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`0022-3565/02/3011-322–332$7.00
`THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
`Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics
`JPET 301:322–332, 2002
`
`Vol. 301, No. 1
`4641/974298
`Printed in U.S.A.
`
`4-(2-Chloro-4-methoxy-5-methylphenyl)-N-[(1S)-2-cyclopropyl-
`1-(3-fluoro-4-methylphenyl)ethyl]5-methyl-N-(2-propynyl)-1,3-
`thiazol-2-amine Hydrochloride (SSR125543A): A Potent and
`Selective Corticotrophin-Releasing Factor1 Receptor
`Antagonist. I. Biochemical and Pharmacological
`Characterization
`
`DANIELLE GULLY, MICHEL GESLIN, LAURENCE SERVA, EVELYNE FONTAINE, PIERRE ROGER, CHRISTINE LAIR,
`VALERIE DARRE, CLAUDINE MARCY, PIERRE-ERIC ROUBY, JACQUES SIMIAND, JOSETTE GUITARD,
`GEORGETTE GOUT, REGIS STEINBERG, DANIEL RODIER, GUY GRIEBEL, PHILIPPE SOUBRIE, MARC PASCAL,
`REBECCA PRUSS, BERNARD SCATTON, JEAN-PIERRE MAFFRAND, and GERARD LE FUR
`Exploratory Research Department, Sanofi-Synthelabo, Toulouse, France (D.G., M.G., L.S., E.F., P.R., C.L., V.D., C.M., P.E.R., M.P.) and
`Strasbourg, France (R.P.); Central Nervous System Department, Sanofi-Synthelabo, Toulouse, France (J.S., J.G., G.G.), Montpellier, France
`(R.G., D.R., P.S.), and Paris, France (G.G.); Discovery Research Division, Sanofi-Synthelabo, Paris, France (B.S., J.P.M., G.L.F.)
`Received October 11, 2001; accepted December 31, 2001
`This article is available online at http://jpet.aspetjournals.org
`
`ABSTRACT
`4-(2-Chloro-4-methoxy-5-methylphenyl)-N-[(1S)-2-cyclopropyl-1-
`(3-fluoro-4-methylphenyl)ethyl]5-methyl-N-(2-propynyl)-1,3-thia-
`zol-2-amine hydrochloride (SSR125543A), a new 2-aminothiazole
`derivative, shows nanomolar affinity for human cloned or native
`corticotrophin-releasing factor (CRF)1 receptors (pKi values of
`8.73 and 9.08, respectively), and a 1000-fold selectivity for CRF1
`versus CRF2␣ receptor and CRF binding protein. SSR125543A
`antagonizes CRF-induced stimulation of cAMP synthesis in hu-
`man retinoblastoma Y 79 cells (IC50 ⫽ 3.0 ⫾ 0.4 nM) and adre-
`nocorticotropin hormone (ACTH) secretion in mouse pituitary tu-
`mor AtT-20 cells. SSR125543A is devoid of agonist activity in
`these models. Its brain penetration was demonstrated in rats by
`using an ex vivo [125I-Tyr0] ovine CRF binding assay. SSR125543A
`displaced radioligand binding to the CRF1 receptor in the brain
`
`(duration of action ⬎24 h).
`with an ID50 of 6.5 mg/kg p.o.
`SSR125543A also inhibited the increase in plasma ACTH levels
`elicited in rats by i.v. CRF (4 ␮g/kg) injection (ID50 ⫽ 1, 5, or 5
`mg/kg i.v., i.p., and p.o., respectively); this effect lasted for more
`than 6 h when the drug was given orally at a dose of 30 mg/kg.
`SSR125543A (10 mg/kg p.o.) reduced by 73% the increase in
`plasma ACTH levels elicited by a 15-min restraint stress in rats.
`Moreover, SSR125543A (20 mg/kg i.p.) also antagonized the in-
`crease of hippocampal acetylcholine release induced by i.c.v.
`injection of 1 ␮g of CRF in rats. Finally, SSR125543A reduced
`forepaw treading induced by i.c.v. injection of 1 ␮g of CRF in
`gerbils (ID50 ⫽ ⬃10 mg/kg p.o.). Altogether, these data indicate
`that SSR125543A is a potent, selective, and orally active CRF1
`receptor antagonist.
`
`Corticotrophin-releasing factor (CRF) is the prime coordi-
`nator of the neuroendocrine and behavioral responses to
`stress (Owens and Nemeroff, 1991). This 41-amino acid pep-
`tide is the major hypothalamic factor responsible for the
`stimulation of corticotrophin (ACTH) secretion from the an-
`terior pituitary, which in turn induces synthesis and release
`of glucocorticoids from the adrenal cortex (Vale et al., 1981).
`The highest density of CRF-containing cell bodies is found in
`the medial paraventricular nucleus of the hypothalamus, a
`brain region that projects
`to the median eminence
`
`(Sawchenko and Swanson, 1991). CRF-containing neurons
`are also found in extrahypothalamic areas, e.g., limbic struc-
`tures (Gray and Bingaman, 1996), suggesting that CRF may
`also play a neurotransmitter role, mediating both stress re-
`sponse and affective behavior (Arborelius et al., 1999). Be-
`cause CRF hypersecretion associated with overactivation of
`the hypothalamo-pituitary-adrenal (HPA) axis has been im-
`plicated in depression and anxiety, the discovery of nonpep-
`tide molecules that selectively inhibit CRF activity is of ma-
`jor clinical interest (Holsboer, 1999).
`
`ABBREVIATIONS: CRF, corticotropin-releasing factor; ACTH, adrenocorticotropin hormone; HPA, hypothalomo-pituitary-adrenal axis; CRF-BP,
`corticotropin-releasing factor-binding protein; DMSO, dimethyl sulfoxide; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; ANOVA,
`analysis of variance; ACh, acetylcholine; R-121919, 3-[6-(dimethylamino)-4-methyl-pyrid-3-yl]-2,5-dimethyl-N,N-dipropyl-pyrazolo[2,3-a]pyrimi-
`din-7-amine; antalarmin, butylethyl-[2,5,6-trimethyl-7-(2,4,6-trimethylphenyl)-7H-pyrrolo[2,3-d]pyrimidin-4-yl]-amine.
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`possess pharmacological activity in experimental models of
`anxiety after oral administration in the 3- to 30-mg/kg
`range (Gutman et al., 2000). Beneficial effects of this com-
`pound have been observed in an open clinical trial per-
`formed in depressed patients, supporting the view that
`CRF1 receptor antagonism could be of therapeutic value in
`the treatment of depression. However R-121919’s develop-
`ment has been stopped because of hepatic toxicity (Zobel et
`al., 2000). In the present study, we report on the charac-
`terization of a new CRF1 receptor antagonist, 4-(2-
`chloro-4-methoxy-5-methylphenyl)-N-[(1S)-2-cyclopropyl-
`1-(3-fluoro-4-methylphenyl)ethyl]5-methyl-N-(2-propy-
`nyl)-1,3-thiazol-2-amine hydrochloride (SSR125543A), ob-
`tained by the optimization of a lead compound discovered
`by random screening of several thousand chemicals. This
`compound belongs to the novel 2-aminothiazole chemical
`family (Fig. 1).
`
`Experimental Procedures
`
`Animals
`Male Sprague-Dawley CD rats and female OF1 mice purchased
`from Iffa Credo (L’Arbresele, France), and male Mongolian gerbils
`from Janvier (Le Genest St. Isle, France) were housed in a controlled
`temperature and light-dark environment with water and chow avail-
`able ad libitum before the experiments. All experimental procedures
`were approved by the Animal Care and Use Committee of Sanofi-
`Synthelabo Recherche and were carried out in accordance with
`French legislation.
`
`Materials
`SSR125543A (Fig. 1) and antalarmin were synthesized by Sanofi-
`Synthelabo Recherche (Toulouse, France). Both compounds were
`solubilized in pure DMSO for the in vitro assays and in 5% DMSO
`and 5% Cremophor EL in saline when administered to mouse, rat,
`and gerbil. Rat/human CRF, ovine CRF, [D-Phe11,His12]Svg(11– 40)
`(antisauvagine-30), and rat/human CRF(6 –33)
`from Neosystem
`(Strasbourg, France) were solubilized in 0.1% acetic acid solution
`containing 1 mg/ml serum bovine albumin. [125I-Tyr0] ovine CRF,
`[125I-Tyr0] rat/human CRF, and [125I-Tyr0] sauvagine were pur-
`chased from PerkinElmer Life Sciences (Boston, MA). Cell culture
`media, antibiotics, and fetal calf serum were obtained from Invitro-
`
`Fig. 1. Chemical structure of the selective CRF1 receptor antagonist
`SSR125543A.
`
`The functional effects of CRF are mediated via the activa-
`tion of two receptor subtypes, CRF1 and CRF2, that are 70%
`homologous in their amino acid sequences but appear phar-
`macologically and anatomically distinct. Both receptor sub-
`types are members of the G protein-coupled receptor super-
`family positively coupled to adenylate cyclase. CRF1 is the
`predominant receptor within the pituitary, cerebellum, and
`neocortex. Two CRF2 isoforms exist: the CRF2␣, which is
`expressed in limbic regions, e.g., lateral septum and dorsal
`raphe nucleus; and the CRF2␤, more abundant in the periph-
`ery (Chalmers et al., 1995). Moreover, a CRF binding protein
`(CRF-BP) binds native rat/human CRF with higher affinity
`than CRF receptors (Behan et al., 1995). CRF-BP is ex-
`pressed in the brain of numerous species, where it might
`regulate CRF-mediated neurotransmission.
`A second CRF receptor endogenous agonist, urocortin, has
`been described (45% homology with CRF) and binds to CRF2
`receptors with a 10-fold higher affinity than CRF. Urocortin
`mRNA expression is prominent in the Edinger-Westphal nu-
`cleus, which does not contain CRF mRNA and is colocalized
`with the CRF2␣ receptor mRNA in the rat lateral septum and
`dorsal raphe nucleus (Vaughan et al., 1995). Recently, uro-
`cortin II, which possesses only 26% homology with CRF, has
`been cloned and found to be a selective agonist at CRF2
`receptors (Reyes et al., 2001).
`The hypothesis that CRF plays a role in the pathophysiol-
`ogy of affective disorders has been put forward on the basis of
`experimental behavioral data, and is consistent with the
`contribution of CRF system alterations to the etiology of
`psychiatric disorders exacerbated or precipitated by stress.
`Thus, high levels of cerebrospinal fluid CRF and an increased
`number of CRF immunoreactive neurons in the hypotha-
`lamic paraventricular nucleus have been measured in pa-
`tients with depressive disorders (Nemeroff et al., 1984). After
`electroconvulsive therapy or antidepressant treatment, HPA
`axis and CRF function normalize, suggesting that CRF over-
`activity may be a marker for human depression (Nemeroff et
`al., 1991). Moreover, intra-amygdala injection of antisense
`oligonucleotides directed against the CRF1 and CRF2 recep-
`tor mRNA in the rat and knock out of the CRF1 receptor gene
`in mice have been associated with reduced levels of anxiety
`and lower anxiogenic responses to i.v. CRF injections (Lieb-
`sch et al., 1995; Heinrichs et al., 1997; Smagin and Dunn,
`2000). Furthermore, CRF1 receptor antagonists have demon-
`strated anxiogenic effects in rodents (Gutman et al., 2000).
`A number of synthetic CRF1 receptor antagonists have
`been identified (Gutman et al., 2000), e.g., butylethyl-[2,5-
`dimethyl-7-(2,4,6-trimethylphenyl)-7H-pyrrolo[2,3-d]pyrimi-
`din-4-yl]-amine (CP-154,526), 5-chloro-N-cyclopropylmethyl-
`2-methyl-N-propyl-N⬘-(2,4,6-trichlorophenyl)-pyrimidin-4,6-
`diamine (NBI 27914), 4-(3-pentylamino)-2,7-dimethyl-8-(2-
`methyl-4-methoxyphenyl)-pyrazolo-[1,5-a ]pyrimidine
`(DMP904), 2-[(N -(2-methylthio-4-isopropylphenyl)-N -
`ethylamino]-4-[4-(3-fluorophenyl)-1,2,3,6-tetrahydropyri-
`din-1-yl)-6-methylpyrimidine (CRA 1000), and R-121919
`(formerly NBI 30775). However, it is of interest to note
`that these molecules have close structural similarities,
`including pyrrolo-, pyrazolo-, and other substituted pyrim-
`idine moieties. The more recent compounds offer better
`solubility and central nervous system penetration than
`their predecessors. For example, R-121919 (Ki value of 3
`nM for the human CRF1 receptor) has been shown to
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`Gully et al.
`
`gen (Cergy Pontoise, France). All other chemicals were from com-
`mercial sources.
`
`(BBN Software Product Corporation, Cambridge, MA) and an inter-
`nal computerized interactive procedure.
`
`Cell Cultures
`CHO cells stably transfected with the human CRF1 receptor
`(hCRF1-CHO cells) or with the human CRF2␣ receptor (hCRF2␣-CHO
`cells) were cultured in Dulbecco’s modified Eagle’s medium supple-
`mented with 10% heat inactivated fetal calf serum, 300 ␮g/ml L-
`glutamine, nonessential amino acids, 100 U/ml penicillin, 100 ␮g/ml
`streptomycin, and 0.17 ␮g/ml amphothericine. Y 79 cells purchased
`from American Type Culture Collection (Rockville, MD) were cul-
`tured in RPMI-1640 medium supplemented with 10% heat-inacti-
`vated fetal calf serum and 300 ␮g/ml L-glutamine.
`AtT-20 cells purchased from American Type Culture Collection
`were cultured in Dulbecco’s modified Eagle’s medium containing
`only in supplement 10% fetal calf serum, 300 ␮g/ml L-glutamine,
`HEPES, and sodium pyruvate. Cells were incubated at 37°C in a
`humidified atmosphere with 5% CO2 except AtT-20, which was in-
`cubated with 15% CO2.
`
`Preparation of Cell Membrane Homogenates
`Cells were cultured to confluence and the flasks were washed with
`10 ml of phosphate-buffered saline (PBS) medium and filled with an
`equal volume of PBS medium. Cells (hCRF1-CHO, hCRF2␣-CHO,
`and AtT-20) were detached from the flask with a cell scraper. Y 79
`cells were cultured in suspension. After centrifugation at 800g for 5
`min, the cell pellet was homogenized at 4°C by using a Polytron
`(setting 6, 2 ⫻ 20 s) in 50 mM Tris-HCl pH 7.4, 2 mM EDTA buffer
`for hCRF1-CHO and hCRF2␣-CHO cells. Homogenization was per-
`formed in 50 mM Tris-HCl pH 7.2, 10 mM MgCl2, 2 mM EDTA, 0.1%
`serum bovine albumin, 8 mg/l aprotinin, and 0.5 mg/ml soybean
`trypsin inhibitor for Y 79 cells. After centrifugation at 40,000g for 20
`min at 4°C, the pellet was homogenized at 4°C by using a Polytron in
`binding buffer (see below). Aliquots obtained from the membrane
`suspension were stored in liquid nitrogen.
`
`Preparation of Brain Membrane Homogenates
`Because the in vivo pharmacological profile of the compound was
`to be characterized in rodents, the inhibitory effects of SSR125543A
`on [125I-Tyr0] ovine CRF binding to rat, mouse, and gerbil brain were
`assessed.
`Mouse, rat, and gerbil were sacrificed by decapitation and brains
`were rapidly removed and homogenized at 4°C by using a Polytron
`(setting 4, 30 s) in 50 mM Tris-HCl pH 7.4, 2 mM EDTA buffer. After
`centrifugation at 40,000g for 20 min at 4°C, the 0.5-mg/ml pellet was
`homogenized at 4°C by using a Polytron in binding buffer (see below).
`Aliquots obtained from the membrane suspension were stored were
`stored at ⫺80°C.
`
`CRF1 Receptor Binding Assay
`[125I-Tyr0] ovine CRF binding was performed with hCRF1-CHO
`cell membranes, Y 79 cell membranes, or rodent brain membrane
`homogenates in the presence of 25 pM radiolabeled CRF in 50 mM
`Tris-HCl pH 7.2, 10 mM MgCl2, 2 mM EDTA, 0.1% serum bovine
`albumin, 8 mg/l aprotinin, and 0.5 mg/ml soybean trypsin inhibitor
`under a final volume of 400 ␮l. Nonspecific binding was determined
`in the presence of 1 ␮M rat/human CRF. Agonists and antagonists
`were added in 1% DMSO (final concentration). After incubation at
`20°C for 2 h, the incubation mixture was filtered on Whatman GF/B
`filters presoaked in 0.5% bovine serum albumin solution for 2 h. The
`filters were washed twice with ice-cold Tris-HCl pH 7.2 buffer and
`the radioactivity was determined with a gamma scintillation counter
`(LKB 1261 multi gamma; EG G Instruments, Evry, France). Specific
`binding was determined as the difference between total and nonspe-
`cific binding. IC50 values were determined using a nonlinear least-
`square regression analysis (Munson and Rodbard, 1980) with RS/1
`
`CRF2 Receptor Binding Assay
`[125I-Tyr0] sauvagine binding was performed using a similar pro-
`tocol as with [125I-Tyr0] ovine CRF binding. In this case, hCRF2␣-
`CHO cell membranes were used at the concentration of 2.5 ␮g of
`protein/tube in presence of 20 pM radiolabeled sauvagine, under a
`final volume of 250 ␮l. Nonspecific binding was determined in pres-
`ence of 1 ␮M unlabeled sauvagine.
`
`CRF-BP Binding Assay
`Displacement of CRF from CRF-BP was measured by a detergent
`phase separation assay. Recombinant human CRF-BP was incu-
`bated at 20°C for 2 h with 30 pM [125I-Tyr0] rat/human CRF in 0.02%
`Nonidet-40 phosphate-buffered saline, pH 7.4. Bound and free CRF
`were then separated by the addition of Triton X-114 (octylphenoxy-
`polyethoxyethanol) buffer stirring and incubation 20 min at 37°C.
`Free CRF segregates to the detergent phase at the bottom of the
`tube, and the CRF/CRF-BP complex remains in the aqueous phase.
`The amount of radioactivity in an aliquot of the aqueous phase was
`determined with a gamma scintillation counter (LKB 1261 multi
`gamma; EG G Instruments). Values were expressed as the mean ⫾
`S.E.M. of at least three determinations performed in triplicate. Spe-
`cific binding was determined as in CRF1 binding assays.
`
`Measurement of Intracellular cAMP Synthesis in Y 79
`Cells
`CRF-induced cAMP synthesis in human retinoblastoma Y 79 cells
`was assessed as described by Hauger et al. (1997). In the present
`article, two types of experiments were performed on Y 79 cells. In the
`first experiment, Y 79 cells were incubated for 15 min at 37°C under
`stirring in presence of 10 nM rat/human CRF with increasing con-
`centrations of SSR125543A in 1 mM isobutylmethyl xanthine sup-
`plemented RPMI buffer, pH 7.2. The intracellular cAMP content was
`measured after lysing the cells by 0.5% ice-cold Triton X-100 by using
`a cAMP 125I scintillation proximity assay kit (Amersham Biosciences
`plc, Little Chalfont, Buckinghamshire, UK). IC50 values were deter-
`mined using a nonlinear least-square regression analysis (Munson
`and Rodbard, 1980) with RS/1 (BBN Software Product Corporation)
`and an internal computerized interactive procedure.
`In the second experiment, Y 79 cells were incubated for 15 min at
`37°C under stirring with increasing concentrations of rat/human
`CRF alone or in presence of three concentrations of SSR125543A.
`Intracellular cAMP synthesis was expressed as the percentage of
`maximal release after subtraction of basal release. Values were
`expressed as the mean of at least three determinations performed in
`duplicate.
`
`Measurement of ACTH Secretion by AtT-20 Cells
`CRF-induced ACTH secretion in mouse pituitary AtT-20 cells was
`previously described by Litvin et al. (1984). A subclone of AtT-20/
`D16v cells was used in this study. Cells were seeded in 12-well plates
`and cultured overnight in their growth medium. They were incu-
`bated for 120 min at 37°C with 2 ml of basal medium, alone or with
`increasing concentrations of rat/human CRF in the presence or ab-
`sence of three concentrations of SSR125543A. ACTH release was
`measured on supernatant samples by using a radioimmunoassay
`(Diasorin, Stillwater, MN). Values were expressed as mean values of
`three determinations performed in triplicate.
`
`Ex Vivo Binding Assay in Rats
`SSR125543A or the corresponding vehicle was administered p.o.
`or i.v. to rats (three per group) at various doses (dose-effect studies)
`and times (time course studies) before rat decapitation and organ
`(brain and pituitary) removal. Tissues were homogenized in 10 ml of
`incubation buffer by using a Polytron (speed 21,500 rpm ⫻ 17 s) then
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`SSR125543A, a New Nonpeptide CRF1 Receptor Antagonist
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`325
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`diluted (1/20) with the same incubation buffer and submitted to a
`[125I-Tyr0] ovine CRF binding assay procedure as previously de-
`scribed. To determine the relative population of CRF1 binding sites
`present in crude homogenates from rat brain and pituitary, binding
`studies were performed in vitro, on naı¨ve brain and pituitary tissue.
`Competition curves were determined for ovine CRF, antisauvagine-
`30, and rat/human CRF(6 –33), two peptides selective for CRF2 recep-
`tor and CRF-BP, respectively (Behan et al., 1995; Ruhmann et al.,
`1998). In the ex vivo binding assay, nonspecific binding was defined
`with 100 nM antalarmin. Values were expressed as the mean per-
`centage of specific binding ⫾ S.E.M. Statistical differences between
`drug- and vehicle-treated groups were assessed by a Student’s t test.
`
`CRF-Induced ACTH Secretion in Rats
`Animals were habituated to the experimental procedure 1 day
`before the experiment. SSR125543A or its vehicle was administered
`p.o. or i.v. to rats (3–7/group) at various doses (dose-effect studies)
`and times (time course studies) before intravenous injection of 4
`␮g/kg rat/human CRF. Thirty minutes later, animals were sacrificed
`by decapitation and trunk blood samples were collected in a 1 mg/ml
`EDTA solution for the determination of ACTH plasma levels by
`radioimmunoassay (Diasorin). Results were expressed as the mean
`values ⫾ S.E.M. Statistical differences between drug- and vehicle-
`treated groups were assessed by a Student’s t test. The median
`inhibitory doses (ID50) with 95% confidence limits were determined
`by fitting of the dose-response curve to the four-parameter logistic
`model according to Ratkowsky and Reedy (1986). The adjustment
`was performed by nonlinear regression by using the Levenberg-
`Marquardt algorithm in the RS/1 software.
`
`Restraint Stress-Induced ACTH Secretion in Rats
`One hour after oral administration of SSR125543A or its vehicle,
`rats were placed into hemicylindrical Plexiglas enclosures (6 cm in
`width and 4 cm in height) for 15 min. After this stress period, the
`animals were placed back in their cages, carried to an adjacent room
`and immediately sacrificed. Nonstressed control animals remained
`in their cage for 15 min before sacrifice. Blood was collected in a 1
`mg/ml EDTA solution for the determination of ACTH plasma levels
`by radioimmunoassay (Diasorin). Values were expressed as the
`mean ACTH levels ⫾ S.E.M. Statistical differences between drug-
`and vehicle-treated groups were assessed by a single factor ANOVA
`or by the nonparametric Kruskall-Wallis test followed by Dunnett’s
`t test or by the Mann-Whitney U test with ␣ adjustment of Holm on
`RS1/software, respectively.
`
`CRF-Induced Hippocampal Acetylcholine Release
`Surgery and Microdialysis. Rats were anesthetized with ure-
`thane (1.4 g/kg i.p.) and then placed in a stereotaxic frame. A micro-
`dialysis probe (CMA-12, length 2 or 3 mm and outer diameter 0.5
`mm; Carnegie Medicine AB, Stockholm, Sweden) was stereotaxically
`implanted in the dorsal hippocampus. The coordinates were 3.5 mm
`posterior to bregma, 2 mm lateral to the midline, and 3.8 mm down
`from the dural surface for the hippocampus (Paxinos and Watson,
`1986). For i.c.v. injection of CRF, ejection pipettes were implanted
`into the left lateral ventricle at the following coordinates: 0.8 mm
`posterior to bregma, 1.5 mm lateral to the midline, and 3.4 mm down
`from the dural surface. The ejection of CRF (1 ␮g/2 ␮l/90 s) was
`performed by applying air pressure with a 1-ml syringe connected to
`the nontapered side of the pipette by Tygon tubing. The probes were
`perfused with a gassed Ringer’s solution containing 125 mM NaCl, 3
`mM KCl, 1.3 mM CaCl2, 1.0 mM MgCl2, 23 mM NaHCO3, and 1.5
`mM KH2PO4, pH 7.4, at a rate of 2 ␮l/min by using a microinjection
`pump (CMA-100; Carnegie Medicine AB). To reduce acetylcholine
`degradation in the dialysate, 1 ␮M neostigmine was added to the
`Ringer’s solution perfused in the hippocampal probe. Microdialysis
`sampling started 90 min after the probe was placed in the hippocam-
`pus. Serial samples were collected at 30-min intervals. SSR125543A,
`
`antalarmin, and vehicle were given intraperitoneally (5 ml/kg of
`body weight) 180 and 30 min before peptide application.
`The time course of the CRF effects was analyzed by ANOVA with
`repeated measures and Dunnett’s t test was used for individual time
`comparisons. The antagonism of the CRF effect was evaluated by
`comparing the area under the curve during the 120 min after peptide
`injection. A statistical analysis was carried out by using the Stu-
`dent’s t test.
`Assay of Acetylcholine (ACh). ACh levels were measured in
`30-min dialysate samples (50 ␮l) by using a high-performance liquid
`chromatography system (Waters, Milford, MA) as previously de-
`scribed by Steinberg et al. (1995) except for the electrochemical
`detection system (Coulochem II; ESA, Chelmsford, MA). Briefly, the
`analytical system for ACh included a trapping precolumn and im-
`mobilized enzyme reactor (BAS.MF-6151). The mobile phase, 35 mM
`phosphate buffer, pH 8.5, supplemented with the antibacterial re-
`agent Kathon (5 ml/l; BAS DF-2150), was pumped at a flow rate of
`0.8 ml/min and replaced with a fresh preparation every 3 days. The
`enzyme postcolumn reactor converted ACh to hydrogen peroxide that
`was electrochemically detected using a platinum electrode (ESA P/N
`55-0183) set at 0.3 V. The chromatographic column and enzyme
`reactor were kept at 35°C. The detection sensitivity was 0.2 pmol/50
`␮l.
`
`CRF-Induced Forepaw Treading in Gerbils
`This test was based on the observation that i.c.v. injection of CRF
`(1 ␮g/2 ␮l) produces forepaw treading (“piano playing”) in gerbils, an
`effect that is prevented by treatment with the CRF1 receptor antag-
`onist R-121919 (Owens and Nemeroff, 1999). Gerbils were placed
`individually in small transparent plastic cages for 30 min. They were
`then pretreated with SSR125543A p.o. or antalarmin i.p. CRF (1 ␮g)
`was injected i.c.v. (free-hand method; Jung et al., 1996) 15 min
`(antalarmin) or 60 min (SSR125543A) later. In each experiment, a
`control group was injected i.c.v. with the vehicle. Forepaw treading
`was measured by an observer unaware of the drug treatment, for 1
`min every 15 min over a 2-h period (8 min in total cumulative times).
`The cumulative forepaw treading time was calculated for each gerbil
`and then expressed as the mean and S.E.M. Comparisons between
`control and treated groups were performed using the Kruskall-Wal-
`lis test, followed by Mann-Whitney U test with ␣ adjustment of
`Holm.
`
`Results
`Affinity of SSR125543A for CRF1 Receptors
`SSR125543A inhibited the specific binding of [125I-Tyr0]
`ovine CRF to human CRF1 receptors expressed in CHO cells
`
`TABLE 1
`Affinity of SSR125543A for CRF receptor subtypes and for CRF1
`receptors of various species
`Values of pKi and Hill coefficients are the means ⫾ S.E.M. of three experiments
`performed in triplicate.
`
`Binding Assay
`CRF1 binding: [125I-Tyr0]ovine
`CRF ligand
`hCRF1-CHO cells
`Y 79 cells
`Rat brain
`Mouse brain
`Gerbil brain
`CRF2␣ binding: [125I-Tyr0]
`ovine sauvagine ligand
`hCRF2␣-CHO cells
`CRF-BP binding: [125I-Tyr0]
`rat/human CRF ligand
`hCRF-BP
`rCRF-BP
`
`Results
`
`pKi
`
`n
`
`1.25 ⫾ 0.10
`8.73 ⫾ 0.15
`0.97 ⫾ 0.17
`9.08 ⫾ 0.20
`0.89 ⫾ 0.12
`8.77 ⫾ 0.23
`1.16 ⫾ 0.13
`8.90 ⫾ 0.10
`0.75 ⫾ 0.11
`9.00 ⫾ 0.00
`Inhibition at 10 ␮M
`
`0%
`Inhibition at 10 ␮M
`
`0%
`0%
`
`4
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`Gully et al.
`
`with a pKi value of 8.73 ⫾ 0.15 (mean ⫾ S.E.M.; Table 1),
`which was comparable to that of antalarmin and higher than
`that of the natural ligand rat/human CRF (pKi ⫽ 8.70 and
`8.22, respectively). It also recognized with high affinity the
`native CRF1 receptors present on the human retinoblastoma
`cell line Y 79 (pKi ⫽ 9.08 ⫾ 0.20). At 10 ␮M, SSR125543A did
`not interact with the human CRF2␣ receptor expressed in
`CHO cells, or human and rat recombinant CRF-BP (Table 1).
`Binding studies performed with [125I-Tyr0] ovine CRF on
`membrane preparations obtained from rodent brains (rat,
`mouse, and gerbil) did not reveal species differences in affin-
`ity because the respective pKi values of 8.77 ⫾ 0.23, 8.90 ⫾
`0.10, and 9.00 ⫾ 0.00 were very close to the pKi for the
`human CRF1 receptor (Table 1). The high selectivity of
`SSR125543A for the CRF1 receptor was demonstrated by its
`lack of activity (inhibition lower than 50%) at 1 or 10 ␮M in
`125 assays performed by Panlabs and Cerep (receptors,
`transporters, enzymes, and ion channels) (Table 2).
`
`CRF1 Receptor Antagonism by SSR125543A: In Vitro
`Studies
`When rat/human CRF was applied to Y 79 cells, which
`express constitutively CRF1 receptors, the intracellular
`cAMP production was increased by ⬃7-fold over basal levels,
`with an EC50 value of 4.0 ⫾ 0.9 nM (mean ⫾ S.E.M).
`SSR125543A did not modify the basal level of cAMP but fully
`blocked the CRF (10 nM) response with an IC50 value of 3.0 ⫾
`0.4 nM (mean ⫾ S.E.M., n ⫽ 3; Fig. 2A). Under similar
`experimental conditions, the IC50 for antalarmin was 0.8 ⫾
`0.1 nM (mean ⫾ S.E.M., n ⫽ 3; data not shown). Increasing
`concentrations of SSR125543A produced a rightward shift of
`the rat/human CRF concentration-response curve (Fig. 2B),
`without modifying the maximal cAMP production obtained
`with rat/human CRF alone. EC50 values for CRF were 2.5 nM
`(2.1–2.9), 8.3 (5.5–12.4), 55.6 (42.5–73.0), and 92.2 (55.7–
`144.9) (means and confidence limits) in the presence or ab-
`sence of 3, 30, and 100 nM SSR125543A, respectively.
`When rat/human CRF was applied to mouse pituitary
`AtT-20 cells, which express CRF1 receptors, ACTH secretion
`was stimulated by ⬃3-fold over basal levels. SSR125543A did
`not modify basal secretion of ACTH but antagonized the
`ACTH secretion induced by increasing concentrations of rat/
`human CRF. Increasing concentrations of SSR125543A also
`produced a rightward shift of the rat/human CRF dose-re-
`sponse curve and a concentration-dependent inhibition of the
`maximal ACTH secretion elicited by rat/human CRF alone
`(Fig. 3). In the course of three experiments, EC50 values for
`CRF were 1.6 (1.4 –1.9), 11.9 (10.0 –14.1), 49.8 (34.2–74.3),
`and 128.1 (104.6 –160.0) (means and confidence limits) in the
`presence or absence of 3, 30, and 100 nM SSR125543A,
`respectively.
`
`Ex Vivo Binding Assay
`The nonspecific binding obtained in the presence of 1 ␮M
`rat/human CRF in the ex vivo [125I-Tyr0] ovine CRF binding
`assay averaged 20%. To determine the real proportion of
`CRF1 binding sites in this model, competition studies were
`performed with ovine CRF, antalarmin, SSR125543A, anti-
`sauvagine-30, and rat/human CFR(6 –33) on crude brain and
`pituitary homogenates prepared from untreated rats. As
`shown in Fig. 4A and Table 3, the binding of radiolabeled
`ovine CRF could be competed by ovine CRF with a biphasic
`
`curve, suggesting two populations of 50 and 30% of the total
`binding sites and respective pKi values of 8.35 and 6.23.
`Antalarmin and SSR125543A displaced only the first popu-
`lation of sites in a monophasic manner and similar pKi values
`of 8.80 and 8.89. Antisauvagine-30 competed weakly with
`iodinated ovine CRF with a shallow monophasic curve (pKi ⫽
`6.77, nH ⫽ 0.67). In contrast, the selective CRF-BP ligand
`rat/human CRF(6 –33) inhibited only 30% of the total binding
`that represents the non-CRF1 component. Its low affinity for
`ovine CRF compared with rat/human CRF explains the weak
`pKi (6.43) measured in this study. Unlike crude brain mem-
`branes, the specific binding of [125I-Tyr0] ovine CRF to crude
`rat pituitary homogenates represented 90% of the total bind-
`ing and was completely displaced by 100 nM antalarmin.
`Taking into account the high selectivity of antalarmin, CRF1
`specific binding to crude tissue homogenates was considered
`as the maximal displacement measured in the presence of
`100 nM antalarmin.
`The blockade of brain and pituitary CRF1 receptors was
`evaluated in binding studies performed on crude tissue ho-
`mogenates prepared from rats treated with SSR125543A (ex
`vivo binding assay). No specific CRF1 binding could be mea-
`sured after a 2-h oral treatment at the dose of 30 mg/kg,
`whereas at 4 h postadministration, the binding was still
`reduced by 76 ⫾ 2% (Fig. 4C). The presence of SSR125543A
`at the pituitary level was also demonstrated in the same
`experiment, by a decrease of 67 ⫾ 1% in binding 1 h after
`SSR125543A oral administration that reached 78 ⫾ 1% at 2
`and 4 h (Fig. 4C). Another experiment performed under sim-
`ilar conditions demonstrated that ligand binding inhibition
`was still present 24 h after SSR125543A treatment, with
`62 ⫾ 14% inhibition in the brain and 80 ⫾ 2% in the pituitary
`(mean and S.E.M., n ⫽ 3). Dose-effect study performed 2 h
`after oral treatment revealed a dose-dependent inhibition of
`brain CRF1 receptor binding with an ID50 of 6.5 (3.2–11.8)
`mg/kg (mean and confidence limits; Fig. 4B). SSR125543A
`also reached both brain and pituitary after i.p. injection, ID50
`determined 2 h post-treatment being 11.7 (6.0 –23.0) mg/kg,
`slightly higher than after oral administration (data not
`shown).
`
`CRF1 Receptor Antagonism by SSR125543A: In Vivo
`Studies
`CRF-Induced ACTH Secretion in Rats. In conscious
`rats, the plasma level of ACTH determined by radioimmuno-
`assay was 34 ⫾ 4 pg/ml (mean ⫾ S.E.M., n ⫽ 12). Oral
`administration of 30 mg/kg SSR125543A, 2 h before blood
`sampling significantly diminished the ACTH level (18 ⫾ 2
`pg/ml, p ⬍ 0.01, n ⫽ 5). CRF (4 ␮g/kg i.v.) injection, 30 min
`before blood sampling, induced a more than 10-fold stimula-
`tion (269 ⫾ 20 pg/ml) of the ACTH secretion. When admin-
`istered orally at the dose of 30 mg/kg, SSR125543A inhibited
`the increase in ACTH secretion induced by CRF injection
`with significant effects from 1 to 6 h (Fig. 5A). Dose-effect
`studies performed after oral administration of SSR125543A,
`2 h before the CRF injection, yielded ID50 values of 4.9
`(3.0 – 8.6) mg/kg (Fig. 5B) (means and confident limits). After
`i.v. injection of 3 mg/kg SSR125543A, the maximal inhibition
`of CRF-induced ACTH secretion was observed at 5 min
`postinjection. The dose-effect study performed at the same
`time yielded an ID50 of 1.3 (1.2–1.4) mg/kg i.v. (Fig. 5C).
`
`5
`
`

`

`TABLE 2
`Screening of the selectivity of SSR125543A
`Targets and tissue/cell sources for binding, t