Introduction

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Drug discrimination has become a standard tool in the experimental laboratory for characterizing physiological and behavioral effects and neuropharmacological actions of drugs (Stolerman et al., 1995). The value of this procedure largely stems from its pharmacological specificity. That is, when animals trained to discriminate a drug from saline (or no drug) are tested with a variety of other drugs, they tend to emit drug-appropriate responses only in the presence of the training drug and other pharmacologically similar drugs and to emit an alternate response in the presence of saline and pharmacologically dissimilar drugs. The pharmacological specificity of drug discrimination responding has been extensively documented in animal studies (Young, 1991).

The pharmacological specificity of drug discrimination responding has been tested in humans, although to a lesser degree than in nonhumans (Kamien et al., 1993). For example, in stimulant abusers trained to discriminate d-amphetamine, 30 mg, and placebo, the stimulants d-amphetamine and methylphenidate fully substituted, and the sedative diazepam and opiate hydromorphone did not substitute, for d-amphetamine (Heishman and Henningfield, 1991; Lamb and Henningfield, 1994). Tests in normal volunteers trained to discriminate diazepam, 10 mg, from placebo have also been consistent with pharmacological specificity: diazepam, triazolam, and lorazepam fully substituted, pentobarbital and buspirone partially substituted, and d-amphetamine and tripelenamine did not substitute for diazepam (Johanson, 1991a,b). Thus, overall, there is cross generalization among stimulants, there is cross generalization among sedatives, and humans discriminate stimulants from sedatives, just as has been shown in the animal laboratory (Young, 1991).

Pharmacological specificity can extend to receptor subclasses. The fact that laboratory animals can discriminate among agonists acting at the mu and kappa opioid receptors (e.g., Herling and Woods, 1981; Picker and Cook, 1997) has made drug discrimination a particularly useful tool for studying opioid pharmacology. Conversely, receptor theory has been useful in developing a basis for interpreting opioid drug discrimination results (Woods et al., 1988). Our laboratory has conducted a series of studies investigating the discriminative stimulus effects of a group of four agonist-antagonist opioids under a variety of training conditions in experienced opioid abusers (Preston et al., 1989, 1992; Preston and Bigelow, 1990, 1994; Jones et al., 1999). Butorphanol, pentazocine, nalbuphine, and buprenorphine have varying degrees of agonist and antagonist activity at the mu and kappa opioid receptors (Reisine and Pasternak, 1996). Under some training conditions these drugs were discriminated as similar to hydromorphone, whereas under other training conditions they were not discriminated as hydromorphone-like, as illustrated in Fig. 1. For example, in volunteers trained to discriminate i.m. hydromorphone versus saline (top panel), butorphanol, pentazocine, nalbuphine, and buprenorphine each fully substituted (i.e., produced 80% of responding by pressing the drug-appropriate key) for the hydromorphone training condition (Preston et al., 1992). However, when tested in volunteers trained to discriminate among hydromorphone, saline, and a third choice of either pentazocine (middle panel) or butorphanol (lower panel), the agonist-agonists rarely substituted for hydromorphone (Preston et al., 1989; Preston and Bigelow, 1994). We have concluded from these studies that the outcome of human drug discrimination studies is dependent on the training conditions of the study and that the three-choice procedure is more useful for differentiating among drugs with overlapping discriminative stimuli than the two-choice, drug versus placebo, procedure.

View larger version (38K): [in this window] [in a new window]   Fig. 1.   Dose-response curves of percentage of drug-appropriate responding on the operant response discrimination measure, from three drug discrimination studies with different training conditions in three previously published studies, as shown [top panel (Preston et al., 1992); middle panel (Preston et al., 1989); bottom panel (Preston and Bigelow, 1994)]. Data are the mean across two presentations (at 60- and 80-min postdrug administration) in each session. Each point is the mean ± 1 S.E. based on one administration of each drug condition in each subject (number depends on study: top panel, five subjects; middle panel, hydromorphone and pentazocine six subjects, butorphanol and nalbuphine five subjects; buprenorphine four subjects; bottom panel, six subjects). The S.E.M. is not shown where it is less than the radius of the symbol.

A peculiarity in the research literature has been that, in humans, three-choice discrimination procedures have been needed to make the pharmacological differentiations among mu agonists and agonist-antagonists that animals make with two-choice procedures. One possible explanation for this difference is that the instructional and training procedures used with humans in two-choice discriminations have inadvertently established a different task from that trained in animals.

In the present study, we evaluated a different instructional set on the outcome of the two-choice discrimination procedure in humans. In the earlier two-choice study (Preston et al., 1992) participants were instructed to learn to discriminate between two different drugs labeled, for example, Drug A and Drug B and to respond to test drugs by pressing the computer key of the most similar training drug. Participants were explicitly trained to discriminate hydromorphone and saline as Drug A and Drug B. In the instructional set of the present study, participants were instructed to learn to discriminate the drug labeled, for example, Drug A, and to respond on the "Drug A" key only if test drugs were identical with Drug A and to respond on a "Not Drug A" key for all other test drugs. They were then explicitly trained to discriminate hydromorphone and saline as Drug A and Not Drug A. Other than this instructional difference, the procedures and test conditions were the same for both studies.

	Materials and Methods

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Subjects. The participants were eight adult male volunteers with opioid-abuse histories but not physically dependent; they gave written informed consent and were paid for their participation. Ages ranged from 31 to 40 years (mean 37.8); weights ranged from 60.5 to 77.3 kg (mean 67.8). Subjects reported illicit opioid use of 6 to 24 years duration (mean 19.2). In addition to their opiate abuse, all reported current or past use of marijuana, alcohol, and cocaine; four of the eight subjects also reported current or past use of hypnotics. All reported current use of tobacco. None were physically dependent on opiates or alcohol at the time of the study as determined by self-report and observation while drug-free in the first 3 days of admission to the research unit. On the basis of physical examination, history, routine laboratory chemistries, and chest X-rays, participants were found to be in good health and without significant psychiatric disturbance other than their drug abuse. One additional participant enrolled but did not complete the study due to a medical condition that contraindicated the administration of the study drugs. His data are not included in this report.

Drugs. Drugs were administered i.m. under double blind conditions in a constant volume of 3 ml. The training drugs were saline (3 ml) and hydromorphone hydrochloride 3 mg/70 kg. Dose-response generalization testing was conducted with hydromorphone (0, 0.125, 0.25, 0. 5, 1, 2, 3, and 4 mg/70 kg), butorphanol (0, 0.375, 0.75, 1.5, 3, and 6 mg/70 kg), pentazocine (0, 4, 8, 16, 32, and 64 mg/70 kg), nalbuphine (0, 1.5, 3, 6, 12, and 24 mg/70 kg), and buprenorphine (0, 0.055, 0.11, 0.22, 0.45, and 0.9 mg/70 kg). Commercially available preparations of each drug were used: hydromorphone hydrochloride (10 mg/ml; Knoll Pharmaceuticals, Whippany, NJ); butorphanol tartrate (2 mg/ml; Bristol Laboratories, Syracuse, NY); pentazocine lactate (30 mg (base)/ml; Winthrop Laboratories, Inc., New York, NY); nalbuphine hydrochloride (10 mg/ml; DuPont Pharmaceuticals, Wilmington, DE); buprenorphine hydrochloride (0.3 mg (base)/ml; Norwich Eaton, Norwich, NY). Hydromorphone hydrochloride, butorphanol tartrate, and nalbuphine hydrochloride doses were calculated on the basis of the salts, and pentazocine lactate and buprenorphine hydrochloride doses were calculated as the drug base. Appropriate volumes of each drug were diluted to the desired concentration with bacteriostatic saline. For generalization testing a range of doses for each drug was selected such that the recommended analgesic dose fell mid-range.

General Methods. Subjects were informed: 1) that they could receive various psychoactive drugs that might have sedative properties, stimulant properties, opioid properties, or opioid blocking properties; 2) that the study involved evaluation of their ability to discriminate one drug from another and evaluation of the subjective, behavioral, and physiological effects of those drugs; and 3) that in each session they could earn money for correct discriminations. Subjects were trained to discriminate the presence of Drug A (hydromorphone 3 mg/70 kg) from the absence of Drug A. Subjects were instructed that they were to learn to identify a drug, identified to them as Drug A, from all others by the way it made them feel. They were instructed that they should respond on the Drug A key when the effects they experienced were exactly like those produced by Drug A and that they should respond on the Not Drug A key if the effects were not those of Drug A. Subjects were told that Drug A would not change during the study. Different letter codes were used for different subjects, but the A/not A terminology is used throughout for convenience.

Experimental Session. Daily sessions were conducted using methods similar to those previously reported (Preston et al., 1989). At the beginning of each experimental session, respiration rate, pulse, temperature, blood pressure, and pupil diameter were recorded, and the subject completed baseline self-report questionnaires [adjective rating scales and the Addiction Research Center Inventory (ARCI)] and the Digit Symbol Substitution Test (DSST) in the experimental room. The scheduled drug or saline was then injected. During the initial training sessions the subject was informed of the drug's identifying letter code at the time of injection. The subject remained under staff observation for 20 min and then returned to the experimental room to complete the postdrug discrimination, subjective effect, and performance testing. Postdrug testing lasted for 90 min (20-110 min postinjection) as described in detail elsewhere (Preston et al., 1989). Subjective effects and DSST performance were assessed five times after drug administration; drug discrimination performance was assessed twice. At the end of the session the staff again recorded pupil diameter and other physiological measures. A sealed envelope was then opened, and the staff informed the patient of the letter-code identity of the administered drug or, on test sessions, that the code could not be revealed.

Discrimination Procedures. Drug discrimination data were collected in three ways. In each of these three procedures only correct responses were converted to monetary reinforcement for the subject. In Discrete Choice the subject indicated whether he thought he had received Drug A or Not Drug A as a single choice. In Point Distribution the subject distributed 50 points between the two training alternatives depending on how certain he was of the identity of the administered drug; this required typing in the numbers. On the Operant Responding measure the subject responded on a fixed interval, 1-s schedule on computer keys designated with Drug A or Not Drug A to earn points for 3 min; points (displayed on the computer screen) could be earned (at a maximum rate of one per second) for each of the training drugs by pressing the key corresponding to that drug.

Subject-Rated Measures. Four questionnaires were completed: 1) visual analog scales (VAS), 2) a pharmacological class questionnaire, 3) an adjective rating scale, and 4) a shortened form of the ARCI. On the VAS, the subject rated the degree of "Any Drug Effects," "Liking," "Good Effects," "Bad Effects," and "High" produced by the drug and rated the similarity of the drug to the training drugs on questions asking "How much is this drug like ... (each of the training conditions, by letter code, e.g., Drug A, Not Drug A)" by placing an arrow along a 100-point line marked at either end with "none" and "extremely." On the pharmacological class questionnaire, the subject categorized the drug effect as being most similar to one of 10 classes of psychoactive drugs (Preston et al., 1989). The adjective rating scale consisted of 32 items, which the subject rated on a 5-point scale from 0 ("no effect") to 4 ("maximum effect"). The items in the adjective list were divided into three subscales (Preston et al., 1989): a 13-item Agonist scale, an 10-item Antagonist, and a 9-item Agonist-Antagonist opioid scale. The ratings of the individual items within each scale were summed to yield a single total score for each scale. The short form of the ARCI consisted of 49 true/false questions and contained five major subscales: Morphine-Benzedrine group (MBG, an index of euphoria); Pentobarbital, Chlorpromazine, Alcohol group (PCAG, an index of sedation); Lysergic Acid Diethylamide (LSD, an index of dysphoric and somatic changes); and Benzedrine group (BG) and Amphetamine scales (empirically derived amphetamine-sensitive scales) (Martin et al., 1971). At baseline, only the adjective rating scales and ARCI were completed; all questionnaires were completed at other time intervals as described above.

Physiological and Performance Measures. Physiological measures consisted of respiration rate, pulse, blood pressure, temperature, and pupil diameter. Vital signs were collected manually. Pupil diameter was measured from photographs taken in constant ambient room lighting using a Polaroid camera with 3× magnification. Psychomotor/cognitive performance was tested using a computerized version of the DSST developed in our laboratory (McLeod et al., 1982).

Data Analysis. Because time-related effects are minimal for these drugs within the 20- to 110-min postdrug assessment period, results of each session were summarized to yield a single value for each measure. Within-session means were calculated for VAS. Percent Drug A- and Not Drug A-appropriate responses were calculated for each discrimination measure. Mean changes from predrug scores were calculated for the ARCI, adjective rating scales, and DSST scores and physiological measures.

	Results

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Test of Acquisition. Subjects reliably discriminated hydromorphone as Drug A and saline as Not Drug A in the test of acquisition sessions after training (sessions 5-8) with 85 to 94% correct responding for each substance. Two-way ANOVA showed that there was a significant main effect of drug but no main session effect or drug by session interaction (Table 1). The results were similar on all three discrimination measures.

Generalization Testing. Results of the generalization testing are summarized in Tables 2 and 3, and selected variables are shown in Figs. 2 through 4. Table 2 summarizes significant effects as well as major trends for P values less than or equal to .10. The arrows indicate the directions of drug effects relative to placebo.

View this table: [in this window] [in a new window]   TABLE 2 Summary of generalization testing Results of individual and across-drug analyses. Individual drug analyses were determined by repeated measures, one factor ANOVA (main effect of dose). Across-drug analyses were determined by repeated measures, two factor ANOVA (drug, dose, and drug × dose interactions). Values represent P values; all P values .10 are shown. Arrows indicate the direction of statistically significant (P  .05) individual drug effects; arrows in parentheses indicate the direction of drug effects with P values greater than .05 and less than or equal to .10. Tukey HSD test lists significant differences between agonist-antagonists and hydromorphone, 4 mg/70 kg, and the direction of the differences (N = 8).

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Dose-response curves of percentage hydromorphone-appropriate responding on the operant response discrimination measure and ratings on the "How much is this drug like Drug A (hydromorphone)?" and the "How much is this drug like Not Drug A?" VAS produced by hydromorphone, pentazocine, butorphanol, nalbuphine, and buprenorphine. Data are the mean of two assessments (at 60 and 80 min postdrug administration) in each session. Each point is the mean ± 1 S.E. based on one administration of each drug condition in each of eight subjects. The S.E.M. is not shown where it is less than the radius of the symbol.

View larger version (29K): [in this window] [in a new window]   Fig. 3.   Dose-response curves of hydromorphone, pentazocine, butorphanol, nalbuphine, and buprenorphine on the Drug Effect, Liking, Good Effects, and Bad Effects VAS. Data are the mean of five assessments in each session. Each point is the mean ± 1 S.E.M. based on one administration of each drug condition in each of eight subjects. The S.E.M. is not shown where it is less than the radius of the symbol.

View larger version (32K): [in this window] [in a new window]   Fig. 4.   Dose-response curves of hydromorphone, pentazocine, butorphanol, nalbuphine, and buprenorphine on the MBG scale of the ARCI, Agonist adjective rating scale, number of correct trials on the DSST, and pupil diameter. Data from the MBG scale, Agonist scale, and DSST are the mean change from baseline across five assessments in each session. Data for pupil diameter are the mean change of a single post session measurement from baseline in each session. Each point is the mean ± 1 S.E.M. based on one administration of each drug condition in each of eight subjects. The S.E.M. is not shown where it is less than the radius of the symbol.

	Discussion

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In the present study, subjects were trained to discriminate Drug A (hydromorphone 3 mg/70 kg administered i.m.) from Not Drug A (saline) and tested with a series of opioid agonist-antagonists. Only hydromorphone and buprenorphine produced significant dose-related increases in hydromorphone-appropriate responses. Pentazocine also produced significant increases, with greater than 80% at 32 mg, but the highest dose tested (64 mg) produced fewer hydromorphone-appropriate responses, resulting in an inverted U-shaped dose-response curve. Neither butorphanol nor nalbuphine produced dose-related or statistically significant increases in hydromorphone-appropriate responses. Thus, in this procedure in which subjects were trained to discriminate hydromorphone as Drug A and instructed to identify all other drugs as Not Drug A and trained to respond on the Not Drug A key with saline, there was clear differentiation among the four agonist-antagonists on the behavioral discrimination measures.

In a previous two-choice study in which subjects were explicitly trained with hydromorphone as Drug A and saline as Drug B (for example) with instructions to respond on the key of the most similar drug, all five test drugs fully substituted for the hydromorphone training condition (Fig. 1, top panel; Preston et al., 1992). Those results are quite different from the present study as well as from several three-choice studies conducted in our laboratory. For example, none of the studied agonist-antagonists fully substituted for hydromorphone in volunteers trained to discriminate among hydromorphone, saline, and pentazocine (Fig. 1, middle panel; Preston et al., 1989), and only buprenorphine fully substituted in volunteers trained to discriminate among hydromorphone, saline, and butorphanol (Fig. 1, bottom panel; Preston and Bigelow, 1994). Furthermore, in subjects trained to discriminate among hydromorphone at 0, 1, and 4 mg, pentazocine, butorphanol, and nalbuphine fully substituted only for the hydromorphone 1-mg training dose, whereas buprenorphine produced intermediate responding and fully substituted for neither 1 nor 4 mg (Jones et al., 1999). Based on the results of these prior studies, we have concluded that the outcome of human drug discrimination studies is dependent on the discrimination training conditions (including number and choice of training drugs used) (Jones et al., 1999). The present study demonstrates that instructions are one of these important procedural factors that must be considered in the design, conduct, and interpretation of drug discrimination studies in humans. Although based on across-study comparisons, the conclusion is strengthened by the fact that the series of studies was conducted in a single laboratory with similar methods and subjects across studies. Research by Smith and Bickel (1999) showing that an instruction-based, novel-response procedure has increased pharmacological selectivity compared with a standard two-choice procedure in human sedative discrimination also supports this conclusion.

Instructions are a feasible experimental manipulation only in human studies. Nevertheless, animal studies also support the more general finding that a number of procedural factors, such as training dose, species, training drug, and context affect discrimination performance, including the differentiation of mu and kappa opioids (Woods et al., 1988; Jarbe, 1989; Ator, 1999). The fact that discrimination performance is influenced by instructions offers the tantalizing possibility that the degree of correspondence between results of animal versus human studies might shed light on what concept or construct is discriminated by animals. This has long been a topic of debate (Overton et al., 1983; Jarbe, 1989).

Given that different generalization profiles are found with different discrimination procedures, the question arises as to which procedure best reflects the receptor activities of the test drugs. One comparison that might be made is to assays in vitro. Mu and kappa receptors are coupled to the G-protein second messenger system; binding of an agonist to the receptor activates the G-protein by stimulating the release of GDP and the binding of GTP. All four agonist-antagonists have at least partial agonist activity at both receptor types and have been tested for their ability to stimulate [³⁵S]GTPS binding in cloned mu and kappa receptors to determine their intrinsic activity (Emmerson et al., 1996; Zhu et al., 1997; Selley et al., 1998; Remmers et al., 1999). Butorphanol, nalbuphine, and pentazocine were reported to have similar low efficacies in a C6 glioma cell line expressing the mu receptors (Emmerson et al., 1996). Selley et al. (1998) determined the intrinsic activity of a series of opioids in mu receptor-transfected Chinese hamster ovary (CHO) cells and rat thalamus; they found buprenorphine to be a partial agonist with moderate efficacy (like meperidine) and nalbuphine low efficacy (like nalorphine and levallorphan). In an assay of kappa agonist activity in CHO cells, Zhu et al. (1997) found a rank order of potencies of pentazocine = nalbuphine > buprenorphine in stimulating [³⁵S]GTPS binding; pentazocine was a partial agonist with maximal responses 23% of full agonists, and nalbuphine and buprenorphine had low levels of agonist activities. Butorphanol had 22% efficacy and nalbuphine had 18% (maximal stimulation divided by maximal stimulation of a full agonist) in a C6 glioma cell line expressing kappa receptors (Remmers et al., 1999). Overall, it appears that buprenorphine has greater mu agonist activity and lower kappa agonist activity than the other three. The relative activities of pentazocine, butorphanol, and nalbuphine at mu and kappa receptors is less clear, although all appear to have low to moderate activity at both receptors.

The results of the present study are generally consistent with the intrinsic activities of the agonist-antagonists tested at the mu and kappa opioid receptors in vitro, with buprenorphine fully substituting for the mu agonist, whereas pentazocine did so at one dose and butorphanol and nalbuphine not at all (Fig. 2). In contrast, all four agonist-antagonists fully substituted for hydromorphone in the two-choice hydromorphone-saline (Drug A versus Drug B) discrimination, which differed from the present study only by instructions (top panel, Fig. 1; Preston et al., 1992). Thus, the hydromorphone/not hydromorphone discrimination resulted in greater pharmacological specificity across opioid receptor types than the hydromorphone-saline discrimination.

Human studies permit assessment of the relationship between discrimination responding and subjective effect measures. The present hydromorphone/not hydromorphone discrimination results showed good agreement with subjective effect measures, particularly as assessed by the Tukey HSD analyses. Butorphanol, which did not substitute for hydromorphone on the discrimination measures, was differentiated from hydromorphone on a substantial number of subjective effect measures, including some effects consistent with kappa agonist activity. Buprenorphine, the only test drug to fully substitute, had few subjective effects significantly different from hydromorphone, and none of them suggested qualitative differences. The inverted U-shaped dose-response curve produced by pentazocine on the discrimination measures was mirrored by changes in subjective effect measures. The subjective effects shown in the present study are consistent with earlier studies showing that mu agonists and mixed action opioids (drugs with mu and kappa agonist activity) produce different patterns both of discrimination and subjective effects (Dykstra et al., 1997). The present hydromorphone/not hydromorphone discrimination results agree better with subjective effect measures than did the hydromorphone-saline discrimination results (Preston et al., 1992) and are similar to results seen with our earlier three-choice studies (Preston et al., 1989; Preston and Bigelow, 1994). As noted above, the hydromorphone-saline discrimination did not differentiate among the agonist-antagonists (Preston et al., 1992; Fig. 1), with each of agonist-antagonists substituting for hydromorphone, even though, as in the present study, the subjective effects profiles varied across the five study drugs. Interestingly, subjects were less likely to identify butorphanol and nalbuphine as opiate-like in the hydromorphone/not hydromorphone discrimination study than in the hydromorphone-saline discrimination study, suggesting that the discrimination categories used by participants influenced their identifications on the pharmacological class questionnaire. Thus, there is good agreement between discrimination responding and subjective effects, particularly with the Drug A/Not Drug A and three-choice discrimination procedures, although there may be some reciprocal influences between discrimination training and subjective effect measures.

Both types of measures, drug discrimination and subjective effects, have utility for evaluating the stimulus effects of drugs in humans. Drug discrimination provides a good mechanism for comparison across drugs, whereas subjective effect measures provide descriptive information about the stimuli individuals experience after drug administration. Determination of the similarity of two drugs based only on subjective effects measures can be difficult because of the number of individual measures that are collected and the unknown contribution of each to the overall effect. On the other hand, subjective effect measures are useful in the interpretation of discrimination results, because drug discrimination responding is significantly influenced by the training conditions. In addition, studies collecting discrimination and subjective effects of drugs in humans have provided evidence supporting the utility of animal drug discrimination paradigm as a model for subjective effects in humans.

In conclusion, the agonist-antagonists were differentially discriminated as hydromorphone-like, with buprenorphine fully substituting, pentazocine fully substituting at one dose, but not at a higher dose, and butorphanol and nalbuphine not fully substituting for hydromorphone in this two-choice, hydromorphone/not hydromorphone discrimination procedure. The results differ from those of a previous two-choice, hydromorphone-saline discrimination study, confirming that instructions to subjects are an important factor in human drug discrimination studies. Thus, the present study confirmed the significant impact of training conditions on the outcome of drug discrimination studies and demonstrated that the two-choice drug/not drug instructional set produced discrimination results that are more consistent with the intrinsic activities of these drugs in in vitro assays of mu and kappa agonist activity and more consistent with their subjective effects than does the drug A/drug B procedure.