Lucio Boglione

Association of ITPA polymorphisms rs6051702/rs1127354 instead of rs7270101/rs1127354 as predictor of ribavirin-associated anemia in chronic hepatitis C treated patients

Functional variants rs7270101 and rs1127354 of inosine triphosphatase (ITPA) were recently found to protect against ribavirin (RBV)-induced hemolytic anemia. However, no definitive data are yet available on the role of no functional rs6051702 polymorphism. Since a simultaneous evaluation of the three ITPA SNPs for hemolytic anemia has not yet been investigated, we aimed to understand the contribution of each SNPs and its potential clinical use to predict anemia in HCV treated patients. A retrospective analysis included 379 HCV treated patients. The ITPA variants rs6051702, rs7270101 and rs1127354 were genotyped and tested for association with achieving anemia at week 4. We also investigated, using multivariate logistic regression, the impact of each single and paired associated polymorphism on anemia onset. All SNPs were associated with Hb decrease. The carrier of at least one variant allele in the functional ITPA SNPs was associated with a lower decrement of Hb, as compared to patients without a variant allele. In multivariate logistic regression analyses the carrier of a variant allele in the rs6051702/rs1127354 association (OR=0.11, p=1.75×10(-5)) and Hb at baseline (OR=1.51, p=1.21×10(-4)) were independently associated with protection against clinically significant anemia at week 4. All ITPA polymorphisms considered were shown to be significantly associated with anemia onset. A multivariate regression model based on ITPA genetic polymorphisms was developed for predicting the risk of anemia. Considering the characterization of pre-therapy anemia predictors, rs6051702 SNP in association to rs1127354 is more informative in order to avoid this relevant adverse event.

Kinetics and prediction of HBsAg loss during therapy with analogues in patients affected by chronic hepatitis B HBeAg negative and genotype D

In patients affected by chronic hepatitis because of HBV infection, long‐term suppressive therapy with nucleos(t)ides analogues in the HBeAg− patients has shown low effects on HBsAg titre (qHBsAg) decrease, and HBsAg loss is difficult to achieve. Thus, in this type of patients the main goals of antiviral therapy is the suppression of HBV‐DNA and ALT normalization.

Sequential therapy with entecavir and PEG-INF in patients affected by chronic hepatitis B and high levels of HBV-DNA with non-D genotypes

Complete eradication of hepatitis B virus (HBV) is rarely achieved. Treatment options include currently available nucleos(t)ide analogues and pegylated interferon. The aim of our exploratory study was to assess the effectiveness of sequential therapy for chronic hepatitis B (CHB) vs the current standard of care. We evaluated an association with entecavir and pegylated interferon alfa‐2a (PEG‐IFN) in 20 patients with hepatitis B, high HBV viremia and genotypes A, B, C and E. Patients received entecavir alone for 12 weeks, then entecavir and PEG‐IFN for 12 weeks, lastly PEG‐IFN alone for 36 weeks. The results were compared with 20 patients (control group) treated in the past with 48 weeks of PEG‐IFN monotherapy. Our results show that complete sustained virological response (SVR) and partial SVR were, respectively, 60% and 80% in the study group and 10% and 30% in the control group; anti‐HBe seroconversion rate were 76.9% vs 15%, and anti‐HBs seroconversion were 20% vs 0%, respectively. We found a correlation among different genotypes and virological and serological outcomes – genotype C has a better virological response, while genotype A had a better serological response, and E genotype had a poor response. These results show that a sequential approach is a promising strategy of treatment in patients with CHB and high viremia in comparison with PEG‐IFN monotherapy. The E genotype seems to have the worse rate of response and requires other treatment strategies.

Significant early higher ribavirin plasma concentrations in patients receiving a triple therapy with pegylated interferon, ribavirin and telaprevir

The new standard of care for treatment for infection with genotype 1a/b of HCV now is the combination of telaprevir (TLV) with ribavirin (RBV) and pegylated interferon (Peg‐IFN). Although this new therapy gives a higher response rate than the Peg‐IFNα plus RBV treatment, a greatly higher rate of anaemia onset has been reported in all clinical trials. Because haemolysis is a typical concentration‐dependent side effect of RBV, modulated by ITPA gene polymorphisms, we aimed to compare the early RBV plasma exposure of nine patients after 2 weeks of treatment with triple therapy with RBV concentrations of 187 patients treated with RBV and Peg‐IFNα over the same time scale; this comparison was performed also stratifying patients according to ITPA polymorphism genotype and anaemia onset after 1 month of treatment. All TLV‐treated patients had unfavourable ITPA genetic profile and developed anaemia. Moreover, both the rate of anaemia onset and the haemoglobin loss at 1 month were significantly higher in patients treated with TLV. This observation has been confirmed also in patients with the same ITPA genetic profile in double therapy. Strikingly, also early RBV plasma concentrations were significantly higher in patients treated with TLV. These unbiased results confirm the observations recently reported and suggest that the high rate of anaemia onset could be mainly due to the increased RBV exposure, probably caused by a ‘boosting effect’ by TLV. These data highlight the great importance of early therapeutic drug monitoring of RBV in the management of anaemia in the triple therapy.

A UPLC-MS/MS method for the simultaneous plasma quantification of all isomeric forms of the new anti-HCV protease inhibitors boceprevir and telaprevir.

HCV infection affects over 170 milions people in the world. Up to now standard-of-care therapy consisted in ribavirin plus interferon-α 2a or 2b. Recently, two new Direct Acting Antivirals (DAA), inhibitors of NS3 HCV protease, have been developed and approved for the routine use on HCV genotype 1 infected patients: boceprevir and telaprevir. Protease inhibitors show complex pharmacokinetics (strong metabolism of both drugs by CYP3A and drug interactions), they require a TID dosage and, furthermore, they are present in plasma patients in two different isomeric forms. In this work, we developed and validated an UPLC-tandem mass spectrometry assay method capable of monitoring the telaprevir and boceprevir concentrations in plasma, discriminating each isomer of both drugs. Blank plasma used for Standards and QCs preparation, was obtained from blood of healthy donors. The calibration curves for each isomer of telaprevir and boceprevir were linear in a range from 46.87 ng/mL to 6000 ng/mL and from 23.43 to 3000 ng/mL, respectively (mean r(2)>0.998 for all analytes). QCs at three different concentration were prepared: High, Medium and Low. Each Standard, QC and patient sample was treated with a protein precipitation protocol with a solution containing acidified acetonitrile and Internal Standard (6,7-Dimethyl-2,3-di(2-pyridyl)quinoxaline). An aliquot of supernatant was diluted and then directly analyzed by reverse-phase UPLC-MS/MS. Accuracy (mean 98.47%), intra-day (mean <5.21%) and inter-day (mean <7.57%) precision for telaprevir and boceprevir quality controls fitted all FDA guidelines. All compounds resulted stable in our method conditions and the extraction procedure showed a recovery near to 100%. Finally, we tested this method by monitoring plasma concentrations in 9 HCV+ patients, for each triple combined therapy, with good results. The observed median ratio between S and R isomers for boceprevir and telaprevir were 1.22 (IQR 1.10-1.33) and 1.52 (IQR 1.21-1.67), respectively. The use of this simple assay method could be an important tool for management of HCV-1 DAAs treated patients.

Development and validation of a useful HPLC-UV method for quantification of total and phosphorylated-ribavirin in blood and erythrocytes of HCV+ patients.

Ribavirin-induced hemolytic anemia is the main cause of discontinuation of the combination therapy with alpha-interferon-2b and ribavirin for the treatment of hepatitis C virus (HCV) infection. The determination of intracellular ribavirin levels in blood, including the levels of its phosphorylated metabolites, might be useful for predicting ribavirin-induced anemia, which could be caused by the accumulation of these molecules within the erythrocytes. In this work, we simplified and validated a previously developed assay method, to make it suitable for routine monitoring of cellular ribavirin. Whole blood diluted with a five-fold volume of ice-cold distilled underwent a process of acid phosphatase digestion to convert phosphorylated ribavirin metabolites to free ribavirin. The resulting mixture, spiked with an internal standard, was treated with a protein precipitation protocol in acetonitrile, followed by reverse-phase high-performance liquid chromatography analysis. The calibration curve for ribavirin levels in whole blood was linear at concentrations from 625 to 320,000 ng/mL (r(2)=0.998). Accuracy, intra-day and inter-day precision for ribavirin and phosphorylated-ribavirin quality controls were all below 9.0%. We tested this method by monitoring blood ribavirin concentrations in 13 HCV+ patients, receiving alpha interferon-plus ribavirin combination therapy.

Role of CYP27B1+2838 promoter polymorphism in the treatment of chronic hepatitis B HBeAg negative with PEG‐interferon

In HBV‐infected patients, the vitamin D deficiency has been related to chronic liver diseases, progression of hepatic fibrosis and poor response to the treatment. The CYP27B1 gene, which encodes the 1‐α‐hidroxylase and involved in the 1,25‐dihydroxyvitamin D synthesis, was recently associated to type‐1 diabetes, autoimmune disorders and treatment response in HCV. Then, we aimed to investigate the role of CYP27B1 polymorphisms in HBV treatment with PEG‐IFN. We retrospectively enrolled 190 patients with chronic hepatitis B HBeAg negative treated for 48 weeks with PEG‐IFN α‐2a. We examined the role of rs4646536 CYP27B1 SNP (CYP27B1+2838) according to virological and serological response. Our results showed that the TT genotype of CYP27B1+2838 was significantly prevalent in patients with end‐of‐therapy virological response (37.6%) vs CT/CC (9.4%) (P < 0.001). Virological relapse was prevalent in patients with CT/CC genotype (12.6%) vs TT genotype (2.1%) (P < 0.001). TT genotype was also related to HBsAg loss (P = 0.004) and anti‐HBs appearance (P = 0.002). In the multivariate analysis, the TT genotype resulted to be a good positive predictor of sustained virological response (OR = 5.632, IC = 1.938–16.368, P = 0.001) and serological response (OR = 6.161, IC = 1.856–20.457, P = 0.003). The CYP27B1+2838 polymorphism may be useful as pretreatment factor to selection of patients with higher probability of response to therapy.

Vitamin D pathway gene variants and HCV-2/3 therapy outcomes.

BACKGROUND The combination of ribavirin and pegylated interferon-α is considered the standard of care for HCV-2/3 genotypes. The immune system plays a key role in the achievement of a sustained virological response (SVR). Vitamin D seems to influence antiviral response in chronic hepatitis C and its pathway is controlled by polymorphic genes such as CYP27B1, CYP24A1 and VDR. In this study, we have investigated the correlation among the treatment outcomes and single nucleotide polymorphisms (SNPs) in the above-mentioned genes and IL28B genes. METHODS A total of 112 HCV-2/3 patients treated with interferon plus ribavirin were retrospectively studied; allelic discrimination was performed by real-time PCR. RESULTS CYP24A1rs2585428, IL28Brs12979860 and rs8099917 SNPs affected treatment failure and body mass index (BMI), Metavir score, IL28Brs8099917TT and CYP24A1rs2585428GG were the only factors able to predict it. SVR was predicted by Metavir score, HCV RNA at baseline and early virological response (EVR). IL28Brs12979860 SNP and HCV RNA were also related to rapid virological response. EVR was predicted by BMI, Metavir score and CYP24A1rs2585428 SNP. IL28Brs8099917TT and FokITT were relapse prediction factors. CONCLUSIONS In addition to non-genetic factors, SNPs in the vitamin D pathway seem to have a role in HCV-2/3 therapy outcomes. This study reveals the likely usefulness of pharmacogenetic-based ribavirin and interferon therapy to help identify patients for whom therapy could be successful or not, also considering new future expensive therapy options. To date, no similar data were published on these viral genotypes, but further studies in different and bigger cohorts are needed.

Role of IL28-B polymorphisms in the treatment of chronic hepatitis B HBeAg-negative patients with peginterferon

Interleukin (IL)28-B polymorphism has been related to interferon response in the treatment of hepatitis C, but its role in chronic hepatitis B (CHB) therapy is still poorly understood. We aimed to investigate the effect of IL28-B polymorphisms in the treatment with pegylated-interferon (PEG-IFN) of patients with CHB. We retrospectively analyzed 190 patients with chronic hepatitis B e antigen (HBeAg) negative, genotype A (22%), B (12%), C (10%), D (33%), E (20%), treated with PEG-IFN alfa-2a for 48weeks; genotype analysis was performed for IL28-B polymorphisms rs12979860, rs8099917 and rs12980275 according to virological, serological and biochemical response. During 2years of follow-up 12 patients (6.3%) cleared hepatitis B surface antigen (HBsAg) with seroconversion, 40 (21%) obtained a negative viral load and 104 (54.7%) gained a biochemical response. We found a difference of distribution of rs12979860 CC genotype among different ethnicity (p=0.013). Rs12979860 CC genotype was significantly associated with serological and virological response (p<0.001); rs8099917 TT and rs12980275 AA genotypes were mostly related with virological response (p<0.001). In multivariate logistic analysis rs12979860 CC was predictive of virological response (OR=4.290; CI=1.589-11.580, p=0.004) and serological response (OR=10.129; CI=2.440-42.044; p<0.001). Rs8099917 TT was predictive only of virological response (OR=3.746, CI=1.235-11.355; p=0.020). The E genotype was a negative predictive factor of virological response (OR=0.057; CI=0.014-0.238; p<0.001). IL28-B polymorphisms are related to different response in the treatment of CHB HBeAg-negative with PEG-IFN, and the E genotype is a novel negative predictive factor.

Development and validation of a useful UPLC-MS/MS method for quantification of total and phosphorylated-ribavirin in peripheral blood mononuclear cells of HCV+ patients.

The current standard-of-care therapy in HCV consists in ribavirin (RBV) plus pegylated-interferon-α 2a or 2b and, for HCV-1 infected patients, also directly acting antivirals (DAAs). Despite the increase in the number of patients who reach sustained virological response (SVR) for HCV-1, a great inter-individual variability in the response to therapy remains. Whether new drugs are available in combination with RBV and Peg-IFN for HCV-1, the treatment of the other viral genotypes remains the same: this issue highlights the lasting importance of RBV and Peg-IFN in anti-HCV treatment. Moreover, a strong limiting factor to the usefulness of anti-HCV treatment remains the occurrence of adverse events, first of all hemolytic anemia, which have increased with the addition of DAAs, but is mainly an RBV-dependent effect. For these reasons, the monitoring of RBV exposure in the various compartments should be important. Since the routinely determination of RBV in the target cells as the hepatocytes is impracticable for of its invasiveness, the quantification in easier to obtain cells could be a good choice. In this work, we developed and validated an ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) assay method to quantify RBV concentrations in peripheral blood mononucleated cells (PBMCs). QCs were prepared with RBV and RBV-monophosphate (RMP). Each sample was divided into two aliquots, which undergone the same extraction procedure: one was treated with acid phosphatase to convert RBV phosphorylated metabolites into free RBV, the other one was not-treated. The extracts were analyzed with reverse-phase column with UPLC-MS/MS. Calibration curves fitted a least squares model (weighed 1/X) for ribavirin levels in a range from 0.1 ng to 200 ng (mean r(2)=0.9993). Accuracy, intra-day and inter-day precision of the methods were in accordance with FDA guidelines. Moreover, phosphorylated QCs were used to assess the correct determination of total RBV concentration. We tested this method by monitoring RBV concentrations in PBMCs from 20 HCV+ patients, receiving alpha interferon-plus RBV combination therapy. This method showed to be reliable, precise, accurate and suitable for evaluation of intracellular RBV concentrations.