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Stereoselective HPLC analysis, pharmacokinetics, serum protein binding and metabolism of mexiletine enantiomers… Kwok, David W. K. 1991

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STEREOSELECTIVE HPLC ANALYSIS, PHARMACOKINETICS, SERUM PROTEIN BINDING AND METABOLISM OF MEXILETINE ENANTIOMERS IN HEALTHY HUMAN SUBJECTS BY DAVID W. K. KWOK M . S c , The University of Brit ish Columbia, 1987 .Sc. (Pharm), The University of Brit ish Columbia, 1984 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Faculty of Pharmaceutical Sciences) (Division of Pharmaceutical Chemistry) We accept this thesis as confirming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1991 0 DAVID W. K. KWOK, 1991 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. The University of British Columbia Vancouver, Canada DE-6 (2/88) i i ABSTRACT M e x i l e t i n e ( M e x i t i l R ) i s an o r a l l y e f f e c t i v e antiarrhythmic drug used c l i n i c a l l y as a racemate of the R ( - ) - and S(+)-enantiomers. A s t e r e o s e l e c t i v e high-performance l i q u i d chromatographic assay was developed f o r the determination of m e x i l e t i n e enantiomers in serum, s a l i v a , red blood c e l l s and u r i n e . The m e x i l e t i n e enantiomers were resolved as t h e i r N-anthroyl d e r i v a t i v e s on a P i r k l e ^ phenylglycine i o n i c c h i r a l HPLC column. The present study examined the serum f r e e (unbound) and t o t a l drug k i n e t i c s f o r the m e x i l e t i n e enantiomers in twelve healthy volunteers f o l l o w i n g o r a l a d m i n i s t r a t i o n of 200 mg of racemic m e x i l e t i n e h y d r o c h l o r i d e . To f u r t h e r c h a r a c t e r i z e serum f r e e m e x i l e t i n e in the body, the concentrations of m e x i l e t i n e enantiomers in s a l i v a and in red blood c e l l s were examined. Since m e x i l e t i n e was l a r g e l y eliminated by metabolic processes, p-hydroxy-mexiletine and hydroxymethyl-mexiletine metabolites were examined in the urine of four healthy s u b j e c t s . Following o r a l drug a d m i n i s t r a t i o n , the d i s p o s i t i o n of m e x i l e t i n e enantiomers was described by one or two-compartment open models. The mean peak serum t o t a l m e x i l e t i n e concentration of 217 ± 68 ng/ml f o r R ( - ) - m e x i l e t i n e was found to be s i g n i f i c a n t l y greater (p<0.01) than a mean value of 196 ± 56 f o r S ( + ) - m e x i l e t i n e . The mean serum t o t a l R ( - ) -m e x i l e t i n e concentrations were a l s o found to be s i g n i f i c a n t l y greater than those f o r S(+)-mexiletine during the f i r s t s i x hours. The a b s o r p t i o n , r a p i d and terminal d i s p o s i t i o n k i n e t i c parameters between 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 . From u r i n a r y d a t a , the mean percentages of m e x i l e t i n e enantiomers recovered from the urine over 72 hours were found to be 3.5 ± 3.4% and i i i 3.7 ± 3.9% f o r R ( - ) - and S ( + ) - m e x i l e t i n e , r e s p e c t i v e l y . The mean terminal e l i m i n a t i o n h a l f - l i v e s were found to be 5.8 ± 1.5 h and 5.6 ± 1.4 h f o r R ( - ) - and S ( + ) - m e x i l e t i n e , r e s p e c t i v e l y . Both the u r i n a r y r e c o v e r i e s and the h a l f - 1 i v e s f o r the 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 . Comparative in vitro studies on the serum p r o t e i n binding of m e x i l e t i n e enantiomers by u l t r a f i l t r a t i o n and by e q u i l i b r i u m d i a l y s i s i n d i c a t e d a serum pH-dependent s t e r e o s e l e c t i v e p r o t e i n binding of m e x i l e t i n e enantiomers. A serum pH range from 6.3 to 9.4 was c o r r e l a t e d with the serum p r o t e i n binding of m e x i l e t i n e enantiomers from «30% to «80%. Within t h i s pH range, the serum f r e e drug R(-)/S(+) r a t i o was found to decrease from 1.0 to 0 . 7 . At serum pH 7 . 4 , the serum p r o t e i n binding of m e x i l e t i n e enantiomers was s i m i l a r , and was not dependent on the t h e r a p e u t i c concentration range of 0.25 to 3.0 fig/m\. The in vivo serum p r o t e i n binding of m e x i l e t i n e enantiomers was found to be n o n - s t e r e o s e l e c t i v e . The mean serum f r e e f r a c t i o n s of 0.57 ± 0.07 and 0.56 ± 0.06 f o r R ( - ) - and S ( + ) - m e x i l e t i n e , r e s p e c t i v e l y , were not s i g n i f i c a n t l y d i f f e r e n t . The o v e r a l l mean serum f r e e R(-)/S(+) m e x i l e t i n e r a t i o of 1.09 was a l s o i n d i c a t i v e of a n o n - s t e r e o s e l e c t i v e binding of m e x i l e t i n e enantiomers. Following the c o l l e c t i o n of unstimulated s a l i v a , the o v e r a l l mean s a l i v a / serum f r e e m e x i l e t i n e area under the concentration-time curve r a t i o s of 6.10 ± 2.82 and 7.49 + 3.48 f o r R ( - ) - and S ( + ) - m e x i l e t i n e , r e s p e c t i v e l y , were found to be s i g n i f i c a n t l y d i f f e r e n t (p<0.01). The o v e r a l l mean s a l i v a R(-)/S(+) r a t i o of 0.89 ± 0.02 (mean ± S . E . ) over 48 hours suggested that the d i s p o s i t i o n of m e x i l e t i n e enantiomers in s a l i v a was s t e r e o s e l e c t i v e . In a d d i t i o n , s a l i v a m e x i l e t i n e concentrations were found to c o r r e l a t e iv poorly with serum f r e e m e x i l e t i n e c o n c e n t r a t i o n s . In vitro studies on the d i s t r i b u t i o n of m e x i l e t i n e enantiomers into red blood c e l l s i n d i c a t e d a d i s t r i b u t i o n e q u i l i b r i u m of «40 minutes. The mean red blood c e l l m e x i l e t i n e area under the concentration-time curve of 2.3 ± 1 . 5 ^g/ml/h and 2 . 8 ± 2.1 //g/ml/h f o r R ( - ) - and S ( + ) - m e x i l e t i n e , r e s p e c t i v e l y , were not s i g n i f i c a n t l y d i f f e r e n t . The o v e r a l l mean red blood c e l l m e x i l e t i n e R(-)/S(+) r a t i o of 0.91 ± 0.13 suggested a s i m i l a r d i s t r i b u t i o n of the enantiomers into the red blood c e l l s . A s t e r e o s e l e c t i v e HPLC assay was developed f o r the simultaneous determination of m e x i l e t i n e , p - h y d r o x y - m e x i l e t i n e , and hydroxymethyl-m e x i l e t i n e enantiomers i n u r i n e . M e x i l e t i n e and hydroxymethyl -m e x i l e t i n e enantiomers were resolved on a P i r k l e R i s o l e u c i n e covalent HPLC column as t h e i r N-anthroyl d e r i v a t i v e s , w h i l e p-hydroxy-mexiletine enantiomers were resolved as t h e i r 0 - e t h y l - N - a n t h r o y l d e r i v a t i v e s . For m e x i l e t i n e and p - h y d r o x y - m e x i l e t i n e , chromatographic r e t e n t i o n was found to favour the R(-)-enantiomer, thus leading to the i n i t i a l e l u t i o n of the S(+)-enantiomer. For hydroxymethyl-mexiletine, the order of e l u t i o n was found to be r e v e r s e d . The mean cumulative amounts of p-hydroxy-m e x i l e t i n e enantiomers recovered from the urine over 72 hours were found to be 1.31 ± 0.33 mg and 1.27 + 0.39 mg f o r the R(-) and S(+)-enantiomers, r e s p e c t i v e l y . The two values were not s i g n i f i c a n t l y d i f f e r e n t . The mean cumulative amounts of R(-)-hydroxymethyl-mexiletine (2.94 ± 1.70 mg) recovered from the urine over 72 hours were found to be s i g n i f i c a n t l y (p<0.01) greater than a value of 1.17 ± 0.60 mg f o r the S(+)-enantiomer, suggesting the presence of a s t e r e o s e l e c t i v e metabolic pathway f o r hydroxymethyl-mexiletine. V TABLE OF CONTENTS Page ABSTRACT i 1 LIST OF TABLES x i i LIST OF FIGURES xv LIST OF ABBREVIATIONS xx ACKNOWLEDGEMENTS xxiii 1. INTRODUCTION 1 1.1 Development of M e x i l e t i n e 1 1.2 Pharmacology 1 1.2.1 Mechanisms of A c t i o n 1 1 . 2 . 2 Dosage and A d m i n i s t r a t i o n 3 1 . 2 . 3 E l e c t r o p h y s i o l o g y 4 1 2 4 Haemodynamic E f f e c t s of M e x i l e t i n e 5 1 . 2 . 5 Neurological A c t i v i t y of M e x i l e t i n e 5 1 . 2 . 6 Comparative Studies with other Antiarrhythmic Drugs 6 1.2.7 Plasma Concentration-Antiarrhythmic Response R e l a t i o n s 7 1 . 2 . 8 Drug I n t e r a c t i o n s with M e x i l e t i n e 7 1 . 2 . 9 Toxicology 9 1.3 Pharmacokinetics in Humans 9 1.3.1 Oral Absorption and B i o a v a i l a b i l i t y 9 1 . 3 . 2 Tissue D i s t r i b u t i o n 10 1 . 3 . 3 E l i m i n a t i o n 10 1 . 3 . 4 E f f e c t s of U r i n a r y pH on Excretion of M e x i l e t i n e 11 1 . 3 . 5 Serum P r o t e i n Binding 12 1 . 3 . 6 Pharmacokinetics of M e x i l e t i n e in Disease States 12 1.3 7 Pharmacokinetics of M e x i l e t i n e in the E l d e r l y 14 1.4 Metabolism 14 1.4.1 Human Studies 14 1 .4 .2 Animal Studies 16 1.5 A n a l y s i s of Racemic M e x i l e t i n e 17 1.6 S t e r e o s e l e c t i v e A n a l y s i s of M e x i l e t i n e Enantiomers 18 1.7 S t e r e o s e l e c t i v e Drug A n a l y s i s 18 1.7.1 Resolution of Enantiomers as Diastereoisomers 18 1.7.2 D i r e c t Separation Using C h i r a l S t a t i o n a r y Phases 20 v i 1 . 7 . 2 . 1 Gas Chromatography 21 1 . 7 . 2 . 2 High Performance L i q u i d Chromatography 22 1 . 7 . 2 . 2 . 1 C y c l o d e x t r i n Phases 22 1 . 7 . 2 . 2 . 2 Serum P r o t e i n Bound Phases 23 1 . 7 . 2 . 2 . 3 C e l l u l o s e Bound C h i r a l S t a t i o n a r y Phases 24 1 . 7 . 2 . 1 . 4 P i r k l e C h i r a l S t a t i o n a r y Phases 25 1.8 Summary of Research Goals 27 1.8.1 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 and M e x i l e t i n e M e t a b o l i t e Enantiomers 27 1 . 8 . 2 Pharmacokinetics of Serum Total and Free M e x i l e t i n e Enantiomers in Healthy Subjects 27 1 . 8 . 3 Metabolism of M e x i l e t i n e Enantiomers 28 1.9 Objectives 29 1.10 Rationale 30 2. EXPERIMENTAL 34 2.1 M a t e r i a l s and Supplies 34 2.1 .1 Chemicals and Reagents 34 2 . 1 . 2 Solvents 35 2 . 1 . 3 Supplies f o r Human Studies 35 2 . 1 . 4 Supplies f o r Serum P r o t e i n Binding Studies 35 2 . 1 . 5 Chromatographic Columns 35 2.2 Equipment 36 2 . 2 . 1 High-Performance L i q u i d Chromatographs 36 2 . 2 . 2 Gas Chromatograph - Mass Spectrometer 36 2 . 2 . 3 Centrifuge 37 2 . 2 . 4 P o l a r i m e t r y 37 2.3 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 37 2 . 3 . 1 Synthesis of 2-Anthroyl Chloride 37 2 . 3 . 1 . 1 Reduction of Anthraquinone-2-carboxylic A c i d with Zinc/Aqueous Ammonia 37 2 . 3 . 1 . 2 Formation of 2-Anthroyl C h l o r i d e 38 2 . 3 . 2 P u r i f i c a t i o n of 2-Anthroyl Chloride by Preparative-HPLC 38 2 . 3 . 3 S t r u c t u r a l C h a r a c t e r i z a t i o n of 2-Anthroyl C h l o r i d e and 2-Anthroyl M e x i l e t i n e D e r i v a t i v e by Mass Spectrometry 39 2 . 3 . 4 S t e r e o s e l e c t i v e HPLC of M e x i l e t i n e Enantiomers 39 2 . 3 . 4 . 1 Assay Method f o r Total ( P r o t e i n Bound + Free) M e x i l e t i n e Enantiomers in Serum 2 . 3 . 4 . 2 Assay Method f o r Free M e x i l e t i n e Enantiomers in Serum 2 . 3 . 4 . 3 Assay Recovery of M e x i l e t i n e from Serum and Serum U l t r a f i U r a t e 2 . 3 . 4 . 4 A c y l a t i o n K i n e t i c s of M e x i l e t i n e with 2-Anthroyl Chloride 2 . 3 . 4 . 5 Determination of Detection L i n e a r i t y of M e x i l e t i n e Enantiomers in Serum and in Serum U l t r a f i l t r a t e 2 . 3 . 4 . 6 Determination of Detector L i n e a r i t y of M e x i l e t i n e Enantiomers i n S a l i v a 2 . 3 . 4 . 7 Determination of Detector L i n e a r i t y of M e x i l e t i n e Enantiomers i n Red Blood C e l l s 2 . 3 . 4 . 8 HPLC Assay I n t r a - and I n t e r - v a r i a b i l i t y In Vitro Serum P r o t e i n Binding and Red Blood C e l l D i s t r i b u t i o n of the M e x i l e t i n e Enantiomers 2 . 4 . 1 The E f f e c t s of Serum C o l l e c t i o n and Storage Methods on M e x i l e t i n e Serum Free F r a c t i o n 2 . 4 . 2 Determination of Serum P r o t e i n Binding C h a r a c t e r i s t i c s f o r M e x i l e t i n e Enantiomers 2 . 4 . 2 . 1 Determination of Nonspecific Binding of M e x i l e t i n e to U l t r a f i l t r a t i o n Units 2 . 4 . 2 . 2 Determination of Nonspecific Binding of M e x i l e t i n e to E q u i l i b r i u m D i a l y s i s Units 2 . 4 . 2 . 3 Determination of D i a l y s i s E q u i l i b r i u m Time f o r M e x i l e t i n e 2 . 4 . 2 . 4 Comparison of Serum P r o t e i n Binding of M e x i l e t i n e Enantiomers Using U l t r a f i l t r a t i o n and E q u i l i b r i u m D i a l y s i s 2 . 4 . 2 . 5 The E f f e c t s of Temperature on Serum P r o t e i n Binding of M e x i l e t i n e 2 . 4 . 2 . 6 The E f f e c t s of Serum pH on the Binding of M e x i l e t i n e Enantiomers Using E q u i l i b r i u m D i a l y s i s 2 . 4 . 2 . 7 The E f f e c t s of Serum pH on the Binding of M e x i l e t i n e Enantiomers Using U l t r a f i l t r a t i o n 2 . 4 . 2 . 8 Comparison on the E f f e c t s of Serum pH Adjustment Methods by Sulphuric A c i d and Phosphate Buffers on M e x i l e t i n e Free F r a c t i o n 2 . 4 . 2 . 9 C o n c e n t r a t i o n - L i n e a r i t y of Serum P r o t e i n Binding of M e x i l e t i n e Enantiomers 2 . 4 . 3 In Vitro Determination of the Extent and Time-Course of M e x i l e t i n e D i s t r i b u t i o n into Red Blood C e l l s 2 . 4 . 4 Pharmacokinetic Study of M e x i l e t i n e Enantiomers in Healthy Human Subjects 2 . 4 . 4 . 1 V a l i d a t i o n of the Potency of M e x i t i l R 200 mg Capsules 50 2 . 4 . 4 . 2 S e l e c t i o n of Healthy Human Subjects 51 2 . 4 . 4 . 3 P h y s i c a l , Urine and Haemetology Examination 52 2 . 4 . 4 4 Drug A d m i n i s t r a t i o n and Serum, S a l i v a , Urine and Red Blood C e l l C o l l e c t i o n 52 2 . 5 Development of a 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 , p - H y d r o x y - M e x i l e t i n e , and Hydroxymethyl-Mexiletine Enantiomers in Urine 53 2 . 5 . 1 P i r k l e Dinitrobenzoyl D-Phenylglycine Ionic C h i r a l HPLC Column 53 2 . 5 . 1 . 1 A c y l a t i o n of M e x i l e t i n e and M e x i l e t i n e Metabolites Using 2-Anthroyl C h l o r i d e or 2-Naphthoyl C h l o r i d e 53 2 . 5 . 1 . 1 . 1 M e x i l e t i n e Enantiomers 53 2 . 5 . 1 . 1 . 2 p-Hydroxy-Mexiletine Enantiomers 54 2 . 5 . 1 . 1 . 3 Hydroxymethyl-Mexiletine Enantiomers 54 2 . 5 . 1 . 2 S i l y l a t i o n of M e x i l e t i n e Metabolites Using N - T r i m e t h y l s i l y l Imidazole Followed by A c y l a t i o n with 2-Naphthoyl C h l o r i d e 55 2 . 5 . 1 . 2 . 1 p-Hydroxy-Mexiletine Enantiomers 55 2 . 5 . 1 . 2 . 2 Hydroxymethyl-Mexiletine Enantiomers 55 2 . 5 . 1 . 3 A c y l a t i o n of M e x i l e t i n e Metabolites Using 2-Anthroyl C h l o r i d e Followed by A l k y l a t i o n with Ethyl Bromide 56 2 . 5 . 1 . 3 . 1 p-Hydroxy-Mexiletine Enantiomers 56 2 . 5 . 1 . 3 . 2 Hydroxymethyl-Mexiletine Enantiomers 57 2 . 5 . 2 P i r k l e Dinitrobenzoyl L - I s o l e u c i n e Ionic C h i r a l HPLC Column57 2 . 5 . 2 . 1 A c y l a t i o n of M e x i l e t i n e and M e x i l e t i n e Metabolites Using 2-Anthroyl C h l o r i d e or 2-naphthoyl C h l o r i d e 57 2 . 5 . 2 . 2 Methylation of M e x i l e t i n e Metabolites Using Diazomethane Followed by A c y l a t i o n Using 2-Anthroyl C h l o r i d e 57 2 . 5 . 2 . 2 . 1 p-Hydroxy-Mexiletine Enantiomers 57 2 . 5 . 3 P i r k l e Dinitrobenzoyl L - I s o l e u c i n e Covalent C h i r a l HPLC Column 58 ix 2 . 5 . 3 . 1 A c y l a t i o n of M e x i l e t i n e and M e x i l e t i n e and Hydroxymethyl-Mexiletine Enantiomers Using the P i r k l e Covalent Isoleucine C h i r a l HPLC Col limn 58 2 . 5 . 3 . 1 . 1 p-Hydroxy-Mexiletine Enantiomers 58 2 . 5 . 3 . 1 . 2 Hydroxymethyl-Mexiletine Enantiomers 59 2 . 5 . 4 Assay Method f o r M e x i l e t i n e , p-Hydroxy-Mexiletine and Hydroxymethyl-Mexiletine Enantiomers in Urine 59 2 . 5 . 5 S t e r e o s e l e c t i v e HPLC of M e x i l e t i n e , Hydroxymethyl-M e x i l e t i n e and p-Hydroxy-Mexiletine Enantiomers in Urine 60 2 . 5 . 6 The Time-Course of A l k y l a t i o n of p-Hydroxy-Mexiletine Enantiomers with Ethyl Bromide 61 2 . 5 . 7 Solvent E x t r a c t i o n Recovery of M e x i l e t i n e , p-Hydroxy-M e x i l e t i n e , and Hydroxymethyl-Mexiletine Enantiomers 61 2 . 5 . 8 Detection Response L i n e a r i t y C a l i b r a t i o n f o r M e x i l e t i n e , p-Hydroxy-Mexiletine and Hydroxymethyl-Mexiletine Enantiomers in Urine 62 2 . 5 . 9 Determination of Assay V a r i a b i l i t y f o r M e x i l e t i n e , p-Hydroxy-Mexiletine and Hydroxymethyl-Mexiletine Enantiomers in Urine 63 2.6 Pharmacokinetic Data A n a l y s i s 63 2 . 6 . 1 Computer Curve F i t t i n g 63 2 . 6 . 2 C a l c u l a t i o n of Pharmacokinetic Parameters 64 2 . 6 . 3 S t a t i s t i c a l Data A n a l y s i s 67 3 . RESULTS AND DISCUSSIONS 68 3.1 Development of 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 Assay f o r M e x i l e t i n e Enantiomers 68 3.1 .1 Synthesis of 2-Anthroyl Chloride 68 3 . 1 . 2 Confirmation of the Structure of 2-Anthroyl C h l o r i d e and M e x i l e t i n e - N - A n t h r o y l D e r i v a t i v e by Mass Spectrometry 69 3 . 1 . 3 The Time-Course of A c y l a t i o n of M e x i l e t i n e Enantiomers with 2-Anthroyl C h l o r i d e 69 3 . 1 . 4 Resolution of M e x i l e t i n e Enantiomers as Their Anthroyl D e r i v a t i v e s on the P i r k l e Ionic Phenylglycine C h i r a l Column 71 3 . 1 . 5 Assay Method f o r the M e x i l e t i n e Enantiomers in Serum, S a l i v a , Red Blood C e l l s and Urine 73 3 . 1 . 6 Assay Recoveries of M e x i l e t i n e Enantiomers from Serum and Serum U l t r a f i U r a t e 76 3.1 .7 L i n e a r i t y of Chromatographic Peak Height Measurements of the M e x i l e t i n e Enantiomers in Serum, Serum U l t r a f i U r a t e , S a l i v a , Red Blood C e l l s , and Urine 76 3 . 1 . 8 HPLC Assay I n t r a - and I n t e r - v a r i a b i l i t y 80 X 3.2 In Vitro Serum Protein Binding and Red Blood C e l l D i s t r i b u t i o n of the M e x i l e t i n e Enantiomers 87 3 . 2 . 1 Serum C o l l e c t i o n and Storage Methods 87 3 . 2 . 2 Comparisons of M e x i l e t i n e Serum Free Drug Measurements Using U l t r a f i l t r a t i o n and E q u i l i b r i u m D i a l y s i s 89 3 . 2 . 3 Determination of D i a l y s i s E q u i l i b r i u m Time 93 3 . 2 . 4 The E f f e c t of Serum pH on the Protein Binding of M e x i l e t i n e Enantiomers 95 3 . 2 . 5 Determination of Concentration-Dependent P r o t e i n Binding of M e x i l e t i n e Enantiomers 98 3 . 2 . 5 In Vitro Determination of the Time-Course of M e x i l e t i n e D i s t r i b u t i o n into Red Blood C e l l s 101 iopc 3 . 2 . 7 V a l i d a t i o n of the Potency of M e x i t i l R 200 mg Capsules 104 3 . 3 Pharmacokinetics of M e x i l e t i n e Enantiomers in Healthy Human Subjects 105 3 . 3 . 1 Pharmacokinetics of M e x i l e t i n e in Serum 105 3 . 3 . 2 The Pharmacokinetics of M e x i l e t i n e in Serum U l t r a f i l t r a t e 123 3 . 3 . 3 Pharmacokinetics of M e x i l e t i n e in S a l i v a 123 3 . 3 . 4 Pharmacokinetics of M e x i l e t i n e in Red Blood C e l l s 137 3 . 3 . 5 Urine Pharmacokinetic Data 147 3.4 M e x i l e t i n e Metabolite D i s p o s i t i o n in Humans 155 3 . 4 . 1 Development of 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 Assay f o r the Enantiomers of M e x i l e t i n e , p-Hydroxy-Mexiletine and Hydroxymethyl-M e x i l e t i n e in Urine 155 3 . 4 . 1 . 1 A c y l a t i o n of M e x i l e t i n e Metabolite Enantiomers 157 3 . 4 . 1 . 2 S i l y l a t i o n / A c y l a t i o n of M e x i l e t i n e Metabolite Enantiomers 159 3 . 4 . 1 . 3 Methylation and A c y l a t i o n of M e x i l e t i n e Metabolite Enantiomers 162 3 . 4 . 1 . 4 A c y l a t i o n and A l k y l a t i o n of M e x i l e t i n e Metabolite Enantiomers 162 3 . 4 . 2 The Time-Course of A l k y l a t i o n of p-Hydroxy-M e x i l e t i n e with Ethyl Bromide 167 3 . 4 . 3 Solvent Recovery f o r M e x i l e t i n e , p-Hydroxy-M e x i l e t i n e , and Hydroxymethyl-Mexiletine from Urine 168 3 . 4 . 4 HPLC Assay Method f o r M e x i l e t i n e , p-Hydroxy-Mexiletine, and Hydroxymethyl-Mexiletine in Urine 168 3 . 4 . 5 Detection C a l i b r a t i o n L i n e a r i t y f o r M e x i l e t i n e , p-Hydroxy-Mexiletine, and Hydroxymethyl-Mexiletine Enantiomers in u r i n e 170 3 . 4 . 6 Urine M e x i l e t i n e Metabolite Pharmacokinetic Data 175 x i 3 .4 . 6 . 1 p-Hydroxy-Mexiletine Enantiomers 175 3 .4 . 6 . 2 Hydroxymethyl-Mexiletine Enantiomers 178 SUMMARY OF RESULTS 190 CONCLUSIONS 199 REFERENCES 200 APPENDIX 213 x i i LIST OF TABLES Page Table 1. The d i e t h y l ether solvent e x t r a c t i o n recovery of R ( - ) -and S(+)-mexiletine from serum at 50 and 100 ng/ml c o n c e n t r a t i o n s . 79 Table 2. HPLC assay c a l i b r a t i o n curve data f o r R ( - ) - and S(+)-m e x i l e t i n e in s a l i v a , red blood c e l l s and u r i n e . 79 Table 3. HPLC assay v a r i a b i l i t y data f o r R ( - ) - and S(+)-mexiletine in serum at 10 and 200 ng/ml c o n c e n t r a t i o n s . 86 Table 4. The e f f e c t of blood c o l l e c t i o n and storage c o n d i t i o n s on serum f r e e f r a c t i o n of R ( - ) - and S(+)-mexiletine. 90 Table 5. A comparison of the serum p r o t e i n binding of m e x i l e t i n e using u l t r a f i l t r a t i o n versus e q u i l i b r i u m d i a l y s i s at 0 . 5 and 2.0 /zg/ml and at 22 and 37 ° C . 94 Table 6. The recovery of R ( - ) - and S(+)-mexiletine from the C e n t r i f r e e m i c r o p a r t i t i o n serum u l t r a f i l t r a t i o n u n i t s . 94 Table 7. The recovery of R ( - ) - and S(+)-mexiletine from e q u i l i b r i u m d i a l y s i s u n i t s . 102 Table 8. The e f f e c t of serum pH adjustments using sodium phosphate b u f f e r s , 0.1 M s u l p h u r i c and 0.1 M phosphoric acids on R ( - ) - and S(+)-mexiletine serum f r e e f r a c t i o n and serum f r e e R(-)/S(+) r a t i o . 102 Table 9. The R ( - ) - and S(+)-mexiletine serum f r e e f r a c t i o n s and serum R(-)/S(+) r a t i o s over a concentration range of 0.25 to 3 . 0 M9/ml• 103 Table 10. The serum t o t a l R ( - ) - and S(+)-mexiletine concentrations over 48 hours from twelve healthy subjects f o l l o w i n g o r a l a d m i n i s t r a t i o n of 200 mg racemic m e x i l e t i n e h y d r o c h l o r i d e . 112 Table 11. The serum f r e e R ( - ) - and S(+)-mexiletine concentrations from twelve healthy subjects over 48 hours f o l l o w i n g o r a l a d m i n i s t r a t i o n of 200 mg racemic m e x i l e t i n e h y d r o c h l o r i d e . 113 Table 12. The serum t o t a l and serum f r e e m e x i l e t i n e R(-)/S(+) r a t i o s over 48 hours from twelve healthy s u b j e c t s . 114 Table 13. The R ( - ) - and S(+)-mexiletine serum f r e e f r a c t i o n s over 48 hours from twelve healthy s u b j e c t s . 117 X111 Table 14. The pharmacokinetic data f o r R ( - ) - and S(+)-mexiletine derived from serum t o t a l drug data from twelve healthy subjects f o l l o w i n g oral a d m i n i s t r a t i o n of 200 mg racemic m e x i l e t i n e h y d r o c h l o r i d e . 119 Table 15. The pharmacokinetic data f o r R ( - ) - and S(+)-mexiletine derived from serum f r e e drug data from twelve healthy subjects f o l l o w i n g o r a l a d m i n i s t r a t i o n of 200 mg racemic m e x i l e t i n e h y d r o c h l o r i d e . 126 Table 16. The R ( - ) - and S(+)-mexiletine concentrations in s a l i v a over 48 hours from twelve healthy subjects f o l l o w i n g o r a l a d m i n i s t r a t i o n of 200 mg racemic m e x i l e t i n e h y d r o c h l o r i d e . 127 Table 17. The pharmacokinetic data f o r R ( - ) - and S(+)-mexiletine derived from s a l i v a drug data from twelve healthy subjects f o l l o w i n g o r a l a d m i n i s t r a t i o n of 200 mg of racemic m e x i l e t i n e h y d r o c h l o r i d e . 129 Table 18. The r e s p e c t i v e R ( - ) - and S(+)-mexiletine s a l i v a / serum f r e e m e x i l e t i n e concentration r a t i o s f o r over 48 hours from twelve healthy s u b j e c t s . 133 Table 19. The s a l i v a R(-)/S(+) m e x i l e t i n e concentration r a t i o s over 48 hours in twelve healthy s u b j e c t s . 134 Table 20. The t h e o r e t i c a l s a l i v a / serum f r e e drug concentration r a t i o s f o r a c i d i c and b a s i c drugs derived from s a l i v a drug concentrations and s a l i v a pH according to the Matin equation. * 136 Table 21. The R ( - ) - and S(+)-mexiletine concentrations in red c e l l s from twelve healthy subjects over 48 hours f o l l o w i n g o r a l a d m i n i s t r a t i o n of 200 mg racemic m e x i l e t i n e h y d r o c h l o r i d e . 142 Table 22 The pharmacokinetic data For R ( - ) - and S(+)-mexiletine derived from m e x i l e t i n e red blood c e l l data from twelve subjects f o l l o w i n g o r a l a d m i n i s t r a t i o n of 200 mg racemic m e x i l e t i n e h y d r o c h l o r i d e . 143 Table 23. The red blood c e l l m e x i l e t i n e R(-)/S(+) concentration r a t i o s over 48 hours from twelve healthy s u b j e c t s . 146 Table 24. The r e s p e c t i v e R ( - ) - and S(+)-mexiletine red blood c e l l / serum f r e e drug concentration r a t i o s in twelve subjects over 48 hours. 149 Table 25. The pharmacokinetic data f o r R ( - ) - and S(+)-mexiletine derived from m e x i l e t i n e urine data from twelve healthy subjects f o l l o w i n g o r a l a d m i n i s t r a t i o n of 200 mg racemic m e x i l e t i n e h y d r o c h l o r i d e . 153 x i v Table 26. The solvent e x t r a c t i o n r e c o v e r i e s of m e x i l e t i n e , p-hydroxy m e x i l e t i n e and hydroxymethyl m e x i l e t i n e enantiomers from urine at adjusted urine pH 9 . 0 . 174 Table 27. HPLC assay 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 , p-hydroxy-mexiletine and hydroxymethyl-mexiletine enantiomers in u r i n e . 180 Table 28. HPLC i n t e r - a s s a y v a r i a b i l i t y f o r R ( - ) - and S(+)-mexiletine in u r i n e . 180 Table 29. HPLC i n t e r - a s s a y v a r i a b i l i t y f o r R ( - ) - and S(+)-p-hydroxy m e x i l e t i n e in u r i n e . 181 Table 30. HPLC i n t e r - a s s a y v a r i a b i l i t y f o r R ( - ) - and S(+)-hydroxymethyl m e x i l e t i n e In u r i n e . 182 Table 31. HPLC i n t r a - a s s a y v a r i a b i l i t y f o r R ( - ) - and S ( + ) - m e x i l e t i n e , p-hydroxy-mexiletine and hydroxymethyl-mexiletine in u r i n e . 183 Table 32. The pharmacokinetic data of R ( - ) - and S ( + ) - m e x i l e t i n e , p-hydroxy-mexiletine and hydroxymethyl-mexiletine derived from urine metabolite data from twelve healthy subjects f o l l o w i n g o r a l a d m i n i s t r a t i o n of 200 mg racemic m e x i l e t i n e h y d r o c h l o r i d e . 184 X V LIST OF FIGURES Page Figure 1. The development of m e x i l e t i n e . 2 Figure 2. The metabolic d i s p o s i t i o n of m e x i l e t i n e in man. 15 Figure 3 . The synthesis of 2-anthroyl c h l o r i d e . 69 Figure 4. The electron-impact mass spectra of (a) 2-anthroyl c h l o r i d e and (b) N-anthroyl d e r i v a t i v e of m e x i l e t i n e . 72 Figure 5. The time-course of a c y l a t i o n of R ( - ) - and S(+)-m e x i l e t i n e by vortex-mixing of the enantiomers with 2-anthroyl c h l o r i d e at ambient room temperature. 74 Figure 6. A diagrammatic representation of the bonding i n t e r a c t i o n s between R ( - ) - m e x i l e t i n e and the d i n i t r o b e n z o y l -D - ( - ) - p h e n y l g l y c i n e HPLC s t a t i o n a r y phase. 75 Figure 7. Representative HPLC chromatograms of (a) serum from a subject and (b) blank serum showing r e s o l u t i o n of the N-anthroyl d e r i v a t i v e s of R ( - ) - m e x i l e t i n e (1) , S(+)-m e x i l e t i n e (2) and the i n t e r n a l standard, KOE-2963 ( 3 ) . 77 Figure 8 . HPLC assay c a l i b r a t i o n curves f o r R ( - ) - and S(+)-m e x i l e t i n e in serum and in serum u l t r a f i l t r a t e . 81 Figure 9. HPLC assay c a l i b r a t i o n curves f o r R ( - ) - and S(+)-m e x i l e t i n e in s a l i v a . 82 Figure 10. HPLC assay c a l i b r a t i o n curves f o r R ( - ) - and S(+)-m e x i l e t i n e in red blood c e l l s . 83 Figure 11. HPLC assay c a l i b r a t i o n curves f o r R ( - ) - and S(+)-m e x i l e t i n e in U r i n e . 84 Figure 12. HPLC assay c a l i b r a t i o n curve f o r S(+)-mexiletine concentrations of 10 to 500 ng/ml in the presence of a constant 500 ng/ml of R ( - ) - m e x i l e t i n e . 85 Figure 13. The time-course of e q u i l i b r i u m d i a l y s i s f o r R ( - ) -and S(+)-mexiletine in serum against (a) i s o t o n i c phosphate b u f f e r , and (b) serum u l t r a f i l t r a t e at 37 ° C . 96 Figure 14. The e f f e c t of serum pH on (a) serum f r e e f r a c t i o n s and (b) serum f r e e m e x i l e t i n e R(-)/S(+) r a t i o s obtained using d i a l y s i s of racemic m e x i l e t i n e against a 0.067 M phosphate b u f f e r at 37 °C f o r 4 hours. 99 xv i Figure 15. The e f f e c t of serum pH on serum f r e e f r a c t i o n s f o r R ( - ) - and S(+)-mexiletine at (a) 500 ng/ml and (b) 2.0 ^g/ml concentrations obtained by u l t r a f i l t r a t i o n of racemic m e x i l e t i n e in serum. 100 Figure 16. P l o t s of the extent and time-course of the d i s t r i b u t i o n of R ( - ) - and S(+)-mexiletine from i s o t o n i c phosphate b u f f e r into red blood c e l l s . 106 Figure 17. Representative semilogarithmic p l o t s of R ( - ) - and S(+)-m e x i l e t i n e concentrations in serum, serum u l t r a f i l t r a t e , s a l i v a and red c e l l s from subject RL f o l l o w i n g o r a l a d m i n i s t r a t i o n of 200 mg of racemic m e x i l e t i n e h y d r o c h l o r i d e . 108 Figure 18. Semilogarithmic p l o t s of the mean ± S.D. serum t o t a l R ( - ) - and S(+)-mexiletine concentrations over 48 hours from twelve healthy subjects f o l l o w i n g oral a d m i n i s t r a t i o n of 200 mg of racemic m e x i l e t i n e h y d r o c h l o r i d e . 109 Figure 19. Semilogarithmic p l o t s of the mean ± S.D. serum f r e e R ( - ) - and S(+)-mexiletine concentrations over 48 hours from twelve healthy subjects f o l l o w i n g oral a d m i n i s t r a t i o n of 200 mg of racemic m e x i l e t i n e h y d r o c h l o r i d e . 110 Figure 20. P l o t s of the mean ± S.D. R ( - ) - / S(+)-mexiletine serum t o t a l and serum f r e e concentration r a t i o s over 48 hours from twelve healthy s u b j e c t s . I l l Figure 21. P l o t s of the mean ± S.D. R ( - ) - and S(+)-mexiletine serum f r e e f r a c t i o n s from twelve healthy subjects over 48 hours. 116 Figure 22. Semilogarithmic p l o t s of the serum t o t a l and serum f r e e R ( - ) - and S(+)-mexiletine concentrations over 48 hours from subject AO f o l l o w i n g o r a l a d m i n i s t r a t i o n of 200 mg racemic m e x i l e t i n e h y d r o c h l o r i d e . 122 Figure 23. Semilogarithmic p l o t s of the mean ± S.D. R ( - ) - and S(+)-mexiletine concentrations in s a l i v a over 48 hours from twelve healthy subjects f o l l o w i n g a d m i n i s t r a t i o n of 200 mg racemic m e x i l e t i n e h y d r o c h l o r i d e . 125 Figure 24. P l o t s of the mean + S.D. r e s p e c t i v e R ( - ) - and S(+)-m e x i l e t i n e s a l i v a / serum f r e e concentration r a t i o s over 48 hours from twelve healthy s u b j e c t s . 131 Figure 25. P l o t s of the mean ± S.D. R/S-mexiletine enantiomer concentration r a t i o s in s a l i v a over 48 hours in twelve healthy s u b j e c t s . 132 xv i i Figure 26. Representative l i n e a r c o r r e l a t i o n of the r e s p e c t i v e c a l c u l a t e d versus observed serum f r e e R ( - ) - and S(+)-m e x i l e t i n e concentrations from subject JG. [Serum f r e e R ( - ) - and S(+)-mexiletine concentrations were c a l c u l a t e d from the r e s p e c t i v e s a l i v a drug concentrations and s a l i v a pH]. 138 Figure 27. Linear c o r r e l a t i o n of the r e s p e c t i v e c a l c u l a t e d versus observed serum f r e e R ( - ) - and S(+)-mexiletine concentrations from ten healthy s u b j e c t s . [Serum f r e e (R-)- and S(+)-mexiletine concentrations were c a l c u l a t e d from the r e s p e c t i v e s a l i v a drug concentrations and s a l i v a pH]. 139 Figure 28. Semilogarithmic p l o t s of the mean ± S.D. R ( - ) - and S(+)-mexiletine concentrations in red blood c e l l s over 48 hours from twelve healthy subjects f o l l o w i n g o r a l a d m i n i s t r a t i o n of 200 mg of racemic m e x i l e t i n e h y d r o c h l o r i d e . 141 Figure 29. P l o t s of the mean ± S.D. R ( - ) - / S(+)-mexiletine red blood c e l l concentration r a t i o s over 24 hours from twelve healthy s u b j e c t s . 145 Figure 30. P l o t s of the mean ± S.D. r e s p e c t i v e R ( - ) - and S(+)-m e x i l e t i n e red blood c e l l / serum f r e e concentration r a t i o s over 36 hours from twelve healthy s u b j e c t s . 148 Figure 31. Representative p l o t s of the cumulative amount of R ( - ) -and S(+)-mexiletine excreted over 72 hours in the urine from subject TK f o l l o w i n g o r a l a d m i n i s t r a t i o n of 200 mg of racemic m e x i l e t i n e h y d r o c h l o r i d e . 151 Figure 32. Representative p l o t s of the cumulative amount of R ( - ) -and S(+)-mexiletine excreted over 72 hours in the urine from subject ST f o l l o w i n g o r a l a d m i n i s t r a t i o n of 200 mg of racemic m e x i l e t i n e h y d r o c h l o r i d e . 152 Figure 33. Representative semilogarithmic p l o t s of the amount of R ( - ) - and S(+)-mexiletine remaining to be excreted over 72 hours in the urine from subject TK f o l l o w i n g o r a l a d m i n i s t r a t i o n of 200 mg of racemic m e x i l e t i n e h y d r o c h l o r i d e . 156 Figure 34. P l o t s of the u r i n a r y e x c r e t i o n rates of R ( - ) - and S(+)-m e x i l e t i n e , urine volume and urine pH from subject CK over 72 hours f o l l o w i n g o r a l a d m i n i s t r a t i o n of 200 mg racemic m e x i l e t i n e hydrochloride in s i x healthy s u b j e c t s . 158 x v i i i Figure 35. HPLC chromatograms of resolved R ( - ) - and S(+)-hydroxymethyl m e x i l e t i n e as t h e i r N,0-anthroyl d i -d e r i v a t i v e s (1) and N-anthroyl mono-derivatives (2) showing aqueous sodium hydroxide h y d r o l y s i s of the N,0-anthroyl d i - d e r i v a t i v e s to the mono-derivatives at (a) room temperature f o r 5 o minutes, (b) at 60 °C f o r 30 minutes, and (c) at 60 °C f o r 40 minutes. 160 Figure 36. HPLC chromatogram of unresolved p-hydroxy-mexiletine enantiomers as t h e i r N,0-anthroyl d i - d e r i v a t i v e s (1) . 161 Figure 37. HPLC chromatogram showing r e s o l u t i o n of R ( - ) - and S(+)-hydroxymethyl m e x i l e t i n e as t h e i r 0 - s i l y l - N - n a p h t h o y l d e r i v a t i v e s (1) and N-naphthoyl d e r i v a t i v e s ( 2 ) . 163 Figure 38. HPLC chromatogram showing r e s o l u t i o n of the N-anthroyl d e r i v a t i v e s of R ( - ) - and S(+)-mexiletine (1) , N-anthroyl d e r i v a t i v e of K0E-2963 ( 2 ) , 0-methyl-N-anthroyl d e r i v a t i v e s of R ( - ) - and S(+)-p-0H m e x i l e t i n e (3) and N-anthroyl d e r i v a t i v e s of R ( - ) - and S(+)-0H-methyl m e x i l e t i n e ( 4 ) . 165 Figure 39. HPLC chromatograms showing r e s o l u t i o n of (a) the 0 - e t h y l - N - a n t h r o y l d e r i v a t i v e s , (b) 0-propyl-N-anthroyl d e r i v a t i v e s and (c) 0 - b u t y l - N - a n t h r o y l d e r i v a t i v e s of R ( - ) - and S(+)-p-hydroxy-mexiletine (1) and R ( - ) - and S(+)-hydroxymethyl-mexiletine ( 2 ) . A l s o shown are the unresolved N-anthroyl d e r i v a t i v e s of hydroxymethyl m e x i l e t i n e enantiomers ( 3 ) . 166 Figure 40. P l o t s of the time-course of a l k y l a t i o n of R ( - ) - and S(+)-p-hydroxy-mexiletine using ethyl bromide at 60 °C over 60 minutes. 169 Figure 41. The ethyl acetate e x t r a c t i o n recovery of R(-)-p-hydroxy-m e x i l e t i n e and R(-)-hydroxymethyl-mexiletine as a p l o t of the chromatographic peak height r a t i o s versus e x t r a c t i o n pH. 171 Figure 42. The dichloromethane / ether (20/80) e x t r a c t i o n recovery of R ( - ) - m e x i l e t i n e , R ( - ) - p - h y d r o x y - m e x i l e t i n e and R ( - ) -hydroxymethyl-mexiletine as a p l o t of the chromatographic peak height r a t i o s versus e x t r a c t i o n pH. 172 Figure 43. A schematic representation of the assay procedure a l l o w i n g simultaneous determination of m e x i l e t i n e , p-Hydroxy-mexiletine and hydroxymethyl-mexiletine enantiomers in u r i n e . 173 Figure 44. Representative HPLC chromatograms of (a) blank urine and (b) urine from a subject showing the r e s o l u t i o n of the N-anthroyl d e r i v a t i v e s of R ( - ) - and S(+)-mexiletine (1) , the N-anthroyl d e r i v a t i v e of KOE-2963 ( 2 ) , the O-ethyl-N-anthroyl d e r i v a t i v e s of R ( - ) - and S(+)-p-hydroxy m e x i l e t i n e ( 3 ) , the 0 - e t h y l - N - a n t h r o y l d e r i v a t i v e s of tyramine ( 4 ) , the N-anthroyl d e r i v a t i v e of methyl amine ( 5 ) , and the N-anthroyl d e r i v a t i v e s of R ( - ) - and S(+)-hydroxymethyl m e x i l e t i n e ( 6 ) . 176 Figure 45. HPLC assay c a l i b r a t i o n curves f o r m e x i l e t i n e , p-hydroxy-m e x i l e t i n e and hydroxymethyl-mexiletine enantiomers in u r i n e . 177 Figure 46. Representative p l o t s of the cumulative amount of R ( - ) -and S(+)-p-hydroxy-mexiletine excreted over 72 hours in the urine from subject TK f o l l o w i n g o r a l a d m i n i s t r a t i o n of 200 mg of racemic m e x i l e t i n e h y d r o c h l o r i d e . 179 Figure 47. Representative p l o t s of the amount of R ( - ) - and S(+)-p-hydroxy-mexiletine remaining to be excreted over 72 hours in the urine from subject TK f o l l o w i n g o r a l a d m i n i s t r a t i o n of 200 mg of racemic m e x i l e t i n e h y d r o c h l o r i d e . 186 Figure 48. Representative p l o t s of the cumulative amount of R ( - ) -and S(+)-hydroxymethyl m e x i l e t i n e excreted over 72 hours in the urine from subject TK f o l l o w i n g o r a l a d m i n i s t r a t i o n of 200 mg of racemic m e x i l e t i n e h y d r o c h l o r i d e . 187 Figure 49. Representative p l o t s of the amount of R ( - ) - and S(+)-hydroxymethyl m e x i l e t i n e remaining to be excreted over 72 hours in the urine from subject TK f o l l o w i n g o r a l a d m i n i s t r a t i o n of 200 mg racemic m e x i l e t i n e h y d r o c h l o r i d e . 189 XX L I S T O F A B B R E V A T I O N S o r a p i d d i s p o s i t i o n rate constant of a 2-compartment model (h-1) ARE amount of drug remaining to be excreted (fig) AUC area under the plasma concentration-time curve (/zg/ml/h) AUTOAN a decision-making pharmacokinetic computer program 13 slow d i s p o s i t i o n r a t e constant of a 2-compartment model (h-1) Cmax maximum plasma concentration achieved f o l l o w i n g oral drug a d m i n i s t r a t i o n (ng/ml) °C degree C e l s i u s C l r renal clearance (ml/min) C l / f apparent o r a l body clearance (ml/min/kg) CSP c h i r a l s t a t i o n a r y phase C V . c o e f f i c i e n t of v a r i a t i o n Dg amount of drug in the body Ex e x c i t a t i o n Em emission F f r a c t i o n of dose a v a i l a b l e to the systemic c i r c u l a t i o n f s s a l i v a f r e e f r a c t i o n g g r a v i t y GC gas chromatography h hour HPLC high-performance l i q u i d chromatography k a f i r s t - o r d e r absorption r a t e constant ( h " l ) K|r terminal e l i m i n a t i o n r a t e constant of a one-compartment model ( h " l ) kf formation rate constant of a drug metabolite ( h _ 1 ) kg kilogram X X I k m u u r i n a r y e x c r e t i o n rate constant f o r a drug metabolite (h_i) k n r non-renal e x c r e t i o n rate constant ( h _ 1 ) L l i t e r M m o l a r i t y of s o l u t i o n MB amount of drug metabolite in the body Mex. m e x i l e t i n e min minute ml m i l l i l i t e r Hq microgram MS mass sprectrometery M+ molecular ion m/z ion mass to charge r a t i o ng nanogram NONLIN computer program f o r n o n - l i n e a r l e a s t square regression a n a l y s i s of pharmacokinetic data ODS o c t a d e c y l s i l a n e OH-methyl hydroxymethyl -OH hydroxy group pg picogram pH - l o g [H+] p-OH p-hydroxy pKa i o n i z a t i o n constant R chromatographic r e s o l u t i o n r c o r r e l a t i o n r^ c o e f f i c i e n t of determination ± S . D . ± one standard d e v i a t i o n ± S . E . ± one standard e r r o r t l / 2 h a l f - l i f e TLC t h i n - l a y e r chromatography t m a x time taken to reach maximum serum concentration f o l l o w i n g o r a l dose UV u l t r a v i o l e t V d a r e a apparent volume of d i s t r i b u t i o n c a l c u l a t e d from AUC (L) Xmm cumulative amount of drug metabolite excreted in urine Xu°° cumulative amount of drug excreted in urine ACKNOWLEDGEMENTS I would l i k e to s i n c e r e l y thank Dr. K e i t h M. McErlane f o r h i s encouragement and support throughout the course of my study. His constant guidance and a s s i s t a n c e in the preparation of t h i s t h e s i s i s much a p p r e c i a t e d . I would a l s o l i k e to thank the members of my research committee, Dr. Frank Abbott, Dr. James Axel son and Dr. John S i n c l a i r f o r t h e i r suggestions and a s s i s t a n c e during the course of my undergraduate and graduate s t u d i e s . The a s s i s t a n c e from Dr. Charles Kerr during the course of the pharmacokinetic study i s a l s o very much a p p r e c i a t e d . Special thanks to Mr. Michael Gentleman f o r h i s endless help on blood c o l l e c t i o n during the pharmacokinetic study. L a s t l y , s p e c i a l thanks to the three most i n f l u e n t i a l people in my l i f e , my parents f o r t h e i r support and encouragement, and Ms. Kathleen Cheng f o r her companionship and understanding during the course of my s t u d i e s . Her a s s i s t a n c e in the preparation of t h i s t h e s i s i s a l s o a p p r e c i a t e d . This p r o j e c t was made p o s s i b l e by grants received from the Medical Research Council of Canada and from Boehringer Ingelheim Canada L t d . 1 1. INTRODUCTION 1.1 Development of M e x i l e t i n e M e x i l e t i n e [l-(2' ,6'-dimethylphenoxy)-2-aminopropane] (Figure 1, I) was developed by Koppe (1977) with the aim of obtaining a non-a d d i t i v e a n o r e c t i c agent based on the s t r u c t u r e of phenmetrazine ( I I ) . The opening of the oxazine r i n g of ( I I ) with i n s e r t i o n of an oxygen between the phenyl and the a l k y l groups l e d to the preparation of 1-aryloxy-2-aminoalkane ( I I I ) . Analogs of s t r u c t u r e III can also be derived from the e l i m i n a t i o n of the -CHOH group from the 6-blocker analog, l-phenoxy-3-aminoisopropanol ( I V ) . V a r i a t i o n s of the s u b s t i t u t e n t s of ( I I I ) f i n a l l y l e d to the discovery of m e x i l e t i n e (US patent 3,659,019) which was found to possess anticonvulsant and antiarrhythmic a c t i v i t i e s . 1.2 Pharmacology 1 . 2 . 1 Mechanisms of A c t i o n M e x i l e t i n e i s a c l a s s l b antiarrhythmic drug. I t s antiarrhythmic actions are mediated through blockade of the f a s t sodium channel, r e s u l t i n g in depression of the maximum r a t e of d e p o l a r i z a t i o n as well as depression of the a t r i o v e n t r i c u l a r and v e n t r i c u l a r conduction v e l o c i t i e s . These e l e c t r o p h y s i o l o g i c a l p r o p e r t i e s r e s u l t in a prolonged e f f e c t i v e r e f r a c t o r y period and a shortened a c t i o n p o t e n t i a l (Singh and Vaughan-Williams, 1972; Vaughan-Wil1iams, 1977). In animal s t u d i e s , m e x i l e t i n e was found to a b o l i s h ouabain and coronary 1 i g a t i o n - i n d u c e d arrhythmias in dogs ( A l l e n et al., 1972). The antiarrhythmic a c t i v i t y of m e x i l e t i n e was a l s o demonstrated in guinea pigs and in anaesthetized r a t models (Singh and Vaughan-Will iams, 1972; Marshall et al., 1981). A d d i t i o n a l pharmacological p r o p e r t i e s of 2 I Figure 1. The development of mexiletine. m e x i l e t i n e include l o c a l anaesthetic and anticonvulsant actions (Singh and Vaughan-Williams, 1972; Danneberg and S h e l l y , 1977). The antiarrhythmic a c t i v i t i e s of the m e x i l e t i n e enantiomers, R ( - ) -m e x i l e t i n e and S ( + ) - m e x i l e t i n e , were studied using e l e c t r i c a l and o c c l u s i o n - i n d u c e d arrhythmia models in the r a t (Igwemezie, Ph.D T h e s i s , 1989). The r e s u l t s of these studies i n d i c a t e d that the a n t i a r r h y t h m i c , c a r d i o v a s c u l a r and n e u r o l o g i c a l a c t i v i t i e s of the R ( - ) - and S(+)-m e x i l e t i n e 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 . 1.2.2 Dosage and A d m i n i s t r a t i o n M e x i l e t i n e hydrochloride i s a v a i l a b l e from Boehringer Ingelheim as M e x i t i l R 100 and 200 mg c a p s u l e s . Paalman et al., (1977) and Bradbook et a / . , (1981) recommended an i n i t i a l o r a l dose of 200 mg three times d a i l y . This may be increased to a maximum of 1200 mg d a i l y in d i v i d e d doses. The authors cautioned that dosage adjustment should proceed in steps of 100 mg d a i l y , and a minimum of three days between each dosage increase may be required to reduce the incidence of adverse e f f e c t s in some p a t i e n t s . A d m i n i s t r a t i o n of m e x i l e t i n e with food was recommended to reduce the incidence of g a s t r o i n t e s t i n a l adverse e f f e c t s . For p a t i e n t s who r e q u i r e r a p i d c o n t r o l of v e n t r i c u l a r arrhythmia, a loading dose of 200 to 250 mg may be administered intravenously followed by a continuous i n f u s i o n of 60 to 90 mg/h (Paalman et al., 1977). To lower the incidence of g a s t r o i n t e s t i n a l and n e u r o l o g i c a l adverse e f f e c t s due to the i n i t i a l high drug plasma c o n c e n t r a t i o n s , the authors suggested that the loading dose should be administered by slow i n f u s i o n over a period of 30 minutes. 4 1 . 2 . 3 E l e c t r o p h y s i o l o g y The reported e l e c t r o p h y s i o l o g i c a l actions of m e x i l e t i n e on the heart are s i m i l a r to those reported f o r l i d o c a i n e and t o c a i n i d e , which are c o n s i s t e n t with the c l a s s i f i c a t i o n of c l a s s - l b agents according to Singh and Vaughan-Williams (1971). In humans, the e l e c t r o p h y s i o l o g i c a l p r o p e r t i e s of m e x i l e t i n e are v a r i a b l e . At therapeutic plasma concentrations of 0.75 to 2.0 /fg/ml, m e x i l e t i n e was found to lack c o n s i s t e n t e f f e c t s on sinus rhythm, s i n o a t r i a l r e f r a c t o r i n e s s or s i n o a t r i a l conduction (Roos et al., 1977; McComish et al., 1977). M e x i l e t i n e was a l s o found to increase the f u n c t i o n a l r e f r a c t o r y period of the AV-node with 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 period of the H i s - P u r k i n j e system; lengthening i t in some p a t i e n t s (Roos et al., 1977) or shortening i t i n others (McComish et al., 1977). In other s t u d i e s of p a t i e n t s with conduction d e f e c t s , m e x i l e t i n e lengthened H i s -P u r k i n j e conduction time and increased i t s r e f r a c t o r i n e s s ; w h i l e in p a t i e n t s with p r e - e x i s t i n g s i c k sinus syndrome, m e x i l e t i n e produced more pronounced depression of the sinus r a t e and/or prolongation of sinus node recovery time (Boehringer, 1986 and Rosen et al., 1972). In i s o l a t e d heart p r e p a r a t i o n s , m e x i l e t i n e was found to decrease conduction v e l o c i t y , depress the maximum r a t e of d e p o l a r i z a t i o n , prolong 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 a l t e r the duration of the c a r d i a c a c t i o n p o t e n t i a l with v a r i a b l e response (Vaughan-Williams, 1977). In r a b b i t a t r i a l and v e n t r i c u l a r myocardial p r e p a r a t i o n s , m e x i l e t i n e was found to slow conduction v e l o c i t y without s i g n i f i c a n t l y a f f e c t i n g the r e s t i n g membrane p o t e n t i a l or the a c t i o n p o t e n t i a l duration ( A l l e n et al., 1972; Singh and Vaughan-Williams, 1972). In i s o l a t e d P u r k i n j e f i b r e s from dogs, m e x i l e t i n e e x h i b i t e d a dose-dependent e f f e c t in 5 decreasing the duration of a c t i o n p o t e n t i a l which r e s u l t e d in shortening of the e f f e c t i v e r e f r a c t o r y period (Yamaguchi et al., 1979). These 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 were c o n s i s t e n t with the actions of m e x i l e t i n e in reducing the maximal r a t e of d e p o l a r i z a t i o n in 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 a c t i o n p o t e n t i a l s . 1 .2 .4 Hemodynamic Effects of Mexiletine Studies on the hemodynamic e f f e c t s of m e x i l e t i n e by Sami and Lisbona (1985) and Rutledge et a / . , (1985) i n d i c a t e d that in p a t i e n t s with normal or abnormal myocardial f u n c t i o n , m e x i l e t i n e had minor e f f e c t s on c a r d i a c output, pulmonary c a p i l l a r y wedge pressure, l e f t v e n t r i c u l a r e n d - d i a s t o l i c p r e s s u r e , pulmonary d i a s t o l i c p r e s s u r e , blood pressure and heart r a t e . However, a study of s i x c a r d i a c p a t i e n t s by Saunamaki (1975) reported evidence of impaired m y o - c o n t r a c t i l i t y in three p a t i e n t s f o l l o w i n g m e x i l e t i n e therapy. Other studies by Bigger (1984), Block and Winkle (1983), as w e l l as Mehta and Conti (1982) reported no s i g n i f i c a n t changes i n c a r d i a c output and l e f t v e n t r i c u l a r pressure; however, the authors cautioned the use of m e x i l e t i n e in p a t i e n t s with hypotension or congestive heart f a i l u r e . 1 .2 .5 Neurological A c t i v i t y of Mexiletine In an animal study, m e x i l e t i n e was c i t e d to possess dose-dependent anticonvulsant a c t i v i t y (unpublished d a t a , Boehringer Ingelheim L t d . ) , Chew et al. (1979). Following o r a l a d m i n i s t r a t i o n of m e x i l e t i n e to r a t s and mice, Chew et al. (1979) noted that m e x i l e t i n e e x h i b i t e d maximal anticonvulsant a c t i v i t y against electroshock and p e n t y l e n e t r a z o l e -induced c o n v u l s i o n s . Although the mechanisms of a c t i o n f o r the anticonvulsant a c t i v i t y of m e x i l e t i n e remain undetermined, i n v e s t i g a t i o n s reported by Danneberg and S h e l l e y (1977) e s t a b l i s h e d that 6 m e x i l e t i n e i n h i b i t e d the b r a i n re-uptake of gama-aminobutyric a c i d (GABA) in e l e c t r i c a l l y stimulated b r a i n s l i c e p r e p a r a t i o n s . Wilder et al. (1973) a l s o reported that the anticonvulsant a c t i v i t y of m e x i l e t i n e was e f f e c t i v e against psychomotor s e i z u r e s , but had no s i g n i f i c a n t e f f e c t on primary g e n e r a l i z e d s e i z u r e s . However, the successful use of m e x i l e t i n e c l i n i c a l l y as an anticonvulsant was l i m i t e d to a s i n g l e report of an i n f a n t with i n t r a c t a b l e e p i l e p s y unresponsive to phenytoin, v a l p r o i c a c i d , diazepam, clonazepam, gama-globulin and carbamazepine therapy (Kohyama et al., 1988). 1.2.6 Comparative Studies with other Antiarrhythmic Drugs The c l i n i c a l antiarrhythmic e f f i c a c y of m e x i l e t i n e has been demonstrated in p a t i e n t s with a v a r i e t y of acute v e n t r i c u l a r arrhythmias (Nimmo, 1977; Campbell et al., 1977; B r e i t h a r d t et al., 1981) as well as in p a t i e n t s with d i g i t a l i s - i n d u c e d arrhythmia and chronic recurrent v e n t r i c u l a r arrhythmias (Trimarco et al., 1983). The study reported by Trimarco et al. (1983) suggested that m e x i l e t i n e and disopyramide were comparable i n t h e i r antiarrhythmic e f f i c a c i e s . Fenster et al. (1983) i n d i c a t e d that m e x i l e t i n e was a l s o as e f f e c t i v e as q u i n i d i n e in reducing the frequency of v e n t r i c u l a r e c t o p i c beats in arrhythimic p a t i e n t s . In a d d i t i o n , Campbell (1975) reported that the use of procainamide and m e x i l e t i n e reduced the incidence of serious v e n t r i c u l a r arrhythmias i n the l a t e - p h a s e of myocardial i n f a r c t i o n . In m u l t i p l e drug therapy, m e x i l e t i n e has been used s u c c e s s f u l l y with p r o p r a n o l o l , q u i n i d i n e or other c l a s s l a antiarrhythmics such as disopyramide or procainamide f o r improved arrhythmic c o n t r o l with reduced incidence of side e f f e c t s ( B i g g e r , 1984; Duff et al., 1983; Greenspan et al., 1985). 7 1.2.7 Plasma Concentration-Antiarrhythmic Response Relationships The t h e r a p e u t i c plasma concentration of m e x i l e t i n e has been found to range from 0.7 to 2.0 /Kg/ml (Talbot et a 7 . , 1973). The authors reported t h a t , w i t h i n t h i s therapeutic plasma concentration range, a greater than 95% reduction of v e n t r i c u l a r e c t o p i c beats was observed in 37 p a t i e n t s . In another study of 149 p a t i e n t s by Campbell et al. (1978), i t was observed that 77% of premature v e n t r i c u l a r c o n t r a c t i o n s were suppressed when p a t i e n t plasma l e v e l s were maintained between 0.7 and 1.0 [ig/m\. In the l a t t e r two s t u d i e s , the incidence of adverse e f f e c t s were found to be c o n c e n t r a t i o n - r e l a t e d with 30% of the p a t i e n t s experiencing adverse e f f e c t s when plasma concentration exceeded 2.0 /ig/ml. 1.2.8 Drug Interactions with Mexiletine M e x i l e t i n e has been used c l i n i c a l l y with d i g o x i n (Sami and Lisbona, 1985), warafin and propranolol (Bigger, 1984) and various d i u r e t i c s (Akhtar, 1984) without evidence of serious untoward e f f e c t s . Since the metabolic clearance of m e x i l e t i n e i s a major component of i t s t o t a l body c l e a r a n c e , the use of hepatic enzyme inducers such as phenytoin, r i f a m p i c i n and phenobarbital have been shown to increase the non-renal clearance and decrease the e l i m i n a t i o n h a l f - l i f e of m e x i l e t i n e in healthy volunteers (Beff et al., 1982; Pentikainen et al., 1982). The authors suggested that metabolic induction of the hepatic mixed-f u n c t i o n oxidase enzymes could be i n v o l v e d , and blood l e v e l monitoring and dosage adjustments were recommended f o r p a t i e n t s during concomitant therapy with known metabolic enzyme inducers. Conversely, the hepatic enzyme i n h i b i t o r , c i m e t i d i n e , was reported to have no s i g n i f i c a n t e f f e c t on the k i n e t i c s of m e x i l e t i n e f o l l o w i n g o r a l or intravenous 8 a d m i n i s t r a t i o n in healthy subjects ( K l e i n et al., 1985; Brockmeyer et al., 1989). The e f f e c t s of metoclopramide on the o r a l absorption of m e x i l e t i n e were reported to cause an increase in the o r a l absorption rates and an increase in the plasma concentrations of m e x i l e t i n e (Wing et al., 1980). In a study with healthy subjects and with p a t i e n t s on long-term m e x i l e t i n e therapy, m e x i l e t i n e was reported to i n h i b i t the e l i m i n a t i o n of c a f f e i n e by about 50% (Joeres et al., 1987). However, during t h i s study, the clearance of m e x i l e t i n e remained unchanged. The authors suggested that the adverse n e u r o l o g i c a l e f f e c t s associated with m e x i l e t i n e therapy could be a t t r i b u t e d p a r t l y to the CNS e f f e c t s of c a f f e i n e . In another report (Katz et al., 1987), t h e o p h y l l i n e serum l e v e l s were found to be elevated from 15.3 to 25.0 fig/m\ when m e x i l e t i n e therapy was i n i t i a t e d during s t e a d y - s t a t e t h e o p h y l l i n e therapy. Uponlii withdrawal of m e x i l e t i n e , t h e o p h y l l i n e serum concentrations returned to 14.2 [iq/m\. The authors suggested that m e x i l e t i n e may have been r e s p o n s i b l e f o r the impairment of hepatic metabolism of t h e o p h y l l i n e , and t h e o p h y l l i n e dosage may need to be reduced during concomitant use with m e x i l e t i n e . The e f f e c t of c i g a r e t t e smoking on the k i n e t i c s of m e x i l e t i n e was studied in healthy subjects a f t e r o r a l a d m i n i s t r a t i o n of m e x i l e t i n e (Grech-Belanger et al., 1985b). The authors reported that c i g a r e t t e smoking d i d not a f f e c t the absorption or d i s t r i b u t i o n of m e x i l e t i n e , but smoking s i g n i f i c a n t l y reduced the e l i m i n a t i o n h a l f - l i f e of m e x i l e t i n e from 11.1 to 7.2 hours. From the same r e p o r t , studies on the m e x i l e t i n e metabolites in urine i n d i c a t e d that c i g a r e t t e smoking induced g l u c u r o n i c a c i d conjugation of m e x i l e t i n e and a l i p h a t i c hydroxylation of m e x i l e t i n e 9 to OH-methyl m e x i l e t i n e ; however, p-hydroxylation of m e x i l e t i n e remained u n a f f e c t e d . 1 . 2 . 9 Toxicology Adverse c a r d i o v a s c u l a r e f f e c t s that have been reported f o r m e x i l e t i n e include worsening of congestive heart f a i l u r e (<3%), proarrhythmia (10%) and heart block (1%) (Cetnarowski and R i h n , 1985) Non-cardiovascular side e f f e c t s have been observed i n 47% to 65% of p a t i e n t s (Cetnarowski and R i h n , 1985). Neurological and 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 are the most common and i n c l u d e : hand tremor, 1ight-headedness, d i z z i n e s s , a t a x i a , p a r e s t h e s i a s , throat numbness, b l u r r e d v i s i o n , nervous a g i t a t i o n , nausea, v o m i t i n g , a n o r e x i a , and heartburn. Less frequent adverse e f f e c t s (1%) include macular erythematous r a s h , abnormal l i v e r f u n c t i o n , lupus syndrome, thrombocytopenia and impotence. 1.3 Pharmacokinetics i n Humans 1 .3.1 Oral Absorption and B i o a v a i l a b i l i t y In healthy v o l u n t e e r s , o r a l absorption of m e x i l e t i n e was reported to be r a p i d with peak plasma concentrations ( C m a x ) a t t a i n e d in two to four hours ( P r e s c o t t et a 7 . , 1977; Campbell et al., 1978; Haselbarth et al., 1981; Chew et al., 1979) f o l l o w i n g adminstration of the drug in e i t h e r s o l u t i o n or in capsule f o r m u l a t i o n . The o r a l absorption rates of the m e x i l e t i n e enantiomers in f i v e healthy subjects were found to be s i m i l a r with absorption h a l f - l i v e s of 23.7 and 21.3 minutes 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 (Igwemezie et al., 1989). The time to achieve peak plasma l e v e l ( t m a x ) , peak plasma c o n c e n t r a t i o n , ( C m a x ) , and area under the serum concentration time p r o f i l e ( A U C 0 _ < ° ) were also found to be s i m i l a r between the two m e x i l e t i n e enantiomers (Igwemwzie et al., 1989). Contrary to these f i n d i n g s , the mean AUC0"00 of S(+)-mexiletine was found to be s i g n i f i c a n t l y greater (p<0.01) than that f o r R ( - ) - m e x i l e t i n e in s i x healthy subjects (Grech-Belanger et al., 1986). The f r a c t i o n of an o r a l dose of m e x i l e t i n e a v a i l a b l e to systemic c i r c u l a t i o n (F) was reported to range from 0 . 8 to 0.9 (Prescott et al., 1977; Campbell et al., 1978; Haselbarth et al., 1981). However, the extent of systemic a v a i l a b i l i t y of the i n d i v i d u a l m e x i l e t i n e enantiomer has not been r e p o r t e d . 1.3.2 Tissue D i s t r i b u t i o n Following intravenous a d m i n i s t r a t i o n of m e x i l e t i n e to healthy s u b j e c t s , m e x i l e t i n e was found to d i s t r i b u t e r a p i d l y and e x t e n s i v e l y to p e r i p h e r a l t i s s u e s with l e s s than 1% of the t o t a l drug remaining in the systemic compartment ( P r e s c o t t et al., 1977). The extensive d i s t r i b u t i o n of m e x i l e t i n e i n t o t i s s u e s i t e s was a l s o r e f l e c t e d by the l a r g e and v a r i a b l e t o t a l volume of d i s t r i b u t i o n (V^), ranging from 5.5 to 9 . 5 1/kg ( P r e s c o t t et al., 1977; Haselbarth et a7., 1981; Campbell et a7., 1978). Studies on the d i s t r i b u t i o n k i n e t i c s of m e x i l e t i n e enantiomers in healthy subjects by Igwemezie et a7. (1989) and by Grech-Bel anger et a7. (1986) found no s i g n i f i c a n t d i f f e r e n c e s in the r a p i d d i s p o s i t i o n h a l f - l i v e s ( t j / 2 a) between R ( - ) - and S ( + ) - m e x i l e t i n e . 1.3.3 E l i m i n a t i o n The d i s p o s i t i o n of m e x i l e t i n e in healthy subjects f o l l o w i n g o r a l adminstration was described e i t h e r by b i - e x p o n e n t i a l or t r i - e x p o n e n t i a l f u n c t i o n s with terminal e l i m i n a t i o n h a l f - l i v e s ranging from 6.2 to 11.8 hours ( P r e s c o t t et al., 1977; Campbell et a7., 1978; Haselbarth et a7., 1981; Igwemezie et a7 . , 1989). The t o t a l body clearance (CLj) of 11 racemic m e x i l e t i n e in healthy subjects was found to range from 6.1 to 11.1 ml/min/kg with a mean renal clearance of 0.6 ml/min/kg at p h y s i o l o g i c a l urine pH (Haselbarth et al., 1981; M i t c h e l l et al., 1983). A study of the k i n e t i c s of m e x i l e t i n e enantiomers in f i v e healthy subjects by Igwemezie et a / . (1989) indicated that the mean t o t a l body clearance values f o r the m e x i l e t i n e enantiomers were s i m i l a r . However, s i g n i f i c a n t d i f f e r e n c e s were observed in the e l i m i n a t i o n h a l f - l i v e s , volumes of d i s t r i b u t i o n , renal c l e a r a n c e s , and in the amount of i n t a c t drug excreted in urine of the enantiomers. Within these pharmacokinetic parameters, the values observed f o r S(+)-mexiletine were s i g n i f i c a n t l y greater than those f o r R ( - ) - m e x i l e t i n e . In contrast to these f i n d i n g s , a study on the k i n e t i c s of m e x i l e t i n e enantiomers in s i x healthy subjects by Grech-Belanger et a / . (1986) reported that the mean e l i m i n a t i o n h a l f - l i v e s f o r 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 . 1.3.4 Effects of Urinary pH on Excretion of Mexiletine The u r i n a r y e x c r e t i o n of m e x i l e t i n e was reported to vary with urine pH. In c o n t r o l l e d a c i d i c urine (pH 5 ) , the mean e l i m i n a t i o n h a l f -l i f e of m e x i l e t i n e in four subjects was found to be 2.8 ± 0 . 3 hours with 58 ± 7% of an intravenous dose excreted unchanged in the u r i n e over 72 hours. In c o n t r o l l e d a l k a l i n e urine (pH 8 ) , the mean e l i m i n a t i o n h a l f -l i f e of m e x i l e t i n e was found to be s i g n i f i c a n t l y (p<0.01) increased to 8 . 6 ± 0 . 1 hours with 0.6 ± 0.1% of an intravenous dose excreted unchanged in the urine over 72 hours (Kiddie et al., 1974). Other i n v e s t i g a t o r s have a l s o reported s i m i l a r e f f e c t s of urine pH on the e l i m i n a t i o n of m e x i l e t i n e ( M i t c h e l l et al., 1985; Kaye et al., 1977). Furthermore, Johnston et al. (1979) suggested that the s t e a d y - s t a t e 12 plasma concentration of m e x i l e t i n e can f l u c t u a t e by more than 50% as a r e s u l t of spontaneous p h y s i o l o g i c a l changes of urine pH. However, due to the l a r g e i n t e r - s u b j e c t v a r i a t i o n s in d i s t r i b u t i o n volume and the v a r i a b l e degree of compensation by non-renal c l e a r a n c e , the e f f e c t s of p h y s i o l o g i c a l changes in urine pH on steady-state m e x i l e t i n e plasma l e v e l s can not be c a u s a l l y g e n e r a l i z e d . 1.3.5 Serum Protein Binding M e x i l e t i n e i s 70% bound to serum p r o t e i n s in healthy subjects (Talbot et a / . , 1973). An i n i t i a l study on the in vitro serum p r o t e i n binding of m e x i l e t i n e enantiomers at uncontrolled serum pH reported a s i g n i f i c a n t s t e r e o s e l e c t i v e binding of R ( - ) - m e x i l e t i n e ( f r e e f r a c t i o n of 19.8 ± 1.5%) i n respect to S(+)-mexiletine (free f r a c t i o n of 28.3 + 1.5%) over the t h e r a p e u t i c concentration range of 0.2 to 2.0 /jg/ml (McErlane et al., 1987). Binding s t u d i e s of m e x i l e t i n e enantiomers using p u r i f i e d 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 i - a c i d g l y c o p r o t e i n p r o t e i n revealed that the serum binding of m e x i l e t i n e could be accounted f o r mainly by binding to albumin and a j - a c i d g l y c o p r o t e i n (Igwemezie Ph.D. T h e s i s , 1989). 1.3.6 Pharmacokinetics of Mexiletine in Disease States A l l a f et al. (1982) reported the influence of renal i n s u f f i c i e n c y on the plasma pharmacokinetics of m e x i l e t i n e in 15 p a t i e n t s . At steady-s t a t e , the plasma concentrations and the e l i m i n a t i o n h a l f - l i v e s of m e x i l e t i n e in p a t i e n t s with c r e a t i n i n e clearances between 10 to 30 ml/min were found to be i n s i g n i f i c a n t l y d i f f e r e n t from those of a c o n t r o l group of nine p a t i e n t s with normal renal and l i v e r f u n c t i o n s . However, m e x i l e t i n e plasma concentrations and e l i m i n a t i o n h a l f - l i v e s were s i g n i f i c a n t l y increased in p a t i e n t s with c r e a t i n i n e clearances l e s s than 10 ml/min. The study concluded that the s t e a d y - s t a t e k i n e t i c s of m e x i l e t i n e are not l i k e l y to be modified in renal i n s u f f i c i e n t p a t i e n t s with c r e a t i n i n e clearances greater than 10 ml/min. In a s i n g l e dose study of m e x i l e t i n e in 14 renal f a i l u r e p a t i e n t s , Wang et a / . (1985) reported that there was no c o r r e l a t i o n between the e l i m i n a t i o n h a l f - l i v e s and the c r e a t i n i n e clearances of m e x i l e t i n e among the p a t i e n t s . Furthermore, in f i v e renal f a i l u r e p a t i e n t s who required heamodialysis, no s i g n i f i c a n t d i f f e r e n c e s in the AUC0 - 0 0 values f o r m e x i l e t i n e were observed between p a t i e n t s who r e c e i v e d , or who had not r e c e i v e d , heamodialysis. The e f f e c t s of v e n t r i c u l a r i n s u f f i c i e n c y on the s t e a d y - s t a t e k i n e t i c s of m e x i l e t i n e was studied by Leahy (1980). The study reported that the mean u r i n a r y recovery of m e x i l e t i n e at urine pH 4 . 5 to 6.0 was 8 . 3 ± 5.1%, which was not s i g n i f i c a n t l y d i f f e r e n t from that observed in normal s u b j e c t s . However, the mean terminal e l i m i n a t i o n h a l f - l i f e of 15.4 + 5.8 h f o r m e x i l e t i n e i n p a t i e n t s with v e n t r i c u l a r i n s u f f i c i e n c y was s i g n i f i c a n t l y higher than the mean h a l f - l i f e of 8.1 ± 1.8 h in normal s u b j e c t s . Leahy (1980) concluded that the prolongation of the e l i m i n a t i o n h a l f - l i f e of m e x i l e t i n e in p a t i e n t s with v e n t r i c u l a r i n s u f f i c i e n c y may allow e f f e c t i v e antiarrhythmic c o n t r o l i n these p a t i e n t s using twice d a i l y dosing i n t e r v a l s . The e f f e c t of an acute myocardial i n f a r c t i o n on the k i n e t i c s of m e x i l e t i n e was studied and was found to increase the e l i m i n a t i o n h a l f - l i f e of m e x i l e t i n e . In a d d i t i o n , the o r a l absorption of m e x i l e t i n e in p a t i e n t s s u f f e r i n g from an acute myocardial i n f a r c t i o n was found to be incomplete and delayed. However, the mechanisms c o n t r i b u t i n g to the delayed o r a l absorption observed in these p a t i e n t s remained uncertain (Pottage et al., 1978; Prescott et al., 1977; Pentikainen et al., 1984). 1 . 3 . 7 Pharmacokinetics of M e x i l e t i n e i n the E l d e r l y The e f f e c t of age on the k i n e t i c s of m e x i l e t i n e has been reported (Grech-Belanger et al., 1989). Following a s i n g l e oral dose of m e x i l e t i n e to seven healthy volunteers (65 to 79 y e a r s ) , the o r a l absorption rates of m e x i l e t i n e were found to be slower than those determined from a group of eight healthy young subjects (19 to 37 y e a r s ) . However, the terminal e l i m i n a t i o n h a l f - l i v e s , o r a l clearances and the amounts of unchanged drug recovered in urine in 48 hours were found not to be s i g n i f i c a n t l y d i f f e r e n t between the two groups. The authors concluded that a g e - r e l a t e d a l t e r a t i o n s in the k i n e t i c s of m e x i l e t i n e would not l i k e l y be c l i n i c a l l y important. 1.4 Metabolism 1.4 .1 Human Studies M e x i l e t i n e i s e x t e n s i v e l y metabolized in humans with mininal f i r s t - p a s s metabolism (Chew et al., 1979). Approximately 9% of an o r a l dose i s recovered i n the urine over 72 hours (Prescott et a / . , 1977; Campbell et al., 1978). In humans, the metabolic transformations of m e x i l e t i n e c o n s i s t of o x i d a t i v e and r e d u c t i v e pathways which give r i s e to nine metabolites (Beckett and Chidomere, 1977). A summary o u t l i n e of the metabolites of m e x i l e t i n e reported from human studies i s shown in Figure 2. The major metabolites include the p-hydroxy-mexiletine (p-OH-m e x i l e t i n e , V ) , hydroxymethyl-mexiletine (OH-methyl- m e x i l e t i n e , V I ) , and t h e i r r e s p e c t i v e deamination alcohol metabolites (VII) and ( V I I I ) , which account f o r approximately 20% of an o r a l dose (Beckett and 15 (H) 0 - CH 9 — C — R. CH, R2 R3 IV H H NH2 mexiletine V H OH NH2 1- (4'-hydroxy-2',6'-dimethylphenoxy-2- aminopropane VI OH H NH2 1-(2'-hydroxymethyl-6'-methyl)phenoxy aminopropane VII H OH OH 1-(4'-hydroxy-2',6'-dimethyl)phenoxy-propan-2-ol VIII OH H OH 1—(2'-hydroxymethyl-6'methyl)phenoxy-propan-2-ol IX H H N H C H 3 N-methy1-mexiletine X H H =0 1-(2',6'-dimethyl)phenoxy-2-one XI H H OH 1-(2',6'-dimethyl)phenoxy-2-ol XII H H NHOH N-hydroxy-mexiletine XIII H H =NOH 1-(2',6'-dimethyl)phenoxypropan-2-one oxime Figure 2. The metabolic d i s p o s i t i o n of m e x i l e t i n e in man. Chidomere, 1977;). Among these m e t a b o l i t e s , p-OH-mexiletine and i t s corresponding alcohol metabolite were suggested by Beckett et al. (1977) to undergo f u r t h e r phase-II metabolic c o n j u g a t i o n . However, the r e l a t i v e percentage of each of the f r e e and conjugated metabolites of m e x i l e t i n e have not been r e p o r t e d . In a d d i t i o n to the suggestion of f u r t h e r metabolic conjugation of o x i d a t i v e m e t a b o l i t e s , 14% of an o r a l dose of m e x i l e t i n e was recovered in the urine as a g l u c u r o n i c a c i d conjugate (Grech-Belanger et al., 1986). The u r i n a r y e x c r e t i o n of m e x i l e t i n e as i t s g l u c u r o n i c a c i d conjugate c o n s i s t e d mainly of the R ( - ) - c o n f i g u r a t i o n with a mean R(-)/S(+) enantiomeric r a t i o of 9.7 over 48 hours (Grech-Belanger et al., 1986). From the same study, the mean R(-)/S(+) AUC0 - 0 5 r a t i o f o r the m e x i l e t i n e conjugate was found t o be 2 . 9 . Although the R ( - ) -m e x i l e t i n e glucuronide conjugate concentrations i n serum and in urine were p e r s i s t e n t l y higher than those f o r S ( + ) - m e x i l e t i n e , the d i f f e r e n c e between the mean urine and mean AUC0_CO r a t i o s suggested that more than one s t e r e o s e l e c t i v e pathway may be involved in the d i s p o s i t i o n of the m e x i l e t i n e glucuronide conjugates. Pharmacological studies on the antiarrhythmic and n e u r o l o g i c a l a c t i v i t i e s of the m e x i l e t i n e metabolites have not been a v a i l a b l e in the 1 i t e r a t u r e . 1.4.2 Animal Studies From in vitro metabolic studies of m e x i l e t i n e in r a b b i t l i v e r homogenate p r e p a r a t i o n s , m-OH-mexiletine and 2 , 6 - d i m e t h y l p h e n o l , have been i d e n t i f i e d as metabolites of m e x i l e t i n e (Grech-Belanger and Turgeon, 1987; Grech-Belanger et al., 1988). From the same s t u d i e s , the authors observed the s t e r e o s e l e c t i v e formation of N-OH-mexiletine, m-OH-17 m e x i l e t i n e , p-OH-mexiletine, and OH-methyl-mexiletine. Among these m e x i l e t i n e m e t a b o l i t e s , except f o r OH-methyl-mexiletine, the S(+)-enantiomer of the metabolites were found in higher concentrations than those of the R(-)-enantiomer. The authors a l s o observed the formation of greater amounts of the R(-)-enantiomer of OH-methyl-mexiletine. 1.5 A n a l y s i s of Racemic M e x i l e t i n e In order to obtain adequate s e n s i t i v i t y f o r therapeutic monitoring of nanogram l e v e l s of m e x i l e t i n e , most gas chromatographic (GC) methods reported f o r m e x i l e t i n e required the formation of halogenated d e r i v a t i v e s followed by e l e c t r o n - c a p t u r e d e t e c t i o n . A simple and s e n s i t i v e GC method f o r m e x i l e t i n e was reported by W i l l c o x and Singh (1976). The method allowed d e t e c t i o n of picogram amounts of the h e p t a f l u o r o b u t y r i c anhydride d e r i v a t i v e of m e x i l e t i n e with an e l e c t r o n -capture d e t e c t o r f o l l o w i n g separation on a g l a s s column packed with 1% cyclohexane dimethanol succinate on 100-200 mesh Gas-Chrom Q. Other GC methods f o r m e x i l e t i n e involved formation of the butylamide d e r i v a t i v e of m e x i l e t i n e followed by d e t e c t i o n with a nitrogen-phosphous detector ( K e l l y et a 7 . , 1973), as well as formation of the pentafluoropropion-amide or t r i f l u o r o a c e t a m i d e d e r i v a t i v e s of m e x i l e t i n e followed by d e t e c t i o n with an e l e c t r o n - c a p t u r e detector ( P e r c h a l s k i et al., 1974). Although most GC methods reported f o r m e x i l e t i n e required pre-column d e r i v a t i z a t i o n , Smith and M e f f i n (1980) reported the d e t e c t i o n of u n d e r i v a t i z e d m e x i l e t i n e at 5 ng/ml in plasma with a nitrogen-phosphous d e t e c t o r . High-performance l i q u i d chromatographic methods, with e i t h e r forward-phase or reverse-phase HPLC columns, have been developed f o r the q u a n t i t a t i o n of m e x i l e t i n e . Using a forward-phase s i l i c a column, Bhamra et al. (1984) reported an HPLC method with fluorescence d e t e c t i o n of u n d e r i v a t i z e d m e x i l e t i n e at 50 ng/ml in plasma. The use of a reverse-phase C-18 column was reported by Grech-Belanger and Turgeon (1984) as w e l l as by K e l l y et a / . (1981). The method reported by Grech-Belanger and Turgeon involved formation of the fluorescamine d e r i v a t i v e of m e x i l e t i n e followed by fluorescence d e t e c t i o n ; whereas the method developed by K e l l y et al. involved u l t r a v i o l e t d e t e c t i o n of u n d e r i v a t i z e d m e x i l e t i n e . 1.6 S t e r e o s e l e c t i v e A n a l y s i s of M e x i l e t i n e Enantiomers Two high-performance l i q u i d chromatographic (HPLC) methods have been reported f o r the a n a l y s i s of m e x i l e t i n e enantiomers (Grech-Belanger et al., 1985a; McErlane and Igwemezie, 1987b). In one method, m e x i l e t i n e enantiomers were reacted with a c h i r a l reagent, 2 , 3 , 4 , 6 -t e t r a - O - a c e t y l - 6 - D - g l u c o p y r a n o s y l isothiocyanate (GITC), to produce two diastereoisomers which were separated by reverse-phase HPLC (Grech-Belanger et al., 1985a). The d e t e c t i o n of the mexiletine-GITC d e r i v a t i v e at 250 nm o f f e r e d a minimum assay l i m i t of 50 ng/ml in plasma. The method by McErlane and Igwemezie (1987b) involved a c h i r a l P i r k l e R phenylglycine i o n i c HPLC column f o r the d i r e c t r e s o l u t i o n of the enantiomers as t h e i r N-naphthoyl d e r i v a t i v e s . The method involved d e r i v a t i z a t i o n of the enantiomers using 2-naphthoyl c h l o r i d e followed by fluorescence d e t e c t i o n ( e x c i t a t i o n / e m i s s i o n 230/340 nm). The minimum assay l i m i t was found to be 5 ng/ml in plasma f o r each of the enantiomers with a s i g n a l - t o - n o i s e r a t i o of 5/1. 1.7 S t e r e o s e l e c t i v e Drug A n a l y s i s 1.7.1 R e s o l u t i o n of Enantiomers as Diastereoisomers Since enantiomers are non-superimposable isomers of i d e n t i c a l chemical and p h y s i c a l p r o p e r t i e s , chromatographic r e s o l u t i o n of enantiomers as t h e i r diastereoisomer d e r i v a t i v e s can be accomplished by d e r i v a t i z a t i o n with an e n a n t i o m e r i c a l l y pure reagent. D e r i v a t i z a t i o n of enantiomers with an appropriate enantiomeric reagent, leading to the formation of two diastereoisomers of d i f f e r e n t chemical and p h y s i c a l p r o p e r t i e s , w i l l f a c i l i t a t e r e s o l u t i o n of the enantiomers by conventional chromatographic techniques. The choice of a c h i r a l d e r i v a t i z a t i o n reagent f o r the r e s o l u t i o n of enantiomers often r e q u i r e s c o n s i d e r a t i o n of the nature of the f u n c t i o n a l groups a v a i l a b l e on the enantiomer f o r d e r i v a t i z a t i o n , the d i f f e r e n c e i n d e r i v a t i z a t i o n rates between the enantiomers, the chemical s t a b i l i t y of the d i a s t e r e o i s o m e r s , the inversion of c h i r a l i t y during d e r i v a t i z a t i o n , and the a v a i l a b i l i t y of e n a n t i o m e r i c a l l y pure reagents (Konig et al., 1977; Frank et al., 1978; L i u and Ku, 1983). However, d e s p i t e these numerous c o n s i d e r a t i o n s , the u t i l i z a t i o n of c h i r a l d e r i v a t i z a t i o n reagents f o r r e s o l u t i o n of enantiomers has been f r e q u e n t l y r e p o r t e d . The GC r e s o l u t i o n of t o c a i n i d e enantiomers as diastereoisomers was acheived with S(-)-2-methoxy-2-(trif1uoromethyl)phenylacetyl c h l o r i d e (S-MTPA) (Gal et al., 1982). Amphetamine and biogenic amines were resolved by GC as d e r i v a t i v e s of N - t r i f l u o r o a c e t y l - S - p r o l y l c h l o r i d e (Westley et al., 1968; W e l l s , 1970). Other c h i r a l reagents used s u c c e s s f u l l y f o r GC a p p l i c a t i o n s included the use of S ( - ) - N -pentafluorobenzoyl p r o l y l - a - i m i d a z o l i d i d e (PFBP) f o r the metabolic 20 i n v e s t i g a t i o n of naltrexone ( C h a t t e r j i e et al., 1974) and the use of S-MTPA f o r the q u a n t i t a t i o n of amphetamine and t o c a i n i d e enantiomers (Gal et al., 1977). GC r e s o l u t i o n of c h i r a l 2 - a r y l a l k a n o i c acids such as ibuprofen, ketoprofen, naproxen, fenoprofen, f l u r b i p r o f e n , p i r p r o f e n , c i c l o p r o f e n , t r i p r o f e n i c a c i d and e t o d o l i c a c i d were achieved f o l l o w i n g d e r i v a t i z a t i o n with (+)- or (-)-amphetamine (Singh et al., 1986) The use of c h i r a l reagents f o r the HPLC r e s o l u t i o n of enantiomers has f r e q u e n t l y been r e p o r t e d . For a c i d i c drugs, a r y l p r o p i o n i c a c i d enantiomers and several n o n - s t e r o i d a l anti-inflammatory analogs were resolved as d i a s t e r e o i s o m e r i c d e r i v a t i v e s with S ( - ) - l - p h e n y l e t h y l a m i n e (Maitre et al., 1984). S(+)-2-0ctanol (Johnson et a / . , 1979; Lee et al., 1984) and S(+)-naproxen ( J a m i l i et al., 1989) were used s u c c e s s f u l l y f o r the r e s o l u t i o n of ibuprofen enantiomers. For b a s i c drugs, S ( - ) - l - p h e n y l e t h y l i s o c y a n a t e was used f o r the r e s o l u t i o n of propranolol enantiomers as t h e i r urea d e r i v a t i v e s (Thompson et al., 1982). Many examples of r e s o l u t i o n of amino a c i d s , biogenic amines and catecholamines were a l s o reported with the use of R(+)-1-phenyl ethyl -isocyanate (PEIC), ( - ) - 2 - m e t h o x y - 2 - ( t r i f l u o r o m e t h y l ) phenylacetyl c h l o r i d e (MTPA), 2 , 3 , 4 , 6 - t e t r a - 0 - a c e t y l - 8 - D - g l u c o p y r a n o s y l isothiocyanate (GITC) and 2 , 3 , 4 - t r i - 0 - a c e t y l - a - D - a r a b i n o p y r a n o s y l isothiocyanate (AITC) ( M i l l e r et al., 1984; Nimura et al., 1981; Nimura et al., 1980). Extensive reviews on the use of c h i r a l reagents f o r r e s o l u t i o n of drug enantiomers are a v a i l a b l e in the l i t e r a t u r e (Lochmuler and Souter, 1975; Tamegai et al., 1979; Lindna, 1982 and 1987). 21 1 .7 .2 D i r e c t Separation Using C h i r a l S t a t i o n a r y Phases The d i r e c t r e s o l u t i o n of enantiomers on a c h i r a l s t a t i o n a r y phase i s mediated by the formation of 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 between the enantiomer and the c h i r a l s t a t i o n a r y phase through n-n e l e c t r o n bonding, hydrogen bonding, as well as e l e c t r o s t a t i c and s t e r i c i n t e r a c t i o n s . A t h r e e - p o i n t i n t e r a c t i o n between the enantiomers and the c h i r a l s t a t i o n a r y phase r e s u l t s i n the formation of a p a i r of t r a n s i e n t diastereoisomers with d i f f e r e n t s t a b i l i t i e s . The enantiomer which r e s u l t s i n 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 with the c h i r a l s t a t i o n a r y phase i s p r e f e r e n t i a l l y r e t a i n e d on the chromatographic column r e l a t i v e to the l e s s s t a b l e d i a s t e r e o i s o m e r i c complex. 1 . 7 . 2 . 1 Gas Chromatography C h i r a l s t a t i o n a r y phases f o r GC a p p l i c a t i o n s can be grouped into diamide, p e p t i d e , and ureide phases. The diamide c h i r a l phase, N - t e r t - b u t y l - L - v a l i n a m i d e p o l y s i l o x a n e ( C h i r a s i l - V a l R ) was the f i r s t commercially a v a i l a b l e c h i r a l s t a t i o n a r y phase f o r GC a p p l i c a t i o n s . This peptide phase was developed by Frank et al. (1978) and was found to r e s o l v e a wide range of amino a c i d enantiomers as t h e i r t r i f l u o r o a c e t a t e (TFA) or heptafluorobutyrate (HFBA) d e r i v a t i v e s . The r e s o l u t i o n of enantiomers on the diamide GC l i q u i d phase was postulated to be due to the formation of 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 i n v o l v i n g hydrogen-bonding and s t e r i c i n t e r a c t i o n s between the enantiomer and the s t a t i o n a r y phase. A p p l i c a t i o n s of t h i s c h i r a l s t a t i o n a r y phase f o r the r e s o l u t i o n of t o c a i n i d e enantiomers was f i r s t reported by McErlane and P i l l a i (1983). The peptide phases were derived from the N - t r i f l u o r o a c e t y l (TFA) d e r i v a t i v e of amino a c i d e s t e r s . This c h i r a l s t a t i o n a r y phase was used mainly f o r the r e s o l u t i o n of amino acids as t h e i r N-TFA d e r i v a t i v e s ( L i u and Ku, 1983). The ureide c h i r a l s t a t i o n a r y phases were g e n e r a l l y derived from carbonyl bis-(amino a c i d e s t e r s ) d e r i v a t i v e s which were mainly used f o r the r e s o l u t i o n of enantiomeric amine d e r i v a t i v e s (Lochmuller et a 7 . , 1972). 1 . 7 . 2 . 2 High Performance L i q u i d Chromatography Over 35 d i f f e r e n t HPLC c h i r a l s t a t i o n a r y phases are a v a i l a b l e commercially. The more f r e q u e n t l y u t i l i z e d c h i r a l s t a t i o n a r y phases include the c y c l o d e x t r i n phases (Armstrong et al., 1984; Armstrong et al., 1986; Han and Armstrong, 1989), the c e l l u l o s e t r i a c e t a t e phases (Hesse and Hegel, 1976), the p r o t e i n bound phases (Allenmark et al., 1983; Hermansson, 1983), and the P i r k l e R amino a c i d phases ( P i r k l e and Welch, 1984; P i r k l e et a 7 . , 1984; Wainer et a 7 . , 1984). Other novel c h i r a l s t a t i o n a r y phases include h e l i c a l polymer phases (Okamoto and Hatada, 1989) and 1igand-exchange phases (Davankov, 1989). Extensive reviews on the a p p l i c a t i o n of each of the c h i r a l s t a t i o n a r y phases are a v a i l a b l e (Louchmuller and Souter, 1975; Blaschke, 1980; L i u and Ku, 1980; Armstrong, 1984; Wainer and Alembik, 1988; K r s t u l o v i c , 1989). 1 . 7 . 2 . 2 . 1 C y c l o d e x t r i n Phases C y c l o d e x t r i n s are c h i r a l macrocyclic polymers derived from the a c t i o n of Bacillus macerans amylase on starch (Armstrong, 1984). Commercially a v a i l a b l e c y c l o d e x t r i n phases include the o j - c y c l o d e x t r i n , B - c y c l o d e x t r i n and q - c y c l o d e x t r i n which vary from s i x to twelve glucose u n i t s with a - ( l , 4 ) - l i n k a g e s . The r e s o l u t i o n of enantiomers, as discussed by Armstrong (1984), involved a t h r e e - p o i n t i n t e r a c t i o n between the enantiomer and the c y c l o d e x t r i n phase c o n s i s t i n g of 23 i n c l u s i o n of the enantiomer w i t h i n the hydrophobic i n t e r i o r of the c y c l o d e x t r i n c a v i t y , together with a minimum of two hydrogen-bonding s i t e s p r o j e c t i n g r a d i a l l y from the mouth of the c y c l o d e x t r i n c a v i t y . Examples of reported r e s o l u t i o n of c h i r a l compounds using the c y c l o d e x t r i n phases i n c l u d e : 6-naphthamides, h e x o b a r b i t a l , a t r o p i n e , c o c a i n e , mephobarbital , verapamil , m e t o p r o l o l , propranolol and the dansyl d e r i v a t i v e s of amino acids (Armstrong et al., 1987; Armstrong et al., 1986; Hinze et al., 1985). An extensive survey of analytes resolved by the c y c l o d e x t r i n phases i s a v a i l a b l e from Han and Armstrong (1989). 1 . 7 . 2 . 2 . 2 Serum P r o t e i n Bound Phases Two HPLC s i l i c a - b a s e d p r o t e i n columns used f o r the r e s o l u t i o n of enantiomers are the bovine serum albumin column (marketed as R e s o l v o s i l R ) and the a j - a c i d g l y c o p r o t e i n column (marketed as E n a n t i o P a c R ) . The use of ovomucoid, an egg white p r o t e i n , as an HPLC s t a t i o n a r y phase has a l s o been described (Miwa et al., 1987a; 1987b). The bovine serum albumin (BSA) s t a t i o n a r y phase c o n s i s t s of BSA, a g l o b u l a r p r o t e i n immobilized onto s i l i c a p a r t i c l e s . BSA i s a hydrophobic p r o t e i n with demonstrated a f f i n i t y towards anions and hydrophobic c a t i o n s (Lewin, 1974). Examples of the r e s o l u t i o n of enantiomers using the BSA R e s o l v o s i l R HPLC column have included numerous b a r b i t u r a t e s (Allenmark, et al., 1988), B-blockers (Kusters and G i r o n , 1986), benzothiadiazine d i u r e t i c s (Wollweber et al., 1976) and benzodiazepinones (Allenmark, 1989). a j - A c i d g l y c o p r o t e i n (orosomucoid) i s a c a t i o n i c binding p r o t e i n which binds to analytes possessing a hydrophobic moiety (Schmid, 1975). The EnantioPac R s t a t i o n a r y phase c o n s i s t s of the g l y c o p r o t e i n c h e m i c a l l y c r o s s l i n k e d and bonded onto s i l i c a p a r t i c l e s . Reports of r e s o l u t i o n of c h i r a l compounds using the EnantioPac R HPLC column have included numerous 6 - b l o c k e r s ( S c h i l l , 1986; Hermansson, 1985) as well as 6-adrenergics such as ephedrine, pseudoephedrine and t e r b u t a l i n e ( S c h i l l , 1986). The ovomucoid s t a t i o n a r y phase, s i m i l a r to the orosomucoid p r o t e i n , i s a g l y c o p r o t e i n s t a b l e over a wide pH range and in a v a r i e t y of organic solvents (Allenmark, 1989). The ovomucoid s t a t i o n a r y phase was found to e x h i b i t strong hydrophobic i n t e r a c t i o n s with hydrophobic s o l u t e s and i t was reported to be s u i t a b l e f o r the r e s o l u t i o n of a wide range of c h i r a l s o l u t e s c o n t a i n i n g an aromatic s u b s t i t u t e n t , i n c l u d i n g numerous 2 - a r y l p r o p i o n i c acids and 6-blockers (Miwa et a 7 . , 1987a,b). 1 . 7 . 2 . 2 . 3 C e l l u l o s e - B a s e d C h i r a l S t a t i o n a r y Phases This c l a s s of c h i r a l s t a t i o n a r y phases c o n s i s t s mainly of c e l l u l o s e and amylose e s t e r s as well as carbamate d e r i v a t i v e s coated on s i l i c a p a r t i c l e s . Hesse and Hagel (1973) proposed that c h i r a l r e c o g n i t i o n by the c e l l u l o s e - d e r i v e d s t a t i o n a r y phases involved i n c l u s i o n of a s o l u t e by two adjacent glucose residues on the s t a t i o n a r y phase. M i c r o c r y s t a l l i n e c e l l u l o s e d e r i v a t i v e s , such as m i c r o c r y s t a l l i n e c e l l u l o s e t r i a c e t a t e have a l s o been used as s t a t i o n a r y phases f o r the r e s o l u t i o n of enantiomers. Okamoto et a7. (1986b) reported that f o r the c e l l u l o s e trisphenylcarbamate d e r i v a t i v e s t a t i o n a r y phase, the s u b s t i t u t e n t s on the phenyl groups g r e a t l y a f f e c t e d the r e s o l u t i o n of enantiomers. To d a t e , approximately nine d i f f e r e n t c e l l u l o s e c h i r a l HPLC s t a t i o n a r y phases are a v a i l a b l e (Shibata et a 7 . , 1989). D i r e c t r e s o l u t i o n of amino-alcohol and B-blocker enantiomers on the t r i ( 3 , 5 ) -dimethylphenyl carbamate c e l l u l o s e s t a t i o n a r y phase were reported by Okamoto (1986a, 1987). This c e l l u l o s e c h i r a l s t a t i o n a r y phase was found to r e s o l v e many B-blockers without pre-column d e r i v a t i z a t i o n , thus providing a convenient method f o r the i s o l a t i o n of pure enantiomers. The d i r e c t r e s o l u t i o n of b a r b i t u r a t e analog enantiomers was a l s o reported using the m i c r o c r y s t a l l i n e c e l l u l o s e t r i a c e t a t e s t a t i o n a r y phase (Blaschke, 1986). The r e s o l u t i o n of 6-lactams enantiomers using the trisphenylcarbamate c e l l u l o s e s t a t i o n a r y phase (Yamada et al., 1987) and the r e s o l u t i o n of d i l t i a z e m , disopyramide and d i h y d r o p y r i d i n e analog enantiomers using the chlorophenyl carbamate s t a t i o n a r y phase were a l s o reported (Shibata and Y u k i , unpublished r e s u l t s , c i t e d by S h i b a t a , 1989). 1 . 7 . 2 . 2 . 4 P i r k l e R C h i r a l S t a t i o n a r y Phases C h i r a l r e c o g n i t i o n models were proposed by P i r k l e et al. (1983) to account f o r the i n t e r a c t i o n s between an enantiomer and a c h i r a l s t a t i o n a r y phase. To achieve r e s o l u t i o n of two enantiomers, a minimum of three simultaneous i n t e r a c t i o n s between the analyte and the s t a t i o n a r y phase was found necessary, with one or more of these i n t e r a c t i o n s i n v o l v i n g s u b s t i t u t e n t s on the c h i r a l center of the a n a l y t e . For the P i r k l e R type-1 c h i r a l phases, a combination of aromatic n-n e l e c t r o n i c i n t e r a c t i o n , hydrogen-bonding, e l e c t r o s t a t i c and s t e r i c i n t e r a c t i o n s were required to generate c h i r a l r e c o g n i t i o n ( P i r k l e et al., 1984). The P i r k l e t y p e - l A HPLC columns are a v a i l a b l e as 3 , 5 -d i n i t r o b e n z o y l d e r i v a t i v e s of D-phenylglycine or L - l e u c i n e , both of which are i o n i c a l l y bonded on s i l i c a - b a s e d HPLC packings f o r forward-phase chromatography. The P i r k l e R type-1 HPLC columns are a v a i l a b l e as c o v a l e n t l y bonded d i n i t r o b e n z o y l d e r i v a t i v e s of D- or L-phenylglycine or L - l e u c i n e f o r 26 reverse-phase HPLC. These f i r s t - g e n e r a t i o n P i r k l e R c h i r a l HPLC phases, with the presence of the d i n i t r o b e n z o y l group a c t i n g as 7 r - e l e c t r o n a c c e p t o r s , were found useful in r e s o l v i n g enantiomers with 7r-electron donating benzoyl or naphthoyl m o i e t i e s . The P i r k l e R t y p e - l A c h i r a l phases were reported to resolve the enantiomers of a l c o h o l s , s u l f o x i d e s , b i - 8 - n a p h t h o l s , hydantoins, s u c c i n i m i d e s , benzodiazepines and B-adenergic agents ( P i r k l e and Tsipouras, 1984; P i r k l e and Hyun, 1984; P i r k l e et al., 1981; Wainer and Doyle, 1983). Following the P i r k l e c h i r a l r e c o g n i t i o n concept, a P i r k l e R type-2 7r-electron donating c h i r a l s t a t i o n a r y phase was designed based on the d e r i v a t i v e s of n a p h t h y l a l a n i n e . For c h i r a l r e c o g n i t i o n by n a p h t h y l a l a n i n e , i t was found necessary f o r an enantiomer to possess an a c i d s i t e , a b a s i c s i t e , and a 7r-electron acceptor aromatic group. The type-2 P i r k l e R HPLC s t a t i o n a r y phases were reported to r e s o l v e c h i r a l amines, amino a c i d s , aminoalcohols and aminophosphonic acids as t h e i r 3 , 5 - d i n i t r o b e n z o y l d e r i v a t i v e s ( P i r k l e et al., 1984). 27 1.8 Summary of Research Goals 1.8.1 Stereoselective HPLC Analysis of the Enantiomers of Mexiletine, p-Hydroxy-Mexiletine and Hydroxymethyl-Mexiletine. A p r e v i o u s l y reported HPLC assay f o r m e x i l e t i n e enantiomers involved r e s o l u t i o n of the enantiomers as t h e i r 2-naphthoyl d e r i v a t i v e s followed by fluorescence d e t e c t i o n with an assay d e t e c t i o n l i m i t of 5 ng/ml in plasma (McErlane and Igwemezie, 1987b). In order to improve the assay l i m i t f o r monitoring serum f r e e drug c o n c e n t r a t i o n s , the present study w i l l d e s c r i b e the synthesis and development of 2-anthroyl c h l o r i d e , which i s expected to allow d e t e c t i o n of the enantiomers at picogram l e v e l s . A s t e r e o s e l e c t i v e assay f o r the determination of m e x i l e t i n e metabolite enantiomers i s l a c k i n g . Since the metabolism of m e x i l e t i n e i s expected to be s t e r e o s e l e c t i v e , the present study w i l l i n v e s t i g a t e various methods to achieve r e s o l u t i o n of the metabolite enantiomers and design an assay method f o r the simultaneous determination of p-OH-m e x i l e t i n e and OH-methyl-mexiletine enantiomers in u r i n e . 1.8.2 Pharmacokinetics of Serum Total and Free Mexiletine Enantiomers in Twelve Healthy Subjects The pharmacokinetics of m e x i l e t i n e enantiomers in healthy subjects have p r e v i o u s l y been reported by Grech-Belanger et al. (1986) and Igwemezie et al. (1989). The l a t t e r study reported s i g n i f i c a n t d i f f e r e n c e s in the volume of d i s t r i b u t i o n , e l i m i n a t i o n and clearance between the enantiomers. These f i n d i n g s were in contrast with an e a r l i e r report of n o n - s t e r e o s e l e c t i v e d i s p o s i t i o n of the enantiomers. This disagreement between the two studies may have been due to the l i m i t e d number of subjects in d i f f e r e n t i a t i n g a small d i f f e r e n c e in the 28 o v e r a l l d i s p o s i t i o n between the enantiomers. The present pharmacokinetic study, therefore w i l l involve twelve subjects and a n t i c i p a t e s a s i m i l a r o v e r a l l d i s p o s i t i o n between the enantiomers. The in vivo serum p r o t e i n binding of m e x i l e t i n e enantiomers has not been i n v e s t i g a t e d . P r e l i m i n a r y in vitro serum p r o t e i n binding studies i n d i c a t e d a pH-dependent s t e r e o s e l e c t i v e binding of the enantiomers. However, binding was n o n - s t e r e o s e l e c t i v e at pH 7 . 4 . With the exception of metabolite binding i n t e r a c t i o n , the in vivo p r o t e i n binding of the enantiomers i s expected to be s i m i l a r . To f u r t h e r c h a r a c t e r i z e the d i s p o s i t i o n of f r e e drug, the d i s p o s i t i o n of the enantiomers i n s a l i v a and in red blood c e l l s w i l l a l s o be examined. 1 . 8 . 3 Metabolism of M e x i l e t i n e Enantiomers The metabolites of m e x i l e t i n e are p-OH-mexiletine, OH-methyl-m e x i l e t i n e and t h e i r corresponding deaminated alcohol m e t a b o l i t e s , which account f o r «20% of an o r a l dose (Beckett and Chidomere, 1977). However, the in vivo s t e r e o s e l e c t i v e d i s p o s i t i o n of each of the metabolites has not been r e p o r t e d . An in vitro study of the metabolism of m e x i l e t i n e enantiomers in r a t l i v e r homogenate preparations revealed that p-hydroxylation of m e x i l e t i n e favors the S(+)-enantiomer, w h i l e the formation of OH-methyl-mexiletine favors the R(-)-enantiomer (Grech-Belanger et a l . , 1988). In l i g h t of the s t e r e o s e l e c t i v e in vitro metabolism of m e x i l e t i n e enantiomers, i t i s proposed that s t e r e o s e l e c t i v e u r i n a r y recovery of p-OH-mexiletine and/or OH-methyl-m e x i l e t i n e enantiomers w i l l occur in vivo. 1.9 Objectives 1. To develop 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 assay allowing d i r e c t r e s o l u t i o n and q u a n t i t a t i o n of serum t o t a l and f r e e m e x i l e t i n e enantiomers f o r 48 hours a f t e r drug a d m i n i s t r a t i o n . 2. To examine the pharmacokinetic parameters of m e x i l e t i n e enantiomers in twelve healthy male volunteers a f t e r a d m i n i s t r a t i o n of a s i n g l e 200 mg o r a l dose of racemic m e x i l e t i n e h y d r o c h l o r i d e . 3 . To examine the serum p r o t e i n binding of m e x i l e t i n e enantiomers in the twelve healthy subjects by q u a n t i t a t i o n of f r e e and t o t a l serum drug c o n c e n t r a t i o n s . 4. To examine the time-course of d i s t r i b u t i o n of m e x i l e t i n e in s a l i v a in the twelve s u b j e c t s . To e s t a b l i s h i f a r e l a t i o n s h i p e x i s t s between serum f r e e drug and s a l i v a drug concentrations and to e s t a b l i s h the e f f e c t s of p h y s i o l o g i c a l v a r i a t i o n s in s a l i v a pH on s a l i v a drug e x c r e t i o n . 5. To examine the time-course of u r i n a r y e x c r e t i o n of m e x i l e t i n e enantiomers in the twelve subjects f o r 72 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 . 6. To examine the time-course of d i s t r i b u t i o n of m e x i l e t i n e enantiomers in red blood c e l l s and to e s t a b l i s h i f a r e l a t i o n s h i p e x i s t s between serum f r e e drug and erythrocyte drug c o n c e n t r a t i o n s . 7. To develop 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 assay f o r the r e s o l u t i o n and q u a n t i t a t i o n of p-OH-mexiletine, and OH-methyl-m e x i l e t i n e in urine 8 . To examine the s t e r e o s e l e c t i v e metabolism of m e x i l e t i n e by q u a n t i t a t i o n of two m e t a b o l i t e s , the OH-methyl-mexiletine and p-OH-m e x i l e t i n e , in the urine of four healthy s u b j e c t s . 1.10 R a t i o n a l e a . Development of 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 f o r the Assay of M e x i l e t i n e Enantiomers. The s t e r e o s e l e c t i v e method developed by McErlane and Igwemezie (1987) employed the use of a c h i r a l P i r k l e R 1A phenylglycine HPLC column f o r the d i r e c t r e s o l u t i o n of m e x i l e t i n e enantiomers. The method involved 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 with 2-naphthoyl c h l o r i d e to form mexiletine-N-naphthoyl d e r i v a t i v e which was required f o r the r e s o l u t i o n of the enantiomers. Fluorescence d e t e c t i o n of the naphthoyl d e r i v a t i v e s of m e x i l e t i n e enantiomers was reported with an assay l i m i t of 5 ng/ml in plasma. In e v a l u a t i o n of our proposal to study the plasma p r o t e i n binding of m e x i l e t i n e enantiomers i n healthy subjects f o l l o w i n g a s i n g l e 200 mg o r a l dose of racemic m e x i l e t i n e h y d r o c h l o r i d e , we a n t i c i p a t e d a m e x i l e t i n e serum l e v e l below the s e n s i t i v i t y of the reported assay. F r o e h l i c h (1985) reported that the fluorescence quantum y i e l d of anthracene was 1 . 5 - f o l d higher than that of naphthalene. The development of 2-anthroyl c h l o r i d e as an a c y l a t i o n reagent was therefore proposed to allow improved fluorescence d e t e c t i o n of m e x i l e t i n e while preserving the n - e l e c t r o n donating c h a r a c t e r i s t i c s of the m e x i l e t i n e d e r i v a t i v e necessary f o r c h i r a l r e c o g n i t i o n of i t s enantiomers. b . A Study of the Pharmacokinetics of M e x i l e t i n e Enantiomers i n Healthy S u b j e c t 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 a racemic mixture of two enantiomers with p o s s i b l y d i s t i n c t pharmacokinetic p r o p e r t i e s . Our present study of the pharmacokinetics of m e x i l e t i n e enantiomers in 31 twelve healthy human subjects w i l l allow us to assess d i f f e r e n c e s in the a b s o r p t i o n , d i s t r i b u t i o n , and e l i m i n a t i o n of the enantiomers. c. Study on the Protein Binding of Mexiletine Enantiomers. M e x i l e t i n e was reported to be 70% bound to plasma p r o t e i n s (Talbot et a l . , 1973). In vitro serum p r o t e i n binding of m e x i l e t i n e enantiomers was reported with a s t e r e o s e l e c t i v e binding of S(+)-mexiletine (McErlane et a 7 . , 1987). Although the pharmacological p r o p e r t i e s of the two enantiomers have not been evaluated in humans, i t i s g e n e r a l l y recognized that pharmacological a c t i o n i s proportional to f r e e drug concentration in the body. The present study of the serum p r o t e i n binding of m e x i l e t i n e enantiomers in healthy subjects w i l l allow an assessment of the in vivo k i n e t i c s of m e x i l e t i n e serum f r e e drug. d. A Study of the Salivary Excretion of Mexiletine Enantiomers. Many drugs were reported to be excreted in s a l i v a (Levy et a 7 . , 1979; Koysooko et a 7 . , 1973; Mucklow, 1982). Therapeutic monitoring of drug concentrations in s a l i v a samples has provided a convenient and non-invasive method of assessing serum f r e e drug concentrations in somes cases (Fraser et a 7 . , 1976; Mucklow et a 7 . , 1977). Our present study w i l l determine i f a r e l a t i o n s h i p e x i s t s between m e x i l e t i n e in s a l i v a and serum f r e e drug c o n c e n t r a t i o n s . In a d d i t i o n , we w i l l examine the e f f e c t s of s a l i v a pH on the e x c r e t i o n of m e x i l e t i n e enantiomers into s a l i v a . e. A Study of the Distr i b u t i o n of Mexiletine Enantiomers in Red Blood C e l l s . The d i s t r i b u t i o n of drugs through the red c e l l membrane was well documented (Schanker et a 7 . , 1964). The d i s t r i b u t i o n of drugs across the erythrocyte membrane i s u s u a l l y governed by an e q u i l i b r i u m with f r e e drug concentration in serum. Therefore, erythrocyte drug concentrations can p o t e n t i a l l y provide a convenient estimation of f r e e drug c o n c e n t r a t i o n s . However, in a d d i t i o n to passive d i f f u s i o n of drugs across the erythrocyte membrane, numerous drugs have been reported to accumulate w i t h i n the red c e l l (Kurata and W i l k i n s o n , 1974; Wallace and Riegelman, 1977; Gorodischer et a / . , 1976) and behaved as a d i s t i n c t pharmacokinetic compartment w i t h i n the systemic c i r c u l a t i o n . Our present study w i l l examine the extent of d i s t r i b u t i o n of m e x i l e t i n e enantiomers across the erythrocyte membrane, and w i l l examine i f a r e l a t i o n s h i p e x i s t between erythrocyte and serum f r e e drug concentrations f o r m e x i l e t i n e enantiomers. f. A Study of the Metabolic Disposition of Mexiletine Enantiomers. M e x i l e t i n e i s 90% e l i m i n a t e d from the body by metabolic o x i d a t i v e and r e d u c t i v e pathways. The major metabolites of m e x i l e t i n e i n humans include the p-OH-mexiletine and OH-methyl-mexiletine as w e l l as t h e i r corresponding deaminated m e t a b o l i t e s , the p-OH-mexiletine alcohol and OH-methyl-mexiletine a l c o h o l . The u r i n a r y e x c r e t i o n of these four metabolites in healthy subjects has been described and the four metabolites have accounted f o r 20% of an o r a l dose (Beckett and Chidomere, 1977a; Beckett and Chidomere, 1977b). Nevertheless, the enantiomeric composition of the o x i d a t i v e metabolites of m e x i l e t i n e has not been r e p o r t e d . With a n t i c i p a t i o n that most hepatic metabolic pathways are governed by s t e r e o s e l e c t i v e mechanisms, the present study on the s t e r e o s e l e c t i v e d i s p o s i t i o n of two m e t a b o l i t e s , the p-OH-m e x i l e t i n e and OH-methyl-mexiletine, w i l l allow us to f u r t h e r understand any d i f f e r e n c e s in the d i s p o s i t i o n between m e x i l e t i n e enantiomers. { 33 g. Development of an Analytical Method f o r Mexiletine Metabolite Enantiomers. The absence of information on the s t e r e o s e l e c t i v e d i s p o s i t i o n of the metabolites of m e x i l e t i n e i s a r e s u l t of a lack of a s t e r e o s e l e c t i v e method f o r the a n a l y s i s of metabolite enantiomers. The requirement f o r HPLC separation of m e x i l e t i n e enantiomers on the P i r k l e R phenylglycine column d i c t a t e s the need f o r a coplanar n-e l e c t r o n donating aromatic r i n g system a l l o w i n g i n t e r a c t i o n with the n-e l e c t r o n accepting d i n i t r o b e n z o y l group of the c h i r a l s t a t i o n a r y phase. Following t h i s requirement, the 7r-basic naphthoyl and anthroyl d e r i v a t i v e s of m e x i l e t i n e were resolved on the P i r k l e R column. Resolution of the OH-methyl-mexiletine and p-OH-mexiletine metabolites on the P i r k l e R column should therefore r e q u i r e d e r i v a t i z a t i o n with naphthoyl or anthroyl c h l o r i d e f o l l o w i n g s i m i l a r stereochemical requirements compared to those f o r m e x i l e t i n e . However the presence of hydroxy groups in the metabolites may cause a d d i t i o n a l hydrogen-bonding between the metabolites enantiomers and the s t a t i o n a r y phase. Should the presence of hydroxy groups a f f e c t the expected r e s o l u t i o n of m e x i l e t i n e metabolite enantiomers, p r o t e c t i o n of these groups w i l l be attempted using a v a r i e t y of d e r i v a t i z a t i o n reagents. 2. Experimental 2.1 M a t e r i a l s and Supplies 2.1.1 Chemicals and Reagents M e x i t i l R 200 mg c a p s u l e s * , ( 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 2 , 1-(2' ,6'-dimethylphenoxy)-2-ethanamine (KOE 2963, i n t e r n a l s t a n d a r d ) 3 , p - O H - m e x i l e t i n e 4 , OH-methyl-mexiletine 5 , t y r a m i n e 6 , methyl amine 7 , ethyl bromide**, 2-naphthoyl c h l o r i d e 9 , anthraquinone-2-carboxylic a c i o x a l y l c h l o r i d e 1 1 , z i n c d u s t 1 2 , concentrated ammonia s o l u t i o n 1 3 , sodium h y d r o x i d e 1 4 , barium hydroxide o c t a h y d r a t e 1 5 , z i n c sulphate heptahydrate 1 ^, h y d r o c h l o r i c a c i d 1 7 , phosphoric a c i d 1 8 , s u l p h u r i c a c i d 1 9 , sodium c h l o r i d e 2 ^ , monosodium phosphate monohydrate 2 1 , sodium b i c a r b o n a t e 2 2 , anhydrous sodium c a r b o n a t e 2 3 , disodium phosphate h e p t a h y d r a t e 2 4 , ethyl b r o m i d e 2 5 , n-propyl bromide2**, n-butyl b r o m i d e 2 7 , methylamine (70% v/v in w a t e r ) 2 8 , t r i m e t h y l s i l y l i m i d a z o l e 2 9 , p y r i d i n e 3 ^ , potassium h y d r o x i d e 3 1 , 2 - ( 2 - e t h o x y e t h o x y ) e t h a n o l 3 2 , N-m e t h y l - N - N i t r o s o - p - t o l u e n e 3 3 and t r i f l u o r o b o r o e t h e r a t e 3 4 were obtained. Chemicals and reagents were obtained as reagent grade and were used without f u r t h e r p u r i f i c a t i o n . 1-5 Boehringer Ingelheim L t d . ( B u r l i n g t o n , O n t a r i o , Canada) 6-11 A l d r i c h Chemical Co. (Milwaukee, Wisconsin, USA) 12-14 A r i s t a r BDH Chemical (Poole, England) 15-24 BDH Chemicals (Vancouver, B . C . , Canada) 25-28 F i s 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) 29 P i e r c e Chemical Co. (Rockford, I l l i n o i s , USA) 30 Supelco L t d . ( B e l l a f o n t e , Pennsylvania, USA) 31 J . T . Baker ( P h i l i p s b u r g , New J e r s e y , USA) 32-34 Sigma Chemical Co. ( S t . L o u i s , M i s s o u r i , USA) 35 2.1.2 Solvents n-Hexane 3 5 , d i e t h y l e t h e r 3 6 , d i c h l o r o m e t h a n e 3 7 , c h l o r o f o r m 3 8 , ethyl a c e t a t e 3 9 , e t h a n o l 4 0 , m e t h a n o l 4 1 , 2-propanol 4 ^ and a c e t o n i t r i l e 4 3 were obtained in g l a s s - d i s t i l l e d HPLC q u a l i t y . 2.1 .3 Supplies f o r Human Studies H e p a r i n 4 4 f o r i n j e c t i o n (1000 u n i t s / m l ) , s a l i n e f o r i n j e c t i o n 4 5 , p l a i n VacutainerR with s i l i c o n coated s t o p p e r 4 6 , p l a i n VacutainerR with non-coated s t o p p e r 4 7 , heparinized V a c u t a i n e r R 4 8 , EDTA V a c u t a i n e r R 4 9 , s t e r i l e g l a s s syringes (10 m l ) , B u t t e r f l y R - 2 1 INT i n d w e l l i n g c a n n u l a 5 ^ . 2.1.4 Supplies f o r Serum Protein Binding Studies A CentrifreeR m i c r o p a r t i t i o n system 5 * with a 30,000 Dalton molecular weight c u t - o f f YMT f i l t r a t i o n membrane was used. E q u i l i b r i u m d i a l y s i s c e l l s were manufactured from p l e x i g l a s s 5 ^ and used with c e l l u l o s e t u b i n g 5 3 with a molecular weight cut o f f of >12,000 d a l t o n s . The tubing was cut and washed i n b o i l i n g d i s t i l l e d water p r i o r to each experiment. 2.1 .5 Chromatographic Columns A Whatman (10 /zm) ODS-2 (M-9) 0.94 i d . x 25 cm HPLC column 5 4 was used f o r p u r i f i c a t i o n of 2-anthroyl c h l o r i d e . P i r k l e R 1-A 35-43 BDH Chemicals L t d . (Vancouver, B . C . , Canada) 44 Glaxo L t d . (Toronto, O n t a r i o , Canada) 46-49 Becton Dickson (Montreal , Quebec, Canada) 45,50 Abbott Laboratories (Toronto, O n t a r i o , Canada) 51 Amicon Canada L t d . ( O a k v i l l e , O n t a r i o , Canada) 52 3-Dimension P l a s t i c (Vancouver, B . C . , Canada) 53 Sigma Chemical Co. ( S t . L o u i s , Mo. , USA) 54 Whatman Inc. ( C l i f t o n , N J . , USA) 36 i o n i c phenyl g l y c i n e 5 5 , P i r k l e R i o n i c l e u c i n e 5 * * and P i r k l e R covalent l e u c i n e 5 7 c h i r a l HPLC columns were used f o r enantiomer r e s o l u t i o n . An A l l t e c h (5 fim) s i l i c a 0.46 i d . x 15 cm HPLC column 5 8 was used in tandem with a P i r k l e R 1-A column. 2.2 Equipment 2 . 2 . 1 High-Performance Liquid Chromatographs A Hewlett-Packard model 1082B l i q u i d chromatograph5 9 was equipped with a Hewlett-Packard model 79850B LC t e r m i n a l , a Rheodyne 20 /zl or 100 fi\ i n j e c t i o n l o o p 6 0 , and a G i l s o n HM-Holochrome v a r i a b l e wavelength UV m o n i t o r 6 1 or a Hewlett-Packard model 1072B F i r e f l y ^ fluorescence d e t e c t o r 6 2 . A G i l s o n model 302 l i q u i d chromatograph6 3 was equipped with a G i l s o n model 811 dynamic m i x e r 6 4 , a G i l s o n model 620 data m o d u l e 6 5 , a Rheodyne 20 /zl i n j e c t o r 6 6 , and a Schoeffel model GM 970 f l u o r o m e t e r 6 7 . Chromatographic data was acquired and stored by an Apple R 11+ computer. 2 . 2 . 2 Gas Chromatograph - Mass Spectrometer A Hewlett-Packard model 5987A quadrupole mass spectrometer 6 8 was i n t e r f a c e d to a Hewlett-Packard model 1000 data a c q u i s i t i o n system. 55-56 Regis Chemical Co. (Morton Grove, I l l i n o i s , USA) 57 Baker Chemicals (Toronto, O n t a r i o , Canada) 58 Applied Science (Rockwood, O n t a r i o , Canada) 5 9 , 6 2 , 6 8 Hewlett-Packard (Avondale, P e n s y l v a n i a , USA) 60,66 Rheodyne Inc. (Berkeley, C a l i f o r n i a , USA) 61,63-65 Mandel S c i e n t i f i c Co. (Edmonton, A l b e r t a , Canada) 67 Kratos Schoeffel Instruments (Westwood, New J e r s e y , USA) 37 2.2.3 Centrifuge A Beckman Js-21 c e n t r i f u g e 6 9 with a 35 degree angle r o t o r head was used f o r u l t r a f i l t r a t i o n experiments. 2 . 2 . 4 Polarimetry A Perkin-Elmer 141 p o l a r i m e t e r 7 0 was operated with a sodium lamp at 589 nm. Sample measurements were made in hexane with a quartz c e l l of 9.99 cm path l e n g t h . 2.3 Stereoselective HPLC Assay f o r Mexiletine Enantiomers 2.3.1 Synthesis of 2-Anthroyl Chloride 2.3.1.1 Reduction of Anthraquinone - 2-carboxylic Acid with Zinc/Aqueous Ammonia Anthraquinone-2-carboxylic a c i d ( 0 . 5 g) was added with s t i r r i n g to 30 ml of a d i l u t e aqueous ammonia s o l u t i o n (14 %). The mixture was r e f l u x e d with s t i r r i n g , and z i n c dust ( 2 . 0 g) was added in small portions over a 30 minute p e r i o d . The r e s u l t i n g dark red mixture, c h a r a c t e r i s t i c of the quinonic s t r u c t u r e , bleached g r a d u a l l y to a c l e a r p a l e - y e l l o w s o l u t i o n when the mixture was r e f l u x e d with slow s t i r r i n g f o r one hour. The z i n c dust was removed by f i l t r a t i o n . The product, 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 r e c i p i t a t e d upon a c i d i f y i n g the f i l t r a t e to pH 3 with h y d r o c h l o r i c a c i d . The crude product was r e c r y s t a l l i z e d in hot ethanol to y i e l d 0.12 g of product (27.2%). The melting point (uncorrected) was found to be 278-279 °C [ l i t . m.p. 276-278 °C (Sanchorawala et a l . , 1963)]. 5 9 , 6 2 , 6 8 Hewlett-Packard (Avondale, Pensylvania, USA) 60,66 Rheodyne Inc. (Berkeley, C a l i f o r n i a , USA) 61,63-65 Mandel S c i e n t i f i c Co. (Edmonton, A l b e r t a , Canada) 67 Kratos Schoeffel Instruments (Westwood, New J e r s e y , USA) 69 Beckman S c i e n t i f i c Instruments ( I r v i n e , C a l i f o r n i a , USA) 70 Perkin-Elmer (Uberlingen, West Germany) 2.3.1.2 Formation of 2-Anthroyl Chloride Oxalyl c h l o r i d e ( 0 . 5 ml) was added to anthracene-2-carboxylic a c i d (0.1 g) in 20 ml of dry benzene with s t i r r i n g . The mixture was slowly heated over a water bath f o r 30 minutes, a f t e r which a second a l i q u o t of 0 . 5 ml o x a l y l c h l o r i d e was added and heating was continued f o r another 30 minutes. A f t e r c o o l i n g , the unreacted a c i d was f i l t e r e d and removed. Excess o x a l y l c h l o r i d e and solvent were removed under reduced pressure to y i e l d 0.04 g (41.8 %) of p a l e - y e l l o w 2-anthroyl c h l o r i d e . R e c r y s t a l l i z a t i o n was c a r r i e d out in warm dry hexane to remove the unreacted anthracene-2-carboxylic a c i d . 2.3.2 P u r i f i c a t i o n of 2-Anthroyl Chloride by Preparative-HPLC The 2-anthroyl c h l o r i d e was p u r i f i e d with reverse-phase preparative HPLC. A Hewlett-Packard model 1082B l i q u i d chromatograph was equipped with a UV monitor set at 230 nm. A 0.9 x 25 cm Whatman 0DS-2 column was used with a 100 % a c e t o n i t r i l e mobile phase at a flow r a t e of 0.7 ml/min. A 500 mg sample of the s y n t h e t i c sample was d i s s o l v e d in 1 ml of a c e t o n i t r i l e and 100 fil a l i q u o t s were i n j e c t e d . The a c e t o n i t r i l e column eluant corresponding to the 2-anthroyl c h l o r i d e chromatographic peak at adjusted r e t e n t i o n time of « 11.0 minutes was c o l l e c t e d and evaporated to dryness with a stream of nitrogen to y i e l d 300 mg of 2-anthroyl c h l o r i d e . The p u r i t y of the HPLC p u r i f i e d 2-anthroyl c h l o r i d e reagent was a l s o evaluated by reverse-phase t h i n - l a y e r chromatography (TLC). A s i n g l e component on the TLC-plate was observed when v i s u a l i z e d under U V - l i g h t at 254 nm. 2 . 3 . 3 Structural Characterization of 2-Anthroyl Chloride and 2-Anthroyl Mexiletine Derivative by Mass Spectrometry A Hewlett-Packard model 5987A quadrupole mass spectrometer was operated with d i r e c t probe sample i n t r o d u c t i o n in the e l e c t r o n - i m p a c t i o n i z a t i o n mode at 70 eV with an emission current of 300 /zA and m u l t i p l i e r voltage of 2500 V. The source temperature was set at 240 °C and the sample probe temperature was programmed from 50 °C f o r 1 minute to 300 °C f o r 10 minutes at a r a t e of 30 "C/min. 2 . 3 . 4 Stereoselective HPLC of Mexiletine Enantiomers A G i l s o n model 302 l i q u i d chromatograph was used with a P i r k l e R 1-A phenyl g l y c i n e (5 fan) 4 . 6 mm x 25 cm c h i r a l column connected i n tandem to an A l l t e c h (5 fan) 4 . 6 mm x 15 cm s i l i c a column. An i s o c r a t i c mobile phase of isopropanol/chloroform/ hexane (35/755/390) was d e l i v e r e d at a flow rate of 0 . 8 ml/min. Fluorescence d e t e c t i o n with a Schoeffel fluorometer was optimized at e x c i t a t i o n and emission wavelengths of 270 nm and 420 nm, r e s p e c t i v e l y . 2 . 3 . 4 . 1 Assay Method f o r Total (Protein Bound + Free) Mexiletine Enantiomers in Serum In d u p l i c a t e , 200 /zl a l i q u o t s of serum were added to 30 /zl of an i n t e r n a l standard s o l u t i o n (KOE-2963, 1 /zg/ml). Serum p r o t e i n s were p r e c i p i t a t e d by the a d d i t i o n of a 200 /zl of barium hydroxide s o l u t i o n (0.15 M) followed by vortex-mixing and the a d d i t i o n of 200 /zl z i n c sulphate s o l u t i o n (0.15 M). The serum was mixed and adjusted to b a s i c pH with 200 /zl of sodium hydroxide s o l u t i o n (2.0 M) and extracted with two, 5 ml p o r t i o n s of d i e t h y l e t h e r . The ether was separated and reduced to « 1 ml under nitrogen in a 37 °C water bath. The ether was a c i d i f i e d with 300 /zl of a h y d r o c h l o r i c a c i d s o l u t i o n (0.1 M) and washed with two portions (2 ml) of d i e t h y l e t h e r . The aqueous l a y e r was readjusted to b a s i c pH with 300 [i\ of sodium hydroxide s o l u t i o n ( 2 . 0 M) and 10 fi\ of 2-anthroyl c h l o r i d e reagent (1 mg/ml) was added and vortex-mixed f o r 3 minutes. The m e x i l e t i n e d e r i v a t i v e s were extracted into 1 ml of methylene c h l o r i d e and the organic l a y e r was separated and reduced to dryness under nitrogen in a 37 °C water bath. The sample was r e c o n s t i t u t e d with 200 [A of HPLC mobile phase p r i o r to a n a l y s i s . 2.3.4.2 Assay Method f o r Free Mexiletine Enantiomers in Serum U l t r a f i l t r a t i o n of serum p r o t e i n s was c a r r i e d out with the C e n t r i f r e e R M i c r o p a r t i t i o n system. In d u p l i c a t e , 1 ml a l i q u o t s of serum were adjusted and maintained at pH 7.4 with the a d d i t i o n of 5.2 mg of sodium phosphate monobasic monohydrate and 43.4 mg of sodium phosphate d i b a s i c heptahydrate. The f i l t r a t i o n u n i t was c e n t r i f u g e d at 1650 g f o r 20 minutes at 37 °C using a Beckman Js-21 c e n t r i f u g e with a 35 degree angle r o t o r head. A f t e r u l t r a f i l t r a t i o n , 400 z*l of the u l t r a f i l t r a t e was added to 30 fi\ of an i n t e r n a l standard s o l u t i o n (K0E-2963, 1 //g/ml) before the sample was b a s i f i e d , e x t r a c t e d , acid-washed, and d e r i v a t i z e d with 10 n~\ of 2-anthroyl c h l o r i d e reagent as described f o r the determination of serum t o t a l drug. 2.3.4.3 Assay Recovery of Mexiletine from Serum and Serum U l t r a f i l t r a t e The r e c o v e r i e s of m e x i l e t i n e enantiomers from serum and u l t r a f i l t r a t e were studied at concentrations of 50 and 100 ng/ml. Duplicate samples were subjected to p r o t e i n p r e c i p i t a t i o n with barium hydroxide and z i n c s u l p h a t e , solvent e x t r a c t i o n with two portions of 5 ml of d i e t h y l e t h e r , and an aqueous acid-wash as p r e v i o u s l y described p r i o r to the a d d i t i o n of 50 ng of i n t e r n a l standard. The samples were acylated with 2-anthroyl c h l o r i d e by vortex-mixing and the m e x i l e t i n e anthroyl d e r i v a t i v e s were extracted into dichloromethane followed by HPLC a n a l y s i s as d e s c r i b e d . The peak height r a t i o s obtained were expressed as a percentage of those obtained from i d e n t i c a l amounts of m e x i l e t i n e and i n t e r n a l standard d i r e c t l y adjusted to b a s i c pH, d e r i v a t i z e d and analyzed. 2.3.4.4 Acylation Kinetics of Mexiletine with 2-Anthroyl Chloride The time-course of a c y l a t i o n of m e x i l e t i n e enantiomers with 2-anthroyl c h l o r i d e was studied over 1, 2, 4 , 6, 8 , and 10 minutes. In d u p l i c a t e , 100 fi\ of ( R , S ) - m e x i l e t i n e stock s o l u t i o n (100 ng/100 #1) was added to s i x tubes. A c y l a t i o n was c a r r i e d out by vortex-mixing of each m e x i l e t i n e sample with 200 /*1 of sodium hydroxide (2 M) and 10 /zl of 2-anthroyl c h l o r i d e (1 mg/ml) f o r the s p e c i f i e d t i m e s . At the end of each r e a c t i o n , 100 /zl of an external standard s o l u t i o n of the anthroyl d e r i v a t i v e of K0E-2963 (30 ng/100 jtzl, prepared as described f o r m e x i l e t i n e ) was added followed by e x t r a c t i o n of the d e r i v a t i v e s into dichloromethane f o r HPLC a n a l y s i s . 2.3.4.5 Determination of Detection Line a r i t y of Mexiletine Enantiomers in Serum and in Serum U l t r a f i l t r a t e Volumes of 50 and 250 zzl of a 0.1 zzg/ml of aqueous racemic m e x i l e t i n e stock s o l u t i o n and volumes of 50, 100, 150 and 200 zzl of a 1.0 /zg/ml of racemic m e x i l e t i n e aqueous stock s o l u t i o n were used to prepare the corresponding d u p l i c a t e samples of 2 . 5 , 12.5, 25, 50, 75, and 100 ng/ml of each enantiomer in serum or in serum u l t r a f i l t r a t e . A 30 /zl a l i q u o t of K0E-2963 i n t e r n a l standard s o l u t i o n (1 /zg/ml) was used f o r serum and serum u l t r a f i l t r a t e c a l i b r a t i o n . The serum and serum u l t r a f i l t r a t e samples were subjected to barium hydroxide and z i n c sulphate serum p r o t e i n p r e c i p i t a t i o n , solvent e x t r a c t i o n and 42 de r i v a t i z a t i o n as previously described in section 2.3.4.1 and 2.3.4.2 for serum t o t a l and serum free drug, respectively. The chromatographic peak height of each of mexiletine enantiomers was expressed as a r a t i o to that of 30 ng of the internal standard (K0E-2963, 1 /zg/ml). Serum and serum u l t r a f i l t r a t e c a l i b r a t i o n curves were determined with each subject's serum and serum u l t r a f i l t r a t e p r ior to the analysis of samples. 2.3.4.6 Determination of Detector Li n e a r i t y of Mexiletine Enantiomers in S a l i v a Racemic mexiletine stock solutions of 0.1 and 1.0 /zg/ml, as used for the preparation of serum c a l i b r a t i o n samples in Section 2.3.4.5, were used to prepare duplicate samples of 2.5, 12.5, 25, 50, 75 and 100 ng/ml of each enantiomer in s a l i v a . A 30 /zl aliquot of K0E-2963 internal standard solution (1 /zg/ml) was used for s a l i v a c a l i b r a t i o n . Saliva samples were subjected to barium hydroxide/zinc sulphate protein p r e c i p i t a t i o n , extraction and der i v a t i z a t i o n as described in section 2.3.4.1. The chromatographic peak height of each mexiletine enantiomer was expressed as a r a t i o to that of 30 ng of the internal standard, KOE-2963. 2.3.4.7 Determination of Detector Li n e a r i t y of Mexiletine Enantiomers in Red Blood C e l l s Racemic mexiletine stock solutions of 0.1 and 1.0 /zg/ml, as used for the preparation of serum c a l i b r a t i o n samples in Section 2.3.4.6, were used to prepare duplicate samples of 2.5, 12.5, 25, 50, 75 and 100 ng/ml of each enantiomer in a red blood c e l l suspension. A 30 /zl of KOE-2963 internal standard solution (1 /zg/ml) as used for c a l i b r a t i o n . The red c e l l s were lyzed with the addition of 0.5 ml of d i s t i l l e d water followed by barium hydroxide/zinc sulphate p r o t e i n p r e c i p i t a t i o n , solvent e x t r a c t i o n and d e r i v a t i z a t i o n as described i n s e c t i o n 2 . 3 . 4 . 1 . The chromatographic peak height of each m e x i l e t i n e enantiomer was expressed as a r a t i o to that of 30 ng of KOE-2963 as an i n t e r n a l standard. Z.3.4.8 HPLC Assay Intra- and I n t e r - v a r i a b i l i t y The v a r i a b i l i t y in q u a n t i t a t i o n of m e x i l e t i n e enantiomers between sample preparations ( i n t e r - a s s a y v a r i a b i l i t y ) was studied with t r i p l i c a t e samples of 10 and 200 ng/ml in serum prepared from the racemic m e x i l e t i n e stock s o l u t i o n s of 0.1 and 1.0 /zg/ml. The v a r i a b i l i t y i n q u a n t i t a t i o n of the enantiomers from repeated HPLC a n a l y s i s of one sample ( i n t r a - a s s a y v a r i a b i l i t y ) was obtained from t r i p l i c a t e a n a l y s i s of a 10 and 200 ng/ml sample. Samples were subjected to serum p r o t e i n p r e c i p i t a t i o n , solvent e x t r a c t i o n , aqueous-wash, d e r i v a t i z a t i o n and HPLC a n a l y s i s as described in Section 2 . 3 . 4 . 1 . 2.4 In Vitro Serum Protein Binding and Red Blood C e l l D i s t r i b u t i o n of Mexiletine Enantiomers 2.4.1 The Effects of Serum Collection and Storage Methods on Mexiletine Serum Free Fraction A t o t a l of 150 ml of venous blood was c o l l e c t e d from one human subject with g l a s s s y r i n g e s . The pooled blood was allowed to c l o t at room temperature f o r 2 hours and was then c e n t r i f u g e d at 2500 g f o r 15 minutes. The serum c o l l e c t e d was d i v i d e d i n t o two, 30 ml a l i q u o t s . To each 30 ml volume of serum, 600 /zl of a racemic m e x i l e t i n e stock s o l u t i o n of 1 or 10 ng/ml was added and mixed f o r 1 minute by s l o w l y i n v e r t i n g the samples on a r o t a t i n g r a c k . The serum samples were then e q u i l i b r a t e d at 37 °C i n a water bath f o r 30 minutes. A l i q u o t s of 6 ml 44 of serum from each set of samples, with concentrations of 20 ng/ml and 2 fig/mi, were used t o examine the e f f e c t s of the f o l l o w i n g sample c o l l e c t i o n / s t o r a g e methods have on serum p r o t e i n drug b i n d i n g : (1) . Indwelling B u t t e r f l y R - 2 1 INT cannula. (2) . P l a i n VacutainerR with s i l i c o n coated stopper. (3) . Glass syringe c o l l e c t i o n . (4) . V a c u t a i n e r R coated with h e p a r i n . (5) . VacutainerR coated with EDTA. (6) . Freezing at -20 *C f o r 72 hours. A l i q u o t s of 6 ml serum from each of the concentrations were f l u s h e d through a B u t t e r f l y R cannula and c o l l e c t e d in a 10 ml p o l y t e t r a f l u o r o e t h y l e n e (PTFE)-lined screw capped tube. A l t e r n a t i v e l y , 6 ml serum samples from each of the concentrations were t r a n s f e r r e d i n t o two VacutainersR and inverted slowly on a r o t a t i n g rack f o r 10 minutes. Another sample was stored at -20 °C f o r 72 hours p r i o r to f u r t h e r a n a l y s i s . Following the various treatment p r o t o c o l s , d u p l i c a t e 1 ml serum samples were used f o r t o t a l ( free + p r o t e i n bound) drug determinations f o l l o w i n g p r o t e i n p r e c i p i t a t i o n as described in Section 2 . 3 . 4 . 1 , w h i l e a second set of d u p l i c a t e samples were used f o r f r e e drug determination f o l l o w i n g u l t r a f i l t r a t i o n . A f t e r u l t r a f i l t r a t i o n , 50 fi\ of the i n t e r n a l standard s o l u t i o n was added to 200 (i\ of the u l t r a f i l t r a t e before the sample was adjusted t o b a s i c pH, extracted and d e r i v a t i z e d as described i n Section 2 . 3 . 4 . 2 . To serve as c o n t r o l s , d u p l i c a t e 1 ml serum samples c o l l e c t e d w i t h g l a s s syringes were a l s o analyzed f o r t o t a l and f r e e m e x i l e t i n e l e v e l s . 2-4.2 Determination of Serum Protein Binding Characteristics for Mexiletine Enantiomers 2.4.2.1 Determination of Nonspecific Binding of Mexiletine to Ultrafiltration Units Determination of n o n s p e c i f i c binding of m e x i l e t i n e in the u l t r a f i l t r a t i o n system was c a r r i e d out with d u p l i c a t e samples of 10 and 100 ng/ml of m e x i l e t i n e in serum u l t r a f i l t r a t e p r e v i o u s l y obtained from u l t r a f i l t r a t i o n of f r e s h l y c o l l e c t e d serum. Duplicate 1 ml serum samples at each of the m e x i l e t i n e concentrations were subjected to u l t r a f i l t r a t i o n with the C e n t r i f r e e R f i l t r a t i o n u n i t s as described in Section 2 . 3 . 4 . 2 . A f t e r u l t r a f i l t r a t i o n , a 500 fi] a l i q u o t of the u l t r a f i l t r a t e was added to 30 fi] of KOE-2963 s o l u t i o n (1 zzg/ml) as an external standard, followed by serum p r o t e i n p r e c i p i t a t i o n , solvent 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 . With the same procedure, d u p l i c a t e a l i q u o t s of 500 fi] serum u l t r a f i l t r a t e at each of 10 and 100 ng/ml was added to 30 fi] of K0E-2963 s o l u t i o n (1 jug/ml) as an external standard followed by sample preparations f o r HPLC a n a l y s i s without u l t r a f i l t r a t i o n . The peak height r a t i o s obtained from each of the concentrations with u l t r a f i l t r a t i o n were expressed as percent of recovery of m e x i l e t i n e from those obtained without u l t r a f i l t r a t i o n . 2.4.2.2 Determination of Nonspecific Binding of Mexiletine to Equilibrium Dialysis Units The n o n s p e c i f i c binding of m e x i l e t i n e enantiomers to e q u i l i b r i u m d i a l y s i s c e l l s was examined at serum racemic m e x i l e t i n e concentrations of 10 and 100 ng/ml. Duplicate 1 ml serum samples at each of the m e x i l e t i n e concentrations were d i a l y s e d against an equal volume of an i s o t o n i c phosphate b u f f e r (pH 7.4) at 37 °C f o r 4 hours. A f t e r d i a l y s i s , the serum retentate and serum d i a l y s a t e volumes were combined and d u p l i c a t e 1 ml a l i q u o t s at each of the concentrations were analyzed f o r m e x i l e t i n e enantiomers f o l l o w i n g the serum p r o t e i n p r e c i p i t a t i o n and the assay procedure as described p r e v i o u s l y . Duplicate 1 ml serum samples at each of the m e x i l e t i n e concentrations were d i l u t e d with an equal volume of i s o t o n i c phosphate b u f f e r . Duplicate 1 ml a l i q u o t s of the d i l u t e d serum at each of the concentrations were analyzed f o r m e x i l e t i n e enantiomers without d i a l y s i s . The peak height r a t i o from each m e x i l e t i n e enantiomer obtained from the d i a l y s e d serum was expressed as percent of drug recovery compared to those obtained from the non-dialysed serum. 2.4.2.3 Determination of D i a l y s i s Equilibrium Time f o r Mexiletine Freshly c o l l e c t e d serum was adjusted to p h y s i o l o g i c a l pH with the equivalence of 0.2 M sodium phosphate s a l t s . Serum samples with 500 ng/ml of each m e x i l e t i n e enantiomer were d i a l y s e d against an equal volume of a 0.067 M sodium phosphate b u f f e r or against an equal volume of serum u l t r a f i l t r a t e obtained by u l t r a f i l t r a t i o n . D i a l y s i s experiments were c a r r i e d at 37 °C and were allowed to proceed f o r 0 . 5 , 1, 1 . 5 , 2, 3 , 4 or 5 hours. A f t e r d i a l y s i s , the d i a l y s a t e from two c e l l s were i n d i v i d u a l l y assayed f o r m e x i l e t i n e . Since a s i g n i f i c a n t v o l u m e - s h i f t between the serum and b u f f e r compartments may lead to a l t e r a t i o n of m e x i l e t i n e serum f r e e f r a c t i o n , f o l l o w i n g d i a l y s i s , the serum d i a l y s a t e and the b u f f e r r e t e n t a t e volumes were t r a n s f e r r e d from the d i a l y s i s c e l l and weighed to monitor f o r apparent s h i f t in volumes or pH between the d i a l y s a t e and the r e t e n t a t e compartments. 47 2.4.2.4 Comparison of Serum Protein Binding of Mexiletine Enantiomers with U l t r a f i l t r a t i o n and Equilibrium D i a l y s i s Experiments were c a r r i e d out with racemic m e x i l e t i n e serum concentrations of 0 . 5 and 2.0 /zg/ml f o r u l t r a f i l t r a t i o n and e q u i l i b r i u m d i a l y s i s . Serum was c o l l e c t e d from one human subject and adjusted to p h y s i o l o g i c a l pH 7.4 using phosphate b u f f e r s a l t s , as described in Section 2 . 3 . 4 . 2 , p r i o r to u l t r a f i l t r a t i o n or d i a l y s i s . U l t r a f i l t r a t i o n of serum was c a r r i e d out with the C e n t r i f r e e ^ M i c r o p a r t i t i o n f i l t r a t i o n u n i t s at e i t h e r 22*C or 37°C at 1650 g f o r 20 minutes. E q u i l i b r i u m d i a l y s i s was c a r r i e d out with p l e x i g l a s s d i a l y s i s c e l l s , d i a l y s e d against an i s o t o n i c phosphate b u f f e r (pH 7.4) at 37 °C f o r 4 hours. A f t e r d i a l y s i s , the serum r e t e n t a t e was removed and the f r e e drug concentration was determined by u l t r a f i l t r a t i o n . The f r e e drug concentrations obtained from a n a l y s i s of serum u l t r a f i l t r a t e was then compared to those obtained from a n a l y s i s of the d i a l y s a t e b u f f e r i n the corresponding d i a l y s i s c e l l . 2.4.2.5 The Effects of Temperature on Serum Protein Binding of Mexiletine The e f f e c t s of ambient room temperature ( 2 2 ° C ) and 37°C on serum p r o t e i n binding of m e x i l e t i n e enantiomers were evaluated with u l t r a f i l t r a t i o n and e q u i l i b r i u m d i a l y s i s of racemic m e x i l e t i n e at concentrations of 0 . 5 and 2 /zg/ml i n serum. Data were obtained from studies of m e x i l e t i n e serum p r o t e i n binding with u l t r a f i l t r a t i o n and e q u i l i b r i u m d i a l y s i s as described in Section 2 . 4 . 2 . 2 . 48 2.4.2.6 The Effects of Serum pH on the Binding of Mexiletine Enantiomers Measured by Equilibrium D i a l y s i s Four, 2 ml aliquots of serum were in d i v i d u a l l y adjusted to a serum pH of 6.34, 7.38, 7.95 or 8.99 with hydrochloric acid (0.1 M), sodium hydroxide (0.1 M), or with the equivalence of 0.2 M sodium phosphate buffer s a l t s . Duplicate aliquots of 1 ml of each serum sample were dialysed against an isotonic buffer of the corresponding pH at 37 *C for 4 hours. Following d i a l y s i s , mexiletine enantiomer concentrations were determined in the buffer dialysate and in the serum retentate. The data were expressed as mexiletine serum free f r a c t i o n as a function of serum pH. 2.4.2.7 The Effects of Serum pH on the Binding of Mexiletine Enantiomers Measured by U l t r a f i l t r a t i o n Six, 2 ml aliquots of serum were in d i v i d u a l l y adjusted to a serum pH of 6.5, 7.0, 7.4, 8.0, 8.5 or 9.0 with hydrochloric acid (0.1 M), sodium hydroxide (0.1 M), or the equivalence of 0.2 M sodium phosphate buffer s a l t s . U l t r a f i l t r a t i o n of duplicate aliquots of 1 ml of serum was carried out at ambient room temperature with the Centrifree^ f i l t r a t i o n unit at 1650 g f o r 20 minutes. Following u l t r a f i l t r a t i o n , mexiletine enantiomer concentrations were determined in the serum u l t r a f i l t r a t e . Serum free fractions f o r mexiletine enantiomers were obtained from the r a t i o of drug in serum u l t r a f i l t r a t e to that in serum before u l t r a f i l t r a t i o n . 2.4.2.8 Comparison on the Effects of Serum pH Adjustment Methods by Sulphuric Acid and Phosphate Buffers on Mexiletine Free Fraction Following the c o l l e c t i o n of serum (pH « 8 . 5 ) , the serum pH was adjusted to a p h y s i o l o g i c a l value of pH 7.4 using h y d r o c h l o r i c a c i d (0.1 M), phosphoric a c i d (0.1 M) or s u l p h u r i c a c i d (0.1 M). Duplicate a l i q u o t s of 1 ml of serum from each of the pH adjustment methods at 500 ng/ml of racemic m e x i l e t i n e was subjected to u l t r a f i l t r a t i o n at 1650 g f o r 20 minutes. Following u l t r a f i l t r a t i o n , the m e x i l e t i n e enantiomer concentrations in the serum u l t r a f i l t r a t e were determined. The serum u l t r a f i l t r a t e drug concentrations were r e l a t e d to t o t a l serum drug l e v e l s p r i o r to u l t r a f i l t r a t i o n and the data were expressed as serum m e x i l e t i n e f r e e f r a c t i o n s and serum R(-)/S(+) r a t i o . 2 .4.2 .9 Concentration-Linearity of Serum Protein Binding of Mexiletine Enantiomers The serum p r o t e i n binding of m e x i l e t i n e enantiomers was examined at concentrations of 0 . 2 5 , 0 . 5 0 , 1 . 0 , 2 .0 and 3 /zg/ml. Duplicate samples of 1 ml of serum at each of the m e x i l e t i n e concentrations were prepared and were adjusted to serum pH 7.4 with phosphate b u f f e r s a l t s . U l t r a f i l t r a t i o n of serum p r o t e i n s was c a r r i e d out at 1650 g f o r 20 minutes with the C e n t r i f r e e R f i l t r a t i o n u n i t s . Following u l t r a f i l t r a t i o n , m e x i l e t i n e enantiomer concentrations i n the serum u l t r a f i l t r a t e were determined and r e l a t e d to t o t a l serum drug l e v e l s p r i o r to u l t r a f i l t r a t i o n . The data obtained were expressed as serum f r e e f r a c t i o n and serum R(-)/S(+) r a t i o f o r each of the enantiomers. 2.4.3 In Vitro Determination of the Extent and Time-Course of M e x i l e t i n e D i s t r i b u t i o n i n t o Red Blood C e l l s Red blood c e l l s were prepared by repeatedly washing f r e s h l y c o l l e c t e d whole blood with an i s o t o n i c s a l i n e s o l u t i o n (pH 7 . 4 ) . A red c e l l suspension was then prepared by r e c o n s t i t u t i o n with an i s o t o n i c phosphate b u f f e r (pH 7.4) to a hematocrit of 0 . 5 . The red c e l l suspension was adjusted to a racemic m e x i l e t i n e concentration of 200 ng/ml and immediately incubated i n a 37 °C water bath f o r 5 , 10, 20, 40, 80, and 120 minutes. A f t e r the s p e c i f i e d incubation t i m e , d u p l i c a t e 1 ml samples were removed and washed with 10 ml of i s o t o n i c phosphate b u f f e r at pH 7.4 ( p r e v i o u s l y warmed to 37 ° C ) . The red c e l l s were immediately c e n t r i f u g e d f o r 30 seconds, and the b u f f e r removed. The red c e l l s were then l y s e d by the a d d i t i o n of 1 ml of d i s t i l l e d water and the m e x i l e t i n e enantiomer concentrations were determined as p r e v i o u s l y described i n Section 2 . 3 . 4 . 1 . 2.4.4 Pharmacokinetic Study of M e x i l e t i n e Enantiomers i n Healthy Human Subjects 2.4.4.1 V a l i d a t i o n of the Potency of M e x i t i l R 200 mg Capsules M e x i t i l ^ capsules were s u p p l i e d with a l a b e l c l a i m of 200 mg. The content of 10 capsules were mixed and d u p l i c a t e samples equivalent to 200 mg m e x i l e t i n e hydrochloride dose were d i s s o l v e d in 200 ml of d i s t i l l e d water. To each a l i q u o t of the aqueous samples equivalent to 100 ng of each enantiomer, 30 ng of KOE-2963 was added as an i n t e r n a l standard and the samples were adjusted to a l k a l i n e pH with sodium hydroxide, extracted with two p o r t i o n s of 3 ml d i e t h y l e t h e r , and d e r i v a t i z e d with 2-anthroyl c h l o r i d e by v o r t e x - m i x i n g . The amount of m e x i l e t i n e enantiomers obtained was expressed as percentage of the l a b e l 5 1 c l a i m . 2.4.4.2 Selection of Healthy Human Subjects Twelve healthy Caucasian male volunteers (19 to 25 years of age) were e n r o l l e d i n t o the study with informed w r i t t e n consent and with Human C l i n i c a l I n v e s t i g a t i o n Ethics approval . The body height and weight of the volunteers were w i t h i n the Metropolitan L i f e measurement l i m i t s (1983). A 12-lead EKG recording was determined f o r each subject p r i o r to the study and at one and o n e - h a l f 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 . P h y s i c a l examinations, as well as blood and u r i n e b i o c h e m i s t r y , were evaluated f o r each subject before and a f t e r the study. 2.4.4.3 Physical, Urine and Haemetology Examination General p h y s i c a l examinations i n c l u d i n g blood pressures and r e s t i n g heart r a t e s were conducted w i t h each of the subjects on the day of the study. Haematological assessments and serum biochemistry t e s t s f o r each of the subjects were conducted no more than 10 days p r i o r t o , and f o l l o w i n g , each study. Urine pH, urine g l u c o s e , urine p r o t e i n , urine 24-hour c r e a t i n i n e c l e a r a n c e , and urine microscopic examinations were a l s o conducted p r i o r t o , and f o l l o w i n g , each study. A l l haematological and u r i n e biochemistry values from each subject were w i t h i n 10% of the c l i n i c a l standard normal values (as determined by the c l i n i c a l l a b o r a t o r y , Acute Care H o s p i t a l , The U n i v e r s i t y of B r i t i s h Columbia). A l i s t of the haematological and urine biochemistry t e s t s performed on each subject i s shown i n Appendix 1. 52 2.4.4.4 Drug Administration and Serum, Saliv a , Urine and Red Blood C e l l C o l l e c t i o n . A 200 mg racemic m e x i l e t i n e hydrochloride c h l o r i d e ( M e x i t i l R ) capsule was administered to each subject with 200 ml of water f o l l o w i n g an overnight (8 hours) f a s t . Venous blood samples (8 ml) were c o l l e c t e d from the forearm at 0 , 0 . 5 , 1, 1 . 5 , 2 , 2 . 5 , 3 , 3 . 5 , 4 , 4 . 5 , 6 , 8 , 10, 12, and 14 hours through an i n d w e l l i n g B u t t e r f l y ^ cannula into V a c u t a i n e r s R with s i l i c o n - c o a t e d stoppers. The cannula was f l u s h e d with 1 ml of a s t e r i l e , i s o t o n i c heparin s o l u t i o n ( d i l u t e d to 50 IU/ml with normal s a l i n e f o r i n j e c t i o n ) a f t e r each blood sampling. A d d i t i o n a l blood samples were c o l l e c t e d at 24, 30, 36, and 48 hours by i n d i v i d u a l venipuncture. Blood samples were allowed to c l o t at room temperature f o r 2 hours, then c e n t r i f u g e d at 2500 g f o r 15 minutes, and the serum was separated and stored at -20 ° C . S a l i v a samples were c o l l e c t e d , without s t i m u l a t i o n , into 20 ml g l a s s v i a l s at the same time as f o r blood samples. The s a l i v a pH was measured by a p o r t a b l e pH-meter f i t t e d with a microelectrode immediately a f t e r c o l l e c t i o n and the samples were stored at -20 °C u n t i l required f o r a n a l y s i s . Urine samples were c o l l e c t e d i n s t e r i l e p l a s t i c W h i r l -Pak R bags at 0 , 1, 2 , 3 , 4 , 5 , 6 , 8 , 10, 12 and 14 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 . A d d i t i o n a l u r i n e samples were c o l l e c t e d at the convenience of the subject f o r up to 72 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 . The u r i n e pH was measured with a portable pH-meter immediately a f t e r each u r i n e sample c o l l e c t i o n . The urine volumes were obtained by m u l t i p l y i n g the weight of each urine sample with the mean s p e c i f i c g r a v i t y of urine from each s u b j e c t . Red blood c e l l samples were c o l l e c t e d at the same s p e c i f i e d times as serum samples. In 53 d u p l i c a t e from each venous blood sample, a d u p l i c a t e of 1 ml sample was t r a n s f e r r e d immediately a f t e r c o l l e c t i o n into a 10 ml i s o t o n i c phosphate b u f f e r s o l u t i o n at pH 7 . 4 . The red c e l l suspension was c e n t r i f u g e d at 1500 g f o r « 1 5 seconds and the supernatant i s o t o n i c b u f f e r was immediately removed and d i s c a r d e d . The r e s u l t a n t red c e l l sample was stored at -20 ° C . 2 . 5 Development of a Stereoselective HPLC Assay f o r Mexiletine, p-Hydroxy-mexiletine, and Hydroxymethyl-Mexiletine Enantiomers i n Urine The r e s o l u t i o n of m e x i l e t i n e , p-OH-mexiletine and OH-methyl-m e x i l e t i n e enantiomers was attempted using three types of P i r k l e R c h i r a l HPLC columns with the f o l l o w i n g s t a t i o n a r y phases: i P i r k l e R d i n i t r o b e n z o y l D-phenylglycine i o n i c s t a t i o n a r y phase. i i P i r k l e R d i n i t r o b e n z o y l L - i s o l e u c i n e i o n i c s t a t i o n a r y phase. i i i P i r k l e R d i n i t r o b e n z o y l L - i s o l e u c i n e covalent s t a t i o n a r y phase. Corresponding to the three HPLC s t a t i o n a r y phases, the f o l l o w i n g experimental procedures are thus d i v i d e d i n t o three s e c t i o n s , each d e a l i n g with d i f f e r e n t attempts to r e s o l v e the enantiomers using various d e r i v a t i z a t i o n methods. 2 . 5 . 1 P i r k l e R Dinitrobenzoyl D-Phenylglycine Ionic Column 2 . 5 . 1 . 1 Acylation of Mexiletine and Mexiletine Metabolites Using 2-Anthroyl Chloride or 2-Naphthoyl Chloride 2 . 5 . 1 . 1 . 1 Mexiletine Enantiomers A 0 . 5 fig sample of racemic m e x i l e t i n e was acylated with 10 /zl of a 2-anthroyl c h l o r i d e (1 mg/ml i n dichloromethane) or 10 /zl of a 2-naphthoyl c h l o r i d e s o l u t i o n (1 mg/ml in dichloromethane) using the Schotten-Baumann r e a c t i o n c o n d i t i o n s (Baumann, 1886) by vortex-mixing 54 the acylating mixture with 0.2 ml of an aqueous sodium hydroxide solution (2.0 M). Acylation was previously found to be completed within 3 minutes at ambient room temperature. Following d e r i v a t i z a t i o n , the mexiletine derivatives were extracted into dichloromethane followed by HPLC analysis of the derivatives on the P i r k l e R ionic phenylglycine HPLC column. 2.5.1.1.2 p-Hydroxy-Mexiletine Enantiomers A 0.5 fig sample of racemic p-OH-mexiletine was acylated with 10 fi\ of 2-anthroyl chloride or 10 fi\ of 2-naphthoyl chloride reagent (1 mg/ml in dichloromethane) by vortex-mixing the acylation mixture with 0.2 ml of an aqueous sodium hydroxide (2.0 M) or 0.2 ml of an aqueous sodium bicarbonate solution (5.0 M, pH 10). Derivatization was carried out f o r 3 minutes at ambient room temperature. After the reaction, the mexiletine metabolite derivatives were extracted into dichloromethane followed by HPLC analysis. 2.5.1.1.3 Hydroxymethyl-Mexiletine Enantiomers A 0.5 fig sample of racemic OH-methyl-mexiletine was acylated with 10 fi\ of 2-anthroyl chloride or 10 fi\ of 2-naphthoyl chloride reagent (1 mg/ml in dichloromethane) by vortex-mixing the acylating mixture with 0.2 ml of an aqueous sodium hydroxide solution (2.0 M) for three minutes. Following the reaction, an aliquot of the sample was removed and the derivatives were extracted into dichloromethane f o r HPLC analysis. The remaining sample was heated at 60 °C for up to 60 minutes in a dry heating block. Aliquots of the heat treated sample were removed at 20, 30, 40 and 60 minutes and the OH-methyl-mexiletine derivatives were extracted into dichloromethane f o r HPLC evaluation of the formation of varying proportions of the mono- and di-acylated 55 d e r i v a t i v e s . 2.5.1.2 S i l y l a t i o n of M e x i l e t i n e Metabolites with N - T r i m e t h y l s i l y l Imidazole (TMSI) Followed by A c y l a t i o n w i t h 2-Naphthoyl C h l o r i d e 2.5.1.2.1 p-Hydroxy-Mexiletine Enantiomers A 0 . 5 fig sample of racemic p-OH-mexiletine was s i l y l a t e d with 10 /zl of TMSI reagent at 60 ° C . A l i q u o t s of the d e r i v a t i z a t i o n sample were removed at 0 . 5 , 1, 2 and 3 hours and the t r i m e t h y l s i l y l d e r i v a t i v e of p-OH-mexiletine was extracted into 2 ml of dichloromethane. In separate experiments, p y r i d i n e (10 /zl) or t r i e t h y l a m i n e (10 /zl) were used as s i l y l a t i o n c a t a l y s t s and heated at 60 °C f o r 1 hour. Following s i l y l a t i o n , the t r i m e t h y l s i l y l d e r i v a t i v e of p-OH-mexiletine was acylated with 10 /zl of 2-naphthoyl c h l o r i d e (1 mg/ml of dichloromethane) by vortex-mixing the r e a c t i o n mixture with 0.2 ml of an aqueous sodium hydroxide s o l u t i o n (2 M) f o r 3 minutes. The r e s u l t i n g s i l y l a t e d / a c y l a t e d d e r i v a t i v e was extracted into 2 ml of dichloromethane. The solvent was evaporated under a stream of nitrogen and the sample r e c o n s t i t u t e d i n 0.2 ml of a HPLC mobile phase p r i o r to a n a l y s i s . 2.5.1 .2.2 Hydroxymethyl-Mexiletine Enantiomers A 0 . 5 fig sample of racemic OH-methyl-mexiletine was s i l y l a t e d with 100 #1 of TMSI reagent i n the presence of 100 /zl of p y r i d i n e at 60 ° C . A l i q u o t s of 50 /zl of the r e a c t i o n mixture were removed at 0 . 5 , 1, 2 and 3 hours and the s i l y l a t e d OH-methyl-mexiletine d e r i v a t i v e was extracted i n t o 2 ml of dichloromethane. The e x t r a c t i o n solvent was removed and evaporated to dryness under a stream of nitrogen i n a 35 °C water bath. The s i l y l a t e d d e r i v a t i v e s were subsequently acylated with 10 /zl of 2-naphthoyl c h l o r i d e (1 mg/ml) by vortex-mixing the reagent with an aqueous sodium bicarbonate s o l u t i o n (5 M) f o r 3 minutes at ambient room temperature. The s i l y l a t e d / a c y l a t e d products of OH-methyl-mexiletine d e r i v a t i v e s were extracted into 2 ml of dichloromethane and the solvent separated and evaporated to dryness under a stream of n i t r o g e n . The sample was r e c o n s t i t u t e d i n 0.2 ml of HPLC mobile phase p r i o r t o a n a l y s i s . 2.5.1.3 Acylation of Mexiletine Metabolites with 2-Anthroyl Chloride Followed by Alk y l a t i o n with Ethyl Bromide 2.5.1.3.1 p-Hydroxy-Mexiletine Enantiomers A 0 . 5 fig sample of racemic p-OH-mexiletine was acylated with 10 fi\ of a 2-anthroyl c h l o r i d e s o l u t i o n (1 mg/ml i n dichloromethane) by vortex-mixing the r e a c t i o n mixture with 0.2 ml of an aqueous sodium bicarbonate s o l u t i o n (5 M) f o r 3 minutes at ambient room temperature. The r e s u l t i n g anthroyl d e r i v a t i v e s of p-OH-mexiletine were extracted i n t o 2 ml of dichloromethane and the solvent was separated and evaporated under a stream of nitrogen i n a 35 "C water b a t h . The anthroyl d e r i v a t i v e s of p-OH-mexiletine were then a l k y l a t e d using 100 (A of e t h y l bromide i n the presence of 50 fi\ of e t h a n o l i c potassium hydroxide (0.1 M) at 60 °C f o r 40 minutes. Following the r e a c t i o n , the v o l a t i l e organic reagents were removed by evaporation under a stream of nitrogen in a 35 °C water bath and the a c y l a t e d / a l k y l a t e d p-OH-m e x i l e t i n e d e r i v a t i v e s were extracted i n t o dichloromethane f o r HPLC a n a l y s i s . 2.5.1.3.2 Hydroxymethyl-Mexiletine Enantiomers A racemic 0 . 5 jttg sample of OH-methyl-mexiletine was acylated with 10 fi\ of 2-anthroyl c h l o r i d e (1 mg/ml in dichloromethane) by vortex-mixing the r e a c t i o n mixture with 0.2 ml of an aqueous sodium bicarbonate s o l u t i o n (5 M) followed by a l k y l a t i o n of the acylated d e r i v a t i v e s with 50 fi\ of ethyl bromide in the presence of 100 ii\ of an ethanol i c potassium hydroxide s o l u t i o n (0.1 M) as described f o r p-OH-mexiletine in Section 2 . 5 . 1 . 3 . 1 . 2 . 5 . 2 P i r k l e R dinitrobenzoyl L-Isoleucine ionic HPLC Column 2 . 5 . 2 . 1 Acylation Mexiletine and Mexiletine Metabolites with 2-Anthroyl Chloride or 2-Naphthoyl Chloride A 0 . 5 fiq sample of racemic m e x i l e t i n e , p-OH-Mexiletine or OH-m e t h y l - m e x i l e t i n e was acylated with 10 (A of 2-anthroyl c h l o r i d e reagent in the presence of 0.2 ml of aqueous sodium bicarbonate s o l u t i o n ( 5 . 0 M) at room temperature as p r e v i o u s l y described in Section 2 . 5 . 1 . 1 . 1 . The acylated products were extracted into dichloromethane f o r HPLC a n a l y s i s using the P i r k l e R L - l e u c i n e column. 2 . 5 . 2 . 2 Methylation of Mexiletine Metabolites with Diazomethane Followed by Acylation Using 2-Anthroyl Chloride 2 . 5 . 2 . 2 . 1 p-Hydroxy-Mexiletine Enantiomers The phenolic-OH group of p-OH-mexiletine was methylated with f r e s h l y generated diazomethane in a c i d i c methanol. Into a 10 ml round bottom f l a s k , 2 ml each of d i e t h y l ether and 2-(2-ethoxyethoxy)ethanol ( C a r b i t o l R ) were added to 30 mg of N-methyl-N-nitroso-p-toluene ( D i a z a l d R ) . A rubber stopper was placed over the f l a s k with an i n l e t l i n e d e l i v e r i n g a constant stream of nitrogen at a r a t e of 0 . 5 ml/min. Diazomethane gas was generated by the a d d i t i o n of 1 ml of potassium hydroxide s o l u t i o n (60% w/v), and the diazomethane was d e l i v e r e d by an o u t l e t l i n e i n t o 1 ml of an a c i d i c methanolic s o l u t i o n c o n t a i n i n g p-OH-m e x i l e t i n e . E s t e r i f i c a t i o n was continued u n t i l a c l e a r yellow c o l o r p e r s i s t e d f o r 2 minutes i n s o l u t i o n . Following the r e a c t i o n , the methanol was removed by evaporation under a stream of nitrogen in a 35 °C water bath. The methylated p-OH-mexiletine d e r i v a t i v e s were subsequently acylated by the a d d i t i o n of 10 /zl of 2-anthroyl c h l o r i d e (1 mg/ml) f o l l o w e d by vortex-mixing the reagent with 0.2 ml of an aqueous sodium hydroxide s o l u t i o n f o r 3 minutes. The methylated and acylated d e r i v a t i v e s of p - m e x i l e t i n e were i s o l a t e d into dichloromethane f o r HPLC a n a l y s i s . 2.5.2.2.2 Hydroxymethyl-Mexiletine Enantiomers Methylation of 0 . 5 /zg of racemic OH-methyl-mexiletine with diazomethane was c a t a l y z e d by the a d d i t i o n of 0.1 ml of t r i f l u o r o b o r o e t h e r a t e at 0°C f o l l o w i n g the procedure as described f o r p-OH-mexiletine i n Section 2 . 5 . 2 . 2 . 1 . 2.5.3 P i r k l e R Dinitrobenzoyl L-Isoleucine Covalent Chiral HPLC Column 2.5.3.1 Acylation of Mexiletine Metabolites with 2-Anthroyl Chloride or 2-Naphthoyl Chloride Followed by Alkylation with Alkyl Hal ides 2.5.3.1.1 p-Hydroxy-Mexiletine Enantiomers A c y l a t i o n of 0 . 5 /zg of racemic p-OH-mexiletine was c a r r i e d out with 10 /zl of 2-anthroyl c h l o r i d e reagent (1 mg/ml in dichloromethane) by vortex-mixing the r e a c t i o n with 0.2 ml of an aqueous sodium bicarbonate s o l u t i o n ( 5 . 0 M). The acylated p-OH-mexiletine d e r i v a t i v e s were e x t r a c t e d i n t o 2 ml of dichloromethane followed by a l k y l a t i o n of the phenolic-OH with 50 /zl of ethyl bromide and 100 /zl of a potassium hydroxide s o l u t i o n (0.1 M i n ethanol) at 60 °C f o r 45 minutes to a f f o r d the N - a n t h r o y l d e r i v a t i v e of ethyl phenyl e t h e r . In separate experiments, 50 /zl of n-propyl bromide or n-butyl bromide were a l s o used as a l k y l a t i n g reagents to examine the s i z e of the a l k y l d e r i v a t i v e s on the chromatographic r e s o l u t i o n of the p-OH-mexiletine enantiomers. 2.5.3.1.2 Hydroxymethyl-Mexiletine Enantiomers A 0 . 5 VQ sample of racemic OH-methyl-mexiletine was acylated with 10 /zl of 2-anthroyl c h l o r i d e reagent by vortex-mixing the r e a c t i o n with 0.2 ml of aqueous sodium bicarbonate s o l u t i o n ( 5 . 0 M). The acylated OH-methyl - m e x i l e t i n e d e r i v a t i v e s were extracted into dichloromethane and i s o l a t e d f o r a l k y l a t i o n by the a d d i t i o n of 50 #1 of e t h y l bromide and 100 /i] of e t h a n o l i c potassium hydroxide as described in Section 2 . 5 . 3 . 1 . 1 . 2.5.4 Assay Method f o r Mexiletine, p-Hydroxy-Mexiletine and Hydroxymethyl-Mexiletine Enantiomers i n Urine Duplicate a l i q u o t s of 0.2 to 1.0 ml of urine were adjusted to pH 9 . 0 with the a d d i t i o n of 0 . 5 ml of phosphate b u f f e r (0.4 M) and extracted two times with 3 ml of dichloromethane/ether ( 2 0 : 8 0 ) . Following e x t r a c t i o n s , the solvent was removed and the aqueous f r a c t i o n was adjusted t o pH>12 with the a d d i t i o n of 0.2 ml sodium hydroxide (2 M). The a l k a l i n e u r i n e was then e x t r a c t e d with 2 ml of d i e t h y l e t h e r . The pooled e x t r a c t i o n solvents were concentrated to « 1 . 0 ml under a stream of nitrogen in a 35 °C water bath followed by the a d d i t i o n of 0.2 ml of a saturated aqueous sodium bicarbonate s o l u t i o n and 10 #1 of 2-anthroyl c h l o r i d e (1 mg/ml i n dichloromethane). A c y l a t i o n of m e x i l e t i n e and i t s hydroxylated metabolites were c a r r i e d out by vortex-mixing the reagents at ambient room temperature f o r 3 minutes. The excess 2-anthroyl c h l o r i d e was reacted with 100 /xl of methyl amine. The sample was then adjusted to pH >12 with 0.2 ml sodium hydroxide (2 M) and the N-anthroyl d e r i v a t i v e s of m e x i l e t i n e and OH-methyl-mexiletine were e x t r a c t e d 2 times with 2 ml of hexane. The aqueous f r a c t i o n was 60 a c i d i f i e d with 0 . 3 ml of h y d r o c h l o r i c a c i d (2 M) and the N-anthroyl d e r i v a t i v e of p-OH-mexiletine was extracted into 2 ml of dichloromethane. The dichloromethane was evaporated to dryness under a stream of nitrogen followed by the a d d i t i o n of 100 (i\ of e t h a n o l i c potassium hydroxide ( 0 . 5 M) and 50 (i\ of ethyl bromide. A l k y l a t i o n of the hydroxy group of p-OH-mexiletine was predetermined to be optimal at 60 °C f o r 40 minutes a f t e r which the a l k y l a t e d product was extracted i n t o 2 ml of dichloromethane. The dichloromethane and hexane e x t r a c t s were pooled, evaporated to dryness under nitrogen and r e c o n s t i t u t e d with 0.2 ml of HPLC mobile phase p r i o r to HPLC a n a l y s i s . 2.5.5 Stereoselective HPLC of Mexiletine, Hydroxymethyl-Mexiletine and p-Hydroxy-Mexiletine Enantiomers in Urine A Hewlett-Packard model 1082B l i q u i d chromatograph was used with a Hewlett-Packard 1062B F i r e f l y R f luorescence detector optimized with e x c i t a t i o n and emission wavelengths of 270 and 420 nm, r e s p e c t i v e l y . A P i r k l e R l e u c i n e covalent c h i r a l column was connected in s e r i e s with an A l l t e c h s i l i c a column (adjacent to the i n j e c t o r ) . The mobile phase c o n s i s t e d of h e x a n e / 2 - p r o p a n o l / e t h a n o l / a c e t o n i t r i l e / e t h y l acetate (90/4/4/1/1) at programmed flow r a t e s of 0 . 6 ml/min from 0 to 30 minutes followed by 2.0 ml/min from 30 to 50 minutes. 2.5.6 The Time-Course of Alkylation of p-Hydroxy-Mexiletine Enantiomers with Ethyl Bromide Stock s o l u t i o n s of p-OH-mexiletine (1.4 mg/100 ml) and KOE-2963 (0.1 /KJ/100 ml) were prepared. An a l i q u o t of 20 fi\ of the p-OH-m e x i l e t i n e stock s o l u t i o n and 100 fi\ of the K0E-2963 i n t e r n a l standard s o l u t i o n were t r a n s f e r r e d to s i x separate tubes. The samples were then adjusted to b a s i c pH (pH » 1 0 ) with a sodium bicarbonate s o l u t i o n and acylated with 2-anthroyl c h l o r i d e at room temperature f o r three minutes followed by the a d d i t i o n of methylamine (100 #1) to destroy the excess anthroyl c h l o r i d e . The N-anthroyl d e r i v a t i v e of p-OH-mexiletine was extracted with 2 ml of dichloromethane and the solvent was t r a n s f e r r e d and evaporated to dryness under a stream of n i t r o g e n . A l k y l a t i o n of the hydroxy group of p-OH-mexiletine with e t h y l bromide (100 (A) in e t h a n o l i c potassium hydroxide (100 jul) was conducted f o r 10, 20, 30, 40, 50, or 60 minutes at 60 *C i n a closed screw-cap g l a s s tube. Following a l k y l a t i o n , the metabolite was extracted i n t o dichloromethane (2 ml) and the solvent removed and evaporated to dryness under a stream of n i t r o g e n . Each sample was r e c o n s t i t u t e d with 1 ml HPLC mobile phase p r i o r to a n a l y s i s . 2.5.7 Solvent Extraction Recovery of Mexiletine, p-Hydroxy-Mexiletine, and Hydroxymethyl-Mexiletine Enantiomers The r e c o v e r i e s of m e x i l e t i n e , p-OH-mexiletine, and OH-methyl-m e x i l e t i n e enantiomers from urine were determined with ethyl a c e t a t e , e t h e r , and dichloromethane/ether (20:80) as e x t r a c t i o n s o l v e n t s . K0E-2963 was acylated with 2-anthroyl c h l o r i d e and was used as an external standard. The r e c o v e r i e s of m e x i l e t i n e and m e x i l e t i n e metabolites from u r i n e were determined using d u p l i c a t e samples of 10 and 100 ng of each of m e x i l e t i n e and the two m e t a b o l i t e s . Solvent e x t r a c t i o n and d e r i v a t i z a t i o n procedures were c a r r i e d out as p r e v i o u s l y described f o r m e x i l e t i n e in Section 2 . 5 . 4 . The data obtained from the extracted samples were expressed as percent recovery of drug and drug metabolites compared to those obtained from the non-extracted samples. 62 2.5.8 Detection Response Li n e a r i t y Calibration f o r Mexiletine, p-Hydroxy-Mexiletine and Hydroxymethyl-Mexiletine Enantiomers in Urine C a l i b r a t i o n data f o r each m e x i l e t i n e enantiomer in urine were obtained using t r i p l i c a t e samples of 10, 25, 50, 100, 250 and 500 ng/ml of u r i n e . To the r e s p e c t i v e sample, 25, 50, 100, 500, 1000 and 1500 ng of each of p-OH-mexiletine and OH-methyl-mexiletine enantiomers were prepared. To each sample, 100 ng of KOE-2963 was used as an i n t e r n a l standard f o r m e x i l e t i n e and OH-methyl-mexiletine, w h i l e 200 ng of tyramine was used as i n t e r n a l standard f o r p-OH-mexiletine. Urine samples were then extracted with dichloromethane/diethyl ether (20/80) and w i t h d i e t h y l ether at the corresponding pH 9 and 12. Following e x t r a c t i o n , m e x i l e t i n e and the two metabolites were d e r i v a t i z e d with 2-anthroyl c h l o r i d e f o l l o w e d by e t h y l bromide as described p r e v i o u s l y i n Section 2 . 5 . 4 . The N-anthroyl d e r i v a t i v e s of m e x i l e t i n e and OH-methyl-m e x i l e t i n e as w e l l as the 0 - e t h y l - N - a n t h r o y l d e r i v a t i v e of p-OH-m e x i l e t i n e were r e c o n s t i t u t e d in 0 . 2 ml of mobile phase p r i o r to HPLC a n a l y s i s . C a l i b r a t i o n of m e x i l e t i n e , p-OH-mexiletine, and OH-methyl-m e x i l e t i n e enantiomers i n urine was performed p r i o r to a n a l y s i s of u r i n e sample from each s u b j e c t . 2.5.9 Determination of Assay V a r i a b i l i t y f o r Mexiletine, p-Hydroxy-Mexiletine and Hydroxymethyl-Mexiletine Enantiomers in Urine The v a r i a b i l i t y i n the determination of m e x i l e t i n e , p-OH-m e x i l e t i h e , and OH-methyl-mexiletine enantiomers between sample preparations ( i n t e r - a s s a y ) was obtained by s i n g l e HPLC a n a l y s i s of each of the t r i p l i c a t e c a l i b r a t i o n samples over the c a l i b r a t i o n range as o u t l i n e d i n Section 2 . 5 . 8 . The v a r i a b i l i t y in sample measurement ( i n t r a - a s s a y ) was examined by t r i p l i c a t e a n a l y s i s of one of the c a l i b r a t i o n samples over the c a l i b r a t i o n range as o u t l i n e d in Section 2 . 5 . 8 . The v a r i a b i l i t y data was expressed as c o e f f i c i e n t of v a r i a t i o n ( C . V . ) . 2 . 6 Pharmacokinetic Data A n a l y s i s 2 . 6 . 1 Computer Curve F i t t i n g The serum, s a l i v a and blood c e l l m e x i l e t i n e enantiomer concentrations from each of the twelve subjects were analyzed using Autoan (Sedman and Wagner, 1976), a program which mathematically describes drug concentration data and provides i n i t i a l estimates of pharmacokinetic parameters of various pharmacokinetic compartmental models. The i n i t i a l estimates from each of the subjects were compared to the values derived from p l o t t i n g the l o g a r i t h m i c drug concentrations versus t i m e . The i n i t i a l k i n e t i c parameters obtained from Autoan were then r e f i n e d by NONLIN (Metzler et a l . , 1974), a nonlinear l e a s t - s q u a r e curve f i t t i n g program, to obtain the f i n a l pharmacokinetic parameters. The apparent f i r s t - o r d e r absorption ( k a ) , r a p i d d i s p o s i t i o n (a) and slow d i s p o s i t i o n rate constant (B) were obtained with equal weighting of a l l the data p o i n t s . The u r i n a r y m e x i l e t i n e and m e x i l e t i n e metabolite data were analyzed by PKCAL (Shumaker, 1984), a pharmacokinetic curve f i t t i n g program, as a p l o t of the amount of drug or metabolite remaining t o be excreted i n urine (ARE) versus t ime. The terminal e l i m i n a t i o n rate constants and e l i m i n a t i o n h a l f - l i v e s f o r m e x i l e t i n e enantiomers derived from u r i n e data were obtained from the terminal slope of the ARE-plots. 64 2 . 6 . 2 C a l c u l a t i o n of Pharmacokinetic Parameters The pharmacokinetic parameters that describe the time-course of a drug in the body are discussed in G i b a l d i and P e r r i e r , (1975; 1982). The area under the serum concentration versus time curve (AUC0 - C O) was c a l c u l a t e d with the f o l l o w i n g equation: A U C o - « = A U C 0 ^ + AUCt'0 0 (1) where t represented the time of l a s t sampling. The f i r s t term, A U C 0 - t , was c a l c u l a t e d by the t r a p e z o i d a l r u l e and the second term was c a l c u l a t e d w i t h : AUCt"00 = C t / 8 (2) where Ct was the concentration of the l a s t c o l l e c t e d sample, and B was the terminal d i s p o s i t i o n r a t e constant. The terminal d i s p o s i t i o n h a l f - l i f e was c a l c u l a t e d from the equation: The o r a l absorption and r a p i d d i s p o s i t i o n h a l f - l i v e s were determined with equation 4 and 5 r e s p e c t i v e l y : 0.693/B (3) 0.693/ka (4) 0.693/a (5) The time to achieve peak serum concentration ( t m a x ) was c a l c u l a t e d from: tmax = [2.303/ka - 8] l o g [ka/6J (6) where ka and 8 were the r e s p e c t i v e absorption and terminal e l i m i n a t i o n r a t e c o n s t a n t s , under the assumption that ka was much g r e a t e r than 8. The peak serum drug concentration ( C m a x ) was estimated from the serum concentration versus time curve. The apparent o r a l clearance (CLoral) was described by the equation: where F was the f r a c t i o n of dose reaching the systemic c i r c u l a t i o n and was not determined due to the l a c k of approval from the Health P r o t e c t i o n Branch of Canada to administer m e x i l e t i n e intravenously to healthy v o l u n t e e r s . The apparent volume of d i s t r i b u t i o n ( V d a r e a ) was c a l c u l a t e d by the r e l a t i o n s h i p : Vdarea/F = Dose/AUC-8 (8) The amount of drug remaining to be excreted in the u r i n e (ARE) during the post-absorption phase of drug d i s p o s i t i o n was described by the f o l l o w i n g r e l a t i o n s h i p s : CLoral/F = dose/AUC°- GO (7) l o g (XUo, - X u t ) = l o g [ F - D B ° - k e / K E ] - K E ' t / 2 . 3 0 3 (9) where (Xu™ - Xut) was the amount of drug remaining to be excreted i n u r i n e ; ke was the u r i n a r y e x c r e t i o n r a t e constant and Dp/ represented the i n i t i a l amount of drug i n the body. The terminal e l i m i n a t i o n h a l f -l i f e ( K E ) , derived from urine d a t a , was c a l c u l a t e d from the terminal slope of the ARE-plot. The amount of drug metabolite remaining to be excreted i n the u r i n e during the post-absorption phase was described by the Sigma-Minus p l o t (Mayersohn and G i b a l d i , 1969): ( M u - - M u t ) » [ M u - k m u / ( k m u - K E ) ] e - K E t - [ M u«K E / ( k M U -K E ) ] e - k m u t (10) where (Mu,,, - Mut) was the amount of metabolite remaining to be excreted i n the u r i n e . K E and k m u were the e l i m i n a t i o n r a t e constants f o r m e x i l e t i n e and the u r i n a r y e x c r e t i o n r a t e constant f o r the m e t a b o l i t e , r e s p e c t i v e l y . The r e l a t i o n s h i p assumed k m u was much greater than K E , and k m u was the only e l i m i n a t i o n process f o r the metabolite i n the body. The o v e r a l l renal clearance ( C L r ) , derived from u r i n e d a t a , was obtained from the equation: CL r - Xu'VAUC0-00 (11) The non-renal e l i m i n a t i o n r a t e constant ( k n r ) was estimated using the r e l a t i o n s h i p : k n r - B ( l - f ) (12) 67 where f was the f r a c t i o n of unchanged drug excreted in the u r i n e . 2 . 6 . 3 S t a t i s t i c a l Data A n a l y s i s Student paired t - t e s t was used f o r evaluation of a s t a t i s t i c a l s i g n i f i c a n t d i f f e r e n c e in the mean pharmacokinetic parameters obtained f o r the two m e x i l e t i n e enantiomers. One-way a n a l y s i s of variance (ANOVA) was used to evaluate the mean values of two or more sample treatment comparisons. Levels of s i g n i f i c a n c e w i t h p<0.05 or p<0.01 were chosen and the mean data presented were expressed as mean ± one standard d e v i a t i o n unless otherwise s t a t e d . 3. RESULTS AND DISCUSSION 3.1 Development of 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 Assay f o r M e x i l e t i n e Enantiomers 3.1.1 Synthesis of 2-Anthroyl C h l o r i d e In order to develop 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 assay f o r the determination of the enantiomers of m e x i l e t i n e in serum, s a l i v a , red blood c e l l s and u r i n e , 2-anthroyl c h l o r i d e was synthesized. This h i g h l y f l u o r e s c e n t d e r i v a t i z a t i o n reagent provided an increase in d e t e c t i o n s e n s i t i v i t y compared to an e a r l i e r report (McErlane et a J . , 1987), and as w e l l , r e t a i n e d the necessary c h i r a l r e c o g n i t i o n requirements f o r enantiomeric r e s o l u t i o n on the c h i r a l s t a t i o n a r y phases. As shown i n Figure 3 , the synthesis of 2-anthroyl c h l o r i d e was c a r r i e d out using commercially a v a i l a b l e anthraquinone-2-carboxylic a c i d (XIV). Reduction of the anthraquinone to anthracene has been reported to y i e l d varying proportions of 9,10-dihydroxy-9,10-dihydroanthracene, 9,10-dihydroxyanthracene, anthrone, a n t h r o l , anthracene, and other minor products (Sanchorawala et a 7 . , 1963). However, the use of a zinc/ammonia s o l u t i o n was found to give reproducible y i e l d s of the d e s i r 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 (XV). A c y l a t i o n of the c a r b o x y l i c a c i d was achieved with an excess of o x a l y l c h l o r i d e to y i e l d 2-anthroyl c h l o r i d e . The 2-anthroyl c h l o r i d e (XVI) reagent was f u r t h e r p u r i f i e d by p r e p a r a t i v e HPLC with an ODS-2 reverse-phase column, the s y n t h e t i c m a t e r i a l was i s o l a t e d with an a c e t o n i t r i l e mobile phase which had been p r e v i o u s l y d r i e d over calcium c h l o r i d e . The p u r i t y of the p u r i f i e d reagent was also ascertained with reverse-phase t h i n - l a y e r chromatography. 69 Figure 3 . The synthesis of 2-anthroyl c h l o r i d e ( XVI) . 3.1.2 Confirmation of the Structure of 2-Anthroyl Chloride and Mexiletine-N-Anthroyl Derivative by Mass Spectrometry Due to the high molecular weight and the n o n - v o l a t i l e nature of 2-anthroyl c h l o r i d e and i t s m e x i l e t i n e d e r i v a t i v e , mass s p e c t r a l s t r u c t u r a l confirmation f o r both 2-anthroyl c h l o r i d e and the m e x i l e t i n e anthroyl d e r i v a t i v e was obtained with d i r e c t - p r o b e sample i n t r o d u c t i o n . The r e s u l t i n g e l e c t r o n - i m p a c t mass spectra of anthroyl c h l o r i d e and i t s m e x i l e t i n e d e r i v a t i v e are shown in Figures 4a and 4b. The [M+] molecular ions f o r 2-anthroyl c h l o r i d e and f o r the m e x i l e t i n e anthroyl d e r i v a t i v e were observed at m/z 240 and 383, r e s p e c t i v e l y . A mass ion at m/z 205, which l i k e l y r e s u l t e d from cleavage adjacent to the heteroatom to give an anthroyl carbonyl fragment, was common in both s p e c t r a . Other mass ions at m/z 151 and 177, a l s o common in both s p e c t r a , were derived from the anthracene r i n g s t r u c t u r e . A summary of the s t r u c t u r e s of the d i a g n o s t i c ion fragments f o r 2-anthroyl c h l o r i d e and f o r the m e x i l e t i n e N-anthroyl d e r i v a t i v e i s shown in Appendix 2. 3.1.3 The Time-Course of Acylation of Mexiletine Enantiomers with 2-Anthroyl Chloride A review on the r e a c t i o n s of a l i p h a t i c and aromatic a c i d c h l o r i d e s has been published (Sonntag, 1952). The r e a c t i o n between an amino compound and an aromatic a c i d c h l o r i d e can be c h a r a c t e r i z e d by a S R 2 r e a c t i o n with the formation of an intermediate a c i d s a l t . Due to the r e l a t i v e l y slow r a t e of aqueous h y d r o l y s i s of the aromatic a c i d c h l o r i d e , the a c y l a t i o n of m e x i l e t i n e with 2-anthroyl c h l o r i d e can be c a t a l y z e d by vortex-mixing the r e a c t i o n mixture with an aqueous sodium hydroxide s o l u t i o n . This r e a c t i o n was f i r s t described by Schotten and Baumann (Baumann, 1886). The a c y l a t i o n of m e x i l e t i n e enantiomers with 71 100 c CD CD > '-i-> _o 40 80 120 160 200 240 280 320 360 400 440 m/z co c CD CD > *-+-« CD cr. 100 50 0 1 7 7 I S I 8 3 V I / X . / 12. l i j lHli toL. 2 0 S H J - J . i 2 6 2 + 2<?2 N M 3 8 3 1 40 80 120 160 200 240 280 320 360 400 440 m/z Figure 4. The electron-impact mass spectra of (a) 2-anthroyl chloride and (b) N-arithroyl derivative of mexiletine. 2-anthroyl c h l o r i d e was found to be completed in 2 to 3 minutes at ambient room temperature. The d e r i v a t i v e s were a l s o found to be s t a b l e f o r 14 days under storage at 5 * C . The time-course of a c y l a t i o n of m e x i l e t i n e enantiomers i s shown in Figure 5. 3.1.4 Resolution of Mexiletine Enantiomers as Their Anthroyl Derivatives on the P i r k l e Ionic Phenylglycine Chiral Column The use of a s i l i c a column i n tandem with the P i r k l e column was necessary to separate an unknown compound which coeluted with S(+)-m e x i l e t i n e . The unknown compound was thought to be an a c y l a t i o n by-product of the Schotten-Baumann r e a c t i o n . Attempts to i s o l a t e and i d e n t i f y the i n t e r f e r i n g compound f a i l e d to y i e l d c o n c l u s i v e r e s u l t s . Chromatographic r e s o l u t i o n of m e x i l e t i n e enantiomers as t h e i r 2-anthroyl d e r i v a t i v e s on the P i r k l e 3 , 5 - d i n i t r o b e n z o y l phenylglycine (1-A) HPLC s t a t i o n a r y phase (CSP) i s thought to be mediated by the formation of t r a n s i e n t diastereomeric complexes between the enantiomers and the c h i r a l s t a t i o n a r y phase. A d i f f e r e n c e i n the s t a b i l i t y of the diastereomeric bonding i n t e r a c t i o n s between the enantiomers and the CSP r e s u l t s i n r e s o l u t i o n of the enantiomers. These diastereomeric i n t e r a c t i o n s are thought to be composed of n - e l e c t r o n bonding, s t e r i c i n t e r a c t i o n , e l e c t r o s t a t i c and hydrogen-bonding which r e s u l t i n c h i r a l r e c o g n i t i o n of an enantiomer. Resolution of m e x i l e t i n e enantiomers i s t h e r e f o r e envisioned by means of 7 t - e l e c t r o n bonding between the d i n i t r o b e n z o y l group on the CSP and the anthroyl r i n g of the m e x i l e t i n e d e r i v a t i v e , amide d i p o l e - d i p o l e i n t e r a c t i o n between the d e r i v a t i v e and the CSP, and s t e r i c i n t e r a c t i o n of the methyl group at the c h i r a l carbon of m e x i l e t i n e with the s t a t i o n a r y phase. As shown i n Figure 6 , R ( - ) -m e x i l e t i n e i s envisioned to form a l e s s s t a b l e diastereomeric complex o o o e o o o • S(+) O R(-) 4-0 2 4 6 8 10 1: Time (min) Figure 5 . The time-course of a c y l a t i o n of R ( - ) - and S(+)-mexiletine by vortex-mixing of the enantiomers with 2-anthroyl c h l o r i d e at ambient room temperature. 74 gure 6. A diagrammatic representation of the bonding i n t e r a c t i o n s between R ( - ) - m e x i l e t i n e and the d i n i t r o b e n z o y l -D - ( - ) - p h e n y l g l y c i n e HPLC s t a t i o n a r y phase. 75 between the enantiomer d e r i v a t i v e and the CSP thus leading to e a r l i e r e l u t i o n of the R(-)-enantiomer. A r e p r e s e n t a t i v e HPLC chromatogram of blank serum and serum from a study subject showing the r e s o l u t i o n of the N-anthroyl d e r i v a t i v e s of m e x i l e t i n e enantiomers in serum i s shown in Figure 7. 3.1.5 Assay Method f o r Mexiletine Enantiomers in Serum and Serum U l t r a f i l t r a t e . McErlane et al. (1987) reported that the recovery of m e x i l e t i n e enantiomers from plasma was found to vary with the methods used f o r serum p r o t e i n p r e c i p i t a t i o n . The authors noted that serum p r o t e i n p r e c i p i t a t i o n procedures using barium hydroxide and z i n c sulphate s o l u t i o n s were required to provide r e l i a b l e recovery of serum t o t a l m e x i l e t i n e enantiomers. In the present study, serum t o t a l m e x i l e t i n e concentrations were determined using the barium hydroxide/zinc sulphate serum p r o t e i n p r e c i p i t a t i o n method. The determination of serum f r e e drug concentrations was c a r r i e d out using the C e n t r i f r e e M i c r o p a r t i t i o n System by c e n t r i f u g a t i o n . Following the c o l l e c t i o n of serum, the pH was found to vary between 7.8 to 8 . 9 . U l t r a f i l t r a t i o n of serum at 37 °C f o r 20 minutes was found to a l t e r serum pH d e s p i t e serum pH adjustment to 7.4 with 0.1 M h y d r o c h l o r i c a c i d p r i o r to u l t r a f i l t r a t i o n . The a d d i t i o n of phosphate b u f f e r s a l t s was found necessary to maintain the serum pH at 7.4 during u l t r a f i l t r a t i o n and t h i s a d d i t i o n of phosphate s a l t s was found not to a f f e c t the serum p r o t e i n binding of m e x i l e t i n e enantiomers. Experimental data on the e f f e c t s of serum pH on the p r o t e i n binding of m e x i l e t i n e enantiomers w i l l be f u r t h e r discussed in Section 3 . 2 . 2 . 4 . 3.1.6 Assay Recovery of Mexiletine Enantiomers from Serum and Serum I-<r t-co t— cr. h- »"~ to _ 10 20 TIME (MINI) Figure 7. Representative HPLC chromatograms of (a) serum from a subject and (b) blank serum showing r e s o l u t i o n of the N-anthroyl d e r i v a t i v e s of R ( - ) - m e x i l e t i n e ( 1 ) , S(+)-m e x i l e t i n e (2) and the i n t e r n a l standard, KOE-2963 ( 3 ) . Chromatographic C o n d i t i o n s : HPLC Column : Mobile Phase: Flow Rate : Detection : P i r k l e Phenylglycine Ionic S t a t i o n a r y Phase 0.45 x 25 cm (10 jm) Hexane / 2-Propanol / Chloroform (78/7/15) 0 . 8 ml/min Fluorescence 270/400 nm (ex/em) 77 U l t r a f i l t r a t e The recovery of m e x i l e t i n e enantiomers from plasma was reported to give s u b s t a n t i a l l y low values when sodium hydroxide s o l u t i o n ( 2 . 0 M) was used f o r p r o t e i n p r e c i p i t a t i o n (McErlane et al., 1987). In a d d i t i o n , the percent of recovery f o r each of the enantiomers from plasma was found to be h i g h l y d i f f e r e n t when a 2.0 M sodium hydroxide s o l u t i o n was used f o r serum p r o t e i n p r e c i p i t a t i o n . Since enantiomers have s i m i l a r s o l u b i l i t y p r o p e r t i e s in s o l u t i o n , a d i f f e r e n c e in the r e l a t i v e recovery of the enantiomers from plasma f o l l o w i n g p r o t e i n p r e c i p i t a t i o n was thought to r e f l e c t the r e l e a s e of varying amounts of m e x i l e t i n e enantiomers from the p r o t e i n binding s i t e s . However, the use of barium hydroxide and z i n c sulphate s o l u t i o n s f o r serum p r o t e i n p r e c i p i t a t i o n , as described by Somogyi (1945), was found to enhance the recovery and maintain the 1:1 r a t i o of a racemic sample of the enantiomers in plasma (McErlane et al., 1987). The recovery of m e x i l e t i n e enantiomers from serum and from serum u l t r a f i l t r a t e were studied at concentrations of 50 and 100 ng/ml. The determination of the recovery of each m e x i l e t i n e enantiomer involved p r o t e i n p r e c i p i t a t i o n w i t h barium hydroxide / z i n c s u l p h a t e , solvent e x t r a c t i o n w i t h d i e t h y l e t h e r , and an a c i d aqueous b a c k - e x t r a c t i o n . As shown i n Table 1, the recovery of m e x i l e t i n e enantiomers from serum and from serum u l t r a f i l t r a t e were found to be approximately 90%. 5.1.7 L i n e a r i t y of Response f o r Mexiletine Enantiomers in Serum, Serum U l t r a f i l t r a t e , S a l i v a , Red Blood C e l l s , and Urine The l i n e a r i t y of chromatographic response f o r m e x i l e t i n e enantiomers i n serum and i n serum u l t r a f i l t r a t e was performed over a concentration range of 2 . 5 to 100 ng/ml f o r each of the enantiomers Table 1. The d i e t h y l ether solvent e x t r a c t i o n recovery of R ( - ) -and S(+)-mexiletine from serum at 50 and 100 ng/ml c o n c e n t r a t i o n s . E x t r a c t i o n Recovery of M e x i l e t i n e (n=2) Control E x t r a c t i o n % Recovery Serum (ng/ml) R(-) S(+) R(-) S(+) R(-) S(+) 50 6 . 1 1 ± 0 . 1 8 5 . 6 5 ± 0 . 2 1 5 . 7 0 ± 0 . 3 0 5 . 4 1 ± 0 . 5 2 93.3 95.8 100 14.08+0.20 1 3 . 1 7 ± 0 . 3 2 1 2 . 6 3 ± 0 . 6 2 1 2 . 1 0 ± 0 . 0 2 89.7 91.9 Table 2. HPLC assay c a l i b r a t i o n curve data f o r R ( - ) - and S(+)-m e x i l e t i n e in s a l i v a , red blood c e l l s and u r i n e . M e x i l e t i n e C a l i b r a t i o n data S a l i v a Red Blood C e l l s Urine y = mx + b R ( . ) S(+) R(-) S(+) R(-) S(+) x - i n t e r c e p t 0.1013 0.1004 0.1859 0.1865 -0.1156 -0.0837 slope (m) 0.0528 0.0591 0.0508 0.0512 0.0290 0.0282 r 2 0.999 0.999 0.994 0.994 0.999 0.999 using 30 ng of an i n t e r n a l standard K0E-2963. This c a l i b r a t i o n range was found to be s a t i s f a c t o r y f o r the determination of m e x i l e t i n e enantiomer concentrations in 0.2 ml of serum or 0.4 ml of serum u l t r a f i l t r a t e f o r 48 hours f o l l o w i n g o r a l a d m i n i s t r a t i o n of 200 mg of racemic m e x i l e t i n e hydrochloride to healthy s u b j e c t s . The serum and the serum u l t r a f i l t r a t e c a l i b r a t i o n curves were found to be l i n e a r with a c o e f f i c i e n t of determination ( r 2 ) of 0.999 f o r both. Representative c a l i b r a t i o n curves f o r m e x i l e t i n e enantiomers in serum and i n serum u l t r a f i l t r a t e are shown in Figure 8 . The l i n e a r i t y of chromatographic response f o r m e x i l e t i n e enantiomers in s a l i v a and in red blood c e l l s was studied over the same concentration range as f o r serum using 30 ng of K0E-2963 as an i n t e r n a l standard. This c a l i b r a t i o n range was found to be s a t i s f a c t o r y f o r the determination of m e x i l e t i n e enantiomer concentrations in 0.2 ml of s a l i v a or from red blood c e l l s from 1 ml of whole b l o o d . As shown in Figures 9 and 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 f o r each of the enantiomers i n s a l i v a and in red blood c e l l s were found to be l i n e a r . C o r r e l a t i o n data f o r the c a l i b r a t i o n curves are shown in Table 2. The l i n e a r i t y of chromatographic response f o r m e x i l e t i n e enantiomers in u r i n e was determined over a concentration range of 10 to 500 ng/ml f o r each of the enantiomers using 100 ng of K0E-2963 as an i n t e r n a l standard. As shown in Figure 11, 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 were obtained f o r each of the enantiomers i n u r i n e . The l i n e a r c o r r e l a t i o n data f o r the c a l i b r a t i o n curves are given in Table 2. Since the N-anthroyl d e r i v a t i v e s of m e x i l e t i n e enantiomers were not completely resolved ( R « 1 . 3 ) on the P i r k l e column, q u a n t i t a t i o n of each of the enantiomers using peak area i n t e g r a t i o n was found to be more 80 4.5 120 Concentration of Enantiomer (ng/ml) Figure 8. HPLC assay c a l i b r a t i o n curves f o r R ( - ) - and S(+)-m e x i l e t i n e in serum and in serum u l t r a f i l t r a t e . v a r i a b l e than peak height q u a n t i t a t i o n . As indicated by the c a l i b r a t i o n data f o r m e x i l e t i n e enantiomers in serum, s a l i v a , blood c e l l s , and urine in Figures 8 to 11, chromatographic peak height measurements were found to provide l i n e a r c o r r e l a t i o n s with drug c o n c e n t r a t i o n s . While the chromatographic peak height c a l i b r a t i o n of a racemic sample of m e x i l e t i n e enantiomers was found to be l i n e a r over the concentration range s t u d i e d , a study was conducted to confirm that the incomplete chromatographic r e s o l u t i o n of m e x i l e t i n e enantiomers, which r e s u l t e d i n overlapping of chromatographic peak h e i g h t s , would not a f f e c t the l i n e a r i t y of q u a n t i t a t i o n of varying concentration r a t i o s of the enantiomers during the pharmacokinetic study. Thus, c a l i b r a t i o n samples ranging from 10 to 500 ng of S(+)-mexiletine were prepared in the presence of a constant amount (500 ng) of R ( - ) - m e x i l e t i n e . As shown in Figure 12, the c o r r e l a t i o n of the peak height of S(+)-mexiletine with drug concentration was l i n e a r with a c o e f f i c i e n t of determination ( r 2 ) of 0 . 9 9 9 . 3 . 1 . 8 HPLC Assay I n t r a - and I n t e r - v a r i a b i l i t y The i n t e r - a s s a y v a r i a b i l i t y of m e x i l e t i n e enantiomers was determined from a n a l y s i s of t r i p l i c a t e samples of 10 and 200 ng/ml in serum w i t h a c o e f f i c i e n t of v a r i a t i o n ( C V . ) of 7.1%. The i n t r a - a s s a y v a r i a b i l i t y of m e x i l e t i n e enantiomers was determined from t r i p l i c a t e i n j e c t i o n s of a s i n g l e sample at 10 and 200 ng/ml in serum with a c o e f f i c i e n t of v a r i a t i o n ( C V . ) of 7.1%. The i n t r a - and i n t e r - a s s a y v a r i a b i l i t y data are shown i n Table 3 . 40 80 Concentration (ng /m l ) 120 Figure 9. HPLC assay c a l i b r a t i o n curves f o r R ( - ) - and S(+)-Mexiletine in s a l i v a . Figure 10. HPLC assay c a l i b r a t i o n curves f o r R ( - ) - and S(+)-Mexiletine in red blood c e l l s . O R(-) • S(+) 100 2 0 0 3 0 0 4 0 0 5 0 0 Concentration (ng/ml) Figure 11. HPLC assay c a l i b r a t i o n curves f o r R ( - ) - and S(+)-M e x i l e t i n e in Urine. D O R(- ) © S( + ) 0 100 2 0 0 3 0 0 4 0 0 5 0 0 Concentration (ng /m l ) Figure 12. HPLC assay c a l i b r a t i o n curve f o r S(+)-mexiletine concentrations of 10 to 500 ng/ml in the presence of a constant 500 ng/ml of R ( - ) - m e x i l e t i n e . Table 3. HPLC assay v a r i a b i l i t y data f o r R ( - ) - and S(+)-mexiletine in serum at 10 and 200 ng/ml c o n c e n t r a t i o n s . M e x i l e t i n e serum Concentration Intra-Assay Inter-Assay (ng/ml) (Peak Height R a t i o , n=3) R( -) S( + ) R( -) S( + ) 10 0. 16 0. 15 0. 16 0. 15 0. 16 0. 14 0. 18 0. 14 0. 17 0. 13 0. 18 0. 16 Mean±S.D. 0. 16±0.01 0. 14±0.01 0. 1 7 ± 0 . 0 1 0. 15±0 C V . 6. 3 7. 1 5. 9 6. 6 200 2. 21 2. 09 2. 38 2. 24 2. 23 2. 16 2. 21 2. 09 2. 34 2. 21 2. 42 2. 40 A v e r a g e ± S . D . 2.26+0.06 2 . 1 5 ± 0 . 0 5 2.34+0.09 2 . 2 4 ± 0 . 1 2 C V . 3.1 2.8 4.7 7.1 3.2 In Vitro Serum Protein Binding and Red blood c e l l D i s t r i b u t i o n of Mexiletine Enantiomers 3.2.1 Serum Collection and Storage Methods Serum f r e e (unbound) drug i s g e n e r a l l y considered an important component of pharmacological and t o x i c o l o g i c a l e f f e c t s . However, i n c o n s i s t e n c i e s i n the serum f r e e drug data f o r l i d o c a i n e (Brown et al., 1984; McNamara et al., 1981; Stargel et al., 1979), propranolol (Brown et al., 1981; Cotham and Shand, 1975), q u i n i d i n e (Edwards et al., 1984), and various dopamine-receptor antagonists ( L a i et al., 1982) have been c i t e d in the l i t e r a t u r e . The discrepancy i n serum f r e e drug data reported f o r each of these therapeutic agents may have been due to the i n t r o d u c t i o n of serum p r o t e i n binding a r t i f a c t s during serum c o l l e c t i o n or during serum f r e e drug determination. Serum f r e e l i d o c a i n e concentrations were reported by Stargel et al. (1979) to vary with blood samples c o l l e c t e d using d i f f e r e n t l o t s of Becton Dickinson Vacutainers. McNamara et al. (1979) confirmed t h i s f i n d i n g and f u r t h e r noted that the type of Vacutainer as well as v a r i a t i o n s between lot-numbers in one type of Vacutainer could cause an increase of 50% in the serum f r e e f r a c t i o n f o r l i d o c a i n e . The authors suggested that the presence of p l a s t i z e r s such as d i e t h y l p h a t h l a t e e s t e r s or tris(2-butyoxyethylJphosphate could have lead to the displacement of l i d o c a i n e from i t s binding s i t e s . In a d d i t i o n , the serum f r e e f r a c t i o n s of l i d o c a i n e , propranolol and diazepam were a l s o reported t o increase when heparin coated Vacutainers were used f o r plasma c o l l e c t i o n (Brown et al., 1981). The a c t i o n of heparin as t r i g l y c e r i d e l i p a s e was found to lead to the production of f r e e f a t t y a c i d s which could have d i s p l a c e d drug bound onto serum p r o t e i n s . 88 S p u r i o u s l y low plasma concentrations of propranolol were reported when heparin-coated Becton Dickinson Vacutainers were used f o r blood c o l l e c t i o n (Cotham and Shand, 1975). The observed reduction in serum t o t a l propranolol concentration was suggested to be due to the presence of p l a s t i z e r s in the stopper of the Vacutainer that reduced serum p r o t e i n binding and r e s u l t e d in a r e d i s t r i b u t i o n of serum f r e e propranolol i n t o e r y t h r o c y t e s . In the present study, in order to obtain accurate and r e l i a b l e data f o r the serum p r o t e i n binding of m e x i l e t i n e enantiomers, the e f f e c t s of the f o l l o w i n g serum c o l l e c t i o n methods and storage conditions on the serum p r o t e i n binding of m e x i l e t i n e enantiomers were examined: (1) . S t e r i l e g l a s s syringe c o l l e c t i o n . (2) . P l a i n Vacutainer with s i l i c o n coated stopper. (3) . Indwelling B u t t e r f l y R - 2 1 INT cannula. (4) . Vacutainer coated with h e p a r i n . (5) . Vacutainer coated with EDTA. (6) . Freezing at -20 °C f o r 72 hours. As shown i n Table 4, the serum f r e e f r a c t i o n s f o r m e x i l e t i n e enantiomers obtained using g l a s s syringe blood c o l l e c t i o n were found not to be s i g n i f i c a n t l y d i f f e r e n t from those obtained using the non-additive Becton Dickinson V a c u t a i n e r R , and from the B u t t e r f l y R i n - d w e l l i n g cannula t r e a t e d serum. Storage of the serum samples f o r 72 hours at -20°C was a l s o found not to s i g n i f i c a n t l y a l t e r the serum p r o t e i n b i n d i n g of m e x i l e t i n e enantiomers. At c o n t r o l l e d serum pH of 7.4, the plasma f r e e f r a c t i o n s obtained from the EDTA and from the heparin-coated V a c u t a i n e r s R were found to be s l i g h t l y lower than those obtained from the n o n - a d d i t i v e V a c u t a i n e r s R , but the d i f f e r e n c e was not s t a t i s t i c a l l y Table 4 . The e f f e c t of blood c o l l e c t i o n and storage c o n d i t i o n s serum f r e e f r a c t i o n of R ( - ) - and S ( + ) - m e x i l e t i n e . Serum Serum Free F r a c t i o n Treatment pH (ng/ml) R(_) s(+) Glass S y r i n g e * 500 (n=2) 0.30 0.46 200 (n=2) 0.29 0.42 80 (n=4) 0.40 0.52 32 (n=4) 0.35 0.42 mean±S.E. 0.34±0.02 0.46±0.02 Non-Additive* 500 (n=2) 0.29 0.42 200 (n=2) 0.35 0.55 80 (n=4) 0.37 0.47 32 (n=4) 0.36 0.50 mean±S.E. 0.34±0.02 0.49±0.03 Indwelling Cannula* 500 (n=2) 0.31 0.45 200 (n=2) 0.29 0.47 80 (n=4) 0.34 0.46 32 (n=4) 0.30 0.42 mean±S.E. 0 . 3 1 ± 0 . 0 1 0 . 4 5 ± 0 . 0 1 -20°C f o r 72 hours* 500 (n=2) 0.34 0.38 200 (n=2) 0.29 0.40 80 (n=4) 0.30 0.47 32 (n=4) <L31 0^40 mean±S.E. 0 . 3 1 ± 0 . 0 1 0 . 4 1 ± 0 . 0 2 N o n - A d d i t i v e * * 7.4 2000 (n=5) 0.52±0.02 0.50±0.03 * Unadjusted pH at 8 . 5 to 8 . 9 * * pH adjustment with sodium phosphate b u f f e r s a l t s . 90 s i g n i f i c a n t . The d i f f e r e n c e i n m e x i l e t i n e serum f r e e f r a c t i o n s between c o n t r o l l e d and uncontrolled serum pH was found to be due to s t e r e o s e l e c t i v e , pH-dependent serum p r o t e i n binding of m e x i l e t i n e enantiomers. The data on pH-dependent serum p r o t e i n binding of m e x i l e t i n e enantiomers w i l l be f u r t h e r discussed in Section 3 . 2 . 2 . 4 . For the present pharmacokinetic study of m e x i l e t i n e enantiomers in healthy v o l u n t e e r s , a s i n g l e l o t of the Becton Dickinson non-additive s i l i c o n e - c o a t e d stopper VacutainersR were used throughout. 3 . 2 . 2 Comparisons of M e x i l e t i n e Serum Free Drug Measurements Using U l t r a f i l t r a t i o n and Using E q u i l i b r i u m D i a l y s i s Methods that are r o u t i n e l y used f o r the determination of f r e e drug in serum include e q u i l i b r i u m d i a l y s i s , u l t r a f i l t r a t i o n , s i z e e x c l u s i o n chromatography and u l t r a c e n t r i f u g a t i o n techniques. The t h e o r e t i c a l and p r a c t i c a l c o n s i d e r a t i o n s on these techniques have been reviewed ( L i n et a 7 . , 1987; Rowland, 1980; Lindup, 1975; C h i g n e l l , 1971). The determination of serum f r e e drug concentrations by d i a l y s i s r e l i e s on the achievement of d i a l y s i s e q u i l i b r i u m between a serum compartment and a b u f f e r compartment separated by a semipermeable membrane of chosen molecular weight c u t - o f f . At e q u i l i b r i u m , the concentration of f r e e drug i n the two compartments are approximately e q u a l , and a determination of the r a t i o of the f r e e drug i n the b u f f e r compartment to the t o t a l drug in the serum compartment w i l l y i e l d serum f r e e f r a c t i o n . The determination of f r e e drug by u l t r a f i l t r a t i o n of serum using the commercial C e n t r i f r e e M i c r o p a r t i t i o n system r e l i e s on u l t r a f i l t r a t i o n of a serum sample through an a n i s o t r o p i c h y d r o p h i l i c YMT-membrane by c e n t r i f u g a t i o n at 1000 to 2000 g. Following 91 c e n t r i f u g a t i o n , determination of the r a t i o of drug concentration in the serum u l t r a f i l t r a t e to the t o t a l drug concentration in serum w i l l y i e l d serum f r e e f r a c t i o n . In a d d i t i o n , the use of the C e n t r i f r e e M i c r o p a r t i t i o n system was reported to provide greater than 99.9% r e t e n t i o n of serum p r o t e i n s and greater than 95% recovery of f r e e L-thyroxine from the serum u l t r a f i l t r a t e (Sophianopoulos et al., 1980). In g e n e r a l , u l t r a f i l t r a t i o n i s a r a p i d procedure which requires 15 to 20 minutes of c e n t r i f u g a t i o n to obtain serum u l t r a f i l t r a t e f o r f r e e drug determination. Numerous comparative s t u d i e s have examined the p r a c t i c a l and the t h e o r e t i c a l b a s i s of u l t r a f i l t r a t i o n and e q u i l i b r i u m d i a l y s i s f o r the determination of serum f r e e drug c o n c e n t r a t i o n s . Bowers et al. (1984) speculated that during u l t r a f i l t r a t i o n , the continuously decreasing serum f r a c t i o n a l volume may t h e o r e t i c a l l y a l t e r the serum f r e e f r a c t i o n and the binding e q u i l i b r i u m of non-1inearly bound drugs. However, the authors found that the continuously changing serum f r a c t i o n a l volumes during u l t r a f i l t r a t i o n were, i n f a c t , independent of the f r e e drug concentration i n the serum u l t r a f i l t r a t e . In studies by Sophianopoulos et al. (1978), and by Whitlam and Brown (1980), mathematical simulations of the mass t r a n s f e r of drugs across a f i l t e r membrane during u l t r a f i l t r a t i o n were used to prove t h a t drug concentration in the serum u l t r a f i l t r a t e remained c o n s t a n t , and that the serum drug binding e q u i l i b r i u m remained unaltered by the reducing serum f r a c t i o n a l volume. In the present study, a comparison of the e q u i l i b r i u m d i a l y s i s method with the u l t r a f i l t r a t i o n method f o r determining serum f r e e drug was c a r r i e d out to examine the v a l i d i t y of serum f r e e m e x i l e t i n e concentrations obtained from the l a t t e r method. The experimental protocol employed was modified from a procedure reported by Bowers et a7. (1984). U l t r a f i l t r a t i o n was c a r r i e d out using C e n t r i f r e e M i c r o p a r t i t i o n u n i t s , while e q u i l i b r i u m d i a l y s i s was conducted at m e x i l e t i n e serum concentrations of 0 . 5 and 2.0 /zg/ml against a phosphate b u f f e r . At d i a l y s i s e q u i l i b r i u m , a l i q u o t s of the b u f f e r d i a l y s a t e and the serum r e t e n t a t e were assayed f o r f r e e and t o t a l drug c o n c e n t r a t i o n s . The serum r e t e n t a t e was f u r t h e r subjected to u l t r a f i l t r a t i o n with the C e n t r i f r e e f i l t r a t i o n u n i t s followed by determination of the f r e e drug concentration in the serum u l t r a f i l t r a t e . Following u l t r a f i l t r a t i o n , the serum u l t r a f i l t r a t e m e x i l e t i n e concentrations were compared to those obtained from the b u f f e r d i a l y s a t e of the corresponding d i a l y s i s c e l l . As shown in Table 5 , the r e s p e c t i v e R ( - ) - and S(+)-mexiletine peak height r a t i o s between the b u f f e r d i a l y s a t e and the serum u l t r a f i l t r a t e were found not to be s i g n i f i c a n t l y d i f f e r e n t at m e x i l e t i n e serum t o t a l concentrations of 0 . 5 and 2.0 /zg/ml. These data suggested t h a t , under the c o n d i t i o n s s t u d i e d , u l t r a f i l t r a t i o n was equivalent to e q u i l i b r i u m d i a l y s i s f o r the determination of serum f r e e m e x i l e t i n e enantiomer c o n c e n t r a t i o n s . For the present pharmacokinetic study of serum p r o t e i n binding of m e x i l e t i n e enantiomers in healthy v o l u n t e e r s , the u l t r a f i l t r a t i o n method was used f o r the determination of serum f r e e drug. In the present study, a c e n t r i f u g e with temperature c o n t r o l was not a v a i l a b l e f o r serum u l t r a f i l t r a t i o n at 37 ° C , and the use of a c e n t r i f u g e r o t o r warmed to 37 °C was found to be inadequate f o r maintaining serum temperature at 37 °C during the 20 minutes required f o r u l t r a f i l t r a t i o n . Therefore, the e f f e c t of temperature on the serum p r o t e i n binding of m e x i l e t i n e enantiomers was determined by e q u i l i b r i u m Table 5. A comparison of the serum protein binding of m e x i l e t i n e using u l t r a f i l t r a t i o n versus e q u i l i b r i u m d i a l y s i s at 0 . 5 and 2.0 /zg/ml and at 22 and 37 ° C . Free Drug (Peak Height Ratio) (n=5) Serum Temp "C Dial vsate U l t r a f i l t r a t e U l t r a -(ug/ml) D i a l y s i s f i l t r a t i o n R(-) S( + ) R(-) S(+) 0 . 5 37 22 0. 21±0. 05 0 . 1 9 ± 0 . 04 0 . 1 7 ± 0 . 0 3 0.15+0.02 2.0 22 22 3 . 20+0. 20 2 . 9 7 ± 0 . 18 3 . 1 5 ± 0 . 3 9 2.83±0.34 2.0 37 37 2. 90±0. 16 2.68+0. 18 3.17+0.15 2 . 7 3 ± 0 . 0 3 Table 6. The recovery of R ( - ) - and S(+)-mexiletine from the C e n t r i f r e e m i c r o p a r t i t i o n serum u l t r a f i l t r a t i o n u n i t s . Serum Peak Height Ratio (n=2) (ng/ml) Control U l t r a f i l t r a t i o n % Recovery R(-) S(+) R(-) S(+) R(-) S(+) 10 0.61 0.55 0.59 0.54 96.7 94.9 100 1.27 1.18 1.21 1.13 95.3 95.8 d i a l y s i s and u l t r a f i l t r a t i o n at 22 or 37 ° C . As shown in Table 5 , at 2 . 0 /zg/ml, the r e s p e c t i v e R ( - ) - and S(+)-mexiletine peak height r a t i o s were found not to be s i g n i f i c a n t l y d i f f e r e n t at these two temperatures. At 0 . 5 /zg/ml, the R ( - ) - and S(+)-mexiletine peak height r a t i o s at 37 ° C , obtained from d i a l y s i s , were a l s o not s i g n i f i c a n t l y d i f f e r e n t from those at 22 * C , obtained from u l t r a f i l t r a t i o n . These data i n d i c a t e d that the serum p r o t e i n binding of m e x i l e t i n e enantiomers were not dependent on the temperatures s t u d i e d . 3.2.3 Determination of D i a l y s i s Equilibrium Time For the determination of serum f r e e drug concentrations using e q u i l i b r i u m d i a l y s i s , serum samples are u s u a l l y d i a l y z e d against an i d e n t i c a l volume of phosphate b u f f e r . However, due the l a r g e concentrations of n o n - d i f f u s a b l e p r o t e i n s in serum, the b u f f e r from the d i a l y s a t e compartment may d i f f u s e towards the serum r e t e n t a t e compartment causing a s h i f t i n volumes between the two compartments. This volume s h i f t may become s i g n i f i c a n t during prolonged d i a l y s i s times and may a l s o a f f e c t the serum f r e e f r a c t i o n s of a drug with concentration-dependent serum p r o t e i n binding (Behm and Wagner, 1980). The use of serum u l t r a f i l t r a t e in the d i a l y s a t e compartment has been suggested by Bowers et al. (1984) as an a l t e r n a t i v e to provide a p h y s i o l o g i c a l medium f o r determining d i a l y s i s e q u i l i b r i u m . The d i a l y s i s of m e x i l e t i n e in serum against sodium phosphate 0.067 M b u f f e r and against serum u l t r a f i l t r a t e were s t u d i e d . Following d i a l y s i s , the contents from both the d i a l y s a t e and the retentate compartments were t r a n s f e r r e d and weighed. No apparent s h i f t in the volume or i n the pH was observed between the two compartments. I t was a l s o found that d i a l y s i s e q u i l i b r i u m was a t t a i n e d w i t h i n three hours as DIALYSATE (n=2) Figure 13. The time-course of e q u i l i b r i u m d i a l y s i s f o r R ( - ) -and S(+)-mexiletine in serum against (a) i s o t o n i c phosphate b u f f e r , and (b) serum u l t r a f i l t r a t e at 37 shown i n Figure 13. 3.2.4 The Effect of Serum pH on the Protein Binding of Mexiletine Enantiomers The influence of serum pH and/or the e f f e c t s of d i f f e r e n t d i a l y s i s b u f f e r s on the serum p r o t e i n binding of t h e o p h y l l i n e (Brors et a 7 . , 1984), q u i n i d i n e (Brors et al., 1984), prazosin (Brors et a 7 . , 1984), imipramine (Kristensen and Gram, 1981), w a r f a r i n ( W i l t i n g et a 7 . , 1980), b e t a - b l o c k e r s (Henry and M i t c h e l l , 1980) and l i d o c a i n e (McNamara et a 7 . , 1981) have been r e p o r t e d . For t h e o p h y l l i n e , q u i n i d i n e and p r a z o s i n , the serum f r e e f r a c t i o n s were found to decrease s i g n i f i c a n t l y with i n c r e a s i n g serum pH. In a d d i t i o n , the binding of q u i n i d i n e to i s o l a t e d human serum albumin was s i g n i f i c a n t l y reduced by h a l i d e ions such as I", B r " , C l " and F. (Brors et al., 1984). For imipramine, serum p r o t e i n binding was reported to l i n e a r l y increase with i n c r e a s i n g pH (Kristensen and Gram, 1982). For w a r f a r i n , W i l t i n g et a7. (1980) reported that the binding constant to human serum albumin was increased t h r e e - f o l d as serum pH increased from 6.1 to 9 . 3 . For the B - b l o c k e r s , a t e n o l o l , p r a c t o l o l , p i n d o l o l , b e t a x o l o l , oxprenolol and p r o p r a n o l o l , the serum p r o t e i n binding was found to be pH-dependent, and the percent of drug binding was found to be increased by up to 8.92% with an increase of each 0.1 pH u n i t . The authors noted that pH-dependent serum p r o t e i n binding of the l i p o p h i l i c B-2 s e l e c t i v e antagonists was greater than those f o r the h y d r o p h i l i c B - l s e l e c t i v e antagonists (Henry and M i t c h e l l , 1980). The pH-dependent drug binding onto serum p r o t e i n s has been p r e v i o u s l y described by a general r e l a t i o n s h i p derived from the pH-p a r t i t i o n theory (Henry et a 7 . , 1980). The e q u i l i b r i u m d i s t r i b u t i o n of 97 f r e e and p r o t e i n bound drug in serum followed the assumption that f r e e drugs were aqueous s o l u b l e and protein-bound drugs were non-aqueous s o l u b l e . The degree of serum p r o t e i n binding was thus found to be d i r e c t l y dependent on the pKa of the drug and serum pH. This r e l a t i o n s h i p was used s u c c e s s f u l l y to describe the pH-dependent serum p r o t e i n binding of propranolol and f u s i d i c a c i d (Henry et a 7 . , 1980). To d a t e , examples f o r the pH-dependent serum p r o t e i n binding of drug enantiomers have not been r e p o r t e d . Since the pKa f o r m e x i l e t i n e enantiomers are i d e n t i c a l , binding d i f f e r e n c e s induced by a change i n serum pH would l i k e l y be caused by conformational changes i n the binding p r o t e i n s . pH-Induced conformational changes of human serum albumin have been reported by W i l t i n g et a7. (1980), who observed that the binding a f f i n i t y of w a r f a r i n increased t h r e e - f o l d as serum pH increased from 6.1 to 9 . 3 . In order t o determine i f pH-dependent serum p r o t e i n binding would a f f e c t the serum f r e e f r a c t i o n of the enantiomers of m e x i l e t i n e during sample c o l l e c t i o n and serum u l t r a f i l t r a t i o n , the pH of serum was c a r e f u l l y monitored. Following blood c o l l e c t i o n and serum s e p a r a t i o n , serum pH was found to range from 8 . 5 to 8 . 9 . During p r e l i m i n a r y experiments, e q u i l i b r i u m d i a l y s i s of a serum sample at pH « 8 . 5 against an equal volume of 0.067 M i s o t o n i c phosphate b u f f e r (pH 7.4) f o r 19 hours f a i l e d to r e s t o r e the serum pH to 7 . 4 , and m e x i l e t i n e serum f r e e f r a c t i o n s were found to be v a r i a b l e . The pH-dependent serum p r o t e i n binding of m e x i l e t i n e enantiomers was therefore studied over a pH range of 6 . 3 to 9.4 using both e q u i l i b r i u m d i a l y s i s and u l t r a f i l t r a t i o n . Over t h i s pH range, m e x i l e t i n e serum f r e e f r a c t i o n s were found to increase from « 30% to « 80%. As shown in Figures 14 and 15, the serum f r e e 0.8 0.6 0.4 0.2 FREE FRACTION (n=2) R(-) - + - S(+) R(-)/S(+) RATIO (n=2) 6.34 7.38 7.95 8.99 PH Figure 1 4 . The e f f e c t of serum pH on (a) serum f r e e f r a c t i o n s and (b) serum f r e e m e x i l e t i n e R(-)/S(+) r a t i o s obtained using d i a l y s i s of racemic m e x i l e t i n e against 0.067 M phosphate b u f f e r at 37 °C f o r 4 hours. 9 9 iZ o o ct> E 00 c o o o E CO 1.0 0.8 --0.6 0.4 0.2 0.0 . o 2 ug/ml ( b ) O R(-) • S(+) o * ° o o 7 8 PH 10 Figure 15. The effect of serum pH on serum free fractions for R(-)- and S(+)-mexiletine at (a) 500 ng/ml and (b) 2.0 /zg/ml concentrations obtained by u l t ra f i l t ra t ion of racemic mexiletine in serum. 100 R(-)/S(+) enantiomeric r a t i o s were found to decrease from « 1.0 to 0 . 7 , i n d i c a t i n g that the serum p r o t e i n binding of m e x i l e t i n e enantiomers was s t e r e o s e l e c t i v e l y dependent on serum pH. This pH-dependent serum p r o t e i n binding of m e x i l e t i n e enantiomers may have been the r e s u l t of changes in the degree of i o n i z a t i o n of the binding p r o t e i n which lead to changes in the conformation of p r o t e i n . At p h y s i o l o g i c a l serum pH, the serum p r o t e i n binding of m e x i l e t i n e was found to be s i m i l a r between the two enantiomers with comparable serum f r e e f r a c t i o n s and R(-)/S(+) r a t i o s approaching u n i t y . To maintain serum pH at 7.4 and to maintain p h y s i o l o g i c a l c o n d i t i o n s during serum u l t r a f i l t r a t i o n , various in vitro methods were i n i t i a l l y evaluated. As shown i n Table 8 , a l l serum adjustment methods were found comparable in maintaining serum pH during the 20 minutes r e q u i r e d f o r the completion of u l t r a f i l t r a t i o n . Following the a d d i t i o n of 0.2 M equivalence of sodium phosphate b u f f e r s a l t s to serum, the serum f r e e f r a c t i o n s f o r m e x i l e t i n e enantiomers were found to be s i m i l a r to those obtained using carbon dioxide/oxygen (5/95) (Igwemezie Ph.D. T h e s i s , 1989). The use of d i l u t e d acids to adjust serum pH was also found to produce s i m i l a r serum f r e e f r a c t i o n s and serum f r e e R(-)/S(+) r a t i o s . For the in vivo pharmacokinetic s t u d i e s , the a d d i t i o n of 0.2 M equivalence of sodium phosphate b u f f e r s a l t was used to adjust serum pH to 7.4 p r i o r to u l t r a f i l t r a t i o n . 3.2.5 Determination of Concentration-Dependent Protein Binding of Mexiletine Enantiomers As shown in Table 9 , the in vitro serum p r o t e i n binding parameters f o r m e x i l e t i n e enantiomers were found to be independent of the serum drug concentrations w i t h i n the t h e r a p e u t i c serum concentration range of 101 Table 7. The recovery of R ( - ) - and S(+)-mexiletine from e q u i l i b r i u m d i a l y s i s u n i t s . Serum Incubated Before A f t e r % Recovery (ng/ml) g l a s s tube D i a l y s i s D i a l y s i s ( 3 7 ° C / 4 hr) (37°C / 4hr) 10 100 Serum Peak Height Ratio (n=2) R(-) S( + ) R(-) S(-f) R(-) S( + ) R(-) S( + ) 0.68 0.64 0.70 0.66 0.62 0.60 88.6 90.9 1.43 1.36 1.41 1.30 98.6 94.3 Table 8 . The e f f e c t of serum pH adjustments using sodium phosphate b u f f e r s , 0.1 M sulphuric and 0.1 M phosphoric a c i d s on R ( - ) - and S(+)-mexiletine serum f r e e f r a c t i o n and serum f r e e R(-)/S(+) r a t i o . Serum 500 ng/ml (pH 7.4) Serum pH Adjustment Free Fraction R/S (n=2) R(-) S(+) Ratio Sodium Phosphate S a l t (0.2 M) 0 . 2 7 ± 0 . 0 0 1 0.29±0.020 S u l p h u r i c a c i d (0.1 M) 0.31+0.010 0.34±0.020 Phosphoric a c i d (0.1 M) 0.29±0.001 0 . 3 1 ± 0 . 0 0 1 0.93 0.91 0.94 Table 9. The R ( - ) - and S(+)-mexiletine serum f r e e f r a c t i o n s and serum R(-)/S(+) r a t i o s over a concentration range of 0.25 to 3.0 fig/ml. Serum Peak Height Ratio (n=2) Free F r a c t i o n * R/S ng/ml Free Drug Total Drug R(-) S(+) R(-) S(+) R(-) S(+) 250 0.54 0. 48 1.04 1.07 0. 52±0 .02 0. 45±0.03 1 .13 500 1.22 1. 02 2.41 2.36 0. 51±0 .01 0. 43±0.01 1 .19 1000 2.56 2. 15 5.40 5.27 0. 48±0 .05 0. 41±0.05 1 .19 2000 4.87 4. 11 9.14 8.99 0. 54±0 .04 0. 47±0.03 1 .18 3000 6.46 5. 65 11.52 11.23 0. 56+0 .01 0. 49±0.02 1 .14 Mean+S.E. 0. 52±0 .01 0. 45+0.01 1 .17 * Mean ± one standard d e v i a t i o n 103 0.25 to 3 /zg/ml. However, small d i f f e r e n c e s in the mean serum f r e e f r a c t i o n s of 0.52 ± 0.01 and 0.45 ± 0.01 were observed, r e s p e c t i v e l y , f o r R(-) and S(+)-mexiletine over the serum concentration range s t u d i e d . The serum f r e e R(-)/S(+) enantiomeric r a t i o s were found to be s i m i l a r over the therapeutic concentration range with a mean value of 1.17. 3 . 2 . 6 In vitro Determination of the Time-Course of M e x i l e t i n e D i s t r i b u t i o n i n t o Red Blood C e l l s T h e ^ d i s t r i b u t i o n of drugs across the erythrocyte membrane has been described using a l i p i d / water p a r t i t i o n model (Schanker et al., 1964). Compounds such as phenol, s u l f a d i a z i n e and s u l f a t h i a z o l e , with r e l a t i v e l y high l i p i d s o l u b i l i t y , were found to e q u i l i b r a t e between the red c e l l s and a suspending s a l i n e s o l u t i o n i n l e s s than 5 minutes (Schanker et al., 1964). These authors a l s o reported that compounds such as h i p p u r i c a c i d and s u l f a n i l i c a c i d , with low l i p i d s o l u b i l i t y , were found to e q u i l i b r a t e more slowly with e q u i l i b r a t i o n times of 1 to 7 hours observed. I t was a l s o reported that organic anions d i s t r i b u t e d across the erythrocytes membrane at a f a s t e r r a t e than c a t i o n s of s i m i l a r 1 i p o p h i l i c i t y (Schanker et al., 1964). The d i s t r i b u t i o n of o r a l l y administered racemic m e x i l e t i n e into erythrocytes was examined i n seven healthy volunteers by Turgeon et al. (1987). The mean AUC r a t i o s of m e x i l e t i n e concentration in e r y t h r o c y t e s , to that in serum, were found to range from 0.69 to 1.43 over 24 hours with an o v e r a l l mean of 0,97 ± 0.27. The authors suggested that m e x i l e t i n e was almost e q u a l l y d i s t r i b u t e d between serum and red blood c e l l s . The 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 of m e x i l e t i n e enantiomers into red blood c e l l s has not been i n v e s t i g a t e d . In order to determine i f 104 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 of m e x i l e t i n e enantiomers into red blood c e l l s o c c u r r e d , a p r e l i m i n a r y in vitro study was conducted to examine the r a t e of m e x i l e t i n e enantiomers d i s t r i b u t i o n into the red blood c e l l s p r i o r to the pharmacokinetic study, Racemic m e x i l e t i n e was added to an i s o t o n i c s a l i n e red blood c e l l suspension. Following i n c u b a t i o n , an a l i q u o t of the suspension was removed, r a p i d l y washed with i s o t o n i c s a l i n e s o l u t i o n followed by determination of the i n t r a c e l l u l a r m e x i l e t i n e enantiomer c o n c e n t r a t i o n s . As shown in Figure 16, the r a t e of d i s t r i b u t i o n of m e x i l e t i n e enantiomers into red blood c e l l s was moderate, and s i m i l a r , with d i s t r i b u t i o n e q u i l i b r i u m achieved w i t h i n «80 minutes. Since 80 minutes was required f o r m e x i l e t i n e enantiomers to reach d i s t r i b u t i o n e q u i l i b r i u m across the red blood c e l l membrane, a subsequent washing time of « 1 minute during red blood c e l l sample c o l l e c t i o n , would not be expected to a f f e c t the r e d i s t r i b u t i o n of m e x i l e t i n e enantiomers to any appreciable e x t e n t . 3.2.7 Validation of the Potency of M e x i t i l R 200 mg Capsules M e x i t i l ^ capsules were supplied by Boehringer Ingelheim (Canada) L t d . , O n t a r i o , Canada with a l a b e l c l a i m of 200 mg racemic m e x i l e t i n e hydrochloride and were analyzed by the s t e r e o s e l e c t i v e HPLC assay used in t h i s study. The R(-)/S(+) r a t i o of m e x i l e t i n e enantiomers was found to be 1.01. The potency of the M e x i t i l R capsules was found to be 101.4 mg and 100.5 mg, r e s p e c t i v e l y f o r R ( - ) - m e x i l e t i n e and S(+)-mexiletine h y d r o c h l o r i d e . 3.3. Pharmacokinetics of Mexiletine Enantiomers i n Healthy Human Subjects 3.3.1 Pharmacokinetics of Mexiletine in Serum In order to determine the pharmacokinetics of m e x i l e t i n e 0.45 0.35 -0.25 © 6 4 i ! O R ( - ) 9 S ( + ) 0 20 40 60 80 100 120 140 160 180 200 Time (min) Figure 16. Plots of the extent and time-course of the d i s t r i b u t i o n of R ( - ) - and S(+)-mexiletine from i s o t o n i c phosphate buffer into red blood c e l l s . 106 enantiomers in healthy s u b j e c t s , an o r a l dose of 200 mg of racemic m e x i l e t i n e hydrochloride was administered to each of twelve healthy s u b j e c t s . M e x i l e t i n e enantiomer concentrations were determined i n serum, serum u l t r a f i l t r a t e , s a l i v a , red blood c e l l s and urine samples as o u t l i n e d in Section 2 . 3 . 4 . 2 . To show the r e l a t i v e concentrations of m e x i l e t i n e enantiomers in serum with respect to those in s a l i v a and red blood c e l l s , r e p r e s e n t a t i v e p l o t s of m e x i l e t i n e concentrations versus time data from subject RL are presented i n Figure 17. For the twelve s u b j e c t s , the mean serum t o t a l and mean serum f r e e m e x i l e t i n e enantiomers concentrations versus time data are shown in Figure 18 and 19 as w e l l as in Table 10 and 11. The mean serum t o t a l drug data i n d i c a t e d a l a r g e but normally d i s t r i b u t e d i n t e r - s u b j e c t v a r i a t i o n over the 48 hour p e r i o d . The mean serum f r e e drug data a l s o showed a s i m i l a r l y l a r g e i n t e r - s u b j e c t v a r i a b i l i t y , but both the mean serum t o t a l and mean serum f r e e drug l e v e l s followed a p a r a l l e l and l o g -l i n e a r d e c l i n e over 48 hours, suggesting that the mean terminal d i s p o s i t i o n h a l f - l i v e s of t o t a l and f r e e m e x i l e t i n e enantiomers i n serum were s i m i l a r among the twelve s u b j e c t s . As shown i n Figure 20 and Table 12, the serum t o t a l and serum f r e e m e x i l e t i n e R(-)/S(+) r a t i o s f o r the twelve subjects were found to be v a r i a b l e but deviated in equal magnitude around the o v e r a l l mean values of 1.04 ± 0.06 and 1.09 ± 0.06 (mean ± S . E . ) over 48 hours f o r serum t o t a l and serum f r e e m e x i l e t i n e , r e s p e c t i v e l y . These s i m i l a r R(-)/S(+) r a t i o s , observed f o r both serum t o t a l or serum f r e e m e x i l e t i n e , i n d i c a t e d that the d i s p o s i t i o n of m e x i l e t i n e enantiomers in the body was s i m i l a r . The o v e r a l l mean serum f r e e m e x i l e t i n e R(-)/S(+) r a t i o of 1.09 f u r t h e r suggested that the in vivo serum p r o t e i n binding of m e x i l e t i n e Mexiletine Concentrat ion ( n g / m l ) << CL CL 3 -5 ->• O 3 O ->• 3" to —' rt-O -5 "5 B> _.. r+ Q. ->• n> o • 3 o -+> ro o o o c -j O 3 70 CD CD ro —' X TD < -2 PJ fD CO &>-.-=! 3 3 r+ D- fD CO - J O - . . fD O < Q- 3 fD O o ro oo fD 3 fD CO £1) i c+ o -h - J - I Q -5 O ju O 3 ~s 3 oo _,. r+ co 3-£_•. CO o fD fD n -5x3 r+ c: —i 3 O 73- r+ r - co CO -h (D o O -S -h 3 3 r+ i fD I Q -S O -h 3 -i -.. a. Co —' —' r+ cry ~i -W + r+—-fD i 3 o C n -O c n NO O o OJ c n o c n O o I o o o l _ -«<l l> • O • • • W TO TO ¥1 3) J/> ^> }S. 70 CD CL o CD —i CD CD oo CD ~ i C 3 o r-t-CD c 3 CO Q_ < " Q Mexiletine Concentration (ng/ml) o o o o o m • O O W • K> tO m K> • m cm » • « m » • m <* on •m m <W K> • CD •*<¥•(» CM ZOl Time (h) Figure 18. Semilogarithmic plots of the mean ± S.D. serum t o t a l R ( - ) - and S(+)-mexiletine concentrations over 48 hours from twelve healthy subjects f o l l o w i n g oral administration of 200 mg of racemic m e x i l e t i n e hydrochloride. o 00 O R( - ) 0 5 10 15 20 25 30 35 40 45 Time (h) Figure 19. Semilogarithmic plots of the mean ± S.D. serum f r e e R ( - ) - and S(+)-mexiletine concentrations over 48 hours from twelve healthy subjects f o l l o w i n g o r a l administration of 200 mg of racemic m e x i l e t i n e hydrochloride. • I I J " t 9 8 o 0-• Free Serun ° Total Serum 0 5 10 15 20 25 30 35 40 45 Time (h) Figure 20. P l o t s of the mean ± S.D. R ( - ) - / S(+)-mexiletine serum t o t a l and serum free concentration r a t i o s over 48 hours from twelve healthy s u b j e c t s . Table 10. The serum t o t a l R ( - ) - and S(+)-mexiletine concentrations over 48 hours from 12 healthy subjects f o l l o w i n g oral administration of 200 mg racemic m e x i l e t i n e h y d r o c h l o r i d e . 00 Total Serum Mexiletine Concentration ( n°,/m0 T t u e 1 I n t TOTAL R ( -torn h o i i i * rKUM PKUu A 0 K I N AO CK CT GR GT JB JG J K RL ST TH TK MEAN S . O . • I H m x i HtlflfllDI iBiscten « « » « « « * « • IMIIIMI IS33S3ISS IltSCBISI 8333S8SS3 3ES33SSS3 333333303 ========= CSS S3 3333 ESCfBSES: 323333333 0 . 0 1 . 0 6 2 . 4 1 5 4 . 4 2 9 9 . 5 9 6 . 0 2 3 0 . 6 8 0 . 2 1 5 4 . 1 1 3 9 . 1 1 2 8 . 3 6 3 . 6 2 1 5 . 7 1 0 8 . 5 1 4 4 . 4 8 7 . 9 1 . 5 8 4 . 4 3 5 7 . 2 2 5 3 . 7 1 1 0 . 3 2 1 5 . 0 1 1 4 . 4 1 7 4 . 6 1 3 0 . 5 2 0 4 . 5 1 1 0 . 2 1 6 2 . 9 1 3 7 . 9 1 7 1 . 3 1 0 0 . 8 2 . 0 1 1 7 . 8 3 1 0 . 4 3 0 6 . 9 1 4 1 . 2 2 0 3 . 5 1 3 6 . 1 1 6 5 . 7 1 5 9 . 5 1 9 5 . 2 1 1 3 . 7 1 6 7 . 7 1 6 7 . 9 1 8 2 . 1 1 0 1 . 4 2 . 5 1 6 7 . 9 2 9 7 . 2 2 7 5 . 7 1 3 9 . 8 2 0 7 . 0 1 5 8 . 0 1 6 2 . 6 1 8 4 . 4 1 4 8 . 1 2 2 7 . 9 1 6 1 . 4 1 7 7 . 5 1 0 3 . 1 3 . 0 1 7 6 . 2 2 1 1 . 8 2 3 7 . 3 7 6 . 6 1 8 3 . 5 1 7 3 . 8 1 4 4 . 8 1 8 3 . 8 1 7 7 . 2 1 4 1 . 3 1 2 0 . 7 1 7 8 . 0 1 6 7 . 1 8 8 . 3 3 . 5 1 8 7 . 6 1 7 5 . 5 2 1 6 . 4 1 3 5 . 5 1 8 2 . 1 " 1 7 1 . 9 1 2 3 . 0 2 4 3 . 3 1 6 7 . 1 1 3 6 . 5 1 0 8 . 9 1 5 2 . 3 1 6 6 . 7 8 7 . 4 4 . 0 1 6 7 . 1 1 8 8 . 2 2 2 0 . 5 1 2 6 . 2 1 7 0 . 1 1 7 7 . 3 1 4 2 . 1 2 1 8 . 2 1 7 1 . 4 1 1 9 . 1 1 0 4 . 7 1 5 1 . 3 1 6 3 . 0 - 8 5 . 2 4 . 5 1 5 9 . 4 1 7 2 . 4 1 9 0 . 8 1 1 3 . 2 1 0 4 . 8 1 7 1 . 5 1 2 8 . 8 2 0 1 . 8 1 7 1 . 4 1 1 4 . 9 9 4 . 9 1 5 2 . 3 1 4 8 . 0 7 7 . 9 6 . 0 1 4 1 . 9 1 5 0 . 9 1 6 5 . 4 7 7 . 4 1 4 8 . 8 1 6 8 . 2 1 0 3 . 5 1 5 0 . 1 1 3 3 . 3 1 0 6 . 6 8 1 . 9 1 4 2 . 1 1 3 0 . 8 6 8 . 7 8 . 0 1 2 3 . 3 1 4 1 . 1 1 2 1 . 0 6 6 . 5 1 2 7 . 5 1 3 3 . 8 9 4 . 6 1 6 8 . 1 1 2 1 . 1 8 9 . 2 6 8 . 1 1 1 6 . 9 1 1 4 . 3 6 0 . 7 1 0 . 0 1 4 3 . 2 1 2 5 . 1 9 9 . 7 6 2 . 2 1 0 7 . 0 1 2 0 . 8 8 3 . 0 1 9 8 . 7 9 7 . 4 6 9 . 9 5 3 . 7 8 8 . 0 1 0 4 . 1 5 8 . 8 1 2 . 0 1 0 0 . 4 : 1 1 2 . 8 1 0 0 . 2 6 5 . 0 9 1 . 5 9 1 . 1 6 4 . 8 1 4 4 . 6 9 5 . 2 6 4 . 1 4 0 . 7 6 8 . 1 8 6 . 5 4 7 . 3 1 4 . 0 3 1 . 0 . 9 4 . 0 9 0 . 4 6 3 . 6 8 0 . 8 8 0 . 4 5 1 . 4 1 7 1 . 0 9 4 . 1 4 3 . 1 2 8 . 3 5 2 . 9 7 3 . 4 4 5 . 4 2 4 . 0 1 6 . 4 5 4 . 6 3 2 . 8 1 3 . 3 4 5 . 4 4 7 . 6 2 6 . 2 5 1 . 5 5 6 . 1 1 5 . 7 1 0 . 3 3 0 . 2 3 3 . 3 2 0 . 4 3 0 . 0 9 . 5 ; 3 7 . 9 2 0 . 4 6 . 8 3 1 . 5 3 8 . 0 2 3 . 5 3 7 . 0 5 8 . 4 1 3 . 6 7 . 6 2 1 . 5 2 5 . 5 1 6 . 7 3 6 . 0 3 . 9 . 3 1 . 8 5 . 1 5 . 4 . 2 2 . 1 2 7 . 8 1 5 . 9 2 6 . 5 3 3 . 6 9 . 1 5 . 6 1 5 . 4 1 6 . 9 1 1 . 4 4 S . 0 1 . 5 ! 1 5 . 6 2 . 1 , 5 . 2 ; 1 0 . 5 2 2 . 2 3 0 . 6 1 8 . 1 1 3 . 5 4 . 4 . 1 0 . 3 1 2 . 2 8 . 6 (h) TIME FRCH DRUG ADMIN •••••III 0 . 0 1 . 0 1 . 5 2 . 0 2 . 5 3 . 0 3 . 5 4 . 0 4 . 5 6 . 0 8 . 0 1 0 . 0 1 2 . 0 1 4 . 0 2 4 . 0 3 0 . 0 3 6 . 0 4 3 . 0 Total Serum Mexiletine Concentration (ng/ml) TOTAL S ( + ) AO CK CT GR GT J B JG JH RL ST TH TK MEAN S . D . 3338*3333 a s s s s s s s s S333333E3 3S33XSBS3 s s c s s s s s s KEEIKSEl ========= SB33S3333 3S3CES333 3333BB3SS ========= ========= CSI93SBC3 ======= 5 4 . 4 1 2 7 . 7 2 5 6 . 2 8 2 . 1 2 3 0 . 6 7 2 . 2 1 2 9 . 0 1 2 0 . 6 1 0 3 . 9 . 5 3 . 5 1 8 0 . 8 8 7 . 6 • 1 2 4 . 9 7 7 . 3 7 9 . 4 2 9 1 . 9 3 1 0 . 0 9 5 . 6 1 8 1 . 4 9 9 . 3 1 6 1 . 2 1 1 9 . 0 1 8 5 . 7 9 2 . 1 1 4 3 . 6 1 1 5 . 8 1 5 6 . 2 9 4 . 1 1 0 8 . 1 2 5 1 . 6 2 1 4 . 6 1 2 5 . 7 1 7 5 . 9 1 2 0 . 3 1 5 7 . 5 1 5 0 . 3 1 7 9 . 7 1 0 1 . 8 1 5 3 . 2 1 3 5 . 1 • 1 5 6 . 2 8 3 . 7 . 1 5 6 . 1 2 4 7 . 3 2 2 6 . 9 1 3 0 . 2 1 7 9 . 1 1 4 4 . 1 1 5 7 . 5 1 7 3 . 0 1 2 7 . 0 1 9 7 . 1 1 4 6 . 6 1 5 7 . 1 8 9 . 6 1 6 1 . 6 1 9 0 . 4 2 0 4 . 9 7 3 . 8 1 7 8 . 6 1 5 8 . 8 1 4 0 . 9 1 6 9 . 9 1 6 3 . 2 1 2 0 . 2 1 0 9 . 5 1 5 6 . 0 1 5 2 . 3 8 0 . 1 1 6 8 . 7 1 8 6 . 1 1 9 3 . 6 1 3 3 . 9 1 7 5 . 4 1 5 8 . 3 1 2 5 . 3 2 3 1 . 7 1 5 8 . 7 1 2 1 . 4 9 4 . 3 1 3 6 . 7 1 5 7 . 0 8 2 . 6 1 5 8 . 6 1 7 2 . 8 1 8 2 . 9 1 2 6 . 4 1 6 3 . 6 1 6 5 . 6 1 4 0 . 5 2 0 6 . 7 1 6 4 . 7 1 0 5 . 2 9 5 . 8 1 4 1 . 5 1 5 2 . 0 7 9 . 0 1 5 7 . 8 1 6 2 . 9 1 6 6 . 2 1 1 2 . 1 1 3 3 . 6 1 6 0 . 7 1 2 4 . 4 2 0 1 . 0 1 6 7 . 7 1 1 5 . 3 9 1 . 5 1 3 6 . 7 1 4 4 . 2 7 5 . 0 1 4 0 . 8 1 3 9 . 6 1 4 8 . 6 7 8 . 4 1 3 4 . 0 1 6 0 . 7 1 0 7 . 4 1 4 4 . 8 1 3 9 . 2 9 7 . 4 7 6 . 9 1 2 5 . 4 1 2 4 . 4 6 5 . 0 1 2 8 . 8 1 1 4 . 5 1 1 3 . 5 6 7 . 8 1 1 9 . 3 1 3 3 . 0 1 0 0 . 9 1 6 2 . 0 1 2 5 . 7 8 1 . 9 6 7 . 0 1 1 1 . 7 1 1 0 . 5 5 8 . 4 1 4 3 . 0 1 1 1 . 1 8 5 . 7 6 5 . 6 1 0 2 . 3 1 2 5 . 5 8 4 . 8 1 9 4 . 9 1 0 3 . 1 6 8 . 5 5 4 . 5 8 5 . 8 1 0 2 . 1 5 7 . 6 1 6 4 . 5 1 0 0 . 2 9 2 . 8 6 7 . 8 9 2 . 2 9 0 . 4 7 3 . 3 1 4 5 . 6 1 0 2 . 4 5 9 . 6 4 2 . 6 6 7 . 0 9 1 . 5 5 1 . 7 3 5 . 3 9 0 . 7 7 9 . 4 5 3 . 6 7 7 . 7 8 0 . 1 5 7 . 2 1 5 0 . 1 9 0 . 5 3 9 . 0 2 9 . 8 5 4 . 7 6 9 . 9 4 1 . 7 1 8 . 8 5 2 . 8 2 6 . 0 1 4 . 5 4 3 . 8 5 0 . 6 2 9 . 6 5 2 . 0 6 5 . 1 1 4 . 1 1 1 . 4 3 5 . 0 3 4 . 5 2 1 . 3 9 . 7 3 6 . 6 1 4 . 8 9 . 3 3 0 . 6 3 8 . 0 2 2 . 3 3 6 . 3 6 6 . 9 1 2 . 1 8 . 5 2 3 . 8 2 5 . 7 1 7 . 5 4 . 4 2 8 . 1 4 . 0 4 . 8 2 1 . 2 2 7 . 8 1 5 . 3 2 5 . 8 3 5 . 6 8 . 7 6 . 1 1 7 . 0 1 6 . 6 1 1 . 2 1 . 2 1 4 . 8 1 . 2 5 . 5 1 0 . 2 2 2 . 2 3 2 . 9 1 5 . 9 1 4 . 0 4 . 5 1 0 . 5 1 2 . 1 8 . 8 Table 11. The serum f r e e R ( - ) - and S(+)-mexiletine concentrations from twelve healthy subjects over 48 hours f o l l o w i n g oral administration of 200 mg ( 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 . ( h ) Free Serum Mexiletine Concentration (ng/ml) TTUC nnc FREE R(-) r g / v i riDlir rKun UKUu AOMIN AO CK CT GR GT JB JG JH RL ST TH TK MEAN S.D. I I I H H M B S E I 3 E S I E S3S3XSES8 33833333= I E S S 3 B S 3 B E B B E B B I I B SSSSBS5VS SBBBtBBBE S I B I E B B B E aSKSStSSBES IBEBBBSSB K S I B 3 I I I 0 . 0 1 .0 4 6 . 0 69.0 90.1 47.7 201.4 6 0 . 9 9 5 . 9 71.4: 6 2 . 3 52.4 121.1 3 8 . 8 79.7 50.5 1.5 62.3 140.7 126.1 46.5 168.0 8 0 . 6 122.5 77.9 105.8 93.1 89.3 5 3 . 8 97.2 5 4 . 8 2 . 0 85.9 121.1 121.9 57.2 133.3 87.3 103.5 42.6 103.0 80.2 9 5 . 3 56.4 9 0 . 6 4 9 . 3 2 . 5 127.8 107.7 8 2 . 7 55.3 129.5 110.7 105.2 91.3 79.8 102.0 6 2 . 9 8 7 . 9 5 0 . 6 3 . 0 134.8 8 9 . 2 78.5 59.1 130.8 118.2 8 6 . 6 99.2 8 5 . 0 77.1 103.7 6 9 . 0 94.3 4 9 . 9 3 . 5 128.4 78/2 73.9 62.3 120.7 122.3 86.1 135.0 8 2 . 5 75.1 72.2 58.4 91.2 4 9 . 3 4 . 0 119.9 6 3 . 8 75.8 59.8 114.3 123.3 86.1 158.8 83.7 67.1 67.5 5 9 . 0 8 9 . 9 5 0 . 0 4 . 5 107.1 73.5 79.5 52.2 108.0 117.1 77.3 151.4 83.1 71.7 56.5 58.4 86.3 47.6 6 . 0 94.3 62.6 67.7 37.0 106.1 115.5 6 4 . 6 123.7 6 9 . 9 6 5 . 6 46.6 52.7 75.5 42.3 8 . 0 8 9 . 6 44.1 64.4 30.1 90.4 105.0 53.2 128.7 67.3 61.8 49.4 4 9 . 0 69.4 3 9 . 9 10.0 92.6 3 6 . 6 52.6 25.6 70.2 96.4 51.9 115.9 6 2 . 3 54.2: 3 9 . 6 3 8 . 2 61.3 36.1 12.0 73.1 . 34.2 37.5 29.4 55.7 72.9 48.5 86.4 4 5 . 2 4 3 . 8 3 0 . 4 42.2 50.0 2 8 . 0 U . O 18.6 29.7 19.5 28.2 56.5 6 9 . 3 3 2 . 2 83.8 4 6 . 0 24.0 23.4: 4 0 . 2 3 9 . 3 24.3 24.0 9 . 9 9.4 13.2 8.2 26.5 40.4 16.7 34.7 17.5 8 . 6 8 . 0 18.2 17.6 11.6 3 0 . 0 6.7 4.1 9 . 0 5.6 17.4 36.5 14.2 12.4 16.3 8.2. 13.9 13.1 8 . 9 3 6 . 0 2.7 2.4 1 .9 3.7 11.1 29.5 10.1 9.1 8.7 3.0: 10.9 8 . 5 6 . 8 4 8 . 0 0 . 8 2.5 4 . 5 23.3 19.0 8.7 6 . 6 3 . 3 13.9 9 . 2 6 . 7 ( h ) Free Serum Mexiletine Concentration (ng/ml) TIME FROM DRUG FREE SC + ) ADMIN AO CK CT GR GT J 8 JG JH RL ST TH TK MEAN s . o . BSE3BIBB1 E==3XS=S= CBBEECBSS "="="= 3ES333B33 S E S B S E E E E S S I E E S E S B B S B B S E S I S BEEBBSBBS E E E 1 S 1 H I 0 . 0 1.0 40.4 5 3 . 0 77.2 36.9 139.5 5 8 , 3 8 0 . 0 62.0 46.7 51.0 9 4 . 5 3 3 . 2 64.4 3 8 . 3 1.5 54.3 109.0 113.2 25.9 123.1 75.9 112.6 67.4 8 9 . 6 85.0 73.5 44.2 81.1 4 5 . 6 2 . 0 68.6 101.5 108.5 47.3 93.6 82.1 9 2 . 3 75.2 85.1 74.2 8 4 . 6 50.0 80.2 42.1 2.5 110.3 91.3 73.8 47.3 102.1 116.9 9 6 . 9 79.2 77.2 9 0 . 2 5 4 . 9 78.4 4 5 . 2 3 . 0 117.2 6 8 . 2 71.7 52.5 110.0 110.4 82.1 86.1 74.7 77.0 8 8 , 2 64.4 83.5 4 3 . 9 3 . 5 120.6 6 3 . 3 67.2 55.8 103.4 113.7 85.5 115.6 75.3 73.5 71.4 53.4 83.2 44.8 4 . 0 112.0 50.6 6 8 . 8 55.8 94.2 119.2 85.4 141.0 76.6 64.7 6 3 . 8 54.8 8 2 . 2 45.7 4 . 5 98.7 67.0 75.0 49.3 92.3 113.1 72.4 139.6 77.3 6 8 . 9 5 5 . 8 53.4 8 0 . 2 44.1 6 . 0 92.4 5 3 . 0 65.4 3 3 . 8 89.0 110.8 60.5 113.1 6 5 . 6 62.7 50.0 50.7 70.6 39.3 8 . 0 84.8 37.2 61.1 27.3 76.5 105.9 55.5 119.5 6 3 . 6 57.2 5 0 . 0 4 5 . 9 65.4 37.6 10.0 8 5 . 9 30.5 5 2 . 2 25.2 59.5 99.5 5 2 . 9 106.9 5 4 . 8 55.7 41.5 3 7 . 9 5 8 . 6 3 4 . 3 12.0 69.2 2 9 . 2 3 4 . 8 2 9 . 8 46.4 72.9 4 9 . 5 79.7 44.1 43.3 31.4 41.1 47.6 26.6 U . O 20.8 20.1 19.9 25.2 52.3 70.1 35.5 77.7 45.4 24.5 2 6 . 9 4 0 . 8 3 8 . 3 23.5 24.0 10.4 3.7 12.8 7.7 23.5 4 2 . 6 18.4 34.5 20.1 8 . 5 8 . 5 19.7 17.5 11.9 3 0 . 0 7.1 1.2 8 . 2 5.1 13.7 3 9 . 9 14.5 12.6 17.1 8.5 14.5 12.9 9 . 4 3 6 . 0 3.1 1.2 1.6 3.1 9.1 3 2 . 8 9 . 5 9.2 12.6 3.1 11.5 8 . 8 7.4 4 8 . 0 0.5 1.8 2.8; 24.1 20.4 8.2 6.1 2 . 9 14.5 9 . 0 7 . 0 Table 12. The serum t o t a l and serum free mexiletine R(-)/S(+) r a t i o s over 48 hours from twelve healthy s u b j e c t s . ••••••• Total Serum Mexiletine R/S Ratio TIME FROM DRUG TOTAL R/S AOMIN AO CK CT GR GT JB JG JH RL ST TH TK MEAN S . O . M I I I B I B I B S S E X I I I S B I E I I X 8 S S s s s s s s s s s 38X3338=3 8 X 8 3 8 3 3 3 3 8 E S 8 3 3 3 S 3 3 3 S 3 S 3 S 8 S 3 8 8 3 = 3 3 3 3 3 3 3 3 8 3 8 3 3 3 3 8 X 8 8 X 3 8 8 S 3 S 8 B S 3 3 S 8 3 3 8 3 S S 3 X S 3 S S 8 8 S 3 8 3 8 8 8 8 X 3 8 0 . 0 0 . 0 0 0 . 0 0 1 . 0 1 . 1 5 1 . 2 1 1 . 1 7 1 . 1 7 1 . 0 0 1 . 1 1 1 . 2 0 1 . 1 5 1 . 2 4 1 . 1 9 1 . 1 9 1 . 2 4 1 . 1 7 0 . 5 8 1 . 5 1 . 0 6 1 . 2 2 0 . 8 2 1 . 1 5 1 . 1 9 1 . 1 5 1 . 0 8 1 . 1 0 1 . 1 0 1 . 2 0 1 . 1 3 1 . 1 9 1 . 1 2 0 . 5 6 2 . 0 1 . 0 9 1 . 2 3 1 . 4 3 1 . 1 2 1 . 1 6 1 . 1 3 1 . 0 5 1 . 0 6 1 . 0 9 1 . 1 2 1 . 0 9 1 . 2 4 1 . 1 5 0 . 5 8 2 . 5 1 . 0 8 1 . 2 0 1 . 2 1 1 . 0 7 1 . 1 6 1 . 1 0 1 . 0 3 1 . 0 7 1 . 1 7 1 . 1 6 1 . 1 0 1 . 0 3 0 . 5 6 3 . 0 1 . 0 9 1 . 1 1 1 . 1 6 1 . 0 4 1 . 0 3 1 . 0 9 1 . 0 3 1 . 0 8 1 . 0 9 1 . 1 8 1 . 1 0 1 . 1 4 1 . 0 9 0 . 5 5 3 . 5 1 .11 0 . 9 4 1 . 1 2 1 . 0 1 1 . 0 4 1 . 0 9 0 . 9 8 1 . 0 5 1 . 0 5 1 . 1 2 1 . 1 6 1 . 1 1 1 . 0 7 0 . 5 3 4 . 0 1 . 0 5 1 . 0 9 1 . 2 1 1 . 0 0 1 . 0 4 1 . 0 7 1 . 0 1 1 . 0 6 1 . 0 4 1 . 1 3 1 . 0 9 1 . 0 7 1 . 0 7 0 . 5 4 4 . 5 1 . 0 1 1 . 0 6 1 . 1 5 1 . 0 1 0 . 7 8 0 . 1 1 1 . 0 4 1 . 0 0 1 . 0 2 1 . 0 0 1 . 0 4 1 . 1 1 0 . 9 4 0 . 5 1 6 . 0 1 . 0 1 1 . 0 8 1 . 1 1 • 0 . 9 9 1 .11 1 . 0 5 0 . 9 6 1 . 0 4 0 . 9 6 1 . 0 9 1 . 0 7 1 . 1 3 1 . 0 5 0 . 5 3 8 . 0 0 . 9 6 1 . 2 3 1 . 0 7 0 . 9 8 1 . 0 7 1 . 0 1 0 . 9 4 1 . 0 4 0 . 9 6 1 . 0 9 1 . 0 2 1 . 0 5 1 . 0 3 0 . 5 2 1 0 . 0 1 . 0 0 1 . 1 3 1 . 1 6 0 . 9 5 1 . 0 5 0 . 9 6 0 . 9 8 1 . 0 2 0 . 9 4 1 . 0 2 0 . 9 9 1 . 0 3 1 . 0 2 0 . 5 1 1 2 . 0 0 . 6 1 1 . 1 3 1 . 0 8 0 . 9 6 0 . 9 9 1 . 0 1 0 . 8 8 0 . 9 9 0 . 9 3 1 . 0 8 0 . 9 6 1 . 0 2 0 . 9 7 0 . 4 9 1 4 . 0 0 . 8 8 1 . 0 4 1 . 1 4 1 . 1 9 1 . 0 4 1 . 0 0 0 . 9 0 1 . 1 4 1 . 0 4 1 . 1 0 0 . 9 5 0 . 9 7 1 . 0 3 0 . 5 2 2 4 . 0 0 . 8 7 1 . 0 4 1 . 2 6 0 . 9 2 1 . 0 4 0 . 9 4 0 . 8 8 0 . 9 9 0 . 8 6 1 . 1 1 0 . 9 0 0 . 8 6 0 . 9 7 0 . 4 9 3 0 . 0 0 . 9 8 1 . 1 3 1 . 3 8 0 . 7 4 1 . 0 3 0 . 9 2 1 . 0 5 1 . 0 2 0 . 8 7 1 . 1 2 0 . 9 0 0 . 9 0 1 . 0 0 0 . 5 1 3 6 . 0 0 . 8 9 1 . 1 3 1 . 2 6 1 . 1 3 1 . 0 5 0 . 8 9 1 . 0 4 1 . 0 3 0 . 9 4 1 . 0 5 0 . 9 1 0 . 9 1 1 . 0 2 0 . 5 2 4 8 . 0 1 . 7 5 0 . 9 6 1 . 0 3 1 . 0 1 0 . 9 3 1 . 1 4 0 . 9 6 0 . 9 8 0 . 9 8 1 . 0 8 0 . 5 6 (h) Free Serum Mexiletine R/S Ratio TIME FROM DRUG FREE R/S MEAN S . D . ADMIN AO CK CT GR GT JB JG JH RL ST TH TK m n i i i i • t i i i i i i i 8 8 3 3 8 8 8 X 3 X 8 8 S 3 3 3 3 X = 8 3 3 8 8 8 3 3 8 8 S 8 8 3 8 8 X 8 X 8 8 8 3 8 3 8 XSS8888S8 8SBXXX33S 3 3 3 8 3 8 X 3 8 8 8 8 3 8 8 8 8 8 S 8 8 B 8 3 8 B B 3 3 8 B 8 3 8 8 X S 8 8 X S X 8 8 8 8 S S 8 8 S 8 S 8 0 . 0 0 . 0 0 0 . 0 0 1 . 0 1 . 1 4 1 . 3 0 1 . 1 7 1 . 2 9 1 . 4 4 1 . 0 4 1 . 2 0 1 . 1 5 1 . 3 3 1 . 0 3 1 . 2 8 1 . 1 7 1 . 2 1 0 . 6 1 1 . 5 1 . 1 5 1 . 2 9 1 . 1 1 1 . 8 0 1 . 3 6 1 . 0 6 1 . 0 9 1 . 1 6 1 . 1 8 1 . 0 9 1 . 2 1 1 . 2 2 1 . 2 3 0 . 6 3 2 . 0 1 . 2 5 1 . 1 9 1 . 1 2 1 . 2 1 1 . 4 2 1 . 0 6 1 . 1 2 0 . 5 7 1 . 2 1 1 . 0 8 1 . 1 3 1 . 1 3 1 . 1 2 0 . 5 8 2 . 5 1 . 1 6 1 . 1 8 1 . 1 2 1 . 1 7 1 . 2 7 0 . 9 5 1 . 0 8 1 . 1 5 1 . 0 3 1 . 1 3 1 . 1 4 1 . 0 3 0 . 5 7 3 . 0 1 . 1 5 1 . 3 1 1 . 1 0 1 . 1 2 1 . 1 9 1 . 0 7 1 . 0 5 1 . 1 5 1 . 1 4 1 . 0 0 1 . 1 8 1 . 0 7 1 . 1 3 0 . 5 7 3 . 5 1 . 0 6 1 . 2 4 1 . 1 0 1 . 1 2 1 . 1 7 1 . 0 8 1 . 0 1 1 . 1 7 1 . 1 0 1 . 0 2 1 . 0 1 1 . 0 9 1 . 1 0 0 . 5 5 4 . 0 1 . 0 7 1 . 2 6 1 . 1 0 1 . 0 7 1 . 2 1 1 . 0 3 1 . 0 1 1 . 1 3 1 . 0 9 1 . 0 4 1 . 0 6 1 . 0 8 1 . 1 0 0 . 5 5 4 . 5 1 . 0 9 1 . 1 0 1 . 0 6 1 . 0 6 1 . 1 7 1 . 0 4 1 . 0 7 1 . 0 8 1 . 0 8 1 . 0 4 1 . 0 1 1 . 0 9 1 . 0 7 0 . 5 4 6 . 0 1 . 0 2 1 . 1 8 1 . 0 3 1 . 1 0 1 . 1 9 1 . 0 4 , 1 . 0 7 1 . 0 9 1 . 0 7 1 . 0 5 0 . 9 3 1 . 0 4 1 . 0 7 0 . 5 4 8 . 0 1 . 0 6 1 . 1 9 1 . 0 6 1 . 1 0 1 . 1 8 0 . 9 9 0 . 9 6 1 . 0 8 1 . 0 6 1 . 0 8 0 . 9 9 1 . 0 7 1 . 0 7 0 . 5 4 1 0 . 0 1 . 0 8 1 . 2 0 1 . 0 1 1 . 0 2 1 . 1 8 0 . 9 7 0 . 9 8 1 . 0 8 1 . 1 4 0 . 9 7 0 . 9 5 1 . 0 1 1 . 0 5 0 . 5 3 1 2 . 0 1 . 0 6 1 . 1 7 1 . 0 8 0 . 9 9 1 . 2 0 1 . 0 0 0 . 9 8 1 . 0 8 1 . 0 3 1 . 0 1 0 . 9 7 1 . 0 3 1 . 0 5 0 . 5 3 1 4 . 0 0 . 8 9 1 . 4 7 0 . 9 8 1 . 1 2 1 . 0 8 0 . 9 9 0 . 9 1 1 . 0 8 1 . 0 1 0 . 9 8 0 . 8 7 0 . 9 9 1 . 0 3 0 . 5 3 2 4 . 0 0 . 9 5 1 . 1 3 1 . 0 3 1 . 0 6 1 . 1 3 0 . 9 5 0 . 9 1 1 . 0 1 0 . 8 7 1 . 0 0 0 . 9 4 0 . 9 3 0 . 9 9 0 . 5 0 3 0 . 0 0 . 9 4 1 . 9 1 1 . 0 9 1 .11 1 . 2 7 0 . 9 2 0 . 9 8 0 . 9 8 0 . 9 5 0 . 9 7 0 . 9 6 1 . 1 0 0 . 5 8 3 6 . 0 0 . 9 0 1 . 9 1 1 . 1 6 1 . 2 0 1 . 2 2 0 . 9 0 1 . 0 7 0 . 9 8 0 . 6 9 0 . 9 7 0 . 9 5 1 . 0 9 0 . 5 8 4 8 . 0 1 . 6 3 1 . 3 6 1 . 5 6 0 . 9 7 0 . 9 3 1 . 0 7 1 . 0 9 1 . 1 1 0 . 9 6 1 . 1 9 0 . 6 2 enantiomers was n o n - s t e r e o s e l e c t i v e . This f i n d i n g was c o n s i s t e n t with the r e l a t i v e l y constant the in vivo serum f r e e f r a c t i o n s f o r m e x i l e t i n e enantiomers as presented in Figure 21 and Table 13. The o v e r a l l mean serum f r e e f r a c t i o n s f o r R ( - ) - and S(+)-mexiletine over 48 hours were found to be 0.57 ± 0.07 and 0.56 ± 0 . 0 6 , r e s p e c t i v e l y (mean ± S . E . ) . As shown in Tables 10 and 11, the serum t o t a l and serum f r e e concentrations of R ( - ) - m e x i l e t i n e were c o n s i s t e n t l y and s i g n i f i c a n t l y (p<0.01) g r e a t e r than those of S(+)-mexiletine during the f i r s t s i x hours f o l l o w i n g o r a l drug a d m i n i s t r a t i o n . However, the d i f f e r e n c e s between the two enantiomers i n both the serum mean t o t a l and mean f r e e drug l e v e l s were found not to be s i g n i f i c a n t past the s i x t h hour. These small but c o n s i s t e n t l y greater mean R ( - ) - m e x i l e t i n e serum concentrations observed during the f i r s t s i x hours f o l l o w i n g o r a l drug a d m i n i s t r a t i o n were c o n s i s t e n t with an e a r l i e r report of greater mean R ( - ) - m e x i l e t i n e plasma concentrations i n the f i r s t 8 to 10 hours f o l l o w i n g o r a l drug a d m i n i s t r a t i o n to f i v e subjects (Igwemezie et a l . , 1989). The reasons f o r the t r a n s i e n t increase i n R ( - ) - m e x i l e t i n e serum t o t a l and serum f r e e drug concentrations remained u n c e r t a i n . The d i s p o s i t i o n of the serum t o t a l m e x i l e t i n e enantiomer concentrations was best described by a one-compartment open model in four s u b j e c t s , w h i l e i n the remaining eight s u b j e c t s , the d i s p o s i t i o n of serum t o t a l m e x i l e t i n e enantiomers was best described by a two-compartment open model. The pharmacokinetic parameters derived from serum t o t a l drug data f o r m e x i l e t i n e enantiomers are presented i n Table 14. The c a l c u l a t e d peak serum t o t a l concentrations ( C m a x ) f o r the twelve subjects were found to range from 141 to 357 ng/ml f o r R ( - ) -m e x i l e t i n e and from 133 to 310 ng/ml f o r S ( + ) - m e x i l e t i n e , with a mean O R(-0 • S(+) 1 . 0 0 n o o Li-CD CD E D L_ CD CO 0 . 5 0 8 o 0 . 0 0 0 1 0 1 5 2 0 2 5 3 0 3 5 4 0 4 5 T i m e ( h ) Figure 21, Plots of the mean ± S.D. R ( - ) - and S(+)-mexiletine serum free f r a c t i o n s from twelve healthy subjects over 48 hours. Table 13. The R ( - ) - and S(+)-mexiletine serum f r e e f r a c t i o n s over 48 hours from twelve healthy s u b j e c t s . (h) R(-) Mexiletine Serum Free.Fraction TIME FREE R/TOTAL R FROM DRUG AOMIN AO CK CT GR GT JB JG JH RL ST TH TK MEAN S . D . IIIEBMII BSS3SS33S S3333S333 : : s = s s=E3 SS3SS33S3 : s : = s s n s SI3S3S33S 33S33S3S3 333333333 333335338 33333333= SSSSBXSBB SSSSSEEB8S BSS383B3S 0 . 0 0 . 0 0 0 . 0 0 1 . 0 0 . 7 4 0 . 4 5 0 . 3 0 0 . 5 0 0 . 8 7 0 . 7 6 0 . 6 2 0 . 5 1 0 . 4 9 0 . 8 2 0 . 5 6 0 . 3 6 0 . 5 8 0 . 3 2 1 . 5 0 . 7 4 0 . 3 9 0 . 5 0 0 . 4 2 0 . 7 8 0 . 7 0 0 . 7 0 0 . 6 0 0 . 5 2 0 . 8 4 0 . 5 5 0 . 3 9 0 . 5 9 0 . 3 2 2 . 0 0 . 7 3 • 0 . 3 9 0 . 4 0 0 . 4 1 0 . 6 6 0 . 6 4 0 . 6 2 0 . 2 7 0 . 5 3 ' 0 . 7 1 0 . 5 7 0 . 3 4 0 . 5 2 0 . 2 8 2 . 5 0 . 7 6 0 . 3 6 0 . 3 0 0 . 4 0 0 . 6 3 0 . 7 0 0 . 6 5 0 . 5 0 : 0 . 5 4 0 . 4 5 0 . 3 9 0 . 4 7 0 . 2 8 3 . 0 0 . 7 7 . 0 . 4 2 0 . 3 3 0 . 7 7 0 . 7 1 0 . 6 8 0 . 6 0 0 . 5 4 0 . 4 8 ; 0 . 5 5 0 . 8 6 0 . 3 9 0 . 5 9 0 . 3 2 3 . 5 0 . 6 8 0 . 4 5 0 . 3 4 0 . 4 6 0 . 6 6 0 . 7 1 0 . 7 0 0 . 5 5 0 . 4 9 0 . 5 5 0 . 6 6 0 . 3 8 0 . 5 5 0 . 2 9 4 . 0 0 . 7 2 0 . 3 4 0 . 3 4 0 . 4 7 0 . 6 7 0 . 7 0 0 . 6 1 0 . 7 3 0 . 4 9 0 . 5 6 0 . 6 4 0 . 3 9 0 . 5 6 0 . 2 9 4 . 5 0 . 6 7 0 . 4 3 0 . 4 2 0 . 4 6 1 . 0 3 0 . 6 8 0 . 6 0 0 . 7 5 0 . 4 8 0 . 6 2 0 . 6 0 0 . 3 8 0 . 5 9 0 . 3 2 6 . 0 0 . 6 6 0 . 4 1 0 . 4 1 0 . 4 8 0 . 7 1 0 . 6 9 0 . 6 2 0 . 8 2 0 . 5 2 0 . 6 2 0 . 5 7 0 . 3 7 0 . 5 7 0 . 3 0 8 . 0 0 . 7 3 0 . 3 1 0 . 5 3 0 . 4 5 0 . 7 1 0 . 7 8 0 . 5 6 0 . 7 7 0 . 5 6 0 . 6 9 0 . 7 3 0 . 4 2 0 . 6 0 0 . 3 2 1 0 . 0 0 . 6 5 0 . 2 9 0 . 5 3 0 . 4 1 0 . 6 6 0 . 8 0 0 . 6 3 0 . 5 8 0 . 6 4 0 . 7 8 0 . 7 4 0 . 4 3 0 . 5 9 0 . 3 2 1 2 . 0 0 . 7 3 0 . 3 0 0 . 3 7 0 . 4 5 0 . 6 1 0 . 8 0 0 . 7 5 0 . 6 0 0 . 4 8 0 . 6 8 0 . 7 5 0 . 6 2 0 . 6 0 0 . 3 2 1 4 . 0 0 . 6 0 0 . 3 2 0 . 2 2 0 . 4 4 0 . 7 0 0 . 8 6 0 . 6 3 0 . 4 9 0 . 4 9 0 . 5 6 0 . 8 3 0 . 7 6 0 . 5 7 0 . 3 2 2 4 . 0 0 . 6 1 0 . 1 1 0 . 4 0 0 . 6 1 0 . 5 9 0 . 8 5 0 . 6 4 0 . 6 7 0 . 3 1 0 . 5 5 0 . 7 8 0 . 6 1 0 . 5 6 0 . 3 1 3 0 . 0 0 . 7 0 0 . 0 8 0 . 4 4 0 . 8 3 0 . 5 5 0 . 9 6 0 . 6 1 0 . 3 3 0 . 2 8 0 . 6 1 0 . 6 5 0 . 5 5 0 . 3 2 3 6 . 0 0 . 7 1 0 . 0 8 0 . 3 8 0 . 7 0 0 . 5 0 1 . 0 6 0 . 6 4 0 . 3 4 0 . 2 6 0 . 3 3 0 . 7 1 0 . 5 2 0 . 3 2 4 8 . 0 0 . 3 8 0 . 4 8 0 . 4 3 1 . 0 5 0 , 6 2 0 . 4 8 0 . 4 9 0 . 7 4 1 . 3 6 0 . 6 7 0 . 3 9 0 . 0 1 . 0 1 . 5 2 . 0 2 . 5 3 . 0 3 . 5 4 . 0 4 . 5 6 . 0 8 . 0 1 0 . 0 1 2 . 0 1 4 . 0 2 4 . 0 3 0 . 0 3 6 . 0 4 8 . 0 S(+) Mexiletine Serum Free Fraction FREE S/TOTAL S AO CK CT GR GT JB JG JH RL ST TH TK MEAN s.o.. llltHSIB S3SS83S88 S333S3SB3 IBIISSBIB BB3BSESSI 33SB3S33S B1EE2EBBS SEBEBB3EE SSS&3&&SS 33BB3SBEB 333SSSBSS 383SEBSB3 s s s s s x a s a 0 . 0 0 ESSBSSBB 0 . 0 0 0 . 7 4 0 . 4 1 0 . 3 0 0 . 4 5 0 . 6 0 0 . 8 1 0 . 6 2 0 . 5 1 0 . 4 5 0 . 9 5 0 . 5 2 0 . 3 8 0 . 5 6 0 . 3 1 0 . 6 8 0 . 3 7 0 . 3 7 0 . 2 7 0 . 6 8 0 . 7 6 0 . 7 0 0 . 5 7 0 . 4 8 0 . 9 2 0 . 5 1 0 . 3 8 0 . 5 6 0 . 3 1 0 . 6 3 0 . 4 0 0 . 5 1 0 . 3 8 0 . 5 3 0 . 6 8 0 . 5 9 0 . 5 0 0 . 4 7 0 . 7 3 0 . 5 5 0 . 3 7 0 . 5 3 0 . 2 8 0 . 7 1 0 . 3 7 0 . 3 3 0 . 3 6 0 . 5 7 0 . 8 1 0 . 6 2 0 . 4 6 0 . 6 1 0 . 4 6 0 . 3 7 0 . 4 7 0 . 2 8 0 . 7 3 0 . 3 6 0 . 3 5 0 . 7 1 0 . 6 2 0 . 7 0 0 . 5 8 0 . 5 1 0 . 4 6 0 . 6 4 0 . 8 1 0 . 4 1 0 . 5 7 0 . 3 0 0 . 7 1 0 . 3 4 0 . 3 5 0 . 4 2 0 . 5 9 0 . 7 2 0 . 6 8 0 . 5 0 0 . 4 7 0 . 6 1 0 . 7 6 0 . 3 9 0 . 5 4 0 . 2 9 0 . 7 1 0 . 2 9 0 . 3 8 0 . 4 4 0 . 5 8 0 . 7 2 0 . 6 1 0 . 6 8 0 . 4 7 0 . 6 1 0 . 6 7 0 . 3 9 0 . 5 5 0 . 2 9 0 . 6 3 0 . 4 1 0 . 4 5 0 . 4 4 0 . 6 9 1 . 0 7 0 . 5 8 0 . 6 9 0 . 4 6 0 . 6 0 0 . 6 1 0 . 3 9 0 . 5 9 0 . 3 2 0 . 6 6 0 . 3 8 0 . 4 4 0 . 4 3 0 . 6 6 0 . 6 9 0 . 5 6 0 . 7 8 0 . 4 7 0 . 6 4 0 . 6 5 0 . 4 0 0 . 5 6 0 . 3 0 0 . 6 6 0 . 3 2 0 . 5 4 0 . 4 0 0 . 6 4 0 . 8 0 0 . 5 5 0 . 7 4 0 . 5 1 0 . 7 0 0 . 7 5 0 . 4 1 0 . 5 8 0 . 3 1 0 . 6 0 0 . 2 7 0 . 6 1 0 . 3 9 0 . 5 8 0 . 7 9 0 . 6 2 0 . 5 5 0 . 5 3 0 . 8 1 0 . 7 6 0 . 4 4 0 . 5 8 0 . 3 1 0 . 4 2 0 . 2 9 0 . 3 7 0 . 4 4 0 . 5 0 0 . 8 1 0 . 6 8 0 . 5 5 0 . 4 3 0 . 7 3 0 . 7 4 0 . 6 1 0 . 5 5 0 . 3 0 0 . 5 9 0 . 2 2 0 . 2 5 0 . 4 7 0 . 6 7 0 . 8 7 0 . 6 2 0 . 5 2 0 . 5 0 0 . 6 3 0 . 9 0 0 . 7 5 0 . 5 8 0 . 3 3 0 . 5 5 0 . 1 0 0 . 4 9 0 . 5 3 0 . 5 4 0 . 8 4 0 . 6 2 •' 0 . 6 6 0 . 3 1 0 . 6 1 0 . 7 5 0 . 5 6 0 . 5 5 0 . 3 0 0 . 7 3 0 . 0 5 0 . 5 6 0 . 5 5 0 . 4 4 0 . 9 7 0 . 6 5 0 . 3 5 0 . 2 6 0 . 7 0 0 . 6 1 0 . 5 3 0 . 3 2 0 . 7 0 0 . 0 5 0 . 4 1 0 . 6 6 0 . 4 3 1 . 0 5 0 . 6 2 0 . 3 6 0 . 3 5 0 . 3 6 0 . 6 8 0 . 5 1 0 . 3 1 0 . 4 1 0 . 3 4 0 . 2 8 1 . 1 0 0 . 6 2 0 . 5 1 0 . 4 4 0 . 6 6 1 . 3 8 0 . 6 4 0 . 3 9 Table 14. The pharmacokinetic data f o r R ( - ) - and S(+)-mexiletine derived from serum t o t a l drug data from twelve healthy subjects f o l l o w i n g oral administration of 200 mg racemic mexiletine hydrochloride. B o d / w e i g h t ( K g ) l l / 2 ka(h) Cmax (19/ml) Tmax (h) t l / 2 o (h) ti/2 6 (h) AUC?(ng/h/m1) CL/F ( m l / m i n / K g ) Vd/F ( t / t g ) SUBJECTS R S R S R S R s R S R S R S R S R S GT JG CK RL GR JH AO CT ST TH TK JB 5 9 . 0 8 1 . 8 7 5 . 0 8 2 . 0 7 3 . 0 6 3 . 5 7 2 . 0 6 8 . 0 6 3 . 0 6 6 . 0 8 0 . 0 6 2 . 7 2 . 7 6 8 5 . 2 2 7 3 . 8 5 9 7 . 8 4 8 1.311 0 . 6 2 3 0 . 8 9 V 1 . 5 9 8 1.461 3 . 7 4 0 0 . 5 3 3 0 . 7 5 3 3 . 7 3 4 3 . 5 1 4 5 . 5 9 8 -6 . 3 6 5 0 . 9 4 3 0 . 5 4 3 0 . 6 0 2 3 . 4 5 3 1 . 1 8 7 1 .974-0 . 5 5 6 0 . 5 6 2 0 . 1 0 . 1 0 . 0 0 . 5 1.1 0 . 7 0 . 4 . 0 . 4 0 . 1 1 . 3 0 . 9 0 . 2 O . i : 0 .1 -0 . 7 1 . 2 1.1 0 . 2 0 . 5 0 . 3 1 . 2 1.2. 2 3 0 . 6 1 7 4 . 6 3 5 7 . 3 2 0 4 . 6 1 4 1 . 3 2 4 3 . 3 1 8 7 . 6 3 5 2 . 7 1 4 8 . 2 2 2 8 . 0 1 6 1 . 5 177 .3 2 3 0 . 6 161.2 2 9 2 . 0 1 8 5 . 8 1 3 3 . 9 2 3 1 . 8 1 6 8 . 8 3 1 0 . 1 1 2 7 . 0 197.1 1 5 6 . 0 1 6 5 . 7 1 . 3 0 . 8 0 . 9 0 . 6 2 . 4 4 . 1 2 . 6 1 . 8 2 . 0 0 . 9 3 . 4 3 . 5 1.1 i . t 0 . 7 0 . 7 2 . 8 . 4 . 4 : 3 . 3 1 . 0 2 . 3 1 . 3 3 . 5 4 . 2 9 . 9 1 . 5 . ; 1-3 3 . 9 ' 2 . 3 5 . 3 9 . 6 -8 . 2 1 . 7 O.4.. 7 . 7 1 . 4 4 . O . , 6 . 4 8.7; 1 0 . 5 5 . 3 1 1 . 3 8 . 3 11.1. 6 . 0 6 . 6 7.8 5 . 0 ' 5 . 0 1 0 . 7 . 11 .0 11.1 6 . 8 1 2 . 2 8 . 4 10.7 5 . 9 6 . 2 7 . 5 3 . 5 6 . 1 10.1 . 3 1 5 4 . 8 2 5 8 4 . 5 3 9 4 5 . 0 3 6 2 1 . 2 1 6 9 0 . 2 4 3 8 1 . 8 2 0 4 1 . 3 3 0 0 9 . 7 1 7 7 9 . 8 1 5 5 9 . 7 2 4 5 6 . 6 3 5 5 7 . 6 2 5 2 0 . 9 1 6 9 6 . 1 3 6 0 9 . 1 3 5 8 4 . 3 1 6 4 5 . 6 4 1 2 0 . 7 2 0 6 5 . 9 2 6 0 0 . 5 1 6 3 1 . 0 1 4 5 1 . 9 2 4 2 8 . 3 3 5 9 1 . 3 7 . 5 6 . 6 4 . 7 4 . 7 1 1 . 3 5 . 0 9 . 3 6 . 8 12.4 1 3 . 6 7 .1 6 . 3 9.4 1 0 . 0 5 . 1 4 .7 1 1 . 6 5 . 3 9.4 7 . 9 1 3 . 6 1 4 . 6 7 .1 6 . 1 5 . 7 6 . 2 2 . 2 4 . 6 8 . 1 4 . 9 4 . 9 3.9 8.4 5 . 9 3 . 1 5 . 8 8.9 9 .7 3 . 0 5 . 1 8.4 5 . 0 4 . 9 4 . 3 8 . 9 4 . 5 3 . 8 5.4 MEAN 2 . 5 5 1 2 . 4 1 9 0 . 5 0 . 6 2 1 7 . 3 1 9 6 . 7 2 . 0 : . 2 . 2 4 . 8 . 4 . 3 B . O ; . 8 . 3 2 8 1 5 . 2 2 5 7 9 . 0 7 . 9 8 . 8 5 . 3 6 . 0 S . O . 2 . 1 5 6 1 . 9 8 5 0 . 4 : ' 0 . 4 6 8 . 9 5 6 . 2 1.1 1 . 3 3 . 5 3 . 1 2 . 3 ' 2 . 5 9 0 2 . 7 8 9 3 . 5 2 . 9 3 . 1 1 .7 2 . 2 value of 217 ± 68 ng/ml and 196 ± 56 ng/ml f o r R ( - ) - and S(+)-m e x i l e t i n e , r e s p e c t i v e l y . The higher mean C m a x f o r serum t o t a l R ( - ) -m e x i l e t i n e was found to be s i g n i f i c a n t l y greater (p<0.01) than that f o r the S(+)-enantiomer. The time to achieve peak serum t o t a l concentrations ( t m a x ) was c a l c u l a t e d and was found to vary from 0.63 to 4.11 hours f o r R ( - ) - m e x i l e t i n e , w h i l e a range of 0.73 to 4.46 hours was obtained f o r S ( + ) - m e x i l e t i n e . The mean t m a x values of 2.06 ± 1.14 h and 2.25 ± 1.35 h , f o r R ( - ) - and S ( + ) - m e x i l e t i n e , r e s p e c t i v e l y , f o r the twelve s u b j e c t s , were found not to be s i g n i f i c a n t l y d i f f e r e n t . The s i m i l a r mean t m a x values observed between R ( - ) - and S(+)-mexiletine a f t e r o r a l drug a d m i n i s t r a t i o n suggested t h a t , i n f a s t i n g s u b j e c t s , the r a t e s of o r a l drug absorption f o r the two enantiomers were l i k e l y to be s i m i l a r . However, the s i g n i f i c a n t l y greater mean serum t o t a l C m a x values f o r R ( - ) - m e x i l e t i n e , compared to those of the S(+)-enantiomer, suggested that the extent of systemic a v a i l a b i l i t y f o r R ( - ) - m e x i l e t i n e was greater than t h a t of S ( + ) - m e x i l e t i n e . Since m e x i l e t i n e i s a low hepatic e x t r a c t i o n drug with minimal f i r s t - p a s s metabolism (Haselbarth et al., 1981; P r e s c o t t et al., 1977), the higher C m a x values f o r R ( - ) -m e x i l e t i n e are not l i k e l y to be due to pre-systemic clearance of S(+)-m e x i l e t i n e . A Comparison of the area under the serum t o t a l concentration time curve (AUC0"*0) data between the two enantiomers revealed small but c o n s i s t e n t l y g r e a t e r values f o r R ( - ) - m e x i l e t i n e i n ten s u b j e c t s , compared to S ( + ) - m e x i l e t i n e . In the remaining two s u b j e c t s , the AUC 0 - " values f o r S(+)-mexiletine were greater than R ( - ) - m e x i l e t i n e . The mean serum t o t a l AUC0"™ value of 2.82 ± 0.90 /zg/ml/h f o r R ( - ) - m e x i l e t i n e f o r the twelve healthy subjects was found to be s i g n i f i c a n t l y greater (p<0.05) than the mean value of 2.57 ± 0.89 /zg/ml/h f o r S ( + ) - m e x i l e t i n e . This s i g n i f i c a n t l y greater mean serum t o t a l AUC0"00 f o r R ( - ) - m e x i l e t i n e was c o n s i s t e n t with the greater i n i t i a l mean serum t o t a l drug concentrations and the greater mean C m a x values observed f o r R ( - ) -m e x i l e t i n e . The o r a l absorption of m e x i l e t i n e was found to be v a r i a b l e among the twelve s u b j e c t s . For subject GT, drug absorption was completed before the f i r s t blood sampling time (1.0 h) and assessment of the o r a l absorption r a t e was not p o s s i b l e . In other s u b j e c t s , blood sampling at 0.50 and 0.75 hour were implemented i n the blood sample c o l l e c t i o n protocol to a l l o w an assessment of the r i s e i n m e x i l e t i n e serum concentrations during the absorption phase. The mean o r a l absorption r a t e constants ( k a ) of 2.5317 ± 2.2513 h " 1 f o r R ( - ) - m e x i l e t i n e and 2.3001 ± 2.032 h " 1 f o r S(+)-mexiletine were observed. These mean f i r s t -order absorption r a t e c o n s t a n t s , and the c a l c u l a t e d mean absorption h a l f - l i v e s of 0.56 ± 0.39 h and 0.65 ± 0.46 h, f o r R ( - ) - and S(+)-m e x i l e t i n e , r e s p e c t i v e l y , were found not to be s i g n i f i c a n t l y d i f f e r e n t . For the seven s u b j e c t s , the mean r a p i d d i s p o s i t i o n r a t e constants (a) of 0.2459 + 0.1703 h " 1 and 0.4036 ± 0.4729 h ' 1 , f o r R ( - ) - and S(+)-m e x i l e t i n e , r e s p e c t i v e l y , were a l s o found not to be s i g n i f i c a n t l y d i f f e r e n t . The f i r s t - o r d e r r a p i d d i s p o s i t i o n r a t e constants were used to c a l c u l a t e the corresponding h a l f - l i v e s with mean values of 4.86 ± 3.36 h and 4.31 ± 2.96 h f o r R ( - ) - and S ( + ) - m e x i l e t i n e , r e s p e c t i v e l y . The long h a l f - l i v e s f o r m e x i l e t i n e enantiomers were found not to be s i g n i f i c a n t l y d i f f e r e n t and the values observed were i n agreement w i t h • those reported by Igwemezie et a7. (1989). However, as shown in Figure 22, the time-course of the serum t o t a l and serum f r e e drug 120 concentrations from subject AO indicated a second r i s e in serum drug concentrations during the post-absorption phase. S i m i l a r r i s e in serum concentrations was observed i n other s u b j e c t s . The reasons f o r t h i s increase in the serum t o t a l and serum f r e e drug concentrations remain u n c e r t a i n . However, f u t u r e studies on the e f f e c t of enterohepatic r e c y c l i n g of m e x i l e t i n e conjugates may allow a d e f i n i t i v e assessment of the underlying mechanisms l e a d i n g to the observed increase in m e x i l e t i n e serum concentrations during the p o s t - a b s o r p t i v e phase of drug d i s p o s i t i o n . The terminal h a l f - l i v e s of m e x i l e t i n e enantiomers were found to be v a r i a b l e among the twelve s u b j e c t s , ranging from 5.0 to 11.3 hours f o r R ( - ) - m e x i l e t i n e , and from 3.6 to 12.6 hours f o r S ( + ) - m e x i l e t i n e . Although the terminal h a l f - l i v e s f o r m e x i l e t i n e enantiomers were found to be v a r i a b l e , the mean values of 8.08 ± 2.23 h and 8.36 ± 2.64 h, f o r R ( - ) - and S ( + ) - m e x i l e t i n e , r e s p e c t i v e l y , were not s i g n i f i c a n t l y d i f f e r e n t . The mean terminal d i s p o s i t i o n rate constants were determined to be 0.0938 ± 0.0282 h " 1 and 0.0936 ± 0.0370 h " 1 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 . These s i m i l a r mean d i s p o s i t i o n rate constants f o r m e x i l e t i n e enantiomers observed i n t h i s study were c o n s i s t e n t with the r e s u l t s reported from an e a r l i e r study in s i x healthy subjects (Grech-Belanger et a 7 . , 1986). However, in another study of f i v e healthy volunteers (Igwemezie et a l . , 1989), the mean terminal d i s p o s i t i o n r a t e constants of 0.0825 ± 0.0255 h ' 1 and 0.0697 ± 0.0236 h " 1 , f o r R(-)-and S ( + ) - m e x i l e t i n e , r e s p e c t i v e l y , were reported to be s i g n i f i c a n t l y d i f f e r e n t (p<0.01). The d i f f e r e n c e in the terminal d i s p o s i t i o n data f o r m e x i l e t i n e enantiomers reported from the l a t t e r study may be due to the smaller study p o p u l a t i o n . 1 0 0 . 0 o c CD O c o o CD JD • • X CD 10 .0 1.0 0.1 • sR[+_] Tota l S e r u m A R(t) Free S e r u m 8 8 0 5 10 15 2 0 25 3 0 3 5 40 4 5 5 0 T i m e (h) Figure 22. Semilogarithmic plots of the serum t o t a l and serum free R ( - ) - and S(+)-mexiletine concentrations over 48 hours from subject AO f o l l o w i n g oral a d m i n i s t r a t i o n of 200 mg racemic mexiletine h y d r o c h l o r i d e . The mean apparent o r a l clearance values (CL/F) f o r the twelve subjects were 7.9 + 2.9 ml/min/kg f o r R ( - ) - m e x i l e t i n e and 8 . 8 ± 3.1 ml/min/kg f o r S ( + ) - m e x i l e t i n e . The two mean values were not s i g n i f i c a n t l y d i f f e r e n t and were c o n s i s t e n t with r e s u l t s reported in the l i t e r a t u r e (Igwemezie et al., 1989; Grech Belanger et al., 1986). Since the o r a l b i o a v a i l a b i l i t y of m e x i l e t i n e was not determined in the s u b j e c t s , clearance values were expressed as a r a t i o of b i o a v a i l a b i l i t y , F. Since the r e l a t i o n s h i p between drug clearance and drug e l i m i n a t i o n i s described by CL = Vn, x Kg, the s i m i l a r mean o r a l clearance values and mean e l i m i n a t i o n constants between m e x i l e t i n e enantiomers suggest that the volume of d i s t r i b u t i o n f o r the enantiomers would a l s o be s i m i l a r . The volume of d i s t r i b u t i o n f o r R ( - ) - m e x i l e t i n e was found to range from 2.2 to 8 . 4 1/kg, w h i l e a range of 3 . 0 . 1 to 9.7 1/kg was observed f o r S ( + ) - m e x i l e t i n e . The mean volumes of d i s t r i b u t i o n of 5.3 ± 1 . 7 1/kg and 6 . 0 ± 2.2 1/kg, f o r R ( - ) - and S ( + ) - m e x i l e t i n e , r e s p e c t i v e l y , were found not to be s i g n i f i c a n t l y d i f f e r e n t . These l a r g e d i s t r i b u t i o n volumes i n d i c a t e d t h a t m e x i l e t i n e enantiomers were e x t e n s i v e l y and e q u a l l y bound to t i s s u e s . In a 70 kg human subject with a systemic c i r c u l a t i o n volume of 5 l i t e r s , a mean volume of d i s t r i b u t i o n of « 5.0 1/kg f o r each m e x i l e t i n e enantiomer suggests that l e s s than 1.0% of the t o t a l amount of drug i n the body i s a v a i l a b l e in the systemic c i r c u l a t i o n . This l a r g e d i s t r i b u t i o n volume f o r m e x i l e t i n e f u r t h e r suggests that small changes in the t i s s u e binding of m e x i l e t i n e may g r e a t l y a f f e c t e i t h e r the clearance and/or the e l i m i n a t i o n of the drug. Since the drug i s >90% e l i m i n a t e d by non-renal mechanisms, the e f f e c t of changing drug d i s t r i b u t i o n on the e l i m i n a t i o n of m e x i l e t i n e may be p a r t i c u l a r l y important in 1 i v e r - i m p a i r e d p a t i e n t s . 3 . 3 . 2 The Pharmacokinetics of Serum Free M e x i l e t i n e The pharmacokinetic parameters f o r m e x i l e t i n e enantiomers, c a l c u l a t e d from serum u l t r a f i l t r a t e drug c o n c e n t r a t i o n s , are presented in Table 15. The pharmacokinetic behavior of serum f r e e m e x i l e t i n e was found to c l o s e l y f o l l o w that of serum t o t a l drug. As shown p r e v i o u s l y in Figure 16, the mean f r e e f r a c t i o n s f o r R ( - ) - m e x i l e t i n e were found to range from 0.47 to 0 . 6 7 , w h i l e those f o r S(+)-mexiletine were found to range from 0.47 to 0,64 among the twelve s u b j e c t s . The o v e r a l l mean serum f r e e f r a c t i o n s over the 48 hours were found to be 0.57 ± 0.07 f o r R ( - ) - m e x i l e t i n e and 0.56 ± 0.06 f o r S(+)-mexiletine (mean ± S . E . ) . These s i m i l a r values i n d i c a t e d that the in vivo serum p r o t e i n binding of m e x i l e t i n e enantiomers was not s t e r e o s e l e c t i v e . In a d d i t i o n , the low serum p r o t e i n binding of m e x i l e t i n e enantiomers, together with t h e i r l a r g e volumes of d i s t r i b u t i o n , would not be expected to lead to c l i n i c a l l y s i g n i f i c a n t drug-drug i n t e r a c t i o n s . 3 . 3 . 3 Pharmacokinetics of M e x i l e t i n e i n S a l i v a The mean s a l i v a r y m e x i l e t i n e concentration versus time data from the twelve subjects f o l l o w i n g o r a l a d m i n i s t r a t i o n of 200 mg of m e x i l e t i n e hydrochloride are shown i n Figure 23 and Table 16. The s a l i v a r y m e x i l e t i n e concentrations among the subjects were found to be v a r i a b l e . Since s a l i v a samples were c o l l e c t e d without s t i m u l a t i o n , the v a r i a b i l i t y observed in the s a l i v a r y m e x i l e t i n e concentrations may be due to spontaneous changes i n s a l i v a pH r e s u l t i n g from v a r i a t i o n s i n s a l i v a f l o w . I t has been reported that s a l i v a pH i s l a r g e l y governed by s a l i v a flow r a t e , and that b a s i c drugs have been found to concentrate i n normally a c i d i c s a l i v a by i r r e v e r s i b l e d i f f u s i o n or e x c r e t i o n . Despite Time (h) Figure 23. Semilogarithmic p l o t s of the mean ± S.D. R ( - ) - and S(+)-mexiletine concentrations in s a l i v a over 48 hours from twelve healthy subjects f o l l o w i n g a d m i n i s t r a t i o n of 200 mg racemic mexiletine h y d r o c h l o r i d e . r o -p. 125 E > , OJ sz o ra r -CD r— CD CO x ^ E E > o ' r— CvJ "T" OJ + 3 <+-•—'+-> o oo - o o 03 J -i o3 d C C0-r-=3 E 5- - O 03 • _ OJ ro cD i— "O ' " CU 03 T -S— i— S— 4- O O $-o ^ - 6 03 - O <4_ <-> E • ' - 3 +-> S_ OJ OJ •r— o o <J $-«J U— E s- -a ro cl) " § . > S-OJ CO ^ - o LO C7>_C c u r- o O "O — >> — -C o +- <D U CD OJ i— •o-<-_Q X =3 OJ OJ o3 r o< s in to w i/i c 00 Of i O OJ •dollANKJMOOO-OOJ p.»-t>(M'OMinrJ'OcoiMS O^ IOlAO-f IT-0(Oin»-rvj »- »- r>i — f - ' -OOOJinNN'-OKIln iAli-tD3O>K1C0O>(Mr-rUOta i OOOOfNJCJ^OOO — • oooor\<oc?ooo»— - o o -o s > O O K» «— C oAN-OtricoK.cOOr' (O-fiOOOOM'-WII lOO>-ON>t>-0 OCOOOOi/">OOOOON-Table 16. The R ( - ) - and S(+)-mexiletine concentrations in s a l i v a over 48 hours from twelve healthy subjects f o l l o w i n g oral administration of 200 mg racemic m e x i l e t i n e hydrochloride. •(h)-• Saliva Mexiletine Concentration (ng/ml) R ( - > FKOn DRUG AOMIN AO CK CT GR GT JB JG JH RL ST TH TK MEAN S . D . •IU8IKI t£3StI333 E8S3833S3 SESSSEBES SS3333333 IC3SESSS3 S33333S38 333333833 338833883 SSS3EB CE3S83E83 EB33IE33) 3S338E3S3 0 . 0 1 . 0 6 7 . 7 1 1 1 . 0 5 9 3 . 2 7 2 0 . 5 9 8 2 . 7 1 2 9 3 . 5 3 , 0 9 7 . 5 : 3 0 4 . 4 4 1 8 . 0 5 3 6 . 7 4 8 3 . 8 2 4 7 . 7 7 3 8 . 1 6 7 7 . 6 1 . 5 3 2 . 1 4 8 9 . 5 2 , 1 1 2 . 9 1 , 2 8 3 . 9 1 , 1 4 6 . 7 1 7 4 1 . 6 1 , 7 9 3 . 3 4 3 6 . 5 8 0 9 . 2 8 0 7 . 4 4 7 4 . 7 1 0 1 1 . 6 6 7 4 . 6 2 . 0 1 4 8 . 9 6 0 3 . 5 5 , 9 1 0 . 3 1 , 1 6 3 . 9 1 , 3 3 8 . 9 1 3 9 3 . 9 1 , 4 4 3 . 1 6 0 0 . 4 6 8 1 . 6 1 , 0 3 4 . 3 1 0 9 6 . 7 7 3 0 . 8 1 3 4 5 . 5 1 2 2 9 . 8 2 . 5 2 4 4 . 5 5 0 3 . 5 4 4 8 . 3 9 3 7 . 9 9 1 1 . 0 1 6 4 2 . 0 1 , 1 0 3 . 1 4 2 5 . 2 5 3 6 . 9 1 , 1 2 1 . 7 7 6 2 . 8 7 8 5 . 2 4 7 9 . 3 3 . 0 2 4 0 . 3 5 1 3 . 1 1 , 9 9 2 . 2 9 3 5 . 3 8 9 4 . 9 1 2 1 6 . 6 9 3 5 . 4 4 6 5 . 2 5 5 5 . 7 9 8 3 . 4 5 5 6 . 2 6 6 6 . 6 8 2 9 . 6 5 2 1 . 6 3 . 5 2 7 1 . 1 5 8 1 . 6 7 9 6 . 5 1 , 0 3 7 . 9 4 4 7 . 5 1 0 4 2 . 6 8 4 8 . 2 3 5 1 . 9 4 2 9 . 0 7 3 4 . 9 3 7 7 . 1 7 2 0 . 6 6 3 6 . 6 3 6 7 . 3 4 . 0 2 6 0 . 8 4 6 0 . 0 6 5 2 . 3 7 1 4 . 0 7 8 9 . 0 1 0 7 5 . 1 7 3 6 . 0 3 9 2 . 4 5 3 8 . 5 3 9 1 . 5 8 0 9 . 0 6 1 9 . 9 3 4 8 . 6 4 . 5 3 1 3 . 7 2 9 0 . 2 4 4 9 . 7 1 , 0 5 9 . 9 2 4 1 . 1 9 6 1 . 5 7 7 1 . 0 3 4 1 . 4 6 7 2 . 0 9 1 9 . 8 5 1 9 . 2 5 8 8 . 6 5 9 4 . 0 3 5 5 . 4 6 . 0 1 3 9 . 8 2 5 4 . 9 4 9 4 . 1 2 7 5 . 9 2 2 3 . 8 8 3 7 . 9 5 5 2 . 7 4 2 8 . 2 4 5 3 . 2 3 6 0 . 2 1 9 7 . 0 6 0 9 . 8 4 0 2 . 3 2 4 4 . 8 8 . 0 1 4 3 . 6 1 8 5 . 6 2 7 2 . 8 6 1 5 . 3 5 6 8 . 0 5 5 7 . 4 2 6 0 . 5 4 0 9 . 2 4 3 6 . 0 2 1 4 . 4 1 5 9 . 0 4 0 4 . 9 3 5 2 . 2 2 1 1 . 1 lO' .O 8 7 . 7 2 1 3 . 1 9 1 . 9 1 3 1 . 4 1 3 1 . 3 3 1 9 . 2 2 9 7 . 0 3 1 8 . 0 4 0 9 . 4 4 6 8 . 9 1 1 2 . 3 3 9 4 . 9 2 4 7 . 9 1 5 5 . 8 1 2 . 0 1 5 2 . 2 1 7 9 . 9 1 7 8 . 2 1 7 0 . 1 2 7 0 . 9 4 4 1 . 8 2 5 5 . 2 3 8 5 . 0 3 4 5 . 2 1 4 4 . 9 1 1 6 . 1 2 6 9 . 9 2 4 2 . 5 1 4 0 . 9 1 4 . 0 1 1 7 . 8 1 5 4 . 0 9 1 . 4 1 3 9 . 8 1 4 7 . 4 4 6 2 . 3 2 0 1 . 4 2 4 9 . 4 3 0 6 . 9 2 4 1 . 7 7 7 . 6 1 7 8 . 6 1 9 7 . 4 1 2 3 . 4 2 4 . 0 3 0 . 8 9 4 . 8 1 5 3 . 3 9 3 . 3 8 3 . 0 2 0 6 . 4 1 6 5 . 3 1 1 9 . 7 1 9 5 . 7 5 8 . 2 3 3 . 7 1 6 5 . 3 1 1 6 . 6 7 1 . 7 3 0 . 0 2 1 . 8 7 0 . 3 3 0 . 2 3 3 . 3 1 4 4 . 9 8 9 . 6 6 3 . 0 1 3 3 . 9 2 6 . 7 1 9 . 9 1 8 1 . 9 7 4 . 1 5 3 . 2 3 6 . 0 1 5 . 2 7 3 . 4 2 4 . 1 1 5 . 2 3 2 . 3 1 1 6 . 8 6 7 . 0 7 3 . 5 1 0 1 . 1 2 1 . 0 1 1 6 . 3 5 9 . 6 4 0 . 2 4 8 . 0 . 6 . 4 5 4 . 6 1 5 . 2 . 2 6 . 0 6 5 . 6 5 1 . 2 4 6 . 7 5 6 . 4 5 2 . 3 4 1 . 6 2 4 . 4 (h) CBl*>Vf no II" Saliva Mexiletine Concentration (ng/ml) S ( + ) rKUr-1 UKUu AOMIN AO CK CT GR GT J B JG JH RL ST TH TK MEAN S . D . • SCSH8SCSS8 83*3X3333 • = = = 3=3=3=33 "===="= " " = " " ========= ========= ========= ========= = ========= 383333838 338SES333 0 . 0 1 . 0 5 9 . 4 1 0 7 . 7 5 7 5 . 2 6 9 7 . 4 9 3 3 . 2 1 2 7 7 . 2 3 , 1 7 9 . 0 2 9 0 . 4 3 9 5 . 2 4 8 9 . 5 4 4 6 . 3 2 2 3 . 4 7 2 2 . 8 6 8 8 . 3 1 . 5 3 1 . 8 4 6 4 . 9 2 , 2 3 1 . 3 1 , 2 9 1 . 7 1 , 1 4 3 . 6 1 7 8 6 . 7 1 , 9 1 5 . 8 4 2 8 . 7 8 4 3 . 7 7 9 8 . 4 4 6 2 . 3 1 0 3 6 . 3 7 0 3 . 1 2 . 0 1 5 0 . 4 5 9 7 . 5 6 , 2 0 1 . 6 1 , 0 9 9 . 1 1 , 3 5 3 . 2 1 4 7 3 . 3 1 , 5 9 8 . 6 5 9 7 . 2 7 3 3 . 1 1 , 0 8 2 . 3 1 1 6 9 . 5 7 5 2 . 5 1 4 0 0 . 7 1 2 9 0 . 8 2 . 5 2 5 6 . 1 5 9 8 . 3 4 9 5 . 5 1 , 0 1 9 . 7 9 5 2 . 4 1 7 5 8 . 3 1 , 2 4 7 . 0 4 3 5 . 4 - 5 7 6 . 6 1 , 2 1 9 . 4 8 0 2 . 3 8 5 1 . 0 5 1 9 . 2 3 . 0 2 5 7 . 8 5 3 2 . 1 2 , 2 1 0 . 5 1 , 0 3 0 . 8 9 5 3 . 9 1 3 2 7 . 3 1 , 0 9 8 . 2 4 7 1 . 1 6 6 7 . 2 1 , 0 5 3 . 0 5 8 0 . 9 6 4 0 . 5 9 0 3 . 6 5 7 5 . 4 3 . 5 2 8 3 . 3 6 0 0 . 9 8 5 9 . 1 1 , 1 5 2 . 7 4 8 8 . 0 1 1 5 1 . 8 9 9 0 . 2 3 7 7 . 9 4 7 9 . 3 7 9 5 . 3 3 9 9 . 3 7 2 7 . 4 6 9 2 . 1 4 0 3 . 6 4 . 0 2 9 7 . 6 4 7 9 . 2 7 1 2 . 2 8 0 9 . 9 8 3 8 . 1 1 1 9 9 . 1 8 9 2 . 6 4 3 9 . 5 6 1 0 . 1 4 1 7 . 4 8 3 7 . 2 6 8 4 . 8 3 8 6 . 1 4 . 5 3 6 2 . 6 2 9 3 . 3 5 4 2 . 9 1 , 2 1 3 . 0 2 6 7 . 4 1 0 8 4 . 5 9 3 0 . 4 . 3 0 1 . 7 7 5 0 . 2 1 , 0 2 1 . 6 5 6 0 . 9 6 2 3 . 7 6 6 2 . 7 4 0 3 . 7 6 . 0 1 7 1 . 2 2 7 1 . 0 5 5 1 . 3 3 2 9 . 3 4 7 3 . 7 9 6 4 . 9 7 2 8 . 6 4 9 5 . 9 5 2 7 . 6 3 9 8 . 9 2 1 9 . 7 6 3 5 . 9 4 8 0 . 7 2 8 6 . 7 8 . 0 1 6 9 . 2 1 9 9 . 0 3 0 2 . 7 7 2 8 . 5 5 8 2 . 9 6 4 5 . 2 3 2 5 . 4 4 7 4 . 8 5 0 5 . 0 2 2 9 . 9 1 7 8 . 7 4 4 0 . 2 3 9 8 . 5 2 3 9 . 3 1 0 . 0 1 1 5 . 5 2 2 5 . 3 9 5 . 0 1 6 1 . 2 1 3 2 . 0 3 7 9 . 2 5 9 7 . 2 3 7 2 . 7 4 7 4 . 6 5 1 6 . 7 1 3 4 . 3 4 3 8 . 9 3 0 3 . 6 1 9 5 . 8 1 2 . 0 1 9 7 . 8 1 8 7 . 7 2 0 5 . 2 2 0 6 . 9 2 9 3 . 0 . 5 3 7 . 9 3 3 4 . 5 4 7 0 . 2 4 1 8 . 5 1 7 6 . 1 1 4 9 . 1 3 0 2 . 6 2 9 0 . 0 1 6 9 . 5 1 4 . 0 1 5 0 . 3 1 6 5 . 9 2 0 0 . 5 1 7 5 . 1 1 7 1 . 4 5 5 7 . 3 2 6 8 . 0 3 1 7 . 3 3 7 8 . 1 2 8 5 . 6 9 7 . 3 2 0 7 . 7 2 4 7 . 9 1 S 1 . 2 2 4 . 0 4 5 . 0 1 0 2 . 8 1 7 1 . 1 1 2 6 . 1 9 4 . 1 2 5 7 . 6 2 6 1 . 5 1 6 3 . 8 2 5 5 . 0 7 7 . 8 4 2 . 8 1 9 5 . 1 1 4 9 . 4 9 3 . 1 3 0 . 0 2 6 . 8 7 8 . 6 3 1 . 5 4 3 . 4 1 8 8 . 8 1 2 6 . 3 7 2 . 9 1 7 4 . 1 3 4 . 6 2 0 . 5 2 1 5 . 2 9 2 . 1 6 6 . 8 3 6 . 0 2 5 . 2 8 1 . 3 2 4 . 3 1 4 . 9 3 5 . 5 1 4 8 . 6 8 8 . 8 9 4 . 6 1 4 1 . 4 2 0 . 6 1 4 4 . 4 7 4 . 5 5 1 . 7 4 8 . 0 6 . 4 . 6 2 . 4 1 5 . 6 2 6 . 1 7 7 . 4 7 1 . 8 6 7 . 6 6 7 . 4 5 4 . 0 4 9 . 9 2 9 . 9 a large v a r i a t i o n in s a l i v a r y m e x i l e t i n e concentrations among the twelve s u b j e c t s , the time-course of the mean s a l i v a r y m e x i l e t i n e concentrations were found to f o l l o w the d i s p o s i t i o n k i n e t i c s of a 2-compartment open model. In eight s u b j e c t s , the k i n e t i c s of s a l i v a r y m e x i l e t i n e concentrations were best described by a 2-compartment open model. In the remaining three s u b j e c t s , the k i n e t i c s of m e x i l e t i n e in s a l i v a were described by a one-compartment open model. In subject JG, due to a l a r g e f l u c t u a t i o n i n s a l i v a r y m e x i l e t i n e c o n c e n t r a t i o n s , the data could not be described by e i t h e r one- or two-compartmental k i n e t i c s . The pharmacokinetic parameters of m e x i l e t i n e enantiomers in s a l i v a from the twelve subjects are presented in Table 17. The mean terminal d i s p o s i t i o n r a t e constant of 0.2426 ± 0.3188 h " 1 f o r R ( - ) - m e x i l e t i n e was found to be s i g n i f i c a n t l y greater than the mean value of 0.1484 ± 0.1083 h " 1 f o r S ( + ) - m e x i l e t i n e . The d i s p o s i t i o n r a t e constants were used to c a l c u l a t e the corresponding h a l f - l i v e s with mean values of 5.84 ± 3.55 h and 7.01 ± 4.06 h f o r R ( - ) - and S ( + ) - m e x i l e t i n e , r e s p e c t i v e l y . The t o t a l amount of m e x i l e t i n e excreted i n t o s a l i v a over 48 hours, as measured by the mean area under the s a l i v a drug concentration-time curve (AUC0 - 0 0) f o r the twelve s u b j e c t s , was found to be 7.6 ± 3 . 9 /jg/ml/h and 9.7 ± 4 . 0 /zg/ml/h f o r R ( - ) - and S ( + ) - m e x i l e t i n e , r e s p e c t i v e l y . Although a l a r g e d e v i a t i o n i n the s a l i v a AUC0 - 0 0 was observed among the twelve s u b j e c t s , the mean s a l i v a AUC0 - 0 0 f o r S(+)-mexiletine was s i g n i f i c a n t l y greater (p<0.01) than that f o r R ( - ) - m e x i l e t i n e . Since the mean serum f r e e f r a c t i o n s f o r R ( - ) - and S(+)-mexiletine were found to be s i m i l a r , and since only serum f r e e drug was a v a i l a b l e f o r d i f f u s i o n i n t o s a l i v a f l u i d s , the s i g n i f i c a n t l y d i f f e r e n t s a l i v a AUC values between the two m e x i l e t i n e enantiomers suggested that a c t i v e e x c r e t i o n of S(+)-Table 17. The pharmacokinetic data for R(-)- and S(+)-mexiletine derived from saliva drug data from twelve healthy subjects following oral administration of 200 mg of racemic mexiletine hydrochloride. A U O n g . h / m l ) B i n " 1 ) t l / 2 B (h) (h"l) t l / 2 M h ) a (h-1) t l / 2 a (h) SUBJECTS R S R S R S R S R S R S R S GT 8 1 5 6 . 9 9 1 8 1 . 9 0.215 0.194 3 . 2 3 . 5 2 . 0 2 6 2.014 0 . 3 0 . 3 0.182 0 . 2 1 7 3 . 8 3 . 1 JG 12208.5 1 5 6 7 3 . 6 - - - - - ' - - - - - - -CK 7 6 5 3 . 6 8 2 7 6 . 5 0.152 0 . 0 9 5 4 . 6 7 . 2 1 . 2 5 3 1 .460 0 . 5 0 . 4 0 . 2 2 3 0 . 1 8 5 3 . 1 3 . 7 RL 12085.4 14980.1 0 . 0 6 2 0 . 0 4 7 11.0 14.5 1 . 3 6 8 1 .138 0 . 5 0 . 6 0 . 3 9 6 - 1 . 7 -GR 8 7 5 3 . 7 1 0 5 8 . 6 0 . 1 9 8 0.119 3 . 5 5 . 8 3 . 4 7 4 2 . 3 3 2 0 . 2 0 . 3 0 . 1 2 5 0 . 1 4 6 5 . 5 4 . 7 JH 9 3 4 0 . 7 11605.7 0 . 0 5 5 0 . 0 4 9 12.4 1 3 . 9 2.591 1 .823 0 . 2 0 . 3 - - - -AO 3 2 7 8 . 7 4 0 5 0 . 7 0 . 0 8 5 0 . 0 7 9 8.1 8 . 6 1 .922 0 . 9 4 6 0 . 3 0 . 7 - - - -CT 10791.6 1 2 3 0 5 . 9 - 0 . 4 3 8 - 1 .5 - 2.041 - 0 . 3 - 0 . 2 5 6 - 2 . 6 ST 8 4 0 3 . 8 9 3 9 4 . 4 1.181 0 . 1 8 7 0 . 5 3 . 6 1 .564 1 . 8 5 0 0 . 4 0 . 3 0 . 3 3 2 0 . 3 9 2 2 . 0 1 .7 TH 4 7 0 3 . 2 5214.1 0 . 2 4 5 0.231 2 . 8 2 . 9 2 . 3 5 9 2.191 0 . 2 0 . 3 0.197 0 . 2 2 6 3 . 5 3 . 0 TK 11716.6 1 2 9 6 2 . 6 0.1.10 0 . 0 8 0 6 . 2 8 . 6 0 . 6 4 8 0 . 9 9 3 1 . 0 0 . 7 0.124 0 . 1 9 9 5 . 5 3 . 4 JB 2 0 4 7 . 9 2 2 3 8 . 8 0.118 0 . 1 0 8 . 5 . 2 6 . 3 5 . 3 7 9 6 . 8 0 0 0.1 0.1 0.155 0.176 4 . 4 3 . 9 . MEAN 8 2 6 1 . 7 8 9 1 1 . 9 0 . 2 4 2 0 . 1 4 8 5 . 8 7 . 0 2 . 2 5 9 2.144 0 . 4 0 . 4 0.2 17 0 . 2 2 5 3 . 7 3 . 3 S . D . 3246.1 4672.1 0.304 0.111 3 . 9 4 . 3 1 .440 1.591 0 . 2 _ 0 . 2 0.12 7 0 . 1 2 0 2 . 0 1.7 129 m e x i l e t i n e into s a l i v a could have o c c u r r e d . As shown i n Table 18, the mean s a l i v a r y m e x i l e t i n e enantiomer concentrations over the 48 hours were found to exceed t h e i r r e s p e c t i v e mean serum t o t a l and serum f r e e drug c o n c e n t r a t i o n s . As shown in Figure 24, the s a l i v a / serum f r e e drug concentration r a t i o s from the twelve subjects were v a r i a b l e but were always greater than u n i t y . The o v e r a l l mean s a l i v a / serum f r e e m e x i l e t i n e AUC r a t i o s over the 48 hours were found to be 6.10 ± 2.82 and 7.49 ± 3.48 f o r R ( - ) - and S ( + ) - m e x i l e t i n e , r e s p e c t i v e l y . The greater s a l i v a / serum f r e e AUC r a t i o s f o r S(+)-m e x i l e t i n e r e l a t i v e to R ( - ) - m e x i l e t i n e suggested that a greater amount of serum f r e e S(+)-mexiletine was excreted into s a l i v a r e l a t i v e to the R(-)-enantiomer. As shown i n Figure 25 and Table 19, one hour f o l l o w i n g drug a d m i n i s t r a t i o n , the mean s a l i v a R(-)/S(+) r a t i o s from the twelve subjects were always below u n i t y . The o v e r a l l mean s a l i v a R(-)/S(+) r a t i o of 0.89 ± 0.02 (mean ± S . E . ) , over the 48 hours, was found to be s i g n i f i c a n t l y d i f f e r e n t (p < 0.01) than that observed from serum t o t a l and serum f r e e drug of 1.05 ± 0.02 and 1.09 ± 0 . 0 2 , r e s p e c t i v e l y . The s i g n i f i c a n t l y s m a l l e r mean s a l i v a R(-)/S(+) m e x i l e t i n e r a t i o with respect to t h a t observed from serum t o t a l and serum f r e e drug data was c o n s i s t e n t with the s i g n i f i c a n t l y smaller mean s a l i v a AUC observed f o r R ( - ) - m e x i l e t i n e i n respect to i t s antipode. The mean s a l i v a R(-)/S(+) data f u r t h e r suggested that a g r e a t e r amount of S(+)-mexiletine was excreted i n t o s a l i v a . The use of s a l i v a drug sampling as a non-invasive and convenient method f o r the determination of serum f r e e drug concentrations has been reviewed (Mucklow et a 7 . , 1977; Mucklow, 1982). For a n t i p y r i n e (pKa o c$9 o o o o in o o 0 5 10 15 20 25 30 35 40 45 Time (h) Figure 24. Plots of the mean ± S.D. r e s p e c t i v e R ( - ) - and S(+)-mexiletine s a l i v a / serum f r e e concentration r a t i o s over 48 hours from twelve healthy s u b j e c t s . 2 0 5 10 15 20 25 30 35 40 45 Time (h) Figure 25. P l o t s of the mean ± S.D. R/S-mexiletine enantiomer concentration r a t i o s in s a l i v a over 48 hours in twelve healthy s u b j e c t s . Table 18. The respective R ( - ) - and S(+)-mexiletine s a l i v a / serum f r e e m e x i l e t i n e concentration r a t i o s f o r over 48 hours from twelve healthy s u b j e c t s . (h) Saliva / Free Serum Mexiletine Concentration Ratio R(-) EDrttf hDiir TKUn UKUu ADMIN AO CK CT GR GT J B JG JH RL ST TH TK MEAN S . O . U l l l l l l t BSSBBIBIS 3IBB3S3BC SSBIBSXES 3383t:;3S c s s s s s s s s SISBSBSSB "="-== B338CC383 33333333E S333S3E31 i sesEsss t SEtSSStSESX i s s s s i t i i 0 , 0 1 . 0 1 . 4 7 1 * 6 1 6 . 5 8 1 5 . 0 8 4 . 8 8 2 1 . 2 2 3 2 . 3 0 4 . 2 6 6 . 7 1 1 0 . 2 3 3 . 9 9 1 0 . 6 5 9 . 9 1 8 . 0 1 1 . 5 0 . 5 1 3 . 4 8 1 6 . 7 5 2 7 . 5 9 6 . 8 2 2 1 . 6 1 1 4 . 6 4 5 . 6 0 7 . 6 4 9 . 0 4 1 2 . 2 1 1 1 . 4 4 7 . 9 3 2 . 0 1 . 7 3 4 C 9 8 4 8 . 4 7 2 0 . 3 2 1 0 . 0 4 1 5 . 9 6 1 3 . 9 4 1 4 . 0 7 6 . 6 2 f 2 . 8 8 1 1 . 5 1 1 3 . 5 7 1 4 . 5 1 1 0 . 9 3 2 . 5 1 . 9 1 4 . 6 7 5 . 4 2 1 6 . 9 4 7 . 0 3 1 4 . 8 3 1 0 . 4 9 5 . 8 8 1 4 . 0 5 7 . 4 8 8 . 8 7 5 . 5 0 3 . 0 1 . 7 8 5 . 7 5 2 5 . 3 7 1 5 . 8 1 6 . 8 4 1 0 . 2 9 1 0 . 8 0 4 . 6 9 6 . 5 3 1 2 . 7 5 5 . 3 6 1 0 . 5 9 9 . 7 1 6 . 5 2 3 . 5 2 . 1 1 7 . 4 3 1 0 . 7 7 1 6 . 6 6 3 . 7 1 8 . 5 3 9 . 8 4 2 . 6 1 5 . 2 0 9 . 7 8 5 . 2 2 1 0 . 4 4 7 . 6 9 4 . 8 1 4 . 0 2 . 1 7 7 . 2 1 8 . 6 0 1 1 . 9 4 6 . 9 0 8 . 7 2 8 . 5 4 2 . 4 7 6 . 4 3 5 . 8 0 1 3 . 8 5 7 . 5 1 4 . 4 3 4 . 5 2 . 9 3 3 . 9 5 6 . 2 8 2 0 . 3 0 2 . 2 3 8 . 2 1 9 . 9 7 2 . 2 5 8 . 0 8 1 2 . 8 2 9 . 1 8 9 . 9 7 8 . 0 1 5 . 3 7 6 . 0 1 . 4 8 4 . 0 7 7 . 3 0 7 . 4 5 2 . 1 1 7 . 2 5 8 . 5 5 3 . 4 6 6 . 4 8 5 . 4 8 4 . 2 3 1 1 . 5 6 5 . 7 9 3 . 5 1 8 . 0 1 . 6 0 4 . 2 1 4 . 2 3 2 0 . 4 4 6 . 2 8 5 . 3 1 4 . 8 9 3 . 1 8 6 . 4 7 3 . 4 7 3 . 2 2 8 . 2 5 5 . 9 6 4 . 5 1 1 0 . 0 0 . 9 5 5 . 8 2 1 . 7 5 5 . 1 1 1 . 8 7 3 . 3 1 5 . 7 2 2 . 7 4 6 . 5 7 8 . 6 5 2 . 8 4 1 0 . 3 2 4 . 6 4 3 . 0 7 1 2 . 0 2 . 0 8 5 . 2 5 4 . 7 5 5 . 7 7 4 . 8 6 6 . 0 6 5 . 2 5 4 . 4 5 7 . 6 3 3 . 3 1 3 . 8 1 6 . 3 8 4 . 9 7 2 . 6 8 1 4 . 0 6 . 3 2 5 . 1 8 4 . 6 8 4 . 9 5 2 . 6 1 6 . 6 7 6 . 2 5 2 . 9 8 6 . 6 7 1 0 . 0 7 3 . 3 1 4 . 4 4 5 . 3 4 3 . 0 2 2 4 . 0 3 . 1 1 1 0 . 0 9 1 1 . 5 9 1 1 . 3 7 4 . 8 1 5 . 1 1 8 . 7 0 3 . 4 4 1 1 . 1 4 6 . 7 8 4 . 2 1 9 . 0 4 7 . 4 5 4 . 3 4 3 0 . 0 3 . 2 5 1 6 . 7 8 3 . 3 5 5 . 8 7 3 . 9 6 5 . 3 4 5 . 0 8 8 . 2 1 3 . 2 3 1 3 . 0 2 6 . 1 9 4 . 5 5 3 6 . 0 5 . 4 8 2 9 . 8 5 1 2 . 4 3 4 . 0 3 2 . 9 1 3 . 9 5 4 . 6 9 8 . 0 6 1 1 . 5 7 6 . 9 1 1 0 . 6 3 9 . 1 4 6 . 8 7 4 8 . 0 6 . 0 3 5 . 7 8 2 . 8 1 5 . 0 3 5 . 3 5 8 . 4 7 3 . 7 5 2 . 1 9 2 . 7 9 (hj Saliva / Free Serum Mexiletine Concentration Ratio S(+) com iMJir rKun UKUU ADMIN AO CK CT GR GT J B JG JH RL ST TH TK MEAN S . D . BBBXBBBBB • = = " « « « • « « • « « « « » « • « » • « « • « « » » • • • • « sxsaastBBBi CCBESE3EI • • « « » » • » BtBtXtSSBtSSS BtSESstasas 0 . 0 1 . 0 1 . 4 7 2 . 0 3 7 . 4 5 1 8 . 8 6 6 . 6 9 2 1 . 8 8 3 9 . 7 1 4 . 6 8 8 . 4 6 9 . 5 8 4 . 7 2 1 0 , 4 7 1 1 . 3 3 9 . 4 0 1 . 5 0 . 5 9 4 . 2 6 1 9 . 7 0 4 9 . 8 4 9 . 2 9 ' 2 3 . 5 3 1 7 . 0 1 6 . 3 6 9 . 4 1 1 0 . 8 5 ' 1 3 . 8 9 1 4 . 9 7 1 1 . 7 3 2 . 0 2 . 1 9 5 . 8 9 5 7 . 1 6 2 3 . 1 9 1 4 . 4 5 1 7 . 9 3 1 7 . 3 1 7 . 9 4 8 . 6 1 1 4 . 5 8 1 3 . 8 1 1 7 . 0 0 1 6 . 6 7 1 2 . 7 9 2 . 5 2 . 3 2 6 . 5 5 6 . 7 1 2 1 . 5 2 9 . 3 2 1 5 . 0 3 1 2 . 8 6 7 . 2 8 1 5 . 7 9 8 . 8 9 1 0 . 6 3 6 . 4 7 3 . 0 2 . 2 0 7 . 8 0 3 0 . 8 3 1 9 . 6 0 8 . 6 7 1 2 . 0 2 1 3 . 3 7 5 . 4 7 9 . 2 0 1 3 . 6 6 6 . 5 8 1 1 . 6 5 1 1 . 7 5 7 . 8 4 3 . 5 2 . 3 5 9 . 4 8 1 2 , 7 8 2 0 . 6 4 4 . 7 2 1 0 . 1 3 1 1 . 5 7 3 . 2 7 6 . 3 6 1 0 . 8 1 5 . 5 9 1 1 . 2 9 9 . 0 8 5 . 7 3 4 . 0 7 . 0 7 9 . 4 7 1 0 . 3 4 1 4 . 5 0 8 . 8 9 1 0 . 0 6 1 0 . 4 4 3 . 1 2 7 . 9 6 6 . 5 4 1 5 . 6 6 9 . 4 6 5 . 2 9 4 . 5 3 . 6 7 4 . 3 8 7 . 2 4 2 4 . 5 8 2 . 9 0 9 . 5 9 1 2 . 8 4 2 . 1 6 9 . 7 0 1 4 . 8 1 1 0 . 0 4 1 1 . 3 8 9 . 4 4 6 . 4 1 6 . 0 1 . 8 5 5 . 1 1 8 . 4 2 9 . 7 4 5 . 3 2 8 . 7 0 1 2 . 0 3 4 . 3 8 8 . 0 4 6 . 3 6 4 . 3 9 1 2 . 5 4 7 . 2 4 4 . 2 6 8 . 0 1 . 9 9 5 . 3 5 4 . 9 6 2 6 . 6 6 7 . 6 1 6 . 0 9 5 . 8 6 ' 3 . 9 7 7 . 9 3 4 . 0 2 3 . 5 7 9 . 5 7 7 . 3 0 5 . 7 6 1 0 . 0 1 . 3 4 7 . 3 9 1 . 8 2 6 . 3 8 2 . 2 2 3 . 8 1 1 1 . 2 8 3 . 4 8 8 . 6 5 9 . 2 6 3 . 2 3 1 1 . 5 6 5 . 8 7 3 . 8 9 1 2 . 0 2 . 8 6 6 . 4 3 5 . 9 0 6 . 9 4 6 . 3 1 7 . 3 7 6 . 7 5 , 5 . 9 0 9 . 4 8 4 . 0 6 4 . 7 5 7 . 3 5 6 . 1 7 3 . 3 0 1 4 . 0 7 . 2 0 8 . 2 3 1 0 . 0 5 6 . 9 3 3 . 2 7 7 . 9 5 7 . 5 5 ' 4 . 0 8 8 . 3 1 1 1 . 6 6 3 . 6 1 5 . 0 9 6 . 9 9 3 . 9 2 2 4 . 0 4 . 3 2 1 1 . 1 8 1 3 . 3 6 1 6 . 3 6 6 . 8 7 6 . 0 4 1 2 . 8 2 4 . 7 4 1 2 . 6 8 9 . 1 0 5 . 0 0 9 . 8 7 1 0 . 2 1 5 . 4 3 3 0 . 0 3 . 7 6 2 1 . 1 5 3 . 8 0 8 . 5 0 4 . 7 3 6 . 8 6 5 . 7 8 1 0 , 1 3 4 . 0 7 1 4 . 8 0 8 . 3 6 5 . 5 9 3 6 . 0 8 . 1 3 6 3 . 0 5 1 4 . 5 6 4 . 7 4 3 . 9 0 4 . 5 3 6 . 0 9 1 0 . 2 1 1 1 . 1 9 6 . 5 5 1 2 . 5 3 1 3 . 2 2 1 3 . 1 7 4 8 . 0 8 . 4 0 9 . 0 9 3 . 2 0 7 . 5 7 8 . 2 4 1 1 . 0 1 3 . 7 1 7 . 3 2 3 . 9 3 Table 19. The saliva R(-)/S(+) mexiletine concentration ratios over 48 hours in twelve healthy subjects. TIME Saliva Mexiletine R / S Ratio FROM DRUG ADMIN AO CK CT GR GT JB JG JH RL ST TH TK MEAN S . D . 0 . 0 1 . 0 1 . 1 4 1 . 0 3 1 . 0 3 1 . 0 3 1 . 0 5 1.01 0 . 9 7 1 . 0 5 1 . 0 6 1 .10 1 . 0 8 1.11 1 . 0 6 0 . 5 3 1 . 5 1.01 1 .05 0 . 9 5 0 . 9 9 1 . 0 0 0 . 9 7 0 . 9 4 1 . 0 2 0 . 9 6 1.01 1 . 0 3 0 . 9 9 0 . 5 0 2 . 0 0 . 9 9 1.01 0 . 9 5 1 . 0 6 0 . 9 9 0 . 9 5 0 . 9 0 1.01 0 . 9 3 0 . 9 6 0 . 9 4 0 . 9 7 0 . 9 7 0 . 4 9 2 . 5 0 . 9 5 0 . 8 4 0 . 9 0 0 . 9 2 0 . 9 6 0 . 9 3 0 . 8 8 0 . 9 8 0 . 9 3 0 . 9 2 0 . 9 5 0 . 9 2 0 . 4 6 3 . 0 0 . 9 3 0 . 9 6 0 . 9 0 0.91 0 . 9 4 0 . 9 2 0 . 8 5 0 . 9 9 0.81 0 . 9 3 0 . 9 6 1 .04 0 . 9 3 0 . 4 7 3 . 5 0 . 9 6 0 . 9 7 0 . 9 3 0 . 9 0 0 . 9 2 0 . 9 1 0 . 8 6 0 . 9 3 0 . 8 9 0 . 9 2 0 . 9 4 0 . 9 9 0 . 9 3 0 . 4 6 4 . 0 0 . 8 8 0 . 9 6 0 . 9 2 0 . 8 8 0 . 9 4 0 . 9 0 0 . 8 2 0 . 8 9 0 . 8 8 0 . 9 4 0 . 9 7 0.91 0 . 4 5 4 . 5 0 . 8 7 0 . 9 9 0 . 9 2 0 . 8 7 0 . 9 0 0 . 8 9 0 . 8 3 1 .13 0 . 9 0 0 . 9 0 0 . 9 3 0 . 9 4 0 . 9 2 0 . 4 6 6 . 0 0 . 8 2 0 . 9 4 0 . 9 0 0 . 8 4 0 . 4 7 0 . 8 7 0 . 7 6 0 . 8 6 0 . 8 6 0 . 9 0 0 . 9 0 0 . 9 6 0 . 8 4 0 . 4 3 8 . 0 0 . 8 5 0 . 9 3 0 . 9 0 0 . 8 4 0 . 9 7 0 . 8 6 0 . 8 0 0 . 8 6 0 . 8 6 0 . 9 3 0 . 8 9 0 . 9 2 0 . 8 9 0.44 1 0 . 0 0 . 7 6 0 . 9 5 0 . 9 7 0.81 1 . 0 0 0 . 8 4 0 . 5 0 0 . 8 5 0 . 8 6 0 . 9 1 0 . 8 4 0 . 9 0 0 . 8 5 0 . 4 3 1 2 . 0 0 . 7 7 0 . 9 6 0 . 8 7 0 . 8 2 0 . 9 2 0 . 8 2 0 . 7 6 0 . 8 2 0 . 8 2 0 . 8 2 0 . 7 8 0 . 8 9 0 . 8 4 0 . 4 2 1 4 . 0 0 . 7 8 0 . 9 3 0 . 4 6 0 . 8 0 0 . 8 6 0 . 8 3 0 . 7 5 0 . 7 9 0.81 0 . 8 5 0 . 8 0 0 . 8 6 0 . 7 9 0 . 4 0 2 4 . 0 0 . 6 9 0 . 9 2 0 . 9 0 0 . 7 4 0 . 8 8 0 . 8 0 0 . 6 3 0 . 7 3 0 . 7 7 0 . 7 5 0 . 7 9 0 . 8 5 0 . 7 9 0 . 4 0 3 0 . 0 0 . 8 1 0 . 8 9 0 . 9 6 0 . 7 7 0 . 7 7 0.71 0 . 8 6 0 . 7 7 0 . 7 7 0 . 8 5 0 . 8 2 0.41 3 6 . 0 0 . 6 1 0 . 9 0 0 . 9 9 1 . 0 2 0 . 9 1 0 . 7 9 0.75 0 . 7 8 0.71 1 . 0 2 0.81 0 . 8 4 0 . 4 3 4 8 . 0 1 . 0 0 0 . 8 7 0 . 9 8 0 . 9 9 0 . 8 5 0.71 0 . 6 9 0 . 8 4 0 . 9 7 0 . 8 8 0.44 « 7 . 5 ) , since the serum p r o t e i n binding was minimal, and the drug remained l a r g e l y unionized at p h y s i o l o g i c a l pH, s a l i v a drug concentrations were w e l l c o r r e l a t e d with plasma drug concentrations (Fraser et al., 1976). For t h e o p h y l l i n e and phenytoin, both with a low degree of i o n i z a t i o n at p h y s i o l o g i c a l plasma pH, s a l i v a drug concentrations were a l s o shown to d i r e c t l y c o r r e l a t e with plasma drug concentrations (Mucklow et al., 1977). For a c i d i c or b a s i c drugs, according to the Matin e q u a t i o n , the s a l i v a / serum f r e e drug concentration r a t i o s , S/Pf, are si f o r a c i d i c drugs at a c i d i c s a l i v a pH, while the S/Pf r a t i o s f o r b a s i c drugs are z.1 at a c i d i c s a l i v a pH. The t h e o r e t i c a l S/Pf r a t i o s derived using the Matin equation f o r drugs of d i f f e r e n t pKa values at d i f f e r e n t s a l i v a pH are presented in Table 20. In the present study, the S/Pf r a t i o s f o r m e x i l e t i n e enantiomers from the twelve subjects are shown i n Table 18. The observed mean S/Pf r a t i o s f o r m e x i l e t i n e enantiomers over the 48 hours were found to exceed u n i t y and were w i t h i n the range of t h e o r e t i c a l values derived from the Matin equation. To determine whether s a l i v a r y m e x i l e t i n e concentrations could be u t i l i z e d to p r e d i c t serum f r e e drug c o n c e n t r a t i o n s , serum f r e e drug data were c a l c u l a t e d based on s a l i v a r y m e x i l e t i n e concentrations and s a l i v a pH using the Matin equation (Matin et a!., 1974): S/P = [1 + 10 (pKa-pHs)j x fp / [1 + 10 (pKa-pHp)j x f s where S and P represent t o t a l drug concentrations i n s a l i v a and in plasma, r e s p e c t i v e l y . pHs and pHp are s a l i v a pH and serum pH (assumed to be 7 . 4 ) , r e s p e c t i v e l y , w h i l e fp and f s represent the f r e e f r a c t i o n s Table 20. The t h e o r e t i c a l s a l i v a / serum f r e e drug concentration r a t i o s f o r a c i d i c and basic drugs derived from s a l i v a drug concentrations and s a l i v a pH according to the Matin equation. * Acid Drugs Basic Drugs S a l i v a pH S a l i v a pH pKa 6 7 8 6 7 8 2 0.04 0.04 3.98 1.00 1.00 1.00 4 0.04 0.04 3.98 1.01 1.00 1.00 6 0.08 0.42 3.87 1.92 1.06 0.97 8 0.81 0.88 3.87 20.28 2.21 0.40 10 1.00 1.00 1.00 25.06 2.51 0.25 12 1.00 1.00 1.00 25.06 2.51 0.25 1 Matin et al., C l i n Pharmacol T h e r . , 16:1052 (1974) of m e x i l e t i n e in plasma and s a l i v a , r e s p e c t i v e l y . Studies have shown that a c i d i c or b a s i c drugs that are e x t e n s i v e l y p r o t e i n bound i n serum e x h i b i t l i t t l e binding to s a l i v a p r o t e i n s , and t h u s , f s was assumed to be 1.0 (Matin et a 7 . , 1974; Mucklow, 1982). Rearrangement of the Matin equation r e s u l t s in the f o l l o w i n g e x p r e s s i o n : [Serum f r e e ] c a i = S x 23.387 / [1 + 10 (8.75-pHs)] where [serum f r e e ] c a ] i s the c a l c u l a t e d serum f r e e drug concentration obtained from the Matin equation based on the corresponding s a l i v a r y drug c o n c e n t r a t i o n , S, and s a l i v a pH. As shown in Figure 26, the c a l c u l a t e d serum f r e e m e x i l e t i n e enantiomer concentrations from one subject were p l o t t e d in r e l a t i o n to the observed serum f r e e m e x i l e t i n e c o n c e n t r a t i o n s . A c o e f f i c i e n t of determination of 0.677 and 0.648 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 , was observed. A composite of the c a l c u l a t e d versus the observed serum f r e e m e x i l e t i n e enantiomer concentrations from 10 subjects are shown in Figure 27. An o v e r a l l c o e f f i c i e n t of determination of 0.551 and 0.643 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 , was observed. Since the c a l c u l a t e d serum f r e e m e x i l e t i n e enantiomer concentrations were poorly c o r r e l a t e d to the observed v a l u e s , the use of m e x i l e t i n e s a l i v a r y concentrations would provide an inaccurate estimation of serum f r e e drug c o n c e n t r a t i o n s . 3.3.4 Pharmacokinetics of Mexiletine in Red Blood C e l l s The mean red blood c e l l m e x i l e t i n e concentration versus time data from the seven subjects are shown in Figure 28 and Table 21. M e x i l e t i n e concentrations in the red blood c e l l s were v a r i a b l e among the s u b j e c t s , C D C o • ^— -+-1 o -+-< c CD o c: o o CD CD E CD CO 200 150-100-50-0 • s(+) O R( - ) o o o o o o o o o o 0 50 100 150 200 Calculated Serum Concentrations (ng/ml) Figure 26. Representative l i n e a r c o r r e l a t i o n of the r e s p e c t i v e c a l c u l a t e d versus observed serum free R ( - ) - and S(+)-mexiletine concentrations from subject JG. [Serum free R ( - ) - and S(+)-mexiletine concentrations were c a l c u l a t e d from the respective s a l i v a drug concentrations and s a l i v a pH]. 2 0 0 1 150-100-5 0 -o R(- ) * S(+) o o o oo o • 8 • o ««o o • • P . O . O IS O • O " ® W O ° C L 0 e o 0 o 250 500 750 Calculated Serum Concentrat ions (ng /ml ) Figure 27. Linear correlation of the respective calculated versus observed serum free R(-)- and S(+)-mexiletine concentrations from ten healthy subjects. [Serum free (R-)- and S(+)-mexiletine concentrations were calculated from the respective saliva drug concentrations and saliva pH]. co 00 C D C c o c CD O c o o 15 O "O o _o CD X I CD LY. 1 0 0 0 i 100 1 0 -• s(+) O R( - ) S 8 • T o T 8 1 T O 1 0 5 10 15 20 25 30 35 40 45 Time (h) Figure 28. Semilogarithmic p l o t s of the mean ± S.D. R ( - ) - and S(+)-mexiletine concentrations in red blood c e l l s over 48 hours from twelve healthy subjects f o l l o w i n g oral administration of 200 mg of racemic m e x i l e t i n e hydrochloride. to Table 21. The R ( - ) - and S(+)-mexiletine concentrations in red c e l l s from'twelve healthy subjects over 48 hours f o l l o w i n g oral administration of 200 mg racemic m e x i l e t i n e hydrochloride. ;- " . (h) Red Blood Cell R(-) Mexiletine Concentration (ng/ml) FROM DRUG ADMIN AO CK CT GR GT JB JG JH RL ST TH TK MEAN I S E B S B f 1 8 STD I B B B S R t S S 0 . 0 0 1 . 0 0 6 2 . 8 7 6 . 9 1 4 2 . 6 1 0 4 . 3 7 1 . 0 . 5 7 . 5 3 2 . 4 6 8 . 4 4 3 . 3 1 . 5 0 1 2 2 . 7 7 1 . 5 1 7 1 . 8 5 2 . 3 1 0 4 . 6 1 5 8 . 7 : 7 5 . 1 2 5 . V 4 8 . 0 1 0 3 . 7 5 6 . 0 2 . 0 0 4 8 . 3 1 0 9 . 8 1 1 0 . 7 6 1 . 2 1 1 9 . 9 5 8 . 2 5 6 . 6 , 7 7 . 4 2 7 . 6 5 1 . 4 7 2 . 1 4 1 . 4 2 . 5 0 5 0 . 3 9 2 . 7 4 9 . 5 3 7 . 8 5 8 . 4 1 1 3 . 8 5 8 . 4 3 4 . 9 8 0 . 2 2 8 . 2 3 6 . 2 5 8 . 2 3 4 . 3 3 . 0 0 5 3 . 7 8 8 . 4 5 5 . 7 8 0 . 5 6 0 . 2 7 4 . 3 7 2 . 7 3 1 . 5 3 3 . 4 6 1 . 1 3 2 . 9 3 . 5 0 5 1 . 7 7 5 . 6 4 1 . 5 5 4 . 0 6 7 . 9 1 4 3 . 9 5 9 . 5 4 9 . 9 7 6 . 7 2 9 . 9 3 1 . 7 6 2 . 0 3 7 . 5 4 . 0 0 5 5 . 4 7 3 . 5 4 1 . 0 3 0 . 6 6 5 . 1 1 2 1 . 3 6 0 . 4 1 1 4 . 9 6 8 . 5 2 7 . 4 3 5 . 8 8 6 . 7 3 7 . 9 4 . 5 0 5 0 . 5 6 2 . 8 4 0 . 7 2 4 . 0 6 9 . 3 1 2 5 . 3 6 1 . 3 5 9 . 9 6 7 . 9 2 8 . 2 5 3 . 6 3 4 . 8 6 . 0 0 4 8 . 8 5 4 . 2 3 6 . 7 6 8 . 4 8 7 . 6 5 3 . 1 4 3 . 8 5 9 . 9 2 6 . 0 2 6 . 7 5 6 . 1 2 8 . 1 8 . 0 0 4 6 . 3 5 2 . 1 2 4 . 2 2 6 . 4 5 7 . 9 5 3 . 4 4 9 . 4 5 6 . 9 2 5 . 8 2 4 . 5 ' 4 1 . 7 2 2 . 9 1 0 . 0 0 4 6 . 2 5 6 . 3 2 3 . 4 5 3 . 7 5 1 . 4 5 2 . 1 5 1 . 6 5 6 . 6 2 3 . 3 1 . 7 4 1 . 6 2 4 . 1 1 2 . 0 0 4 5 . 5 4 7 . 8 2 7 . 0 3 1 . 2 5 2 . 8 5 1 . 7 5 0 . 0 2 7 . 7 4 9 . 1 2 1 . 9 2 4 . 7 4 3 . 0 2 1 . 2 1 4 . 0 0 4 1 . 4 2 9 . 9 4 8 . 9 2 8 . 8 2 8 . 5 2 3 . 9 2 5 . 1 1 6 . 8 2 4 . 0 0 2 7 . 4 5 0 . 5 1 5 . 3 4 6 . 4 1 3 . 8 4 8 . 8 1 9 . 0 2 7 . 6 1 8 . 1 3 0 . 0 0 1 4 . 6 1 2 . 2 1 3 . 4 4 . 8 3 6 . 0 0 1 2 . 0 9 . 4 1 0 . 7 3 . 9 4 8 . 0 0 (h) Red Blood Cell S(+) Mexiletine Concentration (ng/ml) FROM DRUG ADMIN AO CK CT GR GT JB JG JH RL ST TH TK MEAN STD m t l S S B B • B I 3 I E I 2 B B 3 B B 3 S S 8 B 3 S 3 3 3 3 C 3 S B I B 3 E B 8 B 3 s s s s s s s s s S B B B E S S S I B S S B B X X : : 8 3 B B E E 3 B 3 3 S 3 3 B E B E S 3 E B 8 B B B B 3 X E S S S S 3 S X BSaSSGBKB B B B S B S 8 S 3 E S B B E S S 8 B B 0 . 0 0 1.00 5 7 . 7 7 1 . 9 1 4 9 . 5 5 3 . 2 6 6 . 4 5 6 . 9 3 1 . 3 6 0 . 9 4 0 . 2 1.50 1 0 8 . 5 7 4 . 0 1 7 0 . 3 5 5 . 5 5 4 . 4 1 5 7 . 1 6 4 . 0 2 6 . 4 4 7 . 9 9 4 . 7 5 2 . 6 2 . 0 0 5 2 . 2 1 0 6 . 3 5 6 . 1 6 0 . 8 1 3 7 . 1 6 4 . 1 5 6 . 1 7 0 . 2 2 7 . 6 4 8 . 9 6 1 . 8 3 9 . 6 2 . 5 0 5 4 . 0 8 6 . 4 5 0 . 6 4 0 . 8 5 8 . 7 1 3 4 . 0 6 0 . 8 3 7 . 7 7 9 . 2 2 8 . 3 3 3 . 1 6 0 . 3 3 6 . 5 3 . 0 0 5 9 . 1 7 9 . 8 5 7 . 8 8 1 . 3 6 3 . 4 7 5 . 0 7 1 . 2 3 2 . 2 3 5 . 9 6 1 . 7 3 2 . 7 3 . 5 0 5 4 . 6 7 5 . 4 4 5 . 0 5 7 . 6 7 1 . 1 1 7 7 . 9 . 6 4 . 5 5 3 . 2 7 8 . 4 3 1 . 8 3 1 . 8 6 7 . 4 4 3 . 1 4 . 0 0 6 0 . 8 7 0 . 9 4 1 . 2 3 1 . 5 6 5 . 5 1 5 3 . 8 6 4 . 3 1 3 5 . 9 7 1 . 2 3 1 . 7 4 0 . 2 6 9 . 7 4 4 . 1 4 . 5 0 5 4 . 7 5 7 . 7 4 2 . 1 2 6 . 5 7 1 . 8 1 6 7 . 6 6 4 . 5 6 3 . 6 7 2 . 9 2 8 . 9 5 9 . 1 4 1 . 5 6 . 0 0 5 4 . 0 5 1 . 0 3 8 . 9 6 9 . 6 1 0 6 . 9 5 2 . 5 5 2 . 1 6 0 . 3 2 6 . 5 2 9 . 4 6 0 . 1 3 0 . 9 8 . 0 0 5 1 . 0 5 1 . 0 2 7 . 4 3 0 . 9 5 5 . 8 5 8 . 4 5 4 . 9 5 9 . 6 2 7 . 2 2 7 . 5 4 4 . 4 2 4 . 0 1 0 . 0 0 4 9 . 0 5 3 . 3 2 5 . 3 6 8 . 8 6 5 . 0 5 6 . 1 6 5 . 3 5 7 . 3 2 4 . 7 2 7 . 1 4 9 . 2 2 7 . 0 1 2 . 0 0 5 0 . 2 4 4 . 4 3 6 . 5 5 5 . 3 5 4 . 7 5 5 . 5 . 2 8 . 6 5 4 . 7 2 3 . 3 2 7 . 9 4 3 . 1 2 3 . 1 1 4 . 0 0 4 2 . 2 2 8 . 4 3 9 . 8 5 4 . C 2 9 . 7 4 0 . 5 3 6 . 4 2 6 . 1 3 3 . 0 1 9 . 2 2 4 . 0 0 2 7 . 0 5 2 . 7 2 2 . 1 4 9 . 2 1 6 . 0 2 0 . 3 2 3 . 4 1 7 . 2 3 0 . 0 0 1 9 . 4 1 3 . 6 1 6 . 5 6 . 0 3 6 . 0 0 2 1 . 0 1 1 . 9 1 6 . 4 6 . 2 4 8 . 0 0 but the mean drug concentrations were observed to f o l l o w a l o g - l i n e a r d e c l i n e over 48 hours. The red blood c e l l m e x i l e t i n e concentrations were found to be best described by a one-compartment open model in s i x subjects (JG, CK, GR, AO, ST, TK). In subject RL, the h i g h l y v a r i a b l e m e x i l e t i n e concentrations w i t h i n the red blood c e l l s could not be described by e i t h e r one- or two-compartmental k i n e t i c s . In the remaining f o u r s u b j e c t s , red blood c e l l samples were c o l l e c t e d f o r up to 12 hours f o l l o w i n g drug a d m i n i s t r a t i o n and d i s p o s i t i o n r a t e constants were not derived from these s u b j e c t s . The pharmacokinetic data derived from a n a l y s i s of the concentrations of m e x i l e t i n e enantiomers in red blood c e l l s are presented i n Table 22. The mean terminal d i s p o s i t i o n r a t e constants f o r the s i x subjects were found to be 0.0628 ± 0.0322 f r 1 f o r R ( - ) -m e x i l e t i n e and 0.0485 ± 0.0159 h " * f o r S ( + ) - m e x i l e t i n e , with no s i g n i f i c a n t d i f f e r e n c e between the two v a l u e s . The d i s p o s i t i o n r a t e constants were used to c a l c u l a t e the corresponding h a l f - l i v e s which provided mean values of 15.12 ± 9.36 h and 16.95 ± 8.71 h f o r R ( - ) - and S ( + ) - m e x i l e t i n e , r e s p e c t i v e l y . High i n t e r - s u b j e c t v a r i a t i o n s in the h a l f - l i v e s of m e x i l e t i n e in red blood c e l l s were observed among the s i x subjects as i n d i c a t e d by the large standard d e r i v a t i o n s observed. The terminal d i s p o s i t i o n h a l f - l i v e s f o r R ( - ) - and S(+)-mexiletine derived from red blood c e l l drug concentration data were found to be 2 - f o l d higher than those obtained from serum drug d a t a . From another study i n seven healthy subjects (Turgeon et a 7 . , 1987), the h a l f - l i f e of racemic m e x i l e t i n e i n red blood c e l l s was a l s o found to be s i g n i f i c a n t l y higher than that obtained from serum d a t a . However, the mechanisms involved in prolonging the h a l f - l i f e of m e x i l e t i n e i n red blood c e l l s remain Table 22 The pharmacokinetic data For R(-)- and S(+)-mexiletine derived from mexiletine red blood c e l l data from twelve subjects f o l l o w i n g o r a l a d m i n i s t r a t i o n of 200 mg racemic m e x i l e t i n e h y d r o c h l o r i d e . AUCt(ng.h/ml) 6 ( h " 1 ) t l / 2 8. .(h) a ( h " 1 ) SUBJECTS R S R S R S R S GT - . - - - - -JG 1617.2 2153.1 0.122 0.045 • 5.6. : 15.1 0.067> 0.022" CK 1451.3 1472.3 0.078 0.070 8.ay 9 . 8 . ; 0.369 0.376, RL 1188.9 1349.7 - - - - - -GR 707.0 827.6 0.061 0.061 11.2 11.2 - -JH 3005.5 6728.7 - - - - - -AO 3647.0 4388.4 0.020 0.019 34.1 35.9 - -CT 1346.7 1440.9 - - - - - -ST 2457.5 2050.2 0.037 0.047 18.6 14.6 0.369 -TH 2749.5 2388.9 - - - - - -TK 1154.3 1266.8 0.057 0.046 12.1 14.9 0.642 0.714 .'i JB 6226.1 6687.1 - - - - - -MEAN 2322.8 2795.8 0.062 0.048 , 15.1 16.9 / 0.362 0.371 ; S.D. 1583.1 2108.2 0.038 ' 0.026; 10.0 10.4 / 0.207 0.213: 143 u n c e r t a i n . The mean red blood c e l l m e x i l e t i n e AUC0"0 values of 2.3 ± 1.5 pg/ml/h f o r R ( - ) - m e x i l e t i n e and 2.8 ± 2.1 /fg/ml/h f o r S(+)-m e x i l e t i n e , from s i x s u b j e c t s , were not s i g n i f i c a n t l y d i f f e r e n t , suggesting that the d i s t r i b u t i o n of m e x i l e t i n e enantiomers i n t o red blood c e l l s was not s t e r e o s e l e c t i v e . As shown in Figure 29 and Table 23, the o v e r a l l mean red blood c e l l m e x i l e t i n e R(-)/S(+) r a t i o s from the twelve subjects were found to be 0.91 ± 0.13 (mean + S . E . ) over the 48 hours. This data f u r t h e r suggested a n o n - 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 of m e x i l e t i n e enantiomers into red blood c e l l s . Furthermore, the red blood c e l l m e x i l e t i n e AUC values f o r R ( - ) - and S(+)-mexiletine were found not to be s i g n i f i c a n t l y d i f f e r e n t from the r e s p e c t i v e t o t a l serum AUC v a l u e s . Numerous drugs have been reported to d i s t r i b u t e and/or bind to c e l l u l a r components w i t h i n the red blood c e l l s . Red blood c e l l drug concentrations of s a l i c y l a t e (McArthur et a 7 . , 1971), phenobarbital (McArthur et a l . , 1971), phenytoin (Kurata and W i l k i n s o n , 1974), c h l o r t h a l i d o n e (Beermann et a 7 . , 1974), d i g o x i n (Gorodischer et a 7 . , 1975) and acetazolamide (Wallace and Riegelman, 1977) have been reported to exceed t h e i r r e s p e c t i v e serum f r e e drug c o n c e n t r a t i o n s . Acetazolamide has been found to accumulate i n red blood c e l l s due to drug binding to erythrocyte carbonic anhydrase, r e s u l t i n g in red blood c e l l / plasma concentration r a t i o s of « 4 at 24 hours f o l l o w i n g a s i n g l e o r a l dose (McArthur et a 7 . , 1971). The uptake of d i g o x i n into red blood c e l l s during maintenance therapy i n ten infants was found to lead to a mean red blood c e l l / plasma d i g o x i n concentration r a t i o of 3.6 ± 2.0 (Gorodischer et a 7 . , 1975). Red blood c e l l phenytoin concentrations have been found to be l i n e a r l y dependent on the serum f r e e f r a c t i o n s , 2-1 1 -cr o u o _o CD TD CD a : 0-0 10 15 Time (h) 20 25 30 Figure 29. Plots of the mean ± S.D. R ( - ) - / S(+)-tnexiletine red blood c e l l concentration r a t i o s over 24 hours from twelve healthy s u b j e c t s . Table 23. The red blood c e l l mexiletine R(-)/S(+) concentration r a t i o s over 48 hours from twelve healthy s u b j e c t s . TIME rnf**J HDI If* Red B lood Cell Mexi let ine R / S Rat io rKLfl UKUu ADMIN AO CK CT GR GT JB JG JH RL ST TH TK MEAN STD : : : : : : : : : ========= ========= ========= ========= ========= ========= ========= ========= ========= ========= ========= ========= ========= ========= 0 . 0 0 1 . 0 0 1 . 0 9 1 . 0 7 0 . 9 5 1.70 1 . 0 7 0 . 9 8 1.03 0 . 9 9 0 . 5 7 1 . 5 0 1 .13 0 . 9 7 1 .01 0 . 9 4 1 . 6 6 1.01 1 . 0 7 0 . 9 5 1 .00 1 . 2 2 0 . 5 6 2 . 0 0 0 . 9 3 1 . 0 3 1 . 9 7 1.01 0 . 8 7 1 . 6 0 1.01 1 . 0 2 1 . 0 0 1.05 1 . 0 4 0 . 6 2 2 . 5 0 0 . 9 3 1 . 0 7 0 . 9 8 0 . 9 3 0 . 9 9 0 . 8 5 0 . 9 3 0 . 9 8 1 . 0 0 1 . 0 9 0 . 8 9 0 . 4 9 3 . 0 0 0 . 9 1 1.11 0 . 9 6 0 . 9 9 0 . 9 5 0 . 9 9 1 . 0 2 0 . 9 8 0 . 9 3 0 . 9 8 0 . 4 9 3 . 5 0 0 . 9 5 1 . 0 0 0 . 9 2 0 . 9 4 0 . 9 6 0.81 0 . 7 9 0 . 9 4 0 . 9 6 0 . 9 4 1 .00 0 . 9 3 0 . 4 7 4 . 0 0 0 . 9 1 1 . 0 4 0 . 9 9 0 . 9 7 0 . 9 9 0 . 7 9 0 . 8 3 0 . 8 5 0 . 9 3 0 . 8 7 0 . 8 9 0 . 9 1 0 . 4 6 4 . 5 0 0 . 9 2 1 . 0 9 0 . 9 7 0 . 9 1 0 . 9 7 0 . 7 5 0 . 8 8 0 . 9 4 0 . 9 0 0 . 9 8 0 . 8 4 0 . 4 7 6 . 0 0 0 . 9 0 1 . 0 6 0 . 9 4 0 . 9 8 0 . 8 2 0 . 9 2 0 . 8 4 0 . 9 5 0 . 9 8 0.91 1 . 0 3 0 . 4 7 8 . 0 0 0 . 9 1 1 . 0 2 0 . 8 8 0 . 8 6 1 .04 0 . 8 5 0 . 9 0 0 . 8 8 0 . 9 5 0 . 8 9 0 . 9 2 0 . 4 6 1 0 . 0 0 0 . 9 4 1 . 0 6 0 . 9 3 0 . 7 8 0 . 7 9 0 . 8 6 0 . 7 9 1 . 0 2 0 . 9 5 0 . 0 7 0 . 8 2 0 . 4 5 1 2 . 0 0 0 . 9 1 1 . 0 8 0 . 8 6 0 . 9 5 0 . 9 5 0 . 8 2 0 . 9 7 0 . 8 9 0 . 9 4 0 . 8 9 0 . 9 3 0 . 4 6 1 4 . 0 0 0 . 9 8 0 . 9 5 0 . 7 5 0 . 8 4 0 . 9 7 1 . 2 2 0 . 7 8 0 . 9 2 0 . 8 2 0 . 4 7 2 4 . 0 0 1.01 0 . 9 6 0 . 6 9 0 . 9 3 0 . 8 7 0 . 9 3 0 . 6 7 0 . 4 3 3 0 . 0 0 0 . 7 5 0 . 8 9 0 . 8 2 0 . 3 0 3 6 . 0 0 0 . 5 7 0 . 7 9 0 . 6 8 0 . 2 5 4 8 . 0 0 and thus monitoring of red blood c e l l phenytoin l e v e l s was suggested as an a l t e r n a t i v e technique f o r the determination of serum f r e e phenytoin concentrations (Kurata and W i l k i n s o n , 1974). The o v e r a l l mean red blood c e l l / serum t o t a l concentration r a t i o of racemic m e x i l e t i n e has been reported by Turgeon et al, (1987) to be 0.97 ± 0.27 (mean ± S . E . ) in seven healthy s u b j e c t s . However the authors d i d not examine the red blood c e l l d i s t r i b u t i o n of the enantiomers, nor the serum f r e e m e x i l e t i n e c o n c e n t r a t i o n s . In the present study, the red blood c e l l / serum f r e e concentration r a t i o s f o r m e x i l e t i n e enantiomers were determined in eleven healthy s u b j e c t s . As shown i n Figure 30 and Table 24, the mean m e x i l e t i n e red blood c e l l / serum f r e e concentration r a t i o s from the eleven subjects were found to range from 0 . 6 to 1.4 f o r R ( - ) - m e x i l e t i n e and from 0.6 to 1.8 f o r S(+)-m e x i l e t i n e . However, the o v e r a l l mean r a t i o s of 0.85 ± 0.06 and 0.84 ± 0.08 (mean ± S . E . ) over the 48 hours, f o r R ( - ) - and S ( + ) - m e x i l e t i n e , r e s p e c t i v e l y , were both s l i g h t l y but s i g n i f i c a n t l y d i f f e r e n t (p<0.05) from u n i t y . Since serum f r e e m e x i l e t i n e enantiomers were assumed to be a v a i l a b l e f o r d i s t r i b u t i o n i n t o the red blood c e l l s , a red blood c e l l / serum f r e e m e x i l e t i n e r a t i o of l e s s than u n i t y suggested that the d i s p o s i t i o n of m e x i l e t i n e enantiomers into red blood c e l l s may l a r g e l y be a process of passive d i f f u s i o n . These f i n d i n g s a l s o suggested that no s i g n i f i c a n t i n t r a c e l l u l a r binding and accumulation of m e x i l e t i n e enantiomers w i t h i n the red blood c e l l s was not evident from the study. 3 . 3 . 5 Urine Pharmacokinetic Data The cumulative amounts of unchanged R ( - ) - and S(+)-mexiletine i n the urine were found to be v a r i a b l e among the twelve subjects s t u d i e d . Larger q u a n t i t i e s of R ( - ) - m e x i l e t i n e were recovered from the u r i n e of O R( - ) • S(+) o fi 8 0 9 o 9 • o I i i i i i i r 0 5 10 15 20 25 30 35 40 45 Time (h) Figure 30. Plots of the mean ± S.D. respective R(-)- and S(+)-mexiletine red blood cell / serum free concentration ratios over 36 hours from twelve healthy subjects. Table 24. The respective R ( - ) - and S(+)-mexiletine red blood c e l l / serum f r e e drug concentration r a t i o s in twelve subjects over 48 hours. (h) Red Blood Cell / Free Serum Mexiletine Ratio R(-) FROM DRUG ADMIN AO CK CT GR GT JB JG JH RL ST TH TK MEAN ST0 • IH>3S:3 SSX3&SSS3 ========= ========= ========= ========= ========= ========= ======3=3 ========= ========= ========= " " " * " 0 . 0 0 1 . 0 0 0 . 9 1 1 . 6 1 0 . 8 6 1 . 4 9 1 . 4 6 1 .14- 1 . 1 0 1 . 7 4 1 . 2 9 0 . 6 7 1 . 5 0 0 . 8 7 0 . 5 7 3 . 6 9 1 . 3 4 1 . 5 0 0 . 8 1 0 . 2 8 2 . 0 6 1 . 3 9 0 . 9 5 2 . 0 0 0 . 5 6 0 . 9 1 0 . 9 1 1 . 0 7 0 . 6 7 1 . 1 6 1 . 3 7 0 . 5 5 0 . 9 7 0 . 2 9 1 . 3 2 0 . 8 9 0 . 5 0 2 . 5 0 0 . 3 9 0 . 8 6 0 . 6 0 0 . 6 8 0 . 5 4 1 . 0 8 0 . 3 8 1 . 0 1 0 . 2 8 0 . 6 4 0 . 5 9 0 . 3 7 3 . 0 0 0 . 4 0 . 9 9 0 . 7 1 1 . 3 6 0 . 5 7 0 . 8 7 0 . 9 4 0 . 3 0 0 . 5 3 0 . 7 4 0 . 4 3 3 . 5 0 0 . 4 0 . 9 7 0 . 5 6 0 . 8 7 0 . 5 3 1 . 6 7 0 . 4 4 0 . 6 1 1 . 0 2 0 . 4 1 0 . 4 6 0 . 7 2 0 . 4 5 4 . 0 0 0 . 4 6 1 . 1 5 0 . 5 4 0 . 5 1 0 . 5 6 1 . 4 1 0 . 3 8 1 . 3 7 1 . 0 2 0 . 4 1 0 . 6 1 0 . 7 7 0 . 4 7 4 . 5 0 0 . 4 7 0 . 8 5 0 . 5 1 0 . 4 6 0 . 5 8 1 . 6 2 0 . 4 1 0 . 7 2 0 . 9 5 0 . 5 0 0 . 6 4 0 . 4 3 6 . 0 0 0 . 5 2 0 . 8 7 0 . 5 4 1 . 3 6 0 . 4 3 0 . 6 3 0 . 9 1 0 . 5 6 0 . 5 1 0 . 7 0 0 . 4 0 8 . 0 0 0 . 5 2 1 . 1 8 0 . 3 8 0 . 8 8 0 . 5 5 0 . 4 2 0 . 7 3 0 . 9 2 0 . 5 2 0 . 5 0 0 . 6 6 0 . 3 7 1 0 . 0 0 0 . 5 1 . 5 4 0 . 9 1 0 . 5 6 0 . 9 9 0 . 4 5 0 . 8 3 1 . 0 4 0 . 5 9 0 . 0 5 0 . 7 5 0 . 4 6 1 2 . 0 0 0 . 6 2 1 . 3 9 1 . 0 6 0 . 7 3 1 . 0 7 0 . 5 8 0 . 6 1 1 . 1 2 0 . 7 2 0 . 5 8 0 . 8 5 0 . 4 6 1 4 . 0 0 1 . 3 9 1 . 3 9 0 . 9 3 0 . 5 8 0 . 6 3 2 . 0 3 1 . 2 2 0 . 5 9 0 . 9 7 0 . 6 2 2 4 . 0 0 2 . 0 7 1 . 2 5 0 . 8 1 1 . 3 3 0 . 7 9 1 . 0 4 0 . 9 1 0 . 6 4 3 0 . 0 0 0 . 8 7 0 . 7 5 0 . 8 1 0 . 2 9 3 6 . 0 0 0 . 8 4 1 . 0 8 0 . 9 6 0 . 3 5 4 8 . 0 0 (h) c a^j DI if* Red Blnod Cell / Free Serum Mexiletine Ratio S(+) FROM DRUG AOMIN AO CK CT GR GT J B JG JH RL ST TH TK MEAN STO 33.331333 IBS33t3S3 ========= ========= ! " ========= 3333:3333 3C3EXEI33 X833E3EES 3=3333883 S383S83SS 88X838333 833838333 383888888 888883888 0 . 0 0 1 . 0 0 0 . 1 1 1 . 9 5 0 . 9 1 0 . 8 6 1 . 4 2 1 . 1 1 1 . 5 6 0 . 9 9 0 . 6 5 1 . 5 0 1 . 0 0 0 . 6 5 6 . 5 7 0 . 3 8 0 . 8 1 1 . 7 5 0 . 7 5 0 . 3 6 2 . 2 5 1 . 8 1 1 . 4 8 2 . 0 0 0 . 7 6 1 . 0 5 0 . 5 2 1 . 2 8 0 . 7 3 0 . 8 5 0 . 6 6 0 . 9 5 0 . 3 3 1 . 4 7 0 . 7 8 0 . 4 8 2 . 5 0 0 . 4 9 0 . 9 5 0 . 6 9 0 . 8 6 0 . 2 8 0 . 6 7 0 . 4 8 1 . 0 3 0 . 3 1 0 . 6 6 0 . 5 8 0 . 3 6 3 . 0 0 0 . 5 0 1 . 1 7 0 . 8 1 1 . 5 5 0 . 2 2 1 . 0 0 0 . 9 2 0 . 3 7 0 . 6 5 0 . 8 0 0 . 4 8 3 . 5 0 0 . 4 5 1 . 1 9 0 . 6 7 1 . 0 3 0 . 2 6 1 . 0 1 0 . 5 6 0 . 7 1 1 . 0 7 0 . 4 5 0 . 4 9 0 . 7 2 0 . 4 2 4 . 0 0 0 . 5 4 1 . 4 0 0 . 6 0 0 . 5 6 0 . 2 3 0 . B S 0 . 4 6 1 . 7 7 1 . 1 0 0 . 5 0 0 . 7 5 0 . 8 0 0 . 5 1 4 . 5 0 0 . 5 6 0 . 8 6 0 . 5 6 0 . 5 4 0 . 2 4 1 . 1 3 0 . 4 6 0 . 8 2 1 . 0 6 0 . 5 2 0 . 6 1 0 . 3 8 6 . 0 0 0 . 5 8 0 . 9 6 0 . 5 9 0 . 2 4 0 . 8 6 0 . 4 6 0 . 7 9 0 . 9 6 0 . 5 3 0 . 5 8 0 . 7 3 0 . 3 6 8 . 0 0 0 . 6 0 1 . 3 7 0 . 4 5 1 . 1 3 0 . 2 1 0 . 4 9 0 . 8 6 1 . 0 4 0 . 5 4 0 . 6 0 0 . 7 3 0 . 4 3 1 0 . 0 0 0 . 5 7 1 . 7 5 1 . 0 0 0 . 2 7 0 . 6 0 0 . 5 3 1 . 1 9 1 . 0 3 0 . 5 9 0 . 7 1 0 . 8 2 0 . 5 0 1 2 . 0 0 0 . 7 3 1 . 5 2 1 . 2 2 0 . 3 0 0 . 5 4 0 . 7 0 0 . 6 5 1 . 2 6 0 . 7 4 0 . 6 8 0 . 8 3 0 . 4 8 1 4 . 0 0 2 . 1 0 1 . 4 3 0 . 5 5 0 . 7 0 0 . 6 5 1 . 6 7 1 . 3 5 0 . 6 4 1 . 0 1 0 . 6 6 2 4 . 0 0 2 . 1 1 0 . 4 9 0 . 5 3 1 . 4 3 0 . 8 0 1 . 0 3 0 . 8 0 0 . 6 1 3 0 . 0 0 0 . 5 2 0 . 8 0 0 . 6 6 0 . 2 4 3 6 . 0 0 0 . 7 0 0 . 9 5 0 . 8 2 0 . 3 0 4 8 . 0 0 149 three s u b j e c t s , w h i l e in the other nine s u b j e c t s , l a r g e r q u a n t i t i e s of unchanged S(+)-mexiletine were recovered from the u r i n e . Representative p l o t s of the cumulative amounts of unchanged m e x i l e t i n e enantiomers recovered i n the urine over 72 hours from the two groups of subjects are shown in Figures 31 and 32. As shown in Table 25, f o l l o w i n g an o r a l dose, the mean percent of u r i n a r y r e c o v e r i e s of m e x i l e t i n e enantiomers were 3.49 ± 3.35% and 3.68 ± 3.94% f o r R ( - ) - and S ( + ) - m e x i l e t i n e , r e s p e c t i v e l y . The two values were not s i g n i f i c a n t l y d i f f e r e n t . Since the d i s p o s i t i o n of m e x i l e t i n e enantiomers in serum was s i m i l a r , the s i m i l a r q u a n t i t i e s of R ( - ) - and S(+)-mexiletine recovered from urine suggested t h a t the net u r i n a r y e x c r e t i o n of m e x i l e t i n e enantiomers i n urine was not s t e r e o s e l e c t i v e . The combined percentage of the two m e x i l e t i n e enantiomers recovered i n the urine was found to range from 1.20 to 10.38% with a mean value of 7.17 ± 7.45%. This value was found to agree w i t h the mean 72-hour u r i n a r y recovery of 7.9% f o r racemic m e x i l e t i n e reported by P r e s c o t t et a7. (1977), and as w e l l with the recovery range of 7 .5 to 9.2% reported by Haselbarth et a7. (1981) from healthy s u b j e c t s . The renal clearance values f o r the two m e x i l e t i n e enantiomers were corrected to body weight and were found to vary from 0.07 to 1.42 ml/min/kg f o r R ( - ) - m e x i l e t i n e and from 0.06 to 1.12 ml/min/kg f o r S(+)-m e x i l e t i n e . The mean renal clearance f o r S(+)-mexiletine was 0.72 ± 0.85 ml/min/kg, and was not s i g n i f i c a n t l y d i f f e r e n t from the mean value of 0.63 ± 0.85 ml/min/kg f o r R ( - ) - m e x i l e t i n e . This f i n d i n g s of s i m i l a r renal clearance values f o r m e x i l e t i n e enantiomers were c o n s i s t e n t with the r e s u l t s reported by Grech-Belanger et al. (1986) from s i x healthy v o l u n t e e r s . 4000 • s(+) O R( - ) 3000-2000-cP o o o o o o o o o oo i 1000-0 * 0 10 20 30 40 Time (h) 50 60 70 Figure 31. Representative plots of the cumulative amount of R(-)-and S(+)-mexiletine excreted over 72 hours in the urine from subject TK following oral administration of 200 mg of racemic mexiletine hydrochloride. 1E4 8000-(Tjp O dD O O O O o o oo 6000-4000-2000-• s(+) O R( - ) ° 0 10 20 30 40 50 60 70 Time (h) Figure 32. Representative plots of the cumulative amount of R(-)-and S(+)-mexiletine excreted over 72 hours in the urine from subject ST following oral administration of 200 mg of racemic mexiletine hydrochloride. Table 25. The pharmacokinetic data f o r R ( - ) - and S(+)-mexiletine derived from mexiletine urine data from twelve healthy subjects f o l l o w i n g oral administration of 200 mg racemic mexiletine hydrochloride. Xu (mg) CLr % (ml/min/Kg) B (h" 1 ) t l / 2 6 (h) Knr ( h ' 1 ) Dose Excreted SUBJECTS R S R S R S R S R S R S GT 3.09 3.42 0.2 0.3. 0.100' 0.103 6.9 6.7 0.077 0.061 1.8 2.0 JG 3.43 4.33 0.2. 0.5" 0.109:.. 0.102 6.4 6.8 0.064 0.060 2.0 2.5 CK 3.60 3.89 0.2;. 0.2". 0.110 0.110 6.3 6.3 0.125 0.099 2.1 2.0 RL 1.35 1.05 o.o- 0.0., 0.080 0.189 8.7 3.7 0.060 0.054 0.8 0.6 GR 23.69 26.90 3.2 3.7. 0.154 0.139 4.5 5.0 0.071 0.069 14.1 16.0 JH 4.43 4.79 0.2: 0.3 0.125 0.121 5.5 5.8 0.060 0.062 2.6 2.8 AO 3.63 4.93 0.4 0.5. 0.096 0.088 7.2 7.9 0.111 0.113 2.1 2.9 CT 8.89 8.48 0.7 1.8 0.191. 0.194 3.6 3.6 0.098 0.105 5.3 5.0 ST 9.52 6.68 1.4 1.1; 0.210 0.206 3.3 3.4 0.083 0.088 5.6 3.9 TH 0.94 1.07 O.V O.V 0.118- 0.109 5.9 6.4 0.137 0.192 0.5 0.6 TK 2.51 2.87 0.2 0.2 . 0.152' : 0.157 4.6 4.4 0.134 0.113 1.5 1.7 J8 5.06 5.87 0-3 0.4 0.100 0.102.. 6.9 6.8 0.062 0.065 3.0 , 3.5 MEAN 6.34 6.19 0.6 0.8 0.128 . 0.135 5.8 5.6 0.090 0.090: 3.4 3.6 S.D. 5.75 6.57 0.8 1.0 0.038. 0.039 1.5 1.4 0.028 0.037 3.5 3.9 153 Since the renal clearance of a drug i s dependent on i t s glomerular f i l t r a t i o n , a c t i v e t u b u l a r s e c r e t i o n and passive t u b u l a r r e a b s o r p t i o n . A l s o , since serum f r e e drug i s a v a i l a b l e f o r glomerular f i l t r a t i o n , the product of the glomerular f i l t r a t i o n rate (130 ml/min) and the serum f r e e f r a c t i o n can be used to approximate the renal clearance of f r e e d r u g , and provide information on the mechanisms of renal drug e x c r e t i o n . The renal clearance value of 1.00 ml/min/kg f o r f r e e m e x i l e t i n e was c a l c u l a t e d from the product of the glomerular f i l t r a t i o n r a t e (1.79 ml/min/kg i n a 70 kg healthy subject) and the mean m e x i l e t i n e serum f r e e f r a c t i o n ( 0 . 5 6 ) . The observed renal clearance values of <1 ml/min/kg f o r m e x i l e t i n e enantiomers suggested that a net t u b u l a r reabsorption of m e x i l e t i n e may be o c c u r r i n g . Since renal t u b u l a r reabsorption of drugs i s u s u a l l y a passive process that f o l l o w s the p H - p a r t i t i o n r e l a t i o n s h i p , the suggestion of renal t u b u l a r reabsorption of m e x i l e t i n e i s c o n s i s t e n t with the reports of pH-dependent u r i n a r y e x c r e t i o n of m e x i l e t i n e ( M i t c h e l l et a7. 1985; Kaye et a7. 1977). The non-renal e l i m i n a t i o n rate constants f o r m e x i l e t i n e enantiomers from the twelve subjects were c a l c u l a t e d from urine drug d a t a . The mean non-renal e l i m i n a t i o n r a t e constant was 0.0910 ± 0.0288 h " l f o r R ( - ) - m e x i l e t i n e , which was not s i g n i f i c a n t l y d i f f e r e n t from the mean value of 0.0906 + 0.0373 h - 1 observed f o r S ( + ) - m e x i l e t i n e . This f i n d i n g was c o n s i s t e n t with the s i m i l a r non-renal e l i m i n a t i o n rate constants f o r m e x i l e t i n e enantiomers reported p r e v i o u s l y by Igwemezie et a7. (1989). However, from another study (Grech-Belanger et a 7 . , 1986), the s t e r e o s e l e c t i v e g l u c u r o n i d a t i o n of m e x i l e t i n e enantiomers was reported to favor the R(-)-enantiomer. This report of s t e r e o s e l e c t i v e g l u c u r o n i d a t i o n of R ( - ) - m e x i l e t i n e , in r e l a t i o n to the s i m i l a r o v e r a l l 154 non-renal e l i m i n a t i o n rate constant f o r m e x i l e t i n e enantiomers observed in t h i s study, suggested the presence of a s t e r e o s e l e c t i v e non-renal e l i m i n a t i o n pathway f o r S(+)-mexiletine. From urine drug d a t a , the terminal e l i m i n a t i o n r a t e constants f o r m e x i l e t i n e enantiomers were derived from a p l o t of the amount of drug remaining to be excreted in urine over 72 hours (ARE-plot) . Representative ARE-plots of m e x i l e t i n e enantiomers obtained from one subject are presented in Figure 33, showing a p a r a l l e l l o g - l i n e a r r e l a t i o n s h i p of the amount of each enantiomer to be excreted over 72 hours. The mean terminal d i s p o s i t i o n r a t e constants were found to be 0.1287 ± 0.0383 h " 1 and 0.1350 ± 0.0396 h " 1 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 , and the values were not s i g n i f i c a n t l y d i f f e r e n t . The mean terminal d i s p o s i t i o n h a l f - l i v e s were c a l c u l a t e d to be 5 . 8 ± 1.5 h and 5.6 ± 1.4 h, f o r R ( - ) - and S ( + ) - m e x i l e t i n e , r e s p e c t i v e l y , and were not s i g n i f i c a n t l y d i f f e r e n t . An e a r l i e r study ( M i t c h e l l et a / . , 1983) reported that the renal e x c r e t i o n of racemic m e x i l e t i n e was dependent on u r i n a r y pH. In f i v e healthy subjects with induced systemic a c i d o s i s (mean u r i n e pH of 5 . 2 4 ) , the mean renal clearance of m e x i l e t i n e was found t o be 168 ± 18 ml/min. During induced systemic a l k a l o s i s (mean urine pH 7 . 9 7 ) , the mean renal clearance of m e x i l e t i n e was reduced to 4 + 1 ml/min. In the present study, the u r i n a r y e x c r e t i o n rates f o r m e x i l e t i n e enantiomers from subject CK were examined with respect to t h e i r u r i n e volume and urine pH over the 72 hours. As shown in Figure 34, the u r i n a r y e x c r e t i o n rates f o r m e x i l e t i n e enantiomers were found to be v a r i a b l e over the observed u r i n e pH of 4 . 5 to 7.5 and thus a r e l a t i o n s h i p between u r i n e pH and the u r i n a r y e x c r e t i o n of m e x i l e t i n e enantiomers could not be e s t a b l i s h e d . o 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 Time (h) Figure 33. Representative semilogarithmic p l o t s of the amount of R ( - ) - and S(+)-mexiletine remaining to be excreted over 72 hours in the urine from subject TK f o l l o w i n g oral administration of 200 mg of racemic m e x i l e t i n e hydrochloride. i—» cn cn 156 en CD -m D L_ c o o x OJ D C 800 700 600 500 400 300 200 100 0 • s(+) O R( - ) 8 § § 8 • t \ S CD E O > CD C 300 200 100 0 • • • q-pn • • • CD • u a • n ° O L CD C 8 ^ A A A 0 A A A A A A A A l A A A A 10 20 30 40 Time (h) 50 60 70 Figure 34. P l o t s of the u r i n a r y e x c r e t i o n rates of R ( - ) - and S(+)-m e x i l e t i n e , urine volume and urine pH from subject CK over 72 hours f o l l o w i n g o r a l a d m i n i s t r a t i o n of 200 mg racemic m e x i l e t i n e hydrochloride in s i x healthy s u b j e c t s . 3.4 Mexiletine Metabolite Disposition in Humans 3.4.1 Development of a Sensitive and Stereoselective HPLC Assay f o r the Enantiomers of p-Hydroxy-Mexiletine and Hydroxymethyl-Mexiletine in Urine In order to develop a s t e r e o s e l e c t i v e assay method f o r the m e x i l e t i n e metabolite enantiomers, three types of P i r k l e c h i r a l HPLC s t a t i o n a r y phases were evaluated along with the use of appropriate d e r i v a t i z a t i o n reagents. The c h i r a l HPLC s t a t i o n a r y phases i n c l u d e d : i . D-Phenyl g l y c i n e i o n i c s t a t i o n a r y phase. i i . L - I s o l e u c i n e i o n i c s t a t i o n a r y phase. i i i . L - I s o l e u c i n e covalent s t a t i o n a r y phase. While m e x i l e t i n e enantiomers were r e a d i l y resolved as t h e i r 2-naphthoyl or 2-anthroyl d e r i v a t i v e s on these c h i r a l HPLC columns, the presence of the p-OH group and the OH-methyl group precluded the simultaneous r e s o l u t i o n of the metabolite enantiomers on these columns. Thus, to f a c i l i t a t e simultaneous r e s o l u t i o n of the enantiomers of the two m e x i l e t i n e m e t a b o l i t e s , various d e r i v a t i z a t i o n methods f o r the p r o t e c t i o n of the hydroxy groups were i n v e s t i g a t e d . The f o l l o w i n g r e s u l t s were obtained from the d e r i v a t i z a t i o n experiments and chromatographic e v a l u a t i o n s on the three types of P i r k l e c h i r a l HPLC columns. 3.4.1.1 Acylation of Mexiletine Metabolite Enantiomers M e x i l e t i n e enantiomers were acylated with 2-anthroyl or 2-naphthoyl c h l o r i d e . Both the m e x i l e t i n e anthroyl and naphthoyl d e r i v a t i v e s were resolved on the P i r k l e phenylglycine s t a t i o n a r y phase as discussed during the assay development f o r m e x i l e t i n e enantiomers. OH-Methyl-mexiletine enantiomers were acylated with 2-anthroyl c h l o r i d e in the presence of an aqueous sodium hydroxide s o l u t i o n . The r e s u l t i n g chromatogram obtained using the P i r k l e phenylglycine i o n i c column showed two p a i r s of enantiomers as shown in Figure 35. Since a c y l a t i o n at the terminal -NH2 group was expected to be complete, as was found f o r the a c y l a t i o n of m e x i l e t i n e enantiomers, the observation of two p a i r s of enantiomers suggested the formation of mono- and d i -acylated d e r i v a t i v e s due to p a r t i a l a c y l a t i o n of the benzylic-OH group. Since the incomplete a c y l a t i o n of the benzylic-OH group was considered to be due to h y d r o l y s i s of the acyl benzyl e s t e r in the presence of aqueous sodium hydroxide, the r e a c t i o n mixture was f u r t h e r heated to determine i f the r a t i o of the two enantiomeric p a i r s would be a l t e r e d . Heating at 60 °C f o r 40 minutes was found to lead to the disappearance of the f i r s t p a i r of enantiomers, which was assumed t o be due to the d i -acylated d e r i v a t i v e s . Thus a c y l a t i o n with anthroyl c h l o r i d e was not considered to be a v i a b l e method f o r the r e s o l u t i o n of the OH-methyl-m e x i l e t i n e enantiomers due to simultaneous formation and h y d r o l y s i s of the a c y l - e s t e r d e r i v a t i v e . The enantiomers of the p-OH-mexiletine were a l s o acylated with 2-anthroyl c h l o r i d e in the presence of sodium hydroxide. The r e s u l t i n g chromatogram obtained using the P i r k l e phenylglycine column showed a s i n g l e p a i r of enantiomers that were poorly resolved (Figure 3 6 ) . Based on the observation that sodium hydroxide promoted h y d r o l y s i s of the acyl e s t e r d e r i v a t i v e of OH-methyl-mexiletine, the p-OH-mexiletine enantiomers were acylated with 2-anthroyl c h l o r i d e i n the presence of an aqueous sodium bicarbonate s o l u t i o n . However, chromatographic e v a l u a t i o n of the d e r i v a t i z a t i o n mixture revealed two p a i r s of poorly resolved enantiomers which were thought to due to the formation of the 159 c 0 5 1 0 1 5 ~ 2 0 2 5 3 0 3 5 40~(m\n) Figure 3 5 . HPLC chromatograms of resolved R ( - ) - and S(+)-hydroxymethyl mexiletine as t h e i r N,0-anthroyl d i -d e r i v a t i v e s (1) and N-anthroyl mono-derivatives (2) showing aqueous sodium hydroxide h y d r o l y s i s of the N,0-anthroyl d i - d e r i v a t i v e s to the mono-derivatives at (a) room temperature f o r 5 o minutes, (b) at 60 C f o r 30 minutes, and (c) at 60 °C f o r 40 minutes. Chromatographic C o n d i t i o n s : HPLC Column : P i r k l e Phenylglycine Ionic Stationary Phase 0.45 x 25 cm (10 /zm) Mobile Phase: Hexane / 2-Propanol / Chloroform (78/7/15) Flow Rate : 0 . 8 ml/min ' Detection : Fluorescence 270/400 nm (ex/em) 160 1 0 10 20 30 40 (min) Figure 36.(HPLC chromatogram of unresolved p-hydroxy m e x i l e t i n e enantiomers as t h e i r N,0-anthroyl d i - d e r i v a t i v e s (1) . Chromatographic C o n d i t i o n s : HPLC Column : P i r k l e Phenylglycine Ionic S t a t i o n a r y Phase 0.45 x 25 cm (10 nm) Mobile Phase: Hexane / 2-Propanol / Chloroform (78/7/15) Flow Rate : 0 . 8 ml/minute Detection : Fluorescence 270/400 nm (ex/em) mono-acylated and d i - a c y l a t e d p-OH-mexiletine d e r i v a t i v e s . Since a c y l a t i o n of the p-OH-mexiletine enantiomers d i d not provide adequate chromatographic r e s o l u t i o n , t h i s d e r i v a t i z a t i o n protocol was not examined f u r t h e r . 3.4.1.2 S i l y l a t i o n / Acylation of Mexiletine Metabolite Enantiomers In order to form a n o n - e l e c t r o p h i l i c d e r i v a t i v e and t o avoid h y d r o l y s i s and/or incomplete d e r i v a t i z a t i o n at the hydroxy! groups of the m e t a b o l i t e s , s i l y l a t i o n of the metabolite enantiomers was i n v e s t i g a t e d . N - T r i m e t h y l s i l y l imidazole (TMSI) reagent was chosen as i t allows s e l e c t i v e s i l y l a t i o n of hydroxyls without a f f e c t i n g amino groups. S i l y l a t i o n of the benzylic-OH group of OH-methyl-mexiletine with TMSI, followed by a c y l a t i o n of the amino f u n c t i o n with 2-naphthoyl c h l o r i d e , was found to lead to the production of two p a i r s of enantiomeric peaks as shown in Figure 37. The presence of two p a i r s of enantiomers was thought to be due to p a r t i a l s i l y l a t i o n of the OH-methyl group. p-OH-Mexiletine was a l s o s i l y l a t e d with the TSMI reagent, followed by a c y l a t i o n with 2-naphthoyl c h l o r i d e . However, r e s o l u t i o n of p-OH-m e x i l e t i n e enantiomers was not observed chromatographically. 3.4.1.3 Methylation and Acylation To achieve r e s o l u t i o n of p-OH-mexiletine enantiomers, methylation of the enantiomers with diazomethane in a c i d i c methanol was i n v e s t i g a t e d . The time-course of methylation of p-OH-mexiletine with diazomethane was found to be complete at room temperature w i t h i n 5 minutes. Following m e t h y l a t i o n , the p-OH-mexiletine enantiomers were acylated with 2-anthroyl c h l o r i d e in the presence of aqueous sodium bicarbonate s o l u t i o n . As shown in Figure 38, the p-OH-mexiletine 162 Figure 37. HPLC chromatogram showing r e s o l u t i o n of R ( - ) - and S(+)-hydroxymethyl m e x i l e t i n e as t h e i r 0 - s i l y l - N - n a p h t h o y l d e r i v a t i v e s (1) and N-naphthoyl d e r i v a t i v e s ( 2 ) . Chromatographic C o n d i t i o n s : HPLC Column : P i r k l e Phenylglycine Ionic Stationary Phase 0.45 x 25 cm (10 fm) Mobile Phase: Hexane / 2-Propanol / Chloroform (78/7/15) Flow Rate : 0 . 8 ml/min Detection : Fluorescence 270/400 nm (ex/em) 163 ra oo 0 10 20 30 40 (^) '• Figure 38. HPLC chromatogram showing r e s o l u t i o n of the N-anthroyl d e r i v a t i v e s of R ( - ) - and S(+)-mexiletine (1) , N-anthroyl d e r i v a t i v e of KOE-2963 ( 2 ) , 0-methyl-N-anthroyl d e r i v a t i v e s of R ( - ) - and S(+)-p-0H mexiletine (3) and N-anthroyl d e r i v a t i v e s of R ( - ) - and S(+)-OH-methyl m e x i l e t i n e ( 4 ) . Chromatographic Conditions: HPLC Column : P i r k l e Isoleucine Ionic S t a t i o n a r y Phase 0.45 x 25 cm (10 (im) Mobile Phase: Hexane/Ethanol/2-Propanol (95/2/3) Flow Rate : 0 . 8 ml/min Detection : Fluorescence 270/400 nm (ex/em) enantiomers were resolved as t h e i r 0-methyl-N-anthroyl d e r i v a t i v e s on the P i r k l e i s o l e u c i n e i o n i c column. Also shown in t h i s chromatogram are the enantiomers of OH-methyl-mexiletine and m e x i l e t i n e . The l a t t e r two compounds were not methylated, and hence, they were eluted as t h e i r N-anthroyl d e r i v a t i v e s . Although p r o t e c t i o n of the hydroxy group of p-OH-mexiletine using diazomethane was successful in permitting simultaneous r e s o l u t i o n of the enantiomers of m e x i l e t i n e and m e x i l e t i n e m e t a b o l i t e s , the methylation r e a c t i o n was found to be v a r i a b l e . 3 . 4 . 1 . 4 Acylated and A l k y l a t e d D e r i v a t i v e s A c y l a t i o n of the NH2- group of the m e x i l e t i n e metabolites with 2 - a n t h r o y l c h l o r i d e was necessary f o r enantiomeric r e s o l u t i o n and f o r f luorescence d e t e c t i o n as p r e v i o u s l y d i s c u s s e d . However, attempts to protect the hydroxyl groups of the metabolites of m e x i l e t i n e p r i o r to a c y l a t i o n were l a r g e l y u n s u c c e s s f u l . Thus, p-OH-mexiletine enantiomers were i n i t i a l l y acylated with anthroyl c h l o r i d e followed by a l k y l a t i o n of the hydroxy group with e t h y l bromide, n-propyl bromide, and n-butyl bromide. As shown in Figure 39, as the s i z e of the a l k y l group i n c r e a s e d , the chromatographic r e t e n t i o n of the enantiomers a l s o i n c r e a s e d . However, the r e s o l u t i o n of the enantiomers was r e t a i n e d on the P i r k l e i s o l e u c i n e i o n i c column. A l k y l a t i o n of p-OH-mexiletine with ethyl bromide was chosen f o r i t s optimal r e t e n t i o n times. For OH-methyl-mexiletine enantiomers, since t h e i r N-acylated mono-d e r i v a t i v e s were w e l l resolved on the P i r k l e phenylglycine column as discussed i n Section 3 . 4 , 1 . 1 , chromatographic r e s o l u t i o n of the d e r i v a t i v e s on the P i r k l e i s o l e u c i n e i o n i c and the P i r k l e i s o l e u c i n e column covalent columns were a l s o evaluated. Baseline r e s o l u t i o n of the N-anthroyl OH-methyl-mexiletine enantiomers was observed on the P i r k l e 165 Figure 39. HPLC chromatograms showing r e s o l u t i o n of (a) the 0 - e t h y l - N - a n t h r o y l d e r i v a t i v e s , (b) 0-propyl-N-anthroyl d e r i v a t i v e s and (c) 0 - b u t y l - N - a n t h r o y l d e r i v a t i v e s of R ( - ) - and S(+)-p-hydroxy m e x i l e t i n e (1) and hydroxymethyl mexile t ine ( 2 ) . Also shown are the unresolved N-anthroyl d e r i v a t i v e s of hydroxymethyl m e x i l e t i n e enantiomers ( 3 ) . ^ HPLC Column : P i r k l e Isoleucine Ionic S t a t i o n a r y Phase ( •?• 0.45 x 25 cm [lOfim)  ! ' Mobile Phase: Hexane/Ethanol/2-Propanol/Chloroform (95/2/3/5) Flow Rate : 1.0 ml/min Detection : Fluorescence 270/400 nm (ex/em) 166 i s o l e u c i n e covalent s t a t i o n a r y phase. Since the N-acylated d e r i v a t i v e s of m e x i l e t i n e and OH-methyl-m e x i l e t i n e enantiomers were resolved on the P i r k l e i s o l e u c i n e covalent column, and w h i l e p-OH-mexiletine enantiomers were a l s o resolved on the column as t h e i r acylated / a l k y l a t e d d e r i v a t i v e s , a d e r i v a t i z a t i o n scheme f o r a c y l a t i o n of m e x i l e t i n e and the m e x i l e t i n e metabolites followed by s e l e c t i v e a l k y l a t i o n of p-OH-mexiletine would allow simultaneous r e s o l u t i o n of the three compounds. With t h i s approach, the amino groups of m e x i l e t i n e , p-OH-mexiletine and OH-methyl-mexiletine were t h e r e f o r e f i r s t acylated with 2-anthroyl c h l o r i d e . The r e s u l t i n g N-anthroyl d e r i v a t i v e s of m e x i l e t i n e and OH-methyl-mexiletine were separated from the N-anthroyl d e r i v a t i v e of p-OH-mexiletine by e x t r a c t i o n into hexane from a b a s i c (sodium hydroxide) s o l u t i o n . The l a t t e r d e r i v a t i v e was subsequently a l k y l a t e d with e t h y l bromide and then recombined w i t h the former d e r i v a t i v e s f o r simultaneous chromatographic e v a l u a t i o n s . In order to confirm the c o n f i g u r a t i o n of each enantiomer as i t e l u t e d from the HPLC column, reference standards of R ( - ) - and S(+)-m e x i l e t i n e were studied by polarimetry as t h e i r N-anthroyl d e r i v a t i v e s . This confirmed that the r o t a t i o n of plane p o l a r i z e d l i g h t was negative f o r R ( - ) - m e x i l e t i n e and p o s i t i v e f o r the antipode. To determine the e l u t i o n order of m e x i l e t i n e , p-OH-mexiletine and OH-methyl-mexiletine, a composite sample was i n j e c t e d on the i s o l e u c i n e covalent column and eluant f r a c t i o n s were c o l l e c t e d at 29.0 and 29.5 minutes f o r m e x i l e t i n e enantiomers, 34.9 and 35.7 minutes f o r p-OH-mexiletine enantiomers, and at 42.8 and 43.9 minutes f o r OH-methyl-mexiletine enantiomers (Figure 4 0 ) . The o p t i c a l r o t a t i o n of each of the enantiomers i n d i c a t e d t h a t 167 1 S(+) R(- ) cr-E h~ I— N L Figure 40. Representative HPLC chromatograms of (a) blank urine and (b) urine from a subject showing the r e s o l u t i o n of the N-anthroyl d e r i v a t i v e s of S(+)- and R ( - ) - m e x i l e t i n e (1, Rt=29.0 and 29.5 min) , the N-anthroyl d e r i v a t i v e of KOE-2963 (2, Rt=31.7 m i n ) , the 0 - e t h y l - N - a n t h r o y l d e r i v a t i v e s of S(+)- and R(-)-/?-hydroxy m e x i l e t i n e ( 3 , Rt=34.9 and 35.7 min) , the 0 - e t h y l - N - a n t h r o y l d e r i v a t i v e s of tyramine (4, Rt=38.0 min) , the N-anthroyl d e r i v a t i v e of methyl amine ( 5 , Rt=40.8 min) , and the N-anthroyl d e r i v a t i v e s of R ( - ) - and S(+)-hydroxymethyl m e x i l e t i n e (6, Rt=42.8 and 43.9 min). HPLC Column : P i r k l e I s o l e u c i n e Covalent S t a t i o n a r y Phase 0.45 x 25 cm (10 /zm) Mobile Phase: Hexane/Ethanol/2-Propanol/Acetonitrile/ Ethyl acetate/Chloroform (90/4/4/1/1). Flow Rate : 0 . 6 to 2.0 ml/min Detection : Fluorescence 270/400 nm (ex/em) 168 S(+)- and R ( - ) - m e x i l e t i n e eluted at 29.0 and 29.5 minutes, r e s p e c t i v e l y ; S(+)- and R(-)-p-OH-mexiletine eluted at 34.9 and 35.7 minutes, r e s p e c t i v e l y , and R ( - ) - and S(+)-0H-methyl-mexiletine e l u t e d at 42.8 and 43.9 minutes, r e s p e c t i v e l y . The order of e l u t i o n f o r the OH-methyl m e x i l e t i n e enantiomers on the P i r k l e i s o l e u c i n e covalent s t a t i o n a r y phase was opposite to those of m e x i l e t i n e and p-OH-mexiletine enantiomers. The presence of an a d d i t i o n a l i n t e r a c t i o n of the OH-methyl -mexi l e t ine with the s t a t i o n a r y phase was thought to lead to the r e v e r s a l of e l u t i o n order. 3.4.2 The Tine-Course of Alky l a t i o n of p-Hydroxy-Mexiletine with Ethyl Bromide As shown i n Figure 41, the time-course of a l k y l a t i o n of p-OH-m e x i l e t i n e at 60°C i n d i c a t e d that d e r i v a t i z a t i o n was completed i n approximately 40 minutes. During the course of r e a c t i o n t o 60 minutes, the N - a n t h r o y l OH-methyl-mexiletine metabolite d e r i v a t i v e was found to be s t a b l e i n the a l k y l a t i o n c o n d i t i o n s . An optimal a l k y l a t i o n time of 40 minutes was used f o r a l k y l a t i o n of p-OH-mexiletine during a n a l y s i s of the u r i n e samples. 3.4.3 Solvent Recovery f o r Mexiletine, p-Hydroxy-Mexiletine, and Hydroxymethyl-Mexiletine from Urine The e x t r a c t i o n of m e x i l e t i n e into ether/dichloromethane (20/80) was observed to be constant over the pH range of 6 to 10. As shown i n Figure 42 and 43, the solvent e x t r a c t i o n of p-OH-mexiletine and OH-methyl - m e x i l e t i n e into d i f f e r e n t solvents was found to vary as a f u n c t i o n of pH. The e x t r a c t i o n r e c o v e r i e s of OH-methyl- and p-OH-m e x i l e t i n e i n t o ether / dichloromethane and e t h y l acetate were found to increase with i n c r e a s i n g pH. The e f f e c t of pH on the e x t r a c t i o n of the ^ %+l P -OH-Mex i le t ine A R ( - ) - O H - M e t h y l Mexiletine 8 A A A A 10 20 30 40~ 50 60 70 Time (min) Figure 41. Plots of the time-course of a l k y l a t i o n of R ( - ) - and S(+)-p-hydroxy mexiletine using ethyl bromide at 60 °C over 60 minutes. 0.6 A R ( - ) - O H - M e t h y l Mexi le t ine • R ( - ) - p - O H Mexi let ine 0.5 o LY. 0.4 *CD o CD CL 0.3 0.2 0.1 0.0 7 8 10 11 12 13 14 15 Extraction pH Figure 42. The ethyl acetate e x t r a c t i o n recovery of R(-)-p-hydroxy mexiletine and R(-)-hydroxymethyl m e x i l e t i n e as a p l o t of the chromatographic peak height r a t i o s versus e x t r a c t i o n pH. o • R ( - ) —Mexiletine ' • R ( - ) - p - O H Mexiletine • R ( - ) - O H - M e t h y l Mexiletine 6 7 8 9 10 11 Extraction pH Figure 43. The dichloromethane / ether (20/80) e x t r a c t i o n recovery of R ( - ) - m e x i l e t i n e , R(-)-p-hydroxy mexiletine and R ( - j -hydroxymethyl mexiletine as a p l o t of the chromatographic peak height r a t i o s versus e x t r a c t i o n pH. 172 metabolites were more pronounced with p-OH-mexiletine probably due to the i o n i z a b l e phenolic hydroxy group. The e x t r a c t i o n of OH-methyl-m e x i l e t i n e i n t o ethyl acetate was found to be optimal at pH 9. Since solvent e x t r a c t i o n between two enantiomers can be considered s i m i l a r , the solvent recovery data f o r the R(-)-isomers of m e x i l e t i n e and the m e x i l e t i n e metabolites from u r i n e are shown in Table 26. Dichloromethane / ether (20/80) was found to g i v e 98.3%, 61.9% and 60.3% r e c o v e r i e s of the R(-)-isomers of m e x i l e t i n e , p-OH-mexiletine and OH-methyl - m e x i l e t i n e , r e s p e c t i v e l y . To improve the recovery y i e l d s of the metabolites from u r i n e , urine samples were extracted with three portions of 2 ml of solvent as o u t l i n e d in the assay p r o t o c o l . 3 . 4 . 4 HPLC Assay Method f o r M e x i l e t i n e , p-Hydroxy-Mexiletine* and Hydroxymethyl-Mexi l i s t ine i n Urine Following the e x t r a c t i o n protocol developed (Figure 44) , m e x i l e t i n e and i t s metabolites were acylated with 2-anthroyl c h l o r i d e in the presence of sodium bicarbonate s o l u t i o n . A weakly b a s i c sodium bicarbonate s o l u t i o n was found to c a t a l y z e the a c y l a t i o n of -NHg groups w h i l e d e r i v a t i z a t i o n of the OH-methyl and p-OH groups on the metabolites was not observed chromatographically. Following a c y l a t i o n , the excess a c i d c h l o r i d e was reacted with methylamine followed by adjustment to pH>12 with the a d d i t i o n of sodium hydroxide to form the phenoxide ion of the p-OH-mexiletine. The N-acylated d e r i v a t i v e s of m e x i l e t i n e and OH-methyl - m e x i l e t i n e were then extracted i n t o dichloromethane. The remaining aqueous f r a c t i o n , c o n t a i n i n g the N-acylated p-OH-mexiletine metabolite was adjusted t o a c i d i c pH and extracted into hexane. The hexane was evaporated to dryness followed by a l k y l a t i o n with ethyl bromide. The r e s u l t i n g p-OH-mexiletine d e r i v a t i v e was extracted into Urine (0.2 to 1 ml) Adjust to pH 9.0 (0.5 ml P0 4 " buffer) E x t r a c t i o n { 3x [ Add 2 ml CH2CI2/ether (20/80) ] ) { [ Vortex mixing f o r 2 minutes ] } { Evaporate Solvent to » 1 . 0 ml } A c y l a t i o n { Add 0.2 ml NaHC03 ( 0 . 5 M) - ) { Add 0.01 ml 2-anthroyl c h l o r i d e (1 mg/ml) } { Vortex mix f o r 3 minutes } Add 0.1 ml methyl amine (70% in H2O) Vortex mix Add 0.2 ml NaOH (2.0 M) 2x [ Extract with 2 ml hexane ] Aqueous « 1 • Organic A l k y l a t i o n Add 0 . 2 ml HC1 ( 3 . 0 M) 2x [ Extract with CH2CI2 ] Evaporate to dryness Add 0 . 1 ml ethyl bromide Add 0 . 1 ml K0H/Et0H o (0 .05 M) Heat 40 minutes 60 °C A d d . 1 . 0 ml d i s t i l l e d water Evaporate EtOH to dryness Extract with 2 ml CH2CI2 Hexane extract CH2CI2 e x t r a c t Hexane extract Evaporate to dryness Reconstitute in HPLC mobile phase Figure 44. A schematic representation of the assay procedure a l l o w i n g simultaneous determination of m e x i l e t i n e , p-Hydroxy m e x i l e t i n e and hydroxymethyl mexiletine enantiomers in u r i n e . 174 Table 26. The solvent e x t r a c t i o n recoveries of m e x i l e t i n e , p-hydroxy m e x i l e t i n e and hydroxymethyl m e x i l e t i n e enantiomers from urine at adjusted urine pH 9 . 0 . % Recovery of 100 no drug from urine (pH 9.0) p-OH- OH-methyl-E x t r a c t i o n M e x i l e t i n e M e x i l e t i n e M e x i l e t i n e Solvent (n-2) R(-) S(+) R(-) S(+) R(-) S(+) Ethyl acetate Diethyl Ether Dichloromethane/ Diethyl ether (80/20) 88.5 92.9 23.8 95.1 96.8 38.1 98.3 97.2 61.9 22.5 30.8 29.3 35.9 44.2 42.7 57.3 60.3 57.3 175 dichloromethane which was then combined with the m e x i l e t i n e and OH-methyl - m e x i l e t i n e d e r i v a t i v e s . This d e r i v a t i z a t i o n scheme was found to allow r e s o l u t i o n of the enantiomers of m e x i l e t i n e and of the two metabolites on the covalent i s o l e u c i n e s t a t i o n a r y phase. The procedure was found to be reproducible and r e l i a b l e f o r the q u a n t i t a t i o n of m e x i l e t i n e and the two metabolites in u r i n e . A r e p r e s e n t a t i v e HPLC chromatogram showing the simultaneous r e s o l u t i o n of the enantiomers of m e x i l e t i n e , p-OH-mexiletine and OH-methyl-mexiletine in the urine of a subject i s shown in Figure 40. 3.4.5 Detection Response Li n e a r i t y f o r Mexiletine, p-Hydroxy-Mexiletine, and Hydroxymethyl-Mexiletine Enantiomers in Urine The d e t e c t i o n response l i n e a r i t y data f o r m e x i l e t i n e , p-OH-m e x i l e t i n e and OH-methyl-mexiletine enantiomers are shown i n Figure 45. A concentration range from 10 to 500 ng/ml i n u r i n e was found to be s a t i s f a c t o r y f o r the determination of m e x i l e t i n e peak height r a t i o s in u r i n e with 100 ng of K0E-2963 i n t e r n a l standard. For OH-methyl- and p-OH-mexiletine m e t a b o l i t e s , a concentration range from 25 to 1500 ng/ml was used with 200 ng of tyramine as an i n t e r n a l standard. The p r e c i s i o n of the c a l i b r a t i o n data f o r the enantiomers of m e x i l e t i n e and the two metabolites are presented i n Table 27. I n t e r - and intra-sample assay v a r i a b i l i t y data over the c a l i b r a t i o n range f o r the enantiomers of m e x i l e t i n e , p-OH-mexiletine and OH-methyl-mexiletine are shown in Tables 28 to 31, The assay v a r i a b i l i t y , as measured by the c o e f f i c i e n t of v a r i a t i o n ( C V . ) f o r i n t e r - and i n t r a - s a m p l e s , was found to g e n e r a l l y f a l l w i t h i n 10%. Although the inter-sample assay v a r i a b i l i t y f o r m e x i l e t i n e and p-OH-m e x i l e t i n e at one concentration was observed to be 13% and 15% • s(+)1 I R(-) Mexi'e"tine O R(-) 9 S ( + ) O H - M e t h y l Mexi le t ine A S(+) R ( _ ) P - O H - M e x i l e t i n e 300 600 900 1200 Concentration (ng/ml) Figure 45. HPLC assay calibration curves for mexiletine, p-hydroxy mexiletine and hydroxymethyl mexiletine enantiomers in urine. Table 27. HPLC assay 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 , p-hydroxy mexiletine and hydroxymethyl m e x i l e t i n e enantiomers in u r i n e . p-OH OH-Methvl mx + b M e x i l e t i n e M e x i l e t i n e M e x i l e t i n e R(-) S(+) R(-) S(+) R(-) S(+) b - i n t e r c e p t -0.1673 -0.1433 -0.1518 -0.1628 -0.2349 -0.2184 Slope (m) 0.0261 0.0257 0.0025 0.0025 0.0062 0.0057 r 2 0.9997 0.9997 0.9957 0.9961 0.9955 0.9956 Table 28. HPLC i n t e r - a s s a y v a r i a b i l i t y f o r R ( - ) - and S(+)-mexiletine in u r i n e . M e x i l e t i n e Inter-Assay V a r i a b i l i t y Peak Height Ratio C V . (Average ± S . D . . n=31 Urine (ng/ml) R(-) S(+) R(-) S(+) 25 0 .481±0 .028 0 .449±0 .025 5.8 5.6 50 0 . 7 0 1 ± 0 .007 0 .648±0 .005 1.0 0 . 8 100 1 .220±0 .034 1 . 1 4 1 ± 0 .023 2.8 2.0 250 2 .442±0 .322 2 .338±0 .298 13.2 12.7 500 3 .856±0 .094 3 .635±0 .016 2.4 0.4 178 Table 29. HPLC i n t e r - a s s a y v a r i a b i l i t y f o r R ( - ) - and S(+)-p-hydroxy m e x i l e t i n e in u r i n e . P-OH m e x i l e t i n e Inter-Assay V a r i a b i l i t y Urine Peak Height Ratio Concentration (Average ± S . D . , n=3) C V (ng/ml) R(-) S(+) R(-) S( + ) 50 0 . 1 2 8 ± 0 . 0 1 0 0.128+0.050 7.8 4.7 100 0.319±0.048 0.313±0.042 15.0 13.4 500 1 . 8 7 8 ± 0 . 1 0 3 1 . 8 7 4 ± 0 . 1 2 3 0.1 6.6 1000 7 . 6 1 8 ± 0 . 3 3 1 7 .485±0.308 4 . 3 4.1 1500 1 4 . 6 5 ± 0 . 7 3 0 1 4 . 1 8 ± 0 . 6 3 0 5.2 4.4 Table 30. HPLC i n t e r - a s s a y v a r i a b i l i t y f o r R ( - ) - and S(+)-hydroxymethyl mexiletine i n u r i n e . OH-Methvl M e x i l e t i n e Inter-Assay V a r i a b i l i t y Urine Peak Height Ratio Concentration (Average ± S . D . , n=3) C.V. (ng/ml) R(-) S(+) R(-) S(+) 50 0.063±0.001 100 0.085±0.002 500 0.579±0.011 1000 0.910±0.042 0.063±0.001 1.6 1.6 0.088±0.005 2.3 5.6 0.631±0.010 1.8 1.6 0.962±0.047 4.6 4.9 Table 31. HPLC i n t r a - a s s a y v a r i a b i l i t y f o r R ( - ) - and S ( + ) - m e x i l e t i n e , p-hydroxy mexiletine and hydroxymethyl m ex iletin e i n u r i n e . Urine Intra-Assay Var iabi l i ty (ng/ml) Mexiletine p-OH OH-Methvl Mexiletine Mexiletine C.V. C.V. C.V. 50 R(-) 0.697+0.001 0.1 0.092+0.004 4.3 S(+) 0.639±0.006 0.9 0.092±0.004 4.3 100 R(-) 1 . 2 2 3 ± 0 . 0 0 1 0.1 0.286±0.010 3 . 5 0 . 5 9 7 ± 0 . 0 0 7 1.2 S(+) 1.146+0.013 1.1 0.296+0.018 6.1 0.637±0.002 0.3 500 R(-) S(+) 2.008±0.064 3.2 1 .964±0.001 0.1 r e s p e c t i v e l y , the discrepancy of the s i n g l e data point d i d not suggest the presence of any systematic e r r o r . 3.4 .6 Urine M e x i l e t i n e M e t a b o l i t e Pharmacokinetic Data The pharmacokinetic parameters f o r m e x i l e t i n e and f o r the m e x i l e t i n e metabolites derived from metabolite urine data i s presented i n Table 32. 3.4 .6 .1 p-Hydroxy-Mexiletine Enantiomers Representative p l o t s of the cumulative amounts of p-OH-mexiletine enantiomers recovered from the urine of one subject over 72 hours f o l l o w i n g o r a l a d m i n i s t r a t i o n of a 200 mg m e x i l e t i n e hydrochloride are shown i n Figure 46. The cumulative amounts of the p-OH-mexiletine enantiomers recovered from the urine of four subjects over 72 hours were found to range from 0.99 to 1.85 mg f o r the R ( - ) - i s o m e r , and a range of values from 0.92 to 1.88 were observed f o r the S(+)-isomer. The mean cumulative amounts of p-OH-mexiletine enantiomers recovered from the u r i n e of four subjects were 1.31 ± 0.33 mg and 1.27 ± 0.39 mg, r e s p e c t i v e l y , f o r the R ( - ) - and S(+)-enantiomer. The two values were not s i g n i f i c a n t l y d i f f e r e n t . The terminal h a l f - l i v e s f o r the m e x i l e t i n e enantiomers were obtained from the sigma-minus p l o t (Mayerson and G i b a l d i , 1969) by p l o t t i n g the r e s p e c t i v e amounts of each of the metabolites remaining to be excreted in urine over the 72 hours. As shown in Figure 47, the amounts of p-OH-metabolite enantiomers remaining to be excreted in the urine from one of the subjects followed l o g - l i n e a r p a r a l l e l d e c l i n e over 72 hours. The e l i m i n a t i o n rate constants f o r m e x i l e t i n e enantiomers were 0.1223 ± 0.0637 h - 1 and 0.1270 ± 0.0608 h " 1 , r e s p e c t i v e l y , f o r R ( - ) - and S ( + ) - m e x i l e t i n e , and the two values were found to be s i m i l a r . The s i m i l a r terminal d i s p o s i t i o n r a t e constants 3000 • R f } p - O H Mexi let ine O S(+) 2000 8 8 8 8 9 ® o 1000-•o o 0 0 10 20 30 40 Time (h) 50 60 70 Figure 46. Representative plots of the cumulative amount of R(-)-and S(+)-p-hydroxy mexiletine excreted over 72 hours in the urine from subject TK following oral administration of 200 mg of racemic mexiletine hydrochloride. £1000.0 ZD c "O CD -+-> CD CJ X LxJ CD C O 100.0 10.0 1.0 0.1 ? § § § • R( - ) O S ( + ) p - O H - M e x i l e t i n e 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 Time (h) Figure 47. Representative plots of the amount of R ( - ) - and S(+)-p-hydroxy mexiletine remaining to be excreted over 72 hours in the urine from subject TK f o l l o w i n g oral administration of 200 mg of racemic m e x i l e t i n e hydrochloride. Table 32. The pharmacokinetic data of R ( - ) - and S(+)-mexiletine, p-hydroxy mexiletine and hydroxymethyl m e x i l e t i n e derived from urine metabolite data from twelve healthy subjects f o l l o w i n g oral administration of 200 mg racemic m e x i l e t i n e hydrochloride. p-OH M e x i l e t i n e Kel h " 1 t i / 2 (h) z xmu°°(mg) % dose Subjects R(-) S(+) R(-) S(+) R(-) S(+) R(-) S(+) JH 0.047 0.048 14.7 14.3 1.12 0 .92 0.6 10.5 ST 0.214 0.207 3.2 3.3 0.99 0 .93 0.5 0.5 TH 0.146 0.146 4.7 4.7 1.29 1 .36 0.7 0.7 TK 0.082 0.083 8.4 8.3 1.85 1 .88 1.0 1.0 mean ± 0.122 0.127 7.7 7.7 1.31 1 .27 0.7 0.7 S.D. 0.064 0.061 4.4 4.2 0.33 0 .39 0.1 0.2 OH-Methvl M e x i l e t i n e JH 0.047 0.053 14.8 13.0 1.33 0.68 0.7 0.3 ST 0.062 0.072 11.0 9.6 1.14 0.49 0.6 0.2 TH 0.063 0.074 10.9 9.3 4.59 1.63 2.6 0.9 TK 0.070 0.077 9.9 9.0 4.68 1.88 2.6 1.0 mean ± 0 . 0 6 1 * 0.069 11.7 *10.2 2 . 9 4 * 1.17 1.6 * 0.6 S.D. 0.008 0.009 1.8 1.6 1.70 0.60 0.9 0.3 * S i g n i f i c a n t l y d i f f e r e n t (p<0.05) 185 f o r m e x i l e t i n e enantiomers derived using metabolite k i n e t i c data were c o n s i s t e n t with the s i m i l a r terminal r a t e constants observed from the m e x i l e t i n e u r i n a r y d a t a . The mean terminal d i s p o s i t i o n h a l f - l i v e s of 7.79 ± 4.42 h and 7.71 ± 4.27 h, f o r R ( - ) - and S ( + ) - m e x i l e t i n e , r e s p e c t i v e l y , were not s i g n i f i c a n t l y d i f f e r e n t , but the values were found greater than those derived from u r i n a r y m e x i l e t i n e d a t a . 3.4 . 6 . 2 Hydroxymethyl-Mexiletine Enantiomers Representative p l o t s of the cumulative amounts of OH-methyl-m e x i l e t i n e recovered from the urine of one subject over 72 hours are shown in Figure 48. The cumulative amounts of OH-methyl-mexiletine enantiomers recovered from the urine in 72 hours were found to be h i g h l y v a r i e d among the four s u b j e c t s . A high i n t e r - s u b j e c t v a r i a t i o n i n the u r i n a r y recovery of the racemic OH-methyl-metabolite was reported by Beckett and Chidomere (1977). However, the reported urine recovery range from «5 to 7% f o r OH-methyl-mexiletine from two subjects was obtained under induced a c i d i c urine pH. In the present study, the mean cumulative amounts of OH-methyl-mexiletine excreted in the urine were found to be 2.94 ± 1.70 mg and 1.17 ± 0.60 mg f o r the R ( - ) - and S(+)-enantiomers, r e s p e c t i v e l y . The two mean values were found to be s i g n i f i c a n t d i f f e r e n t (p<0.001). This i n d i c a t e d that hydroxylation of the x y l i d i d e methyl group was mediated by s t e r e o s e l e c t i v e mechanisms. Representative p l o t s of the amounts of OH-methyl-mexiletine metabolite enantiomers remaining to be excreted in the urine from one subject were found t o f o l l o w l o g - l i n e a r d e c l i n e over 72 hours as shown in Figure 49. The mean terminal d i s p o s i t i o n r a t e constants of 0.0605 ± 0.0084 h ~ * , and 0.0690 ± 0.0094 h " 1 , derived from the OH-methyl metabolite data f o r R ( - ) - and S ( + ) - m e x i l e t i n e , r e s p e c t i v e l y , were found to be s i g n i f i c a n t l y cn CD c c • — T J CD -+-J CD i _ O X Lxl 6 0 0 0 n O R( - ) • S(+) O H - M e t h y l Mexi let ine 4 0 0 0 - o o o o o o o o c o E < CD > _o E o 2 0 0 0 - o o CP o o 0 0 10 Figure 48. 20 30 40 Time (h) 50 60 70 Representative plots of the cumulative amount of R(-and S(+)-hydroxymethyl mexiletine excreted over 72 hours in the urine from subject TK following oral administration of 200 mg of racemic mexiletine hydrochloride. CO 1E4-, 1000-100-10-1 O O R(- ) • S(+) O H - M e t h y l Mexi let ine O O o 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 Time (h) Figure 49, Representative plots of the amount of R(-)- and S(+)-hydroxymethyl mexiletine remaining to be excreted over 72 hours in the urine from subject TK following oral administration of 200 mg racemic mexiletine hydrochloride. CO 188 d i f f e r e n t (p<0.05). The mean terminal d i s p o s i t i o n h a l f - l i v e s of 11.73 ± 1.86 h and 10.27 ± 1.64 h, derived from OH-methyl-metabolite data f o r R ( - ) - and S ( + ) - m e x i l e t i n e , r e s p e c t i v e l y , were a l s o found to be s i g n i f i c a n t l y d i f f e r e n t (p<0.05) and l a r g e r than those derived from the m e x i l e t i n e u r i n a r y d a t a . The use of m e x i l e t i n e metabolite data t o d e r i v e d i s p o s i t i o n data f o r m e x i l e t i n e using the sigma-minus p l o t (Mayersohn and G i b a l d i , 1969) was based on the f o l l o w i n g schematic k i n e t i c model: kf k m u x ^ Mxg » Xxu Kmuy MyB • Xyu where D B represents the amount of drug in the body. Mx and My correspond to the amount of m e t a b o l i t e - x and m e t a b o l i t e - y i n the body, kf i s the formation r a t e constant f o r metabolite Mx; and k m u x and k m U y are the u r i n a r y e x c r e t i o n r a t e constants f o r m e t a b o l i t e - x and m e t a b o l i t e - y i n the u r i n e . In the present study, the sigma-minus p l o t (Mayersohn and G i b a l d i , 1969) followed the assumption of a consecutive model D B • Mx • Xu. However, f o r the p-OH and the OH-methyl-mexiletine m e t a b o l i t e s , phase-2 metabolic conjugation of the metabolites could be o c c u r r i n g , and thus the e l i m i n a t i o n k i n e t i c data f o r m e x i l e t i n e derived from m e x i l e t i n e metabolite u r i n e data would not be c o n s i s t e n t with those obtained d i r e c t l y from m e x i l e t i n e urine d a t a . Furthermore, since s i m i l a r cumulative amounts of the enantiomers of m e x i l e t i n e and of p-OH-m e x i l e t i n e were recovered from the u r i n e , a s i g n i f i c a n t l a r g e r amount of S(+)-OH-methyl-mexiletine recovered from the urine suggested that e i t h e r s t e r e o s e l e c t i v e conjugation of R(-)-OH-methyl- m e x i l e t i n e or a second s t e r e o s e l e c t i v e d i s p o s i t i o n pathway f a v o r i n g R ( - ) - m e x i l e t i n e may be present. 190 SUMMARY OF RESULTS A s t e r e o s e l e c t i v e high-performance l i q u i d chromatographic assay was developed f o r the determination of m e x i l e t i n e enantiomers in serum, serum u l t r a f i l t r a t e , s a l i v a , red blood c e l l s and u r i n e . The method involved the synthesis of 2-anthroyl c h l o r i d e , a h i g h l y s e n s i t i v e f luorescence d e r i v a t i z a t i o n reagent f o r 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 , followed by the r e s o l u t i o n of m e x i l e t i n e enantiomers as t h e i r N-anthroyl d e r i v a t i v e s on the P i r k l e R phenylglycine c h i r a l HPLC column. Fluorescence d e t e c t i o n of m e x i l e t i n e enantiomers over the concentration ranges studied was found to provide l i n e a r chromatographic peak height measurements of m e x i l e t i n e in serum, s a l i v a , red blood c e l l s and urine with a lower d e t e c t i o n l i m i t of 0 . 5 ng/ml in serum. In vitro studies on the serum p r o t e i n binding of m e x i l e t i n e enantiomers using u l t r a f i l t r a t i o n and/or e q u i l i b r i u m d i a l y s i s techniques provided the f o l l o w i n g f i n d i n g s : a . Serum c o l l e c t i o n using the Becton Dickinson p l a i n VacutainerR, with s i l i c o n coated stopper, through an i n d w e l l i n g B u t t e r f l y ^ cannula, as well as storage of the serum samples at -20 °C f o r 72 hours, were found not to a f f e c t the serum p r o t e i n binding of m e x i l e t i n e enantiomers. b. The e q u i l i b r i u m d i a l y s i s of m e x i l e t i n e in serum against i s o t o n i c phosphate b u f f e r or against serum u l t r a f i l t r a t e was found to be completed from two to three hours. c . The determination of m e x i l e t i n e serum f r e e drug concentrations using the C e n t r i f r e e ^ M i c r o p a r t i t i o n system by c e n t r i f u g a t i o n was found to produce serum f r e e f r a c t i o n s and serum R(-)/S(+) m e x i l e t i n e r a t i o s comparable to those obtained from e q u i l i b r i u m d i a l y s i s of m e x i l e t i n e in serum against i s o t o n i c phosphate b u f f e r . A l s o , the serum p r o t e i n binding of m e x i l e t i n e enantiomers was s i m i l a r at 22 °C or at 37 ° C . d . The serum p r o t e i n binding of m e x i l e t i n e enantiomers was found to be dependent on serum pH. Over the serum pH range from 6.3 to 9 . 4 , the serum p r o t e i n binding of m e x i l e t i n e was found to increase from «30% to «80%. Within t h i s pH range s t u d i e d , the serum f r e e m e x i l e t i n e concentration r a t i o s were found to increase from 0.7 to 1 . 0 . To maintain serum pH at 7.4 during u l t r a f i l t r a t i o n or e q u i l i b r i u m d i a l y s i s , the a d d i t i o n of 0.2 M equivalence of sodium phosphate b u f f e r s a l t s to serum was necessary. The adjustment of serum pH with phosphate b u f f e r s a l t s was found not to a f f e c t the serum p r o t e i n binding of m e x i l e t i n e enantiomers. e. The in vitro serum p r o t e i n binding of m e x i l e t i n e enantiomers was constant over the therapeutic serum concentration range of 0.25 to 3.0 /*g/ml with a mean serum f r e e f r a c t i o n of 0.52 ± 0.01 f o r R ( - ) - m e x i l e t i n e and 0.45 ± 0.01 f o r S(+)-m e x i l e t i n e . In vitro s t u d i e s of the d i s t r i b u t i o n of m e x i l e t i n e enantiomers i n t o red blood c e l l s i n d i c a t e d a moderate rate and extent of e q u i l i b r a t i o n f o r the two enantiomers, with d i s t r i b u t i o n e q u i l i b r i u m achieved w i t h i n 80 minutes of exposure to drug. This moderate r a t e of e q u i l i b r a t i o n of m e x i l e t i n e across the red blood c e l l membrane suggested that the r a p i d red blood c e l l sample c o l l e c t i o n protocol employed in the present study would not cause s i g n i f i c a n t r e d i s t r i b u t i o n of m e x i l e t i n e from the red blood c e l l s . Following o r a l a d m i n i s t r a t i o n of 200 mg racemic m e x i l e t i n e hydrochloride to 12 healthy s u b j e c t s , the d i s p o s i t i o n k i n e t i c s of m e x i l e t i n e enantiomers in the serum were best described by a one-compartment open model in four s u b j e c t s , while in the remaining eight s u b j e c t s , the d i s p o s i t i o n k i n e t i c s of m e x i l e t i n e enantiomers were best described by a two-compartment open model. The pharmacokinetic parameters derived using the serum t o t a l drug concentration data from the twelve subjects are as f o l l o w s : a . The peak serum t o t a l m e x i l e t i n e concentrations (C^gx) were found to range from 141 to 357 ng/ml f o r R ( - ) - m e x i l e t i n e , and from 133 to 310 ng/ml f o r S ( + ) - m e x i l e t i n e , with a mean of 217 ± 68 ng/ml and 196 ± 56 ng/ml f o r R ( - ) - and S ( + ) - m e x i l e t i n e , r e s p e c t i v e l y . The mean ( C m a x ) f o r 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 greater (p<0.01) than that of the S(+)-enantiomer. b. The mean serum t o t a l concentration of 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 greater (p<0.01) than that of S(+)-mexiletine in the f i r s t s i x 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 . However, the d i f f e r e n c e s were found not to be s i g n i f i c a n t past the s i x t h hours. c . The time to achieve peak serum t o t a l concentration ( t m a x ) was found to range from 0.6 to 4.1 hours f o r R ( - ) - m e x i l e t i n e , while a range from 0.7 to 4 . 5 was observed f o r S(+)-m e x i l e t i n e . The mean t m a x of 2.1 ± 1.1 h and 2.3 ± 1.4 h, f o r R ( - ) - and S(+)-mexiletine r e s p e c t i v e l y , were not 193 s i g n i f i c a n t l y d i f f e r e n t . d . The mean AUC of 2.8 ± 0.9 /zg/ml/h f o r R ( - ) - m e x i l e t i n e was found to be s i g n i f i c a n t l y greater (p<0.05) than the mean of 2.6 ± 0.9 /zg/ml/h f o r S(+)-mexiletine. This s i g n i f i c a n t l y greater AUC f o r R ( - ) - m e x i l e t i n e was c o n s i s t e n t with the greater i n i t i a l serum t o t a l drug concentrations and the greater C m a x observed f o r R ( - ) - m e x i l e t i n e . e. The o r a l absorption of m e x i l e t i n e was found to be v a r i a b l e in the f a s t i n g s u b j e c t s . The mean o r a l absorption h a l f - l i v e s of 0 . 6 ± 0.4 h and 0.7 ± 0 . 5 h, f o r the r e s p e c t i v e R ( - ) - and S ( + ) - m e x i l e t i n e , were not s i g n i f i c a n t l y d i f f e r e n t . f . The mean d i s t r i b u t i o n h a l f - l i v e s of m e x i l e t i n e in eight subjects were found to be 4 . 9 ± 3.4 h and 4 . 3 ± 2.9 h f o r R ( - ) - and S ( + ) - m e x i l e t i n e , r e s p e c t i v e l y . The values were not s i g n i f i c a n t l y d i f f e r e n t . g . The terminal h a l f - l i v e s of m e x i l e t i n e from the twelve subjects were v a r i a b l e , ranging from 5.0 to 11.3 hours f o r R ( - ) - m e x i l e t i n e , and from 3.6 to 12.6 hours f o r S(+)-m e x i l e t i n e . The mean h a l f - l i v e s of 8.1 ± 2.2 h and 8.4 ± 2.6 h , f o r R ( - ) - and S ( + ) - m e x i l e t i n e , r e s p e c t i v e l y , were not s i g n i f i c a n t l y d i f f e r e n t . h. The mean apparent o r a l clearance values (CL/F) from the twelve subjects were 15.9 ± 5.8 ml/min/kg f o r R ( - ) - m e x i l e t i n e and 17.4 ± 6.2 ml/min/kg f o r S ( + ) - m e x i l e t i n e . The two values were not s i g n i f i c a n t l y d i f f e r e n t . i . The mean volumes of d i s t r i b u t i o n (Vp,) of 10.6 ± 3 . 5 1/kg and 11.9 ± 4.4 1/kg, f o r R ( - ) - and S ( + ) - m e x i l e t i n e , r e s p e c t i v e l y , were not s i g n i f i c a n t l y d i f f e r e n t , j . The mean percent of unchanged drug recovered from the urine over 72 hours was found to be 3 . 5 ± 3.4% f o r R ( - ) - m e x i l e t i n e and 3.7 ± 3.9% f o r S ( + ) - m e x i l e t i n e , suggesting that the e l i m i n a t i o n of m e x i l e t i n e enantiomers from the body was predominantly by non-renal mechanisms. The r e c o v e r i e s of the two enantiomers from the urine were not s i g n i f i c a n t l y d i f f e r e n t . k. The mean renal clearance of 0.7 ± 0 . 8 ml/min/kg f o r S(+)-m e x i l e t i n e was not s i g n i f i c a n t l y d i f f e r e n t from the mean value of 0.6 + 0 . 8 ml/min/kg f o r R ( - ) - m e x i l e t i n e . 1. The mean terminal h a l f - l i v e s of 5.8 ± 1.5 h and 5.6 ± 1.4 h, derived from urine data f o r R ( - ) - and S ( + ) - m e x i l e t i n e , r e s p e c t i v e l y , were not s i g n i f i c a n t l y d i f f e r e n t . The o v e r a l l d i s p o s i t i o n k i n e t i c s f o r m e x i l e t i n e enantiomers in the twelve subjects were found to be s i m i l a r . However, the pharmacokinetic parameters, derived from serum t o t a l and serum f r e e drug d a t a , were observed with high i n t e r - s u b j e c t v a r i a t i o n s . The d i s p o s i t i o n of m e x i l e t i n e enantiomers in the body was c h a r a c t e r i z e d by t h e i r extensive t i s s u e d i s t r i b u t i o n and extensive non-renal e l i m i n a t i o n . The in vivo serum p r o t e i n binding of m e x i l e t i n e enantiomers was found to be v a r i a b l e among the twelve subjects with the f o l l o w i n g summary of d a t a : a . The mean serum f r e e f r a c t i o n s of 0.57 ± 0.07 and 0.56 ± 0 . 0 6 , f o r R ( - ) - and S ( + ) - m e x i l e t i n e , r e s p e c t i v e l y , were not s i g n i f i c a n t l y d i f f e r e n t . b. The serum t o t a l and serum f r e e m e x i l e t i n e R(-)/S(+) r a t i o s of 1.04 ± 0.06 and 1.09 ± 0 . 0 6 , r e s p e c t i v e l y , were a l s o found not to be s i g n i f i c a n t l y d i f f e r e n t . These s i m i l a r serum f r e e f r a c t i o n s and serum f r e e R(-)/S(+) r a t i o s f o r the two m e x i l e t i n e enantiomers i n d i c a t e d that m e x i l e t i n e was only moderately bound to serum p r o t e i n s , and the in vivo serum p r o t e i n binding of m e x i l e t i n e was not s t e r e o s e l e c t i v e . The moderate serum p r o t e i n binding of m e x i l e t i n e with respect to i t s extensive t i s s u e d i s t r i b u t i o n suggested that drug i n t e r a c t i o n by binding displacement from serum p r o t e i n s would not l i k e l y be of c l i n i c a l s i g n i f i c a n c e . The d i s p o s i t i o n of m e x i l e t i n e in s a l i v a was found to be described by a two-compartment open k i n e t i c model. Pharmacokinetic parameters f o r m e x i l e t i n e enantiomers were derived from s a l i v a drug data and are as f o l l o w s : a . The mean terminal h a l f - l i v e of 6.1 ± 3.1 h f o r R ( - ) -m e x i l e t i n e in s a l i v a was found to be s i g n i f i c a n t l y lower (p<0.01) than a mean value of 7.5 ± 3.8 h f o r S(+)-m e x i l e t i n e . b. A s i g n i f i c a n t l y greater (p<0.01) s a l i v a AUC value f o r S(+)-m e x i l e t i n e was observed compared to that f o r R ( - ) - m e x i l e t i n e , i n d i c a t i n g that a greater amount of S(+)-mexiletine was excreted into s a l i v a . c . The o v e r a l l mean s a l i v a / serum f r e e m e x i l e t i n e AUC r a t i o of 6.1 ± 2.8 f o r R ( - ) - m e x i l e t i n e was found to be s i g n i f i c a n t l y lower (p<0.01) than that of 7.4 ± 3.4 f o r S(+)-mexiletine. The greater s a l i v a / serum f r e e area under concentration-time curve r a t i o s f o r S(+)-mexiletine r e l a t i v e to R ( - ) - m e x i l e t i n e was c o n s i s t e n t with the suggestion of a higher rate of e x c r e t i o n of S(+)-mexiletine into s a l i v a , d . The s a l i v a R ( - ) - and S(+)-mexiletine concentrations and the r e s p e c t i v e s a l i v a pH were used to c a l c u l a t e the corresponding serum f r e e drug concentrations using the Matin equation (Matin et al., 1974). The c a l c u l a t e d values were found to c o r r e l a t e poorly with the r e s p e c t i v e observed serum f r e e drug concentrations with c o e f f i c i e n t s of determination ( r 2 ) of 0.677 and 0.648 f o r R ( - ) - and S(+)-mexiletine, r e s p e c t i v e l y . The d i s p o s i t i o n of m e x i l e t i n e enantiomers in s a l i v a was found to be s t e r e o s e l e c t i v e . The terminal h a l f - l i f e , s a l i v a AUC, and s a l i v a / serum f r e e AUC r a t i o f o r S(+)-mexiletine were c o n s i s t e n t l y greater than the r e s p e c t i v e values f o r R ( - ) - m e x i l e t i n e . The use of s a l i v a m e x i l e t i n e concentrations f o r estimating serum f r e e m e x i l e t i n e concentrations was found to be u n r e l i a b l e . The d i s p o s i t i o n of m e x i l e t i n e concentrations in the red blood c e l l s of s i x subjects was found to f o l l o w the k i n e t i c s of a one-compartment open model. Pharmacokinetic parameters f o r m e x i l e t i n e enantiomers were derived from red blood c e l l drug concentration data and are as f o l l o w s : a . The mean terminal h a l f - l i v e s of 15.1 ± 9.3 h and 16.9 ± 8 . 7 h, f o r R ( - ) - and S(+)-mexiletine r e s p e c t i v e l y , were not s i g n i f i c a n t l y d i f f e r e n t . b. The mean red blood c e l l m e x i l e t i n e AUC value of 2.3 ± 1.5 //g/ml/h and 2.8 ± 2.1 pg/ml/h, f o r R ( - ) - and S ( + ) - m e x i l e t i n e , r e s p e c t i v e l y , were a l s o not s i g n i f i c a n t l y d i f f e r e n t . 197 c . The o v e r a l l mean red blood c e l l m e x i l e t i n e R(-)/S(+) r a t i o s was found to be 0.91 ± 0.13 over 48 hours. d . The o v e r a l l mean m e x i l e t i n e red c e l l / f r e e serum concentration r a t i o s of 0.85 ± 0.06 and 0.84 ± 0.08 f o r R ( - ) -and S ( + ) - m e x i l e t i n e , r e s p e c t i v e l y , were not s i g n i f i c a n t l y d i f f e r e n t . From these f i n d i n g s , the d i s t r i b u t i o n of m e x i l e t i n e into red blood c e l l s was suggested to f o l l o w a slow passive d i f f u s i o n . Since serum f r e e m e x i l e t i n e was a v a i l a b l e f o r d i f f u s i o n into red blood c e l l s , the n o n - 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 of m e x i l e t i n e enantiomers into the red blood c e l l s was c o n s i s t e n t with the s i m i l a r serum f r e e f r a c t i o n s observed f o r m e x i l e t i n e enantiomers. A s t e r e o s e l e c t i v e HPLC assay was developed f o r the simultaneous determination of m e x i l e t i n e , p-OH-mexiletine and OH-methyl-mexiletine enantiomers i n the u r i n e . The m e x i l e t i n e and OH-methyl-mexiletine enantiomers were resolved on a P i r k l e R i s o l e u c i n e covalent HPLC column as t h e i r N-anthroyl d e r i v a t i v e s , w h i l e the p-OH-mexiletine enantiomers were resolved as t h e i r 0 - e t h y l - N - a n t h r o y l d e r i v a t i v e s . P o l a r i m e t r i c measurements of ([a] - 3 . 7 ° ) and ([a] + 2 . 9 ° ) were obtained using pure samples of R ( - ) - and S(+)-N-anthroyl m e x i l e t i n e d e r i v a t i v e , r e s p e c t i v e l y . These p o l a r i m e t r i c r e s u l t s were found to c o r r e l a t e with the f i r s t chromatographic e l u t i o n of the S(+)-enantiomer followed by the R(-)-enantiomer f o r m e x i l e t i n e and p-OH-mexiletine. However, the order of e l u t i o n of the OH-methyl-mexiletine enantiomers was reversed with f i r s t e l u t i o n of the R(-)-enantiomer followed by the S(+)-enantiomer. The fluorescence d e t e c t i o n and chromatographic peak height measurements of m e x i l e t i n e , p-OH-mexiletine and OH-methyl- m e x i l e t i n e enantiomers were found to be l i n e a r over the concentration ranges s t u d i e d , with a lower d e t e c t i o n l i m i t of 0 . 5 ng/ml f o r m e x i l e t i n e in u r i n e . The u r i n a r y d i s p o s i t i o n of the enantiomers of p-OH-mexiletine and OH-methyl-mexiletine was studied in four healthy subjects f o l l o w i n g o r a l a d m i n i s t r a t i o n of 200 mg racemic m e x i l e t i n e h y d r o c h l o r i d e . The u r i n a r y k i n e t i c data f o r the two m e x i l e t i n e metabolites are summarized in the f o l l o w i n g : a . The mean cumulative amounts of p-OH-mexiletine enantiomers recovered from the urine over 72 hours were found to be 1.31 ± 0.33 mg and 1.27 ± 0.39 mg, f o r the R ( - ) - and S(+)-enantiomer, r e s p e c t i v e l y . The two values were not s i g n i f i c a n t l y d i f f e r e n t . b. The mean cumulative amounts of OH-methyl-mexiletine enantiomers recovered from the urine over 72 hours were found to be 2.94 ± 1.70 mg and 1.17 + 0.60 mg, f o r the r e s p e c t i v e R ( - ) - and S(+)-enantiomers. The amount of R(-)-OH-methyl-m e x i l e t i n e recovered from the urine was found to be s i g n i f i c a n t l y greater (p<0.01) than that obtained f o r the S(+)-enantiomer. Since there was no d i f f e r e n c e in the cumulative amounts excreted between the two enantiomers f o r both m e x i l e t i n e and p-OH-mexiletine, a s i g n i f i c a n t greater amount of R(-)-OH-methyl-mexiletine recovered from the u r i n e suggested the presence of a s t e r e o s e l e c t i v e metabolic pathway f o r S(+)-mexiletine or S(+)-OH-methyl-mexiletine. 1 9 9 CONCLUSIONS A s t e r e o s e l e c t i v e high-performance l i q u i d chromatographic assay was developed f o r the determination of m e x i l e t i n e enantiomers. In vitro studies revealed that the serum p r o t e i n binding of m e x i l e t i n e enantiomers was s t e r e o s e l e c t i v e l y dependent on serum pH. Following o r a l a d m i n i s t r a t i o n of 200 mg racemic m e x i l e t i n e hydrochloride to twelve s u b j e c t s , the o r a l a b s o r p t i o n , d i s t r i b u t i o n , e l i m i n a t i o n and u r i n a r y r e c o v e r i e s of 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 , suggesting an o v e r a l l n o n - s t e r e o s e l e c t i v e d i s p o s i t i o n of m e x i l e t i n e enantiomers. The d i s p o s i t i o n of m e x i l e t i n e enantiomers in s a l i v a was s t e r e o s e l e c t i v e . The s a l i v a / serum f r e e AUC r a t i o s f o r S(+)-mexiletine was found to be greater than that f o r R ( - ) - m e x i l e t i n e . However, the s a l i v a m e x i l e t i n e concentrations were found to c o r r e l a t e poorly with serum f r e e drug c o n c e n t r a t i o n s . The d i s t r i b u t i o n of m e x i l e t i n e enantiomers into red blood c e l l s was n o n - s t e r e o s e l e c t i v e , and was found to be l a r g e l y dependent on serum f r e e drug c o n c e n t r a t i o n s . A s t e r e o s e l e c t i v e HPLC assay was developed f o r the simultaneous determination of m e x i l e t i n e , p-OH-mexiletine, and OH-methyl-mexiletine enantiomers. The mean cumulative amounts of p-OH-mexiletine enantiomers recovered from the urine of four subjects were not s i g n i f i c a n t l y d i f f e r e n t . However, the recovery of R(-)-OH-methyl-mexiletine was s i g n i f i c a n t l y (p<0.01) greater than that of the S(+)-enantiomer, suggesting a s t e r e o s e l e c t i v e metabolic pathway f o r R(-)-OH-methyl-m e x i l e t i n e or the presence of a s t e r e o s e l e c t i v e phase II metabolic pathway f o r the S(+)-OH-methyl-mexiletine m e t a b o l i t e . 200 REFERENCES Akhtar M. P r a c t i c a l c o n s i d e r a t i o n s in the treatment of v e n t r i c u l a r arrhythmias with m e x i l e t i n e . Am Heart J , 107:1086 (1984) A l l a f D, P i r o t t e J and C a r l i e r J . The influence of c a r d i a c f u n c t i o n compensation on the biotransformation of drugs. Rev Med L i e g e , 15(37):197 (1982) A l l e n JD, Kofi-Ekue JM, Shanks RG and Z a i d i SA. The e f f e c t of Ko-1173, a new anticonvulsant agent on experimental c a r d i a c arrhythmias. Br J Pharmacol, 45:561 (1972) Allenmark S. In: K r s t u l o v i c AM. ( e d . ) : "Protein Bound Phases". E l l i s Horwood L t d . , New York, pp.286 (1989) Allenmark S, Andersson S and B o j a r s k i J . D i r e c t l i q u i d chromatographic separation of enantiomers on immobilized p r o t e i n s t a t i o n a r y phases: V I . Optical r e s o l u t i o n of a s e r i e s of racemic b a r b i t u r a t e s : Studies of s u b s t i t u t e n t and mobile-phase e f f e c t s . J Chromatogr, 436:479 (1988). Allenmark S, Bomgren B and Boren B. D i r e c t l i q u i d chromatographic seperation of enantiomers on immobilzed p r o t e i n s t a t i o n a r y phases I I I . O p t i c a l r e s o l u t i o n of a s e r i e s of N-aroyl-D,L-amino acids by HPLC on bovine serum albumin c o v a l e n t l y bound to s i l i c a . J Chromatogr, 264:63 (1983). Armstrong DW and DeMond W. C y c l o d e x t r i n bonded phases f o r the l i q u i d chromatographic separation of o p t i c a l , g e o m e t r i c a l , and s t r u c t u r a l isomers. J Chromatogr S c i , 22:411 (1984). Armstrong DW, Han SM and Han Y I . Separation of o p t i c a l isomers of scopolamine, cocaine and homatropine. Anal Biochem, 167:261 (1987) Armstrong DW, Ward TJ , Armstrong RD and Beesley TE. Separation of drug stereoisomers by the formation of b e t a - c y c l o d e x t r i n i n c l u s i o n complexes. S c i e n c e , 232:1132 (1986). Baumann E. Reactions of a c i d c h l o r i d e s . Ber, 3218 (1886) Beemann B, Hellstrom K and Lindstrom B. B i n d i n g - s i t e i n t e r a c t i o n of c h l o r t h a l i d o n e and acetazolamide, two drugs transported by red blood c e l l s . C l i n Pharmacol Ther, 17:424 (1974) Beckett AH and Chidomere. The d i s t r i b u t i o n , metabolism and e x c r e t i o n of m e x i l e t i n e in man. Postgrad Med J , 53(Suppl. 1):60 (1977) Beff EJ , Chinwah PM, Webb C, Day RO and Wade DN. Enhanced metabolism of m e x i l e t i n e a f t e r phenytoin a d m i n i s t r a t i o n . Br J C l i n Pharmacol, 14:219 (1982) Behm HL and Wagner JG. Free f r a c t i o n change with d i f f e r i n g volume r a t i o s in e q u i l i b r i u m d i a l y s i s : case of n o n - l i n e a r drug p r o t e i n b i n d i n g . J Pharm Pharmacol, 33:283 (1980) Bhamra RK, Flanagan RJ and Holt DW. High-performance l i q u i d chromatographic method f o r the measurement of m e x i l e t i n e and f l e c a i n i d e in b l o o d , plasma or u r i n e . J Chromatogr, 307:439 (1984) Bigger JT. The i n t e r a c t i o n of m e x i l e t i n e with other c a r d i o v a s c u l a r drugs. Am Heart J , 107:1079 (1984) Blaschke G. Chromatographic r e s o l u t i o n of c h i r a l drugs on polyamides and c e l l u l o s e t r i a c e t a t e . J L i q Chromatogr, 9:341 (1986) Blaschke G. Chromatographic r e s o l u t i o n of racemates. Angew Chem I n t , ( E n g l . ) 19:13 (1980) Block PJ and Winkle RA. Heamodynamic e f f e c t s of antiarrhythmic drugs. Am J C a r d i o l , 52:14c (1983) Boehringer Ingelheim (Canada) L t d . Product monograph on m e x i l e t i n e , M e x i t i l R . (1986) Bowers WF, Fulton S and Thompson J . U l t r a f i l t r a t i o n vs e q u i l i b r i u m d i a l y s i s f o r determination of f r e e f r a c t i o n . C l i n Pharmacokinet, 9(suppl 1):49 (1984) Bradbook ID, F e l d s c h r e i b e r P, Morrison P J , Rogers HJ and Spector RG. Plasma m e x i l e t i n e concentration f o l l o w i n g combined o r a l and intravenous a d m i n i s t r a t i o n . Eur J C l i n Pharmacol, 19:301 (1981) B r e i t h a r d t G, Seipel L and Abendroth RR. K l i n i s c h - e l e k t r o p h y s i o l o g i s c h e Untersuchung. Zur Z K a r d i o l , 70(7):530 (Eng Abstr) (1981) Brockmeyer NH, Breithaupt H, Ferdinand W, Von Hattingberg M and Ohnhaus EE. K i n e t i c s of o r a l and intravenous m e x i l e t i n e : Lack of e f f e c t of c i m e t i d i n e and r a n i t i d i n e . Eur J C l i n Pharmacol, 36:375 (1989) Brors 0 , N i l sen 0G, Sager G, Sandnes S and Jacobsen S. Influence of pH and b u f f e r type on drug binding in human serum. C l i n Pharmacokinet, 9(suppl 1):84 (1984) Brown JE, K i t c h e l l BB, Bjornsson TD and Shand DG. The a r t i f a c t u a l nature of heparin-induced drug p r o t e i n - b i n d i n g a l t e r a t i o n s . C l i n Pharmacol Ther, 30(5):636 (1981) Campbell NPS, K e l l y JG, Adgey AAJ and Shanks RG. M e x i l e t i n e in normal V o l u n t e e r s . Br J C l i n Pharmacol, 6:103 (1978) Cetnarowski AB and Rihn TL. A review of adverse r e a c t i o n s to m e x i l e t i n e and t o c a i n i d e . Cardiovas Rev Rep, 6:1335 (1985) 202 C h a t t e r j i e N, Fujimoto JM, I n t u r r i s i CE, Roerig S, Wang RIH, Bowen DV, F i e l d FH and Clarke DD. I s o l a t i o n and stereochemical i d e n t i f i c a t i o n of a metabolite of naltrexone from human u r i n e . Drug Metab Dispos, 2:401 (1974) Chew CYC, C o l l e t t J and Singh BN. M e x i l e t i n e : A review of i t s pharmacological p r o p e r t i e s and therapeutic e f f i c a c y in arrhythmias. Drugs, 17:161 (1979) C h i g n e l l CF. Physical methods for studying durg-protein binding. In Brodie and G i l l e t e ( e d s . ) Handbook of experimental pharmacology. Vol 28, pp.187, S p r i n g e r - V e r l a g , B e r l i n , (1971) Cotham RH and Shand D. Spuriously low plasma propranolol concentrations r e s u l t i n g from blood c o l l e c t i o n methods. C l i n Pharmacol Ther, 18(5):535 (1975) Danneberd PB and S h e l l e y JH. The pharmacology of m e x i l e t i n e . Postgrad Med J , 53:25 (1977) Davankov VA. In: K r s t u l o v i c AM. ( e d . ) : "Ligand-exchange phases". E l l i s Horwood L t d . , New York, pp.446 (1989). Duff HJ. M e x i l e t i n e in the treatment of r e s i s t a n t v e n t r i c u l a r arrhythmias: enhancement of e f f i c a c y and reduction of d o s e - r e l a t e d s i d e e f f e c t s by combination with q u i n i d i n e . C i r c u l a t i o n , 63:1124 (1983) Edwards DJ, Axelson JE , Slaughter RL, E l v i n AT and Lalka D. Factors a f f e c t i n g q u i n i d i n e p r o t e i n binding in humans. J Pharm S c i , 73(9):1264 (1984) El A l l a f D, Henrard L, Crochelet L, D e l a p i e r r e D, C a r l i e r J and Dresse A. Pharmacokinetics of m e x i l e t i n e in renal i n s u f f i c i e n c y . Br J C l i n Pharmacol, 14:431 (1982). Fenster P. M e x i l e t i n e and q u i n i d i n e in v e n t r i c u l a r ectopy. C l i n Pharmacol Ther, 34:136 (1983) Frank H, Nicholson GJ and Bayer E. Gas Chromatog. - Mass spectrometric a n a l y s i s of o p t i c a l l y a c t i v e metabolites and drugs on a noval c h i r a l s t a t i o n a r y phase. J Chromatogr, 146:197 (1978). F r o e h l i c h P. Fluorescence of o r a g i n i c compounds. Am Laboratory, March:98 (1985) Gal J . Stereochemistry of metabolism of amphetamines. Use of (-)-gama-methoxy-gama-(trif1uoromethyl)phenylacetyl c h l o r i d e f o r GLC r e s o l u t i o n of c h i r a l amines. J Pharm S c i , 66:169 (1977) Gal J , Devito D and Harper TW. Gas-chromatographic r e s o l u t i o n of enantiomeric secondary a l c o h o l s . S t e r e o s e l e c t i v e reductive metabolism of ketones in r a b b i t - l i v e r c y t o s o l . Drug Metab Dispos, 9:557 (1981) 203 Gorodischer R, Jusko WJ and Yaffe S J . Tissue and erythrocyte d i s t r i b u t i o n of d i g o x i n in i n f a n t s . C l i n Pharmacol Ther, 19(3):256 (1976) Grech-Belanger 0 and Turgeon J . High-pressure l i q u i d chromatographic assay f o r m e x i l e t i n e in serum. J Chromatogr S c i , 22:490 (1984) Grech-Belanger 0 and Turgeon J . High-performance l i q u i d chromatographic assay f o r m e x i l e t i n e enantiomers human plasma. J Chromatogr, 337:172 (1985a). Grech-Belanger 0 , G i l b e r t M, Turgeon J and LeBlanc PP. E f f e c t of c i g a r e t t e smoking on m e x i l e t i n e k i n e t i c s . 37(6):638 (1985b) Grech-Belanger 0 , Turgeon J and G i l b e r t M. S t e r e o s e l e c t i v e d i s p o s i t i o n of m e x i l e t i n e in man. Br J C l i n Pharmacol, 21:481 (1986) Grech-Belanger 0 , Turgeon J and Lalande M. 2,6-Dimethylphenol: a new metabolite of m e x i l e t i n e . Res Commum Chem Path Pharmacol, 58(1):53 (1987) Grech-Belanger 0 , Turgeon J and Lalande M. The s t e r e o s e l e c t i v e metabolism of m e x i l e t i n e . Abstract in press (1988) Grech-Belanger 0 , Barbeau G, Kishka P, F i s e t C, Leboeuf E and Blouin M. Pharmacokinetics of m e x i l e t i n e in the e l d e r l y . J C l i n Pharmacol, 29:311 (1989) Greenspan A. E f f i c a c y of combination therapy with m e x i l e t i n e and a t y p e - l a agent f o r i n d u c i b l e v e n t r i c u l a r t a c h y c a r d i a secondary to coronary a r t e r y d i s e a s e . Am J C a r d i o l , 56:277 (1985) Han SM and Armstrong DW. In: K r s t u l o v i c AM ( e d . ) : "HPLC separation of enantiomers and other isomers with cyclodextrin-bonded phases: rules for chiral recognition". E l l i s Horwood L t d . , New York, pp.209 (1989) Haselbarth V, Doevendans JE and Wolf M. K i n e t i c s and b i o a v a i l a b i l i t y of m e x i l e t i n e in healthy s u b j e c t s . C l i n Pharmacol Ther, 29(6):729 (1981) Henry JA, Dunlop AW, Turner P, M i t c h e l l SN and Adams P. A model f o r the pH-dependence of d r u g - p r o t e i n b i n d i n g . J Pharm Pharmacol, 33:183 (1980) Henry JA and M i t c h e l l SN. E f f e c t of pH on human plasma p r o t e i n binding of a s e r i e s of beta-adrenoceptor a n t a g o n i s t s . Proceedings of the b . p . s . 10-12th p p . H 9 p (1980) Hermansson J . Resolution of racemic aminoalcohols ( b e t a - b l o c k e r s ) , amines and acids as enantiomeric d e r i v a t i v e s using a c h i r a l a j - a c i d g l y c o p r o t e i n column. J Chromatogr, 325:3779 (1985). Hermansson J . D i r e c t l i q u i d chromatographic r e s o l u t i o n of racemic drugs using o q - a c i d g l y c o p r o t e i n as the c h i r a l s t a t i o n a r y phase. J Chromatogr, 269:71 (1983) Hesse G and Hagel R. I n c l u s i o n chromatography and a new r e t e n t i o n mechanism f o r benzene d e r i v a t i v e s . Chromatographia, 9:62 (1976). Hesse G, Hagel R and Eine R. V o l l s t a n d i g e racemattrennung durch elutions-chromatographie an c e l l u l o s e - t r i - a c e t a t . Chromatographia, 6:277 (1973) Hinze WL, Riehl TE, Armstrong DW, DeMond W, Alak A and Ward T. L i q u i d chromatographic separation of enantiomers using a c h i r a l penta-cyclodextrin-bonded s t a t i o n a r y phases and conventional aqueous-organic mobile phases. Anal Chem, 57:237 (1985) Igwemwzie L, Kerr CR and McErlane KM. The Pharmcokinetics of m e x i l e t i n e enantiomers in human s u b j e c t s . X e n o b i o t i c a , 19:677 (1989) Igwemezie L. Ph.D. Thesis : 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 m e x i l e t i n e enantiomers. The U n i v e r s i t y of B r i t i s h Columbia, (1989). Jamali F. Research methodology in NSAID monitoring: plasma concentrations of c h i r a l drugs. J Rheumatol Suppl, 17:71 (1988) Joeres R, K l i n k e r H, Heusler H, Epping J and R i c h t e r E. Influence of m e x i l e t i n e on c a f f e i n e e l i m i n a t i o n . Pharmacol Ther, 33:163 (1987) Johnson DM, Reuter A, C o l l i n s JM and Thompson GF. Enantiomeric p u r i t y of naproxen by l i q u i d chromatographic a n a l y s i s of i t s diastereomeric o c t y l e s t e r . J Pharm S c i , 68:112 (1979). Johnston A, Burgess CD, Warrington S J , Wadsworth J and Hamer NAJ. The e f f e c t of spontaneous changes i n u r i n a r y pH on m e x i l e t i n e plasma concentrations and e x c r e t i o n during chronic adminstration to healthy v o l u n t e e r s . Br J C l i n Pharmacol, 8:349 (1979) Katz A, B u s k i l a D and Sukenik S. Oral m e x i l e t i n e - t h e o p h y l l i n e i n t e r a c t i o n . Inter J C a r d i o l , 17:227 (1987) Kaye CM, Turner P and K i d d i e MA. V a r i a b l e pharmacokinetics of m e x i l e t i n e . Postgrad Med J , 53(suppl. 1):56 (1977). K e l l y R, Christmore D, Smith R, Doshier L and Jacobs SL. A n a l y s i s of m e x i l e t i n e in plasma by high-pressure l i q u i d chromatography. Therap Drug Monit , 3(3):279 (1981) K e l l y JG, Nimrno J , Rae R, Shanks RG and Prescott LF. Spectrophotofluorometric and g a s - l i q u i d chromatographic methods f o r the estimation of m e x i l e t i n e (KOI173) in plasma and u r i n e . J Pharm Pharmacol, 25:550 (1973). Kiddie MA, Kaye CM and Turner P. The influence of u r i n a r y pH on the e l i m i n a t i o n of m e x i l e t i n e . Br J C l i n Pharmac, 1:229 (1974). K l e i n A, Sami M and S e l i n g e r K. M e x i l e t i n e k i n e t i c s in healthy subjects t a k i n g c i m e t i d i n e . C l i n Pharmacol Ther, 37:669 (1985) Kohyama J , Shimohira M, Watanabe S, Fukuda C and Iwakawa Y. M e x i l e t i n e hydrochloride in an i n f a n t with i n t r a c t a b l e e p i l e p s y . Brain Dev, 10:258 (1988) Konig WA, S t o l t i n g K and Kruse K. Gas chromatographic separations of o p t i c a l l y a c t i v e compounds in g l a s s c a p i l l a r i e s . Chromatographia 10:44 (1977) Koppe HG. The development of m e x i l e t i n e . Postgrad Med J , 53(suppl. 1):22 (1977) Koysooko R, E l l i s EF and Levy G. R e l a t i o n s h i p s between drug concentrations in plasma and s a l i v a of man. C l i n Pharmacol Ther, 15(5):454 (1974) Kristensen CB and Gram LF. E q u i l i b r i u m d i a l y s i s f o r determination of p r o t e i n binding of imipramine - Evaluation of a method. Pharmacol et T o x i c o l , 50:130 (1982) K r s t u l o v i c AM, Fouchet MH, Burke JT, G i l l e t G and Durand A. D i r e c t enantiomeric separation of betaxolol with a p p l i c a t i o n s to a n a l y s i s of bulk drug and b i o l o g i c a l samples. J Chromatogr, 28(452):477 (1989) Kurata D and Wilkinson GR. Erythrocyte uptake and plasma binding of diphenylhydantoin. C l i n Pharmacol Ther, 16(2):355 (1974) Kusters E and Giron D. Enantiomeric separation of the b e t a - b l o c k i n g drugs p i n d o l o l and bopindolol using a c h i r a l - i m m o b i l i z e d p r o t e i n s t a t i o n a r y phase. J High Resolut Chromatogr Chromatogr Commun, 9:531 (1986) Lai AA, Fleck RJ , Patzke JV, Glueck BG, Shaskan EG and Rosenberg B J . Influence of blood sampling methods on dopamine-receptor-blocking a c t i v i t i e s as determined by a r a d i o r e c e p t o r assay. Ther Drug Monitor, 4:89 (1982) Leahey EB, G i a r d i n a EGV and Bigger JT. E f f e c t of v e n t r i v u l a r f a i l u r e on steady s t a t e k i n e t i c s of m e x i l e t i n e . C l i n Res, 28:239A (1980). Lee EJD, Williams KM, Graham GG, Day RO and Champion GD. L i q u i d chromatographic determination and plasma concentration p r o f i l e of o p t i c a l isomers of ibuprofen i n humans. J Pharm S c i , 73(11):1542 (1984) Lee EJD, Williwms KM, Graham GG, Day R0, amd Champion D. S t e r e o s e l e c t i v e D i s p o s i t i o n of Ibuprotein Enantiomers in Man. Br J C l i n Pharmacol, 19:669(1985) 206 Levy G, Procknal JA, Olufs R and Pachman LM. R e l a t i o n s h i p between s a l i v a s a l i c y l a t e concentration and f r e e or t o t a l s a l i c y l a t e concentration in serum of c h i l d r e n wirh j u v e n i l e rheumatoid a r t h r i t i s . C l i n Pharmacol T h e r . , 27(5):619 (1980) Lewin S. ( e d . ) , "Displacement of water and its control of biochemical reactions". Academic P r e s s , New York, (1974). L i n JH, Cocchetto DM and Duggan DE. P r o t e i n binding as a primary determinant of the c l i n i c a l pharmacokinetic p r o p e r t i e s of non-s t e r o i d a l anti-inflammmatory drugs. C l i n pharmacokinet, 12:402 (1987) Lindna WF. In: F r e i RW and Lawrence JP ( e d s . ) : "Chemical Derivatization in Analytical Chemistry". Plenum P r e s s , New York, PP.145 (1982). Lindna WF. I n : Z i e f M and Crane LJ ( e d s . ) : "Chromatographic Chiral Separations". Marcel Deker, I n c . , New York, PP. 91 (1987). Lindup WE. Drug-albumin b i n d i n g . Biochem Soc Trans, 3:635 (1975) L i u RH and Ku WW. C h i r a l s t a t i o n a r y phases f o r the g a s - l i q u i d chromatographic separation of enantiomers. J Chromatogr, 271:309 (1983) Louhmuller CH, H a r r i s JM and Souter RW. Chromatographic r e s o u l t i o n of enantiomers in *H and nuclear magnetic resonance studies of hydrogen bonding in c h i r a l ureide ester-amide. J Chromatogr, 71:405 (1972). Lochmuller CH and Souter RW. Chromatographic r e s o l u t i o n of enantiomers s e l e c t i o n review. J Chromatogr, 113:283 (1975) Maitre JM, Boss G and Testa B. High-performance l i q u i d chromatographic separation of the enantiomers of anti-Inflammatory 2-a r y l p r o p i o n a t e s : s u i t a b i l i t y of the method f o r in-vitro metabolic s t u d i e s . J Chromatogr, 229:397 (1984). Matin SB, Wan SH and Karam JH. Pharmacokinetics of tolbutamide: p r e d i c t i o n by concentration in s a l i v a . C l i n Pharmacol Ther, 16:1052 (1974) Mayersohn M and G i b a l d i M. Mathematical methods in pharmacokinetics use of Laplace transform f o r s o l v i n g d i f f e r e n t i a l rate equations. Am J Pharm Educ, 609 (1969). Mehta J and Conti CR. M e x i l e t i n e , a new antiarrhythmic agent f o r treatment of premature v e n t r i c u l a r complexes. Am J C a r d i o l , 49:455 (1982) Metropolitan L i f e Measurements (1983), Society of Actuaries and Association of Life Insurance Medical Directors of America, 1980 M i l l e r K J , Gal J and Ames MM. High-performance l i q u i d chromatographic r e s o l u t i o n of enantiomers of 1-phenyl-2-aminopropanes (amphetamines) with four c h i r a l reagents. J Chromatogr, 307:335 (1984). M i t c h e l l BG, Clements JA, Pottage A and Prescott LF. M e x i l e t i n e d i s p o s i t i o n : i n d i v i d u a l v a r i a t i o n in response to urine a c i d i f i c a t i o n and a l k a l i n i z a t i o n . Br J C l i n Pharmacol, 16:281 (1985) Miwa T, Ichikawa M, Tsuno M, H a t t o r i T, Miyakawa T, Kayano M and Miyake Y. D i r e c t l i q u i d chromatographic r e s o l u t i o n of racemic compounds. Use of ovomucoid as a column l i g a n d . Chem Pharm B u l l (Tokyo), 35:682 (1987a) Miwa T, Miyakawa T, Kayano M and Miyake Y. A p p l i c a t i o n of an ovomucoid-conjugated column f o r the o p t i c a l r e s o l u t i o n of some pharmaceutically important compounds. J Chromatogr, 408:316 (1987b) McComish M, K i t s o n D, Robinson C and J e w i t t DE. 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 s of m e x i l e t i n e . Postgrad Med J , 53(suppl. 1):85 (1977) McErlane KM and P i l l a i GK. G a s - l i q u i d chromatographic r e s o l u t i o n and assay of t o c a i n i d e enantiomers using a c h i r a l c a p i l l a r y column and study of t h e i r s e l e c t i v e d i s p o s i t i o n in man. J Chromatogr, 274:129 (1983) McErlane KM, Igwemwzie L and Kerr CR. S t e r e o s e l e c t i v e serum p r o t e i n binding of m e x i l e t i n e enantiomers in man. Res Commun Chem Path Pharmacol, 56(1):141 (1987a) McErlane KM and Igwemwzie L. S t e r e o s e l e c t i v e a n a l y s i s of the enantiomers of m e x i l e t i n e by high-performance l i q u i d chromatography using fluorescence d e t e c t i o n and study of t h e i r s t e r e o s e l e c t i v e d i s p o s i t i o n i n man. J Chromatogr, 415:335 (1987b) McNamara P J , Slaughter RL, Pieper JA, Wyman MG and Lalka D. Factors i n f l u e n c i n g serum p r o t e i n binding of l i d o c a i n e in humans. Anesthesia and A n a l g e s i a , 60(6):395 (1981) Mucklow JC. The use of s a l i v a in therapeutic drug monitoring - Review. Ther Drug Monit , 4:229 (1982) Nimmo R. The development of m e x i l e t i n e in the treatment of c a r d i a c arrhythmias. Postgrad Med J , 53(suppl . 1):120 (1977) Nimura N, Ogura H and K i n o s h i t a T. Reverse-phase l i q u i d chromatographic r e s o l u t i o n of amino a c i d enantiomers by d e r i v a t i z a t i o n with 2 , 3 , 4 , 6 - t e t r a - O - a c e t y l - B - D - g l u c o p y r a n o s y l i s o t h i o c y a n a t e . J Chromatogr, 202:375 (1980) Nimura N, Kasahaara Y and K i n o s h i t a T. Resolution of enantiomers of norepinephrine and epinephrine by reversed-phase high-performance l i q u i d chromatography. J Chromatogr, 213:327 (1981) N i t s c h J , Doliwa R, Steinbeck G, and L u d e r i t z B. M e x i l e t i n e - s p i e g e l bei patienten mit v e n t r i k u l a r e n arrhthmein und nieren l e b e r oder herzinsugguzienz. Ver Dtsch Ges Inn Med, 87:429 (1981) Okamoto Y and Hatada K. In: K r u s t u l o v i c AM ( e d . ) : "Helical polymers as chiral stationary phases for HPLC: Optically active poly(triphenyl methyl methyacrylate) and poly(diphenyl-2-pyridylmethyl methacrylate)". E l l i s Horwood L t d . , New York, pp.316 (1989) Okamoto Y, Cao ZK, Aburatani R and Hatada K. Optical r e s o l u t i o n of a l c o h o l s as carbamates by HPLC on c e l l u l o s e tris(phenylcarbamate) d e r i v a t i v e s . B u l l Chem Soc Jpn, 60:3999 (1987) Okamoto Y, Kawashima M, Aburatani R, Hatada K, Nishiyama T and Masuda M. O p t i c a l r e s o l u t i o n of b e t a - b l o c k e r s by HPLC on c e l l u l o s e t r i p h e n y l carbamate d e r i v a t i v e s . Chem L e t t , 1237 (1986a) Okamoto Y, Kawashima M and Hatada K. Chromatographic r e s o l u t i o n : XI . c o n t r o l l e d c h i r a l r e c o g n i t i o n of c e l l u l o s e triphenylcarbamate d e r i v a t i v e s supported on s i l i c a g e l . J Chromatogr, 363:173 (1986b) Paalman ACA, Roos JC, S i e b e l i n k J and Dunning AJ. Development of a dosage scheme f o r simultaneous intravenous and o r a l a d m i n i s t r a t i o n of m e x i l e t i n e . Postgrad Med J , 53(suppl. 1):128 (1977) Pentikainen P J , K o i v u l a H and Hiltunen HA. E f f e c t of r i f a m p i c i n treatment on the k i n e t i c s of m e x i l e t i n e . Eur J Pharmacol, 23:261 (1982) Pentikainen P J , Halinen M and H e l i n M. Pharmacokinetics of intraveneous m e x i l e t i n e in p a t i e n t s with acute myocardial i n f a r c t i o n . J Cardiovasc Pharmacol, 6:1 (1984) P e r c h a l s k i RJ , Wilder BJ and Hammer RH. Flame i o n i z a t i o n s and e l e c t r o n -capture GLC determinations of - l - ( 2 , 6 - d i m e t h y l p h e n o x y l ) - 2 -aminopropane in plasma. Pharm S c i , 63:1489 (1974) P i r k l e WH, Finn JM, Schreiner JL and Hamper BC. A widely useful c h i r a l s t a t i o n a r y phase f o r the high-performance l i q u i d chromatography separation of enantiomers. J Am Chem Soc, 103(13):3964 (1981) P i r k l e WH and Hyun MH. A c h i r a l s t a t i o n a r y phase f o r the f a c i l e r e s o l u t i o n of amino a c i d s , amino a l c o h o l s and amines as the N-3,5-d i n i t r o b e n z o y l d e r i v a t i v e s . J Org Chem., 49(17):3043 (1984) P i r k l e WH, Hyun MH, Tsipouras A, Hamper BC and Banks B. A r a t i o n a l approach to the design of h i g h l y e f f e c t i v e c h i r a l s t a t i o n a r y phases f o r the l i q u i d chromatographic separation of enantiomers. J Pharm Biomed A n a l , 2(2):173 (1984) P i r k l e WH and Tsipouras A. D i r e c t l i q u i d chromatographic separation of benzodiazepinone enantiomers. J Chromatogr, 291:291 (1984) P i r k l e WH, Welch CJ and Hyun MH. A c h i r a l r e c o g n i t i o n model f o r the chromatographic r e s o l u t i o n of N-acylated 1-aryl-1-aminoalkanes. J Org Chem, 48:5023 (1983) P i r k l e WH and Welch C J . Chromatographic separation of the enantiomers of acylated amines on c h i r a l s t a t i o n a r y phases. J Org Chem, 49(1):138 (1983) P i r k l e WH and Welch C J . Chromatographic separation of the enantiomers of acylated amines on c h i r a l s t a t i o n a r y Phases. J Org Chem, 49:138 (1984) P i r k l e WH, Welch CJ and Mahler GS. Chromatographic separation of the enantiomers of N-Acylated h e t e r o c y c l i c amines. J Org Chem, 49:2504 (1984). Pottage A, Campbell RW and F i d d l e r G l . M e x i l e t i n e therapy in a c h i l d with a chronic v e n t r i c u l a r arrhythmia. Postgrad Med, 53(suppl. 1):137 (1977) Prescott LF, Pottage A, Clements JA. A b s o r p t i o n , d i s t r i b u t i o n , and e l i m i n a t i o n of m e x i l e t i n e . Postgrad Med J , 53(suppl. 1):50 (1977) Roos JC, Paalman ACA and Dunning AR. E l e c t r o p h y s i o l o g i c a l e f f e c t s of m e x i l e t i n e in man. Postgrad Med J , 53(suppl. 1):92 (1977) Rosen KM, Barwolf C, Ehsani A and Rahimtoola SH. E f f e c t s of l i d o c a i n e and propranolol on the normal and anomalous pathways in p a t i e n t s with p r e - e x c i t a t i o n . Am J C a r d i o l , 30:801 (1972) Rowland M. Plasma p r o t e i n binding and therapeutic drug monitoring. Ther Drug Monit , 2:29 (1980) Rutledge JC. C l i n i c a l evaluation of o r a l m e x i l e t i n e therapy in the treatment of v e n t r i c u l a r arrhythmias. J Am C o l l C a r d i o l , 6:780 (1985) Sami M and Lisbona R. M e x i l e t i n e : long-term e f f i c a c y and heomodynamic a c t i o n in p a t i e n t s with v e n t r i c u l a r arrhythmia. Can J C a r d i o l , 1:251 (1985) Sanchorawala C J , Subba Rao BC, Unni MK and Venkataraman K. Anthroquinone and anthrone s e r i e s . XXVIII. Reduction of anthraquinone to anthracene d e r i v a t i v e s by sodium borohydride and by sodium borohydride in the presence of aluminum c h l o r i d e or boron f l u o r i d e . Indian J Chem, 1:19 (1963) Saunamaki K I . Henodynamic e f f e c t s of a new antiarrhythmic agent, m e x i l e t i n e (Ko 1173), in ischaemic heart d i s e a s e . Cardiovasc Res, 9:788 (1975) Schanker LS, Johnson JM and J e f f r e y J J . Rapid passage of organic anions into human red c e l l . Am J P h y s i o l , 207:503 (1964) S c h i l l G, Wainer IW, Barkan SA. C h i r a l separation of c a t i o n i c drugs on an a j - a c i d g l y c o p r o t e i n bonded s t a t i o n a r y phases. J Chromatogr, 9:641 (1986) Schmid K. a j - a c i d g l y c o p r o t e i n . In: "The Plasma Proteins". V o l . 1, Putnam FW., ( e d . ) , Academic P r e s s , New York, pp.184 (1975). Shumaker RC. Wallace L a b o r a t o r i e s . P.O. Box 1001, Cranbury, NJ, 08512-0181 (1984) Shibata T, Mori K and Okamoto Y. I n : "Polysaccharide Phases". E l l i s Horwood L t d . , New York, pp.336 (1989) Singh BN and Vaughan-Willians EM. E f f e c t of a l t e r i n g potassium concentration on the a c t i o n of 1idocaine and diphenylhydantoin on r a b b i t a t r i a l and v e n t r i c u l a r muscle. C i r c u l a t i o n R e s . , 29:286 (1971) Singh BN and Vaughan-Williams EM. I n v e s t i g a t i o n s of the mode of a c t i o n of a new antidysrhythmic drug, Ko 1173. Br J Pharmacol, 44:1 (1972) Singh NN, Pasutto FM, Coutts RT and Jamali F. Gas chromatogaphic separation of o p t i c a l l y a c t i v e anti-Inflammatory 2 - a r y l p r o p i o n i c acids using (+)- or (-)-amphetamine as d e r i v a t i z i n g reagent. J Chromatogr, 378:125 (1986) Smith KJ and Meffin P J . M e x i l e t i n e a n a l y s i s in blood and plasma using gas chromatography and 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 . J Chromatogr, 181:469 (1980) Sonntag NV. The r e a c t i o n s of a l i p h a t i c a c i d c h l o r i d e s . Chem Rev, 52:324 (1952) Sophianopoulos JA, Durham S J , Sophianopoulos A J , Ragsdale HL and Cropper WP. U l t r a f i l t r a t i o n i s t h e o r e t i c a l l y equivalent to e q u i l i b r i u m d i a l y s i s but much simpler to c a r r y out. Arch Biochem Biophys, 187(1):132 (1978) Sophianopoulos J , Jerkunica I , Lee CM and Sgoutas D. An improved method f o r f r e e thyroxine and t r i i o d o t h y r o n i n e e in serum. C l i n Chem, 26:159 (1980) Talbot RG, J u l i a n DG, Clark RA, Nielson JMM and Prescott LF. Treatment of v e n t r i c u l a r arrhythmias with m e x i l e t i n e . Lancet, 2:399 (1973) Tamegai T, Ohmae M, Kawabe K and Tomoeda M. Separation of o p t i c a l isomers as diastereomeric d e r i v a t i v e s by HPLC. J L i q Chromatogr, 2:1229 (1979) Thompson JA, Holtzman JL , Tsuru M, Lerman C and Holtzman J . Procedure f o r the c h i r a l d e r i v a t i z a t i o n s and chromatographic r e s o l u t i o n of R(+)- and S ( - ) - p r o p r a n o l o l . J Chromatogr, 238:470 (1982) Trimarco B. Disopyramide, m e x i l e t i n e and procainamide in the long-term o r a l treatment of v e n t r i c u l a r arrhythmias. Antiarrhythmic e f f i c a c y and heomodynamic e f f e c t s . Curr Ther Res, 33:472 (1983) Vaughan-Williams EM. M e x i l e t i n e in i s o l a t e d t i s s u e models. Postgrad Med J , 53(suppl. 1):30 (1977) Wainer IW, Doyle TD, Donn KH and Powell JR. The d i r e c t enantiomer determination of ( - ) - and (+)-propranolol in human serum by HPLC on a c h i r a l s t a t i o n a r y phase. J Chromatogr, 9:405 (1984) Wainer IW and Doyle TD. A p p l i c a t i o n of high-performance l i q u i d chromatographic c h i r a l s t a t i o n a r y phases to pharmaceutical a n a l y s i s - d i r e c t enantiomeric r e s o l u t i o n of amide d e r i v a t i v e s of 1-phenyl-2-aminopropane. J Chromatogr, 259:465 (1983) Wainer IW and Alembik MC. In : Z e i f M and Crane LJ ( e d s . ) : "Chromatographic Chiral Separations". Marcel Dekker, I n c . , New York, PP. 355 (1988). Wainer IW, Barkan SA and S c h i l l G. LC/GC Mag, 4:422 (1986) Wallace M and Riegelman S. Uptake of acetazalamide by human e r y t h r o c y t e s . J Pharm S c i , 66(5):729 (1977) Wang T, Wuellner D, Woosley RL and Stone WJ. Pharmacokinetics and n o n d i a l y z a b i l i t y of m e x i l e t i n e in renal f a i l u r e . C l i n Pharmacol Ther, 37:649 (1985) Wells CE. G a s - l i q u i d chromatographic determination of the o p t i c a l isomers of amphetamine. J Assoc Anal Chem, 53:113 (1970) Westley JW, Hal pern B and Karger BL. E f f e c t of solute s t r u c t u r e on separation of diastereomeric e s t e r s and amides by g a s - l i q u i d chromatography. Anal Chem, 40:2046 (1968) Whitlam JB and Brown KF. U l t r a f i l t r a t i o n in serum p r o t e i n binding determinations. J Pharm S c i , 70(2):146 (1981) Wilder B J , Langlois Y and Hammer RH. C l i n i c a l and l a b o r a t o r y s t u d i e s of a new anticonvulsant drug. E p i l e p s i a , 14:104 (1973) W i l l c o x S and Singh BN. S e n s i t i v e gas chromatographic method f o r the estimation of a new antiarrhythmic compound, m e x i l e t i n e (Koll73) in b i o l o g i c a l f l u i d s . J Chromatogr, 128:196 (1976). W i l t i n g J , Van Der Gesen WF, Janssen LHM, Weideman MM and O t a g i r i M. The e f f e c t of albumin conformation on the binding of w a r f a r i n to human serum albumin. J B i o l Chem, 255(7):3032 (1980) Wing LMH, M e f f i n P J , Grygiel J J , Smith KJ and B i r k e t t DJ. The e f f e c t of metoclopramide and atropine on the absorption of o r a l l y administrated m e x i l e t i n e . Br J C l i n Pharmacol, 9:505 (1980) Wollweber H, Horstmann H and Meng K. Stereochemical research on drugs: Part 2. absolute stereochemistry of d i u r e t i c a l l y a c t i v e enantiomers of the mefurside s e r i e s . Eur J Med Chem Chim Ther 11:159 (1976) Yamada T, Suzuki H and Mukaiyama T. The asymmetric a d d i t i o n of the Sn(II) enolates of t h i o e s t e r to a l p h a - i m i n o e s t e r s : A. Convenient synthesis of o p t i c a l l y a c t i v e c i s - s u b s t i t u t e d B-lactams. Chem L e t t , 293 (1987) Yamaguchi I , Singh BN and Mandel WJ. E l e c t r o p h y s i o l o g i c a l actions of m e x i l e t i n e on i s l o a t e d r a b b i t a t r i a and canine v e n t r i c u l a r muscle and P u r k i n j e f i b r e s . Cardiovasc Res, 13:288 (1979) 214 APPENDIX 2 M / Z - J2t 

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