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-Kammer-Aufzeichnungen

  Die Wirkung von Ivacaftor (VX-770) auf die CFTR-vermittelte Cl^--Sekretion wird durch Messung der CFTR-vermittelten I_T in Kammern unter Verwendung von rekombinanten Fisher-Ratten-Schilddrüsenzellen (FRT) charakterisiert, die G551D oder F508del CFTR exprimieren. Die Zellen werden auf Costar Snapwell-Zellkultureinsätzen bei 37 °C vor der Aufzeichnung gezüchtet. Die Zellkultureinsätze werden in eine Ussing-Kammer montiert, um I_T im Voltage-Clamp-Modus (V_hold = 0 mV) aufzuzeichnen. Für FRT-Zellen wird eine basolaterale zu apikalen Cl^--Gradienten an der basolateralen Membran etabliert. Die basolaterale Badlösung enthält 135 mM NaCl, 1,2 mM CaCl₂, 1,2 mM MgCl₂, 2,4 mM K₂HPO₄, 0,6 mM KHPO₄, 10 mM N-2-Hydroxyethylpiperazin-N

• Zelllinien

  FRT and NIH 3T3 cells

• Konzentrationen

  100/25 nM

• Inkubationszeit

  24 h

• Methode

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

• Tiermodelle

  Male Sprague-Dawley rats

• Dosierungen

  3 mg/kg

• Verabreichung

  i.v.