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Stereoselective HPLC analysis, pharmacokinetics, tissue distribution and pharmacodynamics of mexiletine… Igwemezie, Linus Nnamdi 1989

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STEREOSELECTIVE HPLC ANALYSIS, PHARMACOKINETICS, TISSUE DISTRIBUTION AND PHARMACODYNAMICS OF MEXILETINE ENANTIOMERS By LINUS IGWEMEZIE B. Pharm., U n i v e r s i t y o f I F E , N i g e r i a , 1981 M. S c . , U n i v e r s i t y o f B.C., C a n a d a , 1986 A TH E S I S SUBMITTED IN PARTIAL FULFILMENT 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 a l C h e m i s t r y 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 BR I T I S H COLUMBIA November, 1989 ( c ) L i n u s I gwemezie 08 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. P h a r m a c e u t i c a l S c i e n c e s Department of The University of British Columbia Vancouver, Canada Date December 28, 1989 DE-6 (2/88) i i ABSTRACT M e x i l e t i n e [(2',6'-dimethylphenoxy)-2-amino propane] i s a c l a s s 1 a n t i a r r h y t h m i c agent with a s i m i l a r chemical s t r u c t u r e and 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 to those o f l i d o c a i n e . It i s a c h i r a l drug which i s used c l i n i c a l l y i n the racemic form ( i . e . 50:50 r a t i o of two enantiomers). T h i s t h e s i s d e s c r i b e s the s t e r e o s e l e c t i v e HPLC a n a l y s i s , pharmacokinetics, t i s s u e d i s t r i b u t i o n and pharmacodynamics o f m e x i l e t i n e enantiomers. The development of a h i g h l y s e n s i t i v e and s t e r e o s e l e c t i v e HPLC assay f o r m e x i l e t i n e enantiomers, using 2-anthroyl c h l o r i d e as a d e r i v a t i z a t i o n reagent, was attempted. The s y n t h e s i s and c h a r a c t e r i z a t i o n of the a c i d c h l o r i d e was s u c c e s s f u l l y c a r r i e d out. The 2-anthroyl d e r i v a t i v e s o f the enantiomers were r e s o l v e d on a P i r k l e ^ i o n i c (phenyl g l y c i n e ) c h i r a l column using a mobile phase o f e t h y l acetate/2-propanol/Hexane (4:6:90). D e t e c t i o n was accomplished by f l u o r e s c e n s e (ex = 270 nm, em = 400 nm) with a lower l i m i t o f 0.5 ng/ml. However, there was an i n t e r f e r i n g peak c o e l u t i n g with S(+)-mexiletine which could not be r e s o l v e d . T h i s precluded the use of the assay f o r the proposed pharmacokinetic and pharmacodynamic s t u d i e s . A p r e v i o u s l y developed s t e r e o s e l e c t i v e HPLC method, with 2-naphthoyl c h l o r i d e as a d e r i v a t i z a t i o n reagent, was subsequently used. The in vitro p r o t e i n binding o f m e x i l e t i n e enantiomers was examined with human serum, l i p o p r o t e i n d e f i c i e n t serum, albumin and a j - a c i d g l y c o p r o t e i n . The binding o f the enantiomers to human serum was moderate (45 to 50%) w i t h i n the t h e r a p e u t i c range o f m e x i l e t i n e . T h i s binding was due, mainly, to albumin and o ^ - a c i d g l y c o p r o t e i n . The f r e e f r a c t i o n s of the enantiomers decreased s i g n i f i c a n t l y (P<0.05) as pH was i n c r e a s e d from i i i 7.0 t o 8.0. S t e r e o s e l e c t i v e b i n d i n g was a p p a r e n t a t pH 8.0 s u c h t h a t t h e f r e e f r a c t i o n o f S ( + ) - m e x i l e t i n e was s i g n i f i c a n t l y (p<0.05) g r e a t e r t h a n t h a t o f t h e R ( - ) - e n a n t i o m e r . However, s t e r e o s e l e c t i v e b i n d i n g was n o t o b s e r v e d a t p h y s i o l o g i c a l pH 7 . 4 ) . T h e s e r e s u l t s i n d i c a t e d t h a t t h e s e r u m b i n d i n g o f m e x i l e t i n e e n a n t i o m e r s i s p H - d e p e n d e n t . B i n d i n g was n o t c o n c e n t r a t i o n - d e p e n d e n t , n o r was t h e r e any c o m p e t i t i v e b i n d i n g i n t e r a c t i o n b e t w e e n t h e e n a n t i o m e r s , w i t h i n t h e t h e r a p e u t i c r a n g e . S c a t c h a r d a n a l y s i s o f t h e b i n d i n g d a t a o b t a i n e d w i t h s e r u m and a l b u m i n b o t h showed t h e p r e s e n c e o f 2 c l a s s e s of b i n d i n g s i t e s . A h i g h a f f i n i t y , l o w c a p a c i t y s i t e 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 s i t e . In c o n t r a s t , a j - a c i d g l y c o p r o t e i n showed o n l y 1 c l a s s of b i n d i n g s i t e s and t h i s was a h i g h a f f i n i t y , l o w c a p a c i t y s i t e . P h a r m a c o k i n e t i c and t i s s u e d i s t r i b u t i o n s t u d i e s i n r a t s f o l l o w i n g t h e a d m i n i s t r a t i o n o f r a c e m i c m e x i l e t i n e (10 mg/kg) i n d i c a t e d e x t e n s i v e t i s s u e u p t a k e and r a p i d e l i m i n a t i o n o f t h e e n a n t i o m e r s . R ( - ) - M e x i l e t i n e showed a 3 2 % g r e a t e r s y s t e m i c c l e a r a n c e ( 161.8 m l / m i n / k g vs 122.9 m l / m i n / k g ) t h a n t h e S ( + ) - e n a n t i o m e r . The s t e a d y s t a t e - v o l u m e o f d i s t r i b u t i o n was a l s o g r e a t e r f o r t h e R ( - ) - e n a n t i o m e r ( 9 . 0 L / k g v s 7.4 L / k g ) , w h i l e t h e e l i m i n a t i o n h a l f - l i v e s o f t h e e n a n t i o m e r s (1.4 and 1.3 h f o r R ( - ) - and S ( + ) - m e x i l e t i n e , r e s p e c t i v e l y ) were n o t d i f f e r e n t . Maximum t i s s u e c o n c e n t r a t i o n s were o b s e r v e d a t 5 min i n a l l t h e t i s s u e s s t u d i e d ( h e a r t , b r a i n , l u n g , k i d n e y , l i v e r and f a t ) . T h e s e c o n c e n t r a t i o n s were n o t s i g n i f i c a n t l y d i f f e r e n t , e x c e p t f o r t h e l i v e r t i s s u e w here a 2 . 4 - f o l d g r e a t e r c o n c e n t r a t i o n of t h e S ( + ) - e n a n t i o m e r was f o u n d . H i g h t i s s u e / s e r u m r a t i o s (>20) were o b s e r v e d f o r e a c h e n a n t i o m e r i n t h e b r a i n , l u n g s and k i d n e y s . The b r a i n a c c u m u l a t e d 3 - f o l d t h e h e a r t c o n c e n t r a t i o n s of t h e e n a n t i o m e r s . P h a r m a c o d y n a m i c s t u d i e s on t h e r e l a t i v e a n t i a r r h y t h m i c e f f e c t s o f r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s were c a r r i e d o u t u s i n g e l e c t r i c a l a nd i s c h a e m i a - i n d u c e d a r r h y t h m i a s i n r a t s . R a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s s i g n i f i c a n t l y (P<0.05) i n c r e a s e d VFT and ERP. However, t h e d i f f e r e n c e s b e t w e e n t h e e f f e c t s o f t h e 3 d r u g s on t h e s e v a r i a b l e s w ere n o t s t a t i s t i c a l l y s i g n i f i c a n t . R,S-, S ( + ) - and R ( - ) - m e x i l e t i n e c a u s e d s i g n i f i c a n t b r a d y c a r d i a and PR p r o l o n g a t i o n i n b o t h p e n t o b a r b i t o n e a n a e s t h e t i z e d and c o n s c i o u s r a t s . T h e s e e f f e c t s o f t h e d r u g s were a l s o n o t s i g n i f i c a n t l y d i f f e r e n t f r o m e a c h o t h e r . In t h e i s c h a e m i c c o n s c i o u s r a t s , t h e 3 d r u g s d i d n o t s i g n i f i c a n t l y r e d u c e t h e i n c i d e n c e o f VT and VF, t h e number o f PVCs n o r t h e " a r r h y t h m i a s c o r e " when c o m p a r e d t o s a l i n e ( c o n t r o l ) . R a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s p r o d u c e d c o m p a r a b l e CNS t o x i c i t y i n t h e c o n s c i o u s r a t s . V TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES x i i i LIST OF FIGURES xv SYMBOLS AND ABBREVIATIONS x v i i i ACKNOWLEDGEMENT x x i CHAPTER Page 1. INTRODUCTION 1 1.1 Sudden C a r d i a c Death 1 1.1.1 Overview 1 1.1.2 A r r h y t h m i a s 3 1.1.2.1 The C a r d i a c A c t i o n P o t e n t i a l 3 1.1.2.2 E l e c t r o p h y s i o l o g i c a l Changes D u r i n g 6 A c u t e M y o c a r d i a l I s c h e m i a . 1.1.2.3 A r r h y t h m o g e n e s i s 7 1.1.2.3.1 Impulse I n i t i a t i o n ( A u t o m a t i c i t y ) 7 1.1.2.3.2 T r i g g e r e d A u t o m a t i c i t y 8 1.1.2.3.3 Impulse C o n d u c t i o n ( R e - e n t r y ) 9 1.1.2.4 Mode o f A c t i o n o f A n t i a r r h y t h m i c a g e n t s . . . . 10 1.1.2.5 E x p e r i m e n t a l A r r h y t h m i a s 12 1.1.2.5.1 E l e c t r i c a l l y - i n d u c e d a r r h y t h m i a s 12 1.1.2.5.2 C h e m i c a l l y - I n d u c e d A r r h y t h m i a s 13 1.1.2.5.3 M e c h a n i c a l l y - I n d u c e d A r r h y t h m i a s 13 1.1.2.5.4 R e p e r f u s i o n - I n d u c e d A r r h y t h m i a s 14 1.2 M e x i l e t i n e 15 1.2.1 C h e m i s t r y 15 1.2.2 Pharmacology 17 1.2.2.1 Animal S t u d i e s 17 1.2.2.2 C l i n i c a l E f f e c t i v e n e s s 17 v i 1.2.2.3 Plasma Concentration-Clinical Effect Relationships 18 1.2.3 Mechanism of Action ,.19 1.2.4 Comparison with Other Antiarrhythmic Agents 21 1.2.5 Prevention of Sudden Cardiac Death 22 1.2.6 Clinical Indications 22 1.2.7 Toxicology 23 1.2.8 Pharmacokinetic Parameters 23 1.2.8.1 Pharmacokinetics of Racemic Mexiletine 23 1.2.8.2 Effects of Disease on Pharmacokinetics 24 1.8.2.3 Pharmacokinetics of Mexiletine Enantiomers 25 1.2.9 Dosage 25 1.2.10 Metabolism 26 1.2.11 Interactions of Mexiletine with Other Drugs 26 1.2.12 Serum Protein Binding 28 1.2.13 Tissue Distribution of Mexiletine 28 1.2.14 Analytical Methods for Mexiletine 28 1.2.15 Stereoselective Analysis of Mexiletine 29 1.3 Chiral i ty 30 1.3.1 Introduction 30 1.3.2 Stereoselective Drug Disposition 31 1.3.2.1 Pharmacodynamics 31 1.3.2.2 Absorption 32 1.3.2.3 Distribution 32 1.3.2.4 Metabolism 33 1.3.3 Stereoselective Drug Analysis 34 1.3.3.1 Introduction 34 v i i 1.3.3.2 R e s o l u t i o n o f Enantiomers as D i a s t e r e o i s o m e r s 35 1.3.3.3 R e s o l u t i o n o f Enantiomers on C h i r a l S t a t i o n a r y Phases 36 1.3.3.4 R e s o l u t i o n o f Enantiomers U s i n g C h i r a l E l u e n t s 38 1.4 R a t i o n a l e 39 1.5 S p e c i f i c O b j e c t i v e s 43 2. EXPERIMENTAL 44 2.1 S u p p l i e s 44 2.1.1 Drugs 44 2.1.2 C h e m i c a l s and Reagents 44 2.1.3 S o l v e n t s 44 2.1.4 Human Serum P r o t e i n s 45 2.2 C h r o m a t o g r a p h i c S t a t i o n a r y Phases and Columns 45 2.2.1 HPLC Columns 45 2.3 Equipment 46 2.3.1 High - P e r f o r m a n c e L i q u i d Chromatograph 46 2.3.2 Gas-Chromatograph/Mass S p e c t r o m e t e r 46 2.3.3 P o l y g r a p h 46 2.3.4 E l e c t r i c a l S t i m u l a t o r 46 2.4 A s s a y o f M e x i l e t i n e Enantiomers U s i n g 2 - A n t h r o y l C h l o r i d e as a D e r i v a t i z a t i o n Reagent 47 2.4.1 S y n t h e s i s o f 2 - A n t h r o y l C h l o r i d e from A n t h r a q u i n o n e - 2 - c a r b o x y l i c A c i d 47 2.4.1.1 S y n t h e s i s o f A n t h r a c e n e - 2 - c a r b o x y l i c A c i d 47 2.4.1.1 P u r i f i c a t i o n o f A n t h r a c e n e - 2 - c a r b o x y l i c A c i d 48 2.4.1.3 C h a r a c t e r i z a t i o n o f A n t h r a c e n e - 2 - c a r b o x y l i c . A c i d 48 v i i i 2.4.1.4 S y n t h e s i s o f 2 - A n t h r o y l C h l o r i d e 49 2.4.1.5 P u r i f i c a t i o n o f 2 - A n t h r o y l C h l o r i d e 49 2.4.1.6 C h a r a c t e r i z a t i o n o f 2 - A n t h r o y l C h l o r i d e 49 2.4.2 Development o f A s s a y o f M e x i l e t i n e E n a n t i o m e r s 50 2.4.2.1 D e r i v a t i v e o f M e x i l e t i n e E n antiomers w i t h 2 - A n t h r o y l C h l o r i d e 50 2.4.2.2 C h r o m a t o g r a p h i c R e s o l u t i o n o f M e x i l e t i n e E n a ntiomers 50 2.4.2.3 S e n s i t i v i t y 51 2.4.2.4 S t r u c t u r e o f 2 - A n t h r o y l D e r i v a t i v e o f M e x i l e t i n e 51 2.4.2.5 E x t r a c t i o n S o l v e n t 51 2.4.2.6 S e l e c t i o n o f I n t e r n a l S t a n d a r d 51 2.4.2.7 HPLC Assay o f M e x i l e t i n e E n antiomers 52 2.4.2.8 Attempted R e s o l u t i o n / R e m o v a l o f t h e I n t e r f e r r i n g Peak 52 2.5 Assay o f M e x i l e t i n e E n a n t i o m e r s u s i n g 2-Naphthoyl C h l o r i d e as a D e r i v a t i z a t i o n Reagent 54 2.5.1 E x t r a c t i o n , D e r i v a t i z a t i o n and HPLC A n a l y s i s 54 2.5.2 C a l i b r a t i o n Curves and Assay P r e c i s i o n i n Human Plasma 55 2.6 In Vitro Serum P r o t e i n B i n d i n g o f M e x i l e t i n e E n a n t i o m e r s i n Humans 55 2.6.1 Serum C o l l e c t i o n 55 2.6.2 Serum pH Adjustment 55 2.6.3 P u r i f i e d Human Serum P r o t e i n S o l u t i o n s 56 2.6.4 Sample P r e p a r a t i o n 56 2.6.5 U l t r a f i l t r a t i o n 57 2.6.6 A n a l y s i s o f Free and T o t a l M e x i l e t i n e i n Serum and P r o t e i n S o l u t i o n s 57 i x 2.6.7 A n a l y s i s o f B i n d i n g Data 57 2.6.8 S t a t i s t i c a l Data A n a l y s i s 58 2.7 T i s s u e D i s t r i b u t i o n K i n e t i c s o f M e x i l e t i n e E n a n t i o m e r s i n Rats 58 2.7.1 Drug A d m i n i s t r a t i o n and Sample C o l l e c t i o n 58 2.7.2 A n a l y s i s o f Serum and T i s s u e Samples 59 2.7.3 E f f i c i e n c y o f Recovery o f M e x i l e t i n e Enantiomers from T i s s u e Homogenates 59 2.7.4 Serum P r o t e i n B i n d i n g 59 2.7.5 P h a r m a c o k i n e t i c Data A n a l y s i s 60 2.7.6 S t a t i s t i c a l Data A n a l y s i s 60 2.8 A n t i a r r h y t h m i c A c t i v i t y o f Racemic M e x i l e t i n e and i t s Enantiomers 61 2.8.1 E l e c t r i c a l l y - I n d u c e d A r r h y t h m i a 61 2.8.1.1 P r e p a r a t i o n o f Rats 61 2.8.1.2 E x p e r i m e n t a l E n d - p o i n t s '. 61 2.8.1.3 V a r i a b l e s R e l a t e d t o A n t i a r r h y t h m i c E f f e c t s 62 2.8.1.4 O t h e r V a r i a b l e s . . . . 63 2.8.1.5 Drug A d m i n i s t r a t i o n 63 2.8.1.6 A n a l y s i s o f Plasma Samples 63 2.8.1.7 S t a t i s t i c a l A n a l y s i s 64 2.8.2 C o r o n a r y A r t e r y O c c l u s i o n i n C o n s c i o u s Rats 64 2.8.2.1 P r e p a r a t i o n o f Rats 64 2.8.2.2 P r e p a r a t i o n o f O c c l u d e r 65 2.8.2.3 P r e p a r a t i o n o f L i n e s 65 2.8.2.4 I m p l a n t a t i o n o f L i n e s 66 2.8.2.5 I m p l a n t a t i o n o f t h e O c c l u d e r 66 2.8.2.6 I m p l a n t a t i o n o f t h e ECG Leads 67 X 2.8.2.7 C o r o n a r y A r t e r y O c c l u s i o n 68 2.8.2.8 Drug A d m i n i s t r a t i o n and Plasma C o n c e n t r a t i o n Measurement 68 2.8.2.9 Responses t o Drug Treatment and O c c l u s i o n 69 2.8.2.10 O c c l u d e d Zone 69 2.8.2.11 O c c l u s i o n - I n d u c e d A r r h y t h m i a s 70 2.8.2.12 E v a l u a t i o n o f A n t i a r r h y t h m i c E f f i c a c y 70 2.8.2.13 S t a t i s t i c a l Data A n a l y s i s 71 2.8.2.14 E x c l u s i o n C r i t e r i a 72 2.8.3 C o r o n a r y A r t e r y O c c l u s i o n i n P e n t o b a r b i t o n e A n a e s t h e t i z e d Rats 73 3. RESULTS and DISCUSSION 75 3.1 A s s a y o f M e x i l e t i n e E n antiomers by HPLC u s i n g 2 - A n t h r o y l C h l o r i d e as a D e r i v a t i z a t i o n Reagent 75 3.1.1 S y n t h e s i s o f 2 - A n t h r o y l C h l o r i d e . 75 3.1.2 Development o f Assay o f M e x i l e t i n e E n a ntiomers 79 3.1.2.1 D e r i v a t i z a t i o n o f M e x i l e t i n e E n a n t i o m e r s w i t h 2 - A n t h r o y l C h l o r i d e 79 3.1.2.2 S t r u c t u r e o f the 2 - A n t h r o y l D e r i v a t i v e o f M e x i l e t i n e 82 3.1.2.3 R e s o l u t i o n o f M e x i l e t i n e E n a n t i o m e r s 81 3.1.2.4 S e n s i t i v i t y o f the 2 - A n t h r o y l D e r i v a t i v e o f M e x i l e t i n e 85 3.1.2.5 Mechanism o f R e s o l u t i o n o f M e x i l e t i n e E nantiomers 85 3.1.2.6 HPLC Assay o f M e x i l e t i n e E n antiomers 87 3.1.2.7 Attempted R e s o l u t i o n / R e m o v a l o f the I n t e r f e r r i n g Peak 89 3.2 A s s a y o f M e x i l e t i n e Enantiomers by HPLC w i t h 2-Naphthoyl C h l o r i d e as a D e r i v a t i z a t i o n r e a g e n t 92 xi 3.3 In Vitro Serum P r o t e i n B i n d i n g o f M e x i l e t i n e E n a n t i o m e r s 95 3.3.1 F a c t o r s A f f e c t i n g Serum P r o t e i n B i n d i n g 95 3.3.1.1 N o n - s p e c i f i c B i n d i n g 95 3.3.1.2 E f f e c t o f Serum pH 95 3.3.1.3 C o m p e t i t i v e B i n d i n g ( E n a n t i o m e r -Enantiomer I n t e r a c t i o n ) 99 3.3.2 Methods o f Serum pH Adjustment 99 3.3.3 B i n d i n g o f M e x i l e t i n e Enantiomers t o Serum and t o V a r i o u s Serum P r o t e i n s 102 3.3.4 D e t e r m i n a t i o n o f B i n d i n g C o n s t a n t s 104 3.3.4.1 Serum B i n d i n g Data 104 3.3.4.2 o j - A c i d G l y c o p r o t e i n B i n d i n g Data 107 3.3.4.3 Albumin B i n d i n g Data 110 3.4 T i s s u e D i s t r i b u t i o n K i n e t i c s o f M e x i l e t i n e E n a n t i o m e r s i n Rats 110 3.4.1 Serum L e v e l s 112 3.4.2 P h a r m a c o k i n e t i c Parameters 112 3.4.3 Serum P r o t e i n B i n d i n g 115 3.4.4 Recovery o f t h e Enantiomers from T i s s u e s 117-3.4.5 T i s s u e L e v e l s , 117 3.5 A n t i a r r h y t h m i c E f f e c t s o f Racemic M e x i l e t i n e and i t s E n a ntiomers i n Rats 123 3.5.1 E l e c t r i c a l l y - i n d u c e d A r r h y t h m i a i n P e n t o b a r b i t o n e A n a e s t h e t i z e d Rats 123 3.5.1.1 Dosage and Plasma C o n c e n t r a t i o n 124 3.5.1.2 A n t i a r r h y t h m i c E f f e c t s 127 3.5.1.2.1 V e n t r i c u l a r F i b r i l l a t i o n T h r e s h o l d (VFT) 127 3.5.1.2.2 C a r d i a c R e f r a c t o r y P e r i o d 129 3.5.1.3 C a r d i o v a s c u l a r E f f e c t s 134 x i i 3.5.2 C o r o n a r y A r t e r y O c c l u s i o n - I n d u c e d A r r h y t h m i a i n C o n s c i o u s Rats 139 3.5.2.1 Dosage and Plasma C o n c e n t r a t i o n 140 3.5.2.2 O c c l u d e d Zone (OZ) 140 3.5.2.3 A n t i a r r h y t h m i c E f f e c t s 142 3.5.2.4 C a r d i o v a s c u l a r E f f e c t s 152 3.5.2.5 CNS S i d e E f f e c t s 156 3.5.3 C o r o n a r y A r t e r y O c c l u s i o n - I n d u c e d A r r h y t h m i a i n P e n t o b a r b i t o n e A n a e s t h e t i z e d Rats 156 3.5.3.1 A n t i a r r h y t h m i c E f f e c t s o f Racemic M e x i l e t i n e 158 4. SUMMARY and CONCLUSIONS 167 5. REFERENCES 171 x i i i LIST OF TABLES T a b l e Page 1. C a l i b r a t i o n c u r v e d a t a f o r m e x i l e t i n e e n a n t i o m e r s i n human plasma 94 2. P e r c e n t r e c o v e r y o f m e x i l e t i n e e n a n t i o m e r s d u r i n g u l t r a f i l t r a t i o n 96 3. The p e r c e n t f r e e f r a c t i o n s o f m e x i l e t i n e e n a n t i o m e r s from serum c o n t a i n i n g r a c e m i c m e x i l e t i n e o r the i n d i v i d u a l e n a n t i o m e r 100 4. Methods o f serum pH adjustment and t h e c o r r e s p o n d i n g p e r c e n t f r e e f r a c t i o n s o b t a i n e d 101 5. The c o n t r i b u t i o n s o f t h e major drug b i n d i n g p r o t e i n s t o t h e serum b i n d i n g o f m e x i l e t i n e e n a n t i o m e r s 103 6. The p e r c e n t f r e e f r a c t i o n s o f m e x i l e t i n e e n a n t i o m e r s i n serum from h e a l t h y human s u b j e c t s 105 7. B i n d i n g c o n s t a n t s o f m e x i l e t i n e e n a n t i o m e r s i n serum from h e a l t h y s u b j e c t s 108 8. B i n d i n g c o n s t a n t s o f m e x i l e t i n e e n a n t i o m e r s i n i s o l a t e d AAG 109 9. B i n d i n g c o n s t a n t s o f m e x i l e t i n e e n a n t i o m e r s i n i s o l a t e d HSA I l l 10. The p h a r m a c o k i n e t i c parameters o f m e x i l e t i n e e n a n t i o m e r s c a l c u l a t e d from serum c o n c e n t r a t i o n - t i m e d a t a a f t e r a s i n g l e i . v . dose o f r a c e m i c m e x i l e t i n e 114 11. The in vivo f r e e serum c o n c e n t r a t i o n s (ng/ml) and t h e p e r c e n t f r e e f r a c t i o n s ( f f ) o f m e x i l e t i n e e n a n t i o m e r s a f t e r a s i n g l e i . v . dose o f r a c e m i c m e x i l e t i n e 116 12. P e r c e n t r e c o v e r y o f m e x i l e t i n e e n a n t i o m e r s from v a r i o u s t i s s u e homogenates 118 13. The time-dependent c o n c e n t r a t i o n o f m e x i l e t i n e e n a n t i o m e r s i n t h e t i s s u e s o f r a t s f o l l o w i n g a s i n g l e dose o f r a c e m i c m e x i l e t i n e 119 14. P h a r m a c o k i n e t i c p a r a m e t e r s o f m e x i l e t i n e e n a n t i o m e r s i n v a r i o u s t i s s u e s a f t e r a s i n g l e i . v . dose o f r a c e m i c m e x i l e t i n e 120 x i v 15. The in vivo f r e e f r a c t i o n s (%) o f r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s i n p e n t o b a r b i t o n e a n a e s t h e t i z e d r a t s 126 16. P r e - d r u g v a l u e s f o r t h e v a r i a b l e s f o r v e n t r i c u l a r f i b r i l l of1 u t t e r i n p e n t o b a r b i t o n e a n a e s t h e t i z e d r a t s 128 17. The e f f e c t s o f r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s on t h e ECG parameters i n p e n t o b a r b i t o n e a n a e s t h e t i z e d r a t s 135 18. P r e - d r u g v a l u e s f o r t h e haemodynamic par a m e t e r s i n p e n t o b a r b i t o n e a n a e s t h e t i z e d r a t s 136 19. The mean weight and o c c l u d e d zone i n t h e d i f f e r e n t t r e a t m e n t groups i n c o n s c i o u s i s c h a e m i c r a t s ....143 20. The e f f e c t s o f r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s on t h e i n c i d e n c e o f a r r h y t h m i a s f o l l o w i n g c o r o n a r y a r t e r y o c c l u s i o n i n c o n s c i o u s r a t s 146 21. The e f f e c t s o f r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s on t h e PVCs f o l l o w i n g c o r o n a r y o c c l u s i o n i n c o n s c i o u s r a t s 148 22. The e f f e c t s o f drug t r e a t m e n t s on t h e ECG param e t e r s i n c o n s c i o u s i s c h a e m i c r a t s . . 153 23. The i n c i d e n c e o f CNS t o x i c e f f e c t s and t h e c o r r e s p o n d i n g plasma c o n c e n t r a t i o n s i n c o n s c i o u s r a t s o f r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s 157 24. The mean weight and o c c l u d e d zone i n t h e d i f f e r e n t t r e a t m e n t groups i n p e n t o b a r b i t o n e a n a e s t h e t i z e d r a t s ..159 25. The e f f e c t s o f r a c e m i c m e x i l e t i n e on t h e i n c i d e n c e o f a r r h y t h m i a s f o l l o w i n g c o r o n a r y a r t e r y o c c l u s i o n i n p e n t o b a r b i t o n e a n a e s t h e t i z e d r a t s 160 26. The e f f e c t s o f r a c e m i c m e x i l e t i n e on the number o f premature v e n t r i c u l a r c o n t r a c t i o n s ( l o g i Q P V C ) f o l l o w i n g c o r o n a r y a r t e r y o c c l u s i o n i n p e n t o b a r b i t o n e a n a e s t h e t i z e d r a t s 161 XV LIST OF FIGURES F i g u r e Page 1. The s t r u c t u r e o f m e x i l e t i n e 16 2. Scheme f o r t h e s y n t h e s i s o f 2 - a n t h r o y l c h l o r i d e from a n t h r a q u i n o n e - 2 - c a r b o x y l i c a c i d 76 3. The t o t a l i o n mass chromatogram (A) and t h e mass spectrum (B) o f a n t h r a c e n e - 2 - c a r b o x y l i c a c i d 77 4. The t o t a l i o n mass chromatogram (A) and t h e mass spectrum (B) o f 2 - a n t h r o y l c h l o r i d e 78 5. R e a c t i o n scheme f o r t h e d e r i v a t i z a t i o n o f m e x i l e t i n e e n a n t i o m e r s and the i n t e r n a l s t a n d a r d w i t h 2 - a n t h r o y l c h l o r i d e 80 6. The t o t a l i o n mass chromatogram (A) and the mass s p e c t r a (B and C) o b t a i n e d from t h e 2 - a n t h r o y l d e r i v a t i v e o f m e x i l e t i n e 82 7. The major fragment i o n s o f the 2 - a n t h r o y l d e r i v a t i v e o f m e x i l e t i n e 83 8. Chromatogram o f t h e 2 - a n t h r o y l d e r i v a t i v e o f m e x i l e t i n e e n a n t i o m e r s 84 9. The s t r u c t u r e o f t h e P i r k l e ^ i o n i c c h i r a l s t a t i o n a r y phase [ ( R ) - 3 , 5 - d i n i t r o b e n z o y l p h e n y l g l y c i n e i o n i c a l l y bonded t o T-amino p r o p y l s i l i c a ] and t h e 2 - a n t h r o y l d e r i v a t i v e o f m e x i l e t i n e 86 10. Chromatograms o f e x t r a c t s o f d i s t i l l e d w a ter c o n t a i n i n g m e x i l e t i n e e n a n t i o m e r s and t h e i n t e r n a l s t a n d a r d ( A ) , and b l a n k d i s t i l l e d w ater (B) 88 11. Chromatogram o f R ( - ) - m e x i l e t i n e and t h e i n t e r n a l s t a n d a r d d e r i v a t i z e d w i t h 2 - a n t h r o y l c h l o r i d e a f t e r e x t r a c t i o n from an aqueous s o l u t i o n w i t h d i e t h y l e t h e r (A) and the same c o n c e n t r a t i o n o f R ( - ) - m e x i l e t i n e and the i n t e r n a l s t a n d a r d d e r i v a t i z e d w i t h o u t p r i o r e x t r a c t i o n 90 12. Chromatograms o b t a i n e d a f t e r e x t r a c t i o n o f 1 ml a l i q u o t s o f b l a n k d i s t i l l e d water w i t h d i f f e r e n t e x t r a c t i o n s o l v e n t s : d i e t h y l e t h e r ( A ) , hexane (B) and d i c h l o r o m e t h a n e (C) 91 x v i 13. Chromatograms o f m e x i l e t i n e e n a n t i o m e r s and t h e i n t e r n a l s t a n d a r d (100 ng/ml) i s o l a t e d from human plasma (A) and b l a n k plasma (B) 93 14. The r e l a t i o n s h i p between p e r c e n t f r e e f r a c t i o n and serum pH 97 15. A r e p r e s e n t a t i v e " R o s e n t h a l " p l o t o f t h e b i n d i n g o f m e x i l e t i n e e n a n t i o m e r s t o serum from a h e a l t h y male s u b j e c t 106 16. The s e m i l o g a r i t h m i c p l o t o f serum c o n c e n t r a t i o n v e r s u s t i m e f o r m e x i l e t i n e e n a n t i o m e r s i n r a t s f o l l o w i n g t h e a d m i n i s t r a t i o n o f a s i n g l e i . v . dose o f r a c e m i c m e x i l e t i n e 113 17. The c o n c e n t r a t i o n - t i m e p r o f i l e o f m e x i l e t i n e e n a n t i o m e r s i n serum, h e a r t and b r a i n t i s s u e s f o l l o w i n g t h e a d m i n i s t r a t i o n o f a s i n g l e i . v . dose o f r a c e m i c m e x i l e t i n e 122 18. The r e l a t i o n s h i p between dose ( c u m u l a t i v e ) and th e plasma c o n c e n t r a t i o n o f r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s i n P e n t o b a r b i t o n e a n a e s t h e t i z e d r a t s 125 19. C u m u l a t i v e d o s e - r e s p o n s e c u r v e s o f t h e e f f e c t s o f r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s on VFT i n p e n t o b a r b i t o n e a n a e s t h e t i z e d r a t s 130 20. C u m u l a t i v e d o s e - r e s p o n s e c u r v e s o f t h e e f f e c t s o f r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s on ERP i n p e n t o b a r b i t o n e a n a e s t h e t i z e d r a t s 132 21. C u m u l a t i v e d o s e - r e s p o n s e c u r v e s o f t h e e f f e c t s o f r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s on MFF i n p e n t o b a r b i t o n e a n a e s t h e t i z e d r a t s 133 22. C u m u l a t i v e d o s e - r e s p o n s e c u r v e s o f t h e e f f e c t s o f r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s on th e h e a r t r a t e i n p e n t o b a r b i t o n e a n a e s t h e t i z e d r a t s 137 23. C u m u l a t i v e d o s e - r e s p o n s e c u r v e s o f t h e e f f e c t s o f r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s on th e mean a r t e r i a l b l o o d p r e s s u r e i n p e n t o b a r b i t o n e a n a e s t h e t i z e d r a t s 138 24. The plasma c o n c e n t r a t i o n - t i m e p r o f i l e o f r a c e m i c m e x i l e t i n e and t h e i n d i v i d u a l e n a n t i o m e r s i n i s c h a e m i c c o n s c i o u s r a t s a f t e r t h e a d m i n i s t r a t i o n o f a dose o f 20 mg/kg, f o l l o w e d 1.5 h l a t e r by a second dose (20 mg/kg) 141 x v i i 25. The r a t h e a r t showing t h e o c c l u s i o n s n a r e around t h e l e f t a n t e r i o r d e s c e n d i n g c o r o n a r y a r t e r y 144 26. T y p i c a l a r r h y t h m i a s r e s u l t i n g from o c c l u s i o n o f t h e l e f t a n t e r i o r d e s c e n d i n g c o r o n a r y a r t e r y 145 27. The e f f e c t s o f ( R ) - , R,S- and S ( + ) - m e x i l e t i n e and s a l i n e on " a r r h y t h m i a s c o r e " f o r t h e 0-30 min p e r i o d f o l l o w i n g c o r o n a r y a r t e r y o c c l u s i o n i n c o n s c i o u s r a t s 149 28. The e f f e c t s o f ( R ) - , R,S- and S ( + ) - m e x i l e t i n e and s a l i n e on " a r r h y t h m i a s c o r e " f o r t h e 0-4 h p e r i o d f o l l o w i n g c o r o n a r y a r t e r y o c c l u s i o n i n c o n s c i o u s r a t s 150 29. The e f f e c t s o f ( R ) - , R,S- and S ( + ) - m e x i l e t i n e and s a l i n e on h e a r t r a t e i n i s c h a e m i c c o n s c i o u s r a t s 154 30. The e f f e c t s o f ( R ) - , R,S- "and S ( + ) - m e x i l e t i n e and s a l i n e on mean a r t e r i a l b l o o d p r e s s u r e i n i s c h a e m i c c o n s c i o u s r a t s 155 31. The e f f e c t s o f r a c e m i c m e x i l e t i n e and s a l i n e on " a r r h y t h m i a s c o r e " f o r t h e 0-30 min p e r i o d f o l l o w i n g c o r o n a r y a r t e r y o c c l u s i o n i n p e n t o b a r b i t o n e a n a e s t h e t i z e d r a t s 162 32. The plasma c o n c e n t r a t i o n - t i m e r e l a t i o n s h i p f o l l o w i n g a d m i n i s t r a t i o n o f r a c e m i c m e x i l e t i n e (20 mg/kg) ( A ) , and t h e d i s t r i b u t i o n o f major a r r h y t h m i a s (VT + VF) i n i s c h a e m i c p e n t o b a r b i t o n e a n a e s t h e t i z e d r a t s (B) 164 33. The d i s t r i b u t i o n o f major a r r h y t h m i a s (VT + VF) i n i s c h a e m i c p e n t o b a r b i t o n e a n a e s t h e t i z e d r a t s f o l l o w i n g t h e a d m i n i s t r a t i o n o f s a l i n e ( c o n t r o l ) 165 34. The plasma c o n c e n t r a t i o n - t i m e r e l a t i o n s h i p f o l l o w i n g t h e a d m i n i s t r a t i o n o f r a c e m i c m e x i l e t i n e (40 mg/kg) ( A ) , and t h e d i s t r i b u t i o n o f major a r r h y t h m i a s (VT + VF) i n i s c h a e m i c p e n t o b a r b i t o n e a n a e s t h e t i z e d r a t s (B) 166 x v i i i SYMBOLS AND ABBREVIATIONS AAG a j - A c i d g l y c o p r o t e i n ANOVA A n a l y s i s o f v a r i a n c e AP A c t i o n p o t e n t i a l ARP A b s o l u t e r e f r a c t o r y p e r i o d AUC A r e a under t h e plasma c o n c e n t r a t i o n - t i m e c u r v e AUC T A r e a under t h e t i s s u e c o n c e n t r a t i o n - t i m e c u r v e AV A t r i o - v e n t r i c u l a r C C e n t r i g r a d e C V . C o e f f i c i e n t o f v a r i a t i o n CL S y s t e m i c c l e a r a n c e C L i n t I n t r i n s i c c l e a r a n c e CNS C e n t r a l nervous system CSP C h i r a l s t a t i o n a r y phase ECG El e c h o c a r d i o g r a m EI E l e c t r o n impact em E m i s s i o n ERP E f f e c t i v e r e f r a c t o r y p e r i o d ex E x c i t a t i o n u F r e e f r a c t i o n GLC G a s - l i q u i d chromatography . HC1 H y d r o c h l o r i c a c i d HPLC Hi g h - p e r f o r m a n c e l i q u i d chromatography HSA Human serum albumin ht H e i g h t i .d. I n t e r n a l d i a m e t e r i . p . I n t r a - p e r i t o n e a l x i x I.S. I n t e r n a l s t a n d a r d i . v . I n t r a v e n o u s IU I n t e r n a t i o n a l u n i t LAD L e f t a n t e r i o r d e s c e n d i n g LP L i p o p r o t e i n mex M e x i l e t i n e MFF Maximum f o l l o w i n g f r e q u e n c y min M i n u t e s ml M i l l i l i t r e MP Membrane p o t e n t i a l MS Mass s p e c t r o m e t e r msec M i l l i s e c o n d s ng Nanogram NSVF N o n - s p o n t a n e o u s l y r e v e r t i n g v e n t r i c u l a r f i b r i l l a t i o n OZ O c c l u d e d zone PE P o l y e t h y l e n e PTFE P o l y t e t r a f l u o r o e t h y l e n e PVC Premature V e n t r i c u l l a r C o n t r a c t i o n R R e s o l u t i o n r ^ C o e f f i c i e n t o f d e t e r m i n a t i o n RRP R e l a t i v e r e f r a c t o r y p e r i o d R^ . R e t e n t i o n t ime j9 F i r s t o r d e r e l i m i n a t i o n r a t e c o n s t a n t s.d. S t a n d a r d d e v i a t i o n s.e.m S t a n d a r d e r r o r o f the mean sec Seconds SVF S p o n t a n e o u s l y r e v e r t i n g v e n t r i c u l a r f i b r i l l a t i o n XX UV U l t r a v i o l e t VF V e n t r i c u l a r f i b r i l l a t i o n VFT V e n t r i c u l a r f i b r i l l a t i o n t h r e s h o l d vs V e r s u s V s s S t e a d y - s t a t e volume o f d i s t r i b u t i o n VT V e n t r i c u l a r t a c h y c a r d i a wt Weight xs E xcess a F i r s t o r d e r d i s t r i b u t i o n r a t e c o n s t a n t /xl M i c r o l i t r e fig Microgram x x i ACKNOWLEDGEMENT I wish t o e x p r e s s my s i n c e r e g r a t i t u d e t o Dr. K e i t h M c E r l a n e f o r h i s s u p e r v i s i o n and g u i d a n c e t h r o u g h the c o u r s e o f t h i s s t u d y . I would a l s o l i k e t o thank t h e members o f my t h e s i s committee, D r s . F r a n k A b b o t t , J i m O r r , J i m A x e l s o n and C h a r l e s K e r r , f o r a l l t h e h e l p f u l d i s c u s s i o n s . My g r a t i t u d e a l s o goes t o Mr. Gregory Beatch f o r h i s t e c h n i c a l a s s i s t a n c e . The f i n a n c i a l s u p p o r t from B o e h r i n g e r I n g e l h e i m L t d . (Canada) and The Canadian H e a r t F o u n d a t i o n are g r a t e f u l l y acknowledged. DEDICATION T h i s t h e s i s i s d e d i c a t e d t o Dr. J.D. K u l k a r n i , who g r e a t l y i n f l u e n c e d d e c i s i o n t o p u r s u e g r a d u a t e s t u d i e s . 1 1. INTRODUCTION 1.1 Sudden Cardiac Death 1.1.1 Overview Sudden c a r d i a c d e a t h has been d e f i n e d as d e a t h o c c u r r i n g from c o r o n a r y h e a r t d i s e a s e w i t h i n 1 hour o f l a s t b e i n g seen a l i v e ( O l i v e r , 1982). Amstrong et al. (1972) r e p o r t e d t h a t 43% o f a l l d e a t h s o c c u r r i n g w i t h i n 4 weeks o f t h e o n s e t o f a h e a r t a t t a c k t o o k p l a c e w i t h i n one hour and t h a t l e s s than 10% o f t h e s e p a t i e n t s were seen by a me d i c a l p e r s o n . Sudden c a r d i a c d e a t h i s the l e a d i n g cause o f d e a t h i n most o f t h e i n d u s t r i a l i z e d n a t i o n s . A c c o r d i n g t o a r e p o r t by Lown and Graboys (1977), about 450,000 such d e a t h s ( n e a r l y 25 % o f a l l d e a t h s ) o c c u r a n n u a l l y i n t h e U n i t e d S t a t e s a l o n e . The cause o f sudden c a r d i a c d e a t h has been i d e n t i f i e d as v e n t r i c u l a r f i b r i l l a t i o n (VF) (Smith 1939; M i l l e r , 1939; Bashe et al., 1975; S c h a f f e r and Cobb, 1975; Lown and Graboys, 1977; O l i v e r , 1982; Adgey, 1982) and sudden c a r d i a c d e a t h and VF a r e viewed as b e i n g synonymous by many p h y s i c i a n s ( O l i v e r , 1982). VF i s t h e most s e v e r e form o f v e n t r i c u l a r a r r h y t h m i a s , c h a r a c t e r i z e d by a c h a o t i c r a p i d c o n t r a c t i o n o f t h e v e n t r i c u l a r muscle f i b r e s w i t h no b l o o d b e i n g pumped. I t was f i r s t d e s c r i b e d by H o f f a and Ludwig (1850) who r e p o r t e d t h a t e l e c t r i c a l s t i m u l a t i o n o f the mammalian h e a r t l e d t o VF and d e a t h . M c W i l l i a m i n 1889 s u g g e s t e d t h a t VF was t h e l i k e l y c a u s e o f sudden c a r d i a c d e a t h i n p a t i e n t s w i t h A n g i n a P e c t o r i s . The f i r s t e l e c t r o c a r d i o g r a m (ECG) o f VF i n t h e c l i n i c a l s e t t i n g was o b t a i n e d i n 1911 by Hoffman. However, i t was Smith (1939) and M i l l e r (1939) who f i r s t r e p o r t e d VF as t h e cause o f sudden c a r d i a c d e a t h i n man f o l l o w i n g 2 m y o c a r d i a l i n f a r c t i o n (MI). A c u t e r e g i o n a l i s c h a e m i a has been r e c o g n i z e d as t h e b a s i s f o r t h e development o f v e n t r i c u l a r a r r h y t h m i a s l e a d i n g t o sudden c a r d i a c d e a t h ( O l i v e r , 1982). T h i s i s c o n s i s t e n t w i t h t h e f i n d i n g t h a t many p a t i e n t s d y i n g s u d d e n l y had e x t e n s i v e a r t e r o s c l e r o s i s o f one o r more c o r o n a r y v e s s e l ( s ) upon postmortem e x a m i n a t i o n ( K u l l e r et al., 1975; P e r p e r e t al., 1975). However, sudden c a r d i a c d e a t h i s not always a s s o c i a t e d w i t h i d e n t i f i a b l e MI o r c o r o n a r y t h r o m b o s i s a t a u t o p s y ( L o v e g r o v e and Thompson, 1978). In f a c t , ECG e v i d e n c e o f MI i s p r e s e n t i n l e s s t h a n 20% o f t h o s e p a t i e n t s s u r v i v i n g a VF a t t a c k (Cobb et al., 1980). T h i s has l e a d t o t h e s u g g e s t i o n t h a t vasospasm, f o l l o w e d by sudden r e p e r f u s i o n , may be a mechanism f o r t h e i n d u c t i o n o f v e n t r i c u l a r f i b r i l l a t i o n i n man ( H e l l s t r o m , 1979; Lown, 1979). C o r o n a r y a r t e r y spasm has been c l i n i c a l l y d e m o n s t r a t e d i n man ( O l i v a et al., 1973; M a s e r i et al., 1975), and t h e i n d u c t i o n o f VF by c o r o n a r y a r t e r y o c c l u s i o n , f o l l o w e d by sudden r e p e r f u s i o n , i s w e l l e s t a b l i s h e d i n a n i m a l s (Tennent and Wiggers, 1935; Penkoski et al., 1978; Stockman e t al., 1979; K a b e l l e t al, 1980). E p i d e m i o l o g i c a l s t u d i e s o f s u r v i v o r s o f MI have shown t h a t t h e s e p a t i e n t s a r e a t t h e g r e a t e s t r i s k o f d e v e l o p i n g VF where c h r o n i c e c t o p i c a c t i v i t y i s p r e s e n t ( C o r o n a r y Drug P r o j e c t R e s e a r c h Group, 1973; H i n k l e , 1981; The M u l t i c e n t e r P o s t - I n f a r c t i o n R e search Group, 1983). As a r e s u l t o f t h e s e and o t h e r s t u d i e s , many drugs have been d e v e l o p e d f o r th e t r e a t m e n t and p r e v e n t i o n o f a r r h y t h m i a s . However, i n e f f e c t i v e n e s s a g a i n s t a l l forms o f a r r h y t h m i a s , t o x i c i t y , and sometimes u n f a v o u r a b l e p h a r m a c o k i n e t i c s have l i m i t e d t h e t h e r a p e u t i c b e n e f i t o f t h e s e a g e n t s . For example, q u i n i d i n e i n t h e r a p e u t i c doses may i n d u c e v e n t r i c u l a r t a c h y c a r d i a and v e n t r i c u l a r f i b r i l l a t i o n ( S e l z e r and Wray, 1964; K o s t e r 3 and W e l l e n s , 1976). L i d o c a i n e , a n o t h e r a n t i a r r h y t h m i c agent, i s not a d m i n i s t e r e d o r a l l y due t o h i g h h e p a t i c f i r s t - p a s s m e t a b o l i s m ( S i n g h e t a7., 1981). Thus, many novel drugs a r e b e i n g d e v e l o p e d and t e s t e d i n th e c o n t i n u i n g s e a r c h f o r a n t i a r r h y t h m i c agents w i t h b e t t e r b e n e f i t - t o -r i s k r a t i o , as w e l l as c o n v e n i e n t d o s i n g . The s u b j e c t o f t h e p r e s e n t s t u d y , m e x i l e t i n e , i s one o f t h e newer a n t i a r r h y t h m i c agents which was approved f o r c l i n i c a l use i n Canada i n 1985. 1.1.2 A r r h y t h m i a s 1.1.2.1 The C a r d i a c A c t i o n P o t e n t i a l U n d e r s t a n d i n g t h e i o n i c e v e n t s which t a k e p l a c e a c r o s s c a r d i a c c e l l membranes d u r i n g each c a r d i a c c y c l e i . e a c t i o n p o t e n t i a l (AP) i s n e c e s s a r y i n r a t i o n a l i z i n g t h e mechanisms o f f o r m a t i o n o f a r r h y t h m i a s , and a l s o t h e p o s s i b l e mechanisms o f a c t i o n o f a n t i a r r h y t h m i c d r u g s . These i o n i c e v e n t s have been e x t e n s i v e l y r e v i e w e d ( C r a n e f i e l d , 1975; H a u s w i r t h and S i n g h , 1979; P e r r y and II1 s i e y , 1986). The r e s t i n g membrane p o t e n t i a l (MP) o f most c a r d i a c c e l l s i s about -80 t o -90 mV and i s s u p p o r t e d p r i n c i p a l l y by sodium and p o t a s s i u m i o n s . By means o f an a c t i v e t r a n s p o r t mechanism i n v o l v i n g t h e N a + / K + ATPase (sodium pump), th e c e l l m a i n t a i n s a h i g h i n t r a c e l l u l a r - t o - e x t r a c e l l u l a r p o t a s s i u m c o n c e n t r a t i o n and a low i n t r a c e l l u l a r - t o - e x t r a c e l l u l a r sodium c o n c e n t r a t i o n . At r e s t , t h e c a r d i a c c e l l membrane i s r e a d i l y permeable t o p o t a s s i u m i o n s and r e l a t i v e l y impermeable t o sodium, c a l c i u m , and c h l o r i d e i o n s . Thus, e x c e s s i n t r a c e l l u l a r K + can d i f f u s e out o f t h e c e l l unaccompanied by n e g a t i v e l y c h a r g e d i o n s , t h e r e b y , p r o d u c i n g t h e above mentioned n e g a t i v e p o t e n t i a l a c r o s s t he membrane ( i . e . c e l l s a r e n e g a t i v e on t h e i n s i d e w i t h r e s p e c t t o the o u t s i d e ) . 4 The action potential is composed of five phases that represent the changing ionic fluxes and membrane potentials of the cardiac c e l l . Reduction of the membrane potential to a threshold voltage, -60 to -70 mV, due to an applied stimulus or current spreading from pacemaker ce l l s , evokes the fast inward depolarizing current carried by sodium ions (phase 0 of the action potential). The intensity of this inward current is reflected in the maximal rate of depolarization (MRD) of the transmembrane AP. Depolarization to levels positive to -55 mV inactivates the fast sodium channels. However, the membrane continues to depolarize to a level of +25 to +30 mV due to the rapid rate of depolarization from the i n i t i a l portion of phase 0. A second inward current (the slow inward current) carried mostly by calcium ions and activated at a MP of about -35 mV also exists. The kinetics of this current, in terms of activation, inactivation and reactivation, are much slower than those governing the sodium current. After depolarization, repolarization takes place in three phases (phases 1, 2 and 3). Phase 1 results partly from inactivation of the fast inward current and partly from an inward movement of chloride ions. Phase 2 is characterized by inward C a + + and CI" currents and a slow outward K + current. Very l i t t l e net charge flows across the membrane at this point which results in the characteristic plateau of the action potential. Phase 3 is typified by inactivation of the C a + + current and a rapid efflux of K + leading to fu l l repolarization. Phase 4 represents the period between action potentials i . e . the resting phase of the c e l l . The automatic cardiac fibers (section 1.1.3.1) undergo spontaneous depolarization during phase 4. There are two types of cardiac fibres, namely the fast and the 5 slow f i b r e s . The f a s t f i b r e s a r e t h e a t r i a l and v e n t r i c u l a r s p e c i a l i z e d c o n d u c t i n g t i s s u e s , t h e d i s t a l AV node, and a t r i a l and v e n t r i c u l a r muscle (Wits et al., 1974a; Z i p e s , 1984). These f i b e r s g e n e r a t e f a s t inward c u r r e n t AP as d e s c r i b e d above and are c h a r a c t e r i z e d by r a p i d c o n d u c t i o n v e l o c i t y . They a r e s e n s i t i v e ( b l o c k e d ) t o i n c r e a s e d e x t r a c e l l u l a r p o t a s s i u m c o n c e n t r a t i o n , t e t r o d o t o x i n and l o c a l a n a e s t h e t i c s . The slow f i b r e s a r e found i n t h e p r o x i m a l AV node, SA node, f i b r e s o f t h e AV r i n g and t h e m i t r a l and t r i c u s p i d v a l v e l e a f l e t s (Wits et al., 1974a; Z i p e s , 1984). They g e n e r a t e a slow inward c u r r e n t AP which i s me d i a t e d by c a l c i u m , has a r e s t i n g membrane p o t e n t i a l o f -40 t o -60 mV and a r e a c t i v a t e d a t a t h r e s h o l d o f -30 t o 40 mV. The AP i s b l o c k e d by manganese and c a l c i u m a n t a g o n i s t s . The MRD d u r i n g an a c t i o n p o t e n t i a l i s a f u n c t i o n o f t h e membrane p o t e n t i a l from which t h e AP t a k e s o f f . The more n e g a t i v e t h e MP, t h e g r e a t e r t h e MRD. T h i s r e l a t i o n s h i p i s known as membrane r e s p o n s i v e n e s s ( S i n g h e t al., 1981). D u r i n g an a c t i o n p o t e n t i a l , t h e r e i s a p e r i o d o f time d u r i n g which a second a c t i o n p o t e n t i a l cannot be p r o d u c e d . T h i s p e r i o d i s r e f e r r e d t o as t h e r e f r a c t o r y p e r i o d ( P e r r y and I l l s l e y , 1986). The a b s o l u t e r e f r a c t o r y p e r i o d (ARP) c o r r e s p o n d s t o t h e p e r i o d d u r i n g which a s t i m u l u s cannot produce any degree o f d e p o l a r i z a t i o n . A t MP below -50 mV, t r a n s i e n t d e p o l a r i z a t i o n may o c c u r but an a c t i o n p o t e n t i a l cannot be p r o p a g a t e d , t h i s r e p r e s e n t s t h e e f f e c t i v e r e f r a c t o r y p e r i o d (ERP). Below -60 mV a s t r o n g e r than normal s t i m u l u s may g e n e r a t e an a c t i o n p o t e n t i a l o f low a m p l i t u d e . T h i s c o r r e s p o n d s t o the r e l a t i v e r e f r a c t o r y p e r i o d (RRP). A c t i o n p o t e n t i a l s g e n e r a t e d d u r i n g t h i s p e r i o d a r e c o n d u c t e d s l o w l y , c o n s e q u e n t l y , t h e h e a r t i s prone t o a r r h y h t h m i a f o r m a t i o n ( P e r r y and I l l s l e y , 1986). 6 1.1.2.2 E l e c t r o p h y s i o l o g i c a l Changes During Acute Myocardial Ischaemia I t has l o n g b e e n e s t a b l i s h e d t h a t m y o c a r d i a l c e l l s r e l e a s e K + d u r i n g a c u t e i s c h a e m i a ( H a r r i s , et al., 1 9 5 4 ) . H i l l and G e t t e s ( 1 9 8 0) and H i r c h e e t al. ( 1 9 8 0 ) r e p o r t e d i n c r e a s e d e x t r a c e l l u l a r K + w i t h i n 15 s e c o n d s o f c o r o n a r y a r t e r y o c c l u s i o n i n p i g s ( a 3 - f o l d i n c r e a s e was o b s e r v e d by 5-8 m i n ) . The i n c r e a s e i n e x t r a c e l l u l a r K + was a c c o m p a n i e d by a d e c r e a s e i n t h e r e s t i n g MP due t o t h e c h a n g e d g r a d i e n t o f K + . The o b s e r v e d l o s s o f K + f r o m i s c h a e m i c c e l l s has b e e n s p e c u l a t e d t o be due t o a n o x i a - i n d u c e d i n c r e a s e i n K + c o n d u c t a n c e o r i n h i b i t i o n o f t h e s o d i u m pump. S i m u l t a n e o u s t o t h e i n c r e a s e i n e x t r a c e l l u l a r K + i s a d e c r e a s e i n t h e AP a m p l i t u d e , MRD and a c t i o n p o t e n t i a l d u r a t i o n ( A P D ) . T h e s e c h a n g e s w o u l d be e x p e c t e d t o r e s u l t f r o m t h e d e c r e a s e d r e s t i n g MP. However, M o r e n a e t al. ( 1 9 8 0) showed t h a t a d e c r e a s e i n r e s t i n g MP o n l y c o u l d n o t a c c o u n t f o r t h e m a r k ed d e p r e s s i o n o f t h e AP u p s t r o k e c h a r a c t e r i s t i c s o b s e r v e d d u r i n g a c u t e i s c h a e m i a . The r e s u l t s o f t h e i r w ork i n d i c a t e d t h a t t h e c o m b i n a t i o n o f l a c k o f O2 and s u b s t r a t e and l a c k o f w a s h o u t ( i n c r e a s e d e x t r a c e l l u l a r K + and a c i d o s i s ) i s n e c e s s a r y . T h i s c o m b i n a t i o n o f f a c t o r s was shown t o h a v e an e x t r a - d e p r e s s a n t e f f e c t on t h e i o n i c m e c h a n i s m s r e s p o n s i b l e f o r t h e g e n e r a t i o n o f t h e AP u p s t r o k e . T h e c u r r e n t s g e n e r a t i n g t h e d e p r e s s e d u p s t r o k e were i n v e s t i g a t e d by C a r d i n a l e t al. (1981) and f o u n d t o be a " d e p r e s s e d f a s t r e s p o n s e " . T h i s c o n c l u s i o n was d e d u c e d f r o m r e s u l t s o b t a i n e d i n i s o l a t e d h e a r t s w h i c h showed: a) l i d o c a i n e had an e x t r a - d e p r e s s a n t e f f e c t on AP u p s t r o k e c h a r a c t e r i s t i c s , and b) no r e s p o n s e c o u l d be e l i c i t e d when t h e r e s t i n g MP was d e p o l a r i z e d t o -55 mV, a t w h i c h l e v e l t h e f a s t N a + c h a n n e l i s t o t a l l y i n a c t i v a t e d . However, t h e r e i s e v i d e n c e t h a t t h e 7 slow inward c u r r e n t i s e q u a l l y d e p r e s s e d by i s c h a e m i a ( J a n s e , 1982). As a consequence o f t h e r e d u c t i o n i n APD, t h e ERP would be e x p e c t e d t o be s h o r t e n e d . However, ERP i s p r o l o n g e d i n a c u t e l y i s c h a e m i c c e l l s s i n c e ERP c o n t i n u e s beyond t h e p o i n t o f f u l l r e p o l a r i z a t i o n . T h i s phenomenon i s known as p o s t - r e p o l a r i z a t i o n - r e f r a c t o r i n e s s (Downar et al., 1977; L a z z a r a et al., 1978). I t i s i m p o r t a n t t o note t h a t t h e s e e l e c t r o p h y s i o l o g i c a l changes o c c u r i n d i f f e r e n t i s c h a e m i c c e l l s t o d i f f e r e n t d e g r e e s . .Downar et al. (1977), u s i n g p i g s u b e p i c a r d i a l muscle, n o t e d t h a t groups o f c e l l s w i t h a d i f f e r e n c e o f o n l y a few m i l l i v o l t s i n t h e i r r e s t i n g MP v a r i e d i n ERP from 180-500 msec. Thus, a t a c e r t a i n c o u p l i n g i n t e r v a l , t h e l e a s t d e p o l a r i z e d c e l l group may e x h i b i t an AP w i t h a low u p s t r o k e v e l o c i t y which w i l l p r o p a g a t e v e r y s l o w l y ; whereas t h e c e l l s w i t h a s l i g h t l y l o w er r e s t i n g MP a r e a t t h e same time i n e x c i t a b l e , and complete c o n d u c t i o n b l o c k may o c c u r . T h e r e f o r e , slow c o n d u c t i o n and c o n d u c t i o n b l o c k , both o f which a r e n e c e s s a r y c o n d i t i o n s f o r r e - e n t r a n t a r r h y t h m i a s ( s e c t i o n 1.1.2.3 below), a r e p r e s e n t d u r i n g a c u t e i s c h a e m i a . 1.1.2.3 A r r h y t h m o g e n e s i s A r r h y t h m i a s r e s u l t from d i s t u r b a n c e s i n normal i m p u l s e i n i t i a t i o n ( a u t o m a t i c i t y ) , i m p u lse c o n d u c t i o n , o r both (Hoffman and Rosen, 1981). A r r h y t h m i a s i n c l u d e e c t o p i c b e a t s , t a c h y c a r d i a , A-V b l o c k and f i b r i l l a t i o n . 1.1.2.3.1 Impulse I n i t i a t i o n ( A u t o m a t i c i t y ) A u t o m a t i c i t y i s the a b i l i t y o f a c a r d i a c f i b r e t o d e p o l a r i z e s p o n t a n e o u s l y and g e n e r a t e an a c t i o n p o t e n t i a l (Wit et al., 1974a; V e r a 8 and Mason, 1981). Autonomic f i b r e s i n c l u d e t h e SA node, d i s t a l AV node, H i s - P u r k i n j e f i b r e s , s p e c i a l i z e d a t r i a l f i b r e s , m i t r a l and t r i c u s p i d v a l v e l e a f l e t s . The SA node i s n o r m a l l y t h e dominant f i b e r (pacemaker) and e s t a b l i s h e s t h e r a t e o f impulse g e n e r a t i o n . Abnormal a u t o m a t i c i t y r e s u l t s when a h e a r t t i s s u e o t h e r than the SA node t a k e s o v e r t h e pacemaker r o l e . The s i t e o f pacemaker a c t i v i t y may be s h i f t e d by 3 f a c t o r s t h a t a f f e c t t h e r a t e o f d e p o l a r i z a t i o n : a) l e v e l o f maximum d i a s t o l i c p o t e n t i a l , b) l e v e l o f t h r e s h o l d p o t e n t i a l and c) s l o p e o f phase 4 o f t h e a c t i o n p o t e n t i a l (Wit e t al., 1974a; V e r a and Mason, 1981). For example, v a g a l s t i m u l a t i o n i n h i b i t s SA node a u t o m a t i c i t y (by d e c r e a s i n g phase 4 s l o p e and by making t h e d i a s t o l i c p o t e n t i a l more n e g a t i v e ) w i t h o u t a f f e c t i n g t h e P u r k i n j e f i b r e s . Thus pacemaker a c t i v i t y may be s h i f t e d t o t h e s e f i b r e s . Ischaemia i n c r e a s e s phase 4 s l o p e and s h i f t s t h e maximum d i a s t o l i c p o t e n t i a l t o a more p o s i t i v e v a l u e , t h u s e n h a n c i n g a u t o m a t i c i t y . 1.1.2.3.2 T r i g g e r e d A u t o m a t i c i t y C o n s i d e r a b l e a t t e n t i o n has a l s o been d i r e c t e d towards t h e phenomenon o f t r i g g e r e d a c t i v i t y i n the g e n e s i s o f c e r t a i n t y p e s o f a r r h y t h m i a s (Spear and Moore, 1982). T r i g g e r e d a u t o m a t i c i t y has so f a r o n l y been d e m o n s t r a t e d in vitro i n q u i e s c e n t c a r d i a c c e l l s ( F e r r i e r e t al., 1973). C e r t a i n c r i t i c a l c h a r a c t e r i s t i c s o f a r r h y t h m i a s evoked by t r i g g e r e d a u t o m a t i c i t y c l o s e l y resemble t h o s e i n d u c e d by r e - e n t r y , n o t a b l y t h e c o u p l i n g o f d e p o l a r i z a t i o n t o a p r e c e d i n g b e a t . The a f t e r -d e p o l a r i z a t i o n s r e s p o n s i b l e f o r t r i g g e r e d a u t o m a t i c i t y a r e a s s o c i a t e d w i t h e l e v a t e d i n t r a c e l l u l a r C a + + c o n c e n t r a t i o n s . T h i s p r o v o k e s t h e o s c i l l a t o r y r e l e a s e o f C a + + from t h e s a r c o p l a s m i c r e t i c u l u m and 9 a c t i v a t e s membrane c h a n n e l s t h a t p e r m i t t h e passage o f N a + and K +. The n e t f l u x o f t h e s e c a t i o n s c o n s t i t u t e a t r a n s i e n t inward c u r r e n t t h a t i s r e s p o n s i b l e f o r the d e p o l a r i z a t i o n o f t h e c e l l membrane. The a m p l i t u d e o f t h e a f t e r - d e p o l a r i z a t i o n i s , t h u s , i n c r e a s e d by f a c t o r s t h a t r a i s e t h e i n t r a c e l l u l a r C a + + c o n c e n t r a t i o n s ; such as e l e v a t e d e x t r a c e l l u l a r C a + + o r t o x i c l e v e l s o f d i g i t a l i s g l y c o s i d e s . 1.1.2.3.3 Impulse C o n d u c t i o n ( R e - e n t r y ) The c o n c e p t o f c i r c u s movement o r r e - e n t r y as a mechanism u n d e r l y i n g s u s t a i n e d a r r h y t h m i a s was f i r s t i n t r o d u c e d by Mines i n 1913. Recent r e s e a r c h ( A l l e s s i e et al., 1973; 1976 & 1977) has p r o v i d e d d i r e c t e v i d e n c e f o r t h e e x i s t e n c e o f r e - e n t r a n t c i r c u i t s . R e - e n t r a n t a r r h y t h m i a s a r e c h a r a c t e r i z e d by a r e a s o f u n i d i r e c t i o n a l b l o c k such t h a t t h e p r o p a g a t i o n o f t h e normal i m p u l s e s i s b l o c k e d i n an a n t e g r a d e d i r e c t i o n and i s c o n d u c t e d v i a an a l t e r n a t e pathway. I f c o n d u c t i o n a l o n g t h i s pathway i s slow enough t o a l l o w t h e zone o f b l o c k t o r e c o v e r e x c i t a b i l i t y , t hen r e t r o g r a d e c o n d u c t i o n t h r o u g h t h e b l o c k zone w i l l o c c u r and t h e w a v e f r o n t w i l l r e - e x c i t e f i b e r s a t t h e s i t e o f o r i g i n o f t h e i m p u l s e (Wit et al., 1974b; V e r a and Mason, 1981). T h i s t y p e o f r e -e n t r y i s r e f e r r e d t o as macro r e - e n t r y . The c o n d u c t i o n pathway w i l l be f u n c t i o n a l l y l o n g i f c o n d u c t i o n i s d e p r e s s e d . On t h e o t h e r hand, i f t h e r e f r a c t o r y p e r i o d i n t h e a n t e g r a d e pathway i s s h o r t e n e d , then t h e r e -e n t r a n t c i r c u i t need not be l o n g . The l a t e r t y p e o f r e - e n t r y i s c a l l e d m i c r o r e - e n t r y ( S a s y n i u k and Mendez, 1971; Kramer et al., 1985). The r e - e n t r y phenomenon can r e s u l t i n e i t h e r s i n g l e premature b e a t s o r r e p e t i t i v e e c t o p i c a c t i v i t y ( P e r r y and I l l s l e y , 1986). C o n d u c t i o n i s s u f f i c i e n t l y slow i n c a r d i a c f i b e r s w i t h s l o w - c u r r e n t a c t i o n p o t e n t i a l (SA and AV nodes) t o a l l o w r e - e n t r y t o o c c u r , o r i n f a s t f i b e r s where normal f a s t c u r r e n t a c t i o n p o t e n t i a l has been slowed by d i s e a s e o r drugs ( W i t t et al., 1974b). 1.1.2.4 Mode o f A c t i o n o f A n t i a r r h y t h m i c a g e n t s . Most a n t i a r r h y t h m i c agents have major 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 on m y o c a r d i a l c e l l s . Based on t h e s e e f f e c t s , t h e y have been c l a s s i f i e d i n t o 4 main groups (Vaughan W i l l i a m s , 1974 & 1975). C l a s s I a n t i a r r h y t h m i c d rugs have a l o c a l a n a e s t h e t i c a c t i o n on th e n e r v e and m y o c a r d i a l c e l l membranes. However, t h e i r e f f e c t s on c a r d i a c c e l l s a r e o f t e n o b s e r v e d a t lower c o n c e n t r a t i o n s t h a n t h o s e on n e r v e s . Drugs i n t h i s group b l o c k t h e f a s t inward c u r r e n t c a r r i e d by Na +, t h u s d e p r e s s i n g t h e MRD i n c a r d i a c t i s s u e s . The r e d u c t i o n i n MRD i s a s s o c i a t e d w i t h an i n c r e a s e i n t h e t h r e s h o l d o f e x c i t a b i l i t y , a d e p r e s s i o n i n c o n d u c t i o n v e l o c i t y , a p r o l o n g a t i o n i n t h e e f f e c t i v e r e f r a c t o r y p e r i o d and an i n h i b i t i o n o f spontaneous d i a s t o l i c d e p o l a r i z a t i o n i n a u t o m a t i c f i b r e s ( S i n g h , 1978). These a c t i o n s l e a d t o t h e s u p p r e s s i o n o f a u t o m a t i c i t y o r r e - e n t r a n t a r r h y t h m i a s . C l a s s I agents i n c l u d e m e x i l e t i n e , t o c a i n i d e , q u i n i d i n e , l i d o c a i n e , d i s o p y r a m i d e , e n c a i n i d e , f l e c a i n i d e and p r o p a f e n o n e . A f u r t h e r c l a s s i f i c a t i o n o f t h e s e agents has been pr o p o s e d based on t h e i r e f f e c t s on t h e c a r d i a c a c t i o n p o t e n t i a l (Vaughan W i l l i a m s , 1974 & 1975). Q u i n i d i n e , p r o c a i n a m i d e and d i s o p y r a m i d e which p r o l o n g t h e a c t i o n p o t e n t i a l d u r a t i o n form c l a s s IA. The c l a s s IB drugs s h o r t e n t h e a c t i o n p o t e n t i a l d u r a t i o n and i n c l u d e m e x i l e t i n e , t o c a i n i d e and l i d o c a i n e . F l e c a i n i d e and e n c a i n i d e which have l i t t l e e f f e c t on t h e a c t i o n p o t e n t i a l d u r a t i o n make up c l a s s IC. I t i s i n t e r e s t i n g t o note t h a t a more r e c e n t c l a s s i f i c a t i o n based on t h e k i n e t i c s o f t h e i n t e r a c t i o n o f t h e s e drugs w i t h t h e f a s t sodium c h a n n e l s (Vaughan W i l l i a m s , 1984) produced a s i m i l a r g r o u p i n g t o t h a t above. The c l a s s II a n t i a r r h y t h m i c agents c o n s i s t o f drugs t h a t a n t a g o n i z e c a r d i a c s y m p a t h e t i c d r i v e , e i t h e r by p r e - s y n a p t i c r e c e p t o r b l o c k a d e o r by a c e n t r a l a c t i o n ( J e w i t t and S i n g h , 1974). C l i n i c a l l y , l i f e t h r e a t e n i n g a r r h y t h m i a s o f t e n o c c u r a s s o c i a t e d w i t h o v e r - a c t i v i t y o f s y m p a t h e t i c o u t - f l o w (Winslow, 1984). C a t e c h o l a m i n e s can e x a c e r b a t e a f t e r - p o t e n t i a l s i n damaged myocardium and i n c r e a s e t h e s l o p e o f d i a s t o l i c d e p o l a r i z a t i o n , t h u s , p r o v o k i n g t a c h y a r r h y t h m i a s ( J e w i t t and S i n g h , 1974). The major c l i n i c a l e l e c t r o p h y s i o l o g i c e f f e c t o f fi-b l o c k e r s i s t h e d e p r e s s i o n o f phase 4 d e p o l a r i z a t i o n ( S i n g h e t a/., 1981). However, l o n g term t r e a t m e n t w i t h 0 - b l o c k e r s has been r e p o r t e d t o produce a l a r g e p r o l o n g a t i o n o f a t r i a l and v e n t r i c u l a r APD (Vaughan W i l l i a m s , 1978), which may i n i t s e l f be a n t i a r r h y t h m i c . Examples o f d r u g s i n t h i s c l a s s a r e p r o p r a n o l o l , p r a c t o l o l and s o t a l o l . C l a s s I I I drugs d e l a y r e p o l a r i z a t i o n , t h e r e b y , c a u s i n g a p r o l o n g a t i o n o f t h e APD and ARP ( S i n g h and Vaughan W i l l i a m s , 1970; Vaughan W i l l i a m s , 1977). Drugs i n t h i s c l a s s i n c l u d e amiodarone and b r e t y l i u m . C l a s s IV a n t i a r r h y t h m i c agents r e s t r i c t t he slow inward c u r r e n t c a r r i e d by c a l c i u m ( S i n g h and Vaughan W i l l i a m s , 1972a; S i n g h , 1978). They a r e r e f e r r e d t o as c a l c i u m a n t a g o n i s t s and i n c l u d e v e r a p a m i l , n i f e d i p i n e and d i l t i a z a m . The a c t i o n p o t e n t i a l s i n a l l c a r d i a c t i s s u e s , a p a r t from t h e SA and AV nodes, a r e n o r m a l l y m e d i a t e d by f a s t inward sodium c u r r e n t . However, i n i s c h a e m i a o r o t h e r s i t u a t i o n s where p a r t i a l d i a s t o l i c d e p o l a r i z a t i o n and i n a c t i v a t i o n o f f a s t c u r r e n t o c c u r , t h e 12 slow inward c u r r e n t may t a k e o v e r , p e r m i t t i n g t h e a c t i v a t i o n o f s l o w l y c o n d u c t i n g AP which can i n i t i a t e r e - e n t r a n t a r r h y t h m i a s . The a n t i a r r h y t h m i c a c t i o n o f c a l c i u m a n t a g o n i s t s i s a t t r i b u t e d t o a b o l i s h i n g t h e s e abnormal slow d e p o l a r i z a t i o n s . 1.1.2.5 E x p e r i m e n t a l A r r h y t h m i a s A r r h y t h m o g e n i c s t i m u l i i n a n i m a l s f a l l i n t o 3 main c a t e g o r i e s : e l e c t r i c a l , c h e m i c a l and m e c h a n i c a l (Winslow, 1984). 1.1.2.5.1 E l e c t r i c a l l y - i n d u c e d a r r h y t h m i a s . The i n d u c t i o n o f a r r h y t h m i a s by the a p p l i c a t i o n o f e l e c t r i c c u r r e n t i s based on t h e premise t h a t f o l l o w i n g normal e x c i t a t i o n , t h e r e i s a p e r i o d o f i n h o m o g e n e i t y o f r e c o v e r y o f e x c i t a b i l i t y i n t h e c a r d i a c f i b e r s , i . e . t h e v u l n e r a b l e p e r i o d (Mines, 1913; Moe et al, 1964). T h i s c o r r e s p o n d s t o t h e downward s l o p e o f t h e T-wave i n t h e ECG. An e x t r a -s t i m u l u s o f s u f f i c i e n t i n t e n s i t y a p p l i e d d u r i n g t h i s p e r i o d w i l l p r e c i p i t a t e f i b r i l l a t i o n . A n t i a r r h y t h m i c drugs a r e e x p e c t e d t o i n c r e a s e t h e i n t e n s i t y o f t h e c u r r e n t n e c e s s a r y t o evoke f i b r i l l a t i o n . F i b r i l l a t i o n has been produced by s i n g l e o r s e r i a l shocks w i t h p r o g r e s s i v e l y i n c r e a s i n g i n t e n s i t y . The use o f e l e c t r i c a l t h r e s h o l d f o r t h e measurement o f a n t i a r r h y t h m i c e f f e c t s has many ad v a n t a g e s . I t s e f f e c t s a r e f u l l y r e v e r s i b l e ( e x c e p t f o r e x t r e m e l y i n t e n s e s t i m u l i ) , t h e a n a l o g y w i t h t h e n a t u r a l impulse i s c l o s e r than a l l t h e o t h e r methods, each animal s e r v e s as i t s own c o n t r o l , and t h e p a r a m e t e r s c h a r a c t e r i z i n g t h e s t i m u l u s ( s t r e n g t h and d u r a t i o n ) can be a c c u r a t e l y d e t e r m i n e d and c o n t r o l l e d ( S z e k e r e s and Papp, 1971). D e t a i l s o f t h i s a r r h y t h m i a model has been d e s c r i b e d by Wiggers and Wegria (1940) and Han (1969). 1.1.2.5.2 C h e m i c a l l y - I n d u c e d A r r h y t h m i a s Many c h e m i c a l agents a l o n e o r i n c o m b i n a t i o n a r e used t o i n d u c e a r r h y t h m i a s i n a n i m a l s . Lawson (1968) r e p o r t e d t h a t c h l o r o f o r m a l o n e produced VF i n mice. The a n i m a l s were p l a c e d i n a c l o s e d c o n t a i n e r w i t h c o t t o n wool soaked i n c h l o r o f o r m . R e c e n t l y , c h l o r o f o r m - i n d u c e d a r r h y t h m i a s i n r a t s s e n s i t i z e d w i t h t h e o p h y l l i n e has been r e p o r t e d ( B a k e r and E r k e r , 1980). I n t r a v e n o u s a c o n i t i n e has a l s o been shown t o i n d u c e a r r h y t h m i a s i n a n a e s t h e t i z e d r a t s ( S z e k e r e s and Papps, 1971). Many p u b l i c a t i o n s have r e p o r t e d a r r h y t h m i a s i n d u c e d by c a r d i a c g l y c o s i d e s ( u s u a l l y oaubain) i n a n i m a l s i n c l u d i n g g u i n e a p i g s ( S e k i y a and Vaugham W i l l i a m s , 1963), c a t s (Raper and W a l l , 1968), and dogs ( L u c h e s s i and Hardman, 1961). O t h e r c h e m i c a l agents t h a t a r e known t o be a r r h y t h m o g e n i c i n c l u d e barium c h l o r i d e , c a l c i u m c h l o r i d e , a d r e n a l i n e , a d r e n a l i n e and i n s u l i n a l o n e o r i n c o m b i n a t i o n w i t h g l u c o s e and a c e t y l c h o l i n e (Winslow, 1984) 1.1.2.5.3 M e c h a n i c a l l y - I n d u c e d A r r h y t h m i a s O c c l u s i o n o f a major c o r o n a r y a r t e r y ( i s c h a e m i a ) i s known t o l e a d t o a r r h y t h m i a s , i n c l u d i n g VF, i n v a r i o u s a n i m a l s . The most p o p u l a r i s c h a e m i a model i s t h e H a r r i s two-stage c o r o n a r y a r t e r y o c c l u s i o n t e c h n i q u e i n t h e dog ( H a r r i s , 1950). In t h i s model, an i n i t i a l p a r t i a l o c c l u s i o n o f t h e l e f t a n t e r i o r d e s c e n d i n g (LAD) c o r o n a r y a r t e r y was per f o r m e d , f o l l o w e d 30 min l a t e r by complete o c c l u s i o n . A r r h y t h m i a s d e v e l o p w i t h i n 4-7 h and r e a c h a peak 24-48 h a f t e r o c c l u s i o n . The two-s t e p o c c l u s i o n a v o i d s t h e development o f a c u t e VF ( S z e k e r e s and Papp, 1971) and i s m a i n l y used f o r t h e s t u d y o f l a t e a r r h y t h m i a s . Ischaemia-i n d u c e d a r r h y t h m i a s have a l s o been r e p o r t e d i n t h e c a t ( R i t c h i e e t al., 14 1977 & 1979). Meesman and co-workers developed a model of early arrhythmias in the dog in which mortality from VF was 100% (Meesman et al., 1970; Abendroth et al., 1977; Menken et al., 1979). This involved occlusion of the left circumflex coronary artery under morphine-urethane-chlorolose anaesthesia. In the pig, whose coronary tree resembles that of man, occlusion of the LAD coronary artery results in death within 2 hours from VF (Verdouw et al., 1978). However, by lowering blood flow to 25% of normal, a less severe but consistent model was developed. Early studies on ischaemic arrhythmias in anaesthetized rats were carried out by Heimburger (1946) and Selye et al. (1960). In this model, a s i l k suture was placed around the LAD coronary artery following left thoracotomy (under anaesthesia) and the artery occluded. Consistent arrhythmias which included premature ventricular contractions, ventricular tachycardia and f i b r i l l a t i o n were produced . This model has been ut i l ized to assess the antiarrhythmic actions of several agents (Au et al., 1979; Clark et al., 1980; Kane et al., 1980). Recently, ischaemic arrhythmias have been demonstrated in conscious chronically prepared rats (Johnston et al., 1983; Curtis et al; 1984 & 1986). This model has the advantage of circumventing the effects of anaesthesia and recent surgery on the outcome of coronary occlusion. 1.1.2.5.4 Reperfusion-Induced Arrhythmias Arrhythmias have been induced in dogs by sudden reperfusion after complete coronary artery occlusion for 30-45 min (Fiedler et al., 1979; Stockman et al., 1979; Martorana et al., 1980; Kabell et al., 1980). The reported incidence of arrhythmias in this model is 63 to 87%. The arrhythmias are almost immediate in onset, and in the absence of VF 15 t e r m i n a t e w i t h i n 10 min. R e p e r f u s i o n a r r h y t h m i a s have a l s o been d e m o n s t r a t e d i n t h e c a t (Penkoske et al., 1978) and i n L a n g e n d o f f p e r f u s e d r a t h e a r t (Lubbe et al., 1978). The e l e c t r o p h y s i o l o g i c a l b a s i s f o r t h e s e a r r h y t h m i a s seem t o d i f f e r from t h o s e due t o MI ( t h e y a r e a s s o c i a t e d w i t h a r a p i d i d i o - v e n t r i c u l a r r a t e which i s u s u a l l y normal i n MI). I t has been s u g g e s t e d t h a t t h e d i f f e r e n c e s i n t h e r a t e s a t which c e l l s i n t h e i s c h a e m i c zone r e g a i n o r improve t h e i r e l e c t r i c a l a c t i v i t y a f t e r r e p e r f u s i o n may be t h e i m p o r t a n t f a c t o r t h a t t r i g g e r s t h e a r r h y t h m i a s (Downar et al., 1977). 1.2 M e x i l e t i n e 1.2.1 C h e m i s t r y M e x i l e t i n e , 1-(2',6'-dimethylphenoxy)-2-aminopropane, was s y n t h e t i c a l l y d e r i v e d from t h e phenethanolamine compound, Phe n m e t r a z i n e , i n an attempt t o produce an a n o r e x i c agent w i t h r e d u c e d CNS s i d e e f f e c t s (Koppe, 1977). The r e s u l t a n t compound was an agent t h a t e x h i b i t e d a n t i c o n v u l s a n t a c t i v i t y . Subsequent s t u d i e s i n a n i m a l s showed t h a t m e x i l e t i n e a l s o had a n t i a r r h y t h m i c p r o p e r t i e s ( A l l e n et al., 1972). M e x i l e t i n e i s a b a s i c d r u g w i t h a pKa o f 8.8 (Merck Index, 1983). I t i s a c h i r a l compound, composed o f equal p r o p o r t i o n s o f two e n a n t i o m e r s . M e x i l e t i n e i s used c l i n i c a l l y as the h y d r o c h l o r i d e s a l t o f t h e racemate ( M e x i t i l R ) which i s an almost w h i t e c r y s t a l l i n e s u b s t a n c e , s o l u b l e i n water, methanol, e t h a n o l and c h l o r o f o r m and p r a c t i c a l l y i n s o l u b l e i n e t h e r (Merck Index, 1983). 16 CH- CH: 0 - C H 2 - C H - N H 2 F i g u r e 1. The s t r u c t u r e o f m e x i l e t i n e [ ( 2 ' , 6 ' - d i m e t h y l p h e n o x y ) - 2 - a m i n o p r o p a n e ] 1.2.2 Pharmacology 1.2.2.1 Animal S t u d i e s E a r l y s t u d i e s on t h e a n t i a r r h y t h m i c e f f e c t s o f m e x i l e t i n e i n a n i m a l s were c a r r i e d by A l l e n e t al., (1972) and S i n g h and Vaughan W i l l i a m s (1972b). M e x i l e t i n e was found t o be e f f e c t i v e a g a i n s t v e n t r i c u l a r a r r h y t h m i a s i n d u c e d by d i g i t a l i s i n dogs. The e f f e c t i v e plasma c o n c e n t r a t i o n was 0.6 . In the c a n i n e 24 h two-stage c o r o n a r y o c c l u s i o n a r r h y t h m i a , m e x i l e t i n e a d m i n i s t e r e d i n t r a v e n o u s l y r e s t o r e d s i n u s rhythm a t a plasma c o n c e n t r a t i o n o f 5.3 /jg/ml. M e x i l e t i n e was a l s o shown t o be e f f e c t i v e a g a i n s t a r r h y t h m i a s i n d u c e d by h a l o t h a n e and e p i n e p h r i n e i n dogs. F u r t h e r s t u d i e s i n c a n i n e a r r h y t h m i a models (Hashimoto et al., 1984) have c o n f i r m e d t h e a n t i a r r h y t h m i c e f f i c a c y o f m e x i l e t i n e . Minimum plasma c o n c e n t r a t i o n s o f 1.8, 1.9 and 3.7 nq/ml were a s s o c i a t e d w i t h s u p p r e s s i o n o f a r r h y t h m i a s i n d u c e d by d i g i t a l i s , c o r o n a r y o c c l u s i o n and e p i n e p h r i n e , r e s p e c t i v e l y . In a r e c e n t s t u d y ( U p r i c h a r d e t al., 1988), m e x i l e t i n e was not e f f e c t i v e a g a i n s t a r r h y t h m i a s evoked by programmed e l e c t r i c a l s t i m u l a t i o n i n dogs. CNS t o x i c i t y l i m i t e d t h e dose o f m e x i l e t i n e g i v e n t o t h e dogs i n t h i s s t u d y . The o t h e r p h a r m a c o l o g i c a l p r o p e r t i e s o f m e x i l e t i n e i n c l u d e s t r o n g l o c a l a n a e s t h e t i c ( S i n g h and Vaughan W i l l i a m s , 1972b; Danneberg and S h e l l e y , 1977) and a n t i c o n v u l s a n t (Danneberg and S h e l l e y , 1977) a c t i o n s . 1.2.2.2 C l i n i c a l E f f e c t i v e n e s s Numerous human s t u d i e s have r e p o r t e d t h e e f f e c t i v e n e s s o f m e x i l e t i n e a g a i n s t a r r h y t h m i a s o r i g i n a t i n g from d i g i t a l i s i n t o x i c a t i o n , 18 m y o c a r d i a l i n f a r c t i o n and c a r d i a c s u r g e r y (Campbell e t al., 1973 & 1978a; T a l b o t e t al., 1973 & 1976; Z i p e s and Troup, 1978; S i n g h et al., 1980; Bury e t al., 1982 and IMPACT, 1984). 1.2.2.3 Plasma C o n c e n t r a t i o n - C l i n i c a l E f f e c t R e l a t i o n s h i p s T a l b o t e t al. (1973) r e p o r t e d t h a t m e x i l e t i n e c o n c e n t r a t i o n s o f 0.5 t o 2.0 /xg/ml were a s s o c i a t e d w i t h g r e a t e r than 95% r e d u c t i o n o f v e n t r i c u l a r e c t o p i c b e a t s i n 37 p a t i e n t s . However, c l e a r s e p a r a t i o n between t h e r a p e u t i c and t o x i c ranges was not o b s e r v e d i n t h i s s t u d y s i n c e a d v e r s e e f f e c t s sometimes o c c u r r e d a t c o n c e n t r a t i o n s as low as 0.3 /Ltg/ml. The r e l a t i o n s h i p between plasma c o n c e n t r a t i o n s o f m e x i l e t i n e and c l i n i c a l e f f e c t s was i n v e s t i g a t e d i n 149 p a t i e n t s by Campbell e t al., (1978a). S u p p r e s s i o n o f premature v e n t r i c u l a r c o n t r a c t i o n s was o b s e r v e d i n 77% o f t h e p a t i e n t s when plasma c o n c e n t r a t i o n was m a i n t a i n e d between 0.74 and 1.0 /zg/ml• An 80% r e s p o n s e was o b s e r v e d when plasma c o n c e n t r a t i o n s were 2 jig/ml o r above. However, a d v e r s e e f f e c t s d e v e l o p e d i n 30% o f r e c i p i e n t s a t t h e s e l e v e l s w i t h 19% d e v e l o p i n g s e v e r e a d v e r s e r e a c t i o n s such as h y p o t e n s i o n , v o m i t i n g , t r e m o r , t o x i c c o n f u s i o n a l s t a t e s and a t r i o v e n t r i c u l a r d i s s o c i a t i o n . S t u d i e s on t h e e f f i c a c y o f l o n g term o r a l m e x i l e t i n e t r e a t m e n t showed adequate s u p p r e s s i o n o f v e n t r i c u l a r a r r h y t h m i a s a t serum c o n c e n t r a t i o n s o f 0.9 t o 2.6 Mg/ml ( T a l b o t e t al., 1976). F u r t h e r s t u d i e s d u r i n g s h o r t term o r a l m e x i l e t i n e t e s t i n g i n c a s e s w i t h p e r s i s t i n g v e n t r i c u l a r e c t o p i c b e a t s r e p o r t e d m e x i l e t i n e l e v e l s between 0.38 t o 2.76 /ig/ml i n 30 r e s p o n d e r s whose premature v e n t r i c u l a r c o n t r a c t i o n s were r e d u c e d by more t h a n 50%. E f f e c t i v e plasma c o n c e n t r a t i o n s d u r i n g maintenance t r e a t m e n t were i n t h e same range, 0.44 t o 2.0 /jg/ml ( P o d r i d and Lown, 1981). However, serum m e x i l e t i n e c o n c e n t r a t i o n s i n n o n - r e s p o n d i n g p a t i e n t s were not d i f f e r e n t from t h o s e o f t h e r e s p o n d e r s . 1.2.3 Mechanism o f A c t i o n The major e l e c t r o p h y s i o l o g i c a c t i o n o f m e x i l e t i n e i s b l o c k a d e o f t h e f a s t sodium c h a n n e l s . T h i s a c t i o n r e s u l t s i n a d e c r e a s e i n t h e phase 0 maximal u p s t r o k e v e l o c i t y ( V m a x ) o r membrane r e s p o n s i v e n e s s i n a t r i a l , v e n t r i c u l a r and P u r k i n j e AP (Vaughan W i l l i a m s , 1977 and Yamaguchi et al., 1979). The d e c r e a s e i n V m a x i s accompanied by 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 which v a r y i n d i f f e r e n t t i s s u e s . In i s o l a t e d a t r i a l and v e n t r i c u l a r m y o c a r d i a l t i s s u e p r e p a r a t i o n s , m e x i l e t i n e d e p r e s s e d c o n d u c t i o n v e l o c i t y , i n c r e a s e d t h e t h r e s h o l d o f e x c i t a b i l i t y and p roduced a marked p r o l o n g a t i o n o f t h e ERP ( A l l e n et al., 1972; S i n g h and Vaughan W i l l i a m s , 1972b; Vaughan W i l l i a m s , 1977; A r i t a et al., 1979). These a l t e r a t i o n s , o c c u r r i n g w i t h o u t a s i g n i f i c a n t change i n e i t h e r t h e r e s t i n g MP o r APD a r e a s s o c i a t e d w i t h t h e d e p r e s s i o n o f s pontaneous d i a s t o l i c d e p o l a r i z a t i o n i n a u t o m a t i c f i b r e s . In i s o l a t e d P u r k i n j e f i b r e s o f t h e dog, a c o n c e n t r a t i o n dependent d e c r e a s e i n a c t i o n p o t e n t i a l d u r a t i o n was o b s e r v e d ( A r i t a e t a/., 1979; Weld e t al, 1979 and Yamaguchi et al., 1979). T h i s was accompanied by a s h o r t e n i n g o f t h e e f f e c t i v e r e f r a c t o r y p e r i o d (Yamaguchi e t al., 1979) but t h e r a t i o o f t h e ERP t o t h a t o f the APD was c o n s i s t e n t l y i n c r e a s e d . Thus, t h e r e i s l e s s t i m e d u r i n g an a c t i o n p o t e n t i a l f o r an e c t o p i c i m p u l s e t o i n i t i a t e a n o t h e r a c t i o n p o t e n t i a l . I t has been s u g g e s t e d t h a t m e x i l e t i n e may have an e x a g g e r a t e d e f f e c t on p a r t i a l l y d e p o l a r i z e d t i s s u e s i n c e i t b l o c k s a c t i v a t e d and i n a c t i v a t e d sodium c h a n n e l s more than t h e r e s t i n g c h a n n e l s ( A r i t a e t al., 1979; H e r i n g e t al., 1983; 20 H o h n l o s e r e t al., 1982 and Sada et al., 1980). For example, Sada et al. (1980) showed t h a t 5 jug/ml o f m e x i l e t i n e d e c r e a s e d V m a x o f g u i n e a p i g v e n t r i c u l a r muscle o n l y 4% a t a r e s t i n g MP o f -96 mV. However, t h e same c o n c e n t r a t i o n o f m e x i l e t i n e was shown by H o h n l o s e r et al. (1982) t o d e c r e a s e V m a x 24% and 33% a t r e s t i n g MP o f -76 and -69 mV, r e s p e c t i v e l y . I t has a l s o been dem o n s t r a t e d t h a t the sodium channel b l o c k i n g e f f e c t o f m e x i l e t i n e i s p o t e n t i a t e d i n in vitro i s c h a e m i c models and i n h y p o x i c c o n d i t i o n s (Frame et al., 1982). The t h r e s h o l d e f f e c t i v e c o n c e n t r a t i o n s o f m e x i l e t i n e used i n t h e in vitro animal s t u d i e s above ranged from 1 t o 10 /jg/ml (Iwamura et al., 1976; S i n g h and Vaughan W i l l i a m s , 1972b; Weld et al., 1979; A r i t a et al., 1979; Sada et al., 1980; Campbell et al., 1983a & 1983b; H e r i n g et al., 1983). M e x i l e t i n e has no e f f e c t on t h e c a l c i u m channel o r t h e a d r e n e r g i c ^ - r e c e p t o r ( S i n g h et al., 1980). In humans, t h e 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 o f m e x i l e t i n e have been shown t o be somewhat v a r i a b l e . I t was found t o have no c o n s i s t e n t e f f e c t on s i n u s r a t e , a t r i a l r e f r a c t o r i n e s s , and AV o r H i s - P u r k i n j e c o n d u c t i o n t i m e s (Ross et al., 1977; McCormish et al., 1977). However, t h e f u n c t i o n a l r e f r a c t o r y p e r i o d o f t h e AV node was i n c r e a s e d by m e x i l e t i n e , w i t h a v a r i a b l e e f f e c t on t h e e f f e c t i v e r e f r a c t o r y p e r i o d o f t h e H i s -P u r k i n j e system; l e n g t h e n i n g i t i n some p a t i e n t s (Ross et al., 1977), w h i l e s h o r t e n i n g i t i n o t h e r s (McCormish e t al., 1977). However, i f t h e H i s - P u r k i n j e c o n d u c t i o n i s i m p a i r e d by d i s e a s e , the d r u g always l e n g t h e n s t h e AV i n t e r v a l , s u g g e s t i n g t h a t m e x i l e t i n e may produce AV b l o c k i n p a t i e n t s w i t h u n d e r l y i n g c o n d u c t i o n d i s t u r b a n c e s (Ross e t al., 1977; S i n g h e t al., 1981). 1.2.4 Comparison w i t h Other A n t i a r r h y t h m i c A g e n t s . H o r o w i t z e t a7. (1981), i n a c o n t r o l l e d s t u d y , compared m e x i l e t i n e and l i d o c a i n e o v e r a p e r i o d o f 48 h i n p a t i e n t s who d e v e l o p e d v e n t r i c u l a r a r r h y t h m i a s w i t h i n 2 days o f t h e o n s e t o f a c u t e MI. The two drugs comparably r e d u c e d e c t o p i c b e a t s o v e r t h e f i r s t 24 h. However, a s i g n i f i c a n t l y l o w e r f r e q u e n c y o f a r r h y t h m i a s was o b s e r v e d i n p a t i e n t s on m e x i l e t i n e , t h e r e a f t e r . In a n o t h e r c o m p a r a t i v e s t u d y , (Arakawa e t a7., 1984), m e x i l e t i n e (3 mg/kg i . v . ) was e q u i - e f f e c t i v e w i t h p r o c a i n a m i d e (10 mg.kg i . v . ) i n s u p p r e s s i n g v e n t r i c u l a r premature c o n t r a c t i o n s i n c a r d i a c p a t i e n t s . S i m i l a r r e s u l t s have p r e v i o u s l y been r e p o r t e d by J e w i t t e t a7. (1977) and Campbell e t a7. (1981). In t h e s t u d y by Arakawa e t a7., m e x i l e t i n e and p r o c a i n a m i d e were more p o t e n t t h a n ^ - a d r e n o c e p t o r a n t a g o n i s t s i n s u p p r e s s i n g v e n t r i c u l a r a r r h y t h m i a s . F e n s t e r e t a7. (1981) compared t h e e f f i c a c y o f m e x i l e t i n e and q u i n i d i n e i n a d o u b l e - b l i n d d o s e - r a n g i n g s t u d y u s i n g 26 a m b u l a t o r y p a t i e n t s w i t h c h r o n i c v e n t r i c u l a r a r r h y t h m i a s . M e x i l e t i n e was f o u n d t o be as e f f e c t i v e as q u i n i d i n e i n t h e s u p p r e s s i o n o f v e n t r i c u l a r e c t o p i c b e a t s . R e c e n t l y , S i n g h e t a7 (1984) r e p o r t e d comparable a n t i a r r h y t h m i c e f f i c a c y o f o r a l m e x i l e t i n e and q u i n i d i n e i n a s i n g l e - b l i n d randomized t r i a l u s i n g 26 c a r d i a c p a t i e n t s . O t h e r s t u d i e s have r e p o r t e d comparable a n t i a r r h y t h m i c e f f i c a c y f o r m e x i l e t i n e and d i s o p y r a m i d e ( B r e i t h a r d t e t a7., 1982; T r i m a r c o e t a7., 1983). However, a more r e c e n t s t u d y i n 160 p a t i e n t s w i t h c h r o n i c v e n t r i c u l a r a r r h y t h m i a s o f v a r i o u s e t i o l o g i e s found m e x i l e t i n e t o be s l i g h t l y more a c t i v e than d i s o p y r a m i d e (Kato e t a7., 1984). 1.2.5 P r e v e n t i o n o f Sudden c a r d i a c d e a t h The b e n e f i t s o f a n t i a r r h y t h m i c d r u g t h e r a p y w i t h r e s p e c t t o t h e p r e v e n t i o n o f sudden c a r d i a c d e a t h remains t o be c l e a r l y e s t a b l i s h e d . A r e v i e w o f t h e r e s u l t s o f 20 randomized, c o n t r o l l e d , c l i n i c a l t r i a l s showed t h a t b e n e f i c i a l e f f e c t s on p a t i e n t s ' s u r v i v a l c o u l d not be d e m o n s t r a t e d e i t h e r d u r i n g t h e e a r l y h o s p i t a l phase f o l l o w i n g a c u t e m y o c a r d i a l i n f a r c t i o n o r a f t e r d i s c h a r g e ( F u r b e r g et al., 1983). In a s t u d y on t h e e f f e c t o f m e x i l e t i n e on m o r t a l i t y i n 344 p a t i e n t s , C h a m b e r l a i n et al., (1980) were not a b l e t o show any s i g n i f i c a n t r e d u c t i o n i n d e a t h r a t e . R e c e n t l y , i n a n o t h e r s t u d y i n c a r d i a c p a t i e n t s , t h e IMPACT r e s e a r c h group r e p o r t e d t h a t t h e r e were more d e a t h s i n t h e m e x i l e t i n e group (7.6%) when compared t o a p l a c e b o group (4.8%) (IMPACT, 1984). However, t h e d i f f e r e n c e between t h e two g roups 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 . In a n o t h e r s t u d y i n 240 p a t i e n t s w i t h m y o c a r d i a l i n f a r c t i o n ( S m y l l i e et al., 1984), m e x i l e t i n e d i d not p r e v e n t p r i m a r y f i b r i l l a t i o n . The m o r t a l i t y a t 6 weeks i n t h i s s t u d y 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 t h a t o f t h e p l a c e b o group. However, one s t u d y ( S t e i n e t al., 1984) r e p o r t e d a s i g n i f i c a n t r e d u c t i o n i n t h e d e a t h r a t e f o l l o w i n g m e x i l e t i n e t h e r a p y i n p a t i e n t s who had v e n t r i c u l a r t a c h y c a r d i a and f i b r i l l a t i o n w i t h syncope. 1.2.6 C l i n i c a l I n d i c a t i o n s The c l i n i c a l i n d i c a t i o n s f o r m e x i l e t i n e are v e n t r i c u l a r a r r h y t h m i a s such as v e n t r i c u l a r t a c h y c a r d i a and premature v e n t r i c u l a r e c t o p i c b e a t s ( u n i f o c a l premature v e n t r i c u l a r b e a t s , s a l v o s , and R-on-T phenomenon) ( B r a d l e y , 1983; K r e e g e r and Hammill, 1987). M e x i l e t i n e i s 23 a l s o u s e f u l i n t h e p r o p h y l a c t i c t r e a t m e n t o f v e n t r i c u l a r a r r h y t h m i a s a f t e r m y o c a r d i a l i n f a r c t i o n ( B r a d l e y , 1983). 1.2.7 Toxicology Between 47 and 60% o f p a t i e n t s on m e x i l e t i n e t h e r a p y e x p e r i e n c e a d v e r s e e f f e c t s (DiMarco et al., 1981; P o d r i d and Lown 1981; Waspe et al., 1983; C e t n a r o w s k i and Rihn , 1985). D i s c o n t i n u a t i o n o f t h e r a p y has o c c u r r e d i n up t o 30% o f p a t i e n t s due t o a d v e r s e e f f e c t s ( K u p e r s m i t h et al., 1985; S h r a d e r and Bauman, 1986). The n e u r o l o g i c s i d e e f f e c t s a r e d i z z i n e s s , l i g h t h e a d e d n e s s , t r e m o r , a t a x i a and c o n v u l s i o n s . The g a s t r o i n t e s t i n a l s i d e e f f e c t s i n c l u d e nausea, v o m i t i n g and d y s p e p s i a . The c a r d i o v a s c u l a r s i d e e f f e c t s a r e t r a n s i e n t h y p o t e n s i o n and b r a d y c a r d i a . 1.2.8 Pharmacokinetic Parameters 1.2.8.1 Pharmacokinetics of racemic mexiletine M e x i l e t i n e i s r a p i d l y a bsorbed a f t e r o r a l a d m i n i s t r a t i o n w i t h peak plasma l e v e l s n o r m a l l y a c h i e v e d w i t h i n 1 t o 4 h (S i n g h et al. 1981) . U n l i k e l i d o c a i n e , m e x i l e t i n e undergoes minimal (< 10%) f i r s t - p a s s e l i m i n a t i o n (Woosley et al., 1984).. B i o a v a i l a b i l i t y r a n g e s from 80 t o 88% ( P r e s c o t t et al., 1977; H a s e l b a r t h et al., 1981 and Ohashi et al., 1984). The t o t a l volume o f d i s t r i b u t i o n i s l a r g e and h i g h l y v a r i a b l e (5.5 t o 9.5 l / k g ) (Campbell et al., 1978b; P r e s c o t t e t al., 1977; H a s e l b a r t h e t al., 1981), r e f l e c t i n g e x t e n s i v e t i s s u e uptake o f t h e d r u g . The e l i m i n a t i o n h a l f - l i f e ranges from 6.3 t o 11.8 h (Campbell e t al., 1978b; D a n i l l o , 1979; P r e s c o t t e t al., 1977 and H a s e l b a r t h e t al., 1981). The t o t a l body c l e a r a n c e i s l a r g e , 6.1 t o 7.1 ml/min/kg (Paalman et al., 1977; Campbell et al., 1978b). The mean renal clearance in normal subjects is 0.58 ± 0.30 ml/min/kg (Grech-Belanger et al., 1985), and is increased by urinary acidosis. At a urinary pH of 5, renal clearance is reported to increase by a factor of 3 to 4 (Kiddie et al., 1974; Prescott et al., 1977). However, the effect of normal physiologic fluctuation in urinary pH is not conclusive. While Prescott et al. (1977) reported that normal urinary pH fluctuations are c l i n i c a l l y unimportant, Johnston et al. (1979) found a significant change in urinary excretion and an increase of up to 50% in plasma concentrations of mexiletine due to spontaneous fluctuation in urinary pH. Approximately 9% of the administered dose of mexiletine is excreted unchanged in urine of healthy subjects (Prescott et al., 1977 and Campbell et al., 1978b). 1.2.8.2 Effects of Disease on Pharmacokinetics The pharmacokinetic parameters of mexiletine are affected by many disease conditions. Patients with hepatic impairment have been reported to have decreased plasma clearance of mexiletine (Nitsch et al., 1981). This is not surprising since the l iver is the major site of metabolism of the drug. Renal disease has no significant effect on the plasma clearance, except at creatinine clearance values below 10 ml/min (El Al l a f et al., 1982). Delayed and incomplete absorption, as well as increased elimination ha l f - l i f e , were associated with acute myocardial infarction (Prescott et al., 1977; Pottage et al., 1978 and Pentikainen et al., 1984). Prolongation of ha l f - l i fe has also been reported in patients with severe left ventricular failure (Leahey at al., 1980a). 25 1.8.2.3 P h a r m a c o k i n e t i c s o f M e x i l e t i n e E n a n t i o m e r s The p h a r m a c o k i n e t i c s o f m e x i l e t i n e e n a n t i o m e r s has been r e p o r t e d from our l a b o r a t o r y (Igwemezie et al., 1989) and by one o t h e r group ( G r e c h - B e l a n g e r et al., 1986). Both i n v e s t i g a t i o n s i n v o l v e d s i n g l e , o r a l dose s t u d i e s i n h e a l t h y human s u b j e c t s . In our s t u d y , a s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e i n the e l i m i n a t i o n h a l f - l i v e s o f t h e two e n a n t i o m e r s (9.1 ± 2.9 h f o r R ( - ) - m e x i l e t i n e and 11.0 ± 3.8 h f o r S ( + ) - m e x i l e t i n e ) was o b s e r v e d . The a p p a r e n t volume o f d i s t r i b u t i o n o f S ( + ) - m e x i l e t i n e (7.3 ± 2.4 l / k g ) was s i g n i f i c a n t l y g r e a t e r t h a n t h a t o f R ( - ) - m e x i l e t i n e (6.6 ± 2.6 l / k g ) , w h i l e a p p a r e n t o r a l c l e a r a n c e and plasma a r e a under t h e c o n c e n t r a t i o n - t i m e c u r v e (AUC) o f t h e two e n a n t i o m e r s were not s i g n i f i c a n t l y d i f f e r e n t . These r e s u l t s d i f f e r from t h o s e o f G r e c h - B e l a n g e r et al. (1986) who r e p o r t e d a s i g n i f i c a n t d i f f e r e n c e i n serum AUC and no d i f f e r e n c e i n t h e e l i m i n a t i o n h a l f - l i v e s o f t h e e n a n t i o m e r s . However, both s t u d i e s found a s t e r e o s e l e c t i v e r e n a l e x c r e t i o n which f a v o u r e d t h e S ( + ) - e n a n t i o m e r . The s a l i v a r y e x c r e t i o n o f S ( + ) - m e x i l e t i n e was s i g n i f i c a n t l y g r e a t e r than t h a t o f t h e R ( - ) -e n a n t i o m e r (Igwemezie, 1986). 1.2.9 Dosage The r e p o r t e d t h e r a p e u t i c plasma c o n c e n t r a t i o n range o f r a c e m i c m e x i l e t i n e i s 0.5 t o 2.0 /xg/ml (Campbell e t al., 1975; T a l b o t et al., 1976). T h i s c o n c e n t r a t i o n range i s u s u a l l y a c h i e v e d w i t h o r a l doses o f 200 t o 400 mg o f m e x i l e t i n e h y d r o c h l o r i d e g i v e n e v e r y s i x t o e i g h t h o u r s . For i n t r a v e n o u s d o s i n g , P r e s c o t t e t al., (1977) recommended t h e f o l l o w i n g d o s i n g regimen: 150 t o 200 mg i n f u s e d o v e r 2 t o 5 m i n u t e s , f o l l o w e d by a s t e a d i l y d e c r e a s i n g l o a d i n g i n f u s i o n o v e r 11 hours (250 mg i n 30 min, 260 mg i n 2.5 h, 500 mg i n 8 h) and a maintenance dose o f 500 t o 1000 mg o v e r 24 h. 1 . 2 . 1 0 M e t a b o l i s m M e x i l e t i n e i s e l i m i n a t e d from the body by e x t e n s i v e h e p a t i c m e t a b o l i s m and r e n a l e x c r e t i o n o f the m e t a b o l i t e s and unchanged dru g ( B e c k e t t and Chidomere, 1977a & 1977b). The major m e t a b o l i t e s a r e p a r a -hydroxy m e x i l e t i n e , hydroxymethyl m e x i l e t i n e and t h e i r c o r r e s p o n d i n g a l c o h o l s , and m e x i l e t i n e g l u c u r o n i d e ( P r e s c o t t et al., 1977; B e c k e t t and Chidomere, 1977b; G r e c h - B e l a n g e r e t al., 1985). Minor m e t a b o l i t e s i n c l u d e t h e h y d r o x y l a m i n e and ketone, and t h e N - m e t h y l a t e d m e t a b o l i t e s ( B e c k e t t and Chidomere 1977a & 1977b) and 2,6-dimethyl phenol ( G r e c h -Bel anger e t a7., 1987). None o f t h e s e m e t a b o l i t e s has been shown t o p o s s e s s any a n t i a r r h y t h m i c a c t i v i t y (Brown and Shand, 1982). The g l u c u r o n i d a t i o n o f m e x i l e t i n e e n a n t i o m e r s i n humans has been shown t o o c c u r s t e r e o s e l e c t i v e l y i n f a v o u r o f t h e R ( - ) - e n a n t i o m e r ( G r e c h - B e l a n g e r e t al., 1986). The s t e r e o s e l e c t i v i t y o f the o t h e r m e t a b o l i c pathways has not been documented. 1 . 2 . 1 1 I n t e r a c t i o n s o f M e x i l e t i n e w i t h O t h e r Drugs The p o t e n t i a l f o r i n t e r a c t i o n between m e x i l e t i n e and o t h e r d r u g s i s g r e a t because p a t i e n t s who have s i g n i f i c a n t v e n t r i c u l a r a r r h y t h m i a s o f t e n have h e a r t d i s e a s e and o t h e r c a r d i o v a s c u l a r p r o b l e m s . Thus, t h e s e p a t i e n t s c o n c o m i t a n t l y r e c e i v e o t h e r c a r d i o v a s c u l a r , as w e l l as non-c a r d i o v a s c u l a r d r u g s . The a b s o r p t i o n o f m e x i l e t i n e a f t e r o r a l d o s i n g has been shown t o be d e l a y e d by drugs which slow g a s t r i c emptying such as n a r c o t i c a n a l g e s i c s ( P r e s c o t t e t al., 1977), a n t a c i d s (Herzog e t al., 27 1982), and a t r o p i n e - 1 i k e agents (Wing e t a7., 1980). M e t o c l o p r a m i d e , on t h e o t h e r hand, i n c r e a s e s t h e a b s o r p t i o n r a t e (Wing et al., 1980). B i o a v a i l a b i l i t y , however, i s not a l t e r e d by any o f t h e s e a g e n t s . M e x i l e t i n e has no e f f e c t on d i g o x i n serum l e v e l s (Leahey et al., 1980b). T h i s i s i n c o n t r a s t t o many a n t i a r r h y t h m i c drugs i n c l u d i n g amiodarone, p r o p a f e n o n e , q u i n i d i n e , and v e r a p a m i l , which a r e known t o i n c r e a s e t h e plasma c o n c e n t r a t i o n s o f d i g o x i n (Hager et al., 1979; Leahey et al., 1981; Moysey et al., 1981., K l e i n et al., 1982; Pedersen et al., 1981 and Pederson et al., 1982). S i n c e m e x i l e t i n e undergoes e x t e n s i v e h e p a t i c m e t a b o l i s m i n man, drugs which i n d u c e t h e h e p a t i c enzyme systems a r e e x p e c t e d t o enhance i t s n o n - r e n a l c l e a r a n c e . T h i s has been r e p o r t e d f o r r i f a m p i c i n ( P e n t i k a i n e n e t a7., 1982) and p h e n y t o i n (Begg e t a7., 1982). C i m e t i d i n e , c o n t r a r y t o t h e e x p e c t e d i n h i b i t i o n o f m e x i l e t i n e m e t a b o l i s m , was r e p o r t e d t o have no e f f e c t on t h e d i s p o s i t i o n o f o r a l m e x i l e t i n e i n normal s u b j e c t s ( K l e i n et al., 1985). Leahey e t a7. (1980c) o b s e r v e d a b e n e f i c i a l d r u g i n t e r a c t i o n between m e x i l e t i n e and the ^ - a d r e n e r g i c r e c e p t o r b l o c k i n g d r u g , p r o p r a n o l o l . They showed t h a t m e x i l e t i n e and p r o p a n o l o l a r e e f f e c t i v e i n s u p p r e s s i n g v e n t r i c u l a r premature d e p o l a r i z a t i o n and v e n t r i c u l a r t a c h y c a r d i a when m e x i l e t i n e a l o n e had f a i l e d t o do so. P r o p r a n o l o l d i d not s i g n i f i c a n t l y i n c r e a s e t h e plasma c o n c e n t r a t i o n o f m e x i l e t i n e i n t h i s s t u d y , hence t h e a u t h o r s c o n c l u d e d t h a t m e x i l e t i n e and p r o p a n o l o l had " c o o p e r a t i v e " a n t i a r r h y t h m i c e f f e c t s i n v o l v i n g a pharmacodynamic i n t e r a c t i o n . A s i m i l a r b e n e f i c i a l i n t e r a c t i o n between m e x i l e t i n e and q u i n i d i n e has been r e p o r t e d ( D u f f et al., 1983). 1.2.12 Serum P r o t e i n B i n d i n g The serum p r o t e i n b i n d i n g o f r a c e m i c m e x i l e t i n e has been r e p o r t e d t o be 70% i n h e a l t h y s u b j e c t s ( T a l b o t e t al., 1973). In a c u t e m y o c a r d i a l i n f a r c t i o n p a t i e n t s , b i n d i n g was r e p o r t e d t o be 64% ( P e n t i k a i n e n et al., 1984). No s i g n i f i c a n t d i f f e r e n c e was n o t e d i n t h e serum b i n d i n g between t h e a c u t e and r e c o v e r y s t a g e s i n t h e s e p a t i e n t s . B i n d i n g s t u d i e s on t h e i n d i v i d u a l e n a n t i o m e r s c a r r i e d out i n our l a b o r a t o r y showed s t e r e o s e l e c t i v e b i n d i n g t o serum p r o t e i n s w i t h f r e e f r a c t i o n s o f 28.3 ± 1.4% f o r S ( + ) - m e x i l e t i n e and 19.8 ± 1.5% f o r R ( - ) - m e x i l e t i n e ( M c E r l a n e e t al., 1987). 1.2.13 T i s s u e D i s t r i b u t i o n o f M e x i l e t i n e Thus f a r , o n l y one s t u d y on the t i s s u e d i s t r i b u t i o n o f r a c e m i c m e x i l e t i n e i n r a t s has been p u b l i s h e d ( B a r r i g o n e t al., 1983). These a u t h o r s showed t h a t f o l l o w i n g i . v . a d m i n i s t r a t i o n , t h e t i s s u e c o n c e n t r a t i o n s o f m e x i l e t i n e r e a c h e d peak v a l u e s t h a t were 12 t o 2 4 - f o l d h i g h e r t h a n plasma c o n c e n t r a t i o n s i n v a r i o u s p a r t s o f t h e b r a i n , 6 - f o l d h i g h e r i n t h e h e a r t , 3 3 - f o l d h i g h e r i n t h e l u n g s and a comparably h i g h r a t i o i n t h e l i v e r and k i d n e y s . These h i g h t i s s u e / s e r u m r a t i o s r e f l e c t t h e l a r g e volume o f d i s t r i b u t i o n o f m e x i l e t i n e i n both a n i m a l s ( B a r r i g o n e t al., 1983) and man ( P r e s c o t t et al., 1977; H a s e l b a r t h e t al., 1981). The h i g h d i s t r i b u t i o n o f m e x i l e t i n e i n t o the b r a i n c o u l d e x p l a i n t h e CNS s i d e e f f e c t s o f t e n o b s e r v e d d u r i n g m e x i l e t i n e t h e r a p y . 1.2.14 A n a l y t i c a l Methods f o r M e x i l e t i n e The a s s a y o f r a c e m i c m e x i l e t i n e i n human b i o l o g i c a l f l u i d s has been c a r r i e d out by s e v e r a l t e c h n i q u e s . These i n c l u d e a number o f gas-l i q u i d c h r o m a t o g r a p h i c (GLC) and h i g h - p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h i c (HPLC) p r o c e d u r e s . The GLC methods measured m e x i l e t i n e w i t h a n i t r o g e n s e l e c t i v e d e t e c t o r w i t h o u t d e r i v a t i z a t i o n (Smith and M e f f i n , 1980; V a s i l i a d e s e t al., 1984); a f t e r d e r i v a t i z a t i o n w i t h a n i t r o g e n - s e l e c t i v e d e t e c t i o n ( K e l l y et al., 1973; Bradbrook et al., 1977; and E l f i n g e t al., 1981), flame i o n i z a t i o n d e t e c t i o n ( P e r c h a l s k i et al., 1974; K e l l y , 1977; H o l t e t al., 1979; K a c p r o w i c z , 1982 and G r e c h - B e l a n g e r , 1984), and e l e c t r o n - c a p t u r e d e t e c t i o n (Frydman e t al., 1978). The l o w e s t amount d e t e c t e d was r e p o r t e d t o be 5 ng/ml w i t h t h e n i t r o g e n - s e l e c t i v e d e t e c t o r (Smith and M e f f i n , 1980). These GLC methods d i f f e r i n t h e i r mode o f d e t e c t i o n , volume o f sample r e q u i r e d f o r a n a l y s i s , e x t r a c t i o n p r o c e d u r e s , d e r i v a t i z a t i o n r e a g e n t and t h e n a t u r e o f t h e s t a t i o n a r y phase. The method d e s c r i b e d by H o l t e t al., 1979, has been adapted f o r t h e r o u t i n e serum m o n i t o r i n g o f m e x i l e t i n e . The HPLC methods measured m e x i l e t i n e e i t h e r d i r e c t l y u s i n g UV d e t e c t i o n ( K e l l y e t al., 1981); M a s t r o p o l o e t al., 1984) o r a f t e r d e r i v a t i z a t i o n f o l l o w e d by UV ( B r e i t h a u p t and W i l f l i n g , 1982) o r f l u o r e s c e n c e d e t e c t i o n (White and F a r i d , 1983; G r e c h - B e l a n g e r e t al., 1984). Most o f t h e UV t e c h n i q u e s do not have t h e r e q u i r e d s e n s i t i v i t y f o r s t u d y i n g s i n g l e dose k i n e t i c s o f m e x i l e t i n e i n humans. 1.2.15 Stereoselective Analysis of Mexiletine G r e c h - B e l a n g e r e t al. (1985) f i r s t r e p o r t e d a p r o c e d u r e f o r the HPLC measurement o f m e x i l e t i n e e n a n t i o m e r s u s i n g a method o f d e r i v a t i z a t i o n w i t h 2 , 3 , 4 , 6 - t e t r a - 0 - a c e t y l - / J - D - g l u c o p y r a n o s y l -i s o t h i o c y a n a t e ( G I T C ) , f o l l o w e d by r e s o l u t i o n o f t h e e n a n t i o m e r s on a re v e r s e - p h a s e ( o c t a d e c y l s i l a n e ) column and d e t e c t i o n by UV ab s o r b a n c e . 30 The l o w e s t amount o f each e n a n t i o m e r d e t e c t e d was 50 ng/ml. We have d e v e l o p e d a s t e r e o s e l e c t i v e HPLC as s a y p r o c e d u r e ( M c E r l a n e et al., 1987) i n which m e x i l e t i n e e n a n t i o m e r s were r e s o l v e d on a c h i r a l s t a t i o n a r y phase ( P i r k l e ^ i o n i c phenyl g l y c i n e column) w i t h subsequent d e t e c t i o n o f t h e 2-naphthoyl d e r i v a t i v e s by f l u o r e s c e n c e . The l o w e r l i m i t o f d e t e c t i o n i n plasma was 5 ng/ml, which i s a c o n s i d e r a b l e improvement from t h a t r e p o r t e d above. 1.3 C h i r a l i t y 1.3.1 I n t r o d u c t i o n C h i r a l i t y i s t h e p r o p e r t y d i s p l a y e d by any m o l e c u l e which i s not s u p e r i m p o s a b l e on i t s m i r r o r image. The two n o n - s u p e r i m p o s a b l e forms a r e r e f e r r e d t o as e n a n t i o m e r s , enantiomorphs o r o p t i c a l i s o m e r s . A v a s t number o f drugs o f s y n t h e t i c as w e l l as n a t u r a l o r i g i n e x h i b i t t h e p r o p e r t y o f c h i r a l i t y . W h ile t h e n a t u r a l s y n t h e s i s o f c h i r a l d r ugs p r o d u c e s o n l y one o f two p o s s i b l e e n a n t i o m e r s , t h e i r l a b o r a t o r y s y n t h e s i s u s u a l l y produces a racemate (50:50 r a t i o o f t h e two e n a n t i o m e r s ) . Enantiomers are c h a r a c t e r i z e d by t h e same p h y s i c a l and c h e m i c a l p r o p e r t i e s e x c e p t f o r t h e d i r e c t i o n i n which t h e y r o t a t e t h e p l a n e o f p o l a r i z e d l i g h t , and t h e i r i n t e r a c t i o n w i t h o t h e r c h i r a l compounds. D u r i n g e v o l u t i o n , l i v i n g systems p r o g r e s s i v e l y became c h i r a l l y e n r i c h e d such t h a t t h e y c o n t a i n o n l y D-sugars and L-amino a c i d s i n t h e i r c h e m i c a l make-up (Mason, 1984 & 1988). Thus, i t i s not s u r p r i s i n g t h a t l i v i n g o rganisms are a b l e t o d i s c r i m i n a t e between e n a n t i o m e r s a t t h e m o l e c u l a r l e v e l . T h i s o f t e n l e a d s t o d i f f e r e n c e s ( s t e r e o s e l e c t i v i t y ) i n t h e d i s p o s i t i o n o f the two e n a n t i o m e r s o f a c h i r a l d r u g . 1.3.2 Stereoselective drug Disposition 1.3.2.1 Pharmacodynamics The R ( - ) - e n a n t i o m e r o f t h e a n t i a r r h y t h m i c agent, t o c a i n i d e , has been shown t o be t h r e e t i m e s more p o t e n t t h a n t h e S ( + ) - e n a n t i o m e r a g a i n s t c h l o r o f o r m - i n d u c e d a r r h y t h m i a s i n t h e mouse (Bynes e t al., 1979; B l o c k e t al., 1988) and a r r h y t h m i a s evoked by programmed e l e c t r i c s t i m u l a t i o n ( U p r i c h a r d e t al., 1988). (-)Verapamil i s f o u r t i m e s more p o t e n t than i t s o p p o s i t e e n a n t i o m e r i n s u p p r e s s i n g i s c h a e m i a - i n d u c e d a r r h y t h m i a s i n c o n s c i o u s r a t s ( C u r t i s and Walker, 1986). The a n t i a r r h y t h m i c a c t i v i t y o f d i s o p y r a m i d e has been r e p o r t e d t o r e s i d e m a i n l y w i t h t h e S ( + ) - e n a n t i o m e r i n both animal ( M i r r o e t al., 1981) and human ( P o l l i c k e t al., 1982) s t u d i e s . A r e c e n t s t u d y (Lima and Boudoulas, 1987) n o t e d t h a t S ( + ) - d i s o p y r a m i d e has l e s s n e g a t i v e i n o t r o p i c e f f e c t than t h e R ( - ) - e n a n t i o m e r i n humans. The a u t h o r s c o n c l u d e d t h a t i t may be p o s s i b l e t o remove up t o 72% o f t h e n e g a t i v e i n o t r o p i c e f f e c t a s s o c i a t e d w i t h r a c e m i c d i s o p y r a m i d e by the a d m i n i s t r a t i o n o f t h e pure S ( + ) - e n a n t i o m e r . Most o f t h e t h e r a p e u t i c a c t i o n s o f p r o p r a n o l o l , i n c l u d i n g t h e a n t i h y p e r t e n s i v e e f f e c t s , a r e med i a t e d by t h e S ( - ) - e n a n t i o m e r ( N i e s and Ge r b e r , 1980; Rahn e t al., 1974). The a n t i c o a g u l a n t p o t e n c y o f S - w a r f a r i n i s a p p r o x i m a t e l y 6 ti m e s t h a t o f t h e R-enantiomer ( E b l e e t al., 1966). The a c t i o n o f t h e non-s t e r i o d a l a n t i i n f l a m m a t o r y d r u g , i b u p r o f e n , shows s t e r e o s e l e c t i v i t y i n t h a t o n l y t h e S(+)- e n a n t i o m e r i n h i b i t s p r o s t a g l a n d i n s y n t h e s i s (Kazuna e t al., 1974). 32 1.3.2.2 A b s o r p t i o n The a b s o r p t i o n o f t h e e n a n t i o m e r s o f a c h i r a l drug may be s t e r e o s e l e c t i v e i f t h e drug undergoes a c t i v e t r a n s p o r t a c r o s s t h e i n t e s t i n a l mucosa. Examples a r e dopa (Wade e t al., 1973) and m e t h o t r e x a t e (Hendel and B r o t h a g e n , 1984) whose L - i s o m e r s have been shown t o be a b s o r b e d more r a p i d l y than t h e D-isomers. 1.3.2.3 D i s t r i b u t i o n The d i s t r i b u t i o n o f e n a n t i o m e r s may d i f f e r as a r e s u l t o f d i f f e r e n c e s i n e i t h e r t i s s u e and/or plasma p r o t e i n b i n d i n g . I t has been shown t h a t S ( - ) - p r o p r a n o l o l i s more r a p i d l y t a k e n up by t h e h e a r t t i s s u e than i t s a n t i p o d e i n r a t s (Kawashima et al. 1976). S i m i l a r l y , t h e e x t r a v a s c u l a r b i n d i n g o f S ( - ) - p r o p r a n o l o l i s g r e a t e r than t h a t o f the R(+)-enantiomer i n dogs (Bai e t al., 1983). I t has a l s o been d e m o n s t r a t e d t h a t ( - ) t i m o l o l i s more r a p i d l y t a k e n up by l u n g , h e a r t , and b r a i n t i s s u e f r a c t i o n s than t h e ( + ) e n a n t i o m e r . The l a t t e r was found t o b i n d o n l y t o n o n - s p e c i f i c s i t e s (Tocco e t al., 1976). The serum p r o t e i n s c o n s i s t o f c h i r a l amino a c i d s and p o s s e s s e s c h i r a l s e c o n d a r y s t r u c t u r e s . Thus, t h e y p r o v i d e c h i r a l e n v i r o n m e n t s f o r s m a l l b i o a c t i v e m o l e c u l e s . For example, t h e e s s e n t i a l amino a c i d , L - t r y p t o p h a n , b i n d s t o human serum albumin (HSA) w i t h an a f f i n i t y about 100 t i m e s t h a t o f D - t r y p t o p h a n (McMenamy and O n c l e y , 1958). The f i r s t d r u g t h a t was shown t o e x h i b i t s t e r e o s e l e c t i v e b i n d i n g t o HSA was oxazepam s u c c i n a t e . M u l l e r and W o l l e r t (1975) found t h a t (+)oxazepam bound t o HSA w i t h an a f f i n i t y 40 t i m e s t h a t o f i t s e n a n t i o m e r . S - W a r f a r i n has a l s o been shown t o b i n d t o HSA w i t h a g r e a t e r a f f i n i t y t han t h e R-enantiomer (Brown e t a l . , 1977). a ^ - a c i d g l y c o p r o t e i n (AAG) i s t h e major serum b i n d i n g p r o t e i n f o r many b a s i c drugs and i t s b i n d i n g has been shown t o be s t e r e o s e l e c t i v e f o r such agents as p r o p r a n o l o l ( B a i e t al., 1983; W a l l e e t al., 1983; A l b a n i e t al., 1984) and d i s o p y r a m i d e ( V a l d i v i e s o e t al., 1983; Lima e t al., 1984; Lima and Boudoulas, 1986). 1.3.2.4 Metabolism V o g e l g e s a n g e t al. (1984) r e p o r t e d an i n t e r e s t i n g s t e r e o s e l e c t i v e f i r s t - p a s s m e t a b o l i s m f o r v e r a p a m i l . They showed t h a t t h e s y s t e m i c a v a i l a b i l i t y o f t h e more a c t i v e (-)-enantiomer was 2 t o 3 t i m e s s m a l l e r than t h a t o f t h e (+)-enantiomer. Thus, t h e i r d a t a e x p l a i n e d t h e e a r l i e r d i s c r e p a n c y o b s e r v e d by E i c h e l b a u m e t al., (1980) t h a t (±)-verapamil's e f f e c t on a t r i o - v e n t r i c u l a r c o n d u c t i o n i n man i s 2 t o 3 f o l d l e s s a f t e r o r a l a d m i n i s t r a t i o n than a f t e r i . v . a d m i n i s t r a t i o n when doses which produced equal plasma c o n c e n t r a t i o n s o f r a c e m i c d r u g were used. E n a n t i o m e r i c d i f f e r e n c e s i n p r e - s y s t e m i c m e t a b o l i s m have a l s o been r e p o r t e d f o r p r o p r a n o l o l e n a n t i o m e r s i n man ( S i l b e r and Reigelman, 1980; Von Bahr e t al., 1982). The 2 - a r y l p r o p i o n i c a c i d s p r o v i d e a good example o f s t e r e o s e l e c t i v e m e t a b o l i c a c t i v a t i o n . The i n a c t i v e R ( - ) - e n a n t i o m e r s undergo m e t a b o l i c i n v e r s i o n i n humans, in vivo, t o t h e a c t i v e S ( + ) - e n a n t i o m e r (Adams e t al., 1976; K r i p a l a n i e t al., 1976; Bopp e t al., 1979; Lee e t al., 1985). The p r o g r e s s i v e e n r i c h m e n t o f t h e a c t i v e S ( + ) - e n a n t i o m e r means t h a t i t s e n a n t i o m e r i c r a t i o must be d e t e r m i n e d i f m e a n i n g f u l c o n c e n t r a t i o n - e f f e c t r e l a t i o n s h i p s a r e t o be d e t e r m i n e d . Enantiomers may a l s o i n t e r a c t w i t h each o t h e r a t t h e m e t a b o l i c l e v e l . F or example, t h e a n a l g e s i c a c t i v i t y o f levomethorphan i s s i g n i f i c a n t l y enhanced and p r o l o n g e d by t h e c o - a d m i n i s t r a t i o n o f i t s a n t i p o d e , t h e i n a c t i v e d extromethorphan, due t o m e t a b o l i c i n h i b i t i o n (Cooper and A n d e r s , 1974). The r e v i e w p a p e r s p u b l i s h e d by J e n n e r and 34 T e s t a (1980), T e s t a (1986) and C a l d w e l l et al. (1988) c o n t a i n many o t h e r examples o f s t e r e o s e l e c t i v e m e t a b o l i s m . 1.3.3 S t e r e o s e l e c t i v e Drug A n a l y s i s 1.3.3.1 I n t r o d u c t i o n U n t i l r e c e n t l y , t h e r e has been a p a u c i t y o f i n f o r m a t i o n i n t h e l i t e r a t u r e on t h e importance o f s t e r e o c h e m i s t r y i n drug d i s p o s i t i o n . T h i s can be a t t r i b u t e d t o t h e l a c k o f a n a l y t i c a l methodology a p p r o p r i a t e f o r t h e d e t e r m i n a t i o n o f t h e e n a n t i o m e r i c c o m p o s i t i o n o f c h i r a l d rugs a t t h e r a p e u t i c c o n c e n t r a t i o n s . The t r a d i t i o n a l and c l a s s i c a l method o f r e s o l v i n g e n a n t i o m e r s i n v o l v e s t h e i r c o n v e r s i o n t o d i a s t e r e o i s o m e r i c s a l t s by r e a c t i o n w i t h an o p t i c a l l y a c t i v e compound, and subsequent r e s o l u t i o n by f r a c t i o n a l r e c r y s t a l l i z a t i o n . R e s o l u t i o n i s p o s s i b l e because d i a s t e r e o i s o m e r s have d i f f e r e n t c h e m i c a l and p h y s i c a l p r o p e r t i e s , i n c l u d i n g s o l u b i l i t y i n a g i v e n s o l v e n t . T h i s t r a d i t i o n a l method, however, i s o f t e n l a b o r i o u s , i n e f f i c i e n t , does not y i e l d o p t i c a l l y pure samples and i s e s s e n t i a l l y r e s t r i c t e d t o c a r b o x y l i c a c i d s and amines ( B l a s c h k e , 1980). More i m p o r t a n t l y , i t i s not amenable t o p h a r m a c o k i n e t i c s t u d i e s . Thus, most o f t h e r e c e n t advances i n en a n t i o m e r s e p a r a t i o n t e c h n o l o g y have been made i n t h e a r e a o f chromatography. C h r o m a t o g r a p h i c methods have t h e advantage o f s m a l l sample s i z e , speed, e f f i c i e n c y , independence from t h e magnitude o f s p e c i f i c r o t a t i o n and t h e p r e s e n c e o f o t h e r o p t i c a l l y a c t i v e s p e c i e s i n t h e sample ( L o c h m u l l e r and S o u t e r , 1975). En a n t i o m e r s a r e r e s o l v e d c h r o m a t o g r a p h i c a l l y as t h e d i a s t e r e o i s o m e r s on an a c h i r a l s t a t i o n a r y phase o r by d i r e c t r e s o l u t i o n on a c h i r a l s t a t i o n a r y phase (CSP). 1.3.3.2 Resolution of Enantiomers as Diastereoisomers The e n a n t i o m e r s o f t h e a n t i a r r h y t h m i c agent, t o c a i n i d e , have been r e s o l v e d by t h e f o r m a t i o n o f d i a s t e r e o i s o m e r s u s i n g S(-)-2-methoxy-2-t r i f l u o r o m e t h y p h e n y l a c e t y l c h l o r i d e by GLC (Gal et al., 1982). S ( - ) - l -( 4 - N i t r o p h e n y l s u l f o n y l ) p r o l y l c h l o r i d e was s u c c e s s f u l l y a p p l i e d as a c h i r a l d e r i v a t i z a t i o n r e a g e n t f o r t h e s e p a r a t i o n o f amphetamines and e p h e d r i n e s by HPLC ( C l a r k and B l a c k s d a l e , 1984). S i m i l a r l y , t h e 2-a r y l p r o p i o n i c a c i d s were r e s o l v e d w i t h S ( + ) - 2 - o c t a n o l by HPLC (Johnson e t al., 1979; Lee et al., 1984), and w i t h o p t i c a l l y a c t i v e amphetamine by GLC ( S i n g h e t al., 1986). I s o c y a n a t e s such as phenyl e t h y l i s o c y a n a t e , have been u t i l i z e d t o r e s o l v e many amino compounds by HPLC. These i n c l u d e amphetamines ( M i l l e r e t al., 1984), p r o p r a n o l o l ( W i l s o n and W a l l e , 1984) and e p h e d r i n e (Gal and Sedman, 1984). E x t e n s i v e r e v i e w p a p e r s on t h e r e s o l u t i o n o f e n a n t i o m e r s u s i n g c h i r a l d e r i v a t i z a t i o n r e a g e n t s have been p u b l i s h e d by s e v e r a l a u t h o r s (Lochmuler and S o u t e r , 1975; Tamegai e t al., 1979; L i n d n a , 1982 & 1988). C o n s i d e r a b l e advances toward u n d e r s t a n d i n g t h e s e p a r a t i o n mechanism f o r d i a s t e r e o i s o m e r s have been made by Rose et al. ( 1 9 6 6 ) . In t h e i r s t u d y w i t h d i a s t e r e o i s o m e r i c e s t e r s o f 2 - a c e t o x y p r o p i o n i c a c i d , t h e y o b s e r v e d t h a t t h e s i z e o f the s u b s t i t u e n t s a t t h e asymmetric c a r b o n , t h e d i s t a n c e between t h e o p t i c a l c e n t r e s i n t h e e s t e r , as w e l l as t h e p o l a r i t y o f t h e s t a t i o n a r y phase were c r i t i c a l f o r r e s o l u t i o n . T h e r e a r e , however, drawbacks a s s o c i a t e d w i t h t h e r e s o l u t i o n o f e n a n t i o m e r s by f o r m a t i o n o f t h e i r d i a s t e r e o i s o m e r s . These i n c l u d e t h e r e q u i r e m e n t o f an a c t i v e f u n c t i o n a l group on t h e e n a n t i o m e r , d i f f e r e n c e s i n t h e r e a c t i o n k i n e t i c s o f e n a n t i o m e r s w i t h t h e c h i r a l r e a g e n t which may r e s u l t i n q u a n t i t a t i o n e r r o r , and t h e r e q u i r e m e n t o f an o p t i c a l l y pure r e a g e n t ( K o n i g et al., 1977; Frank, ef. a7., 1978; L i u and Ku, 1983). In a d d i t i o n , t h e d i a s t e r e o i s o m e r s must be c h e m i c a l l y and s t e r e o c h e m i c a l l y s t a b l e under t h e c h r o m a t o g r a p h i c c o n d i t i o n s . 1.3.3.3. R e s o l u t i o n o f E n a n t i o m e r s on C h i r a l S t a t i o n a r y Phases The d i r e c t approach t o t h e r e s o l u t i o n o f e n a n t i o m e r s u s i n g a c h i r a l s t a t i o n a r y phase does not have any o f t h e d i s a d v a n t a g e s o f t h e above methods and i n some i n s t a n c e s , t h e e n a n t i o m e r s can be r e s o l v e d w i t h o u t p r i o r d e r i v a t i z a t i o n . R e s o l u t i o n depends on t h e f o r m a t i o n o f t r a n s i e n t d i a s t e r e o i s o m e r i c complexes ( v i a 7r-bond, hydrogen bonds, e l e c t r o s t a t i c bonds and s t e r i c i n t e r a c t i o n s ) between t h e e n a n t i o m e r s and t h e CSP ( F e i b u s h and G r i n b e r g , 1988). The r e l a t i v e s t a b i l i t y o f t h e s e complexes l e a d s t o t h e r e s o l u t i o n o f t h e e n a n t i o m e r s . C h i r a s i l -V a l ^ ( L - v a l i n e - t e r t - b u t y l a m i d e - c a r b o x y a l k y l - m e t h y l - s i l o x a n e ) was t h e f i r s t s t a b l e GLC CSP t o become a v a i l a b l e c o m m e r c i a l l y ( F r a n k e t al., 1977). R e s o l u t i o n o f e n a n t i o m e r s on t h e phase was based m a i n l y on H-bonding and s t e r i c i n t e r a c t i o n s . T h i s c h i r a l GLC phase was s u c c e s s f u l l y used t o r e s o l v e t h e e n a n t i o m e r s o f amino a c i d s , s u g a r s , a r o m a t i c and a l i p h a t i c h y d r o x y - a c i d s and amines ( F r a n k e t al., 1977; Frank e t a7., 1979; Frank e t al., 1980). T h i s phase has a l s o been used i n t h e r e s o l u t i o n o f t h e e n a n t i o m e r s o f t o c a i n i d e i n r e p o r t s from our l a b o r a t o r y ( M c E r l a n e and P i l l a i , 1983). The h i g h thermal s t a b i l i t y o f C h i r a s i l - V a l R made i t p o s s i b l e , f o r t h e f i r s t t i m e , t o employ a mass-s p e c t r o m e t e r , c o u p l e d t o a gas chromatograph, f o r t h e a n a l y s i s o f e n a n t i o m e r i c drugs and m e t a b o l i t e s ( F r a n k e t al., 1978). XE-60 v a l i n e -S - p h e n y l e t h y l amide i s a n o t h e r c o m m e r c i a l l y a v a i l a b l e GLC c h i r a l phase which has been used t o r e s o l v e c h i r a l a l c o h o l s , h y d r o x y a c i d s and c a r b o h y d r a t e s by GLC ( K o n i g and S i e v e r s , 1980; K o n i g et al., 1981; K o n i g et al., 1982). The development of c h i r a l HPLC phases has seen a much f a s t e r growth than GLC phases and more than 22 a r e now c o m m e r c i a l l y a v a i l a b l e . The r e s o l u t i o n o f e n a n t i o m e r s on t h e a- and / J - c y c l o d e x t r i n s ( c y c l o h e p t a a m y l a s e and c y c l o o c t a a m y l a s e ) i n v o l v e s i n c l u s i o n complexes i n which t h e e n a n t i o m e r s e n t e r e x c l u s i v e l y i n t o c h i r a l c a v i t i e s w i t h i n t h e CSPs. S t e r e o s e l e c t i v i t y r e s u l t s from a d i f f e r e n c e i n t h e f i t o f t h e e n a n t i o m e r s (Armstrong and Damond, 1984; Armstrong et al., 1986). A s i m i l a r mechanism has been proposed f o r the c h i r a l phases, c e l l u l o s e t r i a c e t a t e (Hesse and H e g e l , 1976) and p o l y ( t r i p h e n y l m e t h y l m e t h a c r y l a t e ) (Okamoto et al., 1981; Okamoto and Hatada, 1986). The p r o t e i n - b o n d e d CSPs c o n t a i n m a i n l y b o v i n e serum albumin ( A l l e n m a r k et al., 1983) o r ox-a c i d g l y c o p r o t e i n (Hermansson, 1983) a t t a c h e d t o s i l i c a . Both CSPs have been s u c c e s s f u l l y used f o r t h e r e s o l u t i o n o f many e n a n t i o m e r i c drugs (Wainer et al., 1986). The r e t e n t i o n mechanism o f t h e s e phases i n v o l v e a c o m b i n a t i o n o f e l e c t r o s t a t i c , H-bonding and h y d r o p h o b i c i n t e r a c t i o n s ( S c h i l l et al., 1986; A l l e n m a r k , 1986). By f a r , t h e most v e r s a t i l e o f a l l t h e c h i r a l HPLC phases a r e t h e P i r k l e ^ c h i r a l p h a s e s . These columns c o n t a i n amino a c i d d e r i v a t i v e s such as 3 , 5 - d i n i t r o b e n z o y l d e r i v a t i v e s o f ( R ) - and ( S ) - p h e n y l g l y c i n e , ( S ) - l e u c i n e and ( R ) - and ( S ) -n a p h t h y l a l a n i n e . The p r o p o s e d mechanism o f r e s o l u t i o n by t h e s e phases a r e 7 r - a c i d 7 r-base, d i p o l e - d i p o l e and s t e r i c i n t e r a c t i o n s ( P i r k l e and Welch, 1984). The P i r k l e * c h i r a l phases have proven t o be p a r t i c u l a r l y v a l u a b l e f o r t h e r e s o l u t i o n o f a v a r i e t y o f compounds such as amines ( P i r k l e and Welch, 1984; P i r k l e et al., 1984), amino a c i d s and a l c o h o l s ( P i r k l e and Welch, 1984), b e n z o d i a z e p i n e s ( P i r k l e and P s i p o u r a s , 1984) and a c y c l i c a l k y l c a r b i n o l s (Weems and Yang., 1982). In o u r own work, t h e P i r k l e ^ i o n i c (phenyl g l y c i n e ) CSP has been used t o r e s o l v e t h e e n a n t i o m e r s o f m e x i l e t i n e ( M c E r l a n e e t a7., 1987). The a p p l i c a t i o n s o f c h i r a l s t a t i o n a r y phases have been r e v i e w e d ( L o u c h m u l l e r and S o u t e r , 1975; B l a s c h k e , 1980; L i u and Ku, 1980; A r m s t r o n g , 1984; Wainer and Alembik, 1988). 1.3.3.4 R e s o l u t i o n o f E n a n t i o m e r s U s i n g C h i r a l E l u e n t s With a s u i t a b l e c h i r a l component i n t h e m o b i l e phase, e n a n t i o m e r s can be r e s o l v e d on an a c h i r a l HPLC s u p p o r t . Two p o s s i b l e mechanisms may r e s u l t i n e n a n t i o m e r r e s o l u t i o n . The c h i r a l component p r e s e n t i n t h e m o b i l e phase can i n t e r a c t w i t h e n a n t i o m e r s t o form d i a s t e r e o i s o m e r i c complexes o r w i t h t h e s t a t i o n a r y phase t o produce e s s e n t i a l l y a c h i r a l s t a t i o n a r y phase. Examples i n t h e l i t e r a t u r e i n c l u d e t h e r e s o l u t i o n o f D and L amino a c i d s u s i n g an o p t i c a l l y a c t i v e C u + + - p r o l i n e complex ( P o o l e and S c h u e t t e , 1984) and e n a n t i o m e r i c amines w i t h a c h i r a l c o u n t e r i o n o f (+)-10-camphor s u l f o n i c a c i d ( P o o l e and S c h u e t t e , 1984). 1.4 R a t i o n a l e a) R a t i o n a l e f o r development o f a s s a y o f m e x i l e t i n e e n a n t i o m e r s w i t h 2 - a n t h r o y l c h l o r i d e as a d e r i v a t i z a t i o n r e a g e n t . Two s t u d i e s ( G r e c h - B e l a n g e r et al., 1986; M c E r l a n e et al., 1987) have r e p o r t e d t h e r e s o l u t i o n and q u a n t i t a t i o n o f m e x i l e t i n e e n a n t i o m e r s by HPLC. The s e n s i t i v i t y l i m i t ( l o w e r ) o f t h e a s s a y by G r e c h - B e l a n g e r et al. (1986) was 50 ng/ml o f each e n a n t i o m e r . The method d e v e l o p e d by M c E r l a n e et al. (1987) u s i n g 2-naphthoyl c h l o r i d e as a d e r i v a t i z a t i o n r e a g e n t improved s e n s i t i v i t y o f d e t e c t i o n by 1 0 - f o l d ( l o w e r l i m i t o f d e t e c t i o n was 5 ng/ml o f each e n a n t i o m e r ) . However, adequate d e l i n e a t i o n o f t h e p h a r m a c o k i n e t i c s , i n c l u d i n g serum p r o t e i n b i n d i n g , o f m e x i l e t i n e e n a n t i o m e r s i n r a t s where r e l a t i v e l y s m a l l b i o l o g i c a l samples and f a s t e r d r u g e l i m i n a t i o n were a n t i c i p a t e d i n d i c a t e d t h e need f o r a more s e n s i t i v e a s s a y . E v a l u a t i o n o f t h e s t e r e o c h e m i c a l i n t e r a c t i o n s between t h e 2-naphthoyl d e r i v a t i v e s o f t h e e n a n t i o m e r s and t h e P i r k l e ^ c h i r a l s t a t i o n a r y phase (Igwemezie, 1986; M c E r l a n e et al. 1987) l e a d t o t h e p r o p o s a l t h a t s e n s i t i v i t y c o u l d be improved w h i l e p r e s e r v i n g s e l e c t i v i t y by u s i n g 2 - a n t h r o y l c h l o r i d e , i n s t e a d o f 2 - n a p h t h o y l c h l o r i d e , as a d e r i v a t i z a t i o n r e a g e n t ( t h e f l u o r e s c e n c e quantum y i e l d o f t h e a n t h r a c e n e r i n g i s 1 . 5 - f o l d t h a t o f t h e n a p h t h a l e n e r i n g ) . b) R a t i o n a l e f o r t h e s t u d y o f t h e b i n d i n g o f m e x i l e t i n e e n a n t i o m e r s t o serum and i s o l a t e d serum p r o t e i n s . The f r e e form o f a p r o t e i n bound drug i s b e l i e v e d t o be i n e q u i l i b r i u m w i t h the t a r g e t t i s s u e s i t e s s i n c e i t i s t h e form t h a t can d i f f u s e out o f t h e v a s c u l a r system. I n t u i t i v e l y , t h e serum f r e e d r u g c o n c e n t r a t i o n o f drugs s h o u l d c o r r e l a t e b e t t e r w i t h p h a r m a c o l o g i c a l e f f e c t s than t h e t o t a l d r u g c o n c e n t r a t i o n . T h i s has been r e p o r t e d f o r p r o p r a n o l o l ( M c D e v i t t et al., 1976) and d i s o p y r a m i d e (Lima et al., 1981). The serum b i n d i n g o f r a c e m i c m e x i l e t i n e i s r e p o r t e d t o be 70% i n h e a l t h y human s u b j e c t s ( T a l b o t et al., 1973). T h i s s t u d y was c a r r i e d out by u l t r a f i l t r a t i o n t e c h n i q u e w i t h o n l y one drug c o n c e n t r a t i o n . P r e l i m i n a r y s t u d i e s on t h e b i n d i n g i f m e x i l e t i n e e n a n t i o m e r s i n our l a b o r a t o r y showed s t e r e o s e l e c t i v e b i n d i n g w i t h bound f r a c t i o n s o f 81 and 72% f o r R ( - ) - and S ( + ) - m e x i l e t i n e , r e s p e c t i v e l y . However, t h e b i n d i n g p a r a m e t e r s were not d e t e r m i n e d s i n c e t h e range o f c o n c e n t r a t i o n s s t u d i e d was s m a l l (0.2-2 /jg/ml). Both s t u d i e s i n d i c a t e d r e l a t i v e l y h i g h b i n d i n g f o r m e x i l e t i n e . The p r e s e n t s t u d y was pr o p o s e d t o f u l l y c h a r a c t e r i z e t h e b i n d i n g k i n e t i c s o f m e x i l e t i n e e n a n t i o m e r s as w e l l as e v a l u a t e t h o s e f a c t o r s known t o a f f e c t serum p r o t e i n b i n d i n g . F u r t h e r m o r e , most b a s i c d r u g s a r e known t o b i n d t o a l - a c i d g l y c o p r o t e i n ( P i a f s k y and Knoppert, 1978). The c o n c e n t r a t i o n o f t h i s p r o t e i n i s r e p o r t e d t o be i n c r e a s e d i n many d i s e a s e c o n d i t i o n s i n c l u d i n g a c u t e MI. S i n c e m e x i l e t i n e may be used t o c o n t r o l a r r h y t h m i a s i n t h e s e p a t i e n t s , i t i s p e r t i n e n t t o i d e n t i f y t h e p r o t e i n ( s ) r e s p o n s i b l e f o r t h e b i n d i n g o f t h e e n a n t i o m e r s i n serum. T h i s w i l l make i t p o s s i b l e t o p r e d i c t t h e e f f e c t o f d i s e a s e on t h e p h a r m a c o k i n e t i c s o f m e x i l e t i n e e n a n t i o m e r s . c) Rationale for studying tissue distribution kinetics of mexiletine enantiomers. B a r r i g o n et al. (1983) s t u d i e d t he t i s s u e d i s t r i b u t i o n k i n e t i c s o f r a c e m i c m e x i l e t i n e i n t h e r a t and r e p o r t e d e x t e n s i v e uptake i n t o v a r i o u s t i s s u e s . T i s s u e / s e r u m r a t i o s o f 12-24 was found i n d i f f e r e n t p a r t s o f th e b r a i n , 6 i n t h e h e a r t , and up t o 33 i n t h e l u n g s . These h i g h t i s s u e 41 u p t a k e s s u g g e s t t h e p o s s i b l e i n v o l v e m e n t o f a f a c i l i t a t e d uptake mechanism. S t e r e o s e l e c t i v e d i s t r i b u t i o n i n t o t i s s u e s i n c l u d i n g t h e h e a r t has been r e p o r t e d f o r p r o p r a n o l o l (Bai e t a l . 1983) and t i m o l o l (Tocco e t a l . , 1976). However, the r e l a t i v e t i s s u e uptake o f m e x i l e t i n e e n a n t i o m e r s has not been r e p o r t e d . The b r a i n uptake o f t h e e n a n t i o m e r s i s o f p a r t i c u l a r i n t e r e s t s i n c e i t has been r e p o r t e d t h a t a s i g n i f i c a n t number o f p a t i e n t s on m e x i l e t i n e t h e r a p y e x p e r i e n c e CNS s i d e e f f e c t s , n e c e s s i t a t i n g w i t h d r a w a l o f t h e drug i n some o f t h e p a t i e n t s ( K r e e g e r and Hammill, 1987). I f t h e b r a i n uptake o f m e x i l e t i n e e n a n t i o m e r s i s s t e r e o s e l e c t i v e , t h e n , removing t h e e n a n t i o m e r t h a t a c c u m u l a t e s more, may r e d u c e t h e s e v e r i t y o f t h e CNS s i d e e f f e c t s a s s o c i a t e d w i t h m e x i l e t i n e t h e r a p y . d) Rationale for the pharmacodynamic studies on the relative antiarrhythmic act iv i ty of racemic mexiletine and i t ' s enantiomers. I t i s w e l l e s t a b l i s h e d t h a t d r u g e n a n t i o m e r s o f t e n d i f f e r i n t h e i r p h a r m a c o l o g i c a l p r o p e r t i e s ( S i m o n y i , 1984). With r e s p e c t t o m e x i l e t i n e , t h e r e l a t i v e a n t i a r r h y t h m i c a c t i v i t y o f t h e e n a n t i o m e r s has n e v e r been r e p o r t e d . However, t h e a n t i a r r h y t h m i c e f f e c t s o f o t h e r c l a s s I agents which s h a r e t h e same mode o f a c t i o n w i t h m e x i l e t i n e have been shown t o be s t e r e o s e l e c t i v e . F or example, R ( - ) - t o c a i n i d e i s s i g n i f i c a n t l y more p o t e n t t h a n t h e S ( + ) - e n a n t i o m e r i n p r o t e c t i n g a g a i n s t c h l o r o f o r m i n d u c e d a r r h y t h m i a s i n mice (Bynes e t al., 1979; B l o c k e t al., 1988) and e l e c t r i c a l l y - i n d u c e d a r r h y t h m i a s i n dogs ( U p r i c h a r d e t al., 1988). The (+)-en a n t i o m e r o f d i s o p y r a m i d e has been shown t o p r o l o n g a c t i o n -p o t e n t i a l d u r a t i o n i n c a n i n e c a r d i a c p u r k i n j e f i b r e s , w h i l e t h e (-)-enantiomer s h o r t e n s i t ( M i r r o e t a/., 1981). These r e s u l t s s u g g e s t 42 t h a t t h e a n t i a r r h y t h m i c a c t i o n s o f m e x i l e t i n e e n a n t i o m e r s may a l s o be d i f f e r e n t . 1.5 Specific Objectives a) To d e v e l o p a s e n s i t i v e and s t e r e o s e l e c t i v e HPLC a s s a y f o r t h e measurement o f m e x i l e t i n e e n a n t i o m e r s i n b i o l o g i c a l f l u i d s and t i s s u e homogenates. b) To s t u d y t h e b i n d i n g c h a r a c t e r i s t i c s o f m e x i l e t i n e e n a n t i o m e r s t o human serum, i s o l a t e d human serum albumin and a j - a c i d g l y c o p r o t e i n . c) To d e t e r m i n e t h e t i s s u e d i s t r i b u t i o n k i n e t i c s o f t h e e n a n t i o m e r s o f m e x i l e t i n e i n t h e h e a r t , b r a i n , l i v e r , l u n g , k i d n e y and f a t t i s s u e s i n r a t s . d) To d e t e r m i n e t h e r e l a t i v e e f f i c a c y o f t h e e n a n t i o m e r s o f m e x i l e t i n e a g a i n s t e l e c t r i c a l and i s c h a e m i a - i n d u c e d a r r h y t h m i a s i n r a t s , and t o e s t a b l i s h plasma c o n c e n t r a t i o n - e f f e c t r e l a t i o n s h i p s . e) To e v a l u a t e t h e r e l a t i v e e f f e c t s o f m e x i l e t i n e e n a n t i o m e r s on t h e haemodynamic and ECG parameters i n r a t s . 2. EXPERIMENTAL 2.1 S u p p l i e s 2.1.1 Drugs The f o l l o w i n g drugs were used: R , S - m e x i l e t i n e h y d r o c h l o r i d e * , R ( - ) 2 , and S ( + ) - m e x i l e t i n e H y d r o c h l o r i d e 3 , KOE 2963 ( i n t e r n a l s t a n d a r d ) ^ , p e n t o b a r b i t o n e sodium^, h a l o t h a n e 6 , C i c a t r i n 7 , M a r c a i n e 8 and h e p a r i n 9 . 2.1.2 C h e m i c a l s and Reagents The f o l l o w i n g c h e m i c a l s and r e a g e n t s were used: 2-naphthoyl c h l o r i d e 1 0 , a n t h r a q u i n o n e - 2 - c a r b o x y l i c a c i d 1 1 , o x a l y l c h l o r i d e 1 2 , c a l c i u m c h l o r i d e 1 3 , p o t a s s i u m permanganate 1^, t r i c h l o r o a c e t i c a c i d 1 ^ , aluminium h y d r o x i d e ( a l u m i n a ) 1 6 , z i n c d u s t 1 7 , sodium h y d r o x i d e 1 8 , barium h y d r o x i d e o c t a h y d r a t e 1 9 , z i n c s u l p h a t e h e p t a h y d r a t e 2 0 , h y d r o c h l o r i c a c i d 2 1 , sodium c h l o r i d e 2 2 , monosodium phosphate m o n o h y d r a t e 2 3 , d i s o d i u m phosphate h e p t a h y d r a t e 2 ^ , c o n c e n t r a t e d ammonia s o l u t i o n 2 6 and s u l p h u r i c a c i d 2 6 . 2.1.3 S o l v e n t s The f o l l o w i n g s o l v e n t s were used: d i c h l o r o m e t h a n e 2 7 , n - h e x a n e 2 8 , 2 - p r o p a n o l 2 9 , d i e t h y l e t h e r 3 0 , c h l o r o f o r m 3 1 , 1-4 B o e h r i n g e r I n g e l h e i m L t d . , B u r l i n g t o n , O n t a r i o , Canada. e t h a n o l 3 2 , a c e t i c a c i d 3 3 , m e t h a n o l 3 4 , a c e t o n i t r i l e 3 5 , e t h y l a c e t a t e 3 5 . 2.1.4 Human Serum P r o t e i n s These were a l b u m i n 3 ^ , a j - a c i d g l y c o p r o t e i n 3 ^ and l i p o p r o t e i n -d e f i c i e n t s e r u m 3 ^ . 2.2 C h r o m a t o g r a p h i c S t a t i o n a r y Phases and Columns 2.2.1 HPLC Columns a) A P i r k l e R i o n i c c h i r a l column 4*' (25 x 0.46 cm i . d . ) w i t h a s t a t i o n a r y phase c o n s i s t i n g o f ( R ) - N - 3 , 5 - d i n i t r o b e n z o y l p h e n y l g l y c i n e i o n i c a l l y bonded t o T-aminopropyl s i l i c a was used. b) A P i r k l e R i o n i c c h i r a l c o l u m n 4 1 (25 x 0.46 cm i . d . ) w i t h a s t a t i o n a r y phase c o n s i s t i n g o f ( S ) - N - 3 , 5 - d i n i t r o b e n z o y l l e u c i n e i o n i c a l l y bonded t o T-aminopropyl s i l i c a was used. c) A HPLC s i l i c a g u a r d c o l u m n 4 2 (1.5 x 0.32 cm i . d . ) was used i n f r o n t o f t h e P i r k l e R column. d) Whatman P r e p a r a t i v e C18 c o l u m n 4 3 (25 x 0.94 cm i . d . ) . 5 M.T.C. P h a r m a c e u t i c a l s , M i s s a u s a g a , O n t a r i o , Canada. 6 A y e r s t L a b o r a t o r i e s , M o n t r e a l , Quebec, Canada. 7 Wellcome I n c . , K i r k l a n d , Quebec, Canada. 8 Winthrop L a b o r a t o r i e s , A u r o r a , O n t a r i o , Canada. 9 G l a x o L t d . , T o r o n t o , O n t a r i o , Canada 10-14 A l d r i c h Chemical Co., Milwaukee, W i s c o n s i n , USA. 15-16 F i s c h e r S c i e n t i f i c Co. F a i r Lawn, New J e r s e y , USA. 46 2.3 Equipment 2.3.1 H i g h - P e r f o r m a n c e L i q u i d Chromatograph A G i l son model 302 h i g h - p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h 4 4 e q u i p p e d w i t h a G i l s o n model 602 d a t a master, a NEC model PC-8023A-C d a t a t e r m i n a l 4 5 , a 20 /J1 l o o p i n j e c t o r 4 6 and a S c h o e f f e l model 970 f l u o r o m e t e r 4 7 was used. 2.3.2 Gas-Chromatograph/Mass S p e c t r o m e t e r A H e w l e t t Packard model 5987A GC/MS 4 8 e q u i p p e d w i t h a model 5880 gas chromatograph was used. 2.3.3 P o l y g r a p h A model 79D G r a s s p o l y g r a p h 4 9 was used. 2.3.4 E l e c t r i c S t i m u l a t o r A model SD9 G r a s s e l e c t r i c s t i m u l a t o r 6 0 was used. 17-18 BDH C h e m i c a l s L t d . , P o o l e , England, UK. 19-36 BDH C h e m i c a l s L t d . , Vancouver, B r i t i s h Columbia, Canada. 37-38 Terochem L a b o r a t o r i e s L t d . , Edmonton, A l b e r t a , Canada. 39 Sigma Chemical Co., S t . L o u i s , Mo, USA 40-41 R e g i s Chemical Co., Morton Grove, I l l i n o i s , USA. 42 R a i n i n I n s t r u m e n t s I n c . , Woburn, M a s s a c h u s s e t t s , USA. 2.4 A s s a y o f M e x i l e t i n e Enantiomers U s i n g 2 - A n t h r o y l C h l o r i d e as a D e r i v a t i z a t i o n Reagent 2.4.1 S y n t h e s i s o f 2 - A n t h r o y l C h l o r i d e from A n t h r a q u i n o n e - 2 - C a r b o x y l i c A c i d The s y n t h e s i s o f 2 - a n t h r o y l c h l o r i d e was a c c o m p l i s h e d by t h e r e d u c t i o n o f a n t h r a q u i n o n e - 2 - c a r b o x y l i c a c i d w i t h d i l u t e ammonia and z i n c d u s t t o a n t h r a c e n e - 2 - c a r b o x y l i c a c i d , f o l l o w e d by c o n v e r s i o n o f t h e a c i d t o t h e a c i d c h l o r i d e w i t h o x a l y l c h l o r i d e . 2.4.1.1 S y n t h e s i s o f A n t h r a c e n e - 2 - C a r b o x y l i c A c i d A n t h r a q u i n o n e - 2 - c a r b o x y l i c a c i d (1.0203 g) was suspended, w i t h s t i r r i n g , i n 80 ml o f d i l u t e ammonia ( 9 . 5 % ammonia s o l u t i o n ) i n a 250 ml round bottom g l a s s f l a s k . To t h i s s u s p e n s i o n was added 2 g o f z i n c d u s t and t h e m i x t u r e was h e a t e d i n a water bath a t 100°C. The r e a c t i o n m i x t u r e which was i n i t i a l l y b l o o d - r e d i n c o l o u r t u r n e d t o p a l e y e l l o w a t t h e end o f t h e r e a c t i o n ( a p p r o x i m a t e l y 45 m i n u t e s ) . The m i x t u r e was a l l o w e d t o c o o l and was f i l t e r e d t h r o u g h a Whatman (No 1) f i l t e r p a p er. 43 Whatman In c . , C l i f t o n , New J e r s e y , USA. 44-45 Mandel S c i e n t i f i c Co. L t d . , Edmonton, A l b e r t a , Canada. 46 Rheodyne I n c . , B e r k e l e y , C a l i f o r n i a , USA. 47 K r a t o s ( S c h o e f f e l I n s t r u m e n t s ) , Westwood, New J e r s e y , USA. 48 H e w l e t t P a c k a r d , A v o n d a l e , P e n s y l v a n i a , USA. 49-50 G r a s s I n s t r u m e n t s Co., Quincy, M a s s a c h u s s e t t s , USA. The f i l t r a t e was a c i d i f i e d t o pH ~7 w i t h 80 ml o f 2 M HC1, f i l t e r e d and t h e r e s i d u e was d i s c a r d e d . The f i l t r a t e was f u r t h e r a c i d i f i e d t o pH =1 w i t h 20 ml o f 2 M HC1 and a y e l l o w p r e c i p i t a t e o f a n t h r a c e n e - 2 -c a r b o x y l i c a c i d appeared. The aqueous p o r t i o n was f i l t e r e d o ut by s u c t i o n . The r e s u l t i n g a n t h r a c e n e - 2 - c a r b o x y l i c a c i d was p u r i f i e d by HPLC ( s e c t i o n 2.4.1.2 be l o w ) . 2.4.1.2 P u r i f i c a t i o n o f A n t h r a c e n e - 2 - C a r b o x y l i c A c i d The s y n t h e t i c a c i d was p u r i f i e d by r e v e r s e phase HPLC u s i n g a Whatman P r e p a r a t i v e C18 column. The m o b i l e phase was m e t h a n o l / a c e t i c a c i d / c h l o r o f o r m (2:1:97) pumped i s o c r a t i c a l l y a t a f l o w o f 0.5 ml/min. D e t e c t i o n was a c c o m p l i s h e d by UV a t 262 nm. The a n t h r a c e n e - 2 - c a r b o x y l i c a c i d t h u s i s o l a t e d ( R t = 8 min) was s u b s e q u e n t l y r e c r y s t a l 1 i z e d from e t h a n o l ( 9 5 % ) . 2.4.1.3 C h a r a c t e r i z a t i o n o f A n t h r a c e n e - 2 - C a r b o x y l i c A c i d a) M e l t i n g p o i n t : The m e l t i n g p o i n t o f t h e r e c r y s t a l 1 i z e d a n t h r a c e n e - 2 - c a r b o x y l i c a c i d was 278-279°C. b) S t r u c t u r e o f A n t h r a c e n e - 2 - c a r b o x y l i c A c i d : The s t r u c t u r e o f a n t h r a c e n e - 2 - c a r b o x y l i c a c i d was c o n f i r m e d by d i r e c t probe e l e c t r o n -impact m a s s - s p e c t r o m e t r y (EI-MS). The f o l l o w i n g EI-MS c o n d i t i o n s were employed: s o u r c e t e m p e r a t u r e , 240°C; probe t e m p e r a t u r e programme, 50°C f o r 1 min t o 300°C f o r 10 min a t a r a t e o f 30°C p e r min; e l e c t r o n beam v o l t a g e , 70 eV; e m i s s i o n c u r r e n t , 300 uA; and m u l t i p l i e r v o l t a g e , 2500 V. 2.4.1.4 Synthesis of 2-Anthroyl Chloride 2 - A n t h r o y l c h l o r i d e was p r e p a r e d by r e f l u x i n g 25 mg o f a n t h r a c e n e -2 - c a r b o x y l i c a c i d i n 10 ml o f p u r i f i e d anhydrous d i c h l o r o m e t h a n e w i t h 200 / i l o f o x a l y l c h l o r i d e ( f r e s h l y d i s t i l l e d ) f o r 5 hours i n a 250 ml round bottom f l a s k f i t t e d w i t h a r e f l u x c o n d e n s e r and a d r y i n g t u b e . A t the end o f t h e r e a c t i o n , t h e m i x t u r e was a l l o w e d t o c o o l and was f i l t e r e d . E x c e s s r e a g e n t and t h e r e a c t i o n s o l v e n t were removed by r o t a r y e v a p o r a t i o n under r e d u c e d p r e s s u r e . The r e s i d u e , which was g r e e n i s h -y e l l o w , was d r i e d o v e r n i g h t under vacuum (1 mm Hg). The s y n t h e t i c a c i d c h l o r i d e was p u r i f i e d by HPLC ( s e c t i o n 2.4.1.5 b e l o w ) . 2.4.1.5 Purification of 2-Anthroyl Chloride The s y n t h e t i c 2 - a n t h r o y l c h l o r i d e was p u r i f i e d by p r e p a r a t i v e HPLC. The s t a t i o n a r y phase was a Whatman C18 r e v e r s e phase column and th e m o b i l e phase was 100% a c e t o n i t r i l e pumped i s o c r a t i c a l l y a t a f l o w r a t e o f 2 ml/min. D e t e c t i o n o f t h e a c i d c h l o r i d e was a c c o m p l i s h e d w i t h a UV d e t e c t o r s e t a t 230 nm. The R t o f t h e a c i d c h l o r i d e was 12 min. 2.4.1.6 Characterization of 2-Anthroyl Chloride The s t r u c t u r e o f t h e s y n t h e t i c 2 - a n t h r o y l c h l o r i d e was c o n f i r m e d by d i r e c t probe EI-MS. The MS c o n d i t i o n s employed were t h e same as t h o s e used f o r t h e a n t h r a c e n e - 2 - c a r b o x y l i c a c i d . 2.4.2 Development o f A s s a y o f M e x i l e t i n e E n a n t i o m e r s 2.4.2.1 D e r i v a t i z a t i o n o f M e x i l e t i n e E n a n t i o m e r s w i t h 2 - A n t h r o y l C h l o r i d e The d e r i v a t i z a t i o n o f m e x i l e t i n e e n a n t i o m e r s w i t h 2 - a n t h r o y l c h l o r i d e was c a r r i e d out a c c o r d i n g t o t h e Schotten-Baumann r e a c t i o n ( V o g e l , 1964). An aqueous s o l u t i o n o f R , S - m e x i l e t i n e h y d r o c h l o r i d e (200 c o n t a i n i n g 50 ng e q u i v a l e n t o f t h e base, was t r a n s f e r r e d i n t o a 10 ml g l a s s t u b e . The s o l u t i o n was b a s i f i e d w i t h 100 /xl o f 2 M NaOH and 15 /zl o f 2 - a n t h r o y l c h l o r i d e s o l u t i o n (1 mg/ml i n d i c h l o r o m e t h a n e ) was added and mixed by v o r t e x f o r 5 min a t room t e m p e r a t u r e . The d e r i v a t i v e s formed were e x t r a c t e d i n t o 0.5 ml o f m o b i l e phase ( s e c t i o n 2.4.2.2 below) and a n a l y z e d by HPLC. The optimum r e a c t i o n t ime was e v a l u a t e d by d e r i v a t i z a t i o n o f a g i v e n amount o f R ( - ) - m e x i l e t i n e and t h e i n t e r n a l s t a n d a r d o v e r v a r i o u s t i m e i n t e r v a l s (2, 5, 7.5, 10, 15 and 30 min) and comparing t h e a b s o l u t e peak h e i g h t s . 2.4.2.2 C h r o m a t o g r a p h i c R e s o l u t i o n o f M e x i l e t i n e E n a n t i o m e r s The e n a n t i o m e r d e r i v a t i v e s were r e s o l v e d by HPLC u s i n g t h e P i r k l e ^ i o n i c (phenyl g l y c i n e ) c h i r a l column. The optimum m o b i l e phase was 2 - p r o p a n o l / e t h y l a c e t a t e / h e x a n e (6:4:90) pumped i s o c r a t i c a l l y a t a f l o w r a t e o f 1.7 ml/min. T h i s m o b i l e phase was a r r i v e d a t a f t e r an e x t e n s i v e assessment o f many o t h e r s o l v e n t c o m b i n a t i o n s . These i n c l u d e d 2-propanol/hexane, 2 - p r o p a n o l / c h l o r o f o r m / h e x a n e , e t h a n o l / h e x a n e , e t h a n o l / a c e t o n i t r i 1 e / h e x a n e , e t h a n o l / c h l o r o f o r m / h e x a n e , e t h a n o l / a c e t o n i t r i l e / e t h y l a c e t a t e / h e x a n e , e t c . Each o f t h e s e m o b i l e phases was a s s e s s e d a t d i f f e r e n t s o l v e n t r a t i o s t o p roduce d i f f e r e n t p o l a r i t i e s , as w e l l as a t d i f f e r e n t f l o w r a t e s . 2.4.2.3 S e n s i t i v i t y The d e t e c t i o n o f t h e e n a n t i o m e r d e r i v a t i v e s was a c c o m p l i s h e d w i t h a f l u o r e s c e n c e d e t e c t o r u s i n g optimum e x c i t a t i o n and e m i s s i o n w a v e l e n g h t s o f 270 and 400 nm, r e s p e c t i v e l y . These w a v e l e n g t h s were d e t e r m i n e d w i t h a f l u o r e s c e n c e s p e c t r o p h o t o m e t e r 5 1 . 2.4.2.4 S t r u c t u r e o f 2 - A n t h r o y l D e r i v a t i v e o f M e x i l e t i n e The s t r u c t u r e o f t h e 2 - a n t h r o y l d e r i v a t i v e o f m e x i l e t i n e ( m e x i l e t i n e - 2 - a n t h r a m i d e ) was c o n f i r m e d by d i r e c t probe EI-MS u s i n g t h e same c o n d i t i o n s as t h o s e employed f o r the a n t h r a c e n e - 2 - c a r b o x y l i c a c i d . 2.4.2.5 E x t r a c t i o n S o l v e n t The s o l v e n t used f o r t h e e x t r a c t i o n o f m e x i l e t i n e e n a n t i o m e r s and t h e i n t e r n a l s t a n d a r d from aqueous s o l u t i o n s and plasma was d i e t h y l e t h e r . The s u i t a b i l i t y o f t h i s s o l v e n t had been e s t a b l i s h e d i n a p r i o r s t u d y (Igwemezie, 1986). 2.4.2.6 S e l e c t i o n o f I n t e r n a l S t a n d a r d The i n t e r n a l s t a n d a r d used was KOE 2963 [ ( 2 ' , 6 ' - d i m e t h y l p h e n o x y ) -2 -aminoethane], which i s an analogue o f m e x i l e t i n e . The c h o i c e o f t h i s i n t e r n a l s t a n d a r d was based on i t s s t r u c t u r a l s i m i l a r i t y t o m e x i l e t i n e . 52 2.4.2.7 HPLC A n a l y s i s o f M e x i l e t i n e E n a n t i o m e r s An aqueous s o l u t i o n o f R , S - m e x i l e t i n e (1 ml) c o n t a i n i n g 250 ng e q u i v a l e n t o f t h e base was t r a n s f e r r e d i n t o a 10 ml P T F E - l i n e d screw-capped c u l t u r e t u b e . To t h i s tube was added 50 /xl o f an aqueous s o l u t i o n o f t h e i n t e r n a l s t a n d a r d ( c o n t a i n i n g 62 ng e q u i v a l e n t o f t h e b a s e ) . The pH was a d j u s t e d above 12 by t h e a d d i t i o n o f 0.4 ml o f 2 M NaOH and e x t r a c t e d t w i c e w i t h 5 ml p o r t i o n s o f d i e t h y l e t h e r . The o r g a n i c e x t r a c t s were combined and e v a p o r a t e d t o a p p r o x i m a t e l y 1 ml under a g e n t l e s t r e a m o f n i t r o g e n a t 37°C. The r e s u l t i n g s o l u t i o n was shaken w i t h 200 jil o f 0.1 M HC1 and t h e e t h e r l a y e r removed and d i s c a r d e d . The aqueous l a y e r was a g a i n b a s i f i e d w i t h 100 fi] o f 2 M NaOH and 15 /il o f 2 - a n t h r o y l c h l o r i d e (1 mg/ml) was added and mixed v i g o r o u s l y f o r 5 minutes a t room t e m p e r a t u r e . The 2 - a n t h r o y l d e r i v a t i v e s o f t h e e n a n t i o m e r s were e x t r a c t e d i n t o 0.5 ml o f t h e m o b i l e phase and a 20 /il a l i q u o t i n j e c t e d onto t h e HPLC column. The HPLC a n a l y s i s showed t h e p r e s e n c e o f an e x t r a n e o u s peak c o - e l u t i n g w i t h t h e S( + ) - e n a n t i o m e r . 2.4.2.8 Attempted R e s o l u t i o n / R e m o v a l o f t h e I n t e r f e r i n g Peak a) V a r i a t i o n o f M o b i l e Phase P o l a r i t y : The f i r s t a pproach t o s o l v i n g t h e i n t e r f e r i n g peak problem was t o t r y and r e s o l v e i t from t h e en a n t i o m e r peaks. Thus, e x t e n s i v e v a r i a t i o n o f t h e m o b i l e phase 51 P e r k i n Elmer, Norwalk, C o n n e c t i c u t , USA. 53 p o l a r i t y was un d e r t a k e n u s i n g d i f f e r e n t b i n a r y , t e r n a r y and q u a t e r n a r y c o m b i n a t i o n s o f hexane and 2 - p r o p a n o l , e t h y l a c e t a t e , c h l o r o f o r m , a c e t o n i t r i l e and e t h a n o l . The f l o w r a t e was a l s o v a r i e d w i t h each m o b i l e phase. b ) E x t r a c t i o n S o l v e n t : The e x t r a c t i o n s o l v e n t was changed t o hexane and d i c h l o r o m e t h a n e t o d e t e r m i n e i f t h e i n t e r f e r i n g s u b s t a n c e c o u l d be removed by s e l e c t i v e s o l u b i l i t y . c ) P u r i f i c a t i o n o f s o l v e n t s and Reagents: To r u l e out t h e p o s s i b i l i t y o f t h e s o u r c e o f t h e i n t e r f e r i n g peak b e i n g an i m p u r i t y p r e s e n t i n t h e s o l v e n t s o r r e a g e n t s , an e x t e n s i v e p u r i f i c a t i o n o f each s o l v e n t and r e a g e n t was c a r r i e d out a c c o r d i n g t o p r o t o c o l s d e s c r i b e d by P e r r i n e t a7. (1966). i ) O x a l y l c h l o r i d e : by d i s t i l l a t i o n i i ) D i e t h y l e t h e r : by s e q u e n t i a l washing w i t h s t r o n g l y a l k a l i n e p o t a s s i u m permanganate and c o n c e n t r a t e d s u l p h u r i c a c i d , f o l l o w e d by d r y i n g o v e r sodium w i r e and d i s t i l l a t i o n . i i i ) Hexane: by e l u s i o n t h r o u g h a c t i v a t e d a l u m i n a , f o l l o w e d by d i s t i l l a t i o n . i v ) D i c h l o r o m e t h a n e : by t r e a t m e n t w i t h c o n c e n t r a t e d s u l p h u r i c a c i d and sodium h y d r o x i d e , s e q u e n t i a l l y , f o l l o w e d by d r y i n g w i t h c a l c i u m c h l o r i d e and d i s t i l l a t i o n . v) A n t h r a c e n e - 2 - c a r b o x y l i c a c i d : by HPLC ( s e c t i o n 2.4.2.2.). v i ) 2 - A n t h r o y l c h l o r i d e : by HPLC ( s e c t i o n 2.4.2.5.). d) P i r k l e R ( S ) - L e u c i n e C h i r a l Column: The s t a t i o n a r y phase used f o r t h e HPLC a n a l y s i s was changed t o t h e P i r k l e R ( S ) - l e u c i n e c h i r a l column. 2.5 A s s a y o f M e x i l e t i n e Enantiomers u s i n g 2-Naphthoyl C h l o r i d e as a D e r i v a t i z a t i o n Reagent The a s s a y o f m e x i l e t i n e e n a n t i o m e r s u s i n g 2-naphthoyl c h l o r i d e as a d e r i v a t i z a t i o n r e a g e n t has been p r e v i o u s l y d e s c r i b e d (Igwemezie, 1986; M c E r l a n e et al., 1987). 2.5.1 E x t r a c t i o n , D e r i v a t i z a t i o n and HPLC A n a l y s i s An a l i q u o t o f b i o l o g i c a l f l u i d o r t i s s u e homogenate was t r a n s f e r r e d i n t o a 10 ml P T F E - l i n e d screw-capped c u l t u r e t u b e . To t h i s tube was added m e x i l e t i n e and t h e i n t e r n a l s t a n d a r d . The plasma p r o t e i n s were p r e c i p i t a t e d w i t h 1 ml o f barium h y d r o x i d e (0.15 M) and 1 ml o f z i n c s u l f a t e (0.15 M) s o l u t i o n s . The pH o f a l l b i o l o g i c a l samples was a d j u s t e d above 12 by t h e a d d i t i o n o f 0.4 ml o f 2 M NaOH and e x t r a c t e d t w i c e w i t h 5 ml p o r t i o n s o f d i e t h y l e t h e r . The o r g a n i c e x t r a c t s were combined and e v a p o r a t e d i n a water bath a t 37°C under a g e n t l e s t r e a m o f n i t r o g e n t o a volume o f a p p r o x i m a t e l y 1 ml. The r e s u l t i n g s o l u t i o n was a c i d i f i e d w i t h 0.2 ml o f 0.1 M HC1, shaken, and th e e t h e r l a y e r was removed and d i s c a r d e d . The aqueous l a y e r was a g a i n b a s i f i e d w i t h 0.2 ml o f 2 M NaOH and 15 /il o f t h e 2-naphthoyl c h l o r i d e s o l u t i o n was added and mixed v i g o r o u s l y on a v o r t e x m i x e r f o r 2 min. The d e r i v a t i v e s formed were e x t r a c t e d i n t o 0.5 ml o f t h e m o b i l e phase used f o r t h e HPLC a n a l y s i s and a 20 /*L a l i q u o t was i n j e c t e d o n t o t h e HPLC column. The HPLC c o n d i t i o n s were : s t a t i o n a r y phase, P i r k l e R i o n i c (phenyl g l y c i n e ) c h i r a l phase; m o b i l e phase, c h l o r o f o r m / 2 - p r o p a n o l / H e x a n e ( 7 : 7 : 8 6 ) ; f l o w r a t e , 1.2 ml/min; and d e t e c t i o n , f l u o r e s c e n c e a t 230 nm (ex) and 370 nm (em). 2.5.2 C a l i b r a t i o n C u r v e s and A s s a y P r e c i s i o n i n Human Plasma To f i v e 1 ml a l i q u o t s o f b l a n k plasma were added 20, 40, 100, 400 and 1000 ng ( e q u i v a l e n t o f the base) o f r a c e m i c m e x i l e t i n e a l o n g w i t h 50 ng o f KOE 2963 ( i n t e r n a l s t a n d a r d ) . The samples were s u b s e q u e n t l y t r e a t e d as d e s c r i b e d under " E x t r a c t i o n , D e r i v a t i z a t i o n and HPLC A n a l y s i s " ( s e c t i o n 2.5.1). The c a l i b r a t i o n c u r v e s were c o n s t r u c t e d by p l o t t i n g t h e peak h e i g h t r a t i o s o f each e n a n t i o m e r t o t h a t o f t h e i n t e r n a l s t a n d a r d a g a i n s t t h e known c o n c e n t r a t i o n o f t h e e n a n t i o m e r . I n t e r - a s s a y v a r i a b i l i t y was d e t e r m i n e d by t r i p l i c a t e p r e p a r a t i o n and a n a l y s i s o f each o f t h e samples used f o r the s t a n d a r d c u r v e . The s t a n d a r d c u r v e s f o r m e x i l e t i n e e n a n t i o m e r s i n r a t plasma, serum and t i s s u e homogenates were p r e p a r e d i n a s i m i l a r manner. 2.6 In Vitro Serum P r o t e i n B i n d i n g o f M e x i l e t i n e E n a n t i o m e r s i n Humans 2.6.1 Serum C o l l e c t i o n S i x h e a l t h y s u b j e c t s were used f o r t h i s s t u d y . The b l o o d c h e m i s t r y o f t h e s u b j e c t s were a s s e s s e d p r i o r t o sample c o l l e c t i o n . A p p r o x i m a t e l y 100 ml o f venous b l o o d was c o l l e c t e d from each s u b j e c t u s i n g 10 ml g l a s s s y r i n g e s . B l o o d was i m m e d i a t e l y t r a n s f e r r e d i n t o 10 ml g l a s s t u b e s w i t h PTFE l i n e d c a p s , a l l o w e d t o c l o t a t room t e m p e r a t u r e f o r 2 h, c e n t r i f u g e d a t 2500 x g f o r 15 min and t h e serum s e p a r a t e d . 2.6.2 Serum pH A d j u s t m e n t A d j u s t m e n t o f serum pH t o -7.4 was n e c e s s a r y t o e n s u r e t h a t the b i n d i n g e x p e r i m e n t s were done under p h y s i o l o g i c a l c o n d i t i o n s . The f o l l o w i n g a p proaches were used: a) b u b b l i n g 5% CO2/O2 i n t o serum b) b u b b l i n g 100% C 0 2 i n t o serum c) d i s s o l v i n g phosphate b u f f e r s a l t s i n serum. The c o n c e n t r a t i o n o f t h e s a l t s used were 5 . 0 2 mg monobasic sodium phosphate monohydrate and 32.55 mg d i b a s i c sodium phosphate h e p t a h y d r a t e p e r 1 ml o f serum ( 0 . 1 2 M ) . The l a s t a pproach was found t o be t h e most e f f e c t i v e i n m a i n t a i n i n g t h e serum pH a t p h y s i o l o g i c a l v a l u e s and was s u b s e q u e n t l y used f o r t h e b i n d i n g e x p e r i m e n t s . 2.6.3 Purified Human Serum Protein Solutions S o l u t i o n s o f p u r i f i e d human serum p r o t e i n s were p r e p a r e d by d i s s o l v i n g t h e p r o t e i n s i n phosphate b u f f e r (pH = 7.4) t o y i e l d c o n c e n t r a t i o n s o f 4% and 0 . 1 % w/v f o r human serum albumin (HSA) and a j - a c i d g l y c o p r o t e i n (AAG), r e s p e c t i v e l y . The c o n t r i b u t i o n o f serum l i p o p r o t e i n s t o t h e b i n d i n g o f m e x i l e t i n e e n a n t i o m e r s was a s s e s s e d u s i n g l i p o p r o t e i n d e f i c i e n t human serum. 2.6.4 Sample Preparation To 4 ml a l i q u o t s o f serum was added r a c e m i c m e x i l e t i n e i n phosphate b u f f e r (50 u l ) t o y i e l d a s e r i e s o f c o n c e n t r a t i o n s ; 0 . 2 , 0.5, 1 . 0 , 2 . 0 , 1 0 , 40, 1 0 0 , 2 0 0 , 1 0 0 0 , 2 0 0 0 , 3000, and 4000 j a g/ml. The serum was mixed on a r o t a t o r f o r 10 m i n u t e s . D u p l i c a t e 1 ml samples were used t o d e t e r m i n e t h e t o t a l d r u g c o n c e n t r a t i o n s w h i l e a second s e t o f d u p l i c a t e 1 ml samples were s u b j e c t e d t o u l t r a f i l t r a t i o n t o d e t e r m i n e f r e e d r u g c o n c e n t r a t i o n s . The b i n d i n g s t u d i e s were r e p l i c a t e d w i t h phosphate b u f f e r s o l u t i o n s (pH = 7.4) o f i s o l a t e d HSA and AAG. 2.6.5 U l t r a f i l t r a t i o n U l t r a f i l t r a t i o n was c a r r i e d out w i t h t h e MPS-1 m i c r o p a r t i t i o n s y s t e m 5 2 and a 30,000 D a l t o n m o l e c u l a r w e i g h t c u t - o f f YMT u l t r a f i l t r a t i o n membrane 5 3. The c e n t r i f u g e (Beckman Model J 2 - 2 1 ) 5 4 had a 35° a n g l e head r o t o r (Beckman model J A - 1 7 ) 5 5 which was e q u i l i b r a t e d t o 37°C b e f o r e i n t r o d u c t i o n o f the samples. C e n t r i f u g a t i o n t i m e was 15 min a t a r e l a t i v e c e n t r i f u g a l f o r c e o f 1650 x g. The c e n t r i f u g a t i o n p a r a m e t e r s were t h o s e recommended by t h e m a n u f a c t u r e r o f t h e u l t r a f i l t r a t i o n system. 2.6.6 A n a l y s i s o f F r e e and T o t a l M e x i l e t i n e E n a n t i o m e r s i n Serum and P r o t e i n S o l u t i o n s . The c o n c e n t r a t i o n s o f m e x i l e t i n e e n a n t i o m e r s i n serum and p r o t e i n s o l u t i o n s , and t h e i r r e s p e c t i v e u l t r a f i U r a t e s ( f r e e e n a n t i o m e r c o n c e n t r a t i o n s ) were d e t e r m i n e d u s i n g the HPLC method d e s c r i b e d i n s e c t i o n 2.5. 2.6.7 A n a l y s i s o f B i n d i n g Data The f r e e f r a c t i o n , e x p r e s s e d as a p e r c e n t a g e , was t h e r a t i o o f t h e f r e e t o t h e t o t a l e n a n t i o m e r c o n c e n t r a t i o n . I n i t i a l e s t i m a t e s o f t h e e q u i l i b r i u m a s s o c i a t i o n ( a f f i n i t y ) c o n s t a n t (K) and t h e c a p a c i t y c o n s t a n t (N) were d e t e r m i n e d g r a p h i c a l l y f o r t h e e n a n t i o m e r s by f i t t i n g 52-53 Amicon Canada L t d . , O a k v i l l e , O n t a r i o , Canada. 54-55 Beckman I n s t r . I n c . , F u l l e r t o n , C a l i f o r n i a , USA t h e e x p e r i m e n t a l d a t a t o t h e " R o s e n t h a l " e q u a t i o n ( R o s e n t h a l , 1967): N1K1 NoKo B / F = + _ _ - t _ t _ _ . 1 + K j F 1 + K 2 F (where B and F a r e t h e molar c o n c e n t r a t i o n s o f bound and f r e e d r u g , r e s p e c t i v e l y . N i s a l s o t h e p r o d u c t o f t h e number o f b i n d i n g s i t e s p e r mole o f p r o t e i n (n) and the molar c o n c e n t r a t i o n o f t h e p r o t e i n ( s ) ) . Thus, from t h e b i n d i n g d a t a o b t a i n e d from t h e i s o l a t e d p r o t e i n s , n was d e t e r m i n e d . The i n i t i a l e s t i m a t e s o f t h e c a p a c i t y and a f f i n i t y c o n s t a n t s were used by t h e n o n - l i n e a r l e a s t s q u a r e s program, ENZFITTER ( L e a t h e r b a r r o w , 1987), t o y i e l d t h e f i n a l v a l u e s o f t h e s e p a r a m e t e r s . 2.6.8 S t a t i s t i c a l Data A n a l y s i s The S t u d e n t ' s t - t e s t ( p a i r e d and ind e p e n d e n t g r o u p s ) and one way ANOVA (a = 0.05) were used. 2.7 T i s s u e D i s t r i b u t i o n K i n e t i c s o f M e x i l e t i n e E n a n t i o m e r s i n R ats 2.7.1 Drug A d m i n i s t r a t i o n and Sample C o l l e c t i o n Male Sprague-Dawley r a t s (weight = 150-200 g) were a d m i n i s t e r e d 10 mg/kg o f r a c e m i c m e x i l e t i n e t h r o u g h a t a i l v e i n u s i n g a b u t t e r f l y c a n n u l a (30 gauge) w h i l e each r a t was t e m p o r a r i l y r e s t r a i n e d i n a Perspex r e s t r a i n e r . Drugs were i n j e c t e d o v e r a 1 min p e r i o d . Groups o f an i m a l s (4-5 r a t s p e r group) were s a c r i f i c e d by d e c a p i t a t i o n a t 0.08, 59 0.25, 0.5, 1, 2, 4 and 6 hours f o l l o w i n g drug a d m i n i s t r a t i o n and t h e b r a i n , h e a r t , l u n g , l i v e r , k i d n e y and e p i d i d y m a l f a t t i s s u e s were r a p i d l y e x c i s e d . The organs were c u t i n t o s m a l l p i e c e s , b l o t t e d w i t h f i l t e r p a p e r , weighed and homogenized i n i c e c o l d 0.1 M HC1. B l o o d was a l s o c o l l e c t e d a t t h e same time as t h e t i s s u e s and serum was o b t a i n e d f o r f r e e and t o t a l c o n c e n t r a t i o n measurements. The serum and t i s s u e homogenates were s t o r e d a t -20°C u n t i l r e q u i r e d f o r a n a l y s i s . 2.7.2 A n a l y s i s o f Serum and T i s s u e Samples The c o n c e n t r a t i o n s o f t h e e n a n t i o m e r s o f m e x i l e t i n e i n both serum and t h e t i s s u e homogenates were d e t e r m i n e d u s i n g t h e HPLC a s s a y d e s c r i b e d i n s e c t i o n 2.5. 2.7.3 E f f i c i e n c y o f R e c o v e r y o f M e x i l e t i n e E n a n t i o m e r s from T i s s u e Homogenates The e f f i c i e n c y o f r e c o v e r y o f t h e e n a n t i o m e r s from t i s s u e homogenates was d e t e r m i n e d by t h e a d d i t i o n o f 500 and 2000 ng o f R,S-m e x i l e t i n e t o two 1 ml a l i q u o t s o f t h e homogenates ( c o n t a i n i n g 100 mg o f t i s s u e ) . The samples were e x t r a c t e d , d e r i v a t i z e d and a s s a y e d as e a r l i e r d e s c r i b e d e x c e p t t h a t t h e i n t e r n a l s t a n d a r d was added j u s t p r i o r t o t h e d e r i v a t i z a t i o n s t e p . The r e s u l t i n g peak h e i g h t r a t i o s were e x p r e s s e d as a p e r c e n t a g e o f t h o s e o b t a i n e d w i t h i d e n t i c a l amounts o f R , S - m e x i l e t i n e and t h e i n t e r n a l s t a n d a r d which were d e r i v a t i z e d d i r e c t l y w i t h o u t p r i o r e x t r a c t i o n . 2.7.4 Serum P r o t e i n B i n d i n g The in vivo f r e e f r a c t i o n s o f t h e e n a n t i o m e r s were d e t e r m i n e d by u l t r a f i l t r a t i o n t e c h n i q u e as d e s c r i b e d i n t h e serum p r o t e i n b i n d i n g section (2.6). 2.7.5 P h a r m a c o k i n e t i c Data A n a l y s i s The serum and tissue concentration time data were best described by a bi-exponential function of the form C = A e _ Q * + Be~^ (a > 0) where C is the serum or tissue concentration at time, t, A and B are constants, a and /} are the f i r s t order distr ibution and elimination rate constants, respectively. The in i t i a l estimates of the rate constants were obtained using the computer program, AUTOAN (Sedman and Wagner, 1976). These in i t i a l estimates were used by the i terat ive non-linear regression program, NONLIN (Metzler, 1974) to obtain the f inal values. The remaining pharmacokinetic parameters were obtained using formulae reported in Gibaldi and Perrier (1982). The distr ibution h a l f - l i f e L t l / 2 (a ) ] w a s determined from the formula: t\/2(a) = 0-693/a, while the terminal elimination h a l f - l i f e [tiy 2(/})] w a s calculated from the formula: tiy2(/j) = 0.693//J. The area under the serum concentration-time curve (AUC) was determined from the trapezoidal rule with extrapolation to in f in i ty from the last data point. The systemic clearance (CL) was calculated from the formula: CL = dose/AUC. The steady-state volume of distr ibution (V s s ) was determined from the formula: V s s = dose.AUMC/AUC2 where AUMC is the total area under the f i r s t moment curve (Benet and Galeazzi, 1979). 2.7.6 S t a t i s t i c a l Data A n a l y s i s The Student's paired t - test (a=0.05) was used to assess the s ta t is t ica l significance of differences in the serum and tissue concentrations obtained for the enantiomers. 61 2.8 A n t i a r r h y t h m i c A c t i v i t y o f Racemic M e x i l e t i n e and i t s E n a n t i o m e r s The a n t i a r r h y t h m i c e f f e c t s o f r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s were a s s e s s e d u s i n g 2 models. These were e l e c t r i c a l - i n d u c e d a r r h y t h m i a s i n p e n t o b a r b i t o n e a n a e s t h e t i z e d r a t s and i s c h a e m i a - i n d u c e d a r r h y t h m i a s i n both c o n s c i o u s and p e n t o b a r b i t o n e a n a e s t h e t i z e d r a t s . A l l t h e e x p e r i m e n t s were c a r r i e d out b l i n d and randomized. 2.8.1 E l e c t r i c a l l y - I n d u c e d A r r h y t h m i a 2.8.1.1 P r e p a r a t i o n o f Rats Male Sprague-Dawley r a t s (wt = 250-350 g) were a n a e s t h e t i z e d w i t h p e n t o b a r b i t o n e sodium (45 mg/kg i . p . ) . The l e f t c a r o t i d a r t e r y was c a n n u l a t e d f o r b l o o d p r e s s u r e r e c o r d i n g and sample a c q u i s i t i o n , w h i l e a second c a n n u l a e was p l a c e d i n t h e r i g h t j u g u l a r v e i n f o r dru g a d m i n i s t r a t i o n . ECG l e a d s ( l e a d I I ) were i m p l a n t e d s u b c u t a n e o u s l y . A l e f t t h o r a c o t o m y was per f o r m e d on t h e r a t s between t h e 5th and 6th i n t e r c o s t a l r i b s and t h e h e a r t exposed. A P a i r o f t e f l o n c o a t e d s i l v e r w i r e e l e c t r o d e s were p l a c e d 0.3 cm a p a r t i n t h e l e f t v e n t r i c u l a r w a l l . S t i m u l a t i o n o f t h e l e f t v e n t r i c l e w i t h s q u a r e wave p u l s e s was a c c o m p l i s h e d u s i n g a G r a s s E l e c t r i c S t i m u l a t o r . 2.8.1.2 E x p e r i m e n t a l E n d - p o i n t s Permanent r e c o r d s o f t h e ECG and b l o o d p r e s s u r e were o b t a i n e d u s i n g a G r a s s P o l y g r a p h . The ECG was a l s o c o n t i n u o u s l y m o n i t o r e d t h r o u g h o u t t h e e x p e r i m e n t w i t h a d e l a y e d l o o p o s c i l l o s c o p e (Honeywell Type E f o r M). D i s c r i m i n a t i o n o f t h e e n d - p o i n t s was c a r r i e d out u s i n g t h e o s c i l l o s c o p e . 2.8.1.3 V a r i a b l e s R e l a t e d t o A n t i a r r h y t h m i c E f f e c t s The v a r i a b l e s were: C u r r e n t T h r e s h o l d f o r C a p t u r e , C u r r e n t T h r e s h o l d P u l s e Width, Maximum F o l l o w i n g Frequency, E f f e c t i v e R e f r a c t o r y P e r i o d and V e n t r i c u l a r F i b r i l l a t i o n T h r e s h o l d . Each v a r i a b l e was measured 3 t i m e s and a mean v a l u e o b t a i n e d . The p r o c e d u r e f o r t h e measurements has been d e s c r i b e d ( C u r t i s et al., 1984 & 1986; E u l e r and S c a n l o n , 1988). a) C u r r e n t T h r e s h o l d f o r C a p t u r e : C u r r e n t t h r e s h o l d f o r c a p t u r e was d e t e r m i n e d a t 1 ms and 7.5 Hz. I t i s t h e minimum c u r r e n t r e q u i r e d t o c a p t u r e t h e h e a r t . b) T h r e s h o l d P u l s e w i d t h : The t h r e s h o l d p u l s e w i d t h f o r c a p t u r e was d e t e r m i n e d a t 7.5 Hz and t w i c e t h e c u r r e n t t h r e s h o l d . c ) E f f e c t i v e R e f r a c t o r y P e r i o d (ERP): ERP was d e t e r m i n e d by t h e e x t r a - s t i m u l u s method. The h e a r t was paced a t 7.5 Hz and a s i n g l e e x t r a s t i m u l u s was a p p l i e d a t v a r y i n g i n t e r v a l s b e h i n d t h e p a c i n g s t i m u l i . The s h o r t e s t i n t e r v a l between t h e p a c i n g s t i m u l i and t h e e x t r a - s t i m u l u s i n which an e x t r a - s y s t o l e was o b t a i n e d was t a k e n as t h e ERP. d) Maximum F o l l o w i n g F r e q u e n c y (MFF): MFF was d e t e r m i n e d a t t w i c e t h e c u r r e n t and p u l s e w i d t h t h r e s h o l d s . MFF was t a k e n as t h a t p o i n t when t h e h e a r t f a i l e d t o f o l l o w , on a 1:1 b a s i s , a s t e a d i l y i n c r e a s i n g f r e q u e n c y o f s t i m u l a t i o n from 7 t o 20 Hz. T h i s was r e a d i l y seen as a sudden i n c r e a s e i n b l o o d p r e s s u r e a f t e r an i n i t i a l s u s t a i n e d d r o p . e) V e n t r i c u l a r F i b r i l l a t i o n T h r e s h o l d ( V F T ) : VFT was d e t e r m i n e d a t 60 Hz and t w i c e p u l s e w i d t h t h r e s h o l d . The h i g h f r e q u e n c y was used t o e n s u r e t h a t a p u l s e was d e l i v e r e d d u r i n g t h e v u l n e r a b l e p e r i o d , i . e . 63 t h e t e r m i n a l p o r t i o n o f t h e QT i n t e r v a l i n t h e ECG. The maximum c u r r e n t which e l i c i t e d s u s t a i n e d f i b r i l l a t i o n w i t h a p r e c i p i t o u s f a l l i n b l o o d p r e s s u r e was t a k e n as t h e f i b r i l l a t i o n t h r e s h o l d . 2.8.1 .4 O t h e r V a r i a b l e s The o t h e r v a r i a b l e s measured i n c l u d e d h e a r t r a t e , b l o o d p r e s s u r e and t h e ECG para m e t e r s (PR, QRS and QT C i n t e r v a l s ) 2.8.1.5 Drug A d m i n i s t r a t i o n The r a t s were d i v i d e d i n t o 4 t r e a t m e n t groups w i t h 8 r a t s p e r group. Each group r e c e i v e d o n l y one o f t h e t h r e e forms o f m e x i l e t i n e (R,S-, S(+)- and R ( - ) - m e x i l e t i n e ) d i s s o l v e d i n s a l i n e , o r s a l i n e as a c o n t r o l . A f t e r e s t a b l i s h i n g a s t a b l e base l i n e v a l u e f o r t h e measured v a r i a b l e s , a 4 mg/kg dose was g i v e n i n t r a v e n o u s l y o v e r 1 min. B l o o d (0.5 ml) was withdrawn i n t o p l a s t i c v i a l s c o n t a i n i n g a drop o f h e p a r i n (50 IU/ml) 10 min p o s t - d o s e f o r d e t e r m i n a t i o n o f dru g c o n c e n t r a t i o n s . ERP, MFF and VFT were d e t e r m i n e d between 10 and 15 min and a f a s t ECG t r a c e (150 mm/min) was o b t a i n e d f o r t h e d e t e r m i n a t i o n o f h e a r t r a t e and th e ECG p a r a m e t e r s . The subsequent dose was then a d m i n i s t e r e d a t 20 min. Dosages were a s s i g n e d t o produce a c u m u l a t i v e d o u b l i n g o f t h e p r e v i o u s dose i . e . 4, 8, 16 and 32 mg/kg. A f t e r t h e f i n a l measurements were t a k e n , b l o o d (3 ml) was c o l l e c t e d from t h e r a t s f o r d e t e r m i n a t i o n o f t h e serum p r o t e i n b i n d i n g o f r a c e m i c m e x i l e t i n e and t h e i n d i v i d u a l e n a n t i o m e r s . 2.8.1.6 A n a l y s i s o f Plasma Samples The b l o o d c o l l e c t e d from t h e r a t s was c e n t r i f u g e d f o r 5 min and t h e plasma s e p a r a t e d and s t o r e d a t -20°C u n t i l r e q u i r e d f o r a n a l y s i s . The c o n c e n t r a t i o n s o f t h e e n a n t i o m e r s o f m e x i l e t i n e were d e t e r m i n e d u s i n g t h e s t e r e o s e l e c t i v e HPLC a s s a y d e s c r i b e d i n s e c t i o n 2.5. For the r a t s g i v e n r a c e m i c m e x i l e t i n e , t h e i n d i v i d u a l e n a n t i o m e r s were measured and added t o o b t a i n e d t h e c o n c e n t r a t i o n o f t h e racemate. The serum b i n d i n g o f t h e e n a n t i o m e r s was d e t e r m i n e d by u l t r a f i l t r a t i o n as d e s c r i b e d i n s e c t i o n 2.6. 2.8.1.7 S t a t i s t i c a l A n a l y s i s D i f f e r e n c e s i n t h e measured v a r i a b l e s between t h e groups were a s s e s s e d u s i n g "Repeated Measures" ANOVA (a=0.05). The changes from p r e - d r u g v a l u e s were a n a l y z e d as a b s o l u t e v a l u e s ( r a t h e r than as p e r c e n t c h a n g e ) . The in vivo f r e e f r a c t i o n s o f r a c e m i c m e x i l e t i n e and t h e en a n t i o m e r s were compared by one-way ANOVA. 2.8.2 C o r o n a r y A r t e r y O c c l u s i o n i n C o n s c i o u s Rats C o r o n a r y a r t e r y o c c l u s i o n has been used by many i n v e s t i g a t o r s f o r th e assessment o f t h e a n t i a r r h y t h m i c e f f i c a c y o f s e v e r a l d r u g s ( S e l y e e t al., 1960; C l a r k e t al., 1980; Kane e t al., 1980; J o h n s t o n e t al., 1983; C u r t i s e t al., 1984). The p r e s e n t s t u d y was c a r r i e d out i n c o n s c i o u s r a t s a c c o r d i n g t o t h e method d e s c r i b e d by J o h n s t o n e t al. (1983) and C u r t i s (1986). 2.8.2.1 P r e p a r a t i o n o f Rats The e x p e r i m e n t s were c a r r i e d out u s i n g male Sprague-Dawley r a t s (wt = 350-450 g ) . A l l t h e l i n e s , l e a d s and o c c l u d e r were s t e r i l i z e d i n 70% e t h a n o l i n d i s t i l l e d water. A n a e s t h e s i a was i n d u c e d i n t h e r a t s i n 65 a g l a s s j a r w i t h 5% ha l o t h a n e / o x y g e n d e l i v e r e d v i a a v a p o u r i z e r . The r a t s were s u b s e q u e n t l y i n t u b a t e d ( f o r a r t i f i c i a l r e s p i r a t i o n ) w i t h a 14 gauge human i n t r a v e n o u s c a t h e t e r w i t h t h e a i d o f a p a e d i a t r i c l a r y n g o s c o p e and m a i n t a i n e d on 1% h a l o t h a n e t h r o u g h o u t t h e r e s t o f t h e s u r g e r y . 2.8.2.2 P r e p a r a t i o n o f O c c l u d e r The o c c l u d e r was made up o f an 11 cm l e n g t h o f PE-10 ( p o l y e t h y l e n e ) t u b i n g . One end o f t h e t u b i n g was f l a r e d by b r i e f e x p o s u r e t o h e a t . About 1 cm from t h e o t h e r end, a f l a n g e was made by b r i e f l y m e l t i n g t h e tube ( w i t h a w i r e i n s e r t e d t o p r e v e n t b l o c k a g e o f th e lumen) by r o t a t i n g i t i n f r o n t o f a j e t o f hot a i r and th e n p r e s s i n g i t t o g e t h e r ( t h e j e t o f hot a i r was c r e a t e d by p a s s i n g p r e s s u r i z e d a i r t h r o u g h a t h i n c o p p e r tube h e l d o v e r a Bunsen b u r n e r ) . The t u b i n g was used as a g u i d e f o r a 5.0 gauge p r o l e n e s u t u r e t h r e a d e d t h r o u g h w i t h t h e n e e d l e end o f t h e s u t u r e a t t h e f l a r e d end o f the g u i d e . 2.8.2.3 P r e p a r a t i o n o f L i n e s A 14 cm l e n g t h o f PE-50 t u b i n g was welded o n t o a 10 cm l e n g t h o f PE-10 t u b i n g by m e l t i n g t h e i r t i p s u s i n g t h e j e t o f hot a i r d e s c r i b e d above and th e n p r e s s i n g them t o g e t h e r . A l e n g t h o f t h i n w i r e was pa s s e d t h r o u g h t h e t u b i n g s p r i o r t o t h e w e l d i n g p r o c e s s t o p r e v e n t t h e lumen o f th e t u b e s from g e t t i n g b l o c k e d . The PE-10 end o f t h e l i n e was shaped i n t o a c o i l by l o o p i n g i t around a g l a s s r o d and submerging i t i n b o i l i n g w a ter f o r 3 seconds. The c o i l was f i x e d by submerging i t i n i c e - c o l d w a t e r . 2.8.2.4 I m p l a n t a t i o n o f L i n e s F o l l o w i n g m i d l i n e l a p a r o t o m y , t h e i n f e r i o r vena c a v a and t h e abdominal a o r t a were c a n n u l a t e d f o r d r u g a d m i n i s t r a t i o n and b l o o d p r e s s u r e r e c o r d i n g , r e s p e c t i v e l y . The PE-10 ends o f t h e l i n e s were p l a c e d p r o x i m a l t o t h e b l o o d v e s s e l s and t h e s m a l l d i a m e t e r e n s u r e d t h a t t h e a o r t a and vena cava were not b l o c k e d . These v e s s e l s were used because t h e y a r e l a r g e , e a s i l y a c c e s s i b l e v e s s e l s s u i t a b l e f o r c a n n u l a t i o n w i t h c h r o n i c i n d w e l l i n g n o n - o c c l u d i n g l i n e s . The l i n e s were r o u t e d s u b c u t a n e o u s l y and e x t e r i o r i z e d between t h e s h o u l d e r b l a d e s o f t h e animal w i t h t h e a i d o f a t r o c a r . The abdomen was d u s t e d w i t h C i c a t r i n a n t i b i o t i c powder and t h e body w a l l c l o s e d w i t h r u n n i n g s t i t c h e s . The s k i n was s u b s e q u e n t l y c l o s e d w i t h i n t e r r u p t e d s t i t c h e s and t h e wound was i n f i l t r a t e d w i t h C i c a t r i n and M a r c a i n e . A p p r o x i m a t e l y 0.3 ml o f s a l i n e was i n j e c t e d i n t o t h e vena c a v a t h r o u g h t h e e x t e r i o r i z e d end o f the c a n n u l a and t h e l i n e a b r u p t l y clamped w i t h a p a i r o f a t r a u m a t i c f o r c e p s . The open end o f t h e c a n n u l a was t h e n s e a l e d w i t h t h e a i d o f a l i g h t e r f l ame and t h e f o r c e p s were r e l e a s e d . T h i s t r e a t m e n t kept t h e l i n e p a t e n t . The a o r t i c l i n e was t r e a t e d i n a s i m i l a r f a s h i o n . 2.8.2.5 I m p l a n t a t i o n o f t h e O c c l u d e r A l e f t t horacotomy was performed by b l u n t d i s s e c t i o n t h r o u g h t h e 4th i n t e r c o s t a l space w h i l e r a t s were r e s p i r e d a r t i f i c i a l l y ( s t r o k e volume = 10 ml/kg a t a s t r o k e r a t e o f 60/min). R e t r a c t o r s were used t o widen t h e i n c i s i o n and t h e h e a r t exposed. The p r o l e n e s u t u r e o f t h e o c c l u d e r was p l a c e d around t h e l e f t a n t e r i o r d e s c e n d i n g c o r o n a r y a r t e r y such t h a t t h e s u t u r e emerging from t h e g u i d e t u b i n g p a s s e d under t h e a r t e r y and back t h r o u g h t h e t i p o f t h e g u i d e t u b i n g making a l o o s e s n a r e . The n e e d l e end o f t h e s u t u r e was c u t o f f and t h e r e m a i n i n g l e n g t h c a r e f u l l y m e l t e d down t o form a b a l l , t h u s , p r e v e n t i n g t h e s u t u r e from b e i n g p u l l e d back t h r o u g h t h e f l a r e d t i p o f t h e g u i d e t u b i n g . The p r o l e n e s u t u r e extended t h e f u l l l e n g t h o f t h e g u i d e t u b i n g . The s i z e o f t h e s n a r e was a d j u s t e d such t h a t t h e f l a r e d end o f t h e g u i d e t u b i n g was p o s i t i o n e d a d j a c e n t t o t h e a t r i a l appendage. The d i s t a l end o f t h e s u t u r e was t h e n m e l t e d down t o a sma l l b a l l . The pectoral is muscle was s u t u r e d l i g h t l y t o t h e rectus abdominus muscle w i t h s i l k f o r m i n g a p u r s e s t r i n g s u t u r e around t h e o c c l u d e r . A t t h e time t h e c h e s t was c l o s e d , t h e pneumothorax was e v a c u a t e d by a p p l y i n g n e g a t i v e p r e s s u r e t h r o u g h a l e n g t h o f PE-90 t u b i n g . The o c c l u d e r was r o u t e d s u b c u t a n e o u s l y t o t h e s u b s c a p u l a r r e g i o n u s i n g a t r o c a r and e x t e r i o r i z e d between t h e s h o u l d e r b l a d e s . The c h e s t wound was i n f i l t r a t e d w i t h C i c a t r i n R and M a r c a i n e R and t h e s k i n c l o s e d w i t h i n t e r r u p t e d s u t u r e . 2.8.2.6 Implantation of the ECG Leads The ECG l e a d s were p r e p a r e d from t e f l o n c o a t e d s t a i n l e s s s t e e l w i r e . The i n s u l a t i o n was removed from a p p r o x i m a t e l y 1 cm o f one end o f each l e a d w i t h t h e a i d o f a l i g h t e r f l a m e . A t i g h t c o i l was made i n one end o f one l e a d by wrapping i t around a 21 gauge hypodermic n e e d l e . T h i s end was t h e n t i e d t h r o u g h t h e p e c t o r a l i s muscle ( c h e s t l e a d ) and e x t e r i o r i z e d w i t h t h e o c c l u d e r . Limb l e a d s were p l a c e d s u b c u t a n e o u s l y i n t h e 2 f o r e l i m b s and t h e l e f t h i n d l i m b . The ECG measured was a p p r o x i m a t e l y V3. A f t e r i m p l a n t i n g a l l t h e l e a d s , l i n e s and o c c l u d e r , t h e s u b s c a p u l a r wound was i n f i l t r a t e d w i t h C i c a t r i n ^ and M a r c a i n e ^ and t h e r e s p i r a t o r y pump d i s c o n n e c t e d . The l e a d s , l i n e s and o c c l u d e r were e x t e r i o r i z e d no more than 1 cm b e h i n d t h e s h o u l d e r o f t h e a n i m a l . The a n i m a l s were a l l o w e d t o r e c o v e r i n i n d i v i d u a l c a g e s . The i n c i d e n c e o f i n f e c t i o n o r m o r t a l i t y f o l l o w i n g s u r g e r y was l e s s than 1%. 2.8.2.7 C o r o n a r y A r t e r y O c c l u s i o n Rats were a l l o w e d t o r e c o v e r f o r one week. On t h e day o f t h e o c c l u s i o n e x p e r i m e n t , the r a t s were weighed and t h e i r w e i g h t s r e c o r d e d . The l i n e s and t h e ECG l e a d s were c o n n e c t e d and s t a b l e b l o o d p r e s s u r e r e c o r d i n g and ECG were o b t a i n e d f o r a t l e a s t 15 min. Drug was then i n f u s e d o v e r a 10 min p e r i o d and t h e c o r o n a r y a r t e r y o c c l u d e d 5 min a f t e r the end o f d r u g a d m i n i s t r a t i o n . To o c c l u d e t h e c o r o n a r y a r t e r y , t h e o c c l u d e r g u i d e was h e l d by t h e Spenser W e l l s f o r c e p s w i t h t h e a t r a u m a t i c t i p s j u s t above t h e f l a n g e l o c a t e d a p p r o x i m a t e l y 1 cm from t h e end d i s t a l t o t h e h e a r t . A second p a i r o f f o r c e p s was used t o f i r m l y g r i p t h e i n n e r s n a r e o f t h e o c c l u d e r and t r a c t i o n was a p p l i e d s m o o t h l y between the i n n e r s n a r e and t h e o u t e r g u i d e t u b i n g t o produce o c c l u s i o n . When t h e sudden ECG and b l o o d p r e s s u r e changes c h a r a c t e r i s t i c o f o c c l u s i o n were o b s e r v e d , t h e a t r a u m a t i c f o r c e p s were clamped down f i r m l y on t h e o c c l u d e r . The exposed l e n g t h o f t h e i n n e r s n a r e o f t h e o c c l u d e r was t h e n m e l t e d down w i t h a s o l d e r i n g i r o n t o form a b u l b a d j a c e n t t o t h e d i s t a l end o f t h e o u t e r g u i d e t u b i n g , f i x i n g i t i n p l a c e . 2.8.2.8 Drug A d m i n i s t r a t i o n and Plasma C o n c e n t r a t i o n Measurement A 20 mg/kg dose o f drug (R,S- o r R ( - ) - o r S ( + ) - m e x i l e t i n e ) i n s a l i n e , o r s a l i n e as c o n t r o l , was a d m i n i s t e r e d t o each r a t by c o n s t a n t 69 r a t e i n t r a v e n o u s i n f u s i o n o v e r 10 min. A second dose was s t a r t e d a t 1.5 h a f t e r o c c l u s i o n and i n f u s e d o v e r 30 min. B l o o d (»0.3 ml) was c o l l e c t e d a t -5 and 20 min and a t 1, 1.5, 2, 3 and 4 h ( w i t h r e s p e c t t o i n i t i a t i o n o f c o r o n a r y o c c l u s i o n ) i n t o p l a s t i c v i a l s c o n t a i n i n g a drop o f h e p a r i n (50 IU/ml). The plasma was s e p a r a t e d and a n a l y z e d f o r m e x i l e t i n e e n a n t i o m e r c o n c e n t r a t i o n s as d e s c r i b e d e a r l i e r ( s e c t i o n 2.5). 2.8.2.9 Response t o Drug Treatment and O c c l u s i o n The ECG and b l o o d p r e s s u r e were r e c o r d e d f o r 30 min b e f o r e and 4 h r a f t e r c o r o n a r y a r t e r y o c c l u s i o n . F a s t ECG t r a c e s (150 mm/min) were o b t a i n e d j u s t b e f o r e t h e b e g i n n i n g o f drug i n f u s i o n and a t 1 min b e f o r e o c c l u s i o n f o r t h e d e t e r m i n a t i o n o f t h e e f f e c t o f t r e a t m e n t on h e a r t r a t e and ECG p a r a m e t e r s . F a s t t r a c e s were a l s o o b t a i n e d a t 1, 2, 5, 10, 15 and 30 min and a t 1, 1.5, 2, 3 and 4 h a f t e r o c c l u s i o n 2.8.2.10 O c c l u d e d Zone The a n i m a l s were s a c r i f i c e d 4 h a f t e r c o r o n a r y a r t e r y o c c l u s i o n by s t u n n i n g and d e c a p i t a t i o n . The h e a r t was e x c i s e d and p e r f u s e d t h r o u g h t h e a o r t a w i t h s a l i n e . When b l o o d was no l o n g e r p r e s e n t i n t h e p e r f u s a t e , p e r f u s i o n was c o n t i n u e d w i t h s a l i n e c o n t a i n i n g i n d o c y a n i n e g r e e n (0.5 g/1) v i a a 2-way s t o p - c o c k f o r 2-3 min. A f t e r p e r f u s i o n , t h e a t r i a , a o r t a and pulmonary v e s s e l s were e x c i s e d and d i s c a r d e d . The r e m a i n i n g v e n t r i c u l a r myocardium was r e a d i l y d i f f e r e n t i a t e d i n t o normal (green) and o c c l u d e d ( p i n k ) t i s s u e by v i s u a l i n s p e c t i o n . The two zones were s e p a r a t e d w i t h a p a i r o f s c i s s o r s , b l o t t e d w i t h f i l t e r p a p er and weighed. 70 2.8.2.11 O c c l u s i o n - I n d u c e d A r r h y t h m i a The f o l l o w i n g d e f i n i t i o n s were used i n t h e d i a g n o s i s o f a r r h y t h m i a s f o l l o w i n g c o r o n a r y o c c l u s i o n i n r a t s . a) Premature V e n t r i c u l a r C o n t r a c t i o n (PVC): PVC was d e f i n e d as a premature QRS complex o c c u r r i n g i n d e p e n d e n t o f t h e P wave. A PVC was g e n e r a l l y accompanied by a t r a n s i e n t drop i n a o r t i c b l o o d p r e s s u r e . b) V e n t r i c u l a r T a c h y c a r d i a ( V T ) : VT was d e f i n e d as a run o f 4 o r more c o n s e c u t i v e premature v e n t r i c u l a r c o n t r a c t i o n s . No r e s t r i c t i o n was made on t h e a s s o c i a t e d h e a r t r a t e . VT's were a s s o c i a t e d w i t h a d e c r e a s e i n mean a r t e r i a l b l o o d p r e s s u r e which was s u s t a i n e d t h r o u g h t h e d u r a t i o n o f t h e VT. c) V e n t r i c u l a r F i b r i l l a t i o n ( V F ) : VF was d e f i n e d as a c h a o t i c ECG t r a c i n g w i t h o u t r e c o g n i z a b l e QRS complexes and accompanied by a p r e c i p i t o u s f a l l i n b l o o d p r e s s u r e . VF was f u r t h e r s u b d i v i d e d i n t o s p o n t a n e o u s l y r e v e r t i n g (SVF) i . e . f i b r i l l a t i o n l a s t i n g l e s s than 10 sec and n o n - s p o n t a n e o u s l y r e v e r t i n g (NSVF) i . e . t h o s e l a s t i n g more than 10 s e c . In t h e l a t e r c a s e , t h e a r r h y t h m i a was t e r m i n a t e d by p r e c o r d i a l t a p s . 2.8.2.12 E v a l u a t i o n o f A n t i a r r h y t h m i c E f f i c a c y A n t i a r r h y t h m i c e f f i c a c y was e v a l u a t e d by d e t e r m i n i n g t h e i n c i d e n c e o f VT and VF, and t h e number o f PVCs f o l l o w i n g c o r o n a r y o c c l u s i o n . " A r r h y t h m i a s S c o r e " ( J o h n s t o n et al, 1983) was used t o summarize and gr a d e t h e a r r h y t h m i a s . T h i s s c o r e i s as f o l l o w s : 0 = No more than 49 PVCs, 1 = 50 - 499 PVCs 71 2 = No more t h a n 1 e p i s o d e o f SVT o r SVF a n d / o r g r e a t e r t h a n 499 PVCs. 3 = More t h a n 1 e p i s o d e o f VT a n d / o r VF o f l e s s t h a n 60 s e c d u r a t i o n , 4 = VT a n d / o r VF o f 60 - 119 s e c t o t a l d u r a t i o n 5 = VT a n d / o r VF o f g r e a t e r t h a n 119 s e c t o t a l d u r a t i o n 6 = F a t a l VF o c c u r r i n g 15 min - 4 h a f t e r o c c l u s i o n 7 = F a t a l VF o c c u r r i n g 4 min - 14 min 59 s e c a f t e r o c c l u s i o n 8 = F a t a l VF o c c u r r i n g 1 min - 3 min 59 s e c a f t e r o c c l u s i o n 9 = F a t a l VF o c c u r r i n g b e f o r e 1 min a f t e r o c c l u s i o n A l l t h e r e s u l t s w ere s u b s e q u e n t l y d i v i d e d i n t o e a r l y ( 0 - 0 . 5 h) and o v e r -a l l ( 0-4 h) a r r h y t h m i a s . 2.8.2.13 S t a t i s t i c a l Data A n a l y s i s D i f f e r e n c e s i n t h e b i n o m i a l l y d i s t r i b u t e d v a r i a b l e s ( i n c i d e n c e o f a r r h y t h m i a s ) were* a s s e s s e d by C h i - s q u a r e t e s t w i t h t h e a i d o f M a i n l a n d s c o n t i n g e n c y t a b l e ( M a i n l a n d et al., 1 9 5 6 ) . One-way ANOVA was u s e d t o com p a r e t h e n o r m a l l y d i s t r i b u t e d v a r i a b l e s ( a r r h y t h m i a s c o r e , b l o o d p r e s s u r e , h e a r t r a t e and t h e ECG p a r a m e t e r s ) . The number o f PVCs i s n o t a G a u s s i a n d i s t r i b u t e d v a r i a b l e ( J o h n s t o n et al., 1 9 8 3 ) . T h e r e f o r e , d a t a o b t a i n e d f o r t h i s v a r i a b l e was l o g ^ Q t r a n s f o r m e d b e f o r e b e i n g u s e d f o r s t a t i s t i c a l t e s t s . When a s i g n i f i c a n t F r a t i o was o b t a i n e d f r o m ANOVA, s u b s e q u e n t a n a l y s i s u s i n g D u n c a n ' s and Newman K e u l ' s m u l t i p l e c o m p a r i s o n t e s t s were c a r r i e d o u t t o d e t e r m i n e w h i c h t r e a t m e n t g r o u p s w ere s i g n i f i c a n t l y d i f f e r e n t . T he v a l u e o f a was 0.05 f o r a l l t h e s t a t i s t i c a l t e s t s . 2.8.2.14 E x c l u s i o n C r i t e r i a To a v o i d s o u r c e s o f v a r i a n c e t h a t may i n f l u e n c e t h e p r e c i s i o n and a c c u r a c y o f t h e e x p e r i m e n t a l r e s u l t s , a s e t o f e x c l u s i o n c r i t e r i a r e p o r t e d by C u r t i s ( 1 9 8 6 ) was u s e d t o e x c l u d e a n i m a l s f r o m t h e s t u d y . T h e s e t o f c r i t e r i a w e r e a p p l i e d b l i n d l y and r a t s w e r e e x c l u d e d b e f o r e , d u r i n g o r a f t e r o c c l u s i o n b a s e d on t h e f o l l o w i n g a b n o r m a l i t i e s . I) P r e - o c c l u s i o n A b n o r m a l i t i e s T h e s e were d e s i g n e d t o e x c l u d e r a t s w i t h p r e v i o u s o c c l u s i o n . a) The p r e s e n c e o f a Q-wave. T h i s i s b e c a u s e a Q-wave i s o n l y s e e n i n c h e s t l e a d s i n r a t s f o l l o w i n g c o r o n a r y o c c l u s i o n . b) The p r e s e n c e o f more t h a n 5 PVCs d u r i n g t h e 15 min p e r i o d p r i o r t o d r u g a d m i n i s t r a t i o n . T h i s may i n d i c a t e t h e p r e s e n c e o f a l e s i o n i n t h e m y o c a r d i u m w h i c h may i n f l u e n c e t h e outc o m e o f o c c l u s i o n . c ) More t h a n 2 5 % w e i g h t l o s s b e t w e e n s u r g e r y and c o r o n a r y o c c l u s i o n . R a t s were e x c l u d e d i f w e i g h t l o s s was a s s o c i a t e d w i t h o t h e r s i g n s o f i l l n e s s s u c h as d i a r r h e a a n d / o r p r e - d r u g mean a r t e r i a l b l o o d p r e s s u r e o f 85 mmHg o r l e s s . d) S i g n s o f l u n g i n f e c t i o n s u c h as e x u d a t e a r o u n d s n o u t a n d / o r n o i s y r e s p i r a t i o n . e) H y p e r t e n s i o n (mean b l o o d p r e s s u r e g r e a t e r t h a n 125 mmHg f o r l o n g e r t h a n 10 min b e f o r e d r u g a d m i n i s t r a t i o n ) . R a t s were e x c l u d e d o n l y i f p o s t m o r t e m e x a m i n a t i o n r e v e a l e d r e n a l n e c r o s i s . I I ) P o s t - o c c l u s i o n A b n o r m a l i t i e s T h e s e e v a l u a t i o n s were d e s i g n e d t o e x c l u d e r a t s w i t h o u t f u l l o c c l u s i o n , r a t s w h i c h e x p e r i e n c e d r e p e r f u s i o n , and r a t s w i t h m i s p l a c e d o c c l u d e r s . 73 a) No i n c r e a s e i n R wave, a n d / o r ST e l e v a t i o n . R a t s were e x c l u d e d i f t h e y had u n a c c e p t a b l y l o w OZ, i f d e a t h o c c u r r e d w i t h i n 4 h o u r s and pus o r s c a r r i n g was f o u n d i n t h e h e a r t , i f a u t o p s y showed o c c l u d e r was l o o s e , i f s u d d e n ECG c h a n g e s t y p i c a l o f o c c l u s i o n o c c u r r e d l a t e r t h a n 3 m i n a f t e r o c c l u s i o n ( p o s t u r a l l y i n d u c e d f u l l o c c l u s i o n ) . b) L o s s o f R wave i n c r e a s e a n d / o r ST e l e v a t i o n l e a d i n g t o ECG r e s e m b l i n g p r e - o c c l u s i o n ECG w i t h a c c o m p a n y i n g r e c o v e r y o f b l o o d p r e s s u r e . R a t s were e x c l u d e d i f OZ was u n a c c e p t a b l y l o w , i f t h e o c c l u d e r was l o o s e e v e n i f OZ was n o r m a l . c ) D e a t h a s s o c i a t e d w i t h i m m e d i a t e c a l a m i t o u s h y p o t e n s i o n . R a t s were e x c l u d e d i f t h o r a x was f i l l e d w i t h b l o o d and t h e r a t had n o t r e c e i v e d c h e s t t a p s . I l l ) Postmortem A b n o r m a l i t i e s R a t s were e x c l u d e d i f t h e y h a d : a) S m a l l OZ d e f i n e d as l e s s t h a n 2 5 % o f v e n t r i c u l a r w e i g h t . b) S c a r t i s s u e g r e a t e r t h a n 5% o f t h e v e n t r i c l e . c ) Pus o f 1 0 % o r more v e n t r i c u l a r w e i g h t a t t h e o c c l u s i o n s i t e . 2.8.3 C o r o n a r y A r t e r y O c c l u s i o n i n P e n t o b a r b i t o n e A n a e s t h e t i z e d Rats The s t u d y o f t h e a n t i a r r h y t h m i c e f f e c t s o f r a c e m i c m e x i l e t i n e i n p e n t o b a r b i t o n e a n a e s t h e t i z e d r a t s was c a r r i e d o u t as d e s c r i b e d f o r t h e c o n s c i o u s r a t s w i t h t h e f o l l o w i n g m o d i f i c a t i o n s : a) R a t s were a c u t e l y p r e p a r e d u n d e r p e n t o b a r b i t o n e a n a e s t h e s i a (45 mg/kg i . p . ) and a r t i f i c i a l l y r e s p i r e d t h r o u g h o u t t h e d u r a t i o n o f t h e e x p e r i m e n t . b) The c a r o t i d a r t e r y and j u g u l a r v e i n were c a n n u l a t e d f o r b l o o d p r e s s u r e m o n i t o r i n g and d r u g a d m i n i s t r a t i o n , r e s p e c t i v e l y . c ) L e a d I I ECG was r e c o r d e d . d) R e s p o n s e t o c o r o n a r y a r t e r y o c c l u s i o n was m o n i t o r e d f o r o n l y 30 min w h i c h c o v e r e d t h e e a r l y p h a s e o f a r r h y t h m i a s . e) T h r e e g r o u p s o f r a t s (n = 9 p e r g r o u p ) w e r e g i v e n e i t h e r s a l i n e o r R , S - m e x i l e t i n e (20 o r 40 mg/kg) i n a b l i n d and random manner. f ) B l o o d was c o l l e c t e d a t -5, 15 and 30 min p o s t - o c c l u s i o n f o r t h e d e t e r m i n a t i o n o f d r u g l e v e l s . 3. RESULTS and DISCUSSION 3.1 A s s a y o f M e x i l e t i n e E n a n t i o m e r s by HPLC u s i n g 2 - A n t h r o y l C h l o r i d e as a D e r i v a t i z a t i o n Reagent 3.1.1 S y n t h e s i s o f 2 - A n t h r o y l C h l o r i d e The s y n t h e s i s o f 2 - a n t h r o y l c h l o r i d e u n d e r t a k e n i n t h i s s t u d y was n e c e s s a r y s i n c e n e i t h e r t h e a c i d c h l o r i d e n o r t h e p r e c u r s o r a c i d c o u l d be o b t a i n e d c o m m e r c i a l l y . 2 - A n t h r o y l c h l o r i d e was s y n t h e s i s e d by a 2 - s t e p r e a c t i o n a c c o r d i n g t o t h e scheme shown i n f i g u r e 2. T h e f i r s t s t e p i n v o l v e d t h e s e l e c t i v e r e d u c t i o n o f a n t h r a q u i n o n e - 2 - c a r b o x y l i c a c i d t o a n t h r a c e n e - 2 - c a r b o x y l i c a c i d u s i n g d i l u t e ammonia and z i n c d u s t . T h i s r e a c t i o n p r o c e e d e d s m o o t h l y , g o i n g t o c o m p l e t i o n w i t h i n 45 m i n . The p r o d u c t i s o l a t e d by p r e p a r a t i v e HPLC was r e c r y s t a l 1 i z e d f r o m e t h a n o l ( 9 5 % ) and was f o u n d t o have a m e l t i n g p o i n t o f 2 7 8 - 2 7 9 ° C . T h i s a g r e e s w i t h t h e r e p o r t e d l i t e r a t u r e v a l u e w h i c h i s 2 8 0 ° C ( W e a s t , 1 9 7 6 ) . The s t r u c t u r e o f t h e a c i d was c o n f i r m e d by d i r e c t p r o b e EI-MS. The t o t a l i o n c u r r e n t c h r o m a t o g r a m and t h e mass s p e c t r u m a r e shown i n f i g u r e 3. The m a j o r f r a g m e n t i o n s were d i a g n o s t i c o f t h e a c i d w i t h M + (m/z=222), M +-0H (m/z=205), and M +-C00H (m/z=177) mass i o n s p r e s e n t . The s e c o n d s t e p i n t h e r e a c t i o n scheme ( f i g . 2) i n v o l v e d t h e c o n v e r s i o n o f a n t h r a c e n e - 2 - c a r b o x y l i c a c i d t o 2 - a n t h r o y l c h l o r i d e by t r e a t m e n t w i t h o x a l y l c h l o r i d e . The s y n t h e t i c a c i d c h l o r i d e was i s o l a t e d by p r e p a r a t i v e HPLC and t h e s t r u c t u r e c o n f i r m e d by d i r e c t p r o b e EI-MS. F i g u r e 4 shows t h e t o t a l i o n c u r r e n t c h r o m a t o g r a m and t h e mass s p e c t r u m o f 2 - a n t h r o y l c h l o r i d e . The m o l e c u l a r i o n p e a k s were p r e s e n t i n t h e mass s p e c t r u m a t m/z=240 and 242 f r o m t h e n a t u r a l i s o t o p e r a t i o 76 0 I 0 a C - O H Zn/NH. 0 I C — O H 0 a n t h r a q u i n o n e - 2 - c a r b o x y l i c a c i d a n t h r a c e n e - 2 - c a r b o x y ! i c COCl I C O C l a n t h r a c e n e - 2 - c a r b o x y l i c a c i d 2 - a n t h r o y l c h l o r i d e F i g u r e 2. Scheme f o r t h e s y n t h e s i s o f 2 - a n t h r o y l c h l o r i d e f r o m a n t h r a q u i n o n e - 2 - c a r b o x y l i c a c i d . 12000-10900-3906-5 3 8 8 -4000-2080-4.7ft ti J 3.2^ 1 '1 • ! ' 3.0 1.9 2.0 1 . ' ' ' 1 I 1 ' ' 1 . ' • • • 1 ' 1 ] 4.0 5.0 6.0 7.0 8.8 9.3 3200-2 8 8 0 -2400-2900-1600-1200-300-400-8 8 222 177 149 129 i • i 80 120 16ft >00 i 1—i-1—;—1—i—•—r-'—r-1—i— 240 230 320 360 408 440 F i g u r e 3. T h e t o t a l i o n c u r r e n t c h r o m a t o g r a m (A) and t h e mass s p e c t r u m (B) o f a n t h r a c e n e - 2 - c a r b o x y l i c a c i d . The f o l l o w i n g EI-MS c o n d i t i o n s w e r e e m p l o y e d : s o u r c e t e m p e r a t u r e , 2 4 0 ° C ; p r o b e t e m p e r a t u r e programme, 5 0 ° C f o r 1 min t o 3 0 0 ° C f o r 10 min a t t h e r a t e o f 3 0 b C p e r m i n ; e l e c t r o n beam v o l t a g e , 70 eV; e m i s s i o n c u r r e n t , 300 uA and m u l t i p l i e r v o l t a g e , 2500 V. 128888-leeeee-98089-88888-78608-68900-S9000-4 9006-30889-20800-10000-9-1 3.S9 10 12 14 16 18 1S000-1 "5000-14880-12000-10000-SO30-6000" 4806-2900-- I 177 20S 151 i / Mil i i 2 & j 249 / 296 88 i ' i • i • i • i • i • i • i • I • i • i • ; • : • i • i 16& 280 240 289 329 368 498 448 F i g u r e 4. The t o t a l i o n c u r r e n t c h r o m a t o g r a m (A) and t h e mass s p e c t r u m (B) o f 2 - a n t h r o y l c h l o r i d e . The f o l l o w i n g EI-MS c o n d i t i o n s w ere e m p l o y e d : s o u r c e t e m p e r a t u r e , 2 4 0 ° C ; p r o b e t e m p e r a t u r e programme, 5 0 ° C f o r 1 min t o 3 0 0 ° C f o r 10 min a t t h e r a t e o f 3 0 D C p e r m i n ; e l e c t r o n beam v o l t a g e , 70 e v ; e m i s s i o n c u r r e n t , 300 uA and m u l t i p l i e r v o l t a g e , 2500 V. o f c h l o r i n e ( 3 : 1 ) . O t h e r i m p o r t a n t f r a g m e n t i o n s o b s e r v e d were M -CI (m/z=205) and M+-C0C1 (m/z=177). 3.1.2 Development o f A s s a y o f M e x i l e t i n e E n a n t i o m e r s 3.1.2.1 D e r i v a t i z a t i o n o f M e x i l e t i n e E n a n t i o m e r s w i t h 2 - A n t h r o y l C h l o r i d e A number o f p r o p e r t i e s a r e r e q u i r e d o f an i d e a l d e r i v a t i z a t i o n r e a g e n t f o r t h e HPLC r e s o l u t i o n and q u a n t i t a t i o n o f m e x i l e t i n e e n a n t i o m e r s u s i n g t h e P i r k l e ^ i o n i c ( p h e n y l g l y c i n e ) c h i r a l p h a s e . T h e s e p r o p e r t i e s i n c l u d e a f u n c t i o n a l g r o u p t h a t w i l l r e a c t w i t h t h e p r i m a r y amino g r o u p o f m e x i l e t i n e , a s t r o n g f l u o r o p h o r e f o r s e n s i t i v e d e t e c t i o n o f t h e e n a n t i o m e r d e r i v a t i v e s and f u n c t i o n a l g r o u p s t h a t a i d i n t h e c h i r a l r e c o g n i t i o n p r o c e s s on t h e P i r k l e ^ c h i r a l p h a s e . In a p r e v i o u s s t u d y , 2 - n a p h t h o y l c h l o r i d e was f o u n d t o h a v e t h e s e n e c e s s a r y c h a r a c t e r i s t i c s and was s u c c e s s f u l l y u s e d t o r e s o l v e and q u a n t i t a t e m e x i l e t i n e e n a n t i o m e r s u s i n g t h e P i r k l e ^ i o n i c ( p h e n y l g l y c i n e ) c h i r a l c o l u m n ( I g w e m e z i e , 1986; M c E r l a n e et al., 1 9 8 7 ) . E x a m i n a t i o n o f t h e c h i r a l r e c o g n i t i o n p r o c e s s r e q u i r e d f o r t h e r e s o l u t i o n o f t h e 2 - n a p h t h o y l d e r i v a t i v e s o f t h e e n a n t i o m e r s l e a d t o t h e p r o p o s a l t h a t s e l e c t i v i t y c o u l d be p r e s e r v e d , w h i l e i m p r o v i n g s e n s i t i v i t y by t h e u s e o f 2 - a n t h r o y l c h l o r i d e as t h e d e r i v a t i z a t i o n r e a g e n t ( t h e f l u o r e s c e n c e q uantum y i e l d o f n a p h t h a l e n e i s 0.19 w h i l e t h a t o f a n t h r a c e n e i s 0.30 i n c y c l o h e x a n e a t 2 5 ° C ) ( F r o e h l i c h , 1 9 8 5 ) . The d e r i v a t i z a t i o n o f m e x i l e t i n e e n a n t i o m e r s w i t h 2 - a n t h r o y l c h l o r i d e was a c c o m p l i s h e d u s i n g t h e S c h o t t e n - B a u m a n n r e a c t i o n ( V o g e l , 1964) as shown i n f i g u r e 5. The a m ide d e r i v a t i v e s o f t h e e n a n t i o m e r s were i n s o l u b l e i n t h e a q u e o u s Figure 5. Reaction scheme for the derivatization of mexiletine enantiomers and the internal standard with 2-anthroyl chloride 00 o r e a c t i o n medium and t h e r e f o r e r e a d i l y i s o l a t e d by e x t r a c t i o n w i t h t h e m o b i l e p h a s e . 3.1.2.2 Structure of the 2-Anthroyl Derivative of Mexiletine The s t r u c t u r e o f t h e 2 - a n t h r o y l d e r i v a t i v e o f m e x i l e t i n e ( m e x i l e t i n e - 2 - a n t h r a m i d e ) was c o n f i r m e d by d i r e c t p r o b e EI-MS. T he t o t a l i o n c u r r e n t c h r o m a t o g r a m ( f i g . 6A) showed t h e p r e s e n c e o f two p e a k s . T h e m e x i l e t i n e - 2 - a n t h r a m i d e p e a k had a r e t e n t i o n t i m e o f 5.21 m i n and t h e mass s p e c t r u m ( f i g . 6C) e x h i b i t e d m a j o r f r a g m e n t i o n s w h i c h w e r e d i a g n o s t i c as shown i n f i g u r e 7. The i d e n t i t y o f t h e p e a k a t 3.28 min ( f i g . 6A) c o u l d n o t be d e t e r m i n e d f r o m i t ' s mass s p e c t r u m ( f i g . 6 B ) . However, t h e p r e s e n c e o f mass i o n s a t 149 and 279 s u g g e s t e d t h a t i t may be a p h t h a l a t e c o n t a m i n a n t ( W a t s o n , 1 9 8 5 ) . 3.1.2.3 Resolution of Mexiletine Enantiomers F o l l o w i n g d e r i v a t i z a t i o n w i t h 2 - a n t h r o y l c h l o r i d e , t h e e n a n t i o m e r s o f m e x i l e t i n e were r e s o l v e d on a P i r k l e ^ i o n i c ( p h e n y l g l y c i n e ) c h i r a l s t a t i o n a r y p h a s e ( s e e f i g . 9 ) . The optimum m o b i l e p h a s e was e t h y l a c e t a t e / 2 - p r o p a n o l / h e x a n e ( 4 : 6 : 9 0 ) d e l i v e r e d i s o c r a t i c a l l y a t a f l o w r a t e o f 1.7 m l / m i n . T h e r e t e n t i o n t i m e s o f t h e e n a n t i o m e r s w e r e 16.68 and 17.69 min f o r R ( - ) - and S ( + ) - m e x i l e t i n e , r e s p e c t i v e l y ( f i g . 8 ) . T he e l u t i o n o r d e r o f t h e p e a k s was d e t e r m i n e d by c o m p a r i n g t h e r e t e n t i o n t i m e o f t h e i n d i v i d u a l e n a n t i o m e r d e r i v a t i v e s w i t h t h o s e o f t h e r a c e m a t e p e a k s and a l s o f r o m t h e a n t i c i p a t e d s t e r e o c h e m i c a l i n t e r a c t i o n s b e t w e e n t h e e n a n t i o m e r s and t h e s t a t i o n a r y p h a s e ( s e c t i o n 3 . 1 . 2 . 5 ) . T h e 3.28 5.21 32K»-2«8?> 2*l>t«-1281^  19 85 98 \*7 I > I H'l ill llll 1 2 9 in, •83 2»3 2 3 3 ^ - p - — I — T r y , . 28a 294 240 328 SS88-«ee8 358*-3(>8f<-2f.f.<« F*0 e-UU 83 ?l X / 2(2 177 ISI 121 28S 128 II7~ 292 s 383 V 26* -r 1 248 288 328 3 A ' l • I " I «ee F i g u r e 6. The t o t a l i o n c u r r e n t c h r o m a t o g r a m (A) and t h e mass s p e c t r a (B and C) o b t a i n e d f r o m t h e 2 - a n t h r o y l d e r i v a t i v e o f m e x i l e t i n e . The f o l l o w i n g EI-MS c o n d i t i o n s were e m p l o y e d : s o u r c e t e m p e r a t u r e , 2 4 0 ° C ; p r o b e t e m p e r a t u r e programme, 50°C f o r 1 min t o 3 0 0 ° C f o r 10 min a t 3 0 ° C p e r m i n ; e l e c t r o n beam v o l t a g e , 70 eV; e m i s s i o n c u r r e n t , 300 uA and m u l t i p l i e r v o l t a g e , 2500 V 00 ro m/z 3 8 3 ( M + ) m/z 1 2 1 m/z 1 7 7 F i g u r e 7. The m a j o r f r a g m e n t i o n s o f t h e 2 - a n t h r o y l d e r i v a t i v e o f m e x i l e t i n e . <u <u coco vjr--F i g u r e 8. C h r o m a t o g r a m o f t h e 2 - a n t h r o y l d e r i v a t i v e o f m e x i l e t i n e e n a n t i o m e r s . C h r o m a t o g r a p h i c c o n d i t i o n s w e r e : s t a t i o n a r y p h a s e , P i r k l e R i o n i c ( p h e n y l g l y c i n e ) c h i r a l p h a s e ; m o b i l e p h a s e , e t h y l a c e t a t e / 2 - p r o p a n o l / h e x a n e ( 4 : 6 : 9 0 ) ; f l o w r a t e , 1.7 m l / m i n ; d e t e c t i o n f l u o r e s c e n c e a t 270 nm ( e x ) and 400 nm ( e m ) . resolution of the enantiomers achieved was considered satisfactory (R = 1.4) for peak height measurement. 3.1.2.4 Sensitivity of the 2-Anthroyl Derivative of Mexiletine The detection of the enantiomer derivatives was accomplished by fluorescence (ex=270 nm and em=400 nm). The minimum detectable quantity of each enantiomer was 0.5 ng/ml, representing 50 pg at the detector (signal-to-noise ratio of 5:1). Thus, 2-anthroyl chloride provided a 10-fold increase in sensit ivity over a previously reported assay (Igwemezie, 1986; McErlane et a7., 1987) in which mexiletine enantiomers were derivatized with 2-naphthoyl chloride. 3.1.2.5 Mechanism of Resolution of Mexiletine Enantiomers To resolve enantiomers on a chiral stationary phase (CSP), a minimum of 3 simultaneous interactions between the enantiomers and the CSP is necessary (Feibush and Grinberg, 1988). One of these interactions also has to be stereochemically dependent and could be an attraction or a repulsion. These interactions result in the formation of transient diastereoisomeric complexes whose s tab i l i t i es are different, resulting in a difference in the migration rate of the enantiomers through the CSP. Figure 9 shows the structure of the P i rk le R ionic (phenyl glycine) chiral phase and the 2-anthroyl derivative of mexiletine. The enantiomer derivatives are envisaged to interact with the CSP by means of 7T-7T bonding between the 7r-basic anthroyl group and the 7r-acidic 3,5-dinitrobenzoyl group of the CSP, electrostatic bonding of the amide dipoles (broken arrows in figure 9), 86 F i g u r e 9. The s t r u c t u r e o f t h e P i r k 1 e K i o n i c c h i r a l s t a t i o n a r y p h a s e [ ( R ) - 3 , 5 - d i n i t r o b e n z o y l p h e n y l g l y c i n e i o n i c a l l y b o n d e d t o T - a m i n o p r o p y l s i l i c a ] and t h e 2 - a n t h r o y l d e r i v a t i v e o f m e x i l e t i n e . T h e b r o k e n a r r o w s show t h e e n v i s a g e d i n t e r a c t i o n s . and a s t e r i c i n t e r a c t i o n o f t h e m e t h y l and x y l y l o x y m e t h y l g r o u p s a t t h e c h i r a l c e n t r e o f m e x i l e t i n e w i t h t h e p r o x i m a t e p o r t i o n o f t h e CSP. Due t o t h e p r e s e n c e o f t h e b u l k y p h e n y l g r o u p b e l o w t h e p l a n e o f t h e CSP, t h e a n a l y t e m o l e c u l e c a n o n l y a p p r o a c h t h e CSP f r o m t h e t o p . T h e a n a l y t e - C S P i n t e r a c t i o n s w i l l p r o d u c e a more s t a b l e d i a s t e r e o i s o m e r i c c o m p l e x w i t h S ( + ) - m e x i l e t i n e - 2 - a n t h r a m i d e where t h e m e t h y l g r o u p l i e b e l o w t h e p l a n e o f t h e a n a l y t e m o l e c u l e . The R ( - ) - m e x i l e t i n e - 2 -a n t h r a m i d e , w i t h t h e b u l k i e r x y l y l o x y m e t h y l g r o u p b e l o w t h e p l a n e o f t h e a n a l y t e m o l e c u l e , w i l l be more s t e r i c a l l y h i n d e r e d . T h e r e s u l t i s a f a s t e r e l u t i o n o f t h i s e n a n t i o m e r t h r o u g h t h e CSP. T h i s s t e r e o c h e m i c a l model o f i n t e r a c t i o n o f m e x i l e t i n e e n a n t i o m e r d e r i v a t i v e s w i t h t h e P i r k l e ^ CSP was d e r i v e d f r o m t h e " d i p o l e s t a c k i n g " c h i r a l r e c o g n i t i o n model o f P i r k l e and W e l c h ( 1 9 8 4 ) . The model has been u s e d t o d e t e r m i n e t h e e l u t i o n o r d e r o f e n a n t i o m e r i c a m i n e s ( P i r k l e and W e l c h , 1984) and m e x i l e t i n e e n a n t i o m e r s as t h e 2 - n a p h t h o y l d e r i v a t i v e s ( M c E r l a n e e t al., 1987) on t h e P i r k l e R CSP. 3.1.2.6 HPLC A s s a y o f M e x i l e t i n e E n a n t i o m e r s M e x i l e t i n e e n a n t i o m e r s and t h e i n t e r n a l s t a n d a r d were e x t r a c t e d f r o m an a q u e o u s s o l u t i o n w i t h d i e t h y l e t h e r p r i o r t o d e r i v a t i z a t i o n . F i g u r e 10 shows r e p r e s e n t a t i v e c h r o m a t o g r a m s o f e x t r a c t s o f d i s t i l l e d w a t e r c o n t a i n i n g r a c e m i c m e x i l e t i n e and t h e i n t e r n a l s t a n d a r d , and b l a n k d i s t i l l e d w a t e r . The l a t t e r c h r o m a t o g r a m ( f i g . 10B) i n d i c a t e d t h e p r e s e n c e o f an e x t r a n e o u s p e a k w h i c h c o - e l u t e d w i t h t h e S ( + ) - e n a n t i o m e r . 0 5 1 0 1 5 2 0 T i m e ( M i n ) F i g u r e 10. C h r o m a t o g r a m s o f e x t r a c t s o f d i s t i l l e d w a t e r c o n t a i n i n g m e x i l e t i n e e n a n t i o m e r s ( 1 2 5 ng/ml e a c h ) a n d t h e i n t e r n a l s t a n d a r d ( 6 0 ng/ml) ( A ) , and d i s t i l l e d w a t e r ( B ) . C h r o m a t o g r a p h i c c o n d i t i o n s w e r e : s t a t i o n a r y p h a s e , P i r k l e R i o n i c ( p h e n y l g l y c i n e ) c h i r a l p h a s e m o b i l e p h a s e , e t h y l a c e t a t e / 2 - p r o p a n o l / h e x a n e ( 4 : 6 : 9 0 ) ; f l o w r a t e , 1.7 m l / m i n ; d e t e c t i o n , f l u o r e s c e n c e a t 270 nm ( e x ) and 400 nm ( e m ) . 3.1.2.7 Attempted R e s o l u t i o n / R e m o v a l o f t h e I n t e r f e r i n g Peak a) V a r i a t i o n o f M o b i l e Phase P o l a r i t y : E x t e n s i v e v a r i a t i o n s o f t h e m o b i l e p h a s e s p o l a r i t y as d e s c r i b e d i n s e c t i o n 2.4.2.8 d i d n o t s i g n i f i c a n t l y c h a n g e t h e r e t e n t i o n t i m e o f t h e i n t e r f e r i n g compound r e l a t i v e t o t h a t o f t h e e n a n t i o m e r s . The e x t e n t t o w h i c h p o l a r i t y c o u l d be i n c r e a s e d was l i m i t e d by t h e f a c t t h a t t h e P i r k l e i o n i c c h i r a l c o l u m n c a n o n l y t o l e r a t e t h e e q u i v a l e n t o f 2 0 % 2 - p r o p a n o l / h e x a n e w i t h r e s p e c t t o p o l a r i t y . b) E x t r a c t i o n S o l v e n t : A c o m p a r i s o n o f t h e c h r o m a t o g r a m s o b t a i n e d f r o m t h e e x t r a c t e d and u n e x t r a c t e d R ( - ) - e n a n t i o m e r (100 ng/ml) and t h e i n t e r n a l s t a n d a r d (100 ng/ml) d e m o n s t r a t e d t h a t t h e s i z e o f t h e i n t e r f e r i n g compound was s i g n i f i c a n t l y g r e a t e r when an e x t r a c t i o n s t e p was i n v o l v e d ( f i g . 1 1 ) . T h u s , t h e p o s s i b i l i t y o f a r e a c t i o n b e t w e e n t h e 2 - a n t h r o y l c h l o r i d e and an i m p u r i t y i n t h e e x t r a c t i o n s o l v e n t was s u s p e c t e d . Two o t h e r s o l v e n t s , h e x a n e and d i c h l o r o m e t h a n e w e r e i n v e s t i g a t e d . As shown i n f i g u r e 12, t h e i n t e r f e r i n g p e a k was p r e s e n t w i t h e a c h s o l v e n t . c ) P u r i f i c a t i o n o f S o l v e n t s and Reagents: To d e t e r m i n e i f t h e i n t e r f e r i n g p e a k was due t o an i m p u r i t y p r e s e n t i n t h e s o l v e n t s o r r e a g e n t s , e x t e n s i v e p u r i f i c a t i o n o f e a c h s o l v e n t and r e a g e n t was c a r r i e d o u t as d e s c r i b e d i n s e c t i o n 2.4.2.8. T h e s e t r e a t m e n t s d i d n o t e l i m i n a t e t h e i n t e r f e r i n g compound. d) P i r k l e R ( S ) - L e u c i n e C h i r a l Column: In o r d e r t o r e s o l v e t h e i n t e r f e r i n g p e a k f r o m t h o s e o f m e x i l e t i n e e n a n t i o m e r s , t h e s t a t i o n a r y p h a s e was c h a n g e d t o t h e P i r k l e R ( S ) - l e u c i n e p h a s e . However, t h e Time (Min) Figure 11. Chromatograms of R(-)-mexiletine (100 ng/ml) and the internal standard (100 ng/ml) derivatized with 2-anthroyl chloride after extraction from an aqueous solution with diethyl ether (A), and the same concentrations of R(-)-mexiletine and the internal standard derivatized without prior extraction (B). \AJ. / —T— 1 1 10 15 20 T r 10 15 20 - T - r 5 10 15 20. Time ( M i n ) F i g u r e 12. C h r o m a t o g r a m s o b t a i n e d a f t e r e x t r a c t i o n o f 1 ml a l i q u o t s o f d i s t i l l e d w a t e r w i t h d i f f e r e n t e x t r a c t i o n s o l v e n t s : d i e t h y l e t h e r ( A ) , h e x a n e (B) and d i c h l o r o m e t h a n e ( C ) . The a r r o w s i n d i c a t e t h e r e t e n t i o n t i m e o f t h e i n t e r f e r i n g s u b s t a n c e . a n t h r o y l d e r i v a t i v e s o f m e x i l e t i n e e n a n t i o m e r s were u n r e s o l v e d on t h i s s t a t i o n a r y p h a s e . S i n c e a l l a t t e m p t s t o s o l v e t h e i n t e r f e r i n g p e a k p r o b l e m w e r e s u c c e s s f u l , a p r e v i o u s l y d e v e l o p e d s t e r e o s e l e c t i v e a s s a y f o r m e x i l e t i n e e n a n t i o m e r s u s i n g 2 - n a p h t h o y l c h l o r i d e as d e r i v a t i z a t i o n r e a g e n t ( s e e b e l o w ) was u s e d f o r t h e p r o p o s e d p h a r m a c o k i n e t i c and p h a r m a c o d y n a m i c s t u d i e s . 3.2 A s s a y o f M e x i l e t i n e E n a n t i o m e r s by HPLC w i t h 2-Naphthoyl C h l o r i d e as a D e r i v a t i z a t i o n r e a g e n t F i g u r e 13 shows t h e r e s u l t i n g c h r o m a t o g r a m s o f m e x i l e t i n e e n a n t i o m e r s (50 ng/ml e a c h ) and t h e i n t e r n a l s t a n d a r d (100 ng/ml) i s o l a t e d f r o m human p l a s m a and b l a n k p l a s m a and a s s a y e d as t h e i r 2 - n a p h t h a m i d e d e r i v a t i v e s . T h e r e was no e v i d e n c e o f any i n t e r f e r i n g p e a k w i t h t h o s e due t o t h e e n a n t i o m e r s o r t h e i n t e r n a l s t a n d a r d . The minimum d e t e c t i o n l i m i t o f t h e a s s a y was 5 ng/ml o f e a c h e n a n t i o m e r i n p l a s m a , a t a s i g n a l - t o - n o i s e r a t i o o f 5:1. T a b l e 1 shows t h e c a l i b r a t i o n c u r v e d a t a f o r t h e e n a n t i o m e r s i n p l a s m a . A c o e f f i c i e n t o f d e t e r m i n a t i o n ( r 2 ) g r e a t e r t h a n 0.99 was o b t a i n e d f o r b o t h e n a n t i o m e r s and t h e i n t e r - a s s a y v a r i a b i l i t y ( o f 3 d e t e r m i n a t i o n s ) was l e s s t h a n 10%. R e p r e s e n t a t i v e c a l i b r a t i o n c u r v e s o f t h e e n a n t i o m e r s i n r a t p l a s m a ( c o n c e n t r a t i o n r a n g e = 10 t o 250 ng/ml o f e a c h e n a n t i o m e r ) g a v e t h e f o l l o w i n g l i n e a r l e a s t s q u a r e s r e g r e s s i o n e q u a t i o n s : Y = 0.024X + 0.027 ( r 2 = 0 . 9 9 9 ) f o r R ( - ) - m e x i l e t i n e and Y = 0.022X + 0.022 ( r 2 = 0 . 9 9 9 ) f o r S ( + ) - m e x i l e t i n e . S i m i l a r c a l i b r a t i o n c u r v e s were a l s o o b t a i n e d f o r t h e e n a n t i o m e r s i n t h e v a r i o u s r a t t i s s u e h o m o g e n a t e s . A B X X CO CD 1 -+• oo al oo —-i n o m-T oo oooo r~ ~o — o <s my? • • " " - mm CO U7 TO CD CM CO F i g u r e 13. C h r o m a t o g r a m s o f 2 - n a p h t h o y l d e r i v a t i v e s o f m e x i l e t i n e e n a n t i o m e r s ( 5 0 ng/ml e a c h ) and t h e i n t e r n a l s t a n d a r d ( 1 0 0 ng/ml) i s o l a t e d f r o m human p l a s m a ( A ) , and b l a n k p l a s m a ( B ) . T h e c h r o m a t o g r a p h i c c o n d i t i o n s w e r e : s t a t i o n a r y p h a s e , P i r k l e R i o n i c ( p h e n y l g l y c i n e ) c h i r a l p h a s e ; m o b i l e p h a s e , c h l o r o f o r m / 2 - p r o p a n o l / h e x a n e ( 7 : 7 : 8 6 ) ; f l o w r a t e , 1.2 ml/nnir» d e t e c t i o n , f l u o r e s c e n c e a t 230 nm ( e x ) and 370 nm ( e m ) . T a b l e 1. C a l i b r a t i o n c u r v e d a t a f o r m e x i l e t i n e e n a n t i o m e r s i n human p l a s m a Wt o f e a c h P e a k - h t r a t i o ^ C-V** P e a k - h t r a t i o ^ C V e n a n t i o m e r R ( - ) - m e x / I . S . (%) S ( + ) - m e x / I . S . (%) (ng) 500 12.50 ± 0.04 200 4.98 ± 0.14 50 1.24 ± 0.02 20 0.50 ± 0.01 10 0.26 ± 0.02 1 12.10 ± 0.06 1 3 4.82 ± 0.18 4 2 1.18 ± 0.02 2 1 0.48 ± 0.01 2 8 0.24 ± 0.02 9 s l o p e 0.025 0.024 i n t e r c e p t -0.003 -0.014 r 2 0.999 0.999 * Mean ± s . d . , n = 3 d e t e r m i n a t i o n s . ** P e r c e n t c o e f f i c i e n t o f v a r i a b i l i t y . The amount o f i n t e r n a l s t a n d a r d ( I . S . ) u s e d was 50 n g . 3.3 In Vitro Serum P r o t e i n B i n d i n g o f M e x i l e t i n e E n a n t i o m e r s 3.3.1 F a c t o r s A f f e c t i n g Serum P r o t e i n B i n d i n g The in vitro s e r u m p r o t e i n b i n d i n g o f m e x i l e t i n e e n a n t i o m e r s was c a r r i e d o u t u s i n g s e r u m f r o m h e a l t h y human s u b j e c t s as w e l l a s c o m m e r c i a l l y o b t a i n e d o ^ - a c i d g l y c o p r o t e i n ( A A G ) , human s e r u m a l b u m i n (HSA) and l i p o p r o t e i n d e f i c i e n t s e r u m . To e n s u r e t h a t t h e b i n d i n g r e s u l t s w e r e m e a n i n g f u l , m e t h o d o l o g i c a l f a c t o r s w h i c h may i n f l u e n c e s e r u m p r o t e i n b i n d i n g were e v a l u a t e d . T h e s e f a c t o r s i n c l u d e d non-s p e c i f i c a d s o r p t i o n t o t h e u l t r a f i l t r a t i o n s y s t e m , p H - d e p e n d e n t b i n d i n g and c o m p e t i t i v e b i n d i n g . The i n f l u e n c e o f o t h e r f a c t o r s s u c h as b i n d i n g d i s p l a c e m e n t by t r i s ( b u t o x y e t h y l ) p h o s p h a t e f r o m V a c u t a i n e r ^ c a p s and t h e age o f s e r u m were a v o i d e d by c o l l e c t i n g b l o o d i n g l a s s s y r i n g e s and u s i n g t h e s e r u m s h o r t l y a f t e r c o l l e c t i o n . 3.3.1.1 N o n - s p e c i f i c B i n d i n g T a b l e 2 shows t h e p e r c e n t r e c o v e r y o f m e x i l e t i n e e n a n t i o m e r s d u r i n g u l t r a f i l t r a t i o n . T he r e c o v e r y was i n e x c e s s o f 9 0 % f o r b o t h e n a n t i o m e r s i n d i c a t i n g t h a t n o n - s p e c i f i c b i n d i n g t o t h e u l t r a f i l t r a t i o n s y s t e m was m i n i m a l . However, t h e f r e e f r a c t i o n s o b t a i n e d were c o r r e c t e d f o r t h e s m a l l a d s o r p t i v e l o s s . 3.3.1.2 E f f e c t o f Serum pH The e f f e c t o f pH on t h e b i n d i n g o f m e x i l e t i n e e n a n t i o m e r s t o human s e r u m was e v a l u a t e d a t 3 d i f f e r e n t pH v a l u e s ( 7 . 0 , 7.4 and 8 . 0 ) . As shown i n F i g u r e 14, t h e f r e e f r a c t i o n o f e a c h e n a n t i o m e r d e c r e a s e d T a b l e 2. P e r c e n t r e c o v e r y o f m e x i l e t i n e e n a n t i o m e r s d u r i n g u l t r a f i l t r a t i o n C o n c e n t r a t i o n o f R(-)-mex S(+)-mex e a c h e n a n t i o m e r (/zg/ml) 1.00 96 ± 2 94 ± 1 0.25 91 ± 2 92 ± 3 0.10 92 ± 2 91 ± 2 V a l u e s a r e means ± s.e.m o f t r i p l i c a t e d e t e r m i n a t i o n s . P e r c e n t r e c o v e r y was d e t e r m i n e d f r o m t h e r a t i o o f t h e c o n c e n t r a t i o n o f t h e e n a n t i o m e r s ( i n p h o s p h a t e b u f f e r , pH = 7.4) b e f o r e and a f t e r u l t r a f i l t r a t i o n . — • — R(-)-mex - O - S(+)-mex 80 i 20 -10 -6.50 7.00 7.50 8.00 8.50 Serum pH F i g u r e 14. The r e l a t i o n s h i p b e t w e e n p e r c e n t f r e e f r a c t i o n (mean ± s.e.m., n=6 s u b j e c t s ) and s e r u m pH. 98 s i g n i f i c a n t l y (p<0.05) w i t h i n c r e a s i n g pH. B i n d i n g was f o u n d t o be s t e r e o s e l e c t i v e a s t h e pH was r a i s e d a b o v e 7.4 and was g r e a t e r f o r t h e R ( - ) - e n a n t i o m e r . T h e r e were no s i g n i f i c a n t d i f f e r e n c e s b e t w e e n t h e f r e e f r a c t i o n o f t h e e n a n t i o m e r s a t pH 7.4 and 7.0. A c h a n g e i n t h e f r e e f r a c t i o n o f a d r u g o c c u r r i n g w i t h a c h a n g e i n pH c a n be e x p l a i n e d on t h e b a s i s o f c h a n g e s i n t h e i o n i z a t i o n o f t h e b i n d i n g p r o t e i n a n d / o r t h e i o n i z a t i o n o f t h e d r u g (McNamara et al., 1 9 8 1 ) . A s i m i l a r d e c r e a s e i n f r e e f r a c t i o n w i t h i n c r e a s e i n pH was r e p o r t e d f o r l i d o c a i n e , a s t r u c t u r a l a n a l o g u e o f m e x i l e t i n e , by B u r n e y et al. ( 1 9 7 8 ) and McNamara et al. ( 1 9 8 1 ) . B o t h i n v e s t i g a t o r s e x p l a i n e d t h e c h a n g e s i n b i n d i n g t o be due t o a c h a n g e i n t h e i o n i z a t i o n o f l i d o c a i n e as pH was i n c r e a s e d . W i t h r e s p e c t t o m e x i l e t i n e , a c h a n g e i n pH f r o m 8.0 t o 7.0 s h o u l d n o t m a r k e d l y c h a n g e t h e r a t i o o f t h e i o n i z e d t o t h e u n i o n i z e d f o r m s s i n c e t h e p K a o f m e x i l e t i n e i s 8.8 ( M e r c k I n d e x , 1983) ( a t a pH o f 8.0, m e x i l e t i n e i s a p p r o x i m a t e l y 8 4 % i o n i z e d ) . F u r t h e r m o r e , s i n c e e n a n t i o m e r s h a v e t h e same c h e m i c a l p r o p e r t i e s , t h e i r i o n i z a t i o n c o n s t a n t s h o u l d be t h e same and t h e r a t i o o f t h e i r f r e e f r a c t i o n s s h o u l d r e m a i n c o n s t a n t as pH i s d e c r e a s e d f r o m 8.0 t o 7.0. T h e p r e s e n t r e s u l t s , t h e r e f o r e , s u g g e s t t h a t t h e p H - d e p e n d e n t c h a n g e s i n f r e e f r a c t i o n and s t e r e o s e l e c t i v i t y o f t h e e n a n t i o m e r s i s most l i k e l y a r e f l e c t i o n o f p H - d e p e n d e n t c h a n g e s i n t h e b i n d i n g p r o t e i n ( s ) . The r e s u l t s a l s o i n d i c a t e t h a t t o o b t a i n m e a n i n g f u l b i n d i n g d a t a f o r e i t h e r r a c e m i c m e x i l e t i n e o r i t s e n a n t i o m e r s , serum pH must be a d j u s t e d and m a i n t a i n e d a t =7.4 d u r i n g t h e b i n d i n g e x p e r i m e n t . Serum pH as h i g h a s 8.5 was o b s e r v e d i n t h e p r e s e n t s t u d i e s b e f o r e pH a d j u s t m e n t . 3.3.1.3 C o m p e t i t i v e B i n d i n g ( E n a n t i o m e r - E n a n t i o m e r I n t e r a c t i o n ) L i m a (1987) r e p o r t e d c o m p e t i t i v e i n t e r a c t i o n b e t w e e n t h e e n a n t i o m e r s o f d i s o p y r a m i d e r e s u l t i n g i n a g r e a t e r t h a n 2 - f o l d i n c r e a s e i n t h e f r e e f r a c t i o n o f e a c h e n a n t i o m e r . The p o s s i b i l i t y o f s u c h an i n t e r a c t i o n was i n v e s t i g a t e d f o r m e x i l e t i n e e n a n t i o m e r s . T a b l e 3 shows t h e f r e e f r a c t i o n s o f t h e e n a n t i o m e r s o b t a i n e d when b i n d i n g s t u d i e s were c a r r i e d o u t w i t h t h e i n d i v i d u a l e n a n t i o m e r s o r t h e r a c e m a t e . T h e r e were no s i g n i f i c a n t d i f f e r e n c e s i n t h e f r e e f r a c t i o n s o b t a i n e d f o r e a c h e n a n t i o m e r ( f r e e f r a c t i o n s w ere c o m p a r e d by i n d e p e n d e n t g r o u p s t - t e s t ) . T h i s s u g g e s t s t h e a b s e n c e o f e n a n t i o m e r - e n a n t i o m e r i n t e r a c t i o n , w h i c h c o u l d a f f e c t p r o t e i n b i n d i n g , w i t h i n t h e t h e r a p e u t i c r a n g e o f m e x i l e t i n e . 3.3.2 Methods o f Serum pH Adjustment T h e p H - d e p e n d e n t b i n d i n g d e s c r i b e d a b o v e ( s e c t i o n 3.3.1.2) n e c e s s i t a t e d t h e a d j u s t m e n t o f s e r u m pH b e f o r e c a r r y i n g o u t b i n d i n g e x p e r i m e n t s . T a b l e 4 shows t h e m ethods i n v e s t i g a t e d f o r t h e a d j u s t m e n t and m a i n t e n a n c e o f s e r u m pH a t a p p r o x i m a t e l y 7.4 d u r i n g u l t r a f i l t r a t i o n . B o t h 1 0 0 % CO2 and b u f f e r s a l t s a d e q u a t e l y m a i n t a i n e d s e r u m pH a t - 7 . 4 . T h e r e s u l t s o b t a i n e d by t h e s e two m ethods i n d i c a t e d t h a t b u f f e r s a l t s d i d n o t a f f e c t t h e f r e e f r a c t i o n v a l u e s o b t a i n e d . B u f f e r s a l t s w ere s u b s e q u e n t l y u s e d i n t h i s s t u d y b e c a u s e t h i s a p p r o a c h was more a d a p t a b l e t o t h e l a r g e number o f s a m p l e s i n v o l v e d i n t h e b i n d i n g e x p e r i m e n t s . Serum pH was n o t s a t i s f a c t o r i l y c o n t r o l l e d by u s i n g 5% CO2/O2 as shown i n t a b l e 4. 100 T a b l e 3. The p e r c e n t f r e e f r a c t i o n s o f m e x i l e t i n e e n a n t i o m e r s f r o m s e r u m c o n t a i n i n g r a c e m i c m e x i l e t i n e o r t h e i n d i v i d u a l e n a n t i o m e r C o n c e n t r a t i o n o f ^ * e a c h e n a n t i o m e r R(-)-mex S(+)-mex R(-)-mex S(+)-mex (Mg/ml) 1.00 53 ± 1 54 ± 2 59 ± 4 48 ± 3 0.25 49 ± 2 5 0 + 2 57 ± 4 52 ± 3 * F r e e f r a c t i o n s f r o m r a c e m i c m e x i l e t i n e . V a l u e s a r e means ± s.e.m, n = 5-6 s u b j e c t s . T a b l e 4. M e t h o d s u s e d t o a d j u s t s e r u m pH t o p h y s i o l o g i c a l v a l u e {- 7.4) and t h e c o r r e s p o n d i n g p e r c e n t f r e e f r a c t i o n s o b t a i n e d . M e t h o d pH b e f o r e pH a f t e r pH a f t e r F r e e f r a c t i o n a d j u s t m e n t a d j u s t m e n t f i l t r a t i o n R(-)-mex S(+)-mex 5% C0 2 /0 2 8.05 7.47 7.65 ± 0.01 44 ± 1 52 + 2 100% co 2 8.05 7.35 7.41 ± 0.02 52 + 1 58 + 1 p h o s p h a t e * b u f f e r s a l t s 8.05 7.35 7.35 ± 0 48 + 1 51 ± 1 D r u g c o n c e n t r a t i o n d e t e r m i n a t i o n s . = 2 /jg/ml R , S - m e x i l e t i n e . V a l u e s a r e means ± s, .e.rn., n = 5-6 C o n c e n t r a t i o n o f t h e b u f f e r s a l t s were 5.02 mg m o n o b a s i c s o d i u m p h o s p h a t e and 23.55 mg d i b a s i c s o d i u m p h o s p h a t e p e r 1 ml o f serum (0.12 M). 3.3.3 B i n d i n g o f M e x i l e t i n e E n a n t i o m e r s t o Serum and t o V a r i o u s Serum P r o t e i n s T a b l e 5 s u m m a r i z e s t h e d a t a o b t a i n e d f o r t h e f r e e f r a c t i o n s o f m e x i l e t i n e e n a n t i o m e r s when p r o t e i n b i n d i n g was d e t e r m i n e d u s i n g s e rum, l i p o p r o t e i n d e f i c i e n t serum, AAG and HSA. The 2 c o n c e n t r a t i o n s o f t h e e n a n t i o m e r s e v a l u a t e d were w i t h i n t h e t h e r a p e u t i c r a n g e o f m e x i l e t i n e . The r e s u l t s i n d i c a t e d t h a t t h e b i n d i n g o f m e x i l e t i n e e n a n t i o m e r s t o s e r u m was o n l y m o d e r a t e , w i t h f r e e f r a c t i o n s r a n g i n g f r o m 50 t o 55%. T h e s e r e s u l t s a r e i n d i s a g r e e m e n t w i t h t h o s e i n t h e l i t e r a t u r e t h a t r e p o r t e d l o w e r f r e e f r a c t i o n s (24 t o 30%) ( T a l b o t et al., 1973; M c E r l a n e et al., 1 9 8 7 ) . The d i s a g r e e m e n t i s t h o u g h t t o be due t o t h e f a c t t h a t s e r u m pH was n o t c o n t r o l l e d i n t h e s e s t u d i e s . The r e s u l t s o f t h e p r e s e n t s t u d y a l s o i n d i c a t e d t h a t t h e in vitro b i n d i n g o f m e x i l e t i n e e n a n t i o m e r s t o human se r u m i s n o t s t e r e o s e l e c t i v e . As shown i n t a b l e 5, t h e b i n d i n g o f t h e e n a n t i o m e r s t o HSA was a p p r o x i m a t e l y 3 0 % and was n o t s t e r e o s e l e c t i v e . The b i n d i n g o f t h e e n a n t i o m e r s t o AAG showed s i g n i f i c a n t (p<0.05) s t e r e o s e l e c t i v i t y , w i t h b i n d i n g b e i n g g r e a t e r f o r R ( - ) - m e x i l e t i n e . T h i s i s p r o b a b l y due t o t h e s i g n i f i c a n t l y (p<0.05) g r e a t e r b i n d i n g a f f i n i t y c o n s t a n t o f R ( - ) - m e x i l e t i n e r e l a t i v e t o i t s e n a n t i o m e r ( 3 . 3 . 4 . 2 ) . W i t h l i p o p r o t e i n d e f i c i e n t s e rum, b i n d i n g was s l i g h t l y l o w e r i n c o m p a r i s o n t o t h a t o b t a i n e d w i t h s e rum, s u g g e s t i n g a p o s s i b l e s m a l l c o n t r i b u t i o n o f t h e l i p o p r o t e i n s t o t h e b i n d i n g o f m e x i l e t i n e e n a n t i o m e r s . T h i s b i n d i n g was f o u n d t o be s i g n i f i c a n t l y (p<0.05) g r e a t e r f o r S ( + ) - m e x i l e t i n e . The d a t a i n t a b l e 5 show t h a t t h e b i n d i n g o f t h e e n a n t i o m e r s w i t h i n t h e t h e r a p e u t i c r a n g e c a n be a c c o u n t e d f o r m a i n l y by t h e b i n d i n g t o HSA and AAG. r T a b l e 5. T h e c o n t r i b u t i o n s o f t h e m a j o r d r u g b i n d i n g p r o t e i n s t o t h e s e r u m b i n d i n g o f m e x i l e t i n e e n a n t i o m e r s C o n c e n t r a t i o n o f LP e a c h e n a n t i o m e r Serum HSA AAG d e f . (jug/ml) s e r u m 1.0 R(-)-mex 52±1 7 3 ± 2 7 7 ± 1 6 3 ± 0 S(+)-mex 53+2 72+2 8 4 ± 0 * 5 6 ± 1 * 0.5 R(-)-mex 52±1 70±1 74±1 58±1 S(+)-mex 51±1 6 8 ± 1 84+2* 50+0* V a l u e s a r e means ± s.e.m. o f p e r c e n t f r e e f r a c t i o n s (n = 6 e x p e r i m e n t s ) . HSA, AAG and LP d e f . ser u m r e f e r t o human ser u m a l b u m i n , a j - a c i d g l y c o p r o t e i n and l i p o p r o t e i n d e f i c i e n t s e rum, r e s p e c t i v e l y . C o n c e n t r a t i o n s o f t h e e n a n t i o m e r s u s e d were t h o s e w i t h i n t h e t h e r a p e u t i c r a n g e . * S i g n i f i c a n t ( p < 0 . 0 5 ) . S i n c e many d i s e a s e c o n d i t i o n s a r e known t o i n f l u e n c e t h e b i n d i n g o f d r u g s t o AAG and HSA ( R o u t l e d g e , 1985; K r e m e r et al., 1 9 8 8 ) , t h e p r e s e n t r e s u l t s s u g g e s t t h a t t h e b i n d i n g o f t h e e n a n t i o m e r s may c h a n g e i n d i s e a s e s t a t e s . However, P e n t i k a i n e n et al. ( 1 9 8 4 ) f o u n d no s i g n i f i c a n t d i f f e r e n c e s i n t h e f r e e f r a c t i o n s o f r a c e m i c m e x i l e t i n e i n m y o c a r d i a l i n f a r c t i o n p a t i e n t s , d u r i n g t h e a c u t e and r e c o v e r y p h a s e s . The m o d e r a t e b i n d i n g o f t h e e n a n t i o m e r s o b s e r v e d i n t h i s s t u d y , and t h e i r l a r g e v o l u m e o f d i s t r i b u t i o n i n humans ( 6 . 6 ± 2.6 and 7.3 ± 2.4 L / k g f o r R ( - ) - and S ( + ) - m e x i l e t i n e , r e s p e c t i v e l y ) ( I g w e m e z i e et al., 1 9 8 9 ) , s u g g e s t t h a t s e r u m p r o t e i n b i n d i n g may n o t be an i m p o r t a n t f a c t o r i n t h e p h a r m a c o d y n a m i c s o f r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s . 3.3.4 D e t e r m i n a t i o n o f B i n d i n g C o n s t a n t s 3.3.4.1 Serum B i n d i n g D a t a To d e t e r m i n e t h e c o n s t a n t s c h a r a c t e r i z i n g t h e b i n d i n g o f t h e e n a n t i o m e r s t o s e r u m p r o t e i n s , t h e b i n d i n g o f a w i d e r a n g e o f t o t a l d r u g c o n c e n t r a t i o n s ( 0 . 1 - 2 0 0 0 /xg/ml o f e a c h e n a n t i o m e r ) was i n v e s t i g a t e d . The b i n d i n g d a t a o b t a i n e d a r e shown i n t a b l e 6. The r e s u l t s i n d i c a t e t h a t t h e b i n d i n g o f m e x i l e t i n e e n a n t i o m e r s was n o t 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 i n t h e t h e r a p e u t i c r a n g e o f 0.5-2.0 nq/m'] o f r a c e m i c m e x i l e t i n e i n man ( t h e f r e e f r a c t i o n s a t t o t a l e n a n t i o m e r c o n c e n t r a t i o n s o f 0.25, 0.5 and 1.0 /Kj/ml were c o m p a r e d by one-way ANOVA). However, s i g n i f i c a n t i n c r e a s e s i n t h e f r e e f r a c t i o n s o f t h e e n a n t i o m e r s was e v i d e n t a t 50 /ng/ml and a b o v e , i n d i c a t i n g a p o s s i b l e s a t u r a t i o n o f t h e b i n d i n g s i t e s on t h e p r o t e i n ( s ) . F i g u r e 15 i s a r e p r e s e n t a t i v e " R o s e n t h a l " p l o t o f t h e b i n d i n g d a t a o b t a i n e d f o r e a c h e n a n t i o m e r i n a Table 6. The percent free fractions of mexiletine enantiomers in serum from healthy human subjects Concentration of Free Fraction enantiomer in R(-)-mex S(+)-mex serum (M9/ml) 0.10 0.42 + 0.02 0.44 + 0.04 0.25 0.49 + 0.01 0.49 + 0.02 0.50 0.52 ± 0.01 0.52 + 0.01 1.0 0.52 ± 0.01 0.51 + 0.02 5.0 0.55 ± 0.01 0.58 + 0.01 20.0 0.59 ± 0.02 0.60 + 0.01 50.0 0.62 ± 0.02 0.62 + 0.02 100.0 0.62 ± 0.02 0.61 + 0.02 500.0 0.68 ± 0.02 0.65 + 0.02 1000.0 0.70 ± 0.02 0.69 + 0.02 1500.0 0.77 ± 0.02 0.75 + 0.02 2000.0 0.77 ± 0.01 0.75 + 0.01 * Free fraction values are means + s.e.m., n = 6 subjects. 106 2.0 O O 1.0--0.5 T o R(—)-mex • S(+) —mex o o o.o T 1 1 1 1 1 1 -100 0 100 200 300 400 500 Bound ( x10~~ 5 M) F i g u r e 15. A r e p r e s e n t a t i v e " R o s e n t h a l " p l o t o f t h e b i n d i n g o f m e x i l e t i n e e n a n t i o m e r s t o s e r u m f r o m a h e a l t h y s u b j e c t . h e a l t h y s u b j e c t . A n a l y s i s o f t h e d a t a u s i n g t h e n o n - l i n e a r l e a s t s q u a r e s r e g r e s s i o n p r o g r a m "ENZFITTER" ( L e a t h e r b a r r o w , 1987) i n d i c a t e d t h e p r e s e n c e o f two c l a s s e s o f b i n d i n g s i t e s . A h i g h a f f i n i t y , l o w c a p a c i t y c l a s s o f b i n d i n g s i t e s and a lo w a f f i n i t y , h i g h c a p a c i t y c l a s s o f b i n d i n g s i t e s . The a f f i n i t y and c a p a c i t y c o n s t a n t s d e t e r m i n e d f o r t h e s e c l a s s e s o f b i n d i n g s i t e s a r e shown i n t a b l e 7. T h e r e were no s i g n i f i c a n t d i f f e r e n c e s i n t h e b i n d i n g c o n s t a n t s o f t h e e n a n t i o m e r s . 3.3.4.2 aj-Acid Glycoprotein Binding Data A s i m i l a r a n a l y s i s was c a r r i e d o u t on t h e b i n d i n g d a t a o b t a i n e d w i t h AAG. The b i n d i n g c o n s t a n t s d e r i v e d a r e shown i n t a b l e 8. AAG showed o n l y one c l a s s o f b i n d i n g s i t e s . T h i s was a h i g h a f f i n i t y , l o w c a p a c i t y b i n d i n g s i t e and p r o b a b l y a c c o u n t s f o r t h e h i g h a f f i n i t y b i n d i n g o f s e r u m ( t a b l e 8 ) . The b i n d i n g c a p a c i t y o f t h e e n a n t i o m e r s were n o t s i g n i f i c a n t l y d i f f e r e n t (mean v a l u e s were 1.9 x 10" 5M and 1.8 x 10" 5M f o r R ( - ) - and S ( + ) - m e x i l e t i n e , r e s p e c t i v e l y ) . However, R ( - ) - m e x i l e t i n e had a s i g n i f i c a n t l y (p<0.05) g r e a t e r a f f i n i t y t h a n t h e S ( + ) - e n a n t i o m e r (mean v a l u e s were 2.0 x 1 0 4 M _ 1 v s 1.2 x 1 0 4 M _ 1 ) . T he number o f b i n d i n g s i t e s p e r m o l e o f p r o t e i n (n) was a p p r o x i m a t e l y 1 f o r b o t h e n a n t i o m e r s . T h i s i s c o n s i s t e n t w i t h t h e r e p o r t s o f o t h e r s t u d i e s on t h e b i n d i n g o f b a s i c d r u g s t o AAG ( M u l l e r et al., 1985; K r e m e r et al., 1 9 8 8 ) . I t i s g e n e r a l l y a c c e p t e d i n t h e l i t e r a t u r e t h a t o n l y one h i g h a f f i n i t y c l a s s o f b i n d i n g s i t e s e x i s t on AAG ( M u l l e r et al., 1 9 8 5 ) . Table 7. Binding constants o f m e x i l e t i n e enantiomers i n serum from healthy subjects R(-) -mex S(+) -mex Subject m p i K l n 2 P 2 njPj Kl n 2 P 2 K 2 (xlO" 6M) ( x l o V 1 ) (xlO" 3M) (x ioV 1 ) (xl0" 6M) ( x l 0 5 M _ 1 ) (xlO" 3M) (x loV 1 ) 1 1.5 9.9 6.2 1.2 1.7 4.2 7.4 1.1 2 1.3 7.0 4.4 1.9 1.2 12.2 6.2 1.2 3 2.1 3.1 5.0 1.3 2.1 3.1 4.7 1.4 4 3.9 2.3 5.6 0.7 7.8 1.1 5.7 0.8 5 3.0 2.9 5.9 0.9 3.2 2.9 9.7 0.5 6 2.7 2.1 4.7 1.3 3.0 . 1.2 5.4 1.2 mean 2.4 4.5 5.3 1.2 3.2 4.1 6.5 1.0 is.e.m 0.4 1.3 0.3 0.2 1.0 1.7 0.7 0.1 K and nP r e f e r to the a f f i n i t y and c a p a c i t y constants, r e s p e c t i v e l y . Binding constants were generated using the non-linear l e a s t squares r e g r e s s i o n program "ENZFITTER". T a b l e 8. B i n d i n g c o n s t a n t s o f m e x i l e t i n e e n a n t i o m e r s i n i s o l a t e d human a j - a c i d g l y c o p r o t e i n (AAG) nP n K ( x l O " 5 M ) ( x l 0 4 M _ 1 ) R(-)-mex 1.9+0.1 0.8 2 . 0 ± 0 . 1 S(+)-mex 1.8±0.1 0.8 1 . 2 ± 0 . 1 * V a l u e s a r e means ± s.e.m, n = 4 e x p e r i m e n t s . K and nP r e f e r t o t h e b i n d i n g a f f i n i t y and c a p a c i t y c o n s t a n t s , r e s p e c t i v e l y . AAG c o n c e n t r a t i o n u s e d was 0.1% w/v. The m o l e c u l a r w e i g h t o f AAG u s e d f o r t h e c a l c u l a t i o n o f n ( t h e number o f b i n d i n g s i t e s p e r m o l e o f p r o t e i n ) was 44,000 D a l t o n s . * S i g n i f i c a n t ( p < 0 . 0 5 ) . 110 3 .3 .4 .3 Albumin B i n d i n g Data In c o n t r a s t t o a j - a c i d g l y c o p r o t e i n , t h e b i n d i n g d a t a f r o m a l b u m i n showed t h e p r e s e n c e o f two c l a s s e s o f b i n d i n g s i t e s ( t a b l e 9 ) . The l o w a f f i n i t y s i t e s h ad c o m p a r a b l e b i n d i n g c o n s t a n t s t o t h o s e o f t h e c o r r e s p o n d i n g s i t e s i n s e r u m f o r e a c h o f t h e e n a n t i o m e r s . R ( - ) - M e x i l e t i n e had a s l i g h t l y g r e a t e r c a p a c i t y c o n s t a n t t h a n S ( + ) - m e x i l e t i n e (mean v a l u e s were 4.0 and 3.2 x 1 0 " 3 M). However, t h e a f f i n i t y o f R ( - ) - m e x i l e t i n e was s i g n i f i c a n t l y (p<0;05) l e s s t h a n t h a t o f i t s a n t i p o d e (mean v a l u e s were 0.5 and 1.2 x 1 0 2 M " * ) . T h e mean number o f b i n d i n g s i t e s p e r m o l e o f p r o t e i n were 6.6 and 5.4 f o r R ( - ) - and S ( + ) - m e x i l e t i n e , r e s p e c t i v e l y . The h i g h a f f i n i t y c l a s s o f b i n d i n g s i t e s h a d b i n d i n g c o n s t a n t s t h a t w e r e c o m p a r a b l e t o t h e same c l a s s o f b i n d i n g s i t e s i n s e r u m . T h e r e were no s i g n i f i c a n t d i f f e r e n c e s i n t h e b i n d i n g c o n s t a n t s o f t h e e n a n t i o m e r s . The mean number o f b i n d i n g s i t e s o b t a i n e d f o r t h e h i g h a f f i n i t y s i t e s were 0.006 and 0.001 f o r R ( - ) - and S ( + ) - m e x i l e t i n e , r e s p e c t i v e l y . The m a g n i t u d e o f t h e s e n v a l u e s s u g g e s t t h a t t h e h i g h a f f i n i t y s i t e may n o t r e p r e s e n t a t r u e b i n d i n g s i t e on a l b u m i n . I t may be s p e c u l a t e d t h a t t h e h i g h a f f i n i t y b i n d i n g c o u l d h a v e r e s u l t e d f r o m t h e p r e s e n c e o f AAG c o n t a m i n a n t i n t h e HSA. 3.4 T i s s u e D i s t r i b u t i o n K i n e t i c s o f M e x i l e t i n e E n a n t i o m e r s i n Rats The t i s s u e d i s t r i b u t i o n k i n e t i c s o f m e x i l e t i n e e n a n t i o m e r s was s t u d i e d i n m a l e S p r a g u e - D a w l e y r a t s a f t e r a d m i n i s t r a t i o n o f a s i n g l e i . v . d o s e (10 mg/kg) o f r a c e m i c m e x i l e t i n e . T a b l e 9. B i n d i n g c o n s t a n t s o f m e x i l e t i n e e n a n t i o m e r s i n i s o l a t e d human serum a l b u m i n n l p l "1 K l n 2 P 2 n 2 K 2 ( x l O " 5 M ) ( x l 0 5 M _ 1 ) ( x l O ~ 3 M ) (xloV 1) R(-)-mex 3.4+0.7 0.006 0.9+0.3 4.0+0.4 6.6 0.5±0.1 S(+)-mex 2.0+0.3 0.001 1.2+0.2 3.2±0.4 5.4 1.2+0.1* V a l u e s a r e means ± s.e.m, n = 4 e x p e r i m e n t s . Albumin c o n c e n t r a t i o n was 4% w/v. The m o l e c u l a r w e i g h t o f albumin used f o r t h e c a l c u l a t i o n o f n ( t h e number o f b i n d i n g s i t e s p e r mole o f p r o t e i n ) was 66,000 D a l t o n s . * S i g n i f i c a n t (p<0.05). 3.4.1 Serum L e v e l s The mean s e r u m c o n c e n t r a t i o n s o f m e x i l e t i n e e n a n t i o m e r s p l o t t e d as a f u n c t i o n o f t i m e a r e shown i n f i g u r e 16. The c o n c e n t r a t i o n - t i m e d e c a y c u r v e was b i e x p o n e n t i a l . S ( + ) - M e x i l e t i n e had a g r e a t e r s e r u m c o n c e n t r a t i o n a t e a c h s a m p l i n g t i m e t h a n t h e R ( - ) - e n a n t i o m e r b u t t h i s d i d n o t a c h i e v e s t a t i s t i c a l s i g n i f i c a n c e u n t i l 1 h a f t e r d r u g a d m i n i s t r a t i o n . However, t h e r a t i o o f t h e e n a n t i o m e r s r e m a i n e d c o n s t a n t b e t w e e n 1 and 6 h. 3.4.2 P h a r m a c o k i n e t i c P a r a m e t e r s T a b l e 10 shows t h e p h a r m a c o k i n e t i c p a r a m e t e r s d e t e r m i n e d f o r t h e e n a n t i o m e r s . S i n c e b l o o d a t e a c h s a m p l i n g t i m e was c o l l e c t e d f r o m d i f f e r e n t a n i m a l s , o n l y mean p h a r m a c o k i n e t i c p a r a m e t e r s c o u l d be c a l c u l a t e d . The s y s t e m i c c l e a r a n c e ( C L ) o f R ( - ) - m e x i l e t i n e was 3 2 % g r e a t e r t h a n t h a t o f t h e S ( + ) - e n a n t i o m e r ( 1 6 1 . 8 m l / m i n / k g vs 122.9 m l / m i n / k g ) . M e x i l e t i n e i s a r e s t r i c t i v e l y c l e a r e d d r u g ( W i l k i n s o n and S h a n d , 1 9 7 5 ) , t h u s , i t s CL d e p e n d s d i r e c t l y on b o t h t h e i n t r i n s i c c l e a r a n c e (CL^ n^.) and f r e e f r a c t i o n ( f f ) . S i n c e t h e in vivo f f o f t h e e n a n t i o m e r s were n o t m a r k e d l y d i f f e r e n t f r o m e a c h o t h e r ( s e c t i o n 3 . 4 . 3 ) , t h e s e r e s u l t s s u g g e s t t h a t t h e C L ^ n t o f R ( - ) - m e x i l e t i n e may be g r e a t e r t h a n t h a t o f t h e S ( + ) - e n a n t i o m e r . A s i g n i f i c a n t l y g r e a t e r g l u c u r o n i d a t i o n o f R ( - ) - m e x i l e t i n e , r e l a t i v e t o i t s o p p o s i t e e n a n t i o m e r , has b e e n r e p o r t e d i n man ( G r e c h - B e l a n g e r et a / . , 1 9 8 6 ) . R ( - ) - M e x i l e t i n e was a l s o f o u n d t o ha v e a 2 2 % g r e a t e r s t e a d y - s t a t e v o l u m e o f d i s t r i b u t i o n t h a n t h e S ( + ) - e n a n t i o m e r ( 9 . 0 L / k g v s 7.4 L / k g ) . S t e r e o s e l e c t i v e d i f f e r e n c e s i n t h e v o l u m e o f d i s t r i b u t i o n o f e n a n t i o m e r s 1 1 1 1 1 1 1—; 1 0 1 2 3 4 5 6 Time (h) F i g u r e 16. The s e m i l o g a r i t h m i c p l o t o f serum c o n c e n t r a t i o n vs t i m e f o r m e x i l e t i n e e n a n t i o m e r s i n r a t s f o l l o w i n g t h e a d m i n i s t r a t i o n o f a s i n g l e i . v . d o s e (10 mg/kg) o f r a c e m i c m e x i l e t i n e . V a l u e s a r e means ± s.e.m, n * 4-5 r a t s . to 114 T a b l e 10. T h e p h a r m a c o k i n e t i c p a r a m e t e r s o f m e x i l e t i n e e n a n t i o m e r s c a l c u l a t e d f r o m s e r u m c o n c e n t r a t i o n - t i m e d a t a a f t e r a s i n g l e i . v . . d o s e (10 mg/kg) o f r a c e m i c m e x i l e t i n e P a r a m e t e r R(-)-mex S(+)-mex (min) 19.8 24.4 (h ) 1.4 1.3 AUC (ng.h.ml"*) 515.1 677.9 CL (ml/min/kg) 161.8 122.9 V s s (Vkg) 9.0 7.4 can result from differences in their tissue and/or serum protein binding (Williams and Lee, 1985). Due to the small difference in the free fractions of the enantiomers, i t is unlikely that serum protein binding would account for the observed stereoselective volume of d istr ibut ion. The distr ibution h a l f - l i f e of S(+)-mexiletine, 24.4 min, was 24% greater than that of the R(-)-enantiomer, 19.8 min. Thus, the greater volume of distr ibution of R(-)-mexiletine appears to result from i ts greater tissue uptake. Stereoselective tissue uptake of other chiral drugs such as propranolol (Kawashima et al., 1976) and timolol (Tocco et al., 1976) have been reported. The terminal elimination hal f - l ives of the enantiomers were 1.4 and 1.3 h for R(-)- and S(+)-mexiletine, respectively. These values were markedly shorter than those reported in humans (9.1 ± 2.9 and 11.0 ± 3.8 h for R(-)- and S(+)-mexiletine, respectively) (Igwemezie et al., 1989). The expected shorter terminal elimination h a l f - l i f e for R(-)-mexiletine, due to i ts larger systemic clearance, was offset by i ts equally greater volume of d istr ibut ion. This resulted in an apparent lack of a difference in the terminal elimination ha l f - l ives of both enantiomers. 3.4.3 Serum Protein Binding Table 11 shows the in vivo percent free fractions of mexiletine enantiomers in the rats. The mean free fraction of R(-)-mexiletine, 49%, was signi f icant ly greater than that of the S(+)-enantiomer (44%). However, the difference in the free fractions is small and would not be expected to have any pharmacokinetic or pharmacodynamic consequences. Table 11. The in vivo free serum concentrations (ng/ml) and the percent free fractions (ff) of mexiletine enantiomers after a single i . v . dose (10 mg/kg) of racemic mexiletine Time (h) R(-)-mex S(+)-mex ff 0.08 400.1 ± 33.6 52 426.4 ± 40.3 47 0.25 212.8 ± 2.4 48 243.0 ± 3.1 45 0.50 215.6 ± 6.1 49 215.0 ± 6.0 41 1.00 49.7 ± 2.3 47 74.3 ± 10.0 45 mean ± s.e.m. 49 ± 1 * 44 ± 1 Concentrations are means ± s.e.m, n = 4-5 rats. * significant (p<0.05). 117 3.4.4 R e c o v e r y o f t h e Ena n t i o m e r s from t h e T i s s u e s T a b l e 12 shows t h e r e c o v e r y o f t h e e n a n t i o m e r s f r o m t i s s u e h o m o g e n a t e s . R e c o v e r y was a p p r o x i m a t e l y 8 0 % o r more i n a l l t h e t i s s u e s s t u d i e d . 3.4.5 T i s s u e L e v e l s T a b l e 13 p r e s e n t s t h e c o n c e n t r a t i o n o f t h e e n a n t i o m e r s i n t h e v a r i o u s t i s s u e s s t u d i e d . Maximum c o n c e n t r a t i o n s were o b s e r v e d a t 5 m i n , w h i c h was t h e e a r l i e s t s a m p l i n g t i m e . T h e r e were no s i g n i f i c a n t d i f f e r e n c e s i n t h e maximum t i s s u e c o n c e n t r a t i o n s e x c e p t f o r t h e l i v e r w h ere t h e c o n c e n t r a t i o n o f S ( + ) - m e x i l e t i n e , 7.5 ± 2.6 /xg/g, was 2.4 f o l d g r e a t e r t h a n t h a t o f t h e R ( - ) - e n a n t i o m e r (3.1 ± 1.1 ng/g). However, a s i g n i f i c a n t l y (p<0.05) g r e a t e r c o n c e n t r a t i o n f o r S ( + ) - m e x i l e t i n e was e v i d e n t a t 1 h o u r and t h e r e a f t e r i n a l l t h e t i s s u e s s t u d i e d , r e f l e c t i n g t h e c h a n g e s i n t h e e n a n t i o m e r i c r a t i o s i n ser u m ( s e c t i o n 3 . 4 . 1 ) . T h e s e r e s u l t s i n d i c a t e d t h a t t h e r e was a r a p i d u p t a k e o f m e x i l e t i n e e n a n t i o m e r s i n t o t h e h i g h l y p e r f u s e d t i s s u e s ( w h i c h i n c l u d e d a l l t h e t i s s u e s s t u d i e d e x c e p t t h e f a t ) , f o l l o w e d by a s l o w e r d i s t r i b u t i o n , p r e s u m a b l y i n t o t h e d e e p t i s s u e c o m p a r t m e n t . The d i s t r i b u t i o n i n t o t h e d e e p t i s s u e c o m p a r t m e n t a p p e a r e d t o be s i g n i f i c a n t l y g r e a t e r f o r t h e R ( - ) - e n a n t i o m e r w h i c h r e s u l t e d i n t h e s i g n i f i c a n t l y l o w e r s e r u m c o n c e n t r a t i o n s o b s e r v e d f o r t h i s e n a n t i o m e r . The p h a r m a c o k i n e t i c p a r a m e t e r s c a l c u l a t e d f o r t h e e n a n t i o m e r s f r o m t h e t i s s u e c o n c e n t r a t i o n - t i m e d a t a a r e s u m m a r i z e d i n t a b l e 14. The e l i m i n a t i o n h a l f - l i v e s i n t h e t i s s u e s s t u d i e d were n o t s i g n i f i c a n t l y d i f f e r e n t f r o m e a c h o t h e r . The a r e a u n d e r t h e t i s s u e c o n c e n t r a t i o n - t i m e Table 12. Percent recovery of mexiletine enantiomers from tissue homogenates. Enantiomer concentration (ng/ml) l ive r kidney heart fat lung brain 500 R(-)mex 79 83 86 92 82 85 S(+)mex 80 83 86 92 82 85 125 R(-)mex 87 87 87 94 90 90 S(+)mex 84 84 84 84 84 89 Values are the averages of duplicate determinations. 1 ml of tissue homogenate contained 100 mg of tissue. Table 13. The time-dependent concentrations (^g/g) of mexiletine enantiomers in the tissues of rats following a single i.v. dose (10 mg/kg) of racemic mexiletine Time- Brain Heart Lung Kidney Liver Fat R(-) -mex 18.99 ± 0.76 6.23 ± 0.46 24.57 ± 0.64 19. 99 + 0.64 3.10 + 0. 53* 1.03 + 0.04 5 min S(+) -mex 19.18 ± 0.82 6.52 + 0.46 25.86 + 0.79 20. 12 ± 0.66 7.47 + 1. 28 1.10 + 0.05 R(-) -mex 12.44 + 0.06 3.30 ± 0.15 14.05 ± 0.03 8 92 + 0.64 2.06 + 0. 30* 1.04 t 0.04 15 min S(+) -mex 13.33 t 0.06 3.38 ± 0.15 16.21 + 0.03 10. 66 ± 0.76 4.12 ± 0. 50 1.16 i 0.04 R(-) -mex 6.67 ± 0.61 2.58 + 0.46 13.70 ± 0.17 6. 78 i 1.19 0.79 ± 0. 02* 0.78 i 0.14 30 min S(+) -mex 7.00 ± 0.57 2.71 + 0.48 14.75 ± 0.18 7. 58 ± 1.30 1.63 + 0. 01 0.86 ± 0.14 R(-) -mex 3.3 ± 0.18* 1.11 ± 0.23* 6.48 ± 1.09* 2. 53 i 0.44* 0.65 ± 0. 09* 0.42 ± 0.04* 1 hr S( + ) -mex 4.2 ± 0.32 1.43 + 0.24 8.59 ± 1.00 3. 94 ± 0.58 1.31 i 0. 28 0.54 ± 0.06 R(-) -mex 0.84 ± 0.09* 0.41 + 0.10* 2.68 ± 0.66* 0. 55 ± 0.06* 0.23 ± 0. 02* 0.18 t 0.02* 2 hr S( + ) -mex 1.30 ± 0.14 0.55 ± 0.12 3.88 ± 1.04 1. 11 + 0.15 0.38 ± 0. 06 0.23 + 0.02 R(-) -mex 0.18 ± 0.02* 0.10 ± 0.12* 0.47 ± 0.08* 0. 12 i 0.02* 0.03 + 0. 01* 0.02 ± 0.00* 4 hr S( + ) -mex 0.23 ± 0.03 0.13 ± 0.01 0.70 ± 0.11 0. 23 + 0.02 0.07 ± 0. 01 0.03 + 0.00 R(-) -mex 0.05 ± 0.01* 0.03 + 0.01* 0.14 ± 0.02* 0. 04 ± 0.01* 0.02 + 0. 01* 6 hr S( + ) -mex 0.07 ± 0.01 0.04 + 0.01 0.22 ± 0.02 0. 07 + 0.01 0.04 ± 0. 00 * s i g n i f i c a n t (P< 0.05) ** mean + s.e.m., n = 4-5 rats T a b l e 14. P h a r m a c o k i n e t i c p a r a m e t e r s o f m e x i l e t i n e e n a n t i o m e r s f r o m t i s s u e d a t a a f t e r a s i n g l e i . v . d o s e (10 mg/kg) o f r a c e m i c m e x i l e t i n e A U C T ( u g . h . g - 1 ) T i s s u e / S e r u m H e a r t R ( - ) -mex 1.1 4.4 8 S(+) -mex 1.1 5.1 7 B r a i n R ( - ) -mex 1.0 11.9 25 S(+) -mex 1.0 14.0 21 L i v e r R ( - ) -mex 1.1 2 - ° * 4 * S(+) -mex 1.2 4.4 8 Lung R(-) -mex 1.0 22.4 32 S(+) -mex 1.0 27.1 28 K i d n e y R ( - ) -mex 1.1 11.1 26 S(+) -mex 1.0 13.6 22 F a t - R(-) -mex 0.7 1.3 1 S(+) -mex 0.7 1.5 1 * S i g n i f i c a n t ( p < 0 . 0 5 ) , n = 4-5 r a t s . T i s s u e / S e r u m r a t i o s a r e v a l u e s a t t h e o b s e r v e d maximum t i s s u e c o n c e n t r a t i o n s i . e . 5 min a f t e r d r u g a d m i n i s t r a t i o n . A U C j r e f e r s t o a r e a u n d e r t h e t i s s u e c o n c e n t r a t i o n - t i m e c u r v e . c u r v e ( A U C j ) was s m a l l e r f o r R ( - ) - m e x i l e t i n e b u t t h e d i f f e r e n c e s d i d n o t e x c e e d 16%; e x c e p t f o r t h e l i v e r w ere t h e AUC-r was 1 2 0 % g r e a t e r f o r t h e S ( + ) - e n a n t i o m e r . The t i s s u e / s e r u m r a t i o o f t h e e n a n t i o m e r s ( a t t h e maximum o b s e r v e d t i s s u e c o n c e n t r a t i o n ) i n d i c a t e d an e x t e n s i v e t i s s u e u p t a k e . T h e h i g h e s t r a t i o s were f o u n d i n t h e l u n g s w h i c h a c c u m u l a t e d 32 and 2 8 - f o l d t h e s e r u m c o n c e n t r a t i o n o f R ( - ) - and S ( + ) - m e x i l e t i n e , r e s p e c t i v e l y . The b r a i n a c c u m u l a t e d 25 and 2 1 - f o l d ; w h i l e t h e h e a r t a c c u m u l a t e d 8 and 7 - f o l d t h e s e r u m c o n c e n t r a t i o n s o f R ( - ) - and S ( + ) - m e x i l e t i n e , r e s p e c t i v e l y . S i m i l a r h i g h t i s s u e / s e r u m r a t i o s h a v e been r e p o r t e d f o r r a c e m i c m e x i l e t i n e i n r a t s ( B a r r i g o n et a 7 . , 1 9 8 3 ) . T h e h i g h t i s s u e / s e r u m r a t i o s o b s e r v e d i n t h e p r e s e n t s t u d y w e r e a l s o m a i n t a i n e d o v e r t i m e , as shown f o r b r a i n and h e a r t t i s s u e s i n f i g u r e 17. E x c e p t f o r t h e l i v e r and f a t , a l l t h e o t h e r t i s s u e s showed a 1 4 - 1 9 % g r e a t e r a c c u m u l a t i o n o f R ( - ) - m e x i l e t i n e r e l a t i v e t o t h e S ( + ) - e n a n t i o m e r . The l i v e r t i s s u e e x h i b i t e d a 100 % g r e a t e r a c c u m u l a t i o n o f S ( + ) - m e x i l e t i n e w h i l e t h e f a t t i s s u e d i d n o t a c c u m m u l a t e t h e e n a n t i o m e r s . The h i g h t i s s u e / s e r u m r a t i o s o f m e x i l e t i n e e n a n t i o m e r s c a n be a t t r i b u t e d t o a number o f m e c h a n i s m s ; two o f w h i c h may be an a c t i v e t r a n s p o r t p r o c e s s a n d / o r h i g h a f f i n i t y b i n d i n g t o s i t e s i n t h e t i s s u e s . T h e 2 . 4 - f o l d g r e a t e r l i v e r c o n c e n t r a t i o n o f S ( + ) - t n e x i l e t i n e r e l a t i v e t o t h e R ( - ) - e n a n t i o m e r may r e s u l t f r o m s t e r e o s e l e c t i v e a c t i v e t r a n s p o r t a n d / o r t i s s u e b i n d i n g a n d / o r m e t a b o l i s m . However, f r o m t h e d a t a o b t a i n e d i n t h i s s t u d y , i t was n o t p o s s i b l e t o d e t e r m i n e w h i c h m e c h a n i s m ( s ) was i n o p e r a t i o n . The t i s s u e s s t u d i e d e x h i b i t e d r e d i s t r i b u t i o n a t a r a t e g r e a t e r f o r t h e R ( - ) - e n a n t i o m e r , r e f l e c t i n g t h e c h a n g i n g r a t i o o f t h e e n a n t i o m e r s i n t h e s e r u m . T h u s , a s i g n i f i c a n t l y 1 00.000 r, F i g u r e 17. The c o n c e n t r a t i o n - t i m e p r o f i l e o f m e x i l e t i n e e n a n t i o m e r s i n s e r u m , h e a r t and b r a i n t i s s u e s f o l l o w i n g t h e a d m i n i s t r a t i o n o f a s i n g l e i . v . d o s e (10 mg/kg) o f r a c e m i c m e x i l e t i n e . V a l u e s a r e means ± s.e.m. o f r e s u l t s f r o m 4-5 r a t s . 123 g r e a t e r c o n c e n t r a t i o n o f S ( + ) - m e x i l e t i n e was e v i d e n t i n a l l t h e t i s s u e s b e t w e e n 1 and 6 h o u r s b u t t h e e n a n t i o m e r i c r a t i o was c o n s t a n t as i n d i c a t e d by t h e c o m p a r a b l e 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 v e s o f t h e e n a n t i o m e r s i n t h e t i s s u e s . However, much o f t h e e n a n t i o m e r s were a l r e a d y c l e a r e d f r o m t h e t i s s u e s a t t h e t i m e s t e r e o s e l e c t i v i t y became a p p a r e n t , h e n c e o n l y s m a l l d i f f e r e n c e s i n t h e A U C j o f t h e e n a n t i o m e r s was e v i d e n t i n most o f t h e t i s s u e s s t u d i e d ( t a b l e 1 4 ) . T h i s i s n o t l i k e l y t o h a v e any p h a r m a c o k i n e t i c c o n s e q u e n c e s . I t c a n be c o n c l u d e d f r o m t h e p r e s e n t r e s u l t s t h a t t h e u p t a k e o f m e x i l e t i n e e n a n t i o m e r s i n t o t h e t a r g e t t i s s u e , i . e . t h e h e a r t , as w e l l as t h e b r a i n , i s n o t s t e r e o s e l e c t i v e . The h i g h b r a i n l e v e l s o f t h e e n a n t i o m e r s a r e most l i k e l y r e s p o n s i b l e f o r t h e CNS s i d e e f f e c t s i n d u c e d by m e x i l e t i n e i n b o t h a n i m a l s ( s e c t i o n 3.5.2.5; U p r i c h a r d and H a r r o n , 1989) and man ( P a l i l e o et al., 1982, Waspe et al., 1 9 8 3 ) . 3.5 A n t i a r r h y t h m i c E f f e c t s o f Racemic M e x i l e t i n e and i t s E n a n t i o m e r s i n Rats 3.5.1 E l e c t r i c a l l y - i n d u c e d A r r h y t h m i a i n P e n t o b a r b i t o n e A n a e s t h e t i z e d Rats The r e l a t i v e a n t i a r r h y t h m i c a c t i v i t y o f r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s was i n v e s t i g a t e d u s i n g e l e c t r i c a l s t i m u l a t i o n o f t h e h e a r t i n p e n t o b a r b i t o n e a n a e s t h e t i z e d r a t s . To d e t e r m i n e t h e t r u e e f f e c t s o f t h e d r u g s on t h e m e a s u r e d v a r i a b l e s , c h a n g e s due t o s a l i n e ( c o n t r o l ) w ere f i r s t s u b t r a c t e d . The c u m u l a t i v e d o s e - r e s p o n s e c u r v e s g e n e r a t e d w ere a n a l y s e d by " R e p e a t e d M e a s u r e s " ANOVA. 3.5.1.1 Dosage and Plasma Concentration T h e d o s e s o f r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s u s e d were 4, 8, 16 and 32 mg/kg, a d m i n i s t e r e d c u m u l a t i v e l y . T h e o b s e r v e d p l a s m a c o n c e n t r a t i o n s o f r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s i n c r e a s e d l i n e a r l y ( r 2 > 0 . 9 9 ) w i t h d o s e as shown i n f i g u r e 18. R,S- and S ( + ) - M e x i l e t i n e had s i g n i f i c a n t l y (p<0.05) g r e a t e r p l a s m a c o n c e n t r a t i o n s t h a n t h e R ( - ) e n a n t i o m e r . The maximum c o n c e n t r a t i o n s a c h i e v e d were 2.3 ± 0.2, 2.3 ± 0.2 and 1.7 ± 0.1 pg/ml f o r S ( + ) - , R,S-, and R ( - ) - m e x i l e t i n e , r e s p e c t i v e l y . N o r m a l l y , t h e c o n c e n t r a t i o n o f t h e r a c e m a t e w o u l d be e x p e c t e d t o be b e t w e e n t h a t o f t h e two e n a n t i o m e r s . T h e r e f o r e , t h e c o m p a r a b l e p l a s m a c o n c e n t r a t i o n s o f R,S- and S ( + ) - m e x i l e t i n e i n d i c a t e d a p o s s i b l e i n t e r a c t i o n b e t w e e n t h e two e n a n t i o m e r s . T h i s may o c c u r a t t h e s e r u m b i n d i n g , t i s s u e b i n d i n g a n d / o r m e t a b o l i s m l e v e l . The in vivo f r e e f r a c t i o n s d e t e r m i n e d f o r r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s a r e shown i n t a b l e 15. T h e r e w e r e no s i g n i f i c a n t d i f f e r e n c e s i n t h e f r e e f r a c t i o n v a l u e s . T h u s , i t i s u n l i k e l y t h a t s e r u m b i n d i n g i n t e r a c t i o n s w o u l d h a v e b e e n r e s p o n s i b l e f o r t h e o b s e r v e d p l a s m a c o n c e n t r a t i o n s . T h e s e r e s u l t s s u g g e s t t h a t t h e r e may h a v e e x i s t e d an i n t e r a c t i o n b e t w e e n t h e e n a n t i o m e r s o f m e x i l e t i n e a t t h e t i s s u e b i n d i n g a n d / o r m e t a b o l i s m l e v e l w h i c h r e s u l t e d i n g r e a t e r t h a n e x p e c t e d p l a s m a c o n c e n t r a t i o n s o f t h e r a c e m a t e . S u c h an i n t e r a c t i o n a f f e c t i n g m e t a b o l i s m has been r e p o r t e d f o r l e v o m e t h o r p h a n w h e r e i n c r e a s e d p l a s m a l e v e l s and a n a l g e s i c a c t i v i t y w ere o b s e r v e d when t h e d r u g was c o - a d m i n i s t e r e d w i t h i t s o p p o s i t e e n a n t i o m e r , d e x t r o m e t h o r p h a n ( C o o p e r and A n d e r s , 1 9 7 4 ) . However, b a s e d on d a t a o b t a i n e d f r o m t h e p r e s e n t s t u d y , i t was n o t p o s s i b l e t o d e t e r m i n e 125 3000 i 1 1 1 r -0 8 16 24 32 D o s e ( m g / k g ) F i g u r e 18. The r e l a t i o n s h i p b e t w e e n d o s e ( c u m u l a t i v e ) and t h e p l a s m a c o n c e n t r a t i o n o f r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s i n P e n t o b a r b i t o n e a n a e s t h e t i z e d r a t s . B l o o d s a m p l e s w e r e c o l l e c t e d 10 m i n a f t e r e a c h d o s e . V a l u e s a r e means ± s.e.m. o f r e s u l t s f r o m 7-8 r a t s . Table 15. The in vivo percent free fractions of racemic mexiletine and i ts enantiomers in pentobarbitone anaesthetized rats Rat R(-)-mex R,S-mex S(+)-mex 1 51 57 36 2 34 47 47 3 35 43 61 4 41 31 42 5 34 48 56 6 35 - -• mean ± s.e.m. 39 3 45 4 48 4 w h e t h e r t i s s u e b i n d i n g a n d / o r m e t a b o l i s m was r e s p o n s i b l e f o r t h e g r e a t e r t h a n e x p e c t e d p l a s m a c o n c e n t r a t i o n s o f r a c e m i c m e x i l e t i n e . 3.5.1.2 A n t i a r r h y t h m i c E f f e c t s 3.5.1.2.1 V e n t r i c u l a r F i b r i l l a t i o n T h r e s h o l d (VFT) Many e x p e r i m e n t a l s t u d i e s h a v e u s e d VFT as a q u a n t i t a t i v e m e a s u r e o f c a r d i a c v u l n e r a b i l i t y t o f i b r i l l a t i o n ( s e c t i o n 1 . 1 . 2 . 5 . 1 ) . The u n d e r l y i n g a s s u m p t i o n i s t h a t p h y s i o l o g i c o r p h a r m a c o l o g i c i n t e r v e n t i o n s t h a t a l t e r VFT w i l l p r o d u c e a s i m i l a r d i r e c t i o n a l c h a n g e i n t h e v u l n e r a b i l i t y t o s p o n t a n e o u s f i b r i l l a t i o n ( M oore and S p e a r , 1 9 7 5 ) . I t ha s been s u g g e s t e d t h a t e l e c t r i c a l l y i n d u c e d VF i s a r e s u l t o f l o c a l r e - e n t r y ( M a r s h a l l e t al., 1 9 8 3 ) . The p r o p e r t i e s o f a p h a r m a c o l o g i c a l a g e n t r e q u i r e d t o b r e a k o r p r e v e n t a r e - e n t r a n t c i r c u i t i n c l u d e a s l o w i n g o f c o n d u c t i o n , an i n c r e a s e i n membrane t h r e s h o l d a n d an i n c r e a s e i n t h e e f f e c t i v e r e f r a c t o r y p e r i o d ( M a r s h a l l e t al., 1 9 8 3 ) . T h e s e p r o p e r t i e s a r e t h e same as t h o s e p o s s e s s e d by c l a s s I a n t i a r r h y t h m i c a g e n t s ( s e c t i o n 1.1.2.4) and s e v e r a l s t u d i e s h a v e r e p o r t e d e l e v a t i o n o f VFT by many c l a s s I a n t i a r r h y t h m i c d r u g s ( W i g g e r s and W e g r i a , 1940; Yoon e t al., 1974; G e r s t e n b l i t h e t al., 1972; H o d e s s e t al., 1 9 7 9 ) . The d e t e r m i n a t i o n o f VFT was t h e r e f o r e c o n s i d e r e d t o be a s u i t a b l e m ethod f o r a c o m p a r a t i v e s t u d y o f t h e a n t i a r r h y t h m i c a c t i v i t y o f r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s . . In t h e p r e s e n t s t u d y , t h e a v e r a g e VFT v a l u e s o b s e r v e d b e f o r e e a c h d r u g t r e a t m e n t a r e l i s t e d i n t a b l e 16. T h e s e p r e - d r u g v a l u e s w ere n o t s i g n i f i c a n t l y d i f f e r e n t i n any o f t h e g r o u p s . The r e l a t i v e i n c r e a s e s i n Table 16. Pre-drug values for the variables for ventricular f i b r i l l o - f l u t t e r in pentobarbitone anaesthetized rats VFT MFF ERP OA) (Hz) (msec) R(-)-mex 136 ± 26 13.7 ± 0.4 48 + 3 R,S-mex 110 ± 25 14.4 ± 0.6 50 ± 3 S(+)-mex 105 ± 22 15.8 ± 0.7 4 6 + 3 Values are means ± s .e .m. , n = 7-8 rats VFT (% c h a n g e f r o m p r e - d r u g ) f o r e a c h d r u g a r e shown g r a p h i c a l l y i n f i g u r e 19. R a c e m i c m e x i l e t i n e and t h e two e n a n t i o m e r s c a u s e d a d o s e -d e p e n d e n t s i g n i f i c a n t (p<0.05) i n c r e a s e i n VFT. However, t h e d i f f e r e n c e s b e t w e e n t h e d r u g t r e a t m e n t s d i d n o t r e a c h s t a t i s t i c a l s i g n i f i c a n c e . I t was n o t p o s s i b l e t o c ompare t h e r e s u l t s o f t h i s s t u d y w i t h t h o s e o f o t h e r s s i n c e t h e e f f e c t s o f m e x i l e t i n e e n a n t i o m e r s on VFT h a v e n o t b e e n r e p o r t e d . R a c e m i c m e x i l e t i n e , h o w e v e r , h a s b e e n shown t o i n c r e a s e VFT i n a n a e s t h e t i z e d r a t s ( M a r s h a l l e t al., 1981) and d o g s ( A l l e n e t a/., 1977). 3.5.1.2.2 Cardiac R e f r a c t o r y Period The r e f r a c t o r y p e r i o d o f t h e c a r d i a c t i s s u e i s an i m p o r t a n t d e t e r m i n a n t o f t h e i n i t i a t i o n o f a r r h y t h m i a s ( s e c t i o n 1.1.2.2). M o s t m e t h o d s u s e d t o d e t e r m i n e c a r d i a c r e f r a c t o r i n e s s m e a s u r e t h e e f f e c t i v e r e f r a c t o r y p e r i o d (ERP) d i r e c t l y o r i n d i r e c t l y . ERP i s d e p e n d e n t on MRD, t h e MP a t w h i c h t h e i n w a r d s o d i u m c u r r e n t i s r e a c t i v a t e d , t h e d u r a t i o n o f t h e r e p o l a r i z a t i o n p h a s e and t h e t i m e c o u r s e o f t h e r e a c t i v a t i o n p r o c e s s ( W i n s l o w , 1984). S i n g h (1978) r e p o r t e d t h a t t h e d e c r e a s e i n MRD e v o k e d by c l a s s I a n t i a r r h y t h m i c d r u g s was a s s o c i a t e d w i t h an i n c r e a s e i n t h e ERP. T h u s , ERP i s a f u r t h e r i n d e x o f t h e a n t i a r r h y t h m i c a c t i o n s o f c l a s s I d r u g s ( V a u g h a n W i l l i a m s , 1984). In t h i s s t u d y , ERP was m e a s u r e d d i r e c t l y by t h e e x t r a - s t i m u l u s m e t h o d and i n d i r e c t l y by d e t e r m i n i n g t h e maximum f r e q u e n c y a t w h i c h c a r d i a c m u s c l e c a n be i n d u c e d t o f o l l o w s t r o n g e l e c t r i c a l s t i m u l u s ( M F F ) . T h i s p a r a m e t e r has an i n v e r s e r e l a t i o n s h i p w i t h ERP. MFF h a s b e e n u s e d t o 130 F i g u r e 19. C u m u l a t i v e d o s e - r e s p o n s e c u r v e s o f t h e e f f e c t s o f r a c e m i c ' m e x i l e t i n e and i t s e n a n t i o m e r s on v e n t r i c u l a r f i b r i l l a t i o n t h r e s h o l d ( V F T ) i n p e n t o b a r b i t o n e a n a e s t h e t i z e d r a t s . V a l u e s a r e means ± s.e.m. o f r e s u l t s f r o m 7-8 r a t s . The o r d i n a t e r e f e r s t o c h a n g e f r o m p r e - d r u g v a l u e s . q u a n t i t a t i v e l y s t u d y t h e a n t i a r r h y t h m i c a c t i o n s o f o t h e r c l a s s I a g e n t s ( V a u g h a n W i l l i a m s and S z e k e r e s , 1 9 6 1 ) . T h e p r e - d r u g v a l u e s f o r ERP and MFF a r e shown i n t a b l e 16. T h e r e w ere no s i g n i f i c a n t d i f f e r e n c e s b e t w e e n t h e d r u g t r e a t m e n t g r o u p s . U s i n g t h e e x t r a - s t i m u l u s m e thod, r a c e m i c m e x i l e t i n e and t h e e n a n t i o m e r s w ere f o u n d t o i n c r e a s e t h e ERP s i g n i f i c a n t l y (P<0.05) and d o s e -d e p e n d e n t l y ( f i g . 2 0 ) . The d i f f e r e n c e s b e t w e e n t h e d r u g t r e a t m e n t s d i d n o t r e a c h s t a t i s t i c a l s i g n i f i c a n c e . A s i m i l a r l e n g t h e n i n g o f ERP t o t h a t o b t a i n e d i n t h i s s t u d y was r e p o r t e d i n i s o l a t e d c a n i n e v e n t r i c u l a r t i s s u e ( A r i t a et al., 1 9 7 9 ) . As e x p e c t e d , MFF was s i g n i f i c a n t l y (P<0.05) d e c r e a s e d by t h e d r u g s ( f i g . 21) and t h i s was a l s o d o s e -d e p e n d e n t . However, R,S- and S ( + ) - M e x i l e t i n e were s i g n i f i c a n t l y (P<0.05) more p o t e n t t h a n t h e R ( - ) - e n a n t i o m e r b u t R,S- and S ( + ) - m e x i l e t i n e d i d n o t d i f f e r s i g n i f i c a n t l y f r o m e a c h o t h e r . T h e s i g n i f i c a n t l y g r e a t e r e f f e c t s o f R,S- and S ( + ) - m e x i l e t i n e on MFF r e l a t i v e t o t h e R ( - ) - e n a n t i o m e r were n o t c o n s i s t e n t w i t h t h e i r e f f e c t s on ERP, where s i g n i f i c a n t d i f f e r e n c e s c o u l d n o t be d e t e c t e d . I t h a s been r e p o r t e d t h a t MFF i s a l e s s p r e c i s e method o f d e t e r m i n i n g ERP t h a n t h e e x t r a - s t i m u l u s method ( W i n s l o w , 1 9 8 4 ) . T h i s i s b e c a u s e ERP i s a f f e c t e d by f a c t o r s s u c h as t h e f u n d a m e n t a l d r i v i n g f r e q u e n c y ( V a u g h a n W i l l i a m s and S z e k e r e s , 1961; W i n s l o w , 1 9 8 4 ) . F u r t h e r m o r e , t h e e f f e c t s o f r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s on VFT d i d n o t d i f f e r s i g n i f i c a n t l y i n t h i s s t u d y . T h e r e f o r e , t h e c o n c l u s i o n drawn f r o m t h e p r e s e n t r e s u l t s was t h a t t h e e f f e c t s o f r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s on ERP w e r e . n o t m a r k e d l y d i f f e r e n t f r o m e a c h o t h e r . 132 5 0 - 1 0 - 1 1 1 1 1 0 1 0 0 Dose ( m g / k g ) F i g u r e 20. C u m u l a t i v e d o s e - r e s p o n s e c u r v e s o f t h e e f f e c t s o f r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s on t h e e f f e c t i v e r e f r a c t o r y p e r i o d ( E R P ) i n p e n t o b a r b i t o n e a n a e s t h e t i z e d r a t s . V a l u e s a r e t h e means ± s.e.m. o f r e s u l t s f r o m 7-8 r a t s . T he o r d i n a t e r e f e r s t o c h a n g e f r o m p r e - d r u g v a l u e s . 133 1 0 - 5 0 - I , : 1 1 1 0 1 0 0 D o s e ( m g / k g ) F i g u r e 2 1 . C u m u l a t i v e d o s e - r e s p o n s e c u r v e s o f t h e e f f e c t s o f r a c e m i c m e x i l e t i n e a n d i t s e n a n t i o m e r s on t h e maximum f o l l o w i n g f r e q u e n c y (MFF) i n p e n t o b a r b i t o n e a n a e s t h e t i z e d r a t s . V a l u e s a r e means ± s.e.m. o f r e s u l t s f r o m 7-8 r a t s . T he o r d i n a t e r e f e r s t o c h a n g e f r o m p r e - d r u g v a l u e s . 3.5.1.3 Cardiovascular Effects The e l e c t r o c a r d i o g r a p h i c e f f e c t s o f r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s a r e shown i n t a b l e 17. The QRS and QT C i n t e r v a l s were not s i g n i f i c a n t l y a f f e c t e d by drug t r e a t m e n t . The PR i n t e r v a l was s i g n i f i c a n t l y i n c r e a s e d but o n l y a t t h e h i g h e s t c u m u l a t i v e dose (32 mg/kg) compared t o p r e - d r u g v a l u e s . T h e r e were no s i g n i f i c a n t d i f f e r e n c e s i n t h e PR p r o l o n g a t i o n i n d u c e d by t h e d r u g s . The s i g n i f i c a n t i n c r e a s e i n t h e PR i n t e r v a l i n d i c a t e d a s l o w i n g o f AV c o n d u c t i o n . S i n c e i t has been r e p o r t e d t h a t r a c e m i c m e x i l e t i n e does not p o s s e s s any c a l c i u m a n t a g o n i s t p r o p e r t i e s nor a f f e c t t h e / ^ - a d r e n e r g i c r e s p o n s e s ( S i n g h e t al., 1980), i t i s most l i k e l y t h a t t h e o b s e r v e d PR p r o l o n g a t i o n i s a r e s u l t o f sodium channel b l o c k a d e i n t h e p e r i p h e r a l AV nodal t i s s u e . T a b l e 18 shows t h e p r e - d r u g v a l u e s f o r t h e haemodynamic v a r i a b l e s ( h e a r t r a t e and b l o o d p r e s s u r e ) . T h e r e were no s i g n i f i c a n t d i f f e r e n c e s between t h e drug t r e a t m e n t g r o u p s . Racemic m e x i l e t i n e and t h e en a n t i o m e r s caused s i g n i f i c a n t (P<0.05) b r a d y c a r d i a ( f i g . 22) and h y p o t e n s i o n ( f i g . 23) i n t h e r a t s . However, t h e s e e f f e c t s d i d not d i f f e r s i g n i f i c a n t l y between t h e drug t r e a t m e n t g r o u p s . The o b s e r v e d d e c r e a s e i n h e a r t r a t e may have r e s u l t e d from a d e p r e s s i o n o f phase 4 s l o p e o f pacemaker p o t e n t i a l s which has been r e p o r t e d f o r c l a s s 1 a n t i a r r h y t h m i c agents ( S i n g h and H a u s w i r t h , 1974). P r o l o n g a t i o n o f PR i n t e r v a l and d e c r e a s e i n h e a r t r a t e has been r e p o r t e d t o be a common f e a t u r e o f c l a s s 1 a n t i a r r h y t h m i c agents ( M a r s h a l l e t al., 1981). The hy p o t e n s i v e e f f e c t s o f t h e racemate and t h e i n d i v i d u a l e n a n t i o m e r s o f Table 17. The effects of racemic mexiletine and i ts enantiomers on the ECG parameters in pentobarbitone anaesthetized rats PR QRS QTQ (msec) (msec) (msec) R(-)mex pre-drug 45 + 2 30 + 0.8 190 + 9 4 45 + 2 30 + 0.8 190 + 10 8 46 + 1 30 + 0.8 190 + 10 16 45 + 1 31 ± 0.8 182 + 10 32 47 + 1 31 + 0.8 182 + 10 R,S-mex pre-drug 47 + 1 30 + 0.7 195 + 4 4 48 + 1 31 + 0.4 189 + 4 8 48 + 1 31 + 0.4 182 + 3 16 48 + 2 32 + 0.7 184 + 10 32 51 + 2 31 + 1.2 181 + 11 S(+)mex pre-drug 44 + 1 32 + 0.7 189 + 7 4 45 ± 2 32 + 0.8 185 + 7 8 46 + 2 33 ± 0.7 187 ± 8 16 47 + 2 33 + 1.1 180 + 7 32 49 + 3 34 ± 1.2 188 + 8 Values are means ± s. ,e.m., n = 7-8 rats T a b l e 18. P r e - d r u g v a l u e s f o r t h e haemodynamic p a r a m e t e r s i n p e n t o b a r b i t o n e a n a e s t h e t i z e d r a t s HR BP ( b e a t s / m i n ) (mm Hg) R(-)-mex 401 + 10 123 + 6 R,S-mex 388 + 14 122 + 5 S(+)-mex 389 + 16 106 + 6 V a l u e s a r e means ± s.e.m., n = 7-8 r a t s 137 1 0 F i g u r e 22. C u m u l a t i v e d o s e - r e s p o n s e c u r v e s o f t h e e f f e c t s o f r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s on t h e h e a r t r a t e i n p e n t o b a r b i t o n e a n a e s t h e t i z e d r a t s . V a l u e s a r e means ± s.e.m. o f r e s u l t s f r o m 7-8 r a t s . The o r d i n a t e r e f e r s t o c h a n g e f r o m p r e - d r u g v a l u e s . 138 F i g u r e 23. C u m u l a t i v e d o s e - r e s p o n s e c u r v e s o f t h e e f f e c t s o f r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s on t h e mean a r t e r i a l b l o o d p r e s s u r e i n p e n t o b a r b i t o n e a n a e s t h e t i z e d r a t s . V a l u e s a r e means ± s.e.m. o f r e s u l t s f r o m 7-8 r a t s . The o r d i n a t e r e f e r s t o c h a n g e f r o m p r e - d r u g v a l u e s . m e x i l e t i n e were n o t c o n s i s t e n t w i t h r e s u l t s o b t a i n e d i n t h e c o n s c i o u s r a t s ( s e c t i o n 3.5.2.4) where no s i g n i f i c a n t d e c r e a s e i n mean a r t e r i a l b l o o d p r e s s u r e was p r o d u c e d by t h e d r u g s . I t c a n be s p e c u l a t e d t h a t as a r e s u l t o f r e c e n t s u r g e r y and t h e p r e s e n c e o f a n a e s t h e s i a i n t h e s e a n i m a l s , t h e i r c a r d i o v a s c u l a r s t a t u s may be l e s s s a t i s f a c t o r y t h a n t h a t o f t h e c o n s c i o u s r a t s . In summary, t h e r e s u l t s o f t h e p r e s e n t s t u d y h a v e shown a l a c k o f d i s t i n c t d i f f e r e n c e s i n t h e e f f e c t s o f r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s on VFT and ERP, as w e l l as h e a r t r a t e , b l o o d p r e s s u r e and PR i n t e r v a l . A l t h o u g h a s i g n i f i c a n t l y g r e a t e r MFF was o b s e r v e d f o r S ( + ) - and R , S - m e x i l e t i n e r e l a t i v e t o t h e R ( - ) - e n a n t i o m e r , t h e d a t a o b t a i n e d f o r a l l t h e o t h e r v a r i a b l e s do n o t s u p p o r t t h e p r e s e n c e o f s i g n i f i c a n t d i f f e r e n c e s i n t h e a n t i a r r h y t h m i c and c a r d i o v a s c u l a r a c t i o n s o f r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s . 3.5.2 Coronary Artery Occlusion-Induced Arrhythmia in Conscious Rats T h e a n t i a r r h y t h m i c and c a r d i o v a s c u l a r e f f e c t s o f r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s were f u r t h e r i n v e s t i g a t e d u s i n g c o r o n a r y a r t e r y o c c l u s i o n - i n d u c e d a r r h y t h m i a i n c o n s c i o u s r a t s . The c h o i c e o f t h i s model was b a s e d on r e p o r t s i n t h e l i t e r a t u r e w h i c h i n d i c a t e d t h a t t h e s o d i u m c h a n n e l b l o c k i n g a c t i o n o f r a c e m i c m e x i l e t i n e , in vitro, was p o t e n t i a t e d i n i s c h a e m i c and h y p o x i c c o n d i t i o n s ( H o h n l o s e r e t al., 1982; Frame e t al., 1 9 8 2 ) . S i m i l a r r e s u l t s h a v e been o b s e r v e d w i t h l i d o c a i n e ( K u p p e r s m i t h e t al., 1975; L a z a r r a e t al., 1 9 7 8 ) . The u s e o f c o n s c i o u s a n i m a l s c i r c u m v e n t e d t h e i n f l u e n c e o f a n a e s t h e s i a and r e c e n t s u r g e r y on t h e e f f e c t s o f r a c e m i c m e x i l e t i n e and t h e e n a n t i o m e r s . 140 3.5.2.1 D o s a g e a n d P l a s m a C o n c e n t r a t i o n A s i n g l e d o s e o f r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s (20 mg/kg) was u s e d f o r t h e s t u d y . The d o s e was i n f u s e d o v e r 10 m i n and i t p r o d u c e d o v e r t CNS t o x i c e f f e c t s ( a t a x i a ) i n a l m o s t a l l t h e r a t s a t t h e e n d o f t h e i n f u s i o n and c o n v u l s i o n s d e v e l o p e d i n a few o f t h e a n i m a l s . The d o s e o f d r u g u s e d made i t p o s s i b l e t o m e a s u r e t h e p l a s m a c o n c e n t r a t i o n s r e s p o n s i b l e f o r t h e s e CNS e f f e c t s i n t h e r a t s . The t o x i c e f f e c t s w e r e t r a n s i e n t , l a s t i n g u s u a l l y l e s s t h a n 5 m i n . H i g h e r d o s e s p r o d u c e d s e v e r e CNS t o x i c i t y and d e a t h i n t h e r a t s . Due t o t h e r a p i d d i s t r i b u t i o n and e l i m i n a t i o n o f m e x i l e t i n e i n t h e r a t s ( s e c t i o n 3.4) and t h e r e l a t i v e l y l o n g m o n i t o r i n g p e r i o d (4 h) f o l l o w i n g c o r o n a r y a r t e r y o c c l u s i o n , t h e a d m i n i s t r a t i o n o f a s e c o n d d o s e o f d r u g (20 mg/kg) was n e c e s s a r y . T h i s was i n i t i a t e d 1.5 h p o s t - o c c l u s i o n and was i n f u s e d o v e r 30 m i n . T h e p l a s m a c o n c e n t r a t i o n - t i m e r e l a t i o n s h i p s o f r a c e m i c m e x i l e t i n e and t h e e n a n t i o m e r s t h a t were o b s e r v e d a r e shown i n f i g u r e 24. R,S- and S ( + ) - M e x i l e t i n e had s i g n i f i c a n t l y (p<0.05) g r e a t e r mean p l a s m a c o n c e n t r a t i o n s t h a n R ( - ) - m e x i l e t i n e a t e a c h s a m p l i n g t i m e . T h e s e r e s u l t s a r e c o n s i s t e n t w i t h t h o s e o b t a i n e d i n t h e p e n t o b a r b i t o n e a n a e s t h e t i z e d r a t s ( s e e - s e c t i o n - 3 . 5 . 1 . 1 f o r e x p l a n a t i o n o f p l a s m a c o n c e n t r a t i o n s ) . 3.5.2.2 O c c l u d e d Z o n e ( 0 Z ) T h e e x t e n t o f m y o c a r d i a l i s c h a e m i a i s known t o i n f l u e n c e t h e i n c i d e n c e o f a r r h y t h m i a s ( J a n s e , 1 9 8 7 ) . In g e n e r a l , t h e l a r g e r t h e i s c h a e m i c a r e a ( o c c l u d e d z o n e ) , t h e more f r e q u e n t t h e a r r h y t h m i a s . A u s t i n et al. (1982) r e p o r t e d t h a t t h e i n c i d e n c e o f a r r h y t h m i a s • * — R ( - ) - m e x — e — R , S - m e x — * — S ( + ) - m e x F i g u r e 24. The p l a s m a c o n c e n t r a t i o n - t i m e p r o f i l e o f r a c e m i c m e x i l e t i n e and t h e i n d i v i d u a l e n a n t i o m e r s i n i s c h e m i c c o n s c i o u s r a t s a f t e r t h e a d m i n i s t r a t i o n o f a d o s e o f 20 mg/kg, f o l l o w e d 1.5 h l a t e r b y a s e c o n d d o s e (20 mg/kg). V a l u e s a r e means ± s.e.m o f r e s u l t s f r o m 9 r a t s . f o l l o w i n g coronary a r t e r y o c c l u s i o n i n mongrel dogs was c r i t i c a l l y dependent on OZ s i z e . Johnston et al. (1983) i n t h e i r experiments i n conscious r a t s , showed that the "arrhythmia score" (a number s c a l e which summarizes arrhythmias i n terms of i n c i d e n c e and d u r a t i o n ) was l i n e a r l y r e l a t e d to the square root of OZ. Thus, v a r i a b i l i t y i n OZ s i z e among the treatment groups could be a p o t e n t i a l source of e r r o r i n the outcome of coronary a r t e r y o c c l u s i o n . Table 19 shows the percent OZ observed i n r a t s i n the (R)-, S(+)- and R,S-mexiletine and s a l i n e ( c o n t r o l ) treatment groups. No s i g n i f i c a n t d i f f e r e n c e s i n OZ s i z e were observed between the groups. 3.5.2.3 A n t i a r r h y t h m i c Effects The o c c l u s i o n of the l e f t a n t e r i o r descending (LAD) coronary a r t e r y evoked r e p r o d u c i b l e arrhythmias i n the conscious r a t s . Figure 25 shows the o c c l u s i o n snare around the LAD coronary a r t e r y while f i g u r e 26 shows the r e s u l t a n t arrhythmias which i n c l u d e d v e n t r i c u l a r f i b r i l l a t i o n (VF), v e n t r i c u l a r t a c h y c a r d i a (VT) and premature v e n t r i c u l a r c o n t r a c t i o n s (PVCs). The arrhythmias occurred i n two d i s t i n c t phases c o n s i s t e n t with the r e p o r t of Johnston et al. (1983). The e a r l y phase comprised of the p e r i o d 0-30 min p o s t - o c c l u s i o n , while the l a t e phase arrhythmias occurred mainly between 1.5 and 4 h. The arrhythmias observed i n t h i s study are presented as e a r l y and o v e r - a l l (0-4 h) arrhythmias. The data given i n t a b l e 20 show the i n c i d e n c e o f the major arrhythmias (VT & VF) i n the conscious r a t s . Racemic m e x i l e t i n e and i t s enantiomers d i d not s i g n i f i c a n t l y reduce the i n c i d e n c e of these arrhythmias. The e f f e c t s of the d i f f e r e n t treatments on the number of T a b l e 19. T h e mean (± s.e.m.) w e i g h t and o c c l u d e d z o n e (OZ) i n t h e d i f f e r e n t t r e a t m e n t g r o u p s i n c o n s c i o u s i s c h a e m i c r a t s W e i g h t * OZ (9) (%) S a l i n e 337 ± 1 7 3 4 + 1 R(-)-mex 352 ± 2 0 33 ± 2 R,S-mex 339 ± 1 4 37 ± 1 S(+)-mex 324 ± 1 7 3 6 + 1 * w e i g h t o f r a t s i n e a c h g r o u p . OZ r e f e r s t o t h e p e r c e n t a g e o f t h e v e n t r i c l e t h a t was o c c l u d e d . F i g u r e 25. The r a t h e a r t s h o w i n g t h e o c c l u s i o n s n a r e a r o u n d t h e l e f t a n t e r i o r d e s c e n d i n g c o r o n a r y a r t e r y . 145 F i g u r e 26. T y p i c a l a r r h y t h m i a s r e s u l t i n g f r o m o c c l u s i o n o f t h e l e f t a n t e r i o r d e s c e n d i n g c o r o n a r y a r t e r y . P r e m a t u r e v e n t r i c u l a r c o n t r a c t i o n s ( P V C s ) a r e shown i n p a n e l A, s p o n t a n e o u s l y r e v e r t i n g v e n t r i c u l a r t a c h y c a r d i a ( V T ) i n B, and s p o n t a n e o u s l y r e v e r t i n g VT d e g e n e r a t i n g t o v e n t r i c u l a r f i b r i l l a t i o n ( V F ) i n C. The t o p and b o t t o m t r a c e s i n e a c h p a n e l r e p r e s e n t b l o o d p r e s s u r e and ECG, r e s p e c t i v e l y . T a b l e 20. The e f f e c t s o f r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s on t h e i n c i d e n c e o f a r r h y t h m i a s f o l l o w i n g c o r o n a r y a r t e r y o c c l u s i o n i n c o n s c i o u s r a t s VT VF SVF NSVF 0-0.5h 0-4h 0-0.5h 0-4h 0-0.5h 0-4h 0-0.5h 0-4h S a l i n e 6/9 8/9 6/9 8/9 2/9 4/9 5/9 8/9 R(-)-mex 3/9 9/9 2/9 7/9 0/9 8/9 2/9 6/9 R,S-mex 7/9 9/9 4/9 7/9 4/9 5/9 2/9 6/9 S(+)-mex 9/9 9/9 8/9 8/9 5/9 6/9 7/9 8/9 VT = v e n t r i c u l a r t a c h y c a r d i a , VF = v e n t r i c u l a r f i b r i l l a t i o n , SVF = s p o n t a n e o u s l y r e v e r t i n g v e n t r i c u l a r f i b r i l l a t i o n , NSVF = n o n - s p o n t a n e o u s l y r e v e r t i n g v e n t r i c u l a r f i b r i l l a t i o n . PVCs a r e shown i n t a b l e 21. As was t h e c a s e w i t h VT and VF, no s i g n i f i c a n t d i f f e r e n c e s c o u l d be d e t e c t e d when t h e d r u g t r e a t m e n t g r o u p s w e r e c o m p a r e d t o t h e s a l i n e g r o u p . F i g u r e s 27 and 28 show t h e " a r r h y t h m i a s c o r e s " c a l c u l a t e d f o r t h e e n a n t i o m e r s . T h e r e were no s i g n i f i c a n t d i f f e r e n c e s b e t w e e n t h e d r u g t r e a t e d r a t s and t h o s e t h a t r e c e i v e d s a l i n e . In g e n e r a l , d u r i n g t h e e a r l y p h a s e , t h e r e was a t e n d e n c y t o w a r d s l o w e r i n c i d e n c e and number o f a r r h y t h m i a s i n t h e R ( - ) - m e x i l e t i n e g r o u p r e l a t i v e t o s a l i n e ( F i g . 2 7 ) . In c o n t r a s t , t h e S ( + ) - m e x i l e t i n e g r o u p showed a h i g h e r i n c i d e n c e and number o f a r r h y t h m i a s d u r i n g t h e same t i m e p e r i o d . T h e o c c l u s i o n o f t h e LAD c o r o n a r y a r t e r y i n c o n s c i o u s r a t s i s an e s t a b l i s h e d and r e l a t i v e l y s i m p l e model f o r t h e p r o d u c t i o n o f e x p e r i m e n t a l c a r d i a c a r r h y t h m i a s . T h i s model has been u s e d t o t e s t t h e e f f e c t i v e n e s s o f many c l a s s I a n t i a r r h y t h m i c d r u g s , i n c l u d i n g ORG 6001 (Kane et al., 1 9 8 0 ) , l i d o c a i n e and q u i n i d i n e ( J o h n s t o n et al., 1 9 8 3 ) . In t h e p r e s e n t s t u d y , r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s f a i l e d t o p r o t e c t c o n s c i o u s r a t s f r o m i s c h a e m i a - i n d u c e d a r r h y t h m i a s . I t was n o t p o s s i b l e t o compare t h e r e s u l t s o f t h i s s t u d y w i t h t h o s e o f o t h e r s s i n c e t h e e f f e c t s o f m e x i l e t i n e e n a n t i o m e r s on i s c h a e m i c a r r h y t h m i a s i n r a t s h a v e n o t b e e n p r e v i o u s l y r e p o r t e d . However, M a r s h a l l et al. ( 1 9 8 1 ) , u s i n g a s i m i l a r model i n a n a e s t h e t i z e d W i s t a r r a t s , i n v e s t i g a t e d t h e a n t i a r r h y t h m i c a c t i o n s o f r a c e m i c m e x i l e t i n e . T h e s e a u t h o r s r e p o r t e d t h a t 1 mg/kg o f r a c e m i c m e x i l e t i n e a d m i n i s t e r e d 15 min b e f o r e c o r o n a r y a r t e r y o c c l u s i o n c o m p l e t e l y a b o l i s h e d VF i n t h e 30 min p e r i o d p o s t -o c c l u s i o n . T h i s i s i n d i s a g r e e m e n t w i t h r e s u l t s o b t a i n e d i n t h e p r e s e n t s t u d y . B a s e d on t h e r a p i d d i s t r i b u t i o n and p l a s m a c l e a r a n c e o f T a b l e 21. T h e e f f e c t s o f r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s on t h e number o f p r e m a t u r e v e n t r i c u l a r c o n t r a c t i o n s ( l o g ^ V C s ) f o l l o w i n g c o r o n a r y a r t e r y o c c l u s i o n i n c o n s c i o u s r a t s l o g 1 0 P V C s 0-1/2 h 0-4 h s a l i n e 1.63 ± 0.35 2.97 ± 0.12 R(-)-mex 1.24 ± 0.41 2.64 ± 0.15 R,S-mex 1.90 ± 0.23 2.80 ± 0.13 S(+)-mex 2.02 ± 0.12 3.00 ± 0.10 V a l u e s a r e means ± s.e.m, n = 9 r a t s . P r e m a t u r e v e n t r i c u l a r c o n t r a c t i o n s a r e e x p r e s s e d as l o g j o P V C s s i n c e t h e number o f PVCs i s a l o g ^ g n o r m a l l y d i s t r i b u t e d v a r i a b l e . Sal ine R(—)—mex R , S - m e x S ( H - ) - m e x F i g u r e 27. The e f f e c t s o f ( R ) - , R,S- and S ( + ) - m e x i 1 e t i n e and s a l i n e on " a r r h y t h m i a s c o r e " f o r t h e 0-30 min p e r i o d f o l l o w i n g c o r o n a r y a r t e r y o c c l u s i o n i n c o n s c i o u s r a t s . 0 Saline R ( - ) - m e x R , S - m e x S ( + ) - m e x F i g u r e 28. The e f f e c t s o f ( R ) - , R,S- and S ( + ) - m e x i l e t i n e and s a l i n e on " a r r h y t h m i a s c o r e " f o r t h e 0-4 h p e r i o d f o l l o w i n g c o r o n a r y a r t e r y o c c l u s i o n i n c o n s c i o u s r a t s . m e x i l e t i n e e n a n t i o m e r s i n t h e r a t ( s e c t i o n 3 . 4 . 2 ) , i t i s u n l i k e l y t h a t t h e r a p e u t i c c o n c e n t r a t i o n s o f r a c e m i c m e x i l e t i n e w o u l d be a c h i e v e d and m a i n t a i n e d f o r t h e 30 min p e r i o d p o s t - o c c l u s i o n f r o m a 1 mg/kg d o s e a d m i n i s t e r e d 15 m i n b e f o r e o c c l u s i o n . O t h e r s t u d i e s on t h e a n t i a r r h y t h m i c a c t i o n s o f r a c e m i c m e x i l e t i n e h a v e been c a r r i e d o u t i n d o g s u s i n g t h e H a r r i s 2 - s t a g e c o r o n a r y a r t e r y o c c l u s i o n p r o c e d u r e ( A l l e n e t al., 1977; M e r t z and S t e f f e , 1980; H a s h i m o t o et al., 1 9 8 4 ) . H a s h i m o t o et al. (1984) r e p o r t e d t h a t a mean p l a s m a c o n c e n t r a t i o n o f 1.9 Mg/ml s i g n i f i c a n t l y r e d u c e d " a r r h y t h m i a r a t i o s " (number o f PVCs d i v i d e d by t h e t o t a l h e a r t r a t e ) . However, t h e maximum r e d u c t i o n i n " a r r h y t h m i a r a t i o " was l e s s t h a n 50%. T h e i r r e s u l t s w e r e c o n s i s t e n t w i t h t h o s e o f M e r t z and S t e f f e ( 1 9 8 0 ) who u s e d t h e same d o s e o f r a c e m i c m e x i l e t i n e as H a s h i m o t o et al., (1980) (5 mg/kg). The l a t t e r a u t h o r s p o i n t e d o u t t h a t t h e a d m i n i s t r a t i o n o f a h i g h e r d o s e o f m e x i l e t i n e was l i m i t e d by CNS t o x i c e f f e c t s w h i c h i n c l u d e d t r e m o r s and c o n v u l s i o n s . A l l e n et al. ( 1 9 7 7 ) , h o w e v e r , r e p o r t e d t h a t a t a mean p l a s m a c o n c e n t r a t i o n o f 5.33 ± 0.34 / i g / m l , s i n u s r h y t h m was c o m p l e t e l y r e t u r n e d i n c o n s c i o u s i s c h a e m i c d o g s . T h i s s t u d y d i d n o t r e p o r t a n y CNS t o x i c e f f e c t s a t t h i s p l a s m a c o n c e n t r a t i o n i n t h e d o g s . From t h e r e s u l t s o f t h e s e 3 s t u d i e s , i t may be s p e c u l a t e d t h a t g r e a t e r p l a s m a c o n c e n t r a t i o n s t h a n t h o s e a c h i e v e d i n t h e p r e s e n t s t u d y w o u l d be r e q u i r e d f o r t h e a n t i a r r h y t h m i c e f f e c t s o f r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s t o be o b s e r v e d i n t h e r a t . T h i s s p e c u l a t i o n was c o n f i r m e d w i t h a h i g h e r d o s e o f r a c e m i c m e x i l e t i n e i n i s c h a e m i c p e n t o b a r b i t o n e a n a e s t h e t i z e d r a t s ( s e c t i o n 3 . 5 . 3 . 1 ) . P l a s m a c o n c e n t r a t i o n s o f m e x i l e t i n e and i t s e n a n t i o m e r s , s u c h as t h o s e r e p o r t e d by A l l e n e t a 7 . ( 1 9 7 7 ) , w o u l d produce severe CNS toxici ty in the conscious rats (see toxic plasma concentrations in table 23). 3.5.2.4 Cardiovascular Effects Table 22 shows the effects of the different treatments on the ECG parameters in conscious rats. Racemic mexiletine and the enantiomers caused a significant (p<0.05) increase in the PR interval from pre-drug values relative to saline. There were no significant differences between the drug treatment groups. Neither the drugs nor saline affected the QRS interval of the rat heart. The effects of racemic mexiletine and the enantiomers on heart rate and blood pressure are presented in figures 29 and 30, respectively. The drugs significantly (p<0.05) decreased the heart rate from pre-drug values when compared to saline ( f ig . 29) (heart rate at 1 min before coronary artery occlusion were compare by one-way ANOVA). There were no significant differences between the drug treatment groups. When the heart rate over the 4 h observation period was analysed by "Repeated Measures" ANOVA, no s ta t i s t i ca l ly significant differences could be detected between the drug and saline treatment groups. A similar decrease in heart rate following the administration of racemic mexiletine has been reported in dogs (Hashimoto et al., 1984). The mean arterial blood pressure was not significantly changed by either drug or saline treatment ( f ig . 30). The cardiovascular effects of racemic mexiletine and i ts enantiomers in the conscious rats are consistent with those observed in T a b l e 22. T h e e f f e c t o f d r u g t r e a t m e n t on t h e ECG p a r a m e t e r s i n c o n s c i o u s i s c h a e m i c r a t s PR (msec) QRS (msec) p r e - d r u g p o s t - d r u g p r e - d r u g p o s t - d r u g s a l i n e 44.5 + 1.4 44.0 + 1 .4 24.0 + 1.4 24.0 + 1.3 R(-)-mex 39.9 + 1.1 48.0 + 1 .8* 22.8 + 1.0 22.8 + 0.7 RS-mex 43.0 + 2.0 49.3 + 1 .3* 22.0 + 0.8 22.0 + 1.0 S(+)-mex 44.0 + 2.3 53.0 + 1 .4* 22.0 + 1.4 23.0 + 1.0 V a l u e s a r e means ± s.e.m, n = 9 r a t s . * S i g n i f i c a n t (p<0.05) when c o m p a r e d t o s a l i n e w i t h r e s p e c t t o i n c r e a s e f r o m p r e - d r u g . 154 Saline R(—)—mex —•- R,S—mex S(+)—mex 500 -g 400 E -v. CO ro V v. 300 CD rr 2 0 0 CO CD I 100 0 — 15min -1min 1min lOmin 1h Time 1.5h 2h 3h 4h F i g u r e 29. The e f f e c t s o f ( R ) - , R,S- and S ( + ) - m e x i l e t i n e and s a l i n e on h e a r t r a t e i n i s c h a e m i c c o n s c i o u s r a t s . The s.e.m a r e o m i t t e d f o r c l a r i t y o f p r e s e n t a t i o n . 155 Saline R(—)—mex RS—mex S(+)-mex 160 — 15min F i g u r e 30. The e f f e c t s o f ( R ) - , R,S- a n d S ( + ) - m e x i l e t i n e and s a l i n e o n mean a r t e r i a l b l o o d p r e s s u r e i n i s c h a e m i c c o n s c i o u s r a t s . T h e s.e.m a r e o m i t t e d f o r c l a r i t y o f p r e s e n t a t i o n . t h e p e n t o b a r b i t o n e a n a e s t h e t i z e d r a t s , e x c e p t f o r t h e i r e f f e c t on b l o o d p r e s s u r e ( s e e s e c t i o n 3 . 5 . 1 . 3 ) . 3.5.2.5 CNS S i d e E f f e c t s T a b l e 23 shows t h e i n c i d e n c e o f o v e r t CNS t o x i c e f f e c t s ( a t a x i a and c o n v u l s i o n s ) i n t h e c o n s c i o u s r a t s . The c o r r e s p o n d i n g p l a s m a c o n c e n t r a t i o n s a t w h i c h t h e s e s i g n s o f t o x i c i t y a p p e a r e d a r e a l s o p r e s e n t e d . The i n c i d e n c e o f CNS t o x i c e f f e c t s were s i g n i f i c a n t l y (p<0.05) g r e a t e r i n t h e d r u g t r e a t e d r a t s c o m p a r e d t o t h o s e t h a t r e c e i v e d s a l i n e . However, t h e r e were no s i g n i f i c a n t d i f f e r e n c e s b e t w e e n t h e d r u g t r e a t m e n t g r o u p s . The CNS t o x i c e f f e c t s were p r e s u m e d t o be due t o t h e h i g h b r a i n u p t a k e o f m e x i l e t i n e e n a n t i o m e r s i n t h e r a t ( s e c t i o n 3 . 4 . 5 ) . In summary, r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s d i d n o t p r o t e c t c o n s c i o u s r a t s f r o m c o r o n a r y a r t e r y o c c l u s i o n - i n d u c e d a r r h y t h m i a s . The b r a d y c a r d i c and PR p r o l o n g a t i o n e f f e c t s o f r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s were s i g n i f i c a n t b u t d i d n o t d i f f e r f r o m e a c h o t h e r . The d r u g s p r o d u c e d c o m p a r a b l e CNS s i d e e f f e c t s i n c o n s c i o u s r a t s . 3.5.3 C o r o n a r y A r t e r y O c c l u s i o n - I n d u c e d A r r h y t h m i a i n i n P e n t o b a r b i t o n e A n a e s t h e t i z e d I s c h a e m i c Rats To e x p l a i n t h e i n e f f e c t i v e n e s s o f r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s a g a i n s t i s c h a e m i a - i n d u c e d a r r h y t h m i a s i n t h e c o n s c i o u s r a t s , t h e a n t i a r r h y t h m i c e f f e c t s o f a h i g h e r d o s e o f r a c e m i c m e x i l e t i n e (40 mg/kg), w h i c h c o u l d n o t be t o l e r a t e d by c o n s c i o u s r a t s , was i n v e s t i g a t e d . T h e e f f e c t s o f t h e same d o s e o f t h e r a c e m a t e u s e d i n c o n s c i o u s r a t s (20 mg/kg) and s a l i n e ( c o n t r o l ) were a l s o e v a l u a t e d . The T a b l e 23. T h e i n c i d e n c e o f CNS t o x i c e f f e c t s and t h e c o r r e s p o n d i n g p l a s m a c o n c e n t r a t i o n s i n c o n s c i o u s r a t s f o l l o w i n g t h e a d m i n i s t r a t i o n o f r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s (20 mg/kg) CNS t o x i c e f f e c t s P l a s m a c o n c e n t r a t i o n Og/ml) S a l i n e R (-)-mex R,S-mex S(+)-mex 0/9 8/9' 7/9' 8/9' 2.3 ± 0.16 3.8 ± 0.41 3.6 ± 0.36 * S i g n i f i c a n t (p<0.05) when c o m p a r e d t o s a l i n e . ** CNS t o x i c e f f e c t s o b s e r v e d were a t a x i a and c o n v u l s i o n s . P l a s m a c o n c e n t r a t i o n s a r e means ± s.e.m. R,S- and S ( + ) - M e x i l e t i n e had s i g n i f i c a n t l y (p<0.05) g r e a t e r p l a s m a c o n c e n t r a t i o n s t h a n t h e R ( - ) - e n a n t i o m e r . 158 a n i m a l s were m o n i t o r e d f o r 30 min a f t e r c o r o n a r y a r t e r y o c c l u s i o n ( w h i c h c o v e r e d t h e f i r s t p h a s e o f a r r h y t h m i a s ) . T h i s i n v e s t i g a t i o n was c a r r i e d o u t i n p e n t o b a r b i t o n e a n a e s t h e t i z e d r a t s . T h e a r r h y t h m i a s r e s u l t i n g f r o m c o r o n a r y a r t e r y o c c l u s i o n i n p e n t o b a r b i t o n e a n a e s t h e t i z e d r a t s h a v e b e e n shown t o c o r r e s p o n d a p p r o x i m a t e l y w i t h t h o s e s e e n i n c o n s c i o u s r a t s ( J o h n s t o n et al., 1 9 8 3 ) . 3.5.3.1 A n t i a r r h y t h m i c E f f e c t s o f R a c e m i c M e x i l e t i n e T a b l e 24 shows t h e p e r c e n t OZ i n t h e 3 t r e a t m e n t g r o u p s . T h e r e w e r e no s i g n i f i c a n t d i f f e r e n c e s i n t h i s p a r a m e t e r b e t w e e n t h e g r o u p s . The i n c i d e n c e o f VT and VF and t h e number o f PVCs f o l l o w i n g t h e 2 d o s e s o f r a c e m i c m e x i l e t i n e (20 and 40 mg/kg) and s a l i n e a r e shown i n t a b l e s 25 and 26, r e s p e c t i v e l y . A t t h e d o s e o f 20 mg/kg, R , S - m e x i l e t i n e d i d n o t s i g n i f i c a n t l y r e d u c e t h e i n c i d e n c e o f VT and VF n o r t h e number o f PVCs when c o m p a r e d w i t h t h e s a l i n e c o n t r o l g r o u p ( t a b l e s 25 & 2 6 ) . T h e s e r e s u l t s w ere s i m i l a r t o t h o s e o b t a i n e d f o r t h e e a r l y a r r h y t h m i a s i n c o n s c i o u s r a t s . A t t h e h i g h e r d o s e o f 40 mg/kg, R , S - m e x i l e t i n e s i g n i f i c a n t l y (p<0.05) r e d u c e d t h e i n c i d e n c e o f VT and t h e number o f PVCs ( t a b l e s 25 & 26) and c o m p l e t e l y s u p p r e s s e d VF i n a l l t h e r a t s ( t a b l e 2 5 ) . The " a r r h y t h m i a s c o r e s " o b t a i n e d f o r t h e 3 t r e a t m e n t g r o u p s a r e shown i n f i g u r e 3 1 . A s i g n i f i c a n t l y (p<0.05) l o w e r " a r r h y t h m i a s c o r e " was o b s e r v e d i n t h e 40 mg/kg g r o u p when c o m p a r e d t o t h e 20 mg/kg and s a l i n e g r o u p s . The mean " a r r h y t h m i a s c o r e " f o r t h e 20 mg/kg g r o u p , h o w e v e r , was n o t s i g n i f i c a n t l y d i f f e r e n t f r o m t h a t o f t h e s a l i n e g r o u p . T h e p l a s m a c o n c e n t r a t i o n - t i m e r e l a t i o n s h i p f o l l o w i n g t h e a d m i n i s t r a t i o n T a b l e 24. The mean (± s.e.m.) w e i g h t and o c c l u d e d z o n e (OZ) i n t h e d i f f e r e n t t r e a t m e n t g r o u p s i n p e n t o b a r b i t o n e a n a e s t h e t i z e d r a t s W e i g h t * OZ (9 ) (%) S a l i n e 402 + 18 34 ± 1 R,S-mex 410 + 6 36 + 1 (20 mg/kg) R,S-mex 410 + 10 34 + 1 (40 mg/kg) * W e i g h t o f r a t s i n e a c h g r o u p . 0Z r e f e r s t o t h e p e r c e n t a g e o f t h e v e n t r i c l e t h a t was o c c l u d e d . T a b l e 25. The e f f e c t o f R , S - m e x i l e t i n e on t h e i n c i d e n c e o f a r r h y t h m i a s f o l l o w i n g c o r o n a r y a r t e r y o c c l u s i o n i n p e n t o b a r b i t o n e a n a e s t h e t i z e d r a t s VT VF SVF NSVF S a l i n e 9/9 8/9 4/9 6/9 R,S-mex (20 mg/kg) 8/9 4/9 4/9 2/9 R,S-mex (40 mg/kg) * 3/9 * 0/9 * 0/9 * 0/9 * s i g n i f i c a n t (P<0.05) when c o m p a r e d t o s a l i n e . VT, VF, SVF and NSVF ha v e t h e same m e a n i n g as i n t a b l e 20. R a t s were m o n i t o r e d o v e r a 30 min p e r i o d . T a b l e 26. T h e e f f e c t s o f r a c e m i c m e x i l e t i n e on t h e number o f p r e m a t u r e v e n t r i c u l a r c o n t r a c t i o n s ( l o g j Q P V C s ) f o l l o w i n g c o r o n a r y a r t e r y o c c l u s i o n i n p e n t o b a r b i t o n e a n a e s t h e t i z e d r a t s l o g 1 0 P V C s s a l i n e 2.42 + 0.16 R,S-mex 2.80 + 0.06 (20 mg/kg) R,S-mex 1.68 ± * 0.30 (40 mg/kg) V a l u e s a r e t h e means ± s.e.m., n = 9 r a t s . * S i g n i f i c a n t (p<0.05) when c o m p a r e d t o s a l i n e . P r e m a t u r e v e n t r i c u l a r c o n t r a c t i o n s a r e e x p r e s s e d as l o g j p P V C s s i n c e t h e number o f PVCs i s a log - m n o r m a l l y d i s t r i b u t e d v a r i a b l e . R a t s w e r e m o n i t o r e d o v e r a 30 min p e r i o d . 6 I F i g u r e 3 1 . The e f f e c t s o f r a c e m i c m e x i l e t i n e (20 and 40 mg/kg) and s a l i n e on " a r r h y t h m i a s c o r e " f o r t h e 0-30 min p e r i o d f o l l o w i n g c o r o n a r y a r t e r y o c c l u s i o n i n p e n t o b a r b i t o n e a n a e s t h e t i z e d r a t s . * S i g n i f i c a n t (p<0.05) when comp a r e d t o s a l i n e . 163 o f 20 mg/kg o f R , S - m e x i l e t i n e and t h e d i s t r i b u t i o n o f m a j o r a r r h y t h m i a s (VT + VF) d u r i n g t h e 30 min a f t e r c o r o n a r y o c c l u s i o n a r e shown i n f i g u r e 32. The mean p l a s m a c o n c e n t r a t i o n s r a n g e d f r o m 5.0 ± 0.3 M9/ml a t t h e e n d o f d r u g i n f u s i o n (5 min b e f o r e c o r o n a r y a r t e r y o c c l u s i o n ) t o 1.6 ± 0.2 jug/ml 30 min p o s t - o c c l u s i o n ( f i g . 3 2 A ) . T h e r e was a s i g n i f i c a n t l y (p<0.05) l o w e r i n c i d e n c e o f m a j o r a r r h y t h m i a s d u r i n g t h e 0-5 min p e r i o d p o s t - o c c l u s i o n ( F i g . 32B) when c o m p a r e d t o t h e s a l i n e g r o u p ( F i g . 3 3 ) . T h e c o r r e s p o n d i n g mean p l a s m a c o n c e n t r a t i o n , 5 m i n p o s t - o c c l u s i o n , was a p p r o x i m a t e l y 3.5 jug/ml ( F i g . 3 2 A ) . In t h e g r o u p o f r a t s t h a t r e c e i v e d 40 mg/kg o f R , S - m e x i l e t i n e , where s i g n i f i c a n t (p<0.05) s u p p r e s s i o n o f a r r h y t h m i a s was o b s e r v e d t h r o u g h o u t t h e 30 min p e r i o d p o s t - o c c l u s i o n ( F i g . 3 4 B ) , p l a s m a c o n c e n t r a t i o n s r a n g e d f r o m 10.3 ± 0.5 /xg/ml a t t h e e n d o f d r u g i n f u s i o n t o 3.3 ± 0.3 iig/ml 30 min p o s t - o c c l u s i o n ( f i g . 3 4 A ) . T h e s e r e s u l t s c o n f i r m e d t h a t t h e a n t i a r r h y t h m i c e f f e c t s o f r a c e m i c m e x i l e t i n e a r e s e e n a t h i g h e r p l a s m a c o n c e n t r a t i o n s t h a n t h o s e a c h i e v e d i n t h e c o n s c i o u s r a t s . However, t h e s e c o n c e n t r a t i o n s a r e c o m p a r a b l e t o , o r g r e a t e r t h a n t h o s e a s s o c i a t e d w i t h CNS t o x i c e f f e c t s i n c o n s c i o u s r a t s ( t a b l e 2 5 ) . The r e s u l t s o b t a i n e d f r o m b o t h t h e e l e c t r i c a l and i s c h a e m i c a r r h y t h m i a s t u d i e s i n d i c a t e d t h a t t h e a n t i a r r h y t h m i c , c a r d i o v a s c u l a r and CNS t o x i c e f f e c t s o f r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s a r e n o t m a r k e d l y d i f f e r e n t f r o m e a c h o t h e r . E CO ro a O •1.0 -5 O 10 15 20 25 30 35 Time (min) LL > > O V c •g o c 0-5 5-10 10-15 Time (min) 15-30 Figure 32. The plasma concentration-time relationship following the administration of racemic mexiletine (20 mg/kg) (A) and the distr ibut ion of major arrhythmias (VT + VF) in ischaemic pentobarbitone anaesthetized rats (B). The abscissa indicates time (min) with respect to coronary artery occlusion. The plasma concentrations are means ± s.e.m. of results from 9 rats. 165 LL > 1-> <D O C <D "D O C 100 90 -80 -70 60 50 40 r 30 20 10 h 0 0-5 mm • 5-10 10-15 Time (min) 15-30 F i g u r e 33. The d i s t r i b u t i o n o f m a j o r a r r h y t h m i a s (VT + VF) i n i s c h a e m i c p e n t o b a r b i t o n e a n a e s t h e t i z e d r a t s f o l l o w i n g t h e a d m i n i s t r a t i o n o f s a l i n e ( c o n t r o l ) . The a b s c i s s a i n d i c a t e s t i m e ( m i n ) w i t h r e s p e c t t o c o r o n a r y a r t e r y o c c l u s i o n 3 2 1 O -10 -5 O 10 15 20 25 30 35 Time (min) 100 0-5 5-10 10-15 Time (min) 15-30 Figure 34. The plasma concentration-time relationship following the administration of racemic mexiletine (40 mg/kg) (A) and the distr ibution of major arrhythmias (VT + VF) in ischaemic pentobarbitone anaesthetized rats (B). The abscissa indicates time (min) with respect to coronary artery occlusion. The plasma concentrations are means ± s.e.m. of results from 9 rats. 167 4. SUMMARY and CONCLUSIONS The s y n t h e s i s and c h a r a c t e r i z a t i o n o f 2 - a n t h r o y l c h l o r i d e was s u c c e s s f u l l y a c c o m p l i s h e d . D e r i v a t i z a t i o n o f m e x i l e t i n e e n a n t i o m e r s w i t h t h e a c i d c h l o r i d e was r a p i d and t h e e n a n t i o m e r d e r i v a t i v e s f o r m e d were r e s o l v e d on a P i r k l e i o n i c ( p h e n y l g l y c i n e ) c h i r a l c o l u m n by HPLC. 2 - A n t h r o y l c h l o r i d e was f o u n d t o be a h i g h l y s e n s i t i v e f l u o r e s c e n c e d e r i v a t i z a t i o n r e a g e n t . A t t e m p t s a t u s i n g t h i s d e r i v a t i z a t i o n r e a g e n t t o q u a n t i t a t e m e x i l e t i n e e n a n t i o m e r s w e r e - u n s u c c e s s f u l due t o t h e p r e s e n c e o f an i n t e r f e r i n g p eak c o - e l u t i n g w i t h S ( + ) - m e x i l e t i n e w h i c h c o u l d n o t be r e s o l v e d . A p r e v i o u s l y d e v e l o p e d , s t e r e o s e l e c t i v e HPLC a s s a y , w i t h 2 - n a p h t h o y l c h l o r i d e as d e r i v a t i z a t i o n r e a g e n t , was s u b s e q u e n t l y u s e d f o r t h e p h a r m a c o k i n e t i c and p h a r m a c o d y n a m i c s t u d i e s . The s e r u m p r o t e i n b i n d i n g s t u d i e s i n humans r e v e a l e d t h a t t h e b i n d i n g o f m e x i l e t i n e e n a n t i o m e r s was p H - d e p e n d e n t ; w i t h f r e e f r a c t i o n s d e c r e a s i n g s i g n i f i c a n t l y as t h e pH was i n c r e a s e d f r o m 7.0 t o 8.0. T h e s e r e s u l t s h a v e i m p o r t a n t i m p l i c a t i o n s s i n c e when pH was m a i n t a i n e d a t p h y s i o l o g i c a l v a l u e s ( = 7 . 4 ) , t h e o b s e r v e d b i n d i n g o f m e x i l e t i n e was m o d e r a t e and s i g n i f i c a n t l y l o w e r t h a n t h a t r e p o r t e d i n t h e l i t e r a t u r e ( T a l b o t et a7 ., 1973; M c E r l a n e et a7., 1 9 8 7 ) . More i m p o r t a n t l y , t h e b i n d i n g o f t h e e n a n t i o m e r s was n o t s t e r e o s e l e c t i v e a t p h y s i o l o g i c a l pH. T h e s e c h a n g e s i n f r e e f r a c t i o n s a r e most l i k e l y due t o p H - d e p e n d e n t c h a n g e s i n t h e i o n i z a t i o n o f t h e b i n d i n g p r o t e i n ( s ) . The b i n d i n g s t u d i e s d i d n o t r e v e a l any s i g n i f i c a n t i n t e r a c t i o n s b e t w e e n t h e e n a n t i o m e r s a f f e c t i n g b i n d i n g n o r any c o n c e n t r a t i o n - d e p e n d e n t b i n d i n g w i t h i n t h e t h e r a p e u t i c r a n g e o f r a c e m i c m e x i l e t i n e . The e n a n t i o m e r s were d e t e r m i n e d t o b i n d m a i n l y t o AAG and HSA. B i n d i n g t o s e r u m and HSA i n d i c a t e d t h e p r e s e n c e o f two c l a s s e s o f b i n d i n g s i t e s . T h e s e were a h i g h a f f i n i t y , l o w c a p a c i t y 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 c l a s s o f b i n d i n g s i t e s . In c o n t r a s t , b i n d i n g t o AAG showed o n l y one c l a s s o f b i n d i n g s i t e s w h i c h was a h i g h a f f i n i t y , l o w c a p a c i t y s i t e . P h a r m a c o k i n e t i c and t i s s u e d i s t r i b u t i o n s t u d i e s i n r a t s i n d i c a t e d v e r y e x t e n s i v e t i s s u e u p t a k e and r a p i d e l i m i n a t i o n o f t h e e n a n t i o m e r s . R ( - ) - M e x i l e t i n e had a m a r k e d l y g r e a t e r s y s t e m i c c l e a r a n c e and s t e a d y -s t a t e v o l u m e o f d i s t r i b u t i o n t h a n t h e S ( + ) - e n a n t i o m e r . The 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 v e s o f t h e e n a n t i o m e r s were n o t d i f f e r e n t . The u p t a k e o f t h e e n a n t i o m e r s i n t o t h e t i s s u e s s t u d i e d was n o t s t e r e o s e l e c t i v e , e x c e p t f o r t h e l i v e r t i s s u e t h a t showed a much g r e a t e r u p t a k e o f t h e S ( + ) - e n a n t i o m e r . H i g h t i s s u e / s e r u m r a t i o s (>20) were o b s e r v e d i n b r a i n , l u n g s and k i d n e y s . The b r a i n a c c u m u l a t e d 3 - f o l d t h e h e a r t c o n c e n t r a t i o n s o f t h e e n a n t i o m e r s . T h i s r e l a t i v e l y h i g h u p t a k e o f t h e e n a n t i o m e r s i n t o t h e b r a i n t i s s u e c o u l d e x p l a i n t h e CNS t o x i c e f f e c t s o f t e n o b s e r v e d w i t h m e x i l e t i n e t h e r a p y . T he p h a r m a c o d y n a m i c s t u d i e s u s i n g e l e c t r i c a l - i n d u c e d a r r h y t h m i a s i n p e n t o b a r b i t o n e a n a e s t h e t i z e d r a t s showed t h a t r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s s i g n i f i c a n t l y i n c r e a s e d VFT. However, no s i g n i f i c a n t d i f f e r e n c e s i n t h i s e f f e c t was f o u n d b e t w e e n t h e d r u g s . MFF was s i g n i f i c a n t l y d e c r e a s e d and R,S- and S ( + ) - m e x i l e t i n e showed s i g n i f i c a n t l y g r e a t e r e f f e c t s t h a n t h e R ( - ) - e n a n t i o m e r . However, t h e i n c r e a s e i n ERP i n d u c e d by r a c e m i c m e x i l e t i n e and i t s e n a n t i o m e r s d i d n o t show s i g n i f i c a n t d i f f e r e n c e s . The p r o l o n g a t i o n o f PR i n t e r v a l and d e c r e a s e i n h e a r t r a t e due t o t h e d r u g s d i d n o t d i f f e r s i g n i f i c a n t l y f r o m e a c h o t h e r . B a s e d on t h e s e r e s u l t s , i t i s u n l i k e l y t h a t t h e antiarrhythmic effects of racemic mexiletine and i ts enantiomers are markedly different from each other. The 3 drugs produced comparable hypotension in pentobarbitone anaesthetized rats, although, this effect may have been due to the presence of anaesthesia and/or recent surgery in these animals. This conclusion is based on the fact that similar effects were not present in the conscious rats. In the conscious ischaemic rats, racemic mexiletine and the enantiomers did not significantly reduce the incidence and number of arrhythmias nor the "arrhythmia score" when compared to saline. As was the case in the pentobarbitone anaesthetized rats, the significant bradycardic and PR prolongation effects of the drugs were not stereoselective. Comparable CNS toxici ty was induced by racemic mexiletine and the enantiomers in the conscious rats. Studies in the pentobarbitone anaesthetized ischaemic rats using a higher dose of racemic mexiletine (which could not be tolerated by conscious rats) revealed that the effective plasma concentrations of racemic mexiletine were within the toxic range in rats. The results in the anaesthetized rats explained the ineffectiveness of racemic mexiletine and i ts enantiomers in conscious rats. The data obtained from both the electrical and occlusion-induced arrhythmia studies suggested that there were no marked differences in the antiarrhythmic, cardiovascular and CNS toxic effects of racemic mexiletine and i ts enantiomers. The important question raised by the results of this study is whether a truly effective antiarrhythmic plasma concentration of racemic mexiletine can be achieved and maintained in man without the manifestation of severe CNS toxic effects. Although the results of many s t u d i e s c a r r i e d o u t i n a n i m a l s ( m a i n l y d o g s ) h a v e shown t h e a n t i a r r h y t h m i c e f f e c t i v e n e s s o f r a c e m i c m e x i l e t i n e , t h e s e s t u d i e s h a v e u s e d t h e H a r r i s 2 - s t a g e c o r o n a r y a r t e r y o c c l u s i o n p r o c e d u r e . I t i s w e l l known t h a t VF d o e s n o t o c c u r i n t h e s e d o g s ( s e c t i o n 1 . 1 . 2 . 5 . 3 ) . T h u s , t h i s model w o u l d n o t p r o v i d e i n f o r m a t i o n on t h e e f f e c t o f r a c e m i c m e x i l e t i n e on VF, w h i c h i s t h e p r i m a r y c a u s e o f s u d d e n d e a t h . Many i n v e s t i g a t o r s who h a v e s t u d i e d t h e b e n e f i c i a l e f f e c t s o f r a c e m i c m e x i l e t i n e w i t h r e s p e c t t o p r e v e n t i o n o f s u d d e n d e a t h i n MI p a t i e n t s h a v e n o t f o u n d any s i g n i f i c a n t r e d u c t i o n i n d e a t h r a t e i n t h e s e p a t i e n t s when c o m p a r e d t o c o n t r o l p a t i e n t s ( s e c t i o n 1 . 2 . 5 ) . I t i s p l a u s i b l e t h a t t h e i n e f f e c t i v e n e s s o f r a c e m i c m e x i l e t i n e was due t o t h e m a i n t e n a n c e o f s u b - t h e r a p e u t i c p l a s m a c o n c e n t r a t i o n s i n t h e s e p a t i e n t s . 5. 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