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Propafenone pharmacokinetics : GLC-ECD analysis, metabolic induction by phenobarbital in non-smoking… Chan, Grace Lap-Yu 1989

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PROPAFENONE PHARMACOKINETICS: GLC-ECD ANALYSIS; METABOLIC INDUCTION BY PHENOBARBITAL IN NON-SMOKING AND SMOKING HEALTHY VOLUNTEERS; PROTEIN BINDING; PHARMACODYNAMICS IN PATIENTS by GRACE LAP-YU CHAN B.Sc. (Pharm), N a t i o n a l Taiwan U n i v e r s i t y , 1979 M . S c , U n i v e r s i t y o f Saskatchewan, 1984 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY 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 August, 1989 © GRACE LAP-YU CHAN, 1989 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 September 11, 1989  DE-6 (2/88) i i ABSTRACT Propafenone (PF) i s a new c l a s s I a n t i a r r h y t h m i c agent used t o t r e a t s u p r a v e n t r i c u l a r and v e n t r i c u l a r t a c h y a r r h y t h m i a s . T h i s t h e s i s r e p o r t s : a) an in vitro p r o t e i n b i n d i n g s t u d y o f PF i n normal and uremic s e r a ; b) a d r u g - d r u g i n t e r a c t i o n s t u d y o f PF and p h e n o b a r b i t a l i n h e a l t h y human s u b j e c t s and c) a 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 s t u d y o f PF i n p a t i e n t s . In o r d e r t o conduct t h e s e s t u d i e s , i t was n e c e s s a r y t o d e v e l o p a s e n s i t i v e and a c c u r a t e a s s a y method f o r t h e measurement o f PF i n b i o l o g i c a l f l u i d s . A c a p i l l a r y column e l e c t r o n - c a p t u r e d e t e c t i o n g a s - l i q u i d c h r o m a t o g r a p h i c (GLC-ECD) a s s a y was d e v e l o p e d f o r t h e q u a n t i t a t i o n o f PF. The i d e n t i t y o f t h e d e r i v a t i v e formed w i t h 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 was c o n f i r m e d by GLC-mass s p e c t r o m e t r y . The l i m i t o f d e t e r m i n a t i o n o f t h e a s s a y method was 2.5 ng/mL u s i n g 1 mL o f serum. The GLC-ECD method d e v e l o p e d f o r the q u a n t i t a t i o n o f PF was f u r t h e r m o d i f i e d t o measure the major and a c t i v e m e t a b o l i t e o f PF, 5-hydroxy PF. The serum p r o t e i n b i n d i n g o f PF was examined and c h a r a c t e r i z e d in vitro i n serum o b t a i n e d from h e a l t h y human s u b j e c t s u s i n g e q u i l i b r i u m d i a l y s i s . 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 , l o w - c a p a c i t y and one l o w - a f f i n i t y , h i g h - c a p a c i t y , were a p p a r e n t . The serum p r o t e i n b i n d i n g o f PF was found t o be c o n c e n t r a t i o n - d e p e n d e n t , w i t h PF f r e e f r a c t i o n i n c r e a s i n g from 0.03 t o 0.19 as PF c o n c e n t r a t i o n i n c r e a s e d from 0.25 t o 100 /zg/mL. However, no e v i d e n c e f o r s i g n i f i c a n t c o n c e n t r a t i o n - d e p e n d e n t changes i n b i n d i n g were o b s e r v e d w i t h i n the PF c o n c e n t r a t i o n range o f 0.25-1.5 ng/ml, which c o v e r e d a major p o r t i o n o f t h e t h e r a p e u t i c c o n c e n t r a t i o n range (0.5-2 /jg/mL). i i i In p o o l e d uremic serum, the PF f r e e f r a c t i o n was a p p r o x i m a t e l y 50% o f t h a t o f the PF f r e e f r a c t i o n i n normal serum t h r o u g h o u t t h e c o n c e n t r a t i o n range s t u d i e d (1-5 ng/ml). In serum from p a t i e n t s w i t h c h r o n i c r e n a l f a i l u r e , the i n c r e a s e i n PF b i n d i n g r a t i o was p o s i t i v e l y and s i g n i f i c a n t l y c o r r e l a t e d w i t h the i n c r e a s e i n serum a ^ - a c i d g l y c o p r o t e i n (AAG) c o n c e n t r a t i o n , s u g g e s t i n g t h a t 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 PF i n serum. The e f f e c t o f enzyme i n d u c t i o n on the p h a r m a c o k i n e t i c s o f PF and i t s major and a c t i v e m e t a b o l i t e , 5-hydroxy PF, was s t u d i e d i n e i g h t h e a l t h y non-smoking and e i g h t h e a l t h y heavy c i g a r e t t e smoking C a u c a s i a n males (age 20-45 y ) . Each s u b j e c t r e c e i v e d a s i n g l e o r a l dose o f PF (300 mg) on two o c c a s i o n s , s e p a r a t e d by 23 days o f p h e n o b a r b i t a l t r e a t m e n t (100 mg d a i l y at b e d t i m e ) . Except f o r two smokers who were 'slow' m e t a b o l i z e r s , a l l non-smoking and smoking s u b j e c t s were ' r a p i d ' m e t a b o l i z e r s ( i n t r i n s i c c l e a r a n c e , C L ^ n ^ >0.5 L/min) S i n c e t h e r e was g r e a t i n t e r s u b j e c t v a r i a b i l i t y i n most k i n e t i c parameters c a l c u l a t e d , each s u b j e c t s e r v e d as h i s own c o n t r o l . P h e n o b a r b i t a l induced h e p a t i c microsomal enzymes and enhanced the e x t e n t o f t h e f i r s t - p a s s metabolism o f PF. T h e r e was a s i g n i f i c a n t i n c r e a s e i n C L i n t a f t e r p h e n o b a r b i t a l t r e a t m e n t . The i n c r e a s e i n C L ^ n t ranged from 10-831% i n the non-smokers and 23-450% i n the smokers, r e s u l t i n g i n a s u b s t a n t i a l d e c r e a s e i n the s y s t e m i c a v a i l a b i l i t y , as measured by a r e d u c t i o n i n PF peak c o n c e n t r a t i o n ( C m a x ) and the a r e a under the serum c o n c e n t r a t i o n - t i m e c u r v e (AUC). The d e c r e a s e i n C m a x ranged from 0-87% i n the non-smokers and 8-85% i n the smokers w h i l e the d e c r e a s e i n AUC ranged from 10-89% i n the non-smokers and 19-82% i n t h e smokers. Except f o r two smoking s u b j e c t s , the p e r c e n t d e c r e a s e i n serum AUC was s i m i l a r t o t h e p e r c e n t d e c r e a s e i n s a l i v a r y AUC noted a f t e r enzyme i n d u c t i o n i n the i v non-smoking and the smoking subjects. Phenobarbital treatment did not lead to increases in the serum concentration, C m a x or the AUC of 5-hydroxy PF. Furthermore, there was no observed increase in the renal excretion of the conjugates of either 5-hydroxy PF or 5-hydroxy-4-methoxy PF, a subsequent metabolite of 5-hydroxy PF. Twenty-three days of phenobarbital treatment did not cause any change in PF free fraction or serum AAG concentration in the non-smoking and the smoking subjects. A wide range in the extent of metabolic induction of PF by phenobarbital, expressed as percent decrease in AUC, was observed in the non-smokers and the smokers, and in Vapid' and 'slow' metabolizers. Enzyme induction did not convert xslow' metabolizers of PF to 'rapid' metabolizers. Furthermore, the extent of metabolic induction of PF by phenobarbital was independent of the individual's polymorphic phenotype, serum phenobarbital concentration or the apparent initial ability of the individual's liver to metabolize drugs, i.e., CL^ n t c o n t r o l • When compared to the non-smokers, heavy cigarette smokers had a significantly larger CL i n t, a lower C m a x and a smaller AUC. While smoking did appear to increase the clearance of PF, it is difficult to conclude that smoking induced the metabolism of PF, due to the small sample size and the lack of comparison of smokers (serving as their own experimental control) under a nonsmoking circumstance. The concentration-response relationship of PF was studied in 10 patients (age 30-71 y) receiving PF (mean daily oral dose = 650 mg) for treatment of supraventricular arrhythmias. The QRS width measured from signal-averaged electrocardiograms (150 beats) was used as an indicator of V the a n t i a r r h y t h m i c response of PF. The c o r r e l a t i o n between QRS width and s e v e r a l parameters such as PF serum c o n c e n t r a t i o n , 5-hydroxy PF serum c o n c e n t r a t i o n and serum AAG c o n c e n t r a t i o n was examined. Each of these parameters seemed to c o n t r i b u t e to or i n f l u e n c e the o v e r a l l pharmacological e f f e c t o f PF. I t was p o s s i b l e to p r e d i c t QRS width from the v a l u e s o f these parameters us i n g an equation developed from m u l t i p l e stepwise r e g r e s s i o n . The equation was d e s c r i b e d as Y = 0.5Xj + 4.5X2 + ^47X3 + ^9 where Y was QRS w i d t h , Xj was l o g PF serum c o n c e n t r a t i o n , X2 was l o g 5-hydroxy PF serum c o n c e n t r a t i o n and X 3 was the r e c i p r o c a l o f serum AAG c o n c e n t r a t i o n . vi TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES x i i i LIST OF FIGURES xv LIST OF APPENDIX xix LIST OF ABBREVIATIONS xx ACKNOWLEDGEMENTS x x i i i 1. INTRODUCTION 1 1.1 General Background 1 1.2 Therapeutic and Clinical Effects of Propafenone 3 1.3 Comparison of Propafenone to Placebo and Other Antiarrhythmic Agents in the Treatment of Arrhythmias 3 1.4 Antiarrhythmic Drug Classification of Propafenone 4 1.4.1. Class I-Like Action of Propafenone 6 1.4.2. Class II-Like Action of Propafenone 6 1.4.3. Class IV-Like Action of Propafenone 7 1.5. Electrophysiological Effects of Propafenone and 5-Hydroxy Propafenone 7 1.6 Pharmacokinetics of Propafenone 9 1.7 Metabolism of Propafenone 12 1.8 Adverse Effects of Propafenone 15 1.8.1 Cardiac Adverse Effects 16 1.8.2 Neurologic Adverse Effects 17 1.8.3 Gastrointestinal Adverse Effects 17 1.9 Serum Concentration-Response Relationship of Propafenone and Clinical Monitoring of Drug Effect in Patients 17 1.10 Dose and Dosage Forms 20 1.11 Propafenone Drug Interactions 20 1.12 Propafenone-Food Interaction 22 1.13 Rationale 23 1.13.1 Rationale for Development of a GLC-ECD Method for Quantitative Analysis of Propafenone 23 v i i 1.13.2 R a t i o n a l e f o r a Study o f t h e E f f e c t o f Enzyme I n d u c t i o n by C i g a r e t t e Smoke and P h e n o b a r b i t a l 25 1.13.3 R a t i o n a l e f o r a Study o f t h e Serum P r o t e i n B i n d i n g o f Propafenone 29 1.13.4 R a t i o n a l e f o r a Study o f t h e P h a r m a c o l o g i c a l E f f e c t o f Propafenone 31 1.14 O b j e c t i v e s 32 2. EXPERIMENTAL 34 2.1 M a t e r i a l s and S u p p l i e s 34 2.1.1 Drugs, M e t a b o l i t e s and I n t e r n a l S t a n d a r d s 34 2.1.2 C h e m i c a l s and Reagents 34 2.1.3 S o l v e n t s 35 2.1.4 Gases 35 2.1.5 R a d i a l Immunodiffusion P l a t e s 35 2.1.6 U l t r a f i l t r a t i o n D e v i c e 35 2.1.7 E q u i l i b r i u m D i a l y s i s D e v i c e 36 2.1.8 Ot h e r S u p p l i e s 36 2.2 Columns 36 2.2.1 GLC Column 36 2.2.2 HPLC Column 37 2.3 Equipment 37 2.3.1 G a s - L i q u i d Chromatography 37 2.3.2 Gas L i q u i d Chromatography-Mass S p e c t r o m e t r y 37 2.3.3 High-Performance L i q u i d Chromatography 37 2.3.4 M i s c e l l a n e o u s 38 2.4 P r e p a r a t i o n o f St o c k and Reagent S o l u t i o n s 38 2.4.1 Drug, M e t a b o l i t e s and I n t e r n a l S t a n d a r d 38 2.4.2 Reagents and S o l u t i o n s 39 2.5 C a p i l l a r y E l e c t r o n - C a p t u r e D e t e c t i o n G a s - L i q u i d Chromato-g r a p h i c A n a l y s i s o f Propafenone 40 2.5.1 P r e l i m i n a r y Development o f a C a p i l l a r y GLC-ECD Assay Method f o r Propafenone 40 2.5.1.1 E x t r a c t i o n and T r i f l u o r o a c e t i c A n h y d r i d e D e r i v a t i z a t i o n Treatment f o r Propafenone 40 2.5.1.2 N e u t r a l i z a t i o n o f Ex c e s s T r i f l u o r o a c e t i c A n h y d r i d e 41 2.5.2 Optimal D e r i v a t i z a t i o n C o n d i t i o n s F o r Propafenone 41 2.5.2.1 Use o f T r i e t h y l a m i n e as C a t a l y s t and Optimal D e r i v a t i z a t i o n Time 41 2.5.2.2 Q u a n t i t y o f T r i e t h y l a m i n e Used 42 2.5.2.3 Q u a n t i t y o f 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 Used 42 2.5.3 S o l v e n t E x t r a c t i o n E f f i c i e n c y 42 2.5.4 Optimal GLC-ECD C o n d i t i o n s 43 2.5.5 Recovery o f Propafenone 43 2.5.5.1 E x t r a c t a b i l i t y o f Propafenone 43 2.5.5.2 V a r i a b i l i t y i n Recovery o f Propafenone 44 v i i i 2.5.6 Q u a n t i t a t i v e A n a l y s i s o f Propafenone 44 2.5.7 D e t e r m i n a t i o n o f Day-to-Day V a r i a b i l i t y 45 2.6 C a p i l l a r y E l e c t r o n - C a p t u r e D e t e c t i o n G a s - L i q u i d Chromato-g r a p h i c A n a l y s i s o f 5-Hydroxy Propafenone and 5-Hydroxy-4-Methoxy Propafenone , 45 2.6.1 E x t r a c t i o n , 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 Treatment and N e u t r a l i z a t i o n o f Excess 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 45 2.6.2 Optimal D e r i v a t i z a t i o n C o n d i t i o n s f o r 5-Hydroxy Propafenone 46 2.6.2.1 Q u a n t i t y o f 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 Used 46 2.6.2.2 D e r i v a t i z a t i o n Time 47 2.6.3 E x t r a c t i o n E f f i c i e n c y o f S o l v e n t s 47 2.6.4 Optimal GLC-ECD C o n d i t i o n s f o r 5-Hydroxy Propafenone 48 2.6.5 Recovery o f 5-Hydroxy Propafenone 48 2.7 S t r u c t u r a l C o n f i r m a t i o n o f the HFB D e r i v a t i v e s o f Propafenone, 5-Hydroxy Propafenone and l . S . - a by G a s - L i q u i d Chromatography-Mass S p e c t r o m e t r i c A n a l y s i s 49 2.8 Measurement o f Trough Plasma Propafenone C o n c e n t r a t i o n s by E l e c t r o n - C a p t u r e D e t e c t i o n G a s - L i q u i d Chromatography and H i g h - P e r f o r m a n c e L i q u i d Chromatography i n P a t i e n t s R e c e i v i n g Propafenone 49 2.9 In Vitro Serum P r o t e i n B i n d i n g Study 50 2.9.1 E q u i l i b r i u m Time f o r Propafenone 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 50 2.9.2 N o n - S p e c i f i c B i n d i n g o f Propafenone 50 2.9.3 E q u i l i b r i u m D i a l y s i s P rocedure 51 2.9.4 D e t e r m i n a t i o n o f Propafenone B i n d i n g Parameters -R o s e n t h a l A n a l y s i s 53 2.9.5 S c a t c h a r d P l o t o f the B i n d i n g Data 54 2.9.6 Propafenone Free F r a c t i o n i n P o o l e d Uremic Serum 54 2.9.7 E f f e c t o f Uremia and Renal F a i l u r e on the Serum B i n d i n g o f Propafenone 54 2.9.8 Measurement o f Serum P r o t e i n C o n c e n t r a t i o n s 55 2.9.8.1 Serum Albumin C o n c e n t r a t i o n 55 2.9.8.2 Serum a ^ - A c i d G l y c o p r o t e i n C o n c e n t r a t i o n 55 2.10 P h e n o b a r b i t a l Treatment i n Non-Smokers and Smokers: P h a r m a c o k i n e t i c s and B i n d i n g S t u d i e s o f Propafenone and 5-Hydroxy Propafenone 56 2.10.1 Study S u b j e c t s 56 2.10.2 Study P r o t o c o l 58 2.10.3 D o s i n g , B i o l o g i c a l F l u i d Sampling and P h y s i o l o g i c a l M o n i t o r i n g o f Study S u b j e c t s 58 2.10.4 Sample C o l l e c t i o n T e c h n i q u e s 60 2.10.5 A s s u r a n c e o f P h e n o b a r b i t a l Compliance 61 2.10.6 Measurement o f o ^ - A c i d G l y c o 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 P h e n o b a r b i t a l Treatment 61 2.10.7 A n a l y t i c a l P r o c e d u r e s 61 i x 2.10.7.1 Phenobarbital c o n c e n t r a t i o n 61 2.10.7.2 Serum and S a l i v a Samples 61 2.10.7.3 Urine Samples 63 2.10.7.4 Pro t e i n Binding 63 2.10.8 Data A n a l y s i s 64 2.10.8.1 Propafenone Serum Data 64 2.10.8.2 5-Hydroxy Propafenone Serum Data 66 2.10.8.3 Propafenone S a l i v a r y Data 67 2.10.8.4 Urine Data 67 2.10.9 S t a t i s t i c a l A n a l y s i s 68 2.11 E v a l u a t i o n of Propafenone Serum Concentration-Response R e l a t i o n s h i p 68 2.11.1 Study Subjects and Protocol 68 2.11.2 A n a l y t i c a l Procedures 70 2.11.2.1 Serum Samples 70 2.11.2.2 Pro t e i n Binding 70 2.11.3 Measurement of Pharmacological E f f e c t 70 2.11.4 Data A n a l y s i s 70 2.11.5 S t a t i s t i c a l A n a l y s i s 71 3. RESULTS 72 3.1 C a p i l l a r y Electron-Capture Detection Gas-Liquid Chromato-graphic A n a l y s i s of Propafenone 72 3.1.1 P r e l i m i n a r y Results of the C a p i l l a r y GLC-ECD Method of A n a l y s i s f o r Propafenone 72 3.1.1.1 E x t r a c t i o n and TFAA Treatment 72 3.1.1.2 N e u t r a l i z a t i o n of Excess D e r i v a t i z i n g Agent 72 3.1.2 Optimal D e r i v a t i z a t i o n Conditions 75 3.1.3 E x t r a c t i o n E f f i c i e n c y of Solvents 80 3.1.4 Optimal GLC-ECD Conditions 80 3.1.5 Recovery of Propafenone 84 3.1.5.1 E x t r a c t a b i l i t y of Propafenone 84 3.1.5.2 V a r i a b i l i t y i n Recovery of Propafenone 84 3.1.6 V a r i a b i l i t y Test 84 3.1.7 C a l i b r a t i o n Curves 84 3.1.8 Summary of the E x t r a c t i o n , D e r i v a t i z a t i o n Procedure and the R e s u l t i n g Chromatograms f o r Propafenone 88 3.2 C a p i l l a r y Electron-Capture Detection Gas-Liquid Chromato-graphic A n a l y s i s o f 5-Hydroxy Propafenone and 5-Hydroxy-4-Methoxy Propafenone 88 3.2.1 Optimal D e r i v a t i z a t i o n Conditions 88 3.2.2 E x t r a c t i o n E f f i c i e n c y o f Solvents 94 3.2.3 Optimal GLC-ECD Conditions 94 3.2.4 E x t r a c t a b i l i t y o f 5-Hydroxy Propafenone 94 3.2.5 C a l i b r a t i o n Curve 94 3.2.6 Summary of E x t r a c t i o n , D e r i v a t i z a t i o n Procedure and the Re s u l t i n g Chromatograms f o r 5-Hydroxy Propafenone 98 3.2.7 5-Hydroxy-4-Methoxy Propafenone 98 3.3 S t r u c t u r a l Confirmation of HFB D e r i v a t i v e s of Propafenone, X 5-Hydroxy Propafenone and I.S.-a by G a s - L i q u i d Chromatography-Mass S p e c t r o m e t r i c A n a l y s i s . 103 3.4 Measurement o f Plasma Propafenone C o n c e n t r a t i o n s by E l e c t r o n - C a p t u r e D e t e c t i o n G a s - L i q u i d Chromatography and High-Performance L i q u i d Chromatography 114 3.5 In Vitro Serum P r o t e i n B i n d i n g Study 114 3.5.1 E q u i l i b r i u m Time f o r Propafenone 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 114 3.5.2 N o n - S p e c i f i c B i n d i n g o f Propafenone 117 3.5.3 D e t e r m i n a t i o n o f Propafenone B i n d i n g Parameters -Rosenthal A n a l y s i s 117 3.5.4 S c a t c h a r d P l o t o f t h e B i n d i n g Data 117 3.5.5 Propafenone Free F r a c t i o n i n Normal and Po o l e d Uremic Serum 121 3.5.6 E f f e c t o f Uremia and Renal F a i l u r e on the Serum B i n d i n g o f Propafenone 121 3.5.7 C o r r e l a t i o n Between Serum a j - A c i d G l y c o p r o t e i n C o n c e n t r a t i o n and Propafenone B i n d i n g R a t i o 123 3.6. P h e n o b a r b i t a l Treatment i n H e a l t h y Non-Smokers: Pharmaco-k i n e t i c s and B i n d i n g S t u d i e s o f Propafenone and 5-Hydroxy Propafenone 123 3.6.1. Serum Data o f Propafenone 123 3.6.2. S a l i v a r y Data o f Propafenone 126 3.6.3. Serum Data o f 5-Hydroxy Propafenone 128 3.6.4. P r o t e i n B i n d i n g and Serum o ^ - A c i d G l y c o p r o t e i n C o n c e n t r a t i o n 131 3.6.5. R e l a t i o n s h i p Between Serum T o t a l , Serum Free and S a l i v a r y Propafenone C o n c e n t r a t i o n s 135 3.6.6. U r i n a r y Data 135 3.7. P h e n o b a r b i t a l Treatment i n H e a l t h y Smokers: Pharmaco-k i n e t i c s and B i n d i n g S t u d i e s o f Propafenone and 5-Hydroxy Propafenone 140 3.7.1. Serum Data o f Propafenone 140 3.7.2. S a l i v a r y Data o f Propafenone 144 3.7.3. Serum Data o f 5-Hydroxy Propafenone 146 3.7.4. P r o t e i n B i n d i n g and Serum o ^ - A c i d G l y c o p r o t e i n C o n c e n t r a t i o n 148 3.7.5. R e l a t i o n s h i p Between Serum T o t a l , Serum Free and S a l i v a r y Propafenone C o n c e n t r a t i o n s 152 3.7.6. U r i n a r y Data 152 3.8. P h e n o b a r b i t a l Treatment: Comparison o f E f f e c t on Pharmaco-k i n e t i c s and B i n d i n g S t u d i e s o f Propafenone and 5-Hydroxy Propafenone Between H e a l t h y Non-smokers and Smokers 155 3.9. E v a l u a t i o n o f Propafenone Serum 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 158 4. DISCUSSION 170 x i 4.1 C a p i l l a r y E l e c t r o n - C a p t u r e D e t e c t i o n G a s - L i q u i d Chromato-g r a p h i c A n a l y s i s o f Propafenone 170 4.1.1. S p l i t l e s s I n j e c t i o n and ' C o l d T r a p p i n g ' E f f e c t 170 4.1.2. C a p i l l a r y Column 171 4.1.3. E l e c t r o n - C a p t u r e D e t e c t i o n and A c y l a t i o n 171 4.1.4. E x t r a c t i o n and I n j e c t i o n S o l v e n t 173 4.1.5. Optimal GLC-ECD C o n d i t i o n s 174 4.2 E l e c t r o n - C a p t u r e D e t e c t i o n C a p i l l a r y G a s - L i q u i d Chromato-g r a p h i c A n a l y s i s o f 5-Hydroxy Propafenone and 5-hydroxy-4-methoxy Propafenone 175 4.3 S t r u c t u r a l C o n f i r m a t i o n o f the HFB D e r i v a t i v e s o f Propafenone and 5-Hydroxy Propafenone 176 4.4 In Vitro Serum P r o t e i n B i n d i n g Study o f Propafenone 178 4.4.1 P r o t e i n B i n d i n g T e c h n i q u e 178 4.4.2 B i n d i n g C h a r a c t e r i s t i c s and C o n c e n t r a t i o n - D e p e n d e n t Serum P r o t e i n B i n d i n g o f Propafenone 180 4.4.3 C l i n i c a l I m p l i c a t i o n o f the C o n c e n t r a t i o n - D e p e n d e n t Serum P r o t e i n B i n d i n g o f Propafenone 181 4.4.4 E f f e c t o f Uremia and Renal F a i l u r e on t h e Serum P r o t e i n B i n d i n g o f Propafenone 182 4.4.5 Free Drug M o n i t o r i n g o f Propafenone 184 * 4.5 P h e n o b a r b i t a l Treatment i n H e a l t h y Non-Smokers: Pharmaco-k i n e t i c s and B i n d i n g S t u d i e s o f Propafenone and 5-Hydroxy Propafenone 185 4.5.1 Data F i t t i n g 185 4.5.2 S t a t i s t i c a l A n a l y s i s 185 4.5.3 I n t e r i n d i v i d u a l V a r i a t i o n i n K i n e t i c Parameters o f Propafenone 185 4.5.4 I n d u c t i o n o f Propafenone M e t a b o l i s m by P h e n o b a r b i t a l 187 4.5.5 S a l i v a r y E x c r e t i o n o f Propafenone 191 4.5.6 P r o t e i n B i n d i n g o f Propafenone B e f o r e and A f t e r P h e n o b a r b i t a l Treatment 197 4.5.6.1 E f f e c t o f H e p a r i n on P r o t e i n B i n d i n g o f Propafenone 197 4.5.6.2 E f f e c t o f P h e n o b a r b i t a l Treatment on Propafenone Free F r a c t i o n 199 4.6 P h e n o b a r b i t a l Treatment i n H e a l t h y Smokers: Pharmaco-k i n e t i c s and B i n d i n g S t u d i e s o f Propafenone and 5-Hydroxy Propafenone 200 4.6.1 Data F i t t i n g 200 4.6.2 I n t e r i n d i v i d u a l V a r i a t i o n i n K i n e t i c Parameters o f Propafenone 200 4.6.3 I n d u c t i o n o f P h e n o b a r b i t a l M e t a b o l i s m by P h e n o b a r b i t a l 202 4.6.4 S a l i v a r y E x c r e t i o n o f Propafenone 202 4.7 E x t e n t o f M e t a b o l i c I n d u c t i o n o f Propafenone by P h e n o b a r b i t a l i n H e a l t h y Non-smokers and Smokers 203 x i i 4.8 E f f e c t o f C i g a r e t t e Smoking on the P h a r m a c o k i n e t i c s and Serum P r o t e i n B i n d i n g o f Propafenone 207 4.8.1 S t a t i s t i c a l A n a l y s i s 207 4.8.2 E f f e c t o f C i g a r e t t e Smoking on the M e t a b o l i s m o f Propafenone 208 4.8.3 E f f e c t o f C i g a r e t t e Smoking on the Serum P r o t e i n B i n d i n g o f Propafenone 208 4.9 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 o f Propafenone 210 4.9.1 S i g n a l Averaged E l e c t r o c a r d i o g r a m s 210 4.9.2 D e t e r m i n a n t s o f t h e P h a r m a c o l o g i c a l E f f e c t o f Propafenone 210 4.9.3 P o l y m o r p h i c O x i d a t i v e M e t a b o l i s m 212 5. SUMMARY AND CONCLUSION 214 5.1 C a p i l l a r y E l e c t r o n - C a p t u r e D e t e c t i o n G a s - L i q u i d Chromato-g r a p h i c A n a l y s i s o f Propafenone 214 5.2 In Vitro Serum P r o t e i n B i n d i n g Study 215 5.3 P h e n o b a r b i t a l Treatment i n H e a l t h y Non-smokers and Smokers: P h a r m a c o k i n e t i c s and B i n d i n g S t u d i e s o f Propafenone and 5-Hydroxy Propafenone 216 5.4 E v a l u a t i o n o f Propafenone Serum 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 219 6. REFERENCES 221 7. APPENDICIES 242 x i i i LIST OF TABLES Page IA. C l a s s i f i c a t i o n o f a n t i a r r h y t h m i c agents [Vaughan W i l l i a m s , 1970; 5 H a r r i s o n , 1983]. IB. S u b d i v i s i o n o f c l a s s 1 a n t i a r r h y t h m i c agents [Vaughan W i l l i a m s , 1984]. 5 2. A n a l y t i c a l methods f o r propafenone measurement. 24 3. C h a r a c t e r i s t i c s o f non-smoking and smoking s u b j e c t s * . 57 4. Serum p h e n o b a r b i t a l c o n c e n t r a t i o n s o f non-smoking and smoking s u b j e c t s d u r i n g p h e n o b a r b i t a l t r e a t m e n t . 62 5. C h a r a c t e r i s t i c s o f p a t i e n t s u b j e c t s . 69 6. E x t r a c t a b i l i t y o f p ropafenone. 85 7. C a l i b r a t i o n c u r v e d a t a o f propafenone f o r v o l u n t e e r s a l i v a and serum samples and p a t i e n t serum samples. 87 8. E x t r a c t a b i l i t y o f 5-hydroxy p r o p a f e n o n e . 96 9. C a l i b r a t i o n c u r v e d a t a o f 5-hydroxy p r o p a f e n o n e . 97 10. S t e a d y - s t a t e plasma propafenone t r o u g h c o n c e n t r a t i o n o f p a t i e n t s r e c e i v i n g p r o pafenone. 115 11. N o n - s p e c i f i c a d s o r p t i o n o f propafenone t o t h e e q u i l i b r i u m d i a l y s i s membrane, d i a l y s i s c e l l and u l t r a f i l t r a t i o n d e v i c e . 118 12. Propafenone f r e e f r a c t i o n i n normal and p o o l e d uremic serum. 122 13. The k i n e t i c d a t a o f propafenone i n serum b e f o r e and a f t e r p h e n o b a r b i t a l t r e a t m e n t from e i g h t h e a l t h y non-smoking s u b j e c t s . 125 14. The volume o f d i s t r i b u t i o n o f p r o p a f e none b e f o r e and a f t e r p h e n o b a r b i t a l t r e a t m e n t from e i g h t h e a l t h y non-smoking s u b j e c t s . 127 15. The k i n e t i c d a t a o f propafenone i n s a l i v a b e f o r e and a f t e r p h e n o b a r b i t a l t r e a t m e n t from e i g h t h e a l t h y non-smoking s u b j e c t s . 129 16. The k i n e t i c d a t a o f 5-hydroxy propafenone i n serum b e f o r e and a f t e r p h e n o b a r b i t a l t r e a t m e n t from e i g h t h e a l t h y non-smoking s u b j e c t s . 130 17. The s a l i v a / s e r u m c o n c e n t r a t i o n r a t i o o f p r o p a f e n o n e , pH o f s a l i v a and the p e r c e n t o f f r e e PF from e i g h t h e a l t h y non-smoking s u b j e c t s . 134 18. C a l i b r a t i o n c u r v e d a t a o f 5-hydroxy p r o p a f e n o n e and 5-hydroxy-4-x i v methoxy propafenone f o r urine samples 137 19. Renal e x c r e t i o n o f 5-hydroxy propafenone and 5-hydroxy-4-methoxy propafenone conjugates (cumulative) before and a f t e r phenobarbital treatment from eig h t healthy non-smoking s u b j e c t s . 139 20. The k i n e t i c data of propafenone i n serum before and a f t e r phenobarbital treatment from eig h t healthy smoking s u b j e c t s . 141 21. The volume of d i s t r i b u t i o n of propafenone before and a f t e r phenobarbital treatment from eig h t healthy smoking s u b j e c t s . 143 22. The k i n e t i c data of propafenone i n s a l i v a before and a f t e r phenobarbital treatment from e i g h t healthy smoking s u b j e c t s . 145 23. The k i n e t i c data of 5-hydroxy propafenone i n serum before and a f t e r phenobarbital treatment from s i x healthy smoking s u b j e c t s . 147 24. The saliva/serum concentration r a t i o o f propafenone, pH of s a l i v a and the percent of f r e e PF from e i g h t healthy smoking s u b j e c t s . 151 25. Renal e x c r e t i o n o f 5-hydroxy propafenone and 5-hydroxy-4-methoxy propafenone conjugates (cumulative) before and a f t e r phenobarbital treatment from eig h t healthy smoking s u b j e c t s . 154 26. Comparison o f pharmacokinetic parameters of propafenone i n serum between e i g h t non-smokers and eigh t smokers before and a f t e r phenobarbital treatment ( a l l data presented as mean + s.d.). 156 27. Comparison of pharmacokinetic parameters of 5-hydroxy propafenone in serum between eig h t non-smokers and s i x smokers before and a f t e r phenobarbital treatment ( a l l data presented as mean + s.d.). 157 28. The k i n e t i c data of propafenone and 5-hydroxy propafenone i n serum from ten p a t i e n t s . 161 XV LIST OF FIGURES Page IA. Chemical structures of propafenone and propranolol (* is the asymmetric carbon that gives rise to stereoisomerism). 2 IB. Chemical structures of propafenone and other /3-adrenoceptor blockers. 2 2. Metabolic pathways of propafenone in man. 13 3. Scheme of equilibrium dialysis procedure for propafenone. 52 4. Scheme of study protocol. 59 5. Chromatogram of the TFA derivatives of propafenone and l.S.-a. 73 6. Chromatogram of the HFB derivatives of propafenone and l.S.-a. 74 7. Effect of TEA on the HFB-PF response, as estimated by the peak area of HFB-PF. Samples were derivatized with HFBA and incubated at 65°C for various time with (o) or without (A) TEA. Number of samples = 3, duplicate injections. The data are presented as mean + ls.d. 76 8. Effect of derivatization time on the HFB-PF response, as estimated by the peak area of HFB-PF. Number of samples = 3, duplicate injections. The data are presented as mean + ls.d. 77 9. Effect of the volume of TEA (0.003M in toluene) on the HFB-PF response, as estimated by the peak area of HFB-PF. Number of samples = 3, duplicate injections. The data are presented as mean + ls.d. 78 10. Effect of the volume of HFBA on the HFB-PF response, as estimated by the peak area of HFB-PF. Number of samples = 3, duplicate injections. The data are presented as mean + ls.d. 79 11. The extractability of propafenone from serum using different solvents. The extraction efficiency (E) of each solvent was estimated as compared to methanol (unextracted, E = 1). Number of samples = 3, duplicate injections. 81 12. Effect of inlet purge valve activation time on the HFB-PF response, as estimated by the peak area of HFB-PF. The data are presented as mean + ls.d. 82 13. Effect of (A) injection port temperature, (B) detector temperature and (C) make-up gas flow rate on the HFB-PF response, as estimated by the peak area ratio (HFB-PF/HFB-I.S.-a). The data are presented as mean + ls.d. 83 14. Variability in recovery of propafenone. 86 xvi 15. Scheme of e x t r a c t i o n procedure f o r propafenone. 89 16. D e r i v a t i z a t i o n o f propafenone and I.S.-a to y i e l d the HFB d e r i v a t i v e s o f PF and I.S.-a, r e s p e c t i v e l y . 90 17. Chromatograms of e x t r a c t s from (A) blank serum; (B) blank serum spiked with propafenone (0.08 /ig) and I.S.-a (0.07 /xg) and (C) a serum sample (spiked with I.S.-a, 0.07 /xg) from a subject r e c e i v i n g propafenone. 91 18. E f f e c t o f the volume of HFBA on the HFB-5-hydroxy PF response, as estimated by the peak area of HFB-5-hydroxy PF. Number of samples = 3, d u p l i c a t e i n j e c t i o n s . The data are presented as mean + l s . d . 92 19. E f f e c t of d e r i v a t i z a t i o n time on the HFB-5-hydroxy PF ECD de t e c t o r response, as estimated by the peak area of HFB-5-hydroxy PF. Number of samples = 3, d u p l i c a t e i n j e c t i o n s . The data are presented as mean + l s . d . 93 20. The e x t r a c t a b i l i t y of 5-hydroxy propafenone from serum using d i f f e r e n t s o l v e n t s . The e x t r a c t i o n e f f i c i e n c y (E) of each solvent was estimated as compared to methanol (unextracted, E = 1). Number of samples = 3, d u p l i c a t e i n j e c t i o n s . 95 21. Scheme of e x t r a c t i o n procedure f o r 5-hydroxy propafenone. 99 22. D e r i v a t i z a t i o n o f 5-hydroxy propafenone and I.S.-b to y i e l d the HFB d e r i v a t i v e s of 5-hydroxy propafenone and I.S.-b, r e s p e c t i v e l y . 100 23. Chromatograms of e x t r a c t s from (A) blank serum spiked with 5-hydroxy propafenone (0.02 /ng) and I.S.-b (0.08 /ng) and (B) a serum sample (spiked with I.S.-b, 0.08 /ug) from a subject r e c e i v i n g propafenone. 101 24. Chromatogram of e x t r a c t s from blank serum spiked with 5-hydroxy-4-methoxy propafenone. 102 25. GLC-EI-MS of HFB-PF: (A) Total ion chromatogram ( r e t e n t i o n time, 13.70 min) and (B) EI mass spectrum. 104 26. GLC-EI-MS: a proposed fragmentation p a t t e r n of HFB-PF. 105 27. GLC-PICI-MS of HFB-PF: (A) Total ion chromatogram ( r e t e n t i o n time, 11.33 min) and (B) PICI mass spectrum. 106 28. GLC-PICI-MS: a proposed fragmentation pattern of the HFB-PF. 107 29. GLC-NICI-MS of HFB-PF: (A) Total ion chromatogram ( r e t e n t i o n time, 11.42 min); (B) a proposed fragmentation pattern and (C) NICI mass spectrum. 108 30. GLC-EI-MS of HFB-I.S.-a: (A) Total ion chromatogram ( r e t e n t i o n time, 13.16 min) and (B) EI mass spectrum and s e l e c t e d proposed X V I 1 f r a g m e n t a t i o n s t r u c t u r e s . 109 31. GLC-PICI-MS o f HFB-I.S.-a: (A) T o t a l i o n chromatogram ( r e t e n t i o n t i m e , 13.11 min) and (B) PICI mass spectrum and s e l e c t e d proposed f r a g m e n t a t i o n s t r u c t u r e s . 110 32. GLC-NICI-MS o f HFB-I.S.-a.: (A) T o t a l i o n chromatogram ( r e t e n t i o n t ime, 13.12 min); (B) a proposed f r a g m e n t a t i o n p a t t e r n and (C) NICI mass spectrum. I l l 33. GLC-EI-MS o f HFB-5-hydroxy PF: (A) T o t a l i o n chromatogram ( r e t e n t i o n t ime, 10.3 min) and (B) EI mass spectrum. 112 34. GLC-EI-MS: a proposed f r a g m e n t a t i o n p a t t e r n o f HFB-5-hydroxy PF. 113 35. E q u i l i b r i u m time f o r e q u i l i b r i u m d i a l y s i s o f p r o p a f e n o n e . I n i t i a l propafenone c o n c e n t r a t i o n was 0.25 ( o ) and 100 ( A ) ng/ml. 116 36. R e l a t i o n s h i p between the r a t i o o f bound c o n c e n t r a t i o n / f r e e c o n c e n t r a t i o n and bound 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 i n t h e serum o f s i x h e a l t h y male s u b j e c t s ( R o s e n t h a l p l o t ) . 119 37. R e l a t i o n s h i p between [r'/bound c o n c e n t r a t i o n ] and [ r ' ] o f propafenone i n the serum o f s i x h e a l t h y male s u b j e c t s ( S c a t c h a r d p l o t ) ; serum c o n c e n t r a t i o n s o f (A) albumin and (B) AAG were used i n t h e c a l c u l a t i o n o f [ r ' ] . 120 38. R e l a t i o n s h i p between propafenone b i n d i n g r a t i o (bound c o n c e n t r a t i o n / f r e e c o n c e n t r a t i o n ) and AAG c o n c e n t r a t i o n i n serum o b t a i n e d from s i x h e a l t h y s u b j e c t s ( O ), t h r e e mid-range uremic p a t i e n t s ( A ) and f i v e p a t i e n t s w i t h c h r o n i c r e n a l f a i l u r e ( • ). The d a t a a re p r e s e n t e d as mean + l s . d . 124 39. The f r e e f r a c t i o n o f propafenone [2 ( O ) and 4 ( A ) h samples] b e f o r e and a f t e r 23 days o f p h e n o b a r b i t a l t r e a t m e n t i n e i g h t h e a l t h y non-smokers. 132 40. The serum AAG c o n c e n t r a t i o n (0 h sample) b e f o r e and a f t e r 23 days o f p h e n o b a r b i t a l t r e a t m e n t i n e i g h t h e a l t h y non-smokers. 133 41. The c o r r e l a t i o n between (A) PF serum t o t a l c o n c e n t r a t i o n and s a l i v a r y c o n c e n t r a t i o n ( O ); (B) PF serum t o t a l c o n c e n t r a t i o n and serum f r e e c o n c e n t r a t i o n ( • ) and (C) PF s a l i v a r y c o n c e n t r a t i o n and serum f r e e c o n c e n t r a t i o n ( A ) i n e i g h t h e a l t h y non-smokers. 136 42. Chromatograms o f e x t r a c t from (A) b l a n k u r i n e s p i k e d w i t h 5-hydroxy propafenone (6 fig), 5-hydroxy-4-methoxy propafenone (6 fig), propafenone (6 fig) and I.S.-b (7 fig) and (B) a u r i n e sample ( s p i k e d w i t h I.S.-b, 7 fig) from a s u b j e c t r e c e i v i n g p r o p a f e n o n e . 138 43. The f r e e f r a c t i o n o f propafenone [2 ( O ) and 4 ( A ) h samples] x v i i i before and a f t e r 23 days of phenobarbital treatment i n e i g h t healthy smokers. 149 44. The serum AAG concentration (0 h sample) before and a f t e r 23 days o f phenobarbital treatment i n e i g h t healthy smokers. 150 45. The c o r r e l a t i o n between (A) PF serum t o t a l c o n c e n t r a t i o n and s a l i v a r y c o n c e n t r a t i o n ( O ); (B) PF serum t o t a l c o n c e n t r a t i o n and serum f r e e concentration ( • ) and (C) PF s a l i v a r y c o n c e n t r a t i o n and serum f r e e concentration ( A ) i n e i g h t healthy smokers. 153 46. The c o r r e l a t i o n between phenobarbital serum c o n c e n t r a t i o n (day 29) and [(AUCp-AUC c)/AUC c]xlOO% i n eigh t healthy non-smokers ( O ) and e i g h t healthy smokers ( A ). 159 47. The c o r r e l a t i o n between values o f CLj„+ c o n t r o l anc* [(AUC p-AUC c)/AUC c]xl00% i n eight healthy non-smokers ( O ) and ei g h t healthy smokers ( A ). 160 48. The c o r r e l a t i o n between QRS width and log PF serum c o n c e n t r a t i o n o f ten p a t i e n t s . 163 49. The c o r r e l a t i o n between QRS width and l o g 5-hydroxy PF serum c o n c e n t r a t i o n of ten p a t i e n t s . 164 50. The c o r r e l a t i o n between-(A) QRS width and log PF serum t o t a l c o n c e n t r a t i o n and (B) QRS width and l o g PF serum f r e e c o n c e n t r a t i o n of ten p a t i e n t s (two data p o i n t s from each p a t i e n t . 165 51. The c o r r e l a t i o n between serum propafenone f r e e f r a c t i o n and serum AAG concentration o f ten p a t i e n t s . The data are presented as mean + l s . d . 167 52. The c o r r e l a t i o n between measured QRS width and p r e d i c t e d QRS width. QRS width i s pred i c t e d from the f o l l o w i n g equation (obtained by m u l t i p l e stepwise r e g r e s s i o n ) : Y = 0.5Xi + 4.5X? + 347X3 + 7 9 where Y i s QRS width, Xi i s log PF serum c o n c e n t r a t i o n , Xp i s log 5-hydroxy PF serum c o n c e n t r a t i o n and X 3 i s 1/AAG. 168 53. The c o r r e l a t i o n between measured QRS width and p r e d i c t e d QRS width. QRS width i s pred i c t e d from the f o l l o w i n g equation (obtained by m u l t i p l e stepwise r e g r e s s i o n ) : Y = 0.004Xj' + O.O4X2' + 379X3 + 9^ where Y i s QRS width, X j ' i s PF serum c o n c e n t r a t i o n , X2' i s 5-hydroxy PF serum conc e n t r a t i o n . 169 LIST OF APPENDICIES C a l c u l a t i o n of percent of f ree (unbound) drug in serum f o r a bas ic drug. Semi - logar i thmic p l o t s of the propafenone serum concentra t ion versus time curves of e ight non-smoking subjects before ( O ) and a f t e r 23 days of phenobarbital treatment ( # ). Semi - logar i thmic p l o t s of the propafenone s a l i v a r y concentra t ion versus time curves of e ight non-smoking subjects before ( • ) and a f t e r 23 days of phenobarbital treatment ( • ) . A lso shown in the f igures i s the pH of s a l i v a before ( O ) and a f t e r ( • ) phenobarbital treatment. Semi - logar i thmic p lo ts of the 5-hydroxy propafenone serum concentrat ion versus time curves of e ight non-smoking subjects before ( A ) and a f t e r 23 days of phenobarbital treatment ( A ) . Semi- logar i thmic p l o t s of the propafenone serum concentra t ion versus time curves of e ight smoking subjects before ( o ) and a f t e r 23 days of phenobarbital treatment ( • ) . Semi- logar i thmic p lo ts of the propafenone s a l i v a r y concentra t ion versus time curves of e ight smoking subjects before ( • ) and a f t e r 23 days of phenobarbital treatment ( • ) . A lso shown in the f igures i s the pH of s a l i v a before ( O ) and a f t e r ( + ) phenobarbital treatment. Semi- logar i thmic p lo ts o f the 5-hydroxy propafenone serum concentra t ion versus time curves of s i x smoking subjects before ( A ) and a f t e r 23 days of phenobarbital treatment ( A ) . Semi- logar i thmic p l o t s of the propafenone ( O ) and 5-hydroxy propafenone ( A ) serum concentrat ion versus time curves of ten p a t i e n t s . XX LIST OF ABBREVIATIONS AAG a^ -ac id g lycopro te in AUC area under the concentrat ion versus time curve AUC„ area under the concentra t ion versus time curve from time 'O zero to time t oo AUC 0 area under the concentra t ion versus time curve from time zero to i n f i n i t y AUMC area under the f i r s t moment of the concentrat ion versus time curve AV a t r i o v e n t r i c u l a r ft apparent e l i m i n a t i o n rate constant d e s c r i b i n g the terminal por t ion of the serum drug concentrat ion versus time curve C m a x peak concentrat ion o f drug in serum Cp drug concentrat ion in serum at time t C p S S drug serum concentra t ion at s teady-s ta te CL c learance C L c r c r e a t i n i n e c learance CL. j n + c i n t r i n s i c c learance C L Q oral c learance C V . c o e f f i c i e n t of v a r i a t i o n D dose E ex t rac t ion e f f i c i e n c y ECD e lec t ron -capture de tec tor ECG e lectrocardiogram EI e lec t ron- impact F systemic a v a i l a b i l i t y GLC g a s - l i q u i d chromatography GLC-ECD g a s - l i q u i d chromatography-electron-capture de tec t ion GLC-MS g a s - l i q u i d chromatography-mass spectrometry GI g a s t r o i n t e s t i n a l HFB heptaf luorobutyry l HFBA hepta f luorobutyr ic anhydride HP Hewlett-Packard HPLC high-performance l i q u i d chromatography I.D. in terna l diameter l . S . in terna l standard i . v . intravenous IU in te rna t iona l uni t absorpt ion rate constant MRD maximum rate of d e p o l a r i z a t i o n M.W. molecular weight NDPP N-depropyl propafenone NICI negat ive - ion c h e m i c a l - i o n i z a t i o n PAH p o l y c y c l i c aromatic hydrocarbons PF propafenone PFPA penta f luoropropion ic anhydride PICI p o s i t i v e - i o n c h e m i c a l - i o n i z a t i o n PTFE polytetraf1uoroethylene PVCs premature v e n t r i c u l a r cont rac t ions r c o r r e l a t i o n c o e f f i c i e n t RID r a d i a l immunodiffusion rpm revo lu t ions per minute S s t a t i s t i c a l l y s i g n i f i c a n t xxi i SAECGs s igna l -averaged electrocardiograms s . d . standard dev ia t ion SGOT serum glutamic oxa loacet ic transaminase t time t. h a l f - l i f e i t m a x time to reach peak concentrat ion T dosing in te rva l TEA t r i e t h y l amine TFAA t r i f l u o r o a c e t i c anhydride TMA tr imethylamine UHP u l t r a high pur i t y UV u l t r a v i o l e t V c volume of d i s t r i b u t i o n of the centra l compartment V^ volume of d i s t r i b u t i o n ^darea volume of d i s t r i b u t i o n according to area or c learance method VJ25 volume of d i s t r i b u t i o n at s teady-s ta te WPW Wolf f -Parkinson-White xxi i i ACKNOWLEDGEMENTS I would l i k e to s i n c e r e l y thank Dr . James Axelson fo r h is s u p e r v i s i o n , guidance and f i n a n c i a l support . Specia l g ra t i tude fo r h is va luable adv ice , u n f a i l i n g support and unceasing encouragement throughout the study. I would a lso l i k e to thank my committee members Drs. Jim Orr , Char les Ker r , Kei th McErlane and John S i n c l a i r fo r t h e i r encouragement and support throughout the Ph.D. program. Thanks to Drs . C. Kerr and J . Yeung f o r t h e i r superv is ion of study s u b j e c t s . Thanks go to Dr. F. Abbott fo r h is help with the use of the GC-MS and h is capable ass is tance in the i n t e r p r e t a t i o n of GC-MS data . I would a lso l i k e to thank R. Burton for h is extensive help with the GC-MS. My apprec ia t ion to B. McErlane f o r her ass is tance in c o l l e c t i n g blood samples, N. Wong fo r her help in c o l l e c t i n g uremic blood samples and S. Tse f o r her help with the measurement of the binding p r o t e i n s . Thanks to my fe l low graduate students Sun Dong Yoo, J ing Wang, K. . Yeleswaram and Rajesh Mahey and my f r i ends Paul Molund, Roland Burton and Jacque l ine Wal isser fo r t h e i r i n s p i r a t i o n and f r i e n d s h i p . Specia l thanks to Wayne Riggs, Matthew Wright, George Tonn and Sue Panesar fo r t h e i r c o n s t r u c t i v e c r i t i c i s m in the wr i t ing of the t h e s i s and t h e i r constant support and encouragement. Th is pro ject was supported by the B r i t i s h Columbia Heart Foundation, the Kidney Foundation of Canada and Knol l Pharmaceuticals Canada Inc. F i n a l l y , I would l i k e to g r a t e f u l l y acknowledge the r e c e i p t of Tra ineeships from the U n i v e r s i t y of B r i t i s h Columbia, the B r i t i s h Columbia Heart Foundation and the Canadian Heart Foundation. xxi v This thes is is dedicated to my wonderful parents , whose u n s e l f i s h and boundless l o v e , continuous support and encouragement have c a r r i e d me through my Ph.D. program, to my dear s i s t e r and brother and to the memory of my grandfather and grandmother 1 1. INTRODUCTION 1.1 General Background Propafenone (PF) i s a potent , genera l ly w e l l - t o l e r a t e d and o r a l l y e f f e c t i v e new ant iar rhythmic agent with demonstrated e f f e c t i v e n e s s against a v a r i e t y of c a r d i a c arrhythmias [Siddoway et al., 1984a; Harron and Brogden, 1987; Schlepper , 1987]. Propafenone was synthesized in 1970 (Knoll Pharmaceut ica ls , Germany) and has been a v a i l a b l e in Europe s ince 1977 as Rytmonorm^ and in Canada s ince 1987 as Rhythmol^. Propafenone i s a bas ic compound (pK a = 9.0) and i s s l i g h t l y so lub le in water ( s o l u b i l i t y <1%). Propafenone hydrochlor ide appears as c o l o r l e s s c r y s t a l s or a white c r y s t a l l i n e powder with a b i t t e r t a s t e . Propafenone (2 - (2 ' -hydroxy-3 ' -propy lamino-propoxy) -w-pheny l -propiophenone) conta ins an asymmetric carbon and is used as a racemate conta in ing the R and S-enantiomers (Figure 1) . Propafenone i s a weak )8-adrenoceptor b locker and i s s t r u c t u r a l l y s i m i l a r to other ^ - b l o c k e r s such as propranolol (F igure IA) , a l p r e n o l o l , metopro lo l , oxprenolol and p r a c t o l o l (F igure IB) . The subs t i tu ten t at Rj i s usua l ly an isopropyl group but in PF i t i s n - p r o p y l . The s t r u c t u r e - a c t i v i t y r e l a t i o n s h i p of these ^ - b l o c k e r s has been examined. The s i ze and p o s i t i o n of the other s u b s t i t u t e n t s on the benzene r ing (i.e., R 2 and R 3 ) appear to be important in determining the c a r d i o s e l e c t i v e potency of ^-adrenoceptor b l o c k e r s . Compounds with a subs t i tu ten t at R3 (para -pos i t ion) have been found to be more c a r d i o s e l e c t i v e than those with a subst i tu tent at R 2 (ortho pos i t ion ) [Bagwell and Vaughan Wi l l i ams , 1973; Vaughan Wil l iams et al., 1973]. 2 0-CHo-CH-CHo-NH-CH/CH3  L OH NCH-5 propranolol ^ ^r0-CH2-CH-CH2-NH-CH2-CH2-CH3 C0-CH2-CH2-C6H5 propafenone Figure IA. Chemical s t ruc tures of propafenone and propranolol (* i s the asymmetric carbon that gives r i s e to stereoisomer ism) . 0-CH, -CH-CHo-NH-OH Compound R l R 2 R3 Alpreno lo l - C H ( C H 3 ) 2 -CH 2 - CH=CH 2 -H Metoprolol - C H ( C H 3 ) 2 -H -CH 2 -CH 2-0CH 3 Oxprenolol - C H ( C H 3 ) 2 -0CH2-CH=CH2 -H Propafenone - C H 2 ~ C H 2 - C H 3 - C O - C H 2 - C H 2 - C 6 H 5 -H Prac to lo l - C H ( C H 3 ) 2 -H -NH-C0-CH 3 Figure IB. Chemical s t ruc tures of propafenone and other ^-adrenoceptor b l o c k e r s . 3 1.2 Therapeut ic and C l i n i c a l E f f e c t s of Propafenone C l i n i c a l s tud ies have shown that PF i s e f f e c t i v e in t r e a t i n g and suppressing chron ic recurrent supravent r icu la r and v e n t r i c u l a r tachyarrhythmias and ec top ic beats [Waleffe et al., 1981; Ch i l son et al., 1982; Connol ly et al., 1983a; Hodges et al., 1984; Podrid et al., 1984; Rabkin et al., 1984; Salerno et al., 1984; Siddoway et al., 1984b; Dinh et al., 1988]. Propafenone has a lso been shown to be e f f e c t i v e in the short and long-term treatment of supravent r i cu la r tachycardias assoc ia ted with the Wol f f -Park inson-Whi te (WPW) syndrome [Rudolf et al., 1979; Bre i thard t et al., 1984] and in the prevent ion of recurrent a t r i a l f i b r i l l a t i o n [Kerr et al., 1988]. 1.3 Comparison of Propafenone to Placebo and Other Ant iarrhythmic Agents in the Treatment of Arrhythmias Propafenone was shown to be an e f f e c t i v e ant iarrhythmic agent with an acceptable frequency of adverse e f f e c t s when used in pat ients with severe v e n t r i c u l a r arrhythmias who were r e f r a c t o r y to other ant iarrhythmic agents or who had s i g n i f i c a n t in to le rance to these drugs [Podrid and Lown, 1984; Rabkin et al., 1984]. Propafenone was a lso demonstrated to be super ior to placebo in suppressing v e n t r i c u l a r ec top ic a c t i v i t y [Salerno et al., 1982, Hodges et al., 1984; Salerno et a / . , 1984; Soyza et al., 1984; Naccare l l a et al., 1985]. In several d o u b l e - b l i n d crossover s t u d i e s , PF appeared to have comparable or greater e f f i c a c y than other c l a s s I ant iarrhythmic agents. 4 For example, PF showed comparable e f f i c a c y to qu in id ine f o r the contro l of v e n t r i c u l a r arrhythmias in ambulatory pat ients with d ive rse forms of heart d iseases [Dinh et a 7 . , 1985]. In pat ients with frequent v e n t r i c u l a r arrhythmias who had not responded to treatment with procainamide (50 mg/kg/day) or qu in id ine (1200 mg/day), PF exh ib i ted greater e f f i c a c y over e i t h e r drug in the suppression of premature v e n t r i c u l a r cont rac t ions (PVCs). When combined with e i t h e r procainamide or q u i n i d i n e , lower doses of PF could be used e f f e c t i v e l y than with PF alone [K le in et al., 1987]. Propafenone a lso had greater e f f i c a c y over disopyramide in the treatment of frequent and complex PVCs [Naccare l la et al., 1982; Naccare l l a et al., 1985]. Besides c l a s s I agents, PF a lso showed s i m i l a r e f f i c a c y to amiodarone in the short - term treatment of c h r o n i c , i s o l a t e d or r e p e t i t i v e v e n t r i c u l a r e x t r a s y s t o l e s [Fauchier et al., 1986], 1.4 Ant iarrhythmic Drug C l a s s i f i c a t i o n of Propafenone The c l a s s i f i c a t i o n of ant iarrhythmic agents in to four c lasses according to t h e i r i n d i v i d u a l e l e c t r o p h y s i o l o g i c a l and pharmacological ac t ions was f i r s t proposed by Vaughan Wil l iams in 1970 and was l a t e r updated by Harr ison in 1983 (Table IA) . Class 1 agents had fur ther been subdiv ided in to three groups (Table IB), based on the k i n e t i c s ( r a p i d i t y ) of attachment of the drug t o , and i t s detachment from, the sodium channels [Vaughan Wi l l i ams , 1984] or on t h e i r e f f e c t on the durat ion of the act ion po ten t ia l [Harr ison et al., 1985]. Previous studies ind ica ted that PF exerted c l a s s e s I, II and IV ant iarrhythmic act ion and i t was c l a s s i f i e d as p r i m a r i l y a c l a s s IC agent, with l i t t l e or no e f f e c t on myocardial 5 Table IA. C l a s s i f i c a t i o n of ant iarrhythmic agents [Vaughan Wi l l i ams , 1970; H a r r i s o n , 1983], CI ass Mechanism of act ion Examples I sodium channel b lockers depress fas t inward see Table IB current II /3-adrenoceptor b lockers i n h i b i t sympathetic propranolol a c t i v i t y metoprolol III - prolong r e p o l a r i z a t i o n amiodarone brety l ium IV ca lc ium channel b lockers depress slow inward n i f e d i p i n e current verapamil Table IB. 1984]. S u b d i v i s i o n of c l a s s 1 ant iarrhythmic agents [Vaughan Wi l l i ams , CI ass Examples C h a r a c t e r i s t i c s of C lass IA q u i n i d i n e disopyramide procainamide c i b e n z o l i n e a) widens QRS and slows conduction at high concentrat ions b) prolongs QT in te rva l and lengthens durat ion of ac t ion potent ia l c) lengthens r e f r a c t o r y per iods IB 1 idocaine mex i le t ine t o c a i n i d e phenytoin a) l i m i t e f f e c t on QRS and conduction b) shortens r e p o l a r i z a t i o n and QT in terva l c) e levates f i b r i l l a t i o n threshold IC f l e c a i n i d e encain ide l o r c a i n i d e indeca in ide a) widens QRS and slows conduction at low concentrat ion b) s l i g h t e f f e c t on r e p o l a r i z a t i o n and durat ion of act ion potent ia l c) small changes in r e f r a c t o r i n e s s 6 r e p o l a r i z a t i o n [Dukes and Vaughan Wi l l i ams, 1984]. 1.4.1. C lass I -L ike Act ion of Propafenone Propafenone has a strong membrane s t a b i l i z i n g or l o c a l anaesthet ic e f f e c t [ F i l l et al., 1977; Z e i l e r et al., 1984]. It reduces automat ic i ty , c o n d u c t i v i t y and e x c i t a b i l i t y in a l l card iac t i s s u e s . Propafenone causes a dose-dependent decrease in the maximum rate of d e p o l a r i z a t i o n (MRD) and in the overshoot of the act ion potent ia l in a l l t i s s u e s normally depo lar i zed by f a s t sodium cur ren t . In i s o l a t e d rabb i t proximal and d i s t a l Purkin je f i b e r s , the MRD and overshoot of the act ion potent ia l have been demonstrated to reduce to a greater extent than in the v e n t r i c l e or a t r i a l t i s s u e s [Dukes and Vaughan Wi l l i ams, 1984]. Although PF i n h i b i t s the f a s t inward current (c lass I a c t i o n ) , i t a lso e x h i b i t s ac t ions of other c l a s s e s and cannot be considered as a s e l e c t i v e i n h i b i t o r o f the sodium channel . 1.4.2. C lass 11 - Li ke Act ion of Propafenone Propafenone conta ins in i t s molecular s t r u c t u r e , the aryloxy propanolamine s t ruc ture common to j3-blockers. Propafenone e x h i b i t s a compet i t ive antagonism of the chronotropic and i n o t r o p i c e f f e c t s of the /J-agonist isoproterenol {i.e., i n h i b i t i o n of isopro te reno l - induced tachycard ia and decrease in blood pressure) [Ledda et al., 1981; McLeod et al., 1984]. Furthermore, PF has a s e l e c t i v i t y of /^ -adrenoceptors o v e r /J j -adrenoceptors; the / ^ - b l o c k i n g e f f e c t i s three times stronger than the /?2-blocking e f f e c t on the heart [Dukes and Vaughan Wi l l i ams , 1984]. In i s o l a t e d card iac prepara t ions , the ^-adrenoceptor b lock ing and the membrane s t a b i l i z i n g e f f e c t s occur in an approximately equal concentra t ion range [Ledda et al., 1981]. In comparison to p r o p r a n o l o l , the j3-blocking e f f e c t 7 of PF appears to be 2-5% that of propranolol on the bas is of isoproterenol s e n s i t i v i t y t e s t i n g [ M u l l e r - P e l t z e r et al., 1983; McLeod et al., 1984]. 1 .4 .3 . C lass IV-L ike Act ion of Propafenone Propafenone has some depressive e f f e c t s on the calc ium channel (c lass IV a c t i o n ) . Th is i s supported by the c l i n i c a l evidence that PF depresses AV conduct ion and that i t i s e f f e c t i v e against WPW type arrhythmias [Dukes and Vaughan W i l l i a m s , 1984]. In pat ients with the WPW syndrome, PF slows (and, in some c a s e s , completely blocks) the anterograde and retrograde conduction and prolongs the r e f r a c t o r y per iod of the accessory pathway, i r r e s p e c t i v e of the i n i t i a l length [Waleffe et al., 1981; Bre i tha rd t et al., 1984]. The calc ium channel b locking e f f e c t of PF i s extremely weak, being -1% that of verapamil or n i f e d i p i n e [Ledda et al., 1981; Dukes and Vaughan W i l l i a m s , 1984]. 1.5. E l e c t r o p h y s i o l o g i c a l E f f e c t s of Propafenone and 5-Hydroxy Propafenone Propafenone decreases the automat ic i ty of the sinus node and s i g n i f i c a n t l y prolongs corrected sinus node recovery time [Bre i thard t et a / . , 1984]. In c l i n i c a l s tudies in p a t i e n t s , heart rate has been found to be e i t h e r increased [McLeod et al., 1984] or unaffected by therapeut ic doses of PF [Bre i thard t et al., 1984]. In conscious dogs with chron ic a t r i o v e n t r i c u l a r (AV) b lock , PF i s bel ieved to increase a t r i a l ra te by a d i r e c t and r e f l e x v a g o l y t i c act ion at plasma concentrat ions wi th in the therapeut ic range (0.5-2 /zg/mL). However, the bradycardia which occurs at lower PF concentra t ions (<0.33 /zg/mL) seems to be due to i t s membrane 8 s t a b i l i z i n g a c t i v i t y [Li et al., 1985]. When administered in a dose of 0.5-2 mg/kg, PF i n i t i a l l y increases v e n t r i c u l a r ra te through a r e f l e x response to i t s hypotensive e f f e c t . This increase in v e n t r i c u l a r rate i s reversed at high doses (4 mg/kg) and v e n t r i c u l a r bradycardia r e s u l t s due to i t s membrane s t a b i l i z i n g and/or i t s weak ^ - b l o c k i n g e f f e c t s . Propafenone prolongs the r e f r a c t o r y per iod of normal a t r i a , AV node and v e n t r i c u l a r t i s s u e [Connolly et al., 1983a] and e levates the v e n t r i c u l a r f i b r i l l a t i o n threshold [Bre i thardt et al., 1984]. Propafenone a lso suppresses AV nodal conduct ion, His bundle conduction and i n t r a v e n t r i c u l a r conduct ion . Furthermore, i t lengthens i n t r a c a r d i a c conduction t imes, inc lud ing the PA ( i n t r a - a t r i a l ) , AH (AV nodal) and HV (H is -Purk in je system) i n t e r v a l s [Connolly et al., 1983a; Bre i thard t et al., 1984; Fe ld et al., 1987]. These e f f e c t s are shown in e l e c t r o c a r d i o g r a p h i c measurement as a prolonged PR in te rva l (-16%), a widened QRS complex (-18%) and a prolonged QT in te rva l [Connolly et al., 1983a; Hodges et al., 1984; Salerno et al., 1984]. However, prolongat ion of the QT in te rva l i s minor [Connol ly et al., 1983b; Hodges et al., 1984]. The presence of ac t ive metabol i tes found in the plasma of pa t ien ts r e c e i v i n g chronic oral therapy with encainide or l o r c a i n i d e has been suggested to account fo r the e l e c t r o p h y s i o l o g i c a l d i f f e r e n c e s between o r a l l y and in t ravenously administered drugs [Jackman et al., 1982; Echt et al., 1983]. Unl ike l o r c a i n i d e and enca in ide , the e l e c t r o p h y s i o l o g i c e f f e c t s produced by oral dosing of PF has been found to be q u a l i t a t i v e l y s i m i l a r to those produced by intravenous ( i . v . ) dosing [Connolly et al., 1983a]. Th is suggests that i f there were an ac t ive metabol i te accumulating during oral therapy of PF, i t s e l e c t r o p h y s i o l o g i c a l e f f e c t s must be q u a l i t a t i v e l y s i m i l a r to those of PF [Connolly et ah., 1983a]. Studies of 9 the e l e c t r o p h y s i o l o g i c e f f e c t s of 5-hydroxy PF on guinea p ig v e n t r i c u l a r muscle f i b e r s [Valenzuela et al., 1987] and in canine Purk in je f i b e r s [Thompson et al., 1988] have ind icated that the e l e c t r o p h y s i o l o g i c a l e f f e c t s o f 5-hydroxy PF, a major metabol i te of PF, are s i m i l a r to those descr ibed with PF. 1.6 Pharmacokinetics of Propafenone Studies using deuter ium-labeled PF showed that >95% of the administered dose was absorbed from the g a s t r o i n t e s t i n a l (GI) t r a c t a f te r ora l admin is t ra t ion [Hollmann et al., 1983b]. Peak plasma concentra t ion was reached 2 to 3 h a f t e r the dose [ K e l l e r et al., 1978; Seipel and B r e i t h a r d t , 1980; Connol ly et al., 1983b; Hollmann et al., 1983a]. A f t e r admin is t ra t ion o f an intravenous dose of PF to normal v o l u n t e e r s , PF d i s t r i b u t e d r a p i d l y to body t i s s u e s and plasma concentrat ions then d e c l i n e d , with the mean apparent terminal h a l f - l i f e of PF in plasma being approximately 5 h [Connolly et al., 1984]. Propafenone was shown to bind ex tens ive ly yet v a r i a b l y to plasma p r o t e i n . The binding was found to be approximately 97% at PF plasma concentra t ions up to 1.6 /zg/mL, and decreasing s l i g h t l y at higher PF concentra t ions (94% at 7 iig/ml and 88% at 36 ng/ml) [Hollmann, 1983b]. The volume o f d i s t r i b u t i o n descr ib ing the centra l compartment (V c ) was approximately 0.7-1.1 L/kg in normal vo lunteers , a f t e r the admin is t ra t ion of an i . v . bolus of PF. The apparent h a l f - l i f e fo r d i s t r i b u t i o n of drug from the cent ra l compartment was approximately 4 minutes. The steady state volume of d i s t r i b u t i o n ( V ^ ) was approximately 1.9-3 L/kg in healthy 10 subjects [Seipel and B r e i t h a r d t , 1980; Hollmann et al., 1983a]. Th is large d i s t r i b u t i o n volume, which was fa r greater than any actual body space, seemed to i n d i c a t e a considerable binding of PF in per iphera l t i s s u e s . At autopsy, PF was found in the lung at a concentrat ion about t e n - f o l d higher than that in heart muscle or l i v e r and twenty- fo ld higher than that in s k e l e t a l muscle and kidney [Seipel and B r e i t h a r d t , 1980]. The metabo l i te , 5-hydroxy PF, was found in the heart at a concentrat ion comparable to that of PF [ L a t i n i et al., 1987]. While PF was shown to be completely absorbed [Hollmann et al., 1983b], i t s b i o a v a i l a b i l i t y was found to be 5 and 12% fo r the 150 and 300 mg f i l m - c o a t e d t a b l e t s , r e s p e c t i v e l y , due to the extensive presystemic e l i m i n a t i o n of PF a f t e r oral dos ing . Hollmann et al. [1983a] found a n o n - l i n e a r , d ispropor t iona te increase in the area under the concentra t ion versus time curve (AUC) of PF with increas ing dose. In t h e i r s tudy, the systemic b i o a v a i l a b i l i t y of PF increased from 4.8 to 12.1% when the dose of PF was increased from a 150 to a 300 mg t a b l e t . The b i o a v a i l a b i l i t y of PF was f u r t h e r increased to 23.5% when the 300 mg dosage was administered as an ora l s o l u t i o n . Connol ly et al. [1983b] studied the r e l a t i o n s h i p between dose and s teady -s ta te mean plasma concentrat ion of PF during a dose-ranging study in pa t ien ts with arrhythmia. They found that as the dose of PF increased t h r e e - f o l d from 300 to 900 mg, there was a t e n - f o l d increase in the s teady -s ta te mean plasma concent ra t ion . This n o n - l i n e a r r e l a t i o n s h i p between AUC and dose or s teady-s ta te plasma concent ra t ion , a lso seen with l o r c a i n i d e k i n e t i c s [Klotz et al., 1978], was suggested to be a r e s u l t of sa tu ra t ion of f i r s t - p a s s metabolism. The saturable f i r s t - p a s s metabolism could lead to a rap id increase in PF plasma concentrat ion with increas ing dosage beyond the ' s a t u r a t i o n ' point [Siddoway et al., 1984a]. 11 Studies of s i n g l e - d o s e e l im ina t ion k i n e t i c s ind ica ted that the apparent e l im ina t ion h a l f - l i f e of PF was r e l a t i v e l y shor t , being 4.6 h (range, 2 .3 -9 .5 h; 300 mg dose) in healthy volunteers [Hollmann et al., 1983a] and 3.6 h (range, 1.8-4.3 h; 900 mg dose) in pat ients with ca rd iac dysrhythmias [ K e l l e r et al., 1978]. Studies of s teady-s ta te e l i m i n a t i o n k i n e t i c s of PF in pa t ien ts requ i r ing ant iarrhythmic therapy showed longer e l i m i n a t i o n h a l f - l i v e s and greater i n t e r i n d i v i d u a l v a r i a b i l i t y . Connol ly et al. [1983b] reported a wide range of e l im ina t ion h a l f - l i f e of 2.4 to 11.8 h. S i m i l a r l y , Salerno et al. [1984] reported a range of 1.8 to 17.2 h while Siddoway et al. [1983] reported a range of 1.8 to 32.3 h. - Connol ly et al. [1983b] found that the large i n t e r i n d i v i d u a l v a r i a b i l i t y in the e l i m i n a t i o n h a l f - l i f e observed in h is pat ients could not be expla ined by c l i n i c a l f ac to rs such as age, l i v e r d i s e a s e , heart f a i l u r e or the presence of concomitant drug therapy that could i n t e r f e r e with the e l i m i n a t i o n o f a h igh ly metabolized drug. He suggested tha t , l i k e procainamide [Reidenberg et al., 1975] and encain ide [Roden et al., 1980], i nd iv idua l d i f f e r e n c e s in drug metabolism may cause the v a r i a t i o n seen in the e l im ina t ion h a l f - l i f e of PF. Another explanat ion fo r the longer e l i m i n a t i o n h a l f - l i f e of PF dur ing chronic oral dosing was a decrease in the apparent c learance of the drug [Connolly et al., 1984]. Siddoway et al. [1983] reported that PF undergoes metabolism via a polymorphic ox ida t ive pathway which cor re la ted with the debr isoquine ox ida t i ve phenotype. The populat ion was d iv ided into ' r a p i d ' and ' s low ' metabol i z e r s of debr isoquine on the bas is of the ur inary r a t i o of debr isoquine to i t s 4-hydroxy metabol i te a f te r a s i n g l e oral dose. The i n h i b i t i o n of debr isoquine 4-hydroxylat ion by PF was fur ther demonstrated both in vivo and in a human l i v e r microsomal system in vitro [Siddoway et 12 al., 1987]. Th is suggested that PF was metabolized via the same cytochrome P-450 systems which was respons ib le fo r the ox idat ion of debr isoquine [Siddoway et al., 1987]. In pharmacokinetic and pharmacodynamic s tudies of PF in pa t ien ts with arrhythmia [Siddoway et al., 1983; Siddoway et al., 1987], ' s low ' metabol izers of debr isoquine appeared to have a long PF apparent e l i m i n a t i o n h a l f - l i f e (16.8 + 0.6 h or 17.2 + 8.0 h ) , high plasma concentra t ions at s teady-s ta te and a f t e r s i n g l e dosing (2.16 + 0.49 ng/mL/mg dose or 2.5 + 0.5 ng/ml/mg d a i l y dosage) , low oral c learance (0.26 + 0.05 mL/min), proport ional dose-concentra t ion r e l a t i o n s h i p and an absence of detectable 5-hydroxy PF in plasma. On the other hand, ' r a p i d ' metabol izers were charac te r i zed by a r e l a t i v e l y short apparent e l im ina t ion h a l f - l i f e (4.7 + 1.3 h or 5.5 + 2.1 h ) , low plasma concentrat ions at s teady -s ta te and a f t e r s i n g l e dosing (0.81 + 0.51 ng/mL/mg dose or 1.1 + 0.6 ng/mL/mg d a i l y dosage), high oral c learance (1.12 + 1.24 L /min) , d i s p r o p o r t i o n a l dose-concentra t ion r e l a t i o n s h i p and detectable quan t i t i es of 5-hydroxy PF in plasma. 1.7 Metabolism of Propafenone The metabol ic pathway of PF in man i s i l l u s t r a t e d in Figure 2. Fol lowing oral admin is t ra t ion of PF, the drug is subject to extensive f i r s t - p a s s metabolism, with l e s s than 1% of the administered dose excreted unchanged in the ur ine or in the feces [Seipel and B r e i t h a r d t , 1980; Hollmann et al., 1983b; Hege et al., 1984a]. Propafenone i s metabolized to 5-hydroxy PF and hydroxy-methoxy PF via aromatic hydroxy la t ion , a major metabol ic pathway of PF [Hollmann et al., 1983b]. The mechanism is 13 C O - C H 2 - C H 2 - C 6 H 5 OH 2-hydroxy-u-phenyl propiophenone < ^ ^ ) - C 0 - C H 2 - C H 2 - C 6 H 5 O - C H 2 - ( ; H - C H 2 OH OH d i o l d e r i v a t i v e of propafenone (/ V c O - C H 2 - C H 2 - C 6 H 5 0 - C H 2 - ^ H - C00H l a c t i c ac id d e r i v a t i v e of propafenone H O O C - C H 2 - C H 2 - C 6 H 5 3-phenylpropionic acid <^J^CO-CH 2-CH 2-C 6H 5 0-CH ? -CH-CH ? -NH ? OH N-depropyl propafenone f~^-CO-CH2-CH2-C6H5 0-CHo-CH-CH?-NH-CoH7 OH propafenone C0-CH2-CH2-C5H5 0-CHo-CH-CH?-NH-C,H7 OH ' 5-hydroxy propafenone HO C 0 - C H 2 - C H 2 - C 5 H 5 CHoO O-CH0-CH-CH0-NH-C0H7 J L OH C ' glucuronide g l u c u r o n i d e / s u l f a t e g l u c u r o n i d e / s u l f a t e hydroxy-methoxy propafenone F igure 2. Metabol ic pathways of propafenone in man [Hege et al., 1984]. 14 suggested to be s i m i l a r to that of propranolol where an intermediate arene oxide i s formed, fol lowed by opening of the oxiran r i n g to the hydroxylated product in the para p o s i t i o n to the alkoxy oxygen [Walle et a 7 . , 1982]. In a d d i t i o n to ox ida t ive metabolism, PF, 5-hydroxy PF and hydroxy-methoxy PF a lso conjugate with g lucuronic and su lphur ic a c i d s . These conjugates are the primary form in which PF and i t s ox ida t ive metabol i tes are recovered in b i l e , ur ine and feces [Hollmann et al., 1983b; Hege et a / . , 1984a]. The g lucuron ide and s u l f a t e conjugates of 5-hydroxy PF and hydroxy-methoxy PF and PF g lucuronide appear in a r a t i o of 3:2:1 in plasma and ur ine and in a r a t i o of 2:1:1 in b i l e [Hege et al., 1984a]. There i s ind iv idua l v a r i a t i o n in the propor t ion of the excret ion products in the ur ine [Hege et al., 1984a]. The primary route of excret ion of the metabol i tes i s via the f e c e s . Most of the hydroxylated metabol i tes in the feces are found to be con jugates . Although the GI t rac t contains m i c r o f l o r a l ^ -g lucuron idase that i s capable of hydrolyz ing the conjugates and r e l e a s i n g the f ree drug which can then be reabsorbed [Levine, 1978], i t i s be l ieved that enzymatic c leavage of PF may be hindered in the feces and there i s no evidence of enterohepat ic c i r c u l a t i o n [Hege et al., 1984a]. Other monohydroxylated metabol i tes of PF have not yet been i d e n t i f i e d [Hege et al., 1984a]. Di -hydroxylated products (via corresponding arene oxides and d ihydrod io l intermediates) have so fa r been d iscovered only r a r e l y as they are r a p i d l y converted to hydroxy-methoxy d e r i v a t i v e s by ca techol -o -methy l t ransferase [Hege et al., 1984a]. Other minor metabolic pathways of PF i d e n t i f i e d in man include N-dea lky la t ion fol lowed by o x i d a t i v e deamination to form a g lyco l and a l a c t i c ac id d e r i v a t i v e ( th is pathway i s common to many endogenous and exogenous amines and most / 3 -b lockers ) , C-C s p l i t t i n g to y i e l d a r e l a t i v e l y la rge amount of 15 3 -pheny lprop ion ic ac id and cleavage of the ether group to y i e l d 2-hydroxy-w-phenyl propiophenone. The metabol i te 3 -phenylpropionic ac id i s found mainly in the feces and, while i t may be a metabol ic product , i t may a lso be a breakdown product of the o r i g i n a l metabol i te ( u n i d e n t i f i e d , specu la t ion only) dur ing drug ana lys is [Hege et al., 1984a]. Another metabo l i te , N-depropyl PF (NDPP), formed by N - d e a l k y l a t i o n , has been q u a l i t a t i v e l y i d e n t i f i e d [La t in i et al., 1988]. The major metabol i te of PF, 5-hydroxy PF, has been found to be pharmaco log ica l ly a c t i v e in animal models [Ph i l ipsborn et al., 1984; Valenzuela et al., 1987; Thompson et al., 1988]. In i s o l a t e d t i s s u e s (gu inea-p ig a t r i a , ra t a o r t i c s t r i p s ) , 5-hydroxy PF has been demonstrated to have a grea ter negative i n o t r o p i c and calcium channel b lock ing e f f e c t but a weaker ^-adrenoceptor b locking and l o c a l anesthet ic e f f e c t than PF [ P h i l i p s b o r n et al., 1984]. In canine Purkinje f i b e r s , the e l e c t r o p h y s i o -l o g i c a l e f f e c t s of NDPP appears to be s i m i l a r to but l e s s a c t i v e than those o f PF and 5-hydroxy PF [Thompson et al., 1988]. In in vivo s tud ies (dog and r a t ) , 5-hydroxy PF has exhib i ted a more potent ant iarrhythmic e f f e c t and a lower /3-blocking e f f e c t than PF [Ph i l ipsborn et al., 1984]. Two metabo l i t es , 5-hydroxy PF and NDPP, have been found to accumulate in the plasma of pa t i en ts during chronic oral PF therapy f o r frequent v e n t r i c u l a r ectopy, although the mechanism i s unclear [Kates et al., 1985]. The pharmacological and ant iarrhythmic e f f e c t s of NDPP have yet to be examined. 1.8 Adverse E f f e c t s of Propafenone The major adverse e f f e c t s of PF include c a r d i a c , neuro log ic and 16 g a s t r o i n t e s t i n a l e f f e c t s . Other e f f e c t s such as leukopenia , t r a n s i e n t c h o l e s t a t i c h e p a t i t i s and cutaneous rash have been reported but are rare [Seipel and B r e i t h a r d t , 1980]. 1.8.1 Cardiac Adverse E f f e c t s Propafenone seems to prolong c e r t a i n ECG parameters without causing symptoms or s i g n i f i c a n t bradyarrhythmia [de Soyza et al., 1984]. A l l ant iar rhythmic agents have some capaci ty f o r worsening arrhythmias [Ve leb i t et al., 1982]. Arrhythmogenici ty of PF, such as worsening of v e n t r i c u l a r ectopy or aggravation of v e n t r i c u l a r arrhythmia, has been reported [Seipel and B r e i t h a r d t , 1980; Connol ly et al., 1983a; Nathan et al., 1984; Podrid and Lown, 1984]. Other unwanted card iac e f f e c t s observed inc lude depression of s inus node f u n c t i o n , development of conduction abnorma l i t i es , such as SA b lock , f i r s t degree and second degree AV block and bundle branch block and exacerbat ion of congest ive heart f a i l u r e [Hodges et al., 1984; Podrid and Lown, 1984; Salerno et a / . , 1984]. Propafenone has been demonstrated to exert a s i g n i f i c a n t negative i n o t r o p i c e f f e c t on the heart [Karagueuzian, 1984; Salerno et a / . - , 1984] and depress l e f t v e n t r i c u l a r funct ion in pat ients with base l ine myocardial dysfunct ion [Baker et al., 1984]. The magnitude of the negative i n o t r o p i c e f f e c t of PF appears to be only h a l f that of disopyramide in man [Wester and Mousel imis , 1982], In pat ients with myocardial impairment ( e jec t ion f r a c t i o n <50%), PF therapy have been shown to fur ther reduce l e f t v e n t r i c u l a r f u n c t i o n . However, in pat ients with normal e j e c t i o n f r a c t i o n s (50-70%), t h i s drug has no e f f e c t on l e f t v e n t r i c u l a r funct ion [Podrid and Lown, 1984; Podrid et a / . , 1984]. 17 1.8.2 Neurologic Adverse E f f e c t s Central nervous system adverse e f f e c t s of PF inc lude v isua l b l u r r i n g , headache, l ightheadedness, d i z z i n e s s , ve r t igo and paresthes ias [Rabkin et al., 1984; Siddoway et al., 1987]. 1.8.3 G a s t r o i n t e s t i n a l Adverse E f f e c t s G a s t r o i n t e s t i n a l adverse e f f e c t s of PF include m e t a l l i c t a s t e , dry mouth, e p i g a s t r i c d iscomfor t , nausea, vomiting and c o n s t i p a t i o n [Connol ly et al., 1983b; Rabkin et al., 1984]. 1.9 Serum Concentrat ion-Response Re la t ionship of Propafenone and C l i n i c a l Monitor ing of Drug E f f e c t in Pat ients The concentra t ion- response r e l a t i o n s h i p fo r arrhythmia suppression by PF has shown remarkable in te rsub jec t v a r i a b i l i t y and, un for tunate ly , has yet to be e s t a b l i s h e d . Connol ly et al. [1983b] have observed a wide range of ' t h e r a p e u t i c ' plasma concentra t ion of PF (64-1044 ng/mL) in t h e i r p a t i e n t s . Siddoway et al. [1983] have a lso noted a wide range of mean plasma concentrat ions of PF, ranging from 143 to 1992 ng/mL, at an apparent ly e f f e c t i v e PF dosage. S i m i l a r l y , Salerno et al. [1984] have reported that the minimum e f f e c t i v e trough plasma concentrat ions of PF ranged from 91 to 3271 ng/mL in t h e i r p a t i e n t s . Connol ly et al. [1983b] have found an approximately l o g - l i n e a r r e l a t i o n s h i p between s teady-s ta te plasma concentra t ion and ant iarrhythmic response at intermediate concentrat ions (100-1000 ng/mL) in ind iv idua l pat ients undergoing a shor t -term PF oral therapy (no ant iarrhythmic response was seen at concentrat ions 18 l e s s than 100 ng/mL). The therapeut ic concentrat ion of PF, def ined as the concentrat ion at which 90% suppression of PVCs occur red , has been reported to be wi th in 0.5 and 2 ng/ml [Seipel and B r e i t h a r d t , 1980]. However, the a b i l i t y to suppress PVCs does not n e c e s s a r i l y c o r r e l a t e with ant iarrhythmic e f f i c a c y in the treatment of more ser ious arrhythmias with t h i s drug [Connolly et al., 1983a]. The adverse e f f e c t s of PF have been suggested to be dose-dependent, although no c l e a r - c u t r e l a t i o n s h i p has been demonstrated between plasma concent ra t ion and adverse e f f e c t s [Siddoway et al., 1984]. There fore , adjustment of the dose can sometimes show p e r s i s t e n t e f f e c t i v e n e s s with low s ide e f f e c t s . Siddoway et al. [1984] have reported that pat ients with neuro log ic s ide e f f e c t s have plasma concentrat ions greater than 1100 ng/mL. Moreover, the incidence of cent ra l nervous system side e f f e c t s appears to be s i g n i f i c a n t l y higher in x s l o w ' metabol izers than in v f a s t ' metabol izers (67% versus 14%). The n o n - l i n e a r drug accumulat ion, polymorphic metabolism, i n t e r i n d i v i d u a l v a r i a b i l i t y in e l im ina t ion h a l f - l i f e , s teady-s ta te mean plasma concentrat ion and therapeut ic plasma concentrat ion of PF (parameters that a f f e c t the durat ion of ant iarrhythmic response during long term therapy) , ind ica te a need fo r ind iv idua l therapy and contro l [Connolly et al., 1983b]. Propafenone prolongs the PR in te rva l and QRS durat ion at therapeut ic doses and a s i g n i f i c a n t c o r r e l a t i o n has been observed between plasma PF concentrat ion and prolongat ion of the PR in te rva l [ K e l l e r et al., 1978; Meyer -Estor f et al., 1978; Meyer -Estor f et al., 1980; Siddoway et al., 1983]. The c o r r e l a t i o n between plasma PF concentrat ion and pro longat ion of QRS durat ion i s not c l e a r . Siddoway et al. [1987] have 19 observed a c o r r e l a t i o n between PF plasma concentra t ion and QRS durat ion when they excluded the 's low' metabol izers from t h e i r p a t i e n t s . Because of the e x c e l l e n t r e l a t i o n s h i p between PF induced pro longat ion of PR in terva l (or QRS durat ion) and the percent suppression of PVCs, the extent of the increase in the length of the PR in te rva l (or QRS durat ion) can be used as an index of c l i n i c a l e f f i c a c y [Hodges et al., 1984; Salerno et al., 1984]. To guarantee safe ty of treatment, the PR and/or QRS i n t e r v a l s should not be prolonged by more than 20% [Naccare l la et al., 1982]. Excessive pro longat ion of the QT in terva l i s associa ted with arrhythmia aggravation f o r c l a s s I drugs, such as qu in id ine and disopyramide. However, PF appears to have l i t t l e e f f e c t on the QT in te rva l [Hodges et al., 1984; Salerno et al., 1984] and in pat ients with arrhythmia exacerbat ion , excessive pro longat ion of QT in te rva l has not been observed [Connol ly et al., 1983b; Podr id et al., 1984; Buss et al., 1985]. Propafenone i s bel ieved to increase the v e n t r i c u l a r rate at low concentra t ions and decrease the v e n t r i c u l a r ra te at high concent ra t ions . To minimize the r i s k of v e n t r i c u l a r b radycard ia , s p e c i a l care and caut ion should be taken fo r c l i n i c a l use of PF when the drug i s administered to pa t ien ts with congest ive heart f a i l u r e [Podrid and Lown, 1984] or to pa t ien ts with evidence of ser ious conduction system d i s e a s e , p a r t i c u l a r l y i f the drug i s administered by i . v . i n j e c t i o n [Connol ly et al., 1983b; Podrid and Lown, 1984; Li et al., 1985]. Careful observat ion of the p a t i e n t , inc lud ing ECG monitoring f o r AV and i n t r a v e n t r i c u l a r conduction abnorma l i t i es , i s advisable [de Soyza et al., 1984]. 20 1.10 Dose and Dosage Forms Propafenone i s a v a i l a b l e in two dosage forms: f i l m - c o a t e d t a b l e t s conta in ing 150 mg or 300 mg of PF hydrochlor ide and intravenous i n j e c t i o n ampoules conta in ing 70 mg of PF hydrochlor ide and 1.076 g of dextrose in 20 mL of water. A s i n g l e oral dose of 200 or 300 mg or a s i n g l e i . v . dose of 1 mg/kg of PF has been demonstrated to be w e l l - t o l e r a t e d and e f f e c t i v e in the reduct ion (ora l ) or complete repression ( i . v . ) of e x t r a s y s t o l e s or r e s t o r a t i o n of normal sinus rhythm [Koch, 1977; de Soyza et al., 1984]. O c c a s i o n a l l y therapeut ic e f f e c t s are obtained with a s i n g l e i . v . dose of 0.5 mg/Kg. In arrhythmias, the e f f e c t i v e and t o l e r a t e d i . v . dose range i s from 70 to 140 mg [Beck et al., 1975] or 1.5 to 2 mg/Kg depending on the s e v e r i t y of the arrhythmia. The i n j e c t i o n should be given s lowly . If a second i n j e c t i o n is g iven , i t should be 90 to 120 min a f t e r the f i r s t i n j e c t i o n [Bachour and Hochrein, 1977]. In severe c a s e s , PF has been given as an i n f u s i o n over a per iod of up to 3 h; o c c a s i o n a l l y over several days [Bachour and Hochrein , 1977]. Six to e ight ampoules per day are genera l ly s u f f i c i e n t fo r long term i n f u s i o n . Intravenous admin is t ra t ion has often been fol lowed immediately by the oral formula t ion . The most commonly e f f e c t i v e dose of PF for PVCs i s 300 mg every 8 h or 900 mg per day, although some pat ients respond to smal ler doses [Hodges et al., 1984]. 1.11 Propafenone Drug Interact ions Propafenone reduces the clearance and increases the mean s teady-s ta te 21 plasma concent ra t ion of war fa r in . The s i g n i f i c a n t increase in prothrombin time i n d i c a t e s an enhanced ant icoagulant e f f e c t and a requirement f o r dose adjustment [Kates et al., 1987]. L ike qu in id ine and verapamil [Leahey et al., 1980; K l e i n et al., 1982], PF a lso s i g n i f i c a n t l y increases plasma d igox in concent ra t ion [Belz et al., 1983; Hodges et al., 1984; Salerno et al., 1984] but to a l e s s e r degree than both drugs [Belz et al., 1983]. The mechanism of the increase in plasma d igox in concentrat ion by PF i s unknown. Since e levated plasma d igox in concentra t ion may increase the r i s k of g l y c o s i d e t o x i c i t y , care fu l monitor ing of pa t ien ts i s recommended and adjustment of d igox in dosage may be necessary [Belz et al., 1983; Salerno et al., 1984]. L idoca ine diminishes the prolongat ion in a t r i a l and v e n t r i c u l a r r e f r a c t o r i n e s s produced by PF a lone. L idocaine a lso produces mild add i t i ve negat ive i n o t r o p i c e f f e c t s that may.be of hemodynamic s i g n i f i c a n c e , e s p e c i a l l y in pa t ien ts with v e n t r i c u l a r dysfunct ion [Fe ld et al., 1987]. In a s i n g l e dose study in healthy s u b j e c t s , PF has been demonstrated to cause a two- fo ld decrease in the oral c learance o f metoprolol and increased the durat ion of /^-adrenoceptor b locking a c t i v i t y , as measured by reduct ion of exerc ise - induced tachycard ia . When administered to pat ients with c a r d i o v a s c u l a r d isease t reated with metopro lo l , PF increases the s teady -s ta te l e v e l s of metoprolol two to f i v e f o l d . The mechanism i s speculated to be competi t ive i n h i b i t i o n of metoprolol metabolism by PF. It may be necessary to reduce the dose of metoprolol when i t i s co-admin is tered with PF [Wagner et al., 1987]. On the other hand, the k i n e t i c s of PF during chronic therapy seem to be unaf fected by coadmin is t ra t ion of metopro lo l . 22 1.12 Propafenone-Food Interact ion Food intake has been shown to markedly in f luence the presystemic metabolism of c e r t a i n drugs such as propranolol [Melander et al., 1977], metoprolol [Melander et al., 1977], l a b e t a l o l [Daneshmend and Roberts , 1982] and d ixy raz ine [Liedholm et al., 1985], r e s u l t i n g in greater b i o a v a i l a b i l i t y . Food intake had been found to have s i m i l a r e f f e c t on the b i o a v a i l a b i l i t y of PF [Axelson et al., 1987]. The pharmacokinetics of PF were examined and compared in 24 healthy volunteers ( four of the twenty-four subjects were i d e n t i f i e d as ' s low ' metabol izers) in a fas ted s ta te and with a standard b reak fas t . With food, the maximum plasma PF concentra t ion was reached e a r l i e r and was s i g n i f i c a n t l y increased . When data from 's low ' metabol izers were excluded, there was an average increase of 147% in the AUC fo l low ing the standard breakfast . Food intake d i d not a f f e c t the b i o a v a i l a b i l i t y of PF in ' s low ' metabol izers or in subjects with a low i n t r i n s i c c learance (CL^ n^-). There was a s i g n i f i c a n t c o r r e l a t i o n (r = 0.946, p<0.05) between [ ( A U C f e d - A U C f a s t e d ) / A U C f a s t e - d ] and ^ i n t fas ted* ^he authors concluded that pat ients should be advised to take PF in a constant r e l a t i o n s h i p to food to assure cons is ten t b i o a v a i l a b i l i t y [Axelson et al., 1987]. The probable mechanism(s) of the food e f f e c t inc lude food induced t rans ien t increase in hepat ic blood flow r a t e , changes in the hepat ic drug d e l i v e r y r a t e , a l t e r a t i o n s in plasma pro te in b i n d i n g , d i r e c t i n h i b i t i o n of drug metabol is ing enzymes by food components or t h e i r metabol i tes , shunting of blood past the l i v e r and/or e l i m i n a t i o n of the ' th resho ld dose' e f f e c t [Melander et al., 1988]. 23 1.13 Rat iona le 1.13.1 Rat iona le f o r Development of a GLC-ECD Method f o r Quant i ta t i ve A n a l y s i s of Propafenone The a n a l y t i c a l methods a v a i l a b l e fo r the quant i t a t ion of PF and t h e i r l i m i t of determinat ion are l i s t e d in Table 2. P r i o r to the i n i t i a t i o n of the present i n v e s t i g a t i o n , several high performance l i q u i d chromatographic (HPLC) methods [Brode, 1982; Brode et al., 1982; Harapat and Kates, 1982; Kannan et al., 1983; Brode et al., 1984] and a g a s - l i q u i d chromatographic (GLC) method using a packed column and e lec t ron -cap ture de tec t ion (ECD) [Marchesini et al., 1982] were publ ished fo r the a n a l y s i s of PF. The HPLC techniques [Brode, 1982; Brode et a7., 1982; Harapat and Kates , 1982; Kannan et a7., 1983] required a r e l a t i v e l y large sample volume (-1-5 mL) and the l i m i t of determination was -5-20 ng/mL. Unfor tunate ly , these methods lacked the necessary s e n s i t i v i t y to measure t race l e v e l s of drug using small sample volumes such as are encountered dur ing s i n g l e dose k i n e t i c s tud ies and, as w e l l , in prote in binding s t u d i e s . In 1984, Brode et a7. publ ished an HPLC method using f luorescence de tec t ion which d isp layed a l i m i t of determination fo r PF as low as 1 ng/mL in 1 mL of human plasma. Fur ther , a h ighly s e n s i t i v e g a s - l i q u i d chromatography-mass spectrometr ic (GLC-MS) method fo r the quant i ta t ion of PF in plasma, with a l i m i t of determinat ion as low as 1 ng/mL in 0.5 mL of human plasma, was a lso publ ished [Higuchi et a7., 1985]. The GLC method [Marchesini et a7., 1982] using packed-column technology showed greater s e n s i t i v i t y than the ea r l y HPLC methods [Brode, 1982; Brode et a7., 1982; Harapat and Kates , 1982; Kannan et a7., 1983] and permitted the determinat ion of 10 ng/mL of PF. The obta inable s e n s i t i v i t y in modern gas chromatography has been 24 Table 2. A n a l y t i c a l methods f o r propafenone measurement. Author A n a l y t i c a l method Limit of determinat ion Brode, 1982 HPLC 2.5 ng/mL / 2 mL plasma Brode et al., 1982 HPLC 5 ng/mL /2 mL plasma Harapat and Kates, 1982 HPLC 5 ng/mL Marchesini et al., 1982 GLC 10 ng/mL Kannan et al., 1983 HPLC 50 ng/mL Brode et a 7 . , 1984 HPLC 1 ng/mL / l mL plasma L a t i n i et al., 1988 HPLC 30 ng/mL HPLC high-performance l i q u i d chromatography GLC g a s - l i q u i d chromatography 25 improved tremendously through the use of c a p i l l a r y columns and new i n l e t technology using s p l i t l e s s or on-column sample i n t r o d u c t i o n . The development of a s e n s i t i v e , s p e c i f i c and r e l i a b l e GLC method was essen t ia l f o r the ul t imate completion of the proposed s i n g l e dose pharmacokinetic study o f drug-drug i n t e r a c t i o n s with PF and a lso the in vitro and in vivo prote in binding study of t h i s drug. 1.13.2 Rat ionale f o r a Study of the E f f e c t of Enzyme Induction by C igare t te Smoke and Phenobarbital More than 200 drugs and chemicals are known to induce the microsomal drug-metabol iz ing enzymes in the l i v e r [Conney, 1967; Hunter and Chasseaud, 1976; Greim, 1981]. These inducers have been c l a s s i f i e d according to t h e i r e f f e c t s on var ious components of the enzyme system. The most common and simple c a t e g o r i z a t i o n i s c l a s s i f i c a t i o n of inducers into two groups. The f i r s t group, exempl i f ied by phenobarb i ta l , other barb i tura tes and r i f a m p i n , s t imulates cytochrome P-450, the terminal oxidase of the microsomal mixed funct ion ox idases , and induces a wide v a r i e t y of metabolic pathways i n c l u d i n g o x i d a t i o n , reduct ion and g lucuron idat ion [Valer ion et al., 1974; Ioannides and Parke, 1975; Greim, 1981]. The second group, t y p i f i e d by benzo(a)pyrene, 3-methylcholanthrene and p o l y c y c l i c aromatic hydrocarbons (PAH) found in c i g a r e t t e smoke, s t imulates cytochrome P-448 (or Pj-450) and induces a more l i m i t e d group of reac t ions [Beckett and T r i g g s , 1967; Conney, 1971; Jusko, 1978; Jusko, 1979; Vestal and Wood, 1980]. F i n a l l y , a t h i r d group, t y p i f i e d by pregnenolone 1 6 a - c a r b o n i t r i l e and other s t e r o i d s , has been noted to a lso induce cytochrome P-450 [Solymoss et al., 1971].. The enzymes involve in both phase I (b iotransformat ion) and phase II (conjugation) reac t ions e x i s t in mul t ip le forms {i.e., a fami ly of 26 isozymes) [Lu and West, 1980]. The mu l t ip le forms of cytochrome P-450 in the l i v e r microsomes of d i f f e r e n t s p e c i e s , such as r a t , mouse, rabb i t and human, have been i d e n t i f i e d and designated as P450I-P450XXII [Nebert et a / . , 1987]. Enzyme inducers can increase the enzyme a c t i v i t y of a large number of routes of metabolism, or be r e l a t i v e l y s e l e c t i v e f o r ind iv idua l isozymes [Breimer et al., 1977]. Phenobarbital i s one of the most potent barb i tu ra tes known to induce a large number of both microsomal and c y t o s o l i c enzymatic r e a c t i o n s . The metabol ic pathways induced by phenobarbital inc lude aromatic hydroxy!at ion (e.g. 3 ,4 -benz(a)pyrene) , a l i p h a t i c hydroxylat ion (e.g. t e s t o s t e r o n e ) , 0 - d e a l k y l a t i o n (e.g. acetophenet id ine) , N-dea lky la t ion (e.g. aminopyrine) , s u l f o x i d a t i o n (e.g. ch lorpromazine) , dehalogenation (e.g. ha lothane) , n i t r o group reduct ion (e.g. ch loramphenicol ) , azo l i n k reduct ion (e.g. n e o p r o n t o s i l ) , d e - e s t e r i f i c a t i o n (e.g. procaine) and g lucuron ida t ion (e.g. sa l icy lamide ) [Conney et al., 1967]. Branch and Herman [1984] reviewed the complex nature of events surrounding the e f f e c t s of metabolic induct ion of the r a p i d l y c leared /3-adrenergic receptor blockers by phenobarbital and r i f a m p i c i n . It had been well recognized that such changes in pharmacokinetic d i s p o s i t i o n might be very complex s ince inducing agents could in f luence l i v e r s i ze [Fouts and Rogers, 1965; Conney, 1967; A r g y r i s , 1968], l i v e r blood flow [Nies et al., 1976], b i l i a r y flow [Klaassen, 1969; K laassen , 1975] and prote in binding [Bai and Abramson, 1982], in addi t ion to t h e i r e f f e c t on drug metabol iz ing a c t i v i t y [Conney, 1967; Parke, 1975]. Furthermore, enzyme induct ion exh ib i ted both dose and time dependency r e l a t i o n s h i p s [Breckenridge et al., 1972; Mignet et al., 1977; Ohnhaus et al., 1977; Ohnhaus and Park, 1979; Ohnhaus et al., 1983]. With such high c learance drugs i t had been 27 recognized that the e f f e c t of enzyme induct ion was qu i te d i f f e r e n t when the drug was administered int ravenously as compared to the ora l route , due to subs tan t i a l d i f f e r e n c e s in the extent of f i r s t - p a s s metabolism a f t e r d i f f e r e n t routes of admin is t ra t ion [Wilkinson and Shand, 1975]. Pretreatment with phenobarbital was shown to have much l e s s e f f e c t on the k i n e t i c s o f i . v . administered ^ -adrenerg ic b lockers whi le a dramatic reduct ion in peak concentrat ions and AUC values f o r i n t a c t drug was observed a f t e r ora l a d m i n i s t r a t i o n , although there was l i t t l e observed e f f e c t on b i o l o g i c a l h a l f - l i f e [Wilkinson and Shand, 1975]. Drugs such as metoprolol [Bennett et al., 1982], a lpreno lo l [ C o l l s t e et al., 1979] and propranolo l [Herman et al., 1982; Herman et a 7 . , 1983] exh ib i ted increased oral c learance ranging from 50 to 500% a f t e r enzyme i n d u c t i o n . Since PF showed a c l o s e s t r u c t u r a l resemblance to p r o p r a n o l o l , shared modest 0 - b l o c k i n g a c t i v i t y [ M u l l e r - P e l t z e r et a 7 . , 1983; McLeod et a 7 . , 1984] with the p r e v i o u s l y mentioned ^ -b lockers and d isp layed extensive f i r s t - p a s s metabolism a f t e r oral dos ing , i t seemed l i k e l y that i t s oral c learance would be a l t e red by phenobarbital treatment. Tobacco smoke i s a mixture of over 3000 chemicals [ K i l b u r n , 1974; Severson et a 7 . , 1976; Schumaker et a 7 . , 1977]. Numerous compounds found in tobacco smoke, inc lud ing n i c o t i n e [Wenzel and Broadie , 1966; Yamamoto et al., 1966] and PAHs (e .g . benz(a)pyrene, anthracene, benz(a)anthracene, d ibenz(a ,h )anthracene, chrysene, 3 ,4 -benzof luorene , f luoranthene and pyrene, produced from incomplete combustion of tobacco) [Welch et a 7 . , 1969; Severson et a 7 . , 1976], are potent enzyme inducing agents which increase microsomal enzyme a c t i v i t y . Unl ike phenobarbital which a f f e c t s d i f f e r e n t aspects of l i v e r funct ion and blood f low, the e f f e c t of PAHs seems to be l i m i t e d to induct ion of se lec ted drug metabol iz ing enzymes 28 [Conney, 1967; K laassen , 1969; K laassen, 1975; Parke, 1975; Nies et a 7 . , 1976]. Numerous s tud ies [Vestal et a 7 . , 1975; K a p i t u l n i k et al., 1977; Kuntzman et al., 1977; Cusack et al., 1979; Vestal et al., 1979; Wood et a 7 . , 1979b; Grygie l et a 7 . , 1981] and review a r t i c l e s [Jusko, 1978; Jusko, 1979; Vestal and Wood, 1980; Dawson and V e s t a l , 1982; D 'Arcy , 1984] have reported or documented induced metabolic pathways of c e r t a i n drugs in 'heavy ' c i g a r e t t e smokers. However, other drugs which are metabol ized through the same pathways have been reported in two reviews to be unaf fected by smoking [Jusko, 1978; Jusko, 1979], For example, N -dea lky la t ion has been found to be both induced (e.g. t h e o p h y l l i n e , imipramine) and unaffected (e.g. diazepam, meperidine, n o r t r i p t y l i n e ) by c i g a r e t t e smoking. S i m i l a r l y , aromatic hydroxylat ion has a lso be found to be induced (e.g. benzo(a)pyrene, zoxazolamine) and unaffected (e.g. phenytoin , warfar in) by c i g a r e t t e smoke. This i s p o s s i b l y due to the presence of enzyme i n h i b i t o r s (e.g. carbon monoxide, hydrogen cyanide) in c i g a r e t t e smoke which may counteract the enzyme induct ion e f f e c t [Roth and Rubin, 1976]. The e f f e c t of c i g a r e t t e smoke may increase the metabolism of PF in a manner s i m i l a r to that observed fo r propranolol due to the s t r u c t u r a l and k i n e t i c s i m i l a r i t i e s between these two drugs. When the e f f e c t of smoking on propranolo l k i n e t i c s was examined in groups of young and e l d e r l y healthy subjects [Vestal et a 7 . , 1979], there was a t h r e e - f o l d l a r g e r ora l c learance in the young smokers than that seen in the e l d e r l y smokers and non-smokers. These r e s u l t s suggested the p o s s i b i l i t y of a d iminished i n d u c i b i l i t y of drug-metabol iz ing enzyme with aging. In heal thy vo lun teers , an oral dose of PF i s v i r t u a l l y completely metabolized to a number of metabol i tes [Hege et a 7 . , 1984a] by pathways 29 p r e v i o u s l y shown to be subject to the e f f e c t o f phenobarbital and c i g a r e t t e smoke with other drugs [Jusko, 1978]. No reports have yet appeared per ta in ing to the e f f e c t s of enzyme induct ion on the pharmacokinetics of PF and i t s major and ac t ive metabo l i te , 5-hydroxy PF. 1.13.3 Rat ionale f o r a Study of the Serum Prote in Binding of Propafenone A drug in plasma can bind to pro te in (s ) and i t i s widely accepted that only the f ree (unbound) drug can permeate c e l l membranes, reach receptor s i t e ( s ) and exert pharmacological e f f e c t ( s ) [ G o l d s t e i n , 1949], There fore , drug e f f e c t may be more c l o s e l y re la ted to f ree drug concentra t ion than to ta l (bound + unbound) drug concentrat ion in b lood . Th is i s supported by the observat ions that f ree propranolol [McDevitt and Shand, 1975; McDevitt et a 7 . , 1976] and disopyramide [Huang and O i e , 1981; Lima et a 7 . , 1981] concentrat ions in plasma c o r r e l a t e bet ter with the pharmacological e f f e c t than does t o t a l plasma drug concen t ra t ion . Propafenone i s h igh ly plasma pro te in bound and there has been a suggestion that t h i s b inding may be concentrat ion-dependent [Hollmann et a 7 . , 1983b]. Moreover, PF appears to e x h i b i t a steep dose-response e f f e c t [Connolly et a 7 . , 1983b; Siddoway et a 7 . , 1984a], although a good c o r r e l a t i o n between serum concentra t ion and ant iarrhythmic e f f e c t has yet to be e s t a b l i s h e d . If PF does e x h i b i t concentrat ion-dependent b i n d i n g , i t i s p o s s i b l e that the large i n t e r i n d i v i d u a l v a r i a b i l i t y in the PF concentrat ion necessary fo r arrhythmia suppression may be due, in par t , to f l u c t u a t i o n s in f ree PF serum concen t ra t ion . The major ac t ive metabol i te of PF, 5-hydroxy PF, may a lso cont r ibu te to the overa l l pharmacological e f f e c t [Connolly et al., 1983b]. S teady-s ta te PF concentrat ions may be a f fec ted by drug concentrat ion-dependent binding or the concentrat ion of plasma p r o t e i n s . 30 G i l l i s et al. [1985] have shown that PF, l i k e other bas ic drugs, binds to a j - a c i d g lycopro te in (AAG). A l p h a s - a c i d g lycopro te in i s an acute-phase reactant plasma prote in found in concentrat ions of 70-110 mg/dL in healthy i n d i v i d u a l s . It i s charac te r i zed by a non l inear , concentrat ion-dependent and saturable binding which leads to a rap id increase in f ree f r a c t i o n with i n c r e a s i n g concentrat ion of the drug in plasma beyond the ' s a t u r a t i o n p o i n t ' . Although i t s p h y s i o l o g i c a l funct ion i s unc lear , serum AAG concentra t ions are g r e a t l y e levated in pat ients with Crohn's d isease [P ia fsky et al., 1978], a r t h r i t i s [P ia fsky et al., 1978], uremia [Henriksen et al., 1982], traumatic in ju ry [Edwards et al., 1981] and fo l lowing surgery [Aronsen et al., 1972] or myocardial i n f a r c t i o n [Johansson et al., 1972; Snyder et a / . , 1975]. G i l l i s et al. [1985] a lso demonstrated that PF binds to AAG to a greater extent than l i d o c a i n e , verapamil and p r o p r a n o l o l , i n d i c a t i n g that AAG may be an important binding prote in f o r PF. In c e r t a i n d isease s ta tes when the concentrat ion of AAG i s increased [Keyser, 1979; P i a f s k y , 1980; Kremer et al., 1988] such as renal f a i l u r e , PF f ree f r a c t i o n or f ree plasma concentrat ion could be expected to decrease and a potent ia l a l t e r a t i o n in ant iarrhythmic e f f e c t could r e s u l t . A l t e r a t i o n in binding of PF due to a l te red AAG l e v e l s could a lso change the d i s t r i b u t i o n and pharmacokinetic proper t ies of t h i s drug. While an e a r l i e r report [Hollmann et a / . , 1983b] has suggested concent ra t ion dependence of PF b i n d i n g , the few s e l e c t i v e drug concentra t ions studied have not e s t a b l i s h whether or not PF undergoes n o n - l i n e a r binding over the apparent therapeut ic range of drug c o n c e n t r a t i o n . Furthermore, no previous report has d iscussed the prec ise d e t a i l s of the b ind ing , namely, the number of binding s i t e s , the a s s o c i a t i o n constants and the binding capac i ty of the prote in responsib le 31 f o r the b inding of t h i s drug. 1.13.4 Rat ionale f o r a Study of the Pharmacological E f f e c t of Propafenone Many ant iar rhythmic drugs inc lud ing qu in id ine [Data et al., 1976], l i d o c a i n e [Harr ison et al., 1971] and toca in ide [Meff in et al., 1977] are c h a r a c t e r i z e d by steep log dose-response r e l a t i o n s h i p s and evidence suggests that such i s a lso the case fo r disopyramide [Niarchos, 1976; Robert et al., 1978; A i t i o , 1981]. Ear ly s tudies have shown marked i n t e r s u b j e c t v a r i a b i l i t y and an inconc lus ive r e l a t i o n s h i p between PF concent ra t ion and e f f e c t [ K e l l e r et al., 1978]. Most r e c e n t l y , Connol ly et al. [1983b] have demonstrated that PF e x h i b i t s a s i m i l a r degree of i n t e r s u b j e c t v a r i a b i l i t y in concentrat ion-response r e l a t i o n s h i p s to that seen f o r t o c a i n i d e [Meff in et al., 1977]. As mentioned in Sect ion 1 .13 .3 . , theory suggests that f ree concent ra t ion of drug in plasma is more c l o s e l y c o r r e l a t e d with the pharmacological e f f e c t ( therapeut ic and/or tox ic ) than i s to ta l drug c o n c e n t r a t i o n . It i s a lso mentioned in Sect ion 1.13.3. that f l u c t u a t i o n s in f ree PF serum concentrat ion and/or the presence of the ac t ive metabol i te (5-hydroxy PF) in serum may cont r ibute to the wide i n t e r i n d i v i d u a l v a r i a b i l i t y in the PF concentrat ion necessary fo r arrhythmia suppress ion . Since PF shows cons iderab le v a r i a t i o n in i t s pharmacokinetic parameters and pharmacological response, i t i s essent ia l to e s t a b l i s h the concent ra t ion -response r e l a t i o n s h i p of PF in pat ients rece iv ing t h i s drug. 32 1.14 Object ives (a) to develop a s e n s i t i v e and reproducib le f u s e d - s i l i c a c a p i l l a r y GLC-ECD assay method fo r the quant i ta t ion of PF and i t s major and a c t i v e metabo l i te , 5-hydroxy PF, in small volumes of human b i o l o g i c a l f l u i d s ; (b) to i d e n t i f y the HFB d e r i v a t i v e of PF and 5-hydroxy PF by GLC-MS; (c) to v a l i d a t e the developed GLC-ECD method by comparison with a publ ished HPLC method; (d) to apply the developed GLC-ECD method to measure trough plasma PF concentra t ions in pat ients rece iv ing PF fo r treatment of arrhythmia; (e) to study the concentrat ion-dependency of the serum pro te in binding of PF in vitro using equ i l ib r ium d i a l y s i s ; (f) to determine the plasma prote in binding c h a r a c t e r i s t i c s of PF, such as the a s s o c i a t i o n constant and the binding capac i ty of the p r o t e i n ; (g) to study the binding of PF in uremic serum as an example of plasma binding in d i s e a s e ; (h) to inves t iga te the importance of AAG as a binding prote in fo r PF and to study the c o r r e l a t i o n between serum AAG concentra t ion and PF binding r a t i o ; ( i ) to study PF pharmacokinetics and i t s metabol ic induct ion by the prototype enzyme inducer , phenobarb i ta l , in healthy non-smokers and heavy c i g a r e t t e smokers; ( j ) to determine the e f f e c t of twenty-three days of phenobarbital treatment on serum AAG concentrat ion and PF f ree f r a c t i o n in healthy non-smokers and smokers; (k) to determine PF s a l i v a r y concentrat ion and i t s r e l a t i o n s h i p with 33 PF serum t o t a l and f ree concentrat ion and to evaluate the v a l i d i t y of s a l i v a r y PF measurement; and to evaluate the serum concentrat ion-response r e l a t i o n s h i p of PF -c o r r e l a t i o n between pharmacological e f f e c t and PF serum to ta l c o n c e n t r a t i o n , 5-hydroxy PF serum to ta l concentrat ion or PF serum f ree concentra t ion in pat ients r e c e i v i n g PF in the treatment of arrhythmia. 34 2. EXPERIMENTAL 2.1 Ma te r ia ls and Suppl ies 2.1.1 Drugs, Metabol i tes and Internal Standards Propafenone hydroch lo r ide , 5-hydroxy propafenone h y d r o c h l o r i d e , 5-hydroxy-4-methoxy propafenone hydroch lor ide , Li-1115 h y d r o c h l o r i d e , ( in te rna l s tandard , l . S . - a f o r propafenone quant i ta t ion) and Li -1548 hydroch lor ide ( I . S . - b f o r 5-hydroxy propafenone quant i ta t ion) were suppl ied by Knol l Pharmaceuticals Canada Inc . , Markham, Ontar io , Canada. Phenobarbital t a b l e t s (100 mg, Lot K5074HI) were obtained from the Family P r a c t i c e Unit Pharmacy, U . B . C . , Vancouver, B . C . , Canada. Sodium Ch lor ide In ject ion USP (20 mL, 9 mg/mL) was purchased from Abbott Labora tor ies L t d . , Montrea l , Canada and heparin sodium i n j e c t i o n USP (10 mL, 1000 units /mL) from A l l e n & Hanburys, A Glaxo Canada L t d . C o . , Toronto , O n t a r i o , Canada. 2.1.2 Chemicals and Reagents Tr ie thy lamine (TEA) ( S e q u a n a l ™ grade) , t r i f l u o r o a c e t i c anhydride (TFAA), pen ta f luoroprop ion ic anhydride (PFPA) and hep ta f luorobuty r ic anhydride (HFBA) were purchased from Pierce Chemical Co. (Rockford, IL, U . S . A . ) . ACS reagent grade sodium hydroxide was obtained from F i s h e r S c i e n t i f i c C o . , F a i r Lawn, NJ, U . S . A . ; ACS reagent grade monopotassiurn phosphate, disodium phosphate, sodium carbonate and potassium carbonate from BDH Chemicals , Toronto, Ontar io , Canada; ACS reagent grade h y d r o c h l o r i c ac id from American S c i e n t i f i c and Chemical , S e a t t l e , WA, U . S . A . ; ACS reagent grade Ammonia Strong Solut ion from Ma l l inckrodt Inc . , 35 S t . L o u i s , MI, U . S . A . and ACS reagent grade t r i c h l o r o a c e t i c ac id from J . T . Baker Chemical C o . , P h i l l i p s b u r g , NJ , U .S .A . /? -G lucuron idase /a ry lsu l fa tase (from He l i x pomatia) was purchased from Boehringer Mannheim, West Germany. 2 .1 .3 Solvents Benzene, to luene, methanol, dichloromethane and isopropyl a lcohol ( d i s t i l l e d in g lass ) and HPLC grade a c e t o n i t r i l e ( u l t r a v i o l e t (UV) c u t o f f 190 nm) were purchased from Caledon Laborator ies L t d . , Georgetown, Ontar io , Canada. Deionized d i s t i l l e d water was produced in the labora tory using a M i l l i - R 0 R Water System ( M i l l i p o r e C o r p . , Bedford, MA., U . S . A . ) . 2 .1 .4 Gases U l t r a high p u r i t y (UHP) hydrogen and argon/methane (95:5) were purchased from Matheson Gas Products Canada L t d . , Edmonton, A l b e r t a , Canada and n i t r o g e n , U . S . P . , from Union Carbide Canada L t d . , Toronto, Onta r io , Canada. 2 .1 .5 Radial Immunodiffusion Plates NOR-Part igen R AAG rad ia l immunodiffusion (RID) p la tes (12 -we l l , volume = 5 /xL per wel l ) conta in ing monospecif ic antiserum to human AAG in a r e a d y - f o r use agarose-gel l ayer were purchased from Terochem Laborator ies L t d . , Edmonton, A l b e r t a , Canada. 2 .1 .6 U l t r a f i l t r a t i o n Device Disposable u l t r a f i l t r a t i o n devices ( C e n t r i f r e e ™ M i c r o p a r t i t i o n System) were purchased from Amicon Canada L t d . , O a k v i l l e , Onta r io , Canada. Each u l t r a f i l t r a t i o n device cons is ted of a s ing le YMT membrane (molecular 36 weight (M.W.) c u t o f f = 30,000 Daltons) and an 0 - r i n g , sealed between the sample r e s e r v o i r and support base. The r e s e r v o i r was provided with a cap to minimize sample evaporation and pH change due to l o s s of carbon 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 attached to the support base. 2 .1 .7 E q u i l i b r i u m D i a l y s i s Device P l e x i - G l a s s ^ d i a l y s i s c e l l s (0.4 mL and 1.0 mL) were used fo r e q u i l i b r i u m d i a l y s i s . Cellophane d i a l y s i s membrane "sacks" (M.W. c u t o f f = 12,000 Daltons) were purchased from Sigma Chemical C o . , S t . L o u i s , U .S .A . 2 .1 .8 Other Suppl ies Pyrex^ d isposab le g lass cu l tu re tubes (15 mL) were purchased from Corning Glass Works, Corning, 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 Canlab, 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 cannulae were purchased from Abbott L a b o r a t o r i e s , L t d . , Montreal , Canada. Vacutainer^ blood c o l l e c t i o n tubes (without a d d i t i v e ) were obtained from Becton Dickinson Canada Inc . , M i s s i s s a u g a , O n t a r i o , Canada. 2.2 Columns 2.2.1 GLC Column 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.D., was used f o r a l l GLC and GLC-MS analyses (s ta t ionary 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 thickness 0.52 /xm; phase r a t i o 150; Hewlett-Packard, Palo A l t o , CA, U . S . A . ) . 37 2.2.2 HPLC Column An U l t r a s p h e r e ™ - c y a n o column, 15 cm x 4.6 mm I.D. (5 n dp ) , Beckman Instruments, I nc . , A l tex D i v . , San Ramon, CA, U . S . A . , was used f o r HPLC ana lyses . 2.3 Equipment 2.3.1 G a s - L i q u i d Chromatography GLC analyses were performed on a Model 5830A Hewlett -Packard (HP) g a s - l i q u i d chromatograph, equipped with a Model 18835B c a p i l l a r y i n l e t system, a ^ N i e lec t ron -cap ture de tec tor , a Model 18850A GC terminal fo r peak i n t e g r a t i o n and a Model 7671A automatic sampler. 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 with a small plug of s i l a n i z e d glass-wool 3 cm from the column end. Thermogreen™ LB-2 septa (Supelco, I n c . , B e l l e f o n t e , PA, U . S . A . ) , low-bleed septa at high i n l e t temperatures, were used. The septum was changed r o u t i n e l y to prevent leakage r e s u l t i n g from repeated puncturing during automatic sampling. 2 .3 .2 Gas L i q u i d Chromatography-Mass Spectrometry GLC-MS analyses were obtained using an HP Model 5987A g a s - l i q u i d chromatography-MS system. The data were processed by a Ser ies 1000E HP computer and d isp layed on an HP Model 2623A te rmina l . Ident ica l c a p i l l a r y columns were used fo r GLC-MS analyses, as in GLC ana lyses . 2 .3 .3 High-Performance L iqu id Chromatography A HP Model 1090 l i q u i d chromatograph equipped with a HP Model 1040A 38 d i o d e - a r r a y UV detector and a Model 310 HP computer f o r data ana lys is was used. 2 .3 .4 Miscel laneous Other equipment used inc luded: Eppendorf m ic rop ipe t tes , a Vortex-Genie'* mixer (F isher S c i e n t i f i c Indus t r i es , S p r i n g f i e l d , MA, U . S . A . ) , a pH meter and e lec t rode (F isher S c i e n t i f i c C o . , S p r i n g f i e l d , MA, U . S . A . ) , a Model 415-110 Labquake R ro tary shaker (Lab indus t r i es , Berk ley , CA, U . S . A . ) , an incubat ion oven (Isotemp R model 350, F isher S c i e n t i f i c Indus t r i es , S p r i n g f i e l d , MA, U . S . A . ) , an IEC model 2K cent r i fuge (Damon/IEC D i v i s i o n , Needham H t s . , MA., U .S .A . ) and a Behringwerke measuring viewer (Behringwerke AG, Marburg, West Germany). 2.4 Preparat ion of Stock and Reagent So lu t ions 2.4.1 Drug, Metabol i tes and Internal Standard Propafenone hydrochlor ide was accura te ly weighed and d i s s o l v e d in de ion ized d i s t i l l e d water using s e r i a l d i l u t i o n to a f i n a l concentrat ion of -100 ng/mL (-11.07 mg of PF hydrochlor ide i s equivalent to -10 mg of PF f ree base) . 5-Hydroxy propafenone hydrochlor ide was accurate ly weighed and d i s s o l v e d in methanol:deionized d i s t i l l e d water (1:9) using s e r i a l d i l u t i o n to a f i n a l concentrat ion of -100 ng/mL (-11.02 mg of 5-hydroxy PF hydroch lor ide i s equivalent to -10 mg of 5-hydroxy PF f ree base) . 5-Hydroxy-4-methoxy propafenone hydrochlor ide was accurate ly weighed and d i s s o l v e d in methanol:deionized d i s t i l l e d water (1:9) using s e r i a l 39 d i l u t i o n to a f i n a l concentrat ion of -100 ng/mL (-10.94 mg of 5-hydroxy-4-methoxy PF hydrochlor ide i s equiva lent to -10 mg of 5-hydroxy-4-methoxy PF f ree base) . Li -1115 hydrochlor ide was accura te ly weighed and d i s s o l v e d in de ion ized d i s t i l l e d water using s e r i a l d i l u t i o n to a f i n a l concentrat ion of -200 ng/mL (-11.11 mg of Li-1115 hydrochlor ide i s equiva lent to -10 mg of Li -1115 f ree base) . Li -1548 hydrochlor ide was accura te ly weighed and d i s s o l v e d in de ion ized d i s t i l l e d water using s e r i a l d i l u t i o n to a f i n a l concentrat ion of -200 ng/mL (-11.06 mg of Li-1548 hydrochlor ide i s equiva lent to -10 mg of Li-1548 f ree base) . A l l stock and d i l u t e d so lu t ions were protected from sun l igh t by wrapping the g l a s s conta iners in aluminum f o i l and stored at 4°C a f te r p r e p a r a t i o n , f o r up to four months. 2 .4 .2 Reagents and So lu t ions Tr ie thy lamine 0.003 M was prepared by d i l u t i n g t r ie thy lamine with to luene . Four or f i v e p e l l e t s of NaOH were added to the s o l u t i o n . Sodium hydroxide (NaOH) 1 M and 5 M so lu t ions were prepared by d i s s o l v i n g NaOH p e l l e t s in de ionized d i s t i l l e d water. Sodium carbonate (Na2C0 3) 0.1 M s o l u t i o n and potassium carbonate (K2CO3) 5 M s o l u t i o n were prepared by d i s s o l v i n g Na2C03 and K2CO3 powder in de ion ized d i s t i l l e d water. Hydroch lor ic ac id (HC1) 1 M was prepared by d i l u t i n g ACS reagent grade concentrated (37%) HC1 in de ionized d i s t i l l e d water. Ammonium hydroxide (NH4OH) 4% was prepared by d i l u t i n g Ammonia S o l u t i o n Strong (27%) in deionized d i s t i l l e d water. 40 Phosphate buf fe r (pH 7.4) was prepared using the fo l lowing procedures. Monopotassium phosphate (KH2PO4, 2.28 g) was accura te ly weighed and d i s s o l v e d in de ionized d i s t i l l e d water to a f i n a l volume of 250 mL ( s o l u t i o n A ) . Disodium phosphate ( ^ H P G ^ , 9.47 g) was accura te ly weighed and d i s s o l v e d in de ionized d i s t i l l e d water to a f i n a l volume of 1 L ( s o l u t i o n B) . An accurate volume of so lu t ion A (200 mL) was t r a n s f e r r e d in to a c lean 1 - l i t r e volumetr ic f l a s k . The volume was then adusted to 1 L with stock s o l u t i o n B to y i e l d a phosphate buf fer of 0.067 M. The pH of the f i n a l s o l u t i o n was checked and adjusted, i f necessary, to 7.4 with a l i q u o t s o f stock so lu t ions A or B. Phosphate buf fe r (pH 6.0) was prepared by d i s s o l v i n g 2.28 g of KH2PO4 in de ion ized d i s t i l l e d water to a volume of 250 mL (so lu t ion 1) and 0.95 g Na2HP04 to a volume of 100 mL (so lu t ion 2) . The f i n a l buf fe r s o l u t i o n was prepared by combining so lu t ions 1 and 2 (90 mL of s o l u t i o n 1 and 10 mL of s o l u t i o n 2 ) . 2.5 C a p i l l a r y E lect ron-Capture Detect ion Gas-L iqu id Chromatographic A n a l y s i s of Propafenone 2.5.1 Pre l iminary Development of a C a p i l l a r y GLC-ECD Assay Method fo r Propafenone 2 .5 .1 .1 Ex t rac t ion and T r i f l u o r o a c e t i c Anhydride D e r i v a t i z a t i o n Treatment f o r Propafenone An a l i q u o t of blank serum spiked with PF and l . S . - a in des i red concentra t ions was made to a constant volume with water. The s o l u t i o n was 41 al kai i ni zed with 0.5 mL of 1 M sodium hydroxide to pH 11 and extracted with 6 mL of benzene. A f t e r ro tary mixing f o r 20 min and c e n t r i f u g a t i o n , the benzene l a y e r was t r a n s f e r r e d to a c lean g lass tube, to which 2 mL of 1 M h y d r o c h l o r i c ac id was added. The s o l u t i o n was then ro tary mixed fo r 20 min and a f t e r c e n t r i f u g a t i o n , the benzene l a y e r was d iscarded and the aqueous l a y e r was washed with two 4 mL a l i q u o t s of benzene. Then 0.5 mL of 5 M sodium hydroxide and 6 mL of benzene were added to the aqueous layer and the s o l u t i o n ro tary mixed f o r 20 min. A f t e r c e n t r i f u g a t i o n , the benzene l a y e r was t r a n s f e r r e d to a c lean t e s t tube and dr ied under n i t rogen in a 40°C water bath to complete dryness . The d r ied sample was recons t i tu ted with 800 /xL of toluene (0.003 M TEA was added as a c a t a l y s t f o r the d e r i v a t i z a t i o n react ion) and d e r i v a t i z e d with TFAA at 65°C f o r 1 h. 2 .5 .1 .2 N e u t r a l i z a t i o n of Excess T r i f l u o r o a c e t i c Anhydride Hydro lys is of the excess TFAA to TFA-ac id was c a r r i e d out by mixing the sample with 0.5 mL of water on a vortex mixer f o r 10 sec , fol lowed by n e u t r a l i z a t i o n of the ac id with 0.5 mL of 4% ammonium hydroxide fo r 15 sec . 2 .5 .2 Optimal D e r i v a t i z a t i o n Condi t ions For Propafenone 2 .5 .2 .1 Use of Tr ie thylamine as Ca ta lys t and Optimal D e r i v a t i z a t i o n Time Two sets of samples conta in ing equivalent amounts of PF and I.S . -a in methanol were d r ied and recons t i tu ted with 800 /zL of to luene , with TEA (0.003 M) added to only one s e t . The samples were then d e r i v a t i z e d with 50 fit of HFBA f o r var ious times (0 .5 , 1, 2, 3 and 4 h) at 6 5 ° C . The peak areas of the PF d e r i v a t i v e were evaluated to assess the n e c e s s i t y of using TEA as a c a t a l y s t and to determine the optimum 42 d e r i v a t i z a t i o n t ime. 2 .5 .2 .2 Quant i ty of T r i e t h y l amine Used Samples conta in ing equiva lent amounts of PF and l . S . - a in methanol were d r i e d and r e c o n s t i t u t e d with var ious volumes (200, 400, 600 and 800 (il) o f toluene conta in ing 0.003 M TEA. The samples were then d e r i v a t i z e d with 50 ill of HFBA at 65°C fo r 1 h. The peak areas of the HFB-PF d e r i v a t i v e were evaluated to determine the minimum amount of TEA requi red as a c a t a l y s t f o r the d e r i v a t i z a t i o n r e a c t i o n . 2 .5 .2 .3 Quanti ty of Hepta f luorobutyr ic Anhydride Used Samples conta in ing equiva lent amounts of PF and l . S . - a in methanol were d r i e d and r e c o n s t i t u t e d with 800 nl of toluene conta in ing 0.003 M TEA. The samples were then d e r i v a t i z e d with var ious volumes (5, 10, 20 and 40 (il) of HFBA at 65°C fo r 1 h. The peak areas of the HFB-PF d e r i v a t i v e were evaluated to determine the minimum amount of HFBA required for the d e r i v a t i z a t i o n r e a c t i o n . 2 .5 .3 Solvent Ex t rac t ion E f f i c i e n c y The optimal ex t rac t ion solvent was tested by adding i d e n t i c a l concentra t ions o f PF to blank plasma and subsequent ex t rac t ion with four d i f f e r e n t s o l v e n t s , viz., to luene, benzene, hexane or a combination of to luene:d ich loromethane: isopropy l alcohol (7:3:1) [Brode et al., 1984]. A f t e r e x t r a c t i o n , an i d e n t i c a l concentrat ion of l . S . - a in methanol was added to the above extracted samples and also to a sample that contained the same concentra t ion of PF in methanol (not subjected to e x t r a c t i o n ) . 43 The samples were d r ied under n i t rogen and then d e r i v a t i z e d with HFBA. The e x t r a c t i o n e f f i c i e n c y of these solvents was evaluated by comparing the peak area r a t i o s of the extracted samples to the peak area r a t i o of the unextracted sample. 2 .5 .4 Optimal GLC-ECD Condi t ions The optimal column temperature was determined by i n j e c t i n g a sample conta in ing HFB d e r i v a t i v e s of PF and I . S . - a in to the GLC under d i f f e r e n t temperature programming cond i t ions to obtain optimum peak shape and peak symmetry f o r both PF and I . S . - a as well as r e s o l u t i o n of the peaks fo r PF and I . S . - a from peaks of serum endogenous substances. Var ious i n l e t purge a c t i v a t i o n times (10, 20, 40, 60 and 80 s e c ) , i n j e c t i o n port temperatures (200, 210, 220, 230, 240, 250 and 2 6 0 ° C ) , de tec tor temperatures (330, 340 and 350°C) and make-up gas flow rates (30, 40, 50 and 60 mL/min) were examined as mentioned p r e v i o u s l y . The peak areas of HFB-PF or peak area r a t i o ( H F B - P F / H F B - I . S . - a ) , peak shape and symmetry were examined to obtain the optimal value f o r each parameter. 2 .5 .5 Recovery of Propafenone 2 .5 .5 .1 E x t r a c t a b i l i t y of Propafenone Propafenone hydrochlor ide was d i s s o l v e d in water and methanol, r e s p e c t i v e l y , to produce so lu t ions of equal concentrat ions (equivalent to 100 rig/mL of base) . Various volumes (0 .1 , 0 .2 , 0 .4, 0 .6 , 0.8 and 1.0 mL) of these s o l u t i o n s were t rans fe r red into two sets of g lass tubes, r e s p e c t i v e l y . To the set of PF hydrochlor ide samples in water was added human blank serum fol lowed by ex t rac t ion with benzene. The s a l t of the 44 l . S . - a was d i s s o l v e d in methanol to y i e l d a concentrat ion of 200 ng/mL, and 0.35 mL of t h i s s o l u t i o n was added to both sets of samples. The two sets of samples were then subjected to the same d e r i v a t i z a t i o n r e a c t i o n . The amounts of PF extracted from the aqueous so lu t ions by benzene were c a l c u l a t e d from the c a l i b r a t i o n curve of PF hydrochlor ide in methanol and compared with the actual amount of PF added. 2 .5 .5 .2 V a r i a b i l i t y in Recovery of Propafenone Propafenone hydrochlor ide (equivalent to 0.1 mg/mL of base) in d i s t i l l e d water was d i l u t e d to prepare two so lu t ions with concent ra t ions of 10 and 100 ng/mL. Four p a r t s , three p a r t s , two parts and one part o f the 10 ng/mL s o l u t i o n were mixed with one par t , two p a r t s , three par ts and four parts of the 100 ng/mL s o l u t i o n , r e s p e c t i v e l y , to y i e l d s o l u t i o n s with concentra t ions of 28, 46, 64 and 82 ng/mL. One m i l l i l i t e r of each of these s o l u t i o n s , i n c l u d i n g the 10 and 100 ng/mL so lu t ions were then subjected to a n a l y s i s . The amount of PF in each so lu t ion was estimated from a c a l i b r a t i o n curve prepared at the same t ime. The amount of PF found was p lo t ted versus the amount of PF added and the c o r r e l a t i o n was assessed by l i n e a r r e g r e s s i o n . 2 .5 .6 Quant i ta t i ve Ana lys is of Propafenone An a l i q u o t of human b i o l o g i c a l f l u i d (serum or s a l i v a ) spiked with PF standard s o l u t i o n s and l . S . - a was extracted and d e r i v a t i z e d . A 2 fil a l i q u o t of the f i n a l so lu t ion conta in ing the HFBA d e r i v a t i v e s of PF and l . S . - a was i n j e c t e d into the GLC under optimized c o n d i t i o n s . A c a l i b r a t i o n curve was constructed by p l o t t i n g the peak area r a t i o (HFB-PF /HFB- I .S . -a ) versus the amount of PF added, using l i n e a r r e g r e s s i o n . D i f f e r e n t 45 concentra t ion ranges were used fo r var ious samples, viz., 0.5-10 ng/mL fo r s i n g l e dose volunteer s a l i v a samples, 2.5-50 ng/mL fo r s i n g l e dose volunteer serum samples and 10-100 ng/mL f o r s teady-s ta te pa t ien t serum samples. The c a l i b r a t i o n curve thus obtained was used fo r the est imat ion of the unknown concentrat ions of PF in b i o l o g i c a l samples (an equiva lent amount of I . S . - a was added to both the standard so lu t ions and the unknown samples) . A l l standard s o l u t i o n s and unknown samples were analyzed in d u p l i c a t e and each sample was in jec ted twice in to the GLC. 2 .5 .7 Determination of Day-to-Day V a r i a b i l i t y The day- to-day v a r i a b i l i t y of the GLC-ECD method was determined by sp ik ing blank plasma with an equivalent amount of PF which was then stored at - 2 0 ° C . On the day of a n a l y s i s , a s e l e c t i o n of these samples was analyzed along with the standard so lu t ions and unknown plasma samples by sp ik ing them with an equiva lent amount of I . S . - a . The c o e f f i c i e n t s of v a r i a t i o n were c a l c u l a t e d by comparing the amount of PF found to the amount of PF added and the w i th in - run and between-run p r e c i s i o n were determined. 2.6 C a p i l l a r y E lec t ron-Capture Detect ion Gas-L iqu id Chromatographic A n a l y s i s of 5-Hydroxy Propafenone and 5-Hydroxy-4-Methoxy Propafenone 2.6.1 E x t r a c t i o n , Hepta f luorobutyr ic Anhydride Treatment and N e u t r a l i z a t i o n of Excess Hepta f luorobutyr ic Anhydride The GLC-ECD method developed fo r the ana lys is of PF was modif ied fo r the a n a l y s i s of 5-hydroxy PF and 5-hydroxy-4-methoxy PF. An a l i q u o t of blank serum spiked with 5-hydroxy PF or 5-hydroxy-4-methoxy PF and I .S . -b 46 in d e s i r e d concentrat ions was d i l u t e d to a constant volume with water. The s o l u t i o n was a l k a l i n i z e d with 0.15 mL of 0.1 M sodium carbonate to pH 10 and ext rac ted with 6 mL of a solvent mixture, to luenerdichloromethane: isopropy l a lcohol (7 :3 :1 ) . A f t e r rotary mixing fo r 20 min and c e n t r i f u g a t i o n , the organic layer was t rans fe r red to a c lean g l a s s tube, and 2 mL of 1 M hydroch lo r ic ac id were added. The s o l u t i o n s were then ro ta ry mixed f o r 20 min and a f t e r c e n t r i f u g a t i o n , the organic l a y e r was d i s c a r d e d , and the aqueous layer washed with two 4 mL a l i q u o t s of the so lvent mixture . Then 0.5 mL of 5 M potassium carbonate and 6 mL of the so lvent mixture were added to the aqueous layer and the s o l u t i o n ro ta ry mixed f o r 20 min. A f t e r c e n t r i f u g a t i o n , the organic l a y e r was t r a n s f e r r e d to a c lean g l a s s tube and dr ied completely under n i t rogen at 40°C in a water bath . The dr ied sample was reconst i tu ted with 800 [il of toluene (0.003 M TEA was added as a c a t a l y s t fo r the d e r i v a t i z a t i o n reac t ion) and d e r i v a t i z e d with HFBA at 65°C fo r 1 h. Excess HFBA was removed by mixing the sample with 0.5 mL phosphate buf fer (pH 6) fo r 30 s e c . The toluene l a y e r was t r a n s f e r r e d to a c lean autosampler v i a l , d i l u t e d with toluene and capped with a PTFE- l ined s e a l . A volume of 2 p.1 of t h i s mixture was then i n j e c t e d in to the GLC for GLC-ECD a n a l y s i s . 2 .6 .2 Optimal D e r i v a t i z a t i o n Condit ions for 5-Hydroxy Propafenone 2 .6 .2 .1 Quanti ty of Heptaf luorobutyr ic Anhydride Used Samples conta in ing equivalent amounts of 5-hydroxy PF and I . S . - b in methanol were d r i e d under ni t rogen and then recons t i tu ted with 800 [il of to luene conta in ing 0.003 M TEA. The samples were then d e r i v a t i z e d with var ious volumes (25, 50 and 100 /*L) of HFBA at 65°C fo r 1 h. 47 The peak areas of the HFB-5-hydroxy PF d e r i v a t i v e were evaluated to determine the minimum quant i ty of HFBA required f o r the r e a c t i o n . 2 .6 .2 .2 D e r i v a t i z a t i o n Time Two sets o f samples conta in ing equivalent amounts of 5-hydroxy PF and I . S . - b in methanol were d r ied under ni t rogen and subsequently recons t i tu ted with 400 nl of to luene , with TEA (0.003 M) added to only one s e t . The samples were then d e r i v a t i z e d with 20 /iL of HFBA fo r var ious times (5, 15, 30, 45 and 60 min) at 6 5 ° C . The peak areas of the HFB-5-hydroxy PF d e r i v a t i v e were evaluated to assess the n e c e s s i t y of using TEA as a c a t a l y s t and to determine the optimum d e r i v a t i z a t i o n t ime. 2 .6 .3 E x t r a c t i o n E f f i c i e n c y of Solvents The optimal ex t rac t ion solvent was tested by adding i d e n t i c a l concentra t ions o f 5-hydroxy PF to blank plasma and subsequent ex t rac t ion with four d i f f e r e n t s o l v e n t s , viz., to luene, benzene, hexane and a combination of to luene:dichloromethane: isopropyl a lcohol (7 :3 :1 ) . A f t e r e x t r a c t i o n , an i d e n t i c a l concentrat ion of I .S . -b in methanol was added to the ext racted samples and a lso to a sample that contained the same concent ra t ion o f 5-hydroxy PF in methanol (not subjected to e x t r a c t i o n ) . The samples were d r i e d under ni t rogen and then d e r i v a t i z e d . The ex t rac t ion e f f i c i e n c y of these solvents was evaluated by comparing the peak area r a t i o s of the ext racted samples to the peak area r a t i o of the unextracted sample. 48 2 .6 .4 Optimal GLC-ECD Condit ions fo r 5-Hydroxy Propafenone The optimal column temperature was determined by i n j e c t i n g a sample conta in ing the HFB d e r i v a t i v e s of PF, 5-hydroxy PF and I .S . -b in to the GLC under d i f f e r e n t temperature programming cond i t ions to obta in ideal peak symmetry and r e s o l u t i o n for 5-hydroxy PF and I . S . - b , as well as complete separat ion o f 5-hydroxy PF and I .S . -b from each other and from PF and endogenous substances present in b i o l o g i c a l f l u i d s . 2 .6 .5 Recovery of 5-Hydroxy Propafenone The hydrochlor ide s a l t of 5-hydroxy PF was d i s s o l v e d in water and methanol, r e s p e c t i v e l y , to make up so lu t ions of equal concentra t ions (equiva lent to 100 ng/mL of base) . Various volumes (0 .1 , 0 .2 , 0 .3 , 0.4 and 0.5 mL) of these so lu t ions were t rans fe r red in to two sets of g l a s s tubes, r e s p e c t i v e l y . Human blank serum was added to the set of 5-hydroxy PF hydroch lor ide samples in water, fol lowed by ex t rac t ion with to luene: d ichloromethane: isopropyl alcohol (7 :3:1) . The s a l t of I . S . - b was d i s s o l v e d in methanol to y i e l d a concentrat ion of 200 ng/mL, and 0.2 mL of t h i s s o l u t i o n was added to both sets of samples. The two sets of samples were then subjected to the same d e r i v a t i z a t i o n r e a c t i o n . The amount of 5-hydroxy PF extracted from the aqueous s o l u t i o n s by the solvent mixture were c a l c u l a t e d from the c a l i b r a t i o n curve of 5-hydroxy PF hydrochlor ide in methanol and compared with the actual amount of 5-hydroxy PF added. 49 2.7 S t r u c t u r a l Conf irmat ion of the HFB Der iva t i ves of Propafenone, 5-Hydroxy Propafenone and l . S . - a by Gas -L iqu id Chromatography-Mass Spectrometr ic A n a l y s i s S t r u c t u r a l conf i rmat ion of the d e r i v a t i v e s , HFB-PF and H F B - I . S . - a , was c a r r i e d out by GLC-MS with e lect ron- impact (E I ) , p o s i t i v e - i o n chemical -i o n i z a t i o n (PICI) and negat ive - ion c h e m i c a l - i o n i z a t i o n (NICI) as the GLC-MS i o n i z a t i o n sources . HFB-5-hydroxy PF was analyzed by EI-GLC-MS. The GLC-MS operat ing cond i t ions were the same as used in rout ine GLC ana lyses . Methane was used as reagent gas in PICI and NICI and the temperature of the GLC-MS i n t e r f a c e was 270°C. The MS operat ing cond i t ions were: e lec t ron i o n i z a t i o n energy 70, 110 and 130 eV fo r E I , PICI and NICI-MS, r e s p e c t i v e l y ; emission cur ren t , 0.3 mA; ion source temperature, 240°C . 2.8 Measurement of Trough Plasma Propafenone Concentrat ions by E l e c t r o n -Capture Detect ion Gas-L iqu id Chromatography and High-Performance L i q u i d Chromatography in Pat ients Receiving Propafenone Pat ien t samples were obtained during the course of a study examining the e f f e c t i v e n e s s of PF in the treatment of a t r i a l f i b r i l l a t i o n and s u p r a v e n t r i c u l a r t achycard ia . Trough plasma samples were drawn on a treatment day immediately p r i o r to the next d a i l y dose. A l l samples were assayed in d u p l i c a t e by the present GLC-ECD method. Propafenone concent ra t ions of ten pat ient samples were a lso measured by a publ ished HPLC method to t e s t the v a l i d i t y of the GLC-ECD measurement technique. The samples se lec ted were based on the s e n s i t i v i t y of the HPLC method and had a 50 PF concent ra t ion greater than 300 ng/mL. A minor m o d i f i c a t i o n of the method o f Harapat and Kates [1982] was employed. The mobile phase used in the method of Harapat and Kates [ a c e t o n i t r i l e : 0 . 0 0 5 M phosphate b u f f e r , pH 2 .4 , (25:75)] was changed s l i g h t l y [ a c e t o n i t r i l e : 0 . 0 0 5 M phosphate b u f f e r , pH 2 .9 , (38:62)] to reduce a n a l y s i s time and to improve chromatographic peak shape. 2.9 In Vitro Serum Protein Binding Study 2.9.1 E q u i l i b r i u m Time fo r Propafenone During E q u i l i b r i u m D i a l y s i s Phosphate buf fer (pH 7.4) conta in ing PF was d i a l y z e d against an equal volume (1 mL) of blank serum at 37°C fo r 1, 2, 4 and 6 h. The concent ra t ion of PF in the buf fer was measured and c a l c u l a t e d as percentage o f the i n i t i a l PF concentrat ion and p lo t ted against t ime. E q u i l i b r a t i o n i s e s t a b l i s h e d when a p lo t of percent of i n i t i a l concentra t ion versus time d e c l i n e s to an apparent asymptote (i.e., e q u i l i b r a t i o n of f ree PF concent ra t ion between the serum and buf fer chambers of the d i a l y s i s c e l l ) . E q u i l i b r i u m time fo r PF was evaluated at both low (2.5 /zg/mL) and high (100 zzg/mL) PF concent ra t ions . 2 .9 .2 N o n - S p e c i f i c Binding of Propafenone The extent of adsorpt ion of PF to the e q u i l i b r i u m d i a l y s i s membrane was determined by measuring PF concentrat ion before and a f t e r soaking the membrane in buf fer contain ing PF. The extent of adsorpt ion of PF to the e q u i l i b r i u m d i a l y s i s c e l l was also determined by i n j e c t i n g buf fe r conta in ing PF into c e l l s l ack ing the membrane and by measuring PF 51 concent ra t ion in buf fe r before and a f t e r incubat ion in the c e l l fo r 6 h in a 37°C water bath. The n o n - s p e c i f i c binding of PF to the u l t r a f i l t r a t i o n device was determined by measuring PF concentrat ion before and a f t e r u l t r a f i l t r a t i o n {i.e., PF concentra t ion in u l t r a f i U r a t 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 at 25°C with a c e n t r i f u g a l force of 2000 x g f o r 30 min. About 300-400 /zL o f u l t r a f i U r a t e were obtained a f te r f i l t r a t i o n of 1 mL of serum spiked with a small volume (5 /xL) of buf fer conta in ing PF (0 .1 , 0 .5 , 1, 5, 10 and 100 /xg/mL) to minimize d i l u t i o n of prote in concentrat ion in the serum. A l l n o n - s p e c i f i c binding studies were conducted at PF concentrat ions of 0 . 1 , 0 .5 , 1, 5, 10 and 100 /xg/mL and the percentage of PF adsorbed to mater ia l sur faces was c a l c u l a t e d . 2 .9 .3 E q u i l i b r i u m D i a l y s i s Procedure The e q u i l i b r i u m d i a l y s i s procedure is shown in Figure 3. The cel lophane d i a l y s i s membrane was immersed in b o i l i n g d i s t i l l e d water fo r 1 h and then soaked in phosphate buf fer (pH 7.4) fo r another hour before mounting wi th in the d i a l y s i s c e l l s . Extreme care was taken not to touch the sur face of the membranes p r i o r t o , during and a f t e r the mounting procedure. Blank serum was d ia lyzed against an equal volume (0.4 mL fo r the concent ra t ion range of 1 to 100 /xg/mL and 1 mL f o r the concentrat ion range of 0.25 to 0.75 /xg/mL) of phosphate buf fer conta in ing PF in a 37°C water bath with the assembly ro ta t ing at a speed of 14 rpm f o r 6 h. At the end o f d i a l y s i s , a l iquo ts of the buf fer and serum were t r a n s f e r r e d to clean g l a s s t e s t tubes to which equivalent amounts of l . S . - a were added and the samples analyzed fo r PF concentrat ion by GLC-ECD. D i a l y s i s was performed in t r i p l i c a t e at each concent ra t ion . A drop of the d i a l y s i s buf fe r was 52 propafenone hydroch lor ide (= 0.1 mg/mL base) in phosphate buf fe r (0.067 M, pH 7.4) d i l u t i o n with phosphate buf fer blood from each subject t r a n s f e r in to P l e x i - G l a s s R d i a l y s i s c e l l separated by cel lophane membrane I e q u i l i b r i u m d i a l y s i s at 37°C fo r 6 h*3 1 a l iquo ts of buf fer and serum fo r GLC-ECD ana lys is a 0.4 mL f o r PF concentra t ion range of 1-100 itg/mL and 1.0 mL f o r PF concentra t ion range of 0.25-0.75 £Kj/roL b d i a l y s i s was performed in t r i p l i c a t e at each concentrat ion in a l l subjects Figure 3. Scheme of e q u i l i b r i u m d i a l y s i s fo r propafenone 53 added to 0.1 mL of 3% t r i c h l o r o a c e t i c ac id to tes t f o r leakage of prote in across the membrane. The absence of t u r b i d i t y was used as an i n d i c a t o r of membrane i n t e g r i t y . The f ree f r a c t i o n of PF was c a l c u l a t e d from the r a t i o o f e q u i l i b r i u m PF concentrat ion in the buf fe r and serum. 2 .9 .4 Determination of Propafenone Binding Parameters - Rosenthal Ana lys is Venous blood was obtained from 6 non-smoking, heal thy male Caucas ians , aged 22-39 y , weight, 61-80 kg, he igh t , 170-183 cm, using g lass Vacuta iners^ (conta in ing no heparin or other ant icoagulants ) and an indwe l l ing Butterf ly** -19 INT cannula inser ted in the brach ia l v e i n . Precaut ion was taken so that c o l l e c t e d blood d id not come into contact with the red rubber cap of the Vacuta iners^ . Fol lowing coagula t ion and c e n t r i f u g a t i o n of the blood samples, the serum was separated and t r a n s f e r r e d to a g lass tube with a PTFE l i n e d screw cap and stored at - 2 0 ° C . E q u i l i b r i u m d i a l y s i s was performed using the c o l l e c t e d serum within 2 weeks a f t e r blood c o l l e c t i o n . The f ree f r a c t i o n of PF was determined over a concentra t ion range from 0.25 to 100 /zg/mL and was c a l c u l a t e d by d i v i d i n g the PF concentrat ion in buf fe r ( f ree drug concentrat ion) by the PF concent ra t ion in serum ( tota l drug concen t ra t ion ) . The binding r a t i o (bound c o n c e n t r a t i o n / f r e e concentrat ion) of PF was p lo t ted versus the bound c o n c e n t r a t i o n , by the method of Rosenthal [1967]. The computer program NONLIN [Metz ler , 1974] was used to obtain the b inding parameters of PF, namely the number of binding s i t e s , the a s s o c i a t i o n constants and the b inding capac i ty of the p r o t e i n . 'Regression with r e p l i c a t i o n ' was ca r r i ed out to t e s t the l i n e a r i t y of f ree f r a c t i o n over the PF concentra t ion range s t u d i e d ; the leve l of s i g n i f i c a n c e of 0.05 was used. 54 2.9 .5 Scatchard Plot of the Binding Data The binding data from the s i x healthy subjects were a lso p lo t ted by the method of Scatchard , with r ' p lo t ted versus [ r ' / f r e e drug c o n c e n t r a t i o n ] . The parameter r ' i s def ined as the moles of drug bound d iv ided by the moles of prote in ( e . g . , albumin, AAG, l i p o p r o t e i n , e t c . ) . 2 .9 .6 Propafenone Free F rac t ion in Pooled Uremic Serum In preparat ion f o r the pro te in binding study, a l iquo ts of sera obtained p r i o r to hemodialysis from nine uremic d i a l y s i s pat ients ( c r e a t i n i n e c l e a r a n c e , C L c r <10 mL/min) were subjected to GLC a n a l y s i s and shown to be f ree of substances capable of i n t e r f e r i n g with the measurement of PF. These sera were then pooled to y i e l d a l a r g e r volume fo r an e q u i l i b r i u m d i a l y s i s study of the pro te in binding of PF in uremia. The f ree f r a c t i o n of PF was determined over an i n i t i a l t o t a l PF concentrat ion range of 1 to 5 /zg/mL. 2 .9 .7 E f f e c t of Uremia and Renal F a i l u r e on the Serum Binding of Propafenone The sera of three mid-range uremic pat ients ( C L c r = 3 6 + 1 2 mL/min) and sera of f i v e pat ients with chronic renal f a i l u r e ( C L c r = 0 mL/min) obtained from another PF study were used. The f ree f r a c t i o n of PF was determined by equ i l ib r ium d i a l y s i s at an i n i t i a l PF concentra t ion of 1 Mg/mL. 55 2 .9 .8 Measurement of Serum Prote in Concentrat ions 2 .9 .8 .1 Serum Albumin Concentrat ion Serum samples from the s i x healthy subjects were analyzed f o r serum albumin concentrat ion by the D i v i s i o n of C l i n i c a l Chemistry, Department of Pathology, U n i v e r s i t y H o s p i t a l , U . B . C . S i t e , Vancouver, B .C . Albumin concentra t ion was assayed by a c o l o r i m e t r i c t e s t . The measurement was performed at 630 nm using a Kodak 700 Ektachem Control U n i t , Eastman Kodak Company, Rochester , NY, U .S .A . The c o e f f i c i e n t of v a r i a t i o n was 2-3% (within an albumin concentrat ion range of 2 .5 -4 .8 g / d L ) . 2 .9 .8 .2 Serum a j - A c i d Glycoprote in Concentrat ion A l l samples were assayed fo r AAG concentrat ion in our l abor tory using a RID procedure with NOR-Part igen R AAG-RID p l a t e s . The method was based on the p r i n c i p l e that the p r e c i p i t a t i o n r i n g formed due to the formation of the AAG-ant isera complex was in a l i n e a r r e l a t i o n s h i p with the AAG (antigen) concent ra t ion . Fresh undi luted serum samples (5 ill) were t r a n s f e r r e d in to the wel ls of the RID p la tes using an Eppendorf m ic rop ipe t te . Each sample was analyzed in t r i p l i c a t e . The p la tes were allowed to stand t i g h t l y c losed at room temperature fo r 2 days to a t ta in the d i f f u s i o n end-point . The diameters of the p r e c i p i t a t e r ings were measured using a Behringwerke Measuring Viewer and the AAG concentrat ions read from a c a l i b r a t i o n tab le provided with the RID p l a t e s . The accuracy of the method, estimated by using the contro l serum f o r N o r - P a r t i g e n R , was + 5% of the standard value provided by the company. 56 2.10 Phenobarbital Treatment in Non-Smokers and Smokers: Pharmacokinetics and Binding Studies of Propafenone and 5-Hydroxy Propafenone 2.10.1 Study Subjects Eight healthy non-smoking (age 21-38 y , weight 65-82 kg, height 168-184 cm) and e ight healthy smoking (age 25-43 y , weight 64-80 kg, height 173-185 cm) Caucasian males served as study subjects a f t e r g i v i n g wr i t ten informed consent . The recruitment of these subjects was made through Campus b u l l e t i n s and advertisement in l o c a l newspapers. The protocol and the procedures of the present study were approved by the U n i v e r s i t y Human E th ics Committee, U . B . C . Each volunteer received E th ics Committee approved compensation ($210) fo r inconvenience incurred during p a r t i c i p a t i o n in the present research p r o j e c t . None of the non-smoking subjects had a recent h i s t o r y of smoking ( in the past 15 y e a r s ) . A l l smoking i n d i v i d u a l s were habi tual c i g a r e t t e smokers (none admitted marijuana use) who smoked at l e a s t 20 c i g a r e t t e s ( tar content : 8-16 mg, n i c o t i n e content : 0.8-1.1 mg) a day fo r the past 5 y e a r s . A l l subjects admitted to mi ld to moderate a lcohol consumption. None had a h i s t o r y of c a r d i a c , hepat ic or renal d iseases and a l l had normal phys ica l examination, normal e lect rocard iogram (ECG) and b i o c h e m i c a l / • haematological laboratory r e s u l t s at the time of the study (Table 3 ) . A l l subjects were ins t ruc ted to abstain from other medications fo r 2 weeks p r i o r to and during the study. A l s o , they were proh ib i ted from consumption of a lcohol or any ca f fe ine conta in ing beverages fo r 48 h before and during each phase of the study. 57 Table 3. C h a r a c t e r i s t i c s of non-smoking and smoking subjects . Age Weight . A L P a SG0T b C l _ c r c Subject (y) (kg) ( IU/L) ( IU/L) (mL/min/1.73m 2) Non-smokers BK 38 65.4 92 27 82.9 NP 28 70.4 55 25 122.6 CA 21 81.8 80 21 87.9 DA 21 72.7 105 46 92.3 SG 33 75.0 57 32 149.6 MV 24 72.7 85 47 119.9 GP 22 79.5 81 24 152.0 UH 34 65.0 87 27 63.6 Mean 28 72.8 80 31 108.9 ± s . d . ±7 ± 6 . 0 ±17 ±10 ± 3 2 . 2 Smokers JL 43 77.2 66 18 128.5 MA 35 80.0 70 27 141.6 TN 33 63.5 35 20 149.4 DW 25 79.0 58 24 73.8 GE 26 75.0 67 24 95.0 MG 38 77.0 78 34 140.0 SR 35 75.0 96 19 173.4 DB 37 72.7 78 24 150.0 Mean 34 74.9 69 24 131.5 ±s . d . ±6 ± 5 . 2 ±18 ±5 ± 3 2 . 2 a serum a l k a l i n e phosphatase, normal range = 30-110 IU/L b serum glutamic oxa loace t ic transaminase, normal range = 5-47 IU/L c c r e a t i n i n e c learance , corrected fo r body surface area , normal range = 63-173 mL/min/1.73 m^  s . d . standard dev ia t ion * smoked an average of 20 c i g a r e t t e s ( tar content: 8-16 mg; n i c o t i n e content : 0.8-1.1 mg) per day f o r the past 5 years 58 2.10.2 Study Protocol The study protocol i s shown in Figure 4. In the f i r s t phase of the study (control s t a t e ) , subjects received a 300 mg PF hydrochlor ide tab le t o r a l l y . B lood, s a l i v a and ur ine samples were c o l l e c t e d up to 48 h a f t e r PF a d m i n i s t r a t i o n . A f t e r a 5 day washout p e r i o d , subjects rece ived phenobarbital (100 mg o r a l l y ) , at bedtime from day 8 to day 30. The second phase o f the study ( tes t s ta te) was c a r r i e d out on day 29. Aga in , each subject rece ived a s i n g l e ora l dose of PF (300 mg). B i o l o g i c a l f l u i d sampling and other study cond i t ions were conducted in the same manner as employed in the contro l s t a t e . 2.10.3 Dosing, B i o l o g i c a l F l u i d Sampling and P h y s i o l o g i c a l Monitor ing of Study Subjects During the two phases of the study (control s ta te and t e s t s t a t e ) , a l l subjects were under the superv is ion of a c a r d i o l o g i s t in the D i v i s i o n of Card io logy , U n i v e r s i t y H o s p i t a l , U . B . C . S i t e . A l l subjects fasted fo r the 12 h preceding the study. A b u t t e r f l y cannula was inser ted in to the brach ia l vein of each subject on the day of the study fo r m u l t i p l e blood sampl ing. Each subject rece ived one tab le t of PF hydrochlor ide (300 mg) o r a l l y with 200 mL of water and, t h e r e a f t e r , d id not ingest food fo r 4 h or water f o r 3 h a f t e r PF a d m i n i s t r a t i o n . To minimize a l t e r a t i o n s in hepat ic blood flow caused by postural changes, a l l subjects were requi red to remain seated on a c h a i r f o r 60 min p r i o r to and a minimum of 90 min a f t e r dos ing . Blood pressure was measured and an ECG was recorded p r i o r to and 1 h fo l low ing PF a d m i n i s t r a t i o n . Venous blood (10 mL) and s a l i v a (2-4 mL) samples were c o l l e c t e d p r i o r to the dose and at 0.25, 0 .5 , 1, 1.5, 2, 3, 4, 5, 6, 8, 10, 12, 14, 24, 30 and 48 h a f te r PF a d m i n i s t r a t i o n . S e r i a l urine 59 Dav 1 to Day 31 CONTROL - S T A T E -Day 1 H — — r -_ T E S T ^ S T A T E ^ Phenobarbital Treatment (100 mg d a i l y at bedtime) ' ( * blood sampling fo r phenobarbital serum l e v e l ) 15 22 29 30 31 Dav 1 (control state) and Day 29 ( test state) PROPAFENONE (300 mg tab le t & 200mL water) fasted-i subject remain s i t t i n g ECG I I i no Iwaterh— I <—no food—•! I I i ECG I ' I 4-Hour -1 0 1 2 3 4 5 6 7 biood ttHH M M t t & s a l i v a sampling 8 9 10 11 12 13 14 t t -H-24 -H-32 -ih 48 t t ur ine c o l l e c t i o n (0-48h) Figure 4. Scheme of study p r o t o c o l . 60 samples were c o l l e c t e d fo r 48 h. Subjects received a c o n t r o l l e d meal f o r lunch and d inner (two Big Macs R , f rench f r i e s , a S p r i t e R deca f fe ina ted so f t d r ink and an apple pie from a McDonalds R Restaurant) on the f i r s t day of each phase of the study. 2.10.4 Sample C o l l e c t i o n Techniques An indwel l ing b u t t e r f l y cannula was inser ted in to the brach ia l vein to f a c i l i t a t e repeated blood sampling. Blood samples (10 mL) were withdrawn from the cannula using g lass syr inges and 1.5 inch 22 gauge needles . One mL of s a l i n e conta in ing heparin (5 units/mL) was i n j e c t e d in to the cannula a f t e r the withdrawl of each sample to prevent c l o t t i n g . The cannula was removed a f t e r the 14 h blood sample was c o l l e c t e d . The 24, 32 and 48 h blood samples were drawn d i r e c t l y from the vein using g l a s s s y r i n g e s . Blood samples were allowed to c l o t at room temperature and serum obtained a f t e r c e n t r i f u g a t i o n of the sample at 2,500 rpm fo r 20 min. Mechanica l ly unstimulated mixed s a l i v a samples (2-4 mL) were c o l l e c t e d by expectorat ion in to a 20 mL clean g lass v i a l at the same time as blood c o l l e c t i o n . The pH of s a l i v a was measured immediately a f t e r c o l l e c t i o n with a pH meter. S a l i v a samples were cent r i fuged at 2,500 rpm f o r 10 min to remove any i n s o l u b l e n o n - s a l i v a r y substances and c e l l u l a r d e b r i s ; the c l e a r supernatant was subsequently used fo r a n a l y s i s . Urine was c o l l e c t e d in s t e r i l i z e d p l a s t i c Whirl Pak R ur ine sample bags. The pH and the volume of each urine sample were immediately measured and recorded during the time per iod where the subjects were under superv is ion at the h o s p i t a l . Urine from each subject was then pooled to y i e l d 0-12, 12-24 and 24-48 h samples. A l l b i o l o g i c a l samples were kept at -20°C u n t i l analyzed. 61 2.10.5 Assurance of Phenobarbital Compliance S i n g l e blood samples (5 mL) were c o l l e c t e d from a l l subjects on day 15, 22 and 29 (day 15, 21, 25 and 29 fo r some subjects) and quant i ta ted f o r phenobarbital concentra t ion to ensure compliance. The protocol required the serum phenobarbital concentrat ion to exceed 10 ng/ml by day 21 or 22 or the d a i l y dose of phenobarbital would be doubled (Table 4 ) . 2 .10.6 Measurement of a j - A c i d Glycoprote in Concentrat ion Before and A f t e r Phenobarbital Treatment Blood samples from day 1 and day 29 were analyzed f o r serum AAG concentra t ion in a l l subjects to determine the e f f e c t of phenobarbital admin is t ra t ion on AAG concent ra t ion . The procedure used i s the same as in Experimental Sect ion 2 . 9 . 8 . 2 . 2.10.7 A n a l y t i c a l Procedures 2.10.7.1 Phenobarbital concentrat ion Serum phenobarbital concentrat ions were analyzed by the D i v i s i o n of C l i n i c a l Chemistry, Department of Pathology, U n i v e r s i t y H o s p i t a l , U . B . C . S i t e , Vancouver, B .C . A f luorescent p o l a r i z a t i o n immunoassay technique was used and the analyses were performed on an Abbott TDX, Abbott Laborator ies D iagnost ics D i v i s i o n , I r v ing , TX, U .S .A . The c o e f f i c i e n t of v a r i a t i o n was 2.2% (at a phenobarbital concentrat ion of 10 /xg/mL) and 2.8% (at a phenobarbital concentrat ion of 52 /zg/mL). 2 .10.7 .2 Serum and S a l i v a Samples Propafenone in both serum and s a l i v a samples and 5-hydroxy PF in 62 Table 4. Serum phenobarbital concentrat ions of non-smoking and smoking subjects dur ing phenobarbital treatment. Serum phenobarbital concentra t ion (/zg/mL) Subject Day 15 Day 21 Day 22 Day 25 Day 29 Non-smokers BK 12.3 * 13.8 - 16.0 NP 11.0 - 15.4 - 19.2 CA 9.0 - 12.3 - 13.1 DA 10.6 - 14.5 - 16.0 SG 9.4 - 14.2 - 13.8 MV 10.4 - 14.6 - 16.8 GP 12.9 - 14.3 - 16.8 UH 7.9 15.8 - 21.6 20.2 Smokers JL 8.5 _ 11.6 _ 17.4 MA 13.0 - 18.6 - 20.4 TN 11.1 - 17.9 - 18.1 DW 11.4 - 14.6 - 16.9 GE 12.5 18.3 - 19.0 19.5 MG 9.3 12.5 - 12.5 13.0 SR 10.2 10.7 - 11.4 12.3 DB 10.9 13.9 - 15.3 15.1 * serum phenobarbital concentrat ion not measured 63 serum samples were quant i tated using the developed c a p i l l a r y GLC-ECD technique d iscussed in the Experimental Sect ions 2.5 and 2.6 . 2 .10 .7 .3 Urine Samples Glucuronide and s u l f a t e conjugates of 5-hydroxy PF and 5-hydroxy-4-methoxy PF were hydrolyzed using 0 - g l u c u r o n i d a s e / a r y l s u l f a t a s e (Hel ix pomatia, Boehringer Mannheim, West Germany) [Hege et a 7 . , 1984b]. Samples were f i r s t incubated with the enzyme (50 /xL per mL of ur ine) in a 37°C water bath at pH 4.6 for 24 h and then at pH 6.2 fo r another 24 h to hydrolyze 5-hydroxy PF conjugates to 5-hydroxy PF and 5-hydroxy-4-methoxy PF conjugates to 5-hydroxy-4-methoxy PF [Hege et al., 1984b]. I .S . -b was then added to the samples and 5-hydroxy PF and 5-hydroxy-4-methoxy PF were extracted and quant i ta ted using a publ ished HPLC method [Harapat and Kates, 1982]. The absorbance was monitored at 209 nm. The HPLC method was s l i g h t l y modif ied to obtain bet ter r e s o l u t i o n of I . S . - b , 5-hydroxy PF and 5-hydroxy-4-methoxy PF. The modi f ica t ions include using to luene: d ichloromethane: isopropyl alcohol (7:3:1) instead of 1% isoamyl a lcohol in heptane as the ex t rac t ion solvent and a c e t o n i t r i l e : phosphate bu f fe r (pH 2.9) (20:80) as an a l t e r n a t i v e to the a c e t o n i t r i l e : phosphate buf fe r (pH 2.4) (25:75) as the mobile phase. 2 .10.7 .4 Prote in Binding The f ree f r a c t i o n of PF in the 2 and 4 h serum samples obtained from each subject was estimated using the equ i l ib r ium d i a l y s i s technique descr ibed in the Experimental sect ion 2 .9 .3 . The serum AAG concentrat ions of the 0 h (blank) samples were quant i tated by the RID technique descr ibed in the Experimental sect ion 2 . 9 . 8 . 2 . 64 2.10.8 Data A n a l y s i s 2.10.8.1 Propafenone Serum Data A b e s t f i t o f t h e serum c o n c e n t r a t i o n versus time d a t a was o b t a i n e d u s i n g t h e computer programme AUTOAN (Sedman and Wagner, 1976) ( w i t h equal w e i g h t o f a l l d a t a p o i n t s ) t o o b t a i n t he appa r e n t e l i m i n a t i o n r a t e c o n s t a n t d e s c r i b i n g t h e t e r m i n a l p o r t i o n o f the serum drug c o n c e n t r a t i o n versus time c u r v e s (/?). The app a r e n t t e r m i n a l e l i m i n a t i o n h a l f - l i f e ( t ^ j ) was c a l c u l a t e d by d i v i d i n g 0.693 by /?. The a r e a under t h e serum c o n c e n t r a t i o n versus time c u r v e from time co z e r o t o i n f i n i t y (AUC 0) was c a l c u l a t e d by u s i n g E q u a t i o n 1: AUCQ = AUC 0 + AUC™ (1) where t r e p r e s e n t s t he time o f the l a s t sample w i t h d e t e c t a b l e PF c o n c e n t r a t i o n ( C p -] a s+J and ft i s t h e t e r m i n a l e l i m i n a t i o n r a t e c o n s t a n t . The a r e a under t h e f i r s t moment o f the c o n c e n t r a t i o n versus time c u r v e from time z e r o t o i n f i n i t y (AUMC0) was c a l c u l a t e d by u s i n g E q u a t i o n 2. AUMCQ = AUMC0 + AUMC* + (2) The C L I N T o f PF was c a l c u l a t e d by d i v i d i n g t h e dose by the o r a l AUC, assuming t h a t C L ^ N T i s equal t o the o r a l c l e a r a n c e (CL Q ) {i.e., f o r t h i s c o m p l e t e l y absorbed drug and t h a t t he l i v e r i s the s o l e e l i m i n a t i n g organ 65 that the venous e q u i l i b r i u m model of hepat ic drug c learance appl ies ) [Wilkinson and Shand, 1975]. Peak serum concentra t ion ( C m a x ) and the time to reach peak serum concent ra t ion ( t m a x ) were estimated from i n d i v i d u a l ' s serum data . The apparent volume of d i s t r i b u t i o n ( V d a r e a ) , using the compartment model method, was c a l c u l a t e d using Equations 3 and 4. For one compartment model: F x D F x C L 0 v d a r e a = = I ^ fi x AUC fi For two compartment model: F x D k 1 2 + k 2 1 k 1 3 - fi v d a r e a = " x + " ) ( 4 ) c p o k21 k21 where F = systemic a v a i l a b i l i t y D = dose C p o = in te rcept of k a slope with ord inate k j 2 = d i s t r i b u t i o n rate constant fo r t r a n s f e r of drug from cent ra l to per ipheral compartment k 2 j = d i s t r i b u t i o n rate constant f o r t r a n s f e r of drug from per iphera l to centra l compartment kj3 = e l im ina t ion rate constant of drug from centra l compartment The volume of d i s t r i b u t i o n at s teady-s ta te ( V ^ ^ ) , using non-compartment model method, was c a l c u l a t e d using Equation 5 [ P e r r i e r and Mayersohn, 1982]. F x D AUMC 1 V d s s = x ( ) <5) AUC AUC k, 66 The value of F fo r PF i s -0.12 in healthy volunteers [Hollmann et a 7 . , 1983a]. Since F changed a f te r phenobarbital treatment and the absolute values of F are unknown, a l l the volume of d i s t r i b u t i o n terms were expressed as V ^ / F . Because of the great i n t e r i n d i v i d u a l v a r i a b i l i t y in a l l k i n e t i c parameters o f PF, each subject served as h is own c o n t r o l . K i n e t i c parameters such as C L ^ , C m a x and AUC a f te r phenobarbital treatment were compared to the contro l values and the changes ( increase or decrease) expressed as percent of the contro l va lues . (^ L in t p " C L i n t C^ percent change in C L ^ j . = x 100% C L i n t C t . . „ (cmax P " cmax c) percent change in C m a x = x 100% ^max C (AUCp - AUCC) percent change in AUC = x 100% AUC C where C = contro l (untreated) P = phenobarb i ta l - t rea ted 2 .10.8 .2 5-Hydroxy Propafenone Serum Data A best f i t of the serum concentrat ion versus time data was obtained using the computer programme AUT0AN (Sedman and Wagner, 1976) (with equal weight of a l l data points) to obtain (3. The t ^ was c a l c u l a t e d by d i v i d i n g 0.693 by z3. The AUC values were c a l c u l a t e d using Equation 1. The changes 67 ( increase or decrease) in C m a x and AUC a f te r phenobarbital treatment were expressed as percentage of the contro l va lues . ^ n e ^max a n < - *max w e r e estimated from i n d i v i d u a l ' s serum data . 2 .10 .8 .3 Propafenone S a l i v a r y Data The sa l i va /se rum concentrat ion r a t i o of PF was c a l c u l a t e d by d i v i d i n g PF s a l i v a r y concentra t ion by the PF serum concentrat ion determined at the i d e n t i c a l time a f t e r drug admin is t ra t ion . The AUCg was estimated by using Equation 6 and the change ( increase or decrease) in t h i s parameter a f t e r phenobarbital treatment expressed as percentage of the contro l va lues . AUCQ = Jo C p dt (6) The percent of f ree (unbound) PF in serum was c a l c u l a t e d by using Equation 7 [Matin et al., 1974] (the d e r i v a t i o n of Equation 7 i s shown in Appendix 1) and compared to the values obtained from equ i l ib r ium d i a l y s i s . Percent of f ree PF in serum C T 1 + 1 0 P K a " 7 - 4 s a l i v a 1 U = x — x 1 0 0 (7) C 1 + 1 0 P K a - P H s ^p serum 1 + 1 U where pK a = pK a of PF pH $ = pH of s a l i v a 2 .10 .8 .4 Urine Data The renal excre t ion (cumulat ive, 0-12, 12-24 and 24-48 h) of the conjugates of 5-hydroxy PF and 5-hydroxy-4-methoxy PF before and a f t e r phenobarbital treatment was c a l c u l a t e d and expressed as percent of dose of i n t a c t drug (corrected fo r molecular weight d i f f e r e n c e ) . 68 2.10.9 S t a t i s t i c a l A n a l y s i s A l l k i n e t i c parameters a f t e r phenobarbital treatment were compared to the contro l values using the Wilcoxon paired-sample tes t at the 5% leve l of s i g n i f i c a n c e . S t a t i s t i c a l eva luat ion was a lso performed on var ious pharmacokinetic parameters between non-smokers and smokers using the Mann-Whitney t e s t with t i e d ranks at the 5% leve l of s i g n i f i c a n c e . The c o r r e l a t i o n between PF serum to ta l concent ra t ion , PF serum f ree concentra t ion and PF s a l i v a r y concentrat ion was determined by l i n e a r r e g r e s s i o n . 2.11 Eva luat ion of Propafenone Serum Concentration-Response Re la t ionsh ip 2.11.1 Study Subjects and Protocol Ten pat ients (aged 30-71 y) who were r e c e i v i n g PF fo r treatment of a t r i a l f i b r i l l a t i o n p a r t i c i p a t e d in the study (Table 5) . A l l pa t ients had normal l i v e r and kidney funct ion at the time of the study. They were not on any other medicat ion(s) other than PF (mean d a i l y oral dose = 650 mg). S e r i a l blood samples inc lud ing predose (trough l e v e l ) and 4-5 postdose samples (up to 24 h) were c o l l e c t e d . Signal -averaged ECGs (SAECGs) were obtained from each subject at each time of blood sampling using a model 1200-ETX ECG s ignal averager, Arrhythmia Research Technology, Houston, TX, U . S . A . Table 5. C h a r a c t e r i s t i c s of pat ient s u b j e c t s . Age Weight Oral dose of Type of Pat ient (y) (kg) propafenone arrhythmia(s) BN 71 * 300mg q8h A - f l u t t e r , A - f i b , PAF, C 68 53.3 300mg q8h A - f i b JC 42 - 300mg bid A - f i b JL 54 - 150mg q id A - f i b , CAD DT 69 90.0 300mg q8h Recurrent A - f i b LS 67 72.2 150mg t i d V P B ' s , VT GB 65 - 300mg q8h A - f l u t t e r , A - f i b RM 30 94.5 300mg q8h A - f l u t t e r , A - f i b , SVT AP 49 - 300mg q8h A - f i b , APB's BM 57 - 300mg q8h A - f i b , PAF * body weight unavai lab le q8h every e ight hours b id twice a day q id four times a day t i d three times a day A - f l u t t e r a t r i a l f l u t t e r A - f i b a t r i a l f i b r i l l a t i o n AT a t r i a l tachycard ia PAF paroxysmal a t r i a l f i b r i l l a t i o n CAD coronary a r te ry d isease VPB's v e n t r i c u l a r premature beats VT v e n t r i c u l a r tachycard ia SVT supraven t r i cu la r tachycard ia APB's a t r i a l premature beats 70 2.11.2 A n a l y t i c a l Procedures 2.11.2.1 Serum Samples Propafenone and 5-hydroxy PF concentrat ions in serum samples were quant i ta ted using the newly developed c a p i l l a r y GLC-ECD technique d iscussed in the Experimental sec t ion 2.5 and 2 .6 . 2 .11.2 .2 Prote in Binding Two samples were chosen from each subject fo r a prote in binding study (on the bas is that the PF concentrat ions in these samples were c lose to peak and trough l e v e l s ) . The f ree f r a c t i o n of PF in these samples was estimated using e q u i l i b r i u m d i a l y s i s as descr ibed in the Experimental s e c t i o n 2 . 9 . 3 . Serum AAG concentra t ion of these samples were quant i tated by the RID technique given in the Experimental sect ion 2 . 9 . 8 . 2 . 2.11.3 Measurement of Pharmacological E f f e c t The QRS width was used as an i n d i c a t o r of the pharmacological response of PF [Siddoway et al., 1987]. The SAE-ECGs data was generated by averaging 150 beats and QRS widening was determined to wi th in 1 msec by an observer not involved in t h i s study. 2.11.4 Data A n a l y s i s The area under the serum concentrat ion versus time curve of PF over one dosing in te rva l (AUCQ") at s teady-s ta te was c a l c u l a t e d using the t rapezo ida l r u l e . C L i n t was c a l c u l a t e d by d i v i d i n g the dose by AUCQ". In four pa t ien ts (JC, J L , DT and L S ) , the predose PF serum concentrat ion was 71 used as the PF serum concentrat ion at the dosing in te rva l in the est imation of AUC^ and C L ^ n t . The s teady-s ta te serum concentra t ion of PF ( C p s s ) was c a l c u l a t e d by d i v i d i n g AUC^ by the dosing in te rva l ( r ) . The area under the serum concentrat ion versus time curve of 5-hydroxy PF over one dosing in te rva l was a lso c a l c u l a t e d using the t rapezoida l r u l e . 2 .11.5 S t a t i s t i c a l A n a l y s i s L inear regress ion was used to obtain the c o r r e l a t i o n between PF serum concent ra t ion and QRS width and between 5-hydroxy PF serum concentrat ion and QRS width . For the samples f o r which prote in binding was performed, l i n e a r regress ion was a lso used to obtain the c o r r e l a t i o n between PF serum t o t a l concentra t ion and QRS width and, as w e l l , PF serum free concentrat ion and QRS width. M u l t i p l e stepwise regress ion [computer program RS-1 ( B o l t , Baranek and Newman Software Products Corpora t ion , Cambridge, MA, U .S .A . ) ] was used to obta in an equation to p red ic t QRS width as a funct ion of PF serum c o n c e n t r a t i o n , 5-hydroxy PF serum concentrat ion and serum AAG c o n c e n t r a t i o n . 72 3. RESULTS 3.1 C a p i l l a r y E lec t ron-Capture Detect ion Gas-L iqu id Chromatographic A n a l y s i s of Propafenone 3.1.1 Pre l iminary Resul ts o f the C a p i l l a r y GLC-ECD Method of A n a l y s i s fo r Propafenone 3 .1 .1 .1 Ex t rac t ion and TFAA Treatment TFAA has been reported to form highly e lec t ron withdrawing d e r i v a t i v e s with compounds possessing reac t ive hydroxyl and/or amino groups. D e r i v a t i z a t i o n of PF and the l . S . - a with TFAA y i e l d e d two sharp and completely reso lved peaks on the GLC chromatogram. However, the occas ional presence of an unresolved negative peak before the l . S . - a peak (Figure 5) caused a potent ia l e r r o r in peak area in tegra t ion making a TFAA d e r i v a t i v e i n a p p l i c a b l e with t h i s column. This problem was solved by using other a c y l a t i n g agents such as PFPA or HFBA. The s e n s i t i v i t y was fu r ther improved when HFBA, which conta ins four more f l u o r i n e atoms than TFAA, was employed (HFBA y i e l d e d an approximately f i v e - f o l d increase in s e n s i t i v i t y over TFAA). 3 .1 .1 .2 N e u t r a l i z a t i o n of Excess D e r i v a t i z i n g Agent The appearance of ext ra peaks in the GLC chromatogram (Figure 6) suggested that the removal of excess HFBA by h y d r o l y s i s with water and subsequent n e u t r a l i z a t i o n with ammonium hydroxide caused a rap id decomposit ion of the HFB es te rs (HFB acy la t ion of the hydroxyl group) of PF and l . S . - a by a l k a l i h y d r o l y s i s . Figure 5. Chromatogram of the TFA d e r i v a t i v e s of propafenone and I .S. 74 F igure 6. Chromatogram of the HFB d e r i v a t i v e s of propafenone and I . S . - a . 75 Evaporat ion , a widely accepted technique [Walle and Ehrsson, 1971], was used to remove excess HFBA. In t h i s method, the sample was allowed to cool to room temperature a f t e r the d e r i v a t i z a t i o n reac t ion and then dr ied under a gent le stream of ni t rogen in a 35°C water bath. The residue was r e c o n s t i t u t e d with toluene and in jec ted in to the GLC. Another a l t e r n a t i v e method was to n e u t r a l i z e the sample with 0.5 mL of phosphate buf fe r (pH 6) fo r 30 sec [Ehrsson et a / . , 1971]. The toluene l a y e r was then t r a n s f e r r e d and d i l u t e d with toluene to a des i red volume and i n j e c t e d in to the GLC. This method was l a t e r employed as part of a rout ine procedure f o r the ana lys is of PF. 3 .1 .2 Optimal D e r i v a t i z a t i o n Condi t ions Data i l l u s t r a t i n g the need fo r using TEA as a c a t a l y s t and determinat ion of the optimum react ion time are shown in Figure 7. No s i g n i f i c a n t d i f f e r e n c e was observed in the peak area of PF between 0.5 and 4 h when TEA was used, i n d i c a t i n g the reac t ion was maximal in 0.5 h. When TEA was not incorporated into the react ion mixture, the d e r i v a t i z a t i o n was slow and incomplete, as i l l u s t r a t e d by the slow r i s e in the curve and lack of attainment of a p la teau . A fur ther study with incubat ion times of 0, 5, 15, 30, 45 and 60 min ind icated that the reac t ion was complete wi thin 15 min in the presence of TEA (Figure 8 ) , thus permit t ing the use of a shor ter incubat ion t ime. The minimum amount of TEA (0.003 M) required was 400 /iL (Figure 9) and the minimum amount of HFBA required was 20 juL (Figure 10). 76 0_ o CD (D CL 30,000 25,000 m 20,000 L i _ 15,000 -* 10,000 -5000 0 0 o-T/ A 1 T. A /I T -O-i T 1 2 Time, h T -o 1 T - A 1 Figure 7. E f f e c t of TEA on the HFB-PF response, as estimated by the peak area of HFB-PF. Samples were d e r i v a t i z e d with HFBA and incubated at 65°C for var ious time with ( O ) or without ( A ) TEA. Number of samples = 3, dup l i ca te i n j e c t i o n s . The data are presented as mean + l s . d . 77 Lu CL I m Lu X D <U o Q_ 14,000 n 12,000 10,000 -8000 6000 4000 2000 ^ 0 0 T . O -J. —1— 15 T -o-1 — i — 30 45 60 Time, min T -o i Figure 8. E f f e c t of d e r i v a t i z a t i o n time estimated by the peak area of 3, d u p l i c a t e i n j e c t i o n s . The l s . d . on the HFB-PF response, as HFB-PF. Number of samples = data are presented as mean + 78 20,000 1 o. 15,000 I m u_ x o 10,000 -o a! 5000 T 0 ~200 400 600 800 Volume of TEA used, fiL Figure 9. E f f e c t of the volume of TEA (0.003M in toluene) on the HFB-PF response, as estimated by the peak area of HFB-PF. Number of samples = 3, d u p l i c a t e i n j e c t i o n s . The data are presented as mean + l s . d . 79 Lu Cu O a CL 25,000 20,000 m x 15,000 H 10,000 5000 T -o 0 10 20 30 40 Volume of HFBA, JJL F i g u r e 10. E f f e c t o f the volume o f HFBA on the HFB-PF r e s p o n s e , as es t i m a t e d by the peak a r e a o f HFB-PF. Number o f samples = 3, d u p l i c a t e i n j e c t i o n s . The d a t a a re p r e s e n t e d as mean + l s . d . 80 3 .1 .3 E x t r a c t i o n E f f i c i e n c y of Solvents The e x t r a c t i o n e f f i c i e n c y (E) of so lvents was tes ted by measuring the maximal peak area obtained fo r the chromatographic peak o f HFB-PF as a funct ion of the solvent system used. The r e s u l t s are shown in Figure 11. Benzene was found to provide optimal ex t rac t ion of PF (E = 0.81) with toluene showing s l i g h t l y diminished e f f i c i e n c y (E = 0 .76) . Nei ther the l e s s po la r s o l v e n t , hexane, (E = 0.49) nor the s l i g h t l y more po la r solvent combination of to luene:d ichloromethane: isopropyl a lcohol (7:3:1) (E = 0.45) gave s a t i s f a c t o r y r e s u l t s . 3 .1 .4 Optimal GLC-ECD Condi t ions The i n l e t purge a c t i v a t i o n time (-60 s e c , Figure 12) c o n t r o l s sample t r a n s f e r to the column during s p l i t l e s s i n j e c t i o n with minimal l o s s of so lu te (<1%) and s u f f i c i e n t removal of solvent (-5%) to reduce t a i l i n g . No s i g n i f i c a n t d i f f e r e n c e in the peak area r a t i o (HFB-PF/HFB- I .S . -a ) was observed between the d i f f e r e n t i n j e c t i o n port temperatures tested ( 2 0 0 - 2 6 0 ° C , F igure 13A). However, peak broadening occured when the i n j e c t i o n port temperature was lowered to 200°C. There were a lso no s i g n i f i c a n t d i f f e r e n c e s in the response between the de tec tor temperatures tes ted ( 3 3 0 - 3 6 0 ° C , Figure 13B) and between the var ious make-up gas flow rates tes ted (30-60 mL/min, Figure 13C). The optimal temperature programming cond i t ions were: i n i t i a l column temperature, 220°C; r a t e , 4 ° C / m i n ; f i n a l column temperature, 270°C; i n j e c t i o n port temperature, 210°C; ECD temperature, 350°C and make-up gas flow r a t e , 60 mL/min. 81 14,000 -, 12,000 -°\ 10,000 m Li_ x 8000 o S 6000 -j a | 4000 CL 2000 0 E=1 E=0.81 E=0.49 E=0.76 Benzene Hexane Methanol Toluene E=0.45 Toluene: Dichloromethane: Isopropyl alcohol (7:3:1) Figure 11. The e x t r a c t a b i l i t y of propafenone from serum using d i f f e r e n t s o l v e n t s . The ex t rac t ion e f f i c i e n c y (E) of each solvent was estimated as compared to methanol (unextracted, E = 1) . Number of samples = 3, dup l i ca te i n j e c t i o n s . 82 Figure 12. E f f e c t of i n l e t purge valve a c t i v a t i o n time on the HFB-PF response, as estimated by the peak area of HFB-PF. The data are presented as mean + l s . d . 83 o I. c/i T m u. x Q. I m u_ X o v» o l _ a u 1.2 1.0 -0.8 -0.6-0.4-0.2-A 200 220 240 Temperature, °C 260 • I in m X CL I m L_ X o a O V 1.2 1.0 0.8 -I 0.6 0.4 0.2 H B 330 340 350 Temperature, °C o I u\ T ca L_ X CL I ca b_ X o a a 4) 1.8 1.6 ^ 1.4 1.2 -1.0 -0.8 • 0.6 0.4 -I 0.2 0 30 40 50 Flow rate, mL/min —i 60 Figure 13. E f f e c t of (A) i n j e c t i o n port temperature, (B) de tec tor temperature and (C) make-up gas flow rate on the HFB-PF response, as estimated by the peak area r a t i o (HFB-PF/ H F B - I . S . - a ) . The data are presented as mean + l s . d . 84 3 .1 .5 Recovery of Propafenone 3 .1 .5 .1 E x t r a c t a b i l i t y of Propafenone The recovery of PF from plasma a f t e r solvent ex t rac t ion i s shown in Table 6 and was found to be approximately 90% over the concentra t ion range of 10-100 ng/mL. 3 .1 .5 .2 V a r i a b i l i t y in Recovery of Propafenone A s i g n i f i c a n t l i n e a r r e l a t i o n s h i p was demonstrated between the actual and the c a l c u l a t e d amounts of PF obtained during the course of a recovery experiment (F igure 14). The best f i t through the data points was obtained by l i n e a r regress ion and was descr ibed by Y = 0.838X - 0.962 with a c o r r e l a t i o n c o e f f i c i e n t (r) of 0.995. 3 .1 .6 V a r i a b i l i t y Test The day- to-day v a r i a b i l i t y tes t showed that the c o e f f i c i e n t of v a r i a t i o n was 0.5-4.5% and 4.5% fo r w i th in - run ( r e p e a t a b i l i t y ) and between-run ( r e p r o d u c i b i l i t y ) p r e c i s i o n , r e s p e c t i v e l y . The ext racted and d e r i v a t i z e d samples were stable fo r up to one week without any apparent degradat ion when stored at - 2 0 ° C . 3 .1 .7 C a l i b r a t i o n Curves The data for a representat ive c a l i b r a t i o n curve used in the q u a n t i t a t i o n of PF for s ing le dose volunteer s a l i v a and serum samples and s t e a d y - s t a t e pat ient serum samples are presented in Table 7. L i n e a r i t y was observed over the concentrat ion ranges studied (0.5-10 ng/mL fo r volunteer s a l i v a samples, 2.5-50 ng/mL fo r volunteer plasma samples and 10-100 ng/mL 85 Table 6. E x t r a c t a b i l i t y of propafenone. Number o f samples, n = 3 (two i n j e c t i o n s fo r each sample) Propafenone added Propafenone measured Recovery (%) (ng/mL) (ng/mL) (mean + s . d . ) (mean + s . d . ) 10 8.9 + 1.6 89.0 + 16.0 20 18.5 + 1.0 92.5 + 5.0 40 37.1 + 1.7 92.8 + 4.2 60 54.4 + 0.7 90.7 + 1.1 80 70.7 + 3.7 88.4 + 4.6 100 84.7 + 3.9 84.7 + 3.9 Mean + s . d . 89.7 + 3.0 s . d . standard dev ia t ion 86 Figure 14. V a r i a b i l i t y in recovery of propafenone. 87 Table 7. C a l i b r a t i o n curve data of propafenone fo r volunteer s a l i v a and serum samples and pat ient serum samples. Number o f samples, n = 2 (two i n j e c t i o n s fo r each sample) Concentrat ion of Peak area r a t i o C o e f f i c i e n t of propafenone (ng/mL) p ropa fenone / I .S . - a (mean + s . d . ) v a r i a t i o n (%) Volunteer s a l i v a samples 0.5 0.067 ± 0.004 5.8 1.0 0.138 + 0.005 3.7 2.0 0.267 ± 0.008 3.0 4.0 0.511 ± 0.006 1.2 6.0 0.775 ± 0.017 2.2 8.0 1.016 + 0.019 1.9 10.0 1.269 + 0.073 5.8 S t a t i s t i c s : l i n e a r r e g r e s s i o n , Y = 0.126X + 0.010; r = 0.999 Volunteer serum samples 2.6 0.097 ± 0.002 1.8 5.1 0.171 ± 0.001 0.2 10.2 0.313 + 0.027 8.7 20.4 0.581 ± 0.005 0.9 30.6 0.827 ± 0.004 0.5 40.8 1.003 ± 0.014 1.4 51.0 1.335 ± 0.042 3.2 S t a t i s t i c s : l i n e a r r e g r e s s i o n , Y = 0.025X + 0.050; r = 0.998 Pat ient serum samples 10.1 0.191 ± 0.002 1.1 20.3 0.349 ± 0.003 0.9 40.5 0.610 ± 0.002 0.4 60.8 0.866 + 0.006 0.7 81.0 1.131 + 0.029 2.5 101.3 1.334 ± 0.016 1.2 S t a t i s t i c s : l i n e a r r e g r e s s i o n , Y = 0.013X + 0.088; r = 0.999 s . d . standard dev ia t ion 88 f o r pa t ient plasma samples). The best f i t through the data points was obtained by l i n e a r regress ion and was descr ibed by Y = 0.126X + 0.010 with a r of 0.999, Y = 0.025X + 0.050 with a r of 0.998 and Y = 0.013X + 0.088 with a r of 0.999, r e s p e c t i v e l y . The c o e f f i c i e n t of v a r i a t i o n ( C V . ) was l e s s than 10% over the concentrat ion ranges s t u d i e d . 3 .1 .8 Summary of the E x t r a c t i o n , D e r i v a t i z a t i o n Procedure and the Resu l t ing Chromatograms for Propafenone The e x t r a c t i o n and d e r i v a t i z a t i o n procedure fo r PF i s shown in Figure 15. F igure 16 i l l u s t r a t e s the proposed reac t ion of PF and l . S . - a with HFBA to y i e l d the respec t ive HFB d e r i v a t i v e s . Figure 17A and B show representa t ive gas chromatograms from the ex t rac ts of blank serum and blank serum spiked with PF and l . S . - a and Figure 17C shows the chromatogram of a serum sample (spiked with l . S . - a ) from a subject r e c e i v i n g PF. The HFB d e r i v a t i v e o f PF was completely resolved from the HFB d e r i v a t i v e of the l . S . - a . No i n t e r f e r i n g peaks were observed from endogenous serum components of the serum extract (Figure 17A) and an ana lys is time of l e s s than 15 min was obta ined. 3.2 C a p i l l a r y E lect ron-Capture Detect ion Gas -L iqu id Chromatographic A n a l y s i s of 5-Hydroxy Propafenone and 5-Hydroxy-4-Methoxy Propafenone 3.2.1 Optimal D e r i v a t i z a t i o n Condi t ions The minimum amount of HFBA required fo r the d e r i v a t i z a t i o n of 5-hydroxy PF was 20 ill (Figure 18) and the optimal incubat ion time was 30 min (Figure 19). 89 propafenone standard so lu t ion + blank serum serum samples d i s c a r d aqueous layer d i s c a r d benzene layer d i s c a r d aqueous layer l . S . - a 0.5 mL 1 M NaOH 6 mL benzene mix 20 min, c e n t r i f u g e benzene layer 2 mL 1 M HC1 mix 20 min, cen t r i fuge aqueous layer wash twice with 4 mL benzene 0.5 mL 5 M NaOH mix 20 min, cen t r i fuge benzene l a y e r I evaporate to dryness with N 2 at 40°C r e c o n s t i t u t e with 0.4 mL toluene (with 0.03 M TEA) 20 [il HFBA, mix incubate at 65°C fo r 15 min \ neut ra l i ze excess HFBA with 0.5 mL phosphate buf fe r (pH 6) \ t r a n s f e r toluene l a y e r and d i l u t e i t with toluene to the des i red volume \ i n jec t 2 [il in to the GLC Figure 15. Scheme of ex t rac t ion procedure f o r propafenone 90 C 0 - C H 2 - C H 2 - C 6 H 5 HFBA 0-CH 2 -CH-CH 2 -NH-R ' OH C 0 - C H 2 - C H 2 - C 6 H 5 0 -CH 2 -CH-CH 2 -N -R 0 C=0 C 3 F 7 C=0 C 3 F 7 propafenone R = C 3 H 7 I . S . - a R = C 2 H 5 Figure 16: D e r i v a t i z a t i o n of propafenone and I . S . - a to y i e l d the HFB d e r i v a t i v e s of propafenone and I . S . - a , r e s p e c t i v e l y . 91 B in co LU i—i Q -l l CO CQ 3: 3= \ / OTA cap • • t co iis Q_ l CQ N IP Chromatograms of extracts from (A) blank serum; (B) blank serum spiked with propafenone (0.08 fig) and l . S . - a (0.07 ng) and (C) a serum sample (spiked with l . S . - a , 0.07 fig) from a subject r e c e i v i n g propafenone. 92 0 -I , , , , 0 . 10 20 30 40 Volume of HFBA, /xL Figure 18. E f f e c t of the volume of HFBA on the HFB-5-hydroxy PF response, as estimated by the peak area of HFB-5-hydroxy PF. Number of samples = 3, d u p l i c a t e i n j e c t i o n s . The data are presented as mean + l s . d . 93 Q_ I X o m I m X D d) i _ O o Q_ 30,000 "i 25,000 20,000 -15,000 10,000 -5000 0 0 15 30 45 60 Time, min Figure 19. E f f e c t of d e r i v a t i z a t i o n time on the HFB-5-hydroxy PF detector response, as estimated by the peak area of HFB-5-hydroxy PF. Number of samples = 3, d u p l i c a t e i n j e c t i o n s . The data are presented as mean + l s . d . 94 3.2.2 Ex t rac t ion E f f i c i e n c y of Solvents The optimal ex t rac t ion solvent fo r recovery of 5-hydroxy PF from serum was tes ted and the r e s u l t s shown in Figure 20. The more polar so lvent mixture - to luene: d ichloromethane: isopropyl a lcohol (7:3:1) [Brode et al., 1984] was found to provide optimal ex t rac t ion fo r 5-hydroxy PF (E = 0 .80) . Th is was fol lowed by the non-polar s o l v e n t s , benzene (E = 0.70) and toluene (E = 0 .60) . The non-polar solvent hexane gave a very poor e x t r a c t i o n e f f i c i e n c y (E = 0.01) . 3 .2 .3 Optimal GLC-ECD Condit ions The optimal temperature programming cond i t ions fo r rout ine ana lys is o f 5-hydroxy PF were: i n i t i a l column temperature, 205°C ( for 0.8 min); r a t e , 3 ° C / m i n ; f i n a l column temperature, 270°C; i n j e c t i o n port temperature, 210 'C; e l e c t r o n - c a p t u r e detector temperature, 350°C and make-up gas flow r a t e , 60 mL/min. 3 .2 .4 E x t r a c t a b i l i t y of 5-Hydroxy Propafenone The recovery of 5-hydroxy PF, fo l lowing solvent ex t rac t ion from serum, i s shown in Table 8 and was found to be approximately 88% over the concent ra t ion range of 10-50 ng/mL. 3 .2 .5 C a l i b r a t i o n Curve The data fo r a representat ive c a l i b r a t i o n curve used in the q u a n t i t a t i o n of 5-hydroxy PF for s i n g l e dose volunteer serum samples are presented in Table 9. L i n e a r i t y was observed over the concentrat ion range of 2.5-50 ng/mL. The best f i t through the data points was obtained by l i n e a r regress ion and was descr ibed by Y = 0.023X + 0.052 with a r of 95 35,000 -i o_ L 30 ,000 x o >, 25,000 i uo 20,000 m LL. J 15,000 o £ 10.000 a I 5000 0 E=0.70 T E=0.01 E=0.80 E=0.60 Benzene Hexane Methanol Toluene Toluene: Dichloromethane: Isopropyl alcohol (7:3:1) Figure 20. The e x t r a c t a b i l i t y of 5-hydroxy propafenone from serum using d i f f e r e n t so lven ts . The ex t rac t ion e f f i c i e n c y (E) of each solvent was estimated as compared to methanol (unextracted, E = 1). Number of samples = 3, d u p l i c a t e i n j e c t i o n s . 96 Table 8. E x t r a c t a b i l i t y of 5-hydroxy propafenone. Number of samples, n = 3 (two i n j e c t i o n s f o r each sample) 5-Hydroxy propafenone 5-Hydroxy propafenone measured Recovery (%) added (ng/mL) (ng/mL) (mean + s . d . ) (mean + s . d . ) 10.9 9.3 + 1.0 85.7 + 9.4 21.8 20.0 + 0.6 91.9 + 2.9 32.7 29.6 + 0.8 90.5 + 2.4 43.6 37.3 + 1.1 85.6 + 2.5 54.4 45.6 + 2.3 83.8 + 4.2 Mean + s . d . 87.5 + 3.5 s . d . standard dev ia t ion 97 Table 9. C a l i b r a t i o n curve data of 5-hydroxy propafenone. Number of samples, n=2 (two i n j e c t i o n s for each sample) Concentrat ion of Peak area r a t i o 5-hydroxy propafenone 5-hydroxy propafenone / I .S . -b C o e f f i c i e n t of (ng/mL) (mean + s . d . ) v a r i a t i o n (%) 2.5 0.0743 ± 0.0017 2.2 5.0 0.1267 ± 0.0021 1.7 10.0 0.3136 ± 0.0083 2.7 20.0 0.5549 ± 0.0143 2.6 30.0 0.7678 + 0.0137 1.8 40.0 0.9748 ± 0.0115 1.2 50.0 1.1468 + 0.0350 3.1 S t a t i s t i e s : l i n e a r r e g r e s s i o n , Y=0.023X + 0.052; r=0.996 s . d . standard dev ia t ion 98 0.996. The C V . was l e s s than 3.1% over the concentrat ion range s t u d i e d . 3 .2.6 Summary of E x t r a c t i o n , D e r i v a t i z a t i o n Procedure and the Resul t ing Chromatograms f o r 5-Hydroxy Propafenone The scheme of the ex t rac t ion and d e r i v a t i z a t i o n procedure f o r 5-hydroxy PF i s shown in Figure 21. It i s s i m i l a r to the ex t rac t ion procedure f o r PF with only s l i g h t m o d i f i c a t i o n . A solvent mixture [ to luene:d ichloromethane: isopropyl a lcohol (7:3:1)] was used to replace benzene as the ex t rac t ion solvent s ince t h i s solvent mixture i s more polar and was found to g ive a higher ex t rac t ion e f f i c i e n c y . Sodium and potassium carbonate were used to replace sodium hydroxide during the ex t rac t ion to minimize the formation of a water -so lub le sodium s a l t of 5-hydroxy PF which would be l o s t in the aqueous l a y e r dur ing solvent e x t r a c t i o n . Figure 22 i l l u s t r a t e s the proposed reac t ion of 5-hydroxy PF and Li-1548 with HFBA to y i e l d the respec t ive HFB d e r i v a t i v e s . Figure 23A shows the chromatogram of an extracted blank serum spiked with 5-hydroxy PF and I . S . - b . Figure 23B presents the chromatogram of a serum sample (spiked with I .S . -b) from a subject r e c e i v i n g PF. The HFB d e r i v a t i v e s of PF, 5-hydroxy PF and I .S . -b were completely resolved from each o ther . 3 .2.7 5-Hydroxy-4-Methoxy Propafenone Figure 24 shows a chromatogram of the HFB d e r i v a t i v e of 5-hydroxy-4-methoxy PF. The appearance of two peaks i n d i c a t e incomplete d e r i v a t i z a t i o n . The d e r i v a t i z a t i o n was not completed even when a higher incubat ion temperature (up to 100°C) and/or a l a r g e r amount of HFBA (up to 200 ill) were used. 5-hydroxy propafenone standard s o l u t i o n + blank serum serum samples d iscard aqueous layer I .S . -b 0.12 mL 0.1 g Na 2 C0 3 6 mL solvent mix 20 min, cen t r i fuge solvent l ayer d iscard solvent l a y e r 2 mL 1 M HC1 mix 20 min, cen t r i fuge aqueous layer d iscard aqueous layer wash twice with 4 mL solvent 0.5 mL 5 M K 2 C 0 3 mix 20 min, cent r i fuge solvent l a y e r evaporate to dryness with N 2 at 40°C r e c o n s t i t u t e with 0.4 mL toluene (with 0.03 M TEA) 20 nl HFBA, mix incubate at 65°C f o r 30 min i I neut ra l i ze excess HFBA with 0.5 mL phosphate buf fe r (pH 6) I t r a n s f e r toluene l a y e r and d i l u t e i t with toluene to the des i red volume I i n j e c t 2 fil in to the GLC to luene:dichloromethane: isopropyl a lcohol (7:3:1) Figure 21. Scheme of ex t rac t ion procedure f o r 5-hydroxy propafenone 100 C O - C H 2 - C H 2 - C 6 H 5 0-CH 2 -CH-CH 2 -NH-R OH C3F7-CO-O, HFBA C 0 - C H 2 - C H 2 - C 6 H 5 0 - C H 2 - C H - C H 2 - N - R C=0 0 i c=o C 3 F 7 C 3 F 7 5-hydroxy propafenone R = C 3 H 7 I . S . - a R = C 2 H 5 Figure 22: D e r i v a t i z a t i o n of 5-hydroxy propafenone and I .S . -b to y i e l d the HFB d e r i v a t i v e s of 5-hydroxy propafenone and I . S . - b , r e s p e c t i v e l y . 101 Figure 23. Chromatograms of ext racts from (A) blank serum spiked with 5-hydroxy propafenone (0.02 /xg) and I .S . -b (0.08 /xg) and (B) a serum sample (spiked with I . S . - b , 0.08 /xg) from a subject r e c e i v i n g propafenone. 102 Figure 24. Chromatogram of ex t rac ts from blank serum spiked with 5-hydroxy-4-methoxy propafenone. 103 3.3 S t r u c t u r a l Confirmation of HFB Der iva t ives of Propafenone, 5-Hydroxy Propafenone and l . S . - a by Gas-L iqu id Chromatography-Mass Spectrometr ic A n a l y s i s . F igures 25-29 provides the GLC-MS r e s u l t s inc lud ing to ta l ion chromatograms, fragmentation patterns and MS spectrum of the HFB d e r i v a t i v e of PF with EI (Figures 25-26), PICI (Figures 27-28) and NICI (Figure 29) as the i o n i z a t i o n modes, r e s p e c t i v e l y . For EI , the molecular ion (M +) of HFB-PF was at m/e 733 and in order of decreasing i n t e n s i t y , the prominent fragment ions were at m/e 91, 121, 508, 43, 104, 252, 226, 294. For PICI, the pseudomolecular ion (M+l) + f o r HFB-PF was at m/e 734 and in order of decreas ing i n t e n s i t y , the prominent fragment ions were at m/e 508, 734, 520, 265, 294, 133, 252. For NICI, the pseudomolecular ion (M-l)~ fo r HFB-PF was at m/e 732 and in order of decreasing i n t e n s i t y , the prominent fragment ions were at m/e 488, 213, 693, 194, 673, 448, 653. F igures 30-32 provides the GLC-MS r e s u l t s inc lud ing to ta l ion chromatograms, s e l e c t i v e fragmentation s t ruc tu res and MS spectrum of the HFB d e r i v a t i v e of l . S . - a with EI (Figure 30) , PICI (Figure 31) and NICI (Figure 32) as the i o n i z a t i o n modes, r e s p e c t i v e l y . For EI , the molecular ion (M +) f o r H F B - I . S . - a was at m/e 719 and in order of decreasing i n t e n s i t y , the prominent fragment ions were at m/e 91, 494, 169, 121, 226, 280, 104. For PICI, the pseudomolecular ion (M+l) + fo r H F B - I . S . - a was at m/e 720 and in order of decreasing i n t e n s i t y , the prominent fragment ions were at m/e 494, 720, 505, 265, 288. For NICI, the pseudomolecular ion (M-l)~ f o r H F B - I . S . - a was at m/e 718 and in order of decreasing i n t e n s i t y , the prominent fragment ions were at m/e 474, 213, 194, 679, 434, 659. F igures 33-34 shows the GLC-MS-EI r e s u l t s inc lud ing to ta l ion 104 52000-48000-40000-32000-24000H 16000 8000H 13 .78 13.3 10.0 11.0 12.0 13.0 91 100 80 •Z 60 43 40 H 20 A 77 121 226 2 ? 2 294 14? 224 207 200 268 508 466 19 536 400 m/e 600 M 800 F igure 25. GLC-EI-MS of HFB-PF: (A) Total ion chromatogram ( re tent ion t ime, 13.70 min) and (B) EI mass spectrum. Figure 26. GLC-EI-MS: a proposed fragmentation pattern of HFB-PF. 106 A 70000 60000 50000 40000 30000-i 20000 10000-! 11 . 3 3 11 . 6 6 A 10.0 1 1 11 1 1 1 1 i 1 ' 1 11 1 ' 1 : • • • ' ! I ' ' " l i . i . . . . ! 11.0 12.0 13.0 14.0 100 508 80 1 (M+l)' 60 1 40 1 265 520 20 133 294 252 .. 1.11 II 200 400 m/e 600 (M+C 2H 5) (M + C 3 H 5 ) -800 Figure 27. GLC-PICI-MS of HFB-PF: (A) Total ion chromatogram ( re tent ion t ime, 11.33 min) and (B) PICI mass spectrum. Figure 28. GLC-PICI-MS: a proposed fragmentation pattern of the HFB-PF. 108 A 180000 160000 140000 120000-1 100000 80000 60000-| 40000 20000 10.0 TlTo 12.0 13.0 14.0 c 100 1 „ 80 »« >> £ 60 40 H 20 213 194 225 J_u 200 = 733 488 -1 = 732 -2HF = 693 -3HF = 673 -4HF = 653 693 673 448 400 m/e 653 609, 600 (M-l)-800 F i g u r e 29. GLC-NICI-MS o f HFB-PF: (A) T o t a l i o n chromatogram ( r e t e n t i o n t i m e , 11.42 min); (B) a proposed f r a g m e n t a t i o n p a t t e r n and (C) NICI mass spectrum. 109 1 3 . I S 80000 : 70000-60000: 50000-40000-30000-20000-100004 10.0 11.0 12.0 13.0 14.0 15.0 16.0 B 100 -i 1/J c cu > cu 80 -60 4 0 -20 -91 69 7 ' 121 169 c=o+ CH2 0 H H-N*CO I 3 r 7 C H o - C H \J I). 1D4 147 226 C2H5-N —CH 224 20i7 280 254 co i C3F7 200 400 m/e CH 7-CH-0-C0-C 3F 7 C2H5-N — C H 2 CO I 3r7 494 522 C0-CH 2-CH 2-C 6H 5 0-CH,-CH-CH2-N-C2H5 I 0 CO 1 I CO C 3 F ? C 3 F 7 I + M 600 800 F igure 30. GLC-EI-MS of H F B - I . S . - a : (A) Total ion chromatogram ( re tent ion t ime, 13.16 min) and (B) EI mass spectrum and s e l e c t e d proposed fragmentation s t r u c t u r e s . 110 A 360000 320000 280000 240000 200000 160000 120000 80000 40000 13.11 10.0 .1 1 3 . 6 7 .4 "lJLO 12.0 ' 1?.0 ' 14.0 ' 15.0 B 100 c a> CD > 0) 80 -60 40 -20 CH 2 - •CH CH2 CH CH2-CH-0-CO-C3F7 C 2 H 5 - N — C H 2 CO - • ^ 7 C0-CH2-CH2-C5H5 0-CH2-CH=CH-N*C2H5 CO I C3P7 494 1 CO I 3 r 7 265 133 199 288 A 505 200 400 m/e 600 (M+l) + (M+C 2 H 5 ) + ( M + C 3 H 5 ) + 1 800 Figure 31. GLC-PICI-MS of H F B - I . S . - a : (A) Total ion chromatogram ( re tent ion t ime, 13.11 min) and (B) PICI mass spectrum and se lec ted proposed fragmentation s t r u c t u r e s . I l l A 13.13 1200000 IOOOOOO-I 800000 16.0 B 595 213 C 3 F 7 194 494 -HF = 474 -3HF = 434 100 _ 80 >« >> £ 60 40 & 2 0 213 194 200 474 434 400 m/e 679 659 595 600 ( H - l ) ' M~* = 719 800 -1 = 718 -2HF = 679 -3HF = 659 -4HF = 639 Figure 32. GLC-NICI-MS of H F B - I . S . - a . : (A) Total ion chromatogram (retent ion t ime, 13.12 min); (B) a proposed fragmentation pattern and (C) NICI mass spectrum. 112 40000; 36000: 32000 : 28000: 24000: 20000-16000: 12000 : 8000: 4000: 0 5.0 18.3 7 . 7 1 7.0 9.0 11.0 13.0 15.0 100 508 ~ 80 C cu > cu 6 0 " 4 0 -20 -91 43 252 294 104 169 226 333 466 A 359 748 200 400 m/e 600 800 Figure 33. GLC-EI-MS of HFB-5-hydroxy PF: (A) Total ion chromatogram ( re tent ion t ime, 10.3 min) and (B) EI mass spectrum. 113 Figure 34. GLC-EI-MS: a proposed fragmentation pattern of HFB-5-hydroxy PF. 114 chromatogram, fragmentation pattern and MS spectrum of 5-hydroxy PF. In order of decreasing i n t e n s i t y , the prominent fragment ions were at m/e 508, 91, 43, 294, 252, 104, 169, 226, 466, 333, 359, 77, 748. The molecular ion (M +) at m/e 945 was not observed on the EI-MS spectrum. 3.4 Measurement o f Plasma Propafenone Concentrat ions by E lec t ron-Capture Detect ion G a s - L i q u i d Chromatography and High-Performance L i q u i d Chromatography Table 10 shows s teady-s ta te trough plasma PF concentra t ions in pa t i en ts r e c e i v i n g PF fo r contro l of card iac dysrhythmias. A lso contained in Table 10 are the r e s u l t s of a comparison of PF plasma concentra t ions analyzed by our GLC assay and by a modi f ica t ion of the HPLC method reported by Harapat and Kates [1982]. S t a t i s t i c a l ana lys is (paired t - t e s t , l eve l of s i g n i f i c a n c e = 0.05) ind ica tes that there is no s i g n i f i c a n t d i f f e r e n c e between the r e s u l t s obtained using these two independent methods of PF measurement. 3.5 In Vitro Serum Prote in Binding Study 3.5.1 E q u i l i b r i u m Time fo r Propafenone During Equ i l ib r ium D i a l y s i s The r e s u l t s o f the e q u i l i b r a t i o n study to e s t a b l i s h the time necessary to reach dynamic equ i l ib r ium in the d i a l y s i s chambers are presented in F igure 35. E q u i l i b r i u m , which is independent of PF c o n c e n t r a t i o n , was reached between 4 and 6 h. Therefore , 6 h was chosen as 115 T a b l e 10. S t e a d y - s t a t e plasma propafenone t r o u g h c o n c e n t r a t i o n o f p a t i e n t s r e c e i v i n g p r o p a f e n o n e . C o n c e n t r a t i o n measured (ng/mL) (mean + s.d.) P a t i e n t r — number GLC a HPLC D 1 175.6 2 696.7 3 737.5 4 5.3 5 190.8 6 554.3 7 217.9 8 131.4 9 179.4 10 476.7 11 356.4 12 373.6 13 303.3 14 444.9 15 612.9 16 633.8 17 487.3 18 28.7 19 119.2 20 1740.5 + 7.7 * + 6.1 738.2 + 53.4 + 7.7 820.2 ± 5.1 + 0.4 -+ 10.6 -+ 84.4 -+ 6.7 -+ 6.0 -+ 6.8 -+ 23.5 482.1 ± 1.5 + 1.7 -+ 8.7 385.0 + 2.3 + 2.2 328.7 + 10.2 + 2.1 365.6 + 77.3 + 7.2 608.8 + 78.1 + 17.5 719.9 + 12.9 + 35.8 604.5 + 5.8 + 0.4 -+ 6.6 -+ 31.9 1873.8 + 8.5 s.d. s t a n d a r d d e v i a t i o n a number o f samples, n = 2 (two i n j e c t i o n s f o r each sample) b number o f samples, n = 1 (two i n j e c t i o n s f o r each sample), The method i s a s l i g h t m o d i f i c a t i o n o f the HPLC t e c h n i q u e o f Harapat and K a t e s [1982]. * PF c o n c e n t r a t i o n unmeasurable 116 1 0 0 ® 0 1 2 3 4 . 5 6 Time, h Figure 35. Equi lbr ium time for equ i l ib r ium d i a l y s i s of propafenone. I n i t i a l propafenone concentrat ion was 0.25 ( O ) and 100 ( A ) pq/ml. 117 the optimal time to assure attainment of e q u i l i b r i u m . T h e o r e t i c a l l y , the approach to e q u i l i b r i u m i s f a s t e r when d i a l y z i n g buf fe r with serum samples (conta in ing drug already bound to serum p r o t e i n s ) . There fore , 6 h was used as the d i a l y s i s time when measuring the binding of PF in serum samples conta in ing PF. 3 .5 .2 Non-Spec i f i c Binding of Propafenone The extent of the n o n - s p e c i f i c adsorpt ion of PF to the equ i l ib r ium d i a l y s i s membrane, equ i l ib r ium d i a l y s i s c e l l and u l t r a f i l t r a t i o n device i s shown in Table 11 and the mean percentage of PF adsorbed was 2.3 + 1.7 %, 16.6 + 8.0 % and 16.2 + 7.2 %, r e s p e c t i v e l y . 3 .5 .3 Determination of Propafenone Binding Parameters - Rosenthal A n a l y s i s The binding r a t i o (bound concen t ra t ion / f ree concentrat ion) of PF in serum from s i x healthy subjects was p lo t ted versus the bound concentrat ion of PF by the method of Rosenthal [1967] (Figure 36) . Two c l a s s e s of b inding s i t e s were observed over the concentrat ion range s t u d i e d . The a f f i n i t y and capac i ty constants f o r each binding s i t e are a lso shown in Figure 36. The mean values f o r the h i g h - a f f i n i t y , low-capac i ty binding s i t e are Kj = 6.53 x 10 5 M" 1 and rijPj = 1.73 x 10" 4 M; f o r the l o w - a f f i n i t y , h igh -capac i ty s i t e are K 2 = 8.77 x 10 3 M" 1 and n 2 P 2 = 8.57 x 10" 3 M. 3 .5 .4 Scatchard Plot of the Binding Data The binding data from the same s ix healthy s u b j e c t s , p lo t ted as r' versus the r a t i o of r'/free drug concentrat ion (Scatchard p l o t ) , i s shown in F igure 37A and B. Serum albumin (M.W. -65,000) (Figure 37A) and AAG 118 Table 11. N o n - s p e c i f i c adsorpt ion of propafenone to the e q u i l i b r i u m d i a l y s i s membrane, d i a l y s i s c e l l and u l t r a f i l t r a t i o n d e v i c e . Concentrat ion of propafenone Og/mL) Percentage of propafenone adsorbed Equ i l ib r ium d i a l y s i s membrane Equ i l ib r ium d i a l y s i s c e l l U l t r a f i l t r a t i o n device 0.1 5.0 21.6 21.3 0.5 0.8 22.3 25.1 1.0 2.5 23.3 19.7 5.0 3.2 19.5 15.5 10.0 1.7 7.7 8.8 100.0 0.3 5.1 6.9 Mean ± s . d . 2.3 ± 1 . 7 16.6 ± 8 . 0 16.2 ± 7 . 2 s . d . standard dev ia t ion 119 c o o -*-> c CD O c o o <D c o c cu C J c o o X I c o CQ 100-1 80-60 o 40-. 20-a 0 Kj(M- l) r-iPi(M) K 2 (M- l) n 2 P 2 (M) Sub jec t MS ( A ) 2 08 x 10 6 8 51 x 10" 5 1 70 x 10 4 9. 76 x 10" 4 Sub jec t SC ( • ) 1 28 x 106 1 20 x 10" 4 1 80 x 10 4 1 54 x 10" 3 Subject MR (o) 6 60 x 10 4 4 60 x 10" 4 1 .00 x 10 2 4 32 x 10" •2 Subject BJ ( O ) 2 .64 x 105 6 .55 x 10 -5 1 .10 x 10 4 1 33 x 10 -3 Subject KW ( • ) 1 .15 x 105 1 .60 x 10 -4 3 .00 x 10 3 2 .32 x 10 -3 Subject BG ( • ) 1 .12 x 105 1 .50 x 10 -4 3 .50 x 10 3 2 .05 x 10 -3 8 a o S>i«A 0 2 4 6 Bound concentration, Mx10 8 10 12 14 16 18 5 Figure 36. Re la t ionsh ip between the r a t i o of bound c o n c e n t r a t i o n / f r e e concentra t ion and bound concentrat ion of propafenone in the serum of s i x healthy male subjects (Rosenthal p l o t ) . 0.25 0.3 B 10 12 14 Figure 37. Re la t ionsh ip between [ r ' /bound concentrat ion] and [ r ' ] of propafenone in the serum of s i x healthy male subjects (Scatchard p l o t ) ; serum concentrat ions o f (A) albumin and (B) AAG were used in the c a l c u l a t i o n of [ r ' ] . 121 (M.W. -40,000) (Figure 37B) concentrat ions were used in the c a l c u l a t i o n of r ' , r e s p e c t i v e l y . Two c lasses of binding s i t e s were apparent over the concentra t ion range s tud ied . 3 .5 .5 Propafenone Free Frac t ion in Normal and Pooled Uremic Serum In normal serum, the f ree f r a c t i o n of PF was 0.027 + 0.011 at a PF concentra t ion of 0.25 /zg/mL, 0.041 + 0.010 wi th in the therapeut ic concentra t ion range (0.5-2 /zg/mL), 0.138 + 0.012 at a PF concentrat ion of 25 /zg/mL and 0.187 + 0.005 when the PF concentrat ion was increased to 100 /zg/mL (Table 12). Regression of f ree f r a c t i o n over the e n t i r e concentra t ion range studied (0.25-100 /zg/mL) was found to be n o n - l i n e a r (p<0.05). When the concentrat ion range of 0.25 to 1.5 /zg/mL was fu r ther t e s t e d , the c o r r e l a t i o n between f ree f r a c t i o n and concentrat ion was found to be l i n e a r but the slope was not s i g n i f i c a n t l y d i f f e r e n t from zero (p>0.05), i n d i c a t i n g that PF free f r a c t i o n i s concentrat ion- independent w i th in t h i s range. The f ree f r a c t i o n of PF in pooled uremic serum (AAG concentra t ion = 124 mg/dL) was approximately 50% of the serum f ree f r a c t i o n seen in normal serum (AAG concentrat ion range = 53-117 mg/dL) at each PF concentra t ion studied (1-5 /zg/mL). 3 .5 .6 E f f e c t of Uremia and Renal F a i l u r e on the Serum Binding of Propafenone In pat ients with chronic renal f a i l u r e , serum AAG concentrat ion (140 + 39 mg/dL) was twice that in healthy subjects (71 + 24 mg/dL) and mid-range uremic pat ients (72 ± 2 mg/dL). The PF f ree f r a c t i o n ranged from 0.012 to 0.042, with a mean value of 0.024 + 0.012 (corresponding value in heal thy s u b j e c t s , 0.034 + 0.007; mid-range uremic p a t i e n t s , 0.037 + 0.008). 122 Table 12. Propafenone f ree f r a c t i o n in normal and pooled uremic serum. I n i t i a l concentrat ion of propafenone (/zg/mL) Propafenone f ree f r a c t i o n (mean + s . d . ) Normal serum 3 ^ Pooled uremic serum 0 0.25 0.027 + 0.011 * 0.50 0.039 + 0.013 -0.75 0.044 + 0.009 -1.0 0.034 + 0.007 0.016 + 0.001 1.5 0.041 + 0.008 0.026 + 0.001 2.0 0.049 + 0.010 0.029 ± 0.002 2.5 0.055 + 0.014 0.030 ± 0.001 5.0 0.064 + 0.011 0.034 + 0.001 10.0 0.089 + 0.020 -25.0 0.138 + 0.012 -50.0 0.161 + 0.006 -100.0 0.187 + 0.005 -s . d . standard dev ia t ion a mean propafenone f ree f r a c t i o n in serum from normal healthy vo lun teers , n = 6, AAG concentrat ion ranged from 53 to 117 mg/dL b propafenone f ree f r a c t i o n i s concentrat ion- independent wi th in the concentrat ion range of 0.25-1.5 /zg/mL, tested by ' r e g r e s s i o n with r e p l i c a t i o n ' on the l i n e a r i t y ( l i n e a r , p<0.05) and slope (slope not s i g n i f i c a n t l y d i f f e r e n t from 0, p>0.05) c propafenone f ree f r a c t i o n in pooled serum from nine uremic p a t i e n t s , AAG concentrat ion is 124 mg/dL * propafenone free f r a c t i o n not measured 123 3 .5 .7 C o r r e l a t i o n Between Serum a j - A c i d Glycoprote in Concentrat ion and Propafenone Binding Ratio The r e l a t i o n s h i p between serum AAG concentra t ion and the PF binding r a t i o i s shown in Figure 38. There i s a p o s i t i v e and s i g n i f i c a n t c o r r e l a t i o n (r = 0.830, n = 14, p<0.05) between these two parameters. The b e s t - f i t l i n e by l i n e a r regress ion a n a l y s i s is descr ibed by Y = 0.351X + 3.347. 3 .6 . Phenobarbital Treatment in Healthy Non-Smokers: Pharmacokinetics and Binding Studies of Propafenone and 5-Hydroxy Propafenone 3 . 6 . 1 . Serum Data of Propafenone The semi - logar i thmic p lo ts of the serum concentrat ion versus time curves of PF from the e ight healthy non-smoking subjects are shown in Appendix 2. The k i n e t i c data of PF in serum before and a f t e r phenobarbital treatment i s shown in Table 13. A l l non-smoking subjects were ' f a s t ' metabol i z e r s (CL^n^- >0.5 L/min) . Propafenone i s a compound with a high i n t r i n s i c c learance which e x h i b i t s substant ia l i n t e r i n d i v i d u a l v a r i a b i l i t y in t h i s parameter. In our non-smoking sub jec ts , C L ^ n t ranged from 0.8 to 13.3 L/min in the contro l s tate and 1.8 to 69.8 L/min a f t e r phenobarbital t reatment. There was a lso a large i n t e r i n d i v i d u a l v a r i a b i l i t y in other k i n e t i c parameters. For example, a 7 f o l d d i f f e r e n c e in C m a x (range 103-750 ng/mL) and a 16 f o l d d i f f e r e n c e in AUC (range 340.5-5465.4 h.ng/mL) were noted between subjects in the contro l s ta te while a 30 f o l d d i f f e r e n c e in C m a x (range 16-433 ng/mL) and a 39 f o l d d i f f e r e n c e in AUC (range 64.7-124 90-, Linear regression: 80-Y = 0.351 X + 3.347 r = 0.830 70-60-50-1—Q—i 40-T 30-1 A O zfr 20-10-0- i • i — — i • •• • • 60 80 100^  1^0 iTo^  160 180 200 0 20 40 Serum alpha—1—acid glycoprotein concentration, mg/dL Figure 38. Re la t ionship between propafenone binding r a t i o (bound concent ra t ion / f ree concentrat ion) and AAG concentrat ion in serum obtained from s ix healthy subjects ( O ), three mid-range uremic pat ients ( A ) and f i v e pat ients with chronic renal f a i l u r e ( • ) . The data are presented as mean + l s . d . 125 Table 13. The k i n e t i c data of propafenone in serum before and a f t e r phenobarbital treatment from eight healthy non-smoking s u b j e c t s . hp ^max ^max C L i n t AUC ? i increase % decrease in Subject (h) (ng/mL) (h) (L/min) (h.ng/mL) i n C L i n t r max AUC BK C 3.4 194 4 3.7 1227.4 62 28 38 P 3.7 140 5 6.0 756.4 NP C 2.5 217 1.5 4.9 923.7 176 58 64 P 1.9 92 1.5 13.5 333.7 CA C 1.4 177 3 6.2 725.6 176 63 64 P 1.4 66 4 17.1 264.1 DA C 2.1 124 3 7.5 602.7 831 87 89 P 2.3 16 3 69.8 64.7 SG C 4.5 129 2 8.3 542.8 10 26 9 P 2.8 96 3 9.1 496.5 MV C 4.4 750 3 0.8 5465.4 125 42 54 P 4.7 433 3 1.8 2499.5 GP C 1.6 328 2 4.3 1052.4 153 53 61 P 1.7 153 2 10.9 414.0 UH C 2.6 103 1.5 13.3 340.5 22 0 18 p 1.3 103 1.5 16.2 278.2 Mean c 2.8 253 2.5 6.1 1360.1 194 45 50 ± s . d . ± 1 . 2 ±221 ± 0 . 9 ± 3 . 7 ± 1 6 8 3 . 5 ±266 ±27 ±26 p 2.5 137 S 2.9 18 .1 S 638.4 S ± 1 . 2 ±127 ± 1 . 2 ± 2 1 . 5 ± 7 7 8 . 3 C cont ro l (untreated) P phenobarb i ta l - t rea ted S Wilcoxon paired-sample t e s t , compared to the c o n t r o l , s i g n i f i c a n t , p<0.05 s . d . standard dev ia t ion 126 2499.5 h.ng/mL) were observed a f te r phenobarbital treatment. Phenobarbital treatment enhanced the f i r s t - p a s s metabolism of PF, as shown by a s i g n i f i c a n t increase in C L ^ n + c and a s i g n i f i c a n t decrease in C m a x and AUC. The absolute increase in C L ^ n t w i th in the i n d i v i d u a l subject ranged from 10 to 831%, with a mean of 194%. The absolute decrease in AUC ranged from 9 to 89%, with a mean of 50% while the decrease in C m a x ranged from 0 to 87%, with a mean of 45%. In a d d i t i o n , the percent decrease in C m a x was not s i g n i f i c a n t l y d i f f e r e n t from the percent decrease in AUC. The t^p of PF ranged from 1.4 to 4.5 h (mean 2.8 h) in the contro l s t a t e . No s i g n i f i c a n t change in the t ^ was observed a f t e r phenobarbital treatment (range 1.3-4.7 h, mean 2.5 h) The t m a x of PF ranged from 1.5 to 4 h (mean 2.5 h) in the contro l s t a t e . Phenobarbital treatment d id not a f f e c t the t m a x (range 1.5-5 h, mean 2.9 h ) . Table 14 shows the values of V c | a r e a / F and V d s s / F of the non-smokers before and a f te r phenobarbital treatment. In the cont ro l s t a t e , V d a r e a / F ranged from 3 to 38.4 L/kg and V d s s / F ranged from 3.5 to 45.7 L /kg . Except f o r subjects SG and UH, there was a 2 to 10 f o l d increase in both V d a r e a / F and V d s s / F a f t e r phenobarbital treatment. The increase in V d s s / F was s i g n i f i c a n t while the increase in V d a r e a / F was not . The values of V d s s / F were, in g e n e r a l , higher than V d a r e a / F , the d i f f e r e n c e being s t a t i s t i c a l l y s i g n i f i c a n t . 3 . 6 . 2 . S a l i v a r y Data of Propafenone The s a l i v a r y concentrat ion versus time curves of PF from the eight non-smoking subjects are shown in Appendix 3. Most subjects showed s i g n i f i c a n t f l u c t u a t i o n s in s a l i v a r y PF concentrat ions over t ime. However, 127 Table 14. The volume of d i s t r i b u t i o n of propafenone before and a f t e r phenobarbital treatment from e ight healthy non-smoking s u b j e c t s . Subject V d a r e a / F 0 - / ^ * V d s s / F ( L / k g ) * BK C 10.7 15.8 • P 19.1 30.0 NP C 12.9 16.8 P 31.5 38.5 CA C 9.3 14.0 P 25.6 52.6 DA C 18.5 22.2 P 190.0 257.7 SG C 30.6 32.9 P 21.0 30.7 MV C 3.0 3.5 P 7.5 7.6 GP C 5.9 8.8 P 15.1 26.5 UH C 38.4 45.7 P 28.6 42.2 Mean + s . d . C 16.2 ± 12.4 20.0 ± 13.6 P 42.3 ± 60.2 60.7 ± 8 0 . 7 S C contro l (untreated) P phenobarb i ta l - t rea ted * normalized to the i n d i v i d u a l ' s body weight S Wilcoxon paired-sample t e s t , compared to the c o n t r o l , s i g n i f i c a n t , p<0.05 s . d . standard dev ia t ion 128 the reduct ion in s a l i v a r y PF concentrat ions caused by phenobarbital mirrored the changes noted in serum PF concent ra t ions . The k i n e t i c data of PF in s a l i v a before and a f t e r phenobarbital treatment i s shown in Table 15. The sa l i va /serum PF concentra t ion r a t i o s were 0.21 + 0.08 in the contro l state and 0.27 + 0.11 a f t e r phenobarbital t reatment, with la rge i n t e r - and i n t r a i n d i v i d u a l v a r i a t i o n . In two s u b j e c t s , the sa l i va /serum PF concentrat ion r a t i o ranged from 0.03 to 0.17 (subject MV, cont ro l ) and 0.05 to 1.19 (subject UH, p h e n o b a r b i t a l - t r e a t e d ) . The s a l i v a / s e r u m PF concentrat ion r a t i o s were i n v e r s e l y c o r r e l a t e d with the pH o f s a l i v a both in the control s tate (r = -0 .461, n = 72, p<0.05) and a f t e r phenobarbital treatment (r = -0.422, n = 53, p<0.05). The best f i t through the data points was Y = 1.916 - 0.244X (cont ro l ) and Y = 2.473 -0.323X (phenobarb i ta l - t rea ted ) . The s a l i v a r y AUCQ of PF in the contro l s ta te ranged from 70.7 to 391.7 h.ng/mL. There was a s i g n i f i c a n t decrease in AUCQ a f t e r phenobarbital treatment. The absolute reduct ion in AUCQ a f t e r phenobarbital treatment ranged from 1 to 82%, with a mean of 44%. 3 . 6 . 3 . Serum Data of 5-Hydroxy Propafenone The serum concentrat ion versus time curves of 5-hydroxy PF from the e ight non-smoking subjects are shown in Appendix 4. Serum concentrat ions of 5-hydroxy PF were lower than the serum concentrat ions of PF. The k i n e t i c data of 5-hydroxy PF in serum is shown in Table 16. There was a lso a large i n t e r i n d i v i d u a l v a r i a t i o n in the k i n e t i c parameters of the metabo l i te . For example, a 3 f o l d d i f f e r e n c e in C M A X (range 80-245 ng/mL) and AUC (range 412.2-1359.2 h.ng/mL) between subjects was observed in the cont ro l s tate while a 9 f o l d d i f f e r e n c e in C M A X (range 19-177 ng/mL) and an 11 f o l d d i f f e r e n c e in AUC (range 78.3-878.0 h.ng/mL) was noted a f te r 129 Table 15. The k i n e t i c data of propafenone in s a l i v a before and a f t e r phenobarbi ta l treatment from eight healthy non-smoking s u b j e c t s . sa l iva /serum PF t t Subject concentrat ion r a t i o AUCQ (h.ng/mL) % decrease in AUC 0 BK C 0.29 ± 0.09 319.1 54 P 0.23 ± 0.09 147.5 NP C 0.14 ± 0.07 130.1 55 P 0.21 ± 0.13 58.8 CA C 0.28 ± 0.15 170.9 66 P 0.25 ± 0.12 58.7 DA C 0.30 ± 0.15 127.4 82 P 0.44 ± 0.18 23.5 SG C 0.22 ± 0.13 178.8 1 P 0.39 ± 0.32 177.7 MV C 0.09 ± 0.05 391.7 44 P 0.10 ± 0.05 220.0 GP C 0.14 ± 0.09 133.2 44 P 0.22 ± 0.14 74.8 UH C 0.25 ± 0.16 70.7 9 P 0.33 ± 0.38 64.2 Mean c 0.21 190.2 44 ± s . d . +0.08 ± 1 0 8 . 8 ±27 p 0.27 103.2 s ± 0 . 1 1 ± 6 9 . 5 C cont ro l (untreated) P phenobarbi t a l - t r e a t e d s Wilcoxon paired-sample t e s t , compared to the c o n t r o l , s ign i f i c a n t , p<0.05 s . d . standard dev ia t ion 130 Table 16. The k i n e t i c data of 5-hydroxy propafenone in serum before and a f t e r phenobarbital treatment from eight healthy non-smoking s u b j e c t s . H/3 r ^max ^max AUC % decrease % decrease Subject (h) (ng/mL) (h) (h.ng/mL) i n Cmax in AUC BK C 3.1 118 5 826.5 36 49 P 2.4 75 5 425.1 NP C 3.4 130 2 449.3 28 37 P 3.2 94 1.5 285.2 CA C 1.7 105 3 482.8 17 24 P 2.3 87 3 366.9 DA C 3.8 80 1.5 412.2 76 81 P 1.9 19 3 78.3 SG C 4.1 91 2 413.4 59 35 P 4.8 37 3 267.0 MV C 4.8 126 5 1359.2 20 35 P 4.3 101 3 878.0 GP C 2.0 245 2 756.6 60 61 P 2.6 98 2 294.8 UH C 1.9 231 1.5 780.8 23 36 P 1.9 177 1.5 501.8 Mean C 3.1 141 2.8 685.1 40 45 ± s . d . ± 1 . 1 ±62 ± 1 . 5 ± 3 2 3 . 7 ±22 ±18 P 2.9 86 s 2.8 387 .1 S ± 1 . 1 ±47 ± 1 . 1 ± 2 3 4 . 4 C cont ro l (untreated) P phenobarb i ta l - t rea ted S Wilcoxon paired-sample t e s t , compared to the c o n t r o l , s i g n i f i c a n t , p<0.05 s . d . standard dev ia t ion 131 phenobarbital treatment. There was a s i g n i f i c a n t decrease in both C m a x and AUC a f t e r phenobarbital treatment. The absolute decrease in C m a x ranged from 17 to 76%, with a mean of 40%, while the decrease in AUC ranged from 24 to 81%, with a mean of 45%. The t^g ranged from 1.7 to 4.8 h (mean 3.1 h) in the contro l s t a t e . No s i g n i f i c a n t change in the t^g was observed a f t e r phenobarbital treatment (range 1.9-4.8 h, mean 2.9 h ) . The t m a x of 5-hydroxy PF ranged from 1.5 to 5 h (mean 2.8 h) in the cont ro l s t a t e . Phenobarbital treatment d id not change the t m a x (range 1.5-5 h, mean 2.8 h ) . 3 . 6 . 4 . Prote in Binding and Serum a j - A c i d Glycoprote in Concentrat ion The f ree f r a c t i o n of PF (2 and 4 h samples) was 0.027 + 0.011 in the contro l s ta te and 0.038 + 0.032 a f t e r phenobarbital treatment. Serum AAG concentra t ions (0 h samples) in the non-smokers ranged from 69.3 to 108.0 mg/dL (mean 81.1 mg/dL) in the control s ta te and 61.5 to 103.0 mg/dL (mean 77.7 mg/dL) a f t e r phenobarbital treatment. Twenty-three days of phenobarbital treatment d id not cause any s i g n i f i c a n t change in e i t h e r PF free f r a c t i o n (Figure 39) or serum AAG concentrat ion (Figure 40). Table 17 shows the sa l iva /serum PF concentrat ion r a t i o s and s a l i v a r y pH of the 2 and 4 h samples and allows comparison of the values of percent of f ree (unbound) PF in these samples obtained by two d i f f e r e n t methods. The percent of f ree PF in serum, c a l c u l a t e d from the data of PF serum/sa l i va concentrat ion r a t i o , pH of s a l i v a and pK a of PF using Equation 7, was s i g n i f i c a n t l y higher than the values obtained from e q u i l i b r i u m d i a l y s i s . 132 0.15 -i Figure 39. The free f r a c t i o n of propafenone [2 ( O ) and 4 ( A ) h samples] before and a f te r 23 days of phenobarbital treatment in e ight healthy non-smokers. 133 Figure 40. The serum AAG concentrat ion (0 h sample) before and a f t e r 23 days of phenobarbital treatment in e ight healthy non-smokers. 134 Table 17. The sa l i va /se rum concentrat ion r a t i o of propafenone, pH of s a l i v a and percent of f ree PF from eight healthy non-smoking s u b j e c t s . Subject Sampling time (h) Propafenone concentrat ion r a t i o (sa l iva /serum) pH of sal i va Serum pro te in binding (% f ree) observed 3 c a l c u l a t e d ' 3 BK C 2 0.22 6.80 3.8 5.7 4 0.30 6.95 3.0 10.9 P 2 0.17 7.00 2.2 7.0 4 0.27 6.93 1.4 9.1 NP C 2 0.12 7.15 1.9 6.8 4 0.22 7.00 3.0 8.9 P 2 0.14 7.00 3.8 5.7 4 0.26 6.92 2.3 8.8 CA C 2 0.20 7.00 1.1 8.1 4 0.20 7.01 0.9 8.3 P 2 _* 7.07 - -4 0.21 6.97 2.0 7.9 DA C 2 0.28 6.70 2.0 5.7 4 0.23 6.82 2.1 6.2 P 2 0.28 6.63 - 4.9 4 0.33 6.55 - 4.8 SG C 2 0.16 7.05 2.7 7.2 4 0.15 6.92 2.0 5.0 P 2 0.10 7.08 2.7 4.8 4 0.48 6.85 2.4 13.8 MV C 2 0.07 7.32 5.1 5.8 4 0.08 7.22 3.9 5.3 P 2 0.08 7.05 12.6 3.6 4 0.08 7.50 8.5 10.0 GP C 2 0.14 6.94 3.1 4.9 4 0.12 7.35 2.1 10.7 P 2 0.08 6.97 3.6 3.0 4 0.27 6.75 1.8 6.2 UH C 2 0.12 6.87 3.9 3.6 4 0.38 6.83 3.2 10.4 P 2 0.14 6.79 3.2 3.5 4 0.45 6.74 3.0 10.0 C cont ro l (untreated) P phenobarb i ta l - t rea ted a obtained from e q u i l i b r i u m d i a l y s i s , n=3 b c a l c u l a t e d using Equation 7 Wilcoxon paired-sample t e s t , compared to the observed va lues , s i g n i f i c a n t , p<0.05 * values unava i lab le 135 3 . 6 . 5 . Re la t ionsh ip Between Serum T o t a l , Serum Free and S a l i v a r y Propafenone Concentrat ions Figure 41 shows the r e l a t i o n s h i p between PF serum to ta l c o n c e n t r a t i o n , serum free concentrat ion and s a l i v a r y concentrat ion of the 2 and 4 h samples (control and phenobarbi ta l - t reated) in the non-smoking s u b j e c t s . The l i n e a r regress ion and c o r r e l a t i o n s are descr ibed as fo l lows: PF serum to ta l concentra t ion and PF s a l i v a r y concent ra t ion , Y = 0.060X + 13.656, r = 0.702 (n = 31, p<0.05); PF serum to ta l concentrat ion and PF serum f ree concen t ra t ion , Y = 0.055X - 2.309, r = 0.838-(n = 29, p<0.05); PF s a l i v a r y concentra t ion and PF serum free concent ra t ion , Y = 0.353X -1.525, r = 0.435 (n = 29, p<0.05). 3 . 6 . 6 . Ur inary Data The data f o r a representa t ive c a l i b r a t i o n curve used in the q u a n t i t a t i o n of the g lucuronide and s u l f a t e conjugates of 5-hydroxy PF and 5-hydroxy-4-methoxy PF in ur ine samples are presented in Table 18. A representa t ive chromatogram i s shown in Figure 42. The renal excre t ion (cumulat ive, 0-48 h) of the glucuronide and s u l f a t e conjugates of 5-hydroxy PF and 5-hydroxy-4-methoxy PF, expressed as percent of the dose, i s shown in Table 19. A l l subjects showed a decrease in the renal excre t ion of the 5-hydroxy PF conjugates a f t e r phenobarbital t reatment. The renal excret ion of 5-hydroxy PF conjugates was 13.9% of the dose in the contro l s ta te and 10.6% of the dose a f t e r phenobarbital t reatment. Three subjects (BK, CA and UH) d id not show a change while the others showed a minor reduct ion in the renal excret ion of the 5-hydroxy-4-methoxy PF conjugates a f t e r phenobarbital treatment. The renal excre t ion of 136 0 100 200 300 400 500 600 700 800 PF serum total concentration, ng/mL Figure 41. The c o r r e l a t i o n between (A) PF serum to ta l concentra t ion and s a l i v a r y concentrat ion ( O ); (B) PF serum to ta l concentra t ion and serum free concentrat ion ( • ) and (C) PF s a l i v a r y concentrat ion and serum free concentrat ion ( A ) in e ight healthy non-smokers. 137 Table 18. C a l i b r a t i o n curve data of 5-hydroxy propafenone and 5-hydroxy-4-methoxy propafenone f o r ur ine samples. Number of samples, n = 2 (two i n j e c t i o n s fo r each sample) Concentrat ion Peak area r a t i o C o e f f i c i e n t of of metabol i te m e t a b o l i t e / I . S . - b v a r i a t i o n (Mg/mL) (mean + s . d . ) (%) 5-Hydroxy propafenone 1.02 0.165 + 0.012 7.3 2.03 0.323 + 0.009 2.8 4.06 0.625 + 0.024 3.8 6.10 0.957 + 0.019 2.0 8.13 1.286 + 0.025 1.9 10.16 1.627 + 0.058 3.6 S t a t i s t i c s : l i n e a r r e g r e s s i o n , Y = 0.160X - 0.007; r = 0.999 5-Hydroxy-4-methoxy propafenone 1.02 0.129 + 0.007 5.4 2.05 0.299 + 0.027 9.0 4.09 0.622 + 0.033 5.3 6.14 0.959 + 0.068 7.1 8.19 1.399 + 0.069 4.9 10.24 1.738 + 0.110 6.3 S t a t i s t i c s : l i n e a r r e g r e s s i o n , Y = 0.176X - 0.072; r = 0.999 s . d . standard dev ia t ion C V . c o e f f i c i e n t of v a r i a t i o n 138 20 n 15 10 i nil i 6 8 Time (min) 10 12 14 20 15H 10 04 ID cn A i — 1 — 1 — 1 — i — 1 — • — 1 — i — • — 1 — ' — i — • — ' — • — i — • — • — • — i — • — 1 — 1 — i — • — • 1 i 0 2 4 6 8 10 12 14 Time (min) F igure 42. Chromatograms of ex t ract from (A) blank ur ine spiked with 5-hydroxy propafenone (6 tig), 5-hydroxy-4-methoxy propafenone (6 ng), propafenone (6 ng) and I .S . -b (7 fig) and (B) a ur ine sample (spiked with I . S . - b , 7 /xg) from a subject r e c e i v i n g propafenone. 139 Table 19. Renal excre t ion of 5-hydroxy propafenone and 5-hydroxy-4-methoxy propafenone conjugates (cumulative) before and a f t e r phenobarbital treatment from eight healthy non-smoking s u b j e c t s . Renal e x c r e t i o n , % of dose Control (untreated) Phenobarb i ta l - t rea ted Subject 0-12h 12-24h 24-48h Total 0-12h 12-24h 24-48h Total 5-Hydroxy PF conjugates BK 11.1 1.8 0.9 13.8 7.4 1.2 1.4 10.0 NP 11.5 0.6 1.2 13.3 10.1 0.4 0.9 11.4 CA 13.5 1.3 0.7 15.5 10.9 1.4 0.8 13.1 DA 11.0 1.0 0.6 12.6 6.0 0.8 0.4 7.2 SG 11.3 0.7 0.4 12.4 10.0 0.6 0.3 10.9 MV 12.9 5.0 2.1 20.0 10.6 2.4 0.3 13.3 GP 9.6 1.0 0.5 11.1 8.0 0.7 0.6 9.3 UH 12.1 0.2 0.2 12.5 9.2 0.1 0.1 9.4 Mean 13.9 10.6 ± s . d . +2.8 +2.0 5-hydroxy-4-methoxy PF conjugates BK 2.1 1.0 0.7 3.8 1.7 0.8 1.3 3.9 NP 3.4 0.4 1.0 4.8 2.8 0.4 0.8 4.0 CA 3.5 1.2 0.6 5.3 3.2 1.3 0.8 5.3 DA 3.3 0.9 0.4 4.5 2.2 0.8 0.3 3.3 SG 3.6 0.7 0.3 4.6 2.5 0.4 0.2 3.1 MV _* 1.2 0.4 1.6 - 1.3 0.2 1.5 GP 2.4 0.9 0.4 3.7 1.8 0.7 0.7 3.2 UH 3.9 0.1 0.1 4.1 3.8 0.2 0.1 4.1 Mean 4.1 3.6 ± s . d . +1.1 +1.1 s . d . standard dev ia t ion * 5-hydroxy-4-methoxy undetected 140 5-hydroxy-4-methoxy PF conjugates was 4.1% of the dose in the contro l s tate and 3.6% of the dose a f t e r phenobarbital treatment. 3 .7 . Phenobarbital Treatment in Healthy Smokers: Pharmacokinetics and Binding Studies of Propafenone and 5-Hydroxy Propafenone 3 . 7 . 1 . Serum Data of Propafenone The semi - logar i thmic p lo ts of serum concentrat ion versus time curves of PF from the e ight healthy smoking subjects are shown in Appendix 5. The k i n e t i c data of PF in serum before and a f t e r phenobarbital treatment is shown in Table 20. Like the observat ions noted in the non-smoking s u b j e c t s , there was a large i n t e r i n d i v i d u a l v a r i a b i l i t y in a l l k i n e t i c parameters among smokers. For example, £ljnt in the contro l state ranged from 0.4 to 20.7 L/min, with two of the e ight smoking subjects (GE and MG) being ' s low ' metabol i z e r s (CL-jnj. < 0.5 L /min) . Excluding the data of the two 's low ' metabo l i zers , there was s t i l l a la rge i n t e r i n d i v i d u a l v a r i a t i o n in t h i s parameter, ranging from 8.0 to 20.7 L/min. Besides a smal ler C L ^ , the two 's low ' metabol i z e r s had a longer t ^ , a higher C m a x and a l a r g e r AUC when compared to the ' r a p i d ' metabo l i zers . The mean t^j of the two ' s low ' metabol i z e r s (11.5 h) was 4 times longer than that of the ' r a p i d ' metabol i z e r s (2.7 h ) . The mean C m a x of the two 's low ' metabol i z e r s (635 ng/mL) was 6 times higher than that of the ' r a p i d ' metabol izers (107 ng/mL) while the mean AUC of the two ' s low ' metabol izers (9975 h.ng/mL) was 25 times l a r g e r than that of the ' r a p i d ' metabol izers (405 h.ng/mL). Other k i n e t i c parameters a lso showed wide in te rs ub je c t v a r i a t i o n , even a f t e r the data of the two 's low ' metabol izers were 141 Table 20. The k i n e t i c data of propafenone in serum before and a f t e r phenobarbital treatment from e ight healthy smoking s u b j e c t s . Subject (h) r ^max (ng/mL) ^max (h) CLint (L/min) AUC "/ (h.ng/mL) 'o increase i n C L i n t % decrease in cmax A U C JL C P 3.2 3.3 83 58 1 1.5 13.3 23.9 338.6 188.8 80 30 44 MA C P 2.2 2.4 222 139 3 1.5 6.4 7.9 705.8 570.9 23 37 19 TN C P 1.9 2.3 51 41 4 1.5 20.7 34.9 218.6 129.5 69 20 41 DW C P 4.5 2.5 125 19 3 3 8.0 44.0 567.3 102.6 450 85 82 GE C P 11.3 9.5 711 651 3 4 0.4 0.5 11513.1 8932.3 25 8 22 MG C P 11.6 12.6 559 280 3 4 0.5 1.0 8435.9 4549.3 100 50 46 SR C P 2.0 1.9 64 27 1 1.5 18.2 44.0 248.2 102.6 142 58 59 DB C P 2.6 1.8 99 20 2 3 12.8 58.3 353.8 77.5 355 80 78 Mean ± s . d . C 4.9 ± 4 . 1 239 ±253 2.5 ± 1 . 1 10.0 ± 7 . 6 2797.7 ± 4 5 0 8 . 2 156 ±159 46 ±28 49 ±23 P 4.5 ± 4 . 1 154 S ±220 2.5 ± 1 . 1 26 .8 S ± 2 1 . 9 1831.7 S ± 3 2 5 2 . 4 Mean ±s . d . C 2.7 ± 1 . 0 107 ±62 2.3 ± 1 . 2 13.2 ± 5 . 6 405.4 ± 1 9 1 . 4 187 ±174 52 ±27 54 ±24 P 2.4 ± 0 . 5 51 S ±46 2.0 ± 0 . 8 3 5 . 5 S ± 1 7 . 7 195.3 S ± 1 8 7 . 9 C contro l (untreated) P phenobarb i ta l - t rea ted S Wilcoxon paired-samplet t e s t , compared to the c o n t r o l , s i g n i f i c a n t , p<0.05 * mean and s . d . of the k i n e t i c parameters excluding the data of the two ' s low ' metabol izers (subject GE and MG) s . d . standard dev ia t ion 142 excluded. For example, a 4 f o l d d i f f e r e n c e in C m a x (range 51-222 ng/mL) and a 3 f o l d d i f f e r e n c e in AUC (range 218.6-705.8 h.ng/mL) were noted in the cont ro l s ta te whi le a 7 f o l d d i f f e r e n c e in C m a x (range 19-139 ng/mL) and AUC (range 77.5-570.9 h.ng/mL) were observed a f te r phenobarbital treatment. There was a s i g n i f i c a n t increase in the apparent CL^ n 1 - of smokers and a s i g n i f i c a n t decrease in C m a x and AUC a f te r phenobarbital treatment, inc lud ing or excluding the data of the two 's low' metabo l i ze rs . The absolute increase in C L ^ n t ranged from 23 to 450%, with a mean of 156% (or a mean of 187% excluding the ' s low ' metabo l i ze rs ) . The absolute decrease in C m a x ranged from 8 to 85%, with a mean of 46% (or a mean of 52% excluding the ' s low ' metabol izers) while the decrease in AUC ranged from 19 to 82%, with a mean of 49% (or 54% excluding the 's low ' metabo l i ze rs ) . In a d d i t i o n , the percent decrease in C m a x was a lso not s i g n i f i c a n t l y d i f f e r e n t from the percent decrease in AUC in smokers. The t^p of the smokers in the control s tate ranged from 1.9 to 11.6 h. This large i n t e r s u b j e c t v a r i a t i o n was due to the longer t^g of the two ' s low ' metabol izers (when excluding the 's low' metabol i zers , t^p ranged from 1.9 to 4.5 h ) . No s i g n i f i c a n t change in the t^p was observed a f te r phenobarbital treatment (which ranged from 1.8 to 12.6 h or from 1.8 to 3.3 h excluding the ' s low ' metabo l i ze rs ) . The t m a x of PF in the contro l state ranged from 1 to 4 h. Phenobarbital treatment d id not change the t m a x (which ranged from 1.5 to 4 h or from 1.5 to 3 h when excluding the 's low' metabol i z e r s ) , inc lud ing or exc luding the data of the two 's low ' metabo l i zers . Table 21 shows the values of V d a r e a / F and V d s s / F of the smokers before and a f te r phenobarbital treatment. In the contro l s t a t e , V d a r e a / F 143 Table 21. The volume of d i s t r i b u t i o n of propafenone before and a f t e r phenobarbital treatment from e ight healthy smoking s u b j e c t s . Subject V d a r e a / F ( L / k 9 > * V d s s / F ( L / k g ) * JL C 33.6 40.3 P 80.9 130.1 MA C 15.1 20.3 P 20.8 22.7 TN c 53.8 88.0 p 109.4 86.3 DW c 30.7 34.9 p 120.7 162.1 GE c 4.5 4.9 p 4.9 5.2 MG c 5.9 6.3 p 11.7 11.7 SR c 41.9 44.8 p 94.8 105.8 DB c 34.5 41.8 p 123.5 212.1 Mean + s . d . c 27.5 ± 17.5 35.2 ± 26.5 p 70.8 ± 50 .4 S 92.0 ± 75 .4 S ieic Mean + s . d . c 34.9 + 12.8 45.0 + 22.8 p 91.7 ± 3 8 . 3 S 119.9 ± 6 5 . 1 S C contro l (untreated) P phenobarb i ta l - t rea ted S Wilcoxon paired-sample t e s t , compared to the c o n t r o l , s i g n i f i c a n t , p<0.05 * normalized to the i n d i v i d u a l ' s body weight * * mean and s . d . of the k i n e t i c parameters excluding the data of the two 's low ' metabol izers (subjects GE and MG) s . d . standard dev ia t ion 144 ranged from 4.5 to 53.8 L/kg (or 15.1 to 53.8 L/kg excluding the ' s low ' metabol izers) and V d s s / F ranged from 4.9 to 88.0 L/kg (or 20.3 to 88.0 L/kg excluding the ' s low ' metabo l i ze rs ) . There was a 1 to 5 f o l d increase in both V ( j a r e a / F and V Q . S S / F a f te r phenobarbital treatment, the d i f f e r e n c e being s t a t i s t i c a l l y s i g n i f i c a n t . The values of V ^ g / F were not s i g n i f i c a n t l y d i f f e r e n t from the values of V ( j a r e a / F in smokers. Although the i n t e r i n d i v i d u a l v a r i a t i o n s in C m a x , C L ^ n t and AUC were reduced s u b s t a n t i a l l y when the data from the two ' s low ' metabol izers were excluded, there was s t i l l a wide range in the percent increase in CL^ n^. and the percent decrease in C m a x and AUC. 3 . 7 . 2 . S a l i v a r y Data of Propafenone The s a l i v a r y concentrat ion versus time curves of PF obtained before and a f t e r phenobarbital treatment from the e ight smoking subjects are shown in Appendix 6. L ike the non-smokers, the smokers a lso showed f l u c t u a t i o n s in s a l i v a r y PF concent ra t ions , yet the PF s a l i v a r y concentrat ions were reduced a f t e r phenobarbital treatment. The k i n e t i c data of PF in s a l i v a before and a f t e r phenobarbital treatment i s shown in Table 22. The sa l iva /serum PF concentra t ion r a t i o s were 0.36 + 0.20 (or 0.33 + 0.22 excluding the two ' s low ' metabol izers) in the contro l s tate and 0.40 + 0.15 (or 0.40 + 0.18 excluding the two 's low' metabol izers) a f t e r phenobarbital treatment, with la rge i n t e r - and i n t r a i n d i v i d u a l v a r i a t i o n . In two sub jec ts , the sa l i va /se rum PF concentra t ion r a t i o ranged from 0.04 to 0.18 (subject MA, cont ro l ) and 0.06 to 1.06 (subject DB, phenobarb i ta l - t rea ted ) . The sa l i va /se rum PF concentra t ion r a t i o s were inverse ly cor re la ted with the pH of s a l i v a in both the contro l s tate (r = -0 .541, n = 80, p<0.05) and a f te r phenobarbital 145 Table 22. The k i n e t i c data of propafenone in s a l i v a before and a f t e r phenobarbital treatment from e ight healthy smoking s u b j e c t s . sa l i va /serum PF t t Subject concentra t ion r a t i o AUCQ (h.ng/mL) % decrease in AUCQ JL C 0.08 + 0.09 16.9 P 0.21 ± 0.25 24.8 TN C 0.37 + 0.09 72.4 30 P 0.58 + 0.36 50.9 MA C 0.10 ± 0.05 64.5 -P 0.26 + 0.13 156.7 DW C 0.66 + 0.33 353.0 81 P 0.63 + 0.31 67.7 GE C 0.53 + 0.20 6588.2 34 P 0.43 ± 0.19 4378.1 MG C 0.36 + 0.14 3199.3 35 P 0.40 ± 0.25 2093.3 SR C 0.35 + 0.16 82.5 69 P 0.25 ± 0.15 25.5 DB C 0.44 + 0.22 118.3 77 P 0.44 + 0.38 27.2 Mean C 0.36 1311.9 54 + s . d . ± 0 . 2 0 ± 2 3 9 1 . 3 ±24 P 0.40 853.0 ± 0 . 1 5 ± 1 5 9 2 . 9 Mean C 0.33 117.9 64 + s . d . ± 0 . 2 2 ± 1 1 9 . 7 ±23 P 0.40 58.8 +0.18 +51.0 C contro l (untreated) P phenobarb i ta l - t rea ted * subjects (JL and MA) who d id not show a decrease in s a l i v a r y AUC a f t e r phenobarbital treatment, t h e i r data excluding from the c a l c u l a t i o n of the mean % decrease in AUC * * mean and s . d . of the k i n e t i c parameters excluding the data of the two 's low ' metabol izers (subjects GE and MG) s . d . standard dev ia t ion 146 treatment (r = -0 .387, n = 72, p<0.05). The best f i t through the data po ints are Y = 7.000 - 0.630X (contro l ) and Y = 6.868 - 0.464X ( p h e n o b a r b i t a l - t r e a t e d ) . The two 's low' metabol izers a lso had a higher t t s a l i v a r y AUCQ compared to the ' r a p i d ' metabol izers . The mean AUC 0 of the two ' s l o w ' metabol izers (4894 h.ng/mL) was 41 times higher than that of the ' r a p i d ' metabol izers (118 h.ng/mL). Subjects JL and MA showed an increase in s a l i v a r y AUCQ a f te r phenobarbital treatment. Although the change in AUCQ was not s t a t i s t i c a l l y s i g n i f i c a n t , the absolute decrease in AUCQ (excluding subjects JL and MA) ranged from 30 to 81%, with a mean of 54% (or 64% exc luding the two 's low' metabo l i ze rs ) . 3 . 7 . 3 . Serum Data of 5-Hydroxy Propafenone The serum concentra t ion versus time curves of 5-hydroxy PF from the e ight smoking subjects are shown in Appendix 14-15. The serum concentra t ions o f 5-hydroxy PF were a lso lower than the serum concentra t ions of PF in smokers. The k i n e t i c data of 5-hydroxy PF in serum is shown in Table 23. The metabo l i te , 5-hydroxy PF, was quant i tated in 6 smokers. Subject GE and MG had undetectable l e v e l s of 5-hydroxy PF in t h e i r serum, which i s a unique c h a r a c t e r i s t i c of ' s low ' metabol izers . Even a f te r phenobarbital treatment, these two subjects s t i l l had undetectable quant i t i es of serum 5-hydroxy PF. There was a lso a la rge in te rsub jec t v a r i a t i o n in the k i n e t i c parameters of 5-hydroxy PF. For example, a two f o l d d i f f e r e n c e in both C M A X (range 77-165 ng/mL, mean 122 ng/mL) and AUC (range 310.5-673.3 h.ng/mL, mean 460.3 h.ng/mL) was noted among the ' r a p i d ' metabol izers in the contro l s t a t e . There were s i g n i f i c a n t decreases in both C M A X and AUC of 5-hydroxy PF a f t e r phenobarbital treatment. The absolute decrease in C M A X ranged 147 Table 23. The k i n e t i c data of 5-hydroxy propafenone in serum before and a f t e r phenobarbital treatment from s ix heal thy.smoking s u b j e c t s . Subject SP (h) c ^max (ng/mL) ^max (h) AUC (h.ng/mL) % decrease i n cmax % decrease in AUC JL C 5.1 89 1 321.1 37 48 P 5.3 56" 1.5 168.5 MA C 5.0 165 3 673.3 61 56 P 6.9 65 1.5 295.2 TN C 2.9 77 3 364.3 19 67 P 1.2 62 1.5 118.4 DW C 2.7 153 3 596.3 71 72 P 2.2 44 3 167.0 SR C 4.0 108 1 310.5 54 49 P 2.9 50 1.5 159.2 DB C 3.6 137 1.5 496.4 68 66 P 2.0 44 3 169.8 Mean* ± s . d . C P 3.9 ± 1 . 0 3.4 ± 2 . 2 122 ±36 54 S ±9 2.1 +1.0 2.0 ± 0 . 8 460.3 ± 1 5 2 . 4 179.7 s ± 5 9 . 8 52 ±20 60 ±10 C cont ro l (untreated) P phenobarb i ta l - t rea ted S Wilcoxon paired-sample t e s t , compared to the c o n t r o l , s i g n i f i c a n t , p<0.05 s . d . standard dev ia t ion * mean and s . d . of the k i n e t i c parameters excluding the data of the two ' s l o w ' metabol izers (subjects GE and MG). The i r serum 5-hydroxy PF concentra t ions were lower than the detect ion l i m i t . 148 from 19 to 71%, with a mean of 52% while the decrease in AUC ranged from 48 to 72%, with a mean of 60%. The percent decrease in C m a x was not s i g n i f i c a n t l y d i f f e r e n t from the percent decrease in AUC. The tjL£ ranged from 2.7 to 5.1 h (mean 3.9 h) in the contro l s t a t e . No s i g n i f i c a n t change was observed a f t e r phenobarbital treatment (range 1.2-6.9 h, mean 2.1 h ) . The t m a x of 5-hydroxy PF ranged from 1 to 3 h (mean 2.1 h) in the cont ro l s t a t e . Phenobarbital treatment d id not change the t m a x (range 1.5-3 h, mean 2.0 h ) . 3 . 7 . 4 . Prote in Binding and Serum a^ -Ac id Glycoprote in Concentrat ion The f ree f r a c t i o n of PF (2 and 4 h samples) was 0.027 + 0.008 in the cont ro l s ta te and 0.030 + 0.004 a f te r phenobarbital treatment. Serum AAG concentra t ions in the smokers ranged from 57.7 to 113.0 mg/dL (mean 79.1 mg/dL) in the contro l s tate and 61.5 to 122.0 mg/dL (mean 80.1 mg/dL) a f t e r phenobarbital treatment. Twenty-three days of phenobarbital admin is t ra t ion d i d not cause any s i g n i f i c a n t change in e i t h e r PF free f r a c t i o n (Figure 43) or serum AAG concentra t ion (Figure 44). Table 24 shows the data of the sa l i va /se rum PF concentrat ion r a t i o s and s a l i v a r y pH of the 2 and 4 h samples and allows comparison of the values of percent of f ree PF in these samples obtained by two d i f f e r e n t methods. The percent of f ree PF in serum, c a l c u l a t e d from the data of PF s e r u m / s a l i v a concentrat ion r a t i o , pH of s a l i v a and pK a of PF using Equation 7, was s i g n i f i c a n t l y higher than the values obtained from e q u i l i b r i u m d i a l y s i s . 149 0.15 i o 0.12 S ° - 0 9 0.06 H SMOKERS 0.03 0.00 Control (untreated) Phenobarbital-treated Figure 43. The f ree f r a c t i o n of propafenone [2 ( o ) and 4 ( A ) h samples] before and a f te r 23 days of phenobarbital treatment in e ight healthy smokers. 150 1 3 0 n 50 4 1 1 > Control Phenobarbital (untreated) treated Figure 44. The serum.-AAG concentrat ion (0 h sample) before and a f t e r 23 days o f p h e n o b a r b i t a l treatment in eight healthy smokers. 151 Table 24. The sa l iva /serum concentrat ion r a t i o of propafenone, pH of s a l i v a and percent of f ree PF in serum from eight heal thy smoking sub jec ts . Subject Sampling time (h) Propafenone concentrat ion r a t i o (sa l iva /serum) pH of s a l i v a Serum pro te in binding (% f ree) observed 3 ca lcu la ted ' 3 JL C 2 0.04 7.39 3.1 3.9 4 0.03 7.71 4.1 6.0 P 2 0.09 7.40 3.8 9.0 4 0.09 7.45 _* 10.1 MA C 2 0.07 6.90 1.9 2.3 4 0.10 6.96 2.0 3.7 P 2 0.38 6.46 - 4.5 4 0.36 6.65 2.5 6.5 TN C 2 0.21 6.14 _ 1.2 4 0.42 6.60 2.4 6.8 P 2 0.39 6.57 2.7 5.9 4 0.48 6.40 - 4.9 DW C 2 0.29 6.47 3.1 3.5 4 0.75 6.46 3.0 8.8 P 2 0.60 6.46 - 7.0 4 0.83 6.75 - 18.9 GE C 2 0.44 6.57 3.6 6.6 4 0.53 6.65 3.2 9.6 P 2 0.22 6.83 2.9 6.0 4 0.54 6.84 3.2 15.1 MG C 2 0.29 6.68 3.6 5.6 4 0.65 6.57 3.0 9.8 P 2 0.44 6.70 2.9 9.0 4 0.58 6.68 2.7 11.3 SR C 2 0.33 7.00 2.4 13.3 4 0.39 7.12 1.3 20.7 P 2 0.49 6.56 - -4 0.11 6.70 - -DB C 2 0.16 6.72 2.6 3.4 4 0.70 6.51 1.4 9.2 P 2 0.12 6.46 - -4 0.37 6.46 - -C cont ro l (untreated) P phenobarb i ta l - t rea ted a obtained from equ i l ib r ium d i a l y s i s , n=3 b c a l c u l a t e d using equation 7 Wilcoxon paired-sample t e s t , compared to the observed va lues , s i g n i f i c a n t , p<0.05 * values unavai lab le 152 3 . 7 . 5 . Re la t ionsh ip Between Serum T o t a l , Serum Free and S a l i v a r y Propafenone Concentrat ions Figure 45 shows the r e l a t i o n s h i p between PF serum to ta l c o n c e n t r a t i o n , serum f ree concentrat ion and s a l i v a r y concentra t ion of the 2 and 4 h samples (control and phenobarbital ) in the smoking s u b j e c t s . The l i n e a r regress ion and c o r r e l a t i o n s are descr ibed as f o l l o w s : PF serum t o t a l concent ra t ion and PF s a l i v a r y concent ra t ion , Y = 0.495X - 9.907, r = 0.944 (n = 32, p<0.05); PF serum to ta l concentrat ion and PF serum f ree concen t ra t ion , Y = 0.033X - 0.453, r = 0.994 (n = 22, p<0.05); PF s a l i v a r y concentra t ion and PF serum free concent ra t ion , Y = 0.056X + 1.317, r = 0.924 (n = 22, p<0.05). 3 . 7 . 6 . Ur inary Data The renal excre t ion (cumulat ive, 0-48 h) of the g lucuronide and s u l f a t e conjugates of 5-hydroxy PF and 5-hydroxy-4-methoxy PF, expressed as percent of the dose, i s shown in Table 25. Subject TN missed some ur ine samples and h is ur inary data were excluded. Subject GE d id not show any change in the renal excre t ion of the 5-hydroxy PF conjugates whi le the remaining subjects showed a reduct ion a f te r phenobarbital t reatment. The renal excre t ion of 5-hydroxy PF conjugates was 10.7% of dose in the contro l s tate and 8.0% a f t e r phenobarbital treatment. Subjects GE and MG had undetectable l e v e l s of 5-hydroxy-4-methoxy PF conjugates in t h e i r u r i n e . Subject SR d id not show a change whi le the remaining subjects showed a reduct ion in the renal excret ion of the 5-hydroxy-4-methoxy PF conjugates. The renal excret ion of 5-hydroxy-4-methoxy PF conjugates was 5.4% of dose in the contro l s tate and 4.6% of dose a f t e r phenobarbital treatment. 153 PF serum total concentration, ng/mL \ PF salivary concentration, ng/mL Figure 45. The c o r r e l a t i o n between (A) PF serum to ta l concentra t ion and s a l i v a r y concentrat ion ( O ) ; (B) PF serum to ta l concent ra t ion and serum f ree concentrat ion ( • ) and (C) PF s a l i v a r y concentra t ion and serum free concentrat ion ( A ) in e ight healthy smokers. 154 Table 25. Renal excre t ion of 5-hydroxy propafenone and 5-hydroxy-4-methoxy propafenone conjugates (cumulative) before and a f te r phenobarbital treatment from e ight healthy smoking sub jec ts . Renal e x c r e t i o n , % of dose Control (untreated) Phenobarb i ta l - t rea ted Subject 0-12h 12-24h 24-48h Total 0-12h 12-24h 24-48h Total 5-Hydroxy PF conjugates JL 15.8 1.0 1.3 18.1 10.7 0.6 0.7 12.0 MA 16.3 0.6 0.3 17.2 13.8 0.6 0.3 14.7 T N a _* - - - - - - -DW, 10.0 0.2 0.4 10.6 5.7 0.4 0.2 6.3 G E h 2.2 0.4 1.1 3.7 2.5 0.5 0.7 3.7 MGb 3.3 0.8 0.8 4.9 3.1 0.5 1.0 4.6 SR 6.1 0.1 - 6.2 4.3 0.04 - 4.3 DB 13.8 0.4 - 14.2 10.4 0.3 - 10.7 Mean 10.7 8.0 ± s . d . +5.9 ± 4 . 4 5-hydroxy-4-methoxy PF conjugates JL 4.1 0.8 0.7 5.6 3.5 0.6 0.5 4.6 MA 4.8 0.6 0.4 5.8 3.3 0.6 0.2 4.1 T N a - - - - - - - -DWU •3.1 0.1 0.4 3.6 2.1 0.8 0.3 3.2 - - - - - _ _ MGb - - - - - - - -SR 3.4 0.2 - 3.6 3.1 0.4 - 3.5 DB 7.3 1.0 - 8.3 6.0 1.3 0.2 7.5 Mean 5.4 4.6 + s . d . ± 1 . 9 ± 1 . 7 a subject TN missed a ur ine sample and subjects GE and MG had b undetectable concentrat ions of 5-hydroxy-4-methoxy PF conjugates in t h e i r ur ine * values unava i lab le s . d . standard dev ia t ion 155 3 .8 . Phenobarbital Treatment: Comparison of E f f e c t on Pharmacokinetics and Binding Studies of Propafenone and 5-Hydroxy Propafenone Between Healthy Non-smokers and Smokers Table 26 al lows comparison of the pharmacokinetic parameters of PF in serum before and a f t e r phenobarbital treatment between non-smoking and smoking s u b j e c t s . Compared to the non-smokers, the smokers (with the e x c l u s i o n of the data of the two 's low' metabol izers) had a s i g n i f i c a n t l y l a r g e r CL^ n ^ . , a smal ler C m a x and AUC and a l a r g e r V d a r e a / F and V ^ / F in the cont ro l s t a t e . When the values of t ^ and t m a x were compared, there was no s i g n i f i c a n t d i f f e r e n c e between these two groups, inc lud ing or exc luding the data o f the two 's low' metabol izers from the smoking group. No s i g n i f i c a n t d i f f e r e n c e was observed when the k i n e t i c parameters were compared across groups a f t e r the phenobarbital t e s t . However, when the contro l values f o r AUC and C m a x were compared in the non-smoking and smoking groups, phenobarbital was found to cause a s i g n i f i c a n t increase in C L . j n t (194 + 266% in the non-smokers and 156 + 159% in the smokers) and a s i g n i f i c a n t reduct ion in both AUC (50 + 26% in the non-smokers and 49 + 23% in the smokers) and C m a x (45 + 27% in the non-smokers and 46 + 28% in the smokers) of PF. Serum AAG concentrat ions in both non-smoking and smoking subjects were wi th in the normal range and there was no s i g n i f i c a n t d i f f e r e n c e in t h i s parameter between the two groups. There was a lso no s i g n i f i c a n t d i f f e r e n c e in the f ree f r a c t i o n of PF between the non-smokers and the smokers. Table 27 shows comparison of the pharmacokinetic parameters o f 5-hydroxy PF in serum before and a f te r phenobarbital treatment between e ight non-smoking and s i x smoking sub jec ts . When the values of the k i n e t i c 156 Table 26. Comparison of pharmacokinetic parameters o f propafenone in serum between e ight non-smokers and e ight smokers before and a f t e r phenobarbital treatment ( a l l data presented as mean + s . d . ) . Non- smokers (n=8) Smokers (n=8) K i n e t i c Phenobarbi ta l - Phenobarbi ta l -parameter Control t reated Control t reated (h i 2.8 ± 1.2 2.5 ± 1.2 4.9 + 4.1 4.5 + 4.1 (2.7 ± 1.0)* (2.4 ± 0.5) c wmax (ng/mL) 253 ± 221 137 ± 127 239 + 253 (107 + 62 ) S 154 ± 220 (51 ± 46) ^max (h) 2.5 ± 0.9 2.9 ± 1.2 2.5 ± 1.1 (2.3 ± 1.2) 2.5 ± 1.1 (2.0 ± 0.8) C L i n t 6.1 + 3.7 18.1 ± 21. 5 10.0 ± 7.6 (13.2 ± 5 . 6 ) S 26.8 ± 21.9 (L/min) (35.5 + 17.7) AUC 1360 + 1684 638 ± 778 2798 + 4508 1832 + 3252 (h.ng/mL) (405 ± 191) S (195 + 188) V d a r e a ^ * (L/kg) 16.2 + 12 .4 42.3 + 60. 2 27.5 + 17.5 (34.9 + 12 .8 ) S 70.8 + 50.4 (91.7 + 38.3) (L/kg) 20.0 ± 13 .6 60.7 ± 80. 7 35.2 + 26.5 (45.0 + 2 2 . 8 ) S 92.0 + 75.4 (119.9 + 65.1) Serum AAG concent ra t ion (mg/dL) a 81.1 + 19 .1 77.7 ± 14. 2 79.1 ± 19.3 80.1 ± 19.2 PF f ree k f r a c t i o n 0.027 ± 0 .011 0.038 + 0. 032 0.027 ± 0.008 0.030 + 0.004 % increase 194 ± 266 156 + 159 i n C L i n t (187 ± 174) % decrease 45 ± 27 46 ± 28 i n Cmax (52 ± 27) % decrease 50 ± 26 49 + 23 in AUC (54 + 24) s . d . standard dev ia t ion a AAG concentrat ion of 0 h (blank) serum sample b the f ree f r a c t i o n of PF was determined in the 2 and 4 h serum samples from each subject by equ i l ib r ium d i a l y s i s * numbers in brackets are mean and s . d . of the k i n e t i c parameters excluding the data of the two v s l o w ' metabo l i ze rs . * * normalized to the i n d i v i d u a l ' s body weight S Mann-Whitney t e s t , compared to the contro l values of the non-smokers, s i g n i f i c a n t , p<0.05 157 Table 27. Comparison of pharmacokinetic parameters of 5-hydroxy propafenone in serum between eight non-smokers and s i x smokers before and a f t e r phenobarbital treatment (a l l data presented as mean + s . d . ) . Non-•smokers (n=8) Smokers (n=6)* K i n e t i c parameter Control Phenobarbi ta l -t reated Control Phenobarbital -t reated SP (h) 3.1 ± 1.1 2.9 + 1.1 3.9 ± 1.0 3.4 ± 2.2 c max (ng/mL) 141 ± 62 86 + 47 122 ± 36 54 + 9 ^max (h) 2.8 ± 1.5 2.8 + 1.1 2.1 ± 1.0 2.0 + 0.8 AUC 685 ± 324 387 + 234 460 ± 152 180 ± 60 (h.ng/mL) % decrease 40 ± 22 52 + 20 i n cmax % decrease in AUC 45 ± 18 60 + 10 s . d . standard dev ia t ion * mean and s . d . of the k i n e t i c parameters excluding the data of the two ' s l o w ' metabol izers (subjects GE and MG). The i r serum 5-hydroxy PF concentra t ions were lower than the detect ion l i m i t . 158 parameters AUC, C m a x , t ^ and tmax measured in the contro l s ta te were compared between the non-smoking and smoking s u b j e c t s , there was no s i g n i f i c a n t d i f f e r e n c e between these two groups. S i m i l a r r e s u l t s were observed when these parameters were compared across groups a f t e r phenobarbital admin is t ra t ion . However, when the contro l values fo r AUC and C m a x were compared to the t reated values in the non-smoking and smoking s u b j e c t s , phenobarbital caused a s i g n i f i c a n t reduct ion in both AUC (45 + 18% in the non-smokers and 60 + 10% in the smokers) and C m a x (40 + 22% in the non-smokers and 52 + 20% in the smokers). The c o r r e l a t i o n between phenobarbital serum concentra t ion and the extent of metabol ic induct ion of PF by phenobarb i ta l , as expressed by [(AUCp-AUC c ) /AUC c ]xlOO%, i s shown in F igure 46. There was no c o r r e l a t i o n between these two parameters (r = -0 .323, n = 16, p>0.05). The best f i t through the data points was descr ibed by Y = 98.981 - 3.007X. Figure 47 i l l u s t r a t e s the c o r r e l a t i o n between C L i n t c o n t r o l a n d [ (AUC p -AUC c ) /AUC c ] x 100%. No s i g n i f i c a n t c o r r e l a t i o n was observed between these two parameters (r = 0.048, n = 16, p>0.05). The best f i t through the data points was descr ibed by Y = 0.187X + 47.929. 3 .9 . Evaluat ion of Propafenone Serum Concentrat ion-Response Re la t ionsh ip The semi - logar i thmic p lo ts of the serum concentrat ion versus time curves of PF and 5-hydroxy PF from the ten pa t ien ts are shown in Appendix 8. The k i n e t i c data of PF from the ten pat ients i s shown in Table 28. Two of the ten pat ients (GB and BM) were ' s low ' metabol izers 159 a? o o X I — I o o Z> < 100-, 80 4 60 o o < I Q_ O < 40 20 H Linear regression: Y = 98.981 - 3.007X r = - 0 . 3 2 3 o o 2 4 6 8 10 12 14 16 18 20 Serum phenobarbital concentrat ion ( /zg/mL) 22 Figure 46. The c o r r e l a t i o n between phenobarbital serum concent ra t ion (day 29) and [ (AUC p -AUC c ) /AUC c ]x lOO% in e ight healthy non-smokers ( O ) and e ight healthy smokers ( A ) . 100T o 2 80-Linear regression: Y = 0.187X + 47.929 r = 0.048 o o Z> < 60- o o o < i CL O Z) < 40 20-~4 6 8 10 12 14 16 18 20 22 CL int control Figure 47. The c o r r e l a t i o n between values of C L i n t r n n t ™ i a n d [(AUCp-AUC )/AUC c]xlOO% in e ight healthy non-smokers ( O and eight healthy smokers ( A ). 161 Table 28. The k i n e t i c data of propafenone and 5-hydroxy propafenone in serum from ten p a t i e n t s . PF 5-hydroxy PF Pat ient AUCj" (h.ng/mL) C L i n t ( L / m i n ) c p s s (ng/ m L) AUCj" (h.ng/mL) BN 7329.6 0.62 916 1161.7 C 3916.6 1.15 490 1142.0 J C * 5185.1 0.87 432 1482.7 J L * 2416.7 0.93 403 1575.0 DT* 4441.2 1.02 555 836.9 LS* 2258.9 1.00 282 1162.4 GB 14445.5 0.31 1806 ** RM 5157.4 0.88 645 795.0 AP 1374.0 3.29 172 510.4 BM 14617.9 0.31 1827 -Mean + s . d . 6114.3 +4756.4 1.04 ± 0 . 8 4 753 ±596 Mean* ± s . d . 4009.9 +1944.4 1.22 ± 0 . 8 5 487 ±228 1083.3 ± 3 5 6 . 4 * the predose serum PF concentrat ions of these pa t ien ts were used as the serum PF concentrat ions at the dosing in te rva l in the est imat ion of AUCj" and C L i n t * * sub jects GB and BM had undetectable serum 5-hydroxy propafenone # mean and s . d . of the k i n e t i c parameters excluding the data of the two ' s l o w ' metabol izers (pat ients GB and BM) 162 ( C L i n t < 0.5 L /min) . In addi t ion to low C L ^ n t v a l u e s , the two 's low' metabol izers exh ib i ted high C p s s and large AUCQ" va lues . The mean C p s s (1816.7 ng/mL) and AUCj (14531.7 h.ng/mL) values f o r the two 's low ' metabol izers were about four times the mean C p s s (487 ng/mL) and AUCQ" (4009.9 h.ng/mL) values of the ' r a p i d ' metabo l i zers . The 's low ' metabol izers account for some of the considerab le i n t e r i n d i v i d u a l v a r i a t i o n seen in PF k i n e t i c parameters among pat ients r e c e i v i n g the drug. However, a large i n t e r i n d i v i d u a l d i f f e r e n c e in the k i n e t i c parameters s t i l l e x i s t s among the ' r a p i d ' metabol izers . There was a 5 f o l d d i f f e r e n c e in AUCQ~ (range 1374.0-7329.6 h.ng/mL), C L i n t (range 0.62-3.29 L/min) and C p s s (range 172-916 ng/mL) among these p a t i e n t s . The k i n e t i c data of 5-hydroxy PF from the ten pat ients i s a lso shown in Table 28. The metabol i te , 5-hydroxy PF, was measurable in serum of only e ight of the ten pat ients r e c e i v i n g the drug dur ing treatment f o r arrhythmias. Serum 5-hydroxy PF concentrat ions were lower than serum PF concent ra t ions . There was a 3 f o l d d i f f e r e n c e in the AUCQ" (range 510-1575 h.ng/mL) among the p a t i e n t s . Subjects GB and BM had undetectable q u a n t i t i e s o f 5-hydroxy PF, a c h a r a c t e r i s t i c of ' s low ' metabo l i ze rs . F igure 48 shows log PF serum concentrat ion versus QRS width. The best f i t through the data points by l i n e a r regress ion i s Y = 5.094X + 94.249 (r = 0.461, n = 56). Figure 49 shows log 5-hydroxy PF serum concentra t ion versus QRS width. The best f i t by l i n e a r regress ion i s Y = 10.534X + 85.718 (r = 0.527, n = 45). QRS width i s s i g n i f i c a n t l y c o r r e l a t e d to e i t h e r PF serum concentrat ion or 5-hydroxy PF serum c o n c e n t r a t i o n . F igure 50A and B allow comparison of the c o r r e l a t i o n between log PF serum t o t a l concentrat ion and QRS width (Y = 4.367X + 95.929, r = 0.288, 163 125-, 120 o £ 110 SZ •o * 105-1 100H 95 Linear regression: Y = 5.094X + 94.249 r = 0.461 o o cP ° o <LD O < § £ o 0 Q ^ o Q %o 0 o o CP o GSD O O OO 10 100 1000 PF serum concentration, ng/mL 10000 Figure 48. The c o r r e l a t i o n between QRS width and log PF serum concentrat ion of ten p a t i e n t s . 164 1 2 51 120-<u c o 110-1 -t-> -g * 105-j cn or: o 100-1 95 10 Linear regression: Y = 10.534X + 85.718 r = 0.527 A A A A A A A ^ A A ^ A A A A AA A A I i i r -100 5—Hydroxy PF serum concentrat ion, n g / m L 1000 Figure 49. The c o r r e l a t i o n between QRS width and log 5-hydroxy PF serum concentrat ion of ten p a t i e n t s . 165 A CO c 125n 120 CO 10 Jg 115 =S 110 CO cn ° 105 100 100 cP o o o o % Linear regression: Y = 4.367x + 95.929 r = 0.288 1000 PF serum total concentrat ion, n g / m L 10000 B CD c 125 120 CO co ° 115 I 110 co CrT 0 105-1 100 Linear regression: Y = 4.225X 4- 102.327 r = 0.346 • •• 10 100 PF serum free concentrat ion, n g / m L Figure 50. The c o r r e l a t i o n between (A) QRS width and log PF serum to ta l concentrat ion and (B) QRS width and log PF serum f ree concentrat ion of ten pat ients (two data points from each p a t i e n t . 166 n = 18) and log PF serum free concentrat ion and QRS width (Y = 4.225X + 102.327, r = 0.346, n = 18) (two data points from each p a t i e n t ) . QRS width is not s i g n i f i c a n t l y (p>0.05) cor re la ted to e i t h e r PF serum to ta l concentrat ion or PF serum free concent ra t ion . The serum AAG concentrat ions of the pat ients ranged from 43 to 115 mg/dL, which i s wi th in the normal range of serum AAG concent ra t ion . Figure 51 shows the r e l a t i o n s h i p between serum AAG concentra t ion and PF f ree f r a c t i o n . The best f i t l i n e by l i n e a r regress ion i s Y = -0.0004X + 0.070. The c o r r e l a t i o n between serum AAG concentrat ion and PF f ree f r a c t i o n i s negative and s i g n i f i c a n t (r = -0.504, n = 20, p<0.05). The n o n - l i n e a r l e a s t squares method g ives a bet ter f i t as Y = 1/(1 + 0.357X). Using mul t ip le stepwise r e g r e s s i o n , Equations 8 and 9 are obtained which modelled QRS width as a funct ion of PF serum concent ra t ion , 5-hydroxy PF serum concentrat ion and serum AAG concent ra t ion : Y = 0.5Xj + 4 .5X 2 + 347X 3 + 79 (8) Y = 0.004X}' + 0 .04X 2 ' + 379X 3 + 95 (9) where Y is QRS width, Xj i s log PF serum concent ra t ion , X2 i s log 5-hydroxy PF serum concent ra t ion , X3 i s the r e c i p r o c a l of serum AAG concentrat ion (1/AAG), X j ' i s PF serum concentrat ion and l^' ""s 5-hydroxy PF serum concent ra t ion . Figure 52 shows the r e l a t i o n s h i p between measured QRS width and pred ic ted QRS width using Equation 8. The c o r r e l a t i o n is s i g n i f i c a n t (r = 0.536, n = 45, p<0.05). The best f i t l i n e by l i n e a r regress ion i s Y = 0.177X + 74.881. Figure 53 shows the r e l a t i o n s h i p between measured QRS width and pred ic ted QRS width using Equation 9. The c o r r e l a t i o n is s i g n i f i c a n t (r = 0.630, n = 45, p<0.05). The best f i t l i n e by l i n e a r regress ion i s Y = 0.387X + 65.902. 167 q o o cu O c o c 0) o C L O i _ CL O.O8-1 0 .07 -0.06 0.05 H 0.04 0.03 -I 0.02 0.01 0 4'i 1 O T o T o o oo o o o 40 60 80 100 Serum AAG concentrat ion, m g / d L 120 Figure 51. The c o r r e l a t i o n between serum propafenone f ree f r a c t i o n and serum AAG concentrat ion of ten p a t i e n t s . The data are presented as mean + l s . d . 168 X J CO or: o X J <u -M o X J <u CL 100 -, Q) CO o X I c* 96 9 4 -9 2 -90 88 95 Linear regression: Y = 0.177X + 74.881 0.536 r = o o o o c ? o© cP o 0 o o 100 105 110 115 120 Measured QRS width (% baseline) 125 Figure 52. The c o r r e l a t i o n between measured QRS width and pred ic ted QRS width. QRS width i s pred ic ted from the fo l lowing equation (obtained by mul t ip le stepwise r e g r e s s i o n ) : Y = 0.5X^ + 4.5X2 + 347X3 + 79 where Y i s QRS width, Xi i s log PF serum concent ra t ion , Xo i s log 5-hydroxy PF serum concentra t ion and X 3 i s 1/AAG. 169 115-1 CD co o jQ <* 1 1 0 -LY. a ~o CO -+-> O T J CD l_ CL 1 0 5 -100 Linear regression: Y = 0.387X + 65.902 r = 0.630 95 o o o ° ° ° o 0 ° n O o O ( cr o o o o o o 100 105 110 115 120 Measured QRS width (% baseline) 125 Figure 53. The c o r r e l a t i o n between measured QRS width and pred ic ted QRS width. QRS width i s pred ic ted from the fo l lowing equation (obtained by mul t ip le stepwise r e g r e s s i o n ) : Y = 0.004Xi' + 0.04X2' + 379X3 + 95 where Y i s QRS width, Xj' i s PF serum concent ra t ion , l^' i s 5-hydroxy PF serum concent ra t ion . 170 4. DISCUSSION 4.1 C a p i l l a r y E lect ron-Capture Detect ion Gas -L iqu id Chromatographic A n a l y s i s of Propafenone The GLC-ECD assay method developed fo r the quant i ta t ion of PF demonstrated improved s e l e c t i v i t y and s e n s i t i v i t y over the e x i s t i n g GLC method [Marchesini et al., 1982] through the use of a bonded-phase f u s e d - s i l i c a c a p i l l a r y column and the s p l i t l e s s i n j e c t i o n technique. The l i m i t of determination was - 2 . 5 ng/mL using 1 mL of serum. The method was f u r t h e r va l ida ted by comparison of i t s performance to a publ ished HPLC method by Harapat and Kates [1982]. The two methods were shown to y i e l d comparable quant i ta t ion of PF in serum. The GLC-ECD method demonstrated super io r s e n s i t i v i t y to the publ ished HPLC method (UV detect ion) with a lower l i m i t of determination of 2.5 ng/mL using 1 mL of serum. On the other hand, the HPLC method can only be used to quant i ta te PF in samples (2-5 mL) with drug concentrat ions greater than 200 ng/mL (Table 10). 4 . 1 . 1 . S p l i t l e s s In ject ion and ' C o l d Trapping ' E f f e c t The s e n s i t i v i t y of the assay was g rea t ly enhanced by the use of a s p l i t l e s s i n j e c t i o n mode. The s p l i t l e s s mode of i n j e c t i o n involves the in t roduc t ion of a r e l a t i v e l y large amount of sample into the c a p i l l a r y column without s p l i t t i n g o f f any sample to the vent [Freeman, 1981]. When using the s p l i t l e s s technique of sample i n j e c t i o n , the so lutes of the sample must be reconcentrated at the head of the column using e i t h e r a ' s o l v e n t ' or ' c o l d t rapp ing ' e f f e c t . With reconcent ra t ion , the band widths of the e l u t i n g peaks w i l l r e f l e c t column e f f i c i e n c y rather than the volume 171 of the g l a s s i n j e c t i o n port l i n e r [Freeman, 1981]. The bas ic d i f f e r e n c e between these two methods i s the i n i t i a l column temperature. The ' s o l v e n t ' e f f e c t invo lves using an i n i t i a l column temperature which i s lower than the solvent b o i l i n g point [Grob and Grob, 1974]. On the other hand, the ' c o l d t r a p p i n g ' e f f e c t involves using an i n i t i a l column temperature which i s h igher than the solvent b o i l i n g point but lower than the b o i l i n g point of the compound of i n t e r e s t [Freeman, 1981]. The ' c o l d t r a p p i n g ' e f f e c t was used e f f e c t i v e l y f o r PF (Figure 17). 4 . 1 . 2 . C a p i l l a r y Column A bonded-phase f u s e d - s i l i c a c a p i l l a r y column was employed fo r the a n a l y s i s of PF. This type of column has the bene f i t s of e x c e l l e n t thermal and chemical s t a b i l i t y , e f f i c i e n c y , s e l e c t i v i t y and s e n s i t i v i t y . The l i q u i d phase of the column was 5% phenyl methyl s i 1 icone. S u b s t i t u t i o n of 5% of the methyl groups by phenyl moiet ies increases the p o l a r i t y and s e l e c t i v i t y of the l i q u i d phase, as compared to the nonsubst i tuted m e t h y l s i l i c o n e . 4 . 1 . 3 . E lec t ron-Capture Detect ion and A c y l a t i o n The ECD i s a h igh ly s e l e c t i v e and s e n s i t i v e d e t e c t o r . Its high s e l e c t i v i t y comes from i t s d i s c r i m i n a t i o n against non-e lec t ron captur ing m a t e r i a l s , a l lowing i t to respond to only a few types of compounds (e .g . compound conta in ing halogen atoms, anhydrides, a l i p h a t i c amines, e s t e r s , aldehydes and n i t r i l e s ) . Its high s e n s i t i v i t y o f f e r s the p o s s i b i l i t y of q u a n t i t a t i v e a n a l y s i s at the nanogram or picogram l e v e l . The PF molecule conta ins a hydroxyl group and a secondary amine (Figure 1). Reaction of PF with f l u o r i n a t e d acid anhydrides such as TFAA or HFBA forms h igh ly e lectron 172 withdrawing per f luoroacy l d e r i v a t i v e s , making the compound more amenable to a n a l y s i s by ECD. In addi t ion to increased s e n s i t i v i t y and s e l e c t i v i t y , d e r i v a t i z a t i o n a lso has the advantage of reducing peak t a i l i n g , which i s common among compounds conta in ing hydroxy l , c a r b o x y l , amino and imino groups as a r e s u l t of i n t e r a c t i o n with the GLC column [Ahuja, 1976]. The a n a l y s i s time i s a lso shortened due to the decreased p o l a r i t y and increased v o l a t i l i t y of the d e r i v a t i v e . TFAA was the f i r s t agent used to d e r i v a t i z e PF. It was abandoned, however, due to in te r fe rence from a negative peak. HFBA was then used as the acy la t ing agent. Although ECD response i s , in g e n e r a l , d i f f i c u l t to p r e d i c t , there i s evidence that de tec tor response increases as the number of halogen atoms increases [Anggard and Hankey, 1969]. Th is was confirmed with the HFB d e r i v a t i v e of PF where an approximate f i v e - f o l d increase in s e n s i t i v i t y was observed as compared to the TFA d e r i v a t i v e . The re tent ion time was only minimal ly increased . A c y l a t i o n reac t ions can be performed in a non-polar solvent such as benzene or toluene with a t e r t i a r y amine as a c a t a l y s t . The t e r t i a r y amine serves as an ac id acceptor , enhancing r e a c t i v i t y . Trimethylamine (TMA) in benzene i s commonly used as a c a t a l y s t fo r the a c y l a t i o n of amino, a l c o h o l i c and phenol ic compounds [Walle et al., 1970; Ehrsson et al., 1971; Walle et al., 1971]. However, i t has to be f r e s h l y prepared by saturat ion of benzene with gaseous TMA from TMA c h l o r i d e and the TMA content determined t i t r i m e t r i c a l l y [Walle et al., 1971]. Because of the troublesome preparat ion and i n s t a b i l i t y of TMA, TEA in toluene was used to rep lace TMA in benzene in our study. TEA is commercial ly a v a i l a b l e as a reagent and i s chemica l ly s t a b l e . We have demonstrated that by using TEA, the a c y l a t i o n time for PF react ion with HFBA was s u b s t a n t i a l l y shortened (Figure 7 ) . Furthermore, TMA and TEA have the advantage over other basic 173 c a t a l y s t s (such as pyr id ine ) in that they do not cause d is turbances in the gas chromatograms at high EC s e n s i t i v i t y s e t t i n g s . About 50 fil of 0.05 M of TMA has been recommended as a c a t a l y s t fo r a c y l a t i o n of amines [Walle et a 7 . , 1971]. In the ana lys is of PF, a r e l a t i v e l y smal ler proport ion of TEA (i.e. 400 /zL of 0.003M TEA) was used to reduce solvent f ront band-spreading without a f f e c t i n g the a c y l a t i o n react ion (Figure 9 ) . A c y l a t i o n usua l l y proceeds more slowly than other d e r i v a t i z a t i o n reac t ions and i t i s common to heat the samples to between 60°C and 100°C. A c y l a t i o n of PF and I . S . - a c a r r i e d out at 65°C appeared e n t i r e l y s a t i s f a c t o r y . 4 . 1 . 4 . E x t r a c t i o n and In ject ion Solvent Four so lvents with d i f f e r e n t p o l a r i t i e s were tes ted fo r t h e i r e f f i c i e n c y in ex t rac t ing PF. In order of increas ing p o l a r i t y they were: hexane, to luene , benzene and to luene:d ichloromethane: isopropyl a lcohol (7 :3 :1 ) . Benzene was chosen as the ex t rac t ion solvent because i t s e x t r a c t i o n e f f i c i e n c y was higher than that of the other solvents tested (F igure 11). Although the ex t rac t ion e f f i c i e n c y of benzene (E = 0.81) was only s l i g h t l y higher than that of toluene (E = 0 .76) , benzene ex tens ive ly reduced (approximately by ha l f ) the time fo r sample evaporat ion during the a n a l y t i c a l procedure. This was an important t ime-sav ing advantage, due to the la rge number of samples to be analyzed by t h i s method during the pharmacokinetic s tudies of PF c a r r i e d out dur ing t h i s p r o j e c t . Solvents such as methylene c h l o r i d e (dichloromethane) , ch loroform, carbon d i s u l f i d e , d ie thy l e ther , hexane and isooctane are widely used for s p l i t l e s s i n j e c t i o n . Dichloromethane and chloroform cannot be used fo r ECD s ince these so lvents have a high e lec t ron capture response. Because of the iner tness of the bonded phase of modern fused s i l i c a c a p i l l a r y columns, 174 polar and aromatic so lvents can a lso be used because they do not r e s u l t in s i g n i f i c a n t phase s t r i p p i n g due to the improved phase bonding technology in the manufacturing process of c a p i l l a r y columns [Freeman, 1981]. Although benzene was used as the ex t rac t ion so lvent , toluene was used as the d e r i v a t i z i n g and i n j e c t i o n solvent because of i t s higher b o i l i n g p o i n t . Th is has the advantage of considerably reducing the sample d i s c r i m i n a t i o n encountered in the s p l i t l e s s sampling mode due to the high i n j e c t o r port temperature and low b o i l i n g point of the solvent [Schomburg et al., 1981]. 4 . 1 . 5 . Optimal GLC-ECD Condi t ions The GLC-ECD cond i t ions such as the i n l e t purge valve a c t i v a t i o n t ime, i n i t i a l and f i n a l column temperature, temperature programming r a t e , i n j e c t i o n port temperature, ECD temperature and make-up gas flow rate were a l l opt imized to obta in the best peak r e s o l u t i o n , band width and symmetry with the shor tes t re tent ion time (Figure 13). To reduce solvent t a i l i n g during s p l i t l e s s i n j e c t i o n , the i n j e c t i o n port was backf lushed or purged some time a f te r sample i n j e c t i o n {i.e., a temporary conversion of the sample i n l e t from a s p l i t l e s s mode back to a s p l i t c o n f i g u r a t i o n ) . This i n l e t purge valve a c t i v a t i o n t ime, which we determined as 40 seconds fo r PF (Figure 12), allowed most of the solute to pass through the column {i.e., with a loss of <1% of the solute) and removal o f -5% or l e s s of the so lven t , thereby reducing solvent t a i l i n g . Although there was no apparent d i f f e r e n c e in response between the var ious i n j e c t i o n port temperatures tested (200 -260°C) (Figure 13A), a lower temperature (210°C) was chosen. A f t e r sample i n j e c t i o n in the s p l i t l e s s mode, the peaks of i n t e r e s t are a c t u a l l y broadened s l i g h t l y in the i n j e c t i o n i n l e t due to the low column flow rate and then recondensed into 175 narrow bands at the head of the column. At an i n i t i a l low column temperature, the low b o i l i n g components proceed through the i n l e t to the column while the high b o i l i n g components are trapped at the head of the column u n t i l the oven temperature {i.e., column) is i n c r e s e d . As a r e s u l t , a lower i n j e c t i o n port temperature i s often more s a t i s f a c t o r y in that i t f a c i l i t a t e s v c o l d t r a p p i n g ' of high b o i l i n g so lu tes [Rooney, 1985]. The operat ion of an ECD requi res a r e l a t i v e l y high detector temperature to maintain detector c l e a n l i n e s s . Consequently, a detector temperature of 350°C was chosen to minimize contamination (Figure 13B). The ECD requi res the use of a moderating gas to assure an e q u i l i b r i u m concentra t ion of thermal e l e c t r o n s . Th is moderating gas can be used a d d i t i o n a l l y as the sweep gas fo r the column ex i t [Freeman, 1981]. However, an optimum make-up gas flow rate has to be determined so that both s e n s i t i v i t y (favored by low flow rate) and band width (favored by high flow rate) can be obta ined. Genera l ly when using e i t h e r hydrogen or helium as the c a r r i e r gas, a make-up flow rate of e i t h e r n i t rogen or argon/methane (95:5) at 20-60 mL/min appears to be s a t i s f a c t o r y [Freeman, 1981]. In the assay development f o r PF, argon/methane (95:5) was used as the make-up gas and a flow rate of 60 mL/min seemed to g ive the optimum s e n s i t i v i t y and the minimum band width f o r PF (Figure 13C). 4.2 E lec t ron-Capture Detect ion C a p i l l a r y Gas -L iqu id Chromatographic A n a l y s i s of 5-Hydroxy Propafenone and 5-hydroxy-4-methoxy Propafenone Although i t i s d e s i r a b l e to measure the in tac t drug and i t s metabol i te (s ) s imul taneously , o c c a s i o n a l l y separate assay methods are 176 necessary because of the d i f f e r e n t physico-chemical nature of the m e t a b o l i t e ( s ) . The metabo l i te , 5-hydroxy PF, has increased a c i d i t y and p o l a r i t y compared to the parent drug due to the hydroxyl subs t i tu ten t on the benzene r i n g (Figure 2 ) . There fore , simultaneous measurement of both compounds was not p o s s i b l e with the present method without s i g n i f i c a n t s a c r i f i c e of the recovery of PF or i t s primary metabol i te . The GLC-ECD method developed fo r the q u a n t i t a t i o n of PF was, t h e r e f o r e , fu r ther modif ied f o r the measurement of 5-hydroxy PF. The more po lar solvent mixture - to luene:d ichloromethane: isopropyl a lcohol (7:3:1) had the highest ex t rac t ion e f f i c i e n c y (E = 0.81) of the four so lvents tested and was used to replace benzene (E = 0.76) as the ex t rac t ion solvent fo r 5-hydroxy PF (Figure 20). The phenol ic group on 5-hydroxy PF i s a c i d i c and can form a water -so lub le phenolate s a l t with the strong a l k a l i , sodium hydroxide. There fore , sodium and potassium carbonate were used to replace sodium hydroxide during the ex t rac t ion to minimize the formation of such water -so lub le sodium s a l t of 5-hydroxy PF. In add i t ion to the hydroxyl group and the secondary amine, the phenol ic group of 5-hydroxy PF i s a lso acy la ted by HFBA. Temperature programming and other GLC-ECD condi t ions were opt imized to obtain complete separat ion of 5-hydroxy PF from PF, I .S . -b and endogenous components of the serum. 4.3 S t ruc tu ra l Conf irmation of the HFB Der iva t ives of Propafenone and 5-Hydroxy Propafenone The EI-MS fragmentation of the HFB d e r i v a t i v e of PF in the present study exh ib i ted a pattern s i m i l a r to the MS fragmentation of a TFA 177 d e r i v a t i v e of PF reported by Hege et al. [1984a]. The ion at m/e 733 was the molecular ion (M + ) , cons is ten t with the reac t ion of PF with two molecules of HFBA (Figure 25). As compared to EI-MS, the PICI-MS fragmentation of the HFB d e r i v a t i v e of PF had fewer fragment ions . In add i t ion to the pronounced (M+l) + i o n , c h a r a c t e r i s t i c ions of (M + C 2 H g ) + and (M + C-jHg)"1" were a lso found when methane was used as an i o n i z i n g gas (Figure 27). The fragment ions at m/e 508, 294 and 252 appeared in both EI-MS and PICI-MS p r o f i l e s . The NICI-MS fragmentation of the HFB d e r i v a t i v e of PF in the present study fol lowed a pattern s i m i l a r to that of the d e r i v a t i v e s of metoprolol and oxprenolol formed by HFB and reported by Gaudry et al. [1985a,b]. S t r u c t u r a l l y , metoprolol and oxprenolol have s i d e - c h a i n s that have the same molecular formulae as PF (C 3 H 7 -NH-CH 2 -CH(OH) -CH 2 - , +2HFB = 508), and the fragment ions at m/e 488 (508 - HF) and 448 (508 - 3HF) were the common ions in the NICI mass spectra of these three compounds. Other common fragment ions observed were at m/e 213 (C 3 F 7 COO-) and 194 (213 - F ) . The (M- l )" ion (m/e 732) was c h a r a c t e r i s t i c of NICI-MS. The molecular ion l o s t a p r o g r e s s i v e l y increas ing number of HF fragments to y i e l d ions at m/e 693 (733 - 2HF), 673 (733 - 3HF) and 653 (733 - 4HF) (Figure 29). The EI-MS, PICI-MS and NICI-MS of I . S . - a fo l low a s i m i l a r pattern as PF. The EI-MS of 5-hydroxy PF showed a s i m i l a r pattern to the EI-MS of PF. The common ions include those at m/e 508, 91, 43, 294, 252, 104, 226, 466 and 77. The molecular ion (M +) at m/e 945 was not observed in the mass spectrum (Figure 33). The ions at m/e 333, 359 and 748 are c h a r a c t e r i s t i c of the HFB d e r i v a t i v e of 5-hydroxy PF s ince i t i nd ica tes the acy la t ion of the phenol ic funct iona l group of 5-hydroxy PF, in add i t ion to the 178 d e r i v a t i z a t i o n of the hydroxyl and secondary amine. 4.4 In Vitro Serum Prote in Binding Study of Propafenone 4.4.1 Prote in Binding Technique Several methods inc lud ing e q u i l i b r i u m d i a l y s i s , u l t r a 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 , gel f i l t r a t i o n , e t c . have been used f o r the determinat ion of drug prote in binding or f ree f r a c t i o n [review in Kurz et a 7 . , 1977; Wandell and Wi lcox -Tho le , 1983; Barre et a 7 . , 1988]. Among these techniques , equ i l ib r ium d i a l y s i s and u l t r a f i l t r a t i o n are the most commonly used methods in the laboratory s e t t i n g [Kurz et al., 1977; Bowers et al., 1984; Kwong, 1985]. Equ i l ib r ium d i a l y s i s i s of ten regarded as the re ference method and has the advantage of f ree component r e t a i n i n g access to the bound component when e q u i l i b r i u m i s achieved [Briggs et al., 1983]. U l t r a f i l t r a t i o n i s l e s s r e l i a b l e but a lso l e s s time-consuming than e q u i l i b r i u m d i a l y s i s . Although u l t r a f i l t r a t i o n has the advantage of speed and ease, the extens ive adsorpt ion (16.2%) of PF to the u l t r a f i l t r a t i o n device makes t h i s technique unacceptable fo r PF pro te in binding s t u d i e s , s ince i t leads to an underestimation of f ree drug concentra t ion or f ree f r a c t i o n . The n o n - s p e c i f i c binding of PF to the e q u i l i b r i u m d i a l y s i s membrane (2.3%) i s low. Although the adsorpt ion of PF to the equ i l ib r ium d i a l y s i s c e l l i s s i m i l a r to that of u l t r a f i l t r a t i o n device (16.6%) (Table 11), i t does not a f f e c t the c a l c u l a t i o n of f ree f r a c t i o n because both the bu f fe r and the serum were assayed fo r PF concentrat ions a f t e r equ i l ib r ium d i a l y s i s a l lowing fo r the necessary c o r r e c t i o n [Nimmo et al., 1977; Bowers et al., 1984]. The s e n s i t i v i t y of the GLC-ECD method developed fo r PF is 179 e n t i r e l y adequate f o r d i r e c t f ree drug measurement in our in vitro prote in b inding study, the s ing le -dose enzyme induct ion study and the concent ra t ion- response r e l a t i o n s h i p study where, in a l l i n s t a n c e s , low sample volumes were encountered. Although using rad io !abe l1ed drug in e q u i l i b r i u m d i a l y s i s w i l l fu r ther improve the assay s e n s i t i v i t y , experimental accuracy depends on the pur i t y [Bu i lder and S e g e l , 1978] and the s t a b i l i t y of the r a d i o l a b e l e d drug. Furthermore, the r a d i o l a b e l e d substances in the d i a l y s a t e a f te r d i a l y s i s have to be i d e n t i f i e d in order to obta in v a l i d data on the binding of h igh ly bound r a d i o l a b e l e d compounds [Yacobi and Levy, 1975]. Besides n o n - s p e c i f i c adsorpt ion of drugs to d i a l y s i s membranes and apparatus [Briggs et al., 1983], other problems assoc ia ted with the use of e q u i l i b r i u m d i a l y s i s fo r prote in binding study include volume s h i f t [Lima et al., 1983; Tozer et al., 1983; Boundinot and Jusko, 1984], pH s h i f t [Br inkschu l te and B r e y e r - P f a f f , 1979; Lui and Chiou, 1986], and, as w e l l , b a c t e r i a l growth [ I l e t t et al., 1975; Br inkschul te and B r e y e r - P f a f f , 1979; Br iggs et al., 1983]. Volume s h i f t i s the net movement of water from the buf fer chamber in to the serum chamber, probably due to the osmotic e f f e c t of plasma p r o t e i n s , r e s u l t i n g in a reduct ion in the concentrat ion of the binding p r o t e i n s . It i s time-dependent and has been shown to have a s i g n i f i c a n t e f f e c t on binding i f the d i a l y s i s time exceeds -12 h [Lima et al., 1983]. Volume s h i f t can be corrected by use of der ived equations [Lima et al., 1983; Tozer et al., 1983; Boudinot and Jusko, 1984]. It can be attenuated by the i n c l u s i o n of dextran (M.W. 70,000, 55% of the to ta l p ro te in concentra t ion) in the buf fer to decrease the osmotic pressure gradient e x i s t i n g between the prote in and buf fer s ides of the membrane. It can also 180 be attenuated by use of a t h i c k , low M.W. c u t o f f membrane dur ing e q u i l i b r i u m d i a l y s i s [Lima et al., 1983]. Lima et al. s tudied the consequence of volume s h i f t on the binding of c l o f i b r a t e , l i d o c a i n e , d isopyramide, propranolol and diazepam and found that drugs with higher plasma pro te in binding seemed to have lower volume s h i f t . In our prote in binding study, we d id not observe any apparent volume s h i f t , probably due to the short d i a l y s i s time (6 h) and the high prote in binding of PF. Lui and Chiou [1986] have shown that there was no s i g n i f i c a n t change in pH in the d i a l y s i s so lu t ions (phosphate b u f f e r , pH 7.4) i f the d i a l y s i s time was l e s s than 6 h, although they recommended presoaking of the d i a l y s i s c e l l s in 70% ethanol overnight to minimize v a r i a b i l i t y in the pH of the d i a l y s i s s o l u t i o n s . When longer d i a l y s i s times are necessary , b a c t e r i a l growth can cause a pH s h i f t in d i a l y s i s s o l u t i o n s and/or a decrease in the amount of prote in a v a i l a b l e fo r drug binding as a r e s u l t of hydro lyza t ion of plasma p r o t e i n s . Bac ter ia l growth has been shown to reduce the percent of prote in binding of qu in id ine in dog plasma, and s a l i c y l a t e in human plasma [IIet t et al., 1975]. The use of kanamycin to i n h i b i t b a c t e r i a l growth seemed to solve the problem without i n t e r f e r i n g with the pro te in binding of the drugs tested [IIet t et al., 1975]. Another s o l u t i o n was the use of sodium azide as a preservat ive to prevent pH s h i f t but caut ion must be exerc ised i f azide ions i n t e r f e r e with the binding of the drug of i n t e r e s t [Lui and Chiou, 1986]. 4 .4 .2 Binding C h a r a c t e r i s t i c s and Concentration-Dependent Serum Prote in Binding o f Propafenone Two main c l a s s e s of binding s i t e s on serum prote ins were i d e n t i f i e d f o r PF, one with h i g h - a f f i n i t y and low-capaci ty and the other with low-181 a f f i n i t y and h igh -capac i ty (Figure 36). The h i g h - a f f i n i t y , low-capaci ty b inding s i t e i s usua l l y associated with AAG while the l o w - a f f i n i t y , h igh-capac i ty b inding s i t e is genera l ly assoc ia ted with albumin [Javaid et al., 1983]. We have a lso demonstrated that the serum pro te in binding of PF i s concentrat ion- independent in vitro wi th in the concentrat ion range of 0 .25-1 .5 zzg/mL. However, the binding i s concentrat ion-dependent at PF concentra t ions greater than 1.5 /zg/mL (Table 12). Using p u r i f i e d human AAG, G i l l i s et al. [1985] showed that PF was bound to t h i s prote in and there were two c l a s s e s of binding s i t e s on AAG f o r PF, both with high a f f i n i t y . Serum AAG concentrat ion (normal range: 70-110 mg/dL) i s normally 100 times lower than serum albumin concentrat ion (normal range: 3 .9 -5 .5 g / d L ) . Furthermore, AAG represents a h i g h - a f f i n i t y , low-capac i ty binding s i t e which can be r e a d i l y saturated by increas ing drug concentrat ions [Routledge, 1986]. It may expla in why PF exh ib i ted apparent concent ra t ion-dependent serum pro te in binding at higher PF concent ra t ions . 4 .4 .3 C l i n i c a l Impl icat ion of the Concentration-Dependent Serum Prote in Binding of Propafenone PF has been demonstrated to have potent ia l fo r use as an e f f e c t i v e ant iar rhythmic agent [Rudolph et al., 1979; Connol ly et al., 1983a; Bre i thard t et al., 1984; Hodges et al., 1984; Podrid et al., 1984; Salerno et al., 1984]. However, such treatment i s sometimes accompanied by severe s ide e f f e c t s , most notably card iovascu la r in o r i g i n [Schlepper, 1987]. The therapeut ic range of PF i s suggested to be 0.5-2 /zg/mL [Seipel and B r e i t h a r d t , 1980], although a great i n t e r i n d i v i d u a l v a r i a b i l i t y has been shown [Connol ly et al., 1983b; Salerno et al., 1984; Siddoway et al., 1984a] and cons iderab le overlap between the therapeut ic and t o x i c ranges 182 has been demonstrated. In the presence of concentrat ion-dependent b ind ing , the f ree drug concentrat ion i s no longer a constant f r a c t i o n of the to ta l plasma concentra t ion and a s l i g h t change in to ta l drug concentrat ion, may cause a f l u c t u a t i o n in f ree drug concentra t ion leading to an a l t e r a t i o n in the pharmacological e f f e c t ( therapeut ic and/or t o x i c ) . No evidence fo r s i g n i f i c a n t concentrat ion-dependent changes in f ree f r a c t i o n was observed wi th in the PF concentra t ion range of 0 .5 -1 .5 /xg/mL, which covers the greates t propor t ion o f the proposed therapeut ic range (0.5-2 /jg/mL). However, concentrat ion-dependent b inding was demonstrated at PF concentrat ions greater than 1.5 ng/mL. In other words, f l u c t u a t i o n s of f ree drug concentra t ion may occur when PF serum concentrat ions approach the upper end of the proposed therapeut ic range. When there i s an untoward accumulation o f PF in p a t i e n t s , f o r example, as seen with hepat ic dysfunct ion [Lee et a 7 . , 1987], i t would appear that the potent ia l e x i s t s f o r v a r i a b l e PF f ree f r a c t i o n at the upper l i m i t s of c l i n i c a l l y encountered therapeut ic drug concent ra t ions . A l t e r n a t i v e l y , f l u c t u a t i o n s in f ree PF concentra t ion could a lso be a f fec ted by fac to rs such as age, concomitant drug therapy (displacement of PF from prote in binding s i t e ) and c e r t a i n p h y s i o l o g i c a l or pa tho log ica l s ta tes which can cause a q u a l i t a t i v e or q u a n t i t a t i v e change in the binding pro te in (s ) or a l t e r a t i o n s in the d i s t r i b u t i o n of the binding pro te in (s ) in the body [Wandell and Wilcox-T h o l e , 1983; Levy and Morel and, 1984; Kwong, 1985]. 4 .4 .4 E f f e c t of Uremia and Renal F a i l u r e on the Serum Prote in Binding of Propafenone The e f f e c t of renal d isease on plasma binding of basic drugs is heterogeneous. The binding may be increased , unchanged or decreased, 183 depending on the r e l a t i v e con t r ibu t ion of the i n d i v i d u a l plasma prote ins to the t o t a l b inding and the type and s e v e r i t y of d isease [ P i a f s k y , 1978; P i a f s k y , 1980]. Pat ients with terminal uremia have been shown to have increased concentrat ions of AAG in plasma [Henriksen et al., 1982; Docci and T u r c i , 1983; P a c i f i c i et al., 1986]. In our study, the AAG concent ra t ions observed in mid-range uremic pa t ien ts were not s i g n i f i c a n t l y d i f f e r e n t from those observed in healthy s u b j e c t s . However, the AAG concent ra t ions in pat ients with renal f a i l u r e were twice that noted in heal thy vo lunteers and propafenone f ree f r a c t i o n was reduced by approximately 30% (Figure 38). With the exc lus ion o f the data of one renal f a i l u r e p a t i e n t , who had a much lower AAG concentra t ion (98.8 mg/dL) compared to the other pat ients and a PF f ree f r a c t i o n (0.042) which was not s i g n i f i c a n t l y d i f f e r e n t from healthy vo lunteers , the f ree f r a c t i o n of PF was reduced by approximately 44% in serum from pa t ien ts with renal f a i l u r e . In pooled uremic serum, the f ree f r a c t i o n of PF was approximately 50% of that observed in normal serum throughout the concentra t ion range studied (1-5 /jg/mL) (Table 1.2). The decreased PF f ree f r a c t i o n in pa t ien ts with increased serum AAG concent ra t ion , as well as the p o s i t i v e and s i g n i f i c a n t c o r r e l a t i o n between AAG concentrat ion and the b inding r a t i o o f PF observed f u r t h e r impl ies that AAG i s an important binding pro te in fo r PF in serum, as noted by G i l l i s et al. [1985]. This i s in accordance with the f i n d i n g that p ro te in binding of some bas ic drugs, such as diazepam and l i d o c a i n e , which bind l a r g e l y to AAG, was increased in plasma obtained from pat ients with renal f a i l u r e and the increased binding were assoc ia ted with increased AAG concent ra t ion [Grossman et al., 1982], S i m i l a r r e s u l t s were a lso demonstrated between increased AAG concentrat ions and decreased drug free f r a c t i o n f o r l i d o c a i n e [Edwards et al., 1981] and qu in id ine [Edwards et 184 al., 1984] in plasma obtained from pat ients a f t e r traumatic i n j u r y . In a v a r i e t y of inflammatory condi t ions and other d isease s ta tes when AAG concentra t ion may increase four or f i v e - f o l d [Wi lk inson, 1983], a potent ia l increase in pro te in binding can be expected. The plasma prote in binding of propafenone may a lso increase in pat ients with c a r d i a c dysrhythmias complicated by myocardial i n f a r c t i o n , s ince an increase in AAG concentra t ion a f t e r myocardial i n f a r c t i o n i s well documented [Johansson et al., 1972; Snyder et al., 1975], and thus f ree drug monitor ing may be appropr iate in these p a t i e n t s . 4 .4 .5 Free Drug Monitor ing of Propafenone PF i s a high c learance drug and i t s hepat ic e l i m i n a t i o n i s per fus ion r a t e - l i m i t e d . That i s , i t s c learance depends on hepat ic blood flow and i s not a f fec ted by plasma prote in binding changes [Wilkinson and Shand, 1975]. As a r e s u l t , average s teady-s ta te serum to ta l drug concentrat ion i s unaffected by changes in prote in b i n d i n g . However, average s teady-s ta te serum free drug concentrat ion w i l l be a f fec ted [MacKichan, 1984; Rowland, 1984]. Since to ta l drug concentrat ion i s more commonly monitored than f ree drug concen t ra t ion , a change in f ree drug concentra t ion may go undetected. In those instances where a drug is h igh ly pro te in bound, possesses a narrow therapeut ic range, d i s p l a y s a poor d o s e - e f f e c t r e l a t i o n s h i p and exh ib i t s a concentrat ion-dependent b ind ing , the measurement of f ree drug concentrat ion should be considered to improve therapeut ic response and reduce t o x i c i t y [Levy and Moreland, 1984]. Since PF d i s p l a y s most of the above mentioned c h a r a c t e r i s t i c s , i t would appear that t h i s drug may benef i t from the assessment of f ree drug concentrat ions in serum. 185 4.5 Phenobarbital Treatment in Healthy Non-Smokers: Pharmacokinetics and Binding Studies of Propafenone and 5-Hydroxy Propafenone 4.5.1 Data F i t t i n g When the serum concentra t ion versus time data of PF was f i r s t f i t t e d into AUTOAN, the k i n e t i c parameters f o r the best f i t pharmacokinetic open-model were obta ined . These i n i t i a l parameters were then analyzed by NONLIN [Metz ler , 1974] to obtain a bet ter estimate of f3, k a and other parameters. However, in some s u b j e c t s , appreciable e r ro r was noted in the apparent best f i t processed by NONLIN as compared to AUTOAN or hand s t r i p p i n g of the data . Since the value of ft estimated from AUTOAN were not s i g n i f i c a n t l y d i f f e r e n t from values obtained by hand s t r i p p i n g of the data , the estimates from AUTOAN were used. In a d d i t i o n , the c a l c u l a t i o n of most of the k i n e t i c parameters was not a f fec ted by model f i t t i n g s ince C m a x and t m a x were obtained from i n d i v i d u a l serum concentrat ion versus time curves whi le AUC and Vg-ss were obtained by non-compartmental methods. 4.5.2 S t a t i s t i c a l A n a l y s i s In the a n a l y s i s of the d i f f e r e n c e in the k i n e t i c parameters before and a f t e r phenobarbital treatment, the Wilcoxon paired-sample tes t was used. It i s a nonparametric analogue of or a l t e r n a t i v e to the paired t - t e s t (Zar, 1984). Since we can not assume that the d i f f e r e n c e s in the k i n e t i c parameters before and a f t e r phenobarbital treatment are normally d i s t r i b u t e d , the Wilcoxon paired-sample tes t i s more appropr ia te . 4 .5 .3 In te r ind iv idua l V a r i a t i o n in K i n e t i c Parameters of Propafenone There was a large i n t e r i n d i v i d u a l v a r i a t i o n in most of the 186 pharmacokinetic parameters of PF between non-smoking subjects (Table 13). Factors that have been shown to contr ibute to the v a r i a t i o n in drug d i s p o s i t i o n [ V e s e l l , 1982; S e l l e r s et al., 1983] were i d e n t i f i e d or c a r e f u l l y c o n t r o l l e d in our study. These fac to rs inc lude age ( s e l e c t i o n of subjects was r e s t r i c t e d to a populat ion age range of 20-45 y ) , body composit ion (normal weight and normal he igh t ) , r a c i a l background (Caucasian) , sex (male), tobacco exposure (non-smokers who d id not have a h i s t o r y of smoking or who had not smoked a c i g a r e t t e f o r 15 y e a r s ) , a lcohol consumption (mild to moderate) and disease state (hea l thy ) . In a d d i t i o n , subjects fas ted f o r 12 h p r i o r to the study and d id not ingest any food u n t i l 4 h a f t e r PF admin is t ra t ion and the contents of the meals (lunch and dinner) on the f i r s t day of each phase of the study were c o n t r o l l e d ( a l l subjects were given the same type of food) to decrease the in f luence of t h i s f a c t o r on PF metabolism, s ince the b i o a v a i l a b i l i t y of PF was shown to be s i g n i f i c a n t l y in f luenced by food [Axelson et al., 1987]. However, the long-term e f f e c t of d i e t , which has been shown to cont r ibute to i n t e r i n d i v i d u a l v a r i a t i o n s in drug metabolism [ V e s e l l , 1984], was not considered in our study. Furthermore, although a l l non-smoking subjects were ' r a p i d ' metabol izers based on the value of t h e i r C L i n t c o n t r o l (>0.5 L /min) , there was a wide range in t h i s parameter among the subjects (0 .8-13.3 L /min) , i n d i c a t i n g a large i n t e r i n d i v i d u a l v a r i a t i o n in t h e i r a b i l i t y to metabol ize drugs. Therefore , p h y s i o l o g i c a l f a c t o r s ( e . g . n u t r i t i o n a l s t a t u s ) , long-term e f f e c t of d i e t and genet ic f a c t o r (the i n d i v i d u a l ' s a b i l i t y to metabolize drugs) , as well as other environmental f a c t o r s ( e . g . environmental or occupational exposure to drugs and other xenob io t ics ) [Conney et al., 1977; Mucklow, 1988] may have cont r ibuted to the v a r i a t i o n in the pharmacokinetic parameters of PF observed in these 187 s u b j e c t s . 4 .5 .4 Induction of Propafenone Metabolism by Phenobarbital I n t r i n s i c c learance ind ica tes the maximal a b i l i t y of the l i v e r to i r r e v e r s i b l y remove drug by a l l pathways in the absence of any flow l i m i t a t i o n s [Wilkinson and Shand, 1975]. Phenobarbital induces hepat ic metabol iz ing enzyme a c t i v i t y and increases the CL-j n ^ of PF. The increase in C L . j n t of PF a f te r phenobarbital treatment was h igh ly v a r i a b l e among s u b j e c t s , ranging from a 1.1 to a 9.3 f o l d increase in t h i s parameter (Table 13). The e f f e c t of increas ing i n t r i n s i c c learance on the t o t a l blood concentra t ion versus time curve a f t e r oral admin is t ra t ion of a t o t a l l y metabolized high clearance drug was f u l l y descr ibed and d iscussed by Wilk inson and Shand [1975]. For a high c learance drug l i k e PF, induct ion can only increase the ex t rac t ion r a t i o by a small f a c t o r s ince i t s i n i t i a l ex t rac t ion r a t i o i s already very high (c lose to one). As a r e s u l t , there was minimal change in hepat ic c learance and therefore no change in the t^g of PF. However, t h i s change increased even fu r ther the a l ready extensive f i r s t - p a s s metabolism a f t e r oral admin is t ra t ion and decreased the systemic a v a i l a b i l i t y of PF. This was observed in the s i g n i f i c a n t reduct ion in C m a x and AUC of PF a f t e r phenobarbital treatment (Table 13). The percent decrease in C m a x a f t e r enzyme induct ion was very s i m i l a r to the percent decrease in serum AUC in most sub jec ts . The ora l c learance of several ^ -adrenerg ic b l o c k e r s , inc lud ing metopro lo l , a lp reno lo l and p r o p r a n o l o l , has been shown to be increased by 50-500% a f t e r r i f a m p i c i n and phenobarbital treatment [Branch and Herman, 1984]. The oral c learance of PF was increased by 10-831% in the non-smoking subjects a f te r phenobarbital treatment. 188 Since PF undergoes aromatic hydroxy!at ion to 5-hydroxy PF, a pathway known to be induced by phenobarb i ta l , i t was an t ic ipa ted that an increase in the serum concentrat ion of 5-hydroxy PF a f t e r enzyme induct ion would be observed. Contrary to what was expected, the serum l e v e l s , C m a x and AUC of 5-hydroxy PF were reduced a f t e r phenobarbital treatment (Table 16). Since 5-hydroxy PF i s fu r ther metabolized to g lucuronide and s u l f a t e conjugates, an increase in the serum leve l of 5-hydroxy PF may not be observed i f aromatic hydroxyla t ion i s s t i l l the r a t e - l i m i t i n g step a f t e r enzyme i n d u c t i o n . Eichelbaum et a7. [1986] studied the e f f e c t s of admin is t ra t ion of three enzyme inducing agents, a n t i p y r i n e , phenobarbitone and r i f a m p i c i n , on spar te ine metabolism in ' e x t e n s i v e ' and 'poor ' metabol izer s u b j e c t s . They found that spar te ine metabolism was induced in both ' e x t e n s i v e ' and ' p o o r ' metabol izer s u b j e c t s . However, the ox idat ion pathway was not induced s ince there was a decrease in the ur inary accumulation of 2 and 5-dehydro-s p a r t e i n e . Eichelbaum et al. suggested that 2 and 5-dehydrosparteine undergo f u r t h e r metabolism and that t h i s metabolic pathway was induced. They a lso found that in ' p o o r ' metabol izer s u b j e c t s , enzyme induct ion d id not lead to a change in phenotype. They concluded that the regu la t ion of the cytochrome P-450 isozyme involved in polymorphic debr isoqu ine /spar te ine metabolism i s predominantly under genet ic contro l [Inaba et al., 1980; Eichelbaum et al., 1982; Inaba et al., 1982] and that enzyme inducing agents have a minor in f luence [Eichelbaum et al., 1986]. Propafenone undergoes metabolism via a polymorphic ox idat ive pathway, presumably by the same isozyme as fo r debr isoquine and sparte ine [Siddoway et al., 1983; Siddoway et al., 1987]. It i s p o s s i b l e that the cytochrome P-450 isozyme involved in polymorphic ox ida t ive metabolism of PF is n o n - i n d u c i b l e . 189 Glucuron idat ion i s a pathway known to be induced by phenobarb i ta l . A reduct ion in the renal excret ion of the conjugates of 5-hydroxy PF and 5-hydroxy-4-methoxy PF (expressed as percent of dose) was a lso noted (Table 19). Th is can be explained by the fac t that the major excre t ion route for the g lucuronides of 5-hydroxy PF in man i s b i l i a r y excre t ion fol lowed by faeca l excre t ion without appreciable enterohepat ic c i r c u l a t i o n [Hege et al., 1984a]. Furthermore, chronic admin is t ra t ion of phenobarbital and other enzyme inducing agents increases the b i l i a r y excre t ion of c e r t a i n xenob io t i cs and/or metabol i tes [Fujimoto et a / . , 1965; Bernste in etal., 1968; Go lds te in and Taurog, 1968; K laassen, 1970; Lev ine , 1970a, 1970b; Schlede et al., 1970; Levine , 1972; Cooke et al., 1973; Javor et al., 1973; Lev ine , 1974; Whelan and Combes, 1975; Roerig et al., 1976]. Four mechanisms f o r t h i s process have been proposed [Whelan and Combes, 1975]. F i r s t , phenobarbital increases the l i v e r content of a cytoplasmic organic anion b inding p r o t e i n , l i gand in [Reyes et al., 1971], thereby increas ing the hepat ic uptake of drug or metabol i te . Th is w i l l increase the b i l i a r y excre t ion of a compound only i f hepat ic uptake i s the r a t e - l i m i t i n g step in hepat ic t ranspor t from blood to b i l e . Second, phenobarbital induces the microsomal drug-metabol iz ing enzymes and increases the hepat ic metabolism of drugs [Conney, 1967]. This w i l l increase the b i l i a r y excre t ion of a metabol i te i f the metabol i te is excreted into the b i l e at a rate f a s t e r than that of the in tac t drug. T h i r d , phenobarbital increases b i l i a r y flow [K laassen, 1969] and increases the e f f i c i e n c y of t ranspor t of drug or metabol i te from l i v e r c e l l s into b i l e . Fourth , phenobarbital increases l i v e r s i z e [Conney, 1967] and hepatomegaly inc ludes not only hypertrophy but a lso hyperp las ia of hepatocytes [Fouts and Rogers, 1965; A r g y r i s , 1968]. Phenobarbital treatment may r e s u l t in the synthes is of some new 190 c a n a l i c u l a r t ranspor t ing un i ts which f a c i l i t a t e the t ranspor t of a compound in to the b i l e [Whelan and Combes, 1975]. Th is p o s s i b i l i t y i s supported by the f a c t that increased l i v e r s i ze and increased b i l i a r y flow are c h a r a c t e r i s t i c s of phenobarbital treatment, which are not shared by other enzyme inducing agents such as 3-methylcholanthrene and 3,4-benzo(a)pyrene [K laassen, 1969]. For drugs that are metabolized in the l i v e r p r i o r to b i l i a r y e x c r e t i o n , i t i s d i f f i c u l t to i d e n t i f y the t rue mechanism r e s p o n s i b l e f o r enhanced b i l i a r y excret ion s ince the o v e r a l l process inc ludes hepat ic uptake, hepat ic metabolism and t ranspor t from l i v e r c e l l s in to b i l e . Th is i s fur ther complicated by the f a c t that the r a t e - l i m i t i n g step f o r the o v e r a l l process can be d i f f e r e n t f o r d i f f e r e n t compounds. For example, metabolism i s the r a t e - l i m i t i n g step f o r compounds such as methadone [Roerig et al., 1976], 3-methylcholanthrene [Levine, 1972], 3,4-benzo(a)pyrene [Levine, 1970a] and 7,12-dimethylbenzanthracene [Levine, 1974] whi le t ranspor t into the b i l e is the r a t e - l i m i t i n g step f o r compounds such as phenolphtha le in , 4-methyl-umbel 1 i ferone and 8 -hydroxychinol ine [Mulder, 1973]. It was suggested that when phenobarbital enhances the b i l i a r y excre t ion of a compound, more than one of the above mechanisms may be s t i m u l a t e d . If phenobarbital does increase the b i l i a r y excre t ion of the g lucuronides of 5-hydroxy PF and s ince b i l i a r y excre t ion i s the major excre t ion route for t h i s metabol i te , an increase in the amount of g lucuron ides as a r e s u l t of enzyme induct ion may be observed in the feces and not in the u r ine . This would then exp la in the lack of an increase in the propor t ion of dose being excreted by t h i s pathway. C o l l s t e et a / . [1979] examined the in f luence of pentobarbi ta l on the e f f e c t and plasma l e v e l s of a lprenolo l and i t s metabo l i te , 4-hydroxy a l p r e n o l o l . They found that ten days of pentobarbi ta l treatment decreased 191 the plasma concentrat ions of both a lp reno lo l and 4-hydroxy a l p r e n o l o l . The authors concluded that conjugat ion of 4-hydroxy a lp reno lo l or other metabol ic pathways were induced. The f a c t that the induct ion of PF metabolism by phenobarbital d id not r e s u l t in an increase in e i t h e r the plasma concentrat ions of 5-hydroxy PF or the ur inary excre t ion of the conjugates of 5-hydroxy PF and 5-hydroxy-4-methoxy PF may be due to an induct ion of metabol ic pathways other than aromatic hydroxyla t ion and g l u c u r o n i d a t i o n . These other metabol ic pathways may become important a f te r phenobarbital treatment. Indeed, an induct ion of the ' i n d u c i b l e ' pathways can cause a reduct ion in the v n o n - i n d u c i b l e ' pathways, which may exp la in why we observed a decrease in the serum l e v e l s of 5-hydroxy PF a f t e r enzyme i n d u c t i o n . 4 .5 .5 S a l i v a r y Excret ion of Propafenone The excre t ion of drugs in human s a l i v a was reported as ear ly as in 1965 by B o r z e l l e c a and Cher r ick . Since then, the s a l i v a r y excre t ion of a wide v a r i e t y of drugs inc lud ing ant iconvulsants ( e . g . b a r b i t u r a t e s , carbamazepine, phenytoin) , s a l i c y l a t e s , sul fonamides, a n t i p y r i n e , t h e o p h y l l i n e , l i t h i u m , ant iarrhythmic agents ( e . g . procainamide, q u i n i d i n e , d isopyramide, l i d o c a i n e ) , psychotropic agents ( e . g . chlorpromazine, diazepam) and ethanol have been examined and inves t iga ted by d i f f e r e n t researchers [review in Mucklow, 1982]. It genera l l y accepted that the unbound ( free) drug in plasma was in e q u i l i b r i u m with drug in s a l i v a . There fore , most of the previous studies focused on the c o r r e l a t i o n between s a l i v a and plasma drug l e v e l s and f a c t o r s that a f fec ted s a l i v a r y drug l e v e l . For a number of compounds a c l o s e , l i n e a r c o r r e l a t i o n has been e s t a b l i s h e d between plasma and s a l i v a l e v e l s . Measurement of s a l i v a r y drug 192 concentra t ion has the advantage of being a s imple , e a s i l y a c c e s s i b l e and pa in less (non- invasive) way o f obta in ing pharmacokinetic data (e.g. a n t i p y r i n e , s a l i c y l a t e ) [Graham and Rowland, 1972; Fraser et al., 1976], est imat ing drug plasma prote in binding (e.g. amobarbital) [Inaba and Kalow, 1975], assur ing compliance and monitoring drug therapy (e.g. phenytoin, carbamazepine) [Rylance and Moreland, 1981] and e s t a b l i s h i n g the existence of metabol ic induct ion by phenobarbital of the ant iarrhythmic agent, disopyramide [ K a p i l , 1985; Kapi l et al., 1987]. Ki l lmann and Thaysen [1955] demonstrated that the r e l a t i o n s h i p between s a l i v a and plasma concentrat ions of d i f f e r e n t sulphonamides was dependent on the pK a of the drug and the pH of both s a l i v a and plasma. Mucklow and co-workers [1978] have fu r ther demonstrated that fo r weak ac ids with pKa>7 [e.g. phenobarbital (pK a 7 .2 ) , phenytoin (pK a 8.3)] and weak bases with pK a<5.5 [e.g. an t ipyr ine (pK a 1 .4 ) ] , the sa l iva /p lasma drug concentra t ion r a t i o i s constant , i r r e s p e c t i v e of any change in s a l i v a flow rate or pH. However, for a c i d i c drugs with pKa<7 [e.g. chlorpropamide (pK a 4 .8 ) , tolbutamide (pK a 5.4)] and bas ic drugs with pK a>5.5 (e.g. meperidine (pK a 8 .7 ) , propranolol (pK g 9 . 4 ) ] , the r a t i o i s extremely s e n s i t i v e to any change in s a l i v a flow rate or pH. Since PF i s a weak base with a pK a of 9.0 and i s l a r g e l y ion ized at serum pH (7 .4 ) , i t could be an t ic ipa ted that i t s p a r t i t i o n i n g into s a l i v a (pH 5.5-7.8) may be high and dependent on s a l i v a r y pH. This is apparently true as shown by the negative and s i g n i f i c a n t ( c o n t r o l , r = -0 .461; phenobarb i ta l - t rea ted , r = -0.422; p<0.05) c o r r e l a t i o n found between the sa l iva /serum PF concentrat ion r a t i o and the pH of s a l i v a . This ind ica tes that an increase in the pH of s a l i v a w i l l cause a decrease in s a l i v a r y PF concent ra t ion . Our r e s u l t s a lso showed that PF s a l i v a r y concentrat ion in the non-smokers was cons iderab ly 193 higher than PF serum f ree concentrat ion (Table 17). Propafenone s a l i v a r y concentra t ion was approximately 21% (contro l ) and 27% (phenobarbi ta l -t reated) of PF serum to ta l concent ra t ion , with considerable i n t e r - and i n t r a i n d i v i d u a l v a r i a t i o n (Table 15). The mean sa l i va /serum PF concentra t ion r a t i o of the e ight non-smokers in the contro l s ta te was 0.21 + 0.08. Th is i s s i m i l a r to the r e s u l t obtained by Mason et a7. [1987], who found that the s a l i v a / p l a s m a PF concentrat ion r a t i o at s teady-s ta te (300 mg PF every 8 h f o r 6 days) in 24 healthy male subjects was 0.25 + 0.11. Matin et a7. [1974] studied the s a l i v a r y excret ion of to lbutamide. They attempted to c o r r e l a t e s a l i v a and plasma drug concentrat ions with the u l t imate goal of p r e d i c t i n g plasma drug l e v e l s from s a l i v a r y excret ion da ta . Inaba and Kalow [1975] used the equation developed by Matin et a7. [1974], which incorporates sa l iva /serum drug concentrat ion r a t i o , pK a of the drug and pH of s a l i v a and plasma, to c a l c u l a t e the percent of unbound ( f ree) amobarbital in serum and found that the estimated and measured (by e q u i l i b r i u m d i a l y s i s ) values were in good agreement. A s i m i l a r equation developed f o r bas ic drugs [Equation 7, Matin et a 7 . , 1974] was used to c a l c u l a t e the percent of f ree (unbound) PF in serum. If PF concentrat ion in s a l i v a i s t r u l y an i n d i c a t o r of PF free concentrat ion in serum, then by measuring PF serum t o t a l concent ra t ion , PF s a l i v a r y concentrat ion and s a l i v a pH, an est imat ion of PF f ree f r a c t i o n can be obtained without the more time-consuming method of equ i l ib r ium d i a l y s i s . Our r e s u l t s showed that the estimated percent of f ree PF in serum was s i g n i f i c a n t l y higher than the values obtained from equ i l ib r ium d i a l y s i s (Table 17), i n d i c a t i n g that other f a c t o r s may a f f e c t or be involved in the excre t ion of PF in s a l i v a . The d iscrepancy can perhaps be explained by the hypothesis and assumption of which Equation 7 was based on. These inc lude : a) excre t ion 194 i s a simple pass ive d i f f u s i o n process; b) only the non-prote in bound and the unionized form of the drug can d i f f u s e across the ep i the l ium of the s a l i v a r y gland [Ki l lmann and Thaysen, 1955]; c) the unbound drug in serum i s in e q u i l i b r i u m with s a l i v a [Huffman, 1975; Koysooko et al., 1974]; d) e q u i l i b r i u m across the membrane i s reached in s p i t e of the continuous s e c r e t i o n and outf low of s a l i v a and e) drug i s unbound in s a l i v a or prote in b inding of drug in s a l i v a i s n e g l i g i b l e [Kil lmann and Thaysen, 1955; Rasmussen, 1964;.Matin et al., 1974]. Some of the above assumptions may not be t rue f o r PF. B o r z e l l e c a and Putney [1970] have demonstrated, in the dog, that s a l i v a r y excre t ion of s a l i c y l a t e i s more than simple d i f f u s i o n of the drug across membranes. The s a l i v a r y excret ion of s a l i c y l a t e i s best descr ibed by two processes i n c l u d i n g f i l t r a t i o n of drug ( i n c l u d i n g ion ized form) across the basal membrane through aqueous pores in to the e p i t h e l i a l c e l l , fo l lowed by d i f f u s i o n of the drug (only unionized form) across the ap ica l membrane in to the s a l i v a r y f l u i d . The f i r s t process i s governed by a concentra t ion grad ient and i s independent of pH while the second process depends on s a l i v a pH and is r a t e - l i m i t i n g . The p o s s i b i l i t y of ac t i ve t ranspor t cannot be ru led out, although only l i m i t e d evidence i s a v a i l a b l e to suggest a c t i v e secre t ion of drugs (so fa r only l i t h i u m , p e n i c i l l i n , metoprolol and toca in ide ) into s a l i v a [Borze l leca and C h e r r i c k , 1965; Groth et al., 1974; Dawes et al., 1978; P i l l a i et al., 1984], In the case of t o c a i n i d e , s t e r e o s p e c i f i c s a l i v a r y excret ion of t o c a i n i d e enantiomers [R(-) and S(+) isomers] has been observed. The t r a n s f e r of toca in ide enantiomers from plasma to s a l i v a does not c o r r e l a t e with s a l i v a r y pH and is more pronounced f o r the R(-) -enantiomer of the drug [ P i l l a i et al., 1984]. There are two potent ia l sources of e r ror that may cause i n c o r r e c t 195 es t imat ion of percent of f ree drug in serum by Equation 7 [Mucklow, 1982]. F i r s t , the pH of s a l i v a measured a f te r expectorat ion may be higher than the pH of s a l i v a at the moment of s e c r e t i o n , due to the l o s s of carbon d i o x i d e , formed from carbonic ac id in s o l u t i o n , when s a l i v a comes into contact with a i r . Second, drug may bind to p a r t i c u l a t e matter in whole s a l i v a ( e . g . phenytoin) [Anavekar et al., 1978] or to buccal mucosa ( e . g . trimethoprim) [Eatman et al., 1977]. However, they are u n l i k e l y to cause any e r ror in the es t imat ion of percent f ree drug for PF using Equation 7, s ince both should g ive f a l s e l y low estimates of percent of f ree drug in serum f o r a bas ic drug [Mucklow, 1982]. Another source of e r ror i s perhaps the contamination of s a l i v a samples when the drug tested i s administered o r a l l y [Paxton and Foote, 1979]. However, i t should not be a major problem unless the drug was taken as a syrup , chewable t a b l e t s or crushed t a b l e t s (to c h i l d r e n ) . In a d d i t i o n , contamination should only a f f e c t s a l i v a samples c o l l e c t e d s h o r t l y a f t e r admin is t ra t ion of the drug. In our study, contamination was u n l i k e l y s ince the subjects drank 200 mL of water a f t e r the t a b l e t was swallowed. Furthermore, aberrant ly high PF l e v e l s were not observed in s a l i v a samples c o l l e c t e d s h o r t l y a f t e r PF admin is t ra t ion , i n d i c a t i n g that there was no contamination during the process of i n g e s t i o n . It has been demonstrated that many fac to rs can a f f e c t s a l i v a r y e x c re t ion o f drugs. In addi t ion to pK a (degree of i o n i z a t i o n ) , the l i p i d s o l u b i l i t y of the drug ( r e f l e c t e d by the solvent /water p a r t i t i o n c o e f f i c i e n t ) , molecular weight and spa t i a l con f igura t ion may a lso determine the d i f f u s i b i l i t y of the compound across the membrane [Amberson and Hober, 1932; Rasmussen, 1964]. S a l i v a flow rate i s another important f a c t o r that can a f f e c t d i f f u s i o n of the drug and attainment of equi l ibr ium across the 196 e p i t h e l i a l membrane of the s a l i v a r y g lands . Since PF i s ion ized to an apprec iab le extent at serum pH, i t would seem that s a l i v a pH and flow r a t e , ra ther than serum pH, would have a greater e f f e c t on the s a l i v a r y excret ion of PF. The flow rate of whole s a l i v a in man var ies from 0.5-2.1 mL/min, with cons iderab le ind iv idua l v a r i a t i o n [Becks and Wainwright, 1943]. S t i m u l a t i o n , age [Gutman and Ben-Aryeh, 1974], anxiety and p e r s o n a l i t y ( consc ien t iousness , shrewdness and in t rovers ion ) [Costa et al., 1980] can a f f e c t the flow rate of s a l i v a and are respons ib le f o r the v a r i a b i l i t y in both s a l i v a flow rate and s a l i v a pH, s ince the l a t t e r var ies in d i r e c t propor t ion to s a l i v a flow rate [Mucklow et al., 1978]. The pH of s t imulated s a l i v a has been demonstrated to r i s e by as much as two u n i t s , depending on the increase in flow rate [Dawes and Jenk ins , 1964]. The s a l i v a r y concentrat ion versus time curves of PF (Appendix 3) showed that s a l i v a r y concentrat ions of PF in some subjects f luc tua ted over the 48 h p e r i o d . Since mechanical ly unstimulated whole s a l i v a was c o l l e c t e d in our study, the f l u c t u a t i o n s in s a l i v a r y PF concentrat ions observed in some subjects may be due to the c i r c a d i a n rhythm of s a l i v a flow r a t e . The r e p r o d u c i b i l i t y in the ind iv idua l and the cons iderab le i n t e r i n d i v i d u a l v a r i a t i o n in both the c i r c a d i a n and c i rcannual rhythms of s a l i v a flow rate and composit ion have been f u l l y and ex tens ive ly studied [Dawes, 1972; Dawes and Ong, 1973; Ferguson et al., 1973; Ferguson and F o r t , 1974]. However, whether f l u c t u a t i o n s in s a l i v a r y PF concentra t ion fo l low the same pattern as the c i r c a d i a n rhythm of s a l i v a flow rate in the ind iv idua l subject has yet to be e s t a b l i s h e d . The c o r r e l a t i o n between s a l i v a r y PF concentra t ion and serum PF free concentra t ion was s i g n i f i c a n t (p<0.05) but not c lose (r = 0.435). Furthermore, there was a bet ter c o r r e l a t i o n between serum to ta l 197 concentrat ion and serum free concentrat ion (r = 0.838, p<0.001) than that between serum to ta l concentrat ion and s a l i v a r y concentrat ion (r = 0.702, p<0.001) and that between s a l i v a r y concentrat ion and serum f ree concentrat ion (r = 0.435, p<0.02) (Figure 41). Mason et al. [1987] a lso found a s i g n i f i c a n t but not c lose c o r r e l a t i o n between s teady-s ta te PF serum concentrat ion and s teady-s ta te PF s a l i v a r y concent ra t ion . This suggests that PF s a l i v a r y concentrat ion i s not a p a r t i c u l a r l y good i n d i c a t o r of PF serum free concentrat ion and, the re fo re , should be used with caut ion when p r e d i c t i n g the corresponding serum drug l e v e l . One i n t e r e s t i n g observat ion was that the decrease in s a l i v a r y AUC was very s i m i l a r t o , and not 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, the decrease in serum AUC a f t e r phenobarbital treatment. In s i t u a t i o n s when s e r i a l blood sampling i s not p o s s i b l e , s a l i v a sampling may provide an a l t e r n a t i v e way of est imat ing a l t e r a t i o n s in serum AUC. This observat ion with PF seems even more p r e d i c t i v e than that seen when a s i m i l a r study was conducted in our labora tory with disopyramide, where the v a r i a b i l i t y of s a l i v a r y l e v e l s was greater than that seen in the present study [ K a p i l , 1985; Kapi l et al., 1987]. 4 .5 .6 Prote in Binding of Propafenone Before and A f t e r Phenobarbital Treatment 4 .5 .6 .1 E f f e c t of Heparin on Prote in Binding of Propafenone In our study, the use of heparin ( in sa l ine ) to f l u s h the indwel l ing cannula a f t e r blood sampling was necessary to prevent the formation of c l o t s in the cannula . It i s well known that the use of heparin a f f e c t s the pro te in binding of a number of a c i d i c drugs such as warfar in [Routledge et 198 al., 1979] and d igox in [S to rs te in and Janssen, 1976]. Heparin in vivo re leases l i p o p r o t e i n l i p a s e s and increases the serum concentrat ion of n o n - e s t e r i f i e d f a t t y ac id [Ol ivecrona et al., 1977]. The elevated f ree f a t t y ac id compete with the drug fo r the prote in binding s i t e ( s ) on albumin, r e s u l t i n g in an overest imat ion of drug f ree f r a c t i o n . Brown et al. [1981] have shown that the hepar in- induced drug pro te in binding changes are , to a la rge extent , in vitro a r t i f a c t s r e s u l t i n g from the continued in vitro a c t i v i t y of t r i g l y c e r i d e l i p a s e s . The plasma prote in binding of c e r t a i n bas ic drugs , such as propranolol [Wood et a / . , 1979a], qu in id ine [N i lsen et al., 1977; K e s s l e r et al., 1979] and verapamil [Keefe et al., 1981], have been shown to be a f fec ted by the use of hepar in . Changes in concentra t ions of n o n - e s t e r i f i e d f a t t y ac id seem to a f f e c t the plasma pro te in binding of drugs in which albumin i s the major b inding p r o t e i n , but not drugs which bind mainly to AAG [Grossman et al., 1982]. Although p r o p r a n o l o l , qu in id ine and verapamil bind to AAG, they are probably p r i m a r i l y bound to serum albumin. The e f f e c t of heparin admin is t ra t ion on the b inding of PF has not been examined. However, the plasma pro te in binding o f propranolol had been shown to be unaffected by heparin admin is t ra t ion in doses necessary to maintain the funct ion of indwel l ing ca theters even when n o n - e s t e r i f i e d f a t ty ac id concentra t ion was increased as a r e s u l t of the l i p o l y t i c a c t i v i t y of heparin [ S i l b e r et al., 1980]. In our s tudy, only a small amount of heparin (5 un i ts ) contained in s t e r i l e normal s a l i n e f o r i n j e c t i o n was administered a f te r each blood sample was c o l l e c t e d and as a r e s u l t , an e f f e c t on PF binding would not be a n t i c i p a t e d . A l t e r n a t i v e l y , i t i s l i k e l y that the e f f e c t of heparin on the plasma pro te in binding of drugs i s more important when very la rge doses of heparin (several thousand uni ts in a few hours) are required in 199 cardiopulmonary bypass surgery or in hemodialysis [Naranjo et al., 1980a, 1980b]. 4 .5 .6 .2 E f f e c t of Phenobarbital Treatment on Propafenone Free Frac t ion Studies on the e f f e c t of phenobarbital on the pharmacokinetics of propranolo l in dog [Bai and Abramson, 1982; Vu et al., 1983] and monkey [Branch et al., 1974] i l l u s t r a t e d that the major determinants of hepat ic c l e a r a n c e , such as i n t r i n s i c c learance , l i v e r blood flow and pro te in b i n d i n g , can be a f fec ted by enzyme i n d u c t i o n . For example, Branch et al. [1974] found a 45% increase in l i v e r blood flow a f t e r phenobarbital treatment in the monkey. There i s no evidence that a s i m i l a r change occurs in man. In animal s t u d i e s , phenobarbital was found to ,cause a dramatic increase in plasma AAG concentrat ion and a decrease in serum f ree f r a c t i o n of propranolol in dogs [Bai et al., 1982] and an increase in serum prote in b inding of desmethylimipramine in ra ts [Br inkschul te and Breyer , 1982]. In man, the e f f e c t of phenobarbital on serum AAG concentra t ion and drug pro te in binding i s not c l e a r . For example, Routledge et al. [1981] found that e p i l e p t i c s t reated with ant iconvulsants had serum AAG concentra t ions approximately twice that observed in untreated healthy volunteers and the f ree f r a c t i o n of l i d o c a i n e was reduced in the t reated s u b j e c t s . C o n f l i c t i n g r e s u l t s were observed in e p i l e p t i c s t reated with e i t h e r carbamazepine or phenobarbital [Bruguerol le et al., 1984]. Our r e s u l t s showed that phenobarbital treatment d id not cause any change in the prote in b inding ( f ree f r a c t i o n ) of PF in man (Figure 39). In a d d i t i o n , no change in the serum concentrat ion of AAG, which we have proved to be an important b inding pro te in fo r PF [Chan et al., 1989], was shown a f t e r phenobarbital admin is t ra t ion (Figure 40). Th is i s in agreement with the f i n d i n g of Kapil 200 et al. [1987] that phenobarbital (a lso three weeks treatment) caused no s i g n i f i c a n t change in e i t h e r serum AAG concentra t ion or the f ree f r a c t i o n of disopyramide in man. S i m i l a r r e s u l t s were a lso observed by Herman et al. [1983] on the binding of ^-adrenoceptor b l o c k e r . In terspecies v a r i a t i o n s in the response of induct ion of p ro te in synthes is [Branch and Herman, 1984] or se izure - induced changes in AAG concent ra t ion unrelated to drug treatment may expla in the apparent d isc repancy . The volume of d i s t r i b u t i o n ind ica tes the extent of d i s t r i b u t i o n and b inding of the drug in the body. Since phenobarbital treatment d id not a f f e c t the serum prote in binding and f ree f r a c t i o n of PF, the increase in ^darea /^ o r ^dss^ w a s ^ e l y due to the decrease in systemic a v a i l a b i l i t y (assuming that there was no change in the d i s t r i b u t i o n and b inding of PF in t i s s u e s ) . The decrease in systemic a v a i l a b i l i t y of PF a f t e r phenobarbital treatment was responsib le for the marked reduct ions in C m a x and AUC. 4.6 Phenobarbital Treatment in Healthy Smokers: Pharmacokinetics and Binding Studies of Propafenone and 5-Hydroxy Propafenone 4.6.1 Data F i t t i n g The values of pharmacokinetic parameters of PF were obtained a f te r f i t t i n g the serum concentrat ion versus time data of PF of the e ight healthy smokers in to AUTOAN. 4 .6 .2 In te r ind iv idua l Var ia t ion in K i n e t i c Parameters of Propafenone Like the non-smoking sub jec ts , the smoking subjects a lso showed a la rge i n t e r i n d i v i d u a l v a r i a t i o n in the pharmacokinetic parameters of PF 201 (Table 20). Two of the smoking subjects were ' s low ' metabol izers ( C L ^ j . <0.5 L/min) and contr ibuted to the cons iderab le i n t e r i n d i v i d u a l v a r i a t i o n . Even when the data of these two subjects were excluded, the la rge i n t e r i n d i v i d u a l v a r i a t i o n s t i l l e x i s t e d . Smoking i s added as another important environmental f a c t o r which a f f e c t s the d i s p o s i t i o n of drugs in the smoking popu la t ion . However, smoking i s not an ' independent ' f a c t o r i t s e l f . It has been demonstrated that c i g a r e t t e smoking i s assoc ia ted with age and alcohol consumption [Vestal and Wood, 1980; S e l l e r s et al., 1983]. In our study, smoking subjects were a lso c a r e f u l l y se lec ted employing the same c r i t e r i a as the non-smoking s u b j e c t s . The i r c i g a r e t t e consumption was based on the number of c i g a r e t t e s smoked per day (at l e a s t 20 c i g a r e t t e s per day fo r the past 5 y e a r s ) . However, the extent of e f f e c t that smoking has on drug d i s p o s i t i o n i s modif ied by f a c t o r s that determine 'exposure ' to c i g a r e t t e smoke c o n s t i t u e n t s . These inc lude tobacco c h a r a c t e r i s t i c s (e .g. harvest ing and cur ing p r o c e s s ) , smoking c h a r a c t e r i s t i c s (e .g. burn temperature) , f i l t e r performance (e .g. mater ia l and d e s i g n ) , smoking p r o f i l e (e .g. puf f frequency and d u r a t i o n ) , smoking s t y l e (e .g. pulmonary re ten t ion time) and const i tuent k i n e t i c s (e .g. b i o a v a i l a b i l i t y ) [ S e l l e r s et al., 1983]. Excluding the data of the two ' s low ' metabol izers (subjects GE and MG), there was s t i l l a large i n t e r i n d i v i d u a l v a r i a t i o n in C L ^ n t contro l ° ^ PF in the smoking s u b j e c t s , ranging from 8.0 to 18.2 L/min (2.3 f o l d d i f f e r e n c e ) (Table 20). Long term d i e t and other environmental f a c t o r s may have a lso contr ibuted to t h i s large i n t e r s u b j e c t v a r i a t i o n . Enzyme induct ion d id not convert subject GE to a ' r a p i d ' metabol izer (CL^n^- was 0.4 L/min before and 0.5 L/min a f t e r phenobarbital t reatment) . Although the CL. j n t of subject MG was increased to 1.0 L/min a f t e r phenobarbital 202 treatment, i t was s t i l l low compared to the C L ^ n t c o n t r o l ° ^ m o s t v r a p i d ' metabo l i ze rs . In other words, a f t e r enzyme i n d u c t i o n , subjects GE and MG s t i l l d isp layed long e l i m i n a t i o n h a l f - l i f e , high serum concent ra t ion , low oral c learance and absence of detectab le 5-hydroxy PF, which are c h a r a c t e r i s t i c s of a ' s low ' metabol izer . 4 .6 .3 Induction of Phenobarbital Metabolism by Phenobarbital S i m i l a r r e s u l t s were observed in the smokers as in the non-smokers. The decrease in systemic a v a i l a b i l i t y of PF as a r e s u l t of enhanced f i r s t -pass metabolism by phenobarbital treatment caused a remarkable reduct ion in the serum l e v e l s , C m a x and AUC of PF (Table 20). The increase in oral c learance was not as large as that of the non-smokers, ranging from 23 to 450%. A decrease in the serum concentra t ion of 5-hydroxy PF and a decrease in the renal excre t ion of the conjugates of 5-hydroxy PF and 5-hydroxy-4-methoxy PF was a lso observed a f t e r enzyme induct ion in the smokers. Excluding the data of the two 's low ' metabol izers reduced the i n t e r i n d i v i d u a l v a r i a t i o n in the k i n e t i c parameters of PF among the smoking subjects but had no s i g n i f i c a n t in f luence on the ranges of the percent increase in C L ^ n t and the percent decrease in C m a x and AUC. 4 .6 .4 S a l i v a r y Excret ion of Propafenone Like non-smokers, the s a l i v a concentrat ions of PF in the smokers f luc tua ted over the 48 h (Appendix 6 ) . A negative and s i g n i f i c a n t c o r r e l a t i o n a lso ex is ted between sa l i va /serum PF concentrat ion r a t i o and s a l i v a pH ( c o n t r o l , r = -0 .541; p h e n o b a r b i t a l - t r e a t e d , r = -0 .387; p<0.05), i n d i c a t i n g that an increase in the pH of s a l i v a w i l l cause a decrease in PF s a l i v a r y concent ra t ion . Propafenone concentrat ion in s a l i v a was 203 cons iderab ly higher than PF serum free concentrat ion (Table 24). Propafenone s a l i v a r y concentrat ion was approximately 36% (contro l ) and 40% (phenobarb i ta l - t rea ted) of PF serum to ta l concent ra t ion , with large i n t e r -and i n t r a i n d i v i d u a l v a r i a t i o n s (Table 22). The estimated percent f ree PF in serum was a lso s i g n i f i c a n t l y higher than the values obtained from equ i l ib r ium d i a l y s i s (Table 24), i n d i c a t i n g that f a c t o r s other than the pK a of PF or the pH of s a l i v a and serum, a lso cont r ibuted to the excre t ion of PF in s a l i v a . Unl ike the non-smokers, the c o r r e l a t i o n between s a l i v a r y concentrat ion and serum free concentrat ion i s both c lose (r = 0.920) and s i g n i f i c a n t . However, l i k e the non-smokers, there was a lso a be t te r c o r r e l a t i o n between serum to ta l concentrat ion and serum f ree concentra t ion (r = 0.993, p<0.001) than between serum to ta l concentra t ion and s a l i v a r y concentrat ion (r = 0.940, p<0.001) or between s a l i v a r y concentra t ion and serum free concentrat ion (r = 0.940, p<0.001) (Figure 45). 4.7 Extent of Metabol ic Induction of Propafenone by Phenobarbital in Healthy Non-smokers and Smokers The dose and the durat ion of treatment of the enzyme inducing agent are e s s e n t i a l in e l i c i t i n g maximal enzyme induct ion [Breckenridge et al., 1972; Mignet et al., 1977; Ohnhaus et a / . , 1977; Ohnhaus and Park, 1979; Ohnhaus et a / , 1983]. When the dose is too low or when the durat ion of treatment i s too s h o r t , s i g n i f i c a n t induct ion may not be observed [Breckenridge et al., 1972; Eichelbaum et al., 1986]. In genera l , the extent of enzyme induct ion depends on the e l im ina t ion rate of the enzyme 204 inducing agent and the enzyme turnover r a t e . Microsomal enzyme turnover rate (ranged from 1-6 days [Lai et a 7 . , 1978]), as well as the dose and durat ion of treatment, are more important on the reversa l of enzyme i n d u c t i o n . Enzyme induct ion p e r s i s t s as long as the enzyme inducing agent i s admin is tered . The e l im ina t ion h a l f - l i f e of phenobarbital i s about 24 h. Since i t takes about one week ( f i v e to seven times the h a l f - l i f e ) f o r phenobarbital serum concentrat ion to reach s t e a d y - s t a t e , a minimum of two weeks i s necessary f o r maximal induct ion of microsomal enzymes. In order to ensure maximal i n d u c t i o n , we t reated our subjects with phenobarbital at a therapeut ic dose of 100 mg d a i l y fo r three weeks. Blood samples were obtained p e r i o d i c a l l y to ensure serum phenobarbital concentra t ion had reached s teady -s ta te and was between 10-20 pg/ml at the second stage of the study f o r a l l s u b j e c t s . Several authors [Vesel l and Page, 1969; Breckenridege et a 7 . , 1971; Breckenridge and Orme, 1971] have drawn a general conc lus ion that subjects who metabol ize drugs slowly w i l l show a greater enzyme induct ion than subjects who metabol ize drugs r a p i d l y . Our f i n d i n g s do not support the conc lus ion drawn by these authors. The decrease in AUC a f t e r phenobarbital treatment ranged from 9 to 89% in the non-smoking subjects (Table 13) and 19 to 82% in the smoking subjects (Table 20). The two non-smokers who had the lowest (9%, subject DA) and the highest (82%, subject CA) metabol ic induct ion had s i m i l a r C L ^ n t c o n t r o l ( 8 - 3 a n c l 7 , 5 L /min, r e s p e c t i v e l y ) , a parameter that i n d i c a t e s the i n d i v i d u a l ' s a b i l i t y to metabol ize drug. The smoking subject who had the highest metabolic induct ion (82%, subject DW) was not a ' s low ' metabol izer . On the other hand, the two 's low ' metabol i z e r s (subject GE and MG) d id not show a greater enzyme induct ion (22 and 46% r e s p e c t i v e l y ) compared to the ' r a p i d ' metabo l i ze rs . 205 Furthermore, C L i n t c o n t r o l ^ id n o t c o r r e l a t e with the percent decrease in AUC in both non-smoking (r = -0 .425, p>0.05) and smoking subjects (r = 0.319, p>0.05) (combined non-smokers and smokers, r = 0.048, p>0.05) (F igure 47) . Two hypotheses concerning the in te rsub jec t v a r i a t i o n in the extent of metabol ic induct ion between subjects have been proposed. One was that fo l lowing induct ion there w i l l be a smal ler i n te rsub jec t v a r i a t i o n in drug metabol is ing a c t i v i t y than p r i o r to i n d u c t i o n . Th is hypothesis i s an i n t e r p r e t a t i o n of the conc lus ion drawn by the previous mentioned authors [Vesel l and Page, 1969; Breckenridege et al., 1971; Breckenridge and Orme, 1971]. The other hypothesis was that the range of i n te rsub jec t v a r i a t i o n in drug metabol is ing a c t i v i t y should be as large a f t e r induct ion as before induct ion [Herman et al., 1982]. Our study supports the l a t t e r hypothes is . In f a c t , the c o e f f i c i e n t of v a r i a t i o n in C\-\nt in the non-smokers a f t e r induct ion (119%) was twice that before induct ion (61%) (Table 13) while the c o e f f i c e n t of v a r i a t i o n in CL^ n^. in the smokers before and a f t e r induct ion was s i m i l a r , i n c l u d i n g (76% before and 82% a f te r ) or excluding (42% before and 50% a f te r ) the two 's low' metabol izers (Table 20). Another i m p l i c a t i o n of the conclus ion drawn by the previous mentioned authors [Vesel l and Page, 1969; Breckenridege et a / . , 1971; Breckenridge and Orme, 1971] i s that subjects who metabolize drugs slowly w i l l show a higher serum phenobarbital concentrat ion and a greater percent reduct ion in AUC, provided PF and phenobarbital are metabolized by the same or s i m i l a r route or t h e i r metabolism is under a common regula tory contro l (both undergo metabolism via aromatic hydroxy la t ion ) . Our data demonstrated that although phenobarbital treatment caused a reduct ion in C m a x and AUC of PF, there was no apparent c o r r e l a t i o n between serum phenobarbital concentrat ion 206 and induct ion of PF metabolism, as expressed by [ (AUC p -AUC c ) /AUC c ]x l00% (r = -0 .323, p>0.05) (Figure 46). D i f f e ren t subjects (NP and CA) required d i f f e r e n t serum concentrat ions of inducing agents to produce the same induct ion e f f e c t (64%). Although some subjects ( e . g . subjects GE and MA) produced s i m i l a r induct ion e f f e c t s (20%) with s i m i l a r serum phenobarbital concent ra t ion (20 /xg/mL), others ( e . g . subjects CA and SG) produced a d i f f e r e n t induct ion e f f e c t (64 and 9%) with i d e n t i c a l serum phenobarbital concent ra t ion (13 /zg/mL). S i m i l a r r e s u l t s were found between the extent of induct ion of a lp reno lo l metabolism by s p e c i f i c pentobarb i ta l plasma concentra t ions [ C o l l s t e et al., 1979] and l i kewise the extent of induct ion of war far in metabolism by quinalbarbi tone plasma concentrat ions [Breckenridge et al., 1972]. The poor c o r r e l a t i o n between the extent of induct ion of PF by phenobarbital suggests that PF and phenobarbital are metabol ized by d i f f e r e n t isozymes of cytochrome P-450. Kellermann and Luyten-Kellermann [1977] s tudied the e f f e c t of enzyme induct ion by phenobarbital on ant ipyr ine h a l f - l i v e s . These authors suggested that i n t e r i n d i v i d u a l v a r i a t i o n in the metabol ic rate of the inducing agent should be taken into account when conducting induct ion s tud ies in order to obtain comparable r e s u l t s in a l l sub jec ts . However, in t h e i r s t u d i e s , even a f t e r the dose of phenobarbital was adjusted to the i n d i v i d u a l rates of metabolism of the inducing agent, they could not f i nd any c o r r e l a t i o n between the i n i t i a l an t ipyr ine h a l f - l i f e and the percent decrease in plasma h a l f - l i f e . These authors a lso suggested that other f a c t o r s such as i n t e r i n d i v i d u a l d i f f e r e n c e s in the pro te in binding or d i f f e r e n c e s in renal excret ion of phenobarbital may cont r ibute to the observed v a r i a t i o n in enzyme induc t ion . They concluded that s ince the decrease in an t ipyr ine h a l f - l i v e s ranged from 13.3 to 30.6%, the 207 i n t e r i n d i v i d u a l v a r i a t i o n in the magnitudes of phenobarbital -evoked induct ion may be under genet ic c o n t r o l . Considerable i n t e r i n d i v i d u a l v a r i a b i l i t y was a lso observed in the induct ion of metoprolol metabolism by pentobarbi ta l (2-46% decrease in AUC) [Haglund et al., 1979] and in the induct ion of warfar in metabolism by q u i n a l b a r b i t a l (5-64.5% decrease in s teady-s ta te plasma warfar in concentrat ion) [Breckenrideg et a 7 . , 1972]. Both non-smoking and smoking subjects in our study showed a s i m i l a r wide range of percent decrease in C m a x (0-87% in the non-smokers and 8-85% in the smokers) and AUC (9-89% in the non-smokers and 19-82% in the smokers) a f t e r phenobarbital treatment, i n d i c a t i n g that phenobarbital induces PF metabolism to a s i m i l a r extent in both non-smoking and smoking popula t ions . Since we have shown that the decrease in AUC d id not c o r r e l a t e with the i n d i v i d u a l ' s CL^ n^. or serum phenobarbital concent ra t ion , i t seems p o s s i b l e that such v a r i a t i o n s were under both environmental and genet ic c o n t r o l . 4.8 E f f e c t of C igare t te Smoking on the Pharmacokinetics and Serum Prote in Binding of Propafenone 4.8.1 S t a t i s t i c a l Ana lys is In the ana lys is of the d i f f e r e n c e in the k i n e t i c parameters between the non-smoking and the smoking s u b j e c t s , the Mann-Whitney tes t with t i e d ranks was used. It i s a nonparametric analogue of or a l t e r n a t i v e to the unpaired t - t e s t . I n i t i a l F t e s t showed that there were s i g n i f i c a n t d i f f e r e n c e s in the var iances in C m a x and AUC between the non-smokers and the smokers. The unpaired t - t e s t was, the re fo re , not app l icab le s ince the unpaired t - t e s t assumes equa l i t y of the var iances of the two sampled 208 populat ions [Zar, 1984]. 4.8.2 E f f e c t of C igare t te Smoking on the Metabolism of Propafenone Table 26 al lows a comparison of the k i n e t i c parameters of PF between e ight non-smoking subjects and e ight smoking subjects or s ix smoking subjects excluding the two ' s low ' metabo l i ze rs . Comparing the values of the k i n e t i c parameters in the contro l s t a t e , the mean C m a x in the smokers (107 ng/mL) was about h a l f of that of the non-smokers (253 ng/mL) while the AUC in the smokers (405 h.ng/mL) was three f o l d smal ler than that of the non-smokers (1360 h.ng/mL). In a d d i t i o n , the mean CL^ n^. in the smokers (13.2 L/min) was double that of the non-smokers (6.1 L /min) . The mean s a l i v a r y AUC in the smokers (117.9 h.ng/mL) was a lso smal ler than that of the non-smokers (190.2 h.ng/mL). While smoking d id appear to increase the c learance of PF, i t i s d i f f i c u l t to conclude that smoking induced the metabolism of PF, due to the small sample s i z e and the lack of comparison of smokers (serv ing as t h e i r own experimental cont ro l ) under a nonsmoking c i rcumstance. Table 27 provides a comparison of the k i n e t i c parameters of 5-hydroxy PF between the non-smoking and the smoking sub jec ts . Since there was no s i g n i f i c a n t d i f f e r e n c e in both C m a x and AUC of 5-hydroxy PF between the non-smokers and smokers, aromatic hydroxyla t ion was probably not induced by c i g a r e t t e smoking. 4 .8 .3 E f f e c t of C igare t te Smoking on the Serum Prote in Binding of Propafenone In add i t ion to drug-metabol iz ing c a p a c i t y , smoking may a lso a f f e c t drug pro te in b i n d i n g . The extent of l i d o c a i n e binding was found to be 209 greater in serum obtained from smokers than in the serum of non-smokers [McNamara et a / . , 1980]. Th is was probably due to the elevated serum AAG concentra t ion in the smoking populat ion [Hol l inshead et al., 1977; Benedek et al., 1983]. Benedek et a / . [1983] reported that the mean serum AAG concentra t ion in smokers (mean = 84.3 mg/dL, n = 10) was s i g n i f i c a n t l y d i f f e r e n t from that of non-smokers (mean = 62.8 mg/dL, n = 10). However, they a lso found that in contrast to the strong c o r r e l a t i o n between AAG serum leve l and pro te in binding of propranolol in non-smokers, the propranolol unbound f r a c t i o n d id not r e f l e c t e levated serum AAG concent ra t ion . They suggested that other f a c t o r s , inc lud ing d i f f e r e n t serum concentra t ion of l i p o p r o t e i n and albumin, may a f f e c t the binding of propranolol in smokers. A l t e r n a t i v e l y , components in c i g a r e t t e smoke may compete with propranolol fo r the serum prote in binding s i t e s . The under ly ing mechanism(s) behind the e leva t ion in serum AAG concentrat ion in the smoking populat ion i s unknown. Benedek et al. [1983] suggested that smoking may cause an increase in t h i s acute phase reactant prote in e i t h e r in a fashion s i m i l a r to that of d isease s ta tes or by an enzyme induct ion mechanism. On the other hand, the f i n d i n g that smoking d id not a f f e c t the prote in binding of phenytoin [Benedek et al., 1983; Rose et al., 1978] was not s u p r i s i n g s ince phenytoin is an a c i d i c drug which binds to albumin so elevated serum AAG concentrat ions should not a f f e c t i t s b ind ing . Our r e s u l t s d id not support the presence of e levated AAG concentrat ion in the serum of healthy heavy c i g a r e t t e smokers. There were no s i g n i f i c a n t d i f f e r e n c e s in e i t h e r serum AAG concentrat ion or PF free f r a c t i o n between the non-smoking and the smoking sub jec ts . 210 4.9 Concentrat ion-Response Rela t ionship of Propafenone 4.9.1 Signal Averaged Electrocardiograms The problem assoc ia ted with normal surface ECG i s a high s ignal to noise r a t i o , r e s u l t i n g in v a r i a b i l i t y in the measurement of QRS width from beat to beat . In the present study, a ECG s ignal averaging technique was used. It i s a t r a c i n g which r e s u l t s from the computerized averaging of a large number of heart beats . The signal to noise r a t i o decreases in proport ion to the square root of the number of beats averaged. In g e n e r a l , i t i s p o s s i b l e to accura te ly measure QRS width wi th in one m i l l i s e c o n d by averaging 80 or more beats . In our study, we averaged 150 beats to generate the ECG data and to obtain an accurate measurement of QRS width. 4 .9 .2 Determinants of the Pharmacological E f f e c t of Propafenone The c o r r e l a t i o n between PF serum to ta l concentrat ion and QRS width was not s i g n i f i c a n t . It seems l i k e l y that fo r PF, f ree drug concen t ra t ion , rather than to ta l drug concent ra t ion , would bet ter c o r r e l a t e with pharmacological e f f e c t . It could a lso be that more than one f a c t o r may cont r ibute to or in f luence the pharmacological response of PF and account fo r the observed poor concentrat ion-response r e l a t i o n . Already d iscussed in Sect ion 4 .4 .4 . i s the observat ion that PF appears to be a drug which may benef i t from the assessment of f ree drug concentrat ions in serum. In order to j u s t i f y f ree drug leve l moni tor ing , the c o r r e l a t i o n between therapeut ic or tox ic response and f ree drug concentra t ion has to be es tab l i shed [Barre et al., 1988]. The f ree drug concentrat ions of two samples from each pat ient were determined and found not to s i g n i f i c a n t l y c o r r e l a t e with QRS width. However, the c o r r e l a t i o n 211 between serum PF f ree concentrat ion and QRS width (r = 0.346) was bet ter than the c o r r e l a t i o n between serum PF to ta l concentra t ion and QRS width (r = 0.288) . The serum AAG concentrat ion of the ten pa t ien ts were within the normal range. Except fo r the two v s low ' metabol izers who had a steady-sta te serum PF concentrat ion of 1.8 pg/ml, the s teady -s ta te serum concentrat ions of the other pat ients were below 1.5 pg/ml (range 0.17-0.92 pg/ml). Since PF d isp layed concentrat ion- independent b inding below 1.5 pg/ml, i t was p o s s i b l e that fo r these reasons the c o r r e l a t i o n between PF serum f ree concentrat ion and QRS width in these pa t ien ts was not apparent. The major metabol i te of PF, 5-hydroxy PF, has been found to be pharmacologica l ly ac t ive in animal s tudies [ P h i l i p s b o r n et a / . , 1984; Valenzuela et a / . , 1987; Thompson et al., 1988]. Although i t s p rec ise pharmacological e f f e c t i s unclear in man, t h i s metabol i te has been found to accumulate in the plasma of pat ients who rece ived propafenone fo r the treatment of arrhythmia [Kates et a / . , 1985]. The s teady -s ta te serum concentra t ion and AUC of 5-hydroxy PF has been shown to be 1.5-6 times lower than those of PF (Table 28). However, i f 5-hydroxy PF is a lso pharmacologica l ly ac t ive in man, i t can s t i l l con t r ibu te to the overa l l ant iar rhythmic e f f e c t of PF and to the apparently poor c o r r e l a t i o n observed between serum PF concentrat ion and i t s ant iarrhythmic e f f e c t . We have shown that the binding r a t i o of PF i s s i g n i f i c a n t l y c o r r e l a t e d to serum AAG concentrat ion in sera from pa t ien ts with chronic renal f a i l u r e . We have fur ther demonstrated a s i g n i f i c a n t c o r r e l a t i o n between the f ree f r a c t i o n of PF and serum AAG concentra t ion in pat ients with arrhythmias (Figure 51). Even in healthy s u b j e c t s , there i s a wide i n t e r i n d i v i d u a l v a r i a t i o n in the serum concentra t ion of AAG, ranging from 212 33 to 137 mg/dL [Routledge et al., 1980]. Th is v a r i a t i o n is fu r ther magnif ied by c e r t a i n p h y s i o l o g i c a l and pa tho log ica l s ta tes [P iafsky et al., 1978] and concomitant drug therapy [Baumann et al., 1982], which may increase or decrease serum AAG concent ra t ion . Diurnal v a r i a t i o n , on the other hand, accounts fo r some i n t r a i n d i v i d u a l v a r i a t i o n in serum AAG concent ra t ion [Yost and DeVane, 1985]. The r e l a t i o n s h i p between PF f ree f r a c t i o n and AAG appears to be c u r v i l i n e a r (F igure 51), suggesting that a small change in AAG concentrat ion may br ing about a la rge a l t e r a t i o n in PF f ree f r a c t i o n . There fore , in addi t ion to PF serum t o t a l concentrat ion and 5-hydroxy PF serum concent ra t ion , serum AAG concent ra t ion which a f f e c t s PF serum f ree concentrat ion may a lso be an important f a c t o r that in f luences the pharmacological e f f e c t of PF. Using mu l t ip le stepwise r e g r e s s i o n , we have demonstrated that i t i s p o s s i b l e to develop an equation to p red ic t the QRS width as a funct ion of a l l the f a c t o r s that may a f f e c t or cont r ibute to the o v e r a l l pharmacological response of PF, inc lud ing serum PF concen t ra t ion , serum 5-hydroxy PF concentrat ion and serum AAG c o n c e n t r a t i o n . The QRS width pred ic ted from using Equation 8 i s , in g e n e r a l , lower than but s i g n i f i c a n t l y c o r r e l a t e d with the measured QRS width (r = 0.536). Although i t i s more common to use the logari thm of serum drug or metabol i te concentra t ion in the evaluat ion of the concentra t ion- response r e l a t i o n s h i p , the QRS width pred ic ted from Equation 9 i s c l o s e r to and s i g n i f i c a n t l y c o r r e l a t e d with the measured QRS width (r = 0.630) . 4 .9 .3 Polymorphic Oxidat ive Metabolism Pat ients who are 's low' metabol izers of PF appear to exh ib i t a d i f f e r e n t concentra t ion response r e l a t i o n s h i p from that observed in ' r a p i d ' 213 metabol izers [Siddoway et al., 1987]. While the reason(s) fo r t h i s are not c l e a r l y e s t a b l i s h e d , i t i s i n t e r e s t i n g to note that the 's low' metabol izers e x h i b i t apparently higher PF serum concent ra t ions , a smal ler oral c learance and an absence of serum 5-hydroxy PF. Furthermore, un l ike the d i s p r o p o r t i o n a l dose-concentra t ion r e l a t i o n s h i p observed in ' r a p i d ' metabo l i ze rs , ' s low ' metabol izers a lso demonstrate what appears to be a propor t iona l dose-concentra t ion r e l a t i o n s h i p [Siddoway et al., 1987]. The developed equations to c a l c u l a t e QRS width apparent ly only apply to ' r a p i d ' metabo l i ze rs . There fore , i t i s necessary to i d e n t i f y a p a t i e n t ' s phenotype before using these equat ions. The ' s low ' metabol izers can be i d e n t i f e d simply by measuring t h e i r dose-cor rec ted s teady-s ta te plasma concentrat ion o f PF (>2 ng/mL/mg d a i l y PF dose) or by checking f o r the absence of plasma 5-hydroxy PF [Siddoway et al., 1987]. 214 5. SUMMARY AND CONCLUSION 5.1 C a p i l l a r y E lect ron-Capture Detect ion G a s - L i q u i d Chromatographic A n a l y s i s of Propafenone The GLC-ECD method developed fo r the q u a n t i t a t i o n of PF was the f i r s t GLC method employing f u s e d - s i l i c a c a p i l l a r y column and s p l i t l e s s i n j e c t i o n technique. Propafenone was d e r i v a t i z e d with HFBA to increase s e l e c t i v i t y and s e n s i t i v i t y and the funct ional groups involved in the d e r i v a t i z a t i o n r e a c t i o n , inc lud ing hydroxyl and secondary amine, were confirmed by GLC-MS. L i n e a r i t y was observed in the ranges 0.5-10 ng/mL ( s a l i v a samples, s i n g l e -dose s tudy) , 2.5-50 ng/mL (serum samples, s i n g l e - d o s e study) and 10-100 ng/mL (serum samples, s teady-s ta te s tudy ) . The c o e f f i c i e n t of v a r i a t i o n was found to be l e s s than 10% over the concentrat ion ranges s t u d i e d . The accuracy of the GLC-ECD method was confirmed by comparing PF serum concentrat ions of pat ient samples with the developed method and a publ ished HPLC method. The l i m i t of determinat ion was 2.5 ng/mL using 1 mL of serum. The s e n s i t i v i t y and s e l e c t i v i t y of the GLC-ECD method allowed us to accura te ly quant i ta te PF in serum, s a l i v a and d i a l y s a t e in s ing le -dose pharmacokinetic s tudies and prote in b inding s tudies of PF. The r e p r o d u c i b i l i t y and r e p e a t a b i l i t y of the method made i t poss ib le to carry out rout ine ana lys is of PF during plasma to ta l (or f ree) drug monitoring of pa t ien ts r e c e i v i n g PF fo r treatment of arrhythmias. The GLC-ECD method developed fo r the quant i t a t ion of PF was fur ther modi f ied to measure the major and ac t ive metabol i te of PF, 5-hydroxy PF. L i n e a r i t y was observed in the range 2.5-50 ng/mL. The c o e f f i c i e n t of v a r i a t i o n was found to be l e s s than 3.5%. 215 5.2 In Vitro Serum Prote in Binding Study The r e s u l t s of the in vitro p ro te in binding study of PF can be summarized as f o l l o w s : i ) Using serum obtained from healthy human s u b j e c t s , we confirmed in vitro the concentration-dependency of the serum pro te in binding of PF. The f ree f r a c t i o n of PF was 0.027 + 0.011 at a PF concentra t ion of 0.25 pg/ml, 0.041 + 0.010 wi thin the therapeut ic concentra t ion range (0.5-2 pg/ml), 0.138 + 0.012 at a PF concentrat ion of 25 pg/ml and 0.187 ± 0.005 when the PF concentrat ion was increased to 100 pg/ml. No evidence f o r s i g n i f i c a n t concentrat ion-dependent changes in f ree f r a c t i o n was observed wi th in the PF concentrat ion range of 0 .25-1.5 pg/ml. However, concentrat ion-dependent binding was demonstrated at concentrat ions greater than 1.5 pg/ml. A h i g h - a f f i n i t y , low-capaci ty b inding s i t e (Kj = 6.53 x. 10^ M"*; n jPj = 1.73 x 10"^ M) and a l o w - a f f i n i t y , h i g h - c a p a c i t y binding s i t e (K 2 = 8.77 x 10 3 M" 1 ; n 2 P 2 = 8.57 x 1 0 - 3 M) were i d e n t i f i e d . i i ) In pooled uremic serum, the f ree f r a c t i o n of PF was approximately 50% of that of normal serum throughout the concentrat ion range studied (1-5 pg/ml). i i i ) In serum obtained from pat ients with chronic renal f a i l u r e , the increase in PF binding r a t i o was associa ted with the increase in serum AAG concent ra t ions . The p o s i t i v e and s i g n i f i c a n t (p<0.05) c o r r e l a t i o n between these two parameters (r = 0.8302) suggested that AAG was an important binding prote in fo r PF in serum. 216 5.3 Phenobarbital Treatment in Healthy Non-smokers and Smokers: Pharmacokinetics and Binding Studies of Propafenone and 5-Hydroxy Propafenone The e f f e c t of enzyme induct ion on the pharmacokinetics of PF and i t s major and ac t ive metabol i te , 5-hydroxy PF can be summarized and concluded as f o l l o w s : i ) There was considerable i n t e r i n d i v i d u a l v a r i a b i l i t y in the pharmacokinetic parameters, inc lud ing C m a x , C L ^ and AUC of serum PF, s a l i v a / s e r u m concentrat ion r a t i o and AUC of s a l i v a r y PF and C m a x and AUC of 5-hydroxy PF before and a f t e r phenobarbital t reatment. Because of the cons iderab le i n t e r i n d i v i d u a l v a r i a t i o n in these parameters, each subject served as h is own c o n t r o l . i i ) Except for the two smokers who were ' s low ' metabo l i ze rs , a l l non-smoking and smoking subjects were ' r a p i d ' metabol izers ( C L ^ n t >0.5 L /min) . The two 's low' metabol izers were charac te r i zed by low i n t r i n s i c c l e a r a n c e , high PF serum concent ra t ion , low ora l c learance and undetectable 5-hydroxy PF in serum. i i i ) Phenobarbital treatment (100 mg d a i l y f o r 23 days) induced hepat ic microsomal enzymes and enhanced the extent of the f i r s t - p a s s metabolism of PF. There was a s i g n i f i c a n t increase in C L ^ n t a f te r phenobarbital treatment. The increase in C L ^ n ^ ranged from 10-831% (mean 194%) in the non-smokers and 23-450% (mean 156%) in the smokers. This r e s u l t e d in a substant ia l decrease in the systemic a v a i l a b i l i t y , as shown by a s i g n i f i c a n t reduct ion in C m a x and AUC. The decrease in C m a x ranged from 0-87% (mean 45%) in the non-smokers and 8-85% (mean 46%) in the smokers whi le the decrease in AUC ranged from 10-89% (mean 50%) in the non-217 smokers and 19-82% (mean 49%) in the smokers. iv) The percent decrease in C M A X was s i m i l a r to the percent decrease in serum AUC f o r PF in the non-smokers and the smokers. v) Except f o r two smokers, enzyme induct ion a lso caused a substant ia l reduct ion in the s a l i v a r y AUCQ of PF. The decrease in AUCQ ranged from 1-82% (mean 44%) in the non-smokers and 30-81% (mean 54%) in the smokers. v i ) The percent decrease in s a l i v a r y AUCQ of PF mirrored the percent decrease in serum AUC in the non-smoking and the smoking s u b j e c t s . v i i ) Enzyme induct ion d id not a f f e c t t ^ or t m a x of PF in the non-smokers or the smokers. v i i i ) Twenty-three days of phenobarbital treatment d id not cause any change in PF f ree f r a c t i o n or serum AAG concentrat ion in the non-smokers and the smokers. vx) There was a wide range in the extent of metabolic induct ion of PF by phenobarb i ta l , as expressed by the percent decrease in AUC, in the non-smokers and the smokers, and in ' r a p i d ' and ' s low ' metabol izers . x) Enzyme induct ion d id not convert ' s low ' metabol izers of PF to ' r a p i d ' metabo l i ze rs . x i ) The extent of metabol ic induct ion of PF by phenobarbital was independent of the serum concentrat ion of the inducing agent, phenobarb i ta l , or of the i n i t i a l a b i l i t y of the i n d i v i d u a l ' s l i v e r to metabolize drugs ( C L i n t c o n t r o l ) • x i i ) The poor c o r r e l a t i o n between the extent of metabolic induct ion of PF by phenobarbital suggested that PF and phenobarbital were metabolized by d i f f e r e n t isozymes of cytochrome P-450. x i i i ) Phenobarbital treatment decreased the serum concent ra t ion , 218 C m a x and AUC of 5-hydroxy PF. The decrease in C m a x ranged from 17-60% (mean 40%) in the non-smokers and 19-71% (mean 52%) in the smokers while the decrease in AUC ranged from 24-81% (mean 45%)in the non-smokers and 48-72% (mean 60%) in the smokers. x iv) Phenobarbital treatment a lso decreased the renal excre t ion (expressed as percent of dose) of the conjugates of 5-hydroxy PF and 5-hydroxy-4-methoxy PF. xv) The metabol ic pathway of PF which was expected to be induced by phenobarbital could not be i d e n t i f i e d through our f i n d i n g s . Several p o s s i b i l i t i e s have been d i s c u s s e d . The aromatic hydroxylat ion pathway may be noninducible while g lucuron ida t ion may or may not be induced. B i l i a r y excre t ion of PF may be increased by phenobarbital treatment. In such case , an induct ion in g lucuron ida t ion may not be observed s ince an increase in the g lucuronide conjugates of 5-hydroxy PF would appear in the b i l e or the feces and not in the u r i n e . The decrease in 5-hydroxy PF a f te r enyzme induct ion could a lso be the r e s u l t of induct ion of other minor metabol ic pathways of PF. xv i ) Compared to the non-smokers, heavy c i g a r e t t e smokers had a s i g n i f i c a n t l y l a r g e r C L - j ^ , a lower C m a x and a smal ler AUC (Mann-Whitney tes t with t i e d ranks, p>0.05). x v i i ) While smoking d id appear to increase the c learance of PF, i t i s d i f f i c u l t to conclude that smoking induced the metabolism of PF, due to the small sample s i z e and the lack of comparison of smokers (serv ing as t h e i r own experimental con t ro l ) under a nonsmoking c i rcumstance. 219 5.4 Eva luat ion of Propafenone Serum Concentrat ion-Response Re la t ionsh ip The study of the serum concentrat ion-response r e l a t i o n s h i p of PF can be summarized and concluded as f o l l o w s : i ) The QRS width measured from the s ignal averaged ECG data i s an accurate and e a s i l y obtained measure of the PF ant iar rhythmic e f f e c t . i i ) Several f ac to rs such as PF serum c o n c e n t r a t i o n , 5-hydroxy PF serum concentrat ion and serum AAG concentrat ion may cont r ibu te to or in f luence the overa l l pharmacological e f f e c t of PF. We have demonstrated that i t was p o s s i b l e to develop an equation to inc lude a l l these f a c t o r s to p r e d i c t the QRS width. The equat ion, developed using mul t ip le stepwise r e g r e s s i o n , was descr ibed as Y = 0.5Xj + 4.5X2 + 347X3 + 79 where Y i s QRS width, Xj i s log PF serum concent ra t ion , X 2 i s log 5-hydroxy PF serum concentra t ion and X3 i s the r e c i p r o c a l of serum AAG concent ra t ion . i i i ) Unl ike the usual concentrat ion-response r e l a t i o n s h i p which uses the logar i thm of serum drug concent ra t ion , serum concentrat ion of PF and 5-hydroxy PF, rather than the logar i thm of these terms, y i e l d e d a bet ter estimate of QRS width. The equation was descr ibed as Y = 0.004X}' + 0 .04X 2 ' + 379X3 + 9 5 w h e r e Y i s Q R S w i d t n > x i ' i s P F s e r u m concent ra t ion , X 2 ' i s 5-hydroxy PF serum concentrat ion and X 3 i s the r e c i p r o c a l of serum AAG concen t ra t ion . In c o n c l u s i o n , c l i n i c a l monitoring of PF may be necessary in pa t ien ts r e c e i v i n g t h i s drug fo r treatment of arrhythmias s ince PF shows remarkable i n t e r i n d i v i d u a l v a r i a t i o n . It may a lso be necessary to i d e n t i f y the p a t i e n t ' s phenotype s ince 's low' metabol izers have d i f f e r e n t pharmacokinetic c h a r a c t e r i s t i c s of PF and an undetectable leve l of 5-hydroxy PF. 'S low' metabol izers can be simply i d e n t i f i e d by t h e i r high 220 plasma PF concent ra t ions or by the absence of 5-hydroxy PF in plasma. Pat ient monitor ing may a lso be necessary in s i t u a t i o n s when the p h y s i o l o g i c a l and patho log ica l state of the pat ient causes a dramatic increase in serum AAG concent ra t ion , which may r e s u l t in an a l t e r a t i o n in PF f ree f r a c t i o n . 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C a l c u l a t i o n of percent of f ree (unbound) drug in serum f o r a bas ic drug. The Henderson-Hasselbalch equation f o r a bas ic drug i s as fo l lows: pH = pK a + log (Cy /C i ) log (Cjj/Ci) = pH - pK a C ^ = 10 P K a " P H where K a = d i s s o c i a t i o n constant C u = concentra t ion of the unionic moiety C.j = concentra t ion of the i o n i c moiety Concentrat ion of f ree drug in s a l i v a , Cf s a l i va ^u s a l i v a + s a l i v a = C u s a l i v a + ( c u s a l i v a x 1 0 p K a " p H $ ) = C U s a l i v a p K a " p H $ ) Concentrat ion of f ree drug in serum, Cf = C + f . L u serum r ° i serum serum = C + (C Y in " 7.4\ ^u serum T ^ u serum x 1 U / = C (l + 10 P K a ' 1 A \ u u serum v 1 + 1 U > where pHs = pH of s a l i v a 7.4 = pH of serum .-f:«]iY?_ - + 1 0 p K a " p H S ) Cr r n + in pKa-7.4^ u f serum ^u serum ^ + 1 U > Since C u s a l i v a = C u s e r u m [Koysooko et a 7 . , 1974; Huffman, 1975] r = _cr.!?iiyL - _ ! ! _ : _ ! ° _ p K a ~ p H S ) C f serum " " u V I o " ^ 1 7 - V 243 Appendix 1. (Cont 'd) c f s a l i v a c p s a l i v a x ^sal I va Cf serum Cp s e r u m x ^serum where ^ s a l i v a = f r a c t i o n of drug unbound in s a l i v a fserum = f a c t i o n of drug unbound in serum C T • x f -i • 1 + 10 pKa-pHs u p s a l v i a x ' s a l i v a i + iu C x f 1 + 1 0 pKa-7.4 ^p serum A 1 serum i + iu Assume drug e x i s t s t o t a l l y in the unbound form in s a l i v a , ^ s a l i v a = 1 C T 1 + 10 P K a-7 .4 _ p s a l i v a i + iu 'serum = x Y i ^ ' P ^ P ^ " ^p serum i + iu Percent of f ree drug in serum ^serum x C T 1 + 1 0 P K a-7 .4 u p s a l i v a i + iu x x 1 0 0 c l + i o PKa-pHs L p serum i + iu r 244 Appendix 2. Semi- logar i thmic p lots of the propafenone serum concentrat ion versus time curves of e ight non-smoking subjects before ( O ) and a f te r 23 days of phenobarbital treatment ( • ) . 1000 £ 100 10 0.1 SERUM PF FOR CA. (non—smoker) 1000 100 6 Time, h 10 12 10 0.1 0.01 SERUM PF FOR 0-A-oo-°-oN (non—amoker) i * 12 Time, h 1S 20 24 Appendix 2. (Cont'd) 245 1000 e 100 •2 10 o.i / ^ SERUM PF FOR S.G. ( non—smoker ) I 1 12 Time, h 16 20 2+ 10000 E 1000 100 10 SERUM P F FOR M.V. ( n o n - s m o k e r ) 12 16 Time, h 20 2+ 23 32 1000 tS 100 10 SERUM P F FOR G.P. (non-smoker) 6 Time, h 10 12 1000 £ 100 •2 10 0.1 SERUM P F FOR U.H. ( n o n - s m o k e r ) 4 6 Time, h 10 246 Appendix 3. Semi - logar i thmic p lots of the propafenone s a l i v a r y concentra t ion versus time curves of eight non-smoking subjects before ( • ) and a f te r 23 days of phenobarbital treatment ( • ) . A lso shown in the f i g u r e s is the pH of s a l i v a before ( O ) and a f te r ( • ) phenobarbital treatment. Appendix 3. (Cont 'd) 248 Appendix 3. (Cont'd) 80 SALIVARY PF FOR M.V. (non—smoker) •7.8 •7.6 " 7.4 7.2 7.0 • 6.8 - 6.6 6.4 6.2 6.0 Appendix 3. (Cont'd) 250 Appendix 4. Semi- logar i thmic p lo ts of the 5-hydroxy propafenone serum concentrat ion versus time curves of e ight non-smoking subjects before ( A ) and a f t e r 23 days of phenobarbital treatment ( A ). SERUM 5-OH PF FOR B.K. (non-smoker) 1000 100 10 SERUM 5-OH PF FOR N.P. (non-smoker) ~ A . 6 •'Time, h 10 12 6 Time, h 10 12 1000n 100-SERUM 5-OH PF FOR C.A. (non-smoker) A - A 10-1000 g 100 ~ 10 ., c o 0.1 SERUM 5-OH PF FOR O A (non-smoker) 4 6 8 Time, h 10 12 6 Time, h 10 12 Appendix 4. (Cont'd) 2 5 1 1000 £ 100 •2 10 SERUM 5-OH PF FOR S.G. (non—smoker) 0.1 •-fcr-6 a Time, h 10 12 \* 1000 100 10 SERUM 5-OH PF FOR M.V. (non-smoker) «~ 6 8 Time, h 10 12 14 1000i 100 J 10 -i / SERUM 5-OH PF FOR G.P. (non-smoker) 4 6 Time, h 10 1000 100 10 SERUM 5-OH PF FOR U.H. (non-smoker) 4 6 Time, h 10 252 Appendix 5. Semi - logar i thmic p lo ts of the propafenone serum concentrat ion versus time curves of e ight smoking subjects before ( O ) and a f t e r 23 days of phenobarbital treatment ( • ). O.l • 6 8 Time, h 10 12 14 6 8 Time, h 10 12 Appendix 5. (Cont 'd) 253 254 Appendix 6. Semi- logar i thmic p lo ts of the propafenone s a l i v a r y concentrat ion versus time curves of e ight smoking subjects before ( • ) and a f t e r 23 days of phenobarbital treatment ( • ) . A lso shown in the f igures i s the pH of s a l i v a before ( O ) and a f te r ( • ) phenobarbital treatment. 12 10-8.0 • 7.8 7.6 ,J 7.4 Q f7.0 J 6.8 -o 6.6 x I- 6.4 6.2 6.0 5.8 SAUVARY PF FOR J.L (smoker) 50 40 30-20 10-8.0 7.8 7.6 -f7-0 I" -"•6.8 ^ 6.6 T J 6.4 1 6.2 6.0 5.8 SALIVARY PF FOR M.A. (smoker) Appendix 6. (Cont'd) Time, h Time, h Appendix 6. (Cont'd) T 8.0 • 7.a •7.6 -7.4 + • , - I 1 . 1 T - 1 r 0 6 12 18 24 30 36 42 48 Time, h Time, h Appendix 6. (Cont 'd) 258 Appendix 7. Semi- logar i thmic p lo ts of the 5-hydroxy propafenone serum concentra t ion versus time curves of s ix smoking subjects before ( A ) and a f t e r 23 days of phenobarbital treatment ( A ). 100O-, 100-SERUM 5-OH PF FOR J.L. (smoker) 10-6 Time, h 10 12 1000 100 10 SERUM 5-OH PF FOR M A (smoker) 6 Time, h 10 12 1000 E 100 = 10 • SERUM 5-OH PF FOR T.N. (smoker) 0.1 • 6 Time, h 10 12 1000 E 100 10 0.1 SERUM 5-OH PF FOR D.W. (smoker) / 6 Time, h 10 12 SERUM 5-OH PF FOR S.R. (non-smoker) 6 Time, h 10 12 1000 100 10 SERUM 5-OH PF FOR D.B. (non-smoker) 6 8 Time, h 10 12 1 + 259 Appendix 8. Semi- logar i thmic plots of the propafenone ( O ) and 5-hydroxy propafenone ( A ) serum concentrat ion versus time curves of ten pa t i en ts . 10000 i 1000^ = 1001 10-j PATIENT S.M. 10000 -II g 1000 2 100 12 Tim«, h 16 20 24 3 Time, h 10000 £ 1000 •2 100 10-10000 e I O O O 100 i 260 Appendix 8. (Cont'd) 10000 g 1000 too 10000 £ 1000 100 P u b l i c a t i o n s : 1. Wallace SM and Chan GL-Y: In Vitro In teract ion of Aminoglycosides with ^-Lactam P e n i c i l l i n s , Ant imicrob. Agents Chemother. 28(2): 274-281, 1985. 2. Axelson J E , Chan GL-Y, K i r s t e n ED, Mason WD, Lanman RC and Kerr CR: Food Increases the B i o a v a i l a b i l i t y of Propafenone, Br . J . C l i n . Pharmac. 23:735-741, 1987. 3. Chan GL-Y, Axelson J E , Abbott FS, Kerr CR, McErlane KM: Determination of Propafenone in B i o l o g i c a l F l u i d s by F u s e d - S i l i c a C a p i l l a r y Gas Chromatography Using E lect ron-Capture De tec t ion , J . Chromatogr. (Biomed. App l . ) 417:295-308, 1987. 4. Chan GL-Y. Axelson J E , Pr ice JDE, McErlane KM and Kerr CR: In Vitro Serum Prote in Binding of Propafenone in Normal and Uraemic Human Sera , Eur. J . C l i n . Pharmacol . , 36:495-499, 1989. 5. Chan GL-Y. Axelson J E , Abbott FS, McErlane KM and Kerr CR: Determination of 5-Hydroxy Propafenone, an Ac t i ve Metabol i te of Propafenone in B i o l o g i c a l F lu ids by F u s e d - S i l i c a C a p i l l a r y Gas Chromatography Using E lect ron-Capture Detec t ion , (Let ter to the E d i t o r ) , J . Chromatogr., 1989, in p ress . 6. Chan GL-Y. Axelson J E , Yeung J , McErlane KM and Kerr CR: Propafenone Pharmacokinetics and Metabolism: E f f e c t of Inducers on Clearance and B ind ing , to be submitted to Br . J . C l i n . Pharmac, 1989. 

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