Charles Advenier

A potent and selective non-peptide antagonist of the neurokinin a (NK2) receptor

SR 48968 is a potent and selective non-peptide antagonist of the neurokinin A (NK2) receptor. SR 48968 selectively inhibited neurokinin A binding to its receptor and was a competitive antagonist of neurokinin A-mediated contraction of different isolated smooth muscle preparations from various species including human. In vivo, the compound inhibited the bronchoconstriction induced by neurokinin A in guinea pigs. SR 48968 can be used to study the physiological or pathological role of neurokinin A and may be useful in the treatment of neurokinin A-dependent pathology.

Neurokinin A (NK2) receptor revisited with SR 48968, a potent non-peptide antagonist

SR 48968, a new non-peptide antagonist of NK2 receptors, has been tested in a variety of isolated smooth muscle preparations from rats, guinea pigs, rabbits, hamsters and men, in order to assess its selectivity for NK2 receptors as well as its competitivity and specificity. The compound has been found to be inactive as a stimulant or relaxant in all preparations but to exert a potent, competitive antagonism, particularly in tissues obtained from rabbits (pA2 9.8-10.3), guinea pigs (10.5), rats (9.4-9.6), men (9.36-9.6) and hamsters (7.45-8.6). SR 48968 is therefore more active on the NK2A than on the NK2B receptor subtype and the human receptor is close to the NK2A subtype. SR 48968 exerts an antagonism of the competitive type and is therefore suitable for receptor classification despite its slow reversibility in vitro. The sensitivity of NK2A receptors to SR 48968 is at least 1000 times higher than those of NK1 and NK3 receptors. The compound does not affect the effects of bradykinin, angiotensin or bombesin. Because of its activity in human tissues, its potency and long duration of action, SR 48968 is a new promising pharmacologic and possibly therapeutic agent.

Comparison of the effects of epithelium removal and of an enkephalinase inhibitor on the neurokinin‐induced contractions of guinea‐pig isolated trachea

1 The influence of epithelium removal and/or thiorphan on the effects of neurokinins (substance P (SP), neurokinin A (NKA), neurokinin B (NKB)) and related peptides on airway contractility was investigated on the guinea‐pig isolated trachea. 2 Removing the tracheal epithelium significantly enhanced the sensitivity but not the maximum contractile responses to the peptides. 3 After removal of the epithelial layer, the shifts to the left of the log concentration response curves were greater for SP and SP‐OMe (1.62 and 1.94 log units, respectively) than for two SP analogues substituted in position 9 namely [Pro9]SP sulfone and [β‐Ala,4 Sar9]SP(4–11)sulfone (0.66 and 0.68 log units, respectively). The leftward shifts for compounds related to NKA or NKB lay between 0.58 and 0.73 log units. 4 The leftward shifts of the log concentration‐response curves for SP, SP‐OMe, [Pro9]SP sulfone, [β‐Ala4, Sar9]SP(4–11) sulfone and NKA were of similar magnitude after removal of the epithelium or after pretreatment with thiorphan (10−5 m), an enkephalinase inhibitor, in the presence of epithelium. No significant additional shift of the curves to the left was observed with thiorphan plus epithelium removal. 5 The results obtained with the selective agonists for each of the three classes of neurokinin receptor (i.e NK1, NK2, NK3) suggest that the guinea‐pig trachea contains receptors for SP and NKA but few if any for NKB. 6 It was concluded that neurokinins and related peptides (especially SP and analogues not substituted in position 9) are degraded by enkephalinase mainly located in the tracheal epithelium and that the addition of thiorphan or epithelium removal results in an inhibition or loss of enkephalinase activity, thereby increasing similarly the potencies of these peptides. It was, therefore, suggested that the supersensitivity to neurokinins produced by epithelium removal was due neither to the elimination of a permeability barrier nor to reduced production of a relaxant factor, but mainly to reduced peptide degradation.

The role of tachykinin receptor antagonists in the prevention of bronchial hyperresponsiveness, airway inflammation and cough

Several recent observations suggest that tachykinins, such as substance P and neurokinin A, might be involved in the pathogenesis of bronchopulmonary alterations. Progress in investigations on the physiological and pathological roles of tachykinins has been greatly facilitated by the availability of a number of highly selective nonpeptide antagonists for tachykinin neurokinin 1, 2 and 3 (NK1, NK2 and NK3) receptors. The use of selective tachykinin NK2 receptor antagonists suggests that tachykinin NK2 receptor stimulation plays an important role in the development of airway hyperresponsiveness in the guinea-pig. Others studies have also indicated that tachykinin NK1-receptors are involved in immediate or delayed neurogenic inflammation including microvascular leakage and the subsequent increase in plasma protein extravasation. A role for the sensory neuropeptide system has also been proposed in cough, as shown by the observation that the antitussive effect of tachykinin NK2 receptor antagonists has clearly been demonstrated in several experimental conditions, but the effect of tachykinin NK1 receptor antagonists is still debated. Taken together, the results obtained with the various selective receptor antagonists provide pharmacological evidence that tachykinins play a role in delayed bronchopulmonary alterations and suggest that tachykinin receptor antagonists may be useful for investigating mechanisms and possibly reducing airway functional alterations in asthmatic patients.

Relative potencies of neurokinins in guinea pig trachea and human bronchus.

The three endogenous neurokinins, substance P (SP), neurokinin A (NKA) and neurokinin B (NKB), as well as NKA-(4-10), carbachol, acetylcholine and histamine, were tested in guinea pig tracheae and human bronchi in order to compare the activities of peptides and non-peptide agents and to characterize the neurokinin receptors by means of agonists. Neurokinin A and NKA-(4-10) were potent stimulants of the two preparations: pD2 values for NKA-(4-10) averaged 8.62 in the guinea pig trachea and 7.50 in the human bronchus. The rank order of potency of neurokinins was NKA-(4-10) greater than NKA greater than SP greater than NKB in the human bronchus and NKA-(4-10) greater than NKA greater than NKB greater than SP in the guinea pig trachea. SP was 2-3 orders of magnitude less active than NKA and appears to be a partial agonist. NKB is inactive on the human bronchus. Compared to non-peptide agents, NKA had an affinity 2-3 orders of magnitude greater than acetylcholine and histamine but produced only 75-80% of the maximal effect of acetylcholine. The present results indicate that neurokinins contract the human bronchus by activating a NK-A receptor type which is more sensitive to NKA than to SP and is insensitive to NKB. The guinea pig trachea appears to be a complex preparation containing not only NK-A but also other neurokinin receptors.

Contractile activity of three endothelins (ET-1, ET-2 and ET-3) on the human isolated bronchus.

1 The effects of three endothelins: (i) the classical or human/porcine endothelin (ET‐1); (ii) [Trp6, Leu7] endothelin (ET‐2) and (iii) [Thr2, Phe4, Thr5, Tyr6, Lys7, Tyr14] endothelin or rat endothelin (ET‐3) were tested on the human isolated bronchus. 2 ET‐1 produced a concentration‐dependent contraction of the human isolated bronchus that proceeded in two different steps. The first step was observed at very low concentrations (pD2 = 11.01 ± 0.17, n = 10) but corresponded to a low intrinsic activity (Emax = 15.6 ± 1.8% of Emax induced by acetylcholine (ACh) 3 × 10−3 m, n = 10). This effect was potentiated by Bay K 8644 10−7 m (Emax = 26.1 ± 2.9% of ACh 3 × 10−3 m, n = 5, P < 0.05), reduced by nicardipine 10−6 m (Emax = 6.0 ± 2.6% of ACh 3 × 10−3 m, n = 5, P < 0.05) and strongly inhibited in calcium‐free medium. The second step of the action of ET‐1 corresponded to a lesser potency (pD2 = 7.90 ± 0.17, n = 9) but a higher intrinsic activity (Emax = 82.5 ± 4.7% of ACh 3 × 10−3 m). This effect was not significantly modified by nicardipine 10−6 m or by Bay K 8644 10−7 m. Neither of the two effects was modified by indomethacin 3 × 10−6m. 3 The effects of ET‐2 and ET‐3 were qualitatively similar to those of ET‐1 but quantitatively different; for these two steps of contracting activity and for potency and efficacy the ranking was: ET‐1 > ET‐2 = ET‐3. 4 Thus, ET‐1 appears to be the most potent of these three substances in its effect on the human isolated bronchus. Its activity seems to involve the action of voltage‐dependent calcium channels at low concentrations (10−12 to 10−9 m), whereas other mechanisms are involved at higher concentrations (10−8 to 3 × 10−7 m).

Effect of indacaterol, a novel long-acting β2-agonist, on isolated human bronchi

Indacaterol is a novel β2-adrenoceptor agonist in development for the once-daily treatment of asthma and chronic obstructive pulmonary disease. The present study evaluated the relaxant effect of indacaterol on isolated human bronchi obtained from lungs of patients undergoing surgery for lung carcinoma. Potency (-logEC50), maximal relaxant effect (Emax) and onset of action were determined at resting tone. Duration of action was determined against cholinergic neural contraction induced by electrical field stimulation (EFS). At resting tone, -logEC50 and Emax values were 8.82±0.41 and 77±5% for indacaterol, 9.84±0.22 and 94±1% for formoterol, 8.36±0.16 and 74±4% for salmeterol, and 8.43±0.22 and 84±4% for salbutamol, respectively. In contrast to salmeterol, indacaterol did not antagonise the isoprenaline response. Indacaterols onset of action (7.8±0.7 min) was not significantly different from that of formoterol (5.8±0.7 min) or salbutamol (11.0±4.0 min), but it was significantly faster than that of salmeterol (19.4±4.3 min). EFS-induced contractions were inhibited with -logIC50 values of 6.96±0.13 (indacaterol), 8.96±0.18 (formoterol), 7.18±0.34 (salmeterol) and 6.39±0.26 (salbutamol). Duration of action was >12 h for indacaterol and salmeterol, and 35.3±8.8 and 14.6±3.7 min for formoterol and salbutamol, respectively. In isolated human bronchi, indacaterol behaved as a long-acting β2-adrenoceptor agonist with high intrinsic efficacy and fast onset of action.

Tachykinin receptors and the airways

The tachykinins, substance P, neurokinin A and neurokinin B, belong to a structural family of peptides. In mammalian airways, substance P and neurokinin A are colocalized to afferent C-fibres. Substance P-containing fibres are close to bronchial epithelium, smooth muscle, mucus glands and blood vessels. Sensory neuropeptides may be released locally, possibly as a result of a local reflex, and produce bronchial obstruction through activation of specific receptors on these various tissues. Three types of tachykinin receptors, namely NK-1, NK-2 and NK-3 receptors, have been characterized by preferential activation by substance P, neurokinin A and neurokinin B respectively. NK-1 and NK-2 receptors were recently cloned. The determination of receptor types involved in the effects of tachykinins in the airways has been done with synthetic agonists and antagonists binding specifically to NK-1, NK-2 and NK-3 receptors. Although the existence of species differences, the conclusion that bronchial smooth muscle contraction is mainly related to activation of NK-2 receptors on bronchial smooth muscle cell has been drawn. The hypothesis of a NK-2 receptor subclassification has been proposed with NK-2A receptor subtype in the guinea-pig airways. Other effects in the airways are related to stimulation of NK-1 receptors on mucus cells, vessels, epithelium and inflammatory cells. A non-receptor-mediated mechanism is also involved in the effect of substance P on inflammatory cells and mast cells.

Effect of the two tachykinin antagonists, SR 48968 and SR 140333, on cough induced by citric acid in the unanaesthetized guinea pig

It is now well-established that sensory nerves stimulation in the airway induces bronchoconstriction and inflammation, but also protective reflexes, such as coughing. These effects are mediated through the release of tachykinins (substance P and neurokinin A) and we have recently shown that SR 48968, a tachykinin NK2-receptor antagonist, inhibited cough induced by citric acid. In this paper, we have studied the effects of SR 48968 administered by aerosol. We have also investigated the effects of SR 140333, a tachykinin NK1-receptor antagonist, and the combination of both SR 48968 and SR140333 to determine whether tachykinin NK1 receptors are involved in cough. Finally, we have studied the combined effects of SR 48968 and salbutamol to find out whether the antitussive effect of SR 48968 is a consequence of the inhibition of bronchoconstriction. Unanaesthetized guinea-pigs were placed in a transparent chamber and exposed to an aerosol of citric acid (0.4 M). The number of coughs was counted by visual inspection and by determination of sounds and pressure variations in the chamber. By the aerosol route, SR 48968 was an efficient antitussive and 16 times more potent than codeine. SR 140333 (0.1-1 mg.kg-1 i.p.) did not exert any antitussive effect but it potentiated the maximal effect induced by SR 48968. Finally, salbutamol, in a dose (0.3 mg.kg-1) which inhibits bronchoconstriction, but not cough induced by citric acid, did not modify the antitussive effect of SR 48968.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of five different organic calcium antagonists on guinea-pig isolated trachea.

1 The relaxant effects of five organic calcium antagonists (nicardipine, diltiazem, PY 108068, verapamil and bepridil) on guinea‐pig isolated trachea were tested against contractions induced by acetylcholine, histamine, 5‐hydroxytryptamine, potassium chloride (KCl) and tetraethylammonium (TEA) in a medium containing the normal amount of calcium and against calcium dose‐response curves in a calcium‐free, potassium‐enriched medium. These effects were compared with those of spasmolytic or specific acetylcholine or histamine antagonists. 2 In contrast to the other drugs tested, organic calcium antagonists exerted a specific inhibitory effect on KCl‐ and TEA‐induced contractions. Their degree of activity was in the order: nicardipine > diltiazem > PY 108068 > verapamil > bepridil. All calcium antagonists inhibited calcium dose‐response curves at similar concentrations. 3 Organic calcium antagonists therefore seem to exert a specific inhibitory effect on depolarizing agents in the guinea‐pig isolated trachea, unlike other tissues, notably some vascular smooth muscles.