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Antagonist profile of ibodutant at the tachykinin NK2 receptor in guinea pig isolated bronchi
European Journal of Pharmacology 720 (2013) 180–185
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Pulmonary, gastrointestinal and urogenital pharmacology
Antagonist profile of ibodutant at the tachykinin NK2 receptor in guinea pig isolated bronchi Paolo Santicioli n, Stefania Meini, Sandro Giuliani, Alessandro Lecci, Carlo Alberto Maggi Department of Pharmacology, Menarini Ricerche S.p.A., Via Rismondo 12/A, 50131 Florence, Italy
art ic l e i nf o
a b s t r a c t
Article history: Received 12 June 2013 Received in revised form 1 August 2013 Accepted 10 October 2013 Available online 24 October 2013
In this study we have characterized the pharmacological profile of the non-peptide tachykinin NK2 receptor antagonist ibodutant (MEN15596) in guinea pig isolated main bronchi contractility. The antagonist potency of ibodutant was evaluated using the selective NK2 receptor agonist [βAla8] NKA(4-10)-mediated contractions of guinea pig isolated main bronchi. In this assay ibodutant (30, 100 and 300 nM) induced a concentration-dependent rightward shift of the [βAla8]NKA(4-10) concentrationresponse curves without affecting the maximal contractile effect. The analysis of the results yielded a Schild-plot linear regression with a slope not different from unity (0.95, 95% c.l. 0.65–1.25), thus, indicating a surmountable behavior. The calculated apparent antagonist potency as pKB value was 8.31 7 0.05. Ibodutant (0.3–100 nM) produced a concentration-dependent inhibition of the nonadrenergic–noncholinergic (NANC) contractile response induced by electrical field stimulation (EFS) of intrinsic airway nerves in guinea pig isolated main bronchi. At the highest concentration tested (100 nM) ibodutant almost abolished the EFS-induced bronchoconstriction (957 4% inhibition), the calculated IC50 value was 2.98 nM (95% c.l. 1.73–5.16 nM). In bronchi from ovalbumin (OVA) sensitized guinea pigs ibodutant (100 nM) did not affect the maximal contractile response to OVA, but completely prevented the slowing in the fading of the motor response induced by phosphoramidon pretreatment linked to the endogenous neurokinin A release. Altogether, the present study demonstrates that ibodutant is a potent NK2 receptor antagonist in guinea pig airways. & 2013 Elsevier B.V. All rights reserved.
Keywords: Ibodutant MEN15596 NK2 antagonist NANC contraction Bronchi Guinea pig
1. Introduction Tachykinins (TKS) are transmitters of the excitatory NANC peripheral nervous system (Lundberg and Saria, 1987), they are co-localized with other neuropeptides in the sensory unmyelinated C-fibers which innervate all compartments of the airways wall, from the trachea down to the bronchioles (Lundberg and Saria, 1987; Baluk and McDonald, 1998). Guinea pig and human airways are well supplied with tachykinin-containing nerves (Lundberg et al., 1984) and TKS have been described to play an important role in the pathophysiology of airways such as asthma and bronchial hyperresponsiveness (Pernow, 1985; Barnes, 1990; Maggi, 1995; Kraneveld et al., 1997; De Vries et al., 1999; Joos et al., 2001) mediating several processes such as bronchoconstriction (Hua et al., 1984; Advenier et al., 1987), submucosal gland hypersecretion (Gashi et al., 1986), plasma proteins extravasation (Lundberg et al., 1983), recruitment of inflammatory cells (Maggi, 1997 for review; Schuiling et al., 1999) and stimulation of resident immune cells (Brunelleschi et al., 1990).
n
Corresponding author. Tel.: þ 39 55 5680735; fax: þ 39 55 56809954. E-mail address: [email protected] (P. Santicioli).
0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.10.029
The bronchial compartment of airways, characterized by high airflow resistance, is the most sensitive region to neurokinin A, suggesting the importance of this peptide in bronchoconstriction (Venugopal et al., 2004). Although both NK1 and NK2 receptors are capable to elicit contractions of guinea pig bronchus (Maggi et al., 1991a), pharmacological studies using selective NK1 and NK2 tachykinin receptor antagonists have provided evidence that the nonadrenergic–noncholinergic contractile (NANC) responses produced by stimulation of sensory unmyelinated C-fibers by the transient receptor vanilloid 1 (TRPV1) agonist capsaicin, or by electrical stimulation of intrinsic airway nerves, mainly involve the selective activation of tachykinin NK2 receptors with a smaller NK1 receptor contribution (Szolcsányi and Barthó, 1982; Maggi et al., 1991b; Renzetti et al., 1992). Ibodutant (MEN15596) is a potent and selective non-peptide tachykinin NK2 receptor antagonist characterized by subnanomolar antagonist potency in human, guinea pig, and minipig in vitro bioassays (Cialdai et al., 2006; Santicioli et al., 2013) and long lasting antagonist properties both in vivo and in vitro (Cialdai et al., 2006; Meini et al., 2009; Santicioli et al., 2013). The present study was undertaken to characterize the pharmacological properties of ibodutant in blocking the smooth muscle contractility of the guinea pig isolated bronchi in different models of tachykinin NK2 receptor activation.
P. Santicioli et al. / European Journal of Pharmacology 720 (2013) 180–185
2. Materials and methods 2.1. General The experimental procedures employed in this study were approved by the Institutional Animal Care and Use Committee of Menarini Ricerche (Firenze, Italy) and carried out in accordance with the Italian legislation (D.L. 116 27/01/1992), which complies with European Community guidelines (CEE Directive 86/609) for the care and use of experimental animals. Male Dunkin-Hartley guinea pigs (500–700 g, Charles-River Laboratory, Italy) were killed by an intraperitoneal overdose of pentobarbital. The tracheobronchial tree was rapidly excised and placed in ice-cold Krebs solution of the following composition (mmol/l): NaCl 119; NaHCO3 25; KH2PO4 1.2; MgSO4 1.5; CaCl2 2.5; KCl 4.7 and glucose 11. The main bronchi were cleaned of surrounding tissue and two main bronchial transverse ring preparations (3–4 mm in length) were obtained from each animal. Unless otherwise specified in the text, the bronchial rings were deprived of epithelium by gently scraping the luminal surface with the smooth edge of a Dumont forceps and placed in 5 ml organ baths filled with warmed (37 1C) and oxygenated (96% O2 and 4% CO2, pH 7.4) Krebs solution. The tissues were connected to isometric force transducers (Ugo Basile, Varese, Italy) under an initial tension of 10 mN and mechanical activity was digitally recorded by an Octal Bridge Amplifier connected to PowerLab/8sp data acquisition system and analyzed using the Chart 4.2 software (ADInstruments Ltd, Oxford, UK). Depending on the experimental protocol, various antagonists or inhibitors were added to the Krebs solution as described in the individual sections. 2.2. Contractions induced by [βAla8]NKA(4-10) After 120 min equilibration period, the preparations were stimulated using a maximal concentration of histamine (100 μM) to evaluate the contractile response of each preparation. After a further 120 min equilibration period, during which the medium was renewed every 10 min, cumulative concentration-response curves to the tachykinin NK2 receptor selective agonist [βAla8]NKA (4-10), were constructed. One of the two tissue rings obtained from the same animal was pretreated with vehicle (DMSO; 1–3 μl/ml) and used to perform the control curve to [βAla8]NKA(4-10), while the other ring was pretreated with ibodutant (30, 100 or 300 nM) added to the organ bath 60 min before the concentration-response curve to [βAla8]NKA(4-10). In each preparation only a single cumulative concentration-response curve to [βAla8]NKA(4-10) was carried out and only one concentration of the antagonist was tested. These experiments were performed in the presence of atropine (1 μM), indomethacin (3 μM), DL-thiorphan (1 μM) and the selective tachykinin NK1 receptor antagonist SR140333 (1 μM). 2.3. NANC contractions induced by electrical field stimulation In a separate series of experiments, endogenous tachykininergic contractile responses mediated by the NK2 receptor activation were evoked in bronchial rings by electrical field stimulation (EFS) of intramural capsaicin-sensitive afferent nerves (Maggi et al., 1990, 1991b; Heavey et al., 1997). These experiments were performed in the presence of atropine (1 mM) to eliminate the fast cholinergic component of the response to EFS, indomethacin (3 mM) to inhibit the endogenous formation of cyclooxygenase products, SR140333 (1 mM) to selectively block tachykinin NK1 receptors, DL-thiorphan (1 mM) to inhibit TKS metabolism and guanethidine (3 mM) to inhibit adrenergic neurotransmission. After 120 min equilibration period, histamine (100 μM) was administered to evaluate the contractile response of each
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preparation. After 90 min, each preparation was subjected to three cycles of EFS (S1, S2 and S3) consisting of a train of pulses at the frequency of 10 Hz for 10 s (60 V, 0.5 ms pulse width) delivered every 90 min by means of two platinum wire electrodes placed at the top and the bottom of the organ bath and connected to a GRASS S88 stimulator (Grass Medical Instruments, Quincy, MA, USA). After two control responses to EFS (S1 and S2), ibodutant (0.3–100 nM) or its vehicle (DMSO 1–3 ml/ml), were applied 60 min before the third (S3) response to EFS. 2.4. OVA sensitization procedure and assessment of bronchial reactivity The guinea pig sensitization procedure and in vitro functional experiments were performed according to the method previously described by Kohrogi et al. (1991, 1997). Guinea pigs were actively sensitized by intramuscular injection of 35 mg OVA dissolved in 0.3 ml of sterile saline twice with an interval of one week between injections. The animals were used for in vitro experiments from three weeks after the second OVA injection. Bronchial rings with intact epithelium were prepared and set-up for isometric tension measurement as described in the general section. After 120 min equilibration period the preparations were stimulated using a maximal concentration of acetylcholine (1 mM) to evaluate the viability and the contractile response of each preparation. After 60 min, during which the bathing solution was renewed at 10 min intervals, phosphoramidon (10 mM) or its vehicle (distilled water, 1 ml/ml) were added to the tissue bathing solution and after 15 min ibodutant (100 nM) or its vehicle (DMSO 1 ml/ml) were added and left in contact for 60 min. Finally, in order to induce the allergic response, all preparations received 10 mg/ml OVA and the total contractile response was recorded over a period of 100 min. 2.5. Data evaluation and statistical analysis All data in the text and figures were expressed as the mean 7 standard error of the mean (S.E.M.) or 95% confidence limits (95% c.l.) of the given number of experiments (n). Concentration-response curves were analyzed by sigmoidal nonlinear regression fitting using the Prism 4.02 program (GraphPad Software Inc., San Diego, CA, USA) to determine the molar concentration of the agonist producing the 50% (EC50) of its maximal response (Emax). The antagonist potency of ibodutant was expressed as apparent pKB estimated as the mean of the individual values obtained with the equation: pKB ¼log(CR-1) log[B] where CR is the concentration-ratio calculated from equieffective concentrations of agonist (EC50) obtained in the presence and in the absence of antagonist and B is the antagonist concentration used (Kenakin, 1997). Competitive antagonism was checked by the Schild regression analysis by plotting the estimates of log(CR-1) against log[B] to determine the slope of linear regression: a plot with linear regression line and slope not significantly different from unity was considered as proof of competitive antagonism (Arunlakshana and Schild, 1959). In experiments with EFS, the effect produced by ibodutant or its vehicle (DMSO) on the amplitude of contraction was assessed as percentage of variation of the response in the presence of the antagonist (S3) vs. control response (S2). The preparations in which the variation between the two control responses S1 and S2 was 420% were excluded from calculations. The concentration of antagonist causing 50% inhibition (IC50) of contraction amplitude induced by EFS was calculated by sigmoidal nonlinear regression analysis (GraphPad Prism 4.02, San Diego, CA, USA). In experiments with bronchial rings from OVA sensitized guinea pigs the amplitude of contractile response induced by 10 mg/ml OVA
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was evaluated at 1 min intervals during the initial 10 min from application and then every 5 min for a total of 100 min. For each experiment the duration of the contraction induced by OVA was calculated from start until 50% of its baseline recovery (T50%). Since there were no differences in maximum contractile effect induced by OVA between different groups, collected data were normalized to the maximal response induced by OVA in each preparation. Results were compared for significant differences using one- or two-way analysis of variance (ANOVA) followed by Dunnett’s or Bonferroni’s post hoc test. A P value o0.05 was considered statistically significant. 2.6. Chemicals Drugs were obtained from the following sources: ibodutant (MEN15596; 6-methyl-benzo[b]thiophene-2-carboxylic acid [1-(2phenyl-1R-{[1-(tetrahydropyran-4-ylmethyl)-piperidin-4-ylmethyl]carbamoyl}-ethylcarbamoyl)-cyclopentyl]-amide) (batches L2/06 and L3/08) was synthesized at Lusochimica (Menarini Group, Lomagna, Italy); the non-peptide NK1 antagonist SR140333 or: [(S)1-{2-[3-(3,4dichlorophenyl)-1-(3-isopropoxyphenylacetyl) piperidin-3-yl]ethyl};4-phenyl-1-azoniabicyclo [2,2,2] octane chloride] was kindly provided by Sanofi Aventis (Montpellier, France); [βAla8]NKA(4-10) was from EspiKem (Florence, Italy); acetylcholine hydrochloride, atropine sulfate, histamine hydrochloride, indomethacin, ovalbumin (grade V), phosphoramidon and DL-thiorphan were from Sigma–Aldrich Italy (Milan, Italy). Guanethidine sulfate was from ICFI (Milan, Italy). All other reagents used were of analytical grade and purchased from Merck (Darmstadt, Germany).
Fig. 1. (A) Antagonism by ibodutant towards the contractile responses induced by [βAla8]NKA(4-10) in the isolated guinea pig bronchus. Concentration-response curves for [βAla8]NKA(4-10) were constructed as described in Section 2 in the absence (control) and presence of the indicated concentrations of ibodutant. Each value is the mean7 S.E.M. of five experiments. (B) Schild-plot of agonist concentration ratios vs. ibodutant concentrations (slope ¼ 0.95, 95% c.l. 0.65–1.25). Each value is the mean 7 S.E.M. of five experiments.
3. Results 3.1. Antagonism of contractions produced by [βAla8]NKA(4-10) In the presence of atropine (1 μM), indomethacin (3 μM), (1 μM) and SR140333 (1 μM), the selective NK2 receptor agonist [βAla8]NKA(4-10) (0.1 nM to 3 mM) produced a slowly developing concentration-dependent tonic contraction of the guinea pig isolated bronchus whose maximal effect (Emax) was 13.537 1.08 mN, corresponding to 112 72% of that produced by histamine 100 μM (n ¼15). The EC50 value calculated from the control concentration-response curves to [βAla8]NKA(4-10) was 2.85 nM (95% c.l. 2.62–3.12 nM; n ¼15). Ibodutant (30, 100 and 300 nM) was devoid of any effect on the resting tension of the preparation whereas it concentrationdependently antagonized the [βAla8]NKA(4 -1 0)-induced contractile responses, thus producing a parallel rightward shift (Fig. 1A) of the agonist response curves without depressing the agonist Emax being 13.5371.08, 13.3770.58, 14.3771.32 and 12.6571.32 mN in controls and in the presence of 30, 100 and 300 nM of ibodutant, respectively. Schild-plot analysis was consistent with competitive antagonism (slope¼0.95, 95% c.l. 0.65–1.25; n¼15) and a pKB value of 8.3170.05 was calculated (Fig. 1B). DL-thiorphan
3.2. Inhibition of NANC contractions produced by electrical field stimulation In the presence of atropine (1 μM), indomethacin (3 μM), SR140333 (1 μM), DL-thiorphan (1 μM) and guanethidine (3 μM), EFS (10 Hz, 60 V, 0.5 ms for 10 s) induced a slowly developing NANC contraction of the guinea pig isolated bronchus which averaged 5.167 0.28 mN (n ¼34) corresponding to 42 72% of that produced by histamine 100 μM. In the controls, vehicle-treated preparations, no significant reduction of the contractile response
Fig. 2. Concentration-dependent (0.3–100 nM) inhibitory effect of ibodutant on NANC contractions induced by electrical field stimulation (10 Hz, 60 V, 0.5 ms for 10 s) of the isolated guinea pig bronchus. Each value is the mean 7S.E.M. of 3–12 experiments. One-way ANOVA and Dunnett’s post hoc test. *Po 0.05 compared to control (vehicle) value.
to the repeated EFS was observed (% of histamine Emax: S1 ¼42 74, S2 ¼41 7 5 and S3 ¼38 74; n ¼12). Ibodutant (0.3–100 nM), administered 60 min before the third cycle of stimulation (S3), produced a concentration-dependent inhibition of the EFS-induced response (Fig. 2). At the highest concentration used (100 nM) ibodutant almost abolished the contraction induced by EFS (96 74% inhibition; n ¼3; Fig. 2). The calculated IC50 value was 2.98 nM (95% c.l. 1.73–5.16 nM). 3.3. Inhibition of antigen-induced contractions In bronchi from sensitized animals, OVA (10 mg/ml) evoked an immediate contraction which averaged 4.45 70.46 mN corresponding to 63 76% of that produced by 1 mM acetylcholine (n ¼9). After the maximal response was reached the tension declined toward the baseline level during the 100 min of recording period (Fig. 3), at this time the residual contraction being 16 75% of the OVA maximal response and the T50% of baseline recovery was 29 74 min. Phosphoramidon (10 mM), added 75 min before
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Fig. 3. Time-course of the normalized contractile allergic response induced by ovalbumin (OVA, 10 mg/ml, added at time zero) in the bronchi isolated from OVAsensitized guinea pigs: (■) controls, (▲) preparations pretreated with phosphoramidon (10 mM, 75 min before OVA) and (●) preparations pretreated with ibodutant (100 nM, 60 min before OVA) and phosphoramidon (10 mM, 75 min before OVA). Each value is the mean 7 S.E.M. of 9–17 experiments. Two-way ANOVA and Bonferroni’s post hoc test. *Po 0.05 compared to time-matched values in controls and ibodutant pretreated preparations.
the OVA challenge, did not affect the amplitude of the contractile response induced by OVA (10 mg/ml) which averaged 4.067 0.45 mN corresponding to 627 5% of the contraction to 1 mM acetylcholine (n ¼13) but it produced a significant slowing in the fading of the motor response (Fig. 3) that, at the end of the recording period (100 min), was still significantly sustained and averaged 38 73% of the OVA maximal response, the T50% of baseline recovery in phosphoramidon pretreated preparations was 487 5 min (P o0.05 vs. controls). The addition of ibodutant (100 nM, 60 min before the OVA challenge) to preparations preincubated with phosphoramidon (10 mM, 15 min before ibodutant and 75 min before OVA challenge) did not affect the contractile response to OVA which averaged 4.20 70.36 mN corresponding to 59 73% of the contraction to 1 mM acetylcholine (n¼ 17), but completely prevented the slowing in the rate of relaxation induced by phosphoramidon pretreatment (Fig. 3). At the end of the recording period the residual tension was similar to that of controls in the absence of phosphoramidon and averaged 117 3% of the OVA maximal response, the T50% of baseline recovery was 3372 min (P o0.05 vs. phoshoramidon pretreated; P 40.05 vs. controls).
4. Discussion The antagonist activity of ibodutant was investigated in guinea pig bronchial preparations towards contractile responses produced by (1) the exogenous application of the selective NK2 receptor agonist [βAla8]NKA(4-10), (2) the release of endogenous TKS induced by electrical field stimulation of intramural capsaicinsensitive afferent nerves and (3) the antigen challenge in preparations from OVA sensitized guinea pigs. The analysis of ibodutant antagonism towards concentrationresponse curves produced by the NK2 selective agonist [βAla8]NKA (4-10) clearly demonstrates that in guinea pig bronchus ibodutant acts as a competitive NK2 receptor antagonist (Schild-plot slope not different from unity). The apparent antagonist potency value of ibodutant measured in this assay (pKB ¼8.31) is one log unit lower than that previously measured in the isolated guinea pig colon contractility assay (pKB ¼9.3, Cialdai et al., 2006). In previous functional experiments in isolated smooth muscle preparations ibodutant showed a marked species selectivity at the tachykinin NK2 receptor with the highest antagonist potency in human and guinea pig colon (pKB ¼ 9.1 and 9.3, respectively; Santicioli et al., 2013; Cialdai et al., 2006) and human and pig bladder (pKB ¼9.2 and 8.8, respectively; Cialdai et al., 2006) whereas it was less potent in the rat and mouse urinary bladder (pKB ¼ 6.3 and 5.8, respectively; Cialdai et al., 2006). Although marked species-related
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differences have been observed in the tachykinin NK2 receptor pharmacology on the basis of the different pharmacological profile and potency rank order of previously discovered antagonists (Maggi et al., 1992a,b; Maggi, 1995 for review), less frequently examples of intraspecies heterogeneity have been described. Putative intraspecies heterogeneity have been reported when comparing the pharmacology of NK2 receptors expressed in rat spinal cord (Xu et al., 1991; Wiesenfeld-Hallin et al., 1994; Lepre et al., 1994), in sensory nerves in the rat urinary bladder (Morrison et al., 1990; Nimmo et al., 1992) or in guinea pig alveolar macrophages (Brunelleschi et al., 1990, 1992) vs. the pharmacology of NK2 receptors expressed on smooth muscle from these species (Maggi et al., 1993 for review). Several studies have indicated the existence of isoforms of the tachykinin NK1 receptor differing in the length of the C-terminal tail (Fong et al., 1992; Baker et al., 2003). Similarly the organization of the NK2 receptor gene indicates alternative splicing and/or post-translational modifications as a potential mechanisms leading to intraspecies receptor heterogeneity. Candenas et al. (2002) identified a splice variant (NK2β isoform) of the tachykinin NK2 receptor in human uterus and different rat tissues expressing also the wild-type tachykinin NK2 receptor (NK2α isoform). However, although the ratio of tachykinin NK2α/NK2β receptor mRNA levels varies among different rat tissues, the NK2β splice variant does not display functional activity in the conventional transduction pathways (IP production, Ca2 þ mobilization) activated by wild-type tachykinin NK2 receptor (Bellucci et al., 2004). The NK2β splice variant encoded a protein with a truncated, six transmembrane domains structure, and certain membrane receptors have truncated forms that are not inserted in the membrane but remain in solution and are able to interact with ligands (Dannies, 2001; Rose-John and Heinrich, 1994). These findings may suggest that even if not apparently functional, the β isoform may be of physiological relevance and may act as a regulator of tachykinin NK2 receptor function and responsiveness. In addiction the NK2 receptor has been described to adopt different conformations that are linked to different coupling mechanisms (Palanche et al., 2001; Lecat et al., 2002). In this view, a different antagonist potency measured in different tissues of the same animal species may indicate a different participation of transduction mechanisms into the measured responses which may be highlighted by the different selectivity of an antagonist (Maggi et al., 1993 for review). Upon agonist activation, the tachykinin NK2 receptor is known to couple through two different transduction pathways. The first one involves the Gαq-IP3-Ca2 þ pathway that is the main signal leading to smooth muscle contraction. The second one is linked to Gαs and the consequent activation of adenylate cyclase to increase cAMP levels (Maillet et al., 2007). Interestingly, these tachykinin NK2 receptor conformers can be differentially modulated by tachykinin NK2 receptor ligands that might change the relative proportions of receptor conformations with compounds acting as antagonists on cAMP production but without activity on the IP3-Ca2 þ pathway (Valant et al., 2009). NK2 receptor agonists produce a contractile response in both guinea pig colon and bronchial smooth muscle and a different contribution of NK2 receptor-triggered cAMP production in these two tissues cannot be excluded. If in the bronchus the cAMP production induced by NK2 receptor stimulation is more prominent than in the colon, and ibodutant antagonize the excitatory IP3-Ca2 þ pathway more potently than the cAMP production, the estimated potency of this antagonist should be different in these two tissues. This hypothesis is substantiated by the presence of NK2 receptors inducing inhibitory responses mediated by nitric oxide (NO) on smooth muscle contraction both in the guinea pig airways (1; Imasaki et al., 2001) and colon (Zagorodnyuk and Maggi, 1995), although the inhibitory effect in the colon is mediated by neuronal rather than direct smooth muscle activation (Onori et al., 2000).
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The capability of ibodutant to antagonize the endogenous activation of the NK2 receptor in guinea pig bronchus was evaluated in NANC contraction induced by EFS. As already reported the EFS of guinea pig bronchus elicits a NANC contraction mediated by TKS released from unmyelinated sensory nerves (Maggi et al., 1991b). Experiments with selective antagonists demonstrated that both NK1 and NK2 receptors mediate the NANC contraction and that the relative contribution of NK2 receptors is greater than that of NK1 (Maggi et al., 1991b; Renzetti et al., 1992; Heavey et al., 1997). In our study the contractile response of the guinea pig bronchus induced by EFS is fully mediated by stimulation of NK2 receptors as experiments were performed in the presence of atropine and SR140333 to rule out the cholinergic and the NK1 components of the contraction, respectively. In these conditions ibodutant concentration-dependently inhibit the NK2mediated component of the NANC bronchoconstriction induced by EFS further demonstrating its potent antagonistic activity. Sensitization of guinea pigs to OVA is an established animal model for allergic airway diseases and antigen challenge of sensitized airways results in bronchoconstriction due to the action of several different mediators (Barnes et al., 1988; Barnes, 1996). It has been proposed that the effects of antigen challenge comprise the activities of both inflammatory cells and sensory nerves and the interaction among these structures has been suggested (Barnes, 1992; Marshall and Waserman, 1995). TKS are present in sensory nerve fibers in the airways of many species, including guinea pigs, but conflicting results have been reported on the role played by these peptides in the response that occurs after antigen challenge both in vivo and in vitro (Manzini et al., 1987; Ingenito et al., 1991; Kohrogi et al., 1991, 1997; Lai, 1991; Bertrand et al., 1993; Warth Mdo et al., 1995; Cibulsky and Thompson, 1997). The involvement of TKS in the bronchoconstriction induced by application of antigen has been shown by Kohrogi et al. (1991) who reported that in guinea pig bronchi in vitro, the neutral endopeptidase inhibitor phosphoramidon markedly prolonged the contraction following the peak reaction after antigen challenge. This effect was absent in capsaicin-pretreated bronchial tissue and was completely reversed by the pretreatment with the mixed tachykinin NK1 and NK2 receptor antagonist FK224 (Kohrogi et al., 1997) indicating the participation of TKS released from capsaicin-sensitive sensory nerves. Similar results were obtained by in vivo experiments in OVA sensitized guinea pig by Bertrand et al. (1993) who found that small doses of aerosolized OVA produced an increase of pulmonary resistance which was markedly prolonged by phosphoramidon pretreatment and reduced by the NK2 receptor antagonist SR48968. Our findings in experiments performed with bronchi from OVA-sensitized guinea pigs are in agreement with the results of Kohrogi et al. (1997) and show that ibodutant inhibits the tachykinin-mediated component of antigen induced bronchial contraction potentiated by phosphoramidon. In conclusion, the present investigation along with our previous results obtained in in vitro and in vivo, further confirm that Ibodutant is a potent NK2 receptor antagonist being very effective in blocking the NK2 component of the guinea pig bronchoconstriction produced by the stimulation of sensory nerves.
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European Journal of Pharmacology journal homepage: www.elsevier.com/locate/ejphar
Pulmonary, gastrointestinal and urogenital pharmacology
Antagonist profile of ibodutant at the tachykinin NK2 receptor in guinea pig isolated bronchi Paolo Santicioli n, Stefania Meini, Sandro Giuliani, Alessandro Lecci, Carlo Alberto Maggi Department of Pharmacology, Menarini Ricerche S.p.A., Via Rismondo 12/A, 50131 Florence, Italy
art ic l e i nf o
a b s t r a c t
Article history: Received 12 June 2013 Received in revised form 1 August 2013 Accepted 10 October 2013 Available online 24 October 2013
In this study we have characterized the pharmacological profile of the non-peptide tachykinin NK2 receptor antagonist ibodutant (MEN15596) in guinea pig isolated main bronchi contractility. The antagonist potency of ibodutant was evaluated using the selective NK2 receptor agonist [βAla8] NKA(4-10)-mediated contractions of guinea pig isolated main bronchi. In this assay ibodutant (30, 100 and 300 nM) induced a concentration-dependent rightward shift of the [βAla8]NKA(4-10) concentrationresponse curves without affecting the maximal contractile effect. The analysis of the results yielded a Schild-plot linear regression with a slope not different from unity (0.95, 95% c.l. 0.65–1.25), thus, indicating a surmountable behavior. The calculated apparent antagonist potency as pKB value was 8.31 7 0.05. Ibodutant (0.3–100 nM) produced a concentration-dependent inhibition of the nonadrenergic–noncholinergic (NANC) contractile response induced by electrical field stimulation (EFS) of intrinsic airway nerves in guinea pig isolated main bronchi. At the highest concentration tested (100 nM) ibodutant almost abolished the EFS-induced bronchoconstriction (957 4% inhibition), the calculated IC50 value was 2.98 nM (95% c.l. 1.73–5.16 nM). In bronchi from ovalbumin (OVA) sensitized guinea pigs ibodutant (100 nM) did not affect the maximal contractile response to OVA, but completely prevented the slowing in the fading of the motor response induced by phosphoramidon pretreatment linked to the endogenous neurokinin A release. Altogether, the present study demonstrates that ibodutant is a potent NK2 receptor antagonist in guinea pig airways. & 2013 Elsevier B.V. All rights reserved.
Keywords: Ibodutant MEN15596 NK2 antagonist NANC contraction Bronchi Guinea pig
1. Introduction Tachykinins (TKS) are transmitters of the excitatory NANC peripheral nervous system (Lundberg and Saria, 1987), they are co-localized with other neuropeptides in the sensory unmyelinated C-fibers which innervate all compartments of the airways wall, from the trachea down to the bronchioles (Lundberg and Saria, 1987; Baluk and McDonald, 1998). Guinea pig and human airways are well supplied with tachykinin-containing nerves (Lundberg et al., 1984) and TKS have been described to play an important role in the pathophysiology of airways such as asthma and bronchial hyperresponsiveness (Pernow, 1985; Barnes, 1990; Maggi, 1995; Kraneveld et al., 1997; De Vries et al., 1999; Joos et al., 2001) mediating several processes such as bronchoconstriction (Hua et al., 1984; Advenier et al., 1987), submucosal gland hypersecretion (Gashi et al., 1986), plasma proteins extravasation (Lundberg et al., 1983), recruitment of inflammatory cells (Maggi, 1997 for review; Schuiling et al., 1999) and stimulation of resident immune cells (Brunelleschi et al., 1990).
n
Corresponding author. Tel.: þ 39 55 5680735; fax: þ 39 55 56809954. E-mail address: [email protected] (P. Santicioli).
0014-2999/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejphar.2013.10.029
The bronchial compartment of airways, characterized by high airflow resistance, is the most sensitive region to neurokinin A, suggesting the importance of this peptide in bronchoconstriction (Venugopal et al., 2004). Although both NK1 and NK2 receptors are capable to elicit contractions of guinea pig bronchus (Maggi et al., 1991a), pharmacological studies using selective NK1 and NK2 tachykinin receptor antagonists have provided evidence that the nonadrenergic–noncholinergic contractile (NANC) responses produced by stimulation of sensory unmyelinated C-fibers by the transient receptor vanilloid 1 (TRPV1) agonist capsaicin, or by electrical stimulation of intrinsic airway nerves, mainly involve the selective activation of tachykinin NK2 receptors with a smaller NK1 receptor contribution (Szolcsányi and Barthó, 1982; Maggi et al., 1991b; Renzetti et al., 1992). Ibodutant (MEN15596) is a potent and selective non-peptide tachykinin NK2 receptor antagonist characterized by subnanomolar antagonist potency in human, guinea pig, and minipig in vitro bioassays (Cialdai et al., 2006; Santicioli et al., 2013) and long lasting antagonist properties both in vivo and in vitro (Cialdai et al., 2006; Meini et al., 2009; Santicioli et al., 2013). The present study was undertaken to characterize the pharmacological properties of ibodutant in blocking the smooth muscle contractility of the guinea pig isolated bronchi in different models of tachykinin NK2 receptor activation.
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2. Materials and methods 2.1. General The experimental procedures employed in this study were approved by the Institutional Animal Care and Use Committee of Menarini Ricerche (Firenze, Italy) and carried out in accordance with the Italian legislation (D.L. 116 27/01/1992), which complies with European Community guidelines (CEE Directive 86/609) for the care and use of experimental animals. Male Dunkin-Hartley guinea pigs (500–700 g, Charles-River Laboratory, Italy) were killed by an intraperitoneal overdose of pentobarbital. The tracheobronchial tree was rapidly excised and placed in ice-cold Krebs solution of the following composition (mmol/l): NaCl 119; NaHCO3 25; KH2PO4 1.2; MgSO4 1.5; CaCl2 2.5; KCl 4.7 and glucose 11. The main bronchi were cleaned of surrounding tissue and two main bronchial transverse ring preparations (3–4 mm in length) were obtained from each animal. Unless otherwise specified in the text, the bronchial rings were deprived of epithelium by gently scraping the luminal surface with the smooth edge of a Dumont forceps and placed in 5 ml organ baths filled with warmed (37 1C) and oxygenated (96% O2 and 4% CO2, pH 7.4) Krebs solution. The tissues were connected to isometric force transducers (Ugo Basile, Varese, Italy) under an initial tension of 10 mN and mechanical activity was digitally recorded by an Octal Bridge Amplifier connected to PowerLab/8sp data acquisition system and analyzed using the Chart 4.2 software (ADInstruments Ltd, Oxford, UK). Depending on the experimental protocol, various antagonists or inhibitors were added to the Krebs solution as described in the individual sections. 2.2. Contractions induced by [βAla8]NKA(4-10) After 120 min equilibration period, the preparations were stimulated using a maximal concentration of histamine (100 μM) to evaluate the contractile response of each preparation. After a further 120 min equilibration period, during which the medium was renewed every 10 min, cumulative concentration-response curves to the tachykinin NK2 receptor selective agonist [βAla8]NKA (4-10), were constructed. One of the two tissue rings obtained from the same animal was pretreated with vehicle (DMSO; 1–3 μl/ml) and used to perform the control curve to [βAla8]NKA(4-10), while the other ring was pretreated with ibodutant (30, 100 or 300 nM) added to the organ bath 60 min before the concentration-response curve to [βAla8]NKA(4-10). In each preparation only a single cumulative concentration-response curve to [βAla8]NKA(4-10) was carried out and only one concentration of the antagonist was tested. These experiments were performed in the presence of atropine (1 μM), indomethacin (3 μM), DL-thiorphan (1 μM) and the selective tachykinin NK1 receptor antagonist SR140333 (1 μM). 2.3. NANC contractions induced by electrical field stimulation In a separate series of experiments, endogenous tachykininergic contractile responses mediated by the NK2 receptor activation were evoked in bronchial rings by electrical field stimulation (EFS) of intramural capsaicin-sensitive afferent nerves (Maggi et al., 1990, 1991b; Heavey et al., 1997). These experiments were performed in the presence of atropine (1 mM) to eliminate the fast cholinergic component of the response to EFS, indomethacin (3 mM) to inhibit the endogenous formation of cyclooxygenase products, SR140333 (1 mM) to selectively block tachykinin NK1 receptors, DL-thiorphan (1 mM) to inhibit TKS metabolism and guanethidine (3 mM) to inhibit adrenergic neurotransmission. After 120 min equilibration period, histamine (100 μM) was administered to evaluate the contractile response of each
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preparation. After 90 min, each preparation was subjected to three cycles of EFS (S1, S2 and S3) consisting of a train of pulses at the frequency of 10 Hz for 10 s (60 V, 0.5 ms pulse width) delivered every 90 min by means of two platinum wire electrodes placed at the top and the bottom of the organ bath and connected to a GRASS S88 stimulator (Grass Medical Instruments, Quincy, MA, USA). After two control responses to EFS (S1 and S2), ibodutant (0.3–100 nM) or its vehicle (DMSO 1–3 ml/ml), were applied 60 min before the third (S3) response to EFS. 2.4. OVA sensitization procedure and assessment of bronchial reactivity The guinea pig sensitization procedure and in vitro functional experiments were performed according to the method previously described by Kohrogi et al. (1991, 1997). Guinea pigs were actively sensitized by intramuscular injection of 35 mg OVA dissolved in 0.3 ml of sterile saline twice with an interval of one week between injections. The animals were used for in vitro experiments from three weeks after the second OVA injection. Bronchial rings with intact epithelium were prepared and set-up for isometric tension measurement as described in the general section. After 120 min equilibration period the preparations were stimulated using a maximal concentration of acetylcholine (1 mM) to evaluate the viability and the contractile response of each preparation. After 60 min, during which the bathing solution was renewed at 10 min intervals, phosphoramidon (10 mM) or its vehicle (distilled water, 1 ml/ml) were added to the tissue bathing solution and after 15 min ibodutant (100 nM) or its vehicle (DMSO 1 ml/ml) were added and left in contact for 60 min. Finally, in order to induce the allergic response, all preparations received 10 mg/ml OVA and the total contractile response was recorded over a period of 100 min. 2.5. Data evaluation and statistical analysis All data in the text and figures were expressed as the mean 7 standard error of the mean (S.E.M.) or 95% confidence limits (95% c.l.) of the given number of experiments (n). Concentration-response curves were analyzed by sigmoidal nonlinear regression fitting using the Prism 4.02 program (GraphPad Software Inc., San Diego, CA, USA) to determine the molar concentration of the agonist producing the 50% (EC50) of its maximal response (Emax). The antagonist potency of ibodutant was expressed as apparent pKB estimated as the mean of the individual values obtained with the equation: pKB ¼log(CR-1) log[B] where CR is the concentration-ratio calculated from equieffective concentrations of agonist (EC50) obtained in the presence and in the absence of antagonist and B is the antagonist concentration used (Kenakin, 1997). Competitive antagonism was checked by the Schild regression analysis by plotting the estimates of log(CR-1) against log[B] to determine the slope of linear regression: a plot with linear regression line and slope not significantly different from unity was considered as proof of competitive antagonism (Arunlakshana and Schild, 1959). In experiments with EFS, the effect produced by ibodutant or its vehicle (DMSO) on the amplitude of contraction was assessed as percentage of variation of the response in the presence of the antagonist (S3) vs. control response (S2). The preparations in which the variation between the two control responses S1 and S2 was 420% were excluded from calculations. The concentration of antagonist causing 50% inhibition (IC50) of contraction amplitude induced by EFS was calculated by sigmoidal nonlinear regression analysis (GraphPad Prism 4.02, San Diego, CA, USA). In experiments with bronchial rings from OVA sensitized guinea pigs the amplitude of contractile response induced by 10 mg/ml OVA
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was evaluated at 1 min intervals during the initial 10 min from application and then every 5 min for a total of 100 min. For each experiment the duration of the contraction induced by OVA was calculated from start until 50% of its baseline recovery (T50%). Since there were no differences in maximum contractile effect induced by OVA between different groups, collected data were normalized to the maximal response induced by OVA in each preparation. Results were compared for significant differences using one- or two-way analysis of variance (ANOVA) followed by Dunnett’s or Bonferroni’s post hoc test. A P value o0.05 was considered statistically significant. 2.6. Chemicals Drugs were obtained from the following sources: ibodutant (MEN15596; 6-methyl-benzo[b]thiophene-2-carboxylic acid [1-(2phenyl-1R-{[1-(tetrahydropyran-4-ylmethyl)-piperidin-4-ylmethyl]carbamoyl}-ethylcarbamoyl)-cyclopentyl]-amide) (batches L2/06 and L3/08) was synthesized at Lusochimica (Menarini Group, Lomagna, Italy); the non-peptide NK1 antagonist SR140333 or: [(S)1-{2-[3-(3,4dichlorophenyl)-1-(3-isopropoxyphenylacetyl) piperidin-3-yl]ethyl};4-phenyl-1-azoniabicyclo [2,2,2] octane chloride] was kindly provided by Sanofi Aventis (Montpellier, France); [βAla8]NKA(4-10) was from EspiKem (Florence, Italy); acetylcholine hydrochloride, atropine sulfate, histamine hydrochloride, indomethacin, ovalbumin (grade V), phosphoramidon and DL-thiorphan were from Sigma–Aldrich Italy (Milan, Italy). Guanethidine sulfate was from ICFI (Milan, Italy). All other reagents used were of analytical grade and purchased from Merck (Darmstadt, Germany).
Fig. 1. (A) Antagonism by ibodutant towards the contractile responses induced by [βAla8]NKA(4-10) in the isolated guinea pig bronchus. Concentration-response curves for [βAla8]NKA(4-10) were constructed as described in Section 2 in the absence (control) and presence of the indicated concentrations of ibodutant. Each value is the mean7 S.E.M. of five experiments. (B) Schild-plot of agonist concentration ratios vs. ibodutant concentrations (slope ¼ 0.95, 95% c.l. 0.65–1.25). Each value is the mean 7 S.E.M. of five experiments.
3. Results 3.1. Antagonism of contractions produced by [βAla8]NKA(4-10) In the presence of atropine (1 μM), indomethacin (3 μM), (1 μM) and SR140333 (1 μM), the selective NK2 receptor agonist [βAla8]NKA(4-10) (0.1 nM to 3 mM) produced a slowly developing concentration-dependent tonic contraction of the guinea pig isolated bronchus whose maximal effect (Emax) was 13.537 1.08 mN, corresponding to 112 72% of that produced by histamine 100 μM (n ¼15). The EC50 value calculated from the control concentration-response curves to [βAla8]NKA(4-10) was 2.85 nM (95% c.l. 2.62–3.12 nM; n ¼15). Ibodutant (30, 100 and 300 nM) was devoid of any effect on the resting tension of the preparation whereas it concentrationdependently antagonized the [βAla8]NKA(4 -1 0)-induced contractile responses, thus producing a parallel rightward shift (Fig. 1A) of the agonist response curves without depressing the agonist Emax being 13.5371.08, 13.3770.58, 14.3771.32 and 12.6571.32 mN in controls and in the presence of 30, 100 and 300 nM of ibodutant, respectively. Schild-plot analysis was consistent with competitive antagonism (slope¼0.95, 95% c.l. 0.65–1.25; n¼15) and a pKB value of 8.3170.05 was calculated (Fig. 1B). DL-thiorphan
3.2. Inhibition of NANC contractions produced by electrical field stimulation In the presence of atropine (1 μM), indomethacin (3 μM), SR140333 (1 μM), DL-thiorphan (1 μM) and guanethidine (3 μM), EFS (10 Hz, 60 V, 0.5 ms for 10 s) induced a slowly developing NANC contraction of the guinea pig isolated bronchus which averaged 5.167 0.28 mN (n ¼34) corresponding to 42 72% of that produced by histamine 100 μM. In the controls, vehicle-treated preparations, no significant reduction of the contractile response
Fig. 2. Concentration-dependent (0.3–100 nM) inhibitory effect of ibodutant on NANC contractions induced by electrical field stimulation (10 Hz, 60 V, 0.5 ms for 10 s) of the isolated guinea pig bronchus. Each value is the mean 7S.E.M. of 3–12 experiments. One-way ANOVA and Dunnett’s post hoc test. *Po 0.05 compared to control (vehicle) value.
to the repeated EFS was observed (% of histamine Emax: S1 ¼42 74, S2 ¼41 7 5 and S3 ¼38 74; n ¼12). Ibodutant (0.3–100 nM), administered 60 min before the third cycle of stimulation (S3), produced a concentration-dependent inhibition of the EFS-induced response (Fig. 2). At the highest concentration used (100 nM) ibodutant almost abolished the contraction induced by EFS (96 74% inhibition; n ¼3; Fig. 2). The calculated IC50 value was 2.98 nM (95% c.l. 1.73–5.16 nM). 3.3. Inhibition of antigen-induced contractions In bronchi from sensitized animals, OVA (10 mg/ml) evoked an immediate contraction which averaged 4.45 70.46 mN corresponding to 63 76% of that produced by 1 mM acetylcholine (n ¼9). After the maximal response was reached the tension declined toward the baseline level during the 100 min of recording period (Fig. 3), at this time the residual contraction being 16 75% of the OVA maximal response and the T50% of baseline recovery was 29 74 min. Phosphoramidon (10 mM), added 75 min before
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Fig. 3. Time-course of the normalized contractile allergic response induced by ovalbumin (OVA, 10 mg/ml, added at time zero) in the bronchi isolated from OVAsensitized guinea pigs: (■) controls, (▲) preparations pretreated with phosphoramidon (10 mM, 75 min before OVA) and (●) preparations pretreated with ibodutant (100 nM, 60 min before OVA) and phosphoramidon (10 mM, 75 min before OVA). Each value is the mean 7 S.E.M. of 9–17 experiments. Two-way ANOVA and Bonferroni’s post hoc test. *Po 0.05 compared to time-matched values in controls and ibodutant pretreated preparations.
the OVA challenge, did not affect the amplitude of the contractile response induced by OVA (10 mg/ml) which averaged 4.067 0.45 mN corresponding to 627 5% of the contraction to 1 mM acetylcholine (n ¼13) but it produced a significant slowing in the fading of the motor response (Fig. 3) that, at the end of the recording period (100 min), was still significantly sustained and averaged 38 73% of the OVA maximal response, the T50% of baseline recovery in phosphoramidon pretreated preparations was 487 5 min (P o0.05 vs. controls). The addition of ibodutant (100 nM, 60 min before the OVA challenge) to preparations preincubated with phosphoramidon (10 mM, 15 min before ibodutant and 75 min before OVA challenge) did not affect the contractile response to OVA which averaged 4.20 70.36 mN corresponding to 59 73% of the contraction to 1 mM acetylcholine (n¼ 17), but completely prevented the slowing in the rate of relaxation induced by phosphoramidon pretreatment (Fig. 3). At the end of the recording period the residual tension was similar to that of controls in the absence of phosphoramidon and averaged 117 3% of the OVA maximal response, the T50% of baseline recovery was 3372 min (P o0.05 vs. phoshoramidon pretreated; P 40.05 vs. controls).
4. Discussion The antagonist activity of ibodutant was investigated in guinea pig bronchial preparations towards contractile responses produced by (1) the exogenous application of the selective NK2 receptor agonist [βAla8]NKA(4-10), (2) the release of endogenous TKS induced by electrical field stimulation of intramural capsaicinsensitive afferent nerves and (3) the antigen challenge in preparations from OVA sensitized guinea pigs. The analysis of ibodutant antagonism towards concentrationresponse curves produced by the NK2 selective agonist [βAla8]NKA (4-10) clearly demonstrates that in guinea pig bronchus ibodutant acts as a competitive NK2 receptor antagonist (Schild-plot slope not different from unity). The apparent antagonist potency value of ibodutant measured in this assay (pKB ¼8.31) is one log unit lower than that previously measured in the isolated guinea pig colon contractility assay (pKB ¼9.3, Cialdai et al., 2006). In previous functional experiments in isolated smooth muscle preparations ibodutant showed a marked species selectivity at the tachykinin NK2 receptor with the highest antagonist potency in human and guinea pig colon (pKB ¼ 9.1 and 9.3, respectively; Santicioli et al., 2013; Cialdai et al., 2006) and human and pig bladder (pKB ¼9.2 and 8.8, respectively; Cialdai et al., 2006) whereas it was less potent in the rat and mouse urinary bladder (pKB ¼ 6.3 and 5.8, respectively; Cialdai et al., 2006). Although marked species-related
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differences have been observed in the tachykinin NK2 receptor pharmacology on the basis of the different pharmacological profile and potency rank order of previously discovered antagonists (Maggi et al., 1992a,b; Maggi, 1995 for review), less frequently examples of intraspecies heterogeneity have been described. Putative intraspecies heterogeneity have been reported when comparing the pharmacology of NK2 receptors expressed in rat spinal cord (Xu et al., 1991; Wiesenfeld-Hallin et al., 1994; Lepre et al., 1994), in sensory nerves in the rat urinary bladder (Morrison et al., 1990; Nimmo et al., 1992) or in guinea pig alveolar macrophages (Brunelleschi et al., 1990, 1992) vs. the pharmacology of NK2 receptors expressed on smooth muscle from these species (Maggi et al., 1993 for review). Several studies have indicated the existence of isoforms of the tachykinin NK1 receptor differing in the length of the C-terminal tail (Fong et al., 1992; Baker et al., 2003). Similarly the organization of the NK2 receptor gene indicates alternative splicing and/or post-translational modifications as a potential mechanisms leading to intraspecies receptor heterogeneity. Candenas et al. (2002) identified a splice variant (NK2β isoform) of the tachykinin NK2 receptor in human uterus and different rat tissues expressing also the wild-type tachykinin NK2 receptor (NK2α isoform). However, although the ratio of tachykinin NK2α/NK2β receptor mRNA levels varies among different rat tissues, the NK2β splice variant does not display functional activity in the conventional transduction pathways (IP production, Ca2 þ mobilization) activated by wild-type tachykinin NK2 receptor (Bellucci et al., 2004). The NK2β splice variant encoded a protein with a truncated, six transmembrane domains structure, and certain membrane receptors have truncated forms that are not inserted in the membrane but remain in solution and are able to interact with ligands (Dannies, 2001; Rose-John and Heinrich, 1994). These findings may suggest that even if not apparently functional, the β isoform may be of physiological relevance and may act as a regulator of tachykinin NK2 receptor function and responsiveness. In addiction the NK2 receptor has been described to adopt different conformations that are linked to different coupling mechanisms (Palanche et al., 2001; Lecat et al., 2002). In this view, a different antagonist potency measured in different tissues of the same animal species may indicate a different participation of transduction mechanisms into the measured responses which may be highlighted by the different selectivity of an antagonist (Maggi et al., 1993 for review). Upon agonist activation, the tachykinin NK2 receptor is known to couple through two different transduction pathways. The first one involves the Gαq-IP3-Ca2 þ pathway that is the main signal leading to smooth muscle contraction. The second one is linked to Gαs and the consequent activation of adenylate cyclase to increase cAMP levels (Maillet et al., 2007). Interestingly, these tachykinin NK2 receptor conformers can be differentially modulated by tachykinin NK2 receptor ligands that might change the relative proportions of receptor conformations with compounds acting as antagonists on cAMP production but without activity on the IP3-Ca2 þ pathway (Valant et al., 2009). NK2 receptor agonists produce a contractile response in both guinea pig colon and bronchial smooth muscle and a different contribution of NK2 receptor-triggered cAMP production in these two tissues cannot be excluded. If in the bronchus the cAMP production induced by NK2 receptor stimulation is more prominent than in the colon, and ibodutant antagonize the excitatory IP3-Ca2 þ pathway more potently than the cAMP production, the estimated potency of this antagonist should be different in these two tissues. This hypothesis is substantiated by the presence of NK2 receptors inducing inhibitory responses mediated by nitric oxide (NO) on smooth muscle contraction both in the guinea pig airways (1; Imasaki et al., 2001) and colon (Zagorodnyuk and Maggi, 1995), although the inhibitory effect in the colon is mediated by neuronal rather than direct smooth muscle activation (Onori et al., 2000).
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The capability of ibodutant to antagonize the endogenous activation of the NK2 receptor in guinea pig bronchus was evaluated in NANC contraction induced by EFS. As already reported the EFS of guinea pig bronchus elicits a NANC contraction mediated by TKS released from unmyelinated sensory nerves (Maggi et al., 1991b). Experiments with selective antagonists demonstrated that both NK1 and NK2 receptors mediate the NANC contraction and that the relative contribution of NK2 receptors is greater than that of NK1 (Maggi et al., 1991b; Renzetti et al., 1992; Heavey et al., 1997). In our study the contractile response of the guinea pig bronchus induced by EFS is fully mediated by stimulation of NK2 receptors as experiments were performed in the presence of atropine and SR140333 to rule out the cholinergic and the NK1 components of the contraction, respectively. In these conditions ibodutant concentration-dependently inhibit the NK2mediated component of the NANC bronchoconstriction induced by EFS further demonstrating its potent antagonistic activity. Sensitization of guinea pigs to OVA is an established animal model for allergic airway diseases and antigen challenge of sensitized airways results in bronchoconstriction due to the action of several different mediators (Barnes et al., 1988; Barnes, 1996). It has been proposed that the effects of antigen challenge comprise the activities of both inflammatory cells and sensory nerves and the interaction among these structures has been suggested (Barnes, 1992; Marshall and Waserman, 1995). TKS are present in sensory nerve fibers in the airways of many species, including guinea pigs, but conflicting results have been reported on the role played by these peptides in the response that occurs after antigen challenge both in vivo and in vitro (Manzini et al., 1987; Ingenito et al., 1991; Kohrogi et al., 1991, 1997; Lai, 1991; Bertrand et al., 1993; Warth Mdo et al., 1995; Cibulsky and Thompson, 1997). The involvement of TKS in the bronchoconstriction induced by application of antigen has been shown by Kohrogi et al. (1991) who reported that in guinea pig bronchi in vitro, the neutral endopeptidase inhibitor phosphoramidon markedly prolonged the contraction following the peak reaction after antigen challenge. This effect was absent in capsaicin-pretreated bronchial tissue and was completely reversed by the pretreatment with the mixed tachykinin NK1 and NK2 receptor antagonist FK224 (Kohrogi et al., 1997) indicating the participation of TKS released from capsaicin-sensitive sensory nerves. Similar results were obtained by in vivo experiments in OVA sensitized guinea pig by Bertrand et al. (1993) who found that small doses of aerosolized OVA produced an increase of pulmonary resistance which was markedly prolonged by phosphoramidon pretreatment and reduced by the NK2 receptor antagonist SR48968. Our findings in experiments performed with bronchi from OVA-sensitized guinea pigs are in agreement with the results of Kohrogi et al. (1997) and show that ibodutant inhibits the tachykinin-mediated component of antigen induced bronchial contraction potentiated by phosphoramidon. In conclusion, the present investigation along with our previous results obtained in in vitro and in vivo, further confirm that Ibodutant is a potent NK2 receptor antagonist being very effective in blocking the NK2 component of the guinea pig bronchoconstriction produced by the stimulation of sensory nerves.
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