Non-peptide Bradykinin Research Articles

Effects of a nonpeptide bradykinin B2 receptor antagonist, FR167344, on guinea-pig tracheal smooth muscle bradykinin receptors

It is speculated that bradykinin may play an important role in asthma. Thus, bradykinin receptor antagonists may have therapeutic potential against asthma. Orally active bradykinin antagonists would be more desirable for the treatment of the disease. In the present study, we examined the effects of a novel, potent, selective, and orally active nonpeptide bradykinin B2 receptor antagonist, FR167344 (N-[N-[3-[(3-bromo-2-methylimidazo[1,2-a]pyridin-8-yl)oxymethyl]- 2,4-dichlorophenyl]-N-methylaminocarbonylmethyl]-4-(dimethylaminocarbonyl)cinnamylamide hydrochloride), on guinea-pig tracheal smooth muscle bradykinin receptors. FR167344 inhibited [3H]bradykinin binding to bradykinin receptors in epithelium-denuded guinea-pig tracheal membrane with an IC50 of 2.1 nM and a Ki of 0.44 nM. This compound also inhibited bradykinin-induced contraction of epithelium-denuded guinea-pig trachea with a pKB of 10.8, but had no effect on carbachol-induced contraction of the trachea even at 10-6 M. These results indicate that FR167344 has the specific antagonistic activity against guinea-pig tracheal smooth muscle bradykinin receptors.Key words: bradykinin, receptor, antagonist, FR167344, trachea.

A novel class of orally active non-peptide bradykinin B2 receptor antagonists. 3. Discovering bioisosteres of the imidazo[1,2-a] pyridine moiety.

Recently we reported on overcoming the species difference of our first orally active non-peptide bradykinin (BK) B2 receptor antagonists, incorporating an 8-[[3-(N-acylglycyl-N-methylamino)-2, 6-dichlorobenzyl]oxy]-3-halo-2-methylimidazo[1,2-a]pyridine skeleton, leading to identification of the first clinical candidate 4a (FR167344). With this potent new lead compound in hand, we then investigated further refinement of the basic framework by replacement of the imidazo[1,2-a]pyridine moiety and discovered several bioisosteric heterocycles. Extensive optimization of these new heteroaromatic derivatives revealed the detailed structure-activity relationships (SAR) around the imidazo[1, 2-a]pyridine ring and the 2,6-dichlorobenzyl moiety, leading to the discovery of our second clinical candidate 87b (FR173657) which inhibited the specific binding of [3H]BK to recombinant human B2 receptors expressed in Chinese hamster ovary (CHO) cells and guinea pig ileum membrane preparations expressing B2 receptors with IC50's of 1.4 and 0.46 nM, respectively. This compound also displayed excellent in vivo functional antagonistic activity against BK-induced bronchoconstriction in guinea pigs with an ED50 value of 0.075 mg/kg by oral administration. Further modifications of the terminal substituents on the pyridine moiety led to a novel pharmacophore and resulted in the identification of 99 (FR184280), whose IC50 value for human B2 receptors (0.51 nM) was comparable to that of the second-generation peptide B2 antagonist Icatibant.

A novel class of orally active non-peptide bradykinin B2 receptor antagonists. 2. Overcoming the species difference between guinea pig and man.

Recently we reported the identification of a series of 8-[[3-(N-acylglycyl-N-methylamino)-2, 6-dichlorobenzyl]oxy]-3-halo-2-methylimidazo[1,2-a]pyridines as the first orally active non-peptide bradykinin (BK) B2 receptor antagonists (1-3). These compounds inhibited the specific binding of [3H]BK to guinea pig ileum membrane preparations expressing B2 receptors with nanomolar IC50's and also displayed in vivo functional antagonistic activities against BK-induced bronchoconstriction in guinea pigs at 1 mg/kg by oral administration. However, it was found that their affinities for the B2 receptors in human A-431 cells (human epidermoid carcinoma) were much lower. Intensive modifications of the terminal substituents at the glycine moiety elucidated the structure-activity relationships (SAR) for human B2 receptors, leading to an extended basic framework which incorporated a novel key pharmacophore. Thus, we overcame the species difference and identified the first clinical candidate 18c (FR167344) with IC50's of 0.66 and 1.4 nM for guinea pig ileum and human A-431 cells, respectively. This compound displayed in vivo functional antagonistic activity against BK-induced bronchoconstriction in guinea pigs with an ED50 value of 0.17 mg/kg by oral administration. This novel non-peptide B2 antagonist is extremely potent both in vitro and in vivo by oral administration and is expected to be the first member of a new class of drug for the treatment of various inflammatory diseases.

Inhibition of Guinea Pig Skin Allergic Reactions by Nonpeptide Bradykinin B2 Receptor Antagonist FR173657

Background: Kinins, which cause vascular permeability enhancement (VPE) through bradykinin (BK) B₂ receptors, are released at allergic reaction sites; however, the role kinins play in the lesions is still unknown. To determine whether kinins contribute to allergic reactions, the effect of a potent, nonpeptide BK B₂–receptor–specific antagonist, FR173657, on VPE induced in the reaction sites was investigated. Methods: Type I allergic reaction was induced by intradermal injections of dinitrophenol (DNP)–bovine serum albumin (BSA) into guinea pigs having received an intravenous injection of anti–DNP antiserum a week before. Type III allergic reaction was induced by intradermal injections of BSA into animals sensitized with subcutaneous injections of complete Freund's adjuvant–emulsified BSA 2 weeks before. VPE and swelling were measured by the leakage of intravenously injected dye and double thickness, respectively, at allergic reaction sites. FR173657 (4 mg/kg body weight) was administered orally 30 min before antigen injection. Results: In the type I allergy model, FR173657 inhibited 34% of the early–phase VPE, which occurs a few minutes after antigen injection, and 50% of the subsequent swelling at the allergic reaction sites. In the type III allergy model, FR173657 also inhibited 30% of the early VPE but neither the late VPE, which occurs from 1 h to at least 24 h after the antigen injection, nor the following swelling at the allergic reaction sites. FR173657 inhibited BK–induced VPE almost completely without affecting histamine–induced VPE. Conclusions: Kinins contribute significantly to the VPE induced in the animal allergy models. FR173657 may be useful for the therapy of human allergic diseases.

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

1. The nonpeptide bradykinin B2 receptor antagonist, 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 FR173657. A pKB value of 7.9 was calculated. Effects of substance P were unaffected by FR173657. 3. Nociceptive behavioural responses following intraplantar injection of bradykinin in unanaesthetized rats were reduced by 0.3 micromol kg(-1) FR173657 s.c. (P < 0.05), and completely abolished by 3 micromol kg(-1) (P < 0.05). Peroral administration of 5 micromol kg(-1) 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 micromol kg(-1) FR173657 s.c. (P < 0.05), while 300 nmol kg(-1) had an intermediate effect. Hyperalgesia induced by prostaglandin E2 remained unaffected by FR173657. 5. Blood pressure reflexes following i.p. instillation of bradykinin in anaesthetized rats were inhibited by FR173657 s.c. with an ID50 of 1.1 micromol 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 FR173657. 6. 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.

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

1. The nonpeptide bradykinin (BK) B2 receptor antagonist, FR165649 (8-[2,6-dichloro-3-[N-[(E)-4-(N-methylcarbamoyl)cinnamidoacetyl ]-N-methylamino]benzyloxy]-2-methylquinoline), and agonist, FR190997 (8-[2,6-dichloro-3-[N-[(E)-4-(N-methylcarbamoyl) cinnamidoacetyl]-N-methylamino]benzyloxy]-2-methyl-4-(2-pyridyl methoxy)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 FR165649 and FR190997 displaced [3H]-BK binding to B2 receptors in guinea-pig ileum membranes, with an IC50 of 4.7 x 10(-10) M and 1.5 x 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 x 10(-9) M and 9.8 x 10(-10) M, respectively. 3. In guinea-pig isolated ileum-preparations, 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, 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, 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. 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 FR165649 and 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. FR165649 and FR190997 may be useful tools for studying the relationship between ligands and receptors.

A novel class of orally active non-peptide bradykinin B2 receptor antagonists. 1. Construction of the basic framework.

A novel class of potent, selective, and orally active non-peptide bradykinin (BK) B2 receptor antagonists were designed and synthesized starting from 8-benzyloxyimidazo[1,2-a]pyridine derivative 2. The unique screening lead (2) was discovered by a two-step intentional random screening process, involving recognition of the relationship between BK and angiotensin II (Ang II) and the common structural features. Systematic chemical modification of 2 elucidated the structural requirements essential for B2 binding affinity leading to the identification of 8-[[3-(N-acylglycyl-N-methylamino)-2,6-dichlorobenzyl]oxy]-3-halo- 2- methylimidazo[1,2-a]pyridine skeleton as the basic framework of this new series of B2 antagonists. A molecular modeling study suggested the key role of the N-methylanilide moiety at the 3-position of the 2,6-dichlorobenzene ring to allow these compounds to adopt the characteristic active conformation. The representative lead compounds inhibited the specific binding of [3H]BK to guinea pig ileum membrane preparations expressing B2 receptors, with nanomolar IC50S and also displayed in vivo functional antagonistic activities against BK-induced bronchoconstriction in guinea pigs at an oral dose of 1 mg/kg. Pharmacokinetic studies of compounds 47c and 50b in rats highlighted their excellent oral bioavailabilities, indicating that they represent the first orally active non-peptide B2 antagonists reported to date.

Mouse Paw Edema Induced by a Novel Bradykinin Agonist and Its Inhibition by B2-Antagonists

A novel non-peptide bradykinin B2-receptor agonist, FR190997 (8-[2,6-dichloro-3-[N-[(E)-4-(N-methylcarbamoyl) cinnamidoacetyl]-N-methylamino] benzyloxy]-2-methyl-4-(2-pyridylmethoxy) quinoline), induced dose-dependent and longer-lasting swelling than bradykinin in the mouse paw. The swelling, peaking around 30 min, was suppressed dose-dependently by intraperitoneal administration of FR173657, a novel non-peptide B2-receptor antagonist. A known B2-antagonist, Hoe 140, also significantly suppressed this edema. The result indicates that the novel B2-agonist FR190997, being more stable than bradykinin, could induce plasma exudation locally in mice via the B2-receptor as a substitute for bradykinin.

Effects of a nonpeptide bradykinin B2 receptor antagonist, FR167344, on guinea-pig tracheal smooth muscle bradykinin receptors.

It is speculated that bradykinin may play an important role in asthma. Thus, bradykinin receptor antagonists may have therapeutic potential against asthma. Orally active bradykinin antagonists would be more desirable for the treatment of the disease. In the present study, we examined the effects of a novel, potent, selective, and orally active nonpeptide bradykinin B2 receptor antagonist, FR167344 (N-[N-[3-[(3-bromo-2-methylimidazo[1,2-a]pyridin-8-yl)oxymethyl]-2 ,4-dichlorophenyl]-N-methylaminocarbonylmethyl]-4-(dimethylamin ocarbonyl)cinnamylamide hydrochloride), on guinea-pig tracheal smooth muscle bradykinin receptors. FR167344 inhibited [3H]bradykinin binding to bradykinin receptors in epithelium-denuded guinea-pig tracheal membrane with an IC50 of 2.1 nM and a Ki of 0.44 nM. This compound also inhibited bradykinin-induced contraction of epithelium-denuded guinea-pig trachea with a pK(B) of 10.8, but had no effect on carbachol-induced contraction of the trachea even at 10(-6) M. These results indicate that FR167344 has the specific antagonistic activity against guinea-pig tracheal smooth muscle bradykinin receptors.

Effects of a nonpeptide bradykinin B2 receptor antagonist, FR167344, on different in vivo animal models of inflammation.

1. The effects of a novel, potent and orally active nonpeptide bradykinin B2 receptor antagonist, FR167344 (N-[N-[3-[(3-bromo-2-methylimidazo[1,2-a]pyridin-8-yl)oxymethyl]-2 ,4-dichlorophenyl]-N-methylaminocarbonylmethyl]-4-(dimethylamin ocarbonyl) cinnamylamide hydrochloride) were tested in three different in vivo models of inflammation. 2. Oral administration of FR167344 inhibited carrageenin-induced paw oedema in rats (carrageenin: 1%, 0.1 ml per animal, intraplantar), with an ID50 of 2.7 mg kg(-1) at 2 h after carrageenin injection (n=10 or 11). 3. Oral administration of the compound also inhibited kaolin-induced writhing (kaolin: 250 mg kg(-1), i.p.) in mice, with ID50 of 2.8 mg kg(-1) in 10 min writhing and 4.2 mg kg(-1) in 15 min writhing (n=19 or 20). 4. Additionally, oral administration of FR167344 inhibited caerulein-induced pancreatic oedema with an ID50 of 13.8 mg kg(-1) as well as increases in amylase and lipase of blood samples with ID50 of 10.3 and 7.4 mg kg(-1), respectively, in rats (n=10). 5. These results show that FR167344 is an orally active, anti-inflammatory and anti-nociceptive agent in carrageenin-induced paw oedema, kaolin-induced writhing and caerulein-induced pancreatitis. FR167344 may have therapeutic potential against inflammatory diseases by oral administration and it may be a useful tool for studying the involvement of B2 receptors in various in vivo models of inflammation.

Pharmacological characterization of a novel, orally active, nonpeptide bradykinin B 2 receptor antagonist, FR167344

To investigate the pathophysiological role of bradykinin and to develop a drug for inflammatory diseases, we discovered an orally active, nonpeptide bradykinin B 2 receptor antagonist, FR167344, N-[ N-[3-[(3-bromo-2-methylimidazo[1,2-a]pyridin-8-yl)oxymethyl]-2,4-dichlorophenyl]- N-methylaminocarbonylmethyl]-4-(dimethylaminocarbonyl) cinnamylamide hydrochloride. This compound competitively displaced [ 3 H ]bradykinin binding to bradykinin B 2 receptors present in guinea pig ileum membrane with an IC 50 value of 6.6×10 −10 M. In isolated guinea pig ileum preparations, it also antagonized bradykinin-induced contraction with a pA 2 value of 9.3. In human lung fibroblast IMR-90 cells, FR167344 displaced [ 3 H ]bradykinin binding to human bradykinin B 2 receptors with an IC 50 value of 1.3×10 −8 M, but not [ 3 H ]des-Arg 10–kallidin binding to human bradykinin B 1 receptors. In vivo, oral administration of FR167344 inhibited bradykinin-induced bronchoconstriction in guinea pigs and the bradykinin-induced hypotensive response for 6 h in rats. These results show that FR167344 is a potent, selective, orally active and long acting bradykinin B 2 receptor antagonist.

Nonpeptide bradykinin antagonist analogs based on a model of a Sterling-Winthrop nonpeptide bradykinin antagonist overlapped with cyclic hexapeptide bradykinin antagonist peptides

A proposed overlap between cyclic hexapeptide Bradykinin antagonists and nonpeptide Bradykinin antagonists is discussed. Structural variations on both the peptides and nonpeptides support the proposed overlap based on an increase or decrease in the biological activities of the antagonists.

Nonpeptide mimic of bradykinin with long-acting properties at the bradykinin B2 receptor.

Kinins, members of a family of peptides released from kininogens by the action of kallikreins, exhibit a variety of biological activities including vasodilation, increased vascular permeability, contraction of smooth muscle cells, and activation of sensory neurons. However, investigation of the physiological actions of kinins has been greatly hampered because its effects are curtailed by rapid proteolysis in blood, lung, and liver. We describe the pharmacological characteristics of a novel nonpeptide bradykinin receptor agonist FR190997 (8-[2,6-dichloro-3-[N-[(E)-4-(N-methylcarbamoyl)cinnamidoacetyl ]-N-methylamino]benzyloxy]-2-methyl-4-(2-pyridylmethoxy)quinoli ne). FR190997 markedly stimulated phosphatidylinositol hydrolysis in Chinese hamster ovary cells permanently expressing the human bradykinin B2 receptor. The response of phosphatidylinositol hydrolysis was antagonized by the B2 receptor selective antagonist Hoe 140 (D-Arg-[hydroxyproline3,beta-thienylalanine4,D-Tic7,++ +Oic8]bradykinin). In competitive experiments using membranes prepared from Chinese hamster ovary cells expressing the human bradykinin receptor subtypes, FR190997 showed a high affinity binding to the B2 receptor with IC50 value of 5.3 nM and no binding affinity for the B1 receptor. In vivo, FR190997 mimics the biological action of bradykinin and induces hypotensive responses in rats with prolonged duration. Therefore, FR190997 is a highly potent and subtype-selective nonpeptide agonist which displays high intrinsic activity. This compound should represent a powerful tool for further investigation of the physiology and pathophysiology of bradykinin receptors.

Effects of the non-peptide B2 antagonist FR173657 on kinin-induced smooth muscle contraction and relaxation, vasoconstriction and prostaglandin release.

1. The non-peptide bradykinin (BK) antagonist (E)-3-(6-acetamido-3-pyridyl)-N-[N-[2,4-dichloro-3-[(2-methyl-8-quinolin yl) oxymethyl]phenyl]-N-methylaminocarbonylmethyl]acrylamide (FR173657) was tested in intestinal, uterine, tracheal and vascular in vitro preparations. The investigation aimed at determining the antagonistic potency, duration of action, specificity for BK receptors and apparent mode of antagonistic action of FR173657. 2. Contractions of the isolated ileum of the guinea-pig in response to BK were inhibited by FR173657 (10-300 nM) in a concentration-dependent manner. The inhibition lasted for up to 90 min after wash-out of FR173657. Cumulative concentration-response curves to BK were shifted to the right with a concomitant decrease in the maximum effect. A pKB value of 8.7 was determined. FR173657 had no effect on contractions induced by acetylcholine, histamine, 5-hydroxytryptamine, substance P, angiotensin II or caerulein. 3. The concentration-response curves for B2 receptor-mediated relaxations of the rat isolated duodenum induced by BK were shifted to the right together with a concomitant reduction of the maximum BK effect in the presence of FR173657 (10-300 nM). A pKB of 9.0 +/- 0.2 was calculated. FR173657 had no effect on B1 receptor-mediated relaxations in response to des-Arg9-BK. 4. The concentration-response curves for BK-induced contractions of the rat isolated uterus were shifted to the right by FR173657 (3-300 nM) in a concentration-dependent and parallel manner. The Schild plot for the inhibition caused by FR173657 had a slope of -0.98 indicating a competitive mode of antagonism. A pA2 value of 9.1 was determined. 5. Contractions of the circular smooth muscles of the guinea-pig isolated trachea in response to BK were concentration-dependently inhibited by FR173657 (10-100 nM). An affinity estimate of 9.3 was calculated for FR173657. Contractions induced by acetylcholine and relaxations in response to isoprenaline remained completely unaffected by FR173657. 6. In the rabbit isolated perfused ear, BK (0.01-10 nmol) produced a dose-dependent vasoconstriction. In the presence of 30 nM FR173657, the effects of BK were reduced by at least 60%, while FR173657 completely abolished the effects of all BK doses at 300 nM. FR173657 did not affect vasoconstriction induced by noradrenaline or angiotensin II. 7. The arterial injection of BK (10 nmol) into the rabbit isolated perfused ear caused an approximately three fold increase in the release of the prostaglandins E2 and I2 into the venous effluent. The BK-stimulated prostaglandin release was completely abolished in the presence of FR173657 (300 nM) while the basal prostaglandin release was unchanged. 8. In summary, FR173657 was shown to be a highly potent and selective BK antagonist which was active on B2, but not B1, receptors. FR173657 was a competitive antagonist in the rat uterus but showed a deviation from competitive inhibition in the other preparations studied similar to other second generation peptide antagonists. The inhibitory action in vitro was long-lasting, but was fully reversible.

Effects of FR173657, a non‐peptide B2 antagonist, on kinin‐induced hypotension, visceral and peripheral oedema formation and bronchoconstriction

1. Kinins are believed to play a key role in many inflammatory conditions. Therefore, bradykinin antagonists are being developed for potential therapeutic applications. In the present investigation we describe the pharmacology, in vivo, of (E)-3-(6-acetamido-3-pyridyl)-N-[N-[2,4-dichloro-3-[2-methyl-8-quinoliny l) oxymethyl]phenyl]-N-methylaminocarbonylmethyl]acrylamide (FR173657), a novel, non-peptide bradykinin antagonist. 2. The hypotensive effects of i.v. injections of bradykinin (50 pmol) in captopril-pre-treated anaesthetized rats were significantly inhibited by 100 nmol kg-1 FR173657 s.c., and completely abolished by 300 nmol kg-1. The full inhibitory effect developed within 60 min and remained unchanged for at least 4 h. However, the effect was reversible, since 24 h after an injection of 300 nmol kg-1 FR173657 no inhibitory effect could be observed. 3. The plasma protein extravasation into the pancreas and duodenum induced by an i.v. infusion of bradykinin (11 nmol kg-1 within 20 min) in captopril-treated anaesthetized rats was completely abolished by FR173657 at doses of 30 nmol kg-1 s.c. and above, given 60 min before bradykinin. FR173657 3 nmol kg-1 was ineffective, while a dose of 10 nmol kg-1 produced an intermediate effect. 4. The paw oedema induced by the subplantar injection of bradykinin (30 nmol) in anaesthetized rats was inhibited slightly by s.c. injection of FR173657 0.3 mumol kg-1, whereas 1 and 3 mumol kg-1 produced significant inhibition of the bradykinin-induced oedema. The maximum inhibition amounted to about 50% and could not be increased even when the dose of FR173657 was increased to 30 mumol kg-1. FR173657 did not effect the oedema caused by histamine or 5-hydroxytryptamine. 5. Bradykinin (20 nmol kg-1, i.v.) caused increases in pulmonary inflation pressure by 300-600 Pa in anaesthetized, respirated guinea-pigs. The effect was reduced to 58 +/- 9% of the initial value 60 min after the s.c. injection of FR173657 1 mumol kg-1, whereas only 9 +/- 7% remained after 10 mumol kg-1. The bronchoconstrictor actions of histamine remained unaffected by FR173657. 6. In summary, FR173657 is a highly potent and selective bradykinin antagonist. The inhibitory action in vivo lasts for longer than 4 h but is fully reversible. FR173657, or similar compounds, will be a useful tool for the pharmacological investigation of pathophysiological states and may possess a therapeutic potential in diseases involving the endogenous release of kinins.

O-275 - Pharmacological Characterization of a Novel, Orally Active, Nonpeptide Bradykinin B2 Receptor Antagonist, FR167344

O-275 - Pharmacological Characterization of a Novel, Orally Active, Nonpeptide Bradykinin B2 Receptor Antagonist, FR167344

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-Arg 9-bradykinin (pD 2, 7.78 ± 0.13; E max, 87.4 ± 4.3%) and to bradykinin (pD 2, 8.69 ± 0.30; E max, 104.2 ± 4.4%). These responses were abolished by endothelium removal and unaffected by indomethacin whilst N G-nitro- l-arginine reduced the relaxation due to des-Arg 9-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-Arg 9-bradykinin. The bradykinin receptor antagonists, des-Arg 9-[Leu 8]bradykinin, Hoe 140 ( d-Arg-[Hyp 3, Thi 5, d-Tic 7,Oic 8]bradykinin) and NPC 567 ( d-Arg-[Hyp 3, d-Phe 7]bradykinin) antagonized competitively the response to des-Arg 9-bradykinin, giving respective pA 2 values of 6.82 ± 0.34, 6.63 ± 0.28 and 6.48 ± 0.41 whereas the non-peptide bradykinin B 2 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-Arg 9[Leu 8]bradykinin behaved as competitive antagonists towards the relaxation due to bradykinin. In conclusion, both bradykinin B 2 and B 1 receptors are present on the endothelium of large coronary arteries from juvenile pig. The bradykinin B 1 receptor subtype appears partly inducible and is coupled to the synthesis of nitric oxide.

Inhibition of bradykinin‐evoked trigeminal nerve stimulation by the non‐peptide bradykinin B2 receptor antagonist WIN 64338 in vivo and in vitro

1. This study investigated the effect of the recently described non-peptide bradykinin B2 receptor antagonist, WIN 64338 ([[4-[[2- [[bis(cyclohexylamino)methylene]amino]-3-(2-naphthalenyl)-1-oxopropyl] amino]phenyl]methyl]tributylphosphoniumchloride monohydrochloride), in experimental models of bradykinin-evoked sensory nerve stimulation. 2. In the rabbit isolated iris sphincter in vitro, bradykinin-evoked contractile responses are mediated via tachykinins released from peripheral endings of the trigeminal sensory nerve. WIN 64338 (1-10 microM) competitively antagonised contractile responses to bradykinin with a pKB estimate of 6.6 +/- 0.1 (n = 11). The antagonism was selective since WIN 64338 (10 microM) did not significantly inhibit submaximal contractile responses to the direct-acting spasmogens substance P (10 nM), neurokinin A (3 nM), substance P methyl ester (10 nM) or senktide (100 nM); nor by sensory non-adrenergic non-cholinergic nerve stimulation evoked by capsaicin (10 microM), or electrical field-stimulation (3, 10, 30 Hz) (P > 0.05; n = 3-11). 3. Topical application of bradykinin to the conjunctiva and to the nasal mucosa of the guinea-pig in vivo causes plasma extravasation predominantly via the release of tachykinins from peripheral endings of the trigeminal nerve. The increases in plasma extravasation (measured by extravasation of Evans blue dye) induced by bradykinin in the guinea-pig conjunctiva (20 nmol) and nasal mucosa (50 nmol) were markedly reduced (by 81 +/- 3% and 69 +/- 5%, respectively) following pretreatment with WIN 64338 (30 nmol kg-1, i.v.) (n = 5-6; P < 0.05), with almost complete inhibition at a higher dose of WIN 64338 (300 nmol kg-1, i.v.; n = 5-6). This inhibition was selective since at 300 nmol kg-1, WIN 64338 did not inhibit plasma extravasation evoked by substance P in the conjunctiva (5 nmol; P > 0.05; n = 6) or in the nasal mucosa (50 nmol; P > 0.05; n = 5). 4. This study demonstrates that WIN 64338 is a selective and competitive bradykinin B2 receptor antagonist and can be useful for analysing bradykinin-evoked trigeminal nerve stimulation both in vitro and in vivo.

Structure-Based Drug Design: Progress Toward the Discovery of the Elusive Bradykinin Receptor Antagonists

The de nova design of a non-peptide bradykinin receptor antagonist (or the optimization of a lead discovered by random screening) will ultimately require knowledge about the receptor topology. In the absence of an experientally-determined structure of the bradykinin-bradykinin receptor complex, we have attempted to gain insights from other sources. Specifically, we have synthesized conformationally constrained lignads, and completed extensive computer modeling and mutagenesis experiments on the bradykinin receptor. For the former, the recently reported hydroxyproline ether­ containing peptides have been useful in mapping the bradykinin receptor topology. Moreover, using systematic synthetic modifications, we have explored the relative importances of selected amide bonds 1 and side chains in second generation peptides and have made a series of and/or N-methyl 1 substitutions at positions four and five which led to the discovery of two new cyclic peptide antagonists. 1 For the latter, computational simulations led to a proposed model of bradykinin bound to its receptor which was found to be in good agreement with mutagens is results. This model led ultimately to the design and synthesis of D-Arg0-Arg 1 -(12- aminododecanoy1)2-Ser3-D-Tic4-0ic5-Arg6, a pseudo-peptide leading into a third generation of antagonists. Further improvements in tis lead structure were achieved via combinatorial non-peptide libraries.

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.