Vesatolimod (GS-9620) is an effective and specific orally active agonist of Toll-like receptor 7.

TLR7:291 nM(EC50)

Vesatolimod rapidly internalizes into cells and preferentially localizes to and signals from endo-lysosomal compartments. To test this hypothesis, the kinetics of cellular uptake of the compound in Daudi cells using tritiated Vesatolimod (3H-Vesatolimod) is measured. The kinetics of 3H-Vesatolimod accumulation is rapid, reaching concentration-dependent steady-state equilibrium in approximately thirty minutes. Measured intracellular concentration of 3H-Vesatolimod is 5-fold higher than the extracellular concentration of 3H-Vesatolimod used to treat cells. Increases in intracellular 3H-Vesatolimod concentrations are roughly proportional with increasing concentrations of 3H-Vesatolimod[1].

Administering single oral doses of GS-9620 at 0.3 and 1 mg/kg to uninfected chimpanzees resulted in a dose-dependent increase in serum interferon alpha (IFN-α), various cytokines/chemokines, and activation of interferon-stimulated genes (ISG) in both peripheral blood and liver tissues. Specifically, GS-9620 reached peak plasma concentrations (Cmax) of 3.6±3.5 nM at 0.3 mg/kg (n=3), 36.8±34.5 nM at 1 mg/kg (n=3), and 55.4±81.0 nM at 1 mg/kg (n=4), respectively, with peak serum interferon responses recorded 8 hours after dosing. The induced peak serum levels of IFN-α were measured at 66 pg/mL for the 0.3 mg/kg dose and 479 pg/mL for the 1 mg/kg dose. Treatment with GS-9620 at these doses also elevated ISG transcripts, specifically ISG15, OAS-1, MX1, IP-10 (CXCL10), and I-TAC (CXCL11), in peripheral blood mononuclear cells (PBMC) at the lower dose and in both PBMC and liver at the higher dose.

Biochemical assays: Biochemical assays are performed by a broad-coverage, TR-FRET-based kinase binding assay platform.

GS-9620 is dissolved in DMSO and stored, and then diluted with appropriate medium before use[1]. Daudi cells are incubated for indicated times with varying concentrations [3H]GS-9620 (0.7μCi/mL). Cell associated radioactivity is extracted with ice cold 80% ethanol and measured using liquid scintillation counting. The total amount of GS-9620 in cells is calculated from a calibration curve for GS-9620 mass versus radioactivity. Cell volume is measured[1].