SOME BIOCHEMICAL ASPECTS of THALIDOMIDE TOXICITY By

Home , Oxanamide

SOME BIOCHEMICAL ASPECTS OF THALIDOMIDE TOXICITY

DAVID EDWARD HAGUE

Being a Thesis submitted

for the degree of

DOCTOR OF PHILOSOPHY

in the

University of London

January 1969 Department of Biochemistry St. Mary's Hospital Medical School, London I

Thalidomide should be the first drug to be analyzed and investigated beyond the empirical level. Teratologists, pharmaceutical industries, and governmental agencies must pursue this matter until it is understood why this drug, composed of two nontoxic substances, is so toxic and teratogenic to human embryos, but relatively harmless to embryos of other mammals. Until this differential effect is explained and understood, administration of drugs to women of reproductive age remains a precarious matter. One should have expected an outburst of activities along these lines, but,it . seems that after the initial excitement about the thalidomide catastrophe, society has returned to an indifference which is difficult to comprehend. One can imagine what a second thalidomide.like incident would do to the pharmaceutical industry, to responsible governmental agencies, and to medical science.

Report of the Commission on Drug Safety 1964. II

TO MY PARENTS III

ABSTRACT

Chapter I reviews the thalidomide episode, and studies carried out on its embryotoxio properties. One of the puzzling features of thalidomide is that its teratogenio, neurotoxic and sedative properties are species dependent. Chapter II reviews the problem of species variations in response to foreign compounds. The effect of thalidomide in the hamster was studied following its administration to the pregnant animal. Unlike other species previously studied. the drug was found not 14 to be teratogenic in the hamster. The fate of C -thalidomide following its administration to the pregnant hamster was investigated. The absorption and plasma levels of thalidomide. and the persistence and penetration of the teratogen in the embryo are described. and the results are compared to those found in the rabbit, a thalidomide sensitive species. No difference was observed, and it was concluded that the difference in teratogenicity must lie at the site of action of the compound. Chapters IV and V describe some physical and chemical properties of Dlo.thalidomide and its optical antipodes, and their acute toxicity in three strains of mice. The results show that the D. and L...isomers are toxic in both sexes of the three strains of mice whilst the racemate is not. A higher acute toxicity is also associated with the D6, isomer. A comparison of DL-thalidomide and its antipodes indicate that IV this difference in toxicity is related to differences in their physico-chemical properties, for although those of the isomers were identical, they differed markedly from the racemate. For example, the solubilities, molecular weights and rates of hydrolysis were different. A comparison of the rates of excretion, plasma levels and tissue distribution of the three compounds was made following the administration to mice of IBC-labelled DL-. D. and 1-thalidomide. The results are discussed. It was concluded that the racemate exists as a dimer in solution, and that its own toxicity may depend on its dissociation into the free isomers. Finally, in Chapter VII, the free amino acid pattern present in the rabbit embryo from the 6th to the 11th day of pregnancy is described, and the results compared to those found in the maternal plasma during this period. The effect thalidomide, has on this pattern following its administration to the pregnant rabbit is also described. Seventeen amino acids were found in the blastocyst at 6 days. From the 6th to the 8th day, the general pattern of the amino acids altered quite radically, with-large drops in the concentration of some amino acids, namely, threonine, serine, lysine, glycine. isoleucine, leuoine and tyrosine, and arginine could no longer be detected. The changes which occur in the amino acid pattern, and the concentration ratios of the individual amino acids observed in the embryo, yolk sac and maternal plasma are discussed. V

ACKNOWLEDGEMENTS

I wish to thank Professor R.T. Williams for his advice, support and direction throughout the period of my work. It gives me great pleasure to record my thanks to Dr. R.L. Smith for the general supervision of the research and whose personal interest and constant support were invaluable. I should also like to thank Dr. S. Fabro for his suggestions, Mr. Audas and his technical staff for assistance, Miss A. Pitman for typing this thesis, and Andree-for her patience. Finally, I wish to acknowledge the Association for the Aid of Cripple Children (New York) for their-financial support during this research.

CONTENTS Chapter Page 14o,.

I Introduction ...... • • • **0 1 II Species differences in thalidomide teratogenicity ...... 15

0** 16 Introduction Methods O00 33 Results and Discussion •. 35 III Fate of POThaiidomide in the pregnant

hamster ••• ••• ••• ... 38

Methods ••• *60 O** 39

ReSults • • • ••• MOO 44

Discussion ... •.. *410 50 . IV The chemistry of thalidomide and its optical isomers 56

Methods *06 ••. •,•• 61 Results • * • ••• • .. 62 Discussion * 0 . ••• *00 69 V Toxicity of thalidomide and its isomers in different species 73

Methods .••• ••• ••• 79 Results ••• 0•6 ••• 81 Discussion - ••• ••• ••• 90 VI Fate of [14C]-D-, L- and DL-thalidomide in mice 460 *** 000 00* 94 Methods ...... 95 Results and Discussion ... 104 VII Amino acids in embryogenesis ... 117 Methods ...... 127 Results ... ..• ... 133 Discussion ••• ••• 00* 144

Summary 147 References 154 CHAP TER

INTRODUCTION INTRODUCTION

Thalidomide, a-phthalimidoglutarimide, was synthesised by Kurz, Keller and Mtetker (1956). Its pharmacological action as a sedative-hypnotic with low toxicity was demonstrated in animals by Kunz et al. (1956) and later confirmed by Somers (1960), Osterloh and Lagler (1960) and Kuhn and Van Maanen (1961). It had a similar pharmacological activity in man (Jung, 1956) and its action, combined with its apparent lack of toxicity (Esser and Heinzler, 1956;. Stark, 1956; Blasiu, 1958; Salter, Lodge-Patch and Hare, 1959; Winzenried, 1961) made it more suitable as a sedative-hypnotic than other drugs, such as the barbiturates. The drug was widely used in Germany after its release on to the market in 1957, and because it was considered a 'safe' drug, it was obtainable without prescription. It was introduced into Britain in 1958 and later into many other countries, under a variety of trade names. In retrospect, it is perhaps fortunate that the drug became so popular, and so widely prescribed in such a short period of time. Otherwise the detection of the side effects of the drug may not have occurred and it could well have been in use at the present time. The first indication that thalidomide was toxic to humans appeared in various reports published between 1960-1961, - 2 - suggesting that the drug was the cause of peripheral neuritis when used over an extended period (Florence, 1960; Kuenssberg, Simpson and. Stanton. 1961; Fullerton and Kremer, 1961; Scheid, Wieck, Steamier, Kladetsky and Gibbels, 1961). It was Lenz (1961) who first related the increase in congenital malformations with the use of thalidomide. This observation was later confirmed by various other workers (McBride, 1961; Lenz, 1962a, b; Pfeiffer and Kosenow, 1962; Speirs, 1962).

Thalidomide as a Teratogen Table 1 lists the types of congenital abnormalities found in the offspring of women who had taken thalidomide. Although limb deformities were the most commonly observed defect, it can be seen that the drug was not specific with regards to the abnormalities that were manifest. From analysis of various cases where the period of intake of the drug was known, Lenz (1965) considers that man is sensitive to the teratogenic effects of the drug from the 35th to the 50th day of pregnancy, a period of 16 days. Women who took thalidomide outside this period gave birth to normal offspring (Lenz and Knapp, 1962). The sensitive period to thalidomide is during the morphogenio phase of development. Later work carried out on the rabbit (Fabro et al., 1967) confirms this, as it is only when taken during this period of development in the rabbit

3 TABLE 1., Congenital Malformations attributed to thalidomide in Man *

Systems affected Type of Malformation

Locomotor Amelia; phocomelia; micromelia; , oligodactylia; syndactylia; club-hand; club foot. Nervous Hydrocephalus; meningomyelocelel spina bifida. Cutaneous Haemangioma of the unpr lip, glabella and forehead. Ocular Microphthalmia; anophthalmia; coloboma. Auditory Deformed or absent pinna; atresia of the external canal; aplasia of the drum;, low set ears. Respiratory Choanal atresia; saddle nose; cleft palate; bilobular right lung. Cardiovascular Aortic hypoplasia; transposition of the great vessels; perviety of atrial or ventricular septum; stenosis of the pulmonary artery; abnormal pulmonary veins. Gastrointestinal Atresia of the oesophagus: pyloric stenosis: imperforate anus; absence of the gall bladder; atresia of the common bile duct. Urogenital Hypoplasia and aplasia of the kidney; horseshoe kidney; hypoplasia and aplasia of the ureter; atresia of the vagina; bieornate uterus; aplasia of the uterus; recto-vaginal fistula.

* Data compiled from Fabro (1966) - 4 - (8-11 day post coltum) that any malformations result. Similar work was carried out on other species, and wide variations in sensitivity were observed. These findings will be discussed more fully in the next chapter, where the problem of species differences is investigated.

Other Pharmacolosical Properties of Thalidomide Effect on the Central Nervous System: The sedative properties of thalidomide have been investigated by various workers. The administration of small doses in mice, rats and dogs results in a decrease in their activity and a relaxation of the skeletal muscles. However they retain the ability to react to external stimuli. Large doses in mice induce sleep, but they are easily aroused, and at no time do the `animals lose the righting reflex. Thalidomide increases the sedative effect of barbiturates in mice, prolonging the sleeping time of both hexobarbital or barbital. The sedative effect of thalidomide is depressed by stimulators of the central nervous system, such as amphetamine, pipradrol or caffeine (Kuhn? VanMaanen, 1961) and by methylamphetamine or methylphenidate (Somers, 1960). That thalidomide acts directly on the central nervous system is also indicated by experiments which demonstrate that the drug alters the E.E.G. pattern in man (Walkenhorst, 1957), guinea pig (Bergstram et al.. 1963)'and rabbit (Giurgea and Molyersoons, 1963). . 5 - Besides its effect as a sedative, thalidomide has been tested with a wide variety of drugs which act upon the nervous system to see whether any potentiation or depression of their activity occurs. Thalidomide was found to potentiate the action of chlorpromazine, oxanamide, phenobarbital, thiopentone, reserpine and ethanol in rats and mice; and to increase their toxicity (Osterloh, 1959; Somers, 1960; Kuhn and Van Naanen, 1961). Thalidomide is also reported to potentiate the analgesic effect of an aspirin-phenacetin-caffeine combination (Osterloh, 1959), whilst Somers (1960) demonstrated a slight enhancement in the analgesic effect Of morphine and pethidine in mice. Thalidomide was found to be ineffective against the convulsant activity of leptazol and strychnine (Somers, 1960; Fincato, 1957; Kunz et al., 1956). Similarly, the cardiovascular effects of acetylcholine, adrenaline and histamine were unaffected by the drug, and no response was observed when an isolated rabbit heart was perfused with a solution containing thalidomide (Somers, 1960).

Thalidomide as an immunosuppressive agent. There have been various reports in the literature either suggesting or discounting the ability of thalidomide to aid in tissue grafting by inhibiting in some way, the rejection process. Thalidomide was found to prolong the survival time of skin grafts in mice (Turk, Hellman and Duke, 1966; Mouzas, 1966) - 6 - and reduce the graft rejection in chicken embryo (Field et al.. 1966). This led to the suggestion by Hellman that thalidomide may not be a teratogen, but may act as an immune- suppressive agent during pregnancy, allowing the continued development and eventual birth of a malfOrmed foetus, which .would otherwise have been rejected by the mother. However. Playfair et al. (1963), Floersheim (1966). Bore and Soothorne (1966) and Jaffe (1966) failed to observe any immunosuppressive activity of thalidomide in mice. rabbit, and hen. Furthermore. this theory is not supported by evidence obtained.in rabbits (Fabro and Smith. 1966). where an increased incidence in resorptions, and hence rejection, is observed following the administration-of thalidomide during pregnancy.

Thalidomide as a Cancerostatic Agent. Because of the drugs activity on developing embryonic cells, various. workers have checked its therapeutic value on a variety of diseases involving cell' disturbance, Its value as a cancerostatic agent has been studied, but with little success. It .appears to have no effect on the majority of malignant growths (Roe and Mitohley, 1963; Kunz et al., 1956; Juret and Aubert, 1963; Di Paolo and Wenner, 1964; Bach, Bichel and Hejgaard, 1963). However. it did appear to inhibit the growth of a hormone dependent tumour in rat (Mater and More, 1966).

The Lepra Reaction. The drug has apparently been used - 7 - with some success as a therapeutic agent in leprosy, in that it significantly reduces the lepra reaction (Sheskin and Convit. 1966).

Biochemical Studies on Thalidomide as a Teratogen Enzyme studies. It is probable that thalidomide exerts its teratogenic effect by interfering with some biochemical aspect involved in the embryos development. About a dozen enzymes which are known to be important in normal cellular metabolism have been studied to see whether they are affected by thalidomide. Table 2 lists the enzymes studied and the effect thalidomide and its metabolites have upon their activity. It can be seen that thalidomide has no significant effect on any of the systems studied to account for its teratogenic action on the embryo.

Antimetabolite effects. The possible role of thalidomide as an antagonist in the utilisation of coenzymes or their precursors have been investigated. The experiments have usually involved the assessment of the effect of thalidomide on the growth of bacteria which are dependent on one or more of these metabolites (Rauen. 1963, 1964; NystrOm, 1963; Keberle et al., 1965). Thalidomide had no dramatic effect on any of the systems studied, and only slight inhibition in growth was detected in two systems, in Lactobacillus arabinosus, dependent on pantothenic acid (Rauen, 1963) and Lactobacillus TABLE 2. - THE EFFECT OF THALIDOMIDE AND ITS HZTABOLITES ON VARIOUS ENZYMES Enzyme Source Compounds Tested Effects References glutamio decarboxylase Mouse and rat brain I, III-IX, XII, V. moderate - Fabro et al. (1965); Hirschberg XIII competitive et al.-1M4) inhibitor glutamine synthetase Rat brain I-VIII VIII. moderate Fabro et al. (1965) inhibitor glutamic dehydrogenase Mouse brain. liver I, III-IX, XII, VI, VIII, IX. Fabro et al. (1965); Hirschberg & embryo; rat XIII XIII weak e al. (1964); Friedman et al. brain & liver inhibitors (1965) glutamic oxaloacetic transaninase Mouse brain, liver I None Hirschberg et al. (1964) & embryo lactic dehydrogenase Mouse brain& liver I None Hirschberg et al. (1964) glucose oxidase Pure I-V, VII, VIII None • Rauen (1963) r6pamino acid oxidase Pure & rat liver I-V, VII. VIII None Rauen (1963); Friedman et a1.(1965: L-amino acid oxidase Pure I-V, VII, VIII None Rauen (1963) Folic acid reductase Pure I-V, VII, VIII None Rauen (1963) Succinic dehydrogenase Rat liver I None Friedman 21.11. (1965) acid phosphatase Pupae of Tribolium I None Chaudhary & Lemonde (1964) confusom alkaline phosphatase Pupae of Tribolium I None Chaudhary & Lemonde (1964) 1 confusom co ‘talase Rat liver I Yu-Chan Lin et al. (1966)

- 9 -

KEY TQ TABLE 2

Compound No. Nomenclature Thalidomide ' II 4-Fhthalimidoglutaramic acid III 2-Fhthalimido6lutaramic acid IV a-(o-carboxybenzamido)-glutarimide V 2-Phthalimidoglutaric acid VI 4-(0-oarboxybenzamido)-glutaramio acid VII 2-(o-earboxybenzamido)-glutaramic acid VIII 2-(o-oarbotybenzamido)-glutaric acid IX Phthalic acid X a-amino glutarimide XI Isoglutamine XII Glutamine XIII Glutamic acid - 10 - mesenteroides, dependent on glutamate (Rauen and Keberle et al. 1965). Similar experiments in mammalian systems, where coenzymes are known to be essential for absorption of certain compounds, have all failed to demonstrate any antagonism by thalidomide, (Evered and Randall, 1963; Toivanen et al., 1964).

Metabolic effects. A wide spectrum of biological systems have been studied to determine the effect of thalidomide upon their metabolism (Table 3). It can be seen that thalidomide has no apparent effect on any of these systems. It is obviously difficult to draw any conclusions from the negative results obtained from these studies. However, it would appear that the drug is ineffective against the major routes of metabolism which are essential for the functioning of adult tissue. One would expect. that similar systems are working during embryonic development; however, during morphogenesis, metabolism is necessarily geared to differentiation, whioh may involve many important, yet secondary metabolic pathways to this procets, where thalidomide may act.

Embryo toxicity of Thalidomide Analogues. Various investigations have been carried out to assess the embryotoxiot- of chemical analogues of thalidomide. This would demonstrate the specificity of the original compound, and also that part of the molecule which is essential for the teratogenio properties. TABLE 3. THE EFFECT OF THALIDOMIDE AND ITS METABOLITES ON VARIOUS BIOCHEMICAL SYSTEMS Compounds System Tested- Effect References

RNA and DNA levels of regenerating rat liver I None Fabro (1965) Regeneration of rat liver I w Gershbeim (1965) Respiration of glucose and L-glutamate by rabbit foetus I if Fabro (1965) Hepatic glucosamine 6-phosphate synthesis I " Fabro (1965) Hepatic nicotinamide coenzyme levels I & IV " Narrod.and King (1963) Oxygen and glucose uptake and glycogen content of rat diaphragm I " Gershbeim (1965) Oxygen uptake by Tribolium confusom I w Chaudhary and Lemonde (1964) Respiration of pyruvate in Ehrlich tumor cells I w Di Paolo and Wenner (1964) - 12 - Table 4 shows some of the compounds, related to, thalidomide that have been tested for teratogenic activity. It would appear that the N-substituted phthalimido moiety is important for teratogenic activity. Furthermore, whilst substitution on the aromatic ring does not abolish the, activity, replacement of the phthalimido group by eyelohexane-1,2-dicarboximido and a suceinimido moiety does. The group attached to the phthalimido moiety is not so specific. Although the 3t-glutarimido group on the phthalimido ring is the most effective teratogen, replacement of this group does not result in complete loss in activity. Furthermore, Schumacher (1967) claims that N-phthalimido- phthalimide is equally effective. Thus, the glutarimide ring,.onee thought to be the prime cause of the teratogenic activity of thalidomide, because of its relation to the biological substrate glutamine, appears to be unnecessary' for eibryotoxieitY.

Mechanism of Action of Thalidomide. It can be seen that much information concerning the metabolism, toxicity and action of thalidomide as a teratogen has been accumulated. Despite this, the mechanism of embryotoxieity remains obscure. Most biochemical studies have been concerned with metabolic routes operating in adult tissue. This is hardly surprising as little is known about the biochemistry of embryogenesis, and this is especially true of the early stages, such as TABLE 4. EMBRYOTOXIC ACTIVITY OF SOME COMPOUNDS RELATED TO THALIDOMIDE

.Animal References Compound treated Effect

N-butylphthalimide Rat Embryotoxio Bignatiet al. (1962. 1963)

4-phthalimidoglutarimide ) Rat Enbryotoxic Misiti et al. (1963) 3-phthalimido(aspartimide)) Chicken Teratogenic if a-N-(3-hydroxyphthaloyl)glutamine Chicken Teratogenic Boylen et al. (1963. 1964) N-methyl thalidomide Rat and Not teratogenic Lechat et al. (1964) Rabbit 4-Nitrothalidomide Rabbit Teratogenic Fabro et al. (1965) 4.4minothalidomide' Rabbit Teratogenic It II Hexahydrothalidomide Rabbit Not teratogenic if a-Suceintuidoglutarimide Rabbit Not teratogenic to Phthalimidophthalimtde Rabbit Teratogenic Schumacher et al. (1967) - 14.. morphogenesis, when thalidomide'is known to act. One can hardly blame the biochemist, as work on adult tissue poses problems enough, without the added complication of size and differentiation which one has to overcome in any work involving embryonic development. Past failures using adult systems for studying the action of thalidomide have served to illuminate our present problem. If we hope to find an answer concerning the mechanism of action of thalidomide, we must first know. something about the biochemical environment of the embryo during its development. CHAPTER II

SPECIES DIFFERENCES IN

THALIDOMIDE ' TERATOGENI CI TY -15-

(1) Introduction (2) 'Factors determining species differences in the toxicity of foreign compounds '(a) Absorption (b) Intestinal flora (o) Metabolism of foreign compounds (d)Excretion (e)Biliary-exeretion (f)Idiosyncrasies (3) Teratogenicity (4) Investigations of the teratogenicity of thalidomide in the pregnant hamster (a)Materials and Methods (b)Results (o) Discussion -16-

There has been an increasing reliance in recent years on the use of foreign compounds, such as drugs, food additives and preservatives, etc., both in man and other domestic species. This trend will undoubtedly continue into the foreseeable future. Their use has aroused a growing awareness of their possible harmful properties, and increasing emphasis has been placed on the necessity of ensuring that no undesired side effects result. Man in particular is subject to a whole spectrum of these. foreign compounds, and in the present state of civilisation, avoiding them is impossible. There is of course a control on the use of compounds which are toxic to man. However, the thalidomide tragedy showed that, rigid though the controls may be, our lack of knowledge of the mechanisms involved in toxicity make them inadequate. Unfortunately, man is in a predicament. The use of human slaves and prisoners to prove the efficacy of poisons as in past centuries has rightly been prohibited. As the human subject is ineligible to assess the possible harmful properties of a potentially useful compound, we have to rely on laboratory animals. The problem is that species are frequently found to respond differently to any given compound, 'often as a result of a difference in the fate (such as the metabolism) of the drug, following its administration. - 17 - Examples of species differences in toxicity are shown in Table 5. No definite conclusions can be drawn by merely observing whether a particular species is reactive or refractory to a particular drug. However, if we can determine the biochemical basis for the difference, we may be able to forecast the 'unexpected' reaction. Most of the factors which influence the response of an animal to a foreign compound fall into four categories:- absorption; rate and route of metabolism; rate and route of excretion; susceptibility of the animal to the compound or its metabolites.

Absorption Absorption is essential for any drug which acts within the body. and the degree and rate of absorption is often an index of its efficacy. The absorbing membrane, usually the intestine is not equally pervious to foreign compounds, but acts as if it were an inert lipoprotein barrier, permeable to the more liposoluble molecules. The less ionised the molecule, the more liposoluble. Thus, Shanker (1958) and Austin (1967) have found that the ionic state of a compound affects the rate of absorption. Shanker (1960) demonstrated that the absorption of weakly or nonionised moieties occurred more readily than strongly ionised compounds, and molecules which were completely ionised, such as quaternary ammonium compounds and sulphonic acids were poorly absorbed. TABLE 5,. EXAMPLES OF SPECIES DIFFERENCES IN TOXICITY,

.TYpe of Toxicity Toxic Agent Reactive Species Refractory Species References Central nervous Azauracil Man Mouse, dog, rat, Welch „et al. (1960) system monkey Pulmonary oedema a-Naphthylthiourea (A.N.T.U.) Rat, dog Monkey, chicken, Richter (1945) rabbit Pulmonary vaso- Norbimide 5.(a hydroxy -a-2-pyridylbenzyl) -7 -(a- Rat Cat, chicken, dog, Roszkowski (1965) constriction and 2.-pyridylbenzylidene) -5 -norbornene -2,3 - monkey, mouse respiratory dicarboximide. failure Carcinogenic Amino azotoluenes-2,4,-Methyl 4-amino Mouse Rat Crabtree (1949) toluene Hepatic lesion ChlorPromazine Man Rat Popper (1966) Necrosis of Penicillin Hamster, guinea Man, mouse, rabbit Haure et ai..(1943) adrenal gland. pig Schneierson-et al. (1956) Leueopenia Azauridine Dog Man Welch'(1965) Welch et al. (1960) 'Teratogenie Thalidomide Nan Hamster Hague et al. (1967) Fratta et al. (1965) Somers (1963) . 19 -

The degree of ionisation depends on the pK of the compound. For example, a compound with a low pK would tend to be less ionised in acid solution, and this would be expected to facilitate its absorption. As a large number of drugs are weakly ionised, the pH of the intestine must be a factor controlling their absorption. Smith (1965) has shown that the pH at any given area of the alimentary tract varies between species so a difference in the absorption of these compounds between species is to be expected. This could be important With regard to the overall effect of a drug between species, as the therapeutic value and/or toxicity of a compound is related to the plasma and tissue levels, which are directly dependent on the rate of absorption.

Intestinal Flora As the intestinal flora has been shown capable of metabolising constituents of the diet, it has to be taken into consideration when comparing species differences in response to foreign compounds. Smith (1965) examined the bacterial flora, and its relationship to other factors, such as pH, in 20 different species, ranging from chordata-monkey, rat, rabbit, mouse, birds, amphibians and fish, to members of the insect phylum. He demonstrated that the flora of the gut differ between species. Can these differences affect the final form of the drugs eventually absorbed? Gustafsson (1957) and Portman (1962) have found that

-20- species differences in the intestinal flora that metabolise oholic acid result in a species difference in its metabolites which are circulating in the blood. The aromatisation of quinic acid, almost certainly the result of intestinal metabolism, has been examined in 22 species of mammals (Cotran, 1960; Beer etal., 1951; Evans, 1968). Only in man, Old World monkeys and possibly guinea pigs is this reaction observed. Thus man and monkey excrete up to 60% of an administered dose of quinic acid as hippuric acid.

CON HC t-te)C001-1

Quinie Acid Hippuric Acid

Products of metabolism by the intestinal flora may have an effect unlike, that of the original compound. Sometimes this metabolism results in the formation of a toxic product, such as the conversion of chloramphenicol (I) into goitrogenio aryl amines (II and III) (Glazko et al., 1949).

-21-

H a -C-C-C Hest

0=C-c II 1;11i i-p-aminopheny1-2-amino propane C-C-CH 014 1:3 diol. bH

I Chloramphenicol

OOH

III p-aminobenzole acid

Similarly, the intestinal degradation of amygdalin yields cyanide. Hence amygdalin (IV) is toxic when given orally but not when adminittered i.p.

/ CH -CHO 4 HCr4 4 (CILUCCS) 0 C(11-11000 C61-.11,06 IV

Similarly, azauridine (V) a chemotherapeutic agent in the treatment of psoriasis (Colabresi. et al., 1966) and mycosis fungoidee, a malignant skin disease (Zambia et al.., 1963) was also found to be metabolised in the intestine. with rather

4. 22w drastic results. Not well absorbed in man (only about 30% as compared to its excellent absorption in mouse and dog) it is converted by microorganisms of the lower bowel into azauraeil (VI), the free base, which is readily absorbed and causes severe c.n.s. effects (Welch et al., 1960; Sehnider, et al., 1960; Wells et al., 1957). C)

N N RIBOSE 1

Ki aS tbrr.

V VI

Metabolism of Foreign Compounds After absorption the drug comes into contact with the *drug metabolising enzymes'. Their presence is well substantiated; whether they have developed for the metabolism of 'usual' biological material or specifically for 'foreign' compounds is debatable. The general routes of their metabolism has been expounded by Williams (1959). This envisages a two phase reaction; phase 1 being asynthetic. usually oxidation, reduction or hydrolysis; phase 2 is a synthetic pathway giving a conjugated product. The majority of these reactions can be carried out by all

23 - species so far studied. However, due to factors such as enzyme concentration, enzyme inhibitors, reversible reactions and competition between enzymes for the same substrate, the extent of a particular product formed can vary enormously from species to species. The rate of the above reactions normally determine the duration of a drugs action. For example, the enzyme formation of conjugates usually results in the loss of the drugs activity and its subsequent removal from the body. Hence, the ability to form these compounds can determine the efficacy of a drug. Table 6 summarises the major conjugation reactions found, and also some of the minor ones which are found to occur in only a few species. If one considers that many foreign compounds have more than one position at which conjugation can occur, coupled with the number of compounds with which they can conjugate, there is the possibility of enormous variation between species. Metabolism of foreign compounds does not always lead to its inactivation. Some metabolites have a pharmacological activity similar to, and even greater than the parent compound. One example is P-hydroxyacetanilide (VIII), a metabolite of phenacetin (VII), which has analgesic properties similar to the parent compound (Conney et al., 1966).

111 TABLE 6. COMPOUND'S USED IN CONJUGATION *

Main Source Distribution of Compounds or Groups Involved of Conjugating,,_ in ConluRation agent General Special or Rare

Carbohydrate . GlneUronic acid Glucose (insects) N.aoetyl glucosamine (rabbits) Ribose (rats and mice)

Amino Acids 'Glyeine Glutamine (man) Glutathione (eysteine) Ornithine (birds) Methionine (CH3) ' Arginine. Agmattne (ticks and spiders) Glycyltaurine ,(eats) Glycylglyeine (cats) Serine (rats and rabbit

Miscellaneous Acetyl Formyl (rats and dogs) Sulphate Phosphate (dogs and Thio (S. group) insects)

* From R.T. Williams (1967) - 25 - Codeine is partly metabolised to morphine, both compounds being potent analgesics (Adler, 1952; Mannering et al., 1954). Sometimes the pharmacological response is not due to the administered compound, but to one of its metabolites. Thus, it is the metabolite of prontosill sulphanilamide which is responsible for its antibacterial activity (Tre;fonS1 et al., 1935). Lucanthone (IX), a schistosomicidal agent, is converted to the active drug hycanthone (X) in man (Rost et al., 1967a).

kitqcti), NI (Ca 111 toiCcH)m

clipcm

IX X

The reason for the schistosomicidal action of mirasan (XI) in mouse, not reproducable in man and monkey (Gannert. 1961) is thought to be due to its conversion to the active metabolite the former, but not the latter Masi et al., 1967a).

Ce_ CQ

1 INt (CI (ct-t;.)4 N LC,j4s)2 XI - 26 -

The antibacterial action of 21 2-diohloro-N-0-hydroxy- a-(hydroxymethyl)-p(methylsulphiny1)-phenethyl3acetamide (XIII) can be mainly attributed to two products of its metabolism, the sulphide and sulphone (XIV and XV) (Rosi et al., 1965, 1967b).

C I-IO H 6\11100-OH

XIII R = CH380 R' = C12CHCO

XIV R = CH3S RI = C12CHCO sulphide

XV R = CH3S02 = C12CHCO sulphone

Excretion 'of Foreign Compounds Excretion is an important factor to be considered when comparing species difference in drug response. For example barbitone is excreted in dog to an extent of 95 per cent in 75 hr., 20 per cent in 24 hr. (Goldschmidt, 1957; Naynert, 1950); whilst less than 35 per cent is excreted in a week by chicken (Koppanyi, 1934). Thus the toxicity of this compound is far greater in the chicken. Similarly, the toxicity of methylglyoxal-bis-guanyl hydrazone is directly related to the rate of excretion (Oliviero et al., 1963). Urinary excretion depends on the polarity of the compound. - 27 - The highly polar molecules are rapidly excreted, whilst liposoluble ones are reabsorbed at the kidney tubules. The fate of the latter will thus depend on the rate and direction of metabolism to more'polar compounds. Metabolism is not the only factor governing the rate of excretion. Barbitone is not metabolised to any extent, but wide variations are found between species, in the ability to eliminate this compound. Conversely. the toxicity is not always related to the rate of excretion. The drug Abbot 16612, N-(3-ohlor6-4-methylpheny1)- N -(4-t-amylphenoxyhexamethylene)piperazine.hydrochloride, although excreted far more slowly in the monkey than the dog, is far more toxic in the latter (Miller et al.. 1966).

Biliary Excretion Some forms of excretion. such as the removal of volatile gases via the lungs. and the passive removal of material via the faeces have little bearing on species differences in drug response. The bile is also a route for the elimination of foreign compounds and must be taken into account when investigating species differences. The importance of this route of excretion has not yet been fully evaluated. but certainly such compounds as stilboestrol and certain sulphonamides are extensively excreted via the bile (Smith. 1966). Studies so far carried out (Williams et al., 1965a. b; Abou-El•-Makarem et al., 1966) have demonstrated differences between species, but suggest that they are predictable. Thus. - 28 - rats are good, whilst rabbits are poor biliary excretors. This factor will obviously complicate the response of any drug extensively excreted in the bile. A large part of the drug administered would thus be expected to be eliminated via the bile of 'good' biliary excretors such as the rat. There is then the possibility of the compound being further metabolised by bacteria in the intestine and being absorbed or undergoing continual absorption and elimination in the enterohepatic circulation. Either could be a factor in difference in drug response between a good and poor biliary excretor.

Idiosyncrasies in Drug Response Drug toxicity is further complicated by variations in drug response within a species. This is especially pertinent with regards to the use of drugs in man. For example, the extreme reaction to suceinylcholine and other local anaesthetics, a defect seen in 1-2% of the population, is due to the absence of plasma pseudocholinesterase (Harris and Whittaker, 1962; Lehmann and Liddell, 1964; Kalow, 1959, 1965). The toxic effects seen in about half the white population following the administration of isoniazid is the result of differences in the acetyltransferase in the human liver (Evans, 1965). A deficiency of glucose-6-phosphate dehydrogenase results - 29 - in haemolysis following the administration of a number of drugs, such as nitrofurantoin, aminosalicylic acid and sodium sulphoxone (Beutler, 1964). These strain differences have a genetic basis, and the continued use of new drugs will undoubtedly uncover similar idiosyncrasies.

Teratogenicity Teratogenicity is a very important aspect of toxicology. The thalidomide episode has served to focus our attention on the problem. With the continued increase in the use of drugs and other foreign compounds in humans and also domestic species, the danger of repeating the thalidomide tragedy becomes more acute. Trials to investigate teratogenic hazard are hampered by the universal problem of toxicological evaluation, that of species difference in response. Table 7 shows the teratogenic effects of thalidomide in different species. Following the demonstration of its effect in man, its teratogenicity was demonstrated in rabbit (Felisati, 1962; Giroud et al., 1962; Seller, 1962; Somers, 1962; Spencer, 1962), rat (Bignami et l., 1962, 1964). mouse (Giroud et al., 1962; Di Paolo, 1963), chicken (Kemper, 1962; Boylen et al., 1963; Ehmann, 1963; Yang et al., 1963). However, it was not teratogenic in the hamster (Somers, 1963; Fratta et al., 1965). It is difficult to explain these differences as the mechanism of teratogenicity itself is not understood. It is to be expected that the foetus would react differently than the -30-

TABLE 7. Comparative Teratopnicity of Thalidomide - mg/ks/day

"Positive EffeetNegative Effect Species (smallest dose)(largest dose) References Man 0.5 ... 1.0 - Mellin ,et al. (1962) Monkey 10 50 Delahunt et al. (1964) (a) (b) Rabbit 2..5 25 (a)Somers (1962) (b)Seller.(1962) Mouse 31(a) 4000(b) (a)Giroud et al. (1962) (b)Somers (1963) Meuse (1963) (b) Rat 10(a) 4000 (a)King et al. (1962) (b)Brent (196 Armadillo 100 . Marin-Padilla et al. . (1963) Dog 100 'Weidman et al. (1963) Hamster 8000 Somers (1963) Cat - 500 Somers (1963) Chick 0.2 - 1.0 500 Jurand (1963) - 31 - adult to drugs as the former undergoes the delicately controlled process of differentiation. Also, the foetus and the new-born have been shown to lack certain drug metabolising enzymes possessed by the adult (Jondorf et al.. 1958; Kato et al., 1964; Hart et al., 1962; Foutes et al.. 1959). In mammalian foetal development there is a dependence upon the mother for nutritional requirement. Thus maternal metabolism, and the effect of the placenta, have to be taken into consideration. Different placental types are found which could well effect species response. The placenta may have some role in producing congenital malformations. For example, the azo dyes, such as trypan blue, are known to be teratogenic in some species (Gunsberg, 1958; Tuohmann-Luplessis et al., 1959; Beck et al., 1960; Wegner, 1961), and yet it does not reach the foetus, but accumulates in the placenta (Tuchmann- Duplessis, 1965). Other examples of species differences concerning susceptibility to teratogens have been observed. The purine analogue, azathioprine, given to pregnant rabbits causes 50% of'the off-springs to be malformed, whilst it is ineffective in rat and mouse under the same experimental conditions (Tuchmann- DuplesSis. 1965). Cortisone also, whilst producing a teratogenic response in some strains of mice, is without effect in rat. The need to test new drugs for possible toxic effects on the foetus is essential. All animals, and especially man, - 32 - cannot be tested with regards their teratogenic sensitivity to every new compound introduced into social use. We have to rely on the examination of a few selected animals to assess the compounds potential toxicity. However, investigation concerning toxicity are possible, even in nan. It is at this point that an assessment of a drug's potential effect on the foetus should be possible. By comparison of the fate of the compound in different species, any unexpected result, such as the production of an unusual metabolite or the delay in its excretion can be re.examined to assess their possible effect on foetal development. In this and the succeeding chapter, the apparent refractory behaviour of the hamster during embryogenesis was examined. The fate of the drug following its administration to the pregnant hamster was investigated in the hope of finding a reason for its insensitivity to thalidomide. - 33 -

MATERIALS AND METHODS

Thalidomide m.p. 2710C was a gift of Lilly Researchl Laboratories Ltd.

Animals Pregnant European golden hamsters (random breed; 7 years .closed colony 100-150 g.) were purchased (A.F. Longmoor, Sherrard Road, London E.11). They were mated at the breeding centre at'night (11 p.m.,. 2 a.m.) and were sent to our. Animal Department the following day. They were kept in individual cages.and were maintained on diet 41B (E. Dixon & Son, Ware) with water ad lib. For the teratogenic testing thalidomide was administered orally daily, as a suspension (in 0.5% oarboxymethyl cellulose) at dose levels of 1 g. and 2 g./kg. to groups of pregnant hamsters from the beginning of the 4th day until the 12th day inclusive of gestation. Controls received 1 ml. of 0.5% C.M.C.' orally, On the 15th day of gestation, the hamsters were anaesthetised, the uterus exposed, and the number of implantations, resorptions, and viable foetuses, both normal and. abnormal, were counted. The position of the various implantations and resorption sites relative to the uterus were noted. The size and nature of the resorptions, were examined in order to determine approximately the age at which the 'development of the foetus was arrested. The viable foetuses . 34 - were washed of amniotic fluid and the umbilical cord was removed. They were then weighed and examined for external malformations, then fixed in 95 ethanol. After one week, the foetuses were dissected and examined for internal malformations. The skeletons were stained with alizarin according to the method of Chaube (1965). - 35 -

RESULTS AND DISCUSSION

Effect of Thalidomide on the Hamster Pregnancy Table 8 shows that the average number of implantation sites, the litter size, the mean body weight of the 15-day old foetus, and the incidence of resorptions' and malformed foetuses was not affected by the daily oral administration of thalidomide (1 and 2 g./kg.) to pregnant European Golden Hamsters treated daily on days 4-11 of pregnancy. These findings are in accord with those found by Somers (1963) who administered doses of up to 8,000 mg/kg. throughout pregnancy without causing a reduction in litter size, malformations of the young or a significant increase in resorptions. Similar results have been found by Fratta et al. (1965) and by Homburger et 1. (1965) in certain random breed strains of hamster.

Discussion The results presented above demonstrate that in this strain of. hamster, thalidomide does not affect development. How oan we account for this species difference between the hamster and that of rabbits and other species, where thalidomide is such a potent teratogen? The discrepancy could be due to one of the first three factors which were discussed earlier, i.e. absorption, maternal and foetal metabolism, and/or excretion pattern of the drug. If these three possible variants do in fact prove to coincide in hamstei- . TABLE 8. EFFECT OF THALIDOMIDE IN PREGNANT HAMSTERS

No. of Total No..of Average No. of Average Mean foetal Normal Malformed Treatment Animals implantations. implantations Resorptions litter:112e SD body wt. foetuses foetuses + SD (g _. SD)

None 44 370 8.4 ± 3.0 62 7.0 ± 2.4 1.8 ± 0.4 208 0 (16.7%)

Thalidomide (lg/kg) '24 '224 ' 9.3' + 2.7 30 8.1 ±3.2 1.7 ± 0.3 194 0 (13%)

Thalidomide (2g/kg) 22 162 7.4 ± 3.1 26 8.0 ±.2.5 2.0 ± 0.5 176 0 . , (16%)

Thalidomide was orally administered on days 4-12 inclusive of pregnancy. Animals were killed on the 15th day of pregnancy. Foetuses were examined for external and- internal malformations. - 37 - with that of the rabbit, then the difference in the drug's action must be due to a fundamental difference at the point at which the drug acts as a teratogen. CHAPTER III

FATE OF [14C] THALIDOMIDE IN

THE PREGNANT HAMSTER - 38 -

INTROWCTION

In the preceding chapter it was shown that the embryo of the European golden hamster is insensitive to the teratogenie effect of thalidomide. This could be due to a difference in the pharmacodynamic properties of the drug in different species. For example* a poor absorption following its oral administration could result in an insufficient concentration at the site of action. . A rapid, excretion of the drug would have a similar effect. Thalidomide may not be capable of penetrating into the embryo of the hamster'during morphogenesis* or the penetration which occurs may be insufficient to bring about a teratogenie response. An answer to these problems may explain the species differences in the teratogenie response to thalidomide. For this purpose, the absorption, plasma levels and excretion of 14C-thalidomide, and also the penetration and persistanee of the compound in the embryo have been studied in the pregnant hamster, and the results compared with those found in the pregnant rabbit. -39-

MATERIALS AND METHODS

Materials 14 arbonyl- (m.p. 2700, specific activity C 14 0.75 4c/mg) was synthesised from carbonyl- C phthalio 1 anhydride according to the method of Beckmann (1962). (1)-a-(o.carboxybenzamido)-glutarlmide, (±)-2. and 4. phthalamldo glutaramic acids, (±)-2-phthalimidoglutaric acid, (±)-2. and 4.(o-carboxybenzamido) glutaramie acids and 2.(o.carboxybenzamldo)-glutaric acid were prepared previously in this laboratory. Phthalleacld was purchased commercially,-

Animals and Treatment European golden hamsters were mated in the manner previously described in Chapter 2. They were allowed food and water ad lib. 14C-Thalidomlde (150 mg/kg; 20 µc/kg. body weight) was orally administered to the pregnant hamsters on the 204th hour of pregnancy. At this time of the pregnancy, morphogenesis is occurring and 16-20 pairs of somltes are present (Graves, 1945). Furthermore, this is the stage of development when thalidomide is known to be teratogento in other species. One group of three treated animals were housed individually in metabolism cages and urine and faeces were collected over 72 hours. The urine was allowed to run over an inverted round bottom flask into a beaker in such a - 4o - way as to allow its collection without any faecal contamination. Blood samples (0.4 ml.) were withdrawn by cardiac puncture into a heparinised syringe from a second group (of four animals) at 4, 8, 12 and 24 hours after dosing, and the plasma separated by centrifugation at 2000 rev./min. for 10 min. Three groups, each of 4 treated animals, were killed by exsanguination at 4, 12 and 24 hours respectively after dosing. The plasma was obtained as before. The embryos were removed, together with the uterine tissue and transferred to a beaker containing isopentane at -40°C. Embryos free of blood and uterine tissue were then obtained by peeling off the surrounding tissue, whilst the embryos and trophoblasts remained in the frozen state. The embryos were quickly washed in ice-cold saline, dried on blotting paper, weighed and homogenised in groups of three in a mixture of equal parts of methanol and dioxan and the volume adjusted to 10 ml. Portions (2 ml.) were transferred to counting vials containing a scintillation fluid consisting of a mixture of dioxan-ethylene glycol-methanol (88:2:10 by vol.) containing naphthalene (6%), 2,5-diphenyloxazole (0.4%). 1,4-bis- (5-phenyloxazoly1) benzene (0.02%) and 5% thixotropic gel powder (carb-o-sil). The urine was made up to 100 mis. volume with 1:1 methanol:dioxan and 1 ml. samples counted. The faeces were treated with glacial acetic acid (0.1 ml/g. wet material) homogenised in dioxan-methanol mixture as before and the final volume made up to 100 mis. Aliquots . 41 - (1 m1'0 were used to estimate the 14C; the 14C was counted using a Packard Triearb Liquid Scintillation Spectrometer (Model No. 3214): counting efficiency was measured by the twin-channel ratio method (Bush, 1963) or by internal standards.

Identification of 14C-compounds 14 The nature of C compounds in the excreta, maternal plasma and embryos was determined by a reverse isotope dilution technique after a preliminary chromatographic separation (Fabro, Smith and Williams, 1967). Pooled embryos (9-12) were homogenized, and then freeze- dried to a yellowish-red powder. The latter was then extracted three times with methanol (10 mis.) and the methanol extract, after centrifugation, was concentrated at 30° to 2 mis. using a rotary evaporator. Aliquots (0.1-0.2 ml.) of the extract were spotted onto Whatman No. 1 paper. Over this spot 0.1 ml. of a methanolic solution containing 100 :is. of each of the carrier compounds, namely, thalidomide, 2- and 4. phthalidomidoglutaramic acids, 2-phthalimido glutaric acid, a-(o-carboxybenzamido)glutaramide, 2- and 4-(o-carboxybenzamido)- glutaramie acids, 2-(o-carboxybenzamido)glutaric acid, and phthalic acid. The chromatograms were developed in two dimensions using two solvents: i)Butanol:acetic acid:water (10:l:saturated - solvent A) ii)Propanol:pyridinemater (7:7:6 - solvent B) (Schumacher et al., 1965). - 42 After drying, the chromatograms were examined under ultraviolet light before and after hydrazine treatment (Schumacher et al..1965). The compounds were identified by comparison of their values and their colour reaction under ultra-violet light (Table 9). Maternal plasma (4.m1s.) was similarly treated. Urine was applied directly to the paper. Areas of the paper chromatogram containing the compounds were individually cutout and transferred to counting vials containing 20 mis. of scintillation fluid. for estimation of 14C. The rest of the chromatogram was cut out into, sections and also counted as above. The total 14C on the chromatogram was estimated, corrected for background. and the 14C associated with each compound was expressed as a percentage 'of the'total 14C on the paper chromatogram. TABLE 9. CHROMATOGRAPHY OF THALIDOMIDE AND RELATED COMPOUNDS

RF values x 100 in Appearance of spot Compound solvent In ultraviolet light A ; i In ultraviolet light after hydrazine spray +

Thalidomide 86-91;65-70 )dark becoming green . greenish-blue 4-Phthalimidoglutaramie acid 45-49;53-58 )after 4-5 min. exposure greenish-blue 2-Phthalimidoglutaramie acid 35-39:45-51 dark greenish-blue a-(o-Carboxybenzamido)glutarimide- 36-39;39-42 purplish-black - greenish-blue (weak) 2-Phthalimidoglutaric acid 24.29;62.66 dark greenish-blue 4-(o-Carboxybenzamido)glutaramic acid 14-18;24-27 dark greenish-blue (weak) 2..(o...Carboxybenzamido)glutaramic acid 15-19;36.40 dark greenish-blue (weak) 2-(o-Carboxybenzamido)glutaric acid 16-20;43.47 dark greeniSh-blue (weak) Phthaltc acid 56-59;58-61 dark blue (very weak)

Two ways descending chromatography on Whatman No. 1 paper; the first solvent (A) consisted of pyridine; n-amyl alcohol:water (7:7:6 by vol.) and the second solvent (B) of n-butanol:glacial acetic acid (10:1 by vol.) saturated with water. Ultraviolet light (254 m4) from a Hanovia "Chromatolite" lamp; +chromatograms were sprayed with an ethanolic solution of hydrazine (5%) and then heated in an oven at 100° for 10 min. .44.

RESULTS Table 10 shows that the greater part of an oral dOse of thalidomide in pregnant hamsters is absorbed and excreted in the urine (84%) with only about 9% appearing in'the faeces in the 3 days following administration. Furthermore. the greater part of the dose excreted (68-78%) appears in the urine in the first 24 hours. The urinary 14 C at 24 hr. consists of thalidomide (3% of dose), a-(75-carboxybenzamido)- lautarimide (26%), 2- and 4-phthalamidoglutaramic acids (8%), 2-phthalimidoglutario acid (0.2%) and 2- and 4-(o-earboxybenzamido)glutaramic acids plus 2-(o-carboxybenzamido)glutaric acid (27%). These values are the means of three experiments. 14 Fig. 1 shows the C plasma levels at various times after oral 14 dose of C -thalidomide. It reaches a maximum at about 4 hours after dosing and then steadily declinest at 24 hours the level-is about 40% of that at 4 hours. It can be seen from Table 11 that radioactivity passes to the embryo and that 14 the relative concentration of C in the embryo and plasma .are about the same at 4, 12 and 24 hours after dosing. At 4 hours after dosing about 50% of the plasma 14C is thalidomide, the rest is largely a-(o-carboxybenzamido)glutarimide with small amounts of other hydrolysis products. At 12 hoUrs after dosing the plasma level of thalidomide has declined to about 30% of that at 4 hours and the rest consists of hydrolysis products. Similarly, at 4 hr. the embryo contains

- 45

14 TABLE 10. EXCRETION OF C BY PREGNANT HAMSTERS AFTER THE ORAL, ADMINISTRATION OF TP*C3THALIEOMIDE

% of 14C administered found in Hamster Faeces Total No. Urine 0-24 hr. 24-72 hr. 0-24 hr. 24-72 hr.

18 78.4 7.4 2.8 5.3 93.9

19 68.5 2.7 2.8 9.0 83.0

20 77.0 10.4 0 7.6 95.0

. _

14C-thalidomide (150 mg./kg: 20 40/kg) was administered orally to pregnant hamsters on the 204th hour of pregnancy. Cr\ 0 as 10 Disintegrations/min x J) Fig. 1 Plasma Clevelsafterasingleoraldoseof1 thalidomide (150mg/kg,20 14 Time afterDosing(hr.) 8

la -46- 4 c/kg) toPregnantHamsters

14 7 - 47 - 14 TABLE 11. DISTRIBUTION OF C IN THE MATERNAL PLASMA AND

EMBRYO AFTER THE ORAL ADMINISTRATION OF

c14C)THALIEOMIDE TO PREGNANT HAMSTERS t140Thalidomide (150 mg./kg; 20 4C/kg.) was administered orally to groups of pregnant hamsters on the 204th hour of pregnancy and they were killed at the times shown.

Time disintegration/Min./g. after No. of 1 I dosing animals plasma embryo (hr.)

4 4 10,176 11.363 (8532-14820) (4992-19980)

12 4 6,762 7,741 (5956-6044) (6213-7992)

24 4 3.190 2.921 (2850-3624) (2679-3079) - 48 - mainly thalidomide and a7(o-carboxybenzamido)-glutarimide but at 12 hr. the level of thalidomide has declined to 20% of that at 4 hr.. and the rest of the 14C consists of seven hydrolysis products (Table 12). Thalidomide has thus been demonstrated to be capable of penetrating the embryo during morphogenesis. . 49 - TABLE 12. CONCENTRATIONS OF THALIDOMIDE AND ITS METABOLITES IN THE MATERNAL PLASMA AND EMBRYOS AFTER THE 14 ADMINISTRATION OF CJTHALIEOMIDE TO THE PREGNANT HAMSTER

Concentration (peg.) in Compound plasma embryo • 4 hr. 12 hr. 4 hr. 12 hr.

Thalidomide 17.2 5.7 19.1 4.4 a-(o-carboxybenzamido)glutarimide 14.1 8.3 15.0 7.2

2- and 4-phthalimidoglutaramie acids 1.3 4.4 0.1 2.5

2-phthalimidoglutaric acid 0.1 0.1 0.1 0.1

2- and 4-(o-carboxybenzamido)- glutaramie acid + 2-(o-carboxybenzamido)glutaric'acid 0.8 2.2 ' 0.1 10.8

Values are the means of three experiments. - 50 -

DISCUSSION

In the preceding chapter it was found that thalidomide was not teratogenic in the strain of hamster used, At dose levels far in excess of those found to be toxic to the embryo in other species, no malformed foetuses, nor even an increase in resorptions or decrease in foetal body weight was observed. This is a striking contrast to the effect observed in rabbits. When rabbits are treated with thalidomide, at a level of only 150 mg/kg., as opposed to 1 or 2 gms/kg. for the hamster, between 20 to 25% of the foetuses are malformed, the body weight of normal foetuses is down 10%, and nearly 50% of the implantations suffer resorption (Fabro and'Smith„ 1966). Can these results be explained by differences in the pharmacodynamio properties of the drug in the rabbit and hamster?

Excretion This difference is not explained by the absorption pattern of the drug found in the rabbit and hamster. Thus following administration of thalidomide to pregnant rabbits (150 mg/kg.), 75-80% of the dose appears in the urine within 24 hours (Fabro et al, 1967), a very similar finding to that in hamster (70-80%). This suggests that thalidomide is absorbed as easily by the hamster as by the rabbit. Furthermore, there . 51 . appears to be no greater tendency for the drug to be retained by the rabbit, as would be expected if a specific concentration of the compound over a period were required for the teratogenic action to manifest itself. The effectiveness of a given drug as regardt therapeutic and toxic effect is often directly related to its plasma concentration. One would also expect a similar relationship between teratogenic effect and yolk sac concentration. With thalidomide, however, this is not found to be the case. Following an oral dose of 150 mg/kg. in hamster* the plasma and yolk sac concentrations reach a level of 17.2 and 19,1 4g/ml. of thalidomide respectively (Table 13) after four hours. In the rabbit, the sensitive species, the maximum concentration is also reached at 4 hours, but is only 7.6 and 4.2 4g/ml. thalidomide (for the plasma and yolk sac respectively) following the administration of the equivalent dose (Fabro et al., 1967). Although there are no corresponding figures for the levels of thalidomide in the human embryo, the plasma level following a teratogenic dose of the drug (150 mg.) is only 1.5 µg/ml. It is unlikely that the concentration attained in the human embryo is higher than that observed in the plasma. Thus teratogenic activity is observed in the human at a dose level one tenth of that found in the hamster, an unreactive spebies. It is evident that the absorption of the drug across both the intestinal wall and placental membrane occurs as readily in the hamster as in the rabbit. Hence, the passive nature of . 52 . TABLE 13. CONCENTRATION OF THALIDOMIDE IN THE MATERNAL PLASMA AND EMBRYOS IN DIFFERENT SPECIES

Concentrations of Th alidomide (4g/m1) in Species Plasma Embryo 1 4 hrs. 12 hrs. 4 hrs. 12 hrs.

Man* 1.5 1.5 OPP

Rabbit 7.6 4.1 4.2 2.7

Hamster 17.2 5.7 19.1 4.4

* After an oral dose of 150 mg. After an oral dose of 150 mg./kg. - 53 - thalidomide in the pregnant hamster is not due to the failure of the drug to penetrate the embryo in sufficient concentration during its development. The placenta of the hamster (hemachorial) differs in type from that of the rabbit (hemoendothelial) and this may be relevant to the species difference observed. Certainly, in the case of other teratogens, such as the azo dyes, the teratogen itself does not have to cross into the yolk sac to exert its teratogenic effect (Gillman et al., 1948). Table 14 shows the relationship between radioactivity found in the embryo and that found in the maternal plasma of both rabbit and hamster, following the oral, administration (150 mg/kg.) of the labelled drug. It can be seen that after four hours, whilst the ratio, embryo:maternal plasma (e/m) is virtually unity in the hamster, it is only 0.5 in the rabbit, demonstrating that penetration of the embryo by thalidomide occurs more readily in hamster than in rabbit. After four hours, the ratio elm increases quite markedly in rabbit, and continues to do so for at least 24 hours. In hamster, on the other hand, there is a gradual rise up to 12 hours following administration, after which time the e/m ratio falls below unity. This would suggest that the ability to accumulate the compound by the embryo could be the reason why the drug is teratogenic in this species and not in hamster. However, the accumulation is due to the products of hydrolysis, and not to thalidomide itself. Furthermore, these . 54 TABLE 14. COMPARISON OF THE ACCUMULATION OF RADIOACTIVITY IN THE RABBIT AND HAMSTER EMBRYO AFTER THE ORAL 14 ADMINISTRATION OF. C.THALIDOMIDE

14C in embryo Time (hrs.) Ratio %11 after 4."C in maternal plasma treatment Rabbit * Hamster

4 0.54

12 1636

24 2.41

14C-thalidomide (150 mg/kg.) was administered orally to the pregnant animals on the 204th hour (hamsters) and 192nd hour (rabbits) after conception. The animals were killed at the time shown.

* Values from Fabro et al. (1965) - 55 - compounds have been shown to possess no teratogenic properties (Fabro et al., 1965; Smith et al., 1965; Kerberle et al.. 1965a, b). Thus, although this demonstrates a placental difference regarding the passage of the more polar hydrolysis products across the placental membrane of the rabbit and hamster, it does not in itself explain this difference in teratogenic effect between the two species. It was expected. because of the different placenta found in the two species (hemachorial in hamster and hemoendothelial in rabbit), and the difference in the stage of placental development (implantation occurs at L days in hamster, and between 7.8 days in rabbit) that the preceding experiments would have demonstrated differences in the distribution of thalidomide in the embryos to account for this remarkable contrast in teratogenie response. The results, however, have failed to illuminate such a difference, and so it must be concluded that this species difference in response to thalidomide lies within the biochemical processes involved in the development of the embryos. CHAPTER IV

THE CHEMISTRY OF THALIDOMIDE AND ITS

OPTICAL ISOMERS -56-

Thalidomide, a-phthalimidoglutarimide. is a relatively simple substance, systematically described as 1,3-dioxo-2- 0 • (2 .6 -dioxopiperidin-3 -yl)isoindoline. There is an • asymmetric carbon atom on the 3 - column of the glutarimide ring. so that thalidomide exists in three forms, the

4 (

Thalidomide optically active '(+)- and (-)- and optically inactive (±)... form (Fig. 2). Chemical Properties of Thalidomide The presence of four amide bonds. located at positions • 1-2 and 2-3 of the.phthalimido ring'and 1t-2f and ,l -6 of ,the glutarimide ring make3thalidomide particularly susceptible to nucleophilic attack. Hydrolysis of thalidomide Thalidomide is unstable in aqueous solution, and at pH values above 6 spontaneously hydrolyses at a rate that increases 57 i o ocLiir- ,t-reonlodols of the two o”tioal Isomers of thalidomide . 58 - with rise in pH (Schumacher et al.. 1965). There are twelve hydrolysis products (Fig. 3) as a result of secondary, tertiary and quaternary reactions following the initial primary hydrolysis. Reaction with aliphatic diamines Fabro et al. (1967) showed that thalidomide is very reactive with certain aliphatic diamines (Table 15). Thus. whilst thalidomide appears unreactive with simple diamines, and monoamines, it reacted rapidly with the naturally occurring diamines: spermidine, spermine, cadaverine and putresine. The reaction with putresine does not appear to be straight- forward acylation. Linnecar and Smith (1968) have isolated and characterized some of the reaction products. Of these, 1 2 1. the acylation product (N -4-aminobutyl-N -a-glutarimido phthalimide) and II, (N,Ng-bis[o-(a-glutarimido carbamoy1)- benzoyil- 1,4-diaminobutane) have been identified. Some Products of the reaction of thalidomide with putresine

CON11 .CON146-04 (\INC()

C) iJ Comi (CH04 NIL - 59 -

The Sp_ontancou,, Hvlcsis Thallion,ids•

6 0 7;4 o H (I) Thalidomide

„.N

COOH c COOH 0 N 0 o CONH2 6 COOH CONH 2 ( 2) 4 -Phthalimidoglutaramic acid (3) 2-Phtlialimidogluta ramie acid (4) a -( sz-ca rboxyben zo en;do) - gIuta rim'do

(6) 7) O COON COON

(5) 2-Phthalimido9lutaric acid

O e N. COOH NH COOK COOK CONH2 COOH COOH COON CONH2

(6) 4 -(2-ca rboxybonzamido) 2-( 2-ca rboxybcnzamido) (9) Phthalic acid gtuta ramie acid saute ram!c acid

NH2

0 1%.4 0 COOH COOH COOK

(8) 2-(2-carboxybcnzamido)- ( 10) a -arninog/uto rimide glutaric acid •

COOH CCNH CONH2 COOH 2 (I1) Isoglutamine ( 12) Glutamine

4 -

14 N (9) 2 (9) COON COOK ( 13) Glutam'e acid

(9)

• •

-6o-

TABLE 15, ACYLATING ACTIVITY OF THALIDOMIDE TOWARDS VARIOUS ALIPHATIC AMINES

Compounds incubated with 2. of thalidomide Thalidomide d isappearing in 2 hrs. at 370 n-Butylamine 0 n-Octadecylamine 0 Ethylenediamine 3 Putrescine 72 Cadaverine 27 Spermidine 100 Spermine 28 Ethanolamine 0 Ir-Aminobutyric acid 0

Thalidomide (4 x 10-5 m) and the compound under test 0 (10 m) were incubated together in absolute ethanol at 37 for periods up to 10 hrs. (spermine was tested at a concentration of 10-3 m). 61 Physical Properties of DL-, 106- and L-Thalidomide

Experimental Molecular Weights: These were determined by the rest camphor method and by the depression of freezing point of dioxan. Spectra: Infra red spectra were determined using a KBr disc and scanned on the Infracord spectrophotometer. The ultra-violet absorption spectra were measured in water and ethanol using a Unicam S.P.500 spectrophotometer. Solubilities: The solubilities were determined by shaking excess of the compound (at 180) with 50 mis. of the solvent. The solution was filtered, and portions of the filtrate were evaporated to dryness in a tared beaker. The weight of the dissolved thalidomide was then determined gravimetrically, and the solubility expressed as mg/100 mis. of solvent. The solubility in water was measured by taking portions of the saturated solution, suitably diluted, and the extinction at 220 mµ determined. The concentration of thalidomide was calculated from the optical density of the solution and its known extinction coefficient (Greene and Benson, 1961). Hydrolysis at pH 7.4: The rates of hydrolysis of solutions (6 µg/ml.) of DL-, D- and L-thalidomide in 0.2 m- Tris buffer, pH 7.4 at 30, 230 and 370 were determined speotrophotometrically as described by Schumacher et al. (1965). - 62 - RESULTS The main physical properties of DL-. ID- and L-thalidomide are given in Table 16.

Crystal form (±)- (+)-. and (.0 -thalidomide crystallized from aqueous dioxan gave irregular rectangular plate crystals; however. those of (t) thalidomide were much larger than those of the two isomers.

Melting points The melting point of the (1) form was 2710 and that of the (+) and (-) forms were 244° and 244-246° respectively. The melting point of the two isomers depended on the rate of heating. If the temperature was raised slowly the melting point occurred over a wide range because of extensive thermal. racemisation. Mixed melting point curves of the two isomers' showed that when the ratio of the (+)- and (-)- forms was then a racemic compound of m.p. 271° was formed; eutectic points at 242° occurred when the composition was 97 and 3 per cent.

Optical rotation The Ealio" for (-)-thalidamide agrees with the values given by Shealy et al. (1965). The specific rotation for the sample of (+) thalidomide is less than that quoted by Shealy. et-al. -63- TABLE 16. SOME PHYSICAL PROPERTIES OF DL-, Di- AND L- THALIDOMIDE

optical form m.p. 2710 244 244-246 Crystal form large irregular small irregular small irregular ,rectangular rectangular rectangular plates plates p fates D LgA wNo (2% in dilbthyl- formamide) +59 -66 Molecular weight East camphor method 555 284 273 Depression of 255 276 275 freezing point of dioxan Ultra-violet spectra max 221 221 221 221 50.6 51.7 49.6 Rates of hydrolysis 4 at: 3° 131 hrs. 116 hrs. 116 hrs. 23° 48 hrs. 25 hrs. 25 hrs. 37o 5i4hr. 5ihr. 5ihr. Solubility (mg/100m1) in: Water 6 34 34 Ether 6 45 45 Ethanol 16 145 145 Chloroform 270 1450 1400 Acetone 450 2500 2500 Dioxan 5000 8000 8000 n-hexane 1.2 1.6 1.7 - 64 - (1965) of +64° suggesting that it contains about 4>4; of the (-)- form.

Solubilities The compounds are seen to be poorly soluble in polar solvents such as water and ethanol but much more soluble in non-polar solvents. In five of the solvents, namely water, ether. ethanol, chloroform and acetone, the (+)- and (-)- forms were 5-9 times more soluble than the racemio form. However, in dioxan and n-hexOne the solubility of the three forms are similar. The marked difference in solubility of the (±)-, (+)- and (-)- forms is clearly shown if the (I) form is formed-in aqueous solution from the optical isomers. Thus, if'saturated aqueous solutions of (+)- and (-)-thalidomide are mixed, crystals of (t)-thalidomide separate (m.p. 2710) due to its lower solubility.

Molecular weights The molecular weights of the (+)- and (-)- forms determined in dioxan and camphor gave valUes close to that of the empirical weight of 258. In dioxan, the (±)- form also gave a similar value. However, in camphor, the latter gave a molecular weight of 555, more than twice that of the empirical value. suggesting that the compound exists as a dimerie molecule in this solvent. Spectra The ultra-violet spectrum of DL-thalidomide in water is shown in Fig. 4. The spectra of the two isomers are very similar. Two absorption peaks are exhibited in this region of the spectrum, at wavelengths of 221 mµ and 295 mil. At 221 mµE= 50,600, 51,700• and 49, 600 for DL-, D- and. L-thalidomide respectively. The infra red spectrum for the DL-form is seen in Fig. 4. Again, the spectra of the (+)- and (-)- forms are very similar. However, a difference is seen at 3400 (cm-1), where the shoulder is much more pronounced in the spectrum of the D- and L- than the DL-form. Differences in the spectrum of the D- and the L- with the DL- have also been observed by Shealy etal. (1965). When the compounds were recrystallised from D.W.F.-water, differences were observed at 3200 m,b and 3100 m,b (D- and L-) versus 3270 m,b and 3100 w,b.; 1730 sh., 1705 vs., 1690 sh. (D- and L-) versus 1710 v.s.; 850 w., and 830 w. (doublet) for D- and L- versus 850 w. (only) for the DL-. When the compounds were recrystallised from dioxan the spectra were more closely alike, the differences being the absence of the 830 cm-1 band and differences in the relative intensities of a few bands. (N.B. - m = medium; v.s. = very strong; w = weak; b = broad; sh = shoulder) WAVELENGTH (MICRONS) 5 6 7 8 9 10 11 12 13 14 15 I 100 . , 100 -. --- • . • - ' • . '

• • 80 • iihNtlifilTAMIMMIIIIMISTAINVO uJ • i . lanfif 60 . .. - 60 z : i ,. - _ - : : r :_. : , -LIE:, 40 • - • E ' ; ; :-. : - :.; ; ; ; -.;:q1d--_::. -;-_-- 7- --=-:4--.7--1=t_- 40 z - : - E E•.: -:::--- _ _ 7_7:- - , Vj ------,,,_-_-7-.-7,---1---i--_-_-L-- --;_._ 20 20 i . ; -t_ ; ; - . - - :-:•7 - t__ -_-:- 1 0 4000 3000 2000 1500 100p 900 800 700 0 WAVENUMBER (CM" )

(a) Infra-red spectrure of thalidom16e in,KBr (Perkin-Elner Infrccord Spectrophotometer)

tim to VA, UM WANKLINIU 6.),) (b) Ultra-violet spectrum of thalidomide (Unicom S.P.500) -67-

Rates of hydrolysis The rates of hydrolysis of (±)-, (+)- and (-)-thalidomide o at 3 and 23° are shown in Fig. 5. At 37o the rates of hydrolysis for all three compounds are found to be the same, the time taken for the hydrolysis of half the thalidomide (q) is ji hours'. A difference is seen in the rates of hydrolysis of the compounds at 3° and 23°. At both temperatures, the (+)-.and (-)- form "hydrolyse at the same rate. The (--)- form hydrolyses at a slower rate. This difference is more marked at 23°C, with t2 for D- and L- being 25 hours, and 48 hr. o for the racemate. At 3 4,ti is 116 hrs. for the D- and L- and 131 hrs. for the DL-. -68- FIG.5 RATE OF HYDROLYSIS OF DL-, D- AND L-THALIDOMIDE AT pH 7.4 (TRIS BUFFER) (a) At 3°C •

20 40 60 80 100 120 140 Time (Hrs.)

(b) At 23°C

t-40 0 X 20 O x x

lb 214, Time (Hrs.)

KEY: 0-0 D- and L-thalidomide DL-thalidomide - 69 - DISCUSSION Significant differences in the physical properties of the three compounds have been observed. In all experiments, the Li- and L-forms appeared very similar, but differed from the DL- form-. Thus, the melting point of the raoemate is 26° higher than the isomers. This suggests that the crystal lattice of the former is much more stable, as more energy is required to separate the molecules and give the liquid state. The solubilities of the compounds in various solvents also confirm this, indicating that the molecules of Di. and L- together have greater affinity for one another than molecules of the same configuration. Observation of the molecular weights in camphor suggest that the (t)- form can exist as a diner. This indicates the, formation of a relatively stable chemical bond between the (+)- and (-)- forms. This type of interaction would explain ,the differences observed in the rates•of hydrolysis of the DL- and the D6 and L-forms. The formation of a chemical compound consisting of one D- and one L- molecule would be expected to interfere with hydrolysis in either of two ways; by sterically hindering the approach of the hydroxyl ion, or by the chemical interaction strengthening or weakening the electron density at the carbonyl group, the point at which the hydroxyl ion attacks the thalidomide molecule. This would explain the differences in rate of hydrolysis . 70 - observed between the racemate and isomers at 30 and 230. However, at 370 the rates of hydrolysis of the three compounds are the same. This could be explained if,the dimer undergoes partial dissociation at this temperature such that the dimeric interaction is no longer a rate determining step in the hydrolysis. Dissociation of the diner would also explain why the molecular weight of the racemate, measured in dioxan, is the same as the empirical weight. Dioxan must be capable of breaking any chemical association of the (+)- and (-)- forms. This would account for the solubility of the (1)- form in dioxan being similar to those of the optical antipodes. The results indicate that an interaction between'the D- and isomers can occur, resulting in the formation. of a racemic compound in solution. Racemates definitely do exist in the solid state, as is demonstrated by the great difference in melting point between the racemic compounds and the enantimorphs. However, their existence in solution or in the fused state has been doubted. However, other evidence has appeared in the literature suggesting that, like thalidomide, some compounds do exist, at least partially associated as a racemic compound in solution. Brun). et al. (1902, 1904), determining the molecular weights of dimethyltartrate, dimethyldiaccetyltartrate, ammonium hydrogen tartrate and ethyl dl-dibromophenylpropionate, - 71 - concluded that the racemates are at least partly associated in solution. Byk (1904) has shown that solutions of copper tartrate and copper racemate differ in colour, the former being blue, the latter greenish-blue. Viscosity determinations carried out to demonstrate the presence of racemic compounds have yielded conflicting results. Thus, Beck (1904) was able to show the free existence of the racemic compounds of dimethyl tartrate and dimethyl diacetyl- tartrate by freezing point and cryoscopic measurements. but viscosity measurements failed to confirm these results. Thole (1908, 1910) investigated a number of active and inaotive compounds in solution. He found a slight divergence of the viscosity-concentration curve with active and inactive solutions of mandelic acid and amyl acetate, suggesting the existence of. the racemic compound in solution. and that the degree of association depended upon the concentration of the solution. However, Mitchell and Smith (1913) determined the surface tension of solutions of dimethyl-dl tartrate, and could find no evidence for the existence of the raoemate in the liquid state. As the solubility of thalidomide- in water is so low. the demonstration of association Of the (.1)- form as a racemic compound in solution is difficult to prove. Crtainly, the demonstration of the association of a taeemate in solution is an unusual observation. However, the evidence certainly - 72 - indicates that an association of this kind does occur with DL-thalidomide., CHAPTER V

TOXICITY OF THALIDOMIDE AND ITS ISOMERS IN DIFFEHENT SPECIES — 73 -

INTRODUCTION

Thalidomide is non-toxic to man and to various species of laboratory animals. Many cases have been reported which support this. Burley (1960) described 16 cases where doses of up to 4 g. caused no untoward effect. Nehaus and Ibe (1961) described a case where a male patient took over 14 g. of the drug. together with a large amount of alcohol, and survived. A 70 year old man took over 2 g. of thalidomide and suffered only prolonged sedation (De Souza, 1959). The toxicity has also been investigated in other species following oral, subcutaneous and intraperitoneal administration. These results (Table 17) show the drug to be non-toxic to all species studied. In mice, a single dose of 10 g./kg. was given without adverse effect; similar observations. at a lower dose level, were made in rat, guinea pig. hamster, dog and monkey.

Chronic toxicity 100-500 mg/kg. of thalidomide has been given to rats, mice, guinea pigs, hamsters and dogs over periods of 21-294 days without affecting body weight, tissue histology or plasma transaminase (Table 18). Some side-effects were observed, however. following chronic administration of the drug. In adult rats, there was a reduction in the colloid material of the thyroid glands (Somers, 1960); and in weanling rats, 0.5 % thalidomide in the diet led to a depression of growth and - 74 TABLE 17. ACUTE TOXICITY OF THALIDOMIDE IN VARIOUS SPECIES

Dose levels administered without signs of acute toxicity

Dose Species Level Route Reference F/kg. Mouse 5 oral Somers, 1960 4 i.p. Somers, 1960 5 s.c. Kunz, Keller & Mftokter, 1960 10 oral Fabro et al., 1967

Rat 2 oral Smith, 1968 1 i.p. Kuhne & Van Maanen, 1961

Guinea pig 0.65 oral Somers, 1968

Golden hamster 2 oral Fabro et al., 1967

Dog 1.5 oral Kunz et al., 1956 0.1 i.p. Kuhne & Van Maanen, 1961

Monkey 0.1 '1.p. Kuhne & Van Maanen, 1961 TABLE 18. CHRONIC TOXICITY STUDIES ON THALIDOMIDE

Species Dose mg/kg. Duration (days) Effects observed References 500 126 None Osterloh & Lagler., 1960 250 21 ) None — slight reduction 1000 21 ) in thyroid colloid Somers, 1960 material Rat 200 S.0 30 None Kunz et al., 1956 1000 39,182 None .Staemmler & Lagler, 1965 2000.4000 . 39 None -Staemmler-& Lagler, 1965 5000 294 None Staemmler & Lagler, 1965 , Mouse 50.100,200 84 None 'Osterloh & Lagler, 1960- 500 S.0 30 None Kunz et al., 1956'

100 S.0 30 - None Kunz et al., 1956 Guinea pig 1000,3000 46) 2000 ' 105) Loss of body weight Staemmler & Lagler, 1965.

Golden hamster 1000 70. None Staemmler & Lagler,1965 100 S.0 30 None Kunz et al., 1956 Rabbit 250, - 35 None Staemmler & Lagler,,1965 Dog 1000 196 'None Staemmler-&"Lagler, 1965 100 for 65 days Staemmler & Lagler, 1965 then 200 for 20 days 119 Depression of body Kemper, 1962 - Cockerel 100 for 65 days weight; retardation of ,Kemper & Berger, 1962 then 300 for. 5 days 85 sexual development 1000 for 5 days - - 76 . haemoglobin level (Lin et al., 1966). Kemper (1962) and Kemper and Berger (1962) observed retardation in the growth.of testes and secondary sexual characteristics in'the cockerel. Pathological changes in the ganglionic cells of the brain and spinal cord of dogs were noted by Die1el (1963) and Degerring (1966). In man, effects on the nervous system, especially peripheral neuritis, have been described, following chronic dosing of thalidomide (Raffauf, 1961; Scheid et al., 1961; Kuensberg et al.. 1961; Fullerton and Kremer, 1961; Cohen, 1961). The symptoms persist a long while after the drug is removed. Marked proximal muscle weakness, absent in simple peripheral neuropathies, has also been reported as an after- effect of chronic dosage of thalidomide.

Qytotoxio effects There is conflicting evidence concerning the oytotoxicity of thalidomide. Di Paolo and Wenner (1964) observed an increase in the mitotic activity of Erhlioh aseites tumor cells in the presence of thalidomide. Chromosomal damage in human lymphocytes (Tersen, 1965), cessation of oell division in chick embryo (Villa and Eeridium, 1963) and induction of chromosomal abnormalities in root meristems of Vicia faba (Amirkharian, 1966) have all been related to thalidomide. Roath et al. (1962) found that the drug affected the rate of development of maturing human erythrocytes and caused morphological - 77 - abnormalities in these cells.. However, Natarajan and Nilssen (1966) failed.to.confirm the findings of Amirkharian in Vicia tabs.; and Lindahl-Kiessling andBa8k contradicted those of Roath et al. Attempts to demonstrate cytotoxio' aetivity in plants has

resulted in similarly conflicting reports. Giacomello et, al. (1964) found that thalidomide induced chromosomal aberrations and alteration of the mitotic stages in the root apical cells of the common onion (allium cepa), However, a solution of thalidomide appears to have no effect on the growth and development of various plants: tomato,-barley and soya bean. They develop quite normally. Furthermore, the flower and 'seeds of tomato treated in this manner germinate normally. The drug retarded the root elongation of flax seedling, but had no effect-on the elongation of oat coleoptiles induced by indolyl-3 -acetic , acid (Parups and Hoffman, 1965). Some unicellular organisms are, also sensitive to thalidomide. Frank et'al. (1963a, 1963b) report toxic effects in several protozoan'flagellate organisms such as Euglena gracilis, Ochromonas melhameniis„ Cohromonas clarica,' and the ciliate Tetrahymena pyriformis. Boney (1963) demonstrated growth inhibition in marine red algae Callithammion tetrieum Ag., and also that some of those algae that developed showed growth abnormalities. - 78 - That the racemic form of thalidomide is teratogenic is conclusive. The evidence supporting other forms of toxicity seem rather inconclusive, no more than an indication of a potential hazard. However, Fabro et al. (1967) showed the isomers of thalidomide to be acutely toxic in mice. They found the oral LD50 for the D. and L- isomers in the male I.C.I. SAS mice to be 400 and 700 mg/kg. respectively. Thalidomide has always been considered unique in that it is non•«toxio to the adult, but toxic to the embryo. This toxicity of the isomers brings the relative maternal:foetal toxicity ratio more in line with that of other teratogens such as 2 -ethylamino -113,4 thiadiazole, and 6-Meroaptopurine Murphy, 1960). This suggests that the mechanism of foetal .and adult toxicity to thalidomide could well be related. In this and the following chapter the toxicity of the three compounds both to the adult and to the foetus has been investigated, and an attempt has been made to resolve the differences in toxicity between the racemic and isomeric forms. - 79 -

MATERIALS AND METHODS Animals Adult, white mice (body weight 18-22 gms.) of three strains were used in the toxicity experiments. I.C.I. SAS mice were purchased from Scientific Animal Service, Home Farm, Elstree, Herts; T.O. ASL strain from Animal Suppliers Ltd., 685 High Road. London N.12; CFI. strain from A.' Tuck Ltd., Small Animal Breeding Station, Raleigh, Essex. They were allowed food and water-ad lib. In certain experiments to determine the effect of starvation on the drues, toxicity, the animals were without food for 24 hrs. prior'to the administration of the dose.

Compounds, D(+).. and L(.).phthalimidoglutartmide were synthesised in this laboratory using the procedure described in the next chapter. Bemigride (megimide), D-amphetamine sulphate and the monoamine oxidase inhibitor Mo 911 were purchased commercially.

Acute Tioxicity of Thalidomide and its Isomers in Mice DL-, D(+). and L(-)-thalidomide were given as a suspension in 1% methyl cellulose. The compounds were administered orally by stomach tube in 0.5 ml. of suspending fluid. For - 80 - intraperitoneal injections 0.25 ml. was used. Groups of 10 or more mice were treated with the drugs either under normal feeding conditions or following a 24 hour period of starvation. The mice were examined at intervals for 24 hrs. post administration and the degree and duration of the sedation, maintenance of the righting reflex and fatalities were recorded. This was repeated at varying dosages, orally and intraperitoneally, to obtain the minimum doSe at which the drug was toxic, the LD50 and the LD100. The compound was regarded as non-toxic if no fatalities were observed after the administration of 5000 mg/kg.

Antagonists Compounds to be studied as possible antagonists of thalidomide toxicity were administered 1.p. as follows:- Bemigride (5 mg/kg.) was given half an hour after the dose of thalidomide; D(+)-Amphetamine sulphate was given both as a single dose (10 mg/kg.) or as consecutive doses (5 mg/kg.) at to 2 and 4 hours following the drug's administration; Monoamineoxidase inhibitor MO 911 (50 mg/kg.) was given at the same time as thalidomide. Five animals were set up as controls and given the same dose of antagonist as the experimental animals. .81-

RESULTS Acute Toxicity Table 19 shows the results obtained following both oral and intraperitoneal doses given to various strains of mice. The results obtained for the raoemate agree with those found by other workers in that there were no deaths even after an oral dose of 5 gms/kg. or an i.p. dose of 3 gms/kg. The mice , were no more sedated than if given a.low dose of about 150 mg/kg. The test animals recovered over a period of a few hours. During this time they never lost their righting reflex and'were easily disturbed from sleep.

Oral Toxicity This was in complete contrast to the effect observed with the optical isomers. At relatively low oral doses of about 100 to 150 mg/kg., the mice were heavily sedated, and remained so for several hours. However, they still retained the righting reflex and could be aroused from sleep. This was true for all strains of mice studied. At a higher dose level, the response varied with sex and strain of the mouse under test. Orally, the drug was acutely toxic to male I.C.I. SAS and T.O. strains of mice, the LD5O being 700 and 750 mg/kg. .respectively. At this level, the mice were heavily sedated. and many died from marked c.n.s. depression. They retained the 82

TABLE 19. ACUTE TOXICITIES OF Ds., L- AND DL-THALIDOMIDE IN VARIOUS STRAINS OF MICE.

Route of Adm inistration Oral (g/kg) I.P. (g/kg)

Optical form DL- L- DL- D- L-

STRAIN SAS >5 0.7 1.5 > 3 0.3 0.5 >5 1.5 3 > 3 0.5 1.5

TO > 5 0.75 1.0 >3 0.2 0.2 > 5 1.5 1.5 > 3 0.75 1.0

1.0 CFI > 5 >3 >5 > 3 1.5 >5 >3 >5 >3 1.5 1.5

D., L-, and DL-thalidomide were administered orally or by intraperitoneal injection as a suspension in 1% carboxymethylcellulose to groups of at least 10 mice; mortalities were counted at 24 hours after administration. .. 83 - righting reflex. and would move When disturbed. They suffered from hypothermia, and'their limbs were maintained in a rigid position. Those that survived appeared to make'a complete recovery. However, in the CF1 strain, neither male nor female suffered any fatalities even when given 3 to 5 gms/kg. of the

D- or L- isomer. They were heavily sedated, but all made a full recovery within 24 hours. The female of the I.C.I. SAS and'TO strains were less susceptible to both the D- and L- isomer. with LD50 of 1500 mg/kg. (D-) and 2500 (L-) (I.C.I. SAS) and 1500 mg/kg. both .D- and L- isomers (TO strain). Table 20 gives the relationship between the toxicity of the Di-isomer and that of the other two forms. Following oral administration, the D-isomer is seen to be the most toxic compound. It is toxic at a level 1/10 that at which the racemate is without effect. In I.C.I. SAS strains the DI- isomer is 2'to 3 times more toxic than the L.., whilst in TO and CF1 strains it is only slightly more toxic.

Intraperitoneal Administration (1.p.) When the compounds are administered intraperitoneally. differences are observed regarding their relative toxicities in the three strains of mice. Thus. the LE100 for 10,-thalidomide in SAS mice was 1000 mg/kg. when administeret orally, and 750 mg/kg. when given i.p. This would be expected if intestinal absorption is rate limiting. as intraperitoneal -84. TABLE 20. RELATIVE TOXICITY OF DL-. 106-. AND L.THALIDOMIDE IN DIFFERENT STRAINS OF MICE

Ratio of LD of Di. to the other isomeric forms

Strain Oral i.p. DL L DL L D 'T:). D 15

I.C.I. SAS cfi, >10 3 - > 10 1.5 >10 —'2 ?l0 2

TO di > 10 1.5 > 10 1 9 , >10 3. > 10 .--'1

CF1 e --....' 1 1 >10 V ' 1 3. >10 1 - 85 - administration would by-pass the intestine and thus increase the concentration of the drug in the plasma and at the site of action. This difference is even more marked in the CF1 strain of mice, where there were no lethalities following an oral administration of over 5 gms/kg. of the isomers, whilst they were toxic when given i.p. (1000 mg/kg.). The difference in toxicity between the D. and L- isomers is not so marked when the drugs are administered i.p. In the I.C.I. SAS strain, the D6 isomer was three times more toxic than the I,- when given orally, whereas this ratio is only 1.5:1 when administered i.p. A sex difference in susceptibility is still apparent when the drug is given i.p. In the SAS strain, the LD 100 for the D#60 isomer is 750 mg/kg. in the male and 1000 mg/kg. in the female. A similar difference is seen with the L- isomer (LD 100 in the male 750 mg/kg., in the female 1500 mg/kg.). This is not the case in the CF1 strain however, where male and female show very similar dose effect With both isomers.

Antagonism'ot thalidomide'toxicity Table 21 shows a set of typical results obtained when the D- and L- isomers were administered simultaneously, The mice were given a lethal dose (1000 mg/kg. orally; 750 mg/kg. i.p.) of the D6isomer, and a similar dose of lithalidomide. When

- 86 . TABLE 21. ANTAGONISM OF DI- AND L- THALIDOMIDE TOXICITY IN I.C.I SAS MALE MICE

Expt. Dose of Isomers (g/kg.) No. of No. mice "1' mortality D L

1 1.0 (oral) 0 10 100 1.0 (oral) 1.0 (oral) 0

2 0.5 (i.p.)* 1.0 (oral) 10 50 0.5 (i.p.) 0 10 100

3 0.50 (i.p.) 0.75 (oral) 10 10

0.5 (i.p.) 0 10 100

SAS male mice. wt. 20 grms. — 2 grms. * D-thalidomide was administered 15-30 mins. after the oral dose of L-thalidomide. . 87 . both isomers were given orally, no deaths occurred, and all the mice were fully recovered after 24 hours. When the isomers were administered by different routes (orally and i.p.), some deaths did occur. However, this was always less than 100 per cent; and when 1.p. administration was delayed 15 to 30 minutes after oral dose, only one, or two, or even no deaths were recorded. Table 22 shows typical results obtained when a lethal dose of D-thalidomide was given together with a stimulant of the central nervous system, D-amphetamine. . Again, varying results were obtained, depending on the method of administration of the suspected antagonist.. If the amphetamine was given as a single dose, the number of resulting deaths were similar to the controls. However, if smaller doses of the drug were administered at intervals (such as *, 2* and 4* hours after giving thalidomide), the resulting deaths were 50-70 per cent less than the controls, demonstrating a positive antagonistic effect. Table 23 shows the results obtained when the monoamine oxidase inhibitor (MO 911) was given together with D-thalidomide. The treated animals appeared to be as completely sedated as the controls (which were only given thalidomide) and the resulting deaths were comparable with the controls (a mortality rate of 90% or over). - 88 -

TARLP 22. ANTAGONISM OF D.THALIEOMIDE TOXICITY WITH D-AMPHETAMINE

- . Oral Dose No. of D6.thalidomide D-amphetamine Dead Animals+ (mg/kg) (mg/kg)

6 - 3 x 5 mg/kg. 0

10 1000 3 x 5 * 5

10 1000 - .10

Mice were male I.C.I. SAS strain wt. 20 grins. .(± 2 grins.) * The D-amphetamine was given by i.p. injection *, 2* and 42 hours following the oral administration of the D-thalidomide. - 89 -

TABLE 23. ANTAGONISM OF D-THALIDOMIDE TOXICITY USING A MONO AMINE OXIDASE INHIBITOR (MO 911)

No. of Oral dose mice+, DN-thalidomide MO 911* Dead Ong/kg) (mg/kg)

10 1000 50 9

10 50 0

* given i.p. in 1% methyl cellulose in 'nips.' water Mice were male I.C.I. SAS (wt. 20 grms. ± 2 grms) -90-

DISCUSSION

As I have previously described, the racemate. DL- thalidomide has not been- shown to be acutely toxic, whether given orally or by i.p. administration.' Both isomers of the drug however, are acutely toxic'in mice. This difference is not easy to explain and there appears to bejlo parallel to this in the literature. 'It was seen in the previous chapter, however, that the raoemate differed from its isomers in various physico-chemical properties. The solubility of the DL-form is significantly lower both polar and in non-polar solvents, and'this could affect its comentration in the plasma and at its site of action in the central nervous system. Other drugs acting upon the nervous system, such as the muscle relaxant carisoprodol, require a specific plasma level to take effect (Albert-. 1967). ThUt, above a platma level of about 130 µg/ml., oat, rabbit, rat and mouse lose the righting reflex, and below this level the reflex returns. If this were so in the case of thalidomide toxicity, the lol solubility of the raoemate could prevent it from reaching the level required to exert lethal effect, whilst the D- and L. isomers, being more soluble, could do so. A marked sex difference in toxicity is also observed, the VII6. and L. forms being between 1 and 2 times more toxic in the male I.C.I. SAS strain than in the female. Sex differences in drug response are usually attributed to a difference in the - 91 - rate of metabolism of the drug between sexes, due to the effect of androgens on the drug metabolising enzymes. - However, studies on the fate of thalidomide in rabbit (Fabro et al., 1967) and hamster (Hague, Fabro and Smith, 1967) showed that it spontaneously hydrolysed, but none of the products formed involved the drug metabolising enzymes. Thus the rate of removal of thalidomide should be expected to be independent of sex, strain or species. A comparison of the distribution of the drug in the body between the sexes following its administration, could give a clue as to whether the blood-brain barrier plays a part in this difference in susoeptibility. , The results also demonstrated a difference in the toxicity of the antipodes of thalidomide, the D- isomer being the more toxic, of the two in I.C.I. SAS and T.O. strains of mice. This difference cannot be explained by the physico-chemical properties of the two compounds, as they have identical solubilities, rates of'hydrolysis and physical properties. This difference could be due to a stereo-specificity at the absorbing membrane, the D- isomer passing through more readily, thus reaching the .critical concentration for toxicity at a lower plasma level than the L-. The accumulation of the isomers also depends upon their rate of excretion, the greater the latter rate the less chance it has of accumulating. This possibility .could be verified by determining the distribution and excretion of the isomera'following their administration. - 92 An answer to this difference in'toxicity is further complicated by the fact that the D. and L- isomers, administered by the same (oral) or different routes (oral and i.p.), antagonise each other's effect. As both are neurotoxic, competition for the binding site can be disregarded. We must conclude that the lemners, together in solution. associate to form a diner which interferes with their passage across the nerve membrane and so reduces the degree of their neurotoxicity. The results show that the e.n.s. depressant action of the drug can, at least partially, be alleViated by the concurrent administration of amphetamine* Amphetamine is,a powerful central nervous stimulant thought to act in either of two ways. Firstly, by causing the release of noradrenaline from central stores, a theory supported by the fact that it causes a lowering in the level of noradrenaline in the brain (Moore, 1963); or secondly by competitively inhibiting the monoamine oxidase (Blaschko, Richter and Schlessman„ 1937; Iversen, 1967). This indicates that the toxicity of thalidomide is due to its sedative effect on the central nervous system. This inhibition was not 100 per cent effective, probably because of the difficulties involved in titrating the exact amount required to alleviate the toxic effect, without allowing the toxic nature of amphetamine to manifest itself. In conclusion, it appears that'the D- and Li- tom6rs of thalidomide cause depression of the central nervous system. That the racemate is not toxic may be due to its - 93 - poor solubility and/or its possible dimeric molecular form in solution. There is a definite sex and strain difference in the toxicity of these compounds. A difference in 'toxicity between the isomers is also obderved. There is wpossibility that some of these differences can be explained by examining the absorption, distribution and excretion of the three compounds in the mice. This was the aim of the work described in the next chapter. CHAPTER VI

FATE OF [14 C) - D- , -L-, AND - DL-THALIDOMI DE IN MICE - 94 .

INTRODUCTION

In the preceding chapter it was demonstrated that D. and L-thalidomide were much more toxic to the male SAS albino mice than DL-thalidomide. A comparison of'the absorption, excretion and distribution of D., L. and DI,- thalidomide was made In thin strain of mice to see whether differences in these factors might explain the different toxicittes of the three forms of thalidomide. -95-

MATERIALS AND METHODS Materials 14C-, DL-Thalidomide ((1)-3-([carbony1-14Cilphthalimido)- 'glutarimide (specific activity 0.6 µc/mg; m.p. 270°) was synthesised from [carbonyl-14C1] -phthalic anhydride by the method of Beckmann (1962). DL-Thalidomide was a gift of Lilly Research Labs., Ltd.

Synthesis of L-Thalidomide L-glutamine (7.3 g; 0.05 mol.) was dissolved in 85 mis. water containing anhydrous sodium carbonate (14.4 g; 0.136 mol.). N-Carbethoxyphthalimide (12.5 g; 0.575 mol.) was added slowly to the solution and stirred. After stirring for 45 minutes, the reaction mixture was cooled to 0°C in an ice-bath and filtered. The filtrate was acidified to pH2 with 5N.HC1 and allowed to stand at 4°C overnight. The L-2-phthalimidoglutaramic acid that separated was filtered off and dried (yield 11.8 g, m.p. 170°C).

Cyclisation For the cyclisation, L-2-phthalimidoglutaramic acid (5.52 g; 0.02 mol.) was suspended in dry methylene dichloride (50 ml.). Pyridine (1.58 g.), was added at 18°C and stirred for 30 mins. The reaction mixture was cooled in a bath -96- O (-30 ) and stirred whilst thionyl chloride (2.38 g; 1.45 mi.) 0 was added. The bath temperature was allowed to rise to -15 over 20 minutes and the reaction mixture was then cooled to -40°C and triethylamine (2.402 g; 2.77 ml.) was added with stirring. Stirring was continued overnight, during which time the reaction mixture was allowed to reach room temperature. The reaction mixture was evaporated to dryness, with the temperature maintained below 25°C, using a rotary evaporator. The residue was triturated with water (150 ml.), filtered and the precipitate washed with water and dried. The precipitate was recrystailised from 50$ aqueous dioxan to give small white crystals of L-thalidomide m.p. 244-246°, yield 4 g. (78$); [ac] 20 = .66o (c = 2% in dimethylformamide).

Synthesis of D-Thalidomide D-2-Phthalimidoglutaric acid:- D-Glutamic aoid (14.7 g; 0.1 mol.) was dissolved in 127 mls. of water containing anhydrous sodium carbonate (21.2 g; 0.2 mol.). N-Carbethoxyphthalimide (21.9 g; 0.1 mol.) was added slowly and the solution stirred for 2 hours. The solution was cooled to 0°C, filtered and acidified to pH2 with 5N.HC1, and allowed to stand overnight. The reaction mixture was filtered and the precipitate was washed with water and dried. Yield 9 g. (62%) m.p. 163-164°C.

D.2'-Phthalimidoglutaric anhydride: D-2,-Fhthalimido- glutaric acid (7.5 g; 0.027 mol.) was warmed on a steam-bath - 97 .., with acetic anhydride (20 mis.) for 5 minutes until it dissolved. The excess acetic anhydride was distilled off at 50°C under _reduced pressure. The residue was triturated with dry ether. filtered and the product stored under vacuo. Yield 7 g. (95%) m.p. 207.210°.

E.21-PhthalimidoFlutaramic acid: D.2'-Phthalimidoglutarie anhydride (12.95,g; 0.05 mol.) was dissolved in acetone (150 ml. and treated with 0.88 NH3 (13 ml.). The reaction mixture was evaporated to dryness (below 25°C) using a rotary evaporator. The residue was dissolved in water (10 ml.) and the solution acidified to pH3 with cone. HC1. On cooling to 00C the solid that separated was filtered off, washed with water and dried. Yield 6.8 g. (55%) m.p. 165.167° CoOD25 = +34.8 (0=0.5 in ethanol).

D.Thalidomidel D-29 -Phthalimidoglutaramic acid (3.64 g; 0.014 mol.) was then eyellsed using the method described for L-thalidomide. The D.thalidomide was recrystallised from 50% aqueous dioxan to give small white crystals. Yield

2.8 gm. (80%) m.p. 244°C; E c43 D20 +59° (C=2% in dimethylformamide).

Synthesis of L-31.Phthalimido. cu.14 C4clutarimide1 L.[U.14C]Glutamine (400 mg:100 µc) was dissolved in a - 98 - solution of anhydrous sodium carbonate (0.79 g.) in water (4.7 ml.). N-Carbethoxyphthalimide (0.69 g.) was added and the solution stirred for 30 minutes. The reaction mixture was cooled to 0°C and filtered, ,then acidified to pH3 with cone. HC1. On standing at 4°C overnight, white crystals of L-21 phthalimido-[1403-glutaramie acid separated. Yield 420 mg; m.p. 167-170°C. The latter (400 mg.) was suspended in methylene dichloride (3.6.ml.), treated with pyridine' (042 ml.) and the mixture stirred for 20 minutes. The reaction mixture was cooled to -30°C end then treated with thionyl chloride (0.11, m1.). The temperature was allowed ,to rise to -15°C, then cooled to -40°C and triethylamine (0.2 ml.) added. The mixture was stirred for 5 hours, during which time the temperature rose to 10°C. The reaction mixture was evaporated to dryness at 15°C and the residue triturated with water (2 mis.). The solid was filtered off, washed with water, and reerystallised from 50% aqueous dioxan, to give white crystals. Yield = 250 mg;' m.p. 244°C; [41)20 - -59.7 (C = 2% dimethylforamide) specific activity = 0.6 µc/mg.

Synthesis of D6-3' -([l -14c .carbonyoralthalimido)giutarimide

[.1-14C-Carbonyl] Phthalimide. 14C3Phthalic anhydride (600 mg; 500 µe) was heated gently with 0.88 NH3 (0.53 ml.) - 99 - o over a bunsen flame until the temperature had risen to 300 C. The melt was allowed to cool, the solid removed and ground to a fine powder in a small mortar. Yield 540 mg. (90%) m.p. 238°.

2-CarbethoxyC1-14C carbonyi3phthalimide.: Cl4C]Phthalimide (300 mg; 250 pc) was dissolved in dimethylformamide (1.3 ml.) and triethylamine (0.31 ml.). The reaction mixture was cooled to 5°C and ethylchloroformate (0.24 ml.) was added dropwise and the temperature kept' at 5-10°C for 90 minutes. The reaction was allowed to reach room temperature and was then poured into 6 ml. of water chilled with ice. The C14C3earbethoxyphthalimide that separated was filtered off, washed with water, then washed with petroleum ether (b.p. 40-60°). The material was then dried in vacuo over silica gel and recrystallised from - ethanol to give white needle crystals of [14C3carbethoxyphthalimide. Yield 200 mg; . m.p. 84°.

D-2•-t(1-14C carbony1)phthalimido~ lutaric acid: E14C3Carbethoxyphthalimide (170 mg; 100 pc) was added slowly, with stirring, to a solution of anhydrous sodium carbonate (165 mg.) and Di.glutamic acid (105 mg.), in water (2 ml.) and the reaction mixture stirred for 30 mins. The solution was filtered and then acidified to pH2-3 with 5N.01. The D.PC1phtha/imidoglutaric acid that separated was filtered - 100 - off and dried. Yield 145 mg; m.p. 164°C.

D-2*-(E1714C earbonyilphthalimido)glutarie acid anhydride: D-2*-114C Phalimidtiglutarie acid (140 mg; 70 µe) was warmed until it just dissolved in acetic anhydride (0.45 ml.). The excess acetic anhydride was removed by evaporation at 500G under reduced pressure. The residue of D6-21- il4 diphthalimido glutarie acid anhydride was triturated with dry ether and dried. Yield 105 mg; m.p. 194° (shrinks) and melts at 197200°C.

D4.29-([1-14C carbonyilphthalimido)glutaramic acid; D-204140Phthalimidoglutario acid anhydride (100 mg; 46 µc) dissolved in acetone (2 ml.) was treated with 0.88 ammonia (0.2 ml.). The reaction mixture was evaporated to dryness at 25°C. The residue was dissolved in water and the solution acidified to pH2-3 with 5N.HC1. and then cooled in ice. The 110_214140 phthalimidoglutaramie acid that separated was washed with ice cold water and dried. Yield 55 mg; m.p. 167°.

D-2'-[(1-14C carbonyllphthalimiddlglutarimide: D-2°-E14C1Phthalimidoglutaramic acid (55 mg; 27 µa) duspended in dry methylene dichloride (1 ml.) was treated with pyridine (0.016 ml.) and the suspension stirred for 20 minutes. The reaction mixture was cooled to -30°C and treated dropwise with thionyl chloride (0.015 ml.). The temperature was - 101 - allowed to rise to -150C over 15 mins., then cooled to -40°C. and, treated with,triethylamine (0.028 ml.). The temperature was allowed to rise to 10°C over a periOd of 5 hours, and the reaction mixture was evaporated. to dryness under reduced Pressure at leC. The D414C]thalidomide'was recrystallised from 50% aqueous dioxan to give pale yellow crystals. Yield 40 mg; m.p. 244°C; specific activity 0.45 ile/mg. The yield was too small to allow the determination of(a) 20* However, .the pilot experiment using non-radioactive intermediates gave an identical Yield to the above And the [420 of the D.thalidomide wse +58 (C = 2% in ,dimethylformamide).

Animals and Treatment Excretion: Three groups of three male albino I.C.I. SAS strain mice (22 ±2 grms) were given an oral dose of

[140.4)., r or E14C1-DL-thalidomide (150 mg/kgs100 4c/kg). The mice were kept in separate cages with access to food and water; their urine was collected at regular intervals for the first 12 hours. then at 24, 48 and 72 hours.

Tissue Levels and E14C3-DL.thalidomide (200 mg/kg; 50 lic/kg.) were administered orally to three groups of ,twelve mice. Three mice from each group were then killed at 2, 4, 8 and 12 hr. after dosing. Blood samples (0.8-1.2 ml.) were collected in small heparinized tubes and centrifuged at 200 r.p.m. (M.S.E.'Minor centrifuge) and plasma - 102 . samples used for assay of total and [140-thalidomide. The brain and liver were removed immediately, dried .on blotting paper and quickly weighed. They were then homogenized .in water (2-3 mis.) in a Potter homogenizer, and the final volume was adjusted to 5 ml. with water.

14 14 Estimation of C: C in plasma, and homogenates of brain and liver. was estimated by adding aliquots (0.1-1 ml.) to a scintillation fluid of the composition previously described (Fabro et al., 1967). For brain and liver. homogenates 5% Cab-o-sil was included in the scintillation fluid. [14C3 was counted using the Packard Tricarb scintillation'counter (Model No. 3216). Efficiencies (estimated by the twin channel ratio method) were as follows (%): plasma (66); liver (54); brain (63); urine (70) and faeces (45-60). Faeces were homogenised in 50% aqueous dioxan to a final volume of 100 mis. Portions Cl ml.) were counted as above.

14 14 R v14 , Assay of Ci-E1-., . C4-1,- and L CA-DL-Thalidomide in 14 plasma, brain and liver: 1r. Cj-Thalidomide in plasma, brain and liver samples was isolated by solvent extraction with chloroform at pH 7.4 which removed only thalidomide and not its polar hydrolysis products. Plasma samples (0.2 ml.), - 103.. homogenates of liver and brain (4 ml.) were extracted with 5 ml. and then 25 ml. respectively of chloroform. The solvent was separated and [1401-thalidomide estimated in 1 ml. samples by 14 scintillation counting. The recovery of [ 0- and 1-14L CJ-DL-thalidomide added to plasma. brain and liver samples for assay by this procedure was 95-101'70. - 104.. RESULTS The excretion patterns following the oral administration of -L- and -EL-thalidomide to male I.C.I. SAS mice are shown in Table 24 and -Fig. 6. It can be seen that the excretion patterns for, D- and L-thalidomide are similar but differ from that of the DL- form. About 70,6 of the 14C of the optically active forms is excreted in the urine within 24 hrs., whereas in the case of the racemic compound only about 40% can be so accounted for. In contrast, faecal excretion of the DL- form is greater; about 35% appears in the faeces over 3 days, whereas the corresponding values for the D- and Li- forms is about 20-25g. It is thus clear that absorption of the DL-thalidomide is slower and less complete than that of the optically active forms.

Plasma levels and Tissue Distribution Fig. 7 and Tables 25, 26 and 27 show the plasma and tissue 14 levels for total C and El4C1-thalidomide after the oral administration of the labelled isomers (200 mg/kg.). The 14 total C refers to the sum of the thalidomide and its hydrolysis products formed by spontaneous breakdown of the former (Schumacher et al., 1965). The plasma, brain and liver levels of the optically active forms were similar but were different from those of the DL- form. Thus, at 2 and 4 hours after dosing the plasma thalidomide_ levels after administration of the D- and TABLE 24. EXCRETION OF'14 C AFTER THE ADMINISTRATION OF 14C-E61 -L-, and -EL- THALIDOMIDE TO MALE I.C.I. SAS ALBINO MICE 150 mg/kg.- (100 ge/kg) administered orally and urine and faeces collected at the times shown below.

Animali A dose 14C in urine at Faeces Total No. „...... -....."-.....-.....N (i,) 2 6 12 24 4 6 24 . 48 72 DL- 1 . 16.5 10.8 1.1 16.8 - 4.5 . 28 77.7 2 - 29 18.8 10.6 7.8 1.7 5 - 12.7 85.6

L- 1 19.1 22.0 - 16.1 3.2 . 9.8 ' 4.6 c-1 74.8 2 21.3 26.2 . 14.9 1.6 . 9.8 8.3 <1 82.1 3 20.3 22.0 - 25.7 2.9 - 12.2 8.0 <1 91.1

1 36.9 10.0 27.8 1> - 12.9 12 <1 88.8 2 - 34.4 18.6 19.6 1) - 17.7 - 11 <1 101.4 3 - 24.8 19.2 24.8 1> - 3.4 21.8 (1 94.0 FIG. 6. IINARY AND FAECAL EXCRETION OF 14C AFTER THE ORAL ADMINISTRATION OF C LABELLED D-, L- AND DL-THALIDOMIDE TO MALE I.C.I. SAS ALBINO MICE

D 70 Fa. L H X

0 SI

0 4o DL 0 30 Co 0

Time (hr.) after dosing — 107—

Fig. 7.

I I 14 PLASMA LEVELS AND TISSUE DISTRIBUTION OF C AFTER THE ORAL. ADMINISTRATION OF C-. I - OR W.-THALIDOMIDE TO MALE I.C.I. SAS MICE

MICE WERE DOSED WITH "C-. L- OR DL-THALIDOMIDE (.700MG/KG. 50.,C/KG) AND THEY WERE KILLED AT THE TIMES SHOWN. VALUES ARE MEANS OF THREE ANIMALS.

A-4116,-DL- -fHAL 'Dom rK 5 X —X ,D - 414co. Defirg 5 0 —0 , L- ltAL oomrog (e.o..31KI.) 50/At/KA)

/4 iii1AUD0140E C PLASMA 80

2.0 A • cro MAIN 80 0 60 av 4.. 40

z ZO 0 A • I ...... "'"s...... li I

LivER 160

= 120

+0 • 4 2 4 6 8 10 la 2 4 6 8 10 12 AlleR DosiNq (Hrs./ TABLE 25. PLASMA LEVELS OF 14C-16, L.. AND DL- THALIDOMIDE AFTER THEIR ORAL ADMINISTRATION TO MALE•I.C.I. SAS MICE Mice were given orally 14C-D-, L-, or DL-thalidomide (200 mg/kg; 50 µc/kg) suspended in 1% methyl cellulose. Mice were killed in groups of three at the time shown.

µg/ml. Plasma Concentration inA Time DL-Thalidomide L-Thalidomide D-Thalidomide (hrs) 14 14 14 14 14 14 Thalidomide C Total C Thalidomide C Total C Thalidomide C Total C

2 8.2 (7.2-9.6) 19.6 (16.1-21.9) 39.7 (30-47) • 99. (66-138) • 45.9 (38-50) 79.6 (68-89)

4.9 (3.9-5.6) 16.4 (13.2-17.8) 17.8 (7-20) 33.8 (24.7-46.6) 28.3 (17-47) 51.5 (33-77)

8 3.3 (2.8-4.4) 8.2 (7.1-10.5) 7.8 (6.4-9.4) 18.9 (17.2-20.4) 4.2 (3.6-4.6) 15.4 (10-18)

12 2.1 (1.8-2.8) 2.7 (2.4-3.4) <0.5 (-) 3.5 (2.9-4.3) 1.7 (1.4-2.0) 7.8 (5.6-10.3)

0 00 14 TABLE 26. BRAIN LEVELS OF C-136-. L. and DL-THALIDOMIDE AFTER THEIR ORAL ADMINISTRATION TO MALE I.C.I. SAS MICE 14 Mice were given orally C-D-. and DL-Thalidomide (200 mg/kg.. 50 Ac/kg) suspended in 1% methyl cellulose. Mice were killed in groups of 3 at the times shown.

Concentration Thalidomide• - mg/grm. of Brain Time DL-Thalidomide L-Thalidomide D.-Thalidomide (hrs) 14 14 14 14 14 Thalidomide C Total 14C Thalidomide C Total C Thalidomide C ' Total C

2 5.3 (4.3-649) 12.7 (10.5-14.1) 38.2 (19.5-.51.5) 42.8 (20.6-62.0) 35.6 (29.3-43.2) 53.3 (48.1-59.5)

4 5.7 (4.4..7.4) 1645 (10.426.2) 14.0 (11.5-16.8) 25.6 - (19.7-35.0) 22.2 (11.7-38.8) 44.5 (28.3-64.9)

246 (2.3-30) 8.6.(8.2-8.8) 3.5 (2.0-5.2) 14.7 (12.1-17.3) 4.7 (3.8-5.5) 14.1 (10.9-16.7)

12 144 (1.4=.1.4) 5.7 (5.4-5.9) <0.5 8.9 (7.7-10.7) 2.2 (2.1-2.3) 7.3 (6.9-7.8)

14 TABLE 27. LIVER LEVELS OF C-E6., L- AND DL-THALIDOMIDE AFTER THEIR ORAL ADMINISTRATION TO MALE I.C.I. SAS MICE Mice Were given orally 14 C-D6., L. and DL-Thalidomide (200 mg/kg. 50 4o/kg) suspended in 1% methyl cellulose. Mice were killed in groups of three at the times shown.

Concentration Thalidomide - 4g/grm. liver Time DL-Thalidomide 1.0-Thalidomide 10-Thalidomide (hrs) 14 14 14 14 Thalidomide C Total C Thalidomide C Total C Thalidomide 14C Total 14C

2 14.2 (9.1-17.0) 53.9 (42.0-70.1) 84.2 (37.9-109) 130.5 (64.0-164.4) 62.5 (52.5-69.3) 176.7 (168-184)

4. 12.4 (9.1-14.9) 66.9 (50.2-77.6) 35.9 (28.7-46.7) 107.7 (75.4-160.1) 36.7 (22.9-60.6) 150 (105-196)

8 3.8 (2,9.4.2) 20.9 (17.9-25.1) 12.8 (8.4-17.4) 94.7 (33.1-142.1) 7.9 (4.7-10.4) 79 (58-99)

12 0.5 11.0 (8.4-13.3) (0.5 32.0 (19.5-46.4) 2.5 (2.2-2.8) 42 (33-52).

H H - 111 - L- isomers were 4-6 times greater than those seen with the DI,- form though at 12 hours the levels of the three were similar. The plasma and tissue levels of the 10- and L- forms. although initially high due to rapid absorption rapidly declined. By contrast the levels of the DL-form although lower, tend to plateau. indicating a slow rate of absorption. 14 Similar differences can be seen for the total C levels. The results suggest that the D. and. L- isomers are more readily absorbed than the DL- form, and that their higher acute toxicity Is a,reflection'of the higher plasma levels which they attain. D-thalidomide is approximately 2-3 times more toxic to I.C.I. SAS mice than the L- form. The absorption and distribution studies"have shown that this difference is not due to variations in these factors and therefore a higher toxicity is associated with the unnatural D- form than with the L- configuration. Tables 28 and 29 and Fig. 8 show the plasma and tissue levels of thalidomide following the administration of a lethal dose (2000 mg/kg.),of the isomer. At this dose level the mice die 8-12 hours after dosing. It thus appears that a plasma level of L-thalidomide of about 135-150 4g/ml. over a period of 6 hours is lethal. Brain levels reached a peak at 4 hr. (175 µg/ml.) and then declined to 135 1.1,g/m1. at 8 hr. Liver levels are somewhat higher: the level was about TABLE 28. PLASMA. BRAIN AND LIVER LEVELS OF 14,C-L-THALIEOMIrE AFTER THE ADMINISTRATION OF A TOXIC* DOSE (2000 mg/kg) TO MALE SAS ALBINO MICE

Time Plasma Brain Liver after dosing Total 14C Thalidomide 14C ' Total 14C Thalidomide 14C Total 14C Thalidomide 14C (hr.) 4../ml. tig./ml. 4g./gm. 4g./gm. µg./gm. ' pg./gm.

2 250 (167-326) 140 (83-198) 230 (157-305) 77 (50-103) 305 (215-394) 194 (143-245)

4 448 (443-453) 148 (136-161) 238 (224-252) 179 (143-214) 402 (380-424) 196 (187-204)

8 380. 157 194 (165.223) 133 367 (333-401) 196 (145-246)

Values are means of three animals; 'rangesAn parentheses. * At 2000 mg/kg orally at 6-12 hr.'after dosing. TABLE 29. PLASMA, BRAIN AND LIVER LEVELS OF 14C-DL-THALIDOMIDE FOLLOWING A SINGLE ORAL DOSE (2000 mg/kg; 68 uojkg) TO MALE I.C.I. SAS MICE

Time after, Plas6a Brain Liver dosing Tcitir. %143...0 ThalliiMe 14C 14 Thalidomide 14C 14 ThaltilgoVe_14C (hr.) 171,14m.0 5rA4.1,gm

2 122 (115-132) 70 (64-78) 105 (80-135) 69 (41-109) 225 (220-229) 117 (108-133)

4 145 (124-167) 83 (62,104) 118 (85-140 80(45-104) 183 (160-198) 112`(83-128)

8 106 (67-145) 61 (33-89) 94 (57-140 79 (76-82) 206 (190-223) 90 '(66-115)

12 56 (49-67) 30 (20-39) 40 (22-58) 18 (3-34) 84 (42-126) 33. (12-54)

Values are mean of three animals; ranges in parentheses.

1.1:—

PLASMA %NA) TI,,!.31. F.: LEVEL., OF 14C. I.. and 1)1 -1-11A Lipommt: (2()00NIG KG ORALLY/ TO I.C.I. SAS MICE

x—x (l000il)x3) 3111.1 1).600.4)or1i)2 (l000.44 )K3) 0-0 'fiigka.r.30m1)E (2000,N) "rodu.r)orOpe (200o K /4c I4C 'r0SAL Soo • PLastiq

400 0 IrAln

Soo 0 Zoo

"Decrrei

BRAIN +.0.1

3.0 Cr* CF0

Zoo... I. 0 1:12.alx X tistv-rit X leo L. at

z0 LIVER 400 ui

304r) r

-3 cr 2oo- 0 0

too- A

a + 6 8 10 la a 4 6 8 to 1a lrAE ATI-ER bosiNy (itY0 - 115 - 190 4g/g. over the period of 2 hrs. after dosing until 8 hr. These findings are to be contrasted with those obtained with a similar, but non-lethal dose of DL-thalidomide. Plasma levels were much lower; at 2 and 4 hours after dosing the levels were 70 and 80 µg/ml. respectively and by 8 hrs. these had declined to about 55 µg/ml. -Brain and liver levels were higher than those of the plasma. Thus the brain level was 65 and 100 mg/g. at 2 and 8 hr. while the corresponding values for the liver were 115 and 90 µg/g' respectively. It can be concluded that a plasma level of L-thalidomide of 135-145 µg/M1. is lethal to male I.C.I. SAS mice. Plasma and tissue levels of DL-thalidomide of about 'calf those seen with the L-isomer are non-lethal. Also shown on Fig. 8 are the plasma and tissue levels of L-E14Cithalidomide following its oral administration (1000 mg/kg.) together with a similar dose of the unlabelled D-isomer. The values refer only to those of the L-isomer and thus do not include levels of the D-form. Both D- and I,- thalidomide are toxic when given orally at this dose level but when given together they are no longer toxic. It can be seen that the levels of LX4C3thalidomide are higher than those given by the DL-form but less than those given by a larger dose of the L-isomer. Thus, the plasma level of the L-isomer under these conditions was 90, 110 and. 125 µg/ml. at 2, 4 and 8 hrs. after dosing respectively. If it'is assumed that the D-isomer is also absorbed then it is clear that the - 116 - plasma levels of the D- and L- forms combined must exceed those seen with the L-isomer alone and may exceed those given by a single lethal dose of L-thalidomide. The problem thus arises as to how the D- and L- forms when together are no longer toxic. It w'as shown earlier that crystalline DL-thalidomide is a racemic compound in which equal parts of the Di- and L- isomers are nombined together in some way. Furthermore, hydrolysis studies suggested that the thalidomide dimer molecule is stable in aqueous solution. If it is assumed that the dimeric form of thalidomide is non-toxic then the non-toxicity of co-administered Bi- and L-thalidomide may be explained by the formation in the gut, and also the tissues and plasma of the non-toxic dimer. The dimer may exist in equilibrium with its respective dissociated monomeric optically active forms which are pharmacologically active. These results suggest that the low toxicity of DL- thalidomide is related to two factors, both of which are determined by its molecular structure. Firstly, the rate of absorption of DL-thalidomide is slow and incomplete beCause of the low solubility of the dimer. Secondly, it appears that the dimer molecule is stable in aqueous conditions and that it may exist under physiological conditions. While the free isomers are toxic the diner form appears to be much less active, and its own activity may depend on its dissociation into the free isomers. CHAPTER VII

AMINO ACIDS IN EMBRYOGENESIS -117-

INTRODUCTION EMbryogenesis is a delicate and complicated. process'. At present we have to content ourselves with fragments of information, in the hope of assembling them correctly at some later stage. The problem of size is one factor which has to be overcome. The introduction of the electron 'microscope has .helped enormously in the study of the histology of development. With this instrument cellular division and differentiation can be viewed at a macromolecules level. However, it is not the gross change that interests the biochemist so much as the building blocks and inducers that lead to their aggregation. We must track down countless different molecules, with perhaps several or only one molecule of:each type, necessary either to initiate or to coordinate growth and differentiation. A classical example of'the inducer type was the discovery that iodine could initiate metamorphosis in the Colorado oxolotle Amblystoma tigrinum (Hirsohler, 1922; Blacker and Belkin, 1927). The iodine can be compared to an operator switch for a complicated machine. What remains to be solved is how the machine functions! The processes involved in morphogenesis can be described under three main facets (Table 30). They are growth, differentiation and metabolism. Of these, the most oharaoter.. lotto feature of embryonic development is differentiation. - 118 - TABLE 30. FUNDAMENTAL PROCESSES IN MORPHOGENESIS

A. Growth: increase in 1. Multiplicative: increase in spatial dimensions number of nuclei. and in weight 2. Multiplicative: increase in number of cells. 3. Auxetic: increase in size of cells. 4. Accretionary: increase in amount of non-living matter.

B. Differentiation: 1. Increase in number of kinds of increase in cells: complexity and (a) invisible. Determination of organisation fates; gain or loss of competences. etc. (b) visible. Histogenesis. 2. Increase in morphological heterogeneity; assumption of form and pattern; (a) Individuation: due to the primary individuation field of the organism before any of its parts have become functional. (b)Corporative: due to the mutual interactions of functional parts.

C. Metabolism: the 1. Respirative, i.e. oxidation of chemical changes in carbon compounds the organism 2. Fermentation or Glycolysis: the non oxidative catabolism of carbohydrate. 3. Catabolism of protein 4. Catabolism of fat 5. Characteristic chemical activity, e.g. pigment formation. or the synthesis of glycogen

from Needham. J. (1950) . 119 - However. it is difficult to study development in isolation from other aspects, as. for example. the differentiation of the embryo is closely linked with the rate and direction of metabolism, and information obtained about one aspect would undoubtedly shed light on the other. It is only- in recent years that the biochemist has begun to study the chemical processes involved in embryogenesis. An. area of considerable interest is the role of amino acids in this process. However, at present little is known about the types and fluxes of amino acids in the mammalian embryo during this period. We have therefore made a study of the amino acid pattern of the rabbit embryo during the 6-11 day period of morphogenesis. The 6-day period corresponds to a pre-implantation time, and the 7-11 day: interval corresponds to the.transition of the embryo through the blastooyst stage to the time when development commences.

The Origin of Amino Acids in Embryovnetis The amino acid source for the embryo during morphogenesis has been investigated both in mammalian' and non-mammalian species. Rupe and Farmer (1955) believe that for the first 8 days, very few amino acids are taken up from the albumen by the developing. chick embryo. However Walter and Mahler (1958) using radioactively labelled amino acids found that penetration into the embryo did occur. . 120 - Proteins and peptides have also been studied to see if they can be incorporated into the protein of the non-mammalian embryo. 'Walter and Mahler (1958) demonstrated the uptake by 14 the embryo of radioactivity from C-labelled protein introduced into the albumen of the egg. Similarly Schechtman and Knight (1955) and Brierley and Hemmings (1956) have shown that serologically recognisable protein can pass intact_into the chick embryo. However, Winnick and .Winnick (1953) came to the conclusion that albumen proteins are completely hydrolysed before their utilisation for protein synthesis. In mammalian species,,the situation is rather different, as the embryo may also obtain its amino acids'froman external source, the maternal environment. This external supply could influence the morphogenesis of the embryo, as amino acids have been found to control certain processes of ‘differentiation. Greenwold and EVerett (1959) studied the incorporation of 35s methionine into the mouse from the primary zygote to blastocyst stage. They found that no radioactivity was taken up by the zygote during its passage down the tubules, even though the amino acid was present in the milieu of the lumen. They concluded that during this stage, development is independent of external nutrition. This inability of the blastooyst to absorb nutrients' was also studied by Lutwak-Mann (1954, 1960). , Following the intravenous injection of certain nutrients into the 6 day pregnant rabbit, she compared their uptake relative to,the - 121 - plasma concentration. She was unable to demonstrate their absorption into the blastoeyst. However, in vitro studies have shown that an external source of amino acids is essential for the'development of the mammalian embryo even before implantation. Daniel (1965) using 5 day rabbit blastocysts studied the effect of amino acids on development in vitro. By varying the concentration of certain amino acids which had been reported to be present in high concentration in the uterine fluid prior to implantation (Gregoire et al., 1961) he found that an increase in the concentration of alanine,glycine, glutionic acid, threonine and serine resulted in . a better growth of the blastooyst. Similarly. Brinster (1965) demonstrated that albumen and amino acids were required for the development, in vitro of the mouse before implantation.. He found that amino acids could replace the need for albumen if the suspending mediums viscosity was increased by an artificial polymer. Heconcluded'that free amino acids were essential for the development of. the zygote. Thus it would appear that amino acids do pass into the blastocyst before implantation. Proteins have also been examined as a possible amino acid source for the mammalian embryo. Glass (1963) reports that bovine serum protein mill pass into the rabbit blastocyst prior to implantation. However, Zimmerman (1963) working with plasma proteins, demonstrated that in rabbits they could only - 122 - cross into the blastoeyst after 7i days, that is, after implantation. This has been confirmed by Brambell and Hemmings (l949). They attached Evans Blue dye to maternal plasma proteins, and these were detected in the yolk sac fluid after 8 days. Furthermore, they found no evidence that there is any differential permeability operating across the membrane at this stage of development, as the proteins of the yolk sac were the same as those in the maternal plasma. This was confirmed by McCarthy et al. (1949), who followed a similar method, and then separated the proteins of the yolk sac and maternal plasma by electrophoresis.

+Electrophoretie behaviour of Proteins of yolk sac and Maternal Plasma

Electrophoretic Analysis*

Albumin Globulins Fibrin Blastocyst fluid 64 4.0 12.5 12.8 6.6 Rabbit Plasma (1) 63.3 11.5 13.0 4.3 7.9

Rabbit Plasma (2) 76 1.1 10.8 12.2 1110

* Results given as a 5 of total protein After McCarthy et al. (1949) - 123 - The Role of Amino Acids in Embryogenesis Metabolic Role Differentiation results in the formation of histologically and functionally distinct tissues. These differ from one another in some of their proteins, as has now been demonstrated by a number of .workers using immunological techniques (Ebert, 1958). The study of amino acids in embryonic tissue is further complicated by the fact that the mechanisms operating are changing with time and also from cell to cell. For this reason biochemists have restricted their study to adult, differentiated tissue. The last twenty years have seen the introduction of sensitive techniques for the study of amino acids: paper chromatography, microbiological assay, autoanalytical procedure for amino acids and the use of radioactively labelled amino- acids. In spite of this progress, our knowledge of mammalian development is still sparse. For convenience, non-mammalian systems have been studied, as they are free from maternal involvement. However, much of this work may well be relevant to mammalian embryogenesis. Amino Acids in Embryonic Protein Synthesis Most of the research on embryonic protein synthesis has been carried out by following the fate of radioactively labelled amino acids. The synthesis of proteins in the embryo follows a similar pattern to that of adult mammalian tissue. The activation of amino acids by attachment to adenosine - 124 - monophosphate, the first step in protein synthesis in adult mammalian tissue has been observed in chick and Xenopus embryo (Jacobson, 1960; Deuchar, 1961a, b) and in sea urchin eggs (Scarano and Maggio, 1957). Amino acids also accumulate in microsomes prior to their incorporation into protein in Paracentrotus lividus blastulae (Hultin, 1952; Hultin and Bergstrand, 1960; Monroy, 1960). Free Amino Acid 'Pool' during Bmbryogenesis Several investigators have examined the free amino acid 'pool' in the embryo during morphogenesis. 'Holtfreter et al. (1950) analysed the amino acid pattern of a range of tissues from the embryos of several species, and found a similar amino acid pattern in the protein hydrolysates. This would seem to rule out any attempt at relating free amino acid levels to specific protein synthesis. However, several workers have found consistent amino acid variations during embryogenesis. Kutsky et al. (1953) found a fluctuation in the free amino acid levels during cleavage, blastula and mid- neurulation stages of the Rana pipiens embryos. Chen (1956) and Deuchar (1956,.1958) have found reproducible fluctuations ,in certain amino acids, for example the glutamine/glutamio acid concentrations during the development of both Triturus and Xenopus embryos. This suggests a relationship between the amino acid level and the state of embryonic metabolism Amino Acids as Inducing Agents Amino acids have been studied as possible inducing agents - 125 - in the process of embryogenesis. Miller and Heimann (1940) and Miller et al. (1941) found that methionine or cystine increased the rate of cleavage of the rabbit egg in vitro. while proline, hydroxyproline or aspartic acid enhanced its differentiation. However their interpretation of the results was questioned by Deuchar (1962). Phenylalanine appears to induce the formation of the neural crest in the embryo of Ambystoma (Wilde. 1955a, b, 1956). Firstly Wilde showed that neural crest formation was induced when phenylalanine was present; secondly, that various analogues of phenylalanine affected this differentiation. Finally, he demonstrated that neural crest formation could occur when the archenteron roof was present, together with two precursors of phenylalanine. p-2-thienylalanine and phenyllactic acid. He advanced the hypothesis that the mesoderm synthesised phenylalanine from the two precursors, which then passed onto the neural crest cell precursors, initiating the neural crest formation. The use of amino acid analogues has led to other amino acids being identified as possible inducers. Bosco and Monroy (1960) using ethionine found that methionine is required for primary mesenchyme formation in the sea urchin embryo. Somite segmentation in the chick embryo appears to have a specific requirement for leucine as shown by its suppression by a leucine analogue (Herrmann. 1953; Rothfels, 1954). - 126 . Methionine analogues can also block somite segmentation (Herrmann et al.', 1955). The specific requirement for leuoine during somite segmentation in chick and also Xenopus embryo has also been found by Deuchar (1960. 1961). Very little work has been reported regarding the distribution. fluctuation or involvement of amino acids in early mammalian embryogenesis along the lines of Eakin, Chen and. Deuehar described above, and yet it would probably prove fruitful as a basis for continued research on the biochemistry of development. The work described here on the free amino acids in embryogenesis was carried out with this view in mind. Another important reason for choosing this particular aspect, the species (rabbit) and period of development (6th llth day) was the result of studies made on thalidomide. As has been previously shown (Somers, 1962) thalidomide is teratogenic in rabbit. Secondly. the drug is chemically active as an acylating agent (Fabro et al.. 1965; Linnecar et al., 1968) so possibly it may interfere with amino acid metabolism,of the embryo. The following is an investigation of the amino acid composition of the blastocyst fluid of the rabbit during morphogenesis. Experiments were also carried out to determine the effect of teratogenic doses of thalidomide on the rabbit embryo free amino acid pattern. - 127 -

MATERIALS AND METHODS

Estimation of Free Amino Acids The procedure used was essentially the same as that developed by Moore and Stein (1951). The amino acids were separated by chromatography on columns of ion exchange resin (Blowex 50 x 12), and assayed, after elution, by a photometric ninhydrin method.

Instruments A Technicon auto-analyser was used for the assay of all the'amino acid samples. The glass column (130 em.) containing the resin was surrounded by a thermostatically controlled water jacket (at 60°C). The column effluent then passed through a proportionating pump, where it was mixed with the ninhydrin reagent. The colour was allowed to develop whilst passing through a thermostatically controlled oil bath set at 100°C. Finally the extinction of the reaction mixture was measured at 445 mµ and 510 mµ using a spectrophotometer and the values recorded on a Technicon automatic recorder.

Preparation of•the Gradient Buffer The amino acids were eluted using a citrate buffer gradient of increasing ionic strength and pH. Three stock -128— buffers were used in preparing the autogradient: pH 2.875. One litre contained 0.05 M sodium citrate, 0.05 N sodium hydroxide, 5 mis. of thiodiglycol and 10 mis. of 50% Brij 35, The pH was adjusted to the required value using 6N HC1. pH 3.8. The buffer was the same as above, the pH being adjusted to 3.8. pH One litre contained 0.15 N sodium citrate, 2.0 N sodium hydroxide, 0.6 N sodium chloride and 10 mis. of Brij. 35• . The pH was.then adjusted to 5.0 using 6N HC1.

Autogradient: The autogradient consisted of 9 chambers containing mixtures of the three buffers as shown in Table 31.

Ninhydrin Reagent: One litre of ninhydrin reagent was prepared using 204gms. of ninhydrin, 1.5 gms. of hydrindantin added to 650 mis. of 2-methoxyethanol. After bubbling of nitrogen through the solution to remove oxygen, 350 mis. of 4.0 N sodium acetate buffer (pH 5.5) were added. Finally, 500 mis. of the solution were, added to 1.5 litres of 1:1 methoxyethanol and water. - 129 - TABLE 31. AUTOGRADIENT

Volume of Buffer used (mis.) Chamber . Buffer No. 1 Buffer No. 2 Buffer No. 3 pH.2.875 pH.3.8 , pH.5.0 1 70+ - + 2 72 - - 3 75 - 4 75 , . - 5 - 70 5 6 6 9 60 7 - - 75 8 - 75 9 - - 75

Chambers 1 and 2 contained 5 mis. and 3 mis. respectively of methyl alcohol. This enhanced the separation of serine and threonine. - 130 - Standard Amino Acid Mixture: A solution, containing equimolar concentrations of 18 amino acids was supplied by Technicon. A solution containing 0.2 pmole of each amino acid was used to standardise the autoanalyser.

Animals and Treatment Adult New Zealand White does (3-4 kg. body weight) were mated with bucks of the same breed; the time of copulation was considered as hour zero of the pregnancy. The pregnant animals were maintained on diet No. 41 (J. Park & Sons Ltd.) with water ad lib. Thalidomide (150 mg/kg) was administered orally daily for 2 days, before the removal of samples. Groups of three animals, with and without treatment with thalidomidei were killed by a blowOn the head at the 6th, 8th, 9th, 10th and 11th day of pregnancy. Blood samples (10-20 mis.) were collected, and the plasma obtained by centrifuging the blood at 2000 r.p.m. for 10 mins. Embryos (8-11 days) were obtained free from the uterine tissue and blood by a technique similar to that described by Brambell (1954). The uterus containing the embryos was removed from the animal and immediately frozen in isopentane cooled to -40°C using acetone/solid carbon dioxide mixture. After 2-3 min. the frozen uterus was carefully opened and the frozen embryos removed from the site of implantation. At 6 days the uterus was carefully dissected without prior - 131 - freezing, and the whole blastoeyst carefully picked out from the surrounding tissue. -The embryos were separated from the yolk sac fluid by centrifuging at 10,000 r.p.m. for 10 mins. at 0°C using a M.S.E. high speed centrifuge (Model 17).

Preparation of samples for Amino Acid Analysis Maternal plasma (2-5 mis.) and yolk sac fluid (0.5-5 mis.) were treated with 5X the volume of 1% picric acid to precipitate all the protein. After shaking the precipitate for a few moments, the picric acid precipitate was removed by centrifugation at 2,000 r.p.m. Embryos (0.5-2 gms. wet weight) were first washed with ice cold saline to remove excess yolk sac fluid, then were dispersed in a Waring blender with a 10-fold amount of 1 per cent picric acid. The precipitate was then removed by centrifugation. Excess picric acid was removed by passing the protein free extracts through columns of Permutit deacidite (FF.SRA.61- chloride form), 4 ems. high, 2 cm. in diameter. The resin bed and walls of the chromatograph tube were then washed with five 3 ml. samples of 0.02N HC1. The colourless effluents were concentrated to 1-2 mis. on a rotary evaporator, the pH adjusted to 7.8 with 1N Na OH, and allowed to stand at room temperature for 4 hours to oxidise eysteine to oystine. The solution was then made up to a final volume of 5 mis. and 'the pH adjusted to pH2 with IN HCI. -132- Chromatographic Analysis Samples (0.2-0.4 ml.) of tissue extract were then introduced into the Dowex column to assay the amino acids present. Samples not used immediately were stored at -200 A standard analysis was carried out, with 0.2 µ mole of 18 amino acids, with 0.2 µ mole of norleueine AS an internal standard. The concentration of the amino acids in the various extracts was then obtained by comparison of the area under the peak for the particular amino acid with the area for the standard amino acid. - 133 -

RESULTS

Free Amino acids in Maternal plasma, yolk sac fluid and embryo at days 6-11 of _pregnancy Table 32 shows the amino acids found in the three tissues examined, and the typical elution curves obtained from extracts of maternal plasma, yolk sac and embryo are shown in Figs. 9 and 10. 6th day of pregnancy. At the 6th day of pregnancy, the amino acid concentration of the blastooyst is seen to be very high, approximately 1500 moles/100 mis., 2000 µg./ml. Nearly two-thirds of the amino acid nitrogen present consists of glycine, the simplest of the, amino acids. Alanine, serine, lysine and glutamic acid are also present in quite high concentrations. 8-11 day yolk sac fluid. At 8 days there is a large drop in the amino acid concentration in the yolk sac fluid from 1500 to 700 moles/100 mis. fluid. This is largely accounted for by the fall in the concentration of glycine from 1000 moles at 6 days, to a level of 265 moles/100 mis. at 8 days. There is a general fall in other amino acid levels also, although those of glutamic acid, alanine, proline, histidine and , ornithine show a reversal of the general trend. Arginine, present in measurable concentrations at the 6th day (18 gmoles/100 mis.), appears to be completely absent from the 8th day onwards.

1 TABLE ,2.4 CONCENTRATION OF FREE AMINO ACIDS IN THE MATERNAL PLASMA, YOLK SAC FLUID AND EMBRYO LURING DAYS 6-11 OP PREGNANCY iw

.1%.10•111110.60•••••01.0. 6 DAY' 10 DAYS 11 DAYS Amino Acid Yolk sae fluid ernaLPlas Yolk sac flub sae fluid 'Internal Plasma Yolk sac fluid Enbryo al Plasma Yolk sac fluid eleT al Plasma (7.5-8.6) 2.7 (2.0-3.3) 7.4 (5.0-11) 20 (18-22) 8.5 49 (46-49) 2P*8 (2.3.3.0) 9.2 37 (30-43) Aspartio acid 3 2.4 (2.0-3.0) 9.0 (7.0-13.0 3.0 (2.9-3.1) 20 45 (44-47) (16.37) 36 (23-59) 39 (36-41) 48 (41-57) 32 (19-39) (21.5-35) 17 (12-25) 26 (16-33) 45 (31-60) (12-21) reonine 70 (67-76) (34-69) 31 (24-41) 50' (31-64) 90 (66-113) (22-41) 37 $9 (95-96) (26-32) 65 (49-95) 108 (79-120) erine 41 (33-50) 145 (140-150)' 36 (26-55) 51 (65-67) (7.5-11) 47 (26-66) 79 (e3-94) 11.3 (10-13.1) (34-51) (7.0-16) 4.5' .(36-60) 75 (49-100) (13-20) 76 Glutamic acid 17 (14-22) •37 (36-38) (45-48) (29-48) .34 (28-46) (57-60) (14-29) 37.5 57 (55-58) 224.: (19-23) 38 (31-50) 44 (41..48) Froline 47 (42-50) 18 (11-27) 38 (29-48) 37 57 186 (166-176) (94-110) 184 (138-327) 215 (152-255) (1030-1120i 147 (124-184) (250..278) 146 (93-202) 185 (116-260) 200 (160-250) (111-159) 178 Clycine 143 (113-160) 1080 (148-196) 36 (28-40) 190 (170-280) 182 (134-240) (33-47) 162 225 (213-242) 3:.°;' (27-39 157 (152-260) 203 (156.287) Alanine 41 (40.43) 89 (32-95) 31 (27-35) 14. (3.2-4.6) Trace 6 (4-9) Trace ,1 2 Trace Trace 2.4 (1.4-3.2) 6 6 (5-6.9) 3 Trace (2.(-3) 4 qyatine (14-16) 20 (13-24) '14 (11-19) 33 (25-42) (20-27) 16 (11-19) 4 (28-37) (12-19) 16 (12-21) 26 (24-49Tt% Valine 25 (23-28) 19 (16-23) 18 (14-24) 5.2 (4.0-6.5) 6.5 (6-7) 45 (4.5-6) 5 (2.2-9) 6 (6) 10.3 (7-13) 4.6 (3.0-4.0) 8 (7.7-8) 5.6 (5-7) 5.5 (3-8) (3.0-9.5) Methionine 8.3 (8-9.0) 8 (2.5-2.9) (4.9) 3.5 (2-5) 13.5 (11.5-16) (7.5-11) 4.8 (4-6.2) 13 (11-16) (5) 3.6 (2-5) 12 (11-13.5) Isoleucinc 9 (9) 11 (5-18) I 5.6 (5.0-64) 7 1 (11-17) 11 (8.8-12) 29 (27-30) 4i.5 (6-8) 8.5 (6-12) 24 (23-25)` Lauoine 13.6 (11.14) 12 (10-13) 8.9 (7.6-10.0), (5.3-7.5) 11.3 (7-14) 27 (18-35) 1ti "6:: 9 (7-12) 17 (15-18) 6.40-,. (6.0) 10 (8-14) 25 (2.127) (7.5-9) Wrostne 10.3 (10-11) 19 (16.26) 8.1 (6.5-.9.0) (6.2-9.5) 9 (5-12) 8 (6-10) 17 (12-20) (4-8) 4.7 (4-6) 13 (12-14) 14* (4) 7.3 (4.8-11) 16 (i2-19) Fhenylalanine 7 (6-8) 7.3 (7-e) 5.4 (447.6,0) (5-7) 5.1 (3-7) 4;5.5 (4.5.7) 17 (12-21) (90-180) 75 (63-101) 489 (420-530) 140.4, (105-115) 130 (62-200) 420 (370-450) kmonia 134 (133-136) 240 (220-280) 120 (113.124) (150-173) 154 (58-210) 160 (120-310) 500 (390-600) (24-29) 24 (22.25) 87 (66-88) (16-17) 36 (34-39) 50 (43-60) Lysine 27 (25-.29) 72 (65-78) 21 (16-29) (18-26) 19 (13-26) 25 (17-30) 57 (27-79) (12-15) 17 (6-16) 20 (17-22) 100 (10) 19 (17-21) 33 (32-34) Histidine 12.3 (12-13) 9 (8-10) 9.9 (6.9-14) (12-17) 16 (8-25) 11' (9-13) 16.5 (20-41) (17-20) None None (12.5-11.3 None None Ar3inine 17.6 (15-19) 7 (6-8) 13 (11-21) e 16 (15-17) 'Nene None (13-17) 8.5 (6-11) 57 (30-71) Ief (12) 13 (12-14) 53 (36-69) Ornithine 1 16 (14-20) 8.1 (6.4--9.1) 17 (15-18) (10-15) 8 (4-10) '8 (5.4-11) 41 (30-61) ,11./

4New Zealand Uhite does (body wt. 3..4 k6.) itlintained on diet No. 41 Mknank and Sons Ltd.) and vater ate. The results are the mean from three or more animals. Zach of these values was done in duplicate. The concentration of the amino acids is given in umoles/ 1O mla. of maternal plasma, yolk sac fluid or embryo. :ranges are given in brackets. ' -135-

FIG.9 FHLE AMINO ACIDS PTLSENT IN:-

(A) 6 LAY BLASTOCY3T FLUID (0.08 ml)

6DAY BOS*051 (0.08,00

(..i) 9 LAY Ell3HY0 EXTRACT (0.125 ml)

a - 136 -

FIO.10. FREE AMINO ACIDS PRESENT IN:- ( C) 10 DAY MATERNAL PLASMA (0.4 ml)

MM!RM& RIT4tt

( L ) 10 DAY YOLK SAC FLUID (0.4 ml)

- 136(a) - KEY TO THE AMINO ACID PEAKS IN FIGURES 9 AND 10

No. on Chart Amino Acid 1 Aspartic acid 2 Threonine 3 Serine 4 Glutamio acid

5 Praline 6 Glycine Alanine 8 Cystine 9 10 Methionine 11 Isoleucine 12 Leuoine 13 Tyrosine

'14 ..... Phenylalanine 15 Ammonia 16 Lysine 17 Histidine 18 Arginine NL Norleucine - 137 - The amino acid levels of the yolk sac fluid remain relatively constant from the 8th'to 11th day. However. there is a general increase in amino acid level at the 11th day, but this'is not nearly as dramatic as the drop observed between the 6th and 8th day. Amino acids in the embryo. No material was available for the measurement of the amino acids in the- embryo at the 6th and 8th day. From the 9th to llth day. the amino acids present'are seen to be accumulated in the embryo with respect to the yolk sac fluid. The maternal plasma levels are relatively constant irrespective of the stage of pregnancy; no significant variations were observed in the amino acid levels. Generally speaking the plasma levels reflect the general trend observed in the yolk sac; thus, glycine, alanine and serine are present in the greatest concentrations, whilst aspartiO acid, cystine, methionine, isoleucine, tyrosine and phenylalanine are only found in small amounts. Unlike the embryonic tissue, arginine was found in quite measurable amounts (13-18 gmoles/ 100 mis.). Trace amino acids. Besides the amino acids shown on Table 32# various other ninhydrin positive peaks were observed in both maternal and embryonic tissue. By introducing known compound into the sample during analysis, some of these minor peaks were tentatively identified as ethanolamine phosphoric acid, taurine, amino butyric acid and urea. . 138 - The effect of thalidomide on the free amino acid levels. Table 33 shows the amino acids found in the maternal plasma and yolk sae fluid on the 10th day of pregnancy following the treatment of the pregnant rabbit with thalidomide (150 mg/kg., administered on the 8th and 9th day of pregnancy). The amino acid levels are seen to relate quite closely with those found in the untreated animal. There appears to be no significant difference in any of the amino acid levels. Only ornithine reaches a level in the yolk sac fluid of the treated animal outside the ranges observed in the untreated rabbit (17 pmoles and 11 pmoles/100 xis. respectively).

Comparison of the amino acid levels in the maternal plasma. yolk sac and embryo Tables 34, 35 and 36 show_the relationship between the amino acids of the three tissues studied. From the distribution ratio between the amino acids of the maternal plasma and yolk sac fluid (Table 34), certain . patterns emerge. Firstly, where their concentration in the yolk sac is approximately the same as in the plasma, that ie, a ratio of 1. 'This is found with threonine, serine, proline, valine, methionine, leueine,tyrosine, phenylalanine, lysine, histidine, ornithine and glycine. Secondly, certain amino acids are accumulated in the yolk sac fluid at a level 2-6 times that observed in the plasma, namely aspartie acid, glutamic acid and alanine._ Thirdly, - 139 -

TABLE 33. CONCENTRATION OF FREE AMINO ACIDS IN THE MATERNAL PLASMA AND YOLK SAC FLUID AT DAY 10 OF PREGNANCY FOLLOWING ORAL ADMINISTRATION OF THALIDOMIDE Concentration of Amino Acids in Amino Aoid pmole/100 mis. Tissue Maternal Plasma Yolk sac fluid Aspartic acid 3.0 (3.0) 7.5 (5-10) Threonine 26 (21-31) 27 (17-37) Serine 41 (38-44) - 47 (35-59) Glutamic acid 19 (15-23) 40 (26-54) Proline 39 (34.44) 28 (17-40) Glyoine 157 (124-191) 178 (137-218) Alanine 50 (45-55) 130 (89-170) Valine ' 25 (25-26) 13. (6-21) Cystine Timoe Trace Methionine 6.5 (6.0-7.0) 4.5 (3-7) Isoleueine 7.5 (7.0.8.0) 3.5 (2-5) Leucine 12 (12-13) 9.0 (5-13) Tyronine 11 (11) 9.5 (5-14) Phenylalanine 5.5 (4.0-7.0) 2.0 (1.3) Ammonia 141 (139.144) 213 (206-222) Lysine 23 (23-24) 26 (16-36) Histidine 12 (11-14) 11 (7-15) Arginine 14 (11-18) None Ornithine 31 (20-42) 17 (17)

New Zealand White rabbits were treated as in Table 32. Thalidomide was administered orally on days 8 and 9 of pregnancy, 150 mg/kg., as a suspension in 1% methyl cellulose. -140- TABLE 34. AMINO ACID RATIO BETWEEN THE YOLK SAC FLUID AND MATERNAL PLASMA Ratio - Yolk sac fluid. Maternal Plasma Day of Pregnancy 6 8 9 10 11 Aspartio acid 3.8 2.7 2.7 2.8 3.3 Threonine 0.7 0.8 1.5 1.2 2.2 Serine 3.5 1.3 1.3 1.0 1.7 Glutamic acid 2.2. 3.7 4.1 3.3 5.2 Proline 0.4 1.2 0.9 1.5 1.7 Glyeine 7.6 1.8 1.3 1.4 1.9 Alanine 2.2 5.4 5.3 4.3 4.7 eystine 1.0 0.7 0.7 1.5 1.0 Valine 0.8 0.8 0.7 0.7 0.9 Methionine 1.2 1.7 . 0.9 0.8 0.7 Isoleucine 1.2 0.5 0.5 0.5 0.7 Leueine 0.9 0.7 0.7 0.8 1.3 Tyrosine 1.8 1.0 0.9 1.1 1.6 Phenylalanine 1.0 1.1 1.1. 0.9 1.8 Ammonia 1.8 1.3 1.0 0.6 1.1 Lysine 2.7 1.0 1.3 0.9 2.2 Histidine 0.7 1.4 0.7 0.9 1.9 1 1 1 1 Arginine 0.4 m U 7 Tc Ornithine 0.5 0.7 1.0 0.6 1.1 - 141. TABLE 35. RATIO BETWEEN AMINO ACIDS OF THE MATERNAL PLASMA AND EMBRYO AT VARIOUS TIMES OF PREGNANCY

Day of Pregnancy 9 10 11

Aspartic acid 7.4 15.8 13.2 Threonine 1.7 2.7 1.5 Serine' 2.4 2.6 3.7 Glutamio acid - 6.8 4.9 8.7 Proline 1.5 2.2 2.0 Glycine 1.4 1.5 2.2 Alanine '5.1 5.9 6.1 Cystine - - - Valine. "1,;7 1.5 1.6 Methionine 1.0 0.93 1.0 Isoleucine '1.9 1.4 2.4 Leueine '2.1 2.6 3.7 Tyrosine '1,9 2.1 4.1 Phenylalanine 3.3 2.4 4.0 Lysine' '3.0 3.3 3.8 Histidine '1.0 1.5 3.0 . 1 1 1 Arginite 17 a rc Ornithine 5.0 3.9 4'.4 142- TABLE 36. RATIO OF CONCENTRATIONS OF AMINO ACIDS BETWEEN THE EMBRYO AND YOLK SAC FLUID

Ratio - Embryo amino acid Yolk sac amino acid

Day of- Pregnancy 9 10 11 Aspartic acid 2.7 5.6 4.0 Threonine 1.7 2.2 1.1 Serine 1.8 2.5 1.7 Glutamic acid 1.7 1.5 1.7 Proline 1.7 1.5 1.1 Glycine 1.1 1.1 1.1 Alanine 1.0 1.4 1.3 Cystine - - - Valine 2.3 2.1 1.6 Methipnine ' 1.1 1.2 1.2 Isoleueine 3.9 2.7 3.3 Leucine 3.4 2.6 2.8 Tyrosine 2.1 1.9 2.5 Phenylalanine 3.1 2.9 2.2 Ammonia - - 3.2 Lysine 2.3 3.,6 1.4 Histidine 1.5, 1.6 1.7 Arginine - - - Ornithine 5.1 6.7 4.1 - 143 - arginine is not accumulated at all in the yolk sac. Fourthly, a striking difference is observed between the ratios observed at six days and those seen from 8 to 11 days of pregnancy. Thus the levels of serine, glycine and lysine, not accumulated in the later stage of morphogenesis are seen to be much higher in the yolk sae than the plasma at the 6th day of pregnancy. The glycine level in the yolk sac is nearly 8 times that of the maternal plasma, serine is 3i times and lysine nearly 3 times the plasma level.. Secondly, glutamic acid and alanine, accumulated to a level i to 5 times that of the plasma in 8-11 day yolk sac fluid is,accumulated to a much lesser extent at 6 days. Finally, arginine, not detected in the embryonic tissue at 8-11 days, is present in the yolk sac fluid at 6 days (7 moles/100 ml.). Table 36 gives the relationship between amino acids present in the embryo, and those found in the yolk sac from the 9th to the 11th day of pregnancy. The concentration of all the amino acids in the embryonic tissue is at least equivalent to that of the yolk sac fluid. A general accumulation of all the amino acids is observed, and especially aspartic acid, isoleuoine, leucine, phenylalanine and ornithine, with embryo levels 2i to 6 times that found in the yolk sac. Those amino acids not accumulated to any extent are glycine, alanine, nethionine and histidine. Again. arginine is not detected in the embryonid tissue during this period. - 144 -

DISCUSSION

At 6 days post coitum, development of the rabbit embryo has reached the blastocyst stage, consisting of a small mass of undifferentiated cells. The blastocyst is freely suspended in the uterine fluid which has been shown to contain quite high levels of amino acids (Gregoire et al., 1961). There have been conflicting reports as to the ability of the blastocyst to absorb any nutrients from the surrounding medium at this stage, thus neither Greenwold (1959) nor Lutwak-Mann (1960) were able to show penetration into the rabbit blastocyst of a variety of labelled nutrients following their addition to the uterine fluid. However, Daniel (1965) showed that development of the rabbit embryo in vitro was enhanced by the presence of amino acids in the suspending medium. The amino acid level of the 6 day blastocyst is very high (1500 ymoles/100 mis.). If no absorption of amino acids takes place after ovulation, the concentration of amino acids must be even greater, as the volume of the ovum is less than that of the 6 day blastocyst. Lesinksi et al. (1967) have also assayed the amino acids present in the blastocyst of the rabbit at this stage of development. They too found a total amino acid concentration of approximately 1500 ymoles/100 mis. (2000 µg/100 mis.). Their results also showed that glycine

was present at a high concentration (300 400 moles/ 100 mis.). However, they found alanine (600 µmoles/100 mis.) - 145 - to be present at an even higher concentration. He found other amino acids, namely glutamic acid, aspartic acid, threonine, serine. lysine and histidine to be present in quite considerable amounts. His results also demonstrate that 'absorption of amino acids does take place between the 5th and 6th day of development, that is before implantation, as certain amino acids present in only detectable amounts on the 5th day* occur at quite high levels at the 6th day. They are aspartio acid, threonine, histidine, lysine and arginine. The difference in amino acid concentration between the blastocyst and maternal plasma also shows that the blastocyst is able to absorb amino acids selectively, and against a concentration gradient. Thus a control mechanism must be operating to maintain the correct level of amino acids. , Does this control originate from the embryo? From the 8th day of development, a marked drop in the concentration of amino.acids in the yolk sac is observed. However, during the preceding 36 hours of development, several changes occur that could account for this drop in amino acids. Firstly, implantation has occurred, and this is known to affect the permeability of the blastocyst membrane. During this period. the volume of the yolk sac fluid increases rapidly, which could have the effect of diluting the amino acids. present,, unless an equivalent absorption of these compounds occurs. - The amino acids now entering the blastocyst could arise from two sources: direct absorption of the free amino acids, or - 146 - from the serum proteins entering the yolk sac and their subsequent hydrolysis to give the free amino acids. A change in the general pattern of amino acids is also observed after the 8th day. The concentration of seven amino acids, namely threonine, serine, glycine, isoleucine, lysine. leucine and tyrosine, fall to levels * to the value.of the 6 day blastocyst. Very similar results were observed by. Lesinksi et al. With the exception of alanine, the concentration of which rises at the 8th day (from 90 to 170 vmoles/100 mls.), the other amino acid levels remain constant. This change in the amino"acid. pattern. which is maintained through to the llth day of pregnancy. is probably due to the greater permeability of the yolk sac. Asrthe:pattern is now much closer to that of the maternal plasMa. The amino acid levels of the yolk sac from the 8th to,the- llth day still differ in many respects from that of the maternal plasma. This demonstrates that the embryo is still capable of maintaining its own internal environment. The embryonic tissue also. selectively absorbs amino acids from the yolk sac fluid. Thus, from the 9th to the 11th day, '13 amino acids are present in the embryo at a greater concentration than the yolk sac fluid. This is probably a reflection of the degree of synthesis of new protein which is occurring at this time. SUMMARY . 147 - Thalidomide was shown to be responsible for the epidemic of congenitally malformed children in Western Europe during the period 1958-1962. Laboratory studies showed thalidomide to be the teratogen in several common species, such as the rabbit, rat. chicken, mouse and monkey. However, not all species were sensitive to thalidomide, in particular the golden hamster. Pregnant golden hamsters were administered thalidomide, 1 or 2 g/kg. orally, daily from the 4th to the 12th day of gestation. On the 15th day of pregnancy the offsprings were removed by caesarian section and examined for malformation. No malformations were detected, and the incidence of reabsorption was of the same order as for control animals. The atypical response to thalidomide in hamster from that of other animals tested could be due to a fundamental difference at the site of action of the drug. Conversely. It could be a result of a different metabolic fate of the drug in the hamster. Pin-pointing this difference could establish the reason for its teratogenic action in the other species examined. Therefore. the metabolic fate of thalidomide in the golden hamster was investigated in detail, and the findings compared to those observed in the .rabbit. 14C-labelled thalidomide (20 µc/kg.. 150 mg/kg.) was orally administered to pregnant golden hamsters on the 9th day of gestation. The absorption, plasma levels and excretion of thalidomide, and also the penetration and persistence of - 148 - the teratogen and its hydrolysis products in the embryo were examined. The results showed that thalidomide behaved in a similar way in the golden hamster as in the rabbit. About 70-80% of the radioactivity was excreted in the urine in the first 24 hours The plasma levels and yolk sac concentration were found to be even higher in the hamster than the rabbit (17.2 and 19.1 and 7.6 and 4.2 µg/ml. respectively) after 4 hours. The lack of toxicity of the hamster to thalidomide is obviously not due to its inability to penetrate to the.embryo. The fate of thalidomide was followed by chromatography of extracts of yolk sac and plasma fluids. Thalidomide and six of its hydrolysis products were found, and the relative proportions of these compounds were of the same order as observed following a similar administration in the rabbit. It can be concluded that the failure of the hamster to respond to the teratogen is not due to its inability to penetrate the embryo. The answer must lie` at the site of action of the teratogen. Thalidomide is regarded as non-toxic to the adult, mice having recovered from doses of up to 10 gm/kg., and large quantities of the drug having been administered to other species without ill-effect. However, the isomers of thalidomide were shown to be acutely toxic in mice, the oral L10150 for the D. and. L-form in I.C.I. S.A.S. mice being 400 and 700 mg/kg. - 149 - respectively. The toxicity of the isomers has been investigated in mice, and an attempt has been made to resolve the reason for the difference in toxicity between thalidomide end its isomeric forms. Sex and strain differences in response were observed following administration of the isomers. Following oral administration, the isomers were toxic in I.C.I. S.A.S. and

TO but not CF1 strains of mice. The compounds are toxic in all three strains of mice following intraperitoneal administration. It can be concluded that the isomers are . poorly absorbed from the intestine of the CF1 strain. The compounds are also more toxic in the male than the female of the species, and this is pronounced in I.C.I. S.A.S. and TO strains, where the compounds are from 2 to 3 times more toxic in the male than female. A difference in relative toxicity of the isomers was also observed, D-thalidomide being from one to three times more toxic than its antipode. As the physical and chemical properties for the two compounds are Identical, there must be a stereospecificity for the D-isomer, for example at the cell membrane across which the compounds must pass to reach their site of action. This could result in the more rapid accumulation of the D-isomer to the critical concentration. As far as the mechanism of toxicity of the isomers is concerned, various possible antagonists were administered - 150 - together with the isomers, in order to establish the site at which the compound acts. The concurrent administration of amphetamine with the isomers was found to alleviate the c.n.s. depressant action of the drugs, and increasing the up50, indicating that the toxicity of thalidomide is due to a central effect. A surprising observation was that the concurrent administration of D- and L-thalidomide abolished their individual toxicity. Thus, when a lethal dose of both the D- and the L--were administered, either by the same or by different routes, no deaths occurred. It was concluded that the isomers associated in solution to form a dimer which interfered with their penetration and attachment to the site of action.' This conclusion was substantiated by examination of the physical and chemical properties of the isomers and comparing them to those of the racemate. Thus, the results obtained for the molecular weights in camphor, the rate of hydrolysis, and the solubility in various solvents, indicate that the DL- form can exist as a dimer in solution. These results indicate that the difference in toxicity between the lib-. L- and DL-thalidomide is due to differences in the physical and chemical properties of these compounds. These properties would be expected to affect the fate of the compounds following their administration to an animal. Therefore, a comparison of the absorption, excretion and . 151 distribution of DL-, D-, and L-thalidomide was made in the mouse following the oral administration of the 14 C -labelled compounds. 14C -D-, -L- and -DL-thalidomide was administered to male I.C.I. S.A.S. mice at both a non-toxic (200 mg/kg.) and toxic (1000 and 2000 mg/kg.) dose level, and the rate of excretion and tissue distribution determined. The more rapid rate of urinary excretion of the D- and. L- and the greater faecal excretion of the DL- show that the latter is less completely absorbed. The plasma and brain levels of the and L- compounds are four to 'six times higher than the DL- at two and four hours after administration but fall to a similar level as that of the DL- at 12 hours. It is concluded that the isomers are more rapidly absorbed. and that their higher acute toxicity is a reflection of their higher plasma levels. As the plasma levels of the D- and Ie..' are similar, but the D- isomer is from two to three times more toxic than the L-6 a higher toxicity must be associated with the unnatural D-form. From administering a lethal dose of L-thalidomide (2000 mg/kg.) and a similar, though.non-lethal, dose of DL-thalidomide, it was concluded that a plasma level of the L- of 135-145 4g/ml. is lethal. A plasma level of only half this was reached with the DL.-compound. L-Thalidomide (1000 mg/kg.) was administered concurrently - 152 - with an equivalent dose of the D.. At this dose level, both compounds were toxic when given separately, but not when given together. The plasma level of the L. at this dose level was 125 tleml. at 8 hrs. Assuming a similar absorption of the D. isomer, the two together must exceed that of a single lethal dose of the L- isomer. Previous results had already indicated the possibility of dimerisation of the D. and L- forms when together in solution. It was concluded that while the isomers are toxic, the diner is less active, and may depend for its activity on the dissociation into the free isomers. All the research so far carried out has failed to find a mechanism for the teratogenic action of thalidomide. Our knowledge of the biochemical processes involved is very limited and it seems that the chances of finding an answer to the problem remain rather remote until we have a better understanding of embryogenesis. It was considered essential that we expand -our knowledge in this field. For this reason a study was made of the amino acids of the embryo during morphogenesis. The amino acid pattern of the yolk sac, embryo and maternal plasma was determined from the 6th to the 11th day of morphogenesis. and a similar study made following the administration of thalidomide to the pregnant rabbit. The results show that the embryo is capable of selective absorption of amino acids, and that most of the 18 amino acids studied are accumulated by both the yolk sac and embryo. - 153 - At six days the yolk sac fluid is seen to contain certain amino acids in very high concentration, especially glycine (1000 moles/mi.). The general pattern at this time is also unlike that to be found in the maternal tissue. From the 8th to 11th day the pattern alters radically.' At this period, implantation occurs and the change it attributed to the alteration in permeability of the yolk sac membrane. .The general pattern is seen to conform more to its maternal environment. However, differences in levels are still in evidence and the ability to accumulate amino acids against a lower maternal plasma level is retained. Thalidomide did not appear. to affect the amino acid pattern of the yolk sac fluid. . If thalidomide does affect amino acid metabolism, the change is too small to be . detected by,the procedure used fot this assay. At this stage, no conclusion could be drawn as to a specific function. of the amino acids in embryogenesis other than their Utilisation in protein synthesis. However, the ground work has been laid upon which we can expand our knowledge of the role of these compounds in embryogenesis. Their prime function is undoubtedly protein synthesis,.but future research is likely to demonstrate , their importance in secondary, yet essential roles in embryogenesis. REFERENCES - 154 Abou El Makarem, M.M., Millburn, P., Smith, R.L. and Williams, R.T. (1966). Biochem. J.,SZ 3. Adler, T.K. (1952). J. Pharmac. exp. Ther., 106, 371. Amirkhanian. J.D. (1966). Prot. R. Mioroscop. Soc., 1, 153. Austin, F.H. (1967). Fedn. Proc. Fedn. Am. Soos. exp. Biol., 26. 1001. Bath, A., Biohel, J. and Hejgaard, J.J. (1963). Lancet 1, 1271. Beck, C. (1904). Z. phys. Chem., 48, 670. Beck, F., Spencer, B. and Baxter, J.S. (1960). Nature, Lond., 1§2.* 605. Beckmann, R. (1962). Arznelmittel.Forsoh., 12, 1095. Beer, Ca., Dickens, F. and Pearson, J. (1951). Bloohem. J., 88, 222. Bergatr8m, R.M., Bergstrdm, L., Putkonen, P. and Saint, K. (1963). Medna exp., 11. 119. Beutler, E. (1964). J. Am. med. Ass., .1112. 143. Bignami, G., Bovet, D., Bovet-Nitti. F. and Rosnati, V, (1962). Lancet 2, 1333. Bignami, G., Bovet-Nitti. F. and Rosnati, V. (1963). Proceedings of the 3rd International Meeting in Forensic Immunology, Medicine, Pathology and TOxioology, p.124. Bignami. G., Bovet-Nitti, F. and Rosnati, V. (1964). In Excerpta Medioae, Int. Congress Series, No. 80, p.124. Blather. J. and Belkin, R.O. (1927). Trudy Lab. elope B101. mock. Zoopk. p.227. - 155 Blasiu. A.P. (1958). Medsehe. Klin., 12, 800. Bonet'. A.D. (1963). Nature, 12§, 1069. Bore, P.J. and Sounthorne, R.J. (1966). Lancet. 1, 1240. Boson. M. and Monroy, A. (1960). Acta Embryo).* Morph. exp.. 1.• 53. Boylen, J.B., Horne, H.H. and Johnson, W.J. (1963). Lancet, 1, 552. Boylen, J.B., Horne, H.H. and Johnson, W.J. (1964). Caned. J. Biochem., La. 35. Brambell, F.W.R. and Hemmings, W.A. (1949). J. Physiol., Lond.. 108, 177. Brambell, F.W.R. (1954). Cold Spring Harb. Symph. Quanta Biol., 12, 71. Brent, R.L. (1964). J. Pediat., 64. 762. Brierley, J. and Hemmings, W.A. (1956). J. Embryol. exp. Morph., 4, 34. Brinster. R.L. (1965). J. exp. Zool., la., 69. Bruni, T. (1902). Atti Aooad. naz. Lineei Memorie., 11, 212. Iruni, Ts (1904). Atti Mead. naz. Lineei Memorie.. la, 349. Burley. D.M. (1960). Med. Wid., Lond., 21. 26. Bush, E.T. (1963). Analyt. Chem.. 11, 1024. Byk, R. (1904). Z. phys. Chem.. Le31, 682. Calabresi. P. and Turner, R.W. (1966). Ann. intern. Med.. 64. 352. . 156 Chaube. S. (1965). In Teratology Principles and Techniques. eds. Wilson. J.G. and Warkany, J. p.162. Chicago: University of Chicago Press. Chaudhary. K.D. and Lemonde. A. (1964). Biochem. Pharmac.,

Chen, P.S. (1956). Expl. Cell Hes.. 12.. 675. Cohen, S. (1962). New Engi. J. med., lg. 1268. Coney. A.H., Sansur, M. and Soroko, F. (1966). J. Pharmac. exp. Ther.. 111, 133. Cotran, H., Kendrick, M.I. and. Kass, E.H. (1960). Proc. Soc. exp. Biol. Med.. 10, 424. Crabtree, H.G. (1949). Br. J. Cancer, 387. Daniel, J.C. (1965). J. Embryol. Exp. Morph., 1j, 83. De Souza, L.P. (1959). Br. med. J., 2. 635. Degenring, F.H. (1964). Dies Heidelberg Delahunt, C.S. and Lassen, L.J. (1964). Science N.Y.. 1!.$6.., 1300. Di Paolo, J.A. (1963). J. Am. med. Ass.. 104.139. Di Paolo, J.A. and Wenner, C.E. (1964). Science N.Y., 144. 1583. Diezel. P.B. (1963). Med. Woohr. (Minch). Mu. 2265. Emu:harp E.M. (1956). J. EMbryol. ezp. Morph.. 4. 327. Deuchar, E.M. (1958). Expl. Cell Res., 14. 84. Deuchar, E.M. (1960). J. Embryol. ezp. Morph., 8, 259. Deuchar. E.M. (1961a). Nature. Lend.. 121, 1006. Deuchar, E.M. (1961b). Expl. Cell Res., 364. Deuchar, E.M. (1962). Biol. Rev.. 22, 378. . 157 - Ebert, J.D. (1958). The chemical basicofdevelopment, p.526. Ed. McElroy and Glass. " Baltimore:John Hopkins Press. Ehmann, B. (1963). Lancet, 1. 772. , Esser, H. and Heinzler. F. (1956). Therapie Gegenw..'211 374. Evans, D.A.P. (1965). J. Chron. Dis.„ 18.59. Evans* M. (1968). Unpublished observation. Evered, D.F. and Randall, H.G. (1963). Br. med. J., 1. 610. Fabro, S., SchuMacher,'H., Smith, R.L., Stagg, R.B.L. and Williams, R.T. .(1965). Br. J. Pharmac. Chemother.. g.j.* 352._ Fabro, S., Smith, R.L. and Williams, R.T. (1965). Nature, Land., 208,1208. Fabro, S. (1965). In-lEtbryopathic Activity of Drugs'. Eds. Robson, J.M., Sullivan, .F.M. and Smith, R.L. Londonj. & A. Churchill Ltd. Fabro, S. and Smith, R.L. (1966). J. Path. Batt., 22, 511. Fabro, S., Smith, R.L. and Williams, R.T. (1967). Bioohem. J.. Io4, 565 Fabro. S. (1966). Ph.D. Thesis. Fabro, S., Smith, R.L. and Williams, R.T. (1967). Nature, Lond., gm, 296. Fabro. S., Smith, R.L. and Williams, R.T., (1967). Nature, Lond., all. 1208. Felisati, D. (1962). Lancet, 2, 724. - 158 - Field, E.0., Gibbs. J.E., Tucker, D.F. and Hellmann, K. (1966). Nature,.Lond., 211. 1308. . Finoato, M. (1957). Boll. Soc. ital. Biol. Spar., Iz 872. Floersham, G.L. (1966). Lancet. 1, 207. Florence, A.L. (1960). Br. med. J., 2,,1954. Fouts, J.R. and Adamson, R.H. (1959). Science N.Y., lar, 897. Frank, 0., Baker, H., Hutner, S.H. and Sobotka, H. (1963). Pros. Soo. exp. Biol. Med., la. 326. Fratta, I.D., Sigg. E.B. and Matorana, K. (1965). Toxic. appl. Pharmao., 2, 268. Friedman, L. Shue. G.M. and Hove, E.L. (1965). J. Nutr., 309«

Fullerton, P.M. and Kremer, M. (1961). Br. med. J.. 2, 855. Gershbeim, L.L. (1965). Bioohem. Pharmao.. 14, 893. Giaoomello, G., Malatesta. P. and Quaglia. G. (1964). Nature. Loud.. au, 940. Gillman, J., Gillman, T., Gilbert, C. (1948). S. Afr. J. med. Sot.. LI, 47. Giroud. A., Tdohmann-Duplessis, H. and Neroier-Parot. L. (1962). C.r.Se'eno. Soo. Biol., 151, 765. Giurgea, C. and Nolyersoons. F. (1963). Nedna. exp., 8. 66. Glass. L. (1963). Am. Zool., 2, 135-156. Glazko, A.J., Wolf. L.M., Dill, W.A., Bratton. A.C. (1949). 3. Pharmao. exp. Thar., 26, 445. Goldsohmidt, S. and Wehr, R. (1957). Hoppe Seylers Z. Physiol. ahem., 10, 9. 159 - Onnert. H. (1961). Bull. Wid. Hlth. Org., ga, 702. Graves, A.P. (1945). Am. J. Anat., 7, 219. Green, J.N. and Benson, B.C. (1961). J. Pharm. Marmite., U. 117T. Greenwold. G.S. and Everett, N.B. (1959). Anat. Rec.. 171. Gregoire, A.T., Congsakdi, D. and Rukoff, A.E. (1961). Fart. Steril., 4, 322. Gunberg. D.L. (1958). Anat. Rec., ljz 310. Gustafsson. B.E., Bergstrom, S., Lindstedt. and Norman, A. (1957). Proc. Soc. exp. Biol. Med., 24. 467. Hague, D.E., Fabro„ S., Smith. H.L. (1967). J. Pharm. Pharmac.. 12, 603. Hamra, D.M., Rake, G., McKee, C.M. and MaoPhillamy. H.B. (1943). Am. J. med. Set., 206. 642. Hart, L.G., Adamson, R.H., Dixon, R.L. and Fouts, J.R. (1962). J. Pharmace exp. Thera, Da. 103. Harris, H. and Whittaker. M. (1962). Ciba Fdn. Symp. Enzymes and Drug Action, p.301. London:Churchill. Herrmann, H. (1953). J. Embryo'. exp. Morph.. 1291. Herrmann, H., Konigsberg, U.R. and Curry, M.F. (1955). J. exp. Zool.. 128# 359. Hirschberg, E., Osnos, M., Bryant, S. and Ultmann, J.E. (1964). Science N.Y.. 111A 1343. Hirschler, J. (1922). Arch. EntwMeoh. Org.. 51, 482, -160.. Holtfreter, J., Koszalka, T.R. and Miller, L.L. (1950). Expi. Cell Res., 1, 453. Homburger, F., Chaube,' S. Eppenberger, M.. Bogdonoff, P.D. and Dixon, C.W. (1965). Toxic. apple Pharmac., 7,, 686. Hultin, T. (1952). Expl. Cell Res., 2.„ 494. Hultin, T. and Bergstrand. A. (1960). Levl. Biol., 2, 61. &14_1-c&To (few,' - Jacobson, K.B. (1960). Level Biol. Conf. Tallahassee, Florida (Abstr.). - p.113. Jaffe, W.P. (1966). Vet. Rec., L8, ,1 883. Jensen, M.K. (1965). Acta med. scant'', 1.72, 783. Jondorf, W.R., Maickel. R.P. and Brodie, B.B. (1958). Biochem. Pharmac., 1, 352. Juret, P. and Aubert, C.R. (1963). Soc. Biol., 142, 246. Jung, H. (1956). Arzneimittel-Forsch., 6, 430. Jurand, A.J. (1966). J. Edbryol. exp. Morph., 16, 289. Kalow, W. (1959). Ciba Fdn. Symp. Biochemistry of Human Genetics, p.39. London:Churchill. Kalow, W. (1965). Fedn. Proc. Fedn. Am. coos. exp. Biol., 24, 1259. Kato, H., Vasaanelli, P., Frontino, G. and Chiesara, E. (1964). Biochem. Pharmao., 12, 1037. Kemper, F. and Bergere 'H. (1962). Z. Gee. exp. Med«, 1.1.0.A 86. Kemper, F. (1962). Z. Gee. exp. Med., 125, 454. Kemper, F. (1962). Lancet, 2, 836. . 161 - Kerble, H., Faigle, J.V., Fritz, H., Knesel, F., Lonsalot, P. and Schmid; K. (1965a). In 'Embryopathic Activity of Drugs, )p.210. Ed. by Robson, J.M., Sullivan, F. and Smith, A.L. London:J. & A. Churchill Ltd. Kerble, H., Lonsalot, P., Mailer, A.K., Faigle, J.W. and Schmid, K. (1965b). Ann. N.Y. Acad. Sol., 223,, 252. King, C.T.G. and Kendrick. F.J. (1962). Lancet, 2, 1116. Koppanyi, T., Murphy„.W.S. and 'Gray, P.L. (1934). J. Pharmac. exp. Thar.. 20 78. Kuensberg, E.V., Simpson, J.A. and Stanton, J.B. (1961). Br. med. J., 291. Kuhn, W.L. and Van Maanen, E.F. (1961). 3, Pharmac. exp. Ther., 1, 60. Kunz, W., Keller, H. and Mdokter. H. (1956). Arzneimittel- Fosch. 6, 426. Kutsky, P.B. Eakin, A.M., Berg, W.E. and Kavanau, J.L. (1953). J. exp. Zool., 124,, 263. Lehmann, H. and Liddell, J. (1964). In Progress in Medical Genetics, 75. Eds. Steinberg, A.G. and Bern, A.G. New York and London:Grune and Stratton. Lechat, P., Deleau, D., Bane, A. and Bunot, 0. (1964). Th4iapie, 13..1. 1393. Lenz, W. (1961). Dt. med. Wschr.. 86: 2555. Lenz, W. (1962a). Lancet, 1, 45. Lenz, W. (1962b). Lancet, 1, 271. - 162 - Lena, W. (1965). In Embryopathic activity of drugs, p. 182. Eds. Robson, J.M„ Sullivan, P.M. and Smith, R.L. London:J. & A. Churchill Ltd. Lenz, W. and. Knapp, K. (1962). Dt. med. Wschr.. Z. 1232. Lesinski, J., Jajszczak, St., Dentyn, K. and Janczarski, I. (1967). Am. J. Obetet. Gynee., 2z 280. Lin, Y.* Cheng, H. and Yuan, G.'(1966). J. Formosan med. Ass.. 61, :225. Lindahl-Kiessling, K. and BOdk, J.A. (1963). Lancet, 3. 405. Linneoar. D. and Smith, R.L. (1968). Private communication. Lutwak-Mann, C. (1954). J. Eftbryol. exp. Morph.. z 1. Lutwak-Mann, C., Boursnell, J.C. and Bennett, 4.P. (1960). J. Reprod. Pert., 1* 169. Mannering, Dixon, A.C., Baker, E.N. and Asami, T. (1954). J. Pharmac. exp. Ther., 111, 142. Marin-Padilla, M. and Benirechke. K. (1963). Am. J. Path., . 999. Mauss, H.J. and. Stumps, K. (1963). Klin. Wsohr., 41, 21. Maynert, E.W. and Van tyke. H.B. (1950). J. Pharmac. exp. Ther., 211, 184. McBride, W.G. (1961). Lanoet, 2, 1358. McCarthy, E.P. and Kekwick, R.A. (1949). J. Physiol., Lond., 108, 184. Mellin, G.W. and Katzenetein. N. (1962). New Engl. 3. Med., ZAZA 1184. - 163 . Miller, B.J. and Reimann, S.P. (1940). Archs Path., 22. 181. Miller, B.J., Ciacci, V.W. and Heimann, S.P. (1941). Growth, 1, 329, Miller, J.P., Alter, A., Cardinal, B.V., Dalziel, A. and Bauman. P.M. (1966). Toxic. appl. Pharmac., 8, 295. Misiti, D., Rosnati, V., Bignami, G.. Bovet.Nitti. F. and Bovet, Ds (1963). J. medni. pharm. Chem., 6, 464. Mitchell, A.D. and Smith. C. (1913). J. chem. Soc., 122, 489. ?Ionroy, A. (1960). Experientia, 16, 114. Moore, S. and Stein, W.H. (1951). J. biol. Chem., laz 663. Mouzas, G.L. (1966). Lancet, 1, 882. Muckter, H. and More, E. (1966). Arzneimittel-Forseh., 16, 129. Murphy, M.L. (1960). In Congenital MalformationS, Ciba Foundation Symp., p.78. London:J. & A. Churchill Ltd. Narrod, S.A. and. King, C.T.G. (1963). Lance t, 2, 1375. Natarajan, A.T. and Nilsson, R. (1966). Naturwissenshaften, 12, 275. Needham, J. (1950). In Biochemistry and Morphogenesis. Cambridge University Press. Nehaus, G. and Ibe, K. (1961). Die. nerv. Syst., 22, 52. Nystrem, C. (1963). Stand. J. Clin. Lab. Invest.. p.102. Oliviero. V.T.$ Adamson. R.H., Henderson, E.S., and Davidson, J.D. (1963). J. Pharm. exp. Thar., 141, 149. Osterloh. G. and Lagler, F. (1960). Arzneimittel-Forseh.. 10, 985. - 164 - Pfeiffer. R.A. and Kosenow, W. (1962). Lancet. 1, 45. Playfair, J.H.L., Leuchare, E. and Davies, A.J.S. (1963). Lancet, 1.2003. Popper, H. (1966). Therapeutic Agents and the Liver. Eds. McIntyre, N. and Sherlock, S. PhiladelphiasEavies. Portman. 0.W. (1962). Fedn. Proc. Fedn. Am. Soca. exp. Biol., al. 896. Raffauf. H.J. (1961). Dt. med. Wschr., 86. 935. Rauen, H.M. (1963). Arzneimittel-Forschi 12, 1081. Avian. H.M. (1964). Arzneimittel-Forsch., 14, 111. Richter, C.P. (1945). J. Am. med. Asioc., 142, 927. Roath, S.. Elves, M.W. and IsrAels M.C.G. (1962). Lancet, 11. 812. Roe,'F.J.C. and Mitohley, B.C.V. (1963). Nature. Land.. 200. 1016. Rosie D., Merola, and Archer. S. (1967a). Life Sci., 6, 1433. Rosi, D., Guy. D., Braemer, A. and Archer. S. (1967b). Life Set., 6. 1351. Ram,, D., Peruzzotti.E., Dennis, E.W., Berberian, D.A., Freele. H. and Archer, S. (1965). Nature, Lond.. ga. 1005. Roszkowski. A.P. (1965). J. Pharmac. exp. Ther.,14Q, 288. Rothfels, U. (1954). J. exp. Zooi., 115,.. 17. . 165 . Rupe,,C.O. and Farmer, C.J. (1955). J. biol. Chem., 211, 899. Salter, C.E.,,Lodge.Patch, I.C. and Hare, E.M. (1959). J. Clin. exp. psychopath.. 20, 243. Soarano. E. and Maggio, R. (1957). Expl. Cell Res.. la, 403. Schechtman, A.M. and Knight. P.P. (1955). Nature, Lond., 1210 786. Scheid, W., Wieck, H.A., Stammler, A., Kladetzky, A. and Gibbels, E. (1961). Dt. med. Uschr., 86, 938. Schneierson, S.S. and Perlman, E. (1956). Proe. Soo. exp. Biol. Med.. 22... 229° Schumacher, H,, Smith, R.L. and Williams, R.T. (1965). Br. J. Pharmac. Chemother., 324. Schumacher, H.. Blake, A. and Gillette. J.R. (1967). Fedn. Proc. Fedn. Am. Soca. exp. Biol., 26, 730.. Seller, N.J. (1962). Lancet, 2, 249. Shenker, L.S., Todoo, D.J., Brodie, B.B. and Hogben, (1958),., J. Pharmae. exp. Ther.. 81, 123. Shanker, L.S. and Tom), D.J. (1960). J. Pharmae. exp. Ther., 128, 115. Shealy, Y.F., Opliger, C.E. and Montgomery, J.A. (1965). Chemy. Ind., 14 1031. Sheskin. J. and Convit, J. (1966). Hautarzt., 12, 548. Shnider, B.I., Frei, E., Tuohy. J., Gorman, J., Freireich, E., Brindley. C.O. and Clements. J. (1960). Cancer Res., 20, 28. 166 Somers, C.F. (1960). Br. J. Pharmac. Chemother., 15, 111. Somers, G.F. (1962). Lancet, 1, 912. Somers, G.F. (1963). Proceedings of the European Society for . the Study of. Drug Toxicity, 1, 49. Smith, H.,, and Williams (1965). J. Path. Bact.. 2. 95.

Smith, R.L., Fabro, So, Schumacher, R. 'and Williams. R.T. (1965). In Eml*yopathic Activity of Drugs, p.164. Eds. Robson, J.M., Sullivan, F. and Smith, R.L. London: J. & A. Churchill Ltd. Smith. R.L. (1966). Fortschr. Arzneimitt Forsch.. 20 301. Speirs, A.L. (1962). Lancet, 1, 303. Spencer. K.E. (1962)., Lancet,.?, 100. Stark, G. (1956). Praxis, 1.141, 966. . Thole, F.B. (1908). J. chem. Soc., 21 1815. Thole. F.B. (1910)., J. chem. Soc., 22, 1249. Toivanen, A.. Markkanen, T,, Mantyjarvi. R. and TOivanen, P. (1964). Biochem. Pharmac., LI, 1489. Trefonel, J., Mme. Trelfonil, J., Nitti. F. and Bovet, D. (1935). Compt. Rend. Soc. Biol., 120, 756. TUohmann.Luplessis, H. and Mercier-Parot, L. (1959). Biol. med. (Paris), 48, 238. Tuchmann.Luplessis. H. (1965). Symposium on Embryopathic activities of Drugs. Eds. Robson. J., Sullivan, F. and Smith, R.L. London:Churchill. Turk ,J.L. Hellmann, K. and Duke, D.I. (1966). Lancet. 1. 1134. -167.. Villa, L. and Eridani, S. (1963). Haematol. Latina, 6. 217. Waikenhorst, A. (1957). Wien. kiln. Wschr., 62., 334. Walter, H. and Mahler, H.R. (1958). J. biol. Chem., gm, 241. Wegner, t. (1961). Arch. Kreislaufforsoh., 4, 99. Weidman, W.H., Young. H.H. and Zoliman, F.E. (1963). Mayo Clin. Froc..8, 518. Welch, A.D.. Handschumacher, R.E. and Jaffe, J.J.J. (1960). J. Pharmac. exp. Thar., 1320 262. Welch, A.D. (1965), Ann. N.Y. Acad. Sci., 1.21, 19. -Wells. C.E., Azmone-Marsan, C., Frei, E., Tuohy, J.H. and Shnider, B.I. (1957). Electroenceph. Clin. Neurophysiol.. .2.* 325* Wilde, C.E. (1955a). J. exp. Zool., 212, 573. Wilde, C.E. (1955b). J. Morph.. 7, 313. Wilde C.E. (1956). 3. exp. Zool., 12.1, 409. Williams, R.T. (1959). In Detoxication Mechanisms, p.717. London:Chapman & Hall Ltd. Williams, R.T., Millburn. P. and Smith, R.L. (1965a). Ann. N.Y. Acad. Sot., 222, 110. Williams, R.T., Smith. R.L. and Millburn, P. (1965b). Therapeutic Agents and the Liver. p.37. Eds. McIntyre, N. and Sherlock, S.. Oxford:Blackwell. Williams, R.T. (1967). Fedn. Proc. Fedn. Am. Soc. exp. Biol., 26, 1029. Winnick. R.E. and Winniok, T. (1953). J. nat. Cancer Inst.. 14, 519-526. - 168 - Winzenried, F.J.M. (1961)., Medsche Kline, 1046. Yang, T.J., Yang, T.S. and Liang, H.H. (1963). Lancet, 1, 552. Yu-Chan Lin, Hsing Cheng and Guey-Mei Yuan (1966). J. Formosan med. Ass., a, 225. Zaruba, F,, Kuta, A. and Elis, J. (1963). Lancet, le 275. Zimmerman, W., Gottschewski, G.H.M., Flamm, H. and Kunz, C.H. (1963). Devl. Biol., 6, 233.