Metabolite GS-331007 Research Articles

Effect of carvedilol on pharmacokinetics of sofosbuvir and its metabolite GS-331007: role of P-glycoprotein.

Sofosbuvir (SOF) is a P-glycoprotein (P-gp) substrate, and carvedilol (CAR) is an inhibitor of P-gp, suggesting that it may affect the oral pharmacokinetics and safety of SOF. The current study investigated the pharmacokinetic interaction of CAR with SOF and its metabolite, GS-331007, and the possible consequent toxicities in rats. To assess the pharmacokinetics of SOF and GS-331007, rats were divided into three groups; all received a single oral dose of SOF preceded with saline (SAL), verapamil (VER) as a standard P-gp inhibitor, or CAR, respectively. The serosal, plasma, and hepatic tissue contents of SOF and GS-331007 were assessed using LC-MS/MS. Renal and hepatic toxicities were assessed using biochemical and histopathological tests. Serosal and plasma concentrations of SOF and GS-331007 were increased in the presence of CAR, suggesting a significant inhibitory effect of CAR on intestinal P-gp. Simultaneously, the pharmacokinetic profile of SOF showed a significant increase in the Cmax, AUC(0-t), AUC (0-∞), t1/2, and a reduction in its apparent oral clearance. While the pharmacokinetic profile of GS-331007 was not significantly affected. However, this notable elevation in drug oral bioavailability was corroborated by a significant alteration in renal functions. Hence, further clinical investigations are recommended to ensure the safety and dosing of CAR/SOF combination.

Sofosbuvir-velpatasvir in children 3-17 years old with hepatitis C virus infection.

The safety and efficacy of sofosbuvir-velpatasvir in children aged 3-17 years with chronic hepatitis C virus (HCV) infection of any genotype were evaluated. In this Phase 2, multicenter, open-label study, patients received once daily for 12 weeks either sofosbuvir-velpatasvir 400/100 mg tablet (12-17 years), 200/50 mg low dose tablet or oral granules (3-11 years and ≥17 kg), or 150/37.5 mg oral granules (3-5 years and <17 kg). The efficacy endpoint was sustained virologic response 12 weeks after therapy (SVR12). Dose appropriateness was confirmed by intensive pharmacokinetics in each age group. Among 216 patients treated, 76% had HCV genotype 1% and 12% had genotype 3. Rates of SVR12 were 83% (34/41) among 3-5-year-olds, 93% (68/73) among 6-11-year-olds, and 95% (97/102) among 12-17-year-olds. Only two patients experienced virologic failure. The most common adverse events were headache, fatigue, and nausea in 12-17-year-olds; vomiting, cough, and headache in 6-11-year-olds; and vomiting in 3-5-year-olds. Three patients discontinued treatment because of adverse events. Four patients had serious adverse events; all except auditory hallucination (n = 1) were considered unrelated to study drug. Exposures of sofosbuvir, its metabolite GS-331007, and velpatasvir were comparable to those in adults in prior Phase 2/3 studies. Population pharmacokinetic simulations supported weight-based dosing for children in this age range. The pangenotypic regimen of sofosbuvir-velpatasvir is highly effective and safe in treating children 3-17 years with chronic HCV infection.

Ledipasvir/Sofosbuvir in Hepatitis C Virus–Infected Children With Hematological Malignancies: A Pharmacokinetic Study

Children with hematological malignancies and chronic hepatitis C virus (HCV) infection are at a higher risk for rapid progression of liver disease and malignancy relapse due to multiple hepatitis flares and chemotherapy interruption. They are therefore potential candidates for microelimination of HCV infection. This study aimed to assess the effect of acute lymphoblastic leukemia (ALL) on the pharmacokinetic (PK) profile of direct-acting antivirals, namely ledipasvir/sofosbuvir (LDV/SOF) and the SOF major metabolite GS-331007. This was a 24-week, prospective, controlled, open-label, 2-arm PK study of patients receiving 45/200 mg once-daily LDV/SOF orally for 12 weeks. Eligible patients were HCV-RNA-positive, treatment-naive children aged 6 to <12 years and/or weighing 17 to <35 kg with genotype 4 chronic HCV infection without cirrhosis. The primary efficacy and safety end points were the achievement of sustained virologic response for all patients with absence of any adverse events leading to permanent discontinuation of the study drug. Steady-state noncompartmental analysis was performed to determine the PK parameters of SOF, GS-331007, and LDV as the primary PK outcome. Dose suitability was based on the 90% CI of exposure geometric mean ratio percentage within 50% to 200% compared with adults. Ten HCV-infected children with ALL (chemotherapy treatment group) and 12 eligible children with no malignancy (control group) were enrolled and completed the study period. All 22 patients achieved the sustained virologic response with no adverse events leading to interruption or permanent discontinuation of the study drug. Compared with the control group, the ALL group patients had similar SOF, GS-331007, and LDV exposure. Compared with adults, the AUCτ of GS-331007 was lower and the AUCτ and Cmax,ss of SOF and the Cmax,ss of LDV were modestly higher in the ALL group (acceptance limit, 50%-200%). However, the observed efficacy and favorable safety profile made these changes not clinically significant. Weight-based dosing of LDV/SOF (45/200 mg) is highly effective and safe among genotype 4 HCV-infected children weighing 17 to <35 kg and diagnosed with ALL undergoing maintenance chemotherapy. The similarity in the drug exposure, efficacy, and safety clinical end points between patients with and without hematological malignancy support their therapeutic equivalence. Further studies with a larger sample size may be required to confirm the safety of LDV/SOF in patients with ALL and to recommend appropriate dosing in children with hematological malignancies, if needed. gov identifier: NCT03903185.

Efficacy of ultra-short, response-guided sofosbuvir and daclatasvir therapy for hepatitis C in a single-arm mechanistic pilot study.

World Health Organization has called for research into predictive factors for selecting persons who could be successfully treated with shorter durations of direct-acting antiviral (DAA) therapy for hepatitis C. We evaluated early virological response as a means of shortening treatment and explored host, viral and pharmacokinetic contributors to treatment outcome. Duration of sofosbuvir and daclatasvir (SOF/DCV) was determined according to day 2 (D2) virologic response for HCV genotype (gt) 1- or 6-infected adults in Vietnam with mild liver disease. Participants received 4- or 8-week treatment according to whether D2 HCV RNA was above or below 500 IU/ml (standard duration is 12 weeks). Primary endpoint was sustained virological response (SVR12). Those failing therapy were retreated with 12 weeks SOF/DCV. Host IFNL4 genotype and viral sequencing was performed at baseline, with repeat viral sequencing if virological rebound was observed. Levels of SOF, its inactive metabolite GS-331007 and DCV were measured on days 0 and 28. Of 52 adults enrolled, 34 received 4 weeks SOF/DCV, 17 got 8 weeks and 1 withdrew. SVR12 was achieved in 21/34 (62%) treated for 4 weeks, and 17/17 (100%) treated for 8 weeks. Overall, 38/51 (75%) were cured with first-line treatment (mean duration 37 days). Despite a high prevalence of putative NS5A-inhibitor resistance-associated substitutions (RASs), all first-line treatment failures cured after retreatment (13/13). We found no evidence treatment failure was associated with host IFNL4 genotype, viral subtype, baseline RAS, SOF or DCV levels. Shortened SOF/DCV therapy, with retreatment if needed, reduces DAA use in patients with mild liver disease, while maintaining high cure rates. D2 virologic response alone does not adequately predict SVR12 with 4-week treatment. Funded by the Medical Research Council (Grant MR/P025064/1) and The Global Challenges Research 70 Fund (Wellcome Trust Grant 206/296/Z/17/Z).

Rescue use of Pegylated Interferon in Dialysis Patient who Failed to Respond Sofosbuvir.

Rescue use of Pegylated Interferon in Dialysis Patient who Failed to Respond Sofosbuvir.

Effects of aged garlic and ginkgo biloba extracts on the pharmacokinetics of sofosbuvir in rats.

Sofosbuvir is a direct acting antiviral (DAA) approved for the treatment of hepatitis C virus (HCV). Sofosbuvir is a substrate of P-glycoprotein (P-gp). For this reason, inhibitors, or inducers of intestinal P-gp may alter the plasma concentration of sofosbuvir and increase or decrease its efficacy causing a significant change in its pharmacokinetic parameters. The purpose of the study was to evaluate the pharmacokinetic interaction between either aged garlic or ginkgo biloba extracts with sofosbuvir through targeting P-gp as well as possible toxicities in rats. Rats were divided into four groups and treated for 14days with saline, verapamil (15mg/kg, PO), aged garlic extract (120mg/kg, PO), or ginkgo biloba extract (25mg/kg, PO) followed by a single oral dose of sofosbuvir (40mg/kg). Validated LC-MS/MS was used to determine sofosbuvir and its metabolite GS-331007 in rat plasma. Aged garlic extract caused a significant decrease of sofosbuvir AUC(0-t) by 36%, while ginkgo biloba extract caused a significant increase of sofosbuvir AUC(0-t) by 11%. Ginkgo biloba extract exhibited a significant increase of the sofosbuvir t1/2 by 60%, while aged garlic extract significantly increased the clearance of sofosbuvir by 63%. The pharmacokinetic parameters of GS-331007 were not affected. The inhibitory action of ginkgo biloba on P-gp and the subsequent increase in the sofosbuvir plasma concentration did not show a significant risk of renal or hepatic toxicity. Conversely, although aged garlic extracts increased intestinal P-gp expression, they did not alter the Cmax and Tmax of sofosbuvir and did not induce significant hepatic or renal toxicities.

The Metabolic Activation of Sofosbuvir Is Impaired in an Experimental Model of NAFLD.

Simple SummarySteatosis is a disease of the liver characterized by the deposition of lipids in hepatocytes. Although the effect of steatosis on the metabolism of drugs has been investigated, the findings obtained so far are still controversial. We here evaluated the pharmacokinetics of the main inactive metabolite of sofosbuvir, the first direct antiviral agent reaching the market for hepatitis C treatment, called GS-331007. We demonstrated that plasmatic levels of GS-331007 increased significantly in rats with steatosis, whereas the expression of the enzyme UMP-CMPK, responsible for the activation of sofosbuvir to its active metabolite GS-331007-TP, was significantly lower than that of healthy animals. The reduction of UMP-CMPK expression suggests an impairment of sofosbuvir activation, giving a possible explanation for the reduction of its efficacy in patients affected by genotype 3 HCV, which is often associated with liver steatosis. The effect of liver steatosis on drug metabolism has been investigated in both preclinical and clinical settings, but the findings of these studies are still controversial. We here evaluated the pharmacokinetic profile of the main sofosbuvir metabolite GS-331007 in healthy animals and rats with non-alcoholic fatty liver disease (NAFLD) after the oral administration of a single 400 mg/kg dose of sofosbuvir. The plasma concentration of GS-331007 was evaluated by HPLC-MS. The expression of the two enzymes uridine monophosphate-cytidine monophosphate kinase 1 (UMP-CMPK1), and nucleoside diphosphate kinase (ND-PK), responsible for the formation of the active metabolite GS-331007-TP, were measured by qRT-PCR and Western Blot. We demonstrated that in rats with steatosis, the area under the plasma concentration-vs-time curve (AUC) and the peak plasma concentration (Cmax) of GS-331007 increased significantly whereas the expression of UMP-CMPK was significantly lower than that of healthy animals. The reduction of UMP-CMPK expression suggests an impairment of sofosbuvir activation to GS-331007-TP, giving a possible explanation for the reduction of sofosbuvir efficacy in patients affected by genotype 3 Hepatitis C virus (HCV), which is often associated with liver steatosis. Furthermore, since GS-331007 plasma concentration is altered by steatosis, it can be suggested that the plasma concentration of this metabolite may not be a reliable indicator for exposure-response analysis in patients with NAFLD.

Factors Influencing the Intracellular Concentrations of the Sofosbuvir Metabolite GS-331007 (in PBMCs) at 30 Days of Therapy.

Sofosbuvir (SOF) is an HCV NS5B polymerase inhibitor, and GS-331007 is its major metabolite. The aim of this study was to investigate whether clinical and pharmacological factors could influence GS-331007 intracellular (IC) concentrations in peripheral blood mononuclear cells (PBMCs) associated with a sustained virological response in patients treated with SOF and ribavirin (RBV). Drug levels were analyzed using liquid chromatography at different days of therapy, whereas variants in genes encoding transporters and nuclear factors were investigated using real-time PCR. This study enrolled 245 patients treated with SOF; 245 samples were analyzed for pharmacogenetics and 50 were analyzed for IC pharmacokinetics. The GS-331007 IC concentration at 30 days was associated with its plasma concentration determinate at 30, 60 and 90 days of SOF-therapy and with daclatasvir concentrations at 7 days of therapy. No genetic polymorphism affected IC exposure. In linear multivariate analysis, ledipasvir treatment, baseline albumin and estimated glomerular filtration rate were significant predictors of IC exposure. This study presents data on an IC evaluation in a cohort of patients treated with SOF, also considering pharmacogenetics. These results could be useful for regions where SOF–RBV treatment is considered the standard of care; moreover, they could further deepen the knowledge of IC exposure for similar drugs in the future.

Acute interstitial nephritis following treatment with direct-acting antiviral agents in hepatitis C virus-infected patients: A case series.

Hepatitis C virus (HCV) infection has been associated with several extrahepatic adverse clinical outcomes, including an accelerated rate of loss of kidney function in patients with chronic kidney disease (CKD) and an increased mortality in patients with CKD and kidney failure on hemodialysis. Clinical trials using direct-acting antiviral (DAA) agents have uniformly achieved sustained viral response at 12weeks (SVR12) rates of >90% in the general population as well as in patients with CKD/kidney failure on hemodialysis. Sofosbuvir is a DAA prodrug that is phosphorylated into the active metabolite GS-461203, with subsequent dephosphorylation into the inactive metabolite GS-331007. The kidneys clear both sofosbuvir and GS-331007, and its use has been associated with worsening kidney function in some studies. In the HCV-TARGET study, patients with an estimated glomerular filtration rate (eGFR) < 30 mL/min at the initiation of sofosbuvir-based therapy had higher rates of deterioration of kidney function compared to patients with higher eGFR [1]. However, based on recent data demonstrating safety in patients with advanced CKD, the US Food and Drug Administration (FDA) approved the use of sofosbuvir-containing regimens in patients with CKD 4/5 (eGFR of less than 30 mL/min/1.73m2) and end-stage renal disease (ESRD) in late 2019. The current report describes 8 HCV-infected patients who developed acute kidney injury (AKI) with biopsy-proven acute interstitial nephritis (AIN) temporally associated with the use of DAAs. The mean age of the group was 61.3 (±6 years). The most common HCV genotype (GT) was 1a (n=7). The DAA formulations were sofosbuvir/ledipasvir (n=5), elbasvir/grazoprevir (n=2), and sofosbuvir/simeprevir (n=1). All patients achieved an SVR12. The mean serum creatinine at the initiation of DAA treatment was 1.5 mg/dL (± 0.6) and increased to 2.03 mg/dL (± 0.7) by the last day of DAA administration. The kidney biopsies were performed at a mean of 320 days (± 247) after achieving an SVR12, at which point the mean creatinine had increased to 2.3 mg/dL (± 1.4). All patients received a course of high-dose corticosteroids after the diagnosis of AIN was confirmed by biopsy. Serum creatinine levels at 3 and 6months following the completion of steroid therapy were 2.8 (±1.2) and 3.1mg/dL (±1.5), respectively. Three patients had worsening CKD and progressed to kidney failure requiring hemodialysis. These results are consistent with earlier studies demonstrating the efficacy of the DAAs in HCV-infected CKD patients. Of note, the demonstration of AIN in these patients may explain some of the AKI events reported with the use of DAAs in patients with CKD.

Evaluation of the pharmacokinetics and food intake effect of generic sofosbuvir in healthy Chinese subjects .

Sofosbuvir is an NS5B nucleotide inhibitor that was approved for hepatitis C treatment. Generic sofosbuvir has been produced to improve the affordability. The present study investigated the pharmacokinetics (PK) and safety of generic sofosbuvir as well as the effect of food intake on its PK parameters in healthy Chinese subjects. This open-label, randomized, multiple-dose, dose-escalating, and food effect trial enrolled 12 healthy Chinese subjects. The subjects received a single oral dose of 400mg of generic sofosbuvir in fasted state or after a high-fat meal, or 800mg in fasted state, in a three-way crossover design, and then all subjects were administered with 400mg daily for 8days. The PK parameters for sofosbuvir and its metabolites were determined, and the safety was monitored. Sofosbuvir was absorbed rapidly into plasma, with a half-life of 0.46-0.48 hours. Plasma exposure to sofosbuvir and its metabolite GS-566500 was increased in an approximately proportional manner to the increased dose. Repeated dosing did not result in drug accumulation in the blood. Sofosbuvir was mainly excreted as the metabolite GS-331007 in the urine. Drug administration after a high-fat meal increased the plasma sofosbuvir exposure by 1.29-fold, without substantially altering the absorption rate. No serious adverse events were observed, and all subjects tolerated the doses well. This generic sofosbuvir was well absorbed, the plasma concentration was increased with an increased dose, and it was safe in healthy subjects. A high-fat meal appeared to promote the bioavailability of sofosbuvir and the metabolite GS-566500.

Quantification of plasma levels of antiviral drug sofosbuvir and its metabolite GS331007 in patients of chronic hepatitis C with chronic kidney disease using UPLC-MS/MS method

Sofosbuvir based regimens are the only treatment available in India and some other Asian countries for curing hepatitis C viral (HCV) infection. The main excretion route of sofosbuvir is renal so treatment of HCV infection is challenging in patients on hemodialysis. A simple and sensitive UPLC-MS/MS method was developed and validated according to USFDA guidelines on Linear Ion trap Quadrupole tandem mass spectrometer (QTRAP4500) which was applied to estimate the drug concentration of sofosbuvir and its metabolite GS331007 in patients of chronic kidney disease with HCV infection. Clopidogrel was used as internal standard (IS) for this study. All analytes and IS were separated on UPLC C18-HSS column (2.1mmX50mm, 1.7μm) with retention time of 2.07, 0.29 and 1.58 min, respectively, by using mobile phases of 5mM ammonium acetate in 0.2% formic acid in water (A) and 5mM ammonium acetate in 0.2% formic acid in methanol (B) on gradient elution mode at a flow rate of 0.6 mL/min. Calibration curve was plotted over the range of 10-200 μg/mL for both of the analytes and equation was calculated by applying linear regression method. Detections of daughter ions (sofosbuvir-530 to 243m/z, GS331007-261 to 112.1m/z and clopidogrel- 322 to 154.9m/z) were done in multiple reactions monitoring (MRM) mode and weights were analyzed by using Linear ion trap quadrupole mass spectrometer with turbo spray ion source. The developed method has been successfully used for quantification of drug concentration of sofosbuvir and GS331007 to see the safety of sofosbuvir in patients of HCV infection with renal failures.

A liquid chromatography-tandem mass spectrometry method for simultaneous determination of simeprevir, daclatasvir, sofosbuvir, and GS-331007 applied to a retrospective clinical pharmacological study

A highly sensitive liquid chromatography-tandem mass spectrometry (LC–MS/MS) method was developed and validated for simultaneous determination of daclatasvir (DCV), simeprevir (SMV), sofosbuvir (SOF), and its major metabolite GS-331007 in human plasma using stable-isotope-labeled (SIL) analogs as internal standards (IS) to minimize a possible matrix effect. Liquid-liquid extraction (LLE) of the analytes and IS from human plasma was performed using a commercial extraction kit requiring low sample volume (50 μL). The analytes were eluted under a gradient program with mobile phase A (water + 0.1% formic acid) and mobile phase B (methanol + 0.1% formic acid) at a flow-rate of 0.6 mL/min for 10 min. The detection was performed on a Qtrap 5500 triple quadrupole tandem-mass spectrometer using multiple reaction monitoring (MRM) mode via the positive electrospray ionization interface. The method was validated according to the European Medicine Agency (EMA) guidelines over the clinically relevant concentration range of 15.6–2000 ng/mL. The high reproducibility, the low matrix effect associated with the use of SIL-IS, and the need of small sample amounts make this method particularly suited for high-throughput routine analysis. The proposed method was successfully applied to a retrospective clinical pharmacology study involving 67 HIV/HCV co-infected patients treated with a SOF-based therapy. DCV, SMV, SOF, and GS-331007 plasma levels were measured at week 4 of treatment and compared with the patients' clinical and laboratory characteristics. Higher GS-331007 plasma concentrations were observed in female patients compared to males, which can be explained by different anthropometric characteristics between genders. Importantly, patients with high plasma levels of GS-331007 also showed enhanced concentration of DCV and SMV probably due to a specific metabolic/pathological condition. Altogether, our findings indicate that the proposed method is a reliable and accurate new tool for high-throughput screening of large patient cohorts that could be readily used to optimize treatment modalities and reduce drug-related toxicities.

Investigation of Pharmacokinetic Interactions between Doravirine and Elbasvir-Grazoprevir and Ledipasvir-Sofosbuvir.

Doravirine is a non-nucleoside reverse transcriptase inhibitor for the treatment of human immunodeficiency virus type 1 (HIV-1) infection. Due to the high prevalence of HIV-1 and hepatitis C virus (HCV) coinfection and coadministration of HIV-1 and HCV treatment, potential drug-drug interactions (DDIs) between doravirine and two HCV treatments were investigated in two phase 1 drug interaction trials in healthy participants. Trial 1 investigated the effect of multiple-dose doravirine and elbasvir + grazoprevir coadministration (N = 12), and trial 2 investigated the effect of single-dose doravirine and ledipasvir-sofosbuvir coadministration (N = 14). Doravirine had no clinically relevant effect on the pharmacokinetics of elbasvir, grazoprevir, ledipasvir, sofosbuvir, or the sofosbuvir metabolite GS-331007. Coadministration of elbasvir + grazoprevir with doravirine moderately increased doravirine area under the concentration-time curve from 0 to 24 h (AUC0-24), maximal concentration (Cmax), and concentration 24 h postdose (C24), with geometric least-squares mean ratio (GMR) with 90% confidence intervals (CI) of 1.56 (1.45, 1.68), 1.41 (1.25, 1.58), and 1.61 (1.45, 1.79), respectively. Doravirine AUC0-∞, Cmax, and C24 values increased slightly following coadministration with ledipasvir-sofosbuvir (GMR [90% CI] of 1.15 [1.07, 1.24], 1.11 [0.97, 1.27], and 1.24 [1.13, 1.36], respectively). The modest increases in doravirine exposure are not clinically meaningful based on the therapeutic profile of doravirine. Effects are likely secondary to cytochrome P450 3A and P-glycoprotein inhibition by grazoprevir and ledipasvir, respectively. Coadministration of doravirine with elbasvir + grazoprevir or ledipasvir-sofosbuvir was generally well tolerated. Clinically relevant DDIs are not expected to occur between doravirine and elbasvir-grazoprevir or ledipasvir-sofosbuvir at the therapeutic doses.

Quick and Sensitive UPLC-ESI-MS/MS Method for Simultaneous Estimation of Sofosbuvir and Its Metabolite in Human Plasma.

A simple, fast and highly sensitive RP-UPLC-MS/MS method was developed and validated for the simultaneous determination of sofosbuvir (SR) and its metabolite GS331007 in human plasma using ketotifen as an internal standard (IS). The separation was achieved on Acquity UPLC BEH C18 (50 × 2.1 mm, i.d. 1.7 µm, Waters, USA) column using acetonitrile:5 mM ammonium formate:0.1% formic acid (85:15:0.1% v/v/v) as a mobile phase at a flow rate of 0.35 mL/min in an isocratic elution. The Xevo TQD UPLC-MS/MS was operated under the multiple-reaction monitoring mode using positive electrospray ionization. Extraction with dichloromethane was used in the sample preparation. Method validation was performed as per the Food and Drug Administration (FDA) guidelines and the calibration curves of the proposed method were found to be linear in the range of 1–1000 ng/mL for SR and in the range of 10–1500 ng/mL for its metabolite (GS331007) with an elution time of 1.83 min. All validation parameters were within the acceptable range according to the bioanalytical methods validation guidelines. Furthermore, the obtained results of matrix effects indicate that ion suppression or enhancement from human plasma components was negligible under the optimized conditions. The proposed method can be applied in high-throughput analysis required for pharmacokinetic and bioequivalence studies in human samples.

Pharmacokinetics, Safety, and Tolerability of the Direct-acting Hepatitis C Antiviral Sofosbuvir in Healthy Chinese Subjects

PurposeThe purpose of this study was to investigate the safety, tolerability, and pharmacokinetic profile of sofosbuvir and its metabolites after a single dose of sofosbuvir 400 mg and once daily dosing of sofosbuvir 400 mg for 7 days in healthy Chinese subjects. MethodsThis Phase I, open-label, single- and multiple-dose study enrolled 14 Chinese subjects aged 18 to 45 years with an approximately even distribution of healthy male (n = 9) and nonpregnant, nonlactating female subjects (n = 5). Subjects received a single oral dose of sofosbuvir 400 mg (one tablet) (morning, fasted conditions; single-dose treatment). After a 3-day washout, subjects received oral sofosbuvir 400 mg (one tablet) (morning, fasted) for 7 days (multiple dose treatment). FindingsNo significant accumulation of sofosbuvir, GS-566500, or GS-331007 was observed. Steady state of the major metabolite GS-331007 was achieved after 4 days of consecutive dosing with sofosbuvir 400 mg once daily. Sofosbuvir was generally well tolerated. ImplicationsOverall, this study supports the further evaluation of sofosbuvir 400 mg in the Chinese population. The pharmacokinetic properties of sofosbuvir, GS-556500, and GS-311007 were found to be broadly similar in healthy Chinese subjects compared with non-Chinese subjects in previous sofosbuvir studies. ChinaDrugTrials.org.cn identifier: CTR20150249.

Development a validated highly sensitive LC–MS/MS method for simultaneous quantification of Ledipasvir, sofosbuvir and its major metabolite GS-331007 in human plasma: Application to a human pharmacokinetic study

A highly sensitive and rapid LC–MS/MS method was developed, fully optimized and validated for the simultaneous determination of Ledipasvir (LED) and Sofosbuvir (SOF) in the presence of its major metabolite GS-331007 in human plasma using Daclatasvir as internal standard (IS). The extraction of analytes and IS from plasma was performed using liquid-liquid extraction with ethyl acetate. The chromatographic separation of these prepared samples was achieved on Xterra MS C8 column (4.6×50mm,5μm) using gradient elution with a mobile phase of ammonium formate buffer (pH 3.5; 10mM), acetonitrile and methanol pumped at a flow rate 0.7mLmin−1.The detection was performed on API4000 triple quadrupole tandem mass spectrometer using multiple reaction monitoring (MRM) positive electrospray ionization interface. The method was validated according to FDA guidelines for bio-analytical methods with respect to linearity, accuracy, precision, selectivity, carry-over, stability and dilution integrity. Linearity was obtained over a concentration range of 0.1–1000, 0.3–3000 and 3.0–3000ngmL−1 for LED, SOF and GS-331007; respectively by applying weighted least-squares linear regression method (1/x2). The wider range of quantification in a shorter period of separation time less than 5.0min allowed monitoring the serum concentration of analytes up to 144h. The proposed method can be successfully applied for pharmacokinetic and bioequivalence studies in healthy human volunteers.

Simultaneous determination of ledipasvir, sofosbuvir and its metabolite in rat plasma by UPLC-MS/MS and its application to a pharmacokinetic study.

In this work, a rapid and sensitive ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method for the determination of ledipasvir, sofosbuvir and its metabolite GS-331007 in rat plasma was developed. The analytes and the internal standard (diazepam) 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 889.8→130.1 for ledipasvir, m/z 530.3→243.1 for sofosbuvir, m/z 261.5→113.1 for GS-331007 and m/z 285.2→193.1 for diazepam (IS) using a positive electrospray ionization interface. The method was validated over a concentration range of 2-500ng/mL for ledipasvir, 10-2000ng/mL for sofosbuvir and 10-2000ng/mL for GS-331007. Total time for each chromatography 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)<10.2% and the accuracy values ranged from -9.8% to 11.2%. The method was successfully applied to a pharmacokinetic study of ledipasvir, sofosbuvir and GS-331007 in rats.

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.

Development of a sensitive UPLC-ESI-MS/MS method for quantification of sofosbuvir and its metabolite, GS-331007, in human plasma: Application to a bioequivalence study

A rapid and simple LC-MS/MS method was developed and validated for the simultaneous estimation of sofosbuvir (SF) and its metabolite GS-331007 (GS) using famotidine as an internal standard (IS). The Xevo TQD LC-MS/MS was operated under the multiple-reaction monitoring mode using electrospray ionization. Extraction with ethyl acetate was used in sample preparation. The prepared samples were chromatographed on Acquity UPLC HSS C18 (50mm×2.1mm, 1.8μm) column by pumping 0.1% formic acid and acetonitrile (50:50, v/v) in an isocratic mode at a flow rate of 0.3ml/min. Method validation was performed as per the FDA guidelines and the standard curves were found to be linear in the range of 10–2500ng/ml for both SF and its metabolite. The intra-day and inter-day precision and accuracy results were within the acceptable limits. A very short run time of 1.2min made it possible to analyze more than 300 human plasma samples per day. The developed assay method was successfully applied to a bioequivalence study in human volunteers.

Pharmacokinetic, Pharmacodynamic, and Drug-Interaction Profile of the Hepatitis C Virus NS5B Polymerase Inhibitor Sofosbuvir.

Sofosbuvir (SOVALDI(®)), a potent, once-daily, orally administered nucleotide analog prodrug inhibitor of the hepatitis C virus (HCV) NS5B polymerase is approved in the USA, EU, Canada, and other regions for the treatment of HCV infection as a component of an antiviral treatment regimen. Sofosbuvir undergoes intracellular activation to form GS-461203 (active triphosphate, not detected in plasma), and ultimately the inactive, renally eliminated metabolite GS-331007. GS-331007 was identified as the primary analyte of interest for clinical pharmacology studies as it accounted for >90 % of systemic drug-related material exposure, and provided comparable exposure-response relationships for viral kinetics as observed for sofosbuvir. GS-331007 and sofosbuvir exhibit linear pharmacokinetics with minimal accumulation upon multiple dosing. Compared to healthy subjects, HCV-infected patients had modestly lower (39 %) GS-331007 area under the plasma concentration-time curve (AUC) and higher sofosbuvir AUC (60 %). Sofosbuvir can be administered without dose modification in HCV-infected patients with any degree of hepatic impairment or mild to moderate renal impairment. Sofosbuvir has a low propensity for clinically significant drug interactions with common concomitant medications used by HCV-infected patients. Clinically significant alterations in GS-331007 or sofosbuvir exposures are limited to potent inducers of intestinal P-glycoprotein that may lower exposure. In HCV-infected patients, demographic variables do not significantly influence GS-331007 and sofosbuvir exposures and no consistent exposure-response relationships were observed for efficacy or safety. This review focuses on the clinical pharmacokinetics, pharmacodynamics, and pharmacokinetic-pharmacodynamic relationships of sofosbuvir, and summarizes a number of drug interaction studies with important concomitant medications commonly used by HCV-infected patients.