Antinociceptive profile of the pseudopeptide B2 bradykinin receptor antagonist NPC 18688 in mice.

1. The purpose of this study was to investigate the topical and systemic anti-hyperalgesic effect of the newly-developed pseudopeptide B2 receptor antagonist, NPC 18688, in different models of nociception in mice. 2. Given systemically 30 min beforehand, NPC 18688 (10-300 nmol kg-1, i.p.) caused no agonist effect, but produced a dose-related and significant inhibition of abdominal constrictions caused by intraperitoneal injection of acetic acid (0.6%), acetylcholine (ACh, 4.5 mg kg-1) or kaolin (50 mg kg-1). The calculated mean ID50s and the percentages of maximal inhibitions (MI) for these effects were: 77, 34 and > 300 nmol kg-1 and 65 +/- 6, 70 +/- 5 and 40 +/- 3%, respectively. The anti-hyperalgesic effect of NPC 18688 (100 nmol kg-1, i.p.) occurred rapidly (30 min) and lasted for at least 150 min. Hoe 140 (3-30 nmol kg-1, i.p.) given 30 min beforehand also inhibited, in a graded manner, acetic acid and ACh-induced writhing, with mean ID50s and MI of 6 and 9 nmol kg-1 and 56 +/- 7 and 62 +/- 6%, respectively. 3. NPC 18688 (10-300 nmol kg-1, i.p.) caused a graded inhibition of both phases of formalin (2.5%)-induced pain, its effects being more potent in relation to the second phase of the formalin test. The calculated mean ID50s and the MI were > 300 and 60 nmol kg-1 and 20 +/- 3 and 60 +/- 5% against the first and second phases of formalin-induced nociception, respectively. NPC 18688 at the same doses also inhibited, in a dose-related manner, formalin-induced paw oedema (MI of 35 +/- 3%). 4. When injected locally in the mouse paw, NPC 18688 (2, 10 and 20 nmol/paw) had no agonist activity. However, when co-injected with formalin NPC 18688 (2-20 nmol/paw), it produced significant inhibition of both phases of formalin response, with MI of 40 +/- 3 and 33 +/- 2%, respectively. NPC 18688 at 10 nmol/paw also significantly inhibited formalin-induced paw oedema (25 +/- 2%). 5. Given intraperitoneally, NPC 18688 (30-300 nmol kg-1) determined a graded inhibition of the nociceptive response caused by intraplantar injection of capsaicin (1.6 micrograms/paw) (40 +/- 2%). However, NPC 18688 (up to 300 nmol kg-1, i.p.), given 30 min beforehand, had no significant analgesic effect when analyzed in the tail flick and in the hot plate pain models, nor did it change the performance of animals in the rota rod test. 6. The action of NPC 18688 was quite selective for the B2 receptor, and like Hoe 140, (1 to 100 nmol kg-1, i.p.) it caused graded inhibition of bradykinin (BK, 3 mol/paw)-induced increase in mouse paw volume, with mean ID50s of 61 and 6 nmol kg-1, respectively. In addition, at 100 nmol kg-1, the dose at which NPC 18688 significantly antagonized BK (3 nmol)-mediated rat paw oedema in naive animals, it had no significant effect on des-Arg9-BK (100 nmol/paw)-induced oedema in paws that had been desensitized to BK. NPC 18688 (210 nmol kg-1), like Hoe 140 (230 nmol kg-1) given s.c. 30 min beforehand, completely abolished BK (28 nmol)-induced hypotension, without affecting the fall of mean arterial blood pressure induced by i.v. injection of ACh (2 nmol kg-1). Finally, NPC 18688 (1 microM) did not affect ACh-mediated contraction in the guinea-pig ileum or toad rectus abdominii in vitro. 7. These results demonstrate that the newly-developed and selective pseudopeptide B2 receptor antagonist, NPC 18688, although less potent than the available second generation of B2 peptide BK receptor antagonists, exhibits topical and long-lasting systemic anti-hyperalgesic properties when analysed in several models of nociception in mice, making it a useful tool for investigating the participation of BK and related kinins in physiological and pathological processes. Finally, this new class of selective pseudopeptide B2 receptor antagonist may constitute a new strategy for developing the third generation of potent and long-lasting orally-active non-peptide BK antagonists, which may be useful for the management of clinical disorders involving BK and relate     

Effects of WIN 64338, a nonpeptide bradykinin B2 receptor antagonist, on guinea-pig trachea.

We investigated the effect of the nonpeptide bradykinin receptor antagonist, [[4-[[2-[[bis(cyclohexylamino)methylene] amino]-3-(2-naphthalenyl) 1-oxopropyl]amino]-phenyl]-tributyl, chloride, monohydrochloride (WIN 64338), on [3H]-bradykinin binding and on bradykinin-induced contraction of the guinea-pig trachea. This non peptide bradykinin receptor antagonist inhibited [3H]-bradykinin binding with a nanomolar range of affinity, Ki = 50.9 +/- 19 nM and inhibited bradykinin-induced contraction in a non-competitive manner with a KB value of 6.43 10(-8) +/- 2.34 10(-8) M.     

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-phenyl]sulphonyl] -2(S) - [[4 -[4-(aminoiminomethyl)phenylcarbonyl]piperazin-1-yl]carbonyl]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 K_i 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, B_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 μM 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 μM 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 μM) 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.

     

Pharmacological characterization of a nonpeptide bradykinin B2 receptor antagonist,FR165649, and agonist,FR190997

1. The nonpeptide bradykinin (BK) B₂ receptor antagonist, {"type":"entrez-nucleotide","attrs":{"text":"FR165649","term_id":"258310103","term_text":"FR165649"}}FR165649 (8-[2,6-dichloro-3-[N-[(E)-4-(N- methylcarbamoyl)cinnamidoacetyl] -N-methylamino] benzyloxy] -2 - methylquinoline), and agonist, {"type":"entrez-nucleotide","attrs":{"text":"FR190997","term_id":"257934014","term_text":"FR190997"}}FR190997 (8-[2,6-dichloro-3-[N-[(E)-4-(N-methylcarbamoyl) cinnamidoacetyl]-N-methylamino]benzyloxy]-2-methyl-4-(2-pyridylmethoxy)quinoline) have been identified. These compounds have a common chemical structure, and the 2-pyridylmethoxy group is the only structural difference between them.
2. Both {"type":"entrez-nucleotide","attrs":{"text":"FR165649","term_id":"258310103","term_text":"FR165649"}}FR165649 and {"type":"entrez-nucleotide","attrs":{"text":"FR190997","term_id":"257934014","term_text":"FR190997"}}FR190997 displaced [³H]-BK binding to B₂ receptors in guinea-pig ileum membranes, with an IC₅₀ of 4.7×10⁻¹⁰ M and 1.5×10⁻⁹ M, respectively. They also displaced [³H]-BK binding to B₂ receptors in human lung fibroblast IMR-90 cells, with an IC₅₀ of 1.6×10⁻⁹ M and 9.8×10⁻¹⁰ M, respectively.
3. In guinea-pig isolated ileum-preparations, {"type":"entrez-nucleotide","attrs":{"text":"FR165649","term_id":"258310103","term_text":"FR165649"}}FR165649 had no agonistic effect on contraction and caused parallel rightward shifts of the concentration-response curves to BK on contraction. Analysis of the data produced a nominal pA₂ value of 9.2±0.1 (n=5) and a slope of 1.4±0.1 (n=5). On the other hand, {"type":"entrez-nucleotide","attrs":{"text":"FR190997","term_id":"257934014","term_text":"FR190997"}}FR190997 induced concentration-dependent contraction of guinea-pig ilea with a pD₂ of 7.9±0.2 and the contraction was inhibited by a specific peptide bradykinin B₂ receptor antagonist, Hoe 140 (D-Arg-[Hyp³, Thi⁵, D-Tic⁷, Oic⁸]BK) in a non-competitive manner.
4. In IMR-90 cells, {"type":"entrez-nucleotide","attrs":{"text":"FR165649","term_id":"258310103","term_text":"FR165649"}}FR165649 had no agonistic effect on phosphatidyl inositol (PI) hydrolysis and caused parallel rightward shifts (approximately 200 fold shift at 10⁻⁷ M) of the concentration-response curves to BK on PI hydrolysis. {"type":"entrez-nucleotide","attrs":{"text":"FR190997","term_id":"257934014","term_text":"FR190997"}}FR190997 induced concentration-dependent PI hydrolysis in IMR-90 cells with a pD₂ of 8.4±0.1, and this effect was inhibited by Hoe 140.
5. These results indicate that {"type":"entrez-nucleotide","attrs":{"text":"FR165649","term_id":"258310103","term_text":"FR165649"}}FR165649 and {"type":"entrez-nucleotide","attrs":{"text":"FR190997","term_id":"257934014","term_text":"FR190997"}}FR190997 are, respectively, a potent bradykinin B₂ receptor antagonist and agonist, and that the agonistic activity depends on the small part of the nonpeptide ligand. {"type":"entrez-nucleotide","attrs":{"text":"FR165649","term_id":"258310103","term_text":"FR165649"}}FR165649 and {"type":"entrez-nucleotide","attrs":{"text":"FR190997","term_id":"257934014","term_text":"FR190997"}}FR190997 may be useful tools for studying the relationship between ligands and receptors.

     

Effect of FK3657, a non-peptide bradykinin B2 receptor antagonist,on allergic airway disease models

Bradykinin has been suggested to be involved in allergic diseases. In this study, we tested the effect of FK3657 ((E)-3-(6-acetamido-3-pyridyl)-N-[N-[2,4-dichloro-3-[(2-methyl-8-quinolinyl)-oxymethyl]phenyl]-N-methylaminocarbonylmethyl]acrylamide), an orally active non-peptide bradykinin B(2) receptor antagonist, on allergic airway disease models in guinea pigs. FK3657 given orally inhibited bradykinin-induced or dextran sulfate (an activator of kinin-kallikrein cascade)-induced bronchoconstriction and plasma extravasation in the lower airways (trachea and main bronchi) and nasal mucosa of guinea pigs with ED(50) of 0.04-0.23 mg/kg. In the antigen-induced dual asthmatic response model of guinea pigs, FK3657 significantly attenuated the late phase asthmatic response, but not the immediate asthmatic response. FK3657 also significantly inhibited the 2,4-tolylene diisocyanate (TDI)-induced plasma extravasation in nasal mucosa of TDI-sensitized guinea pigs. These results suggest that oral FK3657 may be useful for asthma or allergic rhinitis as a therapeutic drug.     

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%).     

MEN16132, a kinin B2 receptor antagonist, prevents the endogenous bradykinin effects in guinea-pig airways

Kinins have been suggested to be involved in human airway diseases such as asthma and rhinitis. MEN16132 is a non-peptide kinin B(2) receptor antagonist able to inhibit the responses produced by intravenous bradykinin into the airways, as bronchoconstriction and microvascular leakage; we tested the effect of MEN16132 on endogenously generated bradykinin through the dextran sulfate-induced contact activation of kinin-kallikrein cascade in guinea-pigs. After dextran sulfate administration (1.5 mg/kg i.v.), the pulmonary insufflation pressure was monitored and the microvascular leakage of upper and lower airways was assessed using Evans blue as tracer of plasma protein extravasation. Our results demonstrated that topical MEN16132 strongly inhibited the dextran sulfate-induced bronchoconstriction (0.3 mM solution aerosol for 5 min) and plasma protein extravasation in both lower airways (3-10 microM solution aerosol for 5 min) and nasal mucosa (0.3 nmol/nostril); Icatibant, the peptide antagonist of kinin B(2) receptor, exerted a 3-30-fold less potent inhibitory effect than MEN16132. We conclude that local application of MEN16132 into the airways abolishes the responses produced by the endogenous generation of bradykinin and it can be useful as new pharmacological tool to check the role of kinins in human diseases.     

New selective bradykinin receptor antagonists and bradykinin B2 receptor characterization

Substantial progress has been made recently in the field of kinin pharmacology with the identification of sensitive bioassay organs and the discovery of bradykinin B2 receptor antagonists. Data obtained with such compounds in various laboratories support the hypothesis that kinins act on multiple (at least two) receptor types. Domenico Regoli and colleagues review here the basic criteria of receptor characterization as they apply to kinins and present a critical analysis of the bioassay organs and B2 receptor antagonists currently used in kinin pharmacology.     

Icatibant,the bradykinin B2 receptor antagonist with target to the interconnected kinin systems

Introduction: HOE-140/Icatibant is a selective, competitive antagonist to bradykinin (BK) against its binding to the kinin B2 receptor. Substitution of five non-proteogeneic amino acid analogues makes icatibant resistant to degradation by metalloproteases of kinin catabolism. Icatibant has clinical applications in inflammatory and vascular leakage conditions caused by an acute (non-controlled) production of kinins and their accumulation at the endothelium B2 receptor. The clinical manifestation of vascular leakage, called angioedema (AE), is characterized by edematous attacks of subcutaneous and submucosal tissues, which can cause painful intestinal consequences, and life-threatening complications if affecting the larynx. Icatibant is registered for the treatment of acute attacks of the hereditary BK-mediated AE, i.e., AE due to C1 inhibitor deficiency.

Areas covered: This review discusses emerging knowledge on the kinin system: kinin pharmacological properties, biochemical characteristics of the contact phase and kinin catabolism proteases. It underlines the responsibility of the kinins in AE initiation and the potency of icatibant to inhibit AE formation by kinin–receptor interactions.

Expert opinion: Icatibant antagonist properties protect BK-mediated AE patients against severe attacks, and could be developed for use in inflammatory conditions. More studies are required to confirm whether or not prolonged and frequent applications of icatibant could result in the impairment of the cardioprotective effect of BK.     

Conformational properties of a cyclic peptide bradykinin B2 receptor antagonist using experimental and theoretical methods

Abstract: The solution conformation of the cyclic peptide J324 (cyclo^0,6‐[Lys⁰,Glu⁶,d ‐Phe⁷]BK), an antagonist targeted at the bradykinin (BK) B₂ receptor, has been investigated using experimental and theoretical methods. In order to gain insight into the structural requirements essential for BK antagonism, we carried out molecular dynamics (MD) simulations using simulated annealing as the sampling protocol. Following a free MD simulation we performed simulations using nuclear Overhauser enhancement (NOE) distance constraints determined by NMR experiments. The low‐energy structures obtained were compared with each other, grouped into families and analyzed with respect to the presence of secondary structural elements in their backbone. We also introduced new ways of plotting structural data for a more comprehensive analysis of large conformational sets. Finally, the relationship between characteristic backbone conformations and the spatial arrangement of specific pharmacophore centers was investigated.     

The identification of an orally active,nonpeptide bradykinin B2 receptor antagonist,FR173657

1. An orally active, nonpeptide bradykinin (BK) B₂ receptor antagonist, {"type":"entrez-nucleotide","attrs":{"text":"FR173657","term_id":"257935500","term_text":"FR173657"}}FR173657 (E)-3-(6-acetamido-3 - pyridyl) - N - [N - [2 - 4 -dichloro-3-[(2-methyl-8-quinolinyl) oxymethyl]phenyl]-N-methylaminocarbonylmethyl]acrylamide) has been identified.
2. This compound displaced [³H]-BK binding to B₂ receptors present in guinea-pig ileum membranes with an IC₅₀ of 5.6×10⁻¹⁰ M and in rat uterus with an IC₅₀ of 1.5×10⁻⁹ M. It did not inhibit different specific radio-ligand binding to other receptor sites.
3. In human lung fibroblast IMR-90 cells, {"type":"entrez-nucleotide","attrs":{"text":"FR173657","term_id":"257935500","term_text":"FR173657"}}FR173657 displaced [³H]-BK binding to B₂ receptors with an IC₅₀ of 2.9×10⁻⁹ M and a K_i of 3.6×10⁻¹⁰ M, but did not reduce [³H]-des-Arg¹⁰-kallidin binding to B₁ receptors.
4. In guinea-pig isolated preparations, {"type":"entrez-nucleotide","attrs":{"text":"FR173657","term_id":"257935500","term_text":"FR173657"}}FR173657 antagonized BK-induced contractions with an IC₅₀ of 7.9×10⁻⁹ M, but did not antagonize acetylcholine or histamine-induced contractions even at a concentration of 10⁻⁶ M. {"type":"entrez-nucleotide","attrs":{"text":"FR173657","term_id":"257935500","term_text":"FR173657"}}FR173657 caused parallel rightward shifts of the concentration-response curves to BK at concentrations of 10⁻⁹ M and 3.2×10⁻⁹ M, and a little depression of the maximal response in addition to the parallel rightward shift of the concentration-response curve at a concentration of 10⁻⁸ M. Analysis of the data yield a pA₂ of 9.2±0.2 (n=5) and a slope of 1.5±0.2 (n=5).
5. In vivo, the oral administration of {"type":"entrez-nucleotide","attrs":{"text":"FR173657","term_id":"257935500","term_text":"FR173657"}}FR173657 inhibited BK-induced bronchoconstriction dose-dependently in guinea-pigs with an ED₅₀ of 0.075 mg kg⁻¹, but did not inhibit histamine-induced bronchoconstriction even at 1 mg kg⁻¹. {"type":"entrez-nucleotide","attrs":{"text":"FR173657","term_id":"257935500","term_text":"FR173657"}}FR173657 also inhibited carrageenin-induced paw oedema with an ED₅₀ of 6.8 mg kg⁻¹ 2 h after the carrageenin injection in rats.
6. These results show that {"type":"entrez-nucleotide","attrs":{"text":"FR173657","term_id":"257935500","term_text":"FR173657"}}FR173657 is a potent, selective, and orally active bradykinin B₂ receptor antagonist.

     

Pharmacological characterization and radioligand binding properties of a high-affinity, nonpeptide, bradykinin B1 receptor antagonist

Compound A (N-[2-[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]ethyl]-2-[(2R)-1-(2-napthylsulfonyl)-3-oxo-1,2,3,4-tetrahydroquinoxalin-2-yl]acetamide) is a member of a new class of aryl sulfonamide dihydroquinoxalinone bradykinin B1 receptor antagonists that should be useful pharmacological tools. Here we report on some of the pharmacological properties of compound A as well as the characterization of [35S]compound A as the first nonpeptide bradykinin B1 receptor radioligand. Compound A inhibited tritiated peptide ligand binding to the cloned human, rabbit, dog, and rat bradykinin B1 receptors expressed in CHO cells with Ki values of 0.016, 0.050, 0.56, and 29 nM, respectively. It was inactive at 10 microM in binding assays with the cloned human bradykinin B2 receptor. In functional antagonist assays with the cloned bradykinin B1 receptors, compound A inhibited agonist-induced signaling with activities consistent with the competition binding results, but had no antagonist activity at the bradykinin B2 receptor. Compound A was also found to be a potent antagonist in a rabbit aorta tissue bath preparation and to effectively block des-Arg9 bradykinin depressor responses in lipopolysaccharide-treated rabbit following intravenous administration. The binding of [35S]compound A was evaluated with the cloned bradykinin B1 receptors. In assays with human, rabbit, and dog receptors, [35S]compound A labeled a single site with Kd values of 0.012, 0.064, and 0.37 nM, respectively, and with binding site densities equivalent to those obtained using the conventional tritiated peptide ligands. Binding assays with the cloned rat bradykinin B1 receptor were not successful, presumably due to the low affinity of the ligand for this species receptor. There was no specific binding of the ligand detected in CHO cells expressing the human bradykinin B2 receptor. In assays with the cloned human bradykinin B1 receptor, the pharmacologies of the binding of [35S]compound A and [3H][Leu9]des-Arg10-kallidin were the same. The high signal-to-noise ratio obtained with [35S]compound A will allow this ligand to be a very useful tool for future investigations of the bradykinin B1 receptor.     

缓激肽B2受体在缬沙坦抑制心肌细胞肥大中的作用研究

目的探讨缓激肽(BK)B2受体在缬沙坦(valsartan)抑制血管紧张素Ⅱ(AngⅡ)诱导的新生大鼠心肌细胞肥大中的作用及可能机制.方法经差速贴壁法获得新生大鼠心肌细胞,随机分为对照组、AngⅡ组、缬沙坦组、AngⅡ+缬沙坦组、Hoe-140组和AngⅡ+缬沙坦+Hoe-140组.采用流式细胞仪技术检测细胞大小和蛋白含量,硝酸还原酶法测定上清液中NO含量,放射免疫法测定细胞内cGMP水平.结果与对照组相比,10-7 mol·L-1 AngⅡ显著增加心肌细胞大小和蛋白含量,降低心肌细胞NO生成(P＜0.01),10-5 mol·L-1缬沙坦显著降低AngⅡ诱导的心肌细胞肥大和蛋白合成增加(P＜0.01),促进心肌细胞NO(P<0.05)和cGMP生成(P＜0.01),BK B2受体阻断剂Hoe-140可部分阻断缬沙坦的上述作用.结论BK B2受体部分介导了缬沙坦抑制心肌细胞肥大的效应,该作用与NO、cGMP生成有关.     

Blockade of feeding inhibition by neuromedin B using a selective receptor antagonist

The ability of a selective neuromedin B receptor antagonist, D-Nal-cyclo[Cys-Tyr-D-Trp-Orn-Val-Cys]-Nal-NH₂ (BIM-23127), to block suppression of food intake produced by the mammalian bombesin-like peptides neuromedin B and neuromedin C was examined. BIM-23127 completely blocked suppression of intake produced by neyromedin B but not by neuromedin C. These results suggest an independent role for neuromedin B receptors in suppression of food intake by bombesin-like peptides and demonstrate the utility of this group of antagonists for in vivo experiments.     

The nonpeptide B2 receptor antagonist FR173657: inhibition of effects of bradykinin related to its role in nociception

1. The nonpeptide bradykinin B₂ receptor antagonist, {"type":"entrez-nucleotide","attrs":{"text":"FR173657","term_id":"257935500","term_text":"FR173657"}}FR173657 ((E)-3-(6-acetamido-3-pyridyl)-N-[N-(2, 4-dichloro-3-[(2-methyl-8-quinolinyl) oxymethyl] phenyl]-N-methylaminocarbonylmethyl] acrylamide), was tested in models involving bradykinin-induced activation of primary afferent neurones in vitro and in vivo.
2. Bradykinin-induced contractions of the rabbit isolated iris sphincter muscle mediated by tachykinin release from trigeminal afferent neurones were inhibited in a non-competitive manner by {"type":"entrez-nucleotide","attrs":{"text":"FR173657","term_id":"257935500","term_text":"FR173657"}}FR173657. A pK_B value of 7.9 was calculated. Effects of substance P were unaffected by {"type":"entrez-nucleotide","attrs":{"text":"FR173657","term_id":"257935500","term_text":"FR173657"}}FR173657.
3. Nociceptive behavioural responses following intraplantar injection of bradykinin in unanaesthetized rats were reduced by 0.3 μmol kg⁻¹ {"type":"entrez-nucleotide","attrs":{"text":"FR173657","term_id":"257935500","term_text":"FR173657"}}FR173657 s.c. (P<0.05), and completely abolished by 3 μmol kg⁻¹ (P<0.05). Peroral administration of 5 μmol kg⁻¹ {"type":"entrez-nucleotide","attrs":{"text":"FR173657","term_id":"257935500","term_text":"FR173657"}}FR173657 abolished the bradykinin effects (P<0.05); lower doses had no significant effect.
4. Shortening by intraplantar injection of bradykinin of the paw withdrawal latency in response to radiant heat was abolished by 3 μmol kg⁻¹ {"type":"entrez-nucleotide","attrs":{"text":"FR173657","term_id":"257935500","term_text":"FR173657"}}FR173657 s.c. (P<0.05), while 300 nmol kg⁻¹ had an intermediate effect. Hyperalgesia induced by prostaglandin E₂ remained unaffected by {"type":"entrez-nucleotide","attrs":{"text":"FR173657","term_id":"257935500","term_text":"FR173657"}}FR173657.
5. Blood pressure reflexes following i.p. instillation of bradykinin in anaesthetized rats were inhibited by {"type":"entrez-nucleotide","attrs":{"text":"FR173657","term_id":"257935500","term_text":"FR173657"}}FR173657 s.c. with an ID₅₀ of 1.1 μmol kg⁻¹, while the peptidic B₂ antagonist icatibant (Hoe-140; D-Arg⁰-[Hyp³, Thi⁵, D-Tic⁷, Oic⁸]-bradykinin) caused inhibition at significantly lower doses (ID₅₀ 8.5 nmol kg⁻¹ P<0.001). Responses to hydrochloric acid i.p. remained unaffected by {"type":"entrez-nucleotide","attrs":{"text":"FR173657","term_id":"257935500","term_text":"FR173657"}}FR173657.
6. {"type":"entrez-nucleotide","attrs":{"text":"FR173657","term_id":"257935500","term_text":"FR173657"}}FR173657 or similar nonpeptide compounds may be useful for the development of drugs for diseases involving pain induced by the release of endogenous kinins, i.e. especially in acute inflammatory conditions.

     

Intramolecular signal transduction by the bradykinin B2 receptor

To study the intramolecular signal transduction, we performed single point and cassette mutations in transmembranal and intracellular regions of the bradykinin B2 receptor. We studied the influence of the two intramembranal Cys residues at positions 304 and 348, the role of Arg at position 177 in the highly conserved tripeptide sequence Asp-Arg-Tyr, the cytosolic G-protein binding area, and attempted to verify the general hypothesis of an ion tunnel-like interface in GPCRs. Wild type receptor, His-tagged receptor, and His-tagged mutant receptors were expressed in COS-7 cells and functionally compared by bradykinin-induced formation of inositolphosphate and arachidonic acid. To investigate the expression, all mutants were modified at the N-terminus by insertion of two successive His-tags and detected with an anti-poly-His antibody. Replacement of the second and third cytosolic loop by a loop from another membrane protein as well as single replacement of Arg at position 177 by Ala leads to a fully inactive receptor mutant without any ligand binding affinity and stimulatory activity. Mutants with replacement of Cys residues 304 and 348 by Ser showed only moderate effects. Regardless of the replacement of Asp 407 by Ala, the receptor is able to increase the agonist-induced levels of inositolphosphate and of arachidonic acid, indicating that our studies can not verify the postulated ion tunnel hypothesis.     

Inhibition of acute nociceptive responses in rat spinal cord by a bradykinin B1 receptor antagonist

This study used behavioural and in vivo electrophysiological paradigms to examine the effects of systemic and spinal administration of a bradykinin B1 receptor antagonist, compound X, on acute nociceptive responses in the rat. In behavioural experiments, compound X significantly increased the latency to withdraw the hindpaw from a radiant heat source after both intravenous and intrathecal administration, without affecting motor performance on the rotarod. In electrophysiological experiments, both intravenous and direct spinal administration of compound X attenuated the responses of single dorsal horn neurones to noxious thermal stimulation of the hindpaw. These data show that the antinociceptive effects of a bradykinin B1 receptor antagonist are mediated, at least in part, at the level of the spinal cord and suggest a role for spinal bradykinin B1 receptors in acute nociception.     

From bradykinin B2 receptor antagonists to orally active and selective bradykinin B1 receptor antagonists

The bradykinin (BK) B1 receptor is an attractive target for the treatment of chronic pain and inflammation. Starting from a dual B1 and B2 antagonist, novel antagonists were designed that display low-nanomolar affinity for human B1 receptor and selectivity over B2. Initially, potent imidazoline derivatives were studied, but these compounds suffered from low bioavailability. This issue could be overcome by the use of less basic amino derivatives leading to orally active compounds.     

Nephroprotection in Zucker diabetic fatty rats by vasopeptidase inhibition is partly bradykinin B2 receptor dependent

1. Vasopeptidase inhibition (i.e., the simultaneous inhibition of both angiotensin-converting enzyme (ACE) and neutral endopeptidase) can ameliorate diabetic nephropathy. We investigated whether this nephroprotection is mediated by the bradykinin B2 receptor. 2. In all, 43 obese Zucker diabetic fatty (ZDF/Gmi-fa/fa) rats aged 21 weeks were separated into four groups and treated for 26 weeks with either placebo, the bradykinin B2 receptor antagonist icatibant (500 microg kg(-1) day(-1) s.c. infusion), the vasopeptidase inhibitor AVE7688 (45 mg kg(-1) day(-1) in chow), or AVE7688 plus icatibant. Nephropathy was assessed as albuminuria at age 31 and 39 weeks, and by histopathologic scoring at the end of the treatment period. 3. All animals had established diabetes mellitus (blood glucose >20 mmol l(-1)) and marked albuminuria at baseline. Blood glucose was not influenced by any treatment. Icatibant alone did not influence albuminuria (8.6+/-1.6 vs placebo 9.5+/-1.3 mg kg(-1) h(-1)). AVE7688 reduced albuminuria at week 31 markedly to 1.1+/-0.1 mg kg(-1) h(-1) and reduced glomerular and tubulo-interstitial kidney damage at week 47. In the AVE7688 plus icatibant group, proteinuria was significantly higher than in the AVE7688 only group (2.0+/-0.6 mg kg(-1) h(-1)), but still reduced compared to placebo. In addition, icatibant partly antagonized the tubulo-interstitial protection mediated by AVE7688. 4. We conclude that vasopeptidase inhibition provides nephroprotection in rats with type II diabetic nephropathy, which is partly mediated by bradykinin B2 receptor activation.     

Potentiation of 2-deoxy-D-glucose antinociception, but not hyperphagia by zolantidine, a histamine (H2) receptor antagonist.

Antagonism of the histamine (H2) receptor reduces antinociception induced by naloxone-resistant foot-shock, naloxone-sensitive foot-shock, and morphine with a rank-order potency similar to their H2 antagonism. The antimetabolic glucose analog 2-deoxy-D-glucose (2DG) produces antinociceptive and hyperphagic responses that dissociate from each other and are in part mediated by opioid systems. The present study determined the effects of the brain-penetrating H2 receptor antagonist zolantidine (ZOL) on 2DG antinociception on the tail-flick and jump tests, as well as on 2DG hyperphagia, in rats. ZOL (0.01-1 mg/kg) potentiated the antinociceptive responses induced by a moderate (450 mg/kg) dose of 2DG, but had lesser effects upon antinociception induced by a lower (100 mg/kg) 2DG dose. ZOL itself slightly increased jump thresholds, but not tail-flick latencies. Combinations of ZOL and 2DG produced supraadditive antinociception, even though ZOL failed to significantly shift the 2DG dose-response curve to the left. In contrast, ZOL failed to alter basal intake or 2DG hyperphagia, supporting previous evidence implicating the H1 but not the H2 receptor in these effects. These results further dissociate the antinociceptive and hyperphagic effects of 2DG, and also support previous results indicating both pro- and antinociceptive roles for H2 receptors.