pharmacokinetics diuretic іДШЕ studies with chlorthalidone and mefruside in man harry l.j.m. fleuren PHARMACOKINETICS OF DIURETIC DRUGS STUDIES WITH CHLORTHALIDONE AND MEFRUSIDE IN MAN 3 PROMOTORES: PROF. DR. J. M. VAN ROSSUM EN PROF. DR. С. A. M. VAN GINNEKEN 4 PHARMACOKINETICS OF DIURETIC DRUGS STUDIES WITH CHLORTHALIDONE AND MEFRUSIDE IN MAN PROEFSCHRIFT TER VERKRIJGING VAN DE GRAAD VAN DOCTOR IN DE WISKUNDE EN NATUURWETENSCHAPPEN AAN DE KATHOLIEKE UNIVERSITEIT TE NIJMEGEN OP GEZAG VAN DE RECTOR MAGNIFICUS PROF. DR. P.G.A.B. WIJDEVELD VOLGENS BESLUIT VAN HET COLLEGE VAN DECANEN IN HET OPENBAAR TE VERDEDIGEN OP DONDERDAG 6 SEPTEMBER 1979 DES NAMIDDAGS TE 2 UUR PRECIES DOOR HENDRIK LAMBERT JOZEF MARIA FLEUREN GEBOREN TE NIJMEGEN 1979 GRAFISCH BEDRIJF H W. JANSSEN B.V., GENNEP THE NETHERLANDS 5 aan mijn ouders voor Marion, Laurens en Emmeke CONTENTS SECTION I GENERAL INTRODUCTION CHAPTER 1 DIURETIC AGENTS AND PHARMACOKINETICS SULFONAMIDE DIURETICS 21 RENAL SITES OF ACTION OF SULFONAMIDE DIURETICS 23 RENAL TRANSPORT OF SULFONAMIDE DIURETICS 26 Relationship between the effect of diuretic drugs and their own urinary excretion 26 PHARMACOKINETICS: GENERAL REMARKS 31 HUMAN PHARMACOKINETICS OF SULFONAMIDE DIURETICS 36 Volumes o f distribution 36 Uptake in red blood cells 36 Plasma protein binding 38 Glomerular filtration, tubular secretion and reabsorption 38 Plasma clearance 39 Pharmacokinetics and diuretic effects 40 THE INVESTIGATIONS DESCRIBED IN THIS THESIS 42 REFERENCES 43 8 SECTION II ANALYTICAL METHODS CHAPTER 2 GENERAL INTRODUCTION TO ANALYSIS OF DIURETIC DRUGS IN BIOLOGICAL FLUIDS INTRODUCTION 51 SEPARATION AND DETECTION OF DIURETIC DRUGS: REVIEW AND DISCUSSION 53 Detection methods without chromatographic separation 53 Thin-layer chromatography 55 High-pressure liquid chromatography 55 Gas chromatography 56 Concluding remarks 58 EXTRACTIVE METHYLATION 60 THE NITROGEN DETECTOR IN GAS CHROMATOGRAPHY 63 SUMMARY 65 APPENDIX 2.1 66 REFERENCES 69 CHAPTER 3 DETERMINATION OF CHLORTHALIDONE IN PLASMA, URINE AND RED BLOOD CELLS INTRODUCTION 74 MATERIALS AND METHODS 75 Apparatus 75 Reagents 75 Determination of chlorthalidone in plasma 76 Determination of chlorthalidone in erythrocytes 77 Determination of chlorthalidone in urine 77 Preparation of calibration graphs 77 Recovery studies 77 Taking of blood samples 78 In vitro incubations 78 Influence of hemolysis on chlorthalidone plasma concentrations 78 Identification of the methyl derivative of chlorthalidone 79 RESULTS AND DISCUSSION 79 Gas chromatography 79 Determination of chlorthalidone in plasma 81 Determination of chlorthalidone in urine 83 Determination of chlorthalidone in erythrocytes 83 Stability of chlorthalidone in frozen plasma, urine and red blood cells 83 Taking of the blood samples and studies concerning the distribution of chlorthalidone between plasma and erythrocytes 84 Influence of duration of centnfugation on chlorthalidone plasma concentrations 86 Influence of heparin on distribution between plasma and red blood cells 87 Influence of hemolysis on chlorthalidone plasma concentrations 87 Equilibrium distribution between plasma and red blood cells in vivo 89 Identity of the chlorthalidone derivative formed in the extractive alkylation reaction 89 SUMMARY 90 REFERENCES 91 CHAPTER 4 DETERMINATION OF MEFRUSIDE IN PLASMA, RED BLOOD CELLS AND URINE INTRODUCTION 92 MATERIALS 93 Reagents 93 Gas chromatography 94 Other apparatus 94 METHODS 95 Sampling of blood and urine 95 Selection of denvatization time and temperature 95 Analytical procedure 96 DRUG PARTITIONING BETWEEN PLASMA AND RED BLOOD CELLS % Rate of distribution in whole blood at 20 and 370C 96 Influence of temperature upon the distribution equilibrium 97 RESULTS AND DISCUSSION 98 Gas chromatography and specificity 98 10 Derivatization yield 99 Extraction recovery 100 Calibration graphs and reproducibility 101 In vitro uptake rate of mefruside by red blood cells 102 Effect of temperature change upon the distribution ratio of plasma and red blood cell concentrations 104 APPLICATION 106 SUMMARY 108 REFERENCES 108 CHAPTER 5 SEPARATE ASSAY OF MEFRUSIDE METABOLITES IN THE LACTONE AND OPEN ACID CONFORMATION INTRODUCTION 110 MATERIALS AND METHODS 111 Drugs 111 Derivatization and gas chromatography 112 pH-dependent distribution between aqueous and organic phases 113 Procedure for the extraction of 5-oxo-mefruside and its hydroxy acid analogue from biological samples 114 Sampling of blood and urine, in vitro distribution between plasma and red cells 115 Comparison of presumed metabolite with synthetic 5-oxo-mefruside by gas chromatography - mass spectrometry 115 RESULTS AND DISCUSSION 116 Drug partitioning 116 Gas chromatographic determination of 5-oxo-mefruside and its open acid counterpart in plasma, red blood cells and urine 117 Identification of mefruside metabolites in biological samples 117 In vitro distribution of mefruside metabolites between plasma and erythrocytes 121 SUMMARY 122 REFERENCES 123 11 CHAPTER 6 DIFFERENCE POTENTIOMETRIC METHOD FOR DETERMINING DISSOCIATION CONSTANTS OF VERY SLIGHTLY WATER-SOLUBLE DRUGS, APPLIED TO THE SULFONAMIDE DIURETIC CHLORTHALIDONE INTRODUCTION 124 EXPERIMENTAL 125 RESULTS AND DISCUSSION 126 SUMMARY 131 REFERENCES 131 SECTION III PHARMACOKINETICS OF CHLORTHALIDONE CHAPTER 7 BASIC PHARMACOKINETIC CHARACTERISTICS OF CHLORTHALIDONE INTRODUCTION 135 MATERIALS AND METHODS 136 RESULTS 137 Time course of chlorthalidone concentration in plasma and red blood cells 137 Urinary excretion 138 Distribution of chlorthalidone between plasma and red blood cells 139 PHARMACOKINETIC ANALYSIS 141 Linear approach 141 Non-linear model 141 DISCUSSION 147 CONCLUSION 150 SUMMARY 151 APPENDIX 7.1 151 APPENDIX 7.2 153 REFERENCES 155 12 CHAPTER 8 UPTAKE OF CHLORTHALIDONE BY HUMAN ERYTHROCYTES IN VITRO INTRODUCTION 156 MATERIALS AND METHODS 158 Drug partitioning studies 158 Analysis of erythrocyte binding sites 159 RESULTS 160 Rate of distribution of chlorthalidone between plasma and erythrocytes in vitro 160 Red blood cell binding parameters 161 DISCUSSION 163 SUMMARY 165 REFERENCES 165 CHAPTER 9 ABSOLUTE BIOAVAILABILITY OF CHLORTHALIDONE IN MAN, DETERMINED BY COMPARISON OF PLASMA, RED BLOOD CELL AND URINE CONCENTRATIONS AFTER ORAL AND INTRAVENOUS ADMINISTRATION INTRODUCTION 167 MATERIALS AND METHODS 168 Subjects and drug administration 168 Sampling and analytical measurements 171 Pharmacokinetics 172 RESULTS 176 Plasma concentrations following intravenous administration 176 Plasma concentrations following oral administration 176 Red blood cell concentrations after intravenous and oral administration 180 Ratio of the areas under the plasma and red blood cell concentration curves after oral versus intravenous doses of chlorthalidone 186 Computer simulations according to a non-linear chlorthalidone model 186 Urinary excretion of chlorthalidone after intravenous and oral administration 189 13 Faecal excretion 192 DISCUSSION AND CONCLUSIONS 192 Curve-fitting according to 3 versus 2 compartment models 193 Bioavailability measurements 194 Central volume of distribution 195 Plasma versus red blood cell half-lives 196 Evidence for metabolic clearance 198 SUMMARY 198 REFERENCES 199 CHAPTER 10 URINARY EXCRETION OF CHLORTHALIDONE AS A FUNCTION OF DOSE INTRODUCTION 201 METHODS 201 Subjects and dosage 201 Biological samples and assay 202 Pharmacokinetic analysis 202 RESULTS 203 Urinary excretion rate vs time, comparison with plasma and red blood cell concentration decay 203 Cumulative urinary excretion 205 Renal clearance 205 Dependence of renal clearance upon urinary flow and pH 209 DISCUSSION 211 Dose-dependent urinary excretion 211 Fluctuations in renal plasma clearance 212 Therapeutic meaning 213 SUMMARY 213 REFERENCES 214 CHAPTER 11 BILIARY EXCRETION OF CHLORTHALIDONE IN HUMANS INTRODUCTION 216 EXPERIMENTAL PROCEDURE 216 Patients 216 Chlorthalidone assay 218 14 RESULTS 219 DISCUSSION AND CONCLUSIONS 219 SUMMARY 223 REFERENCES 223 CHAPTER 12 SALIVA CONCENTRATIONS OF CHLORTHALIDONE INTRODUCTION 225 METHODS 226 RESULTS AND DISCUSSION 226 SUMMARY 229 REFERENCES 230 CHAPTER 13 PHARMACOKINETICS OF CHLORTHALIDONE DURING CHRONIC ADMINISTRATION INTRODUCTION 231 EXPERIMENTAL 234 Drug administration 234 Sampling and analytical measurement 234 Pharmacokinetic analysis 235 RESULTS 236 Single dose kinetics 236 Multiple dose kinetics 236 Urinary excretion during steady-state 239 Therapeutic effect 240 DISCUSSION 240 Blood pressure response 240 Absorption during chronic administration 240 Capacity-limited red blood cell binding 241 Renal clearance 243 Therapeutic meaning 243 SUMMARY 244 APPENDIX 13 1 245 APPENDIX 13 2 246 REFERENCES 246 15 SECTION IV PHARMACOKINETICS OF MEFRUSIDE CHAPTER 14 ABSORPTION, DISTRIBUTION, METABOLISM AND EXCRETION OF MEFRUSIDE IN MAN INTRODUCTION 251 MATERIALS AND METHODS 252 Drug administration and sampling of blood and urine 252 Assay of mefruside and metabolites 253 Pharmacokinetic analysis 253 RESULTS 254 Pharmacokinetics of mefruside in plasma 254 Uptake of mefruside in red blood cells 257 Urinary excretion of mefruside 258 Concentration of mefruside metabolites in plasma and red blood cells 258 Urinary excretion of 5-oxo-mefruside and its hydroxy-carboxylic acid analogue 260 Urinary excretion of conjugated metabolite 263 DISCUSSION AND CONCLUSIONS 263 Pharmacokinetic parameters 263 Binding to red blood cells 264 Intersubject difference in elimination of mefruside 265 Relationship between pharmacokinetics and diuretic effect 265 SUMMARY 266 REFERENCES 267 CHAPTER 15 DIURETIC EFFECTS OF MEFRUSIDE