Andrew J. Hirsh

The CF Salt Controversy: In Vivo Observations and Therapeutic Approaches

There is controversy over whether abnormalities in the salt concentration or volume of airway surface liquid (ASL) initiate cystic fibrosis (CF) airway disease. In vivo studies of CF mouse nasal epithelia revealed an increase in goblet cell number that was associated with decreased ASL volume rather than abnormal [Cl(-)]. Aerosolization of osmolytes in vivo failed to raise ASL volume. In vitro studies revealed that osmolytes and pharmacological agents were effective in producing isotonic volume responses in human airway epithelia but were typically short acting and less effective in CF cultures with prolonged volume hyperabsorption and mucus accumulation. These data show that (1) therapies can be designed to normalize ASL volume, without producing deleterious compositional changes in ASL, and (2) therapeutic efficacy will likely depend on development of long-acting pharmacologic agents and/or an increased efficiency of osmolyte delivery.

Pharmacological Properties of N-(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

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.

Altering airway surface liquid volume: inhalation therapy with amiloride and hyperosmotic agents

The thin layer of liquid lining the entire respiratory tract is the first line of defense against the continuous insult of inhaled bacteria and noxious chemicals. Many chronic obstructive diseases of the airway may reflect decreased airway surface liquid, which results from imbalances in ion transport and mucus production. Reduction in the thickness of airway surface liquid leads to reduced mucociliary clearance rates, causing mucus accumulation and infection in the airway. In this chapter, two inhalation therapies to replenish airway surface liquid and enhance mucociliary clearance are discussed: (1) aerosolized hyperosmotic agents; and (2) aerosolized sodium channel blockers. The advantages and disadvantages of each therapy are discussed, as well as future directions for improving airway surface liquid volume by inhalation pharmacotherapy.

Effect of Topically Applied Epithelial Sodium Channel Inhibitors on Tear Production in Normal Mice and in Mice with Induced Aqueous Tear Deficiency

PURPOSE Dry eye syndromes affect a significant proportion of the population worldwide with reported prevalence ranging from 6% to more than 34%. Patients with dry eye can experience intense pain due to eye irritation, gritty/scratchy feeling in the eyes, blurry vision, and light sensitivity. Available treatments for dry eye syndromes remain mainly palliative. The purpose of the present study was to test the hypothesis that inhibiting sodium absorption via the epithelial sodium channel (ENaC) will increase ocular hydration in both normal as well as in animals with experimentally induced dry eye. METHODS ENaC inhibitors were dissolved in an aqueous buffer that mimics the composition of tears and were applied topically to the ocular surface of isoflurane-anesthetized mice. The effect of ENaC inhibitors was compared with that of the secretagogue uridine triphosphate (UTP; 1%), a purinergic receptor agonist which was shown to increase tear volume in animals. Tear production was measured for 10 s using phenol red-impregnated cotton threads. Fluorescein staining that assesses ocular surface damage was performed at baseline and then at days 1, 2, and 3 after the induction of dry eye in mice. RESULTS Our data show that the inhibition of ENaC led to a time- and concentration-dependent increase in tear volume in normal mice. The effect of ENaC inhibition after a single application outperformed UTP, as it was long-lasting with tear volume still above baseline values 8 h postdosing. ENaC inhibition, which led to increased tear production, improved fluorescein scores in our dry eye model, when compared with nontreated or animals treated with buffer or UTP. CONCLUSION We conclude that the inhibition of ENaC provides long-lasting increases in ocular surface hydration and that ENaC blockers could provide an effective new therapy for chronic dry eye.