Open Collections

UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Stereoselective HPLC analysis of mexiletine enantiomers : pharmacokinetics and protein binding in humans Igwemezie, Linus Nnamdi 1986

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1986_A6_7 I38.pdf [ 5.93MB ]
Metadata
JSON: 831-1.0096727.json
JSON-LD: 831-1.0096727-ld.json
RDF/XML (Pretty): 831-1.0096727-rdf.xml
RDF/JSON: 831-1.0096727-rdf.json
Turtle: 831-1.0096727-turtle.txt
N-Triples: 831-1.0096727-rdf-ntriples.txt
Original Record: 831-1.0096727-source.json
Full Text
831-1.0096727-fulltext.txt
Citation
831-1.0096727.ris

Full Text

STEREOSELECTIVE HPLC ANALYSIS OF MEXILETINE ENANTIOME RS PHARMACOKINETICS AND PROTEIN BINDING IN HUMANS By LINUS NNAMDI IGWEMEZIE B. Pharm., The U n i v e r s i t y of I F E, 1981 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF • MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES ( F a c u l t y of P h a r m a c e u t i c a l S c i e n c e s , D i v i s i o n of P h a r m a c e u t i c a l Chemistry) We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o the r e q u i r e x L - 6 t a n d a r d THE © UNIVERSITY OF BRITISH COLUMBIA October. 19 86 L i n u s Nnamdi Igwemezie, 1986 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of PHARMACEUTICAL SCIENCES The University of B r i t i s h Columbia 1956 Main M a l l Vancouver, Canada V6T 1Y3 Date ?ZLTH nrTnRFP iQ«fi )E-6 (3/81) i i ABSTRACT M e x i l e t i n e i s a r e l a t i v e l y new c l a s s IB a n t i a r r h y t h m i c a g e n t , s i m i l a r i n s t r u c t u r e and p h a r m a c o l o g i c a l e f f e c t s t o l i d o c a i n e . I t i s e f f e c t i v e m a i n l y a g a i n s t v e n t r i c u l a r a r r h -y t h m i a s and can be a d m i n i s t e r e d by the o r a l r o u t e , m e x i l e t i -ne i s a c h i r a l drug which i s used c l i n i c a l l y as the rac e m i c m i x t u r e . The ena n t i o m e r s of numerous c h i r a l drugs have been shown t o d i f f e r i n t h e i r d i s p o s i t i o n i n the body due t o t h e i r s t e r e o s e l e c t i v e pharmacodynamic and/or p h a r m a c o k i n e t i c p r o p e r t i e s . The r e l a t i v e a n t i a r r h y t h m i c p o t e n c i e s of the i n d i v i d u a l e n a n t i o m e r s of m e x i l e t i n e have n ot been s t u d i e d , nor have t h e i r p h a r m a c o k i n e t i c s been p r o p e r l y e l u c i d a t e d . Thus, the p r e s e n t s t u d y was aimed a t d e v e l o p i n g a h i g h l y s e n s i t i v e and s t e r e o s e l e c t i v e assay f o r m e x i l e t i n e e n a n t i o m e r s which w i l l be u t i l i z e d t o st u d y t h e i r p h a r m a c o k i n e t i c s and i n v i t r o serum p r o t e i n b i n d i n g . A h i g h - p e r f o r m a n c e 1 i q u i d - c h r o m a t o g r a p h i c assay was devel o p e d u s i n g the P i r k l e i o n i c c h i r a l s t a t i o n a r y phase. The ena n t i o m e r s were r e s o l v e d as t h e i r 2-naphthoyl d e r i v a t i v e s . The HPLC m o b i l e phase c o n s i s t e d of 5.5% 2-p r o p a n o l i n hexane and was d e l i v e r e d a t a f l o w r a t e o f 1.4 mL/min. D e t e c t i o n of the e n a n t i o m e r i c d e r i v a t i v e s was a c c o m p l i s h e d w i t h a f l u o r e s c e n c e d e t e c t o r [230 nm (Ex) and i i i 340 nm (Em)]. R e c o v e r y of the e n a n t i o m e r s from plasma a f t e r pH a d j ustment t o above 12 was found t o be s u b s t a n t i a l l y low and s t e r e o s e l e c t i v e l y i n f a v o u r of the S(+)-enantiomer when compared w i t h t h e i r r e c o v e r y from w a t e r . T h i s was a t t r i b u t e d t o a g r e a t e r plasma p r o t e i n b i n d i n g o f the R ( - ) - i s o m e r d e s p i t e the h i g h plasma pH. Recovery was improved ( 8 3 % ) , and t h e n a t u r a l e n a n t i o m e r i c r a t i o r e s t o r e d by p r e c i p i t a t i o n of the plasma p r o t e i n s w i t h barium h y d r o x i d e / z i n c s u l f a t e . 2 L i n e a r c a l i b r a t i o n c u r v e s (r >0.999) were o b t a i n e d i n plasma over the c o n c e n t r a t i o n range 5 t o 750 ng/mL f o r each 2 e n a n t i o m e r . S i m i l a r c o r r e l a t i o n s (r >0.999) were o b t a i n e d i n s a l i v a from 10 t o 1,500 ng/mL and i n u r i n e from 0.25 t o 7.5 ug/ml and 10 t o 500 ng/ml. The i n t e r - and i n t r a - a s s a y c o e f f i c i e n t s o f v a r i a t i o n were l e s s than 4% f o r a l l the b i o l o g i c a l f l u i d s . The minimum d e t e c t a b l e q u a n t i t y of each enantiomer i n plasma was 5 ng/mL a t a s i g n a l - t o - n o i s e r a t i o of 5:1, r e p r e s e n t i n g 100 pg i n j e c t e d onto the column. P r o t e i n b i n d i n g o f the enantiomers was d e t e r m i n e d w i t h serum from f i v e h e a l t h y male s u b j e c t s . The mean p e r c e n t f r e e f r a c t i o n o f R ( - ) - m e x i l e t i n e , 19.80 +_ 2.64% was s i g n i -f i c a n t l y (P<0.001) l e s s t h a n t h a t o f S ( + ) - m e x i l e t i n e , 28.32 +_ 1.45%. B i n d i n g was independent of c o n c e n t r a t i o n over the t h e r a p e u t i c range. i v F i v e h e a l t h y male s u b j e c t s (same as above) were given 300 mg of (_+) -mexi l e t i n e h y d r o c h l o r i d e (capsules) o r a l l y . The plasma c o n c e n t r a t i o n - t i m e data were analyzed by AUTOAN and NONLIN computer programs. The enantiomer k i n e t i c s were best d e s c r i b e d by a t r i e x p o n e n t i a l f u n c t i o n i n three of the f i v e s u b j e c t s and a b i e x p o n e n t i a l f u n c t i o n i n the remaining two. There was no s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e i n the a b s o r p t i o n and d i s t r i b u t i o n r a t e c o n s t a n t s , peak plasma c o n c e n t r a t i o n s , time to peak plasma c o n c e n t r a t i o n s and plasma AUCs of the enantiomers. B i o a v a i l a b i l i t y of the enantiomers was not determined due to l a c k of approval from the Health P r o t e c t i o n Branch (Canada) to a d m i n i s t e r intravenous (+_) - m e x i l e t i n e to h e a l t h y v o l u n t e e r s . The t e r m i n a l e l i m i n a t i o n h a l f - l i f e from plasma data f o r R ( - ) - m e x i l e t i n e , 9.1 +_ 2.9 hours, was s i g n i f i c a n t l y (P<0.02) l e s s than t h a t f o r the S( + )-isomer, 11.0 +_ 3.8 hours. The cumulative u r i n a r y e x c r e t i o n of S ( + ) - m e x i l e t i n e was 9.14 +_ 3.07%, which was s i g n i f i c a n t l y (P<0.01) g r e a t e r than t h a t of R ( - ) - m e x i l e t i n e , 7.40 + 2.40%. Renal c l e a r a n c e of the enantiomers was c o n s i s t e n t with cumulative u r i n a r y e x c r e t i o n , 0.72 +_ 0.26 mL., m i n - 1 . K g - 1 and 0.61 + 0.20 m L . m i n _ 1 . K g - l r e s p e c t i v e l y (P<0.05). The non-renal e l i m i n a t i o n r a t e constant (mainly metabolism) was s i g n i f i c a n t l y (P<0.001) g r e a t e r f o r R ( - ) - m e x i l e t i n e , V 0.0763 +_ 0.0273 h " 1 , than f o r the S( + )-iosmer, 0.0634 _+ 0.0270 h _ 1 . The s a l i v a AUCS were 18.2.+ 6.3 ug.mL "'".hr and 14.1 +_ 5.0 ug. mL "'".hr f o r S ( + ) - m e x i l e t i n e and the R(-)-isomer r e s p e c t i v e l y ( d i f f e r e n c e s i g n i f i c a n t , P<0.01). The enantiomer c o n c e n t r a t i o n r a t i o s (R/S) i n the three b i o l o g i c a l f l u i d s showed evidence of a c r o s s - o v e r [R(-)> S(+) to S(+)>R(-)] between 0 - 2 hours i n u r i n e and s a l i v a and 8 - 1 0 hours i n plasma. T h i s i n d i c a t e d an apparent d i s c r e p a n c y i n the c r o s s - o v e r time i n plasma r e l a t i v e to u r i n e and s a l i v a . However, the f r e e enantiomer c o n c e n t r a t i o n r a t i o s i n plasma were s i m i l a r to the enantiomer r a t i o s i n u r i n e and s a l i v a . Thus the d i s p o s i t i o n of m e x i l e t i n e enantiomers i n man i s s t e r e o s e l e c t i v e w i t h serum p r o t e i n b i n d i n g and metabolism f a v o u r i n g the R(-)-isomer. Renal e l i m i n a t i o n and s a l i v a r y s e c r e t i o n , on the other hand, favour the S(+)-isomer. The s i m i l a r i t y between the f r e e enantiomer r a t i o s i n plasma, u r i n e and s a l i v a suggests t h a t the s t e r e o s e l e c t i v e r e n a l e l i m i n a t i o n and s a l i v a r y s e c r e t i o n of the enantiomers are a r e f l e c t i o n of t h e i r s t e r e o s e l e c t i v e p r o t e i n b i n d i n g . v i TABLE OF CONTENTS ABSTRACT i i TABLE OF CONTENTS v i LIST OF TABLES x LIST OF FIGURES x i i LIST OF SCHEMES x i v SYMBOLS AND ABBREVIATIONS xv ACKNOWLEDGEMENTS x v i i CHAPTER PAGE 1. INTRODUCTION 1 2. BACKGROUND 4 2.1 Chemistry 4 2.2 Pharmacodynamics of M e x i l e t i n e 5 2.2.1 Pharmacology 5 2.2.2 E l e c t r o p h y s i o l o g i c a l E f f e c t s on the Heart 5 2.2.3 Hemodynamic E f f e c t s 7 2.3 C l i n i c a l I n d i c a t i o n s 8 2.4 Adverse E f f e c t s 8 2.5 Pharmacokinetics of M e x i l e t i n e 9 2.5.1 Healthy V o l u n t e e r S t u d i e s 9 2.5.2 E f f e c t s of Disease on Pharmacokinetics 10 2.6 Serum C o n c e n t r a t i o n - C l i n i c a l E f f e c t R e l a t i o n s h i p s 11 2.7 Comparison w i t h Other A n t i a r r h y t h m i c Agents . . . 13 2.8 Metabolism 13 2.9 I n t e r a c t i o n s of M e x i l e t i n e with Other Agents. . . 14 2.10 M e x i l e t i n e Serum P r o t e i n B i n d i n g . 16 v i i PAGE 2.11 S t e r e o s e l e c t i v i t y i n Drug D i s p o s i t i o n 16 2.11.1 Abs o r p t i o n 17 2.11.2 D i s t r i b u t i o n 18 2.11.3 Metabolism 19 2.11.4 Renal E x c r e t i o n 22 2.12 S t e r e o s e l e c t i v e Drug A n a l y s i s 22 2.12.1 R e s o l u t i o n of Enantiomers as Diast e r e o i s o m e r s 23 2.12.2 D i r e c t R e s o l u t i o n of Enantiomers on C h i r a l S t a t i o n a r y Phases 24 2.13 A n a l y s i s of M e x i l e t i n e 27 2.13.1 Assay Methods f o r R , S - M e x i l e t i n e . . . .27 2.13.2 Assay Methods f o r M e x i l e t i n e Enantiomers 28 2.14 Primary O b j e c t i v e s 30 3. EXPERIMENTAL 31 3.1 S u p p l i e s 31 3.1.1 Drugs 31 3.1.2 Chemicals and Reagents . .31 3.1.3 Solvents 32 3.2 Chromatographic S t a t i o n a r y Phases and Columns. 32 3.3 Equipment 3 3 3.3.1 High-Performance L i q u i d Chromatograph 33 3.3.2 Gas Chromatograph/ Mass Spectrometer 33 3.3.3 M i s c e l l a n e o u s 33 3.4 Stock S o l u t i o n s 34 3.4.1 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 34 v i i i PAGE 3.4.2 R ( - ) - , and S ( + ) - M e x i l e t i n e H y d r o c h l o r i d e 34 3.4.3 KOE 2963 H y d r o c h l o r i d e ( I n t e r n a l Standard) 34 3.4.4 Barium Hydroxide S o l u t i o n (0.3N). . . .34 3.4.5 Zinc S u l f a t e Heptahydrate S o l u t i o n (5%) 35 3.4.6 2-Naphthoyl C h l o r i d e 35 3.4.7 I s o t o n i c , 0.067M, pH7.4, Phosphate B u f f e r (Documentia Geigy, 1970) 35 3.4.8 T r i c h l o r o a c e t i c A c i d (10%) 35 3.5 Chromatographic R e s o l u t i o n of M e x i l e t i n e Enantiomers 36 3.5.1 T r i f l u o r o a c e t y l - , and A c e t y l D e r i v a t i v e s . . 36 3.5.2 2-Naphthoyl D e r i v a t i v e 37 3.6 Development of Assay of M e x i l e t i n e Enantiomers. . 37 3.6.1 S e l e c t i o n of I n t e r n a l Standard 37 3.6.2 Optimum Reaction Time 38 3.6.3 S e n s i t i v i t y 38 3.6.4 E x t r a c t i o n Solvent 39 3.6.5 Plasma P r o t e i n P r e c i p i t a t i o n 39 3.7 Assay of M e x i l e t i n e Enantiomers by High-Performance L i q u i d Chromatography with Flu o r e s c e n c e D e t e c t i o n 41 3.7.1 E x t r a c t i o n , D e r i v a t i z a t i o n and HPLC A n a l y s i s 41 3.7.2 C a l i b r a t i o n Curves and P r e c i s i o n of of Assay 42 3.7.3 E f f i c i e n c y of Recovery of M e x i l e t i n e Enantiomers from Plasma 43 3.7.4. S t r u c t u r a l C o n f i r m a t i o n of the D e r i v a t i v e s 44 3.8 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 by U l t r a f i l t r a t i o n Technique 45 3.8.1 Serum C o l l e c t i o n 45 3.8.2 Sample P r e p a r a t i o n 45 3.8.3 U l t r a - f i l t r a t i o n 46 3.8.4 A n a l y s i s of Free and T o t a l M e x i l e t i n e i n Serum 46 i x PAGE 3.9 Pharmacokinetics of M e x i l e t i n e Enantiomers i n Healthy V o l u n t e e r s 47 3.9.1 V o l u n t e e r s 47 3.9.2 P h y s i o l o g i c a l M o n i t o r i n g of Study Subjects 47 3.9.3 In v i v o S t u d i e s 48 3.9.4 A n a l y s i s of Plasma, Urine and S a l i v a Samples 49 3.9.5 Data A n a l y s i s 50 4. RESULTS AND DISCUSSIONS 52 4.1 Chromatographic R e s o l u t i o n of M e x i l e t i n e Enantiomers 52 4.1.1 T r i f l u o r o a c e t y l and A c e t y l D e r i v a t i v e s 52 4.1.2 2-Naphthoyl D e r i v a t i v e 54 4.2 Assay Development 56 4.2.1 D e r i v a t i z a t i o n and HPLC with Fluorescence D e t e c t i o n 56 4.2.2 Mechanism of R e s o l u t i o n of M e x i l e t i n e . .59 4.2.3 L i n e a r i t y and P r e c i s i o n of the Assay. . 62 4.2.4 Recovery of M e x i l e t i n e Enantiomers from Plasma 65 4.2.5 S t r u c t u r a l C o n f i r m a t i o n of the D e r i v a t i v e s 71 4.3 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. .76 4.4 Pharmacokinetics of M e x i l e t i n e Enantiomers i n Healthy V o l u n t e e r s 81 4.4.1 Plasma Data 81 4.4.2 Urine Data 91 4.4.3 S a l i v a Data 94 4.4.4 I n t e r r e l a t i o n s h i p of p r o t e i n b i n d i n g , plasma, u r i n e and s a l i v a data 106 5. SUMMARY AND CONCLUSIONS 115 6. REFERENCES 117 X LIST OF TABLES TABLE PAGE 1. C a l i b r a t i o n Curve Data f o r M e x i l e t i n e Enantiomers i n Plasma 66 2. C a l i b r a t i o n Curve Data f o r M e x i l e t i n e Enantiomers i n Urine (Higher C o n c e n t r a t i o n Range) 67 3. C a l i b r a t i o n Curve Data f o r M e x i l e t i n e Enantiomers i n Urine (Lower C o n c e n t r a t i o n Range) 6 8 4. C a l i b r a t i o n Curve Data f o r M e x i l e t i n e Enantiomers i n S a l i v a 69 5. 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 Enantiomer i n Plasma 70 6. E f f i c i e n c y of Recovery of M e x i l e t i n e Enantiomers from Plasma 73 7. Free F r a c t i o n (%) of M e x i l e t i n e Enantiomers i n Serum 80 8. Plasma C o n c e n t r a t i o n - Time Data F o l l o w i n g O r a l Dose of 300 mg of (+_) - M e x i l e t i n e H y d r o c h l o r i d e i n F i v e Healthy Subjects 83 9. Pharmacokinetic Parameters of M e x i l e t i n e Enantiomers F o l l o w i n g an O r a l Dose of 300 mg of (+)- M e x i l e t i n e H y d r o c h l o r i d e 86 10. Enantiomer C o n c e n t r a t i o n R a t i o s i n Plasma 88 11. Pharmacokinetic Parameters of M e x i l e t i n e Enantiomers Estimated From U r i n a r y E x c r e t i o n Data 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 300 mg of (+)-Mexiletine H y d r o c h l o r i d e 92 12. Data f o r Cumulative U r i n a r y E x c r e t i o n Versus Time, and Amount Remaining to be E x c r e t e d (ARE) Versus Time P l o t i n one Subject (MW) 95 13. Enantiomer C o n c e n t r a t i o n R a t i o s i n U r i n e . 99 x i TABLE PAGE 14. M e x i l e t i n e Enantiomers i n the S a l i v a of F i v e Healthy Subjects F o l l o w i n g an O r a l Dose of 300 mg of (_+) - M e x i l e t i n e H y d r o c h l o r i d e . 101 15. The Observed Saliva/Plasma C o n c e n t r a t i o n R a t i o s of M e x i l e t i n e Enantiomers. . 104 16. M e x i l e t i n e Enantiomer S a l i v a AUCs 108 17. Enantiomer C o n c e n t r a t i o n R a t i o s i n S a l i v a 109 18. Comparison of Enantiomer C o n c e n t r a t i o n R a t i o s (R/S) i n Plasma, Urine and S a l i v a 110 19. Free Enantiomer C o n c e n t r a t i o n R a t i o s i n Plasma. . . 113 x i i LIST OF FIGURES FIGURE PAGE 1. Chromatograms of T r i f l u o r o a c e t y l and A c e t y l D e r i v a t i v e s of M e x i l e t i n e on the P i r k l e Covalent C h i r a l Column 53 2. Chromatograms of the 2-Naphthoyl D e r i v a t i v e o f R M e x i l e t i n e on the P i r k l e Covalent, and P i r k l e I o n i c Columns. 55 3. Time-Dependence of D e r i v a t i v e Formation 57 4. Chromatogram of M e x i l e t i n e Enantiomers and the I n t e r n a l Standard I s o l a t e d from Plasma 60 5. S t r u c t u r e of M e x i l e t i n e Enantiomers 61 6. S t r u c t u r e of the P i r k l e C h i r a l S t a t i o n a r y Phases 63 7. The Stereochemical Interaction. Between S (+ ) - M e x i l e t i n e -2-Naphthamide and the P i r k l e I o n i c C h i r a l S t a t i o n a r y Phase 64 8. Chromatograms Showing the R e l a t i o n s h i p of M e x i l e t i n e E x t r a c t e d from Water, Plasma, and Plasma A f t e r P r e c i p i t a t i o n of Plasma P r o t e i n s 72 9. P o s i t i v e Chemical I o n i z a t i o n (CH.) Mass Spe c t r a of 2-Naphthoyl D e r i v a t i v e s of R,S-Mexiletine and the I n t e r n a l Standard 74 10. Proposed Fragment Ions of the 2-Naphthoyl D e r i v a t i v e s of R,S-Mexiletine and the I n t e r n a l Standard 75 11. T o t a l Ion Current Mass Chromatograms of the 2-Naphthoyl D e r i v a t i v e s of R,S-Mexiletine and the I n t e r n a l Standard 7 7 12. Chromatograms of M e x i l e t i n e Enantiomers I s o l a t e d from Spiked Serum, and the Serum U l t r a f i l t r a t e 78 x i i i FIGURE PAGE 13. Semilogarithmic P l o t of Plasma C o n c e n t r a t i o n Versus Time f o r M e x i l e t i n e Enantiomers A f t e r an O r a l Dose of (+_) - M e x i l e t i n e 84 14. M e x i l e t i n e Enantiomers: Plasma P r o f i l e 30 Hours A f t e r an O r a l Dose of the Racemate 8 5 15. Cumulative U r i n a r y E x c r e t i o n Versus Time P l o t . . . . 96 16. S e m i l o g a r i t h m i c P l o t of Amount Remaining to be Excreted Versus Time 97 17. M e x i l e t i n e Enantiomers: Urine P r o f i l e 30 Hours A f t e r an O r a l Dose of the Racemate 98 18. Sem i l o g a r i t h m i c P l o t s of S a l i v a C o n c e n t r a t i o n Versus Time i n A l l the Subjects 102 .19. M e x i l e t i n e Enantiomers: S a l i v a P r o f i l e 30 Hours A f t e r an O r a l Dose of the Racemate 103 20. C o r r e l a t i o n Between S a l i v a and Plasma L e v e l s of M e x i l e t i n e Enantiomers F o l l o w i n g an O r a l Dose of the Racemate 107 x i v L I S T OF SCHEMES SCHEME PAGE 1. Scheme f o r t h e D e r i v a t i z a t i o n o f M e x i l e t i n e and t h e I n t e r n a l S t a n d a r d w i t h 2 - N a p h t h o y l C h l o r i d e . . .58 XV SYMBOLS AND ABBREVIATIONS AAG a ARE AUC AUTOAN AV 3 CI peak CI r C l TB/F C V . dp/dt Ex Em F GC hr HPLC HSA HV a l p h a s - a c i d g l y c o p r o t e i n d i s t r i b u t i o n r a t e c o n s t a n t amount remaining to be e x c r e t e d area under the plasma c o n c e n t r a t i o n - t i m e curve a decision-making pharmacokinetic computer program a t r i o - v e n t r i c u l a r f i r s t - o r d e r o v e r a l l e l i m i n a t i o n r a t e c o n s t a n t chemical i o n i z a t i o n peak plasma c o n c e n t r a t i o n achieved f o l l o w i n g drug a d m i n i s t r a t i o n r e n a l c l e a r a n c e apparent o r a l body c l e a r a n c e c o e f f i c i e n t of v a r i a t i o n r a t e of development of l e f t v e n t r i c u l a r p r e ssure e x c i t a t i o n emission b i o a v a i l a b i l i t y gas chromatography hour high-performance l i q u i d chromatograhpy human serum albumin H i s - v e n t r i c u l a r x v i i .d. I .S. Ka K nr Mex. min mL MS N ng NONLIN PTFE r r 2 +_S.D. tpeak UV Vd area i n t e r n a l diameter i n t e r n a l standard f i r s t - o r d e r a b s o r p t i o n r a t e constant non-renal e x c r e t i o n r a t e constant m e x i l e t i n e minute m i l l i l i t e r • mass spectrometer, spectrum or s p e c t r a normal nanogram computer program f o r n o n - l i n e a r l e a s t square r e g r e s s i o n a n a l y s i s of pharmacokinetic data p o l y t e t r a f l u o r o e t h y l e n e c o r r e l a t i o n c o e f f i c i e n t of d e t e r m i n a t i o n +_ one standard d e v i a t i o n time taken to reach peak plasma c o n c e n t r a t i o n f o l l o w i n g an o r a l dose u l t r a v i o l e t 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 x v i i ACKNOWLEDGEMENTS I would l i k e to express my s i n c e r e g r a t i t u d e to Dr. K e i t h McErlane f o r h i s guidance and s u p e r v i s i o n d u r i n g the course of t h i s study. My g r a t i t u d e a l s o goes to Drs. Jim Orr and Frank Abbott f o r very h e l p f u l d i s c u s s i o n s . I am g r a t e f u l to Dr. C h a r l e s Kerr f o r h i s s u p e r v i s i o n of the human v o l u n t e e r s t u d i e s , Mrs. Barbara McErlane and Mr. Rowland Burton f o r a l l t h e i r t e c h n i c a l a s s i s t a n c e , and Matthew Wright f o r h i s a s s i s t a n c e i n the computer a n a l y s i s of the pharmacokinetic data. The f i n a n c i a l support by Boehringer Ingelheim L t d . (Canada) i s g r a t e f u l l y acknowledged. DEDICATION T h i s t h e s i s i s d e d i c a t e d t o my p a r e n t s , M r s . & Mr. L.M.C. I g w e m e z i e . 1 1. INTRODUCTION M e x i l e t i n e , M e x i t i l ( B o e h r i n g e r I n g e l h e i m L t d . ) i s a r e l a t i v e l y new c l a s s IB a n t i a r r h y t h m i c agent. I t was f i r s t s y n t h e s i z e d i n an attempt to reduce the CNS e f f e c t s of the a n o r e c t i c agent phenmetrazine by modifying i t s chemical s t r u c t u r e (Koppe, 1977). The r e s u l t was an agent w i t h a n t i -c o n v u l s a n t a c t i v i t y . L a t e r , when other a n t i c o n v u l s a n t agents were found to e x h i b i t a n t i a r r h y t h m i c e f f e c t s , mexi-l e t i n e was found to be e f f e c t i v e i n suppressing v a r i o u s forms of v e n t r i c u l a r arrhythmias ( A l l e n e t a l . , 1972, Singh and Vaughan-Williams, 1972, Campbell et a l . , 1973 and T a l b o t e t a 1 ., 1973 ). M e x i l e t i n e has been i n c l i n i c a l use i n Europe and England s i n c e 1976, however, the o r a l dosage form was i n t r o d u c e d i n t o the Canadian market i n 1985. M e x i l e t i n e resembles l i d o c a i n e i n i t s s t r u c t u r e , e l e c t r o p h y s i o l o g i c a l e f f e c t s on the h e a r t and has the same c l i n i c a l i n d i c a t i o n s (Danneberg and S h e l l e y , 1977). I t s primary advantage over l i d o c a i n e i s i t s o r a l e f f i c a c y and r e l a t i v e l y long e l i m i n a t i o n h a l f - l i f e , hence i t i s u s e f u l i n the treatment of acute and long-term v e n t r i c u l a r a rrhythmias (Vaughan-Williams 1977) . M e x i l e t i n e has a c h i r a l c e n t e r i n 2 i t s molecular arrangement ( i . e . , composed of equal propor-t i o n s of two enantiomers). S e v e r a l drugs i n use today con-t a i n c h i r a l c e n t e r s , and i t has been e s t a b l i s h e d t h a t the enantiomers of a number of these agents e x h i b i t d i f f e r i n g pharmacokinetic and/or pharmacological a c t i v i t i e s (Simonyi, 1984, W i l l i a m s and Lee, 1985, and Walle and Walle, 1986). This i s mainly the r e s u l t of e n a n t i o s e l e c t i v i t y i n t h e i r d i s p o s i t i o n ( a b s o r p t i o n , d i s t r i b u t i o n , metabolism and/or e x c r e t i o n ) i n the body. Thus plasma l e v e l s and pharmaco-k i n e t i c s of racemic drugs do not always r e f l e c t those of the a c t i v e enantiomers. Despite t h i s knowledge, most c h i r a l drugs, i n c l u d i n g m e x i l e t i n e , are used t h e r a p e u t i c a l l y as the racemic mixture. At the present time, there i s no i n f o r m a t i o n on the r e l a t i v e a n t i a r r h y t h m i c a c t i v i t y of the i n d i v i d u a l enantiomers of m e x i l e t i n e , but t h e i r r e s o l u t i o n (Grech-Belanger e t a l . , 1985a), and p r e l i m i n a r y pharmacokinetics (Grech-Belanger et a l . , 1986) have been r e p o r t e d . The r e s o l u t i o n was achieved by c o n v e r t i n g the enantiomers to d i a s t e r e o i s o m e r s and subsequently r e s o l v i n g them by HPLC on a r e v e r s e phase column. There are w e l l documented drawbacks i n t h i s approach, such as the requirement of an e n a n t i o m e r i -c a l l y pure 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 formation of the d i a s t e r e o i s o m e r s , and the q u a n t i t a t i o n e r r o r s t h a t 3 may o c c u r due t o d i f f e r i n g r a t e s o f r e a c t i o n o f e n a n t i o m e r s w i t h o t h e r c h i r a l compounds ( K o n i g e t a l . , 1 9 7 7 , F r a n k e t a l . , 1978 and L i u and Ku, 1 9 8 3 ) . I n a d d i t i o n , t h e a s s a y r e p o r t e d by G r e c h - b e l a n g e r e t a l . (1985a) l a c k e d a d e q u a t e s e n s i t i v i t y f o r a r e l i a b l e p h a r m a c o k i n e t i c s t u d y o f t h e e n a n t i o m e r s i n humans. The p r e s e n t s t u d i e s w e r e t h e r e f o r e a i m e d a t d e v e l o p i n g a h i g h l y s e n s i t i v e and s t e r e o s e l e c t i v e a s s a y f o r t h e r e s o l u t i o n o f m e x i l e t i n e e n a n t i o m e r s d i r e c t l y on a c h i r a l c h r o m a t o g r a p h i c s t a t i o n a r y p h a s e . T h i s a s s a y w o u l d n o t h a v e t h e d r a w b a c k s m e n t i o n e d a b o v e , and w o u l d make i t f e a s i b l e t o f o l l o w t h e c o n c e n t r a t i o n - t i m e c o u r s e o f t h e e n a n t i o m e r s i n humans f o r c o n s i d e r a b l y l o n g e r t h a n t h a t p r e v i o u s l y r e p o r t e d . I n a d d i t i o n t h e i r i n v i t r o s t e r e o s e l e c t i v e p r o t e i n b i n d i n g i n human se r u m w o u l d be s t u d i e d . 4 2. BACKGROUND 2.1 C h e m i s t r y C H 3 O C H 2 C H N H 2 C H 3 C H 3 MEXILETINE M e x i l e t i n e i s a b a s i c d r u g and p o s s e s s e s t h e f o l l o w i n g p h y s i c a l and c h e m i c a l c h a r a c t e r i s t i c s ; p K a , 8.8; m o l e c u l a r f o r m u l a e , C ^ H ^ N O ; m o l e c u l a r w e i g h t , 179 .27; c h e m i c a l name, 1- ( 2 ' , 6 ' - d i m e t h y l p h e n o x y ) -2-a m i n o p r o p a n e , t h e a m i n o g r o u p b e i n g a t t a c h e d t o a c h i r a l c a r b o n atom ( M e r c k I n d e x , 1 9 8 3 ) . I t i s u s e d c l i n i c a l l y as t h e h y d r o c h l o r i d e s a l t ( M e x i t i l ) w h i c h i s an a l m o s t w h i t e , a l m o s t o d o r l e s s c r y s t a l l i n e s u b s t a n c e ( m e l t i n g p o i n t 128-204°C), s o l u b l e i n w a t e r , m e t h a n o l , e t h a n o l and c h l o r o f o r m a nd p r a c t i c a l l y i n s o l u b l e i n e t h e r ( P r o d u c t m o n o g r a p h , B o e h r i n g e r I n g e l h e i m L t d . , 1 9 8 6 ) . 5 2.2 Pharmacodynamics of M e x i l e t i n e 2.2.1 Pharmacology M e x i l e t i n e i s e f f e c t i v e i n the c o n t r o l of a v a r i e t y of v e n t r i c u l a r a r r h y t h m i a s . In animal s t u d i e s , i t was found t o a b o l i s h e x p e r i m e n t a l l y induced arrhythmias such as those i n i t i a t e d by ouabain, and c o r o n a r y - a r t e r y l i g a t i o n i n dogs ( A l l e n e t a l . , 1972), ouabain i n guinea p i g s (Singh and Vaughan-Williams, 1972), a d r e n a l i n e i n a n a e s t h e t i z e d dogs r e s p i r e d with halothane ( A l l e n e t a l . , 1972) , and e l e c t r i c a l s t i m u l a t i o n i n a n a e s t h e t i z e d r a t s ( M a r s h a l l , e t a l . , 1981). In man, m e x i l e t i n e e f f e c t i v e l y suppressed v e n t r i c u l a r e x t r a s y s t o l e s , v e n t r i c u l a r t a c h y c a r d i a and v e n t r i c u l a r f i b r i l l a t i o n (Campbell e t a 1 ., 1973 and T a l b o t et a l . , 1973). Other pharmacological p r o p e r t i e s of m e x i l e t i n e i n c l u d e s t r o n g l o c a l a n a e s t h e t i c (Singh and Vaughan-Williams, 1972 and Danneberg and S h e l l e y , 1977) and a n t i c o n v u l s a n t (Danneberg and S h e l l e y , 1977) a c t i o n s . 2.2.2 E l e c t r o p h y s i o l o g i c a l E f f e c t s on the Heart In t h e r a p e u t i c c o n c e n t r a t i o n s , m e x i l e t i n e b l o c k s the f a s t sodium channel thereby d e p r e s s i n g the maximal r a t e of d e p o l a r i z a t i o n (Vmax) or membrane responsiveness i n a t r i a l , v e n t r i c u l a r and P u r k i n j e a c t i o n p o t e n t i a l s (Vaughan-6 W i l l i a m s , 1977 and Yamaguchi e t a l . , 1979). The decrease i n Vmax i s accompanied by e l e c t r o p h y s i o l o g i c e f f e c t s which vary i n d i f f e r e n t t i s s u e s . In i s o l a t e d 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 t i s s u e p r e p a r a t i o n s , m e x i l e t i n e depressed conduction v e l o c i t y , i n c r e a s e d the t h r e s h o l d of e x c i t a b i l i t y and produced a marked p r o l o n g a t i o n of 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 ( A l l e n et a 1 ., 1972; S i n g h and Vaughan-Wi1liams, 1972; Vaughan-Wi1liams, 1977, and Chew e t  a l . , 1979). These a l t e r a t i o n s , o c c u r r i n g without a s i g n i f i c a n t change i n e i t h e r the r e s t i n g membrane p o t e n t i a l or a c t i o n - p o t e n t i a l d u r a t i o n , are a s s o c i a t e d with the d e p r e s s i o n of spontaneous d i a s t o l i c d e p o l a r i z a t i o n i n automatic f i b r e s . In i s o l a t e d P u r k i n j e f i b r e s of the dog, a c o n c e n t r a t i o n dependent decrease i n a c t i o n p o t e n t i a l d u r a t i o n accompanied by a s h o r t e n i n g of 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 was observed (Yamaguchi et a l . , 1979). However, the r a t i o of 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 to t h a t of the a c t i o n p o t e n t i a l d u r a t i o n was c o n s i s t e n t l y i n c r e a s e d . Thus, there i s l e s s time d u r i n g an a c t i o n p o t e n t i a l f o r an e c t o p i c impulse t o i n i t i a t e another a c t i o n p o t e n t i a l . In humans, the e l e c t r o p h y s i o l o g i c e f f e c t s of m e x i l e t i n e are somewhat v a r i a b l e . M e x i l e t i n e was found to have no c o n s i s t e n t e f f e c t on sinus r a t e , a t r i a l r e f r a c t o r i n e s s , AV or H i s - P u r k i n j e c o n d u c t i o n times (Roos 7 e t a l . , 1977 and McComish e t a l . , 1 9 7 7 ) ; t h e d r u g i n c r e a s e d t h e f u n c t i o n a l r e f r a c t o r y p e r i o d o f t h e AV node w i t h a v a r i a b l e e f f e c t on t h e e f f e c t i v e r e f r a c t o r y p e r i o d o f t h e H i s - P u r k i n j e s y s t e m , l e n g t h e n i n g i t i n some p a t i e n t s (Roos e t a l . , 1977) w h i l e s h o r t e n i n g i t i n o t h e r s (McComish e t a l . , 1 9 7 7 ) . H o w e v e r , when t h e H i s - P u r k i n j e c o n d u c t i o n i s i m p a i r e d by d i s e a s e , t h e d r u g a l w a y s l e n g t h e n s t h e H-V i n t e r v a l , s u g g e s t i n g t h a t m e x i l e t i n e may p r o d u c e AV b l o c k i n p a t i e n t s w i t h u n d e r l y i n g c o n d u c t i o n d i s t u r b a n c e s (Roos e t a l . , 1977 and S i n g h e t a l . , 1 9 8 1 ) . 2.2.3 Hemodynamic E f f e c t s S a u n a m a k i (1975) s t u d i e d t h e a d v e r s e hemodynamic e f f e c t s o f i n t r a v e n o u s m e x i l e t i n e i n s i x c a r d i a c p a t i e n t s and f o u n d no s i g n i f i c a n t c h a n g e s i n h e a r t r a t e , c a r d i a c o u t p u t , s y s t e m i c , p u l m o n a r y o r c a p i l l a r y wedge p r e s s u r e s , and p e r i p h e r a l o r p u l m o n a r y v a s c u l a r r e s i s t a n c e . C a r d i a c f u n c t i o n was d e p r e s s e d i n t h r e e o f t h e p a t i e n t s w i t h h i s t o r y o f c a r d i a c f a i l u r e . O t h e r s t u d i e s by P o z e n e l ( 1 9 7 7 ) , B a n i m e t a l . ( 1 9 7 7 ) , a n d Kuhn e t a l . (1977) o b s e r v e d no s i g n i f i c a n t c h a n g e s i n t h e hemodynamic p a r a m e t e r s a b o v e . B a n i m e t a l . , h o w e v e r , a l s o r e p o r t e d a s i g n i f i c a n t l y r e d u c e d c a r d i a c o u t p u t a n d p e a k l e f t v e n t r i c u l a r d p / d t ( r a t e o f d e v e l o p m e n t o f l e f t v e n t r i c u l a r p r e s s u r e ) d u r i n g 8 h a n d g r i p e x e r c i s e s u g g e s t i n g t h a t c a r d i a c d e p r e s s i o n had o c c u r r e d . A l l t h e s e s t u d i e s h a v e shown t h a t m e x i l e t i n e h a s m i n o r hemodynamic e f f e c t s p r o v i d e d t h e p l a s m a c o n c e n t r a t i o n i s m a i n t a i n e d w i t h i n t h e t h e r a p e u t i c r a n g e . 2 . 3 C l i n i c a l I n d i c a t i o n s M e x i l e t i n e i s e f f e c t i v e a g a i n s t v a r i o u s f o r m s o f v e n t r i c u l a r a r r h y t h m i a s ( B r a d l e y , 1983; P r o d u c t M o n o g r a p h , B o e h r i n g e r I n g e l h e i m L t d . , 1 9 8 6 ) . T h e s e i n c l u d e e p i s o d e s o f v e n t r i c u l a r t a c h y c a r d i a and p r e m a t u r e v e n t r i c u l a r e c t o p i c b e a t s s u c h a s u n i f o c a l p r e m a t u r e v e n t r i c u l a r b e a t s , c o u p l e t s , s a l v o s and R-on-T phenomenon. M e x i l e t i n e a l s o f i n d s u s e i n t h e p r o p h y l a x i s o f v e n t r i c u l a r a r r h y t h m i a s e s p e c i a l l y i n s u r v i v o r s o f a c u t e m y o c a r d i a l i n f a r c t i o n ( T a l b o t e t a l . , 1976) . 2 . 4 A d v e r s e E f f e c t s Common a d v e r s e r e a c t i o n s o f m e x i l e t i n e i n c l u d e s u c h c e n t r a l n e r v o u s e f f e c t s as t r e m o r , d i z z i n e s s and l i g h t h e a d e d n e s s ; g a s t r o i n t e s t i n a l e f f e c t s s u c h a s n a u s e a , v o m i t i n g and d y s p e p s i a ( B r a d l e y , 1983) . The c a r d i o v a s c u l a r e f f e c t s a r e h y p o t e n s i o n , b r a d y c a r d i a and f i b r i l l a t i o n ( B r a d l e y , 1 9 8 3 ) . T h e s e a d v e r s e e f f e c t s a r e u s u a l l y m i l d , and r e d u c i n g t h e d o s e , o r t a k i n g t h e m e d i c a t i o n w i t h f o o d w i l l e l i m i n a t e o r r e d u c e t h e i r s e v e r i t y . 9 2.5 Pharmacokinetics of M e x i l e t i n e 2.5.1 Healthy V o l u n t e e r Studies M e x i l e t i n e i s w e l l absorbed 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 , with peak plasma l e v e l s a c hieved w i t h i n 2 to 4 hours (Singh e t a l . , 1981). B i o a v a i l a b i l i t y i s approximately 90% ( P r e s c o t t e t a l . , 1977, H a s e l b a r t h e t a 1 . , 1981; and O h a s h i e t a l . , , 1 9 8 4 ) . The t o t a l volume o f d i s t r i b u t i o n i s l a r g e and h i g h l y v a r i a b l e , 5.5 to 9.5 L/Kg ( P r e s c o t t e t a l . , 1977; H a s e l b a r t h e t a l . , 1981; Campbell e t a l . , 1978a and 1978b) r e f l e c t i n g e x t e n s i v e t i s s u e uptake of the drug. The d i s p o s i t i o n k i n e t i c s are best d e s c r i b e d by a three-compartment model c o n s i s t e n t with a f a s t and slow d i s t r i b u t i o n phase, and a much slower e l i m i n a t i o n phase ( P r e s c o t t e t a l . , 1977 and H a s e l b a r t h et a l . , 1981). The e l i m i n a t i o n h a l f - l i f e ranges from 6.3 to 11.8 hours (mean 10 hours) ( P r e s c o t t et a l . , 1977; H a s e l b a r t h et a l . , 1981; Campbell e t a l . , 1978b and D a n i l o , 1979). T o t a l body c l e a r a n c e i s l a r g e , 6.5 to 7.1 mL/min/Kg (Paalman e t a l . , 1977 and Campbell e t a l . , 1978a) and the mean r e n a l c l e a r a n c e i s 49 _+ 25 mL/min ( P r e s c o t t e t a l . , 1977). The r e n a l c l e a r a n c e of m e x i l e t i n e i s i n c r e a s e d by u r i n a r y a c i d o s i s . At a u r i n a r y pH of 5, r e n a l e x c r e t i o n i s r e p o r t e d to i n c r e a s e by 40 to 1 0 60%, and r e n a l c l e a r a n c e by a f a c t o r of 3 t o 4 ( K i d d i e e t a l . , 1974 and P r e s c o t t e t a l . , 1977). However, the e f f e c t of normal p h y s i o l o g i c a l f l u c t u a t i o n i n u r i n a r y pH i s not c o n c l u s i v e . W h i l e P r e s c o t t e t a l . , (1977) r e p o r t e d t h a t normal u r i n a r y pH f l u c t u a t i o n i s c l i n i c a l l y u n i m p o r t a n t , J o h n s t o n e t a l . , (1979) found a s i g n i f i c a n t change i n u r i n a r y e x c r e t i o n and an i n c r e a s e of up t o 50% i n plasma c o n c e n t r a t i o n s of m e x i l e t i n e due t o spontaneous f l u c t u a t i o n s i n u r i n a r y pH. The o p t i m a l plasma c o n c e n t r a t i o n range of m e x i l e t i n e i s 0.5 t o 2.0 ug/mL ( T a l b o t e t a l . , 1973 and Campbell e t a l . , 1975) w h i c h i s u s u a l l y a c h i e v e d w i t h o r a l doses of 200 t o 400 mg o f m e x i l e t i n e h y d r o c h l o r i d e g i v e n e v e r y s i x t o e i g h t h o u r s . For i n t r a v e n o u s d o s i n g P r e s c o t t e t a l . (19 77) recommended 150 t o 200 mg o v er 2 t o 5 m i n u t e s , f o l l o w e d by a s t e a d i l y d e c r e a s i n g l o a d i n g i n f u s i o n o v er 11 hours (250 mg i n 30 m i n u t e s , 260 mg i n 2.5 h o u r s , 500 mg i n 8 h o u r s ) , and a maintenance dose of 500 mg t o 1000 mg over 24 h o u r s . T h i s dosage regimen g i v e s adequate plasma c o n c e n t r a t i o n w i t h o u t s i g n i f i c a n t s i d e e f f e c t s . 2.5.2 E f f e c t s of D i s e a s e on P h a r m a c o k i n e t i c s Many d i s e a s e c o n d i t i o n s a f f e c t the p h a r m a c o k i n e t i c s of m e x i l e t i n e . P a t i e n t s w i t h h e p a t i c impairment ( c i r r h o s i s ) 11 have been r e p o r t e d to have decreased plasma c l e a r a n c e of m e x i l e t i n e ( N i t s c h e t a l . , 1981). T h i s i s expected s i n c e the l i v e r i s the major s i t e of m e x i l e t i n e metabolism. Renal d i s e a s e have an i n s i g n i f i c a n t e f f e c t on c l e a r a n c e except at a c r e a t i n i n e c l e a r a n c e lower than 10 mL/min (EL A l l a f e t  a l . , 1982). Delayed and incomplete a b s o r p t i o n as w e l l as i n c r e a s e d e l i m i n a t i o n h a l f - l i f e , were a s s o c i a t e d w i t h acute myocardial i n f a r c t i o n ( P r e s c o t t et a l . , 1977, Pottage e t  a l . , 1978 and P e n t i k a i n e n e t a l . , 1984). P r o l o n g a t i o n of e l i m i n a t i o n h a l f - l i f e was a l s o observed i n p a t i e n t s f r e e of h e p a t i c or r e n a l d y s f u n c t i o n but wit h severe l e f t v e n t r i c u l a r f a i l u r e (Leahey e t a l . , 1980a). 2.6 Serum C o n c e n t r a t i o n - C l i n i c a l E f f e c t R e l a t i o n s h i p s The r e l a t i o n s h i p between plasma c o n c e n t r a t i o n s of m e x i l e t i n e and c l i n i c a l e f f e c t s was i n v e s t i g a t e d i n 149 p a t i e n t s by Campbell e t a l . , (1978a). Suppression 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 was observed i n 77% of the p a t i e n t s when plasma c o n c e n t r a t i o n was maintained between 0.74 and 1.0 ug/mL. An 80% response was seen when plasma c o n c e n t r a t i o n s were 2 ug/mL or above, however adverse e f f e c t s developed i n 30% of r e c i p i e n t s a t t h i s l e v e l with 19% d e v e l o p i n g severe adverse r e a c t i o n s such as hypote n s i o n , v o m i t i n g , tremor, t o x i c c o n f u s i o n a l s t a t e s and 12 a t r i o v e n t r i c u l a r d i s s o c i a t i o n . T a l b o t e t a l . , (1973) e a r l i e r r e p o r t e d m e x i l e t i n e c o n c e n t r a t i o n s of 0.5 to 2.0 ug/mL i n 37 p a t i e n t s having g r e a t e r than 95% r e d u c t i o n of v e n t r i c u l a r e c t o p i c b e a t s . There was, however, no c l e a r s e p a r a t i o n between t h e r a p e u t i c and t o x i c ranges i n the study s i n c e adverse e f f e c t s sometimes o c c u r r e d at c o n c e n t r a t i o n s as low as 0.3 t o 1.0 ug/mL. S t u d i e s on the e f f i c a c y of long term o r a l m e x i l e t i n e treatment showed t h a t adequate supp r e s s i o n of v e n t r i c u l a r arrhythmias was a s s o c i a t e d with serum c o n c e n t r a t i o n s of 0.9 to 2.6 ug/mL (Talbot e t a l . , 1976). F u r t h e r s t u d i e s d u r i n g s h o r t term o r a l m e x i l e t i n e -t e s t i n g i n cases with p e r s i s t i n g v e n t r i c u l a r e c t o p i c beats r e p o r t e d m e x i l e t i n e l e v e l s between 0.38 to 2.76 ug/mL i n 30 responders whose premature v e n t r i c u l a r c o n t r a c t i o n s were reduced by more than 50%. E f f e c t i v e plasma c o n c e n t r a t i o n s d u r i n g maintenance treatment were i n the same range, 0.44 to 2.0 ug/mL (Podri d and Lown, 1981). However, serum m e x i l e t i n e c o n c e n t r a t i o n s i n non-responding p a t i e n t s were not d i f f e r e n t from those of the responders. The t h e r a p e u t i c margin f o r m e x i l e t i n e i s narrow, 0.5 to 2.0 ug/mL (Talbot e t a l . , 1973 and Campbell e t a l . , 1975) and s i d e e f f e c t s sometimes occur a t c o n c e n t r a t i o n s w i t h i n t h i s range. C o n c e n t r a t i o n s g r e a t e r than 2.0 ug/mL are u s u a l l y a s s o c i a t e d with marked s i d e e f f e c t s . 13 2.7 C o m p a r i s o n w i t h o t h e r A n t i a r r h y t h m i c A g e n t s M e x i l e t i n e h a s b e e n r e p o r t e d t o p o s s e s s c o m p a r a b l e e f f i c a c y and t o l e r a n c e w i t h t h a t o f q u i n i d i n e a g a i n s t v a r i o u s f o r m s o f v e n t r i c u l a r a r r h y t h m i a s i n p a t i e n t s w i t h d i v e r s e h e a r t d i s e a s e s ( S i n g h e t a l . , 1 9 8 4 ) . C o m p a r a t i v e s t u d i e s h a v e shown m e x i l e t i n e t o be more e f f e c t i v e t h a n l i d o c a i n e ( C a m p b e l l e t a l . , 1973 and H o r o w i t z e t a l . , 1982) and p r o c a i n a m i d e ( T r i m a r c o e t a l . , 1 9 8 3 ) , and e q u a l l y as e f f e c t i v e as d i s o p y r a m i d e ( T r i m a r c o e t a l . , 1983) , i n t h e c o n t r o l o f v e n t r i c u l a r a r r h y t h m i a s . The g r e a t e r e f f i c a c y o f m e x i l e t i n e r e p o r t e d a b o v e was n o t a s s o c i a t e d w i t h i n c r e a s e d d r u g t o x i c i t y . P r o c a i n a m i d e h a s b e e n shown t o i n d u c e a s i g n i f i c a n t d r o p i n s y s t o l i c b l o o d p r e s s u r e , w h i l e d i s o p y r a m i d e p r o d u c e d m a r k e d i n o t r o p i c e f f e c t s t h a t w e r e s i g n i f i c a n t l y g r e a t e r t h a n t h a t o f m e x i l e t i n e (Angermann and J a h r m a r k e r , 1983).. I t a p p e a r s t h a t m e x i l e t i n e i s g e n e r a l l y w e l l t o l e r a t e d by c a r d i a c p a t i e n t s w i t h m i n i m a l a d v e r s e hemodynamic e f f e c t s r e l a t i v e t o t h e o t h e r a n t i a r r h y t h m i c a g e n t s . 2.8 M e t a b o l i s m M e x i l e t i n e i s e l i m i n a t e d f r o m t h e b o d y by e x t e n s i v e h e p a t i c m e t a b o l i s m , and r e n a l e x c r e t i o n o f t h e m e t a b o l i t e s and u n c h a n g e d d r u g ( B e c k e t t and C h i d o m e r e , 1 9 7 7 a , b ) . P r e -14 systemic e l i m i n a t i o n i s n e g l i g i b l e ( l e s s than 10%) which p a r t i a l l y e x p l a i n s the o r a l e f f i c a c y of m e x i l e t i n e (Baudinet e t a l . , 1980). The major m e t a b o l i t e s are parahydroxy-m e x i l e t i n e , h y d r o x y m e t h y l m e x i l e t i n e , and t h e i r c o r r e s p o n d i n g a l c o h o l s and the conjugate of m e x i l e t i n e which i s h y d r o l y z e d by 3 - g l u c u r o n i d a s e (Beckett and Chidomere, 1977b; P r e s c o t t e t a l . , 1977; and Grech-Belanger e t a l . , 1985b) . Minor metabolic routes i n c l u d e N - o x i d a t i o n to the hydroxylamine, N-methylation and deamination to the ketone (Beckett and Chidomere, 1977a,b). None of these m e t a b o l i t e s has been shown to possess any a n t i a r r h y t h m i c a c t i v i t y (Brown and Shand, 1982) . Approximately 8 to 10% of the a d m i n i s t e r e d dose i s recovered unchanged i n u r i n e of h e a l t h y v o l u n t e e r s i n t h r e e days ( P r e s c o t t e t a 1 . , 19 77 , H a s e l b a r t h e t a 1., 1981, and Singh e t a l . , 1981). 2.9 I n t e r a c t i o n s of M e x i l e t i n e with Other Drugs The p o t e n t i a l f o r i n t e r a c t i o n of m e x i l e t i n e and other drugs i s g r e a t because p a t i e n t s who have s i g n i f i c a n t v e n t r i c u l a r arrhythmias o f t e n have h e a r t d i s e a s e and other c a r d i o v a s c u l a r problems. Thus these p a t i e n t s c o n c o m i t a n t l y r e c e i v e other c a r d i o v a s c u l a r as w e l l as n o n - c a r d i o v a s c u l a r drugs. M e x i l e t i n e a b s o r p t i o n a f t e r o r a l dosing has been shown to be delayed by drugs which slow g a s t r i c emptying 15 s u c h a s n a r c o t i c a n a l g e s i c s ( P r e s c o t t e t a l . , 1 9 7 7 ) , a n t a c i d s ( H e r z o g e t a l . , 1 9 8 2 ) , and a t r o p i n e - l i k e a g e n t s (Wing e t a l . , 1 9 8 0 ) . M e t o c l o r p r a m i d e , on t h e o t h e r h a n d , i n c r e a s e s t h e a b s o r p t i o n r a t e (Wing e t a l . , 1 9 8 0 ) . B i o -a v a i l a b i l i t y , h o w e v e r , i s n o t a l t e r e d by any o f t h e s e a g e n t s . M e x i l e t i n e h a s no e f f e c t on d i g o x i n s e r u m l e v e l s ( L e a h e y e t a l . , 1 9 8 0 b ) . T h i s i s i n c o n t r a s t t o many a n t i a r r h y t h m i c d r u g s i n c l u d i n g a m i o d a r o n e , p r o p a f e n o n e , q u i n i d i n e , a nd v e r a p a m i l w h i c h a r e known t o i n c r e a s e t h e p l a s m a c o n c e n t r a t i o n s o f d i g o x i n ( H a g e r e t a l . , 1979; L e a h e y e t a l . , 1 9 8 1 ; Moysey e t a l . , 1 9 81; K l e i n e t  a l . , 1982 ; P e d e r s e n e t a l . , 1981 and P e d e r s e n e t a l . , 19 8 2 ) . S i n c e m e x i l e t i n e u n d e r g o e s e x t e n s i v e h e p a t i c m e t a b o l i s m i n man, d r u g s w h i c h i n d u c e t h e h e p a t i c enzyme s y s t e m s a r e e x p e c t e d t o e n h a n c e i t s n o n - r e n a l c l e a r a n c e . T h i s h a s b e e n r e p o r t e d f o r r i f a m p i c i n ( P e n t i k a i n e n e t a l . , 1982) and p h e n y t o i n (Begg e t a l . , 1 9 8 2 ) . C i m e t i d i n e , c o n t r a r y t o t h e e x p e c t e d i n h i b i t i o n o f m e x i l e t i n e m e t a b o l i s m , was r e p o r t e d t o h a v e no e f f e c t on t h e d i s p o s i t i o n o f o r a l m e x i l e t i n e i n n o r m a l s u b j e c t s ( K l e i n e t  a l . , 1985) . L e a h e y e t a l . (1980c) o b s e r v e d a b e n e f i c i a l d r u g i n t e r a c t i o n b e t w e e n m e x i l e t i n e a nd t h e 3 - a d r e n e r g i c r e c e p t o r b l o c k i n g d r u g , p r o p r a n o l o l . They showed t h a t m e x i l e t i n e a nd p r o p r a n o l o l a r e e f f e c t i v e i n s u p p r e s s i n g 16 v e n t r i c u l a r premature d e p o l a r i z a t i o n and v e n t r i c u l a r t a c h y c a r d i a when m e x i l e t i n e alone had f a i l e d to do so. P r o p r a n o l o l d i d not s i g n i f i c a n t l y i n c r e a s e the plasma c o n c e n t r a t i o n of m e x i l e t i n e i n t h i s study, hence the authors concluded t h a t m e x i l e t i n e and p r o p r a n o l o l had " c o o p e r a t i v e " a n t i a r r h y t h m i c e f f e c t s - i n v o l v i n g a pharmacodynamic i n t e r a c t i o n . A s i m i l a r b e n e f i c i a l i n t e r a c t i o n between m e x i l e t i n e and q u i n i d i n e has been r e p o r t e d (Duff e t a l . , 1983) . 2.10 M e x i l e t i n e Serum P r o t e i n B i n d i n g M e x i l e t i n e i s 70% bound t o plasma p r o t e i n s ( T a l b o t e t a l . , 1973). The amount bound to plasma p r o t e i n s a f t e r d i s t r i b u t i o n i s small i n r e l a t i o n to the t o t a l amount of drug i n the body; l e s s than 1% of drug remains i n blood ( P r e s c o t t e t a l . , 1977). The r e l a t i v e b i n d i n g of m e x i l e t i n e enantiomers to plasma p r o t e i n s has not been i n v e s t i g a t e d y e t . 2.11 S t e r e o s e l e c t i v i t y i n Drug D i s p o s i t i o n The enantiomers of a c h i r a l drug are c h a r a c t e r i z e d by the same p h y s i c a l and chemical p r o p e r t i e s except f o r the manner i n which they r o t a t e plane p o l a r i z e d l i g h t . However, when enantiomers i n t e r a c t with c h i r a l molecules i n the body 17 ( p r o t e i n s , m e t a b o l i s i n g enzymes and r e c e p t o r s ) , d i f f e r i n g a f f i n i t i e s f o r t h e s e m o l e c u l e s c a n r e s u l t i n d i f f e r i n g p h a r m a c o d y n a m i c a n d / o r p h a r m a c o k i n e t i c p r o p e r t i e s . Many s t u d i e s , u s i n g i s o l a t e d t i s s u e s and i n t a c t a n i m a l s h a v e c l e a r l y d e m o n s t r a t e d t h e s t e r e o s e l e c t i v e n a t u r e o f t h e i n t e r a c t i o n o f d r u g e n a n t i o m e r s w i t h c e l l u l a r c o m p o n e n t s ( P a t i l e t a l . , 1970; S a s t r y , 1973; A r i e n s e t a l . , 1983 and S m i t h , 1 9 8 4 ) . F o r e x a m p l e , R ( - ) - n o r a d r e n a l i n e i s 300 t i m e s more p o t e n t t h a n S ( + ) - n o r a d r e n a l i n e as an a d r e n e r g i c r e c e p t o r a g o n i s t on t h e r a b b i t a o r t a due t o i t s h i g h e r b i n d i n g a f f i n i t y f o r t h e r e c e p t o r ( P a t i l , 1 9 7 1 ) . H o w e v e r , most o f t h e b u l k o f i n f o r m a t i o n i n t h e l i t e r a t u r e c o n c e r n s s t e r e o s e l e c t i v e d r u g d i s p o s i t i o n o r i g i n a t i n g f r o m d i f f e r e n c e s i n t h e p h a r m a c o k i n e t i c s o f e n a n t i o m e r s . T h i s i s m a i n l y b r o u g h t a b o u t by d i f f e r e n c e s i n t h e i r a b s o r p t i o n , d i s t r i b u t i o n , m e t a b o l i s m a n d / o r e x c r e t i o n c h a r a c t e r i s t i c s . 2.11.1 A b s o r p t i o n S t e r e o s e l e c t i v i t y i n d r u g a b s o r p t i o n i s n o t e x p e c t e d s i n c e e n a n t i o m e r s h a v e t h e same l i p i d and a q u e o u s s o l u b i l i t i e s , e x c e p t f o r d r u g s t h a t u n d e r g o a c t i v e t r a n s p o r t a c r o s s t h e i n t e s t i n a l m u c o s a . E x a m p l e s a r e L - d o p a (Wade e t  a l . , 1973) a n d L - m e t h o t r e x a t e ( H e n d e l a n d B r o d t h a g e n , 1984) b o t h o f w h i c h h a v e b e e n shown t o be more r a p i d l y a b s o r b e d 18 than the D-isomers. Both drugs are admi n i s t e r e d c l i n i c a l l y as the L-enantiomer. 2.11.2 D i s t r i b u t i o n The enantiomers of a drug may be d i s t r i b u t e d d i f f e r e n t l y w i t h i n the body as a r e s u l t of d i f f e r e n c e s i n e i t h e r t i s s u e or plasma p r o t e i n b i n d i n g . T i s s u e b i n d i n g would u s u a l l y i n c r e a s e the e l i m i n a t i o n h a l f - l i f e of the more h i g h l y bound enantiomer. T h i s i s the case f o r S ( - ) - p r o p r a n o l o l which has been shown to be more r a p i d l y taken up by the he a r t than i t s antipode i n r a t s (Kawashima e t a l . , 1976). The e x t r a v a s c u l a r b i n d i n g of S ( - ) - p r o p r a n o l o l has a l s o been found to be g r e a t e r than t h a t of R(+)-propranolol i n the dog (Bai e t a l . , 1983). S i m i l a r l y , a study of the t i s s u e d i s t r i b u t i o n of the enantiomers of t i m o l o l i n the r a t showed t h a t ( - ) - t i m o l o l was taken up much more a v i d l y by the p a r t i c u l a t e f r a c t i o n of h e a r t , lung, and b r a i n than the (+)-enantiomer, which was bound only to n o n - s p e c i f i c s i t e s (Tocco et a l . , 1976) . The asymmetric nature of the plasma p r o t e i n s , human serum albumin (HSA) and a l p h a s - a c i d g l y c o p r o t e i n (AAG) g i v e r i s e t o s t e r e o s e l e c t i v i t y i n the manner i n which they b i n d drugs. The f i r s t s t e r e o s e l e c t i v e b i n d i n g i n t e r a c t i o n d e s c r i b e d f o r human serum albumin was with tryptophan 19 (McMenamy and O n c l e y , 1958) . L - t r y p t o p h a n was f o u n d t o b i n d w i t h an a f f i n i t y a b o u t 100 t i m e s t h a t o f D - t r y p t o p h a n . O t h e r e x a m p l e s i n c l u d e o xazepam h e m i s u c c i n a t e whose ( + ) - e n a n t i o m e r b i n d s t o HSA w i t h an a f f i n i t y 40 t i m e s t h a t o f t h e ( - ) - e n a n t i o m e r ( M u l l e r and W o l l e r , 1975) and S - w a r f a r i n w h i c h h a s a s l i g h t l y h i g h e r a f f i n i t y t h a n R - w a r f a r i n (Brown e t a l . , 1 9 7 7 ) . A l e b i c K o l b a h e t a l . , (1979) r e v i e w e d t h e s t e r e o s e l e c t i v e b i n d i n g o f d r u g e n a n t i o m e r s t o HSA. I t a p p e a r s n o t much a t t e n t i o n h a s b e e n p a i d t o t h i s a s p e c t o f p r o t e i n b i n d i n g . The b a s i c d r u g s b i n d m a i n l y t o a l p h a s - a c i d g l y c o p r o t e i n ( P i a f s k y and K n o p p e r t , 1978) and r e c e n t e v i d e n c e shows t h a t t h i s b i n d i n g i s a l s o s t e r e o s e l e c t i v e . AAG h a s b e e n r e p o r t e d t o f a v o u r t h e b i n d i n g o f S - p r o p r a n o l o l o v e r R - p r o p r a n o l o l ( B a i e t a l . , 19 83 ; W a l l e e t a l . , 198 3 a n d A l b a n i e t a l . , 198 4 ) , and S - d i s o p y r a m i d e o v e r i t s R - e n a n t i o m e r ( L i m a e t a l . , 1984 and V a l d i v i e s o e t a l . , 1983) . 2.11.3 M e t a b o l i s m One o f t h e e a r l i e s t o b s e r v a t i o n s o f s t e r e o s e l e c t i v e d r u g m e t a b o l i s m i n man was t h a t o f t h e h y p n o t i c d r u g R , S - h e x o b a r b i t a l ( B r e i m e r and Rossum, 1 9 7 3 ) . R , S - h e x o -b a r b i t a l i s e l i m i n a t e d f r o m t h e b o d y e n t i r e l y by o x i d a t i v e m e t a b o l i s m . When t h e s e p a r a t e e n a n t i o m e r s w e r e a d m i n i s t -20 ered, a very c l e a r c e n t r a l depressant e f f e c t was seen a f t e r S - h e x o b a r b i t a l but not a f t e r R - h e x o b a r b i t a l which was c o n s i s t e n t with potency f i n d i n g s i n r a t s . However, i t was a l s o shown t h a t the c l e a r a n c e of S - h e x o b a r b i t a l was o n l y o n e - t h i r d of t h a t of R - h e x o b a r b i t a l . Thus, the plasma c o n c e n t r a t i o n s of S - h e x o b a r b i t a l were h i g h e r , and the h a l f - l i f e longer than f o r R - h e x o b a r b i t a l . Although the i n t r i n s i c a c t i v i t y of the S-enantiomer may be g r e a t e r than t h a t of i t s antipode i n man, the authors e x p l a i n e d the potency d i f f e r e n c e s on the b a s i s of s t e r e o s e l e c t i v i t y i n h e p a t i c o x i d a t i o n . An i n t e r e s t i n g s t e r e o s e l e c t i v e f i r s t - p a s s metabolism was r e p o r t e d f o r verapamil by V o g e l g e s a n g e t a 1 ., (1984). They showed t h a t the s y s t e m i c a v a i l a b i l i t y of the more a c t i v e (-)- enantiomer was 2 to 3 times s m a l l e r than t h a t of the (+)-enantiomer. Thus, t h e i r data e x p l a i n e d the e a r l i e r d i s c r e p a n c y observed by Eichelbaum e t a l . (1980) t h a t (+_) - v e r a p a m i l ' s e f f e c t on a t r i o v e n t r i c u l a r conduction i n man i s 2 to 3 f o l d l e s s a f t e r o r a l than a f t e r I.V. doses, when doses which produce equal plasma c o n c e n t r a t i o n s of racemic drug were used. A s i m i l a r enantiomeric d i f f e r e n c e i n presystemic metabolism has been r e p o r t e d f o r p r o p r a n o l o l i n dogs (Walle and Walle, 1979) and i n man ( S i l b e r and Riegelman, 1980; and Von Barh et a l . , 1982). In v i t r o s t u d i e s u s i n g human l i v e r 21 microsomes have shown t h a t o x i d a t i o n of p r o p r a n o l o l to 4-hydroxypropranolol and n o r p r o p r a n o l o l f a v o r s the R-enantiomer while g l u c u r o n i d a t i o n i s s t e r e o s e l e c t i v e f o r the S-enantiomer. Numerous other examples of e n a n t i o s e l e c t i v e metabolism have been r e p o r t e d by Jenner and Testa (1980). A novel s t e r e o s p e c i f i c metabolic pathway known as " c h i r a l i n v e r s i o n of c o n f i g u r a t i o n " i s a s s o c i a t e d with the metabolic c l e a r a n c e of the i n a c t i v e R(-)-enantiomer of the 2 - a r y l p r o p i o n i c a c i d s . The i n v i t r o potency d i f f e r e n c e s between the enantiomers of these a c i d s were found to be d r a m a t i c a l l y g r e a t e r than the d i f f e r e n c e s i n i n v i v o models. For example, the S(+)-enantiomer of i b u p r o f e n i s 160 times more potent than the R(-)-enantiomer i n p r o h i b i t i n g p r o s t a g l a n d i n synthetase i n v i t r o whereas the i n v i v o potency r a t i o i s o n l y 1.4 (Adams e t a l . , 1976). When the R-enantiomer was a d m i n i s t e r e d i n man, there was a r a p i d appearance of the S-enantiomer i n the blood, whereas no R-i b u p r o f e n was observed a f t e r a d m i n i s t r a t i o n of S-ibuprofen (Lee e t a l . , 1985). These o b s e r v a t i o n s , together w i t h e a r l i e r data showing t h a t u r i n a r y m e t a b o l i t e s f o l l o w i n g a d m i n i s t r a t i o n of (_+)-ibuprofen were predominantly d e x t r o r o t a t o r y l e d t o the c o n c l u s i o n t h a t a m e t a b o l i c c h i r a l i n v e r s i o n of the i n a c t i v e R ( - ) - i b u p r o f e n to the a c t i v e S(+)-22 enantiomer was t a k i n g p l a c e i n v i v o (Lee e t a l . , 1985) . S t e r e o s p e c i f i c metabolic c h i r a l i n v e r s i o n has a l s o been r e p o r t e d f o r c i c l o p r o f e n ( K r i p a l a n i e t a l . , 1976 and Lan et a l . , 1976) and benoxaprofen (Bopp e t a l . , 1979 and Simmonds et a l . , 1980). 2.11.4 Renal E x c r e t i o n S t e r e o s e l e c t i v e r e n a l e x c r e t i o n may occur f o r a drug which i s a c t i v e l y s e c r e t e d and/or reabsorbed but there are y e t no p u b l i s h e d examples. S t e r e o s e l e c t i v i t y i n f i l t r a t i o n at the glomerulus w i l l depend mainly on s t e r e o s e l e c t i v e d i f f e r e n c e s i n unbound ( i . e . f i l t e r a b l e ) l e v e l s of the drug (Williams and Lee, 1985) 2.12 S t e r e o s e l e c t i v e Drug A n a l y s i s The' t r a d i t i o n a l and c l a s s i c a l method of r e s o l u t i o n of enantiomers i n v o l v e d t h e i r c o n v e r s i o n to d i a s t e r e o i s o m e r i c s a l t s by r e a c t i o n with an o p t i c a l l y a c t i v e compound and subsequent r e s o l u t i o n by f r a c t i o n a l r e c r y s t a l l i z a t i o n . R e s o l u t i o n i s p o s s i b l e because d i a s t e r e o i s o m e r s have d i f f e r e n t chemical and p h y s i c a l p r o p e r t i e s i n c l u d i n g s o l u b i l i t y i n a g i v e n s o l v e n t . However, t h i s t r a d i t i o n a l method i s o f t e n l a b o r i o u s , i n e f f i c i e n t , does not always y i e l d o p t i c a l l y pure samples, and i s e s s e n t i a l l y r e s t r i c t e d 23 t o c a r b o x y l i c a c i d s a n d a m i n e s ( B l a s c h k e , 1 9 8 0 ) . C u r r e n t l y , c h r o m a t o g r a p h i c methods a r e t h e methods o f c h o i c e f o r t h e r e s o l u t i o n o f e n a n t i o m e r s . They h a v e t h e a d v a n t a g e s o f s m a l l s a m p l e s i z e , s p e e d , e f f i c i e n c y , i n d e p e n d e n c e f r o m t h e m a g n i t u d e o f s p e c i f i c r o t a t i o n and o t h e r o p t i c a l l y a c t i v e s p e c i e s i n i t i a l l y p r e s e n t i n t h e s a m p l e , a nd w i d e r a p p l i c a -b i l i t y ( L o c h m u l l e r and S o u t e r , 1 9 7 5 ) . E n a n t i o m e r s c a n be r e s o l v e d c h r o m a t o g r a p h i c a l l y e i t h e r a s a m i x t u r e o f d i a s t e r e o i s o m e r s on an a c h i r a l s t a t i o n a r y p h a s e o r d i r e c t l y on a c h i r a l s t a t i o n a r y p h a s e . 2.12.1 R e s o l u t i o n o f E n a n t i o m e r s as D i a s t e r e o i s o m e r s W i t h p r o p e r d e r i v a t i z a t i o n u s i n g c h i r a l r e a g e n t s , e n a n t i o m e r s c a n be s e p a r a t e d as d i s a s t e r e o i s o m e r s on an a c h i r a l s t a t i o n a r y p h a s e . E x a m p l e s i n c l u d e t h e r e s o l u t i o n o f t o c a i n i d e e n a n t i o m e r s w i t h S ( - ) - 2 - m e t h o x y - 2 - t r i f l u o r o -m e t h y l p h e n y l a c e t y l c h l o r i d e ( M T P A - c l ) by GLC ( G a l e t a l . , 1 9 8 2 ) ; a m p h e t a m i n e s and e p h e d r i n e s w i t h S ( - ) - 1 - [ ( 4 - n i t r o -p h e n y l ) s u l f o n y l ] p r o l y l c h l o r i d e by HPLC ( C l a r k a n d B a r k s d a l e , 1 9 8 4 ) ; t h e a r y l p r o p i o n i c a c i d s w i t h S ( + ) - 2 - o c t a n o l by HPLC ( J o h n s o n e t a l . 1979 and L e e e t a l . , 1984) and m e x i l e t i n e w i t h 2 , 3 , 4 , 6 - t e t r a - 0 - a c e t y 1 - B - D - g l u c o p r a n o s y 1 i s o t h i o c y a n a t e (GITC) b y HPLC ( G r e c h - B e l a n g e r e t a l . , 1985a) . F u r t h e r e x a m p l e s c a n be f o u n d i n t h e a r t i c l e s by 24 T a m e g a i e t a l . ( 1 9 7 9 ) , L o c k m u l l e r a n d S o u t e r ( 1 9 7 5 ) , and L i n d n a (1982) . C o n s i d e r a b l e a d v a n c e s t o w a r d s u n d e r s t a n d i n g t h e s e p a r a t i o n m e c h a n i s m f o r d i a s t e r e o i s o m e r s was made by Rose e t a 1 . , ( 1966 ) . I n t h e i r s t u d y w i t h d i a s t e r e o m e r i c e s t e r s o f 2 - a c e t o x y p r o p i o n i c a c i d s , t h e y o b s e r v e d t h a t t h e s i z e o f t h e s u b s t i t u e n t s a t t h e e s t e r a s y m m e t r i c c a r b o n , t h e d i s t a n c e b e t w e e n t h e o p t i c a l c e n t e r s i n t h e e s t e r , a s w e l l as t h e p o l a r i t y o f t h e s t a t i o n a r y p h a s e were c r i t i c a l f o r r e s o l u t i o n . H o w e v e r , t h e r e a r e many d r a w b a c k s a s s o c i a t e d w i t h t h e r e s o l u t i o n o f e n a n t i o m e r s a s d i a s t e r e o i s o m e r s . T h e s e a r e t h e r e q u i r e m e n t o f an a c t i v e f u n c t i o n a l g r o u p f o r t h e f o r m a t i o n o f t h e d i a s t e r e o m e r i c d e r i v a t i v e s , d i f f e r e n c e s i n r e a c t i o n k i n e t i c s o f e n a n t i o m e r s w i t h c h i r a l r e a g e n t s w h i c h c o u l d l e a d t o e r r o r s i n q u a n t i t a t i v e a n a l y s i s , a n d t h e r e q u i r e m e n t o f an o p t i c a l l y p u r e r e a g e n t ( F r a n k e t a l . , 1978, K o n i g e t a l . , 1977 and L i u and Ku, 1 9 8 3 ) . I n a d d i t i o n , t h e d i a s t e r e o i s o m e r s must be c h e m i c a l l y a n d s t e r e o c h e m i c a l l y s t a b l e u n d e r t h e c h r o m a t o g r a p h i c c o n d i t i o n s . 2.12.2 D i r e c t R e s o l u t i o n o f E n a n t i o m e r s on C h i r a l S t a t i o n a r y P h a s e s T h i s i s a more d e s i r a b l e a p p r o a c h t o e n a n t i o m e r r e s o l u t i o n s i n c e i t d o e s n o t h a v e t h e d r a w b a c k s l i s t e d 25 above. I t i n v o l v e s the formation of t r a n s i e n t d i a s t e r e o -meric complexes (no c o v a l e n t bonds) between the s o l u t e and the c h i r a l s t a t i o n a r y phase (CSP). The r e l a t i v e s t a b i l i t y of these complexes r e s u l t s i n d i f f e r e n t r a t e s of e l u t i o n of the enantiomers. The minimum requirement f o r a s e p a r a t i o n to be achieved on a CSP. i s three simultaneous i n t e r a c t i o n s , one o f which must be s t e r e o c h e m i c a l l y dependent, between the s o l u t e and the CSP ( D a l g l i e s h , 1952) . These are u s u a l l y i n the form of pi-bonding, e l e c t r o s t a t i c bonding, hydrogen bonding and s t e r i c i n t e r a c t i o n s . Enormous s t r i d e s have been made i n rec e n t years i n the f i e l d of c h i r a l bonded s t a t i o n a r y phases l e a d i n g to the commercial a v a i l a b i l i t y of a number of them. C h i r a s i l -V a l ( L - v a l i n e - t e r t - b u t y l a m i d e - c a r b o x y a l k y 1 - m e t h y 1 -s i l o x a n e ) has been used s u c c e s s f u l l y to separate the enantiomers of t o c a i n i d e (McErlane and P i l l a i , 1983), alpha-amino a c i d s , g l y c o l s , alpha-amino a l c o h o l s , aromatic and a l i p h a t i c alpha-hydroxy c a r b o x y l i c a c i d s and amines (Frank e t a l . , 1977, Frank e t a l . , 1979 and Frank e t a l . , 1980) by GLC. The h i g h t h e r m a l s t a b i l i t y of C h i r a s i l -V a l made i t p o s s i b l e f o r the f i r s t time to employ a mass spectrometer coupled to a GLC f o r the a n a l y s i s of enantiomeric drugs and m e t a b o l i t e s (Frank e t a l . , 1978). S i m i l a r l y , XE-60-S-valine-s-pheny1 ethylamide has been used 26 to separate c h i r a l a l c o h o l s , hydroxyacids, and carbohydrate by GLC (Konig and S i e v e r s , 1980; Konig e t a l . , 1981 and 1982) . The development of HPLC c h i r a l phases has seen a much f a s t e r growth than the GLC phases, hence a number of them with d i v e r s e chemical s t r u c t u r e s are now commercially a v a i l a b l e . These i n c l u d e a - and 8 - c y c l o d e x t r i n s ( c y c l o -heptaamylase and cyclooctaamylase r e s p e c t i v e l y ) , p r o t e i n bonded phase (bovine serum albumin or a l p h a s - a c i d g l y c o p r o t e i n attached to s i l i c a ) and the P i r k l e c h i r a l phases (cova l e n t and i o n i c types ) (Armstrong, 1984). The P i r k l e c h i r a l phases ( R ( - ) - N - 3 , 5 - d i n i t r o b e n z o y l p h e n y l g l y c i n e c o v a l e n t l y or i o n i c a l l y bonded to y-amino s i l a n i z e d s i l i c a ) have proven to be the most v e r s a t i l e , r e s o l v i n g the enantiomers of a v a r i e d c l a s s of compounds such as amines ( P i r k l e and Welch, 1984; P i r k l e e t a l . , 1984; Wainer et a l . , 1984 and McErlane e t a l . , 1986), benzodiazepines ( P i r k l e and T s i p o u r a s , 1984), amino a c i d s and a l c o h o l s ( P i r k l e and Welch, 1984) , p o l y c y c l i c aromatic hydrocarbons (Weems e t a l . , 1982) and a c y c l i c a l k y l c a r b i n o l s (Kasai e t a1 . , 1983) . The a p p l i c a b i l i t y of the c h i r a l s t a t i o n a r y phase has been e x t e n s i v e l y reviewed (Lochmuller and Souter, 1975; L i u and Ku, 1983; Blaschke, 1980; and Armstrong, 1984). 27 2.13 A n a l y t i c a l M e t h o d s f o r M e x i l e t i n e 2.13.1 A n a l y t i c a l M e t h o d s f o r R , S - M e x i l e t i n e The a s s a y o f r a c e m i c m e x i l e t i n e i n human b i o l o g i c a l f l u i d s h a s b e e n done by s e v e r a l t e c h n i q u e s i n c l u d i n g a number o f g a s - l i q u i d c h r o m a t o g r a p h i c (GLC) and h i g h - p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h i c (HPLC) p r o c e d u r e s . The GLC methods m e a s u r e d m e x i l e t i n e w i t h a n i t r o g e n s e l e c t i v e d e t e c t o r w i t h o u t d e r i v a t i z a t i o n ( S m i t h a n d M e f f i n , 1 9 8 0 ; and V a s i l i a d e s e t a l . , 1 9 8 4 ) , p o s t - d e r i v a t i z a t i o n w i t h n i t r o g e n - s e l e c t i v e d e t e c t i o n ( K e l l y e t a l . , 1973; B r a d b r o o k e t a l . , 1977; and E l f i n g e t a l . , 1 9 8 1 ) , f l a m e i o n i z a t i o n d e t e c t i o n ( P e r c h a l s k i e t a l . , 1974; K e l l y , 1977; H o l t e t a l . , 1979; K a c p r o w i c z , 1982 and G r e c h -B e l a n g e r e t a l . , 1 9 8 4 ) , and e l e c t r o n - c a p t u r e d e t e c t i o n ( Frydman e t a l . , 1973) . The l o w e s t amount d e t e c t e d was r e p o r t e d t o be 5 ng/mL w i t h t h e n i t r o g e n - s e l e c t i v e d e t e c t o r ( S m i t h a n d M e f f i n , 1 9 8 0 ) . T h e s e GLC m e t h o d s d i f f e r i n t h e i r mode o f d e t e c t i o n , v o l u m e o f s a m p l e r e q u i r e d f o r a n a l y s i s , e x t r a c t i o n p r o c e d u r e s , d e r i v a t i z a t i o n r e a g e n t and t h e n a t u r e o f t h e s t a t i o n a r y p h a s e . The method d e s c r i b e d b y H o l t e t a l . , 1 9 7 9 , h a s b e e n a d a p t e d f o r t h e r o u t i n e s e r u m m o n i t o r i n g o f m e x i l e t i n e . The HPLC methods m e a s u r e d m e x i l e t i n e e i t h e r d i r e c t l y 28 u s i n g UV d e t e c t i o n ( K e l l y e t a l . , 1 9 8 1 ; M a s t r o p a o l o e t  a l . , 1984) o r a f t e r d e r i v a t i z a t i o n f o l l o w e d by UV d e t e c t i o n ( B r e i t h a u p t a n d W i l f l i n g , 1982) , o r f l u o r e s c e n c e d e t e c t i o n ( W h i t e a n d F a r i d , 1983; G r e c h - B e l a n g e r e t a l . , 1 9 8 4 ) . M o s t o f t h e UV t e c h n i q u e s do n o t h a v e t h e r e q u i r e d s e n s i t i v i t y f o r s t u d y i n g s i n g l e d o s e k i n e t i c s o f m e x i l e t i n e i n humans. 2.13.2 A n a l y t i c a l M e t h o d s f o r M e x i l e t i n e E n a n t i o m e r s Thus f a r , o n l y one s t u d y h a s r e p o r t e d t h e r e s o l u t i o n and q u a n t i t a t i o n o f m e x i l e t i n e e n a n t i o m e r s i n humans. T h i s i n v o l v e d t h e c o n v e r s i o n o f t h e e n a n t i o m e r s t o t h e i r d i a s t e r e o i s o m e r s w i t h t h e c h i r a l r e a g e n t , 2 , 3 , 4 , 6 - t e t r a - 0 -a c e t y l - 3 - D - g l u c o p r a n o s y l i s o t h i o c y a n a t e ( G I T C ) , f o l l o w e d by r e s o l u t i o n on an HPLC r e v e r s e p h a s e ODS c o l u m n w i t h UV d e t e c t i o n ( G r e c h - B e l a n g e r e t a 1 . , 1985a).. The l o w e r l i m i t o f d e t e c t i o n f o r t h i s m e t h o d was 50 ng/mL o f e a c h e n a n t i o m e r i n p l a s m a . T h i s s e n s i t i v i t y i s n o t a d e q u a t e f o r a r e l i a b l e m e a s u r e m e n t o f t h e v e r y l o w nanogram l e v e l s o f t h e e n a n t i o m e r s e n c o u n t e r e d i n p h a r m a c o k i n e t i c s t u d i e s . M o r e o v e r , t h e r e a r e w e l l d o c u m e n t e d s e t b a c k s a s s o c i a t e d w i t h t h e r e s o l u t i o n o f e n a n t i o m e r s a s d i a s t e r e o i s o m e r s ( s e e r e s o l u t i o n o f e n a n t i o m e r s as d i a s t e r e o i s o m e r s , 2 . 1 2 . 1 ) . R e c e n t l y , a h i g h - p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h i c 29 method 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 employing a commercially a v a i l a b l e HPLC c h i r a l column ( P i r k l e type 1-A c h i r a l column) was developed i n our l a b o r a t o r y . The enantiomers were r e s o l v e d as t h e i r 2-napthamide d e r i v a t i v e s and the assay was s u c c e s s f u l l y used f o r the simultaneous measurement of m e x i l e t i n e enantiomers i n human plasma and u r i n e f o l l o w i n g a s i n g l e o r a l dose of the racemate (McErlane et a l . , 1986) . The lower l i m i t of s e n s i t i v i t y of the assay was 5 ng/mL f o r each of the enantiomers i n plasma which i s c o n s i d e r a b l y below t h a t e a r l i e r r e p o r t e d f o r c h i r a l a n a l y s i s . 30 2.14 P r i m a r y O b j e c t i v e s (a) To d e v e l o p a r e l i a b l e , 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 h i g h - p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h i c (HPLC) 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 m e x i l e t i n e e n a n t i o m e r s i n human b i o l o g i c a l f l u i d s . (b) To a p p l y the HPLC assay d e v e l o p e d t o s t u d y : ( i ) t he p h a r m a c o k i n e t i c s of m e x i l e t i n e e n antiomers i n h e a l t h y male s u b j e c t s a f t e r o r a l a d m i n i s t r a t i o n of the rac e m i c d r u g . ( i i ) t he i n v i t r o s t e r e o s e l e c t i v e p r o t e i n b i n d i n g o f m e x i l e t i n e e n a n t i o m e r s i n human serum. 31 3. EXPERIMENTAL 3 .1 S u p p l i e s 3.1.1 Drugs The f o l l o w i n g drugs were used: R,S-Mexiletine 1 2 h y d r o c h l o r i d e , R ( - ) - , and S(+)-mexiletine h y d r o c h l o r i d e 3 , R,S-mexiletine h y d r o c h l o r i d e 4 R capsules ( M e x i t i l , l o t no. 44255) and the i n t e r n a l standard: 1-(2',6 1-dimethylphenoxy)-2-aminoethane] (KOE 5 2963) h y d r o c h l o r i d e . 3.1.2 Chemicals and Reagents The f o l l o w i n g chemicals and reagents were used: A c e t i c 6 7 anhydride , t r i f l u o r o a c e t i c anhydride , g p e n t a f l u o r o p r o p i o n i c anhydride , h e p t a f l u o r o b u t y r i c 9 10 anhydride , 2-naphthoyl c h l o r i d e , barium hydroxide 11 12 octahydrate , z i n c s u l f a t e heptahydrate , sodium 13 14 hydroxide , t r i c h l o r o a c e t i c a c i d , monopotassium I- 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 AR, M a l l i n c k r o d t Inc. S t . L o u i s , M i s s o u r i , USA. 7-10 A l d r i c h Chemical Co. Milwaukee, Wisconsin, USA. I I - 13 BDH Chemicals L t d . , Poole, England, U.K. 14 F i s h e r S c i e n t i f i c Co., F a i r Lawn, NJ, USA. 32 p h o s p h a t e 1 5 , d i s o d i u m p h o s p h a t e h e p t a h y d r a t e D 1 6 s o d i u m 17 18 c h l o r i d e , and h y d r o c h l o r i c a c i d 3.1.3 S o l v e n t s The f o l l o w i n g s o l v e n t s w e re u s e d : M e t h y l e n e 19 20 21 c h l o r i d e , n-hexane , 2 - p r o p a n o l and 22 d i e t h y l e t h e r 3.2 C h r o m a t o g r a p h i c S t a t i o n a r y P h a s e s and Columns (a) Two HPLC c o l u m n s w e r e u s e d c o n s i s t i n g o f a 25 x R 2 3 0.46 cm i . d . P i r k l e i o n i c ( t y p e 1-A) , and a R 2 4 P i r k l e c o v a l e n t , H i - C h r o m r e v e r s i b l e H P L C c o l u m n . The s t a t i o n a r y p h a s e i n t h e s e c o l u m n s i s 3 , 5 - d i n i t r o b e n z o y 1 -p h e n y l g l y c i n e i o n i c a l l y o r c o v a l e n t l y bound t o y - a m i n o s i l a n i z e d s i l i c a . 25 (b) A HPLC G u a r d Column c o n s i s t e d o f a 1.5 x 0.32 cm i . d . d i s p o s a b l e s i l i c a c a r t r i d g e , a n d r e u s a b l e h o l d e r . (c) A 12 m x 0.2 mm i . d . f u s e d - s i l i c a h i g h p e r f o r m a n c e 2 6 c a p i l l a r y c o l u m n c o a t e d w i t h c r o s s - l i n k e d m e t h y l s i l i -c o n e (SE 54) as t h e s t a t i o n a r y p h a s e was u s e d . 15-17 A l l i e d C h e m i c a l , M o r r i s t o w n , New J e r s e y , USA. 18-22 BDH C h e m i c a l s L t d . , V a n c o u v e r , B.C., C a n a d a . 23,24 R e g i s C h e m i c a l Co., M o r t o n G r o v e , I l l i n o i s , USA. 25 R a i n i n I n s t . Co. I n c . , Woburn, M a s s a c h u s e t t s , USA. 33 3.3 E q u i p m e n t 3.3.1 H i g h - P e r f o r m a n c e L i q u i d C h r o m a t o g r a p h A H e w l e t t P a c k a r d model 1082B h i g h - p e r f o r m a n c e l i q u i d 2 7 c h r o m a t o g r a p h e q u i p p e d w i t h a H e w l e t t P a c k a r d model 79850B d a t a t e r m i n a l , a 20 uL f i x e d v o l u m e l o o p 2 8 i n j e c t o r , and a S c h o e f f e l model 970 LC 29 f l u o r o m e t e r was u s e d . 3.3.2 G a s - C h r o m a t o g r a p h / M a s s S p e c t r o m e t e r A H e w l e t t P a c k a r d model 5987A GC/MS e q u i p p e d w i t h a model 5880 ga s c h r o m a t o g r a p h ( H e w l e t t P a c k a r d ) was u s e d . 3.3.3 M i s c e l l a n e o u s T h e s e w e r e : v o r t e x m i x e r 3 < ^ ; pH meter 3"'"; d i r e c t 32 33 c o n n e c t u n i v e r s a l c o l u m n p r e f i l t e r and a d a p t o r ; 34 and 0.45 u M i l l i p o r e f i l t e r s . 26 H e w l e t t P a c k a r d , A v o n d a l e , P e n n s y l v a n i a , USA. 27 H e w l e t t P a c k a r d , A v o n d a l e , P e n n s y l v a n i a , USA. 28 Rheodyne I n c . , B e r k e l e y , C a l i f o r n i a , USA. 29 K r a t o s ( S c h o e f f e l I n s t r u m e n t s ) , Westwood, New J e r s e y , USA. 30 S y b r o n , Co., Dubuque, I o w a , USA. 31 O r i o n R e s e a r c h I n c . , C a m b r i d g e , M a s s a c h u s e t t s , USA. 32,33 A l l t e c h . A s s o c i a t e s I n c . , D e e r f i e l d , I l l i n o i s , USA.' 34 M i l l i p o r e C o r p . , B e d f o r d , M a s s a c h u s e t t s , USA. 34 3 . 4 Stock S o l u t i o n s 3.4.1 R,S-Mexiletine H y d r o c h l o r i d e R,S-Mexiletine h y d r o c h l o r i d e stock s o l u t i o n was prepared by d i s s o l v i n g 1.239 mg of R,S-mexiletine hydro-c h l o r i d e i n 100 ml of d e i o n i s e d , d i s t i l l e d water ( y i e l d i n g a s o l u t i o n of m e x i l e t i n e e q u i v a l e n t to 10 ug/mL of the base). D i l u t i o n o f 10 ml of t h i s s o l u t i o n to 100 mL wit h d e i o n i s e d , d i s t i l l e d water gave a lower c o n c e n t r a t i o n s t o c k s o l u t i o n of 1 ug/mL e q u i v a l e n t of the base. 3.4.2 R ( - ) - , and S( + ) - M e x i l e t i n e H y d r o c h l o r i d e Stock s o l u t i o n s of R ( - ) - and S(+)-mexiletine hydro-c h l o r i d e were s e p a r a t e l y prepared by d i s s o l v i n g 1.2039 mg of each i n 100 mL of d e i o n i s e d , d i s t i l l e d water. 3.4.3 KOE 2963 H y d r o c h l o r i d e ( I n t e r n a l Standard) Stock s o l u t i o n of the i n t e r n a l s t a n d a r d was prepared by d i s s o l v i n g 0.4976 mg of KOE 2963 h y d r o c h l o r i d e i n 100 mL of d e i o n i s e d d i s t i l l e d water. 3.4.4 Barium Hydroxide S o l u t i o n (0.3N) Barium hydroxide octahydrate (4.7325 g) was d i s s o l v e d i n 100 mL of d e i o n i s e d , d i s t i l l e d water with v i g o r o u s 35 shaking. 3.4.5 Zinc S u l f a t e Heptahydrate S o l u t i o n (5%) Zinc s u l f a t e heptahydrate (5 g) was d i s s o l v e d i n 100 mL of d e i o n i s e d , d i s t i l l e d water. 3.4.6 2-Napthoyl C h l o r i d e The d e r i v a t i z a t i o n reagent, 2-naphthoyl c h l o r i d e , was prepared by d i s s o l v i n g 1 mg i n 1 mL of methylene c h l o r i d e . 3.4.7 I s o t o n i c , 0.067 M, pH 7.4, Phosphate B u f f e r  (Documentia Geigy, 1970) Monopotassium phosphate (KH 2P0^) (9.07 g) was d i s s o l v e d i n 1 l i t e r of d e i o n i s e d , d i s t i l l e d water to gi v e s o l u t i o n A. Disodium phosphate heptahydrate (Na 2HP0 4.7H 20) (17.87 g) was d i s s o l v e d i n 1 l i t e r of d e i o n i s e d , d i s t i l l e d water to gi v e s o l u t i o n B. S o l u t i o n A (197 mL) was t r a n s f e r r e d i n t o a c l e a n 1 l i t e r v o l u m e t r i c f l a s k and 3.9 g of sodium c h l o r i d e added to i t f o r i s o t o n i c i t y adjustment. The volume was then made up to 1 l i t e r w i t h s o l u t i o n B. The pH of the s o l u t i o n was checked with a pH meter and found to be 7.4. 3.4.8 T r i c h l o r o a c e t i c A c i d S o l u t i o n (10%) 36 T h i s was prepared by d i s s o l v i n g 10 g of t r i c h l o r o a c e t i c a c i d i n 100 mL of d e i o n i s e d , d i s t i l l e d water. 3 . 5 Chromatographic R e s o l u t i o n of M e x i l e t i n e Enantiomers 3.5.1 T r i f l u o r o a c e t y l - and A c e t y 1 - D e r i v a t i v e s To 10.0 ug of (+_)-mexiletine (lmL of the h i g h e r C o n c e n t r a t i o n Stock S o l u t i o n ) c o n t a i n e d i n a 10 mL screw-capped c u l t u r e tube was added 0.5 mL of 2N sodium hydroxide and 5 mL of methylene c h l o r i d e . The tubes were t i g h t l y capped and tumbled f o r 10 min on a r o t a r y shaker. The methylene c h l o r i d e l a y e r was t r a n s f e r r e d i n t o a c l e a n dry c u l t u r e tube and the contents were evaporated to dryness under a g e n t l e stream of n i t r o g e n i n a water bath (37°C). The r e s i d u e was r e c o n s t i t u t e d w i t h 0.2 mL of n-hexane and mixed by v o r t e x i n g f o r a few seconds. T r i f l u o r o a c e t y l c h l o r i d e (or a c e t y l c h l o r i d e ) (30 uL ) was then added to the tube, t i g h t l y capped, and heated of 55°C i n a h e a t i n g b l o c k f o r 30 minutes. The tube was cooled to room temperature and the excess reagent was removed by e v a p o r a t i o n under a g e n t l e stream of n i t r o g e n . The r e s i d u e was r e c o n s t i t u t e d with 0.1 mL of n-hexane and 20 uL of t h i s s o l u t i o n was i n j e c t e d onto the HPLC column. The HPLC c o n d i t i o n s were: s t a t i o n a r y phase, P i r k l e c o v a l e n t c h i r a l phase; mobile phase, 5.5% 37 2-propanol i n hexane; flow r a t e , 0.6 mL/min; and UV d e t e c t i o n at 254 nm. 3.5.2 2-Naphthoyl C h l o r i d e To 1.0 ug of (+_) - m e x i l e t i n e (0.1 mL of the hig h e r C o n c e n t r a t i o n Stock S o l u t i o n ) contained i n a 10 mL screw-capped c u l t u r e tube, was added 0.2 mL of 2N sodium h y d r o x i d e . A 15 uL a l i q u o t of the 2-naphthoyl c h l o r i d e stock s o l u t i o n was added and the mixture v o r t e x e d f o r 2 mintues at room temperature. The 2-naphthamide d e r i v a t i v e of m e x i l e t i n e was e x t r a c t e d with 0.6 mL of 5.5% 2-propanol i n hexane. A 20 uL a l i q u o t o f the top o r g a n i c l a y e r was i n j e c t e d onto the HPLC column. The HPLC c o n d i t i o n s were: S t a t i o n a r y phase, P i r k l e i o n i c c h i r a l phase; mobile phase, 5.5% 2-propanol i n hexane; flow r a t e , 1.4 mL/min; and f l u o r e s c e n c e d e t e c t i o n a t 230 nm ( e x c i t a t i o n ) and 370 nm (emission) wavelength. 3.6 Development of Assay of M e x i l e t i n e Enantiomers 3.6.1 S e l e c t i o n of I n t e r n a l Standard KOE 2963 [1-(2',6'-dimethylphenoxy)-2-ethanamine] was chosen as i n t e r n a l standard based on i t s s t r u c t u r a l 38 s i m i l a r i t y to m e x i l e t i n e . In a d d i t i o n , i t has no c h i r a l c e n t e r i n i t s molecular arrangement, hence i t gave a s i n g l e •symmetrical peak under the HPLC c o n d i t i o n s employed. The other s t r u c t u r a l analogs of m e x i l e t i n e c o n s i d e r e d were KOE 3 5 768 [ 1-(2' , 4 1-dimethylphenoxy)-2-propanamine] and KOE 3 6 1307 [ 1 - 2 ' , 4 1 , 6 ' - t r i m e t h y I p h e n o x y ) - 2 - p r o p a n a m i n e ] . Both were found u n s u i t a b l e because the c h i r a l HPLC column r e s o l v e d t h e i r enantiomers g i v i n g r i s e to two peaks. 3.6.2 Optimum Reaction Time The optimum r e a c t i o n time f o r the 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 and the i n t e r n a l standard was s t u d i e d with s i x samples c o n t a i n i n g the same amounts (1000 ng of R,S-mexiletine and 200 ng of i n t e r n a l standard) of the compounds. D e r i v a t i z a t i o n was c a r r i e d out w i t h 2-naphthoyl c h l o r i d e as d e s c r i b e d above f o r d i f f e r e n t time i n t e r v a l s : 1, 5, 10, 15, 20 and 30 minutes and the samples analysed by a p l o t of peak h e i g h t s a g a i n s t time. 3.6.3 S e n s i t i v i t y 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 g r e a t l y enhanced s e n s i t i v i t y due to i t s f l u o r o g e n i c 35,36 Boehringer Ingelheim L t d . , B u r l i n g t o n , Ont. Canada. 39 p r o p e r t y . S e n s i t i v i t y was f u r t h e r i m p r o v e d by d e t e c t i o n o f m e x i l e t i n e - 2 - n a p h t h a m i d e a t i t s optimum UV e x c i t a t i o n (230 nm), and f l u o r e s c e n c e e m i s s i o n (370 nm) w a v e l e n g t h . T h e s e w e r e r e s p e c t i v e l y d e t e r m i n e d w i t h a Beckman m o d e l 25 s p e c t r -37 o p h o t o m e t e r , and a P e r k m E l m e r model 6 5 0 - l O s 3 8 f l u r o e s c e n c e s p e c t r o p h o t o m e t e r 3.6.4 E x t r a c t i o n S o l v e n t The e x t r a c t i b i l i t y o f m e x i l e t i n e and t h e i n t e r n a l s t a n d a r d f r o m p l a s m a was a s s e s s e d w i t h t h r e e o r g a n i c s o l v e n t s ; m e t h y l e n e c h l o r i d e , d i e t h y l e t h e r and n - h e x a n e . D i e t h y l e t h e r g a v e t h e h i g h e s t r e c o v e r y and was a c c o r d i n g l y c h o s e n f o r t h e e x t r a c t i o n p r o c e d u r e . 3.6.5 P l a s m a P r o t e i n P r e c i p i t a t i o n The p r o t e i n p r e c i p i t a t i o n methods were as f o l l o w s : (a) t r i c h l o r o a c e t i c a c i d : t o 1 mL o f p l a s m a was a d d e d 0.2 mL o f 10% t r i c h l o r o a c e t i c a c i d f r o m t h e s t o c k s o l u t i o n . The p l a s m a was v i g o r o u s l y s h a k e n and t h e pH a d j u s t e d a b o v e 12 w i t h 0.5 mL o f 2N NaOH b e f o r e 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 . 37 Beckman I n s t r u m e n t s , I n c . , F u l l e r t o n , C a l i f o r n i a , USA. 38 P e r k i n E l m e r , N o r w a l k , C o n n e c t i c u t , USA. 4 0 (b) barium h y d r o x i d e / z i n c s u l f a t e : t o 1 mL o f plasma was admixed 1 mL of 0.3N barium h y d r o x i d e f o l l o w e d by 1 mL of 5% z i n c s u l f a t e h e p t a h y d r a t e s o l u t i o n . The m i x t u r e was v i g o r o u s l y shaken and the plasma pH was a d j u s t e d above 12 w i t h 0.4 mL of 2N NaOH b e f o r e 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 . T h i s d e p r o t e i n i z a t i o n method, r e p o r t e d by Somogyi (1945), was used w i t h a s l i g h t m o d i f i c a t i o n ( one mL of each s o l u t i o n , i n s t e a d of 10 mL, was used f o r 1 mL of p l a s m a ) . The method r e q u i r e s t h a t the b a r i u m h y d r o x i d e must n e u t r a l i z e the z i n c s u l f a t e , volume f o r volume, when t i t r a t i o n i s performed w i t h p h e n o l p h t h a l e i n as i n d i c a t o r b e f o r e the s o l u t i o n s a re used f o r p r o t e i n p r e c i p i t a t i o n . For the t i t r a t i o n , 1 mL of the z i n c s u l f a t e s o l u t i o n was i n t r o d u c e d i n t o a 250 mL f l a s k and d i l u t e d w i t h 10 mL o f d i s t i l l e d w a t e r . Two d r o p s o f p h e n o l p h t h a l e i n was added and barium h y d r o x i d e s o l u t i o n r u n i n drop w i s e from a b u r e t t e w i t h c o n s t a n t a g i t a t i o n . The e n d p o i n t was reached when the i n d i c a t o r c o l o r t u r n e d p i n k and p e r s i s t e d f o r a t l e a s t one minute. Based on t h e t i t r a t i o n , t he more c o n c e n t r a t e d s o l u t i o n was d i l u t e d w i t h d i s t i l l e d water t o match the o t h e r . 41 3.7 Assay of M e x i l e t i n e Enantiomers by High-Performance L i q u i d Chromatography with  F l u o r e s c e n c e D e t e c t i o n 3.7.1 E x t r a c t i o n , D e r i v a t i z a t i o n and HPLC A n a l y s i s An a l i q u o t of blank human b i o l o g i c a l f l u i d (plasma, u r i n e or s a l i v a ) i n a 10 mL P T F E - l i n e d screw-capped tube was spiked with a p p r o p r i a t e volumes of the R,S-mexiletine h y d r o c h l o r i d e and the i n t e r n a l standard stock s o l u t i o n s . For plasma samples, p r o t e i n s were p r e c i p i t a t e d w i t h 1 mL of barium hydroxide and 1 mL of z i n c s u l f a t e s o l u t i o n s as e a r l i e r d e s c r i b e d . The pH of a l l b i o l o g i c a l samples was adj u s t e d above 12 by the a d d i t i o n of 0.4 mL of 2N NaOH and e x t r a c t e d twice with 5 mL p o r t i o n s of d i e t h y l e t h e r . The orga n i c e x t r a c t s were combined and evaporated a t 37°C under a g e n t l e stream of c l e a n dry n i t r o g e n to a volume of approximately 1 mL. The r e s u l t i n g s o l u t i o n was a c i d i f i e d with 0.2 mL of 0.IN HC1, shaken, and the ether l a y e r was removed and d i s c a r d e d . The aqueous l a y e r was a g a i n b a s i f i e d w i t h 0.2 mL of 2N NaOH and 15 uL of the 2-naphthoyl c h l o r i d e s o l u t i o n was added and mixed v i g o r o u s l y on a v o r t e x mixer f o r two minutes . The d e r i v a t i v e s formed were e x t r a c t e d i n t o 0.6 mL of the mobile phase used f o r the HPLC a n a l y s i s (5.5% 2-propanol i n hexane) and a 20 uL a l i q u o t was i n j e c t e d onto the HPLC column. For very low c o n c e n t r a t i o n s ( l e s s than 50 42 ng/mL) the d e r i v a t i v e e x t r a c t s were evaporated to dryness under a g e n t l e stream of n i t r o g e n and r e c o n s t i t u t e d with 120 uL of mobile phase before i n j e c t i o n . 3.7.2 C a l i b r a t i o n Curves and Assay P r e c i s i o n 3.7.2.1 Plasma To s i x 1 mL a l i q u o t s of blank plasma were added 10, 100, 200, 500, 1,000 and 1,500 ng ( e q u i v a l e n t of the base) of R,S-mexiletine from the stock s o l u t i o n s along w i t h 0.05 mL of the i n t e r n a l standard stock s o l u t i o n ( e q u i v a l e n t to 200 ng of the base). The samples were subsequently t r e a t e d as d e s c r i b e d under " E x t r a c t i o n , D e r i v a t i z a t i o n and HPLC A n a l y s i s " . The c a l i b r a t i o n c u r ves were c o n s t r u c t e d by p l o t -t i n g the peak h e i g h t r a t i o s of each enantiomer to t h a t of the i n t e r n a l s t a n d a r d a g a i n s t the known c o n c e n t r a t i o n of the enantiomer. I n t e r - a s s a y v a r i a b i l i t y was determined by t r i p l i c a t e p r e p a r a t i o n and a n a l y s i s of each of the samples used f o r the standard curve. I n t r a - a s s a y v a r i a b i l i t y was determined by t r i p l i c a t e i n j e c t i o n s of three of the samples c o n t a i n i n g 100, 500, 1,500 ng of R,S-mexiletine. 3.7.2.2 Urine To s i x 0.2 mL a l i q u o t s of blank u r i n e were added 100, 500, 1,000, 1,500, 2,000 and 3,000 ng ( e q u i v a l e n t of the 43 base) of R, S - m e x i l e t i n e from the stock s o l u t i o n and 0.075 mL of the i n t e r n a l standard stock s o l u t i o n ( e q u i v a l e n t to 300 ng of the base) . The s o l u t i o n s were b a s i f i e d by the a d d i t i o n of 0.4 mL of 2N NaOH and e x t r a c t e d twice with 5 mL p o r t i o n s of d i e t h y l e t h e r . The e t h e r e a l e x t r a c t s were then t r e a t e d as d e s c r i b e d f o r plasma. A second c a l i b r a t i o n curve was prepared w i t h 1 mL a l i q u o t s of u r i n e ; 20, 100, 250, 500, and 1,000 ng of R,S-mexiletine and 200 ng of the i n t e r n a l standard. 3.7.2.3 S a l i v a To s i x 0.5 mL a l i q u o t s of blank s a l i v a were added 20, 100, 500, 1,000, 2,000 and 3,000 ng ( e q u i v a l e n t of the base) of R,S-mexiletine from the stock s o l u t i o n s and 0.05 mL of the i n t e r n a l standard s o l u t i o n ( e q u i v a l e n t to 200 ng of the base) . The s o l u t i o n s were b a s i f i e d by the a d d i t i o n of 0.4 mL of 2N NaOH and e x t r a c t e d twice with 5 mL p o r t i o n s of d i e t h y l e t h e r . The e t h e r e a l e x t r a c t s were then t r e a t e d as d e s c r i b e d f o r plasma. 3.7.3 E f f i c i e n c y of Recovery of M e x i l e t i n e Enantiomers from Plasma The e f f i c i e n c y of recovery of the enantiomers from plasma was determined by the a d d i t i o n of 100, 200, 500, 1,000 and 1,500 ng ( e q u i v a l e n t of the base) of R,S-mexil-44 e t i n e t o f i v e 1 mL a l i q u o t s of plasma. The samples were e x t r a c t e d , d e r i v a t i z e d and assayed as e a r l i e r d e s c r i b e d except t h a t the i n t e r n a l standard was added j u s t p r i o r to the d e r i v a t i z a t i o n procedure. The r e s u l t i n g peak h e i g h t r a t i o s were expressed as a percentage of those o b t a i n e d with i d e n t i c a l amounts of R,S-mexiletine and the i n t e r n a l standard which were d e r i v a t i z e d d i r e c t l y a f t e r b a s i f i c a t i o n w ith 0.2 mL of 2N NaOH without p r i o r e x t r a c t i o n . 3.7.4 S t r u c t u r a l C o n f i r m a t i o n of the D e r i v a t i v e s The s t r u c t u r e of the 2-napththamide d e r i v a t i v e s of R,S-mexiletine, the i n d i v i d u a l enantiomers and the i n t e r n a l standard was confirmed by p o s i t i v e chemical i o n i z a t i o n gas-chromatography/mass-spectrometry. The GC column was a 12 m x 0.2 mm i . d . high-performance fused s i l i c a c a p i l l a r y column coated with c r o s s - l i n k e d m e t h y l s i l i c o n e . The f o l l o w i n g s p l i t l e s s GC c o n d i t i o n s were employed: helium ( c a r r i e r gas) flow r a t e , 1 mL/min; i n j e c t i o n p o r t temperature, 240°C; oven temperature was ramped from 50°C f o r 1 min. to 300°C f o r 10 min. at 30°C/min. The MS c o n d i t i o n s were: i n t e r f a c e temperature, 240^C; e l e c t r o n m u l t i p l i e r v o l t a g e , 2,500V; and emission c u r r e n t , 300 uA. Methane was used as the reagent gas. 45 3.8 M e x i l e t i n e Enantiomer Serum P r o t e i n B i n d i n g Studies by U l t r a f i l t r a t i o n Technique 3.8.1 Serum C o l l e c t i o n Approximately 80 mL of venous blood was c o l l e c t e d from f i v e h e a l t h y male v o l u n t e e r s (same study s u b j e c t s ) u s i n g 10 mL g l a s s s y r i n g e s . Blood was immediately t r a n s f e r r e d i n t o 10 mL g l a s s tubes with T e f l o n l i n e d caps, allowed to c l o t a t room temperature f o r 2 hours, then c e n t r i f u g e d a t 2500 x g f o r 15 minutes and the serum separated. 3.8.2 Sample P r e p a r a t i o n A l i q u o t s of R,S-mexiletine h y d r o c h l o r i d e i n phosphate b u f f e r ( e q u i v a l e n t to 0.15 mg/mL of base) were d i l u t e d w i t h phosphate b u f f e r to y i e l d a s e r i e s of c o n c e n t r a t i o n s ; 15, 37.5, 75, 112.5 and 150 ug/mL. To f i v e 4.44 mL p o r t i o n s of serum from each v o l u n t e e r were added 60 uL of the d i l u t e s o l u t i o n s g i v i n g f i n a l c o n c e n t r a t i o n s of 0.2, 0.5, 1.0, 1.5 and 2.0 ug/mL. The spiked serum was mixed thoroughly on a 39 r o t a t o r f o r 10 minutes and l e f t to e q u i l i b r a t e i n a water bath (37°C) f o r 30 minutes. D u p l i c a t e 1 mL samples were used to determine the t o t a l drug c o n c e n t r a t i o n , 39 P e r k i n Elmer, Norwalk, C o n n e c t i c u t , USA. 46 while a second s e t of d u p l i c a t e 1 mL samples were su b j e c t e d to u l t r a f i l t r a t i o n . 3.8.3 U l t r a f i l t r a t i o n U l t r a f i l t r a t i o n was c a r r i e d out with the MPS-1 micro-40 p a r t i t i o n system and a 30,000 Dalton molecular weight 41 c u t - o f f YMT u l t r a f i l t r a t i o n membrane. The 42 o c e n t r i f u g e (Beckman Model J2-21) had a 35 angle-43 head r o t o r (Beckman Model JA-20) and was e q u i l i b r a t e d to 37°C before i n t r o d u c t i o n of the samples. C e n t r i f u -g a t i o n time was 15 minutes at a r e l a t i v e c e n t r i f u g a l f o r c e of 1650 x g (the c e n t r i f u g a t i o n parameters were recommend-a t i o n s of the manufacturer of the u l t r a f i l t r a t i o n system). 3.8.4 A n a l y s i s of Free and T o t a l M e x i l e t i n e i n Serum The c o n c e n t r a t i o n of m e x i l e t i n e enantiomers i n ng/mL spiked serum and the u l t r a f i l t r a t e (unbound enantiomer) was determined u s i n g the HPLC assay p r e v i o u s l y d e s c r i b e d . The standard curves f o r the unbound enantiomer were prepared i n serum u l t r a f i l t r a t e while those f o r the t o t a l enantiomer were prepared i n serum. The unbound f r a c t i o n , expressed as a percentage, was c a l c u l a t e d by d i v i d i n g the c o n c e n t r a t i o n 40,41 Amicon Canada L t d . , O a k v i l l e , O n t a r i o , Canada. 42,43 Beckman I n s t r . Inc., F u l l e r t o n , C a l . , USA. 47 of the unbound enantiomer by the t o t a l e n a n t i o m e r . 3.9 P h a r m a c o k i n e t i c s o f M e x i l e t i n e Enantiomers i n H e a l t h y V o l u n t e e r s 3.9.1 V o l u n t e e r s F i v e male v o l u n t e e r s between 25-43 y e a r s of age w i t h no h i s t o r y of c a r d i a c , r e n a l , h e p a t i c o r g a s t r o - i n t e s t i n a l d i s e a s e p a r t i c i p a t e d i n t h e s t u d y . The r e c r u i t m e n t of t h e s e v o l u n t e e r s was made th r o u g h r e c r u i t m e n t n o t i c e s p o s t e d a t d i f f e r e n t l o c a t i o n s i n U.B.C. Campus. P r i o r t o ac c e p t a n c e i n t he s t u d y , b l o o d and u r i n e samples were c o l l e c t e d from each v o l u n t e e r and s u b j e c t e d t o h e m a t o l o g i c a l and b i o -c h e m i c a l assessment. A thorough p h y s i c a l e x a m i n a t i o n o f the v o l u n t e e r s was performed and t h e i r e l e c t r o c a r d i o g r a m s were a l s o checked. S u b j e c t s were not a l l o w e d any drugs f o r one week and a l c o h o l f o r 48 hours p r i o r t o , o r d u r i n g , the s t u d y . The v o l u n t e e r s were r e q u i r e d t o s i g n an i n f o r m e d c o n s e n t form b e f o r e the s t u d y commenced. 3.9.2 P h y s i o l o g i c a l M o n i t o r i n g of Study S u b j e c t s A l l v o l u n t e e r s t u d i e s were done under the s u p e r v i s i o n of a c a r d i o l o g i s t a t the Acute Care U n i t , U.B.C. H o s p i t a l . H e a r t r a t e , b l o o d p r e s s u r e and ECG were m o n i t o r e d p r i o r t o and 3 hours f o l l o w i n g 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 . 48 3.9.3. In Viv o Study Study s u b j e c t s were f a s t e d o v e r n i g h t and g i v e n an o r a l dose of 300 mg of (+_)-mexiletine h y d r o c h l o r i d e i n capsule form (one 100 mg p l u s one 200 mg capsule) w i t h approximately 100 mL of water. Venous blood samples were c o l l e c t e d i n t o h e p a r i n i z e d V a c u t a i n e r D R t u b e s 4 4 u ~ v i a a n i n d w e l l i n g 19 45 gauge B u t t e r f l y cannula i n s e r t e d i n t o a v e i n on the back of the forearm. Samples (8 ml) were w i t h - drawn at 0, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 6, 8, 10, 12, 14, 24, 30 and 48 hours. 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 s o l u t i o n of h e p a r i n i n sodium c h l o r i d e (50 i n t e r n a t i o n a l units/mL) a f t e r each withdrawal and the hep a r i n was c l e a r e d i n t o an evacuated tube p r i o r to each b l o o d sampling. Blood samples were c e n t r i f u g e d at 2,500 x g f o r 15 minutes and the plasma was separated. Urine samples were c o l l e c t e d i n p l a s t i c W h i r l P a k R b a g s 4 6 a t 0, 1, 2, 3, 4, 6, 8, 10, 12, 14, 24, 36, 48 and 72 hours and at the convenience of the s u b j e c t a f t e r the 14 hour sample. The u r i n e pH, along with the volume, was measured and recorded. S a l i v a samples were c o l l e c t e d i n t o s c i n t i l l a t i o n v i a l s a t the same time as the b l o o d sample. A l l the samples (plasma, 44,45 Becton D i c k i n s o n and Co., R u t h e r f o r d , NJ, USA. 46 Canlab, Vancouver, B.C., Canada. 49 u r i n e and s a l i v a ) were s t o r e d a t -20°C u n t i l a n alyzed. 3.9.4 A n a l y s i s of Plasma, Urine and S a l i v a Samples Plasma: To 1 mL a l i q u o t s of plasma was added 0.05 mL of the i n t e r n a l standard stock s o l u t i o n , and the plasma p r o t e i n s were p r e c i p i t a t e d w i t h 1 mL each of barium hydroxide and z i n c s u l f a t e s o l u t i o n s . The samples were b a s i f i e d with 0.4 mL of 2N NaOH and t r e a t e d as d e s c r i b e d under " E x t r a c t i o n , D e r i v a t i z a t i o n and HPLC A n a l y s i s " . U r i n e : To 0.2 mL a l i q u o t s of the u r i n e samples was added 0.075 mL of the i n t e r n a l standard stock s o l u t i o n . The samples were b a s i f i e d with 0.4 mL of 2N NaOH and t r e a t e d as d e s c r i b e d under " E x t r a c t i o n , D e r i v a t i z a t i o n and HPLC A n a l y s i s " . For the 48 to 72 hour samples, 1 mL u r i n e a l i q u o t s and 0.05 mL of the i n t e r n a l standard s t o c k s o l u t i o n were used. In order to remove endogenous m a t e r i a l s remaining from a n a l y s i s of u r i n e samples, i t was necessary to wash the column with 10% 2-propanol i n hexane f o r 15 minutes a f t e r every second i n j e c t i o n of the u r i n e e x t r a c t s . S a l i v a : To 0.5 mL a l i q u o t s of the s a l i v a samples was added 0.05 mL of the i n t e r n a l standard stock s o l u t i o n . The samples were b a s i f i e d with 0.4 mL of 2N NaOH and t r e a t e d as d e s c r i b e d under " E x t r a c t i o n , D e r i v a t i z a t i o n and HPLC A n a l y s i s " . 50 3.9.5 D a t a A n a l y s i s The p l a s m a c o n c e n t r a t i o n v e r s u s t i m e d a t a f o r t h e e n a n t i o m e r s w e r e a n a l y z e d w i t h t h e c o m p u t e r p r o g r a m AUTOAN (Sedman and Wagner, 1976) and N o n L i n ( M e t z l e r . , 1974) . The b e s t f i t o p e n - m o d e l and t h e p h a r m a c o k i n e t i c p a r a m e t e r s ; a p p a r e n t f i r s t - o r d e r a b s o r p t i o n (K ) , d i s t r i b u t i o n ( a ) and a p p a r e n t e l i m i n a t i o n (B) r a t e c o n s t a n t s w e r e o b t a i n e d u s i n g e q u a l w e i g h t i n g o f a l l t h e d a t a p o i n t s . The t e r m i n a l e l i m i n a t i o n h a l f - l i f e was c a l c u l a t e d f r o m t h e r e l a t i o n s h i p 0.693 / 8 w h i l e t h e a r e a u n d e r t h e p l a s m a c o n c e n t r a t i o n - t i m e c u r v e s t o i n f i n i t y (AUC ^ ) was e s t i m a t e d by t h e t r a p e z o i d a l r u l e f r o m O t o t h e l a s t m e a s u r e d c o n c e n t r a t i o n p l u s t h e l a s t m e a s u r e d c o n c e n t r a t i o n / 8 . R e n a l c l e a r a n c e ( C L r ) o f e a c h e n a n t i o m e r was o b t a i n e d f r o m t h e f o r m u l a oo oo X /AUC^ , w h e r e X i s t h e c u m u l a t i v e amount u 0- 0 0 u o f u n c h a n g e d e n a n t i o m e r e x c r e t e d i n u r i n e o v e r 72 h o u r s . N o n - r e n a l e l i m i n a t i o n r a t e c o n s t a n t (K ) was e s t i m a t e d by t h e f o r m u l a e 8 ( l - f ) w here f i s t h e f r a c t i o n o f u n c h a n g e d e n a n t i o m e r e x c r e t e d i n u r i n e . O t h e r p h a r m a c o k i n e t i c p a r a m e t e r s , w h i c h i n c l u d e s p e a k p l a s m a c o n c e n t r a t i o n ( C p e a k ) , t i m e t o p e a k p l a s m a c o n c e n t r a t i o n ( t p e a k ) , a p p a r e n t o r a l c l e a r a n c e ( C T B / F ) , and a p p a r e n t v o l u m e o f d i s t r i b u t i o n (Vd /F) w e re e s t i m a t e d a s d e s c r i b e d by cl 3f 6 ci G i b a l d i and P e r r i e r ( 1 9 7 5 ) . 51 D i f f e r e n c e s i n the pharmacokinetic parameters obtained f o r the enantiomers were assessed f o r s t a t i s t i c a l s i g n i f i -cance u s i n g the p a i r e d students t - t e s t ( t w o - t a i l e d ) . P r o b a b i l i t y v a l u e s l e s s than, or equal t o , 0.05 were co n s i d e r e d s i g n i f i c a n t . 52 4.RESULTS AND DISCUSSIONS 4.1 Chromatographic R e s o l u t i o n of M e x i l e t i n e Enantiomers 4.1.1 T r i f l u o r o a c e t y l - and A c e t y 1 - D e r i v a t i v e s The p r e l i m i n a r y attempts a t the r e s o l u t i o n of the enantiomers of m e x i l e t i n e were made u s i n g the P i r k l e cova-len t l y - b o n d e d c h i r a l HPLC column and a mobile phase of v a r y i n g p r o p o r t i o n s of 2-propanol i n hexane. Since m e x i l e t i n e i s a primary amine, a c y l a t i o n of the b a s i c amino group was necessary to f a c i l i t a t e i t s passage through the p i - a c i d i c c h i r a l s t a t i o n a r y phase. A c y l a t i o n a l s o p r o v i d e s f u n c t i o n a l groups which a i d i n the c h i r a l r e c o g n i t i o n p r o c e s s . I n i t i a l l y , r e s o l u t i o n was accomplished f o r the enantiomers of m e x i l e t i n e as t h e i r t r i f l u o r o a c e t y l d e r i v a t i v e s . The r e s o l u t i o n , however, was o n l y p a r t i a l (R-0.5; f i g . IA) and e f f o r t s to improve upon i t by a l t e r a t i o n of the mobile phase p o l a r i t y were u n s u c c e s s f u l . When the . higher homologous d e r i v a t i v e s of m e x i l e t i n e enantiomers . (pentafluoropropiony1 and h e p t a f l u o r o b u t y r y l d e r i v a t i v e s ) were e v a l u a t e d on the same column under s i m i l a r c o n d i t i o n s , no r e s o l u t i o n was obtained (chromatograms not shown). Examination of s t e r i c models f o r the i n t e r a c t i o n of the f l u o r o a l k y l d e r i v a t i v e s with the c h i r a l s t a t i o n a r y phase 53 FIGURE 1 C H R O M A T O G R A M S O F T R I F L U O R O A C E T Y L ( A ) A N D A C E T Y L ( B ) D E R I V A T I V E S O F M E X I L E T I N E O N T H E P I R K L E C O V A L E N T C O L U M N B T — • 1 10 2 0 T I M E (MIN) 0 10 2 0 3 0 T I M E (MIN) C h r o m a t o g r a p h i c c o n d i t i o n s : M o b i l e phase, 5% 2-propanol i n hexane; f l o w r a t e , o.6 mL/min. pumped i s o c r a t i c a l l y a t ambient t e m p e r a t u r e ; d e t e c t i o n , UV (254 nm); c h a r t speed, 0.25 cm/min. 54 r e v e a l e d t h a t the f l u o r i n e atoms of the l a t t e r halogenated d e r i v a t i v e s were r e s p o n s i b l e f o r the decrease i n i n t e r a c t i o n with the s t a t i o n a r y phase due to t h e i r e l e c t r o n e g a t i v i t y and/or t h e i r g r e a t e r bulk. F u r t h e r i n v e s t i g a t i o n s w i t h the non-halogenated acetamide d e r i v a t i v e , as a n t i c i p a t e d , gave very s a t i s f a c t o r y r e s o l u t i o n ( f i g . I B ) . 4.1.2 2-Naphthoyl D e r i v a t i v e The r e s o l u t i o n obtained with the acetamide d e r i v a t i v e , a l t h o u g h s a t i s f a c t o r y , would not have s u f f i c i e n t s e n s i t i v i t y u s i n g UV d e t e c t i o n f o r the pharmacokinetic s t u d i e s planned. A d e r i v a t i z a t i o n reagent which would g i v e good r e s o l u t i o n , while p r o v i d i n g high s e n s i t i v i t y , was t h e r e f o r e needed. This l e d to the 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 . The r e s o l u t i o n obtained was p a r t i a l (R= 0.6; f i g . 2A) but s e n s i t i v i t y was markedly enhanced due to the f l u o r o g e n i c p r o p e r t y of the 2-naphthoyl moiety. Using t h i s d e r i v a t i v e , the comparative e f f e c t i v e n e s s of the P i r k l e c o v a l e n t and i o n i c c h i r a l columns f o r enantiomer r e s o l u t i o n was s t u d i e d . The i o n i c column was found to p r ovide a much b e t t e r r e s o l u t i o n (R= 1.45; f i g . 2B), hence a l l subsequent s t u d i e s were conducted w i t h t h i s column type. 55 FIGURE 2 C H R O M A T O G R A M S O F 2 - N A P H T H O Y L D E R I V A T I V E O F M E X I L E T I N E O N T H E P I R K L E C O V A L E N T ( A ) A N D P I R K L E IONICCB) C O L U M N S A B r — — — — i 1 1 i i 1 1 0 1 0 2 0 3 0 0 1 0 2 0 3 0 T I M E (MIN) T I M E (MIN) C h r o m a t o g r a p h i c c o n d i t i o n s : m o b i l e p h a s e , 5.5%. 2 - p r o p a n o l i n h e x a n e ; f l o w r a t e , 1.4 mL/min pumped i s o c r a t i c a l l y a t a m b i e n t t e m p e r a t u r e ; d e t e c t i o n , f l u o r e s c e n c e 230 nm (Ex) and 370 nm (Em); c h a r t s p e e d , 0.15 cm/min; a t t e n u a t i o n , 2 • 56 A s s a y D e v e l o p m e n t D e r i v a t i z a t i o n and HPLC w i t h F l u o r e s c e n c e  D e t e c t i o n The e n a n t i o m e r s o f m e x i l e t i n e w e re d e t e r m i n e d by HPLC a n a l y s i s a f t e r d e r i v a t i z a t i o n w i t h 2 - n a p h t h o y l c h l o r i d e . D e r i v a t i z a t i o n o f m e x i l e t i n e and t h e i n t e r n a l s t a n d a r d was c a r r i e d o u t u n d e r S c h o t t e n - B a u m a n n r e a c t i o n c o n d i t i o n s ( V o g e l , 1964) . The o p t i m u m r e a c t i o n t i m e was e v a l u a t e d w i t h s a m p l e s c o n t a i n i n g t h e same amount o f m e x i l e t i n e a nd t h e i n t e r n a l s t a n d a r d , e x p o s e d t o t h e d e r i v a t i z a t i o n r e a g e n t f o r d i f f e r e n t t i m e i n t e r v a l s . D e r i v a t i z a t i o n was r a p i d and was e s s e n t i a l l y c o m p l e t e w i t h i n one m i n u t e a s shown by t h e i d e n t i c a l p e a k h e i g h t s o b t a i n e d f o r a l l s a m p l e s ( f i g . 3 ) . A r e a c t i o n t i m e o f two m i n u t e s was c h o s e n a s a s t a n d a r d f o r a l l s u b s e q u e n t d e r i v a t i z a t i o n s . The e x c e s s r e a g e n t was c o n v e r t e d t o t h e w a t e r s o l u b l e s o d i u m - 2 - n a p h t h o a t e by t h e s o d i u m h y d r o x i d e p r e s e n t i n t h e r e a c t i o n m i x t u r e (scheme 1 ) . The 2 - n a p h t h a m i d e d e r i v a t i v e s o f m e x i l e t i n e a n d t h e i n t e r n a l s t a n d a r d w e r e i s o l a t e d by e x t r a c t i o n w i t h 0.6 mL o f t h e m o b i l e p h a s e ( 5 . 5 % 2 - p r o p a n o l i n h e x a n e ) . The d e r i v a t i v e s w e r e f o u n d t o be s t a b l e a t room t e m p e r a t u r e f o r a minimum o f 14 d a y s , as v e r i f i e d b y r e p e a t i n j e c t i o n s w h i c h showed no d e c l i n e i n peak h e i g h t s n o r c h a n g e s i n peak h e i g h t r a t i o s o v e r t h i s p e r i o d . 4.2 4.2.1 57 FIGURE 3 T I M E - D E P E N D E N C E O F D E R I V A T I V E F O R M A T I O N T I M E (MIN) SCHEME 1 REACTION SCHEME FOR DERIVATIZATION OF MEXILETINE AND THE INTERNAL STANDARD R= CH 3 ( M e x i l e t i n e ) R= H ( I . S . ) 59 R e p r e s e n t a t i v e HPLC c h r o m a t o g r a m s a r e shown i n f i g u r e 4 f o r m e x i l e t i n e e n a n t i o m e r s and t h e i n t e r n a l s t a n d a r d i s o l a t e d f r o m p l a s m a ( A ) , a n d b l a n k p l a s m a ( B ) . The e l u t i o n o r d e r o f t h e p e a k s w e r e 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 t h e i n t e r n a l s t a n d a r d ( 3 ) , a l l a s t h e 2 - n a p h t h a m i d e d e r i v a t i v e s . T h i s was d e t e r m i n e d by a n a l y s i s o f i n d i v i d u a l e n a n t i o m e r d e r i v a t i v e s u n d e r t h e same HPLC c o n d i t i o n s and c o m p a r i n g t h e i r r e t e n t i o n t i m e s w i t h t h o s e o f t h e r e s o l v e d p e a k s . No e v i d e n c e o f any i n t e r f e r r i n g p e a k s w i t h t h o s e due t o t h e e n a n t i o m e r s a n d t h e i n t e r n a l s t a n d a r d was f o u n d . The c h r o m a t o g r a m s o b t a i n e d f r o m u r i n e and s a l i v a s a m p l e s were s i m i l a r t o t h a t f r o m p l a s m a . They were g e n e r a l l y f r e e o f many e x t r a n e o u s p e a k s due t o t h e s e l e c t i v e n a t u r e o f f l u o r -e s c e n c e d e t e c t i o n . The minimum d e t e c t a b l e q u a n t i t y o f e a c h e n a n t i o m e r i n p l a s m a was 5 ng/mL a t a s i g n a l - t o - n o i s e r a t i o o f 5:1 r e p r e s e n t i n g 10 pg i n j e c t e d o n t o t h e c o l u m n . 4.2.2 M e c h a n i s m o f R e s o l u t i o n o f M e x i l e t i n e E n a n t i o m e r s R e s o l u t i o n o f e n a n t i o m e r s on a c h i r a l s t a t i o n a r y p h a s e (CSP) r e q u i r e s t h e f o r m a t i o n o f a minimum o f 3 s i m u l t a n e o u s t r a n s i e n t i n t e r a c t i o n s b e t w e e n t h e e n a n t i o m e r s and t h e CSP. One o f t h e s e i n t e r a c t i o n s i s r e q u i r e d t o be s t e r e o c h e m i c a l l y d e p e n d e n t and c o u l d be a t t r a c t i o n o r r e p u l s i o n ( s e e 2 . 1 2 . 2 ) . The e n a n t i o m e r d e r i v a t i v e s o f m e x i l e t i n e ( f i g . 5) a r e 60 FIGURE 4 C H R O M A T O G R A M S O F M E X I L E T I N E E N A N T I O M E R S A N D T H E I N T E R N A L S T A N D A R D I S O L A T E D F R O M P L A S M A ( A ) A N D B L A N K P L A S M A ( B ) 1 1 1 r -0 10 20 30 T I M E (min.) Chromatographic c o n d i t i o n s : Column, P i r k l e i o n i c c h i r a l column; mobile phase, 5.5% 2-propanol i n hexane; flow r a t e , 1 .4 mL/min pumped i s o c r a t i c a l l y a t ambient temperature; d e t e c t i o n , f l u o r e s c e n c e 230 nm (Ex£ and 370 nm (Em); c h a r t speed, 0.15 cm/min; a t t e n u a t i o n , 2 . 61 FIGURE 5 S T R U C T U R E O F M E X I L E T I N E E N A N T I O M E R S R ( - ) - M E X I L E T I N E H 3 C S ( + ) - M E X I L E T I N E 62 e n v i s a g e d t o i n t e r a c t w i t h t h e P i r k l e CSP ( f i g . 6) by means o f p i - b o n d i n g b e t w e e n t h e p i - b a s i c n a p h t h o y l g r o u p and t h e p i - a c i d i c 3 , 5 - d i n i t r o b e n z o y 1 g r o u p o f t h e CSP, e l e c t r o -s t a t i c b o n d i n g o f t h e amide d i p o l e s , and a s t e r i c i n t e r -a c t i o n o f t h e m e t h y l and x y l y l o x y m e t h y l g r o u p s a t t h e c h i r a l c e n t e r o f m e x i l e t i n e w i t h t h e p r o x i m a t e p o r t i o n s o f t h e CSP. As shown i n f i g u r e 7, a more s t a b l e c o m p l e x i s f o r m e d w i t h S ( + ) - m e x i l e t i n e - 2 - n a p h t h a m i d e , w h e r e t h e m e t h y l g r o u p i s b e l o w t h e p l a n e o f t h e a n a l y t e m o l e c u l e . The a n t i p o d e w i l l h a v e t h e b u l k i e r x y l y l o x y m e t h y l g r o u p b e l o w t h e p l a n e o f t h e m o l e c u l e a nd t h e r e f o r e w i l l be more s t e r i c a l l y h i n d e r e d f r o m f o r m i n g a c l o s e f i t w i t h t h e CSP. The r e s u l t i s a more r a p i d e l u t i o n o f t h e R ( - ) - e n a n t i o m e r . T h i s s t e r e o c h e m i c a l i n t e r a c t i o n o f m e x i l e t i n e e n a n t i o m e r s w i t h t h e P i r k l e CSP was d e r i v e d f r o m t h e " d i p o l e s t a c k i n g " c h i r a l r e c o g n i t i o n m odel o f P i r k l e and W e l c h (1984) w h i c h was u s e d t o e x p l a i n t h e e l u t i o n o r d e r o f e n a n t i o m e r i c a m i n e s on t h e P i r k l e c h i r a l s t a t i o n a r y p h a s e s . 4.2.3 C a l i b r a t i o n C u r v e s a nd P r e c i s i o n o f t h e A s s a y The c a l i b r a t i o n c u r v e d a t a and p r e c i s i o n o f t h e a s s a y i n p l a s m a , u r i n e and s a l i v a a r e g i v e n i n t a b l e s 1 t o 5. Peak h e i g h t m e a s u r e m e n t s w e r e f o u n d t o p r o v i d e l i n e a r c a l i b r a t i o n 2 c u r v e r e s u l t s ( r >0.999) o v e r t h e c o n c e n t r a t i o n r a n g e 63 FIGURE 6 S T R U C T U R E O F T H E P I R K L E C H I R A L S T A T I O N A R Y P H A S E S Y= NH (covalent) Y= uita, ( ion i c ) 64 FIGURE 7 T H E S T E R E O C H E M I C A L I N T E R A C T I O N B E T W E E N S ( + ) - M E X I L E T I N E - 2 - N A P H T H A M I D E A N D T H E P I R K L E IONIC C H I R A L S T A T I O N A R Y P H A S E T h e 2 - n a p h t h y l a n d t h e c a r b o x y a m i d e s y s t e m s a r e d e p i c t e d a s b e i n g c o p l a n a r . 65 5 to 750 ng/mL f o r each enantiomer i n plasma. A s i m i l a r 2 c o r r e l a t i o n (r >0.999) was found i n s a l i v a over the c o n c e n t r a t i o n range 10 to 1,500 ng/mL, and i n u r i n e over the c o n c e n t r a t i o n ranges 0.25 to 7.5 ug/mL and 10 to 500 ng/mL. Due to the use of peak h e i g h t measurement, a second u r i n e c a l i b r a t i o n curve was necessary to accommodate the low c o n c e n t r a t i o n s of the enantiomers seen i n u r i n e a f t e r 48 hours. I n t e r - a s s a y v a r i a b i l i t y , determined by t r i p l i c a t e p r e p a r a t i o n and a n a l y s i s of each of the samples used f o r the standard curves, was l e s s than 4% f o r a l l the b i o l o g i c a l f l u i d s . I n t r a - a s s a y v a r i a b i l i t y , determined by t r i p l i c a t e i n j e c t i o n of three of the samples used f o r the plasma c a l i b r a t i o n curves, was a l s o l e s s than 4%. 4.2.4 Recovery of M e x i l e t i n e Enantiomers from Plasma I n i t i a l r e c o v e r y s t u d i e s of m e x i l e t i n e enantiomers from plasma a f t e r pH adjustment to above 12 showed s u b s t a n t i a l l y low r e c o v e r i e s when compared to r e c o v e r i e s from water. In a d d i t i o n , the enantiomeric r a t i o s were a l t e r e d from R/S = 1.06 to R/S = 0.70 ( f i g . 8). Since enantiomers have the same s o l u b i l i t y i n or g a n i c s o l v e n t s , i t was s p e c u l a t e d t h a t the plasma p r o t e i n s may be b i n d i n g the enantiomers, d e s p i t e the h i g h plasma pH, and t h a t t h i s b i n d i n g c o u l d be s t e r e o -s e l e c t i v e . Plasma p r o t e i n p r e c i p i t a t i o n by two d i f f e r e n t TABLE 1 CALIBRATION CURVE DATA FOR MEXILETINE ENANTIOMERS IN PLASMA Weight of each enantiomer (ng) Peak-height r a t i o R ( - ) m e x i l e t i n e / i n t e r n a l standard* C V . (%) ** Peak-height r a t i o S ( + ) m e x i l e t i n e / i n t e r n a l standard* C V . (%) ** 5 50 100 250 500 750 0.028 + 0.001 0.334 + 0.011 0.630 + 0.017 1.571 +_ 0.028 3.003 + 0.079 4.565 + 0.027 3.5 0.027 + 0.001 3.4 0.314 + 0.012 2.6 0.593 + 0.013 1.8 1.481 + 0.033 2.6 2.821 + 0.073 0.6 42.70 •+ 0.054 3.8 3.7 2.1 2.2 2.6 1.2 slope i n t e r c e p t 2 0.0060 0.0226 0.9998 0.0056 0.0250 0.9997 * mean +_ standard d e v i a t i o n , n=3 ** c o e f f i c i e n t of v a r i a b i l i t y TABLE 2 CALIBRATION CURVE DATA FOR MEXILETINE ENANTIOMERS IN URINE (HIGHER CONCENTRATION RANGE) Weight of each Peak-height r a t i o C V . Peak-height r a t i o C V . enantiomer (ng) R ( - ) m e x i l e t i n e / (%)** S ( + ) m e x i l e t i n e / (%)** i n t e r n a l standard* i n t e r n a l s tandard* 50 0.205 +_ 0.003 1.6 0.187 + 0.002 1.7 250 0.982 + 0.002 0.2 0.921 +_ 0.006 0.6 500 2.000 +_ 0.0.78 3.9 1 .879 + 0.071 3.8 750 2.974 + 0.037 1.3 2.796 + 0.046 1.6 1000 3.862 + 0.062 1.6 3.600 + 0.035 1.0 1500 5.831 + 0.044 0.7 5.499 + 0.026 0.5 slope 0.0039 0.0036 i n t e r c e p t 0.0304 0.0201 r 2 0.9998 0.9996 * mean +_ standard d e v i a t i o n , n = 3 ** c o e f f i c i e n t of v a r i a b i l i t y TABLE 3 CALIBRATION CURVE DATA FOR MEXILETINE ENANTIOMERS IN URINE (LOWER CONCENTRATION RANGE) Weight of Peak-height r a t i o C V . Peak-height r a t i o C V . each R ( - ) - m e x i l e t i n e / (%)** S( + ) - m e x i l e t i n e / (%)** enantiomer i n t e r n a l standard* i n t e r n a l s tandard* (ng) 10 2.9569 ^ 0.0932 3.1 2.7786 +_ 0.0650 2.3 50 1.5011 + 0.0233 1.6 1.4351 + 0.0127 0.9 125 0.7433 + 0.0208 2.8 0.7067 + 0.0115 1.6 250 0.3195 +_ 0.0009 0.3 0.3079 + 0.0036 1.2 500 0.0598 + 0.0012 2.0 0.0598 + 0.0018 3.1 slope 0.0059 0.0055 i n t e r c e p t 0.0129 0.0217 r 2 0.9999 0.9999 * mean _+ standard d e v i a t i o n , n = 3 ** c o e f f i c i e n t of v a r i a b i l i t y TABLE 4 CALIBRATION CURVE DATA FOR MEXILETINE ENANTIOMERS IN SALIVA Weight of each Peak-height r a t i o C V . Peak-height r a t i o C V . enantiomer (ng) R ( - ) m e x i l e t i n e / (%)** S( + ) m e x i l e t i n e / (%)** i n t e r n a l standard* i n t e r n a l standard* 10 0.067 + 0.002 2.4 0.065 +_ 0.001 2.0 50 0.352 + 0.010 2.8 0.331 + 0.008 2.5 250 1.547 +_ 0.007 0.5 1.433 +_ 0.022 1.6 500 3.153 + 0.040 1.3 2.961 +_ 0.055 1.9 1000 6.104 +_ 0.147 2.4 5.687 + 0.149 2.6 1500 9.093 + 0.288 3.2 8.482 + 0.210 2.5 slope 0.0060 0.0056 i n t e r c e p t 0.0498 0.0485 r 2 • 0.9998 0.9997 * mean +_ standard d e v i a t i o n , n = 3 ** c o e f f i c i e n t of v a r i a b i l i t y TABLE 5 INTRA-ASSAY V A R I A B I L I T Y OF MEXILETINE ENANTIOMERS I N PLASMA W e i g h t o f e a c h P e a k - h e i g h t r a t i o C V P e a k - H e i g h t r a t i o C V e n a n t i o m e r (ng) R ( - ) m e x i l e t i n e / ( % ) * * S ( + ) m e x i l e t i n e / ( % ) * * i n t e r n a l s t a n d a r d * i n t e r n a l s t a n d a r d * 50 0.324 +_ 0.005 250 1.582 + 0.013 750 4.549 + 0.058 1.5 0.302 +_ 0.006 1.9 0.8 1.491 +_ 0.022 1.5 1.3 4.266 + 0.078 1.9 * mean +_ s t a n d a r d d e v i a t i o n , n = 3 i n j e c t i o n s ** c o e f f i c i e n t o f v a r i a b i l i t y 71 m e t h o d s : 10% t r i c h l o r o a c e t i c a c i d and b a r i u m h y d r o x i d e / z i n c s u l f a t e , b o t h e n h a n c e d r e c o v e r i e s and r e s t o r e d t h e n a t u r a l e n a n t i o m e r i c r a t i o ( f i g . 8, c h r o m a t o g r a m f o r 10% t r i c h l o r o -a c e t i c a c i d n o t shown) c o n f i r m i n g t h e s t e r e o s e l e c t i v e b i n d i n g s p e c u l a t e d a b o v e . T h e s e f i n d i n g s h a v e n e v e r b e e n r e p o r t e d and s u g g e s t t h a t a s s a y methods f o r (+_) - m e x i -l e t i n e , and p o s s i b l y o t h e r c h i r a l d r u g s , may h a v e b e e n r e p o r t i n g v a l u e s w h i c h a r e n o t a t r u e r a t i o o f t h e e n a n t i o m e r s . C o n s e q u e n t l y , p r e c i p i t a t i o n o f p l a s m a (serum) p r o t e i n s i s a s t e p t h a t s h o u l d be c o n s i d e r e d d u r i n g t h e d e v e l o p m e n t o f an a s s a y f o r m o d e r a t e l y t o h i g h l y p r o t e i n - b o u n d c h i r a l d r u g s . B a r i u m h y d r o x i d e / z i n c s u l f a t e was c h o s e n o v e r 10% t r i c h l o r a c e t i c a c i d f o r t h e p r o t e i n p r e c i p i t a t i o n p r o c e d u r e b e c a u s e t h e f o r m e r g a v e l e s s v a r i a b l e r e c o v e r i e s . The e f f i c i e n c y o f r e c o v e r y o f t h e e n a n t i o m e r s was 82.6 _+ 1.1% f o r R ( - ) - m e x i l e t i n e and 84.2 +_ 1.5% f o r S ( + ) - m e x i l e t i n e o v e r t h e c o n c e n t r a t i o n r a n g e 50 t o 750 ng/mL ( t a b l e 6 ) . 4.2.5 S t r u c t u r a l C o n f i r m a t i o n o f t h e D e r i v a t i v e s The p o s i t i v e c h e m i c a l i o n i z a t i o n mass s p e c t r a o f t h e 2-n a p h t h o y l d e r i v a t i v e s o f R , S - m e x i l e t i n e and t h e i n t e r n a l s t a n d a r d a r e shown i n f i g u r e 9A and 9B a l o n g w i t h t h e p r o p o s e d f r a g m e n t a t i o n p a t h w a y s ( f i g . 10A and 1 0 B ) . The 72 FIGURE 8 C H R O M A T O G R A M S OF M E X I L E T I N E E N A N T I O M E R S E X T R A C T E D F R O M W A T E R ( A ) , F R O M PL A S M A(B) AND F R O M P L A S M A A F T E R P R O T E I N P R E C I P I T A T I O N ( C ) B J o ~ i 1 r-10 20 30 —r-0 i 1 r 10 20 30 - i 1 1 r O 10 20 30 R E T E N T I O N T I M E (min.) C h r o m a t o g r a p h i c c o n d i t i o n s : C o l u m n , P i r k l e i o n i c c h i r a l c o l u m n ; m o b i l e p h a s e , 5.5% 2 - p r o p a n o l i n h e x a n e ; f l o w r a t e , 1.4 mL/min pumped i s o c r a t i c a l l y a t a m b i e n t t e m p e r a t u r e ; d e t e c t i o n , f l u o r e s c e n c e 230 nm (Ex£ and 370 nm (Em); c h a r t s p e e d , 0.15 cm/min," a t t e n u a t i o n , 2 . TABLE 6 E F F I C I E N C Y OF RECOVERY OF MEXILETINE ENANTIOMERS FROM PLASMA Peak H e i g h t R a t i o s * Peak H e i g h t R a t i o s * Q u a n t i t y o f R( - ) m e x . R ( - ) m e x . R e c o v e r y S(+)mex. S(+)mex. R e c o v e r y e a c h e n a n -t i o m e r (ng) (No P l a s m a P l a s m a (%) (No P l a s m a ) ( P l a s m a ) (%) 50 0.314 0.256 81 + 2.1 0.288 0.241 84 + 2.5 100 0. 620 0.520 84 + 0.4 0.577 0. 500 87 + 0.8 250 1.509 1.266 84 + 3.7 1.407 1.185 84 +. 3.8 500 3.150 2.592 82 + 0.7 2.919 2.437 84 +_ 0.8 750 4.286 3.511 82 + 1.7 3.958 3.250 82 + 0.4 * mean + s t a n d a r d d e v i a t i o n , n=3 74 FIGURE 9 C l M A S S S P E C T R A O F R, S - M E X I L E T I N E (A) A N D T H E I N T E R N A L S T A N D A R D ( B ) 3 3 4 212 155 123 163 I 2 4 0 I 2SI I 362 374 M/Z B 123 198 3 2 0 155 133 183 348 226 M / Z 75 FIGURE 10 FRAGMENT IONS OF 2-NAPHTHOYL DERIVATIVES OF R.S-MEXILETINE(A) AND THE INTERNAL STANDARD(B) (A) ,CH3 CH 3 0 // V o - C H 2 - C H - N H - C OO M / Z - 334(M*H) M/Z - 3 6 2 ( M * C 2 H 5 ) M/Z - 3 7 4 ( M - C 3 H 5 ) i HCX(_. C H 3 M 2 CH 3 o ft ^ O - C H ^ C H ^ N H - C , CO M / Z - 320 (M + H) M / Z - 3 4 8 ( M 4 C 2 H 5 ) / C H 3 H v N r r^C Q - O H 2 H2Ckc*oX^O ^ T H j H 2 M/Z-123 M / Z - 1 9 8 M / Z - 1 5 5 o II TOO CsCO M/Z - 212 M/Z - 155 (B) 4-H 0 76 t o t a l i o n c u r r e n t c h r o m a t o g r a m s ( f i g . 11A and 11B) d i d n o t c o n t a i n any e v i d e n c e o f o t h e r s u b s t a n c e s . The t o t a l i o n c u r r e n t c h r o m a t o g r a m s , mass s p e c t r a and f r a g m e n t a t i o n p a t t e r n f o r t h e i n d i v i d u a l e n a n t i o m e r d e r i v a t i v e s w e r e i d e n t i c a l and s i m i l a r t o t h a t o f R , S - m e x i l e t i n e - 2 - n a p h t h -a m i d e . 4.3 Serum P r o t e i n B i n d i n g o f M e x i l e t i n e E n a n t i o m e r s F i g u r e 12 shows r e p r e s e n t a t i v e c h r o m a t o g r a m s o b t a i n e d f r o m serum s p i k e d w i t h R , S - m e x i l e t i n e t o a c h i e v e a c o n c e n t r a t i o n o f 1.5 ug/mL(A) and t h e s erum u l t r a f i l t r a t e ( B ) . A number o f m e t h o d o l o g i c a l f a c t o r s h a v e b e e n r e p o r t e d t o i n f l u e n c e d r u g b i n d i n g i n human s e r u m . T h e s e i n c l u d e b i n d i n g d i s p l a c e m e n t by t r i s ( b u t o x y e t h y 1 ) p h o s p h a t e f r o m v a c u t a i n e r c a p s , age and s t o r a g e t e m p e r a t u r e o f t h e s e r u m , and t e m p e r a t u r e a t w h i c h t h e b i n d i n g e x p e r i m e n t was p e r f o r m e d ( B o r g a e t a l . , 1 9 7 7 , P i a f s k y 1980 and McNamara e t a l . , 1 9 8 1 ) . I n a d d i t i o n , a d s o r p t i v e l o s s e s t o t h e u l t r a f i l t r a t i o n e q u i p m e n t and p r o t e i n l e a k a g e t h r o u g h t h e f i l t r a t i o n membrane may i n t r o d u c e e r r o r s . The f o l l o w i n g s t e p s w e r e t a k e n t o a v o i d t h e i n f l u e n c e o f t h e s e f a c t o r s : b l o o d s a m p l e s w e r e d r a w n f r o m t h e v o l u n t e e r s w i t h a l l - g l a s s s y r i n g e s i n t o g l a s s t u b e s and s erum s a m p l e s u s e d i m m e d i a t e l y a f t e r s e p a r a t i o n f r o m b l o o d , a l l t h e b i n d i n g s t u d i e s w e re 77 FIGURE 11 T O T A L ION M A S S C H R O M A T O G R A M S O F 2 - N A P H T H O Y L D E R I V A T I V E S O F R . S - M E X I L E T I N E ( A ) A N D T H E I N T E R N A L S T A N D A R D ( B ) ^ «•»•<>-t 1 • • • • -< V 3 .» 4 .« .*> ».* r.» e .* TIME (MIN) T I M E (MIN) GC/MS c o n d i t i o n s : Helium ( c a r r i e r gas) f l o w r a t e 1 mL/min; Column, SE 54 f u s e d s i l i c a Q c a p i l l a r y (12 ni x 0.2 mm); i n j e c -t i o n p o r t t e m p e r a t u r e , 240 C; mode of i n j e c t i o n , s p l i t l e s s ; ovgn t e m p e r a t u r e , 50°C f o r 1 min. t o 300"C f o r 10 min. a t 30 C/min.; i n t e r f a c e t e m p e r a t u r e , 240 C; i o n s o u r c e t e m p e r a t u r e , 240 C; m u l t i p l i e r v o l t a g e , 2500 V; e m i s s i o n c u r r e n t , 300 mA; and r e a g e n t gas methane. 78 FIGURE 12 C H R O M A T O G R A M S O F M E X I L E T I N E E N A N T I O M E R S F R O M S P I K E D S E R U M ( A ) A N D T H E S E R U M U L T R A F I L T R A T E ( B ) B — i — 10 — I — 2 0 3 0 1 0 2 0 3 0 T I M E (MIN) T I M E (MIN) Chromatographic conditions: Column, P i r k l e i o n i c c h i r a l column; mobile phase, 5.5% 2-propanol i n hexane; flow rate, 1.4 mL/min pumped i s o c r a t i c a l l y at ambient temperature; detection, fluorescence 230 nm (Exl and 370 nm (Em); chart speed, 0.15 cm/min; attenuation, 2 . 79 done a t 37°C, t h e e x t e n t o f a d s o r p t i v e l o s s e s t o t h e u l t r a f i l t r a t i o n e q u i p m e n t was a s s e s s e d p r i o r t o t h e b i n d i n g s t u d i e s by m e a s u r i n g t h e c o n c e n t r a t i o n o f m e x i l e t i n e i n p h o s p h a t e b u f f e r b e f o r e and a f t e r u l t r a f i l t r a t i o n . The mean p e r c e n t r e c o v e r y o f t h e e n a n t i o m e r s ( c o n c e n t r a t i o n a f t e r / c o n c e n t r a t i o n b e f o r e f i l t r a t i o n ) a t 3 d i f f e r e n t c o n c e n t r a t i o n s ( 0 . 2 , 0.5 and 2 ug/mL i n t r i p l i c a t e ) w e r e 92.3 +_ 3.7% and 92.0 _+ 3.6% f o r R ( - ) - m e x i l e t i n e a n d S ( + ) - m e x i l e t i n e r e s p e c t i v e l y . R e c o v e r i e s w e r e h i g h a n d more i m p o r t a n t l y , i d e n t i c a l . The f r e e f r a c t i o n v a l u e s o b t a i n e d w e r e s u b s e q u e n t l y c o r r e c t e d f o r a d s o r p t i v e l o s s e s . P r o t e i n l e a k a g e t h r o u g h t h e f i l t r a t i o n membrane was c h e c k e d by t r a n s f e r r i n g a d r o p o f t h e u l t r a f i l t r a t e t o 0.1 mL o f 10% t r i c h l o r o a c e t i c a c i d . A b s e n c e o f t u r b i d i t y was u s e d as an i n d i c a t i o n o f membrane i n t e g r i t y . The mean e n a n t i o m e r f r e e f r a c t i o n s i n serum a t t h e d i f f e r e n t c o n c e n t r a t i o n s s t u d i e d a r e r e p o r t e d i n t a b l e 7. The d a t a i n d i c a t e a s t e r e o s e l e c t i v e b i n d i n g o f t h e e n a n t i o m e r s t o s e r u m p r o t e i n s w i t h R ( - ) - m e x i l e t i n e ( f r e e f r a c t i o n = 1 9 . 8 0 +_ 2.64%) b e i n g b o u n d more t h a n S ( + ) - m e x i -l e t i n e ( f r e e f r a c t i o n = 28.32 +_ 1.45%) ( p < 0 . 0 0 1 ) . E v i d e n c e o f s t e r e o s e l e c t i v e b i n d i n g o f d r u g s t o human serum p r o t e i n s h a s b e e n r e p o r t e d f o r a number o f d r u g s i n c l u d i n g p r o p r a n o l o l ( A l b a n i e t a 1 ., 1 9 8 4 ) , d i s o p y r a m i d e ( L i m a e t TABLE 7 FREE FRACTION (%) OF MEXILETINE ENANTIOMERS IN SERUM C o n c e n t r a t i o n R (-) M e x i l e t i n e C V . S ( + ) - M e x i l e t i n e C V . E q u i v a l e n t o f f r e e f r a c t i o n * ( % ) * * f r e e f r a c t i o n * ( % ) * * (_+) - M e x i l e t i n e b a s e (ug/mL) 0.2 22.14 + 5.50 0.5 19.26 +_ 5.03 1.0 21.02 + 4.58 1.5 17.42 + 2.96 2.0 19.15 + 2.64 mean 19.80 + 1.49 24.8 30.54 + 6.43 21.1 26.1 27.18 + 5.52 20.3 21.8 28.58 + 6.90 24.1 17.0 26.00 + 4.32 16.6 13.8 29.30 +_ 3.63 11.5 28.32 + 1.45 * mean _+ s t a n d a r d d e v i a t i o n , n=5 s u b j e c t s ** c o e f f i c i e n t o f v a r i a b i l i t y . 81 a l . , 1984) and w a r f a r i n (Toon and T r a g e r , 1 9 8 4 ) . I n t h e p r e s e n t s t u d y , t h e b i n d i n g o f m e x i l e t i n e e n a n t i o m e r s was n o t c o n c e n t r a t i o n d e p e n d e n t o v e r t h e c o n c e n t r a t i o n r a n g e 0.2 t o 2 ug/mL w h i c h c o v e r s t h e t h e r a p e u t i c r a n g e . The t o t a l b i n d i n g o f t h e e n a n t i o m e r s (R+S) was 76%. T a l b o t e t a l . , (1973) r e p o r t e d a v a l u e o f 70% f o r r a c e m i c m e x i l e t i n e . S i g n i f i c a n t i n t e r i n d i v i d u a l v a r i a b i l i t y i n t h e f r e e f r a c t i o n s was o b s e r v e d a t e a c h o f t h e c o n c e n t r a t i o n s s t u d i e d . S i m i l a r v a r i a b i l i t i e s i n t h e f r e e f r a c t i o n o f some b a s i c d r u g s , s u c h a s i m i p r a m i n e and a l p r e n o l o l ( P i a f s k y and B o r g a , 1977) and l i d o c a i n e ( R o u t l e d g e e t a l . , 1980 and S h a n d , 1984) h a v e b e e n e x p l a i n e d on t h e b a s i s o f t h e w i d e i n t r a - and i n t e r s u b j e c t v a r i a b i l i t y i n t h e c o n c e n t r a t i o n o f a l p h a s - a c i d g l y c o p r o t e i n , t h e p r i m a r y b i n d i n g p r o t e i n f o r t h i s c l a s s o f d r u g s ( P i a f s k y and K n o p p e r t 1 9 7 8 ) . S i n c e m e x i l e t i n e i s a b a s i c d r u g , t h e i n t e r - i n d i v i d u a l v a r i a b i l i t y i n t h e f r e e f r a c t i o n o f i t s e n a n t i o m e r s c o u l d be due t o t h e v a r i a b i l i t y i n t h e a l p h a s - a c i d g l y c o p r o t e i n c o n c e n t r a -t i o n i n t h e s e i n d i v i d u a l s . H o w e v e r , t h i s c o u l d n o t be v e r i f i e d s i n c e t h e e q u i p m e n t t o m e a s u r e t h e a l p h a s ~ a c i d g l y c o p r o t e i n l e v e l s i n t h e s t u d y s u b j e c t s was n o t a v a i l a b l e . 4.4 P h a r m a c o k i n e t i c s o f M e x i l e t i n e i n H e a l t h y V o l u n t e e r s 4.4.1 P l a s m a D a t a 82 The plasma c o n c e n t r a t i o n s of m e x i l e t i n e enantiomers at v a r i o u s times a f t e r o r a l a d m i n i s t r a t i o n of 300 mg of (+_) -m e x i l e t i n e h y d r o c h l o r i d e are shown i n t a b l e 8. F i g u r e 13 r e p r e s e n t s the plasma c o n c e n t r a t i o n versus time p r o f i l e of the enantiomers i n one s u b j e c t (MW). F i g u r e 14 i s a chroma-togram from a 30 hour sample from the same s u b j e c t . The pharmacokinetic parameters o b t a i n e d from the plasma data are l i s t e d i n t a b l e 9. The enantiomer k i n e t i c s were bes t d e s c r i b e d by a t r i e x p o n e n t i a l f u n c t i o n i n 3 of the 5 s u b j e c t s and a b i e x p o n e n t i a l f u n c t i o n i n the remaining 2 s u b j e c t s (no d i s t r i b u t i o n phase) . The plasma c o n c e n t r a t i o n s were h i g h l y v a r i a b l e with peak l e v e l s ranging from 130 to 520 ng/mL (285 +_ 144 ng/mL) f o r R ( - ) - m e x i 1 e t i n e and 120 to 385 ng/mL (234 +_ 97 ng/mL) f o r S ( + )-mexi l e t i n e . The mean a b s o r p t i o n r a t e constants (K ) were 1.7564 + 0.5562 h _ 1 f o r the R(-)-isomer and 1.9558 _+ 0.8143 h " 1 f o r the S(+)-isomer. T h i s corresponds to a b s o r p t i o n h a l f - l i v e s of 23.7 minutes and 21.3 minutes f o r the R ( - ) -and S(+)-isomers, r e s p e c t i v e l y . The time to peak plasma c o n c e n t r a t i o n s f o r both enantiomers was approximately the same i n each v o l u n t e e r and ranged from 1 to 4 hours. The d i s t r i b u t i o n r a t e c o n s t a n t (a) was 0.8155+0.5986 h"*1 f o r R ( - ) - m e x i l e t i n e and 0.9 434^1.005 h _ 1 f o r S ( + ) - m e x i l e t i n e . There was no s i g n i f i c a n t d i f f e r e n c e i n the 83 TABLE 8 PLASMA CONCENTRATION (NG/ML) - TIME DATA FOLLOWING ORAL DOSE OF 300 MG OF (+)-MEXILETINE HYDROCHLORIDE IN FIVE HEALTHY VOLUNTEERS. Time MW RL BA MM KM (hrs) R(-) S(+) R(-) S(+) R(-) S(+) R(-) S(+) R(-) S(+) 1 118 .2 76 . 25 521.7 384 .0 220.7 176.4 229 .2 174.1 52.65 37.88 1.5 233 .2 184 .7 440.2 337 .3 217.0 159.4 267.6 1 221.0 1 116.6 103 . 5 2 274 .9 225 .5 350.1 266 .1 233.3 192.6 1 249 .2 1 215.3 130.7 120.1 2.5 269 .4 234 .4 368 .8 293 .1 225.9 186.7 222.9 191.6 130.7 120.8 3 276 .4 239 .7 297.3 268 .9 222.0 196.6 224 .4 195.1 126.9 117.3 3.5 276 .4 254 .1 327.4 267 .7 229 .4 192.7 209.9 181.3 120.4 113.5 4 — — 305.5 252 .6 222.3 206.5 195.0 172.0 118.8 113.7 4 . 5 221 .8 212 .1 313.9 280 .8 222 .5 199.0 171.1 158.8 100.7 98.98 6 196 .4 196 .2 282.8 254 .1 196.2 £. / 185.8 156.8 152.5 91.77 93.54 8 154 .6 164 .8 235.4 215 .0 142.7 131.6 128.0 124.9 79.83 79.83 10 138 .2 160 .9 231.3 211 .4 105.4 108.1 103.5 104.2 61.17 61.50 12 120 .4 138 .1 169.1 160 . 1 86.57 88.85 84.15 87.63 52.65 55.82 14 105 .0 124 . 5 137.3 146 .7 68.69 72.96 6 5.00 73.62 52.35 56.53 ~i 24 54 . 44 74. 44 57.25 69 . 02 28.69 32.63 30.33 39.44 33.54 J 40. 89 30 40. 65 59 . 67 38.24 51. 79 13.56 16.69 15.10 20.74 21.42 26.55 36 32. 25 51. 97 20.68 29. 80 4.653 6.326 6.441 8.625 14.57 18.53 48 15. 99 27. 78 7.226 12. 48 — — — -- 7.120 9.227 1 = sample at 2.25 hr, 2 = sample at 5.5 hr, and 3 = sample at 25 hr. 84 FIGURE 13 S E M I L O G A R I T H M I C P L O T S O F P L A S M A C O N C E N T R A T I O N V E R S U S T I M E F O R M E X I L E T I N E E N A N T I O M E R S 3 0 0 i 10 2 0 3 0 4 0 5 0 T I M E ( H O U R S ) 85 FIGURE 14 M E X I L E T I N E E N A N T I O M E R S : P L A S M A P R O F I L E 3 0 H O U R S A F T E R A N O R A L D O S E O F T H E R A C E M A T E 3 2 i l 1 — i 0 10 2 0 3 0 T I M E (MIN) C h r o m a t o g r a p h i c c o n d i t i o n s : C o l u m n , P i r k l e i o n i c c h i r a l c o l u m n ; m o b i l e p h a s e , 5.5% 2 - p r o p a n o l i n h e x a n e ; f l o w r a t e , 1.4 mL/min pumped i s o c r a t i c a 1 l y a t a m b i e n t t e m p e r a t u r e ; d e t e c t i o n , f l u o r e s c e n c e 230 nm (Ex£ and 370 nm (Em); c h a r t s p e e d , 0.15 cm/min; a t t e n u a t i o n , 2 . 86 TABLE 9 PHARMACOKINETIC PARAMETERS OF MEXILETINE ENANTIOMERS FOLLOWING AN ORAL DOSE OF 300 MG OF (+)-MEXILETINE HYDROCHLORIDE peak cpeak K Ct (h) (ng/mL) * , ( h _ 1 ) (h x> a b Subjects R(-) S ( + ) R( -) S( + ) R(-) S( + ) R(-) S( + ) R( -) S ( + ) MW 2.5 3.0 2. 6 2. 7 275 245 1.2655 1.2935 0. 5197 0.4544 RL 1.0 1.0 1. 6 1. 5 520 385 2.0156 2,3888 — * MM 1.5 1.5 1. 3 1. 3 270 220 2.4639 2.8766 1. 5045 2.0943 BA 3.5 4.0 2. 3 2. 7 230 200 1.1213 0.9247 — * KM 2.0 2.0 1. 9 1. 7 130 120 1.9157 2.2954 0. 4224 0.2814 Mean 2.1 2.3 1. 9 2. 0 285 234 1.7564 1.9558 0. 8155 0.9434 1.0 1.2 0. 5 0. 7 144 97 0.5562 0.8143 0. 5986 1.0050 NS NS NS NS NS a estimated from plasma concentration vs time curve b tpeak = (2.303/K g -3) log (K a/g) NS not s i g n i f i c a n t * d i s t r i b u t i o n phase absent The r e s p e c t i v e age and weight of subjects were: MW (23 y r s , 77.3 kg); RL (26 y r s , 68.1 kg); MM (25 y r s , 88.6 kg); BA (28 y r s , 68.2 kg); KM (43 y r s , 100 kg) . 87 TABLE 9 CONT'D B t 1 / 2 A U C ^ c CL/F Vd/F (h~ -1) (h) (ug.h, .mL_1) (mL.inin kg" 1) (L. Kg" 1) R(-) S( + ) R(-) S ( + ) R(-) S( + ) R(-) S{ + ) R(-) S( + ) .0562 .0444 12.3 15 .6 4.19 5.15 6.41 5.22 6.84 7.05 .0852 .0687 8.1 10 .1 5.08 5.11 6.04 5.97 4.25 5.21 .1017 .0870 6.8 8 .0 2.63 2.70 8.91 8.86 5.26 5.99 .1123 .0992 6.2 7 .0 2.72 2.66 11.19 11.45 5.98 6.92 .0571 .0493 12.1 14 .1 2.04 2.24 10.18 9.27 10.69 11.29 .0825 .0697 9.1 11 .0 3.33 3.57 8.60 6.10 6.60 7. 30 .0255 .0236 2.9 3 .8 1.26 1.43 2.30 2.50 2.60 2.40 P<0. 01 P<0. 02 NS NS P<0 .01 88 p e ak p l a s m a c o n c e n t r a t i o n s , t i m e t o p eak p l a s m a c o n c e n t r a t i o n s and t h e a b s o r p t i o n and d i s t r i b u t i o n r a t e c o n s t a n t s f o r t h e e n a n t i o m e r s . The p l a s m a d a t a i n d i c a t e d t h a t a c r o s s o v e r i n t h e c o n -c e n t r a t i o n r a t i o s o f t h e e n a n t i o m e r s [ R(-)>S(+) t o S ( + ) > R ( - ) ] o c c u r r e d b e t w e e n 8 and 10 h o u r s p o s t d r u g a d m i n -i s t r a t i o n ( T a b l e 1 0 ) . H o w e v e r , t h e AUC f o r R ( - ) - m e x i l e t i n e (3.33 +_ 1.26 ug.mL C h ) was n o t s i g n i f i c a n t l y d i f f e r e n t f r o m t h a t o f S ( + ) - m e x i l e t i n e (3.57 +_ 1.43 u g . mL C h ) . T h e s e r e s u l t s a r e i n d i s a g r e e m e n t w i t h t h e f i n d i n g s b y G r e c h - B e 1 a n g e r e t a l . ( 1 9 8 6 ) who r e p o r t e d t h a t t h e s e r u m c o n c e n t r a t i o n s o f R ( - ) - m e x i l e t i n e 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 o f (+_) - m e x i l e t i n e t o h e a l t h y v o l u n t e e r s w ere s i g n i f i c a n t l y (P<0.001) l o w e r t h a n t h o s e o f t h e S ( + ) -i s o m e r a t a l l t i m e s , and t h a t t h e mean AUC o f R ( - ) - m e x i l e -t i n e was s i g n i f i c a n t l y (P<0.01) l e s s t h a n t h a t o f S ( + ) - m e x i -l e t i n e . T h e s e a u t h o r s a s s a y e d m e x i l e t i n e e n a n t i o m e r s a s t h e d i a s t e r e o i o s o m e r s , w h i c h d i f f e r s f r o m t h e a s s a y u s e d i n t h e p r e s e n t s t u d y , b u t more i m p o r t a n t i s t h e f a c t t h a t t h e y d i d n o t r e p o r t t h e s t e r e o s e l e c t i v e r e c o v e r y o f m e x i l e t i n e e n a n t i o m e r s f r o m p l a s m a (serum) i n t h e a b s e n c e o f p r o t e i n p r e c i p i t a t i o n ( s e e 4 . 2 . 4 ) . I t a p p e a r s t h e l o w e r s e r u m c o n c e n t r a t i o n s o f R ( - ) - m e x i l e t i n e r e l a t i v e t o S ( + ) -m e x i l e t i n e w h i c h was o b s e r v e d by G r e c h - B e l a n g e r e t a l . a t 89 TABLE 10 ENANTIOMER CONCENTRATION RATIOS IN PLASMA MW RL BA MM KM R(-)/S(+) R(-)/S(+) R(-)/S(+) R(-)/S(+) R(-)/S 1 1.55 1.36 1.25 1.32 1.39 1.5 1.26 1.31 1.36 1.21 1.13 2 1.22 1.32 1.21 1.16 1.09 2.5 1.15 1.26 1.21 1.16 1.08 3 1.15 1.11 1.13 1.15 1.08 3.5 1.09 1.22 1.19 1.16 1.06 4 — 1.21 1.08 1.13 1.04 4.5 1.04 1.12 1.12 1.08 1.02 6 1.00 1.11 1.06 1.03 1.01 8 0.94 1.09 1.08 1.02 1.00 10 0.86 1.09 0.98 0.99 0.99 12 0.87 0.99 0.97 0.96 0.94 14 0.84 0.94 0.94 0.88 0.93 24 0.73 0.83 0.88 0.77 0.82 30 0.68 0.74 0.81 0.73 0.81 36 0.62 0.69 0.74 0.75 0.79 48 0.58 0.58 — — 0.77 90 a l l times was due to the recove r y of amounts of t h i s enantiomer l e s s than t h a t which was a c t u a l l y present i n serum. The mean t e r m i n a l e l i m i n a t i o n r a t e constants were 0.0825 +_ 0.0255 h " 1 f o r R (-)-mexiletine and 0.0697 +_ _ i 0.0236 h f o r S ( + ) - m e x i l e t i n e . There was a s i g n i f i c a n t d i f f e r e n c e i n the t e r m i n a l e l i m i n a t i o n h a l f - l i v e s , 9.1 +_ 2.9 hours and 11.0 +_ 3.8 hours (P<0.02). These h a l f - l i f e v a l u e s agree w i t h those r e p o r t e d f o r (+_) -mexi l e t i n e i n the l i t e r a t u r e , 6.3 to 11.8 hours (mean 10 hours) ( P r e s c o t t e t a l . , 1977 , Campbell e t a l . , 1978b, D a n i l o , 1979 and H a s e l b a r t h e t a l . , 1981). The 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 enantiomers was not determined i n t h i s study due to the lac k of p e r m i s s i o n from the Health P r o t e c t i o n Branch, Ottawa, Canada, to a d m i n i s t e r m e x i l e t i n e i n t r a v e n o u s l y to h e a l t h y v o l u n t e e r s . The apparent o r a l body c l e a r a n c e and the apparent volume of d i s t r i b u t i o n were estimated u s i n g the f o l l o w i n g e x p r e s s i o n s ( G i b a l d i and P e r r i e r , 1975): CL /F = Dose Vd /F Dose  a r e a AUC ( t o t a l body c l e a r a n c e and t o t a l volume of d i s t r i b u t i o n c o u l d not be c a l c u l a t e d due to the unknown b i o a v a i l i b i l i t y of the 91 e n a n t i o m e r s i n h u m a n s ) . The mean v a l u e s f o r t h e a p p a r e n t -1 -1 o r a l c l e a r a n c e were 8.6 +_ 2.3 mL.min .kg and -1 -1 8.1 +_ 2.5 mL.min .kg f o r t h e R ( - ) - , and t h e S ( + ) - i s o m e r s r e s p e c t i v e l y ( d i f f e r e n c e n o t s i g n i f i c a n t ) . The mean a p p a r e n t v o l u m e o f d i s t r i b u t i o n was 6.6 +_ 2.6 L . k g 1 f o r R ( - ) - m e x i l e t i n e a n d 7.3 '•+_ 2.4 L . k g 1 f o r S ( + ) - m e x i l e t i n e . T h e r e was a s i g n i f i c a n t d i f f e r e n c e i n t h e a p p a r e n t v o l u m e o f d i s t r i b u t i o n ( P < 0 . 0 1 ) . H o w e v e r , n o t much i n f o r m a t i o n c a n be d e r i v e d f r o m t h e v a l u e s o f t h e a p p a r e n t o r a l c l e a r a n c e and t h e a p p a r e n t v o l u m e o f d i s t r i b u t i o n o f t h e e n a n t i o m e r s due t o t h e unknown v a l u e o f F f o r t h e e n a n t i o m e r s . A l l t h e p h a r m a c o k i n e t i c p a r a m e t e r s f o r t h e e n a n t i o m e r s showed h i g h i n t e r i n d i v i d u a l v a r i a b i l i t y . S i m i l a r i n t e r i n d i v i d u a l v a r i a b i l i t y h a s b e e n r e p o r t e d f o r (+_)-mexi l e t i n e . T h i s was e x p l a i n e d on t h e b a s i s o f i n d i v i d u a l d i f f e r e n c e s i n u r i n a r y pH (Kaye e t a l . , 1977 a n d J o h n s t o n e t a l . , 1979) a n d h e p a t i c m e t a b o l i s m ( B e c k e t t a nd C h i d o m e r e , 1977b a nd Kaye e t a l . , 1977)> H o w e v e r , t h e s e f a c t o r s a r e e x p e c t e d t o h a v e a p a r a l l e l e f f e c t on b o t h e n a n t i o m e r s and t h e r e f o r e w i l l n o t i n f l u e n c e 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 . 92 4.4.2 U r i n e D a t a T a b l e 11 shows t h e p h a r m a c o k i n e t i c p a r a m e t e r s f o r mex-i l e t i n e e n a n t i o m e r s e s t i m a t e d f r o m u r i n a r y e x c r e t i o n d a t a 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 o f 300 mg o f (+_) - m e x i l e t i n e h y d r o c h l o r i d e . The c u m u l a t i v e amount o f S ( + ) - m e x i l e t i n e e x c r e t e d was s i g n i f i c a n t l y g r e a t e r t h a n t h a t o f R ( - ) - m e x i l e t i n e i n a l l t h e s u b j e c t s (P<0.01) w i t h mean v a l u e s o f 9.14 _+ 3.07% and 7.40 +_ 2.40% r e s p e c t i v e l y . T h i s i n d i c a t e s a s t e r e o s e l e c t i v e u r i n a r y e x c r e t i o n o f t h e e n a n t i o m e r s i n f a v o u r o f S ( + ) - m e x i l e t i n e . The mean R/S r a t i o , 0.81 +_ 0.07 was s i m i l a r t o t h a t o b t a i n e d b y G r e c h -B e l a n g e r e t a l . , (1986) (R/S = 0 . 8 0 K K 2 1 ) . The mean amount o f b o t h e n a n t i o m e r s (R+S) e x c r e t e d i n u r i n e was 10.3 +. 3.41 mg r e p r e s e n t i n g 8.27 +_ 2.74% o f t h e a d m i n i s t e r e d d o s e . Mean 72 h o u r r e c o v e r i e s o f 7.9% ( P r e s c o t t e t a l . , 1977) and 7.5 t o 9.2% ( H a s e l b a r t h e t a l . , 1981) o f t h e a d m i n i s t e r e d d o s e h a v e b e e n r e p o r t e d f o r (+_) - m e x i l e t i n e i n h e a l t h y v o l u n t e e r s . The s i g n i f i c a n t l y (P<0.05) h i g h e r r e n a l c l e a r a n c e o f S ( + ) - m e x i l e t i n e (0.72 +_ 0.26 mL. m i n - 1 . k g - 1 ) r e l a t i v e t o R ( - ) - m e x i l e t i n e ( 0.61 _+ 0.20 m L . m i n - 1 . k g - 1 ) i s c o n s i s t e n t w i t h a s t e r e o s e l e c t i v e r e n a l e x c r e t i o n o f t h e e n a n t i o m e r s . The r e n a l c l e a r a n c e v a l u e s d i d n o t e x c e e d t h a t o f t h e g l o m e r u l a r f i l t r a t i o n r a t e (125 m l / m i n ; R o w l a n d and T o z e r , 1980) s u g g e s t i n g t h a t t h e TABLE 11 PHARMACOKINETIC PARAMETERS OF MEXILETINE ENANTIOMERS ESTIMATED FROM URINARY EXCRETION DATA FOLLOWING ORAL DOSE OF 300 MG OF (+_) - M E X I L E T I N E HYDROCHLORIDE. t 1 / 2 ( A R E p l o t ) K n r (h) ( h _ 1 ) R ( - ) / S ( + ) R(-) S(+) R ( - ) S(+) MW 0.38 0.41 7.39 9.89 0.75 9.1 9.9 0.0529 0.0409 RL 0.68 0.84 14.13 17.59 0.80 6.8 7.0 0.0755 0.0590 MM 0.47 0.51 6.61 7.30 0.91 6.1 6.1 0.0963 0.0819 BA 0.89 1.07 9.94 11.68 0.85 4.5 4.7 0.1033 0.0899 KM 0.65 0.78 8.01 10.46 0.74 --* — * 0.0534 O.0452 Mean 0.61 0.72 7.40 9.14 6.6 6.9 0.0763 0.0634 +S.D. 0.20 0.26 2.40 3.07 1.9 2.2 0.0235 0.0218 P<0.05 P<0.01 NS P<0.001 CL X r u ( m L . m i n - 1 k g - 1 ) (mg) S u b j e c t R(-) S(+) R(-) S(+) U r i n e c o l l e c t e d up t o 48 h o u r s o n l y 94 s t e r e o s e l e c t i v e r e n a l e x c r e t i o n was n o t due t o an a c t i v e r e n a l t r a n s p o r t m e c h a n i s m . S i n c e o n l y t h e f r e e d r u g i s f i l t e r e d by t h e g l o m e r u l u s i n t h e k i d n e y , t h e s t e r e o s e l e c t i v e r e n a l e x c r e t i o n c o u l d r e s u l t f r o m a d i f f e r e n c e i n t h e s erum p r o t e i n b i n d i n g o f t h e e n a n t i o m e r s . T h i s a g r e e s w i t h t h e b i n d i n g d a t a d i s c u s s e d e a r l i e r w h i c h showed t h a t S ( + ) - m e x i l e t i n e i s b o und t o a l e s s e r d e g r e e t o s e r u m p r o t e i n s t h a n i t s a n t i p o d e , r e s u l t i n g i n h i g h e r l e v e l s o f f r e e S ( + ) - m e x i l e t i n e b e i n g a v a i l a b l e i n t h e b l o o d t o be f i l t e r e d o u t by t h e k i d n e y . The mean n o n - r e n a l e l i m i n a t i o n r a t e c o n s t a n t s ( w h i c h i n c l u d e s m e t a b o l i c e l i m i n a t i o n ) w e r e 0.0763 +_ 0.0235 h - 1 f o r R ( -)-mexi l e t i n e and 0.0634 +_ 0.0218 h 1 f o r S ( + ) - m e x i l e t i n e . F o r a l l t h e s u b j e c t s , t h e n o n - r e n a l e l i m i n a t i o n r a t e c o n s t a n t f o r t h e R ( - ) - i s o m e r was s i g n i f i c a n t l y g r e a t e r (P<0.001) t h a n t h a t f o r S ( + ) - m e x i l e t i n e . S i n c e m e x i l e t i n e i s e x t e n s i v e l y m e t a b o l i s e d by h e p a t i c m i c r o s o m a l enzymes ( B e c k e t t and C h i d o m e r e , 1 9 7 7 b a n d P r e s c o t t e t a 1 . , 1 9 7 7 ) , t h e n o n - r e n a l e l i m i n a t i o n w i l l i n v o l v e m a i n l y m e t a b o l i c e l i m i n a t i o n and i n d i c a t e s a s t e r e o s e l e c t i v e m e t a b o l i s m o f t h e e n a n t i o m e r s i n f a v o u r o f t h e R ( - ) - i s o m e r . T h i s i s i n a g r e e m e n t w i t h t h e r e s u l t s o b t a i n e d by G r e c h - B e l a n g e r e t a l . , (1986) s h o w i n g t h a t t h e g l u c u r o n i d a t i o n o f m e x i l e t i n e e n a n t i o m e r s i s 95 s t e r e o s e l e c t i v e w i t h R ( - ) - m e x i l e t i n e b e i n g c o n j u g a t e d t o a g r e a t e r e x t e n t t h a n i t s e n a n t i o m e r . T a b l e 12 c o n t a i n s d a t a f o r t h e c u m u l a t i v e u r i n a r y e x c r e t i o n v e r s u s t i m e p l o t ( f i g . 15) and t h e s e m i l o g a r i t h m i c p l o t o f amount r e m a i n i n g t o be e x c r e t e d (ARE) v e r s u s t i m e ( f i g . 16) i n one s u b j e c t (MW). The mean t e r m i n a l e l i m i n a t i o n h a l f - l i v e s e s t i m a t e d u s i n g l o g - l i n e a r r e g r e s s i o n a n a l y s i s o f t h e ARE p l o t d a t a a f t e r t h e d i s t r i b u t i o n p h a s e ( f r o m 10 h o u r s p o s t - d o s e ) w e re 6.6 +_ 1.9 h o u r s and 6.9 +_ 2.2 h o u r s f o r R ( - ) - m e x i l e t i n e a n d S ( + ) - m e x i l e t i n e r e s p e c t i v e l y . The v a l u e s f o r t h e h a l f - l i v e s w e r e g e n e r a l l y l o w e r t h a n t h o s e o b t a i n e d f r o m t h e p l a s m a d a t a and showed no s i g n i f i c a n t d i f f e r e n c e b e t w e e n t h e e n a n t i o m e r s (ARE p l o t i n one o f t h e s u b j e c t s (KM) was n o t done b e c a u s e u r i n e was c o l l e c t e d f o r o n l y 48 h o u r s ) . F i g u r e 17 shows t h e e n a n t i o m e r p r o f i l e i n a 30 h o u r u r i n e s a m p l e i n one o f t h e s u b j e c t s (MW) w h i l e t h e t i m e c o u r s e o f t h e e n a n t i o m e r c o n c e n t r a t i o n r a t i o s (R/S) i n u r i n e a r e shown i n t a b l e 1 3 . The c r o s s - o v e r o f t h e r a t i o s o b s e r v e d i n p l a s m a was a l s o p r e s e n t , b u t o c c u r r e d much e a r l i e r i n a l l t h e s u b j e c t s (2 t o 3 h o u r s p o s t d o s e ) . 4.4.3 S a l i v a D a t a M e x i l e t i n e e n a n t i o m e r c o n c e n t r a t i o n s (ug/mL) i n t h e TABLE 12 DATA FOR CUMULATIVE URINARY EXCRETION VERSUS TIME, AND AMOUNT REMAINING TO BE EXCRETED (ARE) VERSUS TIME PLOTS IN ONE SUBJECT (MW) R( - ) - M e x i l e t i n e S ( + ) - M e x i l e t i n e Time Amount Cumulative Amount Amount Cumulative Amount (hrs) Excreted Amount Remaining Excreted Amount Remaining (mg) Excreted to be (mg) Excr e t e d to be (mg) Excreted (mg) Excr e t e d (mg) (mg) 0 7.3925 9.8915 1 0.2028 0.2028 7.1897 0.1559 0.1559 9.7356 2 1.0062 1.2090 6.1835 0.9958 1.1517 8.7398 3 0.3842 1.5932 5.7993 0.3980 1.5497 8.3418 4.5 0.4904 2.0836 5.3089 0.5315 2.0812 7.8103 6 0.3096 2.3932 4.9993 0.3555 2.4367 7.4548 8 0.4947 2.8879 4.5046 0.6121 3.0488 6.8427 10 0.7676 3.6555 3.7370 1.0003 4.0491 5.8424 12 1.0154 4.6709 2.7216 1.4302 5.4793 4.4122 14 0.0859 4.7568 2.6357 0.1036 5.5829 4.3086 23 2.0053 6.7621 0.6304 3.0790 8.6619 1.2296 27.5 0.0802 6.8423 0.5502 0.1204 8.7823 1.1092 30 0.0614 6.9037 0.4888 0.1023 8.8846 1.0069 34.75 0.1187 7.0224 0.3701 0.2337 9.1183 0.7732 36 0.0208 7.0431 0.3494 0.0372 9.1555 0.7360 46.5 0.2036 7.2467 0.1458 0.4150 9.5705 0.3210 48.2 0.0284 7.2751 0.1174 0.0626 9.6331 0.2584 52.5 0.0167 7.2918 0.1007 0.0338 9.6669 0.2246 53.75 0.0034 7.2952 0.0973 0.0079 9.6748 0.2167 58.75 0.0291 7.3243 0.0682 0.0673 9.7421 0.1494 61. 2 0.0026 7.3269 0.0656 0.006 5 9.7486 0.1429 63 0.0042 7.3273 0.0652 0.0010 9.7496 0.1419 72 0.0652 7.3925 0.0000 0.1419 9.8915 0.0000 97 FIGURE 15 C U M U L A T I V E U R I N A R Y E X C R E T I O N V E R S U S T I M E T I M E ( H O U R S ) 98 FIGURE 16 S E M I L O G A R I T H M I C P L O T O F A M O U N T R E M A I N I N G T O B E E X C R E T E D V E R S U S T I M E 10 -I. i i 1 i 1 i 10 2 0 3 0 4 0 5 0 6 0 TIME (HOURS) 99 FIGURE M E X I L E T I N E E N A N T I O M E R S : 3 0 H O U R S A F T E R A N O R A L 17 U R I N E P R O F I L E D O S E O F T H E R A C E M A T E 3 2 I 1 1 i 0 1 0 2 0 3 0 T I M E (MIN) C h r o m a t o g r a p h i c c o n d i t i o n s : Column, P i r k l e i o n i c c h i r a l column; m o b i l e phase, 5.5% 2-propanol i n hexane; f l o w r a t e , 1.4 mL/min pumped i s o c r a t i c a l l y a t ambient t e m p e r a t u r e ; d e t e c t i o n , f l u o r e s c e n c e 230 nm ( E x l and 370 nm (Em); c h a r t s peed, 0.15 cm/min, a t t e n u a t i o n , 2 . 100 TABLE 13 ENANTIOMER CONCENTRATION RATIOS IN URINE MW MM BA RL KM Time (hrs) R(-)/S(+) R(-)/S(+) R(-)/S(+) R(-)/S(+) R(-)/S 1 1.30 1.12 1.10 1.99 3.41 2 1.01 1.07 0.94 1.25 1.56 3 0.97 0.94 0.90 0.92 0.97 4 0.92 0.96 0.86 0.88 0.96 6 0.87 1.01 0.85 0.86 0.92 8 0.81 0.91 0.81 0.84 0.89 10 0.77 0.82 0.81 0.81 0.89 12 0.71 0.81 0.79 0. 80 0.81 14 0.83 0.80 0.76 0.79 0.78 24 0.65 0.78 0.73 0.68 0.75 30 0.60 0.76 0.64 0.67 0. 70 36 0.56 0.67 0.45 0.66 0.67 48 0.45 0.65 0.61 0.61 0.59 52 0.49 0.70 0.58 0.68 — 72 0.46 0.75 — 0.64 101 s a l i v a 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 o f 300 mg o f (+_)-mexi l e t i n e h y d r o c h l o r i d e a r e shown i n t a b l e 14. F i g u r e 18 shows t h e s e m i l o g a r i t h m i c p l o t s o f t h e s a l i v a c o n c e n t r a t i o n - t i m e p r o f i l e s , w h i l e f i g u r e 19 i s a c h r o m a t o g r a m o b t a i n e d f r o m a 30 h o u r s a l i v a s a m p l e i n one s u b j e c t (MW). No p h a r m a c o k i n e t i c c o n s t a n t s c o u l d be c a l c u l a t e d f r o m t h e s a l i v a c o n c e n t r a t i o n - t i m e p l o t s s i n c e t h e i r t e r m i n a l e l i m i n a t i o n p h a s e s w e r e n o t l i n e a r . S a l i v a r y s e c r e t i o n h a s b e e n r e p o r t e d f o r a number o f b a s i c d r u g s i n c l u d i n g p r o p r a n o l o l ( M u c k l o w e t a l . , 1 9 7 8 ) , t o c a i n i d e ( P i l l a i e t a l . , 1 9 8 4 ) , l i d o c a i n e ( B a r c h o w s k y e t a l . , 1982) and (+_) - m e x i l e t i n e ( B e c k e t t and C h i d o m e r e , 1 9 7 7 b ) . Wide i n t e r - and i n t r a - s u b j e c t v a r i a b i l i t y i n t h e s a l i v a / p l a s m a r a t i o s w e r e o b s e r v e d . T h i s h a s b e e n a t t r i b u t e d t o w i d e v a r i a b i l i t y i n i n t r a - a n d i n t e r - s u b j e c t s a l i v a pH, w h i c h i n t u r n i s l a r g e l y d e p e n d e n t on s a l i v a f l o w ( M u c k l o w e t a l . , 1978) . T h u s , f o r b a s i c d r u g s l i k e m e x i l e t i n e , w h i c h a r e h i g h l y i o n i z e d a t p h y s i o l o g i c a l pH due t o t h e i r h i g h p K a , s a l i v a r y s e c r e t i o n i s v e r y s u s c e p t i b l e t o pH c h a n g e s , and c o n s e q u e n t l y w o u l d n o t a c c u r a t e l y r e f l e c t t h e f r e e d r u g l e v e l i n p l a s m a ( M u c k l o w e t a l . , 1 9 7 8 ) . The o b s e r v e d s a l i v a / p l a s m a c o n c e n t r a t i o n r a t i o s o f m e x i l e t i n e e n a n t i o m e r s g e n e r a l l y e x c e e d e d u n i t y , and showed h i g h v a r i a b i l i t y ( t a b l e 1 5 ) . T h i s may h a v e b e e n due t o t h e pH 102 TABLE 14 MEXILETINE ENANTIOMERS IN THE SALIVA (UG/ML) OF HEALTHY VOLUNTEERS FOLLOWING AN ORAL DOSE OF 300 MG OF (+)-MEXILETINE HYDROCHLORIDE Time MW RL MM BA KM (hrs) R(-) S( + ) R( -) S( + ) R( -) S( + ) R( -) S( + ) R( -) S( + ) 1 0.518 0. 525 2. 807 2. 902 1. 827 1. 832 0. 480 0. 464 0. 193 0. 155 1.5 1.031 1. 106 1. 117 1. 199 2. 699 2 . 829 0. 962 1. 018 0. 549 0. 443 2 1.533 1. 704 2. 877 3. 175 2. 409 2. 601 1. 047 1. 148 0. 566 0. 565 2.5 0.939 1. 096 2. 669 3 . 006 3. 506 3. 884 0. 965 . 1. 069 0. 655 0. 680 3 1.045 1. 243 2. 092 2. 430 1. 515 1. 719 1. 023 1. 148 0. 702 0. 754 3.5 0.761 0. 911 1. 984 2. 288 1. 658 1. 889 0. 723 0. 838 0. 644 0. 698 4 — - 1. 306 1. 527 1. 712 1. 987 0. 873 1. 039 0. 566 0. 617 4.5 0.899 1. 115 0. 825 0. 970 1. 545 1. 796 0. 676 0. 781 0. 466 0. 523 6 0.746 1. 017 0. 232 0. 274 0. 593 0. 714 0. 259 0. 318 0. 416 0. 482 8 0.677 0. 921 1. 254 1. 588 0. 830 1. 025 0. 368 0. 443 0. 312 0. 368 10 0.672 0. 944 0. 862 1. 091 0. 666 0. 869 0. 265 0. 320 0. 258 0. 287 12 0.384 0. 569 0. 436 0. 574 0. 492 0. 644 0. 235 0. 295 0. 181 0. 223 14 0.216 0. 338 0. 369 0. 509 0. 263 0. 344 0. 198 0. 259 0. 223 0. 282 24 0.271 0. 490 0. 164 0. 255 0. 160 0. 227 0. 091 0. 143 0. 201 0. 265 30 0.122 0. 247 — - 0. 077 0. 117 0. 014 0. 026 0. 200 0. 280 36 0.022 0. 080 0. 109 0. 186 0. 044 0. 072 0. O i l 0. 018 0. 070 0. 098 48 0.015 0. 066 0. 016 0. 039 .- — — — 0. 089 0. 135 FIGURE 18 S E M I L O G A R I T H M I C P L O T S O F S A L I V A C O N C E N T R A T I O N V E R S U S T I M E IN A L L T H E S U B J E C T S 4 o Ul 0 10 20 30 40 50 0 10 20 30 40 50 T I M E ( H O U R S ) 104 FIGURE 19 K E X I L E T I N E E N A N T I O M E R S : S A L I V A P R O F I L E 3 0 H O U R S A F T E R A N O R A L D O S E O F T H E R A C E M A T E t • i 1 1 0 10 2 0 3 0 T I M E (MIN) C h r o m a t o g r a p h i c c o n d i t i o n s : Column, P i r k l e i o n i c c h i r a l column; m o b i l e phase, 5.5% 2-propanol i n hexane; f l o w r a t e , 1.4 mL/min pumped i s o c r a t i c a l l y a t ambient t e m p e r a t u r e ; d e t e c t i o n , f l u o r e s c e n c e 230 nm (Ex£ and 370 nm (Em); c h a r t speed, 0.15 cm/min; a t t e n u a t i o n , 2 . 1 0 5 TABLE 15 THE OBSERVED SALIVA/PLASMA CONCENTRATION RATIOS OF MEXILETINE ENANTIOMERS MW RL BA MM KM Time (hrs) R(-) S( + ) R(-) S( + ) R(-) S( + ) R(-) S( + ) R(-) S( + ) 1 4.38 6 .88 5.38 7.56 2.17 2.63 7.97 10.52 3.67 4 .09 1.5 4.42 5.99 2.54 3.55 4.43 6.39 10.09 12.80 4.71 4 .28 2 5.58 7.56 8.22 11.93 4.49 5.96 9.67 12.08 4.33 4 .70 2.5 3.49 4.68 7.24 10.26 4.27 5.73 15.73 20.27 5.01 5 .63 3 3.78 5.19 7.04 9 .04 4.61 5.84 6.75 8.81 5.53 6 .43 3.5 2.75 3.59 6.06 9.55 3.15 4.35 7 .90 10.42 5.35 6 .15 4 — — 4.27 6.05 3.93 5 .03 8.78 11.55 4.76 5 .43 4.5 4.05 5.26 2.63 • 3.45 3 .04 3.92 9 .03 11.31 4.63 5 .28 6 3.80 5.18 0.82 1.08 1.32 1.72 3.78 4.68 4.53 5 .15 8 4.38 5.59 5.33 7.39 2.58 3.37 6.48 8.21 3.91 4 .61 10 4.86 5.87 3.73 5.16 2.51 2.96 6.43 8.34 4.22 4 .67 12 3.19 4.12 2.58 3.59 2.71 3.32 5.85 7.35 3.44 3 .99 14 2 .06 2.71 2.69 3.47 2.88 3.55 4.05 4.70 4.26 4 .99 24 4.98 6.58 2.86 3.69 3.17 4.38 5.28 5.76 5.99 6 .48 30 3.00 4.14 — -- 1.03 1.56 5.10 5.64 9.34 10 .55 36 0.68 1.54 5.27 6.24 2.36 2.85 6.82 8.35 4.80 11 .49 48 0.94 2.38 2.22 3.13 — — — — 12.5 14 .63 106 f a c t o r m e n t i o n e d a b o v e , s i n c e t h e s a l i v a c o l l e c t i o n p r o c e d u r e u s e d was n o t s t a n d a r d i z e d ( " u n s t i m u l a t e d " s a l i v a was c o l l e c t e d f r o m t h e s u b j e c t s b u t s a l i v a f l o w may h a v e b e e n s t i m u l a t e d t o v a r y i n g d e g r e e s d u r i n g c o l l e c t i o n ) . U n f o r t u n a t e l y , i t was n o t p o s s i b l e t o p r e d i c t t h e s a l i v a / p l a s m a r a t i o s u s i n g t h e M a t i n ' s e q u a t i o n ( M a t i n e t a l . , 1974) b e l o w b e c a u s e s a l i v a pH c o u l d n o t be m e a s u r e d t h e t i m e o f c o l l e c t i o n . s = i + i o p K a _ p H . f s -p p i + i o p K a - p H f P s s = c o n c e n t r a t i o n o f d r u g i n s a l i v a p = c o n c e n t r a t i o n o f d r u g i n p l a s m a pKa = pKa o f b a s i c d r u g pHs = s a l i v a pH pHp = p l a s m a pH (7.4) f = f r e e f r a c t i o n i n p l a s m a f p = f r e e f r a c t i o n i n s a l i v a (assumed t o s be 1) However, t h e o b s e r v e d s a l i v a / p l a s m a r a t i o s f a l l w i t h i n t h e r a n g e o f v a l u e s o b t a i n e d b y s u b s t i t u t i n g t h e p h y s i o l o g i c a l s a l i v a pH r a n g e o f 5.5 t o 7.9 (Dawes and J e n k i n s , 1964) i n t h e m a t i n ' s e q u a t i o n s u g g e s t i n g t h a t t h e s e c r e t i o n o f t h e e n a n t i o m e r s i n t o s a l i v a d i d n o t i n v o l v e an a c t i v e t r a n s p o r t m e c h a n i s m . A l t h o u g h t h e e n a n t i o m e r s a l i v a / p l a s m a r a t i o s showed m a r k e d i n t e r - and i n t r a - s u b j e c t v a r i a b i l i t y , a good c o r r e l a t i o n (r= 0.8903 _+ 0.0719 f o r R ( - ) - m e x i l e t i n e and r= 0.8493 + 0.0835 f o r S ( + ) - m e x i l e t i n e ) was o b s e r v e d 107 between the s a l i v a and plasma c o n c e n t r a t i o n s of the enantiomers i n each v o l u n t e e r ( f i g . 20). The s a l i v a AUC of S(+)-mexiletine was s i g n i f i c a n t l y (P<0.01) g r e a t e r than t h a t of R ( - ) - m e x i l e t i n e i n a l l the su b j e c t s ( t a b l e 16) i n d i c a t i n g s t e r e o s e l e c t i v e s a l i v a r y s e c r e t i o n . The mean s a l i v a AUCS were 18.2 +_ 6.3 ug. mL - 1.hr and 14.1 +_ 5.0 ug.mL C h r f o r S(+)-mexi-l e t i n e and the R(-)-isomer r e s p e c t i v e l y . I t i s l i k e l y t h a t the g r e a t e r s a l i v a r y s e c r e t i o n of the S(+)-isomer i s a r e f l e c t i o n of the s t e r e o s e l e c t i v e serum p r o t e i n b i n d i n g of the enantiomers (S(+)-mexiletine i s l e s s bound than R ( - ) -m e x i l e t i n e ) as was the case with t h e i r u r i n a r y e x c r e t i o n . T h i s i s supported by the concept t h a t only the unbound un-i o n i z e d drug i s s e c r e t e d by the s a l i v a r y glands and remains i n e q u i l i b r i u m with the f r e e drug i n the plasma water (Koysooko e t a l . , 1974). Furthermore, the s i m i l a r i t y i n the enantiomer c o n c e n t r a t i o n r a t i o s (R/S) i n s a l i v a ( t a b l e 17) and u r i n e (see t a b l e 13) i m p l i e s a common i n f l u e n c e ( s t e r e o s e l e c t i v e serum p r o t e i n binding) behind the s t e r e o -s e l e c t i v e u r i n a r y e x c r e t i o n and s a l i v a r y s e c r e t i o n of the enantiomers. 4.4.4 I n t e r r e l a t i o n s h i p of P r o t e i n B i n d i n g , Plasma, Urine and S a l i v a Data Table 18 compares the time course of the enantiomer 108 FIGURE 20 C O R R E L A T I O N B E T W E E N S A L I V A A N D P L A S M A L E V E L S O F M E X I L E T I N E E N A N T I O M E R S F O L L O W I N G A N O R A L D O S E O F T H E R A C E M A T E S ( + ) - M E X I L E T I N E r= 0 . 9 1 5 2 1 2 0 0 i 8 0 0 A 4 0 0 H R ( - ) - M E X I L E T I N E r = 0 . 9 4 3 4 1 0 0 2 0 0 P L A S M A C O N C E N T R A T I O N ( n g / m L ) 3 0 0 TABLE 16 MEXILETINE ENANTIOMER SALIVA AUCS ( u g . m L - 1 . h ) S u b j e c t R ( - ) - m e x i l e t i n e S ( + ) - m e x i l e t i n e MW 13.62 20.23 RL 19.89 25.27 MM 18.13 21.75 BA 7.98 9.749 KM 10.84 13.73 o P<0.01 110 T A B L E 17 ENANTIOMER CONCENTRATION R A T I O S I N S A L I V A MW R L MM BA KM T i m e ( h r s ) R ( - ) / S ( + ) R ( - ) / S ( + ) R ( - ) / S ( + ) R ( - ) / S ( + ) R ( - ) / S ( + ) 1 0.99 0.97 1.00 1.03 1.25 1.5 0.9 3 0.93 0.95 0.94 1.24 2 0 . 9 0 0.9 1 0.93 0 . 9 1 1.00 2.5 0.86 0.89 0 . 9 0 0 . 9 0 0.96 3 0.84 0.86 0.88 0.89 0 . 9 3 3.5 0.84 0.87 0.88 0.86 0.92 4 — 0.86 0.86 0.84 0.92 4.5 0 . 8 1 0 . 8 5 0.86 0.87 0.89 6 0.73 0 . 8 5 0.83 0 . 8 1 0.86 8 0.74 0.79 0 . 8 1 0 . 8 3 0 . 8 5 10 0 . 7 1 0.79 0.77 0.8 3 0 . 9 0 12 0 .67 0.76 0.76 0 . 8 0 0.8 1 14 0.64 0.72 0.85 0.76 0.79 24 0 . 5 5 0.64 0. 7 0 0.64 0.76 3 0 0.49 — 0.66 0.54 0 . 7 1 36 0.28 0.59 0 . 6 1 0 . 6 1 0 . 7 1 48 0 . 2 3 0 . 4 1 — — 0.66 I l l T A B L E 1 8 ENANTIOMER CONCENTRATION R A T I O S ( R / S ) I N P L A S M A , U R I N E AND S A L I V A . MW RL BA T i m e ( h r s ) P U s P U S P U S 1 1 . 5 5 1 . 3 0 0 . 9 9 1 . 3 6 1 . 9 9 0 . 9 7 1 . 2 5 1 . 1 0 1 . 0 3 2 1 . 2 2 1 . 0 1 0 . 9 0 1 . 3 2 1 . 2 5 0 . 9 1 1 . 2 1 0 . 9 4 0 . 9 1 3 1 . 1 5 0 . 9 7 0 . 8 4 1 . 1 1 0 . 9 2 0 . 8 6 1 . 1 3 0 . 9 0 0 . 8 9 4 — — — 1 . 2 1 0 . 8 8 0 . 8 6 1 . 0 8 0 . 8 6 0 . 8 4 6 1 . 0 0 0 . 8 7 0 . 7 3 1 . 1 2 0 . 8 6 0 . 8 5 1 . 0 6 0 . 8 5 0 . 8 1 B 0 . 9 4 0 . 8 1 0 . 7 4 1 . 1 1 0 . 8 4 0 . 7 9 1 . 0 8 0 . 8 1 0 . 8 3 1 0 0 . 8 6 0 . 7 7 0 . 7 1 1 . 0 9 0 . 8 1 0 . 7 9 0 . 9 8 0 . 8 1 0 . 8 3 1 2 0 . 8 7 0 . 7 1 0 . 6 7 0 . 9 9 0 . 8 0 0 . 7 6 0 . 9 7 0 . 7 9 0 . 80 1 4 0 . 8 4 0 . 8 3 0 . 6 4 o . 9 4 0 . 7 9 0 . 7 2 0 . 9 4 0 . 7 6 0 . 7 6 2 4 0 . 7 3 0 . 6 5 0 . 5 5 0 . 8 3 0 . 6 8 0 . 6 4 0 . 8 8 0 . 7 3 0 . 6 4 3 0 0 . 6 8 0 . 6 1 0 . 4 9 0 . 7 4 0 . 6 7 — 0 . 8 1 0 . 6 4 0 . 5 4 3 6 0 . 6 2 0 . 5 6 0 . 2 8 0 . 6 9 0 . 6 6 0 . 5 9 0 . 7 4 0 . 4 5 0 . 6 1 4 8 0 . 5 8 0 . 4 5 0 . 2 3 0 . 5 8 0 . 6 1 0 . 4 1 — 0 . 6 1 — P = p l a s m a U = u r i n e S = s a l i v a 112 TABLE 18 CONT'D MM KM p U S P U S 1.32 1.12 1.10 1.39 3.41 1.25 1.16 1.07 0.93 1.09 1.56 1.00 1.15 0.94 0.88 1.08 0.97 0.93 1.13 0.96 0.B6 1.04 0.96 0.92 1.03 1.01 0.B3 1.01 0.92 0.86 1.02 0.91 0.81 1.00 0.89 0.90 0.99 0.82 0.77 0.99 0.69 0.90 0.96 0.81 0.76 0.94 0.81 0.81 0.88 0.80 0.85 0.93 0.78 0.79 0.77 0.78 0.70 0.82 0.75 0.76 0.73 0.76 0.66 0.B1 0.70 0.71 0.75 0.67 0.61 0.79 0.67 0.71 0.65 0.77 0.59 0.66 113 c o n c e n t r a t i o n r a t i o s (R/S) i n p l a s m a , u r i n e and s a l i v a . A c r o s s o v e r i n t h e e n a n t i o m e r c o n c e n t r a t i o n r a t i o s i n t h e t h r e e b i o l o g i c a l f l u i d s was e v i d e n t w i t h an a p p a r e n t d i s c r e p a n c y i n t h e c r o s s o v e r t i m e (0 t o 3 h o u r s i n u r i n e and s a l i v a r e l a t i v e t o 8 t o 10 h o u r s i n p l a s m a ) . S i n c e t h e s t u d y r e s u l t s d i d n o t i n d i c a t e t h e p r e s e n c e o f an a c t i v e u r i n a r y o r s a l i v a r y t r a n s p o r t o f t h e e n a n t i o m e r s , i t was assumed t h a t t h e s e two p r o c e s s e s o c c u r r e d by p a s s i v e d i f f u s i o n and w o u l d t h e r e f o r e d e p e n d on t h e f r e e d r u g c o n c e n t r a t i o n o f t h e e n a n t i o m e r s i n b l o o d . T h i s l e d t o t h e s p e c u l a t i o n t h a t t h e f r e e e n a n t i o m e r c o n c e n t r a t i o n r a t i o s i n p l a s m a w o u l d p r o b a b l y c o r r e l a t e b e t t e r w i t h t h e r a t i o s i n u r i n e and s a l i v a . U n f o r t u n a t e l y , t h e f r e e e n a n t i o m e r s e r u m l e v e l s i n t h e s u b j e c t s were n o t d e t e r m i n e d d u r i n g t h e s t u d y s i n c e t h e n e e d was n o t o b v i o u s . M o r e o v e r , t h e s e n s i t i v i t y o f t h e a s s a y w o u l d h a v e b e e n a m a j o r l i m i t a t i o n t o f o l l o w i n g f r e e l e v e l s f o r 48 h o u r s . H o w e v e r , i t i s f e a s i b l e t o o b t a i n f r e e d r u g c o n c e n t r a t i o n s f r o m t h e p r o d u c t o f f r e e f r a c t i o n d e t e r m i n e d i n v i t r o a nd t h e m e a s u r e d t o t a l d r u g c o n c e n t r a t i o n , p r o v i d e d b i n d i n g i s n o t c o n c e n t r a t i o n d e p e n d e n t ( L e v y and Y a c o b i , 1 9 7 4 ) . T h i s a p p r a o c h h a s b e e n u s e d t o e s t i m a t e t h e f r e e l e v e l s o f w a r f a r i n , i n man ( Y a c o b i e t a l . , 1 9 7 6 ) . The f r e e f r a c t i o n s o f m e x i l e t i n e e n a n t i o m e r s d i d n o t e x h i b i t c o n c e n t r a t i o n d e p e n d e n c e o v e r 114 t h e c o n c e n t r a t i o n r a n g e 0.2 ug/mL t o 2 u g / L ( s e e 4.2) a n d i s n o t l i k e l y t o do s o b e l o w 0.2 ug/mL ( e n a n t i o m e r c o n c e n t r a t i o n s a b o v e 2 ug/mL were n o t e n c o u n t e r e d i n any o f t h e s u b j e c t s ) . A c c o r d i n g l y t h e f r e e e n a n t i o m e r l e v e l s w e r e e s t i m a t e d a s d e s c r i b e d a b o v e u s i n g i n d i v i d u a l f r e e f r a c t i o n v a l u e s . The f r e e e n a n t i o m e r c o n c e n t r a t i o n r a t i o s (R/S) a r e shown i n t a b l e 19. The r a t i o s w e r e f o u n d t o be s i m i l a r t o t h o s e i n u r i n e and s a l i v a c o n f i r m i n g t h a t t h e o b s e r v e d s t e r e o s e l e c t i v e u r i n a r y e x c r e t i o n and s a l i v a r y s e c r e t i o n o f t h e e n a n t i o m e r s i n f a v o u r o f S ( + ) - m e x i l e t i n e was a r e s u l t o f t h e s t e r e o s e l e c t i v e p r o t e i n b i n d i n g o f t h e e n a n t i o m e r s w h i c h f a v o u r s R ( - ) - m e x i l e t i n e . The r e l a t i o n s h i p b e t w e e n p l a s m a and t h e u r i n e and s a l i v a l e v e l s was masked b y m e a s u r i n g t h e t o t a l p l a s m a d r u g l e v e l s . T h u s , a s t h e s e r e s u l t s p o i n t o u t , t h e r e a r e d e f i n i t e a d v a n t a g e s i n d e t e r m i n i n g t h e f r e e d r u g l e v e l s a s o p p o s e d t o t h e t o t a l d r u g . T h i s method h a s r e c e i v e d much s u p p o r t i n t h e l i t e r a t u r e f o r i t s more r e l e v a n t a p p r o a c h ( R o w l a n d , 1980 and L e v y , 1 9 8 3 ) . A l t h o u g h t h e r e n a l e x c r e t i o n o f t h e e n a n t i o m e r s i s s t e r e o s e l e c t i v e , t h i s i s n o t e x p e c t e d t o s i g n i f i c a n t l y a f f e c t t h e e n a n t i o m e r p l a s m a l e v e l s s i n c e r e n a l e l i m i n a t i o n c o n s i t i t u t e s a m i n o r p a r t o f t h e o v e r a l l e l i m i n a t i o n p r o c e s s ( t h e v a l u e s o f n o n - r e n a l e l i m i n a t i o n r a t e c o n s t a n t s f o r t h e e n a n t i o m e r s a p p r o a c h e s t h a t o f t h e i r o v e r a l l e l i m i n a t i o n 115 TABLE 19 FREE ENANTIOMER CONCENTRATION* RATIOS IN PLASMA Time MW RL BA MM KM ( h r s ) R ( - ) / S ( + ) R ( - ) / S ( + ) R ( - ) / S ( + ) R ( - ) / S ( + ) R ( - ) / S ( + ) 1 1.07 0.94 0.80 0.98 1.06 1.5 0.87 0. 90 0.87 0.90 0.86 2 0.83 0.91 0.77 0.86 0.83 2.5 0.79 0.87 0.77 0.86 0.82 3 0. 79 0.77 0.72 0.85 0.82 3.5 0.75 0.85 0.76 0.86 0.81 4 0.84 0.68 0.84 0.79 4.5 0.72 0.78 0.71 0. 80 0.7 7 6 0.69 0.77 0.67 0.76 0.74 8 0.65 0.76 0.69 0.76 0.76 10 0.59 0.76 0.62 0.74 0.76 12 0.60 0.73 0.62 0.71 0.72 14 0.59 0.65 0. 60 0.65 0. 70 24 0. 50 0.58 0.56 0.57 0.62 30 0.47 0.51 0.52 0.54 0.61 36 0.43 0.48 0.48 0.55 0.60 48 0.40 0.40 — — 0.59 * F r e e e n a n t i o m e r c o n c e n t r a t i o n by c o n c e n t r a t i o n t h e mean f r e e c a l c u l a t e d f r a c t i o n f o r by m u l t i p l y i n g e a c h s u b j e c t . t o t a l 116 r a t e c o n s t a n t s ) . H o w e v e r , s t e r e o s e l e c t i v e p r o t e i n b i n d i n g and r e n a l e x c r e t i o n i s e x p e c t e d t o e n h a n c e t h e b o d y c l e a r -a n c e o f t h e S ( + ) - i s o m e r s i n c e m e x i l e t i n e i s a r e s t r i c t i v e l y c l e a r e d d r u g . Thus t h e t o t a l e n a n t i o m e r c o n c e n t r a t i o n r a t i o (R/S) i n p l a s m a s h o u l d a l w a y s be g r e a t e r t h a n u n i t y . T h i s was n o t t h e c a s e 6 t o 8 h o u r s p o s t - d o s e i n t h e s u b j e c t s ( t a b l e 10) s u g g e s t i n g t h a t t h e n o n - r e n a l e l i m i n a t i o n ( m a i n l y m e t a b o l i s m ) o f t h e e n a n t i o m e r s i s s t e r e o s e l e c t i v e w i t h t h e R ( - ) - i s o m e r b e i n g m e t a b o l i z e d a t a f a s t e r r a t e t h a n i t s a n t i p o d e . T h i s i s i n a g r e e m e n t w i t h t h e s i g n i f i c a n t l y (P<0.001) g r e a t e r n o n - r e n a l e x c r e t i o n r a t e c o n s t a n t f o r R ( - ) - m e x i l e t i n e r e l a t i v e t o S ( + ) - m e x i l e t i n e f o u n d i n t h i s s t u d y ( t a b l e 1 1 ) . I t i s a l s o c o n s i s t e n t w i t h t h e r e s u l t s o b t a i n e d by G r e c h - B e l a n g e r e t a l . (1986) s h o w i n g t h a t t h e m e t a b o l i s m ( g l u c u r o n i d a t i o n ) o f m e x i l e t i n e e n a n t i o m e r s i s s t e r e o s e l e c t i v e i n f a v o u r o f R ( - ) - m e x i l e t i n e . 117 5. Summary and C o n c l u s i o n s A h i g h - p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h i c a s s a y f o r m e x i l e t i n e e n a n t i o m e r s was d e v e l o p e d u s i n g t h e P i r k l e c h i r a l s t a t i o n a r y p h a s e and a f l u o r e s c e n c e d e t e c t o r . The a s s a y i s r e l a t i v e l y s i m p l e , d o e s n o t h a v e t h e i n h e r e n t draw-b a c k s a s s o c i a t e d w i t h t h e d e t e r m i n a t i o n o f e n a n t i o m e r s a s d i a s t e r e o i s o m e r s and h a s t h e h i g h s e n s i t i v i t y and s e l e c t i v i t y o f f l u o r e s c e n c e d e t e c t i o n . The a s s a y r e v e a l e d t h a t t h e r e c o v e r y o f m e x i l e t i n e e n a n t i o m e r s f r o m p l a s m a was s t e r e o s e l e c t i v e e v e n a f t e r pH a d j u s t m e n t t o ab o v e 12. P r e c i p i t a t i o n o f t h e p l a s m a p r o t e i n s w i t h b a r i u m h y d r o x i d e / z i n c s u l f a t e r e s t o r e d t h e n a t u r a l e n a n t i o m e r i c r a t i o . T h e s e r e s u l t s i n d i c a t e d t h e ne e d t o c o n s i d e r p l a s m a p r o t e i n p r e c i p i t a t i o n when d e v e l o p i n g an a s s a y f o r m o d e r a t e l y t o h i g h l y p r o t e i n - b o u n d c h i r a l d r u g s . The a p p l i c a b i l i t y o f t h e a s s a y was d e m o n s t r a t e d i n i n v i t r o p r o t e i n b i n d i n g and p h a r m a c o k i n e t i c s t u d i e s i n h e a l t h y m a l e s u b j e c t s . Serum p r o t e i n b i n d i n g was s t e r e o s e l e c t i v e w i t h t h e f r e e f r a c t i o n o f t h e R ( - ) - i s o m e r b e i n g 30% l e s s t h a n t h a t o f t h e S ( + ) - i s o m e r . F o r t h e p h a r m a c o k i n e t i c s t u d i e s , p l a s m a and s a l i v a l e v e l s w e r e f o l l o w e d f o r up t o 48 h o u r s and u r i n e l e v e l s f o r up t o 72 h o u r s . S t e r e o s e l e c t i v e u r i n a r y e x c r e t i o n and s a l i v a r y s e c r e t i o n , b o t h i n f a v o u r o f S ( + ) - m e x i l e t i n e , was o b s e r v e d . 118 T h i s was a r e f l e c t i o n o f t h e s t e r e o s e l e c t i v e p r o t e i n b i n d i n g o f t h e e n a n t i o m e r s . A s t e r e o s e l e c t i v e m e t a b o l i s m o f t h e e n a n t i o m e r s i n f a v o u r o f R ( - ) - m e x i l e t i n e was a l s o o b s e r v e d . The e n a n t i o m e r r a t i o s i n u r i n e and s a l i v a w e r e c o n s i s t e n t w i t h t h e f r e e e n a n t i o m e r r a t i o s i n p l a s m a a s o p p o s e d t o t h e t o t a l e n a n t i o m e r r a t i o s . T h i s e s t a b l i s h e d t h e l i n k b e t w e e n t h e p r o t e i n b i n d i n g o f t h e e n a n t i o m e r s and t h e l e v e l s i n u r i n e and s a l i v a , i n d i c a t i n g t h a t v a l u a b l e i n f o r m a t i o n c a n be o b t a i n e d b y m o n i t o r i n g t h e f r e e l e v e l s o f t h e e n a n t i o m e r s i n p l a s m a ( s e r u m ) . The p h a r m a c o k i n e t i c s o f t h e f r e e e n a n t i o m e r s o f m e x i l e t i n e i n humans w i l l be t h e s u b j e c t o f f u t u r e i n v e s t i g a t i o n s . T h u s , t h e d i s p o s i t i o n o f m e x i l e t i n e e n a n t i o m e r s i s s t e r e o s e l e c t i v e i n man. H o w e v e r , t h e c l i n i c a l i m p l i c a t i o n s o f t h i s 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 c a n n o t be e a s i l y p r e d i c t e d p r e s e n t l y s i n c e t h e r e l a t i v e a n t i a r r h y t h m i c p o t e n c i e s o f t h e i n d i v i d u a l e n a n t i o m e r s i s n o t y e t known. I t h a s b e e n s u g g e s t e d t h a t p l a s m a p r o t e i n b i n d i n g o f e n a n t i o m e r s m i g h t be u s e d a s a n i n d i c a t i o n o f t h e i r r e l a t i v e a c t i v i t i e s a t t h e r e c e p t o r s ( W i l l i a m s and L e e , 1 9 8 5 ) . T h i s h a s b e e n f o u n d t o be t h e c a s e f o r w a r f a r i n ( S e l l e r s a n d . K o c h - w e s e r , 1975) and P r o p r a n o l o l ( W a l l e e t a l . , 1 9 8 3 ) . H o w e v e r , t h i s i s an i d e a t h a t n e e d s a l o t o f e x p e r i m e n t a l p r o o f a nd i s f a r f r o m b e i n g a c c e p t e d . 119 6. REFERENCES Adams SS, B r e s l o f f P, and Mason CG: P h a r m a c o l o g i c a l d i f f e r e n c e s b e t w e e n t h e o p t i c a l i s o m e r s o f i b u p r o f e n : E v i d e n c e o f m e t a b o l i c i n v e r s i o n o f t h e ( - ) - i s o m e r . J . Pharm. P h a r m a c o l . , 28:256 ( 1 9 7 6 ) . A l b a n i F, R i v a R, C o n t i n M, B a r u z z i A: S t e r e o s e l e c t i v e b i n d i n g o f p r o p r a n o l o l e n a n t i o m e r s t o human a l p h a -- a c i d g l y c o p r o t e i n and human p l a s m a . B r . J . C l i n . P h a r m a c o l . , 18:244 ( 1 9 8 4 ) . A l e b i c - K o l b a h T, R e n d i c S, F u k s Z, S u n j i c V and K a j f e z F: E n a n t i o s e l e c t i v i t y i n t h e b i n d i n g o f d r u g s t o s e r u m p r o t e i n s . A c t a P h a r m a c e u t i c a Y u g o s l a v i a . 29:53 (1979) . A l l e n J D , K o f i - E k u e JM, S h a n k s RG and Z a i d i SA: The e f f e c t o f Ko 1 1 7 3 , a new a n t i c o n v u l s a n t a g e n t on e x p e r i m e n t a l c a r d i a c a r r h y t h m i a s . B r . J . P h a r m a c o l . , 4 5 : 5 6 1 ( 1 9 7 2 ) . A n g e r m a n n C a n d J a h r m a r k e r H: C o m p a r a t i v e a s s e s s m e n t o f t h e c a r d i o d e p r e s s a n t a c t i v i t y o f d i s o p y r a m i d e , m e x i l e t i n e and p r o p a f e n o n e . Z e i t s c h r i f t f u r k a r d i o l o g i e . 72:665 (1983) . A r i e n E J , S o u d i j n W and Timmermans PB ( E d s ) : S t e r e o -c h e m i s t r y and b i o l o g i c a l a c t i v i t y o f d r u g s . B l a c k w e l l S c i e n t i f i c P u b l i c a t i o n s , O x f o r d , L o n d o n ( 1 9 8 3 ) . A r m s t r o n g DW: C h i r a l s t a t i o n a r y p h a s e s f o r h i g h p e r f o r m a n c l i q u i d c h r o m a t o g r a p h i c s e p a r a t i o n o f e n a n t i o m e r s : A m i n i - r e v i e w . J . L i q . C h r o m a t o g r . , 7:353 ( 1 9 8 4 ) . B a i SA, W a l l e UK, W i l s o n MJ and W a l l e T: S t e r e o s e l e c t i v e b i n d i n g o f t h e ( - ) - e n a n t i o m e r o f p r o p r a n o l o l t o p l a s m a and e x t r a v a s c u l a r b i n d i n g s i t e s i n t h e d o g . D r u g M e t a b . D i s p o s . , 11:394 ( 1 9 8 3 ) . B a n i m SO, Da S i l v a A, S t o n e D a n d B a l l o n R: O b s e r v a t i o n s o f t h e h e modynamics o f m e x i l e t i n e . P o s t g r a d . Med. J . , 53 ( s u p p l . 1 ) : 7 4 ( 1 9 7 7 ) . B a r c h o w s k y A, S t a r g e l WW, Shand DG and R o u t l e d g e PA: s a l i v a 120 c o n c e n t r a t i o n s o f l i d o c a i n e and i t s m e t a b o l i t e s i n man. T h e r . D r u g M o n i t . , 4:335 ( 1 9 8 2 ) . B a u d i n e t G, H e n r a r d L, Q u i n a u x N, E l A l l a f D, L a n d s h e e r e C, C a r l i e r J a n d D r e s s e A: P h a r m a c o k i n e t i c s o f m e x i l e t i n e i n r e n a l i n s u f f i c i e n c y . A c t a C a r d i o l . , ( s u p p l . ) 25:55 ( 1 9 8 0 ) . B e c k e t t AH and C h i d o m e r e EC: The i d e n t i f i c a t i o n a n d a n a l y s i s o f m e x i l e t i n e and i t s m e t a b o l i c p r o d u c t s i n man. J . Pharm. P h a r m a c o l . , 29:281 ( 1 9 7 7 a ) . B e c k e t t AH and C h i d o m e r e EC: The d i s t r i b u t i o n , m e t a b o l i s m a n d e x c r e t i o n o f m e x i l e t i n e i n man. P o s t g r a d . Med. J . , 53 ( s u p p l . 1) 60 ( 1 9 7 7 b ) . Begg E J , C h i n w a h PM, Webb C, Day RO and Wade DN: E n h a n c e d m e t a b o l i s m o f m e x i l e t i n e a f t e r p h e n y t o i n a d m i n i s -t r a t i o n . B r . J . C l i n . P h a r m a c o l . , 14:219 ( 1 9 8 2 ) . Bhamra RK, F l a n a g a n R J and H o l t DW: H i g h - p e r f o r m a n c e l i q u i d - c h r o m a t o g r a p h i c method f o r t h e measu r e m e n t o f m e x i l e t i n e and f l e c a i n i d e i n b l o o d - p l a s m a o r se r u m . J . C h r o m a t o g r . , 307:439 ( 1 9 8 4 ) . B l a s c h k e G: C h r o m a t o g r a p h i c r e s o l u t i o n o f r a c e m a t e s . Angew. Chem. I n t . E n g l . , 19:13 ( 1 9 8 0 ) . Bopp R J , N a s h J F , R i d o l f o AS and S h e p a r d ER: S t e r e o -s e l e c t i v e i n v e r s i o n o f R ( - ) - b e n o x a p r o f e n t o t h e S ( + ) -e n a n t i o m e r i n humans. D r u g M e t a b . D i s p o s . , 7:356 (1979) . B o r g a 0, P i a f s k y KM and N i l s e n 0G: P l a s m a p r o t e i n b i n d i n g o f b a s i c d r u g s . 1. S e l e c t i v e d i s p l a c e m e n t f r o m a l p h a . - a c i d g l y c o p r o t e i n b y t r i s ( 2 - b u t o x y e t h y 1 ) p h o s p h a t e . C l i n . P h a r m a c o l . T h e r . , 22:539 ( 1 9 7 7 ) . B r a d b r o o k I D , James C and R o g e r s H J : A r a p i d method f o r d e t e r m i n a t i o n o f p l a s m a m e x i l e t i n e l e v e l s b y g a s - c h r o m -a t o g r a p h y . B r . J . C l i n . P h a r m a c o l . , 4:380 ( 1 9 7 7 ) . B r a d l e y G: M e x i l e t i n e and T o c a i n i d e : O r a l l y a c t i v e c o n g e n e r s o f l i d o c a i n e . C l i n . Pharm.,' 2:340 ( 1 9 8 3 ) . B r e i m e r DD and Van Rossum JM: P h a r m a c o k i n e t i c s o f ( + ) - , ( - ) - a n d (+_) - h e x o b a r b i t o n e i n man a f t e r o r a l a d m i n i s -t r a t i o n . J . Pharm. P h a r m a c o l . , 25:762 ( 1 9 7 3 ) . 121 B r e i t h a u p t H a n d W i l f l i n g M: D e t e r m i n a t i o n o f m e x i l e t i n e i n b i o l o g i c a l f l u i d s b y h i g h - p e r f o r m a n c e l i q u i d c h r o m a t o -g r a p h y . J . C h r o m a t o g r . , 230:97 ( 1 9 8 2 ) . Brown NA, J a h n c h e n E, M u l l e r WE and W o l l e r t U: O p t i c a l s t u d i e s on t h e m e c h a n i s m o f t h e i n t e r a c t i o n o f t h e e n a n t i o m e r s o f t h e a n t i c o a g u l a n t d r u g s p h e n p r o c o u m o n and w a r f a r i n w i t h human se r u m a l b u m i n . M o l . P h a r m a c o l . , 13:70 ( 1 9 7 7 ) . Brown J E and Shand DG: T h e r a p e u t i c m o n i t o r i n g o f a n t i a r -r h y t h m i c a g e n t s . C l i n . P h a r m a c o k i n e t . , 7:125 ( 1 9 8 2 ) . C a m p b e l l NPS, K e l l y J G , S h a n k s RG, C h a t u r v e d i NC, S t r o n g J E and P a n t r i d g e J F : M e x i l e t i n e (Ko 1173) i n t h e manage-ment o f v e n t r i c u l a r d y s r h y t h m i a s . L a n c e t t 11:404 (1973) . C a m p b e l l NPS, K e l l y J G , Adgey A A J and S h a n k s RG: The c l i n i c a l p h a r m a c o l o g y o f m e x i l e t i n e . B r . J . C l i n . P h a r m a c o l . , 6:103 ( 1 9 7 8 a ) . C a m p b e l l NPS, K e l l y J G , A d g e y A A J and S h a n k s RG: M e x i l e t i n e i n n o r m a l v o l u n t e e r s . B r . J . C l i n . P h a r m a c o l . , 6:372 ( 1 9 7 8 b ) . C a m p b e l l RWF, D o l d e r MA, P r e s c o t t L F , T a l b o t RG, M u r r a y A and J u l i a n DG: C o m p a r i s o n o f p r o c a i n a m i d e and m e x i l e t i n e i n t h e p r e v e n t i o n o f v e n t r i c u l a r a r r h y t h m i a s a f t e r a c u t e i n f a r c t i o n . L a n c e t 1:1257 ( 1 9 7 5 ) . Chew CYC, C o l l e t J and S i n g h BN: M e x i l e t i n e : A r e v i e w o f i t s p h a r m a c o l o g i c a l p r o p e r t i e s a n d t h e r a p e u t i c e f f i c a c y i n a r r h y t h m i a s . D r u g s 17:161 ( 1 9 7 9 ) . C l a r k RC and B a r k s d a l e JM: S y n t h e s i s and l i q u i d c h r o m a t o -g r a p h i c e v a l u a t i o n o f some c h i r a l d e r i v a t i z i n g a g e n t s f o r r e s o l u t i o n o f amine e n a n t i o m e r s . A n a l . Chem., 56 :958 (1984) . D a l g l i e s h CE: The s y n t h e s i s o f N - f o r m y 1 - D L - k y n u r e n i n e , N-a c e t y 1 - D L - K y n u r e n i n e a n d r e l a t e d c o m p o u n d s , a n d o b s e r v -a t i o n s on t h e s y n t h e s i s o f k y n u r e n i n e . Chem. S o c . J . , 137 (1952) . D a n i l o P: M e x i l e t i n e . Am. H e a r t J . , 97:399 ( 1 9 7 9 ) . D a n n e b e r g PB and S h e l l e y J H : The p h a r m a c o l o g y o f m e x i l -122 e t i n e . P o s t g r a d . Med. J . , 53 ( s u p p l . 1 ) : 2 5 ( 1 9 7 7 ) . Dawes C a n d J e n k i n s GN: The e f f e c t s o f d i f f e r e n t s t i m u l i on t h e c o m p o s i t i o n o f s a l i v a i n man. J . P h y s i o l . , 170:86 ( 1 9 6 4 ) . D o c u m e n t i a G e i g y : I n : S c i e n t i f i c T a b l e s . ( E d s ) : Diem K and L e u t n e r C , J.R. G e i g y , New Y o r k ( 1 9 7 0 ) . D u f f J H , Roden D, Primm RK, O a t e s JA and W o o s l e y RL: M e x i l t i n e i n t h e t r e a t m e n t o f r e s i s t a n t v e n t r i c u l a r a r r h y t h m i a s : E n h a n c e m e n t o f e f f i c a c y and r e d u c t i o n o f d o s e - r e l a t e d s i d e e f f e c t s by c o m b i n a t i o n w i t h q u i n i d i n e . c i r c u l a t i o n . 67:1124 ( 1 9 8 3 ) . E i c h e l b a u m M, B i r k e l P, G r u b e E, Gutgemann U and S o m o g y i A: E f f e c t s o f v e r a p a m i l on P-R i n t e r v a l s i n r e l a t i o n t o v e r a p a m i l p l a s m a l e v e l s f o l l o w i n g s i n g l e i . V . and o r a l a d m i n i s t r a t i o n a n d d u r i n g c h r o n i c t r e a t m e n t . K l i n i s c h e W o c h e n s c h r i f t . 58:919 ( 1 9 8 0 ) . E l A l l a f D, H e n r a r d L, C r o c h e l e t L, D e l a p i e r r e D, C a r l i e r J and D r e s s e A: P h a r m a c o k i n e t i c s o f m e x i l e t i n e i n r e n a l i n s u f f i c i e n c y . B r . J . C l i n . P h a r m a c o l . , 1 4 : 4 3 1 ( 1 9 8 2 ) . E l f i n g SM, S v e n s EH and L e s k i n e n EEA: G a s - l i q u i d c h r o m a t o -g r a p h i c d e t e r m i n a t i o n o f m e x i l e t i n e i n human s e r u m w i t h n i t r o g e n - s e n s i t i v e d e t e c t i o n . J . C l i n . Chem. C l i n . B i o c h e m . , 19:1189 ( 1 9 8 1 ) . F r a n k H, N i c h o l s o n G J and B a y e r E: R a p i d g a s - c h r o m a t o -g r a p h i c s e p a r a t i o n o f a m i n o a c i d e n a n t i o m e r s w i t h a n o v e l c h i r a l s t a t i o n a r y p h a s e . J . C h r o m a t o g r . S c i . , 15:174 ( 1 9 7 7 ) . F r a n k H, N i c h o l s o n G J and B a y e r E: G a s - c h r o m a t o g r a p h i c m a s s - s p e c t r o m e t r i e a n a l y s i s o f o p t i c a l l y a c t i v e m e t a b o -l i t e s a n d d r u g s on a n o v e l c h i r a l s t a t i o n a r y p h a s e . J . C h r o m a t o g r . 146:197 ( 1 9 7 8 ) . F r a n k H, Woiwode W, N i c h o l s o n G J and B a y e r E: i n " s t a b l e i s o t o p e s " ( Eds) K l e i n ER and K l e i n PD. P r o c . T h i r d i n t . C o n f . O a k b r o o k . I L , A c a d e m i c P r e s s , New Y o r k , N.Y., U.S.A. ( 1 9 7 9 ) . F r a n k H, R e t t e n m e i r A, W e i c k e r H, N i c h o l s o n G J and B a y e r E: A new g a s c h r o m a t o g r a p h i c method f o r d e t e r m i n a t i o n o f a m i no a c i d l e v e l s i n human s e r u m . C l i n . C h i m . A c t a . , 123 105:201 ( 1 9 8 0 ) . F rydman A, L a F r a g e J P , V i a l F, R u l l i e r e R and A l e x a n d r e JM: New e l e c t r o n - c a p t u r e g a s - l i q u i d c h r o m a t o g r a p h i c method f o r t h e d e t e r m i n a t i o n o f m e x i l e t i n e p l a s m a l e v e l s i n man. J . C h r o m a t o g r . , 145:401 ( 1 9 7 8 ) . G a l J , F r e n c h TA, Z y s s e t T and H a r o l d s e n P E : D i s p o s i t i o n o f ( R , S ) - t o c a i n i d e : Some s t e r e o s e l e c t i v e a s p e c t s . D r u g M e t a b . T h e r . , 10 ( 4 ) : 3 9 9 ( 1 9 8 2 ) . G i b a l d i M and P e r r i e r D: I n " P h a c o k i n e t i c s " . ( E d ) : S w a r b r i c k J . M a r c e l D e k k e r I n c . ( 1 9 7 5 ) . G r e c h - B e l a n g e r 0, T u r g e o n J and G i l b e r t M: H i g h - p r e s s u r e l i q u i d c h r o m a t o g r a p h i c a s s a y f o r m e x i l e t i n e i n s e r u m . J . C h r o m a t o g r . S c i . , 22:490 ( 1 9 8 4 ) . G r e c h - B e l a n g e r 0: G a s - c h r o m a t o g r a p h i c method f o r r o u t i n e s e rum m o n i t o r i n g o f m e x i l e t i n e . J . C h r o m a t o g r . , 309: 165 (1984) . G r e c h - B e l a n g e r 0, T u r g e o n J a n d G i l b e r t M: H i g h - p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h i c a s s a y f o r m e x i l e t i n e e n a n t i o m e r s i n human p l a s m a . J . C h r o m a t o g r . , 337-172 ( 1 9 8 5 a ) . G r e c h - B e l a n g e r 0, G i l b e r t M, T u r g e o n J and L e B l a n c PP: E f f e c t s o f c i g a r e t t e s m o k i n g on m e x i l e t i n e k i n e t i c s . C l i n . P h a r m a c o l . T h e r . , 3 7 ( 6 ) : 6 3 8 ( 1 9 8 5 b ) . G r e c h - B e l a n g e r 0, T u r g e o n 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 o f m e x i l e t i n e i n man. B r . J . C l i n . P h a r m a c o l . , 21:481 ( 1 9 8 6 ) . H a g e r WD, F e n s t e r p, M a y e r s o h n M, P e r r i e r D, G r a v e s P, M a r c u s F l and Goldman S: D i g o x i n - q u i n i d i n e i n t e r -a c t i o n : P h a r m a c o k i n e t i c e v a l u a t i o n . N. E n g . J . Med., 300:1238 ( 1 9 7 9 ) . H a s e l b a r t h V, D o e v e n d a n s J E and W o l f 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 o f m e x i l e t i n e i n h e a l t h y s u b j e c t s . C l i n . P h a r m a c o l . T h e r . , 29:729 ( 1 9 8 1 ) . H e n d e l J and B r o d t h a g e n H: E n t e r o h e p a t i c c y c l i n g o f metho-t r e x a t e e s t i m a t e d b y u s e o f t h e D - i s o m e r a s a r e f e r e n c e m a r k e r . E u r . J . C l i n . P h a r m a c o l . , 26:103 ( 1 9 8 4 ) . H e r z o g P, H o l t e r m u l l e r KH, K a s p e r W, M e i n e r t z T, T r e n k D and 124 J a h n c h e n E: A b s o r p t i o n o f m e x i l e t i n e a f t e r t r e a t m e n t w i t h g a s t r i c a n t a c i d s . B r . J . C l i n . P h a r m a c o l . 14:746 ( 1 9 8 2 ) . H o l t DW, P l a n a g a n RT, H a y l e r AM and L o i z o u M: S i m p l e g a s -l i q u i d c h r o m a t o g r a p h i c method f o r t h e m e asurement o f m e x i l e t i n e a n d l i g n o c a i n e i n b l o o d - p l a s m a o r s e r u m . J . C h r o m a t o g r . , 169,295 ( 1 9 7 9 ) . H o r o w i t z J D , A n a v e k a r SN, M o r r i s PM, G o b l e A J , D o y l e AE and L o i s WJ: C o m p a r a t i v e t r i a l o f m e x i l e t i n e and l i g n o -c a i n e i n t h e t r e a t m e n t o f e a r l y v e n t r i c u l a r t a c h y a r -r h y t h m i a s a f t e r a c u t e m y o c a r d i a l i n f a r c t i o n . J . C a r d i o v a s c . P h a r m a c o l . , 3:409 ( 1 9 8 1 ) . J e n n e r P and T e s t a B i n " C o n c e p t s i n D r u g M e t a b o l i s m " P a r t A. M a r c e l D e k k e r , I n c . , New Y o r k , N.Y., 1 9 8 0 , p . 5 3 . J o h n s o n DM, R e u t e r A, C o l l i n s JM and Thompson GF: E n a n t i o m e r i c p u r i t y o f N a p r o x e n by l i q u i d c h r o m a t o -g r a p h i c a n a l y s i s o f i t ' s d i a s t e r e o i s o m e r i c o c t y l e s t e r s . J . Pharm. S c i , 68:112 ( 1 9 7 9 ) . J o h n s t o n A, B u r g e s s CD, W a r r i n g t o n S J , W a d s w o r t h J and Hamer NAJ : The e f f e c t o f s p o n t a n e o u s c h a n g e s i n u r i n a r y pH on m e x i l e t i n e p l a s m a c o n c e n t r a t i o n s and e x c r e t i o n d u r i n g c h r o n i c a d m i n i s t r a t i o n t o h e a l t h y v o l u n t e e r s . B r . J . C l i n . P h a r m a c o l . , 8:349 ( 1 9 7 9 ) . K a c p r o w i c z AT: I m p r o v e d g a s - l i q u i d c h r o m a t o g r a p h i c method f o r m easurement o f m e x i l e t i n e i n p l a s m a . C l i n . Chem., 28 : 245 (1982) . K a s a i M, F r o u s s i o s C and Z i f f e r H: On t h e r e l a t i o n s h i p b e t w e e n e l u t i o n o r d e r and a b s o l u t e s t e r e o c h e m i s t r y o f a l k y l a r y l c a r b i n o l s f r o m a P i r k l e c o l u m n . J . O r g . Chem., 48:459 ( 1 9 8 3 ) . K awashima K, L e v y A and S p e c t o r S: S t e r e o s p e c i f i c r a d i o i m -m u n o-assay f o r p r o p r a n o l o l i s o m e r s . J . P h a r m a c o l . E x p . T h e r . , 196:517 ( 1 9 7 6 ) . Kaye CM, T u r n e r P and K i d d i e MA: V a r i a b l e p h a r m a c o k i n e t i c s o f m e x i l e t i n e . P o s t g r a d . Med. J . , 53 ( s u p p l . 1 ) : 5 6 (1977) K e l l y J G , Nimmo J , Rae R, S h a n k s RG and P r e s c o t t L F : S p e c t r o f l u o r o m e t r i c and g a s - l i q u i d c h r o m a t o g r a p h i c 125 methods f o r t h e e s t i m a t i o n o f m e x i l e t i n e (KO 1173) i n p l a s m a and u r i n e . J . Pharm. P h a r m a c o l . , 25:550 ( 1 9 7 3 ) . K e l l y J G : M e a s u r e m e n t o f m e x i l e t i n e p l a s m a c o n c e n t r a t i o n s . P o s t g r a d . Med. J . , 53 ( s u p p l . 1 ) : 4 8 ( 1 9 7 7 ) . K e l l y R, C h r i s t m o r e D, S m i t h R, D o s h i e r L and J a c o b s SL: M e x i l e t i n e i n p l a s m a by h i g h - p e r f o r m a n c e l i q u i d c h r o m a -t o g r a p h y . T h e r . D r u g . M o n i t . , 3:279 ( 1 9 8 1 ) . K i d d i e MA, K a y e CM, T u r n e r P a n d Shaw TRD: The i n f l u e n c e o f u r i n a r y pH on t h e e l i m i n a t i o n o f m e x i l e t i n e . B r . J . C l i n . P h a r m a c o l . , 1:229 ( 1 9 7 4 ) . 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 i n h e a l t h y s u b j e c t s t a k i n g c i m e t i d i n e . C l i n . P h a r m a c o l . T h e r . , 37 (6) : 669 (1985) . K l e i n HO, L a n d R, W e i s s E, D i s e g n i E, L i b h a b e r C, G u e r r e r o J and K a p l i n s k y E: The i n f l u e n c e o f v e r a p a m i l on s e r u m d i g o x i n c o n c e n t r a t i o n . C i r c u l a t i o n . 65:998 ( 1 9 8 2 ) . K o n i g WA, S t o l t i n g K and K r u s e K: Gas c h r o m a t o g r a p h i c s e p a r a t i o n o f o p t i c a l l y a c t i v e compounds i n g l a s s c a p i l l a r i e s . C h r o m a t o g r a p h i a 10:44 ( 1 9 7 7 ) . K o n i g WA and S i e v e r s S: S t r u c t u r a l r e q u i r e m e n t s f o r e n a n t i o s e l e c t i v i t y i n g a s c h r o m a t o g r a p h y o f c h i r a l a l p h a - h y d r o x y a c i d s . J . C h r o m a t o g r . , 200:189 ( 1 9 8 0 ) . K o n i g WA, B e n e c k e I and S i e v e r s S: New r e s u l t s i n t h e g a s c h r o m a t o g r a p h i c s e p a r a t i o n o f e n a n t i o m e r s o f h y d r o x y -a c i d s and c a r b o h y d r a t e s . J . C h r o m a t o g r . , 217:71 (1981) . K o n i g WA, F r a n k e W and B e n e c k e I : Gas c h r o m a t o g r a p h i c e n a n t i o m e r s e p a r a t i o n o f c h i r a l a l c o h o l s . J . C h r o m a t o g r . , 239:227 ( 1 9 8 2 ) . K o p p e H: The d e v e l o p m e n t o f m e x i l e t i n e . P o s t g r a d . Med. J . , 53 ( s u p p l . 1) :22 (1977) . K o y s o o k o R, E l l i s EF a n d L e v y G: R e l a t i o n s h i p b e t w e e n t h e o -p h y l i n e c o n c e n t r a t i o n i n p l a s m a and s a l i v a i n man. C l i n . P h a r m a c o l . T h e r . , 15:454 ( 1 9 7 4 ) . K r i p a l a n i K J , A d d i n ZE, Dean AV and S h r e i b e r EC: S t e r e o -126 s p e c i f i c i n v e r s i o n o f 1 - a l p h a - m e t h y I f l u o r i n e - 2 - a c e t i c a c i d t o i t ' s d - e n a n t i o m e r i n t h e d o g . X e n o b i o t i c a . 6 : 159 (1976) . Kuhn P, K r o i s s A, K a i n d l P, K l i c p e r a M and Z i l c h e r H: A n t i -a r r h y t h m i c and hemodynamic e f f e c t s o f m e x i l e t i n e . P o s t g r a d . Med. J . , 53 ( s u p p l . 1 ) : 8 1 ( 1 9 7 7 ) . L a n SU, K r i p a l a n i K J , Dean AV, E g l i P, D i f a z i o LT and S c h r e i b e r EC: I n v e r s i o n o f o p t i c a l c o n f i g u r a t i o n o f a l p h a - m e t h y l f l o u r i n e - 2 - a c e t i c a c i d ( c i c l o p r o f e n ) i n r a t s a n d monkeys. D r u g M e t a b . D i s p o s . , 4:330 ( 1 9 7 6 ) . L e a h e y EB J r , G i a r d i n a EGV and B i g g e r JT J r : E f f e c t o f v e n t r i c u l a r f a i l u r e on s t e a d y s t a t e k i n e t i c s o f m e x i l e t i n e . C l i n . R e s . , 28:239A ( 1 9 8 0 a ) . L e a h e y EB J r , R e i f f e l J A , G i a d i n a EGV and B i g g e r J T J r : The e f f e c t o f q u i n i d i n e and o t h e r o r a l a n t i a r r y t h m i c d r u g s o n serum d i g o x i n . A p r o s p e c t i v e s t u d y . Ann. I n t e r n . Med. 92:605 ( 1 9 8 0 b ) . L e a h e y EB J r , H e i s s e n b u t t e l RH, G i a r d i n a EGV and B i g g e r JT J r : C o m b i n e d m e x i l e t i n e and p r o p r a n o l o l t r e a t m e n t o f r e f r a c t o r y v e n t r i c u l a r t a c h y c a r d i a . B r . Med. J . , 2 8 1 : 357 ( 1 9 8 0 c ) . L e a h e y EB J r , B i g g e r J T J r , B u t l e r VP J r , R e i f f e l J A , O ' C o n n e l l GC, S c a f f i d i LE and R o t t m a n J N : Q u i n i d i n e -d i g o x i n i n t e r a c t i o n : Time c o u r s e a nd p h a r m a c o k i n e t i c s . Am. J . C a r d i o l , 48:114 ( 1 9 8 1 ) . L e e E J D , W i l l i a m s KM, Graham GG, Day RO and C h a m p i o n DG: L i q u i d c h r o m a t o g r a p h i c d e t e r m i n a t i o n o f p l a s m a c o n c e n -t r a t i o n p r o f i l e o f o p t i c a l i s o m e r s o f i b u p r o f e n i n humans. J . Pharm. S c i . , 73 ( 1 1 ) : 1 5 4 2 ( 1 9 8 4 ) . L e e E J D , W i l l i a m K, Day R, Graham G a n d C h a m p i o n D: S t e r e o -s e l e c t i v e d i s p o s t i o n o f i b u p r o f e n e n a n t i o m e r s i n man. B r . J . C l i n . P h a r m a c o l . , 19:669 ( 1 9 8 5 ) . L e v y G and Y a c o b i A: E f f e c t o f p l a s m a p r o t e i n b i n d i n g ' o n e l i m i n a t i o n o f w a r f a r i n . J . Pharm. S c i . , 63:805 (1974) . L e v y RH: M o n i t o r i n g o f f r e e d r u g l e v e l s : D e t e r m i n a t i o n o f f r e e c o n c e n t r a t i o n v e r s u s f r e e f r a c t i o n . T h e r . D r u g . M o n i t . , 5: 243 (1983) . 127 L i m a J L , J u n g b l u t h GL, D e v i n e T, and R o b e r t s o n LW: S t e r e o -s e l e c t i v e b i n d i n g o f d i s o p y r a m i d e t o human p l a s m a p r o t e i n . L i f e S c i . , 35:835 ( 1 9 8 4 ) . L i n d n a WF: i n " C h e m i c a l D e r i v a t i z a t i o n i n A n a l y t i c a l C h e m i s t r y " . (Eds) F r e i RW and L a w r e n c e J P . P l e n u m P r e s s , New Y o r k , p. 145 ( 1 9 8 2 ) . L i u RH and Ku WW: C h i r a l s t a t i o n a r y p h a s e s f o r t h e g a s -l i q u i d c h r o m a t o g r a p h i c s e p a r a t i o n o f e n a n t i o m e r s . J . C h r o m a t o g r . , 271:309 ( 1 9 8 3 ) . L o c h m u l l e r CH and S o u t e r RW: C h r o m a t o g r a p h i c r e s o l u t i o n o f e n a n t i o m e r s . J . C h r o m a t o g r . , 113:283 ( 1 9 7 5 ) . M a t i n SB, Wan SH and Karam J H : P h a r m a c o k i n e t i c s o f t o l b u t -a m i d e : P r e d i c t i o n b y c o n c e n t r a t i o n i n s a l i v a . C l i n . P h a r m a c o l . T h e r . 16:1052 ( 1 9 7 4 ) . M a r s h a l l J R , M u i r AW and W i n s l o w E: C o m p a r a t i v e a n t i d y s -r y t h m i c a n d hemodynamic e f f e c t s o f o r a l l y o r i n t r a v e n -o u s l y a d m i n i s t e r e d m e x i l e t i n e and ORG 6001 i n t h e a n a e s t h e t i z e d r a t . B r . J . P h a r m a c o l . , 74:381 ( 1 9 8 1 ) . M a s t r o p a o l o W, Holmes DR, O s b o r n e MJ, Rooke J and Moyer TR: I m p r o v e d 1 i q u i d - c h r o m a t o g r a p h i c d e t e r m i n a t i o n o f m e x i l e t i n e , an a n t i a r r h y t h m i c d r u g , i n p l a s m a . C l i n . Chem., 30:319 ( 1 9 8 4 ) . M cComish M, K i t s o n D, R o b i n s o n 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 o f m e x i l e t i n e . P o s t g r a d . Med. J . , 53 ( s u p p l . 1 ) : 8 5 ( 1 9 7 7 ) . M c E r l a n e KM and P i l l a i GK: G a s - l i q u i d c h r o m a t o g r a p h i c r e s o l u t i o n and a s s a y o f t o c a i n i d e e n a n t i o m e r s u s i n g a c h i r a l c o l u m n a nd s t u d y o f 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 s i n man. J . C h r o m a t o g r . , 274:129 ( 1 9 8 3 ) . M c E r l a n e KM, I g w e m e z i e L and K e r r CR: S t e r e o s e l e c t i v e a n a l y s i s o f t h e e n a n t i o m e r s o f m e x i l e t i n e by HPLC u s i n g f l u o r e s c e n t d e t e c t i o n , and s t u d y o f 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 . C h r o m a t o g r . , ( i n p r e s s ) . McMenamy RH and O n c l e y J L : The s p e c i f i c b i n d i n g o f L - t r y p -t o p h a n t o serum a l b u m i n . J . B i o l . Chem., 233:1436 (1958) . 128 McNamara P J , S l a u g h t e r RL, P i e p e r J A , Wyrnan MG and L a l k a D: F a c t o r s i n f l u e n c i n g s erum p r o t e i n b i n d i n g o f L i d o c a i n e i n humans. A n e s t h . A n a l g . 60:395 ( 1 9 8 1 ) . M e t z l e r CM: NONLIN: A c o m p u t e r p r o g r a m f o r p a r a m e t e r e s t i m a t i o n i n n o n - l i n e a r s i t u a t i o n s . K a l a m a z o o , M i c h i g a n . The U p j o h n Co., ( 1 9 7 4 ) . M o y s e y J O , J a g g a r a o NSV, G r u n d y EN a n d C h a m b e r l a i n DA: A m i -o d a r o n e i n c r e a s e p l a s m a d i g o x i n c o n c e n t r a t i o n . B r . Med. J . , 182 : 272 (1981) . M u c k l o w J C , B e n d i n g MR, Kahn GC and D o l l e r y CT: D r u g C o n c e n -t r a t i o n i n s a l i v a . C l i n . P h a r m a c o l . T h e r . , 2 4 ( 5 ) :563 ( 1 9 7 8 ) . M u l l e r WE and W o l l e r t U: H i g h s t e r e o s p e c i f i c i t y o f t h e b e n z o d i a z e p i n e b i n d i n g s i t e on human se r u m a l b u m i n . M o l . P h a r m a c o l . , 11:52 ( 1 9 7 5 ) . N i t s c h J , D o l i w a R, S t e i n b e c k 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 b e i P a t i e n t e n m i t v e n t r i k u l a r e n A r r h t h m e i n und N i e r e n l e b e r O d e r H e r z i n s u f f u z i e n z . V e r h . D t s c h . G e s . I n n . Med., 87:429 ( 1 9 8 1 ) . O h a s h i K, E b i h a r a A, H a s h i m o t o T, H o soda S, Kondo K and Oka T: P h a r m a c o k i n e t i c s and t h e a n t i a r r h y t h m i c e f f e c t o f m e x i l e t i n e i n p a t i e n t s w i t h c h r o n i c v e n t r i c u l a r a r -r h y t h m i a s . A r z n e i m . - F o r s c h . / D r u g R e s . , 3 4 ( 1 ) : 5 0 3 (1984) . P a a l m a n ACA, Roos J C , S i e b e l i n k J and D u n n i n g A J : D e v e l o p -ment o f a d o s a g e scheme f o r s i m u l t a n e o u s i n t r a v e n o u s and o r a l a d m i n i s t r a t i o n o f m e x i l e t i n e . P o s t g r a d . Med. J . , 53 ( s u p p l . 1) :128 (1977) . P a t i l N, L a P i d u s J B a n d Tye A: S t e r i c a s p e c t s b f a d r e n e r g i c d r u g s . J . Pharm, S c i . , 59:1025 ( 1 9 7 0 ) . P a t i l PN, P a t e l DG and D r e l l RD: s t e r i c a s p e c t s o f a d r e n -e r g i c d r u g s . XV. Use o f i s o m e r i c a c t i v i t y r a t i o a s a c r i t e r i o n t o d i f f e r e n t i a t e a d r e n e r g i c r e c e p t o r s . J . P h a r m a c o l . E x p . T h e r . , 1 7 6 : 6 2 2 ( 1 9 7 1 ) . P e d e r s e n KE, D o r p h - P e d e r s e n A, H v i d t S, K l i t g a a r d NH and N i e l s e n - K u d s k F: D i g o x i n - v e r a p a m i l i n t e r a c t i o n . C l i n . P h a r m a c o l . T h e r . , 30:311 ( 1 9 8 1 ) . 129 P e d e r s e n KE, D o r p h - P e d e r s e n A, H v i d t S, K l i t g a a r d NH and P e d e r s e n KK: The l o n g - t e r m e f f e c t o f v e r a p a m i l on p l a s m a d i g o x i n c o n c e n t r a t i o n and r e n a l d i g o x i n c l e a r a n c e i n h e a l t h y s u b j e c t s . E u r . J . C l i n . P h a r m a c o l . , 22:123 ( 1 9 8 2 ) . P e n t i k a i n e n P J , K o i v u l a IH and H i l t u n e n HA: E f f e c t s o f r i f a m p i c i n t r e a t m e n t on t h e k i n e t i c s o f m e x i l e t i n e . E u r . J . C l i n . P h a r m a c o l . , 23:261 ( 1 9 8 2 ) . P e n t i k a i n e n P J , H a l i n e n M and H e l i n M: P h a r m a c o k i n e t i c s o f i n t r a v e n o u s m e x i l e t i n e i n p a t i e n t s w i t h a c u t e m y o c a r -d i a l i n f a r c t i o n . J . C a r d i o v a s c . P h a r m a c o l . , 6:1 (1984) . P e r c h a l s k i R J , W i l d e r B J and Hammer RH: F l a m e - i o n i z a t i o n a n d e l e c t r o n - c a p t u r e g a s - l i q u i d c h r o m a t o g r a p h i c d e t e r m i n a t i o n o f 1 - ( 2 , 6 - d i m e t h y l p h e n o x y ) - 2 - a m i n o p r o p a n e i n p l a s m a . J . Pharm. S c i . , 63:1489 ( 1 9 7 4 ) . P i a f s k y KM and B o r g a 0: P l a s m a p r o t e i n b i n d i n g o f b a s i c d r u g s 1 1 . I m p o r t a n c e o f a l p h a s - a c i d g l y c o p r o t e i n f o r i n t e r i n d i v i d u a l v a r i a t i o n . C l i n . P h a r m a c o l . T h e r . , 22 (5) : 545 (1977) . P i a f s k y KM a n d K n o p p e r t D: B i n d i n g o f l o c a l a n a e s t h e t i c s t o a l p h a - a c i d g l y c o p r o t e i n . C l i n . R e s . , 26:836A (1978T". P i a f s k y KM: D i s e a s e i n d u c e d c h a n g e s i n t h e p l a s m a b i n d i n g o f b a s i c d r u g s . C l i n . P h a r m a c o k i n e t . , 5:246 ( 1 9 8 0 ) . P i l l a i GK, A x e l s o n J E and M c E r l a n e KM: S t e r e o s p e c i f i c s a l i v a r y e x c r e t i o n o f t o c a i n i d e e n a n t i o m e r s i n man. R e s . Commun. Chem. P a t h . P h a r m a c o l . , 43:209 ( 1 9 8 4 ) . P i r k l e WH, W e l c h C J and M a h l e r GS: C h r o m a t o g r a p h i c s e p a r a t i o n o f t h e e n a n t i o m e r s o f N - a c y l a t e d h e t e r o -c y c l i c a m i n e s . J . O r g . Chem., 49:2504 ( 1 9 8 4 ) . P i r k l e WH and W e l c h C J : C h r o m a t o g r a p h i c s e p a r a t i o n o f t h e e n a n t i o m e r s o f a c y l a t e d a m i n e s on c h i r a l s t a t i o n a r y p h a s e s . J . O r g . Chem., 49 ( 1 ) : 1 3 8 ( 1 9 8 4 ) . P i r k l e WH and T s i p o u r a s A: D i r e c t l i q u i d c h r o m a t o g r a p h i c s e p a r a t i o n o f b e n z o d i a z e p i n o n e e n a t i o m e r s . J . C h r o m a t o g r . , 291:291 ( 1 9 8 4 ) . 130 P o d r i d P J and Lown B: M e x i l e t i n e f o r v e n t r i c u l a r a r r h y t h m i a s . Am. J . C a r d i o l . , 47:895 ( 1 9 8 1 ) . P o t t a g e A, C a m p b e l l RWF, A c h u f f SC, M u r r a y A, J u l l i a n DG and P r e s c o t t L F : The a b s o r p t i o n o f o r a l m e x i l e t i n e i n c o r o n a r y c a r e p a t i e n t s . E u r . J . C l i n . P h a r m a c o l . , 1 3 : 393 ( 1 9 7 8 ) . P o z e n e l H: Hemodynamic s t u d i e s o n m e x i l e t i n e , a new a n t i a r -r h y t h m i c a g e n t . P o s t g r a d . Med. J . , 53 ( s u p p l . 1 ) : 7 8 (1977) . P r e s c o t t L F , C l e m e n t s J A and P o t t a g e A: A b s o r p t i o n , d i s t r i b u t i o n a nd e l i m i n a t i o n o f m e x i l e t i n e . P o s t g r a d . Med. J . , 5 3 ( s u p p l . 1 ) : 5 0 ( 1 9 7 7 ) . P r o d u c t M o n o g r a p h : M e x i t i l . B o e h r i n g e r I n g e l h e i m L t d . , C a n a d a , M a r c h ( 1 9 8 6 ) . Rose HC, S t e r n RL and K a r g e r BL: S t u d i e s on t h e m e c h a n i s m o f s e p a r a t i o n o f d i a s t e r e o m e r i c e s t e r s by g a s - l i q u i d c h r o m a t o g r a p h y : E f f e c t o f b u l k d i s s y m m e t r y and d i s t a n c e b e t w e e n o p t i c a l c e n t r e s . A n a l . Chem.,38 ( 3 ) : 469 ( 1 9 6 6 ) . R o s s C J , D u n n i n g A J and P a a l m a n DCA: E l e c t r o p h y s i o l o g i c a l e f f e c t s o f m e x i l e t i n e i n man. P o s t g r a d . Med. J . , 53 ( s u p p l . 1) :92 (1977) . R o u t l e d g e PA, S t a r g e l WW, Wagner GS a n d S h a n d DG: I n c r e a s e d a l p h a s - a c i d g l y c o p r o t e i n and l i d o c a i n e d i s p o s i t i o n i n m y o c a r d i a l i n f a r c t o i n . Ann. I n t . Med., 93:70 ( 1 9 8 0 ) . R o w l a n d M: P l a s m a p r o t e i n b i n d i n g and t h e r a p e u t i c d r u g m o n i t o r i n g . T h e r . D r u g M o n i t . , 2:29 ( 1 9 8 0 ) . R o w l a n d M and T o z e r TN: C l i n i c a l P h a r m a c o k i n e t i c s : C o n c e p t s and a p p l i c a t i o n s . L e a and F e b i g e r , P h i l a d e l p h i a , p.53 (1980) S a s t r y BVR: S t e r e o i s o m e r i s m and d r u g a c t i o n i n t h e n e r v o u s s y s t e m . Ann. Rev. P h a r m a c o l . , 13:253 ( 1 9 7 3 ) . S a u n a m a k i K I : Hemodynamic e f f e c t s o f a new a n t i a r r h y t h m i c a g e n t , m e x i l e t i n e (Ko 1173) , i n i s c h a e m i c h e a r t d i s e a s e . C a r d i o v a s c . R e s . , 9:788 ( 1 9 7 5 ) . 131 S e l l e r s EM and Koch-Weser J : I n t e r a c t i o n o f w a r f a r i n s t e r e o i s o m e r s w i t h human a l b u m i n . P h a r m a c o l . R e s . Commun., 7 : 331 (1975) . S h a n d DG: A l p h a - - a c i d g l y c o p r o t e i n a n d p l a s m a l i d o c a i n e b i n d i n g . C l i n . P h a r m a c o k i n e t . , ( s u p p l . 1 ) : 27 ( 1 9 8 4 ) . S i l b e r B a n d R e i g e l m a n S: S t e r e o s p e c i f i c a s s a y f o r ( - ) - and ( + ) - p r o p r a n o l o l i n human a n d d o g p l a s m a . J . P h a r m a c o l . E x p . T h e r . , 215:643 ( 1 9 8 0 ) . Simmonds RG, Woodage T J , D u f f SM and G r e e n J N : S t e r e o s p e c -i f i c i n v e r s i o n o f R ( - ) - b e n o x a p r o f e n i n r a t and man. E u r . J . D r u g M e t a b . P h a r m a c o k i n e t . , 5:169 ( 1 9 8 0 ) . S i m o n y i M: On c h i r a l d r u g a c t i o n . Med. R e s . R e v . , 4:359 (1984) . S i n g h BN and V a u g h a n - W i l l i a m s EM: I n v e s t i g a t i o n s o f t h e mode o f a c t i o n o f a new a n t i d y s r h y t h m i c d r u g , Ko 1 1 7 3 . B r . J . P h a r m a c o l . , 44:1 ( 1 9 7 2 ) . S i n g h BN, Cho YW and K u e m m e r l e HP: C l i n i c a l p h a r m a c o l o g y o f a n t i - a r r h y t h m i c d r u g s : a r e v i e w a n d o v e r v i e w , P a r t I I . 19 (5) : 185 (1981) . S i n g h J B , R a s u l AM, Shah A, Adams E, F l e s s a s A and K o c o t SL: E f f i c a c y o f m e x i l e t i n e i n c h r o n i c v e n t r i c u l a r a r -r h y t h m i a s c o m p a r e d w i t h q u i n i d i n e : A s i n g l e - b l i n d , r a n d o m i z e d t r i a l . Am. J . C a r d i o l . , 53:84 ( 1 9 8 4 ) . S m i t h DF ( E d ) : CRC h a n d book o f s t e r e o i s o m e r s : D r u g s i n P s y c h o p h a r m a c o l o g y . CRC P r e s s I n c . , B o c a R a t o n , F l o r i d a (1984) . S m i t h K J and M e f f i n P J : M e x i l e t i n e a n a l y s i s i n b l o o d and p l a s m a u s i n g g a s c h r o m a t o g r a p h y 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 . C h r o m a t o g r . , 181:469 ( 1 9 8 0 ) . S o m o g y i M: D e t e r m i n a t i o n o f b l o o d s u g a r . J . B i o l . Chem., 160:69 ( 1 9 4 5 ) . T a l b o t RG, Nimmo J , J u l i a n DG, C l a r k RA, N i e l s o n JMM and P r e s c o t t L F : T r e a t m e n t o f v e n t r i c u l a r a r r h y t h m i a s w i t h m e x i l e t i n e (Ko 1 1 7 3 ) . L a n c e t 11:399 ( 1 9 7 3 ) . T a l b o t RG, J u l i a n DG and P r e s c o t t L F : L o n g - t e r m t r e a t m e n t o f v e n t r i c u l a r a r r h y t h m i a s w i t h o r a l m e x i l e t i n e . Am. 132 H e a r t J . 91:58 (1976) . T a m e g a i T, Ohmae M, Kawabe K and Tomoeda M: S e p a r a t i o n o f o p t i c a l i s o m e r s a s d i a s t e r e o m e r i c d e r i v a t i v e s b y h i g h -p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y . J . L i q . C h r o m a t o g r . 2 (8) : 1229 (1979) . The M e r c k I n d e x : (Ed) W i n d h o l z M. M e r c k and Co., I n c . , New J e r s e y , USA. (1983) . T o c c o D J , Hooke KF, D e l u n a FA and Duncan AEW: S t e r e o -s p e c i f i c b i n d i n g o f t i m o l o l , a b e t a - a d r e n e r g i c b l o c k i n g a g e n t . D r u g . M e t a b . D i s p o s . , 4:323 ( 1 9 7 6 ) . Toon S and T r a g e r WF: P h a r m a c o k i n e t i c i m p l i c a t i o n s o f s t e r e o s e l e c t i v e c h a n g e s i n p l a s m a - p r o t e i n b i n d i n g : W a r f a r i n / s u l f i n p y r a z o n e . J . Pharm. S c i . , 7 3 ( 1 1 ) : 1671 ( 1 9 8 4 ) . T r i m a r c o B, R i c c i a r d e l l i B, D e L u c a N, V o l p e M, S a c c a L, Rengo F and C o n d o r e l l i M: D i s o p y r a m i d e , m e x i l e t i n e and p r o c a i n a m i d e i n t h e l o n g - t e r m o r a l t r e a t m e n t o f v e n t r i c u l a r a r r y t h m i a s : A n t i a r r h y t h m i c e f f i c a c y and hemodynamic e f f e c t s . C u r r e n t T h e r . R e s . , 3 3 ( 3 ) : 472 (1983) . V a l d i v i e s o L, B l a s c h k e T, and G i a c o m i n i K. D i s o p y r a m i d e e n a n t i o m e r s b i n d s t e r e o s e l e c t i v e l y t o human p l a s m a p r o t e i n . 2nd W o r l d C o n f e r e n c e on C l i n i c a l P h a r m a -c o l o g y and T h e r a p e u t i c s . W a s h i n g t o n , D.C. ( 1 9 8 3 ) . V a s i l i a d e s J , K e l l e t t J and Cox RS: Gas c h r o m a t o g r a p h i c d e t e r m i n a t i o n o f m e x i l e t i n e w i t h a n i t r o g e n - s e l e c t i v e d e t e c t o r . Am. J . C l i n . P a t h . , 81:776 ( 1 9 8 4 ) . V a u g h a n - W i l l i a m s EM: M e x i l e t i n e i n i s o l a t e d t i s s u e m o d e l s . P o s t g r a d . Med. J . , 53 ( s u p p l . 1 ) : 3 0 ( 1 9 7 7 ) . V o g e l A I : "A t e x t b o o k o f O r g a n i c C h e m i s t r y . " Longmans, L o n d o n , E n g l a n d . p. 652 (1964) . V o g e l g e s a n g B, E c h i z e n H, S c h m i d t E and E i c h e l b a u m M: S t e r e -o s e l e c t i v e f i r s t - p a s s m e t a b o l i s m o f h i g h l y c l e a r e d d r u g s : S t u d i e s o f t h e b i o a v a i l a b i l i t y o f L- and D-v e r a p a m i l e x a m i n e d w i t h a s t a b l e i s o t o p e t e c h n i q u e . B r . J . C l i n . P h a r m a c o l . , 18:733 ( 1 9 8 4 ) . Von B a h r C, H e r m a n s s o n J and T a w a r a K: P l a s m a l e v e l s o f (+) and ( - ) - p r o p r a n o l o l and 4 - h y d r o x y p r o p r a n o l o l a f t e r 133 a d m i n i s t r a t i o n o f r a c e m i c ( + _ ) - p r o p r a n o l o l i n man. B r . J . C l i n . P h a r m a c o l . , 14:79 ( 1 9 8 2 ) . Wade DN, M e a r r i c k PT and M o r r i s J L : A c t i v e t r a n s p o r t o f L- d o p a i n t h e i n t e s t i n e . N a t u r e . 242:463 ( 1 9 7 3 ) . W a l l e T and W a l l e KU: S t e r e o s e l e c t i v e o r a l b i o a v a i l a b i l i t y o f r a c e m i c p r o p r a n o l o l i n t h e d o g : A GCMS s t u d y u s i n g a s t a b l e i s o t o p e t e c h n i q u e . R e s . Commun. Chem. P a t h . P h a r m a c o l . , 23:453 ( 1 9 7 9 ) . W a l l e KU, W a l l e T, B a i SA and O r a n o f f L S : S t e r e o s e l e c t i v e b i n d i n g o f p r o p r a n o l o l t o human p l a s m a , a l p h a - - a c i d g l y c o p r o t e i n and a l b u m i n . C l i n . P h a r m a c o l . T n e r . , 34 : 718 (1983) . W a l l e T and W a l l e KU: P h a r m a c o k i n e t i c p a r a m e t e r s o b t a i n e d w i t h r a c e m a t e s . T I P S . 7 (4) -.155 ( 1 9 8 6 ) . Weems HB and Yang SK: R e s o l u t i o n o f o p t i c a l i s o m e r s by c h i r a l h i g h - p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y : S e p a r a t i o n o f d i h y d r o d i o l s and t e t r a h y d r o d i o l s o f b e n z o [ a ] p y r e n e and b e n z o [ a ] a n t h r a c e n e . A n a l . B i o c h e m . , 125: 156 ( 1 9 8 2 ) . W h i t e SM and F a r i d NA. D e t e r m i n a t i o n o f m e x i l e t i n e a n d i t s m e t a b o l i t e s i n serum b y 1 i q u i d - c h r o m a o g r a p h y w i t h f l u o r e s c e n c e d e t e c t i o n . J . C h r o m a t o g r . , 375:458 (1983) . W i l l i a m s K and L e e E: I m p o r t a n c e o f d r u g e n a n t i o m e r s i n c l i n i c a l p h a r m a c o l o g y . D r u g s , 30:333 ( 1 9 8 5 ) . Wing LMH, M e f f i n P J , G r y g i e l J J , S m i t h K J and B i r k e t t D J : E f f e c t o f m e t o c l o p r a m i d e and a t r o p i n e on t h e a b s o r p t i o n o f o r a l l y a d m i n i s t e r e d m e x i l e t i n e . B r . J . C l i n . P h a r m a c o l . , 9:505 ( 1 9 8 0 ) . Y a c o b i A, U d a l l J A and L e v y G: Serum p r o t e i n b i n d i n g as a d e t e r m i n a n t o f w a r f a r i n b o d y c l e a r a n c e and a n t i c o a g u l a n t e f f e c t . C l i n . P h a r m a c o l . T h e r . , 1 9 ( 5 ) : 552 (1976) . Y a m a g u c h i I , S i n g h BN and M a n d e l WJ: E l e c t r o p h y s i o l o g i c a l a c t i o n s o f m e x i l e t i n e on i s o l a t e d r a b b i t a t r i a a n d c a n i n e v e n t r i c u l a r m u s c l e and p u r k i n j e f i b r e s . C a r d i o v a s c . R e s . 13:288 ( 1 9 7 9 ) . 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
https://iiif.library.ubc.ca/presentation/dsp.831.1-0096727/manifest

Comment

Related Items