Abstract
The effects of naltrexone on ventilation were examined in three rhesus monkeys maintained on 3.2 mg/kg/day morphine. Before the onset of the daily morphine-dosing regimen, naltrexone had only modest effects on ventilation; a dose of 32 mg/kg increased ventilatory rate in the presence of normal air to 36 ± 1 breaths/min, from a baseline rate of 25 ± 1 breaths/min. Naltrexone did not affect other measures of ventilation in the presence of normal air or 5% CO2. Subsequent to the onset of the daily morphine injection regimen, naltrexone dose-dependently increased ventilatory rate at doses 4 orders of magnitude lower (0.001–0.01 mg/kg) than those effective in nondependent monkeys. A dose of 0.01 mg/kg naltrexone in morphine-maintained monkeys increased ventilatory rate in the presence of normal air to 52 ± 4 breaths/min. Naltrexone also dose-dependently increased ventilatory rate in the presence of 3% and 5% CO2; tidal volume was not affected by naltrexone administration. Doubling the maintenance dose of morphine to 6.4 mg/kg/day further increased the ventilatory effects of naltrexone. Withholding the maintenance dose of morphine also increased ventilatory rate without affecting tidal volumes, in a manner similar to that seen after naltrexone administration. These results are consistent with the view that changes in ventilation can be used to measure precipitated and abstinence-associated opioid withdrawal in monkeys.
Chronic administration of opioid agonists produces a physiological dependence revealed by the appearance of withdrawal signs when the maintenance dose of the agonist is withheld (abstinence-associated withdrawal) or when an opioid antagonist is administered (precipitated withdrawal). The signs of precipitated and abstinence-associated withdrawal are qualitatively similar and may be behavioral as well as physiological. In rodents, opioid dependence-related withdrawal has been characterized by use of behavioral measures, such as irritability and exploratory behaviors, as well as more objective physiological measures, such as defecation and hypothermia (Bläsig et al., 1973; Mucha et al., 1979). In monkeys, studies of opioid dependence-related withdrawal have relied primarily on subjective observations of behaviors such as drowsiness or restlessness (Seevers, 1936; Acetoet al., 1977, 1989). Physiological effects of morphine withdrawal in rhesus monkeys have received much less attention, although opioid withdrawal does produce hypothermia, hyperpnea and tachycardia in monkeys (Holtzman and Villarreal, 1969; Goldberg, 1976). More recently, naltrexone-precipitated withdrawal in monkeys receiving daily morphine also has been studied by drug discrimination procedures, in which either cessation of morphine injections or naltrexone administration occasions responding on a lever associated with naltrexone injection (France and Woods, 1989). Responding on the naltrexone-lever is attenuated by morphine and related agonists, which further suggests that the naltrexone discriminative stimulus is associated with opioid withdrawal and, therefore, dependence.
Many symptoms of withdrawal from morphine-like opioids appear to be opposite to the acute effects of agonist administration. For example, morphine withdrawal is characterized by mydriasis, hypothermia and reports of dysphoria, whereas acute morphine administration is associated with miosis, hyperthermia and reports of euphoria (Heishmanet al., 1989; Preston et al., 1988). The ventilatory effects of acute morphine administration versusmorphine withdrawal differ analogously; that is, ventilation is depressed after acute administration of morphine, whereas increased ventilation has been reported during opioid withdrawal (Martin et al., 1968). Both morphine abstinence and antagonist administration have been reported to produce hyperpnea in morphine-dependent subjects (Martin and Jasinski, 1969; Goldberg, 1976; Heishman et al., 1989). However, withdrawal-related changes in ventilation have not been fully characterized, and parametric relationships between abstinence and ventilation remain to be clarified.
The experiments presented here were designed to examine ventilatory effects of abstinence-associated and antagonist-precipitated withdrawal in rhesus monkeys receiving daily injections of morphine. In these experiments, the effects of the opioid antagonist, naltrexone, as well as the effects of withholding the maintenance dose of morphine for differing periods of time were assessed. These studies were completed in concert with other studies of tolerance to the ventilatory-depressant effects of mu opioid agonists (Paronis and Woods, 1997). The results of the present experiments demonstrate that ventilatory frequency increases during opioid withdrawal in a predictable manner that is consistent with the view that daily single injections of morphine produce opioid dependence. The orderliness of these effects suggests that similar procedures may be useful in future evaluations of opioid withdrawal in primates.
Methods
Subjects
Subjects were two adult male (909B and S67) and one adult female (F2) rhesus monkeys. The weights of the subjects remained relatively stable throughout the study and were 6.5 kg (F2), 7.5 kg (S67) and 9 kg (909B). Subjects 909B and F2 had been used previously in experiments involving opioids. Monkeys were housed singly in a temperature-controlled colony room with a 12-hr light/dark cycle (lights on at 6:00 a.m.). Water was available ad libitum, and the monkeys were fed approximately 30 biscuits (Purina Monkey Chow) daily, supplemented twice weekly with fresh fruit.
Apparatus
The apparatus used was similar to that described by Howellet al. (1988). Subjects were seated in a standard primate restraint chair enclosed within a sound-attenuating chamber. A Plexiglas helmet that was placed over the head of the subject served as a pressure-displacement plethysmograph. Customized Plexiglas plates and rubber dams were used to provide an air-tight seal around the monkey’s neck. A continuous flow, 10 liters/min, of either air or a mixture of 1%, 3% or 5% CO2 in air (hereafter referred to only by the CO2 concentration) was introduced through a port at the front of the helmet, and was extracted at the same rate through a port in the back of the helmet. Changes in flow within the plethysmograph were measured by a pressure transducer connected to a polygraph (model 7E, Grass Instrument Co., Quincy, MA) and computer (IBM/PCjr). Pressure displacement was converted to a volume measure by a polygraph integrator (model 7P122E, Grass Instrument Co.). Minute volume (VE) was determined by integration of changes in flow through the plethysmograph; frequency of ventilation (f) was directly determined; and tidal volume (VT) was calculated as the quotient VT = VE/f.
Experimental sessions generally consisted of four to six consecutive 30-min cycles, each comprising a 23-min exposure to air followed by a 7-min exposure to 5% CO2. Data from the first air/CO2 cycle were used for session-control values. Cumulative dosing procedures similar to those described by Howellet al. (1988) were used. At the start of each test cycle, graded doses of drug were administered i.m. so that the total dose increased by 0.25 or 0.5 log unit increments throughout the session. When ventilatory responses to different concentrations of CO2 were measured, a single dosing procedure was used. In these instances, experimental sessions consisted of two 63-min cycles in which 7-min exposures to increasing concentrations of CO2 alternated with 14-min exposures to air. Animals received an i.m. injection of drug after the end of the first cycle. Data obtained from the first cycle served as session-control values.
Experimental Design
Drug tests were conducted during four phases, baseline, single-dosing, double-dosing and abstinence, during which the subjects were repeatedly exposed to morphine and other mu opioid agonists, the opioid antagonist, naltrexone, and several nonopioid drugs. Experimental sessions were conducted at least twice a week to ensure stable patterns of ventilation, and at least 2 days intervened between drug tests. Five to seven days intervened between tests when the daily dosing schedule was interrupted to assess abstinence-associated withdrawal effects, or when the monkeys received large doses of drugs. The order of the drug tests was randomized between subjects within each phase of the study to minimize the influence of duration of exposure to the maintenance dose of morphine on the responses to the test drug.
Baseline phase.
During the baseline phase, the ventilatory effects of morphine and naltrexone were determined in all monkeys. This phase of the study lasted 14 weeks in S67 and 16 weeks in 909B and F2. Data obtained during this phase are referred to throughout this paper as baseline values, and should be distinguished from the session-control values obtained at the start of individual test sessions.
Single-dosing phase.
During the single-dosing phase, the monkeys received injections of 3.2 mg/kg morphine every morning, either in the test chamber or, on days when they were not tested, in the home cage. Except where noted, tests occurred 24 hr after injection of the maintenance dose of morphine. Only the ventilatory effects of morphine were studied during the first 4 weeks of the single-dosing phase. Subsequently, the ventilatory effects of a range of opioid drugs, including naltrexone, morphine, nalbuphine, butorphanol, ethylketazocine and fentanyl, and several nonopioid drugs (midazolam, flumazenil and β-carboline-3-carboxylic acid; reported in Paroniset al., 1994) were examined. The order in which drugs were studied was randomized among subjects. When naltrexone or a nonopioid drug was studied, the daily morphine dose was administered immediately after the test session. The effects of abstinence-associated withdrawal were assessed by withholding the maintenance dose of morphine for either 48 or 72 hr. This phase of the study lasted 40 weeks in S67 and 57 weeks in 909B and F2.
Double-dosing phase.
During the double-dosing phase, monkeys received two injections of 3.2 mg/kg of morphine daily. The injections were spaced by 12 ± 2 hr, and usually were given at 9:00a.m. and 9:00 p.m. This phase of the study lasted 12 weeks in S67 and 17 weeks in 909B and F2. During this time the ventilatory effects of naltrexone and morphine were examined in all monkeys; except where noted, tests occurred 24 hr after morphine.
Abstinence phase.
During the abstinence phase, daily morphine injections were discontinued and the monkeys were drug-free for 4 weeks. Experiments continued throughout this time to assess any effects of abstinence-associated withdrawal. After at least 28 days, the ventilatory effects of morphine and naltrexone were redetermined in all monkeys.
Data Analysis
Data from the last 3 min of each exposure to air or CO2 were used for analysis of drug effects on ventilation. The ventilatory stimulant effects of 3% and 5% CO2 were calculated as a ventilatory ratio (VECO2/VEAir). Effects of naltrexone and abstinence-associated withdrawal on ventilation were subjected to repeated measures one-way ANOVA, followed by Dunnett’s multiple comparison test. Significance was set at P < .05.
Drugs
Morphine sulfate (Mallinckrodt, Inc., St. Louis, MO) and naltrexone HCl (Endo Laboratories, Inc., Garden City, NY) were dissolved in sterile water and injected i.m. in a volume of 0.1 to1.0 ml. Drug doses are expressed as the weight of the salt.
Results
Baseline.
Ventilatory responses to increasing concentrations of CO2 were obtained before and after vehicle injections on 3 consecutive days. Vehicle injections did not systematically alter ventilation, and data collected before and after the vehicle injections were combined to obtain baseline response values given in table1. Exposure to CO2 increased all measures of ventilation in a concentration-dependent manner in each monkey, and increases in ventilatory rate, tidal volume and minute volume achieved statistical significance during exposure to 3% and 5% CO2(table 1). The responses to CO2 were similar for individual monkeys; the mean ventilatory ratio of 3% CO2 compared with air was 1.82 ± 0.07 and the mean ventilatory ratio in the presence 5% CO2 compared with air was 3.13 ± 0.04.
Cumulative administration of morphine (0.32–32.0 mg/kg) dose-dependently decreased all measures of ventilation in both air and 5% CO2 (data presented in companion paper, Paronis and Woods, 1997). A single injection of 3.2 mg/kg morphine decreased minute volume in the presence of normal air approximately 25% and decreased minute volume in air mixed with CO2 approximately 55%; these effects remained stable throughout the study. In contrast to the ventilation-decreasing effects of morphine, naltrexone had modest ventilation-increasing effects during the baseline phase. Doses of naltrexone lower than 10 mg/kg had no effect on ventilation in the presence of normal air; however, the highest dose tested, 32 mg/kg, increased breathing frequency to 144% of the session-control value (fig. 1). Naltrexone (0.032–32.0 mg/kg) had no effect on ventilation in the presence of 5% CO2.
Precipitated withdrawal.
Naltrexone, administered 24 hr after morphine during the single-dosing phase, increased breathing frequency in an orderly manner. The effects of naltrexone during the single-dosing phase were markedly different in both potency and magnitude compared with its effects during the baseline phase (fig.2). Ventilatory frequency was significantly increased by doses of naltrexone as low as 0.0032 to 0.01 mg/kg, reflecting an increased potency of approximately 4 orders of magnitude. In addition to increased potency, the magnitude of the responses to naltrexone was greater during the single-dosing phase than during the baseline phase. For example, under baseline conditions, 32 mg/kg naltrexone increased respiratory rate in air 44%, from 25 ± 1 to 36 ± 1 breaths/min; in monkeys receiving a single daily injection of 3.2 mg/kg morphine, 0.01 mg/kg naltrexone increased ventilatory frequency in air 62%, from 32 ± 4 to 52 ± 4 breaths/min (fig. 2). Naltrexone had no effect on tidal volumes, thus increases in minute volumes were consequential to the increases in ventilatory rate.
The effects of naltrexone on ventilation in the presence of 3% and 5% CO2 were similar to its effects during exposure to normal air. Naltrexone produced increases in breathing frequency at each concentration of CO2 (fig. 3) but did not alter tidal volume. Because ventilation was increased in the presence of both air and CO2, ventilatory ratios were not affected by naltrexone administration.
There were no appreciable differences between the effects of naltrexone administered as single doses (see fig.3) compared with the same doses given cumulatively at 24 hr after morphine (fig. 4A). Time-dependent changes in the position of the naltrexone dose-response curves were observed at 3, 12 and 24 hr after morphine (fig. 4A). Naltrexone was most potent 24 hr after morphine, and was least potent 3 hr after morphine. Although session-control values at the start of the 3-hr test were lower than at the start of the 24-hr test (reflecting ongoing effects of morphine at 3 hr) 0.01 mg/kg naltrexone nevertheless increased ventilatory frequency above baseline values obtained prior to the onset of the daily dosing regimen. Doses of naltrexone lower than 0.01 mg/kg did not produce significant effects 3 hr after morphine. The position of the naltrexone dose-response curve obtained 12 hr after morphine was intermediate to the responses to naltrexone at 3 and 24 hr after morphine.
The effects of naltrexone at 12 and 24 hr after morphine were increased during the double-dosing phase (figs. 2 and 4B). The effects of naltrexone (0.001–0.01 mg/kg) at 3 hr after morphine were virtually identical during the single- and double-dosing phases, with 0.01 mg/kg naltrexone increasing ventilatory frequency to 46 ± 5 and 44 ± 7 breaths/min during the single- and double-dosing phase, respectively. The naltrexone dose-response curve completed at 3 hr after morphine was extended during the double-dosing phase to include 0.0178 mg/kg naltrexone; this dose produced yet greater increases in ventilatory frequency, the mean rate of ventilation for the three monkeys under these conditions was 79 ± 4 breaths/min (fig. 4B).
The naltrexone dose-response curve was redetermined 4 to 6 weeks after cessation of the daily morphine injections. Naltrexone (0.003–1.0 mg/kg) had no effect on ventilation after terminating the morphine-dosing regimen (fig. 2).
Abstinence-associated withdrawal.
The effects of abstinence-associated withdrawal were assessed during the single-dosing phase by withholding the maintenance dose of morphine for up to 72 hr. Later time points were not tested to avoid further disruption of the chronic dosing regimen. During the double-dosing phase, the maintenance dose of morphine was withheld for up to 48 hr; and, in addition, ventilation was measured during a 4-week period after cessation of the daily morphine treatment. Similar to the effects of naltrexone-precipitated withdrawal, abstinence-associated withdrawal was accompanied by an increase in ventilatory frequency, whereas measures of minute volume and tidal volume were not affected (fig.5). The ventilatory effects of abstinence-associated withdrawal were relatively mild compared with the effects of naltrexone-precipitated withdrawal and were statistically significant only during the double-dosing phase. A peak effect, 47 ± 7 breaths/min, was seen at 48 hr after the last morphine injection. Termination of daily morphine injections did not result in significant changes to the ventilatory responses to 3% CO2 and 5% CO2; this is reflected in the fairly stable ventilatory ratios obtained during the abstinence phase (fig. 6). Although ventilatory measures were obtained for 28 days, recovery appeared complete in all monkeys by 5 to 7 days.
Discussion
Before the onset of the daily morphine dosing regimen, doses of naltrexone up to 32 mg/kg had only modest effects on ventilatory rate in rhesus monkeys. In morphine-maintained rhesus monkeys, however, ventilatory rate was markedly increased by low doses, 0.0032 to 0.01 mg/kg, of naltrexone (precipitated withdrawal) or by discontinuation of morphine treatment (abstinence-associated withdrawal). The ventilatory effects of both naltrexone and morphine abstinence were enhanced when the daily maintenance dose of morphine was increased. Moreover, after cessation of all daily dosing with morphine, baseline ventilatory values and the original effects of naltrexone were recovered. The general similarity in the effects of naltrexone injections and discontinuation of morphine maintenance in the present experiments are in accord with other comparisons of antagonist-precipitated and spontaneous abstinence (Wikler et al., 1953). For example, the administration of nalorphine to human subjects receiving 60 to 240 mg/day morphine produced the same constellation of effects,e.g., tachypnea, nausea and yawning, as were reported after abrupt cessation of the morphine treatment. These findings are consistent with the view that during the chronic dosing phases of the present study, the monkeys were dependent on morphine, and the naltrexone- and abstinence-induced increases in ventilatory rate reflect states of opioid withdrawal.
Previous studies that have measured ventilation during antagonist-precipitated opioid withdrawal have produced mixed results. For example, patients enrolled in methadone treatment programs, and presumably opioid dependent, showed no changes in ventilation after injections of 0.2 mg/kg naloxone (Preston et al., 1988), whereas an earlier study reported that nalorphine increased ventilatory rate in humans dependent on morphine, methadone or heroin (Wikleret al., 1953). Similarly, in an acute opioid-dependence procedure in humans, naloxone administered 6 hr after morphine dose-dependently increased ventilatory rate, but only when specific time and dose conditions were satisfied (Heishman et al., 1989, 1990; Kirby et al., 1990). Results from animal studies have been more consistent, and opioid antagonist-induced increases in respiration have been reported after either continuous or repeated agonist administration in rats, dogs and monkeys (Baraban et al., 1993; Gilbert and Martin, 1976; Goldberg, 1976). For example, injections of 0.2 mg/kg nalorphine in rhesus monkeys maximally dependent on morphine (8–12 mg/kg/day) increased respiration to 40 to 60 breaths/min (Goldberg, 1976). The results of the present experiments are in agreement with and extend previous findings, demonstrating that naltrexone will increase ventilation in morphine-dependent monkeys in a manner that is dependent on both the acutely administered naltrexone dose and the maintenance dose of morphine.
The enhanced potency of naltrexone in increasing ventilatory rate in morphine-maintained monkeys is in general agreement with reports of other behavioral effects of antagonists in opioid-dependent subjects, compared with effects in nondependent subjects. For example, in rats responding under a schedule of food presentation, the dose-response curve of naltrexone was displaced 5000-fold to the left after implantation of morphine-filled osmotic minipumps (Adams and Holtzman, 1990). Similarly, both naloxone and nalorphine were approximately 100-fold more potent in disrupting fixed-ratio performance under a schedule of food presentation in etonitazene-dependent rhesus monkeys than in nondependent monkeys (Tang, 1981). The naltrexone-induced increases in ventilation in the present study demonstrate that the potency with which an opioid antagonist exerts unconditioned physiological effects is also enhanced during a period of morphine maintenance.
Previous investigations have reported that exposure to low doses of opioid antagonists during periods of morphine maintenance may sensitize monkeys to behavioral and autonomic effects of the antagonist in the absence of agonist treatment (Goldberg and Schuster, 1970; Goldberg, 1976). Moreover, Warren and Morse (1989) demonstrated that an enduring sensitization to the effects of naltrexone on food-maintained behavior can develop in nondependent monkeys simply as a result of repeated exposure to the antagonist. In the latter experiments, the enhanced potency of naltrexone was retained for several months, dissipating only when the naltrexone was administered under conditions in which behavior was not maintained by food delivery. It is likely that such sensitization to naltrexone represents, in part, a conditioned response to its initial effects rather than pharmacodynamic changes in the effects of naltrexone (Bergman and Schuster, 1985; Warren and Morse, 1989; Schindler et al., 1990). In the present experiment, exposure to low doses of naltrexone also might have elicited interoceptive cues that modified the responses to subsequent naltrexone injections. However, similar responses to naltrexone were obtained using either cumulative or single-dosing procedures. Moreover, the ventilatory effects of naltrexone were apparent only during chronic morphine treatment, which suggests that the enhanced potency of naltrexone primarily reflected alterations in its pharmacodynamic actions rather than conditioned effects of repeated exposure.
Ventilatory measures were transiently increased after cessation of the daily morphine administration, but recovered to predependent baseline values within 1 week of terminating the daily dosing regimen. The duration of morphine withdrawal as measured by changes in ventilation agree with the results of previous studies of spontaneous opioid withdrawal in rhesus monkeys. In previous investigations in monkeys, chronic morphine regimens that lasted for months were followed by abstinence syndromes that persisted for only 4 to 8 days (Holtzman and Villarreal, 1971, 1973; Bergman and Schuster, 1985). Similarly, a study of opioid withdrawal in humans suggested that signs of abstinence first emerge 6 to 8 hours after cessation of the agonist treatment, peak within 72 hr and then gradually subside over 7 to 10 days (Kolb and Himmelsbach, 1938). In other studies, however, the withdrawal syndrome in humans has been reported to last for several months (Martin et al., 1968; Martin and Jasinski, 1969). Furthermore, in the latter study, the most pronounced respiratory effect of the withdrawal syndrome was an immediate, dramatic, hypersensitivity to CO2 that lasted for several weeks and was then followed by a prolonged hyposensitivity to CO2 which persisted for an additional 23 weeks. These findings are in marked contrast to the present results, showing no change in sensitivity to CO2during either abstinence-associated or antagonist-precipitated withdrawal in monkeys. In addition to possible species differences, it is possible that the contrasting results of the studies by Martin and his colleagues and the present experiments are related to different morphine dosing schedules or the different methods used to assess ventilatory responses.
Overall, the ventilatory effects of naltrexone appear to provide sensitive, quantitative measures of precipitated opioid withdrawal in morphine-maintained monkeys. The magnitude of the ventilatory effects of precipitated withdrawal vary dose-dependently with the dose of the antagonist as well as the maintenance dose of the agonist. Hence, ventilatory measures may be useful in future studies addressing questions of opioid withdrawal in a primate species.
The appearance of withdrawal signs reveals a physiological dependence on the chronically administered drug, and the development of dependence on a drug is frequently accompanied by tolerance to the acute effects of the same drug. As described in a companion paper (Paronis and Woods, 1997) the morphine-dosing regimen used in this study produced tolerance to the antinociceptive effects of morphine; however, no changes were seen in the ventilatory depressant effects of morphine. Taken together, the results presented here and in the companion paper suggest that opioid tolerance and dependence may be dissociable effects of chronic morphine exposure in rhesus monkeys.
Acknowledgments
The authors thank J. Bergman and W.H. Morse for helpful comments on the manuscript and W.Z. Wu for technical assistance.
Footnotes
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Send reprint requests to: Carol A. Paronis, Harvard Medical School, ADARC-McLean Hospital, 115 Mill St., Belmont, MA 02178-9108.
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↵1 This work was supported by USPHS grants DA00254, DA 07268 and DA 05653 from NIDA.
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↵2 Preliminary results were presented at the 57th annual meeting of the College on Problems of Drug Dependence, Scottsdale, AZ, 1995.
- Abbreviations:
- ANOVA
- analysis of variance
- f
- breathing frequency
- VT
- tidal volume
- VE
- minute volume
- Received October 16, 1996.
- Accepted March 31, 1997.
- The American Society for Pharmacology and Experimental Therapeutics