Suppression of hepatitis C virus replication by protein kinase C-related kinase 2 inhibitors that block phosphorylation of viral RNA polymerase

Summary.  Hepatitis C virus (HCV) infection is a serious threat to human health worldwide. In spite of the continued search for specific and effective anti-HCV therapies, the rapid emergence of drug-resistance variants has been hampering the development of anti-HCV drugs designed to target viral enzymes. Targeting host factors has therefore emerged as an alternative strategy offering the potential to circumvent the ever-present complication of drug resistance. We previously identified protein kinase C-related kinase 2 (PRK2) as a cellular kinase that phosphorylates the HCV RNA-dependent RNA polymerase (RdRp). Here, we report the anti-HCV activity of HA1077, also known as fasudil, and Y27632, which blocks HCV RdRp phosphorylation by suppressing PRK2 activation. Treatment of a Huh7 cell line, stably expressing a genotype 1b HCV subgenomic replicon RNA, with 20 μ m each of HA1077 and Y27632 reduced the HCV RNA level by 55% and 30%, respectively. A combination of the inhibitors with 100 IU/mL interferon α (IFN-α) significantly potentiated the anti-HCV drug activities resulting in approximately a 2-log₁₀ viral RNA reduction. We also found that IFN-α does not activate PRK2 as well as its upstream kinase PDK1 in HCV-replicating cells. Furthermore, treatment of HCV-infected cells with 20 μ m each of HA1077 and Y27632 reduced the levels of intracellular viral RNA by 70% and 92%, respectively. Taken together, the results identify PRK2 inhibitors as potential antiviral drugs that act by suppressing HCV replication via inhibition of viral RNA polymerase phosphorylation.     

Interferon-α plus amantadine in chronic hepatitis C resistant to interferon alone: a pilot randomized study

The optimal therapy for patients with chronic hepatitis C who have not responded to interferon (IFN) is still an unsolved issue. The aim of this study was to evaluate the efficacy and tolerability of a high dose of IFN-α2a plus amantadine for chronic hepatitis C patients who were non-responders to a previous course of IFN.
Forty consecutive patients with chronic hepatitis C, genotype 1b, who had not responded to IFN-α, were randomized to receive: (i) IFN 4.5  M U daily plus amantadine 200 mg/day for 4 weeks and then IFN 6  M U thrice weekly plus amantadine 200 mg/day for an additional 5 months (group A) or (ii) IFN alone at the same dosage and duration (group B). After 1 month of therapy, normal alanine aminotransferase (ALT) values were observed in three of 21 (14.3%) patients in group A and in three of 19 (15.8%) in group B; serum hepatitis C virus (HCV)-RNA clearance was observed in one patient (4.8%) in group A and in six (31.6%) in group B. At the end of treatment, six patients (28.6%) in group A and three (15.8%) in group B had normal ALT levels; however, HCV-RNA in serum was detectable in all of them at levels comparable to the basal values; an ALT relapse occurred within 3 months of stopping therapy. The combination of daily IFN plus amantadine was ineffective in this setting.     

Predictive value of IgM antibodies to hepatitis C virus in patients with chronic hepatitis C undergoing interferon-α therapy. Analysis by two different methods

SUMMARY. To determine the predictive value of IgM anti-hepatitis C virus (HCV) testing in patients with chronic hepatitis C infections undergoing interferon-α (IFN-α) therapy, IgM anti-HCV reactivity was analysed by two different methods (non-commercial and commercial) in 19 patients and monitored at times 0 (pre-treatment), 3, 6, 12. and 24 months during follow-up. Eight patients were non-responders, five remained in sustained response 1 year after stopping treatment, and six had a relapse. No correlation between alanine transaminase (ALT) levels and IgM anti-HCV reactivity was found by either method in baseline samples. In addition, neither the presence nor absence of IgM anti-HCV in baseline samples, nor the loss of specific IgM reactivity during treatment, had any predictive value. Finally, no other parameters analysed (age, sex, risk group and histological diagnosis), were significantly associated with IgM anti-HCV reactivity in our study. In summary, these results suggest that baseline detection and monitoring of IgM anti-HCV reactivity are not useful in predicting the sustained response to IFN-α therapy in chronic hepatitis C infection.     

Enhanced antiviral effect in cell culture of type 1 interferon and ribozymes targeting HCV RNA

We have recently shown that the replication of an HCV-poliovirus (PV) chimera that is dependent upon the hepatitis C virus (HCV) 5' untranslated region (UTR) can be inhibited by treatment with ribozymes targeting HCV RNA. To determine the antiviral effects of anti-HCV ribozyme treatment in combination with type 1 interferon (IFN), we analysed the replication of this HCV-PV chimera in HeLa cells treated with anti-HCV ribozyme and/or IFN-α2a, IFN-α2b, or consensus IFN. The anti-HCV ribozyme, or any of the IFNs alone have significant inhibitory effects on HCV-PV replication compared to control treatment (≥ 85%, P  < 0.01). The maximal inhibition due to IFN treatment (94%, P  < 0.01) was achieved with ≥ 50 U/ml for either IFN-α2a or IFN-α2b compared to control treatment. A similar level of inhibition in viral replication could be achieved with a 5-fold lower dose of IFN if ribozyme targeting the HCV 5' UTR was given in combination. For consensus IFN, the dose could be reduced by > 12.5-fold if ribozyme targeting the HCV 5' UTR was given in combination. Conversely, the dose of ribozyme could be reduced 3-fold if given in combination with any of the IFN preparations. Moreover, treatment with low doses (1–25 U/mL) of IFN-α2a, IFN-α2b, or consensus IFN in combination with anti-HCV ribozyme resulted in > 98% inhibition of HCV-PV replication compared to control treatment ( P  < 0.01). These results demonstrate that IFN and ribozyme each have a beneficial antiviral effect that is augmented when given in combination.     

Early Loss of Serum Hepatitis C Virus RNA Can Predict a Sustained Response to Interferon Therapy in Patients with Chronic Hepatitis C

Objectives : We evaluated whether the loss of serum hepatitis C virus (HCV) RNA early in interferon (IFN) therapy would indicate a subsequent response to IFN therapy. Methods : One hundred fourteen patients with chronic hepatitis C were treated with IFN-α for 24 weeks. All patients were positive for anti-HCV antibodies and serum HCV RNA. Serum HCV RNA was measured by highly sensitive and specific RT-PCR (modified Amplicor HCV). Results : Of 114 patients who were treated with IFN-α for 24 weeks, 22 of 29 patients (75.9%) who lost HCV RNA at the first week of treatment, 5 of 14 patients (35.7%) who lost HCV RNA at the second week, and 2 of 16 patients (12.5%) who lost HCV RNA at fourth week were judged as sustained responder (SR). The SR rate was significantly higher in patients who lost HCV RNA at the first week of therapy ( p < 0.05). On the contrary, none of 55 patients who retained HCV RNA during the first 4 weeks of IFN therapy were judged as SR. Concerning the patients who lost HCV RNA at the first week of therapy, there were no significant differences in the SR rate in either HCV genotype (1b, 2a, and 2b). Conclusions : Our study confirms that the early response to IFN (loss of HCV RNA at the end of the first week of IFN therapy) can be a predictor of the subsequent sustained response to IFN therapy. Additionally, positivity of HCV RNA at the fourth week of IFN therapy can be a predictor of the subsequent non-sustained response to IFN therapy.     

Expression of interferon-alpha subtypes in peripheral mononuclear cells from patients with chronic hepatitis C: a role for interferon-alpha5

Interferon (IFN)-α is a family of antiviral proteins encoded by different genes. The biological significance of the existence of various IFN-α subtypes is not clear. We have investigated the interferon system in chronic hepatitis C virus (HCV) infection, a disease that responds to interferon-α2 therapy in only a limited proportion of cases. We analysed the expression of interferon regulatory factor (IRF)-1, IRF-2, and IFN-α subtypes in nonstimulated and Sendai virus-stimulated peripheral blood mononuclear cells (PBMC) from HCV infected patients and healthy controls. We observed that the IRF-1 mRNA and IRF-1/IRF-2 ratios were increased in PBMC from hepatitis C patients with respect to normal subjects. Sendai virus stimulation of PBMC led to a significant increase in the levels of IRF-1, IRF-2 and IFN-α mRNAs and in the production of IFN-α protein with respect to basal values in healthy controls as well as in patients with HCV infection. In addition, we found that while natural HCV infection induced increased IFN-α5 expression in PBMC, in vitro infection of these cells with Sendai virus caused a raise in the expression of IFN-α8 in both patients and normal controls. In summary, our results indicate that virus-induced activation of the IFN system in human PBMC is associated with selective expression of individual IFN-α subtypes, IFN-α5 being the specific subtype induced in PBMC from patients with chronic HCV infection.     

Treatment of chronic hepatitis C virus infection in patients with cirrhosis

Chronic hepatitis C virus (HCV) infection eventually leads to cirrhosis in 20–30% of patients and to hepatocellular carcinoma (HCC) in 1–5% of patients. Rates of sustained virological response with standard interferon-α (IFN-α) are low in patients without cirrhosis (generally < 20%) and are even lower in those with cirrhosis. Combination therapy with IFN and ribavirin improves response rates in patients with chronic hepatitis C without cirrhosis, and the results from subgroups of HCV-infected patients with advanced fibrosis or cirrhosis are encouraging. Importantly, treatment with IFN slows progression of liver fibrosis, regardless of HCV genotype or early response to therapy, and reduces the risk of HCC by two- to fivefold. The risk of development of HCC is also lower in patients who show at least a partial response to IFN therapy compared with those who show no response. There is a clear need for more definitive studies of treatment in patients with chronic hepatitis C and cirrhosis, ideally using therapies with greater efficacy. Nonetheless, based on the potential to slow the progression of liver fibrosis (regardless of treatment response) and to reduce the risk of HCC, a greater number of HCV-infected patients with cirrhosis should be considered as candidates for IFN treatment. Preliminary data indicate that pegylated IFNs have improved virological response rates and may have additional clinical benefits in the prevention or reduction of fibrosis and retardation of progression of cirrhosis and HCC in these patients.     

Prediction of relapse or sustained response in biochemical responders by serum hepatitis C virus RNA monitoring during interferon therapy

Normalization of serum aminotransferase levels is achieved in ≈ 50% of chronic hepatitis C patients treated with interferon (IFN); however, in about one-half of these patients the hepatitis relapses after therapy. In this study we investigated the efficacy of serum hepatitis C virus (HCV) RNA monitoring during IFN therapy to predict the outcome of a biochemical end-of-treatment (ETR) response. Eighty patients with chronic hepatitis C received leucocyte (natural) IFN-α (13 patients) or recombinant IFN-α2a (67 patients). Antiviral therapy was given for 12 months to 43 (53.7%) responders and this group was analysed further. During follow-up, 15 relapsed and 28 showed a sustained response (median follow-up 50 months, range 39–67 months). Viraemia was monitored at baseline, and at months 1, 3, 6, 9 and 12 of treatment, by nested polymerase chain reaction (PCR) (sensitivity 10–100 copies ml^–1). A combination of positive nested PCR and HCV RNA values at the 3rd and 6th months of treatment was 100% predictive of relapse (sensitivity, 66.6%; specificity, 100%). A combination of negative nested PCR and HCV RNA values at the 1st and 3rd months of treatment was 100% predictive of sustained response (sensitivity, 39.3%; specificity, 100%). In conclusion, serum HCV RNA monitoring is an appropriate and reliable tool for predicting early outcome of the biochemical ETR response after IFN discontinuation. This could be useful in the modulation of therapeutic management of chronic hepatitis C.     

Interferon-α-2B and ribavirin in combination for chronic hepatitis C patients not responding to interferon-α alone: an italian multicenter, randomized, controlled, clinical study

Objective: The aim of the study was to assess the efficacy of interferon (IFN)-α-2b and ribavirin in combination in the treatment of chronic hepatitis C (CHC) patients unresponsive to a previous treatment with IFN-α−2b alone.
Methods: We conducted a randomized study in 303 CHC patients. One hundred fifty-two patients received subcutaneous administration of recombinant IFN-α−2b (3 MU thrice weekly) and ribavirin (1000–1200 mg/daily per os ), whereas 151 received IFN-α−2b alone (6 MU thrice weekly). Both ribavirin and IFN-α-2b were given for 24 wk, regardless of treatment response. Alanine aminotransferase (ALT) levels and HCV RNA titer were checked during the treatment period and for a further 24 wk.
Results: Normal ALT levels were observed in 64.5% of the patients treated with IFN-α and ribavirin and in 22.6% of the patients treated with IFN-α alone. In the group of patients receiving IFN-α and ribavirin HCV RNA was not detectable in 40% of patients responders and remained undetectable in 44.2% of sustained responders. In the group of patients receiving IFN-α alone HCV RNA was not detectable in 24.2% of patients responders and remained not detectable in 33.3% of sustained responders.
Conclusion: A 24-wk treatment course with IFN-α and ribavirin given to patients with a previous lack of response to IFN-α alone offers a chance of a sustained biochemical and virological response, at least in a subset of such patients. The role of long-term therapy in inducing prolonged remission still remains to be explored.     

Quantitative assessment of hepatitis C virus RNA in peripheral blood mononuclear cells during therapy with interferon-α2a

A significant number of patients with hepatitis C (HCV) treated with interferon (IFN) will initially clear their serum of HCV RNA, but will then have recurrence of viraemia either during or after therapy. One proposed mechanism for relapse is that HCV may persist in peripheral blood mononuclear cells (PBMCs) and that the PBMCs serve as a 'viral reservoir' that is resistant to IFN. To address this hypothesis, we performed serial, quantitative polymerase chain reaction (PCR) of HCV RNA in serum and PBMCs from 26 consecutive patients treated with IFN-α2a. Of the 26 patients, 11 (42%) did not clear virus from their serum during therapy and were termed non-responders. Five patients (19%) had sustained clearance of virus from serum and were termed complete responders. The remaining 10 patients (39%) initially eliminated HCV RNA from their serum, but had relapse of viraemia. They were termed partial responders. In all 10 partial responders HCV RNA was undetectable in PBMCs at the same time that it was undetectable in serum. When virus recurred in serum, it was preceded by or occurred at the same time as the return of virus in PBMCs. The results of our study indicate that PBMCs did not serve as an IFN-resistant 'viral reservoir' during therapy. Partial responders who transiently cleared virus from serum also cleared virus from PBMCs and the presence or titre of HCV RNA in PBMCs at the initiation of therapy did not predict response to therapy.     

Dynamics of hepatitis C virus quasispecies turnover during interferon-alpha treatment

Summary. Interferon- α (IFN) has been shown to accelerate the evolution of hepatitis C virus (HCV) variants (quasispecies) in nonresponder patients. Different sensitivities of HCV variants to IFN are discussed as a possible mechanism. In the present study, quasispecies were investigated in detail by a newly established and validated direct solid-phase sequencing of the hypervariable region 1 (HVR1), during the initial 3 months of IFN therapy. According to single strand conformation polymorphism (SSCP) analysis, 14 of 26 (54%) virologic nonresponders with quasispecies evolution were identified. Six representative patients with SSCP changes were selected for frequent HVR1 sequencing. Pre-existing variants were identified by cloning and sequencing of the pretreatment serum HCV sample. In one patient the major type was substituted by a minor variant within 3 days of treatment while in the majority of patients the pretreatment major type did not decline before days 26–57 of treatment. Total serum HCV RNA levels remained constant in all patients. In conclusion, although quasispecies evolution during IFN therapy is common, it occurs after a wide range of time intervals after initiation of therapy. Thus, nonresponse to IFN cannot exclusively be explained by changes in the quasispecies.     

Zinc supplementation enhances the response to interferon therapy in patients with chronic hepatitis C

We evaluated the synergistic effect of zinc supplementation on the response to interferon (IFN) therapy in patients with intractable chronic hepatitis C in a pilot study using natural IFN-α with or without zinc. No clinical differences were observed between patients treated with IFN alone ( n =40) and IFN with polaprezinc (IFN + Zn, n =35). All patients were positive for HCV genotype Ib and had more than 10⁵ copies of the virus/mL serum. Ten million units of natural IFN-α was administered daily for 4 weeks followed by the same dose every other day for 20 weeks. In the IFN + Zn group, patients received an additional dose of 150 mg/day polaprezinc orally throughout the 24-week IFN course. No additional side-effects of polaprezinc were noted but four out of 40 IFN alone treatment and three out of 35 IFN + Zn group withdrew because of side-effects. Complete response (CR) was defined as negative HCV RNA in the serum on PCR and normal aminotransferase level 6 months after therapy. Incomplete response (IR) was normal liver enzyme and positive serum HCV RNA. Both of them were evaluated at the 6 months after the completion of the treatment. Patients with higher levels of serum HCV (more than 5 × 10⁵ copies/mL) had little response in both treatment groups. Patients with moderate amount of HCV (10⁵ to 4.99 × 10⁵/mL) showed high response rates in combination group (CR: 11/27, 40.7%; CR + IR 15/27, 64.3%), better than IFN alone (CR: 2/15, 18.2%; CR + IR: 2/15, 18.2%). Serum zinc levels were higher in patients with IFN + Zn group than in the IFN group. Our results indicate that zinc supplementation enhances the response to interferon therapy in patients with intractable chronic hepatitis C.     

Long-term titrated recombinant interferon-α2a in chronic hepatitis C: a randomized controlled trial

Summary. The efficacy and tolerability of 12-month treatment with titrated doses of recombinant interferon-α2a (IFN-α2a) in chronic hepatitis C were studied in 67 consecutively recruited patients randomly assigned either to a starting dose of IFN-α2a 6 MU, subsequently adjusted to the serum alanine aminotransferase (ALT) response ( n = 35), or to no therapy ( n = 32; controls). End-of-treatment ALT levels were normal and hepatitis C virus (HCV) RNA was negative by nested polymerase chain reaction (PCR) in 17 (49%) treated patients compared to none of the controls ( P < 0.001). During the 12 months after stopping treatment the number of patients who remained in remission was eight (23%) and one respectively (4%) ( P = 0.031). Follow-up liver biopsy showed reduced hepatic inflammation in 80% of treated patients and in 29% of controls ( P < 0.001). The eight sustained responders and 2 7 non-responders or relapsers received similar mean total doses of IFN (565 MU vs 545 MU) and had a similar incidence of anti-IFN neutralizing anti-bodys (13% vs 19%). Absence of cirrhosis was the only independent pretreatment parameter that predicted a sustained response. In conclusion, a mean cumulative dose of IFN 549 MU, titrated over 12 months, was well tolerated, and resulted in the long-term clearance of HCV RNA and normal ALT levels in 23% of patients.     

Clinical efficacy of intramuscular human interferon-βvs interferon-α2b for the treatment of chronic hepatitis C

Summary. We have conducted a randomized study to compare the efficacy and tolerance of human interferon (IFN) β vs recombinant IFN-α2b in patients with chronic active hepatitis C. Forty patients were included: 21 received IFN-α (group A) and 19 IFN-β (group B). IFN was administered intramuscularly at a dose of 6 MU three times a week (tiw) for 2 months (induction phase), followed by 3 MU tiw for 4 months. Clinical, epidemiological and pathological features were similar in the two groups. Normal alanine aminotransferase (ALT) values at the end of treatment was regarded as a response to therapy and the response rate was 57% (12/21) in group A and 5.2% (1/19) in group B ( P < 0.01). Both types of IFN induced a significant decrease in mean ALT values by the end of the induction phase ( P < 0.01). When the dose was reduced to 3 MU, a marked, but not significant increase in ALT, was seen in group B, whereas no increase was seen in group A. IFN-β was better tolerated and haematological adverse effects (platelet and leucocyte decrease) were less pronounced with IFN-β. Hence, human IFN-β was less effective than IFN-α in treating chronic hepatitis C virus (HCV). Doses of IFN-β of 3 MU intramuscular (IM) tiw were clearly insufficient and it remains to be established whether higher doses of intramuscularly IFN-β can be useful.     

Effect of treatment with interferon α-2b and ribavirin in patients infected with genotype 2 hepatitis C virus

Aim:  Nearly 20% of chronic hepatitis C (CHC) patients with genotype 2 hepatitis C virus (HCV) infection are not curable, even by interferon (IFN)–ribavirin combination therapy. The aim of this study is to investigate the factors that determine the efficacy of combination therapy in patients with genotype 2 HCV infection.
Methods:  Fifty patients with CHC who underwent a treatment of 6 MU IFN α-2b with ribavirin for 24 weeks were retrospectively analyzed.
Results:  All the patients showed no serum HCV-RNA within 12 weeks after starting the therapy. Forty-one of the 50 patients (82%) achieved a sustained virological response (SVR). The age, sex, genotype (2a vs. 2b) and grade/stage of the liver by histopathology and pretreatment viral load werenot different between the sustained responders and relapsers. Univariate analysis showed that an earlier viral clearance from blood and a larger number of amino acid substitutions in the interferon sensitivity determining region (ISDR) were predictors of SVR. Multivariate analysis showed that a large number of amino acid substitutions in the ISDR was a predictor of SVR.
Conclusion:  The characterization of the amino acid sequences of ISDR may be helpful for predicting a relapse after combination therapy in patients with genotype 2 HCV infection.     

Ribavirin and interferon-α combination therapy vs interferon-α alone in the retreatment of chronic hepatitis C: a randomized clinical trial

Interferon-α (IFN-α) induces sustained remission of chronic hepatitisC in approximately 25% of patients. In patients who are non-responders to the first course of therapy, retreatment with IFN-α is of limited efficacy. Ribavirin has also been used to treat chronic hepatitisC, but it induces only a transient response. In this study, we evaluated the efficacy of ribavirin and IFN-α combination therapy for IFN-α resistant chronic hepatitisC. Twenty-four IFN-α non-responders and 24 relapsers were randomized to receive either ribavirin (1000mg per day) together with IFN-α (3–6million units (MU) thrice weekly) or the same dose of IFN-α alone, for 6months. Both at the end of treatment and 6months later, normal transaminase levels were more common in the patients receiving combination therapy than in the group receiving IFN-α alone: 17 (70.8%) vs seven (29.2%) patients ( P =0.009) and six (25%) vs one (4.2%) patient ( P =0.034), respectively. At the end of treatment and 6months later, serum HCV RNA was no longer detectable in eight (33.3%) and five (20.8%) patients in the combination therapy group and in six (25%) and one (4.2%) patient in the IFN-α therapy group, respectively. Three patients (12.5%) were withdrawn prematurely from combination therapy because of side-effects; ribavirin therapy was ceased or dosage reduced in six other patients (25%), again because of side-effects. In conclusion, this combination treatment was more effective than retreatment with IFN-α, alone, in inducing sustained biochemical remission of chronic hepatitisC that was resistant to a previous course of IFN-α. The combination treatment, however, was frequently associated with significant side-effects.     

Hepatic inflammatory cytokine mRNA expression in hepatitis C virus-human immunodeficiency virus co-infection

Summary.  Although epidemiologic studies have documented that hepatitis C virus (HCV)/human immunodeficiency virus (HIV) co-infected patients have accelerated fibrogenesis, especially those with CD4⁺ cell counts <200 cells/mm³, the pathogenic mechanisms are poorly understood. We investigated whether severe immunodeficiency in co-infection is associated with changes in intrahepatic inflammatory cytokine mRNA levels. We measured interferon (IFN)-γ, tumour necrosis factor-α, transforming growth factor (TGF)-β₁, interleukin (IL)-4, IL-10, IL-12p35 and IL-12p40 mRNA levels by real-time PCR performed on liver samples from HCV mono-infected ( n  = 19) and HCV/HIV co-infected ( n  = 24) patients. Co-infected patients had decreased intrahepatic mRNA levels of IFN-γ ( P  = 0.09), IL-4 ( P  = 0.05) and IL-12p35 ( P  = 0.04) compared with mono-infected patients, while IL-10 was increased ( P  = 0.07). In co-infected patients, IFN-γ mRNA levels increased linearly with increasing peripheral CD4⁺ cell counts by 1.23 times relative to the calibrator for every 100 CD4⁺ cells/mm³ increase ( P  = 0.02). No other cytokines were significantly associated with CD4⁺ cell counts. In conclusion, HIV-induced lymphopenia may result in hepatic inflammatory cytokine suppression in HCV/HIV co-infection. Intrahepatic IFN-γ levels are significantly reduced in patients with advanced immunodeficiency. Further studies are needed to assess whether decreased IFN-γ secretion by HCV-specific CD4⁺ cells may account for accelerated fibrogenesis in these patients.