SSR240600 [(R)-2-(1-{2-[4-{2-[3,5-Bis(trifluoromethyl)-
`phenyl]acetyl}-2-(3,4-dichlorophenyl)-2-morpholinyl]ethyl}-
`4-piperidinyl)-2-methylpropanamide], a Centrally Active
`Nonpeptide Antagonist of the Tachykinin Neurokinin-1
`Receptor: I. Biochemical and Pharmacological Characterization
`XAVIER EMONDS-ALT, VINCENZO PROIETTO, R ´EGIS STEINBERG, FLORENCE OURY-DONAT, XAVIER VIG ´E,
`POL VILAIN, EMMANUEL NALINE, SAMIRA DAOUI, CHARLES ADVENIER, G ´ERARD LE FUR, JEAN-PIERRE MAFFRAND,
`PHILIPPE SOUBRI ´E, and MARC PASCAL
`Sanofi-Synthe ´ labo 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 Faculte´d e M e´ decine
`Paris-Ouest, Paris, France (E.N., S.D., C.A.)
`Received June 11, 2002; accepted July 12, 2002
`ABSTRACT
`The biochemical and pharmacological properties of a novel an-
`tagonist 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-methylpro-
`panamide], were evaluated. SSR240600 inhibited the binding of
`radioactive substance P to tachykinin NK
`1 receptors in human
`lymphoblastic IM9 cells (Ki /H110050.0061 nM), human astrocytoma
`U373MG cells (Ki /H110050.10 nM), and human brain cortex (IC 50 /H11005
`0.017 nM). It also showed subnanomolar affinity for guinea pig
`NK
`1 receptors but was less potent on rat and gerbil NK1 recep-
`tors. SSR240600 inhibited [Sar9,Met(O2)11]substance P-induced
`inositol monophosphate formation in human astrocytoma
`U373MG cells with an IC
`50 value of 0.66 nM (agonist concentra-
`tion of 100 nM). It also antagonized substance P-induced con-
`tractions of isolated human small bronchi with a pIC
`50 value of 8.6
`(agonist concentration of 100 nM). The compound was/H11022100- to
`1000-fold more selective for tachykinin NK 1 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 NK
`1 receptor-mediated hypo-
`tension in dogs (3 and 10/H9262g/kg i.v.), microvascular leakage (1 and
`3 mg/kg i.p.), and bronchoconstriction (50 and 100/H9262g/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 endoge-
`nous tachykinins are suspected to play a major role. In conclusion,
`SSR240600 is a new, potent, and centrally active antagonist of the
`tachykinin NK
`1 receptor, able to antagonize various NK1 receptor-
`mediated pharmacological effects in the periphery and in the
`central nervous system.Substance P belongs to a group of related neuropeptides
`named tachykinins, which includes neurokinin A and neuro-
`kinin B. These peptides are widely distributed in the periph-
`eral 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
`/H9251-helical transmembrane segments, denoted NK 1,N K2, and
`NK3. Substance P is the natural endogenous ligand of tachy-
`kinin NK 1 receptors, whereas neurokinin A and neurokinin
`B are the preferential ligands of tachykinin NK 2 and NK 3
`receptors, respectively (Regoli et al., 1994; Maggi, 1995;
`Quartara and Maggi, 1997).
`Article, publication date, and citation information can be found at
`http://jpet.aspetjournals.org.
`DOI: 10.1124/jpet.102.040162.
`ABBREVIATIONS: NK, neurokinin; SSR240600, ( R)-2-(1-{2-[4-{2-[3,5-bis(trifluoromethyl)phenyl]acetyl}-2-(3,4-dichlorophenyl)-2-morpholinyl]ethyl}-
`4-piperidinyl)-2-methylpropanamide; CHO, Chinese hamster ovary; SR140333, (S)-1-[2-[3-(3,4-dichlorophenyl)-1-[1-[[3-(1-methylethoxy)phenyl]acetyl]-
`3-piperidinyl]ethyl]-4-phenyl-1-azaniabicyclo[2.2.2]octane chloride; SR48968, ( S)-N-methyl-N -[4-(4-acetylamino-4-phenyl piperidino)-2-(3,4-
`dichlorophenyl)butyl]benzamide; FK888, N2-[(4R)-4-hydroxy-1-[(1-methyl-1H -indole-3-yl)carbonyl]-L -propyl]-N -methyl-N -(phenylmethyl)-3-(2-
`naphtyl)-L-alaninamide; CP-99,994, (2S,3S)-3-(2-methoxybenzylamino)-2-phenylpiperidine; ANOVA, analysis of variance. SR144190, (R)-3-{1-[2-(4-
`benzoyl-2-(3,4-difluorophenyl)-morpholin-2-yl)-ethyl]-4-phenylpiperidin-4-yl}-1-dimethylurea; SR142801, ( R)-(N )-[1-[3-[1-benzoyl-3-(3,4-
`dichlorophenyl)piperidin-3-yl]propyl]-4-phenylpiperidin-4-yl]-N-methylacetamide; SSR146977, (R)-(N)-[1-[3-[1-benzoyl-3-(3,4-dichlorophenyl)piperidin-
`3-yl]propyl]-4-phenylpiperidin-4-yl]-N-dimethylurea; GR205171, (2S,3S)-2-methoxy-5-[(5-trifluoromethyl-tetrazol)-1-yl]-benzyl-(2-phenyl-piperidin-3-yl)-
`amine; GR73632, D-Ala-[L-Pro9,Me-Leu8]substance P(7-11).
`0022-3565/02/3033-1171–1179$7.00
`THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 303, No. 3
`Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 40162/1026549
`JPET 303:1171–1179, 2002 Printed in U.S.A.
`1171
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`HELSINN EXHIBIT 2062
`Azurity Pharmaceuticals, Inc. v. Helsinn Healthcare S.A.
`IPR2025-00948
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`Over the past several years, potent nonpeptide antago-
`nists,
`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; Lag-
`ente 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
`NK
`1 receptors may provide a novel treatment of major de -
`pression (Kramer et al., 1998; Rupniak and Kramer, 1999)
`and emesis (Rupniak and Kramer, 1999; Diemunsch and
`Gre ´ lot, 2000). These activities of tachykinin NK
`1 receptor
`antagonists are essentially dependent on their ability to pen-
`etrate the brain (Rupniak et al., 1997; Kramer et al., 1998;
`Diemunsch and Gre´ lot, 2000). We now describe some gen-
`eral biochemical and pharmacological activities of a novel
`nonpeptide tachykinin NK
`1 receptor antagonist,
`SSR240600 [(R )-2-(1-{2-[4-{2-[3,5-bis(trifluoromethyl)phe-
`nyl]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).
`Materials and Methods
`Binding Assays. The affinity of SSR240600 for tachykinin recep-
`tors was evaluated in several receptor-radioligand binding assays: 1)
`binding of [
`125I]Bolton-Hunter region-labeled substance P to tachy -
`kinin NK 1 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 /H9253) to tachykinin NK 2 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 recep-
`tor, cloned and stably expressed in CHO cells. All these binding
`assays were conducted and analyzed as previously described in de-
`tail (Emonds-Alt et al., 1993, 1995, 1997).
`The affinity of SSR240600 for tachykinin receptors was also investi-
`gated 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°Ci n5 0m M
`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/H1100220°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
`/H9262g/ml bacitracin, 4 /H9262g/ml leupeptin, 4 /H9262g/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 /H9262l of assay buffer (50 mM Tris-HCl
`buffer, pH 7.4, containing 0.2 mg/ml bovine serum albumin, 40/H9262g/ml
`bacitracin, 4/H9262g/ml leupeptin, 4/H9262g/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 filtra-
`tion 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
`/H9252liquid
`scintillation counter. Specific binding was determined by subtraction of
`the nonspecific binding, which was determined in the presence of 1
`/H9262M
`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 en-
`zyme 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 NK
`1 receptor activation with spe -
`cific 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 NK
`2 or NK3 receptor in
`response to [Nle 10]neurokinin A-(4-10) or [MePhe 7]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 investi-
`gated by using several functional bioassays specific for the three tachy-
`kinin receptor subtypes (Regoli et al., 1994): [Sar
`9,Met(O2)11]substance
`P-induced endothelium-dependent relaxation of rabbit pulmonary ar-
`tery, precontracted by 0.1
`/H9262M norepinephrine (specific for NK1 recep-
`tors), [/H9252Ala8]neurokinin A-(4-10)-induced contractions of endothelium-
`denuded rabbit pulmonary artery (specific for NK 2 receptors), and
`[MePhe7]neurokinin B-induced contractions of guinea pig ileum (spe-
`cific 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 tachy-
`kinin NK
`1,N K2, and NK3 receptors (Emonds-Alt et al., 1993), prelim-
`inary 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 NK
`1 receptors
`was evaluated by measuring inhibition of [Sar 9,Met(O2)11]substance
`P-induced contractions of human isolated small bronchi (diameter/H110211
`mm) as described by Naline et al. (1996). Bronchial tissues were re-
`moved from patients (25 men, previous smokers, mean age 64 /H110062
`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 Comite ´
`d’Expe ´ rimentation Animale (Animal Care and Use Committee) of
`Sanofi-Synthe ´ labo Recherche and are in accordance with the princi-
`ples of the Declaration of Helsinki. The in vivo pharmacological
`profile of SSR240600 was investigated in three animal models in
`which the role of tachykinin NK
`1 receptor has been well character -
`ized: hypotension, bronchoconstriction, and plasma extravasation
`Fig. 1. Structure of SSR240600 [(R)-2-(1-{2-[4-{2-[3,5-bis(trifluorometh-
`yl)phenyl]acetyl}-2-(3,4-dichlorophenyl)-2-morpholinyl]ethyl}-4-piperidi-
`nyl)-2-methylpropanamide].
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`induced by substance P or specific agonists of the tachykinin NK 1
`receptor (Regoli et al., 1994; Quartara and Maggi, 1998). Further-
`more, 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 intrave-
`nous route), and the anesthetic was infused throughout the experi-
`ments 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, [Sar
`9,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 Honey-
`well PC 156 transducer at the carotid artery. In control experiments,
`repeated injections of [Sar
`9,Met(O2)11]substance P (5 ng/kg) pro -
`duced 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) admin-
`istered by the intraperitoneal route and were pretreated with atro-
`pine (0.5 mg/kg), diphenhydramine (1 mg/kg), and indomethacin (2
`mg/kg) injected intravenously. Bronchoconstriction was induced
`with GR73632 (a tachykinin NK
`1 receptor agonist) (5 ng/kg), admin-
`istered intravenously at 20-min intervals before and after intrave-
`nous 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-Ro ¨ ssler 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 intraperi-
`toneal 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 adminis-
`tration of substance P (0.3
`/H9262g/kg). The animals were killed 5 min
`later, and tissues (trachea, main bronchi, esophagus, urinary blad-
`der) 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 intraperi-
`toneal route at various times before the aerosol challenge. Coughs
`were counted by a trained observer, and recognized by the char-
`acteristic 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-Syn-
`the ´ labo 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.
`Results
`Binding Studies. The inhibition constants (K
`i) for
`SSR240600 obtained in the different binding assays for
`tachykinin receptors are shown in Table 1. SSR240600 in-
`hibited the binding of radioactive substance P to tachykinin
`NK
`1 receptors with subnanomolar Ki values, using estab -
`lished human cell lines as well as human brain membranes.
`SSR240600 also displayed a high affinity for tachykinin NK
`1
`receptors from various animal species, especially guinea pigs.
`In binding assays for tachykinin NK
`2 and NK 3 receptors,
`SSR240600 slightly interfered with the binding of their re-
`spective ligands, with K
`i values always above 10 nM in all
`species studied, including human. Finally, SSR240600 was
`assayed in 100 (mainly human) receptor-binding, ion chan-
`nel-binding, and enzyme assays including adenosine (A
`1,
`A2A,A 3), adrenergic ( /H92511, /H92512, /H92521, /H92522) dopamine (D1, D2), nic -
`otinic, muscarinic (M1,M 2,M 3,M 4,M 5), opiate (/H9262, /H9260, /H9254, opioid
`receptor-like receptor 1) serotonin (5-HT1A, 5-HT-2A, 5-HT2C,
`5-HT3, 5-HT 5A, 5-HT 6, 5-HT 7), angiotensin (AT 1,A T 2), bra -
`dykinin (B1, B2), calcitonin gene-related peptide, cholecysto-
`kinin (CCK
`1, CCK 2), corticotropin-releasing factor (CRF 1,
`TABLE 1
`Inhibition constants (K i) of SSR240600 in radioligand binding assays for tachykinin receptors
`Radioligands: /H20851125I/H20852Bolton-Hunter-labeled substance P for NK1 receptors, except for human brain cortex where /H208513H/H20852substance P was used;/H20851125I/H20852iodohistidyl-neurokinin A for
`NK2 receptors, except for guinea-pig ileum where /H20851125I/H20852neuropeptide/H9253was used; /H20851125I/H20852iodohistidyl-/H20851MePhe7/H20852neurokinin B for NK3 receptors, except for rat brain cortex where
`/H20851125I/H20852eledoisin was used. Tachykinins: substance P, neurokinin A, and/H20851MePhe7/H20852neurokinin B, respectively, for NK1,N K2, and NK3 receptors, except for human brain cortex
`where /H20851Sar9,Met(O2)11/H20852substance P was used. Values are means/H11006S.D. obtained from at least three independent experiments performed in triplicate.
`Ki (nM)
`Receptors Tachykinins SSR240600
`Human brain cortex NK1 0.65 /H110060.23 0.0043 /H110060.0012
`IM9 (human) NK1 0.044 /H110060.008 0.0061 /H110060.0004
`U373MG (human) NK1 0.31 /H110010.06 0.10 /H110060.01
`STTG1 (human) NK1 0.33 /H110060.03 0.09 /H110060.01
`Rat ileum NK1 0.054 /H110060.006 1.07 /H110060.07
`Gerbil ileum NK1 0.042 /H110060.008 1.15 /H110060.02
`Guinea pig ileum NK1 0.059 /H110060.007 0.23 /H110060.03
`Human (CHO cells) NK2 0.48 /H110060.01 24 /H110062
`Guinea pig ileum NK2 2.30 /H110060.49 71 /H110065
`Rat urinary bladder NK2 0.76 /H110060.03 34 /H110063
`Human (CHO cells) NK3 0.34 /H110060.05 206 /H110069
`Guinea pig brain cortex NK3 0.43 /H110060.05 21 /H110062
`Gerbil brain cortex NK3 0.16 /H110060.03 544 /H1100677
`Rat brain cortex NK3 0.053 /H110060.001 /H1102210,000
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`CRF2), galanin (GAL 1, GAL 2), neurotensin (NT 1), vasopres -
`sin (V 1A), hormones (glucocorticoid, estrogen, progesterone,
`testosterone), ion channels (sodium, calcium, potassium and
`chloride), cyclooxygenases (COX 1, COX 2), phosphodiester -
`ases (III and IV), acetylcholinesterase. SSR240600, at con-
`centrations up to 1 /H9262M, was inactive (inhibition less than
`50%), except in /H9268receptor (IC 50 /H110050.21 /H9262M) and sodium
`channel site 2 (IC 50 /H110050.18 /H9262M) assays (data not shown).
`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 NK 1 receptor activation in human astrocytoma
`U373MG cells. Activation of tachykinin NK 1 receptors in
`U373MG cells by three different specific agonists
`Fig. 2. Concentration-response curves for [Sar 9,Met(O2)11]substance P-induced endothelium-dependent relaxation of rabbit pulmonary artery
`precontracted with 100 nM norepinephrine (A), [ /H9252Ala8]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 /H11006S.E.M.
`(n /H110056).
`TABLE 2
`Inhibition by SSR240600 of tachykinin receptor-mediated inositol
`phosphate-1 formation in human astrocytoma U373MG cells (NK 1
`receptors) and in CHO cells expressing either human NK 2 or NK3
`receptors
`Results are given as IC 50 values. Values are means /H11006S.E.M. from at least three
`independent experiments performed in triplicate.
`Receptors Agonists (Concentration) IC50
`nM
`NK1 /H20851Sar9,Met(O2)11/H20852Substance P (0.1/H9262M) 0.66 /H110060.11
`GR73632 (0.1 /H9262M) 0.57 /H110060.08
`Septide (0.1 /H9262M) 0.45 /H110010.07
`NK2 /H20851/H9252Ala8/H20852Neurokinin A(4-10) (10 nM) 140 /H110067
`NK3 /H20851MePhe7/H20852Neurokinin B (10 nM) 1760 /H11006100
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`([Sar9,Met(O2)11]substance P, septide, and GR73632) pro -
`voked the formation of inositol phosphate-1, which was con-
`centration dependently inhibited by SSR240600 regardless of
`the agonist used. In contrast, SSR240600 showed a much
`lower potency to block inositol phosphate-1 formation follow-
`ing the activation of human tachykinin NK
`2 and NK3 recep-
`tors stably expressed in CHO cells. The IC50 values are given
`in Table 2.
`In a classical tachykinin NK1 receptor assay using isolated
`tissues, [Sar9,Met(O2)11]substance P-induced endothelium-de-
`pendent relaxation of rabbit pulmonary artery previously con-
`tracted by norepinephrine, SSR240600 produced a concentra-
`tion-dependent rightward shift of the concentration-response
`curves of [Sar
`9,Met(O2)11]substance P (Fig. 2 A). 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 sig-
`nificantly different from unity and the apparent affinity of
`SSR240600 was thus calculated in terms of p D/H11032
`2 (negative
`logarithm of the molar concentration of antagonist that pro-
`duces a 50% reduction of the maximal response to the agonist).
`The pD /H11032
`2 value was 8.67 /H110060.08 (n /H1100518). The activity of
`SSR240600 was then examined on tissue preparations contain-
`ing tachykinin NK
`2 and NK3 receptors. At concentrations up to
`0.1 /H9262M, it had no effect in bioassays for NK2 ([/H9252Ala8]neurokinin
`A-induced contractions of endothelium-deprived rabbit pulmo-
`nary artery) (Fig. 2B) or NK
`3 ([MePhe7]neurokinin B-induced
`contractions of guinea pig ileum) (Fig. 2C) receptors. At a con-
`centration of 1
`/H9262M, 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 NK
`3 receptors. Finally, in an assay using an isolated human
`tissue, SSR240600 potently inhibited contractions of human
`isolated small bronchi (diameter /H110211 mm) induced by
`[Sar
`9,Met(O2)11]substance P (100 nM) with a pIC50 of 8.6 (Fig.
`3).
`In Vivo Studies.The in vivo activity of SSR240600 was first
`investigated using typical pharmacological responses to tachy-
`kinin NK
`1 receptor agonists ([Sar 9,Met(O2)11]substance P,
`GR73632, or substance P). In anesthetized dogs, intravenous
`injection of [Sar
`9,Met(O2)11]substance P (5 ng/kg) provoked a
`reproducible hypotension of 30 to 40 mm Hg. SSR240600 ad-
`ministered either intravenously (3 – 10
`/H9262g/kg) or intraduode-
`nally (30– 300 /H9262g/kg) produced a dose- and time-dependent in-
`hibition of this [Sar 9,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 anesthe-
`tized 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
`/H9262g/kg). Furthermore, 3 h
`after a single oral administration, SSR240600 (3 mg/kg) inhib-
`ited GR73362-induced bronchoconstriction by 83/H110065% (n /H110055),
`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 admin-
`istration of substance P (0.3
`/H9262g/kg) induced plasma extravasa-
`tion in different guinea pig tissues. After intraperitoneal admin-
`istration, 30 min before substance P, SSR240600 at doses equal
`to or greater than 1 mg/kg inhibited the plasma extravasation
`(Fig. 6).
`SSR240600 was then studied on citric acid-induced cough
`in awake guinea pigs, a model in which endogenous tachyki-
`nins and their receptors are suspected to play an important
`role. Cough was provoked by exposure of animals to an aero-
`sol of aqueous citric acid solution (0.4 M) for 10 min. Intra-
`peritoneal administration of SSR240600 (1–10 mg/kg), 30
`min before the citric acid challenge, resulted in a dose-depen-
`dent 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 pre-
`treatment with 1 mg/kg i.p. SSR240600 was comparable with
`that observed after 30 min pretreatment with 10 mg/kg i.p.
`Discussion
`This paper describes biochemical and pharmacological ac-
`tivities of SSR240600, a new, selective and highly potent
`nonpeptide antagonist of the tachykinin NK
`1 receptor. In
`binding experiments, SSR240600 potently inhibited binding
`of radioactive substance P to human tachykinin NK
`1 recep-
`tors with inhibition constants (K i) in the subnanomolar
`range. Of particular interest is its very high affinity for the
`native tachykinin NK
`1 receptor present in human brain cor -
`tex membrane preparation. The potency of SSR240600 was
`comparable with that previously observed with another
`chemically related tachykinin NK
`1 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 NK
`1 receptors of guinea pigs
`than of rats and gerbils. Such species-dependent affinities
`have been observed for other nonpeptide and peptidomimetic
`NK
`1 receptor antagonists (McLean et al., 1993, 1996; Ar -
`amori et al., 1994; Beattie et al., 1995; Cellier et al., 1996;
`Quartara and Maggi, 1997). The potent antagonism of
`SSR240600 at tachykinin NK
`1 receptors has been further
`demonstrated in different in vitro functional assays for
`tachykinin receptors. First, like SR140333, it blocked with
`high efficacy both tachykinin NK
`1 receptor-mediated inositol
`phosphate-1 formation in human astrocytoma U373MG cells
`Fig. 3. Inhibition by SSR240600 of [Sar 9,Met(O2)11]substance P-induced
`contractions of human isolated small bronchi (diameter /H110211 mm).
`[Sar9,Met(O2)11]Substance P concentration was 100 nM. Results are ex -
`pressed as percentage inhibition of control and values are means /H11006
`S.E.M. (n /H110057– 8). Significant variations from control are shown as /H22810for
`P /H110210.05 and /H22810/H22810for P /H110210.01 (ANOVA followed by Dunnett’s t test).
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`(Oury-Donat et al., 1994) as well as contractions of isolated
`human
`small bronchi (Naline et al., 1996). Second, it also
`potently antagonized [Sar 9,Met(O2)11]substance P-induced
`endothelium-dependent relaxation of rabbit pulmonary ar-
`tery precontracted by norepinephrine, a typical tachykinin
`NK
`1 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 NK
`1 receptor an -
`tagonists (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 NK
`2 and NK 3 receptors re -
`mained very low compared with tachykinin activities or ac-
`tivities displayed by specific antagonists of tachykinin NK
`2
`(SR48968, SR144190) (Emonds-Alt et al., 1992, 1997) or NK3
`(SR142801, SSR146977) (Emonds-Alt et al., 1995, 2002) re-
`ceptors at these receptors. The selectivity of SSR240600 for
`tachykinin NK
`1 receptors was further evidenced in different
`in vitro functional assays for these tachykinin receptors. In
`assays using isolated organ preparations typical for tachyki-
`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.
`[Sar
`9,Met(O2)11]Substance P (5 ng/kg) was injected intrave -
`nously at various times after SSR240600 administration. Re-
`sults are expressed as percentage inhibition of the reduction of
`mean blood pressure (about 30 – 40 mm Hg) induced by
`[Sar
`9,Met(O2)11]substance P before SSR240600 administration.
`Values are means /H11006S.E.M. (n /H110053– 5). Significant variations
`from control are shown as /H22810for P /H110210.05 and /H22810/H22810for P /H110210.01
`(ANOVA for repeated measures followed by Dunnett’st test).
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`nin NK 2 and NK 3 receptors (Regoli et al., 1994), it did not
`interact with these receptors, at concentrations up to 0.1/H9262M.
`Its antagonist activity at these receptors at a concentration of
`1 /H9262M remained limited compared with the activities of spe-
`cific 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 NK
`1
`receptors was confirmed in functional assays using CHO cells
`stably expressing human tachykinin NK
`2 and NK3 receptors.
`In these assays, the inhibition of tachykinin NK2 or NK3 recep-
`tor-mediated inositol phosphate-1 formation by SSR240600 was
`only observed at IC
`50 values about 200-fold higher than those
`obtained in similar experimental conditions for specific NK 2
`(SR 48968, SR144190) (F. Oury-Donat, O. Thurneyssen, and P.
`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
`/H9262g/kg i.v.). Basal
`level was determined in the absence of substance P. Results are ex-
`pressed as tissue content of Evans blue dye. Values are means /H11006S.E.M.
`(n /H110054 – 6). Significant variations from control are shown as /H22810/H22810for P /H11021
`0.01 (ANOVA followed by Dunnett’s t test).
`Fig. 5. Inhibition by SSR240600 of GR73632-induced bronchoconstriction
`in anesthetized guinea pigs. SSR240600 was administered by the intra-
`venous route at various doses. GR73632 (0.5 ng/kg) was injected intra-
`venously 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 /H11006
`S.E.M. (n /H110055). Significant variations