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Icatibant is a well-known kinin B₂ receptor antagonist currently used for angiooedema attacks. MEN16132 is a non-peptide B₂ receptor antagonist, more potent and long lasting than icatibant in different models. Here we studied the reasons for these differences between the two antagonists.Rate of reversibility (over about 3 h) of the functional receptor blockade exerted by the antagonists was compared (inositol phosphates accumulation assay) in CHO cells expressing the human B₂ receptor and in human synovial cells. Antagonist pretreated cells were washed with medium and the time taken to restore bradykinin (BK) response measured. Antagonist affinity was measured by radioligand binding to wild type and mutated B₂ receptors.Recovery of BK-induced responses was slower in cells pretreated with MEN16132 than in those treated with icatibant. The affinity of icatibant (for the [³H]-BK or the B₂ receptor antagonist [³H]-MEN11270 binding site) was compared to that of MEN16132 using a panel of point-mutated receptors with mutations located at the transmembrane regions of the B₂ receptor, previously shown to decrease MEN16132 high affinity interaction. No consistent decrease of icatibant affinity was observed. From the different affinity of MEN16132 derivatives at wild type and W86A (transmembrane 2 region) receptors, and by evaluating its antagonist profile at the D266A/D284A double mutant receptor, a model of the MEN16132-B₂ receptor complex is proposed.MEN16132 dissociated from the B₂ receptor compartment more slowly than icatibant and interacted at a deeper level in transmembrane regions of the receptor.

High affinity peptide ligands for the bradykinin (BK) B2 subtype receptor have been shown to adopt a ß-turn conformation of the C-terminal tetrapeptide ...

We investigated the pharmacological profile of MEN 11270, or H-D-Arg-Arg-Pro-Hyp-Gly-Thi-c(Dab-DTic-Oic-Arg)c(7gamma-10 alpha), a conformationally constrained derivative of the B2 kinin receptor antagonist Icatibant. MEN 11270 bound with high-affinity to the B2 kinin receptor constitutively expressed by WI38 human fibroblasts, inhibiting 3H-bradykinin (BK) with a pKi value of 10.3 +/- 0.08 (n = 5). The rank order of affinity of several peptide and nonpeptide antagonists was also assessed: Icatibant (pKi = 10.6) approximately MEN 11270 (pKi = 10.3) approximately B9430 (pKi = 10.0)B9858 (pKi = 8.0)FR173657 (pKi = 7.6)WIN64338 (pKi = 7.2)Lys-[des-Arg9, Leu8]-BK (pKi6)[des-Arg9,Leu8]-BK (pKi5). MEN 11270 showed a low affinity in inhibiting 3H-Lys-[des-Arg9]-BK binding at the human B1 kinin receptor constitutively expressed by the same cells (pKi 6.0 +/- 0.33; n = 3). MEN 11270 showed no binding affinity (pIC505.5) at 29 different receptors and ion channels. In the human umbilical vein contraction assay, MEN 11270, shifted the concentration-response curve to BK to the right in a concentration-dependent manner (pA2 8.14 +/- 0.22, n = 7). The Schild plot was linear (slope 0.95 +/- 0.11), consistent with a competitive antagonism. In the same bioassay, MEN 11270 (10 microM) did not affect the concentration-response curve to the B1 agonist Lys-[des-Arg9]-BK nor the contractile responses elicited by noradrenaline or serotonin. These findings indicate MEN 11270 as an antagonist at the human B2 kinin receptor, with potency and selectivity comparable to those of the linear peptide antagonist, supporting the hypothesis that a constrained C-terminal beta-turn conformation preserves a high affinity for the interaction of Icatibant with the B2 kinin receptor.

The cyclic pseudopeptides MEN 10,548, MEN 10,581, MEN 10,619, MEN 10,677, MEN 10,777 and MEN 10,867 were studied at tachykinin neurokinin (NK)1, NK2 and NK3 receptors on several in vitro bioassays. All compounds were potent and competitive antagonists at tachykinin NK1 and NK2 receptors of the guinea-pig ileum, rabbit pulmonary artery and hamster trachea, showing the highest affinity for the hamster NK2 receptor (e.g., MEN 10,677: pKB = 9.3). By contrast, none showed affinity for NK3 receptors of the rat portal vein, up to 3 microM. In the guinea-pig isolated bronchus, the pseudopeptide compounds competitively antagonized the NK2 receptor-selective agonist [beta Ala8]-NKA (4-10) with potencies comparable to those shown at the rabbit NK2 receptor. In addition, the pseudopeptides were from 3.5-fold (MEN 10,677) to 16-fold (MEN 10548) more potent antagonists against septide than against [Sar9]SP sulfone, two agonists reportedly selective for two distinct sites/subtypes of the NK1 receptor. In binding experiments at human IM9 cells, the pseudopeptide compounds displaced [3H]substance P from human NK1 receptor, showing similar affinities to those displayed at the NK1 receptor in the guinea-pig ileum or bronchus against substance P methylester or septide, as agonists, respectively. This new class of pseudopeptide antagonists, by showing a comparable and high affinity at both tachykinin NK1 and NK2 receptors, might be proposed for treatment/prevention of airway diseases in which endogenous tachykinins play a role by activation of both receptors.

We recently reported the rational design, synthetics, and structural characterization of the most potent and selective peptide-based neurokinin A antagonist thus far described: cyclo(Met1-Asp2-Trp3-Phe4-Dap5-Leu6)cyclo(2 beta-5 beta). Its bicyclic structure is characterized by a type I and a type II two beta-turn around Trp3-Phe4 and Leu6-Met1, respectively. In order to understand whether the two different beta-turned structures are determined by the bicyclic structure or by the amino acid type at the corner positions, we have synthesized the pseudo-symmetrical analogue cyclo(Phe1-Asp2-Trp3-Phe4-Dap5-Trp6)cyclo(2 beta-5 beta). The structural characterization in the crystal state and in solution, here reported, gives an experimental evidence that the backbone of the bicyclic structure is a rigid scaffold that can be used to build both a type I and type II beta-turn independently from the amino acid composition.

We describe the in vitro and in vivo pharmacological properties of MEN 10,627 or cyclo(Met-Asp-Trp-Phe-Dap-Leu)cyclo(2 beta-5 beta), the first example of a polycyclic peptide tachykinin NK2 receptor antagonist. MEN 10,627 is endowed with high affinity for NK2 receptor expressed in various species with pKB values ranging between 10.1 (hamster trachea) and 8.1 (rabbit pulmonary artery). The antagonism is of competitive type in both functional and radioligand binding assays. A 100- to 10,000-fold selectivity was found vs. NK1 or NK3 receptors expressed in various species. As an NK2 receptor antagonist, MEN 10,627 is 10- to 100-fold more potent than the monocyclic peptide antagonist L 659,877 or cyclo(Met-Gln-Trp-Phe-Gly-Leu). At the hamster NK2 receptor, MEN 10,627 is about 30-fold more potent than the nonpeptide NK2 receptor antagonist SR 48,968 [(S)-N-methyl-N[4-acetylamino-4-phenylpiperidino)-2-(3,4-dichlorophenyl) butyl]benzamide], whereas the converse is true for the rabbit NK2 receptor. Furthermore, MEN 10,627 is, up to micromolar concentrations, devoid of antagonist properties toward a wide range of transmitters of both peptide and nonpeptide nature. In urethane-anesthetized rats in vivo, MEN 10,627 (10-100 nmol/kg i.v.) produced long-lasting inhibition of contraction of the urinary bladder and duodenum produced by i.v. administration of the NK2 receptor agonist [beta Ala8]NKA(4-10), without affecting the responses produced by i.v. administration of the NK1 receptor agonist [Sar9]SP sulfone or acetylcholine. In anesthetized rats, both MEN 10,627 and SR 48,968 blocked urinary bladder contraction induced by the NK2 receptor agonist after intravenous, intranasal or intraduodenal administration. Equieffective doses of MEN 10,627 producing about 50% inhibition of the response to [beta Ala8]NKA(4-10) in the rat urinary bladder in vivo, were 0.01, 0.03 and 3 mumol/kg after intravenous, intranasal and intraduodenal administration, respectively. The corresponding doses of SR 48,968 were 0.03, 0.1 and 1 mumol/kg, after intravenous, intranasal and intraduodenal administration, respectively. In conclusion, MEN 10,627 is a potent and selective NK2 receptor antagonist, endowed with high potency and long duration of action in vivo, which is not restricted to parenteral administration.(ABSTRACT TRUNCATED AT 400 WORDS)

We have compared the in vivo activity of the bradykinin B(2) receptor peptide antagonists MEN 11270 and Icatibant versus the nonpeptide antagonist FR 173657, after intravenous (i.v.) and intratracheal (i.t.) administration, on the bradykinin (BK)-induced bronchoconstriction and hypotension in anesthetized guinea pigs. We have also assessed the affinity of these antagonists for B(2) receptors in guinea pig lung membranes by radioligand binding and the metabolic stability of peptide antagonists in guinea pig plasma and tissue homogenates. The i.v. administration of MEN 11270, Icatibant, or FR 173657 induced a dose-dependent (10-100 nmol/kg) inhibition of both hypotension and bronchoconstriction induced by bradykinin (10 nmol/kg i.v.). The inhibitory effect of MEN 11270 and Icatibant was comparable both in terms of potency and time course, whereas FR 173657 was less potent and shorter acting. After i.t. administration MEN 11270 and Icatibant (10-100 nmol/kg) dose dependently inhibited both bronchoconstriction and hypotension, whereas FR 173657 (10-100 nmol/kg) reduced bronchoconstriction without affecting hypotension. The antibronchoconstrictor effect of MEN 11270 was more prolonged than that of Icatibant and FR 173657, whereas no differences were found between the peptide antagonists in inhibiting hypotension. These findings indicated that, in vivo, the peptide antagonists are more potent and longer lasting than FR 173657 acting on bradykinin B(2) receptors in guinea pig airways and in the vascular system. The greater efficacy of the antagonists in blocking airway compared with vascular B(2) receptors after topical administration suggests that they can block airway B(2) receptors with little systemic effects.

The mammalian tachykinins, neurokinin A (NKA) and NKA(4-10), along with the tachykinin NK2 receptor-selective antagonist MEN 10,376, were compared to their C-terminal free acid derivatives, NKA-OH, NKA(4-10)-OH and MEN 10,456, respectively, on several in vitro bioassays for NK1, NK2 and NK3 tachykinin receptors. NKA-OH and NKA(4-10)-OH were much weaker agonists than NKA or NKA(4-10) in the endothelium-deprived rabbit pulmonary artery (endowed with NK2A receptors) and in the guinea pig isolated bronchus (endowed with NK2A and NK1 receptors), where they produced submaximal contractile responses, and were inactive in the hamster isolated trachea (endowed with NK2B receptors) and in the rat isolated portal vein (endowed with NK3 receptors). At NK1 receptors of the guinea pig isolated ileum, NKA-OH produced weak agonist responses, whereas NKA(4-10)-OH was ineffective. In sharp contrast, MEN 10,456, while maintaining the same antagonist potency of the parent compound MEN 10,376 in the rabbit pulmonary artery and hamster isolated trachea, developed a clear-cut agonist character in the rat isolated portal vein, guinea pig isolated ileum and guinea pig isolated bronchus. The agonist responses produced by MEN 10,456 (10 microM) were reduced by MEN 10,376 in the guinea pig isolated bronchus and by the NK1 receptor antagonist GR 82,334 in the guinea pig isolated ileum. These results, although indicating the importance of C-terminal amidation for the agonist activity of natural tachykinins, suggest that the C-terminal amide group may not be directly involved in stimulation of the tachykinin receptors, but could induce agonist activity through a conformation effect.