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)-N′-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 (koff), 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 koff 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 +/– hypertonic saline for CF lung disease.
Footnotes
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M.R.J. and R.C.B. contributed equally to this work.
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Parts of this work have appeared in abstract form as follows: Hirsh AJ, St George J, Zamurs A, Boucher R, and Johnson RM (2006) Combination therapy using a novel epithelial sodium channel blocker and hypertonic saline (abstract 256); 2006 Nov; Denver, CO. Cystic Fibrosis Foundation, Bethesda, MD.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.107.130443.
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ABBREVIATIONS: CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; ENaC, epithelial sodium channel(s); HS, hypertonic saline; MC, mucociliary clearance; ASL, airway surface liquid; NHE, sodium-proton exchanger; CBE, canine bronchial epithelia; HBE, human bronchial epithelia; KRB, Krebs-Ringer bicarbonate; Rt, transepithelial resistance; Isc, short-circuit current; DMSO, dimethyl sulfoxide; MBS, modified Barth's solution; HTBE, human tracheobronchial epithelia; ETT, endotracheal tube; ALI, air-liquid interface; 99mTc-SC, 99mTechnicium-sulfur colloid; amil, amiloride; frct rcvry, fractional recovery; INS37217, denufosol, [P1-(uridine 5′)-P4-(2′-deoxycytidine 5′)tetraphosphate, tetrasodium salt]; MOLI1901, duramycin.
- Received August 17, 2007.
- Accepted January 23, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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