Behavioral Effects of the Systemically Active Delta Opioid Agonist BW373U86 in Rhesus Monkeys1

Transcription

1 /94/ $3./ Tsz Jouari*i. orpns.acoi.oov AND ExnaiMv.mi THERAPEUTICS Copyright C 1994 by The American Society for Pharmacologj and Experimental Therapeutics JPET , 1994 Vol. 27, No.3 Printed in U.S.A. Behavioral Effects of the Systemically Active Delta Opioid Agonist BW373U86 in Rhesus Monkeys1 S. STEVENS NEGUS,2 EDUARDO R. BUTELMAN, KWEN-JEN CHANG, BRIAN DeCOSTA,3 GAIL WINGER and JAMES H. WOODS Department of Pharmacology, University of Michigan, Mn Arbor, Michigan (S.S.N., E.R.B., G.W., J.H.W.); Burroughs Welcome Co., Research Triangle Park, North Carolina (K.-J.C.); and NaUOn& Instftute of Health, NaUOn& Institute of Diabetes and D!gestwe and Kidney Diseases, Bethesda, MarrIand (B.D.) Accepted for publlcatbn May 9, 1994 ABSTRACT The behavioral effects of methyl-i -piperazinyl)-3-hydroxybenzyl)-n,n-diethylbenzamide dihydrochloride (BW373U86), a nonpeptidic, systemically active, delta opiold agonist, were examined in rhesus monkeys. BW373U86, the mu agonist alfentanil and the kappa agonist U69,593 (5a,7a,8-)-N-methyI-N-(7-(1 -pyrrolidinyl)-1 -oxaspiro-(4,5)dec-8-yi)benzeneacetamide all produced a dose-dependent suppression of response rates maintained under a fixed ratio 3 schedule of food presentation. The rate-suppressing effects of BW373U86 lasted I to 2 hr and were no longer apparent after 4 hr. The selective delta antagonist naftrindole (NTI) antagonized the effects of BW373U86 with relatively high potency (pi(5 = 6.5) and the antagonist effects of NTI against BW373U86 lasted approximately 4 hr. NTI was less potent in antagonizing alfentanil (pk = 5.1) and the highest dose of NTI Opioids are thought to produce their effects by acting at three main types of opioid receptors: the mu, kappa and delta receptore (Martin et al, 1976; Lord et al 1977; Wood et a!. 1981; Mansour et al 1987). Selective agomsts and antagonists have played a critical role in distinguishing these opioid receptor types and, for mu and kappa opioid receptors, these selective agonists and antagonists have included both alkaloid and peptide compounds. For example, the alkaloid alfentanil (Cookson et al, 1983; Yeardon and Kitchen, 1988) and the peptide DAMGO (Handa et al, 1981) have been characterized as selective agonists at mu opioid receptors. Similarly, the alkaloids U69,593 (Lahti et al, 1985) and EKC (Martin et al, 1976) and Received for pub lication February 7, This work was supported by Public Health Service grant DA 254. Prelimmary reports on this work were presented at the 1993 meeting of the American Psychological Association and the 1994 meeting of the College on Problems of Drug Dependence. S Preeentaddress: AlcoholandDrogAbuse ResearchCenter, McLean Hospital, Harvard Medical School, 115 Mill St., Belmont, MA s Present address: New College (Wetmore Hall), 21 Classic Ave., Toronto MSF 23Z; Ontario, Canada. examined (1. mg/kg) did not antagonize U69,593. BW373U86 did not generalize to the discriminative stimulus effects of the mu agonist alfentanil or the kappa agonist ethylketocyclazoclne. BW373U86 also did not produce antlnodceptive effects in the warm-water tail-withdrawal procedure, significant respiratory depressant effects in monkeys breathing either air or 5% CO or reinforcing effects in a self-administration procedure. The highest dose of BW373U86 examined (1.78 mg/kg) produced convulsbus in one monkey. The high relative potency of Nil to antagonize the rate-suppressing effects of BW373U86 was consistent with the characterization of BW373U86 as a systemically active, delta-selective agonist in rhesus monkeys. Lh ider the conditions evaluated in the present study, the delta receptors to which BW373U86 binds do not appear to mediate antinociceptive, respiratory depressant or reinforcing effects in monkeys. the peptide dynorphin A (Chavkin et al, 1982) have been characterized as selective agonists at kappa receptors. Historically, the in vivo effects of the alkaloids have been examined by both peripheral and central administration, whereas the behavioral effects of peptides have been examined primarily by central routes of administration. This bias reflects, at least in part, the assumption that peptides do not distribute as well as some alkaloids across the blood-brain barrier and, hence, will either display low potency or be inactive after peripheral administration. Furthermore, because central drug administration is more difficult in primates than in rodents, the behavioral effects of opioid peptides have been examined primarily in rodents. Until recently, the only agonists available to probe the function of delta opioid receptors were peptidic compounds, such as the highiy selective delta agornsts DPDPE (Mosberg et al 1983) and [D-Ala2]deltorphin II (Erspamer et al, 1989). As with other opioid peptides, the behavioral effects of the delta-selective peptides have been examined primarily after central adminis- ABBREVIATION& BW373U86, (±(a(2s.,5r. al-2,5-dimethy1-1-piperazinyi)-3-hydroxybenzyi)-n,ndethylbenzamlde dihydrochioride; EKC, ethylketocyclazoclne; FR, fixed ratio; I.t., Intrathecal; NTI, nalttlndole; DR. dose ratio; %MPE, percent of maximum possible effect. 125

2 126 Hague st al tration in rodents and the central administration of these compounds has been reported to produce such behavioral effects as antinociception (Heyman et a!., 1987; Kovacs et al, 1988; Calcagnetti and Holtzman, 1991), mild respiratory depression (Kiritsy-Roy et al, 1989), generalization to the discriminative stimulus effects of mu agonists (Shearman and Hen, 1982; Locke and Holtzman, 1986; Ukai and Holtzman, 1988) and reinforcing effects (Shippenberg et a!., 1987; Bals- Kubic et at., 199; Devine and Wise, 1994). However, the sensitivity of these effects to antagonism by delta-selective antagonists has not always been determined (Shearman and Herz, 1982; Ukai and Holtzman, 1988; Devine and Wise, 1994). Furthermore, the generality of these findings to other routes of administration or to other species has not been thoroughly examined. BW373U86 has recently been described as the first systemically active, alkaloid agonist that is selective for delta opioid receptors (Chang et al, 1993). As such, BW373U86 represents a compound that may be useful to characterize the behavioral effects that result from delta receptor activation. In vitro, BW373U86 binds selectively and with high affmity to delta opioid receptors and potently inhibits electrically evoked contractions of the mouse vas deferens, a smooth muscle preparation sensitive to peptidic delta agonists (Chang et al, 1993). Furthermore, on the basis of the ratio of agonist affinity in binding assays to agonist potency in functional in vitro assays, Chang et at. (1993) concluded that BW373U86 displays relatively high efficacy at delta opioid receptors. In vivo, BW373U86 and the peptidic delta agonists produce similar but not identical behavioral profiles. For example, both BW373U86 and peptidic delta agonists have been reported to produce antinociception (Drower et al, 1991; Sofuoglu et a!., 1991; Wild et al, 1993) and locomotor activation (Longoni et al, 1991; Chang et al, 1993) in rodents, and these effects are sensitive to antagonism by the delta-selective antagonist NT! (Portoghese et al, 1988). However, the peptidic delta agonists DPDPE and DSLET did not generalize to BW373U86 in pigeons trained to discriminate BW373U86 from saline (Comer et al, 1993b). Furthermore BW373U86 produces convulsant effects in mice (Comer et al, 1993a) and primates (Dykstra et al, 1993), whereas peptidic delta agonists do not consistently produce convulsions and may produce anticonvulsant effects (Tortella, 1993). Findings such as these suggest that the effects of BW373U86 and the peptidic delta agonists may be mediated by overlapping but distinct mechanisms of action. The purpose of the present study was to examine the behavioral effects of BW373U86 in rhesus monkeys. In a previous study from this laboratory, it was found that BW373U86 suppressed rates of schedule-controlled behavior in rhesus monkeys (Negus et al, 1993). These rate-decreasing effects were antagonized by the opioid antagonist quadazocine in a manner consistent with the hypothesis that the rate-decreasing effects of BW373U86 were mediated by delta opioid receptors. The present study extended these findings in two ways. First, the sensitivity of the rate-decreasing effects of BW373U86, the selective mu agonist alfentanil and the selective kappa agonist U69,593 to antagonism by NT! were evaluated. The hypothesis that the rate-decreasing effects of BW373U86 are mediated by delta receptors predicts that NT! should be more potent as an antagonist ofbw373us6 than ofalfentanil or U69,593. Second, the antinociceptive, respiratory depressant, discriminative stimulus and reinforcing stimulus effects of BW373U86 were Vol. 27 compared with those of agonists selective for mu (alfentanil) or kappa (U69,593 and EKC) receptors. As noted earlier, deltaselective peptides and BW373U86 produce antinociceptive effeds in some tests and antinociception represents an important clinical endpoint for existing opioids. Respiratory depression and stimulus effects, likewise, are important but untoward effects of alfentanil and other clinically used mu agonists (Jaffe and Martin, 1985). Subjects Methods The subjects were 22 male and female rhesus monkeys (Macsos mulatto) that weighed from 5. to 1. kg during the course of the study. The number of monkeys used in each experiment was as follows: schedule-controlled behavior, n = 8; drug discrimination, n = 4; warmwater tail withdrawal, n = 4; respiratory function, n = 4; and selfadministration, n = 3. One monkey was used initially in the experiments on self-administration and, later, in experimento on scheckecontrolled responding. All other monkeys were used exclusively in one experimental procedure. The monkeys were housed individually and maintained on a diet of Purina Monkey Chow, which was provided in the home cage after each daily session. Monkeys in the schedulecontrolled responding and drug-discrimination procedures were fed restricted diet (approximately 15 chows per day) such that they were maintained at approximately 9% to 95% of their free-feeding weighta. All other monkeys had free access to food (2-3 chows per day). This diet was supplemented by fresh fruit twice a week in all monkeys. Water was freely available in the home cage for all monkeys. The rooms in which the monkeys were housed were maintained on a 12-hr light/dark cycle. All monkeys had complex experimental and drug histories, including prior administration of opioid agonists and antagonists. Monkeys in the drug self-administration experiments were implantedwith intravenous catheters, as described later. All procedures were approved by the University of Michigan Committee on the Use and Care of Animals. Schedule-Controlled Responding Apparatus. During experimental sessions, two ofthe eight monkeys used in these experiments were removed from their home cages and seated individually in Plexiglas-aluminum primate restraint chairs that were, in turn, enclosed individually in sound-attenuatingoperant chamber8 In the chamber, a panel of colored 5-W bulbs was located along the top of the front wall and served as stimulus lights. Centered on the front wall was a panel that contained a food receptacle between two primate response levers (modelprl-1, BRS-LVE, Laurel, MD). The food receptacle was fed by an externally mounted pellet dispenser (model G521, Gerbrands, Arlington, MA) that contained 3-mg, banana-flavored pellets (formula G/T, P. J. Noyes, Lancaster, NH). Exhaust fans operated continuously and provided mashing noise. Programming, recording and data collection were accomplished with ifim PCjr computers (International Business Machines) located in an adjacent room. The remaining six monkeys completed their experimental sessions in their home cages. The home cages of these monkeys were modified to include an experimental panel on one wall. This panel supported a centrally mounted food receptacle between two primate response levers identical to those described earlier. Stimulus lights were located above each ofthe two levers and were illuminated by colored 5-W bulbs. The food receptacle was fed by an externally mounted pellet dispenser identical to that described before. As mentioned, programming, recording and data collection were accomplished with IBM PCjr computers located in an adjacent room. Behavioral procedure. All monkeys were used five to six days per week and daily sessions were composed of multiple cycles. Each cycle consisted of a 1-mis time-out period, during which all stimulus lights were off and responding had no scheduled consequences, followed by a a

3 5-mm response period, during which the stimulus lights were illumiziated and monkeys could earn up to 1 food pellets by responding under a FR 3 schedule of food presentation. If all 1 reinforcers were earned before the end ofthe 5-mis response period, the stimulus lights were turned off and responding had no scheduled consequences for the remainder of the 5-mm period. Pellets could be earned by responding on either of the two levers but all monkeys had a preferred lever and earned all their reinforcers by pressing the preferred lever almost exclusively. On training days, monkeys were used for five consecutive cycles and received sterile water or sham injections at the beginning of each cycle (i.e., at the beginning of each time-out period). All monkeys were trained until they responded at rates above 1. response/sec for all five cycles for at least 1 consecutive days and, subsequently, monkeys were tested only after training sessions during which they responded at rates above 1. response/sec for all five cycles. Test sessions were identical to training sessions, except that test compounds were administered as described later. In addition, test sessions could extend for up to seven cycles, although this rarely occurred. Monkeys were tested no more that twice a week, with at least one training session between test sessions. Pharmacological procedure. BW373U86 ( mg/kg), alfentanil ( mg/kg) and U69,593 ( mg/kg) were adniirtiatered sc. at the beginning of each cycle by a cumulative dosing procedure in which the total dose of agonist was increased by.5-log unit increments with each injection. The rate-decreasing effects of NT! alone ( mg/kg) were also evaluated It was found that doses of NT! up to 1. mg/kg had little effect on response rates but a does of 17. mg/kg suppressed response rates in some monkeys. Therefore, in subsequent antagonist experiments, NT! was administered only in doses of 1. to 1. mg/kg. hi these antagonist experiments, NT! was administered 3 mm, 4 hr or 24 hr before the beginning of the first response period and cumulative dose-effect curves for the agonist were then redeterinined. Under all conditions, the cumulative agonist doseeffect curves were designed to cover a dose range beginning with low, ineffective doses during the first cycle and progressing to doses that suppressed response rates to rates below.2 responses/sec in most or all monkeys. Different groups of monkeys were used to test each agonist, but for any given agonist, the same group of monkeys was used to evaluate the effects of the agonist alone and in combination with the various NT! pretreatments. Each of these groups consisted of three (BW373U86 and U69,593) or five (alfentanil) monkeys. In addition, the time course of.32 mg/kg of BW373U86 was evaluated in a set of three monkeys by measurements of the response rat#{149}s in a single cycle beginning either 5 mm before or 1 mis, 1 hr, 2 hr or 4 hr after the injection of BW373U86. The response rates measured before and 1 mm after the administration of.32 mg/kg of BW373U86 were determined in a single session. The response rates measured 1, 2 and 4 hr after the administration of BW373U86 were determined after separate injections. Data analysis. The response rates were Calculated for each cycle. The control response rates for each monkey were calculated as the mean response rate on the training day preceding each test dsy The rates of responding during each cycle of a test session were then converted to a percentage of that monkey s control response rate. Doseresponse curves for each agonist were constructed in which the mean percent ofcontrol response rate (linear scale) was plotted as a function of the dose of the test drug (log scale). The slope of the dose-effect curve and the dose of agonist producing 5% ofthe maximal effect value) were calculated from individual data according to Tallarida and Murray (1981, procedure 8). The 95% confidence limits of the slope and Ao values were also determined. DRs were calculated when antagonist pretreatments produced significant increases in Ao values, as determined by nonoverlapping 95% confidence limits. DRa were calculated by dividing the A for a given test drug administored in the presence of NT! by the A( for the same test drug administered alone. An in vics apparent pa2 value for NT! in combination with BW373U86 was CalCulated according to the methods of Tallarida and Deft. Os In Primates 127 Murray (1981, procedure 14) and the slope (± 95% confidence limits) of the Schild plot was deterinineci The confidence limits of the Schild plot slope included -1, so the apparent pa2 value was redetermined with a slope constrained to -1, according to the methods of TaIIarida and Murray (1981, procedure 16). In vivo apparent PKB values were determined for each dose of NT! in combination with BW373U86 and alfentanil by a modification of the equation DR - 1 = B/(KB) (Tallarida et al, 1979), as described by Negus et al (1993), where B - dose in moles per kilogram of NTL If we rearrange this equation and take the negative logarithm of both sides, we have the following equation for the determination of pkb: PKB -log [B/(DR - 1)). This equation differs from the original equation described by Tallarida et al (1979) in that the variable B equals the dose of antagonist in moles per kilogram rather than the molar concentration of antagonist. Average PKB values ± 95% confidence limits were also calculated for NT! in combination with BW373U86. Data from the time-course experiment were evaluated with one-way, repeated-measures analysis of variance; the pretreatment time was the independent variable. A significant main effect was followed by an individual means comparison with the Duncan post hoc test (Winer, 1971). The criterion for a significant effect was P <.5. Drug Discrimination Apparatus. During experimental sessions, four monkeys were removed from their home cages and seated individually in Plexiglasaluminum primate restraint chairs that were, in turn, enclosed individually in sound-attenuating operant chambers identical to those doscribed before. Behavioral procedure. Two monkeys were trained to discriminate the mu agonist alfentanil (.56 mg/kg) from saline and two other monkeys were trained to-discriminate the kappa agonist EKC (.32 or.56 mg/kg) from saline. All monkeys were used 5 days per week during daily sessions composed of multiple cycles. Each cycle consisted of a 1-mis time-out period, during which all stimulus lights were off and responding had no scheduled consequences, followed by a 5-mis response period, during which the stimulus lights were illuminated and monkeys could earn up to 1 food pellets (three monkeys) or 2 pellets (one alfentanil-trained monkey) by responding under a FR 2 schedule of food presentation. If all available reinforcers were earned before the end of the 5-mis response period, the stimulus lights were turned off and responding had no scheduled consequences for the remainder of the 5-mis period. on training days, monkeys were given a s.c. injection ofeither saline or the training dose of the training drug at the beginning of each cycle (i.e., at the beginningofeach time-outperiod). After the administration of saline, responding on only one of the two available levers (the saline lever) produced food, whereas after administration of the training dose ofthe training drug, only responding on the other lever (the drug lever) produced food. Monkeys were used for one to five cycles and, if the trainingdose ofthe training drugwas administered, it was administered only at the beginning ofthe last cycle. Thus, training days consisted of up to five saline cycles followed by zero to one drug cycles. All monkeys were trained until they met the following three criteria for four consecutive training sessions: 1) fewer than 4 responses before delivery of the first reinforcer, 2) at least 9% injection-appropriate responding for the entire cycle and 3) response rates of at least 1. response/sec during saline cycles. The test sessions with BW373U86 were identical tothe training sessions, exceptthat responding on either leverproduced food and BW373U86 was administered as described later. Training sessions were conducted on Mondays, Wednesdays and Thursdays. If responding on training days met criterion levels of discrimination performance, test sessions were conducted on Thesdays and Fridays. If responding did not meet criterion levels ofdiscrimination performance, then training was continued until criterion levels of performance were attained. Pharmacological procedure. BW373U86 ( mg/kg) was administered s.c. at the beginning of each cycle by a cumulative dosing

4 128 Nsgus.tal. VoL 27 procedure, in which the total dose of agonist was increased by.5-log unit increments with each injection. Under all conditions, the cumulative agonist dose-effect curves were designed to cover a dose range beginning with low, ineffective doses during the first cycle and progreasing to doses that either generalized completely to the training stimulus or suppressed response rates to the point at which monkeys did not receive any food pellets. Data analysis. The percent drug appropriate responding was calculated for each cycle as [(total number responses on drug lever + total number responses on both levers) x 1]. Response rates were calculated as (total number responses on both levers + total cycle time).!n this procedure, test drugs are considered to generalize to the training drug if some dose of the test drug produces at least 9% drug-appropriate responding. Warm-Water Tall Withdrawal Apparatus. The warm-water tail-withdrawal procedure, which has been described in detail elsewhere (Dykstra and Woods, 1986), has been used extensively in this laboratory to characterize the antinociceptive effects of opioid and nonopioid drugs. Four monkeys were seated in Plexiglas and tubular aluminum chairs, which loosely restrained them at the neck. The monkeys arms were loosely restrained to prevent them from rotating in the chairs and their shaved tails were positioned so that they hung free and were readily accessible to the experimenter. Tail-withdrawal latencies from water maintained at 4#{149}C, 5#{149}C and 55C were measured by an immersion of the lower 1 to 12 cm of the monkey s tail into a thermos of water and a measurement of the latency to tail withdrawal. The water was heated to 7#{176}C in a hot water bath and cooled to the final temperature in the thermos bythe addition ofwaterat room temperature. Tail-withdrawal latencies were timed using a push-button switch connected to a personal computer (IBM PCjr) that collected and stored all data. Procedure. Tail-withdrawal latencies from 4#{176}C, 5#{176}C and 55C water were evaluated during multiple-cycle sessions that lasted 2 to 3 hr. Each cycle lasted 3 mis. A drug, saline or sham injection was administered 15 mis into the cycle and tail withdrawal latencies were determined from all three temperatures in random order at the end of the cycle, with approximately 2 mm between presentations of each temperature. A 2-sec cutoff latency was used such that, if a monkey did not remove its tail from the water after 2 see, the water was removed by the experimenter and a latency of 2 sec was recorded. Once consistent base lines had been established, monkeys were tested one to two times each week. On test days, base-line tail-withdrawal latencies from 4#{176}C, 5#{176}C and 55#{176}C water were determined during the first cycle ofthe session and only those monkeys with a 2-sec (cutoff) latency to 4#{176}C water were tested further. Alfentanil ( mg/kg) and BW373U86 ( mg/kg) were administereds.c. by a cumulative dosing schedule in which a cumulative dose of the test drug (a dose that incremented the total dose by.25 to.5 log unit) was administered 15 mm into each cycle (Le., every 3 mm). Alfentanil and BW373U86 were tested from doses that had no effect up to doses that produced overt behavioral effects (respiratory depression for alfentanil or convulsions for BW373U86) that precluded the evaluation of higher doses. Data analysis. The effect of drug dose on tail-withdrawal latency was expressed as the %MPE by the equation %MPE = [(test latency - base Iine)/(cutoff - base line)] x 1, where, for each temperature, test latency - the latency to tail withdrawal during the test cycle, base line - the latency to tail withdrawal during the initial base-line trial and cutoff - 2 sec. Graphs were constructed with the %MPE (linear scale) as a function of drug dose (log scale). We calculated A values for alfentanil by drawing a line between the two points above and below 5% MPE and interpolating the drug dose that would produce 5% MPE. Rsspiratory Function Assay Apparatus. The apparatus used to evaluate respiratory function in rhesus monkeys has been described in detail elsewhere (Howell et al, 1988). Four monkeys were seated in Plexiglas and tubular aluminum primate chairs and placed into sound-attenuating chambers. A helmet was placed over the monkey s head and sealed with rubber dams and sealants to reduce gas leakage around the neck. Ten liters/min of air or 5% CO2 in air was pumped through the helmet, and removed by a vacuum pump. Changes in air flow produced by respiration were measured by a pressure transducer (model P23XL, Spectramed, Oxnerd, CA) and data were recorded and translated from the transducer by an analog-to-digital converter to a microprocessor (IBM PC). The dependent variables were respiratory rate (respirations per minute), tidal volume (milliliters per respiration) and minute volume (milliliters per minute). Procedure. Respiratoryparameters were evaluated during multiplecycle sessions lasting 2 to 3 hr. Each 3-mis cycle consisted of a 21- mis exposure to air followed by a 7-rain exposure to 5% CO2 followed by another 2-mis exposure to air. The respiratory rate, tidal volume and minute volume were determined during the 3-mis periods from 18 to 21 miii of the cycle (air values) and from 24 to 27 rain of the cycle (CO2 values). Test sessions were conducted one to two times per week. On test days, the air and CO2 values for respiratory rate, tidal volume and minute volume determined during the first cycle served as control values. Subsequently, alfentanil ( mg/kg) and BW373U86 ( mg/kg) were administered s.c. at the beginning of each cycle by a cumulative dosing procedure that incremented the total dose by.25 or.5 log units. Alfentanil and BW373U86 were evaluated with the same doses that were evaluated in the tail-withdrawal procedure. Each drug was evaluated in a group of three monkeys. Data analysis. The data collected during test cycles were expressed as a percent of the control values collected at the beginning of each test session. For any given treatment, respiratory rate, tidal volume and minute volume were affected in a similar way and only data for minute volume are shown. Graphs were constructed with the percent control minute volume (linear scale) to drug dose (log scale) under both the air and CO2 conditions. We calculated Ao values for alfentanil by drawing a line between the two points above and below 5% of control minute volume and interpolating the drug dose that would produce a 5% decrease in minute volume. Drug Self-Administration Apparatus. The drug self-administration technique has been doscribed in detail elsewhere (Winger et al, 1989). Three monkeys were anesthetized with ketamine (1. mg/kg) and xylazine (2. mg/kg) and implanted with an i.v. silicone rubber catheter (inner diameter,.8 cm; outer diameter,.24 cm; Rodhelm Reiss, Belle Mead, NJ) into the internal or external jugular, femoral or brachial vein. The catheter was tunneled s.c. from the vein to exit from the midscapular region. After surgery, each monkey was fitted with a stainless steel tubular harness connected to a jointed or flexible spring arm that was, in turn, connected to the back of the monkey s home cage. The catheter was positionedso that it fit inside this protective spring arm, passed through the back of the cage and attached to a second length of catheter that passed through a roller infusion pump (model MHRK55, Watson and Marlow, Falmouth, U.K.) to a drug reservoir. A panel fitted with two monkey levers (model 121-7, BRS-LVE) and stimulus lights (5-W red and green bulbs) was attached to the side of each monkey s home cage within easy reach of the monkey. Procedure. Monkeys were allowed to self-administer the drug during twice-daily sessions; one session was conducted each morning and another, each afternoon. Each 13-min experimental session was divided into four 25-mis cycles of drug availability, signaled by illumination of a red stimulus light, separated by 1-mis time-out periods, during which stimulus lights were not illuminated and responding had no scheduled consequences. During each cycle, drug was available for up to a maximum of 2 i.v. infusions under a FR 3 schedule of reinforcement and a 45-sec time-out followed each infusion. A green stimulus light was illuminated during each drug infusion. The dependent variable was response rate during each cycle. During training, different unit doses of alfentanil were available during each ofthe four

5 1994 cycles. The doses ofalfentanil were changedby a change in the duration of infusion pump activation. One duration was very short (1 msec) and served as a zero dose of alfentanil. The other durations were 1.7, 5 and 16.7 sec. The order of presentation of the different alfentanil doses was varied across training sessions. Alfentanil was evaluated at i.v. unit doses that ranged from.32 to.32 kg injection. BW373U86 (1.-3. &g k#{231} injection i.v.) was substituted for alfentanil during test sessions in each of three monkeys. As with alfentanil, the dose of BW373U86 was varied by changes in the duration of the infusion pump activation. The highest unit dose of BW373U86 tested (3 ig kg injectioni was similar to the acute dose ofbw373u86 that completely suppressed response rates in the schedule-controlled responding procedure (32 &g/kg). Doseeffect curves for both alfentanil and BW373U86 were determined twice in each monkey. Drugs Alfentanil hydrochloride (Janssen Pharmaceuticals, Piscataway, NJ), BW373U86 (Burroughs Welcome, Research Triangle Park, NC), EKC (Sterling Winthrop, Renssalaer, NY), NT! hydrochloride (B. DeCosta, National Institutes of Health, Bethesda, MD) and U69,593 (Upjohn, Kalamazoo, MI) were all dissolved in distilled water and injected s.c. in a volume of.2 to 1. ml for the first four procedures. For the self-administration studies, alfentanil and BW373U86 were dissolved in sterile saline and injected i.v. Results Control response rates for the eight monkeys in the schedulecontrolled responding experiment ranged from 1.33 to 3.63 responses/sec. As in our previous studies with this multiplecycle procedure (Negus et a!., 1993), there was no consistent tendency for response rates to increase or decrease across cycles (data not shown). BW373U86, alfentanil and U69,593 all produced a dose-dependent and complete suppression of schedulecontrolled responding. The relative potencies were U69,593 alfentanil > BW373U86 (fig. 1, table 1). Figure 2 shows the time course in three monkeys of.32 mgi kg of BW373U86, the lowest dose that completely suppressed responding under the cumulative dosing procedure. Analysis of variance revealed a significant main effect ofpretreatment time (F = 4.625, P <.5). In a cycle immediately preceding the administration of BW373U86, monkeys responded at 16% of control rates; however, in the subsequent response period beginning 1 mm after the administration of.32 mg/kg of BW373U86, response rates were suppressed to 3.5% of control rates. Response rates were also suppressed in all three monkeys 1 hr after the administration of this dose of BW373U86. Post hoc analysis indicated that both the lo-min and 1-hr pretreatment times yielded response rates that were significantly lower than response rates during the cycle that preceded BW373U86 administration (P <.5). After 2 hi, the response rates in one of the three monkeys remained suppressed (3.9% of control), whereas the other two monkeys responded at approximately control levels. After 4 hr, all three monkeys responded at 5% or higher of control rates. NT! (1.-1. mg/kg) produced dose-dependent and parallel rightward shifts in the BW373U86 dose-effect curve and significant increases in the BW373U86 A (fig. 1, table 1). The Schild plot of the antagonist effects of NT! had a slope of -1.1 (95% confidence limits, -1.8 to -.33) and yielded a pa2 value of 6.3 (95% confidence limits, ). The pa2 value with the slope constrained to -1 was 6.4 (table 2). Table 2 shows the apparent in vivo PKB values for each dose of NT! in combina- G) (I) C. C C.) Delta Oplolds In Primates Dose BW373U86 (mg/kg) Dose Alfentanhl (mg/kg) Dose U69,593 (mg/kg) pa Agonist Alone -a-- +1.ONT P io.o NTI Fig. 1. Rate-decreasing effects of the delta agonist BW373U86 (A), the mu agonist alfentanhl (B) and the kappa agonist U69,593 (C) and their antagonism by NTI. Absdssa, dose agonist ki milligrams per kilogram (log scale). Ordinate, percent control rate of responding. All points represent mean(± S.E.M.)data from three(a and C)or five (B) monkeys. The insert in the top panel shows the Schild plot for NTI antagonism of BW373U86. In this insert, the abscissa is the negative log doseqn moles per kilogram) of NTI and the ordinate is the log (DR - 1). The unconstrained pa.2 value of 6.3 is indicated as the point where the regression ilne crosses the absdssa. tion with BW373U86, and it can be seen that these PKB values were similar both to each other and to the constrained pa2. NT! (1. mg/kg) also produced a small and parallel rightward shift in the dose-effect curve for alfentanil and a significant, 3.6-fold increase in the alfentanil Ao (fig. 1, table 1). The PKB value for 1. mg/kg NT! in combination with alfentanil was 5.1 (table 2). Pretreatment with 1. mg/kg NT! had no effect on the dose-effect curve for U69,593. 1

6 13 Negusetal. Vol. 27 TABLE 1 A5 values and DRs for BW373U86, alfentanil and U69,593 administered alone or after pretreatment with NTI NTI was always administered 3 menbefore determination of the cumulative agonist dose-effect curve, unless noted otherwise. 5) a) a) C. a) a) C C.) Agonist. I reatment (95% V&ue fjj DR BW373U86 Alone.99 (.5-.19) + 1. NTI.62 ( ) NTI 2.2 (1.-4.9) ONTI 6.4 (2.8-15) ONTI(4hr) 3.7 ( ) NTI (24 hr).21 ( ) ND#{176} Alfentanil Alone.1 1 ( ) + 1. NTI.4 ( ) 3.6 U69,593 Alone.61 ( ) + 1. NTI.28 ( ) ND#{176} a DR not determined because A value for agonist alone was not significantly different from A6 value for agonist + NTI Time (mm) Fig. 2. Time course of the rate-decreasing effects of.32 mg/kg of BW373U86. Abscissa, time after injection of BW373U86 in minutes. Ordinate, percent control rate of responding. The point above -5#{176} represents the percent control rate of responding during a single cycle immediately before the administration of BW373U86. The data represent the mean from three monkeys and error bars show the S.E.M. Significantly different from control (P <.5). TABLE 2 In vivo apparent plc. and pa2 values for NTI in combination with BW373U86, alfentanil and U69,593. pk.(dcse NT Mean Agonist (95% Level) (95% Confidence Level) BW373U ( ) 6.4 ( ) Alfentanil ND#{176} U69,593 ND#{176} ND ND a vaiies were not de termi nod boos use of insufficient an tagonist effects by NTI. The antagonist effects of 1. mg/kg NT! against BW373U86 lasted at least 4 hr but were no longer apparent after 24 hr (fig. 3, table 1). A 4-hr pretreatment with NT! produced an antagonist effect that was not significantly different from the antagonist effect produced by 3-mm pretreament with NT!. However, when NT! was administered 24 hr before BW373U86, the A6 value for BW373U86 was not different from the control A value for BW373U86. 5) 5) a) a. a) C C.) -.- BW373U86 Alone ONTI(.5HR) ONTI(4HR) Dose BW373U86 (mg/kg) -& NTI (24 HR) Fig. 3. Time course of the antagonism of BW373U86 by 1. mg/kg NTI. Abscissa, dose of BW373U86 in milligrams per kilogram (log scale). Ordinate, percent control rate of responding. All points represent mean (± S.E.M.) data from three monkeys. Two monkeys were trained to discriminate alfentanil (.56 mg/kg) from saline and two other monkeys were trained to discriminate EKC (.32 or.56 mg/kg) from saline. Table 3 shows control data from the multiple-cycle training session conducted on the day before testing with BW373U86. It can be seen that monkeys responded exclusively on the saline lever after saline administration and exclusively on the drug lever after administration of the training dose oftraining drug. Table 3 also shows that BW373U86 did not generalize to either alfentanil or EKC up to doses that completely suppressed response rates. Control tail-withdrawal latencies for the four individual monkeys in the warm-water tail-withdrawal procedure ranged from.55 to 1.83 sec for 5#{176}Cwater and from.44 to 1.64 sec for 55#{176}Cwater. The overall mean (± S.E.M.) control tail-withdrawal latencies were.92 ±.15 sec for 5#{176}Cwater and.73 ±.13 sec for 55#{176}C water. Alfentanil produced a dose-dependent increase in tail-withdrawal latencies from 5#{176}C and 55#{176}C water (fig. 4). The A values for alfentanil were.36 mg/kg at 5#{176}C and.54 mg/kg at 55#{176}C. BW373U86 at doses up t#{224} 1.78 mgi kg was completely ineffective at either temperature. Higher doses were not evaluated because 1.78 mg/kg BW373U86 produced a brief convulsion in one of the monkeys. In the assay of respiratory function, control minute volumes for the four individual monkeys during respiration of air ranged from 988 to 2432 ml/min, whereas control minute volumes during respiration of 5% CO2 ranged from 2474 to 56 ml/ mm. The overall mean (± S.E.M.) control minute volumes were 1492 ± 225 ml/min for air and 3412 ± 329 mi/mm for 5% CO2. Alfentanil produced a dose-dependent decrease in respiration of both air and CO2, with A values of.5 mg/kg for air and.24 mg/kg for CO2 (fig. 4). BW373U86 at doses up to 1.78 mg/kg had virtually no effect on respiration of air and all doses of BW373U86 produced only a mild suppression of respiration of CO2. The maximum suppression of respiration was obtained with.32 mg/kg of BW373U86, which reduced air minute volume to 86% of control and CO2 minute volume to 69% of control. Finally, alfentanil produced a dose-dependent increase in the rates of drug-maintained responding over unit doses of.32 to.32 kg injection in all three monkeys (fig. 5). When BW373U86 at unit doses of 1 to 32 g k#{231} injection was

7 1994 D&taOploldslnPrlmates 131 TABLE 3 Effcts of BW373U86 on %DAR In monkeys tralrad to discelminats either the mu agonist alf.ntanll or the kappa agonist EKC from salira The training dose of aifentan was.56 mg/lcg in both monkeys. The training dose of EKC was.32 mg/kg monkey MA and.56 mg/kg In monkey MO. Response rates i responses per second are shown In parentheses. MfentI Monkeys a drug-appro respordng. a%dar data not shown when monkey did not receive at least one food pellet. EKC Monkeys Treatment MonkeylO Monkey P Monkey MA Monkey MO %DAR,.) %DAR %OAR %DAR I,) Saline (2.55) (2.3) (2.5) (2.75) Training drug 1 (1.79) 1 (2.14) 1 (1.84) 1 (2.17) BW373U86 (mg/kg).1 (2.12) (2.14) (1.19) (2.64).32 (2.3) (1.93) () (2.53).1 2 (2.43) (.1) -a (.25).32 () 1 a 2.. U a. a E j I.1 b I Dose (mg/kg) Fig. 4. Antmnociceptlve effects (A) and respiratory depressant effects (B) of BW373U86 and alfentanil. Abscissa, dose agonlet in milligrams per kilogram (log scale). Ordinate (A), tail withdrawal latendes from 5#{176}C and 55#{176}Cwater expressed as %MPE. Ordinate (B), percent control minute volume during respiration of either air or 5% COP. fri points represent mean (± S.E.M.) data from four (A) three (B) monkeys. substituted for alfentanil in these monkeys, it did not maintain response rates different from those maintained by saline. Discussion -.- Alfsntsnll I 5#{176}C --- Alfentanil I 55#{176}C -.-- BW373U86 I 5#{176}C -- BW373U86 I 55#{176}C In a previous study (Negus et al, 1993), it was reported that systemically administered BW373U86 produced dose-dependent decreases in schedule-controlled responding in rhesus monkeys. The potency of the opioid antagonist quadazocine antagonizing those effects suggested that the rate-decreasing effects of BW373U86 were mediated by delta opioid receptors Specif- U Alfsntanll/Alr -a- Msntsnll I BW373U86 I Air -- BW373U86 I I Dose -U--- All -..- BW373U (g/kgflnj) Fig. 5. ReinforcIng effects of BW373U86 and alfentanil In monkeys trained to Self-adminISter alfentanil. Abscissa, unit dose of agonist in micrograms per kilogram per Injection (log scale). Ordinate, response rates In responses per second. The point above #{176} represents the response rates maintained by very brief (1-rnsec) injections that functioned as a #{176} dose of alfentanhl. Al points represent mean (± S.E.M.) data from three monkeys. icaily, quadazocine was found to be most potent as an antagoiiist of the mu agomsts alfentanil and fentanyl, less potent as an antagonist of the kappa agonists U69,593 and EKC and least potent as an antagonist of BW373U86. This relative potency of quadazocine in antagonizing mu agonists, kappa agonists and BW373U86 corresponded to quadazocine s roletive affmity for mu, kappa and delta opioid receptors from rhesus monkey cortex measured in an in vitro receptor binding assay. In the present study, it was shown that the selective delta opioid antagonist NT! also antagonized the rate-decreasing effects of BW373U86; however, the relative potency of NT! as an antagonist of BW373U86, alfentanil and U69,593 was markedly different from the relative potency of quadazocine against these agonists. Unlike quadazocine, NT! was approximately 25 times more potent as an antagonist of BW373U86 than as an antagonist of alfentanil, and even the highest dose of NT! (1. mg/kg) did not antagonize, the kappa agonist U69,593. This is the first report on the opioid receptor selectivity of NT! in rhesus monkeys, although the potency of NT! as an antagonist of alfentanil in morphine-dependent rhesus monkeys (pa2 = 529; France et al, 199) is similar to that reported here (PKB 5.1). Furthermore, in in vitro binding and smooth muscle assays, NT! has been reported to display a similar selectivity for delta > mu > kappa opioid receptors (Rogers et

8 132 Negusetal. at., 199; Woods et at., 199). Therefore, the present findings further support the characterization of BW373U86 as a systemically active, delta-selective agonist in suppressing rates of foodmaintained responding in rhesus monkeys. Comer et al (1993b) examined the opioid antagonist effects of NT! with a drug-discrimination procedure in pigeons, and these authors reported that NT! was approximately 1-fold more potent as an antagonist of the discriminative effects of BW373U86 than as an antagonist of the effects of morphine. In relation to the present study, this high selectivity was due to a greater potency of NT! in antagonizing the discriminative effects of BW373U86 in pigeons (unconstrained pa2 = 8.3) versus the rate-suppressing effects of BW373U86 in monkeys (unconstrained pa2 = 6.4). The potency of NT! in antagonizing the mu agonists morphine or alfentanil was nearly identical across the two studies. The reason for this large difference in NT! s potency as an antagonist of BW373U86 in the two studies is unclear but may reflect differences in species or procedure. The discriminative stimulus effects of BW373U86 in pigeons appear to be unusually sensitive to antagonism by NT! because the potency of NT! in antagonizing the effect of BW373U86 in mice (Comer et at., 1993a; Takasuna et at., 1994), rats (Chang et al, 1993) and squirrel monkeys (Dykstra et at., 1993) is similar to that reported here. The time course of the rate-suppressing effects of the lowest, maximally effective dose of BW373U86 (.32 mg/kg) was approximately 1 to 2 hr. This is similar to the time course reported for the discriminative stimulus effects ofbw373us6 in pigeons (Comer et at., 1993b). A dose of 1. mg/kg of NT! antagonized the effects of BW373U86 for at least 4 hr, but the antagonist effects of NT! were no longer significant after 24 hr. It has been stated that NT! has a long duration of action in mice, antagonizing the antinociceptive effects of the delta-selective peptide DSLET for 18 to 24 hr (Abdeihamid et at., 1991). NT! may have a somewhat shorter duration of action in primates; however, detailed comparisons ofthe duration ofthe antagonist effects of NT! across species have not been conducted. Despite the effectiveness of BW373U86 to in suppressing rates of schedule-controlled responding, BW373U86 was totally ineffective in the production ofalfentanil- or EKC-like discriminative stimulus effects, antinociceptive effects in the warmwater tail-withdrawal procedure or reinforcing effects in the drug self-administration procedure. In addition, BW373U86 produced only a mild suppression of respiration in the head plethysmograph procedure. The observed inactivity of BW373U86 in these procedures was probably not a consequence of insufficient dosing. In the drug-discrimination procedures, BW373U86 was tested up to doses that completely suppressed response rates. In the warm-water tail-withdrawal and head plethysmograph assays, alfentanil produced antinociceptive and respiratory depressant effects at doses approximately 2 to 5 times the Aa() in suppressing rates of schedule-controlled behavior. BW373U86, in contrast, produced little or no effect in either the tail-withdrawal or head plethysmograph procedures at doses up to 18 times the A for the suppression of rates of responding. Furthermore, the highest dose of BW373U86 (1.78 mg/kg) used in the tail-withdrawal and head plethysmograph procedures produced a brief convulsion in one monkey, which indicated that BW373U86 was evaluated up to toxic doses. In the drug self-administration procedure, alfentanil produced reinforcing effects at i.v. unit doses from approximately 1% to 3% ofthe A for the suppression of schedule- Vol. 27 controlled responding, whereas BW373U86 did not maintain drug self-administration at unit doses from 1% to 3% of the A5 for rate suppression. The present findings complement previous studies that examined the behavioral effects of BW373U86 and delta-selective peptides. For example, previous studies that evaluated the ability of delta opioid receptors to mediate antinociceptive effects produced conflicting results that contrasted with the more reliable antinociceptive effects obtained with mu agonists. In general, delta-selective peptides, such as DPDPE, have been found to produce antinociceptive effects in rodents in some assays after i.c.v. or i.t. administration (Heyman et at., 1987; Kovacs et at., 1988; Calcagnetti and Holtzman, 1991; Drower et al, 1991). Similarly, i.t. administration of the moderately delta-selective peptides DADLE and metkephamid were reported to produce delta receptor-mediated antinociception in primates (Yaksh, 1983). It is important to note, however, that peptidic delta agonists do not produce antinociceptive effects in all assays of antinociception. For example, DPDPE has been reported to be effective in the rat hot-plate test of antinociception but not in the rat tail-flick test (Heyman et al, 1988). Procedure-specific antinociceptive effects have been even more prominent with BW373U86. Systemically administered BW373U86 produced antinociceptive effects in the acetic acidinduced writhing test in mice (Wild et at., 1993; Broadbear et al, in press, 1994) but not in the warm-water tail-withdrawal test in mice (Wild et at., 1993), rats (Chang et al, 1993) or rhesus monkeys (present study) or in the shock titration procedure in squirrel monkeys (Dykstra et at., 1993). These differences across assays may reflect differences in the intensity of the noxious stimuli or differences in the role of delta receptors in the modulation of different types of nociception. The antinociceptive effects of BW373U86 may also depend on the route of administration. Wild et al (1993) reported that BW373U86 did not produce antinociception in the warm-water tail-withdrawal test in mice when it was administered systemically or i.c.v., but i.t. administration of BW373U86 did produce antinociceptive effects. This is the first study to evaluate the respiratory effects of a delta agonist in primates, and the results of the present study agree with the findings ofprevious studies conducted in rodents that concluded that delta agonists produce little or no decrement in various respiratory parameters (Kiritsy-Roy et al, 1989; Cheng et al, 1992). For example, Kiritsy-Roy et al (1989) reported that central administration of a high dose (125 nm) of DPDPE produced only a mild acidosis in blood ph and no significant change in PO2 or pco2 in conscious rats. This is in contrast to mu opioid agonists, which can produce severe respiratory depression (Jaffe and Martin, 1985; Howell et al, 1988; Butelman et al, 1993). This is also the first study to evaluate the reinforcing effects of a delta agonist in primates. In the self-administration procedure, BW373U86 did not produce reinforcing effects under conditions in which alfentanil did maintain responding. The inability of BW373U86 to maintain drug self-administration in the present study contrasts with the results in previous reports, which indicate that peptidic delta agonists can maintain selfadministration (Goeders et at., 1984; Devine and Wise, 1994), produce place preferences (Shippenberg et al, 1987; Bals-Kubic et at., 199) and decrease thresholds for electrical brain stimulation (Jenck et at., 1987) in rats. There are a number of procedural differences between the present study and the stud-

9 1994 Dsfta Oplolds In Primates 133 ies conducted in rats, including species, type of delta agonist used, route of administration and assay conditions under which reinforcing effects were evaluated. It is not clear which of these differences accounts for the disparity across studies; however, the inability of BW373U86 to produce reinforcing effects in the present study marks an important point of departure from earlier studies that examined the role of delta opioid receptors in reinforcement processes. The convulsant effects of BW373U86 were not explicitly examined in the present study. As noted earlier, BW373U86 alone, at a dose of 1.78 mg/kg, produced a convulsion in one monkey and, because of these convulsant effects, higher doses of BW373U86 alone were not administered. However, in the schedule-controlled responding procedure, BW373U86 was administered at doses as high as 32. mg/kg after pretreatment with NT!. Convulsions were never observed in these monkeys, which suggests that NT! antagonized the convulsant and the rate-suppressing effects of BW373U86. Other studies have also reported that the convulsant effects of BW373U86 can be antagonized by NT! in mice (Comer et al, 1993a) and squirrel monkeys (Dykstra et al, 1993). In contrast to the consistent convulsant effects produced by BW373U86, peptidic delta agornate have been reported to produce both convulsant (Snead, 1986; Haffmans and Dzoljic, 1987; De Sarro et al, 1992) and anticonvulsant effects (Tortella et al, 1988). For example, DPDPE does not produce convulsions in rats (Tortella et al, 1988) or mice (Corner et al, 1993a) and may produce anticonvulsant effects (Tortella et al, 1988). On the other hand, DSLET has been reported to produce convulsions in rats (Snead, 1986; Haffmans and Dzoljic, 1987) but not mice (Comer et ol, 1993a) and deltorphin II was reported to produce seizures and convulsions in rats (De Sarro et al, 1992). Although existing evidence suggests that BW373U86 is a delta-selective agonist in vivo, the degree of this selectivity has not been precisely determined and previous studies that examined the behavioral effects of BW373U86 in pigeons (Comer et al, 1993b) and mice (Wild et al, 1993) reported that BW373U86 may produce at least some of its effects by acting at mu opioid receptors. For example, Corner et al (1993b) reported partial cross generalization between BW373U86 and mu agonists in pigeons trained to discriminate either morphine or BW373U86 from saline. In the present study, there was no evidence to suggest a role for either mu or kappa opioid receptore in the mediation of the agonist effects of BW373U86. This drug did not elicit any drug-appropriate responding in monkeys trained to discriminate either alfentanil or EKC. Furthermore, unlike either mu or kappa agonists (Dykstra et al, 1987), BW373U86 did not produce antinociception in the warm-water tail-withdrawal test. Finally, unlike mu agornsts, BW373U86 did not produce significant respiratory depression nor did it maintain self-administration. This apparent absence of either mu or kappa agonist effects suggests that BW373U86 is a highly selective delta receptor agonist in rhesus monkeys. In summary, the effects ofbw373u86 on schedule-controlled behavior indicate that BW373U86 is a potent, systemically active and delta-selective agonist in rhesus moneys. Despite its ability to produce these delta agonist effects in the assay of schedule-controlled responding, BW373U86 did not produce thermal antinociceptive effects, reinforcing effects or appreciable respiratory depressant effects. These results suggest that BW373U86 binds to delta opioid receptors that do not mediate these other effects in rhesus monkeys under these conditions. 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