Introduction

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Allergic rhinitis is an inflammatory disease of the upper airway. Symptoms of the disease, which include congestion, rhinorrhea formation, sneezing, itching and loss of smell, are triggered by immunoglobulin E-mediated activation of mucosal mast cells, basophils and eosinophils. Activation of these inflammatory cells causes the release of numerous mediators including PAF, histamine, leukotrienes, cytokines and prostaglandins that can in turn recruit additional inflammatory cells, trigger the release of further inflammatory mediators and stimulate afferent nerves (Belser et al., 1996; Garrelds et al., 1996; Krause 1995; Meltzer 1995; Naclerio et al., 1991; Nonaka et al., 1996).

Of inflammatory mediators potentially involved in allergic rhinitis, PAF is of particular interest because it is perhaps the most potent mediator for inducing vascular leakage, a response that may contribute to rhinorrhea formation and congestion (Evans et al., 1988, 1989; Hwang et al., 1985). Because of its potent proinflammatory properties, PAF has been the subject of several studies that implicate it in bronchial asthma (Braquet et al., 1987; Summers and Albert 1995). However, the role of PAF in allergic rhinitis is less well established. Several studies have been done with a few PAF receptor antagonists in animal models of the disease. CV-3988 blocked vascular permeability and nasal airway resistance increases induced by topical application of PAF, and WEB-2086 and SM-10661 attenuated antigen-induced increase in late-phase nasal airway resistance in guinea pigs (Misawa and Iwamura, 1990; Narita and Asakura, 1993). In the clinical setting, installation of PAF into the nose induces many of the symptoms of rhinitis including increases in nasal airway resistance, rhinorrhea formation, nasal neutrophil influx and nasal hyper-responsiveness to subsequent allergen challenge (Andersson and Pipkorn 1988; Leggieri et al., 1991; Miadonna et al., 1996). PAF, and its metabolite (lyso-PAF), have been detected in nasal fluids and plasma of patients with rhinitis (Labrakis-Lazana et al., 1988; Miadonna et al., 1989; Shirasaki and Asakura, 1990). These results implicate PAF in allergic rhinitis and raise the possibility that PAF antagonists may be useful in the treatment of the disease.

ABT-491(fig. 1) is a recently described novel PAF antagonist that inhibits PAF binding to human PAF receptors with a K_i of 0.6 nM. The antagonist is selective and its activity is correlated with functional antagonism of PAF-mediated cellular responses (calcium mobilization, priming of superoxide generation, aggregation and degranulation) (Albert et al., 1997). In vivo ABT-491 is effective in PAF-challenge models with oral potencies (ED₅₀) between 0.03 and 0.4 mg/kg in rat, mouse and guinea pig. Given its intrinsic potency and in vivo activity, ABT-491 may be useful in the treatment of allergic rhinitis. To further explore this possibility we evaluated the efficacy of ABT-491 in experimental models of rhinitis, focusing on acute-phase responses. The present report describes the effects of ABT-491 on PAF and antigen-induced changes in nasal vascular permeability in rats and in nasal airway resistance in guinea pigs. The efficacy of the PAF antagonist in theses models is compared with the effects of other antipermeability agents (histamine and serotonin antagonists and a leukotriene synthesis inhibitor).

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Structure of ABT-491.

	Materials and Methods

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Materials. ABT-491 (4-ethynyl-N,N-dimethyl-3-[3-fluoro-4-[(2-methyl-1H-imidazo-[4,5-c]pyridin-1-yl)methyl]benzoyl]-1H-indole-1-carboxamide hydrochloride) and A-79175 ((R)-(+)-N-[3-[5-(4-fluorophenoxy)-2-furanyl]-1-methyl-2-propynyl]-N-hydroxyurea) were synthesized at Abbott Laboratories (Brooks et al., 1995; Summers et al., 1996). Other drugs and reagents were purchased from Sigma Chemical Company, St. Louis, MO.

Laboratory animals. Male Hartley guinea pigs (300-350 g) were purchased from Charles River Laboratories, Wilmington, MA. Brown Norway rats (275-325 g) were purchased from Harlan Sprague Dawley, Indianapolis, IN. Animals were maintained in a light/dark cycle and were given continuous access to food and water for at least 1 week after purchase. Before use, animals were fasted overnight. All studies were performed in accordance with protocols approved by the Abbott Laboratories Animal Use and Care Committee and met guidelines approved by the American Veterinary Medical Association.

Statistical methods. The Student's t-test was used for statistical comparison of group means with appropriate control groups. Percent inhibition of the control response was computed for each treatment group and, in the case of dose-response data, was fitted with a straight using the logarithm of the dose as the x-value. Linear regression of the data was used to estimate ED₅₀ values (dose yielding 50% inhibition) in appropriate assays.

Active sensitization to ovalbumin. Guinea pigs were sensitized according to a modified procedure of Herxheimer (Herxheimer, 1952) by intramuscular injections (0.35 ml to each leg) of 50 mg/ml ovalbumin in isotonic saline. Seven days later, each animal received a booster injection (i.p.) of 0.5 ml solution containing 80 µg/ml ovalbumin and 400 mg/ml of Al(OH)₃. Seven to fourteen days after the booster injection, each lot of animals was assessed and screened for reactivity to the antigen by determining the degree of bronchoconstriction after an in vivo antigen aerosol challenge (Malo et al., 1994). Brown Norway rats were immunized and given a booster 7 days later with an injection (i.p.) of 1 mg OA mixed with 100 mg Al(OH)₃ in 1 ml. PAF and antigen-induced nasal vascular permeability studies were conducted with the sensitized rats 18 to 21 days after immunization.

Measurement of nasal vascular permeability in Brown Norway rats. Rats sensitized to OA were anesthetized by an i.p. injection of pentobarbital (50 mg/kg) and the trachea was cannulated with a polyethylene PE-260 tube for breathing. A second cannula was inserted through the esophagus to the posterior part of the nasal cavity to allow perfusion of the nasal mucosa (Takahashi et al., 1990). Access to the nasopalatine from the buccal cavity was sealed with adhesive. After insertion of the cannula and before challenge, Evans blue dye (1%), 4 ml/kg was given intravenously by tail vein. The nasal cavity was then perfused with saline containing either PAF (25 µg/ml) and 0.25% BSA or OA (1%) from the esophageal cannula at a rate of 0.25 ml/min. The perfusate was collected at 15-min intervals, centrifuged and assayed photometrically for dye content. Drugs, prepared in methylcellulose, were administered orally to rats (six to eight animals per group) 75 min before PAF or OA challenge.

Measurement of nasal airway resistance in guinea pigs. Guinea pigs were anesthetized by an i.p. injection of pentobarbital (20-24 mg/kg) and urethane (1.0 g/kg), and the trachea was cannulated with PE-240 tubing in posterior direction for spontaneous breathing. A second cannula was then inserted in the caudal direction (retrograde), and the buccal cavity (mouth) was sealed with adhesive to allow for the isolated ventilation through the nares. This cannula was connected to a small animal ventilator (Harvard Instruments, South Natick, MA) (volume set at 10 ml/kg b.wt.; 45 breaths/min) with an in-line pressure transducer (Millar model MPC 500) to monitor insufflation pressure. The pressure signal was recorded and analyzed (peak) by an MI² data processing workstation (Modular Instruments XYZ System, Southeastern, PA). The animal was maintained at 37°C by being placed supine on a heated table and monitored via a rectal temperature probe.

	Results

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Nasal vascular permeability. Topical administration of PAF to the nasal passages of anesthetized Brown Norway rats provokes an acute inflammatory response that resulted in increased nasal vascular permeability. The response, measured as dye leakage, was evident within 15 min after perfusion with PAF (fig. 2A). After reaching a maximum 30 min after initiation of the infusion, the PAF response diminished to approximately half-maximal by the end of the observation period. The vascular leakage induced by PAF was inhibited by pretreatment with ABT-491 (fig. 2A). The inhibition, based on total dye release, was dose related (fig. 2B). An orally administrated dose of 1 mg/kg of the antagonist, given 75 min before PAF challenge, inhibited the response by 75% and complete inhibition was achieved with a dose of 10 mg/kg. From these and the other results given in the figure 2B, the potency (ED₅₀) of ABT-491 for inhibiting the PAF-induced response was computed as 0.3 mg/kg.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   PAF-induced nasal vascular permeability in Brown Norway Rats and inhibition with ABT-491. (A) PAF (25 µg/ml) was perfused through the nasal airways of rats given drug vehicle (squares) or 1 mg/kg ABT-491 (triangles) orally 75 min before challenge. Negative controls are indicated by circles. Dye content of the nasal perfusate collected at 15-min intervals is expressed as the mean ± S.E.M. (B) Dye content of the nasal perfusate collected during 2 hr from groups given the indicated dose of ABT-491. Percent inhibition of the PAF response is shown above each bar. Statistical comparison vs. antigen control: *P  .05; ***P  .005; ****P  .001.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Antigen-induced nasal vascular permeability in Brown Norway rats and inhibition with ABT-491. (A) Antigen (1% OA) was perfused through the nasal airways of rats given drug vehicle (squares) or 1 mg/kg ABT-491 (triangles) orally 75 min before challenge. Negative controls are indicated by circles. Dye content of the nasal perfusate collected at 15-min intervals is expressed as the mean ± S.E.M. (B) Dye content of the nasal perfusate collected during 2.5 hr from groups given the indicated dose of ABT-491. Percent inhibition of the antigen response is shown above each bar. Statistical comparison vs. antigen control: **P  .01; ***P  .005; ****P  .001.

Nasal airway resistance. PAF (50 µg) instilled into each guinea pig nostril produced a steady increase in nasal resistance as compared with instillation of the 0.1% BSA vehicle alone (fig. 4A). The response was approximately 40% of the maximal nasal resistance achievable, as determined by a total occlusion obtained by pinching closed both nostrils. Pretreatment with ABT-491 (1 mg/kg), as a 2-hr oral pretreatment, significantly inhibited the increase in nasal resistance caused by PAF (fig. 4A). The inhibition, based on area under the curve, was dose-dependent. As shown in figure 4B, the 0.3 mg/kg dose of ABT-491 was not significantly different from the control PAF responses, whereas all remaining doses of ABT-491 (1.0, 3.0 and 10 mg/kg) were significantly inhibited by 63%, 65% and 88%, respectively. From these results, the ED₅₀ of ABT-491 was found to be 1.0 mg/kg. ABT-491 (10 mg/kg) also reduced the amount of rhinorrhea qualitatively observed after a PAF challenge but had no effect on histamine-induced nasal resistance (not shown). In additional studies the 5-lipoxygenase inhibitor (A-79175) was evaluated for activity against the PAF-induced response. When tested at a dose of 10 mg/kg A-79175 resulted in only weak (25%), nonsignificant inhibition of the AUC of the PAF-induced response (38.5 ± 5.1 vs. 50.1 ± 7.7).

View larger version (23K): [in this window] [in a new window]   Fig. 4.   PAF-induced increase in nasal airway resistance of guinea pigs and inhibition with ABT-491. (A) PAF (50 µg) was instilled into the nostril of guinea pigs given drug vehicle (squares) or 1 mg/kg ABT-491 (triangles) orally 2 hr before challenge. Negative controls are indicated by circles. Nasal airway resistance was monitored at 1-min intervals and is expressed as percent change. Values are the mean ± S.E.M. (B) Area under the curve (AUC) for the percent change vs. time plots were computed for groups receiving the indicated dose of ABT-491 and are presented as the mean ± S.E.M. Percent inhibition of the PAF response is shown above each bar. Statistical comparison vs. antigen control: *P  .05; ***P  .005.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Antigen-induced nasal airway resistance of guinea pigs and inhibition with ABT-491. (A) Guinea pigs given drug vehicle (squares) or 10 mg/kg ABT-491 (triangles) orally 2 hr before challenge were exposed to aerosolized antigen for 30 min. Negative controls are indicated by circles. After antigen challenge, nasal airway resistance was monitored at 1-min intervals and is expressed as percent maximum. Values are the mean ± S.E.M. (B) Dose-response relationship. Nasal airway resistance of guinea pigs given vehicle or ABT-491 was measured 30 min after exposure to antigen. Values, expressed as percent maximum, are the mean ± S.E.M. Percent inhibition of the antigen response is shown above each bar. Statistical comparison vs. antigen control: * P  .05.

	Discussion

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Increased nasal vascular permeability to macromolecules may be one of the underlying causes of rhinorrhea, a symptom of rhinitis (Garrelds et al., 1996; Horvath et al., 1991; Krause 1995). Both PAF and antigen cause vascular leakage when applied topically to the nasal cavities of rats. The response to topical administration of PAF observed in the current study agrees well with previous studies demonstrating PAF-induced vascular leakage in the trachea, bronchi and upper airways in rats and guinea pigs (Evans et al., 1989; Kyriacopoulos et al., 1995; Misawa and Iwamura 1990). Similar nasal responses to antigen have also been reported (Misawa and Iwamura 1990; Takahashi et al., 1990). In the present study ABT-491 inhibited both PAF and antigen- induced nasal vascular permeability with approximately the same potency (ED₅₀ values of 0.3 and 0.5 mg/kg, respectively). The equivalence in potency suggests that the protective effect of ABT-491 for the antigen response is likely the result of the compound's ability to compete with PAF for binding to the PAF receptor (Albert et al., 1997) and further supports the role of PAF in antigen response. These observations, coupled with those demonstrating that PAF is a potent mucous secretagogue (Hotchkiss et al., 1993), lend support to the concept that PAF plays a role in the rhinorrhea associated with allergic rhinitis. The present study is also the first to demonstrate oral activity of a PAF receptor antagonist for blocking antigen-induced nasal vascular permeability in a model of allergic rhinitis.

Although the maximum inhibition resulting from administration of ABT-491 was substantial (74%), the PAF antagonist did not provide complete protection against the antigen-induced permeability response. This less-than-complete inhibition suggests that other mediators in addition to PAF are involved in the permeability response. Histamine, a product of activated mast cells, is well established as a mediator of vascular permeability in the lower airways (Evans et al., 1988, 1989). In the present study an antihistamine blocked a significant portion (56%) of antigen-induced nasal vascular permeability, thus supporting the role of histamine in the upper airway response to antigen. This result is in good accord with results from previous studies in rats and guinea pigs (Mizuno et al., 1991; Takahashi et al., 1990). Because antihistamines are commonly used therapy in allergic rhinitis(Krause 1995), the rat and guinea pig models appear to be clinically predictive, at least as far as the role of histamine is concerned.

In addition to PAF and histamine, serotonin and leukotrienes are also involved in nasal vascular leakage induced by antigen in Brown Norway rats, as is evidenced by the effectiveness of a serotonin antagonist and a 5-lipoxygenase inhibitor in the antigen challenge model. The number of mediators potentially involved in the permeability response to antigen raises the possibility of combination therapy. In the current study nearly complete inhibition (93%) was achieved with a combination of the PAF antagonist and the serotonin antagonist. However, in contrast to the nasal resistance response to antigen discussed below, combined dosing of the PAF receptor antagonist and the 5-lipoxygenase inhibitor did not increase inhibition of the permeability response above that was achieved with either agent alone. This suggests that PAF and leukotrienes may share a common pathway in inducing vascular leakage. One possibility suggested previously by others is that the PAF-induced response is mediated in part by the generation of leukotrienes (Misawa and Iwamura 1990; Seeds et al., 1995). In the present study the 5-lipoxygenase inhibitor A-79175, administered at a dose effective against antigen challenge, had no effect on the PAF-induced response. It thus appears that in sensitized rat model vascular leakage induced by PAF in the nasal cavity is independent of leukotriene biosynthesis.

Nasal congestion, in addition to rhinorrhea, is another symptom of the acute allergic response associated with allergic rhinitis (Krause 1995). Increased nasal airway resistance that mimics the nasal blockage seen in allergic rhinitis is induced experimentally by antigen administration to sensitized animals (Mizuno et al., 1991; Narita and Asakura 1993). In the present study we observed a response to PAF administered topically to the nasal passages of guinea pigs comparable in magnitude (40% of maximum) with that induced by inhalation of aerosolized antigen (55-60% maximum). These results, which are similar to those obtained with PAF administered via inhalation (Misawa and Iwamura 1990), indicate a role for PAF in the acute upper airway response to antigen.

The effectiveness of the PAF receptor antagonist ABT-491 in inhibiting both the PAF and the antigen-induced increases in nasal airway resistance (oral ED₅₀ values of 1.0 and 5.5 mg/kg, respectively) further supports the role of PAF in the response of the upper airways to antigen. CV-3988, a first-generation PAF receptor antagonist, given intravenously (10 mg/kg) has previously been shown to inhibit antigen-induced nasal airway resistance (Misawa and Iwamura 1990). The present study extends these results by demonstrating the oral activity of a potent PAF receptor antagonist for inhibiting the acute phase airway response to antigen.

As for antigen-induced vascular leakage, other mediators in addition to PAF have been implicated in the airway response. Histamine and leukotrienes are capable of inducing nasal airway resistance, and leukotrienes may be particularly important in the late-phase response to antigen (Howarth et al., 1993; Knapp 1990; Mizuno et al., 1991; Narita and Asakura 1993). In the present study the 5-lipoxygenase inhibitor A-79175 and the antihistamine mepyramine, agents which were effective in inhibiting antigen-induced vascular leakage, had only weak and not statistically significant effects (43% and 29% inhibition, respectively) on antigen-induced nasal airway resistance. The lack of effect of the antihistamine agrees well with results from previous studies in guinea pigs and with the clinical observation that H₁-antagonists do not alleviate nasal congestion (Krause 1995; Misawa and Iwamura 1990). The marginal effect of the 5-lipoxygenase inhibitor suggests that leukotrienes, which are important mediators of nasal vascular permeability, may play less of a direct role in the acute nasal airway resistance response to antigen. Despite the lack of efficacy when given as monotherapies, both the antihistamine and the 5-lipoxygenase inhibitor, when given in combination with the PAF receptor antagonist, increased the extent of inhibition of the airway response to antigen, compared with treatment with the PAF receptor antagonist alone. These results illustrate the complexity of interaction among inflammatory mediators and the potential utility of combination therapies.

In summary, the present study demonstrates the potent oral activity of ABT-491 in animal models of allergic rhinitis. The PAF receptor antagonist, alone and in combination with other anti-inflammatory drugs, was effective at inhibiting antigen-induced increases in nasal vascular leakage and nasal airway resistance, responses that mimic primary symptoms of allergic rhinitis. These results support the potential clinical utility of an orally active PAF antagonist for the treatment of this disease.