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The antinociceptive pharmacology of N-[[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]methyl]-2-[2-[[(4-methoxy-2,6-dimethylphenyl) sulfonyl]methylamino]ethoxy]-N-methylacetamide fumarate (LF22-0542), a novel nonpeptidic B(1) antagonist, was characterized. LF22-0542 showed high affinity for human and mouse B(1) receptors with virtually no affinity for the human B(2) receptor; a selectivity index of at least 4000 times was obtained when LF22-0542 was profiled throughout binding or cell biology assays on 64 other G-protein-coupled receptor, 10 ion channels, and seven enzymes. LF22-0542 was a competitive B(1) receptor antagonist and elicited significant antinociceptive actions in the mouse acetic acid-induced writhing assay, as well as in the second phases of formalin-induced nociception in mice and in both the first and second phases of the formalin response in rats. LF22-0542 was active after s.c. but not p.o. administration. In B(1) receptor knockout (KO) mice, acetic acid and formalin responses were significantly reduced and LF22-0542 had no additional effects in these animals. LF22-0542 alleviated thermal hypersensitivity in both acute (carrageenan) and persistent inflammatory (complete Freund's adjuvant) pain models in rats. LF22-0542 produced a full reversal of experimental neuropathic thermal hypersensitivity but was inactive in reversing nerve injury-induced tactile hypersensitivity in rats. In agreement with this observation, B(1) KO mice subjected to peripheral nerve injury did not show thermal hypersensitivity but developed nerve injury-induced tactile hypersensitivity normally. The data demonstrate the antihyperalgesic actions of a selective systemically administered B(1) receptor antagonist and suggest the utility of this class of agents for the treatment of inflammatory pain states and for some aspects of neuropathic pain.

We recently described a potent bradykinin B2 receptor agonist (JMV1116) obtained by replacing the d-Tic-Oic dipeptide moiety of HOE140 by a (3S)-amino-5-(carbonylmethyl)-2,3-dihydro-1,5-benzothiazepin-4(5H)-one (D-BT) moiety. This compound inhibited the specific binding of [³H]BK on membranes of CHO cells expressing the human cloned B2 receptor with nanomolar affinity and contracted both isolated rat uterus and human umbilical vein. These data demonstrated that D-BT could be a good mimic of the Pro-Phe dipeptide. In the present study we characterized B1 receptor antagonists containing the D-BT moiety. We prepared an analogue of compound JMV1116 deleting the C-terminal arginine residue. The resulting compound (1) had an affinity of 83 nM for the human cloned B1 receptor. The most remarkable property of 1 is its ability to bind also the B2 receptor with an affinity of 4.4 nM despite the absence of the C-terminal arginine residue. Modifications at the N-terminal part of 1 associated with the substitution of the thienylalanine residue by α-(2-indanyl)glycine resulted in analogues selectively binding to the B1 receptor with an affinity in the picomolar range.

We have previously synthesized a potent and selective B1 bradykinin receptor antagonist, JMV1645 (H-Lys-Arg-Pro-Hyp-Gly-Igl-Ser-D-BT-OH), containing a dipeptide mimetic ((3S)-amino-5-carbonylmethyl-2,3-dihydro-1,5-benzothiazepin-4(5H)-one (D-BT) moiety) at the C-terminal. Analogues of this potent B1 bradykinin receptor antagonist in which the central Pro²-Hyp³-Gly⁴-Igl⁵ tetrapeptide has been replaced by constrained N-1-substituted-1,3,8-triazaspiro[4.5]decan-4-one ring system were synthesized. Among these analogues, compound JMV1640 (1) was found to have an affinity of 24.10 ± 9.48 nM for the human cloned B1 receptor. It antagonized the [des-Arg¹⁰]-kallidin-induced contraction of the human umbilical vein (pA2 = 6.1 ± 0.1). Compound 1 was devoid of agonist activity at the kinin B1 receptor. Moreover, it did not bind to the human cloned B2 receptor. Therefore, JMV1640 constitutes a lead compound for the rational search of nonpeptide B1 receptor analogues based on the BK sequence.

To investigate the molecular basis for the specificity of ligand recognition in human kinin B(1) (B(1)R) and B(2) (B(2)R) receptors, we constructed a series of chimeric receptors by progressively replacing, from the N to the C terminus, the human B(2)R domains by their B(1) counterparts. The chimeric construct possessing the C-terminal tail and the transmembrane domain VII (TM VII) of the B(2)R (construct 6) displayed 7- and 20- fold decreased affinities for the B(1) agonist [(3)H]desArg(10)-kallidin (desArg(10)-KD) and the B(1) antagonist [(3)H]desArg(10)-[Leu(9)]-KD respectively, as compared with the wild-type B(1)R. Moreover, the substitution of the B(1) TM VII by its B(2) homologue TM increased the affinity for the pseudopeptide antagonists, Hoe140 and NPC 567. High affinity for desArg(10)-KD binding was fully regained when the B(2) residue Thr(287) was replaced in construct 6 by the corresponding B(1) Leu(294) residue. When the B(2) residue Tyr(295) was exchanged with the corresponding B(1) Phe(302), high affinity binding for both agonist and antagonist was recovered. Moreover, the L294T and F302Y mutant B(1)R exhibited 69- and 6.5-fold increases, respectively, in their affinities for the B(2) receptor antagonist, Hoe140. Therefore we proposed that Leu(294) and Phe(302) residues, which may not be directly involved in the binding of B(1)R ligands and, hence, their Thr(287) and Tyr(295) B(2) counterparts, are localized in a receptor region, which plays a pivotal role in the binding selectivity of the peptide or pseudopeptide kinin ligands.

The aim of this work was to obtain photoactivatable nonpeptide antagonists of the angiotensin II AT1 receptor. Based on structure−function relationships, two chemical structures as well as appropriate synthetic schemes were chosen as a frame for the design of radiolabeled azido probes. The feasibility of the strategy was first assessed by the synthesis of two tritiated ligands 21 and 22 possessing a high affinity for the AT1 receptor and a low nonspecific binding to membrane or cell preparations. We then prepared two unlabeled azido derivatives 7 and 14 which retained a fairly high affinity for the AT1 receptor. The latter compound proved to be suitable for receptor irreversible labeling and was prepared in its tritiated form 28. This tritiated azido nonpeptide probe displayed a Kd value of 11.8 nM and a low nonspecific binding. It was suitable for specific and efficient covalent labeling of the recombinant AT1A receptor stably expressed in CHO cells. The electrophoretic pattern of the specifically labeled entity was strictly identical to that of purified receptor photolabeled with a biotinylated peptidic photoactivatable probe. This new tool should be useful for the mapping of the nonpeptide receptor binding site. These potential applications are discussed in light of the current knowledge of molecular mechanisms of G-protein coupled receptor activation and inactivation.

We have previously shown that substitution of the d-Tic-Oic dipeptide by a (3S)-[amino]-5-(carbonylmethyl)-2,3-dihydro-1,5-benzothiazepin-4(5H)-one (d-BT) moiety in the bradykinin B2 receptor antagonist HOE 140 resulted in a full potent and selective bradykinin B2 receptor agonist (H-dArg-Arg-Pro-Hyp-Gly-Thi-Ser-d-BT-Arg-OH, JMV1116) exhibiting a high affinity for the human receptor (Ki 0.7 nM). In the present study, we have investigated the effects of replacement of the d-Tic-Oic moiety by various constrained dipeptide mimetics. The resulting compounds were tested for their binding affinity toward the cloned human B2 receptor and for their functional interaction with the bradykinin-induced contraction of isolated human umbilical vein. Subsequently, we have designed novel bradykinin B2 receptor agonists which are likely to be resistant to enzymatic cleavage by endopeptidases and which might represent interesting new pharmacological tools. In an attempt to increase the potency of compound JMV1116, both its N-terminal part and the d-BT moiety were modified. Substitution of the d-arginine residue by a l-lysine residue led to a 10-fold more potent bradykinin B2 ligand [compound 22 (JMV1465) (Ki 0.07 nM)], retaining full agonist activity on human umbilical vein. Substitution of the d-BT moiety by a (3S)-[amino]-5-(carbonylmethyl)-2,3-dihydro-8-methyl-1,5-benzothiazepin-4(5H)-one [d-BT(Me)] moiety led to compound 23 (JMV1609) which exhibited a higher agonist activity (pD2 = 7.4) than JMV1116 (pD2 = 6.8).

A bradykinin analogue (H-Arg-Pro-Pro-Gly-Phe-Ser-d-BT-Arg-OH, 3) in which the Pro-Phe dipeptide was replaced by the (3S)[amino]-5-(carbonylmethyl)-2,3-dihydro-1,5-benzothiazepin-4(5H)-one (d-BT) moiety has been synthesized. The same modification was performed on the potent bradykinin B2 receptor antagonist HOE 140 (H-d-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-d-Tic-Oic-Arg-OH), in which the -d-Tic-Oic- moiety was replaced by d-BT to yield H-d-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-d-BT-Arg-OH, 1 (JMV1116). These compounds were examined in vitro for their binding affinity toward bradykinin B1 and B2 receptors as well as for their ability to interfere with bradykinin-induced contraction of both human umbilical vein and rat uterus. The two compounds 3 and 1 competed with [³H]bradykinin binding to the human cloned B2 receptor giving Ki values of 13 ± 2 and 0.7 ± 0.1 nM, respectively. Unexpectedly, both compounds were full bradykinin B2 receptor agonists on the human umbilical vein (pD2 = 6.60 ± 0.07 for 3 and 6.80 ± 0.08 for 1) and rat uterus (pD2 = 7.20 ± 0.09 for 3 and 7.50 ± 0.09 for 1) preparations with the same efficacy as bradykinin. In addition 1 induced a concentration-dependent phosphoinositide production in CHO cells expressing the human cloned B2 receptor. These data provide evidence for a bioactive conformation of bradykinin constrained at the dipeptide Pro-Phe.

In the present study, we developed an experimental model of cystitis induced by cyclophosphamide (CYP). In order to characterize des‐Arg9‐BK‐induced contraction on the urinary bladder (UB) during the development of inflammation and to quantify kinin B1receptor gene expression using a quantitative RT–PCR technique.In the presence of peptidase inhibitors captopril (10 μM), DL‐thiorphan (1 μM) and DL‐2‐mercaptomethyl‐3‐guanidino‐ethylthiopropanoic acid (MERGEPTA 5 μM), bradykinin (BK) (0.3–3,000 nM) evoked a concentration‐dependent contraction of rat UB which was not different between the CYP‐ and vehicle‐treated groups. Unlike BK, des‐Arg9‐BK (0.3–100,000 nM) did not contract UB from vehicle‐treated rats but contracted vigorously bladder strips from CYP‐treated rats 14, 24 and 168 h after treatment. In UB of 24 h treated rat, the pD2value of des‐Arg9‐BK was 7.3±0.1.The cyclo‐oxygenase inhibitor indomethacin (3 μM) reduced by 30% the maximal response of des‐Arg9‐BK. Both the kinin B1receptor antagonists des‐Arg9‐[Leu8]BK (10 μM) and des‐Arg10‐Hoe 140 (10 μM) produced a rightward shift of the concentration‐response curve to des‐Arg9‐BK yielding pKBvalues of 6.8±0.2 and 7.2±0.1, respectively, whilst the kinin B2receptor antagonist Hoe 140 (1 μM) had no effect.After CYP treatment, mRNA coding for the kinin B1receptor appeared predominantly in UB. In this organ, the induction was progressive, reaching a maximum 48 h after CYP treatment.In conclusion, the present study provides strong evidence for an induction of kinin B1receptors in UB of CYP‐treated rats. This was associated at a molecular level with an increase in mRNA expression of the gene coding for the kinin B1receptor. This kinin receptor displayed the whole features of a classical rat kinin B1receptor.

1The present study addresses the possibility of the existence of different kinin B2receptor subtypes in the guinea‐pig by evaluating the affinity of peptide and nonpeptide receptor antagonists. For this purpose, jugular vein rings, ileum segments, lung parenchymal and trachea strips were set up in organ baths for isometric tension measurements. The experiments were conducted in the presence of indomethacin (3 μm), atropine (10 μm) and captopril (10 μm).2BK contracted jugular vein (JV), ileum (GPI), parenchyma (LP) and trachea (GPT) with an EC50of 13.2 ± 1.4 nM (n= 27), 11.2 + 2.1 (n= 26), 23.6 ± 6.3 nM (n= 26) and 33.0 ± 6.5 (n= 27), respectively. Thiorphan, a neutral endopeptidase (EC 3.4.24.11) inhibitor and MERGETPA (DL‐2‐mercaptomethyl‐3‐guanidinoethylthiopropanoic acid), a carboxypeptidase inhibitor, had no effect on the BK‐induced contractions of JV, GPI and LP. In the GPT, thiorpan potentiated the contractile response to BK and was thus added in the corresponding experiments.3The peptide B2receptor antagonist, Hoe 140 and the nonpeptide compound, WIN 64338, behaved as noncompetitive antagonists against contractile responses to cumulative BK in the four tissues although Hoe 140 appeared as a competitive inhibitor in the GPT only. In order to compare the inhibitory potency of these compounds between tissues, pKBvalues were determined. Mean values of pKBfor Hoe 140 were 8.05 ± 0.07, 8.43 ± 0.11, 8.13 ± 0.18 and 8.52 ± 0.25 in the JV, GPI, GPT and LP, respectively. WIN 64338 gave mean pKBvalues of 6.89 ± 0.10, 7.57 ± 0.12, 7.36±0.12 and 7.51 ± 0.28 in the JV, GPI, LP and GPT, respectively.4D‐Arg [Hyp3, D‐Phe7, Leu8]BK and D‐Arg [Hyp3, D‐Phe7]BK (NPC 567) inhibited in a competitive fashion the concentration‐response curves to BK. Values of pA2for each compound were not significantly different in the four tissues and were between 5.81 and 6.31 for D‐Arg [Hyp3, D‐Phe7, Leu8]BK and between 5.55 and 5.65 for NPC 567.5We conclude that the contractile response to BK in guinea‐pig vascular, intestine and lung tissue is mediated by a unique B2receptor. Thus, our results do not support the existence of a B3receptor in the trachea and we suggest that the previously reported B2Breceptor subtype simply represents the guinea‐pig isoform. The present study addresses the possibility of the existence of different kinin B2receptor subtypes in the guinea‐pig by evaluating the affinity of peptide and nonpeptide receptor antagonists. For this purpose, jugular vein rings, ileum segments, lung parenchymal and trachea strips were set up in organ baths for isometric tension measurements. The experiments were conducted in the presence of indomethacin (3 μm), atropine (10 μm) and captopril (10 μm). BK contracted jugular vein (JV), ileum (GPI), parenchyma (LP) and trachea (GPT) with an EC50of 13.2 ± 1.4 nM (n= 27), 11.2 + 2.1 (n= 26), 23.6 ± 6.3 nM (n= 26) and 33.0 ± 6.5 (n= 27), respectively. Thiorphan, a neutral endopeptidase (EC 3.4.24.11) inhibitor and MERGETPA (DL‐2‐mercaptomethyl‐3‐guanidinoethylthiopropanoic acid), a carboxypeptidase inhibitor, had no effect on the BK‐induced contractions of JV, GPI and LP. In the GPT, thiorpan potentiated the contractile response to BK and was thus added in the corresponding experiments. The peptide B2receptor antagonist, Hoe 140 and the nonpeptide compound, WIN 64338, behaved as noncompetitive antagonists against contractile responses to cumulative BK in the four tissues although Hoe 140 appeared as a competitive inhibitor in the GPT only. In order to compare the inhibitory potency of these compounds between tissues, pKBvalues were determined. Mean values of pKBfor Hoe 140 were 8.05 ± 0.07, 8.43 ± 0.11, 8.13 ± 0.18 and 8.52 ± 0.25 in the JV, GPI, GPT and LP, respectively. WIN 64338 gave mean pKBvalues of 6.89 ± 0.10, 7.57 ± 0.12, 7.36±0.12 and 7.51 ± 0.28 in the JV, GPI, LP and GPT, respectively. D‐Arg [Hyp3, D‐Phe7, Leu8]BK and D‐Arg [Hyp3, D‐Phe7]BK (NPC 567) inhibited in a competitive fashion the concentration‐response curves to BK. Values of pA2for each compound were not significantly different in the four tissues and were between 5.81 and 6.31 for D‐Arg [Hyp3, D‐Phe7, Leu8]BK and between 5.55 and 5.65 for NPC 567. We conclude that the contractile response to BK in guinea‐pig vascular, intestine and lung tissue is mediated by a unique B2receptor. Thus, our results do not support the existence of a B3receptor in the trachea and we suggest that the previously reported B2Breceptor subtype simply represents the guinea‐pig isoform.

LF 7-0156 (2-[[[2-butyl-1-[(4-carboxyphenyl)methyl]-1H-imidazol- 5-yl]methyl]amino]benzoic acid) is a nonpeptide angiotensin II receptor antagonist selective for the type 1 angiotensin receptor. In rabbit aortic rings, LF 7-0156 competitively antagonized angiotensin II-induced contractile responses, with a pA2 value of 8.44. The synthesis of the radiolabeled compound [3H]LF 7-0156 has allowed direct binding studies with several membrane or cell preparations. Consistent with competition experiments, the binding of [3H]LF 7-0156 to purified rat liver membranes was characterized by a Kd value of 12.6 nM and very low pseudospecific or nonspecific binding; this latter characteristic confers to this compound an advantage over the structurally different compound [3H]DuP 753, which is the only commercially available nonpeptide radioligand. [3H]LF 7-0156 also bound to the type 1A angiotensin receptor expressed in Chinese hamster ovary cells, with high affinity (Kd = 3.5 nM) and a total absence of nonspecific binding. Functional antagonism in this cell system was assessed by the ability of LF 7-0156 to reverse angiotensin II-induced inositol phosphate production. These properties make [3H]LF 7-0156 an interesting pharmacological tool and should allow future evaluation of recognition of the nonpeptide ligand by mutated receptors expressed in Chinese hamster ovary cells; it will facilitate the analysis of possible differences in receptor amino acids involved in the binding of peptide and nonpeptide ligands, as well as the extent of spatial overlap between several nonpeptide antagonists displaying different structural properties.