HCV/hepatitis C virus nonstructural protein 5A (NS5A)

Velpatasvir (also known as GS-5816) is a novel pan-genotypic inhibitor of hepatitis C virus (HCV) nonstructural protein 5A (NS5A) with activity against genotype 1 (GT1) to GT6 HCV replicons. It is a selective inhibitor of HCV RNA replication with mean 50% effective concentrations (EC50s) against GT1 to GT6 of 6 to 130 pM.

Pan-genotypic [1]
In the first of several ASTRAL studies, Feld et al conducted a phase 3, double-blind, placebo-controlled 12-week study involving 624 chronic HCV patients from across the globe representing genotypes 1, 2, 4, 5, and 6. Patients were randomly assigned to the treatment group (a once-daily combination tablet of 400 mg sofosbuvir and 100 mg Velpatasvir) or a placebo. At 12 weeks posttherapy, HCV RNA levels were measured to assess treatment efficacy as defined by an SVR-12 rate above 85%. Profiles of patients assigned to the treatment group included compensated cirrhosis (19%), prior treatment experience (32%), which primarily included a Peg-IFN therapy (89%), and a mean age of 54 years. Feld et al reported significantly high SVR rates across all patient populations, whether treatment-naïve or experienced. While the patients assigned to the placebo group elicited no SVR, those assigned to the joint sofosbuvir/velpatasvir therapy had a remarkable 99% SVR. The drug combination was effective in all the HCV genotypes, with SVR rates of 100% among genotypes 2, 4, and 6, 99% among genotype 1b, 98% among genotype 1a, and 97% among genotype 5. Moreover, SVR rates remained high irrespective of whether the patients had cirrhosis and whether they had received previous therapy. Though the primary aim was to assess efficacy, treatment safety was documented through rate of adverse effects and patient-reported outcomes. Only 2 patients receiving treatment, both of whom were infected with genotype 1, had a virologic failure, while roughly 2% of all patients had serious adverse side effects. Furthermore, sofosbuvir/velpatasvir treatment did not increase susceptibility to adverse health events. Difficult-to-cure populations [1]
In order to address potentially difficult-to-cure populations, Foster et al, along with ASTRAL-2 and ASTRAL-3 investigators, conducted two randomized, phase 3, open-label studies. They enrolled genotype 2 or 3 patients with and without treatment experience and with and without cirrhosis. Patients were separated based on genotype and randomly assigned to two treatment groups: a once-daily combination tablet of 400 mg sofosbuvir and 100 mg velpatasvir for 12 weeks or 400 mg of sofosbuvir plus weight-based ribavirin for either 12 or 24 weeks. Patients with genotype 2 in the sofosbuvir/Velpatasvir combination tablet treatment arm achieved a 99% SVR. Sofosbuvir plus weight-based ribavirin combination for 12 weeks is an alternative regimen for genotype 2 HCV patients without cirrhosis with an achievement of SVR in 94% of cases. This same regimen can be applied to genotype 3 HCV patients with 80% SVR achievement and similar tolerability despite extended treatment duration of 24 weeks in this group of patients. In the ASTRAL-3 trial, genotype 3 treatment-naive and experienced patients were separated into two treatment arms, with one group receiving 12 weeks of daily sofosbuvir/velpatasvir and the other group receiving 24 weeks of sofosbuvir plus weight-based ribavirin. In the sofosbuvir/velpatasvir arm, 95% of genotype 3 patients achieved SVR (95% CI, 92%–98%) compared with 80% of SVR achievement (CI, 75%–85%) in the sofosbuvir and weight-based ribavirin group.

Efficacy in cirrhotic patients was also evaluated with cirrhotics in the sofosbuvir/Velpatasvir group, and they achieved a 91% SVR compared with a 66% SVR in the sofosbuvir/ribavirin group. Adverse health events (Table 1) were more likely to occur in the sofosbuvir/ribavirin group than in the sofosbuvir/velpatasvir group (71.3% vs 52.3%). At present, there is no dosage adjustment required for sofosbuvir in patients with chronic kidney disease or creatinine clearance less than 30 mL/min. As was the case with the ASTRAL-1 study, Younossi et al documented patient-reported outcomes during and after therapy. Four weeks into the treatment, statistically and clinically significant improvements were observed in many of the prodomains among sofosbuvir/velpatasvir treated patients, including physical and emotional well-being, bodily pain, and general health. Sofosbuvir/ribavirin therapy yielded varying results, with patients reporting increased body aches, poorer emotional health, and well-being. Patients also reported impaired social functioning and physical well-being, decreased below the baseline level. The immediate benefits provided by sofosbuvir/velpatasvir to whole-body health, especially difficult-to-cure genotype 3 patients, was superior to the sofosbuvir plus weight-based ribavirin therapy.

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Antiviral response to Velpatasvir/VEL in GT1 to GT4 HCV-infected patients. [2]
Three-day monotherapy with VEL produced rapid declines in HCV RNA levels. Among the cohorts dosed with 150 mg of VEL, the median reductions in HCV RNA levels through day 17 were 4.19, 4.29, 4.39, 3.13, and 3.17 log10 HCV RNA IU/ml in the GT1a, GT1b, GT2, GT3, and GT4 groups, respectively (Table 1). GT1a patients dosed with 5, 25, 50, or 100 mg had a median HCV RNA reduction of >3.67 log10, and GT3 patients dosed with 25 or 50 mg had a median reduction of >3.12 log10.

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Effect of NS5A RASs present pretreatment. [2]
For all 70 patients who received Velpatasvir/VEL and 8 placebo-treated patients, sequences were analyzed for the presence of polymorphisms that are known NS5A inhibitor RASs at amino acids 28, 30, 31, 32, 58, 92, and 93. Pretreatment, NS5A RASs (detected at >1%) were present in 24 patients, i.e., 2/8 placebo, 10/35 GT1a, 1/8 GT1b, 4/8 GT2, 5/17 GT3, and 2/2 GT4 patients (Table 1), with some patients having >1 RAS. Seven patients who received VEL had pretreatment RASs at amino acid residue 93 (Y93C/F/H/N) (Table 2). For GT1a, NS5A RASs were observed at positions M28T, Q30H/K/R, L31M/V, H58D, and Y93C/H/F/N. The mean viral load decrease in GT1a patients who were dosed with 150 mg of VEL and had pretreatment NS5A RASs was 2.9 log10 (Table 1), compared with a 4.38-log10 reduction in patients without pretreatment NS5A RASs. The GT1b patient with an NS5A RAS pretreatment (Y93H) had a 4.47-log10 HCV RNA reduction, compared with a mean 4.39-log10 reduction in patients without NS5A RASs (Table 1). In the four GT2 patients with L31M pretreatment, the mean log10 reduction was 4.08 log10, compared with 4.62 log10 in patients without NS5A RASs (Table 1). Of the five GT3 patients with pretreatment NS5A RASs, two were treated with 25 mg of VEL, one was treated with 50 mg of VEL, and two were treated with 150 mg of VEL. The GT3 patients with pretreatment RASs treated with 25 or 50 mg of VEL had a <1-log10 mean HCV RNA reduction, while all of the GT3 patients in these dose groups without pretreatment RASs had >3-log10 reductions. The two patients with RASs treated with 150 mg of VEL had mean HCV RNA reductions of 2.9 log10 and 2.7 log10, compared to 3.54 log10 in patients without NS5A RASs (Table 1; Fig. 1). Both GT4 patients who had variants at positions 30 (one with L30H [45.8%] and L30R [53.7%] and one with L30S [2.4%] and L30H [97.1%]) had a mean HCV RNA reduction of 3.47 log10.

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Substitutions selected in HCV of patients following Velpatasvir/VEL treatment through day 17. [2]
To identify the HCV NS5A variants that are potentially associated with virologic resistance to VEL, the full-length NS5A coding region was analyzed during treatment and posttreatment by deep sequencing with a 1% cutoff. Samples were obtained during treatment (days 2 to 3) or posttreatment (days 4 to 10) and on day 17, respectively, and were analyzed when the viral load was ≥1,000 IU/ml. Of the 46/70 VEL-treated patients without pretreatment NS5A RASs, sequences were available from 40 and 46 on days 2 to 10 and day 17, respectively. All patients (40/40; 100%) with available sequences on days 2 to 10 had emergent NS5A RASs, and 80.4% (37/46) still had RASs on day 17 (Table 3). Emergent NS5A RASs were not detected during the posttreatment period in the two placebo-treated patients whose samples were sequenced. NS5A RASs emerged on treatment at more positions in patients with GT1a than in patients with other GTs and included substitutions at positions M28, Q30, L31, P32, H58, E92, and Y93. RASs at positions Y93, M28, and L31 were the most prevalent in GT1a patients (Table 4). RASs were observed at two NS5A positions in GT1b and GT2 patients (Y93, L31) and three positions in GT3 patients (Y93, L31, E92). In two GT4 patients, NS5A RASs emerged at positions L28, M31, P32L, and Y93 (Table 5). L31M/V and Y93H were the most commonly observed RASs emerging on treatment in GT1b and GT2 patients, and E92K and Y93H/N were the most prevalent RASs emerging in GT3 patients (Table 4).

Viral sequencing.[2]
For every Velpatasvir/VEL-treated patient and 8 of 17 who received a placebo, samples with HCV RNA levels of >1,000 IU/ml at the pretreatment visit and day 4 and/or day 2, 4, 5, 7, 10, or 17 and in follow-up weeks 12, 24, and 48 were used to amplify the gene for HCV NS5A, which was deep sequenced with a 1% assay sensitivity cutoff with the Illumina MiSeq platform (Illumina, San Diego, CA), except for 1 patient with population sequencing at the pretreatment visit. Population sequencing of the full-length HCV NS5A coding region was performed by Janssen Diagnostics (Beerse, Belgium) by reverse transcription-PCR and standard Sanger sequencing of the bulk PCR product. The sensitivity of detection of resistant variants is approximately 10 to 20%. Variants are reported as differences from a genotype-specific reference strain, i.e., GT1b Con1 (AJ238799), GT1a H77 (GenBank accession number NC_004102), GT2 JFH-1 (AB047639), GT3 S52 (GU814263), or GT4 ED43 (GU814265). Deep-sequencing reads were aligned and processed with internally developed software via a multistep method to identify the substitutions present at levels of >1%. Sequencing analysis included NS5A class RASs that were summarized by the HCV Drug Resistance Advisory Group and/or recently observed in clinical trials with LDV, VEL, DCV, ABT-267, ABT-530, and MK-8742 and including positions 24, 28, 30, 31, 32, 38, 58, 92, and 93.

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Transient transfection of replicon RNA into Huh7 cells and EC50 determination.[2]
Resistance mutations were introduced into the GT1a, GT1b, GT2a, GT3a, and GT4a replicons (backbone GT1a H77, GT1b Con1, GT2a JFH-1, GT3a S52, and GT4a ED43, respectively) by site-directed mutagenesis and tested in transient transfections as previously described. Briefly, NS5A mutations were introduced into a plasmid encoding the PI-hRluc replicon with a QuikChange II XL mutagenesis kit in accordance with the manufacturer's instructions. Mutations were confirmed by DNA sequencing. Replicon RNAs were transcribed in vitro from replicon-encoding plasmids with a MEGAscript kit. RNA was transfected into Huh-lunet cells by the method of Lohmann et al. Briefly, cells were trypsinized and washed twice with phosphate-buffered saline (PBS). A suspension of 4 × 106 cells in 400 μl of PBS was mixed with 5 μg of RNA and subjected to electroporation at settings of 960 μF and 270 V. Cells were transferred into 40 ml of prewarmed culture medium and then seeded into 96-well plates (100 μl/well). Compounds were 3-fold serially diluted in 100% dimethyl sulfoxide (DMSO) and added to cells at a 1:200 dilution, achieving a final DMSO concentration of 0.5% in a total volume of 200 μl/well. Cells were treated for 3 days, after which culture media were removed, cells were lysed, and Renilla luciferase activity was quantified with a commercially available assay and a Top Count instrument. The EC50 was calculated as the compound concentration at which a 50% reduction in the level of Renilla reporter activity was observed compared with control samples with DMSO. Dose-response curves and EC50s were generated with the GraphPad Prism software package by nonlinear regression analysis. The replication level of either reference strains (1b-Con1 and 1a-H77) or chimeric replicons derived transiently from clinical isolates was determined as the ratio of the Renilla luciferase signal at day 4 to that at 4 h postelectroporation to normalize for transfection efficiency. The replication capacity of each replicon was expressed as their normalized replication efficiency compared with that of the reference strain (1b-Con1 or 1a-H77) within the same experiment.

Velpatasvir (VEL, GS-5816) is a novel pan-genotypic hepatitis C virus (HCV) nonstructural protein 5A (NS5A) inhibitor with activity against genotype 1 (GT1) to GT6 HCV replicons. In a phase 1b 3-day monotherapy study, patients treated with a 150-mg dose of GS-5816 had a mean maximal HCV RNA decline of ≥3.3 log10 IU/ml in GT1a, -1b, -2, -3, and -4. This report characterizes virologic resistance to VEL in these patients. NS5A resistance-associated substitutions (RASs) were detected by deep sequencing (1% cutoff) pretreatment in 22/70 patients, i.e., 10/35 (29%) patients with GT1a, 1/8 (13%) with GT1b, 4/8 (50.0%) with GT2, 5/17 (29.4%) with GT3, and 2/2 (100.0%) with GT4. In GT1a and GT3 patients, pretreatment RASs were associated with a slightly reduced HCV RNA response compared to that of patients without pretreatment RASs; among patients with GT1b, GT2, and GT4, no significant difference in response was observed in those with or without pretreatment RASs. Following treatment, the pattern of emergent RASs was more complex for GT1a than for the other genotypes. In GT1a, substitutions emerged at positions M28, Q30, L31, P32, H58, E92, and Y93, with the most prevalent substitutions at positions Y93, M28, and L31. RASs were observed at two positions in GT1b and GT2 (Y93 and L31), three positions in GT3 (Y93, L31, and E92), and four positions in GT4 (L28, M31, P32L, and Y93). RASs that were present pretreatment persisted through the 48-week follow-up period; however, RASs emerging during treatment were more likely to decline both in prevalence and in frequency within the viral population during follow-up. (This study has been registered at ClinicalTrials.gov under registration no.

Clinical trial population and study design.[2]
This was a phase 1b double-blind, randomized, placebo-controlled, multicenter study of Velpatasvir/VEL in HCV-infected patients in the United States (ClinicalTrials.gov identifier NCT01740791). Clinical data of this trial have been described previously. A total of 87 patients were enrolled and received treatment in 1 of 11 cohorts, each randomized 4:1 to treatment with VEL or a matching placebo for 3 days (except for the GT4 patients, who were not randomized and received VEL). The actual treatments administered are presented in Table 1. One patient discontinued study treatment because of an adverse event, and two patients discontinued the study (one was lost to follow-up, and one withdrew consent) prior to day 17 assessments (these patients were included in the sequencing analyses). VEL was administered once daily as follows: 5, 25, 50, 100, and 150 mg to GT1a patients; 150 mg to GT1b, GT2, and GT4 patients; and 25, 50, and 150 mg to GT3 patients. Eligible patients had plasma HCV RNA levels of >5 log10 IU/ml pretreatment and were treatment naive. Of the 87 patients in this study, 45 had HCV GT1a, 10 had GT1b, 10 had GT2b, 1 had GT3, 19 had GT3a, 1 had GT4, and 1 had GT4a, GT4b, and GT4c. The study was conducted in compliance with the Declaration of Helsinki. The study protocol and informed consent documents were reviewed and approved by the institutional review board of the participating institution, and informed consent was obtained from all patients before any study-specified procedures.

Absorption, Distribution and Excretion
Oral bioavailability is 25-30%. 94% is excreted in feces, of which 77% is the unchanged drug. 0.4% is excreted in urine. 1.4-1.6 L/kg. Estimated absorption rate is 0.12 L/h/kg [A19175]. Metabolism/Metabolites Partially metabolized by CYP2B6, CYP2C8, and CYP3A4. Biological Half-Life 15 hours.

Effects During Pregnancy and Lactation
◉ Summary of Lactation Use
No studies have been conducted on velpatasvir in breastfeeding women undergoing treatment for hepatitis C. Because it binds to maternal plasma proteins at a rate exceeding 99%, its concentration in breast milk is likely to be very low. Some sources suggest that breastfeeding should be avoided when velpatasvir is used in combination with ribavirin.
Hepatitis C is not transmitted through breast milk, and breast milk has been shown to inactivate the hepatitis C virus (HCV). However, the U.S. Centers for Disease Control and Prevention (CDC) recommends that breastfeeding should be considered if the mother has cracked or bleeding nipples. It is unclear whether this warning applies to mothers undergoing treatment for hepatitis C.
Infants born to HCV-infected mothers should be tested for HCV infection; nucleic acid testing is recommended because maternal antibodies are present in the infant for the first 18 months after birth and before the infant develops an immune response.
◉ Impact on Breastfed Infants
No published information was found as of the revision date.
◉ Effects on lactation and breast milk
No relevant published information was found as of the revision date.

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Protein binding
>99.5% bound to plasma proteins.

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Drug interactions[1]
An open-label study evaluated potential drug interactions between sofosbuvir and velpatasvir. The study included 18 healthy individuals without HCV infection. No clinically significant drug interactions were reported in the study by Mogalian et al. When using sofosbuvir or a sofosbuvir-containing regimen, patients should be advised to avoid rifampin, St. John's wort, or tepranavir, as these drugs can reduce circulating drug concentrations of sofosbuvir, thereby reducing its efficacy (Table 2). Velpatasvir absorption may be reduced if patients are taking antacids and acid-suppressing drugs concurrently. Patients should be informed of all medications they are currently taking and whether they plan to start any other medications, including over-the-counter drugs or herbal supplements.

[1]. Drug Des Devel Ther.2017 Feb 23;11:497-502.

[2]. Antimicrob Agents Chemother.2016 Aug 22;60(9):5368-78.

[3]. Signal Transduct Target Ther. 2021 May 29;6(1):212.

Pharmacodynamics
Velpatasvir is a small-molecule direct-acting antiviral drug used in combination with sofosbuvir to treat hepatitis C. Velpatasvir inhibits viral replication by suppressing non-structural protein 5A (NS5A). Even at doses up to five times the recommended dose, velpatasvir does not prolong the QTc interval to any clinically significant extent. Velpatasvir is a complex organic heteropentacyclic compound and an inhibitor of hepatitis C virus non-structural protein 5A, used in combination with sofosbuvir (trade name Epclusa) to treat patients with chronic hepatitis C of all six major genotypes. It is both an antiviral drug and an inhibitor of hepatitis C virus non-structural protein 5A. It is an organic heteropentacyclic compound belonging to the classes N-acylpyrrolidine, L-valine derivatives, carbamates, imidazoles, cyclic compounds, and ethers. Velpatasvir is a direct-acting antiviral (DAA) used in combination therapy for chronic hepatitis C. Chronic hepatitis C is an infectious liver disease caused by hepatitis C virus (HCV) infection. HCV is a single-stranded RNA virus with nine different genotypes, of which genotype 1 is the most common in the United States, affecting 72% of chronic HCV patients. Velpatasvir is a defective substrate of NS5A (non-structural protein 5A), a non-enzymatic viral protein that plays a crucial role in the replication, assembly, and regulation of the host immune response of hepatitis C virus. Since 2011, significant progress has been made in the treatment options for chronic hepatitis C with the development of direct-acting antiviral agents (DAAs) such as velpatasvir. Notably, velpatasvir has a significantly higher resistance barrier than first-generation NS5A inhibitors, such as [DB09027] and [DB09102], making it a highly effective and reliable alternative therapy for chronic hepatitis C. In a joint guideline published in 2016, the American Association for the Study of Liver Diseases (AASLD) and the Infectious Diseases Society of America (IDSA) recommended velpatasvir in combination with sofosbuvir as a first-line treatment for all six genotypes of hepatitis C virus. Currently, velpatasvir is only marketed as the combination drug Epclusa, which contains velpatasvir and another direct-acting antiviral agent [DB08934]. Treatment goals for Epclusa include cure or achieving sustained virological response (SVR), defined as achieving this response after 12 weeks of daily treatment. SVR and eradication of HCV infection are associated with significant long-term health benefits, including reduced liver-related damage, improved quality of life, reduced incidence of hepatocellular carcinoma, and reduced all-cause mortality and liver transplantation risk. Since June 2016, velpatasvir and [DB08934] have been marketed as a fixed-dose combination drug under the brand name Epclusa. Epclusa is the first approved combination therapy for hepatitis C (HCV) of all genotypes (with or without cirrhosis). It is also currently the most potent HCV antiviral drug on the market, achieving a sustained virological response (SVR) rate of 93-99% after 12 weeks of treatment, depending on genotype and degree of cirrhosis, and exhibiting a high resistance barrier. Guidelines in Canada and the United States recommend Epclusa as a first-line treatment for all HCV genotypes. Velpatasvir is a hepatitis C virus NS5A inhibitor. Velpatasvir's mechanism of action includes inhibition of breast cancer resistance protein, P-glycoprotein, organic anion transport peptide 1B1, organic anion transport peptide 1B3, and organic anion transport peptide 2B1. Velpatasvir is an oral inhibitor of the hepatitis C virus (HCV) nonstructural protein 5A (NS5A) replication complex, with potential activity against HCV genotypes 1-6. Although the exact mechanism of action of velpatasvir is not fully elucidated, it appears to bind to domain I of the NS5A protein after oral administration and cellular uptake. This inhibits NS5A protein activity, leading to the disruption of the viral RNA replication complex, blocking HCV viral RNA production, and inhibiting viral replication. NS5A is a proline-rich, zinc-binding, hydrophilic phosphoprotein that plays a crucial role in HCV RNA replication. HCV is a small, enveloped, single-stranded RNA virus belonging to the Flaviviridae family. VELPATASVIR is a small molecule drug, currently in Phase IV clinical trials (covering all indications), and was first approved in 2016 for the treatment of chronic hepatitis C virus infection, with one investigational indication. It is an open target. Hepatitis C virus (HCV) is a global epidemic, infecting nearly 200 million people worldwide. HCV is the most common blood-borne infection in the United States and can lead to a variety of health problems, including liver fibrosis, cirrhosis, and hepatocellular carcinoma. Traditional genotype-based hepatitis C virus (HCV) treatment regimens, such as interferon, have achieved some success in sustained clearance of the viral genome. A recent clinical trial showed that a once-daily combination of sofosbuvir (a non-structural protein 5B polymerase inhibitor) and velpatasvir (a non-structural protein 5A inhibitor) achieved a sustained virological response rate of approximately 95% in all HCV genotypes, regardless of prior treatment history or presence of cirrhosis. Patients reported improvements in overall health, fatigue, mood, and mental health after completing the combination therapy. This combination therapy is highly effective, but should be used with caution in patients taking certain medications or with certain diseases. This article will review the safety and efficacy of the sofosbuvir/velpatasvir combination regimen for all HCV genotypes. [1]

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The advent of direct-acting antiviral agents (DAAs), designed to selectively target the HCV replication process, has spurred a great deal of scientific research and has yielded encouraging results in various aspects. To date, the potent combination therapy of sofosbuvir (an NS5B protease inhibitor) and velpatasvir (an NS5A inhibitor) has demonstrated sustained virological response (SVR) rates exceeding 94% in all major HCV genotypes, including treatment-resistant genotypes, patients with cirrhosis, and previously treated patients (Table 3). This regimen holds promise for virological cure, as SVR rates of 95% or higher have been reported. Furthermore, a significant proportion of patients have experienced improvements in overall health, mood, mental well-being, and social productivity during and after treatment, further demonstrating the effectiveness of this combination therapy. Currently, insurance companies determine HCV treatment eligibility based on pre-treatment genotyping and liver fibrosis staging. While the sofosbuvir/velpatasvir combination therapy has been successful in all genotypes, offering a simple, safe, and potentially curative treatment option, its accessibility remains limited by cost. Once all patients have access to treatment, this will reduce the healthcare burden, thereby lowering morbidity and mortality, and patients will be able to be cured of hepatitis C virus infection without genotyping. [1] Velpatasvir (VEL, GS-5816) is a novel pangenotypic hepatitis C virus (HCV) nonstructural protein 5A (NS5A) inhibitor that is active against HCV replicons from genotype 1 (GT1) to GT6. In a 3-day phase 1b monotherapy study, patients treated with 150 mg of GS-5816 showed a mean maximum decrease in HCV RNA for GT1a, -1b, -2, -3, and -4 genotypes ≥3.3 log10 IU/ml. This report describes the virological resistance to VEL in these patients. Of the 70 patients, 22 had NS5A resistance-associated mutations (RAS) detected by deep sequencing (1% threshold) before treatment. These included 10 out of 35 GT1a patients (29%), 1 out of 8 GT1b patients (13%), 4 out of 8 GT2 patients (50.0%), 5 out of 17 GT3 patients (29.4%), and 2 out of 2 GT4 patients (100.0%). In GT1a and GT3 patients, those with pre-treatment RAS showed slightly lower HCV RNA responses than those without. However, in GT1b, GT2, and GT4 patients, there was no significant difference in response between those with and without pre-treatment RAS. Post-treatment, the newly emerging RAS pattern in GT1a patients was more complex than in other genotypes. In GT1a, amino acid substitutions were observed at M28, Q30, L31, P32, H58, E92, and Y93 sites, with substitutions at Y93, M28, and L31 being the most common. In GT1b and GT2, resistance-associated mutations (RAS) were observed at Y93 and L31 sites; in GT3, resistance-associated mutations were observed at Y93, L31, and E92 sites; and in GT4, resistance-associated mutations were observed at L28, M31, P32L, and Y93 sites. Pre-treatment resistance-associated mutations persisted during the 48-week follow-up period; however, resistance-associated mutations that appeared during treatment were more likely to decrease in prevalence and frequency in the viral population during the follow-up period. (This study has been registered at ClinicalTrials.gov, registration number NCT01740791.) [2] Phenotypic analysis of RAS detected before treatment or screened after velpatasvir/VEL treatment showed that most RAS in the GT1a replicon did not show resistance (EC50 change ≤ 2.5-fold, Q30L/R/H, Y93F) or low to moderate levels of resistance (EC50 change 2.5 to 100-fold, Q30K/E, L31I/M/V, P32L, H58D, Y93C/S). High levels of resistance (EC50 change > 100-fold) were observed in the Y93H/N/R/W and double mutants. All NS5A RAS in the GT1b replicon conferred VEL resistance < 3.3-fold. Most GT2a, GT3a, and GT4a single mutants showed low or no resistance to velpatasvir (VEL); however, the Y93H mutation in GT3a reduced VEL sensitivity by 723.5-fold. In summary, in a 3-day phase 1b monotherapy study, velpatasvir/VEL demonstrated broad genotypic activity and improved activity against pre-existing resistant variants. Although pre-treatment resistance-associated mutations (RAS) were associated with slightly reduced HCV RNA responses in GT1a and GT3 patients, current HCV treatment strategies based on the combination of direct-acting antiviral agents (DAAs) should be able to overcome any slight effects of pre-treatment RAS. Combination therapy with VEL and sofosbuvir (SOF) may provide an effective treatment option for patients infected with HCV GT1 to GT6. [2]

C49H54N8O8

883.00

882.406

C, 66.65; H, 6.16; N, 12.69; O, 14.49

1377049-84-7

67683363

White to light yellow solid powder.

1.3±0.1 g/cm3

1.643

6.78

4

10

13

65

1690

6

O(C([H])([H])[H])C([H])([H])[C@]1([H])C([H])([H])N(C([C@@]([H])(C2C([H])=C([H])C([H])=C([H])C=2[H])N([H])C(=O)OC([H])([H])[H])=O)[C@]([H])(C2=NC([H])=C(C3C([H])=C([H])C4=C(C([H])([H])OC5=C4C([H])=C4C([H])=C([H])C6=C(C4=C5[H])N=C([C@]4([H])C([H])([H])C([H])([H])[C@]([H])(C([H])([H])[H])N4C([C@]([H])(C([H])(C([H])([H])[H])C([H])([H])[H])N([H])C(=O)OC([H])([H])[H])=O)N6[H])C=3[H])N2[H])C1([H])[H]

FHCUMDQMBHQXKK-CDIODLITSA-N

InChI=1S/C49H54N8O8/c1-26(2)41(54-48(60)63-5)47(59)57-27(3)12-17-38(57)45-51-36-16-14-30-20-35-33-15-13-31(19-32(33)25-65-40(35)21-34(30)43(36)53-45)37-22-50-44(52-37)39-18-28(24-62-4)23-56(39)46(58)42(55-49(61)64-6)29-10-8-7-9-11-29/h7-11,13-16,19-22,26-28,38-39,41-42H,12,17-18,23-25H2,1-6H3,(H,50,52)(H,51,53)(H,54,60)(H,55,61)/t27-,28-,38-,39-,41-,42+/m0/s1

methyl ((R)-2-((2S,4S)-2-(5-(2-((2S,5S)-1-((methoxycarbonyl)-L-valyl)-5-methylpyrrolidin-2-yl)-1,11-dihydroisochromeno[4',3':6,7]naphtho[1,2-d]imidazol-9-yl)-1H-imidazol-2-yl)-4-(methoxymethyl)pyrrolidin-1-yl)-2-oxo-1-phenylethyl)carbamate

GS5816; GS-5816; Velpatasvir; 1377049-84-7; GS5816; KCU0C7RS7Z; Velpatasvir [USAN:INN]; Velpatasvir [INN]; GS 5816; Velpatasvir

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : 100~146.66 mg/mL ( 113.25~166.09 mM )
Water : ~100 mg/mL
Ethanol : ~100 mg/mL

Solubility in Formulation 1: ≥ 2.5 mg/mL (2.83 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (2.83 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (2.83 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly..

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Solubility in Formulation 4: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: ≥ 2.5 mg/mL (2.83 mM)

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 (Please use freshly prepared in vivo formulations for optimal results.)