Ribavirin Quantification in Combination Treatment of Chronic Hepatitis C

Abstract

Ribavirin in combination with alpha 2 interferon is the consensus treatment for chronic hepatitis C. However, recent preliminary pharmacological studies have suggested that the bioavailability of ribavirin displays great interindividual variability. In order to monitor serum ribavirin levels during combination treatment, we developed and validated a quantitative assay using an approach adaptable for routine hospital laboratories. The method involved solid-phase extraction on phenyl boronic acid cartridges followed by high-performance liquid chromatography with a C 18 -bonded silica column and a mobile phase containing 10 mM ammonium phosphate buffer (with the pH adjusted to 2.5) and UV detection (207 nm). The sensitivity, recovery, linearity of the calibration curve, intra-and interassay accuracies, precision, and stability at 4°C were consistent with its use in the laboratory routine. In addition, other nucleoside analogues sometimes used with ribavirin in patients coinfected with hepatitis C virus (HCV) and human immunodeficiency virus did not interfere with the quantification of ribavirin levels. The ribavirin concentration was quantified in 24 serum samples from patients with chronic hepatitis C treated with a combination of ribavirin and alpha 2 interferon. The mean serum ribavirin concentration was 2.67 ؎ 1.06 g/ml (n ‫؍‬ 24) at week 12 of treatment (W12) and 3.24 ؎ 1.35 g/ml (n ‫؍‬ 24) at week 24 of treatment (W24). In addition, ribavirin concentrations displayed high interindividual variabilities: the coefficients of variation of the serum ribavirin concentrations adjusted to the administered dose were 44 and 48% at W12 and W24, respectively. Moreover, the ribavirin concentration was higher in patients with a sustained virological response (n ‫؍‬ 11) than in patients with treatment failure (n ‫؍‬ 13), with significant intergroup differences at W12 (P ‫؍‬ 0.030) and W24 (P ‫؍‬ 0.049). The present study describes a simple analytical method for the quantification of ribavirin in human serum that could be a useful tool for the monitoring of ribavirin concentrations in HCV-infected patients in order to improve the efficacy and safety of therapy with ribavirin plus interferon.

A single mutation in poliovirus RNA-dependent RNA polymerase confers resistance to mutagenic nucleotide analogs via increased fidelity

“…1C suggest that passage of virus at a lower concentration of ribavirin was necessary to allow the accumulation of mutations before the stringent selection for resistant variants provided by passage in 400 M ribavirin. In hepatitis C virus (HCV)-infected patients treated with ribavirin, estimates of the drug concentrations in blood plasma range from 10 to 30 M, though the concentration in hepatocytes may be higher (20,21). Although it is uncertain whether the concentration of ribavirin required to inhibit poliovirus growth is similar to the concentration required to inhibit HCV growth, the results presented here suggest that treatment with low concentrations of the drug could facilitate later selection of resistant variants.…”

High-dose ribavirin in combination with standard dose peginterferon for treatment of patients with chronic hepatitis C

“…7 The probability of response to treatment has been shown to increase with increasing ribavirin concentrations. 8,9 Therefore, we chose a target concentration of Ͼ15 mol/L, based partly on the data of Jen et al 8 and partly on our prior studies in which some patients on standard therapy achieved concentrations above 15 mol/L. We reasoned that the more effective concentrations achieved in a small proportion of patients on standard therapy should be explored under controlled conditions.…”

“…7 Other studies have shown a correlation between the ribavirin concentration and viral response. 8,9 We propose that concentrations greater than 15 mol/L may im-prove the response rate for genotype 1 patients infected with HCV.…”

Early ribavirin pharmacokinetics, HCV RNA and alanine aminotransferase kinetics in HIV/HCV co-infected patients during treatment with pegylated interferon and ribavirin

“…RBV concentration was measured using previously published methods [18][19][20] in an assay consisting of phenylboronic acid solid phase extraction (PBA SPE) followed by highperformance liquid chromatography (HPLC) with minor modifications. In brief, to 200 μl of previously frozen plasma samples were added 5.0 μl internal standard mixture(0.40 μg/μl each of 3-methylcytidine methosulfate [3MCMS, Sigma, St. Louis, MO] and 5-methylcytidine [5MC, Sigma]), and 800 μl of 250 mM ammonium acetate buffer (pH 8.5).…”