Prediction of sustained virological response by ribavirin plasma concentration at week 4 of therapy in hepatitis C virus genotype 1 patients

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1 Short communication Antiviral Therapy 13: Prediction of sustained virological response by ribavirin plasma concentration at week 4 of therapy in hepatitis C virus genotype 1 patients Marianne Maynard 1,2,3, Pierre Pradat 1,2,3, Marie-Claude Gagnieu 4, Claude Souvignet 1,3 and Christian Trepo 1,2,3 * 1 Hospices Civils de Lyon, Hôtel Dieu, Service d hépatologie et de gastroentérologie, Lyon, France 2 INSERM, U871, Lyon, France 3 Université Lyon 1, IFR62 Lyon-Est, Lyon, France 4 Hospices Civils de Lyon, Hôpital Edouard Herriot, UF de Pharmacologie Spécialisée, Fédération de Biochimie, Lyon, France *Corresponding author: christian.trepo@chu-lyon.fr Background: Pegylated interferon/ribavirin combination is currently the standard treatment for chronic hepatitis C virus (HCV) infection. Body weight adjustment of ribavirin is crucial for response. However, previous studies found no relation between ingested dose and plasma concentration. The aim of this study was to define the ribavirin trough plasma concentration at week 4 (W4) associated with sustained virological response (SVR). Methods: Thirty-one HCV genotype 1 patients (8 naive and 23 non-responders to a previous pegylated interferon/ribavirin therapy) were treated with pegylated interferon/ribavirin and assessed by HPLC for ribavirin plasma concentration at W4. Results: Eleven patients (35%) achieved SVR, whereas 20 (65%) were non-responders. The median ribavirin plasma concentration at W4 (1.90 mg/l) varied from 1.62 mg/l in patients with subsequent non-response to 2.28 mg/l in sustained responders (P=0.007). Receiver operating characteristic curve analysis indicated that the 2.01 mg/l threshold gave the best sensitivity and specificity (73% and 80%, respectively, area under the curve =0.80; P=0.007). Sixty-seven percent of patients with median ribavirin plasma concentration >2 mg/l achieved SVR versus only 16% below this level (P=0.007). Multivariate regression analysis indicated that a ribavirin plasma concentration >2 mg/l at W4 was associated with SVR independent of gender, age, weight, baseline viral load and response to previous therapy. Conclusion: These results, which remain to be confirmed in large clinical trials, highlight the potential relevance of ribavirin plasma level monitoring at an early stage of treatment. This monitoring could be of help in guiding antiviral therapy by offering dose adjustment in patients with ribavirin plasma level below the 2 mg/l threshold. Introduction Combination therapy with ribavirin (RBV) and pegylated interferon (PEG-IFN) is the current recommended treatment for chronic hepatitis C virus (HCV) infection [1] with an overall sustained virological response (SVR) rate of about 55% [2,3]. Weight-adjusted doses of RBV appear crucial to optimize response rates [4,5]. Pharmacokinetic studies indicate, however, that there is no relation between RBV-ingested dose and plasma concentration. Furthermore, RBV plasma concentrations display high interindividual variability, 30% of which being explained by age, weight, gender and renal function [6]. This variability could explain the severe haematological side effects or lack of efficacy in some patients. Measurements of RBV plasma levels could allow the therapeutic monitoring of RBV administration according to individual pharmacokinetics of the drug. The aim would be to improve virological response rate, because it has been demonstrated that higher RBV concentrations at week 4 of treatment were associated with a higher SVR rate [6,7]. Furthermore, data previously presented suggested that RBV plasma concentration at week 4 of therapy could be predictive of early virological clearance at week 12 [8]. The aim of this pilot study was to define the target trough RBV concentration at week 4 that is possibly associated with optimal sustained HCV clearance International Medical Press

2 M Maynard et al. Methods HCV genotype 1 patients (naive or non-responders to a previous anti-hcv therapy) were prospectively recruited from the Department of Hepatogastroenterology, Hotel- Dieu, Lyon, France, starting in All patients were treated with PEG-IFN-α2b 1.5 μg/kg/week and RBV at a dose of 800 1,200 mg/day for 48 weeks (in two daily doses during meals). Following recommendations, the dose of RBV was adjusted according to body weight (800 mg if <65 kg, 1,000 mg if kg or 1,200 mg if >85 kg). Treatment compliance was orally assessed for each patient. Patients failing to achieve the 80/80/80 rule were excluded (80% PEG-IFN dose, 80% RBV dose and 80% treatment duration completed). HCV RNA levels were assessed by PCR (COBAS AMPLICOR HCV Test, v2.0 Roche Diagnostics, Melun, France; detection limit <615 IU/ml) at baseline, week 12, 24 and 48 of treatment and at 6 months after the end of therapy. Early virological response was defined as undetectable HCV RNA (or HCV RNA decrease 2 log from baseline) at week 12. SVR was classically defined as undetectable HCV RNA 6 months after the end of treatment. Patients failing to achieve SVR were considered as non-responders. RBV plasma concentration at week 4 under therapy was centrally assessed using a validated HPLC assay with diode array UV detection after immediate centrifugation [7,9]. Blood sampling was performed in the morning before RBV intake (trough concentration, that is, h after dosing). Receiving Operating Characteristic (ROC) curves were performed to estimate the RBV plasma threshold giving the best sensitivity and specificity for the prediction of SVR. RBV plasma concentration was compared between responder and non-responder patients using the non-parametric Mann Whitney U-test. A multivariate regression analysis was conducted to see whether RBV concentration was associated to SVR independently of gender, age, weight, baseline viral load and patient status at inclusion (naive or non-responder). Because of the small number of patients in the study, we assessed the effect of RBV concentration using serial regression models with two covariates each. Statistical analyses were performed using SPSS version 12.0 (SPSS, Chicago, IL, USA). A P- value <0.05 was considered as statistically significant. According to the French legislation, and because this study was conducted during patients routine clinical follow-up without any modification, no ethics committee approval was necessary. Written informed consent, however, was obtained from each patient. Results Thirty-one patients were analysed. None of them suffered from renal insufficiency. Main characteristics at baseline are presented in Table 1. Sixty-one percent of patients were males and mean age was 51 years. At inclusion, 8 patients were naive, whereas 23 were non-responders to a previous PEG-IFN/RBV combination therapy. After treatment, 11 patients (35%) achieved SVR, 6 (19%) relapsed within 6 months after the end of therapy and 14 (45%) were nonresponders. As expected, the SVR rate was higher in treatment-naive patients (62%) compared with patients having failed to respond to a previous therapy (26%), although this difference was not statistically significant (P=0.09). The median RBV plasma concentration at week 4 (1.90 mg/l, range ) varied from 1.58 mg/l in non-responders (range ) to 2.28 mg/l in sustained responders (range ; Mann-Whitney U-test P=0.007). The SVR rate varied from 13% in patients with RBV level <1.56 mg/l (25% quartile) to 67% in patients reaching levels >2.66 mg/l (75% quartile; χ 2 for trend analysis gave P=0.03; Figure 1). Sixteen patients had an RBV dose <13.6 mg/kg/day, whereas Table 1. Patient characteristics at inclusion according to subsequent treatment response SVR (n=11) NR (n=14) Relapse (n=6) Total (n=31) P-value Gender, n (%) 0.40 Male 5 (26.3) 10 (52.6) 4 (21.1) 19 (100%) Female 6 (50.0) 4 (33.3) 2 (16.7) 12 (100%) Mean age, years (range) 48 (41 63) 54 (38 78) 50 (43 60) 51 (38 78) 0.25 Mean baseline viral load, IU/ml (range) 604, ,815 2,349,114 1,084, (5,463 1,621,570) (87,565 2,796,560) (441,179 5,741,600) (5,463 5,741,600) Median ribavirin plasma 2.28 ( ) 1.58 ( ) 1.90 ( ) 1.90 ( ) 0.007* concentration at week 4, mg/l (range) Patient treatment status at inclusion, n (%) 0.11 Naive 5 (62.5) 3 (37.5) 0 8 (100%) Non-responder 6 (26.1) 11 (47.3) 6 (26.1) 23 (100%) *Patients with relapse included in the non-responder group. NR, non-responders; SVR, sustained virological response International Medical Press

3 Ribavirin concentration and treatment response in HCV patients 15 had a dose above this value. Interestingly, the SVR rate did not differ between both groups (37% versus 33%, respectively; P=1). No patient was exposed to a dose of <10.6 mg/kg/day of RBV, which is the minimum recommended dose. A ROC curve analysis indicated that the threshold of 2.01 mg/l gave the best sensitivity and specificity (73% and 80%, respectively; area under the curve =0.80; P=0.007). Of patients with an RBV plasma concentration >2.01 mg/l at week 4, 67% subsequently achieved SVR versus only 16% among patients below this level (P=0.007; Table 2 and Figure 2). All patients with RBV plasma concentration >2.80 mg/l achieved SVR. Five of six patients (83%) with an end-of-treatment response and a virological relapse within 6 months had an RBV plasma concentration <2 mg/l. This differed significantly from sustained responders in whom only 3/11 patients (27%) were <2 mg/l (P=0.049). If the analysis was restricted to patients who had failed to respond to a previous course of PEG- IFN/RBV combination therapy (n=23), those with an RBV plasma level >2 mg/l were more likely to achieve an SVR if treated again compared with patients with an RBV plasma level <2 mg/l (57% versus 13%, respectively; P=0.045). In treatment-naive patients (n=8), the SVR rate was increased by almost 50% in patients with an RBV plasma level >2 mg/l compared with patients <2 mg/l (80 versus 33%, respectively), but the number of patients was too limited to draw any conclusion. Serial multivariate logistic regression models based on two covariates indicated that an RBV plasma concentration >2 mg/l was significantly associated with SVR independent of gender, age, weight, baseline viral load and patient status at inclusion (Table 2). Similarly, using regression models with two covariates, we tried to identify factors possibly associated with an RBV plasma concentration >2 mg/l. However, no factor was found to be associated with high RBV levels (data not shown). A correlation analysis showed that RBV plasma concentration at week 4 was correlated to the haemoglobin drop between inclusion and week 4 (r=0.512, P=0.003). Discussion This study indicates that RBV plasma concentration at week 4 of PEG-IFN/RBV combination therapy is predictive of treatment response with significantly higher plasma levels in early virological responders as well as sustained virological responders. A multivariate analysis showed that this association was independent of gender, age, weight, baseline viral load and patient s Figure 1. Early virological response, end of treatment and sustained virological response rates based on ribavirin plasma concentration at week 4 after initiation of combination therapy (given in quartiles) Virological response, % SVR End of treatment EVR < >2.66 n=8 n=8 n=9 n=6 Ribavirin plasma concentration at week 4, mg/l Early virological response (EVR) is defined as undetectable hepatitis C virus (HCV) RNA or >2 log 10 decrease of HCV viraemia from baseline to week 12. End of treatment response is defined as undetectable HCV RNA at the end of treatment and sustained virological response (SVR) as undetectable HCV RNA 6 months after the end of treatment. *For SVR, χ 2 analysis for trend gives P=0.03. Table 2. Sustained virological response: univariate and multivariate logistic regression analysis Univariate Multivariate EOR (95% CI) P-value EOR* (95% CI) P-value Ribavirin plasma concentration (>2 mg/l versus 2 mg/l) 10.7 ( ) Age 0.92 ( ) ( ) Gender (female versus male) 2.80 ( ) ( ) Patient status (naive versus non-responder) 4.72 ( ) ( ) Weight 0.95 ( ) ( ) Baseline viral load (<800,000 versus >800,000) 1.75 ( ) ( ) The multivariate analysis evaluates the effect of ribavirin concentration using serial regression models with two covariates each. *Each estimated odds ratio (EOR) of the multivariate model reflects the effect of ribavirin independently of each specific covariate. 95% CI, 95% confidence interval. Antiviral Therapy 13:4 609

4 M Maynard et al. treatment status at inclusion. This confirms the fact that RBV plasma concentration during the first few weeks of treatment dramatically effects the subsequent treatment efficacy. As the RBV steady state is usually obtained after 4 weeks of drug exposure [6,10,11] and because no major intra-individual variations are expected afterwards, the choice of week 4 as time point for measurement of RBV plasma concentration seems well adapted. The SVR rate among patients reaching a concentration >2 mg/l at week 4 was increased by 50% compared with patients remaining below this threshold. Interestingly, almost all patients with virological relapse had an RBV plasma concentration below the 2 mg/l threshold. RBV is well known to reduce relapses in patients receiving IFN combination therapy [12]. Our results support the fact that RBV may also play a role on early HCV clearance during the first 3 months of therapy in HCV-monoinfected patients [13,14], as previously shown in HCV/HIV-coinfected patients [15 17]. It has been recently suggested that RBV could improve outcomes by enhancing hepatic gene responses to PEG-IFN [18]. Although the association between RBV plasma concentration and treatment response has been already studied during PEG-IFN/RBV combination therapy [19], this report provides for the first time a cutoff value that could distinguish patients most likely to achieve SVR. In the present study, a week 4 RBV plasma level of 2 mg/l appeared to be a crucial threshold for achieving SVR. Because our population sample included mainly nonresponder patients, it remains possible that a slightly different cutoff value would be observed for treatmentnaive patients. However, this cutoff is in accordance with previous studies that retrospectively investigated RBV plasma levels according to treatment outcomes [7,20]. Further studies in naive patients are needed to confirm our results in this subgroup. Defining such a minimal threshold of plasma RBV is an essential step for therapeutic drug monitoring of RBV. Even if week 4 HCV RNA is known to be a good predictor of SVR, RBV dose adjustment should be relevant in hard-to-treat patients, especially those failing to achieve a rapid virological response. Based on the present results, a state-funded French national multicenter clinical trial in HCV genotype 1 patients was setup to test the effect of RBV dose adjustment on RBV plasma concentrations and its effect on SVR rates. Although higher ingested doses of RBV may increase the frequency and severity of side effects, especially anaemia, increasing exposure to RBV will only target those patients with low RBV plasma concentration. This should preserve an acceptable tolerance in most patients because the present study confirms that side effects, especially anaemia, are correlated to serum levels [20]. In the case of severe side effects, the use of erythropoietin will be considered [21]. Figure 2. Ribavirin plasma concentration at week 4 according to subsequent virological response for all 31 patients Ribavirin plasma concentration at week 4, mg/l Given its high interindividual and low intra-individual variability and its association between concentration and efficacy/toxicity, RBV appears as a good candidate for the assessment of drug concentration [22]. If the present results are confirmed, RBV plasma level monitoring at an early stage of treatment, possibly followed by dose adjustment in patients with plasma level below the 2 mg/l threshold, could be of further help in optimizing antiviral therapy in HCV patients. Acknowledgements We gratefully thank Sylvie Thevenon and Charlène Lachaize who substantially contributed to data collection. Disclosure statement The authors declare no competing interests. References Non-response Virological response Sustained response The dashed line represents the 2 mg/l threshold as defined by the receiving operating characteristic curve analysis. Patients with virological relapse were considered as non-responders. 1. Hoofnagle JH, Seeff LB. Peginterferon and ribavirin for chronic hepatitis C. N Engl J Med 2006; 355: Fried MW, Shiffman ML, Reddy KR, et al. Peginterferon alfa-2a plus ribavirin for chronic hepatitis C virus infection. N Engl J Med 2002; 347: International Medical Press

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