Pharmacological profile of LF 16-0687, a new potent non-peptide bradykinin B2 receptor antagonist.

LF 16-0687 (1-[[2,4-dichloro-3-[[(2,4-dimethylquinolin-8-yl)oxy] methyl]phenyl]sulfonyl]-N-[3-[[4-(aminoimethyl) phenyl] carbonylamino]propyl]-2(S)-pyrrolidinecarboxamide) has been selected from a large-scale medicinal chemistry program for further development. In competition binding studies using [3H]bradykinin (BK), LF 16-0687 bound to the human, rat and guinea-pig recombinant B2 receptor expressed in CHO cells giving K(i) values of 0.67 nM, 1.74 nM and 1.37 nM, respectively. It also bound to the native BK B2 receptor from human umbilical vein (HUV), rat uterus (RU) and guinea-pig ileum (GPI) giving K(i) values of 0.89 nM, 0.28 nM and 0.98 nM, respectively. It inhibited BK-induced IP1, IP2 and IP3 formation in INT407 cells yielding pK(B) values of 8.5, 8.6 and 8.7, respectively. In isolated organs experiments, LF 16-0687 behaved as a competitive antagonist of BK-mediated contractions giving pA2 values of 9.1 in HUV, 7.7 in RU and 9.1 in GPI. Binding and functional studies performed over 40 different receptors revealed that LF 16-0687 was selective for the BK B2 receptor. A continuous intravenous infusion of LF 16-0687 antagonized in a dose-dependent manner and with a rapid onset of action BK-induced hypotensive response. Subcutaneous administration of LF 16-0687 at 1.1 micromol/kg to rats markedly reduced BK-induced edema of the stomach (- 69%), duodenum (-65%) and pancreas (-56%).

Pharmacological and molecular evidence for kinin B1 receptor expression in urinary bladder of cyclophosphamide-treated rats.

1. 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 B1 receptor gene expression using a quantitative RT - PCR technique. 2. In the presence of peptidase inhibitors captopril (10 microM), DL-thiorphan (1 microM) and DL-2-mercaptomethyl-3-guanidino-ethylthiopropanoic acid (MERGEPTA 5 microM), 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 pD2 value of des-Arg9-BK was 7.3+/-0.1. 3. The cyclo-oxygenase inhibitor indomethacin (3 microM) reduced by 30% the maximal response of des-Arg9-BK. Both the kinin B1 receptor antagonists des-Arg9-[Leu8]BK (10 microM) and des-Arg10-Hoe 140 (10 microM) produced a rightward shift of the concentration-response curve to des-Arg9-BK yielding pKB values of 6.8+/-0.2 and 7.2+/-0.1, respectively, whilst the kinin B2 receptor antagonist Hoe 140 (1 microM) had no effect. 4. After CYP treatment, mRNA coding for the kinin B1 receptor appeared predominantly in UB. In this organ, the induction was progressive, reaching a maximum 48 h after CYP treatment. 5. In conclusion, the present study provides strong evidence for an induction of kinin B1 receptors 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 B1 receptor. This kinin receptor displayed the whole features of a classical rat kinin B1 receptor.

Pharmacological characterization of the bradykinin B2 receptor: inter-species variability and dissociation between binding and functional responses.

1. The present study addresses the differences in binding profiles and functional properties of the human and rat bradykinin (BK) B2 receptor using various kinin receptor peptide derivatives as well as the non-peptide receptor antagonists WIN 64338 (phosphonium, [[4-[[2-[[bis(cyclohexylamino)methylene]amino]-3-(2-naphtalenyl)1- oxopropyl]amino]-phenyl]-methyl]tributyl, chloride, monohydro-chloride), and FR173657 (E)-3-(6-acetamido-3-pyridyl)-N-[-N-[2,4-dichloro-3-[(2-methyl-8-quinoli nyl)oxymethyl]-phenyl]N-methylamino carbonyl methyl] acrylamide. 2. [3H]-BK bound with a similar affinity to membranes of Chinese hamster ovary cells (CHO-K1) expressing the cloned human (hB2-CHO) or rat (rB2-CHO) B2 receptor, human embryonic intestine cells (INT407) expressing the native B2 receptor, human umbilical vein (HUV) and rat uterus (RU). WIN 64338 and FR173657 bound with a 3.8-6.6 fold and 7.0-16.3 fold higher affinity the rat than the human B2 receptor, respectively. The affinity values of BK derivatives as well as non-peptide antagonists were reduced by 6-23 fold in physiological HBSS compared to low ionic strength TES binding buffer. 3. BK (0.01-3000 nM) increased inositol triphosphates (IP3) levels in hB2-CHO, rB2-CHO and INT407 cells. The B2 receptor antagonist, Hoe 140 (D-Arg0-[ Hyp3, Thi5, D-Tic7, Oic8]-BK) at 10(-7) M, significantly shifted to the right the IP3 response curves to BK giving apparent pKB values of 8.56, 9.79 and 8.84 for hB2-CHO, rB2-CHO and INT407 cells, respectively. 4. In human isolated umbilical vein, Hoe 140, D-Arg0-[Hyp3, D-Phe7, Leu8]-BK and NPC 567 had a lower potency in functional assays (pKB 8.18, 5.77 and 5.60, respectively) than expected from their affinity in binding studies (pKi 10.52, 8.64 and 8.27, respectively). 5. FR173657 behaved as a high affinity ligand with pKi values of 8.59 and 9.81 and potent competitive antagonist with pKB values of 7.80 and 8.17 in HUV and RU, respectively. FR173657 bound with a similar affinity the cloned and native bradykinin B2 receptor in human (pKi of 8.66 and 8.59, respectively) and in rat (pKi 9.67 and 9.81, respectively). 6. In conclusion, we suggest that the binding buffer composition has to be taken into account when screening new compounds and that inter-species differences should be considered when setting up animal models with the aim of developing bradykinin B2 receptor antagonists as therapeutic agents.

In vitro and in vivo effects of the new nonpeptide bradykinin B2 receptor antagonist, LF 16-0335C, on guinea-pig and rat kinin receptors.

Activation of the kinin-kallikrein system and stimulation of bradykinin (BK) B2 receptors are thought to play an important role in the pathophysiology of inflammation and pain. In the present study, we report the pharmacological properties of a novel nonpeptide bradykinin B2 receptor antagonist, LF 16-0335C, (1-[[3-[(2,4-dimethylquinolin-8-yl) oxymethyl]-2,4-dichloro-phenyl]sulfonyl]-2(S)-[[4-[4- (aminoiminomethyl)-phenylcarbonyl]piperazin-1-yl]carbo nyl]pyrrolidine, 2HCl). In binding studies, LF 16-0335C competed with [3H]bradykinin giving Ki values of 1.65 +/- 0.36 nM and 2.20 +/- 0.30 nM in membrane preparations from rat uterus (RU) and guinea-pig ileum (GPI), respectively. In functional experiments, LF 16-0335C inhibited in a competitive manner BK-induced contractions of both isolated RU and GPI, leading to calculated pA2 values of 7.70 +/- 0.70 and 8.30 +/- 0.30, respectively. The inhibitory effect of LF 16-0335C was fully reversible by washing in the guinea-pig ileum. In vivo, LF 16-0335C given intravenously inhibited in a dose-dependent manner BK-induced hypotension in both animal species, although it was more potent in the guinea-pig than in the rat (ED50, 2.5 +/- 1.6 micrograms/kg versus 22.6 +/- 2.3 micrograms/kg). BK is a potent constrictor of guinea-pig airways and this effect was markedly attenuated by LF 16-0335C. In contrast, LF 16-0335C did not affect histamine- and acetylcholine-induced hypotensive response in the rat. We conclude that LF 16-0335C is a potent and selective nonpeptide B2 receptor antagonist which equally binds to the rat and guinea-pig receptor but displays a different in vivo potency in the two species. Therefore, this drug represents a useful tool to better assess the role of bradykinin in pathophysiological conditions.

LF 16.0335, a novel potent and selective nonpeptide antagonist of the human bradykinin B2 receptor.

1. In the present paper, we describe the in vitro pharmacological properties of LF 16.0335 (1-[[3-[(2,4-dimethylquinolin-8-yl)oxymethyl]-2,4-dichloro-p henyl]sulphonyl] -2(S) - [[4 -[4-(aminoiminomethyl)phenylcarbonyl]piperazin-1-yl]ca rbonyl]pyrrolidine), a novel and potent nonpeptide antagonist of the human bradykinin (BK) B2 receptor. 2. LF 16.0335 displaced [3H]-BK binding to membrane preparations from CHO cells expressing the cloned human B2 receptor, INT 407 cells and human umbilical vein with Ki values of 0.84+/-0.39 nM, 1.26+/-0.68 nM and 2.34+/-0.36 nM, respectively. 3. In saturation binding studies performed in INT 407 cell membranes in the presence or absence of LF 16.0335, max values of [3H]-BK were not significantly changed suggesting that LF 16.0335 behaves as a competitive antagonist. 4. LF 16.0335 had no affinity for the cloned human kinin B1 receptor stably expressed in 293 cells. In addition, this compound at 1 microM did not significantly bind to a range of 40 different membrane receptors and eight ion channels except muscarinic M2 and M1 receptors for which an IC50 value of 0.9 and 1 microM was obtained. 5. BK stimulates in a concentration-dependent manner phosphoinositosides (IPs) production in cultured INT 407 cells. Concentration-response-curves to BK were shifted to the right in the presence of LF 16.0335 (0.1 microM) without reduction of the maximum. LF 16.0335 inhibited the concentration-contraction curve to BK in the human umbilical vein giving a pA2 value of 8.30+/-0.30 with a Schild plot slope that was not different from unity. 6. These results demonstrate that LF 16.0335 is a potent, selective and competitive antagonist of the human bradykinin B2 receptor.

Synthesis and pharmacological evaluation of dimer derivatives of the bradykinin receptor antagonist HOE-140.

The synthesis and pharmacological evaluation of dimer derivatives of the C-terminal fragments of the potent bradykinin antagonist HOE-140, linked through their N-termini, were performed. The influence of peptide moiety length was studied using the succinyl moiety as a linker. Our attention focused on the dimer of the C-terminal tetrapeptide of HOE-140 (compound JMV 980), which displayed some inhibiting activity (IC50 = 247 nM) for bradykinin B2 receptors. Unexpectedly, it was orally active in inhibiting bradykinin-induced hypotension in the rat. Based on this tetrapeptide dimer model, we synthesized pseudotetrapeptide dimer bradykinin antagonists 29 and 33, which exhibited high affinity (Ki = 76 and 61 nM, respectively) for the human cloned B2 receptor. In addition, compound 29 inhibited bradykinin-induced contraction of the human umbilical vein giving a pKB value of 6.45. Compounds 29 and 33 were selective toward B2 receptors because they did not bind to the cloned human B1 receptor up to 10 microM.

Characterisation of bradykinin receptors from juvenile pig coronary artery.

Coronary artery rings from juvenile male farm pigs were incubated for 6 h and precontracted with U46619. The rings relaxed in response to des-Arg9-bradykinin (pD2, 7.78 +/- 0.13; Emax, 87.4 +/- 4.3%) and to bradykinin (pD2, 8.69 +/- 0.30; Emax, 104.2 +/- 4.4%). These responses were abolished by endothelium removal and unaffected by indomethacin whilst NG-nitro-L-arginine reduced the relaxation due to des-Arg9-bradykinin only. Preincubation with cycloheximide or actinomycin had no effect against relaxations mediated by kinins whilst the protein trafficking inhibitor, brefeldin A, reduced by 52% the maximum response to des-Arg9-bradykinin. The bradykinin receptor antagonists, des-Arg9-[Leu8]bradykinin, Hoe 140 (D-Arg-[Hyp3, Thi5, D-Tic7, Oic8]bradykinin) and NPC 567 (D-Arg-[Hyp3,D-Phe7]bradykinin) antagonized competitively the response to des-Arg9-bradykinin, giving respective pA2 values of 6.82 +/- 0.34, 6.63 +/- 0.28 and 6.48 +/- 0.41 whereas the non-peptide bradykinin B2 receptor antagonist, WIN 64338 (phosphonium, [[4-[[2-[[bis(cyclohexylamino)methylene]amino]-3-(2- naphtalenyl) 1-oxopropyl]amino]-phenyl]-methyl]tributyl chloride, monohydrochloride), was inactive. Hoe 140 and WIN 64338 but not des-Arg9[Leu8]bradykinin behaved as competitive antagonists towards the relaxation due to bradykinin. In conclusion, both bradykinin B2 and B1 receptors are present on the endothelium of large coronary arteries from juvenile pig. The bradykinin B1 receptor subtype appears partly inducible and is coupled to the synthesis of nitric oxide.

Pharmacological evidence for a single bradykinin B2 receptor in the guinea-pig.

1. The present study addresses the possibility of the existence of different kinin B2 receptor 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 o indomethacin (3 microM), atropine (10 microM) and captopril (10 microM). 2. BK contracted jugular vein (JV), ileum (GPI), parenchyma (LP) and trachea (GPT) with an EC50 of 13.2 +/- 1.4 nM (n=27), 11.2 +/- 2.1 (n=26), 23.6 +/- 6.3 (n=26), and 33.0 +/- 6.5 (n=27), respectively. Thiorphan, a neutral endopeptidase (EC 3.4.34.11) 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. 3. The peptide B2 receptor 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, pKB values were determined. Mean values of pKB for 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 pKB values of 6.89 +/- 0.10, 7.57 +/- 0.12, 7.36 +/- 0.12 adn 7.51 +/- 0.28 in the JV, GPI, LP and GPT, respectively. 4. 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.