NVP-QBE170: an inhaled blocker of the epithelial sodium channel with a reduced potential to induce hyperkalaemia

Abstract

Background and purpose: Inhaled amiloride, a blocker of the epithelial sodium channel (ENaC), enhances mucociliary clearance (MCC) in cystic fibrosis (CF) patients. However, the dose of amiloride is limited by the mechanism-based side effect of hyperkalaemia resulting from renal ENaC blockade. Inhaled ENaC blockers with a reduced potential to induce hyperkalaemia provide a therapeutic strategy to improve mucosal hydration and MCC in the lungs of CF patients. The present study describes the preclinical profile of a novel ENaC blocker, NVP-QBE170, designed for inhaled delivery, with a reduced potential to induce hyperkalaemia.

Experimental approach: The in vitro potency and duration of action of NVP-QBE170 were compared with amiloride and a newer ENaC blocker, P552-02, in primary human bronchial epithelial cells (HBECs) by short-circuit current. In vivo efficacy and safety were assessed in guinea pig (tracheal potential difference/hyperkalaemia), rat (hyperkalaemia) and sheep (MCC).

Key results: In vitro, NVP-QBE170 potently inhibited ENaC function in HBEC and showed a longer duration of action to comparator molecules. In vivo, intratracheal (i.t.) instillation of NVP-QBE170 attenuated ENaC activity in the guinea pig airways with greater potency and duration of action than that of amiloride without inducing hyperkalaemia in either guinea pig or rat. Dry powder inhalation of NVP-QBE170 by conscious sheep increased MCC and was better than inhaled hypertonic saline in terms of efficacy and duration of action.

Conclusions and implications: NVP-QBE170 highlights the potential for inhaled ENaC blockers to exhibit efficacy in the airways with a reduced risk of hyperkalaemia, relative to existing compounds.

Figure 1

Chemical structures of amiloride (A) and the related ENaC blockers P552-02 (B) and NVP-QBE170 (C).

Figure 2

Duration of action of ENaC blockers on primary HBECs. ENaC-mediated I_SC was blocked with the test compounds and recovery of the current was measured during washout of compound from the apical side of the Ussing chamber. A sample raw data trace (A) illustrates the protocol whereby the magnitude of the ENaC-mediated I_SC was first established with amiloride (20 μM) that was then washed out of the chamber. The vertical arrow illustrates the 100% value for ENaC block. A supra-maximal concentration of the test compound (100× IC₅₀) was then added to the apical bath and incubated for either 2 or 10 min (a 2 min incubation is shown). The compound was then removed from the apical bath by constant perfusion and the duration of compound activity is measured as the recovery of the I_SC. Finally, amiloride was reintroduced to the apical bath to confirm that the recovered I_SC was ENaC mediated. Panels (B) and (C) illustrate mean % recovery data ± SEM (n = 6 independent experiments per compound) following either a 2 or 10 min incubation with test compound respectively.

Figure 3

The in vivo effects of ENaC blockers on guinea pig TPD and blood potassium levels following i.t. dosing. TPD and blood potassium levels were measured in anaesthetized guinea pigs at 4 h after i.t. dosing with test compounds or vehicle. Absolute values for TPD and blood potassium are shown for amiloride (A and D), NVP-QBE170 (B and E), and P552-02 (C and F). +ve refers to the positive control compound in the study (P552-02, 2000 μg·kg⁻¹). * P < 0.05, significantly different from vehicle (compound dose = 0 μg·kg^-1); one-way anova with post hoc Dunnett's test; n = 4–10 animals per group.

Figure 4

The effects of ENaC blockers on blood potassium levels in rats following i.t. dosing. Male Sprague-Dawley rats were dosed with test compounds or vehicle by i.t. administration. In addition to a pre-dose baseline level of blood potassium, samples were taken at 6, 24 and 48 h. The effects of a single dose of amiloride (A), NVP-QBE170 (B) and P552-02 (C) were studied as well as repeated daily dosing of NVP-QBE170 (D) for 10 days. Mean absolute data ± SEM for blood potassium levels are shown. Group sizes of four to six animals were used.

Figure 5

The effects of inhaled NVP-QBE170 and HS on MCC in conscious sheep. Mean retention of ^99mTc labelled sulphur colloid ± SEM was measured by gamma scintigraphy in conscious sheep following administration of either inhaled NVP-QBE170 or HS. The assessment of clearance was started at either 1 h (A) or 4 h (B) after dosing with dry powder NVP-QBE170. The effect of inhaled 7% saline was assessed either immediately after inhalation of the osmolyte or at 4 h after dosing (C). Group sizes of three to five sheep per dose were used. *P < 0.05, significantly different from from vehicle control at either 60 or 120 min after ^99mTc administration.