Introduction

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Cystic fibrosis (CF) is a recessive genetic disease, characterized by pulmonary and reproductive tract dysfunctions, which affects more than 30,000 people in the United States (Davis et al., 1996). CF is caused by mutations in the CF transmembrane regulator (CFTR) gene, which encodes for an apical membrane epithelial protein that functions both as a cAMP-regulated chloride channel and a regulator of the epithelial sodium channel (Boucher, 1994). A defective CFTR leads to abnormal fluid and solute transport across epithelia, which contributes to the formation of viscous, dehydrated mucus in airways. The resulting mucostasis leads to progressive loss of ventilatory function and severe inflammatory responses to chronic bacterial infection (Mickle and Cutting, 1998). Most deaths of patients with CF occur as a consequence of pulmonary disease. Although improved treatment of lung disease has increased longevity, the median age for survival is still only 32 years, and patients have significant morbidity, including frequent hospitalizations, throughout their lives (Ramsey, 1996). Current therapies for CF include inhaled antibiotics, bronchodilators, mucolytics, and physiotherapy. Clearly, new therapeutic approaches are needed for the prevention and treatment of CF lung disease.

An emerging therapeutic paradigm for the treatment of cystic fibrosis is to stimulate alternative modes of chloride secretion in the lung, thereby circumventing the genetic defect in the CFTR channel. Agents that correct the underlying ion transport defects in the airways may prove useful in normalizing airway secretions, leading to the improvement of mucociliary clearance and the prevention of chronic lung infections and progressive lung damage. One such method involves the use of inhaled nucleotides that activate P2Y₂ receptors on the airway epithelial surface. The P2Y₂ receptor is abundant on the luminal surface of polarized epithelial cells, especially those lining mucosal surfaces exposed to the external environment. The pharmacology of the P2Y₂ receptor has been established, and the effects of P2Y₂ agonists on epithelial cell functions are numerous, including stimulation of serosal to mucosal chloride and fluid transport (Knowles et al., 1991; Benali et al., 1994; Tarran et al., 2000), enhancement of mucin secretion from goblet cells (Lethem et al., 1993; Kim et al., 1996), increase in cilia beat frequency (Morse et al., 2001), and promotion of surfactant release from type II alveolar cells (Gobran et al., 1994). Additionally, tracheal mucus velocity (TMV), a measure of mucociliary clearance in a single large airway, has revealed the mucokinetic effects of nucleotides and various other agents in the lung in vivo (Sabater et al., 1996).

The discovery of diadenosine 5'-polyphosphates (Ap_nA, in which "n" = 2-7) (Fig. 1) and their release from platelets has led to many studies on their biological activity and metabolism (Picher and Boucher, 1999; Pintor et al., 1999; Guranowski, 2000; Hoyle et al., 2001). Diadenosine and diuridine polyphosphates have interesting pharmacological effects on nucleotide receptors, the latter class avoiding the liability of adenosine-containing metabolites with activities at adenosine receptors (Lazarowski et al., 1995; Pendergast et al., 2001). Recent advances in nucleotide biology indicate that mono- and dinucleotides are stored and released locally by epithelial cells in concentrations that effectively activate P2Y receptors (Donaldson et al., 2000; Pintor et al., 2002). In fact, shear forces caused by cough or osmotic stress may be sufficient to induce local nucleotide release in concentrations relevant for the activation of the P2Y₂ receptor. As with other extracellular signaling systems, P2 purinergic signals are generally terminated when the nucleotides are hydrolyzed by ectoenzymes. These ectonucleotidases quickly dephosphorylate mononucleotides and cleave the more stable dinucleotide tetraphosphates into nucleoside mono- and triphosphates (Zimmerman, 1996; Picher and Boucher, 2000).

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Structures of UTP, Ap4A, Up4U (INS365), and dCp4U (INS37217).

The P2Y₂ receptor agonist, Up₄U (INS365), has been evaluated in normal healthy volunteers and patients with CF. Clinical safety has been established for inhaled INS365 in normal healthy volunteers (nonsmokers and smokers) in single doses up to 400 mg in the nebulizer. In pediatric and adult CF patients (ages 5-12 and 18-63 years, respectively), single doses up to 40 mg were well tolerated (Shaffer et al., 1998). INS37217, a next-generation dinucleotide P2Y₂ receptor agonist, is closely related to INS365 in terms of chemical structure (Fig. 1). However, we demonstrate that the deoxycytidine group that replaces one of the two uridine moieties imparts INS37217 with a significant resistance to enzymatic hydrolysis. The following in vitro and in vivo studies demonstrate how INS37217 is more suitable for treating CF lung disease in which an enhanced duration of action is believed to constitute a significant therapeutic advantage.

	Materials and Methods

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Test Compounds and Reagents. All compounds were obtained from Sigma-Aldrich (St. Louis, MO), unless otherwise stated. INS37217 was synthesized by Inspire Pharmaceuticals, Inc. (unpublished data). The purity of all nucleotide agonists was established by HPLC (95-99% purity). Fluo-3-AM was obtained from Molecular Probes (Eugene, OR). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, geneticin (G-418), and other cell culture reagents were obtained from the Tissue Culture Facility at the University of North Carolina or from Invitrogen (Carlsbad, CA). 1321N1 human astrocytoma cells stably expressing P2Y₁, P2Y_2, P2Y₄, P2Y₆, or P2Y₁₁ receptors and the wild-type 1321N1 cell were obtained from the University of North Carolina at Chapel Hill.

In Situ Hybridization. Rhesus monkey lung specimens were obtained from Pathology Associates, A Charles River Company (Frederick, MD). Tissues were removed from healthy animals immediately following euthanasia and snap-frozen in embedding medium. Frozen tissues were stored at 80°C prior to cryosectioning. Tissues were cut in 5-µm sections and mounted on SuperFrost Plus microscope slides (Fisher Scientific, Pittsburgh, PA) for H&E staining and in situ hybridization (ISH). Sections stained by H&E were prepared to evaluate the quality and orientation of the tissues studied. Examination of H&E slides indicated that all tissues were suitable for ISH. Slide-mounted tissue sections were kept frozen until all sections were cut and then used immediately for ISH.

Intracellular Calcium Mobilization. 1321N1 human astrocytoma cells stably expressing the human P2Y₁, P2Y₂, P2Y₄, P2Y₆, and P2Y₁₁ receptors were grown in DMEM containing 4.5 g/l glucose, 5% fetal bovine serum, and 600 µg/ml G-418. For intracellular Ca²⁺ measurements, cells were seeded in 96-well black-wall/clear-bottom culture plates (3904; Corning Inc., Corning, NY), at a density of 35,000 cells/well, and assays were conducted 2 days later when the cells had reached confluence.

Chloride Secretion. The posterior membrane of fresh mongrel dog tracheas was excised and dissected free of trachealis muscle, and segments (1.5 cm wide) were cut, mounted in Ussing chambers, and bathed on the mucosal and serosal surfaces with Krebs-Ringer bicarbonate solution. The composition of the bath solution was 140 mM Na⁺, 120 mM Cl, 5.2 mM K⁺, 25 mM HCO₃, 2.4 mM HPO₄², 0.4 mM HPO₄, 1.1 mM Ca²⁺, 1.2 mM Mg²⁺, and 5.2 mM glucose, pH 7.4. Tissue baths were maintained at 37°C and gassed with humidified 95% O₂:5% CO₂.

Mucin Secretion. Primary normal human tracheal/bronchial epithelial cells (donor-specific, nonsmoker), which had been shipped cryopreserved in the presence of retinoic acid, were obtained from Clonetics (East Rutherford, NJ; CC-2540) . The cells were initially seeded on Transwell-Clear culture inserts (Corning-Costar 3460; Corning) and grown in bronchial epithelial growth medium (BEGM) (Clonetics; CC-3170 BEGM BulletKit base media, plus supplements). After 2 to 3 days in culture, cells were switched to air/liquid interface (ALI) culture conditions as has been previously described by Gray et al. (1996). The 17Q2 mucin antibody was purified with a Protein G column (Pierce, Rockford, IL) from ascites fluid (University of California at Davis). Alkaline phosphatase was conjugated to 17Q2 antibody using the EZ-Link maleimide-activated alkaline phosphatase kit (Pierce).

Ciliary Beat Frequency. The effects of INS37217 on ciliary activity were determined on individual human ciliated nasal epithelial cells using techniques described previously (Geary et al., 1995; Morse et al., 2001). Briefly, epithelial cells were recovered from protease digests of human nasal turbinates procured through the Tissue Culture Core Facility of the Cystic Fibrosis/Pulmonary Research and Treatment Center at the University of North Carolina at Chapel Hill. The cells were seeded into 12-mm Costar Transwell-Col cell culture supports at a density of 300,000 cells/cm² and incubated overnight in hormone-supplemented culture medium (Gray et al., 1996) at 37°C in an atmosphere of air (5% CO₂), after which nonadherent cells were washed away to reveal small explants of the superficial epithelium as small clumps of ciliated cells that had attached to the substratum. These preparations were used within 4 days.

Airway Surface and CF Sputum Metabolism. Well differentiated cultures from passage 1 human airway epithelial cells were grown as previously described (Gray et al., 1996). In brief, nasal epithelial cells were harvested from turbinates (Wu et al., 1985). Primary cells were isolated by protein digestion and plated on a collagen-coated tissue culture dish (5-10 days) in LHC9 medium (Biosource International, Camarillo, CA) (Lechner and LaVeck, 1985) containing 25 ng/ml epidermal growth factor, 50 nM retinoic acid, 40 µg/ml gentamicin, 0.5 mg/ml bovine serum albumin, 0.8% bovine pituitary extract, 50 U/ml penicillin, 50 µg/µl streptomycin, and 0.125 mg/ml amphotericin termed BEGM. The cells were trypsinized and subpassaged on porous Transwell-Col filters (diameters: well, 24 mm; pore, 0.45 µm) in ALI medium. ALI is similar to BEGM, except that a 50:50 mixture of LHC Basal and DMEM-H is used as the base, amphotericin and gentamicin are omitted, and epidermal growth factor concentration is reduced to 0.5 ng/ml. After 4 weeks, the cultures were composed of columnar ciliated cells (>90%) and secretory cells covering a layer of basal-like cells (Matsui et al., 2000). Enzyme assays were conducted on cultures of transepithelial electrical resistance 300 /cm². Extracellular lactate dehydrogenase, employed as a test of cellular integrity, was tested 1 day before the nucleotide assays.

Tracheal Mucus Velocity. The Mount Sinai Animal Research Committee, which is responsible for assuring the humane care and use of experimental animals, approved all procedures used in this study. Five adult ewes, 25 to 45 kg in weight, were restrained in an upright position in a specialized body harness adapted to a modified shopping cart. The heads of the animals were immobilized, and local anesthesia of the nasal passage was induced with 2% lidocaine. Following topical anesthesia of the nasal passages with 2% lidocaine solution, the sheep were nasally intubated with an endotracheal tube 7.5 cm in diameter (Mallinckrodt Medical Inc., St. Louis, MO), which had been shortened by 6 cm. The cuff of the tube was placed just below the vocal cords (verified by fluoroscopy) to allow for maximal exposure of the tracheal surface area. After intubation, the animals were allowed to acclimate for a period of 20 min before beginning measurements of TMV. During the course of the experiment, the inspired air was warmed and humidified using a Bennett humidifier (Puritan-Bennett, Lenexa, KS). To minimize possible impairment of TMV caused by inflation of the endotracheal tube cuff, the cuff was deflated throughout the study, except for the period of drug delivery. The sheep were periodically gavaged with tap water to avoid dehydration (Sabater et al., 1999).

	Results

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P2Y₂ Receptor Gene Expression in the Lung. Nonradioisotopic ISH was used to determine the cellular localization of P2Y₂ receptor gene expression in cryosections of rhesus monkey lung (Table 1 and Fig. 2). Cytoplasmic ISH staining, indicative of P2Y₂ gene expression, was observed with the antisense probe in bronchial epithelium, including goblet cells, in bronchiolar and alveolar type I and II epithelium, and in submucosal gland epithelium, but not in submucosal gland ductal epithelium. In addition, vascular endothelial cells, intravascular white blood cells, and selected alveolar macrophages exhibited ISH staining consistent with expression of P2Y₂ mRNA. In contrast, no ISH staining was observed in peribronchial smooth muscle, vascular smooth muscle, mesothelium, or connective tissue stroma with the antisense probe. No staining was observed in any cell type with the negative control P2Y₂ sense probe.

View larger version (132K): [in this window] [in a new window]   Fig. 2.   Representative photomicrographs demonstrating the cellular localization of P2Y2 receptor gene expression in monkey lung epithelium by in situ hybridization. Top left, bronchial epithelium (arrow), antisense probe. Top right, bronchial epithelium, sense probe. Bottom left, submucosal glands (arrows), antisense probe. Bottom right, submucosal glands, sense probe.

INS37217 Activates P2Y₂ and P2Y₄ Receptors. In a concentration-dependent manner, INS37217 induced the mobilization of intracellular calcium in 1321N1 astrocytoma cells stably expressing human P2Y₂ and P2Y₄ receptors with EC₅₀ values of 0.22 and 0.8 µM, respectively (Fig. 3). The efficacy of INS37217 was identical to that of UTP, indicating that INS37217 is a full agonist of P2Y₂ and P2Y₄ receptors. However, the potency of INS37217 at these two receptors is somewhat less than that for the native agonist, UTP. In contrast, INS37217 had little or no calcium-mobilizing activity in 1321N1 cells expressing the P2Y₁ receptor (Fig. 3A). INS37217 was a weak agonist of P2Y₆ receptors in which 100 µM INS37217 produced approximately 70% of the response observed with the natural agonist UDP. In conclusion, INS37217 is a full agonist specific for P2Y₂ and P2Y₄ receptors. Essentially, identical effects were observed in assays of phospholipase C-dependent accumulation of inositol phosphates (not shown).

View larger version (32K): [in this window] [in a new window]   Fig. 3.   Effects of INS37217 on P2Y receptor-mediated calcium mobilization. Changes in intracellular calcium concentrations in response to the indicated concentrations of INS37217 were studied in 1321N1 human astrocytoma cells stably expressing the human P2Y1, P2Y2, P2Y4, or P2Y6 receptors. Control agonists for each P2Y receptor subtype were 2-methylio-ADP (P2Y1 receptor cells), UTP (P2Y2 and P2Y4 receptor cells), and UDP (P2Y6 receptor cells). Calcium measurements were performed as indicated under Materials and Methods. Data shown are the mean values ± S.E.M. from three independent experiments.

INS37217 Stimulates Chloride Secretion in Tracheal Epithelium. Figure 4A shows a representative cumulative concentration-response curve for INS37217 on I_sc in freshly isolated dog tracheal epithelial preparations mounted in Ussing chambers. Both INS37217 and UTP stimulated concentration-dependent increases in I_sc activity over the concentration range of 0.1 to 100 µM (Fig. 4B). Maximal responses were observed with 100 µM INS37217 or 10 µM UTP. The maximal responses to INS37217 and UTP were similar, i.e., approximately 20 µA/cm² above baseline. The EC₅₀ values for stimulation of I_sc activity by INS37217 and UTP were 1.9 and 0.3 µM, respectively. The kinetics of the responses differed somewhat for INS37217 and UTP. Both compounds stimulated immediate increases in I_sc activity, which generally peaked within 7 s of compound addition. The response to INS37217 was sustained, whereas the response to UTP began a decline toward baseline soon after reaching its peak.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Effects of INS37217 and UTP on Isc activity in excised dog tracheal epithelium. Dog tracheal epithelium was isolated and mounted in Ussing chambers as described. A, after a stable baseline of Isc activity was achieved, amiloride (100 µM) and indomethacin (100 µM) were added to suppress epithelial sodium channel and cAMP-stimulated chloride channel activities, respectively. INS37217 was added as indicated. B, stimulants were added and peak changes in Isc were recorded. Net changes in Isc (stimulated Isc minus baseline Isc) were normalized to the maximal response elicited by each stimulant. Data represent the mean ± S.E.M. (n = 4) of the normalized, net responses to INS37217 or UTP. EC50 values calculated from the fitted curve functions were 1.9 and 0.3 µM for INS37217 and UTP, respectively.

INS37217 Stimulates Mucin Secretion from Human Airway Cell Culture. To determine whether activation of P2Y receptors by INS37217 stimulated mucin production, fully differentiated cultures of human airway epithelium grown in an air/liquid interface were treated with the P2Y₂/P2Y₄ agonist. INS37217 stimulated in a concentration-dependent manner the production and release of mucin glycoproteins from these cultures (Fig. 5) with an EC₅₀ of 2.67 µM (average from two independent experiments). The efficacy of INS37217 to stimulate mucin production was identical to that observed with the endogenous agonist UTP (data not shown). The identification of specific mucin genes stimulated by INS37217 was not determined in these experiments. These results suggest that INS37217 is a potent and efficacious mucin secretagogue in human airway epithelium.

View larger version (17K): [in this window] [in a new window]   Fig. 5.   Effects of INS37217 on mucin production in differentiated normal human/tracheal epithelial cells. Normal human/tracheal epithelial cells were grown in air/liquid interface conditions as indicated under Materials and Methods. Fully differentiated cells were treated with the indicated concentrations of INS37217 and the production of mucin estimated as described under Materials and Methods. Data points are the average ± S.E.M. from hextuplicate samples from one experiment repeated on one occasion with identical results. The average EC50 value was 2.67 µM.

INS37217 Stimulates Cilia Beat Frequency in Human Airway Explants. As reported previously (Morse et al., 2001), purinergic challenge of human nasal ciliated cells stimulated ciliary activity above baseline rapidly, with peak responses occurring typically within 1 or 2 min (Fig. 6A). Because the agonist responses varied somewhat from explant to explant, the effects of INS37217 on ciliary activity were compared with a subsequent baseline using 100 µM UTP challenge as an internal control. The baseline frequencies recorded were 7.7 ± 0.2 and 7.3 ± 0.2 Hz, respectively. Peak CBF responses to repeated 100 µM UTP challenges, in the earlier study, were similarly indistinguishable. INS37217 stimulated CBF significantly at concentrations above 1 µM, and these effects saturated at about 100 µM. To account for variability in explant responses to saturating concentrations of agonist, the effects of INS37217 were expressed relative to those of UTP in the same experiment (Fig. 6B). By this analysis, the EC₅₀ was 8.3 µM, and at 100 µM, the peak responses to UTP and INS37217 were 200 and 180% of baseline, respectively.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Effects of INS37217 and UTP on ciliary beat frequency in cultured human airway epithelial cells. A, time course of agonist effects. After determining baseline ciliary activity in Ringer's solution, cells were exposed to a variable concentration of INS37217 for 10 min, following which the compound was washed out, and the cells were allowed to recover for 30 min. A second baseline CBF measurement was collected, and the effect of 100 µM UTP was determined; cell responses to each agonist were normalized to the respective baseline. B, concentration-effect relationships. The peak responses to each concentration of INS37217 are expressed as the ratio of the effect of INS37217 to that of the internal standard, 100 µM UTP. The concentration-response relationship modeled using PRISM (Intersil, Irvine, CA) yielded an EC50 of 8.3 µM for INS37217. Data are expressed as the mean ± S.E.M. of individual determinations from five or more separate experiments for each concentration tested.

INS37217 Is Resistant to Airway Surface Metabolism. We compared the metabolic rates of 0.1 mM UTP, INS365, and INS37217 on the mucosal surface of human nasal epithelial cells in culture. The natural P2Y agonist UTP was rapidly hydrolyzed by the epithelium, with an average initial hydrolytic rate of 0.93 ± 0.12 nmol · min¹ · cm² and a half-life around 3 min (Fig. 7A). In contrast, dinucleotides displayed remarkable stability toward the membrane-bound enzymes. The concentration of INS365 was approximately 40 µM at the end of the 60-min incubation period, which corresponded to initial hydrolytic rates and half-lives of 0.15 ± 0.02 nmol · min¹ · cm² and 50 min, respectively. Substitution of a uridine group by a deoxycytidine group increased the half-life of the dinucleotide to 3 h. The concentration of INS37217 on cell surfaces was approximately 90 µM after 60 min, for an average initial hydrolytic rate of 0.02 ± 0.01 nmol · min¹ · cm². Therefore, INS37217 was approximately 50 times and 6 times more stable than UTP and INS365, respectively, on the mucosal surface of human nasal epithelial cells.

View larger version (16K): [in this window] [in a new window]   Fig. 7.   Metabolic rates for UTP, INS365, and INS37217 on human nasal epithelial cells in culture (A) and in sputum samples from CF patients (B). The cells were grown to confluence on an air/liquid interface and differentiated into a polarized ciliated epithelium over 4 weeks. The cells were preincubated (30 min; 37°C) in Krebs' buffer (0.35 ml for mucosal/2 ml for serosal; pH 7.4). The assays were started with 0.1 mM substrate added to the mucosal buffer. Aliquots of 30 µl were transferred to 0.3 ml of ice-cold water, boiled for 5 min, filtered (0.45 µm), and analyzed by HPLC. B, the sputum was diluted (2:1) in Tris/HCl buffer (pH 7.4) containing 0.3 mM dithiothreitol and 0.3 mg/ml DNase, incubated for 30 min at 37°C, and then centrifuged (2,500 rpm/15 min). The supernatant was centrifuged (14,000 rpm/15 min) and filtered (0.45 and 22 µm). The filtered supernatant was diluted (2:1) with Tris/HCl buffer and aliquots of 0.4 ml were preincubated (15 min; 37°C). The reaction was started with 0.1 mM substrate and stopped by quick freeze on dry ice. The samples were boiled for 5 min, filtered (0.45 µm), and analyzed by HPLC. Data were corrected for the 4-fold dilution and expressed as mean ± S.E.M. concentrations (S.E.M. < 10%; n = 3-5).

INS37217 Increases Tracheal Mucus Velocity in Sheep. Figure 8A illustrates the overall response of TMV to the three different doses of INS37217 compared with saline (placebo). The two highest doses of INS37217 significantly enhanced TMV over the 8-h period with respect to placebo (P < 0.05). In addition, there was a dose-dependent effect of the compound, with the 94-µmol dose showing the greatest stimulation within 0.25 h after treatment. Within 0.25 h of treatment with 94 µmol of INS32717, TMV increased to 160 ± 8% from a baseline value of 7.8 ± 0.4 mm/min. This increase is compared with 141 ± 3% after treatment with 471 µmol (from a baseline of 9.5 ± 0.4 mm/min) and 135 ± 5% (from a baseline of 8.2 + 0.3 mm/min) after treatment with 40 µmol. Saline only produced a 119 ± 3% increase in TMV from a baseline of 8.2 ± 0.5 mm/min (Fig. 8B).

View larger version (23K): [in this window] [in a new window]   Fig. 8.   Effects of INS37217 on TMV in sheep. A, time course of stimulatory effects of INS37217 on TMV. Values are mean ± S.E.M. for five sheep; +, P < 0.05 versus saline; , P < 0.05 versus saline and 40 µmol of INS37217. B, peak increases in TMV (0-0.25 h) after treatment with INS37217. Values are mean ± S.E.M. for five sheep; +, P < 0.05 versus saline; , P < 0.05 versus saline and 40 µmol of INS37217.

	Discussion

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The results from the present study indicate that P2Y₂ receptors are primarily expressed in bronchial, bronchiolar, alveolar, and submucosal gland epithelium, including mucus-secreting goblet cells. In vitro, INS37217 is a submicromolar, selective agonist for human P2Y₂ and P2Y₄ receptors, and stimulates chloride secretion and increases ciliary beat frequency. In vivo, INS37217 stimulates TMV, a marker of lung mucociliary clearance. Although earlier P2Y₂ agonists (INS365) have shown similar effects, the novel finding in this study is the duration of action of INS37217.

P2Y₂ receptor activation modulates several physiological activities that can increase mucociliary clearance. Receptor activation induces chloride secretion and water movement into the airway surface liquid, thereby hydrating mucus and optimizing periciliary fluid viscosity, mucin secretion, and increased ciliary beat frequency. The combined effects of these activities is predicted to enhance airway mucociliary clearance. In vivo studies with UTP and a first-generation P2Y₂ agonist confirmed this hypothesis (Sabater et al., 1999). Our current findings showing that INS37217 effectively improved TMV in sheep are consistent with these previous results. In the present study, INS37217 produced greater peak increases in TMV than did INS365, and the stimulatory effect was more prolonged than was seen with previous P2Y₂ agonists. Furthermore, these in vivo effects were achieved at micromolar concentrations of INS37217 compared with the millimolar concentrations used in the previous study. INS37217 showed a bell-shaped, dose-dependent increase in TMV. The reason(s) that the highest concentration used was not the most effective is not clear from these experiments. Possibilities include differences in baseline TMV values among the trials and the unknown effects of P2Y₄ activation. Nevertheless, the major finding of the present study is that the increase in TMV after dosing was significant compared with placebo for up to 8 h for the two highest doses. This is the longest and largest increase in TMV observed for any nucleotide achieved in this model. Finally, it should be pointed out that TMV has been shown to be a reliable surrogate for whole lung mucociliary clearance when evaluating P2Y₂ agonists.

Although all nucleotides examined in the present work effectively stimulate components of mucociliary clearance in airways, they present important differences in stability on human airway epithelia. The metabolism of extracellular nucleotides was recently reported at the surface of human airway epithelial cells (Picher and Boucher, 2001). These enzymes sequentially dephosphorylate mononucleotides such as UTP into UDP, UMP, and uridine. An alkaline phosphodiesterase activity was also identified on human nasal and bronchial epithelial cells in culture (Picher and Boucher, 2000). This enzyme activity catalyzes the asymmetrical cleavage of dinucleotides (Np_nN) into nucleoside 5'-monophosphate and Np_n1 ("N" = A, U, or G; "n" = 2-6). In the present study, we showed that INS365 (Up₄U) was approximately 10 times more stable than UTP on the mucosal surface of human nasal epithelial cells (Fig. 7). As a result, the half-life of the dinucleotide was 50 min compared with only 3 min for UTP. These findings were supported by the extended duration of INS365 on P2Y receptor-mediated responses. However, such improvement was not satisfactory for outpatient treatments of CF, in which less frequent dosing is desired. Interestingly, we have demonstrated that asymmetry significantly enhanced the metabolic stability of INS37217 (dCP₄U) compared with the symmetrical dinucleotide, INS365 (Up₄U). The half-life of the dinucleotide was extended from 50 min to 3 h by simple substitution of a uridine for a deoxycytidine. Accordingly, the longer duration of INS37217-derived, P2Y-mediated responses with respect to those obtained with INS365 could be explained by a higher metabolic stability in human airways.

Because aerosolized nucleotides and dinucleotides are most likely to interact with the mucus layer before they reach P2Y receptors, we explored the possibility that CF sputum could significantly diminish the effective drug concentration (Fig. 7B). Nucleotide and dinucleotide metabolism was also detected in sputum samples collected from human CF patients. Whereas the rate of UTP hydrolysis was comparable to the cell surface activity, the two dinucleotides were 5 times more stable in sputum. These enzymatic activities could originate from epithelial cell desquamation or as ubiquitous cytosolic enzymes released from lysed cells. These experiments indicated that sputum would not be expected to significantly reduce the effective doses of aerosolized mono- or dinucleotides before they reach their target receptor(s) at the surface of human CF airway epithelial cells.

P2Y₂ agonist therapy represents an approach to the treatment of CF that attempts to bypass defective CFTR function in airway epithelia, taking advantage instead of the integrated actions of this class of agents on mucociliary clearance components not dependent on CFTR. The enhanced duration of action of this compound and its ability to resist metabolism on the airway surface may allow for prolonged activation of the alternative chloride channel, thus, providing more effective treatment of CF lung disease. It is anticipated that INS37217 inhalation solution, via activation of P2Y₂ receptors in the airways, will replenish airway surface liquid volume, restore mucociliary clearance, and thereby promote removal of retained secretions in patients with cystic fibrosis. The potential clinical utility of INS37217 inhalation solution is currently being evaluated in patients with CF.