Introduction

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Mucociliary clearance plays a principal role in the nonspecific host defense mechanism in the lungs, whereby locally produced biological debris and trapped inhaled particles and bacteria are removed from the conducting airways of the respiratory tract (Wanner, 1977). It has been believed that the rate of mucus transport toward the oropharynx depends on the beat frequency and coordination of epithelial cilia, on the physicochemical properties of periciliary fluid and on mucus secretion (Silberberg, 1983; Satir and Sleigh, 1990). Because the mucociliary transport is impaired in various airway diseases, such as chronic bronchitis, bronchiectasis, cystic fibrosis and asthma (Maurer et al., 1982; O'Riordan et al., 1992; Smaldone et al., 1993), stimulation of ciliary activity seems desirable in the treatment of these conditions.

We have recently synthesized the triazolopyridazin derivative TAK-225 (2-ethyl-2-[(7-methyl-[1,2,4] tiazolo [1,5-b] pyridazin-6-yl)-oxymethyl] butanesulfonamide) (fig. 1) and found that p.o. administration of this compound to sensitized guinea pigs potently inhibits allergen-induced bronchoconstriction and recruitment of eosinophils into the airway (Ashida et al., 1995). TAK-225 could thus possess antiasthmatic properties, but its effect on airway mucociliary clearance is unknown. Therefore, in the present study, to determine whether TAK-225 affects mucociliary transport and, if so, whether the alteration of epithelial ciliary function is involved, we measured mucociliary transport in the rabbit tracheal mucosa by the Evans blue method ex vivo and by CBF of the tracheal epithelium in vitro.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Chemical structure of TAK-225.

	Materials and Methods

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Measurement of mucociliary transport. Japanese white rabbits weighing between 2.5 and 3.0 kg were obtained from SLC Japan Co. (Hamamatsu, Japan) and housed in a conventional animal facility at our laboratory. The rabbits received TAK-225 (Takeda Chemical Industries, Osaka, Japan) at a dose of 0.3, 3 or 30 mg/kg. Freshly prepared TAK-225 dissolved in 5% DMSO was administered in 1.0-ml volumes by oral gavage with a 17-gauge feeding tube fitted to a 2.5-ml syringe. In the control experiment, animals received in a similar manner an equal volume of the vehicle (5% DMSO) alone. Our separate study showed that 5% DMSO itself had no effect on tracheal mucociliary transport. After 1 h of TAK-225 administration, the rabbits were anesthetized with i.m. ketamine (50 mg/kg) and exsanguinated by sectioning the abdominal aorta and inferior vena cava. The trachea was then removed, dissected free from the underlying connective tissues and mounted horizontally onto a filter paper soaked with Hanks' balanced salt solution in a moist chamber warmed to 37°C.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Time-dependent changes in Evans blue (EB) dye contents in the rabbit tracheal sections after application of the dye to the mucosal surface 1.5 cm above the carina. The EB level in each tracheal section was expressed as percentage of the total amount of the dye in sections 1 to 4. Values are means ± S.E.; n = 8 for each point. * P < .05, ** P < .01, significantly different from corresponding values at time 0.

Measurement of CBF. The method we used to measure CBF of rabbit tracheal epithelium has been described in detail previously (Tamaoki et al., 1995). Briefly, the mucosa of excised rabbit trachea was cut into small pieces (1-2 mm²) and rinsed several times with Hanks' balanced salt solution. Then the tissues were placed on a cover glass (18 × 24 mm) coated with human placental collagen (5.8 µg/cm², Sigma) in a petri dish and incubated in Ham's nutrient F12 medium containing 10 µg/ml insulin, 5 µg/ml transferrin, 25 ng/ml epidermal growth factor, 7.5 µg/ml endothelial cell growth supplement, 50 U/ml penicillin, 50 µg/ml streptomycin and 50 µg/ml gentamicin at 37°C in a CO₂ incubator (95% air-5% CO₂). On the seventh day of incubation, the cover glass on which the cultured explant was adhered was mounted in a Rose chamber, which was then placed on the stage of a microscope equipped with a phase-contrast condenser and an on-base type of halogen illuminator (Nikon, Optiphoto-XF, Tokyo, Japan). The photometer (Hamamatsu Photonics, NFX-II, Hamamatsu, Japan) with built-in periplanatic eyepiece, a limiting aperture and a lateral focusing telescope were attached to the head of the microscope. Because of the beating action of cilia, light from the illuminator passed through the preparation in varying intensities. These variations in light intensity were detected by the photometer and transduced to voltage impulses, which were recorded by a pen recorder (Panasonic, VP-6213A, Osaka, Japan). Measurements of CBF were averaged from clumps of two or more cells with free borders devoid of debris. Our preliminary experiments showed that the variation in CBF among preparations was less than 0.8 Hz (<7%) and that there were no significant differences in variation between experimental groups. In addition to CBF, we assessed ciliary coordination by the image of the beating pattern recorded on a video camera (Sony, VO-5800, Tokyo, Japan) with a videocassette recorder capable of freeze-frame replay. Ciliary discoordination was defined as the loss of metachronal wave on the free border of the cell clump (Sanderson and Sleigh, 1981; Tamaoki et al., 1989).

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Measurement of intracellular cAMP. To confirm whether the effect of TAK-225 on ciliary motility was associated with cAMP production, we measured intracellular levels of cAMP (Brooker et al., 1979). The epithelial cells were incubated with various concentrations of TAK-225 (10⁷ to 10⁴ M) for 10 min in the presence of 3-isobutyl-1-methylxanthine (10³ M, Sigma) to inhibit cAMP phosphodiesterase activity. The cells were quickly removed from the chamber and placed in ice-cold 10% trichloroacetic acid with ether; the residue was dissolved in acetate buffer. Then cAMP levels were determined in triplicate by [³H]-cAMP (Amershan Life Science Japan, Tokyo, Japan), corrected for ether extraction of 88% recovery and normalized for protein content of the cells as determined by the method of Lowry et al. (1951), with bovine serum albumin as a standard.

Statistics. All values were expressed as means ± S.E. Statistical analysis was performed by ANOVA using Scheffé's F test, and a P value of less than .05 was considered statistically significant.

	Results

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Mucociliary transport. Mucociliary transport in the tracheal mucosa of the rabbits that received the vehicle of TAK-225 alone (5% DMSO) is shown in figure 2. The contents of Evans blue dye in section 2, where the dye had been placed at time 0 on the mucosal surface, gradually decreased and reached a plateau after 10 min (98.1 ± 2.4  42.3 ± 7.4% of the total, P < .01, n = 8). The decrease in Evans blue content in section 2 was accompanied by a corresponding increase in the dye content in section 3 and subsequently in section 4, which indicates that Evans blue was transported from the lower trachea toward the larynx.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Dose-dependent effect of TAK-225 on mucociliary transport in the rabbit trachea. Evans blue (EB) contents in the tracheal sections were determined 10 min after application of the dye to the mucosal surface. The EB level in each tracheal section was expressed as percentage of the total amount of the dye in sections 1 to 4. Values are means ± S.E.; n = 16 for each column. * P < .05, ** P < .01, significantly different from corresponding values for vehicle (5% DMSO) alone.

Ciliary motility. Addition of TAK-225 (10⁵ M) to the chamber elicited a rapid increase in CBF of rabbit tracheal epithelium from the base-line value of 12.3 ± 0.4 to 16.6 ± 0.7 Hz (P < .001, n = 9) within 30 s; this effect was followed by the decline and the subsequent stable response (fig. 4). The CBF value in the presence of TAK-225 was still significantly greater than the base-line CBF (P < .001). In contrast, addition of the vehicle alone had no effect. As shown in figure 5, TAK-225 increased the initial peak response of CBF in a concentration-dependent fashion: the maximal increase from the base-line value was 25.1 ± 4.6% (P < .01, n = 8), and the concentration of the drug required to produce a half-maximal effect (EC₅₀) was 3.1 ± 0.8 × 10⁷ M (n = 8). Discoordination of ciliary beating was not observed in the video recording throughout the experiments.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Time course of the effect of TAK-225 on CBF of rabbit cultured tracheal epithelium. Either TAK-225 (105 M, ) or its vehicle (5% DMSO, ) was added to the chamber at time 0 (). Values are means ± S.E.; n = 9 for each point. ** P < .01, *** P < .001, significantly different from corresponding values for vehicle.

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Concentration-dependent effect of TAK-225 on CBF of rabbit cultured tracheal epithelium. Various concentrations of TAK-225 were added to the chamber, and the initial peak response of CBF to each concentration was determined. Responses are expressed as percent increase in CBF from base-line values determined before addition of the drug. Data are means ± S.E.; n = 8. * P < .05, ** P < .01, significantly different from base-line values.

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View larger version (24K): [in this window] [in a new window]   Fig. 6.   Effects of Rp-cAMPS (104 M) and Ca++-free medium containing BAPTA-AM (5 × 105 M) on CBF of rabbit cultured tracheal epithelium in response to TAK-225. The cells were incubated for 15 min with each blocker, and then TAK-225 (105 M) was added to the chamber. Responses are expressed as percent increase in CBF from base-line values determined before the addition of TAK-225. Data are means ± S.E.; n = 10. *** P < .001, significantly different from the response to TAK-225 alone.

Intracellular cAMP levels. Addition of TAK-225 caused a concentration-dependent increase in intracellular cAMP levels of rabbit tracheal epithelium, the maximal increase being from 33.2 ± 3.6 to 96.0 ± 7.6 pmol/mg protein (P < .01, n = 8; fig. 7), but the vehicle (5% DMSO) alone had no effect.

View larger version (21K): [in this window] [in a new window]   Fig. 7.   Concentration-dependent effect of TAK-225 on intracellular cAMP contents in rabbit cultured tracheal epithelium. The cells were incubated with various concentrations of TAK-225 (closed columns) or its vehicle (5% DMSO) alone (shaded column) for 10 min, and cAMP contents were determined by radioimmunoassay. In the control experiment, no drug was added (open column). Data are means ± S.E.; n = 8 for each column. * P < .05, ** P < .01, significantly different from control values.

	Discussion

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Our studies demonstrate that the triazolopyridazin derivative TAK-225 enhances mucociliary clearance in the rabbit trachea, by a mechanism that probably involves stimulation of the ciliary motility of airway epithelium. In the present experiment, we developed an ex vivo method to evaluate airway mucociliary clearance by determining the transport rate of Evans blue dye that had been placed on the tracheal mucosal surface above the carina. We found that the rate of propulsion of the dye toward the larynx was increased in a dose-dependent fashion by pretreatment of animals with TAK-225, a result that indicates an accelerated tracheal mucociliary clearance.

The antiallergic drug TAK-225 has recently been synthesized in our laboratory and found to reduce various airway reactions associated with IgE-dependent asthma. For example, p.o. administration of TAK-225 to sensitized guinea pigs inhibits allergen-induced infiltration of eosinophils into the airway and the immediate and late phases of bronchoconstriction (Ashida et al., 1995). On the other hand, impairment of tracheobronchial mucociliary clearance has long been suspected of playing a significant role in asthma (Wanner, 1977; O'Riordan et al., 1992). Mezey and co-workers (1978) showed decreased base-line tracheal mucus velocity in ragweed-sensitized asthmatics, which became further impaired after acute bronchospasm had been induced by inhalation of specific antigen. Moreover, Bateman and co-workers (1983) and Pavia and colleagues (1985) reported that mucociliary clearance is impaired in asthmatics with mild, stable disease and in asthmatics in remission, respectively, relative to normal volunteers. Therefore, the TAK-225-induced stimulation of tracheal mucociliary transport observed in the present study suggests that, in addition to its antiallergic activities, this drug might be beneficial in the treatment of asthma.

It has been generally accepted that mucociliary transport is governed by ciliary activity and by the depth and rheologic properties of periciliary fluid (Silberberg, 1983; Satir and Sleigh, 1990). We thus hypothesized that stimulation of ciliary activity could account for the effect of TAK-225 on the transport of Evans blue dye. Consequently, addition of TAK-225 rapidly increased the CBF of rabbit tracheal epithelium in a concentration-dependent manner. However, the effectiveness of ciliary action depends on several characteristics of ciliary beating, of which the CBF is but one. Coordination of the beating pattern, for instance, also plays a role in ciliary performance (Satir and Sleigh, 1990). In the present study, no ciliary discoordination was noted among adjacent cilia on the same cell or several bordering cells in association with the increased CBF in response to TAK-225. Thus it seems reasonable to speculate that the observed increase in CBF can be translated into the enhanced mucociliary transport, as predicted by theoretical models of mucociliary pumping (Ross and Corrsin, 1974), but further studies on the effect of this drug on airway secretion are required.

Ciliary motility of airway epithelium is regulated mainly by cAMP and Ca⁺⁺ (Dirksen and Sanderson, 1990; Lansley et al., 1992; Benedetto et al., 1994). Intracellular cAMP activates glycogenolysis and subsequently stimulates the production of ATP, an energy source of ciliary beating (Satir, 1982), via the Krebs cycle (Tamaoki et al., 1989), and the mobilization of intracellular Ca⁺⁺ apparently acts on the ciliary axoneme via the formation of Ca⁺⁺-calmodulin complexes (Verdugo et al., 1983). In our experiment, the increase in CBF produced by TAK-225 was not altered by pretreatment of cells with Ca⁺⁺-free solution in the presence of the intracellular Ca⁺⁺-chelating agent BAPTA-AM to inhibit both Ca⁺⁺ influx and Ca⁺⁺ release from intracellular stores, but it was greatly attenuated by the cAMP antagonist Rp-cAMPS. This result suggests that the ciliary stimulatory action of TAK-225 is mediated by cAMP. This notion was further supported by the finding that incubation of the tracheal epithelium with TAK-225 increased intracellular cAMP contents in a concentration-dependent manner, but the mechanism by which this triazolopyridazin derivative stimulated cAMP synthesis remains unknown.

In conclusion, the triazolopyridazin derivative TAK-225, a newly developed, orally active antiallergic drug, enhances airway mucociliary transport presumably through cAMP-mediated stimulation of the ciliary motility of airway epithelium. Therefore, TAK-225 could be of value in the treatment of impaired mucociliary clearance, such as occurs in asthma.