Ivacaftor (VX-770)

Ivacaftor (10 μM) significantly increases the forskolin-stimulated Cl^- secretion (I_T) by ~4-fold with an EC50 of 100 nM in the recombinant Fisher rat thyroid (FRT) cells expressing G551D gating mutation of CFTR, and by ~6-fold with an EC50 of 25 nM in the recombinant cells expressing temperature-corrected F508del processing mutation of CFTR. Consistent with the increases in the forskolin-stimulated I_T, Ivacaftor (10 μM) increases the open probability (P_o) of G551D-, F508del-, and wild-type CFTR by ~6-fold, ~5-fold and ~2-fold, respectively, indicating that Ivacaftor acts directly on CFTR to increase its gating activity. In primary cultured human CF bronchial epithelia (HBE) carrying the G551D and F508del CFTR mutations, Ivacaftor (10 μM) potently increases the forskolin-stimulated I_T by ~10-fold from 5% to a maximum level of 48% of that measured in non-CF HBE, with an EC50 of 236 nM displaying ~70-fold more potency compared with the commonly used CFTR potentiator genistein, which has an EC50 of 16 μM. In HBE with F508del homozygous CFTR, Ivacaftor causes a significant increase in the forskolin-stimulated I_T with an EC50 of 22 nM, to a less extent from 4% to 16% of non-CF HBE compared with the effect in G551D/F508del HBE. Due to CFTR potentiation, Ivacaftor inhibits excessive ENaC-mediated Na⁺ and fluid absorption with an IC50 of 43 nM, and decreases the amiloride response, resulting in an increase in the surface fluid and cilia beat frequency (CBF) in G551D/F508del HBE. ^[1]

• Ussing Chamber Recordings

  The effect of Ivacaftor on CFTR-mediated Cl^- secretion is characterized by measuring the CFTR-mediated I_T in chambers using recombinant Fisher rat thyroid (FRT) cells expressing G551D, or F508del CFTR. Cells are grown on Costar Snapwell cell culture inserts maintained at 37 °C before recording. The cell culture inserts are mounted into an Ussing chamber to record I_T in the voltage-clamp mode (V_hold = 0 mV). For FRT cells, the basolateral membrane is a basolateral to apical Cl^- gradient is established. The basolateral bath solution contains 135 mM NaCl, 1.2 mM CaCl₂, 1.2 mM MgCl₂, 2.4 mM K₂HPO₄, 0.6 mM KHPO₄, 10 mM N-2-hydroxyethylpiperazine-N

• Cell lines

  FRT and NIH 3T3 cells

• Concentrations

  100/25 nM

• Incubation Time

  24 h

• Method

  Cells were treated with various concentrations of VX-770.

• Animal Models

  Male Sprague-Dawley rats

• Dosages

  3 mg/kg

• Administration

  i.v.