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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 , 1 9 8 6 A T H E S I S SUBMITTED IN P A R T I A L F U L F I L M E N T OF THE REQUIREMENTS OF DOCTOR OF PHILOSOPHY  FOR THE DEGREE  in THE FACULTY OF GRADUATE S T U D I E S Faculty o f Pharmaceutical Sciences Division o f Pharmaceutical Chemistry We a c c e p t t h i s t h e s i s a s c o n f o r m i n g to t h e required  standard  THE U N I V E R S I T Y OF B R I T I S H COLUMBIA November, 1989 (c)  L i n u s Igwemezie  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.  Department of  Pharmaceutical  The University of British Columbia Vancouver, Canada  Date D e c e m b e r 28, 1 9 8 9  DE-6 (2/88)  Sciences  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 o f 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  of mexiletine  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 o f the acid  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 ethyl acetate/2-propanol/Hexane (4:6:90). Detection 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 c o u l d not be r e s o l v e d . T h i s precluded the use o f the assay f o r the proposed pharmacokinetic and studies.  pharmacodynamic  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  protein binding  of  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 glycoprotein.  The b i n d i n g 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 . due, mainly, to albumin and o ^ - a c i d g l y c o p r o t e i n .  T h i s b i n d i n g was  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 ii  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 . 0 5 ) g r e a t e r that of t h e R(-)-enantiomer. observed  than  H o w e v e r , 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  a t p h y s i o l o g i c a l pH  7.4). These r e s u l t s i n d i c a t e d that the  serum b i n d i n g o f m e x i l e t i n e enantiomers  i s pH-dependent.  B i n d i n g was n o t  concentration-dependent,  n o r was t h e r e a n y 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  between t h e enantiomers,  w i t h i n the t h e r a p e u t i c range.  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 serum and albumin  Scatchard analysis  b o t h showed t h e  presence o f 2 c l a s s e s of b i n d i n g s i t e s .  A high a f f i n i t y , low 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 o f 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 capacity site. Pharmacokinetic  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 ( 1 0 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 uptake and r a p i d e l i m i n a t i o n o f t h e enantiomers.  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 ( 1 6 1 . 8 m l / m i n / k g vs 1 2 2 . 9 m l / m i n / k g ) t h a n the S(+)-enantiomer.  The steady state-volume  greater f o r the R(-)-enantiomer  ( 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 of t h e enantiomers S(+)-mexiletine,  o f d i s t r i b u t i o n was a l s o  ( 1 . 4 a n d 1.3 h f o r R ( - ) - a n d  r e s p e c t i v e l y ) w e r e 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 observed  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 , kidney, l i v e r and f a t ) . These 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 , except f o r t h e l i v e r t i s s u e where 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(+)-enantiomer r a t i o s (>20) w e r e o b s e r v e d kidneys.  f o r each enantiomer  The b r a i n accumulated  was f o u n d .  High  tissue/serum  i n t h e b r a i n , l u n g s and  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  enantiomers. 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 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 and i s c h a e m i a - i n d u c e d  arrhythmias  were c a r r i e d out u s i n g e l e c t r i c a l  in rats.  R a c e m i c m e x i l e t i n e and i t s  enantiomers  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.  differences  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  statistically significant.  R,S-,  S ( + ) - and R ( - ) - m e x i l e t i n e  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 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 .  of  However, the were not  caused  pentobarbitone  These e f f e c t s o f the drugs were a l s o  s i g n i f i c a n t l y d i f f e r e n t from each 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  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,  rats, the  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  saline  (control).  comparable  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 x i c i t y i n the conscious  rats.  produced  not  CNS  V  TABLE OF CONTENTS ABSTRACT LIST OF TABLES LIST OF FIGURES SYMBOLS AND ABBREVIATIONS ACKNOWLEDGEMENT  ii xiii xv xvi i i xxi  CHAPTER  Page  1.  1  1.1  INTRODUCTION Sudden C a r d i a c Death  1  1.1.1  Overview  1  1.1.2  Arrhythmias  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  Acute Myocardial Ischemia. 1.1.2.3  1.2  Arrhythmogenesis  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  Triggered Automaticity  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  Experimental Arrhythmias  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 arrhythmias  12  1.1.2.5.2  Chemically-Induced Arrhythmias  13  1.1.2.5.3  Mechanically-Induced Arrhythmias  13  1.1.2.5.4  Reperfusion-Induced Arrhythmias  14  Mexiletine  15  1.2.1  Chemistry  15  1.2.2  Pharmacology  17  1.2.2.1  Animal S t u d i e s  17  1.2.2.2  Clinical Effectiveness  17  vi 1.2.2.3  Plasma Concentration-Clinical Effect Relationships  1.3  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  Chirality  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 1.3.3.1  Stereoselective Drug Analysis Introduction  34 34  vi i 1.3.3.2  R e s o l u t i o n o f Enantiomers as Diastereoisomers  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  Using  Chiral Eluents  38  1.4  Rationale  39  1.5  Specific Objectives  43  2. 2.1  2.2  EXPERIMENTAL Supplies  2.4  44  2.1.1  Drugs  44  2.1.2  C h e m i c a l s and Reagents  44  2.1.3  Solvents  44  2.1.4  Human Serum P r o t e i n s  45  Chromatographic 2.2.1  2.3  44  S t a t i o n a r y Phases and Columns  HPLC Columns  45 45  Equipment  46  2.3.1  High-Performance  L i q u i d Chromatograph  2.3.2  Gas-Chromatograph/Mass S p e c t r o m e t e r  46  2.3.3  Polygraph  46  2.3.4  Electrical Stimulator  46  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 Anthraquinone-2-carboxylic Acid 2.4.1.1 Synthesis of Anthracene-2-carboxylic Acid  46  47 47 47  2.4.1.1  Purification of Anthracene-2-carboxylic Acid  48  2.4.1.3  Characterization of Anthracene-2-carboxylic. Acid  48  vi i i 2.4.1.4  Synthesis o f 2-Anthroyl Chloride  49  2.4.1.5  P u r i f i c a t i o n o f 2-Anthroyl Chloride  49  2.4.1.6  C h a r a c t e r i z a t i o n o f 2-Anthroyl Chloride  49  2.4.2  Development o f A s s a y o f M e x i l e t i n e Enantiomers  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 Enantiomers with 2-Anthroyl Chloride  50  2.4.2.2  Chromatographic Resolution o f M e x i l e t i n e Enantiomers  50  2.4.2.3  Sensitivity  51  2.4.2.4  Structure o f 2-Anthroyl D e r i v a t i v e o f Mexiletine  51  2.4.2.5  Extraction Solvent  51  2.4.2.6  S e l e c t i o n o f Internal Standard  51  2.4.2.7  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  52  2.4.2.8  2.5  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 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 Reagent 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 2.5.2  2.6  In  C a l i b r a t i o n Curves and A s s a y P r e c i s i o n i n Human Plasma  52 54 54 55  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  Ultrafiltration  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  ix  2.7  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  Tissue Distribution Kinetics of Mexiletine Enantiomers  i n Rats  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  2.8  58  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  Pharmacokinetic  60  2.7.6  S t a t i s t i c a l Data A n a l y s i s  Data A n a l y s i s  60  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 2.8.1  61  E l e c t r i c a l l y - I n d u c e d Arrhythmia  2.8.1.1  P r e p a r a t i o n o f Rats  2.8.1.2  Experimental  2.8.1.3  Variables Related to Antiarrhythmic  End-points  61 61 '.  61  Effects  62  2.8.1.4  Other 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  Statistical Analysis  64  2.8.2  Coronary A r t e r y O c c l u s i o n i n Conscious 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  Preparation o f Occluder  65  2.8.2.3  Preparation of Lines  65  2.8.2.4  Implantation o f Lines  66  2.8.2.5 2.8.2.6  II mm pp ll aa nn tt aa tt ii oo nn oo ff tt hh ee OECG c c l uLeads der  66 67  X  2.8.2.7  Coronary A r t e r y Occlusion  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 Occlusion  69  2.8.2.10  O c c l u d e d Zone  69  2.8.2.11  Occlusion-Induced Arrhythmias  70  2.8.2.12  Evaluation of Antiarrhythmic Efficacy  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  Exclusion C r i t e r i a  72  2.8.3  Coronary Artery Occlusion 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  3. 3.1  RESULTS and DISCUSSION  73 75  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  75  3.1.1  75  Synthesis of 2-Anthroyl Chloride.  3.1.2  Development o f A s s a y o f M e x i l e t i n e Enantiomers 3.1.2.1 D e r i v a t i z a t i o n of M e x i l e t i n e Enantiomers with 2-Anthroyl Chloride 3.1.2.2 S t r u c t u r e o f the 2-Anthroyl D e r i v a t i v e o f Mexiletine 3.1.2.3 Resolution of M e x i l e t i n e Enantiomers 3.1.2.4 S e n s i t i v i t y o f the 2-Anthroyl D e r i v a t i v e of Mexiletine 3.1.2.5 3.1.2.6  79 79 82 81 85  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 Enantiomers  85  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  87  3.1.2.7  3.2  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 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  89 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  Enantiomers  95  3.3.1  95  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  3.3.1.1  Non-specific Binding  95  3.3.1.2  E f f e c t o f Serum pH  95  3.3.1.3  Competitive Binding Enantiomer  99  Methods o f Serum pH A d j u s t m e n t  3.3.3  B i n d i n g o f M e x i l e t i n e Enantiomers  99 to  Serum and t o V a r i o u s Serum P r o t e i n s  102  Determination o f Binding Constants  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  Tissue Distribution Kinetics of Mexiletine Enantiomers  3.5  Interaction)  3.3.2  3.3.4  3.4  (Enantiomer-  i n Rats  110  3.4.1  Serum L e v e l s  112  3.4.2  Pharmacokinetic  3.4.3  Serum P r o t e i n B i n d i n g  3.4.4  Recovery o f t h e Enantiomers  3.4.5  Tissue Levels  Parameters  112 115 from T i s s u e s ,  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 Enantiomers 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 Arrhythmia i n  117117 123  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  Antiarrhythmic Effects  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  Cardiac Refractory Period  129  3.5.1.3  Cardiovascular Effects  134  xi i 3.5.2  Coronary A r t e r y Occlusion-Induced 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  Antiarrhythmic Effects  142  3.5.2.4  Cardiovascular Effects  152  3.5.2.5  CNS S i d e E f f e c t s  156  3.5.3 3.5.3.1  Coronary A r t e r y Occlusion-Induced Arrhythmia 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 A n t i a r r h y t h m i c E f f e c t s o f Racemic  156  Mexiletine  158  4.  SUMMARY and CONCLUSIONS  167  5.  REFERENCES  171  xi i i LIST OF TABLES Table  Page  1.  C a l i b r a t i o n curve data f o r mexiletine e n a n t i o m e r s i n human plasma  94  2.  Percent recovery o f mexiletine enantiomers during 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 enantiomer  100  4. Methods o f serum pH a d j u s t m e n t and t h e corresponding percent free f r a c t i o n s obtained  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 enantiomers  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.  Binding constants o f mexiletine enantiomers i n serum from h e a l t h y s u b j e c t s  108  8.  Binding constants o f mexiletine enantiomers i n i s o l a t e d AAG  109  9.  Binding constants o f mexiletine enantiomers i n i s o l a t e d HSA  Ill  10.  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 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 concentration-time data a f t e r a single i . v . dose o f r a c e m i c m e x i l e t i n e  114  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 racemic mexiletine  116  12.  Percent recovery o f mexiletine enantiomers 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 enantiomers i n the t i s s u e s o f rats 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  Pharmacokinetic parameters o f m e x i l e t i n e enantiomers i n various 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  11.  14.  xiv 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 pentobarbitone anaesthetized rats  126  Pre-drug values f o r the 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 pentobarbitone anaesthetized rats  128  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 pentobarbitone anaesthetized rats  135  18.  P r e - d r u g v a l u e s f o r t h e haemodynamic parameters in pentobarbitone anaesthetized rats  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 treatment groups i n conscious ischaemic rats  16.  17.  20.  ....143  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 following coronary artery occlusion i n conscious rats  146  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 coronary occlusion i n conscious rats  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 parameters 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 enantiomers  157  21.  24.  25.  26.  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 treatment groups i n pentobarbitone anaesthetized rats  ..159  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 incidence o f arrhythmias f o l l o w i n g coronary artery occlusion i n pentobarbitone anaesthetized rats  160  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 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 (logiQPVC) 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 in pentobarbitone anaesthetized r a t s  161  XV  LIST OF FIGURES Figure 1.  The s t r u c t u r e o f m e x i l e t i n 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 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 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  3.  Page 16 76 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 standard with 2-anthroyl c h l o r i d e  80  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-anthroyl d e r i v a t i v e of mexiletine  82  7.  The major fragment i o n s o f the 2 - a n t h r o y l derivative of mexiletine  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 enantiomers  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  Chromatograms o f e x t r a c t s o f d i s t i l l e d water c o n t a i n i n g m e x i l e t i n e enantiomers 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 water (B)  88  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 standard d e r i v a t i z e d with 2-anthroyl 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 extraction  90  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 ether (A), hexane (B) and d i c h l o r o m e t h a n e (C)  91  6.  10.  11.  12.  xvi 13.  Chromatograms o f m e x i l e t i n e enantiomers 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 representative "Rosenthal" p l o t o f the binding o f m e x i l e t i n e enantiomers t o serum from a h e a l t h y male s u b j e c t  106  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 time f o r m e x i l e t i n e enantiomers i n r a t s following the administration of a single i.v. dose o f r a c e m i c m e x i l e t i n e  113  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 enantiomers i n serum, h e a r t and b r a i n t i s s u e s following the administration o f a single i . v . dose o f r a c e m i c m e x i l e t i n e  122  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 the 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 enantiomers i n P e n t o b a r b i t o n e anaesthetized rats  125  Cumulative dose-response curves o f 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 enantiomers 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  Cumulative dose-response curves o f 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 enantiomers 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  Cumulative dose-response curves o f 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 enantiomers 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  Cumulative dose-response curves o f 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 enantiomers on the 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 rats  137  Cumulative dose-response curves o f 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 enantiomers on the mean a r t e r i a l b l o o d p r e s s u r e i n pentobarbitone anaesthetized rats  138  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 enantiomers 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  16.  17.  18.  19.  20.  21.  22.  23.  24.  xvi 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 the l e f t a n t e r i o r descending coronary 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 the l e f t a n t e r i o r descending coronary 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 occlusion i n conscious rats  149  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 occlusion i n conscious rats  150  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 conscious rats  154  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 in ischaemic conscious r a t s  155  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 occlusion i n pentobarbitone anaesthetized rats  162  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 following a d m i n i s t r a t i o n o f racemic 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 of 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  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) in ischaemic pentobarbitone anaesthetized r a t s following the administration of saline (control)  165  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 following the a d m i n i s t r a t i o n o f racemic 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 of 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  28.  29.  30. 31.  32.  33.  34.  xvi i i SYMBOLS AND ABBREVIATIONS AAG  aj-Acid  ANOVA  Analysis of variance  AP  Action potential  ARP  Absolute  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  curve  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  curve  AUC  T  glycoprotein  refractory period  AV  Atrio-ventricular  C  Centrigrade  CV.  Coefficient of variation  CL  Systemic  int CNS  Intrinsic  C e n t r a l n e r v o u s 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  Emission  ERP  Effective refractory period  ex  Excitation  u  Free f r a c t i o n  GLC  G a s - l i q u i d chromatography .  HC1  Hydrochloric  HPLC  High-performance l i q u i d chromatography  HSA  Human serum albumin  ht  Height  i .d.  Internal diameter  i.p.  Intra-peritoneal  C L  clearance clearance  acid  xix I.S. i.v.  Internal  standard  Intravenous  IU  International unit  LAD  Left anterior  LP  Lipoprotein  mex  Mexiletine  MFF  Maximum f o l l o w i n g  min  Minutes  ml  Millilitre  MP  Membrane p o t e n t i a l  MS  Mass  msec  Milliseconds  ng  Nanogram  NSVF  Non-spontaneously r e v e r t i n g v e n t r i c u l a r fibrillation  OZ  O c c l u d e d zone  PE  Polyethylene  PTFE  Polytetrafluoroethylene  PVC R  descending  frequency  spectrometer  Premature V e n t r i c u l l a r C o n t r a c t i o n Resolution  r^  C o e f f i c i e n t of determination  RRP  Relative refractory period  R^.  Retention  j9  F i r s t order elimination rate  s.d.  Standard  s.e.m  S t a n d a r d e r r o r o f t h e mean  sec  Seconds  SVF  Spontaneously 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  time constant  deviation  XX  UV  Ultraviolet  VF  Ventricular  VFT  Ventricular f i b r i l l a t i o n  vs  Versus  V  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  s s  fibrillation threshold  VT  Ventricular  tachycardia  wt  Weight  xs  Excess  a  F i r s t order d i s t r i b u t i o n rate constant  /xl  Microlitre  fig  Microgram  xxi  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 . g r a t i t u d e a l s o goes t o Mr. G r e g o r y Beatch f o r h i s t e c h n i c a l  My  assistance.  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 to pursue graduate s t u d i e s .  1 1. 1.1  INTRODUCTION  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 the 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 medical  person.  Sudden c a r d i a c d e a t h i s the l e a d i n g c a u s e o f d e a t h i n most o f the industrialized nations.  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 the U n i t e d S t a t e s  alone.  The c a u s e 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; 1975; S c h a f f e r and Cobb, 1975; Adgey, 1982)  Bashe et  Lown and Graboys, 1977; O l i v e r ,  al.,  1982;  and sudden c a r d i a c d e a t h and VF are 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). 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 ,  VF i s the most s e v e r e  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 the 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 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 M c W i l l i a m i n 1889 suggested  that  death.  t h a t VF was the l i k e l y c a u s e o f sudden  c a r d i a c death in p a t i e n t s with Angina P e c t o r i s .  The  first  electrocardiogram  (ECG) o f VF i n the 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 the c a u s e o f sudden c a r d i a c d e a t h i n man  following  2  myocardial  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 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).  arrhythmias  This i s c o n s i s t e n t with  the 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; Perper e t al.,  1975).  However, sudden  c a r d i a c d e a t h i s n o t 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 than 20% o f those 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  that  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; Maseri 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 1978;  Stockman e t al., Epidemiological  1979; K a b e l l e t al,  al.,  1980).  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  these  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 (Coronary 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 s e a r c h 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 d r u g s have been d e v e l o p e d f o r the 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 .  a g a i n s t a l l forms o f a r r h y t h m i a s , pharmacokinetics  However, i n e f f e c t i v e n e s s  t o x i c i t y , and sometimes  unfavourable  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  agents.  For example, q u i n i d i n e i n t h e r a p e u t i c d o s e s may induce 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 n o t  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 a7., 1981).  (Singh e t  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  t h 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 1.1.2.1  Arrhythmias 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), +  +  the c e l l maintains a high 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 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 concentration.  potassium sodium  A t 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  to 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 chloride ions.  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 o u t 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 h e 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 t h e 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 c e 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. inward C a  ++  Phase 2 is characterized by  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. inactivation of the C a f u l l repolarization.  ++  Phase 3 is typified by  current and a rapid efflux of K leading to +  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 a r e 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 anaesthetics.  The slow f i b r e s a r e found i n t h e proximal 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  current  AP which i s m e 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. b l o c k e d by manganese and c a l c i u m  The AP i s  antagonists.  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. ( S i n g h e t al.,  T h i s r e l a t i o n s h i p i s known as membrane  1981).  responsiveness  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 .  This  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  to the period  d u r i n g which a s t i m u l u s cannot p r o d u c e any d e g r e e 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 b u t an a c t i o n p o t e n t i a l cannot be propagated, p e r i o d (ERP).  t h i s represents the e f f e c t i v e refractory  Below -60 mV a s t r o n g e r than normal s t i m u l u s may  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 . r e f r a c t o r y p e r i o d (RRP). are conducted  This corresponds  generate  to the r e l a t i v e  Action p o t e n t i a l s generated during t h i s period  slowly, consequently,  f o r m a t i o n ( P e r r y and I l l s l e y , 1986).  t h e h e a r t i s prone t o a r r h y h t h m i a  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 and H i r c h e e t al.  al.,  1954).  +  H i l l and G e t t e s ( 1 9 8 0 )  (1980) 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  +  accompanied  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  +  observed l o s s of 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  to anoxia-induced increase in K pump.  Simultaneous  +  conductance or i n h i b i t i o n of the  to the increase in e x t r a c e l l u l a r K  t h e AP a m p l i t u d e , MRD  due sodium  i s a decrease in  +  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 ) .  These  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. H o w e v e r , 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 e d d e p r e s s i o n o f t h e AP c h a r a c t e r i s t i c s observed during acute ischaemia.  upstroke  The r e s u l t s o f t h e i r  w o r k 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 washout ( 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 .  This  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  upstroke.  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 w e r e i n v e s t i g a t e d by C a r d i n a l e t al.  ( 1 9 8 1 ) and f o u n d t o be a " d e p r e s s e d f a s t  T h i s c o n c l u s i o n was d e d u c e d which showed:  response".  from r e s u l t s obtained i n i s o l a t e d h e a r t s  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,  at which l e v e l the f a s t  Na  +  channel 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 shortened.  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., L a z z a r a et al.,  1978).  1977;  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 lower 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), are present during acute ischaemia. 1.1.2.3  Arrhythmogenesis  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 ) , impulse 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 fibrillation. 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 )  Automaticity i s the a b i l i t y o f a cardiac f i b r e to 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 ( W i t 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 the 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 valve l e a f l e t s .  The SA node i s n o r m a l l y the dominant f i b e r (pacemaker)  and e s t a b l i s h e s the 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 the 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 the 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 the a c t i o n p o t e n t i a l (Wit e t al., 1981).  1974a; V e r a and Mason,  For example, vagal 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 the 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 the 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 the 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 , thus enhancing  automaticity.  1.1.2.3.2  Triggered Automaticity  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 the 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 arrhythmias  (Spear and Moore, 1982).  o n l y been demonstrated al.,  1973).  in  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  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  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 , notably the coupling of d e p o l a r i z a t i o n to a preceding beat.  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 are a s s o c i a t e d with elevated i n t r a c e l l u l a r C a o s c i l l a t o r y release of C a  + +  + +  concentrations.  T h i s provokes  from the s a r c o p l a s m i c r e t i c u l u m and  the  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  amplitude  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 the i n t r a c e l l u l a r C a Ca  + +  + +  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  or t o x i c l e v e l s of 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  (Re-entry)  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  evidence f o r the e x i s t e n c e of r e - e n t r a n t c i r c u i t s .  Re-entrant  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.  If conduction  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 , then 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 type of re-  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 long i f conduction i s depressed.  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  agents.  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  cells.  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 g r o u p s (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 r u g s 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 the nerve 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 observed a t lower c o n c e n t r a t i o n s than t h o s e on nerves. Na , +  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  thus 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 depression i n conduction v e l o c i t y , a prolongation i n the 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  the suppression o f automaticity or re-entrant arrhythmias.  Class 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 , disopyramide,  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 .  c l a s s i f i c a t i o n o f t h e s e agents has been proposed  A further  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). Quinidine, procainamide  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 d r u g s 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 lidocaine.  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 the k i n e t i c s o f the i n t e r a c t i o n o f t h e s e drugs w i t h the f a s t sodium c h a n n e l s W i l l i a m s , 1984) produced  (Vaughan  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).  Clinically,  l i f e threatening arrhythmias often occur associated with o v e r - a c t i v i t y of sympathetic  o u t - f l o w (Winslow, 1984).  Catecholamines  can  exacerbate  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 the 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 , thus, provoking tachyarrhythmias S i n g h , 1974).  ( J e w i t t and  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 the 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 p r o d u c e 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 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 . drugs i n t h i s c l a s s are  (Vaughan  Examples o f  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 the APD and ARP (Singh and Vaughan W i l l i a m s , Vaughan W i l l i a m s , 1977).  1970;  Drugs i n t h i s c l a s s i n c l u d e amiodarone and  bretylium. 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 the slow inward  current  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 are 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, are n o r m a l l y mediated sodium c u r r e n t .  by f a s t inward  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 , the  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  antiarrhythmic action of calcium antagonists i s a t t r i b u t e d to abolishing t h e s e abnormal 1.1.2.5  slow d e p o l a r i z a t i o n s . Experimental  Arrhythmias  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, 1.1.2.5.1  1984).  Electrically-induced arrhythmias.  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 inhomogeneity 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).  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.  This  An e x t r a -  stimulus of s u f f i c i e n t intensity applied during this period will precipitate fibrillation.  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  fibrillation.  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 progressively increasing intensity.  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 a d v a n t a g e s .  Its  e f f e c t s are f u l l y r e v e r s i b l e (except f o r extremely intense s t i m u l i ) , the 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 c o n t r o l l e d ( S z e k e r e s and Papp, 1971).  and  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  Chemically-Induced  Arrhythmias  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 arrhythmias i n animals. produced VF i n mice.  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  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 .  Recently, chloroform-induced  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  Mechanically-Induced  Arrhythmias  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  partial  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 p e r 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 .  Arrhythmias  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 twos 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 morphineurethane-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 u t i l i z e d 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., 1986).  1983; Curtis et al; 1984 &  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.,  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  1978).  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  activity  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., 1.2  1977).  Mexiletine 1.2.1  Chemistry  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 p h e n e t h a n o l a m i n e compound, P h e 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  anticonvulsant activity.  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).  It 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 t h e 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 ) which i s an a l m o s t 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 R  w a t e r , m e t h a n o l , 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-CH -CH-NH 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 propane]  2  [(2',6'-dimethylphenoxy)-2-amino  1.2.2  Pharmacology  1.2.2.1  Animal  Studies  E a r l y s t u d i e s on the 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., W i l l i a m s (1972b).  (1972) and S i n g h and Vaughan  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 induced by d i g i t a l i s i n dogs. plasma c o n c e n t r a t i o n was 0.6  .  The e f f e c t i v e  In the c a n i n e 24 h t w o - s t a g e  coronary occlusion arrhythmia, mexiletine administered  intravenously  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.  Further studies in canine  arrhythmia  models (Hashimoto et al.,  1984) have c o n f i r m e d the a n t i a r r h y t h m i c  efficacy of mexiletine.  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 the dose o f m e x i l e t i n e g i v e n t o the dogs i n t h i s study. The o t h e r p h a r m a c o l o g i c a l  properties of mexiletine include strong  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  Clinical Effectiveness  Numerous human s t u d i e s have r e p o r t e d the 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  myocardial  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  1978a; T a l b o t e t al., 1980; Bury e t al., 1.2.2.3  e t al.,  1973 &  1973 & 1976; Z i p e s and Troup, 1978; S i n g h et  al.,  1982 and IMPACT, 1984). 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 Relationships  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 at 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 al.,  (1978a).  et  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 the p a t i e n t s when plasma c o n c e n t r a t i o n was between 0.74 and 1.0 /zg/ml•  maintained  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 , tremor, 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 the 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 cases with 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 beats 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  responders  whose 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 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 m a i n t e n a n c e t r e a t m e n t were i n the same range, 0.44 t o 2.0 /jg/ml ( P o d r i d and Lown, 1981).  However, serum  mexiletine concentrations  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 the 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 the 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 the  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  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 , Yamaguchi et al.,  1979).  i s blockade of  The d e c r e a s e i n V  m a x  1977  and  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 . 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 depressed conduction  1979).  tissue preparations,  In i s o l a t e d  mexiletine  v e l o c i t y , i n c r e a s e d the 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 r o d u c e d a marked p r o l o n g a t i o n and Vaughan W i l l i a m s ,  in  o f the ERP ( A l l e n et al.,  1972b; Vaughan W i l l i a m s ,  1977;  1972;  A r i t a et  Singh  al.,  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 the r e s t i n g MP o r APD are a s s o c i a t e d w i t h the d e p r e s s i o n spontaneous d i a s t o l i c d e p o l a r i z a t i o n in automatic f i b r e s . P u r k i n j e f i b r e s o f the dog, a c o n c e n t r a t i o n  1979).  In i s o l a t e d  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 et a/., 1979; and Yamaguchi et al.,  of  Weld e t al,  1979  T h i s was accompanied by a s h o r t e n i n g  the 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 et al.,  1979)  of  but the r a t i o  o f the 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 another action 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 the 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  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.  of guinea pig However, the 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 decrease V  m a x  24% and 33% at 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 demonstrated mexiletine is potentiated in  t h a t the sodium channel b l o c k i n g e f f e c t o f 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 the 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  al.,  1983).  et al., 1983a & 1983b; H e r i n g et  M e x i l e t i n e has no e f f e c t on the c a l c i u m channel o r the  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, the e l e c t r o p h y s i o l o g i c e f f e c t s 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, the  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 the 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 the 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 the 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 et al., 1977).  However, i f the  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 drug 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. 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.  The two  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 ^-adrenoceptor antagonists in suppressing ventricular arrhythmias. 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 chronic ventricular arrhythmias.  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 of 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  trial  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 the 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 r e m a i n s t o be c l e a r l y e s t a b l i s h e d . r e v i e w o f the r e s u l t s o f 20 r a n d o m i z e d , c o n t r o l l e d , c l i n i c a l  A  trials  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 myocardial  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.,  study 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 C h a m b e r l a i n et al.,  acute  1983).  In a  patients,  (1980) were not a b l e t o show any s i g n i f i c a n t  reduction in death r a t e .  Recently,  i n a n o t h e r study i n c a r d i a c  p a t i e n t s , the 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 the 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, the d i f f e r e n c e between the two g r o u p s 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 study i n 240 p a t i e n t s w i t h myocardial  i n f a r c t i o n ( S m y l l i e et al.,  primary f i b r i l l a t i o n .  1984), m e x i l e t i n e d i d not  The m o r t a l i t y a t 6 weeks i n t h i s study was  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 the p l a c e b o g r o u p . study ( S t e i n et al.,  prevent not  However, one  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 the 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  Clinical  Indications  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 arrhythmias  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 beats ( 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).  Mexiletine is  23  also useful i n the p r o p h y l a c t i c treatment o f v e n t r i c u l a r arrhythmias 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 6 0 % 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 R i h n , 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 3 0 % 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 bradycardia. 1.2.8 1.2.8.1  Pharmacokinetic Parameters Pharmacokinetics of racemic mexiletine  M e x i l e t i n e i s r a p i d l y a b s o r b e d 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 drug. al.,  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 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., and is increased by urinary acidosis.  1985),  At a urinary pH of 5, renal  clearance is reported to increase by a factor of 3 to 4 (Kiddie et 1974; Prescott et al.,  1977).  al.,  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., Campbell et al., 1.2.8.2  1977 and  1978b). 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 i v e r 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 A l l a f et al.,  1982).  Delayed and incomplete absorption, as well as  increased elimination h a l f - l i f e , were associated with acute myocardial infarction (Prescott et al., et al.,  1984).  1977; Pottage et al.,  1978 and Pentikainen  Prolongation of h a l f - l i f e has also been reported in  patients with severe left ventricular failure (Leahey at al.,  1980a).  25  1.8.2.3  Pharmacokinetics  The p h a r m a c o k i n e t i c s  of Mexiletine  of m e x i l e t i n e enantiomers  from our l a b o r a t o r y (Igwemezie et al., (Grech-Belanger  et al.,  Enantiomers  1986).  has been r e p o r t e d  1989) and by one o t h e r group  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 the 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 than 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 apparent o r a l c l e a r a n c e and plasma a r e a under the 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 the two enantiomers  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  those of Grech-Belanger  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 the e l i m i n a t i o n h a l f - l i v e s o f the 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(+)-enantiomer.  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 the R ( - ) enantiomer 1.2.9  (Igwemezie, 1986). 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 1976).  e t al.,  1975; T a l b o t et  al.,  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 hours.  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 et al.,  f o l l o w i n g d o s i n g regimen:  (1977) recommended the  150 t o 200 mg i n f u s e d o v e r 2 t o 5 minutes,  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. 1.2.10  Metabolism  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 drug ( 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., Chidomere, 1977b; G r e c h - B e l a n g e r e t al., include the hydroxylamine  1985).  Minor m e t a b o l i t e s  and ketone, and t h e N-methylated  ( B e c k e t t and Chidomere 1977a & 1977b) and 2,6-dimethyl Bel anger e t a7., 1987).  1977; B e c k e t t and  metabolites  phenol  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). 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 enantiomers  1986).  The  i n humans has been shown t o  occur s t e r e o s e l e c t i v e l y i n favour of the R(-)-enantiomer e t al.,  (Grech-  (Grech-Belanger  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. 1.2.11  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 Other 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 drugs 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 problems.  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 nonc a r d i o v a s c u l a r drugs.  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 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.,  such  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).  Metoclopramide,  the o t h e r hand, i n c r e a s e s the a b s o r p t i o n r a t e (Wing et al., 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  on  1980). agents.  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, propafenone,  q u i n i d i n e , and v e r a p a m i l , which are known t o i n c r e a s e the  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., 1981; Moysey et al.,  1981., K l e i n et al.,  and Pederson et al.,  1982).  hepatic metabolism  1979; Leahey et  al.,  1982; Pedersen et al.,  1981  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  i n man, drugs which induce the h e p a t i c enzyme systems  are 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).  Cimetidine, contrary to the expected i n h i b i t i o n of m e x i l e t i n e  metabolism,  was r e p o r t e d t o have no e f f e c t on the 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 drug 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 drug, 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 are 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 p r e m a t u r e 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  the 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 the 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  "cooperative"  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 et al., myocardial  1973).  In a c u t e  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 the  serum b i n d i n g between the 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 the 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., 1.2.13  1987).  Tissue Distribution of Mexiletine  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 , the 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 the h e a r t , 3 3 - f o l d h i g h e r i n the l u n g s and a c o m p a r a b l y h i g h r a t i o i n the 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  the 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 the  CNS  side e f f e c t s often observed during m e x i l e t i n e therapy. 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 assay 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 chromatographic (HPLC) p r o c e d u r e s .  (GLC) and h i g h - p e r f o r m a n c e l i q u i d  chromatographic  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; B r a d b r o o k 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; Kacprowicz,  1982 and G r e c h - B e l a n g e r ,  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).  1984), and  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 the 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 procedures, phase.  d e r i v a t i z a t i o n r e a g e n t and the n a t u r e o f the s t a t i o n a r y  The method d e s c r i b e d by H o l t e t al., 1979, has been adapted  the 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 .  for  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); Mastropolo  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 the UV t e c h n i q u e s  not have the 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  Grech-Belanger  et 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 with  2,3,4,6-tetra-0-acetyl-/J-D-glucopyranosyl-  i s o t h i o c y a n a t e (GITC), f o l l o w e d by r e s o l u t i o n o f the e n a n t i o m e r s on a reverse-phase  ( 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 a b s o r b a n c e .  do  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 a s s a y p r o c e d u r e ( M c E r l a n e et al., 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  1987)  stationary  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 s u b s e q u e n t d e t e c t i o n o f the 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 from t h a t r e p o r t e d 1.3  improvement  above.  Chirality 1.3.1  Introduction  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  are 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 d r u g s 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 property of c h i r a l i t y .  While the natural synthesis o f c h i r a l drugs  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 p r o d u c e s a racemate (50:50 r a t i o o f t h e two enantiomers).  E n a n t i o m e r s a r e 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 r g a n i s m s a r e a b l e t o d i s c r i m i n a t e between enantiomers at the molecular l e v e l .  This often leads to 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 t h e two e n a n t i o m e r s o f a c h i r a l drug.  1.3.2 1.3.2.1  Stereoselective drug Disposition 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). 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 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 t h e 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  actions o f p r o p r a n o l o l , i n c l u d i n g the antihypertensive e f f e c t s , are m e d 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 G e 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 t i 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  Absorption  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 - i s o m e r s . 1.3.2.3  Distribution  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 . shown t h a t S ( - ) - p r o p r a n o l o l  I t has been  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. extravascular binding of S(-)-propranolol R ( + ) - e n a n t i o m e r i n dogs ( B a i e t al.,  1976).  S i m i l a r l y , the  i s g r e a t e r than t h a t o f t h e  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 , 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 . 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.,  heart,  The l a t t e r was found 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 secondary s t r u c t u r e s . small b i o a c t i v e m o l e c u l 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 F o r 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 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  was  (+)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 than t h e R-enantiomer (Brown e t a l . , 1977).  a^-acid glycoprotein  (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 d r u g s 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 a g e n t s 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.,  ( V a l d i v i e s o e t al., 1.3.2.4  1983; A l b a n i e t al.,  1983; Lima e t al.,  1984) and d i s o p y r a m i d e  1984; Lima and B o u d o u l a s , 1986).  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 ( - ) - e n a n t i o m e r 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 ( + ) - e n a n t i o m e r .  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 p r o d u c e d 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, 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, a c t i v e S ( + ) - e n a n t i o m e r (Adams e t al., e t al.,  1980;  1979; Lee e t al.,  1985).  in  1976; K r i p a l a n i e t al.,  vivo,  to the  1976; Bopp  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 determined.  E n a n t i o m e r s 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  metabolic l e v e l .  F o r example, t h e a n a l g e s i c a c t i v i t y o f l e v o m e t h o r p h a n  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 e x t r o m e t h o r p h a n , 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 metabolism. 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  Introduction  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 c a n 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 m e t h o d o l o g y 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 drugs a t therapeutic concentrations. r e s o l v i n g enantiomers  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  involves t h e i r conversion to diastereoisomeric  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 .  Resolution i s possible  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 properties, including s o l u b i l i t y i n a given solvent.  This 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 n o t 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). pharmacokinetic enantiomer  studies.  More i m p o r t a n t l y , i t i s n o t amenable t o  Thus, most o f t h e r e c e n t advances i n  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.  Chromatographic  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).  Enantiomers  are resolved  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).  Extensive review  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. (1966). I n t h e i r study with d i a s t e r e o i s o m e r i c e s t e r s of 2-acetoxypropionic 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 . There 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 without 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 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 )  bonds,  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 . Val^  Chirasil-  ( 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 al.,  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 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., F r a n k e t a7., 1979; Frank e t al.,  1980).  1977;  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 t h e r m a l s t a b i l i t y o f  C h i r a s i l - V a l 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 massR  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 d r u g s 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 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  phase  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., et al.,  1981; K o n i g  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 ( A r m s t r o n g and Damond, 1984; A r m s t r o n g et al.,  1986).  A  s i m i l a r mechanism has been p r o p o s e d f o r t h e 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., acid glycoprotein  (Hermansson,  1983) a t t a c h e d t o s i l i c a .  1983) o r oxBoth 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 . 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 (R)- and ( S ) - p h e n y l g l y c i n e , naphthylalanine.  These  columns  derivatives of  ( S ) - l e u c i n e and ( R ) - and ( S ) -  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  are 7 r - a c i d 7r-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 Alembik,  and  1988).  1.3.3.4  R e s o l u t i o n o f Enantiomers Using C h i r a l Eluents  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, 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 . r e s u l t i n enantiomer r e s o l u t i o n .  enantiomers  Two p o s s i b l e mechanisms may  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  Rationale 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 reagent.  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. et al.  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  (1986) was 50 ng/ml o f each e n a n t i o m e r .  M c E r l a n e et al.  The method d e v e l o p e d by  (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 of the stereochemical 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 the anthracene r i n g i s 1.5-fold t h a t of the naphthalene r i n g ) . b)  Rationale f o r the study of the binding of 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 t h e 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 e f f e c t s than t h e t o t a l drug 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 1981).  pharmacological (Lima et al.,  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 parameters were n o t d e t e r m i n e d was s m a l l (0.2-2 /jg/ml). for mexiletine.  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  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  The p r e s e n t s t u d y was proposed t o f u l l y c h a r a c t e r i z e  the 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 enantiomers  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 .  Furthermore,  most b a s i c  drugs 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 the p r o t e i n ( s ) r e s p o n s i b l e f o r the binding o f the enantiomers 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 c)  of mexiletine  enantiomers.  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 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 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 tissues.  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  the 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 into t i s s u e s i n c l u d i n g the  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 ( B a i e t a l . 1983) and t i m o l o l (Tocco e t a l . , 1976).  However, t h e 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 mexiletine therapy. d)  Rationale for the pharmacodynamic studies on the relative antiarrhythmic a c t i v i t y of racemic mexiletine and i t ' s enantiomers.  It i s well e s t a b l i s h e d t h a t drug enantiomers 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 reported.  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 a g e n t s  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 o r 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  potent than the S(+)-enantiomer 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 induced 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). T h e ( + ) - e n 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 canine c a r d i a c p u r k i n j e f i b r e s , while the ( - ) - e n a n t i o m e r 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 different.  1.5  a)  Specific Objectives  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 c)  glycoprotein.  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 rats. 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 e)  relationships.  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 p a r a m e t e r s i n r a t s .  2. 2.1  EXPERIMENTAL  Supplies 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 ( - ) , 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 , KOE 2963 ( i n t e r n a l 2  3  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 , C i c a t r i n , M a r c a i n e 6  7  8  and  heparin . 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 , anthraquinone-2-carboxylic a c i d , oxalyl c h l o r i d e , 1 0  1 1  1 2  calcium c h l o r i d e , potassium permanganate ^, t r i c h l o r o a c e t i c a c i d ^ , 1 3  1  1  aluminium h y d r o x i d e ( a l u m i n a ) , z i n c d u s t , sodium h y d r o x i d e , 1 6  1 7  1 8  barium  hydroxide o c t a h y d r a t e , zinc sulphate heptahydrate , hydrochloric 1 9  2 0  a c i d , sodium c h l o r i d e , monosodium phosphate m o n o h y d r a t e , d i s o d i u m 2 1  2 2  23  phosphate h e p t a h y d r a t e ^ , c o n c e n t r a t e d ammonia s o l u t i o n 2  2 6  and s u l p h u r i c  acid . 2 6  2.1.3  Solvents  The f o l l o w i n g s o l v e n t s were used:  dichloromethane , 27  2-propanol , diethyl ether , chloroform , 2 9  1-4  3 0  3 1  Boehringer Ingelheim Ltd., B u r l i n g t o n , Ontario, Canada.  n-hexane , 28  e t h a n o l , acetic a c i d , methanol , a c e t o n i t r i l e , ethyl a c e t a t e . 3 2  3 3  2.1.4  3 4  3 5  3 5  Human Serum P r o t e i n s  These were 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 3  3  d e f i c i e n t serum ^. 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 a)  HPLC Columns  A Pirkle  i o n i c c h i r a l column *' (25 x 0.46 cm i . d . ) w i t h a  R  4  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 - a m i n o p r o p y l s i l i c a was used. b)  A Pirkle  ionic chiral column  R  (25 x 0.46 cm i . d . ) w i t h a  4 1  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 - a m i n o p r o p y l 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  front of the P i r k l e d)  5 6 7 8 9 10-14 15-16  R  4 2  (1.5 x 0.32 cm i . d . ) was used i n  column.  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 . ) .  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. 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. Wellcome I n c . , K i r k l a n d , Quebec, Canada. W i n t h r o p 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. G l a x o L t d . , T o r o n t o , O n t a r i o , Canada A l d r i c h C h e m i c a l Co., Milwaukee, W i s c o n s i n , USA. 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  High-Performance 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  chromatograph  44  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 , a 20 /J1 l o o p i n j e c t o r 4 5  fluorometer 2.3.2  4 7  4 6  and a S c h o e f f e l model  was used. Gas-Chromatograph/Mass  Spectrometer  A H e w l e t t P a c k a r d model 5987A GC/MS  48  e q u i p p e d w i t h a model 5880  gas chromatograph was used. 2.3.3  Polygraph  A model 79D G r a s s p o l y g r a p h 2.3.4  970  4 9  was used.  Electric Stimulator  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 , E n g l a n d , 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 Anthraqui none-2-Carboxyli c Aci 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 to the a c i d c h l o r i d e with o x a l y l c h l o r i d e . 2.4.1.1  Synthesis of Anthracene-2-Carboxylic Acid  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,  with  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 heated 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 paper.  43 44-45 46 47 48 49-50  Whatman I n c . , C l i f t o n , New J e r s e y , USA. Mandel S c i e n t i f i c Co. L t d . , Edmonton, A l b e r t a , Canada. Rheodyne I n c . , B e r k e l e y , C a l i f o r n i a , USA. 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. 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. 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. suction.  The aqueous p o r t i o n was f i l t e r e d o u t by  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 below). 2.4.1.2  P u r i f i c a t i o n o f Anthracene-2-Carboxylic Acid  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 = 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 t  ethanol (95%). 2.4.1.3  a)  Characterization of Anthracene-2-Carboxylic Acid  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-Anthroyl  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 h o u r s 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 evaporation  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  greenish-  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 HPLC.  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  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  the 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. 2.4.1.6  The R o f t h e a c i d c h l o r i d e was 12 min. t  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 by d i r e c t probe EI-MS.  c h l o r i d e was c o n f i r m e d  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  acid.  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 Enantiomers with 2-Anthroyl 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 ( V o g e l , 1964). (200  reaction  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  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 i m e 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  Chromatographic R e s o l u t i o n of M e x i l e t i n e Enantiomers  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 ( p h e n y l 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-propanol/chloroform/hexane, ethanol/hexane, ethanol/acetonitri1e/hexane, ethanol/chloroform/hexane, 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 r o d u c e 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  Sensitivity  The d e t e c t i o n o f the 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 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  with  emission  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 determined  with a fluorescence  2.4.2.4  spectrophotometer .  S t r u c t u r e of 2-Anthroyl Mexiletine  51  Derivative of  The 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 (mexiletine-2-anthramide)  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 the  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 2.4.2.5  acid.  Extraction Solvent  The s o l v e n t used f o r the 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 ether.  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  Selection of Internal  Standard  The i n t e r n a l s t a n d a r d used was KOE 2963  [(2',6'-dimethylphenoxy)-  2 - a m i n o e t h a n e ] , which i s an a n a l o g u e 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 screwcapped 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 base).  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 discarded.  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 m i n u t e s 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 with the S(+)-enantiomer. 2.4.2.8 a)  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  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 p p r o a c h 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 e n a n t i o m e r peaks.  51  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  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 undertaken 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 . mobile  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  phase. b)  Extraction Solvent:  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  substance  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 o u t 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 o u t 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) ii)  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 Diethyl ether:  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 . iii)  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  distillation. iv)  Dichloromethane:  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) vi) d)  Anthracene-2-carboxylic acid:  by HPLC ( s e c t i o n 2.4.2.2.).  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.). Pirkle  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 column.  R  (S)-leucine chiral  2.5  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 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 - 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 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., 2.5.1  1987).  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 m l . 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 t h 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 - n a p h t h o y l 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 m i n . 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 g l y c i n e ) c h i r a l phase; m o b i l e phase,  R  i o n i c (phenyl  chloroform/2-propanol/Hexane  ( 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 Curves 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  (internal standard).  The samples were  subsequently  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 the peak h e i g h t r a t i o s o f each enantiomer  to that of the  i n t e r n a l s t a n d a r d a g a i n s t the known c o n c e n t r a t i o n o f the 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  enantiomer.  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 the samples used f o r the s t a n d a r d 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  curve. 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 Enantiomers 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 the 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 . Approximately  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 caps, a l l o w e d t o c l o t a t room temperature  f o r 2 h, c e n t r i f u g e d at 2500 x g f o r 15 min and the serum  separated. 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 . T h e f o l l o w i n g a p p r o a c h e s 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 p h o s p h a t e 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 p h o s p h a t e 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 (0.12  M).  The l a s t a p p r o a c h 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 a l b u m i n (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 p h o s p h a t e 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,  10,  40,  100,  200,  1000,  2000,  was mixed on a r o t a t o r f o r 10 m i n u t e s .  3000, and 4000 jag/ml.  The serum  D u p l i c a t e 1 ml samples were used  to determine the t o t a l drug c o n c e n t r a t i o n s while 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 drug concentrations.  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  p h o s p h a t e 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 2.6.5  Ultrafiltration  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 the MPS-1 system  5 2  AAG.  and a 30,000 D a l t o n m o l e c u l a r  u l t r a f i l t r a t i o n membrane . 53  micropartition  weight c u t - o f f  YMT  The c e n t r i f u g e (Beckman Model J 2 - 2 1 )  a 35° a n g l e head r o t o r (Beckman model J A - 1 7 ) 37°C b e f o r e i n t r o d u c t i o n o f the samples.  5 5  5 4  had  which was e q u i l i b r a t e d t o  C e n t r i f u g a t i o n t i m e was 15  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  min  centrifugation  p a r a m e t e r s were t h o s e recommended by the m a n u f a c t u r e r o f the 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  protein  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 concentrations) section  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  in  2.5.  2.6.7  A n a l y s i s of Binding 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 the r a t i o o f the f r e e t o the 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 . 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 ) constant constant  I n i t i a l estimates (K) and the  o f the  capacity  (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 the 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 . Inc., F u l l e r t o n , C a l i f o r n i a , USA  the 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):  B/F  N1K1  =  1 + KjF  +  NoKo  __-t_t__. 1 + K F 2  (where B and F a r e t h e m o l a r c o n c e n t r a t i o n s o f bound and f r e e drug, respectively.  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 t h e m o l a r 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 determined.  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 (Leatherbarrow, 2.6.8  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  parameters.  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 independent  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 i n Rats 2.7.1  Enantiomers  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  a n 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  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  and  the  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 o r g a n s were c u t i n t o small 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.  Blood  a l s o c o l l e c t e d a t the same t i m e as the t i s s u e s and serum was  obtained  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.  was  The serum and t i s s u e  homogenates were s t o r e d at -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 the 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 the t i s s u e homogenates were d e t e r m i n e d u s i n g the HPLC a s s a y described in section 2.7.3  2.5.  E f f i c i e n c y of Recovery of Mexiletine 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 the 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 the 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 the homogenates ( c o n t a i n i n g 100 mg tissue).  of  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 the 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 the d e r i v a t i z a t i o n step.  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 the 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 extraction. 2.7.4 The  Serum P r o t e i n in  vivo  Binding  f r e e f r a c t i o n s o f the 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 the serum p r o t e i n  binding  section (2.6).  2.7.5 The by  Pharmacokinetic Data A n a l y s i s  serum and tissue concentration time data were best described  a bi-exponential function of the form C = A e * + B e ~ ^ (a > 0) where _ Q  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 d i s t r i b u t i o n and elimination rate constants, respectively.  The i n i t i a l  estimates of the rate constants  were obtained using the computer program, AUTOAN (Sedman and Wagner, 1976).  These i n i t i a l  estimates were used by the i t e r a t i v e non-linear  regression program, NONLIN (Metzler, 1974) to obtain the f i n a l values. The  remaining pharmacokinetic parameters were obtained using formulae  reported in Gibaldi and Perrier (1982). L t  l/2(a)]  w  a  s  The d i s t r i b u t i o n  determined from the formula:  terminal elimination h a l f - l i f e formula: tiy2(/j)  =  0.693//J.  [tiy (/})]  t\/2(a)  w a s  2  =  half-life  0-693/a, while the  calculated from the  The area under the serum concentration-time  curve (AUC) was determined from the trapezoidal rule with extrapolation to i n f i n i t y from the l a s t data point.  The systemic clearance (CL) was  calculated from the formula: CL = dose/AUC. distribution (V ) ss  The steady-state volume of  was determined from the formula: V  s s  = dose.AUMC/AUC  where AUMC is the total area under the f i r s t moment curve (Benet and Galeazzi, 1979).  2.7.6 The  S t a t i s t i c a l Data A n a l y s i s  Student's paired t - t e s t (a=0.05) was used to assess the  s t a t i s t i c a l significance of differences in the serum and tissue concentrations obtained for the enantiomers.  2  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 Enantiomers 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 o u t b l i n d and r a n d o m i z e d . 2.8.1  E l e c t r i c a l l y - I n d u c e d Arrhythmia  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 drug administration.  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 thoracotomy was performed on t h e r a t s between t h e 5 t h and 6 t h 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 Pair o f t e f l o n coated 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 square wave p u l s e s was accomplished using a Grass E l e c t r i c S t i m u l a t o r . 2.8.1.2  Experimental  End-points  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 using a Grass Polygraph.  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 o u t u s i n g  the o s c i l l o s c o p e .  2.8.1.3  Variables Related t o Antiarrhythmic 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 . measured 3 t i m e s and a mean v a l u e o b t a i n e d .  Each v a r i a b l e was  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)  Current Threshold f o r Capture:  was d e t e r m i n e d a t 1 ms and 7.5 Hz.  Current threshold f o r capture  I t i s t h e minimum c u r r e n t r e q u i r e d  to c a p t u r e t h e h e a r t . b)  Threshold Pulse width:  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):  e x t r a - s t i m u l u s method.  ERP was d e t e r m i n e d by t h e  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):  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 d e t e r m i n e d a t 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 drop. e)  V e n t r i c u l a r F i b r i l l a t i o n Threshold (VFT):  VFT was d e t e r m i n e d  at 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  Other 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 parameters 2.8.1.5  (PR, QRS and Q T i n t e r v a l s ) C  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 control.  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.  Blood  (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 drug 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 t h 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 enantiomers. 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  the 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 enantiomers  o f m e x i l e t i n e were  determined  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. F o r t h e r a t s given racemic m e x i l e t i n e , the i n d i v i d u a l enantiomers  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 r a c e m a t e .  The serum  binding o f t h e enantiomers  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  Statistical Analysis  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 g r o u p s 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 change).  The  enantiomers  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  were compared by one-way ANOVA.  2.8.2  Coronary A r t e r y Occlusion i n Conscious  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 the a s s e s s m e n t 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 drugs ( S e l y e e t al.,  1960; C l a r k e t al.,  C u r t i s e t al.,  1984).  1980; Kane e t al.,  1980; J o h n s t o n e t al., 1983;  The p r e s e n t s t u d y was c a r r i e d o u t 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 Curtis  (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 o u t 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 w a t e r .  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% halothane/oxygen  delivered v i a a vapourizer.  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 surgery. 2.8.2.2  Preparation o f Occluder  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 (polyethylene) tubing. exposure t o heat.  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  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 the 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 h o t a i r and then 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 h o t 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 t h e g u i d e . 2.8.2.3  Preparation o f Lines  A 14 cm l e n g t h o f PE-50 t u b i n g was welded onto 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 h o t a i r d e s c r i b e d above and then 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 passed  through 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 the 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 t e r f o r 3 seconds. i c e - c o l d water.  The c o i l was f i x e d by submerging i t i n  2.8.2.4  Implantation of Lines  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 c a v a 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 cannulation with chronic indwelling non-occluding 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 Cicatrin stitches.  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 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 a m e and t h e f o r c e p s were r e l e a s e d . t r e a t m e n t kept t h e l i n e p a t e n t .  This  The a o r t i c l i n e was t r e a t e d i n a  similar fashion. 2.8.2.5  Implantation of the Occluder  A l e f t thoracotomy 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). widen t h e i n c i s i o n and t h e h e a r t exposed.  R e t r a c t o r s were used t o  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 through the t i p o f the g u i d e t u b i n g making a l o o s e snare.  The n e e d l e end o f the s u t u r e was c u t o f f and the  remaining  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 the s u t u r e from b e i n g p u l l e d back through the f l a r e d t i p o f the g u i d e t u b i n g . p r o l e n e s u t u r e extended the f u l l l e n g t h o f the g u i d e t u b i n g .  The  The s i z e  o f the s n a r e was a d j u s t e d such t h a t the f l a r e d end o f the 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 the a t r i a l appendage. s u t u r e was then m e l t e d down t o a s m a l l b a l l .  The d i s t a l end o f the  The pectoral is m u s c l e was  s u t u r e d l i g h t l y t o the 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 the o c c l u d e r .  A t the time the 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 through 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  to the  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 the s h o u l d e r blades.  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 and R  Marcaine  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 wire.  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  each l e a d w i t h the a i d o f a l i g h t e r f l a m e .  1 cm o f one end o f  A t i g h t c o i l was made i n one  end o f one l e a d by w r a p p i n g i t around a 21 gauge hypodermic n e e d l e . T h i s end was then t i e d through the 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 the o c c l u d e r .  Limb l e a d s were p l a c e d  i n t h e 2 f o r e l i m b s and the l e f t h i n d l i m b . approximately  subcutaneously  The ECG measured was  V3.  A f t e r i m p l a n t i n g a l l the l e a d s , l i n e s and o c c l u d e r , the 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 the  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 . 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 . 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  The  The i n c i d e n c e o f 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  recorded.  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 drug 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 smoothly 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 occlusion.  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 in place. 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 paper and weighed.  70  2.8.2.11  Occlusion-Induced Arrhythmia  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 arrhythmias f o l l o w i n g coronary 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  p r e m a t u r e 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 d r o p 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 r u n 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 blood pressure.  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 s e c 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 taps. 2.8.2.12  Evaluation of Antiarrhythmic Efficacy  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 grade the arrhythmias. 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 m o r e t h a n 1 e p i s o d e o f SVT o r S V F a n d / o r g r e a t e r t h a n 499 PVCs. 3 = M o r e t h a n 1 e p i s o d e o f VT a n d / o r V F 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 V F 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 V F 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 m i n - 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 m i n - 14 m i n 59 s e c a f t e r occlusion 8 = F a t a l V F o c c u r r i n g 1 m i n - 3 m i n 59 s e c a f t e r occlusion 9 = F a t a l VF o c c u r r i n g b e f o r e 1 m i n 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 e r e 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) a n d o v e r all  ( 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  Differences in the binomially distributed variables (incidence of a r r h y t h m i a s ) were* a s s e s s e d b y 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.,  1956).  One-way ANOVA was u s e d t o  compare 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 a n d t h e ECG p a r a m e t e r s ) .  T h e 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.,  1983).  Therefore,  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 for statistical tests. ANOVA, s u b s e q u e n t comparison  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  a n a l y s i s u s i n g D u n c a n ' s a n d Newman K e u l ' s m u l t i p l e  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 which t r e a t m e n t  were s i g n i f i c a n t l y d i f f e r e n t . statistical  tests.  groups  T h e v a l u e o f a was 0.05 f o r a l l t h e  2.8.2.14  Exclusion Criteria  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 a n d accuracy o f the experimental results, a s e t o f exclusion c r i t e r i a r e p o r t e d b y 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 . The s e t o f c r i t e r i a were a p p l i e d b l i n d l y and r a t s were 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 based 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)  Pre-occlusion Abnormalities  These 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)  T h e 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 seen 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)  T h e 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 m i n  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 o u t c o m e o f o c c l u s i o n . c) occlusion.  More t h a n 2 5 % w e i g h t l o s s between s u r g e r y a n d c o r o n a r y  R a t s w e r e 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 a s 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  blood  p r e s s u r e o f 8 5 mmHg o r l e s s . d)  Signs o f lung i n f e c t i o n such as exudate around  snout  and/or 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 m i n 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 w e r e e x c l u d e d only i f postmortem examination revealed renal n e c r o s i s .  II)  Post-occlusion Abnormalities  These 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 which experienced r e p e r f u s i o n , and r a t s with misplaced 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 w e r e  e x c l u d e d i f t h e y h a d 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 a n d p u s 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 than 3 min a f t e r o c c l u s i o n ( p o s t u r a l l y induced 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 pressure.  recovery of blood  R a t s w e r e 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  normal.  c) Death a s s o c i a t e d w i t h immediate  calamitous  hypotension.  R a t s w e r e 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 a n d t h e r a t h a d n o t received chest taps.  Ill)  Postmortem A b n o r m a l i t i e s  Rats were e x c l u d e d i f t h e y had: 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 site.  2.8.3  Coronary A r t e r y Occlusion i n Pentobarbitone 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 a s d e s c r i b e d f o r t h e conscious rats with the following modifications: a)  Rats were a c u t e l y p r e p a r e d under 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 . ) a n d a r t i f i c i a l l y  r e s p i r e d throughout the duration o f the  experiment. 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 w e r e 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  recorded.  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 r a n d o m m a n n e r . 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  the determination o f drug  levels.  3. 3.1  RESULTS and DISCUSSION  A s s a y o f M e x i l e t i n e Enantiomers 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  Synthesis o f 2-Anthroyl  Chloride  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  necessary since neither the acid chloride nor the precursor acid could be o b t a i n e d c o m m e r c i a l l y .  2-Anthroyl  c h l o r i d e was s y n t h e s i s e d b y 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.  The f i r s t  step involved the s e l e c t i v e reduction o f anthraquinone-2-carboxylic to anthracene-2-carboxylic This r e a c t i o n proceeded  a c i d u s i n g d i l u t e ammonia a n d z i n c d u s t .  smoothly,  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 b y 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 ( 9 5 % ) a n d was f o u n d t o h a v e a m e l t i n g p o i n t o f 2 7 8 - 2 7 9 ° C .  This  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 which i s 280°C (Weast, 1976). 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 b y d i r e c t p r o b e EI-MS. i o n c u r r e n t c h r o m a t o g r a m a n d t h e mass s p e c t r u m 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 +  ethanol agrees The  The t o t a l  a r e shown i n f i g u r e 3.  M -0H ( m / z = 2 0 5 ) , a n d M - C 0 0 H (m/z=177) mass i o n s +  acid  +  (m/z=222),  present.  The s e c o n d s t e p i n t h e r e a c t i o n s c h e m e ( f i g . 2) i n v o l v e d t h e conversion o f anthracene-2-carboxylic treatment with oxalyl c h l o r i d e .  acid t o 2-anthroyl  c h l o r i d e by  T h e 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 b y p r e p a r a t i v e HPLC a n d t h e s t r u c t u r e c o n f i r m e d b y d i r e c t p r o b e EI-MS. spectrum  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 a n d t h e mass o f 2-anthroyl c h l o r i d e .  i n t h e mass s p e c t r u m  The m o l e c u l a r i o n peaks were p r e s e n t  a t m/z=240 a n d 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  0  Zn/NH.  a  I  C—OH  C-OH  0 anthraquinone-2-carboxylic  acid  anthracene-2-carboxy!ic  COCl I  COCl  anthracene-2-carboxylic  acid  2-anthroyl  chloride  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 anthraquinone-2-carboxyl i c acid.  4.7ft  ti 12000-  '1  10900-  3906-  5388-  J 3.2^1  4000-  2080-  1.9  2.0  • ! ' 3.0  1  . ' ' '  4.0  1  I  1  5.0  ' '  1  . ' • • •1'  6.0  1  7.0  8.8  ]  9.3  222 32002880-  24002900177 1600120088  300-  149 129  400-  80  120  i • i  16ft  >00  i —i —;——i—•—r'—r —i— 240 230 320 360 1  -1  1  -  -1  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 ) a n d 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 . T h e f o l l o w i n g EI-MS c o n d i t i o n s were employed: source temperature, 240°C; probe t e m p e r a t u r e programme, 5 0 ° C f o r 1 m i n t o 3 0 0 ° C f o r 10 m i n a t t h e r a t e o f 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 a n d m u l t i p l i e r v o l t a g e , 2500 V . b  128888-  3.S9  leeeee98089888887860868900S90004 9006308892080010000-  9-1  10  1S000-  12  16  14  18  20S  177  1 "5000148801200010000SO30249  6000"  151 /  4806-  -I  2900-  i  i  2  &  /  i 296  j  Mil 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. 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 ) a n d 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 . T h e 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 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 m i n t o 3 0 0 ° C f o r 10 m i n a t t h e r a t e o f 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 e v ; e m i s s i o n c u r r e n t , 300 uA a n d m u l t i p l i e r v o l t a g e , 2500 V. D  of c h l o r i n e (3:1).  O t h e r i m p o r t a n t fragment ions o b s e r v e d were M  -CI  (m/z=205) and M+-C0C1 ( m / z = 1 7 7 ) .  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 of Mexiletine Enantiomers with 2-Anthroyl 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 enantiomers using the P i r k l e ^ i o n i c (phenyl These properties  mexiletine  glycine) chiral  phase.  include a functional group that w i l l react with  p r i m a r y amino group o f m e x i l e t i n e ,  a strong fluorophore  the  for sensitive  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 in the c h i r a l r e c o g n i t i o n process previous  s t u d y , 2 - n a p h t h o y l c h l o r i d e was  c h a r a c t e r i s t i c s and was mexiletine  on t h e P i r k l e ^ c h i r a l p h a s e .  found to have t h e s e n e c e s s a r y  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  enantiomers using the P i r k l e ^ i o n i c (phenyl  c o l u m n ( I g w e m e z i e , 1986; c h i r a l recognition process  M c E r l a n e et  In a  al.,  1987).  quantitate  glycine)  Examination of  required f o r the r e s o l u t i o n of  of 2-anthroyl  while  enantiomers with 2-anthroyl  the Schotten-Baumann r e a c t i o n (Vogel,  use  fluorescence  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  cyclohexane at 25°C) ( F r o e h l i c h , 1985). mexiletine  that  i m p r o v i n g s e n s i t i v i t y by t h e  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  q u a n t u m y i e l d o f n a p h t h a l e n e i s 0.19  the  the  2-naphthoyl d e r i v a t i v e s of the enantiomers lead to the proposal 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 ,  chiral  in  The d e r i v a t i z a t i o n o f c h l o r i d e was  1964)  accomplished  as shown i n f i g u r e 5.  amide d e r i v a t i v e s o f the e n a n t i o m e r s were i n s o l u b l e i n the  aqueous  using The  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 mobile phase. 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 h e t o t a l i o n c u r r e n t chromatogram peaks.  ( f i g . 6A) showed t h e p r e s e n c e o f two  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 a n d 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 . 6B).  H o w e v e r , 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 , 3.1.2.3  1985).  Resolution of Mexiletine Enantiomers  Following d e r i v a t i z a t i o n with 2-anthroyl chloride, the enantiomers o f m e x i l e t i n e w e r e 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 phase (see f i g . 9 ) .  The o p t i m u m m o b i l e p h a s e was  ethyl  a c e t a t e / 2 - p r o p a n o l / h e x a n e (4:6:90) 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 .  The 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 were  16.68  a n d 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 h e 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 time o f the i n d i v i d u a l enantiomer d e r i v a t i v e s with those o f the racemate 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 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  between  3.28 5.21  SS88-  19  2(2 177 28S  32K»«ee8 2«8?>  358*-  85  2*l>t«-  3(>8f<\*7  2f.f.<« 1281^ I > I  H'l 98  ill llll  ISI  129  in,  •83 2»3 - p - — I — T ry, 294  ?l  X  /  121  . 28a  328  e-UU  383  292  V  s  F*0  233^ 240  83  128  II7~  -r 248 1  26*  288  328  3A  ' l • «ee  I "  I  F i g u r e 6. 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 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 . T h e 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 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 a n d m u l t i p l i e r v o l t a g e , 2500 V 00 ro  m/z  m/z  121  383  (M ) +  m/z  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 mexiletine.  177  derivative of  <u  coco  <u  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 enantiomers. Chromatographic 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 i o n i c (phenyl g l y c i n e ) c h i r a l phase; mobile phase, 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 ) . R  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 sensitivity 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 t a b i l i t i e s are different, resulting in a difference in the migration rate of the enantiomers through the CSP. Figure 9 shows the structure of the Pirkle  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. T h e s t r u c t u r e o f t h e P i r k 1 e 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 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 . 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 . K  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. to the presence o f the b u l k y phenyl group below the p l a n e o f the 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 .  Due CSP,  The  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 complex 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 the methyl group l i e below the p l a n e o f the 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 -  anthramide, w i t h the b u l k i e r x y l y l o x y m e t h y l group below the p l a n e o f the 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 . 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.  The r e s u l t i s a This stereochemical  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  (1984).  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 amines  ( 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 1987) on t h e P i r k l e  3.1.2.6  R  al.,  CSP.  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 w e r e e x t r a c t e d f r o m an a q u e o u s  solution with diethyl ether prior to 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  of extracts of 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 water.  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(+)-enantiomer.  0  5  Time  10  15  20  (Min)  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 (60 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 were: s t a t i o n a r y phase, 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 phase m o b i l e phase, 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 (4:6:90); 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 ) . R  3.1.2.7  a)  Attempted Resolution/Removal of the I n t e r f e r i n g Peak  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 :  Extensive variations of  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 change the r e t e n t i o n time o f the i n t e r f e r i n g r e l a t i v e to that of the enantiomers.  compound  The e x t e n t t o w h i c h p o l a r i t y  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  could  ionic chiral  column can o n l y t o l e r a t e the e q u i v a l e n t o f 20% 2 - p r o p a n o l / h e x a n e w i t h respect to b)  polarity. Extraction  Solvent:  A comparison  of the chromatograms  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 and t h e i n t e r n a l  standard  (100 n g / m l )  (100 n g / m l ) 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 . 11).  2-anthroyl suspected.  Thus, the p o s s i b i l i t y o f a r e a c t i o n between the  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 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  investigated.  was  were  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  present  with each 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 R e a g e n t s :  To d e t e r m i n e  i f the  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 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. the i n t e r f e r i n g d)  These treatments  carried  d i d not e l i m i n a t e  compound.  Pirkle  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 peak from those o f m e x i l e t i n e enantiomers, 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)-leucine phase.  the s t a t i o n a r y However, the  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—  10  1  15  1  T  20  10 Time  r  15  -T-r  20  5  10  (Min)  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 water with d i f f e r e n t extraction solvents: d i e t h y l ether (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 time of the i n t e r f e r i n g substance.  15  20.  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 w e r e 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 phase. S i n c e a l l attempts t o s o l v e t h e i n t e r f e r i n g peak problem were successful, a p r e v i o u s l y developed stereoselective assay 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  pharmacodynamic  studies.  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  of mexiletine  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 n g / m l ) 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-naphthamide  derivatives.  T h e r e was no e v i d e n c e o f any  interfering  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 curve data f o r the enantiomers i n plasma.  A coefficient of  d e t e r m i n a t i o n ( r ) 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 2  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 curves o f the enantiomers i n r a t plasma ( 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 following l i n e a r least squares r e g r e s s i o n equations: (r =0.999) 2  Y = 0.024X + 0.027  f o r R ( - ) - m e x i l e t i n e and Y = 0.022X + 0.022 ( r = 0 . 9 9 9 )  S(+)-mexiletine.  2  for  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 the  enantiomers in the various r a t t i s s u e  homogenates.  A  X CO  ~o  CO  U7  •  X CD  -+•  oo  al oo  —-  1  in r~  B  o  m-T —o  •  "  TO  "  oo <s CD  -  oooo my? mm  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 n g / m l ) 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 chromatographic 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 i o n i c (phenyl g l y c i n e ) c h i r a l phase; mobile phase, chloroform/2-propanol/hexane ( 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 ) . R  T a b l e 1.  C a l i b r a t i o n curve data f o r m e x i l e t i n e enantiomers  i n human  plasma  Wt o f e a c h enantiomer (ng)  Peak-ht r a t i o ^ R(-)-mex/I.S.  C-V** (%)  Peak-ht r a t i o ^ S(+)-mex/I.S.  CV (%)  500  12.50  ±  0.04  1  12.10  ± 0.06  1  200  4.98  ±  0.14  3  4.82  ± 0.18  4  50  1.24  ±  0.02  2  1.18  ± 0.02  2  20  0.50  ±  0.01  1  0.48  ± 0.01  2  10  0.26  ±  0.02  8  0.24  ± 0.02  9  slope intercept r  2  0.025  0.024  -0.003  -0.014  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 . u s e d was 50 ng.  The amount o f i n t e r n a l s t a n d a r d  (I.S.)  3.3  In Vitro  3.3.1  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  Enantiomers  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 a s w e l l a s commercially (HSA)  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  and l i p o p r o t e i n d e f i c i e n t serum.  r e s u l t s were meaningful,  methodological  serum p r o t e i n b i n d i n g were e v a l u a t e d .  To ensure t h a t t h e b i n d i n g f a c t o r s w h i c h may i n f l u e n c e These f a c t o r s i n c l u d e d non-  specific adsorption to the u l t r a f i l t r a t i o n and c o m p e t i t i v e b i n d i n g . displacement  albumin  system,  pH-dependent binding  The i n f l u e n c e o f o t h e r f a c t o r s such as b i n d i n g  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 a n d  t h e a g e o f s e r u m w e r e 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 a n d u s i n g t h e serum 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  Non-specific Binding  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 during u l t r a f i l t r a t i o n . enantiomers  enantiomers  T h e 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  indicating that non-specific binding to the 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  for the small adsorptive 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 a n d 8 . 0 ) . A s 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  decreased  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 ultrafiltration Concentration of e a c h e n a n t i o m e r (/zg/ml)  R(-)-mex  S(+)-mex  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 Percent recovery concentration of b e f o r e and a f t e r  ± 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 . 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 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) ultrafiltration.  — • — R(-)-mex  - O-  S(+)-mex  80 i  20 10 6.50  7.00  7.50  8.00  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.  8.50  98 s i g n i f i c a n t l y ( p < 0 . 0 5 ) 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 a n d was g r e a t e r f o r t h e R(-)-enantiomer.  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 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 change i n the 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 changes i n the i o n i z a t i o n o f the b i n d i n g p r o t e i n and/or the i o n i z a t i o n o f t h e d r u g (McNamara et al.,  1981).  A similar decrease in  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 et  al.  (1981).  al.  ( 1 9 7 8 ) a n d McNamara  Both i n v e s t i g a t o r s e x p l a i n e d the changes 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 markedly change t h e r a t i o o f the i o n i z e d t o the u n i o n i z e d forms s i n c e the pK  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)  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 84% i o n i z e d ) .  ( a t a pH o f 8.0,  Furthermore, 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 r e f o r e , s u g g e s t t h a t the pH-dependent  The p r e s e n t r e s u l t s ,  changes 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 enantiomers i s most l i k e l y a r e f l e c t i o n o f pH-dependent  changes i n the 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  indicate t h a t to obtain meaningful binding data f o r e i t h e r racemic 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 m u s t be a d j u s t e d a n d m a i n t a i n e d a t =7.4  during the binding experiment.  S e r u m pH as h i g h a s 8.5  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  adjustment.  was  3.3.1.3  Competitive Binding (Enantiomer-Enantiomer Interaction)  Lima (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 between  the  enantiomers o f disopyramide r e s u l t i n g i n a g r e a t e r than 2 - f o l d i n c r e a s e in the f r e e f r a c t i o n o f each enantiomer.  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 w e r e c a r r i e d out with the i n d i v i d u a l enantiomers or the racemate.  There were  no s i g n i f i c a n t d i f f e r e n c e s 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 were compared  by i n d e p e n d e n t g r o u p s  t-test).  This suggests the absence o f enantiomer-enantiomer i n t e r a c t i o n ,  which  could 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 the t h e r a p e u t i c range o f mexiletine.  3.3.2  Methods o f Serum pH A d j u s t m e n t  The pH-dependent  b i n d i n g d e s c r i b e d above ( 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 experiments.  T a b l e 4 shows t h e m e t h o d s 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 e t h o d s 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 not a f f e c t the f r e e f r a c t i o n values o b t a i n e d .  B u f f e r s a l t s were  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 m o r e 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 . S e r u m 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 i n t a b l e 4.  5%  CO2/O2  as shown  100  T a b l e 3. T h e 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 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 t h e i n d i v i d u a l enantiomer Concentration of each enantiomer (Mg/ml)  R(-)-mex  1.00  53 ± 1  0.25  49 ± 2  ^  S(+)-mex  *  R(-)-mex  S(+)-mex  54 ± 2  59 ± 4  48 ± 3  50+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) corresponding percent free fractions obtained. Method  5% 100%  C0 /0 2  co  2  2  phosphate* buffer salts  and t h e  pH b e f o r e adjustment  pH a f t e r adjustment  pH a f t e r filtration  Free f r a c t i o n R(-)-mex S(+)-mex  8.05  7.47  7.65  ±  0.01  44  ±  1  52  +  2  8.05  7.35  7.41  ±  0.02  52  +  1  58  +  1  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 = 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 determinations. 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 s e r u m (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 Enantiomers t o V a r i o u s Serum P r o t e i n s  t o Serum and  T a b l e 5 summarizes the data o b t a i n e d f o r the 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 enantiomers  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  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 , AAG enantiomers  and HSA.  u s i n g serum,  The 2 c o n c e n t r a t i o n s o f t h e  e v a l u a t e d were 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 .  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  to  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%.  These r e s u l t s are in disagreement  with those in the l i t e r a t u r e that  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%) et  al.,  1987).  The d i s a g r e e m e n t  ( T a l b o t et  al.,  1973;  McErlane  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  binding of mexiletine  enantiomers  vitro  t o human s e 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,  the binding of the enantiomers stereoselective.  t o HSA was a p p r o x i m a t e l y  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  3 0 % and was  not  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(-)-mexiletine.  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)  greater binding a f f i n i t y constant of R(-)-mexiletine r e l a t i v e to i t s enantiomer  (3.3.4.2).  With l i p o p r o t e i n d e f i c i e n t serum, b i n d i n g  s l i g h t l y lower i n comparison  was  t o t h a t o b t a i n e d w i t h serum, s u g g e s t i n g  a  p o s s i b l e small c o n t r i b u t i o n of the l i p o p r o t e i n s to the binding of mexiletine enantiomers.  T h i s b i n d i n g was f o u n d t o be  (p<0.05) g r e a t e r f o r S ( + ) - m e x i l e t i n e . binding of the enantiomers  significantly  The d a t a i n t a b l e 5 show t h a t t h 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 c a n be  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.  accounted  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 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 Concentration of each enantiomer (jug/ml)  Serum  HSA  AAG  1.0  R(-)-mex  52±1  73±2  77±1  63±0  S(+)-mex  53+2  72+2  84±0*  56±1*  R(-)-mex  52±1  70±1  74±1  58±1  S(+)-mex  51±1  68±1  84+2*  50+0*  0.5  LP def. serum  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 a n d LP d e f . s e r u m r e f e r t o human s e r 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 serum, 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 enantiomers used were t h o s e w i t h i n t h e therapeutic range. * S i g n i f i c a n t (p<0.05).  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  (Routledge,  K r e m e r et  1985;  al.,  1988), the  present r e s u l t s suggest that the binding of the enantiomers in disease states.  H o w e v e r , P e n t i k a i n e n et  al.  may  (1984) found  change  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 the f r e e f r a c t i o n s o f racemic m e x i l e t i n e i n myocardial  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  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  observed  phases.  in t h i s study,  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 ± r e s p e c t i v e l y ) ( I g w e m e z i e et  L/kg f o r R ( - ) - and S ( + ) - m e x i l e t i n e ,  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  3.3.4  Determination  3.3.4.1 To d e t e r m i n e enantiomers  Serum Binding  of Binding  factor  enantiomers.  Constants  Data  the constants c h a r a c t e r i z i n g the binding of the  t o serum p r o t e i n s , the b i n d i n g o f a wide range o f t o t a l  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 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. that the binding of m e x i l e t i n e enantiomers  m e x i l e t i n e i n man  drug  investigated.  The r e s u l t s i n d i c a t e  was n o t  concentration-  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  o f 0.25,  al.,  n o t be an i m p o r t a n t  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  2.4  (the f r e e f r a c t i o n s at t o t a l enantiomer  racemic  concentrations  0.5 and 1.0 /Kj/ml w e r e c o m p a r e d by one-way ANOVA).  However,  s i g n i f i c a n t increases in the f r e e f r a c t i o n s of the enantiomers  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  "Rosenthal" p l o t o f the b i n d i n g data o b t a i n e d f o r each enantiomer  in a  Table 6. The percent free fractions of mexiletine enantiomers in serum from healthy human subjects Concentration of enantiomer in serum (M9/ml)  Free Fraction R(-)-mex  S(+)-mex  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 R(—)-mex • S(+) —mex  O  1.0--  0.5  o  T  o.o T -100  1 0  1 100  o 1 200  1 300  1 400  1  500  Bound ( x10~~ M) 5  Figure 15. A r e p r e s e n t a t i v e "Rosenthal" p l o t of the binding m e x i l e t i n e enantiomers to serum from a h e a l t h y s u b j e c t .  of  healthy subject.  Analysis of the data using the non-linear least  squares r e g r e s s i o n program "ENZFITTER" (Leatherbarrow, 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 .  1987) i n d i c a t e d  A high a f f i n i t y , low  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 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 of binding sites.  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.  There were no  s i g n i f i c a n t differences i n the binding constants of the 3.3.4.2  enantiomers.  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 w i t h AAG.  obtained  T h e 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 o n e 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  affinity  b i n d i n g o f serum ( 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 w e r e 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 w e r e 1.9 x 1 0 " M a n d 5  1.8 x 1 0 " M f o r R ( - ) - a n d S ( + ) - m e x i l e t i n e ,  respectively).  5  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 w e r e 2.0 x 1 0 M 4  _ 1  v s 1.2 x 1 0 M ) . T h e 4  _ 1  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 both enantiomers.  1 for  This i s consistent with the reports o f other studies  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.,  1988).  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.,  1985).  Table 7. Binding constants o f m e x i l e t i n e  enantiomers in serum from healthy subjects S(+) -mex  R(-) -mex Subject  mi p  n P  njPj  n P  K  (xlO" M)  (xloV )  (xlO" M)  (xioV )  (xl0" M)  Kl (xl0 M )  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 is.e.m  2.4 0.4  4.5 1.3  5.3 0.3  1.2 0.2  3.2 1.0  4.1 1.7  6.5 0.7  1.0 0.1  K  6  l  2  1  2 3  1  6  5  _1  2  2  (xlO" M) 3  2  (xloV )  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".  1  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 human a j - a c i d g l y c o p r o t e i n (AAG) nP  n  (xlO" M)  in isolated  K (xl0 M )  5  4  _ 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 4 4 , 0 0 0 D a l t o n s . * S i g n i f i c a n t (p<0.05).  110  A l b u m i n B i n d i n g Data  3.3.4.3  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  albumin  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 ) . T h e l o w a f f i n i t y s i t e s had comparable b i n d i n g c o n s t a n t s t o those o f t h e corresponding  s i t e s i n serum f o r each o f t h e  enantiomers.  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  than  S(+)-mexiletine  However, t h e  (mean v a l u e s w e r e 4.0 a n d 3.2 x 1 0 " M). 3  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 antipode  (mean v a l u e s w e r e 0.5 and 1.2 x 1 0 M " * ) . 2  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 w e r e 6.6 and 5.4 f o r R ( - ) - and S(+)-mexiletine,  r e s p e c t i v e l y . The high 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 serum.  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 b i n d i n g  constants o f the enantiomers.  T h e 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 w e r e 0.006 and 0.001 f o r R ( - ) - a n d S(+)-mexiletine,  r e s p e c t i v e l y . The magnitude o f t h e s e n v a l u e s  suggest  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 albumin.  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  3.4  i n t h e HSA.  Tissue Distribution Kinetics of Mexiletine i n Rats  Enantiomers  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  studied i n male Sprague-Dawley 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 ( 1 0 mg/kg) o f r a c e m i c  mexiletine.  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 l l (xlO" M) n  p  "1  5  l (xl0 M ) K  5  _ 1  n P 2  2  n  2  K  2  (xloV )  (xlO~ M)  1  3  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 a s 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 decay  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 h a d 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 each sampling time than t h e R(-)-enantiomer but t h i s did not achieve 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 drug administration. between  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 remained c o n s t a n t  1 and 6 h.  3.4.2  Pharmacokinetic Parameters  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 enantiomers.  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 calculated.  T h e 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 (161.8 ml/min/kg 122.9 m l / m i n / k g ) .  vs  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 cleared drug (Wilkinson  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^ ^.) a n d f r e e f r a c t i o n ( f f ) . n  S i n c e t h e in vivo f f o f t h e  enantiomers were n o t markedly d i f f e r e n t from each o t h e r ( s e c t i o n these r e s u l t s suggest that the C L ^ than t h a t o f the S(+)-enantiomer.  n t  3.4.3),  o f R ( - ) - m e x i l e t i n e may be g r e a t e r  A significantly greater  glucuronidation o f R(-)-mexiletine, r e l a t i v e t o i t s opposite enantiomer, 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 h a 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 volume 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  0  1  2  1  1  1—  1  3  4  5  6  ;  Time ( h ) F i g u r e 16. T h e 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 mexiletine enantiomers in rats following the administration o f a single i . v . d o s e ( 1 0 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 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 . . d o s e ( 1 0 mg/kg) o f r a c e m i c m e x i l e t i n e Parameter  R(-)-mex (min)  (h)  S(+)-mex  19.8  24.4  1.4  1.3  AUC  (ng.h.ml"*)  515.1  677.9  CL  (ml/min/kg)  161.8  122.9  9.0  7.4  V  ss  (Vkg)  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 i s t r i b u t i o n . The d i s t r i b u t i o n h a l f - l i f e of S(+)-mexiletine, 24.4 min, was 24% greater than that of the R(-)-enantiomer, d i s t r i b u t i o n of R(-)-mexiletine tissue uptake.  19.8 min.  appears to result from i t s  have been reported.  1976)  and timolol  1976)  and S(+)-mexiletine,  These values were markedly shorter than those reported in  ± 2.9 and 11.0 ± 3.8 h for R(-)-  respectively)  (Tocco et al.,  The terminal elimination h a l f - l i v e s of the  enantiomers were 1.4 and 1.3 h for R(-)-  humans (9.1  greater  Stereoselective tissue uptake of other c h i r a l drugs such  as propranolol (Kawashima et al.,  respectively.  Thus, the greater volume of  (Igwemezie et al.,  elimination h a l f - l i f e  1989).  and S(+)-mexiletine,  The expected shorter terminal  for R(-)-mexiletine,  due to i t s larger systemic  clearance, was offset by i t s equally greater volume of d i s t r i b u t i o n . This resulted in an apparent lack of a difference in the terminal elimination h a l f - l i v e s of both enantiomers.  3.4.3  Serum Protein Binding  Table 11 shows the in vivo percent free fractions of mexiletine enantiomers in the r a t s .  The mean free fraction of  R(-)-mexiletine,  49%, was s i g n i f i c a n t l y 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  Concentrations are means ± s.e.m, n = 4-5 rats. * significant (p<0.05).  * 44 ± 1  117  3.4.4  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 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 homogenates.  tissue  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 m o r e i n a l l t h e t i s s u e s  studied.  3.4.5  Tissue Levels  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 various tissues studied.  i nthe  Maximum c o n c e n t r a t i o n s w e r e 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 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 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 e r e 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 than t h a t o f t h e R(-)-enantiomer  ( 3 . 1 ± 1.1 ng/g). H o w e v e r , a  s i g n i f i c a n t l y ( p < 0 . 0 5 ) 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 hour 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 the changes  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 serum  ( s e c t i o n 3.4.1).  These  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 enantiomers  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 (which 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 , presumably  i n t o t h e deep t i s s u e compartment.  The d i s t r i b u t i o n i n t o t h e  deep t i s s u e compartment 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(-)-enantiomer which 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 lower concentrations observed f o r t h i s  serum  enantiomer.  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 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 summarized  from  i n t a b l e 14. T h e  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 from each o t h e r .  The area 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  Table 12. Percent recovery of mexiletine enantiomers from tissue homogenates. Enantiomer concentration (ng/ml)  liver  kidney  heart  fat  lung  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.  brain  Table 13.  The time-dependent concentrations (^g/g) of mexiletine a s i n g l e i . v . dose (10 mg/kg) of racemic mexiletine  Time-  Brain  Lung  Kidney  Liver  Fat  S(+) -mex  18.99 ± 0.76 19.18 ± 0.82  6.23 ± 0.46 6.52 + 0.46  24.57 ± 0.64 25.86 + 0.79  19. 99 + 0.64 20. 12 ± 0.66  3.10 7.47  15 min  R(-) -mex S(+) -mex  12.44 + 0.06 13.33 t 0.06  3.30 ± 0.15 3.38 ± 0.15  14.05 ± 0.03 16.21 + 0.03  8 92 + 0.64 10. 66 ± 0.76  2.06 + 0.30* 4.12 ± 0.50  1.04 t 0.04 1.16 i 0.04  30 min  R(-) -mex S(+) -mex  6.67 ± 0.61 7.00 ± 0.57  2.58 2.71  13.70 ± 0.17 14.75 ± 0.18  6.78 i 1.19 7.58 ± 1.30  0.79 ± 0.02* 1.63 + 0.01  0.78 i 0.14 0.86 ± 0.14  R(-) -mex S( + ) -mex  3.3 4.2  0.18* 0.32  1.11 ± 0.23* 1.43 + 0.24  6.48 ± 1.09* 8.59 ± 1.00  2. 53 i 0.44* 3. 94 ± 0.58  0.65 ± 0.09* 1.31 i 0.28  0.42 ± 0.04* 0.54 ± 0.06  2 hr  R(-) -mex S( + ) -mex  0.84 ± 0.09* 1.30 ± 0.14  0.41 + 0.10* 0.55 ± 0.12  2.68 ± 0.66* 3.88 ± 1.04  0.55 ± 0.06* 1. 11 + 0.15  0.23 ± 0.02* 0.38 ± 0.06  0.18 t 0.02* 0.23 + 0.02  4 hr  S( + )  R(-) -mex -mex  0.18 ± 0.02* 0.23 ± 0.03  0.10 ± 0.12* 0.13 ± 0.01  0.47 ± 0.08* 0.70 ± 0.11  0.12 i 0.02* 0.23 + 0.02  0.03 + 0.01* 0.07 ± 0.01  0.02 ± 0.00* 0.03 + 0.00  6 hr  R(-) -mex S( + ) -mex  0.05 ± 0.01* 0.07 ± 0.01  0.03 0.04  0.01* 0.01  0.14 ± 0.02* 0.22 ± 0.02  0.04 ± 0.01* 0.07 + 0.01  0.02 + 0.01* 0.04 ± 0.00  5 min  1 hr  * **  R(-) -mex  Heart  enantiomers i n the tissues of rats following  ± ±  s i g n i f i c a n t (P< 0.05) mean + s.e.m., n = 4-5 rats  + +  + +  0.46 0.48  + +  0.53* 1. 28  1.03 1.10  + +  0.04 0.05  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 ( 1 0 mg/kg) o f racemic mexiletine AUC (ug.h.g ) T  Ti ssue/Serum  - 1  Heart  R(-) -mex S(+) -mex  1.1 1.1  4.4 5.1  8 7  Brain  R(-) -mex S(+) -mex  1.0 1.0  11.9 14.0  25 21  Liver  R(-) -mex S(+) -mex  1.1 1.2  Lung  R(-) -mex S(+) -mex  1.0 1.0  22.4 27.1  32 28  Kidney  R(-) -mex S(+) -mex  1.1 1.0  11.1 13.6  26 22  Fat  R(-) -mex S(+) -mex  0.7 0.7  -  -°* 4.4 2  * 8 4  1.3 1.5  * 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 drug a d m i n i s t r a t i o n . AUCj r e f e r s t o area 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 .  1 1  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 e r e t h e AUC-r was S(+)-enantiomer.  120% g r e a t e r f o r t h e  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  (at the  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 uptake.  The h i g h e s t r a t i o s w e r e 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  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 respectively. accumulated  The b r a i n a c c u m u l a t e d  32  S(+)-mexiletine,  25 and 2 1 - f o l d ; w h i l e t h e h e a r t  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 high t i s s u e / s e r u m r a t i o s have b e e n 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  a7.,  1983).  The 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 % greater accumulation of R(-)-mexiletine r e l a t i v e to the 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  S(+)-enantiomer. of  S ( + ) - m e x i l e t i n e w h i l e the f a t t i s s u e d i d not accummulate the enantiomers.  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  can  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 process and/or high a f f i n i t y b i n d i n g to s i t e s i n the t i s s u e s . The 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 the R(-)-enantiomer  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  and/or t i s s u e b i n d i n g and/or metabolism.  However, from 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 m e c h a n i s m ( s ) was  in operation.  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 at a r a t e g r e a t e r f o r the R(-)-enantiomer, changing r a t i o of the enantiomers  which  i n the serum.  r e f l e c t i n g the  Thus, 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. T h e 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 . d o s e ( 1 0 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 enantiomers in the tissues.  H o w e v e r , much o f t h e e n a n t i o m e r s w e r e  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 , hence o n l y small d i f f e r e n c e s i n the AUCj o f the enantiomers was e v i d e n t i n m o s t 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 not  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 m o s t 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  3.5  ( P a l i l e o et  al.,  1982, Waspe et  al.,  1983).  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 a n d 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 Arrhythmia 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 in pentobarbitone anaesthetized rats.  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  (control)  w e r e 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 e r e 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 w e r e 4, 8, 16 and 3 2 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 .  The o b s e r v e d plasma  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 l i n e a r l y ( r > 0 . 9 9 ) w i t h d o s e as shown i n f i g u r e 18. 2  increased  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 t h a n t h e R(-) e n a n t i o m e r .  concentrations  The maximum c o n c e n t r a t i o n s a c h i e v e d w e r e  2.3 ± 0.2, 2.3 ± 0.2 and 1.7 ± 0.1 pg/ml f o r S ( + ) - , R,S-,  and  R(-)-mexiletine, r e s p e c t i v e l y . Normally, the concentration of the 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,SS(+)-mexiletine enantiomers.  and  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  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  metabolism l e v e l .  and/or  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. s i g n i f i c a n t d i f f e r e n c e s in the free f r a c t i o n values.  There were no Thus, i t i s  u n l i k e l y t h a t serum b i n d i n g i n t e r a c t i o n s would have been r e s p o n s i b l e f o r the observed plasma c o n c e n t r a t i o n s .  These r e s u l t s suggest t h a t there  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 the t i s s u e binding and/or metabolism l e v e l which r e s u l t e d i n g r e a t e r than expected plasma c o n c e n t r a t i o n s o f the racemate.  Such 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 e r e 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 dextromethorphan  with i t s opposite  ( C o o p e r and A n d e r s , 1 9 7 4 ) .  enantiomer,  H o w e v e r , 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  0  1 8  1 16  Dose ( m g / k g )  1 24  F i g u r e 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 ) 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 enantiomers 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 . Blood samples were 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  r  -  32  and t h e plasma in c o l l e c t e d 10 m i n f r o m 7-8 r a t s .  Table 15. The in vivo percent free fractions of racemic mexiletine and i t s 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 than expected  plasma concentrations o f racemic  mexiletine.  3.5.1.2  Antiarrhythmic Effects  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 have used VFT as a q u a n t i t a t i v e measure  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). underlying assumption i s that p h y s i o l o g i c o r pharmacologic  The  interventions  t h a t a l t e r VFT w i l l produce a s i m i l a r d i r e c t i o n a l change i n t h e v u l n e r a b i l i t y t o spontaneous f i b r i l l a t i o n (Moore and Spear, 1975). has b e e n s u g g e s t e d  It  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.,  1983).  The p r o p e r t i e s o f a  pharmacological  agent r e q u i r e d t o break o r prevent a re-entrant c i r c u i t include a slowing o f conduction,  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., p r o p e r t i e s a r e t h e same a s t h o s e p o s s e s s e d agents  1983).  These  by c l a s s I a n t i a r r h y t h m i c  ( s e c t i o n 1.1.2.4) a n d 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 a n d W e g r i a , 1 9 4 0 ; Yoon e t al.,  1974; G e r s t e n b l i t h e t al.,  determination  1972; H o d e s s e t al.,  1979).  o f V F T 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 e t h o d  f o r a comparative m e x i l e t i n e and i t s  study o f the antiarrhythmic a c t i v i t y o f racemic enantiomers..  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 V F T v a l u e s o b s e r v e d drug treatment  The  a r e l i s t e d i n t a b l e 16.  These pre-drug  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 groups.  before  each  values were not  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 OA)  MFF (Hz)  ERP (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  46+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  d e p e n d e n t s i g n i f i c a n t ( p < 0 . 0 5 ) i n c r e a s e i n VFT. d i f f e r e n c e s between the drug treatments significance.  a dose-  However, the  d i d not reach  statistical  I t was n o t p o s s i b l e t o c o m p a r e 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 have not been 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 , has 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., ( A l l e n e t a/., 3.5.1.2.2  1981) and d o g s  1977). Cardiac Refractory 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 determinant  VFT  of the i n i t i a t i o n of arrhythmias  important  ( s e c t i o n 1.1.2.2).  Most  methods used 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 measure the 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) MRD,  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  t h e MP a t w h i c h t h e i n w a r d  sodium c u r r e n t i s r e a c t i v a t e d , the  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 process decrease  i n MRD  ( W i n s l o w , 1984).  Singh  (1978) r e p o r t e d t h a t t h e  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  w i t h an i n c r e a s e i n t h e ERP.  associated  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). I n 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 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  at which c a r d i a c muscle  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 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.  and  MFF  (MFF).  This  has b e e n u s e d  to  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 a n d 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 . T h e 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 values.  q u a n t i t a t i v e l y study the a n t i a r r h y t h m i c a c t i o n s o f other c l a s s I agents (Vaughan W i l l i a m s and S z e k e r e s ,  1961).  T h e p r e - d r u g v a l u e s f o r ERP a n d MFF a r e shown i n t a b l e 16. 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 b e t w e e n t h e d r u g t r e a t m e n t  There  groups.  U s i n g t h e e x t r a - s t i m u l u s method, 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 e r e 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) a n d 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 between t h e drug t r e a t m e n t s d i d not reach 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.,  1979).  A s e x p e c t e d , MFF was  significantly  (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) a n d t h i s was a l s o d o s e dependent.  H o w e v e r , R,S- and S ( + ) - M e x i l e t i n e w e r e 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- a n d S ( + ) - m e x i l e t i n e d i d not d i f f e r s i g n i f i c a n t l y from each o t h e r .  The  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, w h e r e 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 has  b e e n 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 m e t h o d 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 (Winslow,  1984).  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 a n d S z e k e r e s , 1961; W i n s l o w , 1 9 8 4 ) .  Furthermore, 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 a n d i t s e n a n t i o m e r s on V F T 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 study.  T h e r e f o r e , t h e c o n c l u s i o n drawn from 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 a n d 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  50  1  -10-1 1  10  1 100  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 (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 . 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 h e 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 values.  133  10  ,  -50-I 1  :  10  1 100  Dose ( m g / k g )  F i g u r e 21. Cumulative dose-response curves o f t h e e f f e c t s o f racemic 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 ( M F F ) 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 h e 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 values.  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 enantiomers  a r e shown i n t a b l e 17. The QRS and Q T i n t e r v a l s were n o t C  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 b u t 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 .  There were no s i g n i f i c a n t  d i f f e r e n c e s 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 . T h e 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 conduction.  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 n o t  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 n o r 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 ) . between t h e drug t r e a t m e n t g r o u p s .  There were no s i g n i f i c a n t d i f f e r e n c e s Racemic m e x i l e t i n e and t h e  enantiomers  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  hypotension  ( 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 n o t  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). T h e h y 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 t s enantiomers on the ECG parameters in pentobarbitone anaesthetized rats PR  QRS  (msec)  R(-)mex  R,S-mex  S(+)mex  QTQ  (msec)  (msec)  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  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  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 h a e m o d y n a m i c p a r a m e t e r s i n pentobarbitone anaesthetized rats HR (beats/min)  BP (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  10  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 a n d 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 . T h e 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 not 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) w h e r e 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 b e 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 of the conscious 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 interval.  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 w e r e 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 artery occlusion-induced arrhythmia in conscious rats.  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.,  1982).  ( K u p p e r s m i t h e t al.,  S i m i l a r r e s u l t s have been o b s e r v e d w i t h l i d o c a i n e 1975; L a z a r r a e t al.,  1978).  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  Dosage and Plasma 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 was u s e d f o r t h e s t u d y .  (20 mg/kg)  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 end 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  plasma  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 . e f f e c t s were 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 than 5 min. 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 .  The t o x i c  Higher  doses  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 necessary. 30 m i n .  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  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 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 w e r e 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 . 0 5 ) g r e a t e r mean  plasma c o n c e n t r a t i o n s than R ( - ) - m e x i l e t i n e at each sampling time. r e s u l t s are c o n s i s t e n t with those obtained in the pentobarbitone anaesthetized rats (see-section 3.5.1.1 for explanation of -  plasma  concentrations). 3.5.2.2  O c c l u d e d Zone (0Z)  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 (Janse, 1987).  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 the i n c i d e n c e o f a r r h y t h m i a s  These  •*—  R(-)-mex  —e—  R,S-mex  — * —  F i g u r e 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 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 a d m i n i s t r a t i o n o f a d o s e o f 2 0 mg/kg, f o l l o w e d 1.5 h d o s e ( 2 0 m g / k g ) . V a l u e s a r e means ± s.e.m o f r e s u l t s  S(+)-mex  racemic mexiletine rats after the l a t e r b y a second from 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 1 9 . T h e mean (± s.e.m.) w e i g h t a n d o c c l u d e d z o n e ( O Z ) in the d i f f e r e n t treatment groups i n conscious ischaemic rats Weight* (9)  OZ (%)  Saline  337 ± 1 7  34+1  R(-)-mex  352 ± 2 0  33 ± 2  R,S-mex  339 ± 1 4  37 ± 1  S(+)-mex  324 ± 1 7  36+1  * weight o f r a t s i n each group. 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 anterior descending coronary artery.  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 descending coronary a r t e r y . Premature 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 (VT) 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. 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 incidence o f arrhythmias following coronary artery occlusion i n conscious rats  0-0.5h  VT  0-4h  VF 0-0.5h  0-4h  SVF 0-0.5h 0-4h  NSVF 0-0.5h 0-4h  Saline  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 were compared t o the s a l i n e group.  groups  F i g u r e s 27 and 28 show t h e  "arrhythmia scores" c a l c u l a t e d f o r the enantiomers.  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 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 received saline.  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 group r e l a t i v e t o s a l i n e ( F i g . 27).  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 experimental cardiac arrhythmias.  T h i s model has b e e n 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 ( K a n e 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.,  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 p r o t e c t c o n s c i o u s r a t s from ischaemia-induced a r r h y t h m i a s .  6001  1983). f a i l e d to  I t was  not  p o s s i b l e t o compare the r e s u l t s o f t h i s study 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 have not been p r e v i o u s l y r e p o r t e d .  H o w e v e r , M a r s h a l l et  al.  (1981),  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 antiarrhythmic actions of racemic mexiletine.  These authors reported  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 occlusion. study.  This i s in disagreement with r e s u l t s obtained i n the present  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. 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 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 ) following coronary artery occlusion i n conscious rats log PVCs 1 0  0-1/2 h  0-4 h  saline  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 . Premature 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 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 .  since  Saline  R(—)—mex  R,S-mex  S(H-)-mex  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 o n " 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 occlusion in conscious rats.  0  Saline  R(-)-mex  R,S-mex  S(+)-mex  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 o n " 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 occlusion in conscious r a t s .  m e x i l e t i n e enantiomers  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 that  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 a n d maintained  f o r t h e 30 m i n 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  administered  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 have been c a r r i e d o u t i n dogs u s i n g t h e H a r r i s 2-stage coronary a r t e r y o c c l u s i o n procedure e t al.,  1 9 7 7 ; M e r t z a n d S t e f f e , 1 9 8 0 ; H a s h i m o t o et al.,  H a s h i m o t o et al.  (Allen  1984).  ( 1 9 8 4 ) 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  "arrhythmia  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 ) .  r a t i o s " ( n u m b e r o f PVCs  H o w e v e r , 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 5 0 % .  T h e i r r e s u l t s were 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 a n d 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 a s H a s h i m o t o et al.,  (1980) (5 mg/kg).  The l a t t e r  authors  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 A l l e n et al.  and c o n v u l s i o n s .  ( 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 /ig/ml, s i n u s r h y t h m was c o m p l e t e l y in conscious ischaemic dogs.  returned  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 plasma c o n c e n t r a t i o n i n t h e dogs.  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 racemic m e x i l e t i n e and i t s enantiomers observed  in the rat.  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  of racemic mexiletine i n ischaemic pentobarbitone ( s e c t i o n 3.5.3.1). enantiomers,  t o be  anaesthetized rats  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 i t s  s u c h a s 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 ) ,  would  produce severe CNS toxicity 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 i g . 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 t a t i s t i c a l l y 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 i g . 30). The cardiovascular effects of racemic mexiletine and i t s enantiomers in the conscious rats are consistent with those observed in  T a b l e 2 2 . T h e e f f e c t o f d r u g t r e a t m e n t o n t h e ECG p a r a m e t e r s i n conscious ischaemic rats PR ( m s e c ) pre-drug  QRS ( m s e c )  post-drug  pre-drug  post-drug  saline  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 from pre-drug.  154  Saline  R(—)—mex —•- R,S—mex  S(+)—mex  500  -g  400  E -v.  V v.  CO  ro  300  CD  rr  200  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 of presentation.  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 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 . The s.e.m a r e omitted f o r c l a r i t y of presentation.  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 pressure (see s e c t i o n 3.5.1.3). 3.5.2.5  CNS S i d e  Effects  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  c o n c e n t r a t i o n s a t which these s i g n s o f t o x i c i t y appeared presented.  plasma  are also  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 w e r e 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 . H o w e v e r , 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 b e t w e e n the drug treatment groups.  T h e CNS t o x i c e f f e c t s w e r e p r e s u m e d t o be  due t o t h e high b r a i n uptake o f m e x i l e t i n e enantiomers (section  in the r a t  3.4.5).  In summary, 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  d i d not protect  c o n s c i o u s r a t s from coronary 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 a n d 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 a n d i t s enantiomers  were s i g n i f i c a n t but d i d not d i f f e r from each o t h e r .  drugs produced comparable  3.5.3  The  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 .  Coronary A r t e r y Occlusion-Induced Arrhythmia 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 Ischaemic 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 a n d i t s enantiomers  against ischaemia-induced  arrhythmias i n the conscious r a t 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 a higher dose o f racemic m e x i l e t i n e (40 m g / k g ) , 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 investigated.  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  used i n  c o n s c i o u s r a t s ( 2 0 mg/kg) a n d s a l i n e ( c o n t r o l ) w e r e a l s o e v a l u a t e d .  The  T a b l e 2 3 . 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 a n d 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 conscious 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 a n d i t s e n a n t i o m e r s ( 2 0 mg/kg) CNS t o x i c e f f e c t s  Plasma c o n c e n t r a t i o n Og/ml)  Saline  0/9  R(-)-mex  8/9'  2.3 ± 0.16  R,S-mex  7/9'  3.8 ± 0.41  S(+)-mex  8/9'  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 ** CNS t o x i c e f f e c t s o b s e r v e d w e r e a t a x i a 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. 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 R(-)-enantiomer.  saline. and c o n v u l s i o n s . R,S- a n d S ( + ) - M e x i l e t i n e concentrations than the  158 a n i m a l s w e r e m o n i t o r e d f o r 30 m i n 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 covered t h e f i r s t phase o f a r r h y t h m i a s ) . out i n pentobarbitone a n a e s t h e t i z e d r a t 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 The arrhythmias  resulting  from 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 have 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., 3.5.3.1  1983). 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  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 .  There  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 a n d VF a n d 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 ( 2 0 a n d 40 mg/kg) a n d s a l i n e a r e shown i n t a b l e s 25 a n d 2 6 , 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 a n d 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 ) . These r e s u l t s were s i m i l a r t o those 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 in conscious rats.  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 a n d t h e number o f PVCs ( t a b l e s 25 & 26) a n d 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 "arrhythmia s c o r e s " o b t a i n e d f o r t h e 3 treatment 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  groups  "arrhythmia  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 .  T h e 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  group.  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  T a b l e 2 4 . T h e mean (± s.e.m.) w e i g h t a n d o c c l u d e d z o n e (OZ) i n the d i f f e r e n t treatment 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 Weight* (9)  OZ (%)  Saline  402  +  18  34  ±  1  R,S-mex (20 mg/kg)  410  +  6  36  +  1  R,S-mex (40 mg/kg)  410  +  10  34  +  1  * Weight o f r a t s i n each group. 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 2 5 . T h e 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 of arrhythmias following coronary artery occlusion i n pentobarbitone anaesthetized rats VT  VF  SVF  NSVF  Saline  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 . 0 5 ) when c o m p a r e d t o s a l i n e . VT, V F , S V F a n d NSVF h a v e t h e same m e a n i n g a s i n t a b l e 2 0 . R a t s w e r e m o n i t o r e d o v e r a 30 m i n p e r i o d .  T a b l e 2 6 . 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 premature v e n t r i c u l a r contractions (logjQPVCs) following coronary artery occlusion i n pentobarbitone anaesthetized rats log PVCs 1 0  saline  2.42  +  0.16  R,S-mex (20 mg/kg)  2.80  +  0.06  R,S-mex (40 mg/kg)  1.68  ±  * 0.30  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 . 0 5 ) when c o m p a r e d t o s a l i n 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 a r e e x p r e s s e d as l o g j p P V C s t h e number o f PVCs i s a l o g - 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 m i n p e r i o d .  since  6  I  F i g u r e 3 1 . 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 ( 2 0 a n d 40 mg/kg) and s a l i n e o n " a r r h y t h m i a s c o r e " f o r t h e 0-30 m i n p e r i o d f o l l o w i n g c o r o n a r y artery occlusion in pentobarbitone anaesthetized rats. * S i g n i f i c a n t ( p < 0 . 0 5 ) when c o m p 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 a n d 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 + V F ) d u r i n g t h e 30 m i n 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.  T h e 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  at t h e end 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 m i n 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 m i n p e r i o d p o s t - o c c l u s i o n ( F i g . 3 2 B ) 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 ) . I n 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 , w h e r e 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 m i n p e r i o d p o s t - o c c l u s i o n ( F i g . 34B), plasma c o n c e n t r a t i o n s ranged  from  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 m i n post-occlusion ( f i g . 34A). These r e s u l t s confirmed that the 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 a r e seen a t h i g h e r plasma c o n c e n t r a t i o n s than t h o s e a c h i e v e d in the conscious rats.  However, t h e s e c o n c e n t r a t i o n s a r e comparable 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 from both t h e e l e c t r i c a l and ischaemic arrhythmia studies indicated that the antiarrhythmic, c a r d i o v a s c u l a r a n d 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 a n d i t s enantiomers a r e not markedly d i f f e r e n t from each 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 d i s t r i b u t i o n 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 r a t s .  165  100  LL  90  -  80  -  •  mm  70  >  60  1>  50  <D O C <D "D O C  40  r  30 20 10  h  0  0-5  5-10  10-15 Time  15-30  (min)  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 pentobarbitone 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 the 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 artery occlusion  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 d i s t r i b u t i o n 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 r a t s .  167 SUMMARY and CONCLUSIONS  4. 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  successfully accomplished.  c h l o r i d e was  Derivatization of mexiletine  enantiomers  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  were r e s o l v e d on a P i r k l e  i o n i c (phenyl  2-Anthroyl  c h l o r i d e was  formed  g l y c i n e ) c h i r a l c o l u m n b y HPLC.  found t o be a h i g h l y s e n s i t i v e  fluorescence  derivatization reagent.  Attempts at using t h i s d e r i v a t i z a t i o n reagent  to quantitate mexiletine  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  the  p r e s e n c e o f an i n t e r f e r i n g peak c o - e l u t i n g w i t h S ( + ) - m e x i l e t i n e c o u l d not be r e s o l v e d .  which  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 a s 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 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 binding of mexiletine decreasing  e n a n t i o m e r s was  s i g n i f i c a n t l y a s t h e pH was  increased  f r o m 7.0  the  t o 8.0.  M c E r l a n e et a7., 1 9 8 7 ) .  b i n d i n g o f t h e e n a n t i o m e r s was  These  maintained at  (=7.4), the observed b i n d i n g of m e x i l e t i n e  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 ( T a l b o t et a7 ., 1973;  that  pH-dependent; with f r e e f r a c t i o n s  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 physiological values  studies.  was  in the l i t e r a t u r e  More i m p o r t a n t l y ,  the  not s t e r e o s e l e c t i v e at 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 m o s t l i k e l y due t o p H - d e p e n d e n t changes i n the i o n i z a t i o n of the binding p r o t e i n ( s ) .  The  binding  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 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 w i t h i n the t h e r a p e u t i c  range of racemic m e x i l e t i n e .  w e r e 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.  The  Binding  the binding  enantiomers t o s e r u m and HSA  i n d i c a t e d the presence  o f two c l a s s e s o f b i n d i n g s i t e s .  These were a  h i g h a f f i n i t y , low c a p a c i t y and a low a f f i n i t y , h i g h c a p a c i t y c l a s s o f binding sites.  In c o n t r a s t , b i n d i n g t o AAG showed o n l y o n e 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 Pharmacokinetic  site.  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  enantiomers.  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 a t e volume o f d i s t r i b u t i o n than the S(+)-enantiomer.  steady-  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 uptake o f the S(+)-enantiomer. observed  High t i s s u e / s e r u m  i n b r a i n , l u n g s and k i d n e y s .  heart concentrations  o f the enantiomers.  r a t i o s (>20)  were  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 This r e l a t i v e l y high uptake 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 often observed  with mexiletine  therapy.  The pharmacodynamic 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 in pentobarbitone  anaesthetized  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.  H o w e v e r , 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 . s i g n i f i c a n t l y decreased  a n d R,S- and S ( + ) - m e x i l e t i n 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 than the R(-)-enantiomer.  MFF was  showed 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 . decrease  T h e p r o l o n g a t i o n o f PR i n t e r v a l and  i n h e a r t r a t e due t o t h e drugs 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  from each 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 t s 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 toxicity 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 t s 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 t s 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 antiarrhythmic  e f f e c t i v e n e s s o f racemic  used t h e H a r r i s 2-stage coronary  m e x i l e t i n e , these s t u d i e s have  artery occlusion procedure.  I t i s well  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 ) .  Thus,  t h i s m o d e l 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 V F , w h i c h i s t h e p r i m a r y  cause o f sudden death.  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 mexiletine with respect t o prevention  o f s u d d e n d e a t h i n MI p a t i e n t s  have n o t found 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 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 ) . the i n e f f e c t i v e n e s s o f racemic sub-therapeutic  patients  I t i s plausible that  m e x i l e t i n e was d u e t o t h e m a i n t e n a n c e o f  plasma concentrations  i n these  patients.  5. REFERENCES A b e n d r o t h RR, Meesman W, S t e p h a n K, S c h l e y G a n d H u b n e r H. 1. 66:341 ( 1 9 7 7 ) . Adams SS, B r e s l o f f P and Mason CG.  J . Pharm. Pharmacol.,  Kardiol.  28:256 (1976)  Adgey JAA. I n : Adgey JAA ( e d . ) : "Acute Phase o f Ischaemic H e a r t D i s e a s e and M y o c a r d i a l I n f a r c t i o n " . 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