Judith A. St. George

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.

Tracheal submucosal gland development in the rhesus monkey,Macaca mulatta: ultrastructure and histochemistry

SummaryThe submucosal glands are thought to be the primary source of the mucus overlying the primate trachea and conducting airways. This study characterizes the development of submucosal glands in the trachea of the rhesus monkey. Tracheas from 46 age-dated fetal, 8 postnatal and 3 adult rhesus were fixed in glutaraldehyde/paraformaldehyde and slices processed for electron microscopy. The earliest (70 days gestational age (DGA)) indication of gland development was the projection of a group of closely packed electron lucent cells with few organelles and small pockets of glycogen into the submucosa. This configuration was observed up to 110 DGA. In fetuses younger than 87 DGA it was present almost exclusively over cartilaginous areas. Between 80 and 140 DGA, a cylinder of electron lucent cells projected into the submucosal connective tissue perpendicular to the surface. In fetuses younger than 100 DGA, it was restricted to cartilaginous areas. By 90 DGA, some glycogen containing cells in proximal regions contained apical cored granules. By 106 DGA, cells in proximal areas contained apical electron lucent granules. More distal cells had abundant GER and electron dense granules. The most distal cells resembled the undifferentiated cells at younger ages. Ciliated cells were present in the most proximal portions of glands at 120 DGA. This glandular organization was found in older animals, including adults, with the following changes: (1) abundance of proximal cells with electron lucent granules increased; (2) abundance of distal cells with electron dense granules increased; and (3) abundance of distal cells with abundant glycogen and few organelles decreased. We conclude that submucosal gland development in the rhesus monkey: (1) is primarily a prenatal process; (2) occurs first over cartilage; (3) continues into the postnatal period; and (4) involves secretory cell maturation in a proximal to distal sequence with mucous cells differentiating before serous cells.

Hydrogen peroxide induces DNA single strand breaks in respiratory epithelial cells.

The respiratory epithelium is often exposed to oxidant gases, including ozone from photochemical smog and toxic oxygen metabolites released from neutrophils recruited in conditions of airway inflammation. We evaluated DNA single strand break formation by alkaline elution as a measure of oxidant-induced DNA damage to bronchial epithelial cells. Human AdenoSV-40-transformed bronchial epithelial cells (BEAS), subclone R1.4 or nonhuman primate bronchial epithelial cells were cultured in growth factor supplemented Hams F12 medium on polycarbonate filters. DNA was labeled by incubation with [3H]thymidine. Cells were incubated for 1 h in HBSS or HBSS and increasing concentrations of hydrogen peroxide (H2O2). Cells incubated in H2O2 demonstrated dose-dependent increases in strand break formation, and BEAS cells were more sensitive to H2O2-induced injury than primary bronchial epithelial cells. The addition of catalase or preincubation of cells with the iron chelator desferoxamine prevented H2O2-induced strand breakage. DNA strand break formation may be an important mechanism of oxidant injury in respiratory epithelial cells.

Neutrophil adherence to airway epithelium is reduced by antibodies to the leukocyte CD11/CD18 complex

Airway inflammation, including neutrophil influx is commonly seen in human pulmonary diseases. We developed an in vitro system where the adherence of neutrophils to bronchial epithelial cells could be examined. Primary cultures of nonhuman primate brenchial epithelial cells or transformed BEAS human bronchial epithelial cells were grown to confluence on collagen-coated culture plates. Cells were cocultured for 30 min following the addition of human neutrophils and PMA. Cultures were then inverted, fixed with methanol, and adherent neutrophils labeled with 1B4 mouse monoclonal anti-human neutrophil antibody followed by fluoresceinlabeled sheep anti-mouse IgG. Slides were examined using fluorescence microscopy. The 1B4 antibody allowed rapid identification of neutrophils adherent to the epithelial cell monolayers, which were not labeled by this technique. PMA increased the adherence of neutrophils to bronchial epithelial cells. Pretreatment of the neutrophils with anti-CD11/CD18 antibodies prevented the increase in PMA-stimulated adherence. We conclude that PMA-stimulated adherence to airway epithelial cells is in part dependent on the neurrophil CD11/CD18 adherence complex.