Ribavirin in chronic hepatitis C: past and future

Pegylated IFN-α and ribavirin is, at present, the standard of care for chronic hepatitis C, leading to a sustained virological response (SVR) in 45–80% of the patients depending on the genotype [1–3]. Ribavirin is a key factor of this therapeutic strategy, doubling the SVR rate obtained with pegylated interferon alone [2,4]. Ribavirin mainly acts by preventing relapses, especially in genotype 1 patients [5]. The near future for the treatment of chronic hepatitis C is specifically targeted antiviral therapy (STAT-C). However, STAT-C and, notably, protease inhibitors are still used in combination with pegylated interferon and ribavirin. The absence of ribavirin in the drug combination resulted in a relapse rate of 48% [6]. Finally, pharmacological and pharmacodynamic data showed that the current use of ribavirin according to bodyweight (BW) can lead to inadequate individual exposure to ribavirin, which in turn impacts the SVR, suggesting that optimizing individual ribavirin dose by monitoring the drug blood levels might be a key strategy to improve the SVR rate, particularly in difficult-to-treat patients [7–10].

Clinical past of ribavirin

Ribavirin is a guanosine analog synthesized in the 1970s that has a broad-spectrum activity against DNA and RNA viruses. It was initially approved in clinics to treat respiratory syncytial virus infection in children and then Lassa fever [11–13]. In 1991, Reichard et al. were the first to show that ribavirin alone can reduce serum alanine aminotransferase activity in chronic hepatitis C patients [14]. In 1994, Brillanti et al. demonstrated that ribavirin associated with IFN-α gave promising results [15]; these results were confirmed in 1995 by the first randomized study [16], showing that ribavirin could double the SVR rate obtained with IFN-α alone. The benefit was particularly significant for genotype 1 patients. The antiviral effect of ribavirin and pegylated IFN-α was also found to be synergistic [1,2], with ribavirin preventing relapses and breakthroughs [5].

Ribavirin maintenance dose seems to be crucial for SVR. Data from two pivotal clinical trials highlighted that the total daily dose of ribavirin (>10.6 mg/kg of BW), especially during the first 3 months, was predictive of SVR [1,17], and that ribavirin had to be administered for the full treatment duration [5]. A higher starting dose of ribavirin reduced relapse in a controlled trial where patients were randomized to receive either 800 mg or 1000–1200 mg of ribavirin [3]. In a pilot study, Lindhal et al. suggested that the early use of very high doses of ribavirin (mean daily dose: 2.5 g [1.6–3.6 g]) led to a higher SVR rate in genotype 1 patients [18].

Ribavirin mechanisms of action

So far, the mechanisms of action of ribavirin are not well understood. The antiviral mechanism is probably the best documented. Once ribavirin enters the cell, it is phosphorylated to ribavirin mono-, di- and tri-phosphate (RMP, RDP and RTP). The erroneous incorporation of RTP into replicating RNA strands may inhibit chain elongation and cause chain termination [19].

The inhibition of inosine monophosphate dehydrogenase (IMPDH), which is an enzyme essential for the de novo synthesis of GTP, is a potential mechanism of action: it may inhibit RNA synthesis and promote the incorporation of RTP in place of GTP. However, ribavirin’s impact on IMPDH is weaker than that of mycophenolate mofetil (MMF). In the replicon system, the addition of guanosine in excess completely restores the HCV replication rate with MMF, but only partially with ribavirin [20].

The mutagenesis hypothesis has been recently prioritized. Ribavirin would lower the viral infectivity by virus mutagenesis. This mutagenesis might be higher in the presence of interferon. In the HCV replicon system, ribavirin was shown to inhibit the ability of progeny subgenomic replicons to transfect new cells, although the replication rate remained unaffected [21,22]. However, a recent in vivo study undermined this hypothesis [23].

The most attractive hypothesis was recently proposed by Feld et al.[24]. The authors suggested that ribavirin enhances the response of interferon-stimulated genes to pegylated interferon and that pretreatment with ribavirin may heighten this induction, making cells more responsive to interferon and increasing the production of endogenous interferon.

Following the onset of double therapy with interferon and ribavirin, one can observe a two-phase profile of plasma RNA decline: a rapid first phase decline observed for 1–2 days [25] attributed to a reduction in production and or release of new virions due to interferon action, and a slower second phase attributed to the elimination of infected cells. The influence of ribavirin on the first-phase decline of HCV RNA is weak and transient (0.5 log) and only observed in patients treated with ribavirin alone [26]. The impact on the second slope is more probable and would involve mutagenesis and more likely enhancement of the interferon-stimulated genes’ response.

Pharmacokinetics of ribavirin

Individual factors probably influence ribavirin’s pharmacokinetics. There is no significant correlation between ribavirin exposure and total or weight-standardized dose. This reflects the large interindividual variability of the dose–concentration relationship. In a study by Bruchfeld et al., the effect of renal function on ribavirin clearance was only apparent when creatinine clearence was less than 34 ml/min [27]. Other studies showed that ribavirin clearance was affected by BW, gender, age and serum creatinine, but that these four covariates only explained 27–40% of its interindividual variability [27,28]. It is possible that the liver plays a role, although Glue et al. showed that hepatic dysfunction had no substantial influence on the apparent clearance of ribavirin [29]. Finally, high-fat meals can increase ribavirin bioavailability by 46% compared with the fasting state [30].

Data from the literature showed that, following a single oral dose, the plasma concentration of ribavirin exhibits a three-phase profile: a rapid absorption phase with a mean time to the maximum concentration (t_max) of approximately 1.5 h; a rapid distribution phase (half-life of ∼3.7 h); and a long terminal elimination phase, the last measurable concentration time point being at approximately 100 h post-dose [19,31].

Ribavirin & anemia

Hemolytic anemia is the major adverse event that impairs the ability of patients to remain on full doses of the drug.

In nucleated cells, ribavirin phosphorylation is reversible, as opposed to anucleated blood cells where its concentrations can exceed that found in plasma by 60-fold. RTP enters in competition with ATP and the oxydative stress results in the alteration of the erythrocyte membrane fluidity and extravascular hemolysis. Ribavirin half-life in the body is 40 days, equal to the half-life of erythocytes before their destruction in the spleen [32]. This hemolytic mechanism is reversible and dose dependent. Moreover, interferon contributes to anemia by its bone marrow-suppressive effect. The prevalence of anemia during standard bitherapy is high, with hemoglobin (Hb) less than 11 g/dl in 30% and less than 10 g/dl in 9–13% of patients [1,2].

In total, 10–15% of patients present with severe anemia, with a decline of more than 5 g/dl in Hb levels. In real life, anemia is responsible for 36% of the HCV treatment interruptions [33]. Erythropoietin improves the quality of life and can help to maintain the full dose of ribavirin by significantly increasing the Hb level, with acceptable tolerance [34,35]. However, its impact on SVR has not been demonstrated [36].

Monitoring ribavirin blood levels

The aforementioned clinical trials all emphasized the crucial role of the cumulative dose of ribavirin on SVR, especially during the first 3 months [1,2,17]. Anemia is linked to ribavirin concentration, even if this relationship is highly variable [7,9]. Moreover, ribavirin has a typical profile for drug monitoring: its half-life is long and there is a large interindividual variability of its dose–concentration relationship, that is, of its pharmacokinetics, despite dose adjustment on BW [7,9,37].

It has recently been demonstrated that patients with genotype non-2 non-3 HCV who are partial or even complete early responders (HCV RNA level <2 logs IU or undetectable at week 12) have a probability of only 27 and 60% of SVR, respectively, and may greatly benefit from early monitoring of ribavirin concentrations. On the contrary, rapid virological responders (RVRs; HCV RNA undetectable at week 4), who represent only 16% of genotype 1 patients, have a high probability (80–90%) of SVR. These patients, who are very sensitive to the effect of interferon, may not so much require the maintenance of an optimal ribavirin exposure [38,39].

A recent study in 35 HCV–HIV-coinfected patients suggested that the influence of ribavirin concentration on the SVR could be linked to the HCV genotype (i.e., clear influence on the virus response only for genotypes 1 or 4) [40]. However, a Swedish study, the NORDynamic trial, conducted in a large population of 382 genotype 2- and 3-monoinfected patients found a statistical relationship between residual ribavirin concentrations at week 4 of treatment and SVR. The positive predictive value for SVR at week 4 was 91% for a ribavirin concentration more than 2 mg/l (p = 0.02) [41]. Therefore, ribavirin residual concentrations seem to have an impact on SVR in all genotypes but this impact is probably greater in genotype 1 patients.

In the literature, the monitoring of ribavirin blood levels was commonly based on trough concentration measurements at weeks 4, 12 or 24 after the initiation of treatment [7,10]. However, multiparametric statistical analyses showed that the relationship between trough concentration and SVR was less influential than the viral load and of course the genotype [7]. A trough concentration above 2 mg/l at week 4 was predictive of SVR [10]. Arase et al. demonstrated that high ribavirin trough concentrations at week 8 (>3.5 mg/l) were linked with a higher rate of ribavirin discontinuation because of anemia and suggested not to exceed this limit, considered as a security test [42]. Another attempt of a single-point monitoring strategy, measuring at week 12 ribavirin concentration, 2–4 h after the morning dose (close to the peak concentration), showed a significant relationship with SVR in genotype 1 patients [8]. However, all these approaches did not allow early ribavirin dose adjustment.

A patient’s global exposure to ribavirin, as evaluated by the AUC, seems more pertinent in terms of exposure–effect relationship than any single time point. We recently showed, in patients infected by genotype 1 HCV, that ribavirin plasma exposure after the first dose (i.e., interdose AUC_0–12h or abbreviated AUC_0–4h) was significantly and strongly linked with SVR, whereas AUCs determined at week 12 and 24 and trough concentrations at day 0 and week 12 were not [9]. After the first dose, AUC_0–12h was only slightly affected by the elimination phase (due to the very long half-life of the drug), while abbreviated AUC_0–4h was clearly limited to ribavirin absorption and distribution.

Finally, based on receiver operating characteristic (ROC) curve analysis, we proposed a minimum AUC_0–4h threshold of 1755 µg.h/l at day 0 as a target for ribavirin dose adjustment [9]. On a practical purpose, AUC_0–4h can be estimated using three blood samples (0.5, 1 and 2 h after the first dose) and Bayesian estimation.

The relationship between ribavirin concentration and early virological response (EVR) is unclear. In a retrospective study, Donnerer et al. found no correlation between ribavirin levels at week 12 and EVR [43]. By contrast, recent findings in HCV–HIV-coinfected patients suggested that a high ribavirin concentration could improve the EVR rate [44]. Ribavirin plasma AUC_0–12h or AUC_0–4h after the first dose were significantly linked to EVR, but to a lesser extent than to SVR and RVR [9]. Explanations for these discrepancies could be the low positive predictive value of EVR for SVR, the retrospective design of most of the studies and, in some of them, the lack of information on the time of sample collection.

Several analytical methods have been developed for the determination of ribavirin in biological fluids [45], including radioimmunoassays, capillary electrophoresis or liquid chromatography coupled with mass-spectrometric or UV detection.

The choice of plasma or serum for ribavirin concentration measurements might be important: ribavirin concentrations would be more reproducible in plasma than in serum in intensive sampling pharmacokinetic studies [7]. This apparent discrepancy might be due to a poorer stability of serum levels during the phase of blood clotting at ambient temperature. Indeed, as ribavirin can be exchanged between blood cells and serum or plasma in the sampling tube, the samples must be immediately put on ice, taken to the laboratory within 2 h and then immediately centrifuged at +4°C. After that, the plasma or serum samples can be stored at -20°C. Another assumption recently made by Dahari et al. is that measuring intra-erythrocyte ribavirin concentrations could be a more relevant approach than measuring plasma levels, based on the assumption that intra-erythrocyte ribavirin accumulation early on during treatment would be higher in patients more likely to achieve SVR [46]. However, the irreversible intra-erythrocyte accumulation of RTP is probably not representative of the drug turnover in other target cells, such as hepatocytes, where ribavirin activation into triphosphate metabolites is reversible. The intra-erythrocyte determination of ribavirin should certainly be further investigated, as there is no evidence so far that it is a better approach than that of plasma concentrations.

Conclusion

Ribavirin remains a key factor of the standard of care of chronic hepatitis C. In the era of specifically targeted antiviral therapy for HCV (STAT-C) therapy, convincing data suggest that there is no getting away from ribavirin in the near-future in combination therapies for chronic hepatitis C. The purpose should be thus to optimize ribavirin utilization in double and then triple therapies. The best way is probably to individually adjust the dose so that each patient could reach the highest probability of SVR with limited side effects. Global exposure after the first dose of ribavirin is an early and robust pharmacokinetic predictor of SVR [9]. Therapeutic drug monitoring based on this approach will be tested soon in a French prospective, randomized fixed-dose versus concentration-controlled trial. The monitoring of ribavirin might also be useful to prevent relapses and drug mutations in future tri-therapies [6].

The ability of ribavirin trough concentration at weeks 4 or 12 to predict SVR seems to be weaker [37], but measurement of ribavirin trough concentration at week 4 and 8 could be useful to prevent the potential toxicity of ribavirin when administered at high doses in certain individuals in order to reach the target AUC and to check patient compliance.

Ribavirin concentrations can be measured either in plasma or in serum, by either high-performance liquid chromatography coupled to UV or mass-spectrometry detection, but the key points are to keep blood samples on ice and reduce the time between blood sampling and centrifugation to less than 2 h.

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.

No writing assistance was utilized in the production of this manuscript.