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Use of a Dlluto Aqueous Solution (5 ncg/ml) of a Bensimidazole Derivative with Potent Morphine-Like Actions Orally as a Presuaptive Relnforelng Agent in Conditioning of Drug-Seeking Behavior in the Rat

By Abrahaa Wikler, W.D. Phillip C. Green Herbert D. Salth and Frank T. Pescor

From ths
National Institute of Mental Health
Addiction Research Center
U. S. Public liealth Service
Lexington, Kentucky

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Use of a Dilute Aqueous Solution (5 mcg/ml) of a Benzimidazole Derivative with Potent Morphine-Like Actions Orally as a Presumptive-Reinforcing Agent in Conditioning of Drug-Seekting Behavior in.the Rat.

During the past 12 years, one of us has repeatedly called attention to certain aspects of the behavior of addicts which strongly suggest (a) that relapse after "cure" may represent, at least in part, a conditioned response to stimuli that have regularly been assoclated with the periodic rellief of such abstinence distress as develops between doscs of opiates during previous episcdes of addlction (1,2,3,4); and (b) that the probability of such "conditfoned" relapse is directly related to the "effort" ("hustling") expended by the addict in his drug-seeking behavior during previous episodes of addiction (5). For heuristic purposes, these concepts have been expressed formatly in terms of both "classical" (Pavlovian) and "instrumental" (inciuding "operant") models of conditloning (1,6,7,8), the former emphasizing the role that conditioning or the abstinence syndrome may play in relapse, and the latter, the role of the organisa's manipulative activity.

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More recently, a few investigators, operating on hypotheses siailar in some respocts to those diseussed above, have reported evidenco obtained in studies on animeis indicating both that the opiate abstinunce syndrone can becoms soniitioned (y) and. that "preierence" for morphine, persisting for periuds beyond the axpected uuratiea of ths abstiasnce synerose, cen be developed by instrusental conditicning during addiction (lo,ll).

In our iabiratories, rspeated attenpts to condition eitner the moiphine atstinence synarone or drug-seeking behavior in rats have previously ket with failura, apparently becauss of the aversive reactions incaced ly tne paln oi subcuteneous injuctien eí noryiine or the zitter taste el sulutluas of morphine or other optates. Llheviss, a promislng study cn rats in which the anisals deiivered doges of morphine to themselves through an impiznted Iintrzperitoneal cetheter by an operant technique, enued dlsasirously afier a few weeks, beczuse the Intraperitonzal upening of the polyethylene catheter becsas ectludtc by a thich merirane. Not to be deterred by such trifiss, we regar to cunsidar seriorsly the priparation of animels with chronically inplanted intravenous cathetcrs, although the prospects for raintaining sudt: preparatlons intact for several sonths were not bright.

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Meanwhile, however, our attention was directed to the properties of e new drug, 1-(Beta-diethylaninoethy1)-2-(pethoxybenzyl)-5-nitrobenzimidazole methane sulfonate (NIH-7607), which was then under stucy for its addiction llability on the research wards of this Center. This drug, which will be referred to by lts ARC nuaber, I-G-2, in this report, had been synthesized by chemists of the Ciba Pharmaccutical Company in Basel, Switzerland, and was found by their pharmacologists (12,13) and others subsequentiy to exert typical morphine-like actions in animais in extremely small doses. Thus, i-G-2 is 1,000 times more potent than morphine as an analgesic in rats (i3), end 1,500 times In the mouse (14); in addicted monkeys, 1-G-2 is 1,500 times as potent as morphino in suppressing signs of abstinence from morphine (15). In post-addicts, Isbell and Fraser. (16) found that 1-G-2 1s 80-l20 times more powerful than morphine as a eúphoriant in single oral doses, and l.ag of I-G-2 is as effective orally as 60 mg of morphino subcutaneously, in suppressing abstinence froa morphine. Tolerance to repeated doses of I-G-2 deyelops rapidly in the rat (13) and in man (16), and in the latter, the dcgree of "physical dependence" developed is coaparable in intensity with that produced by equivalent doses of morphine (16).

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Such evidence of the morphine-like properties of I-G-2, and of its very much greater potency, suggested to us the posslbility that rats might not be averse to drinking an aqucous solution of this drugs in concentrations that would produce morphine-like effects after consumptlon of sqall quantitles. A "single-blind" taste=discrimination test performed by one of the authors on another, yielded a threshold concentration of 4-5 mcg of I-G-2 per al of water. Hoping that the discriminative capacity of the rat is somewhere in thls vicinity. calibrated drinking tubes filled with 3, 5 or 10 mcg/ml aqueous solution of I-G-2 were offered to rats deprived of water for 2h hours. The rats drank ell the solutions avidly, and within foar to seven minutes after beginning to drink (or after consumption of 6ó-lhh mcg/kg of I-G-2) they exhibited morphinelike effects -- exophthalmos, tail rigidity, stupor (snout sometimes falling into the well of the drinking tube) and/or hyperactivity (qulck, Jerky movements of head, darting about, "exploring," chewing on floor of ths cage). One rat, sfter consuming about 330 ccg/kg of I-G-2, lay prostrate on its side with outstretched hindfeet, breathing lrregularly. Within a few ainutes after subcutaneous injection of l0 mig/kg of nalor= phine, however, it rscovered, assuming the upright position, and rasponding to prodding. Addicted rats (maintained on

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200 mg/kg of morphine by subcutaneous injection oncs dally for several months), likesise showed no apparent aversion to the I-G-2 solutión (5.ocg/al) when water-deprived. These animals were tested just prior to their regular dally dose of morphines when the acute effects of the previous day's dose (tall rigidity, hyperactivity) were no longer apparent, and when they were exhibiting signs of morphine-abstinence, notably, repeated discrete twitches of the skin of the back, resembling the rapid skin-shaking of a wet dog, or of a horse shaking off a fiy (sce below). As in the non-addicted rats, typical morphine-like effects appeared within four to seven minutes after beginning to drink, and conconltantly, tho "wet dog" twitchas ceased, sugges= ting relief of abstinence, which persisted until the time for the regular daily dose of morphine a few bours later. The only differenca observed in the effects of l-G-2 in the addicted rats was that the "sedative" effects were much shorter in duratlon, hyperactivity predominating within an hour after ingestion or the drug. .

These prellainary observatfons indicated that a dilute solution of I-G-2 might well be substituted for morphine as a reinforcing agent by ths oral route, and thereby enable us to circuavent the difficultias that attended our earlier efforts

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to induce rats to drink solutions of morphine or other oplatess or to welcome subcutancous injections of these materials. The present communication Is' in the nature of à "progress report" on two studies that have been initiated, with this purpose in mind,

METHODS

In Study A, 6 "experimental" rats, gradually brought up to a constant daily dose level over a period of six to ten weeks, ,are maintained on once-dally subcutaneous injections of 200 mg/kg of morphine, given about 2 p.a., while 6 "control" rats receive volumetrically equivalent subcutaneous injections of physiological saline solutioa on the sane schedule. All rats ers allowed food ad libitue, but they are deprived of water for 22 hours each day, after the training and testing experiments in the morning. The study Is designed to proceed in thres phases. In Phase 1, all rats are given access each morning to a wire cage 24 x 17.5 x 17.5 cm, divided into two equal compartments by a transpercnt vertical partition from the central entrance to which they can see two drinking tubes, placed vertically at the far end of each compartment. The rat can enter either compartoent, drink fros the tube therein, and also go back around the partitlon to the other compartaent to drink from the tubs there. On three succeasive days both tubes contain water, but on the

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fourth day, one of the tubes contains a 5 mc3/al solution of I-G-2, the coapartment in which this tube is placed being alternated in successive four=day blocks (total of 17 blocks, or 63 days). Dlscriminative stioull (bllnking 1ights, metal strips with alternating black and whits stripes on drinking tube and floor), for later use in Phase Il, are also provided, but in Phase I, the sides. of the compartaent in which they are placed are randonized. Each day, the rats ars allowed to drink for 15 ainutes, and records ars sade of the volumes consused from cach tube every day, during successive three=ainute periods. After cach l5-ainute trial, the rats are returaed to their home cages, where they are allowed to drink water ad llbitua from a metal cup for two hours, efter which all water is removed until the next morning. Tho purposes of Phase I aro to determine whethcr or not, without dlscrlminative tralning, either tha "experimental" or the "control"rats-exhibit preference for, or aversion to I-G-2 in ths concentration used on the basis of taste, and whether or not the discriminative stimuli have appetitive or aversiva properties independent of i-G-2. Since ths grossly viaible effects of l-G-2 begin as early as four minutes after commencement of drinking, the critical measures for taste discrimination ars the relative quantities of I-G-2 solutlon and tap water drunk from s given tube (right or left)

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during the first three minutes. Similarly, the critical measures for the reinforcing properties of the discriminative stimull per se are the relative amounts of water (only) consumed when these stimuli are In the right or.left compartment. In Phase I, the i-G-2 solutlon is presented only every fourth day, to mininize conditloning by effcct and the development of tolcrance. In Phase li, all the rats, maintained on morphine or saline and on food-and-water schedules as before, are given access to a cage similar to that already describsd but constructe of plastic and of larger dimensions (40 x 26 x 35 cm) with an opaqus partition. However, the daily schedule is designed to perzit learning of a discrisination of the l-0-2 solution by effect, using successive six-day block trials as follows. Cn the first four days of each block, only one tuba is presented, containing elther tap water the first and'second days, or the I-G-2 solution ón the third and fourth days, the order deing reversed for cach successive block. The compartment in which the I-G-2 solution is placed is always equipped with the discrtainative stimuli previously nentionsd, and renains the same for a given rat throughout all the blocks, though initiaily, the rats are assigned to one or the other I-G-2 compartment (right or left) in alternation. Also, after

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commencing to drink on each of ths four days, egress from the compartment is prevented by closing a rear gats, to ensure that whatever reinforcing effects tha water or 1-G=2 solution may have, will be associated with that compartment and its diseriminative stimuli (if any) only. Cn the fifth day, the water tube is Invariably presented in its usual compartment, and egress is prevented as on the previous four days. On the sixth day of each block, both water end I-G-2 tubes (the latter with the discriminative stimull) are presented in their respective compartments, the egross gates remain open, and the rats are permitted to drink from elther tube at any time within tho 15-minute period of testing. The critical measure in Phase Il is the relative amounts of 1-G-2 solution and water consumed in the first three minutes by the "experimental" and "control" rats on each of the sixth-day test trials, after correction for whatever appetitive or aversive effects the discriminative stisuli may have been found to exort per se in Phase I.

In Phsse Ill, all rats will be tested for "relapse" sevcral weeks after abrupt withdrawal of morphine, under conditions of water satiatlon and water=deprivation, by the technic described for the sixth day of each block in Phass Il.

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In Study 3, "experiaental" and "control" rats are'prepared and maintained on morphine or saline injections respectively as In Study A. Each morning (about 18 hours after the previous daliy dose of morphine, 200 mg/kg), they are placed in an operant conditioning apparatus equipped with a lever whlch, when depressed, activates a mechanism that uncovers a dipper containing 0.25 al of fluid, set on the floor of cags about 6 ca to the rear of the lever. No triais are run on Seturday or Sunday (although, the daily sorphine or saline fajections are continued), but on the other five days of each weck, the rats are given access to the lever under four conditions which aro randonized and balanced over a period of seven weeks: 22 hours wates deprivatlon, water reinforcement; 22 hours water deprivation, I-G-2 solution (5 mcg/ai) reinforcement; water satiation, water reinforcement; water satiation, I-G-2 reinforcement. Each trial in the operant conditloning apparatus proceeds for 15 sinutes, after which water is supplled ad libitum in the home cages for two hours on days preceding the "water deprivation" runs, and throughout the day and night before the "water satiation" runs, food being available at all times throughout. Diseriminative stimuli in the oparant conditioning apparatus consist of rough (hardwars cloth) surfaces for the floor and lever on trial days with weter

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reinforcenent, and sxooth (plastie) surfaces with l-G-2 reinforcement. In addition, on daya with I-G-2 reinforcemant, each lever press actuates a clicker placed Inside the Skinner Box, to serve as a "sacondary reinforcer" (representing the traditional "bad associate" of narcotie addicts' lore).

The critical measures in this study are the coaparative rates of bar-pressing thronghout the 15-minute trial periods within cach group ("experimental" and "control") for water versus I-G-2 reinforceaent, and coaparative rates of barpressing between the two groups for water or I-G-2 reinforcement, both measures referring to water-satiation days, ln addition, similar comparisons are made of unreinforced barpressing rates on water satiation daya, since both "experimental" and "control" animals engage in such activity (possibly because of uncontrolled secondary reinforcers) as well. Study B is also scheduled to.procoed in three phases, the first'of which has already been described. In the second phase, bar-pressing rates for water and I-G=2 relnforcoments will be compared within and between both groups of animals under conditions of water satiation during the acute morphine withdrawal period, and later, in the third phase, tests for "relapse" wiil be made in both groups, under conditions of water doprivation and water satiation, and the discriminative sticuli and (controlled) secondary reinforcer already described.

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If posltive results are obtained after compietion or Study B, it is planned to conduct a similarly designed Investigation, with a aumber of schedules of reinforcement, to enalyze the Influence of various paraaeters of "effort" or "hustling" on the probability of "rslapse."

RESULTS

At the present timé, only the first phases of both atudies have been coapleted. The data for this phase of Study A have been analyzed only with rogard to the question of the capacity of the animals to discrininate between water and the l-G-2 solution by taste. As shown in Table l, the overall differences obtained seea to indicate that, without discriminntive training. tha "experisental" rats exhibit a slight "preference" end the "control" rats a slight "aversion" to the l=G-2 solutlon, but. these differences are not statistically significant by the chi square test.

Several analyses. were made of the data obtalned in the first phase of Study S. In Table 2 are shown intergroup comparisons of "wet dog" responses per 15-minute trial in experisental and control animals on separate days, under the four conditlons of the experiment. It ls noteworthy that on water satlation days, the number or "wet dog" responses

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exhibited by the experinental animals was greater than those of the control animals to highly significant degrees by the Mann=Whitney U test for intergroup comparisons (17), except for one trial cay although, sven then, ths trend was in the sane direction (mean of 5.20 for the experimental, and 2.60 for the control animals). In contrast, no significant differences were observed on water deprivation days regardless of whether the relnforcement used was water or the I-G-2 solution.

The analyses presented in Table 3\* reveal that on water $3 ^ { 1 }$ deprivation days, the reinforced bar-pressing rates of experimental rats exceeded those of control rats, regardless of whether water or I-G-2 was used as the reinforcenent. On water satiation days, however, the reinforced bar-pressing rates of experlmental animals generally exceeded those of control animals only when reinforcement was provided by l-G-2. Acding perhaps to the theoretical significance of these resuits are the comparisons (Table 3) between reinforced and non-reinforced bar-pressing rates of expericental and controi aninals on water satiation days. When reinforced by I-G-2, the reinforced berpressing rates are significantly different for the two groups of anlmals, but not the non-reinforced rates; ahen reinforced by water,nsither the roinforeed nor the non-reinforced rates aro generaliy different to significant degrees. On the other

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hand, the intragroup coaparisons shown in Table 4indicate $\vdots ^ { \frac { 3 } { 4 } }$ that on water satlstion days, neither the experimental nor the control groups showed significant differences between bar= pressing rates with water and I-G-2 reinforcements, while on water deprivation days both groups exhibitsd signiflcantly greater rates with water than with I-G-2 reinforcement.

DISCUSSION

The data for the first phase of. Study A Indicate that, without discriminative training, neither experiaental nor control animals display either preference for or averslon to a 5-ncg/al solution of I-G-2. This finding is of basic importance for the subsequent phases of both studies, since the hypotheses to be tested predict the development of a "preference" for I-G-2 by the experimental, but not the contral anluals.

The findlngs-so far obtalned In Study B are of interest in several respects. The much greater incidence of "wet dog" responses in the experimental rats, observed at 18 hours of abotinence and up to the time of the reguiar dally dose of morphine, as well as the virtual disappearance of these responses after injection of morphine or ingestion of I-G-2, suggest that they may be rellable indicators of tha morphine abstinence

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symdroma in this species. Earlier observations in this faboratory of a more casual sort, inclcate that "wet dogging" continues at a high rate for two or three days alter abrupt withdrawal of morphina and then gradually subsidas to Incidences exhiblted by nonaddicted rats cver a period of about two to three woeks. However, nore quantitative studies are needed to eatablish the reliability of this measure, and to correlate it with other abstinence phenomens in these animals. Also, the curious findling that water deprivation reduces the incidence of "wet dog" responscs in acutely ebstinent rats to those of control anisals, requiros elucidation.

The generally higher bar-pressing rates of experimental than control enimals in Study S say be cue to factors other than thet postulated theoretically - nezely, that "physical depaadenco" provides a "drive" state through reduction of which, "succesaful" drug seeking behavior becomes reinforced. Seemingly, the operation of such other factors is suggested by the data obtained on wster deprivation days, when the bar-pressing rates with vater relnforcement exceeded those with I-G-2 reinforcement, not only in the control but also in the exporimontal aninals. This, however, may de due to the fact that on water deprivatlon days, both groups of animals

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consume cnough of the I-G-2 solution to produce visibie morphine= lihe effects within four to seven minutes, after which their rates of bar pressing declines sharply. On water satlation days, bar-pressing rates were much lower In both groups, and gencrally, nelther group consused enough I-G=2 to affect the rates of bar pressing. Under such circumstances, certain of the evidence (Tabie 3) indicates that the i-G-2 solution was reinforcing for ths experimeatal group only, but evidense for e neat discrlaination between water and l=G-2 relnforcement conditions by these anisals was not obtained in this phase of the study (Table h). As noted earller, both of these studles are still in progress and it is hoped that more conclusive resuits, either consonant or not consonant with the hypothesis, will be forthcoming in tho near future.

SUMMARY

1. Evidence in the literature Is reviewed supporting the concept that, in part, at lcast, relapse is due to conditloning factors, both of the "classical" and "instrumental" variety operating during previous episodes of addiclion to narcotie drugs.

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2. The morphine-like properties of a new benzimidazole derivative (NIH-7607, ARC I-G-2) are described and avidence is presented that neither morphine addieted nor control rats display either preference for or aversion to a 5-mcg/ai aqueous solution of the drug, without discriainative tralaing.

3. Two studies in progress are described, in which this concentration of I-G-2 is being used as a reinforcing agent in attempts to condition drug-seeking behavior in rats.

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REFERENCES

1. Wikler, A.t Recent Progress in Research on the Neurophysiologicai Basis of Korphine Addietion. Aa. J. Psychiat., 105: 329-338,1948.

2. Wikler, A: A Psychodynamic Study of a Patlent During Self-regulated Readdlction to Morphine. The Psychiat. Ouart., 26: 270-293, 1952.

3. Wikler, A.: Opiate Addiction: Psychological and Neurophyslological Aspects in Relation to Ciinical Problems. Springficld, 11linois. C. C. Thonas, l953.

4. Wikier, A. and Rasor, R. W.: Psychlatric Aspects or Drug Addiction. Aa. J. Med., 14: 566-570, 1953.

5. Wikler, A.: Rationale of the Diagnosis and Treatment of Addictlons. Connecticut State Med. J., 19: 560-559, 1955.

6. Wikler, A.t Memorandum to Director of Rescarch, HIMs Addiction Research Center, Lexlngton, Kentucky, 31 Decenber 1956.

7. Wikler, A.: Mcchanisos of Action of Oplates and Opiate Antagonists. Public Health Monogr. No. 52, PHS Publ. No. 589, Washington, D. C. U. S. Govt. Printing Office, 1958.

8. Wikler, A.: Narcoties. Chapter XX, .pp. 334-355 In The Effect of Pharmacologic Agents on the Nervous System. Publ. Assn. Res. Nerv. Ment. Bis., Vol. 37, Baitiaore. Wllllama and Wi13ins Co., 1959.

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9. Irwin, S. and Seevers, M. H.t Altered Response to Drugs In Post-addict Macaca Mulatta. J. Pharmacol, & Exper. Therap., 116: 31-32, 1956.

10. Nichols, J. R., Headlee, C. P. and Coppock, H. W.t Drug Addiction. I. Addietlon by Escape Training. J. Am. Pharmaceut. A., 45: 788-791, 1956.

11. Boach, H. D.: Morphine Addiction in Rets. Canad. J. Psychol., 11: 10h-112, 1957.

12. Hunger, A. J., Kehrle, J., Rossi, A. and Hoffaan, K.t Synthese basisch substituirter, analgetisch wirksamer Bensimidazol-derivate. Experientia, 13: 401-h03, 1957.

13. Gross, F. and Turrian, H.: Über Benzimidazolderlvate mit starker analgetischer Wirkung. Experientia, 13: 401-405, 1957.

14. Eddy, N. B.I Personal Communication.

15. Dencau, G. A., cCarthy, D. A. and Seavers, M. H.t Physical Dependence Llability Studies in the Monkey. Addendum I, Min. of 20th Meet., Coamittee on Drug Addiction and Narcotics, Div. Med. Sei., Hatl. Res. Couneii, Nati. Acad. Sci., washington, D. C. 10-11 January 1959.

. 16. Isbell, H. and Fraser, H. F.: Personal Communication.

17. Siegel, S.: Non-Paranetrlc Statistics for the Behavioral Sciences. Now York, McGraw-H1ll, 1956.

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FOOTNOTE 1

For computation of intergroup and intragroup differences in bar-pressing rates (Tables 3 and 4), oniy the data obtained during the last 18 days of Phase 1 (Study B) were used, since just previously, a change had been made in the fiuid-relnforcement dispensing mechanism, which resulted in augmentation of the rates of bar pressing of all aniaals, "experimental" and '"control."

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Table 1.

Comparisons of per cent fluid consumed in first three minutes of drinking froa rlght and left tubes, when these contained I-G-2 solution (8 trlals for right, 9 for left), and when both tubes contained tap water (24 trlals for right, 27 for left).

The plus sign (+) indicates consumption of an excess of I-G-2 over water.

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Table 2.

Nean "Wet Dog" Frequencies (f). Intergroup Comparisons Setween Experimental (E) and Control (C) Animais.

CONDITIONS:

0 P NVNVNV .05 22 .01 800 ? .001 N.S. Not significant

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Table 3.

Conditions:

WDw Water deprlvation, water reinforcetent WDI Water deprivation, I-d-2 reinforcenent WSW Water satlation, water relnforcement WS1 Water satiatlon, 1-0-2 reinforcement

A .05 d .01

π# P .001

N.S. Not sIgnIficant

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Table 4-

Lean Reinforced Bar-Pressing Frequencies (F). Intragroup

Comparlsons Between Experlgental and Coutrol Aninals.