Comparison of the molecular interactions of two antagonists, MEN16132 or icatibant, at the human kinin B2 receptor.

BACKGROUND AND PURPOSE: 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. EXPERIMENTAL APPROACH: 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. KEY RESULTS: 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. CONCLUSIONS AND IMPLICATIONS: MEN16132 dissociated from the B2 receptor compartment more slowly than icatibant and interacted at a deeper level in transmembrane regions of the receptor.

Bradykinin analogs containing the 4-amino-2-benzazepin-3-one scaffold at the C-terminus.

High affinity peptide ligands for the bradykinin (BK) B(2) subtype receptor have been shown to adopt a beta-turn conformation of the C-terminal tetrapeptide (H-Arg(1)-Pro(2)-Pro(3)-Gly(4)-Phe(5)-Ser(6)-Pro(7)-Phe(8)-Arg(9)-OH). We investigated the replacement of the Pro(7)-Phe(8) dipeptide moiety in BK or the D-Tic(7)-Oic(8) subunit in HOE140 (H-D-Arg(0)-Arg(1)-Pro(2)-Hyp(3)-Gly(4)-Thi(5)-Ser(6)-D-Tic(7)-Oic(8)-Arg(9)-OH) by 4-amino-1,2,4,5-tetrahydro-2-benzazepin-3-one templates (Aba). Binding studies to the human B(2) receptor showed a correlation between the affinities of the BK analogs and the propensity of the templates to adopt a beta-turn conformation. The L-spiro-Aba-Gly containing HOE140 analog BK10 has the best affinity, which correlates with the known turn-inducing property of this template. All the compounds did not modify basal inositolphosphate (IP) output in B(2)-expressing CHO cells up to 10 microM concentration. The antagonist properties were confirmed by the guinea pig ileum smooth muscle contractility assay. The new amino-benzazepinone (Aba) substituted BK analogs were found to be surmountable antagonists.

In vitro and in vivo activity of analogues of the kinin B2 receptor antagonist MEN1 1270.

In this study, we describe the in vitro and in vivo activities of a series of cyclic peptide analogues of the selective kinin B2 receptor antagonist MEN11270 on Chinese hamster ovary cells expressing the human B2 receptor (hB2R), the human isolated umbilical vein (hUV), the isolated guinea pig ileum (gpI), and bradykinin (BK) induced bronchoconstriction (BC) and hypotension in anaesthetized guinea pigs. Substitutions in the backbone of MEN1 1270 (H-DArg-Arg-Pro-Hyp-Gly-Thi-c(Dab-DTic-Oic-Arg)c(7gamma-10alpha)) aimed to increase the potency in inhibiting bronchospasm versus hypotension following the topical (intratracheal (i.t.)) or systemic (intravenous (i.v.)) application of these antagonists. A series of analogues were left unprotected from N-terminal cleavage by aminopeptidases (MEN12739, MEN13052, MEN13346, and MEN13371): these compounds maintained sizeable affinities for the hB2R (pKi = 9.4, 9.6, 9.7, and 8.6, respectively) and antagonist activities toward BK in the hUV (pA2 = 7.9, 8.3, 8.2, and 7.5) and gpI assays (pK(B) = 7.4, 7.8, 7.9, and 7.9), but the inhibition of BK-induced BC and hypotension in vivo was negligible following either i.v. or i.t. administration. Two analogues (MEN12388 and MEN13405) could be potential substrates of angiotensin-converting enzyme: these have good activity in the hB2R (pKi = 9.5 and 8.9, respectively), hUV (pA2 = 8.2 for MEN12388), and gpI assays (pK(B) = 8.4 and 8.0) but an in vivo activity 10- to 30-fold lower than the parent compound MEN1 1270 (pKi = 9.4, pA2 = 8.1, pKB = 8.3) when given by either the i.v. or the i.t. route. Other analogues were functionalized with a quaternary ammonium Lys derivative (MEN13031, MEN12374, and the previously mentioned MEN13052) or with an ethyl group on Arg (MEN13655 and the previously mentioned MEN13346 and MEN13405) in order to hinder or facilitate local absorption. MEN13346 and MEN13031 (pKi = 9.7and 9.5, pA2 = 8.2 and 7.9, pKB = 7.9 and 8.5, respectively) were 10- to 30-fold less active in vivo than MEN1 1270, without improving the discrimination between BK-induced BC and hypotension after either systemic or topical administration. It is concluded that the decreased in vivo activities of cyclic analogues of MEN11270 on BK-induced BC and hypotension following either their intratracheal or their intravenous routes of administration might be due in large part to metabolic degradation.

Cardiovascular effects of peptide kinin B2 receptor antagonists in rats.

Bradykinin (BK) is a vasoactive peptide reputed to play an important role in cardiovascular homeostasis. In this study, we describe the cardiovascular changes (mean blood pressure (BP) and heart rate (HR)) induced by the i.v. administration (left jugular vein) of two selective kinin B2 receptor antagonist, namely icatibant (0.1-1 micromol/kg as a bolus) and MEN1 1270 (0.1-1 micromol/kg as a bolus or 1 micromol/kg infused in 15 or 60 min), in urethane-anaesthetized or conscious rats with an indwelling catheter implanted in the right carotid artery for BP measurements. In conscious rats, icatibant at 0.1 or 0.3 micromol/kg did not change BP but at 0.1 micromol/kg increased HR at 30 min from administration. MEN1 1270 at 0.1 or 0.3 micromol/kg induced a dose-related increase in BP and a concomitant bradycardia (significant at 0.3 micromol/kg) lasting for 5 or 30 min, respectively. Icatibant at 1 micromol/kg induced a slight (P < 0.05) increase in BP that resolved in 5 min and a biphasic tachycardia (peaks at 30 and 90 min from administration). MEN1 1270 at 1 micromol/kg induced a triphasic change in HR (tachycardia in the first 5 min, bradycardia at 30 min, and tachycardia at 90 and 120 min) and a biphasic change in BP (hypotension at 15 min and hypertension at 30 min). The i.v. infusion of MEN1 1270 (1 micromol/kg in 15 or 60 min) produced hypertension, whereas HR was increased only following the 15-min infusion. In urethane-anaesthetized rats, both icatibant and MEN1 1270 (0.1 micromol/kg as a bolus) increased BP and the onset for this effect was correlated with the time course of the antagonism of BK-induced hypotension, where the effect of MEN1 1270 was more rapid than that of icatibant. These results indicate that kinin B2 receptor antagonists can induce acute cardiovascular effects, and the reason for the different haemodynamic profile between icatibant and MEN1 1270 could be putatively attributed to kinetic characteristics.

Synthesis, activity on NK-3 tachykinin receptor and conformational solution studies of scyliorhinin II analogs modified at position 16.

Two analogs of a tachykinin family peptides - scyliorhinin II (ScyII): [Aib(16)]ScyII and [Sar(16)]ScyII were synthesized by the solid-phase method using Fmoc chemistry. Conformational studies in water and DMSO-d(6) on these peptides were performed using a combination of two-dimensional NMR and theoretical conformational analysis. The solution structure of the peptides studied is interpreted as an equilibrium of several conformers with different statistical weights. The structure of [Sar(16)]ScyII in water appeared to be more flexible, especially in the C-terminal fragment. A better defined structure for this analog was obtained in DMSO-d(6), in which the analysis resulted in a family of conformers with similar shapes. Some of these conformers were characterized by the presence of a 3(10)-helix in the N-terminal fragment and middle part of the molecule. The introduction of the Aib residue in position 16 significantly rigidifies the structure. For [Aib(16)]ScyII in both solvent systems very similar populations of conformations were obtained which are characterized by the presence of a 3(10)-helix in the 13-18 fragment. A common structural motif was found in conformationally constrained Cys(7)-Cys(13) fragment, which resembles the Greek letter 'omega'. The differences in the solution structure of the C-terminal fragment of the peptides studied are responsible for their specificity. [Aib(16)]ScyII showed 25% the agonistic activity of selective NK-3 agonist - senktide, but it also showed antagonist effect vs. this peptide, whereas [Sar(16)]ScyII appeared to be a full agonist of NK-3 tachykinin receptor.

Differences between peptide and nonpeptide B(2) bradykinin receptor antagonists in blocking bronchoconstriction and hypotension induced by bradykinin in anesthetized Guinea pigs.

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.

Agonist activity at the kinin B1 receptor: structural requirements of the central tetrapeptide.

A series of analogues of desArg(9)-Lys-bradykinin (BK), Lys-Arg-X-Ac(n)c-X-Ser-Pro-Phe, in which the spacer X-Ac(n)c-X replaces the central tetrapeptide Pro-Pro-Gly-Phe of BK, have been synthesized and functionally characterized at the B1 receptor. The 1-aminocycloalkane-1-carboxylic acids (Ac(6)c, Ac(7)c, Ac(8)c, Ac(9)c, Ac(12)c) were incorporated to impart conformational constraint and probe the importance of the hydrophobicity of the residue in the central position. The linker is varied in length (X = Gly, betaAla, gammaAbu) to examine the optimal distance between the biologically important residues at the N- and C-termini. The biological assays indicate that the optimal length is obtained with X = Gly, with reduced activities for the longer linkers. Although the size of the central cyclic amino acid does not significantly alter the biological activity, the hydrophobic residue Ac(n)c which may tether the peptide in the membrane environment is required (Lys-Arg-Gly-Gly-Gly-Ser-Pro-Phe is inactive). Two of the analogues, Lys-Arg-Gly-Ac(7)c-Gly-Ser-Pro-Phe and Lys-Arg-gammaAbu-Ac(7)c-gammaAbu-Ser-Pro-Phe, have been structurally characterized in the presence of a zwitterionic lipid environment by high-resolution NMR. Both compounds have similar structural features, differing greatest in the distance between the termini (9 and 15 A for the Gly- and gammaAbu-containing analogues, respectively). The correlation of the smaller distance with activity at the B1 receptor is in complete accord with the results from our previous examination of Lys-Arg-NH-(CH(2))(11)-CO-Ser-Pro-Phe. With the results from this series of compounds we are beginning to define some of the molecular descriptors important for activity at the B1 BK receptor.

Peptide and non-peptide bradykinin B2 receptor agonists and antagonists: a reappraisal of their pharmacology in the guinea-pig ileum.

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 11270 <30 min, Icatibant <60 min, FR173657 >60 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 slopes <1). 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.

Solid-phase synthesis of MEN 11270, a new cyclic peptide kinin B2 receptor antagonist.

An efficient synthesis of the cyclic decapeptide MEN 11270 [H-DArg1-Arg2 Pro3-Hyp4-Gly5-Thi6-Dab7-DTic8-Oic9-Arg10 c(7gamma - 10alpha)] was developed. Two three-dimensional orthogonal strategies were applied and compared: Fmoc/Tos/Boc (procedure A) and Fmoc/Pmc/Dde (procedure B). Both resulted in a 23-step strategy comprising the stepwise solid-phase chain assembly of the linear protected peptide, partial deprotection, solution-phase cyclization and final full deprotection. The stepwise assembly of the linear peptide was optimized by double coupling and acylation time prolongation for critical residues (Tic, Dab, Thi, Pro). O-(7-azabenzotriazol-1-yl)-N,N,N',N' tetramethyluronium (HATU) was preferred as coupling reagent for Dab. In the cyclization step, the partial racemization of Arg10 (31% using 1-ethyl-3-(3'-dimethyl-aminopropyl) carbodiimide/1-hydroxybenzotriazole (EDC/HOBt) as activation system) was reduced to 3% with HATU. The final deprotection was performed in the presence of dimethylsulfide (procedure A) and thiocresol (procedure B) as scavengers, to avoid the sulfation of Hyp side chain. The final compound and the main by-products were characterized by mass spectroscopy (MS), nuclear magnetic resonance (NMR) and racemization test. Procedure B produced operationally simpler and more efficient results than A (28% overall yield versus 4%).

Ala scan analogues of HOE 140. Synthesis and biological activities.

The role of the amino acids contained in the sequence of HOE 140 (H-DArg(1)-Arg(2)-Pro(3)-Hyp(4)-Gly(5)-Thi(6)-Ser(7)-DTic(8)-Oic(9 )-Arg(10)-OH), a potent and selective bradykinin B(2) receptor peptide antagonist, has been investigated by the replacement of each original residue (one by one) with Ala. The resulting set of decapeptides has been tested for the B(2) antagonist activity as well as for competition with the binding of [3H]BK to plasma membranes of the human umbilical vein (hUV). Positive correlations have been established between data obtained with the bioassay and with the binding in the hUV (same species, same tissue) and also between the two bioassays, the guinea-pig ileum (GPI) and the hUV (different species, different tissue). The structure-activity study has shown that the replacement of any of the residues that constitute HOE 140 with Ala is accompanied by a decrease of potency of at least 1 log unit. The analogues can be divided into three groups, with Ala(1) and Ala(7) showing affinities lower than HOE 140 by a factor of 10, Ala(4) and Ala(10) by a factor of 100 and Ala(2), Ala(5), Ala(6), Ala(8) and Ala(9) by a factor higher than 100 (100-1000). To verify the effect of chirality, the DAla(5) and DSer(7) analogues were synthesized and it was found that the substitution with a D-residue in position 5 is not tolerated while that in position 7 is favourable. The DSer(7) derivative is the most potent analogue found in this study: it shows potency as high as that of HOE 140 in the bioassays.

Nonpeptide antagonists for kinin receptors.

Kinins are a family of small peptides acting as mediators of inflammation and pain in the peripheral and central nervous system. The two main 'kinins' in mammals are the nonapeptide bradykinin (BK, Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9) and the decapeptide kallidin (KD, [Lys0]-BK, Lys1-Arg2-Pro3-Pro4-Gly5-Phe6-Ser7-Pro8-Phe9- Arg10). Their biological actions are mediated by two distinct receptors, termed B1 and B2. Kinin B and B2 receptor antagonists may be useful drugs endowed with analgesic and anti-inflammatory properties, with potential use in asthma, allergic rhinitis and other diseases. The first nonpeptide kinin B2 receptor antagonist, WIN 64338, was reported in 1993. Despite its low selectivity, the compound provided a reference for pharmacological and modeling studies. Several quinoline and imidazo[1,2-a]pyridine derivatives have been shown by Fujisawa to possess high affinity and selectivity for kinin B2 receptors. Among them, FR 173657 displayed excellent in vitro and in vivo antagonistic activity, while FR 190997 emerged as the first nonpeptide agonist for B2 receptor. Two structurally related Fournier compounds were recently published. Other kinin B2 receptor ligands were obtained by rational design, through library screening or from natural sources. The only example of a nonpeptide kinin B1 receptor ligand has been reported in a patent by Sanofi.

MEN 11270, A novel selective constrained peptide antagonist with high affinity at the human B2 kinin receptor.

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 (pKi < 6) > [des-Arg9,Leu8]-BK (pKi < 5). 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 (pIC50 < 5.5) 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.

A new class of pseudopeptide antagonists of the kinin B1 receptor containing alkyl spacers.

Four previously reported kinin receptor peptide antagonists, including the B1 receptor-selective peptides desArg10-HOE 140 (H-D-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-D-Tic-Oic-OH) and B-9858 (H-Lys-Lys-Arg-Pro-Hyp-Gly-Igl-Ser-D-Igl-Oic-OH), have been modified by replacement of the central tetrapeptide Pro-Hyp-Gly-Xaa with linear alkyl spacers of variable length. The analogue of desArg10-HOE 140 containing the 11-aminoundecanoic acid as spacer, MEN 11575 [H-D-Arg-Arg-NH-(CH2)10-CO-Ser-D-Tic-Oic-OH], was found to be slightly more potent than the unmodified peptide (pA2 = 7.1) as a kinin B1 receptor antagonist in the rat ileum longitudinal smooth muscle assay. Moreover, MEN 11575 is devoid of residual agonist activity at the kinin B1 receptor (rat ileum) and antagonist activity at the kinin B2 receptor (guinea pig ileum longitudinal smooth muscle). Both these activities are displayed by the parent peptide desArg10-HOE 140. Therefore, despite its greatly simplified chemical structure, MEN 11575 shows an improved pharmacological profile in terms of both potency and selectivity, and it represents a good template for the development of new peptidomimetic kinin B1 receptor antagonists. We also report an attempt to investigate the conformational role of the flexible, linear spacer of MEN 11575 and to design more constrained analogues, possibly locked in the bioactive conformation, using semirigid spacers based on Calpha-tetrasubstituted alpha-amino acids of the family of 1-aminocycloalkane-1-carboxylic acids (Acnc).

Characterization of [3H]MEN 11420, a novel glycosylated peptide antagonist radioligand of the tachykinin NK2 receptor.

[3H]MEN 11420, a radiolabeled glycosylated peptide antagonist of the tachykinin NK2 receptor, has been investigated in ligand-receptor binding assays using membranes of CHO cells transfected with the human tachykinin NK2 receptor. [3H]MEN 11420 bound to a single class of high affinity binding sites: its binding was inhibited by natural tachykinins (potency ranking: NKA >> SP > or = NKB), as well as by peptide (MEN 11420 > MEN 10376 >> R 396) and nonpeptide (SR 48968 > GR 159897) selective NK2 receptor antagonists. These data indicate that [3H]MEN 11420 is a potent radioligand for the human tachykinin NK2 receptor that may represent a useful tool for studying ligand-receptor interactions at the molecular level.

The tachykinin NK1 receptor. Part II: Distribution and pathophysiological roles.

The tachykinin NK1 receptor is widely distributed in both the central and peripheral nervous system. In the CNS, NK1 receptors have been implicated in various behavioural responses and in regulating neuronal survival and degeneration. Moreover, central NK1 receptors regulate cardiovascular and respiratory function and are involved in activating the emetic reflex. At the spinal cord level, NK1 receptors are activated during the synaptic transmission, especially in response to noxious stimuli applied at the receptive field of primary afferent neurons. Both neurophysiological and behavioural evidences support a role of spinal NK1 receptors in pain transmission. Spinal NK1 receptors also modulate autonomic reflexes, including the micturition reflex. In the peripheral nervous system, tachykinin NK1 receptors are widely expressed in the respiratory, genitourinary and gastrointestinal tracts and are also expressed by several types of inflammatory and immune cells. In the cardiovascular system, NK1 receptors mediate endothelium-dependent vasodilation and plasma protein extravasation. At respiratory level, NK1 receptors mediate neurogenic inflammation which is especially evident upon exposure of the airways to irritants. In the carotid body, NK1 receptors mediate the ventilatory response to hypoxia. In the gastrointestinal system, NK1 receptors mediate smooth muscle contraction, regulate water and ion secretion and mediate neuro-neuronal communication. In the genitourinary tract, NK1 receptors are widely distributed in the renal pelvis, ureter, urinary bladder and urethra and mediate smooth muscle contraction and inflammation in response to noxious stimuli. Based on the knowledge of distribution and pathophysiological roles of NK1 receptors, it has been anticipated that NK1 receptor antagonists may have several therapeutic applications at central and peripheral level. At central level, it is speculated that NK1 receptor antagonists could be used to produce analgesia, as antiemetics and for treatment of certain forms of urinary incontinence due to detrusor hyperreflexia. In the peripheral nervous system, tachykinin NK1 receptor antagonists could be used in several inflammatory diseases including arthritis, inflammatory bowel diseases and cystitis. Several potent tachykinin NK1 receptor antagonists are now under evaluation in the clinical setting, and more information on their usefulness in treatment of human diseases will be available in the next few years.

MEN 11420 (Nepadutant), a novel glycosylated bicyclic peptide tachykinin NK2 receptor antagonist.

1. The pharmacological profile was studied of MEN 11420, or cyclo[[Asn(beta-D-GlcNAc)-Asp-Trp-Phe-Dap-Leu]cyclo(2beta-5beta )], a glycosylated derivative of the potent, selective, conformationally-constrained tachykinin NK2 receptor antagonist MEN 10627 (cyclo(Met-Asp-Trp-Phe-Dap-Leu)cyclo(2beta-5beta)). 2. MEN 11420 competitively bound with high affinity to the human NK2 receptor stably transfected in CHO cells, displacing radiolabelled [125I]-neurokinin A and [3H]-SR 48968 with Ki values of 2.5+/-0.7 nM (n = 6) and 2.6+/-0.4 nM (n = 3), respectively. 3. MEN 11420 showed negligible binding affinity (pIC50 < 6) at 50 different receptors (including tachykinin NK1 and NK3 receptors) and ion channels. 4. In the rabbit isolated pulmonary artery and rat urinary bladder MEN 11420 potently and competitively antagonized tachykinin NK2 receptor-mediated contractions (pK(B) = 8.6+/-0.07, n = 10, and 9.0+/-0.04, n = 12; Schild plot slope = -1.06 (95% c.l. = -1.3; -0.8) and -1.17 (95% c.l. = -1.3; -1.0), respectively). MEN 11420 produced an insurmountable antagonism at NK2 receptors in the hamster trachea and mouse urinary bladder. However, in both preparations, the effect of MEN 11420 was reverted by washout and an apparent pK(B) of 10.2+/-0.14, n = 9, and 9.8+/-0.15, n = 9, was calculated in the hamster trachea and mouse urinary bladder, respectively. 5. MEN 11420 showed low affinity (pK(B) < 6) at guinea-pig and rat tachykinin NK1 (guinea-pig ileum and rat urinary bladder) and NK3 (guinea-pig ileum and rat portal vein) receptors. On the whole, the affinities (potency and selectivity) showed by MEN 11420 for different tachykinin receptors, measured either in binding or in functional bioassays, were similar to those shown by the parent compound, MEN 10627. 6. The in vivo antagonism of the contractions produced by [betaAla8]neurokinin A(4-10) (1 nmol kg(-1)) was observed after intravenous (dose range: 1-10 nmol kg(-1)), intranasal (3-10 nmol kg(-1)), intrarectal (30-100 nmol kg(-1)) and intraduodenal (100-300 nmol kg(-1)) administration of MEN 11420. MEN 11420 was more potent (about 10 fold) and longer lasting than its parent compound MEN 10627, possibly due to a greater metabolic stability. 7. A dose of MEN 11420 (100 nmol kg(-1), i.v.), that produced potent and long lasting inhibition of the contraction of the rat urinary bladder induced by challenge with the NK2 selective receptor agonist [betaAla8]neurokinin A(4-10) (10-300 nmol kg(-1)), was without effect on the responses produced by the NK1 receptor selective agonist [Sar9]substance P sulphone (1-10 nmol kg(-1)). 8. These findings indicate that MEN 11420 is a potent and selective tachykinin NK2 receptor antagonist. The introduction of a sugar moiety did not produce major changes in the affinity profile of this antagonist as compared to MEN 10627, but markedly improved its in vivo potency and duration of action. With these characteristics, MEN 11420 is a suitable candidate for studying the pathophysiological significance of tachykinin NK2 receptors in humans.

Effect of several bicyclic peptide and cyclic pseudopeptide tachykinin NK2 receptor antagonists in the human isolated ileum and colon.

The affinities of the monocyclic pseudopeptides MEN10,508, MEN10,573, MEN10,581, MEN10,612, MEN10,619 and MEN10,677, and the bicyclic peptides MEN10,627, MEN10,692, MEN10,771, MEN10,882 and MEN10,993 were evaluated at the tachykinin NK2 receptors of the human isolated ileum and colon circular muscle preparations, by using [betaAla8]neurokinin A(4-10) as an agonist. All of the antagonists tested produced a concentration-dependent and competitive antagonism of [betaAla8]neurokinin A(4-10)-mediated contractions in both preparations. MEN10,612 (pKB = 8.1) and MEN10,627 (pKB = 8.4-8.8) were among the most potent analogs within their chemical classes. In general, the bicyclic peptide antagonists were more potent than the monocyclic peptide compounds, showing a nanomolar affinity for the human NK2 receptor. By comparing the affinities shown by the antagonists under study at NK2 receptors of the human gut with the affinities measured at NK2 receptors of the rabbit isolated pulmonary artery and hamster isolated trachea, a high degree of pharmacological homology was found between human and rabbit NK2 receptors. The present results point out the class of NK2 receptor antagonists bearing a bicyclic peptide structure, like MEN10,627, as candidates for testing in pathological conditions characterized by exaggerated gut motility, in which tachykinins might play a role as non-cholinergic excitatory neurotransmitters.

The tachykinin NK1 receptor. Part I: ligands and mechanisms of cellular activation.

The tachykinin NK1 receptor is widely expressed in the mammalian central and peripheral nervous system. Powerful pharmacological tools (agonists and antagonists) are now available to elucidate the physiological role of NK1 receptors at these levels, as well as to understand their role in diseases and establish the possible therapeutic usefulness of NK1 receptor antagonists for treatment of human diseases. The structure-activity studies that have led to the development of potent peptide and non-peptide ligands for the tachykinin NK1 receptor are here reviewed. Among the peptide agonists and antagonists, linear and cyclic sequences have been developed. The non peptide antagonists belong to different chemical classes, i.e. steroids, perhydroisoindolones, quinuclidines, piperidines and tryptophane derivatives. The first non peptide antagonists for NK1 receptors have been obtained by random screening of chemical compounds large collections. The resulting leads were optimized with 'classic' structure activity approaches, aiming at identifying 'common' motifs for interaction with the receptor by ligands of different chemical classes. The results derived from the recent application of molecular biology techniques were useful to drive the design of new ligands toward a precise structural definition of ligand-receptor bi-molecular interactions. Studies on mutant receptors have established that the sites of interaction of peptide agonists and non peptide antagonists with the tachykinin NK1 receptor are largely non overlapping. Moreover, data obtained from mutagenesis of the NK1 receptor further indicate that some amino acid residues in the NK1 receptor sequence are critical for determining the binding affinity of some but not all ligands. Therefore, different antagonists discovered from random screening may not possess common points of interaction or common structural and conformational characteristics for their interaction with the tachykinin NK1 receptor. The tachykinin NK1 receptor couples with G-proteins to determine its biological effects in target cells. Several G-proteins both sensitive (Go, Gi) and insensitive (Gq, G11) to pertussis toxin can mediate the action of NK1 receptors. Moreover, several second messanger signalling systems (elevation of intracellular calcium, stimulation of phosphoinositol turnover, arachidonic acid mobilization, cAMP accumulation) have to be activated following NK1 receptor signalling. Also a direct modulation of certain ion channels at membrane level has been proposed. The NK1 receptor undergoes prompt and significant tachyphylaxis upon exposure to the agonist: this has been shown to be linked with receptor internalization which also occurs physiologically when the NK1 receptor is stimulated by endogenous tachykinins.

Effect of several bicyclic peptide and cyclic pseudopeptide tachykinin NK2 receptor antagonists in the human isolated urinary bladder.

1. We have studied several tachykinin NK2 receptor antagonists, bearing a monocyclic pseudopeptide (MEN 10,508, MEN 10,573, MEN 10,581, MEN 10,612, MEN 10,619 and MEN 10,677), or bicyclic peptide (MEN 10,627, MEN 10,692, MEN 10,771, MEN 10,882 and MEN 10,993) structure, on the human isolated urinary bladder detrusor muscle against neurokinin A as an agonist, and compared their affinities in this preparation with those for NK2 receptors expressed in the rabbit isolated pulmonary artery and hamster isolated trachea. 2. In the human bladder, all the antagonists tested produced a concentration-dependent and competitive antagonism of neurokinin A-mediated contractions: among the cyclic pseudopeptides MEN 10,677 (pKB = 8.0) was the most potent antagonist, while among the bicyclic analogues it was MEN 10,993 (pKB = 8.8). 3. In general, the bicyclic peptide antagonists tested were more potent than the monocyclic pseudopeptide compounds, either in the human urinary bladder or in the rabbit pulmonary artery or hamster trachea, showing a nanomolar affinity for the human NK2 receptor. 4. A highly significant correlation was found between the estimated pKB values of all the antagonists tested in the human urinary bladder and rabbit pulmonary artery (r2 = 0.94, n = 12, P < 0.01), whereas no linear correlation was found between pKB values measured in the human urinary bladder and hamster trachea (r2 = 0.52, n = 12, P > 0.05): these observations provide further pharmacological evidence for receptor homology between the human and rabbit NK2 receptor. 5. The present results point out the class of NK2 receptor antagonists bearing a bicyclic peptide structure, like MEN 10,627, as candidates for testing in pathological conditions, such as bladder hyperactivity, for which preclinical evidence indicates that a therapeutic effect could result from the block of the tachykinin NK2 receptor.

A review of the design, synthesis and biological activity of the bicyclic hexapeptide tachykinin NK2 antagonist MEN 10627.

We review the reported data on the design, the conformational features and the pharmacological properties of the bicyclic peptide tachykinin NK2 receptor antagonist MEN 10,627 or cyclo(Met-Asp-Trp-Phe-Dap-Leu)cyclo(2 beta-5 beta). MEN 10,627 possesses a highly constrained structure characterized by two consecutive beta-turns, as confirmed by the almost coincident results of NMR and X-ray analyses. The compound has been efficiently synthesized by solid-phase methodology using either Boc or Fmoc strategies. It is quite stable to metabolic degradation and is endowed with high affinity and selectivity for NK2 receptor expressed in various species. At the hamster NK2 receptor MEN 10,627 is about 30-fold more potent than the nonpeptide NK2 receptor antagonist, SR 48,968, while the converse is true for the rabbit NK2 receptor. MEN 10,627 and SR 48,968 show comparable affinities for the human NK2 receptor. MEN 10,627 produces a long lasting inhibition of the response to the selective NK2 receptor agonist [beta Ala8]NKA(4-10) in the rat urinary bladder in vivo after intravenous, intranasal and intraduodenal administration. Therefore different administration routes are possible for this compound that overcomes the usual drawbacks for the application of peptides as drugs.