(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
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
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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
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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 pressure9 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 (Ki=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 human13 14 and rabbit3 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 receptors1 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.
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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 findings3 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).
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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).
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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).
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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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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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