| Protocol | In Vitro | In vitro activity: the antiviral activity of simeprevir (TMC435350) in Huh7-Luc cells is dose dependent, and the EC50 and EC90 values determined for TMC435350 are 8 nM and 24 nM, respectively. Inhibition of TMC435350 on NS3/4A protease is time dependent, and the overall Kis are estimated to be 0.5 nM for genotype 1a and 0.4 nM for genotype 1b, respectively. TMC435350 is a potent inhibitor of HCV NS3/4A protease (Ki=0.36 nM) and viral replication (replicon EC50=7.8 nM). Kinase Assay: the in vitro inhibitory activity of simeprevir against NS3/4A is determined using a fluorescence resonance energy transfer cleavage assay with the RetS1 peptide substrate, derived from the genotype 1a NS4A-4B junction, and bacterially expressed full-length NS3 protease domain, supplemented with an NS4A peptide. Briefly, NS3/4A is preincubated in the presence of TMC435350 for 10 min, and then the RetS1 substrate is added and fluorescence is continuously measured for 20 min (excitation, 355 nm; emission, 500 nm). Cleavage of the substrate is expressed as a percentage of the cleavage seen with the vehicle control. Cell Assay: Huh7-Luc cells are seeded at a density of 2,500 cells/well in a 384-well plate in Dulbecco's modified Eagle's medium plus 10% fetal calf serum and incubated with a range of concentrations of serially diluted simeprevir (TMC435350), in a final DMSO concentration of 0.5% in the absence of G418. After 72 h of incubation, Steady Lite reagent is added in a 1:1 ratio to the medium, and luciferase signal is measured using a ViewLux reader. |
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| In Vivo | In rats, TMC435350 (40 mg/kg, p.o.) is extensively distributed to the liver and intestinal tract (tissue/plasma area under the concentration-time curve ratios of >35), and the absolute bioavailability is 44%. |
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| Animal model | 24 male specific-pathogen-free Sprague-Dawley rats, weighing between 200 and 300 g at the time of dosing, are divided into eight groups of three rats each. |
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| Dosages | 3 mg/kg |
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| Administration | PO; single dosage |
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These protocols are for reference only. InvivoChem does not
independently validate these methods.
Preparing Stock Solutions
| Solvent volume to be added |
Mass (the weight of a compound) |
| Mother liquor concentration |
1mg | 5mg | 10mg | 20mg |
| 1mM | 1.2955 mL | 6.4774 mL | 12.9547 mL | 25.9094 mL |
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| 5mM | 0.2591 mL | 1.2955 mL | 2.5909 mL | 5.1819 mL |
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| 10mM | 0.1295 mL | 0.6477 mL | 1.2955 mL | 2.5909 mL |
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| 20mM | 0.0648 mL | 0.3239 mL | 0.6477 mL | 1.2955 mL |
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Quality Control Documentation
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