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(Hypertension. 1999;33:823-829.)
© 1999 American Heart Association, Inc.

Scientific Contributions

Kinin B₁ Receptor Antagonists Containing -Methyl-L-Phenylalanine: In Vitro and In Vivo Antagonistic Activities

Fernand Gobeil, Jr; Stéphanie Charland; Catherine Filteau; Stéphan I. Perron; Witold Neugebauer; Domenico Regoli

From the Department of Pharmacology, Medical School, Université de Sherbrooke, Sherbrooke (Québec), Canada.

Correspondence to Dr Fernand Gobeil Jr, Department of Pharmacology, Medical School, Université de Sherbrooke, 3001 12th Ave North, Sherbrooke (Québec) J1H 5N4, Canada. E-mail fgobei01{at}courrier.usherb.ca

	Abstract

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Abstract—To protect from metabolism and to improve potency of the AcLys-[D-ßNal⁷,Ile⁸]desArg⁹-bradykinin (BK) (R 715), we prepared and tested 3 analogues containing -methyl-L-Phe ([Me]Phe) in position 5: these are the AcLys-[(Me)Phe⁵,D-ßNal⁷,Ile⁸]desArg⁹BK (R 892), Lys-Lys-[(Me)Phe⁵,D-ßNal⁷,Ile⁸]desArg⁹BK (R 913), and AcLys-Lys-[(Me)Phe⁵,D-ßNal⁷,Ile⁸]desArg⁹BK (R 914). The new compounds were tested against the contractile effect induced by desArg⁹BK on 2 B₁ receptor bioassays, the human umbilical vein, and the rabbit aorta. Their antagonistic activities were compared with those of the early prototypes (Lys-[Leu⁸]desArg⁹BK and [Leu⁸]desArg⁹BK) and with other recently described peptide antagonists. The 3 (Me)Phe analogues showed high antagonistic potencies (pA₂) at both the human (8.8, 7.7, and 8.7, respectively) and rabbit (8.6, 7.8, and 8.6, respectively) B₁ receptors. No antagonistic effects (pA₂<5) were observed on the B₂ receptors that mediate the contractile effects of BK on the human umbilical vein, the rabbit jugular vein, and the guinea pig ileum. Moreover, these new B₁ antagonists were found to be resistant to in vitro degradation by purified angiotensin-converting enzyme from rabbit lung. The N-acetylated forms, R 892 and R 914, were resistant to aminopeptidases from human plasma. In vivo antagonistic potencies (ID₅₀) of B₁ receptor antagonists were evaluated in anesthetized lipopolysaccharide-treated (for B₁ receptor) and nontreated (for B₂ receptor) rabbits against the hypotensive effects of exogenous desArg⁹BK and BK. R 892 efficiently inhibited (ID₅₀ 2.8 nmol/kg IV) hypotension induced by desArg⁹BK without affecting that evoked by BK (ID₅₀ >600 nmol/kg IV). Conversely, the peptide antagonists Lys-Lys-[Hyp³,Igl⁵,D-Igl⁷,Oic⁸]desArg⁹BK (B 9858) and DArg-[Hyp³,Thi⁵,D-Tic⁷,Oic⁸] desArg⁹BK (S 0765) showed dual B₁/B₂ receptor antagonism in vitro and in vivo. It is concluded that R 892 and congeners provide selective, highly potent, and metabolically stable B₁ kinin receptor antagonists that can be useful for the assessment of the physiological and pathological roles of kinin B₁ receptors.

Key Words: receptors, bradykinin • human • rabbit • bioassay • antagonists

	Introduction

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DesArg⁹-bradykinin (desArg⁹BK) and Lys-desArg⁹BK, 2 endogenous autacoids acting on the kinin B₁ receptor, have been proposed as factors involved in physiological and pathological processes.^{1 2} Their role has been studied by use of selective B₁ receptor antagonists that were obtained in the late 1970s through the replacement of the Phe in position 8 with a Leu (Lys-[Leu⁸]desArg⁹BK and [Leu⁸]desArg⁹BK).³ Through their extensive use during the past 20 years, a body of experimental evidence has been obtained to show the contribution of B₁ receptors in some experimental diseases such as hypertension, diabetes, septicemia, myocardial ischemia, and in inflammation.² However, these peptide antagonists show some limitations, in part because of their susceptibility to enzymatic degradation by various tissue and plasma peptidases; they also have putative residual agonistic activities. This latter property has been observed inasmuch in vitro (on smooth muscle myotropic assays: the mouse stomach fundus,⁴ the rat duodenum,⁵ the rat ileum,⁶ the rabbit stomach fundus, and the rat portal vein [F. Gobeil, personal observation]; on electrogenic assays: the dog colon mucosa,⁷ the rat colon⁸ ) as in vivo (eg, canine blood pressure^{9 10} ). Use of these peptidic compounds may be the source of inconclusive interpretations, especially when experiments are performed on whole animals, and point to the need of new pharmacological tools for studying B₁ receptor functions. Proteases such as the angiotensin-converting enzyme ([ACE] EC 3.4.15.1), the neutral endopeptidase 24.11 ([NEP] EC 3.4.24.11), and the aminopeptidases P ([AmP] EC 3.4.11.9) and M types ([AmM] EC 3.4.11.2) are thought to be the most efficient in vivo in the inactivation of peptides related to Lys-desArg⁹BK.¹¹ To date, little information is available on the physiological significance of AmM on lysyl-kinin metabolism. It has been found, however, that amastatin, the most potent inhibitor (K_i=25 nmol/L) of AmM, is able to increase the vasodepressor effect of Lys-desArg⁹BK in vivo.¹² The inactivating role of AmM appears to be important in humans; in fact, this enzyme is expected to convert lysyl-B₁ receptor agonists (eg, Lys-desArg⁹BK) as well as antagonists (eg, Lys-[Leu⁸]desArg⁹BK) into metabolites (devoid of the N-terminal Lys), which have been shown to be rather weak on the human^{13 14} and rabbit^{3 15 16} B₁ receptors. The major kinin degradation pathway is provided, however, by ACE (alias kininase II), which plays a predominant role in vivo.^{1 11} ACE acts also on B₁ receptor agonists and antagonists by releasing the C-terminal tripeptidyl (Ser-Pro-Phe [or Leu]) or tetrapeptidyl fragments (Phe-Ser-D-ßNal-Ile) from AcLys-[D-ßNal⁷,Ile⁸]desArg⁹BK (R 715) (C. Filteau, personal observation), a newly described B₁ receptor antagonist.^{16 17} Inhibition of ACE by captopril has been shown to increase the duration of action of B₁ receptor agonist (Lys-desArg⁹BK)¹² and antagonist (Lys-[Leu⁸]desArg⁹BK)¹⁸ in lipopolysaccharide (LPS)-treated rabbits. In the present study, R 715, a potent and selective B₁ antagonist,^{16 17} was taken as a template to design novel compounds containing an (Me)-L-Phe in position 5 and a protecting group (acetyl) at the N-terminal to prevent or reduce the degradation of the peptides by ACE and AmM. Indeed, [(Me)Phe⁵]BK and [(Me)Phe⁸]BK have been shown to be resistant to ACE¹⁹ , and acetylation of the N-terminal lysyl residue of Lys-[Leu⁸]desArg⁹BK is also a suitable modification against AmM inactivation.¹⁸ These new compounds were tested and compared with other B₁ receptor antagonists in biological in vitro and in vivo assays. Their degradation by human plasma and purified ACE was also investigated.

	Methods

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Peptides Synthesis
All peptides were assembled with a peptide synthesizer (Applied Biosystems 430 A) by use of Merrifield-type resins with the first amino acid attached. Amino acids were activated by dicyclohexylcarbodiimide/HOBT (Peptides International, Louisville, Ky) in 1-methyl-2-pyrrolidinone. Peptides were deprotected and cleaved from the resins with anhydrous hydrogen fluoride in the presence of appropriate scavengers. The resulting crude peptides were purified by medium-pressure reversed-phase (C₁₈) chromatography and, if necessary, by HPLC. Purity of the peptides was assessed by analytical HPLC, and their identity was confirmed by electrospray mass spectrometry (VG Quattro). Boc-L-(-methyl)phenylalanine was prepared by treating the corresponding amino acid with di-tert-butyl dicarbonate in the presence of tetramethylguanidine, according to Turk et al.²⁰

Biological Assays on Isolated Tissues
Tissues were taken from New Zealand White rabbits (1.5 to 2.5 kg) and Dunken Hartley guinea pigs (250 to 350 g) of either sex killed by stunning and exsanguination. Umbilical cords were taken from healthy women 22- to 35-years-old after spontaneous delivery at term. The care of animals and all research protocols conformed to the guiding principles for animal experimentation of the Canadian Council on Animal Care and were approved by the Ethical Committee on Animal Research of the Sherbrooke Medical School. Tissues used in the present study were prepared according to procedures previously described¹⁶ ; these are the rabbit jugular vein (rbJV), the rabbit aorta (rbA), the guinea pig ileum (gpI), and the human umbilical vein (hUV). All tissues were rapidly removed, cut into strips, cleaned of fat and connective tissues, mounted vertically in siliconized organ baths, and attached with stainless steel hooks between an anchor and the isometric tension transducer (model FT03C, Grass Instruments). The baths were filled with oxygenated (95% O₂–5% CO₂) and thermoregulated (37°C) Krebs solution (pH 7.4) of the following composition (mmol/L): NaCl 118.1, KCl 4.7, CaCl₂ · 6H₂O 2.5, KH₂PO₄ 1.2, MgSO₄ · 7H₂O 1.2, NaHCO₃ 25, and D-glucose 5.5. After an equilibration period of 60 to 90 minutes during which the tissues were repeatedly washed (every 15 to 20 minutes), contractions were induced with BK or desArg⁹BK, the reference agonists for the B₂ and the B₁ receptor, respectively. The effect of BK and desArg⁹BK was measured in the presence of antagonists applied 10 minutes earlier unless otherwise specified. On the assumption of competitive ligand-receptor interaction, antagonist affinities were estimated in terms of pA₂, the -Log₁₀ of the concentration (mol/L) of antagonist that reduces the effect of a double dose of agonist to that of a single dose.²¹ The potential residual agonistic activities of antagonists were determined by applying a high concentration (10 µg/mL) of each compound. Residual activities are expressed as a fraction of the maximum effects (^E) of BK and desArg⁹BK, respectively, on the B₂ or the B₁ receptor.

Peptide Degradation Assays
The metabolic stabilities of kinin-related peptides were evaluated in vitro by incubating the peptides in the presence of purified ACE from rabbit lungs (Sigma, St. Louis) or human plasma based on protocols previously described.^{16 18} Under the experimental conditions described below, the rates of enzymatic hydrolysis were directly proportional to the time of incubation and to the amount of enzyme preparation.

Assays with Purified ACE
Briefly, the enzymatic extract was dissolved in PBS (50 mmol/L, pH 7.5, containing 300 mmol/L NaCl and 10 µmol/L ZnCl₂; the final concentration of ACE was 45 µg/mL). Enzymatic velocities were calculated from the initial steady state after 0, 5, 15, 30, and 60 minutes of exposure at 37°C of individual kinin analogs (200 µmol/L, 32 to 50 µL) with ACE (8.5 µL) (total volume medium, 187.5 µL). The hydrolysis reaction was stopped by immersing samples into boiling water and then cooling them on ice. Separation of peptide substrates and their metabolites was achieved by reverse-phase HPLC on a C₁₈ µBondapak column (4.6 mmx25 cm) (Waters Associates) with a linear gradient of 5% to 65% of water/acetonitrile (both containing 0.05% TFA) at 2 mL/min over a period of 20 minutes. For degradation studies, 50 µL of each aliquot was injected, and rates of peptide metabolism were calculated from the decrease of peptide substrate concentration. The elution positions of these peptides were determined by following the absorbance at 214 nm (441 UV detector, Waters). Integration of peak areas and quantification of peptide substrate were made with a computer software program (Baseline 810, Waters).

Assays with Human Plasma
Blood (5 mL) was withdrawn by venipuncture from healthy volunteers (men and women, aged 20 to 30 years) and put into heparinized (200 U) tubes. The blood samples were centrifuged at 1500 rpm for 15 minutes in a refrigerated tabletop centrifuge. The peptides (200 µmol/L, 65 to 100 µL) were placed in a PBS buffer (50 mmol/L, pH 7.5, containing 300 mmol/L NaCl) and incubated for 5 minutes at 37°C with 50 µL of human plasma (total volume of the medium, 375 µL). Reaction was ended as described above. Previous experiments have shown that (1) 70% of the kininase activity contained in human plasma is abolished by amastatin (1 µmol/L, preincubated for 10 minutes), a bacterial peptide inhibitor of aminopeptidases and that (2) the electrospray mass spectrometry analysis performed on the degradation mixture of Lys-[Leu⁸]desArg⁹BK shows the presence of [Leu⁸]desArg⁹BK (not shown). Hence, in the experimental conditions used in the present study, the human plasma was considered to be a medium rich with AmM. The enzymatic assays and HPLC analysis of the peptide digest were carried out under the same conditions described for ACE.

In Vivo Experiments. Antagonistic Effects of Several Peptides on the Blood Pressure of Anesthetized Rabbits
Assays on B₁ receptors were performed on New Zealand White rabbits (1.3 to 1.6 kg, pathogen-free) of either sex pretreated with LPS for the in vivo induction of B₁ receptors^{1 2} (50 µg/kg IV) 5 hours before inducing the anesthesia with sodium pentobarbital (Abbott Laboratories) that was given initially at 30 mg/kg IV through the auricular vein and was supplemented when required.^{12 22} The following procedure was used for the in vivo experiments on kinin B₁ and B₂ receptors. Xylocaine (2%) was applied locally at the site of incision at the beginning of every surgical operation. The animals, placed in supine position, were artificially ventilated with room air (6 mL/kg, 50 strokes per minute) through an endotracheal tube (pericardial effusion [PE] 330) with a Harvard pump (model 683, Harvard Apparatus). A polyethylene catheter (PE 90) filled with heparin sodium (1000 U/mL) to prevent clotting was inserted in the right carotid artery and pushed into the aorta to monitor continuously the mean arterial blood pressure (MAP) with a transducer (model TDX-300, Micro-Med Inc) connected to a blood pressure analyzer (model BPA-100c, Micro-Med Inc). A second arterectomy was performed on the left carotid artery for bolus injection of kinin agonists (BK or desArg⁹BK) into the aorta. The jugular vein was also intubated (PE 50) for bolus injection of kinin B₁ receptor antagonists. Pharmacological agents were administered at 10- to 15-minute intervals. At the beginning of each experiment, an average of 10 to 20 minutes of equilibration time was allowed to ensure stabilization of the blood pressure. All drugs were diluted in sterile isotonic saline (0.9%) and administered as bolus injections (0.1 mL, 20 to 30 s) via a 3-way valve connector attached to the aorta catheter. After each injection, the catheter was flushed with 0.2 to 0.4 mL of saline.

Under these experimental conditions, the anesthetized LPS-treated rabbit showed MAP of 115±5 mm Hg, systolic pressure of 130±5 mm Hg, diastolic pressure of 100±5 mm Hg, and heart rate of 345±10 bpm. Similar hemodynamic basal values have been observed in untreated rabbits used for assays on B₂ receptors. To test antagonists, submaximal standard doses of desArg⁹BK (1 µg per animal) or BK (100 ng per animal) were administered repeatedly. No tachyphylaxis occurred. Each injection of desArg⁹BK and BK were followed by transient hypotensive episodes averaging -31±2 mm Hg (1 mm Hg=133.3 Pa) and -25±4 mm Hg (MAP), respectively, and lasting <2 minutes. The antagonists were then injected intravenously 2.5 minutes before the submaximal dose of agonist, which was given intra-arterially. The reduction of the agonist effect, observed in the presence of the antagonist, was expressed in percent of the control. At least 3 doses of antagonist were injected in each animal to calculate antagonist affinities that are expressed as ID₅₀ (the dose of antagonist that reduces by 50% the effect of the standard agonist dose). ID₅₀ values were obtained by extrapolation from a linear regression curve designed for each antagonist. The duration of action of the antagonists (tested at their ID₅₀ values) was estimated from the time intervals required for desArg⁹BK (for B₁ receptor study) or BK (for B₂ receptor study) (both injected every 10 minutes) to regain its full and original activity. Similar protocols have been used in the past to study the in vivo B₂ receptor antagonism of several pharmacological agents.²³

Drugs
Concentrated solutions (1 mg/mL) of peptides were made in bidistilled and deionized water and kept at -20°C until used. Abbreviations for amino acids follow the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature.²⁴ Other abbreviations are described as follows: Hyp, trans-4-hydroxy-L-proline; Thi, ß-(2-thienyl)-L-alanine; Tic, L-(1,2,3,4-tetrahydroisoquinoline-3- carboxylic acid; Oic, L-(3aS,7aS)-octahydro-indol-2-carboxylic acid; ßNal, ß-3-(2-naphthyl)-alanine; Igl, (2-indanyl)-glycine; and Cpg, cyclopentyl-glycine. All chemical agents were obtained from either Bachem or Novabiochem. Captopril was purchased from Squibb. The LPS extracted from Escherichia coli (serotype 0127:B8) was purchased from Difco Laboratories and dissolved at 50 µg/mL in isotonic saline.

Statistical Analysis
Data of in vitro functional experiments are expressed as mean±SEM. In vivo results are mean±SE. Statistical analysis of pA₂ values (for in vitro studies) and ID₅₀ values (for in vivo studies) were made with Student's t tests for unpaired data. Differences were considered significant at values of P<0.05.

	Results

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Pharmacological Characterization of B₁ Receptor Antagonists in Isolated Tissues
The abbreviated structures of the antagonists used in the present study are presented in Table 1. Three reference compounds were used: the antagonists of the first generation [Leu⁸]desArg⁹BK (R 733) and Lys-[Leu⁸]desArg⁹BK (R 732) described by Regoli et al³ and AcLys-[D-ßNal⁷,Ile⁸]-desArg⁹BK (R 715),^{16 17} which served as template for 3 novel analogues, namely, the AcLys-[(Me)Phe⁵,D-ßNal⁷, Ile⁸]desArg⁹BK (R 892), Lys-Lys-[(Me)Phe⁵,D-ßNal⁷, Ile⁸]desArg⁹BK (R 913), and AcLys-Lys-[(Me)Phe⁵,D-ßNal⁷,Ile⁸]desArg⁹BK (R 914). They were designed to improve antagonistic potency and metabolic stability by replacing the Phe in position 5 with an (Me)Phe and extending the N-terminal with Lys residues (acetylated or not). For comparative purpose, other peptidic compounds described by other investigators are included in Table 1. These are AcLys-[(^NMe)Ala⁶,Leu⁸]desArg⁹BK,¹⁸ D-Arg-[Hyp³,Thi⁵,D-Tic⁷, Oic⁸]desArg⁹BK (S 0765),²⁵ Lys-Lys-[Hyp³,Igl⁵,D-Igl⁷,Oic⁸]desArg⁹BK (B 9858),²⁶ and Lys-Lys-[Hyp³,Cpg⁵,D-Tic⁷,Cpg⁸]desArg⁹BK (B 9958).²⁶ Pharmacological activities were evaluated in 2 B₁ receptor bioassay systems, the hUV and the rbA, to estimate their potencies (pA₂) and putative residual agonistic activities (^E) of antagonists. Results presented in Table 1 indicate that (1) all compounds have per se no residual agonistic activities (^E=0) in the 2 preparations; (2) however, they show important differences in their antagonistic potencies; and (3) there is a good correlation between data obtained in the 2 preparations. Thus, R 733 is much weaker (by at least 50-fold) than R 732 at both the human and rabbit B₁ receptors, confirming early findings^{3 16} that point to the importance of a lysyl residue at the N-terminal. The N-acetylation of the N-terminal side conjointly with the replacement of the Pro⁷ and Phe⁸ by a D-ßNal and Ile, as in R 715, increases the antagonistic potency (by 3-fold on the human B₁ receptor), as already reported by Gobeil et al.¹⁶ An additional substitution of the Phe⁵ with an (Me)Phe as in peptide R 892 enhances further (by 2-fold) the antagonistic potency in the 2 B₁ receptor assays. Addition of a lysyl residue in the N-terminal of R 892, as in peptide R 914, does not increase the antagonistic potency further. The N-acetylation of (Me)Phe analogues is important because the nonacetylated form R 913 is 10-fold less potent than R 914. R 892 and R 914 are, indeed, the most potent B₁ receptor antagonists of this series. Further, R 892 (17 µmol/L) was found to be specific for kinins as it did not affect the myotropic responses of the rbA to angiotensin II (1 nmol/L) and noradrenalin (0.1 µmol/L) (data not shown). Interestingly, R 892 has also been found to be a selective and highly potent antagonist, devoid of any agonistic activity (^E=0), at the murine stomachal B₁ receptor (pA₂ 8.06±0.07, n=4; S. Nsa Allogho, personal communication). Other B₁ receptor antagonists reported in the literature were tested for comparison: the AcLys-[(^NMe)Ala⁶,Leu⁸]desArg⁹BK (peptide 7) showed weak affinities comparable to R 733. The desArg⁹ derivative of HOE 140, S 0765, showed pA₂ values ranging from 6.6 to 7.3. Like R 892, B 9858, an Igl-substituted analogue, showed comparable high antagonistic potencies in the 2 B₁ receptor functional assays (pA₂ values from 8.5 to 8.7). B 9958, a Cpg-containing peptide antagonist, was also found to be very active (pA₂ of 8.96) on the human B₁ receptor. With the exception of B 9858, the inhibitory effects of all antagonists are rapidly reversible (<15 minutes) after wash out, suggesting that they act in a competitive manner (not shown).

View this table: [in this window] [in a new window]   Table 1. In Vitro Antagonistic Potencies of Peptides on Human and Rabbit B1 Receptors

To assess their potential selectivities for the B₁ receptors, the antagonists presented in Table 1 were tested on 3 B₂ receptor bioassays, namely, hUV, rbJV, and gpI. The latter 2 preparations have been extensively used in our laboratory to classify B₂ receptor subtypes.²⁷ The results indicate that peptides 1 to 6 do not exert antagonistic (pA₂<5.0) or agonistic (^E=0) activities at the human and rabbit B₂ receptors (not shown). Some compounds show moderate B₂ receptor antagonism, for instance, S 0765 and B 9858 that are able to block the myotropic responses elicited by BK in the rabbit vascular tissue with pA₂ values of 7.50±0.10 and 6.7±0.09, respectively. B 9858 also impeded (pA₂ 5.56±0.12, n=4) the response elicited by BK in the gpI. Applied in high concentrations (10 µmol/L), antagonists such as R 715 and B 9958 have strong stimulating contractile effects (^E 0.4 and 0.6, respectively) in the guinea pig ileal preparation. The nature of these stimulatory effects remains unknown. In fact, the effects remain unchanged in tissues pretreated with HOE 140 (8 µmol/L), a potent B₂ receptor antagonist (data not shown).

Degradation of Antagonists by Human Plasma and Purified ACE
All peptide antagonists were incubated in vitro with human plasma or with purified ACE from rabbit lung to evaluate their metabolic stability (see Methods). The results summarized in Table 2 indicate that R 733 is broken down by ACE but not by human plasma, whereas R 732 is sensitive to both enzyme preparations, especially to ACE. In agreement with data reported by Drapeau et al,^{18 28} R 733 is catabolized twice more rapidly than R 732. ACE is also able to hydrolyze R 715 although at a slower rate, whereas R 892 and R 914 are resistant to both enzymes. R 913 is slowly degraded by AmM. Peptides S 0765 and AcLys-[(^NMe)Ala⁶,Leu⁸]desArg⁹BK are completely resistant to degradation, whereas B 9858 and B 9958 are sensitive to plasma AmM. In complementary experiments, R 892 (0.1 mmol/L) (preincubated for 10 minutes with ACE) was also tested as an inhibitor of ACE and was found not to hamper (IC₅₀ 20% of ACE activity) the degradation of desArg⁹BK by this enzyme (C. Filteau, personal observation).

View this table: [in this window] [in a new window]   Table 2. Metabolic Rate of Peptide Antagonists Incubated With Human Plasma and Purified ACE

In Vivo Pharmacological Assays of B₁ Receptor Antagonists on Rabbit Blood Pressure
All peptide antagonists were evaluated in vivo against the hypotensive responses induced by exogenous desArg⁹BK (in LPS-treated animals) or BK (in nontreated animals) (see Table 3). All B₁ receptor antagonists administered intravenously showed dose-dependent inhibition of the hypotensive effect of desArg⁹BK (not shown). Results indicate that R 733 is a weak antagonist at the B₁ receptor (ID₅₀ 491±74 nmoL/kg) and is inactive (ID₅₀ >790 nmoL/kg) when tested against BK. R 732 is a selective B₁ receptor antagonist whose potency is in the same range as those of R 715, R 913, and B 9958. Worth mentioning is the high in vivo antagonistic potencies of R 892, R 914, and B 9858, the most potent B₁ receptor inhibitory agents in the present study. S 0765 and B 9858 exhibited in vivo antagonistic activities at the B₂ receptor in accord with the results obtained in vitro (see Table 1). Despite their in vitro resistance to AmM and ACE degradation (see Table 2), the AcLys-[(^NMe)Ala⁶, Leu⁸]desArg⁹BK, S 0765, R 715, R 892, and R 914, along with the other labile compounds, did not show prolonged effects in vivo; hypotensive responses induced by desArg⁹BK or BK could be fully recovered within a time period of 10 to 20 minutes (data not shown). With the exception of R 733 (measured at its ID₅₀, -20±5 mm Hg, n=3), all antagonists tested were devoid of residual agonistic activity on rabbit blood pressure (not shown).

View this table: [in this window] [in a new window]   Table 3. In Vivo Antagonistic Activities of Kinin B1 Receptor Antagonists in Anesthesized Rabbits

	Discussion

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As emphasized by Fauchère and Thuriau,²⁹ "... the development of peptide-based drugs for basic and clinical research is strongly impaired by their susceptibility to proteolysis." Several chemical ways are available to prevent peptide metabolism, such as substitution of natural residues by D-amino acids, introduction of peptide-bound surrogates, or partial or end-to-end cyclization; one can surmise that the resulting modified peptides will perhaps display altered conformations and unexpected pharmacological properties.²⁹ In the present study, 3 novel (Me)Phe⁵ analogues of R 715 were prepared in an attempt to prevent their degradation by kininase II while preserving high affinities for the kinin B₁ receptors. Indeed, peptides in which a methyl group has been substituted for the hydrogen on an -carbon have restricted rotational mobility around the adjacent bounds and are less susceptible to proteolytic degradation.¹⁹ Use of a C-methylated phenyl residue is also known to induce ß-turn and helix in the peptide backbone.³⁰ This chemical modification has already been used successfully in the past to identify kinin receptor agonists (eg, [(Me)Phe⁵]-BK and [(Me)Phe⁸]-BK) that were found to be resistant to ACE in vivo.¹⁹ Some of these (Me)Phe⁵-substituted kinin analogues were also protected at the N-terminal with a N-acetyl lysyl group (eg, R 892 and R 914) to prevent the hydrolytic activity of AmM.¹² Since it is now well established that the presence of a lysyl residue at the N-terminal greatly optimizes the biological activities of agonists and antagonists for the human B₁ receptor,^{13 14} any modification made to prevent the activity of AmM should be compatible with full pharmacological activity. The addition of a second lysyl residue was also made on peptides R 913 and R 914 on the basis of previous reports indicating that an N-terminal extension by 2 lysyl residues on desArg⁹BK derivatives is conducive to an increased affinity of antagonists for the B₁ receptors as well as affording some resistance to ACE hydrolysis.^{28 31}

The results from bioassays show the critical role of AcLys for B₁ receptor antagonism since peptide R 914 shows higher affinities (by 1 log unit) than peptide R 913 in both the human and rabbit B₁ receptors (see Table 1). Extension of the N-terminal end by another basic lysyl residue is not favorable to achieve higher antagonistic potency (compare potencies of R 892 and R 914). The present results confirm and further validate previous data obtained with a series of R 715 analogues in which the N-terminal residues DArg, DLys, Sar, Lys, or AcLys were used.¹⁶ Again, the most potent antagonist was the N-acetylated derivative R 715, AcLys-[D-ßNal⁷,Ile⁸] desArg⁹BK.¹⁶ Improvement of the biological activities (by 2-fold) of R 715 on the human and rabbit B₁ receptors was obtained by substituting the Phe with an (Me)Phe in position 5. R 892 is the most active antagonist at both the human and rabbit B₁ receptors. Interestingly, R 892 also shows a high affinity (pA₂ value of 8.06) without any intrinsic agonistic activity (^E=0) on the B₁ receptor of the mouse (S. Nsa Allogho, personal communication), a species in which the classical B₁ receptor antagonists R 732 and R 733 cannot be used as antagonists because of their strong agonistic activities (^E values of 0.70 and 0.64, respectively).⁴ Therefore, R 892 can be considered a pure B₁ receptor antagonist and can be recommended for use in the mouse and possibly in other species (eg, the rat) that possess a B₁ receptor subtype whose major features are a high sensitivity to desArg⁹BK and the partial agonistic character of the classical B₁ antagonists. Antagonistic potency of R 892 is comparable to those of B 9858 and B 9958 designed by Stewart et al²⁶ who used a variety of unnatural amino acids such as Igl, Tic, Oic, and Cpg conjugated with a Lys-Lys extension of the N-terminal. However, B 9858 maintains some antagonistic activity at the B₂ receptor of the rabbit, and B 9958 displays a fairly high contractile activity of unknown nature on the gpI. Peptide S 0765, the desArg⁹ derivative of HOE 140, is a weak agent and nonselective since it acts as an antagonist on the rabbit B₂ receptor both in vitro and in vivo (see Tables 1 and 3). Peptide 6, in which the Ser in position 6 was replaced with (^NMe)Ala to prevent degradation by ACE, is a poor B₁ receptor antagonist in humans, at least in vitro, both in functional (present study) and binding assays.³²

The presence of an (Me)Phe in position 5 and of an AcLys at the N-terminal end prevents degradation by two of the most active peptidases, ACE and AmM, which are widely distributed in the plasma membrane of various cells,¹¹ the most favorable location to inactivate cationic peptides like the kinins, which cannot cross the cell membrane. Complete protection from ACE is obtained with R 892 in which the sole structural difference from R 715 is represented by the (Me)Phe substitution in position 5. Protection from AmM through AcLys is also evident when comparing R 892 with R 913 as well as with B 9858 and B 9958, all of which have a Lys-Lys at the N-terminal (see Table 2). From the above analysis, R 892 emerges as the compound of choice for antagonizing the B₁ receptor because (1) it is a potent agent (even in the mouse); (2) it is selective since it does not interact with either the rabbit or the guinea pig B₂ receptors; (3) it is resistant to degradation by ACE and plasma AmM; and, finally, (4) it is completely devoid of any intrinsic agonistic activities in all contractile B₁ and B₂ receptor assays used (even for the mouse B₁ receptor), in contrast with other potent antagonists such as R 715, B 9858, and B 9958. When tested in vivo, R 892 was found to be potent and selective for the B₁ receptor, similar to R 732, R 715, R 913, and R 914. Increase of potency in vivo for R 892, however, has been lower than expected despite the demonstration that this compound is less inactivated than the others (see Table 2). When we tried to estimate the duration of action in vivo (with submaximal doses; ID₅₀ values), we found little difference (perhaps 5 to 10 minutes) between R 732 that is not protected, R 715 that is partially protected, and R 892 that is completely resistant to AmM and ACE (not shown). These observations are in agreement with those of Drapeau et al¹⁸ who used other stable B₁ receptor antagonist derivatives (eg, AcLys[(^NMe)Ala⁶, Leu⁸]desArg⁹BK) whose actions were found to last <30 minutes in vivo. Hence, resistance to ACE and AmM appears to play little role, if any, for prolonging in vivo activity of B₁ receptor antagonists, at least in this septicemic rabbit model. Obviously, other enzymes (eg, AmP, NEP, endopeptidase 24.15) in addition to those investigated herein may intervene in the metabolism of B₁ receptor antagonists. Our preliminary results have, however, demonstrated that (Me)Phe-substituted peptides R 892 and R 914, in contrast to R 715, are also resistant to degradation by the NEP 24.11 from rabbit kidney extract¹⁸ (unpublished data). In contrast, a peptide such as HOE 140 has been found to exert B₂ receptor antagonism for >60 minutes in the rat,³³ 90 minutes in the rabbit,²³ and 90 minutes in the guinea pig²³ when given intra-arterially; such a prolonged effect has been attributed to the noncompetitive interaction exerted by HOE 140 at the B₂ receptors of several species.^{23 34} HOE 140 (containing a large hydrophobic moiety [D-Tic, Oic] in its C-terminal part) probably binds strongly and dissociates slowly from the B₂ receptor, thus acting as a noncompetitive antagonist and exerting a long-lasting antagonism.^{23 35} Conversely, the desArg⁹BK variant of HOE 140, S 0765, has been described as a competitive antagonist in vitro (at both the B₁ and B₂ receptors)^{27 35} with low persistent action in vivo (<20 minutes) (present study). According to Jarnagin et al,³⁶ the type-II ß-turn orientation between residues 6 and 9 of HOE 140 is believed to be important in the B₂ receptor bond conformation of this peptide. This strengthened conformation of HOE 140 may be partially lost when the arginyl residue in position 9 is missing as in the desArg⁹-HOE 140 derivative, S 0765.³⁶ Such a difference suggests that kinin peptide analogues (such as those described in the present study) that act as competitive antagonists dissociate rapidly from the receptors (antagonists are not internalized) and may be rapidly excreted into the urine. From the above, it emerges that a prolonged receptor occupation plays an instrumental role in the duration of action of kinin receptor antagonists. Therefore, we conclude that peptide B₁ receptor antagonists should be designed to achieve noncompetitive antagonism and long-lasting effect in vivo. Indeed, under the same experimental condition in anesthetized rabbits, HOE 140 harbors a duration of action in vivo that is significantly longer (>90 minutes)³⁷ than even that of the nonpeptide and competitive³⁸ B₂ receptor antagonist FR 173657 (20 minutes).³⁷

	Acknowledgments

 
This study was supported by the Heart and Stroke Foundation of Canada (HSFC) and the Medical Research Council of Canada (MRCC). F. Gobeil, Jr, holds a studentship from the HSFC, and Dr D. Regoli is a Career Investigator of the MRCC. The authors thank Dr John M. Stewart and Dr Eric T. Whalley for their generous gifts of compounds coded B 9958 and B 9858. The peptide AcLys-[(^NMe)Ala⁶,Leu⁸]desArg⁹BK was kindly donated by Dr Guy Drapeau (Center de Recherche Hôtel-Dieu de Québec, Canada). The authors also thank the nursing and resident staffs of the Obstetric Department of the Université de Sherbrooke for providing the umbilical cords.

Received August 31, 1998; first decision September 22, 1998; accepted November 13, 1998.

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