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NEUROPHARMACOLOGY

WAY-163909 [(7bR, 10aR)-1,2,3,4,8,9,10,10a-Octahydro-7bH-cyclopenta-[b][1,4]diazepino[6,7,1hi]indole], a Novel 5-Hydroxytryptamine 2C Receptor-Selective Agonist with Anorectic Activity

Discovery Neuroscience (J.D., H.M., J.Z., S.K., E.S., S.S., L.S., S.R.-L.) and Chemical and Screening Sciences (A.L.S., R.L.V., G.S., B.L.H.), Wyeth Research, Princeton, New Jersey

Received August 2, 2004; accepted September 20, 2004.

	Abstract

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The pharmacological profile of WAY-163909 [(7bR, 10aR)-1,2,3,4,8,9,10,10a-octahydro-7bH-cyclopenta-[b][1,4]diazepino[6,7,1hi]indole], a novel 5-hydroxytryptamine (HT)_2C (serotonin) receptor-selective agonist is presented. WAY-163909 displaced [¹²⁵I]2,5-dimethoxy-4-iodoamphetamine binding from human 5-HT_2C receptor sites, in Chinese hamster ovary (CHO) cell membranes, with a K_i value of 10.5 ± 1.1 nM. Binding affinities determined for the human 5-HT_2A and 5-HT_2B receptor subtypes were 212 and 485 nM, respectively. In functional studies, WAY-163909 stimulated the mobilization of intracellular calcium in CHO cells stably expressing the human 5-HT_2C receptor with an EC₅₀ value of 8 nM, and E_max relative to 5-HT of 90%. WAY-163909 failed to stimulate calcium mobilization in cells expressing the human 5-HT_2A receptor subtype (EC₅₀ » 10µM) and was a 5-HT_2B receptor partial agonist (EC₅₀ 185 nM, E_max 40%). WAY-163909 exhibited negligible affinity (<50% inhibition at 1 µM) for other receptor sites examined, including human 5-HT_1A, D2, and D3 receptors, and the 5-HT transporter binding site in rat cortical membranes. WAY-163909 exhibited weak affinity for the human D4 (245 nM) and 5-HT₇ (343 nM) receptor subtypes and the 1 binding site in rat cortical membranes (665 nM). WAY-163909 produced a dose-dependent reduction in food intake in normal Sprague-Dawley rats (ED₅₀ = 2.93 mg/kg), an effect blocked by a 5-HT_2C receptor antagonist but not by a 5-HT_2A or 5-HT_2B receptor antagonist. In addition, WAY-163909 decreased food intake in obese Zucker rats and diet-induced obese mice with ED₅₀ values of 1.4 and 5.19 mg/kg i.p., respectively, consistent with the potential utility of 5-HT_2C receptor agonists as anti-obesity agents.

With specific reference to obesity, evidence from both transgenic mice with a targeted deletion of the 5-HT_2C receptor subtype (Tecott et al., 1995) and pharmacological studies using 5-HT_2C receptor ligands (for review, see Bickerdike, 2003) supports a potential therapeutic utility of 5-HT_2C receptor agonists as antiobesity agents. 5-HT_2C receptor knockout mice are obese, hyperphagic, have impaired satiety, have elevated insulin and leptin levels and have impaired glucose utilization (Tecott et al., 1995; Heisler et al., 1998; Nonogaki et al., 1998). Moreover, these mice are insensitive to the hypophagic effects of the nonselective 5-HT₂ receptor agonist mCPP (Tecott et al., 1995). mCPP, the most widely studied agonist at 5-HT_2C receptors, decreases food intake in several species, including humans (Samanin et al., 1979; Walsh et al., 1994; Cowen et al., 1995; Kennett et al., 1997; Sargent et al., 1997). Studies using 5-HT antagonists that differ in selectivity among the 5-HT receptor subtypes have provided evidence supporting a role for the 5-HT_2C receptor in the regulation of this mCPP response (Kennett and Curzon, 1988, 1991). More recently described 5-HT_2C agonists such as Ro 60-0175, WAY-161503, VER-3323, PNU-22394, YM348, and WAY-629 have been reported to reduce food intake and body weight in animal models (Martin et al., 1998; Rosenzweig-Lipson et al., 2000; Vickers et al., 2000, 2003; Welmaker et al., 2000; McCall et al., 2001; Bickerdike, 2003; Hayashi et al., 2004; Kimura et al., 2004; Sabb et al., 2004). Conversely, pharmacological agents exhibiting 5-HT_2C antagonist activity such as antipsychotics increase food intake in rodents and cause weight gain in humans (Kennett and Curzon, 1988; Allison et al., 1999; Masand, 2000; Whitaker, 2000). Together, these results clearly support the therapeutic potential of 5-HT_2C receptor agonists as antiobesity drugs.

In the course of our efforts to identify novel 5-HT_2C receptor agonists, we discovered a new heterocyclic ring system initially exemplified by WAY-162545. Chiral resolution of this racemic compound yielded the eutomer WAY-163909, a potent 5-HT_2C receptor-selective agonist. In this report, we describe the in vitro pharmacological profile of WAY-163909. In addition, we demonstrate that WAY-163909 reduces food intake in a number of animal models consistent with a potential therapeutic utility of 5-HT_2C receptor agonists as antiobesity agents.

	Materials and Methods

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5HT_2C Receptor Radioligand Binding. Stable Chinese hamster ovary (CHO) cell lines expressing each of the human 5-HT₂ receptor subtypes were generated in-house using standard protocols. 5-HT_2C receptor binding affinities were determined using the displacement of agonist 2,5-dimethoxy-4-iodoamphetamine ([¹²⁵I]DOI) or antagonist ([³H]mesulergine) radioligand binding to human 5-HT_2C receptor sites in CHO cell membranes. Cell membrane suspensions were prepared in 50 mM Tris-HCl containing 0.1% ascorbic acid, 10 µM pargyline, and 4 mM CaCl₂ and stored at a protein concentration of 1 to 2 mg/ml. Binding experiments were performed in 96-well microtiter plates in a total volume of 200 µl of 50 mM Tris-HCl buffer containing 4 mM CaCl₂, pH 7.4, in the presence of a concentration of radioligand equivalent to the dissociation constant; 0.4 and 0.8 nM for [¹²⁵I]DOI and [³H]mesulergine, respectively, as determined by saturation binding analysis (data not shown). Reactions were initiated by the addition of 100 µl of membrane suspension (50 µg of protein final) and incubated for 60 ([¹²⁵I]DOI) or 120 ([³H]mesulergine) min at room temperature followed by rapid filtration to terminate. Nonspecific binding was determined in the presence of 1 µM DOI and 1 µM mianserin, respectively. Filter plates were dried and radioactivity was determined after addition of 40 µl of Microscint-20 using a Packard TopCount. Analysis of binding data was performed by nonlinear regression using GraphPad Prism.

5HT_2A/B Receptor Radioligand Binding. 5-HT_2A and 5-HT_2B receptor binding affinities were determined using the displacement of agonist ([¹²⁵I]DOI and [³H]5-HT, respectively) radioligand binding to human 5-HT_2A or 5-HT_2B receptor sites in CHO cell membranes. Binding experiments were performed in 96-well microtiter plates in a total volume of 200 µl. Incubation buffer for 5-HT_2A receptor binding studies comprised 50 mM Tris-HCl buffer containing 4 mM CaCl₂, pH 7.4, in the presence of 0.5 nM [¹²⁵I]DOI (approximate K_d concentration). 5-HT_2B receptor binding studies used 50 mM Tris-HCl containing 4 mM CaCl₂, 0.1% ascorbate, 10 µM pargyline, and 20 nM [³H]5-HT (approximate K_d). Nonspecific binding was determined in the presence of 1 µM DOI and 10 µM 5-HT for 5-HT_2A and 5-HT_2B, respectively, and assays were processed as described above for the 5-HT_2C protocol.

Functional Studies. Stable CHO cell lines expressing either the human 5-HT_2A, 5-HT_2B, or 5-HT_2C receptor subtype were used for functional studies using the measurement of agonist-stimulated mobilization of intracellular calcium with the fluorometric imaging plate reader (FLIPR). Cells were maintained and passaged upon reaching approximately 80% confluence. Cells were plated 24 h before the experiment in poly-D-lysine-coated 96-well plates at a density of approximately 60,000 cells per well. In preparation of the assay, the confluent monolayer of cells was washed twice with Hanks' buffered saline solution supplemented with 20 mM HEPES and 2.5 mM probenecid (FLIPR buffer), and then the cells were loaded by adding 4 µM Fluo-4 AM (Molecular Probes, Eugene, OR) in FLIPR buffer for 1 h at 37°C. After loading, the cells were then rinsed twice with FLIPR buffer, and intracellular calcium increases after agonist application were detected by measuring increases in fluorescence with the FLIPR. For evaluation of antagonist activity, compounds were included during the dye-loading step and subsequently stimulated by the addition of an EC₈₀ concentration of 5-HT. Concentration-response data were fit to a four-parameter logistic function for generation of EC₅₀ and IC₅₀ values using Kaleidagraph.

Ancillary Binding Studies. Selectivity binding assays for the human 5-HT_1A, 5-HT₇, and dopamine D2, D3, and D4 receptors were performed using similar protocols to those described above for 5-HT₂ receptor binding studies. In all cases, membranes derived from clonal CHO cell lines were used as receptor source. Radioligands and displacers for nonspecific binding were as follows: 5-HT_1A, [³H]8-hydroxy-2-dipropylaminotetralin, 10 µM 5-HT; 5-HT₇, [³H]lysergic acid diethylamide, 10 µM methiothepin; D2 and D3, [³H]spiperone, 1 µM d-butaclamol; and D4, [³H]spiperone, 10 µM clozapine. Affinity for the 5-HT transporter and the 1 receptor was evaluated by measuring the displacement of ligand ([³H]paroxetine and [³H]prazosin respectively) from these sites labeled in rat cortical membrane preparations.

Food Intake Studies in Rats. Male Sprague-Dawley rats weighing 290 to 370 g or obese Zucker rats weighing 670 to 985 g were individually housed in wire hanging cages. Each cage was equipped with a water spigot attached to an automated watering system that allowed free access to water at all times. Animals were acclimated for a 2-week period to powdered chow (Purina Rat Chow; Ralston Purina, St. Louis, MO) before experiments. Food cups containing powdered chow were removed from the rats' home cage for 24 h before 2-h test sessions. Rats were usually fasted on Monday and Thursday nights, and rats were tested for effects of compounds on food intake on Tuesday and Friday. For the i.p. studies, vehicle and three doses of WAY-163909 (1–10 mg/kg i.p.) in Sprague-Dawley rats (n = 8) and 0.3 to 3 mg/kg i.p., in obese Zucker rats (n = 8) were evaluated in a group of rats in pseudorandom order. For the p.o. studies, vehicle and three doses of WAY-163909 (3–30 mg/kg p.o.) were administered in separate groups (n = 6 per group) of rats. WAY-163909 was dissolved in 0.9% saline and was administered i.p. in a volume of 1 ml/kg or p.o. in a volume of 2 ml/kg immediately before the food cups were replaced in the home cage. Dose calculations were based on active moiety. Data were analyzed using either a one-way analysis of variance (ANOVA) or a repeated measures ANOVA as appropriate. Post hoc tests comparing vehicle to doses of WAY-163909 were conducted using contrasts in a least-squares model. Where appropriate, ED₅₀ values (dose decreasing food intake to 50% of vehicle values) were calculated using nonlinear regression models.

Food Intake Antagonism Studies in Rats. Four dose combinations were evaluated: 1) vehicle + vehicle, 2) vehicle + 10 mg/kg WAY-163909, 3) antagonist + vehicle, and 4) antagonist + 10 mg/kg WAY-163909. Doses, pretreatment times, and route of administration for antagonists were determined based on literature (SB-242084, a selective 5-HT_2C receptor antagonist, Kennett et al., 1997; SB-215505, a selective 5-HT_2B receptor antagonist, Kennett et al., 1998) or other in-house studies (3 mg/kg ketanserin, a nonselective 5-HT_2A receptor antagonist, completely blocks the anorectic effects of 1 mg/kg 5-HT_2A receptor agonist DOI; data not shown). For the studies with SB-242084 (1 mg/kg i.p., 30' pre) or ketanserin (3 mg/kg i.p., 30' pre), vehicle and WAY-163909 (10 mg/kg i.p., 0' pre) were evaluated in Sprague-Dawley rats (n = 8) in pseudorandom order. For the studies with SB-215505 (3 mg/kg p.o., 60' pre), four groups of rats (n = 5 per group) were used. SB-242084 was dissolved in 10% Tween/0.5% methylcellulose, SB-215505 was dissolved in 5% dextrose, and ketanserin was dissolved in sterile water. Dose calculations were based on active moiety. Data were analyzed using either a one-way ANOVA or a repeated measures ANOVA as appropriate. Post hoc tests comparing the groups were conducted using contrasts in a least-squares model.

Food Intake Studies in Mice. Male C57Bl6 mice weighing (54–69 g) had been maintained on a high fat diet for a period of greater than 1 year and were considered to be obese. The mice were individually housed in wire hanging cages. Each cage was equipped with a water spigot attached to an automated watering system that allowed free access to water at all times. Animals were acclimated for a 2-week period to powdered high fat chow before experiments. Food cups containing powdered chow were removed from the mouses' home cage for 24 h before 2-h test sessions. Mice were usually fasted on Monday and Thursday nights and were tested for effects of compounds on food intake on Tuesday and Friday. In all studies, vehicle and three doses of WAY-163909 (3–30 mg/kg i.p.) were evaluated in a group of mice in pseudorandom order. WAY-163909 was dissolved in 0.9% saline and was administered i.p. in a volume of 10 ml/kg immediately before the food cups were replaced in the home cage. Dose calculations were based on active moiety. Data were analyzed using a repeated measures ANOVA. Post hoc tests comparing vehicle to doses of WAY-163909 were conducted using contrasts in a leastsquares model. Where appropriate, ED₅₀ values (dose decreasing food intake to 50% of vehicle values) were calculated using nonlinear regression models.

Chronic Study in Rats. Male Sprague-Dawley rats weighing 195 to 225 g at the start of the study were individually housed in stainless steel cages and were acclimated for 10 days. Animals were weighed and assigned to dosage groups in a manner that minimized mean body weight differences among dosage groups. Food and water were available ad libitum. WAY-163909 (3–30 mg/kg p.o.) was administered once daily, and food intake and body weight were monitored for 10 days. Twenty-four hour food intake was evaluated on day 10. Body weights were taken on days 4, 7, and 10. At the end of the study, blood samples were drawn and triglyceride levels were determined. Data were analyzed using either a one-way ANOVA or repeated measures ANOVA as appropriate. Post hoc tests comparing the groups were conducted using contrasts in a least-squares model.

	Results

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Radioligand Binding Studies. [¹²⁵I]DOI and [³H]mesulergine represent agonist and antagonist radioligands, respectively, suitable for 5-HT_2C receptor binding studies in cells transfected with the 5-HT_2C receptor in the absence of 5-HT_2A and 5-HT_2B receptor sites also labeled by these agents. Figure 1 illustrates the structures of WAY-162545, the originally identified racemic compound, and the enantiomer WAY-163909, generated via chiral high-performance liquid chromatography methodology. Potent 5-HT_2C receptor binding affinity was determined for WAY-162545 for the displacement of [¹²⁵I]DOI (K_i = 6.6 nM), whereas the compound was >50-fold less potent in displacing [³H]mesulergine binding (Table 1). Demonstration of potent binding affinity for WAY-162545 prompted the chiral separation toward the enantiomers to evaluate stereospecificity. 5-HT_2C receptor binding affinities for the enantiomers are shown in Table 1, indicating that WAY-163909 represents the more active enantiomer, whereas WAY-163907 was found to be a relatively weak 5-HT_2C receptor ligand (K_i in micromolar range). As observed for WAY-162545, WAY-163909 was 20-fold less potent in displacing [³H]mesulergine binding compared with [¹²⁵I]DOI binding.

View larger version (10K): [in this window] [in a new window]   Fig. 1. Chemical structures of WAY-162545 (racemic) and WAY-163909.

Primary selectivity determinations for the three compounds using 5-HT_2A and 5-HT_2B receptor radioligand displacement studies are presented in Table 2. In each case, the compounds exhibited weaker affinity for the 5-HT_2A and 5-HT_2B receptor binding sites, e.g., for WAY-163909, the compound was found to display 20- and 46-fold higher affinity toward the 5-HT_2C receptor compared with the 5-HT_2A and 5-HT_2B receptor, respectively. It was further observed that the stereospecificity observed in 5-HT_2C receptor binding affinity was retained in both the 5-HT_2A and 5-HT_2B receptor binding studies, with WAY-163909 representing the more potent enantiomer (Table 2). Ancillary binding studies addressing selectivity of WAY-163909 toward a number of biogenic amine binding sites (Table 3) and a broad spectrum of receptor, transporter, and ion channel targets (Supplemental Data; Table 1) failed to reveal potent binding affinity for any of the targets examined, indicating the compound to be highly selective for the 5-HT_2C receptor subtype.

Functional Studies. Further pharmacological characterization of WAY-163909, WAY-162545, and WAY-163907 was performed using measurements of the stimulation of intracellular calcium mobilization in stable CHO cell lines expressing each of the human 5-HT₂ receptor subtypes. Figure 2 illustrates the concentration-response curves for WAY-162545-(A), WAY-163909-(B), and WAY-163907 (C)-stimulated calcium signaling. WAY-163909 exhibited potent (EC₅₀ = 8 nM) and highly efficacious and best described as a full agonist (E_max = 90 ± 6%) 5-HT_2C receptor agonism, weak (EC₅₀ = 185 nM) 5-HT_2B receptor partial agonism (E_max = 40%) and failed to activate 5-HT_2A receptors. A similar functional profile was observed for the racemate WAY-162545, albeit with slightly weaker potency for activation of 5-HT_2C receptors, whereas WAY-163907 was a relatively weak 5-HT_2C receptor agonist, consistent with the weaker binding affinity and stereospecificity (Table 4). Notably, WAY-163907 exhibited 5-HT_2B receptor partial agonist activity, yet failed to significantly displace agonist radioligand binding.

View larger version (11K): [in this window] [in a new window]   Fig. 2. Effects of WAY-162545 (A), WAY-163909 (B), and WAY-163907 (C) on calcium signaling in stable CHO cells expressing human 5-HT2 receptor subtypes. Agonist-stimulated calcium signaling was measured in cells loaded with the fluorescent calcium indicator dye Fluo-4. Data are expressed as percentage of 10 µM 5-HT maximum response and represent the mean ± S.E.M. from three independent experiments.

Lack of 5-HT_2A agonism exhibited by WAY-163909 prompted us to evaluate its ability to antagonize 5-HT-stimulated calcium mobilization in 5-HT_2A receptor-expressing cells. Results are presented in Fig. 3 indicating that the compound failed to antagonize 5-HT-stimulated calcium signaling, whereas potent antagonism was observed with the selective 5-HT_2A receptor antagonist MDL100907 (IC₅₀ = 0.15 nM). The inability to demonstrate a functional interaction of WAY-163909 with 5-HT_2A receptors in light of its displacement of [³H]5-HT binding to these receptor sights is an obvious discrepancy unexplained at the present time.

View larger version (14K): [in this window] [in a new window]   Fig. 3. Effects of WAY-163909 and MDL100907 pretreatment on 5-HT-stimulated calcium signaling in a stable CHO cell line expressing the 5-HT2A receptor subtype. 5-HT (100 nM)-stimulated calcium was measured after a 60-min preincubation with WAY-163909 or MDL100907. Data are expressed as percentage of 100 nM 5-HT response measured in cells not pretreated with ligand and represent the mean ± S.E.M. from three independent experiments.

Food Intake Studies. WAY-163909 (1–10 mg/kg i.p.) produced dose-dependent decreases in 2-h food intake in Sprague-Dawley rats [F(3,28) = 29.04; p < 0.0001; Fig. 4]. Food intake was significantly decreased by 52% (p = 0.0001) and 96% (p < 0.0001) at 3 and 10 mg/kg i.p., respectively. Similarly, oral administration of WAY-163909 (3–30 mg/kg p.o.) produced dose-dependent decreases in 2-h food intake intake in Sprague-Dawley rats [F(3,23) = 5.56; p = 0.0061; Fig. 4]. Food intake was significantly decreased by 72% (p < 0.0001) at 30 mg/kg p.o. The ED₅₀ values were 2.93 mg/kg (95% CI, 2.1–4.1 mg/kg) and 19.6 mg/kg (95% CI, 11.0–34.8 mg/kg) after i.p. and p.o. administration, respectively.

View larger version (14K): [in this window] [in a new window]   Fig. 4. Effect of WAY-163909 on 2-h food intake in 24-h fasted normal Sprague-Dawley rats. WAY-163909 was evaluated using i.p. or p.o. routes of administration immediately before placement of food cups in the home cage. Data are expressed as grams of food consumed in 2-h period and represent mean ± S.E.M. (n = 8 and 6, respectively, for i.p. and p.o. routes).

In diet-induced obese mice, WAY-163909 (3–30 mg/kg i.p.) produced dose-dependent decreases in 2-h food intake [F(3,36) = 12.78; p < 0.0001; Fig. 5, left]. Food intake was significantly decreased by 46% (p = 0.0028) and 59% (p = 0.0008) and 84% (p < 0.0001) at 3, 10, and 30 mg/kg i.p., respectively. The ED₅₀ value was 5.19 mg/kg (95% CI, 2.4–11.4 mg/kg).

View larger version (17K): [in this window] [in a new window]   Fig. 5. Effect of WAY-163909 on 2-h food intake in 24-h fasted diet-induced obese mice and obese Zucker rats. WAY-163909 was evaluated using i.p. route of administration immediately before placement of food cups in the home cage. Data are expressed as milligrams (mice) or grams (rats) of food consumed in 2-h period and represent mean ± S.E.M. (n = 8).

In obese Zucker rats, WAY-163909 (0.3–3 mg/kg i.p.) produced dose-dependent decreases in 2-h food intake [F(3,28) = 8.99; p = 0.0002; Fig. 5, right]. Food intake was significantly decreased by 73% (p < 0.0001) at 3 mg/kg i.p. The ED₅₀ value was 1.4 mg/kg (95% CI, 0.84–2.4 mg/kg).

Vehicle + WAY-163909 (10 mg/kg i.p.) decreased 2-h food intake in the studies with the 5-HT_2C (SB-242084), 5-HT_2A (ketanserin), and 5-HT_2B (SB-215505) antagonists (p < 0.001 in all three studies; Fig. 6, left, middle, right). SB-242084 (3 mg/kg i.p.; Fig. 3, left), but not SB-215505 (3 mg/kg p.o.; Fig. 3, middle) or ketanserin (3 mg/kg i.p.; Fig. 6, right) blocked the effects of WAY-163909 (10 mg/kg i.p.) on 2-h food intake (p < 0.001).

View larger version (16K): [in this window] [in a new window]   Fig. 6. Effect of 5-HT2 receptor antagonists on the WAY-163909 mediated reduction in 2-h food intake in 24-h fasted normal Sprague-Dawley rats. Antagonists were administered i.p. 30 min (SB242084 and ketanserin) or 60 min (SB215505) before treatment with 10 mg/kg i.p. WAY-163909 was administered coincident with the introduction of food cups for 2 h. Data are expressed as grams of food consumed in 2-h period and represent mean ± S.E.M. (n = 8).

Chronic Study. Twenty-four hour food intake was evaluated at day 10 after once-daily dosing with WAY-163909 (3–30 mg/kg p.o.). As illustrated in Fig. 7 WAY-163909 produced a dose-dependent decrease in food intake [F(3,35) = 7.37; p = 0.0007]. Post hoc tests revealed that both 10 mg/kg (–12%; p = 0.022) and 30 mg/kg (–21%; p = 0.0001), significantly decreased food intake. WAY-163909 also produced decreases in body weight gain [F(3,32) = 10.24; p < 0.0001; Fig. 8]. After vehicle administration, rats gained 62.6 g over the 10-day study, whereas after 30 mg/kg WAY-163909, rats gained only 28.6 g (46% relative to controls). Post hoc tests revealed that 30 mg/kg WAY-163909 decreased body weight gain relative to vehicle on days 4, 7, and 10. Triglyceride levels were also decreased by WAY-163909 (p < 0.01; Table 5). After vehicle administration, triglyceride levels were 263.11 ± 41.58 mg/dl. WAY-163909 at the 30 mg/kg dose produced a statistically significant (p < 0.01) reduction in triglyceride levels by 40% to 157.56 ± 8.35 mg/dl.

View larger version (17K): [in this window] [in a new window]   Fig. 7. Effect of WAY-163909 on 24-h food intake on day 10 of administration of WAY-163909 in normal Sprague-Dawley rats. WAY-163909 was evaluated after 10 days of p.o. administration. Data are expressed as g food consumed in 24-h period and represent mean ± S.E.M. (n = 9 per group).

View larger version (11K): [in this window] [in a new window]   Fig. 8. Effect of daily WAY-163909 administration on body weight in normal Sprague-Dawley rats. WAY-163909 was administered once daily and body weight was evaluated on days 4, 7, and 10 of p.o. administration. Data are expressed as grams of body weight and represent mean ± S.E.M. (n = 9 per group).

	Discussion

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In the present study, we provide a pharmacological characterization of WAY-163909 as a novel and selective 5-HT_2C receptor agonist. The novel heterocyclic ring system represented in WAY-163909 and WAY-162545 evolved from structure-activity relationship studies with compounds identified in a focused screening effort using a pharmacophore model based on compounds with 5-HT_2C receptor agonist activity. WAY-163909 exhibited good binding selectivity compared with the closely related 5-HT_2A and 5-HT_2B receptor subtypes and excellent binding selectivity across a broad spectrum of targets, including both related biogenic amine receptor subtypes and unrelated receptor, ion channel, and enzyme targets. In functional studies using the agonist-stimulated mobilization of intracellular calcium, WAY-163909 also exhibited excellent functional selectivity. This separation is critically important given the postulated side effect liabilities associated with 5-HT_2A and 5-HT_2B receptor activation. Specifically, activation of 5-HT_2A receptors has been linked to the hallucinogenic properties of lysergic acid diethylamide and under certain conditions 5-HT_2B receptor activation has been implicated in primary pulmonary hypertension and cardiac valvulopathy (Fitzgerald et al., 2000; Rothman et al., 2000, Launey et al., 2002). WAY-163909 was observed to be a relatively weak and low intrinsic activity partial 5-HT_2B receptor agonist and was devoid of functional activity at the 5-HT_2A receptor subtype. With respect to both 5-HT_2C binding and functional receptor selectivity WAY-163909 seems to be the most selective agent identified to date compared with other compounds reported in the literature, including Org 37684 (Leyson and Kelder, 1998), Ro 60-0175 (Martin et al., 1998), WAY-161503 (Rosenzweig-Lipson et al., 2000; Welmaker et al., 2000), PNU-22394 (McCall et al., 2001), VER-3323 (Bickerdike, 2003), YM348 (Kimura et al., 2004), and WAY-629 (Sabb et al., 2004). Specifically, although many of the newly reported agents have equivalent low nanomolar affinity for the 5-HT_2C receptor, they have similar high affinity and most significantly equipotent functional activity at the 5-HT_2B receptor subtype. WAY-163909 has distinct advantages over reported 5-HT_2C agonist compounds with respect to both binding and functional selectivity and decreased intrinsic activity at the 5-HT_2A and 5-HT_2B receptors. These enhanced properties would suggest that WAY-163909 will have a low propensity for 5-HT_2A/B receptor-mediated side effects.

From the early pharmacological studies demonstrating anorectic effects of mCPP in multiple species (Samanin et al., 1979; Kennett and Curzon, 1988: Kennett and Curzon, 1991; Walsh et al., 1994; Cowen et al., 1995; Sargent et al., 1997) to more recent data with novel 5-HT_2C agonists that demonstrate effects on both food intake and body weight (Martin et al., 1998; Rosenzweig-Lipson et al., 2000; Welmaker et al., 2000; Vickers et al., 2000, 2003; Hayashi et al., 2004; Sabb et al., 2004), the pharmacological data supporting a role for 5-HT_2C agonists in obesity is compelling. The pharmacological data are supported by studies in mice lacking the 5-HT_2C receptor. 5-HT_2C knockout mice are obese, hyperphagic, have impaired satiety, have elevated insulin and leptin levels, and have impaired glucose utilization (Tecott et al., 1995; Heisler et al., 1998; Nonogaki et al., 1998). Moreover, these mice are insensitive to the hypophagic effects of the serotonergic agonist mCPP (Tecott et al., 1995). The present studies investigated the effects of WAY-163909 on food intake in fasted animals. WAY-163909 produced dose-dependent decreases in food intake in fasted male Sprague-Dawley rats after both intraperitoneal and oral administration with ED₅₀ values of 2.93 and 19.6 mg/kg, respectively. These results confirm previous findings demonstrating anorectic effects of 5-HT_2C agonists and extend these findings to a class of compounds that is structurally distinct from all 5-HT_2C agonists. The pharmacological specificity of the effects of WAY-163909 was demonstrated by the selective antagonism of the anorectic effects of WAY-163909 by the 5-HT_2C receptor antagonist SB-242084. In contrast, antagonists at the 5-HT_2A receptor (ketanserin) or 5-HT_2B receptor (SB-215505) were not able to block the effects of WAY-163909. In additional studies, the anorectic effects of WAY-163909 were also shown in two animal models of obesity (obese Zucker rats and diet-induced obese mice).

Several studies have investigated the effects of 5-HT_2C agonists after repeated administration in rats. mCPP, Ro 60-0175, WAY-161503, and YM348 decrease food intake and/or body weight gain in either lean or obese rats (Vickers et al., 2000; Rosenzweig-Lipson et al., 2000; Hayashi et al., 2004). After chronic administration, WAY-163909 produced decreases in both food intake and body weight gain, and we observed a 40% reduction in triglyceride levels at the end of the study. Similar decreases in food consumption were observed during days 1 through 5 (–23%) and days 6 through 10 (–21%), suggesting that there was no tolerance to the anorectic effects of WAY-163909. Over a 10-day period, body weight gain was reduced by 56%. By comparison, in the chronic studies conducted with Ro 60-0175 (Vickers et al., 2000) and YM348 (Hayashi et al., 2004), the anorectic effect of both these agents was lost by day 10 of the study, in contrast to our observations with WAY-163909, and the reductions in body weight gain were more modest in the region of 20 to 25% compared with 56% for WAY-163909.

In conclusion, WAY-163909 is a novel 5-HT_2C receptor-selective agonist. The anorectic effects of WAY-163909 in multiple animal models coupled with the decreased body weight and the decreased triglyceride levels provide compelling rationale for the potential utility of this compound in the treatment of obesity.

	Acknowledgements

 
We thank Drs. James Barrett and Paul McGonigle for support and Dr. Dave Clarke for assistance with the chronic feeding study.

	Footnotes

 
doi:10.1124/jpet.104.075382.

ABBREVIATIONS: 5-HT, 5-hydroxytryptamine (serotonin); mCPP, 1-(m-chlorophenyl)piperazine; Ro 60-0175, (S)-2-(6-chloro-5-fluoroindol-1-yl)-1-methylethylamine; WAY-161503, (4aR)-8,9-dichloro-2,3,4,4a-tetrahydro-1H-pyrazino[1,2-a]quinoxalin-5(6H)-one; VER-3323, ((2S)-1-[6-bromo-2,3-dihydroindolyl)]-2-propylamine, PNU-22394, 6-methyl-1,2,3,4,5,6-hexahydroazepino[4,5-b]indole; YM348, (S)-2-(7-ethyl-1H-furo[2,3-g]indazol-1-yl)-1-methylethylamine; WAY-629, 1,2,3,4,8,9,10,11-octahydro[1,4]diazepino[6,5,4-jk]cabazole; CHO, Chinese hamster ovary; DOI, 2,5-dimethoxy-4-iodoamphetamine; FLIPR, fluorometric imaging plate reader; ANOVA, analysis of variance; WAY-163909, (7bR,10aR)-1,2,3,4,8,9,10,10a-octahydro-7bH-cyclopenta-[b][1,4]diazepino[6,7,1hi]indole; SB-242084, 6-chloro-5-methyl-1-[2-(2-methylpyridyl 3-oxy)pyrid-5-yl carbamoyl]indoline; SB-215505, 6-chloro-5-methyl-1-(5-quinolyl carbamoyl) indoline; MDL100907, (±)-2,3-dimethoxyphenyl-1-[2-4-(piperidine)-methanol]; CI, confidence interval; Org 37684, 3-(5-methoxy-indan-4-yloxy)-pyrrolidine.

The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.

Address correspondence to: Dr. John Dunlop, Discovery Neuroscience, Wyeth Research, CN 8000, Princeton, NJ 08543. E-mail dunlopj{at}wyeth.com

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