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From the National Institute of Mentai Health Addiction Research Center, U. S. Publie Health Service Hospital, Lexington, Kentueky Cross Toleranec BetweenLSD and Psiloeyhin

-: HARRIs ISBELL, A. B. WOLDACI, A. WIKLER nd E. J. MINER {Reccired December 10, 1960)

Recently it has been shown (HoFa ef al., 195Sa; DEzx ef al., 1958; Isa, I959) that O-PhosphoryI-4-hydroxy.N-dimethyl tryptanine (hereafter referred to as psilocybin), a compound isolated (HortN et al., i958b) fron certain species of mushrooms that are used eeremoníally by Mexican Indians (WAssoy and Wassox, 1957), has psychotounimetic properties similar to those of the diethylamide of lysergic acid (LSD-25). The close resemblance of the patterns of symuptoms induced by LSD and psiloevbin suggested that these drugs produce mental aberrations by some common action or by affecting different mechanisms sharing a common final pathway. Since the effects of LSD diminish rapidly when the drug is given daily (LseLl ef al., 195G), it was felt that if the LSD and psiloeybin syndromes have a conumon mechanisin, this hypothesis could be further tested by determining if "eross toleranee" betweeu the two drugs existed. In other words, if the degree of the reaction induced by a given dose of psilocybin was significantly- less in a person tolerant to LSD, cross tolerance would be said to exist; and, conversely, the reaction to a given dose of LSD should be redueed in a person tolerant to psilocybin. In the latter case it is impliedl that "direet" tolerance to psilocybin can be developed.

Mothods

Experiments. Two experiments were periormed at difierent simes. Expériment Il was earried out to determine if administration of a larger doso of psiloeybin given over a longer period of time than in Experi. ment I would create a greater degree of tolerance and cross tolerance.

A "cross-over" design using each patient as his oun control was employed in both experiments and is summarized in Tahle 1.

Both experiments consisted of seven periods: (l) first control, in whieh measurements were obtained after the test doses of psiioeybin and LSD, (2) first chronic administration, in whiet patients roceivedl either psiloeybin or LSD once daily in doses increasing to the test level over a period of G—12 days, (3) first test of tolcrance and cross tolcrance, in which patients were "challenged" with the drug they had been taking

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(test of "direet toleranee") and on the subsequent day with the drug they had not been taking (test of "cross" tolcrane), (4) a withdrawal or "washout" period, in which the patients reccived placebos (Experiment I) or no drug (Experiment II) in order to lose tolerance, (5) a second ¿control period, in which the test doses of LSD and psilocybin were repeated, in order to replicate the control data obtained in the first control- perigd and to determine if tolerance had been completely lost, (6) second chronic administralion, in which the patients received daily doses of the altermate drug that they had not taken in the first period of chronie administration (cross-over"), and (7) finally, the second challenge, wíth tesz doses of LSD and psilocybin as in period 3.

Drugs aud Doses. LSD and psilocybin were given in 30 ee of cherry syrup at S a.m. with the patients fasting. The syrup, which was used to mask the bitter taste of the psiloeybin, served as the placebo. In the first and second control periods the patients received in randomized order 1.ō meg kg of LSD, placebo, and 150 megkg (Experiment I) or 210 meg/kg of psilocybin (Experiment II) before chronie administration of the drugs was begun. Detailed observations were made on these test days. These. control experiments were condueted at intervals of at least five days in order that any tolerance conferred by the first drug would be lost.

During the first and second periods of chronie administration the patients in Experiment I received 0.25 meg,kg of LSD or 25 megkg of psilocybin on the firss day. These doses were inereased 0.25 meg/kg (LSD) or 25 megkg (psiloeyhin) daily antil the patients were receiving 1.5 meg/kg of LSD or 150 meg/kg of psilocybin on the sixth dlay. These doses were maintained until the tests of tolerance and eross toierance were performed. In Experiment I the patients received 0.15 megkg of LSD or 21 meg kg of psiloeybin ou the first day of chronie administration, inereasing by 0.15 meg/kg of LSD or 21 meg/kg of psiloeybin daily until the patients were receiving 1.5 uneg/kg of LSD or 210 megkg of psilocybin on the tenth day. These doses were maintained througi the twelfth day. The order in which the patients reccived the drugs in the first and seeond periods of ehronie adninistration was randomized in both Experiments I and II. During these periods of ehronic adninistration, detailed observations were not made,

On the fírst day after comupletion of the period of chronic adnuinistration the patients were "challenged" with the dose of drug they had been receiving (test of direet toleranee). On the seeond day, they were

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1 We are indehted to Des. R. BircnzR andl C. IENzs of Sandoz Pharmaceuticals, Hanover, New Terser, for supplies of psilocybin and diethyiamide ot lysergie acid tartrate (LSD.25). 77

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challenged with the test dose of the alternate drug (test of cross toleränce).
Oa both of these days detailed mucasurements were made.

The patients then received placebos for 7-10 days (Experiment I) or no drug for 13 days (Experiment II). It was presumed that the patients would lose any tolerance they had developed, since in previous experiments (IsBELu et al., 1956) tolerance was largcly dissipated within three days after discontinuation of LSD.

Following this withdrawal period, second control measurements were obtained after the patients hadl received in randomized order placebo (Experiments Land H), 1.5 mcg, kg of LSD (Experiments I and II) and 150 megkg (Experiment I) or 210 nieg kg of psiloeybin (Experiment II). with at least five days intervening between adminstration of LSD and psilocybin. .

The patients then again reccived the drugs chronically, those parientz who had taken LSD in the first period of chronie adininistration were given psilocybin according to the sehedules described above andl vice versa. They were then "challenged" with LSD and psiloeybiu in the manner described above.

Preliminary Assay. Experiment II. Siuce the test dose of psilocybin (150 mcg,'kg) had a lesser degree of effect than the test dose of LSD (1.5 meg/kg), a preliminary assay was carried out prior to Experiment II, The dose-response curves obtained by Isnett (1959) were extended and 210 megkg of psilocybin were estimated to be equal to 1.5 meg, kg of LSD. Aecordingly, the above doses of LSD and psiloeybin were admínistered on two occasions at intervals of seven days in random order to 10 subjects. Statistical analyses (see below for method) reveaied no significant differences in any of the comparisons made (Taule 3, Assay Study).

Subjeets. The subjects who volanteered for both experiments were former opiate addicts who were serving sentences for vioiation of the United States narcotic laws. Their ages varied between 25 to 35 years all were physically healthy males, and none presented any evicence of the major psychoses. All had psychiatric diagnoses of charaeter or personality disorders, and all had received LSD in ptevious experiments. Ten subjects served in Experiment I. and 9 in Experiment I.

General Conditions. Subjects were housed in & special ward devoted to clinical research, Temperature, respiratory rate and blood pressure were measured three times daily after tho patients had rested quictly in bed during days on which speeial measuements were not being made, The patients were observed by specially trained aides with long experienee in deteeting drug-induced cluanges in behavior.

Observations. During each day of the control periods and the periods of ehronic drug administration during which the patíents were "ehal-

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lenged" with placebo, LSD or psiloeyhin, the following observations were made at hourly intervals, after 10 minutes rest in bed, twice beforo and eight times after adutinistration oí drugs: rectal temperature, palse rate, systolic blood pressure, pupillary sizo and threshold for clicitatiou of tho knee jerk. The methods usedl were those previously deseribed (IaELL et al., 1956; IsBELL et al, 1959; IsDELL, 1959). Iu audition, the ; patients (with the help of an aide) completed a special questionnaire at hourly intervals from 7:30 a.a. to 3:30 p.m. At these saue times, general notes on behavior were written. Clinical grades of the intensity of the reaetion were assigned on the basis of the system of IsueLL et al. (1956).

Analvsis of Data. The elanges in reetal temperature, pulse and respiratorý rates, pupillary size, blood pressure, and threshokd for elicitation of the knee jerk after atministration of placebo and drags were calenlated by subtraeting the average of the two pre-drug observations fron the values obtained at various hours. The areas under the time-sction curves for each particular nieasurement couposed of thcse figures were caleulated by the method of WER and FLaTE (1950), thus converting all the data ou a particular subjeet, a partieular drug. a particular measurement, and a partieular day to one figure termned "degree-hours" (temperature), "rate-honrs" (pulse rate), ete. The total number of positivo respouses on the questionnaire were counted over the entire period, eliminating answers which were also seored positively before the drug had been given. Means and standard errors of the means were caleulated aceording to standard statistical techniques.

The difference in the various mcasurenents after placebo, 1.3 meg, kg of LSD, and 150 or 210 meg kg of psiloeybin (each individual drug against itself) in the first andi second controls were evaluated by a t-test for paired observations (Epwands, 19+6). In Experiment I the only statistically significant difference found between the two sets of controls was a decrease in the pytetogenie effect of psiloeybin (Table 2). In Experiment II, significant deercases in tlie numiber of positive responses on the questionnaire oecurred in the seconu control (Table 3) after both LSD and psilocybin. Beeause of these differences in the two controls. the ehanges in response to the test doses of psilveybin and LSD after chronie administration of either drug were evaluaçed by comparing the effeets of LSD and psiloeçbin after the first and second periods of chronic drug administration with the correspouuiing first or second eoutrol. In adtition, enleulations were made using the averages of the two controls. The latter procedure did not aiter the siguificance of the differences gteatly, so oniy the tables showing the lifferences calmulated with the iadivilual first and second controls are presented heroin,

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Figures represent the mean differvnces  standard errors of the differonees between tesponsés to the samne doae of the samue drug (placeho, 1.5 mcgkg of I.S D.25 and 150 meg/kg of psilocybin) on the first and seconrl controls on 10 subjeets. Notte of the differences exeept that for temnetature change after psiloeybin were significant.

: + Indicates that the average measurepeat was increased on the second control. — Indicstes that it was decreased,

= P < .05.

The differenees in the effects of the two individual drugs (LSD vs psilocybin) were also calculated for both control periods using the sanie statistical technique for paired observations (Tables 4 and 5).

As explained above, the differences in the response after chronic administration of both LSD and psiloevbin were ealeulated by comparing the responses after first and second chronic admiuistrations of LSD and'or psiloeybin with their respective first andl second controls. Four different conparisons were madle: (1) respouse to LSD after chronic administratiou of LSD ("direet" toleranee to LSD), (2) response to psilocybin after ehronie administration of LSD ("crosa" tolerance to psilocybin), (3) rosponse to psiloeybin after chronie administration of

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psiloepbin ("direet" tolerance to psilocybin), and (4) response to LSD after chronic admninistration of psiloeybin ("eross" tolerance to LSD). The signs of the differenees were so arranged that a minus (—) sign indicated a deerease in the measurements after chronic administration as cempared with control, and a plus (+) sign indicated an inerease. ren h

Since psiloeybin has a shorter duration of action than LSD, the differenees (exeept elinical grade) were also evahuated, using values obtained at the peak of both LSD and psiloeybin reaetions rather than using the area (integráted tiue action eurves) as deseribed above. fn addition, the differenees were evaluated by a non-parmetrie rank order test for paired observations (Wtcoxoy, 1949). The signifieanee of the differenees by these statistical techniques agreed weil witht thuse obtained by the t-test on the tine-action (area) figares, so only the differences obtained by the area method are reportesl in this paper.

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Results

Controls. The differences in the responses to the same doses of the same drug in first and second controls after placobo, LSD) and psilocybin are shown in Tables 2 (Experiment-I) and 3 (Experiment I). In Experiment I, the only ehange that was statistically significant (p< 0.05) was a decline in elevation of body tempcrature after the second control ¿dose of psiloeybin. In Experiment II, a significant decline occurred in the number of positive responses on the questionnaire following the second eontrol doses of both LSD and psilocybin. This might indicate that some-degree of residual tolerance was still present after 13 days. i she ssnnses to the two

Equivalénée of Dosage. . The differences in the responses to vne two different active drugs (LSD and psilocybin) are presented in Tables 4 (Experiment I) and 5 (Experiment Ii). In Esperiment I the responses were generally greater, as indicated by the preponderance of positive signs in Table \*, and these differences were statistically significant on three measures in both the first and second controls. Therefore, in Experiment I, the test dose of psilocybin (150 meg:kg) was weaker than the test dose of LSD (1.5 meg, kg). In Experiment I, comparisons were made on three occasioņs — "assay study", first, and second controls. The majority of the sigus in Table 5 are positive, indicating that ou the average the effects of 1.5 meg kg of LSD were somewhat greater than thosc of 210 megkg of psiloeybin. The differences were, however, statistically significant only in the case of the pulse rate in the first control and the pupillary change in the second control. Since the failure to demonstrate statistically significant differences may have been due to the large variability in some of the measures, the effcets of 210 meg, kg of psiloeybin in Experiment II may, therefore, still have been weaker , than those of 1.5 megkg of LSD.

. Tolerance and Cross Tolerance. The differences in the responses to LSD and psilocybin after chronic administration of either drug an their respective first and second controls are shown in Tables 6 (Experiment I) and 7 (Experiment II). In both tables, the second columa shows the difference in response to LSD as compared with the corresponciiag first or second control after chronie administration of LSD, and reflects "direet" tolerance to LSD. The third eolumn shows the difference in response to psilocybin as compared with the appropriate controi after chronic administration of LSD, and reflcets "eross" teleronce to psilocybin. Similarly, the fourth column presents- measures of "direet" tolerance to psilccybin, and the fifth cohumn "eross" tolerance to LSD. 11- -ene rorr similar in the

Inspection of the tables shows thuat results were very simuar n wie two experiments. All signs are negative in boah tables, indicating an average decrcase in response on ail measures. In the case of "direet" tolerance to LSD (sceond columns), the dlifferences reached statistical

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significance on six of seven measures in both experiments. Iu the-caso of "eross" tolerance to psiloeybin (third columns) the differenees were statistically significant in fivé of seven measures (Experimcnt I), and four of seven measures (Experiment II). In the ense of "direet" tolerance to psiloeybin (fourth columns), statisticaily signifieant change occurred in four measures (Experirent I), and in thrce measures (Esperiment II). In the case ot "eross" tolerançe to LSD (fifth columns):signifieant degrçes of change oecurred in four parameters in both experiments. The nucasures whieh refleeted "direet" tolerance and "eross" tolerance most clearly were the pupillazr diameter, responses on questionnaire and the elinical grades.

Diseussion

The data show that a considerable degree of "dircet" toleranceto LSD was developed in both Experinients I and II, and that patients "directly" tolerant to LSD aiso had a considerable degree of "cross" tolerance to psiloeybin. Although statistically siguificant deereases did pot oceur on as manv measures. the data indicate that definite "direct"tolerance to psilocvbin was developed and that patients tolerant to psiloerbin were "eross" tolerant to LSD. However, under the conditions of these oxperiments. the degrees of direet tolerance to psilocybin and cross tolerance to LSD were Iess than the degrees of direct tolerance to LSD and eross toleranee to psilocybin, In this connection, the fact that the direction of change was negative (reciuction in the degree of reaponse) may be important even though the differences did not reach statistically significant levels in all parameters. Inereasing the dosage and length of time during which psilocvbin was administered (Esperiment II) did pot result in the development of any greater degree of direct toler. anee to psilocybin and eross tolerance to LSD than occurredt with the lower dosage and shorter period of chronic administration in Experiment I.

The finding that "direet" toleranee to psiloeybin and cross tolerance to LSD could not be shown on as many measures might he due to one. or a çombination of several factors. In Experimuent I. the effects of the dose of psiloeybin were definitely less than the effeets of the dose of LSD employed, and in Experiment II the effects of the dose of psilocybin preseribed were probably weaker than those of the LSD. Thus the stimuius for the development of tolerance cluring ehronie atlininistra. tion of psiloeybin may have been weaker than was the ease with LSD. The length of aetion of psiloeybin is shorter than that of LSD) and, since only one dose of eaeh drug was given dlaily, the stimulus for the development of toleranee was not present for as loug a tinio during chronic administration of psiloeybin, and the timne during which tolerance nuight be declining, due to laek of sustained drug effeet, was grenter. Tolerance

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to different effeets of the two drugs uight develop at different rates.. Such differential rates of tolerance developmeut occur; for example, with morphine (rapid and nearly comuplete tolerance to the analgesic effeets, slower and only partial tolerance to the miotic and respiratory depressant effeets). One might also postulate that LSD and psilocybin have somewhat different mechanismns of action or aet on different receptors. It is also possible that failure to dlenonstrate tolerance and cross toleranee refleets nothing more than tho high variability in certain of the measures used (temperature, pulse rate, blood pressure, knee jerk and, responses on the questionnaire). The data are not sufficient for a determination of the relative roles of any of these hypothe. tical factors.

CeaLErT (195S) did not obserye direct tolerance to the pyretogenic effeet of psiloeybin on daily adiuinistration to rabbits, but did find that rabbits "directly" tolerant to LSD were also "eross" tolerant to the temperature-clevating action of psiloeybin.. Thus the results in the rabbit are similar to those observed in man, and do not help in deciding which of the possible explanations given in the preceding paragraph is the most likely.

BaLEstRr (1960) did not observe direct tolerance to psiloeybin or cross tolerance to psiloevbin in patients receiving LSD chronically. The- details in BaLEsTRIenr's paper are not sufficient for a proper evaluation, but the mumber of patients used was small and the doses of psiloeybin employed were low.

The development of "cross" tolerance hetween LSD and psiloeybin reinforces the idea derived fron the siniilarity of clinical effects (IsBELL, 1959) that LSD and psilocybin indluce psyehie disturbances by some common meebanism, or by different mechanisms which aet through a common final pathway. The data, of course, shed no light on the possible nature of such a presumed common action. Biochemical, chemical, neurophysiological or psyelological mechanisius (or some combination of them). couid be involved.

Summary

1. In two experiments, using a cross-over design, the development of "direet" tolerance to LSD and psiloeybin was measured after 10 (Experiment I) or 9 (Experiment II) volunteers had taken LSD in doses inereasing to 1.5 meg kg over the course of 6—7 days (Experiment I) or 13 days (Experiment II). On another oceasion. the same patients received psilocybin in doses inereasing to l50 megikg over the course of 6-7 days (Experiment I) or 210 meg kg over the course of 13 days (Expcriment II).

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2. The development of "cross" tolerance to psilocybin in patients "direetly" tolerant to LSD was measured by "challenging" the paticnts, after they had received LSD ehronically, with 150 meg/kg (Experiment I) or 210 meg/kg (Experiment II) of psiloeybin. "Cross" tolerance to LSD was evaluated by "challenging" the patients, after they had received psiloeybin chronieally, with 1.5 mcg;kg of LSD.

3. A high degree of "direct" tolerance to LSD developed in both experiments, as manifested by statistically signifieant reductions in six of the seven parameters of response. Patients "direotly" tolerant to LSD wére also "eross" tolerant to psiloeybin on five (Experiment I) or four (Experiment H) parameters.

f Definite "direct" tolerance aiso developed after chronie adnuinistration of psiloevbin in both experiments, but statistically significant redúctions occurred in fewer parameters of respouse (fourin Experiment I and three in Experiment II) than was the case with LSD. Patients chronically treated with psilocybin were also "cross" tolerant to LSD on four (Experiment I) or three (Experiment II) measurements. The degree of "direet" tolerance to psilocybin was less than the degree of "direct“ tolerance to LSD.

5. The development of "cross" tolerance between LSD and psilocybin reinforces the idea that these two drugs eause psychie disturbances by acting on some common meekanism, or on mechanisms acting through a eommon final pathway.

Referenees

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1

ISBELL, H., R.E. BELLEVLLE, H. F. FRaSER, A. WIkLER and C. R, LOGAN: Studlies on Iysergie acid diethylamide (LSD.23). L. Effeeta in former morphine adiicta and development of toterence during churonie inroxication. Areh. Neurol. Psvehiat. (Chicago) 76, 168-478 (1950). n à

—, C. R. Loca and E. J. Mrsza: Stadies on lysergic acid diethyfamide (LSD.25. III. Attempts to attenuate the LSD-renction in man by pretreatnient with neutohumoral blocking agents. Areh. Ncurol. Psychiat. (Chieago) 81, 2027 (1959). [Lvvvj "

Wassox, T. P., and R. G. Wassox: Mushroons. Russia and history. New York: Pagtheon Books 1957.

Wicoxox, F.: Some rapid approximate statistical proeeduree. New York: American Cyanamid Compant 1949. :

W, C. A, and L. FLTaER: Sulies on eptazone {6-morpholino-4.4-diphenyl-3 heptanone hydrecliloride) in comparison witli other analgesie agenta. J, Pharmacol. exp, Ther. 08. 305—317 1050).

Dr. HaRRis IaBELL NIH Addietion Research Center U.S. Publię Health Service Hospital Lexington, Kenrucky,U.S..