; body weight 70 to 77 kg (2.2 kg per lb); mean ALT 3.5 x upper limit of normal; mean HAI score I-III, 5.0-6.5, IV 2.2-2.7. Baseline HCV viral load: 5.9 log, 5.94 log, and 5.7 log, respectively.
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We randomized 34 patients infected with chronic hepatitis C genotype 1 virus to receive either peginterferon -2a with (n= 10) or without ribavirin (n= 17) or combination treatment of standard interferon plus ribavirin (n= 7) with similar baseline characteristics between the groups.
Already 14 days after initiating treatment, transaminase levels in patients from both combination regimens were significantly lower than those seen in patients treated with peginterferon alfa-2a alone (P = .026 for ALT, P = .006 for AST.
A similar but less obvious and more delayed trend was observed for viremia. Especially in patients treated with peginterferon alfa-2a plus ribavirin, serum HCV RNA levels were significantly lower than those seen in patients treated with peginterferon alfa-2a alone (P = .048 at week 8 after initiating treatment). Serum viral load for the treatment groups of peginterferon -2a alone and standard interferon -2b plus ribavirin were comparable with a trend to be more favorable for peginterferon alfa-2a alone between weeks 2 and 4 and less favorable for peginterferon alfa-2a alone at week 8.
A more detailed look at the individual viral kinetics revealed that 1 of 10 and 1 of 17 patients treated with peginterferon alfa-2a plus ribavirin and peginterferon alfa-2a alone, respectively, showed a very fast decay with undetectable HCV RNA 10 days after starting treatment and later on. In 8 of 9 patients treated with peginterferon alfa-2a plus ribavirin, 9 of 16 patients treated with peginterferon alfa-2a alone, and 4 of 7 patients treated with standard interferon alfa-2b plus ribavirin, a pronounced third phase of viral decay was observed.
Discussion by authors
We randomized 34 patients infected with chronic hepatitis C genotype 1 virus to receive either peginterferon alfa-2a with or without ribavirin or combination treatment of standard interferon plus ribavirin. Recent studies showed that treatment with pegylated interferon alfa considerably enhances the end-of-treatment and, less pronounced, the sustained virologic response rates compared with standard interferon alfa. Ribavirin, however, mainly affects the sustained virologic response rates by reducing relapse rates after the end-of-treatment.12-14 Here, 100%, 71%, and 29% in patients treated with peginterferon -2a plus ribavirin, peginterferon alfa-2a alone, and standard interferon alfa plus ribavirin, respectively, showed an ETR. These rates differ in comparison with those from the whole study, in which 69%, 59%, and 52%, respectively, in patients of all genotypes showed an ETR20 (genotypic ETR rates were not reported). Besides the inclusion of all genotypes in the
rates of the whole study, reasons for the improved rates for the peginterferon groups may be that we did not consider patients who dropped out here and that all patients were treated at very experienced centers. The sustained virologic response rates in the present study were 50%, 12%, and 14% in patients treated with peginterferon -2a plus ribavirin, peginterferon alfa-2a alone, and standard interferon plus ribavirin, respectively, and were more in accordance with sustained virologic response rates of 46%, 21%, and 36%, respectively, for genotype 1-infected patients in the whole study.
Differences in the ETR rates for the peginterferon -2a groups were mainly due to the fact that 7 of 10 patients and 12 of 17 patients for the peginterferon alfa-2a plus ribavirin and for the peginterferon alfa-2a alone group showed only a moderate decay during the first 48 hours and no further decay in the following week. Nevertheless, all of the patients from the peginterferon alfa-2a plus ribavirin group but only 7 of 12 patients in the peginterferon -2a alone group showed an accelerated viral decay after 7 to 28 days.
Several possible mechanisms of action have been proposed for ribavirin when the drug is used in the treatment of viral diseases, including depletion of the
intracellular guanosine triphosphate pools, synthesis of RNA with abnormal 5« cap structures, inhibition of viral polymerase activity, immune responses with a shift from TH2 to TH1, and an increased mutagenesis. The first suggested mechanism has been disproved, and the second is not applicable because of cap-independent replication of the hepatitis C virus. Preliminary analyses of initial viral kinetics suggested some synergistic antiviral activity between ribavirin and interferon in patients treated with 3 MU interferon alfa but not in patients treated with 3 MU interferon alfa daily or 6 MU interferon alfa given 3 times per week. Combination treatment with ribavirin did not show adverse effects on the infected cell loss rate in spite of improved decreases of ALT and AST levels.
As an alternative explanation of a 3-phasic viral kinetics and to model an increased mutagenesis possibly induced by ribavirin, a compartment model with 2 free viral compartments was used to describe the data of a representative patient treated with peginterferon -2a plus ribavirin (Fig. 2E). In this model, one viral compartment dominated during the steady-state situation before starting therapy, and the other viral compartment represents mutant virus with a substantially decreased infectivity. Here, the second phase was difficult to interpret but, again, the third-phase decay reflected the infected cell loss. Therefore, to account for the treatment-dependent third-phase decay, the data of the present study are best explained by an immune-mediated mechanism of ribavirin, e.g., activation of cytotoxic T cells leading to a treatment-enhanced elimination of productively infected cells. With respect to the rapidly improved ALT and AST levels in patients treated with ribavirin, this enhanced infected cell loss has to be due to elimination processes with only slight effect on transaminase serum levels, e.g., by apoptosis or cure of infected cells.
Alternative explanations such as a further improved antiviral activity on viral production at a delayed time could be described with a different kinetic model that shows 4 phases of viral decay, of which the first and the third ones are relatively short and faster than the almost identical second and fourth ones. Similarly, inclusion of nonhomogenous viral compartments with different sensitivities to interferon and/or ribavirin would typically result in a multiphasic exponential decay function in which the decay rates slow down with time. Both approaches do not reflect our data situation here. Treatment enhanced mutagenesis instead could explain a delayed observation of a more rapid third phase of viral decline but lead to alternative interpretations of the second-phase decay. Indeed, our observation of comparable second-phase decays and treatment-specific, third-phase decays fits well with the assumption that ribavirin improves the immune response already after the first week of treatment but also enhances mutagenesis, leading to a delayed observation of the immune response effect.
The predictive value of the initial decline of serum HCV RNA is more significant than all known baseline parameters including HCV genotype and pretreatment viremia. The assessment of HCV kinetics within the first weeks after initiation of treatment is anticipated to offer the possibility of individualized therapy. The present study confirms that the third-phase decay provides major predictive information. However, because the third phase of decline starts as late as 4 weeks after initiating therapy in some patients, individualizing therapy in patients with chronic hepatitis C based on viral dynamics will require sampling and HCV RNA quantification for the initial 6 to 8 weeks of therapy.
Editorial (excerpts)
Analysis of hepatitis C virus (HCV)-RNA levels in serum with initiation of antiviral therapy for hepatitis C has provided virologic and mechanistic insights into this important chronic viral infection and its therapy. With luck, such findings ultimately will be clinically helpful.
In the seminal article on viral kinetics in hepatitis C, Neumann et al. used 3 differential equations to resolve the decrease in HCV-RNA levels during daily interferon therapy into 2 distinct phases. The first phase was the initial sharp decay in HCV-RNA levels that occurred within the first 24 hours of therapy. The researchers related this first phase decline to the antiviral "efficacy" (e) of interferon in blocking viral production and secretion. The value of e was calculated to vary from 0 (no blocking) to 1 (100% blocking). The second phase decline described the subsequent, more gradual decrease in HCV-RNA levels, which the researchers attributed to the rate of killing or clearance of virally infected cells (f), superimposed on . The differential equations also provided estimates of the spontaneous clearance of
HCV RNA from serum (viral half-life) and the rate of virus production. These mathematic formulae accurately predicted both group and individual data during therapy and revealed that HCV has a high rate of replication (1012 virions/d) and short half-life in serum (1.5 to 4.6 hours), particularly in comparison with hepatitis B virus (1011/d and 24 hours) and human immunodeficiency virus (1010/d and 5.8 hours) analyzed in a similar manner.
Herrmann et al. from Germany analyzed HCV-RNA levels from 34 patients participating in the large, multicenter, registration trial of peginterferon alfa-2a and ribavirin in chronic hepatitis C. Patients received either peginterferon alfa-2a alone, peginterferon alfa-2a with ribavirin, or standard interferon alfa-2b and ribavirin. The investigators focused on 2 issues: (1) what was the effect of addition of ribavirin on decay of HCV-RNA levels during interferon therapy? and (2) were the patterns of virus decay different with pegylated compared with standard interferon in combination therapy? The investigators found that ribavirin did not affect e but did affect f kinetics, increasing it 2-fold. Second, the investigators found that the decay of HCV RNA followed a triphasic pattern in a large proportion (61%) of patients. This pattern was characterized by a typical first phase, a flattened second phase of variable duration, and a third phase that ultimately correlated with viral clearance. This triphasic pattern was not unique to peginterferon therapy. The investigators hypothesized that the third phase represented immune clearance of HCV-infected hepatocytes.
The second report by Layden et al. (in the same Hepatology issue) from the United States analyzed HCV-RNA levels from 35 patients with chronic hepatitis C treated with either standard interferon alone or with interferon combined with ribavirin. African-American and white patients were analyzed separately. These investigators focused on 2 issues: (1) what was the effect of adding ribavirin to interferon on the decay of HCV-RNA levels? and (2) were the kinetic patterns different between African-American and
white patients? The investigators reported that ribavirin had no apparent effect on either e or f, and that in comparison to white patients, African-American patients had lower values for both e (0.89 vs. 0.98) and f (0.13 vs. 0.20).
These results are interesting and important and support previous studies that have reported diminished response rates and diminished viral inhibition by interferon among African-American patients. However, these results are different from those reported by Herrmann et al., in that Layden-Almer et al. found no significant effect of ribavirin on f and reported no evidence for a triphasic response. The potential reasons for these differences are many, but they most likely represent differences in interferon preparations, dose and dosing schedules, and problems in statistical analyses.
Most importantly, the study by Layden et al. used high daily doses of interferon (10 million U of interferon daily for the first month), whereas the study by Herrmann et al. used standard doses and dosing (3 million U thrice weekly of standard interferon and 180 µg weekly of peginterferon alfa-2a). Thus, the unusual flat second phase and new third phase patterns reported by Herrmann et al. could be interpreted as being caused by intermittence of interferon dosing and consequent intermittence of full interferon action (breakthrough). The differential equations used assume a constancy of the of interferon action in blocking viral production. Therefore, one interpretation of these results is that the current dosing recommendations for standard and peginterferon are not optimal. In support of this were the
reported results for in the German patients of 0.36, 0.63, and 0.67 compared with that in the U.S. white patients of 0.98.
A second problem with both studies was the small number of patients studied and the potential for type II statistical error. Thus, the lack of effect of ribavirin in the study by Layden-Almer et al. may have been due to the low numbers of patients. Indeed, the author showed that the results for e and f were not distributed normally: a sizeable proportion of patients had little or no second phase response (flat response), and some had neither a first nor second phase response (null response). Including results from these patients to calculate an average to compare groups for e or f is, therefore, problematic. When the investigators analyzed patients with a flat second phase response separately, they found little or no difference between African-American and white patients. From this one can conclude
that differences between the races were categorical: that the frequency of a flat or null response to interferon therapy was higher among African-American than white patients, but the quality of response (and the e or f) among those who had a response was similar in the 2 racial groups. These analyses have important clinical implications because they suggest that no alteration in interferon dose or dosing will be adequate to overcome this true resistance to interferon and that the mechanisms underlying this problem require separate focus, new approaches, and new drugs.
What is to be learned and what is to be done with the results from these 2 studies? The differences in results point to the major issues that need to be addressed.
First, these 2 studies point out the need for standardization of nomenclature and presentation of results in this field. The terms effectiveness and killing of virally infected cells are confusing and may be inaccurate, and should be modified to accurately reflect the assumptions made in the kinetic model. Furthermore, investigators should state clearly whether they are presenting first and second phase viral decline (i.e., the absolute decline in viral load over a specific period) or e and f (which are calculated from the differential equations) because they are not the same. Finally, changes in viral levels are better expressed as log10 decreases rather than percentages: thus, 90% is 1.0, 99% is 2.0, and 99.9% is a 3.0 log decline.
Second, these studies suggest that further analysis of peginterferon dosing is needed, perhaps comparing twice weekly with weekly dosing (keeping the total weekly dose constant) and reassessing the patterns of decline and response rates (including both end-of-treatment and sustained response rates). Such studies should be performed in patients with features that predict a poor response such as those with genotype 1, high levels of HCV RNA, higher body mass index, and older age.
Finally, adequately powered studies are needed to assess the clinical use of viral kinetics in predicting ultimate response or nonresponse to therapy. These studies should use a dose regimen that is clinically relevant rather than high-dose daily standard interferon therapy that may be better for accurately measuring kinetics. Viral kinetics also might be used to assess the optimal dose and duration of therapy and whether these can be tailored to the individual patient (perhaps by assessing e and f). It is time for the clinical relevance of viral kinetics to be tested in prospective, controlled, adequately powered, and clinically valid studies.
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