I E •F AMPI-1 TAMI ES by a Thesis Submitted for the Degree of Doctor

I E •F_AMPI-1 TAMI ES VARIOUS SPECIES by JOHN CALDWELL a thesis submitted for the degree of Doctor of Philosophy in the University of London June 1972 Department of Biochemistry St. Mary's Hospital Medical School London, W.2. 2 ABSTRACT 14 Using 0 labelled material, the metabolism of the sympathomimetic drug methamphetamine ((-0-2-meihylnaono-1-phenyl- u propane) has been investigated in the rat, guinea pig and man. Administered radioactivity was eliminated rapidly from all 3 species, between 50 and 80% of the dose appearing on the first day. In the rat and man this was found almost exclusively in the urine; in the guinea pig, at a dose of 45 mg/kg about 18% was in the faeces with 69% in the urine but with a dose of 10 mg/kg the excreted radioactivity was almost all in the urine. There are marked differences in the metabolism of methamphetamine between the three species examined. The rat metabolized the compound principally by N-demethylation and ring hydroxylation, while the guinea pig utilized side chain breakdown. In both species, at a dose of 45 mg/kg, sidechain hydroxylation was an important metabolic option, and it would seem that in the guinea pig the metabolism of methamphetamine varies with dose, sidechain hydroxylation being of significance at a high dose but deamination giving a precursor of benzylmethyl ketone being an important pathway at a lower dose (10mg/kg). The guinea pig produced an unknown metabolite at a dose of 45 mg/kg but not at the lower dose. The biliary excretion of amphetamine and methamphetamine in the rat was shown to occur, and the principal biliary compounds were the 0-glucur- onides of the ring hydroxylated metabolites. It was concluded that an appreciable enterohepatic circulation of the biliary metabolites of both drugs occurred in the rat. In man, the metabolism of methamphetamine was very different from that of amphetamine, ring hydroxylation being the major metabolic route and side chain breakdown being very small. Sidechain hydroxylation of both amphetamine and methamphetamine was shown to a minor extent. The results of the metabolic studies have been employed in the construction of an hypothesis to explain the developmentof tolerance to methamphetamine. 3 FOREWORD The work described in this thesis was carried out between October 1969 and January 1972 in the Biochemistry Department of St. Mary's Hospital Medical School. I wish to thank Professor R.T. Williams, F.R.S., for giving me the opportunity to work in his department, and for the great interest he has shown throughout this study. His vast knowledge and experience have been drawn upon extensively, and I am most grateful. I owe a great debt to Dr. Graham Dring, whose practical advice, helpful discussions and personal involvement with this project have acted as invaluable stimulus to me. The other members of the department have been of great help to me in discussion and with specialized techniques at different times. I must mention in this regard Dr. Peter Millburn for his aid with the work described in, and in the preparation of, Chapter 4. I wish to thank Mr. Audas and his technical staff for their assist- ance, and especially Harold Downer for his help with the preparation of biliary cannulated rats. I extend my gratitude to Miss Rhylva Offer for the typing of this thesis, and to Miss Maria Rees for her aid with the preparation of the illustrations. I can never adequately recompense my parents for the constant encouragement and great sacrifices they have made on my behalf throughout my education. I hope that the result in some way matches up to their aspirations Finally, and most importantly, I must thank my wife, Jill, for her patience, encouragement and kindness, which has meant so much to me during my work for my first degree and at St. Mary's. I trust that she, too, finds in the result something of my gratitude to her. 4 INDEX Chapter One : Introduction 5 Chapter Two : Materials and Methods 45 Chapter Three : The Metabolism of Methamphetamine in the Rat and the Guinea Pig 61 Chapter Four : The binary Excretion of Amphetamine and Methamphetamine in the Rat 96 Chapter Five : The Metabolism of Methamphetamine and Amphetamine in Human Volunteers 112 Chapter Six Discussion 127 Appendix Organic Mass Spectroscopy . 151 Bibliography : 158 5 CHAPTER ONE INTRODUCTION 6 Chapter One Introduction The Amphetamines - an historical perseective 8 The Physiology of the Adrenergic Nerve Ending 10 The Concept of the Chemical Neurotransmitter 10 The Identity of the Sympathetic Transmitter 11 Biosynthesis of Noradrenaline 11 Control of Noradrenaline Synthesis 12 Storage of the synthesised Noradrenaline 15 Release of Noradrenaline 15 Action of Noradrenaline 16 Inactivation of the Released Noradrenaline 16 (i) Metabolic Degradation 16 (ii)Uptake of Noradrenaline into the Nerve Endings 17 The Mode of Action of MethaTphetamine 21 (a) Monosmine Oxidase Inhibition 21 (b) Liberation of Noradrenaline 21 (c) Direct Action on Noradrenaline Receptors 21 (d) Inhibition of Uptake 22 7 Pharmacology 23 Peripheral Actions 23 Central Actions 24 Therapeutic Uses 25 Peripheral Actions 25 Central Actions 25 Metabolism of the Amphetamines 26 Amphetamine 26 Aromatic Hydroxylation of Amphetamine 31 Ephedrine 33 Ring Substituted Amphetamines 33 Sidechain Methylated Amphetamines 37 N-Alkylated Amphetamines 39 N.I.H. Shift 41 Interactions between Amphetamine and Other Drugs 42 Scope of the present work 43 8 The Amphetamines - an historical perspective Around the year 1900, there was great interest in the world of physiology in the workings of the autonomic nervous system, eventually leading to the identification of the endogenous transmitters, noradrenaline in the sympathetic and acetylcholine in the parasympathetic system. However, long before the conclusive identification of the transmitters, the pharmacological importance of these compounds had been realised and exploited. Oliver and Schaefer (1895) demonstrated the pressor effects of suprarenal extracts, and Abel and Crawford (1897) isolated the active principle and termed it epinephrine (adrenaline). After some controversy, C R 0 N, Jowett (1904) was able to confirm that the elemental formula was 9 13 3 and proposed the structure to be: HO HO CH-CH -NRCH 2 3 OH This structure was confirmed by synthesis in the work of Stolz (1904) and Dakin (1905). The potential medicinal value of this and related compounds must soon have been realised, for the literature quickly contained many synthetic methods for these materials. It will be noted that the carbon bearing the alcoholic hydroxyl group is an asymmetric centre, and it was found that the synthetic adrenaline (which was racemic) had a lesser physiological activity than the natural suprarenal extracts, known to be laevo rotatory. Cushny (1908,1909) showed that natural (-)-adrenaline had twice the pressor activity of synthetic (+)-adrenaline, and that the (4)-form had only one-twelfth of the activity of the (-)-isomer. The finding that one optical isomer has a greater biological activity than another has often been repeated in work using optically active compounds of this type. 9 As a result of the great number of structural variations on the theme of adrenaline carried out since 1905, in 1910 Barger and Dale were able to publish their classical paper relating chemical structure to the biological activity of this type of amine. They coined the term sympathomimetic, meaning "an action simulating that of true sympathetic nervous systems", and made general conclusions about the structure-activity re- lationships of sympathomimetic amines. The structure for optimal activity has a benzene ring, with a two carbon sidechain, with a terminal primary or secondary amino group. Activity is enhanced by two phenolic hydroxyl groups at 3' and 4' on the ring, and by an alcoholic hydroxyl group on the first carbon of the sidechain. From this time onwards until the present day, structural analogues of,adrenaline have been investigated for potentially useful pharmacological activity. Ephedrine (2-methylamino -1 -phenylpropan -1 -01), an alkaloid from Ephedra species plants isolated by Nagai (1887), was found by Chen and Schmidt (1924) to have a weak but prolonged sympathomimetic action, and to be active orally. Ephedrine has two configurations, each of which can exist in a pair of optical isomers, and the most active form was (-) -ephedrine. The oral activity of this phenylisopropylamihe led' to investigation of other related compounds, and amphetamine (2 -amino -1.;phenylpropane), first synthesised by Edeleanu (1887) as a chemical exercise, was found to be a pressor agent by Pines's et al.(1930). Methamphetamine (2-methylamino -1-phenylpropane), which was synthesised by Schmidt (1914) as part of investigations into the structure of ephedrine, was also found to be a potent sympathomimetic by Hauschild (1938). The activity of these drugs when given orally is due to their resistance to monoamine oxidase, the enzyme which breaks down phenylethyl- amines (Blaschko et al ,1937). The work described in this thesis is principally concerned with methamphetamine, and in addition some work has been performed on amphetamine. This chapter, then, is a review of the physiology of the sympathetic nerve ending, the mode of action and pharmacology of methamphetamine and the metabolism of the amphetamines in general. 10 The Physiology of the Adrenergic Nerve Ending Methamphetamine is a sympathomimetic amine, and' as such has its action at the adrenergic nerve ending. To understand its mode of action it is first necessary to consider the physiology and biochemistry of the adrenergic terminal in some detail. The Concept of the Chemical Neurotransmitter After the isolation and synthesis of adrenaline in the decade 1895-1905, many workers, notably Lewandowsky (1898) and Langley (1901) observed the close similarity between the effects of sympathetic nerve stimulation and the physiological actions of injected adrenaline.

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