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.)
 |
Introduction |
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 
-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
-opioids has been examined less extensively, in part because the
only selective -agonists available for study until recently were
peptidic compounds with poor bioavailability.
Studies with these peptidic -agonists demonstrated that tolerance
may develop under some conditions. For example, in vitro studies
indicate that peptidic -agonists such as
[D-Pen2,D-Pen5]-enkephalin
(DPDPE) can rapidly desensitize and down-regulate -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 -agonists did not confer cross-tolerance to µ- and
-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 -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 -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 -agonists continues in part because of
their potential for the treatment of pain. We and others have reported
that SNC80 and other -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 -opioids have not been adequately
characterized, and these issues have potential importance for the
continued therapeutic development of -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-, -, and -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 -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.
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Materials and Methods |
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 -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
-opioid agonist SNC162 (a piperazinyl benzamide structurally similar
to SNC80), the µ-opioid agonist morphine, or the -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 -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 -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 -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.
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Results |
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;  , 3.2 mg/kg SNC80;
 , 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.
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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; , 10.0 mg/kg SNC80 (5 h); , 10.0 mg/kg SNC80 (1 day);
 , 10.0 mg/kg SNC80 (3 day); , 10.0 mg/kg SNC80 (7 day).
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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 -, µ-, and -Opioids after Acute SNC80
Treatment.
Figure 3 shows the
effects of the DOR-selective agonist SNC162, the MOR-selective agonist
morphine, and the -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; ,
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
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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; , 1.0 mg/kg SNC80
daily; , 3.2 mg/kg SNC80 daily; , 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
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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; 10.0 mg/kg SNC80 daily; , post-SNC80,
week 1; , post-SNC80, week 2; , post-SNC80, week 5.
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Effects of -, µ-, and -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; , chronic; ,
postchronic.
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View this table:
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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
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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; , chronic; , postchronic.
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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 -opioid withdrawal. Moreover, there was no
evidence of any other behavioral signs that might be specific to
-opioid withdrawal (data not shown).
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Discussion |
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 -selective antagonist naltrindole to attenuate
SNC80-induced tolerance, 2) the development of cross-tolerance to other
-selective agonists, and 3) the lack of cross-tolerance to µ- or
-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 -agonist in rhesus
monkeys; however, other studies have shown that tolerance can develop
to -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 -agonist. Similarly,
previous in vitro studies have demonstrated that rapid desensitization
can occur after the administration of -selective agonists. For
example, in C6 glioma cells stably expressing the rat DOR, cells
treated with DPDPE or SNC80 showed a decreased agonist-stimulated
GTP
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 -agonist
administration, and this desensitization may contribute to the rapid
development of tolerance with -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 -agonists administered acutely requires further study. In particular, the interest in -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 -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 -agonists SNC162 and
SNC243A. In contrast, cross-tolerance did not develop to the
rate-decreasing effects of the µ-agonist morphine or the -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
-agonists have demonstrated that tolerance is pharmacologically selective in that tolerance to -agonists confers little
cross-tolerance to µ- or -agonists (Iyengar et al., 1987; Suh and
Tseng, 1990; Stevens and Yaksh, 1992). For example, chronic infusion of
the -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 -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 -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 -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 -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 -agonists (Gmerek et al., 1987; France and Gerak, 1994; Brandt
and France, 1998). Together, these data suggest that -agonists might
produce lower levels of dependence than µ- and -agonists. These
results also indicate that -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.
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.
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-Opioid Agonist
SNC80 in Rhesus Monkeys Responding under a Schedule of Food
Presentation
Top
Abstract
Introduction
-acetylmethadol (LAAM) in rhesus monkeys: tolerance and cross-tolerance to the antinociceptive, ventilatory and rate-decreasing effects of opioids.
J Pharmacol Exp Ther
294:
166-178.
-funaltrexamine in normal and morphine-dependent rhesus monkeys: observational studies.
J Pharmacol Exp Ther
235:
296-301
