Impact of Serum Level of Vitamin B12 on Response to Combined Interferon and Ribavirin Therapy for Chronic HCV Infection

Transcription

1 Med. J. Cairo Univ., Vol. 84, No. 1, June: , Impact of Serum Level of Vitamin B12 on Response to Combined Interferon and Ribavirin Therapy for Chronic HCV Infection REDA M. AMER, M.D.*; MONA A. AMIN, M.D.*; DINA SABRY, M.D.** and AMRO ABD ELAZIZ M.Sc.* The Departments of Internal Medicine* and Medical Biochemistry**, Faculty of Medicine, Cairo University, Egypt Abstract Introduction: Chronic HCV infection affects about 180 million individuals world-wide. Untreated, it leads to liver fibrosis, cirrhosis with increased risk of developing hepatocellular carcinoma. Combination of pegylated interferonribavirin was considered the standard of care for chronic HCV infection. Previous studies showed that B12 inhibits the translation of the HCV polyprotein by interference with the internal ribosome entry site. Aim of Work: To evaluate any discriminative impact of serum vit.b 12 levels on response to interferon-ribavirin therapy in patients with chronic HCV. Methods: 89 patients with chronic HCV receiving interferon-ribavirin therapy. All were subjected to thorough clinical, laboratory evaluation, liver biopsy, measuring serum vit.b 12. Comparing serum level of vit.b12 between responder and non-responders to therapy. Results: Serum Vit.B12 was significantly higher in responders than non-responders. ALT and AST showed significantly lower levels in responders. No significant difference in fibrosis stage, degree of inflammation in liver histopathology or pretreatment viral load. Conclusion: Serum vitamin B 12 can independently predict response to combined interferon-ribavirin therapy in patient with chronic HCV infection. Key Words: HCV Vit.B12 Interferon. Introduction HEPATITIS C Virus (HCV) is a major cause of progressive liver disease with approximately million people infected worldwide. HCV induces chronic infection in up to 85% of infected individuals. Untreated, chronic HCV infection leads to liver fibrosis, and it is estimated that as many as 20-30% of infected individuals subsequently develop cirrhosis [1]. Patients with cirrhosis caused by HCV have substantially increased risk Correspondence to: Dr. Reda M. Amer, The Department of Internal Medicine, Faculty of Medicine, Cairo University, Egypt of developing Hepatocellular Carcinoma (HCC) [2], and HCV-related liver complications are the most common indication for liver transplantation in the Western world [3]. The combination of pegylated interferon (peg- IFN)-a and ribavirin, has been considered the standard of care for chronic HCV infection, with Sustained Virological Response (SVR) rates varying from 50% to 90%, depending on genotype. So this raised the need for more effective treatment as well as better tools to predict treatment outcome [4]. Cyanocobalamin (vitamin B12) is stored in hepatocytes and is essential for the formation of red blood cells as well as for the function of the nervous system [5]. Previous in vitro studies have demonstrated that B 12 inhibits the translation of the HCV polyprotein by interference with the Internal Ribosome Entry Site (IRES), a structure in the HCV 5 -nontranslated region [6]. Serum B12 (S-B 12) reflects the amount stored in tissues and organs, predominantly the liver [7]. Recently, significant better response to pegylated interferon plus ribavirin has been reported at all time-points during therapy, if combined with vitamin B12 supplementation [8]. This correlates with reports on vitamin B 12 being able to inhibit HCV in vitro. Any clinical implication of serum B 12 levels in the setting of HCV treatment has not been widely studied. As the above-mentioned in vitro results suggested B12 may influence HCV replication. In the study presented here, we investigated the potential effect of serum vitamin B 12 levels on the response to combined interferon and ribavirin therapy in patients with chronic HCV infection. 529

2 530 Impact of Serum Level of Vitamin B12 on Response to Combined Interferon Patients and Methods Between October 2013 and October 2014, we prospectively enrolled 89 patients with chronic HCV infection at Internal Medicine Department Kasr El-Aini Hospital. Ethical Committee Approval and written informed consents from the patients were obtained. The patients' age ranged from years eligible for combined therapy of Pegylated interferon (PEG-IFN) and Ribavirin. All patients were subjected to: - Clinical evaluation: (History and physical examination) assessing history of smoking, alcohol consumption, presence of chronic diseases (DM, hypertension...), and past history of dental intervention, surgery or blood transfusion. - Laboratory investigations: Including, ALT, AST, ALP, CBC, Albumin, Bilirubin (Total and Direct), Alpha Fetoprotein (AFP), Prothrombin Time (PT), Antinuclear Antibody (ANA), Anti-schistosomal antibody test. - Quantitative HCV RNA. Before starting therapy and after 12 weeks of treatment with combined PEG-IFN/Ribavirin therapy, to detect the Early Virologic Response (EVR) was done. Complete EVR is considered if the HCV RNA level is undetectable. Incomplete EVR is considered when a greater than 2-log-fold reduction in HCV RNA level is present. - Abdominal sonography. - Ultrasound guided percutaneous liver biopsy. - Histopathological examination of the liver biopsy with staging of fibrosis and grading of inflammation using Metavair score. - Measuring serum level of vitamin B 12. Inclusion criteria: HCV antibodies and HCV RNA positive by PCR, liver biopsy in the with Metavir score over A2 and over or equal to F 1, or over or equal A 1 and over F2, patients never treated with ribavirin, IFN alpha or PEG-IFN alpha, AFP under or equal to 3 times the normal range for the laboratory, HBs antigen negative, Hb over or equal 11 g/dl for male and, over or equal 10g/dL for female, TLC over or equal 3000/dL, Neutrophils over or equal 1500/dL, Platelets over or equal /dL, normal TSH, ANA titre under 1/160, fasting blood sugar between mg/dL (if glucose intolerance or diabetes: HbA1C <= 8,5%), normal ophthalmologic examination, effective contraception and no breastfeeding during the treatment period during the study period. Exclusion criteria: Patients were excluded if co-infected with HBV, HIV. Other causes for chronic liver disease were also ruled out. Other exclusion criteria included neutrophil count <1500/dL, platelet count <90000/ dl or Haemoglobin (Hb) <10mg/dL for female and <1 1g/dL for male. Patients were excluded if they had positive auto-antibodies, overt active autoimmune or thyroid disorders, history of severe psychiatric disease, seizure disorder, organ transplantation, or severe cardiac or pulmonary disease, significant retinal abnormalities, clinical gout, decompensated cirrhosis, focal lesion on abdominal ultrasonography, elevated a-fetoprotein, HCC or were a substance abuser (alcohol or I.V. drugs) or showed any medical condition requiring systemic steroids. Pregnat female patients were excluded by negative serum pregnancy test and no breast feeding during the study. After an initial assessment, patients were treated with pegylated interferon alpha2a, at a dose of 180µg per week or alpha2b at a dose of 1.5 µg per kilogram body weight per week. In addition to ribavirin at a dose of mg daily as per body weight-1000mg if <_75kg and 1200 mg if >_75 kg-for 48 weeks. Clinical, biochemical and virological parameters were collected pretreatment. HCV RNA was measured by quantitative PCR at end of 12th week. So, patients were divided into 2 groups: 1- : Those are patients whose PCR results showed complete EVR. 2- Non-responders: Those are patients whose PCR results didnot show complete EVR. Methods: The serological and biochemical tests were done at Clinical Pathology Department of Cairo University Hospital. Quantitative HCV RNA was done using Real Time PCR technique. Liver histopathology: For the liver biopsies of all patients by pathologists at Pathology Department of Cairo University Hospital. The liver histopathology was staged on using METAVIR scoring system. Vitamin B12 (Vit.B 12, Cyanocobalamin) BioassayTM ELISA Kit (Human), Catalog number: , United States biological, P.O.Box 261, Swampscott, Massachusetts Statistical analysis: The data were coded, and processed on an IBM- PC compatible computer using Statistical Packages for Social Sciences (SPSS). Data were expressed

3 Reda M. Amer, et al. 531 as mean and Standard Deviation (SD) unless otherwise stated. Student- t-test was used to ascertain the significance of difference between mean values of two continuous variables and Mann-Whitney test was used for non-parametric distribution. Chi- Square analysis was performed to test for differences in proportions of categorical variables between 2 or more groups. In 2 x 2 tables, the Fisher exact test (two-tailed) was used instead of Chi- Square, in particular, when sample size was small. One-way analysis of variance (ANOVA) and nonparametric Kruskal Wallis one-way analysis of variance (ANOVA) was employed for comparison of several group means and to determine the presence of significant differences between group means. The Pearson's correlation coefficient was used to evaluate the strength association between two variables. The level p<0.05 was considered as the cut-off value for significance. Multivariate logistic regression analysis was performed. Results From eighty nine chronic HCV patients were included in our study, their age raged between (21 and 57 years) with mean age of 39 years. All patients received antiviral therapy Peg-interferon and ribavirin. According to their response to therapy, we divided our patients into two groups: 1- (44 patients): Their age ranged between (23 and 57 years) with mean age of (41.6±9 years). They are 15 females representing 34% of responders and 29 males representing 66%. 2- Non-responders (35 patients): Their age ranged between (21 and 57 years) with mean age of (42.2± 10 years). They are 25 females representing 60% of non-responders and 20 males representing 40%. Table (1) shows demographic data of both groups. There were no statistically significant difference between both groups as regard age and sex distribution. BMI is below 30 in both groups and responders has lower BMI than non-responders with statistically significant difference. Also responders had a significantly lower rate of diabetes mellitus compared to non-responders (p-value= 0.03). Regarding the laboratory data among the studied groups, the responders had lower baseline of transaminases (ALT and AST) with statistically significant difference compared to the non-responders. Table (1): Demographic data are presented as mean ± SD, frequency (percent), and median (IQR). Patients (n=89) (n=44) (49.44%) Non-responders (n=45) (50.56%) p- value Age 41.6±9 42.2± Gender: Male 29 (66%) 20 (40%) 0.08 Female 15 (34%) 25 (60%) BMI 27± ± Diabetes 1 (2%) 7 (16) 0.03 Pretreatment viral load (HCV RNA measured by PCR) is higher in non-responders than responders but the difference is statistically insignificant. Platelets and WBCs are also higher in responders but with no statistically significant difference. There was no statistically significant difference between both groups as regard bilirubin, alkaline phosphatase, INR, serum albumin levels, TSH and ANA levels (Table 2). Table (2): Baseline laboratory data of both groups. Data are presented as mean ± SD. (n=44) Non-responders (n=45) p - value AST 28.9± ± ALT 28.7± ± Bil 0.77± ± Alb 4.3± ± Alk 110.7± ± PT 13.1± ± INR 1.1 ± ± TSH 3.5± ± HB 13.7± ± TLC 7.59± ± PLT 220x10 3 ± x10 3 ± ve ANA 0 (0%) 1 (2%) 0.5 Pretreatment ± ± 0.74 PCR Regarding pretreatment liver histopathology, both groups were similar in pre-treatment inflammation grade, fibrosis stage, with no statistically significant difference. Of note, none of our patients had liver cirrhosis (F4), (Table 3). Table (3): Comparison between the two groups regarding inflammation grade and fibrosis stage according to metavir scoring system. Non- p - responders value Fibrosis stage 1.5 (1,2) 2 (1,3) 0.5 Inflammation grade 1 (1, 1) 1 (1,2) 0.8

4 532 Impact of Serum Level of Vitamin B12 on Response to Combined Interferon With respect of serum vitamin B 12 level among the studied groups, it was higher in the responder group compared to non-responder group with statistically significant difference ( p-value <0.001) (Table 4) and Fig. (1). Table (4): Baseline serum vit.b12 of both groups. Presented as mean ± SD. (n=44) Non-responders p- (n=45) value 0.0 B12 level 247±78 123± Specificity Sensitivity Fig. (3): Receiver-Operating Characteristic (ROC) curves showing the ability of the serum B 12 level to predict successful response to interferon. AUROC=0.97. B Non-responders Response Fig. (1): Serum B12 levels in responders and non-responders groups of patients. Both range and median values of serum vitamin B 12 is significantly higher in responders than nonresponders Fig. (2). B Responder Non-responder Response Fig. (2): Serum B12 levels in responders and non-responders groups of patients. Expressed as median (horizontal line), 25-75% percentiles (box), and range (bar). Area Under Receiver Operating Characteristic (AUROC) curve was calculated for the ability of serum B12 level to detect patient response to interferon therapy, (AUROC=0.97), sensitivity was 86% and specificity was 97% at cutoff value Pg/L. Fig. (3), (Table 5). Table (5): Cutoff values for serum B 12 level for detection of good response to Peg-interferon and ribavirin therapy. Serum B12 level B Sensitivity 97.7% 93% 86.4% Specificity 75.6% 87% 97.8% Correlation studies between serum vitamin B 12 and different laboratory data failed to show any significant correlation with any of the studied laboratory parameters. It is only negatively correlated with ALT and AST levels (Table 6). Table (6): Correlation between serum B 12 and other laboratory values. Pearson correlation coefficient There was no significant correlation between serum vitamin B 12 level and the fibrosis stage in liver histopathology (Table 7). Table (7): Correlation between fibrosis stage and serum vitamin B 12. Pearson correlation coefficient p-value AST ALT Bilirubin Albumin Alkaline phosphatase PT ANA TSH HB TLC PLT PCR p-value 0.79

5 Reda M. Amer, et al. 533 Multivariate analysis showed that serum vitamin B 12 and pretreatment ALT are independent predictors of virological response to Peg-interferon and ribavirin therapy, while other factors that showed significant difference in univariate analysis are statistically nonsignificant in stepwise multivariate logistic regression analysis (Table 8). Table (8): Multivariate stepwise logistic regression analysis of factors affecting response to interferon-ribavirin therapy. p- Odds 95% confidence interval value ratio Lower Upper Serum B ALT Discussion In this study of patients with chronic hepatitis C, we found a strong correlation between serum vitamin-b12 and virological response of chronic hepatitis C infection to combined interferon ribavirin therapy, and multivariate analysis showed that serum level of vitamin-b12 (s-b 12) was independent of other found predictors of response to interferon-ribavirin therapy. Our findings agree with previous study by Rosenberg and Hagen [9], where they reported that strong correlation between s-b 12 at base-line and on-treatment virological response, and multivariate analysis showed that s-b 12 was independent of previously described predictors of response. In patients with ETR, mean pretreatment s-b 12 was 331 Pico mole (pm), while pretreatment s-b 12 was significantly lower in non-responders with a mean value of 262pm (p=0.012). In our study, the patients (both responders and non-responders) had lower level of serum vitamin B 12 than that was reported by Rosenberg and Hagen [9]. In responders, mean serum vitamin B 12 was 247pm, while in the nonresponders, it was 123pm. So our results clearly indicate that non-responders had significantly lower serum vitamin B12 than responder patients. We have shown that taking 183.9pm as a cutoff value for serum vitamin B 12 for detection of good response to interferon gives Sensitivity of 86.4% and specificity of 97.8%. Our findings are also in line with Rocco et al. [8], they found that the rates of complete early viral response (p=0.03), end-of-treatment viral response (p=0.03) and SVR (p=0.001) were significantly higher in patients receiving interferon-ribavirin plus vitamin B12 than in patients receiving only interferon-ribavirin. They concluded that vitamin B12 supplementation significantly improves SVR rates in HCV-infected patients, naïve to antiviral therapy. However, they did not measure the serum level of vitamin B12 in their patients either at baseline or during therapy, and did not demonstrate the effect of supplementation on serum vitamin B 12 during interferon therapy. Our results are consistent with previous in vitro studies that have showed that B 12 at high concentrations inhibits the translation of HCV-RNA. Because the liver is the organ containing the highest concentrations of B 12, there is a possibility of B 12 interfering with HCV RNA translation in vivo. Lott et al. [10] have demonstrated that B12 inhibits the translation of the HCV polyprotein by interference with the Internal Ribosome Entry Site (IRES), a structure in the HCV 5 -nontranslated region. So, vitamin B 12 has a negative effect on IRES-dependent translation of HCV protein in a concentration dependent manner. HCV viral protein translation is solely IRES dependent (Non-CAP dependent translation). Although, the exact molecular mechanism on why vitamin B12 exhibits opposite IRES-regulation remains elusive, these observations suggest vitamin B 12 has specificity to the structure of IRES. Indeed, vitamin B 12 does not suppress other IRES-dependent viruses such as Encephalomyocarditis Virus or Classical Swine Fever Virus [11]. Second, it has been demonstrated in cultured hepatocytes that structural proteins of HCV genotype 1a bind to and possibly also co-internalize with the Asialoglycoprotein Receptor (ASGP-R) and that this binding is partially prevented by ASGP-R ligands [12]. This is interesting as ASGP- R mediate hepatocyte uptake of haptocorrin-bound B12 [13]. In addition to its direct antiviral effect, it has been proposed that vitamin B 12 indirectly influences HCV pathogenesis through deregulation of lipid metabolism. Insufficient vitamin B 12 leads to the impaired production of succinyl-coa and methionine, resulting in the increased risk of hepatic steatosis. The formation of lipid droplets in hepatocytes offers a foundation for the HCV replication complex [14], thereby insufficient vitamin B12 status presumably enhances the HCV lifecycle through the formation of lipid droplets where the HCV replication complex is formed [15]. Another study showed that HCV patients treated with peg-ifn and ribavirin reached significantly

6 534 Impact of Serum Level of Vitamin B12 on Response to Combined Interferon higher SVR if their homocysteine levels were low [14]. This is in line with our findings, suggesting a possible relation between B12 and treatment outcome. Vitamin B 12 is a cofactor for methionine synthase, (methyltransferase enzyme), which transfers a methyl group from 5-methyltetrahydrofolate to homocysteine, thereby generating Tetrahydrofolate (THF) and methionine. This functionality is lost in vitamin B12 deficiency, resulting in an increased homocysteine level. Approximately 80% of the circulating B12 is bound to haptocorrin, and the levels of haptocorrin bound B 12 are in equilibrium with B 12 stored in organs and tissues. In this study, we analysed B12 in serum, as it is previously shown to reflect the amount of B12 stored in the liver [16]. Increased serum levels of B 12 can be seen in myeloproliferative disorders such as chronic myelogenous leukemia or primary polycythaemia, acute fulminant hepatitis, the rare disease hypereosinophilic syndrome and sometimes in renal failure [17]. None of the patients in the present study had a diagnosis or clinical signs of any of these conditions. We have shown that serum vitamin-b12 is significantly and independently correlated to viral response to combined interferon-ribavirin therapy in chronic HCV infection, thus indicating that s- B 12 may be of importance in a clinical setting. At this point it cannot be ruled out that the benefit of vitamin B12 may only exist in the setting of interferon plus ribavirin therapy. Given that vitamin B 12 has been found to be antivirally active in vitro, there is a likely chance it will also be beneficial in all oral regimens against HCV. Concerning the in vitro data, the combined effects of vitamin B12 with direct antiviral with or without interferon can be demonstrated in vitro. It will be important to evaluate the role of vitamin B12 in interferon-free regimens. Given the fast development of highly active antiviral therapy regimens, which are expected to eradicate HCV in most patients within 12 weeks or even shorter treatment duration in over 90% (expected up to 100%) of patients, an approach that increases response rate to a side-effect-loaded therapy. Given the low drug cost for vitamin B12 compared with direct antivirals, it might be difficult to find pharmaceutical sponsors to evaluate these approaches further. However, the development of such approaches might be more attractive for resource-limited settings, and in areas where direct antivirals are further away from daily clinical practice due to longer regulatory processes. Conclusion: Serum vitamin-b12 is significantly and independently correlated to viral response to combined interferon-ribavirin therapy in chronic HCV infection, thus indicating that s-b 12 may be of importance in a clinical setting. Recommendations: Whether measuring serum level of vitamin B 12 before starting interferon-ribavirin therapy (as a predictor of response to therapy) or supplementation with B12 to maintain high s-b 12 levels either before starting or throughout the whole treatment period could improve virological response rates to antiviral therapy. Further studies are needed to prove value of serum vit. B 12 in the setting of the new oral directly acting antiviral drugs for Chronic HCV infection. References 1- ASCIONE A., TARTAGLIONE T. and DI COSTANZO G.G.: Natural history of chronic hepatitis C virus infection. Dig. Liver Dis., 39: S4-S7, STRAUSS R., TORNER A., DUBERG A.S., HULT- CRANTZ R. and EKDAHL K.: Hepatocellular carcinoma and other primary liver cancers in hepatitis C patients in Sweden-a low endemic country. J. Viral Hepat., 15 (7): 531-7, PERZ J.F., ARMSTRONG G.L., FARRINGTON L.A., HUTIN Y.J. and BELL B.P.: The contributions of hepatitis B virus and hepatitis C virus infections to cirrhosis and primary liver cancer worldwide. J. Hepatol., 45 (4): , HADZIYANNIS S.J., SETTE H J.R., MORGAN T.R., et al.: Peginterferon-alpha2a and ribavirin combination therapy in chronic hepatitis C: A randomized study of treatment duration and ribavirin dose. Ann. Intern. Med., 140 (5): , REYNOLDS E.: Vitamin B 12, folic acid, and the nervous system. Lancet Neurol., 5 (11): , LOTT W.B., TAKYAR S.S., TUPPEN J., et al.: Vitamin B 12 and hepatitis C: Molecular biology and human pathology. Proc. Natl. Acad. Sci. USA, 98 (9): , ERMENS A.A., VLASVELD L.T. and LINDEMANS J.: Significance of elevated cobalamin (vitamin B12) levels in blood. Clin. Biochem., 36 (8): , ROCCO A., COMPARE D., COCCOLI P., ESPOSITO C., DI SPIRITO A., et al.: Vitamin B 12 supplementation improves rates of sustained viral response in patients chronically infected with hepatitis C virus. Gut., 62: , ROSENBERG P. and HAGEN K.: Serum B12 levels predict response to treatment with interferon and ribavirin in patients with chronic HCV infection. Journal of Viral Hepatitis, 18 (2): , 2011.

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