3 الفا دیفنیل متوکسی تروپان 3α Diphenylmethoxytropaneآنالوگ کوکائین
سه الفا دی فنیل متوکسی تروپان یک آنالوگ ماده مخدر کوکائین است که دارای ساختاری مشابه با کوکائین است
سه الفا دی فنیل متوکسی تروپان یک آنالوگ ماده مخدر کوکائین است که دارای ساختاری مشابه با کوکائین است
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| مخدر 3 الفا دیفنیل متوکسی تروپان |
Abstract
Assessment of the Influence of Histaminergic Actions on Cocaine-Like Effects of 3α-Diphenylmethoxytropane Analogs
Abstract
Previous studies demonstrated that analogs of benztropine (BZT) possess high affinity for the dopamine (DA) transporter (DAT) but generally have behavioral effects different from those of cocaine, suggesting either unique actions at the DA transporter or that another action of these drugs interferes with cocaine-like effects. Because the parent compound has histamine-antagonistic effects, the affinity of its analogs for histamine H1, H2, and H3 receptors were compared with DA transporter affinity to assess whether those differences predicted the amount of cocaine-like activity. All of the compounds displaced [3H]mepyramine from H1, [125I]iodoaminopotentidine from H2, and [3H]N-α-methylhistamine from H3 histamine receptors with affinities ranging from 15.7 to 37,600, 218 to >4430, and 4040 to >150,000 nM, respectively. Affinities at histamine H1 receptors were, respectively, approximately 25- or 300-fold greater than those at H2 or H3 histamine receptors. Relative affinities for H1 and DAT binding did not reliably predict the degree of cocaine-like stimulation of locomotor activity. In addition, interactions of various histaminic agents with cocaine assessed whether an action at any of the histamine sites could interfere with cocaine-like effects. None of the histaminic agents fully substituted for cocaine in rats trained to discriminate 10 mg/kg cocaine from saline nor did any of the compounds antagonize or otherwise diminish the discriminative stimulus effects of cocaine. The results suggest that affinity for histamine receptors cannot account for the diminished cocaine-like effects of the BZT analogs and suggest alternatively that these compounds have actions different from those of cocaine but likely mediated by their interaction with the DAT.
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A variety of analogs of benztropine (BZT) have been synthesized that have affinity for the dopamine (DA) transporter and inhibit the uptake of DA (for review, see Newman and Kulkarni, 2002). Despite these in vitro actions, most of these compounds lack cocaine-like behavioral effects, including those that are predictive of drug abuse (Katz et al., 1999, 2004). One possible explanation of the differences between behavioral effects of cocaine and the BZT analogs is that these latter compounds interact with the DA transporter in a manner that is different from the manner in which cocaine and its analogs act (Vaughan et al., 1999; Reith et al., 2001). An alternative explanation is that BZT analogs have affinity for other receptors that may interfere with or in some way alter their cocaine-like behavioral effects. Prominent actions of the parent compound BZT are antimuscarinic and antihistaminic effects (Richelson, 1981; McKearney, 1982a). The present study was conducted to assess the potential of actions at histamine receptors to explain these anomalous effects of BZT analogs.
In previous studies, we have examined the influence of antimuscarinic actions on the behavioral effects of BZT analogs. In one study, the antimuscarinics atropine and scopolamine were examined in combination with cocaine to assess whether the effects of this combination was similar to the effects of the BZT analogs. More specifically, it was hypothesized that if the antimuscarinic effects of the BZT analogs interfere with their cocaine-like behavioral effects, then atropine and scopolamine should noncompetitively antagonize the effects of cocaine. Rather than an antagonism of the effects of the BZT analogs, both atropine and scopolamine added to the effects of cocaine, shifting the cocaine dose-effect curve to the left (Katz et al., 1999). This leftward shift was obtained with both the discriminative-stimulus effects and the locomotor stimulant effects of cocaine. In addition, a second generation of compounds was synthesized that had reduced muscarinic affinity (Agoston et al., 1997). These compounds also possessed a reduced cocaine-like spectrum of behavioral effects (Katz et al., 2004), suggesting that the antimuscarinic effects of the BZT analogs do not interfere with their cocaine-like effects and focusing attention in the present study on histaminic actions.
In the present study, we examined the binding of a series of BZT analogs at histamine receptors. The parent compound has reported affinity for H1 histamine-antagonistic activity; however, its actions at other subtypes of histamine receptors have not been reported. Therefore, we examined the affinity of the drugs for H1, H2, and H3 histamine receptors. Although values varied across a wide range, the BZT analogs had affinity for the DA transporter and histamine receptors and varied with regard to their effectiveness in stimulating locomotor activity (Katz et al., 1999, 2004). We hypothesized that if activity at histamine receptors opposes the cocaine-like effects resulting from DA transporter binding, then relative affinities at histamine sites and the DA transporter should predict behavioral effects. More specifically, those compounds with greater affinity for the DA transporter than for histamine receptors should have cocaine-like locomotor-stimulant effects. Conversely, those with greater affinity for histamine receptors than the DA transporter should not show cocaine-like locomotor stimulant effects. For this assessment, we examined the relative affinities of the various BZT analogs and compared them with their effects in stimulating locomotor activity. We further examined the interactions of cocaine and various histamine receptor agents in animals trained to discriminate cocaine from saline injections. If the histaminic effects of the BZT analogs are interfering with the expression of effects mediated by their actions at the DA transporter, then histaminic drugs should antagonize the discriminative-stimulus effects of cocaine. We examined histamine H1 antagonists, and agonists and antagonists at H2 and H3 histamine receptors as the actions of BZT analogs at these sites have not been characterized.
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Materials and Methods
[3H]Mepyramine Binding Assay. Frozen rat whole brain including cerebellum was thawed on ice and homogenized in 30 volumes of ice-cold 50 mM Na-K buffer (37.8 mM Na2HPO4, 12.2 mM KH2PO4, pH adjusted to 7.5 at 25°C) using a Brinkman Polytron (setting 6 for 20 s) and centrifuged at 25,000g for 10 min at 4°C. The supernatant was discarded, and the pellet was washed once more by resuspension in ice-cold Na-K buffer and centrifuged. The resulting pellet was then resuspended in Na-K buffer to give 200 mg/ml wet weight final volume.
Binding assays were conducted in 50 mM Na-K buffer on ice. Each concentration of this assay was done in triplicate with the total volume in each tube being 0.5 ml. The final concentration of [3H]mepyramine (specific activity 20 Ci/mmol) was 2 nM per tube. Nonspecific binding was measured using promethazine HCl 100 μM final concentration as the displacer. This was allowed to incubate for 60 min at room temperature. Binding affinity was determined by incubating 20 mg of tissue and [3H]mepyramine in the absence or presence of the compound being tested. The incubation was ceased by the addition of ice-cold buffer and rapid filtration with a Brandel R48 filtering manifold, through GF/B glass fiber filter paper that was presoaked in 0.3% polyethylenimine in water to reduce nonspecific binding. The filters were washed twice with 5 ml of ice-cold buffer and then transferred to scintillation vials. Three ml of scintillation cocktail (Beckman Ready Safe) was added to the vials, and the vials were counted the following day using a Beckman 6000 liquid scintillation counter.
[125I]Iodoaminopotentidine Binding Assay. Frozen guinea pig striatum was thawed on ice and homogenized in 30 volumes of ice-cold 50 mM Na-K buffer (37.8 mM Na2HPO4, 12.2 mM KH2PO4, pH adjusted to 7.5 at 25°C) using a Brinkman Polytron (setting 6 for 20 s) and centrifuged at 260g for 10 min at 4°C. The supernatant was saved and respun at 25,000g for 20 min at 4°C. The resulting pellet was then resuspended in Na-K buffer containing 0.1% gelatin to give 25 mg/ml wet weight final volume.
Binding assays were conducted in 50 mM Na-K buffer containing 0.1% gelatin on ice. Each concentration of this assay was done in triplicate with the total volume in each tube being 0.5 ml. The final concentration of [125I]iodoaminopotentidine (specific activity 2000 Ci/mmol) was 2.5 pM per tube. Nonspecific binding was measured using tiotidine 0.1 mM final concentration as the displacer. This was allowed to incubate for 150 min at room temperature. Binding affinity was determined by incubating 2.5 mg of guinea pig striatum and [125I]iodoaminopotentidine in the absence or presence of the compound being tested. The incubation was ceased by the addition of ice-cold buffer and rapid filtration with a Brandel R48 filtering manifold, through GF/B glass fiber filter paper that was presoaked in 0.3% polyethylenimine in water to reduce nonspecific binding. The filters were washed twice with 5 ml of ice-cold buffer and then transferred to 12 × 75 polystyrene tubes. This was counted in an MP Biomedicals Micromedic 10/600 Plus gamma counter.
[3H]N-α-Methylhistamine Binding Assay. Frozen Sprague-Dawley rat frontal cortex was thawed on ice and homogenized in 30 volumes of ice-cold 50 mM Tris HCl buffer (pH adjusted to 7.5 at 25°C) using a Brinkman Polytron (setting 6 for 20 s) and centrifuged at 1000g for 10 min at 4°C. The supernatant was saved and respun at 7000g for 10 min at 4°C. The resulting pellet was then resuspended in Tris buffer to give 50 mg/ml wet weight final volume.
Binding assays were conducted in 50 mM Tris HCl buffer on ice. Each concentration of this assay was done in triplicate with the total volume in each tube being 0.5 ml. The final concentration of [3H]N-α-methylhistamine (specific activity 82 Ci/mmol) was 1 nM per tube. Nonspecific binding was measured using 2 μM (R)-α-methylhistamine. This was allowed to incubate for 30 min at 30°C. Binding affinity was determined by incubating 5 mg of rat frontal cortex membrane and [3H]N-α-methylhistamine in the absence or presence of the compound being tested. The incubation was ceased by the addition of ice-cold buffer and rapid filtration with a Brandel R48 manifold through GF/B glass fiber filter paper that was presoaked in 0.3% polyethylenimine in water to reduce nonspecific binding. The filters were washed twice with 5 ml of ice-cold buffer, and then the filters were transferred to scintillation vials. Three milliliters of scintillation cocktail (Beckman Ready Safe) was added to the vials, and the vials were counted the following day using a Beckman 6000 liquid scintillation counter.
[3H]WIN 35,428 Binding Assay. Male Sprague-Dawley rats (200-250 g) obtained from Taconic Farms (Germantown, NY) were decapitated, and their brains were removed to an ice-cold dish for dissection of the striatum. The striatum was then homogenized in 30 volumes of ice-cold Krebs-HEPES buffer (15 mM HEPES, 127 mM NaCl, 5 mM KCl, 1.2 mM MgSO4, 2.5 mM CaCl2, 1.3 mM NaH2PO4, 10 mM d-glucose, pH adjusted to 7.4 at 25°C) and centrifuged at 20,000g for 10 min at 4°C. The supernatant was discarded, and the pellet was washed two more times by resuspension in ice-cold Krebs-HEPES buffer and centrifuged at 20,000g for 10 min at 4°C. After the second wash, the pellet was resuspended to give 25 mg/ml wet weight final volume.
Binding assays were conducted in Krebs-HEPES buffer on ice. Each concentration of this assay was done in triplicate with the total volume in each tube being 0.5 ml. The final concentration of [3H]WIN 35,428 was 1.5 nM. Nonspecific binding was determined by using cocaine HCl 100 μM final concentration as the displacer.
Binding affinity was determined by using triplicate samples of membranes incubated in the presence and absence of the test drug. There was a 5-min preincubation period during which buffer, test drug, and tissue were in the tube. After 5 min, [3H]WIN 35,428 was added to the tube and allowed to incubate for 60 min on ice. The incubation ended with the addition of ice-cold buffer and rapid filtration through a Brandel R48 (Brandel Inc., Gaithersburg, MD) filtering manifold, through GF/B glass fiber filter paper that was presoaked in 0.1% bovine serum albumin in water to reduce nonspecific binding. The filters were washed twice with 5 ml of ice-cold buffer and then transferred to scintillation vials. Three milliliters of scintillation cocktail (Beckman Ready Safe) was added to the vials, and the vials were counted the following day using a Beckman 6000 liquid scintillation counter (Beckman Coulter Instruments, Fullerton, CA). Data for some of the drugs have been published previously (Katz et al., 1999, 2004). For the histaminergic compounds, the assay was modified slightly, and as described in a previous article (Houlihan et al., 2002). For these compounds the assay conditions were as described above, with the exception that a sucrose-phosphate buffer at pH 7.4 was used, and tubes containing 0.5 nM [3H]WIN 35,428 and 1.0 mg of striatal tissue were incubated for 120 min before filtration.
Cocaine Discrimination. Male Sprague-Dawley rats (Taconic Farms) weighing 300 to 360 g were individually housed (12-h light/dark cycle, 7:00 AM/7:00 PM) and maintained at 85% of their unrestricted feeding weights. The rats had free access to water and were fed 10 to 15 g of “Bacon Lover's Treats” (BioServ, Frenchtown, NJ) 1 h after testing daily. All rats were experimentally naive at the start of the study. All experimental sessions were conducted between 1:00 PM and 4:00 PM in an Association for Assessment and Accreditation of Laboratory Animal Care International-accredited facility that was run in accordance with National Institutes of Health Policy Manual 3040-2, Animal Care and Use in the Intramural Program.
Experiments were conducted with subjects in an operant-conditioning chamber (modified ENV-001, inside dimensions 29.2 × 24.2 × 21 cm) produced by Med Associates (St. Albans, VT) containing two response keys (levers requiring a force of 0.4 N through 1 mm to register a response) and a centrally located food dispenser that delivered one 45-mg pellet as a reinforcer. A pair of green and a pair of yellow light-emitting diodes were arranged horizontally above each lever. The chamber was enclosed in a larger outer compartment that provided light and sound attenuation. White noise was delivered to the chamber at all times to mask extraneous noises.
Initially, rats were trained to respond on both keys and were eventually trained to respond on one after cocaine (10 mg/kg i.p.) and the other after saline (i.p.) injection. Each response produced an audible click. The ratio of responses to food pellets (fixed ratio or FR) was gradually increased until, under the final conditions, the completion of 20 consecutive responses on the cocaine- or saline-appropriate key produced food. Incorrect responses reset the FR response requirement. The right versus left assignment of cocaine- and saline-appropriate keys was counterbalanced among subjects.
Subjects were placed in chambers immediately after injection. There was a 5-min time-out period during which lights were off and responses produced an audible click, but no other scheduled consequences. After the time-out, the house light was turned on until the completion of the FR 20 response requirement. Food presentation was followed by a 20-s time-out during which all lights were off, and responses had no scheduled consequences other than the feedback click. Sessions ended after 20 food presentations or 15 min, whichever occurred first. Sessions were conducted 5 days per week, and cocaine or saline sessions were scheduled to in a double-alternation sequence (cocaine-saline-saline-cocaine). Training continued until subjects met the criteria on four consecutive sessions of at least 85% cocaine- or saline appropriate responding over the entire session as well as the first FR.
Once these criteria were met, testing began. Test sessions were conducted with the administration of different doses of cocaine, histaminic agents, or their combination before sessions. Test sessions were identical to training sessions with the exception that responses on either key were reinforced with the completion of the FR requirement. All data collection and programming of behavioral contingencies was accomplished with software from Med Associates.
Locomotor Activity. Male Swiss-Webster mice (30-35 g) were studied in Digiscan activity monitors (Omnitech Electronics, Columbus, OH). The monitors consisted of 40-cm3 clear acrylic chambers equipped with photoelectric detectors placed 2.56 cm apart along the walls of the chamber. Mice were injected and placed immediately in the chamber with one subject per chamber. Activity was assessed for 1 h, and data during the 30-min period during which activity was maximally stimulated were analyzed. Data for some of the drugs have been previously published (Katz et al., 1999, 2004), and these data are identified in Table 1.
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TABLE 1
Binding of BZT and its analogs at histamine receptors and at the dopamine transporter, and maximal stimulation of locomotor activity Values for S.E.M. are shown for compounds tested and having significant effects in the present study.
Drugs. The following drugs with the exceptions noted were obtained from Sigma-Aldrich (St. Louis, MO): (-)-cocaine HCl; benztropine mesylate; H1 histaminergics promethazine HCl, triprolidine, (S)-(+)-chlorpheniramine maleate, and mepyramine maleate; H2 histaminergics dimaprit dihydrochloride, zolantidine HCl (Tocris Cookson Inc., Ellisville, MO), ranitidine HCl, and ICI-162,846 (Tocris Cookson Inc.); and H3 histaminergics (R)-(-)-α-methylhistamine dihydrobromide (Tocris Cookson Inc.), thioperamide maleate, and clobenpropit dihydrobromide (Tocris) were obtained from Sigma/RBI (St. Louis, MO). All BZT analogs were synthesized in the Medicinal Chemistry Section, National Institute on Drug Abuse, Intramural Research Program, and have been described previously. Their structures are provided in Fig. 1. All drugs were dissolved in either 0.9% physiological saline or sterile water and administered i.p. in a volume of 1.0 ml/kg body weight. All drugs were injected immediately before testing.
Data Analyses. Radioligand displacement data were analyzed using the computer program GraphPad Prism (GraphPad Software Inc., San Diego, CA) using the models for competitive binding. Data from at least three replicate experiments were individually analyzed to determine Ki values, and means and SEMs were calculated. The range of doses tested for each compound was 0.1 nM to 100 μM in triplicate. Reported Ki values are those for the unlabeled ligand using the Cheng-Prusoff equation (Cheng and Prusoff, 1972). Some of the previously published data for displacement of [3H]WIN 35,428 were analyzed with the computer program LIGAND and are indicated as such in Table 1.
For the studies of locomotor activity, data from the 30-min period in which maximal stimulation of horizontal ambulatory activity was observed were selected for analysis. Each dose-effect curve was analyzed using standard analysis of variance and post hoc testing to determine the significance of the effects at individual doses. For those compounds that produced a significant stimulation in activity, the horizontal activity counts at the dose producing the maximal effect were determined. A Fisher's exact test was used to determine whether there was an association between compounds with greater than 2-fold differences in affinity for H1 and DA transporter sites and the observation of significant elevations in locomotor activity. Because optimally localized H2 and H3 receptors might mitigate their lower affinities, we further examined whether the ratio of affinities predicted the degree of stimulant effect. Results of these analyses were considered significant if a two-tail P value was less than 0.05.
For the cocaine discrimination experiments, rates of responding and the percentage of responding on the cocaine-appropriate lever were used to generate dose-effect curves. The data were analyzed using analysis of variance and linear regression techniques to generate ED50 values and 95% confidence limits (Snedecor and Cochran, 1967). Experiments were conducted on three different groups of rats, and the effects of cocaine were assessed in each. The effects of cocaine in all of the subjects were averaged and displayed in Fig. 2. For comparisons of drug interactions, the effects of the drugs in combination with cocaine were compared with the effects of cocaine alone in the corresponding subset of subjects in which the interactions were assessed (Figs. 3, 4, 5; Tables 2, 3, 4). The significance of interactions were determined by an analysis of relative potency (Finney, 1964), which is considered significant if the 95% confidence limits of the relative potency value do not include 1.0. The ED50 values were not determined if there was a nonsignificant linear regression. If response rate was less than 0.02 responses per second in at least half of the subjects then the number of observations was considered to be too few for a reliable calculation of key selection, and therefore the percentage of drug responses was not graphed at that dose. All data points for response rates are shown on the graph.
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