THE METABOLISM OF1 CHLOROQUINE PHOSPHATE BY RAT LIVER CELL FRACTIONS A THESIS PRESENTED TO THE UNIVERSITY OF GHANA BY' PHTLIp OWIREDU YEBOAH IN CANDIDATURE FOR THE DEGREE OF DOCTOR OF PHILOSOPHY, BIOCHEMISTRY DEPARTMENT OF BIOCHEMISTRY, UNIVERSITY OF GHANA, LEGON. JUNE, 1976(. ; C 5 i 1$ ' i z fits, DECLARATION The experimental work described in this thesis was carried out by me at the Department of Biochemistry University of Ghana, and the Department of Chemical Pathology, University of Leeds. The work has been under the joint supervision of Professor G.S. Asante and Dr. Colin Toothill (external supervisor). Drs. Larway and Gyang are members of the supervisory, committee. I certify that this work has not previously been accepted for any degree, and it is not being concurrently submitted in candidature for any other degree. SIGNED CANDIDATE SUPERVISOR' SUPERVISOR ACKNOWLEDGEMENTS I am greatly indebted to Professor G.S. Asante, Professor and Head, Department of Biochemistry, Legon, for suggesting the topic and for his encouragement and direction throughout the work. I am most grateful too, to Drs. P.F. Larway, F.N. Gyang, members of my supervisory committee, and the entire academic staff of the Department of Biochemistry, Legon, from October 1972 to June, 1976, for their advice, encouragement and useful discussions and criticisms throughout the project. Special thanks also go to Professors P.N. Campbell, former Head, Department of Biochemistry, G.H. Lathe, Head, Department of Chemical Pathology and S.R. Stitch, Head, Division of Steroid Endocrinology, all of the University of Leeds, for their hospitality and for the use of their laboratories. To Drs. Colin Toothill and Tom Scott of the University of Leeds, Department of Chemical Pathology and Biochemistry, respectfully, I wish to express my gratitude for their guidance and encouragement towards this work during my stay in Leeds, and also for their continued interest. I't was a pleasure to work with Dr. Toothill and his staff, especially Mrs. Graham, Mrs. Doreen Jobbins, Mrs. Betty Sharp and Mr. John Wiselogle. iii I am also deeply indebted to Professor A. A. Kwapong,-. and Professor D.A. Bekoe former, and current Vice- Chancellors, University of Ghana respectively, whose advice and encouragement helped guide my thinking towards learning to work under difficult conditions. Special thanks also go to Mr. /^.SOdoom, Senior Assistant Registrar at the Universities of Ghana Office, London; Dr. I.A. Mensah and Mr. J.S. Jackson, tutor and ex-senior tutor respectively, of Mensah Sarbah Hall, Legon, for the many discussions and advice I' had with them. I also wish to thank the Inter-University Council for Higher Education Overseas, for sponsoring my trip to Leeds, and also for providing equipment and other research materials to enable me continue the research project in Ghana. And, to the University of Ghana Postgraduate Scholarships Committee, I am most grateful for the award of a post-graduate scholarship. To Dr. S. Dakubu and the staff of the Medical Physics Unit, K'orle Bu; to Mr. Ayim and the staff of the electron microscope laboratory, Korle Bu; to Dr- R.E. Oakey and his staff of the Division of Steroid Endocrinology, Leeds, and to Mr. Defencer Anyetei, Department of Biochemistry, Legon, I wish to express my gratitude for their assistance in the iv use of various scientific equipment'. I am also grateful to Dr. E. Herbert and the technicians of the Department of Chemistry, University of Leeds, for the diverse ways they helped me during the synthesis of the metabolites of chloroquine. To the animal breeding section of the Ghana Medical School, Korle Bu and to Mrs. Kordylas of the Food Research Institute, I wish to express many thanks for providing experimental animals to enable me continue this project at Legon. Special thanks also go to Mr. J.A. Ashong, Department of Biochemistry, Legon, for actually supervising the establishment of an animal breeding unit in this Department to enable me have a constant supply of experimental animals. Mr. Kiya Hinidza of the Registrar's Offices, Legon, Mr. Alexander Narh of Highway Authority, Accra, and Mr. E.K. Antwi, Ghana Atomic Energy Commission, Accra, also deserve special thanks for typing this manuscript. Once again, it is a pleasure to thank the many colleagues, lecturers, students and relatives at home and abroad, whose kind comments, sympathy and encouragement have aided me greatly to bring this work to completion. If there is anybody whose name has not been mentioned, it is not because the help he rendered is insignificant. v ABBREVIATIONS AND INTRODUCTORY NOTES 1. The abbreviations used in the text are as follows EMSO = Diem thy 1 sulpbox icie G6P = Glucose-6-Phosphate G6PD = GLucose-6-Phosphate dehydrogenase I.R. = Infrared NAD+ = Oxidized form of Nicotinamide-adenine dinucleotide ;NADP+' = Oxidized form of Nicotinamide-adenine dinucleotide phosphate NADPH = Seduced form of Nicotinamide-adenine dinucleotide pitesplnnJS- P.P.O. = 2-5-diphenyl-oxazol P.O.P.O.P. = 1,4-di-(2-(5-phenyl-oxazcyl)-benzene). SKF 525-A = p-diethylaminoethyl diphenyl-^ l- propylacetate TCA = Trichloroacetic acid TLC = Thin layer chromatography U.V. = Ultraviolet 2. All melting points (M.P.) are quoted in degrees centigrade and are uncorrected. 3. 'SN1 stands for Survey number; and the number that follows it identifies the various drugs in the files of the survey of Antimalarial Drugs cotmittee on Medical Research; U.S. CDNIENTS PART I PaSe INTRODUCTION AMD LITERATURE REVIEW ... 1 Enzymic Basis of Drug metabolism ... 4 Microsomal Drug Metabolism ... 4 Conjugation Reactions ... 6 Factors affecting hepatic microscmal enzyme activity... 7 (i) Species Variation in Drug metabolism ... 8 (ii) Environmental factors influencing Drug metabolism ... 11 (iii) The effect of sex, age, diet and nutritional status, diurnal variation and liver pathology on hepatic microsomal enzyme activity ... 19 Chloroquine ... 21 lyfetabolism of Chlorciquine: N-dealkylation ... 22 PART II MATERIALS AND METHODS ... 28 I&terials ... 29 General tfethods ... 31 Preliminary studies: Standardization of isolation procedures ... 31 ML: Preparation of subcellular fractions of the rat liver ... 37 M2: Incubation of cell fraction with chloro­ quine and other 4-amino quinoline derivatives ... 41 vii Page M3: Extraction Procedures ... 42 M5: Chromatography ... 43 MB: Estimation of Acetaldehyde ... 43 PART III: RESULTS A3® DISCUSSIONS ... 47 SECTION I: Jfetabolism of Chloroquine ... 48 Preliminary Investigations ... 48 (A) Liver incubation studies ... 48 (B) Analysis of Urine of chloroquine- treated humans ... 49 (C) Rats on chloroquine .. ^ 64 SECTION II: Subcellular distribution of chloroquine ;C5 i < aa 14SECTION III: Metabolism of C-labelled chloroquine phosphate by rat liver microsomes ... 71 SECTION IV: Synthesis of metabolites of chloroquine ... 75 I Introduction ... 75 1 l-hydroxy-4-pentanone oxime ... 78 2 4-amino-l -pentanol ... 84 3 4- (41 -hydroxy—1 Wnethylbutylamino) -7 -ehlo- roquinoline ... 84 4 Conversion of the 'Carbinol' to the 4'-aldehyde derivative ... 96 5 The chloride derivative and its conversion to metabolite E ... 97 (a) Preparation of the chloride derivative ... 97 (b) Conversion of the chloride derivative to metabolite E ... 100 viii SECTION V: Identification of II. V.-absorbing spots and the in vitro metabolism of specific quinoline derivatives (A) Identification of U.V. -absorbing spots on chromatograms (B) In vitro studies on the metabolian of reference quinoline compounds used as standards in Section VA SECTION VI: Investigations into the mechanism of the in vitro degradation of chloroquine by hepatic microsomal fraction of rat liver homogenate (A) Distribution of products of hepatic microsomal incubation mixture of chloroquine between unchanged drug and metabolites thereof, in presence or absence of SKF 525-A (B) Acetaldehyde estimation in the hepatic microsomal incubation mixtures of chloroquine (C) f'fcasurements of rate of oxygen consumption in hepatic microsomal incubation mixtures of chloroquine PART IV: CONCLUSIONS Suggestion for further studies References LIST OF FIGURES FIGURES 1 METABOLISM OF CHLOROQUINE IN VIVO (tfcChesney et al, 1966) 2 Standard calibration curve for chloroquine phosphate in 0.1M acetic acid 3 Schematic representation for fractionation of rat liver cell components 4 Standard calibration curve for estimation of acetaldehyde (Modified method of Gupta and Robinson, 1971). 5 Ultraviolet spectrum of eluate (in 0.1M HC1 in methanol) of spot I (Rf 0.04) on thin layer chromatogram of urine extract 6 Ultraviolet spectrum of eluate (in 0.1M HC1 in methanol) of spot II (Rf 0.19) on thin layer chromatogram of urine extracts 7 Ultraviolet spectrum of eluate (in 0.1MHC1 in methanol) of spot III (Rf 0.47) on thin layer chromatogram of urine extracts 8 Ultraviolet spectrum of eluate (in 0.1MHC1 in methanol) of spot V (Rf 0.65) on thin layer chromatogram of urine extracts 9 Ultraviolet spectrum of eluate (in 0.1MHCL in methanol) of spot VIII (Rf 0.78) on thin layer chromatogram of urine extracts 10. Ultraviolet spectrum of eluate (in 0.1MHC1 in methanol) of spot XII (Rf 0.89) on thin layer chromatogran of uri^ FIGURES 11 Scheme for the synthesis of known metabolites of chloroquine 12 Infra red spectrum of l-hydroxy-4-pentanone oxime in Nujol 13 Infra red spectrum of 4-amino-l-pentanol (KBr disc) 14 Infra red spectrum of the 4-hydroxy- derivative : 4- (4 ’ -hydroxy-1' -methylbuty- lamino)-7-chloroquinoline (in Nujol) 15 Ultraviolet spectrum of the 4'-hydroxy derivative of chloroquine in 0.1MHCL 16 Ultraviolet spectrum of the 4'-aldehyde derivative in CC14 17 Infra red spectrun of the 4'-aldehyde derivative of chloroquine (metabolite E) in Nujol 18 Infra red spectrum of BMSO 19 Infra red spectrum of 4-(4'-chloro-l'- methylbutylamino)-7-chloroquinollne in Nujol 20 Infra red spectrum of 4- (4' -amino-l1 methylbutylamino) -7 -chloroquinoline 21 Ultraviolet spectrum of the primary amine derivative (Fig. 19) in 0.1MHC1 22 Infra red spectrum of 4-amino-7-chloro- quinoline FIGURES 23 lyfess spectrum of eluate of spot VII on thin layer chromatogram, of extract frcm incubation mixtures with chloroquine 24 Proposed pathway for the metabolism of chloroquine phosphate LIST OF TABIES TABLE 1 2 Subcellular distribution of chloroquine degradation - (a) Thin layer chromatograms of ether-extract, pH 10.5, of incubation mixtures of various subcellular fraction. 67 (b) Incubation mixtures of various subcellular fractions extracted with iso-amyl alcohol- dichloroethane (10:90) mixture, pH 10.5 , 68 14Quantitative pattern of metabolism of (3- C) chloroquine by rat liver microscmes using various extracting solvents; pH 10.5 * 73 (a) The identities of U.V. -absorbing areas on the chromatogram of the extract of the incubated mixture containing chloroquine and the microsome/cytoplasm system as shown by matching standards . 110 (b) TLC analysis of the extracts of the incubated mixture containing the microsome/ cytoplasm system, and a reference quinoline compound as substrate . 112 xii ABSTRACT 1. The metabolisn of chloroquine phosphate by the various subcellular fractions of the rat liver honnogenate has been studied. (a) Both the mitochondrial and soluble fractions were found to contain enzyme systems that degraded chloroquine to varying degrees. (b) The maximum activity of chloroquine degradation was however found in the microsomal fraction, 2. In vitro studies on the metabolisij of (ring-3-^C) chloroquine led to the conclusion that the microsomal subcellular fraction was capable of breaking chloroquine down to a minimum of eight metabo­ lites in which the 4-aminoquinoline nucleus remained intact. 3. A number of 4-aminoquinoline derivatives, including the 4'-hydroxy, the 4'-aldehyde, the primary amine (SN 13617) deriva­ tives of chloroquine have been successfully synthesized and characterized, 4. Using the synthesized derivatives of chloroquine as chromatographic standards, four of the eight metabolites of chloroquine (see ' 2 * above) were identified as the primary amine derivative (SN 13617), the 4'-hydroxy and the 4'-aldehyde deri­ vatives and 7 -chloro-4-aminoquinoline. (xiii) 5. Based on in vitro studies on the metabolism of both chloroquine and the synthesized derivatives by the hepatic microsomal fraction of rat, it has been tentatively suggested that the metabcLlic degradation of chloroquine proceeds by the formation of the following metabolit.££.• in the order: i) The hydroxychloroquine, HCQ (ie. the analogue of chloroquine in which a hydroxyl group has been substituted in the 2-position of N-ethyl groups). ii) The secondary amine derivative (SN 13616); iii) The primary amine derivative (SN 13617); iv) The 4'-aldehyde derivative, which is largely converted into the 4'-hydroxy derivative. 6. An alternative pathway for the secondary amine derivative ('SN 13616) has also been proposed. Through this pathway, chloroquine may be converted through SN 13616 and an unidentified intermediate V, to a final product 4-amino-7-chloroquinoline. It has also been demonstrated that in presence of SKF 525A, the metabolism of chloroquine is apparently activated through the latter alternative pathway. 7. Evidence is also presented to show that the proposed pathway for chloroquine metabolism might be common to both the rat and man. (x iv) 1PART 1 INTRODUCTION AND LITERATURE REVIEW 2INTRODUCnCN MD LITERATURE REVIEW Drug metabolism refers to the chemical alterations of a drug produced by the biological environment, and represents one aspect of the physiological disposition or fate of the agent. Cue main natural function of drug metabolism is the formation of more polar and water soluble derivatives which leads to an accelerated termination of the pharmacological activity of the drug and its rapid excretion. Brodie (1964) points out that if there were no process such as drug metabolian, it would take the body about a hundred years to terminate the action of pentobarbital, which is lipid-soluble and cannot be excreted without being metabolised. Although most drug metabolites are less biologically active than their parent compounds, in quite a few instances, a more toxic metabolite is produced. This has been shown to be true with sulphadiazine and sulphamerazine (Williams, 1959). The anti-malarial drug primaquine has also been shown to be., metabo­ lized in man to a compound of high oxidant properties^ which, though an active antimalarial, is very toxic and induces hemolysis in individuals lacking G6PD (TOO, 1967). Thus drug metabolian does not necessarily imply detoxica­ tion or loss of pharmacological activity. In numerous other instances, the administered drug is really only a "pro-drug" which is biotransformed into a more pharmacologically active metabolite. The danethylation of imipramine to deanethylimi- praminejthe desulphuratxon of parathion to paraoxon; the oxidation of diethylene glycol to oxalate, and the conversion of "Active 0^ " 3) "Active 02 + Krug — •— > Oxidized Drug + A + ELO Overall Reaction NADPH + 02 + Drug = NADP1 + igO + Oxidized Drug. Major components of the microsomal drug metabolizing system include the following: 1) NADPIi -cytochrome c reductase, the flavin enzyme involved in the oxidation of RALPH. This reductase is currently thought to reduce cytochrome P-450 riirpct.lv or indirectly through a non-hone iron protein or sane other unidentified carrier. 2) Cytochrome P-450 - a hemoprotein, which^ in its oxidized f orrn^ represents ’A' in equations 1 and 3 in the preceeding paragraph. ‘ Evidence for the involvenent of cytoehrane P-450 in drug metabolism includes the fact that certain substances such as deoxychlolate and steapsin (Gillette et al, 1957; Itosner et al, 1961), which destroy the drug meta­ bolizing activity of microsones, have also been found to convert cytochrane P-450 to the inactive form of the hemoprotein, cyl.ochrome P-420 (Qnura & Sato 1964). 3) NADPH - cytochrome P-450 reductaso which functions in the reduction of oxidized cytochrane P-450. A wide variety of oxidation reactions that have been shown to occur in microsones includes deamination, 0-,N- and S-dealkylation, hydroxyl.ations of alkyl and aryl hydrocarbons, epoxidation, N-hydroxylation etc. (Brodie et al, 1958; Gillette, 1963; 1966), Conjugation Reactions: Conjugation reactions or what has been classified as syntheses may occur when the drug contains a group, usually OH, OOOH,' Mig or- SH, which is suitable for caribining with a natural compound provided by the body to form readily excreted water - soluble metabolites. If the drug does not contain these groups, it may acquire than through the initial phase I reactions, -i „ v\-tr -"^ '-'ition or hydrolysis. Thus whereas phenol 7which contains a hydroxyl group,can undergo conjugation directly, benzene, which does not contain a group suitable for conjugation, acquires one by being oxidized to phenol in the body. The compounds provided by the body for conjugation (i.e. conjugating agents) are derived fran materials Involved in normal carbohydrate, protein and fat metabolism, and include glucuronic acid, glycine, cysteine, imethionine for methylation, sulphate, acetic acid and thiosulphate for sulphur. Apart fran these, sane species provide glucose (insects), glutamine (man), and ornithine (birds) for ranjugation. The conjugating agents, however, do not, as a rule, react directly with the drug or its phase I metabolite, but do so either in an "activated form" or with an "activated form" of the drug. These activated forms are usually nucleotides, and the reaction between the nucleotide and the drug or conjugating agent as the case may be is catalysed by an enzyme, Ihus for glucuronido synthesis, the glucuronic acid is used in the form of the nucleotide, uridine diphosphate glucuronic acid (UDPGA) which^ under the influence of the enzjme glucuronyl transferase, transfers its glucuronyl residue to the drug, Factors Affecting Hepatic Microsomal. Enzyme Activity Both physiological and environmental factors are known to influence the most likely metabolic pathways of an agent, These are listed below ^ IftgfeRlteSgi and only two have been discussed in detail, 8(1) Species and strain of animal used, (2) Interaction of other drugs and environmental contaminants. (3) Age, sex and weight of animal, (4) Dose and frequency of administration of the drug. (5) Diet and nutritional status, (6) Route of administration, (7) Time of administration (circadian light, seasonal changes), (8) Subsequent interaction with other enzymes (^ glucuronidase, sulphjtase, deacetylase for which drug conjugates may be substrates). (9) Pregnancy and physiological abnormalities. (1) Species Variation in Drug metabolism Species variation in the metabolism of foreign compounds tp* not unexpected, since such variations often occur in the metabolism of same natural metabolites of the body and are explicable in enzymic terms. For instance, the inability of man to synthsize L-ascorbic acid is due to the lack of the liver (or kidney in some other species) microsomal enzyme, L-gulonolactone oxidase, which converts L-gulonolactone to 2-Keto-L-gulonolactone which then isomerises to ascorbic acid (Chatterjee et al, 1961). 9Species variation in metabolism of a drug may appear as: (a) qualitative differences in the actual pathways of metabolisn and/or; (b) quantitative differences in pathways of metabolisn which are cannon to several species. The qualitative differences probably result fran the presence in a particular species of an enzyme which is not found in others, or the absence in a particu­ lar species of an enzyme otherwise generally distributed in the animal world, Quantitative differences could be due to variations in the amount of an enzyme or its inhibitor, or to the occurrence of an enzyme possibly located in another tissue, reversing the reaction, or to an enzyme canpetingi'for the same substrate. Thus the in vitro denethylation of ethylmorphine by liver microsanal preparations of mouse, rat, guinea pig and rabbit shows a four fold variation in rate} the mouse preparation being the most active and the rabbit the least active (Davies et al, 1969), These workers also showed that at the enzymic level, species differences parallel differences in NADPH-cytochrane P-450 reductase and may be related to the rate of reduction of the cytochrane P-450 substrate complex in different species. Although the main causes of species variation in drug metabolisn seen to be associated with the tissue enzymes, it has been found that other factors such as the gastrointestinal flora may be involved in certain cases (Scheline, 1968). In spite of the fact that the gut bacteria are cannon to many 10 species, they have been found to vary in numbers and location in the inteptstine of different species and they could vary within the same species according to the nature of the diet. Thus since many foreign compounds are taken orally, it is possible for species variation in the meta­ bolism of seme compounds to depend partly upon the gut flora. A striking example of species variation influenced by gut bacteria is to be found in the metabolism of homoprotocate- chuic acid. This acid is dehydroxylated to m-hydroxyphenyt _1wmtem acid in the rat and rabbit, but the excretion of this metabolite is suppressed almost to zero if the animals are pretreated with antibiotics (Williams, 1971), Aromatic dehydroxylation of this compound seems therefore to depend on the gut flora, Itomoprotocatechuic- acid, however, is also methylated in these animals to 4-hydraxy-3-methoxyphe- nylacetic acid (homovanillic acid) but the output of this metabolite is -unaffected by pretreatment with antibiotics. The rabbit dehydroxylates twice as much as the rat (Dacre et al, 1968) a clear indication that the rabbit differs from the rat in its gut flora (Scheline, 1968), and that species differences in the extent of dehydroxylation apparently depend^ on the gut flora. Attempts to explain species variation in drug metabolism are usually made through in vitro studies; though these studies serve as an important guide to what happens in vivo, it is desirable that both types of studies are made together so that such factors as absorption, tissue distribution, biliary and urinary excretion, enterohepatic circulation and the role of gut bacteria can be taken into account in the overall picture of species variation. Species variation in the conjugation reactions depends on the occurence of the conjugating agent or the ability to form the necessary nucleotide or the amount of the transferring enzyme. It is well known for example, that the cat has a defect in glucuronide synthesis, a defect not in the animal!s inability to provide glucuronic acid, or to make UDPGA, but in a deficiency of the enzyme glucuronyl transferase (Williams, 1971). Man is peculiar in being able to convert phenylacetic acid into phenylacetylglutamine. The rat is unable to do this because although it can make the inter­ mediate nucleotide derivative phenylacetylCoA, it does not possess the necessary transferring enzyme which, however, occurs in the human liver and kidney (Williams, 1971), (2) Environmental factors influencing'Drug metabolism Studies have shown that the duration and intensity of action of many drugs in animals depend on the activity of 11 12 drug-metabolizing enzymes localized in the microsomal fraction of liver homogenate which is derived from the endoplasmic reticulum of the liver cell. Many drugs and environmental chemicals stimulate or inhibit microsomal enzyme function in animals, and this is reflected in vivo by an altered metabolism and action of drugs, carcinogens and various noimal body consti­ tuents, such as steroid hormones, thyroxine and bilirubin (Conney, 1967; Conney and Kuntzman, 1971). Chemicals found in man’s environment that alter the action of drugs by stimulating microsomal enzyme activity in animals include halogenated hydrocarbon insecticides (Hart and Fouts, 1965; Conney et al, 1967), urea herbicides (RLnoshita and TXiBois, 1970), volatile oils (Jori et al, 1970), polycyclic aromatic hydrocarbons (Conney 1967; Arcos et al, 1961, Welch et al, 1969; Nerbert et al 1969), dyes used as colouring agents (Radomski, 1961; Levin and Conney, 1967), nicotine and other alkaloids (Wenzel and Brodie, 1966), food preservatives (Gilbert and Golberg, 1965'; Creaven et al, 1966): Botham et al, 1970), Xanthines (Mitoma et al, 1969), flavones (Miller et_ al, 1954); that occur in food. The stimulatory effect of halogenated hydrocarbon insecticides on drug metabolism was discovered accidentally by Hart and Fouts (1965), after their animal living quarters were treated with chlordane. Studies on the metabolism and action of drags were d&aiuptod, and the effects of chlordane persisted for several weeks, even though insecticide treatment had stopped. Examples of other insecticides that stimulate drug metabolism in rats include DDT, methoxychlor, aldrin, dieldrin, heptachlor and benzene hexachloride (Conney et al, 1967). People exposed to DDT and lindane in a pesticide factory metabolized antipyrine about twice as rapidly as control population (Kolmodin et al, 1969). Ihe effect of intensive occupational exposure to only DDT on drug and steroid metabolism has also been studied. In a population of DDT - factory workers with a serum and fat concentration of DDT-related substances 20 to 30-fold higher than control population, the half life of phenylbutazone in the serum was 19 percent lower, and the urinary excretion of 6 ^ -hydroxycortisol was 57 percent higher than in the control population (Poland et al, 1970). The stimulatory effect of polycyclic aromatic hydrocarbons on the metabolism and action of drugs in animals is evident fran the observation that the smoking of cigarettes enhances the metabolism of nicotine in man (Beckett and Triggs, 1967), lowers the concentration of phenacetin in the plasma (Pantuck et al, 1972) and induces enzymes in the placenta that hydroxylate benzol( a p pyrene (Welch et al, 1971^-Welch et al, 1968). Benzo (a) pyrene and other polycyclic hydrocarbons are present in cigarette smoke. 13 Environmental chemicals that inhibit microsomal function in animals include organophoSphorus insecticides (Welch et al, 1967; Welch et al, 1969), carbon tetrachloride (Dingell and Heimberg, 1968; Levin et al, 1970), Ozone [Palmer et al, 1971), and carbon monoxide (Cooper et al, 1965; Conney et al, 1968). Changes in atmospheric pressure and respiratory oxygen can also influence drug metabolism (Kitawaga, 1968; Merritt and Medina, 1968) The persistent administration of one drug can also affect the pharmacologic activity of another drug by stimulating or inhibiting its metabolic inactivation. Patients are often given several drugs at the same time and sometimes the combination results in an undesirable effect because one drug may inhibit or stimulate the metabolism of the other. For example, phenylbutazone and chloramphenicol inhibit the metabolic inacti­ vation of tolbutamide in man, and this effect can result in hypoglycemia. Phenobarbital, on the other hand, decreases the action of other drugs by increasing the rate at which they are metabolized to pharmacologically inactive substances. The magnitude of the change in drug action that may occur after enzyme induction in animals is illustrated by data obtained from rats given the muscle-relaxant drug zoxazolamine, which is metabolized by the liver microsomal hydroxylase to the inactive compound, 6-hydroxyzoxazolamine. A high dose of 14 zoxazolamine paralyses rats for more than 11 hours, but if they are treated with phenobarbital for 4 days before being given zoxazolamine, they are paralysed for only 105 minutes; if they are treated with benzopyrene 24 hours before the test they are paralysed for only 17 minutes with the same dose of zoxazolamine (Conney et al, 1960). Ethanol, when consumed habitually, stimulates its own metabolisn and the metabolism of various drugs in man. Alcoholics have been shown to metabolize tolbutamide more rapidly than nonalcoholics (Kater et al, 1969), and Cbnney and \ Burns (1972) have suggested that this should be considered when alcoholics are given tolbutamide for the treatment of diabetes, Alcoholics also have an increased concentration of the hepatic enzyme that metabolizes pentobarbital and other barbiturates and sedatives (Rubin and Lieber, 1968), and this helps to explain the increased tolerance of alcoholics to barbiturates and other sedatives when sober. A single large dose of ethanol, however, inhibits drug metabolisn and this effect - in addition to the central depressant action of ethanol - helps explain the enhanced sensitivity of inebriated individuals to barbiturates and other sedatives (Rubin and Lidaer, 1971). 16 Not only does long-term administration of drugs stimulate the metabolian of other compounds, but often the drug stimulates its own metabolian. Examples of drugs that stimulate their own metabolian in dogs include phenyl­ butazone, tolbutamide, probenecid, chlorcyclizine and hexobar- bital (Welch et al, 1967). Welch and his associates actually demonstrated the accumulation of a denethylated metabolite, norchlorcyclizine, after 4SanrtR. administration of the parent compound - chlorcyclizine, to dogs. The development of tolerance to glutethimide in man results from the ability of glutethimide to stimulate its own metabolism and this may be also true for ethanol and meprobamate (Douglas et al, 1963; Schnid.i. et al» 1964). Tblerancc to narcotics such as morphine and meperidine,, on the other hand, does not result from their metabolism being enhanced (Conney and Bums, 1972). The same factors that affect the metabolism of drugs in man have been shown to influence the metabolism of steroids (Conney, 1967; Conney and Kuntzman, 1971), lipid-soluble vitamins (Dent, et al, 1970; i?owe and Pdchens, 1970p; Hunter et al, 1971; Bergstedt et al, 1972; Halm et al, 1972) and other normal body constituents (Maurer et al, 1968). Treatment of rats with, phenobarbital for several days increased the activity of enzymes in liver micro semes that hydroxylate- androgens, estrogens, progestational steroids and adrenocortical 17 steroids (Levin et al, 1968). Several structurally unrelated chemicals that stimulate the activity of drug metabolizing enzymes also stimulate steroid hydroxylase activity. Examples of such compounds foclud'e phenobarbital, diphenylhydantoin, chlorcyclizine, norchlorcyclizine, orphenadrine , phenylbuta­ zone and several halogenated hydrocarbon insecticides (Cbmey and Klutch, 1963; Kuntanan et al, 1964; Penney et al, 1966; Levin et al, 1968; Welch et al, 1971J,). Cbnney and Burns (1972).have even suggested that the effect of insecticides on fertility in animals may be .explained, at leasts in partj by the induction of enzymes that metabolize progesterone and other steroids. Compounds that inhibit drug ^metabolizing enzymes in liver microsomes ftlsjM inhibit steroid metabolism and augpient the action of steroids in animals. SKF 525-A, which potentia­ tes the action, of many drugs by inhibiting their microsomal metabolian (Axelrod et al, 1954), also potentiates the action, of estrone on,the rat uterus and the induction by cortisol of hepatic tyrosine transaminase in the rat (Kupfer and Peets, 1967), Symptons of vitamins D and K deficiences have been observed following therapy with anticonvulsant drugs (Rowe and Richens, 1970b) • long term anticonvulsant therapy was associated with osteomalacia and with reduced concentration of calcim and elevated concentrations of alkaline phosphatase 18 in the serum; suggesting that drug-mediated enzyme induction might be responsible for the osteomalacia by causing increased metabolic inactivation- of vitamin D (Dent et al, 1970; Fowe and .Eichens „ 1970a). Administration of vitamin D to patients receiving anticonvulsants resulted in a return towards normal concentrations of calcium and activity of alkaline phosphatase in the serum. Farther evidence that enzyme, induction can decrease the action of vitamin D came ircm the finding that rats treated with phenobarbital are protected frcm loss of weight, hypercalcemia and renal calcinosis produced by the administration of a single large dose of calciferol (P SUPERNAIE SUPERNAIE SEDIMENT (mitochondria) centrifuged at 5,000 xg for 10 minutes 1/ SUPEPM3E (b) mixed with supemate (a) SEDIMEOT centri- at 100.OOOxg for 60 Iwiinutes (microsones) (soluble fracfci 39 The homogenate was centrifuged at 24,000 x g for 10 min. to sediment nuclei, -unbroken liver cells, red blood cells, and mito­ chondria. Other liver cell components were fractionated according to the scheme in Figure 3. (a) Isolation of nuclear-debris fraction: The sediment obtained after centrifugation at 24,000 x g was made to the original volume of the initial homogenate in cold isotonic sucrose. Half of this solution was layered over an equal volume of cold 0.34M sucrose. The mixture was centrifuged at 700 x g (in a bench centrifuge) for 10 min. The supernatant fluid was relayered over an equal volume of 0.34M sucrose and centrifugation at 700 x g ^ The loosely sedimented pellets in these two consecutive centrifugations were resuspended in 0.25M sucrose.arid recentrifuged at 700 x g for 10 mins. The supernatant fluid was removed and the sedimented nuclei-debris were washed with two portions of 0.25M sucrose solution. The washed pellets were then suspended in isotonic buffer pH 7,4 (l,157o KC1 - 0.01M Na^ /K+ phosphate). (b) Isolation of Mitochondria: The supernatant fluid from the separation of nuclei-debris was centrifuged for 10 mins at 5,000 x g. The supemate was carefully drawn off and the brownish red mito­ chondria were washed twice by resuspending in 0.25M sucrose solution followed by centrifugation at 5,0U0 x g, The mitochondrial pellet was then resuspended in isotonic-K Cl to the original volume of the hanogenate. 40 (c) Isolation of Microsomes: The sapemate obtained in the centri­ fugation at 24,000 x g (a) and the post-mitochondria fraction in stage (b) were pooled and centrifuged at 100,000 x g for 60 mins. The fatty material at the top was removed. The supernatant fluid (known in the text as soluble fraction) vias also removed and diluted with isotonic KC1 to the original volume of the homogenate, The microsomal pellet was washed once by resuspending it in isotonic KC1 and recentrifuging at 100,000 x g for 60 mins. The washed microsomes were resuspended in isotonic KC1 to the same volume as the. original homogenate. B. Preparation of Mcroscmes from .9,000 x g Supemate The whole homogenate (containing 250ttg of liver/ml) prepared in MIA above was centrifuged at 9,000 x g for 10 mins. The 9,000 x g supemate was centrifuged at 105,000 x g for 60 mins. The fatty material at the top was gently removed and discarded, The supernatant fraction was removed and stored as the soluble fraction. The microsomal pellet was resuspended in 10 ml of 0.05M Na’*’/K*" phosphate buffer pH 7.4; and made up to the original volume of the crude homogenate. 41 M2 Incubation of Cell Fractions with Chloroquine and other quinoline derivatives The incubation system that was used to study the metabolism of chloroquine by various cell fractions prepared in method 1A was made up of the following: Phosphate buffer 0.2M pH 7.4 Chloroquine phosphate 10 pmole NADP 2 pmole Glucose-6-Phosphate 25 jamole Magnesium chloride 75 pmole Nicotinamide 100 umole In a total volume of 2.0 ml To this was added a 3.0 ml aliquot of each cell fraction. Incubation was carried out in a shaking water bath, in an atmosphere of oxygen in 25 ml conical flasks. The flasks were incubated for 1 hour at 37°C, after which the reaction was stopped f.__ by addition of 2.0 ml 1 0 % trichloroacetic acid (TCA). 42 M3. Extraction Procedures■ (a) Incubation Systems (McChesriey et al, 1967) Ten minutes after the addition of the TCA solution to the incubation system, the solution was centrifuged (3,000 x g: 10 min.) and the supemate collected and adjusted to pH 10.5 with NaOH (25% w/v). The resulting alkaline solution was then extracted with either three volumes of iso-amyl alcohol/ethylene dichloride (10:90, v/v) or ether. (b) Urine of chloroquine treated humans Human urine samples were adjusted to pH 11.0 with 1 in 5 of 0.88 NHj solution. Extractions of 150 ml portions were made with 3 x 200 ml aliquots of iso-amyl alcohol: ethylene dichloride (10:90, v/v) mixture by vigorous shaking. After each extraction, the phases were separated by centrifugation (3,000 x g: 10 min.). (c) Liver Homogenate from■Rats on■Chloroquine (i) Direct method (McChesney et al, 1967) An aqueous homogenate of the liyers was subjected to direct ether extraction, initially at pH 5.5 ('acidic fraction') and then after adjusting the homogenate to pH 11 ('basic fraction*). (ii) Acid Digest Method (Clarke, 1969) To 20 ml of the aqueous liver homogenate was added 20 ml concentrated HC1. The resulting slurry was diluted with 40 ml distilled water and heated in a boiling water bath for 1 hour. 43 This was filtered and the residue washed with water. The filtrate was first extracted with ether ('acid fraction')» adjusted to pH 11.0 with NaOH and then re-extracted with three volumes of either ether or iso-amvl alcohol: ethylene dichloride mixture. Mfr. ChrCTriatography All extracts were evaporated to dryness and the residue taken up in 1.0 ml methanol. Exactly 30 microlitres of the methanol extract was applied to a thin layer plate of silica gel, and the chromatogram was developed in the appropriate solvent system, M5. Estimation of Acetalddiyde Acetaldehyde was determined by a modification of the diffusion method of Gupta and Robinson (ly66), Warburg flasks with quick-fit ground glass stoppers were used in place of the specially designed flasks described by Gupta and Bobinson (1966). In the main part of the flask were placed 2.0 ml of 107° TCA, and 3.0 ml distilled water. A 0,2 mL sample containing 0,1 - 0,54 prole of paracetalde- UlOS hyde was added to the main part of the flask which stoppered. The centre well contained 0,6 ml 0,015M semicarbazide HC1 in 0.16M phosphate buffer pH 7,0. Precooled containers were used throughout to minimise the loss of acetaldehyde, The flasks were incubated at 37 C for 2 hours. A 0,4 ml aliquot of the acetaldehyde semicarbazide solution in the centre well was diluted with water f I G U R E f y - ACETALDEHYDE (yMOLES) 45 FIGURE 4 STANDARD CALIBRATION CURVE FOR THE ESTIMATION OF ACETALDEHYDE - A MODIFIED METHOD OF GUPTA & ROBINSON (19$?) 46 to 3.0 ml and the absorbance read at 224 mm against a blank containing an equivalent of semicarbazide solution. A standard curve was obtained (Fig. 4). In the experimental estimation of acetaldehyde from incubation systems, the main part of the flask contained the incubation mixture. Two millilitres of 10% TCA was placed in the side-arm. After thirty minutes incubation, the TCA was added to the main part of the flask by carefully tilting the flask. The mixture was then incubated for an additional 2 hours at 37°C, after which 0.4 ml aliquot of the acetaldehyde semicarbazide solution in the centre well was taken and diluted to 3.0 ml and the absorbance read at 224 nm. The acetaldehyde concentration in the incubation system was obtained from the calibration curve (Eig. 4). 47 PACT III RESULTS AND DISCUSSIONS 48 PART III SECTION 1 METABOLISM OF CfflflROQUINE PH3SPHA.TE Prd.iminary Investigations The ability to separate drugs and their metabolites from biological samples is an essential retirement of almost all investigations of drug disposition. Qualitative studies designed to determine whether a drug is metabolised, and if so, the identity of the metabolites have usually posed serious problems to investigators. The following preliminary investiga­ tions were therefore conducted to evolve a rapid and sensitive means of separating and identifying chloroquine and its metabolites from biological tissues, 1-A Liver Incubation Studies: Crude liver homogenate and its 9.000 x g supemate prepared according to general method MEB were incubated as previously described tinder general methods M2. After the reaction had been stopped, the mixture was extracted by the methods of McChesney et al (1967) and also by that of Warhurst (197^ ) .M3. The extracts were examined using TLC .plates without 4-9 fluorescence indicator; the chromatograms were examined under U.V. light and also with the iodoplatinate spray. Results and Discussion : In eight such experiments, only one U.V. - absorbing spot corresponding to chloroquine phosphate was observed with Rf 0.47; and this produced a bluish-brown complex with the iodoplatinate spray. Kb spots corresponding to any metabolites of chloroquine flpi found. These findings suggest that either (i) the amounts of chloroquine metabolites, formed in the incubation systems were too snail to be detected; in \5 V(0.b9.) VI(0.72) VII(0.78) Crude homogenate . .+ + + : y,000 x g supernate + +.' : + .' Mitochondria +' ■ + + + . + + Microsomes + : + .+ ; + + ■ + + MLcrgsomes soluble fraction + + + + + + + Soluble fraction + + + •f + Urine .+ . + + .+ .. + ■ + + + implies spots absorbed strongly under U.V. light. a. viwi. ***** sws not always observed on chromatograms. 68 Ob) Incubation mixtures of various subcellular fractions extracted with iso-amyl alcohol - dichloroethane (10:90) mixture at pH 10.5. In brackets are the corresponding Rf values of ultraviolet absorbing compounds isolated from urine of human volunteers losing the same extraction system (lYfcChesney et al, 1966). Subcellular Ultraviolet - absorbing areas on various chromatograms and their corresponding Rf’s. I. 0.04 II 0.19 0.17) Ila 00.22 (0.19) lib 0.33 (0.31) III 0.47 IV 0,b5 (0.63) V . 0.69 VI 0.72 VII 0.78 Crude homogenate; + + . 9,000 xg supemate + .+ . . . .+ : ■ : Mitochondria + . + + + ± MLcrosomes+ Soluble Fraction + + + + + + + + + Soluble Rraction + + + + ‘ + . + - Urine + + ' + + + 1 * + + implies spots absorbed strongly under U.V. light. + indicates that spots are not always observed on chromatograms. 69 Tables 1(a) and (b) show that the pattern of metabolites isolated from an incubation mixture in this experiment depends on the extracting solvent system: compounds contained in the U.V. absorbing areas corresponding to spots Ila and lib (table lb) are apparently insoluble in ether at pH 10.5. Elution of spots Ila and lib, however demonstrated that the substances present in than had the same ultraviolet absorbing spectra as that of the quinoline derivatives (maxima at 325 and 343 nm in 0.1M-HC1 in methanol). Another observation from tables 1(a) and (b) was that chloroquine was degraded to many more metabolites by the microsomal or microsome + soluble fractions. Thin layer chromatographic analyses of extracts of !'Blank” incubation systems (containing - subcellular fraction, NADP, G6P but no chloroquine) showed no U.V. - absorbing spots. This was an indication that chloroquine was the precursor of all the U.V. - absorbing spots. It was also observed that chloroquine was metabo­ lized in the microsomal + soluble fraction incubation system to at least seven metabolites. Tables 1(a) and (b) show that all the ultra violet - absorbing areas on chromatograms of incubation systems containing microsomes/ soluble fraction, matched seven of the twelve ultra-violet-absorbing spots found on chromatograms of urine extracts. In either case, and, particularly; the extracts from the various incubation 70 systems, the ultimate precursor of UV - absorbing compounds was chloroquine, Hence the observation that the metabolic pattern of chloroquine degradation by rat liver fractions in Vitro is similar to that of humans in vivo suggests that chloroquine metabolism may follow a pathway ccnjmon to both man and the rat. SECTION III 1 / jyfet^ holian of' (ring - 3 - C) chloroquine pbdspliate Kesults obtained fro® section II indicated that microsomes or a solution of microsomes + soluble fraction contained enzymes that metabolize chloroquine to at least seven ultraviolet « absorbing compounds. It was therefore necessary to confirm that all the U.V, - absorbing areas were really derived frcm chloro- quine, This was done by using (ring-3- C) chloroquine in the incubation systan involving microsomes plus soluble fraction, A 3.0 ml solution of microscmes plus soluble fraction (1,5 ml of each) was incubated aerobically for 30 minutes with 10 pool (16 jiCi) of (ring-3-^C) chloroquine at 37°C, in the presence of the NARPH- generating system previously described in general methods M2. A similar incubation system serving as the 'control' contained microsome plus soluble fraction (3.Qnl),NADP, G6P and unlabelled chloroquine. Hie reaction was stopped at the end of 30 minutes by the addition of 2.0 ml 10% TCA. 'The pH of the. incubation mixture was adjusted to 10.5 using 1 in 5, 0.88 NHg solution in water, and the duplicate incubations weTe extracted with three 25 ml portions of ether , and with iso-amyl alcohol/dichloroethane mixture. Ihe extracts for each solvent fMgr pooled; and 25 ml portion of the organic phase was evaporated to dryness under a stream of nitrogen. The residue was dissolved in 1,0 ml methanol, 72 One hundred microlitre portion of the methanol extract was applied to a thin layer plate of silica gel; and the chromatogram was developed using the ethylacetate/iso-propanol/ 1 in 5, 0.88 NH^ in water (79: 15: 6 v/v/v/) solvent system. Three chromatograms were run for each extract. For the quantitative determination of labelled chloroquine and its metabolites, fluorescent spots were scraped from the developed TLC plate. The silica gel was carefully transferred to counting vials and mixed with 10 ujl scintillation fluid containing Dioxane 1 litre, Naphthalene 120 g, PPO 4g, POPOP, 75 mg, (Butler, 1961). The radioactivity was measured in a liquid scintillation counter and the results were expressed as % of total radioactivity extracted (Table 2). The inhibitory effect of SK'F 525-A on the metabolism- of labelled chloroquine was also studied using methods described above. The incubation system contained 2.0 piole SK'F 525-A, labelled chloroquine and the NADPH- generating system described in general methods. 73 QUANTITATIVE PATTERN OF METABOLISM OF (3-^0 CHLOROQUINE BY RAT LIVER MTCROSOMES* USING VARIOUS EXTRACTING SOLVENTS TABLE 2 EXTRACTING QUANTITATIVE PATTERN OF METABOLITIES AS % OF SOLVENT TOTAL RADIOACTIVITY EXTRACTED Rf** I (0.04; II (0.19) Ila (0.22) lib (0,33:) Ill (0.47) IV (0.65) . v (0.69) VE (0.72) VII (0.78) Ether 2.55 40.50 1.62 40.20 - - 86.34 ■40,60 4.70 +0.51 2.70 +0.22 2.09 +0.15 - Iso-amyl alcohol: dicholo- roethane (10:90w/v) 2.77 40.02 3.14 40.55 1.42 43.02 5.09 40.05 66.21 )^.08 4.78 +0.06: 13.32 +0.40 3.22 tp.06 " 3.05 Ether extracts* of incuba­ tion mixture of micro- somes1U + C-chlo- roquine 42 umole SKF 525-A 3.94 40.14 4.04 40.14 - - - 64.93 +0,06 3.34 +0.08 L4.32 K). 08 0.60 H3.05 ■ 4.00 $.06 Hie data in Table 2 represents the mean and standard error of three determinations of three experiments, where each experiment was conducted with the pooled livers frctn three rats, *About 5% of the quinoline bases did not move from the origin and could not be characterized. **m brackets are the corresponding Rf values. Results and Discussion-. The quantitative distribution of the metabolites of chloroquine extracts using two different extraction media is shown in Thble (2). All spots (I-VII) fluoresced under the U.V. light. ' Their Rf values were identical to those in Tables 1(a) and (b). It can^ also be seen in Table 2 that SKF 525-A is apparently activating the metabolisn of chloro­ quine through the formation of metabolite V. Table 2 shows that the compounds contained in the U.V. absorbirg areas are all radioactive. This suggests that compounds contained in the eluates of the'- various U.V. - absorbing spots are metabolites of chloroquine. These deriva­ tives of chloroquine have variable solubilities in the different extracting solvents used - metabolites 11(a) and 11(b) are not extracted into ether whereas appreciable amounts of these metabolites - 1.42% and 5.09%' respectively, are extracted into the iso-amyl alcohol; dichloroethane solvent system. The results of the ether extraction M however shown in Table 2, as a more efficient solvent for extracting chloroquine from such incubation systems than the iso-amyl alcohol/dichloroethane mixture- Ihe latter solvent on the other hand, extracted comparatively substantial amount of metabolite V which contained 13.32% of the total radioactivity. 74 SECTION IV 75 SYNTHESIS OF METABOLITES OF CHLOROQUINE INTRODUCTION: Unsuccessful attempts were made to isolate the metabolites On a [a* —COOHSU-LpilOxidft* g ^ 4.50,012 CHj-CHCH^jCH^CHjCl CHXH^ LTY LTLTYLTFLTYEQTY NH C Hg-CHCHjCHjCHjNHqHy NH 77 SCHEME FOR IHE SYNTHESIS OF SOME K M N METABOLITES OF CHLOROQUINE KEY to figure: A: 4- (4' -hydroxy-1' -methylbutyl amino;-7-chloroquinoline or the 4'-hydroxy derivative B: Ihe 4- (-7' -Chloro-4' -quinonylamim) -4-methyl-l-butanal D: 4- (4' -ethylamino-11 ^ ethylbutylamino)7 -chloroquinoline or the Secondary amine derivative (*SN 13616). E: The Primary amine derivative (*SN 13617). *SN(for Survey Number) identifies the drug in the files of the Survey of Antimalarial Drugs; Ccranittee on jyfedical Research. U.S.A. FIGURE 11 78 This was preferred to the direct reaction with liquid anmonia (Carmack et al, 1947), because the available laboratory facilities were not adequate to supply the high pressure conditions. Moreover, the route via the phthalimido derivative is known to be reliably' efficient for all conversions of organic halides into amino derivatives (Sheeman and Bothofer, 1950). EXPEEtfMENT: Acetobutyrolactone (99%) and 4, 7-dichloroquinoline were purchased frcm Aldrich Chemical Company Limited, (1) 1 -Hydroxy-4-pentanone oxime (Fig. 11) A solution of 200g< of acetobutyrolactone in 60Qnl of 57o hydrochloric acid was stirred and heated on a mantle -under reflux in a round bottcm flask for three hours. The colourless mixture turned yellow during the course of the refluxing and then dark brown later in the experiment. The solution was then cooled and treated with 150.6g of hydraxylamine hydrochloride and 144g. sodium carbonate monohy­ drate. To this mixture, 80g solid sodium hydroxide was added with stirring and cooling to maintain the temperature below 60°C. The final mixture was left to stand at room temperature for 24 hours.. WAVENUMBER (CM-1) 80 Infra red spectrum of 1- hydrclxy -4 - peritarione oxime in Mijol Position of absorption CM -i 32U0-335U Strong intermolecular H-bonded 0-H (str.) 2y50 Sijol peak probably obscuring saturated C-H and C-C (Str.) 1660 C = N (Star,) 1450-1480 C-H (deformations) 1270-1280 0-H (bending)probably due to -O-H of Qsine 1010-1280 C-0 (Str,) Ihe information given above together with the recorded m.p„ (which agrees with literature) is consistent with the following structure, CH3 CCH, CH2 CH OH. FIGURE 12 N-OH 81 The report of Carmack et al. (1947) gives the impression that the pH of the mixture after standing for 24 hours should be on the alkaline side, but contrary to their observation, the pH of the mixture was 6.3, The pH was therefore adjusted to 7.4 (j W *) (as reccranended by Carmack et al/ with -18g KaOH pellets, and the crude l-hydroxy-4-pentanone oxime was extracted with ether for another 24 hours in a liquid - liquid extraction apparatus. Ether was then removed in a vacuum evaporator. The crude oxime recovered from. the ether was distilled as a pale yellow viscous liquid, b.p, 106-108°C (l.OQnm). The yield from acetobutyro- lactone was 807o. The oxime crystallized after 14 days standing at roan temperature and a sample recrystallised from ethyl J acetate formed colourless crystals, 1 Carmack et al. (1947) reported that the oxime crystallised spontaneously upon standing. The crystals were chrcmatographically pure, giving only one spot, Rf 0.42, when developed with iodine vapour. These data together with the infra-red spectrum (Fig, 12) indicated that a hydroxyoxime had been farmed. TR AN S MU lA N Cl !00 - 4000 3000 F I G U R E 4 3 •V". t 2000 1600 1 1200 800 WAVE NUMBER ( CM"1) FIGURE 13 83 Infra red spectrum of 4-anino-l-pentqnbl (k Be disc) Basition of absorption CM-1 3280-3350 Strong intermolecular Jhydrogen-bonded -0-H (Str,); or N-H (unsymnetrical and symmetrical Str.) of primary amines. 2960-2860(s) Saturated C-H and C-C (.Str,) probably associated with CH^ and - CH^ 1670 (weak) C-N- Stretch of unreacted oxime 16oO (s) N-H (Bending) 1380 0-H (Bending) 1060 This peak may be given by C-Q (Str .) Ihe information above together with the recorded m.p. of the compound synthesized (which compares favourably with the \Ja1*£- literature) is consistent with the following structure; A CH3 CH CH, CHg CE^- OH 84 (2) 4-amino-1-pentanol (Fig. 11) Distilled but not recrystalised 1-hydroxy-4-pentanone cecime (70 g) was hydrogenated over about 3,0g Raney nickel catalyst at an initial pressure of 125 atmospheres. No solvent was used. The temperature was carefully raised during 30 minutes to 60°C, and eventually to 75°C. Carmack et ali / , & (1947) stopped the consumption of hydrogen after one mole equivalent of it had been consumed in their reaction. In the absence of a device to measure the rate of uptake of hydrogen, hydrogenation was carried out for 24 hours. The decision to carry out hydrogenation for 24 hours was based on evidence from TLC and infra red studies on several trial hydrogenations that 24 hours was the minimum time required for an almost complete hydrogenation of the oxime under the experimental conditions. The product of hydrogenation was distilled under reduced pressure from water pump at 118-120°C. The yield was 72%. The product was confirmed by infra red spectrum (Figure 13). (3)  -i (4'   1 ’ -methylbutylajii ino)‘ 7*diloroquinoline- metabolite A (Refer to Fig. 11)’ A mixture of 9.8 g (0.1 mole) of 4-amino-l-pentanol and 10 g (0.05 mole) of 4, 7-dichloroquinoline was carefully heated in a 250 ml round bottomed flask fitted with an absorbent cotton wool plug - through which passed a thermometer. , 7 1 4 8 I G , , 1 4 0 2 F I G U R E 1 K b WA V E N UM B E R ( C M ' 1) 86 Infra red spectrum of the 4'-hydroxy derivative; 4- (4' -hydroxy-1 ■ -methylbutylamino) -7 chloroquinoline (in Mljol) -1 Position of absorption CM-A 3,400 - 3,500 Hydrogen bonded - O-H (Str,) 2990 - 3,000 Nujol peaks I60U N-H (Str.) for 2° amines 600 C - Cl (Str.) FIGURE 14 jb so '>A °/ ,c fi J=7CURB j £ W 'y\ 'S jL L /V < rft* A J IL L fM fC /Z O T y S FIGURE 15 Ultra violet spectrum of the 4' -hydroxy derivative of chloroquine in 0.1M-HC1 max .. at 2ZQ, 330 and 343 run; are characteristic of the spectrum of the quinoline nucleus of chloroquine. This together with information in Figure 14 suggest the formula of the compound synthesized to be, By means of an oil bath, the mixture was gradually brought to 152° ± t , and held at this temperature for four hours. The flask was occasionally agitated to ensure thorough mixing. No sign of uncontrollable violence, reported by Carmack et al, (1947) was observed in this reaction. After 4 hours of heating, the mixture was cooled to 100°C and poured into water. The liquid product immediately solidified into a sticky mass. The solid product was then left in cold water overnight to induce crystallisation. The solid product was washed with water, filtered and recrystallised twice from 95% ethanol, m.p. 178-181°C. The m.p. in literature is 175-178° (McChesney et al, 1966) and 179-181°C (Carmack et al, 1947) with yield of 90%. TLC of the recrystallised product using ethyl acetate: iso-propanol: 1 in 5, 0.88 NH^ (79: 16: 6 v/v/v/) mixture yielded only one U.V. absorbing spot, Rf: 0.65. The product was confirmed by infra red (Figure 14) and ultra violet (Figure 15) spectra. The U.V. spectrum was identical to that of chloroquine having maxima at 343,:350 nm, characteristic of the quinoline nucleus of chloroquine. 89 ab so rb an ce o V E D G ' p l£ WAVELENGTH MILLIMICRONS ULTRA VIOLET SPECTRUM OF IHE 4'-ALDEHYDE DERIVATIVE m CC14 FIGURE 16 T R A N S M IT T A N C E F I G U R E 17 W A V E N U M B E R ( C M ' 1) 93 Infra red spectrum of the 4'- aldehyde derivative of chloroquine in Mijol Position of absorption GM-1 3,300 Probably N-H (Str.) of quinoline ring 2900 Nujol peak 1705 C = 0 (Str.) of carbonyl group 1525-1610 Probably N-H (Str.) of 2° amines 1520-1600 Aromatic C-H vibration frequencies, 800 Peak of EMSO (Figure 18) absent; indicating no contamination by EMSO, i ' ^ %! ^ Structure CHj - CH - CK^ - CF^ - CHQ, $ NH FIGURE 17 F I G U R E I S W A V E N U M B E R ( C M ' 1) 95 FIGURE 18 jaSFRflRED SPECTRUM OF TMSO 96 4. Conversion of metabolite A (the carbinol) to metabolite B (the 4'-aldehyde derivative, Figure 11) A mixture of 0.5g of the carbinol, 3.2 ml of DMSO and 2.0 ml of acetic anhydride were left to stand at room temperature for three days. The product was dissolved in 15.0 ml chloroform. The chloroform solution was washed twice with 20.0 ml water. The solution was dried over anhydrous sodium sulphate and evaporated to dryness leaving a yellow solid. Thin layer chromatography, of the product yielded two spots - one corres­ ponding to the carbinol Rf. 0.65; and the other to the 4'-aldehyde f ’ - * . Rf. 0.72 (see Fig. 11). The product was confirmed by comparing I.R. spectra of the solid product (Figure 17) and that of DMSO (Figure 18). U.V. spectra of the compound was also obtained (Figure 16). 5. The c h l o r i d e derivative, (4' -chloro-1' Hnethylbutylamino) -7 -chlOroquinolirie. and its conversion to metabolite (E) (F ig . 11, page JQ) (a) Preparation Of the chloride derivative Eleven grams of the carbinol (recrystallised) in 30 ml of chloroform was treated with 6, 0 g SOCH^ in 5 ml chloroform, and the temperature was allowed to rise to the boiling point. As soon as the solid had dissolved, the reagent was removed by distillation. Bccess solvent was removed using a rotary evaporator. The product at this stage was a dark brown viscous oil: 95% alcohol was added and the mixture was shaken to dissolve the oil. Ekcess 4M NaOH was added. Mich shaking was required to solidify the oil. The dirty brown solid was recrystallised by first boiling in alcoholic solution containing activated charcoal, filtering the hot solution, and adding excess 4M-NaOH to induce crystallisation. Colourless crystals were obtained herein referred to as the chloride derivative, m.p. 130-132°C compared with m.p. in litera­ ture 129-130°C (Carmack et al, 1947). TLC of an alcoholic solution of this chloride derivative with ethylacetate: iso-propanol: 1 in 5, 0.88 NH^ (79: 16: 5) as running solvent showed a spot under U.V. lamp (254 nm) with Rf of 0.80. On the same plate, the carbinol was located with Rf 0.65. The product was confirmed with infra red spectrum. The disappearance of -OH stretch at 3400 cm-'*' (Figure 19) suggestf that conversion, from carbinol to chloride was complete. 97 w > A B S O R w w A B c F I G U R E i f WAVE N U M B E R (CM-1) INFRA. RED SPECTRUM OF 4- (4’ -CHLORQ-1' -METHYLBUTYL MOO)-7-CHIOROQUINOLINE IN NUJOL Position of absorption (CM-1) 1620 - 1560 N -> H - bending for 2° amine, or quinoline H H 2950 - 2890 Nujol peaks 805 C - Cl (Str,) for quinoline nucleus. FIGURE 19 100 (<$»}. Conversion of chloride derivative to metabolite E (Fig. 11) Potassium phtbalimide (0.7 g: 0.027 mole) was added to a solution of l.Og (0.025 mole) of the chloride in 2.5 ml dimethylformamide. The mixture was refluxed on a water bath for 3 hours. TWenty millilitres of chloroform were added, and the mixture was poured into 70 ml water. The aqueous phase was separated and extracted with 10 ml portions of chloroform. Ihe combined chloroform layertwas washed with 0.2M NaOH (to remove unreacted phthalimide) and 20 ml of water. After drying overnight over anhydrous sodium sulphate, the chloroform was removed in a rotary vacuum evaporator (at 70°C). The oily product was treated with ether and a small amount of insoluble material was removed by filtration. The resultant yellowish green oil solidified on standing overnight. Seventeen millilitres of methanol and 0,4 ml of 8570 aqueous hydrazine hydrate solution were added to the solid and the mixture refluxed for one hour. After cooling, 8,0 ml of water was added and the methanol was ranoved in a rotary vacuum evapora­ tor. Concentrated HC1 (0,8 ml) was added to the residual aqueous suspension and the mixture was heated tinder reflux for another one hour. After cooling to 0°C, crystalline phthalhydrazine was removed by filtration. TR AN SM IT T A N C E F I G U R E 20 Q 1--------- *---------------------- 1----------------------------------- 1----------------- 1------------------1__ _________ i____________i____________i________ i 5 0 0 0 3 0 0 0 1 8 0 0 1 4 0 0 IOOO 650 W A V E N U M B E R ( C M ' I ) 102 FIGURE 20 Infra red spectrum of the primary amine derivative: 4- (.4' -amino-1 ’methylbutylamino) -1 -ehlbrbquinoline in Nujol Position of absorption CM-1 2990 Nujol peak possibly obscuring C-H (Str.) 3500-3505 N-H (Str.) for 1° amines 1610 -H-H (Str.) for 2° amines 1590 Probably quinoline - NH (Str.) 800 C - Cl, A B S O R B A N C E F ! G U R E 21. ,7 1 4 8 I G ,,1 4 0 2 104 FIGURE 21 ULTRA VIOLET SPECTRUM OF THE PRIMARY (1°) AMINE DERIVATIVE IN O.lM H CL The maxima at 220, 330 and 343 nm are similar to those of the quinoline nucleus of chloroquine. This together with information frcm the infra red spectrum (Figure 20 ) suggest? the formula of the compound i' v V r ' prepared to be CH^ - CH-Cf^ — — CR, — ^ CH^ - NI^ The filtrate was then concentrated under reduced pressure to remove most of the HC1, and the moist residue was dissolved in 17 ml water. A small amount of insoluble material was removed by filtration, and 2M NaOH was added to precipitate the product. Thin layer chromatography of the product with the ethyl acetate system showed two spots with Rf of 0.04 and 0.80 respectively. The chloride and the carbinol derivative run on the same plate were located and gave spots with Rf s of 0.9 and 0.65 respectively. This indicated that the spot with Rf. 0.80 was probably a side-product of the hydrolysis reaction, but not any of the precursors of the 4(4'-amino-l'methylbutylamino) - 7-chloroquinoline (metabolite E). The product was purified by nmning two-way preparative TLC on 2 mm thick plates, using ethylacetate-iso-propanol: 1 in 5, 0.88 NH^ and n-butanol saturated with 10% NH^ in water, as running solvents respectively. The U.V. absorbing spot (Rf: 0,04)^ was scraped from the plate and was eluted with O.IM HC1 in ethanol. Two molar NaOH was added to precipitate the product as a pale yellow solid, >m.p. 98-100°C and yield 52%. The identity of the product was determined by U.V. and I.R. spectra (Figures 20 and 21). 105 F I G U R E 23 . - WAVELENGTH ( C M ' 1) 107 INFRA RED SPECTRUM OF 4-AMINO-7-CHIjORQQUINOLINE (KBr Disc)* Position of absorption (cm-1) 154U N-H bending for 2° amine (probably of quinoline N -> H.) 1340-1280 N-H (str.) Primary aromatic amines. 805 C - Cl (Str.) FIGURE 22 P^urchased from Rhone Chemicals, Polence - Paris, SECTION V IDENTIFICATION.OF U.V. . . ABSORBING .SPOTS .AND THE :IN VITRO METABOLISM OF SPECIFIC QUINOLINE DERIVATIVES A. IDENTIFICATION OF U.V. - ABSORBING■ SPOTS ON; CHROMATOGRAMS The identity of the substances located at the ultrayiolet- absorbing areas on the various chromatograms was established by chromatographing samples of known reference compounds simultaneously. With exception of 7- chloro - 4 - aminoquinoline, all the reference compounds used as standards were synthesized, purified and characterized as in section TV. These include, the 4'-alde­ hyde derivative, and 4'-hydroxy derivative and the primary amine derivative, SN 13617 (see page 77). 7-chloro-4-aminoquinoline was purchased from Rhone-Poulence-Paris, and had the following physical properties. It was a white crystalline solid m.p. 149-150°C, Cl, 19.85%; N, 15.68% (calc), and C, 64.47% (calc). It was insoluble in water but soluble in CCl^ , dimethyl formamide, ethanol and ethylene glycol. Thin layer chromatographic analysis of extracts of the incubated mixture containing chloroquine and the microsome/ soluble fraction system was carried out as in section IT (page 66) with the reference compounds listed above as standards. Alcoholic solutions of the reference compounds were used. 108 FISPHE £.3 mass gptsgnaiB of ■nvtsm OF spoi VXI on Tim? MW& cm rm ATO fVRm <& ' x p r a c t s o f i t ? c u p # H O W . E D g i g g F H8H 337-75/1 8AS® PRAK ICX^ PSD j/B flOSKRCTION a 321 270 227 ifi 180 178 151 143 140 139 138 137 136 134 125 123 117 116 115 111110 109 107 105 103 101 99 98 XL 0.9 1.2 B 5.2 10.0 32.1 6.7 7.0 35.8 21. 93.8 9.8 f9919 ft 100.0 ABD 5.3 7.5 5.6 11.4 6*6 18.2 3.4 15.5 21f 8.7 9.* 16.0 17.5 R 7.9 22.8 0 I I 1- I I- I I- I I" I I- 1 I- I- I- 6 9 10 f9 110 The identities ■ of compounds'oii the;chromatogram of the extract Of the incubated mixture'containing chloroquine and the miigrdsome/cytoplasm system, as shown by matching standards TABLE 3a U.V. -absorbing spot* Spots from reaction mixture Rf. Corresponding matching standard 0,04 The primary amine derivative (SN 13617 IT!' 0.47 Unchanged chloroquine IV 0.65 4-(4'-hydroxyl-1' -methylbutylamino) - 7-chloro-quinoline or the 41-hydroxy derivative VI 0.72 The 4' -aldehyde derivative VII 0.78 7-chloro-4-aminoquinoline ** U.V. - absorbing spots are numbered I to VII in order of increasing Rf values as in Table la (See page 66) This had already been identified in Section I (page 51) as Ill RESULTS The identities of the compounds on the chromatogram of extract of the incubated mixture containing chloroquine and microsome/cytoplasn system as shown by matching standards is presented in Table 3a. The identity of spot VII was further confirmed by running a mass spectrum on the eluate of spot VII. TTl/The resulting spectra in Figure 23 show peaks at e ratio of 180 and 178 in the proportion of 1:3.2, thus corresponding to 37 357 - chloro-4-aminoquinoline in which Cl is Cl and Cl isotopes respectively. Incidentally, this is the normal isotopic composi­ tion of Cl and all its derivatives, ie. ^Cl: ^C1 (Williams and Fleming, 1973). Four of the U.V. absorbing spots were not identified in this experiment. These are spots II' (Rf 0.19), Ila (Rf 0.22), lib (Rf 0.33) and V, Rf 0.69 (See page 68). B. :IN -VITRO. STUDIES.ON THE METABOLISM .OF .REFERENCE QUINOLINE COMPOUNDS USED AS' STANDARDS ■' IN SECTION VA In another experiment to study the sequence of metabolism of chloroquine and its derivatives, a 0.1% (w/v) solution of each of the reference compounds (in ethanol) used in Section VA was incubated with the microsome/cytoplasm system using the incubation system M2 given in the general methods (page 44). Incubation was carried out for one hour at 37°C; and each incubation mixture was 112 TLC analysis of the extracts of the Incubated mixture containing the microsonio/cytoplasm system with a reference quinoline compound sis substrate TABLE 3b Reference quinoline substrate Observation on thin layer chromatogram of extract 7-chloro-A-- aminoquino- line Only ana U. V.-absorbing spot Rf 0.78, and corresponding to unchanged 7-chloro-4-amino- quinoline was observed. 7-Chloro-4-aminoquinoline was probably not metabolized by rat liver microsomes to any U.V.- absorbing derivative. Ihe 4'-ald­ ehyde deri­ vative. Ttoo U. V.-absorbing spots were abserved - a faint one at Rf 0.72 , corresponding to the unchanged 4'-aldehyde; and another strong U. V.-absorbing spot at Rf 0.65, corresponding to the 4 ' -hydroxy derivative. Cbmparis^ SSff 0 the intensities of the U.V.-absorbing spots, it appears that most of the 4'-aldehyde derivative is converted in vitro by rat liver microsomes into the |4' -hydroxy derivative Only one U. V.-absorbing spot The 4'-hy- was observed at Rf 0.65, droxy corresponding to the derivative unchanged 4'-hydroxy derivative. The 4'-hydroxy derivative was probably not further metabolized in vitro by the rat liver microsome/cytoplasm system. Ihe pri­ mary amine derivative (SN 13617) Two U. V.-absorbing spots corresponding to the 4'-hydroxy derivative, Rf 0.65, the unmetabolized primary amine derivative, Rf 0.04 and a weak U. V.- absorbing spot of the 4'- aldehyde derivative Rf 0.72 were observed. Other ultraviolet-absorbing spot S were detected on the plates, but the ultraviolet spectra of their eluates did not correspond to that of any known compound of the 4-aminoquinoline series. The primary amine derivative SN 13617 (page 6$) is metabolized in vitro by the rat liver micro- some/cytoplasm system into at least two other metabolites, namely: the 4'-hydroxy and the 4'-aldehyde derivatives. In the absence of a labelled atcm in the nucleus of the primary amine derivative, it would be difficult to establish the precursor of the compounds contained in the eluates of the other U.V,-absorbing spots. 113 extracted with ether and also with dichloroethane: iso-amyl alcohol mixture. Each extract was subjected to thin layer chromatographic analysis as in section VA and the results obtained are summerised in table 3b. SECTION VI 114 Investigations into the .mechanism of :the in vitro degradation of chloroquine by hepatic microsomal fraction of rat liver homogenate The in Vitro oxidation of tertiary amine drugs and other foreign compounds has been extensively studied over the past three decades; and, of the different pathways by which tertiary amines can be oxidized, oxidative N-demethylation has been thoroughly studied (Gillette, 1969). The work; of McMahon and Sullivan (1964) suggests a mechanism requiring a direct oxidative attack on the methyl group of an \T-methyl tertiary amine to form the N-hydroxy methyl derivative which would readily decompose to the secondary amine and formaldehyde. On the other hand, the experiments of Fish et al (1955) and the subsequent kinetic studies of Ziegler and Pettit (1964) suggest that, the initial oxidative attack occurs on the nitrogen atom to yield an intermediate N-exide. This oxygenated intermediate is then dealkylated by a second enzyme system. Upon subsequent studies by the latter investigators on N, N-dimethyl- aniline for example, Ziegler and Pettit suggested further that the microsomal system catalysing the oxidative N-dealkylation of lipid soluble dialkylaryl amines could be separated into two 115 partial reactions (i.e. the N-oxide synthesizing and the N-oxide dealkylating systems). Based on the subsequent demonstration that the enzymic dealkylation of the N-oxides of simple methylarylamines is inhibited by SKF 525-A (Machinist et_al, 1966), Zeigler and Pettit (1966), demon­ strated the accumulation of the N-oxide of dimethylaniline in presence of SKF 525-A. In part, the extensive investigations on the pathway of metabolism of simple methyl arylamines via N-oxides can be attributed to the use of a sensitive spectrophotcmetric assay for the estimation of formaldehyde developed by Nash (1953). AL though the N-oxidation of tertiary amines has been demon­ strated in many vertebrates (tighleke and Stahn^ l966) including man (Ktmtzmari et al, 1967), studies on the in Vitro N-oxidation of certain other alkylamines, such as ethyl arylamines have been hampered by lack of sensitive micro methods for the estimation of acetaldehyde - a side product of N-dealkylation of most ethylamines and for the detection of a wide variety of amine oxides. This chapter discusses attempts made to demonstrate the probable formation of the N-oxide of chloroquine and the con­ comitant production of acetaldehyde when chloroquine is incubated with the microsomal fraction of rat liver homogenates. (a) Distribution of the products of the iiicobation of j}4c]~ Chloroquine with hepatic microsomes between the unchanged drug a n d metabolites thereof, in presence of SKF 525-A Ten micromoles of (ring-3-^ C) chloroquine (16 fid) was incubated for 30 minutes at 37°C with the hepatic microsomal fraction in presence of SKF 525-A (2 -pinole). An ethereal extract of the incubated mixture was then analysed by thin layer chroma­ tography and quantitative determination of the U.V. absorbing areas was made by the method in section IIT (page 71). The motive behind this experiment was that if the oxidative dealkyla- tion of chloroquine really proceeded through the formation of the N-oxide intermediate, and if the subsequent oxidation of the N-oxide formed was inhibited by SKF 525-A, then, the presence of SKF 525-A in the incubation system should lead to the accumulation of the U.V. - absorbing spot corresponding to the N-oxide. Results: Table 2 (page 73) shows that the ether extract of incubation systems containing SKF 525-A yielded a mean of 64.93% of the 4-aminoquinoline bases as the unchanged drug. About 5% of the quinoline bases did not move from the origin and could not be characterised. In addition, however, the same extract contained 14.32% of 4-aminoquinoline bases in the form of the compound contained in spot J . 116 In a similar extract of incubation systems containing no SKF 525A, the compound in the U.V. - absorbing spot V accounted for only 2.70% of the total 4-aminoquinoline bases. There were also slight increases in the percentage of compounds contained in spots II and I", and corresponding decreases in those in spots IV and VI in extracts of incubation mixtures containing SKF 525A, compared to the levels of these compounds from extracts of incuba­ tion systems without the SKF 525A. f-fetabolite VII accounted for 47o of the total quinoline bases in the extract of the former incubation system whilst no trace of it was found in extracts of the latter incubation systems. (b) Acetaldehyde estimation in hepatic microsomal incubation mixtures, of chloroquine Acetaldehyde is one of the products of dealkylation reactions involving ethylaimnes (Williams, 1959), Attempts were therefore made to estimate the rate of acetaldehyde production in the microsomal incubation systems using a modified method of Gupta and' Kobinson (1966) as outlined under method (M5). Acetaldehyde concentrations measured losing this method not sensitive enough to measure low rate of acetaldehyde production and allow any subsequent kinetic studies oh chloroquine degradation, based on acetaldehyde measurements, to be made. 118 Ttoo other unsuccessful attempts were made in a search for sensitive methods for the estimation of acetaldehyde. Hie first was a modification of the method of Nash (1953) for the estimation of foCTa.ldeh.yde. Ihis is based on the formation of a corresponding pyridine ring, diethyl 1, 4-dihydrocollidine, frcm a reaction between acetalddiyde, acetylacetone and NH^. Nash (1953) reported a yield of 51% for the. pyridine compound formed frcm this reaction, and that the compound had an absorp­ tion maximum at 353 ran. Estimation of acetalddiyde by this method might have had an advantage over the diffusion method of Gupta and Robinson (1966) in that acetaldehyde was trapped as it was formed in the solution to form the pyridine compound. However, the rate of acetaldehyde production measured by this method was still too low for it to be used in studies of the microsomal degradation of chloroquine. The other unsuccessful attempt on the estimation of acetalde­ hyde was based on the micro diffusion method of E. Cbnway (1960). This method was however found unsuitable since thin layer chromato­ graphic analysis of extracts of incubation mixtures on which acetaldehyde estimation using this method was made did not reveal any U.V. - absorbing spot. The method was therefore abandoned on the grounds that the use of vigorous conditions such as 0.66N I^ SO^ and tungstic acid probably destroyed the quinoline nucleus of chloroquine - the substrate. (c) Measurements of rate of Cfrygen consumption in hepatic 119 mi rrngfma 1 incubation mixtures of chloroquine In a further search for analytical methods for conducting kinetic studies on chloroquine oxidation, attempts were made to measure the rate of oxygen-consumption in the hepatic microsomal incubation system. Ihe dealkylation enzymes are presumed to catalyse the consumption of one molecule of oxygen per molecule of substrate consumed (lYksan, 1957, 1965). Ckygen measurements were made using an oxygen electrode (Bank model), Ihe sensitivity of measurements made by this method was so negligible that tio subsequent kinetic studies on the metabolism of chloroquine based on the rate of oxygen consumption could be made. PART iV.. 120 CO N C L D S I ON, S •Ihe data on the subcellular distribution of chloroquine degradation by the hepatic cellular fractions (table&La and lb) is very interesting especially in view of the fact that a lot of the efforts of most biochemical pharmacologists has been directed towards the solubilization of lipid-soluble microsomal enzyme systems. Thin layer chromatographic analysis of both the ether and the dichloroethane - iso-amyl alcohol extracts of incubation mixtures of chloroquine shows that, the soluble fraction (105,000 x g supemate) of rat liver homogenates contains enzymes responsible for the in vitro degradation of chloroquine. Ihe soluble fraction could therefore be used for metabolic studies involving chloroquine. Also important in the results presented in tables 1(a) and 1(b) is the further observation that, the mitochondrial fraction of the rat liver homogenate degraded chloroquine to t\ some extent. This possibility has been mentioned by La IXi et al (1971) . However, consistent with current thinking, the maximum activity for chloroquine degradation has been found (tables la and lb) to be localised in the microsomal fraction, Ihe data on the metabolism of (ring-3-^ C) chloroquine (table 2) indicates that chloroquine was the precursor of all the 121 nine ultraviolet absorbing spots which appeared on chromato­ grams of the dichloroethane - isQ-amyl alcohol extracts. This implies that the microsomal fraction of the rat liver homogenate contains enzyme systems that metabolize chloroquine into a minimum of eight metabolites in which the 4-aminoquino- line base is intact - one of the nine ultraviolet absorbing spots (Rf 0.47) was identified as unchanged chloroquine. Four other U.V. - absorbing spots were also identified using reference 4-aninoquinoline derivatives as standards. These are the primary amine derivative (Rf 0.04), the 4■-hydroxy derivative (Rf 0,65), the 4'-aldehyde derivative (Rf 0,72) and 7-chloro-4-aminoquino- line (Rf 0.78) (see table 3a page BIO), These results are also consistent with the work of M:Chesney et al £ l9b6 )j in which they observed eight -ultraviolet absorbing, spots on thin layer chromatograms of urine extracts fran monkeys on chloroquine. Using the same solvent system, McChesney and his associates located all eight U. V. -absorbing spots at the position (Rfs) as those of the eight U.V. spots observed in these experiments. (table 2). It is therefore not surprising that the identity of compounds contained in some of the U.V, - absorbing areas in these experiments (section III) parallels that made by MbChesney and his associates (1966) - spot I (Rf;0,04), being the primary amine; spot III (Rf,0,47), the unchanged chloroquine; spot XV (Rf, 0.65), the 4'-hydroxy derivative and spot VII (Rff0.78), the 7-chloro-4-anino quinoline. In their proposed metabolic pathway for the in vivo degradation of chloroquine, McChesney and his associates alluded to the 4'-aldehyde derivative as a very important intermediate which, to a minor extent, is apparently reduced to the 4'-hydroxy derivative. For the first time, the 4'-aldehyde has not only been identified as a metabolite of chloroquine (table 3a, page 110) but also, its subsequent metabolism to the 4 -hydroxy derivative has actually been demonstrated (table 3b) (page 112). In fact, it has been further demonstrated through in vitro studies in this thesis that, the 4'-aldehyde is largely converted to the 4'-hydroxy derivative by the microsomal fraction of the rat liver (refer to Table 3b page 112). This latter finding appears contrary to the proposal of McChesney and his associates, that, the 4'-aldehyde is a major metabolite in vivo which, to a minor extent, is reduced to the 4' h^ydroxy derivative. The disagreement in the results in this thesis and that proposed by McChesney and his associated (1966) is however not unexpected. It has already been stated elsewhere in the literature review (page 11) that such factors as absorption, tissue distribution, the role of gut bacteria etc. dictate the overall picture of species variation in drug metabolism; and that investigations based on either in vivo or in vitro studies alone are not sufficient to account for species variations in drug metabolism. Perhaps the differences in the results recorded in this thesis and that proposed by JVfcChesney et al. (1966) might be a clear example of this fact. 122 It is however evident from both results that the 4 -aldehyde is not only an intermediate in chloroquine metabolism, but that it is to sane extent converted to the 4'-hydroxy derivative. No attempt was however made to detect the 4'-carboxy derivative, which has been shown to be the principal metabolite in the monkey (McChesney et al, 1966); its probable formation therefore cannot be ruled out. The observation that the 4'-hydroxy derivative is not further metabolized by hepatic microsomal enzyme systems is contrary to the conjecture by McChesney e£ aZ, (1966) that the 7-chloro-4-amino- quinoline is formed by the successive degradation of the butylamino side chain through the 4' -hydroxy derivative was not metabolized further by the microsomal enzyme system, it appears the 7-chloro- 4-gminiquinoline observed on thin layer chromatograms of chloroquine might have been formed directly from chloroquine by an alternative pathway. The 7"Chloro-4-aminoquinoline was also not metabolized further by the microsomal fraction; and it could probably be the end product of the alternative pathway being proposed. The secondary amine (SN 13616) could not however be synthesized in this laboratory for lack of suitable glassware to withstand the high pressure conditions required for the reaction between ethylamine and 4-(4'-chloro-1' -methylbutylamino)-7-chloro- quinoline (Fig. 11). It is however interesting to note that the Rfs of the compounds so far indentified in these experiments are 123 identical in most cases to the Rfs of the s a m e compounds identified b y M c C h e s n e y and his associates who used the s a m e solvent system. For e x a m p l e , in both these experiments, and that of M c C h e s n e y and his associates, the unchanged drug has been identified at Rf 0.47, the 4-amino-7-chloroquinoline at Rf 0.78. The 4'- h y d r o x y derivative which was located by M;Chesney and his associates at RE 0,63, was however observed in these experiments at Rf 0.65. McChesney et al (1966) also located the secondary a m i n e (SN 13616) derivative at Rf 0.17. Spot II in these experiments (tables la, lb and 2) with Rf 0.19 is close to the location for SN 13616; and, this, together with the fact that the U.V.-spectrum of spot II (Fig.6) has identical characteristics (max at 330 and 343 nm) as the 4-aninoquinoline bases, suggestSthat spot II in tables la, lb and 2 is probably the secondary amine derivative (SN 13616) . By a similar argument, one could also speculate that spot Ila, R f 0.33 (tables la and 2) is probably hydroxychloroquine (i.e. the analogue of chloroquine in which a hydroxyl group has been substituted in the 2-,position of one N-ethyl group).. McChesney and his associates using the same solvent system, identified authentic hydroxy-chloroquine at RE 0,31, The implication of hydroxychloroquine as a metabolite of chloroquine would not contradict modem theories of drug metabolism. 124 It would only support the mechanism proposed by McMahon and Sullivan (1964) that the initial process in N-dealkylation of tertiary amines involves a direct attack on the alkyl group of N-alkyl tertiary amines to form the N-hydroxy alkyl derivative', which in turn readily decomposes to give a secondary amine and the corresponding aldehyde. The possibility of hydroxychloroquine being an intermediate of chloroquine metabolism is further supported by the further observation of McChesney et al (1966) that thin layer chromatograms of urine extracts of humans on hydroxychloroquine•indicated the following composition: hydroxychloroquine, 49%; SN 13616 - the secondary amine, 21%; SN 13617 - the primary amine, 6%. MeChesney and his associates also demonstrated through in yiyo experiments that SN 13616 is the precursor of SN 13617, the 4-hydroxy- derivative and the 4-amino-7-chloroquinoline, together with small amounts of other unidentified compounds. It has also been shown in these experiments that the primary amine, SN 13617 is metabolized in vitro, to the 4'-aldehyde and the 4'-hydroxy derivatives. In the light of these findings, it is being proposed that the metabolic degradation of chloroquine in vitro by the hepatic microsomal enzyme systems of rat proceeds by the formation of the following metabolites in order of: 125 126 (i) the hydroxychloroquine, (ii) the secondary amine (.SN 13616) (iii) the 4' - aldehyde which is largely converted to the 4' - hydroxy derivative. But for the addition of hydroxychloroquine, the present proposed mechanism is in accord with the concept proposed by lyfcChesney et al (1966). The results in table 1 (b) that all the six metabolites frcm; the extract of the microsomal incubation system matched six of the twelve U.V. absorbing spots from the urine extract of humans on chloroquine, is also interesting in view of species differences danonstrated on the metabolic fate of certain drugs (Dingel|_,et al , 19$4). These data in table 1 (b) therefore suggest that chloroquine metabolism at least, in part, follows qualitatively similar pathways in both man and the rat. The result on the effect of SKF 525A on the in vitro metabolism of - labelled chloroquine is rather puzzling, though, not contradictory to current theories on drug metabolism. In table 2, SKF 525A is apparently activating the metabolisn of chloroquine through the formation of both metabolite V, and the 4-atnino-7-chloroquinoline. This observation is contradictory to the report made by Gaudette and Ooatney (1961) that SKF 525A caused a 50% inhibition of chloroquine metabolism in their in vitro experiments, in which they incubated chloroquine with the 9,000 x g supemate of rabbit liver microsomes. They assayed their enzyme activity by measuring the disappearance of substrate, chloroquine from an n-heptane extract (PH 9.0) using • ..."■ spectrophotofluorimeter at peak activation of 330 and 440 nm. It has however been demonstrated in this laboratory that the n^ heptane extract (pH 9.0) of rat liver microsomal incubation system is a mixture of the unchanged drug, the secondary amine (SN 13616) and the primary amine (SN 13617), Further, since the authors did not study the effect of SIT 525-A on the metabolic pattern of chloroquine in their incubation system, it would be very difficult to compare their results with the work in this thesis. SKF 525-A is a known inhibitor of most N-dealkylation reactions. The metabolism-of a number of compounds such as, the N-dealkylation of N-methylaniline, O-dealkylation of phenacetin, sulphoxidation of chloropromazine and the reduction of aromatic nitro compounds, are however, not inhibited by SKF 525-A (IVknnering, 1969). Since in these experiments, the amounts of metabolite V and 4-amino-7- chloroquinoline are.shown to accumulate on incubation of the hepatic microsomal fraction with chloroquine in presence of SKF 525-A (Table 2), it is being further proposed that the metabo­ lism of chloroquine in vitro may also follow an alternative pathway through metabolite V to produce the 4-amino-7-chloroquinoline. 127 It is also suggested that it is probably this latter pathway in the rat which is activated by the presence of SKF 525-A. Further investigations to prove this hypothesis were not possible because of lack of more labelled chloroquine and other experimental materials. The difference between these results and that of Gaudette and Coatney (1961) might be explained as due to species differences in the metabolic fate of chloroquine in the rat and the:.' rabbit; and that probably chloroquine is not metabolized through metabolite V in the rabbit. Rats for example, haye been shown to metabolize imipramine by demethylation, whilst rabbit microsomes are reported to metabolize this drug through hydroxyimipramine (Dingel e£ al, 1964). In man, chloroquine is slowly metabolized and excreted; and 600 mg chloroquine base have been shown to provide suppression of malaria for at least 20 days (Gaudette and Coatney 1961). Individuals receiving single doses of 300 mg base for 26 weeks experienced suppression for at least 16 days after the last treatment. Yfet in a program where drugs are used for the eradication of malaria, longer parasite-free intervals would be desirable. Chloroquine is known to exert its antimalarial activity directly in yiyo (Olatunde, 1971); and a prolongation of this activity might be obtained if its metabolic transformation could be retarded. It is hoped the observations 128 FIGURE 24 PROPOSED PATHWAY FOR THE METABOLISM OF CHLOROQUINE BY RAT HEPATIC MICROSOMAL ENZYME SYSTEM F I G U R E 2 if i s o . C Hs-CH-CH«.CHl CH»Nr"CaHs NH Ci HUr.. |YW R.R. (1968), J. Pharm. Sci. 57_: 2021-2035. *Schmid, K., Cornn, F., Enhof, P. and Kerberle (1964), Schweiz. Med. Wbchensch 94: 235-240. Scheeman, J.C. and J^ thofer, W'.A. (1950), J. Chan. Soc. 72_: 2786. *Stem, L., Khanna, N.W., Yaffe, S.Y. (1970), Aner. J. Dis. Child 120: 26. Thompson, R.P.H. § Williams, R.J. (1967), Lancet U: 646. Titus, E.O. Craig, L.C., Golumbic, G., Mighton, H.R., Wenjpin, L.M., and Elderfield, R.C. (1948), J. Org, Chem. 13_: 3962. Uehleke, H,, and Stahn, V. (1966), Arch. Exp. Path. Pharmak 255: 287. Vesell, E.S, (1968), Ann. N.Y, Acad. 151: 900-912. *Von Jagow, R. 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Williams, R.T, (1967) Biogenesis of Natural Compounds, ed. p. Bemfeld, pp.589-639, Pergamon Press, N'.Y. Sec. Ed. Williams, R.T. (1971). In Fundamentals of Drug Metabolism and Drug Disposition: La Du, B.N. Mande'l, G.H., and Way, E.L. (eds) page 187, The William and Wilkins Co. Baltimore. U.S. WH3. Tech. Rept, Ser. (1967) Standardisation of Procedures for the Study of G6PD. Yaffe, S.J., Levy, G. Matsuzawa. T., Ba Yah, T. (1966) New Eng. J. Med. 275: 1461. Zanca, A., and Benatti, M. 1959, Arch. Ital. Derm. Vener. 29: 462, Abstr, from Brit. J. Derm. 73; 83 1961. Ziegler, D.M. and Pettite, F.W. (1964) Biochem Biophys. Res. Common. 15: 188 Ziegler, D.M- and Pettit, F.H. (1966), Biochemistry 5: 2932.