key: cord-0792257-jfnsq525 authors: Helal, Gouda Kamel; Gad, Magdy Abdelmawgoud; Abd‐Ellah, Mohamed Fahmy; Eid, Mahmoud Saied title: Hydroxychloroquine augments early virological response to pegylated interferon plus ribavirin in genotype‐4 chronic hepatitis C patients date: 2016-05-25 journal: J Med Virol DOI: 10.1002/jmv.24575 sha: 35ae4e496d587366add22f862da2480fa0613b98 doc_id: 792257 cord_uid: jfnsq525 The therapeutic effect of pegylated interferon (peg‐IFN) alfa‐2a combined with ribavirin (RBV) on chronic hepatitis C Egyptian patients is low and further efforts are required to optimize this therapy for achievement of higher rates of virological response. This study aimed to evaluate the safety and efficacy of hydroxychloroquine (HCQ) in combination with pegylated interferon plus ribavirin on early virological response (EVR) in chronic hepatitis C Egyptian patients. Naïve 120 Egyptian patients with chronic hepatitis C virus infection were divided into two groups. Group 1 have administered the standard of care therapy (pegylated interferon alfa‐2a plus ribavirin) for 12 weeks, (n = 60). Group 2 have administered hydroxychloroquine plus standard of care therapy for 12 weeks, (n = 60). Therapeutics included hydroxychloroquine (200 mg) oral twice daily, peginterferon alfa‐2a (160 μg) subcutaneous once weekly and oral weight‐based ribavirin (1000–1200 mg/day). Baseline characteristics were similar in the two groups. The percentage of early virological response was significantly more in patients given the triple therapy than in patients given the standard of care [54/60 (90%) vs. 43/60 (71.7%); P = 0.011; respectively]. Biochemical response at week 12 was also significantly higher in patients given the triple therapy compared with the standard of care [58/60 (96.7%) vs. 42/60 (70%); P < 0.001; respectively]. Along the study, the observed adverse events were mild and similar across treatment groups. Addition of hydroxychloroquine to pegylated interferon plus ribavirin improves the rate of early virological and biochemical responses in chronic hepatitis C Egyptian patients without an increase in adverse events. J. Med. Virol. 88:2170–2178, 2016. © 2016 Wiley Periodicals, Inc. The therapeutic effect of pegylated interferon (peg-IFN) alfa-2a combined with ribavirin (RBV) on chronic hepatitis C Egyptian patients is low and further efforts are required to optimize this therapy for achievement of higher rates of virological response. This study aimed to evaluate the safety and efficacy of hydroxychloroquine (HCQ) in combination with pegylated interferon plus ribavirin on early virological response (EVR) in chronic hepatitis C Egyptian patients. Na€ ıve 120 Egyptian patients with chronic hepatitis C virus infection were divided into two groups. Group 1 have administered the standard of care therapy (pegylated interferon alfa-2a plus ribavirin) for 12 weeks, (n ¼ 60). Group 2 have administered hydroxychloroquine plus standard of care therapy for 12 weeks, (n ¼ 60). Therapeutics included hydroxychloroquine (200 mg) oral twice daily, peginterferon alfa-2a (160 mg) subcutaneous once weekly and oral weight-based ribavirin (1000-1200 mg/day). Baseline characteristics were similar in the two groups. The percentage of early virological response was significantly more in patients given the triple therapy than in patients given the standard of care [54/60 (90%) vs. 43/ 60 (71.7%); P ¼ 0.011; respectively]. Biochemical response at week 12 was also significantly higher in patients given the triple therapy compared with the standard of care [58/60 (96.7%) vs. 42/60 (70%); P < 0.001; respectively]. Along the study, the observed adverse events were mild and similar across treatment groups. Addition of hydroxychloroquine to pegylated interferon plus ribavirin improves the rate of early virological and biochemical responses in chronic hepatitis C Egyptian patients without an increase in adverse events. J. Med. Virol. 88:2170 -2178 , 2016 . [Wantuck et al., 2014] . There are about 170 million infected individuals all over the world, representing about 3% of total population [Khattab et al., 2011] . Most of HCV patients are chronically infected and are at risk of development of HCV-related complications such as hepatic cirrhosis and hepatocellular carcinoma (HCC) [Kamal and Nasser, 2008] . The prevalence of chronic hepatitis C (CHC) in Egypt is extremely high, affecting about 15% of the population [Guerra et al., 2012] . Six major genotypes and a series of subtypes of HCV have been identified [Simmonds et al., 2005] . The most prevalent HCV genotype in Egyptian patients is [Ray et al., 2000] . HCV Genotype is one of the most important predictors of response to HCV standard therapy [Schaefer et al., 2004] . Until 2011, pegylated interferon-alfa plus ribavirin combination was the standard of care therapy for HCV infection [Manns et al., 2001] . The rate of sustained virological response (SVR) achieved as a result of this dual therapy (DT) for HCV genotypes 2 and 3 was between 70% and 80%, and for HCV genotypes 1 and 4 was between 45% and 60% [Muir et al., 2004] . The standard duration of PegIFN and RBV therapy has been 48 weeks, except in slow responders (detectable HCV RNA at 12 weeks but undetectable HCV RNA at 24 weeks after treatment), in whom increasing the dual therapy duration to 72 weeks may obtain higher rates of SVR [Pearlman et al., 2007] . After 2011, new oral compounds known as directly acting antiviral agents (DAAs) have been introduced in the treatment of chronic HCV infection with SVR rates of between 90% and 100%; explaining that we might soon have the ability to cure all patients with HCV (treatment-na€ ıve, relapsed patients on previous dual therapy and resistant patients) [Kowdley et al., 2014] . Since 2011, telaprevir and boceprevir were approved as first generation NS3 protease inhibitors as a new standard line of therapy for genotype 1 HCV patients in addition to standard classical therapy, but relapsers and previous non-responders to dual therapy have shown low SVR rates to this new therapy in addition to observation of many side effects, especially in patients with advanced grade of hepatic fibrosis [Aghemo et al., 2013] . Since 2014, FDA has approved sofosbuvir (SOF), simeprevir (SIM), and daclatasvir (DCV) as new generations of DAAs of higher SVR rates with fewer side effects and shorter duration of treatment [Abdel-Razek and Waked, 2015] . The previously mentioned DAAs can be used in combination with or without PegIFN and/or RBV combination with different duration of therapy based on the used combination. The optimal regimen in IFN eligible patients is a combination of Peg-IFN and RBV plus SOF, SIM and DCV for 12 weeks but in IFN ineligible patients, the best treatment course is a combination of SOF/RBV for 24 weeks, or a combination of SOF-SIM or SOF-DCV with or without RBV for 12 weeks [Mohamed et al., 2015] . Because of HCV exists as a heterogeneous pool of genetic variants within the infected patient before treatment and the expanded use of DAAs in the near future anticipates that a part of patients will develop resistance and fail to achieve SVR [Ahmed and Felmlee, 2015] . So, there is a need to develop anti-HCV agents, which are more efficacious and cost effective with lower resistance and minimal adverse effects. Hydroxychloroquine (HCQ) is a 4-aminoquinoline known since 1934 and was reported to be used as an antimalarial agent [O'Neill et al., 1998 ]. Hydroxychloroquine was found to have immunomodulatory properties that have enabled its use in the treatment of autoimmune diseases such as rheumatoid arthritis [Wozniacka et al., 2007] . In addition to its antimalarial effect and immunosuppressive activity, HCQ has shown some biochemical characters that rendered it to be active against some viral infections [Savarino et al., 2006] . It has been found that HCQ exerts direct antiviral effects against several viruses including members of the flaviviruses, retroviruses, and coronaviruses through inhibition of pH-dependent steps of their replication inside host cells [Savarino et al., 2003] . Moreover, it has been reported that HCQ has antiviral actions against 12 human pathogenic viruses including hepatitis A, B, and C viruses [Chandramohan et al., 2007] . Hydroxychloroquine was found to block the entire replication cycle of hepatitis A virus through inhibition of its uncoating step inside hepatocytes [Bishop, 1998] . Furthermore, it has been suggested in several in vitro studies that HCV replication uses process involving cellular autophagic proteolysis which can be inhibited by HCQ [Meertens et al., 2006] . In this study, we aimed to investigate the efficacy of hydroxychloroquine as an add-on therapy together with the standard-of-care therapy on early virological response in chronic hepatitis C Egyptian patients. This study was conducted on 120 Egyptian patients with chronic hepatitis C in accordance with the ethical principles that originated in conformance with the Declaration of Helsinki. The study protocol was approved by Viral Hepatitis Treatment Centers, Ministry of Health, Egypt. All patients provided written informed consent before participation in this study. Patients were considered eligible for enrollment in this study if they were agreeable to the following criteria: male or non-pregnant female Egyptian patients with chronic active hepatitis C (genotype 4), aged 18-60 years old, negative HBsAg, positive anti-HCV, white blood cell count (WBC) >3,000/mm 3 , neutrophil count >1,500/mm 3 , platelets >80,000/ mm 3 , hemoglobin content (Hb) !12 gm/dl in males and 11 gm/dl in females, serum creatinine (SC) <1.2 mg/dl and of evidence of chronic hepatitis established by liver biopsy performed within 12 months before commencing of the study according to Metavir scoring system. Exclusion criteria included decompensated liver disease as well as any other cause of liver disease than HCV, body mass index (BMI) >30 kg/m 2 , severe cardiovascular, retinal and thyroid disorders, Hb <10 gm/dl, absolute neutrophil count (ANC) <1,500/mm 3 , platelet count <80,000/mm 3 , F0 and F4 on liver biopsy according to Metavir scoring system for both grades (necroinflammation) and stages (degree of fibrosis) and patients who have administered antiviral or immunosuppressive therapy within the 6 months prior to therapy. The present work is prospective, randomized, controlled, interventional, single-blind study conducted at a single center (Hepatic Viruses Care Unit in Fayoum General Hospital), Fayoum governorate, Egypt, from January 2014 to November 2014. The enrolled 120 patients were divided into two groups according to the received HCV therapy for 12 weeks: Group 1: 60 patients were administered dual therapy of pegylated interferon (Peg-INF-alfa-2a) plus ribavirin (RBV) (standard of care) for 12 weeks. Group 2: 60 patients were administered triple therapy of hydroxychloroquine (HCQ) in combination with the standard of care therapy for 12 weeks. Pegylated interferon 160 mg (Reiferon Retard Vial 1 , Rhein-Minapharm Pharmaceutical Company, Cairo, Egypt) was administered as a weekly subcutaneous injection. Ribavirin 200 mg (Ribavirin Capsules 1 , Minapharm Pharmaceutical Company, Cairo, Egypt) was administered orally twice per day with food in the day and night. Ribavirin doses were adjusted based on the patient body weight as follows: for patients with a body weight lower than 75 kg, 1,000 mg of ribavirin were administered daily, but patients with a body weight higher than 75 kg, 1,200 mg ribavirin were administered daily. Hydroxychloroquine 200 mg (Hydroquine Tablets 1 , Minapharm Pharmaceutical Company) was administered orally twice daily in the morning and evening. The main efficacy parameter was early virological response (EVR), plasma samples were collected for determination of plasma HCV-RNA levels at baseline and at the end of the study (at week 12) using the Roche-COBAS TaqMan Hepatitis C Virus test version 2.0 with a lower limit of quantitation (LLOQ) of 25 IU/ml. EVR is defined as undetectable HCV RNA (90% against most HCV genotypes [Gutierrez et al., 2015] . Although the first generation of DAAs has increased the rate of SVR when administered in combination with pegylated interferon, but many side-effects remained. The approval of next-generation DAAs such as sofosbuvir, simeprevir, and daclatasvir to be in the antiviral therapy of HCV has led to interferon-free regimens in the clinical application [Douam et al., 2016] . Because of most of these drugs have a low barrier to resistance, multiple obstacles will likely appear in the future against the use of them. Furthermore, natural polymorphisms in certain HCV genotypes and subtypes have been reported in addition to resistant mutations to multiple DAAs have already been characterized in NS3-4A, NS5A, and NS5B [Sarrazin, 2016] . The high cost of these medications especially in low-income countries such as Egypt with a high prevalence of HCV, have urged a growing need for developing new, more effective antiviral agents with fewer side effects and can be combined with the standard of care for successful HCV treatment. We aimed in this study to investigate the efficacy of adding hydroxychloroquine to pegylated interferon and ribavirin on EVR in chronic hepatitis C Egyptian patients. The rationale for choosing EVR to be the primary efficacy parameter was based on what have been documented about the predictive value of EVR toward the chances of achieving SVR. It has been illustrated that patients without an EVR have a very little chance of achieving SVR [Ferenci et al., 2005] . Similarly, EVR was an excellent predictor of treatment outcome, whereas the absence of EVR was associated with very low chance (0-3%) of achieving SVR in Egyptian patients [Elefsiniotis et al., 2009 ]. In the present study, the extent of EVR was significantly more in patients received triple therapy of peginterferon alfa-2a, ribavirin, and hydroxychloroquine than those received the standard of care [54/60 (90%) vs. 43/60 (71.7%); P ¼ 0.011]. Also, the extent of virologic failure was significantly lower in hydroxychloroquine patients compared with standard of care patients [[6/60 (10%) vs. 17/60 (28.3%)]. This improvement in EVR in HCQ-treated group was assumed to be due to the reported antiviral activity of HCQ that augmented the inhibitory action of the standard of care on hepatitis C viral replication in hepatocytes. The antiviral activity of HCQ was attributed to the fact that HCQ is a weak base that has a greater tendency to be captured by acidic organelles and accumulated inside them resulting in an increase of their pH [Savarino et al., 2003] . The accumulation of HCQ within the acidic organelles, including endosomes, lysosomes, and Golgi vesicle and thereby the increase of their pH is the main cause of HCQ antiviral activity [Sundelin and Terman, 2002] . For explanation, HCV entry into the host cell is a pHdependent process which requires a low pH to perform some conformational changes that are essential for fusion, penetration, uncoating, and endocytosis which occurs within the endosomal pathway [Ashfaq et al., 2011] . Moreover, HCQ might interfere with post-translational modifications of HCV envelope glycoproteins through inhibition of proteases and glycosyltransferases activities within the trans-Golgi network and endoplasmic vesicles which are responsible for those modifications [Rolain et al., 2007] . As these enzymes require a low pH for their activity, HCQ might therefore lead to decreased viral infectivity through impaired envelope maturation [Randolph et al., 1990] . Based on the previous explanation, it could be suggested that HCQ increased EVR by suppression of HCV replication inside hepatocytes. It was shown that pretreating hepatic cells with HCQ-inhibited hepatitis C virus (HCV) entry through clathrin-mediated endocytosis and fusion within an acidic endosomal compartment [Blanchard et al., 2006] . Moreover, HCQ was also found to be active against hepatitis A and hepatitis B viruses [Offensperger et al., 1991] . It is important to notify that HCQ not only has the potential to inhibit the replication of hepatic viruses but also it has antiviral activity against all viruses of low pH-dependent entry and replication [Thom e et al., 2013] . For instance, it has been reported that lysosomotropic activity of HCQ exert direct antiviral effects against several RNA viruses including coronaviruses, influenza A virus, flaviviruses, and human immunodeficiency virus (HIV) [Savarino et al., 2006 ]. The present study shows that HCQ has a valuable effect on ALT normalization. This is because 96.7% of patients received HCQ in addition to the standard of care have a normal ALT level at week 12 and this is a high percentage compared with ALT normalization achieved in only 70% of patients received the standard of care alone. These obtained data are in accordance with what have been reported when chronic active hepatitis B virus patients have been treated with HCQ for a median of 12 months and it was noticed that ALT in all patients has been returned to normal values [Kouroumalis and Koskinas, 1986] . Additionally, it has been stated that low dose of chloroquine (CQ), analog of HCQ, can normalize ALT level in patients with chronic hepatitis C [Schuppan et al., 1998 ]. In the present work, multivariate analysis revealed that there was no significant association between early virological response of the study patients and age, sex, BMI, baseline viral load, baseline ALT, and fibrosis score of them either across all study patients or across patients of each group separately. This may support that EVR achieved in this study is strongly related to the administered therapy alone without any effect induced by other factor or variable. Those findings are consistent with study what have been explained previously that age, sex, baseline viral load, and BMI were not predictive of virological response [Shehab et al., 2014] . Regarding the safety and tolerability of the study therapy including HCQ and SOC, the results of monitoring of patients for adverse events (AEs), vital signs, physical examinations, clinical and laboratory measurements throughout the study have displayed that the administered therapy was safe with no worsening or abnormalities in the previously measured parameters induced by SOC therapy alone or by the addition of HCQ to SOC but all patients results were within normal range. The half century long use of HCQ for treatment of rheumatoid arthritis and malaria demonstrates the safety of administration of HCQ to human beings [Michaelides et al., 2011] . Likewise, it has been shown that administration of HCQ for rheumatoid arthritis (RA) patients after treatment with cyclosporine A (CSA) was associated with returning the mean levels of complete blood count testing, urine analysis results, liver enzymes, urea nitrogen, and serum creatinine to normal after significant increase of their levels during CSA treatment [Kim et al., 2001] . In addition, evaluation of the toxic effects of HCQ on different organs of albino rats through assessment of liver and kidney functions through determination of serum glutamate oxaloacetate transaminase (SGOT), serum glutamic pyruvate transaminase (SGPT), alkaline phosphatase (ALP), total bilirubin (TB), serum creatinine, and histopathological changes in liver, kidney, and heart have shown that HCQ was safe and well-tolerated medication [El Shishtawy et al., 2015] . Moreover, the assessment of the safety of temsirolimus (TEM) with HCQ through testing of complete blood count (CBC), liver and renal functions has clarified that the combination of standard doses of TEM with the highest doses of HCQ used in clinical practice was safe and tolerable [Rangwala et al., 2014] . In the current study, the most frequently reported adverse events (AEs) were mild (grade I) and similar in both groups, and were consistent with typical IFN, RBV, and HCQ-induced systemic symptoms such as headache, fatigue, influenza-like illness, and gastrointestinal disturbance. The therapy was well tolerated and of excellent adherence, no dropout and no AEs leading to discontinuation of treatment. The observed safety and tolerability HCQ may be attributed to the administered dose of HCQ and the duration of its use because they are the two main factors affecting frequency and severity of adverse effects and clinical abnormalities including hepatic, renal, and hematological abnormalities associated with HCQ use [Ruiz-Irastorza et al., 2008] . The selected a dose of hydroxychloroquine in this study was 400 mg/day, based on the 6.5 mg/kg/day which was recommended to be the maximum safe dose for long-term use in RA patients, and to be with no evidence retinopathy which is the main toxic effect associated with the long-term use of HCQ [Block, 1998] . Furthermore, it was indicated by the American Academy of Ophthalmology that the cumulative dose 1,000 gm HCQ is most important risk for retinopathy but this cumulative dose is reached only after 7 years of HCQ use with a typical daily dose of 400 mg [Geam anu et al., 2014] . Importantly, the selected dose of HCQ was also based on the clinical trials in which HCQ has achieved marked antiviral activity against HIV infection and has potentiated the therapeutic outcome of antiretroviral agents when HCQ was used in combination with them [Paton and Aboulhab, 2005] . Similarly, it was reported that HCQ is a well-tolerated therapy and all the clinical adverse events associated with its use were mild (grade I) and the most concerning side effect is ocular toxicity which is thought not to occur in adults if the dose remains less than 6.5 mg/kg/day [Klinger et al., 2001] . The rate of dose modification in this study was 3.8% compared with 14-42% which represent the accepted percentage of standard of care dose modification [Manns et al., 2001] . This reassuring safety profile may support the rationale for trying HCQ at higher doses for longer duration in future trials. Limitations of the present study include the small number of patients compared with the very large number of Egyptian patients infected with HCV (15% of the Egyptian population). Also performing this study in a single center in Fayoum governorate, Egypt is one of this study limitations because HCV Egyptian patients are settling in several areas in all Egypt governorates. Another limitation is the lack of this study for assessment of rapid virological response (RVR) defined as HCV RNA negativity at week 4 of treatment although RVR is a strong predictor of SVR [positive predictive value (PPV) >96%] and failure to achieve EVR was a strong predictor of non SVR [negative predictive value (NPV) >70%], independent of patient's pretreatment (9). This was because the cost of RVR assessment was very high and was not currently funded by the Egyptian Ministry of Health dependent on assessment of EVR was sufficient to predict the treatment outcome. This study ended at week 12 only without continuous administration of HCQ along with IFN an RBV for 48 weeks then off therapy for 24 weeks according to the standard of care protocol for evaluation of antiviral activity of HCQ on end of treatment virological response (ETR) and SVR and this can be regarded as one of the limitations, but this was based on consideration of this study to be a first step in evaluating the safety and efficacy of HCQ on SVR if HCQ safety and efficacy on EVR is achieved. Other limitations of this study include the absence of doubl blinding and lack of a placebo control which should be avoided in a further larger confirmatory trial. So, multicenter studies with a larger number of patients and assessment of HCQ effect in combination with the standard of care on ETR and SVR are recommended. 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Also, the authors would like to express their deepest gratitude to Minapharm Pharmaceutical Company, Cairo, Egypt for kind support of hydroxychloroquine.