Behavioral effects of the R-(+)- and S-(−) enantiomers of the D1-likepartial receptor agonist SKF 83959 in monkeys

Rajeev I. Desai1, John L. Neumeyer1,2, Carol A. Paronis1,2, Phong Nguyen1, and JackBergman1,2

1 McLean Hospital/Harvard Medical School, 115 Mill Street, Belmont, MA 02478, USA

2 Brain Research Laboratories, Inc., 115 Mill Street, Belmont, MA 02478, USA

Abstract—Dopamine D1-like partial receptor agonists such as SKF 83959 have been proposed as potentialcandidates for the treatment of cocaine addiction. The present studies were conducted to furthercharacterize SKF 83959 by pharmacologically evaluating effects of its R-(+)- and S-(−) enantiomers,MCL 202 and MCL 201, respectively, on overt behavior (eye blinking) and schedule-controlledperformance in squirrel monkeys. MCL 202, like the D1 full receptor agonist SKF 82958, produceddose related increases in eye blinking and decreases in rates of fixed-ratio responding. However, themagnitude of effects of MCL 202 on eye blinking was less than observed with SKF 82958. In contrastto the effects of its R-(+) enantiomer, MCL 201 was relatively devoid of behavioral activity up todoses that were approximately 10-fold greater than MCL 202. Pretreatment with the selective D1-like receptor antagonist SCH 39166 dose-dependently antagonized increases in eye blinkingproduced by MCL 202, confirming the involvement of D1 mechanisms in its effects. A dose-ratioanalysis of the antagonism of effects of MCL 202 by SCH 39166 revealed an apparent pA2 value of7.675 with a slope of −0.78 ± 0.04. In further studies, pretreatment with MCL 202 antagonized theeffects of SKF 82958 on eye blinking and, like SCH 39166, schedule-controlled behavior in a dose-related manner. A dose-ratio analysis of the antagonist effects of MCL 202 on the SKF 82958-inducedincreases in eye blinking revealed ratios of 2.7, 4.8 and 31.1 for 0.1, 0.3 and 1.0 mg/kg dose of theantagonist, respectively, indicative of a significant change in the potency of SKF 82958. These resultssuggest that MCL 202, like its parent compound SKF 83959, has both D1 receptor-mediated agonistand antagonist properties, consistent with its characterization as a partial agonist at the D1-likereceptor. In addition, the inactivity of MCL 201, the S-(−)-enantiomer, suggests that the behavioraleffects of SKF 83959 can be attributed primarily to the activity of its R-(+)-enantiomer.

KeywordsDopamine D1-like receptors; Enantiomers of SKF 83959; Partial agonist; Eye blinking; Schedule-controlled behavior; SKF 82958; Squirrel monkeys

1. IntroductionA considerable body of pharmacological evidence suggests that dopamine D1-like mechanismsmay be involved in the reinforcing and other abuse-related behavioral effects of cocaine and

Corresponding author: Jack Bergman, Preclinical Pharmacology Laboratory, McLean Hospital/Harvard Medical School, 115 Mill Street,Belmont, MA 02478, USA. Fax: +1-617-8552464, Tel: +1-617-8552417, e-mail: jbergman@hms.harvard.edu.Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customerswe are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resultingproof before it is published in its final citable form. Please note that during the production process errors may be discovered which couldaffect the content, and all legal disclaimers that apply to the journal pertain.

NIH Public AccessAuthor ManuscriptEur J Pharmacol. Author manuscript; available in PMC 2008 July 9.

Published in final edited form as:Eur J Pharmacol. 2007 March 8; 558(1-3): 98–106.

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other monoaminergic psychomotor stimulants (e.g. Bergman et al., 1990; Corrigall and Coen,1991; Self and Stein, 1992; Morelli et al., 1993; Weed and Woolverton, 1995; Spealman et al.,1997; Weed et al., 1997; Singh et al., 1997; Tidey and Bergman, 1998; Abrahams et al.,1998a, 1998b; Henry et al., 1998). This evidence has led to the suggestion that D1-like receptorligands may be candidate medications for the treatment of cocaine dependence (Mendelsonand Mello, 1996; Platt et al., 2002). In this regard, D1 ligands with partial agonist effects havebeen of particular interest for several reasons. First, D1 ligands that are presumed to be partialagonists (e.g., SKF 38393, SKF 77434, SKF 83959) do not appear to have reinforcing effectsassociated with D1 full receptor agonists such as SKF 82958 or R-6-BrAPB and may producea less pronounced disruption of motor behavior than observed with D1 receptor blockers suchas SCH 39166 (Weed and Woolverton 1996; Platt et al., 2000, 2001; Mutschler and Bergman,2002). Second, these drugs also appear to attenuate the actions of cocaine or other psychomotorstimulants in studies of their discriminative stimulus and behavioral stimulant effects in rodentsand monkeys (Spealman et al., 1997; Katz et al., 1999; Platt et al., 2001). As well, acutetreatment with a range of D1 partial receptor agonists, including SKF 38393, SKF 75670, SKF83959 and SKF 77434 has been reported to reduce or antagonize i.v. self-administration ofcocaine in rats and/or monkeys (Bergman and Rosenzweig-Lipson, 1992; Katz and Witkin,1992; Caine et al., 1999; Mutschler and Bergman, 2002). Third, D1 ligands such as SKF 38393and SKF 83959 can inhibit the reinstatement of extinguished cocaine-seeking behavior, raisingthe possibility that these compounds may be effective in attenuating clinical relapse (Spealmanet al., 1999).

The relative efficacy of D1-like receptor agonists generally has been determined using bothin vitro and in vivo studies (for review see Jutkiewicz and Bergman, 2004). In vitro studieshave characterized dopamine D1-like receptors by their positive coupling to adenylyl cyclase(AC) stimulation (Kebabian and Calne, 1979), and more recently to phosphoinositide (PI)hydrolysis (Mahan et al., 1990; Undie and Friedman, 1990; Undie, 1999; Undie et al., 1994,2000). In these types of studies, efficacy can be estimated by comparing the maximal effectsof a D1 ligand to the effects of dopamine itself (e.g. Anderson and Jansen, 1990). Unfortunately,a robust correspondence has not been obtained between the behavioral effects of D1-likereceptor agonists and their efficacy in stimulating AC and/or PI activity (e.g. Daly andWaddington, 1992; Gnanalingham et al., 1995a, 1995b; Fornaguera et al., 1999; Katz et al.,1999; Platt et al., 2001; Sinnott and Nader, 2001; Desai et al., 2003a, 2005). This lack ofassociation between in vitro and in vivo efficacy estimates for D1-like receptor agonists isexemplified by the comparison of biochemical and behavioral data obtained for the D1 ligandSKF 83959. In in vitro studies, this compound serves as a potent antagonist of AC-coupleddopamine D1-like receptors in both rats (Arnt et al., 1992; Gnanalingham et al., 1995c) andprimates (Andringa et al., 1999). In behavioral studies, however, the effects of SKF 83959 inboth rats and primates are more comparable to those of dopamine D1-like full receptor agonists.Thus, SKF 83959, like a range of D1 receptor agonists, induces vacuous chewing and enhancedgrooming in rats (Downes and Waddington, 1993) and dose-related increases in eye-blinkingin monkeys (Jutkiewicz and Bergman, 2004). Further, SKF 83959, like other D1 receptoragonists, can reduce motor deficits in MPTP-lesioned rats and monkeys (Gnanalingham et al.,1995b, 1995c; Andringa et al., 1999b; Cools et al., 2002) and may induce dyskinesias inhaloperidol-sensitized monkeys (Peacock and Gerlach, 2001). Finally, the profile of behavioraleffects of SKF 83959, like those of the dopamine D1-like receptor agonist A 68930, whichstimulates AC with dopamine-like efficacy, are significantly retained in D1-like receptorknockout mice (Clifford et al., 1999). Taken together, these findings suggest that our currentunderstanding of the cellular mechanisms that mediate D1 efficacy in vivo is incomplete, andthat behavioral procedures currently provide the most useful means for evaluating the efficacyof D1-like receptor ligands.

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Recent regulatory guidelines in the USA and Europe have recommended the development ofsingle enantiomers of compounds that have clinical applications. This recommendation isbased on the idea that single enantiomers may offer clinical advantages over the racemate interms of potency, efficacy and/or tolerability (reviewed by Caldwell, 2001). With regard todopamine D1 receptor agonists, Neumeyer et al. (1992, 2003) have synthesized and developeda number of novel substituted phenylbenzazepine congeners of the dopamine D1-like receptorpartial agonist SKF 83959 including the R(+)- and S(−)-enantiomers, MCL 202 and MCL 201,respectively. Receptor binding studies have revealed that, in vitro, both MCL 202 and MCL201 are highly selective for D1-like receptors relative to D2-like receptors (Neumeyer et al.,2003). Further, MCL 202 is approximately 2-fold more potent than its racemic mixture SKF83959 at the D1 receptor, whereas MCL 201 is 18-fold less potent than SKF 83959 (Neumeyeret al., 2003). The present study was designed to extend these observations with the R(+)- andS(−)-enantiomers of SKF 83959 by characterizing their effects on eye blinking and scheduledcontrolled behavior in squirrel monkeys. The induction of eye blinking is a robust behavioralmeasure of D1-like receptor agonist activity and appears to be useful for distinguishingefficacy-related differences in the effects of dopamine D1-like receptor agonists (e.g. Bergmanet al., 1995; Elsworth et al., 1991; Kleven and Koek, 1996; Jutkiewicz and Bergman, 2004).The effects of D1 receptor agonists on schedule-controlled responding also have beenextensively documented in monkeys, and serves as a second means for evaluating the efficacy-related effects of D1 receptor agonists (Bergman et al. 1991; 1995; Katz et al., 1995).

In the present studies, the agonist effects of MCL 202 and MCL 201 on eye blink rate and ratesof responding maintained under stimulus-shock termination first were compared with theeffects of the well-established dopamine D1-like full receptor agonist SKF 82958. The agonisteffects of MCL 202 on eye-blinking also were studied after treatment with different doses ofthe selective dopamine D1-like receptor antagonist, SCH 39166 to quantitatively examine theinvolvement of D1 receptor-mediated mechanisms in these effects. Finally, the antagonisteffects of MCL 202 were studied by determining how it modified the effects of SKF 82958 oneye blink rate and scheduled controlled responding. Overall, results of these studies indicatethat D1 activity of SKF 83959 resides in its R-(+)-enantiomer (MCL 202) and that its behavioraleffects are consistent with its characterization as a D1 partial agonist.

2. Materials and Methods2.1. Subjects

Eight experimentally naïve male squirrel monkeys (Saimiri sciureus), weighing 700 to 950 gwere used as subjects. All monkeys were individually housed in a temperature- and humidity-controlled vivarium. Monkeys had unrestricted access to water and were fed a daily allotmentof high protein monkey chow (Purina Monkey Chow, St. Louis, MO), supplemented with fruitand multivitamins. The monkeys were randomly divided into two groups. The subjects, s-2,s-3, s-7 and s-9 were used in observational studies that measured eye blinking and the remainingfour monkeys, s-4, s-13, s-8, and s-11, were studied in experiments involving schedule-controlled behavior. All experiments were conducted between 08:00 AM and 06:00 PM underprotocols that were approved by the Institutional Animal Care and Use Committee at McLeanHospital. Monkeys were maintained in accordance with guidelines provided by the Committeeon Care and Use of Laboratory Animals of the Institute of Laboratory Animals Resources,National Institutes of Health. The facility in which subjects were housed and studies wereconducted is licensed by the U.S. Department of Agriculture.

2.2. Eye blinking: apparatus and experimental methodsAll testing was conducted in a specifically constructed chamber (20.2″ × 10″ × 10″). Monkeyssat in a customized Lexan chair with blackened side and back walls to provide color contrast.

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The front wall of the chair was removed in order to facilitate videotaping with a compact videocamera (JVC, model GR-AX10), equipped with a telephoto lens located at a distance ofapproximately 2 feet in front of the seated monkey. Observation of eye blink responses wasfacilitated by a neck plate that was attached to the chair and maintained a relatively constantorientation of the monkeys head. In order to view eye blink responses when the monkey turnedits head, a curved piece of mirror Plexiglas was placed behind the monkeys head. Optimumillumination was provided by positioning two vertical fluorescent lights on the side walls ofthe chamber. A computer-driven program (ATI Multimedia Software, ATI Player) capturedthe camera image which was viewed on a 20″ monitor and recorded on 6 hour videotapes usinga VHS VCR (JVC, model HR-VP69U).

During test sessions, the effects of various does of drugs or vehicle were examined in eachmonkey for four or five successive 15-min components. Each component comprised a 10-minhabituation period followed by a 5-min period during which eye blinking was videotaped.During the entire videotaping session the image of the monkey head was transmitted by thevideo camera and displayed on the computer screen; this allowed for continuous observationof the subject from an adjacent room. The videotape of each 5-min session component wasscored by observers blind to the treatment conditions. An eye blink was defined as a visibleclosure and opening of the eyelid. Each component was scored by at least one trained observer.To evaluate scoring reliability, test components were periodically rescored by a second trainedobserver for whom inter-observer reliability criteria (>90% concordance in blink rate duringpreceding scoring sessions) had been met.

2.3 Schedule-controlled behavior: apparatus and experimental methodsDuring experimental sessions, subjects sat in Plexiglas chairs (Kelleher and Morse, 1968) inventilated, sound-attenuating chambers that delivered white noise to the chambers at all timesto mask extraneous sounds. While seated, monkeys faced a panel containing colored stimuluslights serving as a visual stimuli and a lever set at 4 inches above the waist plate. Each pressof the lever with a force greater than 0.2 N produced an audible click and was recorded as aresponse. Cumulative recorders were used to track and record patterns of responding over thesession. Prior to each session a shaved portion of the monkey’s tail was coated with electrodepaste and placed under brass electrodes through which a brief, low-intensity shock stimulus (3mA for 200 ms) could be delivered.

All subjects were trained to respond under a 30-response fixed ratio schedule of stimulus-termination. Under this schedule, the illumination of stimulus lights on the front panel starteda program under which shock stimuli were delivered every 30-s. The completion of 30consecutive responses turned off the stimulus lights and the associated program of shockdelivery, and initiated a timeout (TO) period of 10-s during which all stimulus lights were offand responding had no scheduled consequences. Sessions were terminated after delivery offive shock stimuli in any one component. Subjects were considered to be trained under terminalschedule contingencies when responding reliably terminated visual stimuli within 30-sfollowing their illumination. Daily sessions were comprised of five sequential components.Each component comprised a 9-min period during which no stimuli were presented andresponding had no programmed consequences (TO 9′) followed by a 3-min period during whichthe FR 30, TO 10″ schedule of stimulus–termination was in effect.

2.4 Eye Blinking: drug testingCumulative dosing procedures were used to determine the effects of drugs on eye blinking nomore than once or twice weekly. In these experiments, incremental doses of a drug wereadministered at the beginning of the 15-min components of the test session, permitting theevaluation of up to 5 cumulative doses during a single session. In some instances, a complete

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dose-effect function could be determined in a single session. In other instances, the completedose-effect was determined by testing two or three overlapping dose ranges over two or threetest sessions. Using these procedures, the effects of SKF 82958 (0.03–1.0 mg/kg), MCL 201(0.003–10.0 mg/kg), and MCL 202 (0.0003–1.0 mg/kg) on eye blinking were determined infour monkeys; the order of drug testing varied irregularly among individual subjects. Inseparate experiments, the time course of MCL 202-induced changes in eye blinking wasdetermined by videotaping 5-min periods at 5, 10, 15, 30, 45, 60, and 120 min following asingle i.m. injection of 0.1 mg/kg MCL 202. For drug combination studies, each of severaldoses of SCH 39166 (0.1 – 1 mg/kg) was administered i.m. 5 min before test sessions in whichthe effects of MCL 202 (0.0003 – 1 mg/kg) was re-determined. The effects of SKF 82958 (0.03– 1 mg/kg) in the presence of MCL 202 (0.1–1.0 mg/kg) were similarly studied.

2.5 Schedule-controlled behavior: drug testingCumulative dosing procedures described above also were used to determine the effects of SKF82958 (0.03–1.0 mg/kg), MCL 201 (0.1–3.0 mg/kg), MCL 202 (0.01–3.0 mg/kg) onresponding maintained under the schedule of stimulus-termination. In these experiments,incremental doses of a drug were administered at the beginning of sequential 9-min TO periods,and up to five cumulative doses could be studied in a single session. The effects of pretreatmentwith SCH 39166 (0.3–1.0 mg/kg) or MCL 202 (0.03–1.0 mg/kg) were studied by administeringeach pretreatment dose at the start of the session and administering cumulative doses of SKF82958 in successive components beginning with the second component (i.e. 12 min after theinitial treatment).

2.6 DrugsSKF 82958 ((±)-6-chloro-7,8-dihydroxy-3-allyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine HBr) was purchased from Research Biochemicals International Sigma/RBI,(Natick, MA). SCH 39166 was generously supplied by Schering Plough (Kenilworth, NJ). Thesynthesis of enantiomers of SKF 83959, MCL 201 [S(−)–enantiomer] and MCL 202 [R(+)–enantiomer] has been described previously (Neumeyer et al., 1992, 2003). The basic chemicalstructures of dopamine D1 receptor-selective phenylbenzazepines are shown in Fig. 1.

2.7 Data analysisEye blink rates are expressed as blinks per minute and were computed as total number of eyeblinks divided by the duration of time that the subject’s eyes were visible to the observersduring the 5-min test component. Response rates under the schedule of stimulus-terminationare expressed as responses per second and were calculated for each session component bydividing the total number of lever press responses by the duration of the component minus thetime the chamber was darkened (timeout periods). In both observational procedures andexperiments involving schedule-controlled responding, saline injections did not appreciablyalter, respectively, rates of eye blinking or lever press responding across session components.Therefore, mean control values for rates of eye blinking and rates of responding (mean ±S.E.M) for each subject were determined by averaging rates across components of sessionsduring which the effects of saline were determined. For individual monkeys, the effects of eachcumulative dose in both procedures were calculated as response rate after treatment and as apercentage of mean control rate. Data for saline and all drugs are presented in this report asmean values for the group of monkeys. Mean values (± S.E.M) are expressed as rates of eyeblinking (blinks per minute) or lever-press responding (responses per second) or, alternatively,as percentages of control rates of eye blinking or lever-press responding. The effects of a givendose of drug on eye blinking or lever-press responding were considered significant when themean values for that dose lay outside the 99% confidence interval for mean control values forthe given measure.

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Each dose-response curve was analyzed using standard ANOVA and linear regressiontechniques. ED50 values and their 95% confidence limits were determined from data using thelinear portions of the dose-effect curves (Snedecor and Cochran, 1967). One-way analyses ofvariance (ANOVAs) and Dunnett’s t-tests were conducted where appropriate. Relative potencyestimates were obtained by parallel-line bioassay techniques; if 1.0 was not included in the95% confidence limits, then a significant difference in potency was assumed (Finney, 1964).A significance level of P < 0.05 was assumed throughout.

The apparent pA2 value was calculated as described by Dykstra et al. (1988). Dose-ratios weredetermined by dividing each of the ED50 values of MCL 202 in combination with SCH 39166by the ED50 value of MCL 202 alone. A Schild plot representing each dose ratio on the ordinateas log (dose ratio − 1) and expressed as a function of the negative logarithm of the dose ofantagonist which produced that ratio is generated. The point at which the regression lineintercepts the x-axis represents the apparent pA2 value. A competitive antagonism is indicatedwhen the 95% confidence limits for the slope of the Schild plot included the value −1.

3. Results3.1 Eye Blinking

The effects of control rates of eye blinking are shown in Table 1. The control values for eyeblink rates remained consistent throughout the four session components for all monkeys, exceptmonkey Ss 7. Monkey Ss 7 displayed greater variability in control blink rates, with eye blinkingdecreasing from 8.01 (± 1.30) blinks per min during the first component to 3.93 (± 0.62) blinksper min in the fourth component. The control values for eye blink rates failed to exceed 12blinks/min across components for any individual monkey, and the eye blink rates for the groupof monkeys averaged 6.07 (± 0.5 S.E.M) across the four components.

As previously reported, the dopamine D1-like receptor agonist SKF 82958 produced dose-dependent increases in rates of eye blinking (F4,15 = 9.97; p < 0.05; Fig. 2, open circles). Furtheranalysis revealed that the highest doses of SKF 82958 tested, 0.3 and 1 mg/kg, significantlyincreased eye blink rates to approximately 10- and 16-fold above control values, respectively.As with SKF 82958, cumulative doses of the R[+] enantiomer of SKF 83959, MCL 202 (0.003– 1 mg/kg), produced dose-related increases in rates of eye blinking (F8,27 = 18.84; p < 0.05).However, the maximal effects of MCL 202 were less than those achieved with SKF 82958(Fig. 2, downward triangles). Thus, rates of eye blinking were increased to approximately 4-fold above control values following the cumulative dose of 0.03 mg/kg of MCL 202, and thiseffect plateaued as the cumulative dose of MCL 202 increased to as much as 1.0 mg/kg. Post-hoc Dunnett’s test revealed that the increase in eye blink rates produced by 0.01 – 1 mg/kgMCL 202 was significantly above control values (ps < 0.05). In contrast to MCL 202, the samerange of doses of the S[−] enantiomer of SKF 83959, MCL 201, failed to increase eye blinkrates above control values (ps > 0.05). ANOVA revealed a significant effect of dose (F5,18 =4.55; p < 0.05) that was due to the high cumulative dose of 10 mg/kg MCL 201 which increasedrates of eye blinking only to approximately 2-fold of control values (Fig. 2, upward triangles).

Fig. 3 shows the effects of MCL 202 on eye blinking when administered alone and incombination with several doses of the dopamine D1-like antagonist SCH 39166 (0.1 – 1 mg/kg). SCH 39166 dose-dependently antagonized the effects of MCL 202 on eye blinking, asindicated by a parallel rightward shift in the dose-effect function (Fig. 3, open symbols). The0.1 mg/kg dose of SCH 39166 shifted the SKF 82958 dose-effects function to the right by afactor of about 11, whereas 0.3 and 1.0 mg/kg SCH 39166 shifted the SKF 82958 dose-responsecurve approximately 25- and 57-fold to the right, respectively (Fig. 3; Table 2). Relativepotency analysis indicated that all three doses of SCH 39166 significantly shifted the dose-effect curve for SKF 82958-induced eye blinking to the right in a dose-related manner (Table

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2). A Schild plot analysis of the antagonist effects of SCH 39166 generated an apparent pA2value of 7.675 (slope: −0.78 ± 0.04).

The activity of MCL 202 was further characterized by examining its ability to antagonize theeffects of SKF 82958 on eye blinking. Similar to SCH 39166, pretreatment with several dosesof MCL 202 produced a rightward shift in the dose-effect curve for SKF 82958-inducedincreases in rates of eye blinking (Fig. 4, open symbols). A dose-ratio analysis of the antagonisteffects of MCL 202 on the SKF 82958-induced increases in eye blinking revealed ratios of 2.7,4.8 and 31.1 for 0.1, 0.3 and 1.0 mg/kg dose of the antagonist, respectively (Table 2). A relativepotency analysis indicates that all three doses of MCL 202 significantly shifted the dose-effectcurve for SKF 82958-induced eye blinking to the right in a dose-related manner (Table 2).Schild plot analysis of the antagonist effects of MCL 202 revealed a slope of −1.26 ± 0.28 forthe regression of dose ratio values against antagonist concentration, precluding thedetermination of an apparent pA2 value.

3.2 Scheduled Controlled BehaviorTable 3 shows control rates of responding maintained under a schedule of stimulus-terminationin individual monkeys. All subjects performed consistently throughout the session componentswith a group mean of 3.10 (± 0.07 S.E.M) responses/s.

Cumulative doses of SKF 82958 (0.03 – 1.0 mg/kg) produced dose-dependent and significant(F4,10 = 31.74; p < 0.05) decreases in lever-press responding under the schedule of stimulus-termination (Fig. 5, filled circles). The highest cumulative dose of 1.0 mg/kg SKF 82958, whichhad maximally increased rates of eye blinking, virtually eliminated responding. The R[+]enantiomer of SKF 83959, MCL 202, also produced a dose-related decrease in rates ofresponding (F6,18 = 8.70; p < 0.05; Fig. 5, open triangles). Maximal decrease in response rates,to 27% of control rates, was achieved after administration of the highest dose of MCL 202 (3mg/kg). In contrast, cumulative doses of the S[−] enantiomer of SKF 83959, MCL 201, failedto appreciably alter rates of responding (F4,14 = 2.74; p > 0.05; Fig. 4, open squares) and, atthe highest dose (3 mg/kg), decreased response rates only to approximately 68% of controlvalues.

Additional experiments were conducted to compare antagonism of the behavioral effects ofSKF 82958 by the D1 receptor blocker SCH 39166 and the D1 agonist MCL 202. As shownin Fig. 6, doses of the D1 receptor blocker SCH 39166 that decreased rates of responding (0.03and 0.1 mg/kg) also shifted the SKF 82958 dose-effect function to the right in a dose-dependentmanner (Fig. 6, open squares and open triangles, respectively). The highest dose of SCH 39166(0.1 mg/kg) markedly reduced response rates to 16.4 (± 8.40) % of control values (Fig. 6, opentriangle) and produced a 3-fold shift to the right in the dose-effect curve, indicative ofsurmountable antagonism (Fig. 6, open triangles). However, it should be noted that SKF 82958(0.03 – 0.3 mg/kg) may be antagonizing the rate decreasing effects of SCH 39166.

Like SCH 39166, MCL 202 (0.3 – 1.0 mg/kg) antagonized the effects of SKF 82958 onresponse rate by dose-dependently shifting the dose effect curve to the right (Fig. 7, opensquares and open triangles). The magnitude of antagonism produced by MCL 202 was greaterthan observed with the highest dose of SCH 39166, perhaps reflecting the more disruptivedirect effects of SCH 39166 than of MCL 202 on schedule-controlled responding (Fig. 6, closedsymbols above MCL 202). Thus, the higher dose of MCL 202 (1.0 mg/kg) produced anapproximately 10-fold shift to the right in SKF 82958’s dose-effect function (Fig. 7, opentriangles).

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4. DiscussionThe present results in squirrel monkeys reveal interesting similarities and differences in thepharmacology of SKF 82958 and MCL 202, the R-(+)-enantiomer of SKF 83959. Consistentwith previous reports, the dopamine D1-like full receptor agonist SKF 82958 produced dose-related increases in eye blinking and decreases in rates of responding (Elsworth et al., 1991,Bergman et al., 1995; Kleven and Koek, 1996, Jutkiewicz and Bergman, 2004). MCL 202 alsoproduced increases in eye blink rate and decreases in response rates. Pretreatment with theselective dopamine D1-like receptor antagonist, SCH 39166, surmountably antagonized theeffects of MCL 202 on eye blinking and the effects of SKF 82958 on response rates. Further,pA2 analysis of the shift in the MCL 202 dose-effect curve revealed a slope of −0.78 (± 0.04)and a pA2 value of 7.675, suggestive of competitive antagonism at the D1-like receptor. Theseresults are consistent with the report that SCH 39166 competitively antagonized the effects ofR-(+)-6-BrAPB (Jutkiewicz and Bergman, 2004). Together these findings support the viewthat the behavioral effects of both drugs are mediated by D1 receptor mechanisms, and arehighly consistent with the results of earlier in vivo studies with other dopamine D1-likereceptor agonists (Bergman et al., 1995; Jutkiewicz and Bergman, 2004) and with those of inin vitro binding studies. In the latter studies, both drugs have been shown to competitively bindto D1-like receptors with high affinity and selectivity (approximately 800-fold for the D1-likeover the D2-like receptors (Arnt et al., 1992; Andersen and Jansen, 1990; Neumeyer et al.,2003).

In contrast to SKF 82958 and MCL 202, the S-(−)-enantiomer of SKF 83959, MCL 201, failedto alter measures of either eye blinking or scheduled-controlled behavior in the present study.In previous in vitro binding studies, Neumeyer et al. (2003) have reported a 20-fold differencein the potencies with which MCL 201 and its parent compound, SKF 83959, bind to D1-likereceptors, and only a 2- to 3-fold difference in their potency for binding to D2-like receptors(Neumeyer et al., 2003). Taken together, these findings indicate that the behavioral effects ofSKF 83959 reside in the R-(+) enantiomer. Based on the limited effects of MCL 201 in thepresent studies, the S-(−) enantiomer of SKF 83959 appears to be relatively inactive atdopamine D1-like receptors.

Although the effects of both SKF 82958 and MCL 202 appear to be mediated by D1-likereceptor mechanisms, the effects of the two drugs differed in the two behavioral assays withrespect both to potency and efficacy. For example, based on ED50 values, MCL 202 wasapproximately 87-fold more potent than SKF 82958 in increasing rates of eye blinking butapproximately 3-fold less potent than SKF 82958 in decreasing rates of responding. Moreover,in both types of study the effects of MCL 202 were at levels below those attained with SKF82958. In conjunction with previous findings in studies of eye blinking, these results areconsistent with differences in pharmacological efficacy for the two drugs (Jutkiewicz andBergman 2004).

Previous studies have demonstrated that SKF 83959, the parent compound of MCL 202, canproduce both agonist and antagonist like effects (Gnanalingham et al., 1995a, 1995b, 1995c;Waddington et al., 1995). The agonist effects of SKF 83959 are readily observed in rats andmonkeys, both in studies of overt behavior and in studies of anti-Parkinsonian effects (Downesand Waddington 1993; Deveney and Waddington, 1995; Gnanalingham et al., 1995a, b or c;Waddington et al., 1995; Jutkiewicz and Bergman 2004; Desai et al., unpublished data).Moreover, agonist effects of SKF 83959 appear to be significantly conserved in D1-likereceptor knockout mice (Clifford et al., 1999). However, recent studies of D1-mediated eyeblinking in monkeys showed that SKF 83959 also could partially antagonize the effects of R-(+)-6-Br-APB, a D1-like receptor agonist with higher efficacy (Jutkiewicz and Bergman,

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2004). These findings, indicating that SKF 83959 has both agonist and antagonist effects, areconsistent with its characterization as a partial agonist at D1-like receptors.

In light of the present findings, it is likely that MCL 202, like its parent compound SKF 83959,also may function as a D1-like partial agonist. In this regard, the effects of MCL 202 on eyeblink rates were comparable to those of SKF 83959 (Jukiewicz and Bergman, 2004). Bothdrugs produced dose-related increases in eye blinking that plateaued at approximately 400%of control values (Jukiewicz and Bergman, 2004). Both drugs antagonized the effects of D1-like receptor agonists with presumably higher efficacy (Jukiewicz and Bergman, 2004). In thepresent study, for example, MCL 202 dose-dependently antagonized the increased eye blinkingand the decrease in response rates produced by SKF 82958, which is fully efficacious instimulating AC activity. Together, these results suggest that MCL 202, like its parent SKF83959, has both agonist and antagonist like properties, and therefore can function as a partialagonist at the D1-like receptor.

Acknowledgements

The authors gratefully acknowledge the assistance of Susan Trofimow and Heather Millette. This work was supportedby USPHS grants DA 13311 (CAP), DA 145251 (JLN), and DA 03774 (JB).

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Fig. 1.Chemical structures of dopamine D1-like receptor-selective phenyl-benzazepines.

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Fig. 2.The effects of dopamine D1-like receptor agonists, SKF 82958, MCL 201, and MCL 202 oneye blinking in squirrel monkeys. Ordinates, eye blink rates as a percentage of that obtainedafter vehicle injections. Abscissa, cumulative dose of drugs in mg/kg. Each point representsthe average effect determined in all four monkeys. Results are presented as mean % control (+S.E.M). Note that the R[+] enantiomer of SKF 83959, MCL 202 significantly increased ratesof eye blinking, whereas the S[−] enantiomer of SKF 83959, MCL 201 failed to induced eyeblinking in squirrel monkeys, and that both these compounds were less effective than SKF82958. For all drugs, when apparently absent, error bars are within the symbol. * indicatessignificantly different from vehicle group (Dunnetts t-test, P < 0.05, on means derived frommain effect of dose).

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Fig. 3.Changes in the MCL 202 dose-effect curve for rates of eye blinking produced by pretreatmentswith several doses of the dopamine D1-like receptor antagonist SCH 39166. Ordinates, eyeblink rates as a percentage of that obtained after vehicle injections. Abscissa, cumulative doseof MCL 202 in mg/kg. Data are presented at as mean % control (+ S.E.M) of n = 4 animals.Schild plot analysis of the results reveals a slope of -0.96 (± 0.08) and a pA2 value of 8.01.Note that pretreatment with SCH 39166 produced a dose-related rightward shift in the MCL202-induced dose-effect curve.

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Fig. 4.Changes in the SKF 82958 dose-effect curve for rates of eye blinking produced bypretreatments with several doses of the R[+] enantiomer of SKF 83959, MCL 202. Ordinates,eye blink rates as a percentage of that obtained after vehicle injections. Abscissa, cumulativedose of SKF 82958 in mg/kg. Schild plot analysis of the data shows a slope of −0.79 (± 0.11)and a pA2 value of 7.07. Note that pretreatment with MCL 202 produced a dose-relatedrightward shift in the SKF 82958-induced dose-effect curve. Other details as in Fig. 2.

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Fig. 5.The effects of dopamine D1-like receptor agonists, SKF 82958, MCL 201, and MCL 202 onscheduled controlled behavior. Ordinates, response rates expressed as a percentage of thatobtained after vehicle injections. Abscissa, cumulative dose of drugs in mg/kg. Each pointrepresents the average effect determined in three to four monkeys. Results are presented asmean % control (+ S.E.M). Note that the R[+] enantiomer of SKF 83959, MCL 202significantly decreased response rates, whereas the S[−] enantiomer of SKF 83959, MCL 201failed to alter rates of responding in squirrel monkeys, and that both these compounds wereless effective than SKF 82958. * indicates significantly different from vehicle group (Dunnettst-test, P < 0.05, on means derived from main effect of dose).

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Fig. 6.Changes in the SKF 82958 dose-effect curve for response rates of stimulus shock controlproduced by pretreatments with several doses of the dopamine D1-like receptor antagonistSCH 39166. Ordinates, response rates expressed as a percentage of that obtained after vehicleinjections. Abscissa, cumulative dose of SKF 82958 in mg/kg. Other details as in Fig. 4. Notethat pretreatment with SCH 39166 produced a dose-related rightward shift in the SKF 82958-induced dose-effect curve.

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Fig. 7.Changes in the SKF 82958 dose-effect curve for response rates of stimulus shock controlproduced by pretreatments with several doses of the R[+] enantiomer of SKF 83959, MCL202. Ordinates, response rates expressed as a percentage of that obtained after vehicleinjections. Abscissa, cumulative dose of SKF 82958 in mg/kg. Other details as in Fig. 4. Notethat pretreatment with MCL 202 produced a dose-related rightward shift in the SKF 82958-induced dose-effect curve.

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Table 2Effects of interactions of MCL 202 and SKF 82958 with SCH 39166 and MCL 202, respectively, on eye blinking.

Drug Treatment Eye BlinkingED50 value (mg/kg) (95% CL) Relative Potency

MCL 202 alone 0.01 (0.007 – 0.02)MCL 202 with 0.1 mg/kg SCH 39166 0.11 (0.06 – 0.19) 0.07 (0.03 – 0.18)aMCL 202 with 0.3 mg/kg SCH 39166 0.25 (0.19 – 0.35) 0.01 (0.006 – 0.03)aMCL 202 with 1.0 mg/kg SCH 39166 0.57 (0.42 – 0.92) 0.003 (0.001 – 0.006)a

SKF 82958 alone 0.20 (0.11 – 0.42)SKF 82958 with 0.1 mg/kg MCL 202 0.54 (0.37 – 0.72) 0.43 (0.19 – 0.95)SKF 82958 with 0.3 mg/kg MCL 202 0.97 (0.30 – 48.82) 0.21 (0.09 – 0.45)SKF 82958 with 1.0 mg/kg MCL 202 6.29 (2.36 – 416.64) 0.07 (0.02 – 0.20)

aValue is only an estimate because there was a significant effect of preparations.

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