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1.
Pulm Pharmacol Ther ; 27(1): 76-82, 2014 Feb.
Article in English | MEDLINE | ID: mdl-23932897

ABSTRACT

Although the airway surface is the anatomic target for many lung disease therapies, measuring drug concentrations and activities on these surfaces poses considerable challenges. We tested whether mass spectrometric analysis of exhaled breath condensate (EBC) could be utilized to non-invasively measure airway drug pharmacokinetics and predicted pharmacological activities. Mass spectrometric methods were developed to detect a novel epithelial sodium channel blocker (GS-9411/P-680), two metabolites, a chemically related internal standard, plus naturally occurring solutes including urea as a dilution marker. These methods were then applied to EBC and serum collected from four (Floridian) sheep before, during and after inhalation of nebulized GS-9411/P-680. Electrolyte content of EBC and serum was also assessed as a potential pharmacodynamic marker of drug activity. Airway surface concentrations of drug, metabolites, and electrolytes were calculated from EBC measures using EBC:serum urea based dilution factors. GS-9411/P-680 and its metabolites were quantifiable in the sheep EBC, with peak airway concentrations between 1.9 and 3.4 µM measured 1 h after inhalation. In serum, only Metabolite #1 was quantifiable, with peak concentrations ∼60-fold lower than those in the airway (45 nM at 1 h). EBC electrolyte concentrations suggested a pharmacological effect; but this effect was not statistical significant. Analysis of EBC collected during an inhalation drug study provided a method for quantification of airway drug and metabolites via mass spectrometry. Application of this methodology could provide an important tool in development and testing of drugs for airways diseases.


Subject(s)
Amiloride/analogs & derivatives , Mass Spectrometry/methods , Sodium Channel Blockers/pharmacokinetics , Urea/metabolism , Administration, Inhalation , Amiloride/administration & dosage , Amiloride/pharmacokinetics , Amiloride/pharmacology , Animals , Biomarkers/metabolism , Breath Tests , Female , Sheep , Sodium Channel Blockers/administration & dosage , Sodium Channel Blockers/pharmacology , Tissue Distribution
2.
J Ocul Pharmacol Ther ; 28(4): 433-8, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22455658

ABSTRACT

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.


Subject(s)
Dry Eye Syndromes/drug therapy , Sodium Channel Blockers/therapeutic use , Tears/metabolism , Administration, Topical , Amiloride/pharmacology , Animals , Coloring Agents , Diuretics/pharmacology , Dogs , Dry Eye Syndromes/pathology , Epithelial Sodium Channels/metabolism , Female , Fluorescein , Mice , Mice, Inbred BALB C , Purinergic P1 Receptor Antagonists/pharmacology , Sodium Channel Blockers/administration & dosage , Sodium Channel Blockers/pharmacokinetics , Tears/chemistry , Tears/drug effects , Uridine Triphosphate/pharmacology
3.
Am J Respir Cell Mol Biol ; 39(2): 190-7, 2008 Aug.
Article in English | MEDLINE | ID: mdl-18367727

ABSTRACT

Adenosine (ADO) signaling is altered in both asthma and chronic obstructive pulmonary disease, and the A(2B) adenosine receptor (A(2B)-R) may drive pulmonary inflammation. Accordingly, it has been proposed that specific inhibition of the A(2B)-R could treat inflammatory lung diseases. However, stimulation of the cystic fibrosis transmembrane conductance regulator (CFTR) by ADO may be crucial in permitting the superficial epithelium to maintain airway surface liquid (ASL) volume, which is required to ensure hydrated and clearable mucus. Our goal was to determine which ADO receptor (ADO-R) underlies ASL volume regulation in bronchial epithelia. We used PCR techniques to determine ADO-R expression in bronchial epithelia and used nasal potential difference measurements, Ussing chambers studies, and XZ-confocal microscopy to look at Cl- secretion and ASL volume regulation. The A(2B)-R was the most highly expressed ADO-R in donor specimens of human bronchial epithelia, and inhibition of ADO-R in vivo prevented activation of CFTR. A(2B)-R was the only ADO-R detected in cultured human bronchial epithelial cells and inhibition of this receptor with specific A(2B)-R antagonists resulted in ASL height collapse and a failure to effect ASL height homeostasis. Removal of ADO with ADO deaminase and replacement with 5'N-ethylcarboxamide adenosine resulted in dose-dependent changes in ASL height, and suggested that the cell surface (ADO) may be in excess of 1 microM, which is sufficient to activate A(2B)-R. A(2B)-R are required for ASL volume homeostasis in human airways, and therapies directed at inhibiting A(2B)-R may lead to a cystic fibrosis-like phenotype with depleted ASL volume and mucus stasis.


Subject(s)
Adenosine/physiology , Bronchi/physiology , Immunity, Innate , Mucus/physiology , Receptor, Adenosine A2B/physiology , Respiratory Mucosa/physiology , Adenosine/pharmacology , Adenosine A2 Receptor Antagonists , Adenosine Deaminase/pharmacology , Adenosine-5'-(N-ethylcarboxamide)/pharmacology , Bronchi/immunology , Calcium/metabolism , Cells, Cultured , Chlorides/metabolism , Cyclic AMP/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/metabolism , Humans , Receptor, Adenosine A2B/biosynthesis , Respiratory Mucosa/immunology
4.
J Pharmacol Exp Ther ; 325(1): 77-88, 2008 Apr.
Article in English | MEDLINE | ID: mdl-18218832

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 (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 +/- hypertonic saline for CF lung disease.


Subject(s)
Cystic Fibrosis/drug therapy , Epithelial Sodium Channel Blockers , Mesylates/pharmacokinetics , Sodium Channel Blockers/pharmacokinetics , Absorption , Animals , Biotransformation , Dogs , Humans , Lung Diseases , Mesylates/pharmacology , Mesylates/therapeutic use , Respiratory Mucosa/metabolism , Sodium Channel Blockers/chemistry , Sodium Channel Blockers/pharmacology
5.
Biochemistry ; 46(36): 10373-83, 2007 Sep 11.
Article in English | MEDLINE | ID: mdl-17696452

ABSTRACT

Adenosine is a multifaceted signaling molecule mediating key aspects of innate and immune lung defenses. However, abnormally high airway adenosine levels exacerbate inflammatory lung diseases. This study identifies the mechanisms regulating adenosine elimination from the apical surface of human airway epithelia. Experiments conducted on polarized primary cultures of nasal and bronchial epithelial cells showed that extracellular adenosine is eliminated by surface metabolism and cellular uptake. The conversion of adenosine to inosine was completely inhibited by the adenosine deaminase 1 (ADA1) inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA). The reaction exhibited Km and Vmax values of 24 microM and 0.14 nmol x min(-1) x cm(-2). ADA1 (not ADA2) mRNA was detected in human airway epithelia. The adenosine/mannitol permeability coefficient ratio (18/1) indicated a minor contribution of paracellular absorption. Adenosine uptake was Na+-dependent and was inhibited by the concentrative nucleoside transporter (CNT) blocker phloridzin but not by the equilibrative nucleoside transporter (ENT) blocker dipyridamole. Apparent Km and Vmax values were 17 microM and 7.2 nmol x min(-1) x cm(-2), and transport selectivity was adenosine = inosine = uridine > guanosine = cytidine > thymidine. CNT3 mRNA was detected throughout the airways, while CNT2 was restricted to nasal epithelia. Inhibition of adenosine elimination by EHNA or phloridzin raised apical adenosine levels by >3-fold and stimulated IL-13 and MCP-1 secretion by 6-fold. These responses were reproduced by the adenosine receptor agonist 5'-(N-ethylcarboxamido)adenosine (NECA) and blocked by the adenosine receptor antagonist, 8-(p-sulfophenyl) theophylline (8-SPT). This study shows that adenosine elimination on human airway epithelia is mediated by ADA1, CNT2, and CNT3, which constitute important regulators of adenosine-mediated inflammation.


Subject(s)
Adenosine Deaminase/metabolism , Adenosine/metabolism , Cell Polarity , Epithelial Cells/enzymology , Lung Diseases/pathology , Membrane Transport Proteins/metabolism , Respiratory System/cytology , Cell Membrane Permeability , Cells, Cultured , Cytokines/metabolism , Epithelial Cells/metabolism , Humans , Inflammation , Isoenzymes/metabolism , Kinetics , Lung Diseases/enzymology , Receptors, Purinergic P1/metabolism , Respiratory System/enzymology , Respiratory System/metabolism
6.
J Med Chem ; 49(14): 4098-115, 2006 Jul 13.
Article in English | MEDLINE | ID: mdl-16821771

ABSTRACT

Amiloride (1), the prototypical epithelial sodium channel (ENaC) blocker, has been administered with limited success as aerosol therapy for improving pulmonary function in patients with the genetic disorder cystic fibrosis. This study was conducted to synthesize and identify more potent, less reversible ENaC blockers, targeted for aerosol therapy and possessing minimal systemic renal activity. A series of novel 2-substituted acylguanidine analogues of amiloride were synthesized and evaluated for potency and reversibility on bronchial ENaC. All compounds tested were more potent and less reversible at blocking sodium-dependent short-circuit current than amiloride. Compounds 30-34 showed the greatest potency on ENaC with IC(50) values below 10 nM. A regioselective difference in potency was found (compounds 30, 39, and 40), whereas no stereospecific (compounds 33, 34) difference in potency on ENaC was displayed. Lead compound 32 was 102-fold more potent and 5-fold less reversible than amiloride and displayed the lowest IC(50) value ever reported for an ENaC blocker.


Subject(s)
Bronchitis, Chronic/drug therapy , Cystic Fibrosis/drug therapy , Guanidines/chemical synthesis , Pyrazines/chemical synthesis , Sodium Channel Blockers/chemical synthesis , Sodium Channels/drug effects , Animals , Bronchi/drug effects , Bronchi/physiology , Combinatorial Chemistry Techniques , Dogs , Epithelial Sodium Channels , Guanidines/chemistry , Guanidines/pharmacology , Models, Molecular , Pyrazines/chemistry , Pyrazines/pharmacology , Respiratory Mucosa/drug effects , Respiratory Mucosa/physiology , Sodium Channel Blockers/chemistry , Sodium Channel Blockers/pharmacology , Sodium Channels/physiology , Stereoisomerism , Structure-Activity Relationship , Tissue Culture Techniques
7.
J Pharmacol Exp Ther ; 311(3): 929-38, 2004 Dec.
Article in English | MEDLINE | ID: mdl-15273255

ABSTRACT

Epithelial sodium channel (ENaC) blockers have been proposed as a therapy to restore mucus clearance (MC) in cystic fibrosis (CF) airways. The therapeutic effects of the first generation ENaC blocker, amiloride, in CF patients, however, were minimal. Because the failure of amiloride reflected both its low potency and short duration of action on airway surfaces, we investigated whether the increased potency of benzamil and phenamil would produce more favorable pharmacodynamic properties. In vitro potency, maximal efficacy, rate of recovery from maximal block of ENaC, and rate of drug absorption were compared for amiloride, benzamil, and phenamil in cultured human and ovine bronchial epithelial cells. In both human and ovine bronchial epithelia, the rank order of potency was benzamil > phenamil >> amiloride, the maximal efficacy was benzamil = phenamil = amiloride, the recovery to baseline sodium transport was phenamil < benzamil << amiloride, and the rate of drug absorption was phenamil > benzamil >> amiloride. Based on greater potency, benzamil was compared with amiloride in in vivo pharmacodynamic studies in sheep, including tracheal mucus velocity (TMV) and MC. Benzamil enhanced MC and TMV, but acute potency or duration of effect did not exceed that of amiloride. In conclusion, our data support the hypothesis that ENaC blocker aerosol therapy increases MC. However, rapid absorption of benzamil from the mucosal surface offset its greater potency, making it equieffective with amiloride in vivo. More potent, less absorbable, third generation ENaC blockers will be required for an effective aerosol CF pharmacotherapy.


Subject(s)
Amiloride/analogs & derivatives , Amiloride/therapeutic use , Cystic Fibrosis/drug therapy , Lung Diseases/drug therapy , Sodium Channel Blockers/therapeutic use , Absorption , Amiloride/pharmacokinetics , Animals , Bronchi/drug effects , Bronchi/metabolism , Cystic Fibrosis/complications , Electrophysiology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Epithelial Sodium Channels , Epithelium/drug effects , Epithelium/metabolism , Female , Humans , In Vitro Techniques , Lung Diseases/etiology , Mannitol/metabolism , Microscopy, Confocal , Mucus/physiology , Muscle Contraction/drug effects , Sheep , Sodium/metabolism , Sodium Channels/metabolism , Viscosity
8.
Am J Respir Crit Care Med ; 169(5): 645-53, 2004 Mar 01.
Article in English | MEDLINE | ID: mdl-14670800

ABSTRACT

It is controversial whether mutations in cystic fibrosis transmembrane conductance regulator intrinsically dysregulate inflammation. We characterized passage 2 human tracheobronchial epithelial cell cultures morphologically and physiologically and determined whether cytokine production or nuclear factor-kappaB activation was systematically altered in cystic fibrosis (CF) cells. Non-CF and CF cells originating from a total of 33 and 25 lungs, respectively, were available for culture on plastic or at an air-liquid interface until well differentiated. Forskolin-stimulated short-circuit currents were present in representative polarized non-CF cultures and were absent in CF cultures, whereas uridine 5'-triphosphate-stimulated currents were present in both. Constitutive or interleukin (IL)-1beta-induced IL-8 or IL-6 secretion or nuclear factor-kappaB activity was not significantly different between non-CF and CF cells. The cytokines regulated upon activation, normal T cell expressed and secreted (RANTES) and IL-10 were not detectable. Stimulation with tumor necrosis factor-alpha or a synthetic toll-like receptor 2 agonist or variable doses and times of Staphylococcus aureus culture filtrate revealed a single dose- and time-dependent difference in IL-8 production by CF cells. Interestingly, although IL-8 secretion after stimulation with Pseudomonas aeruginosa filtrates was not greater in CF cells in the absence of human serum, it was variably greater in its presence. Thus, although exaggerated responses may develop under certain conditions, our results do not support an overall intrinsically hyperinflammatory phenotype in CF cells.


Subject(s)
Cystic Fibrosis/immunology , Cytokines/metabolism , Respiratory Mucosa , Adolescent , Adult , Aged , Cell Culture Techniques/methods , Cell Differentiation , Cells, Cultured , Chemokine CCL5/immunology , Chemokine CCL5/metabolism , Child , Child, Preschool , Cystic Fibrosis/genetics , Cystic Fibrosis/metabolism , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cytokines/immunology , Female , Genotype , Humans , Inflammation , Interleukin-10/immunology , Interleukin-10/metabolism , Interleukin-8/immunology , Interleukin-8/metabolism , Male , Middle Aged , Mutation/genetics , NF-kappa B/genetics , NF-kappa B/immunology , Phenotype , Respiratory Mucosa/cytology , Respiratory Mucosa/immunology , Respiratory Mucosa/metabolism , Tumor Necrosis Factor-alpha/immunology
9.
J Biol Chem ; 278(15): 13468-79, 2003 Apr 11.
Article in English | MEDLINE | ID: mdl-12560324

ABSTRACT

In human airways, extracellular adenosine regulates epithelial functions supporting mucociliary clearance, an important airway defense mechanism against bacterial infection. Thus, defining the mechanisms of adenosine generation is critical for elucidating the role of this nucleoside in airway homeostasis. In this study, we identified the source of adenosine on the mucosal surface of human airway epithelia. Polarized primary cultures of human nasal or bronchial epithelial cells were assayed for transepithelial transport, cytosolic and cell surface adenosine production. Ussing chamber experiments indicated that serosal 1 microM [(3)H]adenosine was not transported to the mucosal compartment. Messenger RNA for the cytosolic AMP-specific 5'-nucleotidase (CN-I) was not detected in human bronchial epithelial cells, suggesting that mucosal adenosine did not originate from intracellular pools. In contrast, extracellular 0.1 mm ATP was rapidly dephosphorylated into adenosine on the mucosal epithelial surface. We identified two ectonucleotidases that mediated the conversion of AMP to adenosine: ecto 5'-nucleotidase (ecto 5'-NT, CD73) and alkaline phosphatase (AP). Both mucosal and serosal epithelial surfaces displayed ecto 5'-NT activity (K(m) = 14 microM, V(max) = 0.5 nmol x min(-1) x cm(-2)), whereas AP activity was restricted to the mucosal surface (K(m,)(high) = 36 microM, V(max) = 1.2 nmol x min(-1) x cm(-2); K(m,)(low) = 717 microM, V(max) = 2.8 nmol x min(-1) x cm(-2)). In bronchial cultures and tissues, ecto 5'-NT accounted for >80% of total activity toward 0.01 mm AMP, compared with <15% for 5 mm AMP. The proximal airway AP isoform was identified as nonspecific AP (NS AP) by levamisole sensitivity and mRNA expression. The two ectoenzymes presented opposite airway distributions, ecto 5'-NT and NS AP mRNA dominating in higher and lower airways, respectively. Collectively, these experiments support a major role for extracellular nucleotide catalysis and for ecto 5'-NT and NS AP in the regulation of adenosine concentrations on airway surfaces.


Subject(s)
5'-Nucleotidase/metabolism , Adenosine Monophosphate/metabolism , Alkaline Phosphatase/metabolism , Respiratory Mucosa/enzymology , 5'-Nucleotidase/genetics , Alkaline Phosphatase/genetics , Base Sequence , Bronchi , Cell Membrane/enzymology , Cells, Cultured , DNA Primers , Humans , Hydrolysis , Kinetics , Nose , Substrate Specificity
10.
Adv Drug Deliv Rev ; 54(11): 1445-62, 2002 Dec 05.
Article in English | MEDLINE | ID: mdl-12458154

ABSTRACT

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.


Subject(s)
Amiloride/therapeutic use , Cystic Fibrosis/drug therapy , Mucus/metabolism , Respiratory System/drug effects , Sodium Channel Blockers/therapeutic use , Administration, Inhalation , Amiloride/administration & dosage , Cystic Fibrosis/genetics , Cystic Fibrosis/physiopathology , Epithelium/drug effects , Epithelium/physiopathology , Humans , Mannitol/administration & dosage , Mannitol/therapeutic use , Mucociliary Clearance/drug effects , Osmotic Pressure , Respiratory System/physiopathology , Saline Solution, Hypertonic/administration & dosage , Saline Solution, Hypertonic/therapeutic use , Sodium Channel Blockers/administration & dosage
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