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The in vitro characterization of the drug-protein binding of racemic propafenone, and its active metabolite… Tonn, George Roger 1990

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THE IN VITRO CHARACTERIZATION OF THE DRUG-PROTEIN BINDING OF RACEMIC PROPAFENONE, AND ITS ACTIVE METABOLITE 5-HYDROXYPROPAFENONE IN HUMAN SERUM, AND IN SOLUTIONS OF ISOLATED HUMAN SERUM PROTEINS. by GEORGE ROGER TONN B.Sc. (Pharm), U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1988 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTERS OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES ( F a c u l t y o f P h a r m a c e u t i c a l S c i e n c e s ) ( D i v i s i o n o f P h a r m a c e u t i c s ) We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA O c t o b e r , 1990 © GEORGE ROGER TONN In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of PHARMACEUTICAL SCIENCES The University of British Columbia Vancouver, Canada Date O c t o b e r 10, 1990  DE-6 (2/88) i i ABSTRACT An a c c u r a t e plasma c o n c e n t r a t i o n - r e s p o n s e r e l a t i o n s h i p f o r p r o p a f e n o n e ( P F ) , a p o t e n t c l a s s 1 a n t i a r r h y t h m i c agent, has not y e t been d e f i n e d . A g e n e r a l p h a r m a c o l o g i c a l premise s u g g e s t s t h a t o n l y t he f r e e d r u g i s a v a i l a b l e t o c o n t r i b u t e t o the o b s e r v e d p h a r m a c o l o g i c a l r e s p o n s e . I t has p r e v i o u s l y been shown t h a t PF i s h i g h l y bound t o a - l -a c i d g l y c o p r o t e i n (AAG) which r e s u l t s i n a low f r e e PF c o n c e n t r a t i o n . The c o r r e l a t i o n o f f r e e PF c o n c e n t r a t i o n and r e s p o n s e f a i l e d t o a d e q u a t e l y d e s c r i b e t h e dose r e s p o n s e r e l a t i o n s h i p . I t has s u b s e q u e n t l y been shown t h a t upon c h r o n i c d o s i n g , two a c t i v e m e t a b o l i t e s , namely 5-hydroxypropafenone (5-OH-PF), and n - d e p r o p y l p r o p a f e n o n e ( n - d e p r o p y l - P F ) accumulate i n humans t r e a t e d w i t h PF. I t i s h i g h l y l i k e l y t h a t t h e f r e e c o n c e n t r a t i o n o f PF, i n a d d i t i o n t o t h o s e o f 5-OH-PF and n - d e p r o p y l - P F , c o n t r i b u t e s t o the o b s e r v e d p h a r m a c o l o g i c a l e f f e c t f o l l o w i n g a d m i n i s t r a t i o n o f PF a t s t e a d y - s t a t e . To d a t e , no a c c u r a t e e s t i m a t i o n o f 5-OH-PF b i n d i n g i n serum has been e s t a b l i s h e d . T h i s t h e s i s examines the b i n d i n g c h a r a c t e r i s t i c s o f PF and 5-OH-PF and t h e i r i n t e r a c t i o n i n human serum, and i n s o l u t i o n s o f AAG, human serum albumin (HSA), h i g h d e n s i t y l i p o p r o t e i n s (HDL), low d e n s i t y l i p o p r o t e i n s (LDL), and v e r y low d e n s i t y l i p o p r o t e i n s (VLDL) u s i n g e q u i l i b r i u m d i a l y s i s . The b i n d i n g o f PF (2.0 pg/mt) and 5-OH-PF (0.5 ag/ml) was examined i n serum when both drug and m e t a b o l i t e were p r e s e n t . The f r e e f r a c t i o n (FF) o f PF and 5-OH-PF i n serum was 0.063 ± 0.004 and 0.232 ± 0.020, r e s p e c t i v e l y . Both PF and 5-0H-PF were found t o b i n d t o a h i g h a f f i n i t y , low c a p a c i t y b i n d i n g s i t e on AAG, i n a d d i t i o n PF showed a second low a f f i n i t y , h i g h c a p a c i t y b i n d i n g s i t e . PF d i s p l a y e d a 10 f o l d g r e a t e r a f f i n i t y f o r the h i g h a f f i n i t y b i n d i n g s i t e on AAG when compared t o 5-OH-PF. Both PF and 5-OH-PF showed o n l y one low a f f i n i t y , h i g h c a p a c i t y s i t e on HSA o f s i m i l a r a f f i n i t y . The i n t e r a c t i o n o f PF and 5-OH-PF w i t h HDL, LDL, and VLDL appeared t o be due t o s o l u b i l i z a t i o n , r a t h e r than a " t r u e " d r u g - p r o t e i n b i n d i n g i n t e r a c t i o n , s i n c e i t c o r r e l a t e d w e l l w i t h t h e c o n c e n t r a t i o n o f c h o l e s t e r o l w i t h i n t h e l i p o p r o t e i n complex (PF, r^=0.85; 5-OH-PF, r^=0.96). However, PF appeared t o show s a t u r a b l e b i n d i n g t o t h e HDL complex. The uptake o f PF and 5-OH-PF was g r e a t e s t i n LDL f o l l o w e d by HDL, and f i n a l l y VLDL. In serum PF d i s p l a y e d both a h i g h a f f i n i t y , low c a p a c i t y , and a low a f f i n i t y , h i g h c a p a c i t y b i n d i n g s i t e s , a l t h o u g h a s i m i l a r o b s e r v a t i o n was e x p e c t e d f o r 5-OH-PF, o n l y one b i n d i n g s i t e c o u l d be e x p e r i m e n t a l l y i d e n t i f i e d . The uptake o f 5-OH-PF by r e d b l o o d c e l l s (RBC) appeared t o be a p p r o x i m a t e l y 5 f o l d g r e a t e r than t h a t o f PF ( i . e . The r a t i o o f PF and 5-OH-PF c o n c e n t r a t i o n i n the r e d b l o o d c e l l / p l a s m a was 0.7 ± 0.1 and 3.2 ± 0.5, r e s p e c t i v e l y ) . When the b i n d i n g o f PF and 5-OH-PF was c o n s i d e r e d s e p a r a t e l y , the b i n d i n g p r o f i l e s were s i m i l a r , t h a t i s , both d rugs showed h i g h a f f i n i t y b i n d i n g t o AAG, and low a f f i n i t y b i n d i n g and/or n o n - s p e c i f i c b i n d i n g t o o t h e r serum p r o t e i n s such as HSA, HDL, LDL, and VLDL. However, when both d r u g and m e t a b o l i t e were p r e s e n t , t h e b i n d i n g o f 5-OH-PF t o AAG was found t o be r e d u c e d . T h i s i s thou g h t t o o c c u r as a r e s u l t o f the d i s p l a c e m e n t o f 5-OH-PF by PF from AAG. Thus, t he b i n d i n g o f 5-OH-PF i v was n o t e d t o be more dependent on HSA, and l i p o p r o t e i n s when compared t o PF. On the o t h e r hand, t h e b i n d i n g o f PF (2.0 ng/ml), even w i t h t h e a d d i t i o n o f 5-OH-PF, was dependent l a r g e l y on t h e c o n c e n t r a t i o n o f AAG. A l t h o u g h t h e b i n d i n g o f 5-OH-PF was a p p a r e n t l y not a l t e r e d by the a d d i t i o n o f PF i n serum, a d e c r e a s e i n the b i n d i n g o f 5-OH-PF by t h e a d d i t i o n o f PF was o b s e r v e d . I t i s hoped t h a t t h e u n d e r s t a n d i n g g a i n e d from t h i s t h e s i s w i l l p r o v i d e i n f o r m a t i o n r e g a r d i n g t h e r e l a t i v e i m p o r t a n c e o f f r e e PF and 5-OH-PF plasma c o n c e n t r a t i o n i n f u t u r e pharmacodynamic s t u d i e s o f PF. TABLE OF CONTENTS PAGE ABSTRACT i i LIST TABLES x i i i LIST OF FIGURES x i v LIST OF APPENDICES x v i i LIST OF ABBREVIATIONS x v i i i ACKNOWLEDGEMENTS xx 1. INTRODUCTION 1 1.1 PROPAFENONE 1 1.1.1 PHARMACOLOGY OF PROPAFENONE 1 1.1.2 THERAPEUTIC EFFICACY OF PROPAFENONE 2 1.1.3 PHARMACOKINETICS OF PROPAFENONE 3 1.1.3.1 ABSORPTION OF PROPAFENONE 3 1.1.3.2 DISTRIBUTION AND PLASMA PROTEIN BINDING OF PROPAFENONE 3 1.1.3.3 METABOLISM OF PROPAFENONE 4 1.2 DRUG-PROTEIN BINDING 7 1.2.1 ULTRAFILTRATION 9 1.2.2 EQUILIBRIUM DIALYSIS 10 1.3 SERUM PROTEINS 12 1.3.1.HUMAN SERUM ALBUMIN 13 1.3.2 a -1-ACID GLYCOPROTEIN 14 1.3.3 SERUM LIPOPROTEINS 15 1.3.4 UPTAKE OF DRUGS INTO RED BLOOD CELLS 16 1.3.5 CHEMICAL BASIS FOR DRUG-PROTEIN INTERACTION 17 1.4 QUANTITATIVE DESCRIPTION OF THE DRUG-PROTEIN BINDING SITE 18 1.5 RATIONALE 22 1.6 AIMS 25 v i 2. EXPERIMENTAL 27 2.1 MATERIALS AND SUPPLIES 27 2.1.1 DRUGS, METABOLITES, AND INTERNAL STANDARDS 27 2.1.2 CHEMICALS AND REAGENTS 27 2.1.3 PROTEINS 28 2.1.4 SOLVENTS 28 2.1.5 GASES 29 2.1.6 EQUILIBRIUM DIALYSIS CELLS 29 2.1.7 ULTRAFILTRATION DEVICES 29 2.1.8 ELECTROPHORESIS 30 2.1.8.1 POLYACRYLAMIDE ELECTROPHORESIS GELS 30 2.1.8.2 AGAROSE ELECTROPHORESIS GELS 30 2.1.9 RADIAL IMMUNO DIFFUSION PLATES 30 2.1.10 OTHER SUPPLIES 30 2.2 EQUIPMENT 31 2.2.1 GAS LIQUID CHROMATOGRAPH 31 2.2.2 SPECTROPHOTOMETER 32 2.2.3 ELECTROPHORESIS EQUIPMENT 32 2.2.4 OTHER 32 2.3 PREPARATION OF STOCK AND REAGENT SOLUTIONS 33 2.3.1 DRUGS, METABOLITES, AND INTERNAL STANDARDS 33 2.3.2 REAGENTS AND SOLUTIONS 34 2.3.3 PROTEIN SOLUTIONS 35 2.3.4 ESTABLISHED TECHNIQUES USED IN RESEARCH 36 2.3.4.1 GAS LIQUID CHROMATOGRAPHY OF PROPAFENONE AND 5-HYDROXYPROPAFENONE 36 2.3.4.2 LOWRY PROTEIN ASSAY 37 2.3.4.3 POLYACRYLAMIDE GEL ELECTROPHORESIS 37 2.3.4.4 LIPOPROTEIN AGAROSE ELECTROPHORESIS 38 2.4 PROTEIN BINDING OF 5-HYDROXYPROPAFENONE AND PROPAFENONE 39 2.4.1 PRELIMINARY EXPERIMENTS 39 2.4.1.0 PREPARATION OF EQUILIBRIUM DIALYSIS DEVICES 39 2.4.1.1 TIME TO EQUILIBRIUM 40 2.4.2.2 NON-SPECIFIC BINDING OF 5-HYDROXYPROPAFENONE TO EQUILIBRIUM DIALYSIS CELLS AND MEMBRANES. 2.4.2.3 NON-SPECIFIC BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE TO AMICON MPS-1 ULTRAFILTRATION DEVICES. 2.4.3 pH DEPENDENT BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE 2.4.3.1 pH DEPENDENT BINDING OF PROPAFENONE IN SERUM 2.4.3.2 pH DEPENDENT BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE IN ISOLATED PROTEIN SOLUTIONS (HSA AND AAG) 2.4.3.3 pH DEPENDENT PARTITIONING IN TOLUENE AND HEXANE OF PROPAFENONE AND 5-HYDROXYPROPAFENONE. 2.4.4 BUFFER STRENGTH 2.4.5 CHEMICAL DEGRADATION OF 5-HYDROXYPROPAFENONE DURING DIALYSIS 2.4.6 VOLUME SHIFTS 2.4.7 OSMOLARITY OF 5-HYDROXYPROPAFENONE SOLUTIONS 2.4.8 LOSS OF PROTEIN DURING DIALYSIS 2.4.9 TEST FOR DIALYSIS MEMBRANE INTEGRITY FOLLOWING DIALYSIS 2.5 BINDING OF 5-HYDROXYPROPAFENONE IN HUMAN SERUM 2.5.1 BINDING IN NORMAL SERUM 2.5.2 ROSENTHAL PLOTS OF 5-HYDROXYPROPAFENONE BINDING IN SERUM 2.5.3 PROTEIN BINDING OF 5-HYDROXYPROPAFENONE IN SERUM DETERMINED BY EQUILIBRIUM DIALYSIS IN THE PRESENCE OF PROPAFENONE 2.5.4 BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE USING THE METHOD OF ULTRAFILTRATION 2.6 BINDING OF 5-HYDROXYPROPAFENONE AND PROPAFENONE TO ISOLATED PROTEINS 2.6.1 BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE TO HSA AND AAG 2.6.2 BINDING TO PROPAFENONE AND 5-HYDROXYPROPAFENONE TO ALBUMIN AND FREE FATTY ACID FREE ALBUMIN. 2.6.3 BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE TO LIPOPROTEIN DEFICIENT SERUM 2.6.4 ELECTROPHORESIS OF ISOLATED PROTEIN SOLUTIONS 2.6.5 DRUG-DRUG DISPLACEMENT OF PROPAFENONE AND 5-HYDROXYPROPAFENONE FROM SERUM PROTEINS BY DISOPYRAMIDE, AND IBUPROFEN 2.6.6 DRUG-DRUG DISPLACEMENT OF PROPAFENONE AND 5-HYDROXYPROPAFENONE FROM HSA AND AAG BY DISOPYRAMIDE AND IBUPROFEN 2.7 CHARACTERIZATION OF BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE IN PROTEIN SOLUTIONS 50 50 51 2.7.1 CHARACTERIZATION OF BINDING TO POOLED HUMAN SERUM, AAG, HUMAN ALBUMIN, HDL, LDL, AND VLDL SOLUTIONS 2.7.2 ELECTROPHORESIS OF LIPOPROTEINS 51 52 2.8. DATA ANALYSIS 2.8.1 STATISTICAL ANALYSIS OF DATA. 2.8.2 BINDING PARAMETER MODELS AND DATA FITTING 2.8.3 MATHEMATICAL BINDING R E C O N S T I T U T E STUDIES 2.9 RED BLOOD CELL DISTRIBUTION 2.9.1 PREPARATION OF RED BLOOD CELLS 2.8.2 TIME TO DISTRIBUTION EQUILIBRIUM FOR PROPAFENONE AND 5-HYDROXYPROPAFENONE UPTAKE INTO HUMAN RED BLOOD CELLS 2.8.3 UPTAKE OF PROPAFENONE AND 5-HYDROXYPROPAFENONE BY HUMAN RED BLOOD CELLS. 3. RESULTS 52 52 53 54 55 56 56 57 58 3.1 PRELIMINARY IN VITRO PROTEIN BINDING EXPERIMENTS FOR PROPAFENONE AND 5-HYDROXYPROPAFENONE 1 TIME TO EQUILIBRIUM 3.1.3 -SPECIFIC BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE TO EQUILIBRIUM DIALYSIS CELLS AND MPS-1 ULTRA-FILTRATION DEVICES pH DEPENDENT BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE. 58 58 58 61 3.1.3.1 pH DEPENDENT BINDING OF PROPAFENONE IN HUMAN SERUM, AND SOLUTIONS-OF HUMAN SERUM ALBUMIN, AND HUMAN ALPHA-1-ACID GLYCOPROTEIN. 61 ix 3.1.3.2 pH DEPENDENT BINDING 5-HYDROXYPROPAFENONE IN HUMAN SERUM, A SOLUTION OF HUMAN SERUM ALBUMIN, AND OF HUMAN ALPHA-1-ACID GLYCOPROTEIN. 61 3.1.3.3 THE INFLUENCE OF pH ON THE PARTITIONING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE IN A TOLUENE PHOSPHATE BUFFER AND HEXANE PHOSPHATE BUFFER SYSTEM. 64 3.1.4 THE EFFECT OF BUFFER STRENGTH ON BINDING OF 5-HYDROXYPROPAFENONE. 64 3.1.5 DEGRADATION OF 5-HYDROXYPROPAFENONE DURING EQUILIBRIUM DIALYSIS. 68 3.1.6 VOLUME SHIFTS 68 3.1.7 OSMOLARITY OF 5-HYDROXYPROPAFENONE STOCK SOLUTIONS USED IN EQUILIBRIUM DIALYSIS STUDIES. 69 3.1.8 PROTEIN LOSS DURING EQUILIBRIUM DIALYSIS 69 3.1.9 TEST FOR DIALYSIS MEMBRANE INTEGRITY FOLLOWING DIALYSIS 69 3.2 PROTEIN BINDING OF 5-HYDROXYPROPAFENONE IN SERUM OF HEALTHY MALE VOLUNTEERS 70 3.2.1 PROTEIN BINDING OF THE METABOLITE, 5-HYDROXYPROPAFENONE, IN THE PRESENCE OF THE PARENT COMPOUND PROPAFENONE AT A THERAPEUTIC CONCENTRATION AS DETERMINED BY EQUILIBRIUM DIALYSIS AND ULTRAFILTRATION. 75 3.3 BINDING PROFILES OF PROPAFENONE AND 5-HYDROXYPROPAFENONE TO ISOLATED HUMAN SERUM PROTEINS, LIPOPROTEIN DEFICIENT SERUM, AND NORMAL SERUM. 76 3.3.1 BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE TO ISOLATED HUMAN SERUM PROTEINS. 76 3.3.2 BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE TO PURE ISOLATED HUMAN SERUM PROTEINS DISSOLVED IN EITHER BUFFER OR SERUM ULTRAFILTRATE. 78 3.3.3 THE BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE TO FREE FATTY ACID FREE ALBUMIN COMPARED TO NORMAL ALBUMIN. 81 3.3.4 BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE TO LIPOPROTEIN DEFICIENT SERUM v s . NORMAL SERUM 81 3.3.5 DISPLACEMENT OF PROPAFENONE AND 5-HYDROXYPROPAFENONE FROM PROTEIN BINDING-SITES BY IBUPROFEN AND DISOPYRAMIDE. 84 X 3.3.6 DISPLACEMENT OF PROPAFENONE AND 5-HYDROXYPROPAFENONE FROM ISOLATED HUMAN SERUM PROTEINS BY DISOPYRAMIDE AND IBUPROFEN 85 3.3.7 PURITY ASSURANCE OF ISOLATED PROTEIN IN BINDING EXPERIMENTS 88 3.4 BINDING CHARACTERISTICS OF PROPAFENONE AND 5-HYDROXYPROPAFENONE. 88 3.4.1 BINDING CHARACTERISTICS OF PROPAFENONE AND 5-HYDROXYPROPAFENONE IN ALPHA-1-ACID GLYCOPROTEIN SOLUTION. 88 3.4.2 BINDING CHARACTERISTICS OF PROPAFENONE AND 5-HYDROXYPROPAFENONE IN HUMAN SERUM ALBUMIN SOLUTION 90 3.4.3 BINDING CHARACTERISTICS OF PROPAFENONE AND 5-HYDROXYPROPAFENONE IN POOLED HUMAN SERUM 94 3.4.4 BINDING CHARACTERISTICS OF PF AND 5-HYDROXYPROPAFENONE IN A SOLUTION OF HIGH DENSITY LIPOPROTEINS. 94 3.4.5 BINDING CHARACTERISTICS OF PROPAFENONE AND 5-HYDROXYPROPAFENONE IN A SOLUTION OF LOW DENSITY LIPOPROTEINS. 97 3.4.6 BINDING CHARACTERISTICS OF PROPAFENONE AND 5-HYDROXYPROPAFENONE IN A SOLUTION OF VERY LOW DENSITY LIPOPROTEINS. 97 3.4.7 MATHEMATICAL BINDING EQUATIONS FOR PROPAFENONE AND 5-HYDROXYPROPAFENONE. 99 3.4.8 PROTEIN QUALITY ASSURANCE DURING BINDING CHARACTERIZATION EXPERIMENTS 104 3.5 UPTAKE OF PROPAFENONE AND 5-HYDROXYPROPAFENONE INTO RED BLOOD CELLS 104 3.5.1 TIME TO EQUILIBRIUM FOR RED BLOOD CELL UPTAKE OF PROPAFENONE AND 5-HYDROXYPROPAFENONE. 104 3.5.2 UPTAKE OF PROPAFENONE AND 5-HYDROXYPROPAFENONE BY RED BLOOD CELLS 104 4. DISCUSSION 107 4.1.1 PROTEIN BINDING AND PROPAFENONE 107 4.1.2 EQUILIBRIUM DIALYSIS TIME TO EQUILIBRIUM 108 4.1.3 NON-SPECIFIC BINDING OF PF AND 5-HYDROXYPROPAFENONE 110 4.1.4 pH DEPENDENT BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE 111 4.1.5 THE EFFECT OF BUFFER STRENGTH ON THE DRUG PROTEIN BINDING OF 5-HYDROXYPROPAFENONE. 113 xi 4.1.6 DEGRADATION OF 5-HYDROXYPROPAFENONE DURING EQUILIBRIUM DIALYSIS 114 4.1.7 VOLUME SHIFTS DURING EQUILIBRIUM DIALYSIS OF PROPAFENONE AND 5-HYDROXYPROPAFENONE. 114 4.1.8 OSMOLARITY MEASUREMENTS OF 5-HYDROXYPROPAFENONE DRUG SOLUTIONS. 115 4.1.9 PROTEIN LOSS DURING EQUILIBRIUM DIALYSIS OF PROPAFENONE AND 5-HYDROXYPROPAFENONE 115 4.1.10 MEMBRANE INTEGRITY DURING EQUILIBRIUM DIALYSIS 116 4.2 PROTEIN BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE 117 4.2.1 PROTEIN BINDING OF 5-HYDROXYPROPAFENONE IN SERUM OF HEALTHY MALES 117 4.2.2 PROTEIN BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE IN SERUM ESTIMATED BY EQUILIBRIUM DIALYSIS AND ULTRAFILTRATION. 124 4.3 BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE TO ISOLATED SERUM PROTEINS 126 4.3.1 BINDING DIFFERENCES BETWEEN PROPAFENONE AND 5-HYDROXYPROPAFENONE IN VARIOUS PROTEIN SOLUTIONS. 126 4.3.2 THE EFFECT OF BUFFER AND SERUM ULTRAFILTRATE ON THE BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE. 129 4.3.3 THE EFFECT OF THE REMOVAL OF FREE FATTY ACIDS FROM ALBUMIN ON THE BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE. 130 4.3.4 THE EFFECT OF THE REMOVAL OF LIPOPROTEINS FROM SERUM ON THE BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE. 131 4.3.5 DISPLACEMENT OF PROPAFENONE AND 5-HYDROXYPROPAFENONE IN SERUM BY DISPLACING AGENTS IBUPROFEN AND DISOPYRAMIDE. 132 4.3.6 DISPLACEMENT OF PROPAFENONE AND 5-HYDROXYPROPAFENONE FROM INDIVIDUAL HUMAN SERUM PROTEINS. 133 4.4. BINDING CHARACTERISTICS PROPAFENONE AND 5-HYDROXYPROPAFENONE TO ISOLATED PROTEINS 135 4.4.1 BINDING CHARACTERISTICS OF PROPAFENONE AND 5-HYDROXYPROPAFENONE IN A SOLUTION OF AAG. 135 4.4.2 THE BINDING CAPACITIES PROPAFENONE AND 5-HYDROXYPROPAFENONE IN A SOLUTION OF ALBUMIN. 138 4.4.3 CHARACTERIZATION OF PROPAFENONE AND 5-HYDROXYPROPAFENONE BINDING IN SERUM. 138 4.4.4 BINDING CHARACTERISTICS OF PROPAFENONE AND 5-HYDROXYPROPAFENONE TO LIPOPROTEINS. 140 4 . 4 . 5 CALCULATED BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE BY MATHEMATICAL R E C O N S T I T U T E OF THE DRUG-BINDING SYSTEM. 4.5 UPTAKE OF PROPAFENONE AND 5-HYDOXYPROPAFENONE BY HUMAN RED BLOOD CELLS. 4.6 SUMMARY CONCLUSION REFERENCES APPENDICES xi i i LIST OF TABLES PAGE 1. COMPARISON OF NON-SPECIFIC BINDING OF 5-HYDROXYPROPAFENONE AND PROPAFENONE TO THE ACRYLIC EQUILIBRIUM DIALYSIS APPARATUS FOLLOWING INCUBATION FOR 8 HOURS AT 37°C AND TO THE MPS-1 ULTRAFILTRATION DEVICES AT 25°C. 60 2. CHANGE IN TOTAL PROTEIN AND ALPHA-1-ACID GLYCOPROTEIN IN PLEXIGLASS DIALYSIS CELLS AND CELLULOSE DIALYSIS MEMBRANES DURING DIALYSIS. 70 3. BINDING PARAMETERS OF 5-HYDROXYPROPAFENONE IN SERUM OF HEALTHY HUMANS 71 4. 5-HYDROXYPROPAFENONE FREE FRACTION IN NORMAL SERA WITH INCREASING CONCENTRATIONS OF 5-HYDROXYPROPAFENONE 75 5 ASSESSMENT OF THE BINDING OF THE METABOLITE 5-HYDROXYPROPAFENONE IN VITRO BY EQUILIBRIUM DIALYSIS AND ULTRAFILTRATION WITH AND WITHOUT THE ADDITION OF THERAPEUTIC CONCENTRATIONS OF THE PARENT COMPOUND PROPAFENONE. 76 6. DISPLACEMENT OF PROPAFENONE AND 5-HYDROXYPROPAFENONE FROM HUMAN SERUM PROTEIN BINDING SITES BY DISOPYRAMIDE AND IBUPROFEN 84 7. THE COMPOSITION OF DIFFERENT CLASSES OF LIPOPROTEINS [VLDL, LDL, AND HDL] 141 LIST OF FIGURES S t r u c t u r e o f propaf e n o n e and the a c t i v e m e t a b o l i t e s 5 - h y d r o x y p r o p a f e n o n e and n - d e p r o p y l - p r o p a f e n o n e Time r e q u i r e d t o a t t a i n e q u i l i b r i u m f o r the e q u i l i b r i u m d i a l y s i s o f 5-hydroxypropafenone. The pH dependent b i n d i n g o f propafenone (2.0 /jg/mL) i n serum, and s o l u t i o n s o f HSA and AAG. The pH dependent b i n d i n g o f 5-hydroxypropafenone (0.5 /Kj/mL) i n serum, and s o l u t i o n s o f albumin and AAG. I n f l u e n c e o f a l t e r a t i o n s i n pH on the p a r t i t i o n i n g o f pr o p a f e n o n e (2.0 jLtg/mL) between aqueous and o r g a n i c l a y e r s i n a t o l u e n e phosphate b u f f e r s ystem. I n f l u e n c e o f a l t e r a t i o n s i n pH on the p a r t i t i o n i n g o f 5-hydroxypropafenone (0.5 fig/ml) between aqueous and o r g a n i c l a y e r s i n v a r i o u s o r g a n i c s o l v e n t s . The e f f e c t o f molar s t r e n g t h o f phosphate b u f f e r on the f r e e f r a c t i o n o f 5-hydroxypropafenone i n serum ± SO. The r e l a t i o n s h i p between t he r a t i o o f bound d r u g c o n c e n t r a t i o n / f r e e drug c o n c e n t r a t i o n ± SD v s . bound d r u g c o n c e n t r a t i o n o f 5-hydroxypropafenone i n serum. c o n t . c o n t . Comparison o f b i n d i n g o f propafenone (2.0 /jg/mL) and 5-hydroxypropafenone (0.5 /zg/mL) i n AAG, HSA, HSA + AAG, serum and l i p o p r o t e i n d e f i c i e n t serum. The e f f e c t o f b u f f e r v s . human serum u l t r a f i 1 t r a t e as s o l v e n t s t o d i s s o l v e AAG, HSA, and AAG + HSA on t h e f r e e f r a c t i o n (± SD) o f propaf e n o n e (2.0 /zg/mL). The e f f e c t o f b u f f e r v s . human serum u l t r a f i 1 t r a t e as s o l v e n t s t o d i s s o l v e AAG, HSA, and AAG + HSA on t h e f r e e f r a c t i o n (± SD) o f 5-hydroxy-propafenone (0.5 ng/ml). XV 12. Comparison between t h e f r e e f r a c t i o n (± SD) o f pro p a f e n o n e (2.0 ug/ml) + 5-hydroxypropafenone (0.5 /xg/mL) i n f r e e f a t t y a c i d f r e e albumin, and i n HSA. 82 13. Comparison o f the f r e e f r a c t i o n ( + SD) o f propafenone (2.0 ug/ml) and 5-hydroxypropafenone (0.5 ug/ml) i n l i p o p r o t e i n d e f i c i e n t serum, and normal serum. 83 14. The e f f e c t o f t h e a d d i t i o n o f i b u p r o f e n (50.0 ug/ml) on t h e f r e e f r a c t i o n (± SD) o f propafenone (2.0 jug/mL) and 5-hydroxypropafenone (0.5 ug/ml) 86 15. The e f f e c t o f the a d d i t i o n o f d i s o p y r a m i d e (8.0 ug/ml) on t h e f r e e f r a c t i o n ± SD o f propafenone (2.0 ug/ml) and 5-hydroxypropafenone (0.5 ug/ml). 87 16. R e l a t i o n s h i p between t h e r a t i o o f bound drug c o n c e n t r a t i o n / f r e e drug c o n c e n t r a t i o n v s . bound drug c o n c e n t r a t i o n o f propafenone and 5-hydroxypropafenone i n a p h y s i o l o g i c a l c o n c e n t r a t i o n o f AAG (19.3 uM) i n 0.1 phosphate pH 7.40 b u f f e r . 89 17. R e l a t i o n s h i p between the r a t i o o f bound drug c o n c e n t r a t i o n / f r e e drug c o n c e n t r a t i o n v s . bound drug c o n c e n t r a t i o n o f propafenone and 5-hydroxypropafenone i n a p h y s i o l o g i c a l c o n c e n t r a t i o n o f HSA (630.0 jM) i n 0.1 phosphate pH 7.36 b u f f e r . 91 18. R e l a t i o n s h i p between t h e r a t i o o f bound drug c o n c e n t r a t i o n / f r e e drug c o n c e n t r a t i o n v s . bound d r u g c o n c e n t r a t i o n o f propafenone and 5-hydroxypropafenone i n serum. 95 19. R e l a t i o n s h i p between the r a t i o o f bound drug c o n c e n t r a t i o n / f r e e drug c o n c e n t r a t i o n v s . bound drug c o n c e n t r a t i o n o f propafenone and 5-hydroxypropafenone i n a p h y s i o l o g i c a l c o n c e n t r a t i o n o f HDL (15.3 uM complex, 1.72 mg/mL t o t a l p r o t e i n ) i n 0.1 M phosphate b u f f e r pH 7.32. 96 20. R e l a t i o n s h i p between the r a t i o o f bound drug c o n c e n t r a t i o n / f r e e drug c o n c e n t r a t i o n v s . bound drug c o n c e n t r a t i o n o f PF and 5-hydroxypropafenone i n a p h y s i o l o g i c a l c o n c e n t r a t i o n o f LDL (1.8 /zM complex, 0.84 mg/mL t o t a l p r o t e i n ) i n 0.1 phosphate b u f f e r pH 7.32. 98 21. R e l a t i o n s h i p between the r a t i o o f bound drug c o n c e n t r a t i o n / f r e e drug c o n c e n t r a t i o n v s . bound d r u g c o n c e n t r a t i o n o f propafenone and 5-hydroxypropafenone i n a p h y s i o l o g i c a l c o n c e n t r a t i o n o f VLDL (0.17 / J M complex, 0.09 mg/mL t o t a l p r o t e i n ) i n 0.1 M phosphate b u f f e r pH 7.32 100 XVI 22. F r e e f r a c t i o n ± SD o f propa f e n o n e a t v a r i o u s c o n c e n t r a t i o n s i n s o l u t i o n s o f human serum p r o t e i n s . 92 23. Fr e e f r a c t i o n ± SD o f 5-hydroxypropafenone a t v a r i o u s c o n c e n t r a t i o n s i n s o l u t i o n s o f human serum p r o t e i n s . 93 24. A d i r e c t p l o t o f propafenone b i n d i n g (bound v s . f r e e ) c a l c u l a t e d by m a t h e m a t i c a l l y r e c o n s t r u c t i n g b i n d i n g c o n t r i b u t i o n s o f serum p r o t e i n c o n s t i t u e n t s and subsequent removal o f b i n d i n g c o n t r i b u t i o n s o f i n d i v i d u a l serum p r o t e i n s . 102 25. A d i r e c t p l o t o f 5-hydroxypropafenone b i n d i n g (bound v s . f r e e ) c a l c u l a t e d by m a t h e m a t i c a l l y r e c o n s t r u c t i n g b i n d i n g c o n t r i b u t i o n s o f serum p r o t e i n c o n s t i t u e n t s and subsequent removal o f b i n d i n g c o n t r i b u t i o n s o f i n d i v i d u a l serum p r o t e i n s . 103 26. Comparison o f the r a t i o (drug c o n c e n t r a t i o n i n RBC/drug c o n c e n t r a t i o n i n s u p e r n a t a n t ) o f p r o p a f e n o n e and 5-hydroxypropafenone w i t h b u f f e r , serum, and plasma as s u p e r n a t a n t s . 106 27. T h e o r e t i c a l Rosenthal p l o t o f d r u g - p r o t e i n b i n d i n g f i t t i n g a b i n d i n g model f o r a system c o n t a i n i n g one drug b i n d i n g s p e c i f i c s i t e + n o n - s p e c i f i c b i n d i n g s i t e . The b i n d i n g c a p a c i t y o f the h i g h a f f i n i t y s i t e i s 1.0 and the n o n - s p e c i f i c b i n d i n g (Kns) i s h e l d c o n s t a n t a t 2; o n l y t h e a f f i n i t y o f the h i g h a f f i n i t y s i t e was a l t e r e d . 122 28. T h e o r e t i c a l p l o t o f f r e e f r a c t i o n o f d r u g - p r o t e i n b i n d i n g f i t t i n g a model c o n t a i n i n g one s p e c i f i c s i t e + n o n - s p e c i f i c b i n d i n g . B i n d i n g c o n s t a n t s ( i e . the c a p a c i t y o f the h i g h a f f i n i t y s i t e i s 1.0, and n o n - s p e c i f i c b i n d i n g i s 2) are h e l d c o n s t a n t . 123 TABLE OF APPENDICES PROTEIN COMPOSITION OF NORMAL VOLUNTEERS FOR BINDING STUDY PROTEIN CONTENT (TOTAL PROTEIN AND a-l-ACID GLYCOPROTEIN) IN SOLUTIONS ISOLATED PROTEINS USED IN BINDING EXPERIMENTS. COMPOSITION OF HUMAN SERUM ULTRAFILTRATE ELECTROPHORESIS RESULTS FOR ISOLATED PROTEINS USED IN BINDING EXPERIMENTS. LANE 1 STANDARD PROTEIN SOLUTIONS, LANE 2 HUMAN SERUM ULTRAFILTRATE, LANE 3 AAG (UNDILUTED), LANE 4 HSA, LANE 5 HSA + AAG, LANE 6 NORMAL CONTROL SERUM, LANE 7 LIPOPROTEIN DEFICIENT SERUM PROTEIN CONCENTRATIONS IN SOLUTIONS OF ISOLATED HUMAN SERUM PROTEINS (AAG, HSA, HDL, LDL, AND VLDL) USED IN EXPERIMENTS IN WHICH BINDING WAS CHARACTERIZED AGAROSE ELECTROPHORESIS OF LIPOPROTEINS BEFORE AND AFTER EQUILIBRIUM DIALYSIS FOR 8 HOURS AT 37 °C. LIST OF ABBREVIATIONS A.V. a t r i o v e n t r i c u l a r AAG a - l - a c i d g l y c o p r o t e i n ACS American Chemical S o c i e t y ANOVA one way a n a l y s i s o f v a r i a n c e BSA b o v i n e serum albumin D f r e e drug (unbound) DP d r u g - p r o t e i n complex ECD e l e c t r o n c a p t u r e d e t e c t i o n EDTA e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d EM e x t e n s i v e m e t a b o l i z e r s FF f r e e f r a c t i o n GLC g a s - l i q u i d chromatography HDL h i g h d e n s i t y l i p o p r o t e i n HP H e w l e t t Packard HSA human serum albumin I.S. i n t e r n a l s t a n d a r d IDL i n t e r m e d i a t e d e n s i t y l i p o p r o t e i n Ka b i n d i n g a s s o c i a t i o n c o n s t a n t Kns b i n d i n g c o n s t a n t f o r n o n - s p e c i f i c b i n d i n g t o p r o t e i n LDL low d e n s i t y l i p o p r o t e i n M.w. m o l e c u l a r weight n number o f homogeneous b i n d i n g s i t e s NP c a p a c i t y o f b i n d i n g p r o t e i n P f r e e p r o t e i n (unbound) PF p ropafenone X I X PM poor m e t a b o l i z e r s PTFE p o l y t e t r a f l u o e t h y l e n e r molar r a t i o o f drug bound over t o t a l p r o t e i n RBC r e d b l o o d c e l l RID r a d i a l i m m u n o d i f f u s i o n rpm r e v o l u t i o n s per minute SD s t a n d a r d d e v i a t i o n SDS-PAGE sodium d o d e c y l s u l f a t e p o l y a c r y l a m i d e e l e c t r o p h o r e s i s TCA t r i c h l o r o a c e t i c a c i d Ti e l i m i n a t i o n h a l f - l i f e volume o f d i s t r i b u t i o n VLDL v e r y low d e n s i t y l i p o p r o t e i n XX ACKNOWLEDGEMENTS I would l i k e t o s i n c e r e l y thank Dr. James Axel son f o r h i s s u p e r v i s i o n , g u i d a n c e , and f i n a n c i a l s u p p o r t . I am a l s o t h a n k f u l f o r t h e o p p o r t u n i t y t o attempt a MSc degree i n Dr. Axel sons l a b o r a t o r y . I would a l s o l i k e t o thank my committee members D r s . J i m O r r , F r a n k A b b o t t , Bob Rangno, J a c k Diamond f o r t h e i r a d v i c e and g u i d a n c e t h r o u g h o u t my s t u d y . My a p p r e c i a t i o n t o Sue Panesar, Andras S e i t z , J u d i t Orbay, J i n g Wang and Bruce A l l e n f o r t h e i r t e c h n i c a l h e l p and e m o t i o n a l s u p p o r t . A s p e c i a l thank-you t o Matt W r i g h t f o r h i s c r i t i c a l e v a l u a t i o n o f my e x p e r i m e n t s , and my t h e s i s , and h i s f r i e n d s h i p . Thanks t o a l l the g r a d u a t e s t u d e n t s p a r t i c u l a r l y Swamy Yeleswaram, M a r i o n Wong, Ahmad D o r o u d i a n , Seema Ga d k a r i f o r t h e i r f r i e n d s h i p , s u p p o r t and h e l p . A s p e c i a l thank-you t o my w i f e Mieke, and my f a m i l y f o r t h e i r s u p p o r t and u n d e r s t a n d i n g . T h i s p r o j e c t was s u p p o r t e d by Canadian H e a r t F o u n d a t i o n . I would f i n a l l y l i k e t o thank B.C. H e a r t F o u n d a t i o n and t h e M e d i c a l Research C o u n c i l o f Canada f o r the f i n a n c i a l s t u d e n t s h i p s u p p o r t . T h i s t h e s i s i s d e d i c a t e d t o my l o v i n g and s u p p o r t i v e f a m i l y . S. Dad i f you c o u l d o n l y see me now! 1 1. INTRODUCTION 1.1 PROPAFENONE 1.1.1 PHARMACOLOGY OF PROPAFENONE Propafenone (PF) i s a p o t e n t c l a s s 1C [Vaughan-Wi11iams] a n t i a r r h y t h m i c agent ( F i g u r e 1 ) . In s e v e r a l in vitro s t u d i e s PF was fo u n d t o a c t p r i m a r i l y on the f a s t sodium c h a n n e l s , c a u s i n g a d e c r e a s e i n t h e r a t e o f t h e r i s e o f phase 0 o f the a c t i o n p o t e n t i a l [Dukes and Vaughan-Wi11iams, 1984]. In a d d i t i o n , PF d i s p l a y s m i l d b e t a - b l o c k i n g e f f e c t s (1/8 p o t e n c y o f p r o p r a n o l o l i n g u i n e a - p i g c a r d i a c t i s s u e ) [ H a r r o n and Brogden, 1987], p r o l o n g s a c t i o n p o t e n t i a l d u r a t i o n [Dukes and V a u g h a n - W i l l i a m s , 1984], and has v e r y m i l d c a l c i u m channel b l o c k i n g e f f e c t s (1/100 p o t e n c y o f v e r a p a m i l i n g u i n e a - p i g v e n t r i c u l a r s t r i p s ) [ H a r r o n and Brogden, 1987]. T h e r a p e u t i c c o n c e n t r a t i o n s i n humans showed PF t o have the same a c t i v i t y as PF in vitro [ C o n n o l l y et al., 1983; M u l l e r - P e l t z e r , 1983]. These a c t i o n s i n c r e a s e t h e PR, QT, and HV i n t e r v a l s , i n d i c a t i n g a s l o w i n g o f the a c t i o n p o t e n t i a l t r a n s m i s s i o n t h r o u g h t he h e a r t . PF was a l s o found t o i n c r e a s e e f f e c t i v e and f u n c t i o n a l r e f r a c t o r y p e r i o d s , and t o i n c r e a s e A-V nodal c o n d u c t i o n t ime [ C o n n o l l y et al., 1983; M u l l e r - P e l t z e r , 1983; K e r r et al., 1988]. Propafenone i s marketed as a racemate, t h a t i s , c o n s i s t i n g o f an equal amount o f (+)-S-propafenone and (-)-R-propafenone randomly mixed. I t has been shown t h a t both e n a n t i o m e r s produce s i m i l a r 2 f r e q u e n c y - d e p e n d e n t d e p r e s s i o n o f maximum u p s t r o k e o f phase 0 i n c a n i n e P u r k i n j e f i b e r s [Kroemer et al., 1989]. In a d d i t i o n , t h e (+)-S-PF e n a n t i o m e r was shown t o have a 100 f o l d g r e a t e r a f f i n i t y f o r human lymphocyte / ^ - r e c e p t o r as compared to the (-)-R-PF e n a n t i o m e r [Kroemer et a7., 1989]. 1.1.2 THERAPEUTIC EFFICACY OF PROPAFENONE PF has been found t o be e f f e c t i v e i n t h e t r e a t m e n t o f a number o f d i f f e r e n t a r r h y t h m i a s , i n c l u d i n g r e c u r r e n t a t r i a l f i b r i l l a t i o n [ K e r r e t al., 1988; Ludmer, 1987], a t r i a l t a c h y a r r h y t h m i a s a f t e r c a r d i a c s u r g e r y [ C o n n o l l y e t al., 1987], W o l f f - P a r k i n s o n - W h i t e Syndrome [Ludmer, 1987], paroxysmal s u p r a v e n t r i c u l a r a r r h y t h m i a s [Hammill e t al., 1988], v e n t r i c u l a r premature complexes [Rabkin et al., 1987], and c h r o n i c v e n t r i c u l a r a r r h y t h m i a s [ S c h l e p p e r , 1987]. As w i t h o t h e r a n t i a r r h y t h m i c a g e n t s , PF has a d v e r s e e f f e c t s ; however, when compared to o t h e r c l a s s IC a n t i a r r h y t h m i c a g e n t s , t h e s e t e n d t o be l e s s common. The most s e r i o u s o f t h e s e a d v e r s e e f f e c t s a r e t h e c a r d i o v a s c u l a r ; a r r y t h m o g e n e s i s , a g g r a v a t i o n o f a r r h y t h m i a s , d e c r e a s e d l e f t v e n t r i c u l a r f u n c t i o n ( n e g a t i v e i n o t r o p i c e f f e c t s ) , and c o n d u c t i o n b l o c k s [ S c h l e p p e r , 1987]. S i d e e f f e c t s o f a l e s s s e r i o u s n a t u r e are nausea, GI d i s t r e s s , change i n t a s t e p e r c e p t i o n , d i z z i n e s s , p a r e s t h e s i a s , and v i s u a l b l u r r i n g [ S c h l e p p e r , 1987]. A p p r o x i m a t e l y t h r e e p e r c e n t o f p a t i e n t s w i t h a d v e r s e e f f e c t s r e q u i r e d i s c o n t i n u a t i o n o f t h e r a p y [ H a r r o n and Brogden, 1987]. 3 1.1.3 PHARMACOKINETICS OF PROPAFENONE 1.1.3.1 ABSORPTION OF PROPAFENONE A b s o r p t i o n o f PF i n normal s u b j e c t s i s a l m o s t c o m p l e t e . In a s t u d y u s i n g d e u t e r a t e d PF, 95% o f t h e o r a l dose was a b s o r b e d , w i t h o n l y 1% o f t h e dose e x c r e t e d unchanged i n t h e f a e c e s [Hege et al., 1984]. O r a l b i o a v a i l a b i l i t y o f PF appears t o be dose-dependent [Hollmann e t a/.-, 1983]. For example, b i o a v a i l a b i l i t y f o r 150mg, 300mg, and 450mg o r a l doses o f PF was 13%, 32%, and 55% r e s p e c t i v e l y , i n d i c a t i n g p o s s i b l e s a t u r a b l e f i r s t - p a s s m e t a b o l i s m [Hollmann et al., 1983]. In a d d i t i o n , i t was a l s o found t h a t f o o d s u b s t a n t i a l l y i n c r e a s e d (mean v a l u e 147%) the b i o a v a i l a b i l i t y i n e x t e n s i v e m e t a b o l i z e r s [ A x e l s o n et a7., 1987]. 1.1.3.2 DISTRIBUTION AND PLASMA PROTEIN BINDING OF PROPAFENONE The volume o f d i s t r i b u t i o n f o r PF i s e s t i m a t e d t o be between 2-4.5 L/kg o f body mass [ S c h l e p p e r , 1987]. PF i s a l s o e x t e n s i v e l y plasma p r o t e i n bound (~ 95-97%) at t h e r a p e u t i c c o n c e n t r a t i o n s [Chan e t al., 1989b]. Two b i n d i n g s i t e s f o r PF have been i d e n t i f i e d i n whole serum, one l o w - c a p a c i t y h i g h - a f f i n i t y s i t e t h o u g h t t o be a s s o c i a t e d w i t h a l p h a - l - a c i d g l y c o p r o t e i n (AAG), and one h i g h - c a p a c i t y low-a f f i n i t y b i n d i n g s i t e b e l i e v e d t o be a s s o c i a t e d w i t h serum albumin [Chan et al., 1989b]. PF a l s o undergoes c o n c e n t r a t i o n - d e p e n d e n t plasma p r o t e i n b i n d i n g in vitro, a t l e v e l s w e l l above the t h e r a p e u t i c c o n c e n t r a t i o n [Chan et al. 1989b]. PF and i t s major m e t a b o l i t e 5-4 h y d r o x y p r o p a f e n o n e (5-OH-PF) were found t o d i s t r i b u t e p r i m a r i l y t o l i v e r , l u n g , and h e a r t [ L a t i n i et al., 1987]. 1.1.3.3 METABOLISM OF PROPAFENONE PF m e t a b o l i s m e x h i b i t s g e n e t i c polymorphism, i n t h a t s u b p o p u l a t i o n s o f (-90%) e x t e n s i v e m e t a b o l i z e r s (EM) and (-10%) poor m e t a b o l i z e r s (PM) e x i s t f o r the drug [Harron and Brogden, 1987]. PMs were c h a r a c t e r i z e d by h i g h c o n c e n t r a t i o n s o f PF, e x i s t e n c e o f a l i n e a r d o s e - r e s p o n s e r e l a t i o n s h i p , l o n g T^ (17.2 ± 8.0 h ) , low o r a l c l e a r a n c e (264 ± 48 mL/min), and v e r y l i t t l e a c c u m u l a t i o n o f 5-h y d r o x y p r o p a f e n o n e (5-OH-PF) [Siddoway et al., 1987]. EMs, on t h e o t h e r hand, were c h a r a c t e r i z e d by lower PF l e v e l s a t s t e a d y - s t a t e , no l i n e a r d o s e - r e s p o n s e r e l a t i o n s h i p , s h o r t T^ (5.5 ± 2.1 h ) , h i g h o r a l c l e a r a n c e (1115 ± 1238 mL/min), and d e t e c t a b l e l e v e l s o f 5-OH-PF [Siddoway et al., 1987]. EMs showed the g r e a t e s t i n c r e a s e i n b i o a v a i l a b i l i t y w i t h t he consumption o f f o o d , w h i l e a PM showed v i r t u a l l y no i n c r e a s e i n b i o a v a i l a b i l i t y due t o f o o d [ A x e l s o n e t al., 1987]. As w e l l , w i t h c h r o n i c d o s i n g o f PF, the r a t i o o f 5-OH-PF/PF d e c r e a s e s from t he s i n g l e dose s i t u a t i o n . T h i s may be due t o t h e s a t u r a t i o n o f t h e m e t a b o l i c p r o c e s s [ G i a n i et al., 1988]. Propafenone a l s o undergoes s t e r e o s e l e c t i v e d i s p o s i t i o n i n p a t i e n t s on l o n g term t h e r a p y [Kroemer et al., 1989]. I t has been d e m o n s t r a t e d t h a t t he r a t i o o f ar e a under t he c u r v e f o r (+J-S-PF t o (-)-R-PF was 1.73 ± 0.15 [Kroemer et a7., 1989]. 5 PF i s e x t e n s i v e l y m e t a b o l i z e d , p r i m a r i l y t o 5-OH-PF, 4-methoxy-5-OH-PF, and N-d e p r o p y l - P F , f o l l o w e d by c o n j u g a t i o n and e x c r e t i o n o f the r e s p e c t i v e g l u c u r o n i d e o r s u l f a t e . The g l u c u r o n i d e and s u l f a t e o f 5-OH-PF a r e , by f a r , t he most common m e t a b o l i t e s o f PF, f o l l o w e d i n q u a n t i t a t i v e i m portance by the c o n j u g a t e s o f 4-methoxy-5-hydroxy-PF, and PF i t s e l f . The major r o u t e o f e x c r e t i o n o f m e t a b o l i t e s ( c o n j u g a t e d and no n - c o n j u g a t e d ) i n humans i s via t h e f a e c e s (-53%) [Hege et al., 1984]. U r i n a r y e x c r e t i o n o f c o n j u g a t e d and u n c o n j u g a t e d drug and m e t a b o l i t e s a c c o u n t s f o r a p p r o x i m a t e l y 18% o f the t o t a l dose [Hege e t a/., 1984]. The most prominent m e t a b o l i t e i n t h e u r i n e i s 5-OH-PF [Hege et al., 1984]. I t has a l s o been o b s e r v e d t h a t t h e m e t a b o l i t e s 5-OH-PF and N-d e p r o p y l - P F accumulate d u r i n g c h r o n i c d o s i n g i n p a t i e n t s i n which a r r h y t h m i a s were t h e o n l y d i s e a s e s t a t e r e p o r t e d [ K a t e s et al., 1985]. Plasma l e v e l s o f 5-OH-PF and N- d e p r o p y l - P F accumulate t o 23% and 18% o f t h e t o t a l s t e a d y - s t a t e plasma l e v e l o f PF, r e s p e c t i v e l y [ K a t e s et al., 1985] ( F i g u r e 1 ) . OH HO F i g u r e 1. S t r u c t u r e o f pr o p a f e n o n e and t h e a c t i v e m e t a b o l i t e s 5-hy d r o x y p r o p a f e n o n e and n - d e p r o p y l p r o p a f e n o n e . 6 The m e t a b o l i t e , 5-OH-PF, a l s o has much t h e same a c t i v i t y as t h e p a r e n t drug in vitro. In vitro s t u d i e s i n d i c a t e t h a t 5-OH-PF has a somewhat g r e a t e r n e g a t i v e i n o t r o p i c and c a l c i u m channel b l o c k i n g a c t i v i t y than PF [Dukes and Vaug h a n - W i l l i a m s , 1984]. However, i n whole animal s t u d i e s 5-OH-PF was found t o be t w i c e as e f f i c a c i o u s as PF. T h i s may be a t t r i b u t e d t o t h e s m a l l e r volume o f d i s t r i b u t i o n (V^) f o r 5-OH-PF. N-depropyl-PF i s a l s o a c t i v e , but, i t s a c t i v i t y has been l e s s w e l l d e f i n e d [ M a l f a t t o et al., 1988]. S i m i l a r r e s u l t s f o r 5-OH-PF have been r e p o r t e d i n c a n i n e P u r k i n j i e f i b e r s [Thompson et al., 1987] and i n g u i n e a p i g v e n t r i c u l a r muscle [Rouet et al., 1989]. To d a t e t h e r e have been no a c c u r a t e c o r r e l a t i o n s between dru g c o n c e n t r a t i o n and obs e r v e d p h a r m a c o l o g i c a l r e s p o n s e o f p r o p a f e n o n e . I t has been shown t h a t t h e e f f e c t i v e dosage i s not s i g n i f i c a n t l y d i f f e r e n t between EMs and PMs, even though t he c o n c e n t r a t i o n o f PF i s s u b s t a n t i a l l y h i g h e r i n PMs [Siddoway et al., 1987]. I t has been shown t h a t propafenone has two a c t i v e m e t a b o l i t e s which accumulate upon c h r o n i c d o s i n g , namely 5-OH-PF and n - d e p r o p y l - P F [Kates et al., 1985]. Both o f t h e s e m e t a b o l i t e s have been shown t o p o s s e s s s i m i l a r a c t i v i t y t o t h a t o f propafenone [ M a l f a t t o et al., 1988]. F u r t h e r , i t was shown t h a t when the c o n c e n t r a t i o n s o f PF, 5-OH-PF, and AAG were used t o p r e d i c t t h e QRS w i d t h , a b e t t e r c o r r e l a t i o n was o b s e r v e d than when o n l y the c o n c e n t r a t i o n o f PF was used [Chan, 1989c]. I t appears t h a t both t h e c o n c e n t r a t i o n o f propafenone and 5-hydr o x y p r o p a f e n o n e , i n a d d i t i o n t o the d r u g - p r o t e i n b i n d i n g , must be c o n s i d e r e d t o a r r i v e a t a r e a s o n a b l e e s t i m a t e f o r the d o s e - r e s p o n s e r e l a t i o n s h i p f o r 7 p r o p a f e n o n e i n EMs. However, t o d a t e t h e r e i s no e s t i m a t i o n o f 5-OH-PF b i n d i n g w i t h t h e a d d i t i o n o f t h e p a r e n t drug PF, and t h u s t h e f r e e c o n c e n t r a t i o n o f 5-OH-PF. 1.2 DRUG-PROTEIN BINDING The b i n d i n g o f drugs t o plasma and t i s s u e p r o t e i n s can have a l a r g e i n f l u e n c e on both the p h a r m a c o k i n e t i c s and pharmacodynamics, and t h e r e f o r e , a d r u g ' s t h e r a p e u t i c e f f e c t , o r t o x i c i t y [ W i l k i n s o n , 1983]. I f d r u g - p r o t e i n b i n d i n g exceeds 90 %, a l t e r a t i o n s i n b i n d i n g can have l a r g e e f f e c t s on t h e v a r i a b i l i t y o f t h e e s t i m a t e d p h a r m a c o k i n e t i c p a r a m e t e r s o f drugs ( i . e . a 10 % d e c r e a s e i n the b i n d i n g w i l l i n c r e a s e t h e f r e e drug c o n c e n t r a t i o n by a p p r o x i m a t e l y 100%) [Svensson et al., 1986]. For example, s i n c e the e x t e n t o f b i n d i n g i s d i r e c t l y c o r r e l a t e d t o t h e h e p a t i c c l e a r a n c e o f a low c l e a r a n c e d r u g , a l t e r a t i o n s i n b i n d i n g w i l l d i r e c t l y a f f e c t the c l e a r a n c e o f t o t a l d r u g , and t h e r e f o r e , the t o t a l drug c o n c e n t r a t i o n . Once e q u i l i b r i u m has been a t t a i n e d , the f r e e drug c o n c e n t r a t i o n o f a low c l e a r a n c e drug w i l l be s i m i l a r t o t h a t o b s e r v e d b e f o r e the changes i n b i n d i n g . On t h e o t h e r hand, s i n c e the c l e a r a n c e o f a h i g h c l e a r a n c e drug i s i n d e p e n d e n t o f the f r e e drug c o n c e n t r a t i o n , a l t e r e d b i n d i n g may not s i g n i f i c a n t l y a l t e r the t o t a l drug c o n c e n t r a t i o n but can i n c r e a s e o r d e c r e a s e the f r e e drug c o n c e n t r a t i o n [Svensson et al., 1986]. I t i s g e n e r a l l y a c c e p t e d t h a t the f r e e drug c o n c e n t r a t i o n i s r e s p o n s i b l e f o r the m a j o r i t y o f the o b s e r v e d p h a r m a c o l o g i c a l r e s p o n s e , s i n c e i t i s f r e e t o d i s t r i b u t e t o the s i t e o f a c t i o n . S i n c e the f r e e 8 drug c o n c e n t r a t i o n o f a h i g h c l e a r a n c e drug may be changed due t o a l t e r a t i o n s i n b i n d i n g , the o b s e r v e d pharmacodynamics o f t h i s d r u g may a l s o be a l t e r e d [Svensson e t a/., 1986]. These changes i n f r e e d r u g would be p a r t i c u l a r l y i m p o r t a n t f o r drugs w i t h a s t e e p d o s e - r e s p o n s e r e l a t i o n s h i p , such as l i d o c a i n e [ H a r r i s o n and Alderman, 1971]. F u r t h e r m o r e , d e p e n d i n g on the c h e m i c a l n a t u r e o f t h e d r u g ( l i p o p h i l i c o r h y d r o p h i l i c ) , b i n d i n g can a l s o i n f l u e n c e p h a r m a c o k i n e t i c p a r a m e t e r s such as t h e volume o f d i s t r i b u t i o n [ L i n d u p , 1987; W i l k i n s o n , 1983]. Thus, a l t e r a t i o n s i n b i n d i n g can d i r e c t l y i n f l u e n c e p r i m a r y p h a r m a c o k i n e t i c parameters such as c l e a r a n c e and d i s t r i b u t i o n [ L i n d u p , 1987], S i n c e a l t e r a t i o n s i n t h e s e p h a r m a c o k i n e t i c p a r a m e t e r s may have l a r g e e f f e c t s on the f r e e drug c o n c e n t r a t i o n , and t h e r e f o r e , o b s e r v e d t h e r a p e u t i c e f f e c t o r t o x i c i t y , i t i s i m p o r t a n t t o c h a r a c t e r i z e t h e d r u g - p r o t e i n b i n d i n g i n t e r a c t i o n s i n o r d e r t o be a b l e t o p r e d i c t b i n d i n g a l t e r a t i o n s due t o the p r e s e n c e o f o t h e r d r u g s , and/or d i s e a s e . Many t e c h n i q u e s , such as c i r c u l a r d i c h r o i s m [Gamel-El e t al., 1982; C h i g n e l l , 1969], f l u o r e s c e n t probes [ E s s a s s i e t al., 1989], c a l o r i m e t r i c s t u d i e s [ A k i a n d and Yamamoto, 1989], g e l f i l t r a t i o n , and u l t r a c e n t r i f u g a t i o n [Kwong, 1985] are a v a i l a b l e t o e v a l u a t e d r u g -p r o t e i n i n t e r a c t i o n s . T e c h n i q u e s such as, c i r c u l a r d i c h r o i s m , f l u o r e s c e n t p r o b es, and c a l o r i m e t r i c s t u d i e s are g e n e r a l l y used t o d e t e r m i n e the n a t u r e o f the d r u g - p r o t e i n i n t e r a c t i o n , whereas g e l f i l t r a t i o n , u l t r a c e n t r i f u g a t i o n , u l t r a f i l t r a t i o n , and e q u i l i b r i u m d i a l y s i s p r o v i d e more q u a n t i t a t i v e i n f o r m a t i o n o f t h e d r u g - p r o t e i n 9 b i n d i n g i n t e r a c t i o n ( e . g . f r e e f r a c t i o n o f d r u g i n serum). D e s p i t e the l a r g e number o f a v a i l a b l e methods t o s t u d y the d r u g - p r o t e i n i n t e r a c t i o n , the most commonly used methods a r e u l t r a f i l t r a t i o n and e q u i l i b r i u m d i a l y s i s [ L i n d u p , 1987; Kwong, 1985]. Both e q u i l i b r i u m d i a l y s i s and u l t r a f i l t r a t i o n must be r i g o r o u s l y v a l i d a t e d , and c a r e f u l l y c o n t r o l l e d t o p r o v i d e a c c u r a t e and r e p r o d u c i b l e e s t i m a t e s o f d r u g - p r o t e i n b i n d i n g [ L i n d u p , 1987; Kwong, 1985]. 1.2.1 ULTRAFILTRATION The method o f u l t r a f i l t r a t i o n r e l i e s on a p r e s s u r e g r a d i e n t , u s u a l l y due t o c e n t r i f u g a t i o n , which f o r c e s plasma water and d i s s o l v e d s o l u t e s ( d r u g s , i o n s , and s m a l l endogenous compounds) t h r o u g h a semipermeable membrane. The membrane i s assumed t o be " i d e a l " , i n t h a t i t s h o u l d not d i s c r i m i n a t e between dr u g and water m o l e c u l e s [Kwong, 1985]. One o f the drawbacks to u s i n g u l t r a f i l t r a t i o n , p a r t i c u l a r l y i f the m o l e c u l a r weight o f the l i g a n d o f i n t e r e s t i s g r e a t e r than 300, i s m o l e c u l a r s i e v i n g [Kwong, 1985]. T h i s o c c u r s as a r e s u l t o f p r o t e i n s packed on t o the membrane due t o c e n t r i f u g a t i o n which d i s c r i m i n a t e s between water m o l e c u l e s and l i g a n d , such t h a t w a t er m o l e c u l e s pass through the membrane u n o b s t r u c t e d i n comparison t o d r u g m o l e c u l e s [Kwong, 1985]. U l t r a f i l t r a t i o n i s a f a s t and easy method f o r the d e t e r m i n a t i o n o f d r u g - p r o t e i n b i n d i n g p r o v i d e d t h a t , i n a d d i t i o n t o m o l e c u l a r s i e v i n g , f a c t o r s such as pH, t e m p e r a t u r e , and n o n - s p e c i f i c b i n d i n g are a c c u r a t e l y a c c o u n t e d f o r , o r c o n t r o l l e d [ L i n d u p , 1987; Kwong, 1985]. 10 1.2.2 EQUILIBRIUM DIALYSIS E q u i l i b r i u m d i a l y s i s i s c o n s i d e r e d the " c l a s s i c a l " method f o r t h e d e t e r m i n a t i o n o f d r u g - p r o t e i n b i n d i n g [ L i n d u p , 1987]. E q u i l i b r i u m d i a l y s i s works on the p r i n c i p l e t h a t o n l y f r e e o r unbound dr u g i n serum w i l l e q u i l i b r a t e a c r o s s a semi-permeable membrane. Thus, once e q u i l i b r i u m has been a t t a i n e d , drug c o n c e n t r a t i o n i n t h e b u f f e r e q u a l s t h e f r e e drug c o n c e n t r a t i o n i n the serum compartment. I f a l i q u o t s o f serum and b u f f e r a r e measured and b i n d i n g c a l c u l a t e d , n o n - s p e c i f i c b i n d i n g does not c o n t r i b u t e t o the c a l c u l a t e d b i n d i n g [Kwong, 1985]. As w i t h u l t r a f i l t r a t i o n , e q u i l i b r i u m d i a l y s i s must be c a r e f u l l y o p t i m i z e d and c o n t r o l l e d [Kwong, 1985; L i n d u p , 1987]. I n i t i a l l y , time t o e q u i l i b r i u m must be e s t a b l i s h e d . Time t o e q u i l i b r i u m i s d i r e c t l y a f f e c t e d by t h e pore s i z e o f t h e membrane, t h e s u r f a c e (membrane) t o volume r a t i o [ L i n d u p , 1987], and t h e s i d e t o which t h e d r u g i s added [McNamarra and Bogardu, 1982]. T h e o r e t i c a l l y , i f d r u g i s added t o the serum compartment, e q u i l i b r i u m w i l l be o b t a i n e d e a r l i e r . E q u i l i b r i u m can u s u a l l y be e s t a b l i s h e d w i t h i n 4-18 hours f o r most drugs [Kwong, 1985; T o z e r e t a l . , 1981]. A l o n g w i t h l o n g e r d i a l y s i s t i m e s comes unique time dependent p r o b l e m s , such as, volume s h i f t s , and pH s h i f t s . Volume s h i f t s o c c u r as a r e s u l t o f unequal osmotic and c o l l o d i a l p r e s s u r e between the b u f f e r and serum compartment o f the d i a l y s i s c e l l [Kwong, 1985; L i n d u p , 1987]. Serum c o n t a i n s many i o n s and endogenous compounds, i n a d d i t i o n t o l a r g e macromolecules, which c o n t r i b u t e t o o s m o t i c 11 p r e s s u r e . The a d d i t i o n o f a b u f f e r o f a lower o s m o t i c p r e s s u r e t o t h e o t h e r s i d e o f the d i a l y s i s membrane can r e s u l t i n an o s m o t i c p r e s s u r e g r a d i e n t . T h i s g r a d i e n t may p u l l water from the b u f f e r compartment i n t o t h e serum, thus d i l u t i n g serum p r o t e i n s , and r e s u l t i n g i n lower c a l c u l a t e d b i n d i n g [Kwong, 1985]. Volume s h i f t s can be d i r e c t l y measured by comparing volumes p r e - and p o s t - d i a l y s i s , o r by measuring a n o n - d i f f u s i b l e marker, such as serum p r o t e i n c o n c e n t r a t i o n , p r e - and p o s t - d i a l y s i s . G e n e r a l l y s m a l l volume s h i f t s s t a r t t o appear a f t e r 6-8 hours o f d i a l y s i s [Lima e t al., 1981]. A l o n g d i a l y s i s time (24 hours) may s i g n i f i c a n t l y i n c r e a s e t h e pH i n the d i a l y s i s system, due t o the l o s s o f d i s s o l v e d c a r b o n d i o x i d e i n t h e serum compartment [ L u i and C h i o u , 1986]. S i n c e many drugs are known t o undergo pH dependent b i n d i n g t h e b u f f e r chosen must have a l a r g e enough c a p a c i t y t o oppose t h i s s h i f t [ L i n d u p , 1987; Kwong, 1985]. However, b u f f e r s t r e n g t h and c o m p o s i t i o n have been shown t o a f f e c t t he b i n d i n g o f a number o f d r u g s , such as p r o p r a n o l o l and t h e o p h y l l i n e [Kwong, 1985]. D i s c u s s i o n has f o c u s e d on the use o f p h y s i o l o g i c a l b u f f e r s ; however, t h e i r use has been shown not t o o f f e r much improvement t o the p r e s e n t l y used " n o n - p h y s i o l o g i c a l " phosphate b u f f e r s , s i n c e a r t i f i c i a l i n t e r v e n t i o n i s u s u a l l y r e q u i r e d t o c o n t r o l pH and a d j u s t pH p r i o r t o d i a l y s i s [ K r i s t e n s e n and Grams, 1982]. Donnan e f f e c t s , which a r e t h e r e s u l t o f an unequal d i s t r i b u t i o n o f c h a r g e d compounds between the b u f f e r and serum compartment o f t h e d i a l y s i s c e l l have a l s o been shown t o a l t e r d r u g - p r o t e i n b i n d i n g [Kwong, 1985; L i n d u p , 1987]. S i n c e p r o t e i n s a t p h y s i o l o g i c a l pH a r e 12 c h a r g e d , and u s u a l l y c o n f i n e d by the semipermable membrane, unequal d i f f u s i o n o f c harged p a r t i c l e s can l e a d t o a l t e r a t i o n s i n b i n d i n g . T h i s i s p a r t i c u l a r l y t r u e f o r compounds which are weakly bound, and s t r o n g l y i o n i z e d i n the d i a l y s i s system [Kwong, 1985]. T h i s can u s u a l l y be c o r r e c t e d by the a d d i t i o n o f d i f f u s i b l e i o n s such as sodium and c h l o r i d e t o the b u f f e r i n o r d e r t o e l e c t r o s t a t i c a l l y e q u i l i b r a t e the d i a l y s i s system [Kwong, 1985]. P r o t e i n l e a k a g e as a r e s u l t o f membrane r u p t u r e , and/or improper mounting o f the d i a l y s i s membrane must a l s o be c o n s i d e r e d . The p r e s e n c e o f i m p u r i t i e s , p a r t i c u l a r l y i n r a d i o c h e m i c a l s t u d i e s , must be r u l e d out s i n c e the p o s s i b l e d i s p l a c e m e n t o f the d r u g o f i n t e r e s t by an i m p u r i t y may r e s u l t i n e r r o n e o u s d r u g - p r o t e i n b i n d i n g d a t a [ K r i s t e n s e n and Gram, 1982]. When t h e s e f a c t o r s are o p t i m i z e d and c o n t r o l l e d , e q u i l i b r i u m d i a l y s i s can p r o v i d e a c c u r a t e , and some s u g g e s t , more p r e c i s e measurements o f b i n d i n g compared t o o t h e r methods, such as u l t r a f i l t r a t i o n [ K u r t z e t a l . , 1977; Kwong, 1985] 1.3 SERUM PROTEINS S e v e r a l p r o t e i n s , normal c o n s t i t u e n t s o f serum, can b i n d both endogenous, and exogenous compounds. These p r o t e i n s a r e , human serum albumin (HSA), a - l - a c i d g l y c o p r o t e i n (AAG), l i p o p r o t e i n s , p r e - a l b u m i n , 6 - g l o b u l i n s , a - l - a n t i t r y p s i n , and v a r i o u s o t h e r s p e c i f i c t r a n s p o r t p r o t e i n s [ R o u t l e d g e , 1986; Kremer e t a l . , 1988; Kragh-Hansen, 1981]. 13 However, f o r most drug compounds examined, HSA, AAG and l i p o p r o t e i n s a c c o u n t f o r the m a j o r i t y o f o b s e r v e d b i n d i n g [ R o u t l e d g e , 1986]. 1.3.1 HUMAN SERUM ALBUMIN Human serum albumin (HSA) ( m o l e c u l a r weight 69000 D a l t o n s ) , a c c o u n t s f o r a p p r o x i m a t e l y 60% o f the t o t a l plasma p r o t e i n c o n c e n t r a t i o n [Svensson e t al., 1986]. HSA i s t h o u g h t t o be i m p o r t a n t i n t h e s t o r a g e and t r a n s p o r t o f v a r i o u s endogenous l i g a n d s i n serum ( f r e e f a t t y a c i d s , c h l o r i d e i o n s , b i l i r u b i n , s t e r o i d s , and t r y p t o p h a n ) , t r a n s p o r t amino a c i d s t o t i s s u e s [Kragh-Hansen, 1981], and t o p r o v i d e c o l l o i d o s m o t i c p r e s s u r e i n t h e b l o o d . Over 20 g e n e t i c v a r i a n t s o f HSA have been i d e n t i f i e d i n the human p o p u l a t i o n ; however, t h e s e v a r i a n t s are r a r e i n t h e European p o p u l a t i o n . I t i s not c l e a r i f t h e s e v a r i a n t s cause a l t e r e d b i n d i n g o f endogenous and exogenous l i g a n d s [Kragh-Hansen, 1981]. HSA i s c o n s i d e r e d t o be the most i m p o r t a n t d r u g - b i n d i n g p r o t e i n i n plasma due t o i t s l a r g e c o n c e n t r a t i o n ( a p p r o x i m a t e l y 600 uH)[Rout!edge, 1986]. In a d d i t i o n , albumin may a l s o p l a y a r o l e i n t h e t i s s u e b i n d i n g o f drugs, s i n c e a p p r o x i m a t e l y 60% o f albumin i s c o n t a i n e d i n e x t r a v a s c u l a r t i s s u e [ R o u t l e d g e , 1986]. S i x s t r u c t u r a l l y s p e c i f i c b i n d i n g s i t e s have been i d e n t i f i e d on t h e HSA m o l e c u l e . HSA i s known to b i n d a c i d i c , b a s i c and n e u t r a l compounds, but i s g e n e r a l l y c o n s i d e r e d t o b i n d m a i n l y a n i o n i c ( a c i d i c ) l i g a n d s w i t h h i g h a f f i n i t y [Kragh-Hansen, 1981]. I t has been shown t h a t t h e c o n f o r m a t i o n o f HSA 14 can change due t o changes i n pH, and upon b i n d i n g o f c e r t a i n s p e c i f i c a l l y bound l i g a n d s [Kragh-Hansen, 1981]. 1.3.2 a- l - A C I D GLYCOPROTEIN a r - l - a c i d g l y c o p r o t e i n (AAG) i s one o f the 90 o t h e r g l o b u l i n p r o t e i n s p r e s e n t i n serum [ R o u t l e d g e , 1986]. The e x a c t p h y s i o l o g i c a l r o l e o f AAG i s not w e l l u n d e r s t o o d ; however, i t has been shown t o i n c r e a s e up t o 2-3 f o l d i n t r a u m a t i c i n j u r i e s [ R o u t l e d g e , 1986; Kremer et al., 1988]. AAG has a l s o been shown t o be e l e v a t e d i n c e r t a i n c h r o n i c d i s e a s e s i n c l u d i n g c a n c e r , rheumatoid a r t h r i t i s , c h r o n i c r e n a l f a i l u r e and Crohn's d i s e a s e [Kremer et al., 1988]. T h i s has l e d t o s p e c u l a t i o n t h a t AAG i s a m e d i a t o r o f normal c o a g u l a t i o n , i m m u n o l o g i c a l and t i s s u e r e p a i r p r o c e s s e s [Kremer et al., 1988]. AAG appears t o be an imp o r t a n t b i n d i n g p r o t e i n o f b a s i c and n e u t r a l drugs both i n serum and i n t i s s u e , s i n c e a p p r o x i m a t e l y 40% o f the p r o t e i n i s i n t i s s u e [ R o u t l e d g e , 1986]. A p p r o x i m a t e l y 40% o f the AAG m o l e c u l e i s c a r b o h y d r a t e , some o f which i s composed o f s i a l i c a c i d , which r e s u l t s i n the AAG m o l e c u l e b e i n g a c i d i c w i t h a low pKa i n serum [Kremer et a7., 1988]. D e s p i t e t he a c i d i c n a t u r e o f t h e p r o t e i n , t h e m a j o r i t y o f d r u g - p r o t e i n b i n d i n g i n t e r a c t i o n s appear t o be h y d r o p h o b i c i n n a t u r e [ M u l l e r , 1989; R o u t l e d g e , 1986; Kremer et al., 1988]. I t i s not y e t c l e a r as t o the number o f b i n d i n g s i t e s p r e s e n t on t h e AAG m o l e c u l e ; however one h i g h a f f i n i t y , low c a p a c i t y s i t e f o r 15 b a s i c and n e u t r a l drugs has been i d e n t i f i e d ( b i n d i n g a f f i n i t y (Ka) ~ 1 0 4 - 1 0 7 M" 1 and c a p a c i t y » 1 0 " 5 M) [ R o u t l e d g e , 1986]. Thus, f l u c t u a t i o n s i n AAG c o n c e n t r a t i o n can have a l a r g e a f f e c t on the d e g r e e o f d r u g - p r o t e i n b i n d i n g o f most b a s i c d r u g s . A c i d i c d r u g s , such as w a r f a r i n , have a l s o been shown t o b i n d t o AAG and compete f o r b i n d i n g s i t e s w i t h b a s i c drugs [ O t a g i r i et al., 1987]. 1.3.3 SERUM LIPOPROTEINS L i p o p r o t e i n complexes a r e m i x t u r e s o f p r o t e i n , p h o s p h o l i p i d s , c h o l e s t e r o l and t r i g l y c e r i d e s . The f u n c t i o n o f t h e s e complexes i s t h e t r a n s p o r t o f l i p i d s w i t h i n t h e body. High, low and v e r y low d e n s i t y l i p o p r o t e i n s make up the complex t r a n s p o r t system w i t h i n the b l o o d stream, whereas c h y l o m i c r o n s a r e l a r g e l y r e s p o n s i b l e f o r the t r a n s p o r t o f d i g e s t e d f a t s from the i n t e s t i n a l mucosa to the b l o o d s t r e a m [Ganong, 1985]. Very low d e n s i t y l i p o p r o t e i n s (VLDL) are formed i n t h e l i v e r and s e r v e t o t r a n s p o r t t r i g l y c e r i d e s from the l i v e r t o e x t r a h e p a t i c t i s s u e . Thus, VLDLs are composed o f low c o n c e n t r a t i o n s o f p r o t e i n , p h o s p h o l i p i d , c h o l e s t e r o l , and v e r y h i g h c o n c e n t r a t i o n s o f t r i g l y c e r i d e s . Once the t r i g l y c e r i d e s are l a r g e l y removed, t h e VLDL becomes an i n t e r m e d i a t e d e n s i t y l i p o p r o t e i n ( I D L ) . The IDL p i c k s up e x c e s s c h o l e s t e r o l from the h i g h d e n s i t y l i p o p r o t e i n s (HDL), and then becomes the low d e n s i t y l i p o p r o t e i n ( L D L ) . The purpose o f the LDL i s t o p r o v i d e t i s s u e w i t h c h o l e s t e r o l ; t h e r e f o r e , the c o m p o s i t i o n o f the LDL has a h i g h e r c o n c e n t r a t i o n o f c h o l e s t e r o l i n comparison t o the o t h e r l i p o p r o t e i n s examined [Ganong, 1985]. F i n a l l y , the HDL p r o v i d e s a means f o r removing e x c e s s c h o l e s t e r o l from c e l l s ( t h e c o n c e n t r a t i o n 16 o f p r o t e i n s i n much h i g h e r i n the HDL i n comparison t o t h e o t h e r l i p o p r o t e i n s ) [Ganong, 1985]. L i p o p r o t e i n s can be e l e v a t e d i n v a r i o u s t y p e s o f d i s e a s e s , such as f a m i l i a l h y p e r l i p i d e m i a s . F o r example i n f a m i l i a l h y p e r c h o l e s t e r o l e m i a h e t e r o z y g o u s form D, t h e c o n c e n t r a t i o n o f LDL may be s i g n i f i c a n t l y e l e v a t e d [Brown and G o l d s t e i n , 1985]. I t has been ob s e r v e d t h a t both l i p o p h i l i c and c a t i o n i c drugs t e n d t o b i n d , o r are s o l u b i l i z e d w i t h i n t h e l i p o p r o t e i n complex [ R o u t l e d g e , 1986]. I t s h o u l d be no t e d t h a t not much i s known about t h e a c t u a l d r u g - p r o t e i n / 1 i p i d i n t e r a c t i o n f o r l i p o p r o t e i n s . 1.3.4 UPTAKE OF DRUGS INTO RED BLOOD CELLS The uptake o f drug by the r e d b l o o d c e l l may c o n t r i b u t e t o t h e d i s t r i b u t i o n o f a drug, and t h e r e f o r e , i n f l u e n c e t h e p h a r m a c o k i n e t i c p a r a m e t e r s dependent on d i s t r i b u t i o n . I t i s g e n e r a l l y c o n s i d e r e d t h a t drug i n t r i n s i c c l e a r a n c e e x p r e s s e d i n terms o f plasma, i s a p p r o x i m a t e l y t h e same as b l o o d i n t r i n s i c c l e a r a n c e ; however, when drug uptake by r e d b l o o d c e l l s i s g r e a t e r than t h a t o b s e r v e d i n plasma, a l a r g e o v e r e s t i m a t i o n i n the plasma i n t r i n s i c c l e a r a n c e w i l l o c c u r [ G i b a l d i and P e r r i e r , 1982]. The a c c u r a t e e s t i m a t i o n o f drug p r e s e n t i n t h e r e d b l o o d c e l l can have i m p l i c a t i o n s on the p h a r m a c o k i n e t i c parameters c a l c u l a t e d , and t h e r e f o r e , must be i n v e s t i g a t e d . 17 1.3.5 CHEMICAL BASIS FOR DRUG-PROTEIN INTERACTION As i d e n t i f i e d above, a number o f d i f f e r e n t c h e m i c a l i n t e r a c t i o n s can a c c o u n t f o r the d r u g - p r o t e i n i n t e r a c t i o n ( i o n i c bonding, hydrogen bon d i n g , Van d e r Waals f o r c e s , and h y d r o p h o b i c i n t e r a c t i o n s ) [ L i n d u p , 1987]. The degree t o which each b i n d i n g f o r c e c o n t r i b u t e s t o the o v e r a l l d r u g - p r o t e i n i n t e r a c t i o n i s dependent on both t h e drug and the p r o t e i n i n v o l v e d i n t h i s i n t e r a c t i o n . In f a c t , some c h e m i c a l f o r c e s may even redu c e t he s t r e n g t h o f the d r u g - p r o t e i n i n t e r a c t i o n ( e . g . i o n i c r e p u l s i o n ) . HSA, f o r example, t e n d s t o undergo a number o f v a r i o u s c h e m i c a l i n t e r a c t i o n s w i t h l i g a n d s , s i n c e t h e r e a re s i x w e l l d e f i n e d s p e c i f i c b i n d i n g s i t e s on HSA. The diazepam b i n d i n g s i t e t e n d s t o f a v o r l i g a n d s w i t h an a r o m a t i c c a r b o x y l group and an e l o n g a t e d h y d r o p h o b i c group. The a r y l p r o p i o n a t e s , such as i b u p r o f e n , f e n o p r o f e n , and naproxen, t e n d t o f a v o r b i n d i n g t o t h i s s i t e on HSA. T h i s would s u g g e s t t h a t both i o n i c and h y d r o p h o b i c i n t e r a c t i o n s a re i m p o r t a n t i n th e b i n d i n g o f t h e s e drugs t o t h i s p a r t i c u l a r HSA b i n d i n g s i t e [Kragh-Hansen, 1981]. The b i n d i n g o f b a s i c drugs t o the AAG m o l e c u l e tends t o be d e t e r m i n e d l a r g e l y by s p e c i f i c h y d r o p h o b i c i n t e r a c t i o n s . I t was shown t h a t t h e d e s i a l a t i o n o f AAG does not a f f e c t t he b i n d i n g o f p r o p r a n o l o l [Kremer e t al., 1988; M u l l e r , 1989]. T h i s would s u g g e s t t h a t t he s i a l i c a c i d r e s i d u e s on the p r o t e i n do not c o n t r i b u t e t o the b i n d i n g o f p r o p r a n o l o l . 18 The b i n d i n g i n t e r a c t i o n w i t h l i p o p r o t e i n s may be l a r g e l y d i c t a t e d by a s o l u b i l i z a t i o n p r o c e s s . I t s h o u l d however be p o i n t e d out t h a t the b i n d i n g o f q u i n i d i n e was found t o be s a t u r a b l e , i n d i c a t i n g a p o s s i b l e " c l a s s i c a l " b i n d i n g i n t e r a c t i o n [ N i l s e n , 1976]. The c o n f o r m a t i o n and c o n f i g u r a t i o n o f a d r u g may a l s o d e t e r m i n e t h e s t r e n g t h o f the b i n d i n g i n t e r a c t i o n . Both AAG and HSA have shown s t e r e o s e l e c t i v e d r u g - p r o t e i n i n t e r a c t i o n s . T h i s i n d i c a t e s t h a t v e r y s p e c i f i c b i n d i n g r e g i o n s , which can d i s c r i m i n a t e between t h e c o n f i g u r a t i o n o f d r u g s , are p r e s e n t on AAG and HSA [Kremer et a?., 1988; Kragh-Hansen, 1981]. 1.4 QUANTITATIVE DESCRIPTION OF THE DRUG-PROTEIN BINDING SITE A l t h o u g h i t i s i m p o r t a n t t o d e t e r m i n e the c h e m i c a l n a t u r e o f t h e d r u g - p r o t e i n i n t e r a c t i o n , i t i s a l s o i m p o r t a n t t o d e t e r m i n e the q u a n t i t a t i v e n a t u r e o f the d r u g - p r o t e i n i n t e r a c t i o n . Q u a n t i t a t i v e d r u g - b i n d i n g , i n a d d i t i o n t o p r o v i d i n g i n f o r m a t i o n r e g a r d i n g the f r e e drug c o n c e n t r a t i o n , a l s o s e r v e s as a p r e d i c t i v e t o o l . F a c t o r s i n c l u d i n g , at what c o n c e n t r a t i o n b i n d i n g t o a p r o t e i n becomes non-l i n e a r , the changes i n b i n d i n g due t o a l t e r e d p r o t e i n c o n c e n t r a t i o n s , and t h e p o s s i b l e o c c u r r e n c e o f b i n d i n g d i s p l a c e m e n t s due t o t h e a d d i t i o n o f a second drug may be p r e d i c t e d w i t h q u a n t i t a t i v e d r u g -p r o t e i n d a t a [Svensson et al., 1986]. The b i n d i n g c a p a c i t y and a f f i n i t y ( s t r e n g t h o f the d r u g - p r o t e i n i n t e r a c t i o n ) can be d e r i v e d from f i r s t p r i n c i p l e s . Any r e v e r s i b l e d r u g - p r o t e i n b i n d i n g 19 i n t e r a c t i o n i n serum o r t i s s u e can be d e s c r i b e d by e q u a t i o n 1. K l [D] + [P] 5^ [DP] (1) K2 where D and P a r e the unbound d r u g and p r o t e i n , r e s p e c t i v e l y , and DP i s t h e dru g p r o t e i n complex. The c o n s t a n t s f o r t h e f o r w a r d and backward r e a c t i o n s are K l and K2, r e s p e c t i v e l y . F o l l o w i n g t he law o f mass a c t i o n , a t e q u i l i b r i u m the a p p a r e n t a s s o c i a t i o n c o n s t a n t ( K a ) , t h e s t r e n g t h o f the b i n d i n g i n t e r a c t i o n , can be g i v e n by e q u a t i o n 2. Ka = K l = r/DPl (2) K2 [D][P] From e q u a t i o n 2 i t can be shown t h a t t h e c o n c e n t r a t i o n o f bound drug can be g i v e n by the p r o d u c t o f the a f f i n i t y c o n s t a n t and the c o n c e n t r a t i o n s o f f r e e drug and p r o t e i n ([DP] = K a [ D ] [ P ] ) . B i n d i n g can be r e p r e s e n t e d as the molar r a t i o o f dr u g bound ( r ) ( E q u a t i o n 3) r = [DP] (3) [DP] + [P] T h i s r a t i o i s the c o n c e n t r a t i o n o f bound dru g o r p r o t e i n o v e r the t o t a l p r o t e i n c o n c e n t r a t i o n ( P t ) [ P t ] = [DP] + [ P ] . S u b s t i t u t i n g f o r [DP], s i n c e [DP] cannot be measured d i r e c t l y , e q u a t i o n 4 i s d e r i v e d . r = K a f D U P l = K a f D l (4) Ka[D][P] + [P] 1 + Ka[D] T h i s can be s i m p l i f i e d t o show t h e bound c o n c e n t r a t i o n [DP] as i n 20 e q u a t i o n 5 [DP] = K a f P t U D l (5) 1 + Ka[D] and f o r ( i ) number o f d i f f e r e n t b i n d i n g s i t e s ( e q u a t i o n 6 ) . [DP] = 2 ( n ^ P t U D l K a ) j = l i + Ka[D] (6) In e q u a t i o n 5 and 6 t h e c a p a c i t y o f t h e b i n d i n g s i t e i s t h e number o f b i n d i n g s i t e s p e r p r o t e i n m o l e c u l e (n) m u l t i p l i e d by t h e a c t u a l c o n c e n t r a t i o n o f t h a t p r o t e i n . Due t o the n o n - l i n e a r n a t u r e o f the e q u a t i o n s r e p r e s e n t i n g d r u g -p r o t e i n b i n d i n g ( e q u a t i o n 5 ) , i t has become d i f f i c u l t t o g r a p h i c a l l y d e t e r m i n e both t he c a p a c i t y and the a f f i n i t y o f the b i n d i n g s i t e s . S e v e r a l t r a n s f o r m a t i o n s o f the b i n d i n g d a t a have been p r e s e n t e d t o overcome t h i s problem. The K l o t z r e c i p r o c a l p l o t ( e q u a t i o n 7) i s a n a l o g o u s t o the Lineweaver-Burke p l o t o r the Langmuir a d s o r p t i o n i s o t h e r m . The problem e n c o u n t e r e d w i t h t h i s t r a n s f o r m a t i o n i s t h a t i t p l a c e s more emphasis on the s m a l l e r v a l u e s o f r which p o t e n t i a l l y have the g r e a t e s t amount o f e x p e r i m e n t a l e r r o r [Svensson et al., 1986; K l o t z , 1 9 8 3 ] ] . 1 = 1 + 1 (7) r n[D]Ka n 21 The most w i d e l y used t r a n s f o r m a t i o n o f b i n d i n g d a t a has been t h e S c a t c h a r d t r a n s f o r m a t i o n [ S c a t c h a r d , 1 9 4 9 ] ( E q u a t i o n 8 ) . r = nKa - rKa (8) [D] The use o f t h i s t r a n s f o r m a t i o n has been w i d e s p r e a d , and i n many c a s e s , n e g l e c t i n g t h e assumptions made d u r i n g the d e r i v a t i o n o f t h e S c a t c h a r d p l o t , t h a t : 1. The drug p r o t e i n i n t e r a c t i o n i s r e v e r s i b l e 2. A l l b i n d i n g s i t e s behave as i f they have the same a f f i n i t y . 3. Data must be o b t a i n e d a t e q u i l i b r i u m 4. B i n d i n g o f one m o l e c u l e o f l i g a n d does not n e g a t i v e l y o r p o s i t i v e l y a f f e c t the b i n d i n g o f subsequent m o l e c u l e s o f l i g a n d . 5. No b i n d i n g a r t i f a c t s are p r e s e n t . The t h i r d t r a n s f o r m a t i o n i s the Rosenthal p l o t p r e s e n t e d i n e q u a t i o n 9. f P P l = n [ P t ] K a - [DP]Ka (9) [D] When more than one d i f f e r e n t b i n d i n g s i t e i s p r e s e n t f o r a l i g a n d , t h e s e t r a n s f o r m a t i o n s l o s e t h e i r l i n e a r i t y , and any p o t e n t i a l b e n e f i t i n g r a p h i c a l l y d e t e r m i n i n g the b i n d i n g parameters i s l o s t [ K l o t z , 1982; K l o t z , 1983; B u r g i s s e r , 1984]. T h i s has l e d t o t h e e s t i m a t i o n o f b i n d i n g parameters ( c a p a c i t y and a f f i n i t y ) by n o n - l i n e a r 22 f i t t i n g o f e i t h e r S c a t c h a r d and R o s e n t h a l e q u a t i o n s . The use o f S c a t c h a r d and R o s e n t h a l a n a l y s i s , has shown t o be i n c o r r e c t from the s t a t i s t i c a l s t a n d p o i n t , s i n c e the y o b s e r v a t i o n i s not i n d e p e n d e n t o f t h e x o b s e r v a t i o n [Svensson e t a l . , 1986]. T h a t i s , r a t h e r than h a v i n g y as t h e f u n c t i o n o f x (y = f ( x ) ) t h e R o s e n t h a l and S c a t c h a r d a n a l y s i s have y as a f u n c t i o n o f both x and y (y = f ( y / x ) ) . An a l t e r n a t i v e method i s n o n - l i n e a r c u r v e f i t t i n g o f t h e o r i g i n a l b i n d i n g i s o t h e r m ( e q u a t i o n 5 and 6 ) . U s i n g programs such as LIGAND R, ENZFITTER R, and N0NLIN R, e q u a t i o n s 5 and 6 can be e n t e r e d and t h e d a t a f i t t o the most a p p r o p r i a t e e q u a t i o n . T h i s t r u l y a n a l y z e s t h e dependent vs. the independent parameters t o a r r i v e a t the e s t i m a t i o n o f the b i n d i n g parameters [ B u r g i s s e r , 1984; K l o t z , 1982; K l o t z , 1983]. 1.5 RATIONALE L i k e many o t h e r a n t i a r r h y t h m i c s , PF appears t o d i s p l a y a s t e e p d o s e - r e s p o n s e r e l a t i o n s h i p [ C o n n o l l y e t a l . , 1983b; Siddoway e t a l . , 1984]. T h i s s u g g e s t s t h a t small changes i n PF c o n c e n t r a t i o n may r e s u l t i n d i s p r o p o r t i o n a t e changes i n e f f e c t , which c o u l d l e a d t o t o x i c i t y o r l a c k o f p h a r m a c o l o g i c a l e f f e c t . T h e r e f o r e , f a c t o r s which d i r e c t l y a f f e c t PF c o n c e n t r a t i o n , such as the e l i m i n a t i o n and d i s p o s i t i o n o f PF, must be documented and c a r e f u l l y examined. Propafenone i s h i g h l y bound (-96% within the therapeutic range of 0.5-2.0 ug/ml ) i n serum o f normal h e a l t h y v o l u n t e e r s [Chan et al., 23 1989b]. Moreover, i t has been shown t h a t PF b i n d i n g i n t h e serum i s l a r g e l y dependent on the c o n c e n t r a t i o n o f a - l - a c i d g l y c o p r o t e i n (AAG) [Chan et a l . , 1989b; G i l l e s et a l . , 1985], which may i n c r e a s e 2-3 f o l d i n r e s p o n s e t o trauma and v a r i o u s d i s e a s e s t a t e s [ R o u t l e d g e , 1986]. S i n c e , PF i s c o n s i d e r e d a flow-dependent d r u g , changes i n p r o t e i n b i n d i n g o f PF ( i . e . d i s p l a c e m e n t and a l t e r e d c o n c e n t r a t i o n s o f AAG) would not be e x p e c t e d t o a l t e r the p h a r m a c o k i n e t i c parameters o f PF u n l e s s s i g n i f i c a n t changes i n d i s t r i b u t i o n o c c u r r e d i n r e s p o n s e t o t h e s e changes i n f r e e f r a c t i o n . A l t h o u g h f r e e drug c o n c e n t r a t i o n may i n c r e a s e i n r e s p o n s e t o d i s p l a c e m e n t , o r d e c r e a s e as a r e s u l t o f i n c r e a s i n g AAG c o n c e n t r a t i o n , the t o t a l plasma drug c o n c e n t r a t i o n may a ppear unchanged. T h i s i s i m p o r t a n t c o n s i d e r i n g t h e g e n e r a l l y a c c e p t e d p h a r m a c o l o g i c a l premise t h a t f r e e drug i s u s u a l l y r e s p o n s i b l e f o r p h a r m a c o l o g i c a l a c t i v i t y . T h e r e f o r e , i t seems i m p o r t a n t t o e s t a b l i s h not o n l y t h o s e f a c t o r s which e l i c i t changes i n t o t a l plasma PF c o n c e n t r a t i o n s , but a l s o changes i n plasma f r e e drug c o n c e n t r a t i o n o f PF. I n f o r m a t i o n r e g a r d i n g f a c t o r s c o n t r o l l i n g the c o n c e n t r a t i o n o f f r e e (pharmacologically active) drug i n the plasma would c o n t r i b u t e s u b s t a n t i a l l y t o the u n d e r s t a n d i n g o f the as y e t , u n d e f i n e d plasma c o n c e n t r a t i o n vs. e f f e c t r e l a t i o n s h i p f o r PF. E x p e r i m e n t s u s i n g p e r f u s e d r a b b i t h e a r t s have f u r t h e r d e m o n s t r a t e d t h a t unbound PF l a r g e l y d e t e r m i n e s t h e uptake o f PF i n t o m y o c a r d i a l t i s s u e . I t was shown t h a t PF uptake i n t o c a r d i a c t i s s u e was c l o s e l y c o r r e l a t e d t o the c o n c e n t r a t i o n o f AAG, and t h u s , t h e amount o f f r e e PF i n the p e r f u s a t e [Kates and G i l l e s , 1986]. As the c o n c e n t r a t i o n o f AAG i n c r e a s e d , the uptake o f PF and subsequent 24 p h a r m a c o l o g i c a l a c t i v i t y were reduced [Kates and G i l l e s , 1986]. Thus, the i n f l u e n c e o f p r o t e i n b i n d i n g o f PF may be i m p o r t a n t w i t h r e s p e c t t o changes i n AAG c o n c e n t r a t i o n and d i s p l a c e m e n t o f PF from AAG b i n d i n g s i t e s by o t h e r c o - a d m i n i s t e r e d d r u g s . A s i m i l a r s i t u a t i o n may a l s o e x i s t f o r t h e a c t i v e m e t a b o l i t e , 5-OH-PF, a l t h o u g h t h i s h y p o t h e s i s remains u n t e s t e d . L i p o p r o t e i n c o n c e n t r a t i o n s t e n d t o v a r y s i g n i f i c a n t l y between s u b j e c t s and even w i t h i n a s u b j e c t depending on d i s e a s e and d i e t [Getz and Hay, 1979]. In a d d i t i o n , many g e n e t i c d i s o r d e r s e x i s t where one o r more su b - t y p e o f l i p o p r o t e i n s may be s i g n i f i c a n t l y e l e v a t e d , as i n f a m i l i a l h y p e r l i p i d e m i a s [ B r u n z e l l and M i l l e r , 1981]. Due t o the v a r i a b l e n a t u r e o f l i p o p r o t e i n c o n c e n t r a t i o n , t h e i n f l u e n c e o f f a c t o r s a f f e c t i n g PF and 5-OH-PF b i n d i n g t o l i p o p r o t e i n complexes r e q u i r e s f u r t h e r s t u d y . In a d d i t i o n t o t h e b a s i c s c i e n c e c o n t r i b u t i o n o f t h e p r e s e n t e d work, t h i s s t u d y may p r o v i d e an e s t i m a t e o f the r e l a t i v e p r o p o r t i o n o f th e f r e e d r u g , (PF and 5-OH-PF) a v a i l a b l e f o r d i s t r i b u t i o n i n t o t i s s u e s ( p o s s i b l e s i t e s o f p h a r m a c o l o g i c a l a c t i o n ) a f t e r the a d m i n i s t r a t i o n o f PF. The p r o t e i n p r o f i l e s i n human serum can change as a r e s u l t o f d i s e a s e , s t r e s s , and e n v i r o n m e n t a l f a c t o r s [Kremer e t al., 1988]. Thus, t he e x t e n t t o which PF and 5-OH-PF b i n d t o v a r i o u s serum p r o t e i n s w i l l p r o v i d e u s e f u l i n f o r m a t i o n which may be used t o p r e d i c t b i n d i n g i n c o n d i t i o n s where c o n c e n t r a t i o n s o f b i n d i n g p r o t e i n s i n serum may be a l t e r e d . 25 1.6 AIMS The purpose o f the p r e s e n t e d work i s t o d e t e r m i n e t h e d i f f e r e n c e s i n the c h a r a c t e r i s t i c s o f the d r u g - p r o t e i n i n t e r a c t i o n o f PF and 5-OH-PF i n human serum and t o i s o l a t e d human serum p r o t e i n s by t e s t i n g t h e f o l l o w i n g h y p o t h e s i s : THE IN VITRO PROTEIN BINDING CHARACTERISTICS OF PROPAFENONE ARE DIFFERENT THAN THE BINDING CHARACTERISTICS OF IT'S ACTIVE METABOLITE, 5-HYDROXYPROPAFENONE IN SERUM AND IN SOLUTIONS OF ISOLATED HUMAN SERUM PROTEINS. In o r d e r t o t e s t t he above h y p o t h e s i s , t h e f o l l o w i n g l i s t o f o b j e c t i v e s have been completed: 1. E s t a b l i s h an in vitro method f o r the a c c u r a t e and r e p r o d u c i b l e d e t e r m i n a t i o n o f d r u g - p r o t e i n b i n d i n g o f PF and 5-OH-PF i n serum and i n s o l u t i o n s o f i s o l a t e d serum p r o t e i n s , and t o i d e n t i f y p o s s i b l e s o u r c e s o f e r r o r . 2. P r o v i d e an a c c u r a t e e s t i m a t e o f the b i n d i n g o f the m e t a b o l i t e , 5-OH-PF i n serum w i t h and w i t h o u t the p r e s e n c e o f the p a r e n t compound, pr o p a f e n o n e . 3. Determine t he importance o f AAG, HSA, and l i p o p r o t e i n s i n t h e b i n d i n g o f PF i n the p r e s e n c e o f the m e t a b o l i t e 5-OH-PF, and the b i n d i n g o f 5-OH-PF i n the pr e s e n c e o f PF. 26 4. Examine t he e f f e c t o f probe d i s p l a c e r s such as, d i s o p y r a m i d e and i b u p r o f e n , t o b e t t e r u n d e r s t a n d the b i n d i n g i n t e r a c t i o n o f PF and 5-OH-PF w i t h serum, AAG and HSA. 5. C o n t r a s t t h e b i n d i n g c h a r a c t e r i s t i c s o f PF and 5-OH-PF t o AAG, HSA, HDL, LDL, and VLDL i n b u f f e r , and o f PF and 5-OH-PF i n serum. 6. M a t h e m a t i c a l l y c h a r a c t e r i z e t h e b i n d i n g o f PF and 5-OH-PF t o i n d i v i d u a l b i n d i n g p r o t e i n s , and t o t r y t o model t h e b i n d i n g o f PF and 5-OH-PF i n serum based on the models i d e n t i f i e d f o r t h e i n d i v i d u a l d r u g - p r o t e i n i n t e r a c t i o n s . 7. To c h a r a c t e r i z e t h e uptake o f PF and 5-OH-PF by r e d b l o o d c e l l s . 27 2. EXPERIMENTAL 2.1 MATERIALS AND SUPPLIES 2.1.1 DRUGS, METABOLITES, AND INTERNAL STANDARDS PF h y d r o c h l o r i d e , 5-OH-PF h y d r o c h l o r i d e , L i -1115 h y d r o c h l o r i d e , ( i n t e r n a l s t a n d a r d , I.S. f o r PF q u a n t i t a t i o n ) and L i -1548 h y d r o c h l o r i d e ( I . S . f o r 5-OH-PF q u a n t i t a t i o n ) were s u p p l i e d by K n o l l P h a r m a c e u t i c a l s Canada Inc., Markham, Ont., Canada. I b u p r o f e n and d i s o p y r a m i d e were o b t a i n e d from Sigma Chemical Co., S t . L o u i s , MO., U.S.A. 2.1.2 CHEMICALS AND REAGENTS H e p t a f l u o r o b u t y r i c a n h y d r i d e (HFBA) was p u r c h a s e d from P i e r c e Chemical Co. ( R o c k f o r d , I L . , U.S.A.). American c h e m i c a l s o c i e t y r e a g e n t grade sodium h y d r o x i d e , monopotassium phosphate, d i s o d i u m phosphate, sodium c a r b o n a t e , p o t a s s i u m c a r b o n a t e , g l a c i a l a c e t i c a c i d , c o p p e r s u l f a t e , and h y d r o c h l o r i c a c i d were o b t a i n e d from BDH C h e m i c a l s , T o r o n t o , Ont., Canada. Reagent grade (ACS) t r i c h o l o r a c e t i c a c i d was p u r c h a s e d from J.T. Baker Chemical Co., P h i l l i p s b u r g , NJ., U.S.A. B a r b i t a l b u f f e r pH 8.6, bromophenol b l u e , b e t a -m e r c a p t o e t h a n o l , Sudan red No. 7B, sodium p o t a s s i u m t a r t r a t e , and phenol r e a g e n t were a c q u i r e d from Sigma Chemical Co., S t . L o u i s , MO., U.S.A. Sodium d o e d e c y l s u l f a t e , Coomasie b l u e , a c r y l a m i d e , and N,N' m e t h y l e n e b i s a c r y l a m i d e were o b t a i n e d from B i o Rad L a b o r a t o r i e s , M i s s i s s a u g a , Ont. G l y c e r o l , r e a g e n t grade was o b t a i n e d from Anachema 28 Chemical Co., Richmond, B.C., Canada, and Sudan b l a c k was o b t a i n e d from A l d r i d g e Chemical Co., Milwaukee, WI., U.S.A. 2.1.3 PROTEINS C r y s t a l l i z e d and l y o p h i 1 i z e d e s s e n t i a l l y g l o b u l i n f r e e (<1 %) human serum albumin (A8763), e s s e n t i a l l y (< 0.005%) f a t t y a c i d f r e e , g l o b u l i n f r e e (<1 %) human serum albumin (A3783), a - l - a c i d g l y c o p r o t e i n (G9885), and l i p o p r o t e i n d e f i c i e n t human serum (S5519) were o b t a i n e d from Sigma Chemical Co., S t . L o u i s , MO., U.S.A. High d e n s i t y l i p o p r o t e i n s , low d e n s i t y l i p o p r o t e i n s , and v e r y low d e n s i t y l i p o p r o t e i n s w i t h o u t e t h y l e n e d i a m i n e t e t r a a c e t i c a c i d (EDTA) from human s o u r c e were o b t a i n e d as a s p e c i a l o r d e r from C a l b i o c h e m Co., San Di e g o , CA., U.S.A. Serum u l t r a f i l t r a t e was p r e p a r e d w i t h t he a i d o f an Amicon u l t r a f i l t r a t i o n d e v i c e w i t h an Amicon PM 30 membrane. Normal serum was added t o the u l t r a f i l t r a t i o n d e v i c e , and f i l t e r e d w i t h t he a i d o f p r e s s u r i z e d n i t r o g e n as the d r i v i n g f o r c e f o r f i l t r a t i o n . 2.1.4 SOLVENTS P e s t i c i d e grade t o l u e n e , methanol, hexane, d i c h l o r o m e t h a n e , and i s o p r o p y l a l c o h o l ( d i s t i l l e d i n g l a s s ) were p u r c h a s e d from Caledon L a b o r a t o r i e s L t d . , Georgetown, Ont., Canada. D e i o n i z e d d i s t i l l e d w a t e r was produced i n the l a b o r a t o r y u s i n g a M i l l i - R 0 R Water System ( M i l l i p o r e Corp., B e d f o r d , MA., U.S.A.). 29 2.1.5 GASES U l t r a h i g h p u r i t y (UHP) hydrogen and argon/methane (95:5) were p u r c h a s e d from Matheson Gas P r o d u c t s Canada L t d . , Edmonton, A l t a . , n i t r o g e n , and medic a l a i r U.S.P., from Union C a r b i d e Canada L t d . , T o r o n t o , Ont. 2.1.6 EQUILIBRIUM DIALYSIS CELLS P l e x i - G l a s s ^ d i a l y s i s c e l l s (1.0 mL) were used f o r e q u i l i b r i u m d i a l y s i s . C e l l o p h a n e d i a l y s i s membrane " s a c k s " ( M o l e c u l a r Weight (M.W.) c u t o f f = 12,000 D a l t o n s ) were p u r c h a s e d from Sigma Chemical Co., S t . L o u i s , MO., U.S.A. 2.1.7 ULTRAFILTRATION DEVICES TM U l t r a f i l t r a t i o n d e v i c e s (MPS-1 M i c r o p a r t i t i o n System) were p u r c h a s e d from Amicon Canada L t d . , O a k v i l l e , Ont. Each u l t r a f i l t r a t i o n d e v i c e c o n s i s t e d o f a s i n g l e YMT membrane ( m o l e c u l a r w e i g h t (M.W.) c u t o f f = 30,000 D a l t o n s ) and an 0 - r i n g , s e a l e d between the sample r e s e r v o i r and s u p p o r t base. The r e s e r v o i r was p r o v i d e d w i t h a cap t o m i n i m i z e sample e v a p o r a t i o n and pH change due t o l o s s o f ca r b o n d i o x i d e . A removable f i l t r a t e c o l l e c t i o n cup was a t t a c h e d t o the s u p p o r t base. 30 2.1.8 ELECTROPHORESIS 2.1.8.1 POLYACRYLAMIDE ELECTROPHORESIS GELS P o l y a c r y l a m i d e g e l s were p r e p a r e d by h e a t i n g a m i x t u r e o f a c r y l a m i d e and N,N'-methylene b i s - a c r y l a m i d e ; a c a t a l y s t was then added t o t h e m i x t u r e . S t a c k i n g g e l s which are used t o f o c u s t he p r o t e i n sample b e f o r e s e p a r a t i o n ( 3 . 5 % a c r y l a m i d e c o n t e n t ) , and the r e s o l v i n g g e l s which are used t o s e p a r a t e t he p r o t e i n s on the b a s i s o f m o l e c u l a r w e i g h t ( g r a d i e n t o f 5 t o 20% a c r y l a m i d e ) were poured i n t o t h e g e l mold and a l l o w e d t o c o o l . 2.1.8.2 AGAROSE ELECTROPHORESIS GELS U n i v e r s a l EP F i l m a g a r o s e (470100) UNIV R was purchased from C i b a C o r n i n g D i a g n o s t i c s Corp., P a l o A l t o , CA., U.S.A. 2.1.9 RADIAL IMMUN0 DIFFUSION PLATES N 0 R - P a r t i g e n R AAG r a d i a l i m m u n o d i f f u s i o n (RID) p l a t e s (12-w e l l , volume = 5 / J L per w e l l ) c o n t a i n i n g m o n o s p e c i f i c a n t i s e r u m t o human AAG i n a r e a d y - f o r use a g a r o s e - g e l l a y e r were purchased from Terochem L a b o r a t o r i e s L t d . , Edmonton, A l t a . , Canada. 2.1.10 OTHER SUPPLIES p P y r e x ^ d i s p o s a b l e g l a s s c u l t u r e tubes (15 mL) were 31 p u r c h a s e d from C o r n i n g G l a s s Works, C o r n i n g , NY., U.S.A., and p o l y t e t r a f l u o r o e t h y l e n e (PTFE) l i n e d screw caps from C a n l a b , Vancouver, B.C., Canada. V e n i s y s t e m s ™ B u t t e r f l y R - 1 9 INT c a n n u l a e were p u r c h a s e d from A b b o t t L a b o r a t o r i e s , L t d . , M o n t r e a l , Que., Canada. V a c u t a i n e r R b l o o d c o l l e c t i o n t u bes ( w i t h h e p a r i n and w i t h o u t a d d i t i v e ) were o b t a i n e d from B e c t o n - D i c k i n s o n Canada I n c . , M i s s i s s a u g a , Ont. 2.2 EQUIPMENT 2.2.1 GAS LIQUID CHROMATOGRAPH A bonded-phase f u s e d - s i l i c a c a p i l l a r y column, 25 m x 0.31 mm I n t e r n a l d i a m e t e r , was used f o r a l l gas l i q u i d chromatography (GLC) a n a l y s e s ( s t a t i o n a r y phase, c r o s s - l i n k e d 5% p h e n y l m e t h y l - s i l i c o n e , f i l m t h i c k n e s s 0.52 nm; phase r a t i o 150:1; H e w l e t t - P a c k a r d (HP), P a l o A l t o , CA, U.S.A.). GLC a n a l y s e s were performed on Model 5830A and 5840 HP ( P a l o A l t o , CA., U.S.A.) g a s - l i q u i d c hromatographs, e q u i p p e d w i t h Model 18835B c a p i l l a r y i n l e t systems, * ^Ni e l e c t r o n - c a p t u r e d e t e c t o r s , Model 18850A GC t e r m i n a l s f o r peak i n t e g r a t i o n , and Model 7671A a u t o m a t i c s a m p l e r s . A s p l i t l e s s i n j e c t i o n mode was used, employing a f u s e d - s i l i c a i n l e t l i n e r w i t h a small p l u g o f s i l a n i z e d g l a s s - w o o l 3 cm from t he column end. Thermogreen R LB-2 s e p t a ( S u p e l c o , I n c . , B e l l e f o n t e , PA., U.S.A.), l o w - b l e e d a t h i g h i n l e t t e m p e r a t u r e s , were used. The septum was changed r o u t i n e l y t o p r e v e n t l e a k a g e r e s u l t i n g 32 from r e p e a t e d p u n c t u r i n g d u r i n g a u t o m a t i c i n j e c t i o n . 2.2.2 SPECTROPHOTOMETER A H e w l e t t Packard 8452A d i o d e a r r a y s p e c t r o p h o t o m e t e r e q u i p p e d w i t h a H e w l e t t Packard V e c t r a R computer i n t e r f a c e was used f o r a l l s p e c t r o p h o t o m e t r i c measurements. 2.2.3 ELECTROPHORESIS EQUIPMENT A P r o t e i n I I R e l e c t r o p h o r e s i s tank w i t h a model 1000/500 power s u p p l y was used t o run the p o l y a c r y l a m i d e g e l s ( B i o Rad L a b o r a t o r i e s , M i s s i s s a u g a , Ont., Canada). A C i b a C o r n i n g e l e c t r o p h o r e s i s power s u p p l y and g e l s u p p o r t were used t o run t h e a g a r o s e g e l s . 2.2.4 OTHER Ot h e r equipment used i n c l u d e s : Eppendorf m i c r o p i p e t t e s , a V o r t e x - G e n i e ^ mixer ( F i s h e r S c i e n t i f i c Co., S p r i n g f i e l d , MA., U.S.A.), a pH meter and e l e c t r o d e ( F i s h e r S c i e n t i f i c Co., S p r i n g f i e l d , MA., U.S.A.), a Model 415-110 Labq u a k e R r o t a r y shaker ( L a b i n d u s t r i e s , B e r k l e y , CA., U.S.A.), an i n c u b a t i o n oven ( I s o t e m p R model 350, F i s h e r S c i e n t i f i c I n d u s t r i e s , S p r i n g f i e l d , MA., U.S.A.), an IEC model 2K c e n t r i f u g e (Damon/IEC D i v i s i o n , Needham Hts, MA., U.S.A.), and a B e h r i n g w e r k e m e a s u r i n g v i e w e r (Behringwerke AG, Marburg, West Germany). A model AE 163 b a l a n c e was used t o weigh a l l c h e m i c a l s , d r u g s , and r e q u i r e d p r o t e i n s ( M e t t l e r Instrument Co. H i g h t s t o w n , NJ., 33 U.S.A.). An Advanced Wide Range Osmometer 3WII (Advanced I n s t r u m e n t s In c . , Needham H e i g h t s , MI. U.S.A.) was used t o measure the o s m o l a r i t y o f t h e s t o c k s o l u t i o n s . 2.3 PREPARATION OF STOCK AND REAGENT SOLUTIONS 2.3.1 DRUGS, METABOLITES, AND INTERNAL STANDARDS PF h y d r o c h l o r i d e was a c c u r a t e l y weighed and d i s s o l v e d i n d e i o n i z e d d i s t i l l e d w ater. S e r i a l d i l u t i o n s were used t o a r r i v e a t th e f i n a l c o n c e n t r a t i o n o f 100 ng/mL f r e e base (-11.07 mg o f PF h y d r o c h l o r i d e i s e q u i v a l e n t t o 10 mg o f PF f r e e b a s e ) . The m e t a b o l i t e , 5-OH-PF h y d r o c h l o r i d e , was a c c u r a t e l y weighed and d i s s o l v e d i n a m i x t u r e o f methanol : d e i o n i z e d d i s t i l l e d w ater ( 1 : 9 ) . S e r i a l d i l u t i o n s were used t o a r r i v e a t a f i n a l c o n c e n t r a t i o n o f 100 ng/mL f r e e base (-11.02 mg o f 5-OH-PF h y d r o c h l o r i d e i s e q u i v a l e n t t o 10 mg o f 5-OH-PF f r e e b a s e ) . Li-1115 h y d r o c h l o r i d e was a c c u r a t e l y weighed and d i s s o l v e d i n d e i o n i z e d d i s t i l l e d water. S e r i a l d i l u t i o n s were made t o a r r i v e a t a f i n a l c o n c e n t r a t i o n o f 200 ng/mL f r e e base (-11.11 mg o f L i - 1 1 1 5 h y d r o c h l o r i d e i s e q u i v a l e n t t o 10 mg o f L i -1115 f r e e b a s e ) . L i -1548 h y d r o c h l o r i d e was a c c u r a t e l y weighed and d i s s o l v e d i n d e i o n i z e d water. U s i n g s e r i a l d i l u t i o n s t o g i v e a f i n a l c o n c e n t r a t i o n o f 200 mg/mL f r e e base (-11.06 mg o f L i 1548 h y d r o c h l o r i d e i s e q u i v a l e n t t o 10 mg o f L i -1548 f r e e b a s e ) . A l l s t o c k and d i l u t e d s o l u t i o n s were p r o t e c t e d from s u n l i g h t by wrapping t he g l a s s c o n t a i n e r s i n aluminum f o i l and were s t o r e d them a t 4 °C a f t e r p r e p a r a t i o n , f o r up t o f o u r months. 2.3.2 REAGENTS AND SOLUTIONS T r i e t h y l a m i n e (0.003 M) was p r e p a r e d by d i l u t i n g t r i e t h y l a m i n e w i t h t o l u e n e . Four o r f i v e p e l l e t s o f NaOH were added t o t h e s o l u t i o n . Sodium h y d r o x i d e (NaOH) 1 M and 5 M s o l u t i o n s were p r e p a r e d by d i s s o l v i n g NaOH p e l l e t s i n d e i o n i z e d d i s t i l l e d water. Sodium c a r b o n a t e (Na2C03) 0.1 M s o l u t i o n and p o t a s s i u m c a r b o n a t e (K2CO3) 5 M s o l u t i o n were p r e p a r e d by d i s s o l v i n g Na2C03 and K2CO3 powder i n d e i o n i z e d d i s t i l l e d water, r e s p e c t i v e l y . H y d r o c h l o r i c a c i d (HCL) 1 M was p r e p a r e d by d i l u t i n g ACS r e a g e n t grade c o n c e n t r a t e d (37% W/W) HCL i n d e i o n i z e d d i s t i l l e d water. Phosphate b u f f e r (pH 7.4) was p r e p a r e d u s i n g t h e f o l l o w i n g p r o c e d u r e s . Monopotassium phosphate (KH2PO4, 2.69 G ), d i s o d i u m p h osphate (Na 2 H P 0 4 , 11.40 G), and sodium c h l o r i d e (NaCl 1.62 G) were a c c u r a t e l y weighed and d i s s o l v e d i n d e i o n i z e d d i s t i l l e d water t o a f i n a l volume o f 1000 mL to y i e l d a phosphate b u f f e r o f 0.10 M. The pH 35 o f t h e f i n a l s o l u t i o n was checked and a d j u s t e d , i f n e c e s s a r y , t o 7.4 by u s i n g a l i q u o t s o f 1.0 M NaOH o r HC1. Phosphate b u f f e r (pH 6.0) was p r e p a r e d by d i s s o l v i n g 2.28 g o f KH2PO4 i n d e i o n i z e d d i s t i l l e d water t o a volume o f 250 mL ( s o l u t i o n 1) and 0.95 g Na 2HP0 4 t o a volume o f 100 mL ( s o l u t i o n 2 ) . The f i n a l b u f f e r s o l u t i o n was p r e p a r e d by com b i n i n g s o l u t i o n s 1 and 2 (90 mL o f s o l u t i o n 1 and 10 mL o f s o l u t i o n 2 ) . Sodium c a r b o n a t e (2 g) was d i s s o l v e d i n 100 mL o f 0.1 M NaOH t o y i e l d a 2% sodium c a r b o n a t e s o l u t i o n i n 0.1 M NaOH. Copper s u l f a t e (CuS04'5H20; 1 g) and sodium p o t a s s i u m t a r t r a t e (2 g) were d i s s o l v e d i n 100 mL d i s t i l l e d d e i o n i z e d w a t e r . Phenol r e a g e n t was d i l u t e d 1:1 w i t h d i s t i l l e d d e i o n i z e d water a t each use. 2.3.3 PROTEIN SOLUTIONS Human serum albumin (HSA) and e s s e n t i a l l y (< 0.005%) f r e e f a t t y a c i d f r e e albumin (4.5 g) were d i s s o l v e d i n 100 mL o f pH 7.4 i s o t o n i c phosphate b u f f e r t o y i e l d a f i n a l c o n c e n t r a t i o n o f 45 mg/mL which would approximate p h y s i o l o g i c a l c o n c e n t r a t i o n s o f HSA. a - l - a c i d g l y c o p r o t e i n (AAG) (0.09 g) was d i s s o l v e d i n 100 mL o f pH 7.4 i s o t o n i c p hosphate b u f f e r t o y i e l d a f i n a l c o n c e n t r a t i o n o f 0.9 mg/mL which would approximate p h y s i o l o g i c a l c o n c e n t r a t i o n s o f a - l - a c i d g l y c o p r o t e i n . High d e n s i t y l i p o p r o t e i n s (HDL), low d e n s i t y l i p o p r o t e i n s 36 (L D L ) , and v e r y low d e n s i t y l i p o p r o t e i n s (VLDL) i n i s o t o n i c pH 7.2 Krebs b u f f e r were d i l u t e d from the o r i g i n a l c o n c e n t r a t i o n w i t h 0.1 M phosphate pH 7.4 b u f f e r t o y i e l d f i n a l p h y s i o l o g i c a l c o n c e n t r a t i o n s o f 3.0 mg/mL, 3.0 mg/mL, and 1.5 mg/mL o f HDL, LDL, and VLDL, r e s p e c t i v e l y . B o v i n e serum albumin (BSA) 100 mg was d i s s o l v e d i n 100 mL o f d i s t i l l e d d e i o n i z e d water t o p r o v i d e a f i n a l c o n c e n t r a t i o n o f 1 mg/mL. 2.3.4 ESTABLISHED TECHNIQUES USED IN RESEARCH 2.3.4.1 GAS LIQUID CHROMATOGRAPHY OF PROPAFENONE AND 5-HYDR0XYPR0PAFEN0NE S e n s i t i v e and s e l e c t i v e c a p i l l a r y gas l i q u i d c h r o m a t o g r a p h i c t e c h n i q u e s employing e l e c t r o n c a p t u r e d e t e c t i o n [Chan et al., 1988; Chan e t al., 1989a] were used t o q u a n t i f y PF and 5-OH-PF. The PF assay employed a l i q u i d - l i q u i d e x t r a c t i o n u s i n g t o l u e n e as the e x t r a c t i o n s o l v e n t . F o l l o w i n g e x t r a c t i o n , PF samples were e v a p o r a t e d under a g e n t l e stream o f n i t r o g e n gas a t 40 °C. The samples were then r e c o n s t i t u t e d w i t h t r i e t h y l a m i n e i n t o l u e n e , and d e r i v a t i z e d w i t h h e p t a f l u r o b u t y r i c a n h y d r i d e . These d e r i v a t i z e d samples were then i n j e c t e d i n t o t h e gas chromatograph and q u a n t i f i e d . The 5-OH-PF a s s a y employed s i m i l a r t e c h n i q u e s , but d i f f e r e d i n the e x t r a c t i o n s o l v e n t [ t o l u e n e 7 : methylene c h l o r i d e 3 : i s o p r o p y l a l c o h o l 1 vs. t o l u e n e ] and the bases [ 0.1 M sodium c a r b o n a t e vs. 1.0 37 M NaOH, and 5 M p o t a s s i u m c a r b o n a t e vs. 5 M NaOH] used. 2.3.4.2 LOWRY PROTEIN ASSAY The Lowry p r o t e i n a s s a y method was used t o q u a n t i f y p r o t e i n s p r e s e n t i n samples used i n the dru g p r o t e i n b i n d i n g e x p e r i m e n t s . A s t a n d a r d c u r v e u s i n g b o v i n e serum albumin (BSA) was p r e p a r e d (0 - 100 /xg/0.1 mL BSA). The a p p r o p r i a t e d i l u t i o n s o f t h e p r o t e i n samples were made and 0.1 mL o f t h e s e s o l u t i o n s were p i p e t t e d i n t o a c l e a n c u l t u r e t u b e . A 1.0 mL a l i q u o t o f 0.01% CuSO^Sr^O, 0.02% sodium p o t a s s i u m t a r t r a t e , 2.0% NaC0 3 i n 0.1 M NaOH was added, then t h e s o l u t i o n was v o r t e x e d . F o l l o w i n g a 15 minute i n c u b a t i o n s t e p at room t e m p e r a t u r e , 0.1 mL o f a 1.0 M phenol s o l u t i o n was added and t h e s o l u t i o n i m m e d i a t e l y v o r t e x e d . T h i s m i x t u r e was a l l o w e d t o s t a n d a t room t e m p e r a t u r e f o r 30 m i n u t e s . The samples were then p i p e t t e d i n t o o p t i c a l g l a s s c u v e t t e s and r e a d a t 650 nm on the s p e c t r o p h o t o m e t e r . 2.3.4.3 POLYACRYLAMIDE GEL ELECTROPHORESIS P r e p a r e d g e l s were p l a c e d i n t o the e l e c t r o p h o r e s i s tank, and submerged i n e l e c t r o p h o r e s i s b u f f e r (0.1 M sodium phosphate b u f f e r pH 7.0 w i t h 1% sodium d o d e c y l s u l f a t e ) . A p p r o p r i a t e amounts o f p r o t e i n (25.0 ng) were p i p e t t e d i n t o sample b u f f e r (20 p a r t s [0.5 M phosphate b u f f e r pH 7.0, 1.0% sodium d o d e c y l s u l f a t e , and 40% G l y c e r o l ] : 5 p a r t s [bromophenol b l u e s o l u t i o n ] : and 5 p a r t s [ b e t a -m e r c a p t o e t h a n o l s o l u t i o n ] ) , t h e samples were then h e a t e d f o r 90 38 s e c o n d s , c o o l e d , and c a r e f u l l y a p p l i e d t o the s t a c k i n g g e l . The g e l s were run at a c o n s t a n t c u r r e n t o f 25 mA u n t i l t h e t r a c k i n g dye was a p p r o x i m a t e l y 2 cm from the bottom o f the g e l . The samples were then removed from t he g e l a p p a r a t u s , and p l a c e d i n s t a i n i n g s o l u t i o n (0.25% Coomassie B r i l l i a n t B lue R250, 45% methanol, 9% g l a c i a l a c e t i c a c i d , and 45% d i s t i l l e d w a t e r ) . A f t e r 1.0 hour i n the s t a i n i n g s o l u t i o n , t h e g e l was p l a c e d i n t o t h e f i r s t d e s t a i n i n g s o l u t i o n ( 4 5 % methanol, 10% g l a c i a l a c e t i c a c i d , and 45% d i s t i l l e d water) and g e n t l y mixed. A f t e r 1.0 hour t he g e l s were p l a c e d i n t o t h e second d e s t a i n i n g s o l u t i o n ( 2 0 % methanol, 5% a c e t i c a c i d , and 75% d i s t i l l e d w a t e r ) . The g e l remained i n t h i s s o l u t i o n f o r 24 hours u n t i l t h e background o f the g e l was r e l a t i v e l y c l e a r . 2.3.4.4 LIPOPROTEIN AGAROSE ELECTROPHORESIS A l i q u o t s o f 5.0 ul o f sample l i p o p r o t e i n s o l u t i o n s were a p p l i e d i n t o the p r e p a r e d w e l l s on the agarose g e l a l o n g w i t h one c o n t r o l l a n e c o n t a i n i n g o n l y bromophenol b l u e . The p l a t e was then put i n t o t h e e l e c t r o p h o r e s i s a p p a r a t u s , and the b u f f e r w e l l s f i l l e d w i t h 0.05 M b a r b i t a l pH 8.5 b u f f e r . E l e c t r o p h o r e s i s was c a r r i e d out f o r 20 minu t e s a t a c o n s t a n t v o l t a g e o f 90 v o l t s . The a g a r o s e p l a t e was then removed and washed f o r 5 minutes w i t h 1% g l y c e r o l i n 55% e t h a n o l . A f t e r t h e g l y c e r o l wash, the p r o t e i n s were f i x e d a t 70-80 °C f o r 1.5 t o 2 h o u r s . F o l l o w i n g t h i s , t h e p l a t e s were c o o l e d and s t a i n e d f o r a p p r o x i m a t e l y 10 minutes w i t h a s t a i n e r (200 mL s t o c k s o l u t i o n [225 mg F l a t B Red i n 946 mL e t h a n o l ] , 40 mL 0.1 M NaOH, and 8 drops o f T r i t o n X-100). F o l l o w i n g s t a i n i n g , p l a t e s were r i n s e d i n 2% g l y c e r o l f o r 30 39 s e c o n d s , and d r i e d a t 70-80 °C f o r 15 m i n u t e s . 2.4 PROTEIN BINDING OF 5-HYDROXYPROPAFENONE AND PROPAFENONE 2.4.1 PRELIMINARY EXPERIMENTS 2.4.1.0 PREPARATION OF EQUILIBRIUM DIALYSIS DEVICES C e l l o p h a n e d i a l y s i s membranes were immersed i n b o i l i n g d i s t i l l e d w a t er f o r 1 hour, and then soaked i n i s o t o n i c phosphate b u f f e r (pH 7.4) o v e r n i g h t at 4 °C. The membranes were then c u t and mounted on t h e p l a s t i c e q u i l i b r i u m d i a l y s i s a p p a r a t u s . Extreme c a r e was t a k e n t o a v o i d c o n t a c t o f the membrane w i t h p o s s i b l e c o n t a m i n a t i n g s u r f a c e s ( f i n g e r s , l a b bench, d i r t y t o o l s , e t c . ) . A f t e r placement o f t h e membrane u s i n g f o r c e p s , the two h a l v e s o f the matched c e l l s were the n t i g h t l y f a s t e n e d t o g e t h e r t o a v o i d l e a k a g e d u r i n g t h e d i a l y s i s e x p e r i m e n t s . Equal volumes o f the d e s i r e d s o l u t i o n s (serum, b u f f e r , o r p r o t e i n s o l u t i o n s ) were then added t o both s i d e s o f the membrane, and the c e l l s s e a l e d . The c e l l s were then immersed and r o t a t e d a t 14 rpm i n the water bath a t 37 °C f o r e i g h t h o u r s . F o l l o w i n g d i a l y s i s , both b u f f e r and serum were t r a n s f e r r e d t o c l e a n t e s t t u b e s , and f r o z e n . 2.4.1.1 TIME TO EQUILIBRIUM I s o t o n i c phosphate b u f f e r (pH 7.4) c o n t a i n i n g 0.5 /zg/mL 5-OH-PF was d i a l y z e d a g a i n s t an equal volume (0.8 mL) o f b l a n k serum at 40 37 °C f o r 2, 4, 6, 8, and 10 hou r s . The c o n c e n t r a t i o n o f 5-OH-PF i n th e b u f f e r and serum s i d e o f the c e l l was measured, u s i n g a s e n s i t i v e and s e l e c t i v e GLC-ECD method. The f r e e f r a c t i o n was then p l o t t e d a g a i n s t t i m e . E q u i l i b r a t i o n was e s t a b l i s h e d when the p l o t o f f r e e f r a c t i o n r e a c h e d a p l a t e a u ( i e . the f r e e f r a c t i o n f a i l s t o d e c r e a s e f u r t h e r ) . The time t o a t t a i n e q u i l i b r i u m was e v a l u a t e d a t low (0.1 /ig/mL) and h i g h (45.0 ug/ml) c o n c e n t r a t i o n s o f 5-OH-PF. Measurements o f pH were made i n both t he serum and the b u f f e r f o l l o w i n g d i a l y s i s . 2.4.1.2.1 NON-SPECIFIC BINDING OF 5-HYDROXYPROPAFENONE TO EQUILIBRIUM DIALYSIS CELLS AND MEMBRANES. The e x t e n t o f a d s o r p t i o n o f 5-OH-PF t o the s u r f a c e o f t h e e q u i l i b r i u m d i a l y s i s a p p a r a t u s was dete r m i n e d by measuring t he c o n c e n t r a t i o n o f 5-OH-PF s p i k e d b u f f e r b e f o r e and a f t e r d i a l y s i s . F o l l o w i n g d i a l y s i s a t 37 °C f o r 8 hours, a l i q u o t s o f b u f f e r were removed, a n a l y z e d by the above d e s c r i b e d GLC-ECD method, and compared t o u n d i a l y z e d s p i k e d b u f f e r . N o n - s p e c i f i c b i n d i n g e x p e r i m e n t s were c o n d u c t e d a t 0.1, 0.2, 0.5, 1.0, 5.0, and 25.0 ug/ml. The p e r c e n t a g e o f n o n - s p e c i f i c a d s o r p t i o n was then c a l c u l a t e d by e q u a t i o n 10: % NON-SPECIFIC BINDING = (CONC. BEFORE - CONC.AFTER) (10) (C0NC. BEFORE) where CONC. BEFORE and CONC. AFTER are the c o n c e n t r a t i o n s o f 5-OH-PF o f s p i k e d b u f f e r b e f o r e and a f t e r d i a l y s i s , r e s p e c t i v e l y . 41 2.4.1.2.2 NON-SPECIFIC BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE TO AMICON MPS-1 ULTRAFILTRATION DEVICES. I s o t o n i c phosphate pH 7.4 b u f f e r s p i k e d w i t h PF and 5-OH-PF was added t o t h e u l t r a f i l t r a t i o n d e v i c e s . The d e v i c e s were then capped and p l a c e d i n t o an u l t r a c e n t r i f u g e , e q u i l i b r a t e d t o a t e m p e r a t u r e o f 25 °C, and spun f o r 20 minutes a t 2000 g a t a f i x e d a n g l e o f 1 8 ° . A l i q u o t s from the u l t r a f i l t r a t i o n cup were a n a l y z e d f o r both PF and 5-OH-PF. The p e r c e n t a g e l o s s was c a l c u l a t e d u s i n g e q u a t i o n 1, where CONC. BEFORE and CONC. AFTER a r e the c o n c e n t r a t i o n s o f s p i k e d b u f f e r b e f o r e and a f t e r u l t r a f i l t r a t i o n . 2.4.3 pH DEPENDENT BINDING OF PF AND 5-HYDROXYPROPAFENONE 2.4.3.1 pH DEPENDENT BINDING OF PROPAFENONE IN SERUM I s o t o n i c phosphate b u f f e r s r a n g i n g from a pH o f 6.0, 7.0, 7.2, 7.4, 7.6, 7.8, 8.0, t o 9.0 were p r e p a r e d and s p i k e d w i t h 5-OH-PF or PF t o a r r i v e a t a f i n a l c o n c e n t r a t i o n o f 0.2 /jg/mL. The pH o f the serum was a d j u s t e d by the d r o p - w i s e a d m i n i s t r a t i o n o f 0.1 N sodium h y d r o x i d e , o r 0.1 N h y d r o c h l o r i c a c i d . The pH o f both t h e b u f f e r and serum s o l u t i o n s were measured b e f o r e and a f t e r d i a l y s i s a t 37 °C f o r 8 h o u r s . F o l l o w i n g d i a l y s i s , a l i q u o t s o f both the b u f f e r and serum compartments o f t h e d i a l y s i s c e l l were removed and a s s a y e d f o r 5-OH-PF o r PF by GLC-ECD. The f r e e f r a c t i o n o f PF and 5-0H-PF i n serum was p l o t t e d a g a i n s t the serum pH a f t e r d i a l y s i s . T o t a l serum p r o t e i n was 42 a l s o measured u s i n g a Lowry p r o t e i n a s s a y method d e s c r i b e d above. 2.4.3.2 pH DEPENDENT BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE IN ISOLATED PROTEIN SOLUTIONS (ALBUMIN AND AAG) The pH dependent b i n d i n g o f PF and 5-OH-PF t o HSA and AAG was e s t a b l i s h e d . Both AAG and HSA were d i s s o l v e d i n i s o t o n i c phosphate b u f f e r o f v a r y i n g pH (7.0, 7.2, 7.4, 7.6, and 7.8) a t t h e p h y s i o l o g i c a l c o n c e n t r a t i o n s d e s c r i b e d i n s e c t i o n 2.3.3. These s o l u t i o n s were then s p i k e d w i t h PF o r 5-OH-PF t o r e s u l t i n c o n c e n t r a t i o n s o f 2.0 ug/ml and 0.5 ug/ml, r e s p e c t i v e l y . The p r o t e i n s o l u t i o n s were then d i a l y z e d a g a i n s t b l a n k i s o t o n i c phosphate b u f f e r o f a c o r r e s p o n d i n g pH. A f t e r d i a l y s i s was co m p l e t e d , samples were removed, pH measured, and the p o s t - d i a l y s i s b u f f e r and p r o t e i n compartments a s s a y e d f o r both PF and 5-OH-PF. The f r e e f r a c t i o n was c a l c u l a t e d and p l o t t e d as a f u n c t i o n o f pH. T o t a l p r o t e i n and c o n c e n t r a t i o n o f AAG were measured by the Lowry method and r a d i a l i m m u n o d i f f u s i o n p l a t e s , r e s p e c t i v e l y . 2.4.3.3 pH PARTITION COEFFICIENT IN TOLUENE AND HEXANE OF PROPAFENONE AND 5-HYDROXYPROPAFENONE. B u f f e r s were p r e p a r e d a t v a r i o u s pH (7.0, 7.2, 7.4, 7.6, 7.8, and 8.0) s p i k e d w i t h e i t h e r PF o r 5-OH-PF. A l i q u o t s o f s p i k e d b u f f e r (1.5 mL) were added t o 5 mL o f e i t h e r hexane o r t o l u e n e i n a c l e a n t e s t t u b e . These tubes were then capped and mixed f o r 20 m i n u t e s . F o l l o w i n g m i x i n g , o r g a n i c and aqueous l a y e r s were s e p a r a t e d , 43 and t r a n s f e r r e d i n t o c l e a n t e s t t u b e s . PF and 5-OH-PF were measured i n b o t h t h e o r g a n i c , and aqueous l a y e r s . The d i s a p p e a r a n c e o f drug from t h e aqueous l a y e r was p l o t t e d vs. pH f o r t h e r e s p e c t i v e s o l v e n t and d r u g . 2.4.4 BUFFER STRENGTH I s o t o n i c phosphate b u f f e r s a t v a r i o u s i o n i c s t r e n g t h s were p r e p a r e d (0.067, 0.100, 0.150 M). Each b u f f e r was s p i k e d t o a l l o w a f i n a l c o n c e n t r a t i o n o f 0.5 /ig/mL 5-OH-PF. Equal volume (0.8 mL) a l i q u o t s o f b u f f e r and serum were a p p l i e d t o o p p o s i t e s i d e s o f the d i a l y s i s a p p a r a t u s , and then d i a l y z e d as d e s c r i b e d above. A f t e r 8 hours o f d i a l y s i s , a l i q u o t s o f both t h e serum and b u f f e r compartments were a s s a y e d f o r 5-OH-PF. The f r e e f r a c t i o n s ( f r e e / t o t a l drug c o n c e n t r a t i o n ) were compared a t v a r i o u s b u f f e r s t r e n g t h s u s i n g a non-p a r a m e t r i c t e s t (Mann Whitney U-Test) w i t h a s i g n i f i c a n c e l e v e l o f p=0.05. 2.4.5 CHEMICAL DEGRADATION OF 5-HYDROXYPROPAFENONE DURING DIALYSIS A l i q u o t s o f serum and b u f f e r were s p i k e d w i t h 5-OH-PF t o y i e l d a f i n a l c o n c e n t r a t i o n o f 0.5 jug/mL. A sample was removed b e f o r e i n c u b a t i o n t o s e r v e as a c o n t r o l sample. The r e s t o f the samples were i n c u b a t e d f o r 1, 2, 4, 7.4, and 8 hours a t 37 °C. A l i q u o t s from t h e v a r i o u s samples were removed and assayed f o r 5-OH-PF. The c o n c e n t r a t i o n s o f 5-OH-PF were then compared t o c o n c e n t r a t i o n o f 5-OH-PF from t h e sample removed b e f o r e t h e i n c u b a t i o n , u s i n g the K r u s k a l l -44 W a l l i s t e s t w i t h a s i g n i f i c a n c e l e v e l o f p=0.05. 2.4.6 VOLUME SHIFTS An a l i q u o t (0.8 mL) o f b u f f e r and an equal volume o f serum were added t o the d i a l y s i s c e l l . T h i s was then d i a l y z e d f o r 8 hours at 37° C. F o l l o w i n g t h e d i a l y s i s , t h e t o t a l volume on each s i d e o f the c e l l was removed, u s i n g a 1.0 mL Ham i l t o n s y r i n g e . The volumes on each s i d e o f the c e l l were c a r e f u l l y measured, and compared t o t h e volume p r i o r t o d i a l y s i s ( a c o r r e c t i o n f o r t he dead volume i n t h e n e e d l e was made b e f o r e c o m p a r i s o n ) . These r e s u l t s were then compared t o a c o n t r o l e x p e r i m e n t i n which o n l y b u f f e r was added t o both s i d e s o f t h e e e l 1. 2.4.7 OSMOLARITY OF 5-HYDROXYPROPAFENONE SOLUTIONS The o s m o l a r i t y o f phosphate pH 7.4 b u f f e r s p i k e d w i t h v a r i o u s c o n c e n t r a t i o n s o f 5-OH-PF (0.1 t o 45 /xg/mL) was d e t e r m i n e d . 2.4.8 LOSS OF PROTEIN DURING DIALYSIS Measurements o f t o t a l p r o t e i n and AAG i n serum, measurements o f t o t a l p r o t e i n i n an albumin s o l u t i o n , and measurements o f AAG i n a s o l u t i o n o f AAG were made b e f o r e and a f t e r 8 hours o f d i a l y s i s a t 37 °C t o a s s e s s t h e l o s s o f p r o t e i n s d u r i n g e q u i l i b r i u m d i a l y s i s. 45 2.4.9 TEST FOR DIALYSIS MEMBRANE INTEGRITY FOLLOWING DIALYSIS To d e t e r m i n e t h e i n t e g r i t y o f t h e d i a l y s i s c e l l membranes f o l l o w i n g d i a l y s i s , s m a l l a l i q u o t s o f p o s t - d i a l y s i s b u f f e r were added t o a s o l u t i o n o f 3% t r i c h l o r o a c e t i c a c i d . I f p r e c i p i t a t i o n was ob s e r v e d t h e samples were d i s c a r d e d due t o t h e p r e s e n c e o f p r o t e i n i n th e b u f f e r . 2.5 BINDING OF 5-HYDROXYPROPAFENONE IN HUMAN SERUM 2.5.1 BINDING IN NORMAL SERUM Bloo d from seven h e a l t h y male E u r o p i d s was c o l l e c t e d i n t o r e d t op v a c u t a i n e r s (no a d d i t i v e s ) from t h e median c u b i t a l v e i n w i t h t h e a i d o f an i n d w e l l i n g B u t t e r f l y - 1 9 R I n t c a n n u l a . Extreme c a r e was tak e n t o a v o i d c o n t a c t o f the b l o o d w i t h t h e r e d p l a s t i c s t o p p e r . The b l o o d was a l l o w e d t o c l o t by s t a n d i n g at room t e m p e r a t u r e f o r 30 m i n u t e s . The b l o o d was then c e n t r i f u g e d f o r 15 minutes at 2000 rpm a t a f i x e d a n g l e o f 20 ° . The serum was then c a r e f u l l y removed w i t h b o r o s i l i c a t e p i p e t t e s , and t r a n s f e r r e d t o c l e a n t e s t t u b e s . The t o t a l p r o t e i n c o n c e n t r a t i o n and the c o n c e n t r a t i o n o f AAG was de t e r m i n e d from r e p r e s e n t a t i v e samples from each v o l u n t e e r . The tubes were capped w i t h p o l y t e t r a f l u o r o e t h y l e n e l i d s and f r o z e n f o r a maximum o f one week b e f o r e use. A l i q u o t s o f serum were then thawed and added t o the serum s i d e o f t h e e q u i l i b r i u m d i a l y s i s a p p a r a t u s . S p i k e d b u f f e r was added t o t h e o t h e r s i d e o f t h e d i a l y s i s c e l l a t c o n c e n t r a t i o n s o f 0.1, 0.2, 0.5, 1.0, 5.0, 10.0, 25.0, and 45 ng/mi. The c e l l s were then s e a l e d , 46 p l a c e d i n a water bath a t 37 °C, and r o t a t e d f o r 8 hours a t 14 rpm. A f t e r t h e d i a l y s i s had been completed, a l i q u o t s o f both b u f f e r and serum were removed and a n a l y z e d f o r 5-OH-PF w i t h GLC-ECD. The f r e e f r a c t i o n o f 5-OH-PF a t t o t a l drug c o n c e n t r a t i o n s ( t h e c o n c e n t r a t i o n i n th e serum s i d e o f the d i a l y s i s c e l l ) was r e p o r t e d . 2.5.2 ROSENTHAL PLOTS OF 5-HYDROXYPROPAFENONE BINDING IN SERUM Data o b t a i n e d from the above s e t o f e x p e r i m e n t s was p l o t t e d by the method o f Rosenthal ( e . g . b i n d i n g r a t i o [ b o u n d / f r e e ] vs. c o n c e n t r a t i o n o f bound d r u g ) . The computer program ENZFITTER^ ( E l s e v i e r - B I O S O F T ) was used t o det e r m i n e the b e s t n o n - l i n e a r r e g r e s s i o n f i t from t h e unt r a n s f o r m e d d a t a (Bound drug vs. F r e e d r u g ) . From t h i s i n f o r m a t i o n , b i n d i n g parameters f o r 5-OH-PF such as number o f b i n d i n g s i t e s , a s s o c i a t i o n c o n s t a n t s , and the b i n d i n g c a p a c i t y o f serum p r o t e i n s c o u l d be d e t e r m i n e d . 2.5.3 PROTEIN BINDING OF 5-HYDROXYPROPAFENONE IN SERUM DETERMINED BY EQUILIBRIUM DIALYSIS IN THE PRESENCE OF PROPAFENONE The e f f e c t o f the a d d i t i o n o f PF on 5-OH-PF serum b i n d i n g was a s s e s s e d by measuring t h e b i n d i n g o f 5-OH-PF i n serum from h e a l t h y male v o l u n t e e r s s p i k e d w i t h PF (2.0 /Kj/mL) and 5-OH-PF (0.5 /zg/mL). A l i q u o t s o f s p i k e d serum were added to the d i a l y s i s c e l l s and d i a l y z e d f o r 8 hours a t 37 °C a g a i n s t b l a n k b u f f e r . F o l l o w i n g d i a l y s i s , a l i q u o t s o f both serum and b u f f e r were a n a l y z e d f o r PF and 5-OH-PF, u s i n g t h e GLC-ECD method d e s c r i b e d p r e v i o u s l y . The f r e e f r a c t i o n o f 47 5-OH-PF i n t h e p r e s e n c e o f PF was c a l c u l a t e d and compared t o t h e c o n t r o l f r e e f r a c t i o n o f 5-OH-PF a l o n e , u s i n g t h e n o n - p a r a m e t r i c Mann-Whitney U - t e s t , a t a s i g n i f i c a n c e l e v e l o f p=0.05. 2.5.4 BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE USING THE METHOD OF ULTRAFILTRATION Serum (0.8 mL) samples from h e a l t h y male v o l u n t e e r s were s p i k e d w i t h PF and 5-OH-PF t o y i e l d c o n c e n t r a t i o n s o f 2.0 ng/ml and 0.5 lig/ml, r e s p e c t i v e l y . The samples were then p l a c e d i n t o t h e u l t r a f i l t r a t i o n d e v i c e s . The u l t r a f i l t r a t i o n d e v i c e s were then p l a c e d i n t o t h e u l t r a c e n t r i f u g e , e q u i l i b r a t e d t o a t e m p e r a t u r e o f 25 °C, and r o t a t e d a t 2000 g f o r 20 minutes a t a f i x e d a n g l e o f 18 ° . The r e s u l t i n g f i l t r a t e volume was a p p r o x i m a t e l y 0.5 mL. A l i q u o t s o f s p i k e d serum b e f o r e c e n t r i f u g a t i o n , and u l t r a f i l t r a t e were th e n a s s a y e d f o r both PF and 5-OH-PF. These r e s u l t s were compared t o r e s u l t s o b t a i n e d by e q u i l i b r i u m d i a l y s i s . 2.6 BINDING OF 5-HYDROXYPROPAFENONE AND PROPAFENONE TO ISOLATED PROTEINS 2.6.1 BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE TO HSA, AND AAG The b i n d i n g o f PF and 5-OH-PF t o pure HSA, pure human AAG, and both HSA p l u s AAG was a s s e s s e d w i t h p r o t e i n d i s s o l v e d i n i s o t o n i c phosphate pH 7.4 b u f f e r , and serum u l t r a f i l t r a t e . HSA, AAG, and AAG 48 p l u s HSA were d i s s o l v e d i n both b u f f e r and human serum u l t r a f i l t r a t e a t p h y s i o l o g i c a l c o n c e n t r a t i o n s . The p r o t e i n s o l u t i o n s were then s p i k e d w i t h PF and 5-OH-PF t o y i e l d a f i n a l c o n c e n t r a t i o n o f 2.0 uq/ml and 0.5 /zg/mL, r e s p e c t i v e l y . A l i q u o t s (0.8 mL) o f t h e above s o l u t i o n s were then d i a l y z e d a g a i n s t b l a n k i s o t o n i c phosphate pH 7.4 b u f f e r f o r 8 hours a t 37 °C. F o l l o w i n g d i a l y s i s , t h e b u f f e r and serum samples were removed, pH measured, and the c o n c e n t r a t i o n s o f PF and 5-OH-PF measured as d i s c u s s e d above. The f r e e f r a c t i o n o f PF and 5-OH-PF i n each t r e a t m e n t c a s e was c a l c u l a t e d . D i f f e r e n c e s i n b i n d i n g between human serum u l t r a f i l t r a t e and b u f f e r were compared u s i n g t h e two sample T - t e s t w i t h a s i g n i f i c a n c e l e v e l o f p=0.05. C o n c e n t r a t i o n s o f t o t a l p r o t e i n and AAG were measured. 2.6.2 BINDING TO PROPAFENONE AND 5-HYDROXYPROPAFENONE TO ALBUMIN AND FREE FATTY ACID FREE ALBUMIN. B i n d i n g o f PF and 5-OH-PF t o both albumin and (< 0.005%) f r e e f a t t y a c i d f r e e albumin were a s s e s s e d . Both albumin and f r e e f a t t y a c i d f r e e albumin were d i s s o l v e d i n i s o t o n i c phosphate pH 7.4 b u f f e r t o y i e l d p h y s i o l o g i c a l c o n c e n t r a t i o n s . These s o l u t i o n s were then s p i k e d w i t h both PF and 5-OH-PF t o y i e l d f i n a l c o n c e n t r a t i o n s o f 2.0 /zg/mL and 0.5 /zg/mL, r e s p e c t i v e l y . A l i q u o t s o f each s o l u t i o n were d i a l y z e d a g a i n s t an equal volume o f b l a n k i s o t o n i c phosphate pH 7.4 b u f f e r f o r 8 hours a t 37 °C. F o l l o w i n g d i a l y s i s , a l i q u o t s o f b u f f e r and serum were removed from t he d i a l y s i s c e l l s , pH, and the c o n c e n t r a t i o n o f PF and 5-OH-PF were measured. S t a t i s t i c a l c o m parisons were made u s i n g a two sample T - t e s t w i t h a s i g n i f i c a n c e 49 l e v e l o f p=0.05. C o n c e n t r a t i o n s o f t o t a l p r o t e i n were measured by the method o f Lowry. 2.6.3 BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE TO LIPOPROTEIN DEFICIENT SERUM The b i n d i n g o f PF and 5-OH-PF t o c o n t r o l serum and l i p o p r o t e i n d e f i c i e n t serum was measured. Both c o n t r o l serum and l i p o p r o t e i n d e f i c i e n t serum were s p i k e d w i t h PF and 5-OH-PF t o y i e l d f i n a l c o n c e n t r a t i o n s o f 2.0 /ig/mL and 0.5 /xg/mL, r e s p e c t i v e l y . Equal volumes o f s p i k e d serum and l i p o p r o t e i n d e f i c i e n t serum were d i a l y z e d o a g a i n s t b l a n k i s o t o n i c phosphate pH 7.4 b u f f e r f o r 8 hours a t 37 C. F o l l o w i n g d i a l y s i s , samples were removed, pH measured, and the c o n c e n t r a t i o n s o f PF and 5-OH-PF were measured i n t h e samples, as d e s c r i b e d p r e v i o u s l y . S t a t i s t i c a l c o mparisons were made u s i n g t h e two sample T - t e s t w i t h a s i g n i f i c a n c e l e v e l o f p=0.05. C o n c e n t r a t i o n s o f t o t a l p r o t e i n and AAG were measured, as d e s c r i b e d p r e v i o u s l y . 2.6.4 ELECTROPHORESIS OF ISOLATED PROTEIN SOLUTIONS P u r i t y o f the i s o l a t e d p r o t e i n s o l u t i o n s was a s s e s s e d by u s i n g sodium d o d e c y l s u l f a t e p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s (SDS PAGE). Samples o f AAG, HSA, HSA + AAG, l i p o p r o t e i n d e f i c i e n t serum, and normal serum were s u b j e c t e d t o SDS PAGE t o both c o n f i r m and a s s u r e p r o t e i n p u r i t y . 50 2.6.5 DRUG-DRUG DISPLACEMENT OF PROPAFENONE AND 5-HYDROXYPROPAFENONE FROM SERUM PROTEINS BY DISOPYRAMIDE, AND IBUPROFEN The e f f e c t on the b i n d i n g o f PF and 5-OH-PF by t h e a d d i t i o n o f d i s p l a c e r s such as the a c i d i c d r u g , i b u p r o f e n , and t h e amine d r u g , d i s o p y r a m i d e , was a s s e s s e d . Serum s p i k e d w i t h both PF (2.0 ug/ml) and 5-OH-PF (0.5 ug/ml) w i t h t he a d d i t i o n o f t h e r a p e u t i c c o n c e n t r a t i o n s o f e i t h e r i b u p r o f e n (50 ug/ml) o r d i s o p y r a m i d e (8.0 ug/ml) was d i a l y z e d a g a i n s t b l a n k i s o t o n i c pH 7.4 phosphate b u f f e r f o r 8 h o u r s . A l i q u o t s o f serum and b u f f e r were removed and a s s a y e d f o r PF and 5-OH-PF by GLC-ECD. The f r e e f r a c t i o n o f PF and 5-OH-PF were c a l c u l a t e d and compared t o c o n t r o l samples u s i n g t h e K r u s k a l l - W a l l i s t e s t w i t h a s i g n i f i c a n c e l e v e l o f p=0.05. 2.6.6 DRUG-DRUG DISPLACEMENT OF PROPAFENONE AND 5-HYDROXYPROPAFENONE FROM HSA AND AAG BY DISOPYRAMIDE AND IBUPROFEN D i s p l a c e m e n t o f PF and 5-OH-PF from i n d i v i d u a l p r o t e i n s was d e t e r m i n e d by u s i n g two probe d i s p l a c i n g a g e n t s d i s o p y r a m i d e , and i b u p r o f e n . P r o t e i n samples, HSA and AAG, were d i s s o l v e d i n i s o t o n i c phosphate pH 7.4 b u f f e r at p h y s i o l o g i c a l c o n c e n t r a t i o n s . Both PF and 5-OH-PF were then added t o g i v e f i n a l c o n c e n t r a t i o n s o f 2.0 ug/ml and 0.5 ug/ml. The samples were s p i k e d w i t h e i t h e r d i s o p y r a m i d e 8.0 Mg/mL, o r i b u p r o f e n 50 ug/ml. A l i q u o t s o f p r o t e i n s o l u t i o n s c o n t a i n i n g a m i x t u r e o f PF and 5-OH-PF m i x t u r e , and e i t h e r i b u p r o f e n o r d i s o p y r a m i d e were then added t o the d i a l y s i s c e l l s and d i a l y z e d a g a i n s t b l a n k i s o t o n i c phosphate pH 7.4 b u f f e r f o r 8 hours at 37 °C. 51 F o l l o w i n g d i a l y s i s , samples were removed from t h e d i a l y s i s c e l l s , pH measured, and t h e c o n c e n t r a t i o n o f PF and 5-OH-PF measured. C o n c e n t r a t i o n s o f t o t a l p r o t e i n and AAG were a s s e s s e d . 2.7 CHARACTERIZATION OF BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE IN PROTEIN SOLUTIONS 2.7.1 CHARACTERIZATION OF BINDING TO POOLED HUMAN SERUM, AAG, HSA, HDL, LDL, AND VLDL SOLUTIONS P r o t e i n b i n d i n g o f PF and 5-OH-PF was c h a r a c t e r i z e d by e q u i l i b r i u m d i a l y s i s i n p o o l e d human serum, and i n p h y s i o l o g i c a l c o n c e n t r a t i o n s o f AAG, HSA, and human l i p o p r o t e i n s [HDL, LDL, and VLDL] d i s s o l v e d i n i s o t o n i c phosphate pH 7.4 b u f f e r . B u f f e r was s p i k e d w i t h v a r y i n g c o n c e n t r a t i o n s o f e i t h e r PF [0.25, 0.50, 1.0, 2.0, 5.0, 10.0, 25.0, 50.0, 75.0, and 100.0 Mg/mL] o r 5-OH-PF [0.20, 0.50, 0.75, 1.00, 2.00, 5.00, 10.0, 20.0, 30.0, and 45.0 /ig/mL]. S p i k e d b u f f e r was d i a l y z e d a g a i n s t equal volumes (0.8 mL) o f b l a n k p r o t e i n s o l u t i o n s a t p h y s i o l o g i c a l c o n c e n t r a t i o n s p r e v i o u s l y d i s c u s s e d . F o l l o w i n g 8 hours o f e q u i l i b r i u m d i a l y s i s a t 37 °C, samples were removed, pH measured, and c o n c e n t r a t i o n s o f e i t h e r PF o r 5-OH-PF measured. B i n d i n g r a t i o ( b o u n d / f r e e ) was p l o t t e d a g a i n s t c o n c e n t r a t i o n o f bound drug t o o b t a i n a Rosenthal p l o t f o r the b i n d i n g o f PF o r 5-OH-PF t o each p r o t e i n s o l u t i o n d e s c r i b e d above. The f r e e f r a c t i o n s o f PF and 5-OH-PF were p l o t t e d a g a i n s t t h e r e s p e c t i v e t o t a l d r u g o r m e t a b o l i t e c o n c e n t r a t i o n measured. A n a l y s i s o f b i n d i n g p a r a m e t e r s o f PF o r 5-OH-PF t o each p r o t e i n s o l u t i o n was c a r r i e d out 52 by n o n - l i n e a r r e g r e s s i o n a n a l y s i s o f s e v e r a l d e f i n e d b i n d i n g models (see s e c t i o n 2.8.2 BINDING PARAMETER MODELS AND DATA FITTING) u s i n g the computer program ENZFITTER^. C o n c e n t r a t i o n s o f t o t a l p r o t e i n and AAG were a s s e s s e d as d e s c r i b e d p r e v i o u s l y . 2.7.2 ELECTROPHORESIS OF LIPOPROTEINS To e n s u r e t h a t t he c o m p o s i t i o n o f the l i p o p r o t e i n complex would not d e t e r i o r a t e d u r i n g d i a l y s i s , a l i q u o t s o f l i p o p r o t e i n were s u b j e c t e d t o e l e c t r o p h o r e t i c s e p a r a t i o n on an a g a r o s e g e l b e f o r e and a f t e r t h e d i a l y s i s p r o c e d u r e . L i p o p r o t e i n s (LDL, VLDL, and HDL) were a p p l i e d t o t h e agaro s e g e l and run as d e s c r i b e d p r e v i o u s l y . 2.8. DATA ANALYSIS 2.8.1 STATISTICAL ANALYSIS OF DATA. The d a t a i n t h e s e e x p e r i m e n t s were a n a l y z e d by a number o f d i f f e r e n t s t a t i s t i c a l t e c h n i q u e s . These s t a t i s t i c a l t e c h n i q u e s a r e s t a t e d where a p p l i e d . Where assumptions o f normal d i s t r i b u t i o n , and equal v a r i a n c e c o u l d not be demonstrated n o n - p a r a m e t r i c t e s t s were used. The n o n - p a r a m e t r i c e q u i v a l e n t f o r the two sample t - t e s t i s t h e Mann-Whitney U - t e s t , and f o r the one-way a n a l y s i s o f v a r i a n c e (ANOVA), i s t h e K r u s k a l l - W a l l i s t e s t . I f n o n - p a r a m e t r i c t e s t s a r e i n a d v e r t e n t l y used, and the assumptions o f n o r m a l i t y , and equal v a r i a n c e a re met, t h e y a re a p p r o x i m a t e l y 95% as p o w e r f u l as t h e i r p a r a m e t r i c c o u n t e r p a r t s [ Z a r , 1987]. 53 2.8.2 BINDING PARAMETER MODELS AND DATA FITTING S e v e r a l models, d e r i v e d from f i r s t p r i n c i p l e s o f p r o t e i n -l i g a n d i n t e r a c t i o n s ( e q u a t i o n 1 ), were used t o model the b i n d i n g o b s e r v e d f o r 5-OH-PF i n serum samples o f h e a l t h y v o l u n t e e r s , and f o r both PF and 5-OH-PF i n s o l u t i o n s o f i s o l a t e d p r o t e i n s . [DP] [D] + [P] (1) The s i m p l e s t b i n d i n g model used was t h a t o f t o t a l n o n - s p e c i f i c b i n d i n g ( E q u a t i o n 11), f o l l o w e d by 1 s i t e - s p e c i f i c b i n d i n g ( E q u a t i o n 12), 1-s i t e s p e c i f i c + n o n - s p e c i f i c b i n d i n g ( E q u a t i o n 13), and f i n a l l y 2 s i t e - s p e c i f i c l i g a n d b i n d i n g ( E q u a t i o n 14). [Db] = K n s * [ D f ] (11) The e q u a t i o n f o r n o n - s p e c i f i c b i n d i n g ( e q u a t i o n 11) i s merely a s i m p l i f i c a t i o n o f the e q u a t i o n f o r the 1 s i t e b i n d i n g model. That i s , i f t h e a f f i n i t y f o r b i n d i n g o f a drug t o t h i s s i t e i s v e r y low, e q u a t i o n 12 c o l l a p s e s to the p r o d u c t o f the b i n d i n g a f f i n i t y and t h e b i n d i n g c a p a c i t y ( i . e . Kns = Ka*NP^) [ G l a s s o n e t a/., 1980]. [Db] = N P l * K a l * [ D f l = N P l * K a l * [ D f ] i f K a l « 1 (12) (1 + K a l * [ D f ] ) [Db] = ( N P l * K a l * r D f l ) + K n s * [ D f ] (13) (1 + K a l * [ D f ] ) [Db] = ( N P l * K a l * r D f 1 ) + ( N P 2 * K a 2 M D f l ) (14) (1 + K a l * [ D f ] ) (1 + K a 2 * [ D f ] ) 54 Where [Db] i s the molar bound drug c o n c e n t r a t i o n , [Df] i s the m o l a r f r e e d r u g c o n c e n t r a t i o n , NP1 i s the c a p a c i t y o f the f i r s t d r u g b i n d i n g s i t e ( m o l a r ) , K a l i s the a s s o c i a t i o n c o n s t a n t o f the f i r s t b i n d i n g s i t e ( m o l a r " * ) , NP2 i s the c a p a c i t y o f t h e second b i n d i n g s i t e ( m o l a r ) , and Ka2 i s t h e a s s o c i a t i o n c o n s t a n t o f the second b i n d i n g s i t e (molar"-'-). The n o n - t r a n s f o r m e d b i n d i n g d a t a (bound vs. f r e e ) was f i t w i t h t h e n o n - l i n e a r c u r v e f i t t i n g computer program ENZFITTER^ t o one o f the above b i n d i n g e q u a t i o n s . The "goodness o f f i t " was d e t e r m i n e d from the s t a n d a r d d e v i a t i o n o f the e s t i m a t e s , and the random equal d i s t r i b u t i o n o f t h e y - a x i s r e s i d u a l s . N o n - l i n e a r c u r v e f i t t i n g w i t h the ENZFITTER R program employs t h e enhanced a l g o r i t h m o f Marquart [ L e a t h e r b a r r o w , 1987] t o f i t t h e d a t a , p The ENZFITTER^ program a l s o has r o b u s t w e i g h t i n g . Robust w e i g h t i n g a l l o w s extreme o u t l i e r s t o be e l i m i n a t e d based on s t a t i s t i c a l p r i n c i p l e s r a t h e r than s u b j e c t i v e e l i m i n a t i o n . Robust w e i g h t i n g employs the a l g o r i t h m o f M o s t e l l e r and Tukey [ L e a t h e r b a r r o w , 1987] t o s t a t i s t i c a l l y r e d u c e the importance o f o u t l i e r s i n the d a t a u n d e r g o i n g n o n - l i n e a r r e g r e s s i o n . For q u a l i t a t i v e p u r p o s e s , d a t a was p l o t t e d by the method o f R o s e n t h a l , as opposed to S c a t c h a r d . T h i s i s due t o the v a r i e t y o f d i f f e r e n t p r o t e i n s p r e s e n t i n serum making t h e use o f t o t a l p r o t e i n c o n c e n t r a t i o n , as used i n S c a t c h a r d , i n c o r r e c t . 2.8.3 MATHEMATICAL BINDING R E C O N S T I T U T E STUDIES 55 B i n d i n g o f PF and 5-OH-PF i n serum w i l l be e s t i m a t e d based on the sum o f t h e b i n d i n g c o n t r i b u t e d by each p r o t e i n as i n e q u a t i o n 15: n [ D b ] s e r u m = 2 [ D b ] n (15) i = l where [ D b ] s e r u m i s t h e t h e o r e t i c a l l y c a l c u l a t e d b i n d i n g o f e i t h e r PF or 5-OH-PF from t he sum o f the c o n t r i b u t i o n s o f b i n d i n g by i n d i v i d u a l b i n d i n g p r o t e i n s . The term n i s t h e number o f p r o t e i n s c o n t r i b u t i n g t o b i n d i n g o f drug, and [ D b ] n i s the b i n d i n g c o n t r i b u t i o n due t o each i n d i v i d u a l p r o t e i n . The next s t e p i s t o s y s t e m a t i c a l l y s u b t r a c t t h e c o n t r i b u t i o n o f i n d i v i d u a l p r o t e i n s , and then c a l c u l a t e t h e c o n c e n t r a t i o n o f bound drug (see e q u a t i o n 16). n-1 [Dblserum n = 2 ™ n ( 1 6 ) i=5 where [ D b ] s e r u m n i s t h e c o n c e n t r a t i o n o f bound drug c a l c u l a t e d f o r a s p e c i f i c c o n c e n t r a t i o n o f f r e e drug [ D f ] , as the b i n d i n g c o n t r i b u t i o n o f i n d i v i d u a l c o n s t i t u e n t p r o t e i n s i s s y s t e m a t i c a l l y s u b t r a c t e d ( i n the o r d e r VLDL, LDL, HDL, HSA). F i n a l l y [ D b ] s e r u m , [ D b ] s e r u m 4, [ D b ] s e r u m 3, [ D b ] s e r u m 2 » and [ D b ] s e r u m ± are p l o t t e d as a f u n c t i o n o f f r e e drug i n o r d e r t o a s s e s s t h e r e l a t i v e i mportance o f each p r o t e i n t o t h e o v e r a l l b i n d i n g o f PF and 5-OH-PF. 2.9 RED BLOOD CELL DISTRIBUTION 56 2.9.1 PREPARATION OF RED BLOOD CELLS Bl o o d was c o l l e c t e d from h e a l t h y v o l u n t e e r s i n t o r e d top v a c u t a i n e r s . The caps were i m m e d i a t e l y removed, whereupon 2 mL a l i q u o t s o f b l o o d were t r a n s f e r r e d t o c l e a n t e s t t u b e s , and d i l u t e d w i t h 8 mL o f i s o t o n i c pH 7.4 phosphate b u f f e r . The c o n t e n t s o f the t u b e s were then g e n t l y mixed f o r 10 minutes, and t h e tubes were c e n t r i f u g e d a t -1000 g f o r 5 m i n u t e s . The top aqueous l a y e r was removed and d i s c a r d e d , a n o t h e r 8 mL o f f r e s h i s o t o n i c pH 7.4 phosphate b u f f e r was added, and t h e p r o c e d u r e was r e p e a t e d . T h i s washing p r o c e d u r e was r e p e a t e d f o r a t o t a l 3 washes. D u r i n g t h e f i n a l wash s t e p , t h e c e l l s were spun f o r 20 minutes a t -1000 rpm. The aqueous l a y e r was then removed, l e a v i n g packed r e d c e l l s . The r e d c e l l s (0.55 mL) were r e c o n s t i t u t e d i n i s o t o n i c pH 7.4 phosphate b u f f e r (0.45 mL) and g e n t l y mixed f o r 5 minutes. 2.8.2 TIME TO DISTRIBUTION EQUILIBRIUM FOR PROPAFENONE AND 5-HYDR0XYPR0PAFEN0NE UPTAKE INTO HUMAN RED BLOOD CELLS R e c o n s t i t u t e d red b l o o d c e l l s u s p e n s i o n s were s p i k e d w i t h PF t o g i v e a f i n a l c o n c e n t r a t i o n o f 2.0 ug/ml and 5-OH-PF t o g i v e 0.5 ug/ml. These samples were i m m e d i a t e l y p l a c e d i n a water bath a t 37 °C f o r v a r i o u s time p e r i o d s (2, 4, 6, ...30 minutes) t o a s s e s s the time r e q u i r e d t o a t t a i n d i s t r i b u t i o n a l e q u i l i b r i u m . The samples were then i m m e d i a t e l y c e n t r i f u g e d f o r 2 minutes, and the s u p e r n a t a n t removed. The r e d b l o o d c e l l s were then hemolyzed by adding d i s t i l l e d water, and v o r t e x i n g the m i x t u r e . The s u p e r n a t a n t and hemolyzed r e d c e l l s were 57 a n a l y z e d f o r both PF and 5-OH-PF. The r a t i o o f drug c o n t a i n e d i n r e d b l o o d c e l l s ( c o n c e n t r a t i o n o f drug i n r e d c e l l s / c o n c e n t r a t i o n o f drug i n s u p e r n a t a n t ) was c a l c u l a t e d and p l o t t e d w i t h r e s p e c t t o t i m e . 2.8.3 UPTAKE OF PROPAFENONE AND 5-HYDROXYPROPAFENONE BY HUMAN RED BLOOD CELLS. Red b l o o d c e l l s u s p e n s i o n s were p r e p a r e d i n e i t h e r i s o t o n i c phosphate pH 7.4 b u f f e r , o r serum. Whole b l o o d was a l s o used t o e s t i m a t e d r u g uptake by r e d b l o o d c e l l s . These s u s p e n s i o n s were then s p i k e d w i t h PF and 5-OH-PF t o y i e l d f i n a l c o n c e n t r a t i o n s o f 2.0 fig/ml and 0.5 ^g/mL, r e s p e c t i v e l y . These samples were then i n c u b a t e d a t 37 °C f o r 30 m i n u t e s . The samples were then removed and c e n t r i f u g e d f o r 10 minutes a t -1000 g. A l i q u o t s o f s u p e r n a t a n t and packed r e d b l o o d c e l l s were then a n a l y z e d f o r PF and 5-OH-PF. The r a t i o o f drug c o n c e n t r a t i o n i n r e d b l o o d c e l l s o v e r t he drug c o n c e n t r a t i o n i n the s u p e r n a t a n t were c a l c u l a t e d as i n e q u a t i o n 8: Red b l o o d c e l l uptake r a t i o = TP r e d b l o o d e e l 11 (17) [D s u p e r n a t a n t ] Where [P r e d b l o o d c e l l ] i s t h e c o n c e n t r a t i o n o f e i t h e r PF o r 5-OH-PF p r e s e n t i n r e d b l o o d c e l l s , and [D s u p e r n a t a n t ] i s the drug c o n c e n t r a t i o n o f the s u p e r n a t a n t . T h i s r a t i o was c a l c u l a t e d f o r PF and 5-OH-PF f o r each t r e a t m e n t . 58 3. RESULTS 3.1 PRELIMINARY IN VITRO PROTEIN BINDING EXPERIMENTS FOR PROPAFENONE AND 5-HYDROXYPROPAFENONE 3.1.1 TIME TO EQUILIBRIUM The time t o r e a c h b i n d i n g e q u i l i b r i u m was e s t a b l i s h e d t o o c c u r between 6 and 7 hours ( F i g u r e 2a and 2b) when both h i g h (45.0 ng/ml) and low (0.1 ng/ml) c o n c e n t r a t i o n s o f 5-OH-PF were d i a l y z e d a g a i n s t 0.1 M i s o t o n i c phosphate b u f f e r . E i g h t hours was chosen as t h e optimum d i a l y s i s time t o a t t a i n e q u i l i b r i u m . The pH o f t h e serum compartment f o l l o w i n g d i a l y s i s d i d not v a r y s i g n i f i c a n t l y o v e r t h e time r e q u i r e d t o r e a c h e q u i l i b r i u m . 3.1.2 NON-SPECIFIC BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE TO EQUILIBRIUM DIALYSIS CELLS AND MPS-1 ULTRA-FILTRATION DEVICES The e x t e n t o f n o n - s p e c i f i c b i n d i n g o f 5-OH-PF t o both t he d i a l y s i s c e l l and the membrane was de t e r m i n e d o v e r a range o f c o n c e n t r a t i o n s . The e x t e n t o f n o n - s p e c i f i c b i n d i n g o f 5-OH-PF was h i g h o v e r t h e e n t i r e c o n c e n t r a t i o n range s t u d i e d ( T a b l e 1 ) . The t o t a l l o s s t o the c e l l w i t h t h e membrane used f o r e q u i l i b r i u m d i a l y s i s was 28.2 ± 6.8% (Mean ± SD). TIME (hours) 0.000 -a.CGO E 2 7.W0 TIME (houra) F i g u r e 2. Time r e q u i r e d to a t t a i n e q u i l i b r i u m f o r the e q u i l i b r i u m d i a l y s i s o f 5-hydroxypropafenone. a) 0.1 ug/ml of 5-hydroxypropafenone b) 45.0 ug/ml of 5-hydroxypropafenone The pH o f the serum compartment o f the d i a l y s i s c e l l f o l l o w i n g d i a l y s i s i s p r e s e n t e d as a bar graph. (pH + SD) 60 TABLE 1. COMPARISON OF NON-SPECIFIC BINDING OF 5-HYDROXYPROPAFENONE AND PROPAFENONE TO ACRYLIC EQUILIBRIUM DIALYSIS APPARATUS FOLLOWING INCUBATION FOR 8 HOURS AT 37°C AND MPS-1 ULTRAFILTRATION DEVICES AT 25°C. CONCENTRATION {ng/ml) 5-HYDROXYPROPAFENONE ULTRAFILTRATION PERCENT LOST EQUILIBRIUM DIALYSIS PERCENT LOST IN MPS-1 0.10 0.50 1.00 5.00 25.00 25.6 32.0 36.5 28.5 18.4 15.6% 9.6% 3.0% 9.2% 2.4% 25.0 ± 7.2% 28.6 ± 5.7% NOTE: EXPERIMENTS WERE CARRIED OUT USING 5-HYDROXYPROPAFENONE AND PROPAFENONE SPIKED 0.1 M ISOTONIC PHOSPHATE BUFFER. NOTE: NO STATISTICALLY SIGNIFICANT (P > 0.05) DIFFERENCE BETWEEN THE NON-SPECIFIC LOSS OF 5-HYDROXYPROPAFENONE TO DIALYSIS APPARATUS, AND MPS-1 ULTRAFILTRATION DEVICE AT DIFFERENT CONCENTRATIONS OF 5-HYDROXYPROPAFENONE. KRUSKALL-WALLIS TEST. CONCENTRATION ( f j g M ) PROPAFENONE 2.00 19.8 ± 5.3% 10.00 23.0 ± 3.3% The n o n - s p e c i f i c b i n d i n g o f PF and 5-OH-PF t o MPS-1 u l t r a f i l t r a t i o n d e v i c e s was co n d u c t e d w i t h PF and 5-OH-PF s p i k e d 0.1 M phosphate b u f f e r . Samples were spun i n an u l t r a c e n t r i f u g e a t 2000 g f o r 20 minutes at 25 °C a t a f i x e d a n g l e o f 18°. Both PF and 5-OH-PF showed h i g h n o n - s p e c i f i c b i n d i n g ( T a b l e 1) at both low and h i g h c o n c e n t r a t i o n s o f d r u g . The o v e r a l l n o n - s p e c i f i c l o s s o f 5-OH-PF and PF due t o u l t r a f i l t r a t i o n were 27.5% and 21.5% r e s p e c t i v e l y . 61 3.1.3 pH DEPENDENT BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE. 3.1.3.1 pH DEPENDENT BINDING OF PROPAFENONE IN HUMAN SERUM, AND SOLUTIONS OF HUMAN SERUM ALBUMIN, AND HUMAN ALPHA-1-ACID GLYCOPROTEIN. PF d i s p l a y e d pH dependent b i n d i n g t o serum, HSA (45 mg/mL), and AAG (0.9 mg/mL) d i s s o l v e d i n 0.1 M i s o t o n i c phosphate b u f f e r . As the pH o f t h e serum, HSA, and AAG i n c r e a s e d , the f r e e f r a c t i o n o f PF was d e c r e a s e d ( e . g . i n c r e a s e d b i n d i n g ) ( F i g u r e 3 ) . Over a range o f 0.6 pH u n i t s , t h e f r e e f r a c t i o n o f PF i n serum d e c r e a s e d by 50% ( f r e e f r a c t i o n was 0.090 a t pH 7.2 and 0.045 at pH 7.8). S i m i l a r f i n d i n g s o f pH s e n s i t i v e b i n d i n g f o r PF were appa r e n t f o r HSA, and AAG s o l u t i o n s ( F i g u r e 3 ) . 3.1.3.2 pH DEPENDENT BINDING 5-HYDROXYPROPAFENONE IN HUMAN SERUM, A SOLUTION OF HUMAN SERUM ALBUMIN, AND OF HUMAN ALPHA-1-ACID GLYCOPROTEIN. The m e t a b o l i t e , 5-OH-PF, d i s p l a y e d pH dependent b i n d i n g s i m i l a r t o t h a t o f the p a r e n t compound, PF ( F i g u r e 4 ) . The f r e e f r a c t i o n o f 5-OH-PF i n serum and i n a s o l u t i o n o f AAG was d e c r e a s e d ( i n c r e a s e d b i n d i n g ) as t h e pH i n c r e a s e d . L i k e t h e r e s u l t s o b s e r v e d f o r the p a r e n t compound, th e f r e e f r a c t i o n o f 5-OH-PF i n serum d e c r e a s e s ( i n c r e a s e d b i n d i n g ) by 50% o v e r a pH range o f 0.6 pH u n i t s . That i s , a t a low pH o f 7.2 the f r e e f r a c t i o n o f 5-OH-PF i n serum was 0.125 and a t pH 7.8 the f r e e f r a c t i o n was 0.065. A s i m i l a r t r e n d i n pH s e n s i t i v e b i n d i n g t o HSA was not a p p a r e n t . 62 0.500 -T-0.000 -| 1 1 1 1 1 1 7.000 7.200 7.400 7.600 7.800 8.000 8.200 p H of p r o t e i n s o l u t i o n o f t e r d i a l y s i s F i g u r e 3. The pH dependent b i n d i n g o f pr o p a f e n o n e (2.0 /zg/mL) i n serum, and s o l u t i o n s o f HSA and AAG. Human AAG 0.90 mg/mL ( o ) HSA 45.0 mg/mL (•) Normal human serum ( A ) (pH o f p r o t e i n s i d e o f d i a l y s i s c e l l f o l l o w i n g d i a l y s i s ± SD vs. f r e e f r a c t i o n ± SD). 63 OJ ii: 0.200 • 7.200 7.400 7.600 7.800 p H of p r o t e i n s o l u t i o n o f t e r d i a l y s i s F i g u r e 4. The pH dependent b i n d i n g o f 5-hydroxypropafenone (0.5 ng/ml) i n serum, and s o l u t i o n s o f HSA and AAG. Human AAG 0.90 mg/mL ( o ) HSA 45 mg/mL ( • ) Normal human serum ( A ) (pH o f p r o t e i n s i d e o f d i a l y s i s c e l l f o l l o w i n g d i a l y s i s ± SD vs. f r e e f r a c t i o n ± SD). 64 3.1.3.3 THE INFLUENCE OF pH ON THE PARTITIONING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE IN A TOLUENE:PHOSPHATE BUFFER AND HEXANE:PHOSPHATE BUFFER SYSTEM. The i n f l u e n c e o f pH on the p a r t i t i o n i n g o f PF o r 5-OH-PF from t he aqueous l a y e r i n t o t h e o r g a n i c l a y e r o f two o r g a n i c / a q u e o u s f l u i d systems ( t o l u e n e : p h o s p h a t e b u f f e r and- hexane:phosphate b u f f e r ) as a f u n c t i o n o f pH was a s s e s s e d . P a r t i t i o n i n g o f PF from t h e aqueous l a y e r i n t h e hexane system was not o b s e r v e d ; however, p a r t i t i o n i n g o f PF from t h e aqueous l a y e r o f t h e t o l u e n e : p h o s p h a t e b u f f e r system was s u b s t a n t i a l ( F i g u r e 5 ) . As pH o f t h e b u f f e r i n the o r g a n i c / a q u e o u s f l u i d system was i n c r e a s e d , t h e f r a c t i o n o f PF i n t h e aqueous l a y e r d e c r e a s e d s i g n i f i c a n t l y ( F i g u r e 5 ) . T h i s c o r r e l a t e d w e l l w i t h t h e c a l c u l a t e d r a t i o o f i o n i z e d vs. n o n - i o n i z e d PF. These c a l c u l a t i o n s were based on a known pKa v a l u e o f 9.0 f o r PF. The p a r t i t i o n i n g o f 5-OH-PF from t he aqueous l a y e r o f the to!uene:phosphate b u f f e r system was s u b s t a n t i a l , as l e v e l s 5-OH-PF i n the aqueous phase became u n d e t e c t a b l e a f t e r pH 7.6 ( F i g u r e 6 ) . The p a r t i t i o n i n g o f 5-OH-PF from t he aqueous l a y e r o f the hexane:phosphate b u f f e r system was l e s s i n comparison w i t h the l o s s o f 5-OH-PF from the aqueous l a y e r o f the t o l u e n e : phosphate b u f f e r system ( F i g u r e 6 ) . 3.1.4 THE EFFECT OF BUFFER STRENGTH ON BINDING OF 5-HYDROXYPROPAFENONE. The b i n d i n g o f 5-OH-PF i n serum was a s s e s s e d i n t h e p r e s e n c e o f a 0.067 M, 0.10 M, and 0.15 M pH 7.4 phosphate b u f f e r ( F i g u r e 7 ) . No s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e i n the f r e e 65 0.400 • 100 0.300 - -0.200 - -• 8 0 4- 60 - r 40 0.100 - f 0 .000 • 6.500 o X a 7.500 pH OF PHOSPHATE BUFFER 20 • 0 I.500 F i g u r e 5. I n f l u e n c e o f a l t e r a t i o n s i n pH o f the p a r t i t i o n i n g o f p r o p a f e n o n e (2.0 tzg/mL) between aqueous and o r g a n i c l a y e r s i n a to l u e n e : p h o s p h a t e b u f f e r system. F r a c t i o n o f pro p a f e n o n e r e m a i n i n g i n aqueous l a y e r ± SD vs. pH ( o ). C o n t r a l a t e r a l y - a x i s d e p i c t s the c a l c u l a t e d r a t i o o f i o n i z e d / n o n i o n i z e d p r o p a f e n o n e vs. pH ( • ). 66 — t . O O O -r-U l 6.500 7.500 8.500 pH OF P H O S P H A T E B U F F E R F i g u r e 6. I n f l u e n c e o f a l t e r a t i o n s i n pH on the p a r t i t i o n i n g o f 5-hydro x y p r o p a f e n o n e (0.5 izg/mL) between aqueous and o r g a n i c l a y e r s i n v a r i o u s o r g a n i c s o l v e n t s . t o l u e n e r p h o s p h a t e b u f f e r ( o ) system, hexane:phosphate b u f f e r ( 9 ) system. F r a c t i o n o f 5-hydroxypropafenone r e m a i n i n g i n the aqueous l a y e r ± SD vs. pH. 67 0,200 70 0.100 > o 0.000 0.000 P H O S P H A T E B U F F E R S T R E N G T H ( m o l a r ) F i g u r e 7. The e f f e c t o f molar s t r e n g t h o f phosphate b u f f e r on the f r e e f r a c t i o n o f 5-hydroxypropafenone i n serum ± SD. 68 f r a c t i o n between t he t h r e e d i f f e r e n t b u f f e r s t r e n g t h s was o b s e r v e d ( K r u s k a l l - W a l l i s T e s t ; p > 0.05). The 0.10 M pH 7.4 i s o t o n i c phosphate b u f f e r was used f o r a l l subsequent d i a l y s i s e x p e r i m e n t s . 3.1.5 DEGRADATION OF 5-HYDROXYPROPAFENONE DURING EQUILIBRIUM DIALYSIS. E x p e r i m e n t s t o a s s e s s t he degree o f c h e m i c a l d e g r a d a t i o n o f 5-OH-PF i n serum and b u f f e r d u r i n g an e i g h t hour d i a l y s i s p e r i o d were c o n d u c t e d . A l i q u o t s o f serum and b u f f e r were s p i k e d w i t h 5-OH-PF and i n c u b a t e d i n the d i a l y s i s bath a t 37 °C. Samples were removed h o u r l y and t h e c o n c e n t r a t i o n o f 5-OH-PF was d e t e r m i n e d . No d e g r a d a t i o n o f 5-OH-PF o c c u r r e d o v e r t he e n t i r e 8 hour time p e r i o d r e q u i r e d t o r e a c h b i n d i n g e q u i l i b r i u m . 3.1.6 VOLUME SHIFTS F o l l o w i n g a l l e q u i l i b r i u m d i a l y s i s e x p e r i m e n t s , t h e volumes o f the p r o t e i n (serum) compartment and the b u f f e r compartment were measured t o a s s u r e t h a t s i g n i f i c a n t {- > 10%) [Lima e t al., 1983] f l u i d s h i f t s d i d not o c c u r from one f l u i d compartment t o a n o t h e r . I t was o b s e r v e d t h a t s i g n i f i c a n t volume s h i f t s ( > 10% ) d i d not o c c u r d u r i n g t he e i g h t hour d i a l y s i s t i m e . However, volume s h i f t s o f a p p r o x i m a t e l y 5% were commonly e n c o u n t e r e d . 69 3.1.7 OSMOLARITY OF 5-OH-PF STOCK SOLUTIONS USED IN EQUILIBRIUM DIALYSIS STUDIES. The o s m o l a r i t y o f the s t o c k s o l u t i o n s o f 5-OH-PF was a s s e s s e d . Over the m e t a b o l i t e c o n c e n t r a t i o n range o f 0.1 t o 45.0 /Ltg/mL no s i g n i f i c a n t changes i n o s m o l a r i t y were o b s e r v e d . The o s m o l a r i t y o f the s t o c k s o l u t i o n s was 267.3 ± 1.3 mOsm (Mean ± SD). 3.1.8 PROTEIN LOSS DURING EQUILIBRIUM DIALYSIS Ex p e r i m e n t s were conducted t o a s s e s s t he r e d u c t i o n i n t h e amount o f p r o t e i n due t o d e c o m p o s i t i o n , d i l u t i o n , and l e a k a g e d u r i n g e i g h t hours o f e q u i l i b r i u m d i a l y s i s . I t was found t h a t t o t a l p r o t e i n c o n c e n t r a t i o n d i d not change s i g n i f i c a n t l y f o r serum, HSA s o l u t i o n s , and h i g h d e n s i t y l i p o p r o t e i n s s o l u t i o n s ; however, s m a l l changes were o b s e r v e d i n the v e r y low d e n s i t y l i p o p r o t e i n s o l u t i o n (< 2.5%), and the low d e n s i t y l i p o p r o t e i n s o l u t i o n (< 8.5%), as measured by a Lowry p r o t e i n a s s a y ( T a b l e 2 ) . F u r t h e r , i t was shown t h a t w h i l e no change i n AAG o c c u r r e d i n serum, a l o s s o f a p p r o x i m a t e l y 12% o c c u r r e d when AAG was d i a l y z e d a l o n e ( T a b l e 2 ) . 3.1.9 TEST FOR DIALYSIS MEMBRANE INTEGRITY FOLLOWING DIALYSIS To a s s e s s t he i n t e g r i t y o f the d i a l y s i s membranes f o l l o w i n g each d i a l y s i s e x p e r i m e n t , a small a l i q u o t o f the d i a l y z e d b u f f e r was added t o 0.4 mL o f 3% t r i c h l o r o a c e t i c a c i d (TCA). I f p r e c i p i t a t i o n was o b s e r v e d 70 TABLE 2. CHANGE IN TOTAL PROTEIN AND ALPHA-1-ACID GLYCOPROTEIN IN PLEXIGLASS DIALYSIS CELLS AND CELLULOSE DIALYSIS MEMBRANES DURING DIALYSIS. START TOTAL PROTEIN BY LOWRY ASSAY ALBUMIN 39.7 mg/mL SERUM 62.0 mg/mL HDL 1.72 mg/mL (complex = 44-47% p r o t e i n ) LDL 0.84 mg/mL (complex = 19-22% p r o t e i n ) VLDL 0.09 mg/mL (complex = 5-12% p r o t e i n ) FINISH % CHANGE 39.9 ± 1.8 mg/mL ND 61.8 ± 2.7 mg/mL ND 1.81 ± 0.06 mg/mL 5.0% 0.78 ± 0.02 mg/mL -6.0% 0.09 ± 0.01 mg/mL ND ALPHA-1-ACID GLYCOPROTEIN BY RADIAL IMMUNODIFFUSION PLATES SERUM 0.62 mg/mL 0.63 ± 0.02 mg/mL ND ALPHA-1-ACID 0.89 mg/mL 0.78 ± 0.06 mg/mL -12.0% GLYCOPROTEIN NOTE: DUE TO THE COEFFICIENT OF VARIATION, IF % CHANGE WAS BELOW 5% NO CHANGE COULD BE DETECTED. * SAMPLES WERE DIALYZED FOR EIGHT HOURS AT 37 °C. f o l l o w i n g t h e TCA t r e a t m e n t , t h e samples were d i s c a r d e d ; t h i s o c c u r r e d i n l e s s than 1% o f the samples. 3.2 PROTEIN BINDING OF 5-HYDROXYPROPAFENONE IN SERUM OF HEALTHY MALE VOLUNTEERS E q u i l i b r i u m d i a l y s i s b i n d i n g e x p e r i m e n t s were c o n d u c t e d u s i n g serum i n d i v i d u a l l y c o l l e c t e d from 5 h e a l t h y male v o l u n t e e r s . The d a t a 71 were p l o t t e d a c c o r d i n g t o the method o f R o s e n t h a l [ b o u n d / f r e e vs. bound] ( F i g u r e s 8 a - e ) . The b i n d i n g r a t i o ( b o u n d / f r e e ) a t low c o n c e n t r a t i o n s i n comparison t o h i g h e r c o n c e n t r a t i o n s o f 5-OH-PF was g e n e r a l l y found t o be h i g h l y v a r i a b l e ( F i g u r e s 8 a - e ) . T o t a l p r o t e i n and AAG c o n c e n t r a t i o n s f o r each o f the v o l u n t e e r s a r e p r e s e n t e d i n Appendix 1. A n a l y s i s o f the no n t r a n s f o r m e d d a t a w i t h t h e a i d o f t h e n o n - l i n e a r c u r v e f i t t i n g program, ENZFITTER R w i t h r o b u s t w e i g h t i n g , r e s u l t e d i n 5 s u b j e c t s f i t t i n g t h e 1 b i n d i n g - s i t e model ( T a b l e 3 ) . TABLE 3: BINDING PARAMETERS OF 5-HYDROXYPROPAFENONE IN HEALTHY HUMAN SERUM SUBJECT BINDING AFFINITY BINDING CAPACITY (KA) (M ) ± SD (NP) (M) ± SD 1 1.18 X 104 ± 2.74 X 101 2.88 X 10"4 ± 5.59 X 10"7 2 7.97 X 103 6.10 X 10"4 ± 1.77 X 103 ± 1.17 X 10"4 3 1.43 X 104 ± 1.06 X 101 3.10 X 10 - 4 ± 1.91 X 10"7 4 3.43 X 104 ± 4.98 X 103 1.81 X 10 - 4 ± 1.84 X 10' 5 5 1.78 X 104 ± 2.22 X 103 3.43 X 10"4 ± 3.34 X 10"5 mean v a l u e 1.72 X 10; 3.46 X 10"; (± SD) 1.02 X 1 0 4 1.59 X 1 0 " 4 The average f r e e f r a c t i o n o f 5-OH-PF ov e r a wide range o f c o n c e n t r a t i o n s (0.12 - 35.3 ng/ml) was not s t a t i s t i c a l l y d i f f e r e n t ; however, a t the l o w e s t c o n c e n t r a t i o n o f 5-OH-PF (0.06 ng/ml) the f r e e f r a c t i o n o f 5-OH-PF was s t a t i s t i c a l l y d i f f e r e n t from t h e f r e e f r a c t i o n a t a l l o t h e r c o n c e n t r a t i o n s . A s l i g h t i n c r e a s e i n f r e e f r a c t i o n was o b s e r v e d as the c o n c e n t r a t i o n o f 5-OH-PF was i n c r e a s e d ; however, t h i s 20 40 60 ao BOUND 5—HY0ROXYPROPA5TNONE (x 1 OE—6) MOUrt 20 40 60 BOUND 5-HYDROXYPROPAFENONE (x 1 0E - 6 ) MOLAR 80 100 F i g u r e 8. The r e l a t i o n s h i p between the r a t i o o f bound drug c o n c e n t r a t i o n / f r e e drug c o n c e n t r a t i o n ± SD vs. bound drug c o n c e n t r a t i o n o f 5-hydroxypropafenone i n serum. a) Rosenthal p l o t o f s u b j e c t 1. b) Rosenthal p l o t o f s u b j e c t 2. 20 40 60 80 100 BOUND 5-HYDROXYPROPAFENONE (x 1 0 E - 6 ) MOLAR 20 40 60 80 100 BOUND 5-HYDROXYPROPAFENONE (x 1 0 E - 6 ) MOLAR F i g u r e 8. con t . The r e l a t i o n s h i p between the r a t i o o f bound drug c o n c e n t r a t i o n / f r e e drug c o n c e n t r a t i o n + SD vs. bound drug c o n c e n t r a t i o n o f 5-hydroxypropafenone i n serum. c) Rosenthal p l o t o f s u b j e c t 3. d) Rosenthal p l o t o f s u b j e c t 4. 20 40 60 80 100 120 B O U N D . 5 - H Y D R O X Y P R O P A F E N O N E (x 1 0 E - 6 ) MOLAR F i g u r e 8. c o n t . The r e l a t i o n s h i p between the r a t i o o f bound drug c o n c e n t r a t i o n / f r e e drug c o n c e n t r a t i o n + SD vs. bound drug c o n c e n t r a t i o n o f 5-hydroxypropafenone i n serum. e) Rosenthal p l o t o f s u b j e c t 5. 75 d i d not r e a c h s t a t i s t i c a l s i g n i f i c a n c e ( t a b l e 4) TABLE 4. 5-HYDROXYPROPAFENONE FREE FRACTION IN NORMAL SERA WITH INCREASING CONCENTRATIONS OF 5-HYDROXYPROPAFENONE TOTAL CONCENTRATION OF MEAN FREE FRACTION OF 5-HYDROXYPROPAFENONE (/zg/mL) 5-HYDROXYPROPAFENONE 0.06 ± 0.01 0.094 ± 0.013 0.12 ± 0.02 0.154 ± 0.038 0.28 ± 0.03 0.187 ± 0.036 0.55 ± 0.06 0.180 ± 0.016' 3.52 ± 0.43 0.179 ± 0.044 8.01 ± 2.32 0.181 ± 0.035 17.26 ± 3.29 0.193 ± 0.011 35.30 ± 5.31 0.211 ± 0.034 ** S t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e u s i n g K r u s k a l 1 - W a l 1 i s t e s t w i t h p < 0.05 3.2.1 PROTEIN BINDING OF THE METABOLITE, 5-HYDROXYPROPAFENONE, IN THE PRESENCE OF THE PARENT COMPOUND PROPAFENONE AT A THERAPEUTIC CONCENTRATION AS DETERMINED BY EQUILIBRIUM DIALYSIS AND ULTRAFILTRATION. The b i n d i n g o f the m e t a b o l i t e 5-OH-PF a t a c o n c e n t r a t i o n o f 0.5 /zg/mL remained u n a l t e r e d by the a d d i t i o n o f a t h e r a p e u t i c c o n c e n t r a t i o n o f 2.0 /zg/mL o f the p a r e n t compound, PF ( t a b l e 5 ) . The r e s u l t s o b t a i n e d i n t h e s t u d y o f p r o t e i n b i n d i n g o f PF.and 5-OH-PF u s i n g u l t r a f i l t r a t i o n were c o n s i s t e n t l y lower compared t o the r e s u l t s o b t a i n e d from e q u i l i b r i u m d i a l y s i s ( T a b l e 5 ) . 76 TABLE 5 ASSESSMENT OF THE BINDING OF THE METABOLITE 5 -HYDROXYPROPAFENONE IN VITRO BY EQUILIBRIUM DIALYSIS AND ULTRAFILTRATION WITH AND WITHOUT THE ADDITION OF THERAPEUTIC CONCENTRATIONS OF THE PARENT COMPOUND PROPAFENONE. TREATMENT FREE FRACTION EQUILIBRIUM DIALYSIS ± SD FREE FRACTION ULTRAFILTRATION ± SD 5-HYDROXYPROPAFENONE ALONE (0.5 nq/ml) 5-HYDROXYPROPAFENONE (0.5 M9/mL) WITH PROPAFENONE (2.0 ,ug/mL) PROPAFENONE (2.0 M9/mL) WITH 5-HYDROXY-PROPAFENONE (0.5 Mg/mL) 0.223 ± 0.015 0.232 ± 0.020 0.063 ± 0.004 0.160 ± 0.003 0.154 ± 0.008 0.019 + 0.003 3.3 BINDING PROFILES OF PROPAFENONE AND 5-HYDROXYPROPAFENONE TO ISOLATED HUMAN SERUM PROTEINS, LIPOPROTEIN DEFICIENT SERUM, AND NORMAL SERUM. 3.3.1 BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE TO ISOLATED HUMAN SERUM PROTEINS. The d i f f e r e n c e s i n the e x t e n t o f b i n d i n g o f PF and 5-OH-PF t o i s o l a t e d human serum p r o t e i n s i n a system c o n t a i n i n g t h e r a p e u t i c c o n c e n t r a t i o n s o f both PF and 5-5-hydroxypropafenone was a s s e s s e d . Large d i f f e r e n c e s i n the f r e e f r a c t i o n o f PF and 5-OH-PF were o b s e r v e d i n s o l u t i o n s o f AAG d i s s o l v e d i n b u f f e r , AAG p l u s HSA d i s s o l v e d i n b u f f e r , serum, and l i p o p r o t e i n d e f i c i e n t serum ( F i g u r e 9 ) . A s m a l l e r d i f f e r e n c e was o b s e r v e d between t he b i n d i n g o f PF and 5-OH-PF i n a s o l u t i o n o f HSA d i s s o l v e d i n b u f f e r ( F i g u r e 9 ) . • 5 -HYDROXYPROPAFENONE E S PROPAFENONE 3 CD Q O < I I < X Z L i J t — O rr -a. o CJ >-3 rr < I I < I o rr CL o CJ Q_ >-3 CD _ J _ J — ' < < O Z O 2 rr L U rr o 3 0 - U J w Q C O 0.400 • 0.000 • TREATMENT GROUP F i g u r e 9. Comparison o f b i n d i n g o f propafenone (2.0 ng/mL) and 5-hyd r o x y p r o p a f e n o n e (0.5 Mg/mL) i n AAG, HSA, HSA + AAG, serum and l i p o p r o t e i n d e f i c i e n t serum. HSA (39.0 mg/mL) i n 0.1 M phosphate pH 7.51 ± 0.01 b u f f e r . Human AAG (0.71 mg/mL) i n 0.1 M phosphate pH 7.55 ± 0.05 b u f f e r . HSA (39.0 mg/mL) and Human AAG (0.68 mg/mL) i n 0.1 M phosphate pH 7.50 0.01 b u f f e r . Serum (47.0 mg/mL t o t a l p r o t e i n ) pH 7.40 ± 0.05. L i p o p r o t e i n d e f i c i e n t serum (36.5 mg/mL t o t a l p r o t e i n ) pH 7.52 ± 0.03. ( F r e e f r a c t i o n ± SD). 78 3.3.2 BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE TO PURE ISOLATED HUMAN SERUM PROTEINS DISSOLVED IN EITHER BUFFER OR SERUM ULTRAFILTRATE. Pure human serum p r o t e i n s (HSA, AAG, and HSA p l u s AAG) were d i s s o l v e d i n e i t h e r b u f f e r o r serum u l t r a f i l t r a t e ( f o r the c o m p o s i t i o n o f serum u l t r a f i l t r a t e see Appendix 3 ) . The b i n d i n g ( f r e e f r a c t i o n ) o f PF and 5-OH-PF t o t h e s e p r o t e i n s was then a s s e s s e d . The d i f f e r e n c e i n b i n d i n g o f PF t o AAG i n b u f f e r vs. serum u l t r a f i l t r a t e d i d re a c h s t a t i s t i c a l s i g n i f i c a n c e [two sample t - t e s t p < 0.05] ( F i g u r e 10). F u r t h e r , no d i f f e r e n c e i n pH between the b u f f e r and serum u l t r a f i l t r a t e , which c o u l d a c c o u n t f o r the o b s e r v e d b i n d i n g d i f f e r e n c e o f PF i n AAG, was o b s e r v e d . No s t a t i s t i c a l d i f f e r e n c e was o b s e r v e d between PF b i n d i n g t o HSA d i s s o l v e d i n b u f f e r and serum u l t r a f i l t r a t e ( F i g u r e 10). However, a s t a t i s t i c a l d i f f e r e n c e was a p p a r e n t [two sample t - t e s t p < 0.05] between b u f f e r and u l t r a f i l t r a t e when both AAG and HSA were d i s s o l v e d t o g e t h e r ( F i g u r e 10). The d i f f e r e n c e i n pH between the AAG t o g e t h e r w i t h HSA i n e i t h e r b u f f e r o r u l t r a f i l t r a t e a l s o r e a c h e d s t a t i s t i c a l s i g n i f i c a n c e [two sample t - t e s t p < 0.05]. The use o f b u f f e r o r u l t r a f i l t r a t e t o d i s s o l v e t he pure i s o l a t e d human serum p r o t e i n s d i d not r e s u l t i n any s i g n i f i c a n t d i f f e r e n c e s i n the b i n d i n g o f 5-OH-PF [two sample t - t e s t p=0.05] t o AAG, HSA, and HSA t o g e t h e r w i t h AAG ( F i g u r e 11). 79 0.800 - r 3 CD I o QL I CL < O X O °- >-, _ J —' < o < I T < X Q_ o D_ O o >-L~Z3 SERUM ULTRAFILTRATE BUFFER < < o • * TREATMENT GROUP F i g u r e 10. The e f f e c t - o f b u f f e r vs. human serum u l t r a f i l t r a t e as s o l v e n t s t o d i s s o l v e AAG, HSA, and AAG + HSA on the f r e e f r a c t i o n (± SD) o f p r o p a f e n o n e (2.0 /xg/mL). Human AAG (0.71 mg/mL) i n 0.1 M phosphate pH 7.50 ± 0.01 b u f f e r , and (0.67 mg/mL) i n human serum u l t r a f i l t r a t e pH 7.48 ± 0.08. HSA (39.0 mg/mL) i n 0.1 M phosphate pH 7.51 ± 0.01 b u f f e r , and (40.0 mg/mL) i n human serum u l t r a f i l t r a t e pH 7.40 ± 0.07 HSA (39.0 mg/mL) + human AAG (0.68 mg/mL) i n 0.1 M phosphate pH 7.50 ± 0.01 b u f f e r , and 39.1 mg/mL and 0.71 mg/mL, r e s p e c t i v e l y , i n human serum u l t r a f i l t r a t e pH 7.32 ± 0.04. * S t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e ; two sample t - t e s t w i t h p=0.05 between t r e a t m e n t groups o f same drug. 80 3 m < Q z •AC TEI Q z l o X O LJ 1— *— ce (— < 1 o_ 5 1 o < o z m I o | CL CL >-j < o 1 < LBU LPH LYC < < o CZD SERUM ULTRAFILTRATE BUFFER TREATMENT GROUP F i g u r e 11. The e f f e c t o f b u f f e r vs. human serum u l t r a f i l t r a t e as s o l v e n t s t o d i s s o l v e AAG, HSA, and AAG + HSA on the f r e e f r a c t i o n (± SD) o f 5 - h y d r o x y - p r o p a f e n o n e (0.5 ng/ml). Human AAG (0.71 mg/mL) i n 0.1 M phosphate pH 7.50 + 0.01 b u f f e r , and (0.67 mg/mL) i n human serum u l t r a f i l t r a t e pH 7.48 ± 0.08. HSA (39.0 mg/mL) i n 0.1 M phosphate pH 7.51 ± 0.01 b u f f e r , and (40.0 mg/mL) i n human serum u l t r a f i l t r a t e pH 7.40 ± 0.07 HSA (39.0 mg/mL) + human AAG (0.68 mg/mL) i n 0.1 M phosphate pH 7.50 ± 0.01 b u f f e r , and 39.1 mg/mL and 0.71 mg/mL, r e s p e c t i v e l y , i n human serum u l t r a f i l t r a t e pH 7.32 ± 0.04. * S t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e ; two t - t e s t w i t h p=0.05 between t r e a t m e n t g r o u p s o f same dru g . 81 3.3.3 THE BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE TO FREE FATTY ACID FREE HSA COMPARED TO NORMAL ALBUMIN. The b i n d i n g ( f r e e f r a c t i o n ) o f PF and 5-OH-PF t o f r e e f a t t y a c i d f r e e HSA and HSA w i t h endogenous f r e e f a t t y a c i d s was a s s e s s e d . The d i f f e r e n c e i n t h e f r e e f r a c t i o n s o f PF and 5-OH-PF between f r e e f a t t y a c i d f r e e HSA and HSA was s t a t i s t i c a l l y s i g n i f i c a n c e [two sample t - t e s t p=0.05]. The f r e e f r a c t i o n o f both PF and 5-OH-PF d e c r e a s e d i n t h e f r e e f a t t y a c i d HSA group compared t o the normal HSA group. I t s h o u l d be not e d t h a t t h e pH d i f f e r e n c e between t he f r e e f a t t y a c i d f r e e HSA and HSA c o u l d a c c o u n t f o r the o b s e r v e d s t a t i s t i c a l d i f f e r e n c e i n t h e f r e e f r a c t i o n o f PF and 5-OH-PF i n t h e s e t r e a t m e n t groups ( F i g u r e 12). The pH i n the HSA group was 7.51 ± 0.03 vs. 7.60 ± 0.05 i n t h e f r e e f a t t y a c i d f r e e HSA group. 3.3.4 BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE TO LIPOPROTEIN DEFICIENT SERUM vs. NORMAL SERUM D i f f e r e n c e s i n b i n d i n g due t o the p r e s e n c e o r absence o f human l i p o p r o t e i n s i n serum were a s s e s s e d . There was no s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e [two sample t - t e s t p > 0.05] between t he f r e e f r a c t i o n o f PF i n l i p o p r o t e i n d e f i c i e n t serum vs. normal serum ( F i g u r e 13). However, a s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e was a p p a r e n t between t h e f r e e f r a c t i o n o f 5-OH-PF i n l i p o p r o t e i n d e f i c i e n t serum vs. normal serum [two sample t - t e s t p < 0.05] ( F i g u r e 13). 82 C D 5-HYDROXYPROPAFENONE S 3 PROPAFENONE TREATMENT F i g u r e 12. Comparison between the f r e e f r a c t i o n (± SD) o f propafe n o n e (2.0 ug/ml) + 5-hydroxypropafenone (0.5 ug/ml) i n f r e e f a t t y a c i d f r e e albumin, and i n HSA. HSA (39.0 mg/mL) i n 0.1 M phosphate pH 7.51 ± 0.01 b u f f e r . F r e e f a t t y a c i d f r e e HSA (35.8 mg/mL) i n 0.1 M phosphate pH 7.60 ± 0.02 b u f f e r . * S t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e ; two t - t e s t w i t h p=0.05 between t r e a t m e n t groups o f same dru g . 83 • 5 -HYDROXYPROPAFENONE K S PROPAFENONE 0.400 - r TREATMENT F i g u r e 13. Comparison o f the f r e e f r a c t i o n (± SD) o f pr o p a f e n o n e (2.0 /xg/mL) and 5-hydroxypropafenone (0.5 /xg/mL) i n l i p o p r o t e i n d e f i c i e n t serum, and normal serum. Serum: T o t a l p r o t e i n 46.8 mg/mL, AAG 0.69 mg/mL pH 7.40 ± 0.05. L i p o p r o t e i n d e f i c i e n t serum: T o t a l p r o t e i n 36.5 mg/mL, AAG 0.90 mg/mL pH 7.52-. * S t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e ; two t - t e s t w i t h p=0.05 between t r e a t m e n t groups o f the same dru g . 84 3.3.5 DISPLACEMENT OF PROPAFENONE AND 5-HYDROXYPROPAFENONE FROM PROTEIN BINDING SITES BY IBUPROFEN AND DISOPYRAMIDE. The e f f e c t s o f the a d d i t i o n o f drugs c a p a b l e o f d i s p l a c i n g o t h e r d r u g s from serum p r o t e i n b i n d i n g s i t e s were e v a l u a t e d f o r t h e b i n d i n g o f PF and 5-OH-PF i n serum. I b u p r o f e n , a n o n - s t e r o i d a l a n t i - i n f l a m m a t o r y d r u g commonly used as a probe t o d i s p l a c e a c i d drugs from HSA, was found t o i n c r e a s e t he f r e e f r a c t i o n o f PF i n serum by 37% and 35% compared t o c o n t r o l a t c o n c e n t r a t i o n s o f 2.0 and 50.0 /zg/mL r e s p e c t i v e l y ( T a b l e 6 ) . F u r t h e r , i b u p r o f e n was a l s o found t o i n c r e a s e t h e f r e e f r a c t i o n o f 5-OH-PF i n serum by 21 % and 27% compared t o c o n t r o l a t c o n c e n t r a t i o n s o f 2.0 and 50.0 zzg/mL r e s p e c t i v e l y ( T a b l e 6). TABLE 6. DISPLACEMENT OF PROPAFENONE AND 5-HYDROXYPROPAFENONE FROM HUMAN SERUM PROTEIN BINDING SITES BY DISOPYRAMIDE AND IBUPROFEN TREATMENT MEAN FREE FRACTION OF 5-HYDR0XY-PR0PAFEN0NE ( ± SD) MEAN FREE FRACTION OF PROPAFENONE ( ± SD) CONTROL 0.215 ± 0.005 0.063 ± 0.004 IBUPROFEN 2.0 ug/ml IBUPROFEN 50.0 ug/ml 0.260 ± 0.016* 0.273 + 0.010 0.086 ± 0.013* 0.084 ± 0.003 DISOPYRAMIDE 2.0 ug/ml DISOPYRAMIDE 8.0 ug/ml 0.275 ± 0.013* 0.265 ± 0.033 0.110 ± 0.006* 0.133 ± 0.008 * S t a t i s t i c a l l y s i g n i f i c a n t from c o n t r o l u s i n g a Kruskal1-Wal 1 i s t e s t w i t h p < 0.05 Di s o p y r a m i d e , i s an amine a n t i a r r h y t h m i c d r u g known t o b i n d s p e c i f i c a l l y w i t h h i g h a f f i n i t y t o AAG [Kremer e t al., 1988], thus 85 d i s o p y r a m i d e was used as a probe f o r d i s p l a c i n g b a s i c drugs such as PF and 5-OH-PF from s p e c i f i c AAG b i n d i n g s i t e s . At c o n c e n t r a t i o n s o f 2.0 and 8.0 /zg/mL, d i s o p y r a m i d e was found t o i n c r e a s e t h e f r e e f r a c t i o n o f both 5-OH-PF and PF. The i n c r e a s e i n f r e e f r a c t i o n o f 5-OH-PF was 28% and 23% o f c o n t r o l a t c o n c e n t r a t i o n s o f 2.0 and 8.0 fig/ml o f d i s o p y r a m i d e r e s p e c t i v e l y . The i n c r e a s e i n the f r e e f r a c t i o n o f PF was s i g n i f i c a n t l y l a r g e r than 5-OH-PF (74% and 111% o f c o n t r o l at 2.0 and 8.0 ng/ml o f d i s o p y r a m i d e r e s p e c t i v e l y ) . 3.3.6 DISPLACEMENT OF PROPAFENONE AND 5-HYDROXYPROPAFENONE FROM ISOLATED HUMAN SERUM PROTEINS BY DISOPYRAMIDE AND IBUPR0FEN The i n f l u e n c e o f the a d d i t i o n o f the b i n d i n g d i s p l a c e r s , such as i b u p r o f e n and d i s o p y r a m i d e , t o i s o l a t e d p r o t e i n s was a s s e s s e d . S t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e s i n the f r e e f r a c t i o n o f PF were o b s e r v e d i n a l l groups ( HSA, AAG) t r e a t e d w i t h t h e d i s p l a c i n g agent d i s o p y r a m i d e i n comparison t o c o n t r o l ( F i g u r e 15) [two sample t - t e s t p < 0.05]. N o t a b l e d i f f e r e n c e s i n pH which c o u l d a l t e r b i n d i n g were o b s e r v e d o n l y i n the HSA group. The a d d i t i o n o f 50 ng/ml o f i b u p r o f e n a l s o r e s u l t e d i n s t a t i s t i c a l l y s i g n i f i c a n t [two sample t - t e s t p=0.05] d i f f e r e n c e s i n the f r e e f r a c t i o n o f PF when compared t o c o n t r o l ( F i g u r e 14) . B i n d i n g o f 5-0H-PF i n HSA and AAG was a l t e r e d by the a d d i t i o n o f d i s o p y r a m i d e o n l y i n the HSA group [two sample t - t e s t p < 0.05] ( F i g u r e 15) . Th e r e was no ap p a r e n t d i f f e r e n c e s between t he b i n d i n g o f 5-0H-PF w i t h t h e a d d i t i o n o f d i s o p y r a m i d e i n the AAG t e s t group. A pH d i f f e r e n c e i n the HSA was o b s e r v e d between the c o n t r o l and 86 a. 3 S3 PROPAFENONE o a: z o o irk • o in o o o or + I—I 5 - H Y D R O X Y -PROPAFENONE o.ooa - 1 1 ' 1 1 1 ' — TREATMENT GROUP F i g u r e 14. The e f f e c t o f the a d d i t i o n o f i b u p r o f e n (50.0 ug/ml) on the f r e e f r a c t i o n (± SD) o f propafenone (2.0 ug/ml) and 5-h y d r o x y p r o p a f e n o n e (0.5 ug/ml) Serum: T o t a l p r o t e i n 46.5 mg/mL, AAG 0.69 mg/mL pH ( t e s t ) 7.43 and ( c o n t r o l ) 7.40. HSA: T o t a l p r o t e i n 39.0 mg/mL i n 0.1 M phosphate pH ( t e s t ) 7.51 and ( c o n t r o l ) 7.50 b u f f e r . AAG: AAG 0.71 mg/mL i n 0.1 M phosphate pH ( t e s t ) 7.50 and ( c o n t r o l ) 7.55 b u f f e r . * S t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e ; two t - t e s t w i t h p=0.05 between t r e a t m e n t groups o f the same drug. ** S t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e ; Mann Whitney U - t e s t w i t h p=0.05 between t r e a t m e n t groups o f the same dru g . 87 E 3 PROPAFENONE CD 5-HYDROXY-PROPAFENONE TREATMENT GROUP F i g u r e 15. The e f f e c t o f the a d d i t i o n o f d i s o p y r a m i d e (8.0 /ig/mL) . on t h e f r e e f r a c t i o n ± SD o f propafenone (2.0 ug/ml) and 5-hy d r o x y p r o p a f e n o n e (0.5 ug/ml). Serum: T o t a l p r o t e i n 46.5 mg/mL, AAG 0.69 mg/mL pH ( t e s t ) 7.40 and ( c o n t r o l ) 7.41. HSA: T o t a l p r o t e i n 39.0 mg/mL i n 0.1 M phosphate pH ( t e s t ) 7.60 and ( c o n t r o l ) 7.50 b u f f e r . AAG: AAG 0.71 mg/mL i n 0.1 M phosphate pH ( t e s t ) 7.50 and ( c o n t r o l ) 7.55 b u f f e r . * S t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e ; two t - t e s t w i t h p=0.05 between t r e a t m e n t groups o f the same d r u g . ** S t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e ; Mann Whitney U - t e s t w i t h p=0.05 between t r e a t m e n t groups o f the same drug. 88 d i s o p y r a m i d e group. The a d d i t i o n o f i b u p r o f e n a l t e r e d t h e f r e e f r a c t i o n o f 5-OH-PF o n l y i n the HSA group [two sample t - t e s t p < 0.05] ( F i g u r e 1 4). The a d d i t i o n o f 50 Mg/mL o f i b u p r o f e n d i d not r e s u l t i n any s i g n i f i c a n t d i f f e r e n c e i n the b i n d i n g o f 5-OH-PF t o AAG [two sample t - t e s t p > 0.05]. 3.3.7 PURITY ASSURANCE OF ISOLATED PROTEIN IN BINDING EXPERIMENTS The c o m p o s i t i o n and c o n c e n t r a t i o n s o f p r o t e i n s i n each o f the p r o t e i n s o l u t i o n s used i n the above e x p e r i m e n t s was d e t e r m i n e d and i s p r e s e n t e d i n Appendix 2. The c o m p o s i t i o n o f t h e u l t r a f i l t r a t e s o l u t i o n i s compared t o normal serum i n Appendix 3. F i n a l l y , t h e SDS PAGE s e p a r a t i o n s t u d i e s t o a s s u r e p u r i t y r e s u l t s o f the i s o l a t e d p r o t e i n b i n d i n g s o l u t i o n s used i n the mentioned b i n d i n g e x p e r i m e n t s a r e p r e s e n t e d i n Appendix 4. 3.4 BINDING CHARACTERISTICS OF PROPAFENONE AND 5-HYDROXYPROPAFENONE. 3.4.1 BINDING CHARACTERISTICS OF PROPAFENONE AND 5-HYDROXYPROPAFENONE IN ALPHA-1-ACID GLYCOPROTEIN SOLUTION. The b i n d i n g i n t e r a c t i o n o f PF and 5-OH-PF w i t h AAG was q u a l i t a t i v e l y e v a l u a t e d u s i n g a R o s e n t h a l p l o t . B i n d i n g o f PF and 5-OH-PF was measured o v e r a wide range o f dru g c o n c e n t r a t i o n s , and t h e b i n d i n g r a t i o ( b o u n d / f r e e ) was p l o t t e d a g a i n s t t h e c o n c e n t r a t i o n o f bound drug ( F i g u r e 16a and 16b). Curve f i t t i n g was done u s i n g t he 89 +0 - r „ 20 - °& 20 - -oo 10 - -0.000 oag 0.020 0.030 0.040 40 - r BOUND PROPAFENONE CONCENTRATION (mmolcr) 3 0 - -10 - -0 .010 BOUND 5 - H Y D R O X Y P R O P A F E N O N E CONCENTRATION (mmolar) u -r 0 .000 0 . 010 0 . 0 2 0 0 . 0 3 0 0 . 0 4 0 F i g u r e 16. R e l a t i o n s h i p between the r a t i o o f bound drug c o n c e n t r a t i o n / f r e e drug c o n c e n t r a t i o n vs. bound drug c o n c e n t r a t i o n o f propafe n o n e and 5-hydroxypropafenone i n a p h y s i o l o g i c a l c o n c e n t r a t i o n o f AAG (19.3 fiH) i n 0.1 phosphate pH 7.40 b u f f e r . R o s e n t h a l p l o t o f p r o p a f e n o n e ( o ) R o s e n t h a l p l o t o f 5-hydroxypropafenone ( A ) 90 program ENZFITTER R o f t h e n o n - t r a n s f o r m e d d a t a [ L e a t h e r b a r r o w , 1987]. PF appears t o have two d i s t i n c t t y p e s o f b i n d i n g s i t e s on AAG; one h i g h a f f i n i t y , low c a p a c i t y (Kaj 2.94 X 1 0 6 M" 1, NPj 1.1 X 1 0 " 5 M, Nj 5.7 X 1 0 " 1 ) , and one low a f f i n i t y h i g h c a p a c i t y ( K a 2 3.20 X 1 0 2 M" 1, NP 2 6.6 X 1 0 " 4 M, N 2 3.40 X 1 0 1 ) , whereas 5-OH-PF o n l y shows one d i s t i n c t t y p e o f b i n d i n g s i t e (Ka 3.13 X 1 0 5 M" 1, NP 9.4 X 1 0 " 5 M, N 4.87 X 10" 1) on AAG. PF b i n d i n g t o AAG shows c o n c e n t r a t i o n - d e p e n d e n t b i n d i n g a t low c o n c e n t r a t i o n s , f o l l o w e d by a sharp r i s e i n t h e f r e e f r a c t i o n a t h i g h drug c o n c e n t r a t i o n s ( F i g u r e 2 2 ) . A l t h o u g h 5-OH-PF shows a s i m i l a r sharp r i s e i n t h e f r e e f r a c t i o n a t h i g h c o n c e n t r a t i o n s o f d r u g , t h e r e i s l i t t l e e v i d e n c e t o s u g g e s t t h a t 5-OH-PF d i s p l a y s c o n c e n t r a t i o n dependent b i n d i n g t o AAG a t l o w e r c o n c e n t r a t i o n s s i m i l a r t o t h a t o b s e r v e d f o r PF ( F i g u r e 2 3 ) . 3.4.2 BINDING CHARACTERISTICS OF PROPAFENONE AND 5-HYDROXYPROPAFENONE IN HUMAN SERUM ALBUMIN SOLUTION Both PF and 5-OH-PF te n d t o b i n d t o a l e s s e r d e g r e e i n human serum HSA s o l u t i o n as e v i d e n c e d by t h e i r r e s p e c t i v e R o s e n t h a l p l o t s ( F i g u r e s 17a and 17b). F o l l o w i n g n o n - l i n e a r r e g r e s s i o n w i t h ENZFITTER R o f the n o n - t r a n s f o r m e d d a t a [ L e a t h e r b a r r o w , 1987], t h e 1 s p e c i f i c s i t e model d i s p l a y e d the b e s t f i t w i t h the b e s t random d i s t r i b u t i o n o f y - a x i s r e s i d u a l s . The b i n d i n g o f both PF and 5-OH-PF t o HSA was low a f f i n i t y and h i g h c a p a c i t y (PF - Ka 5.3 X 1 0 2 M" 1, NP 2.5 X 1 0 " 3 M, N 3.9; 5-OH-PF - Ka 2.1 X 1 0 2 M" 1, NP 7.8 X 1 0 " 3 M, N 1.24 X 1 0 1 ) . PF and 5-OH-PF a l s o do not show c o n c e n t r a t i o n dependent b i n d i n g t o HSA as would be e v i d e n c e d by s t e a d i l y i n c r e a s i n g f r e e f r a c t i o n as t h e c o n c e n t r a t i o n 91 \ Q Z O c n 10 - r -v7 0 .000 10 rr U -\ a z 3 O CD. 2 0 .020 0 .040 0 .060 0 .080 0 .100 BOUND PROPAFENONE CONCENTRATION (mmo la r ) 0 .120 + + . 0 0 0 0 . 0 2 0 0 . 0 4 0 0 . 0 6 0 0 . 0 8 0 0 . 1 0 0 B O U N D 5 - H Y D R O X Y P R O P A F E N O N E CONCENTRATION ( m m o l a r ) 0 . 1 2 0 F i g u r e 17. R e l a t i o n s h i p between the r a t i o o f bound drug c o n c e n t r a t i o n / f r e e drug c o n c e n t r a t i o n vs. bound d r u g c o n c e n t r a t i o n o f pro p a f e n o n e and 5-hydroxypropafenone i n a p h y s i o l o g i c a l c o n c e n t r a t i o n o f HSA (630.0 juM) i n 0.1 phosphate pH 7.36 b u f f e r . R o s e n t h a l p l o t o f propafenone ( v ) R o s e n t h a l p l o t o f 5-hydroxypropafenone ( •) 1.400 - r 1000 1E4 L O G C O N C E N T R A T I O N O F P R O P A F E N O N E ( n g / m L ) F i g u r e 22. Free f r a c t i o n + SD o f pr o p a f e n o n e a t v a r i o u s c o n c e n t r a t i o n s i n s o l u t i o n s o f human serum p r o t e i n s . HSA (630 izM) 40.9 mg/mL ( A ) pH 7.36. Human AAG (19.3 /zM) 0.88 mg/mL ( 0 ) pH 7.40. Serum ( t o t a l p r o t e i n 48.9 mg/mL, Human AAG 0.79 mg/mL) ( o ) pH 7.47. ; HDL complex (15.3 /xM) 3.8 mg/mL (•) pH 7.32. LDL complex (1.8 iM) 4.2 mg/mL (•) pH 7.32. V l d l complex (0.17 juM) 1.2 mg/mL (•) pH 7.32. 1.000 - r Z o 0.800 0.600 — < L L . L j j 0 .400 L d Lu 0 .200 0.000 100 1000 1E4 L O G C O N C E N T R A T I O N O F 5 - H Y D R O X Y P R O P A F E N O N E F i g u r e 23. Free f r a c t i o n + SD o f 5-hydroxypropafenone a t v a r i o u s c o n c e n t r a t i o n s i n s o l u t i o n s o f human serum p r o t e i n s . HSA (630 /zM) 40.9 mg/mL ( A ) pH 7.36. Human AAG (19.3 uH) 0.88 mg/mL (•) pH 7.40. Serum ( t o t a l p r o t e i n 48.9 mg/mL, Human AAG 0.79 mg/mL) ( o ) pH 7.47. HDL complex (15.3 /zM) 3.8 mg/mL (•) pH 7.32. LDL complex (1.8 uM) 4.2 mg/mL ( A ) pH 7.32. V l d l complex (0.17 uM) 1.2 mg/mL ( • ) pH 7.32. ID CO 94 i n c r e a s e s ( F i g u r e s 22 and 23) 3.4.3 BINDING CHARACTERISTICS OF PROPAFENONE AND 5-HYDROXYPROPAFENONE IN POOLED HUMAN SERUM. The R o s e n t h a l p l o t o f PF i n d i c a t e d t h a t two d i s t i n c t t y p e s o f b i n d i n g s i t e s were p r e s e n t i n p o o l e d human serum ( F i g u r e 18a). F o l l o w i n g c u r v e f i t t i n g (ENZFITTER^) o f the n o n - t r a n s f o r m e d d a t a two d i s t i n c t b i n d i n g s i t e s were appa r e n t (Kaj 9.16 X 1 0 4 M" 1, NPj 9.51 X 10" 5 M, K a 2 2.31 X 1 0 3 M" 1, NP 2 1.66 X 10" 3 M) f o r PF i n serum. While the R o s e n t h a l p l o t o f 5-OH-PF b i n d i n g i n p o o l e d serum shows some s c a t t e r ( F i g u r e 18b), f o l l o w i n g c u r v e f i t t i n g 1 b i n d i n g s i t e was a p p a r e n t (Ka 2.06 X 1 0 4 M " 1 , NP 2.20 X 1 0 " 4 M). C o n c e n t r a t i o n dependent b i n d i n g o f PF i n p o o l e d human serum, was o b s e r v e d p a r t i c u l a r l y a t h i g h e r c o n c e n t r a t i o n s ( F i g u r e 2 2 ) . The m e t a b o l i t e , 5-OH-PF, does not seem t o undergo c o n c e n t r a t i o n dependent b i n d i n g , as e v i d e n c e d by the r e l a t i v e l y c o n s t a n t f r e e f r a c t i o n o v e r the c o n c e n t r a t i o n s examined ( F i g u r e 23). 3.4.4 BINDING CHARACTERISTICS OF PF AND 5-HYDROXYPROPAFENONE IN A SOLUTION OF HIGH DENSITY LIPOPROTEINS. The Rosenthal p l o t o f PF b i n d i n g t o h i g h d e n s i t y l i p o p r o t e i n s shows t h e p o s s i b l e p r e s e n c e o f two d i s t i n c t t y p e s o f b i n d i n g s i t e s ( F i g u r e 19a). However, f o l l o w i n g c u r v e f i t t i n g o f t h e n o n - t r a n s f o r m e d d a t a (ENZFITTER R) o n l y one s p e c i f i c s a t u r a b l e s i t e was a p p a r e n t (Ka 1.1 X 1 0 4 M" 1, NP 2.6 X 1 0 " 4 M, N 1.69 X 1 0 1 ) . Over a wide c o n c e n t r a t i o n range, PF ( i n a s o l u t i o n o f h i g h d e n s i t y l i p o p r o t e i n s ) U J o o m 70 60 50 40 -r A 30 - k 10 A A 0.000 70 • 60 0.050 0.100 0.150 B O U N D P R O P A F E N O N E CONCENTRATION ( m m o l a r ) 0.200 50 - -40 30 20 10 0 i t + -i 0.000 0.050 0.100 0.150 0.200 B O U N D 5 - H Y D R O X Y P R O P A F E N O N E CONCENTRATION ( m m o l a r ) F i g u r e 18. R e l a t i o n s h i p between the r a t i o o f bound drug c o n c e n t r a t i o n / f r e e drug c o n c e n t r a t i o n vs. bound d r u g c o n c e n t r a t i o n p r o p a f e n o n e and 5-hydroxypropafenone i n serum. Serum t o t a l p r o t e i n 49.0 mg/mL, AAG 0.79 mg/mL (19.3 txM) pH 7.47. Ro s e n t h a l p l o t o f propafenone ( A ) Ros e n t h a l p l o t o f 5-hydroxypropafenone ( A ) 96 10 -r \ Q 2 3 O 4 + " 0 3 2 0 0 . 0 0 0 10 -r Q Z 3 O m 2 - -a a • 0 0 .000 + 0 . 0 2 5 0 . 0 5 0 0 . 0 7 5 0 . 1 0 0 0 . 1 2 5 B O U N D P R O P A F E N O N E CONCENTRATION ( m m o l a r ) 0 . 0 2 5 0 .050 0 . 0 7 5 0 . 1 0 0 0 . 1 2 5 0 . 1 5 0 0 .150 BOUND 5—HYDROXYPROPAFENONE CONCENTRATION (mmola r ) F i g u r e 19. R e l a t i o n s h i p between the r a t i o o f bound drug c o n c e n t r a t i o n / f r e e drug c o n c e n t r a t i o n vs. bound dr u g c o n c e n t r a t i o n o f pro p a f e n o n e and 5-hydroxypropafenone i n a p h y s i o l o g i c a l c o n c e n t r a t i o n o f HDL (15.3 izM complex, 1.72 mg/mL t o t a l p r o t e i n ) i n 0.1 phosphate pH 7.32 b u f f e r . R o s e n t h a l p l o t o f propafenone ( Y ) Ros e n t h a l p l o t o f 5-hydroxypropafenone ( • ) 97 te n d s t o show a g r a d u a l r i s e i n f r e e f r a c t i o n which would s u g g e s t c o n c e n t r a t i o n - d e p e n d e n t b i n d i n g ( F i g u r e 2 2 ) . The R o s e n t h a l p l o t o f 5-OH-PF i n the HDL s o l u t i o n shows d a t a s c a t t e r ; however, o n l y one type o f b i n d i n g s i t e seems a p p a r e n t . F o l l o w i n g c u r v e f i t t i n g o n l y n o n - s p e c i f i c b i n d i n g s i t e was a p p a r e n t ( K n o n . S p e c ^ f - j c 3.08). When the a f f i n i t y o f t h e b i n d i n g s i t e i s v e r y s m a l l , t h e e q u a t i o n f o r t h e one s i t e b i n d i n g model c o l l a p s e s such t h a t the ( n o n - s p e c i f i c ) b i n d i n g i s the p r o d u c t o f t h e a f f i n i t y c o n s t a n t and the b i n d i n g c a p a c i t y ( s e e E q u a t i o n 11 S e c t i o n 2.8.2 [ G l a s s o n et al. 1980]). T h e r e i s no c o n c e n t r a t i o n - d e p e n d e n t b i n d i n g o f 5-OH-PF t o h i g h d e n s i t y l i p o p r o t e i n s ( F i g u r e 2 3 ) . 3.4.5 BINDING CHARACTERISTICS OF PROPAFENONE AND 5-HYDROXYPROPAFENONE IN A SOLUTION OF LOW DENSITY LIPOPROTEINS. The Rosenthal p l o t o f both p r o p a f e n o n e and 5-OH-PF i n a s o l u t i o n o f LDL tends t o s u g g e s t the p r e s e n c e o f o n l y one t y p e o f b i n d i n g s i t e ( F i g u r e 20a and 20b). In f a c t , o n l y n o n - s p e c i f i c b i n d i n g was a p p a r e n t f o r both PF ( K n o n . s p e c i f i c 13.9) and 5-OH-PF ( K n o n . s p e c i f i c 12.3) a f t e r m o d e l i n g and c u r v e f i t t i n g . The f r e e f r a c t i o n o f both PF and 5-OH-PF o v e r a wide range o f c o n c e n t r a t i o n s i n a s o l u t i o n o f low d e n s i t y l i p o p r o t e i n s i s v e r y low, and does not change w i t h i n c r e a s i n g c o n c e n t r a t i o n s . 3.4.6 BINDING CHARACTERISTICS OF PROPAFENONE AND 5-HYDROXYPROPAFENONE IN A SOLUTION OF VERY LOW DENSITY LIPOPROTEINS. The b i n d i n g o f PF t o v e r y low d e n s i t y l i p o p r o t e i n s was 98 5 0 - r Id z Z> o m 40 • 0 •> 3 0 - -2 0 o o p 10 -o o 0 • 0 . 0 0 0 50 • 1 1 1 • 1 0 . 0 5 0 0 . 1 0 0 0 . 1 5 0 0 . 2 0 0 B O U N D P R O P A F E N O N E CONCENTRATION ( m m o l a r ) 1 0 . 2 5 0 40 — 3 O 0 .000 0 .050 0 .100 0 . 1 5 0 0 .200 0 .250 BOUND 5 - H Y D R O X Y P R O P A F E N O N E CONCENTRATION (mmola r ) F i g u r e 20. R e l a t i o n s h i p between the r a t i o o f bound drug c o n c e n t r a t i o n / f r e e drug c o n c e n t r a t i o n vs. bound dr u g c o n c e n t r a t i o n o f PF and 5-hydroxypropafenone i n a p h y s i o l o g i c a l c o n c e n t r a t i o n o f LDL (1.8 /xM complex, 0.84 mg/mL t o t a l p r o t e i n ) i n 0.1 phosphate pH 7.32 b u f f e r . R o s e n t h a l p l o t o f propafenone ( 0 ) R o s e n t h a l p l o t o f 5-hydroxypropafenone ( A ) 99 v e r y low, and thus r e s u l t e d i n a poor s p r e a d o f d a t a on t h e x - a x i s o f the R o s e n t h a l p l o t ( F i g u r e 21a). A l t h o u g h t h e b i n d i n g 5-OH-PF was a l s o v e r y low, i t was not as low as PF b i n d i n g t o v e r y low d e n s i t y l i p o p r o t e i n s ( F i g u r e 21b). Both drugs d i s p l a y e d low n o n - s p e c i f i c b i n d i n g (PF - K n o n . s p e c i f i c 0.065, 5-OH-PF - K n o n _ s p e c i f i c 0.593). The f r e e f r a c t i o n o f PF was v e r y h i g h and n o n - u n i f o r m ( F i g u r e 2 2 ) . While t h e f r e e f r a c t i o n o f 5-OH-PF ov e r a wide c o n c e n t r a t i o n range was a l s o v e r y h i g h ( a p p r o x i m a t i n g 1 ), i t showed s i g n i f i c a n t l y l e s s s c a t t e r ( F i g u r e 2 3 ) . 3.4.7 MATHEMATICAL BINDING EQUATIONS FOR PROPAFENONE AND 5-HYDR0XYPR0PAFEN0NE. E q u a t i o n s d e s c r i b i n g the b e s t f i t o f PF and 5-OH-PF b i n d i n g t o i n d i v i d u a l p r o t e i n s were summed t o a r r i v e a t e q u a t i o n s t h a t would attempt t o approx i m a t e the b i n d i n g o f both PF and 5-OH-PF i n serum. PF bound = H O . O i l ) ( 2 9 4 3 ) ( D F ) + ( 0 . 3 2 ) ( 0 . 6 6 ) ( D F ) l a a 9 + (18) (mM) (1+2943(DF)) (1+0.32(DF)) [ ( 4 . 7 2 ) ( 0 . 0 6 ) ( D F ) + 0 . 6 9 3 ( D F ) ] a l b + (1+4.72(DF)) r ( 1 1 . 0 2 ) ( 0 . 2 6 ) ( D F ) l n d 1 + (1+11.02(DF) [ 1 3 . 9 ( D F ) ] 1 d 1 + [ 0 . 0 7 ( D F ) ] v 1 d l 100 o -f^-o.ooo + + 0 .010 0 .020 BOUND PROPAFENONE CONCENTRATION ( m m o l a r ) 0 .030 6 2 - -0 ^ -0.000 0.010 C.020 BOUNO S-HYDROXYPROPAFENONE CONCENTRATION (mmolar) 0.030 F i g u r e 21. R e l a t i o n s h i p between the r a t i o o f bound drug c o n c e n t r a t i o n / f r e e drug c o n c e n t r a t i o n vs. bound drug c o n c e n t r a t i o n o f prop a f e n o n e and 5-hydroxypropafenone i n a p h y s i o l o g i c a l c o n c e n t r a t i o n o f VLDL (0.17 iiM complex, 0.09 mg/mL t o t a l p r o t e i n ) i n 0.1 phosphate pH 7.32 b u f f e r . R o s e n t h a l p l o t o f propafenone ( 0 ) R o s e n t h a l p l o t o f 5-hydroxypropafenone ( v ) 101 5-OH-PF = r(313H0.0094HDFn a a9 + (19) bound (mM) (1+313(DF) r ( 0 . 2 1 H 7 . 8 3 U D F ) l a 1 b + (1+0.21(DF) [ 3 . 0 8 ( D F ) ] n d l + [ 1 2 . 3 ( D F ) ] l d l + [ 0 . 5 9 ( D F ) ] v 1 d l where DF r e p r e s e n t s t h e f r e e PF o r 5-OH-PF i n mMolar. The b i n d i n g a f f i n i t y and c a p a c i t y c o n s t a n t s were e s t i m a t e d from b i n d i n g o f PF and 5-OH-PF i n s o l u t i o n s o f i s o l a t e d serum p r o t e i n s (AAG, HSA, HDL, LDL, and VLDL) i n t h e p r e v i o u s s e c t i o n s . The amount o f bound PF and 5-OH-PF was c a l c u l a t e d and compared t o t h e a c t u a l d a t a p o i n t s F i g u r e s 23 and 24, r e s p e c t i v e l y . S t e p w i s e t he b i n d i n g c o n t r i b u t i o n o f i n d i v i d u a l p r o t e i n s was removed ( i e . PF bound - c o n t r i b u t i o n o f v e r y low d e n s i t y l i p o p r o t e i n s f o l l o w e d by PF bound - c o n t r i b u t i o n o f VLDL and LDL e t c . u n t i l t h e c o n t r i b u t i o n o f o n l y AAG remained) and the amount o f bound PF and 5-OH-PF c a l c u l a t e d and p l o t t e d ( F i g u r e s 24 and 25, r e s p e c t i v e l y ) . The b i n d i n g c a l c u l a t e d w i t h t h e mat h e m a t i c a l r e c o n s t i t u t i o n e q u a t i o n (composed o f the sums o f i n d i v i d u a l b i n d i n g c o n t r i b u t i o n s ) was c o n s i s t e n t l y h i g h f o r both PF and 5-OH-PF when compared t o the a c t u a l d a t a p o i n t s . Upon the removal o f both VLDL and LDL, the c a l c u l a t e d b i n d i n g more c l o s e l y resembled t h e a c t u a l b i n d i n g o f PF and 5-OH-PF ( F i g u r e s 24 and 25). 102 01 5 o 2 ^ 0.020 or Q Q Z o m 0.000 0.000 o.oot FREE DRUG (MMOLAR) 0.600 o 0.400 o r> rr Q Q z o m 0.200 o.ooo 0.002 0.022 F R E E D R U G ( M M O L A R ) F i g u r e 24. A d i r e c t p l o t o f propa f e n o n e b i n d i n g (bound vs. f r e e ) c a l c u l a t e d by m a t h e m a t i c a l l y r e c o n s t r u c t i n g b i n d i n g c o n t r i b u t i o n s o f serum p r o t e i n c o n s t i t u e n t s and subsequent removal o f b i n d i n g c o n t r i b u t i o n s o f i n d i v i d u a l serum p r o t e i n s . AAG + HSA + HDL + LDL + VLDL ( A ) AAG + HSA + HDL + LDL - VLDL ( A ) AAG + HSA + HDL - LDL - VLDL ( • ) AAG + HSA - HDL - LDL - VLDL ( 0 ) AAG - HSA - HDL - LDL - VLDL ( v ) CONTROL SERUM DATA ( o ) 103 rr o o rr Q ZD O CQ 0.600 0.200 3 o s o Z> or o 0.000 0.000 JUL 113= 89 0.001 FREE DRUG (MMOLAR) 0.000 0.002 0.022 FREE DRUG (MMOLAR) F i g u r e 25. A d i r e c t p l o t o f 5-hydroxypropafenone b i n d i n g (bound vs. f r e e ) c a l c u l a t e d by m a t h e m a t i c a l l y r e c o n s t r u c t i n g b i n d i n g c o n t r i b u t i o n s o f serum p r o t e i n c o n s t i t u e n t s and subsequent removal o f b i n d i n g c o n t r i b u t i o n s o f i n d i v i d u a l serum p r o t e i n s . AAG + HSA + HDL + LDL + VLDL ( A ) AAG + HSA + HDL + LDL - VLDL ( A ) AAG + HSA + HDL - LDL - VLDL (• ) AAG + HSA - HDL - LDL - VLDL ( 0 ) AAG - HSA - HDL - LDL - VLDL ( v ) CONTROL SERUM DATA ( o ) 104 3.4.8 PROTEIN QUALITY ASSURANCE DURING BINDING CHARACTERIZATION EXPERIMENTS T o t a l p r o t e i n c o n c e n t r a t i o n s , and the c o n c e n t r a t i o n o f AAG used i n the b i n d i n g c h a r a c t e r i z a t i o n e x p e r i m e n t s a re p r e s e n t e d i n Appendix 5. R e s u l t s from t h e a g a r o s e e l e c t r o p h o r e s i s s e p a r a t i o n b e f o r e and a f t e r d i a l y s i s o f the l i p o p r o t e i n s s u g g e s t t h a t t h e l i p o p r o t e i n complex remains i n t a c t d u r i n g d i a l y s i s (Appendix 6 ) . 3.5 UPTAKE OF PROPAFENONE AND 5-HYDROXYPROPAFENONE INTO RED BLOOD CELLS 3.5.1 TIME TO EQUILIBRIUM FOR RED BLOOD CELL UPTAKE OF PROPAFENONE AND 5-HYDROXYPROPAFENONE. I n i t i a l e x p e r i m e n t s t o d e t e r m i n e t he time c o u r s e o f r e d b l o o d c e l l u ptake o f PF and 5-OH-PF were c o n d u c t e d . The uptake e q u i l i b r i u m f o r PF and 5-OH-PF was v e r y r a p i d . Samples taken w i t h i n 5 minutes a f t e r the s t a r t o f i n c u b a t i o n showed uptake e q u i l i b r i u m was a t t a i n e d almost i m m e d i a t e l y , s i n c e t he c o n c e n t r a t i o n i n the r e d b l o o d c e l l s had not changed f o r the e n t i r e 35 minute i n c u b a t i o n t i m e . An i n c u b a t i o n time o f 30 minutes was chosen t o c o n d u c t a l l r e d b l o o d c e l l uptake e x p e r i m e n t s . 3.5.2 UPTAKE OF PROPAFENONE AND 5-HYDROXYPROPAFENONE BY RED BLOOD CELLS The uptake r a t i o ( c o n c e n t r a t i o n o f drug i n r e d b l o o d 105 c e l l s / c o n c e n t r a t i o n o f drug i n s u p e r n a t a n t ) o f both PF and 5-OH-PF i n r e d b l o o d c e l l s was s u b s t a n t i a l when b u f f e r was used as t h e s u p e r n a t e n t ( F i g u r e 2 6 ) . The uptake r a t i o o f 5-OH-PF was almost d o u b l e t h a t o f PF i n t h e b u f f e r s u p e r n a t e n t system. T h i s o b s e r v a t i o n d i d r e a c h s t a t i s t i c a l s i g n i f i c a n c e [two sample t - t e s t p > 0.05]. The uptake r a t i o i n r e d b l o o d c e l l : serum s u p e r n a t a n t system o f PF and 5-OH-PF was s u b s t a n t i a l l y l e s s than t h a t o b s e r v e d f o r t h e b u f f e r system ( F i g u r e 2 6 ) . The uptake r a t i o o f 5-OH-PF was alm o s t s i x f o l d g r e a t e r t h a t PF [two sample t - t e s t p < 0.05]. The uptake r a t i o o f PF and 5-0H-PF i n a whole b l o o d system showed s i m i l a r r e s u l t s when compared t o the system i n which serum was used as th e s u p e r n a t e n t ( F i g u r e 2 6 ) . The uptake r a t i o o f 5-OH-PF was alm o s t 5 f o l d g r e a t e r t h a n the uptake r a t i o o f PF [two sample t - t e s t p < 0.05]. 106 B U F F E R 10 I I 5 — H Y D R O X Y P R O P A F E N O N E P R O P A F E N O N E S E R U M W H O L E B L O O D T R E A T M E N T G R O U P F i g u r e 26. Comparison o f the r a t i o (drug c o n c e n t r a t i o n i n RBC/drug c o n c e n t r a t i o n i n s u p e r n a t a n t ) o f propafenone and 5-hydroxypropafenone w i t h b u f f e r , serum, and plasma as s u p e r n a t a n t s . RBC 45% v/v s u s p e n s i o n * S t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e ; two t - t e s t w i t h p < 0.05 between t r e a t m e n t g r o u p s o f the same drug. 107 4. DISCUSSION 4.1 PRELIMINARY BINDING EXPERIMENTS 4.1.1 PROTEIN BINDING AND PROPAFENONE PF i s a t y p e IC a n t i a r r h y t h m i c agent which as been found t o be u s e f u l i n t h e t r e a t m e n t o f a number o f d i f f e r e n t a r r h y t h m i a s [ S c h l e p p e r , 1987]. As w i t h most a n t i a r r h y t h m i c a g e n t s , PF i s b e l i e v e d t o d i s p l a y a s t e e p - d o s e r e s p o n s e c u r v e [ C o n n o l l y e t al., 1983b; Siddoway e t al., 1984], s u g g e s t i n g t h a t s m a l l a l t e r a t i o n s i n serum drug c o n c e n t r a t i o n may have d i s p r o p o r t i o n a t e p h a r m a c o l o g i c a l e f f e c t s . F u r t h e r , s i n c e i t i s g e n e r a l l y a c c e p t e d t h a t the f r e e drug c o n c e n t r a t i o n b e t t e r c o r r e l a t e s t o the o b s e r v e d p h a r m a c o l o g i c a l e f f e c t ( f r e e drug d i s t r i b u t e s t o t h e s i t e o f a c t i o n ) [Svensson e t al., 1986], f a c t o r s a f f e c t i n g f r e e d r u g c o n c e n t r a t i o n o f drugs must be c a r e f u l l y c h a r a c t e r i z e d . PF i s a h i g h c l e a r a n c e drug [Holloman e t al., 1984; A x e l s o n e t al., 1987]; t h e r e f o r e , a l t e r a t i o n s i n b i n d i n g , p r o v i d e d t h a t t h e volume o f d i s t r i b u t i o n remains u n a l t e r e d , would not be e x p e c t e d t o a l t e r p h a r m a c o k i n e t i c parameters o f t o t a l d r u g ( e . g . c l e a r a n c e o f a h i g h c l e a r a n c e d r u g i s not dependent on f r e e drug c o n c e n t r a t i o n ) . However, de p e n d i n g on the d i r e c t i o n o f the d r u g - p r o t e i n b i n d i n g i n t e r a c t i o n ( e . g . i n c r e a s e o r d e c r e a s e ) the c o n c e n t r a t i o n o f f r e e , drug would be a l t e r e d [Svensson e t al., 1986]. S i n c e PF i s h i g h l y bound a t t h e r a p e u t i c c o n c e n t r a t i o n s t o AAG i n serum [ G i l l e s e t al., 1985; Chan e t al., 1989b], s m a l l a l t e r a t i o n s i n b i n d i n g may r e s u l t i n v e r y l a r g e changes i n t h e f r e e f r a c t i o n o f the d r u g , and t h e r e f o r e , e f f e c t . Thus, i t i s v e r y 108 i m p o r t a n t t o a c c u r a t e l y c h a r a c t e r i z e t h e b i n d i n g o f PF and t o be a b l e t o p r e d i c t t h e e f f e c t o f a l t e r e d b i n d i n g on f r e e drug c o n c e n t r a t i o n , and f u r t h e r p h a r m a c o l o g i c a l e f f e c t . The m e t a b o l i t e , 5-OH-PF, has been found t o have s i m i l a r a c t i v i t y t o t h e p a r e n t compound ( e . g . c l a s s 1C a n t i a r r h y t h m i c p r o p e r t i e s ) [ M a l f a t t o et al., 1988; Rouet et al., 1989]. S i n c e , 5-OH-PF i s b e l i e v e d t o c o n t r i b u t e t o the p h a r m a c o l o g i c a l e f f e c t o b s e r v e d f o l l o w i n g s y s t e m i c a d m i n i s t r a t i o n o f PF [K a t e s et a l . , 1985], f a c t o r s a f f e c t i n g t h e f r e e d r u g c o n c e n t r a t i o n o f 5-hydroxypropafone must be c a r e f u l l y s t u d i e d . F u r t h e r , no e f f o r t s have y e t been made t o c a r e f u l l y c h a r a c t e r i z e t h e b i n d i n g o f PF and 5-OH-PF, and t o d e t e r m i n e p r o t e i n s which account f o r th e m a j o r i t y o f b i n d i n g a t t h e r a p e u t i c c o n c e n t r a t i o n s . T h e r e f o r e , i t i s th e aim o f t h i s s t u d y t o c o n s i d e r f a c t o r s a f f e c t i n g t h e b i n d i n g o f both PF and i t s a c t i v e m e t a b o l i t e , 5-OH-PF. 4.1.2 EQUILIBRIUM DIALYSIS: TIME TO EQUILIBRIUM Many t e c h n i q u e s a re now a v a i l a b l e f o r the s e p a r a t i o n o f f r e e and p r o t e i n bound d r u g ; however, u l t r a f i l t r a t i o n and e q u i l i b r i u m d i a l y s i s a r e most commonly employed due t o the ease o f use and proven r e l i a b i l i t y [Kwong, 1985]. U l t r a f i l t r a t i o n , a r a p i d method f o r measuring drug-p r o t e i n b i n d i n g , l i k e e q u i l i b r i u m d i a l y s i s has s e v e r a l d i s a d v a n t a g e s , such as, t e m p e r a t u r e c o n t r o l , l i m i t e d c a p a c i t y t o run l a r g e numbers o f samples, and n o n - s p e c i f i c b i n d i n g [Kwong, 1985]. However, u l t r a f i l t r a t i o n can p r o v i d e r e l i a b l e e s t i m a t e s o f d r u g - p r o t e i n b i n d i n g i f t h e method i s r i g o r o u s l y v a l i d a t e d and c o n t r o l l e d [Kwong, 1985]. 109 E q u i l i b r i u m d i a l y s i s , d e s p i t e i t s p o t e n t i a l d i s a d v a n t a g e s , such as Donnan e f f e c t s [ L i n d u p , 1987], volume s h i f t s [Lima et a l . , 1983], non-s p e c i f i c b i n d i n g [Kwong, 1985], and pH s h i f t s d u r i n g d i a l y s i s [ L u i and C h i o u , 1985], s t i l l remains t h e s t a n d a r d method f o r e v a l u a t i n g d r u g -p r o t e i n b i n d i n g . E q u i l i b r i u m d i a l y s i s can p r o v i d e r e p r o d u c i b l e e s t i m a t e s o f d r u g - p r o t e i n b i n d i n g i f the e x p e r i m e n t a l c o n d i t i o n s a r e o p t i m i z e d , and s t e p s taken t o c o r r e c t f o r the above mentioned problems [ K r i s t e n s e n and Gram, 1981]. I n i t i a l l y i n e q u i l i b r i u m d i a l y s i s s t u d i e s , the time r e q u i r e d t o r e a c h d r u g - p r o t e i n b i n d i n g e q u i l i b r i u m must be o p t i m i z e d . The time t o a t t a i n e q u i l i b r i u m i s dependent on many f a c t o r s , namely, t h e s u r f a c e t o volume r a t i o o f t h e d i a l y s i s system, the pore s i z e o f t h e membrane [ L i n d u p , 1987], and whether the drug i s added t o t h e serum o r b u f f e r compartment o f t h e d i a l y s i s c e l l [McNamara and Bogardu, 1982]. I t i s i m p o r t a n t t o o p t i m i z e the d i a l y s i s time i n o r d e r t o r e d u c e o r a v o i d p r o b l e m s , such as pH s h i f t , volume s h i f t s , and b a c t e r i a l growth [Kwong, 1985]; however, i f not enough time t o r e a c h e q u i l i b r i u m i s p r o v i d e d the v a r i a n c e o f the b i n d i n g measurement w i l l be g r e a t e r ( f i g u r e 2b), and d r u g - p r o t e i n b i n d i n g measurements may be i n c o r r e c t . The time r e q u i r e d t o r e a c h e q u i l i b r i u m f o r PF was found t o be 6 hours [Chan et a l . , 1989b], w h i l e the m e t a b o l i t e , 5-OH-PF, reach e d e q u i l i b r i u m i n 8 hours ( f i g u r e 2a & 2 b ) . These may be c o n s i d e r e d i n t e r m e d i a t e t i m e s , s i n c e e q u i l i b r i u m d i a l y s i s times have been r e p o r t e d t o range from l e s s than 4 hours [ L i n d u p , 1987] t o as h i g h as 16 hours [ T o z e r 1981]. F o r t u n a t e l y , s h o r t and i n t e r m e d i a t e d i a l y s i s times reduce the d e g r e e o f time dependent problems such as, volume s h i f t s [ T o z e r et a/., 1981], pH 110 s h i f t s [ L u i and C h o i u , 1986], and b a c t e r i a l growth [Kwong, 1985] 4.1.3 NON-SPECIFIC BINDING OF PF AND 5-HYDROXYPROPAFENONE S i n c e both PF [Chan e t al., 1989b] and 5-OH-PF ( T a b l e 1) showed h i g h and v a r i a b l e n o n - s p e c i f i c b i n d i n g w i t h e q u i l i b r i u m d i a l y s i s and u l t r a f i l t r a t i o n , t h e use o f e q u i l i b r i u m d i a l y s i s t o measure d r u g - p r o t e i n b i n d i n g would be c o n s i d e r e d advantageous o v e r u l t r a f i l t r a t i o n . T h i s i s so because a c o r r e c t i o n f a c t o r t o a c c o u n t f o r the d r u g l o s t t o the u l t r a f i l t r a t i o n d e v i c e s i s r e q u i r e d b e f o r e drug p r o t e i n b i n d i n g can be c a l c u l a t e d . Thus, an a d d i t i o n a l e r r o r due t o the v a r i a b i l i t y o f t h e n o n - s p e c i f i c b i n d i n g e s t i m a t e i s i n t r o d u c e d t o the measurement o f d r u g -p r o t e i n b i n d i n g . The same i s not t r u e f o r e q u i l i b r i u m d i a l y s i s , s i n c e t h e b i n d i n g c a l c u l a t i o n ( f r e e f r a c t i o n ) d e t e r m i n e d u s i n g the p o s t -d i a l y s i s serum and b u f f e r drug c o n c e n t r a t i o n a u t o m a t i c a l l y a c c o u n t s f o r t h e n o n - s p e c i f i c b i n d i n g . That i s , t h e drug c o n c e n t r a t i o n measured i n t h e serum compartment r e p r e s e n t s both the p r o t e i n bound and the f r e e drug i n serum. At e q u i l i b r i u m , the b u f f e r drug c o n c e n t r a t i o n w i l l r e p r e s e n t t h e f r e e drug c o n c e n t r a t i o n i n the serum s i d e o f the d i a l y s i s c e l l . In both o f t h e s e measurements, s i n c e n o n - s p e c i f i c a l l y bound drug i s not i n c l u d e d , o n l y the drug p r o t e i n b i n d i n g independent o f t h e non-s p e c i f i c b i n d i n g w i l l be measured [Kwong, 1985]. T h i s would s t i l l be t r u e i n t h e s i t u a t i o n where n o n - s p e c i f i c b i n d i n g i s unequal between the b u f f e r and t h e serum s i d e o f the d i a l y s i s c e l l , u n l e s s the drug which i s n o n - s p e c i f i c a l l y bound c o u l d a l t e r t h e e q u i l i b r i u m o f the f r e e d r u g , which i s u n l i k e l y . I l l I t s h o u l d be noted t h a t t h e t o t a l d r u g c o n c e n t r a t i o n i s not equal t o t h e c o n c e n t r a t i o n o f drug i n i t i a l l y added t o the d i a l y s i s c e l l . C a l c u l a t i o n s o f d r u g - p r o t e i n b i n d i n g made u s i n g the c o n c e n t r a t i o n o f drug added t o t h e d i a l y s i s c e l l would f a i l t o a c c o u n t f o r both t h e amount o f drug which i s n o n - s p e c i f i c a l l y bound, and t h e amount o f drug which has d i f f u s e d t o t h e b u f f e r compartment o f t h e d i a l y s i s c e l l . These c a l c u l a t i o n s would i n c o r r e c t l y o v e r e s t i m a t e t h e b i n d i n g [Kwong, 1985]. 4.1.4 pH DEPENDENT BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE D i f f e r e n c e s i n d r u g - p r o t e i n b i n d i n g r e l a t e d t o pH have been o b s e r v e d f o r many d r u g s , i n c l u d i n g o x p r e n o l o l and b e t a x o l o l [Henry and M i t c h e l l , 1980], t o l m e t i n [Matsuyama et a l . , 1987], t h e o p h y l l i n e and q u i n i d i n e [Bors et a/., 1984]. In the p r e s e n t s t u d y , PF p r o t e i n b i n d i n g i n serum i n c r e a s e d p r o p o r t i o n a l l y t o i n c r e a s e s i n pH o v e r t h e range s t u d i e d (pH 7.0 - 8 . 0 ) ( F i g u r e 3 ) . S i m i l a r pH dependent a l t e r a t i o n s i n d r u g - p r o t e i n b i n d i n g were a p p a r e n t i n s o l u t i o n s o f AAG and HSA ( F i g u r e 3 ) . The m e t a b o l i t e , 5-OH-PF, d i s p l a y s s i m i l a r b i n d i n g p a t t e r n s i n serum and i n a s o l u t i o n o f AAG ( F i g u r e 4 ) , w h i l e d r u g - p r o t e i n b i n d i n g t o HSA d i d not show pH dependence; r e a s o n s f o r t h i s unexpected b e h a v i o u r were not a p p a r e n t . The o b s e r v e d pH dependent d r u g - p r o t e i n b i n d i n g c o u l d r e s u l t from pH i n d u c e d c h e m i c a l , p h y s i o c h e m i c a l , o r s t r u c t u r a l changes o f the d r u g , p r o t e i n , and/or b o t h . The d e c r e a s e i n the degree o f i o n i z a t i o n i n PF and 5-OH-PF (as demo n s t r a t e d by a r e d u c t i o n i n the f r a c t i o n o f PF and 5-112 OH-PF i n t h e aqueous l a y e r o f an o r g a n i c s o l v e n t / b u f f e r p a r t i t i o n system) w i t h i n c r e a s e s i n pH ( F i g u r e 5 and 6) would s u g g e s t t h a t the b i n d i n g o f PF and 5-OH-PF i s dependent to a l a r g e degree on h y d r o p h o b i c r a t h e r than i o n i c i n t e r a c t i o n s between the drug and t h e p r o t e i n . I t a p p e a r s t h a t s i n c e s i m i l a r amounts o f PF and 5-OH-PF are u n - i o n i z e d a t pH 7.4, o t h e r f a c t o r s i n c l u d i n g , s t e r i c and c h e m i c a l i n t e r a c t i o n s due t o t h e h y d r o x y ! a t i o n o f PF, may account f o r the o b s e r v e d b i n d i n g d i f f e r e n c e s between the drug and m e t a b o l i t e . The c o n t r i b u t i o n o f pH dependent changes i n p r o t e i n ( c o n f o r m a t i o n a l , c h e m i c a l and p h y s i o c h e m i c a l ) t o the d r u g - p r o t e i n b i n d i n g o f PF and 5-OH-PF i s not known. However, HSA has been shown t o undergo pH dependent c o n f o r m a t i o n a l t r a n s i t i o n s , which c o u l d p o t e n t i a l l y a l t e r d r u g - p r o t e i n b i n d i n g [Kremer et al., 1988]. I t i s not known whether AAG undergoes pH s e n s i t i v e changes a f f e c t i n g d r u g - p r o t e i n b i n d i n g [ i o n i z a t i o n , o r c o n f o r m a t i o n a l c h a n g e s ] . Thus, i t seems l i k e l y t h a t a pH i n c r e a s e i n d u c e d d e c r e a s e i n the degree o f i o n i z a t i o n o f both PF and 5-OH-PF c o n t r i b u t e s t o t h e i r i n c r e a s e d b i n d i n g i n serum and such i s o l a t e d serum p r o t e i n s as AAG and HSA. I t i s not c l e a r t o what de g r e e , i f any, pH mediated a l t e r a t i o n s o f serum p r o t e i n s (HSA, AAG, and l i p o p r o t e i n s ) p l a y i n the o b s e r v e d pH dependent d r u g - p r o t e i n b i n d i n g o f PF and 5-OH-PF. 4.1.5 THE EFFECT OF BUFFER STRENGTH ON THE DRUG PROTEIN BINDING OF 5-HYDROXYPROPAFENONE. When pH dependent d r u g - p r o t e i n b i n d i n g i s p r e s e n t , i t i s i m p o r t a n t t o use a b u f f e r w i t h s u f f i c i e n t b u f f e r c a p a c i t y t o c o n t r o l t h e pH d u r i n g t h e c o u r s e o f d i a l y s i s [ L i n d u p , 1987]. S i n c e both PF and 5-OH-PF undergo s i g n i f i c a n t pH dependent b i n d i n g , an i n c r e a s e i n b u f f e r s t r e n g t h from 0.067 M t o 0.100 M was r e q u i r e d t o m a i n t a i n c o n s t a n t pH d u r i n g the d i a l y s i s e x p e r i m e n t . However, b u f f e r s t r e n g t h and c o m p o s i t i o n can have l a r g e e f f e c t s on d r u g - p r o t e i n b i n d i n g as measured by e q u i l i b r i u m d i a l y s i s [ L i n d u p , 1987; Kwong, 1985]. The a d d i t i o n o f h i g h c o n c e n t r a t i o n s o f i n o r g a n i c phosphate i o n s , 7 t o 10 f o l d h i g h e r than i n normal serum [Hansten, 1986], may r e s u l t i n c o n f o r m a t i o n a l s h i f t s i n serum p r o t e i n s which may a l t e r t he d r u g - p r o t e i n b i n d i n g . The i n t r o d u c t i o n o r removal o f v a r i o u s i n o r g a n i c i o n s i n serum, as a r e s u l t o f e q u i l i b r i u m d i a l y s i s , may r e s u l t i n c o n f o r m a t i o n a l changes i n the p r o t e i n which can a l t e r d r u g - p r o t e i n b i n d i n g . I t has been shown t h a t both c h l o r i d e i o n s [Kremer et al., 1985], and c a l c i u m i o n s [ L i n d u p , 1987] a l t e r d r u g - p r o t e i n b i n d i n g . As molar s t r e n g t h o f the phosphate b u f f e r i s i n c r e a s e d , the amount o f sodium c h l o r i d e r e q u i r e d t o m a i n t a i n i s o t o n i c i t y i s r e d u c e d by o v e r 50%, l e a d i n g t o an o v e r a l l r e d u c t i o n o f t h e amount o f c h l o r i d e i o n i n the b u f f e r . I t has a l s o been s u g g e s t e d t h a t phosphate p r e s e n t i n the b u f f e r may b i n d c a l c i u m i o n s , and thus r e d u c e the i n f l u e n c e o f c a l c i u m on d r u g - p r o t e i n b i n d i n g [Kwong, 1985]. The s t r e n g t h o f phosphate b u f f e r d i d not a f f e c t t he b i n d i n g o f 5-OH-PF i n serum ( F i g u r e 7 ) ; however, the same may not be t r u e w i t h r e s p e c t t o t h e d r u g - p r o t e i n b i n d i n g t o i s o l a t e d serum p r o t e i n s , such as HSA and 114 AAG. I s o l a t e d p r o t e i n s i n s o l u t i o n do not have o t h e r p r o t e i n s n o r m a l l y p r e s e n t i n serum t o accommodate the l i b e r a t e d f r e e drug due t o b i n d i n g a l t e r a t i o n s ; t h e r e f o r e , a l t e r e d b i n d i n g t o i n d i v i d u a l serum p r o t e i n s due t o t h e p r e s e n c e o f b u f f e r may go u n n o t i c e d i n serum. 4.1.6 DEGRADATION OF 5-HYDROXYPROPAFENONE DURING EQUILIBRIUM DIALYSIS I m p u r i t i e s may r e s u l t i n h i g h l y e r r o n e o u s d r u g - p r o t e i n b i n d i n g r e s u l t s due t o t h e d i s p l a c e m e n t o f the drug o f i n t e r e s t from i t s p r o t e i n b i n d i n g s i t e by an i m p u r i t y p r e s e n t i n the d i a l y s i s system [ K r i s t e n s e n and Gram, 1982]. These i m p u r i t i e s may be the r e s u l t o f drug d e g r a d a t i o n d u r i n g d i a l y s i s . A l t h o u g h pure 5-OH-PF was used t o co n d u c t b i n d i n g e x p e r i m e n t s , t h e p o s s i b i l i t y o f i m p u r i t i e s due t o d e g r a d a t i o n o f 5-OH-PF d u r i n g d i a l y s i s r e q u i r e d c o n s i d e r a t i o n . No d e g r a d a t i o n o f 5-OH-PF was o b s e r v e d d u r i n g t h e c o u r s e o f the d r u g - p r o t e i n b i n d i n g e x p e r i m e n t . 4.1.7 VOLUME SHIFTS DURING EQUILIBRIUM DIALYSIS OF PROPAFENONE AND 5-HYDROXYPROPAFENONE. Volume s h i f t s , the movement o f f l u i d from t h e b u f f e r compartment t o t h e serum compartment d u r i n g e q u i l i b r i u m d i a l y s i s and the subsequent e f f e c t s on the i n t e r p r e t a t i o n o f d r u g - p r o t e i n b i n d i n g d a t a has been r e v i e w e d a t l e n g t h [Lima et al., 1981]. Volume s h i f t s a r e time dependent and o c c u r as a r e s u l t o f o s m o t i c d i f f e r e n c e s between t h e b u f f e r and serum compartments i n an e q u i l i b r i u m d i a l y s i s c e l l . L a r g e volume s h i f t s can d i l u t e p r o t e i n s i n the serum s i d e o f the d i a l y s i s c e l l , and th u s l e a d t o an u n d e r e s t i m a t i o n o f the de g r e e o f d r u g - p r o t e i n 115 b i n d i n g ( e . g . a 30% u n d e r e s t i m a t i o n i n t h e b i n d i n g o f p r e d n i s o l o n e has been o b s e r v e d ) [ T o z e r et al. 1981]. The volume s h i f t s measured f o r PF and 5-OH-PF, r e s p e c t i v e l y , were a p p r o x i m a t e l y 5%, and would not be e x p e c t e d t o r e s u l t i n an a p p r e c i a b l e u n d e r e s t i m a t i o n o f b i n d i n g . In a s i m i l a r s t u d y w i t h t h e amine a n t i a r r h y t h m i c d r u g , d i s o p y r a m i d e , a volume s h i f t o f 12% was r e q u i r e d t o be a b l e t o d e t e c t a change i n the d r u g -p r o t e i n b i n d i n g [Lima e t al., 1983]. A l a c k o f a s i g n i f i c a n t volume s h i f t i n the p r e s e n t experiment can be f u r t h e r d e m o n s t r a t e d by t h e minimal change i n the p r o t e i n c o n c e n t r a t i o n b e f o r e and a f t e r d i a l y s i s (See T a b l e 2 ) . 4.1.8 OSMOLARITY MEASUREMENTS OF 5-HYDROXYPROPAFENONE DRUG SOLUTIONS. S i n c e o s m o t i c d i f f e r e n c e s c o n t r i b u t e s i g n i f i c a n t l y t o volume s h i f t s between b u f f e r and serum compartments o f d i a l y s i s c e l l s , the e f f e c t o f ad d i n g d i f f e r e n t amounts o f drug (0.1 t o 45.0 zzg/mL 5-OH-PF) t o t h e b u f f e r compartment was c o n s i d e r e d . The f a c t t h a t o s m o l a r i t y remained r e l a t i v e l y c o n s t a n t w i t h t h e a d d i t i o n o f v a r i o u s amounts o f drug c o u l d h e l p t o e x p l a i n t h e c o n s t a n t n a t u r e o f t h e volume s h i f t s r e g a r d l e s s o f the drug c o n c e n t r a t i o n s t u d i e d . 4.1.9 PROTEIN LOSS DURING EQUILIBRIUM DIALYSIS OF PROPAFENONE AND 5-HYDR0XYPR0PAFEN0NE The r e d u c t i o n i n the c o n c e n t r a t i o n o f p r o t e i n may o c c u r as a r e s u l t o f volume s h i f t s [Lima e t a l . , 1983; T o z e r e t a l . 1981], non-s p e c i f i c b i n d i n g o f p r o t e i n s t o d i a l y s i s c e l l s u r f a c e s and the d i a l y s i s 116 membrane, and e n z y m a t i c o r chemical d e g r a d a t i o n o f t h e p r o t e i n [Kwong, 1985]. The r e d u c t i o n i n the p r o t e i n c o n c e n t r a t i o n f o l l o w i n g d i a l y s i s i n t h i s e x p e r i m e n t was m i n i m a l , w i t h the e x c e p t i o n o f AAG d i s s o l v e d i n b u f f e r ( T a b l e 2 ) . S i n c e an e q u i m o l a r r e d u c t i o n i n t h e c o n c e n t r a t i o n o f AAG d i d not o c c u r i n serum, i t may be s p e c u l a t e d t h a t e i t h e r d e g r a d a t i o n o f AAG d u r i n g d i a l y s i s i n the b u f f e r , o r n o n - s p e c i f i c b i n d i n g o f AAG t o t h e d i a l y s i s c e l l , and/or membrane, r a t h e r than sample d i l u t i o n due t o volume s h i f t s was r e s p o n s i b l e f o r the o b s e r v e d r e d u c t i o n i n AAG f o l l o w i n g d i a l y s i s . 4.1.10 MEMBRANE INTEGRITY DURING EQUILIBRIUM DIALYSIS A n o t h e r p o s s i b l e c o m p l i c a t i o n o f e q u i l i b r i u m d i a l y s i s may be p r o t e i n l e a k a g e d u r i n g d i a l y s i s as a r e s u l t o f membrane r u p t u r e and improper mounting o f the d i a l y s i s membranes. The membrane used i n t h e s e e x p e r i m e n t s proved to be r o b u s t , s i n c e v e r y few p o s t - d i a l y s i s b u f f e r samples t e s t e d p o s i t i v e f o r p r o t e i n . I t s h o u l d be remembered, t h a t t h i s t e s t i s v i s u a l r a t h e r than s p e c t r o p h o t o m e t r i c , and t h e r e f o r e l a c k s s e n s i t i v i t y . O n l y l a r g e amounts o f p r o t e i n i n the b u f f e r can be d e t e c t e d ; however, i f a membrane r u p t u r e s , o r l e a k s , the amount o f p r o t e i n i n the b u f f e r would be s u b s t a n t i a l ( a p p r o x i m a t e l y h a l f o f the serum p r o t e i n c o n c e n t r a t i o n ) , and t h e r e f o r e , the v i s u a l t e s t would be a dequate. 4.2 PROTEIN BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE 117 4.2.1 PROTEIN BINDING OF 5-HYDROXYPROPAFENONE IN SERUM OF HEALTHY MALES The d r u g - p r o t e i n b i n d i n g i n t e r a c t i o n can be d e s c r i b e d by the e q u i l i b r i u m i n e q u a t i o n 1: k l [Df] + [P] ^ZT [D*P] (1) k 2 where [ D f ] , [ P ] , and [D*P] are the c o n c e n t r a t i o n s o f f r e e d r u g , f r e e p r o t e i n , and bound drug and p r o t e i n , r e s p e c t i v e l y . T h i s i n t e r a c t i o n between dru g and serum p r o t e i n can be d e s c r i b e d by the a f f i n i t y a s s o c i a t i o n c o n s t a n t (Ka) or the s t r e n g t h o f the i n t e r a c t i o n as i n e q u a t i o n 2. Ka = rD*P1 = k i (2) [D]*[P] k 2 B i n d i n g can a l s o be d e s c r i b e d by the number o f b i n d i n g s i t e s a v a i l a b l e , o r c a p a c i t y ( n [ P t ] ) f o r drug i n the d r u g - p r o t e i n b i n d i n g system where n i s the number o f b i n d i n g s i t e s on the p r o t e i n , and [ P t ] i s t h e t o t a l amount o f p r o t e i n p r e s e n t i n the system. Upon m a n i p u l a t i o n , e q u a t i o n 2 can d e s c r i b e the b i n d i n g o f drug t o p r o t e i n by a b i n d i n g i s o t h e r m ( E q u a t i o n 4 ) . r = [D*P1 = n*Ka*rDl (4) [ P t ] 1 + Ka[D] where r i s the molar r a t i o o f drug bound. Upon m u l t i p l y i n g by the t o t a l p r o t e i n c o n c e n t r a t i o n [ P t ] , e q u a t i o n 4 can be t r a n s f o r m e d t o 118 r e p r e s e n t t he amount o f bound drug ( e q u a t i o n 5) [D*P] = n * K a * f P t l * r D l ( f o r one b i n d i n g s i t e ) (5) 1 + Ka*[D] For m u l t i p l e b i n d i n g s i t e s the c o n t r i b u t i o n o f each b i n d i n g s i t e i s summed. P [ D * P ] t o t a 1 = 2 [ D * P ] p ( f o r p number o f b i n d i n g s i t e s ) (6) The d i f f i c u l t y e n c o u n t e r e d i n the e s t i m a t i o n o f b i n d i n g p a r a m e t e r s , due t o the n o n - l i n e a r n a t u r e o f e q u a t i o n 5 has r e s u l t e d i n the p r e s e n t a t i o n o f s e v e r a l t r a n s f o r m a t i o n s t o l i n e a r i z e b i n d i n g d a t a , o f which the most common are S c a t c h a r d [ S c a t c h a r d , 1949], and R o s e n t h a l [ R o s e n t h a l , 1967]. S c a t c h a r d r = n*Ka - r*Ka (8) t r a n s f o r m a t i o n D R o s e n t h a l rD*Pl = n*Ka*[Pt] - [D*P]*Ka (9) t r a n s f o r m a t i o n [D] S e v e r a l problems can be e n c o u n t e r e d w i t h t h e s e t r a n s f o r m a t i o n s . I f more than two d i f f e r e n t homogeneous ty p e s o f b i n d i n g s i t e s a r e p r e s e n t ( E q u a t i o n 6 ) , the l i n e a r t r a n s f o r m a t i o n s becomes c u r v e d . T h e r e f o r e , any advantage g a i n e d by t r a n s f o r m i n g t he d a t a i s l o s t , s i n c e t h e para m e t e r s cannot be d e t e r m i n e d g r a p h i c a l l y . The S c a t c h a r d and R o s e n t h a l a n a l y s i s a l s o t e n d t o be s t a t i s t i c a l l y i n v a l i d s i n c e the y - o b s e r v a t i o n s a r e not in d e p e n d e n t o f t h e x o b s e r v a t i o n s [ B u r g i s s e r , 1984]. R o s e n t h a l and S c a t c h a r d p l o t s , commonly used i n d e s c r i b i n g b i n d i n g d a t a , may o n l y p r o v i d e a q u a l i t a t i v e d e s c r i p t i o n o f t h e b i n d i n g i n t e r a c t i o n . 119 The a l t e r n a t i v e approach i s n o n l i n e a r r e g r e s s i o n w i t h computer programs, such as ENZFITTER R, N0NLIN R, and LIGAND R, o f the u n t r a n s f o r m e d d a t a ( E q u a t i o n 4 and 5) [ B u r g i s s e r , 1984]. T h i s method does not r e l y on t r a n s f o r m a t i o n s which may emphasize d a t a p o i n t s at lower d r u g c o n c e n t r a t i o n s where the p o t e n t i a l e r r o r i s g r e a t e r , such as the K l o t z r e c i p r o c a l p l o t [Svensson et a l . , 1986]. F u r t h e r t h i s method o f f i t t i n g u n t r a n s f o r m e d b i n d i n g d a t a i s s t a t i s t i c a l l y r i g o r o u s , s i n c e a l l the y-o b s e r v a t i o n s are i n d e p e n d e n t o f x o b s e r v a t i o n s (eg. y = f ( x ) r a t h e r than y = f ( y / x ) ) [ K l o t z , 1982]. D e s p i t e t h e advantages o f f i t t i n g d a t a by t h i s method, i t s h o u l d a l s o be c o n s i d e r e d t h a t the a c c u r a c y o f f i t t e d p a r a m e t e r s depends on the q u a l i t y o f the d a t a [ K l o t z , 1983; B u r g i s s e r , 1984]. Thus, d a t a w i t h c o n s i d e r a b l e s c a t t e r w i l l not p r o v i d e the most a c c u r a t e e s t i m a t e s o f the d r u g - p r o t e i n b i n d i n g p a r a m e t e r s . The b i n d i n g a f f i n i t i e s o f t h e h i g h c a p a c i t y s i t e on p r o t e i n s i n serum f o r PF and 5-OH-PF d i f f e r c o n s i d e r a b l y ( K a ^ p ^ = 6.53 X 1 0 5 M" 1 [Chan e t a l . , 1989b] vs. K a 1 ( 5 _ 0 H _ P F ) = 1.72 X 1 0 4 N T 1 ) ; however a s i m i l a r b i n d i n g c a p a c i t y has been r e p o r t e d ( N P ^ p p ^ = 1.73 X 1 0 " 4 M [Chan e t a l . , 1989b] vs. N P ( 5 _ 0 H _ p F ) = 3.46 X 10" 4 M) ( T a b l e 3 ) . T h i s r a i s e s the q u e s t i o n : Are the b i n d i n g c o n s t a n t s e s t i m a t e d f o r 5-OH-PF a m i x t u r e o f two homogeneous b i n d i n g s i t e s , such t h a t the o b s e r v e d Ka and NP a r e h y b r i d c o n s t a n t s , o r does 5-OH-PF i n f a c t p o s s e s s o n l y one homogeneous t y p e o f b i n d i n g s i t e i n human serum which has a lower a f f i n i t y but s i m i l a r c a p a c i t y t o t h a t o b s e r v e d f o r PF i n serum. The d r u g - p r o t e i n b i n d i n g parameters d e t e r m i n e d by n o n - l i n e a r 120 r e g r e s s i o n o f b i n d i n g d a t a i n serum, such as b i n d i n g a f f i n i t y and c a p a c i t y o f b i n d i n g s i t e s , must be c o n s i d e r e d c a u t i o u s l y s i n c e t h e s e a r e macro c o n s t a n t s composed o f the sum o f b i n d i n g a f f i n i t i e s and b i n d i n g c a p a c i t i e s o f numerous p r o t e i n s p r e s e n t i n serum. S i m i l a r l y , as numerous b i n d i n g s i t e s o f s i m i l a r a f f i n i t i e s and c a p a c i t i e s appear homogeneous i n serum, i f the d a t a p o i n t s a re s c a t t e r e d , t he b i n d i n g a f f i n i t i e s and c a p a c i t i e s o f two d i f f e r e n t homogeneous groups o f b i n d i n g s i t e s may appear as one homogeneous s i t e . T h i s i s p a r t i c u l a r l y e v i d e n t when the d i f f e r e n c e between the a f f i n i t y f o r the h i g h and low a f f i n i t y b i n d i n g s i t e s i s s m a l l . In f i g u r e 27 can be seen a t h e o r e t i c a l d e m o n s t r a t i o n o f the e f f e c t o f d e c r e a s i n g t h e b i n d i n g a f f i n i t y o f t h e h i g h a f f i n i t y b i n d i n g s i t e ( K a j ) , t h u s r e d u c i n g t he d i f f e r e n c e between t h e a f f i n i t i e s f o r the h i g h and low a f f i n i t y b i n d i n g s i t e . The b i n d i n g c a p a c i t y o f the h i g h a f f i n i t y b i n d i n g s i t e i s u n a l t e r e d , and the second low a f f i n i t y s i t e p r o v i d e s a c o n s t a n t c o n t r i b u t i o n t o b i n d i n g . As t h e t h e o r e t i c a l b i n d i n g a f f i n i t y i s d e c r e a s e d , i t becomes i n c r e a s i n g l y d i f f i c u l t t o g r a p h i c a l l y d i s t i n g u i s h between the two b i n d i n g s i t e s . F u r t h e r m o r e , i f d a t a i s e x c e s s i v e l y s c a t t e r e d i t may become d i f f i c u l t , i f n ot i m p o s s i b l e , f o r n o n l i n e a r r e g r e s s i o n t o r e l i a b l y d i f f e r e n t i a t e between t h e s e two d i f f e r e n t b i n d i n g s i t e s . Such may be the case w i t h 5-OH-PF b i n d i n g . A n a l y s i s u s i n g t he Rosenthal p l o t , i n a d d i t i o n t o n o n l i n e a r f i t t i n g o f the u n t r a n s f o r m e d d a t a show o n l y one homogeneous s e t o f b i n d i n g s i t e s ; however, t h e r e i s c o n s i d e r a b l e s c a t t e r i n the d a t a a t l o w e r c o n c e n t r a t i o n s ( F i g u r e s 8 a - 8 e ) . T h e r e f o r e , b i n d i n g c o n s t a n t s r e p o r t e d f o r 5-OH-PF may not be c o m p l e t e l y a c c u r a t e . A n o t h e r problem e n c o u n t e r e d d u r i n g c h a r a c t e r i z a t i o n o f the b i n d i n g 121 5-OH-PF was t h e poor s o l u b i l i t y o f t h i s compound. While e t h a n o l i c s o l u b i l i z a t i o n o f t h e drug might o f f e r some advantages i n c e r t a i n e x p e r i m e n t a l c i r c u m s t a n c e s , t h e a d d i t i o n o f 5% e t h a n o l was found t o a f f e c t t h e b i n d i n g o f p e r a z i n e by 12% i n a s o l u t i o n o f AAG [Kremer e t al., 1988]. To a v o i d s i m i l a r i n t e r f e r e n c e s i n the p r e s e n t b i n d i n g s t u d y no exogenous c h e m i c a l s were added. The maximum c o n c e n t r a t i o n o f 5-OH-PF used i n b i n d i n g e x p e r i m e n t s was 45.0 ug/mL. T h i s p r o v i d e s a s m a l l e r c o n c e n t r a t i o n range o f 5-OH-PF, compared t o PF, ov e r which t he b i n d i n g can be c h a r a c t e r i z e d . T h i s f u r t h e r i n c r e a s e s t h e d i f f i c u l t y t o e x p e r i m e n t a l l y d i f f e r e n t i a t e between two d i f f e r e n t b i n d i n g s i t e s i n serum [ K l o t z , 1982]. A g r a d u a l r i s e i n the f r e e f r a c t i o n o f 5-OH-PF was o b s e r v e d o v e r t h e range o f c o n c e n t r a t i o n s s t u d i e d ( T a b l e 4 ) ; however, t h e g r a d u a l i n c r e a s e i n f r e e f r a c t i o n c o u l d not be d i f f e r e n t i a t e d s t a t i s t i c a l l y . As the d i f f e r e n c e between the h i g h and low a f f i n i t y s i t e a f f i n i t y b i n d i n g c o n s t a n t s becomes s m a l l e r a r i s e i n f r e e f r a c t i o n i s l e s s pronounced, whereas, i f t h i s d i f f e r e n c e i s l a r g e , t h e f r e e f r a c t i o n would remain low u n t i l s a t u r a t i o n o f the h i g h a f f i n i t y b i n d i n g s i t e i s r e a c h e d ( F i g u r e 2 8 ) . F o l l o w i n g s a t u r a t i o n o f the h i g h a f f i n i t y b i n d i n g s i t e , t h e f r e e f r a c t i o n would show an a b r u p t i n c r e a s e . Such b e h a v i o u r would s u g g e s t t h a t t h e compound undergoes a b r u p t n o n - l i n e a r b i n d i n g o v e r a narrow range o f c o n c e n t r a t i o n s , whereas, a g r a d u a l i n c r e a s e , a s - o b s e r v e d f o r Ld UJ O CD 200 180 160 140 120 100 80 60 40 20 0 ° Ka - 10 * Ka = 50 • Ka = 200 ° o o O o o o c A » o tM o cm O o m o t» o BOUND F i g u r e 27. T h e o r e t i c a l Rosenthal p l o t o f d r u g - p r o t e i n b i n d i n g f i t t i n g a b i n d i n g model f o r a system c o n t a i n i n g one drug b i n d i n g s p e c i f i c s i t e + n o n - s p e c i f i c b i n d i n g s i t e . The b i n d i n g c a p a c i t y o f the h i g h a f f i n i t y s i t e i s 1.0 and the n o n - s p e c i f i c b i n d i n g (Kns) was h e l d at 2; o n l y the a f f i n i t y o f the high a f f i n i t y s i t e i s a l t e r e d . A f f i n i t y c o n s t a n t s Ka: 10 ( o ) , 50 ( A ) , and 200 ( • ) . 123 o < rr 0.300 o Ka = 10 * Ka = 50 • Ka = 200 o 0.200 UJ UJ 0 .100 0 .000 TOTAL DRUG Fi g u r e 28. T h e o r e t i c a l p l o t o f f r e e f r a c t i o n o f d r u g - p r o t e i n b i n d i n g f i t t i n g a model c o n t a i n i n g one s p e c i f i c s i t e + n o n - s p e c i f i c b i n d i n g . B i n d i n g c o n s t a n t s ( i e . the c a p a c i t y o f the high a f f i n i t y s i t e i s 1.0, and n o n - s p e c i f i c b i n d i n g i s 2) are h e l d c o n s t a n t ; o n l y the a f f i n i t y o f the h i g h a f f i n i t y s i t e i s a l t e r e d . A f f i n i t y c o n s t a n t s Ka: 10 ( o ) , 50 ( A ) , and 200 ( © ) . 124 compounds were the d i f f e r e n c e i n b i n d i n g a f f i n i t i e s o f the low and h i g h a f f i n i t y s i t e s are s m a l l , may not appear n o n - l i n e a r even though the h i g h a f f i n i t y s i t e i s a l s o s a t u r a t e d . A l t h o u g h t h e f r e e f r a c t i o n o f 5-OH-PF at h i g h e r c o n c e n t r a t i o n s does v a r y s t a t i s t i c a l l y , t h e f r e e f r a c t i o n a t t h e l o w e s t c o n c e n t r a t i o n (0.1 Mg/mL) was shown t o be s t a t i s t i c a l l y d i f f e r e n t compared t o the o t h e r o b s e r v e d v a l u e s . The e x a c t r e a s o n f o r t h i s i s not known; however, c o n s i d e r i n g t h a t drug c o n c e n t r a t i o n measurements, p a r t i c u l a r l y from the b u f f e r compartment, were c l o s e t o the l i m i t o f s e n s i t i v i t y o f the a s s a y method, i n c r e a s e d e x p e r i m e n t a l e r r o r i n m e a s u r i n g the 5-OH-PF c o n c e n t r a t i o n may have c o n t r i b u t e d t o t h i s low o b s e r v a t i o n . 4.2.2 PROTEIN BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE IN SERUM ESTIMATED BY EQUILIBRIUM DIALYSIS AND ULTRAFILTRATION. The d r u g - p r o t e i n b i n d i n g o b s e r v e d when c o n t r o l serum i s s p i k e d w i t h a d r u g , may not c o r r e l a t e w e l l w i t h the d r u g - p r o t e i n b i n d i n g o f t h e same drug when b i n d i n g i s measured f o l l o w i n g the s y s t e m i c a d m i n i s t r a t i o n o f t h e d r u g . One p o s s i b l e e x p l a n a t i o n f o r t h i s may be t h a t the b i n d i n g o f t h e p a r e n t compound i s i n h i b i t e d by the m e t a b o l i t e f o l l o w i n g s y s t e m i c a d m i n i s t r a t i o n o f the d r u g . For example, t h e b i n d i n g o f both d i s o p y r a m i d e and v e r a p a m i l were found t o d e c r e a s e by the a d d i t i o n t o t h e i r r e s p e c t i v e m e t a b o l i t e s , m o n o - n - d e a l k y l a t e d d i s o p y r a m i d e [Bredensen e t al., 1982], and n o r v e r a p a m i l [ B a t e s e t al., 1980]. The p o s s i b i l i t y o f d e c r e a s e d b i n d i n g o f PF due t o t h e p r e s e n c e o f m e t a b o l i t e (5-OH-PF), i n a d d i t i o n t o a s l i g h t y h i g h e r c o n c e n t r a t i o n o f AAG may h e l p e x p l a i n the d i f f e r e n c e i n f r e e f r a c t i o n a t t h e r a p e u t i c 125 c o n c e n t r a t i o n s o f PF o b s e r v e d e a r l i e r [Chan e t al. 1989b] (0.041 ± 0.011), and r e s u l t s c u r r e n t l y r e p o r t e d (0.063 ± 0.004). In the p r e s e n t e x p e r i m e n t t h e r a p e u t i c c o n c e n t r a t i o n s o f propafenone (2.0 ng/ml) and 5-h y d r o x y p r o p a f e n o n e (0.5 /jg/m±) which a r e n o r m a l l y seen f o l l o w i n g m u l t i p l e o r a l d o s i n g were used [Kates e t al., 1985]. C o n v e r s e l y , i f m e t a b o l i t e b i n d i n g i s i n v e s t i g a t e d , t h e p a r e n t compound may d i s p l a c e t h e m e t a b o l i t e from d r u g - p r o t e i n b i n d i n g s i t e s i n serum. The d r u g - p r o t e i n b i n d i n g o f 5-0H-PF appeared u n a l t e r e d by the a d d i t i o n o f t h e r a p e u t i c c o n c e n t r a t i o n s o f t h e p a r e n t d r u g , PF. T h i s was unexpected, c o n s i d e r i n g t h e d i f f e r e n c e s i n b i n d i n g a f f i n i t y between PF and 5-OH-PF t o serum p r o t e i n s , however, o t h e r serum p r o t e i n s may be a b l e t o accommodate t h e l i b e r a t e d 5-0H-PF. U l t r a f i l t r a t i o n c o n s i s t e n t l y o v e r e s t i m a t e d the b i n d i n g o f both PF and 5-OH-PF. One e x p l a n a t i o n f o r the c o n s i s t e n t o v e r e s t i m a t i o n o f b i n d i n g by u l t r a f i l t r a t i o n c o u l d be the h i g h n o n - s p e c i f i c b i n d i n g o f both PF and 5-OH-PF which was not c o r r e c t e d f o r , i n the u l t r a f i l t r a t i o n s t u d y . However, even i f the n o n - s p e c i f i c b i n d i n g c o n t r i b u t i o n i s e s t i m a t e d and i n c l u d e d , the b i n d i n g o f PF i s s t i l l o v e r e s t i m a t e d compared t o e q u i l i b r i u m d i a l y s i s . The t e m p e r a t u r e d i f f e r e n c e between u l t r a f i l t r a t i o n and e q u i l i b r i u m d i a l y s i s c o u l d o f f e r a n o t h e r p o s s i b l e e x p l a n a t i o n f o r the o v e r e s t i m a t e d b i n d i n g o b s e r v e d w i t h u l t r a f i l t r a t i o n as compared t o e q u i l i b r i u m d i a l y s i s . The d r u g - p r o t e i n b i n d i n g i n t e r a c t i o n i s mediated by h y d r o p h o b i c i n t e r a c t i o n s , i o n i c bonds, Van d e r Waal's f o r c e s , and hydrogen bonding [ L a i d e r e t al., 1983; L a z n i c e k e t al., 1987; L i n d u p , 126 1987]. These b i n d i n g i n t e r a c t i o n s a r e a l l i n f l u e n c e d , t o a d i f f e r i n g d e g r e e , by changes i n te m p e r a t u r e [ L a i d e r et al., 1983]. In f a c t , t h e a s s o c i a t i o n c o n s t a n t f o r r e v e r s i b l e p r o t e i n - 1 i g a n d b i n d i n g i n t e r a c t i o n s can be w r i t t e n as a f u n c t i o n o f t e m p e r a t u r e , such t h a t , as te m p e r a t u r e i s i n c r e a s e d , t h e a s s o c i a t i o n c o n s t a n t d e c r e a s e s [ L a i d e r et al., 1983]. T h i s would s u g g e s t t h a t as the t e m p e r a t u r e o f a d r u g - p r o t e i n b i n d i n g system i s i n c r e a s e d t h e d r u g - p r o t e i n b i n d i n g i s d e c r e a s e d . T h i s has been shown f o r d r u g s , such as p r o p r a n o l o l [Paxton et a/., 1983], q u i n i d i n e [ N i l s e n and Odd, 1976], and w a r f a r i n [ O e s t e r et al., 1973]. S i n c e u l t r a f i l t r a t i o n was c a r r i e d out a t 25 °C and e q u i l i b r i u m d i a l y s i s was c a r r i e d out a t 37 °C, te m p e r a t u r e dependent b i n d i n g c o u l d c o n t r i b u t e t o t h e o b s e r v e d d i f f e r e n c e i n b i n d i n g between u l t r a f i l t r a t i o n , and e q u i l i b r i u m d i a l y s i s . Temperature dependent b i n d i n g , i n a d d i t i o n t o n o n - s p e c i f i c b i n d i n g , c o u l d p r o v i d e a r e a s o n a b l e e x p l a n a t i o n f o r the l a r g e o b s e r v e d d i f f e r e n c e i n d r u g - p r o t e i n b i n d i n g between u l t r a f i l t r a t i o n and e q u i l i b r i u m d i a l y s i s . 4.3 BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE TO ISOLATED SERUM PROTEINS. 4.3.1 BINDING DIFFERENCES BETWEEN PROPAFENONE AND 5-HYDROXYPROPAFENONE IN VARIOUS PROTEIN SOLUTIONS. , Large d i f f e r e n c e s i n the f r e e f r a c t i o n a r e a p p a r e n t between PF and 5-OH-PF i n serum, l i p o p r o t e i n d e f i c i e n t serum, and AAG s o l u t i o n ( F i g u r e 9 ) . AAG appears t o account f o r a l a r g e p o r t i o n o f t h e b i n d i n g o f PF a t low c o n c e n t r a t i o n s , i n the p r e s e n c e o f the m e t a b o l i t e , 5-OH-PF. T h i s i s 127 not an unexpected o b s e r v a t i o n s i n c e t h e b i n d i n g o f PF i n serum was shown t o c o r r e l a t e w e l l w i t h the amount o f AAG p r e s e n t [Chan et a l . , 1989b]. In a d d i t i o n , i t has been r e p o r t e d t h a t PF b i n d s w i t h h i g h ' a f f i n i t y t o AAG [ G i l l e s et a l . 1985]. F u r t h e r , i t was shown t h a t AAG a c c o u n t s f o r th e m a j o r i t y o f b i n d i n g o f the weakly b a s i c amine d r u g s p r o p r a n o l o l and p r a z o s i n a t lower c o n c e n t r a t i o n s [Sager et al., 1989]. The b i n d i n g o f 5-OH-PF does not approach t h e e x t e n t o f b i n d i n g o b s e r v e d i n serum f o r PF. T h i s may be a r e s u l t o f c h e m i c a l and/or c o n f o r m a t i o n a l d i f f e r e n c e s i n t r o d u c e d t o the drug m o l e c u l e as a r e s u l t o f m e t a b o l i s m ( i . e . the a d d i t i o n o f a p h e n o l i c hydroxy g r o u p ) . T h i s m e t a b o l i s m i n d u c e d d i f f e r e n c e may i n c r e a s e t h e b i n d i n g a f f i n i t y o f t h e m e t a b o l i t e s as i s the case w i t h d i s o p y r a m i d e [Bredensen et al., 1982], o r c o n v e r s e l y m e t a b o l i s m may reduce t h e b i n d i n g a f f i n i t y o f t h e m e t a b o l i t e as su g g e s t e d f o r 5-OH-PF ( T a b l e 4 ) . The e x t e n t o f 5-OH-PF b i n d i n g t o AAG may a l s o be reduced due t o d i s p l a c e m e n t by t h e p a r e n t compound, PF. In f a c t , the b i n d i n g o f 5-OH-PF t o AAG i n t h e p r e s e n c e o f PF i s lo w e r than t h a t observed when PF i s absent (FF w i t h o u t PF 0.32 ± 0.08, and FF w i t h PF i s 0.55 ± 0.10). Thus, c h e m i c a l and s t e r i c d i f f e r e n c e s may reduce the b i n d i n g a f f i n i t y o f 5-OH-PF, and s u b s e q u e n t l y make i t more prone t o d i s p l a c e m e n t from AAG by the p a r e n t drug PF. The b i n d i n g o f PF and 5-OH-PF i n a s o l u t i o n o f i s o l a t e d HSA i s low, d e s p i t e t h e f a c t t h a t an approximate 30 f o l d g r e a t e r m o l ar c o n c e n t r a t i o n o f HSA compared t o AAG was used, t h e r e b y p r o v i d i n g a much g r e a t e r abundance o f p o t e n t i a l d r u g - p r o t e i n b i n d i n g s i t e s . The m a j o r i t y o f PF, a t the low c o n c e n t r a t i o n s s t u d i e d , was bound t o AAG, whereas the 128 5-OH-PF shows much l o w e r b i n d i n g t o AAG. T h i s would s u g g e s t t h a t HSA i s r e l a t i v e l y more i m p o r t a n t i n the b i n d i n g o f 5-OH-PF as compared t o PF a t t h e r a p e u t i c c o n c e n t r a t i o n s (-2.0 ng/ml o f PF and~0.5 izg/mL 5-OH-PF). I t would appear t h a t a s o l u t i o n o f i s o l a t e d AAG a t a s i m i l a r c o n c e n t r a t i o n t o t h a t e n c o u n t e r e d i n normal human serum can a c c o u n t f o r t h e same de g r e e o f d r u g - p r o t e i n b i n d i n g o f PF found i n serum a t t h e low c o n c e n t r a t i o n s s t u d i e d ( F i g u r e 9 ) . Thus, t h e a d d i t i o n o f p h y s i o l o g i c a l c o n c e n t r a t i o n s o f HSA would not be e x p e c t e d t o a l t e r t he b i n d i n g ; however, when such an experiment was c o n d u c t e d an i n c r e a s e i n t h e f r e e f r a c t i o n was o b s e r v e d ( F i g u r e 9 ) . The r e a s o n f o r t h i s i s not known; however, one e x p l a n a t i o n c o u l d be t h a t the a d d i t i o n o f i m p u r i t i e s i n t r o d u c e d by the HSA due t o p u r i f i c a t i o n may p l a y a r o l e . Heavy metal i o n s such as mercury, s i l v e r , copper, and i r o n have been shown t o d e c r e a s e t h e b i n d i n g o f v a r i o u s l i g a n d s t o AAG i n a n o n - c o m p e t i t i v e manner [Kerkay and Westphal, 1969]. A l t e r n a t i v e l y , p r o t e i n - p r o t e i n i n t e r a c t i o n s o f t h e s e i s o l a t e d p r o t e i n s i n a b u f f e r s o l u t i o n may r e s u l t i n a l t e r e d c o n f o r m a t i o n and t h e r e f o r e a c c o u n t o f the o b s e r v e d d e c r e a s e i n b i n d i n g . A l t h o u g h the pH i n the pure AAG s o l u t i o n i s h i g h e r than t h a t i n the AAG + HSA s o l u t i o n ( i e . as pH i n c r e a s e s , b i n d i n g i n c r e a s e s ) , t h i s d i f f e r e n c e i n pH c o u l d not account f o r the o b s e r v e d d i f f e r e n c e i n b i n d i n g . The a d d i t i o n o f p h y s i o l o g i c a l c o n c e n t r a t i o n s o f HSA t o AAG d e c r e a s e d the f r e e f r a c t i o n o f 5-OH-PF. T h i s p r o v i d e s f u r t h e r e v i d e n c e t h a t HSA, i n a d d i t i o n t o AAG, i s an i m p o r t a n t b i n d i n g p r o t e i n f o r 5-OH-PF a t t h e c o n c e n t r a t i o n s s t u d i e d . The f r e e f r a c t i o n o f 5-OH-PF o b s e r v e d 129 i n t h e AAG + HSA s o l u t i o n approximated the f r e e f r a c t i o n o b s e r v e d i n t h e l i p o p r o t e i n d e f i c i e n t serum ( F i g u r e 9 ) . I t would appear t h a t the b i n d i n g o f PF a t t h e r a p e u t i c c o n c e n t r a t i o n s c o u l d be a c c o u n t e d f o r l a r g e l y by AAG, s i m i l a r t o t h e b i n d i n g p r o f i l e s o b s e r v e d f o r p r o p r a n o l o l [ G l a s s o n et al., 1980; S ager et a l . , 1989]. In c o n t r a s t , b i n d i n g o f 5-OH-PF appears t o be more dependent on v a r i o u s o t h e r serum p r o t e i n s as seen w i t h q u i n i d i n e [ N i l s e n et a l . , 1976]. The r e a s o n f o r the d i f f e r e n c e i n the degree t o which PF and 5-OH-PF b i n d t o v a r y i n g degrees t o d i f f e r e n t serum p r o t e i n s i s not c l e a r . However, c h e m i c a l , p h y s i o c h e m i c a l , and s t r u c t u r a l d i f f e r e n c e s due t o m e t a b o l i s m , and subsequent d i s p l a c e m e n t o f 5-OH-PF by PF, due t o i t s l o w e r a f f i n i t y , from AAG may r e s u l t i n the b i n d i n g o f 5-OH-PF becoming more dependent on o t h e r serum p r o t e i n s a t low c o n c e n t r a t i o n s . 4.3.2 THE EFFECT OF BUFFER AND SERUM ULTRAFILTRATE ON THE BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE. B u f f e r c o m p o s i t i o n and s t r e n g t h can have s i g n i f i c a n t e f f e c t s on the b i n d i n g o f v a r i o u s l i g a n d s , as d i s c u s s e d p r e v i o u s l y ( S e c t i o n 4.1.6) [Kremer et al., 1988; Lindup, 1987]. The d i f f e r e n c e i n b i n d i n g o f PF between b u f f e r and serum u l t r a f i l t r a t e i n the AAG + HSA s o l u t i o n may be, i n p a r t , due t o d i f f e r e n c e s i n pH between t h e two s o l u t i o n s ( F i g u r e 10). That i s , w i t h an i n c r e a s e i n pH, the b i n d i n g o f PF i n the AAG + HSA d i s s o l v e d i n u l t r a f i l t r a t e s o l u t i o n i n c r e a s e s t o y i e l d a lower o b s e r v e d f r e e f r a c t i o n . However, pH s e n s i t i v e b i n d i n g i s not a f a c t o r i n the o b s e r v e d d i f f e r e n c e s f o r the drug p r o t e i n b i n d i n g o f PF between AAG 130 d i s s o l v e d i n e i t h e r t h e b u f f e r o r serum u l t r a f i l t r a t e . O t h e r f a c t o r s such as t h e d i f f e r e n c e s i n i o n i c c o m p o s i t i o n , and s t r e n g t h l i k e l y p l a y a r o l e . The l a c k o f a s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e i n the f r e e f r a c t i o n o f 5-OH-PF between AAG d i s s o l v e d i n e i t h e r b u f f e r o r serum u l t r a f i l t r a t e c o u l d be a r e s u l t o f the v a r i a b i l i t y i n t h e b i n d i n g measurements. T h i s l a c k o f a d i f f e r e n c e f o r 5-OH-PF b i n d i n g between AAG d i s s o l v e d i n e i t h e r serum u l t r a f i l t r a t e o r the b u f f e r c o u l d a l s o be a r e s u l t o f t h e r e l a t i v e l y minor importance o f AAG i n the b i n d i n g o f 5-OH-PF compared t o PF. I t appears t h a t t h e c o m p o s i t i o n o f the s o l v e n t used t o d i s s o l v e AAG a f f e c t s t h e d r u g - p r o t e i n b i n d i n g o f PF but not 5-OH-PF. T h i s would t e n d t o sug g e s t t h a t AAG i s more s e n s i t i v e t o non-p h y s i o l o g i c a l s o l v e n t s , such a s ^ O . l M phosphate b u f f e r , s i n c e t h e b i n d i n g o f PF ( h i g h l y bound t o AAG) was a f f e c t e d by b u f f e r c o m p o s i t i o n , whereas the b i n d i n g o f 5-OH-PF ( s l i g h t l y bound t o AAG) was u n a l t e r e d . 4.3.3 THE EFFECT OF THE REMOVAL OF FREE FATTY ACIDS FROM ALBUMIN ON THE BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE. Fr e e f a t t y a c i d s o f v a r i o u s c h a i n l e n g t h s b i n d t o v a r i o u s s i t e s on the HSA m o l e c u l e [Krahg-Hansen, 1981]. I n c r e a s e s i n the c o n c e n t r a t i o n o f f r e e f a t t y a c i d s have been shown t o d e c r e a s e t h e b i n d i n g o f a number o f d i f f e r e n t endogenous l i g a n d s such as t r y p t o p h a n [Krahg-Hansen, 1981], and v a r i o u s drugs such as, diazepam [ V a l l n e r , 1977] and w a r f a r i n [ N i l s e n et a l . , 1974]. C o n v e r s e l y , f r e e f a t t y a c i d s may a l s o have a s t a b i l i z i n g e f f e c t on t h e HSA m o l e c u l e and r e s u l t i n i n c r e a s e d b i n d i n g o f c e r t a i n l i g a n d s such as t e s t o s t e r o n e and p r o g e s t e r o n e [Kragh-Hansen, 1981]. The b i n d i n g d i f f e r e n c e o f propafenone between HSA and f r e e f a t t y a c i d f r e e 131 HSA may be p a r t i a l l y e x p l a i n e d by d i f f e r e n c e s i n pH between t h e two groups ( F i g u r e 12). That i s , as pH i n c r e a s e s , b i n d i n g i n c r e a s e s i n a p r o p o r t i o n a t e manner o v e r t he pH range s t u d i e d ( F i g u r e 3 and 4 ) . S i n c e t h e pH i n t h e f r e e f a t t y a c i d group was g r e a t e r , a d e c r e a s e i n f r e e f r a c t i o n was e x p e c t e d . T h i s o b s e r v e d d i f f e r e n c e i n t h e b i n d i n g between f r e e f a t t y a c i d f r e e HSA and HSA may be an a r t i f a c t o f pH, and t h e r e may i n f a c t be no d i f f e r e n c e i n the b i n d i n g between f r e e f a t t y a c i d f r e e HSA, and HSA. S i m i l a r l y , t h e a d d i t i o n o f the f r e e f a t t y a c i d , p a l m i t i c a c i d , d i d not a f f e c t t h e b i n d i n g o f p r o p r a n o l o l t o HSA u n t i l a 10:1 r a t i o o f f r e e f a t t y a c i d t o HSA was a c h i e v e d [ G l a s s o n et al., 1980]. In c o n t r a s t t o PF, i t i s u n l i k e l y t h a t pH i s the o n l y c o n t r i b u t i n g f a c t o r i n v o l v e d i n the o b s e r v e d b i n d i n g d i f f e r e n c e s o f 5-OH-PF, s i n c e s i m i l a r pH dependent d i f f e r e n c e s i n b i n d i n g were not o b s e r v e d f o r t h e b i n d i n g o f 5-OH-PF. 4.3.4 THE EFFECT OF THE REMOVAL OF LIPOPROTEINS FROM SERUM ON THE BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE. L i p o p r o t e i n s p l a y a r o l e i n the b i n d i n g o f many b a s i c and n e u t r a l d r ugs i n c l u d i n g v a r i o u s t r i c y c l i c a n t i d e p r e s s a n t agents [ R o u t l e d g e , 1986], p r o p r a n o l o l [ T i l l e m e n t et a l . , 1980], t i a n e p t i n e [ T i l l e m e n t et al., 1990] and q u i n i d i n e [ N i l s e n et a l . , 1976]. S i n c e PF b i n d i n g i n serum a t t h e c o n c e n t r a t i o n s s t u d i e d appears t q ^ b e dominated by AAG, the removal o f l i p o p r o t e i n s was not e x p e c t e d t o r e s u l t i n a d i f f e r e n c e i n b i n d i n g ( F i g u r e 13). U n l i k e PF, i t appears t h a t d r u g - p r o t e i n b i n d i n g o f 5-OH-PF i s dependent on a number o f d i f f e r e n t serum p r o t e i n s ; t h e r e f o r e , the removal o f l i p o p r o t e i n s was e x p e c t e d t o r e s u l t i n an i n c r e a s e i n the 132 f r e e f r a c t i o n o f 5-OH-PF. The removal o f l i p o p r o t e i n s l e a d s t o an a p p r o x i m a t e l y 40% i n c r e a s e i n the m e t a b o l i t e f r e e f r a c t i o n a t t h e c o n c e n t r a t i o n s s t u d i e d . T h i s s u g g e s t s t h a t t h e i n c r e a s e i n f r e e 5-OH-PF c o u l d not be accommodated by a d d i t i o n a l b i n d i n g by HSA and AAG, i n the p r e s e n c e t o PF. T h i s o b s e r v a t i o n p r o v i d e s f u r t h e r e v i d e n c e f o r d i f f e r e n c e s between t he b i n d i n g p r o f i l e s o f the p a r e n t d r u g and m e t a b o l i t e when p r e s e n t t o g e t h e r a t t h e r a p e u t i c c o n c e n t r a t i o n s . That i s , t h e b i n d i n g o f PF 2.0 /xg/mL i n t h e p r e s e n c e o f t h e m e t a b o l i t e , 5-OH-PF, 0.5 /zg/mL i s dominated by AAG, whereas the b i n d i n g o f 5-OH-PF i n the p r e s e n c e o f PF i s dependent on a l l o f t h e i n v e s t i g a t e d serum p r o t e i n s (AAG, HSA, and l i p o p r o t e i n s ) . 4.3.5 DISPLACEMENT OF PROPAFENONE AND 5-HYDROXYPROPAFENONE IN SERUM BY DISPLACING AGENTS IBUPROFEN AND DISOPYRAMIDE. I b u p r o f e n has a high a f f i n i t y f o r HSA [Kragh-Hansen, 1981]; t h e r e f o r e , i t i s not s u r p r i s i n g t h a t t h e a d d i t i o n o f i b u p r o f e n i n serum at a t h e r a p e u t i c c o n c e n t r a t i o n (-40 f o l d g r e a t e r than t h e molar c o n c e n t r a t i o n o f PF) i n c r e a s e d the f r e e f r a c t i o n o f both PF and 5-OH-PF i n serum ( T a b l e 6 ) . These i n c r e a s e s i n f r e e f r a c t i o n may a c t u a l l y be m i s l e a d i n g , s i n c e they do not c o r r e s p o n d t o the a c t u a l amount o f drug d i s p l a c e d ( i e . a 35% i n c r e a s e i n f r e e f r a c t i o n o f PF c o r r e s p o n d s t o a -3% d e c r e a s e i n the bound f r a c t i o n o f PF whereas a 27% i n c r e a s e i n the f r e e f r a c t i o n o f 5-OH-PF c o r r e s p o n d s t o a - 10% d e c r e a s e i n t h e bound f r a c t i o n ) . The l a r g e r d e c r e a s e i n the bound f r a c t i o n o f 5-OH-PF compared t o PF may be due t o the f a c t t h a t HSA, as shown p r e v i o u s l y , may be more i m p o r t a n t i n terms o f b i n d i n g 5-OH-PF a t t h e r a p e u t i c 133 c o n c e n t r a t i o n s . D i s o p y r a m i d e has been shown t o b i n d t o AAG w i t h a l a r g e a f f i n i t y [Kremer e t a/., 1988]. The a d d i t i o n o f d i s o p y r a m i d e as a d i s p l a c i n g agent s p e c i f i c f o r AAG, was e x p e c t e d t o i n c r e a s e t h e f r e e f r a c t i o n o f PF t o a l a r g e r e x t e n t , s i n c e i t appears t h a t AAG i s i m p o r t a n t t o t h e b i n d i n g o f PF i n serum. The d e c r e a s e i n b i n d i n g f o r both PF and 5-OH-PF was a p p r o x i m a t e l y ~ 7 %. T h i s d e m o n s t r a t e s t h a t both PF and 5-OH-PF may appear t o be d i s p l a c e d from AAG t o a s i m i l a r d e g r e e . S i n c e 5-OH-PF may a l r e a d y have been d i s p l a c e d from AAG by PF, thus t h e a d d i t i o n o f d i s o p y r a m i d e i s not e x p e c t e d t o r e s u l t i n any f u r t h e r l a r g e i n c r e a s e i n the f r e e f r a c t i o n o f 5-OH-PF. F u r t h e r , t h e i n c r e a s e s i n f r e e PF and 5-OH-PF l i b e r a t e d from t he b i n d i n g s i t e s may be accommodated by o t h e r low a f f i n i t y p r o t e i n s i n serum such as HSA and l i p o p r o t e i n s (low a f f i n i t y , h i g h c a p a c i t y ) m a i n t a i n i n g t h e f r e e f r a c t i o n o f PF and 5-OH-PF o b s e r v e d i n serum r e l a t i v e l y c o n s t a n t . 4.3.6 DISPLACEMENT OF PROPAFENONE AND 5-HYDROXYPROPAFENONE FROM INDIVIDUAL HUMAN SERUM PROTEINS. As a n t i c i p a t e d , a l a r g e p e r c e n t a g e o f bound o f PF was d i s p l a c e d from AAG ( i n c r e a s e i n f r e e f r a c t i o n ) w i t h t he a d d i t i o n o f d i s o p y r a m i d e ; however, a s i m i l a r d e c r e a s e i n b i n d i n g ( i n c r e a s e i n f r e e f r a c t i o n ) was not o b s e r v e d f o r 5-OH-PF. Because PF may a l r e a d y have d i s p l a c e d s i g n i f i c a n t q u a n t i t i e s o f 5-OH-PF from AAG b i n d i n g s i t e s , t h e f u r t h e r a d d i t i o n o f d i s o p y r a m i d e may not r e s u l t i n a f u r t h e r i n c r e a s e i n the 134 f r e e f r a c t i o n o f 5-OH-PF. Furthermore, the l a r g e r v a r i a b i l i t y o f t h e b i n d i n g measurement f o r 5-OH-PF may a l s o overshadow a s m a l l e r d i f f e r e n c e i n t he b i n d i n g o f 5-OH-PF to AAG due t o the a d d i t i o n o f d i s o p y r a m i d e . I b u p r o f e n , used as a d i s p l a c i n g agent f o r HSA, a l s o i n c r e a s e d the f r e e f r a c t i o n o f PF bound t o AAG. T h i s can be e x p l a i n e d by the l a r g e d i f f e r e n c e i n t h e c o n c e n t r a t i o n o f PF and i b u p r o f e n , and t h a t a c i d i c d r u g s a r e a l s o known t o b i n d t o AAG t o a c e r t a i n d e g r e e [ U r i e n e t al., 1982]. Most a c i d i c drugs b i n d t o AAG w i t h a much lo w e r a f f i n i t y than t h a t commonly r e p o r t e d f o r b a s i c and n e u t r a l d r u g s . I t was found t h a t drugs t h a t b i n d t o s i t e I o f HSA and a l s o l a c k a c a r b o x y l i c group g e n e r a l l y b i n d t o AAG w i t h a h i g h e r a f f i n i t y than a c i d i c d rugs c o n t a i n i n g a c a r b o x y l i c group [ U r i e n e t al., 1982]. S i n c e i b u p r o f e n c o n t a i n s such a c a r b o x y l i c group i t i s s p e c u l a t e d t h a t i t s h o u l d b i n d t o AAG w i t h a low a f f i n i t y ; however, t h i s has n e v e r been d e m o n s t r a t e d . The e x t e n t o f b i n d i n g i s dependent on both the c o n c e n t r a t i o n o f i b u p r o f e n and t h e a f f i n i t y ; t h e r e f o r e , d e s p i t e the lower a f f i n i t y o f i b u p r o f e n f o r AAG, i b u p r o f e n may s t i l l b i n d . Thus, the i n c r e a s e i n f r e e f r a c t i o n o f PF o b s e r v e d i n the i s o l a t e d s o l u t i o n o f AAG may be due t o i b u p r o p f e n c o m p e t i t i v e , o r n o n - c o m p e t i t i v e b i n d i n g d i s p l a c e m e n t o f PF. A l t h o u g h 5-OH-PF a l s o shows a d e c r e a s e i n b i n d i n g w i t h t h e a d d i t i o n o f i b u p r o f e n , t h i s d i f f e r e n c e does not r e a c h s t a t i s t i c a l s i g n i f i c a n c e . The b i n d i n g o f both PF and 5-OH-PF t o HSA was shown t o i n c r e a s e w i t h t h e a d d i t i o n o f the amine drug, d i s o p y r a m i d e ( d e c r e a s i n g f r e e f r a c t i o n ) . The r e a s o n f o r t h i s o b s e r v a t i o n was not e x p l a i n e d . S i n c e i t i s u n l i k e l y t h a t c o n f o r m a t i o n changes i n HSA w i l l o c c u r due t o the 135 b i n d i n g o f d i s o p y r a m i d e t o HSA, t h i s s u g g e s t s t h a t d i f f e r e n c e s i n pH between t h e c o n t r o l and d i s o p y r a m i d e t r e a t m e n t groups may p a r t i a l l y c o n t r i b u t e . A s i m i l a r , but unexpected, o b s e r v a t i o n was made f o r 5-OH-PF b i n d i n g t o HSA w i t h the a d d i t i o n o f i b u p r o f e n . I b u p r o f e n may b i n d t o a d i f f e r e n t s i t e o f t h e HSA m o l e c u l e than 5-OH-PF, and t h e r e f o r e cause c o n f o r m a t i o n a l s h i f t s which c o u l d p o s s i b l y expose h y d r o p h o b i c s i t e s which can f u r t h e r b i n d 5-OH-PF p r e s e n t i n b u f f e r . T h i s , appears t o be t r u e o n l y i n b u f f e r and not i n serum, s i n c e t h e f r e e f r a c t i o n i n serum was i n c r e a s e d when i b u p r o f e n was added. A n o t h e r e x p l a n a t i o n , a l t h o u g h u n l i k e l y , i s t h a t t h e d i s p l a c e m e n t o f 5-OH-PF may o c c u r from o t h e r p r o t e i n s r a t h e r than HSA and AAG, as o r i g i n a l l y s p e c u l a t e d . The f r e e f r a c t i o n o f PF i n a s o l u t i o n o f HSA was found t o i n c r e a s e w i t h t h e a d d i t i o n o f i b u p r o f e n , as o r i g i n a l l y e x p e c t e d ( F i g u r e 14). The r e s u l t s f o r PF i n i s o l a t e d p r o t e i n s HSA and AAG a r e q u a l i t a t i v e l y s i m i l a r t o the o b s e r v a t i o n s i n serum. 4.4 BINDING CHARACTERISTICS OF PROPAFENONE AND 5-HYDROXYPROPAFENONE TO ISOLATED PROTEINS 4.4.1 BINDING CHARACTERISTICS OF PROPAFENONE AND 5-HYDROXYPROPAFENONE IN A SOLUTION OF AAG. B i n d i n g o f PF t o AAG appears t o be q u i t e s p e c i f i c and s a t u r a b l e , s i m i l a r t o t h a t shown f o r p r o p r a n o l o l , PF, l i d o c a i n e , v e r a p a m i l [ G i l l e s e t a l . , 1985], p r o p r a n o l o l [ G l a s s o n e t al., 1980], i s o p r o t e r e n o l , n o r e p i n e p h r i n e , e p i n e p h r i n e [Sagar e t al., 1987], and many o t h e r b a s i c d r ugs [ M u l l e r e t al., 1983]. Much c o n t r o v e r s y e x i s t s as t o the number 136 o f b i n d i n g s i t e s f o r drugs on AAG. Many a u t h o r s r e p o r t o n l y one b i n d i n g s i t e from i n d i r e c t b i n d i n g measurements, whereas o t h e r s r e p o r t two d i f f e r e n t d r u g - p r o t e i n b i n d i n g s i t e s u s i n g d i r e c t b i n d i n g s t u d i e s ( e . g . one h i g h a f f i n i t y , low c a p a c i t y b i n d i n g s i t e , and one low a f f i n i t y h i g h c a p a c i t y s i t e . ) [Kremer et a/., 1988]. In agreement w i t h e a r l i e r r e s u l t s [ G i l l e s et al., 1985], the b i n d i n g o f PF t o AAG appears t o have two d i f f e r e n t b i n d i n g s i t e s ( F i g u r e 16a). A l t h o u g h i t i s g e n e r a l l y not v a l i d t o compare b i n d i n g a f f i n i t i e s from d i f f e r e n t s t u d i e s , q u a l i t a t i v e o b s e r v a t i o n s between e x p e r i m e n t s c o n d u c t e d i n s i m i l a r e x p e r i m e n t a l c o n d i t i o n s may be a p p r o p r i a t e . The magnitude o f the h i g h a f f i n i t y b i n d i n g s i t e f o r PF r e p o r t e d e a r l i e r , c o r r e l a t e d w e l l w i t h t h e a f f i n i t y c o n s t a n t measured [ G i l l e s e t al., 1985]. The 6 f o l d d i f f e r e n c e measured between t h e a f f i n i t y o f t h e p r e s e n t s t u d y and the p r e v i o u s r e p o r t e d v a l u e c o u l d l i k e l y be due t o d i f f e r e n c e s i n e x p e r i m e n t a l t e c h n i q u e [Kremer e t al., 1988] ( i e . a r i g o r o u s method f o r the f u r t h e r p u r i f i c a t i o n o f AAG was c o n d u c t e d i n the e a r l i e r s t u d y [ G i l l e s e t a l . , 1 9 8 5 ] ) . Thus, d i r e c t comparisons between b i n d i n g parameters must be made w i t h c a u t i o n . In c o n t r a s t t o the e a r l i e r s t u d i e s [ G i l l e s et a/., 1985], a 10 f o l d l a r g e r d i f f e r e n c e i n the c a p a c i t y and a 35 f o l d d e c r e a s e i n the a f f i n i t y o f the low a f f i n i t y b i n d i n g s i t e f o r PF on AAG were measured [ G i l l e s e t a l . , 1985]. B i n d i n g o f i s o p r o t e r e n o l , n o r e p i n e p h r i n e , and e p i n e p h r i n e show a low a f f i n i t y s i t e w i t h a l a r g e c a p a c i t y , s i m i l a r t o what has been o b s e r v e d f o r the low c a p a c i t y s i t e f o r PF [ S a g e r e t a l . , 1987]. However, d e s p i t e the f i n d i n g s f o r c a t e c h o l a m i n e s , the c a p a c i t y f o r PF a t the low a f f i n i t y s i t e i s s u s p i c i o u s l y h i g h . 137 The s t u d y o f 5-OH-PF b i n d i n g t o AAG d i d not s u g g e s t t h e p r e s e n c e o f more tha n one d r u g - p r o t e i n b i n d i n g s i t e . T h i s may r e s u l t from t he r e l a t i v e l y s m a l l d i f f e r e n c e between t he h i g h and low a f f i n i t y b i n d i n g s i t e on AAG. I f t h e a f f i n i t i e s f o r t h e s e s i t e s c l o s e l y a pproximate each o t h e r , i t may be d i f f i c u l t i f not i m p o s s i b l e t o d i s t i n g u i s h between t he two b i n d i n g s i t e s . T h i s would r e s u l t i n h y b r i d b i n d i n g c o n s t a n t s , as d i s c u s s e d e a r l i e r ( F i g u r e 16b). T h i s , however, may not be t h e c a s e f o r 5-OH-PF s i n c e t h e c a p a c i t y o f the h i g h a f f i n i t y s i t e o f PF i s s i m i l a r t o t h a t o b s e r v e d f o r 5-OH-PF. The major d i f f e r e n c e between t h e s e two s i t e s i s t h e b i n d i n g a f f i n i t y ( K a l PF i s 10 f o l d g r e a t e r t h a t t h e o b s e r v e d Ka f o r 5-OH-PF). S i n c e t h e a d d i t i o n o f 2.0 /zg/mL o f PF t o a s o l u t i o n o f 0.5 /zg/mL 5-OH-PF d e c r e a s e s t h e m e t a b o l i t e b i n d i n g by ~ 20%, i t seems l i k e l y t h a t PF and 5-OH-PF share a common b i n d i n g s i t e on AAG. T h i s d i s p l a c e m e n t appears t o be c o m p e t i t i v e i n n a t u r e s i n c e the a d d i t i o n o f 0.5 /zg/mL o f 5-OH-PF t o 2.0 /zg/mL o f PF r e s u l t s i n a d i s p l a c e m e n t o f ~ 2% ( i e . t h e d i s p l a c e m e n t i s mutual, but PF w i t h t h e h i g h e r a f f i n i t y c o n s t a n t appears t o d i s p l a c e 5-OH-PF more r e a d i l y . A t f i r s t t h i s would not appear t o c o r r e l a t e w e l l w i t h the p r e v i o u s l y o b s e r v e d r e s u l t s s u g g e s t i n g t h a t 5-OH-PF i s not h i g h l y d i s p l a c e d by d i s o p y r a m i d e from AAG. However, i t s h o u l d be c o n s i d e r e d t h a t PF p r e s e n t i n the e a r l i e r s t u d i e s may a l r e a d y have d i s p l a c e d a s i g n i f i c a n t q u a n t i t y o f 5-OH-PF p r i o r t o t h e a d d i t i o n o f d i s o p y r a m i d e . I f changes i n f r e e f r a c t i o n a r e c o n s i d e r e d , as the c o n c e n t r a t i o n o f d r u g i n c r e a s e s and approaches t he c a p a c i t y o f AAG, the f r e e f r a c t i o n , as e x p e c t e d from f i g u r e 28, d e m o n s t r a t e s an a b r u p t r i s e . T h i s a b r u p t i n c r e a s e i n t h e f r e e f r a c t i o n o f both PF and 5-OH-PF o c c u r s as t h e 138 c o n c e n t r a t i o n s u r p a s s e s the c a p a c i t y o f the h i g h a f f i n i t y s i t e on AAG ( F i g u r e 22 and 23). 4.4.2 THE BINDING CHARACTERISTICS OF PROPAFENONE AND 5-HYDROXYPROPAFENONE IN A SOLUTION OF HUMAN SERUM ALBUMIN. As w i t h many b a s i c d r u g s , such as, p r o p r a n o l o l [ G l a s s o n et a l . , 1980], q u i n i d i n e [ N i l s e n , 1976], and v a r i o u s c a t e c h o l a m i n e s [Sager et al., 1987], both PF and 5-OH-PF d i s p l a y n o n - s a t u r a b l e b i n d i n g t o HSA ( F i g u r e 16a and 16b). The b i n d i n g a f f i n i t y measured f o r PF and 5-OH-PF t o AAG i s 6000 and 1500 f o l d g r e a t e r t h a t t h e a f f i n i t y t o HSA f o r PF and 5-OH-PF, r e s p e c t i v e l y . The low a f f i n i t y and h i g h c a p a c i t y b i n d i n g s i t e s a r e i n k e e p i n g w i t h HSA b i n d i n g o b s e r v e d f o r v a r i o u s o t h e r b a s i c o r n e u t r a l compounds [Kremer et al., 1988]. S i n c e a h i g h c a p a c i t y s i t e was o b s e r v e d f o r PF and 5-OH-PF t o HSA, c o n c e n t r a t i o n dependence i n the b i n d i n g o f 5-OH-PF and PF would not be e x p e c t e d as was o b s e r v e d ( F i g u r e s 22 and 2 3 ) . 4.4.3 CHARACTERIZATION OF PROPAFENONE AND 5-HYDROXYPROPAFENONE BINDING IN SERUM. PF shows two b i n d i n g heterogeneous s i t e s i n serum ( F i g u r e 18a). T h i s would be e x p e c t e d c o n s i d e r i n g t h e -6000 f o l d d i f f e r e n c e i n a f f i n i t i e s between AAG and HSA. The h i g h a f f i n i t y b i n d i n g s i t e i n serum i s a p p r o x i m a t e l y 30 f o l d lower than t h a t i n pure AAG. T h i s c o u l d be a r e s u l t o f endogenous c o m p e t i t i v e o r n o n - c o m p e t i t i v e b i n d i n g i n h i b i t o r s p r e s e n t i n serum. A n o t h e r e x p l a n a t i o n c o u l d be t h a t s i n c e t h e h i g h 139 a f f i n i t y s i t e i n serum i s a h y b r i d c o n s t a n t , composed o f , i n a d d i t i o n t o AAG, o t h e r p r o t e i n s w i t h lower a f f i n i t y which may re d u c e t h e o b s e r v e d a f f i n i t y c o n s t a n t . The b i n d i n g a f f i n i t y o f the low a f f i n i t y s i t e i n serum c o r r e s p o n d s w e l l w i t h t h e a f f i n i t y r e p o r t e d f o r PF b i n d i n g t o HSA i n s e c t i o n 4 . 4 . 4 . The b i n d i n g c o n s t a n t s f o r PF i n serum a l s o c o r r e s p o n d w e l l w i t h e a r l i e r r e s u l t s [Chan et a7., 1989b]. The same r e l a t i o n s h i p o b s e r v e d f o r PF does not h o l d f o r 5-OH-PF s i n c e o n l y one b i n d i n g s i t e was ap p a r e n t f o r the m e t a b o l i t e i n serum ( F i g u r e 18b). I t has been shown f o r 5-OH-PF t h a t a h i g h a f f i n i t y s i t e p r e s e n t on AAG i s a p p r o x i m a t e l y 1500 f o l d g r e a t e r a f f i n i t y than t h e low a f f i n i t y s i t e p r e s e n t on HSA; t h e r e f o r e , a s i m i l a r r e l a t i o n s h i p as seen f o r PF i n serum was e x p e c t e d . However, c o m p e t i t i v e and n o n - c o m p e t i t i v e b i n d i n g i n h i b i t o r s p r e s e n t i n serum c o u l d r e s u l t i n t h i s d i f f e r e n c e b e i n g r e d u c e d s u b s t a n t i a l l y , as was seen w i t h PF. T h i s , i n t u r n , c o u l d r e s u l t i n the d i f f e r e n c e between t he h i g h and low a f f i n i t y b i n d i n g c o n s t a n t s b e i n g t o o small t o d e t e c t two d i s t i n c t b i n d i n g s i t e s ( F i g u r e 2 7 ) . I t i s h i g h l y l i k e l y t h a t 5-OH-PF, w i t h o u t t h e a d d i t i o n o f PF, d i s p l a y s a s i m i l a r b i n d i n g p r o f i l e t o PF i n serum. However, the i n h e r e n t s c a t t e r i n the 5-OH-PF d a t a , i n a d d i t i o n t o the sm a l l d i f f e r e n c e between the h i g h and low a f f i n i t y b i n d i n g s i t e s r e s u l t s i n t h e i n a b i l i t y o f one t o e x p e r i m e n t a l l y d i f f e r e n t i a t e t h e p r e s e n c e o f two b i n d i n g s i t e s i n serum. Thus, t he r e p o r t e d b i n d i n g c o n s t a n t s f o r 5-OH-PF i n serum a r e l i k e l y t o be a h y b r i d c o n s t a n t o f two d i f f e r e n t b i n d i n g s i t e s . PF undergoes c o n c e n t r a t i o n b i n d i n g ; an a b r u p t r i s e i n the f r e e 140 f r a c t i o n i s o b s e r v e d as the c o n c e n t r a t i o n i n c r e a s e s p a s t t he c a p a c i t y o f t h e h i g h a f f i n i t y b i n d i n g s i t e ( F i g u r e 22). S i n c e the c a p a c i t y o f t h e h i g h a f f i n i t y s i t e c o r r e l a t e s w e l l w i t h t he c a p a c i t y o f AAG, t h e c o n c e n t r a t i o n dependent b i n d i n g o f PF can be l i k e l y a t t r i b u t e d t o t h e s a t u r a t i o n o f AAG. A l t h o u g h a s i m i l a r s a t u r a t i o n o f AAG by 5-OH-PF i s e v i d e n t , c o n c e n t r a t i o n dependent b i n d i n g i n serum i s not a p p a r e n t . S i n c e t he Ka f o r 5-OH-PF i s s i g n i f i c a n t l y l e s s than t h a t o f PF, the g r a d u a l r i s e i n f r e e f r a c t i o n p r e d i c t e d i n f i g u r e 28 may not be a p p a r e n t when compared t o the a b r u p t r i s e i n f r e e f r a c t i o n o f PF w i t h i n c r e a s i n g t o t a l d r u g c o n c e n t r a t i o n i n serum. The i n h e r e n t s c a t t e r o f the 5-OH-PF f r e e f r a c t i o n d a t a may r e s u l t i n an i n c r e a s e i n f r e e f r a c t i o n b e i n g overshadowed. S i n c e i t was shown t h a t o t h e r p r o t e i n s i n t h e serum b i n d 5-OH-PF, t h e s e o t h e r p r o t e i n s (HSA and l i p o p r o t e i n s ) may accommodate t h e 5-OH-PF once i t has s a t u r a t e d AAG. 4.4.4 BINDING CHARACTERISTICS OF PROPAFENONE AND 5-HYDROXYPROPAFENONE TO LIPOPROTEINS. L i p o p r o t e i n s a r e complex m i x t u r e s o f p r o t e i n s and v a r y i n g amounts o f l i p i d . In a d d i t i o n , t h e s e complexes d i f f e r s i g n i f i c a n t l y between v a r i o u s c l a s s e s o f l i p o p r o t e i n s (see T a b l e 7 ) . 141 TABLE 7: THE COMPOSITION OF DIFFERENT CLASSES OF LIPOPROTEINS [VLDL, LDL, AND HDL] COMPOSITION VLDL LDL HDL TOTAL LIPID 90% 75% 50% TRIGLYCERIDES 60% 10% 11% CHOLESTEROL 17% 60% 32% PHOSPHOLIPID 20% 30% 50% TOTAL PROTEINS -12% -25% -50% APO-LIPOPROTEIN TYPE MAJOR B100 B100 A-I C'S - A-11 E MINOR A-I C'S C'S A - I I E D'S B48 - E TABLE 7 FROM Scanu, (1985) C o n s i d e r i n g t h e d i v e r s i t y o f v a r i o u s c l a s s e s o f l i p o p r o t e i n s , i t i s not u n r e a s o n a b l e t o e x p e c t d i f f e r e n t d e g r e e s o f d r u g - p r o t e i n b i n d i n g o r i n th e c a s e o f l i p o p r o t e i n s , s o l u b i l i z a t i o n , i n v a r i o u s l i p o p r o t e i n s d e p e n d i n g on the c h a r a c t e r i s t i c s o f the drug i n q u e s t i o n . Many b a s i c and n e u t r a l d r u g s , such as c a t e c h o l a m i n e s [Sager e t a l . , 1987], q u i n i d i n e [ N i l s e n , 1976], a m i t r i p t y l i n e , n o r t r i p t y l i n e , d o x e p i n , d e s m e t h y l d o x e p i n [ P i k e e t al., 1984], t i a n e p t i n e [ Z i n i e t al., 1990], and p r o p r a n o l o l [ G l a s s o n e t al., 1980] b i n d t o l i p o p r o t e i n s w i t h d i f f e r i n g d e g r e e s . I t i s g e n e r a l l y found t h a t drug b i n d i n g t o l i p o p r o t e i n s f o l l o w s t he g e n e r a l o r d e r o f i m p o r t a n c e : HDL > LDL > VLDL [ G l a s s o n e t a l . , 1980; Z i n i e t a l . , 1990]. 142 PF b i n d i n g t o HDL u n l i k e p r e v i o u s s t u d i e s w i t h o t h e r b a s i c d r u g s appears t o have a s a t u r a b l e b i n d i n g s i t e w i t h i n t h e HDL complex ( F i g u r e 19a). T h i s s a t u r a b l e b i n d i n g o f PF t o the HDL complex c o u l d be due t o th e l a r g e c o n c e n t r a t i o n o f p r o t e i n s i n the HDL complex as compared t o th e o t h e r complexes. I t s h o u l d a l s o be c o n s i d e r e d t h a t t h e n o n - s p e c i f i c b i n d i n g c o n t r i b u t i o n t o PF HDL-binding may have been o b s c u r e d due t o t h e l i m i t e d s o l u b i l i t y o f PF i n pH 7.4 b u f f e r ; t h e r e f o r e , b i n d i n g c o u l d not be c h a r a c t e r i z e d i n c o n c e n t r a t i o n s o f PF beyond 100 /xg/mL t o c o m p l e t e l y s a t u r a t e t h e ap p a r e n t s a t u r a b l e b i n d i n g s i t e on the HDL complex. The b i n d i n g o f both PF and 5-OH-PF t o the LDL complex was e x t r e m e l y h i g h . R e s u l t s from f i g u r e s 22 and 23 s u g g e s t t h e LDL c o u l d a c c o u n t f o r a l a r g e r degree o f b i n d i n g than o b s e r v e d f o r both PF and 5-OH-PF i n serum. The re a s o n f o r t h i s i s not known, but i t may be s p e c u l a t e d t h a t t h e b i n d i n g i n t e r a c t i o n o f PF and 5-OH-PF w i t h LDL i s one o f s o l u b i l i z a t i o n o f PF and 5-OH-PF, r a t h e r than t h e c h a r a c t e r i s t i c d r u g - p r o t e i n i n t e r a c t i o n . In f a c t , n o n - s p e c i f i c b i n d i n g was shown f o r ac c o u n t f o r the b i n d i n g i n t e r a c t i o n o f PF and 5-OH-PF t o both LDL and VLDL. N o n - s p e c i f i c b i n d i n g i s the p r o d u c t o f the c a p a c i t y and the b i n d i n g a f f i n i t y c o n s t a n t [ G l a s s o n et a l . 1980]. S i n c e the c a p a c i t y o f LDL has been shown t o be l a r g e , i t i s h i g h l y l i k e l y t h a t the b i n d i n g a f f i n i t y c o n s t a n t i s v e r y low. B i n d i n g o f PF and 5-OH-PF t o VLDL was v e r y low i n comparison t o LDL. The m e t a b o l i t e , 5-OH-PF, d i s p l a y e d a l m o s t a 10 f o l d g r e a t e r b i n d i n g t o VLDL than the p a r e n t d r u g , PF. I f th e i n t e r a c t i o n o f PF and 5-OH-PF w i t h VLDL i s one o f s o l u b i l i z a t i o n o f the drugs r a t h e r than c l a s s i c a l d r u g - p r o t e i n b i n d i n g , the h i g h e r s o l u b i l i t y o f 5-OH-PF i n hexane and t o l u e n e a t pH 7.4 would s u g g e s t t h a t 143 5-OH-PF can p a r t i t i o n i n t o t h e VLDL w i t h g r e a t e r ease than PF, due t o t h e c h e m i c a l d i f f e r e n c e s between the p a r e n t drug and m e t a b o l i t e . I t i s l i k e l y t h a t t h e i n t e r a c t i o n o f PF and 5-OH-PF w i t h l i p o p r o t e i n s i s l a r g e l y due t o s o l u b i l i z a t i o n s i n c e the l o g o f n o n - s p e c i f i c b i n d i n g (Ka*NP) shows a v e r y good c o r r e l a t i o n (r=0.92 f o r PF and r=0.98 f o r 5-OH-PF) t o the amount o f c h o l e s t e r o l p r e s e n t i n the l i p o p r o t e i n complex ( i e . LDL has t h e h i g h e s t c o n c e n t r a t i o n o f c h o l e s t e r o l i n the complex f o l l o w e d by HDL and VLDL [see t a b l e 7 ] ) . T h i s d e m o n s t r a t e s t h a t l i p o p r o t e i n s p l a y a r o l e i n the b i n d i n g / s o l u b i l i z a t i o n o f both PF and 5-OH-PF. The d r u g - p r o t e i n i n t e r a c t i o n t o t h e l i p o p r o t e i n s s t u d i e d would l i k e l y be due t o s o l u b i l i z a t i o n and i n some c a s e s , such as PF and HDL b i n d i n g , the c l a s s i c a l d r u g - p r o t e i n i n t e r a c t i o n . 4.4.5 CALCULATED BINDING OF PROPAFENONE AND 5-HYDROXYPROPAFENONE BY MATHEMATICAL RECONSTITUTION OF THE DRUG-BINDING SYSTEM. The comparison o f d a t a o b t a i n e d i n p r o t e i n s o l u t i o n s d i s s o l v e d i n b u f f e r cannot be q u a n t i t a t i v e l y compared to t h a t o b t a i n e d i n serum, s i n c e serum i s a c o m p l i c a t e d s o l u t i o n / s u s p e n s i o n o f v a r i o u s p r o t e i n s , endogenous c h e m i c a l s , and i o n s . However, q u a l i t a t i v e comparisons may be u s e f u l i n d e t e r m i n i n g the r e l a t i v e importance o f v a r i o u s p r o t e i n s a t s p e c i f i c c o n c e n t r a t i o n s o f PF and 5-OH-PF. The c a l c u l a t e d b i n d i n g o f PF and 5-OH-PF are g r e a t l y o v e r e s t i m a t e d ( f i g u r e s 24 and 25), t h i s appears to be l a r g e l y due t o t h e v e r y h i g h a p p a r e n t s o l u b i l i z a t i o n o f drug i n LDL. As the c o n t r i b u t i o n s o f LDL and VLDL t o the o v e r a l l b i n d i n g o f PF 144 and 5-OH-PF are removed, t he b i n d i n g more c l o s e l y c o r r e l a t e s t o t h a t o b s e r v e d i n serum. The r e a s o n f o r t h e l a r g e uptake o f drug by the LDL complex i s not c o m p l e t e l y known; however, i t i s s p e c u l a t e d t h a t t h e b i n d i n g t o LDL appears t o be a s o l u b i l i z a t i o n phenomena o f v e r y h i g h c a p a c i t y . W ithout o t h e r p r o t e i n s a v a i l a b l e t o b i n d d r u g , and th u s c o u n t e r a c t f o r c e s o f t h e s o l u b i l i z a t i o n o f drug i n t o LDL, t h e LDL complex appears t o a c t as a r e s e r v o i r f o r d r u g . T h a t i s , t h e LDL complex t a k e s up l a r g e q u a n t i t i e s o f both PF and 5-OH-PF. When o t h e r p r o t e i n s w i t h h i g h e r a f f i n i t y a r e not p r e s e n t , drug e n t e r s t h e l i p o p r o t e i n complex unimpeded u n t i l t h e c o n c e n t r a t i o n g r a d i e n t , t h e d r i v i n g f o r c e f o r s o l u b i l i z a t i o n , i s r e d u c e d . A n o t h e r e x p l a n a t i o n c o u l d be t h a t the model chosen i s a g r o s s o v e r s i m p l i f i c a t i o n o f t h e a c t u a l b i n d i n g p r o c e s s . That i s , b i n d i n g c o n t r i b u t i o n s f o r i n d i v i d u a l p r o t e i n s are c a l c u l a t e d independent o f one a n o t h e r when, i n f a c t , b i n d i n g i s a dynamic e q u i l i b r i u m between f r e e d r u g , and a l l t h e serum p r o t e i n s a r e i n v o l v e d i n b i n d i n g o f drug . Endogenous b i n d i n g i n h i b i t o r s and enhancers p r e s e n t i n serum, but not i n our b i n d i n g model, may a l s o have t o be taken i n t o c o n s i d e r a t i o n . AAG, by i t s e l f , a t lower c o n c e n t r a t i o n s appears t o be i m p o r t a n t i n the b i n d i n g o f both PF and 5-OH-PF; c o n v e r s e l y , t h e b i n d i n g o f 5-OH-PF appears t o c o r r e l a t e b e t t e r w i t h t h a t o b s e r v e d i n serum w i t h t h e a d d i t i o n o f HSA a t i n t e r m e d i a t e c o n c e n t r a t i o n s . No d i f f e r e n c e i s ap p a r e n t i n the b i n d i n g o f PF w i t h t h e a d d i t i o n o f HSA. T h i s appears t o sug g e s t t h a t HSA and AAG are i m p o r t a n t b i n d i n g p r o t e i n s i n serum f o r 5-OH-PF a t lower c o n c e n t r a t i o n s , whereas o n l y AAG appears t o be i m p o r t a n t i n t h e b i n d i n g o f PF a t lower c o n c e n t r a t i o n s . 145 At h i g h c o n c e n t r a t i o n s o f 5-OH-PF i t i s found t h a t t h e HSA and AAG c o n t r i b u t i o n s t o b i n d i n g c o r r e l a t e w e l l w i t h the serum, but tend t o u n d e r e s t i m a t e t h e b i n d i n g o b s e r v e d . T h i s tends t o s u g g e s t t h a t l i p o p r o t e i n b i n d i n g may be i m p o r t a n t a t h i g h c o n c e n t r a t i o n s o f 5-OH-PF. C u r i o u s l y , i t appears t h a t HDL p l a y s a r o l e i n the b i n d i n g o f PF a t i n t e r m e d i a t e and h i g h c o n c e n t r a t i o n s , but s t i l l u n d e r e s t i m a t e s t h e b i n d i n g o f PF. T h i s would tend t o su g g e s t t h a t l i p o p r o t e i n s p l a y a i m p o r t a n t r o l e i n the s o l u b i l i z a t i o n o f PF a t h i g h c o n c e n t r a t i o n s . At f i r s t t h e s e r e s u l t s may appear t o c o n t r a d i c t t h e e a r l i e r o b s e r v a t i o n s , s t a t i n g t h a t AAG had a minor r o l e i n the b i n d i n g o f 5-OH-PF, but i t s h o u l d be c o n s i d e r e d t h a t PF, which can d i s p l a c e 5-OH-PF from AAG, was p r e s e n t i n t h e e a r l y s t u d i e s . 4.5 UPTAKE OF PROPAFENONE AND 5-HYD0XYPR0PAFEN0NE BY HUMAN RED BLOOD CELLS. The time r e q u i r e d f o r d i s t r i b u t i o n o f PF and 5-OH-PF i n t o human r e d b l o o d c e l l s (RBC) was v e r y r a p i d , s i m i l a r t o t h a t r e p o r t e d f o r d i s o p y r a m i d e [ G a r r e t t e t a l . , 1985], and p r o p r a n o l o l [Ogata et a l . , 1984]. The e x t e n t o f uptake i n t o r e d b l o o d c e l l s suspended i n o n l y b u f f e r was v e r y h i g h , w i t h 5-OH-PF d i s t r i b u t i o n s t a t i s t i c a l l y h i g h e r t h a n t h a t o f PF. T h i s o b s e r v a t i o n p a r a l l e l s t h e o b s e r v a t i o n t h a t 5-OH-PF d i s t r i b u t e s i n t o VLDLs more r e a d i l y than PF, and f u r t h e r the p a r t i t i o n i n g o f 5-OH-PF i n hexane a t pH 7.4 i s much g r e a t e r than PF. The a d d i t i o n o f serum p r o t e i n s d e c r e a s e s t h e d r i v i n g f o r c e f o r drug t o d i s t r i b u t e i n t o t h e r e d b l o o d c e l l s . T h i s i s s i m i l a r t o the s i t u a t i o n 146 s p e c u l a t e d f o r l a r g e b i n d i n g o f PF and 5-OH-PF t o LDL i n b u f f e r . The drug c o n c e n t r a t i o n r a t i o i n RBC/supernatant (serum) f o r PF and 5-OH-PF c o r r e s p o n d s w e l l w i t h t h e drug c o n c e n t r a t i o n r a t i o i n RBC/supernatant o f o b s e r v e d i n whole b l o o d . The whole b l o o d r a t i o f o r PF was s i m i l a r t o t h a t o b t a i n e d f o r d i s o p y r a m i d e [ G a r r e t t e t al., 1985] and p r o p r a n o l o l [Ogata, 1989]. The r a t i o o b s e r v e d f o r 5-OH-PF was a l m o s t t w i c e t h a t f o r PF, t h i s may be due t o the ob s e r v e d c h e m i c a l d i f f e r e n c e s between t h e s e two compounds as a r e s u l t o f m e t a b o l i s m . 4.6 SUMMARY Due t o the h i g h degree and v a r i a b i l i t y o f n o n - s p e c i f i c b i n d i n g d i s p l a y e d by PF and 5-OH-PF, e q u i l i b r i u m d i a l y s i s was f e l t t o be t h e s u p e r i o r method f o r t h e d e t e r m i n a t i o n o f d r u g - p r o t e i n b i n d i n g f o r t h e s e two compounds. Many f a c t o r s must be o p t i m i z e d and c o n t r o l l e d i n o r d e r f o r e q u i l i b r i u m d i a l y s i s t o p r o v i d e both a c c u r a t e and r e p r o d u c i b l e e s t i m a t e s o f d r u g - p r o t e i n b i n d i n g . The time t o e q u i l i b r i u m was o p t i m i z e d , and many p o s s i b l e f a c t o r s which c o u l d i n t r o d u c e s i g n i f i c a n t e r r o r t o the b i n d i n g e s t i m a t e were c o n s i d e r e d . Both PF and 5-OH-PF were found t o d i s p l a y pH dependent b i n d i n g i n serum and i n a s o l u t i o n o f i s o l a t e d AAG. A l t h o u g h PF showed pH dependent b i n d i n g i n a s o l u t i o n o f i s o l a t e d HSA, the same was not t r u e f o r 5-OH-PF. The pH dependent b i n d i n g o f both PF and 5-OH-PF appeared t o c o r r e l a t e w e l l w i t h the degree o f i o n i z a t i o n , and the p a r t i t i o n i n g between t o l u e n e and phosphate b u f f e r , as a f u n c t i o n o f pH. T h i s s u g g e s t s t h a t t h e b i n d i n g o f both PF and 5-OH-PF i s l a r g e l y h y d r o p h o b i c 147 ( i . e . as t h e de g r e e o f i o n i z a t i o n d e c r e a s e s t h e b i n d i n g i n c r e a s e s ) . The a d d i t i o n o f the p a r e n t d r u g , PF, d i d not appear t o a l t e r t h e b i n d i n g o f 5-OH-PF. AAG appeared t o acc o u n t f o r the m a j o r i t y o f PF b i n d i n g even i n the p r e s e n c e o f the m e t a b o l i t e , 5-OH-PF. However, i t appears t h a t t h e a d d i t i o n o f PF d e c r e a s e s the b i n d i n g o f 5-OH-PF t o AAG. There appears t o be no d i f f e r e n c e i n the b i n d i n g o f PF w i t h t h e a d d i t i o n o f 5-OH-PF i n HSA, and vice versa. The removal o f l i p o p r o t e i n s from serum does not red u c e t h e b i n d i n g o f PF; however, l a r g e d e c r e a s e s i n the b i n d i n g o f 5-OH-PF were o b s e r v e d . The b i n d i n g c h a r a c t e r i s t i c s o f PF and 5-OH-PF were d e t e r m i n e d i n s o l u t i o n s o f AAG, HSA, HDL, LDL, and VLDL. Both PF and 5-OH-PF showed a hig h a f f i n i t y , low c a p a c i t y b i n d i n g s i t e on AAG; however, PF was shown t o e x h i b i t a second low a f f i n i t y , h i g h c a p a c i t y b i n d i n g s i t e . The a f f i n i t y f o r PF t o the h i g h a f f i n i t y , low c a p a c i t y s i t e on AAG was a p p r o x i m a t e l y 10 f o l d g r e a t e r than t h a t o b s e r v e d f o r 5-OH-PF. The b i n d i n g o f PF and 5-OH-PF t o HSA showed one low a f f i n i t y , h i g h c a p a c i t y b i n d i n g s i t e . The d i f f e r e n c e i n a f f i n i t y and c a p a c i t y between t h i s s i t e f o r PF and 5-OH-PF was s m a l l . The b i n d i n g o f PF and 5-OH-PF t o HDL, LDL, and VLDL appeared t o be due t o s o l u b i l i z a t i o n r a t h e r than a " t r u e " d r u g - p r o t e i n b i n d i n g i n t e r a c t i o n , s i n c e t h e degree o f uptake o f e i t h e r PF and 5-OH-PF was c l o s e l y c o r r e l a t e d t o the amount o f c h o l e s t e r o l p r e s e n t w i t h i n t h e l i p o p r o t e i n complex. The o n l y e x c e p t i o n was PF b i n d i n g o f HDL, which appeared t o d i s p l a y s a t u r a b l e b i n d i n g . When 148 s i m i l a r c u r v e f i t t i n g was c a r r i e d o u t i n serum, as e x p e c t e d , PF showed two b i n d i n g s i t e s , one h i g h a f f i n i t y , low c a p a c i t y , and one low a f f i n i t y , h i g h c a p a c i t y . A l t h o u g h a s i m i l a r s i t u a t i o n was e x p e c t e d f o r 5-OH-PF, s i n c e i t a l s o showed a h i g h a f f i n i t y b i n d i n g s i t e on AAG and a number o f lower a f f i n i t y b i n d i n g s i t e s i n s o l u t i o n s o f i s o l a t e d p r o t e i n s , o n l y one s i t e c o u l d be d e s c r i b e d . I t i s h i g h l y l i k e l y t h a t due t o i n h e r e n t s c a t t e r o f t h e d a t a , i n a d d i t i o n t o t h e r e l a t i v e l y s m a l l d i f f e r e n c e between t he a f f i n i t i e s f o r the low and h i g h a f f i n i t y s i t e , two b i n d i n g s i t e s i n serum c o u l d n o t be e x p e r i m e n t a l l y d i s t i n g u i s h e d . Thus, t h e b i n d i n g parameter e s t i m a t e d f o r 5-OH-PF i n serum was a h y b r i d macro c o n s t a n t o f two t h e o r e t i c a l b i n d i n g s i t e s . The a d d i t i o n o f d i s o p y r a m i d e f u r t h e r d e m o n s t r a t e s t h a t PF and 5-OH-PF may sh a r e a common b i n d i n g s i t e on AAG, s i n c e t h e a d d i t i o n o f d i s o p y r a m i d e d i s p l a c e d PF from AAG, but d i s p l a c e d r e l a t i v e l y l i t t l e 5-OH-PF. However, i t s h o u l d be c o n s i d e r e d t h a t t h e 5-OH-PF was most l i k e l y d i s p l a c e d by PF p r i o r t o the a d d i t i o n o f d i s o p y r a m i d e . The uptake o f 5-OH-PF by r e d b l o o d c e l l s was two f o l d g r e a t e r than t h e uptake o f PF i n whole b l o o d . T h i s c o r r e l a t e s w e l l w i t h t h e p a r t i t i o n i n g o f both PF and 5-OH-PF between hexane and b u f f e r (5-OH-PF was found t o p a r t i t i o n b e t t e r i n t o t h e hexane at pH 7.4 than P F ) . Thus, i t appears t h a t PF b i n d i n g i n serum a t t h e r a p e u t i c c o n c e n t r a t i o n s i s d i c t a t e d by AAG, w i t h HSA and l i p o p r o t e i n s a v a i l a b l e t o b u f f e r f r e e PF l i b e r a t e d as a r e s u l t o f a p o t e n t i a l d i s p l a c e m e n t i n t e r a c t i o n , o r s a t u r a t i o n o f the AAG h i g h a f f i n i t y s i t e . I t would f u r t h e r appear t h a t a l t e r a t i o n s i n the c o n c e n t r a t i o n o f AAG can have marked e f f e c t s on t h e f r e e f r a c t i o n o f PF, and t h e r e f o r e , i t s 149 p h a r m a c o l o g i c a l e f f e c t . The a d d i t i o n o f 5-OH-PF does not appear t o a l t e r t h e b i n d i n g o f PF t o AAG s i g n i f i c a n t l y . The a d d i t i o n o f a p h e n o l i c 5 - h y d r o x y l group t o propafenone as a r e s u l t o f m e t a b o l i s m d e c r e a s e s t h e a f f i n i t y o f 5-OH-PF f o r AAG, i n a d d i t i o n t o the f o u r f o l d g r e a t e r c o n c e n t r a t i o n o f PF t o 5-OH-PF, r e s u l t s i n the d i s p l a c e m e n t o f 5-OH-PF by PF. As a r e s u l t o f t h i s c o m p e t i t i o n f o r AAG, t h e b i n d i n g o f 5-OH-PF i s d i s t r i b u t e d more t o o t h e r serum p r o t e i n s , such as HSA, and l i p o p r o t e i n s . Thus, HSA and l i p o p r o t e i n s , p r e s e n t i n serum ac c o u n t f o r a s i g n i f i c a n t degree o f the b i n d i n g o f 5-OH-PF a t t h e r a p e u t i c c o n c e n t r a t i o n s when PF i s p r e s e n t . The b i n d i n g o f 5-OH-PF t o HSA and l i p o p r o t e i n s appears t o be l a r g e l y n o n - s p e c i f i c . T h e r e f o r e , i t i s u n l i k e l y t h a t d i s p l a c e r s w i l l f u r t h e r i n c r e a s e the f r e e f r a c t i o n o f 5-OH-PF. Fu r t h e r m o r e , t h e b i n d i n g o f 5-OH-PF would appear t o remain r e l a t i v e l y c o n s t a n t w i t h f l u c t u a t i o n s i n the c o n c e n t r a t i o n o f AAG when compared t o PF. S i n c e t h e f r e e f r a c t i o n o f 5-OH-PF i s a p p r o x i m a t e l y t h r e e f o l d g r e a t e r than PF, i t appears t h a t 5-OH-PF may p l a y a s i g n i f i c a n t r o l e i n o v e r a l l p h a r m a c o l o g i c a l e f f e c t f o l l o w i n g a d m i n i s t r a t i o n o f PF s y s t e m i c a l l y . However, i t s h o u l d be c o n s i d e r e d t h a t i n a d d i t i o n t o 5-OH-PF, n-d e p r o p y l PF the o t h e r a c t i v e m e t a b o l i t e , may a l s o accumulate and c o n t r i b u t e t o the p h a r m a c o l o g i c a l e f f e c t o f PF [Kates e t a l . , 1985]. 150 5. CONCLUSION 1. A e q u i l i b r i u m d i a l y s i s method was e s t a b l i s h e d t o measure the d r u g - p r o t e i n b i n d i n g o f PF and 5-OH-PF i n human serum, and i n s o l u t i o n s o f i s o l a t e d human serum p r o t e i n s (AAG, HSA, HDL, LDL, and VLDL). T h i s method was found t o be both r e p r o d u c i b l e and c o r r e l a t e w e l l w i t h p r e v i o u s b i n d i n g s t u d i e s u s i n g PF. The d r u g - p r o t e i n b i n d i n g o f b o t h PF and 5-OH-PF was found t o be pH dependent. O t h e r p o s s i b l e s o u r c e s o f e r r o r were m i n i m i z e d , o r appeared t o be r e l a t i v e l y i n s i g n i f i c a n t . 2. The f r e e f r a c t i o n o f 5-OH-PF was found t o be 0.23 ± 0.02 and d i d not change s i g n i f i c a n t l y w i t h t h e a d d i t i o n o f the p a r e n t drug, PF. 3. At t h e r a p e u t i c c o n c e n t r a t i o n s (2.0 /xg/mL PF w i t h 0.5 /xg/mL 5-0H-PF ) , PF appears t o b i n d m a i n l y t o AAG. The b i n d i n g o f 5-OH-PF t o AAG was much l o w e r than t h a t o b s e r v e d f o r PF. The b i n d i n g o f PF and 5-OH-PF t o albumin was low. The removal o f l i p o p r o t e i n s from serum d e c r e a s e d t h e b i n d i n g o f 5-OH-PF, whereas no change was seen f o r PF. 4. D i s o p y r a m i d e and i b u p r o f e n d i s p l a c e d both PF and 5-OH-PF i n serum. The a d d i t i o n o f d i s o p y r a m i d e t o a s o l u t i o n o f i s o l a t e d AAG and HSA r e s u l t e d i n an i n c r e a s e i n the f r e e f r a c t i o n o f PF, whereas the 151 f r e e f r a c t i o n o f 5-OH-PF was u n a l t e r e d . I b u p r o f e n was found t o d e c r e a s e t h e f r e e f r a c t i o n o f 5-OH-PF and PF i n a s o l u t i o n o f HSA, but an i n c r e a s e i n the f r e e f r a c t i o n o f PF was o b s e r v e d i n a s o l u t i o n o f AAG. 5. PF and 5-OH-PF both d i s p l a y e d a h i g h a f f i n i t y , low c a p a c i t y s i t e on AAG; however, PF a l s o d i s p l a y e d a low a f f i n i t y , h i g h c a p a c i t y s i t e . The a f f i n i t y o f PF and 5-OH-PF t o the low a f f i n i t y , h i g h c a p a c i t y s i t e on HSA were s i m i l a r . The i n t e r a c t i o n o f PF and 5-OH-PF w i t h l i p o p r o t e i n s i s s p e c u l a t e d t o be due t o s o l u b i l i z a t i o n r a t h e r than " t r u e " p r o t e i n - b i n d i n g , e x c e p t i n t h e case o f PF b i n d i n g w i t h HDL, which appeared t o d i s p l a y s a t u r a b l e b i n d i n g . The s o l u b i l i z a t i o n o f PF and 5-OH-PF was found t o be g r e a t e s t i n the LDL, f o l l o w e d by HDL, and the n t h e VLDL complexes. 5. The mathematical c h a r a c t e r i z a t i o n PF and 5-OH-PF b i n d i n g i n s i m u l a t e d serum r e s u l t e d i n e s t i m a t e s c o n s i s t e n t l y h i g h e r than t h a t o f a c t u a l d a t a . When the b i n d i n g c o n t r i b u t i o n o f VLDL and LDL was e l i m i n a t e d , t h e b i n d i n g more c l o s e l y resembled t h a t o b s e r v e d i n c o n t r o l serum. 152 7. The uptake o f 5-OH-PF by r e d b l o o d c e l l s was g r e a t e r than t h a t o b s e r v e d f o r PF. The r a t i o o f drug c o n c e n t r a t i o n i n the r e d b l o o d c e l l / d r u g c o n c e n t r a t i o n i n plasma was almost 5 f o l d g r e a t e r f o r 5-OH-PF than PF. 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PROTEIN CONTENT (TOTAL PROTEIN AND a-l-ACID GLYCOPROTEIN) IN SOLUTIONS ISOLATED PROTEINS USED IN BINDING EXPERIMENTS. SAMPLE TOTAL PROTEIN ALPHA-1-ACID GLYCOPROTEIN FREE FATTY ACID 35.8 mg/mL ~ FREE ALBUMIN ALBUMIN IN 40.0 mg/mL ULTRAFILTRATE ALBUMIN IN 39.0 mg/mL BUFFER ALPHA-1-ACID GLYCO- 39.0 mg/mL 0.68 mg/mL PROTEIN AND ALBUMIN IN BUFFER ALPHA-1-ACID GLYCO- 39.1 mg/mL 0.71 mg/mL PROTEIN AND ALBUMIN IN ULTRAFILTRATE LIPOPROTEIN DEFICIENT 36.5 mg/mL 0.90 mg/mL SERUM SERUM 46.8 mg/mL 0.69 mg/mL ALPHA-1-ACID GLYCO- -- 0.71 mg/mL PROTEIN IN BUFFER ALPHA-1-ACID GLYCO-PROTEIN IN ULTRA-FILTRATE 0.67 mg/mL APPENDIX 3. COMPOSITION OF HUMAN SERUM ULTRAFILTRATE TEST PRE-FILTRATION POST-FILTRATION TOTAL PROTEIN 81.0 ± 4.9 ** (9/L) ALBUMIN 47.2 ± 1 . 1 ** (g/L) BILIRUBIN 3.2 ± 0.8 ** (umol/L) N a + 140 ± 1.0 130 ± 11 (mmol/L) K + 4.2 ± 0.1 4.0 ± 0.4 (mmol/L) C I " 104 ± 1 98 ± 6 (mmol/L) C 0 2 23 ± 1 18.6 ± 3.3 (mmol/L) UREA 4 .8 ± 1.1 4 .5 ± 1.2 (mmol/L) CREATININE 98 ± 4 81.9 ± 12.6 (/zmol/L) GLUCOSE 5.5 ± 1.0 5.0 ± 1.0 (mmol/L) CHOLESTEROL 5.97 ± 1 . 1 7 ** (mmol/L) TRIGLYCERIDES 2.15 ± 1.59 0.07 ± 0.05 (mmol/L) ** NOT DETECTABLE i Appendix 4. ELECTROPHORESIS RESULTS FOR ISOLATED PROTEINS USED IN BINDING EXPERIMENTS. LANE 1: STANDARD PROTEIN SOLUTIONS, LANE 2: HUMAN SERUM ULTRAFILTRATE, LANE 3: AAG (UNDILUTED), LANE 4: HSA, LANE 5: HSA + AAG, LANE 6: NORMAL CONTROL SERUM, LANE 7: LIPOPROTEIN DEFICIENT SERUM. (KDa k i l o d a l t o n s ) . 165 APPENDIX 5. PROTEIN CONCENTRATIONS IN SOLUTIONS OF ISOLATED HUMAN SERUM PROTEINS (AAG, HSA, HDL, LDL, AND VLDL) USED IN EXPERIMENTS IN WHICH BINDING WAS CHARACTERIZED. TOTAL PROTEIN MEASUREMENTS ALBUMIN SERUM HIGH DENSITY LIPOPROTEINS LOW DENSITY LIPOPROTEINS VERY LOW DENSITY LIPOPROTEINS 40.9 mg/mL 48.9 mg/mL 1.72 mg/mL 0.84 mg/mL 0.09 mg/mL ALPHA-1-ACID GLYCOPROTEIN SERUM ALPHA-1-ACID GLYCOPROTEIN 0.79 mg/mL 0.85 mg/mL CD CD If •' CD 01 co ro CO co J : APPFNPIX 6: A g a r o s e e l e c t r o p h o r e s i s o f l i p o p r o t e i n s b e f o r e (c) and a f t e r e q u l i b r i u m d i a l y s i s ( 1 - 6 ) . Ch 

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