Selectivity in the Generalization Profile in Baboons Trained to Discriminate Lorazepam: Benzodiazepines, Barbiturates and Other Sedative/Anxiolytics 1

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1 /97/ $03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 282, No. 3 Copyright 1997 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 282: , 1997 Selectivity in the Generalization Profile in Baboons Trained to Discriminate Lorazepam: Benzodiazepines, Barbiturates and Other Sedative/Anxiolytics 1 NANCY A. ATOR and ROLAND R. GRIFFITHS Division of Behavioral Biology, Department of Psychiatry and Behavioral Sciences, Johns Hopkins School of Medicine, Baltimore, Maryland Accepted for publication May 27, 1997 In drug discrimination studies, differential reinforcement procedures are used to train subjects to make one response when a particular dose of a drug has been administered and to make a different response when it has not. When good control of responding is shown under training conditions, the drug is said to serve a discriminative stimulus function for that behavior. Tests with drugs other than the training drug generally show that administration of pharmacologically related drugs results in the same lever selection as the training drug but that administration of pharmacologically disparate drugs does not (Jarbe, 1989). This outcome has made the drug discrimination procedure valuable for studying commonalities of subjective drug effects in animals and for exploring the functional relevance of particular molecular mechanisms of action. Received for publication June 3, About a third of this work was supported by grant DA01147 and the rest was supported by grant DA04133 from the National Institute on Drug Abuse. A portion of these findings was reviewed in Ator and Griffiths, 1989a; preliminary data for the drugs tested in conjunction with CPDD were presented by Brady et al. (1990). ABSTRACT The discriminative stimulus effects of benzodiazepines often have been indistinguishable from those of barbiturates or other sedative/anxiolytics. However, baboons and rats trained to discriminate lorazepam did not reliably generalize to pentobarbital in previous studies, although animals comparably trained to discriminate pentobarbital reliably generalized to lorazepam. The present study investigated the generalization profile for a variety of anxiolytic, sedative and other drugs in baboons trained to discriminate oral lorazepam (1.8 mg/kg). Triazolam, alprazolam, diazepam, midazolam, bromazepam, temazepam and nordiazepam occasioned 80% of total responses on the lorazepam-paired lever, in that order of potency, 60 min after oral dosing; chlordiazepoxide did so in three of five baboons. However, barbiturates (amobarbital, hexobarbital, methohexital, pentobarbital, phenobarbital, secobarbital) and methyprylon occasioned lorazepam-appropriate responding in only one or two baboons. Testing barbiturates at different pretreatment times (amobarbital, hexobarbital, pentobarbital or secobarbital) or by an i.m. route of administration (methohexital, pentobarbital) did not produce an increase in generalization. Neither other classic sedatives/anxiolytics (chloral hydrate, clomethiazole, ethanol, methaqualone, meprobamate, triclofos), nor anticonvulsants (phenytoin, valproic acid), nor drugs from other pharmacological classes shared discriminative-stimulus effects with lorazepam. These results, together with those from previous studies in which lorazepam or another benzodiazepine served as the training stimulus, indicate that lorazepam training results in a more selective generalization profile with respect to sedative/anxiolytic drugs than does training with other benzodiazepines. Benzodiazepines potentiate GABAergic synaptic transmission by binding at a modulatory site on the GABA A -receptor complex (Haefely et al., 1985). Clinically useful Bzs differ primarily in their potencies for producing the anticonvulsant, muscle relaxant, anxiolytic and hypnotic effects characteristic of this class of drugs. There generally have not been qualitative behavioral differences among Bzs across a variety of behavioral paradigms (Dantzer, 1977; Sanger and Blackman, 1981); and in drug discrimination studies, animals and people trained to discriminate a particular Bz generalize to other Bzs (Ator, 1990; Kamien et al., 1993; Rush et al., 1996). Behavioral effects of Bzs are similar in many ways to those of the barbiturates, which they have replaced as anxiolytics and hypnotics in clinical practice (Lader, 1995). The similarity is not surprising, because barbiturates also enhance GABA, albeit through a different site on the GABA A -receptor complex. Animals trained to discriminate a Bz typically generalize to barbiturates and vice versa (Ator, 1990; Ator and Griffiths, 1989a). In fact, training with either Bzs or barbiturates generally has resulted in most sedatives and anxiolytics occasioning the drug-paired response. Thus, such train- ABBREVIATIONS: Bz, Benzodiazepine; 9 -THC, 9 -tetrahydrocannabinol; EtOH, ethanol; GABA, -aminobutyric acid; ND, no drug; PCP, phencyclidine HCl; p.o., per os. 1442

2 1997 Lorazepam Generalization Profile 1443 ing conditions have not seemed highly selective for differentiating among these compounds (Colpaert et al., 1976; Overton, 1984). In the first study with lorazepam as a training drug, however, generalization to pentobarbital occurred in only one of four baboons, even though pentobarbital doses were tested that clearly were behaviorally active (Ator and Griffiths, 1983a). The same study showed that pentobarbital-trained baboons did generalize to lorazepam. A subsequent study in rats, which also manipulated lorazepam and pentobarbital training doses, replicated this asymmetrical generalization profile (Ator and Griffiths, 1989b). In later studies, novel, nonbz, ligands for the Bz modulatory site did occasion lorazepam-lever responding in baboons and rats, which showed that lorazepam-trained animals would generalize to some types of nonbz GABAergic drugs (but they did not generalize to the direct GABA agonist THIP; Ator and Griffiths, 1986, 1992; Griffiths et al., 1992; Sannerud et al., 1992). Neuroactive steroids, which enhance GABA through a nonbz, nonbarbiturate site on the GABA A complex, however, did not produce reliable generalization in lorazepam-trained rats but did so in rats trained to discriminate diazepam, pentobarbital and EtOH (Ator et al., 1993). Thus, animals trained to discriminate lorazepam showed selectivity for compounds that enhance GABA through the Bz site rather than generalizing to compounds that enhance GABA per se. To date, however, the generalization profile for a range of barbiturates and other classic sedative/anxiolytic compounds has not been characterized in lorazepam-trained animals. Such a profile is essential for fully evaluating the selectivity of lorazepam training conditions. Particularly in the context of emerging data on the heterogeneity of the GABA A -receptor and the selectivity of its subtypes, including ones that form the putative 1 /Bz 1 and 2 /Bz 2 subtypes (Langer and Arbilla, 1988; Sieghart, 1995), a full profile of effects of classic sedative/anxiolytic, anticonvulsant compounds is a prerequisite for understanding whether apparent selectivity of the training drug condition matches its neuropharmacological selectivity. The present paper presents a generalization profile for baboons trained to discriminate lorazepam. A range of Bzs, barbiturates and other compounds that have anxiolytic, anticonvulsant, anesthetic, muscle relaxant and/or sedativehypnotic effects were tested; drugs from other classes that share certain characteristics with sedatives also were tested, as well as compounds not believed to share any effects with sedative/anxiolytics. The use of a reliable p.o. dosing procedure in the baboon permitted study of virtually all test drugs across a wide dose range via the route by which most of the test drugs usually are used by humans. Where negative results occurred with barbiturates, route of administration and/or time of testing were manipulated to determine whether the probability of lorazepam-lever responding would increase. Materials and Methods Subjects Six adult male baboons (Papio anubis except that baboon LO was Papio cynocephalus; Primate Imports, New York, NY) were housed individually, with water available at all times. All had served in different studies of i.v. drug self-administration (LE: cocaine; LO, MS, RA: cocaine, nicotine; ML: heroin; RF: cocaine, nicotine, methohexital, baclofen); and all had served in other drug discrimination studies in which tests were conducted with Bz-site ligands, pentobarbital, azapirones, pentylenetetrazole and caffeine (Ator, 1990; Ator and Griffiths, 1983a, 1985, 1986; Ator et al., 1989; Griffiths et al., 1992; Sannerud et al., 1991, 1992, 1993). Rations of laboratory monkey chow were supplemented daily with a multivitamin and fresh fruit. Feeding occurred at approximately the same time each day, which was 30 to 75 min after the regular experimental session; on days when the drug time course was evaluated, the ration of monkey chow was omitted. Ketamine hydrochloride (HCl) preceded by atropine sulfate (SO 4 ) was administered i.m. approximately every 2 weeks to permit weighing the baboons. The baboons were not maintained at reduced weights compared with a free-feeding weight (Ator, 1991). Weights generally increased across the 5 to 7 years in which the data were collected. The range of weights (rounded to the nearest kilogram) was: 24 to 32 for LE, 29 to 35 for LO; 30 to 36 for ML, 27 to 38 for MS and 28 to 34 for RA and RF. Apparatus Two systems were used at different times during the studies. At the beginning, sessions with five of the baboons were conducted when they had been adapted to restraint chairs (Findley et al., 1971) and placed in a sound-attenuating chamber equipped with an intelligence panel. Some of the data for alprazolam (LO, ML, RA), bromazepam (ML, RA) diazepam (ML), temazepam (LO, ML, RF) and phenobarbital (LO, RA) and all data (except LE s) for triazolam and i.m. pentobarbital were obtained with the chair apparatus. All other dose-effect curves were obtained with the baboons seated on a bench in front of the intelligence panel, which formed the rear wall of a standard stainless steel primate cage (see fig. 4 in Ator, 1991). The cage was enclosed in a moderately sound-attenuating chamber for all except the methohexital determinations; for those, visual isolation from the baboons on either side was provided, during sessions, by masonite panels. The intelligence panels (constructed in the laboratory) contained two jewel lights mounted over either two Lindsley levers (Gerbrands Corp., Arlington, MA) or two stainless steel levers (constructed in the laboratory), which closed microswitches when operated. The levers were approximately 15 cm apart on the lower left half of the panel and within easy reach. A drinkometer (Kandota Instruments, Sauk Centre, MN), used for p.o. drug delivery, was mounted on the left above the levers. A food hopper, illuminated coincident with pellet delivery, was located in the upper middle of the panel. An electromechanical feeder (various sources) was used to deliver 1-g banana-flavored pellets (P.J. Noyes, Lancaster, NH or BIO-Serv, Inc., Frenchtown, NJ) and was mounted above the chamber as was a tone generator, which delivered white noise and tones through a speaker mounted on the back of the intelligence panel. A cm translucent white plexiglas panel, which could be transilluminated, was mounted in the upper right quadrant of the panel. Experimental conditions were programmed and data collected by use of PDP8 computers (Digital Corp., Maynard, MA) programmed in SUPERSKED (State Systems, Kalamazoo, MI). Graphic records of each session s performance were collected with cumulative recorders (Gerbrands Corp., Arlington, MA). Procedure Training sessions. A 60-min time-out, which coincided with the lorazepam pretreatment time, preceded each experimental session. During time-out, the translucent panel was illuminated and lever responses were counted but had no programmed consequences. White noise was turned on at the beginning of this time-out and continued until the end of the session. When the time-out ended, the panel light was extinguished, both jewel lights were illuminated, a 3-s tone sounded and a 20-min period of food pellet availability began. A response on either lever in the presence of the jewel lights produced a 0.1-s tone. In training sessions, food pellet delivery depended on a fixed number of consecutive responses on the lever

3 1444 Ator and Griffiths Vol. 282 appropriate to the drug or ND condition in effect. Responses on the inappropriate lever reset the response requirement. The lever paired with the drug and ND conditions was counterbalanced across baboons. Completion of the response requirement turned off the jewel lights, operated the feeder, illuminated the food tray for 1 s and initiated a 6-s time-out. The response requirement for each baboon was determined empirically to be that which best maintained criterion performance. It was 10 or 15 responses for MS, 15 or 20 responses for LO, 20 responses for LE and ML, 35 responses for RF and 40 responses for RA. Lorazepam and ND training sessions generally alternated. Most of the data to be reported were obtained when the baboons were trained to discriminate 1.8 mg/kg lorazepam p.o., administered 60 min before the session, from the ND condition. Four baboons (LE, ML, MS, RA) originally had been trained to discriminate lorazepam 1.0 mg/kg i.m., with the same pretreatment time, but the p.o. route was adopted after the procedure for p.o. drug delivery was perfected to spare the baboons frequent i.m. injections. Baboons LO and RF were trained from the beginning to discriminate p.o. lorazepam. All except baboon RF experienced training with 1.0 mg/kg p.o.; but this dose did not maintain criterion level performance reliably in some baboons, and the 1.8 mg/kg dose was adopted for all. Comparison of dose-effect curves obtained during 1.0 mg/kg training, whether i.m. or p.o., with those obtained or redetermined during 1.8 mg/kg p.o. training revealed no differences between them; likewise, no differences in drug lever responding or response rates could be attributed to the type of apparatus used. Thus, these procedural differences will not be noted further. Test sessions. A test session was conducted only if responding in the training sessions met the criteria that 1) 95 to 100% of the total responses had been on the correct lever and 2) before the first food delivery of the session, completion of the required number of consecutive responses on the correct lever had not been preceded by the same number of consecutive responses on the incorrect lever. Before study of the dose-effect relationship for each drug, test sessions first were conducted with the training dose of lorazepam and with vehicle to confirm the reliability of the trained discrimination. Test sessions were identical with training sessions except that the length of the presession time-out corresponded to the test drug pretreatment time and completing the usual required number of consecutive responses on either lever produced a food pellet. The order of drug and ND training sessions between test sessions was counterbalanced so that test sessions were preceded equally as often by a lorazepam as by a ND training session. At least one drug and one ND training session occurred between tests with novel drug doses. If criterion performance was not shown in a training session, training sessions alternated until criterion performance occurred in four consecutive sessions. Experimental sessions generally were conducted 5 to 6 days a week at the same time each morning, except that sessions were omitted for 1 or more days after higher doses of drugs or after tests with drugs with the longer elimination half-lives (see below). To study the time course of drug action, additional sessions were conducted on some test days. That is, the test session was turned on again, usually at 1- or 2-h intervals after the first session began. These later sessions were 10 min, and the presession time-out before them was 5 min. Design and data analysis. Data are reported on 31 drugs. A single-subject design (Sidman, 1960) was used, in which each baboon served as his own control for determining whether each test drug shared discriminative stimulus effects with lorazepam and whether response rates were affected. Not all drugs were studied in all baboons; the range was 16 (baboon LE) to 31 (baboon MS). The dose at which individual baboons showed effects different from vehicle sometimes varied as much as a 0.5 log 10 unit or more. Thus, this design not only conserved the amount of drug needed in these large animals but also avoided subjecting individual baboons to doses unnecessarily high for the purposes of the study. Order of testing was mixed except that i.m. pentobarbital was studied first and methohexital was studied last. Two or more observations were made at each dose for some or all of the baboons. The dose range encompassed one or more low doses that occasioned 20% drug-lever responding and one or more high doses that occasioned at least 80% druglever responding. For drugs that did not engender lorazepam responding, a log 10 range or greater was studied with almost all to encompass a range of possible effects and to try to include at least one dose that produced a decrease in the rate of lever pressing. A portion of the determinations with bromazepam, clomethiazole, diazepam, methaqualone, temazepam and triclofos were conducted blind in conjunction with the development of a sedative-stimulant screening program by the Committee (now College) on Problems of Drug Dependence. The primary dependent variables were percentage of total test session responses on the lorazepam-paired lever and overall rate of responding on both levers combined. Numbers of responses on each lever were collected pellet by pellet across each session, however; and the lever on which the response requirement was first met and a pellet obtained also was assessed. Effects of test drugs on overall percentages of lorazepam-lever responding were judged in relation to training criterion levels. Because each baboon had to exhibit not only criterion performance in training sessions before each test but also clear evidence of stimulus control by the training dose versus vehicle under test conditions before (and in some cases during) each dose-effect evaluation, test session responding that was less than 95% and greater than 5% on the drug and ND levers, respectively, was, by definition, significantly different from the stimulus control established by the training conditions. Consistent with the drug discrimination literature, however, lorazepam-lever responding of 80 to 95% was not interpreted to indicate effects qualitatively different from the lorazepam training dose, given that at least one food pellet had been produced in the test session (i.e., the lever choice had been made). Conversely, 5 to 20% lorazepam-lever responding was not considered to be qualitatively different from the ND condition. Test drugs that produced increasing individual mean dose-effect functions with maximums of 80% or more in at least four baboons were concluded to share discriminative stimulus effects with lorazepam. For drugs that produced a maximal effect (i.e., full generalization by the definition given above), the ED 50 and ED 80 values for the doses at which drug-lever responding was 45 to 55% and 80 to 100%, respectively, were determined from the generalization gradients for each baboon (cf. Barry, 1974). Overall response rates (i.e., rate on both levers combined, excluding lever presses during time-outs) in test sessions during a dose-effect evaluation were converted to percentages of each baboon s mean rate in the criterion ND training sessions that most closely preceded each test during the evaluation. Drugs and dosing. Doses are expressed as the form of the drug listed below. The following drugs were donated: alprazolam and triazolam, Upjohn Co., Kalamazoo, MI; chlordiazepoxide HCl, diazepam, methyprylon, midazolam maleate and nordiazepam, Hoffmann-LaRoche, Inc., Nutley, NJ; lorazepam and meprobamate, Wyeth Laboratories, Philadelphia, PA; Ketamine HCl, Warner-Lam-

4 1997 Lorazepam Generalization Profile 1445 bert Co., Ann Arbor, MI and also purchased as Ketaset (100 mg/ml), Fort Dodge Laboratories, Inc., Fort Dodge, IA; temazepam, Sandoz, Inc., East Hanover, NJ; d-amphetamine SO 4, Smith, Kline, & French, Philadelphia, PA; mesocarb, PCP and 9 -THC, National Institute on Drug Abuse, Research Triangle Park, NC; phenytoin, Parke-Davis, Ann Arbor, MI. The following drugs were purchased: chloral hydrate, haloperidol, hexobarbital, pentobarbital sodium (Na), and secobarbital Na, Sigma Chemical Co., St. Louis, MO; methaqualone, Lemmon Pharmacal Co., Sellersville, PA; amobarbital Na, and phenobarbital Na, Ganes Chemicals, Inc., Pennsville, NJ; methohexital Na (as Brevital), Eli Lilly and Co., Indianapolis, IN; morphine SO 4, Mallinckrodt, Inc., St. Louis, MO; valproic acid, Saber Laboratories, Inc., Morton Grove, IL. Bromazepam, clomethiazole HCl and triclofos were obtained exclusively through the Committee on Problems of Drug Dependence. Solutions of 10% EtOH weight/volume were prepared by adding distilled water to 95% volume/volume EtOH (both at room temperature). All drugs were given p.o. except haloperidol, ketamine and morphine, which were given i.m. only. Chlordiazepoxide, pentobarbital and methohexital were studied i.m. and p.o. Doses for p.o. administration were blended for 3 min into a volume (generally 60 ml) of a matrix prepared with 1 to 2 g/l of BIO-Serv Agent K in distilled water flavored with orange-drink powder (52 g/l). When the vehicle contained quinine SO 4, the concentration was 0.32 mg/ml. Because the 9 -THC appeared to adhere to the glass container used for blending, it was injected into an orange, which was given to the baboon (and readily consumed). Ketamine, morphine, pentobarbital and chlordiazepoxide were dissolved in 0.9% saline and given in one or two injections. Haloperidol was dissolved in two or three drops of lactic acid and then diluted with 0.9% saline to 1.5 ml. Methohexital was dissolved in sterile water at a concentration of 20 mg/ml, and the total dose was given in one to three injections. Maximum volume per i.m. injection to a single site was 2 ml. All drug solutions or suspensions made from powder were prepared immediately before administration except for the stock solutions of methohexital, which were used within 2 weeks. The pretreatment time was 10 min for EtOH, 15 min for methohexital and 30 min for 9 -THC, d-amphetamine, clomethiazole, haloperidol, morphine and PCP. It was 60 min for all other drugs; however, amobarbital, hexobarbital, secobarbital, pentobarbital and triclofos also were tested 30 min after dosing. During study of chlordiazepoxide, diazepam and nordiazepam, no training sessions were conducted for 1 to 6 days after tests to lower the likelihood that training would be affected by residual active drug. Sessions also often were omitted for at least 1 day after the highest doses of other drugs out of similar concerns and to provide recovery time from possible long-lasting drug effects (even though none might be apparent). Drug-free periods of several days often were interspersed between study of various drugs as well. The baboons had been trained in the p.o. dosing procedure by habituating them to a bitter taste with quinine SO 4 (Turkkan et al., 1989). When it was determined that p.o. dosing could be accomplished as well without the quinine in these experienced baboons, routine use of quinine was omitted. Most vehicle tests throughout the study were conducted with the quinine-adulterated suspension, however, as a form of taste control for drug. The baboons readily drank most doses of all drugs except EtOH. They were induced to accept it by providing the orange-flavored drink solution as a daily dose with a gradually increasing concentration of EtOH. Onset of dosing was paired with illumination of the white lights on the drinkometer. Spout contact operated a solenoid valve and permitted fluid flow; simultaneously, the white lights went out and the green lights on the drinkometer were illuminated. Dosing typically was accomplished in 5 min, but an arbitrary maximum dosing time of 15 min was used to restrict variability in drug pretreatment time. The dose was followed immediately by unadulterated orange-flavored suspension to flush the line. Consumption of the dose and flush was observed directly by the research assistant. The time-out for training or test sessions followed immediately afterward. Results Stimulus control was well maintained at criterion level under the training conditions and was readily reestablished after periods of absence from the procedure. Each baboon s range of response rates in the lorazepam training sessions generally was the same as or only slightly higher than the range of response rates in the ND sessions. However, for one baboon, LO, response rates in the lorazepam training sessions generally were 0.5 to 1 response (r)/s higher than those in the ND sessions such that the ranges did not overlap or did so only slightly in most of the studies. Benzodiazepines Lorazepam. Three lorazepam dose-effect determinations were made across a 4- to 8-month period and are shown in figure 1. All baboons generalized to doses lower than the 1.8 mg/kg lorazepam training dose, but they differed in the probability that doses as low as 0.1 or 0.32 mg/kg would occasion responding on the lorazepam lever. As shown in table 1, the ED 50 for lorazepam ranged from 0.1 to 0.56 mg/kg across baboons; and the ED 80 ranged from 0.32 to 1.8 mg/kg. Only the generalization gradients for baboon MS were quantal (i.e., responding was essentially 100% on one or the other lever at all doses). The other baboons produced intermediate percentages of responding (i.e., between 10 and 90%) on the lorazepam lever in some determinations at doses lower than the training dose. Although baboons LO and RF had the highest ED 50 values (0.56 mg/kg), baboon LO s generalization gradient was very similar to that of the baboon with the lowest ED 50 (0.1 mg/kg for baboon RA). In both LO and RA, 0.1 mg/kg, which occasioned 0% responding in the other three baboons, occasioned considerable lorazepam lever responding on some tests. For all baboons, the dose higher than the training dose, 3.2 mg/kg, occasioned 100% responding on the lorazepam lever. In tests with vehicle, response rates did not differ from those in the ND control sessions (fig. 1, lower panels). Lorazepam did not decrease response rates, compared with rates in ND control sessions, except in baboons ML and RA at 3.2 mg/kg. Baboon LO s response rates were a generally increasing function of dose in all three sets of lorazepam determinations and were about 300% of control at 1.8 and 3.2 mg/kg (in control sessions, the highest rate was 155% of control). These results for LO are consistent with the fact that, unlike the other baboons, response rates in his lorazepam training sessions during determination of the curves shown in figure 1 were higher than rates in the ND sessions (ranges were, respectively, r/s and r/s). Alprazolam, bromazepam, diazepam, midazolam, temazepam, triazolam. Eight Bzs other than lorazepam were studied. Dose-effect determinations were conducted in six baboons with diazepam and in five baboons with the other Bzs; each dose was studied one to three times in each baboon. Group means for lorazepam and six other Bzs, all administered by the p.o. route and assessed at the same interval after dosing, are shown in figure 2. The triazolo-bzs, triazolam and alprazolam, were more potent than lorazepam in occasioning lorazepam-lever responding; and the 1,4-Bzs diazepam, mi-

5 1446 Ator and Griffiths Vol. 282 Fig. 1. Percentages of responding on the lorazepam lever and overall response rates in test sessions 60 min after administration of lorazepam or its vehicle (V) to baboons trained to discriminate lorazepam (1.8 mg/kg p.o.) from the ND condition. Individual points are data from single test sessions in the first ( ), second ( ) and third (E) determinations of the dose-response relationship across a 4-month (baboons MS, RA) or 8-month (LO, ML, RF) period; solid lines indicate the means. Response rates are given as a percentage of the mean rate in control ND sessions. Control mean response rates (r/s) for each baboon were: LO, 0.6; ML, 2.7; MS, 1.4; RA, 3.1; and RF, 2.7. TABLE 1 ED 50 and ED 80 values for lorazepam-lever responding a Drug b dazolam, bromazepam and temazepam were less potent than lorazepam. This order roughly held for individual baboons as well, as shown by the ED 50 and ED 80 values in table 1. Responding peaked at 100% on the lorazepam lever for all those drugs and for each baboon tested except for diazepam for one baboon. Baboon RF did not make 80% lorazepam lever responses at any dose of diazepam, although the other five baboons had ED 50 values of 0.32 to 1.0 mg/kg and showed full generalization (ED 80 ) at 1.0 or 3.2 mg/kg (table 1). The group diazepam generalization gradient in figure 2 peaks at 100% at 5.6 mg/kg because baboon RF was not tested at that dose. Diazepam dose was increased to 18 and then 32 mg/kg for this baboon (data not shown), which occasioned 25% and 65% lorazepam-lever responding, respectively. Neither time course studies nor redetermination of the p.o. diazepam curve 7 years later produced a different qualitative result in total session percentages of lorazepam-lever responding for baboon RF. Diazepam also was given to baboon RF i.m. (in the commercial injectable form, 5 mg/ml concentration); but Baboons LE LO ML MS RA RF Mean ED 50 ED 80 ED 50 ED 80 ED 50 ED 80 ED ED 50 ED 80 ED 50 ED 80 ED 50 ED 80 Triazolam Alprazolam Lorazepam Diazepam NG Midazolam Bromazepam Temazepam NG Nordiazepam NG Chlordiazepoxide NG c NG 18.0 NG NG a ED 50 is the lowest dose tested that produced lorazepam-lever responding between 45 and 55%; if no such intermediate responding occurred, the ED 50 is the quarter log 10 dose between one that produced 45% and the one that produced 55%. The ED 80 is the lowest dose occasioning at least 80% lorazepam-lever responding. b Drugs are listed from most to least potent in terms of mean ED 80. c NG no generalization (i.e., peak mean percentage of lorazepam-lever responding was 80%). it occasioned no lorazepam-lever responding up to 1.0 mg/kg, a dose that had occasioned 100% lorazepam-lever responding in baboons ML and RA (Ator and Griffiths, 1983a). Although baboon RF was the only baboon whose initial training was with 1.8 mg/kg lorazepam p.o., a generally lessened sensitivity to the discriminative stimulus effects of the other Bzs with which he was tested was not shown except for temazepam (see table 1). [However, this same baboon was the only one of five baboons that did not show maximal lorazepamlever responding with the 1 /Bz 1 ligand zolpidem (Griffiths et al., 1992)]. Response rates (fig. 2, bottom panel) were decreased on average to about 50% of the ND control rate by the highest dose of all Bzs except lorazepam; this was a decrease well below the range of rates in control sessions for all baboons. The baboon that failed to generalize completely to diazepam (RF) did show response rate decreases below the range of control rates at all p.o. diazepam doses greater than 1.0 mg/kg, and the rate was 50% of control at 32 mg/kg. In

6 1997 Lorazepam Generalization Profile 1447 Fig. 2. Mean percentages of responding on the lorazepam lever and overall response rates (as a percentage of the control ND rate) in test sessions 60 min after p.o. administration of Bzs to lorazepam-trained baboons. Each data point represents the mean of four or five baboons, except the points for the lowest dose of temazepam and for the lowest and highest doses of diazepam and bromazepam are for two baboons (see text for 32 mg/kg diazepam results, not shown). The ranges of mean control ND response rates (r/s) for individual baboons during these curves were: 1.5 to 2.6 for alprazolam, 1.3 to 2.6 for bromazepam, 0.5 to 3.1 for diazepam, 0.6 to 3.1 for lorazepam, 0.6 to 2.9 for midazolam, 1.5 to 2.4 for temazepam and 1.4 to 2.5 for triazolam. The lorazepam curve is the grand mean of the data in figure 1. In vehicle test sessions (not shown), percentages of lorazepam-lever responding were 5%; and response rates ranged between 80 and 120% of the ND control rate for individual baboons. contrast to the other baboons, baboon LO s response rates often were increased in Bz tests (e.g., as shown in fig. 1) and not decreased below control at the high doses. Thus the data for baboon LO contribute substantially to the increased response rates shown in figure 2. The reason for this unusual sensitivity is not clear. This baboon s control ND response rates were less than 1.0 r/s during many of the curves, whereas those for the other baboons were about 1.5 r/s or higher. However, when his control ND range increased and became similar to those of the other baboons, his response rates still were increased by Bzs. Baboon LO s response rates in lorazepam training sessions were consistently about 0.5 r/s higher than those in ND training sessions, but response rates in Bz test sessions were increased even when all responding was on the ND lever. Nordiazepam and chlordiazepoxide. The group curves for nordiazepam and chlordiazepoxide are not shown in figure 2, because those results are not well characterized by the group means. Neither of the mean generalization gradients reach 80% lorazepam-lever responding. The peak for each drug was at 18 mg/kg: 61% for nordiazepam and 64% for chlordiazepoxide. However, both Bzs did share discriminative stimulus effects with lorazepam in most of the baboons tested (fig. 3). Nordiazepam occasioned 100% drug-lever responding in four of the five baboons tested, but the effective dose varied substantially across baboons (fig. 3, table 1). For example, 0.1 mg/kg occasioned 94% lorazepam lever responding in baboon LO; but generalization from lorazepam to nordiazepam did not occur except at 56 mg/kg for baboon RA. Baboon MS s mean curve peaks only at 67%. This baboon made 86% and 91% lorazepam-lever responses when tested at 10 and 18 mg/kg, respectively, but retests with those doses twice more resulted in lower, intermediate, percentages of lorazepam-lever responding. Retests with 10 mg/kg in baboons ML or RF, however, replicated the original result. For RF, both tests at 10 mg/kg occasioned 90% lorazepam-lever responding, but the single test at 18 mg/kg occasioned 62%. Spacing between nordiazepam tests was 7 days or more. The generalization gradient for nordiazepam was an inverted U for two of the five baboons, which was unusual (as described above, the inverted U for diazepam in fig. 2 was an artifact of one baboon s failure to generalize). Response rates again were increased above the control ND range for baboon LO but were below the control range for lorazepam training sessions during the nordiazepam study (i.e., % of ND rates). Chlordiazepoxide shared discriminative stimulus effects similar to lorazepam in only three of five baboons (fig. 3, table 1; baboon RF failed to consume even low doses of this drug).

7 1448 Ator and Griffiths Vol. 282 For the two baboons that did not generalize, the gradient was an inverted U with a maximum of 31% at 10 mg/kg for LE; for baboon LO, there was a dose-dependent increase to a maximum of 57% at 18 and 32 mg/kg. Multiple tests with 10 and 18 mg/kg for baboon LE occasioned 63% lorazepam-lever responding in the first 10 mg/kg test and 0% in the second; in four determinations at 18 mg/kg, the range was 4 to 45%. For baboon LO, 32 mg/kg occasioned 56 and 59% lorazepam-lever responding in two tests. Time course studies did not reveal an increase to 80% or more at any longer intervals after dosing. Because chlordiazepoxide is water soluble up to relatively high concentrations, it was feasible to test a wide range of doses i.m. to determine whether generalization might occur in more baboons via this route and to compare i.m. and p.o. potencies (fig. 4). The percentage of lorazepam-lever responding 60 min after i.m. chlordiazepoxide was much less than after p.o. The group mean i.m. gradient peaked at 5% at 32 mg/kg; maximum lorazepam-lever responding for any baboon in any i.m. test was 19% at 32 mg/kg in RA. The interval between i.m. injection and testing was shortened to 30 min in a test at 10 mg/kg for baboons LE, ML and RF (data not shown); but the highest percentage of lorazepam lever responding was 25% by RF (response rate decreased to 17% of control). Thus, chlordiazepoxide clearly was more efficacious as a discriminative stimulus p.o. than i.m. in baboons trained with p.o. lorazepam. Barbiturates and Other Sedatives/Anxiolytics Amobarbital, hexobarbital, methohexital, methyprylon, pentobarbital, phenobarbital, secobarbital. Neither barbiturates nor methyprylon, a piperidinedione sedative-hypnotic, occasioned maximal lorazepam-lever responding in most baboons. Group generalization gradients for five barbiturates and for methyprylon, all tested 60 min after dosing, are shown in figure 5. These gradients generally were a monotonically increasing function of dose, but peaked between 20% (hexobarbital) and 50% (phenobarbital). The Fig. 3. Percentages of responding on the lorazepam lever and overall response rates in test sessions 60 min after nordiazepam or chlordiazepoxide and their vehicles (V) in lorazepamtrained baboons. Each data point generally represents a single determination for each baboon, except: Nordiazepam: for RF, V, 3.2, 10 two tests; for ML, V, 10 three tests, 1.0 two and 3.2 four tests; for MS, V, 10, 18 three tests, 3.2 two. Chlordiazepoxide: for RA, 3.2 two tests; for LE, 10 two and 18 four tests; for LO, 32 two tests; for MS, V, 32 two tests; 10, 18 three tests. Control mean response rates (r/s) were: LE, 2.3; LO, 0.6; and MS, 1.7; they differed across curves for three baboons and were: ML, 2.2 and 2.3; RA, 3.0 and 2.7; and RF, 1.6 and 2.5 for, the left and right panels, respectively. intermediate mean percentages of lorazepam-lever responding for drugs shown in figure 5 generally resulted from each drug s occasioning a maximum of 80 to 100% lorazepam-lever responding in one or two baboons and between 10% and 90% in one to three others. Figure 6 presents the results for baboons individually and shows that phenobarbital and methyprylon each occasioned full dose-dependent generalization in two baboons. The maximum percentage of lorazepamlever responding after amobarbital, secobarbital and pentobarbital was at the 18 mg/kg dose and was in baboon ML for both amobarbital and pentobarbital (respectively, 62% and 55%) and in baboon LE for secobarbital (74%). (Baboon RF was not tested above 18 mg/kg amobarbital and phenobarbital nor with the other two barbiturates because he did not reliably consume those drugs.) The drugs generally did decrease response rates below the range of rates in ND control sessions at 18 mg/kg and above (see also fig. 5). As with the Bzs, baboon LO s response rates increased well outside the range of rates in control ND sessions at one or more doses except with phenobarbital (fig. 6). Amobarbital also was tested at a 30-min and secobarbital at a 30-min and a 45-min interval after dosing to determine whether probability of generalization across baboons would increase (data not shown). The results did not differ from those shown in figures 5 and 6, except that the response rate dose-effect curve was shifted to the left for secobarbital at both shorter pretreatment times. Because of interest in whether manipulation of route of administration or pretreatment time change the probability of generalization, the pentobarbital results from figure 6 are replotted in figure 7 with those after i.m. administration and also with those for tests 30-min after both i.m. and p.o. administration (the i.m. results for the 60-min pretreatment time for baboons LE, ML, MS and RA were reported in Ator and Griffiths, 1983a). Regardless of pretreatment time and route of administration, pentobarbital did not produce generalization in more than two baboons via the p.o. route nor one baboon via the i.m. route; but pentobarbital was some-

8 1997 Lorazepam Generalization Profile 1449 Fig. 4. Percentages of responding on the lorazepam lever and overall response rates in test sessions 60 min after chlordiazepoxide or vehicle (V) p.o. or i.m. in lorazepam-trained baboons. Symbols represent each of the same baboons as in figure 3, and the solid or dashed lines represent the group mean. For the p.o. doses, the data points are the same as those in figure 3. For the i.m. doses, each data point represents a single determination. what more efficacious at the 30-min interval p.o. in baboon ML. That is, the generalization gradient was shifted to the left and the maximum was increased compared with the 60-min pretreatment time p.o. In addition, a second baboon (LO) generalized from lorazepam to pentobarbital when the p.o. pretreatment time was 30 min rather than 60 min, and the response rate was decreased after 30 min p.o. but increased after 60 min p.o. Baboon RF had not been tested with higher pentobarbital doses p.o. because he failed to consume even low doses reliably. In tests with i.m. pentobarbital, baboon RF showed 46% lorazepam responding 60 min after 18 mg/kg i.m., with the response rate only suppressed to 47% of control. Other baboons generally failed to complete the response requirement at or above 10 mg/kg pentobarbital i.m. Hexobarbital produced full generalization only in baboon ML and virtually no lorazepam-lever responding in any other baboon (figs. 5 and 8). Because hexobarbital is considered a short-acting barbiturate, it also was tested 30 min after dosing to determine whether more lorazepam-lever responding would occur than at 60 min, but the qualitative result was unchanged across the doses for which direct comparisons could be made. However, 56 mg/kg p.o. occasioned 66% lorazepam-lever responses in baboon MS at the 30-min interval, a dose that decreased response rate to 26% of control. (During the 30 min after dosing, baboon MS was noted to be ataxic and uncoordinated and to show tremor.) At 60 min after dosing, hexobarbital decreased overall response rates below control less reliably across baboons than did the other drugs in figures 5 and 6. At 30 min after dosing, hexobarbital 32 mg/kg decreased responding in more baboons than after 60 min, but it also increased responding above the ND control range for baboon LO. The ultra-short-acting barbiturate methohexital was tested via p.o. and i.m. routes in four baboons (fig. 9; the group data were not included in fig. 5 because the p.o. tests were 15 rather than 60 min after dosing). After p.o. administration, little evidence for behavioral activity was seen except for response rate decreases for baboons ML and RF. (ML also was noted to be slightly sedated at 3.2 mg/kg.) After i.m. administration, however, the baboons showed individual peak mean percentages of lorazepam-lever responding that ranged from 17 to 46%. The peak occurred at 3.2 mg/kg for three baboons and at 1.0 mg/kg for the other. In one of the two tests with the peak dose, each baboon showed 34 to 82% lorazepam-lever responding and 4% or less in the other. A baboon that generalized in the initial determination (ML) was tested a third time, and the result was 53% lorazepam lever responding. After the session at 3.2 mg/kg i.m., but not after any p.o. methohexital, baboon ML vomited. For baboon RF, ataxia and slight sedation were noted after 1.8 mg/kg i.m.; and he was ataxic 5 to 8 min after injection with 3.2 mg/kg in the first test at that dose. At the second 3.2 mg/kg i.m. determination, baboon RF was noted to be heavily sedated and unable to stay upright within 7 min of the injection. He did not begin responding on the lever until 15 min into the session (i.e., 30 min after injection). Thus, the barbiturates and methyprylon shown in figures 5 through 9 were tested at doses that were behaviorally active, but they did not occasion reliable and complete generalization in most of the baboons nor in a particular subset of baboons. Although the lorazepam-lever responding that did occur usually did so at doses that affected response rates, compared with vehicle, these two effects were not perfectly correlated. Chloral hydrate, clomethiazole, EtOH, meprobamate, methaqualone and triclofos. Table 2 presents the results of tests with six other sedative-hypnotics. Although they were tested across a wide range of doses, only methaqualone occasioned greater than 80% drug lever responding

9 1450 Ator and Griffiths Vol. 282 in any baboon tested (92% for baboon LE at 10 mg/kg; response rate was 144% of control). Chloral hydrate, clomethiazole and methaqualone decreased response rates outside the control range in all or all but one baboon and the median percentage of the ND control rate was between 50 and 60%. However, EtOH did so in only one of the three baboons tested; and the anxiolytic meprobamate and the sedative/ hypnotic triclofos did not do so in any baboon, even though doses of 100 and 180 mg/kg were tested. Time course studies were conducted out to 4, 7, 9 and 15 h after dosing with triclofos, clomethiazole, chloral hydrate and meprobamate and methaqualone, respectively; but no later onset of lorazepam-lever responding occurred. Drugs Not Classified as Sedatives or Anxiolytics Table 2 also shows the results of testing nonsedative/anxiolytic drugs, some of which share some pharmacological effects with the sedatives and anxiolytics described above. Although tested at behaviorally active doses, neither the narcotic analgesic morphine, nor the dissociative anesthetics ketamine, phencyclidine, 9 -THC, nor the anticonvulsants phenytoin and valproic acid, nor the neuroleptic haloperidol, Fig. 5. Mean percentages of responding on the lorazepam lever and overall response rates after various barbiturates and methyprylon in lorazepamtrained baboons. See figures 6, 7 and 8 for further details. nor the stimulants mesocarb and d-amphetamine produced 80% lorazepam-lever responding in any baboon. In fact, the median peak (i.e., maximum) percentage of lorazepam lever responding in the baboons tested was not greater than 5%. The drugs were tested up to doses that produced response rate decreases below the range of ND control rates in at least half of the baboons tested (the median percentage of the ND control rate for those baboons was less than 50% in most cases). Time course studies were conducted up to 7 h after dosing with 9 -THC and 15 h with the anticonvulsants and no later onset of lorazepam-lever responding occurred. First-Pellet Analysis Certain results were at variance with those typically obtained for animals trained to discriminate Bzs. Conclusions about drugs sharing discriminative stimulus effects with lorazepam were evaluated further in terms of initial lever selection. For all test sessions, the lever on which the baboon s responding first produced a food pellet was assessed to determine whether this evaluation would have yielded a different conclusion about whether the drug did or did not share discriminative stimulus effects with lorazepam. This

10 1997 Lorazepam Generalization Profile 1451 Fig. 6. Percentages of responding on the lorazepam lever and response rates in test sessions 60 min after administration of barbiturates, methyprylon and their vehicles (V) to lorazepam-trained baboons. Data points generally represent single determinations for each baboon, except that they are means of two for some tests with methyprylon (10 32 mg/kg for LE) and phenobarbital (10 mg/kg for MS and 18 mg/kg for RA). The grand means of these data are in figure 5. The control mean response rates (r/s) for each baboon across these curves were: LE, 2.6 to 3.0 and LO, 0.5 or 0.6, except both were 1.5 during phenobarbital; ML, 2.2 to 2.8; MS, 1.6 to 2.1, except 1.1 during phenobarbital; RA, 1.7 to 3.1; and RF, 2.4 to 2.8, except 1.6 during methyprylon. analysis did not change the conclusion about those drugs that did or did not share discriminative stimulus effects with lorazepam, nor did it reveal greater consistency across baboons for curves in which there was a great deal of intersubject variability, but it did reveal that results of single tests would have been seen differently. Lorazepam lever selection analysis of the data for the non-bzs shown in figure 6 revealed: amobarbital-nonmonotonic lever selection for baboon LE, but not for the other baboons, phenobarbital-nonmonotonic lever selection for baboon RF rather than zero drug-lever responding, and lever selection at 32 mg/kg for baboon MS, compared with partial

11 1452 Ator and Griffiths Vol. 282 generalization; secobarbital-lever selection by baboon LE at 18 mg/kg, compared with partial generalization; methyprylon-nonmonotonic lever selection for baboon MS, rather than an orderly function, and lever selection at 56 mg/kg for baboon RF rather than partial generalization. For hexobarbital (fig. 8), there was lorazepam lever selection at 56 mg/kg p.o. for baboon MS 30 min after dosing, but there was no lorazepam lever selection for the one baboon (ML) that had otherwise been shown to generalize. For methohexital (fig. 9), the conclusion of partial generalization for baboon ML with i.m. dosing would change to none. Lorazepam lever selection data also were evaluated for the Bzs. For the baboon (RF) that showed a maximum of 65% lorazepam responding in any test with diazepam, the first food pellet of the test sessions at 18 and 32 mg/kg was obtained after responding exclusively on the lorazepam lever. Thus, such an assessment results in a conclusion that diazepam shared discriminative stimulus effects with lorazepam in all six baboons. Two baboons failed to generalize completely to chlordiazepoxide (fig. 3). For one (LO), the first test with 32 mg/kg resulted in lorazepam-lever selection; but the second resulted in selection of the ND lever (overall percentages in the two tests were 56 and 59%, respectively). Thus, averaging the results of the tests in terms of lever selection also would have yielded a 50% choice. For the other baboon (LE) that failed to generalize to chlordiazepoxide, selection of the lorazepam lever occurred in two of the four tests with 18 mg/kg p.o., which mirrors the total percentage of lorazepam lever responding in the four tests (i.e., 4 45%). Thus, conclusions about generalization to chlordiazepoxide were not altered by use of a lever selection analysis. Even for Bzs for which full generalization was shown (including lorazepam), there were tests in which total lorazepam-lever responding was high but the ND lever was selected initially. Fig. 7. Percentages of responding on the lorazepam lever and response rates in test sessions 30 or 60 min after administration of p.o. or i.m. pentobarbital to lorazepam-trained baboons. Each symbol represents a different baboon (see fig. 3 for key) and the solid or dashed lines represent the group means. Each data point represents a single determination, except that the points for the open squares in the righthand panels represent the mean of two determinations (three at 5.6 mg/kg). Discussion The present study in baboons is the first to explore systematically the generalization profile for lorazepam-trained animals by testing a wide range of sedative/anxiolytic and other drugs. The lorazepam generalization gradients replicate the initial mean p.o. lorazepam gradients previously presented for those same baboons (Ator and Griffiths, 1986). The present mean gradients generally showed a 0.25 to 0.5 log 10 unit shift to the left compared with those earlier gradients. However, within the three dose-effect determinations conducted across a 4- to 8-month period for the present study, there was no consistent probability of a shift to the right or left with subsequent determinations. Thus, lorazepam stimulus threshold is concluded to fall within the range of doses encompassed between the one that never occasioned lorazepam responding and the one that always did (cf. Ator, 1990). The nine Bzs tested differed in their chemical structure (i.e., classical 1,4-Bzs, such as diazepam; the 1,4 triazolobzs, triazolam and alprazolam; the 1,4-imidazoleBz, midazolam). They differed in their usual clinical uses (e.g., antianxiety, intravenous anesthesia, hypnotic), bioavailability, routes of metabolism, and formation of active metabolites (review in Rall, 1990). All were tested via the oral route and at the same pretreatment time as the training drug, lorazepam. Despite the fact that these Bzs have been shown, in humans, to have different rates of uptake and elimination, and to differ in their formation of active metabolites (Ellinwood et al., 1985; Garzone and Kroboth, 1989; Greenblatt et al., 1989; Kaplan et al., 1976), most occasioned 100% lorazepam-lever responding in all baboons tested. The strongest exception was chlordiazepoxide, which was concluded to share discriminative stimulus effects with lorazepam in only three of five baboons, despite manipulation of the route of administration and study of the time course of the drug effect. The biotransformation of chlordiazepoxide begins shortly after administra-