Abstract
The pharmacological effects were assessed for a series of 3α-diphenylmethoxy-1αH,5αH-tropane analogs which have structural similarities to cocaine. Like cocaine, these compounds displaced [3H]WIN 35,428 binding from rat caudate and had affinities ranging from approximately 10-fold greater than cocaine (Ki=11.8 nM) to relatively low affinity (Ki=2000 nM). The compounds also inhibited dopamine uptake with potencies corresponding to their affinities for WIN 35,428 binding sites. Like the parent compound, benztropine, the 3α-(diphenylmethoxy)tropane analogs displaced [3H]pirenzepine from muscarinic M1 receptors with affinities ranging from 2 to 120 nM. Cocaine produced dose-related increases in locomotor activity (horizontal ambulation) in Swiss Webster mice, whereas the 3α-(diphenylmethoxy)tropane analogs generally had lower efficacy than cocaine. Compounds with fluoro-substituents in the phenyl rings generally were among those with efficacy approaching that of cocaine; those with chloro- and bromo-substituents were markedly less efficacious, despite having binding affinities comparable to those of the corresponding fluoro-substituted compounds. The 3α-(diphenylmethoxy)tropane analogs were also examined in rats trained to discriminate saline from cocaine (10 mg/kg, i.p.). Cocaine produced a dose-related increase in responding on the cocaine-appropriate lever, reaching 100% at 10 mg/kg. Only the 4′,4"-difluoro-substituted analog produced effects similar to those of cocaine; the other compounds showed markedly reduced efficacy compared to cocaine. Drug interaction studies showed that the antimuscarinics, atropine and scopolamine, potentiated rather than attenuated the locomotor stimulant and cocaine-like discriminative-stimulus effects of cocaine, indicating that the antimuscarinic effects of the 3α-diphenylmethoxytropane analogs did not contribute to their diminished cocaine-like activity. Studies of the time course of selected compounds indicated that their reduced cocaine-like efficacy was likely not due to behavioral observations being conducted at an inopportune time period. Because none of the 3α-diphenylmethoxytropane analogs studied showed evidence that they were binding to more than one site, and because the structure activity relationships among these drugs are distinctly different from those obtained with cocaine, these data suggest that the 3α-diphenylmethoxytropane analogs are accessing a different binding domain than that accessed by cocaine. Binding to this domain may produce a behavioral profile that is distinct from that of the cocaine-like dopamine uptake inhibitors.
Cocaine inhibits the reuptake of serotonin, norepinephrine, and dopamine by binding to their respective transporters and acts as a local anesthetic by binding to sodium channels. Despite this multiplicity of actions, most of the behavioral effects of cocaine are thought to be mediated by the inhibition of dopamine transport (Heikkila et al., 1979); those actions are thought to be critical components in the abuse of cocaine (Kuhar et al., 1991). This latter hypothesis is supported by several findings. First, dopamine antagonists appear to specifically increase rates of cocaine self-administration in laboratory animals, whereas noradrenergic antagonists do not (deWit and Wise, 1977). Furthermore, lesions of dopamine rich sites in the brain lead to changes in cocaine self-administration that are not produced by lesions in other areas (Roberts et al., 1977; Koob et al., 1987). Moreover, the potencies of monoamine uptake inhibitors in maintaining self-administration behavior are directly related to their affinities for the dopamine transporter and more closely related to those affinities than their affinities for the norepinephrine or serotonin transporters (Ritz et al., 1987; Bergman et al., 1989). Taken together, these results suggest that the inhibition of dopamine reuptake is the critical action of cocaine that leads to its abuse.
Despite considerable evidence supporting the dopamine hypothesis of the abuse liability of cocaine, some conflicting evidence remains. One apparent inconsistency is that not all dopamine uptake inhibitors are subject to abuse as is cocaine (Rothman, 1990). Both bupropion and benztropine (BZT) are dopamine uptake inhibitors that are used therapeutically but are seemingly devoid of any significant abuse liability. The reason for the differences between these drugs and cocaine is currently not clear, although there are several potential explanations. BZT has clinical use in the treatment of Parkinson’s disease and has several known actions in addition to its dopamine uptake inhibiting effects (Coyle and Snyder, 1969). Notable among other actions of BZT are its antimuscarinic effects (Richelson, 1979).
BZT has some stuctural similarities to cocaine, particularly theN-methyl-tropane ring. Rather than the 3β-ester-linked phenyl ring of cocaine, BZT has a diphenyl ether system (see Fig.1). This functional group is also a component of another dopamine uptake inhibitor, GBR 12909. Initial studies of analogs of BZT have indicated that their structure-activity relations differ from those of cocaine analogs (Newman et al., 1994,1995; Meltzer et al., 1994, 1996). For example, the tropane ring of the BZT analogs lacks a substituent at the 2-position that is necessary for high-affinity binding of cocaine to the dopamine transporter (Carroll et al., 1997; Xu et al., 1997). In addition, when the diphenyl ether system of the BZT analogs is in the axial (α) configuration, higher affinity binding results than when that moiety is in the equatorial (β) configuration. Conversely, the benzoyl ester of cocaine is preferred in the equatorial conformation. Furthermore, substitutions on the phenyl rings of BZT alter the binding affinity more dramatically than do similar substitutions on the cocaine analog, WIN 35,065-2 (Newman et al., 1994). Moreover, the displacement of [3H]WIN 35,428 binding by 3α-(diphenylmethoxy)tropane analogs models better for a single site than it does for two sites, whereas the binding of cocaine and several of its analogs is better fit by a two-site model than by a single-site model (Madras et al., 1989; Izenwasser et al., 1994). Finally, initial reports indicated differences between BZT analogs and cocaine with regard to their behavioral effects. For example, the 4′-Cl substituted analog of BZT had minimal efficacy as a locomotor stimulant and did not substitute for cocaine in rats trained to discriminate cocaine from saline (Newman et al., 1994). Similar results have been obtained with the parent compound, BZT (Colpaert et al., 1979; Acri et al., 1996), and BZT has been reported to be less efficacious thand-amphetamine in producing stereotyped behavior (Scheel-Krüger, 1972). These structural points of comparison between BZT and cocaine, along with the divergence in pharmacology, led to the present study in which we examined the pharmacology of a series of substituted phenyl ring analogs of BZT.
Materials and Methods
[3H]WIN 35,428 Binding Assay.
Details of the procedures used have been published previously (Izenwasser et al., 1993). Briefly, male Sprague-Dawley rats (200–250 g; Taconic Farms, Germantown, NY) were decapitated and their brains removed to an ice-cooled dish for dissection of the caudate putamen. The tissue was homogenized in 30 volumes of ice-cold modified Krebs-HEPES buffer (15 mM HEPES, 127 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 2.5 mM CaCl2, 1.3 mM NaH2PO4, and 10 mMd-glucose, pH adjusted to 7.4) using a Brinkman polytron and centrifuged at 20,000g for 10 min at 4°C. The resulting pellet was then washed two more times by resuspension in ice-cold buffer and centrifuged at 20,000g for 10 min at 4°C. Fresh homogenates were used in all experiments.
Binding assays were conducted in modified Krebs-HEPES buffer on ice. The total volume in each tube was 0.5 ml and the final concentration of membrane after all additions was 0.5% (w/v) corresponding to 200 to 300 mg of protein/sample. Triplicate samples of membrane suspension were preincubated for 5 min in the presence or absence of the compound being tested. [3H]WIN 35,428 (final concentration 1.5 nM) was added and the incubation was continued for 1 h on ice. The incubation was terminated by the addition of 3 ml of ice-cold buffer and rapid filtration through Whatman GF/B glass fiber filter paper (presoaked in 0.1% bovine serum albumin in water to reduce nonspecific binding) using a Brandel Cell Harvester (Gaithersburg, MD). The filters were washed with three additional 3-ml washes and transferred to scintillation vials. Absolute ethanol (0.5 ml) and Beckman Ready Value Scintillation Cocktail (2.75 ml) were added to the vials, which were counted the next day at an efficiency of about 36%. Under these assay conditions, an average experiment yielded approximately 6,000 dpm total binding per sample and approximately 250 dpm nonspecific binding, defined as binding in the presence of 100 μM cocaine. Each compound was tested with concentrations ranging from 0.01 nM to 100 μM for competition against binding of [3H]WIN 35,428 in three independent experiments; each experiment was performed in triplicate.
Displacement data were analyzed by the use of the nonlinear least-squares curve-fitting computer program LIGAND (Munson and Rodbard, 1980). Data from replicate experiments were modeled together to produce a set of parameter estimates and the associated standard errors of these estimates. For each compound, the data were modeled for one- or two-site binding, and a two-site binding model is reported if it fit significantly better than a one-site model according to the F-test at p < .05. The Kivalues reported are the dissociation constants derived for the unlabeled ligands. Data for some of these compounds have been previously published (Newman et al., 1995).
[3H]Dopamine Uptake Assay.
[3H]Dopamine uptake was measured in a chopped tissue preparation as described previously (Izenwasser et al., 1990). Briefly, rats were sacrificed by decapitation and their brains were removed to an ice-cooled dish for dissection of the caudate putamen. The tissue was chopped into 225-μm slices on a McIllwain tissue slicer with two successive cuts at an angle of 90°. The strips of tissue were suspended in oxygenated modified Krebs-HEPES buffer (see above), which was pregassed with 95% O2/5% CO2 and warmed to 37°C. After rinsing, aliquots of tissue slice suspensions were incubated in buffer in glass test tubes at 37°C to which either the drug being tested or no drug was added, as appropriate. After a 5-min incubation period in the presence of the test drug, [3H]dopamine (final concentration 15 nM) was added to each tube. After 5 min the incubation was terminated by the addition of 2 ml of ice-cold buffer to each tube and filtration under reduced pressure over glass fiber filters (presoaked in 0.1% polyethylenimine in water). The filters were rinsed and placed in scintillation vials to which 1 ml of methanol and 2 ml of 0.2 M HCl were added to extract the accumulated [3H]dopamine. Radioactivity was determined by liquid scintillation spectrometry at an efficiency of approximately 30%. The reported values represent specific uptake from which nonspecific binding to filters was subtracted.
Uptake data were analyzed using standard analysis of variance (ANOVA) and linear regression techniques (Snedecor and Cochran, 1967). If there was a significant deviation (p < .05) from linearity, the concentration-effect curve was resolved, if possible, into two linear components that were analyzed independently. IC50 values were calculated only on curve components that exhibited a significant linear regression (p < .05). Data for some of these compounds have been previously published (Newman et al., 1995).
[3H]Pirenzepine Binding Assay.
Muscarinic M1 binding was carried out as modified from methods described by Luthin and Wolfe (1984) and Potter et al. (1988). Frozen whole rat brains were prepared as described previously (Bowen et al., 1989). Briefly, whole brain excluding cerebellum (Taconic Farms) was thawed in ice-cold buffer (10 mM Tris-HCl, 320 mM sucrose, pH 7.4) and homogenized with a Brinkman polytron in a volume of 10 ml/g tissue. The homogenate was centrifuged at 1000g for 10 min at 4°C. The resulting supernatant was then centrifuged at 10,000g for 20 min at 4°C. The resulting pellet was resuspended in a volume of 1.53 ml/g in 10 mM Tris buffer (pH 7.4).
Assays were conducted in binding buffer (10 mM Tris-HCl, 5 mM MgCl2). The total volume in each tube was 0.5 ml and the final concentration of membrane after all additions was approximately 200 to 300 mg of protein/sample. Triplicate samples of membrane suspension were preincubated for 5 min in the presence or absence of the compound being tested. [3H]Pirenzepine (specific activity, 73.9 Ci/mmol, from New England Nuclear, Boston, MA, final concentration 3 nM) was added and the incubation was continued for 1 h at 37°C. The incubation was terminated by the addition of 5 ml of ice-cold buffer (10 mM Tris-HCl, pH 7.4) and rapid filtration through Whatman GF/B glass fiber filter paper (presoaked in 0.5% polyethylenimine in water to reduce nonspecific binding) using a Brandel Cell Harvester. The filters were washed with two additional 5-ml washes and transferred to scintillation vials. Absolute ethanol (0.5 ml) and Beckman Ready Value Scintillation Cocktail (2.75 ml) were added to the vials which were counted the next day at an efficiency of about 36%. Under these assay conditions, an average experiment yielded approximately 15,000 dpm total binding per sample and approximately 900 dpm nonspecific binding, defined as binding in the presence of 10 μM quinuclidinyl benzilate. Each compound was tested with concentrations ranging from 0.01 nM to 100 μM for competition against binding of [3H]pirenzepine, in at least three independent experiments, each performed in triplicate. The data obtained were analyzed as described above for data collected in the WIN 35,428 binding assay.
Locomotor Activity.
Ambulatory activity of Male Swiss Webster mice (Taconic Farms) were studied in 40-cm3 clear acrylic chambers. The acrylic chambers were placed inside monitors (Omnitech Electronics, Columbus, OH) that were equipped with light-sensitive detectors spaced 2.5 cm apart along two perpendicular walls. Mounted on the opposing walls were infrared light sources that were directed at the detectors. One count of horizontal activity was registered each time the subject interrupted a single beam. Mice were injected and immediately placed in the apparatus for 60 min, with horizontal activity counts collected every 10 min. Intraperitoneal injections were administered in volumes of 1 ml/100 g. Each dose was studied in eight mice, and mice were used only once. In some experiments, the interaction of atropine or scopolamine with cocaine was assessed by administering the drugs with cocaine (i.p.) immediately before placing the subjects inside the monitors. Horizontal activity was counted as above for a 60-min time period. In other studies, the time course of the effects on locomotor activity of selected compounds was assessed. Mice were injected and immediately placed in the apparatus for 8 h. All other aspects of these experiments were identical with those which assessed activity for 60 min.
For the 60-min studies, the data from the 30-min period in which maximal stimulation of horizontal ambulatory activity was observed were selected for presentation. For those compounds that did not significantly stimulate activity, some dose significantly decreased activity; the data are shown for the time period in which that maximal effect was observed. Each dose-effect curve was analyzed using standard ANOVA and post hoc testing to determine the significance of the effects at individual doses. The ED50 values and their 95% confidence limits (Snedecor and Cochran, 1967) were calculated either for stimulation or depression of activity. For stimulation of activity, the maximum effect produced by any dose was considered the theoretical maximum stimulation for that compound, and an ED50 value was calculated by linear regression as the dose producing 50% of the maximum activity. For this analysis, points on the linear part of the ascending portion of the dose-effect curve were used. For those compounds that did not significantly stimulate locomotor activity, an ED50 value was calculated for the locomotor depressant effect. This ED50 value was calculated as the dose that decreased activity to 50% of control values, and was calculated by linear regression of the data points on the linear part of the descending portion of the dose-effect curve. The data from the 8-h observation period were analyzed using two-way ANOVA and post hoc testing to determine significance of effects at individual doses, time period, and their interaction. ED50 values for locomotor stimulant and depressant effects of these compounds were assessed at selected time periods to assess differences in effects of these drugs based on time since administration.
Cocaine Discrimination.
Male Sprague-Dawley rats (Charles River Breeding Laboratories, Inc., Wilmington, MA) weighing 310 to 385 g were individually housed with free access to water under a 12-h light/dark cycle (lights on at 7:00 AM). The rats were experimentally naive at the beginning of the study. All testing was performed between 9:00 AM and 1:00 PM. Rats were fed 15 g of Purina chow 30 min after testing daily.
Rats were tested in operant-conditioning chambers (modified BRS/LVE, model RTC-022, Laurel, MD and modified Med Associates model ENV 007, St. Albans, VT) housed within light- and sound-attenuating enclosures with white noise present throughout testing. Ambient illumination was provided by lamps mounted at the top of the front panel of the chamber. Two response keys (levers) were set 17 cm apart, with three stimulus lights above each. A force of 0.4 N through 1 mm was required to register a response, and each response produced an audible click from a relay mounted behind the front panel of the chamber. Reinforced responses produced one 45-mg pellet (Bio-Serv, Inc., Frenchtown, NJ) delivered from a dispenser mounted behind the front panel into a food tray located centrally between the response keys.
Rats were initially trained to press both keys under a fixed ratio (FR) schedule of food reinforcement. Responding on each key was trained separately in a mixed order; the active key on a given training session was indicated by illumination of the lamps positioned directly above the lever. Rats were subsequently trained to discriminate i.p. injections of cocaine (10 mg/kg) from i.p. injections of saline. After cocaine injections, responses on only one key were reinforced; after saline injections, responses on the alternate key were reinforced. The assignment of cocaine- and saline-appropriate keys was counterbalanced across rats. Immediately after injection, rats were placed inside the experimental chambers and a 5-min time-out period was initiated, during which all stimulus lamps were extinguished and responding produced feedback clicks but had no other scheduled consequences. All lamps were then illuminated and responses on the appropriate key were reinforced. The FR value was increased to 20 over several training sessions. Responses on the inappropriate key reset the FR response requirement on the appropriate key. Each food presentation was followed by a 20-s time-out period during which all lamps were off, and responding had no scheduled consequences other than the feedback clicks. Sessions ended after 20 food presentations or 15 min, whichever occurred first. As the FR value reached 20, training sessions for which cocaine and saline injections were administered were ordered in a cocaine-saline-saline-cocaine-cocaine-saline… sequence; test sessions were conducted after consecutive saline-cocaine or cocaine-saline training sessions.
In test sessions, different doses of cocaine or doses of the novel compounds were substituted for cocaine or saline. A test session was conducted if the subject met criteria on both of the immediately preceding saline and cocaine training sessions. The criteria were at least 85% cocaine- or saline-appropriate responding overall and during the first FR of the session. Test sessions were identical with training sessions, with the exception that 20 consecutive responses on either key were reinforced.
On some test sessions, the time course of the discriminative-stimulus effect of selected 3α-diphenylmethoxytropane analogs was assessed. Rats were returned to their home cages immediately after injection, and were placed inside the experimental chambers at various times from 15 to 115 min after the injection. With the programmed time-out period that started the session, these procedures resulted in pretreatment intervals of 20 to 120 min.
On-line experimental control and data collection were by MS-DOS computers operating Med Associates software. For each rat, the overall response rate on both keys and the percentage of responses occurring on the cocaine-appropriate key were calculated. The mean values were calculated for each measure at each drug dose tested. If fewer than three rats responded at a particular dose, no mean value was calculated for percentage of cocaine-appropriate responding at that dose. The data were analyzed using standard ANOVA and linear regression techniques to calculate ED50 values and their 95% confidence limits (Snedecor and Cochran, 1967). In addition to the above values, other indications that have been used in the literature to assess the substitution of the test drug for the training drug were examined, including 1) the average percentage of responses on the cocaine-appropriate key before the delivery of the first reinforcer of the session, 2) the percentage of individual subjects selecting the cocaine-appropriate key, based on data from the entire session (an individual subject was considered to have selected a key if the percentage of responses on that key over the entire session was greater than 80%), and 3) the percentage of subjects selecting the cocaine-appropriate key on the first FR of the session. A subject was considered to have “selected” a key if the percentage of responses on that key during the first FR was greater than 80%.
Drugs.
The drugs tested were (−)-cocaine HCl (Sigma Chemical Co.); GBR 12909 diHCl (Research Biochemicals, Inc., Natick, MA), atropine sulfate (Sigma), methylatropine (Sigma), scopolamine (Sigma), methylscopolamine (Sigma), and the diphenylmethoxytropane analogs synthesized in our laboratories (Newman et al., 1994, 1995). The basic skeleton of the diphenylmethoxytropane analogs is shown in Fig. 1. Substitutions examined in the present study were exclusively on the 4′-, 4"-, or 3′-positions. All drugs were dissolved in water or 0.9% NaCl. The drugs were administered i.p. at 1 ml/kg b.wt. All drugs were injected immediately before testing, with the exceptions as noted below.
Results
[3H]WIN 35,428 Binding Assay.
All of the drugs fully displaced [3H]WIN 35,428 from caudate putamen membranes. The affinities (Ki values) for this activity ranged from 11.8 to 2000 nM (Table1). The highest affinity was obtained with the 4′,4"-difluoro-substituted compound which had an affinity for the dopamine transporter that was 10-fold greater than that for the parent compound, BZT. In addition, this compound had an affinity that was 3-fold greater than that of cocaine (Izenwasser et al., 1994). The comparison to cocaine is based on the high-affinity component of WIN 35,428 binding, as WIN 35,428 binding to the dopamine transporter can be modeled significantly better for two sites than one (Madras et al., 1989; Izenwasser et al., 1993). None of the present 3α-diphenylmethoxytropane analogs produced a displacement profile that was better fit to a two-site model than a one-site model.
[3H]Dopamine Uptake Assay.
Each of the compounds also inhibited the uptake of dopamine in chopped caudate-putamen tissue (Table 1). The potencies for this effect ranged from 24 nM (IC50 value) for the 3′,4′-di-chloro,4"-fluoro-substituted compound to 3520 nM for the 4-chloro-substituted analog with the diphenyl ether system in the β conformation. Previous studies have indicated that the inhibition of dopamine uptake produced by cocaine and several cocaine analogs under these procedures is not linear with respect to concentration (Izenwasser et al., 1990). In contrast to the effects obtained with cocaine and its congeners, the results with the present diphenylmethoxytropane analogs did not significantly deviate from linearity. In general, the potencies of these compounds for the inhibition of dopamine uptake were well correlated with their affinities for the dopamine transporter labeled with [3H]WIN 35,428 (r2 = 0.770;p < .001).
[3H]Pirenzepine Binding Assay.
All of the drugs displaced [3H]pirenzepine from whole-brain membranes (Table 1). The affinities (Ki values) for this activity ranged from 2.1 nM for the parent compound, BZT, to 120 nM for the 4′,4"-dimethoxy-substituted analog. All of the compounds displaced [3H]pirenzepine with a profile that was best fit to a one-site model. The relation between structure and affinity for the M1 muscarinic site was distinctly different from that between structure and affinity for the dopamine transporter as indicated by a lack of a significant correlation between the two groups of affinities (r2 = 0.126,p = .137).
Locomotor Activity.
Cocaine, as has been demonstrated previously, increased horizontal ambulatory activity, and under the present conditions 29.4 μmol/kg produced a maximum of approximately 15,200 counts over 30 min, with higher doses producing less stimulation (Fig. 2, filled circles; see Table 1 for absolute count numbers). The diphenylmethoxytropane analogs showed different degrees of stimulant activity (Fig. 2). The compounds with apara-fluoro substitution as a group (Fig. 2A) had the highest efficacy in stimulating activity among the 3α-diphenylmethoxytropane analogs, with the 4′,4"-difluoro compound showing the greatest effect. Compounds with apara-chloro substituent were generally less efficacious than the corresponding fluoro-substituted analogs (Fig. 2B). For example, 4′-fluoro-3α-(diphenylmethoxy)tropane produced a maximum stimulation to approximately 10,200 counts over 30 min, whereas 4′-chloro-3α-(diphenylmethoxy)tropane (4′-Cl-BZT) produced a maximum stimulation to approximately 7,800 counts. Similarly, the maximum stimulation produced by 4′,4"-difluoro-3α-(diphenylmethoxy)tropane (4′,4"-diF-BZT) was approximately 12,400 counts, but the maximum stimulation produced by 4′,4"-dichloro-3α-(diphenylmethoxy)tropane (4′,4"-diCl-BZT) was 5,500. As noted previously (Newman et al., 1994), 4′-Cl-BZT, which has the diphenyl-ether system attached to the tropane ring with an axial (α) stereochemistry, had higher affinity than its stereoisomer with the diphenyl-ether system in the equatorial (β) configuration (Table 1). In the present study the 4′-chloro-3β-(diphenylmethoxy)tropane [4′-Cl-BZT (β)] produced less stimulation than its α analog, 4′-Cl-BZT (Table 1); this is consistent with its lower affinity for the dopamine transporter. Various other substituents, including methyl and methoxy, yielded compounds with minimal, if any, stimulant activity. (“Minimal” stimulant activity did not achieve statistical significance across a range of doses, from those having no effect to those virtually eliminating locomotor activity.) (Fig. 2C; italicized ED50 values in Table 1).
Eleven compounds produced a significant increase in locomotor activity; for these, a stimulant ED50 value was calculated (Table 1). The relation between stimulant potency of these compounds and affinity for the dopamine transporter was examined by linear regression of the log stimulant ED50 values and log Ki values for displacement of [3H]WIN 35,428. The regression was statistically significant (r2 = 0.432;p = .028), and the slope of the regression line was −0.354, indicating an inverse relation between binding at the dopamine transporter and potency for stimulation of locomotor activity. The linear regression of depressant log ED50 and dopamine transporter Ki values for the eight compounds that only decreased locomotor activity (italicized ED50 values in Table 1) was not significant (r2 = 0.151, p = .341). Linear regression of the stimulant log ED50values and log Ki values for displacement of [3H]pirenzepine was not statistically significant (r2 = 0.028; p= .624). This lack of correlation was obtained despite the efficacy of atropine and scopolamine in stimulating locomotor activity. As can be seen in Fig. 3, both antimuscarinic agents increased locomotor activity in a dose-related manner, although with efficacy less than that obtained with cocaine (compare maximal effects shown in Fig. 3 to maximal effects of cocaine shown in Fig. 2and Table 1). Scopolamine was approximately 10-fold more potent than atropine in stimulating locomotor activity; this relative potency relation is in accord with published differences for the in vivo potency of these two compounds for the antagonism of centrally-mediated muscarinic effects (e.g., McKeon, 1967; Malick and Barnett, 1975).
In contrast to the regression of stimulant potencies, there was a significant relation between potency in decreasing locomotor activity and affinity for M1 muscarinic sites. The linear regression of values for M1 logKi value and depressant log ED50 value for the 3α-diphenylmethoxytropane analogs that did not stimulate locomotor activity (italicized values in Table 1) was significant (r2 = 0.548,p = .036), although the relation was an inverse relation; compounds with higher affinity for M1muscarinic receptors had lower potency in decreasing locomotor activity. These results suggest that affinity for M1 muscarinic receptors interferes with the decreases in locomotor activity obtained with these compounds.
Cocaine Discrimination.
As has been shown in the past, subjects trained to discriminate 10.0 mg/kg (29.4 μmol/kg) cocaine showed a dose-related increase in the percentage of responses emitted on the cocaine-appropriate response key as dose was increased up to the training dose (Fig. 4, filled circles). In contrast, none of the 3α-diphenylmethoxytropane analogs produced a maximum level of drug appropriate responses that exceeded 70% (Fig. 4, A and B; Table 2; column A). The most efficacious of the 3α-diphenylmethoxytropane analogs were generally those with a para-F substitution on at least one ring of the diphenyl ether system (Fig. 4A; Table 2, column A). The greatest efficacy was achieved with 3′,4′-dichloro, 4"-F-3α-(diphenylmethoxy)tropane, which had a maximal effect of 68% cocaine-appropriate responding (Fig. 4A, triangles) with evidence of a plateau occurring at doses of 7 and 13 μmol/kg. Other compounds (e.g., 4′,4"-diF-BZT and 3′,4′-difluoro-3α-(diphenylmethoxy)tropane) had comparable efficacy at the highest dose at which reliable responding was obtained (Table 2). Compounds with paraBr, Cl, or methoxy substitutions were generally less efficacious than those with at least one F substituent in producing cocaine-appropriate responses (Fig. 4B; Table 2, column A). The exceptions to this generalization are 4′-Br,4"-F-BZT, which was among the least efficacious of compounds with a fluoro substituent (Fig. 4A, hexagons; Table 2), and 4′-Cl-BZT, which showed a maximal effect of 55% cocaine-appropriate responses (Fig. 4B, squares). Among the remaining compounds, the maximum effect was the 35% cocaine-appropriate responses produced by 4′,4"-dibromo-3α-(diphenylmethoxy)tropane (4′,4"-diBr-BZT) (Fig. 4B, open circles; Table 2, column A).
In addition to the average among individual subjects of their percentages of responses on the cocaine-appropriate key over the entire session (Table 2, column A), also examined were 1) the average percentage of responses on the cocaine-appropriate key during the first FR of the session (Table 2, column B); 2) the percentage of individual subjects selecting the cocaine-appropriate key based on the data from the entire session (Table 2, column C); and 3) the percentage of subjects selecting the cocaine-appropriate key during the first FR of the session (Table 2, column D). Each of these other measures, as did the average number of responses on the cocaine-appropriate key (Table2, column 1), indicated that none of the compounds had an efficacy comparable to that of cocaine. The correspondence of all of these methods of assessing efficacy in substitution for cocaine is reflected in the generally high and significant correlations among the measures, as shown in Table 2. Each of these other measures further substantiated that the 3α-diphenylmethoxytropane analogs did not fully substitute for cocaine, and that the para-F-substituted compounds generally were more efficacious than the others.
Full cocaine-like discriminative stimulus effects of the 3α-diphenylmethoxytropane analogs could have been obscured by other effect(s) of the drugs. One primary candidate effect of these drugs would be that mediated by actions at muscarinic receptors. BZT is known for its antimuscarinic actions, and all of the compounds had affinity for M1 receptors (Table 1). Antimuscarinic effects could produce disruptions in responding at doses below those that would produce cocaine-like activity; as a result, the relevant doses for cocaine substitution may not have been tested. Had disruptions in responding mediated by antimuscarinic M1 actions obscured cocaine-like discriminative stimulus effects, there would be a relationship between the potencies of the 3α-diphenylmethoxytropane analogs for decreasing rates of responding and the affinity for M1 muscarinic receptors. The log ED50 values for the decreases in response rates (Fig. 4, C and D) were not linearly related to the log Ki values for displacement of [3H]pirenzepine (r2= 0.012; p = .732). The correlation between the log ED50 value for the decreases in response rates and the log Ki value for displacement of [3H]WIN 35,428 was higher than that for the log ED50 value and the log M1affinity, although it also was not significant (r2 = 0.099; p = .321). These results, with those for muscarinic M1receptor binding, suggest that the affects of these drugs on response rates were not related in a simple manner to affinity at either M1 muscarinic or dopamine transporter binding sites.
Prior treatment with either atropine or scopolamine produced a decrease in the calculated ED50 value for cocaine in stimulating locomotor activity in mice (Table3) without altering the maximal effect of cocaine (one-way ANOVA, F3,28 = 1.90;p = .15). These changes in ED50values, however, did not achieve statistical significance (95% confidence limits overlapped).
Antimuscarinic effects of the 3α-diphenylmethoxytropane analogs could have produced a “perceptual masking” of the cocaine-like discriminative stimulus effects that was not due to a simple disruption in the ability of the subject to respond. According to that interpretation, the subjective effects produced by the antimuscarinic actions interferes with the identification of the cocaine stimulus. Accordingly, a muscarinic antagonist should be capable of attenuating the discriminative stimulus effects of cocaine. Figure5 shows the interaction of atropine with cocaine. Atropine, when administered alone, generally produced only saline-appropriate responses (Fig. 5, top, triangles) across the range of doses from those that had minimal effects on response rates to those virtually eliminating responding (Fig. 5, bottom, triangles). Atropine, in combination with the 10 mg/kg training dose of cocaine, did not appreciably alter the discriminative effects of cocaine (Fig. 5, top, diamonds), but generally added to the disruptive effects of cocaine on response rates (Fig. 5, bottom, diamonds). Similarly, atropine was ineffective in attenuating the discriminative effects of a lower (5.6 mg/kg) dose of cocaine (Fig. 5, top, circles), and also potentiated the disruptive effects of cocaine on rates of responding (Fig. 5, bottom, circles), a result consistent with the trend toward potentiation of the effects of cocaine on locomotor activity (Table 3).
Figure 6 shows the alteration of the cocaine dose-effect curve for discriminative-stimulus effects produced by atropine (A) or scopolamine (B). Both antimuscarinic compounds shifted the cocaine dose-effect curve to the left. Although the ED50 value for cocaine was decreased, the shift to the left of the dose-effect curve produced by atropine only approached significance (Table 4; note overlap of 95% confidence limits). Scopolamine also shifted the cocaine dose-effect curve to the left; this shift was dependent on scopolamine dose, and was significant at 0.3 mg/kg (Fig. 6B, compare diamonds with circles; Table 4).
Time Course of Behavioral Effects.
The study of the time course of the effects on locomotor activity of selected 3α-diphenylmethoxytropane analogs revealed lasting locomotor stimulant effects of 4′,4"-diF-BZT that were of a greater duration than those for cocaine (Fig. 7; compare A and B). Maximal stimulant effects of cocaine across the 8-h study were obtained in the first 30 min (Fig. 7A, shaded portion), as they were in the 1-h study, and as would be expected, the parameters of the effect (maximal effects and ED50 values) were similar in the two studies (Table 5). Two-way ANOVA of results of the 8-h study revealed significant effects of time, cocaine dose, and the interaction of the two [F(time)47,1680 = 51.0, p < .001; F(dose)4,1680 = 9.1,p < .001; F(txd)188,1680 = 2.7,p < .001]. As can be seen (Fig. 7A), at the doses of cocaine (29 and 59 μmol/kg) that produced maximal stimulant effects during the 1-h study, the stimulant effects were brief, clearly diminishing over the first 30-min period (shaded portion of Fig. 7A). In addition, the highest dose (118 μmol/kg; Fig. 7A, diamonds) produced comparable stimulation during the subsequent 30-min period, with the degree of stimulation decreasing thereafter, but lasting up to 150 min after injection.
Two-way ANOVA of the effects of 4′,4"-diF-BZT also revealed significant effects of time, dose, and the interaction of these two variables [F(time)47,1680 = 8.0, p < .001; F(dose)4,1680 = 563.2, p < .001; F(txd)188,1680 = 4.6, p < .001]. As can be seen (Fig. 7B; triangles), at the dose (25 μmol/kg) producing maximal stimulant effects during the 1-h study, the stimulant effects were long-lasting. Shaded portions of Fig.7 indicate portions of the 8-h observation period in which maximal stimulant efficacy was observed. Significant increases in locomotor activity at this dose were obtained from 20 min after the injection generally throughout the 8-h observation period. At a lower dose (7.6 μmol/kg; Fig. 7B, squares), significant stimulant effects were obtained generally from 100 to 200 min after the injection. At a higher dose (76 μmol/kg; Fig. 7B, diamonds), stimulant effects were generally obtained from 250 min to the end of the 8-h observation period.
Because the effects of 4′,4"-diF-BZT on locomotor activity increased over the first several time periods, it is possible that the 1-h study (Fig. 2) did not capture the maximal effect of 4′,4"-diF-BZT. As a result, the efficacy of 4′,4"-diF-BZT may have been underestimated in the 1-h study. Table 5 shows an analysis of dose effects of 4′,4"-diF-BZT during 30-min periods during which the drug had its greatest effects in the 8-h study (those periods for which data are presented in Table 5 are indicated by the cross-hatched portions of Fig. 7). ANOVA across these time periods indicated no difference in maximal stimulant effects of 4′,4"-diF-BZT [F3,28 = 0.075, p > .8]. In addition, the ED50 values over these same time points were not significantly different (overlapping 95% confidence limits; Table 5). Thus, both maximal effect and the potency of 4′,4"-diF-BZT remained the same up to approximately four hours after injection.
The time courses for three other compounds, 4′,4"-diCl-BZT, 4′-MeO-BZT, and 4′-CH3-BZT, were also examined in 8-h studies (Fig. 7, C–E). None of these compounds significantly increased locomotor activity in the initial 1-h study (Fig. 2), although there were some suggestions of a potential increase based on insignificant increases in locomotor activity that were obtained at intermediate doses in the 1-h study. These inflections in the dose-effect curves suggested that a slow onset stimulant effect might have been obtained with these compounds if testing was conducted for a period longer than 1 h after injection.
Two-way ANOVA in effects of 4′,4"-diCl-BZT revealed significant effects of time, dose, and interaction of these two variables [F(time)47,1680 = 5.2, p < .001; F(dose)4,1680 = 40.9, p < .001; F(txd)188,1680 = 1.3, p = .003]. After an initial high-dose suppression of activity, a modest stimulation was evident and sustained over approximately 6 h (Fig.7C, diamonds). However, significant stimulation of activity was not consistent, occurring after an injection of 24 μmol/kg between 170 to 250 min after the injection, and after an injection of 73 μmol/kg only occasionally between 170 and 390 min after the injection. Consistent with the effects obtained during the 1-h observation period, the effects over the 8-h period were of smaller magnitude compared with those obtained with cocaine and 4′,4"-diF-BZT (Fig. 7, compare C to A and B). Table 5 shows the maximal stimulant effects of 4′,4"-diCl-BZT. From 160 to 190 min after injection, maximal stimulation to approximately 8800 counts was obtained; this was well below that obtained with cocaine. At a later time point (from 340 to 370 min after injection), smaller but still significant stimulation was obtained (see also shaded portions of Fig. 7C).
Neither 4′-MeO-BZT nor 4′-CH3-BZT produced significant sustained increases in locomotor activity during the 8-h session (Fig. 7, D and E). Two-way analyses of variance in effects of each of these compounds revealed significant effects of time, dose, and the interaction of the two [4′-MeO-BZT: F(time)47,1680 = 22.3, p < .001; F(dose)4,1680 = 23.3, p < .001; F(txd)188,1680 = 1.3, p = .006; 4′-CH3-BZT: F(time)47,1680= 15.1, p < .001; F(dose)4,1680= 37.1, p < .001; F(txd)188,1680= 1.3, p = .004]. Occasional increases were obtained with 4′-MeO-BZT generally at the highest dose well after the initial 60 min. Increases occurring with 4′-MeO-BZT were small compared with those occurring with cocaine (Table 5).
Time course of discriminative stimulus effects of cocaine and several 3α-diphenylmethoxytropane analogs are shown in Fig.8. As was shown in Fig. 2, when administered 5 min before the session, cocaine produced a dose-related substitution with 100% cocaine-appropriate responding at the training dose of 10 mg/kg (29 μmol/kg; Fig. 8A, filled circles). The greater the period between cocaine injection and testing, the farther the dose-effect curve was shifted to the right. When administered 60 min before testing, a time at which locomotor stimulant effects were noticeably diminished (Fig. 7A), there was a marked decrease in the discriminative efficacy of cocaine (Fig. 8A, triangles).
When tested 5 min after injection, 4′,4′-diF-BZT produced little cocaine-appropriate responding (Fig. 8B, filled circles). However, when tested between 30 and 90 min after injection (Fig. 8B, squares, diamonds, and inverted triangles), 4′,4"-diF-BZT produced a much greater level of cocaine-appropriate responding, with full substitution for cocaine with a 90-min pretreatment time. When tested 120 min after the injection, 4′,4"-diF-BZT did not reliably substitute for cocaine.
The efficacy of 4′,4"-diCl-BZT in producing cocaine-like discriminative stimulus effects did not increase with time since its injection over a 2-h period (Fig. 8C). As was shown in Fig. 4, 4′,4"-diCl-BZT produced a maximum level of cocaine-appropriate responding of approximately 20% (filled circles), which did not increase with pretreatment times of up to 90 min. Although the mono-substituted 4′-Cl-BZT initially had greater cocaine-like efficacy, its efficacy was not improved with time since injection (Fig. 8D). Rather, increasing pretreatment times of up to 90 min with 4′-Cl-BZT produced decreases in cocaine-like discriminative efficacy.
Discussion
The present study examined the pharmacology of some novel 3α-diphenylmethoxytropane analogs. Like cocaine, these compounds had varying affinities for the dopamine transporter and inhibited dopamine uptake in vitro. However, as shown with 4′-Cl-BZT in our initial report (Newman et al., 1994), the behavioral effects of these drugs differed from those of cocaine. Generally, the 3α-diphenylmethoxytropane analogs had varying efficacies for stimulating locomotor activity, but none had an efficacy equivalent to that of cocaine. Furthermore, with the exception of the 4′,4"-difluoro substituted compound, the drugs commonly had lower efficacy than cocaine in producing discriminative-stimulus effects in rats trained to discriminate cocaine from saline.
The present data correspond to previous studies that have indicated differences between the behavioral effects of BZT and cocaine. For example, McKearney (1982) showed that both BZT and cocaine increased operant behavior; however, cocaine generally had greater efficacy (see also Acri et al., 1996). Furthermore, several studies have indicated that BZT did not fully substitute for cocaine in rats trained to discriminate cocaine from saline (Colpaert et al., 1979; Acri et al., 1996). Differences from cocaine in the behavioral effects of BZT (Newman et al., 1994; Acri et al., 1996) or other dopamine uptake inhibitors (Rothman, 1990) have prompted suggestions that some of these compounds may serve as leads for the development of pharmacological treatments for cocaine abuse. Moreover, dopamine uptake inhibitors that have in vivo effects that differ from those of cocaine may serve to better our understanding of the pharmacological mechanisms that underlie the behavioral effects of cocaine and lead to its abuse.
Compounds with fluoro substitutions in the para- andmeta-positions generally were among the compounds with the highest affinity for the dopamine transporter and the highest potency for inhibition of dopamine uptake. These compounds generally had behavioral effects most closely resembling those of cocaine, producing the greatest stimulation in locomotor activity and the highest efficacy in substituting for the discriminative stimulus effects of cocaine. In contrast, several of the chloro-substituted analogs exhibited relatively lower behavioral efficacy but had affinities at the dopamine transporter comparable to those of the corresponding fluoro-substituted compounds. This comparison suggests that cocaine-like in vivo effects were not related to affinity at the dopamine transporter in a simple manner. Consistent with that conclusion is the finding of an inverse relation between the affinity of these drugs at the dopamine transporter and potency for producing locomotor stimulation. Among close structural analogs of cocaine and WIN 35,428 there is typically a direct relation between affinity at the dopamine transporter and potency for locomotor stimulation (Cline et al., 1992;Izenwasser et al., 1994). Although this relationship is drawn across species in the study by Izenwasser et al. (1994), it was drawn within species by Cline et al. (1992). In agreement with the present findings,Vaugeois et al. (1993) and Izenwasser et al. (1994) showed that among dopamine uptake inhibitors that are structurally dissimilar to cocaine, the relationship between dopamine transporter affinity and in vivo behavioral effects is distinctly different from that for analogs of cocaine (see also Rothman et al., 1992). There currently is no mechanistic explanation for the differences between cocaine-like dopamine uptake inhibitors and other dopamine uptake inhibitors with respect to these relations.
The 3α-diphenylmethoxytropane analogs that have effects most like those of cocaine all contain at least one fluoro group in thepara-position of one of the phenyl rings of the diphenyl ether moiety. These substituents are sterically small halogens that appear to provide the optimum combination of volume and electronic character to achieve maximum behavioral response. In addition, these compounds are among those in this series with the highest affinity at the dopamine transporter. Cocaine-like activity is somewhat reduced in the compounds that have at least one chloro group in thepara-position of one of the phenyl rings, despite comparable binding affinities (see above). Additional chloro groups (e.g., 4′,4"-diCl-BZT), or in particular substitution with a sterically bulkier bromo group (e.g., 4′-Br,4"-F-BZT), results in diminished cocaine-like activity as compared to the analogous fluoro-substituted compounds. The larger halogenated compounds demonstrate relatively lower binding potencies at the dopamine transporter. The least efficacious compounds in this series do not have small halogens on the phenyl rings but rather have electronically neutral (CH3) or electron withdrawing groups (NO2, CF3, CN), some of which are relatively bulky in size as well. Clearly, these compounds did not produce a cocaine-like stimulant profile. In addition, these compounds were relatively less potent in binding to the dopamine transporter. Therefore, the compounds in this series most like cocaine possess at least one fluoro group in the para-position of one of the phenyl rings. Interestingly, themeta-Cl-substituted compound has significantly greater cocaine-like efficacy than its para-substituted analog, both in stimulation of locomotor activity and in producing cocaine-like discriminative-stimulus effects (Kline et al., 1997). However, this observation may be attributed to the fact that the 3′-Cl analog, in addition to its relatively high potency at the dopamine transporter, is also a potent muscarinic M1 ligand.
The relatively high affinity of fluoro-substituted analogs concurrent with their relatively greater cocaine-like efficacy compared to analogs substituted with other moieties suggests that the general absence of cocaine-like effects in the latter compounds might be due to a lack of sufficient affinity for the dopamine transporter. Mitigating against this interpretation are the findings that several of the compounds showing little or no cocaine-like behavioral activity had affinities for the dopamine transporter that were similar to those of several of the relatively efficacious fluoro-substituted 3α-diphenylmethoxytropane analogs. For example, 4′-Cl-BZT, 4′,4"-diCl-BZT, and 3′,4′-dichloro-3α-(diphenylmethoxy)tropane) had affinities for the dopamine transporter ranging from 20 to 30 nM. In comparison, the affinities of the fluoro-substituted analogs ranged from 11.8 to 32 nM, encompassing those of the chloro-substituted compounds. Thus, affinity for the dopamine transporter alone cannot account for the reduced cocaine-like efficacy of many of the 3α-diphenylmethoxytropane analogs.
One objective of the present study was to examine potential mechanisms contributing to the in vivo differences between the 3α-diphenylmethoxytropane analogs and cocaine. The comparative actions of these drugs at the dopamine transporter were examined using displacement of [3H]WIN 35,428 binding and the inhibition of dopamine uptake. Because BZT is known for its antimuscarinic effects, these compounds were also examined for their affinity at muscarinic M1 receptors; that activity was examined as a potential reason for the differences between these compounds and cocaine. In addition, the durations of action of several of the drugs were assessed in order to determine if cocaine-like behavioral effects could be obtained with a longer time for absorption and distribution to relevant sites of action.
It is tempting to speculate that the reduced cocaine-like activity of the compounds, particularly those with lower affinity for the dopamine transporter, was due to other prepotent effects predominating and interfering with the expression of cocaine-like behavioral effects. For example, cocaine-like behavioral effects of meperidine can be obscured by prepotent opioid activity that decreases response rates. When the opioid activity is blocked by coadministration of an opioid antagonist, higher meperidine doses, which produce cocaine-like effects, can be administered (Izenwasser et al., 1996). For the present compounds one possible action that might interfere with the expression of cocaine-like effects is antagonist effects at muscarinic receptors. Antimuscarinic effects of these drugs might decrease response rates at doses lower than those that produce cocaine-like effects, obscuring their cocaine-like pharmacology. Support for this interpretation, however, was not provided by the linear regression analysis of M1 affinity and ED50values. Furthermore, more direct evidence indicates that antimuscarinic effects did not preclude the expression of cocaine-like behavioral activity. The muscarinic antagonists, atropine and scopolamine, stimulated locomotor activity, albeit with less efficacy than cocaine. This result is inconsistent with an interference with cocaine-like locomotor stimulant effects and suggests that, if anything, antimuscarinic activity of the 3α-diphenylmethoxytropane analogs would potentiate rather than diminish cocaine like activity. Evidence for a potentiation of cocaine-induced stimulation of locomotor activity by the antimuscarinics was obtained in the studies of the interactions among these drugs. Further in the cocaine-discrimination procedure, atropine was ineffective in attenuating the discriminative stimulus effects of cocaine. Moreover, in further studies of these interactions, both atropine and scopolamine potentiated rather than antagonized the discriminative stimulus effects of cocaine. This potentiation occurred at doses of the two muscarinic antagonists that are consistent with their relative in vivo potencies for producing other antimuscarinic effects (McKeon, 1967; Malick and Barnett, 1975), suggesting that the potentiation was indeed due to antimuscarinic effects. These results are consistent with several previous studies that clearly demonstrate a similar potentiation of stimulants by antimuscarinics. For example, atropine and scopolamine augment the stimulation of avoidance behavior produced by cocaine (Scheckel and Boff, 1964). Together with the present findings, these results indicate that the antimuscarinic actions of 3α-diphenylmethoxytropane analogs are incapable of attenuating any cocaine-like actions that they might have.
As with the stimulation of locomotor activity, the potencies of the 3α-diphenylmethoxytropane analogs for decreasing response rates in the cocaine-discrimination studies were not related to theirKi values for displacement of [3H]WIN 35,428. These results suggest that neither of these behavioral effects are simply related to activity at the dopamine transporter, as might have been expected. However, the relation between transporter binding and ED50value for decreasing response rates approached significance (p = .099), suggesting that activity at the dopamine transporter is a factor influencing the effects of these drugs on rates of responding.
Initial observations that 4′-Cl-BZT lacked cocaine-like discriminative stimulus effects (Newman et al., 1994) led to studies of time course of action to determine whether the relatively lower efficacy of these compounds was due to an insufficient time for distribution of drug to relevant central nervous system sites of action. 4′,4"-diF-BZT produced a long-lasting stimulation of locomotor activity, although it was not significantly greater than the stimulation obtained in the first 60 min after injection, and it was less than that produced by cocaine. For those compounds that have been studied to date, there is no evidence that the present estimates of locomotor stimulant potency or efficacy would be increased with longer pretreatment times. With the exception of 4′,4"-diF-BZT, a similar conclusion could be made for the discriminative stimulus effects of these drugs. In contrast to the other compounds studied, 4′,4"-diF-BZT showed increased efficacy in the cocaine-discrimination procedure when the time between injection and testing was between 30 and 90 min compared to when subjects were tested 5 min after injection. Structurally 4′,4"-diF-BZT, more than the other 3α-diphenylmethoxytropane analogs, resembles the selective dopamine uptake inhibitor, GBR 12909, which also has a 4′,4"-difluoro-substituted diphenyl ether system. GBR 12909 also has been reported to share discriminative stimulus effects with cocaine (e.g., Witkin et al., 1991). However, it is currently unclear what mechanism may account for the enhanced cocaine-like pharmacology of 4′,4"-diF-BZT compared to others of the 3α-diphenylmethoxytropane analogs.
The absence of substitution for cocaine by the other 3α-diphenylmethoxytropane analogs in the discrimination procedure and the low efficacy of these drugs in stimulating locomotor activity contrasts with their affinity for the dopamine transporter and their in vitro inhibition of dopamine uptake. Previous studies with BZT (e.g.,Church et al., 1987) and preliminary studies with 4′-Cl-BZT (Tolliver et al., 1998) also indicate that these drugs can inhibit dopamine uptake in vivo. These results suggest behavioral effects similar to those of cocaine. It is possible that other actions or pharmacological properties of the 3α-diphenylmethoxytropane analogs interfered with their cocaine-like activity; however, it is also possible that the regulation of behavioral activity through their actions at the dopamine transporter is different for the 3α-diphenylmethoxytropane analogs and cocaine-like drugs. Consistent with that interpretation is the finding that, in contrast to analogs of cocaine and WIN 35,428, the binding of 3α-diphenylmethoxytropane analogs to the dopamine transporter modeled better for single-site binding than it did for two-site binding. As mentioned above, for the cocaine and WIN 35,428 analogs there is a good correlation between affinity for the dopamine transporter and in vivo potency (e.g., Cline et al., 1992; Izenwasser et al., 1994); however that relationship is not found with structurally diverse dopamine uptake inhibitors (the present study; Vaugeois et al., 1993; Izenwasser et al., 1994). Finally, the structure-activity relations among these drugs are distinctly different from those obtained with cocaine and WIN 35,428 analogs (Newman et al., 1994,1995), suggesting that these compounds are accessing a different binding domain than that accessed by cocaine. Identification of the precise domain on the dopamine transporter accessed by different classes of small molecular structures and the functional consequences of these binding interactions will greatly advance our understanding of how different dopamine uptake inhibitors act, why some are subject to abuse and others are not, and may provide leads for the development of pharmacotherapies for the treatment of cocaine dependence.
Acknowledgments
We thank S. Carter, B. Campbell, D. French, R. Mitkus, R. Loeloff and P. Terry for technical support and data analysis, M. J. Forester for conduct of some of the locomotor activity studies, and Patty Ballerstadt for administrative and clerical support. We especially thank Dr. F. Vocci for continuing support and encouragement.
Footnotes
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Send reprint requests to: Jonathan L. Katz, Psychobiology Section, National Institute on Drug Abuse Intramural Research Program, P.O. Box 5180, Baltimore, MD 21224. E-mail:jkatz{at}intra.nida.nih.gov
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↵1 Some of the locomotor activity data were provided through a contract (NO1DA-7–8076; M. J. Forester, PI) with the National Institute on Drug Abuse Medications Development Division. These studies were supported in part by an IntraAgency Agreement with the National Institute on Drug Abuse Medications Development Division and by the National Institute on Drug Abuse Intramural Research Program.
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↵2 Current address: Department of Neurology, University of Miami School of Medicine, 1501 NW 9th Ave., Room 4061, Miami, FL 33136.
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↵3 Current address: Medications Development Division, National Institute on Drug Abuse, National Institutes of Health, 5600 Fishers Lane, Room 11A-55, Rockville, MD 20857.
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↵4 Current address: Rhone-Poulenc Rorer, New Leads Discovery Section/Analytical Science, NMR S1362, 500 Arcola Road, H37, P.O. Box 5096, Collegeville, PA 19426-0800.
- Abbreviations:
- BZT
- 3α-(diphenylmethoxy)tropane (benztropine)
- 4′
- 4"-diF-BZT, 4′,4"-difluoro-3α-(diphenylmethoxy)tropane
- 4′-Cl-BZT
- 4′-chloro-3α-(diphenylmethoxy)tropane
- 4′-Cl-BZT (β)
- 4′-chloro-3β-(diphenylmethoxy)tropane
- ANOVA
- analysis of variance
- FR
- fixed ratio
- 4′-Cl-BZT (β)
- 4′-chloro-3β-(diphenylmethoxy)tropane
- 4′
- 4"-diBr-BZT, 4′,4"-dibromo-3α-(diphenylmethoxy)tropane
- Received April 1, 1998.
- Accepted August 21, 1998.
- The American Society for Pharmacology and Experimental Therapeutics