Relaxin counteracts asthma-like reaction induced by inhaled antigen in sensitized guinea pigs.

In previous studies, the peptide hormone relaxin (RLX) was found to inhibit mast cell secretion and platelet activation. It has been established that the release of mediators from these cells plays a central pathogenic role in allergic asthma. This prompted us to ascertain whether RLX may counteract the respiratory and histopathological abnormalities of the asthma-like reaction to inhaled antigen in sensitized guinea pigs. Guinea pigs were sensitized with ovalbumin and challenged with the same antigen given by aerosol. Some animals received RLX (30 microg/kg BW, twice daily for 4 days) before antigen challenge. Other animals received inactivated RLX in place of authentic RLX. Respiratory abnormalities, such as cough and dyspnea, were analyzed as were light and electron microscopic features of lung specimens. RLX was shown to reduce the severity of respiratory abnormalities, as well as histological alterations, mast cell degranulation, and leukocyte infiltration in sensitized guinea pigs exposed to ovalbumin aerosol. RLX was also found to promote dilation of alveolar blood capillaries and to reduce the thickness of the air-blood barrier. This study provides evidence for an antiasthmatic property of RLX and raises the possibility of new therapeutic strategies for allergic asthma in humans.

Pharmacodynamic profile of the new potent antibronchospastic agent 7-[(2,2-dimethyl)propyl]-1-methyl xanthine.

The pharmacodynamic profile of a new xanthine derivative, 7-[(2,2-dimethyl)propyl]-1-methyl xanthine (CAS 155006-67-0, MX2/120), was investigated in comparison with theophylline. The compound reduces in vitro the bronchospastic tone induced by carbachol or histamine in guinea-pig bronchi, with a potency 11 and 5 fold greater than theophylline, respectively. MX2/120 is significantly more active and long-lasting than theophylline in in vivo experiments toward spasmogens such as acetylcholine (ED50 over 5 h = 15 mumol/kg p.o. vs 230 mumol/kg p.o.) or histamine (ED50 over 5 h = 122 mumol/kg p.o. vs 500 mumol/kg p.o.) while being almost equiactive to theophylline toward antigen and capsaicin induced cough strokes. MX2/120, if administered by i.p. route reduces hyperresponsiveness to histamine induced by PAF and extravasation of protein into bronchoalveolar lavage fluid induced by capsaicin. These anti-inflammatory effects of MX2/120 are of similar extent when compared to theophylline. Unlike theophylline, MX2/120 up to 275 mumol/kg p.o. possesses little or no CNS excitatory effects in mice in terms of reduction of sleeping time induced by chlordiazepoxide, increase in mortality and convulsions induced by pentetrazol and increase in locomotor activity. This reduced neuroexcitatory action is probably related to its lack of affinity to adenosine receptors that could also explain the absence of effect on basal gastric secretion. Chronotropic effects of MX2/120 in conscious rats are similar to those of theophylline while the effects of both drugs on blood pressure are of minor extent. The overall pharmacodynamic properties of MX2/120 are superior to those of theophylline in relation to its antibronchospastic activity and lack of excitatory effects on CNS.

A new class of antibronchospastic agents: 7-(2,2-dimethyl)propyl substituted xanthines.

A series of 7-(2,2-dimethyl)propyl substituted xanthines were synthesized and tested for their antibronchospastic activity in comparison with theophylline. In vitro, the inhibition of carbachol-induced increase in bronchial tone was determined. In vivo, the inhibition of antigen-induced bronchoconstriction in guinea pigs was determined 1, 3 and 5 h after oral administration. Central side effects were evaluated. In vitro, the majority of compounds were more effective than theophylline. In vivo, three compounds 2, 14 and 15 showed an effect comparable to theophylline but longer lasting. A mild sedative effect was generally observed. Compound 2, 7-(2,2-dimethyl)propyl-1-methyl xanthine, coded MX2/120, was selected for a deeper evaluation.

Hyperresponsiveness to non-adrenergic, non-cholinergic vagal stimulation following multiple antigen challenge in guinea-pigs.

The role of airway inflammation, induced by weekly antigen challenge, in the airway hyperresponsiveness to vagal (whole and NANC components) nerve stimulation and to neurotransmitters (acetylcholine and selective agonists for tachykinin NK1 and NK2 receptors) has been studied in the guinea-pig. Primarily, the time course (3, 7 and 14 days following the last challenge) of the effects of repeated aerosol antigen challenge on airway inflammation and bronchoalveolar fluid cellular composition was investigated. At 7 days following the last antigen challenge a maximal (as compared to 3 and 14 days) inflammatory response, in terms of a diffuse mild to marked infiltration of eosinophils, neutrophils and lymphocytes, was evident throughout pulmonary tissues. Only at this time some evidence of eosinophilia and neutropenia was detectable in BAL fluids. In these animals there was a normal bronchial responsiveness to iv administration of acetylcholine, selective synthetic agonists for the tachykinin NK2 receptors and capsaicin. On the other hand a remarkable airways hyperresponsiveness to iv administration of selective agonists for tachykinin NK1 receptors, as well as electrical stimulation of the vagal nerves (in presence and in absence of atropine), was detected. As a whole, these data indicate that at the peak of the inflammatory airway response following multiple antigen challenge there is a selective hyperresponsiveness to stimulation of vagal (mainly the non-adrenergic, non-cholinergic component) nerves associated with an increase in tachykinins (NK-1)-mediated bronchospasm.

Tachykinin NK1 and NK2 receptors mediate the non-cholinergic bronchospastic response to capsaicin and vagal stimulation in guinea-pigs.

1. The antibronchospastic activity against acetylcholine, capsaicin, electrical vagal stimulation and the selective tachykinin agonists ([beta Ala8]NKA-(4-10) and [Sar9]SP sulfone) of a novel NK2 receptor antagonist, MEN10,627 and/or the known NK1 receptor antagonist (+/-)-CP96,345 was studied in anaesthetized guinea-pigs. 2. MEN10,627 (0.1 mumol kg-1 i.v.) and (+/-)-CP96,345 (3 mumol kg-1 i.v.) selectively reduced the bronchospasm induced by NK2 and NK1 tachykinin receptor agonists, respectively, without affecting the other tachykinin receptor agonist- or acetylcholine-induced bronchospastic response. 3. MEN10,627 (0.1 mumol kg-1 i.v.), in a dose-dependent manner, reduced the non-cholinergic response induced by bilateral stimulation of the vagi or by intravenous capsaicin. 4. The administration of (+/-)-CP96,345 (3 mumol kg-1 i.v.) alone did not affect these responses but, when administered in association with the NK2 antagonist, (+/-)-CP96,345, was able to potentiate its inhibitory effect. 5. It is concluded that both NK1 and NK2 receptors are involved in the non-cholinergic bronchoconstriction induced by capsaicin or by stimulation of the vagi, although the NK2 receptor contribution is prominent.

Antibronchospastic activity of MEN10,627, a novel tachykinin NK2 receptor antagonist, in guinea-pig airways.

The antibronchospastic activity against acetylcholine, antigen, histamine plus platelet-activating factor (PAF) or the selective tachykinin neurokinin (NK)1 and NK2 receptor agonists of the novel tachykinin NK2 receptor antagonist, MEN10,627 (cyclo(Met-Asp-Trp-Phe-Dap-Leu)cyclo(2 beta-5 beta)), was studied in anesthetized guinea-pigs. MEN10,627 (30-100 nmol/kg i.v.) reduced in a dose-dependent manner the bronchospasm induced by the tachykinin NK2 receptor agonist [beta Ala8]neurokinin A-(4-10) and the effect of the highest dose lasted up to 5 h from its administration. Conversely, airway constriction induced by the NK1 receptor agonist [Sar9]substance P sulfone or acetylcholine was unaffected by MEN10,627 up to a dose of 3 mumol/kg i.v. In animals sensitized with ovalbumin and pretreated with the endopeptidase inhibitor phosphoramidon, the aerosolized antigen produced a bronchospasm which was inhibited by MEN10,627 (30-100 nmol/kg i.v.) but not by the tachykinin NK1 receptor antagonist, (+/-)-CP96,345 ([2R,3R-cis- and [2S,3S)-cis-2-(diphenylmethyl)-N-[(2-methoxyphenyl)-methyl]-1- azabicyclo[2.2.2]octan-3-amine]) (3 mumol/kg i.v.). Both MEN10,627 (30-100 nmol/kg i.v.) and (+/-)-CP96,345 (30-300 nmol/kg i.v.) reduced the PAF-induced hyperresponsiveness to histamine, without affecting the hypotension induced by PAF or the bronchospasm induced by histamine in guinea-pigs not exposed to PAF, showing the involvement of both tachykinin NK1 and NK2 receptors in this model. In summary, MEN10,627 behaves as a potent, selective and long-lasting tachykinin NK2 receptor antagonist in vivo. Further, tachykinin NK2 receptors could be activated during allergic responses and in the development of airway hyperresponsiveness.

Isbufylline, a xanthine derivative, inhibits bronchoconstrictor responses produced by stimulation of capsaicin-sensitive sensory nerves in guinea-pig: 'In vitro' and 'in vivo' evidence.

Isbufylline (1,3-dimethyl-7-isobutylxanthine) is a new xanthine derivative claimed to possess remarkable antibronchospastic properties coupled to reduced pro-convulsive side-effects. In guinea-pig bronchial preparations, isbufylline showed a differential and more pronounced, as compared to theophylline, relaxant activity on tonic bronchial contractions evoked by exogenous administration of equieffective concentrations of capsaicin (0.3 microM), neurokinin A (0.1 microM) and carbachol (0.3 microM) (in the presence of indomethacin 5 microM and thiorphan 10 microM). Isubfylline gave an IC50 of 21 (19-25, 95% confidence limits) microM on capsaicin-evoked contractile effects, and 36 (30-43) microM on carbachol-produced contractile effects, whilst it was almost ineffective in inhibiting neurokinin A-induced bronchospasm (IC50 not evaluable, > 100 microM). 'In vitro' studies were also performed using electrical field stimulation (EFS) to produce non-adrenergic non-cholinergic (NANC)- or cholinergic nerves-mediated contractions in guinea-pig isolated bronchi or trachea. Isbufylline (10-90 microM) produced a concentration-dependent inhibition of the NANC response (EFS: 20 Hz, supramaximal voltage, 0.5 ms pulse, width for 10 s) of bronchi (IC50 = 47 microM) without affecting the cholinergic contractile response in tracheal smooth muscle (EFS: 0.5 up to 32 Hz, supramaximal voltage, 0.5 ms pulse, for 15 s every min). The activity of isbufylline was also confirmed in anaesthetized guinea-pig showing a greater antibronchospastic activity towards capsaicin (8 nmol/kg i.v.) or vagal non-cholinergic (10 V, 1 ms, 20 Hz for 20 s) stimulation as compared to the inhibition exerted against acetylcholine (50 nmol/kg i.v.) or neurokinin A (1 nmol/kg i.v.).(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of ruthenium red on the bronchoconstriction induced by capsaicin and by selective tachykinin receptor agonists in anaesthetized guinea-pig.

1. The effect of systemic administration of ruthenium red on bronchospasm induced by acetylcholine, capsaicin or selective tachykinin receptor agonists ([Sar9]SP sulfone or [beta Ala8]-neurokinin A (NKA)-(4-10) for NK-1 and NK-2 receptors, respectively) was studied in anaesthetized guinea-pigs. 2. The bronchospasm induced by capsaicin was reduced by ruthenium red, which did not affect the response induced by acetylcholine. Atropine, which totally blocked the response to acetylcholine, also partially blocked the bronchospasm induced by capsaicin. 3. The inhibitory action of atropine and ruthenium red on the bronchospasm produced by capsaicin was additive, independently from the order of administration of the two antagonists. 4. Ruthenium red induced an increase in [Sar9]SP sulfone-bronchospasm and a marked enhancement of the bronchomotor response to [beta Ala8]NKA-(4-10). This latter was antagonized by the prior administration of the selective NK-2 receptor antagonist MEN 10,376. 5. Pretreatment with guanethidine or propranolol increased the airway constriction induced by [beta Ala8]NKA-(4-10). Furthermore, pretreatment with guanethidine prevented the enhancement induced by ruthenium red, showing that activation of NK-2 receptors influences the sympathetic bronchodilator drive to the airways. 6. It is concluded that ruthenium red antagonizes selectively the in vivo excitatory effect of capsaicin in guinea-pig airways. Furthermore, the additivity of the blocking action of ruthenium red and atropine indicates that two distinct mechanisms take place in bronchospastic response to i.v. capsaicin in this species.

Effects of selective tachykinin receptor antagonists on capsaicin- and tachykinin-induced bronchospasm in anaesthetized guinea-pigs.

Bronchospasm induced by i.v. injection of equieffective doses of acetylcholine, capsaicin or selective tachykinin receptor agonists ([Sar9]SP sulfone or [beta-Ala8]neurokinin A (NKA-4-10)) (for NK1 and NK2 receptors, respectively) was studied in anaesthetized guinea-pigs. The NK1 and NK2 receptor antagonists, (+/-)-CP96,345 (3 mumol/kg i.v.) and MEN 10,376 (3 mumol/kg i.v.), selectively abolished the bronchoconstriction induced by the respective agonist, showing that both NK1 and NK2 receptors mediate bronchoconstriction in guinea-pig airways and that they are activated independently. Capsaicin-induced bronchospasm was inhibited by atropine (1.5 mumol/kg i.v.) and MEN 10,376 (3 mumol/kg i.v.), but unaffected by (+/-)-CP96,345 (3 mumol/kg i.v.). Hexamethonium (79 mumol/kg i.v.), propranolol (17 mumol/kg i.v.) and physostigmine (0.9 mumol/kg i.v.) enhanced the airway constriction induced by acetylcholine, capsaicin, [Sar9]SP sulfone or [beta-Ala8]NKA-(4-10) while guanethidine (67 mumol/kg s.c. for two days) increased only bronchoconstriction induced by capsaicin or the selective NK2 receptor agonist. In hexamethonium-treated animals, MEN 10,376 still abolished the increase in insufflation pressure induced by [beta-Ala8]NKA-(4-10) and reduced the increase elicited by capsaicin. In summary, in anaesthetized guinea pig i.v. capsaicin-induced bronchospasm through activation of postjunctional NK2 (but not NK1) receptors along with activation of cholinergic pathways. This motor response is moderated by the simultaneous stimulation of a sympathetic bronchodilating mechanism(s), possibly through activation of NK2 receptors localized in sympathetic ganglia.

Repeated antigen challenge induced airway hyperresponsiveness to neurokinin A and vagal non-adrenergic, non-cholinergic (NANC) stimulation in guinea pigs.

The effect of repeated weekly antigen challenges by aerosol on bronchopulmonary responses to ACh, histamine, neurokinin A or atropine-resistant (NANC) component of vagal stimulation, has been studied in guinea pigs. Bronchospastic responses were measured in anaesthetized animals, 7 days after the last challenge with antigen (or vehicle). No difference was observed between control and antigen challenged guinea pigs in their responsiveness to acetylcholine (1-300 mumol kg-1 i.v.) or histamine (1-300 mumol kg-1 i.v.). On the other hand, amplitude of bronchospasm induced by neurokinin A (1-3 mumol kg-1 i.v.) or NANC vagal stimulation (20 Hz, 1 msec, 10 V, trains of 5-20 sec) was significantly increased in guinea pigs previously challenged with antigen, as compared to controls. These results suggest that repetitive antigen exposure in sensitized guinea pigs generates an increase in the responsiveness to exogenously administered or endogenously released tachykinins, at a time when no generalized hyperresponsiveness to other spasmogens could be observed.

In vivo evidence for tachykininergic transmission using a new NK-2 receptor-selective antagonist, MEN 10,376.

We have studied the effects of two newly developed tachykinin antagonists, MEN 10,207 and MEN 10,376, which are highly selective for NK-2 tachykinin receptors on tachykinin-induced contraction in the rat urinary bladder and guinea pig airways in vivo. MEN 10,207 exhibited antagonism only at doses which produced agonist effects. By contrast, MEN 10,376 was devoid of significant agonist activity at i.v. doses (1-3 mumol/kg) which selectively antagonized the effects of an NK-2 agonist [beta-Ala8]-neurokinin A(4-10) (bladder contraction in rats, bronchoconstriction in guinea pigs) without affecting the response to an NK-1 agonist [Sar9]-substance P sulfone (hypotension, salivation and bladder contraction in rats, bronchoconstriction in guinea pigs). At these NK-2-selective blocking doses, MEN 10,376: 1) did not affect urodynamic parameters at cystometry in normal rats but reduced amplitude of micturition contractions following induction of chemical (intravesical xylene) cystitis and 2) reduced by a maximum of 50% the noncholinergic bronchoconstrictor response to vagal nerve stimulation. These findings provide the first evidence for involvement of NK-2 receptors in physiological responses in the urinary and respiratory tracts.

Pharmacodynamic profile of isbufylline, a new antibronchospastic xanthine devoid of central excitatory actions.

1,3-Dimethyl-7-isobutylxanthine (isbufylline, TE/06; CAS 90162-60-0) is a newly synthetized xanthine derivative. This compound exhibits remarkable antibronchospastic properties both in in vitro and in vivo (after oral or intravenous administration) experimental models. Isbufylline is significantly more effective than theophylline in antagonizing bronchospasms elicited by spasmogens (capsaicin, arachidonic acid, PAF and antigen) which mainly act by a local release of biologically active substances proposed to be involved in the pathogenesis of asthma. Isbufylline, unlike theophylline, possesses little or no CNS excitatory properties. This reduced neuroexcitatory action is probably related to the poor affinity of isbufylline, as compared to theophylline, to A1 purinoceptor. Indeed, isbufylline is ineffective in antagonizing 2Cl-adenosine-induced EEG synchronization. In normotensive and hypertensive rats oral administration of isbufylline resulted in small and transient positive chronotropic and hypotensive response, markedly lower than those elicited by theophylline or enprofylline. Finally isbufylline exhibits phosphodiesterase inhibitory properties, although at concentration 50-100 times higher than those exerting spasmolytic effects in isolated bronchial tissues. As a whole the pharmacodynamic profile of isbufylline is promising and, in the clinical setting, this compound might exert enhanced antiasthma effects coupled to a low incidence of central and cardiovascular adverse effects.

2-Chloroadenosine induction of vagally-mediated and atropine-resistant bronchomotor responses in anaesthetized guinea-pigs.

1. The bronchoconstrictor effects of intravenous administration of adenosine derivatives in anaesthetized non-curarized guinea-pigs have been studied. 2. 2-Chloroadenosine (2-Cl-Ade), 5'-N-ethylcarboxamideadenosine (NECA) and L-N6-phenylisopropyl-adenosine (L-PIA) all produced dose-dependent, transient increases in tracheal insufflation pressure, with an order of potency (NECA greater than or equal to 2-Cl-Ade much greater than L-PIA) typical of A2-receptor mediated biological responses. 3. 2-Chloradenosine-induced bronchoconstrictor responses disappeared after vagotomy or topical application of tetrodotoxin (TTX) on cervical vagal trunks. 4. 2-Chloradenosine-induced bronchospasm was unaffected by atropine (1 mg kg-1 i.v.), physostigmine (50 micrograms kg-1 i.v.) and hexamethonium (30 mg kg-1 i.v.) but was significantly reduced by theophylline (25 mg kg-1 i.v.). 5. The magnitude of 2-Cl-Ade-induced bronchospasm was significantly reduced by acute (10 micrograms kg-1 i.v.) or chronic (55 mgkg-1 s.c. four days before the experiment) pretreatment with capsaicin. 6. Guanethidine (20 mg kg-1 s.c on two consecutive days), prazosin (10 micrograms kg-1 i.v.), diphenhydramine (1 mg kg-1 i.v.) and indomethacin (1 mg kg-1 i.v.) failed to block the bronchomotor response to 2-Cl-Ade. In contrast, cyproheptadine (1-5 mgkg-1 i.v.) markedly reduced, but did not abolish the bronchospasm elicited by the purine derivative. 7. We conclude that in anaesthetized non-curarized guinea-pigs, a transient vagally-mediated bronchospasm can be induced by stimulation of A2-purinoceptors. This effect is complex and involves, at least in part, stimulation of capsaicin-sensitive sensory nerves and 5-hydroxytryptamine release. This experimental model might be useful for the further study of the potential role of adenosine in asthma, and for the evaluation of new antiasthma drugs.

In vivo pharmacology of [beta Ala8]neurokinin A-(4-10), a selective NK-2 tachykinin receptor agonist.

We studied the effect of [beta Ala8]neurokinin A-(4-10), a newly developed selective NK-2 tachykinin receptor agonist, on various parameters in anaesthetized rats (blood pressure, urinary bladder motility, plasma extravasation) and guinea-pigs (salivation, increase of pulmonary insufflation pressure) as compared to the response produced by tachykinins. [beta Ala8]Neurokinin A-(4-10) was as active as, or more active than, neurokinin A (NKA) or NKA-(4-10) in producing rat bladder contraction or bronchospasm in guinea-pigs, two effects known to involve activation of NK-2 receptors. On the other hand, the synthetic peptide was weakly active, if active at all, in producing hypotension or plasma extravasation in the rat bladder as well as salivation in guinea-pigs, effects known to involve activation of NK-1 receptors. These findings provide evidence that [beta Ala8]NKA-(4-10) acts as a selective NK-2 agonist in vivo and that it can be used to explore the distribution and function of NK-2 receptors.

Arachidonic acid-induced bronchomotor responses are partially mediated by release of sensory neuropeptides from capsaicin-sensitive structures.

Administration of arachidonic acid (AA) both in vitro and in vivo elicited prominent contractile responses in guinea-pig airways, which were markedly reduced after capsaicin desensitization. Furthermore, AA superfusion elicited a significant calcitonin gene-related peptide-like immunoreactivity release from isolated bronchi. It is suggested that at least part of the bronchomotor actions of AA rely upon stimulation of capsaicin-sensitive primary afferents.

Neurokinin A-(4-10): a potent bronchospastic agent virtually devoid of sialologic properties in anaesthetized guinea-pigs.

The ability of substance P, neurokinin A and its C-terminal fragment, neurokinin A-(4-10), to elicit NK-1 (salivation) and NK-2 (bronchospasm) receptor-mediated responses was investigated in anaesthetized guinea-pigs. Neurokinin A-(4-10) produced a dose-related increase in tracheal insufflation pressure and its maximal effect was similar to that elicited by neurokinin A and significantly greater than that of substance P. On the other hand substance P induced a potent dose-dependent increase of salivation while neurokinin A was significantly less potent. Neurokinin A-(4-10) did not exert any sialologic effect even at the dose of 100 nM/kg. These findings indicate that neurokinin A-(4-10) might be a valuable pharmacological tool for characterizing the involvement of NK-1 and NK-2 receptors in physiological responses in vivo.

Blockade by theophylline of capsaicin-induced motor effects in guinea-pig airways.

The effect of theophylline was tested on the capsaicin-induced increase in insufflation pressure in anaesthetized guinea-pigs and contraction of isolated tracheal spirals. Theophylline (6.25-25 mg/kg i.v.) inhibited in a marked, prolonged and dose-related manner the increase in insufflation pressure elicited by i.v. injection of capsaicin (2-10 micrograms/kg). Theophylline, even at plasma concentrations as low as 15-23 micrograms/ml (which are within or slightly higher than the therapeutic range in humans) produced an inhibition of about 30-50% of capsaicin-induced bronchospasm. Theophylline was significantly less effective in antagonizing the bronchospasm elicited by maximally effective dose (100 micrograms/kg i.v.) of acetylcholine as well as by a dose equieffective to capsaicin 10 micrograms/kg (i.e. acetylcholine 50 micrograms/kg). Theophylline (30 microM-1 mM) also inhibited potently and concentration dependently the capsaicin-induced contraction in vitro and, again, it was significantly less effective against cholinomimetic-induced responses. These data suggest that theophylline can interfere, at therapeutic doses, with some process(es) selectively involved in capsaicin-induced neuropeptide-mediated bronchoconstriction. Since capsaicin-sensitive sensory fibers are thought to be involved in the pathogenesis of bronchial hyperreactivity and asthma, these inhibitory actions of theophylline might be relevant for its therapeutic effects.