INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

There is increasing evidence that neural mechanisms may play a role in asthma. Inflammation at many sites in the body can lead to an increased sensitivity of nervesespecially the nonmyelinated C fibersto sensory stimuli. One view is that asthma represents a similar phenomenon in the airways. Mediators, released from a variety of inflammatory cells, can stimulate sensory nerves. These nerves trigger, via a local axon reflex, the release of neuropeptides, which themselves in turn induce further inflammation, as well as bronchospasm and mucous secretion.

It has been suggested that disodium cromoglycate and nedocromil may act to an appreciable extent by interfering with neural pathways. In support of this, these drugs are clinically effective against challenges that act over neural pathways such as bradykinin (1) or adenosine (2).

Epinastine is a novel, orally active antiallergic agent introduced for the treatment of asthma (3). Preclinically it showed, in comparison with other antiallergic antihistamines tested (ketotifen and azelastine), an unusually high degree of activity against bronchospasm induced by bradykinin challenge in guinea pigs (4). One object of this work was to investigate the action of epinastine in an animal model of bronchospasm in which neural pathways play an important role. This model used a special rat strain, the BDE rat.

Like humans with asthma, the BDE rat is able to respond to adenosine receptor agonists with bronchospasm (5). Instead of adenosine, which is rapidly metabolized and combines with several adenosine receptor subtypes, the stable adenosine A₃ receptor agonist N ⁶-2-(4-aminophenyl)ethyladenosine (abbreviated APNEA, 0.1 or 0.2 µmol/kg) was used to challenge the animals. Bronchospasm induced by APNEA is substantially reduced by combined cervical vagotomy and atropine treatment (or by administering a neurokinin₂ [NK₂] receptor antagonist), suggesting a major neural component. Bronchospasm is accompanied by release of mast cell mediators such as histamine or 5-hydroxytryptamine (5-HT) and is reduced by pretreating the rats with compound 48/80 to deplete granular cells such as mast cells (6).

In a few studies, for purposes of comparison and to obtain an insight into the mode of action of epinastine, a second adenosine receptor agonist, adenosine A₁-selective 2-chloro-N ⁶-cyclopentyladenosine (CCPA), was used to challenge rats. Although CCPA also induces bronchospasm in BDE rats, the mechanism is different from that of APNEA. CCPA causes only a limited release of biogenic amines and its effect is not blocked by compound 48/80 pretreatment (6).

5-Hydroxytryptamine has been reported to play a role both in the stimulation of tracheal nerve fibers, and in the control of the pulmonary excitatory nonadrenergic noncholinergic response (7, 8). In vitro studies with isolated guinea pig trachea have suggested that noncholinergic contraction is blocked by epinastine acting on a prejunctional 5HT₁-like receptor (8). The binding characteristics of epinastine at different 5-HT receptor subtypes were therefore characterized, and the possible role of 5-HT receptors in the mechanism by which epinastine produces its effects in the in vivo rat model were investigated. In view of the suggestion that an atypical 5-HT receptor can modulate noncholinergic bronchoconstriction by opening a Ca²⁺-activated potassium channel (7), epinastine was also tested in the presence of iberiotoxin, which blocks this channel.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Drugs

Epinastine (WAL 801 CL, 3-amino-9,13b-dihydro-1H-dibenz[c,f  ]imi-dazo[1,5-a]azepine hydrochloride), WAL 1097 CL (3-amino-3H-dibenz- [c,f  ]imidazo[1,5-a]azepine hydrochloride), disodium cromoglycate, MDL 72222 (bemesetron), BIMU 1 [endo-3-ethyl-2,3-dihydro-N-(8- methyl-8-azabicyclo[3.2.1]oct-3-yl)-2-oxo-1H-benzimidazole-1-carboxamide monohydrochloride], ondansetron, [-Ala⁸]-NKA(4-10) (L-aspartyl-L-seryl-L-phenylalanyl-L-valyl--alanyl-L-leucyl-L-methionine), and L-659877 [cyclo(L-glutaminyl-L-tryptophyl-L-phenylalanylglycyl-L-leucyl- L-methionyl)] were synthesized in the laboratories of Boehringer Ingelheim (Ingelheim am Rhein, Germany, and Milan, Italy). Epinastine as used clinically is a mixture of two enantiomers. All the studies described here were carried out with racemic material as used clinically. APNEA was synthesized by Professor C. Woenckhaus (Gustav Embden Zentrum der Biologischen Chemie, Frankfurt, Germany). Ketotifen fumarate, chlorpheniramine, mepyramine, and 5-hydroxytryptamine were purchased from Sigma (Steinheim, Germany). Iberiotoxin, CCPA (2-chloro-N  ⁶-cyclopentyladenosine), 5-carboxamidotryptamine maleate, and ketanserin tartarate were purchased from RBI Research Biochemicals (Natick, MA).

In Vitro Binding Studies (5-HT Receptor Subtypes)

The receptor source (membrane preparation) from either rat or other species was incubated at the stated temperature in duplicate with a specific ligand in the presence of appropriate concentrations of drug (first testing was at 10 µM drug; if significant binding was seen at this concentration four to eight lower concentrations were tested). A rapid filtration technique onto glass fiber filters, presoaked when necessary with polyethyleneimine to reduce nonspecific binding, was used to separate bound and free ligand. The 50% inhibitory concentration (IC₅₀) for the displacement of radiolabeled ligand was determined, e.g., by graphical extrapolation, and K_i values were calculated after correction for the radioligand occupancy shift by the Cheng and Prusoff equation.

5-HT_1a. Affinity constants for the 5-HT_1a receptor were determined by displacement of 1.0 nM 8-OH-[³H]DPAT (8-hydroxy-2- [di-n-propylamino]tetralin; specific activity, 160 Ci/mmol; Amersham) from rat hippocampus membranes in 50 mM Tris-HCl buffer, pH 7.4, over 15 min at 30° C. Nonspecific binding was determined in the presence of 100 µM 5-HT.

5-HT_1b. A modification of the method of Hoyer and colleagues (9) was used. Affinity constants were determined by displacement of 150 pM [¹²⁵I]iodocyanopindolol from striatal membranes of Sprague-Dawley rats, in a buffer of composition 4 mM CaCl₂, 100 µM pargyline, 6 µM ()isoproterenol, 5 mM Tris (pH 7.4), over a period of 15 min at 37° C. Nonspecific binding was determined in the presence of 10 µM 5-HT.

5-HT_1D. Membrane preparations were made from CHO cells expressing cloned human 5-HT_1D receptors as described by Van Sande and coworkers (10). Affinity constants were determined by displacement of 10 nM 5-[³H]HT (60 Ci/mmol; New England Nuclear, Boston, MA) in a buffer containing 50 mM Tris-HCl, 10 mM MgCl₂, 0.2 mM EDTA, 10 µM pargyline, and 0.1% ascorbate, pH 7.4, over a period of 30 min at 37° C. Nonspecific binding was determined in the presence of 10 µM 5-HT.

5-HT_1D. A modification of the method described by Waeber and coworkers (11) was used. Affinity constants were determined by displacement of 0.75 nM 5-[³H]carboxamidotryptamine (20-70 Ci/mmol) from bovine striatal membranes in 50 mM Tris-HCl buffer, pH 7.7, containing 4 mM CaCl₂, 100 nM 8-OH-DPAT, 100 nM mesulergine, 10 µM pargyline, and 0.1% ascorbic acid for 60 min at 25° C. Nonspecific binding was determined in the presence of 1.0 µM 5-carboxamidotryptamine.

5-HT_2a. Affinity constants were determined by displacement of 1.0 nM [³H]ketanserin (60-90 Ci/mmol; Amersham) from rat cerebral cortex membranes in 50 mM Tris-HCl buffer, pH 7.4, over 10 min at 30° C. Nonspecific binding was determined in the presence of 100 µM methysergide.

5-HT₃. Displacement experiments were performed using a receptor-bearing cell line (12). A homogenate of mouse neuroblastoma × rat glioma hybrid (NG 108-15) cell membranes at a protein concentration of about 150 µg/ml, was incubated for 30 min at 30° C with different concentrations of drug and 0.3 nM [³H]Itasetron (88 Ci/mmol; New England Nuclear) in 50 mM HEPES, pH 7.4. Nonspecific binding was determined in the presence of 3 µM MDL 72222.

5-HT₄. Binding to a homogenate of porcine caudate nuclei was determined essentially as described by Schiavi and others (13). Displacement experiments were performed for 30 min at 30° C in the presence of 0.1 nM [³H]GR 113808 (Amersham). The assay buffer contained 50 mM HEPES, pH 7.4. Nonspecific binding was determined by displacement with 10 µM BIMU 1.

5-HT₇. Binding of 2.0 nM [³H]LSD (60-80 Ci/mmol) to a rat 5-HT₇ receptor expressed in CHO cells (14) was measured for 60 min at 37° C in 5 mM Tris-HCl buffer, pH 7.4, containing 1 mM MgCl₂ and 0.05 mM EDTA. Nonspecific binding was determined in the presence of 0.1 µM 5-carboxamidotryptamine.

In Vivo Studies

Animals. BDE/Han strain rats were bred in the laboratories of Boehringer Ingelheim, and fed Altromin R8014 diet.

Measurement of bronchospasm. Each rat was anesthetized by intraperitoneal injection of 60 mg/kg pentobarbital sodium (Nembutal; Sanofi). The trachea was cannulated with a 1.5-mm-diameter polyethylene catheter; the jugular vein and carotid artery were cannulated with 0.5-mm catheters. The rat was laid on a small-animal operating table and its temperature (measured with a rectal thermometer) maintained between 36-37° C by use of an electrically heated mat. Tracheal airflow was recorded by means of a Fleisch type 00000 pneumotachograph tube attached to a Validyne MP-45-22-871 differential pressure transducer and an MA6 carrier amplifier (Modular Instruments, Malvern, PA). Esophageal pressure was recorded via a fluid-filled catheter and a Statham p23XL pressure transducer attached to an MA2 transducer amplifier. Tidal volume (VT) was calculated by integration of the flow signal, and breathing rate determined from peak-to-peak measurements on the volume waveform. Pulmonary resistance (RL) was calculated from pressure and flow signals measured at isovolumetric points during inspiration and expiration, and dynamic compliance (Cdyn) from the zero flow points. Blood pressure (systolic, diastolic) was also recorded via a Statham p23XL pressure transducer attached to an MA2 transducer amplifier. Mean arterial blood pressure was the arithmetic average of all pressure measurements between successive pressure troughs. The rat was allowed to breath spontaneously throughout the study.

Rats were allowed to remain undisturbed until breathing and blood pressure were stable (approximately 5 min) then challenged with APNEA, CCPA, or 5-HT in a volume of 1 ml/kg via the indwelling jugular vein catheter. The dose of agonist was selected to give an approximately two- to threefold increase in RL in the control group, i.e., for APNEA either 0.1 or 0.2 µmol/kg according to the sensitivity to APNEA of a particular group of rats. One or more controls were always run on the same day as the experimental animals and with the same APNEA dose. Flow and pressure measurements were recorded continuously before and (except for an initial period of approximately 5 s during which the challenge substances were injected) 5 min after challenge. For calculation purposes, control values represent average values recorded in the half-minute before injection of adenosine receptor agonist and are compared with maximum (pulmonary resistance) or minimum (blood pressure, heart rate, air flow, tidal volume, dynamic compliance) measurements after injection of adenosine receptor agonist. Postchallenge maxima and minima were identified by a sliding average over a 5-s period.

Challenge with drugs modulating agonist effects. In tests for pharmacological modulation of APNEA-, CCPA-, 5-HT-, or [-Ala⁸]- NKA(4-10)-induced bronchospasm by intravenous epinastine or comparison drugs, a volume of 1 ml/kg epinastine or comparison drug was administered via an indwelling jugular vein catheter 2 min before the agonist used to induce bronchospasm. In those studies testing the ability of L 659877 to modulate the inhibition of APNEA-induced bronchospasm by epinastine, the neurokinin receptor antagonist was injected together with the epinastine. In those studies testing the ability of iberiotoxin to modulate the inhibition of APNEA-induced bronchospasm by epinastine, the potassium channel blocker drug was injected intravenously 8 min before the epinastine, that is, 10 min before the APNEA challenge. Vehicle used was saline, except in the studies with L 659877, when polyethylene glycol (PEG) 200 was employed. Vehicle controls were run alongside the test substances. For the studies involving oral administration, epinastine, ketotifen, or vehicle was administered by gavage in 10 ml/kg water 2 h before challenge. For studies with intratracheal administration the substance was administered in 0.4 ml/kg 0.45% saline solution 10 min before challenge, again with appropriate vehicle control.

Statistical analysis; pulmonary function parameters. The values measured after administration of APNEA, 5-HT, or [-Ala⁸]-NKA(4-10) were expressed as percent increase for the parameter lung resistance and as percent reduction for the parameters tidal volume and dynamic compliance related to the value measured before administration of APNEA or 5-HT. Wilcoxon tests calculating exact p values by the means of a permutation test procedure were composed, comparing the doses of the test compounds (e.g., epinastine, chlorpheniramine, or disodium cromoglycate; Figure 1) with the control. When more than one group treated with the same potential antagonist was to be compared with control (e.g., epinastine and ketotifen; Figure 2), a Jonckheere test for trend (reduction or increase of the parameter decreased with increasing doses) was applied. In the case of a statistically significant trend exact Wilcoxon tests were calculated. Because of the significant trend a method of ordered hypotheses was performed. In the first step the highest dose of the test compound was compared with the control at the significance level  = 0.05. If a statistically significant difference could be found (p value less than 0.05) the next lower dose was tested in the same way and so on until the p value of a comparison exceeded 0.05. In this case the procedure stopped. The logarithms of dose and the medians of each dose were fitted to straight lines for all test compounds, using the nonparametric method introduced by Theil. The slopes of epinastine and ketotifen were compared. As no evident difference could be seen a common slope was assumed and the 50% effective dose (ED₅₀) values were taken from the straight lines as that dose of the respective test compound, which causes an effect of 50% related to the median of the control.

View larger version (29K): [in this window] [in a new window]   Figure 1.   Effects of epinastine (10 µg/kg, intravenous), chlorpheniramine (10 µg/kg, intravenous), and disodium cromoglycate (30 µg/kg, intravenous) on increase in total lung resistance (RL) induced by 0.1 µmol/kg APNEA. Columns shown are medians (with upper and lower quartiles) of results from 12 rats (control, 43 rats). Significance values determined by Wilcoxon test. The RL value was similar (Kruskal-Wallis test, p > 0.05) in all groups before treatment (median, 0.38 cm H2O/ml/s; quartiles, 0.34-0.45; n = 79).

View larger version (22K): [in this window] [in a new window]   Figure 2.   Effect of epinastine (solid squares) or ketotifen (open circles) given orally 2 h before challenge with 0.2 µmol/kg APNEA on (a) the percentage increase induced by APNEA in total lung resistance (RL), (b) the percentage decrease induced by APNEA in dynamic compliance (Cdyn), and (c) the percentage decrease induced by APNEA in tidal volume (TV). Each point is the median of measurements on 10 individual rats. The horizontal line shows the median of measurements on 39 control rats given only vehicle before APNEA. Regression lines for the relationship between percentage change in each pulmonary parameter and logarithm of drug dose were calculated using the nonparametric method described by Theil. The regression line for epinastine is shown by a solid line, and for ketotifen by a dotted line.

For the parameters mean arterial blood pressure and heart rate, an analysis of variance was calculated. The effects of antagonists on the adenosine agonist or 5-HT-induced effects were determined by comparison with the vehicle control, using a t test and taking the error term of the analysis of variance as an estimate of the variance.

In RESULTS, values of blood pressure and heart rate are quoted as means with standard error of the mean and changes of total lung resistance as medians with upper and lower quartiles (in parentheses).

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Binding Studies (5-HT Receptor Subtypes)

Epinastine showed high affinity for the 5-HT_2a and 5-HT₇ receptor subtypes, modest binding to the 5-HT₃ receptor subtype, and no biologically significant binding to 5-HT_1a, 5-HT_1b, 5-HT_1D, 5-HT_1D, and 5-HT₄ subtypes (see Table 1). In comparison with epinastine, ketotifen and chlorpheniramine showed only weak binding to the 5-HT₇ receptor. Ketotifen shared with epinastine a modest binding to the 5-HT₃ receptor, whereas the standard antihistamine chlorpheniramine lacked biologically meaningful binding to this receptor. A small structural modification (a 9,13 double bond) caused a considerable loss of binding activity to the 5-HT₃ and 5-HT₇ receptors (Table 2).

Inhibition of Adenosine Receptor Agonist-induced Bronchospasm

Intravenous epinastine. Epinastine (10 µg/kg) given intravenously 2 min before 0.1 µmol/kg APNEA challenge significantly (p < 0.01) reduced the ability of this adenosine agonist to induce bronchospasm as assessed by increased lung resistance, decreased lung compliance, or decreased tidal volume. The effect of epinastine (10 µg/kg) on APNEA-induced increase in lung resistance was comparable to that produced by 30 µg/kg disodium cromoglycate administered intravenously, but 10 µg/kg chlorpheniramine was ineffective (Figure 1). Epinastine (10 µg/kg) had no effect on APNEA-induced changes in heart rate (mean decrease 65% for both epinastine-treated rats and controls). Even at a dose as high as 100 µg/kg, epinastine did not significantly decrease CCPA-induced changes in the measured pulmonary parameters.

Oral epinastine. Epinastine given orally 2 h before challenge with 0.2 µmol/kg APNEA dose relatedly and significantly (Jonckheere test, p < 0.0005) blocked the adenosine agonist-induced changes in all the pulmonary function parameters that were assessed. The ED₅₀ of epinastine for inhibition of APNEA-induced increase in RL was 0.47 mg/kg, for decrease in Cdyn 1.88 mg/kg, and for decrease in VT 0.69 mg/kg (see Figure 2a, b, and c). Comparison substance ketotifen, although having some activity, was weaker, the respective ED₅₀ values being 4.55 mg/kg (RL), 12.28 mg/kg (Cdyn), and 2.23 mg/kg (VT).

Inhibition of APNEA-Induced Bronchospasm by Epinastine in the Presence and Absence of NK₂ Receptor Antagonist L 659877

Not only was the increase in pulmonary resistance induced by 0.1 µmol/kg APNEA substantially reduced by prior treatment with intravenously administered 10 µg/kg epinastine (see preceding section), the greater part (about three-quarters) of the response to this dose of APNEA could also be blocked by prior administration of an NK₂ receptor antagonist, L 659877. L 659877 (1 mg/kg), administered intravenously 2 min before 0.1 µmol/kg APNEA, reduced the subsequent median increase in pulmonary resistance from 201% (123-284%) to 55% (39-85%) (n = 6/group; statistically significant difference p < 0.05). Intravenously administered epinastine (10 µg/kg) was also tested against that part of APNEA-induced bronchospasm not blockable by 1 mg/kg L 659877. Because the extent of the bronchospasm induced by 0.1 µmol/kg APNEA in the presence of L 659877 was too small to permit accurate measurement of its inhibition, this study was carried out using an APNEA dose of 1.0 µmol/kg. With this high dose the percentage increase in resistance induced by APNEA even in the presence of 1 mg/kg L 659877 was 159% (62-781%, n = 12/ group). When the APNEA challenge was preceded 2 min beforehand by intravenously administered 10 µg/kg epinastine the percentage increase in RL after APNEA was 258% (96- 528%, n = 12/group). The difference between the effects of APNEA challenge with and without 10 µg/kg epinastine was, in the presence of the NK₂ receptor antagonist, not statistically significant (p > 0.05).

Inhibition of APNEA-Induced Bronchospasm by Epinastine in the Presence and Absence of the Potassium Channel Blocker Iberiotoxin

There was a statistically significant difference (p < 0.01, Wilcoxon) between the degree of APNEA-induced bronchospasm (increase in RL) in animals treated with intravenously administered 10 µg/kg epinastine in combination with intravenously administered 20 µg/kg iberiotoxin, and those treated with 10 µg/kg epinastine alone. Epinastine had no significant protective activity in the iberiotoxin-pretreated animals (Figure 3). Iberiotoxin, at 20 µg/kg, did not alone have a significant effect on APNEA-induced bronchospasm.

View larger version (29K): [in this window] [in a new window]   Figure 3.   Increase in total lung resistance (RL) after injection of 0.2 µmol/kg APNEA and pretreatment with epinastine (10 µg/kg), iberiotoxin (20 µg/kg), or a combination of these two substances. Columns shown are medians (with upper and lower quartiles) of results from 12 rats per group. Control (open bars); epinastine (hatched bars). Significance values determined by Wilcoxon test. The RL value was similar (Kruskal-Wallis test, p > 0.20) in all groups before APNEA treatment (median, 0.38 cm H2O/ml/s; quartiles, 0.34-0.43; n = 48).

Effect of High Doses of a Substance with Antihistamine Activity Comparable to Epinastine, but Lacking Significant Anti-5-Hydroxytryptamine Activity

Chlorpheniramine, 0.01 or 1.0 mg/kg, administered intravenously 2 min before 0.2 µmol/kg APNEA challenge, had no significant effect on APNEA-induced bronchospasm. Median values for percentage increase in RL (n = 12 for all groups) were as follows: control, 215% (upper and lower quartiles, 127-346%); chlorpheniramine (10 µg/kg, administered intravenously), 353% (115-799%); chlorpheniramine (1 mg/kg, administered intravenously), 375% (184-606%) (Kruskall-Wallis, p < 0.05). A second antihistamine, mepyramine, 1 mg/kg administered intravenously, was similarly ineffective.

Effect of Comparison Substances with 5-HT Agonistic and Antagonistic Activity

Ketanserin, 0.1 mg/kg, administered intravenously 2 min before 0.2 µmol/kg APNEA, markedly and significantly (p < 0.005) reduced APNEA-induced bronchospasm. Ondansetron (0.1 mg/kg) also reduced APNEA-induced bronchospasm, but the effect reached statistical significance (p < 0.05) only when the ondansetron was given in the presence of ketanserin (Figure 4). 5-Carboxamidotryptamine, 0.01 mg/kg administered intratracheally, was also effective in blocking APNEA-induced bronchospasm (Figure 5).

View larger version (29K): [in this window] [in a new window]   Figure 4.   Increase in total lung resistance (RL) after injection of 0.1 µmol/kg APNEA and pretreatment with vehicle, ketanserin (0.1 mg/kg), ondansetron (0.1 mg/kg) or ketanserin plus ondansetron (both 0.1 mg/kg). Columns shown are medians (with upper and lower quartiles) of results from 12 rats (control, 13 rats). Significance values determined by Wilcoxon test. The RL value was similar (Kruskal-Wallis test, p > 0.05) in all groups before APNEA treatment (median, 0.39 cm H2O/ml/s; quartiles, 0.33-0.44; n = 49).

View larger version (27K): [in this window] [in a new window]   Figure 5.   Increase in total lung resistance (RL) after injection of 0.2 µmol/kg APNEA and pretreatment 2 min before the adenosine agonist with vehicle or 0.01 mg/kg carboxamidotryptamine given by intratracheal instillation. Columns shown are medians (with upper and lower quartiles) of results from 24 rats per group. Significance is by Wilcoxon test.

Inhibition of Bronchospasm Induced by 5-Hydroxytryptamine

Unlike the adenosine agonists, 5-HT could be administered repeatedly without a marked loss of bronchospasm-inducing activity. It was therefore possible to make a study in which the same animal received the same dose of agonist on several occasions. The dose of 5-HT was between 20 and 60 µg/kg, and set for each individual animal in a preliminary dose titration. The 5-HT challenges were separated by a period of 10 min. There was no significant difference (p < 0.05, Wilcoxon) between the increase in RL after the first and second 5-HT challenges (control phase). The rats were then pretreated either with intravenously administered saline or epinastine at 0.001, 0.01, or 0.1 mg/kg 2 min before a third 5-HT challenge (n = 6/ dose level). The epinastine dose relatedly inhibited the 5-HT-induced increase in RL (Jonckheere-Terpstra test, p < 0.005). The ED₅₀ was 13.8 mg/kg administered intravenously. A fourth challenge 10 min later was also dose-dependently inhibited (Jonckheere-Terpstra test, p < 0.001); the ED₅₀ was 3.2 mg/kg administered intravenously.

Effect on Bronchospasm Induced by an NK₂ Receptor Agonist

Epinastine, at the same dose (10 µg/kg, intravenous) at which it inhibited the ability of APNEA to increase the RL, did not effect the ability of the NK₂ receptor agonist [-Ala⁸]-NKA(4-10) to induce bronchospasm. The standard NK₂ receptor antagonist L 659877 (1 mg/kg, intravenous) was effective when tested in the same rats (positive control). These results are illustrated in Figure 6.

View larger version (27K): [in this window] [in a new window]   Figure 6.   Increase in total lung resistance (RL) after intravenous injection of 300 µg/kg [-Ala8]-NKA(4-10). The NK2 receptor agonist was injected three times, with 20 min between injections. The first injection (open bar) was preceded (2-min pretreatment) by saline, the second (hatched bar) by 10 µg/kg epinastine (intravenous) and the third (cross-hatched bar) by 1 mg/kg L-659,877 (intravenous). Columns shown are medians (with upper and lower quartiles) of results from 12 rats per group. Significance of the comparison to the control is by Wilcoxon test.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

This study demonstrates that even at low doses epinastine hydrochloride is active in blocking bronchospasm in an animal model in which standard antihistamines such as chlorpheniramine are inactive. The notable difference between the activity of epinastine and chlorpheniramine here observed contrasts with the similar affinities of these two drugs for the histamine H₁ receptor (15). The oral ED₅₀ of only 0.47 mg/kg seen here for inhibition by epinastine of APNEA-induced bronchospasm compares with an ED₅₀ of 2.2 mg/kg reported previously for inhibition by epinastine of histamine skin wheal in rats (16). Such results suggests that, whatever mechanism is involved, the nonantihistamine activity of epinastine, responsible for its ability to inhibit bronchospasm in this model, operates at epinastine levels on the same order of magnitude as those relevant for the antihistamine effect.

The Inhibition of Neurally Mediated Mechanisms by Epinastine

An important feature of the model here investigated is that it is a so-called indirect, neurally mediated model (5, 6). Although bronchospasm induced by APNEA was not quite completely blocked by the NK₂ receptor antagonist L 659877, the studies in which epinastine and L 659877 treatment were combined demonstrated that the part of the bronchoconstrictor response to APNEA blocked by the NK₂ receptor antagonist was also the part of the response to APNEA that was blocked by low-dose epinastine. The inhibition of bronchospasm in this neurally mediated model agrees with previous reports showing inhibition by epinastine of bronchospasm induced by electrical field stimulation both in vitro (8) and in vivo (17). Inhibition of bronchospasm cannot be explained by a direct antagonism of the NK₂ receptor by epinastine, as the drug does not block bronchospasm induced by the NK₂ receptor agonist [-Ala⁸]-NKA(4-10).

The ability to interfere with neurally modulated bronchospasm is not unique to epinastine. Azelastine and ketotifen have both been reported to inhibit electrical field stimulation-induced contraction of tracheal muscle (18, 19), and azelastine has been shown to block release of the neuropeptide substance P into the bronchoalveolar fluid of asthmatic subjects after allergen provocation (20). However, the low, pharmacologically relevant, doses at which epinastine produced its effects in this study make epinastine particularly noteworthy.

Modulation of neurally mediated bronchospasm has also been reported to account for the activity in animal models of drugs of a number of classes including GABA_B agonists, µ-opioid receptor agonists, and neuropeptide Y agonists. Stretton and others (21) have proposed that many of the drugs that indirectly inhibit excitatory nonadrenergic noncholinergic neural responses in airways do so via a common inhibitory mechanism involving activation of prejunctional large conductance Ca²⁺-activated K⁺ channels. These channels can be blocked by iberiotoxin. It is therefore interesting that in the presence of 20 µg/kg iberiotoxin epinastine could not significantly inhibit APNEA-induced bronchospasm.

Our model, in which APNEA is used as challenge agent, is known to be dependent not only on neural mechanisms but also on the presence either of mast cells or a closely related cell type (6). If the same strain of rat (the BDE rat) is challenged with a different kind of adenosine receptor agonist (CCPA), which has high affinity for the adenosine A₁ receptor but lacks affinity for the adenosine A₃ receptor subtype, then bronchospasm can also be produced, but in this case the effect is not associated with much release of mast cell mediators and is not blocked by pretreatment of the animals with compound 40/80, suggesting that mast cells are not involved (6). Epinastine at low doses did not block bronchospasm induced by CCPA, showing that epinastine does not nonspecifically prevent contraction of the bronchial musculature. The inhibition by low-dose epinastine of APNEA- but not CCPA-induced bronchospasm is in accord with the concept that a major mechanism of action of epinastine relates to mast cells, and is compatible with an effect of epinastine on mast cell-nerve interaction. This might be through inhibiting mast cell mediator release (22) or by blocking an effect of mast cell mediators on nerves.

The ability of mast cell products to stimulate peripheral nerves has been described in a number of publications. A number of possible mast cell mediators (e.g., leukotrienes, histamine, 5-HT) have been implicated. 5-HT is of particular interest because it has been proposed that activation of an atypical 5-HT receptor can block excitatory nonadrenergic noncholinergic bronchoconstriction by a mechanism that is blocked by the K⁺_Ca channel antagonist charybdotoxin (7). Further, in vitro data suggest that epinastine can inhibit bronchoconstriction induced by electrical field stimulation by activation of an atypical 5-HT₁-like receptor (8).

Epinastine Interacts with 5-HT Receptors

Epinastine has previously been reported to bind both -adrenergic and 5-HT₂ receptors (16). The study here described confirms the previously reported interaction with the 5-HT_2a receptor, and describes a new binding activityto the 5-HT₇ receptor. Epinastine has also been found to possess a modest but significant binding to the 5-HT₃ receptor subtype. The ability of epinastine to bind the 5-HT₃ and 5-HT₇ receptors is sensitive to small changes in molecular structure. Thus the epinastine metabolite WAL 1097, although differing from epinastine itself in only one double bond, has much lower binding activity for these receptors than epinastine itself. Further, epinastine discriminates in its binding behavior between 5-HT₇ and several related 5-HT receptor subtypes. Thus epinastine has no binding activity for the 5-HT_1a, 5-HT_1b, 5-HT_1D, and 5-HT_1D receptor subtypes, although several other ligands for the 5-HT₇ receptor (e.g., 5-carboxamidotryptamine) have appreciable binding activity for some or all of these related 5-HT receptor subtypes.

In rat airways, 5-HT can exert both a direct contractile effect on the smooth muscle of trachea and bronchus and an indirect action via the action of 5-HT on sensory nerves. In general, much higher doses are required to stimulate smooth muscle directly than to activate neural pathways of bronchoconstriction. For physiologically relevant doses of 5-HT, direct stimulation of smooth muscle is probably unimportant relative to indirect, neurally mediated effects (23). The dose of epinastine active in our APNEA-induced model (10 µg/kg, intravenous) was considerably lower than that (> 3 mg/kg) required to block the same degree of bronchoconstriction induced by a high dose of intravenous 5-HT. It is for this reason that the ability of low (pharmacologically relevant) doses of epinastine to modulate bronchospasm probably involves 5-HT receptors controlling the functioning of nerves rather than 5-HT receptors directly on muscles.

In previous published studies, a role in the control of nonadrenergic noncholinergic mediator release or action has been proposed for 5-HT₃ and 5-HT₂ receptor subtypes and possibly for an unidentified 5-HT₁-like receptor subtype.

Significance of the 5-HT₃- and 5-HT₂-Binding Activity of Epinastine

Whereas in some previous published studies no activity of 5-HT₃ or 5-HT₂ receptor subtypes could be identified (7, 24), other studies have suggested (both in guinea pig and humans) a role for 5-HT₃ receptors in the prejunctional modulation of at least the cholinergic component of bronchoconstriction following electrical field stimulation (25). There have also been suggestions concerning a role for 5-HT_2a and 5-HT₃ receptors in the neurally mediated component of the response to exogenous 5-HT, although not in all systems can activity of both 5-HT₂ and 5-HT₃ receptors be shown (23, 26).

To determine whether 5-HT₃ receptor binding might contribute to the ability of epinastine to block APNEA-mediated bronchospasm a known 5-HT₃ receptor antagonist, ondansetron, was tested in the BDE rat model. Although ondansetron is a relatively potent 5-HT₃ receptor antagonist (K_i in this receptor binding assay, 4.6 nM) it was not alone able to reduce APNEA-induced bronchospasm significantly. However, when ondansteron was combined with the 5-HT_2a antagonist ketanserin, then the two drugs combined were more active than ketanserin alone, suggesting that under particular circumstances the 5-HT₃ receptor might still have a role.

Significance of the 5-HT₇-Binding Activity of Epinastine

The pharmacological profile of the 5-HT₇ receptor, to which epinastine binds, fits in with the atypical 5-HT₁-like receptor that Ward and others (7) and Pype and others (24) described as being involved in the regulation of nonadrenergic noncholinergic- bronchoconstriction in airways, and the known binding specificities of the 5-HT₇ receptor are compatible with those of the prejunctional 5-HT₁-like receptor, activation of which has been suggested to account for the ability of epinastine to block bronchospasm induced by electrical field stimulation in guinea pig trachea (8). Further, a known 5-HT₇ agonist, 5-carboxamidotryptamine, is here reported to be active at low doses (10 µg/kg, intratracheal) in our APNEA-induced bronchospasm model. However, the lack of specificity of the pharmacological agonists and antagonists available at present means that caution should be exercised in interpreting these results. Conclusive proof must await the availability of tools such as 5-HT₇ knockout animals or specific receptor antisense nucleotides.

Relevance of the Results with This Animal Model to the Clinical Situation

Orally administered epinastine was effective against APNEA-induced bronchospasm (reduction in RL) with an ED₅₀ of 0.47 mg/kg, which compares with a recommended daily dose of epinastine in humans of 20 mg. On the basis of the known pharmacokinetics of epinastine in humans and rats (3, 27), the doses at which effects of epinastine were seen seem pharmacologically relevant.

The rat does differ in an important respect from humans in that in the rat, the mast cells contain appreciable amounts of 5-HT, whereas the major biogenic amine in human mast cells is histamine. Histamine can also stimulate C fibers and induce axon reflexes, as well as having a direct effect on smooth muscle. The potent antihistamine activity of epinastine is likely to be important for its clinical action. However, just because 5-HT does not occur in human mast cells, a role for 5-HT in human allergic reactions and asthma should not be excluded. Although the 5-HT content of the human lung is not as high as that of the rat, some is present (28). As well as in platelets, in humans and rats 5-HT is also localized (together with various neuropeptides) in a class of granulated epithelial cells termed pulmonary neuroendocrine cells (PNECs). These are located throughout the lung from the trachea to the alveoli, but mostly in smaller conducting airways. There is an intimate connection between this system of 5-HT-releasing cells and the nonadrenergic noncholinergic system (29, 30). PNECs are most prominent in neonates, but are also present, although less clustered, in adults (31). Clinically, an increased number of PNECs is associated with several pulmonary diseases, especially those associated with chronic airflow obstruction and hypoxia (30). Experimental studies (guinea pigs) have shown increased numbers of PNECs in the bronchial wall after allergic sensitization, and degranulation after antigen challenge (32).

We speculate that the interaction of 5-HT₇ receptors in the lung with 5-HT released from PNECs or mast cells might lead to modulation of local axon reflexes, which in the absence of the drug would have caused contraction of bronchial muscle. Most attention in 5-HT₇ receptor research to date has centered on receptors in the brain but 5-HT₇ receptor mRNA is also widely distributed peripherally. The low lipophilicity and marked hydrogen-bonding ability of epinastine prevent its penetration of the blood-brain barrier, and in clinical studies with therapeutically relevant doses, epinastine showed no central effects (33). Only the effects of epinastine on peripheral 5-HT₇ receptors are likely to be relevant to its therapeutic profile.

No drugs whose mode of action is primarily as a 5-HT agonist or antagonist are used in asthma, and the one drug in this class tested, ketanserin, did not affect pulmonary function at rest or after exercise (34). Ketanserin did, however, reduce adenosine-induced bronchospasm in asthmatic subjects (35). We suggest that the interaction of epinastine with 5-HT receptors should not be ignored. Rather, the activity of epinastine and other antiallergic drugs should be considered in relation to their complete spectrum of activities against biogenic amine (histamine and 5-HT) receptors. The binding of epinastine to 5-HT₇, 5-HT₂, and 5-HT₃ receptors may, in addition to its already published antihistamine activity and mast cell-stabilizing properties, contribute to the antiallergic activity of epinastine by modulation of local neuropeptide-mediated neural responses.