HCV NS5A protein [1].

Samatasvir (IDX719) does not cause cross-resistance to HCV protease, nucleotide, and non-nucleoside polymerase inhibitor classes, and it maintains full action in the presence of HIV and hepatitis B virus (HBV) antiviral medications [1].

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Samatasvir is a selective inhibitor of HCV replication with broad genotypic activity. It inhibits genotype 1a and 1b HCV replicons at picomolar concentrations, with mean EC50 values of 4.1 pM (H1a-luc) and 2.4 pM (Zluc), respectively. Against replicons bearing NS5A sequences from genotypes 1 to 5 or a genotype 2a JFH-1 infectious virus, EC50s ranged from 2 to 24 pM. The EC90/EC50 ratio was low at 2.6 [1].
Samatasvir reduced genotype 1a replicon RNA in a dose-dependent manner over 14 days. Maximum reductions were 1.5 log10 at 4 pM (1× EC50) and 4.2 log10 at 40 nM (10,000× EC50). Maximal suppression occurred at 7 to 14 days, depending on the dose, with no evidence of viral rebound [1].
Samatasvir was tested against 17 RNA and DNA viruses and showed weak activity only against bovine viral diarrhea virus (EC50 33.4 μM) and yellow fever virus (EC50 16.5 μM), and was inactive against all others, demonstrating high specificity for HCV [1].
In combination studies with other anti-HCV agents in genotype 1b replicon cells, Samatasvir demonstrated an overall additive effect when combined with interferon alfa, ribavirin, HCV protease inhibitors (simeprevir), nucleoside inhibitors (IDX184), and nonnucleoside polymerase inhibitors (TMC647055), as determined by Bliss Independence and Loewe Additivity models [1].
Samatasvir retained full activity in the presence of anti-HIV and anti-HBV agents (Kaletra, zidovudine, efavirenz, raltegravir, lamivudine, tenofovir, telbivudine) at 5× Cmax equivalent concentrations, with fold change values of < 2 in EC50 [1].
Resistance selection experiments with genotype 1a replicons identified amino acid substitutions within the first 100 amino acids of NS5A at loci M28, Q30, L31, P32, and Y93 as conferring resistance to Samatasvir. The Y93H substitution was the most predominant, conferring a 4,400-fold shift in activity. Other substitutions like Q30E (420-fold), L31V (420-fold), and P32L (170-fold) also conferred high-level resistance in genotype 1a. In genotype 1b replicons, the resistance levels were generally lower (e.g., Y93H: 93-fold, L31M: 3.6-fold). Samatasvir was not cross-resistant with HCV protease (e.g., R155K, A156T, D168V), nucleoside (S282T), or nonnucleoside polymerase inhibitors (C316Y, M414T, M423T) [1].

For HCV replication activity assays, Zluc (genotype 1b), NS5A IGT, or H1a-luc (genotype 1a) cells were seeded in 96-well plates. After at least 4 hours, drug treatment was initiated and cells were incubated for 3 days. Luciferase activity was measured using ONE-Glo luciferase assay reagent to determine EC50 values. For combination assays, Zluc and Huh-7 cells were seeded and treated with drug dilutions in a 5-by-5 checkerboard design. Cytotoxicity was measured in parallel using CellTiter-Blue cell viability assay solution. Data were analyzed for synergy/antagonism using Bliss Independence (MacSynergy II), Loewe additivity (CombiTool), and combination index (CalcuSyn) models [1].
For the extended-treatment replicon assay, H1a cells were seeded in 6-well plates in medium containing compound and incubated. Cells were split every 3-4 days in fresh compound-containing medium. RNA was extracted on days 0, 3, 7, 10, and 14 using a High Pure RNA isolation kit. Replicon RNA was measured by real-time quantitative RT-PCR using TaqMan One-Step RT-PCR master mix with primers and probe specific for the 5'-UTR of the replicon, and normalized to human GAPDH endogenous control [1].
For the HCV in vitro infection core ELISA, HPC cells were seeded in 96-well plates. After 4 hours, JFH-1 HCV virus stock and serial dilutions of drug were added. After 16 hours, the inoculum was removed and cultures were treated with drug for an additional 4 days. Cells were fixed, blocked, and incubated with an anti-HCV core protein antibody, followed by an HRP-conjugated secondary antibody. Color development was initiated with OPD solution and absorbance measured at 490 nm [1].
For cytotoxicity assays, various cell lines (HepG2, HepaRG, CAKI-1, CCRF-CEM, COLO-205, SJCRH30, SNB-78) were seeded and treated with serial dilutions of compound for 3 days (or 14 days for HepaRG). Cell viability was determined using CellTiter-Blue, CellTiter-Glo, WST-1, or the In Cytotox toxicity test system (for HepG2, measuring LDH, glucose consumption, XTT, and acid phosphatase) [1].
For non-HCV antiviral activity assays, standard cytoprotection (CPE), reporter gene (HIV), or plaque reduction assays were used depending on the virus. An aliquot of virus with a predetermined titer was diluted and added to each well. Appropriate positive controls for virus inhibition were used for each virus [1].
For resistance selection, H1a-luc cells were cultured in the presence of 0.25 mg/ml G418 and increasing concentrations of Samatasvir (starting at 2× EC50, increasing to 256× EC50) for 90 days to generate resistant cell lines. Population sequencing of the NS3 to NS5b genomic region and clonal sequencing of the NS5A amino-terminal region (amino acids 1-100) were performed [1].
For transient-transfection assays, HPC cells were electroporated with wild-type or mutant replicon RNA and plated. Approximately 4 hours later, serial dilutions of drug were added and cells incubated for 4 days to determine EC50 values [1].

The literature notes that in clinical studies, Samatasvir was initially evaluated at doses of 1 to 100 mg daily. The pharmacokinetics was dose proportional, with a terminal half-life of approximately 24 hours, supporting once-a-day dosing [1].
Protein binding experiments showed that in the presence of 40% human serum, Samatasvir activity decreased 10.4-fold in the genotype 1b replicon. Using EC50 and EC90 values obtained in 10% to 50% human serum and linear regression analysis, the 100% serum-adjusted mean EC50 and EC90 values were extrapolated to 55.5 pM and 214 pM, respectively [1].

Samatasvir demonstrated a favorable cytotoxicity profile. The CC50 in the Zluc genotype 1b replicon cell line was >100 μM, providing a selectivity index of >5 × 10⁷. In a panel of human liver (HepG2, HepaRG) and non-liver cell lines (CAKI-1, CCRF-CEM, COLO-205, SJCRH30, SNB-78), CC50 values were >50 μM or >100 μM after 3 or 14 days of treatment. In HepG2 cells, a multiparameter cytotoxicity assay showed no measurable cytotoxicity up to 100 μM across four different endpoints (LDH release, glucose consumption, XTT metabolism, acid phosphatase activity). No cytotoxicity was observed when Samatasvir was combined with other anti-HCV, anti-HIV, or anti-HBV agents [1].

[1]. In vitro activity and resistance profile of samatasvir, a novel NS5A replication inhibitor of hepatitis C virus. Antimicrob Agents Chemother. 2014;58(8):4431-4442.

Samatasvir has been used in trials investigating the treatment of hepatitis C, chronic hepatitis C virus, and chronic hepatitis C infection.

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Samatasvir (IDX719) is a novel, potent, and selective pan-genotypic HCV NS5A inhibitor designed to have enhanced activity across HCV genotypes 1-5, with a narrow range of EC50 values (2-24 pM) [1].
NS5A is a nonstructural protein involved in HCV replication and virion maturation. Although its exact functions are not fully understood, it is a clinically validated target for anti-HCV therapy [1].
In clinical studies, following 3 daily doses of 25 to 100 mg Samatasvir, viral load reductions of up to 4 log10 were observed in subjects with HCV genotypes 1 through 4. A reduced antiviral response was observed in HCV genotype 2-infected subjects who had the NS5A M31 polymorphism at baseline. Samatasvir has been generally safe and well tolerated in all clinical trials to date [1].
The lack of cross-resistance to other classes of HCV direct-acting antivirals and additive combination data in vitro support its ongoing development as part of all-oral, interferon-free regimens for HCV treatment [1].

C47H48N8O6S2

885.07

884.313

1312547-19-5

58310140

White to off-white solid powder

1.4±0.1 g/cm3

1.675

10.3

4

10

13

63

1620

4

CC(C)[C@@H](C(=O)N1CCC[C@H]1C2=NC3=C(N2)C=C(C=C3)C4=CSC5=C4SC=C5C6=CC=C(C=C6)C7=CN=C(N7)[C@@H]8CCCN8C(=O)[C@@H](C9=CC=CC=C9)NC(=O)OC)NC(=O)OC

ATOLIHZIXHZSBA-BTSKBWHGSA-N

InChI=1S/C47H48N8O6S2/c1-26(2)38(52-46(58)60-3)44(56)55-21-9-13-37(55)43-49-33-19-18-30(22-34(33)50-43)32-25-63-40-31(24-62-41(32)40)27-14-16-28(17-15-27)35-23-48-42(51-35)36-12-8-20-54(36)45(57)39(53-47(59)61-4)29-10-6-5-7-11-29/h5-7,10-11,14-19,22-26,36-39H,8-9,12-13,20-21H2,1-4H3,(H,48,51)(H,49,50)(H,52,58)(H,53,59)/t36-,37-,38-,39+/m0/s1

methyl N-[(1R)-2-[(2S)-2-[5-[4-[6-[2-[(2S)-1-[(2S)-2-(methoxycarbonylamino)-3-methylbutanoyl]pyrrolidin-2-yl]-3H-benzimidazol-5-yl]thieno[3,2-b]thiophen-3-yl]phenyl]-1H-imidazol-2-yl]pyrrolidin-1-yl]-2-oxo-1-phenylethyl]carbamate

Samatasvir IDX-18719 IDX 18719

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

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

DMSO : ~50 mg/mL (~56.49 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (2.82 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 (2.82 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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 (Please use freshly prepared in vivo formulations for optimal results.)