Abstract
2β-Propanoyl-3β-(2-naphthyl)-tropane (WF-23) is a potent cocaine analog with activity at dopamine and serotonin transporters. The purpose of these experiments was to characterize the time course of effects of acute administration of WF-23 on spontaneous locomotion and biogenic amine transporters. Rats received injections i.p. with WF-23 (1 mg/kg), cocaine (30 mg/kg) or vehicle and locomotor activity was measured at various times postinjection. Animals were killed immediately after behavioral activity. Locomotor activity was significantly increased by WF-23 administration, reaching maximum at 4 hr and persisting for 24 hr. Cocaine-elicited elevations in locomotor activity occurred only at the earliest times. WF-23 decreased DA transporter binding in striatal membranes ([125I]RTI-55 binding), with >50% loss in binding for up to 49 hr postinjection. WF-23 increased the Kd of the high affinity site, with no effect on Bmax. Cocaine depressed binding (20%) only at the earliest times. WF-23 decreased levels of [3H]WIN 35,428 binding sites up to 95% of control in both dorsal and ventral striatum with a similar time-course when assessed autoradiographically. WF-23 also reduced [3H]citalopram binding to serotonin transporter sites throughout the brain. The slow onset and very long duration of action of WF-23, taken together with its actions at dopamine and serotonin transporters, suggest a potential role for treatment of disorders characterized by their involvement of these neural systems.
The primary action of cocaine in brain is the blockade of the reuptake of the monoamines, dopamine (Heikkila et al., 1979; Mooreet al., 1977), norepinephrine (Hertting et al., 1961; Moore et al., 1977) and serotonin (Ross and Renyi, 1967). Cocaine binds relatively equipotently to all three transporters, although its behavioral effects have been largely attributed to its actions at dopamine transporters (cf. Colpaert et al., 1987; DeWit and Wise, 1977; Miczek and Yoshimura, 1982; Ritzet al., 1987). One important characteristic of cocaine is its rapid onset of action when given by the routes of administration by which it is generally abused. Peak plasma concentrations have been reported to be achieved within the first 30 minutes when cocaine is administered either intranasally or intravenously (Shuster, 1992). In addition to its rapid onset, cocaine’s duration of action is also relatively short. Estimates of its half-life vary with route of administration, but have been calculated to be between 15-20 min following intravenous administration and 60-70 minutes following intraperitoneal or subcutaneous administration in rats (Nayak et al., 1976). These pharmacokinetic characteristics have been hypothesized to be in part responsible for cocaine’s intense reinforcing effects and the frequency of its repeated use.
One recent strategy for investigation of the neurobiological actions of cocaine has been the use of cocaine derivatives that bind to dopamine and serotonin transporters with greater affinity and slower dissociation rates than cocaine. Several groups have synthesized different series of such tropane analogs (Kosikowski et al., 1992; Abraham et al., 1992; Boja et al, 1994; Carroll et al, 1992 a, b, c; Lewin et al., 1992; Carroll et al., 1993; Meltzer et al., 1993,1994, 1996) including Davies and colleagues (1993, 1994), who have prepared a series of novel analogs using a unique synthetic scheme based on the reaction of vinylcarabenoids with pyrroles. This approach has allowed greater chemical flexibility which has resulted in the synthesis of a number of analogs not available with other synthetic schemes. One feature of these analogs is the removal of both esters of cocaine. Because 90% of cocaine metabolism occurs by cleavage of ester bonds (Shuster, 1992), the absence of these ester groups increases the metabolic stability of these compounds in vivo. Analogs in this series have been shown to have longer durations of action in behavioral assays (Porrino et al., 1994, 1995), and their administration has resulted in long lasting increases in the concentrations of extracellular dopamine (Hemby et al., 1995). Many of these compounds are highly potent, and the slow rates at which such compounds generally dissociate from their binding sites would also contribute to their extended durations of action. From a therapeutic standpoint, synthesis of compounds with slow onsets and long durations of action is a potential strategy for the development of pharmacological treatments. The use of such long-acting compounds could result in reduced drug-seeking behavior and they might, therefore, serve as substitutes for cocaine in much the same way that methadone is used in the treatment of opioid dependence.
The goal of these studies was to characterize the actions of WF-23 (fig. 1), a highly potent, nonselective tropane in the series synthesized by Davies and his colleagues (1993, 1994). This analog contains a 2-naphthyl moiety that replaces the phenyl ring at the 3-position of cocaine. In addition both esters of cocaine have been replaced: the benzyl ester is replaced by a direct connection between the tropane and phenyl at the 3-position and the methyl ester at the 2-position of cocaine is replaced by an ethyl ketone. In [125I]RTI-55 binding displacement studies, WF-23 binds to dopamine transporters with an IC50 value of 0.12 nM, and as such it is approximately 1400 times more potent than cocaine at dopamine transporters (Bennett et al., 1995). In addition, theKi in displacing [3H]paroxetine binding at serotonin transporters is 0.39 nM, making WF-23 approximately 770 times more potent than cocaine at 5HT transporters (Bennett et al., 1995). Finally, this compound displays similar IC50 values in inhibiting uptake of [3H] dopamine and [3H] serotonin; 0.65 and 0.32 nM, respectively (Bennett et al., 1995). WF-23, therefore, is a relatively nonselective, yet highly potent, tropane. The purpose of these studies was first to measure the effects of WF-23 on spontaneous locomotor activity as an assay of its action in vivo in rats, and second to determine the time course of its occupancy at dopamine and serotonin transporters.
Structures of (-)-cocaine and WF-23.
In our studies, WF-23 was administered to groups of rats and spontaneous locomotor activity was tested at a variety of time points from 1 hr to 10 days postadministration. Each group of rats was killed immediately after behavioral testing. Dopamine and serotonin transporter occupancy were assessed in brains from the same groups of rats. Our results indicate that the behavioral actions of WF-23 persist for up to 24 hr after treatment, but that occupancy of transporters persists beyond that time point, not returning to control levels until up to 10 days postadministration.
Materials and Methods
Animals
Male Sprague-Dawley rats (Harlan, Indianapolis, IN), weighing 200 to 300 g at the time of the experiment, were used in all studies. Animals used for behavioral and in vitro binding studies were housed individually in a climate-controlled room with a 12-hr light/dark cycle. Food and water were available ad libitum. All animals were adapted to vivarium conditions for 5 days before testing, which was conducted during the light phase (7:30a.m.-4:30 p.m.) of the cycle. Injections were given so that all testing occurred during the light phase of the cycle.
Materials
WF-23 was synthesized as previously described (Davies et al., 1994), and was dissolved in 50 mM Tris, which served as the vehicle. The dose of WF-23 used in the present study was based on a preliminary study in our laboratory that demonstrated a robust behavioral response at 1 mg/kg. Cocaine HCl (National Institute on Drug Abuse) was dissolved in physiologic saline. All drugs were administered i.p..
[125I]RTI-55 (2200 Ci/mmol), [3H]WIN 35,428 (83.5 Ci/mmol), also referred to as [3H]CFT and [3H]citalopram (85.7 Ci/mmol) were purchased from New England Nuclear Corp. (Boston, MA). Paroxetine HCl was generously donated by SmithKline Beecham Pharmaceuticals (Worthing, Sussex, UK). Unlabeled RTI-55 was obtained from NIDA Drug Supply System. Mazindol was purchased from Research Biochemicals International (Natick, MA). All other reagent grade chemicals were obtained from Sigma Chemical Co. (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA).
Behavioral Testing
Locomotor activity was assessed in open-field plexiglass test chambers (42 × 42 × 30 cm). Locomotion was measured by electronic counters that detected interruptions of eight independent photocell beams (Omnitech, Columbus, OH). The following measures were recorded and stored in 10-min intervals: horizontal activity or the total number of horizontal beam interruptions, forward locomotor or ambulatory activity, vertical activity or rearing and stereotypy.
Animals were habituated to the chamber for 2 consecutive days before testing, for 60 min each day. On the third (test) day, they received a single i.p. injection of WF-23 (1 mg/kg), cocaine (30 mg/kg) or saline, in the home cage. The animals were removed from their home cage and placed into the locomotor chamber where their locomotor activity was recorded for 60 min starting at 1, 4, 8, 12, 24, 48 and 240 hr after injection. At each time point locomotor activity was assessed in treatment groups consisting of WF-23 (n = 6), cocaine (n = 3) and saline (n = 3).
Tissue Preparation
Immediately after behavioral testing animals were deeply anesthetized with sodium pentobarbital (150 mg/ml) and decapitated, and the brains were removed and hemisected. Tissue for homogenate and autoradiographic binding studies, therefore, was collected at 2, 5, 9, 13, 25, 49 and 241 hr after treatment. Striata from one hemisphere were dissected on ice, frozen and stored for DA transporter binding studies. The remaining hemisphere was frozen at -40°C and stored at -80°C for autoradiographic experiments. Coronal sections (20 μm) for autoradiography were cut at -20°C in a cryostat and thaw-mounted onto gelatin/chrome alum-coated slides. Slide mounted sections were desiccated at 4°C for 24 hr then stored at -80°C until processed.
Dopamine Transporter Binding
Rats were killed immediately after the collection of locomotor data. Using a modification of the procedure of Boja and colleagues (1991), dopamine transporter binding in striatal membranes was determined with [125I]RTI-55 as previously described (Bennett et al., 1995). Tissue was homogenized in 10 volumes of RTI-55 assay buffer (0.32 M sucrose, 10 mM sodium phosphate buffer, pH 7.4) with a Polytron (setting 6, 20 sec), and centrifuged at 48,000 × g for 10 min, then the pellets were resuspended in assay buffer. To minimize dissociation of WF-23 from isolated membranes, these pellets were not subjected to the additional wash steps previously described (Bennett et al., 1995). For [125I]RTI-55 assays, tubes contained 0.5 mg (original wet weight) of membranes and 10 pM [125I]RTI-55, in a final volume of 2 ml. Tubes were incubated for 50 min at 25°C, and the reaction was terminated by rapid filtration with 3 × 5 ml of cold Tris buffer through Whatman GF/B glass fiber filters which had been pre-soaked for at least 1 hr in Tris buffer containing 0.1% bovine serum albumin. Nonspecific binding was determined by the addition of 1 μM WF-23. Protein values were determined by the protein dye-binding method (Bradford, 1976).
In Vitro Quantitative Autoradiography
[3H]CFT.
With minor modifications, the procedure described by Canfield and colleagues (1990) was used to determine [3H]CFT binding to the dopamine transporter. Briefly, to assess total binding, tissue sections were incubated for 2 hr at 4°C in buffer (50 mM Tris-HCl, 100 mM NaCl, pH 7.4) containing 5 nM [3H]CFT. Adjacent sections were incubated under identical conditions in the presence of 30 μM cocaine in order to determine nonspecific binding. Slides were rinsed in buffer once for 10 sec, then twice for 1 min, followed by a 10-sec dip in distilled water, all at 4°C. They were immediately dried under a stream of cool air and apposed to Hyperfilm (Amersham, Arlington Heights, IL) for 4 wk along with tritium standards (Amersham). Preincubations were omitted to prevent washout of WF-23 bound to the tissue sections.
Occupancy of the DA transporter by WF-23 was estimated from reductions in [3H]CFT binding. Percent differences in baseline binding levels in sections from saline-treated animals (as a measure of total occupancy of available transporter binding sites) and levels of binding in WF-23-treated animals were taken to signify percent occupancy of these sites by WF-23.
[3H]citalopram.
Procedures for labeling serotonin transporters were adapted from those described byD’Amato and colleagues (1987). Slide mounted tissue sections were incubated for 1 hr at 25°C in buffer (50 mM Tris, 120 mM NaCl, 5 mM KCl, pH 7.4) containing 1 nM [3H]citalopram to assess total binding. Adjacent sections were incubated under identical conditions in the presence of 20 μM paroxetine to determine nonspecific binding. Sections were then rinsed twice in buffer for 10 min each followed by a 10-sec rinse in distilled water, all at 4°C. They were immediately dried under a cool stream of air and apposed to Hyperfilm (Amersham) for 4 wk along with tritium standards (Amersham). Preincubations were excluded to prevent washout of bound WF-23.
Densitometry
Autoradiographic analysis of [3H]CFT binding to the DAT and [3H]Citalopram binding to the 5HTT was conducted by quantitative densitometry with a computerized image processing system (MCID, Imaging Research, St. Catharine’s, Ontario, Canada). Tissue equivalent values (fmol/mg of wet weight tissue) were determined from the optical densities and from a calibration curve generated by densitometric analysis of the autoradiograms of tritium standards. Specific binding was determined by subtracting nonspecific binding values from the total binding values, as measured in adjacent sections.
Data Analysis
Statistical comparisons of behavioral data were made by two-way analyses of variance (treatment × time), followed by Bonferronit tests for multiple comparisons to determine differences between groups at each time-point.
Statistical significance for [125I]RTI-55 binding data was determined by analysis of variance followed by a nonpaired two-tailed Student’s t test, using JMP (SAS Institute, Cary, NC). Nonlinear regression analysis of concentration-effect curves was also performed with JMP using an iterative model.
Measurements of the concentrations of [3H]CFT binding sites were made in 4 brain regions and concentrations of [3H]citalopram binding sites were made in 12 distinct brain regions. Statistical analysis of binding for each ligand in each brain region was made independently by two-way analysis (treatment × time), followed by Bonferroni t tests for multiple comparisons to determine differences between groups at each time-point.
Pearson product moment correlations were calculated between measures of locomotor activity and estimates of DA transporter occupancy by WF-23 in caudate and nucleus accumbens core and shell.
Results
Effect of WF-23 on locomotor activity.
The dose of WF-23 chosen for these experiments was based on pilot studies that evaluated the effects of 0.03, 0.1, 0.3 and 1 mg/kg of WF-23 on horizontal locomotor activity . It was determined that a 1-mg/kg dose of WF-23 reliably produced robust increases in locomotor activity, the onset of which occurred approximately 60 min postinjection (Daunais JB, Hart SL and Porrino LJ, unpublished observations). In our study, therefore, locomotor activity was measured beginning at 60-min postinjection. The effects of a single i.p. administration of WF-23 (1 mg/kg) or cocaine (30 mg/kg) on horizontal locomotor activity are illustrated in figure2. Administration of WF-23 produced significant time-dependent increases in the total number of horizontal photocell beam interruptions, the onset of which were observed to occur at approximately 50 to 60 min postinjection. Between 1 and 2 hr after the acute administration of WF-23, locomotor activity was increased to 314% of control (fig. 2). Activity peaked between 4 and 5 hr after administration (337% of control) and remained significantly elevated for up to 24 hr (206% of control) when compared to saline-treated rats. Similar effects were observed when vertical activity, stereotypic activity and forward locomotion were evaluated after administration of WF-23 (data not shown). In contrast, a single injection of 30 mg/kg cocaine elicited a robust behavioral response which was observed to begin within 10 min postinjection, peaked by 1 hr and was not significantly different from control at any later time point (fig. 2). Although administration of WF-23 resulted in a behavioral response that was more intense and of longer duration than that elicited by cocaine, the behaviors were qualitatively similar when rats from both groups were visually compared.
Time course of the effects of acute i.p. administration of WF-23 (○, 1 mg/kg) or cocaine (•, 30 mg/kg) on horizontal activity expressed as percent of total photocell beam interruptions by vehicle controls at each time point. Activity was measured for 60 min starting at 1, 4, 8, 12, 24, 48 and 240 hr after injection. Data are mean ± S.E.M. Data were analyzed with a two-way analysis of variance followed by Bonferroni ttest for multiple comparisons comparing the effects of each treatment with vehicle at each time point. *P < .05, different from vehicle. +P < .05, different from cocaine.
Effect of WF-23 on [125I]RTI-55 binding to dopamine transporters in striatal membranes.
After assessing locomotor activity for 60 min at various times after WF-23 or cocaine administration, rats were killed and the brains were removed and hemisected. Residual dopamine transporter binding was determined with [125I]RTI-55 in striatal membranes from one hemisphere. Results of a time course, from 2 hr to 10 days after drug administration, are shown in figure 3. In membranes from rats treated with WF-23, [125I]RTI-55 binding was decreased by 90% 2 hr after a single exposure to WF-23. [125I]RTI-55 binding remained significantly decreased (approximately 50% of vehicle levels) between 9 and 49 hr. Five days after WF-23 treatment, [125I]RTI-55 binding began to approach control levels but was still significantly decreased by 25%. Binding of [125I]RTI-55 to DA transporters recovered to control levels 10 days after the WF-23 injection. In contrast, in membranes from rats that received a single injection of cocaine, [125I]RTI-55 binding was decreased slightly (20%) at 2 to 5 hr after cocaine injection. Binding returned to normal levels after 5 hr and remained at levels comparable to those seen in saline-treated rats for the duration of the study.
Time course of [125I]RTI-55 binding in striatal membranes at various times after acute i.p. administration of vehicle, cocaine (•, 30 mg/kg) and WF-23 (○, 1.0 mg/kg). Data represent mean values ± S.E.M. of triplicate samples determined from four to five animals in each treatment group. Assessments were made 2, 5, 9, 13, 25, 49 and 241 hr after injection.
To determine the effect of WF-23 administration on the parameters of [125I]RTI-55 binding to dopamine transporters, detailed Scatchard analyses were performed on striatal membranes 49 hr after drug administration, a time when cocaine was no longer present. The results of this Scatchard analysis are demonstrated in table1. The plots for cocaine-treated animals (not shown) were identical to those of the control animals. As previously described (Boja et al., 1992; Staley et al., 1994), the binding of radiolabeled tropanes best fits a two-site model. The effect of WF-23, as determined 49 hr after a single injection, appeared to be primarily mediated through a decrease in the high affinity binding site, with little discernible effect on the low affinity site. This was confirmed by LIGAND analysis from three to four rats per group (table 1). WF-23 increased theKd value of the high affinity site by approximately 2-fold, but had no significant effect on theKd of the low affinity site, or on the Bmax values for either the high or low affinity sites. Data from cocaine-treated rats (table 1), determined 49 hr after drug administration, showed no effect of cocaine on any parameter for high or low affinity [125I]RTI-55 binding sites compared to vehicle controls.
Scatchard analysis of [125I]RTI-55 binding in striatal membranes 49 hours after treatment with WF-23
Effects of WF-23 on [3H]CFT binding.
After determining [125I]RTI-55 binding to dopamine transporters in membranes from one brain hemisphere, the other hemisphere was sectioned in the coronal plane to determine [3H]CFT and [3H]citalopram binding. Binding of [3H]CFT to DA transporters was assessed in sections collected through the caudate nucleus and the NAc (fig.4). [3H]CFT binding sites were heterogeneously distributed in the brains of normal rats. High levels of binding were present in portions of the striatum, and were expressed in what appeared to be a lateral to medial gradient, with highest levels laterally (fig. 4). This pattern was consistent with the pattern of [3H]CFT binding described by Coulter and colleagues (1995). Binding was less pronounced in the nucleus accumbens (fig. 4). The time course of effects of a single injection of WF-23 on [3H]CFT binding in the caudate and nucleus accumbens is illustrated in figure5. Acute i.p. administration of WF-23 produced persistent, time-dependent reductions in [3H]CFT binding. Two hours after administration of WF-23, [3H]CFT binding was decreased in the dorsal striatum (-92%), NAc core (-93%) and NAc shell (-87%) when compared to control. Binding remained significantly decreased for up to 49 hr in all areas, and was slower to recover in the caudate when compared to the nucleus accumbens. Binding returned to control levels in all areas by day 10 (fig. 5).
Effects of acute i.p. administration of WF-23 (1 mg/kg) on [3H]CFT binding to dopamine transporter binding sites at 2, 5, 9, 13, 25, 49 and 241 hr after injection and compared to vehicle-treated control. Shown are color-coded transformations of autoradiograms of coronal sections of rat brain at the level of the striatum in which each color represents a range of densities of [3H]CFT binding to DA transporters in fmol/mg we weight tissue. Note that binding remains depressed for up to 49 hr after a single administration of WF-23.
Time course of effects of a single i.p. administration of WF-23 on [3H]CFT binding to DA transporters in caudate and core and shell portions of the nucleus accumbens. *P < .05, different from vehicle. Note that [3H]CFT binding remains significantly reduced for up to 49 hr after a single administration of WF-23. Measurements were made 2, 5, 9, 13, 25, 49 and 241 hr after treatment.
Levels of [3H]CFT binding were not significantly altered by the acute administration of cocaine in any of the areas examined when compared to saline-treated rats (not shown). This is in sharp contrast to the time-dependent decreases in [3H]CFT binding produced by WF-23.
Occupancy of the DA transporter by WF-23, as estimated by measuring reductions in [3H]CFT binding, was significantly correlated with behavioral activation in the caudate (rxy = 0.72, d.f. 27, P < .0001) (fig. 6) and core of the NAc (rxy = 0.74, d.f. 27, P < .0001), as well as in the NAc shell (rxy = 0.54, d.f. 27, P < .002) such that higher estimated occupancy rates were associated with higher levels of behavioral activation (fig. 6).
Correlation between horizontal locomotor activity (total photocell interruptions during 1-hr test session) and dopamine transporter occupancy by WF-23 in caudate. Occupancy was estimated by measuring the percent reduction in [3H]CFT binding sites in sections from WF-23 treated rats as compared to those from vehicle treated controls. Values of Pearson product-moment correlation coefficient are shown. Note the significant positive correlation between dopamine transporter occupancy and spontaneous locomotor activity in the caudate. The dashed line represents the mean number of photocell beam breaks in saline-treated animals.
Effects of WF-23 on [3H]citalopram binding.
Binding of [3H]citalopram to serotonin transporters was determined in twelve forebrain and midbrain structures of the hemisected brain. [3H]citalopram binding sites were localized heterogeneously throughout the brain with high basal levels expressed in the medial septum, olfactory tubercle, basolateral amygdala, substantia nigra and superior colliculus of control animals. Moderate levels of [3H]citalopram binding sites were found in the hypothalamus and lateral geniculate, whereas, low levels were detected in frontal cortex, hippocampal complex, NAc core and NAc shell, and in the dorsal striatum. Figure7 demonstrates representative binding in sections collected through the striatum at the level of the nucleus accumbens. The acute i.p. administration of WF-23 produced long-lasting, time-dependent reductions in levels of [3H]citalopram binding. Decreases in binding were widespread throughout the brain, with the greatest effects occurring at the two hour time point (figs. 7 and8). Decreased [3H]citalopram binding was observed in the caudate (-90%), NAc shell (-91%), NAc core (-93%), prefrontal cortex (-90%), amygdala (-95%), olfactory tubercle (-94%), medial septum (-95%), hippocampus (-88%), lateral hypothalamus (-94%), substantia nigra (-92%), lateral geniculate (-91%) and superior colliculus (-91%). These decreases were followed by a gradual, time-dependent restoration toward normal levels in most areas, such that binding was at control values in amygdala, prefrontal cortex, hippocampus, hypothalamus, substantia nigra and superior colliculus by 49 hr, and in all other areas by day 10. The rate of recovery of [3H]citalopram binding varied depending on the area examined. Recovery was slowest in striatal areas, which continued to exhibit decreases of up to 52% of control at 49 hr, as well as in the substantia nigra. In comparison, the amygdala, prefrontal cortex and olfactory tubercle had recovered to control levels at this time (fig. 8). The reasons for differences in recovery to normal binding levels are unclear, but it does not appear to depend on the original levels of binding, since as demonstrated the olfactory tubercle and substantia nigra express similar basal levels of binding.
Effects of acute i.p. administration of WF-23 (1 mg/kg) on [3H]citalopram binding to 5HT transporter binding sites at 2, 5, 9, 13, 25, 49 and 241 hr after injection and compared to vehicle-treated control. Shown are color-coded transformations of autoradiograms of coronal sections of rat brain at the level of the striatum in which each color represents a range of densities of [3H]citalopram binding to 5HT transporters in fmol/mg wet weight tissue. Note that binding remains depressed for up to 49 hr after a single administration of WF-23.
Time course of effects of a single i.p. administration of WF-23 on [3H]citalopram binding to serotonin transporters in 6 brain regions. *P < .05, different from vehicle. Note that [3H]citalopram binding is slowest to recover in the caudate and nucleus accumbens core when compared to other brain regions. Measurements were made 2, 5, 9, 13, 25, 49 and 241 hr after treatment.
In contrast to the robust decreases in [3H]citalopram binding that resulted from administration of 1 mg/kg WF-23, the acute administration of 30 mg/kg cocaine did not significantly alter [3H]citalopram binding (not shown). This was true for all of the areas examined at all time points.
Discussion
The acute administration of the novel tropane analog, WF-23, produced time-dependent increases in spontaneous locomotor activity that were far more intense than those elicited by a high dose of cocaine. These increases in locomotion were concomitant with persistent decreases in binding at DA and 5HT transporters. These findings clearly demonstrate that WF-23, which is approximately 230 times more potent than cocaine at inhibiting DA uptake, and 1500 times more potent at inhibiting 5HT uptake in vitro (Bennett et al., 1995), is also highly potent when administered in vivo.
WF-23 evoked robust increases in horizontal activity that were more intense than cocaine-induced activities. When animals from both groups were placed in adjacent locomotor chambers, behaviors elicited by WF-23 were qualitatively indistinguishable from those elicited by cocaine, such that a blind observer could discern differences based only onintensity of the response. The potency of WF-23 in producing these effects however, is less than would be predicted from in vitro studies. This discrepancy may result from the tropane’s high degree of lipophilicity compared to cocaine, a phenomenon which would reduce overall bioavailability. Moreover, the behavioral characteristics of WF-23 are unlike those of the DA-selective analog PTT, which produced stereotypic behavior different from cocaine (Porrino et al., 1994). In that study, rats treated with PTT exhibited stereoytypies that were not only much more intense than those elicited by cocaine, but were qualitatively different. For example, high doses of cocaine resulted in typical head bobs and rearing activity, whereas, PTT elicited intense circular head movements. In contrast, the overall behavioral profile of WF-23 was virtually identical to that of cocaine.
The effect of the acute administration of WF-23 on dopamine transporter binding, as measured by [125I]RTI-55 binding in striatal membranes, was profound and long-lasting, with significant decreases in [125I]RTI-55 binding observed up to 5 days after the drug administration. The recovery of binding after WF-23 treatment was defined by a relatively complex pattern exhibiting several distinct phases. First, there was a relatively rapid recovery of dopamine transporter binding between 2 to 5 hr in which binding recovered from a 90% decrease (vs. saline) to approximately 50% decrease. This level of binding then remained relatively constant until 49 hr. After that point, binding gradually recovered to normal between 5 and 10 days after the WF-23 injection. Such a complex pattern of recovery could be explained by a depot effect of the drug, in which the decrease in binding that occurred beyond 49 hr might be associated with the slow release of the lipophilic drug from fatty depot sites in brain membranes.
The long-acting effect of WF-23 was in sharp contrast with that of i.p. cocaine treatment, in which [125I]RTI-55 binding returned to control levels 5 hr after cocaine injection. This difference in recovery of dopamine transporter binding between cocaine and WF-23 can be attributed in part to three important structural differences between WF-23 and cocaine. First, in binding studies, WF-23 (racemic form) is 1400 times more potent than cocaine in binding to dopamine transporters (Davies et al., 1993). Because the dissociation rate of a drug is thought to be directly proportional to its Kd , this high affinity would suggest a much slower clearance rate for WF-23 compared to cocaine. Unfortunately, WF-23 has not been radiolabeled, and a direct assessment of its dissociation rate in vitro is not yet available. Although WF-23 has not yet been radiolabeled, another tropane analog from this series of compounds, PTT, has been radiolabeled and was found to have a t1/2 of 4 min (Letchworthet al., 1997). In comparison, the dissociation rate of cocaine is only a few seconds (Reith et al., 1986). A second important factor determining the time course of action of these two drugs is metabolic stability. Cocaine is rapidly metabolized after i.p. injection, and approximately 80 to 90% of its metabolism is due to hydrolysis of the two ester groups on the cocaine molecule (Shuster, 1992). The removal of these two ester groups on WF-23 should significantly increase the metabolic stability of these tropanes. The relatively long-acting in vivo effects of PTT (Hembyet al., 1995; Porrino et al., 1995), another potent tropane with a lower affinity for dopamine transporters compared to WF-23, further supports the role of metabolic stability in determining the duration of action of these drugs. A final factor in determining the time course of these drugs is lipophilicity. WF-23 is much more lipophilic than cocaine: the calculated log P value for WF-23 is 4.0, compared to 2.4 for cocaine (R. Ehrenkaufer, unpublished observations). This increase in lipophilicity is predictable since the phenyl group of cocaine is replaced with a 2-naphthyl, and the esteratic groups are absent. This higher degree of lipophilicity may contribute to the purported depot effect observed after administration of WF-23.
The Scatchard analysis of membrane dopamine transporter binding, as determined 49 hr after a single injection of WF-23 (table 1), showed a significant increase in the Kd for the high affinity [125I]RTI-55 binding site, but no effect on the Bmax of the high affinity sites and no effect on any parameter for the low affinity sites. These data are consistent with a competitive action of WF-23 on the high affinity site of the dopamine transporter. The lack of effect of WF-23 on the Bmax of the high affinity sites further suggests that this drug can be effectively displaced by related tropanes (like [125I]RTI-55) and is, therefore, not irreversible, despite its long-lasting effects in blocking [125I]RTI-55 binding. Thus, the recovery in binding noted between 5 and 10 days after WF-23 administration probably does not require the synthesis of new dopamine transporters, as would be the case for an irreversible ligand such as RTI-76 (Fleckensteinet al., 1996).
Because of methodological differences, some variations between membrane transporter binding and autoradiography are to be expected. First, the additional washing step involved in membrane preparation could have dissociated WF-23 to a greater degree than in autoradiographic experiments. This is why the washing steps were kept to a minimum in the membrane preparations (see ‘Materials and Methods‘). Another difference in the two assays is that two different radioligands were used: [125I]RTI-55 in membranes and [3H]CFT in autoradiography. [125I]RTI-55 was chosen because its high specific activity required ten times less striatal tissue than a [3H] ligand; this was crucial because the experimental design required the hemi-sectioning of each brain, with only one-half of a striatum available for membrane binding studies. The saturation analyses reported in this study could not have been done with [3H]CFT using this amount of tissue. [3H]CFT was used for autoradiography because the neuroanatomical resolution of a [3H] ligand is much superior to that of an [125I] ligand on film. Despite these methodological differences, there was remarkable agreement between the time course observed in striatum for both assays (e.g., compare fig. 3 with fig. 5). However, because these methodological differences cannot be controlled, it is best to interpret the data within each assay and not between assays.
The acute i.p. administration of WF-23 produced persistent, time-dependent reductions in [3H]CFT binding in the striatum, NAc shell and NAc core that closely paralleled the [125I]RTI binding in striatal homogenates. The recovery of [3H]CFT binding however, was regionally specific. Binding was slower to recover in the caudate and NAc core when compared to the shell of the NAc. This finding suggests that DA transporters in the shell may be regulated differently from those in the core or caudate. This is supported by a number of findings. The shell and core differ in dopamine content (Deutch and Cameron, 1992), dopamine input (Zahm and Brog, 1992; Brog et al., 1993; Tan et al., 1995), and differ as well in their response to pharmacological manipulation (Pierce and Kalivas, 1995; Pontieri et al., 1995; Marcus et al., 1996). Additionally, the shell is less susceptible to damage by injections of ibotenic acid or 6-hydroxydopamine into the ventral mesencephalon that results in an almost complete loss of tyrosine hydroxylase in the core (Zahm, 1991). Finally, DA innervation to the core undergoes degeneration after methamphetamine treatment, while the shell is spared (Broening et al., 1997).
[3H]citalopram binding, as a result of the acute administration of WF-23, appeared to follow a similar time-course of recovery as [3H]CFT binding. This is most likely due to the fact that WF-23 is relatively equipotent at 5HT as well as DA transporters (Bennett et al., 1995). Again, there were regional differences, with the striatum being slower to recover to control levels than other areas. This may relate to the possibility that WF-23 acts like a depot type drug, due to its lipophilic nature. This idea is supported by the finding that areas of the brain that exhibit the slowest rates of recovery are also those that contain the highest content of white matter.
Increases in locomotor activity induced by WF-23 are consistent with the finding that stimulant-induced increases in locomotor activity are DA-mediated events (Kaddis et al., 1993; McCreary and Marsden, 1993; McGregor and Roberts, 1993; Mattingly et al., 1994; Daunais and McGinty, 1996). Increases in activity were significantly correlated with DA transporter occupancy, as estimated by reductions in [3H]CFT binding, in all areas. This was particularly true in the caudate (rxy = 0.72, d.f. 27, P < .0001) and core of the nucleus accumbens (rxy = 0.74, d.f. 27, P < .0001), as well as in the NAc shell (rxy = 0.54, d.f. 27, P < .002). The similarity between the NAc core and the caudate is not surprising given that the core is considered to be a functional extension of the neostriatum, while the shell is thought to be a transition zone between the striatum and the extended amygdala (Heimer et al., 1991).
In our study, occupancy of at least 50% of DA transporters in the caudate appeared to be necessary to produce increases in locomotor activity as demonstrated by figure 6. When less than 50% of the transporters were occupied, spontaneous locomotor behavior was not elevated over normal levels. This is consistent with previous findings that indicate a relationship between potency at DA transporters and psychomotor stimulant effects of cocaine and cocaine analogs (Ritzet al., 1987; Bergman et al., 1989, Spealmanet al., 1989; Cline et al., 1992; Rothmanet al., 1992; Vaugeois et al., 1993; Porrinoet al., 1994). Our findings also agree with the recent findings of Volkow and colleagues (1997) which demonstrated that approximately 50% of DA transporters had to be blocked in order for human subjects to perceive the reinforcing effects (rush) of cocaine.
The intense reinforcing effects of cocaine have been attributed to its rapid brain entry and dopamine transporter occupancy, in addition to its short duration of action. These pharmacokinetic characteristics are thought to lead to repeated drug intake over short periods of time in the form of binges. One strategy for the development of medications for cocaine addiction has been the synthesis of slow onset, long duration inhibitors of the dopamine transporter to act as cocaine substitutes or replacements. This strategy is based on the effective use of slow-onset, long acting compounds such as methadone or LAAM for the treatment of opiate addiction and nicotine patches for treatment of tobacco-related addictions. The present data suggest that WF-23 may be a potential candidate for use as a treatment for cocaine addiction, since it fulfills the criteria of slower onset and long duration of action.
Footnotes
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Send reprint requests to: Dr. Linda J. Porrino, Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157.
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↵1 This work was supported by NIDA Grants P50 DA06634, DA 07246 (J.B.D.) and DA06301 (H.M.L.D.).
- Abbreviations:
- WF-23
- (2β-propanoyl-3β-(2-naphthyl)-tropane)
- DA
- dopamine
- 5HT
- serotonin
- NAc
- nucleus accumbens
- LAAM
- L-α-acetylmethadol
- Received September 18, 1997.
- Accepted February 16, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
References
In this issue
Long-Acting Blockade of Biogenic Amine Transporters in Rat Brain by Administration of the Potent Novel Tropane 2β-Propanoyl-3β-(2-Naphthyl)-Tropane
