Introduction

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The tachykinins, or neurokinins, are a family of small peptides: the main mammalian members are substance P (11 amino acids), neurokinin A (NKA; 10 amino acids), and neurokinin B (NKB; 10 amino acids), which are distributed in both the central (CNS) and peripheral nervous systems. A major location of the tachykinins is in capsaicin-sensitive, primary afferent neurons (unmyelinated sensory fibers) that are predominant in various sites, including the lung, skin, and gastrointestinal tract (Otsuka and Yoshioka, 1993; Maggi, 1996). It has been speculated that the tachykinins play a pathophysiological role in several diseases (Maggio, 1988; Nakanishi, 1991; Maggi et al., 1993; Maggi, 1995). The diverse biological effects of the tachykinins are mediated via three tachykinin receptor subtypes, neurokinin-1 (NK-1R), NK-2R, and NK-3R, which belong to the superfamily of G protein-coupled, seven transmembrane-spanning receptors (Nakanishi, 1991; Gerard et al., 1993; Maggi, 1995).

Of the tachykinins receptors the NK-3R has been the least widely studied, and minimal information exists on the potential relevance of this receptor to disease etiology. A key event in the NK-3R research area occurred in 1995 with the identification of the first potent and selective, nonpeptide NK-3R antagonist, SR 142,801 (Emonds-Alt et al., 1995; Oury-Donat et al., 1995). This was followed soon thereafter with the report of a novel chemical class of potent, competitive, and selective nonpeptide NK-3R antagonists, based on the 4-quinolinecarboxamide backbone (Giardina et al., 1996). Members of this class include SB 223412 (Sarau et al., 1997) and SB 222200 (Sarau et al., 2000). SB 223412 is a moderate CNS-penetrant compound, whereas SB 222200 more effectively crosses the blood-brain barrier.

The NK-3R is distributed in the rodent CNS (Ding et al., 1996; Shughrue et al., 1996; Mileusnic et al., 1999a,b) and in the human brain (Buell et al., 1992; Mileusnic et al., 1999a,b). There is also evidence, from electrophysiological, biochemical, and pharmacological analyses, for the presence of this receptor in the peripheral nervous system, including in guinea pig bronchial parasympathetic ganglia (Myers and Undem, 1993; Myers et al., 1996), guinea pig ileum (Maggi et al., 1990; Yau and Mandel, 1992), rabbit iris sphincter muscle (Medhurst et al., 1997), guinea pig gall bladder (Mawe, 1995), rat kidney (Chen and Hoover, 1993), and rat portal vein (Dion et al., 1987). However, to date there have been no reports of the presence of functional NK-3Rs in human lung. The evaluation of the potential pathophysiological roles of the NK-3R in the peripheral nervous system, and the possible therapeutic utility of NK-3R antagonists in peripheral diseases, will be assisted significantly by the identification of tool compounds that have very limited ability to enter the CNS, certainly lower than SB 223412, SB 222200, and SR 142,801. Herein, we describe the pharmacological and pharmacokinetic profile of an analog of SB 223412 and SB 222200, ()-(S)-N-(-ethylbenzyl)-3-(carboxymethoxy)-2-phenylquinoline-4-carboxamide (SB 235375; Fig. 1), which is a potent and selective, orally active NK-3R antagonist and is a low CNS-penetrant compound in the rat and mouse. SB 235375 is effective against citric acid-induced cough and airways hyper-reactivity in guinea pigs, providing further support for a significant influence of lung NK-3Rs in modulating pulmonary function in this species (Daoui et al., 1997, 1998, 2000).

View larger version (15K): [in this window] [in a new window]   Fig. 1.   Structure of SB 235375.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

All in vivo procedures involving guinea pigs, rabbits, and mice were performed in accordance with protocols approved by the GlaxoSmithKline Institutional Animal Care and Use Committee, and met or exceeded the standards of the American Association for the Accreditation of Laboratory Animal Care, the United States Department of Health and Human Services, and all local and federal animal welfare laws.

Materials. ¹²⁵I-[MePhe⁷]-NKB, ¹²⁵I-NKA, and ¹²⁵I-substance P (all with specific activities of 2200 Ci/mmol) were obtained from PerkinElmer Life Sciences (Boston, MA). NKA, NKB, substance P, and [MePhe⁷]-NKB were purchased from Peninsula Laboratories (Belmont, CA) and senktide {succinyl-[Asp⁹ MePhe⁸]-SP(6-13)}was purchased from California Peptide Research, Inc. (Napa, CA). PEG-400 was purchased from Aldrich Chemical (Milwaukee, WI). Carbachol and atropine were obtained from Sigma Chemical (St. Louis, MO). SB 235375 isomers and racemate, (S)-(+)-N-{{3-[1-benzoyl-3-(3,4-dichlorophenyl)piperidine-3-yl]prop-1-yl}-4-phenylpiperidin-4-yl}-N-methylacetamide (SR 142,801), (S)-N-methyl-N[4-(4-acetylamino-4-phenyl piperidino)-2-(3,4-dichlorophenyl)butyl]benzamide (SR 48,968), and (+)-(2S,3S)-cis-(2-methoxybenzylamino)-2-phenylpiperidine dihydrochloride (CP 99,994) were synthesized in the Department of Medicinal Chemistry, SmithKline Beecham S.p.A, Milan, Italy.

Cloning and Expression of Human and Mouse Tachykinin Receptors. The human (h) NK-1R, hNK-2R, and hNK-3R, and mouse (m) NK-3R and mNK-2R were isolated, cloned, and expressed in Chinese hamster ovary (CHO) or human embryonic kidney (HEK) 293 cell lines (Sarau et al., 1997, 2001). The human receptors were stably expressed in CHO cells for binding experiments and HEK 293 cells for calcium mobilization studies. The clonal cell line expressing the highest number of receptors per cell for each receptor was used in the ligand binding and functional assays. The murine receptors were expressed transiently in HEK 293 cells for both binding and calcium mobilization experiments.

Radioligand Binding Assays. Receptor binding assays were performed with crude membranes from CHO cells stably expressing the hNK-1R (CHO-hNK-1R), hNK-2R (CHO-hNK-2R), or hNK-3Rs (CHO-hNK-3R) and membranes from HEK 293 cells transiently expressing the mNK-3R (HEK 293-mNK-3R) or mNK-2R (HEK 293-mNK-2R), as detailed previously (Sarau et al., 1997, 2001). Competition binding studies for mNK-2R were performed using ¹²⁵I-NKA (0.15 nM) binding to HEK 293-mNK-2R membranes incubated in 150 µl of 25 mM Tris, pH 7.4, containing 4 mM MnCl₂, 1 µM phosphoramidon, and 0.1% ovalbumin, with or without antagonist, for 90 min at 25°C. Incubations were stopped by rapid filtration with a Packard Filtermate 96-well harvester (Packard Instrument Co., Meriden, CT) through Packard GF/C filters that were presoaked for 30 min in 0.1% polyethylenimine. Membranes were washed with 10 ml of ice-cold 20 mM Tris, pH 7.4, containing 0.1% bovine serum albumin, and then 50 µl of Microscint-20 was added to each well, and the radioactivity was counted in a Packard Topcount scintillation counter. Concentration-response curves for each compound were run using duplicate samples in at least three independent experiments. Specific binding was determined by subtracting nonspecific binding from total binding, which was assessed as the binding in the presence of 1 µM cold NKA.

Calcium Mobilization Assay. The functional antagonist activity of SB 235375 was determined by assessing its effects against tachykinin-induced Ca²⁺ mobilization in HEK 293 cells stably expressing the hNK-1R (HEK 293-hNK-1R), hNK-2R (HEK 293-hNK-2R), or hNK-3R receptor (HEK 293-hNK-3R), and in HEK 293 cells transiently expressing the mNK-3R (HEK 293-mNK-3R) or mNK-2R (HEK 293-mNK-2R) as outlined previously (Sarau et al., 1997, 2001).

Senktide-Induced Contraction in Rabbit Isolated Iris Sphincter Muscle. The effect of SB 235375 on senktide-induced contraction of rabbit iris sphincter muscle strips from male rabbits (2-3 kg of body weight; H.A.R.E. Rabbitry, Hewitt, NJ) was determined as outlined previously (Medhurst et al., 1997). Tissues were exposed to SB 235375 (0.03-3 µM) or vehicle (distilled water) for 30 min before cumulative concentration-effect curves to senktide were obtained; atropine (1 µM) was present during construction of the senktide concentration-effect curve. Responses to senktide were expressed as a percentage of the contraction to carbachol (10 µM) added at the start of the experiment, which served as the reference response. The antagonist potency was expressed as pA₂ (log of the dissociation constant), determined from Schild plot analysis of the results (Arunlakshana and Schild, 1958). The dissociation constant, K_b, for the antagonist-NK-3R complex was calculated from the equation K_b = [B]/X  1, where X is the ratio of the concentration of agonist used in the presence and absence of antagonist.

Senktide-Induced Contraction in Guinea Pig Ileal Circular Smooth Muscle. Ileum was removed from male Hartley guinea pigs (weight range 450-650 g; Charles River, Portage, MI) and placed in modified Krebs-Henseleit solution (113 mM NaCl, 4.8 mM KCl, 25 mM NaHCO₃, 1.2 mM KH₂PO₄ · 2H₂O, 2.5 mM CaCl₂, 1.2 mM MgSO₄·7H₂O, and 5.5 mM glucose) overnight at 4°C to diminish spontaneous contractile activity. On the day of the experiment rings of circular smooth muscle (4-6 mm in length) were prepared and placed in 10-ml organ baths containing Krebs-Henseleit solution, which was gassed with 95% O₂, 5% CO₂ and maintained at 37°C and pH 7.4. Preparations were connected via stainless steel hooks and silk suture to Grass FT03C force-displacement transducers. Mechanical responses were recorded isometrically by MP100WS/Acknowledge data acquisition system (BIOPAC Systems, Santa Barbara, CA) run on Macintosh computers. Tissues were equilibrated under 1.5-g resting load (Maggi et al., 1990) for at least 1 h before the start of each experiment and washed every 15 min. After the equilibration period, tissues were contracted with 10 µM carbachol and then rinsed three times over 15 min. The addition of carbachol and rinse was repeated twice to confirm the maximal response of the tissue. The mean of the three carbachol contractions served as a reference contraction for data analysis. Tissues were then rinsed three times over 30 to 40 min before the start of the experiment. Phosphoramidon (10 µM), a neutral endopeptidase inhibitor, and 1 µM CP 99,994, a NK-1R antagonist, were added to each bath before starting the experiment to block enzymatic breakdown of peptides and substance P-mediated contractile effects, respectively.

Citric Acid-Induced Cough in Guinea Pigs. Assessment of cough in male Hartley guinea pigs (500-800 g; Charles River) followed methodology previously described (Kotzer et al., 2000). Briefly, cough was induced by inhalation of an aerosol of 0.4 M citric acid, which has been shown previously to induce the cough reflex in guinea pigs (Forsberg et al., 1992). The aerosol was administered to the animals via a small-volume ultrasonic nebulizer (AeroSonic model 5000D; DeVilbiss, Somerset, PA) connected to the bias flow port immediately before the exposure chamber inlet. A volume of 2 ml of citric acid solution was placed into the ultrasonic nebulizer, and during the 1-min aerosolization period approximately 0.5 ml of the solution was nebulized. The incidence of cough over 13 min (aerosolization time + observation time) was recorded. Animals were used for only one citric acid challenge due to tachyphylaxis of the cough response (Kotzer et al., 2000). SB 235375 was administered i.p. at doses of 3, 5, 10, and 30 mg/kg, 30 min before cough challenge with citric acid.

Citric Acid-Induced Airways Hyper-reactivity in Guinea Pigs. Male Hartley guinea pigs (500-800 g) were treated i.p. with SB 235375 or vehicle, 5 min after administration of thiorphan (1 mg/kg i.p.), the neutral endopeptidase inhibitor, and 30 min before exposure to a 20-min aerosol of 0.4 M citric acid or saline. Twenty-four hours later, the animals were anesthetized with urethane (1.2 g/kg i.p.) approximately 10 min before surgery, and the jugular vein and trachea were cannulated with 50 and 260 polyethylene tubing, respectively. The animals were placed into a whole-body plethysmograph, paralyzed with i.v. succinylcholine chloride (2 mg/kg), and ventilated at 60 breaths/min and 3.75 ml/breath by a Harvard rodent respirator (model 683; Harvard Instruments, South Natick, MA). Transpulmonary pressure was measured with a differential pressure transducer (±80 cm of H₂O, model MP 45; Validyne Engineering, Northridge, CA) that was connected on the positive pressure side to a side arm pressure tap from the trachea and on the negative pressure side to a 16-gauge needle inserted parallel to the heart into the thoracic cavity. Flow through the pneumotachograph was measured with a differential pressure transducer (±2 cm of H₂O, model MP 45; Validyne Engineering). Flow and pressure signals were used to calculate R_L and C_dyn throughout the experiment with Modular Instruments Hardware and BioWindows software (Modular Instruments, West Chester, PA). An ascending noncumulative dose-response curve to i.v. acetylcholine (10, 20, 50, and 100 µg/kg) was determined for each animal.

[Nle¹⁰]-NKA(4-10)-Induced Bronchoconstriction in Guinea Pigs. Male Hartley guinea pigs (600-700 g) were treated with SB 235375 (10 mg/kg i.p), the NK-2R antagonist SR 48968 (10 mg/kg i.p), or vehicle, 10 min before anesthesia with ketamine/rompum (60 mg/kg/10 mg/kg i.m.) followed by vascular and tracheal catheterization as described above. Animals were paralyzed with pancuronium bromide (0.1 mg/kg i.v.), ventilated, and treated with the neutral endopeptidase inhibitor phosphoramidon (1 mg/kg i.v.). Five minutes later (approximately 35 min after i.p. vehicle, SB 235375, or SR 48968), the NK-2R-selective agonist [Nle¹⁰]-NK A(4-10) (0.3 and 1.0 nmol/kg) was administered i.v., and bronchospasm was recorded as described above.

Senktide-Induced Miosis in Rabbits. Senktide (25 µg in 0.2 ml of 5% dimethyl sulfoxide/95% saline) was injected via i.v. bolus in the marginal ear vein. Before injection, baseline pupil diameter measurements were taken with a Finescale Comparator focusing magnifier; the data were recorded in millimeters. Measurements were made and recorded at 2.5-, 5-, 7-, 10-, 15-, and 20-min post-senktide administration (Medhurst et al., 1997). SB 235375 (0.25-1 mg/kg i.v.) or vehicle (0.2 ml of phosphate-buffered saline) was given 2.5 min before administration of senktide.

Senktide-Induced Behavioral Activity in Mice. Male BALB/c inbred mice (20-25 g), obtained from Charles River Breeding Laboratories (Raleigh, NC), were maintained in a barrier-sustained facility. For these experiments, mice were anesthetized using an isoflurane mixture (95% oxygen, 5% isoflurane), the head was shaved, and a midline incision was made in the scalp. Injections into the right lateral ventricle were made at set coordinates from the skull landmark bregma (2 mm posterior, 2 mm lateral, and 2 mm below the skull surface) by using a 27-gauge needle and micromanipulator. Senktide (0.05 nmol) or vehicle (sterile isotonic saline; 5-µl volume) was administered i.c.v. 30 min after administration of oral SB 235375 (3-30 mg/kg) or vehicle (water for SB 235375 or 50% PEG-400/1% methylcellulose for SB 222200). Immediately after the mice were challenged with i.c.v. senktide, the head twitches (i.e., a vigorous shake response) and/or tail whips (i.e., typically counted individually as a rattle that consists of several twitches in tandem) were counted over 15 min (Stoessl et al., 1987, 1990; Sarau et al., 1997). For comparison, the effect of oral administration of SB 222200 (3 mg/kg), the high CNS-penetrant NK-3R antagonist (Sarau et al., 2000), against responses to i.c.v. senktide was explored. For these experiments, the mean and S.E.M. for each group were determined, and data were analyzed using one-way ANOVA with Dunnett's post hoc test; a p value of 0.05 or lower was considered significant.

Pharmacokinetic Studies in Rat. Oral bioavailability evaluations in the rat were carried out using i.v./p.o. crossover experimental design. Indwelling femoral vein (for drug infusion) and artery catheters (for blood sampling) were placed in male Sprague-Dawley rats (300-400 g; n = 6) under ketamine/xylazine anesthesia a week before the studies. On study day 1, each rat received a single i.v. infusion of SB 235375 (1.2 mg/kg/h; 30 min; 80% PEG/20% ethanol). On study day 2, each rat was crossed over to receive a single oral solution dose of SB 235375 (3.4 mg/kg). Blood samples were collected at various times over 24 h after dosing, and plasma was prepared by centrifugation and stored at 30°C until analysis for plasma concentrations of SB 235375 by using high-performance liquid chromatography with triple quadrupole mass spectrometric detection. The lower limit of quantitation was 10 ng/ml for 50 µl of rat plasma.

CNS Penetration Experiments in Rat and Mouse. The CNS penetration study in rats involved i.v. infusion of SB 235375 (1 mg/kg/h) for 6 h to approach steady-state conditions. Blood samples (two 50-µl plasma aliquots) were collected, into heparinized microcentrifuge tubes, from each mouse at 30-min intervals during the final 2 h of infusion, placed on crushed ice, and then centrifuged to isolate plasma. Immediately upon completion of the infusion, the animals were euthanized, and the entire brain was removed and then homogenized in saline. The brain from each mouse was weighed and placed in a volume of chilled isotonic saline equal to 4 times the weight of tissue. Each sample was homogenized individually with a Polytron homogenizer (Brinkmann Instruments, Westbury, NY) and frozen on dry ice. Plasma and brain tissue homogenate samples were stored at 30°C until analysis for concentrations of SB 235375 by using quantitative liquid chromatography with triple quadrupole mass spectrometric detection analysis. Plasma concentrations of SB 235375 are expressed as nanograms per milliliter, whereas brain concentrations are given as nanograms of SB 235375 per gram of total brain weight.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Pharmacological Characterization

In Vitro Studies. Binding experiments: NK-3Rs. SB 235375 produced enantioselective inhibition of the binding of ¹²⁵I-[MePhe⁷]-NKB to CHO-hNK-3R cell membranes. Thus, the active S-enantiomer, SB 235375, inhibited the binding of ¹²⁵I-[MePhe⁷]-NKB to CHO-hNK-3 cell membranes with a K_i of 2.2 ± 0.3 nM (n = 6), whereas the racemate, SB 280765, and the less potent R-isomer, SB 283352, had K_i values of 4.1 ± 1.1 nM (n = 3) and 251 ± 37 nM (n = 3), respectively (Fig. 2A).

View larger version (24K): [in this window] [in a new window]   Fig. 2.   A, competition binding of 125I-[MePhe7]-NKB to CHO-hNK-3 membranes by [MePhe7]-NKB, SR 142801, the enantiomers, and racemate of SB 235375. B, competition binding of 125I-NKA to CHO-hNK-2 membranes by NKA, SR 48968, the enantiomers, and racemate of SB 235375. In A, competition binding to CHO hNK-3 membranes for [MePhe7]-NKB, racemic, R-, and S-isomers of SB 235375 was performed as described under Experimental Procedures. , [MePhe7]-NKB; , (S)-SB 235375; , (R)-SB 283352; , (R,S)-SB 280765-A; , SR 142,801. Values presented are the mean ± S.E.M. of three to seven experiments. In B, competition binding to CHO hNK-2 membranes for NKA, SR 48,968, R- (SB 283352) and S-isomers (SB 235375) and racemate (SB 280765-A) of SB 235375 was performed as described under Experimental Procedures. , NKA; , SR 48,968; , (S)-SB 235375; , (R)-SB 283352; , (R,S)-SB 280765. Values presented are the mean ± S.E.M. of three to six experiments.

Ca²⁺ mobilization studies: NK-3Rs. Cellular functional NK-3R antagonist activity of SB 235375 was determined in Ca²⁺ mobilization studies by using HEK 293-hNK-3R cells. SB 235375 inhibited Ca²⁺ mobilization induced by 1 nM NKB (EC₇₅ for NKB) with an IC₅₀ of 81.9 ± 4.0 nM (n = 3). SB 235375 (33 nM-1 µM) produced a concentration-dependent, surmountable inhibition of NKB-induced Ca²⁺ mobilization in HEK 293-hNK-3 cells. Schild plot analysis of the data revealed a pA₂ of 7.9 (K_b = 12 nM; n = 2) and a slope of 1.1, i.e., not significantly different from 1, indicative of competitive antagonism (Fig. 3). Similar to the results of binding experiments, SB 235375 was a weak inhibitor of NKB (1 nM)-induced Ca²⁺ mobilization in HEK 293-mNK-3R with an IC₅₀ = 3600 nM (n = 2).

View larger version (26K): [in this window] [in a new window]   Fig. 3.   Competitive nature of cellular functional activity of SB 235375. Concentration-response curves were generated for NKB using fura-2-loaded HEK 293 hNK-3 cells in the absence () and presence of 33 nM (), 100 nM (), 330 nM (), and 1000 nM () SB 235375. Results presented are a representative of two experiments.

Selectivity profile. Selectivity of SB 235375 for the hNK-3R relative to other tachykinin receptors was determined from assessment of its effects in competitive binding experiments by using binding of ¹²⁵I-NKA and [³H]substance P to membranes prepared from CHO-hNK-2R and CHO-hNK-1R cell membranes, respectively. SB 235375 had moderate, enantioselective potency for inhibition of ¹²⁵I-NKA binding to CHO-hNK-2R with a K_i = 209 ± 14 nM (n = 6) (Fig. 2B) but was without effect, in concentrations up to 100 µM, on the binding of [³H]substance P to CHO-hNK-1R (n = 2).

Reversibility and time dependence of antagonist activity. The NK-3R antagonist activity of SB 235375 in the Ca²⁺ mobilization assay was rapidly reversible and not time-dependent. Thus, treatment with various concentrations of SB 235375 for 5 min, followed by two washes and resuspension in fresh buffer without antagonist over 30 min, resulted in a significant loss of the inhibitory activity for SB 235375 against NKB (1 nM)-induced calcium mobilization in HEK 293-NK-3R cells [washed, IC₅₀ = 7800 nM; n = 2; control (no washing) = 82 nM; n = 3]. Furthermore, the inhibition of NKB (1 nM)-induced calcium mobilization by SB 235375 was similar with 5-s (IC₅₀ = 146 nM; n = 2) or 5-min incubation (IC₅₀ = 218 nM; n = 2).

Senktide-induced contraction in rabbit isolated iris sphincter muscle and guinea pig ileal circular smooth muscle. NK-3R agonists, such as senktide, potently and effectively contract rabbit isolated iris sphincter muscle (Medhurst et al., 1997) and guinea pig ileal circular smooth muscle (Maggi et al., 1990). SB 235375 (0.03-3 µM) produced a concentration-dependent and surmountable antagonism of senktide-induced contractions in rabbit isolated iris sphincter muscle. Schild plot analysis of the results revealed a pA₂ of 8.1 and a slope of 1.0, indicative of competitive antagonism (n = 5) (Fig. 4). Similar results were obtained with SB 235375 (0.1-10 µM) against senktide-induced contractions in guinea pig ileal circular smooth muscle: pA₂ = 8.3 (slope = 0.8; n = 4) (data not shown).

View larger version (22K): [in this window] [in a new window]   Fig. 4.   Antagonism of senktide-induced contraction in rabbit isolated iris sphincter muscle. Concentration-response curves were generated for senktide in the absence () and presence of SB 235375 (, 30 nM; , 300 nM; and , 3 µM). Results are expressed as a percentage of the response to 10 µM carbachol and are expressed as the mean ± S.E.M. of five experiments. The inset represents the Schild plot analysis of the data.

In Vivo Studies. Senktide (NK-3R)-induced miosis in rabbits. A rabbit model of senktide-induced miosis (pupil constriction) was developed, based upon the contractile effects of NK-3R agonists in the rabbit isolated iris sphincter muscle preparation (Medhurst et al., 1997). Intravenous SB 235375 (0.25-1 mg/kg) produced a potent, dose-related inhibition of i.v. senktide (25 µg)-induced miosis in the rabbit with an ED₅₀ of 0.56 mg/kg (n = 3) (Fig. 5).

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Effects of SB 235375 (0.25-1.0 mg/kg i.v.) against senktide-induced miosis in rabbit. Animals were treated with vehicle (0.2 ml of Dulbecco's phosphate-buffered saline) or SB 235375 (0.25, 0.5, or 1.0 mg/kg i.v.) 2.5 min before exposure to senktide. Results are expressed as a percentage of the control response as the mean ± S.E.M. of three experiments.

Citric acid-induced cough in guinea pigs. Aerosol administration of citric acid to guinea pigs produces cough; this model is used routinely to evaluate potential antitussives. In this study, exposure of saline-treated animals to 0.4 M citric acid for 1 min resulted in 16 ± 0.8 coughs in the 13-min measurement period (n = 8; data not shown). Prior treatment with SB 235375 (3-30 mg/kg i.p.) produced a dose-related inhibition of citric acid-induced cough in guinea pigs with an ID₅₀ of 14.2 mg/kg (Fig. 6A).

View larger version (18K): [in this window] [in a new window]   Fig. 6.   Effect of SB 235375 on citric acid-induced cough (A) and airways hyper-reactivity (B) in guinea pigs. A, animals were treated with vehicle or SB 235375 (3-30 mg/kg i.p.), 30 min before exposure to aerosolized citric acid (0.4 M) for 1 min. The results are expressed as percentage of inhibition of the citric acid-induced cough response compared with vehicle-treated animals. The results are expressed as the mean ± S.E.M; n = 5. B, animals were treated with SB 235375 (, 10 mg/kg i.p.) or vehicle () 5 min after administration of thiorphan (1 mg/kg i.p.) and 30 min before exposure to a 20-min aerosol of 0.4 M citric acid. An aerosol control group was exposed to 0.9% sterile saline for 20 min (). Twenty-four hours later, the responsiveness to acetylcholine (10, 20, 50, and 100 µg/kg i.v.) was assessed. Results are expressed as mean ± S.E.M; n = 4 to 5; *P < 0.05, ANOVA.

Citric acid-induced airways hyper-reactivity in guinea pigs. Exposure to citric acid (0.4 M for 20 min) resulted in a significantly enhanced response to i.v. acetylcholine (50 or 100 µg/kg i.v.), assessed 24 h after citric acid challenge, compared with aerosol saline-exposed animals (Fig. 6B; n = 4-5, P < 0.05). When animals were pretreated with SB 235375 (10 mg/kg i.p., 35 min before citric acid), the citric acid-induced hyper-responsiveness to acetylcholine was attenuated; thus, the acetylcholine dose response was normalized to that observed in saline-exposed animals (Fig. 6B; n = 5, P > 0.05).

Nle¹⁰-NKA (4-10) (NK-2R)-induced bronchospasm in guinea pigs. To ensure that the in vivo effects of SB 235375 were not due to NK-2R antagonism, the activity of the compound against NK-2R-mediated bronchoconstriction in guinea pigs was evaluated. The NK-2R-selective agonist [Nle¹⁰]-NKA (4-10), produced dose-related increases in airway resistance when administered intravenously (0.3-1 nmol/kg) to anesthetized, ventilated guinea pigs that had been treated with vehicle (Fig. 7). [Nle¹⁰]-NKA (4-10)-induced bronchoconstriction was abolished by pretreatment with the NK-2R antagonist SR 48968, but unaffected by SB 235375 (10 mg/kg i.p.) (Fig. 7).

View larger version (27K): [in this window] [in a new window]   Fig. 7.   Effect of SB 235375 on [Nle10]-NKA (4-10)-induced bronchoconstriction in guinea pigs. Animals were treated with vehicle, SB 235375 (10 mg/kg i.p.), or SR 48968 (10 mg/kg i.p.) before anesthesia, cannulation, and ventilation, and then challenged with the NK-2R-selective agonist [Nle10]-NKA (4-10) (0.3 and 1.0 nmol/kg i.v.) 35 min later. Bronchoconstriction was determined as lung resistance changes from baseline (cm of H2O/ml/s) and expressed as mean ± S.E.M. (n = 6-7, *P < 0.001, ANOVA).

Senktide (NK-3R)-induced behavioral responses in mice. Administration of NK-3R-selective ligands, such as senktide, results in a characteristic set of serotonin-mediated behaviors in rodents (Stoessl et al., 1987, 1990). In the current study the effects of oral SB 235375 against behavioral responses induced by i.c.v. senktide in mouse was explored. Oral SB 235375 (3-30 mg/kg; 30-min pretreatment) was without significant effect on the increase in head shakes and tail whips induced by i.c.v. senktide (0.05 nmol); there was a trend toward inhibition at the highest dose of SB 235375 (Fig. 8). In contrast, a low oral dose of SB 222200 (3 mg/kg), a high CNS-penetrant NK-3R antagonist, significantly inhibited, by about 50%, senktide-induced behavioral responses (Fig. 8).

View larger version (25K): [in this window] [in a new window]   Fig. 8.   Effect of oral administration of SB 235375 on behavioral effects of central NK-3R stimulation by i.c.v. senktide. Oral SB 235375 (3-30 mg/kg; 30-min pretreatment) was without effect on the increase in head shakes (A) and tail whips (B) induced by i.c.v. senktide (0.05 nmol). In contrast, oral SB 222200 (3 mg/kg), a high CNS-penetrant NK-3R antagonist, inhibited senktide-induced behavioral responses. Results are given as the mean ± S.E.M.; n = 5 to 8 mice/group; *P < 0.05.

Pharmacokinetic Characterization

The pharmacokinetic profile of SB 235375 in rats (n = 6) was assessed after oral administration (3.4 mg/kg; solution dose) and i.v. infusion (1.2 mg/kg). Systemic plasma clearance of SB 235375 in the rat was moderate (22.0 ± 3.5 ml/min/kg) with a volume of distribution approximately 3 times total body water (2.7 ± 1.0 l/kg), and the terminal half-life was approximately 3 to 5 h. After oral administration, maximum plasma concentrations were obtained at approximately 75 min after dosing, and oral bioavailability was determined to be 23 ± 4%. No significant amounts of circulating metabolites were detected by high-performance liquid chromatography/UV analysis of plasma samples. Preliminary assessment in dogs and monkeys revealed a similar pharmacokinetic profile of SB 235375 in these species: low clearance and oral bioavailability of 40% in monkeys and moderate clearance and oral bioavailability of 39% in dogs (data not shown).

CNS penetration of SB 235375 was assessed in the rat (n = 3). After a 6-h continuous i.v. infusion of SB 235375 (1 mg/kg/h), brain tissue concentrations of SB 235375 were below the limit of detection. Plasma concentrations of SB 235375 were relatively constant during the last 2 h of the infusion (range 670-1400 ng/ml), indicating that steady-state conditions had been achieved.

After oral administration of SB 235375 (10 mg/kg) to mice, plasma concentrations >100 ng/ml were obtained at all times points (0.5, 1, 2, and 4 h), whereas no SB 235375 was detected in brain homogenates at any time postdosing (Fig. 9). At the time used to assess the effects of SB 235375 (10 mg/kg) against senktide-induced behavioral effects, i.e., 30 min after administration, plasma concentrations of SB 235375 were 654 ± 77.6 ng/ml, which is equivalent to 1.4 µM.

View larger version (29K): [in this window] [in a new window]   Fig. 9.   Comparison of plasma and brain concentrations of SB 235375 after oral administration in the mouse. Mice were administered 10 mg/kg SB 235375 by gavage. Plasma () and brain concentrations () of SB 235375 were measured at 0.5, 1, 2, or 4 h as described under Experimental Procedures. Results are expressed as mean ± S.E.M.; n = 4. NQ, not quantifiable, i.e., below the limit of detection.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

It is only relatively recently that potent and selective, nonpeptide NK-3R antagonists have been identified (Emonds-Alt et al., 1995; Oury-Donat et al., 1995; Giardina et al., 1996; Sarau et al., 1997). It is anticipated that these tool compounds will assist greatly in the elucidation of the potential pathophysiological roles of the NK-3Rs, for which there is currently limited information. NK-3Rs have been demonstrated in both the CNS and the peripheral nervous system, where their activation modulates the release of various neurotransmitters (Maggi et al., 1990; Stoessl et al., 1990; Arenas et al., 1991; Schemann and Kayser, 1991; Ramirez et al., 1994). Electrophysiological analysis has also provided evidence of a neuromodulatory role of the NK-3R in the guinea pig tracheal and bronchial parasympathetic ganglia (Myers and Undem, 1993; Myers et al., 1996) and gall bladder (Mawe, 1995). The elucidation of the physiological and pathophysiological roles of the NK-3R requires the utility of compounds with different abilities to enter the CNS. SR 142,801 (Emonds-Alt et al., 1995; Oury-Donat et al., 1995), SB 222200 (Sarau et al., 2000), and, to a lesser extent, SB 223412 (Sarau et al., 1997) effectively enter the CNS. The current study identifies SB 235375 as a potent and selective NK-3R antagonist that is a low CNS-penetrant compound in the rat. Accordingly, SB 235375 would appear to be an appropriate tool compound with which to assess the role of NK-3Rs in peripheral diseases.

SB 235375 belongs to the class of nonpeptide NK-3R antagonists that are based on the 4-quinolinecarboxamide backbone (Giardina et al., 1996). Functional and binding studies indicate that SB 235375 is a high-affinity antagonist for the hNK-3R: pA₂ = 7.9 for inhibition of NKB-induced calcium mobilization in HEK 293-NK-3R cells, and K_i = 2.2 nM for inhibition of ¹²⁵I-MePhe⁷-NKB binding to CHO-hNK-3R cell membranes. In addition, the data indicate that SB 235375 is a selective NK-3R antagonist. Thus, with respect to the tachykinin receptors, SB 235375 has about 100-fold selectivity for the hNK-3R versus the hNK-2R and over 100,000-fold selectivity versus the hNK-1R. Furthermore, SB 235375 was without effect, in concentrations of 1 or 10 µM, in 68 receptor binding, ion channel, and enzyme assays. Overall, SB 235375 has a pharmacological and selectivity profile similar to those of SB 223412 (Sarau et al., 1997) and SB 222200 (Sarau et al., 2000).

Various techniques have demonstrated the presence of NK-3Rs in mammalian CNS, including humans, and also in the peripheral nervous system (Dion et al., 1987; Maggi et al., 1990; Buell et al., 1992; Yau and Mandel, 1992; Chen and Hoover, 1993; Myers and Undem, 1993; Mawe, 1995; Ding et al., 1996; Myers et al., 1996; Shughrue et al., 1996; Medhurst et al., 1997; Mileusnic et al., 1999a,b). NK-3R activation modulates the release of several neurotransmitters, including acetylcholine in the CNS (Arenas et al., 1991) and the gastrointestinal tract (Maggi et al., 1990; Schemann and Kayser, 1991; Ramirez et al., 1994). This suggests that NK-3R antagonists may have therapeutic utility in diseases that are characterized by dysfunction in neuronal inputs. However, there is no clinical information available on the effects of NK-3R antagonists in any disorder and minimal data from the evaluation of compounds in animal models of disease. Regarding peripheral diseases, SR 142,801, the NK-3R antagonist (Emonds-Alt et al., 1995), has demonstrated efficacy in guinea pig (Mazelin et al., 1998) and rat models of gastrointestinal tract diseases (Julia et al., 1999).

There is increasing evidence that NK-3Rs play a significant role in controlling pulmonary function in guinea pigs via an influence on nerve inputs. For example, comprehensive electrophysiological analysis of guinea pig tracheal or bronchial parasympathetic ganglia, including desensitization experiments with an NK-3R-selective agonist and the use of SR 142,801, revealed that exogenous administration of tachykinins or endogenous release using capsaicin increases membrane depolarization by activation of NK-3Rs (Myers and Undem, 1993; Myers et al., 1996). Thus, the evidence suggests that tachykinins released from afferent nerve fibers modulate nerve inputs, including parasympathetic drive, via NK-3R stimulation. These results form part of the growing body of data that local ganglionic reflexes occur in the airways. In vivo studies provide additional support for the in vitro findings. For example, aerosol administration of NKB and NK-3R-selective agonists elicited airways hyper-responsiveness in guinea pigs (Daoui et al., 2000). In addition, in guinea pigs SR 142,801 inhibited substance P-induced airways hyper-reactivity (Daoui et al., 1997) and significantly attenuated citric acid-induced cough, and the airways hyper-reactivity and potentiation of histamine-induced microvascular permeability induced by citric acid (Daoui et al., 1998). The mechanism for the inhibition of airways hyper-responsiveness by the NK-3R antagonists is not clear. The citric acid-induced hyper-responsiveness may involve a serial process involving more than one tachykinin receptor as suggested previously (Daoui et al., 2000). It has been demonstrated that NK-3R-induced airways hyper-responsiveness is not related to bronchoconstriction because the former phenomenon is observed with selective NK-3R agonists at doses that are not bronchoconstrictive (Daoui et al., 2000). In the current study, SB 235375, which is chemically distinct from SR 142,801, also inhibits citric acid-induced cough and airways hyper-responsiveness in guinea pigs. Although SB 235375 has moderate potency at human and guinea pig NK-2Rs, it had had no effect on bronchoconstriction elicited by the NK-2R-selective agonist [Nle¹⁰]-NKA (4-10) in guinea pigs. Overall, the findings suggest that the inhibitory effects of SR 142,801 and SB 235375 in these guinea pig models are indeed due to blockade of NK-3Rs.

A key issue relates to the site of action (peripheral versus central?) for the inhibitory actions of SR 142,801 and SB 235375 against citric acid-induced cough and airways hyper-responsiveness in guinea pigs. Based upon direct assessment of their abilities to enter the brain and/or the potent inhibition of NK-3R-mediated CNS effects in rodents, SR 142,801 (Emonds-Alt et al., 1995), as well as SB 223412 (Sarau et al., 1997) and SB 222200 (Sarau et al., 2000), effectively enter the CNS. Accordingly, they are not appropriate tool compounds to explore exclusively the peripheral roles of the NK-3R. In contrast, the current findings indicate that SB 235375 has limited ability to enter the CNS. Thus, the brain/plasma ratio for SB 235375 in the rat was very low (brain concentrations were below the limit of detection), and significantly less than for SB 222200 (Sarau et al., 2000) and SB 223412 (Sarau et al., 1997). Furthermore, despite possessing about a 2-fold higher affinity for the mNK-3R (K_i = 82 nM) than SB 222200 (K_i = 174 nM; Sarau et al., 2000), concomitant with about 2-fold higher plasma concentrations of SB 235375 over SB 222000 at 30 min after oral administration in the mouse, SB 235375, in oral doses up to 30 mg/kg, was without significant effect on i.c.v. senktide-induced behavioral effects in this species, whereas SB 222200 produced significant inhibition at 3 mg/kg p.o. Direct measurement after oral administration in the mouse demonstrated that SB 235375 concentrations in the brain were below the limit of detection, confirming that it is a low CNS-penetrant compound; in this study high plasma concentrations of SB 235375, equivalent to >1 µM at 30 min postdosing, were detected. The similar effects of SR 142,801 (Daoui et al., 1997, 1998) and SB 225375 against citric acid-induced cough and airways hyper-reactivity in guinea pigs, despite what appears to be marked differences in their abilities to enter the CNS, suggest that it is the lung, rather than the CNS, that is their site of action for producing these effects. However, this hypothesis requires additional investigation. For example, it is possible that, although SB 235375 is a poor CNS penetrant compound, it may enter the brain in sufficient concentrations to exert inhibitory effects on pulmonary function. Pharmacokinetic investigation in rats, dogs, and monkeys demonstrated that SB 235375 is a low-to-moderate clearance molecule with oral bioavailabilities ranging from 23 to 40%. Thus, SB 235375 may also have utility in evaluating the role of peripheral NK-3Rs in animal models in these three species.

The inhibitory effects of SB 235375 in calcium mobilization experiments in HEK 293-hNK-3R are not time-dependent and are reversed rapidly by washout, and the antagonism by SB 235375 of NKB-induced calcium mobilization and senktide-induced contraction in rabbit isolated iris sphincter smooth muscle and guinea pig ileal circular smooth muscle is surmountable. These data indicate the competitive nature of the antagonism by the chemical class to which SB 235375 belongs; this includes the previously described SB 223412 (Sarau et al., 1997) and SB 222200 (Sarau et al., 2000). In contrast, SR 142,801, a member of a structurally distinct chemical class (Emonds-Alt et al., 1995; Oury-Donat et al., 1995), appears to be a noncompetitive antagonist; the inhibitory effects of SR 142801 are time-dependent, insurmountable, and reversed very slowly by washout (Patacchini et al., 1995; Sarau et al., 2000).

In summary, the current study indicates that SB 235375 is a high-affinity, selective, reversible, and competitive antagonist of the hNK-3R. It is a low CNS-penetrant compound and is effective against citric acid-induced airways hyper-reactivity and cough in guinea pigs. The preclinical pharmacodynamic profile of SB 235375 suggests that it will be a useful compound to assist in the elucidation of the pathophysiological roles of NK-3R activation, especially in the peripheral nervous system.