Introduction

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For more than 20 years, LAAM, a synthetic derivative of d-methadone (Pohland et al., 1949), has been proposed as an alternate to methadone in maintenance therapy. In July 1993, LAAM was approved in the United States for use in opioid substitution therapy (Federal Register, 1993). Four decades ago, Fraser and Isbell (1952) characterized the pharmacological actions and assessed the abuse liability of LAAM. They found the effects of a single oral dose of LAAM to be morphine-like. Furthermore, 30 to 60 mg of LAAM, given orally, relieved abstinence in morphine-dependent subjects (400 mg/day). A single, 40 to 60-mg dose of LAAM orally substituted for morphine (60 mg q.i.d.) and suppressed the abstinence syndrome for up to 72 hr. Morphine-like effects of LAAM required 4 to 6 hr to emerge after subcutaneous or intravenous injection and 1.5 hr to appear after oral administration, suggesting the formation of active metabolites.

The initial preclinical pharmacological, metabolic and toxicological studies of LAAM and its metabolites included rodents, dogs and monkeys among the species tested (Archer, 1976). Metabolites of LAAM, particularly the N-demethylated compounds, nor-LAAM and dinor-LAAM, had more rapid onsets of action than LAAM, tended to be more potent than the parent compound (i.e., particularly nor-LAAM) and persisted in animals longer than the metabolites of methadone. For these reasons, the formation of nor metabolites was postulated to confer the long duration of action of LAAM (Archer, 1976; Wolven and Archer, 1976). Except for two analgesic assessments of nor-LAAM (Gruber and Babtisti, 1962; Houde et al., 1962), no other pharmacodynamic data exist for nor- and dinor-LAAM in humans; consequently, their activity profiles must be inferred from animal models. The chronic spinal dog is a validated model for assessing the agonistic actions and dependence-producing properties of morphine-like opioids (Martin and Jasinski, 1977). Using this model, studies were designed to compare the actions and relative potencies of nor-LAAM and dinor-LAAM to morphine, methadone and LAAM. Pharmacological profiles based on single-dose effects, suppression studies of morphine withdrawal and precipitated abstinence were used to define the opiate-like nature of nor-LAAM and dinor-LAAM.

	Materials and Methods

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All animal procedures were approved by the Institutional Animal Care and Use Committee of the NIDA Addiction Research Center (Lexington, KY) and were in accordance with the "Guide for the Care and Use of Laboratory Animals" of the National Institutes of Health.

Animals. The study included 12 beagle-type, T10 chronic spinal dogs ranging in age from 2 to 6 years and weighing between 7 and 12 kg. One group of six animals was used to evaluate acute opiate effects, and the second group of six came from a colony of morphine-dependent animals and were used to assess the suppression of opiate withdrawal. All dogs had been used in previous studies of opiate-related compounds but had different drug histories.

Physiological and behavioral measurements. The following autonomic, reflex and behavioral measures were acquired using methods that have been previously published (Martin et al., 1974, 1976; Vaupel et al., 1986). The dogs, which were trained to the laboratory setting, were positioned on their right sides and loosely restrained at the neck and abdomen. Vertical pupil diameter and lateral nictitating membrane width of the left eye were measured from photographs taken with a Polaroid close-up camera, and rectal body temperature was recorded continuously. Heart rate and respiration were counted by auscultation and visual observation, respectively. For these five autonomic measures, changes for a selected time period (e.g., 300 min) were based on AUCs: change in response = (AUC_treatment  AUC_{pretreatment baseline control})/minutes_AUC.

Single-dose studies and precipitated abstinence. Subjects were six nondependent chronic spinal dogs. Their past experience included use in opiate-related studies, including dose-ranging experiments for the present study, but their drug histories were not identical. In the present study, the acute, single-dose effects of the three methadols were compared with those of morphine and methadone in two series of crossover experiments in which all dogs received all treatments using the following design. In the first series, the effects of morphine (0.5 and 2.0 mg/kg), LAAM (0.25 and 1.0 mg/kg), nor-LAAM (0.05 and 0.2 mg/kg), dinor-LAAM (0.1 and 0.4 mg/kg), methadone (0.125 and 0.5 mg/kg) and a double-distilled water vehicle control (administered twice) were studied over a 12-week period using a randomized block design. The design incorporated six blocks, and each block was 2 weeks in length. Within each block, dogs received a pair of different drug treatments (one treatment per week). Treatment pairs were randomly assigned to dogs across the blocks to counterbalance for time. When a preliminary analysis indicated that higher doses of the methadols were needed to produce more complete dose-response curves, a second, 6-week series of experiments was added. The six treatments [LAAM (4.0 mg/kg), nor-LAAM (0.8 mg/kg), dinor-LAAM (1.6 mg/kg), morphine (0.125 and 2.0 mg/kg) and double distilled water] were tested using a 6 × 6 (dogs × doses) crossover design. In retrospect, a higher dose of methadone should have been included in the second series. However, in making a preliminary assessment of the data, we overestimated the efficacy of methadone. This error limited our data interpretation with respect to the efficacy and relative potency of methadone and its comparison with the LAAM compounds. An additional component to the second series of experiments was the use of a precipitated withdrawal paradigm to assess the development of acute physical dependence 5 hr after drug administration.

Suppression studies. Suppression of morphine abstinence was studied in a second group of six chronic spinal dogs who were physically dependent on morphine. An induction phase of 8 to 10 weeks was required to gradually increase daily subcutaneous and oral doses of morphine until animals were dependent on 125 mg/day morphine sulfate. Thereafter, the maintenance schedule consisted of 25 mg s.c. administered at 8:00 a.m. and 100 mg p.o. administered at 4:00 p.m. The dogs had been used in previous suppression and precipitation studies evaluating the agonist, partial agonist and antagonist effects of opiates.

Data analysis. For the single-dose studies, significance of drug effects was tested against the mean of the three vehicle control experiments using Dunnett's test. Relative potencies and confidence limits were calculated using standard ANOVA methods and Fieller's theorem for parallel line bioassays, which were modified for crossover data (Finney, 1978). Relative potency estimates and 95% confidence limits are presented only for valid bioassay ANOVAs, which were defined by a significant regression term, a nonsignificant deviations from parallelism (parallelism term) and a nonsignificant preparations term (no difference in the mean treatment effects). For some bioassays with a significant preparations term, potency estimates without confidence limits were presented for qualitative purposes. Geometric mean values were calculated for the purpose of summarizing the overall relative potency a drug but used only estimates from statistically valid bioassays; the geometric means have no associated confidence limits. The presence of acute physical dependence in the single-dose study and the suppression of withdrawal in dogs chronically dependent on morphine were determined by analyzing total abstinence or suppression scores using a two-way ANOVA (dogs × doses) and a Dunnett's test (one-tailed, P < .05).

Drugs. The drugs used were morphine sulfate and the hydrochloride salts of dl-methadone, LAAM, nor-LAAM and dinor-LAAM and were kindly provided by the National Institute on Drug Abuse (Rockville, MD). All doses are expressed in terms of the salts. Double-distilled water was the vehicle for all compounds.

	Results

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Single-dose study. The five opiate treatments had pronounced effects on nociception (see figs. 1, 2, 3) and temperature (see fig. 4), whereas changes in pupil diameter and nictitating membrane width were not as robust. Respiration and heart rate were not affected, but the P value for heart rate (.0655) approached the criterion for significance. Analyses to determine which measures were significantly affected in the single-dose study are summarized in table 1. A significant "between animals" term, indicating a difference in animal responsiveness, was associated with every measure (i.e., the comparison of mean responses, collapsed over all treatment conditions for each animal, indicated that some animals had relatively high sensitivity to the opiates and others were relatively less sensitive). Segregation of this significant source of variability emphasized the power of using a crossover design.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   Flexor reflex, low stimulus. Dose-effect curves comparing the antinociceptive effects of morphine, methadone, LAAM, nor-LAAM and dinor-LAAM over 5 hr on the flexor reflex evoked by the low-pressure stimulus toe pinch (4.5 p.s.i.). Each point represents the mean ± S.E.M. percent of maximum antinociception determined in six dogs. The horizontal line represents the mean water vehicle response, and the dashed line indicates the upper 95% confidence limit (CL). , Responses that differ significantly (P < .01) from the water control value.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Flexor reflex, high stimulus. Dose-effect curves comparing the antinociceptive effects of morphine, methadone, LAAM, nor-LAAM and dinor-LAAM over 5 hr on the flexor reflex evoked by the high-pressure stimulus toe pinch (18 p.s.i.). Each point represents the mean ± S.E.M. percent of maximum antinociception determined in six dogs. The horizontal line represents the mean water vehicle response, and the dashed line indicates the upper 95% confidence limit (CL). , Responses that differ significantly (P < .01) from the vehicle value.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Skin twitch reflex. Dose-effect curves comparing the antinociceptive effects of morphine, methadone, LAAM, nor-LAAM and dinor-LAAM over 5 hr on the skin twitch reflex elicited by a radiant heat stimulus. Each point represents the mean ± S.E.M. percent of maximum antinociception determined in six dogs. The horizontal line represents the mean water vehicle response, and the upper and lower dashed lines indicate the upper 95% confidence limits (CL). *P < .05 and P < .01, significant differences between vehicle control values and drug responses, respectively.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Temperature. Dose-effect curves comparing the hypothermic effects of morphine, methadone, LAAM, nor-LAAM and dinor-LAAM over 5 hr on rectal temperature. Each point represents the mean ± S.E.M. change in temperature determined in six dogs. The horizontal line represents the mean water vehicle response, and the dashed line indicates the lower 95% confidence limit (CL). , Responses that differ significantly (P < .01) from the vehicle control value.

View larger version (28K): [in this window] [in a new window]   Fig. 5.   Spinal cord and autonomic signs of precipitated withdrawal. Naltrexone, 1 mg/kg, was infused over 4 min at 300 min after dosing with 4 mg/kg LAAM, 0.8 mg/kg nor-LAAM, 1.6 mg/kg dinor-LAAM or the water vehicle. Emergence of stepping reflex movements, tachycardia and mydriasis represent effects contributing to significant precipitated abstinence scores (see fig. 6). Despite the absence of hyperthermic responses, which would be positive signs of opiate abstinence within the 30-min scoring period, the reversal of hypothermia by naltrexone is clearly evident. Points for each AUC are the mean values for six dogs. The dashed line parallel to the abscissa represents the mean base-line value, and the vertical dashed line indicates the administration of naltrexone. In the flexor reflex panel, only the reflex amplitude measured 5 min after naltrexone dosing is presented because the emergence of fragmentary and continuous stepping reflex movements, indicated by oscillating lines, precluded accurate measurement of the flexor reflex. The fraction adjacent to each oscillating line after naltrexone represents the proportion of animals developing fragmentary or continuous stepping reflex movements within the 30-min evaluation period.

Precipitated withdrawal studies. Five hours after the administration of LAAM, its metabolites and morphine, the injection of naltrexone elicited a statistically significant withdrawal syndrome, indicating the development of acute physical dependence (figs. 5 and 6). Quantitatively, the withdrawal syndromes for 4 mg/kg LAAM, 0.8 mg/kg nor-LAAM and 1.6 mg/kg dinor-LAAM were statistically equivalent to the syndrome obtained after both 0.125 and 2 mg/kg morphine, as determined by a two-tailed Dunnett's test. The most consistent signs typifying acute precipitated withdrawal were rhinorrhea, salivation, tremors, restlessness, whining, urination, fragmentary or continuous stepping reflex movements (fig. 5), mydriasis (fig. 5) and tachycardia (fig. 5). The effects of LAAM, nor-LAAM and dinor-LAAM on the flexor reflex (fig. 5) are presented as "% Control Amplitude" rather than "% Maximal Antinociception" to more clearly demonstrate reversal by naltrexone and emergence of the stepping reflex as a sign of precipitated withdrawal. Heart rate, although not significantly depressed by LAAM and its metabolites (table 1), is presented because rebound tachycardia after naltrexone was a primary contributor to the withdrawal scores. A low level of hyperthermia developed during withdrawal from morphine but not during withdrawal from LAAM, nor-LAAM or dinor-LAAM, although naltrexone almost reversed their hypothermic effects within the 30-min period for scoring withdrawal (fig. 5).

View larger version (41K): [in this window] [in a new window]   Fig. 6.   Precipitated withdrawal. The development of acute physical dependence was assessed in six dogs by administering a naltrexone challenge (1 mg/kg i.v.) 5 hr after drug administration. Precipitated abstinence scores were calculated for a 15-min period. Significance of withdrawal scores (*P < .05, P < .01) for each group was based on a comparison with the water vehicle (two-way ANOVA, post hoc one-tailed Dunnett's test).

Suppression studies. The metabolites nor-LAAM and dinor-LAAM and LAAM, as well as morphine and methadone, effectively suppressed withdrawal from morphine in 40-hr abstinent dogs [two-way ANOVA (dogs × doses); F(14,56) = 1.99; P < .05]. An apparent ceiling effect was reached in suppressing abstinence in dogs dependent on a daily dose of 125 mg of morphine by morphine, LAAM, nor-LAAM and dinor-LAAM (fig. 7). It could not be determined whether a ceiling effect was attained for methadone because a dose of >0.5 mg/kg was not tested. The relative potency relationships of these opioids in suppressing morphine withdrawal are presented in table 3. Nor-LAAM was approximately one order of magnitude more potent than LAAM or dinor-LAAM in suppressing the canine morphine abstinence syndrome.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Suppression studies. The ability of each compound to substitute for morphine was determined by suppressing the abstinence syndrome in morphine-dependent dogs. Dogs were maintained on a 125 mg/day total dose of morphine administered in two doses. A withdrawal syndrome was produced by withholding the morphine dosing for 40 hr. The 40-hr morphine-abstinent animals were given a substitute opioid to suppress withdrawal, and suppression scores were computed for the following 90 min. Each point or the vehicle control line represents the mean of five animals. The horizontal line represents the mean water vehicle response, and the dashed line indicates the lower 95% confidence limit (CL). The dashed segments of four dose-response curves represent ceiling effects. Therefore, the highest doses for each of these curves were not used to determine relative potency estimates shown in table 3. Significant (*P < .05, P < .01) suppression of withdrawal for each treatment was based on a comparison with the water vehicle (two-way ANOVA, post hoc one-tailed Dunnett's test).

	Discussion

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The acute physiological actions of LAAM, nor-LAAM and dinor-LAAM were identical to those of morphine and methadone in the dog. Previous studies in the chronic spinal dog model indicated that morphine produced miosis, bradycardia, hypothermia and depression of nociceptive reflexes (Martin et al., 1976; Martin and Eades, 1964). In the present study, the abilities of these five opiates to depress the flexor and skin twitch reflexes and to lower temperature were pronounced and statistically significant. The absence of bradycardia was not entirely unexpected because this action was not particularly marked in earlier studies. On the contrary, the variable nature of the miotic effects was somewhat puzzling, particularly the maximal effects observed at intermediate doses. The development of tolerance to pupillary constriction does not appear to be a viable explanation because the flexor reflex, skin twitch reflex and temperature responses were proportional to the size of the administered dose. With respect to the 2-mg/kg dose of morphine, the decreases in body temperature we measured were 0.5°C to 1.0°C smaller than those reported in an earlier study (Martin et al., 1976). It is possible that this group of animals had autonomic responses (i.e., miosis and hypothermia) that were less sensitive to opiates than in previous studies. In contrast to the autonomic responses, depression of nociceptive reflexes observed in the present study was comparable to that seen in previous studies (Gilbert and Martin, 1976; Martin et al., 1976).

Based on comparisons with previously established single-dose profiles, the actions of LAAM and its nor and dinor metabolites were representative of those produced by opiate agonists, characterized primarily as mu and typified by morphine and methadone, as opposed to kappa-type opiate agonists. The acute pharmacological actions of the kappa agonists l-ketocyclazocine and U50488H have been established in the dog (Vaupel and Cone, 1991). Although kappa agonists share certain actions, such as depression of nociceptive reflexes and pupillary constriction, with mu agonists, in contrast, they elevate body temperature, relax the nictitating membrane, induce sleep and produce an inability to attend to auditory and visual stimuli. Moreover, U50488H, which is a more selective kappa agonist than l-ketocyclazocine (Lahti et al., 1982; Tang and Collins, 1985), produces additional effects at the highest dose tested. Stimulatory signs, consisting of whining, the emergence of the stepping reflex and occasional myoclonic movements, are accompanied by nystagmus. The appearance of nystagmus, another high-dose effect of U50488H, is produced by the dissociative anesthetic phencyclidine and N-allylnormetazocine (Vaupel et al., 1986). Although mu and kappa opiate activity in the dog can be differentiated on the basis of selective antagonism by naltrexone (Vaupel et al., 1986; Vaupel and Cone, 1991), no attempt was made to demonstrate a selective antagonism of LAAM and its metabolites in this study. Nonetheless, the pharmacologies of LAAM, nor-LAAM and dinor-LAAM are typically mu- or morphine-like in the dog, and there is minimal evidence to suggest the presence of kappa opiate activity.

Initial studies in humans have indicated that LAAM has a delayed onset of action, regardless of whether it was administered intravenously or subcutaneously (Fraser and Isbell, 1952), and this observation subsequently has been attributed to the production of active metabolites. Both nor- and dinor-LAAM have been identified as urinary metabolites of LAAM in dogs, rats and monkeys (Archer, 1976; Mulé and Misra, 1993). However, there are a number of other identified metabolites (Archer, 1976). In contrast to its slow onset of action in humans after intravenous administration, LAAM has a rapid onset in the dog when given by the same route. Furthermore, the onset of action of LAAM in the dog is not distinguishable from that of the nor and dinor metabolites, morphine or methadone. The difference in the onset of action of LAAM in the dog compared with human subjects indicates that the dog is not an appropriate pharmacokinetic model for these opioid effects in humans. Nevertheless, the dog remains an appropriate model to evaluate the pharmacodynamic properties of LAAM and its metabolites. Based on acute drug effects, nor-LAAM and dinor-LAAM were similar in efficacy to LAAM, although they differed in potency. Estimates of the potency of nor-LAAM and dinor-LAAM relative to LAAM found the nor metabolite to be 0.5 to 1.5 orders of magnitude more potent than the parent compound, whereas dinor-LAAM appeared to be equally potent to LAAM.

Although we did not initiate primary addiction studies with LAAM and its metabolites in nondependent animals, the administration of naltrexone after acute single doses of LAAM, nor-LAAM and dinor-LAAM demonstrated the development of acute physical dependence within 5 hr of administration for all three compounds. Acute physical dependence to morphine also developed, and it tended to be dose dependent. Previous studies in the dog demonstrated signs of acute dependence after a 7-hr intravenous infusion of morphine (2 mg/kg/hr) (Martin and Eades, 1964) when nalorphine (20 mg/kg s.c.) was used to precipitate withdrawal. The precipitated abstinence syndromes in acutely and chronically physically dependent low spinal dogs can be differentiated by the shorter duration of action, less intense spinal cord signs and the absence of a hyperthermic response in the acute precipitated withdrawal syndrome. Some signs, such as mydriasis and tachycardia, are equal in magnitude. Because precipitated withdrawal is a more sensitive indicator of physical dependence than withdrawal abstinence (Martin and Jasinski, 1977), there is little reason to doubt that primary addiction studies with nor- and dinor-LAAM would have produced a prominent withdrawal syndrome.

In single-dose suppression tests conducted in morphine-dependent (morphine sulfate 3 mg/kg s.c. q.i.d.) rhesus monkeys that were abstinent for 14 to 15 hr, LAAM, nor-LAAM and dinor-LAAM completely suppressed withdrawal signs. The potencies of LAAM and morphine were estimated to be equal. However, the onset of LAAM was somewhat slower, and its duration of action was longer relative to morphine (Aceto et al., 1992). nor-LAAM was estimated to be 6 times as potent as morphine or LAAM. At comparable high doses, the duration of action of nor-LAAM was 2.5 hr longer than that of morphine (Aceto et al., 1991). Also, nor-LAAM did not precipitate withdrawal when administered to morphine-dependent monkeys, suggesting the absence of opiate antagonist-like effects. The potency of dinor-LAAM was estimated to be equivalent to those of morphine and LAAM in this species (Aceto et al., 1992; Jacobson, 1991, 1992). Based on ability to alleviate morphine withdrawal, the estimated potencies of LAAM, nor-LAAM and dinor-LAAM in the morphine-dependent rhesus monkey (1:6:1) (Aceto et al., 1992; Jacobson, 1992), respectively, were similar to the relative potencies calculated for the morphine-dependent chronic spinal dog (1:9:1) in the present study. These ratios also are consistent with the corresponding 1:9:2 potency ratio for the acute, morphine-like effects in the dog reported herein.

The importance of N-dealkylation metabolites in the pharmacology of opiates has been addressed by Fraser et al. (1978a, 1978b, 1980), who categorized their pharmacologies into the following four groups: (1) morphine-like activity without significant nonmorphine-like activity (e.g., nor-LAAM and dinor-LAAM); (2) limited morphine-like activity and conspicuous non-morphine-like activity (nor-meperidine); (3) little or no activity, like or unlike morphine (nor-methadone) and (4) morphine-like activity plus non-morphine-like activity (nor-morphine). Clearly, morphine-like activity of both nor- and dinor-LAAM was identified in several tests in the dog, and the occurrences of non-morphine-like actions of nor- and dinor-LAAM were judged to be minimal. These data support the inclusion of both metabolites in category 1 listed above.

LAAM appeared to be pharmacologically active in the dog based on the absence of any consistent differences in the onsets of action between the nor and dinor metabolites and LAAM after intravenous administration. This assumption is consistent with the ability of LAAM to inhibit contractions of the guinea pig ileum-myenteric plexus, an in vitro bioassay for determining opiate agonist activity (Nickander et al., 1974), but apparently differs in humans in whom there is a delayed onset for intravenous or oral LAAM (Fraser and Isbell, 1952), which is attributed to the enzymatic biotransformation to two active metabolites.

Among the primary active metabolites of LAAM, nor-LAAM is more potent than the dinor metabolite according to consistent data from primates and dogs. Both metabolites produce opiate-like effects when administered acutely, including acute physical dependence. In morphine-abstinent animals that exhibit physical dependence, both metabolites are capable of fully suppressing the opiate withdrawal syndrome. Historically, drugs demonstrated to have morphine-like properties in animals have been found to be morphine-like in humans (Martin and Jasinski, 1977). Thus, it is reasonable to conclude that nor-LAAM and dinor-LAAM will have the properties of narcotic analgesics in humans. In the dog, the nor and dinor metabolites retained full efficacy compared with the parent LAAM using the intravenous route of administration. Active metabolites that possess full morphine-like pharmacological activity represent one factor contributing to the prolonged action of LAAM, distinguishing it from methadone, whose metabolites lack pharmacological activity (Pohland et al., 1971). The reported half-lives in humans for LAAM and nor-LAAM (2.6 and 2.0 days, respectively) are relatively long, whereas that of dinor-LAAM is even longer (4.0 days) (Henderson et al., 1977; Kaiko and Inturrisi, 1975; Marion, 1995), permitting less frequent dosing than required for methadone. Other processes likely contributing to the prolonged action of LAAM and its metabolites are binding to tissue proteins and enterohepatic circulation (Henderson et al., 1977). The newly available LAAM substitution therapy takes advantage of these pharmacokinetic and pharmacodynamic properties and allows patients to visit treatment sites every other day or three times a week compared with the FDA regulations for methadone, which require at least six visits per week during the first 3 months of treatment. Because LAAM and its metabolites are full agonists, one would anticipate additive or synergistic interactions to develop with other opiates. The ability of the opiate antagonist naltrexone to precipitate acute withdrawal suggests that under conditions in which patients are maintained on long-term, chronic LAAM treatment, the administration of an opiate having antagonist or partial agonist properties (mixed agonists/antagonists) could precipitate opiate withdrawal. Indeed, the potential of these effects have been carefully noted in guidelines for LAAM therapy (Marion, 1995).