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The pharmacological profile of MEN15596 or (6-methyl-benzo[b]thiophene-2-carboxylic acid [1-(2-phenyl-1R-{[1-(tetrahydropyran-4-ylmethyl)-piperidin-4-ylmethyl]-carbamoyl}-ethylcarbamoyl)-cyclopentyl]-amide), a novel potent and selective tachykinin NK2 receptor antagonist endowed with oral activity, is described. At the human recombinant tachykinin NK2 receptor, MEN15596 showed subnanomolar affinity (pKi 10.1) and potently antagonized (pKB 9.1) the neurokinin A-induced intracellular calcium release. MEN15596 selectivity for the tachykinin NK2 receptor was assessed by binding studies at the recombinant tachykinin NK1 (pKi 6.1) and NK3 (pKi 6.4) receptors, and at a number of 34 molecular targets including receptors, transporters and ion channels. In isolated smooth muscle preparations MEN15596 showed a marked species selectivity at the tachykinin NK2 receptor with the highest antagonist potency in guinea-pig colon, human and pig bladder (pKB 9.3, 9.2 and 8.8, respectively) whereas it was three orders of magnitude less potent in the rat and mouse urinary bladder (pKB 6.3 and 5.8, respectively). In agreement with binding experiments, MEN15596 showed low potency in blocking selective NK1 or NK3 receptor agonist-induced contractions of guinea-pig ileum preparations (pA2

The pharmacological outline of a novel and original antagonist at the human tachykinin NK 2 receptor is presented, namely MEN13510 ( N - N ′-bis-[2-(1 H -indol-3-yl)-ethyl]- N , N ′-bis-(3-thiomorpholin-4-yl-propyl)-phthalamide). MEN13510 retained nanomolar affinity for the human tachykinin NK 2 receptor ( K i 6.4 nM), and micromolar affinity for the human tachykinin NK 1 and NK 3 receptors. A competitive antagonism is indicated by the Schild analysis (p K B 7.8, slope − 0.94) of concentration–response curves of NKA induced inositolphosphates accumulation in Chinese hamster ovary (CHO) cells expressing the human NK 2 receptor in the presence of MEN13510 (30–300 nM concentration range). The MEN13510 interaction with the human NK 2 receptor was evaluated by means of heterologous inhibition binding experiments, by using agonist and antagonist radioligands ([ 125 I]NKA, [ 3 H]nepadutant, [ 3 H]saredutant) at a series of mutant receptors having single aminoacidic substitutions of residues located in transmembrane (TM) segments 3, 4, 5, 6, and 7. MEN13510 affinity was not affected by the mutations in TM 3 and 4 (Q109A, F112A, T171A, C167G), and it was reduced by 10-fold at the I202F mutant, but not at the Y206A (TM4). Amongst the investigated mutants bearing the mutated residues in TM6 (F270A, Y266F, W263A) only F270A decreased the MEN13510 affinity by 7-fold. Even mutations in TM7 did reduce MEN13510 affinity by 32-fold (Y289T, but not Y289F) and 13-fold (F293A). Studied mutations represent the human tachykinin NK 2 receptor discriminants involved in the binding of previously reported peptidic and nonpeptidic antagonists, against which results obtained with MEN13510 are compared. Results indicate that the binding site of this antagonist is, at least in part, overlapping to that described for NKA or saredutant. Finally we show that MEN13510 retains nanomolar affinity for the recently discovered splice variant of the human tachykinin NK 2 receptor, namely β isoform, as it has been described for the nonpeptide antagonist saredutant.

In the present study, we have investigated, by binding and functional experiments, the pharmacological profile of a new human tachykinin NK(2) receptor splice variant named beta isoform. Neurokinin A, nepadutant, SR48968 [(S)-N-methyl-N[4-(4-acetylamino-4-phenyl piperidino)-2-(3,4-dichlorophenyl) butyl]benzamide] and substance P have been tested for binding on the receptor expressed in whole CHO transfected cells. Only SR48968 binds, but with an affinity about sixfold lower in respect to the alpha isoform. Moreover, neurokinin A was unable to inhibit the [(3)H]SR48968 binding to the beta isoform up to microM concentrations. In cells expressing the human tachykinin NK(2) receptor beta isoform, contrary to those expressing the alpha isoform, natural or selective tachykinin receptor agonists (1 microM) were unable to produce a significant activation of inositol phosphate (IP) production or increase of intracellular calcium concentration [Ca(2+)](i). The recently discovered tachykinins, endokinins C and D, did not activate IP production or [Ca(2+)](i) increase in cells expressing the alpha or beta isoform of the human tachykinin NK(2) receptor. The present data indicate that the human tachykinin NK(2) receptor beta isoform is poorly or not expressed on the cell membrane surface and that it may possibly act as a regulator of tachykinin NK(2) receptor function. We cannot exclude the possibility that this receptor could interact with other presently unknown ligands.

The pharmacology of peptide and non-peptide bradykinin B2 receptor ligands was evaluated in the inositol phosphate (IP) production assay in CHO cells expressing the human bradykinin B2 receptor. The effect of single and double alanine mutation of D266 and D284 residues at the human bradykinin B2 receptor was evaluated on the agonist profile of bradykinin (H-Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg-OH) and the synthetic agonist FR190997 (8-[2,6-dichloro-3-[N-methylcarbamoyl)cinnamidoacetyl]-N-methylamino]benzyloxy]-2-methyl-4-(2-pyridylmethoxy)quinoline). Bradykinin potency (EC50 0.5 nM at the wild-type receptor) was reduced by 16-fold at D266A and D284A mutants and by 2300-fold at the D266A/D284A double mutant. None of the mutants affected the potency or the efficacy of FR190997. Peptide antagonists, Icatibant (H-DArg-Arg-Pro-Hyp-Gly-Thi-Ser-Dtic-Oic-Arg-OH) and MEN11270 (H-DArg-Arg-Pro-Hyp-Gly-Thi-c(Dab-DTic-Oic-Arg)c(7gamma-10alpha)) (100 nM) similarly antagonized the concentration-response curve to bradykinin or FR190997 (pA2 values 8.5 and 8.4 versus bradykinin and 8.2 and 8.4 versus FR190997) at the wild-type receptor. Non-peptide antagonists FR173657 ((E)-3-(6-acetamido-3-pyridyl)-N-[N-[2,4-dichloro-3-[(2-methyl-8-quinolinyl) oxymethyl]phenyl]-N-methylaminocarbonyl methyl]acrylamide) and LF16-0687 (1-[[2,4-dichloro-3-[(2,4-dimethylquinolin-8-yl)oxy] methyl]-phenyl]sulfonyl]-N-[3-[[4-(aminoiminomethyl)-phenyl]carbonylamino]propyl]-(S)-pyrrolidine carboxamide) (100 nM) showed an equivalent potency values in blocking the IP production induced by bradykinin or FR190997 (pA2 values 8.7 and 8.8 versus bradykinin and 8.8 and 8.6 versus FR190997). Whilst the antagonist potency of FR173657 and LF16-0687 was not affected by D266A/D284A double mutation (IP production induced by the synthetic agonist), that of Icatibant and MEN11270 was reduced by 50- and 200-fold. The antagonist potency of [Ala1]-Icatibant and [Ala2]-Icatibant (pA2 values at wild-type 7.7 and 6.4) was significantly less reduced (20-fold and 13-fold, respectively) by the D266A/D284A double mutation. Our results highlight a crucial role for two aspartic residues, D266 and D284, located at the top of transmembrane segments 6 and 7, in the high-affinity interaction of peptide antagonists with the human bradykinin B2 receptor. An interaction of these receptor residues with the N-terminal basic residues of Icatibant is hypothesized.

In this study we have characterized the pharmacological profile of the non-peptide tachykinin NK(2) receptor antagonist ibodutant (MEN15596) in guinea pig isolated main bronchi contractility. The antagonist potency of ibodutant was evaluated using the selective NK(2) 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) concentration-response 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 pK(B) value was 8.31 ± 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 (95 ± 4% inhibition), the calculated IC(50) 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 NK(2) receptor antagonist in guinea pig airways.

We have characterized the pharmacological profile of the nonpeptide tachykinin NK2 receptor antagonist ibodutant (MEN15596) through radioligand binding and contractility assays in the human colon smooth muscle. The antagonist affinity of ibodutant was evaluated through concentration-dependent inhibition curves at the [125I]NKA specific binding by using membranes prepared from human colon smooth muscle. In this assay the affinity of ibodutant (pKi 9.9) was compared to that of other two selective NK2 receptor antagonists, nepadutant (pKi 8.4) and saredutant (pKi 9.2). The antagonist potency of ibodutant was evaluated towards the [βAla8]NKA(4-10)-mediated contractions of human colon smooth muscle strips. In this assay ibodutant (3, 10, 30 and 100 nM) induced a concentration-dependent rightward shift of the [βAla8]NKA(4-10) concentration-response curves without depressing the maximal contractile effect. The analysis of the curves yielded a Schild-plot linear regression with a slope not different from unity (1.02), thus indicating a surmountable antagonist behavior. The calculated apparent antagonist potency as pKB value was 9.1. No sex related differences were observed in NK2 receptor pharmacology for [βAla8]NKA(4-10) or ibodutant in colonic strips obtained from male or female patients. Reversibility experiments of tachykinin NK2 receptor blockade indicated that the inhibition of the agonist-induced contractions in preparations pre-exposed to ibodutant, and afterwards subjected to repeated washing cycles remained almost constant showing no sign of recovery during the 3 h observation period. Overall, the present study indicates ibodutant as a potent tachykinin NK2 receptor antagonist in the human colon tissue, also endowed with a persistent duration of action.

The tachykinin NK3 receptor agonist, senktide, produces concentration-dependent contraction of the circular muscle of the guinea-pig ileum (EC50 2.59 nM). In the presence of the blocker of neuronal type of voltage-sensitive calcium channels, omega-conotoxin (0.1 microM), the contractile response to a low concentration of senktide was converted to an inhibitory effect on spontaneous activity of the ileum. This inhibitory effect was further enhanced in the presence of atropine (1 microM) and was abolished by tetrodotoxin (1 microM), indicating its neural origin. In the presence of atropine and omega-conotoxin, the inhibitory response to senktide (1 nM) was greatly inhibited or even abolished by L-nitroarginine (30 microM), its effect being prevented by L-arginine but not by D-arginine (300 microM in each case). Apamin (0.1 microM) failed to significantly affect the inhibitory response to senktide. Apamin enhanced spontaneous activity of the preparation while L-nitroarginine had no effect. Neither apamin nor L-nitroarginine affected the inhibitory response to isoprenaline. These findings indicate that inhibition of circular muscle activity produced through NK3 receptor stimulation in the guinea-pig ileum is mediated through a neuronal pathway involving nitric oxide or a nitric oxide-like substance(s) generation.

Several species-related differences have been reported in kinin B(2) receptor pharmacology. The present study aimed to evaluate the affinity of the bradykinin B(2) receptor antagonist MEN16132 for the rabbit and pig B(2) receptor, and radioligand binding experiments using [(3)H]bradykinin and membranes of rabbit and pig ileum smooth muscle were conducted. The [(3)H]bradykinin binding was characterized by homologous displacement curves indicating K(d) values of 0.65 and 0.33nM in rabbit and pig, respectively. The B(2) receptor specificity of [(3)H]bradykinin binding was shown by the low affinity (>microM) displayed by agonists ([desArg(9)]bradykinin and Lys[desArg(9)]bradykinin) and antagonists [Leu(8),desArg(9)]bradykinin and Lys[Leu(8),desArg(9)]bradykinin) selective for the B(1) receptor. The affinity of MEN16132 and other antagonists was determined by inhibition curves (pK(i) values in the rabbit and pig assay, respectively): MEN16132 (10.4 and 10.3) and peptide compounds such as icatibant (10.1 and 9.9) and MEN11270 (10.3 and 10.1) displayed subnanomolar potency in both assays; the nonpeptide LF16-0687 (8.4 and 8.5) and FR173657 (8.2 and 9.1) exhibited a different affinity pattern, whereas WIN64338 displayed low affinity (5.7 and

The pharmacological profile of novel antagonists endowed with high affinity for the human tachykinin NK(2) receptor is presented. MEN13918 (Ngamma[Nalpha[Nalpha(benzo[b]thiophen-2-yl)carbonyl]-1-aminocyclohexan-1-carboxy]-d-phenylalanyl]-3-cis-aminocyclohexan-1-carboxylic-acid-N-(1S,2R)-2-aminocyclohexyl)amide trifluoroacetate salt) and MEN14268 (Nalpha[Nalpha(benzo[b]thiophen-2-yl)carbonyl)-1-aminocyclopentane-1-carboxyl]-d-phenylalanine-N-[3(morpholin-4-yl)propyl]amide trifluoroacetate salt) were more potent in blocking neurokinin A (NKA, His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH(2)) induced contraction in human, which induced greater contraction in human (pK(B) 9.1 and 8.3) than rat (pK(B) 6.8 and6) urinary bladder smooth muscle preparation in vitro. In agreement with functional data, in membrane preparations of CHO cells stably expressing the human NK(2) receptors, both MEN13918 and MEN14268 potently inhibited the binding of agonist ([(125)I]NKA, K(i) 0.2 and 2.8 nM) and antagonist ([(3)H]nepadutant, K(i) 0.1 and 2.2 nM, [(3)H]SR48968 K(i) 0.4 and 6.9 nM) radioligands. Using site-directed mutagenesis and radioligands binding we identified six residues in the transmembrane (TM) helices that are critical determinants for the studied antagonists affinity. To visualize these experimental findings, we constructed a homology model based on the X-ray crystal structure of bovine rhodopsin and suggested a possible binding mode of these newly discovered antagonist ligands to the human tackykinin NK(2) receptor. Both MEN13918 and MEN14268 bind amongst TM4 (Cys167Gly), TM5 (Tyr206Ala), TM6 (Tyr266Ala, Phe270Ala), and TM7 (Tyr289Phe, Tyr289Thr). MEN13918 and MEN14268 diverging binding profile at Y289 mutations in TM7 (Tyr289Phe, Tyr289Thr) suggests a relation of their different chemical moieties with this residue. Moreover, the different influence on binding of these two ligands by mutations located deep along the inner side of TM6 (Phe270Ala, Tyr266Ala, Trp263Ala) indicates a nonequivalent positioning, although occupying the same binding crevice. Furthermore, binding data indicate the Ile202Phe mutation, which mimics the wild-type rat NK(2) receptor sequence, as a species selectivity determinant. In summary, data with mutant receptors describe, for these new tachykinin NK(2) receptor antagonists, a binding site which is partially overlapping either with that of the cyclized peptide antagonist nepadutant (cyclo-[[Asn(beta-d-GlcNAc)-Asp-Trp-Phe-Dpr-Leu]cyclo(2beta-5beta)] or the nonpeptide antagonist SR48968 ((S)-N-methyl-N[4-(4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl)butyl]benzamide).

We have compared the pharmacology of different antagonists, Icatibant (H-DArg-Arg-Pro-Hyp-Gly-Thi-Ser-DTic-Oic-Arg-OH), MEN 11270 (H-DArg-Arg-Pro-Hyp-Gly-Thi-c(Dab-DTic-Oic-Arg)c(7 gamma-10 alpha)), and FR173657 ((E)-3-(6-acetamido-3-pyridyl)-N-[N-[2, 4-dichloro-3-[(2-methyl-8-quinolinyl)oxymethyl]phenyl]-N-methyl aminocarbonylmethyl]acrylamide) at bradykinin B2 receptors expressed in the guinea-pig ileum by using bradykinin and the non-peptide FR190997 ((8-[2,6-dichloro-3-[N-[(E)-4-(N-methylcarbamoyl)cinnamidoacety l]-N -methylamino]benzyloxy]-2-methyl-4-(2-pyridylmethoxy)quinoline) as agonists. In organ bath experiments, Icatibant and FR173657 exerted a non-competitive antagonism (pKB 9.5 and 9.2, respectively) of the contractile response to bradykinin, whereas MEN 11270 showed competitive antagonism (pKB 8.3, slope -0.90). The profile of action and apparent affinities of the three antagonists did not change if contact time was prolonged. The inhibition by the three antagonists of the contractile response to bradykinin was differently reverted by washout (MEN 1127030 min, Icatibant60 min, FR17365760 min). The non-peptide ligand FR190997 acted as partial agonist if applied cumulatively to the bath (pD2 8.06, Emax 43% of maximal contractility), but as a full agonist when a maximally effective concentration was added (Emax 83%). FR173657 produced non-competitive antagonism of the response to FR190997 with apparent affinity similar to that measured toward bradykinin. On the contrary, Icatibant and MEN 11270 (300 nM both) competitively antagonized the contractile activity exerted by FR190997 with lower apparent pA2 value (6.9 and 7.2, respectively). In radioligand binding experiments, MEN 11270 and Icatibant displaced the [3H]bradykinin binding with pKi of 10.2 and 10.5 (Hill slope not different from unity), respectively. The non-peptide ligands displaced the [3H]bradykinin binding with similar affinity, their pKi being 8.7 and 8.6 for FR173657 and FR190997, respectively (both Hill slopes1). The present study indicates the difference in the antagonism type (competitive vs. non-competitive) by Icatibant, MEN 11270, and FR173657, as mainly ascribable to their different reversibility from the bradykinin B2 receptor, and affected by the kinetic of the response induced by the different agonists. Results are discussed in view of a different interaction of peptide and non-peptide agonist at the receptor.

The application of nociceptin (5-50 nmol/rat) onto the serosa in the urinary bladder of urethane-anaesthetized rats, with the intravesical volume kept below threshold for activation of the micturition reflex, induced a low amplitude tonic contraction (local, i.e., resistant to ganglionectomy) with high amplitude phasic contractions (reflex, i.e., abolished by ganglionectomy) superimposed. The pharmacology of the local contraction was studied in animals with acute bilateral ablation in the pelvic ganglia: the combined administration of tachykinin NK(1) (S)1-¿2-[3-(3, 4-dichlorophenyl)-1-(3-isopropoxyphenyl-acetyl)-piperidin-3-yl]eth yl¿-4-phenyl-1-azoniabicyclo[2.2.2.]octane chloride (SR 140333) and NK(2) c¿[(beta-D-GlcNAc)Asn-Asp-Trp-Phe-Dpr-Leu]c(2beta-5beta++ +)¿ (MEN 11420) receptor antagonists (given at doses of 1+0.1 micromol/kg, intravenous (i.v.), respectively) abolished the local bladder contraction induced by topical nociceptin (50 nmol/rat). These results indicate that the topical application of nociceptin onto the bladder evokes a tachykinin-mediated contraction.

We investigated the role of bradykinin B1 receptors in inducing urinary bladder contraction and maintaining bladder compliance in anaesthetized rats following cyclophosphamide-induced bladder inflammation and the influence of dexamethasone treatment on these responses. In the group treated with cyclophosphamide the amplitude of the contraction induced by the selective bradykinin B1 receptor agonist des-Arg9-bradykinin was larger than that in controls and dexamethasone prevented the up-regulation of this response induced by inflammation. The specific binding of [ 3 H ]des-Arg10-kallidin to bladder membranes was only detected in cyclophosphamide-treated rats: this binding was prevented by dexamethasone pretreatment. The bladder contraction induced by des-Arg9-bradykinin in cyclophosphamide-treated rats was antagonized by the bradykinin B1 receptor antagonist des-Arg9- d -Arg-[Hyp3,Thi5, d -Tic7,Oic8]bradykinin (des-Arg10-Hoe 140). Cyclophosphamide treatment increased the bladder weight and dexamethasone reversed this effect. Bladder compliance was decreased in the bladder inflammation group and this effect was partially reversed by dexamethasone pretreatment. Neither des-Arg10-Hoe 140 nor the combined administration of des-Arg10Hoe 140 and the selective bradykinin B2 receptor antagonist d -Arg-[Hyp3,Thi5, d -Tic7,Oic8]bradykinin (Hoe 140) affected bladder compliance, thus excluding a role of kinins in the maintenance of bladder tone during inflammation. These results indicate that: (1) dexamethasone pretreatment ameliorates cyclophosphamide-induced bladder inflammation; (2) dexamethasone pretreatment prevents cyclophosphamide-induced up-regulation of bradykinin B1 receptors; (3) kinins do not contribute to the increased vesical tone during inflammation.

Porcine endothelin (1 nM-0.3 microM) produced a concentration-dependent contraction of the isolated sphincter muscle of the pig iris. Neither atropine (1 microM) nor tetrodotoxin (1 microM) nor pre-exposure of the preparation to 10 microM capsaicin for 15 min affected the endothelin-induced response. We propose that the endothelin-induced contraction of the iris sphincter muscle of the pig is largely due to a direct action on smooth muscle cells and is unrelated to neurogenic myotic mechanisms.