A thesis submitted for the Degree of Doctor of Philosophy in the University of London DISPOSITION OF THEO.A1YLLINE AND AMINOPHYLLINE IN MAN by Terrence J. Monks, B.Sc. Department of Biochemical and Experimental Pharmacology, St. 77ary's Hospital Medical School, London. November 1978 2 Ai!.STRACT 14 The metabolism and pharr,acokinetics of ( C)-theophylline have been studied after intravenous doses of 100mg to healthy volunteers keeping to their usual diets, after 7 days abstention from methylxanthine-containing foods and beverages and again after such abstention while ingesting theophylline and caffeine in tablet form. The metabolism of orally administered (14C)-theophylline has also been investigated. Metabolites were separated and quantitated by ion-exchange column chromatography, ion-exchange paper chromatography and liquid scintillation counting. Three major metabolites were found in urine in addition to theophylline, namely 3-methylxanthine, 1,3-dimethyluric acid and 1-methyluric acid, and two minor metabolites were detected but not identified. The kinetics of the elimination of the metabolites were studied after intravenous administration, and theophylline, 1,3-dimethyluric acid and 1-methyluric acid were eliminated by first-order kinetics while that of 3-methylxanthine was described by Michaelis-Menten kinetics. Abstention from methylxanthine-containing foods and beverages led to a significant decrease in the elimination half- life of 14C due to increases in the pharmacokinetic constants describing-the elimination of theophylline, 3-methylxanthine and 1,3-dimethyluric acid. When the methylxanthine content of the methylxanthine-containing foods and beverages was replaced by theophylline and caffeine in tablet form the pharmacokinetics and metabolism of intravenously administered (14C)-theophylline were unchanged from those seen on the volunteers usual diets. The metabolism and pharmacokinetics of intravenously administered (14C)-theophylline ethylenediamine (aminophylline) have also been studied. The nature and elimination kinetics of the metabolites identified were the same as those observed after the intravenous administration of L,)-theophylline. Abstention from methylxanthine-containing foods and beverages had no effect on the disposition of intravenously administered (14C)-aminophylline. The implications of the results are discussed with reference to pharmacokinetics and drug metabolism,drug-diet interrelations and the effects of dietary methylxanthines on drug metabolism. CONTENTS Page No. ABSTRACT 2 TABLE OF CONTENTS 3 LIST OP TABLES 6 LIST OF FIGURES 9 ACKNOWLEDGEMENTS 13 CHAPTER 1 INTRODUCTION 14 THE METHYLXANTHINES: Source and History 15 Chemistry 20 Aminophylline 20 Pharmacological Properties 31 Metabolism of the xanthines 33 and methylxanthines CLINICAL PHARMACOLOGY OF THEOPHYLLINE 39 FACTORS AFFECTING LRUG YETABOLISM: 45 Genetic Factors:- Genes of large effect 47 Genes of small effect 55 Environmental Factors:- Environmental chemicals 55,58 Socially used drugs 58 Nutritional factors 64 Other factors 73 AIMS AND SCOPE OF THE THESIS 78 CHAPTER 2 MATERIALS AND METHODS 80 COMPOUNDS 81 EXPERIMENTAL PROTOCOL 81 Oral Administration 81 Intravenous Administration:- Theophylline Normal diet 81 Methylxanthine-deprived diet 81 Methylxanthine-supplemented diet 82 Caffeine/theophylline 82 replacement diet Aminophylline 82 CONTENTS curt. Page No. TREATMENT OF URINE SAMPLES 83 Dowex 2 X8 Anion Exchange Column Chromatography:- 83 On column conditioning 83 Application of urine 83 Ion-exchange paper chromatography 84 RADIO-CHEMICAL TECHNIQUES 84 TREATMENT OF BLOOD SAMPLES 86 REVERSE ISOTOPE-DILUTIONS 86 KINETIC ANALYSIS OF URINARY METABOLITE DATA 86 Hanes-type Plots for the Elimination of Theophylline 86 and its Metabolites Determination of Elimination Rate Constants for 87 Theophylline and its Metabolites by the "Sigma-Minus" Method CHAPTER 3 METABOLISM OF ORALLY ADMINISTERED (140-THEOPHYLLINE 90 INTRODUCTION 91 RESULTS 92 DISCUSSION 97 CHAPTER METABOLISM AND PHARMACOKINETICS OF INTRAVENOUSLY 100 ADMINISTERED (14C)-THEOPHYLLINE TO VOLUNTEERS ON THEIR NORMAL DIETS INTRODUCTION 101 RESULTS 102 Urinary Kinetics 111 DISCUSSION 121 CHAPTER 5 METABOLISM AND PHARMACOKINETICS OF INTRAVENOUSLY 139 ADMINISTERED (11+C)-THEOPHYLLINE TO VOLUNTEERS ON VARIOUS DIETS INTRODUCTION 140 RESULTS 141 DISCUSSION 154 CHAPTER 6 METABOLISM AND PHARMACOKINETICS OF INTRAVENOUSLY 161 ADMINISTERED (14C)-AMINOPHYLLINE INTRODUCTION 162 RESULTS 163 Normal Diet 163 Methylxanthine-deprived Diet 172 DISCUSSION 174 CON TE= Page No. CHAPTER 7 DISCUSSION 181 PHARMACOKINETICS AND DRUG METABOLISM 182 Plasma Kinetics or Urinary Metabolite Kinetics? 182 Which Method of Analysis? 185 Considerations and Implications of Data Derived 186 from Urinary Metabolite Studies DRUG-DIET INTERRELATIONS 191 EFFECTS OF DIETARY METHYLXANTHINES ON DRUG METABOLISM: 193 IMPLICATIONS APPENDIX 195 REFERENCES 216 6 LIST OF TAF IES Table No. Title Page No. 1.1 The natural and dietary sources of the 16 methylxanthines. 1.2 The relative pharmacological activity 18 of the methylxanthines. 1.3 Some physical properties of the methyl- 23 xanthines. 1.4 A summary of substances used to solubilise 24 methylxanthines. 1.5 Some stable complexes of theophylline. 27 1.6 Some stable complexes of caffeine. 28 1.7 The final step of purine catabolism in 34 various species. 1.8 Excretion of unchanged caffeine after its 37 oral administration to various species. 1.9 Plasma elimination half-life of caffeine 38 in various species. 1.10 Factors known to influence drug metabolism. 41 1.11 Theophylline elimination rates in various 42 groups. 1.12 General pathways of drug metabolism. 46 1.13 Some compounds known to act as conjugating 48 agents in drug metabolism. 1.14 Drugs whose metabolism is determined by 50 genes of large effect. 1.15 Some drugs known to cause microsomal 56 enzyme induction in man. 1.16 Some drugs known to cause inhibition of 59 drug metabolism in man. 1.17 Environmental chemicals known to affect drug 61 metabolism in man. 1.1b Socially-accepted drugs known to affect drug 65 metabolism in man. 1.19 Nutritional factors known to affect drug 70 metabolism in man. Table No. Title Page No. 1.20 Miscellaneous factors known to influence 74 drug metabolism in man. 3.1 Quantitation of theophylline and its 93 metabolites in 0 - 24 hour urine of volunteers after the oral administration of 100mg (10pCi) theophylline. 3.2 Quantitation of theophylline and its 96 metabolites, by various authors. 4.1 Recovery of radioactivity in urine 102 follyTing the intravenous administration of ( C)-theophylline. 4.2 Quantitation of theophylline and its 105 metabolites in 0 - 24 hour urine follloTing the intravenous administration of ( C)-theophylline. 4.3 Pharmacokinetic parameterp describing the 116 urinary elimination of ( C)-theophylline. 4.4 Pharmacokinetic parameter? describing the 117 urinary elimination of ( fC)-theophylline determined by the "Sigma-minus" method. 4.5 Some novel products of methylxanthine 123 metabolism. 4.6 Evidence for capacity-limited drug 130 metabolism. 4.7 Consequences of first-order drug 135 elimination. 4.8 Implications of capacity-limited drug 135 elimination. 5.1 Quantitation of theophylline and its 142 metabolites in 0 - 24 hour urine follioring the intravenous administration of ("t)-theophylline to volunteers on various diets. 5.2 Pharmacokinetic parameteyp describing the 148 urinary elimination of ( fC)-theophylline to volunteers on various diets. 5.3 Percentage saturation of the enzyme system(s) 151 responsible for the conversion of theophylline to 3-methylxanthine in volunteers on their normal and methylxanthine-deprived diets. Table No. Title Page No. 5.4. Personal detail:: and dietary 153 methylxanthine intake of the subjects. 6.1 Urinary recovery of radioactivity 163 following the intravenous administRtion 14 of either (k C)-aminophylline or ( C)- theophylline to volunteers on their normal diets. 6.2 Quantitation of theophylline and its 165 metabolites in the total 24 hour urine aqer either (1 C)-aminophylline or ( C)-theophylline. 6.3 Pharmacokinetic parameters describinf,the • 170 urinary elimination of intravenous ( '4-0- aminophylline or (14C)-theophylline. 1 Urinary recovery of radioactivity fV.lowing 172 the intravenous administration of ( C)- aminophylline to volunteers on their normal and methylxanthine-deprived diets. 6.5 Quantitation of theophylline and its 175 metabolites in the total 24 hour une of volunteers receiving intravenous ( C)- aminophylline on their normal and methylxanthine-deprived diets. 6.6 Pharmacokinetic parametefs describing the 176 urinary elimination of ( 'C)-aminophylline in volunteers on their normal and methylxanthine-deprived diets. 6.7 Drugs for which formulation has been shown 178 to affect biological availability to an important extent. LIST OF MURES Figure No. Title Page No. 1.1 The structural formlaae of the xanthines. 21 1.2 The two tautomeric forms of uric acid. 22 1.3 The composition of aminophylline according 29 to the British Pharmacopoeia. 1.4 The major metabolic routes of the dietary 35 methylxanthines, caffeine, theophylline and theobromine. 1.5 Frequency distribution curves of a unimodally and 49 bimodally distributed characteristic. 2.1 Elution of radioactivity from a Dowex 2 X8-100 85 anion exchange column following the application of urine (containing approx. 350,000 dpm) from an individual dosed with (14-0-theophylline. 3.1 DE 81 Ion-exchange paper radiochromatogram 94 scan of the concentrated Dowex 2 X8-100 / 14 \ anion