ENaC Inhibitor
BI-8668
BI-8668 is a highly potent and well characterized epithelial sodium channel (ENaC) inhibitor, being structurally distinct from amiloride derived compounds. It possesses a high aqueous solubility, a high microsomal and hepatocyte stability, and a moderate Caco permeability. It is suitable for in vitro and in vivo use. The structurally analogous BI-0377 can be used as a negative control.
More information
The epithelial Na+ channel ENaC is expressed at the apical surface of epithelia of the lung, colon, kidney, salivary gland ducts, and sweat glands1. ENaC mediates the diffusion of luminal sodium and water across the apical membrane of epithelial cells. It plays an essential role in electrolyte and blood pressure homeostasis, as well as in airway surface fluid homeostasis, which is important for proper clearance of mucus. Further it controls the reabsorption of sodium in the kidney, colon, lungs, and eccrine sweat glands2.
The currently solved cryo-EM structure shows that ENaC is a heterotrimeric transmembrane protein composed of α, β, and γ-subunits that form a pore with high selectivity for Na+ and Li+ over K+. Each subunit is encoded by a different gene, namely SCNN1A, SCNN1B, and SCNN1G3.
In cystic fibrosis airways, the lack of cystic fibrosis transmembrane conductance regulator CFTR and increased ENaC activity lead to mucus dehydration that causes mucus obstruction, neutrophilic infiltration, and chronic bacterial infection.
ENaC inhibitors are active in cellular water resorption assays due to their mode of action. in vivo effects (rat lung water resorption models or mucociliary clearance model (MCC) in sheep) however, are only seen with suitably optimized compounds4.
Suitable PK properties are crucial to achieve long duration of action and to avoid renal side effects, also upon inhalative administration. ENaC inhibition is generally seen to be a potential treatment of cystic fibrosis5,6.
3D structure of ENaC as revealed by cryo-electron microscopy (PDB code: 6BQN; transmembrane helices shown as dashed lines as these were not fully resolved in the structure determination.3
BI-8668 effectively inhibits the Na+ current on human airway epithelium in an Ussing Chamber setup with an IC50 of 17 nM. Further, BI-8668 displays 81% inhibition in an M-1 water resorption assay at 3 µM.
| Probe name / negative control | BI-8668 | BI-0337 |
| MW [Da]a | 523.5 | 503.1 |
| Inhibition of M-1 water resorption (%Inhib) @3 µM [%]b | 81 | n.a. |
| Ussing chamber(IC50) [nM]c | 17 | >10.000 |
a For the salt form you will get, please refer to the label on the vial and for the molecular weight of the salt, please refer totheFAQ
bMeasurement of ENaC-mediated water permeability through cell monolayers of the cell line M-1 (M-1 cells: mouse kidney tubules cells) and the blocking capacity of ENaC-inhibitors. The transported volume will be determined with tritiated water. A potent ENaC-inhibitor results in reduced water transport leading to more remaining liquid in the apical (upper) compartment of the cell layer.
cInhibition of Na+ current by Ussing Chamber - MucilAir™ P0: IC50 were derived form a 5-steps concentration curve. (1 nM -> 10,000 nM; n=6)
in vivo DMPK and CMC parameters for BI-8668 and the negative control BI-0337 are tabulated below. BI-8668 possesses a high aqueous solubility, a high microsomal and hepatocyte stability and a moderate Caco permeability. It is devoid of cytochrome P450 inhibition and shows low plasma protein binding.
| Probe name / negative control | BI-8668 | BI-0337 |
| logD pH 2 / pH 7.4 / pH 11 | -0.5/ 0.2/ 0.58 | -0.8/ n.a. / n.a. |
| Solubility in PBS pH 7.4 [µg/ml] | 110 | 81 |
| Caco-2 permeability AB @ pH 7.4 [*10-6 cm/s] | <0.3 | 0.1 |
| Caco-2 efflux ratio | 1.2 | 0.9 |
| Microsomal stability (human/mouse/rat) [% QH] | <23 / <23 / <22 | <23 / <23 / <22 |
| Hepatocyte stability (human/mouse/rat) [% QH] | 23.5 / <12 / 19.5 | <4 / <12 / 4 |
| Plasma Protein Binding (human/mouse/rat) [%] | 38 / 43 / 29 | n.a. / 24 / 20 |
| CYP 3A4 (IC50) [µM] | >50 | >50 |
| CYP 2C8 (IC50) [µM] | >50 | >50 |
| CYP 2C9 (IC50) [µM] | >50 | >50 |
| CYP 2C19 (IC50) [µM] | >50 | >50 |
| CYP 2D6 (IC50) [µM] | >50 | n.d. |
in vivo DMPK parameters for BI-8668 are tabulated below.
| Probe name | BI-8668 |
| Clearance (mouse,rat) [ml/(min*kg)] | 46 / 59 |
| Mean residence time after i.v. doseRT (mouse, rat) [h] | 0.39 / 0.17 |
| Vss (mouse,rat) [L/kg] | 1.10 / 0.58 |
| tmax (mouse,rat) [h] | - / 0.1 |
| Cmax (mouse,rat) [nM] | -/ 34 |
ai.v. dose: 0.3 mg/kg
bi.t. dose: 0.3 mg/kg
BI-8668 at 3 µg/kg displays up to 33% inhibition of fluid absorption compared to control animals in an airway fluid absorption assay in wistar rats.
| Probe name | BI-8668 |
| Inhibition of airway fluid absorption (%Inhib) @ 0.3 µg/kg [%]a | 27 |
| Inhibition of airway fluid absorption (%Inhib) @ 3.0 µg/kg [%]a | 33 |
a Ringer Lactat solution (control) or test solution (0.1mg/kg cpd) was instilled into the trachea of male wistar rats. After 3 hours, the absorption of fluid from the lungs was determined by measuring difference between control and compound treated lungs (weight of both lungs lobes). Concomitantly the serum levels of aldosterone were determined as a measure of systemic ENaC inhibition. The compound effect was expressed in percent inhibition of fluid absorption compared to control animals.
in vivo aldosterone stimulation in rat airway fluid absorption assay (0.3 µg/kg and 3 µg/kg dosing) after 3 h is below 50%, indicating that there is no relevant ENaC inhibition in kidney.
The molecule BI-0337 is structurally very close to BI-8668. Nonetheless, it offers a >500-fold lower potency in the Ussing chamber assay (MucilAir™) on human airway epithelium and therefore can be used as an in vitro negative control.
BI-0337 which serves as a negative control
BI-8668 was tested on 50 targets in a selectivity panel and showed ≥1,000-fold selectivity for 47/50 targets (≤ 50% inhibition @ 10 µM). For three panel targets (M3, M2, α1), percent inhibition data indicate at least 50-fold selectivity. The negative control BI-0337 showed more than 50% inhibition @ 10 µM in 4 out of 44 targets.
| SELECTIVITY DATA AVAILABLE | BI-8668 | BI-0337 |
SafetyScreen44™ with kind support of  | Yes | Yes |
| Invitrogen® | No | No |
| DiscoverX® | No | No |
| Dundee | No | No |
Download selectivity data:
BI-8668_selectivityData.xlsx
BI-0337_selectivityData.xlsx
P-552-02 is an ENaC inhibitor based on the amiloride pharmacophore.7
BI-8668 is a potent and well-characterized in vitro and in vivo tool compound, structurally distinct from amiloride derived ENaC inhibitors. A structurally analogous negative control BI-0377 is also available.
Design, synthesis, and structure− activity relationships of novel 2-substituted pyrazinoylguanidine epithelial sodium channel blockers: drugs for cystic fibrosis and chronic bronchitis
Hirsh A. J., Molino B. F., Zhang J., Astakhova N., Geiss W. B., Sargent B. J., Swenson B. D., Usyatinsky A., Wyle M. J., Boucher R. C., Smith R. T., Zamurs A., Johnson M. R.
Journal of Medicinal Chemistry 2006, 49(14), 4098-4115.
When you plan a publication, please use the following acknowledgement:
BI-8668 was kindly provided by Boehringer Ingelheim via its open innovation platform opnMe, available at https://www.opnme.com.