HCV NS3 protease(IC50≈0.2 nM-3.5 nM)
The target of Asunaprevir is hepatitis C virus (HCV) NS3/4A protease complex. It has a Ki value of 0.4 nM against recombinant enzyme of HCV genotype 1a (H77) and 0.24 nM against genotype 1b (J4L6S)[2]
The target of Asunaprevir is HCV NS3 protease[3]
The target of Asunaprevir is HCV NS3/4A protease complex. It has a Ki value of 0.4 nM against recombinant enzyme of HCV genotype 1a (H77) and 0.24 nM against genotype 1b (J4L6S)[4]
The target of Asunaprevir is HCV NS3 protease[5]

Asunaprevir (ASV) inhibits the NS3 proteolytic activity of genotype 1a (H77 strain) and genotype 1b (J4L6S strain), with IC50s of 0.7 and 0.3 nM, respectively. The EC50s of ASV against replicons encoding the NS3 protease domains representing genotypes 1a, 1b, and 4a, range from 1.2 to 4.0 nM.

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1. Activation of innate immune signaling pathways: Asunaprevir treatment (1, 10, 100 nM) activates ISRE and IFN-β promoter-luciferase activities in Huh 7.5.1 cells in a dose-dependent manner. It increases the expression and phosphorylation of STAT1, STAT2, and IRF3, and upregulates downstream ISGs such as MxA and ISG-15, activating JAK-STAT, MAVS, and TRIF signaling pathways[1]
2. Inhibition of viral replication: Asunaprevir (1, 10, 100, 250, 500 nM) reduces the RNA and protein levels of DENV-2 NS3 in Huh 7.5.1 and HepG2 cells. It also decreases HCV RNA level in JFH-1-infected Huh 7.5.1 cells and inhibits HCV and DENV virus production. The antiviral effect is attenuated in MAVS-knockdown cells, indicating dependence on MAVS-mediated innate immunity[1]
3. No inhibition of DENV protease: Asunaprevir does not inhibit the protease activity of DENV NS2B3, as shown by unchanged NS3 processing from NS2B3 in Huh 7.5.1 cells transfected with Flag-DV2 NS2B3[1]
1. Inhibition of HCV protease activity: Asunaprevir competitively binds to HCV NS3/4A protease complex, with no significant activity against GB virus-B NS3 protease or human serine/cysteine proteases[2]
2. Antiviral activity against HCV replicons: It inhibits replication of HCV genotypes 1 and 4 replicons with EC₅₀ ranging from 1 to 4 nM, and has weaker activity against genotypes 2 and 3 (EC₅₀: 67 to 1162 nM). It shows no activity (EC₅₀ > 12 μM) against other RNA viruses[2]
3. Combination activity: It exhibits additive or synergistic activity with alfa interferon, ribavirin, NS5A inhibitors, or NS5B inhibitors[2]
Asunaprevir shows additive-synergistic effects on HCV replicon inhibition when combined with NS5A inhibitor daclatasvir or NS5B inhibitor BMS-791325. Triple combination therapy enhances synergies and reduces resistance frequency compared to dual therapy[3]
1. Inhibition of HCV protease activity: Asunaprevir competitively binds to HCV NS3/4A protease complex, with no significant activity against GB virus-B NS3 protease or human serine/cysteine proteases[4]
2. Antiviral activity against HCV replicons: It inhibits replication of HCV genotypes 1 and 4 replicons with EC₅₀ ranging from 1 to 4 nM, and has weaker activity against genotypes 2 and 3 (EC₅₀: 67 to 1162 nM). It shows no activity (EC₅₀ > 12 μM) against other RNA viruses[4]
3. Combination activity: It exhibits additive or synergistic activity with alfa interferon, ribavirin, NS5A inhibitors, or NS5B inhibitors[4]
1. Resistance analysis in HCV replicons: For genotype 1a, primary NS3 substitutions R155K, D168G, I170T confer 5-21-fold resistance. For genotype 1b, substitutions at D168 (D168A/G/H/V/Y) confer 16-280-fold resistance with impaired replication capacity[5]
2. Susceptibility of patient-derived variants: Baseline NS3-Q80K polymorphism does not significantly affect susceptibility to Asunaprevir in enzyme assays[5]
3. Cross-susceptibility: Asunaprevir-resistant replicons remain susceptible to NS5A inhibitors[5]

Asunaprevir (ASV, 3-15 mg/kg, p.o.) displays a hepatotropic disposition (liver-to-plasma ratios ranging from 40- to 359-fold across species) in several animal species. Twenty-four hours postdose, liver exposures across all species tested are ≥110-fold above the inhibitor EC50 observed with HCV genotype-1 replicons. Plasma and tissue exposures in vivo in several animal species indicated that ASV/Asunaprevir displayed a hepatotropic disposition (liver-to-plasma ratios ranging from 40- to 359-fold across species). Twenty-four hours postdose, liver exposures across all species tested were ≥110-fold above the inhibitor EC(50)s observed with HCV genotype-1 replicons. Based on these virologic and exposure properties, ASV holds promise for future utility in a combination with other anti-HCV agents in the treatment of HCV-infected patients.[4]

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Luciferase assay[1]
To monitor IFN signaling directed by ISRE, plasmids pISRE-luc (500 ng/well) expressing firefly luciferase and pRL-TK (50 ng/well) expressing Renilla luciferase as an internal control were co-transfected in Huh 7.5.1 cells (1 × 105). To monitor IFN-β promoter signaling, pGL-4 IFNβ-Luc (pIFNβ/FLuc) (500 ng/well) expressing firefly luciferase and pRL-TK (50 ng/well) expressing Renilla luciferase as an internal control were used (Chang et al., 2006, 2009). Huh 7.5.1 cells were transfected with Lipofectamine 2000 Transfection Reagent following the manufacturer's protocol. Briefly, cells cultured in 12-well plates were transfected with a DNA precipitate containing plasmid DNA (amounts as indicated and had been adjusted by the vector control to be the same among samples of the experimental group). Five hours after transfection, cells were replenished with culture medium, and then incubated for various time points. Relative luciferase activity was assessed by the Promega dual-luciferase reporter assay system.

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Enzyme-based selectivity assays. [2]
To assess in vitro selectivity, compounds were counterscreened against GBV-B NS3/4A protease, human neutrophil elastase, porcine pancreatic elastase, human α-chymotrypsin, human cathepsin A, and human liver cathepsin B. Compounds were diluted in assay buffer in 10% dimethyl sulfoxide (DMSO). GBV-B NS3/4A protease (0.3 nM) replaced HCV-NS3/4A protease in the FRET-based assay, as described above. All other assays were performed in 96-well plates with 1% DMSO and measured continuous substrate hydrolysis at 405 nm on a Spectramax plate spectrophotometer. Human neutrophil elastase, porcine pancreatic elastase, and human pancreatic α-chymotrypsin reaction mixtures contained 50 mM Tris-HCl (pH 8.0), 50 mM NaCl, 0.1 mM EDTA, 0.05% Tween 20, and 20 nM human elastase, 25 nM porcine elastase, or 1 nM chymotrypsin, respectively. Reactions were initiated by addition of substrate (elastases, 5 μM succinyl-AAPV-amino-4-methylcoumarin (AMC); chymotrypsin, 5 μM LLVY-AMC) and monitored fluorimetrically at an excitation wavelength (Ex) of 360 nm and an emission wavelength (Em) of 480 nm. Cathepsin A assays were performed per the manufacturer's instructions with (7-methoxycoumarin-4-yl)acetyl-RPPGFSAFK(dinitrophenyl)-OH as the substrate; reactions were monitored at Ex/Em of 565/580 nm. Cathepsin B assay mixtures contained 50 mM sodium acetate (NaOAc) (pH 5.5), 1 mM tris(2-carboxyethyl)phosphine, 5 nM cathepsin B, and 2 μM Z-FR-AMC as the substrate, and reactions were monitored at Ex/Em of 360/460 nM. Factor XA assay mixtures contained 100 mM NaPO4 (pH 7.5), 0.5% polyethylene glycol (PEG) 8000, 200 mM NaCl, 1 nM human factor XA , and 400 μM S-2222 as the substrate. Factor XIA assay mixtures contained 50 mM HEPES (pH 7.5), 0.1% PEG-8000, 5 mM KCl, 145 mM NaCl, 0.14 nM human factor XIA, and 250 μM S-2366 as the substrate. Factor VIIA assay mixtures contained 50 mM HEPES (pH 7.5), 150 mM NaCl, 5 mM CaCl2, 0.1% PEG-8000, 1.8 nM factor VIIA, and 1 mM S-2288 as the substrate. Kallikrein assay mixtures contained 50 mM Tris (pH 7.5), 100 mM NaCl, 0.5% PEG-8000, 0.2 nM kallikrein (Enzyme Research Laboratories), and 400 μM S-2302 as the substrate. Thrombin assay mixtures contained 100 mM sodium phosphate (pH 7.5), 200 mM NaCl, 0.5% PEG-8000, 0.2 nM thrombin, and 200 μM S-2366 as the substrate. Trypsin assay mixtures contained 100 mM NaPO4 (pH 7.5), 200 mM NaCl, 0.5% PEG-8000, 0.332 nM trypsin, and 200 μM S-2222 as the substrate.

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Asunaprevir (ASV) inhibits the NS3 proteolytic activity of genotype 1a (H77 strain) and genotype 1b (J4L6S strain), with IC50s of 0.7 and 0.3 nM, respectively. The EC50s of ASV against replicons encoding the NS3 protease domains representing genotypes 1a, 1b, and 4a, range from 1.2 to 4.0 nM.

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1. HCV NS3/4A protease inhibition assay: Prepare reaction mixtures containing recombinant HCV NS3/4A protease (genotype 1a H77 or 1b J4L6S), fluorescent substrate, and different concentrations of Asunaprevir. Incubate the mixtures at appropriate temperature and measure the rate of fluorescence change to reflect protease activity. Calculate Ki values using Lineweaver-Burk plots and apparent Km values at different drug concentrations[2]
2. Selectivity assay: Perform similar protease inhibition assays with GB virus-B NS3 protease and a panel of human serine/cysteine proteases to evaluate the selectivity of Asunaprevir[2]
1. HCV NS3/4A protease inhibition assay: Prepare reaction mixtures containing recombinant HCV NS3/4A protease (genotype 1a H77 or 1b J4L6S), fluorescent substrate, and different concentrations of Asunaprevir. Incubate the mixtures at appropriate temperature and measure the rate of fluorescence change to reflect protease activity. Calculate Ki values using Lineweaver-Burk plots and apparent Km values at different drug concentrations[4]
2. Selectivity assay: Perform similar protease inhibition assays with GB virus-B NS3 protease and a panel of human serine/cysteine proteases to evaluate the selectivity of Asunaprevir[4]
Enzyme assay for patient-derived variants: Isolate NS3 protease sequences from HCV genotype 1a/1b-infected patients (with Q80 or K80 polymorphism). Express recombinant proteases and perform inhibition assays with Asunaprevir to compare susceptibility[5]

Cell culture virology assays.[2]
For HCV replicons containing an RLuc reporter, compound antiviral activities (50% effective concentration [EC50]), were determined as described previously. For replicons lacking a reporter gene, activity was measured using a FRET-labeled peptide substrate (10 μM), as described previously. BVDV assays were performed as described previously (36), except that incubations were maintained for 4 days.
Antiviral susceptibility of HIV, HRV2, and HCoV was determined by incubation with serial dilutions of compound. For recombinant HIVs expressing RLuc, antiviral activity was evaluated by luciferase assay 5 days postinfection using a dual-luciferase kit. The diluted passive lysis solution was premixed with both the luciferase assay substrate and the Stop & Glo substrate (2:1:1 ratio). To each aspirated sample well, 40 μl of the mixture was added, and luciferase activity was measured immediately on a Wallac TriLux. For determination of activities against HRV2 and HCoV OC43, compound and virus (multiplicity of infection, 0.1) were incubated with cells for 96 h, and cell protection was used as a measure of virus production. For the cell protection assay, 17 μl of Cell-Titer Blue reagent was added to each well. Plates were then incubated for 2 h at room temperature before fluorescence was measured using a Spectramax Gemini EM instrument set to Ex/Em of 530/580 nm.
Cytotoxicity is determined by incubating cells (3,000 to 10,000 cells/well) with serially diluted test compounds or DMSO for 5 days (MT-2 cells) or 4 days (all other cell types). Cell viability is quantitated using an MTS assay for MT-2 or a Cell-Titer Blue reagent assay for HEK-293, HuH-7, HepG2, and MRC5 cells, and 50% cytotoxic concentrations (CC50s) are calculated. For the HCV and BVDV replicon assays, CC50s are determined from the same wells that are later used to determine EC50s.

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1. Luciferase reporter assay: Co-transfect Huh 7.5.1 cells with pISRE-luc (or pIFNβ/FLuc) and pRL-TK plasmids. After 48 hours, treat with 1-100 nM Asunaprevir for 3-48 hours. Measure firefly and Renilla luciferase activities using dual-luciferase assay system to assess ISRE and IFN-β promoter activity[1]
2. Immunoblotting: Treat Huh 7.5.1 cells with 0.1-50 nM Asunaprevir for 48 hours. Lyse cells, separate proteins by SDS-PAGE, transfer to PVDF membranes, and probe with antibodies against STAT1, phospho-STAT1, STAT2, phospho-STAT2, MAVS, TRIF, IRF3, phospho-IRF3, MxA, ISG-15, HCV core, HCV NS3, DENV-2 NS3, and β-actin. Detect signals with ECL kit and analyze densitometry[1]
3. Real-time qPCR: Infect Huh 7.5.1 or HepG2 cells with DENV-2 or JFH-1 HCV. Treat with Asunaprevir for 24 hours. Isolate total RNA, reverse transcribe to cDNA, and perform qPCR with specific primers for DENV-2, HCV, and HPRT (internal control) to quantify viral RNA levels[1]
4. siRNA knockdown assay: Transfect Huh 7.5.1 cells with MAVS or TRIF siRNA (or negative control siRNA) for 48 hours. Treat with Asunaprevir for 24 hours, then perform immunoblotting or qPCR to assess the role of MAVS/TRIF in antiviral activity[1]
5. Immunofluorescence assay: Collect supernatants from Asunaprevir-treated DENV/HCV-infected Huh 7.5.1 cells. Infect naive Huh 7.5.1 cells with the supernatants for 48 hours. Fix cells, stain with anti-DENV-NS3 or anti-HCV core antibody and DAPI, and observe fluorescence signals to evaluate viral production[1]
6. DENV NS2B3 protease activity assay: Transfect Huh 7.5.1 cells with pcDNA3.1 Flag-DV2 NS2B3 (or protease-dead mutant) for 24 hours. Treat with Asunaprevir for 24 hours, then perform immunoblotting with anti-NS3 antibody to assess protease activity[1]
1. HCV replicon inhibition assay: Culture HCV replicon cells (genotypes 1-4) in the presence of serial concentrations of Asunaprevir. Incubate for a specified period, then measure HCV RNA levels or reporter gene activity to calculate EC₅₀ values[2]
2. Combination assay: Treat HCV replicon cells with Asunaprevir combined with alfa interferon, ribavirin, NS5A inhibitors, or NS5B inhibitors. Evaluate the combination effect using appropriate methods to determine additive or synergistic activity[2]
3. Selectivity assay: Treat various RNA virus-infected cells with Asunaprevir (up to 12 μM) and measure viral replication to assess selectivity[2]
1. HCV replicon combination assay: Culture genotype 1b HCV replicon cells with Asunaprevir (NS3 inhibitor) combined with daclatasvir (NS5A inhibitor) or BMS-791325 (NS5B inhibitor) at 5×, 10×, 30× EC₅₀. Incubate for 4 weeks, stain with crystal violet to visualize colonies and evaluate replicon clearance[3]
2. Resistance selection assay: Culture HCV replicon cells with dual or triple combinations of inhibitors to select resistant colonies. Analyze genotypic changes in NS3, NS5A, or NS5B genes[3]
1. HCV replicon inhibition assay: Culture HCV replicon cells (genotypes 1-4) in the presence of serial concentrations of Asunaprevir. Incubate for a specified period, then measure HCV RNA levels or reporter gene activity to calculate EC₅₀ values[4]
2. Combination assay: Treat HCV replicon cells with Asunaprevir combined with alfa interferon, ribavirin, NS5A inhibitors, or NS5B inhibitors. Evaluate the combination effect using appropriate methods to determine additive or synergistic activity[4]
3. Selectivity assay: Treat various RNA virus-infected cells with Asunaprevir (up to 12 μM) and measure viral replication to assess selectivity[4]
1. HCV replicon resistance selection: Culture genotype 1a and 1b HCV replicon cells with Asunaprevir at different concentrations. Select resistant colonies/populations, sequence NS3 protease gene, and identify resistance-associated substitutions[5]
2. Transient-transfection susceptibility assay: Clone resistant NS3 protease sequences into HCV replicon plasmids. Transfect cells, treat with Asunaprevir, and measure EC₅₀ to determine fold resistance[5]
3. Replication capacity assay: Compare the replication capacity of resistant replicons with wild-type replicons by measuring HCV RNA levels over time[5]

In vivo exposure studies.[2]
\n\\nThe amorphous free acid form of Asunaprevir (ASV) was used in all studies. Plasma and tissue exposures were characterized in male FVB mice (20 to 25 g), male Sprague-Dawley rats (300 to 350 g; Hilltop Lab Animals, Scottsdale, PA) with indwelling cannulas implanted in the jugular vein or common bile duct, male beagles (ca. 9 to 12 kg) bearing vascular access ports, and male cynomolgus monkeys bearing vascular access ports (3.1 to 5.7 kg). All animal procedures were performed under protocols approved by the Institutional Animal Care and Use Committee of the test facility. In all studies, coagulated blood samples taken after dosing were centrifuged at 4°C (1,500 to 2,000 × g); plasma samples were stored at −20°C until analyzed. Tissue samples were rinsed, blotted dry, weighed, and stored frozen. ASV in plasma and tissue samples was analyzed by LC-MS/MS. The lower limit of quantification (LLOQ) of ASV was 1 nM in plasma samples from all species, 5 nM in mouse and rat tissues, and 50 nM in dog and monkey tissues.[2]
\n\\n\\nMice (n = 9 per group; overnight fast) received ASV by oral gavage (5 mg/kg; vehicle of PEG-400–ethanol, 9:1). Blood samples (∼0.2 ml) were obtained by retro-orbital bleeding at 0.25, 0.5, 1, 3, 6, 8, and 24 h after dosing. Within each group, three animals were bled at 0.25, 3, and 24 h, three at 0.5 and 6 h, and three at 1 and 8 h, resulting in a composite pharmacokinetic profile. Livers and brains were also removed from mice at the terminal sampling points.[2]
\n\\n\\nRats (n = 3 per group; overnight fast) received ASV (amorphous free acid) by oral gavage (3, 5, 10, and 15 mg/kg) in PEG-400–ethanol (9:1). Serial blood samples (∼0.3 ml) were obtained from the jugular vein predosing (0 h) and at 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 24, and 48 h postdosing. To assess tissue exposure, rats were orally administered ASV (5 or 15 mg/kg, same vehicle as above), and blood, liver, and heart samples from two rats/group were obtained at 0.17, 0.5, 1, 2, 4, 6, 8, 24, 48, and 72 h after dosing.[2]
\n\\n\\nDogs (n = 3; overnight fast) were administered ASV by oral gavage at 3 or 6 mg/kg (3 mg/ml in 85% PEG-400–15% water). Serial blood samples were collected from vascular access ports at 0.08, 0.167, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 24, and 48 h postdose. To assess tissue exposure, six male dogs (8.4 to 12.5 kg) were orally administered ASV (6 mg/kg), and blood, liver, and spleen samples were obtained from one dog each at 1, 3, 7, 24, 48, and 72 h postdosing.[2]
\n\\n\\nMonkeys (n = 3 males; overnight fast) were administered ASV by oral gavage at 3 mg/kg (3 mg/ml in 85% PEG-400–15% water). Blood samples were collected at 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, and 24 h postdose. To assess tissue exposure, three male and six female cynomolgus monkeys (2 to 5 kg) were orally administered ASV (10 mg/kg), and blood, liver, and spleen samples were obtained from one male each at 2, 8, and 24 h postdosing and from one female each at 0.5, 2, 4, 8, 24, and 30 h postdosing.[2]

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\\n\\nAsunaprevir (ASV) is administered orally to mice (n = 9 per group; overnight fast; vehicle: PEG-400-ethanol, 9:1). Retro-orbital bleeding is used to obtain blood samples (-0.2 mL) at 0.25, 0.5, 1, 3, 6, 8, and 24 hours following dosing. To create a composite pharmacokinetic profile, three animals in each group are bled at 0.25, 3, and 24 hours, three at 0.5 and 6 hours, and three at 1 and 8 hours. At the terminal sampling points, mice are also deprived of their livers and brains.Amorphous free acid (ASV) is given orally to rats (n=3 per group; overnight fast) at doses of 3, 5, 10, and 15 mg/kg in PEG-400-ethanol (9:1). The jugular vein is used to draw serial blood samples (-0.3 mL) prior to dosing (0 h) and at 0.25, 0.5, 0.75, 1, 2, 4, 6, 8, 24, and 48 h after dosing. Rats are given ASV (5 or 15 mg/kg, same vehicle as above) orally in order to measure tissue exposure. Samples of the rats' liver, heart, and blood are taken at intervals of 0.17, 0.5, 1, 2, 4, 6, 8, 24, 48, and 72 hours following dosing.

Absorption, Distribution and Excretion
In preclinical studies, Asunaprevir showed a high area under the curve (AUC) of hepatic plasma concentration to plasma concentration. It is rapidly absorbed within 30 minutes after administration. Clinical pharmacokinetic studies showed a time to peak concentration (Tmax) of 2–4 hours. Its pharmacokinetic characteristics are dose-proportional; at a 100 mg dose, the steady-state peak plasma concentration (Cmax) and AUC are 572 ng/mL and 1887 ng·h/mL, respectively. The absolute bioavailability is 9.3%. Food can increase the absorption of Asunaprevir. Asunaprevir is primarily excreted in feces. Of the administered dose, 84% is excreted in feces primarily as metabolites, and less than 1% is excreted in urine as metabolites. Only 7.5% of the administered dose is recovered as unmetabolized Asunaprevir in feces. The steady-state volume of distribution is 194 liters. Clinical pharmacokinetic studies showed a mean oral clearance of 302-491 L/h. Metabolites/Metabolites Asunaprevir is metabolized in the liver. Its metabolism primarily occurs via CYP3A-mediated oxidation. Asunaprevir appears to have a weak endogenous metabolism, with only about 5% of the administered dose converted to metabolites during circulation. Asunaprevir metabolites are formed via mono- and di-oxidation, N-dealkylation, isoquinoline ring demethylation, and O-demethylation. All metabolic reactions produce approximately 15 metabolites. Studies have shown that the major metabolic activities are carried out by CYP3A4 and CYP3A5, while CYP2A6, CYP2B6, CYP2C9, CYP2C19, and CYP2D6 also possess some activity. Biological Half-Life Clinical pharmacokinetic studies indicated a mean terminal half-life of 15-20 hours.

Protein Binding
Asunaprevir has a very high protein binding rate, exceeding 99% of the administered dose regardless of dosage. In vitro human Caco-2 cell studies have shown that Asunaprevir is a substrate of P-gp, OATP1B1, and OATP2B1.

[1]. Front Microbiol. 2017; 8: 668

[2]. Antimicrob Agents Chemother.2012 Oct;56(10):5387-96.

[3]. Antimicrob Agents Chemother. 2012 Oct;56(10):5230-9.

[4]. Antimicrob Agents Chemother. 2012 Oct;56(10):5387-96.

[5]. Antimicrob Agents Chemother. 2012 Jul;56(7):3670-81.

Asunaprevir is an oligopeptide. Asunaprevir, also known as BMS-650032, is a potent inhibitor of the hepatitis C virus (HCV) NS3 protease. Studies have shown that Asunaprevir has extremely high efficacy in patients with chronic HCV genotype 1b infection treated in combination with daclatasvir. Developed by Bristol-Myers Squibb Canada, it was approved by Health Canada on April 22, 2016. However, a year later, on October 16, 2017, the commercialization application for Asunaprevir was withdrawn. Asunaprevir is an orally bioavailable NS3 nonstructural protein inhibitor with potential anti-HCV activity. After administration, Asunaprevir binds to the active site of HCV NS3, preventing NS3 protease-mediated maturation of the polyprotein. This interferes with the processing of viral proteins required for HCV replication. NS3 is a serine protease essential for the proteolytic cleavage of HCV polymers and plays a crucial role in HCV viral RNA replication. HCV is a small, enveloped, single-stranded RNA virus belonging to the Flaviviridae family. Drug Indications Asunaprevir, used in combination with other drugs, is indicated for the treatment of adult patients with chronic hepatitis C who are infected with hepatitis C virus type 1 or 4 and have compensated cirrhosis. Hepatitis C is a liver disease caused by the hepatitis C virus. Chronic hepatitis C accounts for 60-80% of all cases, with an approximately 15-30% risk of developing cirrhosis within 20 years. In the United States, hepatitis C virus type 1 is the most common and most difficult to treat type. Treatment of Chronic Hepatitis C Mechanism of Action Asunaprevir is a highly potent HCV NS3 protease inhibitor. The HCV genome has a positive-strand polarity, allowing it to be translated into proteins within host cells without further transformation. However, the resulting proteins need to be cleaved into individual proteins by the NS3 protease to exert their enzymatic activity or structural function. Therefore, since NS3 is crucial for viral replication, the inhibitory effect of Asunaprevir gives it potent antiviral activity.

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Pharmacodynamics
In vitro studies have shown that Asunaprevir has significant antiviral activity in HCV replicon cell systems, with EC50 values of 4 nM and 1 nM against HCV genotypes 1a and 1b, respectively. These studies indicate that Asunaprevir has limited activity against genotypes 2 and 3. This characteristic makes Asunaprevir a highly selective anti-HCV drug, ineffective against viruses closely related to HCV. Asunaprevir can significantly reduce HCV RNA levels in patients infected with HCV genotype 1. Clinical studies have shown that Asunaprevir is well-tolerated, with a mean maximum reduction in HCV RNA levels from baseline of approximately 2.87 log10 IU/ml. Clinical studies of Asunaprevir monotherapy showed that when the dose was increased from 10 mg to 600 mg, the mean maximum reduction in HCV RNA was in the range of 0.28-2.87 log10 IU/ml. When Asunaprevir was used in combination therapy, 83-92% of patients achieved sustained virological response (no viremia 24 weeks after the end of treatment). Asunaprevir is a direct-acting antiviral (DAA) originally developed as an HCV NS3 protease inhibitor. It activates MAVS-dependent innate immunity in hepatocellular carcinoma cells, thereby exerting its anti-HCV and DENV activity independently of direct inhibition of HCV protease [1]. Asunaprevir is an HCV NS3 protease inhibitor that is effective in patients with chronic HCV genotype 1 infection when used in combination with α-interferon and/or daclatasvir. Its hepatic tropism ensures a high liver exposure, thus supporting its anti-HCV efficacy [2]. Asunaprevir is part of HCV combined DAA therapy. Combination with NS5A or NS5B inhibitors reduces the development of resistance and enhances replicon inhibition, thus addressing the resistance problem caused by monotherapy [3]. Asunaprevir is an HCV NS3 protease inhibitor that is effective in patients with chronic HCV genotype 1 infection when used in combination with alpha interferon and/or daclatasvir. Its hepatic tropism ensures high liver exposure, thus supporting its anti-HCV efficacy [4]. Asunaprevir has shown efficacy in patients with HCV genotype 1b infection in dual therapy with daclatasvir. Resistance-related amino acid substitutions vary by HCV genotype and there is cross-sensitivity to NS5A inhibitors, which supports its use in combination therapy [5].

C35H46CLN5O9S

748.29

747.27

C, 56.18; H, 6.20; Cl, 4.74; N, 9.36; O, 19.24; S, 4.28

630420-16-5

16076883

White to off-white solid powder

1.4±0.1 g/cm3

145-155 ºC

1.616

4.02

3

10

14

51

1470

5

ClC1C([H])=C([H])C2C(=C([H])N=C(C=2C=1[H])O[C@@]1([H])C([H])([H])N(C([C@]([H])(C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])N([H])C(=O)OC(C([H])([H])[H])(C([H])([H])[H])C([H])([H])[H])=O)[C@@]([H])(C1([H])[H])C(N([H])[C@]1(C(N([H])S(C2([H])C([H])([H])C2([H])[H])(=O)=O)=O)C([H])([H])[C@@]1([H])C([H])=C([H])[H])=O)OC([H])([H])[H]

XRWSZZJLZRKHHD-WVWIJVSJSA-N

InChI=1S/C35H46ClN5O9S/c1-9-19-16-35(19,31(44)40-51(46,47)22-11-12-22)39-28(42)25-15-21(49-29-24-14-20(36)10-13-23(24)26(48-8)17-37-29)18-41(25)30(43)27(33(2,3)4)38-32(45)50-34(5,6)7/h9-10,13-14,17,19,21-22,25,27H,1,11-12,15-16,18H2,2-8H3,(H,38,45)(H,39,42)(H,40,44)/t19-,21-,25+,27-,35-/m1/s1

1,1-dimethylethyl ((1S)-1-{((2S,4R)-4-(7-chloro-4-methoxyisoquinolin-1-yloxy)-2-({(1R,2S)-1-((cyclopropylsulfonyl)carbamoyl)-2-ethenylcyclopropyl}carbamoyl) pyrrolidin-1-yl)carbonyl}-2,2-dimethylpropyl)carbamate

BMS-650032; BMS 650032; BMS650032; S9X0KRJ00S; 1,1-dimethylethyl ((1S)-1-{((2S,4R)-4-(7-chloro-4-methoxyisoquinolin-1-yloxy)-2-({(1R,2S)-1-((cyclopropylsulfonyl)carbamoyl)-2-ethenylcyclopropyl}carbamoyl) pyrrolidin-1-yl)carbonyl}-2,2-dimethylpropyl)carbamate; trade name in Japan and Russia: Sunvepra

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 mg/mL ( ~133.63 mM )
Ethanol : 20~100 mg/mL(~26.73 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.34 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 (3.34 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 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 (3.34 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: 2.5 mg/mL (3.34 mM) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 5: 2 mg/mL (2.67 mM) in 10% EtOH + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear EtOH 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 6: ≥ 2 mg/mL (2.67 mM) (saturation unknown) in 10% EtOH + 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 20.0 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 7: 10% DMSO+40% PEG300+5% Tween-80+45% Saline: ≥ 2.5 mg/mL (3.34 mM)

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