Ledipasvir Research Articles

DEVELOPMENT AND VALIDATION OF STABILITY-INDICATING REVERSE-PHASE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR THE ESTIMATION OF LEDIPASVIR IN BULK AND TABLET DOSAGE FORM

Objective: The objective of this study was to develop a stability-indicating reverse-phase high-performance liquid chromatography (RP-HPLC) method for the estimation of ledipasvir (LDP) in bulk and tablet formulation.
 Methods: Stability-indicating RP-HPLC method was developed and validated for the estimation of LDP in bulk and tablet formulation. RP-HPLC was carried out on HiQ SiL C18 columns (250 mm × 4.6 mm, 5 μ particle size) using mobile phase acetonitrile:1 mM ammonium acetate buffer in the ratio of 90:10 v/v at a flow rate of 1 ml/min. The analytes were monitored using MD 2010 PDA detector at 333 nm.
 Results: The retention time was found to be 3.843 min. The proposed method was found to be having linearity in the concentration range of 5–30 μg/ml. The number of theoretical plates obtained was 4236.50 which indicate the efficient performance of the column. The limit of detection was 0.305 μg/ml and limit of quantification was 0.923 μg/ml, which indicate the sensitivity of the method; the high percentage recovery indicates that the proposed method is highly accurate. The developed method has been validated according to the ICH guidelines and found to be simple, specific, precise, and accurate.
 Conclusion: The proposed method is precise, accurate, and stability indicating. Therefore, the proposed method can be used for routine quality control and analysis of LDP during stability studies in bulk samples and tablet dosage forms.

Novel determination of a new antiviral combination; sofosbuvir and velpatasvir by high performance thin layer chromatographic method; application to real human samples

Sofosbuvir (SOF) and velpatasvir (VEL) represent a brand new antiviral combination. A novel, simple and economical high performance thin layer chromatographic separation coupled with densitometric quantification was designed for the pharmaceutical and biomedical analysis of SOF and VEL. Simultaneous quantitation of the studied drugs in their newly introduced pharmaceutical formulation and human plasma; using ledipasvir (LED) as an internal standard; was achieved. Protein precipitation technique using acetonitrile was adopted, utilizing HPTLC as a clean-up step. Separation was achieved on HPTLC silica gel 60 F254 aluminum plates with a green developing system consisting of ethyl acetate-isopropanol (90:10, v/v). Densitometric scanning was carried out at 260 and 302 nm for SOF and VEL, respectively. Peak purity was evaluated through the winCATS® software spectral correlation method to ascertain the specificity of the proposed method. Method validation was assessed as per the US-FDA guidelines. The proposed method represents a simple, green and cost-effective alternative to the only existing more complicated reported LC-MS/MS technique. The method would afford a competent tool for therapeutic drug monitoring and bioavailability studies of SOF and VEL. The method could be applied to the quality control and routine analysis of SOF and VEL in their pure forms and pharmaceutical formulations. Furthermore, the application was extended to simultaneous quantitation of SOF and VEL in real human plasma.

Comparison of Naturally Occurring Resistance-Associated Substitutions Between 2008 and 2016 in Chinese Patients with Chronic Hepatitis C Virus Infection.

Aims: The presence of pre-existing hepatitis C virus (HCV) resistance-associated substitutions (RASs) could attenuate viral susceptibility to direct-acting antiviral agents. The aim of this study was to better understand the differences among HCV RASs over time. We compared the prevalence and characteristics of naturally occurring HCV RASs in the NS3, NS5A, and NS5B genes between 2008 and 2016 in Chinese patients chronically infected with HCV genotypes (GT) 1b, 2a, 3a, 3b, and 6a. Methods: HCV RNA was extracted after serum samples were collected from 242 patients at treatment baseline, including 120 samples in 2008 and 122 samples in 2016. Reverse transcription and nested PCR were performed, and the PCR products of the NS3, NS5A, and NS5B regions were sequenced using the Sanger sequencing method. Finally, RASs were identified from the different viral strains. Results: In GT1b, the overall frequency of NS5A RASs in 2016 was significantly higher than that in 2008 (42.0% vs. 18.4%; p = 0.002). Among NS5A RASs, the most frequently detected RAS was Y93H (5.3% in 2008 vs. 15.9% in 2016; p = 0.035), which confers medium- to high-level resistance to the NS5A inhibitors: daclatasvir (DCV), ledipasvir (LDV), ombitasvir (OMV), and elbasvir. The frequency of NS5A L28 (low-level resistance to DCV/LDV/OMV) in 2016 was also higher than that in 2008 (11.6% vs. 1.3%; p = 0.027). In addition, the highest frequency of clinically relevant NS3 RASs was S122G/A/T (69.7% in 2008 and 72.5% in 2016) in HCV GT1b isolates, which had medium-level resistance to simeprevir and asunaprevir, followed by Y56F (7.9% in 2008 and 14.5% in 2016), which confers resistance to paritaprevir. Although NS5B C316N had the highest substitution rate in GT1b (80.2% in 2008 and 91.3% in 2016), it was associated with low-level resistance to sofosbuvir and dasabuvir. However, HCV RASs were rarely detectable at baseline in other genotypes or subtypes except GT1b in this study. Conclusion: The frequency of NS5A RASs in 2016 was significantly higher than that in 2008, especially at the L28 and Y93 substitution positions, which may be due to their better fitness compared with wild-type viruses.

Quantification of second generation direct-acting antivirals daclatasvir, elbasvir, grazoprevir, ledipasvir, simeprevir, sofosbuvir and velpatasvir in human plasma by UPLC-MS/MS

Direct-acting antivirals (DAA) have markedly improved the treatment of hepatitis C, with a series of DAA combinations available for treatment. A sensitive method by ultra-high performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) was developed for the simultaneous quantification of seven commonly used DAAs, daclatasvir (DAC), elbasvir (ELB), grazoprevir (GZR), ledipasvir (LDV), simeprevir (SIM), sofosbuvir (SOF), velpatasvir (VEL) and the primary analyte of interest of SOF (GS-331007) in EDTA plasma.Adequate chromatographic separation with well-defined peaks for all eight analytes was achieved with an Acquity UPLC BEH C18 column (1.7 μm 2.1 × 50 mm). Runtime for the final assay was 12 min. Protein precipitation in the sample preparation and stable isotope- labelled internal standards resulted in a robust sensitive and rapid method.Method validation was performed in accordance with the “guideline on bioanalytical method validation” of the European Medicines Agency (EMA). No interferences from endogenous substances were observed and carry-over in the blank sample was <20% of the LLOQ of each analyte and <5% of the IS after injection of the HLOQ sample. Validation was established over the range of 10–5000 μg/L for DAC and SIM, 3.0–1500 μg/L for ELB and GZR, 7.5–1500 μg/L for LDV and VEL, 5.0–2500 μg/L for SOF and 25–5000 μg/L for GS-331007. Within-day accuracy values for all analytes, ranged from 94% to 109%, and precision expressed as residual standard deviation % (RSD) was <9.3%. Between-day accuracy ranged from 99 to 107% with a RSD of <5.3%. For the lower limit of quantification, the percent deviation from the nominal concentration and the relative standard deviation was <20%.The method has been applied successfully in clinical evaluation of patients treated with DAA and to describe the pharmacokinetics of DAAs in clinical studies.

Correlation Between Tenofovir Drug Levels and the Renal Biomarkers RBP-4 and ß2M in the ION-4 Study Cohort.

BackgroundConcomitant dosing of ledipasvir (LDV) and tenofovir disoproxil fumarate (TDF) results in an increased tenofovir (TFV) area under the curve (AUC). The aim of this study was to examine whether there was a correlation between the renal biomarkers retinol binding protein–4 (RBP-4) and β2 microglobulin (β2M) and tenofovir AUC.MethodsThe ION-4 trial enrolled HIV/hepatitis C virus–coinfected patients on nonpharmacologically boosted antiretroviral regimens with TDF-containing backbones. We assessed for a correlation between tenofovir AUC and urinary biomarkers and also for changes in serologic biomarkers with respect to clinically relevant changes in renal function (creatinine clearance decrease >25%, change in creatinine >0.2 mg/dL, change in proteinuria from negative/trace to ≥1+).ResultsThree hundred thirty-five patients were enrolled in the ION-4 study; their demographic characteristics have been previously described. Both RBP-4 and β2M exhibited positive correlations with tenofovir AUC. Baseline and study levels of RBP-4 and β2M were higher for patients with increases in urine proteinuria and an absolute creatinine increase.ConclusionsTFV exposure is associated with increased proximal tubule urine biomarkers in participants on ledipasvir/sofosbuvir and nonpharmacologically boosted TDF-based antiretroviral regimens. Baseline proximal tubule biomarkers may predict nephrotoxicity risk if events are prevalent. Further studies assessing the predictive role of these urine biomarkers may help guide medical decision-making and risk/benefit assessments in patients with risk factors for renal dysfunction.

Baseline Characteristics and Treatment Cost of Hepatitis C at Hospital for Tropical Diseases, Ho Chi Minh City, Vietnam in Direct-Acting Antiviral Treatment Era

Background: Direct-Acting Antivirals (DAAs) are recommended as first-line of drugs for the treatment of chronic hepatitis C virus (HCV) infection in Vietnam in 2016. Since then, Hospital for Tropical Diseases (HTD), Ho Chi Minh City, Vietnam introduced DAAs based treatment for all newly presented chronic HCV patients. Here, we report the sociodemographic, clinical, biochemical, and virologic characteristics of patients and the direct medical cost associated with DAAs treatment. Methods: We conducted a retrospective cross-sectional study among chronic HCV patients attended at HTD from March 2016 to October 2017 and treated with DAAs. We used an extract of the patient’s electronic medical record containing demographics, clinical presentations, laboratory results, drug prescription, and cost of treatment at the hospital for data analysis. Results: 2817 chronic HCV patient received DAAs treatment during the study period. The mean age was 55.0 years, and 54.9% (1546/2817) of the patients were female. HCV genotype 1, 2, 3 and 6 prevalence was 32.1% (904/2817), 12.7% (359/2817), 0.4% (10/2817), and 54.7% (1542/2817) respectively. The mean HCV viral load was 3.1 × 106 copies/ml, including 46.9% (1322/2817) had ≥106 copies/ml. 70.64% (1990/2817) and 16.15% (455/28817) of the patients received Sofosbuvir (SOF)/Ledipasvir (LDV) ± Ribavirin (RBV) and SOF/Daclatasvir (DCV) ± RBV therapy respectively. The average drug cost for a 12-week of SOF/LDV ± RBV and SOF/DCV ± RBV treatment was US$2068 - 2230 and US$2417 - 2472, respectively. Conclusion: Genotype 6 was the most predominant genotype in southern Vietnam. The preferred treatment for chronic HCV infection was SOF/LDV ± RBV for 12 weeks.

AN INNOVATIVE METHOD DEVELOPMENT AND FORCED DEGRADATION STUDIES FOR SIMULTANEOUS ESTIMATION OF SOFOSBUVIR AND LEDIPASVIR BY RP HPLC

Objective: To develop an innovative, rapid, simple, cost effective, stability indicating reverse phase-high performance liquid chromatography (RP-HPLC) method for simultaneous estimation of ledipasvir (LP) and sofosbuvir (SB) in combination pill dosage form.&#x0D; Methods: The method was developed using C8 column, 250 mm x 4.6 mm, 5mm using mobile section comprising of 0.1% (v/v) orthophosphoric acid buffer at pH 2.2 and acetonitrile in the ratio of 45:55 that was pumped through the column at a flow rate of 0.8 ml/min. Temperature was maintained at 30 °C, the effluents were monitored at 260 nm with the help of usage of PDA detector.&#x0D; Results: The retention time of LP and SB were found to be 2.246 min and 3.502 min. The approach was found to be linear with the variety of 9-36 µg/ml and 40-240 μg/ml for LP and SB respectively, the assay of estimated compounds were found to be 99.65% and 99.73% w/v for LP and SB respectively.&#x0D; Conclusion: The pressured samples changed into analyzed and this proposed a technique turned into determined to be particular and stability indicating as no interfering peaks of decay compound and excipients were observed. Hence, the approach was easy and economical that may be efficiently applied for simultaneous estimation of both LP and SB in bulk and combination tablet system.

Sustained Viral Response in Genotype 4 Chronic Hepatitis C Virus-infected Children and Adolescents Treated With Sofosbuvir/Ledipasvir.

Recently, direct acting antivirals (DAAs), sofosbuvir (SOF) combined with ledipasvir (LED), were approved for treatment of hepatitis C virus (HCV)-infected children 12 years of age and older or weighting at least 35 kg for all HCV genotypes. The aim of this study was to assess the safety and efficacy of SOF/LED in genotype 4 HCV-infected Egyptian children and adolescents. This observational study included 40 consecutive HCV-infected children of age 12 to <18 years old or weighing >35 kg, both treatment-naive and treatment-experienced. All of the children were hepatitis B virus-negative and had normal renal functions and heart rate. Patients received oral, fixed-dose combination tablet of SOF/LED (400 mg SOF, 90 mg LED [Harvoni]) once daily for 12 weeks. Potential side effects were recorded at weeks 4, 8, and 12 weeks of treatment. The study primary outcome was sustained virological response 12 weeks (SVR12) after end-of-treatment. The study included 40 children and adolescents, 24 were boys (60%); their age ranged between 11.5 and 17.5 years (mean 13.9 ± 1.5). Baseline viral load ranged between 9630 and 24,600,000 IU/mL. HCV RNA became negative in 39 patients (97.5%) at 4 weeks and in all patients (100%) at weeks 8, 12, and SVR12. Asthenia was the commonest side effect, reported in 52.5% followed by headache in 47.5%. Treatment with all-oral DAAs (SOF/LED) for 12 weeks was well tolerated in Egyptian children and adolescents infected with genotype 4 HCV, with 100% SVR12 and negligible side effects.

Cost-effectiveness analysis of Daclatasvir/Sofosbuvir for the treatment of the HCV patients failed after the first line with second generation of DAAs in Italy

ABSTRACT Background: Daclatasvir (DCV) combinated with Sofosbuvir (SOF) has shown good efficacy and safety profile for HCV patients. The aim was to evaluate the cost-effectiveness of DCV/SOF regimen versus HCV alternative treatments for patients who failed to achieve the SVR12 after a first DAA treatment from Italian perspective (PITER cohort). Methods: A Markov model of HCV chronically infected patients was used to develop two scenarios: 1) DCV+ SOF versus Ledipasvir (LDV)+ SOF in Genotype (Gt)1 and Gt4; 2) DCV+ SOF versus no retreatment option in Gt1, Gt3, and Gt4. The percentage of patients who failed the first line with SOF/Simeprevir/Ribavirin (RBV) or SOF/RBV and were retreated or not according to evidences from PITER cohort, were used to populate the model. HCV resources consumption and SVR rates were quantified using PITER data. Transition probabilities and utility rates were derived from the literature. The outcomes were expressed in terms of Quality adjusted life years (QALYs). Probabilistic sensitivity analysis (PSA) was performed considering a cost-effectiveness threshold of € 30,000/QALY. Results: In the base-case analysis, DCV+ SOF represents a cost-effectiveness therapy with ICERs lower than the threshold. The PSA showed robust results, ICERs remain below the threshold in 94% and 99% simulations in Scenario 1 and 2, respectively.

Direct acting antiviral agent for Chinese patients with chronic hepatitis C genotype 1b infection——a real world experience

Objective To evaluate the effectiveness and safety of direct-acting antiviral agents (DAA) treatment in Chinese chronic hepatitis C (CHC) patients with genotype (GT) 1b HCV infection in a real world setting. Methods The consecutive GT1b CHC Chinese patients treated with sofosbuvir (SOF) plus daclatasvir (DCV) (n=62) or SOF plus ledipasvir (LDV) (n=171) were enrolled from July 2014 to December 2016 at 302 Military Hospital of China. The treatment duration for all the patients was 12 weeks. All the clinical parameters were measured at baseline and then 4-weekly till 12 weeks after the end-of-treatment (EOT). Baseline clinical characteristics, treatment efficacy, safety and tolerance were compared. Serum HCV RNA concentration was detected by means of COBAS TaqMan assay with a lower detection limit of 15 IU/mL, and liver stiffness was measured using FibroScan®. Sustained virologic response (SVR) was defined as HCV RNA under the lower limit of quantification 12 weeks after EOT (SVR12). Students′ t-test, pearson χ2 test, Spearman rank correlation analysis and Fisher exact test were used for comparison between groups when appropriate. Results Among 233 patients, 173 cases had baseline HCV RNA level ≥6.0 lg IU/mL and 97 cases hade liver stiffness measurement (LSM)≥17.5 kPa. The baseline liver inflamation, liver fibrosis, and HCV RNA load of patients in the two groups were not significantly different (all P>0.05). The HCV RNA of all the 233 patients was undetectable at the end of 12-week treatment, while 2 patients relapsed after 12 weeks of EOT with the overall SVR12 of 99.1%. HCV RNA decline was significantly faster in patients with lower LSM than those with higher LSM (ρ=0.233, P=0.001), and SVR12 was higher in those with lower LSM. In terms of other clinical characteristics of SOF+ DCV and SOF+ LVD groups, alanine transaminase declined from (68.0±60.1) and (70.1±56.1) U/L to (21.1±10.9) U/L and (15.3±9.5) U/L, respectively, total bilirubin declined from (21.3±17.3) and (18.2±14.0) μmol/L to (13.2±6.7) and (10.2±4.6) μmol/L, respectively, AFP declined from 19.6 (10.6, 62.3) and 15.0 (12.0, 25.0) μg/L to 6.5(4.5, 18.7) and 7.8(5.3, 15.4) μg/L, respectively, LSM declined from 17.6 (8.9, 25.4) and 15.7 (7.8, 23.9) kPa to 13.9(6.5, 21.4) and 9.1(5.6, 19.9) kPa, respectively, serum album elevated form (37.5±5.8) and (38.7±5.5) g/L to (41.3±4.7) and (42.8±5.1) g/L, respectively, platelet elevated from (120.9±78.2) ×109/L and (136.6±65.8)×109/L to (139.5±71.8)×109/L and (149.7±71.4)×109/L, respectively. Reports of adverse events were low in both groups. Conclusions Both SOF+ DCV and SOF/LDV therapy are highly effective with >98% of SVR12 and reduce LSM value significantly with good safety for CHC GT1b Chinese patients. Key words: Hepatitis C, chronic; Treatment; Direct-acting antiviral agents

Multitarget Direct-Acting Antiviral Therapy Is Associated With Superior Immunologic Recovery in Patients Coinfected With Human Immunodeficiency Virus and Hepatitis C Virus.

Patients coinfected with human immunodeficiency virus (HIV) and hepatitis C virus (HCV) have higher levels of immune activation, impaired antigen‐specific responses, and accelerated fibrogenesis compared to patients monoinfected with HCV. Whether different direct‐acting antiviral (DAA) combinations have differential effects on immunophenotypes and functions following successful HCV therapy remain unknown. Therefore, we aimed to assess the peripheral T‐cell immunophenotypes and functions in patients coinfected with HIV/HCV who were successfully treated with combination DAA treatment regimens. We analyzed peripheral blood mononuclear cells (PBMCs) at baseline and at the time of sustained viral response (SVR) from subjects treated with three different combination DAA regimens: daclatasvir (DCV) and asunaprevir (ASV) for 24 weeks (CONQUER 2‐DAA), DCV/ASV/beclabuvir (BCV) for 12 weeks (CONQUER 3‐DAA), and sofosbuvir (SOF) and ledipasvir (LDV) for 12 weeks (ERADICATE study). We used flow cytometry to assess T‐cell phenotypes (activation and exhaustion) and HCV‐specific T‐cell functions (cytokine secretion and cytotoxicity). Statistical analyses were conducted using the Wilcoxon matched‐pairs signed‐rank test with P < 0.05 considered significant. Overall, there was an improvement in T‐cell exhaustion markers, a decrease in T‐cell activation, an increase in the effector memory population, and improved T‐cell function after achieving SVR, with the largest effects noted with CONQUER 3‐DAA treatment. Conclusion: Treatment with DCV/ASV/BCV in patients coinfected with HIV/HCV resulted in greater restoration of the T‐cell impairments and perturbations associated with HIV/HCV coinfection to an extent that was greater than that observed in either two‐drug regimens. We showed that different DAA‐based therapies have different immunologic outcomes after successful HCV treatment in patients coinfected with HIV/HCV. This information will be beneficial for providers when selecting the regimens for patients coinfected with HIV/HCV.

Autoimmune Hepatitis During Ledipasvir/Sofosbuvir Treatment of Hepatitis C: A Case Report.

We report the case of a woman with chronic hepatitis C and idiopathic thrombocytopenic purpura (ITP) who developed autoimmune hepatitis (AIH) during antiviral therapy with ledipasvir (LDV)/sofosbuvir (SOF). The onset of acute hepatitis rose two weeks after starting treatment with LDV/SOF when HCV‐RNA tested negative, suggesting a link between rapid HCV clearance and de novo autoimmune diseases. Conclusion: This case report proposes new immunologic scenarios in patients with hepatitis C virus (HCV) with laboratory or clinical signs of autoimmunity during direct‐acting antiviral (DAA) therapy.

Graphite/Nanocrystalline Zeolite Platform for Selective Electrochemical Determination of Hepatitis C Inhibitor Ledipasvir

AbstractA simple and efficient procedure is described for the electrochemical determination of ledipasvir (LDP) in presence of co‐formulated drug sofosbuvir (SOF). Herein, a highly sensitive, low‐cost electrochemical protocol was utilized to fabricate a zeolite modified carbon paste electrode (ZY/CPE) through mixing of zeolite nanostructures with graphite powder. The fabricated sensor displayed high sensitivity, allowing optimal charge transfer/electrode kinetics. Different experimental and chemical factors, including electrode composition, effect of pH, scan rate and amount of ZY were evaluated carefully to obtain the highest electrochemical response. Under optimized conditions and using square wave voltammetry (SWV), the current response of the ZY/CPE electrochemical sensing platform exhibited a detection limit of 7.5×10−9 M and a large linear range from 5.0×10−8 to 1.0×10−4 M. The practical applicability of the suggested electrochemical platform was verified in the assessment of LDP in pharmaceutical formulations with excellent recoveries in the range of 99.50–98.87 %. Moreover, a biological relevance of the developed sensor was established by the analysis of LDP in human urine and plasma samples with satisfactory recoveries of 99.00 and 99.68 %, respectively. Due to the simplicity and ease of preparation of the proposed sensor, it can be used in quality control laboratories and for clinical analysis.

Generic sofosbuvir-based interferon-free direct acting antiviral agents for patients with chronic hepatitis C virus infection: a real-world multicenter observational study

Real-world data regarding the effectiveness and safety of generic sofosbuvir (SOF)-based interferon-free direct acting antiviral agents (DAAs) for patients with chronic hepatitis C virus (HCV) infection remain limited. A total of 517 chronic HCV-infected patients receiving 12 or 24 weeks of SOF-based therapies were retrospectively enrolled in 4 academic centers in Taiwan. The rate of sustained virologic response at week 12 off-therapy (SVR12) and that of treatment completion were assessed. The baseline characteristics and on-treatment HCV viral kinetics to predict SVR12 were analyzed. By evaluable population (EP) analysis, the SVR12 rate was 95.4% (95% confidence interval [CI]: 93.2–96.9%). The SVR12 was achieved in 29 of 34 patients (85.3%, 95% CI: 69.6–93.6%), 130 of 139 patients (93.5%, 95% CI: 88.2–96.6%), 119 of 124 patients (96.0%, 95% CI: 90.9–98.3%) and 215 of 220 patients (97.7%, 95% CI: 94.8–99.0%) who received SOF in combination with ribavirin (RBV), ledipasvir (LDV), daclatasvir (DCV) and velpatasvir (VEL), respectively. Of 517 patients, 514 (99.4%) completed the scheduled treatment. All 15 patients with true virologic failures were relapsers. Two decompensated cirrhotic patients had on-treatment deaths which were not related to DAAs. All 7 patients who were lost to follow-up had undetectable HCV RNA level at the last visit. The SVR12 rates were comparable in terms of baseline patient characteristics and viral decline at week 4 of treatment. In conclusion, generic SOF-based regimens are well tolerated and provide high SVR12 rates in patients with chronic HCV infection.

Efficacy and Safety in Difference Combination of Sofosbuvir in Treatment of Chronic Hepatitis C Virus

AbstractBackground: Hepatitis C virus infects about 185 million people equating 2.8% of worldwide population. Management of chronic HCV patients traditionally depended on combination of peg-interferon (IFN) with ribavirin but this regimen showed many serious side effects beside its non-satisfactory efficacy. In 2013, a second generation of direct acting antiviral agents (DAAs) gave a promising efficacy and safety. Although many IFN free regimens were approved, further evaluations are needed for these regimens.Aim of Study: To compare safety and efficacy of Sofosbuvir in combination with Daclatasvir (DCV) or Ledipasvir (LDV) or Simeprevir (SIM) in treatment of chronic HCV patients.Patients and Methods: This is a prospective study con-ducted on 150 patients of chronic HCV who visited Al-Ahrar Educational Hospital in Zagazig National Committee for the Control of Viral Hepatitis (NCCVH) from January to Septem-ber of 2017 and were selected according to the inclusion and exclusion criteria set by the (NCCVH).Patients were assigned into three groups: 50 patients received (SOF/DCV ± RBV), 50 patients received (SOF/LDV ± RBV) and 50 patients received (SOF/SIM ± RBV). Three regimens were given for 12 weeks.Results: In this study, the mean age of the 50 patients of each group was (52.6±9 years) in SOF/DCV group, (48.2±13.5 years) in SOF/LDV group and (50.26±10.6 years) in SOF/SIM. A total of 150 patients including 93 (62%) males & 57 (38%) females.Adverse events occurred in (18%) of SOF/DCV group, (18%) of SOF/LDV group and (40%) of SOF/SIM group. The most common adverse events occurred in three groups were; hyperbilirubinemia (20%) in SOF/SIM group, (8%) in SOF/ DCV group and (4%) in SOF/LDV group (Table 2), thus SOF/SIM group showed a higher incidence of adverse events occurrence but adverse events in three groups were mild (not sever enough to cause treatment discontinuation).Sustained Virological Response (SVR) rate was nearly similar in three groups: (100%) of SOF/LDV group, while it was achieved (98%) of SOF/SIM group and (98%) of SOF/DAC group. This results showed no statistically signif-icant difference.Conclusions: Sofosbuvir based antiviral regimen in com-bination with (DCV, LDV and SIM) were tolerable with no obvious side effect and showed high efficacy in management of chronic HCV with nearly similar SVR.

Clinical significance of detectable and quantifiable HCV RNA at the end of treatment with ledipasvir/sofosbuvir in GT1 patients.

AASLD/IDSA treatment guidelines for hepatitis C virus (HCV) infection state that testing for quantitative HCV RNA can be considered at the end of antiviral treatment (EOT) with interferon-free regimens. However, it remains unclear how to respond to a detectable or even quantifiable HCV RNA result. The aim of this study was to analyse the frequency and predictive value of detectable and quantifiable HCV RNA results at the EOT in patients with HCV genotype 1 infection treated with ledipasvir (LDV) and sofosbuvir (SOF)±ribavirin (RBV) in a large real-world cohort. A retrospective analysis of the DHC-R (Deutsches Hepatitis C-Register, German Hepatitis C-Registry) cohort was performed including all patients who were treated with LDV/SOF±RBV and in whom HCV RNA testing was done with either the Roche COBAS AmpliPrep/COBAS TaqMan (CAP/CTM) or the Abbott RealTime HCV assay (ART). The frequency of detectable HCV RNA at the EOT was 7% in this real-world study involving 471 patients. Furthermore, 3% of the patients (n=14/471) even had quantifiable viral load at the EOT. Detectable and quantifiable results were more frequent if the ART was used for testing. However, SVR was achieved by 32/33 patients (97%) with detectable and even by all 14 patients (100%) with quantifiable HCV RNA results at the EOT. Detectable and even quantifiable HCV RNA results are quite frequent if highly sensitive HCV RNA assays are used. However, treatment prolongation is not indicated, as SVR rates remain high in these patients.

Computer analysis of resistance mutations to HCV NS5A antivirals

Background: Hepatitis C virus (HCV) is characterized by a high replication rate associated with a high rate of genetic errors because viral RNA polymerase lacks correction systems. It is therefore not surprising that mutations that occur naturally in the HCV genome in some cases coincide with positions involving amino acids responsible for resistance to direct-acting antivirals (DAAs). Thus, it is important to determine whether mutations produce conformational changes that interfere with drug-target binding to prevent potential therapeutic failures. To develop computational analysis of the mutations of resistance to DAAs in the NS5A gene to identify conformational change of the protein Methods & Materials: Resistance mutations to DAAs described in the literature were represented in the three-dimensional structure of domain I of the NS5A protein. We used the program PyMol (http://pymol.sourceforge.net). We also used the structure Con1 isolate of HCV-1b extracted from the database Protein DataBank (www.ebi.ac.uk/pdbsum), with code (3FQQ). Results: Resistance mutations have been described in the N-terminal region of domain I of NS5A at amino acid positions 23, 28, 30, 31, 32, 54, 58 and 93. The mutations associated with resistance to DAAs were found where the surfaces of the two monomers forming NS5A dimer I were contacted. Conclusion: The mutations associated with the analyzed resistance were located close to the binding site of daclatasvir (DCV), and ledipasvir (LDV), which would affect the drug-target binding and would have a possible impact on the treatment with DAAs.

Pharmacokinetic evaluation of daclatasvir and ledipasvir in healthy volunteers using a validated highly sensitive spectrofluorimetric method.

A simple, highly sensitive and selective spectrofluorimetric method has been developed and fully validated for the determination of daclatasvir (DAC) and ledipasvir (LED) in tablets and human plasma. The method is based on measurement of the native fluorescence in methanol at λem 384nm after excitation at λex 318nm for DAC and in acetonitrile at λem 402nm after excitation at λex 340nm for LED. The fluorescence intensity (FI) concentration plot was rectilinear over the ranges 1.2-12, 0.1-18ngml-1 and 9-90, 1-100ngml-1 with a good correlation of r=0.9994 to r=0.9997 in standard solution and human plasma for DAC and LED, respectively. The extraction of analytes from plasma was performed using methanol and acetonitrile as a precipitating agent with lower limit of quantification (LLOQ) of 0.1 and 1.0ngml-1 for DAC and LED; respectively. The proposed method was validated according to the US Food and Drug Administration (FDA) guidelines and successfully applied for estimating the pharmacokinetic parameters of DAC and LED following oral administrations of their tablets.

ε-MnO2-modified graphite electrode as a novel electrochemical sensor for the ultrasensitive detection of the newly FDA approved Hepatitis C antiviral drug ledipasvir

A novel, simple and sensitive electrochemical method for the determination of ledipasvir (LED), the newly FDA approved Hepatitis C antiviral drug was developed and validated using ε-MnO2-modified graphite electrode. Two different MnO2 polymorphs (γ- and ε-MnO2 nanoparticles) were synthesized and characterized using X-ray powder diffraction (XRD), Fourier transform infrared (FTIR), energy dispersive X-ray (EDX) and thermogravimetric analysis (TGA). Surface area measurements show that ε-MnO2 NPs have large surface area of 345 m2/g, which is extremely high if compared to that of γ-MnO2 NPs (38 m2/g). In addition, a comprehensive study of the difference in the electrochemical behavior of LED while using pencil graphite electrode (PGE) modified with either γ- or ε-MnO2 NPs is carried out. It was found that surface area and percentage of surface hydroxyls of MnO2 NPs are the key factors governing the sensitivity of the fabricated electrode toward the oxidation of the positively charged LED. Scanning electron microscopy (SEM) was employed to investigate the morphological shape of MnO2 NPs and the surface of the bare and modified electrodes. Moreover, cyclic voltammetry and electrochemical impedance spectroscopy (EIS) were used for the surface analysis of the modified electrodes. Based on the obtained results, ε-MnO2/PGE was applied as a selective and sensitive electrode for determination of LED. Under the optimized experimental conditions, ε-MnO2/PGE provides a linear response over the concentration range of 0.025–3.60 μmol L−1 LED with a low limit of detection, which was found to be 5.10 nmol L−1 (4.50 ng mL−1) for the 1st peak and 9.20 nmol L−1 (8.10 ng mL−1) for the 2nd one. In addition, the oxidation behavior of LED is discussed with a full investigation of the oxidized product using FT-IR and LC/MS. The fabricated sensor exhibits a good precision, selectivity and stability and was applied successfully for the determination of LED in its tablets and real rat plasma samples with a good recovery using a simple extraction technique.

Earth-friendly spectrophotometric methods for simultaneous determination of ledipasvir and sofosbuvir: Application to average content and uniformity of dosage unit testing

Simple, rapid, sensitive, accurate, precise and earth-friendly spectrophotometric methods were developed for the simultaneous analysis of ledipasvir (LED) and sofosbuvir (SOF) without interference of both sunset yellow dye and copovidone excipients (the most probable interferents) in their combined dosage form.These proposed methods were based on measurement of LED in synthetic mixtures and combined dosage form by first derivative (1D) spectrophotometry at 314 nm over the concentration range of 2–50 μg mL−1 with coefficient of determination (R2) > 0.9999, mean percentage recovery of 99.98 ± 0.62.On the other hand, SOF in synthetic mixtures and combined dosage form was determined by five methods. Method I is based on the use of 1D spectrophotometry at 274.2 nm (zero crossing point of LED). Method II involves the application of conventional dual wavelength method (DW) at the absolute difference between SOF zero order amplitudes at 261 nm (λmax of SOF) and 364.7 nm. At these wavelengths, the absolute difference between LED zero order amplitudes was observed to equal zero. Method III depends on isosbestic point method (ISP) in which the total concentration of both drugs was measured at isosbestic point at 262.7 nm. Concentration of SOF could be obtained by subtraction of LED concentration. While, method IV depends on absorbance correction method (absorption factor method), which is based on determination of SOF concentration at 262.7 nm (λISP) and LED at 333 nm (λmax of LED). Finally, method V depends on absorbance ratio method (Q-analysis) in which 262.7 nm (λISP) and 261 nm (λmax of SOF) were selected to determine SOF concentration. The linearity range for all methods for SOF determination was 2–50 μg mL−1 with coefficient of determination (R2) > 0.9999.Methods I, II & III were also applied for determination of SOF concentration in single dosage form. Their mean percentage recoveries were 100.35 ± 1.85, 99.97 ± 0.54 and 100.03 ± 0.49, for the three methods respectively.The proposed methods were validated according to international conference of harmonization (ICH) requirements and statistically compared to published reference methods. The ANOVA test confirmed that there is no significant differences between the proposed methods, and can be used for routine analysis of LED and SOF in commercial tablets. These developed methods were applied to estimate the average content and uniformity of dosage unit for LED/SOF combined dosage form and SOF single dosage form according to British pharmacopeia (BP) requirements.