Ivacaftor (VX-770)

Ivacaftor (VX-770) (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, this compound increases the open probability (P_o) of G551D-, F508del-, and wild-type CFTR by ~6-fold, ~5-fold and ~2-fold, respectively, indicating that it 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 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, it 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, this compound inhibits excessive ENaC-mediated Na⁺ and fluid absorption with an IC50 of 43 nM, and decreases the response, resulting in an increase in the surface fluid and cilia beat frequency (CBF) in G551D/F508del HBE. [1]

• Ussing-kameropnames

  Het effect van Ivacaftor (VX-770) op CFTR-gemedieerde Cl^- secretie wordt gekarakteriseerd door de CFTR-gemedieerde I_T te meten in kamers met behulp van recombinante Fisher-ratten schildklier (FRT) cellen die G551D of F508del CFTR tot expressie brengen. Cellen worden gekweekt op Costar Snapwell celkweekinzetstukken die op 37 °C worden gehouden vóór de opname. De celkweekinzetstukken worden gemonteerd in een Ussing-kamer om I_T op te nemen in de voltage-clamp modus (V_hold = 0 mV). Voor FRT-cellen wordt een basolateraal tot apicaal Cl^- gradiënt gecreëerd aan de basolaterale membraan. De basolaterale badoplossing bevat 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

• Cellijnen

  FRT and NIH 3T3 cells

• Concentraties

  100/25 nM

• Incubatietijd

  24 h

• Methode

  Cells were treated with various concentrations of Ivacaftor (VX-770).

• Dierlijke modellen

  Male Sprague-Dawley rats

• Doseringen

  3 mg/kg

• Toediening

  i.v.