Pharmacological Properties ofN-(3,5-Diamino-6-chloropyrazine-2-carbonyl)-N′-4-[4-(2,3-dihydroxypropoxy)phenyl]butyl-guanidine Methanesulfonate (552-02), a Novel Epithelial Sodium Channel Blocker with Potential Clinical Efficacy for Cystic Fibrosis Lung Disease

Abstract

Amiloride improves mucociliary clearance (MC) by blocking airway epithelial sodium channels (ENaC) and expanding airway surface liquid (ASL). However, the low potency and rapid absorption of amiloride by airway epithelia translated into a short duration of efficacy as an aerosolized therapy for cystic fibrosis (CF) patients. To improve ENaC blocker CF pharmacotherapy, a more potent and durable ENaC blocker tailored for aerosol delivery was synthesized. Parion compound N-(3,5-diamino-6-chloropyrazine-2-carbonyl)-NЈ-4-[4-(2,3-dihydroxypropoxy)phenyl]butyl-guanidine methanesulfonate (552-02) was tested for potency and reversibility of ENaC block, epithelial absorption and biotransformation, selectivity, durability of ASL expansion under isotonic and hypertonic conditions in canine and human CF bronchial epithelial cells, and drug dissociation on ENaC in Xenopus oocytes. Short-circuit current assessed compound potency and reversibility, patch-clamp recordings of ENaC current assessed drug off-rate (k off ), a gravimetric method and confocal microscopy measured mucosal water retention and ASL height, and drug absorption and biotransformation were assessed using liquid chromatography-mass spectrometry. Amiloride and 552-02 were tested in vivo for MC activity in sheep immediately and 4 to 6 h after aerosol dosing. Compared with amiloride, compound 552-02 was 60 to 100-fold more potent, it was 2 to 5-fold less reversible, it was slower at crossing the epithelium, and it exhibited a 170-fold slower k off value. 552-02 exhibited greater ASL expansion over 8 h in vitro, and it was more effective than amiloride at increasing MC immediately and 4 to 6 h after dosing. When combining hypertonic saline and 552-02, a synergistic effect on ASL expansion was measured in canine or CF bronchial epithelia. In summary, the preclinical data support the clinical use of 552-02 ϩ/Ϫ hypertonic saline for CF lung disease.The pulmonary disease in patients with cystic fibrosis (CF) reflects genetic mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene that produce defective epithelial ion transport. The CF airway epithelial ion transport abnormalities lead to a well described pathophysiological cascade that adversely affects the innate defense Article, publication date, and citation information can be found at

Non-animal models of epithelial barriers (skin, intestine and lung) in research, industrial applications and regulatory toxicology

“…These cells express basal cell markers including cytokeratin 5 and the transcription factor p63 (Hackett et al, 2011a) and have been used as models of basal cells. al., 2010), mucociliary transport (Seagrave et al, 2012), airway toxicology (Mathis et al, 2013;Watson et al, 2010), electrophysiology (Hirsh et al, 2008), and bacterial and viral infection studies (Deng et al, 2014;Mitchell et al, 2011;Palmer et al, 2012;Ren and Daines, 2011;Ren et al, 2012). The MucilAir cultures have been characterized electrophysiologically and are fully functional: the activity of the main epithelial ionic channels, such as CFTR, EnaC, Na/K ATPase, etc.…”

The Amiloride-Sensitive Endothelial Sodium Channel and Vascular Tone

“…9,13 EnNaC may present a specific target for therapeutic intervention if its inhibition can be functionally separated from EpNaC, and thus avoiding or at least minimizing impairment of renal and intestinal K + secretion. Recent developments of novel 2-acyl-amiloride derivatives 18,19 may provide a potential avenue for further development. An interesting analogue would have reasonable bioavailabilty (oral, sublingual, or transdermal), with hepatobiliary rather than renal clearance, and effectiveness as a noncompetitive inhibitor against EnNaC at subnanomolar concentrations.…”

CFTR: cystic fibrosis and beyond

“…At the same time, efforts are ongoing that focus on strategies for durable ENaC inhibition to improve efficacy in chronic CF airways disease. These efforts include: 1) the design of more potent and long-acting ENaC blockers [100,101]; 2) inhibition of ENaCactivating proteases [44,102]; 3) ENaC silencing with specific short interfering RNAs [103]; and 4) highcontent screens to identify novel ENaC regulators that may be druggable in CF lung disease [104]. Since ENaC is also expressed in the kidney, where it plays an important role in electrolyte balance, strategies are warranted that reduce systemic absorption of ENaC inhibitors effectively to prevent renal exposure and electrolyte imbalance including hyperkalaemia [105].…”