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BEHAVIORAL PHARMACOLOGY

Interactions between and µ Opioid Agonists in Assays of Schedule-Controlled Responding, Thermal Nociception, Drug Self-Administration, and Drug versus Food Choice in Rhesus Monkeys: Studies with SNC80 [(+)-4-[(R)--((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide] and Heroin

Alcohol and Drug Abuse Research Center, McLean Hospital, Harvard Medical School, Belmont, Massachusetts (G.W.S., S.S.N.); and Laboratory of Medicinal Chemistry, National Institute on Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland (J.E.F., K.C.R.)

Received for publication December 27, 2004
Accepted March 23, 2005.

	Abstract

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Interactions between and µ opioid agonists in rhesus monkeys vary as a function of the behavioral endpoint. The present study compared interactions between the agonist SNC80 [(+)-4-[(R)--((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide] and the µ agonist heroin in assays of schedule-controlled responding, thermal nociception, and drug self-administration. Both SNC80 (ED₅₀ = 0.43 mg/kg) and heroin (ED₅₀ = 0.088 mg/kg) produced a dose-dependent and complete suppression of response rates in the assay of schedule-controlled responding. Heroin also produced thermal antinociception (ED_5°C = 0.18 mg/kg) and maintained drug self-administration under both a fixed ratio schedule [dose-effect curve peak at 0.0032 mg/kg/injection (inj)] and under a food versus heroin concurrent-choice schedule (ED₅₀ = 0.013 mg/kg/inj), whereas SNC80 did not produce thermal antinociception or maintain self-administration. Fixed ratio mixtures of SNC80 and heroin (1.6:1, 4.7:1, and 14:1 SNC80/heroin) produced additive effects in the assay of schedule-controlled responding and superadditive effects in the assay of thermal nociception. Also, SNC80 did not enhance the reinforcing effects of heroin, indicating that mixtures of SNC80 and heroin produced additive or infra-additive reinforcing effects. These results provide additional evidence to suggest that /µ interactions depend on the experimental endpoint and further suggest that agonists may selectively enhance the antinociceptive effects of µ agonists while either not affecting or decreasing the sedative and reinforcing effects of µ agonists.

Interactions between and µ agonists have also been reported for other behavioral endpoints, such as bladder motility, convulsions, Straub tail, respiratory depression, and sedation (Sheldon et al., 1989; O'Neill et al., 1997; Su et al., 1998; Stevenson et al., 2003). Importantly, and µ agonists often either did not modify or attenuated each other's nonantinociceptive effects. In rats, for example, the agonists BW373U86, [D-Pen²,D-Pen⁵]-enkephalin, and H-Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH² (deltorphin II) attenuated the respiratory depressant effects of the µ agonist alfentanil without affecting alfentanil-induced thermal antinociception (Su et al., 1998). In rhesus monkeys, mixtures of the agonist SNC80 and µ agonists methadone, fentanyl, morphine, and nalbuphine produced superadditive effects in an assay of thermal nociception, but only additive or subadditive effects in an assay of food-maintained responding (Stevenson et al., 2003). These results suggest that agonists might enhance or maintain some clinically useful effects of µ agonists (e.g., analgesia) without enhancing, or while even decreasing, undesirable effects (e.g., respiratory depression and sedation).

One undesirable effect that limits the clinical utility of µ agonists is their abuse liability (O'Brien, 2001); however, interactions between the reinforcing effects of µ and agonists have not been examined. Accordingly, the purpose of the present study was to evaluate the effects of the agonist SNC80 on the abuse-related effects of µ agonists. SNC80 (Negus et al., 1998) and the µ agonist heroin (Negus et al., 2003) were studied alone and in combination in a series of four experiments in rhesus monkeys. First, the effects of SNC80 and heroin were determined in an assay of schedule-controlled responding for food reinforcement. , µ, and opioid agonists all produce dose-dependent decreases in response rates in this procedure (Negus et al., 1993a), and as a result, this procedure was used to determine the relative potencies of the agonists and to provide an empirical basis for the relative proportions of drugs subsequently used in drug mixtures. Interactions between SNC80 and heroin were then examined in an assay of thermal nociception in rhesus monkeys that has been used to examine the antinociceptive effects of , µ, and opioids administered alone (Dykstra et al., 1987; Walker et al., 1995; Negus et al., 1998). We demonstrated previously that SNC80 enhanced the antinociceptive effects of other µ agonists in this procedure (Stevenson et al., 2003), and SNC80/heroin interactions were evaluated to confirm that SNC80 would also enhance the antinociceptive effects of heroin. Finally, interactions between SNC80 and heroin were examined in two assays of drug self-administration. In the first assay, there was only a single active response key, drug injections were the only reinforcer available, and the primary dependent variable was response rate. This type of procedure has been used extensively to assess the reinforcing effects of opioids administered alone (Wurster et al., 1977; Killian et al., 1978; Mello et al., 1983; Negus et al., 1995a; Winger and Woods, 2001). However, rates of opioid self-administration under these conditions may be influenced not only by the reinforcing effects of the drug but also by direct effects (e.g., sedative effects) of the self-administered drug (Mello and Negus, 1996). Consequently, interactions between the rate-decreasing effects of drugs may confound analysis of interactions between their reinforcing effects. To address this issue, interactions between the reinforcing effects of SNC80 and heroin were also evaluated in a food versus drug choice procedure. In this procedure, two response keys were simultaneously active, food delivery and drug injections were both available as reinforcers, and the primary dependent variable was a measure of food versus drug choice. One advantage of this procedure is that drug choice provides a measure of reinforcement that is relatively independent of drug effects on response rates (Griffiths et al., 1976; Woolverton and Balster, 1979; Negus, 2003, 2004b).

	Materials and Methods

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Subjects. Five male rhesus monkeys (Macaca mulatta) were used in studies of schedule-controlled responding, three male monkeys were used in studies of thermal nociception, three male and female monkeys were used in the single-key assay of drug self-administration, and three male monkeys were used in studies of food versus drug choice. Subjects weighed 4.5 to 12 kg during the course of these studies. All monkeys had prior exposure to drugs (primarily dopaminergic and opioid compounds) and to the behavioral procedures in which they were tested. The subjects were individually housed, and water was freely available. Their diet consisted of PMI Feeds Jumbo monkey diet (two to six biscuits per day; PMI Feeds, Inc., St. Louis, MO). This diet was supplemented with fresh fruit twice daily. In addition, monkeys in the assays of schedule-controlled behavior and food versus drug choice could earn additional food pellets during experimental sessions. A 12-h light/12-h dark cycle was in effect (lights on from 7:00 AM to 7:00 PM).

Animal maintenance and research were conducted in accordance with the guidelines provided by the National Institutes of Health Committee on Laboratory Animal Resources. The facility was licensed by the United States Department of Agriculture, and protocols were approved by the Institutional Animal Care and Use Committee. The health of the monkeys was monitored daily by technical staff and periodically by a consulting veterinarian. Monkeys had visual, auditory, and olfactory contact with other monkeys throughout the study. Monkeys also had access to puzzle feeders, mirrors, and chew toys to provide environmental enrichment. Nature videotapes or music were played daily in all housing rooms.

Behavioral and Pharmacological Procedures. Behavioral studies were conducted using four procedures: 1) an assay of schedule-controlled responding for food presentation, 2) an assay of thermal nociception using warm water as the noxious stimulus, 3) a single-key assay of drug self-administration, and 4) an assay of food versus drug choice. In each assay, the effects of the agonist SNC80 and the µ agonist heroin were examined alone and/or in various mixtures. SNC80 was used as the representative agonist in these studies because it functions as a potent, selective, and systemically active agonist in rhesus monkeys (Negus et al., 1998). Heroin was used as the representative µ agonist because it functions as a selective and relatively high-efficacy µ agonist in rhesus monkeys (Bowen et al., 2002; Negus et al., 2003) and because rhesus monkeys had already been trained to self-administer heroin in our laboratory under both the drug-alone and drug versus food choice procedures.

Assay of Schedule-Controlled Responding. Experiments were conducted in each monkey's home cage (dimensions 60 x 65 x 75 cm). Each cage was modified to include an operant response panel (28 x 28 cm) mounted on the front wall. Three circular translucent response keys (5.1 cm in diameter) were arranged 2.5 cm apart in a horizontal row 3.2 cm from the top of the operant panel. Each key could be transilluminated by red, green, or yellow stimulus lights. The operant panel also supported an externally mounted pellet dispenser (model G5210; Gerbrands, Arlington, MA) that delivered 1 g of fruit-flavored food pellets (P. J. Noyes Co., Lancaster, NH) to a food receptacle mounted on the cage beneath the operant response panel. The panel was controlled by a MED-PC interface and an IBM compatible computer programmed in MEDSTATE Notation (MED Associates, East Fairfield, VT).

Experimental sessions were 75 min in duration and consisted of five 15-min cycles. Each cycle consisted of two components: a 10-min time-out period followed by a 5-min response period. During the time-out period, no stimulus lights were illuminated, and responding had no scheduled consequences. During the response period, the center key was transilluminated yellow, and the subjects could respond for up to 10 food pellets under a fixed ratio 30 (FR30) schedule of reinforcement. If all 10 food pellets were earned before 5 min had elapsed, the lights were turned off, and responding had no scheduled consequences for the remainder of that response period. All monkeys were trained until they responded at rates greater than 1.0 response/s during all five cycles for 10 consecutive days, and we have shown previously that monkeys respond at relatively stable rates across successive response periods in this procedure (Negus et al., 1993a; Stevenson et al., 2003).

Sessions were conducted 5 days a week. Test sessions were usually conducted on Tuesdays and Fridays, and training sessions were conducted on Mondays, Wednesdays, and Thursdays. In addition, test sessions were conducted only after a training session during which the monkeys responded at rates greater than 1.0 response/s for all five cycles. During training sessions, monkeys received either no injection or saline injections at the beginning of each cycle. During test sessions, test compounds were administered using a cumulative dosing procedure, in which doses of the test drug or drug mixture were administered at the beginning of each cycle, and each dose increased the total cumulative dose by one-fourth or one-half log units.

Initially, complete dose-effect curves were determined for each drug administered alone, and each drug was tested twice. Tests of heroin alone were separated by at least 3 days, and tests of SNC80 alone were separated by at least 7 days. Subsequently, three mixtures of SNC80 in combination with heroin were examined, and the proportions of each drug in the mixtures were based on the relative potency of the drugs (see Data Analysis for details). Each mixture was tested once, and 1 week separated each test. Heroin, SNC80, and all mixtures were tested up to doses that eliminated responding in most or all monkeys.

Assay of Thermal Nociception. The monkeys were seated in acrylic restraint chairs so that their tails hung down freely. The bottom 10 cm of each monkey's shaved tail was immersed in a thermal container of warm water, and the latency to tail withdrawal was recorded. If the subject did not withdraw its tail within 20 s, the tail was removed from the water by the experimenter, and a latency of 20 s was assigned to that measurement. During each cycle of measurements, tail-withdrawal latencies were measured from four different temperatures. Usually, these temperatures were 38, 42, 46, and 50°C. When high heroin doses were tested, 54°C was used and 38°C was omitted. Temperatures above 54°C were not tested to avoid any possibility of tissue damage. The order in which the temperatures were presented varied from one set of measurements to the next according to a modified Latin square sequence.

Each tail-withdrawal session consisted of multiple 30-min cycles. Before the first cycle, baseline latencies to tail withdrawal from 38, 42, 46, and 50°C water were determined. During cumulative dosing experiments, a single drug dose was administered at the start of each subsequent cycle, and each dose increased the total cumulative dose by one-fourth or one-half log units. Starting 20 min after each injection, tail-withdrawal latencies from different water temperatures were determined as described above.

Tail-withdrawal sessions were conducted on Tuesdays and Fridays. Initially, complete dose-effect curves were determined for SNC80 and heroin administered alone, and each drug was tested twice. Tests of heroin alone were separated by at least 3 days, and tests of SNC80 alone were separated by at least 7 days. Subsequently, three mixtures of SNC80 in combination with heroin were examined, and the proportions of each drug in the mixtures were identical to those examined in the assay of schedule-controlled behavior described above. Each drug mixture was tested once, and 1 week separated each test. Heroin and all mixtures were tested up to doses that produced robust antinociceptive effects (T10 > 5°C; see Data Analysis) or that produced potentially problematic undesirable effects (e.g., respiratory depression). SNC80 was tested up to a maximal dose of 10 mg/kg as described previously (Stevenson et al., 2003).

Single-Key Assay of Drug Self-Administration. For intravenous drug administration, a double-lumen catheter was implanted into a jugular or femoral vein under aseptic conditions as described previously (Negus et al., 1995a). One lumen of the double-lumen catheter was used for i.v. drug self-administration, and the second lumen was used for intermittent saline administration (0.1 ml every 20 min) to maintain catheter patency. Each monkey was fitted with a nylon vest attached to a flexible stainless steel cable; the other end of the cable was attached to a fluid swivel (Lomir Biomedical, Montreal, QC, Canada). This tether system protected the i.v. catheter and allowed freedom of movement for the animal. Catheter patency was monitored periodically by i.v. administration of ketamine (5 mg/kg) or the short-acting barbiturate methohexital (4 mg/kg). The catheter was considered patent if i.v. ketamine or methohexital administration resulted in loss of muscle tone within 10 s.

Experimental sessions were conducted in each monkey's home cage (76.5 x 66 x 92 cm). The front wall of each cage was adapted for the attachment of an operant response panel identical to that described above. Two infusion pumps (model B5P-1E; Braintree Scientific, Braintree, MA; or model 98021, Harvard Apparatus Inc., South Natick, MA) were mounted above each cage for delivery of saline or drug solutions through the two lumens of the intravenous catheters. The schedules of reinforcement were controlled, and data were collected with an IBM-compatible computer and interface (MED Associates, Georgia, VT).

Experimental sessions were conducted from 11:00 AM to 1:00 PM 7 days per week. Monkeys were initially trained to respond for 0.01 mg/kg/injection heroin under a FR30 timeout 60-s schedule until the total number of reinforcers per session varied by 20% for 3 consecutive days. Saline was then substituted for heroin until the number of injections per day decreased to fewer than five. Subsequently, training sessions alternated with test sessions. During training sessions, either saline or 0.01 mg/kg/injection heroin was available for self-administration. During test sessions, a test dose of heroin alone, SNC0 alone, or a drug mixture was available. Test sessions were separated by at least two training sessions and were conducted only after training sessions in which 0.01 mg/kg/injection heroin was available. A range of heroin doses (0.001-0.032 mg/kg/injection) and SNC80 doses (0.0032-0.1 mg/kg/injection) was tested twice in each monkey. Subsequently, dose-effect curves for three SNC80/heroin mixtures were determined, and each dose of each mixture was tested once in each monkey.

Assay of Food versus Drug Choice. The experimental apparatus was identical to that used in the single-key assay of drug self-administration, and experimental methods were based on a previously described assay of food versus drug choice (Negus, 2003, 2005). Behavioral sessions began daily at 11:00 AM and were conducted 7 days a week. The terminal choice schedule consisted of five 20-min response periods separated by 5-min time-out periods (total session duration of 120 min). During all response periods, the left, food-associated key was illuminated with red stimulus lights, and completion of the FR requirement resulted in the delivery of a food pellet. In addition, Table 1 shows that different doses of heroin were introduced in conjunction with different stimulus conditions on the right, heroin-associated key. The heroin doses available during the five response periods were 0, 0.0032, 0.01, 0.032, and 0.1 mg/kg/inj. These doses were presented in ascending order, and the dose was varied by varying the duration of pump activation and the resulting volume of each injection (Table 1). Stimulus conditions on the heroin-associated key were also varied by flashing yellow stimulus lights, and the duration of the flash corresponded to the magnitude of the available heroin dose (Table 1). Thus, longer flashes (and shorter interflash intervals) were associated with higher available heroin doses. Completion of the FR requirement on the heroin-associated key resulted in delivery of the available dose of heroin. The final ratio values were FR100 on the food-associated key and FR10 on the heroin-associated key for all monkeys. These FR values were used because they usually resulted in a switch from food to heroin choice at an intermediate dose of heroin (0.01 or 0.032 mg/kg/inj), and as a result, it was possible to observe either leftward or rightward shifts in the drug choice dose-effect curve.

During each response period, responding on the food- or drug-associated key reset the ratio requirement of the other key, and monkeys could complete up to 10 total ratio requirements of the food- and heroin-associated keys. Completion of each ratio requirement initiated a 30-s time-out, during which all stimulus lights were turned off, and responding had no scheduled consequences. If all 10 ratio requirements were completed before the 20-min response period had elapsed, then all stimulus lights were extinguished and responding had no scheduled consequences for the remainder of that 20-min response period. Choice training was considered to be complete when the ED₅₀ value of the dose-effect curves for heroin choice (see Data Analysis) varied by less than 2-fold for 3 consecutive days.

After determination of the heroin choice function was complete, choice curves were determined for three mixtures of SNC80 in combination with heroin, and the proportions of each drug in the mixtures were identical to those examined in the assay of schedule-controlled behavior described above. Each mixture was tested for 5 days, and the last 3 days were used for data analysis. Heroin alone was reinstated between tests for at least 3 days and until baseline choice behavior recovered. To test for the possibility of tolerance to the effects of 5 days administration of SNC80, choice behavior maintained by the 14:1 SNC80/heroin mixture (the mixture with the highest proportion of SNC80) was compared for the first and last day of availability. SNC80 was not tested alone in the choice procedure, because SNC80 failed to maintain self-administration in the single-key procedure in the present study, in which no alternative reinforcer was available.

Data Analysis. For the assay of schedule-controlled responding, operant response rates from each cycle were converted to percentage of control using the average rate from the previous training day as the control value. The ED₅₀ for each drug or drug mixture in each monkey was defined as the dose that produced a 50% decrease in the percentage of control rate of responding. Individual ED₅₀ values were calculated by interpolation when only two data points were available (one above and one below 50% control response rate) or by linear regression when at least three data points were available on the linear portion of the dose-effect curve. For drug mixtures, a related quantity, Z_mix, was also calculated for each monkey. Z_mix was defined as the total drug dose (i.e., dose SNC80 + dose heroin) that produced a 50% decrease in responding.

For the assay of thermal nociception, tail-withdrawal data were analyzed as described previously using a method that emphasizes drug-induced shifts in complete temperature-effect curves rather than drug-induced changes in tail-withdrawal latencies at any one temperature (Negus et al., 1993b). For each set of tail-withdrawal latency measurements collected during any one cycle of a test session, a temperature-effect curve was constructed that related tail-withdrawal latency to water temperature. A T10 value was determined from each temperature-effect curve by fitting a line to the two points that fell immediately above and below 10 s and by calculating the temperature in degrees centigrade that corresponded to a tail-withdrawal latency of 10 s. On some occasions (i.e., with the highest doses of heroin in some monkeys), tail-withdrawal latencies were greater than 10 s at all water temperatures up to and including 54°C, and as noted above, temperatures above 54°C were not tested to avoid any possibility of tissue damage. In these cases, the T10 was estimated using the experimentally determined value at 54°C, and a value of "0" at 58°C (i.e., the next 4°C increment). Thus, the maximum possible T10 value was 56°C (i.e., a 20-s latency from 54°C water). The T10 from each monkey's baseline test cycle was subtracted from the T10 determined from each subsequent test cycle, which provided a measure of change relative to the baseline of each animal, or T10. Antinociception was operationally defined as rightward shifts in the temperature-effect curves and increases in the T10 values. The dose of each drug or drug mixture that produced a T10 of 5°C (ED_5°C) was interpolated from the linear portion of the dose-effect curve for each monkey. For drug mixtures, Z_mix was also calculated for each monkey as the total drug dose (i.e., dose SNC80 + dose heroin) that produced a T10 of 5°C.

For the single-key assay of drug self-administration, the principal dependent variable was the response rate in responses per second. To calculate ED₅₀ values, response rates were normalized to a percent maximal rate (%MR) for each monkey using the equation %MR = [(test rate - saline rate) ÷ (maximal heroin rate - saline rate)] x 100. The ED₅₀ value was defined as 50% MR, or the unit dose of drug or drug mixture on the ascending limb of the dose-effect curve that maintained response rates midway between the rate maintained by saline and the peak rate maintained by heroin alone. For each monkey, ED₅₀ values were calculated by interpolation when only two data points were available (one below and one above 50% control) or by linear regression when at least three data points were available on the ascending limb of the dose-effect curve.

For the assay of food versus drug choice, the primary dependent variables for each response period were 1) percentage of drug choice, defined as (number of ratios completed on the drug-associated key ÷ total number of ratios completed) x 100; 2) response rate, defined as the total number of responses ÷ total time responses had scheduled consequences; and 3) total number of ratios completed. The ED₅₀ value of the dose-effect curve for drug choice was defined as the dose of drug or drug mixture that produced 50% drug choice. For each monkey, ED₅₀ values were calculated by interpolation when only two data points were available (one below and one above 50% control) or by linear regression when at least three data points were available on the ascending limb of the dose-effect curve.

Because drug doses were incremented on a logarithmic scale, ED₅₀, ED_5°C, and Z_mix values were converted to their log values for calculation of means and standard errors and for statistical analysis, and these values were then converted back to linear values for presentation in tables and text.

Interactions between SNC80 and heroin were assessed using both graphical and statistical approaches (Woolverton, 1987; Tallarida, 2000; Negus, 2005) as described previously (Stevenson et al., 2003). Graphically, mean ED₅₀ and ED_5°C values [95% confidence limits (CL)] for SNC80 administered either alone or as part of a mixture were plotted as a function of the ED₅₀ or ED_5°C value of heroin. This data presentation format is known as an isobologram, and the line in an isobologram that connects the data points for each drug alone shows predicted data points for drug mixtures assuming additivity. Points that fall above the line of additivity (away from the origin) are suggestive of subadditivity, whereas points that fall below the line (toward the origin) are suggestive of superadditivity.

Statistical evaluation of drug interactions was accomplished by comparing the experimentally determined ED₅₀ or ED_5°C values for each mixture (Z_mix) with predicted additive ED₅₀ or ED_5°C values (Z_add) as described by Tallarida (2000). Z_mix values were determined empirically as described above. For the assay of schedule-controlled responding, in which all drugs were equieffective, Z_add values were calculated individually for each monkey from the equation:

(1)

where A is the ED₅₀ for SNC80 alone, B is the ED₅₀ for heroin alone, and f is the fractional multiplier of A in the computation of the additive total dose. Any choice of f is related to the proportion of drug A (A) in a mixture according to the equation:

(2)

The present study examined effects produced by mixtures in which f = 0.25, 0.5, and 0.75. Thus, fl = 0.25 leads to _A = A/(A/3 + B), and the ratio of the SNC80 concentration to the heroin concentration is [(A/B) ÷ 3]:1; f = 0.5 leads to _A = A/(A + B), and the ratio of the SNC80 concentration to the heroin concentration is A/B:1; and f = 0.75 leads to _A = 3A/(3A + B), and the ratio of the SNC80 concentration to the heroin concentration is [(A/B) x 3]:1.

For the assays of thermal nociception and drug self-administration, SNC80 alone was ineffective, and the hypothesis of additivity predicts that SNC80 would not contribute to the effects of a mixture of SNC80 in combination with heroin. As a result, the equation for Z_add reduces to:

(3)

Mean predicted and experimental values were compared by a t test, and the criterion for a significant effect was p < 0.05.

View larger version (13K): [in this window] [in a new window]   Fig. 1. Effects of heroin alone, SNC80 alone, and SNC80 + heroin mixtures on schedule-controlled responding. Top, dose-effect curves for heroin alone and SNC80 alone. Abscissa, dose of drug in milligrams per kilogram (log scale). Ordinate, percentage of control response rate. Middle, dose-effect curves for heroin alone and three mixtures of SNC80 + heroin. Abscissa, dose of heroin in milligrams per kilogram (log scale). Ordinate, percentage of control response rate. Bottom, isobologram for each SNC80 + heroin mixture. Abscissa, ED50 value of heroin (milligrams per kilogram). Ordinate, ED50 value of SNC80 (milligrams per kilogram). All points show mean data (±S.E.M.) for five monkeys.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Assay of Schedule-Controlled Responding
Control Response Rates and Effects of Opioid Agonists Alone. The average control response rate ± S.E.M., determined on the days before test days throughout the study, was 1.99 ± 0.09 responses/s. Response rates were similar during each of the five cycles of the multiple cycle sessions (1.97 ± 0.12, 2.20 ± 0.12, 2.00 ± 0.14, 1.94 ± 0.11, and 1.83 ± 0.11 responses/s).

The µ agonist heroin and the agonist SNC80 each produced a dose-dependent decrease in rates of responding (Fig. 1, top), and the ED₅₀ values for both compounds are shown in Table 2. Heroin was approximately 4.7-fold more potent than SNC80 in decreasing rates of responding. This relative potency value was used to determine the relative proportions of SNC80 and heroin in the subsequent drug mixtures. Specifically, SNC80/heroin mixtures of 1.6:1, 4.7:1, and 14:1 were studied.

View this table: [in this window] [in a new window]   TABLE 2 ED50 or ED5°C values in milligrams per kilogram (95% CL) for the µ agonist heroin and the agonist SNC80, in assays of schedule-controlled responding for food reinforcement, thermal nociception, drug self-administration, and food versus drug choice in rhesus monkeys

Effects of SNC80 in Combination with Heroin. The effects of heroin administered alone or in combination with SNC80 are shown in Fig. 1 (middle). All SNC80 + heroin mixtures produced dose-dependent decreases in response rates. Addition of SNC80 produced leftward shifts in the dose-effect curve for heroin, and the magnitude of these left shifts was dependent on the proportion of SNC80 in the mixture. Isobolographic display of ED₅₀ values for SNC80 + heroin indicated that mixtures of SNC80 + heroin decreased response rates with ED₅₀ values that fell close to the line of additivity (Fig. 1, bottom). Statistical analysis confirmed that experimentally determined ED₅₀ values for SNC80 + heroin mixtures (Z_mix) were similar to predicted ED₅₀ values for these mixtures (Z_add; Table 3).

Assay of Thermal Nociception
Baseline T10 Values and Effects of Opioid Agonists Alone. Under baseline conditions, maximum tail-withdrawal latencies (i.e., 20 s) were typically obtained with temperatures of 38 and 42°C. As the temperature was increased to 46 and 50°C, tail-withdrawal latencies decreased to 0.99 ± 0.18 and 0.76 ± 0.10 s, respectively. The average baseline T10 value ± S.E.M. throughout the study was 44.10 ± 0.01°C.

The µ agonist heroin produced a dose-dependent increase in the T10 (Fig. 2, top), and the ED_5°C value for heroin is shown in Table 2. The agonist SNC80 was without effect in this assay (i.e., there was no statistically significant change in the T10 across a 100-fold dose range, and an ED_5°C could not be determined; Fig. 2, top). Doses up to and including 10 mg/kg SNC80 were tested (data not shown). Given that SNC80 was ineffective in the assay of thermal nociception, the relative potencies from the assay of schedule-controlled responding were used to determine the relative proportions of SNC80 and heroin in the subsequent drug mixtures.

View larger version (11K): [in this window] [in a new window]   Fig. 2. Effects of heroin alone, SNC80 alone, and SNC80 + heroin mixtures on thermal nociception. Top, dose-effect curves for heroin alone and SNC80 alone. Abscissa, dose of drug in milligrams per kilogram (log scale). Ordinate, change in threshold for acute thermal nociception (T10). Middle, dose-effect curves for heroin alone and three mixtures of SNC80 + heroin. Abscissa, dose of heroin in milligrams per kilogram (log scale). Ordinate, change in threshold for acute thermal nociception (T10). Bottom, isobologram for each SNC80 + heroin mixture. Abscissa, ED5°C value of heroin (milligrams per kilogram). Ordinate, ED5°C value of SNC80 (milligrams per kilogram). All points show mean data (±S.E.M.) for three monkeys. *, experimentally determined values significantly different from additivity (see Table 3). *, experimentally determined value approaches significance (p = 0.08; see Table 3).

View larger version (16K): [in this window] [in a new window]   Fig. 3. Effects of heroin alone, SNC80 alone, and SNC80 + heroin mixtures in the single-key self-administration procedure. Top, dose-effect curves for heroin alone and SNC80 alone. Abscissa, dose of drug in milligrams per kilogram per injection (log scale). Ordinate, response rate (responses per second). Bottom, dose-effect curves for heroin alone and three mixtures of SNC80 + heroin. Abscissa, dose of heroin in milligrams per kilogram per injection (log scale). Ordinate, response rate (responses per second). All points show mean data (±S.E.M.) for three monkeys.

Effects of SNC80 in Combination with Heroin. The reinforcing effects of heroin available alone or in combination with SNC80 are shown in Fig. 3 (bottom). All SNC80 + heroin mixtures produced shallow inverted U-shaped dose-effect curves with an apparent peak at 0.0032 mg/kg/inj. Thus, addition of SNC80 produced downward shifts in the dose-effect curve for heroin (p < 0.05). However, SNC80 + heroin interactions could not be quantitatively assessed in the assay of drug self-administration, because combinations of SNC80 + heroin did not maintain response rates above those maintained by saline (p > 0.05). Consequently, ED₅₀ values for combinations of SNC80 + heroin could not be determined and compared with predicted additive ED₅₀ values.

Assay of Drug versus Food Choice
Effects of Opioid Agonists Alone. The µ agonist heroin produced a dose-dependent increase in percentage of drug choice (Fig. 4, top) and a dose-dependent decrease in response rate (Fig. 5, top). When the unit dose of heroin available for self-administration was relatively low (0 and 0.0032 mg/kg/inj), nearly all responding was allocated to the food-associated key. When a higher unit dose of 0.032 mg/kg/inj heroin was available, responding was reallocated exclusively to the heroin-associated key. When the highest unit dose of 0.1 heroin was available, monkeys continued to allocate all responses to the heroin-associated key; however, response rates declined to very low levels, and monkeys did not always earn the maximum number of reinforcers (data not shown). The ED₅₀ for heroin in the choice procedure is shown in Table 2. Given that the agonist SNC80 was without effect in the single-key assay of drug self-administration, it was not tested alone in the assay of drug versus food choice.

View larger version (12K): [in this window] [in a new window]   Fig. 4. Effects of heroin alone and SNC80 + heroin mixtures in the concurrent drug versus food choice procedure. Top, dose-effect curve for heroin alone. Abscissa, dose of heroin in milligrams per kilogram per injection (log scale). Ordinate, percentage of drug choice. Middle, dose-effect curves for heroin alone and three mixtures of SNC8O + heroin. Abscissa, dose of heroin in milligrams per kilogram per injection (log scale). Ordinate, percentage of drug choice. Bottom, isobologram for each SNC80 + heroin mixture. Abscissa, ED50 value of heroin (milligrams per kilogram per injection). Ordinate, ED50 value of SNC80 (milligrams per kilogram per injection). All points show mean data (±S.E.M.) for three monkeys.

View larger version (15K): [in this window] [in a new window]   Fig. 5. Effects of heroin alone and SNC80 + heroin mixtures on response rate in the choice procedure. Top, dose-effect curve for heroin alone. Abscissa, dose of heroin in milligrams per kilogram per injection (log scale). Ordinate, response rate (responses per second). Bottom, dose-effect curves for heroin alone and three mixtures of SNC80 + heroin. Abscissa, dose of heroin in milligrams per kilogram per injection (log scale). Ordinate, response rate (responses per second). All points show mean data (±S.E.M.) for three monkeys.

Figure 6 shows choice behavior maintained by the 14:1 SNC80/heroin mixture (the mixture with the highest proportion of SNC80) comparing the first and last day of availability. Figure 6, top, shows that the dose-effect curve for percentage of drug choice was identical on both days, suggesting that the addition of SNC80 did not alter the reinforcing effects of heroin at any time. However, Fig. 6, middle, shows that the dose-effect curve for response rates was shifted slightly to the right on day 5 in comparison with day 1, suggesting that some tolerance may have developed to the rate-decreasing effects of the mixture. Similarly, Fig. 6, bottom, shows the dose-effect curve for total reinforcers delivered during availability of each dose of the mixture, and again, there was a trend for a rightward shift in the dose-effect curve on day 5 in comparison to day 1. Overall, the effects of the SNC80/heroin combinations on drug choice did not change during the 5 days of testing, but there was a trend for the rate-decreasing effects of self-administered SNC80/heroin mixtures to decrease during that same period of time.

View larger version (14K): [in this window] [in a new window]   Fig. 6. Effects of the 14:1 SNC80/heroin mixture (the mixture with the highest proportion of SNC80) in the choice procedure on days 1 and 5 of a 5-day period of availability. Abscissa, dose of heroin in milligrams per kilogram per injection (log scale). Top ordinate, percentage of drug choice. Middle ordinate, response rate (responses per second). Bottom ordinate, number of reinforcers per component. All points show mean data (±S.E.M.) for three monkeys.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
This manuscript evaluated interactions between the agonist SNC80 and the µ agonist heroin in four different behavioral procedures in rhesus monkeys. The main finding was that SNC80 alone did not produce reinforcing effects, and addition of SNC80 to heroin did not enhance the reinforcing effects of heroin. In terms of dose-addition analysis, the reinforcing effects of SNC80 and heroin were additive. The present study also provided additional evidence to indicate that combinations of SNC80 with µ opioid agonists produce only additive effects on a measure of schedule-controlled responding for food reinforcement, but superadditive effects in an assay of thermal nociception. Together, these results suggest that SNC80 may selectively enhance a clinically desirable effect of µ agonists (antinociception) without enhancing other, potentially undesirable effects of µ agonists (sedation and abuse potential).

/µ Interactions in the Assays of Schedule-Controlled Responding and Thermal Nociception. In agreement with previous studies (Negus et al., 1998, 2003; Stevenson et al., 2003), both heroin and SNC80 produced dose-dependent rate-decreasing effects in the assay of food-maintained responding. In the present study, heroin was more potent than SNC80 in decreasing rates of responding, and the relative potencies of heroin and SNC80 provided an empirical basis for determining the ratios of both compounds to be tested in subsequent mixtures (see Results). Also in agreement with previous studies (Negus et al., 1998, 2003; Stevenson et al., 2003), heroin produced antinociceptive effects in the warm water tail-withdrawal procedure, whereas SNC80 was ineffective. It is important to note that, in the present study, heroin was used as a representative µ opioid agonist to facilitate subsequent assessment of the reinforcing effects associated with + µ interactions. Pharmacological studies suggest that the behavioral effects of heroin in rhesus monkeys are mediated primarily by µ receptors (Bowen et al., 2002; Negus et al., 2003).

In the present study, mixtures of SNC80 + heroin produced additive effects in the assay of schedule-controlled responding and superadditive effects in the assay of thermal nociception. These results confirm and extend our previous findings that mixtures of SNC80 in combination with the other µ agonists methadone, fentanyl, morphine, and nalbuphine also produced additive effects in an assay of schedule-controlled responding, and superadditive effects in an assay of thermal nociception in rhesus monkeys (Stevenson et al., 2003). Superadditive antinociceptive interactions between and µ agonists have also been reported in rodents and squirrel monkeys (Heyman et al., 1989; Adams et al., 1993; Dykstra et al., 2002).

Reinforcing Effects of SNC80 and Heroin Alone. In the present study, heroin alone maintained self-administration in the single-key self-administration procedure and produced a characteristic inverted U-shaped dose-effect curve. In the drug versus food choice procedure, heroin alone produced a dose-dependent increase in percentage of drug choice. These results are consistent with previous studies demonstrating the reinforcing effects of heroin in both single-key (Wurster et al., 1977; Killian et al., 1978; Mello et al., 1983; Negus et al., 1995a; Winger and Woods, 2001) and choice procedures (Griffiths et al., 1976, 1981; Negus, 2005) in nonhuman primates. In the present study, heroin was less potent in the choice procedure than in the single-key procedure. The decreased potency of heroin in the choice procedure may be due to the availability of an alternative reinforcer (i.e., food), thereby requiring higher doses of heroin to maintain choice responding. Cocaine has also been found to be less potent in the choice procedure versus the single-key procedure (Caine et al., 2000; Negus, 2003).

In contrast to heroin, SNC80 at unit doses up to 0.1 mg/kg/injection did not maintain responding in the single-key drug self-administration procedure. This finding is consistent with previous reports in which substitution of SNC80, or the other agonist BW373U86, did not maintain self-administration in rhesus monkeys maintained on the µ agonist alfentanil or cocaine (Negus et al., 1994, 1995b, 1998; Negus, 2004a). SNC80 alone was not tested in the choice procedure, because drugs are usually less potent in choice procedures than in single-key procedures (see above) and because SNC80 unit doses >0.1 mg/kg/injection decrease response rates (Negus et al., 1998).

Reinforcing Effects of SNC80/Heroin Mixtures. The main purpose of this manuscript was to evaluate the ability of SNC80 to alter the reinforcing effects of heroin. In the single-key assay, addition of SNC80 produced a downward shift in the heroin self-administration dose-effect curve. This downward shift could reflect a decrease in reinforcing effects relative to heroin alone. However, an alternative explanation for the downward shift is that self-administered SNC80 + heroin mixtures produced additive rate-decreasing effects without altering the reinforcing effects of heroin alone. This would be consistent with the findings of the assay of schedule-controlled responding (see above), in which SNC80 and heroin each decreased rates of food-maintained responding, and combinations of SNC80 and heroin produced additive effects. These results illustrate the potential difficulty of dissociating drug-induced reinforcing effects from rate-altering effects in drug self-administration assays that rely on rate-based measures as the primary dependent variable. At the very least, these results suggest that SNC80 did not increase the reinforcing effects of heroin.

In an effort to dissociate interactions between the reinforcing and rate-altering effects of SNC80 and heroin, the effect of SNC80 + heroin mixtures were also examined in a food versus drug choice procedure. In the choice procedure, the measure of percentage of drug choice provides a measure of reinforcing efficacy that is relatively independent of response rate (Negus, 2003). Thus, studies in drug choice allowed for the assessment of SNC80/heroin interactions on measures of reinforcement independent of SNC80/heroin interactions on overall response rate. In the choice procedure, heroin alone produced a dose-dependent increase in drug choice, and the addition of SNC80 to heroin had no effect on the heroin dose-effect curve. In terms of dose-addition analysis, the effects of SNC80 and heroin were additive (i.e., SNC80 had no reinforcing effect on its own, and it did not enhance the reinforcing effects of heroin). This is in marked contrast with the results of the thermal antinociception assay, in which SNC80 was also inactive alone but significantly enhanced the antinociceptive effects of heroin. Thus, SNC80 enhanced the antinociceptive but not the reinforcing effects of heroin.

Mechanisms of Opioid Interactions. The neural mechanisms that underlie interactions between and µ agonists in rhesus monkeys are not known. These behavioral interactions could reflect drug interactions at physically coupled and µ receptors, intracellular interactions mediated by separate and µ receptors located on common neurons, or intercellular interactions mediated by and µ receptors located on separate neurons in a common neural circuit (Mansour et al., 1988, 1995; Cheng et al., 1997; Jordan et al., 2000). The behavioral selectivity of /µ interactions suggests that there is some neuroanatomical selectivity in the substrate that mediates those interactions. The results of the present study, for example, suggest that substrates capable of mediating synergistic /µ interactions may exist in neural systems underlying opioid-induced antinociception, but not in neural systems underlying opioid-induced decreases in operant responding, or opioid self-administration. Regarding the possibility of physically coupled and µ receptors, these dimers seem to mediate both cooperative binding of and µ ligands and synergistic /µ interactions in functional assays. However, the pharmacology of /µ interactions mediated by /µ dimers in vitro (Gomes et al., 2000; Jordan et al., 2000) does not correspond precisely with /µ antinociceptive interactions observed in the present study. For example, synergistic /µ interactions in vitro could be obtained with some antagonists, but not with SNC80 (Gomes et al., 2000). Conversely, the present study found /µ antinociceptive interactions with SNC80. Together, these results suggest that opioid receptor dimers identified to date from in vitro studies may not contribute to opioid antinociceptive interactions in rhesus monkeys.

Rate Effects in the Choice Procedure and the Possibility of Tolerance. In the assay of schedule-controlled responding, heroin alone produced dose-dependent decreases in rates of responding, and addition of SNC80 to heroin produced dose-dependent leftward shifts in the heroin dose-effect curve. In the assay of drug versus food choice, heroin self-administration also produced dose-dependent decreases in response rate. However, addition of SNC80 to heroin had no effect on heroin's rate-decreasing effects (i.e., addition of SNC80 did not shift the heroin dose-effect curve for response rate in the choice assay). This discrepancy could be due to the different dosing schedules used in these assays. Specifically, in the assay of schedule-controlled responding, SNC80/heroin mixtures were tested acutely, whereas in the assay of drug versus food choice, SNC80/heroin mixtures were tested for five consecutive days (with the last 3 days averaged for statistical analysis). We have reported previously that tolerance develops rapidly to the rate-decreasing effects of SNC80 (Brandt et al., 2001). Thus, there is a possibility that tolerance also developed to the rate-decreasing effects of SNC80 in the drug versus food choice procedure. This tolerance could account for the lack of leftward shifts in the rate curves with the addition of increasing amounts of SNC80. In support of this possibility, comparison of the rate-decreasing effects of the 14:1 SNC80/heroin mixture (the mixture which contained the highest concentration of SNC80) on day 1 and day 5 in the choice procedure shows a slight rightward shift in the rate curve on day 5 relative to day 1.

Dependence of Drug Interactions on Experimental Endpoint. A growing body of evidence indicates that the nature of an interaction between two drugs depends in part on the experimental endpoint under evaluation. For example, we reported previously that interactions between the antinociceptive effects of SNC80 and µ agonists were superadditive, whereas interactions between the rate-decreasing effects of SNC80 and µ agonists were only additive or subadditive (Stevenson et al., 2003). Similarly, studies in rats have reported different interactions between and µ agonists on measures of antinociception compared with measures of convulsant activity, respiratory depression, and Straub tail (O'Neill et al., 1997; Su et al., 1998). The present study extends these findings to demonstrate only an additive interaction between the reinforcing effects of SNC80 and the µ agonist heroin. Together, these results provide evidence to suggest that it may be possible to exploit drug interactions to design drugs or drug mixtures that maximize clinically desirable effects (e.g., analgesia) while minimizing undesirable side effects (e.g., sedation and abuse liability).

	Acknowledgements

 
We thank Peter Fivel and Sam McWilliams for excellent technical assistance.

	Footnotes

 
This work was supported by Grants R01-DA11460, T32-DA07252, and P01-DA14528 from the National Institute on Drug Abuse, National Institutes of Health.

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.104.082685.

ABBREVIATIONS: BW373U86, (±)-4-[(R^*)--((2S^*,5R^*)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-hydroxybenzyl]-N,N-diethylbenzamide; SNC80, (+)-4-[(R)--((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide; FR, fixed ratio; inj, injection; CL, confidence limits.

Address correspondence to: Dr. S. Stevens Negus, Alcohol and Drug Abuse Research Center, Harvard Medical School, McLean Hospital, 115 Mill St., Belmont, MA 02478-9106. E-mail: negus{at}mclean.harvard.edu

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