THE METABOLIC FATE OF LYSERGIC ACID 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 lysergic acid 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 Hallucinogens 6 History of Ergot 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 group 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, psychotomimetic, psychedelic and hallucinogen 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 magic 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 MUscarine ci130 CH2C H "2 2 0H3 O 3 Yescaline Harmine 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: Psilocin met hylcarbinolamide e) RI=OPO3H; R2=H: Psilocybin a) R1=e2H5; R2=C225: (+)—LSD OH c5 HII (n) (n) CIL CH3 ZS,9—Tetrahydrocannabinol 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). Amanita muscaria, 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).