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. 2016 Feb;173(3):459-70.
doi: 10.1111/bph.13365. Epub 2016 Jan 13.

Low Free Drug Concentration Prevents Inhibition of F508del CFTR Functional Expression by the Potentiator VX-770 (Ivacaftor)

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Free PMC article

Low Free Drug Concentration Prevents Inhibition of F508del CFTR Functional Expression by the Potentiator VX-770 (Ivacaftor)

Elizabeth Matthes et al. Br J Pharmacol. .
Free PMC article

Abstract

Background and purpose: The most common cystic fibrosis (CF) mutation F508del inhibits the gating and surface expression of CFTR, a plasma membrane anion channel. Optimal pharmacotherapies will probably require both a 'potentiator' to increase channel open probability and a 'corrector' that improves folding and trafficking of the mutant protein and its stability at the cell surface. Interaction between CF drugs has been reported but remains poorly understood.

Experimental approach: CF bronchial epithelial cells were exposed to the corrector VX-809 (lumacaftor) and potentiator VX-770 (ivacaftor) individually or in combination. Functional expression of CFTR was assayed as the forskolin-stimulated short-circuit current (Isc ) across airway epithelial monolayers expressing F508del CFTR.

Key results: The potentiated Isc response during forskolin stimulation was increased sixfold after pretreatment with VX-809 alone and reached ~11% that measured across non-CF monolayers. VX-770 (100 nM) and genistein (50 μM) caused similar levels of potentiation, which were not additive and were abolished by the CFTR inhibitor CFTRinh -172. The unbound fraction of VX-770 in plasma was 0.13 ± 0.04%, which together with previous measurements in patients given 250 mg p.o. twice daily, suggests a peak free plasma concentration of 1.5-8.5 nM. Chronic exposure to high VX-770 concentrations (>1 μM) inhibited functional correction by VX-809 but not in the presence of physiological protein levels (20-40 mg·mL(-1) ). Chronic exposure to a low concentration of VX-770 (100 nM) together with VX-809 (1 μM) also did not reduce the forskolin-stimulated Isc , relative to cells chronically exposed to VX-809 alone, provided it was assayed acutely using the same, clinically relevant concentration of potentiator.

Conclusions and implications: Chronic exposure to clinically relevant concentrations of VX-770 did not reduce F508del CFTR function. Therapeutic benefit of VX-770 + VX-809 (Orkambi) is probably limited by the efficacy of VX-809 rather than by inhibition by VX-770.

Figures

Figure 1
Figure 1
Rescue and potentiation of F508del CFTR currents in well‐differentiated primary HBE cells. (A) Protocol used to measure functional expression. (B) Representative recordings of Isc responses to sequential Fsk (10 μM), Gst (50 μM) and CFTRinh‐172 (172, 10 μM) after 24 h pretreatment with vehicle (0.1% DMSO) or VX‐809 (1 or 5 μM). (C) Maximum activation of Isc by Fsk + Gst after pretreatment with 1 or 5 μM VX‐809. (D) Representative Isc responses to Fsk + Gst and Fsk + VX‐770. (E) Summary of Fsk responses after potentiation by Gst (50 μM) or VX‐770 (100 nM). Means ± SEM, n = 3–4, **P < 0.01, ***P < 0.001, # not different P > 0.05.
Figure 2
Figure 2
Effect of chronic VX‐770 exposure on the maximum functional response. (A) Well‐differentiated primary HBE cells were pretreated with VX‐809 (1 μM) alone or in combination with VX‐770 (100 nM), then stimulated by Fsk (10 μM) and acutely exposed to the potentiators VX‐770 (100 nM) or Gst (50 μM). (B) Summary showing there was no difference in the maximum activation when cells were pretreated with corrector alone for 24 h followed by acute potentiation with 100 nM VX‐770 or with corrector +100 nM VX‐770 for 24 h. (C) Concentration dependence of potentiation by chronic VX‐770 assayed as the Fsk response after 24 h pretreatment with corrector and different concentrations of VX‐770 (1 nM–10 μM). Note inhibition by the highest concentration of VX‐770 (10 μM). Means ± SEM, n = 3–4, *P < 0.05, **P < 0.01, ***P < 0.001, # not different P > 0.05.
Figure 3
Figure 3
Robust functional correction by VX‐809 in the presence of 100 nM VX‐770. Experiments were performed using well‐differentiated primary HBE cells from three patients, CFBE, and VX‐770 from two sources. (A) Responses to Fsk and sequential Fsk + Gst measured using cells from three different patients following 24 h pretreatment with VX‐809 alone or with VX‐809 + VX‐770 (100 nM). (B) Fsk and Fsk + Gst induced small currents after cells had been pretreated for 24 h with 100 nM VX‐770 from GSK (VX‐770‐GSK) or SelleckChem (−VX‐770‐SC) or with VX‐809 alone, which were not significantly greater than the response to vehicle (0.1% DMSO, # not different P > 0.05), although Gst caused significant potentiation after VX‐809 alone (**P < 0.01). Pretreating cells with VX‐809 + VX‐770 from either source for 24 h resulted in larger Fsk‐stimulated currents compared with VX‐809 alone (***P < 0.001), which were not further increased by Gst. (C) Protocol used previously to demonstrate inhibition of functional expression by VX‐770 (Veit et al., 2014). Cells were pretreated with VX‐809 (1 μM) alone or in combination with VX‐770 (100 nM) for 24 h. Functional expression was assayed by acutely adding 10 μM VX‐770 and then Fsk to maximally stimulate rescued F508del CFTR. (D) Maximum currents induced by acute 10 μM VX‐770 + 10 μM Fsk were reduced by 40% when cells were pretreated with corrector + VX‐700 (100 nM). Means ± SEM, n = 3, *P < 0.05, ***P < 0.001. (E) A similar 30% inhibition was observed in CFBE cells using this protocol. Means ± SEM, n = 9, *P < 0.05, **P < 0.01, ***P < 0.001, # not different P > 0.05.
Figure 4
Figure 4
Inhibition of functional expression by 5 μM VX‐770 is prevented in the presence of albumin. (A) Concentration‐dependent inhibition by VX‐770. Cells were pretreated with moderate concentrations of VX‐770 and VX‐809 (100 nM and 1 μM, respectively) or high concentrations (5 and 5 μM, respectively), then stimulated with Fsk. Gst was used as a potentiator in the absence of VX‐770. Currents were enhanced by pretreatment with corrector and 100 nM VX‐770 but inhibited by pretreatment with corrector and 5 μM VX‐770. (B) Adding 40 mg·mL−1 BSA during the 24 h pretreatment period to mimic plasma concentration abolished the inhibitory effect of 5 μM VX‐770. Means ± SEM, n = 5‐6, *P < 0.05, **P < 0.01, # not different P > 0.05. (C, D) Inhibition by chronic 5 μM VX‐770 was also prevented by including 20 mg·mL−1 BSA during the pretreatment period to mimic interstitial protein. Means ± SEM, n = 3, ***P < 0.001, # not different P > 0.05.
Figure 5
Figure 5
Concentration dependence of potentiation by VX‐770 in CFBE cells. (A) Acute potentiation of Fsk‐stimulated current following rescue of F508del CFTR by 24 h pretreatment with 1 μM VX‐809. (B) Calculation of the EC50 for acute potentiation by VX‐770. Data shown are means ± SEM, n = 3.
Figure 6
Figure 6
Concentration‐dependent potentiation of rescued F508del CFTR current in CFBE cells by chronic (24 h) pretreatment with VX‐770. (A) Effect of prolonged exposure to low concentrations (0.03 pM–30 nM VX‐770). Fsk‐stimulated currents are shown as well as total activated currents after addition of 50 uM Gst. (B) Calculation of the EC50 for chronic potentiation by VX‐770 based on Fsk responses after 24 h pretreatment with low concentrations of VX‐770. (C, D) Same as panels A and B but using higher concentrations of VX‐770 (0.1 nM–1 μM).

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