Micellar high performance liquid chromatographic method for separation and validation of two anti-hepatitis C- virus drugs in pure form, human plasma and human urine.

OBJECTIVES: In the present study, an eco- friendly micellar liquid chromatographic technique was validated for separation and quantification of two drugs; namely ribavirin (RIV), and sofosbuvir (SBV) in pure form, pharmaceuticals containing them, human plasma and human urine. These drugs are administered co-administered for treatment of Hepatitis C virus (HCV) that causes hepatitis C in humans. MATERIAL AND METHODS: These drugs were separated using Nucleosil 100-5 phenyl column. Sodium dodecyl sulphate (SDS) solution (0.05M, pH 7.0) containing triethylamine (0.3%) and n-butanol (10%) was used as a mobile phase with 1.2 mLmin-1 flow rate and 215nm detection wavelength. Nine minutes were required for resolving the two drugs from the matrix. RESULTS: The method showed good linearity for RIV and SBV with correlation coefficients (r2) more than 0.9996 within the concentration ranges of (20-400) and (40-400) ngmL-1 in pure form, (30-300) and (50-300) ngmL-1 in human plasma and (20-400) and (40-400) ngmL-1 in human urine, respectively. CONCLUSION: The recommended method was applied for examination of RIV and SBV in pure and pharmaceuticals. The obtained results were statistically matched with reported methods with no significant differences. Also, the recommended method was effectively applied for estimation of both drugs in spiked human urine and plasma without purification or extraction steps and real samples of plasma and urine of humans having therapy of RIV and SBV, as well as, performing tablets dissolution-rate tests with satisfactory results.

The effect of genetic variations on ribavirin pharmacokinetics and treatment response in HCV-4 Egyptian patients receiving sofosbuvir/daclatasvir and ribavirin.

PURPOSE: This study aimed to investigate the effect of single nucleotide polymorphisms (SNPs) of genes involved in ribavirin (RBV) transport (SLC28A2 gene, ABCB1 gene and ABCB11 gene) on the clinical outcome and pharmacokinetics of ribavirin in HCV- 4 Egyptian patients. METHOD: 100 patients treated with sofosbuvir/daclatasvir and ribavirin for 12 weeks. The SNP genotyping was performed by real-time PCR using high resolution melting analysis. Ribavirin plasma trough concentrations were determined at week 4 of therapy using a liquid chromatography/tandem mass spectrometry (LC-MS/MS). For clinical outcomes, sustained virological response (SVR), liver function tests (ALT and AST), total bilirubin, albumin, serum creatinine, hemoglobin, leukocyte count, and platelet count were measured. RESULTS: Concerning RBV pharmacokinetics, ABCB1 2677 G > T SNP and ABCB11 1331 T > C SNP were statistically associated with RBV Ctrough levels after 4 weeks of therapy. ABCB11 1331 T > C SNP revealed significant association with clinical outcomes (SVR). SLC28A2-146 A > T SNP has not showed any statistically significant association with RBV plasma levels or response. CONCLUSION: SNP genotyping for ABCB1 and ABCB11 genes can help in better personalized medicine for maximizing response for ribavirin as explored by the significant association between polymorphism in ABCB1 and ABCB11 genes and ribavirin pharmacokinetics and the significant association of ABCB11 1331 T > C SNP with clinical response.

Validated RP-HPLC Method for Simultaneous Determination of Ribavirin, Sofosbuvir and Daclatasvir in Human Plasma: A Treatment Protocol Administered to HCV Patients in Egypt.

Egypt has the highest prevalence of hepatitis C virus (HCV) in the world thus it launched a national program for eliminating HCV aiming to treat 300,000 HCV patients per year. Three anti-HCV co-administered drugs; ribavirin (RBV), sofosbuvir (SF) daclatasvir (DAC) were simultaneously determined in human plasma by a validated, simple and sensitive RP-HPLC method using propyl paraben as an internal standard. Liquid-liquid extraction using ethyl acetate was used for samples extraction. Chromatographic separation was achieved on Scharlau® C18 column (250 × 4.6 mm2, 5 µm). Gradient elution was employed with a mobile phase mixture of water and acetonitrile at a flow rate 1 mL/min. UV detection using photodiode array detector was carried out at 207, 260 and 312 nm for RBV, SF and DAC, respectively. Method validation was performed according to the FDA guidelines for bioanalytical method validation. The calibration curves were linear over the ranges (0.5-80, 0.1-40 and 0.5-80 µg/mL) with average recoveries (100.64-108.28%, 98.48-105.91% and 97.68-101.38%) for RBV, SF and DAC, respectively. The intra-day and inter-day precision and accuracy results were within the acceptable limits. Stability assays revealed that the three studied analytes were stable during sample storage, preparation and injection. The method can be successfully applied in routine analysis of plasma of HCV patients treated with this combination therapy which aids in therapeutic drug monitoring and patients' follow-up especially in Egypt and other developing countries fighting HCV.

Comparative study of six sequential spectrophotometric methods for quantification and separation of ribavirin, sofosbuvir and daclatasvir: An application on Laboratory prepared mixture, pharmaceutical preparations, spiked human urine, spiked human plasma, and dissolution test.

In accordance with International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) guidelines, six novel, simple and precise sequential spectrophotometric methods were developed and validated for the simultaneous analysis of Ribavirin (RIB), Sofosbuvir (SOF), and Daclatasvir (DAC) in their mixture without prior separation steps. These drugs are described as co-administered for treatment of Hepatitis C virus (HCV). HCV is the cause of hepatitis C and some cancers such as liver cancer (hepatocellular carcinoma) and lymphomas in humans. These techniques consisted of several sequential steps using zero, ratio and/or derivative spectra. DAC was first determined through direct spectrophotometry at 313.7 nm without any interference of the other two drugs while RIB and SOF can be determined after ratio subtraction through five methods; Ratio difference spectrophotometric method, successive derivative ratio method, constant center, isoabsorptive method at 238.8 nm, and mean centering of the ratio spectra (MCR) at 224 nm and 258 nm for RIB and SOF, respectively. The calibration curve is linear over the concentration ranges of (6-42), (10-70) and (4-16) µg/mL for RIB, SOF, and DAC, respectively. This method was successfully applied to commercial pharmaceutical preparation of the drugs, spiked human urine, and spiked human plasma. The above methods are very simple methods that were developed for the simultaneous determination of binary and ternary mixtures and so enhance signal-to-noise ratio. The method has been successfully applied to the simultaneous analysis of RIB, SOF, and DAC in laboratory prepared mixtures. The obtained results are statistically compared with those obtained by the official or reported methods, showing no significant difference with respect to accuracy and precision at p = 0.05.

Ultrasensitive spectrofluorimetric method for rapid determination of daclatasvir and ledipasvir in human plasma and pharmaceutical formulations.

Direct-acting antivirals (DAAs) represent a revolution in the treatment of chronic hepatitis C which have emerged at an extremely rapid pace over the past few years. DAAs act directly on the hepatitis C virus at various points in the viral life cycle to inhibit viral production. Among these novel DAAs, are daclatasvir (DCS) and ledipasvir (LDS). Herein, a novel, fast, simple, ultrasensitive and cost-effective spectrofluorimetric method was designed for determination of DCS and LDS in miscellaneous matrices. The method is based on investigation of the native fluorescence of the cited drugs. The relative fluorescence intensity (RFI) was measured at λex/λem equal to 315/381 nm for DCS and 332/387 nm for LDS. Under the optimum conditions, the linear ranges of calibration curves were 0.2-30 and 6-120 ng mL-1 for DCS and LDS, respectively with correlation coefficients ≥0.9998. The detection limits were 0.047 and 1.939 ng mL-1 for DCS and LDS, respectively indicating ultrasensitivity of the proposed method. Consequently, this permits in vitro and in vivo application of the proposed method in spiked and real human plasma with good percentage recovery (96.6-103.6%). The method was validated in compliance with ICH guidelines and US-FDA guidelines. Furthermore, the application was extended to analysis of DCS and LDS in its pharmaceutical formulations (either alone or in presence of other co-formulated drugs) and in synthetic mixture with sofosbuvir or ribavirin.

Direct antiviral treatment of chronic hepatitis C in heart transplant recipients.

Direct-acting antiviral agents (DAAs) are a safe and effective treatment for chronic hepatitis C (CHC). This may be particularly valuable for patients with severe comorbidities or baseline conditions, including non-liver solid organ transplant. We report cases of two heart transplant recipients with CHC treated with DAAs (sofosbuvir and daclatasvir) achieving sustained virological response. Treatment was well tolerated and no relevant side effects were observed. The drug-drug interactions and graft function were carefully monitored.

Ribavirin steady-state plasma level is a predictor of sustained virological response in hepatitis C-infected patients treated with direct-acting antivirals.

BACKGROUND: In the era of highly effective direct-acting antivirals (DAAs) for treatment of patients with chronic hepatitis C virus (HCV) infection, ribavirin (RBV) is still considered beneficial in certain patients. AIM: To assess the association between RBV steady-state plasma levels and sustained virological response (SVR). METHODS: Consecutive HCV-infected patients treated with DAAs plus RBV from four Dutch academic medical centres were enrolled. RBV steady-state plasma levels were prospectively measured at treatment week 8 using validated assays. Logistic regression analyses were performed to assess the influence of RBV steady-state plasma level on SVR, and RBV therapeutic range was explored using area under the ROC curve analyses. RESULTS: A total of 183 patients were included, of whom 85% had one or more difficult-to-cure characteristics (ie treatment experienced, HCV genotype 3, cirrhosis). The majority was treated with a sofosbuvir-based regimen and 163 (89%) patients achieved SVR. Median RBV dose was 12.9 (interquartile range 11.2-14.7) mg/kg/d, and median RBV steady-state plasma level was 2.66 (1.95-3.60) mg/L. In multivariable analyses, higher RBV steady-state plasma level (adjusted odds ratio 1.79 [95% CI 1.09-2.93]) was an independent predictor of SVR. With regard to the optimal RBV therapeutic range, 2.28 mg/L was the optimal lower cut-off for achieving SVR and 3.61 mg/L was the upper cut-off for preventing significant anaemia (Haemoglobin < 10 g/dL). CONCLUSION: In this cohort of mainly difficult-to-cure patients treated with DAAs plus RBV, higher RBV steady-state plasma level was an independent predictor of SVR.

Increased warfarin requirements in a patient with chronic hepatitis C infection receiving sofosbuvir and ribavirin.

PURPOSE: A case of increased warfarin requirements during treatment with sofosbuvir and ribavirin for chronic hepatitis C virus (HCV) infection is reported. SUMMARY: A 63-year-old white man receiving long-term anticoagulation with warfarin for atrial fibrillation and a history of cardioembolic stroke was initiated in September 2014 on a 12-week course of sofosbuvir 400 mg orally daily and weight-based ribavirin 600 mg orally twice daily for HCV genotype 2 infection. Before starting this treatment regimen, the patient had been stable on warfarin 52.5 mg weekly, with therapeutic International Normalized Ratio (INR) values. During the 12-week course of sofosbuvir and ribavirin, the patient's dose of warfarin progressively increased from 52.5 to 77.5 mg weekly due to subtherapeutic INRs, with the first adjustment in the warfarin dose occurring 9 days after initiation of HCV treatment. Three weeks after completion of the sofosbuvir and ribavirin regimen, the patient's INR was 3.06, and his warfarin dose was then decreased to 70 mg weekly. The patient continued with this warfarin dosage until 18 weeks after completion of his HCV regimen. The dosage was then decreased to 65 mg weekly after an INR of 3.86. Three weeks later, his INR was 2.19, and warfarin 65 mg weekly was continued. As of June 2016, the patient has continued to require warfarin 62.5-65 mg weekly to maintain a therapeutic INR. CONCLUSION: A 63-year-old man on a stable dose of warfarin experienced a decrease in INR values after the initiation of a 12-week course of sofosbuvir and ribavirin for the treatment of chronic HCV infection.

Variant Inosine Triphosphatase Phenotypes Are Associated With Increased Ribavirin Triphosphate Levels.

Individuals with lower inosine triphosphatase (ITPA) enzyme activity have a reduced likelihood of experiencing hemolytic anemia during hepatitis C virus (HCV) treatment containing ribavirin (RBV). Because ITPA degrades purines and RBV is a purine analogue, it is conceivable that ITPA activity may affect intracellular RBV concentrations. Here we assessed the association between ITPA activity phenotype and concentrations of RBV triphosphate (RBV-TP) in red blood cells (RBCs) during HCV treatment. RBV-TP was quantified in the RBCs of 177 HCV-infected individuals at a median (range) of 84 (19 to 336) days into HCV treatment that included RBV. Mean (SD) RBV-TP concentrations were 92.8 (51.6), 101.3 (53.5), 184.8 (84.5), and 197.7 (64.6) pmol/106 cells for 100%, 60%, 30%, and ≤10% ITPA activity groups, respectively. Overall, RBV-TP was approximately 2-fold higher in patients with ≤30% ITPA activity compared to 100% activity (P < .0001). Despite higher RBV-TP levels, individuals with variant ITPA phenotypes had less anemia. The 100% activity group had, on average, a -2.20 g/dL drop in hemoglobin vs -1.43 g/dL (P = .04) for 60% activity, -1.14 g/dL (P = .008) for 30% activity, and -0.70 g/dL (P = .06) for ≤10% activity. This finding of higher RBV-TP concentrations in RBCs in ITPA variants was unexpected given that ITPA activity-deficient individuals have a reduced likelihood of RBV-induced anemia. It also refutes the hypothesis that the mechanism by which ITPA variants are protected against anemia is due to lower RBV-TP levels in RBCs.

Population pharmacokinetics of paritaprevir, ombitasvir, dasabuvir, ritonavir and ribavirin in hepatitis C virus genotype 1 infection: analysis of six phase III trials.

AIM: The aim of the current study was to characterize the population pharmacokinetics of a triple direct-acting antiviral (DAA) regimen (3D) (ombitasvir, paritaprevir-ritonavir and dasabuvir) and adjunctive ribavirin, and estimate covariate effects in a broad spectrum of subjects with hepatitis C virus (HCV) genotype 1 infection. METHODS: Pharmacokinetic data from six phase III studies and one phase II study in subjects receiving the currently approved doses of the 3D ± ribavirin regimen for treating HCV genotype 1 infection for 12 weeks or 24 weeks were characterized using separate population pharmacokinetic models, built using each component of the regimen from nonlinear mixed-effects methodology in NONMEM 7.3. In the models, demographic and clinical covariates were tested. Models were assessed via goodness-of-fit plots, visual predictive checks and bootstrap evaluations. RESULTS: The population pharmacokinetic models for each component of the 3D ± ribavirin regimen (DAAs and ritonavir, n = 2348) and ribavirin (n = 1841) adequately described their respective plasma concentration-time data. Model parameter estimates were precise and robust, and all models showed good predictive ability. Significant covariate effects associated with apparent clearance and volume of distribution included age, body weight, gender, cirrhosis, HCV subtype, opioid or antidiabetic agent use, and creatinine clearance. CONCLUSION: The population pharmacokinetics of the 3D ± ribavirin regimen components in HCV-infected patients were characterized using phase II and III HCV clinical trial data. Although several statistically significant covariates were identified, their effects were modest and not clinically meaningful to necessitate dose adjustments for any component of the 3D regimen.

Influence of Ribavirin Serum Levels on Outcome of Antiviral Treatment and Anemia in Hepatitis C Virus Infection.

BACKGROUND: Ribavirin blood levels vary considerably between patients with standard weight-based dosing. Their impact on sustained virological response (SVR) with pegylated interferon and ribavirin is controversial, but has mostly been studied before the IL28b gene polymorphism as a possible confounder was discovered. METHODS: The impact of serum ribavirin trough levels at week 4, at the end of treatment and of mean levels across the entire antiviral treatment with pegylated interferon and ribavirin on relapse, SVR rates and anemia was retrospectively studied by univariate and multivariable logistic regression analyses in 214 patients with HCV genotype 1-4 infection, including 88 patients with available IL28b genotyping. RESULTS: Mean ribavirin levels varied between 0.68-5.65 mg/l and significantly differed between patients with or without SVR. By multivariable regression including age, sex, HCV viral load, HCV genotype, liver fibrosis stage, prior treatments, immunosuppression and IL28b genotype, ribavirin levels consistently displayed significant influence on SVR and relapse without indication for a specific importance of higher concentrations early or late in the treatment course. Although hemoglobin decline was on average more pronounced in patients with higher ribavirin levels, hemoglobin remained relatively stable in a significant proportion of these, indicating that ribavirin levels alone are insufficient to predict anemia. CONCLUSION: While data are scarce to draw conclusions applicable for modern DAA therapies, these results support ribavirin treatment based on serum levels instead of purely weight-based dosing in combination with pegylated interferon.

Randomized Trial Evaluating the Impact of Ribavirin Mono-Therapy and Double Dosing on Viral Kinetics, Ribavirin Pharmacokinetics and Anemia in Hepatitis C Virus Genotype 1 Infection.

In this pilot study (RibaC), 58 hepatitis C virus (HCV) genotype 1 infected treatment-naïve patients were randomized to (i) 2 weeks ribavirin double dosing concomitant with pegylated interferon-α (pegIFN-α), (ii) 4 weeks ribavirin mono-therapy prior to adding pegIFN-α, or (iii) standard-of-care (SOC) ribavirin dosing concurrent with pegIFN-α. Four weeks of ribavirin mono-therapy resulted in a mean 0.46 log(10) IU/mL HCV RNA reduction differentially regulated across IL28B genotypes (0.89 vs. 0.21 log(10) IU/mL for CC and CT/TT respectively; P = 0.006), increased likelihood of undetectable HCV RNA week 4 after initiating pegIFN-α and thus shortened treatment duration (P<0.05), and decreased median IP-10 concentration from 550 to 345 pg/mL (P<0.001). Both experimental strategies impacted on ribavirin concentrations, and high levels were achieved after one week of double dosing. However, by day 14, double dosing entailed a greater hemoglobin decline as compared to SOC (2.2 vs. 1.4 g/dL; P = 0.03). Conclusion: Ribavirin down-regulates IP-10, and may have an anti-viral effect differently regulated across IL28B genotypes.

[Association between ribavirin plasma concentration and sustained virologic response in treatment of patients with genotype 1b chronic hepatitis C with pegylated interferon-α-2b and ribavirin].

OBJECTIVE: To investigate the association between the dose and plasma concentration of ribavirin (RBV) and sustained virologic response (SVR) during the anti-hepatitis C virus (HCV) treatment with pegylated interferon-α-2b (PEG-IFN-α-2b) and RBV. METHODS: A total of 40 patients with chronic hepatitis C (CHC) who were treated with PEG-IFN-α-2b and RBV as the antiviral treatment were enrolled, and according to the therapeutic effect (SVR was defined as HCV RNA maintained below the lower limit of detection at 24 weeks after drug discontinuation in patients who achieved virologic response at the end of treatment, and recurrence was defined as HCV RNA turning positive), these patients were divided into SVR group (20 patients aged 19-55 years, including 10 male patients) and recurrence group (20 patients aged 21-76 years, including 12 male patients). The HPLC-MS/MS was used to measure the RBV plasma concentration at weeks 4, 12, 24, and 48 of treatment. The t-test and receiver operating characteristic (ROC) curve were used for statistical analysis. RESULTS: During the antiviral treatment, the dose of RBV showed a significant difference between the two groups (15.01 ± 1.21 mg/kg vs 10.28 ± 2.81 mg/kg,t= 6.908,P= 0.000). The area under the ROC curve reached 0.96 (95%CI0.00-1.00,P= 0.000), suggesting that the dose of RBV had a high value in predicting SVR. When the dose of RBV was higher than 13.05 mg/kg (sensitivity 100%; specificity 85%), the possibility of achieving SVR was also increased. The RBV plasma concentrations in the SVR group at weeks 4,12, 24, and 48 of treatment were 1 894.8 ± 740.7 ng/ml, 2 029.9 ± 547.7 ng/ml, 2 011.8 ± 354.2 ng/ml, and2 093.5 ± 540.3 ng/ml, respectively, and those in the recurrence group were 1 223.1 ± 722.7 ng/ml, 1 286.9±685.4 ng/ml, 1304.7 ± 692.0 ng/ml, and 1 221.3 ± 655.3 ng/ml, respectively. The RBV plasma concentration at each time point showed significant differences between the two groups (t= 2.903,P= 0.006;t= 3.787,P= 0.001;t= 4.068,P= 0.000;t= 4.593,P= 0.000). The results of ROC analysis showed that the areas under the ROC curve at weeks 4, 12, 24, and 48 of treatment were 0.76 (95%CI0.61-0.92,P= 0.005), 0.83 (95%CI0.68-0.97,P= 0.000), 0.83 (95%CI0.69-0.98,P= 0.000), and 0.86 (95%CI0.72-1.00,P= 0.000), respectively, suggesting that the RBV plasma concentration had a high value in predicting SVR. When the cut-off values of RBV plasma concentration at weeks 4, 12, 24, and 48 of treatment were higher than 1262.5 ng/ml (sensitivity 90%; specificity 60%), 1432 ng/ml (sensitivity 100%; specificity 65%), 1427 ng/ml (sensitivity 100%; specificity 65%), and 1610 ng/ml (sensitivity 95%; specificity 80%), respectively, there was a greater possibility of achieving SVR. CONCLUSION: During the antiviral treatment with PEG-IFN-α-2b and RBV, the dose and plasma concentration of RBV have a high value in predicting the recurrence of CHC and the possibility of SVR.

Ritonavir-boosted danoprevir plus peginterferon alfa-2a and ribavirin in Asian chronic hepatitis C patients with or without cirrhosis.

BACKGROUND AND AIM: Chronic hepatitis C is an important public health problem in Asia. We evaluated the safety, efficacy, and pharmacokinetics of fixed-dose ritonavir-boosted danoprevir plus peginterferon alfa-2a/ribavirin in treatment-naive Asian patients with chronic hepatitis C virus (HCV) genotype (G)1 infection. METHODS: Treatment-naive G1 patients in Taiwan, Thailand, and Korea with serum HCV-RNA level ≥ 105 IU/mL received ritonavir-boosted danoprevir 125/100 mg twice daily plus peginterferon alfa-2a/ribavirin for either 12 (noncirrhotic patients: Arm A, n = 34) or 24 weeks (cirrhotic patients: Arm B, n = 27) in this phase II open-label study. Sustained virologic response was defined as HCV-RNA < 25 IU/mL 12 weeks after end of treatment (SVR12). RESULTS: Similar SVR12 rates were achieved in Arms A (88.2%; 95% confidence interval, 73.4-95.3%) and B (88.9%; 71.9-96.2%). Most patients had G1b infection, among whom SVR12 rates in Arms A and B were 96.7% and 91.7%, respectively. The overall SVR12 rate was 94.0% in noncirrhotic Taiwanese patients (100% in the subset of G1b patients). No patients withdrew for safety reasons. Three (11%) cirrhotic patients (Arm B) experienced serious adverse events, none of which was considered to be related to treatment. No Grade 3/4 alanine aminotransferase elevations were reported. The pharmacokinetic properties of danoprevir were broadly overlapping in noncirrhotic and cirrhotic patients both on Days 1 and 14. CONCLUSIONS: Ritonavir-boosted danoprevir plus peginterferon alfa-2a/ribavirin produced sustained virologic response rates > 90% after 12 weeks' treatment in noncirrhotic and 24 weeks' treatment in cirrhotic Asian patients with G1b infection and was well tolerated. These regimens are well suited to countries where G1b predominates.

Pharmacokinetics, efficacy prediction indexes, and residue depletion of ribavirin in Atlantic salmon's (Salmo salar) muscle after oral administration in feed.

Ribavirin is an antiviral used in human medicine, but it has not been authorized for use in veterinary medicine although it is effective against infectious salmon anemia (ISA) virus, between others. In this study, we present a pharmacokinetic profile of ribavirin in Atlantic salmon (Salmo salar), efficacy prediction indexes, and the measure of its withdrawal time. To determine the pharmacokinetic profile, fishes were orally administered with a single ribavirin dose of 1.6 mg/kg bw, and then, plasma concentrations were measured at different times. From the time-vs.-concentration curve, Cmax = 413.57 ng/mL, Tmax  = 6.96 h, AUC = 21394.01 µg·h/mL, t1/2  = 81.61 h, and K10  = 0.0421/h were obtained. Ribavirin reached adequate concentrations during the pharmacokinetic study, with prediction indexes of Cmax /IC50  = 20.7, AUC/IC50  = 1069.7, and T>IC50  = 71 h, where IC is the inhibitory concentration 50%. For ribavirin depletion study, fishes were orally administered with a dairy dose of 1.6 mg/kg bw during 10 days. Concentrations were measured on edible tissue on different days post-treatment. A linear regression of the time vs. concentration was conducted, obtaining a withdrawal time of 1966 °C days. Results obtained reveal that the dose of 1.6 mg/kg bw orally administered is effective for ISA virus, originating a reasonable withdrawal period within the productive schedules of Atlantic salmon.

Adherence to ribavirin in chronic hepatitis C patients on antiviral treatment: Results from a randomized controlled trial using real-time medication monitoring.

BACKGROUND AND OBJECTIVE: Adherence is essential in antiviral therapy for chronic hepatitis C. We investigated the effect of real-time medication monitoring on adherence to ribavirin. METHODS: In this randomized controlled trial, patients in the intervention group received a medication dispenser that monitored ribavirin intake real-time during 24 weeks PEG-interferon/ribavirin±boceprevir or telaprevir. Patients in the control group received standard-of-care. Adherence was also measured by pill count. RESULTS: Seventy-two patients were assigned to either intervention (n=35) or control groups (n=37). Median adherence by pill count was 96% (range: 43%-100%) with 30 (94%) of patients exhibiting≥80% adherence. Perfect adherence (i.e. 100%) was similar in intervention and control groups: 22 (85%) vs. 15 (75%) (P=0.47). Adherences by real-time medication monitoring and by pill count did not correlate (R=0.19, P=0.36). No predictors of poor adherence could be identified. Ribavirin trough levels after 8 weeks (median: 2.4 vs. 2.7mg/L, P=0.30) and 24 weeks (median: 3.0 vs. 3.0mg/L, P=0.69), and virological responses did not differ between intervention and control groups. CONCLUSIONS: Adherence to ribavirin during PEG-interferon containing therapy in chronic hepatitis C is high. Real-time medication monitoring did not influence adherence to ribavirin, plasma ribavirin levels or virological responses.

Concentration Monitoring of Plasma Ribavirin: Validation of a Liquid Chromatography-Mass Spectrometric Method and Clinical Sample Collection.

BACKGROUND: A liquid chromatography-tandem mass spectrometric (LC-MS/MS) method was developed for routine measurement of ribavirin concentrations in EDTA-anticoagulated plasma. METHODS: After protein precipitation, we used a bridged ethylene hybrid (hydrophilic interaction) chromatography column, 0.1 mmol/L ammonium formate pH 3.0, and a gradient of 85%-96% acetonitrile to achieve baseline separation of ribavirin from isobaric uridine. Quantitation was assured using both primary (m/z 245.3 > 113.0) and secondary transitions (m/z 245.3 > 96.0) of the protonated species. Chromatographic separation and column washing also negated interference from major phospholipid species. RESULTS: There was a linear relationship between concentration and response to 10 mg/L, with a minimum detectable level and a minimum level of quantitation both of 0.1 mg/L. Imprecision within the assay was <10% at 0.1 mg/L and <6% between assays for concentrations >0.4 mg/L. Bias was <4%. In clinical samples (n = 12), there was no difference in ribavirin concentrations obtained by an established liquid chromatographic assay with ultraviolet detection. Ribavirin concentrations were stable in plasma stored at room temperature for 3 days but then decreased significantly on day 7. Plasma concentrations were stable for 15 weeks at -20 °C. Concentrations in plasma separated from whole blood at room temperature fell by a median of 19.4% at 4 hours and then rose substantially (median 251% by 3 days). Dose-normalized ribavirin concentrations reached a steady state after a mean of >6 weeks treatment in 76 patients with hepatitis C. CONCLUSIONS: A hydrophilic interaction liquid chromatography-tandem mass spectrometric method to measure ribavirin in plasma was developed. Samples for ribavirin estimation should be kept at 4 °C, separated within 2 hours of collection and stored at 4 °C before analysis, with long-term storage at -20 °C. This method was applied to a study of the ribavirin therapeutic monitoring in patients with hepatitis C.

The impact of ribavirin plasma concentration on the efficacy of the interferon-sparing regimen, sofosbuvir and ribavirin.

BACKGROUND: Ribavirin augments sustained virological response when administered with pegylated interferon for the treatment of chronic HCV infection. The impact of ribavirin plasma concentration on outcome in individuals receiving interferon-free regimens has not been evaluated. METHODS: Stored plasma samples were retrieved for 47 treatment-naive subjects who received sofosbuvir and weight-based ribavirin for 12-24 weeks in the Phase IIb QUANTUM study. Week 1, 4 and 8 ribavirin plasma concentrations (mg/l) were quantified using high-performance liquid chromatography with UV detection. RESULTS: Sustained virological response at 12 weeks post treatment was observed in 55% with all treatment failures due to relapse. The median ribavirin plasma concentration increased from week 1 (1.58 mg/l, IQR 1.44-2.24) to week 4 (2.23 mg/l, IQR 1.69-2.87) and week 8 (2.67 mg/l, IQR 2.10-3.26) with wide variability at steady state. Median week 4 ribavirin plasma concentration was 2.25 mg/l (IQR 1.63-3.05) in those with a sustained virological response as compared to 2.07 mg/l (IQR 1.79-2.86) in those with treatment failure (OR 1.35; 95% CI 0.76, 2.39; P=0.3). No significant association between ribavirin plasma concentration and treatment response was noted at weeks 1 or 8. CONCLUSIONS: We found no evidence of an association between ribavirin plasma concentrations and relapse suggesting that, as opposed to interferon-based therapy, suboptimal ribavirin plasma concentrations did not explain the high rate of virological failure with this regimen. Our findings suggest that in interferon-free ribavirin-containing regimens, concerns over ribavirin dosing to achieve previously determined target plasma concentrations are unnecessary.

Evaluation of a rapid method for the simultaneous quantification of ribavirin, sofosbuvir and its metabolite in rat plasma by UPLC-MS/MS.

A rapid and sensitive ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the determination of ribavirin, sofosbuvir and its metabolite GS-331007 in rat plasma was established. The analytes and the internal standard (midazolam) were separated on an Acquity UPLC BEH C18 chromatography column (2.1mm×50mm, 1.7µm) using gradient elution with a mobile phase of acetonitrile and 0.1% formic acid in water at a flow rate of 0.4mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode to monitor the precursor-to-product ion transitions of m/z 245.1→113.1 for ribavirin, m/z 530.3→243.1 for sofosbuvir, m/z 261.5→113.1 for GS-331007 and m/z 326.2→291.1 for midazolam (IS) using a positive electrospray ionization interface. The method was validated over a concentration range of 5-1000ng/mL for ribavirin, 10-2000ng/mL for sofosbuvir and 10-2000ng/mL for GS-331007. Total time for each chromatograph was 3.0min. The intra- and inter-day precision and accuracy of the quality control samples at low, medium, and high concentration levels exhibited relative standard deviations (RSD) <10.0% and the accuracy values ranged from -10.6% to 11.6%. The method was successfully applied to a pharmacokinetic study of ribavirin, sofosbuvir and GS-331007 in rats.