Zaigham Abbas ( Department of Medicine, The Aga Khan University Hospital, Karachi, Pakistan. )
Sajjad Raza ( Department of Medicine, The Aga Khan University Hospital, Karachi, Pakistan. )
Saeed Hamid ( Department of Medicine, The Aga Khan University Hospital, Karachi, Pakistan. )
Wasim Jafri ( Department of Medicine, The Aga Khan University Hospital, Karachi, Pakistan. )
Objectives: To evaluate the efficacy and safety of triple combination regimens comprising of interferon alpha-2b (IFN-alpha) and ribavirin plus either IFN-gamma or amantadine in genotype 3 patients, responders or relapsers to interferon plus ribavirin combination.
Methods: Patients were randomized to receive IFN-alpha 3MU thrice a week, ribavirin 800-1200 mg per day with either IFN-gamma 2 MU thrice a week or amantadine 100 mg twice daily. Treatment was continued for 48 weeks in patients showing complete or partial (2 log reduction) early virological response (EVR) at 12 weeks and negative PCR at 24 weeks.
Results: Total enrollments were 44; 25 were previously non-responders out of them 12 were in the IFN-gamma arm. Nineteen were relapsers, out of them 10 received IFN-Gamma. Overall EVR with triple regimens was 61.4% (27/44). The EVR for IFN-gamma arm was 72.7% (16/22) and for amantadine arm 50% (11/22) (p=0.089). Sustained virological response (SVR) was 50% (11/22) in the gamma arm and 27.3% (6/22) in the amantadine arm (p=0.122). This figure was 60% (6/10) and 44% (5/9) for relapsers (p=0.845), and 41.6% (5/12) and 7.7% (1/13) for non-responders (p=0.046).Treatment was well tolerated by most of the patients in both arms.
Conclusions: About one third of patients responded to the triple regimens. However IFN-gamma was a better option. Its combination with pegylated interferon and ribavirin needs further evaluation. (Trial Registration: ClinicalTrials.gov Identifier NCT00538811).
Keywords: Interferon gamma, Interferon alpha, Ribavirin, Hepatitis C, Non responders, Relapsers (JPMA 62: 338; 2012).
The current standard treatment for chronic hepatitis C virus (HCV) infection is combination of pegylated interferon (PEG-IFN) and ribavirin. The treatment is given for a period of six months for genotype 2 and 3 and one year for genotype 1 and 4.1 This combination gives a sustained virological response (SVR) of 54%-56%; 42-52% of patients with genotype 1 and 76-84% of those with genotypes 2 and 3.2 Comparing PEG-IFN with standard interferon, there is much better response by using PEG-IFN for the treatment of genotype 1. For genotype 3, the response rate with standard interferon and ribavirin is equally good, cost effective3,4 and recommended by the different societies.5,6
Current standard therapy for chronic hepatitis C with interferon alpha (standard or pegylated) and ribavirin is still less than ideal and the problem of the non-responding patients to the combination therapy remains.SVR remains a difficult goal to achieve in many patients. After a relapse from standard interferon plus ribavirin therapy, retreatment with PEG-IFN plus ribavirin for one year may increases the response rate to 40-50%. However, patients who have failed to respond to combination of standard interferon and ribavirin, re-treatment with PEG-IFN plus ribavirin therapy has a response rate of only 10%.2 Different strategy is, therefore, needed for patients who are considered as non-responders to interferon and ribavirin therapy. Due to the limitations of antiviral drugs, there is a need to explore other avenues to enhance the immune system\\\'s ability to fight HCV.
In the context of discovery of IL-28 B gene polymorphism coding for lambda interferon and its importance as an independent predictor of response to interferon alpha based therapy,7,8 there is a resurgence of interest and enthusiasm about the role of interferon lambda and gamma in clearing the virus. Interferon gamma (IFN-gamma) inhibits HCV virion production by an effect on viral RNA and protein synthesis, enhancement of immune lysis of HCV infected cells, and inhibition of hepatic fibrosis by an effect on TGF-beta.9 IFN- alpha and IFN-gamma induce distinct patterns of gene expression10 and differential actions of IFN-alpha and IFN-gamma has its implications in the context of therapeutic intervention.
Considering the antiviral effects of IFN-gamma and expected favourable effects if used in combination with IFN- alpha, we started an investigator driven study to evaluate the efficacy of a regimen combing IFN-gamma with IFN-alpha plus ribavirin in patients who had not responded to the combination of last two drugs.
Patients and Methods
Trialdesign: This was a single centred investigator initiated, open label, parallel randomized controlled trial. The patients were divided into two arms namely experimental (IFN-gamma) and active comparator (amantadine). Randomization was done by opaque sealed envelope method. Non-responders and relapsers to previous treatment with standard interferon and ribavirin were separately randomized.Primary end point was the efficacy in terms of sustained virological response (SVR) defined as undetectable HCV RNA 24 weeks after treatment discontinuation (week 72). Secondary end points were normalization of ALT at week 72, and tolerance and safety. No changes were made in methods after the trial commencement.
Participants: The study was conducted between July 2008 and December 2010. The patients for this were recruited at the Aga KhanUniversityHospital, Karachi, Pakistan. The target patients were adult males and females patients infected with HCV genotype 3, ranging in age from 18-70 years, who had previously received standard interferon alpha 2a of 2b 3MU thrice a week in combination with ribavirin (800-1200 mg) for 24 weeks and had not shown a response as depicted by disappearance of HCV RNA from serum done in the last month of therapy (non-responders) or who relapsed at six months post-treatment (relapsers).
Other inclusion criteria were Hb > 10 g/dL (females) and > 11 g/dL (males), Platelets count > 100 x 109/L, neutrophil count > 1.5 x 109/L, at least one abnormal ALT values in the last year, normal TSH, non-pregnant adult females and absence of drug or alcohol abuse. Exclusion criteria were antiviral treatment in the last three months, hepatitis B or HIV co-infection, severe renal dysfunction or creatinine clearance less than 50 ml/min, pregnant or breast feeding women, suspected hypersensitivity to Interferon alpha, gamma or ribavirin, decompensated liver cirrhosis, history or any evidence of other concomitant causes of chronic liver disease, active malignant disease, any known pre-existing medical condition that could interfere with subject\\\'s participation or completion of study.
Interventions: Experimental arm received IFN- alpha 2b 3 MU thrice a week, ribavirin 800-1200 mg per day, and IFN-gamma 200 MU thrice a week. Patients of less than 70 kg of weight received 800 mg of ribavirin, while 70 kg or above received 1200 mg daily. Active Comparator arm received amantadine 100 mg twice a day in place of IFN-gamma. Duration of therapy was 48 weeks.
Baseline evaluation: The following base line evaluation was conducted for each patient: written informed consent, medical history, physical examination including height and weight, serum HCV RNA (quantitative) to measure the viral load (RT-PCR assay: Cobas Amplicor Monitor v2.0, Roche Diagnostics). HCV genotype determined by Innolipa kits, abdominal ultrasound, and alpha-fetoprotein levels. A liver biopsy was done prior to therapy. Hepatic inflammation and fibrosis were assessed by METAVIR scoring system. For METAVIR system, stage of fibrosis was assessed on a scale of 0-IV and activity was graded on a scale of 0-3.Once patient became PCR negative with the above mentioned quantitative assay, reverse transcription polymerase chain reaction assay with a sensitivity of 100 copies per millimeters was used in the follow up visits.
Follow-up evaluation: Patients were followed up in the clinic at week 2 and 4 and then at monthly intervals until the end of treatment (48 weeks). Patients were able to contact the investigators during the course of the treatment to report any adverse events or side effects and for their queries if any.Following evaluation was performed at each visit. a. physical examination b. assessment of any adverse event. c. Concurrent medication d. Verbal check of compliance e. CBC and biochemistry. Determination of serum HCV RNA was performed at 0, 12, 24 and 48 weeks, and then12 and 24 weeks post treatment (72 weeks). PCR at baseline and 12 weeks was quantitative.
Outcomes: Patients were evaluated for response to therapy which was defined as clearance or 2 log reduction of HCV RNA levels at 12 weeks (early virological response or EVR) and negativity of serum HCV RNA by PCR at 24 weeks. Patients who were considered as responders continued the study till the end of treatment (48 weeks). Non-responders at 24 weeks were withdrawn from the study. Patients who successfully completed 48 weeks of the study were seen at 12 weeks and 24 weeks after treatment discontinuation and were evaluated for the sustained response.No changes to trial outcomes were made after the trial commencement.
Randomization: Simple randomization was done using sealed envelope method. Non-responders and relapsers to previous treatment with standard interferon and ribavirin were separately randomized. Randomization, enrollment of participants and assignment of participants to interventions was done by the principal investigator. It was an open label study.
Safety and ethical issues: Treatment was aimed to be interrupted if subject\\\'s health or wellbeing was considered threatened by continuation of the treatment, occurrence of serious adverse events or any unmanageable factors, and withdrawal of consent by the subject. Adverse events were graded as mild, moderate, severe and life threatening. Therapy was permanently discontinued for life-threatening events. Doses of both interferons were reduced to half if the neutrophil count fell between 0.75 and 0.5 x 106/L and/or platelets fell below 60 x 106/L. If neutrophil count fell below 0.5 x 106/L or platelets below 30 x 109/L therapy was stopped. Ribavirin dose was lowered to half if haemoglobin levels fell below 8.5 g/dl and stopped if level fell below 7.5 g/dl.
Ethical Review Committee of the Institute approved the study. Written informed consent was obtained from all the patients before enrolling the patients in the study after explaining the study objectives and methodology.
Statistical methods: Data was analyzed by the Statistical Package for Social Sciences SPSS 16.0 for Windows (SPSS inc. Chicago, IL). Means with standard deviations were calculated for continuous variables, and compared through Wilcoxon rank-sum test Proportions were calculated for categorical variables, and compared using the Chi square test with Yate\\\'s correction. The statistical significance level of two-sided tests was set at P=0.05.
Participant flow: The total number of patients included in this study were 44 out of which 28 (63.6%) were male and 16 (36.4%) female; mean age 43.6± 9.3years (range 28-60). Twenty two were in the experimental arm and 22 in the active comparator group, Out of the 44 patients,25 were previously non-responders and 12 of them were in the IFN-gamma arm. Nineteen were relapsers; 10 received IFN-gamma.
Baseline data: Out of the total recruited patients 9 (22.3%) were diabetic, 8 (20 %) hypertensive, 12 (31.6%) had hepatomegaly and 8 (20.5%) had splenomegaly. Liver biopsy revealed no fatty change in 16 (57.1%), mild fatty change in 8 (28.6%), moderate fatty change in 4 (14.3%) patients. F3 or F4 fibrosis was seen in 14 (34%). Baseline characteristics are summarized in the Table-1.
Virological response: By intention-to-treat analysis, the overall early virological response (EVR) with triple regimens was 61.4% (27/44). The EVR for IFN-gamma arm was 72.7% (16/22) and for amantadine arm 50% (11/22) (p = 0.089). In the subgroup analysis, this figure was 90% (9/10) and 55% (5/9) for relapsers (p=0.119), and 46.1% (7/12) and 8% (6/13) for non-responders in both arms respectively (p=0.543).
The overall end of treatment response (ETR) was 40.9% (18/44). The ETR was 50% (11/22) and 31.8% (7/22) for IFN-gamma and amantadine arms respectively (p= 0.220). In the subgroup analysis, this figure was 60% (6/10) and 55% (5/9) for relapsers (p=0.845), and 41.6% (5/12) and 15.3% (2/13) for non-responders to previous treatments in both arms respectively (p=0.144).
Sustained virological response (SVR) with both triple regimens was seen in 38.6% (17/44). SVR was 50% (11/22) in IFN-gamma arm and 27.27% (6/22) in amantadine arm (p=0.122). In the subgroup analysis, this figure was 60% (6/10) and 44% (5/9) for relapsers (p=0.845), and 41.6% (5/12) and 7.69% (1/13) for non-responders in both arms respectively (p=0.046).
Biochemical response: The normalization of ALT at week 12 was seen in 84.1% (37/44) patients. It was 86.3% (19/22) and 81.1% (18/22) for IFN-gamma and amantadine arms respectively (p= 0.680). In the subgroup analysis, this figure was 100% (10/10) and 77.7% (7/9) for relapsers (p=0.115), and 75% (9/12) and 84.6% (11/13) for non-responders to previous treatments in both arms respectively (p=0.548).
Normalization of ALT at the end of treatment was seen in 41% (18/44) patients. It was 50% (11/22) and 31.8% (7/22) for IFN-gamma and amantadine arms respectively (p= 0.220). In the subgroup analysis, this figure was 70% (7/10) and 55% (5/9) for relapsers (p=0.515), and 33.3% (4/12) and 15.3% (2/13) for non-responders to previous treatments in both arms respectively (p=0.294).
The sustained normalization of ALT at six months post treatment was seen in 38.6% (17/44) patients. It was 50% (11/22) and 27.3% (6/22) for IFN-gamma and amantadine arms respectively (p= 0.122). In the subgroup analysis, this figure was 60% (6/10) and 55% (5/9) for relapsers (p=0.845), and 41.6% (5/12) and 7.6% (1/13) for non-responders to previous treatments in both arms respectively (p=0.047).
Assessment of adverse events: Treatment was well tolerated in both arms.The 80/80/80 rule i.e. 80% of the drug taken for 80% of the scheduled time was well followed. Mild adverse events included fever, headache, body ache, joint pain, weakness, disturbed sleep, retrosternal burning, decreased appetite, hairloss, weight loss, abdominal discomfort, nausea, vomiting, burning sensation in hands and feet, disturbed sleep and bloating. Treatment was stopped in two patients due to severe adverse events including severe neutropenia in one patient and arthralgia involving knee joint in the other. Both these patients belonged to the active comparator (amantadine) group. Dose reduction was done in one patient in gamma arm because of complaints of hand tremors and jerky movements.
This is the first study to examine IFN-gamma in combination with IFN-alpha and ribavirin for difficult to treat hepatitis C patients. Previously, in a small pilot study of IFN-gamma monotherapy given for four weeks, in the doses of 100-400 microgram thrice weekly, the drug was well tolerated. It did not show any effect on the serum HCV RNA levels.11 However, another study of monotherapy given for 24 weeks showed improvement in liver fibrosis.12 There was successful eradication of virus with a combination of IFN-gamma, interferon alfacon-1, and ribavirin in a nonresponder HCV patient to pegylated interferon therapy.13
Combining IFN-gamma with IFN-alpha appears logical. When administered simultaneously, IFN-alpha together with IFN-gamma results in dramatic enhancement of antiviral activity against hepatitis C.14 There is a distinct pattern of gene expression by two interferons.10 The synergistic effect is likely to be due to differential cell surface receptors and signaling pathways employed.15 IFN-gamma enhances the production of IFN-alpha from immature plasmocytoid dendritic cells.16 Moreover, IFN-alpha can inhibit production of IL-12, a potent activator of STAT4 and IFN-gamma production17 which again stresses the importance of IFN-gamma supplementation in anti-HCV regimens.
Direct anti-HCV effect of IFN-gamma in cell culture is, at least in part, mediated through the Ras-MAPK signaling pathway, which possibly involves a direct or indirect modulation of NS5A protein phosphorylation.18 The mechanism of action of IFN-gamma in inhibiting the HCV infection may involve the down regulation of Claudin-1 expression and HCV receptors\\\' distribution.19 Ribavirin has shown to enhance IFN-gamma levels in patients with chronic hepatitis C treated with interferon-alpha.20 So immune responses may further be enhanced with better efficacy by combining IFN-gamma with IFN-alpha plus ribavirin for treatment of chronic hepatitis C. IFN-gamma has potent anti-fibrotic effects on stellate cells. Even if the combination does not clear HCV, it would slow or prevent fibrosis.12,21
In our study we used standard interferon instead of pegylated interferon for the retreatment. In our country the cost of pegylated interferon based therapy is exorbitant and out of the reach of an ordinary person. Moreover, pegylated interferon with ribavirin is not an effective choice for retreatment of failures of standard interferon plus ribavirin therapy.2 In the comparator group, we used amantadine. Role of amantadine in combination with interferion and ribavirin for the treatment of hepatitis C remains controversial. Previous studies related to retreatment of difficult to treat patients have shown contradictory results; some studies favour22,23 while others refute its role.24,25 We considered it unethical to retreat patients with dual combination of standard interferon with ribavirin again and compare it with a triple regimen.
The strength of this study is that first time IFN-gamma was used in combination with IFN-alpha plus ribavirin to treat difficult to treat hepatitis C patients. The combination was found to be safe and effective especially in non-responders to previous treatment. There are certain weaknesses in this trial which include using standard interferon instead pegylated interferon, small sample size of the study and including genotype 3 only. This was an investigator initiated trial, with limited financial support. As IFN-gamma is not registered in our country, we were granted special permission by the Ministry of Health to import this drug once for this study. We did not include genotype 1, as preferred interferon for this genotype even for naïve patients is pegylated interferon. We included in this study all the patients regardless of degree of fibrosis as the goals of therapy are to prevent complications and death from HCV infection; regardless of the stage of fibrosis.26 Moreover patients with advanced fibrosis need prompt intervention.27,28
In conclusion, this single centre randomized study indicates that half of the patients with HCV genotype 3 infection who were retreated with a combination of IFN-gamma with interferon alpha plus ribavirin achieved a sustained virological response. This response rate was better than comparator triple regimen which included amantadine. Whether our strategy of including IFN-gamma for retreatment is indeed effective, needs further evaluation by combining it with pegylated interferon plus ribavirin in a bigger study recruiting all genotypes. It may be worthwhile evaluating IFN-gamma in combination with the standard of care for patients who had unfavourable IL-28 B genotype polymorphisms
Conflict of interests and Acknowledgements
The authors do not have any financial relationships to disclose related to this study. The study includes the off-label use of the drug. The study was approved by the ERB of the Institute and permission was taken from the Ministry of Health to import and use interferon gamma for hepatitis C patients. The cost of the drugs and the PCR based investigations were funded by the Genetech Biopharm Pakistan. No limitations on publication were imposed by the sponsor. The company does not market gamma interferon in Pakistan.
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