Potential Antidepressant Effects of Novel Tropane Compounds, Selective for Serotonin or Dopamine Transporters 1

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1 /97/ $03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 282, No. 2 Copyright 1997 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 282: , 1997 Potential Antidepressant Effects of Novel Tropane Compounds, Selective for Serotonin or Dopamine Transporters 1 S. E. HEMBY, 2 I. LUCKI, G. GATTO, A. SINGH, C. THORNLEY, J. MATASI, N. KONG, J. E. SMITH, H. M. L. DAVIES and S. I. DWORKIN Departments of Physiology and Pharmacology, Bowman Gray School of Medicine (S.E.H., J.E.S., S.I.D.), and Chemistry (C.T.), Center for the Neurobiological Investigation of Drug Abuse, Wake Forest University, Winston-Salem, North Carolina; Departments of Psychiatry and Pharmacology (I.L., A.S.), University of Pennsylvania, Philadelphia, Pennsylvania; and Department of Chemistry (J.M., N.K., H.M.L.D.), State University of New York at Buffalo, Buffalo, New York Accepted for publication April 28, 1997 ABSTRACT The forced swimming test (FST) predicts the efficacy of clinically effective antidepressants. In the present study, using the FST we examined the antidepressant potential of three novel tropane analogs: 8-methyl-2 -propanoyl-3 -(4-(1-methylethyl)- phenyl)-8-azabicyclo[3.2.1] (WF-31) and 2 -propanoyl-3 -(4- (1-methylethyl)phenyl)-8-azabicyclo[3.2.1]octane (WF-50), selective inhibitors of serotonin uptake, and 8-methyl-2 -propanoyl- 3 -(4-(1-methylphenyl)-8-azabicyclo[3.2.1] octane (PTT, WF-11), a selective inhibitor of dopamine uptake. Fluoxetine and GBR were used as controls for selective inhibitors of serotonin and dopamine, respectively. Drugs were administered three times in a 24-hr period between pretest and test sessions. Intraperitoneal administration of WF-31 ( mg/kg), WF-50 ( mg/kg) and fluoxetine ( mg/kg) dose-dependently decreased immobility while increasing swimming. In contrast, WF-11 ( mg/kg) dose-dependently decreased immobility and increased both swimming and climbing, whereas GBR Several new tropane monoamine uptake inhibitors have been developed in an effort to further characterize the cocaine binding sites in the brain and identify the structural requirements that contribute to the specificity and potency of the respective transport processes (Davies et al., 1993, 1994). These monoamine uptake inhibitors were prepared using a unique synthetic strategy that uses the reaction of vinylcarbenoids with pyrroles to yield novel 2 -methyl ketone and 2 -ethyl ketone tropane compounds (Davies et al., 1991, Received for publication September 24, This research was supported in part by United States Public Health Service Research Grants DA (H.M.L.D., J.E.S., S.I.D.), DA (J.E.S.), DA (H.M.L.D.) and MH (I.L.). 2 Present address: Department of Pharmacology, 37 John Morgan Building, 36th and Hamilton Walk, University of Pennsylvania Medical Center, Philadelphia, PA ( mg/kg) decreased immobility, increased climbing but did not affect swimming. In a separate experiment, WF-11 (1.0 mg/kg) increased locomotor activity, whereas a higher dose of WF-11 (3.0 mg/kg) and GBR (30.0 mg/kg) produced stereotypic behaviors, suggesting that the effects in the FST may have been attributable to increases in general activity. However, the effects of WF-11 on swimming in the FST indicate that WF-11 produces antidepressant-like effects in addition to motor stimulation. These results confirm previous results that behavioral patterns manifested in the FST are characteristic of specific monoamine uptake inhibitors. In addition, these results demonstrate that WF-31 and WF-50 produce behavioral patterns similar to fluoxetine in the FST without accompanying decreases in motor activity, suggesting a potential antidepressant action. Based on comparisons with fluoxetine, the data suggest WF-31 and WF-50 may be therapeutically useful as potential antidepressant medications. 1993). These compounds are more resistant to esterase degradation and have been shown to have a longer duration of action in vivo (Hemby et al., 1995). Three compounds prepared using this strategy are WF-31, WF-50 and WF-11 (PTT) (fig. 1). As reported previously, WF-11 binds with high affinity to the dopamine transporter and with lower affinity for the serotonin and norepinephrine transporters (table 1; Davies et al., 1996). In contrast, WF-31 binds with higher affinity to the serotonin transporter than to the dopamine or norepinephrine transporters (Bennett et al., 1995; Davies et al., 1996). Demethylation of WF-31 yields the compound WF- 50, which binds with even higher affinity to the serotonin transporter while binding with low affinity to the dopamine and norepinephrine transporters. The presence of an isopropyl vs. a methyl constituent at the para position on the 3-aryl ABBREVIATIONS: FST, forced swimming test; WF-11, 8-methyl-2 -propanoyl-3 -(4-(1-methylphenyl)-8-azabicyclo[3.2.1]octane; WF-31, 8-methyl- 2 -propanoyl-3 -(4-(1-methylethyl)phenyl)-8-azabicyclo[3.2.1]; WF-50, 2 -propanoyl-3 -(4-(1-methylethyl)phenyl)-8-azabicyclo[3.2.1]octane; ANOVA, analysis of variance. 727

2 728 Hemby et al. Vol. 282 TABLE 1 Ligand affinities for dopamine, serotonin and norepinephrine transporters Potencies of all tropane compounds in displacing [ 125 I]RTI-55 binding in rat striatal membranes are expressed as IC 50 values due to the multiphasic nature of tropane binding to striatal membranes. The potencies of these compounds in displacing [ 3 H]paroxetine in rat frontal cortex membranes and [ 3 H]nisoxetine in rat whole brain (minus brainstem and cerebellum) are expressed as K i. [Binding data have been presented previously (Davies et al., 1996)]. Tropane compound [ 125 I]RTI-50 (dopamine; IC 50 ) [ 3 H]Paroxetine (serotonin; K i ) [ 3 H]Nisoxetine (norepinephrine; K i ) WF WF ,000 WF Values are mean S.E.M. ring results in a 50-fold decrease in potency at the dopamine transporter and a 5-fold increase in potency at the serotonin transporter (WF-31 vs. WF-11). The elimination of the ester group between the aryl group at the 3 position of WF-31, nm Fig. 1. Chemical structures of WF-31 (A), WF-50 (B), fluoxetine (C), WF-11 (D) and GBR (E). Note the single structural difference between WF-31 and WF-11 is the substitution of the isopropyl for the methyl constituent at the para position on the phenyl ring, significantly increasing serotonin transporter selectivity, and the single structural difference between WF-31 and WF-50 is the N- demethylation at the 8 position. WF-50 and WF-11 and the tropane structure result theoretically in greater metabolic stability than related tropanes (i.e., cocaine), as evidenced by the increased duration of behavioral and neurochemical actions (Hemby et al., 1995). Dopamine and serotonin are thought to be involved in a variety of psychiatric illnesses, including substance abuse (Dackis and Gold, 1985; for a review, see Hemby et al., 1997) and depression (Asberg and Martensson, 1993; Brown and Gershon, 1993). Sites on the dopamine and serotonin transporters have become targets for the development of new compounds to treat these disorders. Due to the high affinity and selectivity of the aforementioned novel tropanes at the dopamine and serotonin transporters, the efficacy of these compounds was assessed as potential antidepressant agents in the FST in the present study. The FST, developed by Porsolt et al. (1978a) is one of the more widely used behavioral screens for antidepressant effects (for a review, see Porsolt and Lenegre, 1992). This procedure measures the development of behavioral immobil-

3 1997 Novel Tropanes as Potential Antidepressants 729 ity after a rodent has been placed in a tank of water for a 5-min test session. The development of immobility is facilitated by a 15-min pretest session 24 hr earlier. Antidepressant drugs administered between the pretest and test sessions decrease the duration of behavioral immobility in the FST, the principal dependent measure. The FST is sensitive to all major classes of antidepressant drugs, including tricyclic antidepressants, monoamine oxidase inhibitors and atypical antidepressants (for a review, see Borsini and Meli, 1988), and can distinguish antidepressants from compounds of other therapeutic classes. Recently, a scoring system for the FST was introduced that uses a behavior sampling procedure to quantify the frequency of behaviors (swimming and climbing) and immobility during the test session (Detke et al., 1995). This scoring system was shown to be sensitive for detecting a behavioral pattern for serotonin uptake inhibitors in the FST, which decreased immobility and increased swimming. The system also distinguished serotonin from norepinephrine uptake inhibitors, which decreased immobility and increased climbing but not swimming. The present study examined the potential antidepressantlike effects of WF-11, WF-31 and WF-50 in the FST because of their affinity for monoamine transporter sites. These novel tropane compounds were compared with fluoxetine and GBR because of their selective inhibition of the neuronal uptake of serotonin and dopamine, respectively. The effects of these compounds on motor activity were also assessed to determine whether drugs that decreased immobility in the FST produced corresponding increases in motor activity. Materials and Methods Subjects Adult male Sprague-Dawley rats ( days old; g; Sasco, Inc., Lincoln, NE) were housed two per cage and were maintained on a 12-hr reversed light/dark cycle (lights off, 7:00 a.m.) with food and water available ad libitum in the home cage. Rats were tested in a separate room under dim lighting during the dark phase of the cycle. This strain is more sensitive than some others to the effects of antidepressants in the FST (Porsolt et al., 1978b). All research was conducted according to the Guide for the Care and Use of Laboratory Animals as promulgated by the National Institutes of Health and approved by the Bowman Gray Animal Care and Use Committee. FST Methods and Apparatus Rats were handled for 10 min each for 2 days before initiation of experimentation. Swim sessions were conducted by placing rats in a 13.2-liter Pyrex cylindrical tank (22 46 cm; Fisher Scientific) containing water (at 25 C) for 15 min. The water depth was 35 cm from the bottom, a depth at which rats could not touch the bottom with their tails. The initial 15-min swim pretest was followed 24 hr later by a 5-min test. Rats were removed from the tanks after each swim session, towel dried, placed in a heated cage for 15 min and then returned to their home cages. After the pretest session, rats were assigned randomly to groups to receive either saline (n 26), propylene glycol/saline vehicle (n 10), WF-31 (0.1, 0.3, 3.0 and 10.0 mg/kg; n 10/dose), WF-50 (0.3, 3.0 and 10.0 mg/kg; n 8, 10 and 10/dose, respectively), fluoxetine (0.3, 3.0 and 10.0 mg/kg; n 10/ dose), WF-11 (0.3, 1.0 and 3.0 mg/kg; n 10/dose) or GBR (3.0, 10.0 and 30.0 mg/kg; n 10/dose). Rats received three injections between the pretest and the test swim. At 15 min after the initial swim, rats were injected with saline, vehicle or a dose of one of the five compounds before being returned to the home cage. The following day, rats were injected with saline, vehicle or drug 5 hr and 1 hr before the 5-min test swim. Thus, each rat received three injections before the swim test on the second day. Scoring of the FST For scoring purposes, the test session was divided into 60 five-sec bins, and each bin was assigned a score of immobility, swimming or climbing, according to the scale of Detke et al. (1995). The test sessions were videotaped and the tapes were later scored by an observer who was unaware of the experimental conditions. Locomotor Activity and Stereotypy Rating Several reports suggest that some compounds that potentiate monoaminergic transmission may yield false-positive effects in the FST (for a review, see Borsini and Meli, 1988). These potential false-positives can be detected by evaluating their effects on locomotor activity. For this reason, the effects of these compounds on locomotor activity were evaluated after an identical treatment regimen used in the FST. Subjects were randomly assigned to groups and were administered intraperitoneal saline (n 8), propylene glycol/saline vehicle (n 8), WF-31 (10.0 mg/kg; n 8), WF-50 (10.0 mg/kg; n 8), fluoxetine (10.0 mg/kg; n 8), WF-11 (1.0 and 3.0 mg/kg; n 8/dose) or GBR (3.0 and 30.0 mg/kg; n 8/dose). For each compound, the dose that produced the largest effect in the FST was selected initially. If those doses stimulated motor activity, then the lowest effective dose in the FST was evaluated for a particular compound. The locomotor chambers were clear, square Plexiglas boxes ( cm) placed inside an activity monitor (Med Associates, St. Albans, VT). Each apparatus was equipped with 16 optical sensors spaced 2.5 cm apart on two perpendicular sides located 3.8 cm above the floor. A second set of optical sensors was located 17 cm above the floor and was used to record vertical activity. Sequential beam disruptions were required to register locomotor activity or vertical activity. Successive breaks of the same beam were recorded as repetitive activity. Activity was recorded by an IBMcompatible computer with a data collection program (Med Associates). On the first day, rats were placed in locomotor chambers for 15 min, analogous to the pretest in the FST experiment. The drug treatment regimen was identical to the one used for the FST. On the following day, subjects were placed in the locomotor chambers and activity was recorded for 5 min, analogous to the test day in the FST experiment. Stereotypic behaviors were rated immediately after the test session by an observer who was unaware of the experimental conditions. The observer rated the behavior of each subject in 1-min intervals for 5 min for stereotypy according to the scale of Ellinwood and Balster (1974). Drugs WF-11, WF-31 and WF-50 were prepared according to established procedures (Davies et al., 1994, 1996). The fumarate salts were prepared for the in vivo studies in the following manner. WF-11 fumarate salt (1:1 ratio). Fumaric acid (1.12 g, 9.65 mmol) was added to a solution of the tropane (2.624 g, 9.67 mmol) in 2-propanol (50 ml). All solids were dissolved by heating the mixture carefully. The solvent was removed thoroughly in vacuo. The residue was triturated with dry ether, and the solid thus obtained was collected by filtration (3.064 g, 81% yield), m.p. 142 to 144 C: 1 H NMR (D 2 O) 7.23 (d, J 8.1 Hz, 2 H), 7.15 (d, J 8.1 Hz, 2 H), 6.67 (s, 2 H), 4.07 (br. s, 1 H), 4.04 (br. s, 1 H), 3.56 (ddd, J 13.6, 5.9, 5.2 Hz, 1 H), 3.46 (dd, J 5.3, 1.5 Hz, 1 H), 2.82 (s, 3H), 2.65 (br. dd, J 13.9, 13.8 Hz, 1 H), (m, 5 H), 2.3 (s, 3H), 1.97 (ddd, J 15.0, 3.8, 3.8 Hz, 1 H), 1.43 (dq, J 19.7, 7.0 Hz, 1 H), 0.59 (t, J 7.0 Hz, 3H). Analysis calculated for C 20 H 25 NO 3 C 2 H 4 O H 2 O: C, 65.90; H, 7.67; N, Found: C, 66.03; H, 7.68; N, WF-31 fumarate salt (1:1 ratio). Fumaric acid ( g, 3.05 mmol) was added to a solution of WF-31 ( g, 3.05 mmol) in

4 730 Hemby et al. Vol propanol (25 ml). All solids were dissolved by heating the mixture carefully. The solvent was removed thoroughly in vacuo. The residue was triturated with dry ether, and the solid thus obtained was collected by filtration (1.192 g, 94% yield), m.p. 130 to 132 C: 1 H NMR (D 2 O) 7.31 (d, J 8.2 Hz, 2 H), 7.19 (d, J 8.2 Hz, 2 H), 6.74 (s, 2 H), 4.07 (br. m, 1 H), 4.04 (br. m, 1 H), 3.57 (ddd, J 13.3, 5.5, 5.5 Hz, 1 H), 3.46 (dd, J 5.3, 1.2 Hz, 1 H), 2.87 (septet, J 6.9 Hz, 1 H), 2.67 (br. dd, J 13.5, 13.5 Hz, 1 H), (m, 6 H), 1.40 (dq, J 19.7, 7.0 Hz, 1 H), 1.19 (d, J 6.9 Hz, 6 H), 0.58 (t, J 7.0 Hz, 3 H). Analysis calculated for C 20 H 28 NO 3 C 2 H 4 O H 2 O: C, 68.63; H, 8.03; N, Found: C, 68.53; H, 8.01; N, WF-50 fumarate salt (1:1 ratio). Fumaric acid (0.227 g, 1.96 mmol, 1.0 equiv.) was added to a solution of WF-50 (0.559 g, 1.96 mmol) in 2-propanol (30 ml). All solids were dissolved by heating the mixture carefully. The solvent was removed thoroughly in vacuo. The residue was triturated with dry ether, and the solid thus obtained was collected by filtration and washed with dry ether (0.577 g, 86% yield), m.p. 179 to 180 C: 1 H NMR (D 2 O) 7.18 (d, J 7.4 Hz, 2 H), 7.02 (d, J 7.4 Hz, 2 H), 6.52 (s, 1 H), 4.10 (br. s, 1 H), 4.02 (br. s, 1 H), 3.40 (ddd, J 13.2, 6.1, 5.4 Hz, 1 H), 3.21 (dd, J 6.2, 1.8 Hz, 1 H), 2.70 (septet, J 6.5 Hz, 1 H), 2.40 (ddd, J 13.9, 13.9, 2.6 Hz, 1 H), (m, 5 H), 1.79 (br. dd, J 14.7, 14.7 Hz, 1 H), 1.21 (dq, J 19.6, 7.0 Hz, 1 H), 1.02 (d, J 7.0 Hz, 6 H), 0.70 (t, J 7.0 Hz, 3 H). Fluoxetine HCl was generously supplied by Eli Lilly Corporation (Indianapolis, IN), and GBR HCl was purchased from Research Biochemicals (Natick, MA). WF-11, WF-31, WF-50 and fluoxetine were dissolved in 0.9% sterile saline, and GBR was dissolved in 30% propylene glycol and 0.9% saline. The doses of WF-11, WF-31, WF-50 and fluoxetine are expressed as the weight of the free base; doses of GBR are expressed as the weight of the salt. Drugs were prepared fresh daily and administered intraperitoneally in a volume of 1 ml/kg. All doses were cryptically encoded and decoded after the behavior of all subjects was recorded or rated. Statistical Analysis FST data were analyzed using a one-way ANOVA with dose as the main effect. Motor activity data were analyzed using Student s t test for WF-31, WF-50 and fluoxetine and by one-way ANOVA for WF-11 and GBR Drug effects were compared with saline (fluoxetine, WF-11, WF-31 and WF-50) or propylene glycol/saline (GBR 12909). Post hoc analyses were conducted using Dunnett s test (Neter et al., 1985), and results were considered statistically significant when P.05. Fig. 2. The effects of treatment with WF-31 in the FST. Bars represent the mean S.E.M. counts for the test session. ANOVA revealed a significant effect of dose on immobility [F(4,49) 14.32, P.0001] and swimming [F(4,49) 25.40, P.0001]. P.05 from saline, # P.05 from preceding dose. Fig. 3. The effects of treatment with WF-50 in the FST. Bars represent the mean S.E.M. counts for the test session. ANOVA revealed a significant effect of dose on immobility [F(4,51) 3.86, P.0086] and swimming [F(4,51) 4.60, P.0032]. P.05 from saline, # P.05 from preceding dose. Results FST. All five compounds tested decreased immobility in the FST compared with vehicle controls. WF-31 produced a dose-dependent decrease in immobility [F(4,49) 14.32, P.0001) and a corresponding dose-dependent increase in swimming [F(4,49) 25.40, P.0001] without significantly affecting the frequency of climbing [F(4,49).44, P NS; fig. 2]. WF-50, the demethylated analog of WF-31, decreased immobility [F(4,51) 3.86, P.0086] and increased swimming [F(4,51) 4.60, P.0032] without significantly affecting climbing [F(4,51).81, P NS; fig. 3]. Fluoxetine administration produced a pattern similar to that of WF-31 (fig. 4): a dose-dependent decrease in immobility [F(3, 45) 6.46, P.0011] with a corresponding increase in swimming [F(3, 45) 10.16, P.0001]. Climbing was not significantly affected by fluoxetine administration [F(3, 45).66, P NS]. Administration of the dopamine uptake inhibitors produced patterns of behavior distinguishable from those produced by WF-31, WF-50 and fluoxetine. WF-11 administration produced a significant dose-dependent decrease in immobility [F(3,45) 57.38, P.0001] with corresponding increases in both swimming [F(3,45) 14.03, P.0001] and climbing [F(3,45) 23.32, P.0001; fig. 5]. WF-11 induced significantly more swimming at 1.0 than at 3.0 mg/kg (P.05), whereas climbing was increased significantly at 3.0 mg/kg (P.05). GBR dose-dependently decreased immobility [F(3,39) 71.85, P.0001] and dose-dependently increased climbing [F(3,39) 11.76, P.0001] but exerted no significant effect on swimming [F(3,39) 1.264, P NS; fig. 6]. Locomotor activity and stereotypy rating. Several doses that were effective in decreasing immobility were assessed for their ability to stimulate locomotor activity to determine whether decreases in immobility in the FST corresponded with increases in general activity (table 2). At the doses tested, neither WF-31, WF-50 nor fluoxetine increased

5 1997 Novel Tropanes as Potential Antidepressants 731 Fig. 4. The effects of treatment with fluoxetine in the FST. Bars represent the mean S.E.M. (n 10/dose). ANOVA revealed a significant effect of dose on immobility [F(3,45) 6.46, P.0001] and swimming [F(3,45) 10.16, P.0001]. P.05 from saline, # P.05 from preceding dose. Fig. 5. The effects of treatment with WF-11 in the FST. Bars represent the mean S.E.M. (n 10/dose). ANOVA revealed a significant effect of dose on immobility [F(3, 45) 57.38, P.0001], swimming [F(3, 45) 14.03, P.0001] and climbing [F(3,45) 23.32, P.0001]. P.05 from saline, # P.05 from preceding dose. ambulatory or vertical activity or stereotypic rating. Fluoxetine decreased locomotor activity (t 2.46, df 14, P.027) and vertical activity (t 2.30, df 14, P.037) compared with saline. As previously demonstrated, WF-11 increased locomotor activity [F(2,23) 5.03, P.016] and ratings of stereotypic behavior [F(2,23) 38.83, P.0001; Hemby et al., 1995]. Post hoc analysis revealed 1.0 mg/kg significantly stimulated locomotor activity compared with saline, whereas 3.0 mg/kg significantly increased repetitive activity [F(2,23) 9.16, P.0014]. Both doses of WF-11 increased stereotypy rating. After the administration of GBR 12909, neither locomotor activity nor vertical activity was significantly different from propylene glycol/saline controls at either of the doses tested. However, there was a significant effect of drug on repetitive activity [F(2,23) 44.82, P.0001] and stereotypy rating [F(2,23) 67.76, P.0001]. Post hoc analysis revealed 30.0 mg/kg GBR significantly increased repetitive activity, whereas both 3.0 and 30.0 mg/kg increased stereotypy rating scores. Fig. 6. The effects of treatment with GBR in the FST. Bars represent the mean S.E.M. (n 10/dose). ANOVA revealed a significant effect of dose on immobility [F(3,39) 71.85, P.0001] and climbing [F(3,39) 11.76, P.0001]. P.05 from saline, # P.05 from preceding dose. Discussion The effects of the novel tropanes selective for the serotonin (WF-31 and WF-50) and dopamine (WF-11) transporters were compared with fluoxetine and GBR in the FST. All of the compounds tested decreased immobility in the FST, albeit to varying degrees. Moreover, administration of the selective serotonin uptake inhibitors WF-31, WF-50 and fluoxetine produced a behavioral pattern qualitatively different from the pattern observed after administration of the dopamine uptake inhibitors WF-11 and GBR WF-31, WF-50 and fluoxetine increased swimming while not affecting climbing, whereas WF-11 increased climbing and swimming and GBR increased climbing while not affecting swimming. In addition, the selective dopamine uptake inhibitors WF-11 and GBR administration increased indices of motor activity and stereotypy, an effect not observed for the selective serotonin uptake inhibitors. The effect of the latter may have been due to increased general activity observed after identical treatment regimens. The possibility remains that the doses selected for the locomotor activity tests may not have included the dose(s) produced the greatest amount of stimulation for each drug. WF-31 and WF-50 induced patterns of behavioral effects similar to fluoxetine in the FST (decreased immobility, increased swimming and no change in climbing). This pattern has been reported previously after subcutaneous administration of the serotonin uptake inhibitors fluoxetine, paroxetine and sertraline (Detke et al., 1995). It should be noted that others have reported that fluoxetine is a false-negative in the FST (Borsini, 1995). The discrepancy may be due in part to the sensitivity of the behavioral scoring system and/or the specific parameters for the FST (cylinder diameter, water depth, etc.) used in the present study. Comparison of the serotonin uptake inhibitors WF-31, WF-50 and fluoxetine revealed both similarities and subtle differences among the compounds. WF-31 and fluoxetine were equipotent at decreasing immobility and increasing swimming, although the least effective dose for WF-31 (0.3 mg/kg) produced effects comparable with 3.0 mg/kg fluoxetine. These findings are interesting for two reasons. WF-31 is less potent than fluoxetine in inhibiting [ 3 H]serotonin uptake in vitro and is less

6 732 Hemby et al. Vol. 282 TABLE 2 Effect of intraperitoneal administration of fluoxetine, WF-31, WF-50, GBR and WF-11 on motor activity and stereotypic behaviors Data are presented as mean activity S.E.M. The effects of fluoxetine, WF-31, WF-50 and WF-11 were compared with saline, whereas the effects of GBR were compared with propylene glycol/saline. Locomotor activity and stereotypy rating for the manner in which repetitive activity and stereotypy ratings were determined. Locomotor activity Vertical activity Repetitive activity Stereotypy rating Saline Propylene glycol/saline Fluoxetine (10.0 mg/kg) a a WF-31 (10.0 mg/kg) WF-50 (10.0 mg/kg) GBR mg/kg a 30.0 mg/kg a a WF mg/kg a a 3.0 mg/kg a a a P.05 compared with control values; n 8 rats/group. selective for the serotonin transporter, being 20 times less potent at the serotonin vs. the dopamine transporters (Davies et al., 1996). These data suggest fluoxetine should have been more efficacious than WF-31 in the FST. Second, WF-50 is more potent than WF-31 in inhibiting [ 3 H]serotonin uptake in vitro 3 and is more selective for the serotonin transporter, being 6 times more potent in binding to the serotonin vs. the dopamine transporter. However, WF-50 is 30 times less efficacious than WF-31 in the FST. Differences in in vivo potencies may be due to the duration of action of the parent compound or the metabolites of WF-31 and WF-50 inasmuch as they are less susceptible metabolism from plasma esterases due to the lack of ester linkage between the tropane and phenyl structures of these compounds. Experiments have been initiated to determine the duration of action of WF-31 on in vivo extracellular serotonin concentrations. In addition, metabolites of WF-31 and fluoxetine may potentiate serotonergic transmission and participate in the behavioral effects of these drugs. For example, fluoxetine is metabolized to norfluoxetine, an effective serotonin uptake inhibitor, which has a long half-life in rats (14 hr; Caccia et al., 1990). The relative efficacies of WF-31, WF-50 and their respective metabolites compared with fluoxetine and norfluoxetine warrant further study. Apart from the similarity of effects in the FST, fluoxetine and WF-31 differ in other behavioral effects. At the doses tested, fluoxetine decreased locomotor and vertical activity, whereas neither WF-31 and WF-50 was significantly different from controls. Decreases in motor activity in laboratory animals after the administration of several serotonin uptake inhibitors have been reported (Detke et al., 1995) and appear to be a common feature of currently available compounds with this action. The doses of fluoxetine that were effective in the FST have been shown to decrease food maintained responding, whereas doses of WF-31 effective in the FST do not. 4 These data suggest that although the potencies of WF- 31, WF-50 and fluoxetine are similar in the FST, important differences exist between these compounds in their actions, especially for behaviors unrelated to their antidepressant effects. The lack of effect of WF-31 on both unconditioned and conditioned behaviors is a unique feature of this compound and may prove to be a desirable characteristic in the development of future antidepressant medications. WF-11 and GBR differed from fluoxetine, the reference antidepressant, in several ways. Unlike fluoxetine, the decrease in immobility induced by GBR and WF-11 was accompanied by an increase in climbing for GBR and by an increase in climbing and swimming for WF-11. Increased climbing has been reported after the administration of desipramine and maprotiline, antidepressants that are selective inhibitors of noradrenergic uptake (Detke et al., 1995). However, amphetamine, a psychomotor stimulant and false-positive on many antidepressant tests, also increased climbing in the FST, and a secondary test of locomotor activity was necessary to distinguish amphetamine from the tricyclic antidepressants (Detke et al., 1995). Both WF-11 and GBR increased motor activity at the doses tested, whereas fluoxetine decreased motor activity. These findings lead to the consideration that WF-11 and GBR are false-positives in the FST because the decreases in immobility induced by WF-11 and GBR administration may be attributable to the motor-stimulating effects of these compounds and not a reflection of their potential antidepressant effects. However, WF-11 differed from GBR in that increases in both swimming and climbing behavior were observed. This dual-response pattern in the FST has also been observed after venlafaxine, an antidepressant drug that potently inhibits both norepinephrine and serotonin uptake. 5 It is reasonable to speculate that potent effects of WF-11 at norepinephrine and dopamine uptake sites contribute to producing increases in climbing behavior and locomotor activity and that potent effects at serotonin uptake sites contribute to increased swimming behavior. Thus, the description of behaviors in the FST lends supports the suggestion that WF-11 produces antidepressant-like effects in addition to motor stimulation. Over the past 20 years, several atypical antidepressant compounds have been developed. Some of the main advantages of these compounds in comparison to the tricyclics are that they are less toxic, exert fewer adverse side effects and are also efficacious in other clinical indications (Baldessarini, 1996). For example, fluoxetine is an equiefficacious antidepressant compared with imipramine but lacks the antimus- 3 S. R. Childers, unpublished observations. 4 S. I. Dworkin, unpublished observations. 5 J.-P. Reneric and I. Lucki, unpublished observations.

7 1997 Novel Tropanes as Potential Antidepressants 733 carinic side effects of that tricyclic (e.g., decreased gastrointestinal motility, tachycardia, dry mouth, and so on) and has proved to be useful in the treatment of panic disorder, obsessive-compulsive disorder and eating disorders. The development of tropanes using the synthesis strategy developed by Davies et al. (1991) may yield compounds with high affinity and selectivity for the respective monoamine transporter sites not previously available with existing synthesis strategies. One of the clinical uses of such compounds may be their activity as antidepressants. In addition, structural modifications may enhance or prolong the pharmacological action of these compounds (i.e., absence of the ester group). The development of selective serotonin uptake inhibitors using this synthesis strategy will also provide a new class of compounds for studying the functional roles of the serotonin in the brain. References ASBERG, M. AND MARTENSSON, B.: Serotonin selective antidepressant drugs: Past, present, future. Clin. Neuropharm. 16: S32 44, BALDESSARINI, R. J.: Drugs and the treatment of psychiatric disorders: Depression and mania. In The Pharmacological Basis of Therapeutics, 9th ed., ed. by J. G. Hardman, L. E. Limbird, P. B. Molinoff, R. Ruddon and A. G. Gilman, pp , McGraw-Hill, New York, BENNETT, B., WICHEMS, C. H., HOLLINGSWORTH, C. K., DAVIES, H. M. L., THORN- LEY, C., SEXTON, T., CHILDERS, S. R.: Novel 2-substituted cocaine analogs: Uptake and ligand binding studies at dopamine serotonin and norepinephrine transport sites in the rat brain. J. Pharmacol. Exp. Ther. 272: , BORSINI, F.: Role of the serotonergic system in the forced swimming test. Neurosci. Biobehav. Rev. 19: , BORSINI, F. AND MELI, A.: Is the forced swimming test a suitable model for revealing antidepressant activity? Psychopharmacology 94: , BROWN, A. S. AND GERSHON, S.: Dopamine and depression. J. Neural Transm. 91: , CACCIA, S., CAPPI, M., FRACASSO, C. AND GARATTINI, S.: Influence of dose and route of administration on the kinetics of fluoxetine and its metabolite norfluoxetine in the rat. Psychopharmacology 100: , DACKIS, C. A. AND GOLD, M. S.: New concepts in cocaine addiction: the dopamine depletion hypothesis. Neurosci. Biobehav. Rev. 9: , DAVIES,H.M.L.,KUHN, L., THORNLEY, C., MATASI, J. J., SEXTON T. AND CHILDERS, S. R.: Synthesis of 3 -aryl-8-azabicyclo[3.2.1]octanes with high binding affinities and selectivities for the serotonin transport site. J. Med. Chem. 39: , DAVIES, H. M. L., SAIKALI, E., HUBY, N. J. S., GILLIATT, V. J., MATASI, J. J., SEXTON, T. AND CHILDERS, S. R.: Synthesis of 2 -acyl-3 -aryl-8- azabicyclo[3.2.1]octanes and their binding affinities at dopamine and serotonin transport sites in rat striatum and frontal cortex. J. Med. Chem. 37: , DAVIES, H. M. L., SAIKALI, E., SEXTON, T. AND CHILDERS, S. R.: Novel 2-substituted cocaine analogs: Binding properties at dopamine transport sites in rat striatum. Eur. J. Pharmacol. 244: 93 97, DAVIES,H.M.L.,SAIKALI,E.AND YOUNG, W. B.: Synthesis of ( )-ferruginine and ( )-anhydroecognine methyl ester by a tandem cyclopropanation/cope arrangement. J. Org. Chem. 56: , DETKE, M. J., RICKELS, M. AND LUCKI, I.: Active behaviors in the rat FST differentially produced by serotonergic and noradrenergic antidepressants. Psychopharmacology 119: 47 54, ELLINWOOD, E. H., JR., BALSTER, R. T.: Rating the behavioral effects of amphetamine. Eur. J. Pharmacol. 28: 35 41, HEMBY, S. E., CO, C., REBOUSSIN, D., DAVIES, H. M. L., DWORKIN, S. I. AND SMITH, J. E.: Comparison of a novel tropane analog of cocaine, 2 -propanoyl-3 -(4- tolyl) tropane (PTT) with cocaine HCl in rats: Nucleus accumbens extracellular dopamine concentration and motor activity. J. Pharmacol. Exp. Ther. 273: , HEMBY, S. E., JOHNSON, B. A. AND DWORKIN, S. I.: Neurobiological basis of drug reinforcement. In Drug Addiction and Its Treatment: Nexus of Neuroscience and Behavior, ed. by B. A. Johnson and J. D. Roache, pp , Raven Press, New York, NETER, J., WASSERMAN, W. AND KUTNER, M. J.: Applied Linear Statistical Models: Regression, Analysis of Variance, and Experimental Designs, 2nd ed., Irwin, Homewood, IL, PORSOLT, R. D., ANTON, G., BLAVET, N. AND JALFRE, M.: Behavioral despair in rats: A new model sensitive to antidepressant treatments. Eur. J. Pharmacol. 47: , 1978a. PORSOLT, R. D., BERTIN, A. AND JALFRE, M.: Behavioral despair in rats and mice: Strain differences and the effect of imipramine. Eur. J. Pharmacol. 51: , 1978b. PORSOLT, R. D. AND LENEGRE, A.: Behavioral models of depression. In Experimental Approaches to Anxiety and Depression, ed. by J. M. Elliot, D. J. Heal and C. A. Marsden, pp , John Wiley and Sons, New York, Send reprint requests to: Dr. Scott E. Hemby, Department of Pharmacology, 37 John Morgan Building, 36th and Hamilton Walk, University of Pennsylvania Medical Center, Philadelphia, PA hemby@pharm.med.upenn.edu