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THE METABOLIC FATE OF

LYSERGIC DIETHYIAMIDE (LSi7)

Iii LABORATORY AI,ItiALS

by

ZAHID HUSSAIN SIDDIK

a thesis submitted for the degree of

Doctor of Philosophy

in the

University of London

September, 1975 Department of Biochemistry, St. Mary's Hospital Medical School, London, 42 ABSTRACT

The metabolism of [i14lk C]-LSD has been studied in the Rhesus monkey, guinea pig and the rat. Marked species differences were observed in its excretion and metabolism. Rats excreted 71, of the dose (1 mg/kg intraperitoneally) in the faeces in 96 h, 16-, in the urine and 3.42, as 14CO2. Guinea pigs eliminated 40';‘, of the intraperitoneally administered 14C (1 mg/kg) in the faeces in 96 h, with 282, in the urine and as 14CO2. Monkeys receiving LSD (0.12 or 0.15 mg/kg) intra- muscularly, however, excreted 39-412c in the urine in 96 h and only 22-24;i, in the faeces. Biliary excretion of radioactivity (dose 1.33 mg/kg intravenously) in the rat was very rapid and 682i; appeared in 5 h whereas guinea pigs eliminated only 522L- (dose 0.5 mg/kg intravenously) in 6 h. Ring hydroxylation was extensive in the rat, glucuronides of 13- and 14- hydroxy-LSD being identified as the main excretory products. A third metabolite was identified as 2-oxo-LSD. 13-Hydroxy-LSD was a minor biliary metabolite as was monoethylamide (LAE) in the urine. Metabolites found in vivo were also present in the bile from isolated perfused livers in addition to some LSD. The perfusate and liver contained mostly LSD but also some glucuronides, 2-oxo-LSD, LAE, nor-LSD and probably "aromatised" 2-oxo-LSD. Ring hydroxylation and 2-oxidation, giving 14-hydroxy-LSD (as the glucuronide) and 2-oxo-LSD respectively, were major transformation products of LSD in the guinea pig. The glucuronide of 13-hydroxy-LSD, LAE and LSD were formed in small quantities. The monkey urine contained very little 13- and ik-hydroxy-LSD glucuronides. Some LAE, LSD and probably "aromatised" 2-oxo-LSD were also present. Of the fourteen compounds tested, seven, including LSD, altered the EEG pattern of the rabbit after intravenous administration. The stability of LSD and the fluorescence properties of LSD and a number of the metabolites have been investigated. 3

;EEO;EMEt

The work described in this thesis was carried out between October 1972 and June 1975 in the Department of Biochemistry, St. Mary's Hospital Medical School. I wish to thank Professor R.T. Williams, F.R.S., for the great interest he has shown throughout the course of this project. To Dr. L.G. Dring and Professor R.L. Smith, I am deeply grateful for their encouragement and helpful advice over the last three years. I will always appreciate the willingness of my fellow research workers, members of staff and the technical staff to assist practically whenever necessary and in providing helpful information. I will remember always Dr. R.B. Franklin for being a constant source of encouragement and practical advice during my first two years. I am indebted to Dr. R.D. Barnes, not only for carrying out the difficult task of synthesising the authentic compounds, but also for his many helpful discussions. I also wish to thank Dr. J.H.J. Durston for his supervision during the EEG study and for his analysis of the results. Miss Joan Caudrey and Miss Emilia Szigeti provided technical assistance during the BEG recordings and I am most grateful. My sincere thanks to Miss Rene Anderson for so capably typing this thesis. Finally, words can never adequately express my appreciation to members of my family for the understanding, patience and encouragement shown to me at all

times.

This work was carried out during the duration of a Research and Development Contract (NRC71/917) between the Medical Research Council and the Department of Biochemistry, St. Mary's Hospital Medical School, London, W2. 4

INDEX

Abstract 2

Acknowledgements 3

Chapter One Introduction 5

Two Materials and Methods 67

Three Metabolism of [14C]-LSD in the Rat, Guinea Pig and Rhesus Monkey 101

Four The Fluorescence of LSD and its Derivatives and the Stability of LSD 175

Five The Effects of LSD and its Derivatives upon the Electroencephalogram (EEG) of Rabbits 204

Six Concluding Remarks and Scope for Future Metabolic Study of LSD 212

Appendix 216

References 225 5

CHAPTER ONE

Introduction

Contents page

Introduction 6 Naturally Occurring 6 History of and the Discovery of LSD-25 10 The Psychic Effects of LSD 12 Illicit Use of LSD 13 Therapeutic Use of LSD 17 Toxicological Studies of LSD 20 Behavioural Effects of LSD 24 Pharmacological Effects of LSD 26 The Mechanism of Action of LSD 28 Biochemical Effects of LSD 37 Structure-Activity Relationship 38 Chemical and Physical Aspects of LSD 41 General Aspects of the Metabolism of Foreign Compounds 43 Absorption, Binding and Distribution of LSD in the Body 46 Metabolism of LSD 51 In Vivo Studies 51 In Vitro Studies 54 Inhibitors of the Metabolism of LSD 57 The Metabolism of Compounds Structurally Related to LSD 57 Scope of the Present Study 66 Introduction

(+)-Lysergic acid diethylamide (LSD, LSD-25, delysid, lysergide) is the most potent of a of compounds which produce changes in thought, perception and mood and elicit visual and auditory hallucinations. These compounds were collectively named "phantastica" by Lewin in 1924. This designation never gained popularity especially among the English-speaking countries and so Hoffer and Osmond (1967) suggested the term "hallucinogens"; producers of hallucinations. True hallucinations, however, were rarely observed in the drug-induced states and this led to the designation "psychotomimetics", coined by Gerard in 1955. But the drugged state did not quite mimic the naturally occurring psychosis as suggested by the term psychotonimetic and it also became inappropriate. "Psychedelics", meaning mind-manifesting, was the term suggested by Osmond (1957) "because it is clear, euphonious and uncontaminated by other associations". "Psychotaraxics" (Caldwell, 1958), meaning mind-disturbers, "psychodysleptics" (Delay, 1959), "psycholytics", "mysticomimetics" (Valzelli, 1973), "psychotogens" and "illusinogen" (Cohen, 1970) are other designations that have been used. Of the above mentioned synonyms, , psychedelic and are most often used in the scientific literature. kiaturally occurring Hallucinogens Most of the naturally occurring hallucinogens are of plant origin and the respective plants have been used as or sacred drugs since time immemorial. Primitive cultures, where sickness and death are usually ascribed to a supernatural cause, have long accorded hallucinogenic plants a high, even sacred, rank in their magic, medical and religious practices (Schultes, 1969). Aside from this, human beings in their persistent drive to acquire unusual states of consciousness have always used drugs. Some major hallucinatory constituents of the psychotomimetic plants are shown in Fig. 1,1.

Fig. 1.1. Structures of some major hallucinogenic constituents of psychotomimetic plants

0 on 11 cEr 0„,--CP2N(5)3 o 3 Muscimole

ci130 CH2C H "2 2

0H3 O 3

Yescaline

CF2CH2N(OH3)2

a) 111=H; R2=H: N,N—Dimethyltryptamine b)R I=H; R2=0H: Eufotenine c)R I=H; R2= CC 5—Nethoxy—N,11— a)R 1=H; R2=H: (+)—Lysergic acid amide dimethyltryptamine b)R I=CH0RCH3; R2=H: (-0—Lysergic acid d) RI=OH; R2=H: met hylcarbinolamide e) RI=OPO3H; R2=H: a) R1=e2H5; R2=C225: (+)—LSD OH

c5 HII (n) (n)

CIL CH3

ZS,9— A8—Tetrahydrohydrocannabinol 8

Of the hallucinogens, 'soma' is the most ancient. According to the old Sanskrit manuscripts, the drug, used several thousand years ago in religious rites in India and Central Asia, "made one feel like a God". It is no longer known from what plant soma was obtained (De F6lice, 1936). The use of the mild hallucinogen hashish (Cannabis sativa, marihuana, Indian hemp) dates back more than 2500 years. According to Herodotus (500 B.C.), the Scythians, Who inhabited what is now the Soviet Union, threw hemp seeds on to hot stones and intoxicated themselves by inhaling the vapours (Lewin, 1924). During the course of time, hashish became widely used throughout the world. In thirteenth century Asia Minor, the hashishins were political murderers who, by ingesting hashish, would carry out murder for pay; assassin is derived from this Arabic term. The drug became very popular in France around the middle of the nine- teenth century, especially among poets and writers in Paris (Caldwell, 19(0). The interest was such that "Club des Hachichins" was formed in Faris. members of the club included people such as Gautier and Baudelaire, the latter describing his experiences with hashish in 1860 in his famous book "Les Paradis Artificiels". Today, the drug is eaten by millions, especially in Moslem areas of North Africa and the Near East. It is in India, however, that hashish assumes extraordinary religious significance. The ancient Atharva-Veda called it a "liberator of sin" and "heavenly guide" and it is still used in temples as a sacred plant (Schultes, 1969). In addition to its religious use, it is valued by the poor of India in folk medicine and as an aphrodisiac. The hallucinogenic action of hashish is mainly due to its content of tetra- hydrocannabinols (Hofmann, 1968). , also called "fly agaric", is a mushroom that grows in Europe, Asia and North America. Koryak nomads, from the Kamchatkan peninsula in northeastern Asia, discovered its hallucinogenic property and found that the voided urine after ingesting the mushroom was also hallucinogenic. Its activity in the central nervous system is probably primarily due to muscimole although other minor constituents like musearine and bufotenine may help in potentiating the effect (L'arnsworth, 1960). Zany of the plants that are hallucinogenic are to be found in Ilexico and South America. Teonanacatl, peyote and ololiuqui are of Ilexican origin and , vinho de Jurema, yopo and virola snuffs are found in South America. Hofmann (1963), .arnsworth (1968) and Schultes (1969) give fuller accounts of the history of these and aplther "magic" drugs. Teonanacatl, meaning "God's flesh" or "sacred mushroom", was used as far back as 1500 3.C. by the Aztecs, the original inhabitants of aexico. i.ushroom- shaped stone statues about a foot high have been found in auateaala, and were probably part of some religious cult (liasson and asson, 1957). The cult has continued ever since and Wasson and Wasson (1957) have reported the hallucino- genic effect of the teonanacati which they experienced during a secret nocturnal mushroom ceremony in Oaxaca. The main active components of teonanaaatl

(silocybe) are psilocybin and a small amount of psilocin.

Peyote (Lophophora williamsii), containing the active principle , has been widely used by the Indian tribes of Mexico and North .mrierica. De 3ahagun was the first to report on the effect of the peyote cactus plant in the sixteenth century (see Lewin, 1924). Bven today peyote is ingested by certain nexican Indian tribe6 during religious festivals. In the United States the Native American Church, which includes around 200,000 Indians as its members, uses the drug as part of its ritna1 (Schultes, 1969). Gloliugui

(rivea corymbosa) seeds were also used by the Aztecs in their magico-religious rites. Hernandez wrote that, through ololiuqui, Aztec priests "communed with their gods when a thousand visions and satanic hallucinations appeared to them" (Schultes, 1969). It is still used today for this same purpose by certain Indian tribes in Mexico. The hallucinogenic activity of the ololiuqui is attributed to the presence of the ergot alkaloids (+)-lysergic acid amide and

(+)-lysergic acid methylcarbinolamide. These are of particular interest since they are structurally related to LSD (see Fig. 1.1). Indians of the Amazon have access to another hallucinogen, a drink known as ayahuasca, caapi or yage. It is prepared from the stems of the Banisteriopsis caapi vine and contains the main alkaloid harmine. Ayahuasca was employed by the Indians for purposes of prophecy and divination and to prepare the male adolescent for the painful rites of initiation into manhood (Farnsworth, 1968). Vinho de Jurema, another South American magic beverage, is prepared from the seeds of Mimosa hostilis plant. It contains N IN-dimethyl- and is also used in religious ceremonies by the Pancaru Indians in Brazil (Hofmann, 1968). Snuffs are another form in which the hallucinogenic plants are used to promote communal friendliness, convulsive dance rhythms or a state of religious conviction. Among the most commonly inhaled by the Indians of the Colombian Amazon are yopo (also called cohoba) and the virola snuffs. These preparations contain the active chemicals bufotenine, 14,U-dimethyltryptamine and 5-methoxy-M,N-dimethyltryptamine (Schultes, 1969). Many of the drugs mentioned have come to be closely associated with religious rites and initiation ceremonies; they have become an inter al part of faith in primitive religions. Although quite powerful, none of the drugs are as active in producing hallucinations as the semi-synthetic compound LSD. History of Ergot and the Discovery of LSD-25 LSD-25 is simply known as LSD in the scientific literature. The initials LSD come from its German name lyserg saure diethylamid and the 25 means that it was the 25th in a set of closely related compounds to be synthesised at the Sandoz Laboratories in Switzerland. Ergot is a very important source of lysergic acid (Fig. 1.2), which is used in the synthesis of LSD. The history ,C2H5 COOH "C2H5 Co -CH3 /r

H Lysergic acid Fig. 1.2 of ergot and ergot poisoning has been exhaustively described by Barger (1931).

that is commonly called ergot is the rhizomorph of the parasitic fungus

Claviceps purpurea, which grows in the ears of various grains such as rye and wheat. In the Middle Ages, the condition known as ergotism occurred in

Europe and was caused by consumption of bread made from rye contaminated with ergot. Symptoms of ergotism ranged from gangrene of the feet and hands, miscarriages, hallucinations and uncontrollable feelings of terror, a desire to achieve total madness and death. In the Middle Ages such epidemics were quite frequent and the disease was called "St. Anthony's Fire". The major epidemic in France in 994 killed about 40,000 people and 12,000 died in the

Cambrai region in 1129. The cause of the epidemics became known in the seventeenth century. Since then there have been only sporadic outbreaks of ergot poisoning. The most recent one was in 1951 when people of a village of Font-Saint-Espirit in France were afflicted with ergotism through the consumption of the local bread (Fuller, 1969).

The medical importance of ergot was realised as early as 1582, when

Lonicer used the extracts obtained from the mycelium portion of the fungus as an aid for quickening childbirth (see reviews by Stoll, 1965 and Mines, 1968). Research on the isolation of the active principles of ergot soon followed but it was not until 1906 when ergotoxine became the first of the pharmacologically useful ergot alkaloids to be identified. This was soon followed by the isolation of in 1918 and (ergonovine) in 1935. Since 1935, extensive investigations have been carried out on the chemistry of ergot alkaloids especially at Sandoz in Switzerland. Stoll and Hofmann (1943) made various derivatives of lysergic acid, which is the characteristic nucleus of all the alkaloids of ergot and can be obtained by alkaline hydrolysis of these alkaloids. One of the compounds that Hofmann synthesised in 1938 was LSD Adth the aim of producing an analeptic like the structurally similar nikethamide. However, pharmacological experiments revealed LSD to be a strong oxytocic agent. In the course of these experiments and five years after it 12

was first synthesised, an accidental observation led Hofmann to the discovery of the powerful hallucinogenic action of LSD (see Hofmann, 1968). Since then, many papers have been published on the clinical, pharmacological, neurophysio- logical, behavioural and biochemical aspects of LSD. The Psychic Effects of LSD

The psychic effects of LSD have been reviewed by Rothlin (1957a), Hofmann (1968), Cohen (1970) and Hoffer (1965). The minimal effective oral dose in humans according to these authors is about 25 ug or less than 0,5 kg, but the optimum dose varies between 100-1000 ig depending on the individual.

However, intense hallucinations appear in normal subjects with an average dose of about 150 pg. sympathetic excitatory effects, such as dilation of the pupils, slight intensification of the heart beat and rise in , appear in about 20 minutes after ingestion. These changes may be accompanied by signs of parasympathetic stimulation, such as salivation, nausea and vomiting. The onset of hallucinations take longer, between 40-60 minutes, and 2-3 hours may elapse before the peak is reached. Between 8 and 12 hours is the mean duration of these actions. The effects of LSD can be speeded ur depending on the route of administration. The intramuscular route produces effects slightly faster than the oral route. The effects begin within a few minutes when LSD is given intravenously and instantly after intraspinal administrations but maximal effects do not occur until after about one hour. The authors report that the psychological effects in man vary and depend upon a number of variables, including the personality of the subject and the setting in which the LSD is taken. The most notable of the effects is the alteration in perception. Colours appear more intense, motionless objects and surfaces move in a wavelike manner and faces of those present seem to be distorted. True visual hallucinations, namely the appearance of images not really there, are the strongest form of change in perception but these are very rare. LSD increases the power of hearing but auditory hallucinations seldom occur. Abnormal smell or taste perceptions play only a secondary role; abnormalities in the sense of touch, however, are not unusual. During the peak of the reaction the body may appear out of shape and time appears to "stand still". Depersonalisation (the disruption in the awareness of the self) and derealisation (the feeling of unreality of the environment) have also been reported. Impairment in thought process occurs due to lowered attentiveness and difficulties in the capacity of concentration. Attempts to quantitate this have been mostly unsuccessful due to the reduced attention and motivation to perform in intelligence tests. The mood is predominantly euphoric and accompanied by compulsive laughter, talkativeness and feelings of ecstasy. However, moods may change suddenly to depression and fear. The following is a part of the original experiences of Hofmann (1968) and is representative of a typical normal reaction of LSD ingestion. "I lay down and sank in a kind of drunkenness which was not unpleasant and which was characterised by extreme activity of imagination. As I lay in a dazed condition with my eyes closed (I experienced daylight as disagreeably bright) there surged upon me an uninterrupted stream of fantastic images of extra- ordinary plasticity and vividness and accompanied by an intense kaleidoscope- like play of colours. I had great difficulty in speaking coherently, my field of vision swayed before me and objects appeared distorted like images in curved mirrors. Moreover, all objects appeared in unpleasant, constantly changing colours, the predominant shades being sickly green and . A remarkable feature was the manner in which all acoustic perceptions (like the noise of a passing car) were transformed into optical effects, every sound causing a corresponding coloured hallucination constantly changing in shape and colour like pictures in a kaleidoscope. The faces of those around me appeared as grotesque, coloured masks. Occasionally, I felt as if I were out of my body." Illicit Use of LSD

After the discovery of LSD it wasn't long before the drug became widely used in the scientific field. The main reason for this was the remarkable ability of the drug to produce intense hallucinations in microgram quantities and provide "model psychoses" for the study of mental illnesses such as schizophrenia (Osmond, 1973). At the same time, a number of artistic people also used the drug to enhance creativity and inspiration (Cohen, 1969). In the early 1960's, Timothy Leary of Harvard University in America became the leading advocate for the use of LSD outside the medical circle (Cohen, 1970) and this attracted much publicity., The mass media began to publicise the drug as the answer to life, instant happiness, the way to increase the ability to solve problems in school and work and to achieve creativity in art and music (Ungerleider and Fuller, 1973). In 1965 LSD use spread and became known in America and Britain as the "acid scene" (Leech, 1970). In August 1966 the concern over the widespread abuse prompted the United nations Commission on Narcotic Drugs to recommend immediate action by governments to control the production, distribution and use of LSD (Leech and Jordan, 1974). Since then, it has become illegal to own, use, buy, sell, import or export the drug illicitly in most countries. The profits in LSD dealings are high and smuggling continues in spite of the severe penalties which include imprisonment. In Britain, the peak of LSD abuse was the summer of 1967 (Leech, 1970). This was preceded by the much attention drawn to LSD by the "pop" singers and groups such as the infamous Beatles with their song "Lucy in the Sky with Diamonds". However, reports of "bad trips" (adverse reactions), chromosomal damage and strict police control reduced the popularity of illicit use in Britain and America (Leech, 1970; Ungerleider and Fuller, 1973). nevertheless, the beginning of 1969 saw a revival of LSD abuse in London but only on a small scale. The "drug scene" in London is concentrated in the Soho, Piccadilly and Notting Hill areas. The "acid scene", however, is most popular among the "hippies" in the Notting Hill district (Leech, 1970). The problem of LSD abuse in Britain is not as grave as that of cannabis judging by the figures issued in the Home Office Press notice (1974). The number of persons found guilty of drug offences (see Table 1.1) indicates that although the use of cannabis has been increasing over the last few years, Table 1.1. Number of Persons Found Guilty of an Offence Involving

Drugs (After Home Office Press iotice, 1974)

Drur, 1970 1971 1972 1-2L

Opium 57 52 88 1 86 226 500 532 460 Cannabis 6682 8212 9316 11246 112 107 179 194 LSD 774 1537 1306 1273

' Others 2758 3404 3189 3286

Total 10579 13812 14610 16645

abuse of LSD is generally on the decline. However, the total extent of L;D abuse in this country is unknown. The majority of the users are young people under 25. ilost of them are males and come from all social backgrounds (Lacune and Hensman, 1971; Forrest and Tarala, 1973a). It would appear that the "drug scene" is normally associated with students (Dewhurst and iatrick, 1972) and a survey in a provincial university in England in the late 1960's revealed that among 2424 students, 9.4, had used some drugs. Most (8> of the total) had restricted themselves to cannabis and and only 1,- of the

total students had used LSD (Leech, 1970). However, in America, a survey on .1 ,91` a national sample showed that therate of conversion to drug use does not occur in colleges and universities, as might be expected, but in the army

(acune, 1972). In the illicit market the LSD circulates as a liquid soaked on a sugar cube, on blotting paper or coloured pills (Leech, 1970). One of the biggest

problems facing the LSD user is the question of purity of these preparations.

In Canada, an investigation revealed that of the 176 street samples, only 97 contained relatively pure LSD (Mattke and Steinigen, 1972). A study in Munich i6

by Mattke and Steinigen (1972) emphasised the dilemma. Among 89 LSD street samples, 58 were pure, 19 preparations of large surface area (sugar cubes, dextrose tablets, powder capsules, blotting paper and solutions) were decomposed and contaminated with substances such as , 11 contained inert materials or harmless compounds (saccharine, dextrose, lactose, urea and potassium sulphate tablets) and one liquid sample was actimlly identified as 60 sulphuric acid. The amount of LSD in samples kmown to contain the drug varied between 10 and 250 g. In New York similar results on LSD purity have been found, with 252, of the samples being adulterated most frequently with amphetamine and (Zacune, 1972). In England, the LSD being circulated in the streets comes entirely from the illicit manufacture of the drug and most of it is smuggled from America

(Adler, 1973; Report by the Advisory Committee on Drug Dependence, 1970). After its synthesis in clandestine, make-shift laboratories, the drug is mixed with four parts of a buffering mixture to minimise oxidative decomposition and sold at about i20 per gram of the mixture (Adler, 1973). After purchase, this is further diluted with glucose and chalk until a suitable concentration is obtained to turn the mixture into tablet or capsule form. The 250 pg tablets and the 600 ig capsules in bulk cost around £250 and :800 per thousand respectively. These are then passed on to the street "pusher" for distribution to his customers. The severe penalties carried in the Misuse of Drugs Act,

1971 for importing, "pushing" or being in possession of the drug and the careful watch placed at international airports and docks for smugglers have contributed to the decline in the use of LSD. After a "trip", the user normally has to wait a few days to obtain the intense effects again from the same dose of LSD. This is due to the rapid development of tolerance and if the same dose is taken daily its effect decreases so that by the fourth day practically nothing is noticed (Cohen 1969). Tolerance is also lost quickly, usually within a few days. True physical addiction is not known among the LSD users, since withdrawal symptoms do not occur. However, dependence on the hallucinogenic effects of LSD is well known. Therapeutic Use of LSD LSD has found increasing medical use especially as an adjunct to traditional psychotherapy interviews. The early interest was to use LSD to construct "model psychoses" for the study of illnesses such as schizophrenia (Osmond, 1973). The repeated and regular use of LSD during psychotherapy has been termed "psycholytic therapy", during which a low dose of LED is given initially but this builds up in subsequent sessions. This use arose from the claims that the drug reduced the patient's defensiveness and allowed access to previously repressed or unconscious facts. This mode of treatment was also reported to shorten the time of treatment (Hofmann, 1968). A number of investigators have used the psycholytic therapy for a variety of emotional disorders with impressive results. Some of these are summarised in Table 1.2. Leuner (1967) reported on 82 patients of which 64 were either greatly improved or fully recovered after 2-8 years of psycholytic treatment. Of the 22 patients with diagnoses including neurotic depression, anxiety states, character disorders and borderline schizophrenia, were classed as having improved in follow-up periods of 6-17 months (Eisner and Cohen, 1958). Eandison and Whitelaw (1957) treated 94 patients, who had failed to respond to conventional therapy, with psycholytic therapy. A follow-up study of between 6 months and about 3 years revealed that 61,- were recovered or improved, while no change was noted in the rest of the patients. Although there have been other reports of successful psycholytic treatment (see Table 1.2), Soskin (1973) found from his study on 11 psychosomatic and 3 non-psychotic psychiatric patients, that LSD was of minimal value. "Psychedelic therapy", administration of a single large dose to produce intense LSD effects followed by intensive psychotherapy over a number of weeks, has also been employed to treat patients. Savage and HcCabe (1973) used a single dose of 300-450 lig on 37 chronic heroin abusers for the psychedelic Table 1.2. Summary of Some Psycholytic and Psychedelic Therapy Studies with LSD (Adapted from Savage et al., 1973

Population Sample Size Dose Range Frequency No. of Improvement Reference (fig) of Sessions Sessions

Depressives 15 20-100 Daily 30 47 Savage (1952) Neurotics 6 20-250 Unknown 1-250 100 Abramson (1956) Mixed 94 25-400 Weekly 1-58 65 Sandison and Whitelaw (1957)

Mixed 22 25-150 Weekly 1-16 73 Eisner and Cohen (1950 Alcoholics 40 200-400 Once One 67 Chwelos et al. (1959) Mixed 110 25-150 Mostly biweekly 1-26 80 Chandler and Hartman A960) Mixed 25 100200 Once One 84 Sherwood et al. (1962)

Mostly neurotics 43 40-175 Biweekly 1-120 79 Ling and Buckman (1963) Neurotics and depressives 77 200-300 Once One 80 Savage et al. (1966) Alcoholics 10 800 Once One 70 Smart et al. (1967) Mixed 82 Unknown Twice weekly, 27 64 Leuner (1967) weekly, biweekly Alcoholics 117 50 or 450 Once One 53; High dose Kurland et al. (1971) 33; Low dose

Heroin addicts 37 300-450 Once One 33 Savage and McCabe (1973) Mostly 14 50-250 Biweekly 5 Minimal Soskin (1973) 0-4. psychosomatics 00 19

treatment. Of these patients, 33;.; abstained from taking heroin for one year after the treatment, compared to 5 in the control group which did not receive 15D (Table 1.2). A short-term initial benefit from LSD psychedelic therapy in the treatment of chronic alcoholics has been reported by Kurland et al. (1971). They administered a single dose of either 50 or 450 pg to alcoholics and reported that at the 6 months follow up, 51: of the patients receiving the high dose but only 33% of those receiving the low dose (controls) were considered greatly improved. However, 18 months after the treatment, improvement rate of about 50, was found for the experimental and the control groups. A recent review of the literature on the psycholytic and psychedelic LSD treatment in alcoholism by Abuzzahab and Anderson (1971) has led these authors to conclude that "the overall effectiveness of this controversial treatment of alcoholics remains disappointing". Kest and Collins (1964) have reported on the analgesic effect of LSD on 50 terminal cancer patients. They found that 100 pg of LSD gave a more prolonged relief of pain. than either 100 mg of meperidine or 2 mg of dihydro- morphinone hydrochloride (Dilaudid). This property of LSD has been adopted in the psychedelic therapy for alleviating psychological stress and physical pain experienced by the dying cancer patient. Grof et al. (1973) claimed that of the 31 patients given 200-500 pg LSD, 29> showed a dramatic improvement from emotional and physical distress, a moderate improvement was noted in 42 remained unchanged and 6;, became worse. They also found that there was an increased acceptance of death by patients who reported "deep religious and mystical experiences". Improvement of homosexual patients has been claimed following LSD therapy. Ball and Armstrong (1961) treated 10 homosexuals with LSD and 2 improved. One homosexual treated with 8 LSD sessions became well and was still normal 6 years later (Martin, 1963). Adolescent boys with behavioural problems, criminal psychopaths and autistic, schizophrenic children have also been treated successfully with the drug (Hoffer, 1965). Use of LSD in conjunction 20

with hypnosis and psychotherapy (hypnodelic therapy) to treat narcotic addicts has been reported by Ludwig and Levine (1965) to be a better form of treatment than LSD and psychotherapy, LSD alone, psychotherapy alone or hypnotherapy alone. Although LSD therapy has been used with success, it becomes apparent that the therapy affects different individuals in different ways; the outcome depending on the patient - therapist relationship (Savage et al., 1973). It appears that apart from a few isolated cases where adverse reaction has occurred (Savage et al., 1973), the worst that can happen to the patients is that they are no better than they were before the start of the LD treatment. Nevertheless, the drug is no longer used therapeutically in Britain.

Toxicological Studies of LSD The LD50 of LSD varies with the species (Rothlin, 1957a). It is 46 mg/kg intravenously for the mouse, 16.5 mg/kg in the rat and only 0.3 me/he in the rabbit. In chronic experiments, rats tolerated a daily intravenous dose of 2.5 mg/kg for 30 days. The rats showed no development of tolerance to the lethal dose whereas rabbits acquired tolerance after repeated exTosure to the drug. In a study by Tauberger and Kilmer (1968), an intravenous dose of 1.6 mg/kg to unanaesthetised cats caused death, although West et al. (1962) state that intravenous doses of as high as 6.5 mg/kg are required to kill a cat. In an attempt to induce an elephant syndrome "musth", an elephant received an of 297 mg (0.1 mg/kg) of LSD. The animal collapsed 5 minutes after the injection and died within 2 hours (West et al., 1962). No lethal dose in man has been recorded, although Hoffer (1965), by inter- polation of the toxic doses in animals, has suggested an LD50 of 0.2 mg/kg or 14 mg for man. This is 150 times the dose required to produce intense LSD effects. The LD50 for other laboratory animals have not been quoted in the literature, although monkeys (Macaca mulatta) have tolerated doses of 1 mg/kg intravenously (Evarts, 1956) and guinea pigs, 1 mg/kg intraperitoneally (this thesis). 21

In 1967, Cohen et al. (1967a) first demonstrated in vitro that the addition of LSD to cultured human leucocytes increased the incidence of chromosomal breakage. This finding was also shown to occur in human leucocytes in vivo (Cohen et al., 1967b). Subsequent investigations concerning whether LSD is capable of producing chromosomal aberrations, however, have been conflicting. The vast amount of results arising from these studies have been reviewed extensively by Dishotsky et al. (1971) and Long (1972). Although Egozcue et al. (1968), Corey et al. (1970) and a number of other workers (see Long, 1972) have confirmed the original in vivo and in vitro findings of Cohen and coworkers, studies by Sturelid and Kihlman (1969), Loughman et al. (1967), Bender and bankar (1968) and Fernandez et al. (1973) on human leucocytes were among the many that failed to show any significant increase in chromosome damage caused by LSD. Similar contradictory results have been reported on the leucocytes of other animals (Long, 1972). Opponents of the suggestion that LSD is responsible for the damage to chromosomes often quote the results of Kato and Jarvik (1969), who have demonstrated that acetylsalicylic acid () and LSD induce chromosomal breakage rate in leucocytes to the same extent. A few studies have suggested that LSD could be a potential causing leukaemia (Cohen et al., 1967a; Irwin and Egozcue, 1967; Grossbard et al., 1968) and two cases of leukaemia have been reported in patients treated with pure LSD (Tylden, 1968; Garson and Robson, 1969). There is no definite evidence at the present, however, to associate neoplasia with LSD use (Dishotsky et al., 1971). Mutagenesis was studied in drosophila (Grace et al., 1968; Vann, 1969) and fungi (Zetterberg, 1969). The results suggested that LSD was a weak but in doses which were 300,000 times greater than the normal dose of 100 pg used by humans. Wagner (1969) and Smythies and Antun (1969) have shown that LSD intercalates between the bases of the DNA helix causing conformational changes unlikely to account for chromosomal breakage. This interaction, however, does not affect the capacity of the human leucocytes to repair -damaged DNA as would be expected (Dorrance et al., 1974). 22

The Dia binding of LSD has been refuted by Brady et al. (1971) and Snit and Borst (1971), who rresented results supporting their claim. But if the interaction does occur, it may provide the physical mechanism for the weak mutagenic effects of LSD in drosophila and fungi (Dishotsky et al., 1971). Much attention has been focussed on the possible teratogenic effect of I.D. In their studies, Alexander et al. (1967, 1970) showed that LSD treatment of rats resulted in an increased incidence of resorptions, stunted embryos and stillbirths, but no malformations were detected. Furthermore, they reported that the abnormality rate in the births was even greater if offspring from two LSD-treated mothers mated. The drug has also been reported to be teratogenic in hamsters (Leber, 1967), mice (Auerbach and Rugowski, 1967; Hanaway, 1969) and rhesus monkeys (Kato et al., 1970). Idanpaan-Heikkila and Schoolar (1969a, 1969b) have demonstrated that LSD can easily penetrate into the embryos of hamsters and mice via the placenta. This may account for the teratogenicity and also the chromosome breakage in human foetuses in utero reported by Cohen et al. (1968). Gant and Dyer (1971) hypothesise that the toxic effects could be due to the of the blood vessels of the umbilical cord by

L:U,- resulting in a decreased foetal blood flow and . However, other workers have failed to show the teratogenic effect of LSD in rats (Foux et al., 1970; `Jarkany and Takacs, 1968), hamsters (Di Paolo et al., 1968; ).oux- et al., 1970), mice (Roux et al., 1970), rabbits (Fabro and Sieber, 1968) and developing chick embryos (Bergman and Gardner, 1974). There have been 7 reports of malformed children following parental ingestion of illicit LSD before and dtring pregnancy (Long, 1972). s.cG1othlin 1-ate et al. (1970) reported a spontaneous abortion(of, 3W,, in patients who had ingested both illicit and pure LSD. The rate was within the normal range of 15-202-. when the patients were exposed only to pure LSD. In their recent study, Jacobson and Berlin (1972) reported that 10 of the children, born to parents admitting to the use of LSD prior to or during pregnancy, had major congenital defects, mainly of the US, compared to the normal incidence of between Q.5 23

and 1.0'i. They also found that of the women who enrolled in the study during of ptervacy the first 12 weeks 43r/0 had spontaneous abortions, twice the normally accepted figure. However, these adverse reactions may have been caused by the toxic contaminants, such as diethylamine, present in the LSD obtained illicitly and to multiple drug use. A possibility that LSD could prevent pregnancy has arisen by the demonstration that the drug inhibits prolactin secretion in rats, either by a direct affect on the pituitary or by hypothalmic stimulation which causes the release of the prolactin inhibiting factor (Quadri and Meites, 1971; Earfknecht et al., 1974). A direct result of inhibition of prolactin secretion is to ova implantation and affect pregnancy. Surveys of the literature suggest that there is very little evidence to link LSD with chromosonal darage or to describe the drug as a carcinogen, a mutagen or a teratogen in man (Dishotsky et al., 1971; Long, 1972). A number of other complications have also been associated with the illicit use of LSD. These include prolonged psychosis (Hoffer, 1965), recurrent "- backs" (return to the hallucinatory state long after the immediate effects of the drug have worn off) (Smart and Bateman, 1967), anxiety, depression, confusion, convulsions (Ungerleider et al., 1966; Cohen, 1967) and suicide (raker, 1970). Forrest and Tarala (1973b) report that 60 LSD users were admitted to one hospital in Edinburgh in under 3 years, suffering from an untoward reaction to the drug. Furthermore, there have been 5 reported cases of homicides committed under the influence of LSD (Klepfisz and Racy, 1973). The drug has recently been implicated in the increased incidence of infectious disease among the users (Naught 1973). This arises from the work of Voss et al. (1973a, 1973b) and Winkelhake et al. (1974), who have demchstrated that LfK) inhibits the production of antibodies of cultured rabbit lymphoid cells by incorporation of a modified form of LSD into the protein. In spite of these hazards, LSD continues to be popular among the psychiatrists who favour the therapeutic use of the drug as an adjunct to psychotherapy. They believe that the hazards have been overemphasised (Deuhurst and Hatrick, 1972). 24

Behavioural Effects of LSD LSD has central actions and one of the consequences of this is the observed alteration in normal behaviour, which is usually dose dependent. The drug not only affects the behaviour of animals but also of fish and insects. Different strains of fish behave differently when placed in a bowl of water with added LSD. Keller and Umbreit (1956a) reported that under LSD, the goldfish swam backwards, the cave fish remained stationary and the guppy continued to swim against the wall of the container, completely unaware that no progress was being made. The green sunfish became more aggressive and the Siamese fighting fish assumed a nose up, tail down position in solutions of

LSD (Cohen, 1967). The studies on the spider's ability to webs have shown that the drug normally leads to a disruption in the symmetry of the webs

(Longo, 1972). Evarts (1956) reported that monkeys (Macaca mulatta) given a high dose of LSD assumed an initial prone position, with no desire to walk or climb. This was followed by ataxic movements from which they recovered after about one hour. The animals also failed to respond to visual or painful stimuli and were unusually tame. Blindness was characterised by the apparent inability of the monkeys to avoid bumping into objects placed in their path. aeduction in motor activity and gait have also been reported in the baboon after the administration of a low dose of LSD (Laguitina et al., 1964). In addition, the baboons exhibited signs of hallucinating since "they would begin to catch at things in the air, glance around and, as if frightened, jump about the chamber and try to escape". In contrast to the docility in monkeys, LSD induced aggression in cats, which have been reported to exhibit "rage" reaction (Elder and Dille, 1962). Aggression in nice, ants and newts has also been demonstrated (Appel, 1963). Mice treated with LSD persistently exhibited a violent head shaking movement when the back of the head was touched very lightly (Keller and

Umbreit, 1956b). Dixon reported that, following LSD administration, rats 25

demonstrated persistent sniffing accompanied by an increase in locomotor activity, which was subsequently impaired. When in contact with each other, the rats became aggressive. The mating behaviour in the rat was affected by LSD. Administration of a low dose increased the number of ejaculations and a high dose reduced the number of copulation preceding ejaculation (Appel, 1968). In situations involving competition for food or an oestrous female, LSD inhibited dominating behaviour in rats and increased submission. The conditioned responses of animals were affected by LSD. Low doses in rats improved performances and increased exploratory behaviour and high doses had an inhibitory effect on the conditioned response (Torre and Fagiani, 1968). Generally, a depressed rate of positive conditioned response for food was shown, which may be related to the reduction in the animal's disposition to eat, as bas been reported for rats, birds and other species, or to a direct interference in the animal's ability to remember the cue, but not to locomotor inhibition (Appel, 1968). Conditioned response (pulling a ring with the mouth in order to get food) was enhanced in the hungry rabbit (Longo, 1972). However, if the animal had to discriminate between two different cues for reward, then the number of positive responses was reduced. This indicated that LSD impaired proper memory functioning. Winter and Flataker (1956) reported that in rats trained to climb a rope, LSD produced a slowing of climbing time. If, however, rats were exposed to the drug repeatedly, tolerance developed to the behavioural effects and performances gradually returned to normal levels (Tilson and Sparber, 1972). Under LSD, the human subjects became docile and attempted to cope with the unusual state (Cohen, 1967). Hyperflexia, tremor of the extremities, incoordination, ataxia and shivering have also been reported (Valzelli, 1973). In a study of speech rhythms, LSD increased the duration of pauses in monologues and dialogues in patients under psychotherapeutic treatment (Jaffe et al., 1973). However, behavioural effects like aggression and panic depend entirely on the individual and are expressions of his immediate and unique experience under LSD. 26

Behavioural and hallucinogenic effects can be modified by other drugs

(Elder and Dille, 1962; Dixon, 1968; Hoffer, 1965). Pretreatment with steroid hormones, such as , 2-bromo-LSD (a 5-hydroxytryptamine antagonist) and the , perpherazine and , inhibited the LSD effects in man and animals. Inhibition was also demonstrated in animals by pretreatment with , an blocking agent, and 0(-methyl-p-, an inhibitor of catecholamine synthesis but without effect on the 5-hydroxytryptamine levels. (blocks autonomic response to LSD) and agents, and , had no effect on the behaviour. A potentiation of effect was obtained following pretreatment with reserpine. Amphetamine potentiated the hallucinogenic action in man and forms the basis for the common adulteration of illicit LSD with the stimulant drug.

Pharmacological Effects of LSD The pharmacological effects of LSD can be divided into two groups: the peripheral and the central actions. These have been reviewed extensively by Rothlin (1957a) and Cohen (1967) and concisely by Hofmann (1968). LSD had direct peripheral actions on the smooth muscles. It contracted the uterus of the rabbit, both in vivo and in vitro, and this oxytocic effect is characteristic of the ergot alkaloids. Vasoconstriction has been demonstrated in the perfused blood vessels of the human umbilical cord (Gant and Dyer, 1971), rabbit ear (Savini, 1956), rat kidney and in the spinal cat (Rothlin, 1957b). However, When the innervation in the cat was intact, the effect of LSD on the CPIS predominated, lowering the vasomotor tone and resulting in the fall of the blood pressure (Rothlin, 1957a). The pronounced antagonism of LSD to 5-hydroxytryptamine (5HT) on the isolated uterus of the rat was first observed by Gaddum (1953). This antagonism was also shown to occur in other smooth muscles, for example on the guinea pig ileum, artery of isolated rabbit ear (Gaddum and Hameed, 1954) and bronchial muscles of the cat and the guinea pig in vivo (Herxheimer, 1955; 27

Konzett, 1956). However, not all 5HT and LSD interactions are antagonistic.

Gyerrnek and Suni (1963) have demonstrated that the cardiovascular and respiratory reflex actions of 5HT (blood pressure fall, and apnea) in the cat were potentiated by the hallucinogenic drug. It is the blocking action to 5HT which is of importance since it has been postulated as a mechanism of action of LSD (Gaddum and Hameed, 1954; Wooley and Shaw, 1954).

The inhibition of the stimulation actions of and noradrenaline on various smooth muscle preparations and on the isolated rabbit heart has also been demonstrated (Hoagland, 1957). Interrelationship between LSD and the brain amines will be discussed separately with regard to the mode of action of the drug.

The central effects of LSD are numerous. It stimulated the midbrain, particularly the hypothalamus, giving rise to the sympathetic effects which predominated in the LSD-treated animal. Nydriasis was the most prominent feature and has been reported in various species including man (Rothlin, 1957a). Animals, such as the cat, dog and the rabbit, also manifested a rise in body temperature (Horita and Dille, 1954). The rabbit was particularly sensitive, requiring doses of as low as 0.5 - 1.0 ps/kg to produce hyperpyrexia, While in the rat lower doses decreased and larger doses increased the body temperature (Rothlin, 1957a). Other sympathetic effects included piloerection, hyperglycaemia and . LSD also evoked parasympathetic activity and produced salivation and lacrimation. In higher doses the drug stimulated the vomiting centre in the medulla oblongata in addition to reducing the vasomotor activity. The latter effect was responsible for the hypotension and bradycardia observed in the cat, since in the spinal cat, LSD raised the blood pressure and bradycardia was absent. Respiration was dose dependent; low doses produced stimulation and higher doses caused death due to respiratory paralysis.

The somatomotor effects of LSD observed in the cat and the dog included ataxia and paralysis. The drug inhibited the 5HT and the reserpine potentiating actions to hypnotics in mice (Rothlin, 1957a). By itself, LSD potentiated 28

the effect of barbiturates (Hofmann, 1968) and the amphetamine excitation in mice (Rothlin, 1957a) and altered the LEG pattern in animals and in man

(Hothlin, 1957a). The hechanism of Action of LSD

The mechanism of action of LaD appears to be by an effect on the normal functions of the brain. -avidence for this has been the observed changes in the spontaneous cortical activity (LIGG) and the facilitation or inhibition of synaptic transmission following LSD administration to cats, dogs and monkeys

(Hothlin, 1957a; Hoffer, 1964; Aghajanian 1972). Visual hallucinations may be regarded as a result of an unusually large number of stimuli which enter and overload the visual system causing bizarre sensory manifestations

(“inters and 'ivallach, 1970). Partial evidence for this has come from the electrophysiological work of kurpura (1956), who demonstrated an increased visual evoked response in the visual cortex of the cat following La . alectrophysiological studies with rats using a number of compounds, including nucleic acid metabolites, have led Cowen et al. (1972) to postulate that LDD "acts by modulating brain nucleic acid metabolism by a process that requires

5-T synthesis". 13D easily crosses the blood-brain barrier (Rothlin, 1957a) ancl an unequal distribution within the brain of the squirrel monkey has been reported (-nyder and R4rich, 1966). Higher concentrations were found in certain regions and these included the thalamus, hypothalamus, limbic system, auditory and visual reflex areas, pineal gland and the pituitary. 1.61) found in the squirrel monkey brain and the rat brain (preedman and Coquet, 1965; £)iab et al., 1971; Faragalla, 1972) was in the neuronal bound form and the free form. ainding- sites for LSD have been demonstrated in vitro in the rat cerebral cortical synaptosomes and synaptic membranes (Farrow and Vunakis, 1973). This study has shown that a number of structurally different hallucinogens produced a competitive inhibition of LSD binding, whereas the non-hallucinogenic congeners had no significant effect, suggesting a common site where the 29

hallucinogenic drugs act. It is interesting to note that chlorpromazine, Which is known to reduce the hallucinogenic effect of LSD, inhibited the binding but reserpine, which potentiates the effect, increased the amount of

LSD that was bound. These results suggested that the drug produces its effects by binding to specific sites in the cerebral cortex. The lengthy time lag after oral ingestion of LSD and Onset of action led to the theory that the action of the drug was due to an active LSD metabolite produced by the liver (Rothlin, 1957a).. This is unlikely, however, since intraspinal administration of LSD to man produced an instantaneous effect

:Indicative of a direct action. It has also been suggested that the drug acted by triggering off biochemical reactions in the brain, because it was thought J. that by the time the drug started to act, it had already left the brain of the rat and mice. However, it was demonstrated in both rabbits and humans that measurable levels of LSD persist in the blood and brain for cker f? hours

(Cohen, 1970). The structural similarity between LSD and 5HT ('i<;. 1.3) and

--C2H5 CO H N-H N-CH 3

HO\/N___/7 11 N H H

LSD Fig. 1.3 5HT

the experiments showing the antagonism of 5HT activity byA.ddum W)53) and riooley and 'haw (1954) on the isolated rat uterus and the guinea gig ileum, led these workers to postulate that LSD might act by antagonism G 5HT in the brain. This was refuted by the demonstration that 2-bromo-LSD not only crossed the blood-brain barrier and possessed no hallucinogenic property, but was more

potent than LSD in antagonising ,5HT in vivo and in vitro (Cerletti and Rothlin, 1955; Rothlin, 1957a). 30

Subsequent studies suggest that the action of the drug may well involve the neurotransmitter 5HT, as well as the other brain amines. Freedman and Giarman (1962) have reported a significant increase in cerebral 5HT of rats treated with 0.13-1.5 De/kg of LSD, and Rosecrans et al. (1967) showed that this increase was accompanied by a fall in the concentration of 5-hydroxy- indoleacetic acid (5HIAA), the principle enzymic degradation product of 5HT (Fig. 1.4). Freedman and Giarman (1962), however, found that LSD did not inhibit the enzymes involved in the degradation of 5HT and concluded that, since the increase in 5HT was only in the particulate fractions, LSD enhanced the neuronal 5HT binding. Anden et al. (1968) demonstrated that the drug reduced the turnover of the neurotransmitter. However, in oontrast to the single acute dose, chronic administration has produced conflicting results. Diaz and Huttunen (1971) reported that administration of 20 pg/kg to rats for one month produced an increase in the 5HT in the brain, an increase in 5HT turnover and an unchanged 5HIAA level. Peters (1974), in contrast, has found that this dose for 14 days increased brain 5HT but decreased 5HT turnover and 5HIAA concentration. Dose of 100 pc/kg for 14 days did not alter 5HT content but increased 5HIAA level and 5HT turnover. That the increase in 5HT levels was due to the reduction in the release of the transmitter substance from its bound form, was supported by the finding that LSD prevented the reserpine- induced 5HT depletion (Tonge and Leonard, 1969), and it has been demonstrated that depletion of free 5HT by reserpine potentiated the; LSD effects in man (Resnick et al., 1965) and rats (Votava et al., 1967). Conversely, elevating 5HT levels by pretreatment with a monoamine oxidase (14A0) inhibitor or by administering the precursor, 5-hydroxytryptophan (see Fig. l.4), attenuated the LSD state (Cohen, 1967). And& et al. (1968) and Peters (1974) reported that the drug increased the turnover rate of noradrenaline and caused a decrease in the brain concentration of this amine. This may be associated with a slight rise in the brain concentration (Leonard, 1972). Histamine levels have also 31

CH2 CHNH HO CH CHNH 2 21 2 COOH hydroxylase COOH H

L-Tryptophan 5-Hydroxytryptophan

5-Hydroxytryptophan decarboxylase 1 HO CH COON HO CH CH NH 2 Monoamine 2 2 2 oxidase (MAO) N H H 5-Hydroxyindoleacetic acid (5HIAA) 5-Hydroxytryptam.ine (5HT)

Fig. 1.4. Biosynthesis of 5HT and its major metabolic pathway (After Valzelli, 1973)

COOH CCOH COOH CH2CHNH CH2CHNH2 CH CHNH 2 2 2 Phenylalanine Tyrosine hydroxylase hydroxylase

OH OH

L -Phenylalanine L-Tyrosine L-DOPA DOPA decarboxylase CH CH NH 2 2 2

COMT

CH 0 OH 3 3,4 -Dihydroxy - Dopamine Methoxytyramine phenylacetic acid Dopamine 73-hydroxylase HO-CHCOOH HO-CHCH NH HO-CHCH NH 2 2 2

MAO COMT

CH 0 OH 3 3,4 -Dihydroxy- L-Noradrenaline Normetanephrine mande lic acid

Fig. 1.5. Biosynthesis of Dopamine and Noradrenaline and their main Breakdown Products (After Valzelli, 1973) COMT=Catechol-0-methyltransferase MAO=Monoamine oxidase 32

been reported to decline (Cohen, 1967). The change in 5HT, noradrenaline and dopamine levels was reflected in the increased and decreased concentrations of the precursors tyrosine and tryptophan respectively in the brain (Leonard, 1972). Recently, Messiha and Grof (1973) have shown that LSD intake of 200- 300 ig by man resulted in significant urinary excretion of dopamine and 5HT. however, excretion of noradrenaline, its metabolite 3,4-dihydroxymandelic acid

(Fig. 1.5), 3,4-dihydroxyphenylacetic acid (metabolite of dopamine) and 5RIAA as unaltered. The concentration of adrenaline has also been reported not to change following LSD administration (Hollister and hoore, 1967). It appears, therefore, that the action of LSD may be the result of an imbalance in the brain concentration of the neurotransmitters 5HT, noradrenaline and dopamine and probably other amines such as histamine. It is of some significance that structurally different hallucinogens, mescaline, (anticholinergic agent) and (analgesic agent), produce similar changes in the brain but structurally similar non-hallucinogenic analogues do not (Leonard, 1972). Horita and Hamilton (1973) have shown that pretreatment of rabbits with op-c-methyl-p-tyrosine, a catecholamine-depleting agent, attenuated the behavioural actions of LSD which were restored on administration of L-dihydroxy- phenylalanine (L-DOIA), a precursor of dopamine and noradrenaline. Since sodium diethyldithiocarbamate, which blocks the conversion of dopamine to noradrenaline, did not inhibit the restorative action of L-DOEA, they suggested that dopamine, rather than noradrenaline, was involved in the behavioural actions of LSD in rabbits. This conclusion was also reached by Dixon (1968) using rats. However, in the rat, o<-methyl-p-tyrosine has also been repotted to have no effect on the behavioural effects of LSD (Leonard and Tonge, 1969).

Evidence which linked LSD directly with the dopamine receptors came from recent studies. Eieri and Pieri (1974) and lieri et al. (1974) demonstrated that in rats, whose striatum was unilaterally depleted of dopamine by either 5-hydroxy- dopamine or 5,6-dihydroxytryptamine, the dopamine induced the animals to rotate towards the non-lesioned side, whereas , 33

which acted by releasing dopamine, caused an ipsilateral rotation. LSD resembled apomorphine in its effect on rotation. , which blocks the dopamine receptors, inhibited the rotation induced by LSD, apomorphine and methamphetamine. 2-Bromo-LSD and were unable to induce circling in the animals. The conclusion reached was that LSD acted as an agonist on the dopamine receptors in the striatum. The authors, however, pointed out that some ergot alkaloids, such as the non-hallucinogenic ergometrine, also acted on the dopamine receptors to induce contralateral turning. In vitro studies on the rat brain by Von Hungen et al. (1974) showed that LSD mimicked dopamine in stimulating adenyl cyclase activity in the striatum, but inhibited this effect caused by dopamine in the cerebral cortex. This indicated that LSD not only acted as an agonist but also as an antagonist at central dopamine receptors. By itself, the gross effect of LSD was to increase adenyl cyclase activity, which was reflected in the increased cyclic 3',5'-adenosine mono- phosphate (cyclic AMP) level in the rat brain (Uzunov and 4eiss, 1972). In the rat brain, the catecholamines, noradrenaline and adrenaline, are very potent in elevating the levels of cyclic AMP, which has been sufzested to play an important role in postsynaptic transmission (Harris et al., 1974). The cyclic AMP response to catecholamines was blocked by classic alpha and beta adrenergic blocking agents as well as by several phenothiazines and 5HT, which suggested that the rat brain adenyl cyclase receptor for adreneitic argines was relatively non-specific (Fulmer and Burks, 1971). It has also been demonstrated that adrenergic blocking agent, chlorpromazine, and LSD bind to nerve ending fractions which contain high adenyl cyclase activity (De Robertis, 1971). Palmer and Burks (1971) found that LSD and 2-bromo-LSD antagonised the cyclic AMP levels produced by addition of noradrenaline to rat brain slices. These researchers further demonstrated that LSD and 2-bromo-LSD antagonised the adrenergic response to noradrenaline in dog isolated arteries (alpha receptors) and in rabbit isolated hearts (beta receptors). It was, therefore, suggested that some of the central and 34

peripheral actions of these two drugs were due to a non-specific blockade. Substance P, a biologically active polypeptide present in the brain tissue, has also been considered as a possible neurotransmitter (Valzelli, 1973) and it has been shown that LSD, but not 2-bromo-LSD, inhibited the disappearance of this substance when incubated with guinea pig brain extract (Giarman and Freedman, 1965). The substance F has been demonstrated to potentiate the effect of LSD on the guinea pig ileum (Giarman and Freedman, 1965) and the drug-induced tremor in mice (Stern, 1973). However, it is doubtful whether substance F is involved in the production of the LSD effects since its concentration in the mouse brain has not been found to alter (Giarman and Freedman, 1965). A large volume of literature has been published on the 5HT involvement in the brain. This is strengthened by the reported accumulation of LSD in the rat brain area which specifically contains 5HT (Diab et al., 1971). 5HT applied by microiontophoresis had an inhibitory effect on some cells of the brain stem of cats and an excitatory effect on others (Aghajanian, 1972). loakes at al. (1970) have consistently demonstrated that in cats, LSD blocked the excitatory effects of 5HT only, whereas the slightly hallucinogenic or non-hallucinogenic analogues, methysergide and 2-bromo-LSD, although strong antagonists of the peripheral effects of 5HT in vitro and in vivo, had little effect. It was shown that electrical stimulation of midbrain raphe nuclei of the rat increased brain 5HT turnover resulting in a reduced 5HT concentration and an increase in 5HIAA (Aghajanian, 1970). LSD reduced the turnover of 5HT, suggesting that the drug inhibited the spontaneous firing of neuronal units only in the raphe nuclei, by either inhibiting the impulse flow from the nuclei to the 5HT containing neurones or by inhibiting 5HT release from the neurones. 2-Eromo-LSD had only iii of the inhibitory effect on the raphe nuclei (Aghajanian, 1972). The firing of neurones outside the nuclei was either unaffected by LSD or only slightly increased. Since MAO inhibitors and L-tryptophan caused an increase in 5HT and depressed the rate of firing 35

of raphe- neurones, Auden et al. (1968) suggested that LSD acted on the 5HT receptor sites and inhibited the 5HT neurones by a negative feed-back inhibition due to an apparent excess of the transmitter substance. It has, therefore, been speculated that hallucinations may result by a reduction in the receptor stimulation by 5HT (Leonard, 1972). Recently, Berridge and Prince (1974) demonstrated that LSD mimicked 5HT and stimulated fluid secretion in vitro from salivary glands of blow flies (Calliphora). But in contrast with 5HT, LSD continued to stimulate the secretion even after considerable washing of the glands. It was, therefore, suggested that the mode of action of LSD could be by a much longer occupation of the 5HT receptor. Hallucinogens, such as mescaline and dimethoxyamphetamine, and also the non-hallucinogenid 2-bromo-LSD, had the same effect on the secretion. An action of LSD by inhibition of cholinesterases is also a possibility and an increase in brain level in the guinea pig has been found following the administeration of the drug (Rothlin, 1957a). However, subsequent in vivo and in vitro results have been conflicting. Some workers have reported inhibition of various cholinesterases by LSD, while others have

either not detected any inhibition or have shown that in very small amounts

the drug increased the activity of the enzymes (Giarman and Freedman, 1965). Furthermore, 2-bromo-LSD had the same effect as LSD on the cholinesterases.

Recently, Kabes et al, (1972) have reported that the rise of acetylcholine in rat brain caused by LSD may also be due to the increased synthesis of the

transmitter substance. The mode of action of LSD may also be due to a different mechanism involving other biochemical changes. LSD increased oxidation of glucose and

glutamate in cerebral homogenates but reduced it in homogenates of cerebellum,

Whereas oxidation of citrate, succinate and -aminobutyric acid were increased

in both fractions (Cohen, 1967). .-ilrainobutyric acid, which has been implicated as an inhibitory neurotransmitter, was also reported to decrease 36

eEvQ.is in vivo in the rat brain andicorrelated with the behavioural effects of the drug due to an increased CMS excitation (Leonard, 1972). The levels of glycogen phosphorylase in the rat brain were reduced by LSD and other hallucinogens but 2-bromo-LSD and tranquillisers caused an increase (Cohen, 1967). It was suggested that LSD may act by increasing the conversion of adrenaline to adrenochrome (Fig. 1.6), which has been reported to be HO 0 CH-OH CH-OH

CH -0 2 HN N- 1 I CH CH3 3 L-Adrenaline Adrenochrome Fig. 1.6 hallucinogenic (Hoffer, 1965). Although this reaction was demonstrated enzymatically in vitro, the occurrence of adrenochrome in nornal or hallucinating subjects seems doubtful (Giarman and Freedman, 1965). It was initially reported that LSD does not alter circulation of blood through the brain (Hoffer, 1965). Recently, however, Goldman et al. (1975) have demonstrated that LSD affected the blood flow to specific areas of the conscious rat brain. Blood flow was significantly higher to the cerebellum, the frontal and parietal cortex and slightly reduced to the hippocampus 20 minutes after LSD administration. These changes were followed soon after by maximal LSD induced behavioural effects. 2-methyl-LSD, nor-LSD and 2409-LSD had no effect on the animal behaviour and did not alter regional blood flow. The possibility exists, therefore, that the alteration in blood flow could play a role in the manifestations of the LSD effects by affecting the normal metabolic functions of the brain. It would appear that although much is known about the effects of LSD in the brain, the theories regarding its mechanism of action still remain speculative. However, the hallucinogenic action may well be due to its 37

interference with the various transmitters at the receptor and the enzyme level. Diochemical Effects of LSD The biochemical effects pertaining to the mechanism of action of LSD have already been discussed. Plasma free fatty were increased following LSD administration (Hollister and Moore, 1965). Increased ammonia excretion, decrease in the excretion of creatinine, phosphate, sodium, potassium and total amines and marked reductions in keto acids and urea excretion in the urine of rats have also been reported (Hoffer, 1965). A marked reduction in urinary phosphate excretion has also been found in LSD-treated normal subjects (Rothlin, 1957a). LSD increased blood sugar level in normal rabbits (Rothlin, 1957a). However, Mayer-Gross et al. (1953) reported that, in subjects/, LSD produced a small fall in blood sugar and a concomitant increase in blood hexose mono- phosphate level. Similarly, studies in guinea pig showed that the drug stimulated glucose oxidation but decreased the breakdown of hexose mono- phosphate in brain and liver tissues. This suggests that LSD stimulates some enzymes and inhibits others. These results of Wer-Gross and coworkers (1953) have not been confirmed by other workers (Bain, 1957). Liddell and Ue4-Malherbe (1953) studied the sugar and adrenaline levels in the blood of patients suffering from various mental disorders. They found that LSD had no significant effect on the blood sugar concentration but raised the adrenaline levels. Chronic use of the drug in large doses has an anorexigenic effect and there are reports of reduced weights in rats due to decreased intake of food (Rothlin, 1957a; Hoffer, 1965). However, following administration of a low dose of 20 jfkg of LSD for one month, Diaz and Huttunen (1971) did not find any significant change in the body weight of rats compared to the controls. Chronic administration of large doses to dogs has been reported to result in hypoglycaemia and hyperbilirubinaemia with degenerations in the parenchymal cells of the liver and the kidney. 38

Structure-Activity Relationship Of the four possible stereoisomers (Fig. 1.8), only (+)-LSD possesses the rotent hallucinogenic and the anti-5HT activity (Hoffer, 1965). Although the similarity in structures of 5HT and LSD (Fig. 1.3) has been recognised, and the molecular orbital calculalions on 5HT have shown that LSD contains some of the essential features necessary for binding at a 5HT receptor (Kier, 1971), it is not known why the non-hallucinogenic stereoisomers are not 5HT antagonists. The potency of LSD has been attributed to the presence of an energetic highest occupied molecular orbital (HOMO), which is an index of the electron donating capacity of the Tr or resonating electrons of the molecule (Snyder and Richelson, 1970). The TI electrons in the 9,10 double bond can resonate with the electrons in the indolic ring and accounts for the energetic HOI,L. The prediction that reduction of this double bond would decrease conjugation and HOMO and reduce hallucinogenic activity has been itafitea for 9,10-dihydro-

'S1) and lumi-LSD. The HOMO energy of LSD is localised at the 2-position and substitution with to form 2-bromo-LSD reduces HOMO and potency. However, the model does not explain why the other stereoisomers of LSD, which probably have the same HOMO energy, are not hallucinogenic. From a study of the molecular configuration using X-ray diffraction techniques, Baker et al. (1972) believe that LSD is active in the protonated form, the positive charge

being on the in the 6-position. No correlation exists between hallucinogenic potency and anti-5HT activity among the many LSD derivatives (Hofmann, 1968). 2-Bromo-LSD and

1-methyl-2-bromo-LSD are stronger antagonists of 5HT than LSD but have little or no effect in eliciting hallucinations. It is reasonable, therefore, to hypothesise that different parts of the molecule in a fixed conformation are responsible for the two actions. Kumbar and Sankar (1973) have partially

correlated molecular orbital properties of LSD and derivatives with hallucino-

genic property and suggest that the entire molecule of LSD may be important in producing hallucinations. A hydrophobic region in the 5HT receptor is 39

predicted and the hydrophobicity of the amide has been suggested to be important in anti-5HT potency (Dunn and Lederka, 1974). A survey of the hallucinogenic potency of the LSD derivatives by Kumbar and Lankar (1973) shows that 1) the hallucinogenic activity of double substituted derivatives (one in the ring and one in the side chain) is greatly reduced compared to the monosubstituted derivatives (either in the ring or in the side chain); 2) the non-polar groups such as 0E3 at position-1 cause a treater reduction in the hallucinogenic activity than the polar groups such as COOH3; 3) the increase or decrease in the number of in either or both of the two ethyl chains attached to the amide nitrogen leads to a considerable loss of hallucinogenic potency. Generally, any change in the

molecular structure of LSD by substitution of groups leads to a reduced hallucinogenic response. An exception to this is (4.)-1-acetyl-Lo:) which is

as active as IUD (Hofmann, 1968). This derivative is de-acetylated in the

body and forms LSD, which accounts for the high potency. In direct contrast, substitutions can either reduce or increase the anti-5HT activity. Ab. 1.7 compares the pharmacological and hallucinogenic activity of 16D and some of

its derivatives. The left-hand side indicates the hallucinogenic activity and

the right-hand side shows the pharmacological effects. The syndrome of excitation (total E-syndrome, dotted line) is caused by sympathetic stimulation

and results in mydriasis, piloerection, hyperthermia and so on. oath compounds

numbers 1-9 the hyperthermic effect (I, strong continuous line) parallels the

syndrome of excitation. In most of the remaining compounds the hyperthermic

effect ,is weaker than the other symptoms of sympathetic stimulation. The thinner line in the figure represents the anti-5HT activity. The importance of the figure is that it reveals the correlation between excitation syndrome

or hyperthermic effect (except 2-bromo-LSD) and hallucinogenic activity.

LSD appears to be of optimum structure as it stands for producing intense hallucinations, as no structural variations are known which would increase the

activity. 40 diethylamino E- syndrome dimethylamino (P) F_3 pyrrolidino

Rs /--morpholino = ethylamino L 6 CO iso 7 8

9 Anti-5HT-value

lumiBr 10 Br, CH, 11 12 Total E-syndrome 'CH, 13 COCH3 111,11 CH,OH 1141 N. / ,ethylamide-- --CH '1 " 1 <,-,:_ethylamide--COCH,

100 10 1 10 100 1000

No. Lysergic acid derivative No. Lysergic acid derivative

1 d-Lysergic acid diethylamide (LSD) 11 1-Methyl-2-promo-LSD 2 d-Lysergic acid dimethylamide 12 Di-LSD-disulfide 3 d-Lysergic acid pyrrolidide 13 1-Methyl-LSD 4 d-Lysergic acid morpholide 14 1-Acetyl-LSD 5 d-Lysergic acid monoethylamide 15 1-1-1ydroxymethyl-LSD 6 /-Lysergic acid diethylamide 16 1-Methyl-d-lysergic acid monoethyl- 7 d-Isolysergic acid diethylamide amide 8 Dihydrolysergic acid-(1) diethylamide 17 1-Acetyl-d-lysergic acid monoethyl- 9 Lumilysergic acid-(I) diethylamide amide 10 2-Bromo-LSD 18 1-Methyl-d-lysergic acid pyrrolidide

Fig. 1.7. Correlation between hallucinogenic and pharmacological activity of lysergic acid derivatives (After Hofmann, 1968)

N(C H ) 2 5 2 1-1_ COCO H_ _ CO N-CH 3 H

5S:8R ,(-)-iso-LSD

N(C H ) 2 5 2 OC H

5R:8S, (+)-iso-LSD

Fig. 1. 8. Stereoisomers of LSD 41

Chemical and Physical Aspects of LSD LSD is a white crystalline solid, m.p. 82-83° (Stoll et al., 1954, on optical activity H;ii9 of +17° in (Hofmann, 1964) and a UVmax. of 312 nm in mildly acidic aqueous solutions (Martin and Alexander, 1967). The free bone is not very soluble in water but its tartrate salt dissolves readily. The conjugated double bond system in the molecule gives it a very strong fluorescence on exposure to UV light, a property which has been utilised in the fluorimetric assay of the drug (Axelrod et al., 1957). LSD contains two asymmetric carbon atoms at C5 and C8. Therefore, four stereoisomers are possible (Fig. 1.8). Unless specified, LSD always refers to the highly active (+)-LSD with the 5R:8R configuration having the diethylamide side chain and the at C5 in the f -equatorial positions. The other three , namely (+)-iso-LSD, (-)-iso-LSD and (-)-LSD, have practically no hallucinogenic activity. LSD does not occur naturally and is made from lysergic acid, which has been synthesised by Kornfeld et al. (1954). However, the method is tedious requiring 13 steps and the yields are low. Lysergic acid is normally obtained commercially by the alkaline hydrolysis of ergot alkaloids, which are produced by submerged cultures of the ergot fungus, (Pharmaceutical Journal, 1971). LSD is synthesised by the reaction of diethylamine with a derivative of lysergic acid (see Fig. 1.9). A number of derivatives have been used a) lysergic acid azide (Stoll et al., 1954); b) lysergic acid chloride (Sandoz Ltd., 1961; Johnson et al., 1973); c) lysergic acid sulphuric acid anhydride (Garbrecht, 1959); d) lysergic acid trifluoroacetic anhydride (Pioch, 1956); e) lysergic acid - imidazole complex (Cerny and Semonsky, 1962). In all of these methods, LSD is not obtained as a pure product; some iso-LSD is always formed. Purification of the LSD is achieved using solvent extraction, chromatography and recrystallisation.

42

C H 5 N— C2H "C2H5 CO CO H- - _ H - N, N -CH3 N-CH /' 3 C2H5 -'C 2H5 _V Diethylamine v N,IJ H H

Lysergic acid derivative LSD

Fig. 1.9

LSD is not a very stable compound. Aqueous solutions undergo decomposition at to give a number of products, including iso-LSD, whose formation is enhanced by alkaline conditions (Fig. 1,10).

C H ,,C2H5 2 5 N C2H5 --C2H5 H CO COH If N-CH 3

„) H H LSD Iso -LSD

Fig. 1.10. (After Stoll, 1965)

In acidic solutions, UV light converts LSD into 10-hydroxy-9,10-dihydro-LSD (lumi-LSD) by the addition of one molecule of water across the 9,10 double bond (Genest and Parmilo, 1964; Blake et al., 1973).

43

General Aspects of the Metabolism of Foreign Compounds

Man is continuously exposed to a diversity of chemicals that are foreign to the body. nany are naturally occurring in the diet and have no apparently useful nutritive value. Others are synthetic compounds, such as drugs and food additives, which may enter the body either intentionally or by accident. however, many of these may have a deleterious effect if allowed to accumulate in the body. As a safeguard against this, animals possess biochemical mechanisms which metabolise drugs and other foreign compounds taken in from the environment. The overall consequence of this is that compounds are converted to metabolites which are more polar or water-soluble and which can be readily excreted in the urine and the faeces. Generally, metabolism leads to the inactivation of the compound but formation of toxic metabolites, such as cyclohexylamine from cyclamate (Renwick and alliams, 1972), is well known.

The majority of the foreign compounds are metabolised, usually by the enzymes present in the liver, but compounds such as 5,5'-methylenedisalicylic acid (williams, 1974) and saccharin (Ball et al., 1974), shown in Fig. 1.11, are

HOOC 0

OH /2 CH2 EH S /1 02 5. I-meth 3.enedi.c acid Saccharin

Fig. 1.11 already highly polar and are found to be excreted unchanged. Therefore, the duration of action and the of a compound are often related to the rate at which the compound can be metabolised and excreted from the body.

In the body, the biochemical transformation of the compound usually occurs in two phases. In I the oxidative, reductive and hydrolytic reactions occur, during which OH, COOH, NH2 and occasionally 6H groups are introduced into the molecule (see Table 1.3). The products of phase I may then proceed to

44

the phase II synthetic reactions when conjugation with glucuronic acid, sulphate and amino acids takes place. The pattern of metabolism can be summarised as follows:

Phase I Oxidation, Phase II Synthetic or reduction, )- conjugation Xenobiotic hydrolysis Enzymes products. Enzymes products. (After Williams, 1974). A number of factors are known that can affect the metabolism of the compound and these can be listed as follows: Species Size of dose Strain Sex Age Stress Chronic administration Temperature Presence of other foreign compounds Time of day Route of administration Route of excretion (urine, bile) Disease Gut flora Diet Season Altitude (After Williams, 1974). The screening of a drug is normally carried out on laboratory animals, such as mice, rats guinea pigs, rabbits, cats, dogs and monkeys before being tested in man. However, the activity and toiicity of the compound are not always the same in animals as they are in man. One reason for this is that the metabolism of a compound in laboratory animals often differs from that in man both qualitatively and quantitatively. Variation in the metabolism of foreign compounds among the species may be viewed from two main aspects: a) variation due to differences in the rate of transformation along a common route of metabolism, and b) variations due to differences in the actual routes of metabolism (Williams, 1964). The rate of transformation may depend on variations in the amount of an enzyme that metabolises the compound, 45

on the amount of a natural inhibitor of the enzyme, on the activity of an enzyme reversing the reaction and on the extent of competi4g reactions for the same foreign substrate Gilliams, 1974).

Table 1.3. Examples of Phase I and Phase II Reactions in Man (After Smith, 1971)

Reaction Type Examples

Phase I: Oxidation Hydroxylation N- and 0-dealkylation Deamination Replacement of S by 0 Cleavage of Oxidation of thio ethers to sulphoxides

Reduction Reduction of nitro and ketone groups Reduction cleavage of azo links

Hydrolysis Hydrolysis of esters and amides

Phase II: Synthetic Glucuronide synthesis (conjugations) conjugation Glutamine conjugation Mercapturic acid synthesis Methylation Acetylation Ethereal sulphate Thiocyanate formation 46

Absorption, Binding, and Distribution of LSD in the Body

LSD was readily absorbed from the gut when given orally to man and the effects of the drug became apparent in about 20 minutes. When the drug was added to the blood of the rat, no loss in its activity was found as measured by its antagonism to 5HT. This demonstrated that either the drug was not bpund to the plasma proteins or if bound, it still retained the activity (Rothlin, 19571). When LSD was added to homogenates of liver and muscle of rats, its antagonism to 5HT was decreased by about 50, in a few minutes and the reduction was only slightly greater after 17 hours. In brain homogenates, the loss in activity was 60,;;- after 10 minutes and 80(;.. after 17 hours. The decrease in activity was similar at 30C or 380C indicating that binding and not metabolic transformation was responsible for this effect. Binding of LSD to plasma protein has been reported in the cat by Axelrod et al. (1957) and in the rat by Boyd (1959). Boyd found that 70;L of the radioactivity in the plasma was bound 5 minutes after administration of [14c]-LSD and the binding decreased to about 40. after 3 hours. The distribution of LSD has been studied in mice, rats and cats. Stoll et al. (1955) gave mice an intravenous injection of 50 )1g of [111t].-LSD and presented results for the distribution of radioactivity in various tissues over a two hour period (Fig. 1.12). The radioactivity in the blood disappeared fairly rapidly with a half-life of 7-10 minutes. The maximal activity in most of the other tissues examined occurred after 10-20 minutes with one exception. The radioactivity in the small intestine continued to increase rapidly and contained about 50L of the total dose after 2 hours, indicating that most of the excretion probably takes place via the biliary route. The amount in the different tissues in decreasing order was small intestine, liver, kidney, adrenal, lung, spleen, pancreas, large intestine, heart, muscle, skin and brain. Reverse dilution procedure (addition of unlabelled LSD to homogenates followed by solvent extraction, chromatography and crystallisation to constant activity) showed that unchanged LSD in the brain, liver and small

113L000 2 SMALL INTESTINE :3 LIVER 4 KIDNEY. ADRENAL 5 LUNG. SPLEEN, PANK R. 6 GREAT INTESTINE 100.0 7 HEART 6 MUSCLE, SKIN 9 BRAIN /Lung

t 10.0 Derain 4,1.1usrie / ' Med

c1.1 is

R- 1.0 0 co

I-7

0.1

Co

O O

0.01 1.0 3.0 10.0 0 h I 2 Dose mg/kg

14 Fig. 1.12. Tissue distribution of [ C]--LSD in mice (After Fig. 1.12. Variation of blood and tissue Stoll et. al. 1955) concentration of LSD with dose in rats (After Doepfner, 1962) 48

intestine represented about 102: of the total activity, in each of the organs, after 2 hours. It would appear that most of the LSD had been metabolised and that the metabolites were also present in the brain, which raises the possibility of a metabolite involvement in the production of some of the LSD effects. An autoradiographic study in mice using [14c]-LSD also indicated that the radioactivity passed very quickly from the blood into the tissues (Idanpaan-Heikkila and Schoolar, 1969b). In addition to the brain, the adrenal, hypophysis, kidney, liver, lung, salivary and lacrimal glands showed the highest uptake, and it was confirmed that the most important route of elimination of the radioactivity was the bile. Lanz et al. (1955) administered an intravenous dose of 35 mg/kg of LSD into mice and worked out the tissue distribution by first extracting the total LSD and any metabolites, and then measuring the extent of antagonism of the extract to 5HT on the isolated rat uterus. They found results which were similar to those obtained using [ 140]-LSD but quoted a half-life for the blood as 37 minutes. These results, however, should be treated with caution since no mention was made to what extent, if any, the metabolites affected the bioassay. Slaytor and Wright (1962) found that the two major glucuronide metabolites of LSD in the rat had 5;::- and 0.5 of the activity of LSD on the uterus. So if the tissue contained either a very large percentage of the dose as the metabolites or very little unchanged LSD only, the extent of antagonism may be the same and this would give a false quantitative distribution. [1401-LSD has been used to determine the distribution of radioactivity in the rat (Boyd, 1959). Boyd examined the effect of intraperitoneal and intra- venous administration of the drug on the distribution and reported that either route produced the same distribution pattern, although the levels of radio- activity were greater after the intravenous dosing. The distribution pattern was basically the same as that obtained in mice by Stoll et al. (1955), with a rapid decline in radioactivity in the blood, accompanied by an increase and then a gradual decrease in the organs examined. Boyd (1959) estimated that 49

only about 0.01,, of the dose probably reached the brain after the intra- peritoneal injection of 1 mg/kg of LSD. Furthermore, the levels in the brain were virtually the same after 20 minutes irrespective of varying the dose between 0.01 and 1 mg/kg. However, Doepfner (1962) demonstrated that there was a proportionate increase in the brain content of LSD after 20 minutes on increasing the dose from 1 mg/kg to 10 mg/kg (Fig. 1.13). Similar increases were also found in the lung, muscle and blood. It is important to mention that Boyd (1959) was measuring total radioactivity, whereas Doepfner extracted specifically for LSD from the tissues and estimated the concentration fluorimetrically.

Oith the aid of a specific and sensitive method for the estimation of unchanged LSD only in biological material, Axelrod et al. (1957) reported the tissue distribution of the drug in the cat. The assay involved extraction of the drug into an organic phase, then an acid phase and followed by estimation of the drug in the acid phase using a fluorimeter. 90 minutes after 1 mg/kg of LSD was given intravenously to a cat, the drug was found to be localised in the bile and the plasma to a considerable extent (see Table 1.L). The concentration of LSD in the tissues in decreasing order was as follows: lung, liver, kidney, brain, intestine, spleen, cerebrospinal fluid, heart, muscle and fat. It has already been shown that in mice and rats, LSD disappeared fairly rapidly from the blood. Axelrod and coworkers confirmed that LSD was metabolised fairly rapidly in mice, with 50;- of the drug in the entire animal remaining unchanged after only 7 minutes. Plasma decay curves for monkey (Eacaca mulatta) and cat following intravenous administration of 0.2 mg/kg of

LsD, indicated a plasma half-life of 100 and 130 minutes respectively. Aghajanian and Bing (1964) used the fluorimetric method to determine the half- life of LSD in humans. Subjects were given 2 pg/kg of LSD intravenously and from the plasma decay curve (Fig. 1.14)lestimated half-life 430-s 175 minutes. This indicated that in humans, contrary to previous suggestions, LSD does not disappear from the body before the onset of the effects (the "trigger"

5 0

Plasma level of LSD-25 ( plasma ) l) o

---- Performance test scores 0 tr rt, 10- E on

;r1 f c 9- t o a 8- 20 en E Perc

(

4-) 7- a. — -0, 40

6- ores CV 9

If) 5- t sc -J

60 tes 4- a) T rn 3- nce

rts 2- 80 rma z fo r 1- Pe

1 8 Time in hours

Fig. 1.14. Relationships between plasma LSD levels and performance in humans (After Aghajanian and Bing, 1964)

Tissue LSD concentration (mg/kg)

Plasma 1.75 Cerebrospinal fluid 0.36

Brain 0.52 Liver 0.67 Kidney 0.53

Muscle 0.20 Heart 0.30 Lung 0.87 Spleen 0.38 Intestine 0.39 Fat 0.20

Bile 1.85

Table 1.4. Tissue distribution of LSD in cat (After Axelrod et. al. , 1957) 51

mechanism). The researchers also found that there was an inverse relation between performance test scores and the plasma level of LSD (Fig. 1.14). Metabolism of LSD Before the synthesis of [ 40]-LSD was achieved, the study of the metabolic fate had received very little attention. This was probably due to the high toxicity of the drug, and only low doses could. be used which made isolation and identification of the excreted products from the animals extremely difficult. However, the use of i4Q -LSD was only partially successful and most of the information has come from studies using in vitro techniques and unlabelled LSD. More important, in vitro studies have not produced results that have been found in vivo. In Vivo Studies According to Axelrod et al. (1957), rhesus monkeys given 0.2 mg/kg of LSD intravenously excreted less than of the unchanged drug in urine or faeces. This suggested that the compound underwent almost complete metabolic trans- formation in the body. Using P-401- LSD, with the label in the ethyl group, Stoll et al. (1955) found that the excretion of LSD and metabolites in mice takes place predominantly via the liver and the bile. Idanpaan-Heikkila and Schoolar (1969b) not only demonstrated the importance of bile in the elimination of the radioactivity in mice, but also reported that after 24 hours the major part of the radioactivity represented the LSD metabolites. Rats with cannulated bile ducts excreted 70;, of the dose in the bile in the first 6 hours (Stoll et al., 1955). Apart from LSD (less than 1G of the dose), 3 radioactive compounds were present in the bile which were highly water soluble in contrast to LSD itself. Boyd (1959) also reported that rats receiving 1 mg/kg of [14U]-1,6D intravenously excreted about 70.. of the dose in the bile in 4 hours. Further- more, intact rats receiving this dose intraperitoneally excreted 80;.- of the radioactivity in the faeces in 60 hours, with 8L. in the urine in 36 hours and

4(i-; as radioactive in the expired air in 12 hours. The similarity in the excretion figures for the bile and the faeces and confirmatory experiments 52

led Boyd to believe that no enterohepatic circulation of the drug takes place. The confirmatory experiment involved the collection of bile containing the radioactive products from one rat and injection into the upper intestine of the rat, Whose bile was then collected for 4 hours. Only about 5v of the radioactivity received by the second rat vas excreted in the bile in this time, However, it is possible that larger amounts of radioactivity would have been excreted over a longer time period. Boyd (1959) confirmed the findings of Stoll and coworkers (1955) by also reporting the presence of 4 compounds in the rat bile. however, Boyd could not detect any unchanged [1401-LSD. The compound, which may have been wrongly assumed as LSD by Stoll et al. from the similar chromatographic characteristics, failed to give the characteristic colour with Van Urk p-dimethylaminobenzaldehyde reagent, which normally produces blue colours with LSD and the metabolites with a free 2-position. Apart from the most polar compound, the metabolites, like LSD, fluoresced blue under UV light. None of these compounds corresponded to 2-oxo-LSD or lysergic acid monoethylamide (LAE), but the two major metabolites, Which represented about 60,;:"6 of the total radioactivity in the bile in the first 2 hours, were reported to be conjugated with glucuronic acid. The hydrolysis of the conjugates with (.-glucuronidase produced less polar radioactive products but one of the aglycones was very unstable. Examination of the urine revealed 7 radioactive compounds, two of which were identified as the glucuronides present in the bile. Of the other five compounds, which were of the same polarity as LSD, one of them could have been LAB since expiration of 14002 is indicative of de-ethylation of the diethylamide side chain. The presence of the two glucuronide metabolites of LSD in the rat bile was confirmed by Slaytor and Wright (1962). These workers, however, used unlabelled LSD and were unable to detect any further metabolites. On hydrolysis, the two aglycones were shown to give a distinct purple colour with diazotised sulphanilamide which indicated the presence of a phenolic hydroxyl group in the LSD molecule. When either of the glucuronideswas incubated in phosphate buffer

53

/,C2H5 C2H5 --C2H5 1 --C2H5 OC H H CO \ \\I

N-CH3 N-CH3 >

c6H906--0

H

12 -hydroxy-LSD glucuronide 12-hydroxy-iso-LSD glucuronide

CH CH3 3 1 HN-CHCH2OH HN-CHCH2OH 1 H, CO OC H

N-CH3 N -CH3

C6H906--0 /A, C6H906--0 V,/,/ /

c N\ \ N \\°>.„ H H

12-hydroxyergometrine 12-hydroxyergometrinine glucuronide glucuronide

Fig. 1.15.

of pH 5.5 at 36°C for 72 hours, a mixture of the two glucuronides was obtained. This interconversion led Slaytor and Wright to believe that the two major metabolites were derivatives of LSD and iso-LSD. These workers also studied the metabolism of ergometrine and identified glucuronide conjugates of 12-hydroxyergometrine and 12-hydroxyergometrin1ne as two of the metabolites and these could also be interconverted (Fig. 1.15). By analogy, they suggested

54

that the two metabolites of LSD were conjugates of 12-hydroxy-LSD and 12-hydroxy-iso-LSD. Recently, an attempt was made to study the metabolism of L3D in the human. raed and McLeod (1973) gave an oral dose of 200 Kgto two patients and following hydrolysis of the urine with p-glucuroniaase„ the method of Axelrod et al. (1957) was used to extract the "LSD-like material". This compound, which was fluorescent under UV light, was not present in the control urine. If the compound was a metabolite of LSD, it would appear that it was still detectable in the urine even on the fourth day after ingestion. However, since urine samples from the subjects who had received ergotamine or methysergido also produced this compound, it is doubtful that it is derived from LSD. In Vitro Studies Axelrod et al. (1956, 1957) used guinea pig liver microsomes in their in vitro preparations and demonstrated that LSD was metabolised to a compound that failed to give a blue colour with Van Urk reagent. This metabolite was identified as 2-oxo-LSD (Fig. 1.16) by its chemical synthesis. However, it

,,C2H5 ,,C2H5 2H5 --C2H5 co CO

guinea pig N-CH3 ti N-CH3 LSD / or \\\ 7 microsomes \ // z .7-,. / N.- / H /7 I . 1 \ \ /.1,, 0 N \s/ N H 2-oxo-LSD

Fig. 1.16. was not stated which of the two possible isomers of 2-oxo-LSD was the metabolite. bro lysergic acid or nor-LSD could be detected. The in vitro system of Axelrod and associates required and a reduced adenine dinucleotide 55

phosphate (NAME) generating system, and guinea pig brain, kidney, spleen and muscle preparations were incapable of metabolising LSD. The guinea pig microsomal system has been used by Szara (1963a) to metabolise LSD to a compound which gave a fast blue colour with p-dimethyl- aminobenzaldehyde reagent compared with LSD. The metabolite was postulated to be 13-hydroxy-LSD for two main reasons: 1) it gave an immediate red colour with diazotised sulphanilic acid in acid which is a characteristic reaction of the 6-hydroxy-indole derivatives; 2) using 111N- (D T), Szara found that only the rat liver microsomes, but not guinea pig microsomes, were able to form the 6-hydroxy-DiT metabolite (Fig. 1.17). The same species

C2H5 C2H5 CO C2H5 )--- N-C H N-CH3 2 5 //

1 ! 1 i1 HO \ \ N HO \\\\\y / N/'

13 -hydroxy-LSD 6-hydroxy-JET

Fig. 1.17. difference was also found when LSD was used as a substrate and by analogy, the position of the hydroxyl group was assigned to the 13-position. Although Axelrod et al. (1957) were unable to detect nor-LSD, the product of demethylation at position-6, recent work suggests that dealkylation at position-6 and in the side chain at position-8 also play an important role in the in vitro metabolism of LSD. Niwaguchi et al. (1974) used liver microsomes of rats, guinea pigs and rabbits in their in vitro preparations and two fluorescent compounds besides LSD were isolated. These metabolites were 56

unequivocally identified as LAE and nor-LSD (Fig. 1.18). The LAE was always

C2H5 HN C2H5 N -C2H5 CO co

N-CH 'N) N-H 3 •N,

Lysergic acid Nor-LSD monoethylamide (LAE) Fig. 1.18. formed4in greater amounts than nor-LSD (see Table 1.5). These workers also

------Rat Qu5nea Pig iw.bbit 1.nzyme activity 27.3 25.4 21.7 (nmole LSD metabolised/mg protein/120 min.) Letabolites formed: (nnole/mg protein/120 min.) 9.4 4.7 3.2 :;or-LSD (nmole/mg protein/120 min.) 1.4 0.6 0.8

Table 1.5. (After iTimguchi et al., 1974) noted a species difference, with the rat liver microsomes forming about twice as much LAE and nor-LSD as the liver microsomes from either the guinea pig or the rabbit. However, as is apparent from Table 1.5, Niwaguchi and coworkers only accounted for about 20-40 of the total metabolites formed and it is possible that the remainder may have been 13-hydroxy-LSD and 2-oxo-LSD, These results refute the suggestion of Axelrod et al. (1957) that 2-oxo-LSD was the only transformation product in vitro. 57

Inhibitors of the Metabolism of LSD

Axelrod et al. (1957) examined the effect of 5HT, reserpine, chlorpromazine and SKF 525A on the metabolism of LSD and found that all four compounds inhibited the metabolic transformation of the hallucinogenic drug in vitro. However, chlorpromazine and SKI? 525A were very potent in this respect. 'Alen chlorpromazine was administered to mice 5 minutes prior to the injection of LSD, the biologic half-life of the latter drug was increased from 7 to 15 minutes. Niwaguchi and associates (1974) confirmed the inhibitory actions of these four compounds but, in addition, demonstrated that nitrazepam and inhibited markedly the enzymic conversion of LSD, iproniazid and noradrenaline inhibited moderately, while little inhibition was observed with

acetylcholine. The Metabolism of Compounds Structurally Related to LSD

LSD is related structurally to indole and metabolism of indole and indolic compounds has been studied extensively. Using carbon-14 labelled indole, King et al. (1966) examined the metabolism in rat in vivo and in vitro. They found that indole was metabolised to a number of compounds Which are summarised in Fig. 1.19, Similar to the in vitro metabolism of LSD, indole was also converted ox to a 2-oxo metabolite, oxindole, which was further converted to 5-hydroxypdole. Products of ring cleavage were also found, and ring opening is also observed in the metabolism of tryptophan (Meister, 1965). Although an endogenous enzymic mechanism exists in the body Which hydroxylates tryptophan to 5-hydroxytryptophan (Meister, 1965) and oxindole is hydroxylated to 5-hydroxyindole in vivo (king et al., 1966), it is apparent from the literature that in vivo and in vitro hydroxylation of a majority of the indolic compounds occurs in the 6-position. This position corresponds to position-13 in the LSD molecule and 13-hydroxy-LSD has been suggested as a metabolite (Szara, 1963a). The compounds that are metabolised by hydroxylation in the 6-position include tryptamine, its analogues li,JA-dimethyltryptamine and 14,N -diethyltryptamine, skatole and melatonin (see Fig. 1.20). Szarak(1962) OH

OH

H H

Indoxyl 63% Isatin 6% (conjugated) COOH

NH CHO

N-Formylanthranilic acid 0. 5%

Indole 1-Aminophenyl acetic acid COOH [HCOOH] NH 2

Anthranilic acid HO V CO2

0 2% in expired air H 5-Hydroxyoxindole 3% (conjugated)

Fig. 1.19. Metabolism of indole by the rat. Hypothetical metabolites are in brackets (After King et al., 1966)

59

CH CH NH 2 2 2 CH2CH2N(CH 3)2

Tryptamine * N, N-Dimethyltryptamine *

CH C OOH 2 CH CH N(C H ) 2 2 2 5 2

Indoleacetic acid ** N, N-Diethyltryptamine *

CH CH 0 CH CH NHCOCH 3 2 2 3

N H Skatole :* Melatonin

CH3

CH CHNH CH CH N(C H ) 2 2 2 2 3 7 2

or -Met hyltryptamine * N, N- *

Fig. 1.20. Indole Derivatives that are Hydroxylated in the 6-position

After Szara et al. (1962) After Jepson et al. (1962) After Horning et al. (1959) After Taborsky et al. (1965); Kopin et al. (1961); Kopin et al. (1960) 60

used various dialkyl derivatives of tryptamine and demonstrated that L,N-diethyltryptamine was converted by rat liver microsomes to the 6-hydroxylated metabolite more rapidly than were the other homologues. In vivo studies showed a considerable species difference in the ability to form 6-hydroxy-N,11-diethyl- tryptamine (Szara, 1963b). Table 1.6 shows that the rat is the most active in forming this metabolite, with very little 6-hydroxylation occurring in guinea pigs, man and monkeys.

Species Dose of DET (mg/ kg) No. 6-0H-DET in urine dose

Rats 3-8 33 51.5 t 15.6 Mice 20 50 40.6 ± 2.1 Rabbits 3-5 6 12.0 ± 4.3 Guinea Vies 1 15 1.6 ± 0.9 Monkeys 1 2 7.1, 13.2 Man 1 10 5.0 * 1.6

Table 1.6. Species Difference in the 6-hydroxylation of Diethyltryptamine (DET) (After Szara, 1963b) * 24 h urine specimen in animals, 9 h specimen in man. 6-0H-DET determined after incubation with p -glucuronidase.

7-Hydroxylation of indole has been reported in only two indole derivatives. Heacock and Mahon (1964) demonstrated that what was initially thought tole 6-suiphatoxyskatole in human urine was in fact a mixture of the sulphate conjugates of 5-, 6- and 7-hydroxyskatole. When 3-(2-acetoxyethyl)-5-methoxy- indole (Fig. 1.21) was given to rats, a single metabolite was excreted in the rat urine. This metabolite was a hydroxy-5-methoxyindole-3-acetic acid, hydroxylation having occurred in the ring. Since the metabolite did not possess the chromatographic characteristics of the synthetic 4-hydroxy- and 6-hydroxy-5-methoxyindole-3-acetic acid, Delvigs et al, (1967) concluded that the metabolite was 7-hydroxy-5-methoxyindole-3-acetic acid.

61

0 CH2CH2OCCH3 CH2COOH 1 1 rat

H

dole 7-hydroxy-_5-methoxyindole-3- acetic acid Fig. 1.21.

Metabolism of a few closely related ergot alkaloids has been studied but in very little detail. Bianchine and Friedman (1970) reported that when 1414c] -methyl-(+)-lysergic acid butanolamide (methysergide, Fig. 1.22)'was given to humans, about 50% of the radioactivity appeared as 14002 in the expired air within the first hour and 30% of the dose was excreted in the urine in 48 hours. However, only 8%, of the total radioactivity in the urine was unchanged methysergide and the remainder was unaccounted for. When randomly tritiated was administered orally to rats, dogs, monkeys and humans, between 60-80%. of the radioactivity appeared in the urine in 4 days with 10-20% in the faeces (hreamone et al., 1972). In addition to a small amount of unchanged nicergoline in the urine of the four species, there were two principle metabolites which were identified as 1,6-dimethy1-8p

C2H5 1

1 CH2OH OC

MethYsergide

Fig. 1.22. 62

hydroxymethy1-100(-methoxyergoline (A in Fig. 1.23) and 813-hydroxymethyl- 1004-methoxy-6-methylergoline (B). Although hydroxylation in the benzene ring was not detected in the metabolism of nicergoline, the same group of workers, Arcamore et al. (1971), reported that a close derivative, , formed a minor metabolite that was hydroxylated in the 12- position (Fig. 1.24). The urine also contained unchanged metergoline, 1-demethylmetergoline and unknown compounds. The species difference in the urinary and faecal excretion of the radioactivity was apparent. The rat, guinea pig and the dog excreted about 15, of the dose in the urine, while the monkey (Patas) excreted 30i,- and man 45,. Most of the remainder of the activity was found in the faeces. 6laytor and Wright (1962) studied the metabolism of ergometrine in rats and reported the excretion of 8 metabolites in the bile. The two major metabolites were very polar and were identified as glucuronide conjugates of 12-hydroxy- ergometrine and 12-hydroxyergometrinine (Fig. 1.15). However, Axelrod et al. (1957) found that ergotanine, like LSD, was metabolised by 2-oxidation to 2-oxo-ergotanine (Fig. 1.25). The piperidine ring and the diethylamide side chain of LSD closely resemble the structure of nikethanide and the metabolism of the latter compound has been studied in detail (Fig. 1.26). Rats, cats, dogs, rabbits and man deethylate the compound to give nicotinamide initially (Ellinger and Abdel Kader, 1949). ,dcotinamide may subsequently be converted to other compounds, such as nicotinic acid, before excretion. By analogy, LSD would be transformed to lysergic acid but this compound was not detected by Axelrod et al* (1957). The ability of the body to deethylate diethylamide grouping has also been demonstrated with 1-diethylcarbainyl-4-methylpiperazine (hetrazan). Faulkner and Smith (1971) reported that the rat was capable of deethylating this compound to de-ethyl- hetrazan. U-demethylation also occurred and formed de-methyl-hetrazan as a minor metabolite (Fig. 1.27). N- formation has aroused much interest recently due to the potential toxicity of these metabolites (Gorrod, 1971) and it appears that the N-oxidation 63

CH OH CH2 OH 2 - H3 N- CH 3 CH 3O CH 0- rat, dog, monkey, 3 ± human >

CH 3 Nicergoline A B Fig. 1.23.

HNC 00CH C 11 HNC 00CH C H HNC 00CH C H 2 6 5 2 6 5 2 6 5 1 CH CH 2 2 N-CH N-CH3TT 3

+ HO

H

Metergoline 1-De-methyl metergoline 12 -Hydroxymetergoline

Fig. 1.24.

H3C\vOLE/ .,,N,,,- HN HN R N 0 I CO 1:) CO CH C H 2 6 5

E rgotamine 2 -Oxo-ergotamine Fig. 1.25. 64

N(C NH 2 H5 ) 2 2 OH CO CO CO

rat, cat, dog, rat \\N \\N rabbit, man

Nikethamide Nicotinamide Nicotinic acid

guinea pig, rat, mouse, rat, mouse, rat hamster hamster

Y N(C H ) • NH2 OH 2 5 2 I I CO CO CO

\\N-0

Nikethamide -N-oxide Nicotinamide-N -oxide Nicotinic acid -N-oxide

Fig. 1. 26.

/ rat \ CH -N NC ON(C H ) CH -N NC ONH C H 2 5 2 3 \ / 2 5 / Hetraz an De -ethyl -het raz an

rat rat

0 / NC ON(C HN NCON(C H C 2 H5 )2 2H5)2 3 \ /

Hetrazan -N -oxide De -methyl-hetrazan

Fig. 1.27.

65

OCH,1 CH3 - 3 CO CO

3 N-CH 0 / 3

Are c oline Are coline -N-oxide Fig. 1.28.

CH CH N(CH ) 2 2 3 2

CH CH N. 2 2 CH N N-Dimethyltryptamine 1 3 (DMT) H rabbit 34- N-Methyltryptamine microsomes Fe , pH 1. 3, heat (not microsomal)

,CH3 CH CH N-,-0 2 2 -■ CH3

,,„CH3 rabbit CH CH N--)--0 2 2 DMT -N-oxide microsomes CH 3 HO

6-Hydroxy-DMT-N-oxide

Fig. 1.29. 66

plays a major role in the metabolism of hetrazan (Faulkner and Smith, 1971). Iiikethamide, nicotinamide, nicotinic acid (Corrod, 1971) and (rhillipson, 1971; Fig. 1.28) are also metabolised by the rat and other species by F-oxidation. Since LSD contains a tertiary nitrogen in the piperidine ring, it is not impossible that the drug may form an H-oxide metabolite. ihis may be an intermediate in the N-demethylation reaction since N vb-dimethyltryptamine- -oxide has been suggested as an intermediate in the formation of -methyl- tryptamine (Fish et al., 1955). However, Szara and Axelrod (1959/ rc;„).o.12i.:Jd that, although the rabbit liver microsomes could form N-methyltryptanine and b,4;-dimethyltryptamine-N-oxide from N,N-dimethyltryptamine, it could not convert

...„4-dimethyltryptamine-N-oxide to N-methyltryptamine but instead formed 6-hydroxy- i.,.,-dimethyltryptamine-N-oxide (Fig. 1.29).

,,,cope of the ITesent Study Although the metabolic fate of L3D has been studied by several group: of wormers, the of its metabolites, as is apparent from the foreL%ein review, is still poorly defined. The aim of the present study was therefcze to study the metabolism of LSD in a few species and attempt to identify some of the metabolites, study their fluorescent intensity variation with pH and investigate whether they still possessed any ONS activity. Due to the -eneral belief in the literature that LSD is unstable, it was felt desirable to briefly investigate this property. It would appear from the reviews on the metabolism of LSD and related compounds that the possible routes of metabolism may involve: 1) deethylation and deamination of the side chain at position-8; 2) demethylation at Position-6; 3) N-oxidation at position-6; 4) aromatic hydroxylation at positions 12, 13 and 14; 5) oxidation at position-2. However, other routes, such as ring fission, are also possible. Combinations of these reactions could also occur. 67

CHAFFER TWO

Materials and Methods

Contents Page.

COMPOUNDS 69 Synthesis of: (+)-Lysergic Acid Di[14C]ethylamide ([14c]-LSD) 70 (+)-Isolysergic Acid Di[14C]ethylanide ([146]-iso-LSD) 73 (+)-Lysergic Acid Diethylarnide (LSD) 73 (+)-Lysergic Acid Uonoethylamide (LAE) 74 10-Hydroxy-9,10-Dihydro-(+)-LSD (luni-LSD) 75 6-Demethyl-(+)-LSD (nor-LSD) 75 12-Hydroxy-(+)-L3D 77 2-0xo-2,3-Dihydro-(+)-LSD (2-oxo-LSO) 78 6- and 7-Hydroxyindoles 79 rurification of [2-3H]-(+)-LSD 79 METABOLIC STUDIES 80 Animals 80 Administration of Compounds 80 Collection of Urine and Faeces 80 Collection of Radioactive Carbon Dioxide in the Expired Air 81 Preparation of Rats and Guinea Pigs with Biliary Fistulae 81 4uantity Production of LSD Metabolites 81 1. Using bile-duct-cannulated rats 81 2. Using isolated perfused rat livers 82 Perfusion medium 82 Perfusion apparatus 82 Operative technique 86 Addition of LSD to the perfusate 90 68

Contents (continued)

Concentration of Metabolites 90 1. From the rat bile 90 2. From the monkey urine 91 3. From the perfusate and liver homogenate from perfusion experiments 91 Isolation of Metabolites 91 Storage of LSD and its Derivatives 92 Paper and Thin-Layer Chromatography 92 Ultraviolet (UV) Light 93 Spray Reagents 93 Enzyme Hydrolysis 94 Methylation of Phenolic Compounds 95 Radiochemical Techniques 95 Spectra 96 Folarimetry 97 Measurement of pH 97 ELECTROENCEPHALOGRAPHIC (EEG) STUDIES 97 Animals 97 Cannulation of the Marginal Vein 97 Administration of Compounds 98 EEG Recordings 98

FLUORESCENCE STUDIES OF LSD AND ITS DERIVATIVES 98 Preparation of Solutions 98 S pectrophotofluorimeter 99 Stability of LSD 99 69

Compounds (+)-Lysergic acid was obtained from two sources, Koch-Light Ltd., Colnbrook, Bucks., U.K. and Sigma Chemical Co., St. Louis, Missouri, U.S.A. Melting points were unobtainable due to the blackening of the samples at 190-200°C. The lysergic acid was used without putification except in the synthesis of[1401-LSD when it was purified by dissolving in warm 2M-MR4OM followed by precipitation with 21.1-acetic acid (Smith and Timis, 1936).

Sodium [14Ci] -acetate was obtained from The Radiochemical Centre, Amersham, Bucks., U.K. /1,1V-Carbonyldiimidazole, m.p. 112-115°C (Fluka A.G., Buchs S.G., Switzerland), disuiphur dichloride, n 2/)/3'11.680 (B.D.H. Chemicals Ltd., Poole, Dorset, U.K.), 5-hydroxyindole, m.p. 105-107°C, 6-benzyloxyindole, m.p. 114- 115°C and 7-benzyloxyindole, m.p. 69-71°C (Sigma Chemical Co., St. Louis, Missouri, U.S.A.) were purchased. Potassium nitrosodisulphonate was synthesised using the method of Rozantzev (1970). Cyanogen bromide was synthesised using the method in Organic Syntheses (1943) and extracted into dichioromethane. All melting points are uncorrected. (+)-Lysergic acid diethylamide tartrate (LSD-25) and [2-31-i]-(+)-lysergic acid diethylamide tartrate ([3111-LSD; specific activity 68 liCi(mg) were gifts from Sandoz A.G., Basle, Switzerland. The Pill-LSD was supplied in glass ampoules containing 1 mg of the compound in i ml of the aqueous solution. Preparative thin-layer chromatography (t.1.c.) was used extensively in the isolation and purification of the required products of chemical syntheses. The preparation of the t.l.c. plates and the elution of the compounds are described later under the heading "Isolation of Metabolites". Other t.l.c. plates to obtain RF values are described under "Paper and Thin-Layer Chromatography". Evaporation of solvents in vacuo was always carried out at temperatures below 40°C to avoid decomposition. Melting points, in most cases, and C, H N analyses of the compounds were not possible. The reason was that, due to the extremely high cost of the starting materials, most of the compounds were prepared in small (milligram) quantities only and so could 70

not be crystallised in pure form to give accurate or C, H and I values. The compounds were judged to be pure when a single spot was obtained on t.l.c. plates developed in at least 3 different solvent systems. The "IF values of the synthesised LSD derivatives are given in Table 2.1. 1:0-Lysergic Acid Di[litlethylamide ([140]-LSD) The synthesis of [14c] -LSD from sodium [14011-acetate was achieved using the following schemes SOC12 EtKH2 14 011314000ha aH314coca. > CH3 cc",t

LIA]H4 li,W-carbonyldiimidazole and 14c -LSD CH14CH 2,AEt1I St lysergic acid No purification of the intermediates was necessary. However, the yields of the N-ethylacetamide and diethylamine were checked by liquid chromatography on a Hewlett iackard F. and N. Scientific 402 High Efficiency as 0hromato3raph fitted with a flame ionisation detector. A 1.5 m glass column (i.d. 3 mm) packed with glycol 6000 (1(v4 w/w) and KOH w/w) on aromsorb G (80-100 mesh) was used. Operating conditions were for N-ethylacetamide, injection port temperature, 1800C, oven temperature, 15000, nitrogen flow rate, 50 ml/min, retention time, 3.3 min; for diethylAmine, injection port temperature, 16000, oven temperature, 75°0, nitrogen flow rate, 30 ml/min, retention time, 1.5 min. In both cases the nitrogen, air and hydrogen were 40, 25 and 20 p.s.i. respectively. Thionyl chloride (160 mg) in dry (1 ml) was added to sodium [14C]- acetate (7.25 mg; 690 pCi/mg) dried over P205 under vacuum. The mixture was stirred at room temperature and after 20 min dry sodium acetate (63 mg) was added. After stirring for a further 20 min the solvent was removed and the solid residue washed with dry ether (3 x 1 ml). The pooled ether solution was stirred, ethylamine (300 mg) in dry ether (2 ml) was added slowly dropwise and after cooling to 0°C the precipitate was filtered off. The filtrate was then evaporated at 0°C by a stream of dry nitrogen and the residue taken up in dry Table 2.1. EF Values of LSD and the Synthetic Derivatives on Paper and Aluminium-Backed T.L.C. Elate

raper Chromatosraphy Thin-Layer Chromatography Compound Solvent.... A B C A C D E F G H

Lysergic acid 0.61 - - - - 0.05 0.00 0.19 - 0.00

LSD 0.83 0.85 0.92 0.46 0.77 0.67 0.53 0.52 0.83 0.47

Iso-LSD 0.84 0.85 0.92 - 0.76 0.40 0.19 0.33 0.62 0.45

LAE 0.72 0.72 0.90 0.46 0.76 - 0.37 - 0.75 0.29

Iso-LB 0.83 0.85 0.92 - - - 0.58 - 0.83 0.61

Lump LSD 0.74 0.73 0.91 - - 0.23 - 0.68 0.41

2-0xo-LSD 0.75 0.72 0.85 0.39 0.76 - 0.50 - 0.83 0.26

"Aromatised" 2-oxo-LSD 0.91 0.95 0.91 0.71 0.76 - 0.72 - 0.91 0,58

2,3-Dihydro-LSD - - - 0.31 0.62 - 0.40 - - 0.17

12-Hydroxy-LSD 0.73 0.72 0.90 0.45 0.76 - 0.40 - 0.81 0.36

Nor-LSD 0.84 0.87 0.90 0.57 0.77 - 0.21 - 0.65 0.17 72

ether (2 ml). Analysis showed a chemical yield of N-ethylacetanide of 55, (g.l.c.) and a radiochemical yield of 48;0 (liquid scintillation counting). The solution of Is-ethyl- [14C1] in dry ether (1.5 ml) was added dropwise to a stirred suspension of LiA1114 (160 mg) in dry ether (4 n1) at 20°C. The mixture was stirred at room temperature for 16 h and then boiled hider reflux for 3 h. After cooling to 0°C, water was added dropwise to decompose the excess Lik1H4. A violent reaction occurred with loss of some radioactivity from the top of the condenser. ",:hen the reaction sto-)-cd, the mixture was transferred to a stoppered test tube, washed in with 10, (w/v) and saturated with The mixture was then shaken vigorously and the ether layer removed after it had separated. The aqueous phase was re-extracted with ether (4 x 2 ml) and the pooled ether solution dried over anhydrous La2SO4. Analysis gave a chemical yield of diethylamine of 31;: and a radiochemical yield of 32;:. 03 (+)-Lysergic acid', dried over 1205 under vacuum, in dry dimethylfornamide (3 ml) and 1:,L'-carbonyldiimidazole (252 mg) were stirred for 30 rain in the dark at room temperature. [140]-diethylamine (19 mg; 34.2 )1Glin,-,, in dry ether (9 ml) was then added dropwise followed by a further 7 mg of diethylamine. After 2.5 h of stirring, the solvent was evaporated in vacuo and the residue dissolved in (+)-tartaric aeld (2,, w/v; 30 m1). The solution (a, 4) extracted with ether t ethanol (9:1, v/v; 4 x 25 ml). The pH of the aqueous solution was adjusted to 9 with 10, Ea0H and then extracted with the ether and ethanol mixture (6 x 25 ml). The pooled extract was taken down to dryness under reduced pressure. The residue, which contained LSD and iso-L.,13, was dissolved in a minimum of and the two compounds separated by preparative t.l.c. in solvent system D. The [1140]-1,1) was purified by crystallisation from diisopropyl ether after exhaustive drying over l'205 under vacuum. Yield 29 mg (26; specific activity 13.6 IICVmg). '1.1.e. in acetone : (4:1, v/v; RF 0.27), methanol : dioxan (3:2, v/v; 0.57) and system D gave a single peak on radiochromatogram scanning and counting sections 73

gave a radiochemical purity of about 97,. The mass spectrum (Appendix) was identical with a reference LSD sample obtained from Sandoz A.G. The 1.r. spectrum shown in the Appendix was identical to the published spectrum of authentic LSD (Cromp and Turney, 1967). The above method of synthesis of [111C]-LSD has recently been published by Barnes (1974). 1;)-Isolysergic Acid Di[14C1ethylamide ([14C1-iso-LSD) In the above synthesis of [14C]-LSD, [140]- iso-LSD was obtained as a by- product contaminated with imidazole and a little [14C]-LSD. Preparative t.l.c. in acetone s : aq. NH3 (sp. gr. 0.88) (100:20:1, by vol.) separated the composite band of iso-LSD and LSD (RF 0.36) from the imidazole 0.15). second preparative t.l.c. of the mixture of iso-LSD and LSD in system 2. was used to isolate the [14C]-iso-LSD. It was dissolved in a minimum of ether and precipitated as a maleate by the addition of maleic acid in ether. precipitate of iso-LSD maleate contaminated with maleic acid was filtereJ and washed with ether. T.I.c. in systems D, E and F followed by scanning gave a single radioactive peak corresponding to the RF of a known sample of iso-1_ ,D which had been obtained as a by-product in the synthesis of unlabelled L ard characterised by . The mass spectrum of iso-1,.D was identical to that of LSD (see Appendix). The radiochemical purity of the compound, estimated by scintillation counting of sections of the t.l.c. plates, Was greater than 95. The specific activity of the [140]-iso-LSD maleate was 0.78 pei/mg. Since the maleate salt was contaminated with maleic acid, it was assumed, for the purpose of eal culation of doses in the metabolic studies, that the specific activity of the [140]-iso-LSD as a free base was the same as that of [14c] -LSD. This assumption was valid as the iso-LSD and LSD were formed during the same reaction. 1:0-Lysergic Acid Diethylamide (LSD) Unlabelled LSD was synthesised using the general method for the synthesis of (+)-lysergic acid amides reported by Johnson et al. (1973). 74

A slurry of (+)-lysergic acid (2 g) and diethylamine (6.2 ml, 4.4 g) in chloroform (100 ml) was heated to . The heat was removed and phosphorus oxychloride (1.4 ml, 2.28 g) was added over a 2 min period at a rate that just maintained the slurry at boiling point. The mixture was then boiled for 4-5 min. After cooling to room temperature, any lysergic acid still remaining was extracted with 1M-NH4OH (4 x 30 m1). The chloroform solution was dried over anhydrous Na2SO4, filtered and concentrated under reduced pressure at 35-40°C. Methanol (100 ml) and water (200 ml) were added to the residue, the solution cooled to 0°C and 1M-NH4OH (100 ml) was then added. After 3 h at 0°C the brown solid which had been deposited was filtered off, washed with water and dried in vacuo over P205 overnight. The crude LSD (yield 1.54 g) was recrystallised from diisopropyl ether. Yield of pure LSD, 1.31 g (58). M.p. 81-83°C (uncorr.) (Stoll et al., 1954; give 82-83°C). The optical rotation in pyridine was [0(I89 = +16° (C = 0.5) (Hofmann, 1964, gives +170). The mass spectrum and the i.r. spectrum were identical to those obtained for [14C]-16D (see Appendix) and t.l.c. in all the solvent systems gave a single spot of identical RF to the standard sample obtained from Sandoz

A.G. (+)_Lysergic Acid 1onoethylamide (LAE) The method of Johnsonet al. (1973) for the preparation of lysergic acid amides was used. (+)-Lysergic acid (1 g) was suspended in chloroform (50 ml) and ethylamine (10 ml, 6.9 g). Phosphorus oxychloride (1 ml) was added dropwise and when the violent reaction ceased, the suspension was heated to boiling. A further 1 ml of phosphorus oxychloride was then added dropwise and this caused the almost complete solution of lysergic acid. When cooled to room temperature, a mass of fine white crystals of ethylamine hydrochloride was deposited. This was filtered off and discarded. The remaining lysergic acid in the filtrate was removed by extraction with 1M-M40H (4 x 15 m1). The organic layer was dried over anhydrous Na2SO4 and evaporated to dryness under reduced pressure. The residue contained lysergic acid monoethylamide (LAE) and the 75

isolysergic acid monoethylamide (iso-LAE). The two compounds were separated by preparative t.l.c. in solvent system L. The LAE and the iso-LAE were eluted with methanol, dried in vacuo and redissolved in a minimum of diisopropyl ether

(approx. 5 ml). Oxalic acid in diisopropyl ether was added to the solution to precipitate the respective oxalate salts as grey powders containing oxalic acid as an impurity. These gave single blue fluorescent spots under UV light

(254 nm) after t.l.c. in solvent systems Er G and H and a blue colour with Van Urk reagent. The mass spectra of LAE (Chapter 3) and iso-LAE (Appendix) were the same and were identical with the published mass spectrum of LAE (Niwaguchi et al., 1974). 10-HydroxY-9.10-Dihydro-(+)-Lysergic Acid Diethylamide (Lumi-LSD)

The method of Blake et al. (1973) was used for the preparation of lumi-LSD. LSD (100 mg) was dissolved in 100 ml of 10i,:1, (Vv) acetic acid and irradiated with a vapour lamp (Hanovia Chromatolite, Slough, Bucks., U.K.) from a distance of 10 cm. A large surface area was obtained by using a long narrow trough (46 x 3 cm). After 2 h the dark green solution was adjusted to pH 9 with aq. NH3 (sp. gr. 0.88), extracted with dichioromethane (5 x 50 ml) and the extract evaporated to dryness in vacuo. The black residue contained a little LSD but the major component was the non-fluorescent (254 nm) lumi-LSD. Diisopropyl ether (3 x 20 ml) was used to extract the residue and the orange- yellow extract subjected to preparative t.l.c. in solvent system L. The lumi- LSD was eluted with methanol from the , evaporated to dryness in vacuo and obtained as a light brown powder (30 mg, 28L). The compound gave a single spot on t.l.c. in systems Fa, G and H, was non-fluorescent, gave a blue colour with Van Urk reagent and a mass spectrum (see Appendix) consistent with that expected for lumi-LSD. 6-Demethyl-(+)-Lysergic Acid Diethylamide (6-Demethyl-LSD, Dior-LSD)

The 3 stage synthesis of nor-LSD from LSD has been reported by Eakahara and Nimaguchi (1971). In another study on the N-demethylation of the ring, Fehr et al. (1970) hydrolysed the 6-cyano intermediate directly to the

76

6-demethyl compound by heating with and acetic acid and so avoiding one intermediate stage of the former workers. A combination of these two methods has been used to synthesise nor-LSD. The reaction sequence is as follows:

DrC1,; LSD 6-cyano -6-demethyl -LSD

Zn OH- in acetic in dioxan acid liaNO2 6-demethyl-LSD < 6-carbamy1-6-demethyl-LSD

LSD (40 mg) was dissolved in dichloromethane (5 ml) and a solution of cyanogen bromide (200 mg) in dichioromethane (5 ml) was added to the stirred solution at room temperature over a period of 10 min. The solution was then refluxed for 1 h and after cooling, the dark green solution was extracted with ici& (N/v) (+)-tartaric acid (4 x 25 ml) to remove any remaining LSD. The dichloromethane solution containing the 6-cyano-6-demethyl-LSD was dried over anhydrous Ea2SO4 and concentrated under reduced pressure, The compound was purified by preparative t.l.c. in solvent system H. The mass spectrum of 6-cyano-6-demethyl-LSD (see Appendix) was identical to the published spectrum (Inoue et al., 1972). The 6-cyano-6-demethyl-LSD was dissolved in 5 ml acetic acid. hater (1 ml) and 1 g zinc dust (A.R. grade; Fisons Scientific Apparatus, Lough- borough, Leicestershire, U.K.) were added and the mixture boiled for 4 h. After cooling and filtering, the solution was diluted with water (5 ml) and adjusted to pH 9 with aq. LH3 (sp. gr. 0.88). Sodium chloride was added to saturate the solution which was then extracted with ether (4 x 10 m1). The ether solution was taken down to dryness in vacua to yield pure nor-LSD. T.l.c. of the compound in solvent systems A, E, G and H gave a single blue fluorescent spot under UV light (254 nm) and a blue colour with Van Urk reagent. The mass spectrum (Chapter 3) was identical to the published spectrum of nor-LSD (Inoue et al., 1972). 77

12-Hydroxy-(+)-Lysergic Acid Diethylamide (12_Hydroxy-LSD)

The synthesis of 12-hydroxy-LSD was achieved in two stages. The LSD was first converted to 2,3-dihydro-LSD which was then oxidised to the required product. The published method of Stadler et al. (1964) was used. However, instead of adding HC1 to a mixture of LSD and zinc as reported, the zinc dust was added to LSD in acid. This gave better yields of the intermediate 2,3- dihydro-LSD as revealed by t.l.c. in systems E and H. LSD (0.8 g) was dissolved in 7.5 H-HC1 (320 ml). The solution was cooled to 10°C and nitrogen passed through it for 15 min to remove dissolved oxygen. The solution was then vigorously stirred and zinc dust (A.R. grade; 120 g) was added in portions over 2 h. T.l.c. at this stage showed that all the LSD had reacted. The excess zinc was filtered off, the filtrate made basic with aq. NH3 (sp. gr. 0.88) until the initial precipitate of zinc hydroxide redissolved and then extracted with dichloromethane (6 x 100 ml). The combined extracts were dried over anhydrous Na2SO4, filtered and evaporated to dryness in vacuo. Diisopropyl ether was used to exhaustively extract the residue. Evaporation of the diisopropyl ether in vacuo gave a pale yellow powder (0.5 g). T.l.c. of this in systems E and H showed the presence of 2 non-fluorescent (254 nm) compounds which gave slowly developing yellow colours with Van Urk reagent. The major component (about 80%) when purified on preparative t.l.c. in system E gave a mass spectrum (see Appendix) consistent with 2,3-dihydro- LSD. However, the mixture of the 2 compounds from subsequent experiments was used without purification, a process which reduced the yield substantially. The yellow powder (0.5 g) was dissolved in a mixture of acetone (10 ml), water (25 ml) and saturated laHCO3 solution (13 ml) by warming. After cooling to 20°C, potassium nitrosodisuiphonate (0.82 g) in water s saturated baHCO3 (30:3 ml) was added quickly to the vigorously stirred solution. After 5 min, the dark brown solution was extracted with ethyl acetate (6 x 20 ml), the organic extract dried 0a2SO4) and evaporated to dryness in vacuo to give a dark brown oily residue. T.l.c. in system E and H showed that this consisted 78

of mostly 2,3-dihydro-LSD with some LSD and 12-hydroxy-LSD. The latter compound was separated and purified on preparative t.l.c. plates in system and then preparative t.l.c. in system L. The methanol eluate of the silica gel was evaporated to dryness to give a very low yield of pure 12-hydroxy-L3D (3.6 mg). This compound gave a single spot on t.l.c. plates developed in systems A, E and Ti, a positive phenolic test with diazotised 4-nitroaniline and a mass spectrum (Chapter 3) consistent with an hydroxy-LSD. The position of hydroxylation was confirmed following n.m.r. analysis (see Appendix). 2-Qxo-2,3-Dihydro-(+)-Lysergic Acid Diethylamide (-0xo-L3D)

Hinman and Bauman (1964) describe a method of converting 3- substituted indoles to the corresponding oxindoles by reaction with E-bromosuccinimide in t-butanol. This method was used to convert LSD to 2-oxo-LSD. LSD (55 mg) was dissolved in 95;:, t-butanol (10 ml) and L-bromosuccinimide (32 mg) was added to the stirred solution. The colour gradlIally turned yellow- orange over a period of 30 min. The mixture was stirred for 2.5 h and then the volume was reduced to 5 ml by a stream of nitrogen. A solid was deposited and after filtering and washing with t-butanol it was dried in vacuo over The yield of the pure pale orange solid was 18 mg. T.l.c. in systems 0 and H gave a single spot. The compound had a weak blue fluorescence under UV light (254 nm) and gave a slowly developing yellow colour with Van Urk reagent. The mass spectrum (see Chapter 3) was consistent with the presence of an additional oxygen in the LSD molecule. The i.r. spectrum (Appendix) gave an additional -, peak over LSD corresponding to the new carbonyl grouping (1720 cm ) and was similar to the published spectrum (Troxler and Hofmann, 1959). 2-oxo-LSD in solutions at room temperature was transformed in 2-3 days to a yellow compound having a yellow fluorescence under UV light. The compound gave a pale orange colour with Van Urk reagent and a mass spectrum (Appendix) having the molecular ion at Mie 337, 2 mass units less than the molecular ion of 2-oxo-LSD. The yellow derivative was identified as "aromatised" 2-oxo-LSD, which has been obtained as a by-product in the synthesis of 2-oxo-LSD by Troxler and Hofmann

(1959). 79

Attempts at synthesising 2-oxo-LSD by the two published methods (Freter et al., 1957; Troxler and Hofmann, 1959) were unsuccessful. 6- and 7-Hydroxvindoles The 6- and 7-hydroxyindoles were obtained by catalytic debenzylation of the respective benzyloxyindoles. Benzyloxyindole (10 mg) was dissolved in methanol (1 ml) and hydrogen was bubbled through at room temperature and atmospheric pressure in the presence of 5% palladium on charcoal (10 mg). After 4 In complete conversion to the hydroxyindole occurred. The catalyst was filtered off and the solvent removed under reduced pressure. The hydroxy- indoles were prepared as required and used as soon as possible due to their unstable nature. Purification of [2-3H]-(+)-Lysergic Acid Diethylamide (PHI-LSD)

T.1,c, of the [3H]-LSD (obtained from Sandoz A.G.) in solvent systems L, and H followed by radiochromatogram scanning showed an extra peak at the origin due to an impurity. Sdintillation counting of 1 cm sections indicated that the LSD was only 92-94ig pure and purification was necessary. The LSD solution (1 ml) and ethanol (5 ml) were evaporated to dryness under reduced pressure and the distillate counted for radioactivity. The L6D residue was dissolved in methanol (1 ml) and chromatographed on glass t.l.c. plate (Silica Gel HF254 0 0.25 mm) in system F. The band corresponding to the LSD was eluted with methanol and evaporated to dryness in vacuo at 35-40°0. The [374-LSD (radioactive yield 68i0 gave a single peak following chromatography in systems E, F and H and radiochromatogram scanning. Counts of sections of ta.c. plates indicated PHI-LSD to be about M pure, It was found that 14 of the initial radioactivity was present in the distillate indicating loss of tritium as 3H20 formed by a mechanism involving hydrogen and tritium exchange (see Sandberg, 1970, p.6). 80

Metabolic Studies

Animals Female Wistar albino rats (150-220 g), female Dunkin-Hartley albino guinea pigs (260-360 g) and a male (5 kg) and a female (6 kg) rhesus monkey (Maraca mulatta) were used in the metabolic studies. The rats (Allington Farms, Porton Down, Salisbury, Wiltshire, U.K.) and the guinea pigs (Redfern Animal Breeders, Brenchley, Kent, U.K.) were fed respectively on 41b nuts and RGF pellets supplied by Labsure Animal Diets, Poole, Dorset, U.K. The monkeys were fed hazuri Primate Diet (Cooper Nutrition Products Ltd., Witham, Essex, U.K.) in addition to oranges, bananas, apples, grapes and Farley's rusks (Glaxo-Farley Foods, Plymouth, U.K.). Administration of Compounds [14C1-LSD or [114C1-iso-LSD was dissolved in 0.5. (w/v) (-0-tartaric acid and administered intravenously (femoral vein) into anaesthetised bile-duct- cannulated rats and intraperitoneally to intact rats and guinea pigs and anaesthetised bile-duct-cannulated guinea pigs. One bile-duct-cannulated rat received [311]-LSD in the tartaric acid solution intravenously. Before the intramuscular administration of [1401-LSD to monkeys, the solution was sterilised by passing through a Millex disposable filter unit (0.22 pm; Millipore S.A., France). Each of the 2 monkeys received 3 separate doses of [14c] -LSD with an interval of 1-2 weeks between each administration. Collection of Urine and Faeces The rats and the guinea pigs were kept singly in glass metabolism cages (Metabowls; Jencons, Hemel Hempstead, Herts., U.K.) which enabled the urine and the faeces to be collected separately. Monkeys were housed in cages with a suitable tray for the separate collection of urine and faeces. The tray and the flask were treated. with a solution of mercuric chloride to prevent any bacterial breakdown of the excreted products. All the animals were allowed free access to water but food was withheld for the first 24 h to avoid contamination of the urine. Urine and faeces were collected daily. 81

Collection of Radioactive Carbon Dioxide in the Expired Air

A pump was used to draw the expired air from the metabowls through a series of 3 wash bottles. The first contained. CaC12 to remove moisture and the next two contained 250 ml and 150 ml respectively of a carbon dioxide absorbant. The absorbent consisted of 2-methoxyethanol and ethanolamine (2:1, v/v) as described by Jeffay and Alvarez (1961). The air passing through the absorbant was at a rate which just prevented condensation inside the metabowls. The carbon dioxide absorbant was replaced after the first 8 h and then after every 24 h. The calcium chloride was replaced twice daily. Preparation of Rats and Guinea Pigs with Binary Fistulae

The animals were anaesthetised with intraperitoneal thiopentone sodium (Pentothaa; Abbot Laboratories Ltd., 4ueensborough, Kent, U.K.; 70 ngo/kg for rats and 50 mg/kg for guinea pigs). The size of the polythene cannulae (Portex Ltd., Hythe, Kent, U.K.) and the procedure for cannulation of the common bile duct were as described by Abou-El-Makarem et al. (1967). The cystic duct in the guinea pig was tied off to ensure that hepatic bile only was collected. The bile from the guinea pig was collected at hourly intervals for 6 h and, if necessary, animals were given 15 mg/kg thiopentone sodium to maintain anaesthesia. After administration of the dose solution, rats were kept in restraining cages and bile was collected at hourly intervals for 5 h and finally after 24 h. The rats were kept warm with suitable lamps above the cages and allowed free access to water. Urine was collected in plastic trays under the cages. Quantity Production of LSD Metabolites 1. UsingL bile-duct-cannUlated rats Between 5 and 20 bile-duct-cannulated rats were used at a time. The animals received 0.5 mg of LSD intraperitoneally every hour for 4 h. Bile was collected continuously and for at least 4 h after the final dose. One rat also received [14C1-LSD (1 mg/kg; 13.6 p4/mg). The animals were kept anaesthetised by the injection of 20 mg/kg thiopentone sodium whenever necessary. The pooled 82

bile was freeze-dried and the metabolites extracted exhaustively with methanol until 85-9W of the radioactivity was removed. The methanol extract was evaporated to dryness under reduced pressure at 35-40°C, the residue taken up in a minimum of water (10-20 ml) and the metabolites isolated (see under "Concentration of Metabolites" and "Isolation of Metabolites"). This method involved extraction of a few milligram of metabolites from as much as 150 ml of bile and proved very tedious and unsatisfactory for minor metabolites. 2. Using isolated perfused rat livers The perfusion medium and the operative technique described below were slight modifications of those of Hems et al. (1966). Perfusion medium Aged human blood, stored 4-6 weeks at 4PC and no longer suitable for transfusion, was used in the preparation of the perfusate. Blood (100 ml) was centrifuged in an MSE "Major" centrifuge for 10 min at 1500 g and the plasma and buffy layer were removed by suction. The red blood cells were washed twice with 75 ml of Krebs and Henseleit (1932) bicarbonate buffer pH 7.4 containing 0.2% glucose and saturated with 02:CO2 (95:5) mixture. The washing medium was removed by centrifugation for 5 min at 1,500 g. A 5.2;:, (w/v) solution (50 ml) of bovine serum albumin powder fraction V (Sigma Chemical Co., St. Louis, Missouri, U.S.A.) in Krebs and Henseleit medium and readjusted to pH 7.4 with 0.5 M-NaOH, was added to the washed cells. The resuspended cells were made up to 100 ml with the Krebs and Henseleit solution to give the required perfusion medium. The perfusate was prepared fresh whenever required and 50 ml of the medium was used for perfusion experiment. The perfusate was placed in the perfusion apparatus 30 min before the start of the operation on the rat to equilibrate at 35°C with the 02:CO2 (95:5) gassing mixture. Perfusion apparatus The design and the arrangement of the apparatus are shown in Pigs. 2.1 and 2.2. The apparatus was similar to those of Miller et al. (1951), 4 Schimassek (1963) anpems et al. (1966). The entire arrangement was housed 83

Heater and Fan

Motor

Oxygenator and Reservoir

Overflow Filter side arm Thermostat Constant head unit

Roller pump

Thermometer

Bile-duct Probe cannula-3-

Tele Thermometer

Fig. 2. 1. Perfusion Apparatus 84

Fig. 2.2. Photograph of the liver perfusion apparatus 85

in a Perspex cabinet (42 x 44 x 44 cm) with hinged doors at the front and heated by a thermostatically controlled fan heater. The temperature of the cabinet and of the circulating medium was 35°C. This provided the temperature of the liver to be maintained at 37°C as measured by a YSI No, 46 Tele Thermometer with a No. 402 Probe (Yellow Springs Instrument Co., Inc., Yellow Springs, Ohio, U.S.A.). The reservoir (12 x 6 x 6 cm deep) for the perfusate and its cover were made of Perspex and also enabled oxygenation of the perfusion medium. The 02:CO2 (95:5) mixture, saturated by bubbling through water in a Perspex cylinder (7 x 3 cm dia.) inside the cabinet was passed into the reservoir at a rate of 75-100 m1/min for oxygenation. The thin film for oxygenation inside the reservoir was provided by rotation of 16 equally spaced flat polytetra- fluoroethylene discs (dia. 4.5 cm) which also provided a means of continuous stirring of the medium. The oxygenator was rotated at 48 revs/min by a constant speed RBT 2407 motor (Holtzer-Cabot, Boston, Mass., U.S.A.). The perfusate from the reservoir was driven by a roller pump (Fletaloglass, Inc., Boston, Mass., U.S.A.) operating at a constant speed of 50 rev./min (providing 300 pulsations/min) to a glass "constant head" arrangement (5 x 2.5 cm dia.) held firmly by a clamp fixed to a central vertical rod. The height of the constant head arrangement was adjusted to provide maximal perfusate pressure of 15 cm of water. The perfusion medium inside the constant head unit was filtered by passing through a plastic mesh filter, taken from a BR-10 Blood Administration Set (Baxter Laboratories Ltd., Thetford, Norfolk, U.K.) and the excess was returned to the reservoir by an overflow side arm. The remainder of the medium passed through the liver placed in a porcelain crucible contained inside a circular Perspex dish (7 cm dia. x 3 cm deep) clamped to the vertical rod in a position below the constant head unit. The porcelain crucible and the Perspex dish had a central 1 cm and 1.5 cm dia. hole respectively to allow passage of a return cannula in the vena cava. After leaving the vena =vat the perfusate returned to the combined oxygenator and 86

reservoir via a small chamber (3 x 2.5 cm dia.) leading out from the bottom of the perspex dish. The chamber enabled the perfusate flow rate to be determined by estimating the time taken to fill it to a 6 ml mark when the return to the reservoir was prevented by means of a polytetrafluoroethylene

tap. Soft transparent vinyl tubing (lortex Ltd., Hythe, Kent, U.K.; i.d. 3.2 mm, e.d. 6.35 mm) was used throughout to connect the individual pieces of

apparatus. However, the end of the tubing entering the porcelain crucible

was connected to a 3.5 cm length of cortex polythene tubing (i.d. 3.99mm, e.d. 5.95 mm), which was heated and drawn out to an external diameter of 4 rum to form a Luer fitting with a size 13 g Luer needle attached to the portal vein

cannula and the liver (see "Operative Technique"). A screw clip was placed around the soft tubing between the constant head unit and the porcelain

crucible to adjust the rate of flow of the perfusate through the liver.

Before each experiment, the entire apparatus was sterilised by circulating

a solution (50 ml) of sodium hypochlorite (containing w/v, of available ) for 10 min. The removal of the hypochlorite solution was followed by thorough rinsing by circulation of water (5 x 80 ml) and then Krebs and Henseleit solution (2 x 50 m1). Bacterial growth was further minimised by the

addition of 20 mg/1 of Terramycin (oxytetracycline hydrochloride; Pfizer Ltd., Sandwich, U.K.) to the perfusate as suggested by Schimassek (1963) and Brauer et al. (1951).

Operative Technique Brauer et al. (1951) have reported that increased survival time of the nerfused livers was obtained if the body weight of the donor rat was high. In the larger animals, however, the blood vessels were difficult to isolate

for cannulation because of the high fat content. For this reason, rats

weighing 200-220 g were used. The livers from these animals weighed 9.0 1.0 g. The animal was anaesthetised for the operation by intraperitoneal

administration of 0.1 ml per 100 g body weight of a solution of liembutal(6%,w/v) 87

(pentobarbitone sodium; Abbot Laboratories Ltd., Ctueensborough, Kent, U.K.). The abdomen was opened through a mid-line incision, and two transverse incisions each to left and right of the mid-line tore made, taking care not to damage the liver. Bleeding-from the major vessels of the abdomen caused by transverse incisions was greatly reduced by clamping the abdominal wall with large (20 cm) artery forceps, which were removed after the incisions were completed. The intestines were placed on the left side of the animal enabling the liver, portal vein, inferior vena cava, bile duct and right kidney to be exposed. The thin strands of connective tissue between the lobes of the liver and holding the liver to the diaphragm and the digestive tract were cut very carefully using scissors with a rounded end to avoid puncturing the lobes. The connective tissue between the right lobe of the liver and the vena cave was cut and to loose ligatures were placed around the cave above the right renal vein. The liver was kept moistened with Krebs and Henseleit solution using a cotton swab.

1,iext, the branch of the hepatic artery, the superior pancreatico-duodenal artery, which supplies blood to the duodenum and the mesoduodenum, was cut between two tight ligatures near the branching point. This ensured that bleeding was minimal on separating the bile duct from the connective tissues.

A loose ligature was placed around the common bile duct, which was then cannulated by a 20 cm length of Portex polythene tubing (i.d. 0.4 ram, e.d. 0.8

mm) cut at an angle to provide a sharp point. The cannula was inserted into the bile duct via a small incision and pushed to the point just before the duct branches and secured by tightening the ligature. The intestines were moved to the animal's right and the pyloric vein

identified. This vein, being a branch of the portal vein nearest to the liver, was cut between two tight ligatures. This ensured that no loss of the perfusion medium occurred from the pyloric vein if the tip of the portal vein cannula failed to reach past this branching point. A single loose ligature was placed around the portal vein away from the liver but just before the pyloric vein. 88

A second loose ligature was placed around the vein at a point distal to the liver. The portal vein was then cannulated using the following technique. A 3 cm length of Portex polythene tubing (i.d. 2.18 mm, e.d. 3.25 mm), which had been drawn at one end to an outside diameter of 1.6-1.8 mm after heating in a gas flame, was used as the cannula. The narrow end was cut at an anad,of 45° to facilitate entry into the vein and a circular constriction made 2.5 mm from the tip using a piece of strong cotton thread. Jhen the ligature around the portal vein was guided into this constriction and tightened, it would have prevented the cannula from slipping out of the vein. A size 13 g Luer syringe filling needle (Holborn Surgical Instrument Co. Ltd., London, U.K.), 2 cm in length, was inserted 0.5 cm into the wider end of the cannula. A 3.5 cm length of Portex polythene tubing (i.d. 3.99 mm, e.d. 5.95 mm) was drawn out at one end to an external diameter of 4.0 mm (this piece was identical to that connected to the end of the soft transparent vinyl tubing entering the porcelain crucible as described under "Perfusion Apparatus"). This allowed the narrow end to fit firmly inside the Luer fitting of the filling needle. The entire portal vein cannula arrangement was connected by a 300 cm length of Portex polythene tubing (i.d. 2.18 mm, e.d. 3.25 mm) to a bottle approximately 100 cm above the operating platform and containing Krebs and Henseleit solution. A central 100 cm of the original 300 cm length of tubing was coiled and placed in a water bath at 40°C. This raised the temperature of the Krebs and Henseleit medium, passing down the tube and then dripping out of the portal vein cannula, to 30-33°C. The rate of flow of the Krebs and Henseleit solution through the cannula was adjusted by a screw clip around the polythene tubing so that the flow was minimal. It was crucial that the medium inside the tubing be free of any air bubbles prior to cannulation of the portal vein. A small incision in the portal vein was made at this stage, the cannula inserted and secured by guiding the ligature into the constriction. After the portal vein was cannulated, the liver changed to a pale brown colour due to the displacement of blood with the Krebs and Henseleit medium. The second ligature around the portal vein distal to the liver was tied to shut off the blood supply from the viscera to the vein, Immediately after the cannulation of the portal vein, the thorax was opened by a longitudinal incision on each side of the animal towards the head and a transverse incision of the diaphragm above the vena cave. The xiphoid cartilage was clamped by a 20 cm artery forceps and the chest wall flapped back towards the head to reveal the heart and the inferior vena cava. Any fatty tissue around the cave was carefully removed using a pair of 12 cm plain dissecting forceps. Two loose ligatures were placed aroun4 the vena cava, one near the remains of the diaphragm and one close to the heart. At this stage the 2 loose ligatures around the abdominal vena eava above the right renal vein were tied and the vein between the ligatures was cut. The ligature around the cava close to the heart was tied and a small incision made in the vein between the heart and the diaphragm. The outflow cannula, very similar to that used for the portal vein, was placed inside the thoracic vena cava and the ligature guided around the constriction near the tip and tied. The outflow of the Krebs and Henseleit solution through the cannula began immediately. The liver was rapidly removed from the animal by cutting the diaphragm below the vena cave., the oesophagus and any other tissues holding the liver. Using a small (12 cm) pair of artery forceps, the portal vein cannula was clamped and so preventing the passage of the Krebs and Henseleit medium through the liver. The Luer needle was disconnected from the supply of the Krebs and Henseleit solution, leaving the needle, the portal vein cannula and the liver still connected to each other.

The liver was then placed diaphragm side down into the porcelain crucible with the return cannula in the vena cava passing through the central holes and into the graduated chamber. The perfusate supply was attached to the Luer needle ensuring that no air bubbles were trapped, and the artery forceps around the portal vein cannula released to start the perfusate flow through the liver.

The flow rate was adjusted to 10-15 ml/min. The liver regained its natural red colour and bile flow began immediately. The total time of the operation 90

was usually 30-40 min and the liver was without an active blood flow for 6-8 min. It was crucial to keep the ischaemia interval well below 10 min otherwise the performance and survival of the liver was greatly reduced. Addition of LSD to the Perfusate The [14C]- LSD (specific activity 0.3 pCi/mg; 1-2 mg LSD/g of liver weight) and tartaric acid (15-30 mg) were dissolved in Krebs and Henseleit medium (5 ml). The liver was allowed to reach a steady state (15 min) before the LSD solution was added to the perfusate in the reservoir-oxygenator. Due to the vasoconstrictor action, addition of the drug immediately reduced the rate of flow of perfusate through the liver to 3-4 ml/min, but this returned to normal after 30-45 min. The flow rate was not altered in control experiments Which were identical in every respect but only LSD was excluded. Bile was collected every hour in graduated tubes until the bile flow rate became very low (usually after 4-5 h). At the end of the experiment, the 14C-activity in the bile, perfusate, liver and washing was determined (see under "Radiochemical Techniques"). Metabolites in the bile, perfusate and liver (as a homogenate) were subsequently isolated. Concentration of Metabolites 1. From the rat bile The metabolites in the bile from liver perfusion or in the extract of the bile from the bile-duct-cannulated rats were concentrated using columns of non-ionic Amberlite XAD-2 exchange resin (B.D.H. Chemicals Ltd., i'cole, Dorset, U.K.) washed as described by Mule et al. (1971). The column size varied depending on the volume to be treated. Uslially, either a 25 ml or a 50 ml butette (i#d. 10-12 mm) was used and the XAD-2 columns were 15 cm or 40 cm in height respectively. The smaller column was most often used for the bile (2-5 ml) from the isolated perfused livers. The bile or extract was passed through the column at a rate of i ml/ min followed by distilled water (100-150 ml) at 5 ml/min to remove the unadsorbed radioactivity (4-5). The column was allowed to run "dry" and excess water expelled by blowing nitrogen 91

through it for 5 min. The adsorbed radioactivity was eluted with methanol (150-200 ml) until 85-90i, was present in the eluate. The methanol solution was evaporated to dryness in vacuo at 35-40°C, the residue dissolved in a minimum of methanol and chromatographed on preparative t.l.c. plates for the isolation of the metabolites. 2. From the monkey urine The 24 h urine samples from the monkeys were pooled and the metabolites concentrated using XAD-2 resin as described above. The XAJJ-2 column was 35 x 3 cm dia. The rate of flow for the urine (350 ml per column) was 2 ml/min, for water (1000 ml) it was 10 ml/min and methanol (500 ml) was used for the elution of the adsorbed metabolites. Recovery of the radioactivity in the methanol solution was 85-90. T.l.c. of methanol eluate developed in system S followed by radiochromatogram scanning showed that the same metabolites were present as those in the "whole" urine. 3. From the perfusate and liver homogenate from perfusion experiments The liver was homogenised with 50 ml water in an Ultra-Turrax homogeniser (Janke-Kunkel KG, Staufen i. Breisgau, West Germany) and pooled with the respective perfusate. The mixture (about 100 ml) was either adjusted to Ida 10 with aq. 11- 13 (sp. gr. 0.88) and extracted with chloroform (2 x 100 ml) or freeze dried and then extracted exhaustively with methanol. The extract was evaporated to dryness in vacuo at 35-40°C. The residue was redissolved in a minimum of methanol and the metabolites isolated by preparative t.l.c. Isolation of Metabolites The concentrated metabolites were chromatographed on preparative t.l.c. plates. They were prepared by forming a slurry of 100 g Silica Gel MF254 (E. Merck A.G., Darmstadt, Germany) in 230 ml water and spreading on glass plates to give a thickness of 0.8 mm. After drying, the plates were heated at 100°C for 1 h, cooled and pre-eluted with methanol before being used for the chromatography of the compounds. The solvent system C was used for the development of t.l.c. plates containing biliary metabolites. System s was 92

used to separate monkey urinary metabolites and metabolites from pooled nerfusate and liver homogenate. The compounds were located under UV light (254 nm) or by radiochromatogram scanning, the corresponding area of silica gel scraped off and eluted with methanol (3 x 3 vol.). After filtering through sintered glass, the eluates were pooled and evaporated to dryness in vacuo at below 40°C. When necessary, the metabolites were further purified on preparative t.l.c. plates using the same solvent system or other appropriate solvent systems such as A, G or H. LQD and its Derivatives Urine, faeces, bile, perfusate and perfused liver were analysed the same day, if possible, or within the next 2 days. When not required for analysis, these samples, LSD and the derivatives were stored at -20°C. [14C]..L6D, however, was kept in liquid nitrogen at -196°C. laper and'Thin-Layer Chromatography The solvent systems used were as follows: A. Butan-1-ol : acetic acid : water (4:1:2, by vol.) B. Buten-1-01 : formic acid (98-1004 : water (4:1:2, by vol.) C. iropan-i-ol aq. E1.13 (sp. gr. 0.88) (7:3, v/v) D. Chloroform : methanol (3:2, v/v) Chloroform ; methanol (4:1, v/v) Iethanol chloroform (4:1, v/v) G. hethanol : chloroform : water (5:5:1, by vol.) H. Acetone : eq. 1H3 (sp. gr. 0.88) (100:1, v/v) Paper chromatograms were developed in systems A, B and C only using the descending technique. Tate. was carried out in all the above solvent systems except B. Bile (0.003-0.2 ml), urine (0.05-0.2 ml) or faecal homogenate (0.3-0.4 ml) was chromatographed on either Whatman No. 1 or 3 I11 paper for solvent systems A and B and on only 3 NN paper for system C. lir values were approximately the same on both papers. For t.l.c. of bile (0.002-0.02 ml), urine (0.03-0.05 ml) or pure compounds, alun5nium-backed Silica Gel 60 i254 93

Plates (E. Merck A.G., Darmstadt, Germany; 0.2 mm thick) were used. Silica Gel GF254 or HP254 (E. Merck A.G.) coated on glass plates (0.25 mM thick) were also used. Ultraviolet (UV) Light LSD and most of its derivatives were seen as blue fluorescent spots under

-0 light (254 nn; Hanovia Chromatolite, Slough, pucks., U.K.). few of the derivatives were not highly fluorescent and quenched the fluorescent baciz,ound-r of the silica gel plates. Spray Reagents 1. Van Urk reagent (Merck, 1971, p.32) 4-Dimethylaminobenzaldehyde (1 g) dissolved in 50 ral concentrated MCi and 50 ml ethanol. Reacts with a free 2-position of indoles to give coloured (usually blue) spots. 2. Ehrlich reagent (Heacock and Mahon, 1965) 4-Dimethylaminobenzaldehyde (1 g) dissolved in a mixture of 25 ml concentrated HC1 and 75 ml methanol. Reacts with a free 2-position of indoles to Give coloured spots. 3. DMCA (Heacock and liahon, 1965) 4-Dimethylaminocinnama7.dehyde (DMQA; 2 g) dissolved in a mixture of 100 ml 611-HC1 and 100 ml ethanol. The solution was diluted with 5 volumes of ethanol immediately before use. Gives blue or purple spots with indole derivatives. 4, Xanthydrol reagent (Merck, 1971, p.103) Xanthydrol (0.1 g) in 90 ml ethanol and 10 ml concentrated ITC1, prepared immediately before use. Gives various colours with indole derivatives. 5. Gibb's reagent 2reshly prepared 2,6-dichlorobenzoquinone-4-M-chloroimine (0.05j in ethanol. The spots were oversprayed with saturated iyeHCO3. Mor the detection of , which give a variety of coloured products with the reagent. 94

6. 1-Litroso-2-naphthol (Udenfriend et al., 1958) The plate was first sprayed with a solution of 1-nitroso-2-naphthol in 95.. ethanol. After drying, it was sprayed with freshly prepared 214 HCl (5 ml) containing 0.2 ml of LaN0,. Specific for 5-hydvuxyindoles which appear as violet spots on a faint yellow background. Unfortunately, the sensitivity of the test is low, at least 10-20 pg being required for detection. 7. Diazotised sulphanilic acid (SA) in HC] (Heacock and Mahon, 1965) Ten volumes of a solution of sulphanilic acid (9 g) in concentrated HC1 (90 ml) and water (900 ml) were mixed at 0°C with one volume of laNO2. The excess nitrous acid was destroyed after 5 min by the addition of excess ammonium sulphamate. 6-Hydroxyindoles react specifically to give an immediate red colour. S. Diazotised sulphanilic acid ,(DSA) in la9CO3 (Smith, 1960) One volume of a solution of sulphanilic acid (9 g) in concentrated 1C1 (90 ml) and water (900 ml) were mixed at room temperature with one volume of The mixture was allowed to stand for 4-5 min and then two volumes of 10';:, Ea2CO3 were added. The chromatograms were sprayed immediately. Phenols give intense reddish-brown colours with this reagent. 9. Diazotised 4-nitroaniline 4Nitroaniline (0.25 g) was dissolved in 25 ml 11 -HC1 and 25 ml ethanol. ',..a1;02 (0.5 g) was added and after 0.5 rain the plate was sprayed. The chromatograms were oversprayed 5 min later with 0.5N-NaOH in ethanol. Phenols show up as reddish-brown spots with diazotised reagent. .;nzyme Hydrolysis Bile (0.02-0.05 ml) or urine (0.5 ml) was adjusted to pH 5 with 0.2M- acetic acid and 1 ml of pH 5 acetate buffer (0.2M) and 1 ml of p-gluctrconidaze (Ketodase; William R. Warner 4 Co. Ltd., Eastleigh, Hants., U.K.) or 0.5 ml sulphatase (Type 11-2; Sigma Chemical Co., London, U.K.) were added. The mixture was then incubated at 37°C for 2-48 h. Purified glucuronide metabolites of LSD (0.3-0.5 mg) were dissolved in 1 ml H 5 buffer, an equal volume of 95

Ketodase added and incubated for 4 h. Although Ketodase contaned no suiphatase, the suiphatase preparation contained some f-glueuronidase. Jmever saccharo-1,4-lactone (2 mg; Sigma Chemical Co., London, U.K.) was added to the suiphatase incubations to inhibit the f).-elucuronidase activity. Controls contained boiled enzyme, saccharo-1,4-lactone or phenolphthalein elucuronide. The formation of phenolphthalein from its glucuronide, indicated by the pink colouration on addition of a base, ensured that the Ketodase was working. Methanol (25 ml) was added to the incubation mixture at the end to precipitate the protein, and the suspension filtered through sintered glass. The filtrate was reduced in volume in vacuo at 35-40°c before being chromato- graphed, Hydrolysis with acid or alkali was net attempted since this would have involved boiling and cause degradation of the LSD derivatives. i,ethylation of Phenolic Compounds The phenolic compound (0.2-0.5 mg) was dissolved in 1 ml methanol. 1,-methylN-nitroso-4-toluenesulphonamide (Diazald; Ralph K. .Emanuel Ltd., Ilembley, U.K.; 1 g) in ether (50 ml) was treated with 5/:, ethanolic KOH (5 ml) to generate diazomethane, which was bubbled directly into the methanolic solution using nitrogen as a carrier gas. The extent of methylation was shown on t.l.c. plates developed in solvent system and E. Uswany 20-60 min were required for almost complete (>95/,) conversion to the methylated derivative. :itrogen was then used to expel the excess of diazomethanc and evaporate the solution to dryness. Alen complete conversion to the methylated derivative did not occur, preparative t.l.c. was used in solvent system E or II to senarate and purify the methoxy-derivative. Radiochemical Techniques The 14C in the samples was determined using 1ackard Tri-Uarb Scintillation spectrometers (models 3214 and 3320) and a dioxan prepared as described by Bridges et al. (1967). Urine (0.2-1.0 ml), bile (0.005-0.2 ml), perfusate (0.05 ml), perfused liver homogenate (0.1 ml) and cage washing (0.5- 1.0 ml) were counted directly. However, faecal homogenate samples (0.1-1.0 ml), 96

rrenared by homogenisation in water (50-150 ml) using an Ultra-Turrax homogeniser, were counted as suspensions in the dioxan scintillator containing (w/v) of Cab-0.611 (thixotropic gelling agent; Koch-Light Laboratories Ltd., Bucks., U.K.). Cab-O-Sil increased counts in the faecal samples by 15-50 although no significant change in counts were obtained with urine or other tissue samples. The radioactivity in the expired carbon dioxide was estimated by counting 3 ml of the absorbent in 15 ml of a toluene : 2-methoxy- ethanol (2:1, v/v) scintillation medium containing 2,5-diphenylemzole (1Y0, 5.5 g/l). Tritium in the samples was counted in a toluene base scintillation nedium (Capel et al., 1974). The efficiency of the counting was determined by the channels-ratio method or by the atiAttion of [3H]- or [14C]-toluene (The :ladlochemical Centre, Amertham, U.K.) internal standard. Counting efficiency for tritium was 35-39% and for carbon-14 it was 50-74 for faecal homogenates, 65-7% for urine, perfusate, liver homogenate and cage wash, 64-69;,- for the carbon dioxide absorbent and 75-83% for bile. Paper chromatograms and t.l.c. plates were scanned in a Packard P.adio- chromatogram Scanner (model 7200). Sections (0.5 or 1 cm wide) of the paTer chromatograms or t.l.c. plates were counted in the scintillation counter to enable quantitation of the metabolites and detection of any minor radioactive compounds. Spectra Infra-red (i.r.) spectra were recorded as liquid paraffin mulls or K.-3r discs on a Perkin-Elmer Infracord 137 Spectrophotometer. Mass spectra of compounds using the direct insertion technique were recorded on either a Varian MAT CH5 or an AEI MS 902 Mass spectrometer: probe temperature, 120-250°C; chamber temperature, 220°C; ionising energy, 70 eV; ionising current, 300 A. Nuclear magnetic (n.m.r.) spectra were recorded on a Bruker ;1YX 90 Spectrometer with Fourier transform (Bruner Magnetics, Burlington, Ma., U.S.A.) at King's College, University of London. 97

Polarimetry Optical activity was determined on a Perkin-Elmer Model 141 Polarimeter at of 546 and 589 nm. The rotations were measured in cells of 10 cm path length using pyridine as the solvent.

Eeasurement of •pH pH readings were obtained on a lye-Unicam pH Teter (model 291). :aectroenceRhalographic (EEG) Studies Animals Fourteen male chinchilla rabbits (Hylyne Commercial Rabbits, Lorthwich, Cheshire, U.K.), weighing 1.5-3 kg, were used in the experiment. The animals were maintained on a diet of RAF pellets (Labsure Animal Diets, Poole, Dorset, U.K.) and used in pairs. Each pair was allowed 3-4 weeks recovery after the experiment before being reused. Caulanc2LnnLo kei- marinalveina ior the intravenous administration of compounds, the marginal ear vein of the rabbit was cannulated as follows. The animal was first restrained by being wrapped in a thick cotton cloth (100 x 100 cm) so that only the head and the ears were exposed. The hairs around the vein were shaved and 0.02-0.05 ml of a local anaesthetic, lignocaine hydrochloride (Lidothesin, (w/v); iharmaceutical llanufacturing Co., Epsom, U.K.) was placed subcutaneously

directly next to the vein. was used to dilate the . A puncture was made in the vein using a Gillette Scimitar disposable hypodermic needle (19 g x 5 cm) and the cannula inserted 2-3 cm into the vessel. The cannula was held in position with an adhesive plaster. The cannula was made from a 20 cm length of Portex polythene tubing (i.d. 0.76 mm, e.d. 1.22 mm) which had been heated in an oven at 100°C and drawn out at one end to an external diameter of 0.5 mm. The narrow end was cut at an angle to provide a sharp point. The wider end was attached to a hypodermic needle (21 g x 3.8 cm) and syringe containing saline with added 500 I.U./ml of heparin (:wines and Byrne Ltd., Greenford, U.K.). This enabled the cannula to

98

be kept filled with the saline medium. After cannulation, the cannula was clamped with a small (12 cn) pair of artery forceps to -nrevent the oetflow of blood. The injection needle and the syringe wore disconnected from the cannula and replaced by a 2 cm length of a suitable vire to act as a plug for '6he cannula. The artery forceps were then released. Administration of compounds All the compounds tested were given at a dose level of 77 I:tole:kg. 'cy were dlosolved in a normal saline nediue, containing 0.05, (w/v) (+)- tartaric acid, to give a concentration of 770 nmole/nl of the compound. The wire plug in the cannula was removed to enable intravenous administration. EEG recordings The EEG recordings were made in the Neurology Department, : ary's I ospital, .eaddington, London, under the supervision of Dr. J. Durstoe. :our disposable hipodermie needles (25 g x 1.6 cm) were placed subdernally in the scalp and attached to the leads of the BEG recorder to produce two chame= bieolar recordings giving right and left antero-posterior tracings. fifth electrode placed at the back of the head acted as the . The recordings were made from two rabbits simultaneously on an Elema-Schbnander Eingograf 224- /6 Electroencephalograph (aierex Ltd., Wembley, Middlesex, U.1%) immediately before and after administration of the compound, and then after ever: 5 min for 0.5 min until 35-40 min had elapsed following administration. At the end of the recordings, the electrodes and the cannula were removed and the animals returned to their cages. Fluorescence Studies of LSD and its Derivatives For the fluorescence study, the purified compounds only were used. Preparation of solutions Stock solutions were prepared by dissolving 0.2-0.5 mg of the compound in a minimum of methanol (0.02-0.05 ml) and diluted with glass distilled water to a concentration of 0.25 mg/mi. For the pH-fluorescence studies, aliquots of the stock solutions were diluted with solutions of 99

appropriate pH to a desired concentration (0.1-1.0 pg/ml) and examined immediately. To obtain solutions of various pH values, the glass distilled water was titrated with 0.1-18 M-H2SO4 or 0.1-10 M-NaOH. Solutions of pH 2-12 were checked with a Pye-Unicam pH Neter (model 291). Solutions of pF. 0-2 and 12-14 were made by the addition of calculated amounts of acid or alkali. Below 0 and above 14, the acidity function Ho was used, the molarity of the solution being obtained from Paul and Long (1957) and Bowden (1966). The excitation and fluorescent wavelengths were adjusted to give maximum luorescent intensity reading for each solution. The readings were made within 30 of exposure to the exciting light. For the variation of fluorescent intensity with fluorescence or excitation , a neutral aqueous solution or a 3N-HC1 solution of the compound was used. The pKa values were estimated from the fluorescence-pH curves (see Bridges et al., 1966). Spectrophotofluorimeter Fluorescence of the solutions was obtained on an Aminco-Bowman Spectrophotofluorimeter (American Instrument Co., Silver Springs, Ed., U.S.A.) using cuvettes with 1 cm pathlength. The excitation and fluorescence wavelengths were calibrated by using a pen ray quartz lamp as described by Udenfriend (1962). Stability of LSD The stability of LSD solution of concentration 0.1 or 1 pg/ml was determined at various PH values. Solutions of different pH values were obtained as described previously but HC1 instead of H2SO4, was used. The LSD solution (20 ml) was placed in a stoppered test tube and left at room temperature in the dark, stored at 4°C, left uncovered in the room or incubated at 37°0 in the dark. Phosphate buffers (0.1, 0.01 and 0.001 Ii, pH 7.4) were also used as solvents. For the stability study under a mercury vapour lamp, the Eanovia Chromatolite lamp was used Without the filter. Irradiation was carried out in quartz cuvettes containing 3 ml of the La solution or in 10 ml beakers 100

containing 5 ml of solution with the lamp 10 cm above the surface of the solution. The change in the fluorescence intensity readings in the Aminco-Bowman fluorimeter was used as a measure of the stability of the LSD solutions. The conversion of LSD to iso-LSD in solid form at room temperature in the dark was studied using the Perkin-ilmer polarimeter. The decomposition of solid [14Cj-LST (13.6 pOi/mg) at -20°C and in liquid nitrogen (-196°C) was determined by t.l.c. in solvent system D or 1 followed by radiochromatogram scanning. 101

CHAPTER THREE

Metabolism of [14C)-LSD in the Rat, Guinea Pig and Rhesus Monkey

Contents Page

RESULTS 102 Rat 102 Excretion of Radioactivity 102 Isolated Perfused Rat Liver 102 Chromatographic Distribution of [140]-LSD Metabolites 103 Chromatographic Distribution of [2-31-1]-LSD Metabolites 105 Chromatographic Distribution of [140]-iso-LSD Metabolites 125 Fluorescence of Metabolites under UV Light and Reaction with Van Urk Reagent 125 Identification of LSD Metabolites 126 Bile and urine 126 1-erfusate and liver from perfusion experiments 131

Faeces 137 Identification of Iso-LSD Metabolites 137 Guinea Pig 138 Excretion of Radioactivity 138 Chromatographic Distribution and Identification of PO LSD Metabolites 138 Rhesus Monkey 149 Excretion of Radioactivity 149 Chromatographic Distribution and Identification of [14C]-LSD Metabolites 149 DISCUSSION 158 102

Metabolism of [140]-LSD in the Rat, Guinea Yis and Rhesus Monkey The metabolism of [1401-LSD has been studied in the rat, guinea pig and Rhesus monkey. The results arising from the study are reported in this chapter.

Results

Rat Excretion of Radioactivity When [140]-LSD (1 mg/kg; 13.6 uCi/mg) was administered intraperitoneally to rats, 71,, of the dose was excreted in the faeces in 96 h, 16.1, in the urine and 3.4;,; as 14002 in the expired air (Table 3.1). About 80;- of the total 140 (2.72,;- of the dose) in the expired air in 96 h was excreted in the first 8 h and the major part of the radioactivity in the urine and faeces was excreted in 48 h. The 14C-labelled drug (1.33 mg/kg) was injected intravenously into bile- duct-cannulated rats and in 24 h, of the 140 was found in the bile, with 7.k., in the urine and 0.7 in the faeces (Table 3.2). Excretion in the bile was very rapid with 6 of the dose appearing in the first 5 h. Elimination of radioactivity in the bile-duct-cannulated rats receiving intravenously either [14C]-iso-LED (3.71 p0i/animal; 1.2 mg/kg assuming the specific radio- activity to be 13.6 pOi/mg) or [2-311]-L6D (9.80 pa administered to one animal) was almost identical (Table 3.2). The [2-2a]-LED was purified prior to use. The exact specific activity was unknown since a considerable loss in the radioactivity (as tritiated water) occurred due to tritium and hydrogen exchange. However, the specific activity was estimated to be about 40 uCi/mg which would make the dose of [3H]-LSD administered to the rat as 1 mg/kg. Isolated Perfused Rat Liver In an attempt to obtain larger quantities of metabolites, isolated rat livers were perfused with LSD. Since LSD produces vasoconstriction of the blood vessels (see Chapter One under "Pharmacological Effects of LSD"), the 103

perfusate flow was greatly reduced on addition of the drug. The flow, however, was restored after 30-45 minutes. Since bile flow was dependent on the flow rate of the perfusate, the amount of bile excreted in the first hour was lower as compared to the controls (see Table 3.3). When the rate of perfusion was restored to its initial value, bile flow increased and was similar to the control figures. Bile flow rate was important as it was directly related to the rate of biliary excretion of the radioactivity. Thus, when [1401-LSD (1 mg/g liver) was added to the perfusate, excretion of 114C in the bile in the first hour was low but increased subsequently so that 44 of the added radio- activity was excreted in 4.5 h. The major part of the total 140 excreted in the bile appeared in the second, third and fourth hour. In one experiment When the amount of LSD perfused was 2 mg/g liver, excretion in the bile in 4.5 h was only 29 although bile flow rate was unusually high (Table 3.3). This was indicative of saturation of the biliary excretory mechanism of perfused livers for LSD and its metabolites. Total recovery of 14C in all the perfusion experiments was 85-87iL, (Table 3.4). Thus, substantial de-ethylation of the diethylamide side chain of LSD occurred resulting in the elimination of radioactivity as 14002. Although elimination of radioactivity as 14CO2 was not studied, the product of de-ethylation, lysergic acid monoethylamide, was subsequently identified (see later). Chromatographic Distribution of [140]-LSD Metabolites Chromatography of the bile, urine, faecal homogenate and extract of pooled perfusate and liver homogenate on parer and thin-layer plates revealed several metabolites. The RF values, fluorescence under UV light (254 nm) and colour reactions with Van Urk reagent of these compounds and other LSD derivatives are summarised in Tables 3.5, 3.7, 3.8 and 3.9. The metabolites were quantitated by scintillation counting of 0.5 or 1.0 cm sections of the chromatograms. Since resolution of the metabolites in most cases was not complete in any one system, results from two or more chromatograms developed in different solvent systems were used for the quantitative estimation. The 104

excretion figures of individual metabolites of LSD are given in Tables 3.6 and 3.7. Bile from bile-duct-cannulated rats dosed with [14C]-LSD contained at least 7 metabolites (R1-7) (see Tables 3.5 and 3.6) as revealed by paper chromatography followed by radiochromatogram scanning and scintillation counting of sections of chromatograms. The radiochromatogram scans and the corresponding histograms following paper chromatography in systems A and C are shown in Figs. 3.1 and 3.2. The excretion of the two major polar compounds, R4 and R5, was 52';', of the dose in 5 h (Table 3.6). Radiochromatogram scans and histograms of urine from intact animals showed that the distribution pattern was very similar to that of bile but R2 was absent. However, an additional radioactive compound, R8, was detected on the urine chromatograms developed in system A (Fig. 3.3). Again, the kajor metabolites were R4 and 1R5 and accounted for 9 of the dose in 24 h. At least 6 compounds were present in the faecal homogenate of intact rats (Table 3.7). Five of these were detected on paper chromatograms developed in system A (Fig. 3.4). metabolites R4, R5 and R6 found in the rat bile were present in addition to 3 new compounds R12, R13 and R14. Unchanged LSD was not detected in the bile, urine or faecal homogenate. Paper chromatograms of bile from isolated perfused rat livers gave a similar distribution pattern to that from bile-duct-cannulated animals. Pig. 3.5 shows the radiochromatogram and histogram of perfused bile chromatographed on paper in system B. a7 was present in the bile in isomeric forms which were resolved on paper in system B. The isomers of R7 will be discussed later in detail. The excretion of R6 (Table 3.6) appeared to be dose dependent. When the amount of LSD perfused was 2 mg/g liver, R6 was present in the bile but When it was 1 mg/g liver the metabolite was no longer detectable on paper or thin-layer plates. An additional radioactive compound, R11, was observed on paper and thin-layer chromatograms. This was identified as unchanged LSD (see later). Excretion of LSD in the bile was quite substantial when the larger 105

amount of the drug was perfused. Consequently, the radioactive LSD could be detected easily on a paper chromatogram developed in system C (see Fig. 3.6). However, when 1 mg LSD/g liver was perfused, excretion of LSD in the bile was greatly reduced and was detectable on thin-layer plates developed in system El C or H. A radiochromatogram scan and a histogram of bile from perfused liver chromatographed in system H are shown in Fig. 3.7. As is apparent from Fig. 3.7, the major metabolites in the bile were R4, R5 and R7. The metabolites in the pooled perfusate and liver homogenate from isolated rat liver -eerfusion experiments were also examined. The perfusate and liver homogenate were freeze-dried, extracted exhaustively with methanol, reduced in volume in vacuo and the residue subjected to t.l.c. in systems E, C. and H followed by radio- chromatogram scanning (Fig. 3.8). Biliary compounds R4, R5, R7 and R11 (LSD) were present in the extract. Three additional radioactive metabolites, R8, R9 and RIO, not present in the bile, were also detected. When the pooled perfusate and liver homogenate was adjusted to pH 10 with aq. NH3, 30r, of the radioactivity could be extracted with chloroform. The extract was chromato- graphed on t.l.c. plates in systems Es G and H. Only metabolites R7, R8, R9, R10 and R11 were found to be present in the chloroform extract. If the mixture (5 ml) of perfusate and liver homogenate was made basic with 1N-NaOH (0.5 ml), saturated with NaC1 and then extracted with i isoamyl

(98:2, v/i; 25 ml), only compounds R10 and R11 could be extracted as revealed by t.l.c. in system Al B or H. Chromatographic Distribution of [2-311]-LSD Metabolites One rat was dosed with [2-1I]-LSD in order to compare the chromatographic distribution pattern of the bile with that of bile from rats given 14,1{ C] LSD. fig. 3.9 shows the radiochromatogram of bile following [3H]-LSD administration. Comparison with Fig. 3.1 reveals that the distribution of tritiated-metabolites is very similar to that of 14C-metabolites. Thus, the compounds R2, R4, R5, R6 and R7 were all present in the bile from rats dosed with [311]-LSD but R1 and R3 were not detected. This was probably due to the insensitivity of the methods

cont. p,I25 Table 3.1. Cumulative Excretion of Raiioactivity by Rats Receiving [14C]-LSD

Three rats were dosed intraperitoneally with [14C]-LED (1 mg/kg; 2.55 ± 0.10 pCt/animal). Average values are given with ranges in parentheses. Values are expressed as of administered 14C.

Urine Faeces 14e Time after dosing (h) ••■•••■•■■•■••=2 Total

8 - - 2.7 (2.0-3.3) 2.7 (2.0-3.3)

24 12.7 (8.1-16.3) 62.2 (41.6-82.8) 3.1 (2.3-3.6) 78.0 (58.5-93.2)

48 14.7 (10.6-17.1) 72.1 (64.1-87.6) 3.2 (2.4-3.8) 90.1 (84.0-100.6)

72 15.8 (12.9-17.4) 72.8 (65.2-88.0) 3.3 (2.5-3.9) 91.9 (85.8-103.4)

96 16.1 (13.4-17.5) 73.1 (65.5-88.3) 3.4 (2.6-4.0) 92.7 (86.6-104.3) Table 3.2. Cumulative Excretion of Radioactivity by Bile-Duct-Cannulated Rats ReceivingLSD,[14k.;-j-1 [2-3111-LSD or C1-4C1-iso-LSD

The [14C]-LSD (1.33 mg/kg; 2.78 t 0.03 pei/animal), [2-31-1]-ISD (about 1 mg/kg; 9.80 )1C1 to one animal) or [140]-iso-LSD (1.2 mg/kg; 3.71 pcijaninal) was administered intravenously (femoral vein). Average values are given, where applicable, with ranges in parentheses.

Time after dose after administration of dosing (h) [14C1-LSD (n=3) 112-3H1-LSD (n=1) [14cl -leo -LSD (n=4)

Bile 1 52.1 (49.7-55.8) 45.6 39.1 (36.7-44.3)

n (gg. 2 k„., o) 58.8 54.1 (51.6-59.3) 3 66.1 (57.7-72.1) 63.4 61.7 (58.8-65.8) 4 67.4 (59.5-73.2) 65.8 (62.9-69.2) 5 67.9 (59.9-73.7) 67,9 (64.4-71.1) 24 70.9 (62.9-76.9) 73.4 74.4 (71.1-76.8) Urine 24 7.4 (4.0-12.6) 5.7 8.3 (4.0-11.5) Faeces 24 0.7 (0.0-1.6) 0.2 0.8 (0.2-1.9) Total 140 recovered 79.0 (76.1-82.5) 79.3 83.5 (81.1-90.2) Table 3.3. Cumulative Excretion of Radioactivity in Bile of Isolated Perfused Pat Livers

The [14C]-LSD (1 or 2 mg/g liver) was dissolved in Krebs and Henseleit solution and added directly to the perfusate. Average values from 3 experiments are given (with ranges in parentheses) when the perfused LSD was 1 mg/g liver. Only one experiment was performed when the amount of LSD perfused was 2 mg/g liver. Control experiments (n=2) were identical in every respect except that LSD was not added.

2 ng Control 1 mg [140]-LSD/g liver [1401 -LSD/g liver

Time after Cumulative Cumulative ": of added Cumulative ,el.of added LSD addition excretion of excretion of 140 in bile excretion of '"O in bile (h) bile (ml) bile (ml) bile (ml)

1 0.35, 0.45 0.16 (0.14-0.17) 5.0 (3.1-6.2) 0.50 7.8

2 0.65, 0.80 0.44 (0.38-0.53) 21.8 (20.8-23.4) 1.00 17.7

3 0.97, 1.07 0.66 (0.55-0.83) 33.4 (31.1-34.7) 1.50 26.2 4 - - 0.82 (0.69-1.01) 41.0 (38.5-42.8) 1.62 27.6 4.5 1.19, 1.32 0.89 (0.75-1.10) 44.2 (41.3-46.0) 1.77 29.1 109

Table 3.4. Recovery of Radioactivity from Isolated Rat Liver Perfusion ExTeriments for 4.5 h

The amount of LSD perfused was as in Table 3.3. Values are expressed as (;6 of added 14G. Ranges, where applicable, are in parentheses.

1 Mg [14C] -LSD/g liver 2 mg [14C]-LSD/g liver

Bile 44.2 (41.3-46.0) 29.1

Perfusate 19.5 (17.8-21.4) 22.4

Liver 19.7 (19.1-20.4) 24.7

Washing 3.5 (2.6-4.1) 9.1

Total 14c recovered 87.0 (85.6-88.6) 85.3

Table 3.5. Values of [114U] Ilet.abolites the at 1s ii ail Urine

n.d. - not detected. The darT.c-blue colours with Van '.irk reacent appeared within 5 min of spraying. The yellow colour of metabolite T7 aq)eared in about 15 min.

:'apes Cterora tom Thin-Layer Chronate,--xaphy 21uorescence Colour with Van , 3olvents.... A B C A C .T G ....v under UV Urk reaent Metabolite Ri - - 0.00 - - - - 0.00 - n.d. n.d. R2 0.14 0.13 0.11 - - - - 0.11 - n.d. n.d. R3 0.22 0.25 0.25 - - - 0.24 - n.d. n.d. immediate R4 0.27 0.33 0.37 0.1i', 0.30 0.00 0.39 0.41 0.00 light-blue light-blue R5 0.33 0.40 0.47 0.22 0.34 0.00 0.40 0.49 0.00 dark-blue mauve-4-dark-blue R6 0.65 0.72 0.i33 ------n.d. n.d. 0.62, 0.63, R7 0.80 0.73e 0.23 0.41 0.30 - 0.00 weak-blue yellow 0.86 0.75 0.73 R8 0.72 0.72 0.90 0.46 0.7S 0.37 - 0.75 0.29 dark=blue mauve dark-blue R9 u.84 0.8? 0. 0 1.57 0.77 0.21 - D.65 0.17 da.tic-Ylue 11,2.uNrc,.---..-drrk-blue R10 0.91 0.95 0.1 0.71 0.76 0.72 - 0.91 0.5,:, n.d. n.d. R11 0.83 0.3!) 0.92 3.44:.; 0.',,7 0.53 0.52 .,,.,., (-.)., -, 0.y da:±-7,:lue mauve-4-dr.rk-blue

Table 3.6. Lotabolitrs of [14'-13D in the Pat Urine and 3110 and in Isolated Ferfused Pat Livers

See Tables 3.1--3.3 for dose of LaD. Average values are given, where applicable, with ranges in parentheses. U = Unknown.

Ferfused livers Intact rats CannUlated rats of dose of dose (1 ng/g liker) in 4.5 h Probable of dose in of dose in (2 mg/g liver) in Bile perfusate Metabolite identity urine in 24 h bile in 5 h bile in 4.5 h and liver

R1 U 0.1 (0.0-0.2) 0.9 (0.6-1.3) 1.5 2.0 (1.8-2.1) R2 U 4.0 (2.9-5.1) 0.3 1.9 (1.6-1.9)

R3 U 0.4 (0.2-0.7) 3.5 (2.3-4.5) 0.2 1.4 (1.0-1.7) R14, Glucuronide of 20.3 (15.7-24.2) 3.1 8.2 (7.0-9.0) 1.7 (0.5-2.5) 13-hydroxy-L5D 5.5 (3.7-7.1)

R5 Glucuronide of 3.5 (2.1-5.0) 31.4 (29.3-33.9) 11.5 20.9 (20.2-21.3) 1.6 (1.1-2.1) 14-hydroxy-LSD R6 13-aydroxy-LSD 0.8 (0.6-1.0) 1.6 (1.4-1.0) 4.0 R7 1.5 (1.0-1.8) 5.0 (4.c-7.o) 4.1 7.4 (6.6-8.3) 5.0 (4.6-52)

R8 Lyserr'ic acid 0.5 (0.2-0.8) 2.1 (1.7-2.5) Tionoethylamide (IAE)

119 Ler -LS:,) 4.0 (3.3-4.6) 1110 "Aromatised" 2-oxo-LSD 4.1 (3.8-4.7) R11 La) 3.1 1.0 (0.8-1.2) 17.6 (14.7-21.1) Total of above metabolites 12.3 (7.8-15.7) 66.7 (58.3-74.5) 27.8 42.8 (41.5-44.2) 36.1 (33.9-38.8) Table 3.7. EF Values and Excretion of Faecal Metabolites of__[146-LSO in the Rat

See Table 3.1 for dose of LSD. Average values are given for the excretion with ranges in parentheses. Colours under UV light or with Van Urk reagent were not detected. U ffi Unknown.

Papar Chromatography Metabolite Solvent.... A of dose_ in 24 h Probable Identity

R4 0.27 - 2.5 (1.6-3.1) Glucuronide of 13 -hydroxl.-LSD

R5 0.33 - 6.1 (4.34.6) Glucuronide of 14hydroxy-ISD

R6 0.69 0.84 7.9 (5.340.5) 13-Hydroxy-7,SD

R12 0.00 0.00 7.1 (4.8-9.6) U

R13 - 0.76 3.8 OA -4.3) U

R14 0.80 0.95 31.0 (20.6-112.0) Decomposed aglycone of R5

Total of above metabolites 58.4 (40.7-76.5) Table 3.8. Ap Values of 12-, 13- and /44(ydroxy-LSD and their Methylated Derivatives

The final colours with Van Urk reagent appeared within 5 min of spraying.

Paper Chromatography Thin-Layer Chromatography Fluorescence Colour with Van Solvent... A B C A C E F G H under UV Urk reagent Compound 12-Hydroxy4ASD 0.73 0.72 0.90 0.45 0.76 0.40 - 0.81 0.36 dark-blue mauve flight-blue dark-blue

13 -Hydroxy-LSD 0.68 0.71 0.86 0.42 0.73 0.39 0.52 0.79 0.40 light-blue immediate light - from R4 blue

14-Hydroxy-LSD - - - 0.44 0.66 0.34 0.52 0.78 0.30 dark-blue mauve-i.dark -blue from R5 -p-dark-grey-blue

12- Methoxy4.SD 0.80 0.82 0.90 0.46 0.77 0.45 0.85 0.40 dark-blue mauve plight-blue --dark-blue

13-Methoxy-LSD 0.80 0.82 0.90 0.46 0.79 0.55 0.85 0.45 lig1A-blue immediate light- blue-o-light-green- blue

14-Methoxy-LSD 0.80 0.82 0.90 0.46 0.79 0.55 0.85 0.45 dark-blue mauve-...dark-blue -3-dark-grey-blue Table 3.9. BF Values and Binary Excretion of Metabolites of [1401-Iso-LSD in the Rat

See Table 3.2 for dose of iso-LSD. Average values are given for the excretion with ranges in parentheses. The final colours with Van Urk reagent appeared within 5 min of spraying. U to Unknown; n.d.. not detected.

Paper Thin-Layer Chromatography Chromatography % of dose Fluorescence Colour with Van Solvent.... A in. 5h under UV Urk reagent Probable Identity Metabolite

R15 - - 0.00 - - 3.6 (2.3-4.1) n.d. n.d. U

R16 0.17 0.06 0.06 - - 6.4 (2.4-9.2) n.d. n.d. U

RI? 0.28 0.27 0.17 - - 5.5 (4.2-7.6) n.d. n.d. U

R18 0.37 0.36 0.36 0.24 0.20 25.3 (20.5-28.4) light-blue immediate Glucuronide of light-blue 13 -hydroxy-iso -LSD

R19 0.37 0.44 0.45 0.24 0.28 14.2 (13.8-14.6) dark-blue mauve--, Glucuronide of dark-blue 14-hydroxy-iso-LSD 0.4 n.d. n.d. 2-0xo-iso-LSD R20 0.82 0.873, 0.83 - - 9.8 (7.6-11.8)

Aglycone 0.24 0.52 light-blue immediate 13-Hydroxy-iso-LSD of R18 light-blue

Aglycone IMP 0.24 0.52 IMO dark-blue mauve --,- 14-Hydroxy-iso-LSD of R19 dark-grey-blue Total of above metabolites 64.8 (62.4-67.1) 115

10

CD 1-1

5

0 10 20 30 cm

Fig. 3.1. Radiochromatogram scan and histogram of bile from rat dosed intra- venously with [14C]-LSD (1.33 mg/kg; 13.6 tiCi/mg) and chromatographed on Whatman No.1 paper in solvent system A. See Table 3.6 for possible identity

of metabolites R2-R7.

0 = Origin S. Solvent front 116

0 10 cm 20 30

Fig. 3.2. Radiochromatogram scan and histogram of bile from rat dosed intra- venously with [14C]-LSD (1.33 mg/kg; 13.6 MCi/mg) and chromatographed on Whatman 3MM paper in solvent system C. See Table 3.6 for possible identity

of metabolites R1-R7.

0 = Origin S. F. = Solvent front 117

O

I

6

4

2

0 10 20 30

Fig. 3.3. Radiochromatogram scan and histogram of 24 h urine from rat dosed 14 intraperitoneally with [ C]-LSD (1 mg/kg; 13.6 tiCi/mg) and chromatographed on Whatman No.1 paper in solvent system A. See table 3.6 for possible identity of metabolites R2-R8.

0 = Origin S. F. = Solvent front 118

0 10 20 30 cm

Fig. 3.4. Radiochromatogram scan and histogram of faecal homogenate from rat 14 dosed intraperitoneally with [ CJ-LSD (1 mg/kg; 13.6 MCi/mg) and chromato- graphed on Whatman No.1 paper in solvent system A. See Table 3.7 for possible identity of metabolites

0 = Origin S. F. = Solvent front 119

10 20 30 40 cm

Fig. 3.5. Radiochromatogram scan and histogram of bile from isolated rat liver perfused with [14C]-LSD (1 mg/g liver; 0.3 ptCi/mg) and chromatographed on

Whatman No.1 paper in solvent system B. See Table 3.6 for possible identity of the metabolites.

0 = Origin S. F. = Solvent front 120

0 10 20 30 cm

Fig. 3. 6. Radiochromatogram scan and histogram of bile from isolated rat liver perfused with [14C]-LSD (2 mg/g liver; 0.3 ACi/mg) and chromatographed on Whatman 3MM paper in solvent system C. See Table 3.6 for possible identity of the metabolites.

0 = Origin S. F. = Solvent front 121

1

cm

Fig. 3.7. Radiochromatogram scan and histogram of bile from isolated rAtliver perfused with [14C]-LSD (1 mg/g liver; 0. 3 jiCi/mg) and chromatographed on aluminium- backed t.l. c. plate in solvent system G. See Table 3. 6 for possible identity of the metabolites.

0 = Origin S. F. = Solvent front 122

R11

Fig. 3. 8. Radiochromatogram scans of methanolic extract of freeze-dried perfusate and liver homogenate from perfusion experiments. Solvent systems used were (a) E, (b) H and (c) G. See Table 3.6 for possible identity of the metabolites.

0 = Origin S. F. = Solvent front 123

Fig. 3. 9. Radiochromatogram scan and histogram of bile from rat dosed intra- 3 venously with [ H]-LSD (1 mg/kg; 40 12Ci/mg) and chromatographed on Whatman No. 1 paper in solvent system A.. See Table 3.6 for possible identity of the metabolites.

0 = Origin S. F. = Solvent front 124

S. F.

R18

S. F.

30 40 0 10 20 CM

Fig. 3.10. Radiochromatogram scan and histogram of bile from rat dosed intra- 14 venously with [ q-iso-LSD (1.2 mg/kg; 13.6 iCi/mg) and chromatographed on Whatman No.1 paper in solvent system B. See Table 3. 9 for possible identity of metabolites R16-R20.

0 = Origin S. F. = Solvent front 125

in detecting low levels of tritium. 4uantitation of the metabolites was not attempted since recovery of counts after chromatography was only 50-60 due probably to exchange of tritium with the hydrogen in solvent systems used to develop the paper chromatograms and thus rendering the results unreliable. Chromatographic Distribution of [14C]-iso-LSD Metabolites Excretion of metabolites of [14C]-iso-LSD is shown in Table 3.9. The metabolites in the rat bile were well separated on paper chromatograms developed in system B (Fig. 3.10). Five radioactive compounds (R16-R20) were revealed on the radiochromatogram scan and histogram. A radiochrornatogram and a histogram following paper chromatography in system A gave only 4 radioactive peaks. These corresponded to R16, R17, a composite peak consisting of R18 and H19 and a single peak for R20 (see Table 3.9). A sixth metabolite, R15, was only present on paper chromatograms developed in system C. The major metabolites in the bile were R18 and R19. The similarity in Figs. 3.5 and 3.10 indicates that the metabolism of [iAC]-iso-LSD and [14C]-LSD occurs along common pathways. The RF values on paper chromatograms of the major LSD metabolites, R4 and R5, in the rat bile were very similar to the major metabolites R18 and R19. However, clear distinction in the chromatographic characteristics of the 4 compounds (R4, R5, R18 and R19) was achieved on thin- layer plates in solvent system G (see Tables 3.5 and 3.9). Fluorescence of Metabolites under UV Light (254 nm) and Reaction with Van Urk Reagent Fluorescence of the LSD or iso-LED metabolites under UV light and their colour reactions with Van Urk spray reagent are included in Tables 3.5, 3.8 and 3.9. Most of the metabolites, whose fluorescence could be detected view:131y, gave dark-blue colours like that of LSD. However, a distinct light blue colour was observed for metabolites R4 and R18 and a weak-blue fluorescence was shown by R7. With Van Urk reagent, LSD and most of the visual3y detectable metabolites gave rapid mauve colours turning to dark-blue within 5 min. Metabolites R4, R7 and R18 again differed, with R4 and R18 giving immediate 126

light-blue colours and R7 giving a slowly developing yellow colour. The majority of the metabolites, however, were produced in very small ouantities and could not be detected under the UV light or with Van Urk reagent. This was also due to a large extent, especially in the case of the faecal homogenate, to the small amount of sample, that could be chromatographed. identification of I,a Metabolites Silo and urine incubation of bile or urine with 13-glucuronidase followed by paper chromatography in systems A, 1; and C and t.l.c. in C and h showed that the intensity of peaks on the radiochromatogram scans corresponding to and 35 were greatly reduced. no hydrolysis was observed with the controls or the sulphatase preparation. It was concluded that the metabolites 34 and 1-15 were glucuronide conjugates. When the conjugates in the bile were hydrolysed with e-glucuronidase for 2-4 h followed immediately by t.l.c. in system or •i , the less polar aglycones were detectable under UV light and with k an U-ok reagent. The aglycone of R4 had the same light-blue fluorescence as the conjugate and gave an immediate light-blue colour changing to light-green-blue in 5 min with Van Urk spray. The aglycone of 35 still eossessed the dark blue fluorescence of the corresponding conjugate, but with Van Urk reaent, the aglycone gave a rapid mauve-colour changing to dark-blue and then to dark-grey- blue in 5 min. If the hydrolysis was carried out for 1L-40 hp the aglycone of R5 only could not be detected on t.l.c. plates in any system. It appeared from the results that the aglycone of 35 was unstable. The values lee the aglycones are given in Table 3.3. In order to identify the two aglycones, the glucuronides were isolated from bile of either bile-duct-cannalated rats or isolated perfused rat livers as described in "Materials and Methods" under the headings "Concentration of Metabolites" and "Isolation of Metabolites". Hydrolysis of i)4 and _z5 with f -glucuronidase for 3-4 h followed by ereparative t.l.c. in system H. allowed isolation of the aglycones in a pure form. The aglycones were phenblic, since 127

they gave brownish-purple colours with dtamtised 4-nitroaniline on t.l.c. plates. The stable aglycone from R4 was subjected to mass spectrometry and gave a molecular ion at pie 339 suggesting a hydroxy-LSD. The mass spectrum was identical with that of a sample of authentic 12-hydroxy-LSD (see Fig. 3.11). "n hen the aglycone of R4 and 12-hydroxy-LSD were methylated with diazomethane, their mass spectra gave identical molecular ions at We 353, corresponding to methoxy-derivatives of LSD. Treatment of LSD with diazomethane did not shift the parent ion from p/e 323 to m/e 337. The enzymic-hydrolysis product of R5 was unstable, as already mentioned, and its mass spectrum was not available. However, when the aglycone was treated with diazomethane immediately after isolation, a stable methylated product amenable to mass spectrometry was obtained. Its mass spectrum was identical to those of methoxy-LSD obtained from metabolite R4 and 12-hydroxy-LSD and also gave a parent ion at p/e 553 (see Fig. 3.12). The only significant difference in the mass spectra was that the ion at all/e 338 was consistently negligible in the mass spectrum of the methoxy-LSD from R4 as compared to the ion from either of the other two methylated hydroxy-derivatives of LSD. However, the mass spectra suggested that both aglycones of metabolites R4 and R5 were hydroxy-LSD. Since the three hydroxylated forms of LSD had different chromatographic characteristics and gave different colour reactions with Van Urk reagent (see Table 3.8), neither of the two aglycones were 12-hydroxy-LSD. hydroxylation only at position 12, 13 or 14 of LSD would give a and it was concluded, by a process of elimination, that metabolites R4 and R5 were glucuronides of 13- and 14-hydroxy-LSD respectively or vice versa. In order to determine the exact position of hydroxylation in the aglycones of the glucuronide metabolites, the three hydroxy-derivatives of LSD and authentic 5-, 6- and 7-hydroxyindole were sprayed with a number of chromogenic reagents including Van Urk reagent (the 5-, 6- and 7-position in indole correspond to positions 12, 13 and 14 in LSD respectively). It was hoped that the corresponding hydroxy-compounds would give one colour reaction with the same reagent. The results are 128

summarised in Table 3.10. In most cases, however, the colour reactions of authentic 12-hydroxy-LSD and 5-hydroxyindole with the same reagent were very different, which was contrary to the single colour expected for both compounds. .thus, the information from colour reactions was not expected to provide conclusive evidence which would indicate the positions of hydroxylation in the

aglycones. Eowever, comparison of colour reactions between the three hydroxy-

indoles and the three hydroxy-LaD with Van Urk reagent tended to sug gest that the aglycone of a4 was 13-hydroxy-LSD (corresponding to 6-hydroxyindolc) and that of R5 was 14-hydroxy-L3D (corresponding to 7-hydroxyindole). This was

confirmed by the colour reactions with diazotised sulphanilic acid. in '201

which gives immediate red colours with 6-hydroxyindoles. Only 6-hydroxyindole and aglycone of R4 gave the specific bright red colours immediately, indicating that this aglycone was 13-hydroxy-L3D. Therefore, the aglycones of a4 and R5

were identified as 13- and 14-hydroxy-LSD respectively.

The Rv. value on paper in system A of aglycone of R4 (13-hydroxy-L. -) compared favourably with that of metabolite R6 (see Tables 3.5 and 3.3). Then _() was eluted with methanol from the paper chromatograms developed in system A and rechromatographed on paper in systems 13 and C, the 2i, values corresponded

to those of 13-hydroxy-LSD. This suggested that R6 was 13-hydroxy-I3D. The metabolites in the bile from the perfused livers were adsorbed onto an 2:AD-2 column, eluted with methanol, concentrated in vacuo and chromatographed on preparative t.l.c. plates in system C as described in Chapter Two. live fluorescent spots were visualised under UV light and these were eluted off the

silica separately with methanol. The one at the highest RF was compound R11

;Lich was identified as unchanged LSD by comparison of chromatographic characteristics on paper and t.l.c. plates with an authentic sample of L.D in all the solvent systems. In addition, the mass spectra of LSD (rig. 3.11) and

R11 were identical. The two metabolites (R4 and R5) at the low values were

the glucuronide conjugates of 13- and 14-hydroxy-LSD. The two metabolites at

2, 0.62 and 0.75 gave a slowly developing yellow colour with Van Uri: reagent Table ,52.10. Colour Reactions of 5-4. 6- and 7-Hydroxyindole and 12-. 13- and 1441ydroxy-LSD on T.L.C. Plate

See Chapter 2 for details of spray reagents. The initial colour produced by the particular reagent is reported together with any subsequent major colour changes. Most colours appeared within 30 min. R4 aglyrone: R3 aglycone: 541ydroxy- 6-Hydroxy- 7-Hydroxy- 12 -Hydrox7- 13-HydroxY- 14-Hydroxy- Spray Reagent indole indole indole LSD LSD LSD LSD

Ehrlich reagent Blue-purple Grey-blue Dark-blue Mauve-,-light- Immediate light- Mauvedark- Mauve-o-purple blue-,-dark-blue blue-3-light- blue-).-dark green-blue grey-blue

Van Urk reagent Purple Blue-green Grey-blue Mauve-,-light- Immediate light- Mauve--dark- Mauve-o-dark-blue blue-,-dark-blue blue --light- blue--dark- green-blue grey-blue

4-Dimethylamino- Purple Dark-blue Blue-green Purple Blue-green Blue Blue-purple cinnamaldehyde

Xanthydrol Purple Purple Purple Blue Grey Green Light-purple

Gibb's reagent Blue Brown Blue-purple Grey-green Brown Light-blue Yellow

Diazotised sulphanilic Brown Immediate Brown-orange Light-brown Immediate Yellow Yellow acid in HC1 red red

Diazotised sulphanilic Brown Light-brown Brown Orange Red-orange Orange-yellow Light-orange acid in Na2CO3

1-Nitros0-2- Blue (red Blue Blue Blue (red Grey-blue Green-blue naphthol after 24 h) after 24 h) 130

like that with R7. The two former metabolites appeared to be interconvertible since repeated purification on preparative t.l.c. plates in system C or G of either compound still produced a mixture of the two. Only one spot was obtained on paper chromatograms in the solvent systems A and C and the RF corresponded to metabolite R7. It was concluded that R7 was present in the bile and urine in isomeric forms. Paper chromatography of bile or urine in system B resolved R7 into two peaks (see Fig. 3.5). The mass spectrum of R7 gave a parent ion at We 339 indicating an additional oxygen in the LSD molecule. Since the metabolite gave a yellow colour with Van Urk reagent, it indicated that the 2-position in the LSD molecule was no longer free. The conclusion was that the compound was 2-oxo-LSD whose mass spectrum was very similar to that of a sample of synthetic 2-oxo-LSD (Fig. 3.13). However, neither of the two isomers of metabolite R7 had the same chromatographic propertieS as that of synthetic 2-oxo-LSD (see Tables 2.1 and 3.5). Metabolites R1 (detected only on paper chromatograms developed in system WI R2 and R3 were excreted in very small amounts making visualisation under UV light and with Van Urk reagent very difficult. These compounds were very polar and were not adsorbed onto XAD-2 columns, being easily eluted with water. Thus, t.l.c. in system G of methanolic eluate from the column contained all the metabolites except the three very polar compounds. Due to the low biliary excretion, it was not possible to isolate these compounds in sufficient quantities to enable their characterisation. However, it has been noted that excretion of R3 in the bile of cannulated rats increased with time while that of R5 (glucuronide of 14-hydroxy-L3D) decreased proportionately. Thus, and 48.0,, of the total 14C in the first hour bile sample was aceountedby 1'3 and R5 respectively. Excretion of R3 increased to 17.1, of the total 14C in the bile sample collected in the fifth hour and that of R5 decreased to 21.5;.... Excretion of R6 (13-hydroxy-LSD) also increased with time, being 5.0;:, in the first hour and 12.0Vi% in the fifth hour, but that of R4 (glucuronide of 13- hydroxy-LSD) and the other metabolites remained constant. The significance 131

of these figures would have been understood, more fully if the nature of metabolite a3 had been known. when the bile which had been stored at -20°C was rechromatographed on paper in systems A, .3 and C 6 months later, the subsequent radiochromatogram scans showed a large increase in the peaks corresponding to metabolites R1 and R2 with a concomitant decrease in the _ea': at RF of R7. 1;io significant changes were noted in the peaha corresponding to the Glucuronides a4 and R5. It would appear, therefore, that ,i and Rel: are decomposition products. 'ilhether they arise directly from the decomposition of

1:7 was not investigated but it would seem to be the case. 1-erfusate and liver from perfusion experiments The pooled perfusate and liver homogenate contained metabolites 2-t, R9,

.L10 and 1111 as well as the identified metabolites R4, a5 and a7 found in the 'wile. The pool was not examined for R1, R2 and R3 but they were probably

T,resent since the compounds were detected in the bile from the isolated sr2used liver. iletabolites R7, R8, R9, RID and R11 were extracted with chloroform from the mixture made basic with amuonia and separated on preparative t.l.c. plates in system E and further purified on plates in system Compound R11 was identified as unchanged LSD by co-chromatography with a known sample of L3.3 in all the systems and by its mass spectrum ($1g. 3.11). similarly, R3 and R9 were identified as lysergic acid monoethylanide (lAd) and nor-LCD (6-demethyl-LCD) by comparison of chromatographic characteristics

(Tables 2.1 and 3.5) and mass spectra (Figs. 3.14 and 3.15) with those of authentic samples. netabolite 1110, which appeared to be less polar than L3D, was thought to be "aromatised" 2-oxo-L0D since the values of the metabolite and a sample of "aromatisee 2-oxo-L3D (see Chapter Two under "synthesis of

2-oxo-L0L;) were identical when co-chromatographed on t.l.c. plates in system

, or H. Zurthermore, the metabolite and naromatised" 2-oxo-LCD, unlike most other LCD derivatives, could be extracted into heptane : isoamyl alcohol

(98:2, v/v) from a basic aqueous solution saturated with "naCl. This method was initially reported by Axelrod et al. (1957) to be highly specific for the 100 323 221 (a) LSD 181 207

196

0 it ill 111111

100 4->,, 237 339 197 (b) Aglycone of R4 c a) 223 C 212 a)

a) 1111lilit, .11111 0

100 1 339 237 (c) 12-hydroxy-LSD

197 223

0 411L___., atilt di , I 150 200 250 300 350

Fie:. 3.11. Mass spectra of (a) LSD, (b) l3-hydro.xy-LSD (aglycone of R4 from bile of rats) and (c) 12-Hydroxy-LSD 100 251 (a) Methylated aglycone of R4 353 211

226 2372

222

Ill! ICU )11114 _IA VIII,

(b) Methylated aglycone of R5 353 237 251 211 338 222 , 226 1111[11 1111111 )1111111 i 11111 1 1111.1. , 4,

100 (C) 12-methoxy-LSD 353

211 237 251 338

222 226 0 150 200 250 300 350 m/e

Fig. 3.12. Mass spectra of (a) Methylated aglycone of R4 (13-methox-y-LSD),(b) Methylated aglycone of R5 (14-niethoxy-LSD) and ( c) 12-Methoxy-LSD 239 100 - 237

(a) Metabolite R7 from bile 3.3 9 of rats

221

196 209 167 181

0 Jim 1I11111, .0111111 .11111I1i .11 )1 c.) 1 237 -4 100- 239 C.)

c.) 339 (h) Synthesised 2-oxo-LSD

209 167 196 181 221

0 111111 dill 1111 Mill Iej I I 1

300 350 150 200 250 m/e

Fig. 3.13. Mass spectra of (a) metabolite R7 (2-oxo-LSD) from bile of rats and (b) synthesised 2-oxo-LSD 295 100 —

(a) Synthesised LAE 221 181 196 207

154 167

295

221 (b) Metabolite RS from 181 isolated perfused 207 rat livers 196

154 167

0 1111 11 Ill" I iii 'ft ,11 100 150 200 250 300 m/e

Fig. 3.14 Mass spectra of (a) synthesised LAE (lysergic acid monoethylamide) and (b) metabolite R8 (LAE) from isolated perfused rat livers

309 100 — 207 (a) Synthesised nor-LSD

182

167 192 280 154

221 237

ity I 0 14 '1 Ia I

tens 207 in

100 — e iv t (b) Metabolite 110 from isolated

la :309 perfused rat Livers Re

182

154 167 1 J2 221 237 280 0 100 150 200 250 300 m/e

lig. 3.15. Mass spectra of (a) synthesised nor-LSD (6-demethyl -LSD) and (b) metabolite 119 (nor-LSD) from isolated

• perfused rat livers 137

extraction of LSD from biological fluids. Attempts to nurify a sufficient quantity of R10 on nreparative t.l.c. plates in system A, 2 or 11 after

heptane isoamyl alcohol extraction of pooled perfusate and liver homogenate, were unsuccessful. Thus, the mass spectrum of the metabolite was unobtainable.

Faeces Paper chromatography of the faecal homogenate in systems A and 0

indicated the presence of at least 6 radioactive compounds (see Table 3.7). Throe of these were the biliary metabolites R4, R5 (previously identified as glucuronide conjugates of 13- and 14-hydroxy-LSD respectively) and. 16

(suggested as being 13-hydroxy-LSD). The conjugates R4 and R5 in the faeces,

detected only in system A (see Fig. 3.4), were in much smaller amounts than

in the bile, indicating deconjugation in the gut (probably bacterial) giving

7:7ise to aglycone 116 and R14 respectively. Metabolite R14„ however, was not the aglycone of 115 since the latter compound was very unstable. It was concluded that R14 was a principle breakdown product of the unstable aglycone.

This was confirmed by paper chromatography in systems A and C of a known. decomposed sample of the aglycone of R5. The radiochromatogram scans showed major peak corresponding to R14 in addition to two minor peaks at - and 0.72 detected on the chromatogram developed in system A only. The break-

down products were non-fluorescent under UV light but, like the a7lycone, gave dark-grey-blue colours with Van Urk reagent. Metabolite R14 was the malor radioactive compound in the faeces. R12 was probably the radioactivity bound to the faecal material and R13, detected only in system C, was not identified.

The presence of any R7 (2-oxo-LSD) was not investigated since this would have involved t.l.c. which was not possible with the faecal homogenate. However,

sone 117 was :probably present since it was excreted in the- bile of cannulated

Identification of Iso-LSD Metabolites

The bile from rats dosed with [1401-iso-LSD was incubated with

-glucuronidase and then ehromatographed on a t.l.c. plate in system G. 138

The radiochromatogram scan showed that the peaks corresponding to metabolites

and R19 were greatly reduced and a single major peak at 0.52 arreared. It was concluded that the major biliary metabolites of iso-D3D were Flucuronide conjugates. The glucuronides gave colours under UV light and with Van Urk reagent which were very similar to the biliary glucuronide metabolites (R4 and of LSD (Tables 3.5 and 3.9). Thus, by analogy with the metabolism of LSD, tP and B19 were identified as glucuronide conjugates of 13- and 14-hydro::y-

!_so-LSD respectively. Similarly, iso-LSD metabolites Rt5, R16, R1? cnd 720 corresponded to the L'ID metabolite R1, R2, 13 and R7 (2-oxo-LSD) resrectiyely. he corresponding iso-LSD metabolite of R6 could not be detected. This "u:;:rested that R20 was 2-oxo-lso-L$D and R15, R16 and 1217 were unnowns. an were very similar in that both existed in isomeric forms 1Mel- were eesolyed on parer chromatogram developed in oysters (compare Figs. 3.5 nna

1.10).

3ile-duct-cannulated guinea pigs given intraperitoneel injections of

[141]-T3D (0.5 mg/hg; 13.6 pCi/ng) excreted of the dose in the bile An

h (Table 3.11). Over a half of this excretion occurred ir the first boys. [14.1]-I ID (1 1.1.1]/hc) was also administered intraperitoneally to intact animals. 7n 96 h, of 14C was eliminated, 18, asa 14002 In the exrired air, 2r1- in

--ine and 1 0 4n faeces (Table 3.12). :;:z:oroicnis ididentification of [1.4c] -TSD retallelltes The _7,- values and the biliary and urinary excretion of the metabolites are shown In ?able 3.13. Radiochronatogram scans and the corresponding histograms following paper chromatography in systems A, B and C (see Figs.

3.16-3.21) revealed at least nine compounds (G1-39). Additional radioactive metabolites, two in the urine (G10 and G11) and one in the bile (.11), were detected on t.l.c. plates developed in systems E and H. netabolites ?-_resent in the bile were also excreted in the urine. G5 and G8 were the major 139

compounds in the bile, Incubation of bile with /3-glucuronidase followed by t.l.c. in system G and radiochromatogram scanning showed that the intensity of peaks corresponding to the metabolites G4 (RF 0.41) and G5 (RF 0.49) decreased whilst a major peak appeared at RF 0.78. It was concluded that the two metabolites were glucuronide conjugates. Concentration of the conjugates following Chromatography of bile on an XAD-2 column and then t.l.c. in system

G of the methanolic eluate, allowed visualisation of the two metabolites. G4 appeared light-blue and G5 dark-blue under UV light. With Van Urk reagent, G4 gave an immediate light-blue colour and G5 gave an initial mauve colour changing to dark-blue in 5 min. The RF values of G4 and G5 on paper in systems A, B and C and on t.l.c. plates in system G were identical with those of the rat metabolites R4 and R5 respectively. It was concluded that G4 and G5 were glucuronide conjugates of 13- and 14-hydroxy-LSD respectively. Metabolite G8 had a weak-blue fluorescence and gave a slowly developing yellow colour with Van Urk reagent. This compound was identified as the

2-oxo-LSD metabolite (R7) formed by the rat, since the chromatographic characteristics of R7 and G8 on paper and t.l.c. plate in all the solvent systems were identical. The radiochromatogram scan and histogram following t.l.c. of bile or urine in systems E and H revealed several peaks (Table 3.13). The two minor compounds, G10 and G11, not detectable on the paper chromatograms, were tentatively identified as LSD and lysergic acid monoethylamide (LAE) respectively by comparison of RF values with those of authentic compounds. The LAE excreted was much lower than predicted from the extent of de-ethylation resulting in the high content of 14002 in the expired air (Table 3.12). The low excretion of the other metabolites prevented their characterisation and visualisation under UV light and with Van Urk reagent. However, compounds G1-G3 correspond to Ri-R3 excreted in the rat bile by comparison of RF values on the paper Chromatograms.

Attempts to chromatograph faecal homogenates on paper were unsuccessful due to the very low counts only that could be chromatographed. 140

Table 1.11. Cumulative Biliar, Excretion of Radioactivity by Bile-Duct- Cannulated Guinea Pigs receiving [14C1-LSD

Three guinea pigs were dosed intraperitoneally with [1-40]-1,SD (0.5 mg/kg; 2.59 t 0.15 pCi/animal). Average values are given with ranges in parentheses. Values are expressed as % of administered 14C.

Time after dosing (h) X, of dose in bile

1 30.3 (15.7-38.6)

2 41.5 (27.7-49.2)

3 46.4 (33.4-53.9)

48.7 (36.0-56.3)

5 50.6 (38.1-58.0)

52.1 (39.6-59.1) Table 3.12. Cumulat ve Excretion of Radioactivity by Intact Guinea Pigs Receiving [14C]-1,SD,

The [14C] LSD (1.0 mg/kg; 4.12 t 0.07 pCi/arimal) was administered intraperitoneally to 3 guinea pigs. Average values are given with ranges in parentheses. Values are expressed as of administered dose.

Time after dosing (h) Urine Faeces 14002 Cage wash Total 140

8 12.6 (11.6-13.4) ••• 12.6 (11.6-13.14

24 23.5 (20.9..26.5) 9.1 (2.5-17.4) 16.5 (15.2-17.7) 4WD 49.1 (45.6-55.8)

48 26.3 (22.8-29.5) 34.0 (29.7-38.2) 17.5 (16.2-18.7) 411D 77.8 (71.2-81.4)

72 27.6 (23.6-30.1) 39.5 (37.3-41.2) 17.7 (16.4-19.0) 84.8 (79.9-89.1)

96 27.8 (23.8-30.2) 40.3 (38.6-41.6) 17.8 (16.4-19.2) 0.3 (0.2-0.6) 86.2 (82.2-89.8) Table 3.13. R^ Values and Billary and Urinary Excretion of Metabolites of [14C}-LSD in the Guinea Pig

See Tables 3.11 and 3.12 for dose of LSD. Average values are given for the excretion of metabolites, with ranges in parentheses. See text for fluorescence under UV light (254 nm) and colour reaction with Van Urk reagent. U Unknown,

Parer Thin-Layer Chromatography Chromatography of dose in g of dose in Solvent... A. B C E II bile in 4, h . urine in 24 h Probable. Identity Metabolite G1 0.00 0.00 0.00 0.00 0.00 0.5 (0.2-0.8) 0.4 (0.2-0.6) U G2 0.12 0.12 0.05 0.00 0.00 1.6 (1.2-2.0) 0.4 (0.2-0.6) U G3 0.18 0.22 0.20 0.00 0.00 2.4 (2.3-2.6) 0.5 (0.3-0.6) U G4 0.27 0.33 0.37 0.00 0.00 2.7 (1.4-4.3) 1.4 (1.3-1.5) Glucuronide of 13-hydroxy4M G5 0.33 0.40 0.47 0.00 0.00 14.8 (9.0-18.8) 3.5 (1.4-5.0) Glucuronide of 14-bydroxy49D G6 0.55 0.53 0.57 0.00 0.00 2.7 (1.9-3.5) 3.9 (2.4-4.8) U G7 0.76 0.73 0.75 0.16 0.04 1.3 (0.7-1.7) 2.8 (2.1-3.2) 0.73, G8 0.81 0.87 0.84 0.30 0.00 14.1 (11.8-16.6) 2.5 (2.2-2.9) 2-Oxo-LSD G9 0.76 0.73 0.84 0.30 0.14 4.6 (2.6-5.9) 3.7 (3.1-4.6) U G10 - - - 0.53 0.47 0.4 (0.0-0.9) 0.6 (0.4-0.7) LSD Gil - - - 0.37 0.29 - 0.6 (0.2-0.9 Lysergic acid monoethylamide (LAE) Total of above metabolites 45.1 (32.7-52.1) 20.3 (17,3.23.2) 143

0 10 20 30 CM

Fig. 3.16. Radiochromatogram scan and histogram of bile from guinea pig dosed 14 intraperitoneally with [ C1-LSD (0. 5 mg/kg; 13.6 liCi/mg) and chromatographed on Whatman No. 1 paper in solvent systern..A. See text for the possible identity of metabolites G1-G9.

0 = Origin S. F. = Solvent front 144

G5

0 10 20 30 40

Fig. 3.17. Radiochromatogram scan and histogram of bile from guinea pig dosed 14 intraperitoneally with [ C1-LSD (0. 5 mg/kg; 13.6 peCi/mg) and chromatographed on Whatman No.1 paper in solvent system B. See text for the possible identity of metabolites G1-G9.

0 = Origin S. F. = Solvent front 145

10

0 ,-i

E

5

0 10 20 30 40 CM

Fig. 3.18. Radiochromatogram scan and histogram of bile from guinea pig dosed 14 intraperitoneally with [ C]-LSD (0. 5 mg/kg; 13.6 i/Ci/mg) and chromatographed on Whatman 31VITvi paper in solvent system C. See text for possible identity of metabolites G1-G9.

0 = Origin S. F. = Solvent front 146

0 10 20 30 cm

Fig. 3.19. Radiochromatogram scan and histogram of 24 h urine from guinea pig dosed 14 intraperitoneally with [ M-LSD (1 mg/kg; 13.6 ptCi/mg) and chromatographed on Whatman No. 1 paper in solvent system A. See text for possible identity of metabolites G1-G9.

0 = Origin S. F. = Solvent front 147

0 10 20 30 CM

Fig. 3.20. Radiochromatogram scan and histogram of 24 h urine from guinea pig 14 dosed intraperitoneally with [ C]-LSD(1 mg/kg; 13.6 ',Xi/mg) and chromatographed on Whatman No.1 paper in solvent system B. See text for possible identity of metabolites G1-G9.

0 = Origin S. F. = Solvent front 148

G8, G9

0 10 20 30 cm

Fig. 3. 21. Radiochromatogram scan Sand histogram of 24 h urine from guinea pig 14 closed intraperitoneally with [ C]-LSD(1 mg/kg; 13.6 pCi/mg) and chromatographed on Whatman 3MM paper in solvent system C. See text for possible identity of metabolites G1-G9.

0 = Origin S. F. Solvent front 149

11=211L1 Excretion of Radioactivity

[1401-LSD (10 )1eil 13.6 3iCi/mg) was administered to a male (dose 0.15 mg/kg) and a female (0.12 mg/kg) monkey. Results of the excretion of 140 are summarised in Table 3.14. There was no significant difference in the elimination of radioactivity between the male and the female monkey. In 96 h, 38-39 of the dose was excreted in the urine, with 22-24% in the faeces. Total recovery of 140 was 63% for each of the two monkeys. Two repeat experiments in the same animals consistently gave recoveries of 56-59% in 48 h, which compared favourably with recovery of 55-58t in the first experiment in the same time. In only one case out of the six was the total excretion lower than 55%. It was concluded that de-ethylation of the diethylamid.e side chain of LSD was probably the cause of the low recoveries.

Chromatographic Distribution and Identification of [1401-LSD Metabolites Chromatography of the monkey urine revealed a number of metabolites as

Shown in Figs. 3.22-3.26. The RF and quantitative excretion values of the metabolites are listed in Table 3.15. Altogether, ten radioactive peaks (M1-1410) were detected of which three (M8-M10) were detectable on t.l.c. plates only (see Figs. 3.25 and 3.26). Metabolite 147 was the major urinary metabolite. 143 and M4 were identified as the glucuronide conjugates of 13- and 14-hydroxy- LSD respectively by comparison of their RF values with those of R4 and R5 formed by the rat (see Tables 3.5 and 3.15). M3 and M4 appeared as a composite peak at an unusually high RF value (0.63) when urine was chromatographed on paper in system C (see Fig. 3.24). It was also noted that R4 and R5 in the rat urine also appeared as a single peak of RF 0.63 in this system. The cause of this phenomenon was not investigated. When the composite peak was eluted with methanol from rat urine chromatograms developed in system C and rechromato- graphed on paper in the same system, resolution into the two glucuronides occurred. The metabolites M3 and M4 were present in very small amounts and could not be detected visually under UV light or with Van Urk reagent. This 150

was also true for the other monkey metabolites except for M7. Compound M7 was isolated from the urine as described in Chapter Two under the headings "Concentration of Metabolites" and "Isolation of Metabolites". The metabolite quenched the background fluorescence of the t.l.c. plate and gave a slowly-developing yellow colour with Van Urk reagent. I17, however, was not the metabolite 2-oxo-LSD (R7) nor the synthetic 2-oxo-LSD since their chromatographic characteristics were very different (see Tables 2.1, 3.5 and 3.15). It was not a phenol since a very pale brown colour, like that with LSD, was obtained with diasotised konitroaniline* However, a pink solution was obtained when the metabolite (10 bag) was treated with nitrous acid followed, by 11-(1-naphthyl) ethylenediamine dihydrochloride solution exactly as described by Bretton and Harebell (1939). The pink colour was not obtained when LSD, LA4 or the metabolite 2-oxo-LSD were similarly treated. This indicated the presence of a primary group in M7. The ultra-violet spectrum of the pink solution, obtained on a Pye-Unicam SP 1800 UV Spectrometer, showed a broad plateau in the wavelength range 490..530 um. Metabolite 1°,7, repeatedly purified on t.l.c. plates in system A, (RF 0.29) and E, was subjected to mass spectrometry but the mass spectrum was unobtainable* The metabolite was unstable if left at room temperature for several weeks and two decomposition products have been noted at RF 0.00 and 0.06 on thin-layer chromatograms developed in system E. The product at RF 0.06, like M7, was non-fluorescent under UV light and gave a slowly-developing yellow colour with Van Urk reagent. T.l.c. of urine in system E or H produced a number of peeks as can be seen in Figs. 3.25 and 3.26. That at the origin was a composite peak and when eluted with methanol and chromatographed on paper in systems A, B and C it resolved into compounds 111-M5. Comparison of RF values (see Tables 3.13 and 3.15) indicated that M6 and M7 corresponded to the unknown guinea pig metabolites G7 and G9 respectively. The three compounds M8-M10 were not detected on paper chromatograes. Co-chromatography of monkey urine with LkE on tale°. plates in systems E or H gave a single spot at the RF of M8. Thus, M8 was tentatively Table 3.14. Excretion of Radioactivity by the Rhesus Monkey after Administration of [1401-LSD

Three separate doses of [140] -LSD (0.12 and 0.15 mgikgi 10 uCi/animal) were administered intramuscularly to two monkeys. Individual cumulative values, as 2 of 14C dose, are given.

Time after dosing (h) Urine Faeces Cage wash Total 14C

First dose 24 35.60 35.5 11.6. 3.1 0.7, 2.0 47.9, 40.6

48 37.50 38.0 19.5,45.3 0.8, 2.1 57.8, 55.4

72 37.7, 38.7 22.8, 20.0 0.8, 2.2 61.3, 60.9

96 37.9, 39.1 24.1, 21.5 0.8, 2.2 62.8, 62.8

Second dose 24 42.0, 40.6 .. .., -

48 43.7, 43.4 14.0, 4.4 1.0, 0.3 58.7, 48.1

Third dose 24 35.51 40.5 ...

48 36.8, 43.2 20.3, 12.8 0.0, 0.3 57.1, 56.3 Table 3.15. AF Values and Excretion of Urinary Metabolites of [140]-LSD in the Rhesus Monkey

See Table 3.14 for dose of LSD. Individual excretion values from two monkeys are quoted. Values are expressed as % of administered i4C. See text for UV fluorescence and colour reaction with Van Uric reagent of M7. Other metabolites were not detected visually. U = Unknown.

Thin-Layer Chromatography, Chromatography of dose in Solvent.... A B C E H urine in 24 h Possible Identity Metabolite - - 0.00 0.00 0.00 1.3, 0.8 M2 - 0.17 0.08 0.00 0.00 1.7, 2.4 M3 0.27 0.33 0.37 0.00 0.00 1.6, 2.7 Glucuronide of 13-hydroxy-LSD M4 0.33 0.40 0.47 0.00 0.00 2.3, 3.4 Glucuronide of 14-hydroxy-LSD M5 0.63 0.60 0.81 0.00 0.00 5.2, 6.1 M6 0.77 0.72 0.75 0.16 0.07 5.74 5.o M7 0.77 0.72 0.85 0.31 0.16 11.4, 11.2 118 - - - 0.39 0.33 1.1, 0.6 Lysergic acid monoethylamide (LA) M9 - - - 0.53 0.48 1.6, 1.0 LSD M10 - - - 0.68 0.55 1.2, 0.6 "Aromatised" 2-oxo-LSD Total of above metabolites 33.1, 33.8 1),2 153

M6, M7

Fig. 3.22. Radiochromatogram scan and histogram of 24 h urine from monkey 14 dosed intramuscularly with [ C1-LSD (0. 12 and 0.15 mg/kg; 13. 6 pCi/mg) and chromatographed on Whatman Nol paper in solvent system A. See Table 3.15 for possible identity of metabolites M3-M7.

0 = Origin S. F. = Solvent front 154

0 10 20 30 cm Fig. 3. 23. Radiochromatogram scan and histogram of 24 h urine from Rhesus 14 monkey dosed intramuscularly with [ C]-LSD (0.12 and 0.15 mg/kg; 13.6 pCi/mg) and chromatographed on 7.1hatman No.1 paper in solvent system B. See Table 3.15 for possible identity of metabolites M2-M7.

0 = Origin S. F. = Solvent front 155

M7

15

0 10 20 30 cm

Fig. 3.24. Radiochromatogram scan and histogram of 24 h urine from monkey

dosed intramuscularly with [14C]-LSD (0.12 and 0.15 mg/kg; 13.6 pCi/mg) and chromatographed on Whatman 3MM paper in solvent system C. See Table 3.15 for possible identity of metabolites M1-M7.

0 =Origin S. F. = Solvent front 156

M1-M5 M6 M7

0 5 10 15 cm

Fig. 3. 25. Radiochromatogram scan and histogram of 24 h urine from Rhesus 14 monkey dosed intramuscularly with [ C1-LSD (0.12 and 0..15 mg/kg; 13.6 tCi/mg) and chromatographed on aluminium-backed t.1. c. plate in solvent system E. See Table 3.15 for possible identity of metabolites Ml-M10.

0 = Origin S. F. = Solvent front 157

Fig. 3. 26. Radiochromatogram scan and histogram of 24 h urine from Rhesus 14 monkey dosed intramuscularly with [ C1-LSD (0.12 and 0.15 mg/kg; 13.6 iCi/mg) and chromatographed on aluminium-backed t.l. c. plate in solvent system H. See Table 3.15 for possible identity of metabolites Ml-M10.

0 = Origin S. F. = Solvent front 158

identified as LAE, the product of de-ethylation of the diethylamide side chain. When the monkey urine (5 ml) was either adjusted to pH 10 with aq. NH, (sp. gr. 0.88) and extracted with ether (25 ml) or made basic with 1M.NaCH (0.5 ml), saturated with NaC1 and then extracted with heptane isoamyl alcohol (9812„ vivs 25 ml), compounds 119 and M10 only were obtained as shown by t.l.c. in systems A (RF 0.42 and 0.72 respectively), E and H. M9 was idemtified as unchanged LSD since authentic LSD and the metabolite gave a single spot following co-chromatography on ta.c. plates in the three systems. Compound M9 could be extracted into heptane s isoamyl alcohol, a method claimed to be very specific for LSD by Axelrod eta. (1957), and this provided. partial confirmation that 149 was LSD* The RF values of M10 on tel.c. plates developed, in system A Or 042), E and H (see Tables 2.1 and 3.15) were very similar to those of naromatised" 2-oxo-LSD and both were unusual in that they could be extracted into the heptane s isoamyl alcohol mixture. Thus, M10, like RIO, was tentatively suggested as the aromatic form of 2-oxo-LSD. However, 2-oxo- LSD (metabolite R7 or the synthetic compound.) could not be detected with the available techniques. Metabolites Iii and 142 corresponded to the unknown rat biliary metabolites R1 and R2 respectively by comparison of Rg values obtained on the paper chromatograms. The UV fluorescence, colour with Van Uric reagent and the nature of the other monkey metabolites could not be studied since they were present in low amounts in the urine. The faecal homogenate could not be chromatographel since it contained insufficient radioactivity.

Disioussion

The metabolism of LSD has previously been reported but in very little detail and the nature of the metabolites were poorly defined. The metabolism of the druglixtut in mice and rat only was reported before because of the relatively high doses that could be used in these missals. However, the 114c] LSD synthesised in the present study was of high specific activity and allowed 159

the study to be extended from the rat to the guinea pig and the monkey. Species differences were apparent in the metabolism of 11401-LSD and the elimination of radioactivity. When rats received LSD, excretion of radioactivity in the bile or the faeces predominated, some appeared in the urine but very little de-ethylation was observed, as indicated by the appearance of 14002 in the expired air. Using LSD labelled similarly in the diethylamide side chain, Boyd (1959) reported the excretion in rat which was very similar to those obtained in the present study. The excretion in the guinea pig faeces in 96 h was almost a half of that found in the rat faeces. However, the radioactive content of the guinea pig urine and expired air was respectively two-fold and five-fold greater than in the rat. Elimination of 140 in the Rhesus monkey differed in that two-thirds of the dose recovered was found in the urine with one-third in the faeces. Comparing monkey with rat, the 14C in the monkey urine was over twice that found in the rat urine. The radioactive content of the rat faeces was over three times greater than that of the monkey faeces. Facilities were not available to examine the expired air of the monkey for 14CO2 but since the total recoveries were consistently low, it was concluded that do-ethylation was probably greater than in the rat or the guinea pig. A species difference in the urinary and faecal excretion of the radioactivity has also been observed with metergoline (Fig. 1.24), which is closely related to LSD (Arcamore et al., 1971). The three species showed a rapid excretion rate and 90..97% of the total

14C excreted in 96 h appeared in the first 48 h. The radioactivity appearing in the bile of rats was 68R; in 5 h whereas guinea pig bile collected for 6 h contained only The lower excretion in the bile of guinea pigs was probably due to the higher excretion in the urine and expired air observed in the intact animal. It should, however, be stressed that the differences observed in the three species may have been due to the differences in the dose of [14C]-LSD and the route of administration. For example, the rat received

1.0 or 1.33 mg/kg of LSD intraperitoneaily (intact animals) or intravenously 160

(bile-duct-cannUlated animals), the guinea pig was given intraperitoneally either 0.5 (cannUlated animals) or 1.0 mg/kg (intact animals) and either 0.12 or 0.15 mg/kg was administered intramuscularly to the monkeys. The similarity in the excretion between the bile from cannulated rats and faeces from the intact animal indicated that no enterehepatic circulation occurred, as has been suggested by Boyd (1959). However* preliminary studies in this department by R.J. Parker (unpublished data) have shown that entero- hepatic circulation does occur in the rat. Examination of the guinea pig data would lead to the conclusion that enterohepatic circulation also occurs in this species since the lit in the faeces was lower than in the bile. When an isolated rat liver was perfused with 2 mg LSD/g liver, the bile flow rate (0.5 ml/h) in the first three hours was almost equal to that encountered in bile-duct-cannulated rats (about 0.8 ml/b) and the excretion of radioactivity in the bile was approximately linear with time. When a lower amount of LSD (1 mg/g liver) was used, the excretion, however, was not linear, being maximal in the second. hour. Nevertheless, in terms of absolute quantities, the radioactivity equivalent to 404 mg of LSD was eliminated in 0.9 ml of bile in 4.5 h When the low dose was used and 5.8 mg in 1.8 ml bile in the same time When the dose was increased two-fold. These results suggest that there is no linear relationship between the amount of 140 excreted in the bile and the dose. Also, the total volume of bile collected appears not to be of major importance in the extent of biliary excretion. This conclusion is also reached when the data with only the low dose is considered.. With this dose, the total bile flow in 4.5 h ranged between 0.75 and 1.10 ml Whereas the % of 140 in the bile ranged between 41 and 461g only. An important conclusion is that there is a maximal rate of clearance of 140 in the bile of perfused livers since the amount (in terms of mg equivalent of LSD) was only slightly increased with a two-fold increase in the doses It is estimated that the maximal rate of clearance is equivalent to about 2 mg LSD/h. Boyd (1959) from his studies has suggested that in vivo the maximal rate at which the rat liver can clear LSD is about 0.9 mg/h. 161

The importance of the isolated perfused liver preparations in the metabolic study of very toxic compounds has been demonstrated and all the metabolites produced in vivo were also present in the bile from the perfusion experiments. However, there were quantitative differences in the relative amounts of

metabolites in the bile from the two sources and this was probably due to the

high amounts of LSD (equivalent to a dose of 50 or 100 mg/kg in vivo) that were utilised.

Chromatography of the rat bile showed six metabolites instead of the four as reported by Stoll et ate. (1955) and Boyd. (1959). The presence of six compounds in the bile has been confirmed using [2(n)-311]-LSD by D.J. Back and J.K.G. Singh of the Department of and Therapeutics, University of Liverpool (personal communication), Boyd (1959) also found "five labelled

compounds with high RI, values in the same region as LSD" when rat urine was

chromatographed on paper in butan-1-ol acetic acid : water (611:5 by vol.). In the present study, however, only three (13-hydroxy-LSD„ LAE and 2-oxo-LSD) could be detected in that region on paper in system A. The main metabolites in the rat were glucuronides of 13- and 14-hydroxy-

LSD which accounted for about 70-75% of the total radioactivity in the 5 h bile

or the 24 h urine samples. In the bile the 13- and 14-hydroxy-LSD glucuronide were in the ratio 2:3 but the ratio was 3:2 in the urine. That the two main metabolites were glucuronide conjugates confirmed the finding of Boyd (1959)

and Slaytor and Wright (1962). The latter workers, however, suggested that

the metabolites were glucuronides of i2-hydroxy-ISD and 12-hydroxy-iso-LSD since interconversion occurred between the two compounds and hydroxylation was reported in the 12-position by analogy with the metabolism of ergometrine,

The present study refutes the finding of these workers. The glucuronides of 13- or 14-hydroxy-LSD could not be interconverted using conditions which were identical to those reported by Slaytor and Wright (1962). In addition, the metabolism of LSD and iso-LSD was shown to occur along common pathways but the

13- and 14-hydroxy-iso-LSD glucuronides formed had different chromatographic 162

properties from the hydroxy.LSD glucuronides. No explanation can be put forward at present to explain the interconversion encountered by the previous workers. The 13-hydroxy-LSD gave a light-blue fluorescence under UV light (254 rim), an immediate light-blue colour with Van Urk (4•dimtthylaminobenzaldehyde) reagent and an immediate red colour with diazotised sulphanilic acid in HU. Confirmation of the hydroxylation in the 13-position of LSD comes from a number of studies:- 1) Szara (1963a) has reported the formation of 13-hydroxy-LSD in vitro by rat liver microsomes. 2) The metabolism of most of the exogenous indolic compounds occurs primarily via hydroxylation in the 6-position which corresponds to the 13-position of LSD (see Chapter One under "The Metabolism of Compounds Structurally Related to LSD"). 3) Szara (1963a) has shown that an immediate blue colour reaction occurred between 4-dimethylaminobenzaldehyde (Ehrlich reagent) and 13-hydroxy-LSD. An immediate light-blue colour has also been reported for 6-hydroxyindoles with this reagent but other colours are obtained with 5- or 7-hydroxyindoles (Jepson et al., 1962). 4) The indoles hydroxylated in the 6-position give an immediate spedific red colour with diazotised sulphanilic acid in HC1 (Jepson et al., 1962; Heacock and Mahon, 1965) as does the 13-hydroxy-LSD reported by Szara (1963a). The major metabolite in the rat was identified as the glucuronide of 14- hydroxy-LSD since its aglycone was neither 12- nor 13-hydroxy-LSD. The 14. hydroxy-LSD was found to be unstable, a fact which was also reported by Boyd (1959) but not by Slaytor and Wright (1962). It was discovered that the 14- hydroxy-LSD completely decomposed overnight even at -20°C. The corresponding 7-hydroxyindoleacetic acid has also been reported to be unstable in solution (Marchelli 2 al., 1969). Thus, overnight hydrolysis of the 14-hydroxy-LSD 163

glucuronide to produce the aglycone was not possible. Commercially, the 5-hydroxyindole is readily available but due to the instability of the 6- and

7-hydroxyindole, they are pirchased as the stable benzyloxyindole. Similarly, the 14-hydroxy-►LSD was stabilised by immediate treatment with excess of diazo- to form the stable 14-methoxy-LSD. The 14hydroxy-LSD glucuronide, its aglycone and 14-methoxyLSD gave the same dark-blue fluorescence under UV light and formed a dark-grey-blue oelour with Van Urk reagent. This is consistent with the reported grey-blue colour reaction of 7-hydroxyskatole with Ehrlich reagent (Heacock and Mahon, 1965), the composition of which was very similar to the Van Urk reagent. There were significant differences in the extent of hydroxylation in the

13- and IA-position of LSD between the guinea pig and the rat. The major metabolite in the guinea pig bile was 14.41ydroxy-LSD glucuronide but this was only a half of that found in the bile of °annulated rats. This was possibly due to the overall low excretion of in the bile of guinea pigs in addition to the greater extent of 2-oxidation than that observed in the rat. It should also be remembered that the dose of LSD and the route of administration were different for the two species. Very little of the 13-hydroxy4ZD glucuronide was formed by the guinea pig as compared with the rat. It was concluded that the guinea pigs were poor hydroxylators in the 13-position. This conclusion was also reached by Szara (1963b) who demonstrated that rats excreted about

5O of the aftimstered 2,M-diethyltryptamine (DET) as a 6-hydroxy derivative in the urine whereas guinea pigs formed less than 2% of this metabolite (see Table 1.6). The urinary content of the glucuronides of 13- and 114-hydroxy-LSD was comparatively lower in the guinea pig or rat urine than in the bile. Similarly, the glucuronides were also present in small amounts in the monkey urine. It is highly probable that the monkey excreted relatively higher proportions of the conjugates in the bile than in the urine by analogy with the other two species.

It is interesting to note that the order of excretion of lit in the urine was 164

monkey> guinea Pig> rat whereas the % of glucuronides in the urine was rat> guinea pig, monkey. Thus, the metabolism of LSD by hydroxylation was most important in the rat but appeared to be of lesser importance in the monkey. As mentioned previously, this may have been due to the differences in the dose and the route of administration. Hydroxylation in the 12-position of LSD was not observed in any of the three species and it would appear that the enzymic 5-hydroxylation of trypto- phan in the synthesis of 5-hydroxytryptamine is not related to the liver detoxication mechanism. For eleotrophilic aromatic substitution, the general order of positional reactivity of the neutral indole molecule has been reported as 3> 2 > 6 >4 >5 >7 (Sandberg, 1970, p. 83). By analogy, the order of positional reactivity for hydroxylation in the benzene ring of LSD would be 13 >12> 14 (Fig. 3.27). Hydrimrlation does occur at the site C2H5 N- C2H5 C=0

4 5

I 3 6 , 7 N 2 7 H

LSD Indole

Fig. 302.7 predicted (C-13) but to a greater extent at position-14. It would appear that the introduction of substituents at the 3- and 4-position of indole to give

LSD changes the positional reactivity to make C-14 the most reactive site. It is probable that the enzymically selective hydroxylation at C-13 and C-14 involves formation of an epoxide intermediate (Smith, 1973). The epoxide would then rearrange to give either 13- or 14-hydroxy-LSD (Fig. 3.28). ,C2H5 /C91-1< N C2H5 C2H5 !\C2H5 -`C2H5 C=0 C=0 C=0

/ N-CH N-CH N-CH 3 3 3 rearrange rearrange

HO

OH

13-Hyaroxx7.LSD LSD epoxide 14-Hxdroxiy-LSD

Fig. 3.28

Only very little 13-hydroxy-LSD was found in the rat urine, bile from

cannulated rats or bile from isolated livers perfused with the higher dose but none could be detected in the guinea pig, monkey or when 1 mg LSWg liver was perfused. Any 14hydroxy-LSD would not be detected due to its very unstable nature. Thus, conjugation with glucuronic acid in the rat liver occurs

extensively immediately after hydroxylation and this is in preference to

conjugation with sulphate. The 2-position in indole is very reactive and 2-oxo-LSD has been identified

as a metabolite in the rat and the guinea pig. The metabolite gave a slowly- developing yellow colour with Van Urk reagent. The 4-dimethylaminobensaldehyde

in the Van Urk reagent reacts with a free 2-position of LSD (or other indoles) to give blue products usually (Look, 1967) but when this position is blocked by oxidation, a yellow colour is obtained. The C-2 , C-3 double bond is

obligatory for the blue colour since 2,3-dihydro-LSD gives a yellow colour

reaction. Oxidation in the 2-position of LSD creates a new asymmetric centre at C-3 and thus two isomers of 2-ox .LSD are possible (Fig. 1.16). Since the 166

metabolite Zoxo-LSD and the synthetic 2-oxo-LSD had different chromatographic characteristics, it would, axoar that they were 2.2-oxo-LSD andX.. .2-oxo.LSD respectively or vice versa. The metabolite 2-oxo-LSD itself existed in an isomeric form as did 2-oxo-iso-LSD. The RF values of the interconv-evtible isomers of 2-oxo-I45D on paper in system B and on tJ..c. plate in system C corresponded with those of the isomers of 2-oxo-iso4SD (BF 0.63 and 0.75 on t.l.c. plate) suggesting that 2-oxo-LSD and 2-oxo-iso-LSD were interconvertible and existed as mixtures of each other. It is also possible that isomerisation may occur about the asymmetric carbon at position-5 which would mean that the two forms of metabolite 2-oxo4iSD were different from those of 2-oxo-iso-LSD. This is probably so since the RF values of 0.52 and 0.58, which have been obtained on t.l.c. plate in system G for the 2-oxo-iso-LSD isomers, were different from those of the 2-oso-LSD tamers. It is not possible to give absolute structures of these products of 2-oxidation on the experimental results obtained. The metabolism of [2-31]-LSD was studied in one rat to see whether the 2-oxo-LSD would retain the tritium label. Theoretically, if 2-oxidation occurred, the tritium label would be lost and the peak on the radiochromatogram scan corresrondihg to the metabolite would not be observed. Contrary to expectation, the metabolite retained the tritium label suggesting that either it was not 2-ono-LSD or that the radioactive label was involved in a NIX shift 1967) during which the tritium is moved to another position in the LSD molecule. Since the yellow colour with Van Ork reagent and the mass spectrum of the metabolite were indicative of 2-oxo-LSD, NIM shift was probably the cause of tritium retention. Altertatively, the tritiated LSD was not labelled specifically in the 2-position. The metabolism of indoles by 2-oxidation has been reported ,fin =c2 and u, zuwg by several workers and the 2-oxo metabolites of LSD and indole have been reviewed in Chapter One. In addition, formation of 3-methyloxindole was reported by Heacock and Mahon (1963) using a model oxidation system consisting 167

of ferrous sulphate, ascorbic acid, EDTA and air. The metabolism of LSD by the formation of 2-oxo-LSD emphasises the importance of this route also in the metabolism of exogenous indoles. Its importance has been demonstrated by the liver perfusion experiments which showed that 2-oxidation occurred to a greater extent than 13-hydroxylation. In vlvo, however, the bile-duct-cannulated rats did not form very much of the 2-oxo-LSD. This was probably due to the low dose (1.33 mg/kg) used in the animals and the high dose (1 or 2 mg/g liver) in isolated perfused livers. It is very probable that hydroxylation of the benzene ring of LSD is a preferential detoxication route of the drug and when this route becomes saturated with high doses as in the isolated livers, 2-oxidation assumes greater importance. If it were possible to administer a high dose of LSD into bile-duct-cannulated rats, it is probable that the extent of 2-oxidation would be greater than with a low dose. The 2-oxo-LSD was formed to a greater extent in the guinea pig than in the rat and it was probably the same metabolite of LSD as that formed in vitro using guinea pig microsomes (Axelrod et al., 1957). The monkey differed considerably from the other two species in that 2-oxo-LSD could not be detected in the monkey urine. Since the rat and the guinea pig excreted the major part of the 2-oxo-LSD in the bile with very little in the urine, it is possible that some 2-oxo-LSD could have been present in the monkey faeces. This may be unlikely since its molecular weight (339) is below the molecular weight threshold for excretion into the bile (Smith, 1973). A probable metabolite detected in the pooled perfusate and liver (as a homogenate) from isolated liver perfusion experiments and in the monkey urine was the aromatised form of 2-oxo-LSD. It was not detected in vivo either in the rat or in the guinea pig. This compound was also formed from the synthetic 2-oxo-LSD. A clear methanolic solution of the synthetic compound eventually turned yellow (after several days at room temperature or -20°C) due to the conversion of the compound to the yellow "aromatic" 2-oxo-LSD (see Fig. 3.29). 168

,C2H5 11/C2H5 'C2H5 N/C2H5C H NC2H5 `2 5 co CO co

N-CH3 N-CH3 N-CH3 .71•■••■••■)/M.

\\ 7\ N /() H H

"Aromatic" 2-oxo-LSD 1.1-2-oxo-LSD

Fig. 3.29

The synthetic 2-oxo-LSD, however, was not detected in any of the three species. If it is assumed that the synthetic and the metabolite 2-oxo-LSD were isomers arising from the asymmetry of the C-3 atom and if they behaved similarly in solution, then these would give rise to the same "aromatic" 2-oxo-LSD as shown in Fig. 3.29. This may explain why 2-oxo-LSD was not detected in the monkey urine since it was probably transformed to the "aromatic" 2-oxo-LSD, which was detected. Since the in the perfusate and liver homogenate and in the monkey urine was identical to the decomposition product of the synthetic 2-oxo-LSD, it provided partial evidence that the metabolite 2-oxo- LSD and the synthetic compound were eitherZ-2-oxo-LSD and Z-2-oxo-LSD respectively or vice versa. However, it should be mentioned that the conversion of the metabolite 2-oxo-LSD isolated from rat bile to the yellow product was not observed. This was probably due to the precautions taken to ensure that the LSD derivatives were not stored as a solution. In solid form the synthetic 2-oxo-LSD also did not transform to the aromatic compound. Beckett and Morton (1966) and King et al* (1966) have reported that hydroxylation of 2-oxindoles occurred at the 5-position in the rat in vivo and

169

by rat, guinea pig and rabbit liver microsomes in vitro. King et al. (1966) studied the metabolism of indole in vivo in rat and identified oxindole (1% of the dose) and 5-hydroxyoxindole (3%) as two of the metabolites in the urine. By analogy, it is very possible that the 2-oxo1SD may be further metabolised to 12-hydroxy-2-oxo-LSD but whether this was formed in the rat, guinea pig or

monkey could not be investigated since an authentic sample was not available.

It is probably not formed to any significant level in the rat since an extra peak would have been detected in the higher RF region on the radiochromatogram scans following paper chromatography. The unknown metabolite (M7 or G9) detected in the guinea pig bile and urine and the monkey urine was isolated from the monkey urine. The compound

was not fluorescent, gave a slowly-developing yellow colour with Van Urk (4-dimethylaminobensaldehyde) reagent indicating 2-oxidation but it did not possess any phenolic nature. However, the yellow colour with 4-dimethylamino-

benzaldehyde can also be obtained with the products of pyrrole ring cleavi#e

of indoles (King et al., 1966; Heacock and Mahon, 1963). By analogy with the metabolism of indole (King et al., 1966; see Fig, 1.19) and tryptophan (Meister, 1965), the structure of the unknown metabolite in the guinea pig and.

monkey would be that shown in Fig. 3.30 (structure B), formed by oxidative

LSD

A Fig. 3.30 170

cleavage between C-2 and 0-3. This structure would be consistent with the obtained result which indicates the presence of an aromatic primary amine in

the compound. The metabolite, if it is formed, may arise from structure A non-enzymically since it has been reported that N-formy1-2-aminoacetophenone

(analogous to A) readily decomposed to give 2-aminoacetophenone (analogous to

structure B) (Heacock and Mahon, 19631 Abmanovitch and Ahmed, 1961). Conversion of A to B may also be enzymic by analogy with the conversion of E-formylkynurenine (from tryptophan metabolism) to (Meister, 1965). The metabolism of LSD leading to structures A and then B may involve 2-oxo-LSD as the intermediate.

With a high dose, other mechanisms of detoxication also become important.

With the low dose of LSD (1 mg/kg) used, in the intact rat, the extent of de-

ethylation was 3.4% of the dose in 96 h and 0.5% was detected as lysergic acid menoethylamide (LAE) in the 24 h urine sample. In the liver perfusion experiment, when a total of 8-10 mg of LSD was used, the recovery was only 87% of 140 indicating a much greater amount of de-ethylation than encountered in vivo with the comparatively lower dose. Since only 2% was detected in the

perfusate and liver as LAE, this would only account for a loss of 2% as i4CO2, leaving over 10% unaccounted far. Since nicotinamide is the major metabolite of nikethainide (NIN-liethylnicotinazide) (see Chapter One under "The Metabolism

of Compounds Structurally Related to LSD"), it is speculated that loss of both ethyl groups in the diethylamide side chain of LSD also takes place. This

would increase the elimination of radioactivity as 14CO2 and lead to a loss of label in the resulting LSD metabolite (probably lysergic acid amide). However, the production of 14CO2 by the isolated perfused liver-vas not studied in order to verify or repudiate the proposal. The metabolic study of LSD labelled with

14C in the 6-methyl would also indicate whether an additional radioactive peak

probably correspond rig to lysergic acid amide is detectab&on the radiochromato- r gram scans. The synthesis of L6-14 CH3]-LSD could be carried out by reacting nor-LSD with Kali and 14CH3I (see Nakabara and Niwaguchi, 1971). 171

De-methylation at the 6-position has also been identified as a minor route for the metabolism of LSD since nor-LSD was formed by the isolated

perfused rat liver. for-LSD, however, was not detected in vivo. If the studies were to be performed with un LSD, any 6-de-methylation by the intact animals would be detected as 14CO2 in the expired air. LAE and nor-LSD were recently reported to be metabolites of LSD in vitro

by Niwaguchi et al. (1974) using liver microsomes of rats, guinea pigs and rabbits. These workers, however, did not look for any lysergic acid amide.

In contrast to their report that LAE was always formed in greater amounts than nor-LSD (see Table 14), the isolated perfused rat liver produced more nor-LSD than LAE. Since the integrity of the liver was maintained in the perfusion experiments, it is probable that further de-ethylation of ME occurred to decrease the amount of the latter compound whereas this may not have been

possible with the in vitro microsomal preparations. The present study, however, confirmed that deaikylation plays a minor role in the metabolism of LSD by the rat.

De-ethylation of the diethylamide side chain of LSD in the guinea pig (18% in 96 h) was about five times that in the rat as indicated by the excretion of 14CO2. However, LAE was detected to a very small extent (0.6% in the 24 h urine sample). This would indicate that LAE was further de- ethylated to produce more 14002 and probably lysergic acid amide. The constant low recoveries of 140 in the experiments with the monkey was probably due to de-ethylation with loss of 14002 in the expired air. This would. be verified if the expired CO2 could be collected from this animal. As in the guinea pig, the monkey excreted very little LAE to account for the predicted substantial de-ethylation. This is in accord with the proposal that loss of both ethyl groups also occurred in the monkey. It is therefore predicted that significant amounts oflysergic acid amide are formed from LSD imam in the guinea pig and monkey. If this is so, then the metabolism of LSD by de-ethylation of both the ethyl groups would be one of the major routes in the detoxication of 172

of the drug in the guinea pig and monkey and a minor route in the rat. LSD was extensively metabolised In vivo in the three species since the unchanged drug was either undetected as in the rat or excreted in very low amounts (about 0.5-1.5 of the dose) as in the guinea pig and the monkey. This confirmed the finding of Axelrod et al. (1957) who reported that less than 1i of the drug was excreted unchanged in the monkey urine. That the rat is capable of excreting LSD in the bile was demonstrated by the liver perfusion experiment. When the low amount of LSD (1 mg/g liver) was used, the elimination of the unchanged drug in the bile was IS of the dose in 4.5 h but with 2 mg/g liver, the unchanged drug accounted for over 3%. It is not known what the other metabolites in the rat, guinea pig and the monkey are. Some of these could be decomposition products of the metabolites and the observed instability of ik-hydroxy-LSD and the unknown metabolite M7 or G9 demonstrates this possibility. The solvent systems used probably did not induce decomposition of the stable compounds since a single peak was obtained when 0.401-LSD was chromatographed on paper or t.l.c. plates in all the solvent systems. It should be mentioned, however, that iso- lysergic acid monoethylamide (transformation product of LAB) and lumi-LSD (a possible metabolite) were not detected in any of the species.

The results also serve to exemplify the molecular-weight threshold for appreciable biliary excretion. It has been proposed that the threshold molecular weights for the rat, guinea pig and the rabbit are about 325 t 50,

400 * 50 and 475 t 50 respectively, with the Rhesus monkey closely resembling the rabbit in this respect (Smith, 1973). The molecular weight of the metabolites that have been identified in the bile of rat or guinea pig are of the right order for biliary excretion. However, LAE (mol. wt. 295) and nor- LSD (moli wt. 309), although within the threshold 'limit in the rat, are not excreted in the bile of rat, guinea pig or isolated perfused rat liver. In contrast, LSD (moli wt, 323) was detected in the guinea pig bile although it would not be expected to be present there. Even so, it is highly probable 173

that the molecular weight of the unidentified biliary metabolites fall in the range 300-500. The molecular weight of the unknown 147 or G9 is probably of the order 300-350. This figure was arrived at by considering the similar

binary and urinary excretion of the metabolite in the guinea pig and a

predicted lower excretion in the monkey bile than in the monkey urine. It can be concluded that there is a marked species variation, qualitative and quantitative, in the metabolism and excretion of LSD. There is a general trend to excrete more 140 in the urine and the expired air and less in the faeces when the rat, guinea pig and the monkey are considered in that order.

The main routes of metabolism in the rat and the guinea pig are by 1) hydroxylation of the benzene ring to form phenols, which are excreted, conjugated with glucuronic acid, mainly in the bile and 2) 2-oxidation to form 2-oxo-LSD. The major metabolite in the monkey urine was unidentified.

De-ethylation is more important in the guinea pig and probably the monkey compared with the rat and a small amount of LAE was detected in all three species. Nor-LSD is probably formed in the rot when the other routes become saturated. The metabolic routes of LSD so far elucidated are summarised in Fig. 3.31. N(C H ) N(C H ) 2 5 2 2 5 2 CO CO N(C H ) 2 5 2 CO

rat, guinea rat pig, HO monkey guinea pig, monkey

13-Hydroxy-LSD 14-Hydroxy-LSD (conjugated) (conjugated) LSD HNC H N(C H ) rat, 2 5 2 5 2 rat, guinea pig, CO CO guinea pig rat monkey

N(C H ) N(C H ) NH 2 5 2 2 5 2 2 CO CO CO

Lysergic acid 2 -Oxo-LSD monoethylamide (LAE)

"Aromatic" 2-oxo-LSD Nor-LSD Lysergic acid amide

Fig. 3.31. The metabolic routes of LSD 175

CHAPTER FOUR

The Fluorescence of LSD and its Derivatives and the Stability of LSD

Contents Fage

INTRODUCTION 176 RESULTS AND DISCUSSION 177 Fluorescence and Excitation Spectra 177 nil-Fluorescence Studies 184 Lysergic Acid 184 LSD, LAE and Nor-LSD 185 12- and 13-Hydroxy-LSD 190 12-, 13- and 14-Methoxy-LSD 196 Stokes Shifts 198 Stability of LSD 199 Stability of LSD Solutions 199 Photodecomposition of Non-Buffered LSD Solutions 199 Conversion of LSD to Iso-LSD 202 Stability of [14C]-LSD 203 X76

The Fluorescence of LSD and its Derivatives and the Stability of LSD

LSD and its derivatives contain conjugated double bond systems Which enable the compounds to fluoresce very strongly on exposure to UV light (see review by Williams and Bridges, 1964). This property has been utilised in the

present study to investigate their fluorescence characteristics in the Aminco-

Bowman Spectrophotofluorimeter. A number of reports have dealt with the fluorimetric assay of the drug in

biological fluids (Axelrod et al., 1957: Aghajanian and Bing, 1964; Upshall and Wailling, 1972) but the literature on the fluorescence variation with pH

of solutions of LSD and its derivatives is very limited. Dal Cortivo et al.

(1966) noted that the fluorescence intensity of LSD was pH dependent. They found that in acidic solutions, maximum intensity was obtained at between pH 3

and 5. It has been reported that the fluorescence of aqueous LSD solutions was slightly greater at alkaline pH than in acidic solutions (Mueller and Lang,

1973). The same authors also found that there was a slight shift in the

excitation and fluorescence wavelengths to the shorter wavelength region when the 0.01M-HCl solution of LSD was replaced with an alkaline 0.05M-Na2H104 solution. However, Gillespie (1969) did not find any significant differences

in the wavelengths When the 0.001M-H2SO4 medium was replaced with 1M-VH4OH but in 1M-NaOH, only the fluorescence wavelength of the LSD solution altered from

435 to 536 nm. It was suggested without proof that this unusually large shift

was probably due to degradation of LSD. Studies with solutions of ergotamine (structurally resembling LSD) of pH

between 1 and 14 have bhown that the compound has maximal fluorescence at pH 10.8 (Hooper et al., 1974). In acid medium, however, the compound had about one-fifth of the fluorescence intensity. Bridges and Williams (1968) reported

that the indole derivatives, tryptophan, tryptamine and H,N-dimethyltryptamine,

had a similar maximal fluorescence intensity at pH 10.4.

This Chapter deals with the fluorescence characteristics of LSD and its

derivatives with a study on the stability of LSD. 177

The excitation and fluorescence wavelengths quoted are those which give maximal fluorescence of the compound and are instrumental values unless other- wise stated. Only the observed fluorescence intensities are quoted. A pH 9 solution of LSD was given an arbitary value of 1.00 and all other intensities were based on this, making allowances for molecular weight. 4uenching of fluorescence of the compounds was not observed at the concentrations used (0.1-1.0 pg/m1). All fluorescence readings were obtained at room temperature

(about 20-22°C).

Results and Discussion

FlogmenandExcitatioSctramelgu g4 The fluorescence and excitation spectra of LSD and its derivatives are shown in Figs. 4.1 - 4.3. These results are summarised in Table 4.1 which includes the relative fluorescence intensities and the Stokes shifts (Bridges et al., 1966; Bridges and Williams, 1968) of the compounds studied. In Table 4.1, the first thirteen compounds have excitation and fluorescence wavelengths of 318-344 and 402-442 nm respectively with minor excitation wave- lengths ranging between 246 and 260 nm. There was no difference in the excitation and fluorescence wavelengths between lysergic acid, lysergic acid monoethylamide (LAE) and LSD which indicates that the side chains in these compounds do not influence the fluorescent properties to any extent. It should, however, be mentioned that iso- LSD or iso -LAE had different excitation and fluorescence wavelengths from their epimeric counterparts although the side chains in the epimers were identical. The side chain appeared to have a significant effect on the relative fluorescence intensities of the three compounds, being about equal for lysergic acid, LAE and iso -LAE and comparatively

15% higher for LSD and iso.LSD. Substitution in the ergoline ring of the LSD molecule alters the wavelengths for maximal fluorescence and reduces the fluorescence intensity of the parent molecule due to localisation of 7'1-electrons.

These changes were minimal for nor-LSD as the de-methylation at position-6 178

Exc. Fl.

(a)

■ nce oresce lu F

300 400 500 Wavelength (nm)

E.Nc. Fl.

(b) ity s ten in

nce sce luore F

300 400 500 Wavelength (nm)

Fig. 4.1. Fluorescence (F1.) and excitation (Exc.) spectra of (a) LSD ( iso-LSD ( and lysergic acid (—.—•) and (h) LAE (lysergic acid mono- ethylamide) ( ), iso-LAE (— . .) and nor-LSD (G-demethyl-LSD) )• The spectra were obtained in water at pH 7. Intensity values are arbitarv, that of LSD at p11 9 being taken as 100. intensity. as 100.Thefluorescenceof12-hydroxy-LSD isshownattwotimesitsactual water atpH7.Intensityvaluesarearbitary, that ofLSDatpH9beingtaken meth oxy-LSD(—-—-)and14-trethoxy( (- ---)and13-hydroxy-LSD( P.1 Fig. 4.2.Fluorescence(Fl.)andexcitation(Exc.) spectraof(a)12-hydroxy-LSD Fluorescence intensity 20 0 200 200 -

.• • ... — -'

• • • I 300 300

./I

I I

/1

/

/

/

e '--•

• Exc.

• ...... ,

•

•

% •

1

) and(b)12-methoxy-LSD(----),13- / • Wavelength (nm) % ,

\ /

),

Wavelength (nm)

. /

/

/

/

/

/ 400 400

/

• ). Thespectrawereobtainedin % • \ • • Fl. • • •

. ‘ ... 500 500 (a) 179

180

Exc. FL

(a)

ity ENC. 11. ns (•\. te / • in I \ t

e _'Si \ 1

nc • e

c I‘ 11 1 \ 1 / " / N \

ores N ,... \ lu s \ / 1 F

0 200 300 400 500 600 700 Wavelength (rim)

Exc. Fl. 5-

ty i tens in orescence lu F

0 r 200 300 400 500 Wavelength (nm)

Fig. 4.3. Fluorescence (Fl.) and excitation (Exc.) spectra of (a) the metabolite 2-oxo-LSD from bile of rats ( ), synthesised 2-oxo-LSD (----) and "aromatised" 2-oxo-LSD (-----) and (h) indole ( ) and lumi-LSD (- - The spectra were obtained in water at p11 7. Intensity values are arbitary, that of LSD at pH 9 being taken as 100. The fluorescences arc shown at ten times their actual intensities for synthesised 2 oxo-LSD, two times for "aromatised" 2-oxo-LSD and four times for lumi-LSD. Table 441. Fluorescence of LSD and its Derivatives

The fluorescence and excitation wavelengths were obtained in water at pH 7. Fluorescence intensity was compared with that of LSD at pH 9, which was given an arbitrary value of 100.

Wavelength (nm) Of minor Of max. Of max. excitation excitation fluorescence Relative Stokes Conc. used fluorescence shift Compound (ugiml) Obs. Corr. Ote. Corr. Obe. Corr. intensity (cm' ), Lysergic acid 0.1 248 240 318 313 420 430 70.0 8693 LSD 0.1 250 242 318 313 420 430 81.0 8693 Iso-LSD 0.1 255 24.7 326 321 437 447 79.0 8781 LAE 0.2 246 238 318 313 420 430 69.2 8693 Iso-LAE 0.1 255 247 325 320 1127 437 68.0 8367 Nor-LSD 0.3 24/ 239 322 317 425 435 64.8 8557 12-hydroxyL3D 1.0 255 247 329 324 412 422 4.3 7168 13-hydroxy-LSD 0.1 248 240 344 339 442 451 19.2 7326 12-methoxy-LSD 0.2 250 242 327 322 406 415 19.0 6959 13-methoxy-LSD 0.3 245 237 337 332 433 443 14.1 754/ 14-methoxy-LSD 0.3 250 2112 318 313 402 411 36.8 7618 Metabolite 2-oxo-LSD 1.0 260 252 322 317 418 428 7.0 8181 Synthesised 2-oxo-LSD 1.0 - - 322 317 422 432 0.6 8397 "Aromatised" 2-oxo-LSD 1.0 384 379 270 263 548 558 2.8 8464 Lumi-LSD 1.0 235 227 290 283 346 354 1.1 7087 Indole 1.0 230 223 280 273 340 348 5.0 7895 182

probably does not have a great influence on the conjugated double bond system. However, substitution in the part of the molecule containing these double bonds has a relatively marked effect on the wavelengths and the fluorescence intensity.

The latter was reduced markedly in the case of 12-hydroxy-LSD and 2-oxo-LSD. The synthesised and the metabolite 2-oxo-LSD (see Chapter Three) had an identical major excitation wavelength and very similar fluorescence wavelengths

Which were not very different from those of LSD (see Table 4.1). This further demonstrates that the two 2-oxo-LSD are very closely related as suggested in Chapter Three. However, the metabolite was about twelve times as fluorescent as the synthesised compound. If the compounds were isomers about position-8, a possibility suggested in Chapter Three, they would be expected to have about equal fluorescence intensities by analogy with LSD and iso-LSD and LAL and iso- LLE. The large difference in the relative fluorescence intensities of the two

2-oxo-LSD provides additional evidence that they are not epimers about position -8. The low fluorescence intensity of the 2-oxo-LSD is to be expected since the reduction of the C-2, C-3 double bond reduces the conjugated double bond system (see Williams and Bridges, 1964). That the metabolite 2-oxo-LSD with a reduced number of double bonds has a greater fluorescence than 12-hydroxy-LSD

can be partially explained. It is suggested that hydroxylation in the 12- position has a greater localising effect on the Tr-eiectrons. When the 12- methoxy-LSD is examined, it is found that the fluorescence is increased by over four-fold as compared to 12-hydroxy-LSD. This would suggest that the

methoxyl group does not localise the electrons to the same extent as the hydroxyl group in the 12-position. The hydroxyl or methoxyl group in position

-13 gives the compound about the same fluorescence intensity as 12-methoxy-ISD indicating a similar localising effect in these three compounds. The 14-

methoxy-LSD behaves differently from 12- or 13-methoxy-LSD in that the former compound has about twice the fluorescence intensity of the latter. 183

It appears that the excitation and fluorescence wavelengths of 318-344 and 402-442 nm respectively for the LSD derivatives are characteristic of the conjugated double bonds in the benzene ring and the C-9, C-10 position. The evidence for this comes from the results obtained for 2-oxo-LSD and lumi-LSD.

For 2-oxo-LSD, as mentioned previously, the wavelengths of maximal fluorescence are very similar to those of LSD (Table 4.1) although the pyrrole ring cannot contribute to the fluorescence property due to the reduction of the C-2, C-3 double bond. However, reduction of the C-9, C-10 double bond to form lumi-LSD alters the wavelengths to 290 nm for excitation and 346 nm for fluorescence which compare with 280 (exc.) and 340 nm (fl.) for indole. Thus, lumi-LSD behaves like indole and fluoresces in the ultraviolet. The lumi-LSD and 2-oxo-

LSD have a relatively low fluorescence intensity as compared to LSD which suggests that a simultaneous presence of double bonds in the indole ring and the C-9, C-10 position is essential for a greater fluorescence intensity.

The "aromatised" 2-oxo-LSD had different excitation and fluorescence wavelengths from those of synthesised 2-oxo-LSD from which the former compound was derived. This is due to the presence of a -type conjugated double bond system in the aromatised compound. The compound had a major excitation wavelength of 270 nm with a wavelength of excitation of 384 nm being a minor one (Fig. 4.3). This is a reversal of what is normally found for indole and LSD-like compounds where the minor excitation wavelength occurs in the 230-260 nm region. It should, however, be noted that the minor excitation wavelength of the metabolite 2-oxo-LSD produces the fluorescence intensity which is nearly that from the major excitation wavelength. The fluorescence of the "aromatised" 2-oxo-LSD occurs at 548 nm which is very much different from those of the other LSD derivatives. This is to be expected since the conjugated double bond system in the "aromatised" 2-oxo-LSD is dissimilar from the other compounds. The excitation and fluorescence wave- lengths compare favourably with the literature value of 542 (fl.) and 380 nm

(exc.) for the aromatised compound (Troxler and Hofmann, 1959). The 184

"aromatised" 2-oxo-LSD has about five times the fluorescence intensity of the synthesised 2-oxo-LSD. phi-Fluorescence St es

The variation of fluorescence intensity with pia of the compounds was studied between Ho -3.9 and 16.1. None of the compounds showed any detectable fluorescence below Ho -1. The results are shown in Figs. 4.4 - 4.7 and Table 4.2. Except for lysergic acid, the compounds studied possessed three nitrogen atoms which may be protonated simultaneously to form trication species.

However, the following results suggest that only the protonation of nitrogen atoms in positions 1 and 6 have any effect on the pH-fluorescence characteristics. Survey of the literature failed to reveal the pKa of nitrogen in the amide side chain of LSD or its derivatives but the pKa would probably be low since amides are generally weakly basic (Albert and Serjeant, 1962). Thus, the trication species may be present in strong acid solutions. However, for purposes of discussion, it has been assumed that the nitrogen in the amide group is in a neutral form within the pH range discussed below. Lysergic Acid The pH-fluorescence characteristics of lysergic acid are shown in Fig. 4.4. COOR COOH COO +N„, ,„. -1 CH3 < -- 1 CH 3

ji H

Dication Cation Zwitterion COO COO-

N-CH I:-CH 3 e' 3

Dianion 185

The fluorescence of this compound does not appear until pH 0. Below this pH the lack of fluorescence may be due to either the non-fluorescent dication by analogy with indole (Bridges and Williams, 1968) or quenching by H+ or S042- ions. Decomposition may also be involved but this is unlikely since fluorescence of lysergic acid was regained When a Ho -1 solution was adjusted to pH 7. The cation fluoresces between pH 0 and 3. At pH 4 there is a sharp increase in intensity and a change in the excitation and fluorescence wave- lengths from 321 (exc.) and 427 nm (fl.) to 318 (exc.) and 420 nm (fl.). This is attributed to the formation of the zwitterion. The ionisation of the carboxyl group in indol-3-ylacetic acid is also known to increase the fluorescence of this compound (Bridges and Williams, 1968). The pKa determined from the fluorescence intensity curve for this ionisation of lysergic acid is

3.4 and compares favourably with the pKa of 3.2 reported by Craig et al. (1938).

The zwitterion fluoresces maximally between pH 5 and 8 and the fluorescence of the anion appears at pH 8. The change in the ionic species did not alter the fluorescence intensity but the excitation and fluorescence wavelengths shifted to 315 and 412 nm respectively. The pKa of the change from the zwitterion to the anion from the shift in wavelengths is 8.5 which compares closely with the reported pKa of 8.0 (Craig et al., 1938). Above pH 9 the fluorescence decreases and reaches zero at Ho 15.3. At pH 14, the fluorescence appears at a new wave- length of 490 nm with no change in the excitation wavelength. The species responsible for this is probably the dianion. The fluorescence could not be detected at Ho 16.1 and this is probably due to decomposition since the normal fluorescence of lysergic acid did not appear when the pH of this solution was adjusted to 7. Decomposition, however, also occurs at pH 14 and Ho 15.1 but not to a great extent.

LSD,. LAE and Nor-LSD

Figs. 4.4 and 4.5 show that the pH-fluorescence characteristics of LSD,

LAE and nor-LSD are very similar to each other but very different from those of lysergic acid. The difference may arise due to the presence of the amide 186

side chain in the former three compounds instead of the carboxylic group which is present in lysergic acid. Identical results were obtained from LAE or nor- LSD obtained metabolically or synthetically. The characteristic shape of the

curves obtained for LSD, LAE, nor-LSD and other LSD derivatives (see later)

are similar to those obtained for ergotamine (Hooper et al., 1974), tryptophan,

tryptamine and NA-dimethyltryptamine (Bridges and Williams, 1968).

/,C H 2 5 /C2H5 H 2 5 /C2H5 R1 R1 E 1 CO co CO CO H N 2-R2 N 2 132

1 1 ! I ,

Dication Cation Neutral molecule Anion

The fluorescence of LSD (Il1eaC2H5; .112..CH3), LAE (RrH; R2=CH3) and nor- LSD (R1iC2H51 R2=H) begins at pH 0 and this coincides with the appearance of the cation from the non-fluorescent dication. The excitation wavelength of 322 nm at this pH was the same for the three compounds under consideration but the fluorescence wavelength was 415 nm for LSD and 426 nm for LAE and nor-LSD.

However, the fluorescence wavelength changed to 429 nm at PH 1 for the three

compounds without a corresponding change in the excitation wavelength. This change may be due to the appearance of the fluorescence of the cation which reaches maximal fluorescence at pH 4 for LSD and nor-LSD and at pH 5 for LAE.

The intensity-pH curves give the pKa values of the change from the dication to the cation of 1.4 for LSD, 1.5 for LAE and 1.6 for nor-LSD (see Table 4.2).

These values agree well with the reported pga of 1.6 and 1.7 for a similar ionisation of tryptamine or dimethyltryptamine and indole respectively

(Bridges and Williams, 1968). 187

The fluorescence of the three compounds is diminished slightly above pH 4 or 5 and that of the neutral molecule appears above pH 6 for LSD and LAE and above pH 7 for nor-LSD. The change in the molecular species corresponds with a rise in fluorescence intensities of the three compounds and accompanied with a shift in the excitation and fluorescence wavelengths to 318 and 420 nm respectively. The shift in wavelengths gives the pKa for this change of 6.5 for LSD and LAE and 7.5 for nor-LSD. Stoll et al. (1954b) have reported a pKa of 6.4 for LSD. The higher pKa for nor-LSD is to be expected since piperidine has a pKa Which is greater than that of N-methylpiperidine by over one unit (Albert and Serjeant, 1962). These values compare closely with the pKa of 6.40-6.90 for protonation of the N-6 nitrogen in a number of ergot alkaloids Which are structurally similar to LSD and have similar amide side chains

(Maulding and Zoglio, 1970). The fluorescence is maximal at pH 8 for LAE, pH 9 for nor-LSD and between pH 9 and 10 for LSD and is greater than at any other pH value. This maximal fluorescence is due to the neutral molecule. The fluorescence begins to decline above these pH values and reaches zero at Ho -15.3. As with lysergic acid, the three compounds under consideration here show a shift in only the fluorescence wavelength above pH 13 to 490 nm for LSD, 494 nm for nor-LSD and 498 nm for LAE and these shifts may be due to the appearance of the anion. Gillespie (1969) has suggested that the large shift in the fluorescence wavelength of LSD may be due to decomposition. In the present study, however, some decomposition was noted but it was not complete since the characteristic intense fluorescence of LSD could be reobtained on neutralising the pH 14 solution. As with lysergic acid, the fluorescence of the three compounds could not be detected at Ho 16.1 due to decomposition. The relative fluorescence intensities of the various species and their pKa values are given in Table 4.2.

188

100 -

Ci a 0 50 C)

La O

0

500 - (b) Fl.

450 -

— nm) **1■- ( h t or-

ng 400 - le Wave

350 -

Sti --fr I Exc. 300 -1 0 2 4 6 8 10 12 14 16 Ho pH Ho 1)-4

Fig. 4.4. Variation of (a) fluorescence intensity and (13) fluorescence (FL ) and excitation (Exc.) wavelength with pH and Ho of LSD (--0--) and lysergic acid (- - -A- - -). Intensity values are arbitary, that of LSD at pH 9 being taken as 100.

189

(a)

ity tens in

nce 50 - sce ore Flu

0

500- (b)

450 -

err-groli■ z

a) 400 -

350 -

Exc.

300 1 1 -1 0 2 4 6 8 10 12 14 16 Ho pH Ho 4

Fig. 4.5. Variation of (a) fluorescence intensity and (b) fluorescence (FL) and excitation (Exc.) wavelength with pH and Ho of LAE (lysergic acid monoethylamide) (- - A—) and nor-LSD (6-demethyl-LSD) (--e---). Intensity values are arbitary, that of LSD at pH 9 being taken as 100. 190

12- and /3-Hydroxy-LSD

The synthesised 12-hydroxy-LSD and the aglycone (13-hydroxy-LSD) of the LSD metabolite identified as the glucuronide of 13-hydroxy-LSD in the rat (see Chapter Three) possess phenolic hydroxyl groups which influence the pH-

fluorescence characteristics as shown in Fig. 4.6. The curve for 13-hydroxy- LSD is similar to that of LSD NA that of 12-hydroxysISD is very different.

,C2H5 ,C2H5 _,, C2H5 C H H --C 2 5 2 5 c2 x5 CO CO CO r,CH3 CH3 N, +"- -H +N-

HO

H Dication Cation Neutral Molecule C H Iv y 2 5 2 5 u -. 21-1.5 _.C2H5 CO co

H

Dianion Anion

The fluorescence of the 12-hydroxy-LSD appears above Ho -1 with excitation and fluorescence wavelengths of 331 and 408 nm respectively. The fluorescence wavelength shifts to 414 nm at pH 1. The fluorescence of the 13-hydroxy-LSD,

however, starts above pm 0 at longer wavelengths of excitation (350 nm) and fluorescence (450 nm). The ionic species responsible for the fluorescence is the cation which has maximal gluorescence at pH 2-3 for 12-hydroxy-LSD and 191

pH 3-4 for the 13-isomer. The pKa values for the change from the dication to the cation are 0.7 for 12-hydroxy-LSD and 1.2 for 13-hydroxy-LSD. As with LSD, LAE and nor-LSD, there is a slight decline in fluorescence above these pH

values but the fluorescence intensity of 13-hydroxy-LSD starts to increase

above pH 6 due to the appearance of the neutral molecule. This coincides with

a shift in the excitation and fluorescence wavelengths to 344 and 442 run respectively. The 12-hydroxy-LSD, however, behaves differently and the fluorescence continues to decline even above pu 6 even though the highly fluorescent neutral molecule is formed as indicated by the change in wave-

lengths to 329 nm for excitation and 412 nm for fluorescence. The failure for the fluorescence to increase in intensity is probably due to decomposition which has been noted in alkaline solution as revealed by t.l.c. in solvent system E. Furthermore, unlike other LSD derivatives which were fairly stable

on exposure to light from the lamp of the fluorimeter, the 12-hydroxy-

LSD photodecomposed under these conditions with a half-life of 9.5 min for a 1 ug/m1 solution and 12 min for a 5 peal solution. The half-life was the same at pH 3 or 10. The 12-hydroxy-LSD, like the 14-hydroxy-LSD, is stabilised by forming the methoxy derivative whose pH-fluorescence curve is found to

behave like that of LSD (see later). The pKa value for the change from the cation to the neutral molecule deduced from the shift in wavelengths, is 6.5 for both compounds and this value is identical with that of LSD or LAE, The 13-hydroxy-LSD has maximal fluorescence at pH 9 and this is probably due to the neutral molecule. There is a slight shift in only the fluorescence wavelength to 438 nm between pH 8 and 9 which is not due to the anion since this would coincide with an expected fall in fluorescence intensity instead of the observed maximal fluorescence at pH 9.

At pH values above 9, there is a rapid decline in fluorescence which is ascribed to the ionisation of the hydroxyl group to form the anion. This is accompanied by a change in the excitation and fluorescence wavelengths of

12-hydroxy-LSD to 324 and 405 nm respectively. There was no detectable similar

192

(a)

20- A' -A.

A

10- cence ores

lu X F

0 s &

500 - (b) Fl.

450 - A- A.

El.

Aar ** Exc. 350 - A.

0••■■• Exc.

300 I I I I I i I I 1 -1 0 2 4 6 8 10 12 14 16 Ho pH Ho

Fig. 4.6. Variation of (a) fluorescence intensity and (b) fluorescence (Fl.) and excitation (Exc.)

wavelength with pH and Ho of 12-hydroxy-LSD (-0--) and 13-hydroxy-LSD (- - A — — ) Intensity values are arbitary, that of LSD at pH 9 being taken as 100. The fluorescence of 12-hydroxy-LSD is shown at two times its actual intensity. inethoxy-LSD wavelength withpHandIt Fig. 4.7.Variationof(zt)fluorescenceintensityand (b)fluorescence(11.)andexcitation(Exc.) Fluorescence 300 350- 450- 500- 5r — -1

11 0

Nr 0

A -•

2

Intensity valuesarcarbitary,thatofLSDatpll9being takenas100. o of12-rnethoxy-LSD(---A---),13-methoxv-LSD 4

1,--_...... 6 Am _

••;.,-. •• pIi 4 ii. _

S

10

12

-----

•

t 9 r •6--•) and14- 1 I

- • 11 0 •

•

In Fl. Exc. Fl. 193 Table 4.2. Fluorescence of LSD and its Derivatives

The fluorescence intensity was compered with that of LSD at pH 9, which was given an arbitrary value of 100.

Wavelength (nm) Of max. Of max, excitation fluorescence Stokes Relative pH of Conc. used Molecular shift. fluorescence maximum Compound (Ug/m1) species Obs. Corr. Obs. Corr. ,(pm71), intensity fluorescence Lysergic acid 0.1 Dication ------Cation 321 316 427 437 8762 - - 3.4 Zwitterion 318 313 420 430 8693 70.0 5-8 8.5 Anion 315 310 412 421 8506 70.0 9 13.5 Dianion 315 310 490 496 12097 - - L3D 0.1 Dication ------1.4 Cation 322 317 429 439 8766 84.1 If 6.5 Neutral form 318 313 420 430 8693 too 9-10 13.5 Anion 318 313 490 496 11787 - - - LAE 0.2 Dication ------1.5 Cation 322 317 429 439 8766 71.0 5 6.5 Neutral form 318 313 420 430 8693 73.9 8-9 13.5 Anion 318 313 498 504 12107 - - - Nor-LSD 0.2 Dication ------1.6 Cation 322 317 429 439 8766 72.5 If 7.5 Neutral form 318 313 420 430 8693 75.0 9 13.5 Anion 318 494 500 11948 _ - - ,...i. 313 = wa Table 4.2 cont.

- - - 0.7 12-hydroxy-LS1) 1.0 Dication - - - - 2-3 6.5 Cation 331 326 414 424 7090 5.5 - - Neutral form 329 324 412 422 7168 9.5 - Anion 324 319 405 414 7193 - - - Dianion 320 315 40 420 7937 - - - 1.2 13-hydroxy-I.SD 0.1 Dication ------Cation 350 345 450 459 7199 18.0 3-4 6.5 Neutral form 344 339 442 451 7326 21.7 9 — 9.5 - - Anion 344 339 438 447 7127 - - _ Dianion 364 359 480 486 7279 - - - 1.2 12-methoxy-WD 0.2 Dication - - - - - Cation 330 325 413 423 7129 18.0 4 6.5 - Neutral form 327 322 406 415 6959 21.3 9-10 - - Anion 327 322 484 490 10647 - 13-methoxy-La 0.3 Dication ------1.1 4 Cation 342 337 444 453 7598 13.3 6.5 - Neutral form 337 332 430 440 7393 15.5 9-10 Aniohl 337 332 422 432 6972 - - - , _ - 1.4 14-netboxy4SD es6 Dication - - - - - Cation 323 318 413 422 7750 45.0 4 6.5 Neutral form 318 313 402 411 7618 38.5 9-10 - - Anion 318 313 402 411 7618 - - 196

shift for 13-hydroxy-LSD. The pKa for this ionisation is 9.5 for 12-hydroxy-

LSD and approximately the same value for the 13-hydroxy-LSD. This compares closely with the pKa of phenol which is about 10.0 (Albert and Serjeant, 1962).

The fluorescence of 12-hydroxy-LSD is not detectable above pH 12 due to decomposition and that of 13-hydroxy-LSD reaches sero at pH above 14. The shift in wavelengths between pH 11 and 12 for the two compounds is probably

due to the appearance of the dianion which is formed at a lower pH than with

LSD, LAE or nor-LSD. The excitation and fluorescence wavelengths, the relative fluorescence intensities and the pKa values of the various ionic species are

included in Table 4.2. 12-, 13- and 14-Methoxy-LSD The 12-, 13- and 14-methoxy-LSD were obtained as described in Chapters

Two and Three. Fig. 4.7 shows the pH-fluorescence characteristics of the three

,,C015 . 2H 5 /25CH ,,C H C H - 1 25 `- C2 5 -C2H5 CO co co

N-CH E-CH E-CH 3 \- 3 3

Ie CH3o H A B C

methoxy-LSD. The fluorescence curves of 12- and 13-methoxy-L5D are typical of

LSD derivatives with maximal fluorescence occurring in an alkaline solution.

However, the 14-methoxy-La shows an uncharacteristic fluorescence curve with fluorescence being greatest at pm 4. One reason for the difference may lie in the type of resonance forms contributing to the fluorescence. Bridges and

Allis= (1968) have Teoposed main quinoid resonance forms contributing to 197

fluorescence for the 5-, 6- and 7-benzyloxyindoles and, by analogy, it is suggested that structure A is responsible for the fluorescence of 12-methoxy- LSD, B for 13-methoxy-LSD and C for 14-methoxy-LSD. Bridges and Williams (1968) have suggested that structures of the type C would produce a phenolic

fluorescence since only the benzene ring is involved in the quinoid form

Whereas in A and B both rings contribute to the resonant structures. Thus, 12- and 13-methoxy-LSD behave like LSD and 14-methoxy-LSD differently. This is further evidence that the main metabolite of LSD in the rat is a glucuronide

of 14-hydroxy-LSD.

The ionic species of the methoxy-LSD contributing to the fluorescence at

various pH values are similar to those of LSD. The cation of 14-methoxy-LSD appears at pH 0 and fluoresces at 396 nm with an excitation wavelength of 337 nm. However, this shifts to 323 nm (exc.) and 413 nm (fl.) at pH 1. The

fluorescence of 12- and 13.methoxy-LSD begins at pH 1 due to the cation. The cation of 12-methoxy-LSD has excitation and fluorescence wavelengths of 330 and 413 nm respectively with 342 and 434 nm respectively for 13-methoxy-LSD. C"J

the fluorescence wavelength of the latter compound shifts to 444 nm at pH 2 as shown in Fig. 4.7. The cations of the three compounds reach maximal fluorescence at pH 4 with that of 14-methoxy-LSD having between two and three times the fluorescence intensity of the other two isomers. The pKa values of the change from the dication to the cation estimated from the pH-fluorescence curves are 1.2 for 12-methoxy-LSD, 1.1 for 13-methoxy-LSD and 1.4 for the 14- isomer. As with the other LSD derivatives, the fluorescence begins to decline slightly above pH 4 until the appearance of the neutral molecule at about pH 6 When the fluorescence intensity again begins to rise and reaches a maximum at pH 9. This change coincides with a shift in the excitation and fluorescence wavelengths of the three compounds. The new wavelengths are 327 (exc.) and 406 nm (fl.) for 12-methoxy-LSD, 337 (ems) and 430 urn (fl.) for 13-methoxy- LSD and 318 (exc.) and 402 nm (fl.) for 14-methoxy-LSD. The shifts in wave- 198

lengths allow PECia values of the change from the cation to the neutral molecule to be estimated and these are 6.5 for the three compounds concerned as would

be expected for a LSD derivative. The fluorescence intensities begin to decline above pH 10 and that of

13-methoxy-LSD reaches zero at pH 14 but the other two compounds become non- fluorescent at Ho 16.1 due to decomposition since the fluorescence could not be regained on neutralisation of this solution. The ionic species involved above le 12 is probably the anion. There is a shift in the fluorescence wave-

length of 13-methoxy-LSD to 422 nm without a corresponding change in the excitation wavelength and the anion of this compound becomes non-fluorescent at pH 14. A shift in the fluorescence wavelength only to 484 nm also occurs for 12-methoxy-LSD at pH 14 but no changes in the wavelengths were detected for the 14-methoxy-LSD. The pft values for these changes are given in Table 4.2. 5-Hydroxyindoles or 5-methoxyindoles differ from the 6- or the 7-isomer by Showing a green fluorescence at about 550 nm in strong acidic solutions

(Bridges and Williams, 1968; Chen, 1968). However, this characteristic

fluorescence of the corresponding 12-hydroxy-LSD or 12-methoxy-LSD could not be detected in the present study although it was shown by 5-hydroxyindole and

5-hydroxytryptamine in 314-HC1.

Stolces Shifts The Stokes shift (Bridges et al., 1966) is a measure of the energy difference between the and the excited state. Tables 4.1 and 4,2

include the Stokes shift for the compounds investigated and was calculated from the corrected excitation and fluorescence wavelengths using the expressions

Stokes shift (cm-1) m 107 (ib\exc. "- 1/X fl.) The Stokes shifts were similar for the compounds but still greater than 2000- 5000 cel which is expected for molecules in which no excited state reaction occurs. In strong alkaline solutions, however, a few ionic species showed a relatively larger shift. The large Stokes shifts for the LSD derivatives and indole are probably not due to excited state ionisation but to interaction between solvent and solute molecules in the excited state (Van Duuren, 1963). 199

Stabilityp of Ls? Stability of LSD Solutions

Table 4.3 shows the extent to which decomposition occurs in the 0.1 and 1.0 pg/m1 aqueous non-buffered solutions of pH 7.4. With the exception of the

solution kept unptotected on the bench, the 1.0 ml solutions showed no decrease in fluorescence intensity under the conditions employed and up to the time stated in the table when the experiment was discontinued. The loss in fluorescence of the solution of low concentration was apparent in all cases,

being 2% in the solution kept in dark at 4°C, 7°A in solution kept in dark at room temperature or at 37°C and over 201i6 in the solution left unprotected from light. The concentrated solution, which was kept on the bench, gave about decrease in fluorescence intensity after 18 h and 4% after 42 h but this figure had risen to 17% after 90 h. In contrast to the non-buffered solution, the sodium phosphate solutions

buffered at VI 7.4 had a much greater loss of fluorescence. Only the solutions of low concentration were investigated. For the solutions kept in the dark at room temperature or 37°C, the loss was very similar, being 13% in 0.001M solution, 1E4 in 0.01M solution and about 20% in 0.1M buffer. However, the unprotected 0.001M solution showed a greater deterioration, the decrease in fluorescence being 34 as compered to 27% in 0.01M solution and 22% in 0.1M- sodium phosphate. quenching of fluorescence (15%) was also noted in the 0,1M solution but not in the 0.001M and 0.0/M solutions.

The results suggest that low concentrations of LSD solutions undergo decomposition irrespective of the conditions. The temperature of 400, however, tends to minimise degradation. The solution is stabilised by increasing the concentration to 1 pg/m1 or over and keeping it protected from light. The results also suggest the possibility of LSD undergoing decomposition in the

body leading to the excretion of artifact metabolites.

Photodecomposition of Non-Buffered LSD Solutions

Fig. 4.8(m) shows the decomposition curves of LSD solution by irradiation Table 4.3. Stability of LSD Solutions

The pH of the solutions ,was 7.4.

% Decrease in Fluorescence Intensity of Solution

Comm. of Sodium phosphate Condition solution (p ml) Time (h) Pion-buffered buffer.... 0.001M 0.01M 0.1M...

Kept in dark at 0.1 24 7 13 16. 21 room temperature 1.0 90 0 410

Kept unprotected at 0.1 24 21 34 27 22 room temperature 1.0 18 1.5 ••■■

42 4 MI/ IMO

90 17 IMO OM.

Incubated at 370C 0.1 24 7 13 16. 19 in dark 1.0 48 0 - - -

Kept at 4°C 0.1 48 2 - - - in dark 1.0 120 0 - - - taken immediatelyalterirradiationfor45seconds withlightfromamercury vapour lamp.Intensityvaluesarearbitary,that ofLSDatpH9beingtakenas100. Fig. 4.8.(a)PhotodecompositioncurvesofLSD duetoirradiationwithlight from amercuryvapourlamp.—e--1µg/ml,pH 5.-El-0.1pg/ml,pH — Fluorescence intensity

0 • 0.1µg/ml,pH9. (b) VariationoffluorescenceintensitywithpH ofLSD.Readingswere 2

4

2. 6 pH Time (mm.)

8

3 10

4

12

14 5 201 202

with light from a mercury vapour lamp. Under the conditions used, the fluorescence intensities fell to half the initial values at 3.2 min for 1 pg/m1 pH 5 solution, 1.2 min for 0.1 pg/m1 phi 5 solution and 0.6 min for OA Rival pH 9 solution. Thus, the LSD solutions of low concentration decompose more rapidly in a NaOH solution than in HC1. Fig. 4.8(b) shows the pH-fluorescence curve following irradiation of a 0.1 pg/m1 solution of LSD of pH values 0-14. The fluorescence intensity is

greatest at pH 5. Normally, the pH-fluorescence curve of a 0.1 }2g/ml solution of LSD is that shown in Fig. 4.4 with maximal fluorescence occurring at pH 9-10. The extent of photodecomposition due to irradiation was 20% at pH 1, V at pH 5, 36% in solution of pH 941 and 8% at pH 12. Niwaguchi and Inoue (1971) have

also demonstrated that photodecomposition for a 50 pgiml solution of LSD was faster in 0.114-HC1 (half-life about 15 min) than in 0.114-NR4OH (half-life

about 70 min) due to a faster rate of conversion to the lumi-LSD in the acidic conditions. The faster rate of deterioration of 0.1 pg/ml pH 9 solution is not fully understood but it may be due to the instability of the neutral molecule in the excited state at this pH leading to break-up of the ring system. At higher or lower pa values, the LSD is in an ionic form which may not

decompose as readily. Conversion of LSD to Iso-LSD Solid LSD (140 mg) was stored at room temperature in the dark and 5 mg samples were taken at 2-3 weeks intervals and dissolved in 1 ml pyridine for determination of optical activity of the sample. Since the reported optical

activity Lcxjr 120 of LSD and iso-LSD in pyridine are respectively +17° and 589 +219° (Hofmann, 1964), it was thought that any significant formation of the F0(120 latter compound would be seen quite easily. However, optical activity L J 589 1 20 and [c,ci (c = 0.5) remained constant at +16° and +30.5° respectively for up to three months when the experiment was discontinued. T.l.c. in solvent

system D, nevertheless, has indicated that a little iso-LSD is formed. 203

Stability of Solid [14C]-LSD Solid [!4C]-LSD (29 mg; specific activity 13.6 uCi/mg) was stored at -2000 and after six months a sample was chromatographed on a t.l.c. plate in system D. Radiochromatogram scanning revealed three additional radioactive peaks (RF 0.06, 3%1 0.25, 4r4 0.40, 3%) to that of LSD (RF 0.67, mo. The compound of RF 0.40 was probably iso-LSD since it had an identical RF to a known sample of iso-LSD on a t.l.c. plate developed in either system D or E. The other two decomposition products are unknown. When the pure [1.40]-1,SD was stored in liquid nitrogen (-19600), no decomposition products were detected on t.l.c. plates for over 18 months. The above results suggest that LSD is unstable under certain conditions but the decomposition can be minimised in concentrated solutions. Storing the solutions in dark or at low temperatures also minimises decomposition. Solid LSD is test stored in liquid nitrogen. 204

CHAPTER FIVE

The Effects of LSD and its Derivatives upon the Electroencephalogram (EEG) of Rabbits

Contents Page

INTRODUCTION 205 RESULTS 206 Pharmacological Observations 206 EEG Observations 206 Control Tracings 206 Experimental Tracings 208 LSD 208 LAE 208 12-Hydroxy-La 209 12-Methoxy-LSD 209 13-Hydroxy-LSD 209 13-Methoxy-LSD 209 13-Hydroxy-LSD Glucuronide 209

DISCUSSION 210 205

The Effects of LSD and its Derivatives upon the Electroencephalogram (EEG) of Rabbits

The effect of LSD on the EEG of man and animals has been reported by several groups of workers (see Rothlin, 1957a). In conscious unrestrained cats, LSD elicited low amplitude fast activity. In man, the drug increased the alpha and beta frequency (Hoffer, 1965) and produced a low amplitude, fast type EEG (Cohen, 1967). The effect on the EEG of rabbits appeared to be dose dependent. Low doses of LSD (3-15 nmol/kg) decreased the amplitude and increased the frequency of the EEG in rabbits whereas slightly higher amounts of the drug (30 nmol/kg) elicited continuous alert patterns (Rinaldi and

Himwich, 19551 Himwich et al., /959). Monroe and Heath (1961) compared the EEG activity of a number of LSD derivatives (dose 40-500 }zg/kg) in monkeys (Macaca mulatta) and none of the . compounds altered the EEG pattern markedly as did LSD or the hallucinogenic 1-acetyl-(+)-LSD. The derivatives that altered the pattern slightly were

(*)-lysergic acid dimethylamide (DAM), (+)-lysergic acid pyrrolidide (LPD), 1-methyl-(+)-LSD (MLD) and (+)-lysergic acid morpholide (LSM) and these compounds also possessed hallucinogenic activity. The derivatives that were not hallucinogenic and did not alter the EEG pattern were (-)-LSD, 2-bromo- (+)-LSD (BOL) and l-methyl-(+)-lysergic acid butanolamide (UHL). This indicated that there was a correlation between the EEG activity and the hallucinogenic response. Schweigerdt et al. (1966) studied the effect of LSD and nine of its analogues on EEG of rabbits at dose levels of 60-600 nmol/kg administered intravenously. They reported that sustained drug-induced EEG arousal patterns were obtained with LSD, lysergic acid monoethylamide (LAE), LSM, MLD and DAM but not with UML, (+)-iso-LSD, (-)-LSD and BOL. All the compounds which altered the EEG pattern were hallucinogenic, but the reverse was not found to be so since one hallucinogen, (+)-lysergic acid amide, failed to elicit the

EEG arousal pattern. 206

The present study was undertaken to determine whether the metabolites of LSD had any effect on the EEG of rabbits. All the compounds were examined at a dose level of 77 nmol/kg and were obtained and identified as described in Chapters Two and Three. The di-LSD disulphide was synthesised as reported by

Freter et al. (1957). The compounds were administered intravenously as described in Chapter Two.

Results

Pharmacalervationsgolag=:=xas

after an injection of LSD, seven rabbits out of eight showed clinical changes which included lip smacking, limb twitching, tachypnoea, tachycardia, sneezing and shivering. These were seen from 1-20 minutes after administration. One animal showed no clinical changes. From 20-25 minutes after injection of LAE, tachypnoea and tachycardia were observed in four experiments in three animals. Tachypnoea was also seen after 25 minutes in two of the four pv4mA1s given 13-methoxy-LSD. No changes in the clinical state of the rabbits were observed after injection of solvent or any other compounds. At no time during the experiments did the animals appear to be asleep.

EEG Observations Control Tracings A total of 42 tracings were recorded from 14 rabbits. In nine of these the wave forms were irregular. All 42 tracings showed a background rhythm of 2.5-6 Hz. In most cases the amplitudes were in the range 20-60 }1V (Fig. 5.1.1a).

In one tracing the amplitude ranged between 5 and 10 iV and in tw000thers between 50 and 100 pV. In 15 of the control recordings, spindles of faster activity were seen in which the frequencies ranged between 9 and 14 Hz (Fig. 5.1.1b). These occurred at intervals of 2-9 seconds and lasted for 1-3 seconds. Their amplitudes ranged from 5-60 3iV and were lower than those of the backggound

RAP = Right Antero— Posterior Trace 207

LAP = Left Antero- Posterior Trace

RAP

1* LAP

RAP

lb LAP rifj

Pi \44.41. 44.4' ., , a liP_ I ! 1\ I iN,ifikrr,v/ 7\e.AN'j • fity.''r' : i rfi, I ' 1 1

AP, • •ri .,...„..H..... ,,... A „... 0, , , s, A. ... -.k ,44 .4 - - .,-* ...' ;-.- y4 'S A...An - ,,. q- ; 1 , •4".' .14 lb

0 i i AP I i. ki 11 lif IVt.'• Ii.;..4.1Yrol.Yiii.■. 6,4,4/r.401?;: .4 ! 1 • i , : ,

11,1 Ivry

Fig. 5.1. EEG Arousal Pattern of Rabbit Evoked by LSD (77nmol/kg)

la and lb; Control tracings

2a and 3a; Control tracings immediately prior to administration of LSD

2b and 3b; Tracings showing the effects of LSD. Note the episodic high voltage slow

activity, flattening (in 2b), increase in frequency and decrease in amplitude of dominant rhythm. 208

rhythm in eight tracings and higher in seven. The same animals often showed spindles in some control recordings and not in others. Experimental Tracings No electrical changes were observed after injection of solvent, (+)- lysergic acid, di-LSD disulphide, nor-LSD (6-de-methyl-LSD), 14-hydroxy-LSD glucuronide, 14-methoxy -LSD, lumi-LSD and metabolite 2-oxo-LSD (see Chapter

Three). LSD Eight experiments were conducted in six animals. Figs. 5.1.2a and

5.1.2b (or 5.1.3a and 5.1.3b) show the control and experimental tracings respectively from the same animal. The control recordings were obtained immediately prior to administration of LSD. The electrical changes were noticeable five minutes after administration and continurCfor the 30 minute period of observation. The dominant rhythm became more regular with decreased amplitudes and increased frequencies. Episodes of flattening occurred at irregular intervals, each lasting 1-2 seconds. In two animals in which spindles were seen in the control tracings immediately before injection of LSD, these were abolished by the drug. In addition to these changes short runs of high voltage slow activity appeared irregularly. Their amplitudes reached 300 pV and their frequencies were in the 2.5-4 Hs range.

LAE

pour experiments in three animals showed that the tracings became more regular 5-10 minutes after injection of LAE. There was a gradual decrease in amplitude and the frequencies became faster in the dominant rhythms. Episodic flattening for 1-2 seconds occurred at irregular intervals. In two animals, spindles were seen in the control tracings but these disappeared after LAE administration. In one animal, however, the spindles reappeared after 20 minutes. 209

12-Hydroxy-LSD

In two experiments in two animals with 12-hydroxy-LSD, there was a

gradual increase in frequency and decrease in amplitude of the dominant rhythm.

In one animal, spindles were seen in the control tracing and these were abolished for 25 minutes after injection. Episodic irregular flattening for

1-2 seconds was seen in one animal.

12-Methoxy-LSD

Two experiments in two rabbits were conducted with 12-methoxy-LSD.

In one animal no changes were observed. In the other, spindles observed in the control tracings were abolished for 25 minutes. There was a gradual

decrease in amplitude and increase in frequency.

13-Hydroxy-LSD

In two experiments in two animals, the 13-hydroxy-LSD caused a

gradual increase in frequency and decrease in amplitude. Spindles were seen

after injection but not before.

13-Methoxy-LSD

Four rabbits were each given a single dose of 13-methoxy-Li) and the

EEG traces showed a gradual increase in frequency with three revealing a

decrease in amplitude also. In ensanimal, spindles were seen in the control tracing and these persisted after injection. In two animals, episodic

flattening for 1-2 seconds occurred irregularly after 25-30 minutes. After

15-20 minutes, short bursts of high voltage slow activity, each lasting 2-3 seconds, occurred in the same two animals. Their frequencies were 3-5 Hz and their amplitudes reached 100 ulf.

13-Hydroxy-LSD Glucuronide

A single dose of the LSD metabolite identified as 13-hydroxy-LSD

glucuronide in rat (see Chapter Three) was administered to each of two animals.

There was a gradual increase in frequency and decrease in amplitude after

injection. In one Anima spindles were seen prior to administration and these

were abolished by the compound. Episodic flattening occurred at irregular

intervals in the same animal. 210

Discussion

The EEG study of LSD and some of its metabolites was performed in order to investigate whether or not the derivatives possessed any CNS activity.

Earlier studies have involved isolated tissues such as rat uterus and the activity of the compounds have been expressed in terms of percentage inhibition of the response to 5-hydroxytryptamine (5HT). Slaytor and Wright (1962) used the isolated rat uterus preparation and reported that the glucuronide metabolites A (more polar) and B (less polar) of LSD in rat had respectively about 5% and 0.5% of the activity of LSD. It is well known (see Chapter One under "Structure-Activity Relationship") that anti-5HT activity does not necessarily mean that the compound is active in the CNS and it was, therefore, felt desirable to investigate the effect of LSD and its derivatives on the EEG

pattern. The small amounts of the compounds available required that the rabbit be used in the present study since the animal is reported to be the most sensitive of the laboratory animals to LSD (see Chapter One under "Pharmaco- logical Effects of LSD").

In the present study, the slow wave activity in the control tracings in conscious rabbit was similar to that described by Schweigerdt et al. (1966). The absence or presence of spindles suggested a variation in the degree of alertness of the animals. After injections of LSD, LAE, 12-hydroxy-LSD,

12-methoxy-LSD, 13-hydroxy-LSD glucuronide and 13-methoxy-LSD, a persistent alerting record was seen as indicated by an increase in frequency, lowering of amplitude and abolition of spindles. Equivocal results were obtained with 13-hydroxy-LS1) in that spindles appeared after injection and not before, although there was an increase in frequency and decrease in amplitude. The occurrence of episodic high voltage slow wave activity after administration of LoD or 13-methoxy-LSD may have been related to the tachypnoea observed clinically. A respiratory alkalosis is known to cause slimier electrical changes in man (personal communication with J.H.J. Durston, Neurology

Department, St. Mary's Hospital, London, W.2). 211

The persistence of an alerting response after injection of LSD agrees

with the results of Bradley (1958) and Schweigerdt et al. (1966). After injection of LAE, the rabbits showed an electrical response at a dose which was about a half of that reported by Schweigerdt et al. (1966) to be minimal in evoking an EEG arousal pattern. In addition, a response was obtained

earlpir than reported by these authors. The activity shown by 13-hydroxy-LSD glucuronide was not expected since

it was felt that the polar compound would not cross the blood-brain barrier. It is possible that the compound may be hydrolysed to the active 13-hydroxy-

LSD in the body. Slaytor and Wright (1962) have also reported this glucuronide to be active, having of the anti-5HT activity of LSD. The results indicate that LAE, 12-hydroxy-LSD, 12-methoxy-L6D, 13-hydroxy- LSD, 13-methoxy-LSD and 13-hydroxy-LSD glucuronide produce a persistent alerting response in rabbits similwe to that caused by LSD itself. Of these

compounds, LSD and LAE are hallucinogenic (see Chapter One under "Structure- Activity Relationship") and it is possible that the other five LSD derivatives in this group may also possess hallucinogenic activity. However, as the number of experiments were small further work is necessary, possibly with modification in dosage, but human trials would be necessary eventually for

confirmation. It would appear from the present study that none of the compounds tested were as active as LSD in altering the EEG of the rabbit. No changes were observed after injection of lysergic acid, di-LSD disulphide, nor-LSD, 14-hydroxy-LSD glucuronide, 14-methoxy-LSD, lumi-LSD or

metabolite 2-oxo-LSD in identical doses. It is probable that none of the compounds in this group are hallucinogenic since the 2-oxo-LSD reported by Axelrod et al. (1957), lumi-LSD and di LSD disulphide do not possess any hallucinogenic activity in man (see Chapter One, Fig. 1.7). It is possible that changes in the EEG pattern may appear with larger doses of some of these compounds. 212

CHAPTER SIX

Concluding Remarks and Scope for Future Metabolic Study of LSD 213

Concluding Remarks and Scope for Future Metabolic Study_of LSD

It has been shown in Chapter Three that there are marked differences in the metabolism of LSD and its excretion by the rat, guinea pig and Rhesus

monkey. It would be convenient to predict the metabolism of the drug in man

but the species variation makes extrapolation to humans very difficult. However, humans would probably resemble the monkey more than the rat or the guinea pig. This would be in accordance with the general belief that monkeys

are better models for man for the metabolic studies of foreign compounds (Smith

and Williams, 1974; Williams, 1974). Thus, humans given [140]-LSD would ,i3oss;111 excrete about 40-45% of the radioactivity in the urine, some 20-25% in the

faeces and the rest appear in the expired air as 14002. Presence or the lack of 14CO2 in the humans would support or refute the suggestion made in 14 Chapter Three that monkeys excreted a substantial amount of C in the expired air. The metabolites of LSD in man would probably include the glucuronide conjugates of 13- and 14-hydroxy-LSD since these were formed by the rat,

guinea pig and the monkey. Hydroxylation in the 13-position of LSD is to be expected since a number

of indole derivatives (Fig. 1.20) are hydroxylated in vivo in the corresponding 6-position. However, microsomal hydroxylation in the 7-position of indoles

(dorresponding to 14-hydroxy-LSD) has been reported to occur in only two other indolic compounds (see Chapter One under "Metabolism of Compounds Structurally

Related to LSD"). It is possible that 7-hydroxyindoles are formed but are more unstable than the 5- or 6-hydroxy derivatives and may preclude identification.

In the present study, the instability of the 14-hydroxy-LSD was realised and steps were taken to make the compound more stable. This was achieved by

converting the compound to 14-methoxy-LSD.

The metabolic study of LSD in animals proved difficult due to the high CNS toxicity of the drug and the low doses that could be administered. However, in the case of the rat, the difficulty was overcome by the isolated

perfused rat liver preparations which enabled larger quantities of LSD to be 214

metabolised, the metabolites including all those encountered in vivo. This clearly demonstrates the usefulness of the technique in the study of the metabolism of toxic compounds. However, for moat foreign compounds, which are relatively non-toxic, it is much easier to administer large quantities to the animals than to use the isolated perfused liver which is not a simple technique. It may be necessary in the future to prepare isolated perfused guinea pig livers in order to examine the nature of the unknown LSD metabolites in this species. Today, it is very difficult to identify any LSD excreted in urines of suspected subjects for forensic purposes (A.C. Moffat, Home Office Central Research Establishment, Aldermaston, Berks.; personal communication). This is probably due to the very low quantity, if any, of the unchanged drug present in the urine of LSD abusers. The low quantity would require a highly sensitive technique, such as radioimmunoassay (Loeffler and Pierce, 1973; Taunton-Rigby et al., 1973), for detection. Extrapolation of the results from the monkey would suggest that humans probably excrete a very small amount of LSD in the urine as unchanged material. This would need immediate analysis since the drug in low concentration undergoes rapid deterioration as has been demonstrated

(see Chapter Four). Its decomposition may be a reason for the failure to detect

LSD excreted in human urine. Another reason could be that the drug may be totally metabolised and it has been shown in Chapter Three that the rat, guinea pig and the monkey metabolise LSD extensively. Thus, it would be useful to identify a major metabolite of LSD in the human urine so that a specific assay

(radioimmunoassay, for example) could be developed for its detection. A possible metabolite that has been postulated to occur in substantial amounts in the monkey urine is lysergic acid amide. Future studies in the three species would need to be duplicated with [6-14CH3]-LSD in order to detect this postulated metabolite, if formed. The administration of [6.14CH3]-LSD to rat and guinea pig would also show whether any de-methylation occurs in vivo since the radioactive label would appear as 14002 in the expired air. 215

The problems involved in the study of the metabolism of LSD would decrease

in magnitude if a number of authentic possible metabolites were available. The synthesis of the various LSD derivatives proved very difficult in the

present study. It would require extensive attention in the future if some of

the unknown metabolites were to be identified unequivocally.

It is tentatively suggested that other LSD-like compounds probably

undergo metabolism along pathways similar to those of LSD. Since this appears to be the case for iso-LSD (see Chapter Three), it may be more convenient to use this relatively non-toxic compound as a model for the metabolism of LSD in future studies.

The pH-fluorescence study (Chapter Four) added further weight to the idea that the two major rat biliary metabolites R4 and R5 are glucuronides of 13- and 14-hydroxy-LSD respectively. Some of the compounds investigated in this study also altered the EEG of rabbits (see Chapter Five). Since the pKa for

protonation of the neutral compounds found to be active in the CNS was about 6.5, as deduced from the pH-fluorescence characterics, they will be 89% unionised at pm 7.4. Thus, it can be concluded that these compounds probably elicit EEG arousal patterns in their neutral forms. 216

APPEbDIX

Contents Page

Interpretation of liMR Spectra of LSD and 12-Hydroxy-LSD 217 Mass Spectral Fragmentation Pattern 217 Mass Spectra of (a) LSD or [14CT-LSD and (b) Iso-LSD or [14C]-Iso-LSD 218 Mass Spectra of (a) Iso-LAE (Iso-Lysergic Acid Monoethylamide) and (b) 2,3-Dihydro-LSD 219 Mass Spectrum of Lumi-LSD 220 Mass Spectra of (a) 6-Cyano-6-Demethyl-LSD and (b) "Aromatised" 2-0xo-LSD 221 liMR Spectrum of LSD 222 EMR Spectrum of 12-Hydroxy-LSD 223 Infra-Red Spectra of (a) LSD and (b) Synthesised 2-0xo-LSD 224 217

Interpretation of Nuclear Magnetic Resonance (NMR) Spectra of LSD and 12-Hydroxy-LSD The NMR spectra of a number of dialkyl amide derivatives of lysergic acid have been reported to show very little differences between each other (Bailey et al., 1973). The spectrum of LSD in CDC13 obtained in the present study is very similar to the spectrum of lysergic acid dipropylamide in CDC13 published by these authors. The NMR spectrum of 12-hydroxy-LSD in CJ3UD is slightly more complex than that of LSD due to signals from the impure solvent. However, the aromatic benzene ring region contains four peaks at 6.61, 6.70, 6.95 and 7,04 ppm which are typical of two ortho (Dyke et al., 1971), indicating that the compound is not substituted in the 13-position. Calculation of the coupling constant of the two protons at 6.61 and 6.70 ppm or 6.95 and 7.04 ppm gives a J value of 8.55 Hz. This indicates substitution at the 12-position by comparison with the coupling constants of indole which has a J5,6 value of 7.0 Hz and J6,7 of 8.0 Hz (Black and Heffernan, 1965). Mass Spectral Fragmentation Pattern The mass spectral fragmentation patterns of LSD and of its derivatives are very similar. Typical fragmentation patterns may be found in reports by Bellman (1968), higam and Holmes (1969), Crawford (1970), 'hakahara and lawaguchi (1971) and Inoue et al. (1972). 323 100 221 I-I 181 (a) LSD or I CI-LSD

207

196 167 280 265

rT

3)3 221 (b) Iso-LSD or 114C I-Iso-LSD

280

0 150 200 250 300 350 m/e

14 14 Mass spectra of (a) LSD or [ C_J- LSD and (b) Iso-LSD or [ Cl-iso-LSD 295 100 — 181 (a) Iso-LAE 221

196 207

167 L '. (,

ity s n 0 te

in 325

(b) 2, 3-Dihydro-LSD 223 225

1G7 182 194 209 0 150 200 250 300 350 m/e

Mass spectra of (a) iso-LAE (iso-lysergic acid monoethylamide) and (b) 2,3- dihydro-LSD 170 100 -

Lurni-LSD 197 I-12

50 -

129

151 2 )3 323 311

183 207 199

0 100 150 200 230 300 350 in/c

1\lass spectrum al lumi- LSD (10 -hydroxy- 9, 10- clihydro-( -)-1yscrgic acid dicthylamide) 335 100 — 193

207 (a) 6- Cyan° -6 -de methyl-LSD

234

167

219

235 c) 100 — 237 337 (b) "A romati sed " 2- oxo -LSD

221 209 167 182 19,1 11111,7 api 1 1

150 200 250 300 350 m/c

Mass spectra of (:1) 6- cyano- 6- demethyl- LSD rind (b) "aromatiscd" 2- oxo -LSD

1000 6 1 4 2

2

NMR SPECTRUM OF LSD

jiL 1

10 ppm

2 10 IS

OR 0 2 2 NMR SPECTRUM OF

12-HYDROXY-LSD

Jortho = 8. 55 Hz JUL JL

8 6 4 2 ppm 0 224

WAVELENGTH (MICRONS) 6 7 8 9 10 11 12 13 14 15

0 4000 3000 2000 1500 100,0 900 800 WAVENUMBER (CM- '

WAVELENGTH (MICRONS) 6 7 8 9 10 11 12 13 14 15 100 100

(b) . SYNTHESISED 2-01<0-LSD 80 80

60 60

40

2 20

0 4000 3000 2000 1500 100,0 900 800 700 WAVENUMBER (CM-

Infra-red spectra of (a) LSD and (b) synthesised 2-oxo-LSD 225

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