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Vol. 299, Issue 2, 629-637, November 2001


Studies of Tolerance and Dependence with the delta -Opioid Agonist SNC80 in Rhesus Monkeys Responding under a Schedule of Food Presentation

Michael R. Brandt1 , M. Scott Furness, Kenner C. Rice, Bradford D. Fischer and S. Stevens Negus

Alcohol and Drug Abuse Research Center, Harvard Medical School-McLean Hospital, Belmont, Massachusetts (M.R.B., B.D.F., S.S.N.); and Laboratory of Medicinal Chemistry, National Institute of Diabetics and Digestive and Kidney Diseases and National Institutes of Health, Bethesda, Maryland (M.S.F., K.C.R.)

    Abstract
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Tolerance and dependence after acute or chronic administration of the selective delta -opioid agonist SNC80 were assessed in rhesus monkeys (Macaca mulatta) responding under a schedule of food presentation. SNC80 dose dependently decreased response rates. These effects waned after 5 h. When administered as an acute 24-h pretreatment, SNC80 (1.0-10.0 mg/kg) produced tolerance as evidenced by dose-dependent rightward shifts in the SNC80 dose-effect curve. Pretreatments of 3.2 or 10.0 mg/kg SNC80 increased the SNC80 ED50 by 4- or 25-fold, respectively. Tolerance to acute SNC80 was also time-dependent as evidenced by increased ED50 values when administered as a 5-h (14-fold), 24-h (25-fold), or 3-day (11-fold) pretreatment. The SNC80 dose-effect curve was similar to control after a 7-day pretreatment. The selective delta -antagonist naltrindole (1.0 mg/kg) partially blocked tolerance to acute SNC80. Chronic SNC80 (1.0-10.0 mg/kg/day) also produced dose-dependent rightward shifts in the SNC80 dose-effect curve. Chronic SNC80 was more effective than acute SNC80 in producing tolerance. Moreover, tolerance to chronic SNC80 waned more slowly than to acute SNC80. Acute or chronic SNC80 (10.0 mg/kg/day) also produced cross-tolerance to the rate-decreasing effects of other delta -agonists (SNC162 and SNC243A) but not to µ- (morphine) or kappa  (U-50,488)-agonists. Changes in response rates or behavioral signs of withdrawal were not observed after the administration of opioid antagonists (i.e., naltrindole or naltrexone) in monkeys treated with SNC80. These data suggest that a pharmacologically selective tolerance develops to delta -agonists after both acute and chronic administration of SNC80 with little or no dependence.

    Introduction
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

The repeated administration of a drug may result in the development of tolerance to that drug and cross-tolerance to other drugs that have pharmacologically similar mechanisms of action. Moreover, repeated administration can produce physical dependence, which can be quantified by the type and severity of withdrawal signs that emerge after the discontinuation of drug treatment or the administration of a pharmacological antagonist. In contrast to the well characterized development of tolerance and dependence to the behavioral effects of µ- and kappa -opioid agonists (Holtzman and Villarreal, 1973; Gmerek and Woods, 1985; Gmerek et al., 1987; Brandt and France, 1998, 2000), the development of tolerance and dependence to the behavioral effects of delta -opioids has been examined less extensively, in part because the only selective delta -agonists available for study until recently were peptidic compounds with poor bioavailability.

Studies with these peptidic delta -agonists demonstrated that tolerance may develop under some conditions. For example, in vitro studies indicate that peptidic delta -agonists such as [D-Pen2,D-Pen5]-enkephalin (DPDPE) can rapidly desensitize and down-regulate delta -opioid receptors (DORs) in cells expressing DORs (Trapaidze et al., 1996; Breivogel et al., 1997; Remmers et al., 1998; Okura et al., 2000). Similarly, in vivo studies report that tolerance can develop to the antinociceptive effects of DPDPE after repeated i.c.v. administration in rodents (Kovacs et al., 1988; Suh and Tseng, 1990; Zhao and Bhargava, 1997). Moreover, tolerance was pharmacologically selective in so far as tolerance to delta -agonists did not confer cross-tolerance to µ- and kappa -agonists (Iyengar et al., 1987; Suh and Tseng, 1990; Stevens and Yaksh, 1992).

Few studies have assessed whether physical dependence develops after chronic treatment with delta -agonists, and it is unclear whether the withdrawal behaviors observed in these studies are exclusively DOR-mediated. For example, a relatively large, nonselective dose of naloxone (3.0 mg/kg) that produced hypothermia and withdrawal jumping in mice treated subchronically with i.c.v. morphine only elicited mild hyperthermia and no withdrawal jumping in mice treated subchronically with i.c.v. DPDPE (Kovacs et al., 1988). In rats receiving i.c.v. DPDPE via osmotic mini-pumps for 70 h, 3.0 mg/kg naloxone precipitated withdrawal signs similar to signs observed in rats receiving i.c.v. morphine (Cowan et al., 1988). However, the magnitude of these behaviors was smaller in DPDPE-treated subjects than in morphine-treated subjects. Although DPDPE is selective for DORs, it is unclear whether withdrawal from DPDPE was mediated by actions solely at DORs. Recent studies indicated that the antinociceptive effects of DPDPE were not fully attenuated with antisense directed at the cloned rat DOR and that antinociception could be attenuated with the selective µ-antagonist D-Phen-c[-Cys-Tyr-D-Trp-Orn-Thr-Pen]-Thr-NH2 (Fraser et al., 2000b). Moreover, the potency of DPDPE for producing antinociception is decreased in µ-opioid receptor knockout mice compared with wild-type mice (Hosohata et al., 2000). These results suggest that assessing the selectivity of dependence and withdrawal by using DPDPE may be confounded by effects mediated by a receptor distinct from DORs, likely MORs.

More recently, nonpeptidic delta -opioids have been developed that have greater bioavailability relative to the peptidic compounds. For example, the piperazinyl benzamide SNC80 is systemically active and more than 800-fold selective for DORs versus MORs (Calderon et al., 1994, 1997). Interest in delta -agonists continues in part because of their potential for the treatment of pain. We and others have reported that SNC80 and other delta -agonists have antinociceptive effects in both rodents and nonhuman primates under conditions of chemically induced allodynia (Stein et al., 1989; Stewart and Hammond, 1994; Butelman et al., 1995; Fraser et al., 2000a; Brandt et al., 2001). SNC80 and related compounds also produce other behavioral effects, such as decreases in rates of schedule-controlled responding for food (Negus et al., 1994, 1998). However, the magnitude of tolerance and dependence produced by novel, nonpeptidic delta -opioids have not been adequately characterized, and these issues have potential importance for the continued therapeutic development of delta -agonists as analgesics.

Therefore, the purpose of the present study was to assess the degree to which tolerance and dependence develop after acute and chronic administration of SNC80 in rhesus monkeys (Macaca mulatta). Studies were conducted in an assay of fixed ratio schedule-controlled responding for food presentation. This procedure has been used extensively to characterize the pharmacology of opioids and is sensitive to changes in opioid effects due to both tolerance and dependence (Holtzman and Villarreal, 1973; Craft et al., 1989; Picker et al., 1991; Picker and Yarbrough, 1991; Gerak and France, 1997; Brandt and France, 1998, 2000). µ-, kappa -, and delta -Opioid agonists produce dose-dependent decreases in response rates in this procedure, whereas opioid antagonists are relatively ineffective and decrease response rates only at high doses (Adams and Holtzman, 1990; Negus et al., 1993, 1994, 1998; Gerak and France, 1997). Numerous studies have shown that chronic treatment with either µ- or kappa -agonists produces tolerance to the rate-decreasing effects of the treatment drug and pharmacologically selective cross-tolerance to other similarly selective opioid agonists (Craft et al., 1989; Picker et al., 1991; Brandt and France, 2000). In contrast, chronic agonist treatment can increase sensitivity to the rate-decreasing effects of opioid antagonists, and this enhanced sensitivity to opioid antagonists has been interpreted as a withdrawal sign indicative of opioid dependence (Thompson and Schuster, 1964; Holtzman and Villarreal, 1973; Adams and Holtzman, 1990; Picker et al., 1991; Brandt and France, 1998, 2000). The present study extended this line of investigation by examining the effects of acute and chronic treatment with SNC80 on the rate-decreasing effects of opioid agonists and antagonists. To assess the possible development of physical dependence on SNC80, evaluations of opioid antagonist effects on schedule-controlled behavior were supplemented by observational studies that assessed the incidence of overt behavioral signs of withdrawal.

    Materials and Methods
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Subjects. One male and two female rhesus monkeys had free access to water and were maintained on a diet of fresh fruit and vegetables, multiple vitamins, and 5-10 Lab Diet Jumbo Monkey biscuits daily (PMI Feeds, Inc., St. Louis, MO). In addition, monkeys could earn between 50 and 70 1-g food pellets (Precision Primate Pellets Formula L/I Banana Flavor; P.J. Noyes Co., Lancaster, NH) during daily operant sessions (see below). 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 laboratory facility was licensed by the United States Department of Agriculture, and research protocols were approved by the McLean Hospital Institutional Animal Care and Use Committee. A consulting veterinarian periodically monitored the health of the monkeys. Monkeys had visual, auditory, and olfactory contact with other monkeys throughout the study. Operant procedures for food-maintained responding provided an opportunity for environmental enrichment.

Apparatus. Monkeys were individually housed in stainless steel cages (56 × 71 × 69 cm). An operant panel (28 × 28 cm) was mounted on the front of each home cage. Each panel contained a horizontal row of three response keys (6.4 × 6.4 cm) that were arranged 2.5 cm apart and 3.2 cm from the top of the operant panel. The center key could be transilluminated with a yellow stimulus light. An externally mounted pellet dispenser (model G5210; Ralph Gerbrands Co., Arlington, MA) delivered 1-g banana-flavored pellets to a food receptacle mounted on the cage beneath the operant panel. Control of experiments and data recording were accomplished with a microprocessor, interface, and software (MED Associates, St. Albans, VT) located in a separate room.

Schedule-Controlled Training. Monkeys were trained under a multiple-cycle procedure during experimental sessions conducted 5 days each week. Each training cycle consisted of a 10-min pretreatment period followed by a 5-min response period. During the pretreatment period, stimulus lights were not illuminated and responding had no scheduled consequences. During the response period, the center key was illuminated yellow, and subjects could respond for up to 10 food pellets under a fixed ratio 30 schedule of food presentation. If all 10 food pellets were earned before 5 min had elapsed, the light was turned off, and responding had no scheduled consequences for the remainder of the response period. The left and right keys were inactive, and responding on these keys had no scheduled consequences. Training sessions consisted of five consecutive cycles, and testing began once individual monkeys responded at rates greater than 1.0 responses/s during all five cycles for 10 consecutive days.

Single Doses of SNC80 Testing. The acute effects of SNC80 were assessed using two experimental procedures: a time course procedure and a cumulative dosing procedure. In the time course procedure, a single dose of the test compound was administered i.m., and 5-min response cycles identical to those described above began at 10, 30, 100, and 300 min after the injection. In the cumulative dosing procedure, test sessions were identical to training sessions except that saline was administered i.m. during the first minute of the first cycle, and cumulative doses of drug, increasing in one-quarter or one-half log unit increments, were administered i.m. during the first minute of subsequent cycles (i.e., 15-min interinjection interval).

Three series of experiments were conducted to assess the changes in sensitivity to the effects of opioids after the acute administration of SNC80. The first series of experiments assessed the effects of a single dose of SNC80 on the SNC80 dose-effect curve determined 24 h later. Saline, or a single dose of SNC80 (1.0-10.0 mg/kg) was administered using the time course procedure to determine the duration of action of SNC80. Twenty-four hours later, increasing doses of SNC80 were administered under the cumulative dosing procedure to determine whether the SNC80 dose-effect curve was modified by the earlier dose of SNC80. Testing was suspended for a minimum of 7 days after all tests that assessed the behavioral effects of SNC80.

Additional studies assessed the duration of behavioral effects produced by a single dose of SNC80. On four separate occasions, 10.0 mg/kg SNC80 was administered, and the SNC80 dose-effect curve was redetermined either 5 h, 1 day, 3 days, or 7 days later by using the cumulative dosing procedure. Training sessions were conducted between test sessions that were separated by more than 1 day. In addition, to determine whether the acute effects of SNC80 could be blocked, 1.0 mg/kg of the delta -selective antagonist naltrindole was administered as a 30-min pretreatment to 10.0 mg/kg SNC80 before the time course procedure. The SNC80 dose-effect curve was then redetermined 24 h later under the cumulative dosing procedure. Tests were separated by a minimum of 7 days.

In the second series of experiments, the development of cross-tolerance was assessed. Similar to previous experiments, saline or 10.0 mg/kg SNC80 was administered, and response rates were assessed using the time course procedure. Twenty-four hours later, increasing doses of the delta -opioid agonist SNC162 (a piperazinyl benzamide structurally similar to SNC80), the µ-opioid agonist morphine, or the kappa -opioid agonist U-50,488 were administered under the cumulative dosing procedure. Testing was suspended for a minimum of 7 days after all tests.

In the third series of experiments, the ability of a single dose of SNC80 to produce acute dependence was assessed by evaluating changes in sensitivity to the rate-decreasing effects of the selective delta -antagonist naltrindole. In the absence of any SNC80 treatment, doses of naltrindole (0.1-10.0 mg/kg) were administered using the cumulative dosing procedure. The naltrindole dose-effect curve was then redetermined 24 h after a dose of 10.0 mg/kg SNC80. After the last dose of naltrindole (10.0 mg/kg), the behavior of monkeys was videotaped and scored for signs of withdrawal (see below for details). Previous studies have demonstrated that monkeys can become more sensitive to opioid antagonists after repeated administration of high doses (France and Morse, 1989; Negus et al., 1993). Therefore, the naltrindole dose-effect curve was redetermined a third time in the absence of SNC80. Each of the three tests with cumulative doses of naltrindole was separated by 13 days.

Chronic SNC80 Testing. After tests of the acute behavioral effects of SCN80, three additional series of studies were conducted to assess changes in sensitivity to the effects of opioids before, during, and after chronic daily administration of SCN80. The first series of experiments evaluated the dose-effect curves for the rate-decreasing effects of drugs in the absence of any SNC80 treatment. The rate effects of drugs were determined using the cumulative dosing procedure. The drugs and order of testing were morphine, SNC80, naltrexone, naltrindole, the delta -selective agonist SNC243A (a piperazinyl benzamide structurally similar to SNC80 and SNC162), and U-50,488.

The second series of experiments consisted of the daily administration of SNC80 and a redetermination of the rate effects of drugs. SNC80 was administered daily to monkeys 5 h before training sessions identical to those described previously. The dose of SNC80 was increased weekly with monkeys receiving 1.0, 3.2, and 10.0 mg/kg/day SNC80 for 7 days each. On the 7th day of treatment with each dose of SNC80, sensitivity to the rate-decreasing effects of SNC80 was redetermined using the cumulative dosing procedure. Saline was administered on the first cycle and cumulative doses of SNC80 were administered on subsequent cycles, up to a maximum dose of 10.0 mg/kg. A dose of 10.0 mg/kg/day SNC80 was administered daily during weeks 4 to 7, and the dose-effect curves for the rate-decreasing effects of drugs were redetermined. The drugs and order of testing were morphine, SNC243A, U-50,488, naltrindole, and naltrexone. During tests with naltrexone and naltrindole, the behavior of monkeys was evaluated for signs of precipitated withdrawal. Monkeys were videotaped for 5 min before the session and again for 5 min immediately after the administration of the last dose of naltrindole (3.2 mg/kg) or naltrexone (1.0 mg/kg). These videotapes were later scored for signs of withdrawal (see below for details).

The third series of studies reassessed the rate effects of drugs after the termination of chronic SNC80 treatment. Daily SNC80 treatment was terminated at the end of week 7 (i.e., after 5 weeks of daily 10.0 mg/kg SNC80 treatment). The dose-effect curve for SNC80 was determined weekly to determine when sensitivity to the rate-decreasing effects of SNC80 would return to prechronic control. During weeks 8 to 9 (i.e., 1-2 weeks after the termination of daily SNC80 treatment), the rate-decreasing effects of SNC80 were assessed up to a maximum dose of 10.0 mg/kg. The maximum dose of SNC80 administered was decreased to 3.2 mg/kg during weeks 10 to 13 (i.e., 3-6 weeks after the termination of daily SNC80 treatment).

After the return to prechronic sensitivity to the rate-decreasing effects of SNC80, the dose-effect curves for the rate-decreasing effects of drugs were redetermined during weeks 14 to 16 (i.e., 7-9 weeks after the termination of daily SNC80 treatment). The drugs and order of testing were morphine, SNC243A, U-50,488, naltrindole, and naltrexone.

Behavioral Signs of Withdrawal. Videotapes were evaluated by an experienced observer who was blind to experimental conditions. Behavioral signs were scored as either present or absent during the 5-min observation periods. Behavioral signs were categorized as general appearance (e.g., unusual location in cage, cataleptic, anesthetized/asleep, piloerection, muscle twitches, tremor, and convulsions), general behavior (hyper- or hypoactivity, holds abdomen, vocalization, retching, vomiting, coughing, grimacing, lying on side, masturbation, wet dog shakes, yawning, scratching, restless pacing, excessive grooming), and response to investigator (abnormal aggressive display, abnormal withdrawal response, rejects food pellet). The observer was also instructed to record any other nontypical behaviors. Many of these behavioral endpoints are commonly observed during µ- and/or kappa -opioid withdrawal in monkeys (Gmerek and Woods, 1985; Gmerek et al., 1987; Brandt and France, 1998).

Data Analyses. Operant response rates from each cycle were converted to percentage of control by using the average rate from the previous training day as the control value (i.e., average of 5 cycles). ED50 values were defined as the dose of a drug that produced a 50% decrease in control response rates. Individual ED50 values were calculated by linear regression when at least three data points were available on the linear portion of the dose-effect curve or by interpolation when only two data points (one above and one below 50%) were available. Individual ED50 values were converted to their log values for calculation of means and 95% confidence limits and then converted back to linear values for presentation. When an ED50 value for an individual monkey could not be determined because response rates did not decrease to below 50% of control response rates, the highest dose tested was assigned as the ED50 value.

To assess changes in response rates before, during, and after chronic SNC80 treatment, weekly response rates for individual subjects were calculated by averaging daily response rates (excluding test days). Weekly averages were calculated for the week immediately before SNC80 treatment through 4 weeks after SNC80 treatment. Statistical analysis of response rates was done using one-way repeated measures analysis of variance. Significant main effects were analyzed further by subsequent paired comparisons using the Student-Newman-Keuls method. The criterion for significance was p < 0.05.

Drugs. Naltrindole, SNC80, SNC162, and SNC243A were synthesized by Kenner C. Rice and colleagues (National Institutes of Health, Bethesda, MD). Naltrexone HCl and morphine sulfate were supplied by the National Institute on Drug Abuse (Bethesda, MD). (±)-trans-U-50,488 methanesulfonate was purchased from Sigma/RBI (Natick, MA). The free-base forms of SNC80, SNC162, and SNC243A were dissolved in 3% lactic acid and sterile water to a final concentration of 50 mg/ml and dilutions were made with sterile water. All other compounds were dissolved in sterile water. Drugs were administered i.m., and the sites of injection were rotated so that the same site was never used on two consecutive days. Doses were based on the free base or salt forms described above.

    Results
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References

Control Performance and Effects of Acute SNC80. The mean control rate of responding in untreated monkeys was 1.35 ± 0.19 responses/s. Figure 1, left, shows the potency and time course of the rate-decreasing effects of SNC80. Injections of saline did not substantially modify rates of responding during the 5-h session. A dose of 1.0 mg/kg SNC80 decreased rates of responding for 100 min, and response rates gradually returned to near control rates (89% control) after 300 min. Larger doses of SNC80 (3.2 and 10.0 mg/kg) produced larger decreases in response rates up to 100 min and greater rate suppression after 300 min (70-81% of control). Response rates recovered to control levels 24 h after saline and all doses of SNC80. Figure 1, right, also shows the cumulative dose-effect curves for SNC80 determined 24 h after saline or 1.0 to 10.0 mg/kg SNC80. ED50 values are shown in Table 1. After 24-h pretreatment with saline, cumulative doses of SNC80 dose dependently decreased response rates, and a dose of 1.0 mg/kg eliminated responding in all monkeys. Twenty-four hour pretreatment with SNC80 produced dose-dependent rightward shifts in the SNC80 dose-effect curve. A dose of 1.0 mg/kg SNC80 produced a small 3-fold increase in the SNC80 ED50 value. Larger doses of SNC80 produced progressively greater increases in the ED50, with a dose of 10.0 mg/kg SNC80 producing a 25-fold increase in the SNC80 ED50.


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  		Fig. 1.   Dose- and time-dependent effects of single doses of SNC80. Abscissa (left), time in minutes after the administration of SNC80. Abscissa (right), dose of SNC80 in milligrams per kilogram. Ordinates, mean rates of responding presented as a percentage of control response rates. All points show mean data (±1 S.E.M.) from three monkeys. , saline; , 1.0 mg/kg SNC80; triangle , 3.2 mg/kg SNC80; diamond , 10.0 mg/kg SNC80.


                              
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  		TABLE 1
Mean ED50 values in mg/kg (± 95% confidence limits) and dose ratios for SNC80 after acute pretreatment with saline or SNC80 (1.0-10.0 mg/kg)

All values show mean data from three monkeys.

Figure 2 shows the initial rate-decreasing effects of 10.0 mg/kg SNC80 and the time course of tolerance produced by this acute dose of SNC80. SNC80 ED50 values for these time course studies are shown in Table 1. The rate-decreasing effects of 10.0 mg/kg SNC80 were similar after repeated administrations with dosing intervals greater than 7 days (Fig. 2, left). However, at shorter intervals, redeterminations of the SNC80 dose-effect curve were shifted to the right in a time-dependent manner (Fig. 2, right). The onset of tolerance to acute SNC80 was evident within 5 h after administration. The maximal shift in the SNC80 dose-effect curve and increase in the ED50 value (Table 1) were observed after 24 h. Rightward shifts in the SNC80 dose-effect curve were still evident after 3 days, whereas the dose-response function and ED50 value were similar to control after 7 days.


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  		Fig. 2.   Time course of single doses of 10.0 mg/kg SNC80. Abscissa (left), time in minutes after the administration of SNC80. Abscissa (right), dose of SNC80 in milligrams per kilogram. Ordinates, mean rates of responding presented as a percentage of control response rates. All points show mean data (±1 S.E.M.) from three monkeys. , saline; triangle , 10.0 mg/kg SNC80 (5 h); diamond , 10.0 mg/kg SNC80 (1 day); down-triangle, 10.0 mg/kg SNC80 (3 day); , 10.0 mg/kg SNC80 (7 day).

The rate-decreasing effects of SNC80 were attenuated by the DOR-selective antagonist naltrindole. Rates of responding at 10 and 100 min were higher (34 and 72% of control, respectively) after a pretreatment with 1.0 mg/kg naltrindole compared with 10.0 mg/kg SNC80 alone (0 and 6% of control, respectively). Moreover, naltrindole decreased the magnitude of the rightward shift in the SNC80 dose-effect curve and attenuated the increase in the ED50 value 24 h after administration (Table 1).

Effects of delta  -, µ-, and kappa -Opioids after Acute SNC80 Treatment. Figure 3 shows the effects of the DOR-selective agonist SNC162, the MOR-selective agonist morphine, and the kappa -opioid receptor-selective agonist U-50,488 24 h after pretreatment with saline or 10.0 mg/kg SNC80. ED50 values are shown in Table 2. SNC162 dose dependently decreased rates of responding after saline pretreatment and had a potency similar to SNC80. When administered 24 h after 10.0 mg/kg SNC80, the SNC162 dose-effect curve was shifted to the right of control, and the ED50 value was increased by more than 28-fold. Morphine and U-50,488 also dose dependently decreased rates of responding. However, 24-h pretreatment with SNC80 did not modify the dose-effect curve or ED50 value for either morphine or U-50,488 (Table 2).


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  		Fig. 3.   Effects of opioid agonists alone and after pretreatment with 10.0 mg/kg SNC80. Abscissa, dose of drug in milligrams per kilogram. Ordinates, mean rates of responding presented as a percentage of control response rates. All points show mean data (±1 S.E.M.) from three monkeys. , 24 h after saline; diamond , 24 h after 10.0 mg/kg SNC80.


                              
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  		TABLE 2
Mean ED50 values in milligrams per kilogram (±95% confidence limits) and dose ratios for SNC162, morphine, or U-50,488 after acute pretreatment with saline or 10.0 mg/kg SNC80

Sensitivity to naltrindole was also assessed 24 h after pretreatment with saline or 10.0 mg/kg SNC80. When administered alone, naltrindole did not modify rates of responding in two monkeys up to a dose of 10.0 mg/kg (data not shown). However, this dose of naltrindole decreased response rates to 65% of control rates in the third monkey. Twenty-four hours after 10.0 mg/kg SNC80, a cumulative dose of 10.0 mg/kg naltrindole eliminated responding in two of the monkeys. To determine whether the sensitivity of monkeys to the rate-decreasing effects of naltrindole was changing after repeated naltrindole administration, the naltrindole dose-effect curve was redetermined in the absence of SNC80. A dose of 3.2 mg/kg naltrindole decreased rates of responding to less than 50% in one monkey and a dose of 10.0 mg/kg naltrindole eliminated responding in all monkeys. Because sensitivity to the rate-decreasing effects of naltrindole appeared to be increasing after repeated high-dose administration, subsequent studies assessed the effects of naltrindole up to a maximum dose of 3.2 mg/kg. Scoring of behavior from videotapes revealed no changes in behavior when naltrindole was administered 24 h after SNC80 (data not shown).

Control Response Rates and Effects of SNC80 during Chronic SNC80 Treatment. Weekly rates of responding during training sessions were calculated before, during, and after chronic SNC80. The average rate of food-maintained responding the week before SNC80 treatment was 1.47 ± 0.19 responses/s. Rates of responding were slightly lower during a week of daily 1.0 mg/kg (1.31 ± 0.26 responses/s), 3.2 mg/kg (1.13 ± 0.11 responses/s), or 10.0 mg/kg (1.18 ± 0.14 responses/s) SNC80 administered 5 h before sessions. There was a trend for decreasing rates of responding with increasing doses of SNC80, however, this trend was not significant (p = 0.09).

Figure 4 and Table 3 show the effects of SNC80 before, during, and after chronic treatment with SNC80. After 7 days of treatment with 1.0 mg/kg/day SNC80, the SNC80 dose-effect curve was shifted to the right of the "baseline" (i.e., nondrug) control, and the ED50 was increased by 2.7-fold. Seven days after an increase in the daily dose to 3.2 mg/kg SNC80, the SNC80 dose-effect curve was shifted further to the right and responding was eliminated at a dose of 32.0 mg/kg SNC80 in only two monkeys. After an additional 7 days of treatment with a larger dose of 10.0 mg/kg SNC80, there was no further rightward shift in the dose-effect curve for SNC80.


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  		Fig. 4.   SNC80 dose-effect curves determined weekly during daily SNC80 treatment. The rate-decreasing effects of SNC80 were determined on the 7th day of treatment with each dose of SNC80 (1.0, 3.2, and 10.0 mg/kg/day). Dose-effect curves were determined 5 h after the daily injection of SNC80. Abscissa, dose of drug in milligrams per kilogram. Ordinates, mean rates of responding presented as a percentage of control response rates. All points show mean data (±1 S.E.M.) from three monkeys. , baseline; triangle , 1.0 mg/kg SNC80 daily; , 3.2 mg/kg SNC80 daily; diamond , 10.0 mg/kg SNC80 daily.


                              
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  		TABLE 3
Mean ED50 values in milligrams per kilogram (±95% confidence limits) and dose ratio for SNC80 before, during, and after chronic treatment with daily administration of 10.0 mg/kg SNC80

Compared with the week before daily SNC80 treatment (1.47 ± 0.19 responses/s), rates of responding were significantly lower during the week after the termination of 10.0 mg/kg/day SNC80 treatment (0.95 ± 0.11 responses/s). However, response rates after the termination of SNC80 treatment were not different from those obtained during the last week of 10.0 mg/kg/day SNC80 treatment (1.01 ± 0.02 responses/s). Rates of responding gradually increased after the termination of SNC80 treatment but remained slightly lower (1.24 ± 0.34 responses/s) than pre-SNC80 control values after 4 weeks.

Sensitivity to SNC80 required many weeks to recover after termination of SNC80 treatment. Cumulative doses of 10.0 mg/kg SNC80 did not decrease response rates to below 50% in all monkeys for the first 2 weeks after the termination of daily SNC80 treatment (Fig. 5; Table 3). Because sensitivity to the rate-decreasing effects of SNC80 was not recovering in all monkeys, the maximum dose tested weekly was decreased to 3.2 mg/kg SNC80. Only after 5 weeks post-SNC80 treatment (with the exception of weekly SNC80 determinations up to a maximum dose of 3.2 mg/kg) did individual ED50 values for the rate-decreasing effects of SNC80 return to prechronic levels in all monkeys.


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  		Fig. 5.   Recovery of sensitivity to SNC80 after chronic treatment. SNC80 rate-decreasing effects were determined weekly (on the 7th day) after the discontinuation of chronic treatment. Doses larger than 10.0 mg/kg (weeks 1 and 2) and 3.2 mg/kg (weeks 3-5) were not administered. SNC80 dose-effect curves for weeks 3 and 4 are not shown for clarity. Abscissa, dose of drug in milligrams per kilogram. Ordinates: mean rates of responding presented as a percentage of control response rates. All points show mean data (±1 SEM) from three monkeys. , baseline; triangle  10.0 mg/kg SNC80 daily; , post-SNC80, week 1; diamond , post-SNC80, week 2; down-triangle, post-SNC80, week 5.

Effects of delta -, µ-, and kappa -Opioid Agonists during Chronic SNC80 Treatment. Figure 6 and Table 4 show the effects of the DOR-selective agonist SNC243A, morphine, and U-50,488 before, during, and after chronic treatment with 10.0 mg/kg SNC80. SNC243A, morphine, and U-50,488 dose dependently decreased response rates before chronic SNC80 treatment. During chronic treatment with 10.0 mg/kg SNC80, the SNC243A dose-effect curve was shifted to the right (Fig. 6, left), and the ED50 was increased by 8.4-fold. In contrast, chronic SNC80 treatment did not modify the dose-effect curves or ED50 values for either morphine or U-50,488. For all agonists, redeterminations of dose-effect curves and ED50 values after chronic SNC80 treatment were similar to those determined before chronic SNC80 treatment.


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  		Fig. 6.   Effects of opioid agonists before, during, and after chronic treatment with 10.0 mg/kg/day SNC80. Abscissa, dose of drug in milligrams per kilogram. Ordinates, mean rates of responding presented as a percentage of control response rates. All points show mean data (±1 S.E.M.) from three monkeys. , prechronic; triangle , chronic; , postchronic.


                              
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  		TABLE 4
Mean ED50 values in milligrams per kilogram (±95% confidence limits) and dose ratios for drugs before, during, and after chronic treatment with daily administration of 10.0 mg/kg SNC80

Effects of Opioid Antagonists during Chronic SNC80 Treatment. Figure 7 and Table 4 show the effects of naltrindole and naltrexone before, during, and after chronic treatment with 10.0 mg/kg SNC80. Up to the largest dose tested (3.2 mg/kg), naltrindole did not decrease rates of responding in untreated monkeys (Fig. 7, left). However, during daily SNC80, a cumulative dose of 3.2 mg/kg naltrindole eliminated responding in two of three monkeys. After the termination of SNC80 treatment, two monkeys remained slightly more sensitive to the rate-decreasing effects of naltrindole compared with before treatment. In these monkeys, 3.2 mg/kg naltrindole eliminated responding in one monkey and response rates were decreased to 66% of control in a second monkey. Only one of these monkeys showed increased sensitivity to the rate-decreasing effects of naltrindole during both the chronic and postchronic conditions. Naltrexone dose dependently decreased response rates, and 1.0 mg/kg naltrexone eliminated responding in all monkeys under prechronic conditions (Fig. 7, right). Daily administration of SNC80 did not modify the rate-decreasing effects of naltrexone. The dose-effect curve for naltrexone was shifted slightly to the right after chronic SNC80 treatment, but the 95% confidence limits of the ED50 values overlapped.


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  		Fig. 7.   Effects of naltrindole (NTI) and naltrexone (NTX) before, during, and after chronic treatment with 10.0 mg/kg/day SNC80. Abscissa, dose of drug in milligrams per kilogram. Ordinates, mean rates of responding presented as a percentage of control response rates. All points show mean data (±1 S.E.M.) from three monkeys. , prechronic; triangle , chronic; , postchronic.

Behavioral signs of physical withdrawal were evaluated 5 min before the experimental sessions and 5 min after the administration of the last dose of either naltrindole (3.2 mg/kg) or naltrexone (1.0 mg/kg). For all monkeys, there was no evidence of behavioral signs commonly observed during µ- or kappa -opioid withdrawal. Moreover, there was no evidence of any other behavioral signs that might be specific to delta -opioid withdrawal (data not shown).

    Discussion
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Abstract
Introduction
Materials and Methods
Results
Discussion
References

The present series of studies examined the rate-altering effects of opioids in an assay of schedule-controlled behavior before, during, and after either acute or chronic SNC80 treatment. Tolerance developed rapidly to the rate-decreasing effects of SNC80 as evidenced by dose- and time-dependent rightward shifts in the SNC80 dose-effect curve. Moreover, tolerance produced by SNC80 was DOR-mediated as evidenced by 1) the ability of the delta -selective antagonist naltrindole to attenuate SNC80-induced tolerance, 2) the development of cross-tolerance to other delta -selective agonists, and 3) the lack of cross-tolerance to µ- or kappa -selective opioid agonists. Neither acute nor chronic SNC80 produced substantial changes in sensitivity to opioid antagonists. In addition, no behavioral signs of withdrawal were observed after antagonist administration. These findings suggest that SNC80 produced little dependence when administered under the current dosing conditions. Together, these findings indicate that SNC80 can produce pharmacologically selective tolerance with little or no evidence of dependence in rhesus monkeys. These results also provide further evidence to suggest that SNC80 acts as a selective DOR agonist in rhesus monkeys.

Tolerance to SNC80. In an assay of schedule-controlled behavior, SNC80 produced dose- and time-dependent decreases in response rates. The potency and time course of SNC80 for decreasing response rates in the present study were similar to results obtained from previous studies in rhesus monkeys (Negus et al., 1998; Brandt et al., 1999). Acute SNC80 produced tolerance as evidenced by dose- and time-dependent rightward shifts in the SNC80 dose-effect curve. The onset of tolerance was apparent as soon as the acute agonist effects of SNC80 had waned (i.e., 5 h after SNC80 administration), and tolerance lasted between 3 and 7 days. This is the first study to demonstrate tolerance to the effects of a delta -agonist in rhesus monkeys; however, other studies have shown that tolerance can develop to delta -agonist effects in rodents. For example, tolerance developed to the antinociceptive effects of centrally administered DPDPE in rats (Kovacs et al., 1988; Suh and Tseng, 1990) and to the convulsant effects of systemically administered BW373U86 in mice (Comer et al., 1993) after treatment with a single dose of the delta -agonist. Similarly, previous in vitro studies have demonstrated that rapid desensitization can occur after the administration of delta -selective agonists. For example, in C6 glioma cells stably expressing the rat DOR, cells treated with DPDPE or SNC80 showed a decreased agonist-stimulated GTPgamma S binding, as well as an attenuated inhibition of forskolin-stimulated cAMP accumulation (Remmers et al., 1998). Moreover, [3H]naltrindole binding was substantially decreased in cells incubated with DPDPE or SNC80, suggesting that rapid internalization of DORs occurs during agonist treatment (Remmers et al., 1998; Okura et al., 2000). Together, these results suggest that rapid desensitization occurs after delta -agonist administration, and this desensitization may contribute to the rapid development of tolerance with delta -selective agonists.

The present study also assessed tolerance to the effects of chronic SNC80 administration. The ED50 value for "prechronic" SNC80 (2.14 mg/kg; Table 3) was somewhat higher than the baseline ED50 value for SNC80 in the initial acute tolerance study (0.37 mg/kg; Table 1), suggesting that a small degree of tolerance to SNC80 was already apparent at the beginning of the chronic dosing studies. Despite this elevated baseline ED50, chronic SNC80 was slightly more effective than acute SNC80 in producing tolerance. For example, 1.0 mg/kg SNC80 produced a substantial rightward shift in the SNC80 dose-effect curve after 1 week of chronic treatment but not after acute treatment. Similarly, 3.2 mg/kg SNC80 produced a greater rightward shift after chronic treatment than after acute treatment. Chronic treatment with a higher dose of 10.0 mg/kg SNC80 did not produce a further rightward shift in the SNC80 dose-effect curve, suggesting that some limit to tolerance had been reached. However, tolerance had a longer time course and waned more slowly after chronic treatment than after acute treatment. Tolerance to acute 10.0 mg/kg SNC80 waned in less than 1 week, but tolerance was still apparent 1 week after chronic treatment with doses up to and including 10.0 mg/kg SNC80. Indeed, the maximal dose of SNC80 used during weekly probes of SNC80 potency during the postchronic phase was reduced from 10.0 to 3.2 mg/kg in an effort to facilitate recovery of the SNC80 dose-effect curve. Taken together, these results suggest that although single doses of SNC80 produced profound levels of tolerance, tolerance was achieved even more readily by administering SNC80 repeatedly as little as once a day for as little as 1 week. Similar results were obtained in evaluations of tolerance to the consultant effects of BW373U86 in mice (Comer et al., 1993). Although acute treatment with 1.0 mg/kg BW373U86 did not produce tolerance to convulsions, daily treatment with 1.0 mg/kg BW373U86 for 1 week did produce tolerance. Consistent with these findings, studies with µ-agonists have also demonstrated greater magnitudes of tolerance after chronic treatment than after acute treatment (Young et al., 1991; Gerak and France, 1997).

In view of evidence in the present study for tolerance to the rate-decreasing effects of SNC80 in rhesus monkeys, it is important to recognize that the development and expression of tolerance may vary depending on parameters such as the type of agonist and the behavioral endpoint. For example, it has been reported that tolerance develops more readily to the antinociceptive effects of µ-agonists than to their respiratory depressant, constipating, or pupillary effects (Ling et al., 1989; Paronis and Woods, 1997; Brandt and France, 2000). In the present study, tolerance to the rate-decreasing effect of acute SNC80 was produced by doses of SNC80 (3.2-10.0 mg/kg) approximately 10- to 30-fold higher than the ED50 value (0.37 mg/kg), and this tolerance waned in less than a week. In contrast, significant tolerance to the convulsant effects of acutely administered BW373U86 in mice was produced by a dose approximately equal to the ED50 value, and pretreatment with a dose 10 times higher than the ED50 produced profound tolerance that lasted between 1 and 2 weeks (Comer et al., 1993). Procedural differences make it difficult to directly compare these studies, but these findings suggest that tolerance may develop more readily to the convulsant effects of BW373U86 in mice than to the rate-decreasing effect of SNC80 in rhesus monkeys. The relative importance of different parametric variables in determining tolerance to delta -agonists administered acutely requires further study. In particular, the interest in delta -agonists as potential analgesics (Brandt et al., 2001) will make it important to examine the degree to which tolerance may also develop to the antinociceptive effects of delta -agonists.

Pharmacological Specificity of SNC80-Induced Tolerance. In the present study, tolerance to 10.0 mg/kg SNC80 was attenuated by the DOR-selective antagonist naltrindole. In addition, both acute and chronic treatment with SNC80 produced cross-tolerance to the rate-decreasing effects of the other selective delta -agonists SNC162 and SNC243A. In contrast, cross-tolerance did not develop to the rate-decreasing effects of the µ-agonist morphine or the kappa -agonist U-50,488. These results confirm and extend previous studies demonstrating that SNC80 acts as a selective DOR agonist in rhesus monkeys (Negus et al., 1998; Brandt et al., 1999, 2001).

Similar to the present study, previous studies that used peptidic delta -agonists have demonstrated that tolerance is pharmacologically selective in that tolerance to delta -agonists confers little cross-tolerance to µ- or kappa -agonists (Iyengar et al., 1987; Suh and Tseng, 1990; Stevens and Yaksh, 1992). For example, chronic infusion of the delta -agonist [D-Ala2,D-Leu5]-enkephalin (DADLE) produced a 33-fold increase in the ED50 for DADLE but only a 1.3-fold increase in the ED50 for morphine (Stevens and Yaksh, 1992). Similarly, selective µ- and kappa -agonists can produce pharmacologically selective tolerance and cross-tolerance (Gmerek et al., 1987; Craft et al., 1989; Picker et al., 1991; Stevens and Yaksh, 1992; Brandt and France, 2000). For example, chronic infusion of the µ-agonist morphine produced a 55-fold increase in the ED50 for morphine but only a 2.7-fold increase in the ED50 for DADLE (Stevens and Yaksh, 1992). Taken together with the results of the present study, these findings demonstrate that studies of tolerance and cross-tolerance can be used to evaluate the pharmacological selectivity of DOR agonists.

Sensitivity to Antagonists during SNC80 Treatment. Acute administration of µ- or kappa -agonists can result in an increased potency for the rate-decreasing effects of opioid antagonists. For example, a single administration of µ-agonists produced a 100- to 250-fold increase in the potency for the rate-decreasing effects of naltrexone (Adams and Holtzman, 1990). Although not as robust, the administration of a single dose of a kappa -agonist also produced an increase (10-fold) in the potency of naltrexone (Adams and Holtzman, 1990). In addition, chronic treatment with µ-agonists can produce up to 5000-fold increases in antagonist potency (Adams and Holtzman, 1990; Picker et al., 1991; Picker and Yarbrough, 1991; Gerak and France, 1997). Increases in antagonist potency produced by agonist administration have been associated with dependence and withdrawal, because withdrawal-like behaviors are often observed at doses that decrease rates of responding (Adams and Holtzman, 1990; Brandt and France, 1998). In contrast to these µ- and kappa -agonist studies, acute or chronic SNC80 did not modify the potency of naltrexone under the current dosing conditions. Although small increases in the potency of naltrindole were observed in the current study, this increase did not appear to be related to dependence, because increases in the potency of naltrindole were subsequently observed in the absence of SNC80 treatment. Instead, these results suggest that sensitization to high doses of naltrindole occurred, an effect that has been observed with other opioid antagonists (e.g., naltrexone, France and Morse, 1989; quadazocine, Negus et al., 1993). Moreover, there were no behavioral signs that indicated a withdrawal syndrome at times when naltrexone has been shown to produce withdrawal effects in monkeys maintained on µ- and kappa -agonists (Gmerek et al., 1987; France and Gerak, 1994; Brandt and France, 1998). Together, these data suggest that delta -agonists might produce lower levels of dependence than µ- and kappa -agonists. These results also indicate that delta -agonists can produce tolerance with little or no evidence of dependence in rhesus monkeys. However, it should be noted that treatment with higher or more frequent doses of SNC80 could produce dependence.

    Footnotes

Accepted for publication August 2, 2001.

Received for publication May 17, 2001.

1 Current Address: Wyeth-Ayerst Research, Wyeth Neuroscience, CN-8000, Princeton, NJ 08543-8000. E-mail: brandtm{at}war.wyeth.com

This study was supported in part by Grants RO1-DA11460, P50-DA04059, T32-DA0752, and KO5-DA00101 from National Institute on Drug Abuse, National Institutes of Health. We also thank National Institute on Drug Abuse for partial support for the Laboratory of Medicinal Chemistry, National Institute of Diabetics and Digestive and Kidney Diseases and National Institutes of Health.

Address correspondence to: Steve Negus, Ph.D., Alcohol and Drug Abuse Research Center, Harvard Medical School, McLean Hospital, 115 Mill St., Belmont, MA 02178-9106. E-mail: negus{at}mclean.org

    Abbreviations

DPDPE, [D-Pen2,D-Pen5]-enkephalin; DOR, delta -opioid receptor; MOR, µ-opioid receptor; DADLE, [D-Ala2,D-Leu5]-enkephalin.

    References
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Abstract
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Materials and Methods
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References

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