INTRODUCTION

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Platelet-activating factor (PAF) is an ether-linked, phospholipid (1-O-alkyl-sn-glycero-3-phosphocholine) that is derived after the metabolism of membrane phospholipid and released from a variety of cells, including platelets, neutrophils, basophils, macrophages, and, of specific relevance to asthma, in large amounts from eosinophils (1). PAF exhibits several biologic actions that could be relevant to the pathogenesis of asthma. Its actions include the recruitment and activation of eosinophils (2), release of mediators such as leukotriene C₄ from eosinophils, and stimulation of mucus secretion (3). When inhaled by normal human subjects PAF induces bronchoconstriction and increases airway responsiveness (4, 5), although this has not been found in all studies (6, 7). PAF is present in bronchoalveolar lavage fluid in asthmatic patients under basal conditions (8), and increased levels of PAF were detected in the plasma of patients exhibiting a late asthmatic response after allergen challenge (9). Thus, there is evidence that this mediator may have a potential role in the genesis of airway inflammation in asthma. Despite this, previous studies with PAF receptor antagonists have failed to show any effect on the allergen-induced responses in asthmatic subjects, casting doubt on the importance of PAF as a significant mediator in asthma (10, 11).

SR27417A is a new and more potent PAF receptor antagonist. On intact rabbit platelets the affinity of SR27417A for the PAF receptor was more than five times greater than PAF itself and more than 50- to 60-fold as active as the best synthetic PAF receptor antagonist tested. Additionally, this compound selectively and competitively inhibited the specific binding of the radiolabeled PAF antagonist [³H]WEB2086 to its high affinity receptors on washed rabbit platelets (12). Furthermore, in Phase I studies of healthy male volunteers, the potency of SR27417A was further confirmed by ex vivo studies on leukocyte binding and PAF-induced platelet aggregation both of which were significantly inhibited by single doses of SR27417A. This effect was long-lasting, and complete inhibition for 24 h was achieved with a single dose of 2.5 mg (13). SR27417A appears to be the most potent PAF antagonist yet developed. To evaluate its therapeutic potential in asthma, we studied the effects of 7 d of administration in patients with a demonstrable late asthmatic response (LAR) to allergen inhalation challenge. We chose this model as the allergen-induced late asthmatic response and subsequent airway hyperresponsiveness are associated with airway inflammation as demonstrated by increased influx and activation of inflammatory cells within the airways (14). The response to SR27417A treatment was evaluated examining baseline lung function and airway responsiveness before and after treatment, as well as the responses to allergen challenge in terms of the early asthmatic response, late asthmatic response, and allergen-induced changes in airway responsiveness.

	METHODS

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Subjects

Twelve male subjects with mild atopic asthma participated in the study after giving written informed consent. The study was approved by the Ethics Committee of the Royal Brompton Hospital/NHS Trust. All subjects were admitted to the Clinical Studies Unit of the Royal Brompton Hospital. All were atopic as defined by skin prick testing to common aeroallergens (Dermatophagoides pteronyssimus, mixed grass pollen, cat hair). None had suffered an exacerbation of wheeze nor a respiratory infection within the previous 6 wk. Each subject had clinical features of asthma, which were controlled with ₂-adrenoceptor agonists therapy alone, and had a baseline FEV₁ > 70% of their predicted value (individual characteristics are summarized in Table 1). Inhaled sympathomimetics and caffeinated beverages were withheld for at least 8 h prior to each study visit.

Study Design

This was a randomized double-blind, placebo-controlled, crossover study evaluating 7 d of treatment with SR27417A or matched placebo. The SR27417A dosage was 10 mg daily taken by mouth as a single morning dose.

Study Protocol

Screening. Before study entry, all subjects underwent an initial visit to demonstrate normal physical examination, ECG, and laboratory tests (hematology, clinical chemistry, and urinalysis) before undergoing baseline pulmonary function and a screening allergen inhalation challenge.

All subjects entered into the study were required to show dual asthmatic responses to the screening allergen challenge. The early asthmatic response (EAR) was defined as a fall in FEV₁ from diluent value > 15% within 60 min, and the late asthmatic response (LAR) was defined as a fall in FEV₁ > 15% between 4 and 10 h after allergen challenge.

Visits 2 and 4. After the screening visit subjects attended for a further five visits. At Visits 2 and 4, subjects were prescribed either placebo or SR27417A (as per randomization) and were instructed to take the treatment once a day prior to breakfast for a period of 7 d. Pretreatment PC₂₀ histamine challenges were performed at these visits.

Visits 3 and 5. Allergen challenge was performed on both Visits 3 and 5, i.e., after the treatment periods. Subjects were admitted to the Clinical Studies Unit for 24 h for these visits. Provided albuterol had been withheld for 8 h prior to the visit, subjects underwent histamine challenge. After recovery of lung function to baseline within 2 h, allergen challenge was commenced. The dose of allergen administered as a single bolus was the cumulative dose that had provoked a dual asthmatic response at screening. Measurements of FEV₁ were taken according to the standard operating procedure of the laboratory for as long as 10 h after allergen inhalation. Lung function was recorded again at 24 h prior to a second PC₂₀ histamine challenge. After Visit 3 there was a minimum washout period of 28 d.

Visit 6. A follow-up visit was arranged for 1 wk after the second allergen challenge. A full clinical assessment in addition to lung function measurements was done. Routine hematology, biochemistry, and urinalysis were rechecked.

Measurement of Pulmonary Function and PC₂₀

Pulmonary function was measured with a dry wedge spirometer (Vitalograph, Buckingham, UK). A standard challenge protocol was used for histamine provocation tests. On arrival in the laboratory each subject rested for 15 min prior to three measurements of FEV₁ taken at 1-min intervals, the best of which was taken as the baseline. The challenges were performed using a nebulizer attached to a breath-activated dosimeter (Dosimeter MB3; MEFAR Electromedical, Bovezzo, Italy). The nebulizer delivered particles with an aerodynamic mass median diameter of 3.5 to 4 µm at an output of 9 µl per breath. The nebulizer was set to nebulize for 1 s with a pause time of 6 s at a pressure of 22 psi. Initially, subjects inhaled five breaths of saline-control by inspiring slowly from FRC to TLC then breathholding for 5 s. FEV₁ was measured 2 min after inhalation of the saline. Unless a fall in FEV₁ of > 10% was observed after saline, subjects inhaled five breaths of serially doubling increments of histamine at 3-min intervals until a 20% fall in FEV₁ was recorded from the postsaline value. A log dose-response curve was constructed, and the provocative concentration causing a 20% fall in FEV₁ (log₁₀ PC₂₀) was calculated by linear interpolation.

Allergen Challenge

Allergen challenge was commenced after recovery from the preallergen PC₂₀ bronchial challenge and was administered as five nebulizations from the dosimeter similar to the other airway challenges. Freeze-dried allergen extracts were used. The allergen giving the strongest skin prick response was selected for airway challenge in each subject. Known dilutions of the allergen were made to give final concentrations of 200, 1,000, 2,500, 5,000, 12,500, 25,000, and 50,000 IU/ml. For the screening challenge the initial dose for the allergen inhalation test was 200 IU/ml, and FEV₁ was measured 5 and 10 min after each allergen dose. Serially increasing doses of allergen were inhaled and the cumulative dosage resulting in a 15% reduction within 10 min was recorded and constituted an adequate challenge and the early asthmatic response (EAR). The FEV₁ was recorded at 5, 10, 20, 30, 45, 60, and 90 min and hourly thereafter until 10 h.

For subsequent allergen challenges the cumulative dose of allergen at screening resulting in a dual response was given as a single bolus.

Materials and Drugs

On each study day fresh solutions of histamine (Sigma Chemicals, Poole, UK) and allergen (Aquagen SQ, ALK; Allergologisk Laboratium A/S DK-2970, Horsholm, Denmark) were made in 0.9% saline. The concentration range for histamine was 0.03 to 32 mg/ml. Freeze-dried allergen extracts were used to make dilutions from a stock solution of 100,000 IU/ml. The allergen used for each subject was predicted by the response to skin prick testing.

Analysis of Data

All airway data are expressed as the means and SEM. The extent of the LAR was assessed as the maximal fall in FEV₁ from baseline (expressed as percentage change) and as the area under the FEV₁/time curve between 4 and 10 h (AUC LAR_4-10h).

All PC₂₀ values were log transformed for analysis but expressed as geometric means and geometric standard errors of the mean. Changes in airway responsiveness after allergen challenge are further shown as doubling dose changes of PC₂₀ histamine and calculated using the formula:

[log₁₀PC₂₀(preallergen)  log₁₀PC₂₀ (postallergen)]  log₁₀2

All data were analyzed using Student's t test for matched pairs, and the AUC data were analyzed using ANOVA. For comparison of treatment effects, 95% confidence intervals for the mean difference between placebo and SR27417A are shown for both mean percent maximum fall in FEV₁ and AUC LAR_4-10h. A p value < 0.05 was considered significant.

	RESULTS

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Treatment with SR27417A was well tolerated in all subjects, and there were no clinically significant adverse effects during either period. All laboratory parameters remained stable during the study. The randomization of treatment was balanced and there was no order or carryover effect.

Baseline Lung Function

During the screening period the mean baseline FEV₁ was 4.13 ± 2.4 L/min (88.4 ± 2% predicted). Treatment with SR27417A did not alter baseline lung function. Before and at the end of treatment with SR27417A, baseline FEV₁ values were 3.99 ± 0.13 and 4.02 ± 0.18 L/min, respectively. Similarly, before and after placebo treatment, baseline FEV₁ values were 4.23 ± 0.15 and 4.04 ± 0.22 L/min, respectively.

Allergen-induced Early and Late Responses

All patients had significant EAR at the end of both treatment limbs. AUC EAR: 30.9 ± 7 arbitrary units after placebo, 34.4 ± 9 units after SR27417A treatment (Table 2 and Figure 1).

View larger version (12K): [in this window] [in a new window]   Figure 1.   Allergen-induced responses. % change FEV1 against time. Placebo (dashed line), and SR27417A (solid line).

Eight of 12 patients exhibited profound late asthmatic responses after both placebo and SR27417A treatments. Analysis of all the data from both groups showed that treatment with SR27417A resulted in significant attenuation of the LAR. The fall in FEV₁ measured as AUC LAR_4-10h was 107 ± 24 after placebo and 79 ± 17 after SR27417A (p < 0.05), and the mean maximum percent fall in FEV₁ was 29 ± 6 and 23.5 ± 5.4% for placebo and SR27417A, respectively (p < 0.05) (Table 2 and Figures 1 and 2).

View larger version (10K): [in this window] [in a new window]   Figure 2.   Late asthmatic responses to allergen after placebo and SR27417A expressed as (a) mean maximal fall in FEV1 between 4 and 10 h; (b) area under the curve for % change FEV1 time between 4 and 10 h (AUC LAR4-10h).

The mean improvement on SR27417A with 95% confidence intervals for the maximum percent fall in FEV₁ was 5.5 ± 1.5% (2.2 to 8.8%) and for the AUC LAR_4-10h it was 29 ± 11 (4 to 54).

Effect of Treatment on Airway Responsiveness

Baseline airway responsiveness to histamine was unchanged after 7 d of either treatment; geometric mean PC₂₀ was 0.78 mg/ml before and 0.87 mg/ml after placebo, 0.89 mg/ml before and 0.79 mg/ml after SR27417A (Figure 3a).

View larger version (14K): [in this window] [in a new window]   Figure 3.   Airway responsiveness (showing geometric means) after placebo and SR27417A; (a) before and after 1 wk of treatment, and (b) before and 24 h after allergen challenge.

Allergen challenge resulted in increased airway responsiveness in both treatment groups, showing doubling dose changes in PC₂₀ from preallergen values of 0.74 ± 0.38 for placebo and 0.47 ± 0.44 for SR27417A (geometric mean PC₂₀: placebo, 0.59 mg/ml; SR27417A, 0.66 mg/ml). These changes from preallergen values were significant only for placebo, and, although the change in airway responsiveness was less in the SR27417A group, there were no significant differences between treatments (Figure 3b).

	DISCUSSION

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Our results show that treatment for 1 wk with the potent PAF-antagonist SR27417A significantly reduced the late asthmatic response to allergen challenge as measured by both the maximum percent fall in FEV₁ 4 to 10 h after challenge and the time-integrated fall during this period as measured by AUC. However, allergen-induced EAR, allergen-induced airway responsiveness, baseline lung function, and baseline airway responsiveness were unaltered after SR27417A. This is the first study to demonstrate attenuation of the LAR with a PAF antagonist. The reduction in late response was relatively small, indicating that the role of PAF in allergen-induced responses is likely to be limited.

Despite in vitro and in vivo data in humans that would support a role for PAF in asthma, previous human studies with PAF antagonists, in particular the PAF antagonists WEB2086 and UK74505, have failed to show an effect on either allergen-induced responses (10, 11) or clinical studies of asthmatic patients (15, 16). WEB2086 inhibits immediate bronchoconstriction to inhaled PAF (17), although it has no effect on the EAR, LAR, or allergen-induced increases in airway responsiveness (10). Furthermore, a 12-wk study examining the effects of WEB2086 in atopic asthmatic subjects showed no benefits in terms of lung function, rescue medication usage, or inhaled steroid requirements (15). Similarly, UK74505 abolished acute bronchoconstriction to inhaled PAF for 24 h (18), but in another study it had no effect on allergen responses or allergen-induced changes in airway hyperresponsiveness (11). UK80067, the racemate of UK74505, was studied over a 28-d period in adult asthmatic subjects and showed no benefits on lung function, symptoms, or airway responsiveness (16). These studies and negative studies with other PAF antagonists, BN52063 (19), BN5202 (20), and MK287 (21), suggest that PAF is not an important mediator in asthma.

The data with SR27417A show attenuation of the LAR and, contrary to the previous data, support a role for this mediator. As already discussed, SR27417A shows greater affinity for the PAF receptor than any other PAF antagonist (12), and it is a potent antagonist at both platelet and polymorphonuclear leukocyte PAF receptors (13). Evidence from both human and animal work suggests that there is heterogeneity of PAF receptors (22), and the level of PAF antagonism may depend upon an individual antagonist's potency at each receptor subtype. The lack of effect of the older PAF antagonists might be explained by their lack of antagonism for each PAF receptor subtype (23) and failure to block the effects of endogenous PAF, which may be generated in high concentrations locally within the airways. Furthermore, only a small percentage of PAF generated is released to act on extracellular receptors, and the importance of intracellular receptors is not established (24). The effects seen with SR27417A might be accountable to both membrane-bound receptor antagonism as well as switching off signal transduction pathways following antagonism of intra-cellular receptors.

The lack of effect of SR27417A on baseline lung function and airway responsiveness warrants further discussion. Previous studies have shown PAF-induced bronchoconstriction and heightened airway responsiveness (4, 5), and Y24180, an orally active PAF antagonist, protected against methacholine challenge in atopic asthmatic subjects (25). However there are data from studies examining both PAF itself (6, 7) and PAF antagonists (10, 11) that contradict these findings. The control of bronchial tone and airway responsiveness is complex and under the influence of a number of factors. PAF may influence airway functions via indirect mechanisms such as cellular recruitment and priming of functions, although PAF itself is not a direct bronchoconstrictor per se. Given the efficacy of SR27417A this study supports the view that resting bronchial tone and, in particular, airway responsiveness are dependent on a variety of factors, and antagonism of PAF alone does not have any influence. However, this study shows that after allergen challenge PAF may have a more central role and therefore an effect on protecting lung function.

In the absence of data showing changes in allergen-induced inflammation after treatment with SR27417A, it is not possible to categorically attribute attenuation of the LAR seen in this study to an anti-inflammatory effect. Nevertheless, these results in parallel with previous data support this conclusion, and further experiments with SR27417A examining airway inflammation would clarify this question further. Irrespective of the mechanism, the importance of these findings should be carefully interpreted. In the knowledge of the superior potency of SR27417A, it might be assumed that previous studies have failed to block PAF rather than demonstrating that PAF is not an important mediator after allergen challenge. However, the effect seen amounts to only a 20% inhibition of the LAR. This study serves to demonstrate that although PAF is implicated in the etiology of the LAR, it plays a limited role, and likelihood that PAF antagonists could be useful in the treatment of asthma remains small.