Sanofi-Synthélabo Recherche, Montpellier, France (X.E.-A.,
V.P., R.S., F.O-D., X.V., P.V., G.L., J.-P.M., P.S., M.P.) and
Faculté de Médecine Paris-Ouest, Paris, France (E.N., S.D.,
C.A.)
The biochemical and pharmacological properties of a novel
antagonist of the tachykinin neurokinin 1 (NK1) receptor,
SSR240600 [(R)-2-(1-{2-[4-{2-[3,5-bis(trifluoromethyl)phenyl]acetyl}-2-(3,4-dichlorophenyl)-2-morpholinyl]ethyl}-4-piperidinyl)-2-methylpropanamide], were evaluated. SSR240600 inhibited the binding of radioactive substance P to tachykinin NK1 receptors in human
lymphoblastic IM9 cells (Ki = 0.0061 nM), human astrocytoma U373MG cells (Ki = 0.10 nM), and human brain cortex (IC50 = 0.017 nM).
It also showed subnanomolar affinity for guinea pig NK1
receptors but was less potent on rat and gerbil NK1
receptors. SSR240600 inhibited [Sar9,Met(O2)11]substance
P-induced inositol monophosphate formation in human astrocytoma U373MG
cells with an IC50 value of 0.66 nM (agonist concentration
of 100 nM). It also antagonized substance P-induced contractions of
isolated human small bronchi with a pIC50 value of 8.6 (agonist concentration of 100 nM). The compound was >100- to 1000-fold
more selective for tachykinin NK1 receptors versus tachykinin NK2 or NK3 receptors as evaluated in
binding and in vitro functional assays. In vivo antagonistic activity
of SSR240600 was demonstrated on tachykinin NK1
receptor-mediated hypotension in dogs (3 and 10 µg/kg i.v.),
microvascular leakage (1 and 3 mg/kg i.p.), and bronchoconstriction (50 and 100 µg/kg i.v.) in guinea pigs. It also prevented citric
acid-induced cough in guinea pigs (1-10 mg/kg i.p.), an animal model
in which central endogenous tachykinins are suspected to play a major
role. In conclusion, SSR240600 is a new, potent, and centrally active
antagonist of the tachykinin NK1 receptor, able to
antagonize various NK1 receptor-mediated pharmacological
effects in the periphery and in the central nervous system.
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Introduction |
Substance
P belongs to a group of related neuropeptides named tachykinins, which
includes neurokinin A and neurokinin B. These peptides are widely
distributed in the peripheral and central nervous systems where they
exert various biological actions as neuromodulators or
neurotransmitters. Biological activities of tachykinins are mediated by
three different, but related, G-protein-coupled receptors with seven
-helical transmembrane segments, denoted NK1,
NK2, and NK3. Substance P
is the natural endogenous ligand of tachykinin
NK1 receptors, whereas neurokinin A and
neurokinin B are the preferential ligands of tachykinin
NK2 and NK3 receptors, respectively (Regoli et al., 1994
; Maggi, 1995; Quartara and Maggi, 1997).
Over the past several years, potent nonpeptide antagonists, selective
for the different tachykinin receptors, have been described and have
provided tools to investigate the physiopathological role of
tachykinins and their receptors both in the periphery and in the
central nervous system (Regoli et al., 1994; Quartara and Maggi, 1997,
1998; Lagente and Advenier, 1998; Stout et al., 2001). Based on the
more recent pharmacological data, confirmed by preliminary clinical
trials, it has emerged that blockade of tachykinin
NK1 receptors may provide a novel treatment of
major depression (Kramer et al., 1998; Rupniak and Kramer, 1999) and emesis (Rupniak and Kramer, 1999; Diemunsch and Grélot, 2000). These activities of tachykinin NK1 receptor
antagonists are essentially dependent on their ability to penetrate the
brain (Rupniak et al., 1997; Kramer et al., 1998; Diemunsch
and Grélot, 2000). We now describe some general biochemical and
pharmacological activities of a novel nonpeptide tachykinin
NK1 receptor antagonist, SSR240600 [(R)-2-(1-{2-[4-{2-[3,5-bis(trifluoromethyl)phenyl]acetyl}-2-(3,4-dichlorophenyl)-2-morpholinyl]ethyl}-4-piperidinyl)-2-methylpropanamide] (Fig. 1). Its activity in various tests
predictive of an antidepressant activity is described in the
accompanying paper (Steinberg et al., 2002).
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Fig. 1.
Structure of SSR240600
[(R)-2-(1-{2-[4-{2-[3,5-bis(trifluoromethyl)phenyl]acetyl}-2-(3,4-dichlorophenyl)-2-morpholinyl]ethyl}-4-piperidinyl)-2-methylpropanamide].
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Materials and Methods |
Binding Assays.
The affinity of SSR240600 for tachykinin
receptors was evaluated in several receptor-radioligand binding assays:
1) binding of [125I]Bolton-Hunter
region-labeled substance P to tachykinin NK1
receptors of rat cortex, guinea pig, and gerbil ileum, human
lymphoblast cells (IM9), and human astrocytoma cells (U373MG, STTG1);
2) binding of [125I]iodohistidyl-neurokinin A
(or [125I]neuropeptide 
) to tachykinin
NK2 receptors of rat or hamster urinary bladder
or guinea pig ileum as well as to human receptors, stably expressed in
CHO cells; and 3) binding of
[125I]iodohistidyl-[MePhe7]neurokinin
B (or [125I]eledoisin) to rat, guinea pig, and
gerbil brain cortex tachykinin NK3 receptors and
human NK3 receptor, cloned and stably expressed in CHO cells. All these binding assays were conducted and analyzed as
previously described in detail (Emonds-Alt et al., 1993, 1995, 1997).
The affinity of SSR240600 for tachykinin receptors was also
investigated on the binding of [3H]substance P
to tachykinin NK1 receptors of human brain
cortex. Brain cortex was obtained from a 49-year-old man, 48 h
after death due to pulmonary edema. Tissue was homogenized at 4°C in
50 mM Tris-HCl buffer, pH 7.4, containing 5 mM KCl and 10 mM EDTA. The homogenate was centrifuged at 28,000g for 15 min at 4°C.
The pellet was homogenized and incubated for 30 min at 4°C in 50 mM
Tris-HCl buffer, pH 7.4, containing 300 mM KCl and 10 mM EDTA. This
homogenate was centrifuged at 40,000g for 15 min and the
pellet was suspended in 50 mM Tris-HCl buffer, pH 7.4. Membranes were
stored at 
20°C until use. Before use in binding assays, the
membranes were diluted at 4°C in 50 mM Tris-HCl buffer, pH 7.4, and
centrifuged at 40,000g for 15 min. The final pellet was
suspended in 50 mM Tris-HCl buffer, pH 7.4, containing 0.2 mg/ml bovine
serum albumin, 40 µg/ml bacitracin, 4 µg/ml leupeptin, 4 µg/ml
chymostatin, and 3 mM MnCl2. Binding assays were
conducted in "low binding" tubes (NUNC A/S, Roskilde, Denmark).
Human brain cortex membranes (10 mg) and
[3H]substance P (0.5 nM) in 500 µl of assay
buffer (50 mM Tris-HCl buffer, pH 7.4, containing 0.2 mg/ml bovine
serum albumin, 40 µg/ml bacitracin, 4 µg/ml leupeptin, 4 µg/ml
chymostatin, 3 mM MnCl2) were incubated at 25°C
for 60 min with various concentrations of SSR240600. At the end of the
incubation, separation of bound and free ligand was done after dilution
[3 ml of cold (4°C) 50 mM Tris-HCl buffer, pH 7.4, containing 0.2 mg/ml bovine serum albumin] and rapid filtration on Whatman
(Maidstone, Kent, UK) GF/C filters pretreated with 50 mM Tris-HCl
buffer, pH 7.4, containing 0.2 mg/ml bovine serum albumin and 0.25%
polyethylenimine. The filters were washed three times at 4°C with 50 mM Tris-HCl buffer, pH 7.4, containing 0.2 mg/ml bovine serum albumin.
The radioactivity was counted in a 
liquid scintillation counter.
Specific binding was determined by subtraction of the nonspecific
binding, which was determined in the presence of 1 µM unlabeled
[Sar9,Met(O2)11]substance P.
In addition, the selectivity of SSR240600 was evaluated in a large
variety of ion channel- and receptor-binding assays as well as enzyme
assays. This was performed by MDS Panlabs Pharmacology Services
(Bothell, WA) and Cerep (Celles L'Evescault, France).
In Vitro Functional Assays.
Antagonistic activity of
SSR240600 was first determined by measuring inhibition of inositol
phosphate-1 formation elicited by tachykinin NK1
receptor activation with specific agonists
([Sar9,Met(O2)11]substance
P, septide, and GR73632) in human astrocytoma U373MG cells. The
effect of SSR240600 on inositol phosphate-1 formation was also measured
using CHO cells stably expressing either human tachykinin
NK2 or NK3 receptor in
response to [Nle10]neurokinin A-(4-10) or
[MePhe7]neurokinin B, respectively. All these
assays were conducted as previously described in detail (Oury-Donat et
al., 1994, 1995).
The in vitro pharmacological profile of SSR240600 was then
investigated by using several functional bioassays specific for the
three tachykinin receptor subtypes (Regoli et al., 1994): [Sar9,Met(O2)11]substance
P-induced endothelium-dependent relaxation of rabbit pulmonary artery,
precontracted by 0.1 µM norepinephrine (specific for
NK1 receptors),
[Ala8]neurokinin A-(4-10)-induced
contractions of endothelium-denuded rabbit pulmonary artery (specific
for NK2 receptors), and
[MePhe7]neurokinin B-induced contractions of
guinea pig ileum (specific for NK3 receptors).
All these assays were conducted and analyzed as previously described in
detail (Emonds-Alt et al., 1993). As already reported and discussed for
other nonpeptide antagonists of the tachykinin
NK1, NK2, and
NK3 receptors (Emonds-Alt et al., 1993),
preliminary experiments have indicated that full activity of SSR240600
was only observed after prolonged incubation with the tissue.
Therefore, SSR240600 was added 120 min before the agonist in all experiments.
Finally, SSR240600 antagonist activity for tachykinin
NK1 receptors was evaluated by measuring
inhibition of
[Sar9,Met(O2)11]substance
P-induced contractions of human isolated small bronchi (diameter <1
mm) as described by Naline et al. (1996). Bronchial tissues were
removed from patients (25 men, previous smokers, mean age 64 ± 2 years) with lung cancer at the time of the surgical operation. Just
after resection, segments of bronchi with an inner diameter of 0.5 to 1 mm were taken from an area as far as possible from the malignancy and
stored overnight at 4°C in Krebs-Henseleit solution.
In Vivo Assays.
All protocols have been approved by the
Comité d'Expérimentation Animale (Animal Care and Use
Committee) of Sanofi-Synthélabo Recherche and are in accordance
with the principles of the Declaration of Helsinki. The in vivo
pharmacological profile of SSR240600 was investigated in three animal
models in which the role of tachykinin NK1
receptor has been well characterized: hypotension, bronchoconstriction, and plasma extravasation induced by substance P or specific agonists of
the tachykinin NK1 receptor (Regoli et al., 1994;
Quartara and Maggi, 1998). Furthermore, the activity of SSR240600 was
studied in a model of cough provoked by inhalation of citric acid,
where endogenous tachykinins and their receptors play an important role (Advenier et al., 1993; Ujiie et al., 1993; Girard et al., 1995; Yasumitsu et al., 1996; Daoui et al., 1998).
Hypotension in Dogs.
Mongrel dogs of either sex (10-20 kg)
were anesthetized with sodium pentobarbital (30 mg/kg by intravenous
route), and the anesthetic was infused throughout the experiments at a
rate of 5 mg/kg per hour. The animals were intubated with an
endotracheal cannula and allowed to breathe spontaneously. After an
equilibration period,
[Sar9,Met(O2)11]substance
P (5 ng/kg) was repeatedly injected via the femoral vein at 15-min
intervals before and after intravenous or intraduodenal administration
of SSR240600. Mean blood pressure was calculated on the basis of
systolic and diastolic blood pressure values recorded with a Honeywell
PC 156 transducer at the carotid artery. In control experiments,
repeated injections of
[Sar9,Met(O2)11]substance
P (5 ng/kg) produced a reproducible hypotension of 30 to 40 mm Hg
(Emonds-Alt et al., 1993).
Bronchoconstriction in Guinea Pigs.
Male tricolored guinea
pigs (200-250 g) were anesthetized with urethane (1.25 g/kg)
administered by the intraperitoneal route and were pretreated with
atropine (0.5 mg/kg), diphenhydramine (1 mg/kg), and indomethacin (2 mg/kg) injected intravenously. Bronchoconstriction was induced with
GR73632 (a tachykinin NK1 receptor agonist) (5 ng/kg), administered intravenously at 20-min intervals before and after
intravenous administration of SSR240600. In control experiments,
repeated injections of GR73632 produced a reproducible
bronchoconstriction. Bronchoconstriction, quantified as a reduction of
tidal volume, was evaluated according to a modified Konzett-Rössler method (Emonds-Alt et al., 1993).
Plasma Extravasation in Guinea Pigs.
Tricolored, male or
female guinea pigs (250-400 g) were anesthetized with urethane (1.25 g/kg by the intraperitoneal route) and prepared for cannulation of the
jugular vein. SSR240600 was administered by the intraperitoneal route
30 min before intravenous injection of Evans blue dye (30 mg/kg), used
as a marker for plasma extravasation. One minute later, plasma
extravasation was provoked by intravenous administration of substance P
(0.3 µg/kg). The animals were killed 5 min later, and tissues
(trachea, main bronchi, esophagus, urinary bladder) were removed and
weighed. Evans blue dye was extracted by incubating the tissues in
formamide at 60°C for 18 h and measured photometrically. Plasma
extravasation was expressed as nanograms of dye per milligram
wet-weight tissue (Qian et al., 1993).
Citric Acid-Induced Cough in Guinea Pigs.
Tricolored, awake,
unrestrained male or female guinea pigs (250-400 g) were placed in a
body plethysmograph. They were then exposed for 10 min to an aerosol of
either an aqueous solution of citric acid (0.4 M) or saline as a
control. SSR240600 was administered by the intraperitoneal route at
various times before the aerosol challenge. Coughs were counted
by a trained observer, and recognized by the characteristic animal
posture and the pressure variation in the body plethysmograph (Advenier
et al., 1993; Girard et al., 1995; Daoui et al., 1998).
Chemicals.
SSR240600 (Fig. 1) was synthesized at
Sanofi-Synthélabo Recherche and was used as its hydrochloride
salt. It was dissolved in organic solvents (ethanol or dimethyl
sulfoxide) and diluted in distilled water when interference from
organic solvents was observed. For oral and intraduodenal
administration, it was suspended in water with 0.6%
carboxymethylcellulose. Radioactive ligands were purchased from
Amersham Biosciences Inc. (Les Ulis, France) and PerkinElmer Life
Sciences (Paris, France). All peptides were obtained from Bachem
(Bubendorf, Switzerland), and were dissolved in organic solvents
(ethanol or dimethyl sulfoxide) and then diluted in water.
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Results |
Binding Studies.
The inhibition constants
(Ki) for SSR240600 obtained in the
different binding assays for tachykinin receptors are shown in Table
1. SSR240600 inhibited the binding of
radioactive substance P to tachykinin NK1
receptors with subnanomolar Ki values,
using established human cell lines as well as human brain membranes. SSR240600 also displayed a high affinity for tachykinin
NK1 receptors from various animal species,
especially guinea pigs. In binding assays for tachykinin
NK2 and NK3 receptors,
SSR240600 slightly interfered with the binding of their respective
ligands, with Ki values always above
10 nM in all species studied, including human. Finally, SSR240600 was
assayed in 100 (mainly human) receptor-binding, ion channel-binding,
and enzyme assays including adenosine (A1, A2A, A3), adrenergic
(1, 2,
1, 2) dopamine (D1,
D2), nicotinic, muscarinic (M1,
M2, M3,
M4, M5), opiate (µ, 
,
, opioid receptor-like receptor 1) serotonin
(5-HT1A, 5-HT-2A,
5-HT2C, 5-HT3,
5-HT5A, 5-HT6,
5-HT7), angiotensin (AT1,
AT2), bradykinin (B1, B2), calcitonin gene-related peptide, cholecystokinin (CCK1,
CCK2), corticotropin-releasing factor
(CRF1, CRF2), galanin
(GAL1, GAL2), neurotensin
(NT1), vasopressin (V1A),
hormones (glucocorticoid, estrogen, progesterone, testosterone), ion
channels (sodium, calcium, potassium and chloride), cyclooxygenases
(COX1, COX2),
phosphodiesterases (III and IV), acetylcholinesterase. SSR240600, at
concentrations up to 1 µM, was inactive (inhibition less than 50%),
except in 
receptor (IC50 = 0.21 µM) and
sodium channel site 2 (IC50 = 0.18 µM) assays (data not shown).
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TABLE 1
Inhibition constants (Ki) of SSR240600 in
radioligand binding assays for tachykinin receptors
Radioligands: [125I]Bolton-Hunter-labeled substance P
for NK1 receptors, except for human brain cortex where
[3H]substance P was used;
[125I]iodohistidyl-neurokinin A for NK2
receptors, except for guinea-pig ileum where
[125I]neuropeptide was used;
[125I]iodohistidyl-[MePhe7]neurokinin B
for NK3 receptors, except for rat brain cortex where
[125I]eledoisin was used. Tachykinins: substance P,
neurokinin A, and [MePhe7]neurokinin B, respectively,
for NK1, NK2, and NK3 receptors,
except for human brain cortex where
[Sar9,Met(O2)11]substance P was
used. Values are means ± S.D. obtained from at least three
independent experiments performed in triplicate.
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In Vitro Functional Studies.
Antagonistic activity of
SSR240600 at human tachykinin NK1 receptors was
studied by measuring inhibition of inositol phosphate-1 formation
provoked by NK1 receptor activation in human
astrocytoma U373MG cells. Activation of tachykinin
NK1 receptors in U373MG cells by three different
specific agonists
([Sar9,Met(O2)11]substance
P, septide, and GR73632) provoked the formation of inositol
phosphate-1, which was concentration dependently inhibited by SSR240600
regardless of the agonist used. In contrast, SSR240600 showed a much
lower potency to block inositol phosphate-1 formation following the
activation of human tachykinin NK2 and
NK3 receptors stably expressed in CHO cells. The
IC50 values are given in Table 2.
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TABLE 2
Inhibition by SSR240600 of tachykinin receptor-mediated inositol
phosphate-1 formation in human astrocytoma U373MG cells
(NK1 receptors) and in CHO cells expressing either human
NK2 or NK3 receptors
Results are given as IC50 values. Values are means ± S.E.M. from at least three independent experiments performed in
triplicate.
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In a classical tachykinin NK1 receptor
assay using isolated tissues,
[Sar9,Met(O2)11]substance
P-induced endothelium-dependent relaxation of rabbit pulmonary artery
previously contracted by norepinephrine, SSR240600 produced a
concentration-dependent rightward shift of the concentration-response curves of
[Sar9,Met(O2)11]substance
P (Fig. 2A). However, SSR240600 also
induced a reduction of the maximal response to the agonist, suggesting
that SSR240600 antagonism was not purely competitive. The slope of the
Schild plot (0.65) was significantly different from unity and the
apparent affinity of SSR240600 was thus calculated in terms of
pD'2 (negative logarithm of the molar
concentration of antagonist that produces a 50% reduction of the
maximal response to the agonist). The
pD'2 value was 8.67 ± 0.08 (n = 18). The activity of SSR240600 was then examined
on tissue preparations containing tachykinin NK2 and NK3 receptors. At concentrations up to 0.1 µM, it had no effect in bioassays for NK2
([Ala8]neurokinin A-induced contractions of
endothelium-deprived rabbit pulmonary artery) (Fig. 2B) or
NK3 ([MePhe7]neurokinin
B-induced contractions of guinea pig ileum) (Fig. 2C) receptors. At a
concentration of 1 µM, SSR240600 produced a rightward shift of the
agonist concentration-response curve in the two bioassays, with a
reduction of maximal response to the agonist in bioassay for
NK3 receptors. Finally, in an assay using an
isolated human tissue, SSR240600 potently inhibited contractions of
human isolated small bronchi (diameter <1 mm) induced by
[Sar9,Met(O2)11]substance
P (100 nM) with a pIC50 of 8.6 (Fig.
3).
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Fig. 2.
Concentration-response curves for
[Sar9,Met(O2)11]substance
P-induced endothelium-dependent relaxation of rabbit pulmonary artery
precontracted with 100 nM norepinephrine (A),
[Ala8]neurokinin A-induced contractions of
endothelium-deprived rabbit pulmonary artery (B), and
[MePhe7]neurokinin B-induced contractions of guinea pig
ileum (C) in the absence and in the presence of SSR240600. Values are
means ± S.E.M. (n = 6).
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Fig. 3.
Inhibition by SSR240600 of
[Sar9,Met(O2)11]substance
P-induced contractions of human isolated small bronchi (diameter <1
mm). [Sar9,Met(O2)11]Substance P
concentration was 100 nM. Results are expressed as percentage
inhibition of control and values are means ± S.E.M.
(n = 7-8). Significant variations from control are
shown as  for P < 0.05 and for
P < 0.01 (ANOVA followed by Dunnett's
t test).
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In Vivo Studies.
The in vivo activity of SSR240600 was
first investigated using typical pharmacological responses to
tachykinin NK1 receptor agonists
([Sar9,Met(O2)11]substance
P, GR73632, or substance P). In anesthetized dogs, intravenous
injection of
[Sar9,Met(O2)11]substance
P (5 ng/kg) provoked a reproducible hypotension of 30 to 40 mm Hg. SSR240600 administered either intravenously (3-10 µg/kg) or
intraduodenally (30-300 µg/kg) produced a dose- and time-dependent
inhibition of this
[Sar9,Met(O2)11]substance
P-induced hypotension (Fig. 4). At the
doses tested, SSR240600 itself had no effect on mean blood pressure.
SSR240600 also potently antagonized GR73632-induced bronchoconstriction in anesthetized guinea pigs (Fig. 5).
Intravenous injection of GR73632 (0.5 ng/kg) produced a reproducible
bronchoconstriction that was dose dependently inhibited by pretreatment
with intravenously administered SSR240600 (50 and 100 µg/kg).
Furthermore, 3 h after a single oral administration, SSR240600 (3 mg/kg) inhibited GR73362-induced bronchoconstriction by 83 ± 5%
(n = 5), indicating both oral bioavailability and a
long-lasting effect of the compound. SSR240600 itself did not modify
the resting bronchial tone at the doses tested. Finally, intravenous
administration of substance P (0.3 µg/kg) induced plasma
extravasation in different guinea pig tissues. After intraperitoneal administration, 30 min before substance P, SSR240600 at doses equal to
or greater than 1 mg/kg inhibited the plasma extravasation (Fig.
6).
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Fig. 4.
Inhibition by SSR240600 of
[Sar9,Met(O2)11]substance
P-induced hypotension in anesthetized dogs. SSR240600 was administered
by the intravenous (A) or intraduodenal (B) route.
[Sar9,Met(O2)11]Substance P (5 ng/kg) was injected intravenously at various times after SSR240600
administration. Results are expressed as percentage inhibition of the
reduction of mean blood pressure (about 30-40 mm Hg) induced by
[Sar9,Met(O2)11]substance P
before SSR240600 administration. Values are means ± S.E.M.
(n = 3-5). Significant variations from control are
shown as for P < 0.05 and for
P < 0.01 (ANOVA for repeated measures followed by
Dunnett's t test).
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Fig. 5.
Inhibition by SSR240600 of GR73632-induced
bronchoconstriction in anesthetized guinea pigs. SSR240600 was
administered by the intravenous route at various doses. GR73632 (0.5 ng/kg) was injected intravenously at the indicated times after
SSR240600 administration. Results are expressed as percentage
inhibition of bronchoconstriction induced by GR73632 before
administration of SSR240600. Values are means ± S.E.M.
(n = 5). Significant variations from control
bronchoconstriction before SSR240600 administration are shown as for P < 0.05 and for P < 0.01 (ANOVA for repeated measures followed by Dunnett's
t test).
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Fig. 6.
Inhibition by SSR240600 of substance P-induced
microvascular leakage in anesthetized guinea pigs. Saline (control) or
SSR240600 was administered by the intraperitoneal route at various
doses, 30 min before Evans blue dye (30 mg/kg i.v.). One minute after
dye administration, plasma extravasation was provoked by substance P
(0.3 µg/kg i.v.). Basal level was determined in the absence of
substance P. Results are expressed as tissue content of Evans blue dye.
Values are means ± S.E.M. (n = 4-6).
Significant variations from control are shown as for
P < 0.01 (ANOVA followed by Dunnett's
t test).
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SSR240600 was then studied on citric acid-induced cough in awake guinea
pigs, a model in which endogenous tachykinins and their receptors are
suspected to play an important role. Cough was provoked by exposure of
animals to an aerosol of aqueous citric acid solution (0.4 M) for 10 min. Intraperitoneal administration of SSR240600 (1-10 mg/kg), 30 min
before the citric acid challenge, resulted in a dose-dependent
inhibition of cough (Fig. 7A). This inhibition was highly time dependent (Fig. 7B), increasing with the
length of the pretreatment. The cough inhibition following 120 min
pretreatment with 1 mg/kg i.p. SSR240600 was comparable with that
observed after 30 min pretreatment with 10 mg/kg i.p.
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Fig. 7.
Inhibition by SSR240600 of cough provoked by exposure
of conscious guinea-pigs to an aerosol of citric acid solution (0.4 M)
for 10 min. A, SSR240600 was administered at different doses by the
intraperitoneal route, 30 min before the citric acid challenge. B,
SSR240600 (1 mg/kg i.p.) was administered at different times before the
citric acid challenge. Results are expressed as percentage inhibition
of control and are means ± S.E.M. (n = 4-10). Significant variations from control are shown as for
P < 0.05 and for P < 0.01 (ANOVA followed by Dunnett's t test).
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Discussion |
This paper describes biochemical and pharmacological activities of
SSR240600, a new, selective and highly potent nonpeptide antagonist of
the tachykinin NK1 receptor. In binding
experiments, SSR240600 potently inhibited binding of radioactive
substance P to human tachykinin NK1 receptors
with inhibition constants (Ki) in the
subnanomolar range. Of particular interest is its very high affinity
for the native tachykinin NK1 receptor present in
human brain cortex membrane preparation. The potency of SSR240600 was
comparable with that previously observed with another chemically related tachykinin NK1 receptor antagonist,
SR140333 (Emonds-Alt et al., 1993), except that its affinity was
typically species-dependent. Contrary to SR140333, it was more active
on tachykinin NK1 receptors of guinea pigs than
of rats and gerbils. Such species-dependent affinities have been
observed for other nonpeptide and peptidomimetic NK1 receptor antagonists (McLean et al., 1993,
1996; Aramori et al., 1994; Beattie et al., 1995; Cellier et al., 1996;
Quartara and Maggi, 1997). The potent antagonism of SSR240600 at
tachykinin NK1 receptors has been further
demonstrated in different in vitro functional assays for tachykinin
receptors. First, like SR140333, it blocked with high efficacy both
tachykinin NK1 receptor-mediated inositol
phosphate-1 formation in human astrocytoma U373MG cells (Oury-Donat et
al., 1994) as well as contractions of isolated human small
bronchi (Naline et al., 1996). Second, it also potently antagonized
[Sar9,Met(O2)11]substance
P-induced endothelium-dependent relaxation of rabbit pulmonary artery
precontracted by norepinephrine, a typical tachykinin NK1 receptor assay (Regoli et al., 1994). Like
SR140333 (Emonds-Alt et al., 1993), SSR240600 antagonism was not purely
competitive. A similar profile was reported with other peptidomimetic
or nonpeptide tachykinin NK1 receptor antagonists
(Beattie et al., 1995; Cirillo et al., 1998).
The selectivity of SSR240600 for tachykinin NK1
receptors has also been clearly demonstrated in our binding and in
vitro functional studies. Indeed, the affinities measured in binding
assays for tachykinin NK2 and
NK3 receptors remained very low compared with tachykinin activities or activities displayed by specific antagonists of tachykinin NK2 (SR48968, SR144190)
(Emonds-Alt et al., 1992, 1997) or NK3 (SR142801,
SSR146977) (Emonds-Alt et al., 1995, 2002) receptors at these
receptors. The selectivity of SSR240600 for tachykinin
NK1 receptors was further evidenced in different
in vitro functional assays for these tachykinin receptors. In assays using isolated organ preparations typical for tachykinin
NK2 and NK3 receptors
(Regoli et al., 1994), it did not interact with these receptors, at
concentrations up to 0.1 µM. Its antagonist activity at these
receptors at a concentration of 1 µM remained limited compared with
the activities of specific antagonists of these receptors in the same
assays (Emonds-Alt et al., 1992, 1995, 1997, 2002). This limited effect
of SSR240600 on tachykinin receptors other than
NK1 receptors was confirmed in functional assays
using CHO cells stably expressing human tachykinin
NK2 and NK3 receptors. In these assays, the inhibition of tachykinin
NK2 or NK3
receptor-mediated inositol phosphate-1 formation by SSR240600 was only
observed at IC50 values about 200-fold higher
than those obtained in similar experimental conditions for specific
NK2 (SR 48968, SR144190) (F. Oury-Donat, O. Thurneyssen, and P. Soubrié, unpublished results) or
NK3 (SR142801, SSR146977) (Oury-Donat et al.,
1995; Emonds-Alt et al., 2002) receptor antagonists. Finally, the
selectivity of SSR240600 for tachykinin NK1
receptors was also shown by its lack of activity at concentrations up
to 1 µM in almost all binding assays for various ion channels and for
neurotransmitter, neuropeptide, and hormone receptors as well as in
enzyme assays. In the few cases where an interaction was detected, this
was always with IC50 values above 0.1 µM.
In vivo, SSR240600 has been shown to exert a highly potent antagonism
at tachykinin NK1 receptors. Its potency was
first demonstrated in animal models where direct activation of
tachykinin NK1 receptors was provoked by
substance P or specific agonists of these receptors. Like SR140333 and
other tachykinin NK1 receptor antagonists
(Emonds-Alt et al., 1993; Hirayama et al., 1993; Cellier et al., 1996;
McLean et al., 1996; Cirillo et al., 1998), SSR240600 very potently
inhibited tachykinin NK1 receptor-mediated
hypotension, bronchoconstriction, and plasma extravasation, three
typical effects of substance P and its analogs (Regoli et al., 1994;
Quartara and Maggi, 1998). It was active by the oral route and had
long-lasting effects.
SSR240600 was then studied in an animal model where endogenous
tachykinins through their respective receptors are suspected to play a
major role: citric acid-induced cough in unanesthetized guinea pigs
(Widdicombe, 1995; Advenier and Emonds-Alt, 1996; Advenier et al.,
1997). Either tachykinin NK2 (Advenier et al., 1993; Girard et al., 1995; Yasumitsu et al., 1996; Emonds-Alt et al.,
1997) or NK3 (Daoui et al., 1998; Emonds-Alt et
al., 2002) receptor antagonists have been reported to have potent
antitussive activity in this model. Regarding the effect of nonpeptide
tachykinin NK1 receptor antagonists in this
model, the results are controversial. No inhibitory activity was
observed for a nonpeptide antagonist such as SR140333 (Girard et al.,
1995), whereas an antitussive effect was reported for a peptidomimetic
antagonist (FK888) (Yasumitsu et al., 1996). However, another
nonpeptide tachykinin NK1 receptor antagonist,
CP-99,994 (McLean et al., 1993), was shown to block cough induced by
capsaicin in unanesthetized guinea pigs as well as by mechanical
stimulation of trachea in anesthetized cats (Bolser, 1996; Bolser et
al., 1997). In the present study, SSR240600 was clearly shown to
potently inhibit citric acid-induced cough in unanesthetized guinea
pigs. Moreover, in the same model and under the same experimental
conditions used for SR140333 and SSR240600, another tachykinin
NK1 receptor antagonist, GR205171 (Gardner et al., 1996), also displayed potent antitussive activity (C. Advenier,
E. Naline, and S. Daoui, unpublished results).
The antitussive activity of SSR240600 in guinea pigs may be related to
its ability to readily penetrate into the brain. Indeed, the
antitussive activity of CP-99,994 was reported as probably mainly
mediated by a central action (Bolser, 1996; Bolser et al., 1997). On
the other hand, there is some parallelism between antitussive activity
and other centrally mediated activities of the tachykinin NK1 receptor antagonists. SR140333 was shown to
have several activities in the rat central nervous system (Jung et al.,
1994), but it was also reported to lack activity in some models, in
particular, models for emesis, in which brain penetration of the
compound is essential (Rupniak et al., 1997; Diemunsch and
Grélot, 2000). On the contrary, GR205171 was shown to have a
potent broad-spectrum anti-emetic activity (Gardner et al., 1996;
Diemunsch and Grélot, 2000). Similarly, CP-99,994 also showed
potent anti-emetic activity as well as other typical centrally mediated
effects (Rupniak et al., 1997). Moreover, a potent antidepressant-like
activity of SSR240600 in guinea pigs was clearly demonstrated
(Steinberg et al., 2002) as for centrally active tachykinin
NK1 receptor antagonists (Rupniak and Kramer,
1999). A preliminary pharmacokinetic study (C. Briot, unpublished
results) also showed an efficient brain penetration of the
compound in guinea pigs with slow kinetics (peak plasma level of 650 ng/ml obtained at 1 h, brain tissue level of 70 ng/g reached at
4 h, and stable between 4 and 8 h after oral administration
at 10 mg/kg), explaining its time-dependent antitussive activity.
However, the results reported for the evaluation of FK888 brain
penetration are opposite (Yasumitsu et al., 1996; Rupniak et al.,
1997), but together, they suggest a low brain penetration, if any. The
antitussive activity of FK888 in guinea pigs could be due to a
peripheral effect related to its peptidomimetic structure since a low
brain-penetrant nonpeptide antagonist, SR140333, was completely inactive.
In conclusion, SSR240600 is a novel, highly potent nonpeptide
antagonist of the tachykinin NK1 receptor. It is
active by the oral route with long-lasting effects and can penetrate
into the brain.
We thank Dr. Christophe Briot (Sanofi-Synthélabo
Recherche, France) for the preliminary pharmacokinetic study of
SSR240600 in guinea pigs.
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Abstract
Introduction
