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 of 25-30%.
94% excreted in feces with 77% as parent compound. 0.4% excreted in urine.
1.4-1.6 L/kg.
Estimated 0.12 L/h/kg [A19175.

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Metabolism / Metabolites
Some metabolism by CYP2B6, CYP2C8, and CYP3A4.

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Biological Half-Life
15h.

Effects During Pregnancy and Lactation
◉ Summary of Use during Lactation
Velpatasvir has not been studied in nursing mothers being treated for hepatitis C infection. Because it is greater than 99% bound to maternal plasma proteins, amounts in breastmilk are likely to be very low. Some sources recommend against breastfeeding when velpatasvir is used with ribavirin.
Hepatitis C is not transmitted through breastmilk and breastmilk has been shown to inactivate hepatitis C virus (HCV). However, the Centers for Disease Control recommends that mothers with HCV infection should consider abstaining from breastfeeding if their nipples are cracked or bleeding. It is not clear if this warning would apply to mothers who are being treated for hepatitis C.
Infants born to mothers with HCV infection should be tested for HCV infection; because maternal antibody is present for the first 18 months of life and before the infant mounts an immunologic response, nucleic acid testing is recommended.
◉ Effects in Breastfed Infants
Relevant published information was not found as of the revision date.
◉ Effects on Lactation and Breastmilk
Relevant published information was not found as of the revision date.

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Protein Binding
>99.5% bound to plasma proteins.

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Drug–drug interactions [1]
Potential drug–drug interactions between sofosbuvir and velpatasvir were evaluated in an open-label study. Eighteen non-HCV, healthy individuals were enrolled, and there were no clinically significant drug–drug interactions reported in this study by Mogalian et al. When administering sofosbuvir or sofosbuvir-containing regimens, patients should be cautioned to avoid rifampin, St John’s wort, or tipranavir because these agents decrease the efficacy of sofosbuvir by decreasing circulating drug levels of sofosbuvir (Table 2). Velpatasvir can have decreased absorption if the patient is on concomitant antacids and acid-reducing medications. Patients should be asked to report all medications they are currently on and if they are going to start on any additional medications including over-the-counter medications 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 used in the treatment of hepatitis C in combination with sofosbuvir. Velpatasvir prevents viral replication by inhibiting non-structural protein 5A (NS5A). At a dose 5 times the recommended dose, velpatasvir does not prolong QTc interval to any clinically relevant extent.

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Velpatasvir is a complex organic heteropentacyclic compound that is a hepatitis C virus nonstructural protein 5A inhibitor used in combination with sofosbuvir (under the brand name Epclusa) for treatment of patients with chronic hepatitis C of all six major genotypes. It has a role as an antiviral drug and a hepatitis C virus nonstructural protein 5A inhibitor. It is an organic heteropentacyclic compound, a N-acylpyrrolidine, a L-valine derivative, a carbamate ester, a member of imidazoles, a ring assembly and an ether.
Velpatasvir is a Direct-Acting Antiviral (DAA) medication used as part of combination therapy to treat chronic Hepatitis C, an infectious liver disease caused by infection with Hepatitis C Virus (HCV). HCV is a single-stranded RNA virus that is categorized into nine distinct genotypes, with genotype 1 being the most common in the United States, and affecting 72% of all chronic HCV patients. Velpatasvir acts as a defective substrate for NS5A (Non-Structural Protein 5A), a non-enzymatic viral protein that plays a key role in Hepatitis C Virus replication, assembly, and modulation of host immune responses. Treatment options for chronic Hepatitis C have advanced significantly since 2011, with the development of Direct Acting Antivirals (DAAs) such as velpatasvir. Notably, velpatasvir has a significantly higher barrier to resistance than the first generation NS5A inhibitors, such as [DB09027] and [DB09102], making it a highly potent and reliable alternative for treatment of chronic Hepatitis C. In a joint recommendation published in 2016, the American Association for the Study of Liver Diseases (AASLD) and the Infectious Diseases Society of America (IDSA) recommend Velpatasvir as first line therapy in combination with sofosbuvir for all six genotypes of Hepatitis C. Velpatasvir is currently only available within a fixed dose combination product as Epclusa with [DB08934], another direct acting antiviral. Goals of therapy for Epclusa include the intent to cure, or achieve a sustained virologic response (SVR), after 12 weeks of daily therapy. SVR and eradication of HCV infection is 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 risk of requiring a liver transplant. Since June 2016, Velpatasvir has been available as a fixed dose combination product with [DB08934], as the commercially available product Epclusa. Epclusa is the first combination HCV product indicated for the treatment of all genotypes of Hepatitis C with or without cirrhosis. It is also currently the most potent HCV antiviral medication on the market with a sustained virologic response (SVR) after 12 weeks of therapy of 93-99% depending on genotype and level of cirrhosis and a high barrier to resistance. Both Canadian and American guidelines list Epclusa as a first line recommendation for all genotypes of HCV.

Velpatasvir is a Hepatitis C Virus NS5A Inhibitor. The mechanism of action of velpatasvir is as a Breast Cancer Resistance Protein Inhibitor, and P-Glycoprotein Inhibitor, and Organic Anion Transporting Polypeptide 1B1 Inhibitor, and Organic Anion Transporting Polypeptide 1B3 Inhibitor, and Organic Anion Transporting Polypeptide 2B1 Inhibitor.
Velpatasvir is an orally available inhibitor of the hepatitis C virus (HCV) non-structural protein 5A (NS5A) replication complex, with potential activity against HCV genotypes 1-6. Although the exact mechanism of action of velpatasvir has not yet been completely determined, upon oral administration and intracellular uptake, it appears to bind to domain I of the NS5A protein. This inhibits the activity of the NS5A protein and results in the disruption of the viral RNA replication complex, blockage of viral HCV RNA production, and inhibition of viral replication. NS5A, a zinc-binding and proline-rich hydrophilic phosphoprotein, 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 with a maximum clinical trial phase of IV (across all indications) that was first approved in 2016 and is indicated for chronic hepatitis c virus infection and has 1 investigational indication. Open Targets.

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Hepatitis C virus (HCV) is a global pandemic, with nearly 200 million infected patients worldwide. HCV is the most common blood-borne infection in the US with numerous health implications including liver fibrosis, cirrhosis, and hepatocellular cancer. Traditional genotype-based HCV therapies with interferon resulted in moderate success in the sustained elimination of viral genome. Recent clinical trials of the once-daily combination tablet of sofosbuvir, a nonstructural (NS) 5B polymerase inhibitor, and Velpatasvir, an NS5A inhibitor, demonstrate sustained virologic response rates of about 95%, regardless of prior treatment experience or presence of cirrhosis across all HCV genotypes. Patients reported improvements in general health, fatigue, and emotional and mental well-being after completing combination therapy. The combination treatment is effective, but does need to be administered with caution in patients receiving certain medications or with certain diseases. Herein, we review the safety and efficacy of sofosbuvir/velpatasvir combination regimen for all HCV genotypes.[1]

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The introduction of DAAs to selectively target the HCV replication process has yielded a plethora of scientific research, with promising results at each end. Thus far, the potent combination of sofosbuvir, an NS5B PI, and velpatasvir, an NS5A inhibitor, has shown >94% SVR rates among all major HCV genotypes, including the difficult-to-cure genotypic, cirrhotic, and treatment-experienced populations (Table 3). The virologic cure is a possibility with this regimen as SVR rates have been reported at or above 95%. In addition to improvements in general health, emotional and mental well-being and social productivity throughout therapy and posttherapy in a sizeable number of patients illustrate the efficacy of the combination treatment. Currently, pretreatment genotype testing and stage of liver disease fibrosis determine which patients will receive therapy for HCV by the insurance companies.31 The pan-genotypic success achieved with sofosbuvir/velpatasvir makes therapy simple, safe, and curative, but access to therapy is limited by cost. Once therapy is accessible to all patients, this will decrease health care disease burden with a resultant decrease in morbidity and mortality, and then patients can be cured from the HCV virus without having to undergo pregenotype testing.[1]

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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. NCT01740791.).[2]

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Phenotypic analyses of the RASs detected pretreatment or selected following Velpatasvir/VEL treatment showed that most of the RASs in the GT1a replicon demonstrated no (≤2.5-fold EC50 change, Q30L/R/H, Y93F) or low- to midlevel resistance (2.5- to 100-fold EC50 change, Q30K/E, L31I/M/V, P32L, H58D, Y93C/S). High levels of resistance (>100-fold EC50 change) were observed in Y93H/N/R/W and double mutants. All of the NS5A RASs in the GT1b replicon conferred <3.3-fold resistance to VEL. The majority of the GT2a, GT3a, and GT4a single mutants displayed no or low levels of resistance to VEL; however, Y93H in GT3a conferred 723.5-fold reduced susceptibility to VEL.
In summary, Velpatasvir/VEL demonstrated broad genotypic activity and improved activity against preexisting resistant variants in a phase 1b 3-day monotherapy study. Although pretreatment RASs were associated with a slightly reduced HCV RNA response in GT1a and GT3 patients, current HCV treatment strategies are based on the combined use of DAAs and should overcome any minor impact of pretreatment RASs. Combination treatment with VEL and 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.)